Pallaval Veera Bramhachari Editor

Dynamics
of Immune
Activation in
Viral Diseases
Dynamics of Immune Activation in Viral
Diseases
Pallaval Veera Bramhachari
Editor

Dynamics of Immune
Activation in Viral Diseases
Editor
Pallaval Veera Bramhachari
Department of Biotechnology
Krishna University
Machilipatnam, Andhra Pradesh, India

ISBN 978-981-15-1044-1    ISBN 978-981-15-1045-8 (eBook)

https://doi.org/10.1007/978-981-15-1045-8

© Springer Nature Singapore Pte Ltd. 2020

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Preface

Viral infectious diseases signify an essential portion of global public health concern
with millions of deaths annually. Viruses produce disease directly by killing the host
cells they infect or by liberating toxins that can cause tissue damage and functional
derangements in neighboring or distant cells and tissues. In the field of viral immu-
nology, the researchers continue to make noteworthy and exhilarating contributions
to our understanding of the fundamental biology of the immune system. Yet the
practical translational applications of this fascinating and enthralling area of science
are little disappointing with regard to the recurrent viral outbreaks.
The development of an infectious disease in an individual involves complex
interactions between the virus and the host. These interactions are a dynamic inter-
play of host mechanisms aimed at eliminating infections and viral strategies
designed to permit survival in the face of beneficial activation of innate and adaptive
immune responses. Different types of infectious viral agents stimulate distinct types
of immune responses and have evolved unique mechanisms for evading immunity.
Noteworthy, in many instances, virus-induced proteins produced by emerging
viruses can trickily antagonize specific immune recognition mechanisms or circum-
vent cell intrinsic restriction factors, thus influencing the host antiviral immune
responses and complicating the viral replication kinetics. However, there is little
understanding of within-host immunological processes underlying the reservoir
host virus interactions, and this question is hardly addressed in several emerging
viral diseases. Moreover, the viral genome itself plays an imperative role in the
rapid evasion of adaptive immune responses, with the generation of diverse viral
quasi-species that is an inherent consequence of error-prone RNA replication mech-
anisms. Moreover, elite immune controllers do not eliminate the virus from their
system, because of the eventual emergence of virus variants that manage to escape
the determined efforts of the immune system.
From serious pandemics and highly contagious infections to common influenza
episodes, clinical prognosis often relies on early detection of the infectious agent.
The recently developed viral diagnostic methods are reshaping the field of clinical
microbiology and could contribute to reducing the prevalence of serious infectious
diseases. Fortunately, the aspects of modernization that help drive pathogen emer-
gence can also propel scientific innovation and current significant scientific advances
have warranted our skill set to address the challenge of emerging viruses. From
advanced genomic sequencing to novel methods in structural biology, we now have

v
vi Preface

an increasingly classy toolkit with which to facilitate the detection and possible
control of emerging viral diseases. Still, current outbreaks of viral diseases serve as
powerful reminders of our ongoing vulnerability to emerging viral pathogens.
Nonetheless, a number of additional viral diseases are considered for inclusion in
the priority list every year. These events underscore the need for concerted efforts to
develop and implement new interventions while continuing to invest in proven pub-
lic health measures.
The field of viral immunology seeks to understand the mechanisms of virus–host
interaction with a view of applying this knowledge to the design of effective vac-
cines to control viral infections. Whereas several viral infections are still in need of
successful prophylactic vaccines, in many instances we also require therapeutic
vaccines that could boost inadequate immunity. This book primarily emphasizes
several areas of the field that hold substantial promise for translation, but where
further work is critically required to find solutions. We emphasize that our funda-
mental understanding of virus–host relationships is moving in leaps and bounds, but
we lag behind in applying this knowledge to the successful control of many viral
infections. Nevertheless, elucidating the nature of immune responses in individual
natural hosts may inform our understanding of how virus–host equilibria are estab-
lished without substantially impacting host health. Furthermore, this may provide
insights into the mechanisms of disease pathogenesis and immunity in humans.
We strongly believe that this book would provide enough insights into the cur-
rent understanding of adaptive and innate immune response in viral diseases. This
book is an attempt to compile the novel information available on recent advance-
ments on various aspects of differential regulation of each immune cell during
viral pathogenesis and immune evasion. This book aims to revitalize the interac-
tion between fields of virology and immunology in order to advance our under-
standing of dynamics of viral immune pathogenesis, as well as innate and adaptive
immune responses elicited by the host. The book also elucidates a comprehensive
yet representative description of a large number of challenges associated with
immune sensors, namely TLRs, DNA and RNA sensors, and other immune cells
during viral infections, as it is indispensable to possess updated information on
emerging viral diseases. This book could be an essential reading for the novice
and experts in the field of viral immunology, immune interventions, and viral
immunodiagnostics, including latest developments in vaccine research. With
these objectives in mind, the content of this textbook has been arranged in a logi-
cal progression from fundamental to more advanced concepts. Finally, this book
also outlines the most advanced immune techniques used in diagnostics of viral
diseases and also primarily focuses on advancements of vaccine development
research for emerging viral diseases.
We hope that this book stimulates your creativity and wish you success in your
experiments. This book is a stunning reflection of the seriousness with which the
several scientific minds are dedicated to the welfare of the scientific community. I
am extremely thankful to the contributors for paying continuous attention to my
request and showing faith in my capabilities. I shall always remain highly obliged
to all of them forever. These words cannot justify the worthiness of their efforts.
Preface vii

We successfully compiled our creative and thoughtful research work due to gen-
uine concern and painstaking effort of many more well-wishers whose names are
not mentioned, but they are still in our heart. So, the reward is surely worth for their
efforts. I want to dedicate this book to my mother, S. Jayaprada (late). Myself and
the contributing authors hope from the bottom of our hearts that this book will be a
good guidebook and compass for research studies in diagnostic virology and
immunotechnology.

Machilipatnam, India Pallaval Veera Bramhachari

Acknowledgments

My sincere thanks are extended to all the academicians and scientists who have
contributed the galaxy of topics in the form of chapters and happily agreed to share
their work on Dynamics of Immune Activation in Infectious Viral Diseases in this
volume.
This book is a stunning reflection of the seriousness with which the several sci-
entific minds are dedicated from the immunology and virology scientific commu-
nity. I am extremely thankful to the contributors for paying continuous attention to
my requests and showing obsolete faith in my competencies and capabilities. I shall
always remain highly obliged and indebted to all of them forever. These words can-
not justify the worthiness of their untiring efforts. We appreciate the excellent work
of the authors and coauthors who were invited to contribute diversified chapters in
this book. The credit for making this book a reality goes to them. Me as an editor
and the review team for the chapters especially appreciate sharing expertise with the
contributors. Each chapter is informative and written as a stand-alone chapter, so the
reader can begin anywhere in the book depending upon his or her interests and
needs.
At the same time, I also express my deepest gratitude to my family members,
especially my wife (Ramadevi Ramaswamy) and my kids (Ruthvik and Jayati), for
their kind support which has prompted me to complete the assignment on time. I am
also thankful to the Department of Biotechnology, Krishna University, for the sup-
port. I am equally thankful to the Springer Nature Publishing group for their full
cooperation during the peer review and production of the volume.
I am thankful to my beloved teachers and mentors for their constant support and
motivations at all stages of progress.

ix
About the Book

Dynamics of Immune Activation in Viral diseases is an authoritative reference book

in virology, which provides the current understanding of adaptive and innate
immune response in viral diseases. This book also illustrates the differential regula-
tion of each immune cell during viral pathogenesis and immune evasion. This book
aims to revitalize the interaction between fields of virology and immunology in
order to advance our understanding of dynamics of viral immune pathogenesis, as
well as innate and adaptive immune responses elicited by the host. Recent advance-
ments in immune intervention and viral immunodiagnostics, including latest devel-
opments in vaccine research, are discussed. It also covers a new area of immune
biology where innate part of immune system helps adaptive part through cross talk
with adaptive immune system. This book primarily emphasizes on the recent chal-
lenges of immune sensors, namely TLRs, DNA and RNA sensors, and other immune
cells during viral infections, as it is indispensable to possess updated information on
emerging viral diseases. Apart from the current understanding of immune response
in human viral diseases, this book outlines the most advanced immune techniques
used in diagnostics of viral diseases and also primarily focuses on advancements of
vaccine development research for emerging viral diseases.

xi
Contents

Part I Role of Innate Immunity in Combating Viral Diseases

1 Significance and Dynamics of Immune Responses During Viral
Pathogenesis����������������������������������������������������������������������������������������������    3
Pallaval Veera Bramhachari
2 Antibody-Dependent Enhancement of Viral Infections ����������������������    9
Ruta Kulkarni
3 Inflammation During Virus Infection: Swings and Roundabouts������   43
Sankar Bhattacharyya
4 Interferons and Their Role in Viral Infection ��������������������������������������   61
Suji George, Gururaj Rao Deshpande, and Gajanan N. Sapkal
5 Role of Cytokines in Infectious Viral Disease����������������������������������������   81
Pavani Sanapala and Sudhakar Pola
6 B Cells and Their Role in Combating Viral Diseases���������������������������� 99
Devanabanda Mallaiah and Pallaval Veera Bramhachari
Part II Role of Adaptive Immunity in Combating Viral Diseases
7 Significant Role of Cellular Activation in Viral Infections������������������ 115
Bojjibabu Chidipi, Samuel Ignatious Bolleddu, Ganugula Mohana
Sheela, and Alavala Matta Reddy
8 Macrophages/Microvesicles and Their Task in Viral Diseases������������ 125
Bojjibabu Chidipi, Samuel Ignatius, Madhavi Maddala, Pallaval
Veera Bramhachari, and Alavala Mattareddy
9 T Cells in Viral Infections: The Myriad Flavours of Antiviral
Immunity�������������������������������������������������������������������������������������������������� 139
Achanta Jagadeesh, A. M. V. N. Prathyusha, Ganugula Mohana
Sheela, and Pallaval Veera Bramhachari
10 Potentiality of Toll-Like Receptors (TLRS) in Viral Infections ���������� 149
A. M. V. N. Prathyusha, Prudhvi Lal Bhukya, and Pallaval Veera
Bramhachari

xiii
xiv Contents

11 Activation of Complement System During Viral Infections:

Prospects and Future Challenges ���������������������������������������������������������� 161
Prudhvi Lal Bhukya and Pallaval Veera Bramhachari
12 MicroRNAs and Their Role in Viral Infection�������������������������������������� 167
Divya Tiraki
13 Evolution, Distribution, and Diversity of
Immunodeficiency Viruses���������������������������������������������������������������������� 187
Harika Sai Vemuri, Surekha Challa,
and Nageswara Rao Reddy Neelapu
14 Role of Immune Cells in Hepatitis B Infection�������������������������������������� 205
Prakriti Sinha and Parul Sahu
15 Significance of RNA Sensors in Activating Immune System
in Emerging Viral Diseases���������������������������������������������������������������������� 229
Preethika Nair and Siddhesh U. Sapre
16 Potentiality of DNA Sensors in Activating Immune System
in Emerging Viral Infectious Diseases���������������������������������������������������� 243
Siddhesh U. Sapre and Preethika Nair
17 Advanced Immunotechnological Methods for Detection
and Diagnosis of Viral Infections: Current Applications
and Future Challenges���������������������������������������������������������������������������� 261
Pallaval Veera Bramhachari, Ganugula Mohana Sheela,
A. M. V. N. Prathyusha, M. Madhavi, K. Satish Kumar,
Neelapu Nageswara Rao Reddy, and Chanda Parulekar Berde
18 Current Advances in Multi-Epitope Viral Vaccines Development
and Research�������������������������������������������������������������������������������������������� 277
A. M. V. N. Prathyusha and Pallaval Veera Bramhachari
Editor and Contributors

About the Editor

Pallaval Veera Bramhachari is currently a Senior

Faculty member of the Department of Biotechnology at
Krishna University, Machilipatnam, India. He has
earlier served as Postdoctoral Research Scientist at
­
the Bacterial Pathogenesis Laboratory, Queensland
Institute of Medical Research (QIMR-Berghofoer
Research Center), Brisbane, Australia, and DBT
Postdoctoral Fellow at the Department of Microbiology
and Cell Biology, Indian Institute of Science (IISc),
Bangalore. He completed his Ph.D. in Microbiology
from Goa University, India. His major research inter-
ests are investigations on the applied research in micro-
biology and cell biology including biochemical
mechanism of bacterial-biofilm formations, structure-
function relationship of bacterial rhamnolipids, basic
and translational cancer research. He has published
more than 125 research articles including 5 ­international
books in prestigious peer-reviewed international
Scopus-indexed journals and presented 47 abstracts at
various national and international conferences.
Dr. Bramhachari served in the Editorial Board member
and as Reviewer for a number of national and interna-
tional peer-reviewed journals. He is a member of many
international scientific societies and organizations,
most importantly Indian Science Congress and Society
of Biological Chemists, India. He successfully com-
pleted two major research projects.
Dr. Bramhachari was awarded a Travel scholarship
from QIMR Australia in 2007 for attending the 4th
Indo-Australian Biotechnology Conference at Brisbane,
Australia, and also awarded with young scientist travel

xv
xvi Editor and Contributors

fellowship (2007) from the DST, Govt. of India for

attending the XVII Lancefield International Symposium
at Porto Heli, Greece, 2008. Dr. Bramhachari was con-
ferred with various prestigious awards, notably Science
Education Research Board (SERB), Government of
India-Young Scientist award (2011) with a Research
grant, Nominated as Associate Fellow of Andhra
Pradesh Academy of Sciences (APAS) 2016, MASTER
TRAINER: Andhra Pradesh, English Communications
Skills Project, by the British Council & Andhra Pradesh
State Council of Higher Education (APSCHE) in 2017,
Andhra Pradesh State Best Scientist award by APCOST
2017, and Dr. V. Ramalingaswamy Memorial award in
Biomedical Sciences 2019 by Science city of Andhra
Pradesh and Andhra Pradesh Academy of Sciences. He
also obtained two Indian patents in 2017. He has more
than 13 years of teaching and research experience at the
university level.

Contributors

Chanda Parulekar Berde Gogate Jogalekar College, Ratnagiri, Maharashtra,

India
Sankar Bhattacharyya Vaccine and Infectious Disease Research Centre,
Translational Health Science and Technology Institute, Faridabad, Haryana, India
Prudhvilal Bhukya Hepatitis Division, National Institute of Virology, Pune,
Maharashtra, India
Samuel Ignatious Bolleddu Molecular Pharmacology and Physiology, University
of South Florida, Tampa, FL, USA
Pallaval Veera Bramhachari Department of Biotechnology, Krishna University,
Machilipatnam, Andhra Pradesh, India
Surekha Challa Department of Biochemistry and Bioinformatics, Institute of
Science, Gandhi Institute of Technology and Management (GITAM) (Deemed to be
University), Rushikonda, Visakhapatnam, Andhra Pradesh, India
Bojjibabu Chidipi Molecular Pharmacology and Physiology, University of South
Florida, Tampa, FL, USA
Gururaj Rao Deshpande National Institute of Virology, Pune, Maharashtra, India
Suji George National Institute of Virology, Pune, Maharashtra, India
Editor and Contributors xvii

Samuel Ignatius Molecular Pharmacology and Physiology, University of South

Florida, Tampa, FL, USA
Achanta Jagadeesh Tumor Microenvironment Global Core Research Center,
College of Pharmacy, Seoul National University, Seoul, South Korea
Ruta Kulkarni Department of Communicable Diseases, Interactive Research
School for Health Affairs (IRSHA), Bharati Vidyapeeth (Deemed to be University),
Pune, Maharashtra, India
Madhavi Maddala Department of Zoology, Nizam College, Osmania University,
Hyderabad, Telangana, India
M. Madhavi Department of Zoology, Nizam College, Osmania University,
Hyderabad, Telangana, India
Devanabanda Mallaiah Department of Biotechnology and Bioinformatics, Yogi
Vemana University, Kadapa, Andhra Pradesh, India
Alavala Mattareddy School of Life Sciences, Adikavi Nannaya University,
Rajahmundry, Andhra Pradesh, India
Ganugula Mohana Sheela Department of Biotechnology, Krishna University,
Machilipatnam, India
Preethika Nair NMIMS School of Science, Mumbai, India
Nageswara Rao Reddy Neelapu Department of Biochemistry and Bioinformatics,
Institute of Science, Gandhi Institute of Technology and Management (GITAM)
(Deemed to be University), Rushikonda, Visakhapatnam, Andhra Pradesh, India
Sudhakar Pola Department of Biotechnology, Andhra University, Visakhapatnam,
Andhra Pradesh, India
A. M. V. N. Prathyusha Department of Biotechnology, Krishna University,
Machilipatnam, Andhra Pradesh, India
Neelapu Nageswara Rao Reddy Department of Biochemistry and Bioinformatics,
GITAM Institute of Science, Gandhi Institute of Technology and Management
(GITAM) (Deemed-to-be-University), Visakhapatnam, Andhra Pradesh, India
Parul Sahu National Institute of Immunology, New Delhi, India
Pavani Sanapala Department of Biotechnology, Andhra University,
Visakhapatnam, Andhra Pradesh, India
Gajanan N. Sapkal National Institute of Virology, Pune, Maharashtra, India
Siddhesh U. Sapre National Institute of Virology, Pune, India
K. Satish Kumar Department of Zoology, Adikavi Nannaya University,
Rajamundry, Andhra Pradesh, India
xviii Editor and Contributors

Prakriti Sinha National Institute of Immunology, New Delhi, India

Divya Tiraki Department of Communicable Diseases, Interactive Research School
for Health Affairs (IRSHA), Bharati Vidyapeeth Deemed University, Pune,
Maharashtra, India
Harika Sai Vemuri Department of Biochemistry and Bioinformatics, Institute of
Science, Gandhi Institute of Technology and Management (GITAM) (Deemed to be
University), Rushikonda, Visakhapatnam, Andhra Pradesh, India
Abbreviations

ABCF1 ATP-binding cassette subfamily F member 1

ACLF Acute-on-chronic liver failure
ADCC Antibody-dependent cytotoxic cells
ADCP Antibody-dependent cellular phagocytosis
ADE Antibody-dependent enhancement
AIC Anti-inflammatory cytokines
AIDS Acquired immune deficiency syndrome
AKT Serine/threonine kinases
ALRs AIM2-like receptors
AP Alternate pathway
AP-1 activation protein-1
APCs Antigen-presenting cells
APOBEC3 Apolipoprotein B Editing Complex
ARDS Acute respiratory distress syndrome
ATF-2 Activating transcription factor 2
BBB Blood–brain barrier
BCR B-cell receptors
BIV Bovine immunodeficiency virus
CARDs Caspase activation and recruitment domains
CCHF Crimean–Congo hemorrhagic fever
CCHFV Crimean–Congo hemorrhagic fever virus
CCL Chemokine
CD Clusters of differentiation
CDC Complement-dependent cytotoxicity
CDNs Cyclic di-nucleotides
cGAMP Cyclic guanosine monophosphate-adenosine monophosphate
cGAS Cyclic GMP-AMP synthase
cGAS Cyclic GMP-AMP synthase
CH25H Cholesterol-25-hydroxylase
CHIKV Chikungunya virus
CIFN Consensus IFN
CLIA Chemiluminescent immunoassay
CLRs C-type lectin receptors
CML Complement-mediated lysis

xix
xx Abbreviations

CMV Cytomegalovirus
CNS Central nervous system
CP Classical pathway
CpG Unmethylated deoxycytidylate-phosphate-deoxyguanylate
CPPs Cell-penetrating peptides
CS Complement system
CSF Colony-stimulating factor
CTL Cytotoxic T lymphocytes
DAA Directly acting antivirals
DAI Z-DNA binding protein
DAMPS Damage-associated molecular patterns
DCs Dendritic cells
DDX DExD/H-box
DDX41 DEAD box polypeptide 41
DENV Dengue virus
DHF Dengue hemorrhagic fever
DIV Dog immunodeficiency virus
DNAse Deoxyribonuclease
DSS Dengue shock syndrome
EBOV Ebola virus
EBV Epstein–Barr virus
ECL Electrochemiluminescence
EGF Epidermal growth factor
EPO Leptin and erythropoietin
Fab Fragment antigen binding
Fc Fragment crystalline
FEB Field Effect Biosensing
FGF Fibroblast growth factor
FIV Feline immunodeficiency virus
FO Follicular
FPIA Fluorescence polarization immunoassay
GC Germinal center
G-CSF Granulocyte CSF
GEFs Guanine nucleotide exchange factors
GH Growth hormone
GM-CSF Granulocyte macrophage colony-stimulating factor
HA Hemagglutinin
HBcAg Hepatitis B core antigen
HBsAg Hepatitis B surface antigen
HCC Hepatocellular carcinoma
HCV Hepatitis C
HeV Hendra virus
HFD Hemagglutinin fusion domain
HIN H-inversion
HIV Human immunodeficiency virus
Abbreviations xxi

HLA Human leukocyte antigen

HMGB High mobility group B protein
HPV Human papilloma virus
HSC Hematopoietic stem cells
IF Immunofluorescence
IFI16 IFN-γ-inducible protein 16
IFIT IFN-induced protein with tetratricopeptide repeats ()
IFIT Interferon-inducible transmembrane protein
IFITM families IFN-induced transmembrane protein family
IFN Interferon
IFN-γ Interferon gamma
IKK IκB kinase
IL Interleukin
IRF interferon regulatory factor
ISGs Interferon-stimulated genes
ISGs Interferon-stimulated genes
ISREs IFN-stimulated response elements
JEV Japanese encephalitis virus
JNK Jun N-terminal kinase
KCs Kupffer cells
LASV Lassa virus
LBP LPS-binding protein
LFIA Lateral flow immunoassay
LGP2 laboratory of genetics and physiology 2
LILR-B1 Leukocyte immunoglobulin-like receptor B1
LP Lectin pathway
LPS Lipopolysaccharide
LRRFIP1 Leucine-rich repeat flightless-interacting protein 1
LRRs Leucine-rich repeats
mAbs Monoclonal antibodies
MAC Membrane attack complex
MagLISA Magnetic nano(e)nzyme-linked immunosorbent assay
MALDI Matrix-assisted laser desorption ionization
MAP Mitogen-activated protein
MASPs MBL-associated proteins
MAVS Mitochondrial antiviral signaling protein, a.k.a. IPS-1/VISA/
Cardif
MDA-5 Melanoma differentiation-associated antigen 5
MDA-5 Melanoma differentiation-associated antigen 5
mDC Myeloid dendritic cells
MEIA Micro-particle immune assay
MHC Major histocompatibility complex
MIA Multiplex microsphere immunoassay
mNGS Metagenomic next-generation sequencing assay
MNPs Magnetic nanoparticles
xxii Abbreviations

MNV Murine norovirus

Mɸ Macrophages
MRE11A Meiotic recombination 11 homolog A
MS Mass spectrometry
MZ Marginal zone
NA Neuraminidase
NAbs Neutralizing antibodies
NEMO NF-κB essential modifier
NF-AT Nuclear factor of activated T cell
NF-κB Nuclear factor κ-B
NGS Next-generation sequencing
NIRS Near-infrared spectroscopy
NiV Nipah virus
NLR Neutrophil to lymphocyte ratio
NLRB Baculovirus inhibitor of apoptosis repeats
NLRC Caspase activation and recruitment domain CARD
NLRP Pyrin domain PYD
NLRs NOD-like receptors
NTCP Sodium taurocholate co-transporting polypeptide
OAS Original antigenic sin
OAS RNase L/oligoadenylate synthase
PAMPS Pathogen-associated molecular patterns
PBMCs Peripheral blood mononuclear cells
PCRs Polymerase chain reactions
pDC Plasmacytoid dendritic cells
PDGF Platelet-derived growth factor
PI3K Phosphatidyl-inositol 3 kinase
PIC Pro-inflammatory cytokine
PK Protein kinase
PKC Protein kinase C
PKR Protein kinase R
PML Promyelocytic leukemia protein
PRL Prolactin
PRR Pattern recognition receptors
PRRs Pattern recognition receptors
py Pyrin
PYHIN Pyrin and H inversion domain
qPCR Quantitative PCR
RCs Replication complexes
RdRp RNA-dependent RNA polymerase
RFFIT Rapid fluorescent focus inhibition test
RIDT Rapid influenza diagnostic test
RIG-I Retinoic acid-inducible gene I
RIG-I Retinoic acid-inducible gene I product
RLRs RIG-I-like receptors
Abbreviations xxiii

RNAse Ribonuclease
RT-LAMP Reverse transcription loop-mediated isothermal amplification
RT-RPA Reverse transcription recombinase polymerase amplification
assay
SARM Sterile-alpha and Armadillo motif-containing protein
SARS-CoV Severe Acute Respiratory Syndrome Coronavirus
SCF Stem cell factor
SGPs Small G-protein
SIV Simian immunodeficiency virus
SNPs Single nucleotide polymorphisms
SOCS3 Suppressor of cytokine signaling 3
SPR Surface plasma resonance spectroscopy
ssRNA Single-stranded RNA
STAT1 Signal transducer and activator of transcription 1
STING Stimulator of interferon genes
Syk Spleen tyrosine kinase
TBK1 TANK binding kinase 1
TEM Transmission electron microscopy
TFH cells Typically follicular T helper cells
TGF Transforming growth factor
TIR Toll/IL-1 receptor
TLRs Toll-like receptors
TNF Tumor necrosis factor
TNP T-cell epitope of nucleoprotein
TPR Tetra tricopeptide
TRAF TNF receptor-associated factor
TRAIL TNF-related apoptosis-inducing ligand
Tregs T regulatory cells
TREX1 Three-primer repair endonuclease 1
TRIF TIR domain-containing adaptor protein-inducing IFN-β
TYK2 Tyrosine kinase2
VEEV Venezuelan equine encephalitis virus
VEGF Vascular endothelial growth factor
VHF Viral hemorrhage fever
VHSV Viral hemorrhagic septicemia virus
VSV Vesicular stomatitis virus
WNV West Nile virus
YFV Yellow fever virus
ZIKV Zika virus
 Part I
Role of Innate Immunity in Combating Viral
Diseases
Significance and Dynamics of Immune
Responses During Viral Pathogenesis 1
Pallaval Veera Bramhachari

Abstract
Immune system is a homeostatic system which is active against numerous invad-
ing pathogens. Host immune system uses multiple immune responses (innate,
adaptive, and complement system) to eliminate virus/viral particles. Additionally
immune system is also well resourced with sensors that detect invading patho-
gens and direct responses for clearing numerous copies of virus recruits. Modern
technologies help prevent pathogen emergence as well as thrust scientific
improvements in understanding the viral immune responses; moreover, recent
scientific advances in diagnostic virology have undeniably transformed the abil-
ity to address challenges of numerous emerging intricate viruses. The journey of
diagnostic virology has started from serology, nucleic acid sequence-based
amplification techniques, and genomic sequencing techniques to most advanced
innovative methods (e.g., structural biology spectroscopy, NGS, microfluidics,
metagenomics, CRISPR/Cas system, nanotechnology and structural biology),
the world progressed way beyond with several classy diagnostic methods to help
tackle the diagnosis and control of emerging viral diseases. However, the techni-
cal competencies alone are inadequate if not sustained by health promotion strat-
egies to raise awareness of the significance of early detection and diagnosis,
outbreak, and spread of virus. Yet, current outbreaks of viral diseases across the
world dole out authoritative reminders to emerging viral pathogens.

Keywords
Immune system · Viruses · Diagnostic methods · Emerging viral diseases

P. V. Bramhachari (*)
Department of Biotechnology, Krishna University, Machilipatnam, Andhra Pradesh, India

© Springer Nature Singapore Pte Ltd. 2020 3

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_1
4 P. V. Bramhachari

1.1 Introduction

Infectious diseases are reported to account for ~20% of global mortality, of which
one-third of deaths are caused by viral diseases. The viral infections pose major public
health risks and warrant further research and development, including surveillance and
diagnostics. All the lethal viral diseases present an absurdity in mounting the patho-
genesis there by killing their hosts, which is noticeably of no advantage to the virus.
Viruses are infectious agents consisting of nucleic acids coated in a simple protein
casing, infect, replicate in host cells, and cause acute, chronic infections. Since viral
infection is an intricate and highly vibrant process, noticeably affected by the physical
and chemical environment, studies into infectious viral biology ought to preferably
occur in advanced research settings. Viruses replicate by hijacking the host cell’s
machinery and making host cells a huge virus factory and erupt as virus after… virus
after… virus after… virus.
The immune system is also well resourced with sensors that detect invading
pathogens and direct responses for clearing numerous copies of virus recruits. On
the entry of virus, immune system of the host typically elicits both nonspecific
innate and “specific” adaptive immune responses against foreign pathogens.
Activation of varied immune responses, time, and the extent of response rely on
how virus interacts and spreads within host cells. Host immune system uses multi-
ple immune responses (innate, adaptive, and complement system) to eliminate
virus/viral particles. The immune system employs most efficient mechanisms
depending on the distinctiveness of infectious agents. Diverse actions occur during
viral infections, equally toward free viral particles in addition to infected cells.
Race between virus and immune response establishes: whether the intruder will
eliminate or establish a persistent infection. The host cell can also be damaged
unswervingly by virus or by viral immune response. However, balance between
good and bad antiviral immune response relies on the amount of viral load, chronic-
ity of infection, and magnitude of tissues infected (Zinkernagel 1996). Therefore, a
balance exists between immune activation vis-à-vis immune suppression during the
setting of chronic infection. Viruses evolved numerous strategies to escape immune
system to institute a chronic infection. Few viruses endure in definite cell types and
hide from immune system, while some encode specific genes that target infected
cells or immune system. A few viruses limit their replication, thus restricting acces-
sibility of antigen to alert the immune system. However, a few viruses are error
prone in their replication, escalating the possibility of making escape viral mutants.
Nonetheless, these immune evasion strategies of virus are unprejudiced by regula-
tion within the immune system (Finlay and McFadden 2006).

1.2 Innate Immune Responses by Virus

During early stages of infection “innate” response limits virus multiplication, which
implicates synthesis of diffusible proteins called interferons, cytokines, and chemo-
kines and stimulation of “natural killer” and dendritic lymphocytes (French and
Yokoyama 2003). In order to limit viral replication, first line of defense system has
1 Significance and Dynamics of Immune Responses During Viral Pathogenesis 5

to sense the pathogens/pathogen-associated molecules (Paladino et al. 2006). This

provides temporary protection against the viral onslaught. This fundamental pro-
cess is accomplished by sensing virus-associated nucleic acids in infected cells
using a variety of pattern recognition receptors (PRRs). PRRs recognize distinct
pathogens conserved structures known as pathogen-associated molecular patterns
(PAMPs). There are several classes of PRRs, namely TLRs (toll-like receptors),
RLRs (retinoid acid-inducible gene-I (RIG-I)-like receptors), NLRs (nucleotide
oligomerization domain (NOD)-like receptors), and many DNA and RNA sensors
(Mogensen 2009). Upon interaction with cognate viral ligands, TLRs, NLRs, and
RLRs stimulate production of interferons, pro-inflammatory cytokines (NF-κB-­
dependent response), and chemokines that limit virus replication and dissemination
(Shayakhmetov et al. 2010). Infected cells produce different classes of interferons
such as INF-α, INF-β, and INF-γ. Interferons mediate the most effective innate
immune response during viral infection by warning nearby cells about viral pres-
ence (Haller et al. 2006). This signals neighboring cells to increase MHC class I
molecules on their surfaces and to facilitate surveying T cells to identify and elimi-
nate viral infection. Interferons also limit viral replication by the activation of NK
cells which is associated with the alterations in expression of SLA by infected cells.
NK cells’ cytotoxic mechanism against viral infected cells is very effective and does
not depend on antigen (NK cells lack TCR) (Paul and Lal 2017). However, comple-
ment activation of an alternative pathway has the effect of activating destruction of
viral particle. Innate defensive mechanism generates a severe evolutionary pressure
on viruses to evolve efficient mechanisms to evade and also strategies permitting for
subversion of host antiviral immune systems (Finlay and McFadden 2006).

1.3 Adaptive Immune Responses by Virus

Another important second line of antiviral response is adaptive immune system that
includes CD8+ and CD4+ T cells and neutralizing antibodies which act against viral
particles and infected cells in combination. Days to weeks are essential to mount an
adaptive immune response tailored for specific virus. Adaptive immune response
possesses two components: humoral response (utilities virus-specific antibodies by
B lymphocytes) and cell-mediated response (specific cytotoxic T lymphocytes
(CTL) that kill infected cells) (Kim et al. 1999). Both the components of adaptive
defense system ensue production of long-lived “memory cells” that allow much
more quick response for consequent infection with similar virus (Campos and
Godson 2003). Antibodies are most vital mechanisms against viral particles, while
cytotoxic mechanisms are noteworthy against infected cells.

1.3.1 Humoral Immunity

Virus or virus-infected cells stimulate B lymphocytes which synthesize IgG, IgM,

and IgA Abs (specific for viral antigens). Antibody neutralization is a significant
mechanism by which virus occurs in huge fluid spaces (e.g., serum) or on mucous
6 P. V. Bramhachari

surfaces (e.g., the gastrointestinal and respiratory tracts). Antibody can neutralize
virus by (1) preventing host cell—virus interactions or (2) distinguishing viral anti-
gens on infected cells that results in antibody-dependent cytotoxic cells (ADCC) or
complement-mediated lysis (CML) (Parkin and Cohen 2001; Chaplin 2010).

1.3.2 Cell-Mediated Immunity

Viruses engage diverse strategies to restrain the presentation of virus-derived pep-

tides. One of the important strategy entails the modulation of proteasome activity
that produces peptides from full-length proteins; if there is adequate binding affin-
ity, peptides bind to MHC class I molecules. However, a few viruses directly inter-
act with and inhibit proteasome machinery likely for generation of peptides.
Specialized immune cells, the CTL cells, recognize virus-infected cells with the
help of specialized proteins (TCRs) on their surface and release cytotoxic factors to
destroy infected cell and, therefore, avert survival of invading virus. The cytotoxic
cells are specially armed with preformed mediators, namely perforins (forms pores
in cell membranes) and granzymes (stored in and released from granules), permit
entry of other factors into virus-infected cell to facilitate their destruction (Rosendahl
Huber et al. 2014). Furthermore, CTL cells synthesize and release additional pro-
teins, called cytokines, including interferon-ɤ and tumor necrosis factor-α, and
transfer a signal from T cell to infected cell, or other neighboring cells, to further
enhance killing mechanisms. Furthermore, cytokine signals, NK cells, act as evolu-
tionary bridge linking innate and adaptive immunity (Belardelli and Ferrantini
2002; Sun and Lanier 2009).
It is well established that there is a mechanism for inhibiting immune response
during chronic infection. In fact this mechanism is executed for two reasons: (1) To
prevent immunopathology. CD8+ T-cell effector function can cause high levels of
tissue damage through killing of infected cells and release of inflammatory cyto-
kines. In reality, cytotoxicity and secretion of cytokines such as tumor necrosis fac-
tor (TNF) are often decreased if not lost in CD8+ T cells in a phenomenon known
as T-cell exhaustion (Ou et al. 2008). (2) To prevent excessive proliferation of virus-­
specific T cells. During acute infection, virus-specific T cells can increase tenfold
each day. This level of proliferation is dangerous and is therefore greatly reduced
upon continued exposure to antigen (Thimme et al. 2012).

1.4 Significance

Viruses are intracellular pathogens that invade and infect host cells. Immune system
is a homeostatic system which is active against numerous invading pathogens inside
the body to clear infections. Propitiously, the aspects of recent modernization tech-
nologies that helped prevent pathogen emergence can also impel scientific improve-
ments; moreover, recent scientific advances in diagnostic virology undeniably
transformed the ability to address the challenges of numerous emerging intricate
1 Significance and Dynamics of Immune Responses During Viral Pathogenesis 7

viruses across the globe. Starting from serology, nucleic acid sequence-based ampli-
fication techniques, genomic sequencing techniques to most advanced innovative
methods (structural biology spectroscopy, NGS, microfluidics, metagenomics,
CRISPR/Cas system, nanotechnology, and structural biology), the world has pro-
gressed way beyond with more classy diagnostic methods to help tackle the detec-
tion and control of emerging viral diseases. However, the technical competencies
alone are inadequate if not sustained by health promotion strategies to raise aware-
ness of the significance of early detection, outbreak, and spread of virus. Still, cur-
rent outbreaks of viral diseases across the world dole out powerful reminders of our
ongoing susceptibility to emerging viral pathogens. Reflecting the diversity of
viruses and viral diseases, these are notoriously difficult drug targets since they
modify and adapt themselves quickly to build up resistance and emerge as new
serotypes. Nonetheless, better perceptiveness of host–pathogen interactions, viral
protein, and nucleic acid functions led to additional rational drug designs, resulting
in important therapeutic advances against viral diseases. Furthermore, novel plat-
forms for vaccine design, namely nanoparticles and virus-like particles, have
embarked avenues for development of new vaccine targets. Treatment strategies for
viral infections comprise hindering binding of virus to host cells by inhabiting on
host cell receptor with an additional molecule, or use of vaccine developed for a
particular virus or an analogous target of diverse viruses. Serious actions should
accentuate the call for strenuous efforts to develop and execute novel interventions
in viral disease diagnosis and vaccine development keeping public health perspec-
tive in view.
Dynamics of Immune Activation in Viral Diseases is an authoritative reference
book in virology, which provides the current understanding of adaptive and innate
immune response in viral diseases. This book also illustrates about differential regu-
lation of each immune cells during viral pathogenesis and immune evasion. This
book aims to revitalize the interaction between fields of virology and immunology
in order to advance our understanding of dynamics of viral immune pathogenesis,
as well as innate and adaptive immune responses elicited by host. Recent advance-
ments in immune intervention and viral immunodiagnostics, including latest devel-
opments in vaccine research, will be discussed. It also covers new areas of immune
biology where innate part of immune system helps adaptive part through cross talk
with adaptive immune system. This book primarily emphasizes on the recent chal-
lenges of immune sensors, namely TLRs, DNA and RNA sensors, and other immune
cells during viral infections, as it is indispensable to possess updated information on
emerging viral diseases. Apart from the current understanding of immune response
in human viral diseases, this book also outlines the most advanced immune tech-
niques used in diagnostics of viral diseases and also primarily focuses on advance-
ments of vaccine development research for emerging viral diseases.

Acknowledgement DR. PVBC is grateful to Krishna University, Machilipatnam, for the support
extended.

Conflict of Interest The author declares that he has no competing interests.

8 P. V. Bramhachari

References
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nity. Trends Immunol 23(4):201–208
Campos M, Godson DL (2003) The effectiveness and limitations of immune memory: understand-
ing protective immune responses. Int J Parasitol 33(5–6):655–661
Chaplin DD (2010) Overview of the immune response. J Allergy Clin Immunol 125(2):S3–S23
Finlay BB, McFadden G (2006) Anti-immunology: evasion of the host immune system by bacte-
rial and viral pathogens. Cell 124(4):767–782
French AR, Yokoyama WM (2003) Natural killer cells and viral infections. Curr Opin Immunol
15(1):45–51
Haller O, Kochs G, Weber F (2006) The interferon response circuit: induction and suppression by
pathogenic viruses. Virology 344(1):119–130
Kim JJ, Nottingham LK, Tsai A, Lee DJ, Maguire HC, Oh J, Dentchev T, Manson KH, Wyand MS,
Agadjanyan MG, Ugen KE, Weiner DB (1999) Antigen-specific humoral and cellular immune
responses can be modulated in rhesus macaques through the use of IFN-γ, IL-12, or IL-18 gene
adjuvants. J Med Primatol 28(4–5):214–223
Mogensen TH (2009) Pathogen recognition and inflammatory signaling in innate immune
defenses. Clin Microbiol Rev 22(2):240–273
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tion and immune-mediated pathology by E3 ubiquitin ligase Cbl-b during chronic viral infec-
tion. J Virol 82(7):3353–3368
Paladino P, Cummings DT, Noyce RS, Mossman KL (2006) The IFN-independent response to
virus particle entry provides a first line of antiviral defense that is independent of TLRs and
retinoic acid-inducible gene I. J Immunol 177(11):8008–8016
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Paul S, Lal G (2017) The molecular mechanism of natural killer cells function and its importance
in cancer immunotherapy. Front Immunol 8:1124
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viral infections–opportunities for peptide vaccination. Front Immunol 5:171
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host responses to viral gene therapy vectors. Mol Ther 18(8):1422–1429
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and adaptive immunity? Eur J Immunol 39(8):2059–2064
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in controlling hepatitis C virus infection. FEMS Microbiol Rev 36(3):663–683
Zinkernagel RM (1996) Immunology taught by viruses. Science 271(5246):173–178
Antibody-Dependent Enhancement
of Viral Infections 2
Ruta Kulkarni

Abstract
Antiviral antibodies constitute an important component of the host immune
response against viral infections and serve to neutralize and reduce infectivity of
the virus. However, these antibodies, intended to protect the host, may some-
times prove beneficial to the virus, by facilitating viral entry and replication in
the target cell. This phenomenon, known as antibody-dependent enhancement
(ADE) of infection, is a result of interaction of virus–antibody immune com-
plexes with Fcγ and/or complement receptors on certain types of host cells and
promotes viral entry into the host cells. The internalized immune complexes then
modulate host immune response so as to enhance viral replication and aggravate
disease severity. The possibility of induction of ADE remains a concern in the
development and implementation of viral vaccines and immunotherapeutics.

Keywords
Viral infection · Neutralization · Antibody-dependent enhancement · Immune
response · Vaccine · Immunotherapeutic

2.1 Introduction

Antibody-dependent enhancement (ADE) of infection represents a paradoxical phe-

nomenon in host–pathogen biology, in which, antibody, an important pillar of the
host defense against invading pathogen, actually allows entry of the pathogen into
host territory. This traitorous behavior of the antibody further serves to weaken the
host defense system and thus generates an environment conducive for enhanced

R. Kulkarni (*)
Department of Communicable Diseases, Interactive Research School for Health Affairs
(IRSHA), Bharati Vidyapeeth (Deemed to be University), Pune, Maharashtra, India

© Springer Nature Singapore Pte Ltd. 2020 9

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_2
10 R. Kulkarni

growth of the pathogen and consequently exacerbates disease in the host. This phe-
nomenon has far-reaching implications for disease control and prevention, as thera-
peutic antibodies deployed to protect the host may aid the pathogen instead.
Similarly, antibodies induced by vaccination may actually increase the risk and/or
severity of disease in subsequent host–pathogen encounters.
This chapter aims to provide insights into the current knowledge on ADE of viral
infections, with a focus on its molecular mechanisms and contribution to viral
pathogenesis and disease, as well as implications for disease control strategies.

2.2 Antibody-Mediated Control of Viral Infections

Antibodies are an important component of the host adaptive immune response

against infecting virus. Virus neutralization is considered to be the key mechanism
of antibody-mediated protection. The antiviral activity of antibody is further aug-
mented by Fc-mediated effector mechanisms such as complement-mediated lysis of
viral particles, phagocytosis, and antibody-dependent cellular cytotoxicity (ADCC)
(Braciale et al. 2013).
Neutralization occurs when antibody molecule(s) bind to the virus particle at its
surface epitope and block viral attachment (to cellular receptor) and entry into host
cell, and/or post-entry processes such as fusion and uncoating, thus resulting in loss
of virion infectivity (Fig. 2.1). Two models have been proposed to describe the
kinetics of neutralization. According to the single-hit model, binding of a single
antibody molecule to a critical site on the virion is sufficient for neutralization. The
more accepted multi-hit model postulates that neutralization can be achieved only
when the virion is bound by multiple antibodies, exceeding the stoichiometric
threshold of neutralization (Klasse and Sattentau 2002). The potency of neutraliza-
tion is determined by the affinity of antibody binding and accessibility of the neu-
tralization epitope(s) on the viral surface (Dowd et al. 2011).

2.3 The Antibody-Dependent Enhancement Phenomenon

The phenomenon of antibody-dependent enhancement (ADE) was discovered in

flaviviruses during the 1960s–1970s. While observational studies on dengue virus
(DENV) infections revealed a significant association between severe illness and
antibody responses in secondary infection (Halstead et al. 1967), in vitro experi-
ments demonstrated increased infectivity of Murray Valley encephalitis virus
(Hawkes 1964; Hawkes and Lafferty 1967) and DENV (Halstead et al. 1973a) in the
presence of antibody. In vivo studies in rhesus monkeys showing association of
antibody response with higher dengue viremia offered further supportive evidence
for this phenomenon (Halstead et al. 1973b; Halstead 1979; Marchette et al. 1973).
ADE ensues when antibodies binding to the virus particle fail to efficiently neu-
tralize the virus (Halstead 2003; Taylor et al. 2015). This may occur due to the non-­
neutralizing nature of the antibody (binding to viral epitopes other than those
2 Antibody-Dependent Enhancement of Viral Infections 11

Fig. 2.1 Schematic representation of effect of antibody on virus–host cell interaction. (a) In the
absence of virus-specific antibody, virus enters the host cell via interaction with cell surface (virus-­
specific) receptor, followed by replication and release of progeny virions. (b) In the presence of
non-neutralizing or sub-neutralizing concentrations of antibody, an additional pathway of entry
into host cell is available to the virus. Virus–antibody immune complexes are internalized via anti-
body interaction with cell surface Fc receptor (FcR), resulting in antibody-dependent enhancement
(ADE) of viral infection and replication. Internalized immune complexes bring about suppression
of cellular innate antiviral immune response further boosting viral replication. (c) In the presence
of neutralizing concentrations of antibody, virus attachment and entry into host cell is blocked

involved in cell attachment and entry) or the presence of sub-neutralizing concen-

trations of antibodies (binding to viral epitopes below the threshold for neutraliza-
tion). Either way, these antibodies aid entry of the virus into target cells, resulting in
enhancement of viral infection, known as antibody-dependent enhancement
(Fig. 2.1.). The mechanism of ADE involves internalization of virus–antibody
immune complexes into cells via interaction of the antibody Fc region with the cel-
lular Fc receptors (FcRs). Consequently, FcR-bearing myeloid cells such as mono-
cytes, macrophages, dendritic cells, and certain granulocytes are permissive to ADE
of infection, through phagocytic uptake of the immune complexes. Such ADE is
principally mediated by IgG antibodies, however, IgM along with complement, and
IgA antibodies have also been shown to be capable of ADE (Hawkes and Lafferty
1967; Cardosa et al. 1983; Janoff et al. 1995; Kozlowski et al. 1995).
Initially, the role of antibody in enhancing viral infection was believed to be
limited to facilitation of virus entry into host cells, resulting in increase in the num-
ber of infected cells and, consequently, higher virus production, in a process termed
 12

Table 2.1 Summary of key features of antibody-dependent enhancement (ADE) of viral infections
Virus (family) Mechanism of ADE Clinical significance of ADE Implications for vaccines
Dengue virus 1. Antibodies against viral EDI, EDII, prM 1. Association of ADE with severe 1. Increased risk of severe dengue among
(family proteins involved in ADE dengue (DHF/DSS) during secondary seronegative Dengvaxia (CYD-TDV)
Flaviviridae) 2. Extrinsic ADE: FcR-mediated virus heterotypic infections has been vaccine recipients led to WHO
internalization of virus–antibody immune demonstrated recommending adoption of a pre-vaccination
complexes into monocytes, macrophages, 2. Severe disease during primary screening strategy, and vaccine
dendritic cells infections among infants born to administration only to dengue seropositives
3. Intrinsic ADE: Modulation of host dengue-­immune mothers has been 2. Identification of stronger correlates of
antiviral response by LILR-B1 co-ligation, attributed to ADE protection, development of more robust
downregulation of TLR-­dependent, RIG-I/ assays for estimation of neutralizing/
MDA5 signaling pathways, induction of enhancing ability, long-term follow-up of
IL-10 production vaccine recipients in clinical trials suggested
4. Antibody-mediated enhancement of viral to better predict vaccine-related ADE
fusion suggested 3. Subunit EDIII, NS1 vaccines, chimeric
5. ADE associated with massive release of vaccines with replaced DENV pr gene,
inflammatory cytokines leading to alteration T-cell response-­inducing vaccines suggested
of vascular permeability and plasma leakage to reduce ADE, but in vivo protection not yet
which are the hallmarks of severe dengue demonstrated
Human Antibodies against viral gp41 and gp120 Positive correlation of enhancing ADE indicated by observation of higher
immunodeficiency enhance viral entry into T cells, monocytes/ antibody level with plasma viral load infection rate/risk of infection among
virus (family macrophages, and granulocytic cells, by the and negative correlation with CD4 vaccine recipients in AIDSVAX, RV144
Retroviridae) following mechanisms: cell count suggest association of ADE clinical trials, but not yet confirmed
1. Complement-mediated, C-ADE: with accelerated immunosuppression
Interaction of antibody-opsonized virus with and disease progression
complement component C3d,g, cellular
complement receptor CR2 and virus-specific
receptor CD4, co-receptor CXCR4
2. FcR-mediated, FcR-ADE: By CD4-
dependent or independent process
R. Kulkarni
 2

Influenza virus 1. Antibodies against viral hemagglutinin Increased risk of medically attended ADE suggested to be responsible for
(family (HA) and neuraminidase (NA) mediate virus illness among individuals with prior vaccine-associated enhanced respiratory
Orthomyxoviridae) uptake via FcRs into macrophages, possibly influenza-like illness during 1918 disease in immunized pigs and ferrets, but
leading to increased antigen presentation and pandemic, and among seasonal mechanism not yet clearly understood
T-cell activation influenza vaccine recipients during
2. Enhancement of viral fusion by anti-HA2 2009 H1N1 pandemic, is suggestive
antibodies suggested of ADE
Respiratory Antibodies against viral glycoproteins G and Clinical relevance remains unclear in Enhanced disease in formalin-inactivated
syncytial virus F mediate virus uptake via FcRs into light of contradictory reports RSV vaccine recipients initially attributed to
(family monocytes, macrophages, dendritic cells regarding association of maternal ADE, but other mechanisms also recently
Paramyxoviridae) leading to immune response modulation antibody-induced ADE with severe suggested
disease in infants
Ebola virus (family Antibodies against viral glycoprotein (GP) Clinical relevance not yet understood Vaccine-related ADE not yet demonstrated,
Filoviridae) promote virus internalization by FcR- but remains a concern. Avoiding induction of
mediated or complement component C1q/ known infectivity-enhancing antibodies,
C1q receptor-mediated process into while retaining T-cell epitopes in vaccines
monocytes/macrophages, endothelial, proposed for ADE mitigation
epithelial cells, and hepatocytes
SARS— Antibodies against viral spike (S) Clinical relevance still debated Impact on vaccine safety not yet understood
Coronavirus glycoprotein mediate virus uptake via FcRs
Antibody-Dependent Enhancement of Viral Infections

(family into immune cells such as B cells,

Coronaviridae) monocytes, macrophages
Chikungunya virus FcR-mediated internalization into B cells, Clinical relevance not yet understood Impact on vaccine safety not yet understood
(family monocytes suggested
Togaviridae)
EDI envelope domain I, EDII envelope domain II, prM precursor membrane, FcR Fc receptor, LILR-B1 leukocyte immunoglobulin-like receptor B1, TLR Toll-
like receptor, RIG-I retinoic acid-inducible gene I, MDA5 melanoma differentiation-associated antigen 5, DHF dengue hemorrhagic fever, DSS dengue shock
syndrome, EDIII envelope domain III
13
14 R. Kulkarni

as “extrinsic ADE.” However, studies on Ross River Virus suggested that internal-
ized immune complexes further serve to enhance viral replication by suppression of
the cellular innate antiviral immune responses (Lidbury and Mahalingam 2000;
Suhrbier and La Linn 2003). This mechanism is termed as “intrinsic ADE” and has
been subsequently observed in flaviviruses as well (Chareonsirisuthigul et al. 2007;
Ubol et al. 2010). Thus, ADE is a complex phenomenon comprising of extrinsic and
intrinsic components, which together contribute to augmentation of viral infection
and replication. The consequence of this increased virus production is the massive
release of inflammatory and vasoactive mediators by host cells, which ultimately
leads to exacerbation of viral pathogenesis and disease severity.
Prior sensitization of the humoral immune response is a prerequisite for ADE
(Halstead 2003; Taylor et al. 2015). This phenomenon is thus widely observed dur-
ing secondary infection with a heterotypic virus of the same genus, wherein preex-
isting antibodies against the primary (sensitizing) infection bind to the (secondary)
virus, but fail to neutralize it. The pathogenicity and outcome of secondary infection
is influenced by the time interval between primary/secondary infections, with
increasing time being associated with more severe disease, and may be explained by
the waning of broadly neutralizing antibodies over time. Passively acquired anti-
bodies are also capable of inducing ADE, as indicated by the enhancement of viral
disease by preexisting maternal antibody in infants born to dengue-immune mothers
(Kliks et al. 1988; Chau et al. 2008, 2009).
The ADE phenomenon has implications for the use of antiviral immunoglobulins
as therapy against viral infection, due to the associated risk of enhancement of dis-
ease (Taylor et al. 2015). ADE also poses a major challenge for implementation of
vaccination programs, as vaccine-induced antibodies may enhance subsequent viral
infection, thus placing vaccine recipients at increased risk of severe disease. Indeed,
concerns regarding the safety of the world’s first licensed dengue vaccine
“Dengvaxia” have forced authorities to reconsider the mass vaccination strategy
and issue specific recommendations for safe implementation of the vaccine in den-
gue control programs (Wilder-Smith et al. 2019).
ADE has been exploited by a variety of viruses belonging to different virus fami-
lies. The mechanism and clinical significance of ADE of selected viruses will be
reviewed in this chapter and are also summarized in Table 2.1. The ADE phenom-
enon has been most extensively studied in dengue virus and will be considered in
detail here.

2.4 Dengue Virus

Dengue virus (DENV) is a member of family Flaviviridae, genus Flavivirus, and is

transmitted to humans through the bite of Aedes mosquitoes, mainly Aedes aegypti.
Infection with any of the four serotypes of this virus, DENV-1 to DENV-4, may be
asymptomatic, or lead to dengue fever with symptoms ranging from mild to high-­
degree fever with headache, myalgia, arthralgia, rash, and retro-orbital pain. In
some patients, the illness may progress to life-threatening severe dengue [earlier
2 Antibody-Dependent Enhancement of Viral Infections 15

classified as dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS)],

characterized by increased vascular permeability, plasma leakage, extensive pleural
effusion, severe hemorrhages, respiratory distress, and organ failure (WHO 2009).
Dengue disease is endemic in more than 125 countries and is considered a major
public health problem worldwide, with an estimated 96 million cases and 20,000
deaths reported annually (Bhatt et al. 2013). In the absence of effective antivirals,
dengue treatment mainly relies on symptomatic interventions. Though a licensed
vaccine is now available, implementation of mass immunization programs remains
complicated due to ADE, and is discussed in later sections.
The DENV genome is a single-stranded, positive-sense RNA molecule (~11 kb
in size) and contains a single open reading frame coding for a large polyprotein,
which is subsequently processed into three structural proteins, capsid (C), mem-
brane (M), and envelope (E), and at least seven nonstructural proteins, NS1, NS2A,
NS2B, NS3, NS4A, NS4B, and NS5 (Lindenbach et al. 2013). The E glycoprotein
is the major surface-exposed region of the virus and displays the viral antigenic
determinants. It functions to bind cellular receptors and fuse with host cell mem-
branes during virus penetration and also directs viral assembly and budding. The E
protein monomer can be divided into three structural/functional domains—EDI, the
central region; EDII, the site of fusion; and EDIII, the site of receptor binding
(Modis et al. 2004). The M protein is initially expressed as a membrane precursor
(prM), which is present in the intracellular immature virus particle. Virion assembly
and maturation involves cleavage of the precursor (pr) peptide, resulting in release
of the mature virus particle containing the M protein into the extracellular environ-
ment (Li et al. 2008; Junjhon et al. 2010).

2.4.1  pidemiological and Experimental Evidence

E
for DENV-ADE

The possibility of immune enhancement of DENV infection was first suggested by

observation of strong association of severe disease (DHF/DSS) with secondary
infection among dengue patients in Bangkok, Thailand, during 1962–1964 (Halstead
et al. 1967; Guzman et al. 2013). These initial findings were supported by prospec-
tive sero-epidemiological studies showing a higher rate of DHF/DSS during sec-
ondary infections (Sangkawibha et al. 1984; Graham et al. 1999; Halstead 2008).
The association of secondary heterotypic DENV (different serotype) infection with
ADE and severe dengue was further strengthened by reports of DHF/DSS cases in
Cuba during the 1981/1997 DENV-2 outbreaks and 2001–2002 DENV 3 outbreak
in a population immune to DENV-1 (1981/1997) and DENV-1/2 (2001–02), respec-
tively (Guzman et al. 1990, 2000; Alvarez et al. 2006). Further, it has been demon-
strated that such secondary DHF/DSS cases have higher viremia and levels of
pro-inflammatory cytokines, suggesting that greater infected cell mass and subse-
quent increase in cytokine release contribute to disease severity in these patients
(Vaughn et al. 2000; Wang et al. 2006; Rothman 2011). However, interestingly,
tertiary/quaternary DENV infections have been rarely associated with severe
16 R. Kulkarni

disease, presumably due to sufficient cross-protective immunity acquired after two

(primary/secondary) different DENV infections (Gibbons et al. 2007).
The most compelling evidence for ADE is provided by observation of severe
dengue during primary infection in infants born to dengue-immune mothers (Kliks
et al. 1988; Chau et al. 2008, 2009). During the first 3–4 months after birth, pas-
sively acquired maternal antibodies have been shown to protect infants from symp-
tomatic dengue. Thereafter, the maternal antibodies begin to decline and reach
sub-neutralizing levels, at which the antibodies are capable of enhancing DENV
infection. Such enhancing antibodies persist till ~12 months of age, placing the
infants at increased risk of severe dengue. Indeed, this enhancing activity of the
infant sera has been demonstrated in vitro.
DENV-ADE is supported extensively through in vitro experiments. Enhancement
of DENV infection by anti-DENV monoclonal antibodies or polyclonal human
immune sera has been observed in primary human myeloid cells as well as cell
lines such as FcγR-expressing BHK-21 cells, monocytic U937, THP-1, K562 cells,
and macrophage-like P388D1 cells (Halstead et al. 1973a; Morens et al. 1987;
Cardosa 1987; Kliks 1990; Goncalvez et al. 2007; Moi et al. 2013; Guzman et al.
2013; Acosta and Bartenschlager 2016). Moreover, in vitro ADE by human sera
has been associated with severe dengue in patients. The ADE phenomenon has also
been demonstrated in animal studies, with higher viremia being recorded during
experimental secondary infection in rhesus monkeys as well as in passively immu-
nized monkeys (Halstead et al. 1973b; Halstead 1979; Marchette et al. 1973;
Goncalvez et al. 2007). Further, DENV-specific antibodies have been shown to
enhance DENV infection and disease severity in AG129 mouse model (Zellweger
et al. 2010). In another study using AG129 mice, maternally acquired anti-DENV
antibodies were found to enhance viremia and vascular leakage, leading to earlier
death (Ng et al. 2014).
The ultimate verification for DENV-ADE in humans has been provided only
recently in a long-term pediatric cohort study in dengue-endemic Nicaragua
(Katzelnick et al. 2017). This study observed highest risk for severe dengue among
individuals with suboptimal anti-DENV antibody titers, and protection from symp-
tomatic dengue at high antibody titers, thus proving that the level of preexisting
anti-DENV antibodies is directly associated with the severity of secondary dengue
disease in humans.

2.4.2 Molecular Mechanism of DENV-ADE

2.4.2.1 Antibodies Enhancing DENV Infection

Studies using monoclonal antibodies obtained from primary/secondary dengue
patients have identified DENV surface proteins E and prM as major targets of host
immune response (Serafin and Aaskov 2001; Crill and Chang 2004; Beltramello
et al. 2010; Matsui et al. 2010; Wahala and Silva 2011; Fibriansah et al. 2015; Gan
et al. 2017). These studies have suggested that highly neutralizing antibodies are
predominantly raised against the complex quaternary epitopes of the viral envelope
2 Antibody-Dependent Enhancement of Viral Infections 17

and the EDIII (lateral ridge) region involved in cellular attachment. These antibod-
ies are mostly serotype-specific; however, antibodies raised against the quaternary
“envelope dimer epitope” (EDE) have been found to potently neutralize all four
DENV serotypes (Dejnirattisai et al. 2015). Such protective antibodies represent
only a small subset of the antibody repertoire in dengue patients, with the immune
response being dominated by cross-reactive, non-neutralizing antibodies targeting
EDI, EDII (fusion loop) and prM, which have the potential to enhance viral infec-
tion (Smith et al. 2014). Moreover, all neutralizing antibodies are also capable of
enhancing infection when present at sub-neutralizing concentration.
The role of antibodies against prM protein is particularly important because of its
ability to enhance infection of immature virions, which are otherwise noninfectious.
The prM protein, present in the intracellular immature virus particle, covers the
fusion domain of the E protein, thus preventing its fusion with the host cell mem-
brane. Cleavage of the precursor (pr) peptide from the prM protein during the pro-
cess of virus maturation renders the released mature virus particle “infectious” to
host cells for further rounds of replication (Li et al. 2008; Junjhon et al. 2010).
Inefficient processing of the prM protein results in the production of immature or
partially immature virus particles, which are normally noninfectious due to their
inability to fuse with host cell. However, studies using human monoclonal antibod-
ies obtained from persons with secondary dengue have revealed that these immature
virus particles are capable of infecting host cells in the presence of the non-­
neutralizing anti-prM antibodies, and are thus infectious during secondary infec-
tions with the possibility of contributing to severe disease (Dejnirattisai et al. 2010;
Schmidt 2010). Similarly, antibodies specific to the fusion loop (FL) in EDII have
also been shown to enhance the infectivity of immature DENV particles (Rodenhuis-­
Zybert et al. 2011).

2.4.2.2 Fcγ Receptors and Their Role in ADE

The Fcγ receptors (FcγRs) present on the surface of cells of the myeloid lineage
function to internalize antibody-opsonized virus via receptor-mediated endocytosis
or phagocytosis and thus aid in clearance of the virus from blood circulation.
However, these FcγRs can also contribute to ADE of DENV infection, by offering
the virus an alternative pathway of entry into its target cells, the FcγR-bearing
monocytes, macrophages, and dendritic cells (Taylor et al. 2015; Gan et al. 2017).
There are three classes of human FcγRs, namely, FcγRI, FcγRII, and FcγRIII. FcγRI
is a high-affinity activating receptor that can bind to monomeric IgG molecules
(Vogelpoel et al. 2015). FcγRII is a class of low-affinity receptors that require high
avidity binding by IgG immune complexes. Of these, FcγRIIA and FcγRIIC are
activating receptors, while FcγRIIB is an inhibitory receptor (Guilliams et al. 2014).
The activating receptors are involved in intracellular signal transduction via the
immunoreceptor tyrosine-based activation motif (ITAM), either accessory (for
FcγRI) or present within the receptor cytoplasmic domain (for FcγRIIA), while the
inhibitory receptor contains immunoreceptor tyrosine-based inhibitory motif
(ITIM) in its cytoplasmic domain (Boonnak et al. 2013). Activating receptors effi-
ciently internalize antibody-opsonized virus, and can be involved in neutralization
18 R. Kulkarni

or enhancement, depending on the antibody concentration and receptor properties.

FcγRI being a high-affinity receptor requires significantly less antibody for com-
plete neutralization and thus plays a predominant role in DENV neutralization
(Chawla et al. 2013). In contrast, FcγRIIA has been shown to be an effective media-
tor of ADE, with its ITAM-containing cytoplasmic tail playing a crucial role
(Boonnak et al. 2013).
The monocytes, which are the major site of DENV infection and replication,
express FcγRI and FcγRIIA at high levels and FcγRIIB at intermediate levels.
FcγRIII is expressed on only ~10% of the monocyte population and has little impact
on DENV neutralization or enhancement (van de Winkel and Anderson 1991).
Macrophages express FcγRI, FcγRII, and FcγRIII. Dendritic cells express FcγRIIA
and FcγRIIB, but FcγRIIB decreases with maturity (Guilliams et al. 2014).

2.4.2.3 Antibody-Mediated Viral Entry into Target Cell

Cells of the mononuclear phagocyte lineage, like monocytes, macrophages, and
dendritic cells, are the main targets for DENV infection in humans. Attachment of
DENV E protein (EDIII) to putative cell surface receptors such as heparan sulfate,
C-type lectins, heat shock protein 70/90, and phosphatidylserine receptors consti-
tutes the first step of viral infection (Castilla et al. 2015; Cruz-Oliveira et al. 2015).
This is followed by penetration of the host cell by receptor-dependent, clathrin- and
dynamin-mediated endocytic pathway. Acidic pH of the endocytic vesicle then trig-
gers fusion of the viral envelope with the endosomal membrane, thus facilitating
virion uncoating and release of the viral genome into the cellular cytoplasm.
During ADE, the antibody-opsonized DENV binds to the FcγRs present on the
surface of the target cells and is internalized by FcγR-mediated endocytosis/phago-
cytosis. The entry pathway is similar to that of free virus; however, it shows slight
alterations, depending on the cell type as well as the FcγR engaged. In vitro studies
in monocytic cell lines U937 and K562 have shown that FcγRII-mediated entry
occurs through clathrin-coated vesicles, while FcγRI-mediated entry is clathrin-­
independent. Also, as FcγRII translocates into lipid rafts upon immune complex
binding, entry via this receptor is affected by the membrane cholesterol content
(Carro et al. 2018). In macrophage-like P388D1 cells, antibody-opsonized virus is
shown to be internalized by phagocytic uptake facilitated by actin-mediated mem-
brane protrusions which actively search and capture the antibody-bound virus par-
ticles, possible by chemotaxis (Ayala-Nunez et al. 2016).
In addition to FcγR, other primary cell receptors may also be required for cell
penetration. In fact, one theory suggests that FcγR plays an auxiliary role in concen-
trating the immune complexes to the cell surface and viral entry is then mediated by
the primary cellular receptors (Chotiwan et al. 2014).

2.4.2.4 Suppression of Innate Antiviral Immune Response in ADE

The suppression of the host antiviral immune response to facilitate higher virus pro-
duction during ADE of DENV infection was first suggested in 2007 through studies
in monocytic THP-1 cells, which revealed upregulation of anti-­inflammatory cyto-
kine production (IL-6, IL-10) but downregulation of anti-DENV free radicals (nitric
2 Antibody-Dependent Enhancement of Viral Infections 19

oxide) and pro-inflammatory cytokine production (IL-12, IFN-γ, TNF-α)

(Chareonsirisuthigul et al. 2007). Subsequent studies in THP-1 cells offered further
insights into the mechanism of this “intrinsic” DENV-ADE (Ubol et al. 2010;
Modhiran et al. 2010; Tsai et al. 2014; Chan et al. 2014). Overall, these studies have
suggested Type I interferon and pro-inflammatory cytokine production as the main
targets of immune suppression during DENV-ADE, with four mechanisms being pro-
posed for the same: (1) leukocyte immunoglobulin-like receptor B1 (LILR-B1) co-
ligation and consequent inhibition of FcγR-signaling and Type I interferon-­stimulated
gene (ISG) expression, (2) downregulation of toll-like receptor (TLR)-dependent
antiviral signaling pathway, (3) downregulation of retinoic acid-inducible gene I
(RIG-I)/melanoma differentiation-associated antigen 5 (MDA-5) signaling pathway
resulting in suppression of Type I interferon (IFN) production, and (4) induction of
IL-10 production resulting in suppressor of cytokine signaling 3 (SOCS3)-mediated
suppression of pro-inflammatory cytokines important for antiviral response.
Binding of immune complexes to cellular activating FcγRs is known to trigger
ITAM-spleen tyrosine kinase (Syk)-signal transducer and activator of transcription
1 (STAT1) signaling pathway which leads to ISG induction and inhibition of viral
replication. Inhibition of this FcγR-dependent ISG transcription is thus crucial for
successful DENV replication and is proposed to be brought about by co-ligation of
LILR-B1 by the FcγR-bound antibody-opsonized DENV (Chan et al. 2014;
Nimmerjahn and Lux 2014). LILR-B1 belongs to a family of ITIM-containing
inhibitory receptors and is expressed on immune effector cells including mono-
cytes, macrophages, and dendritic cells. At sub-neutralizing antibody concentra-
tions, the DENV–antibody immune complex binds to two cellular receptors: FcγR
(through antibody) and LILR-B1 (through DENV E protein). Binding to LILR-B1
recruits Src homology phosphatase-1 (SHP-1) which dephosphorylates and inacti-
vates Syk and prevents ISG expression. SHP-1 signaling also prevents rapid acidifi-
cation and lysosomal degradation of DENV (Ong et al. 2017). On the contrary, at
high antibody concentrations, DENV E protein epitopes are completely blocked by
the antibody; consequently, LILR-B1 binding is not possible, resulting in neutral-
ization of the FcγR-internalized virus.
The TLR-dependent pathway is a key mediator of the innate antiviral immune
response and stimulates production of Type I IFN and pro-inflammatory cytokines
via nuclear factor-κβ (NF-κβ) family of transcription factors. Studies using mono-
cytic cell lines and patient peripheral blood mononuclear cells (PBMCs) have
shown that this pathway is suppressed during DENV-ADE (Modhiran et al. 2010).
Engagement of FcR by DENV–antibody immune complexes or FcR-mediated entry
of immune complexes into target cells results in downregulation of expression of
TLR-3,-4,-7 and TLR signaling molecules. This is accompanied by activation of
Sterile-alpha and Armadillo motif-containing protein (SARM) and TRAF family
member-associated NF-κβ activator (TANK), which are negative regulators of the
TLR adaptor molecule, TIR-domain-containing adapter-inducing interferon-β
(TRIF), and effector molecule, TNF receptor-associated factor 6 (TRAF6), respec-
tively. SARM/TANK activation thus results in suppression of TLR-dependent IFN
stimulating pathway.
20 R. Kulkarni

The RIG-I/MDA-5 antiviral signaling pathway is also downregulated during

DENV-ADE, as demonstrated in vitro and observed among patients of severe sec-
ondary DENV infection (Ubol et al. 2010). Entry of immune complexes into target
cells activates the negative regulators, dihydroxyacetone kinase (DAK) and
autophagy-­related 5–autophagy-related 12 (Atg5-Atg12), which then disrupt the
RIG-I/MDA-5 signaling cascade. This results in suppression of Type I IFN produc-
tion and IFN-mediated antiviral responses.
IL-10 is known to induce T-helper cell 2 (Th2) cytokine response and inhibit
production of pro-inflammatory cytokines such as IFN-γ, IL-12, TNF-α as well as
nitric oxide radicals, which are potent inhibitors of DENV replication. IL-10
induction during DENV-ADE thus exerts an immunosuppressive effect and favors
viral replication. Indeed, high IL-10 levels have been associated with severe dis-
ease in secondary dengue patients (Nguyen et al. 2004; Ubol et al. 2010). The
mechanism of IL-10-mediated immune suppression has been demonstrated through
in vitro studies in monocytic cell lines and patient PBMCs. These studies have
shown that IL-10 induction during ADE occurs through enhanced Syk-regulated
phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB)/glycogen synthase
kinase (GSK)-3β/cyclic adenosine monophosphate response element-binding
(CREB) signaling (Tsai et al. 2014). IL-10 then stimulates expression of SOCS3,
which subsequently inactivates Janus kinase (Jak)-STAT signaling, resulting in
suppression of pro-­inflammatory cytokine production. In addition, IL-10 also sup-
presses inducible nitric oxide synthase (iNOS) gene expression and thus nitric
oxide production, by inhibiting STAT-1 and Interferon Regulatory Factor 1 (IRF-1)
activity (Chareonsirisuthigul et al. 2007; Ubol et al. 2010).
Majority of the studies elucidating the mechanism of intrinsic ADE have been
carried out in monocytic cell lines. Experiments using primary monocyte-derived
human macrophages have demonstrated lower IFN-β and higher IL-6 and SOCS3
levels during DENV-ADE, consistent with the observations in monocytic cells
(Rolph et al. 2011). However, IL-10 induction and RIG-I/MDA-5 downregulation
were not observed. Thus, IL-6 has been proposed as critical regulator of DENV-­
ADE in macrophages, responsible for SOCS-3-mediated suppression of pro-­
inflammatory cytokines, indicating that mechanisms of intrinsic ADE may be cell
type-dependent.
Contradictory to the well-accepted mechanism of ADE-mediated immune sup-
pression, a recent study observed that entry of DENV immune complexes in pri-
mary human macrophages did not trigger immunosuppressive signaling, but resulted
in enhanced fusion of viral envelope with the host cell membrane (Flipse et al.
2016). Further investigations into the different mechanisms involved in ADE of
DENV infection are thus warranted.

2.4.2.5 Enhancement of Disease Severity: ADE and Cytokine Storm

The extrinsic and intrinsic mechanisms of ADE together contribute to enhancement
of viral replication and consequently higher viremia levels, which correlate with the
incidence of severe dengue in patients (Vaughn et al. 2000). ADE is also associated
with triggering of the “cytokine storm” which involves massive release of
2 Antibody-Dependent Enhancement of Viral Infections 21

inflammatory cytokines and other chemical mediators at high levels (Pang et al.
2007). ADE of DENV infection and replication results in increased presentation of
viral antigens on the surface of infected cells, leading to activation and proliferation
of T cells sensitized during a prior infection. This results in release of cytokines
such as IFN-γ, TNF-α, IL-2, IL-6, IL1-β, IL-8 and immunoregulators such as
IL-12p70, which directly act upon the vascular endothelial cells resulting in plasma
leakage, the hallmark of severe dengue. Increased levels of such cytokines have
been associated with plasma leakage in severe dengue patients (Chaturvedi et al.
2000), while effect of the cytokine response on alteration of vascular permeability
has been demonstrated in vitro and in mice models, with disruption of the apical
junction complexes in endothelial cells being the suggested mechanism (Dewi et al.
2004; Appanna et al. 2012; Puerta-Guardo et al. 2013).

2.4.3 Implications for Dengue Vaccines

Generation of a long-term protective immune response against all four serotypes of

DENV is a prerequisite for a dengue vaccine and assumes greater importance due to
the associated risk of vaccine-induced ADE of DENV infection and disease. During
the six-decade long quest for an effective dengue vaccine, several candidates such
as live-attenuated, inactivated, recombinant subunit, virus-like particle (VLP)-
based, and DNA vaccines have been explored, with Sanofi Pasteur’s “Dengvaxia” or
CYD-TDV, a chimeric yellow fever 17D-tetravalent DENV vaccine, National
Institutes of Health’s TV003/TV005, and Takeda Pharmaceutical’s TAK-003, both
live attenuated tetravalent vaccines, leading the development pipeline (McArthur
et al. 2013). Of these, CYD-TDV has been approved for human use and is licensed
in 20 countries, while TV003/TV005 and TAK-003 are currently undergoing Phase
III trials.
CYD-TDV, the world’s first licensed dengue vaccine, was approved by the World
Health Organization (WHO) in May 2016, following Phase III trials in dengue-­
endemic regions (Capeding et al. 2014; Villar et al. 2015; Hadinegoro et al. 2015).
These trials had demonstrated high vaccine efficacy among 9–16-year-olds during
the first 2 years after vaccine (first of three doses) administration, but indicated
increased risk of hospitalized dengue in the 2–5 year age group during the third year
of follow-up. These results suggested the possibility of the vaccine sensitizing den-
gue seronegatives to subsequent enhanced DENV infection and disease. However,
considering the population-level benefits in reducing disease burden, the vaccine
was approved by WHO Strategic Advisory Group of Experts (SAGE) on immuniza-
tion, with recommendations for use in dengue-endemic regions with ≥70% serop-
revalence and administration to individuals in the age range of 9–45 years. This
approval was followed by additional studies to investigate the long-term benefit–
risk ratio of dengue vaccination in seronegative population. These studies revealed
that vaccine performance was indeed affected by serostatus, with vaccine efficacy
being significantly higher in seropositives than seronegatives. Furthermore, an
increased risk of hospitalized, severe dengue was observed among seronegative
22 R. Kulkarni

vaccine recipients as compared to seronegative controls (Sridhar et al. 2018). In

light of this new evidence regarding vaccine-related ADE, WHO-SAGE issued
updated recommendations on the use of CYD-TDV vaccine in April 2018 (Wilder-
Smith et al. 2019). For countries considering vaccination as part of their dengue
control program, WHO now recommends a “pre-vaccination screening strategy,” in
which all potential vaccine recipients are tested for anti-DENV IgG, preferably by
ELISA, and only dengue-seropositive persons are vaccinated. Further, for trials of
new vaccines, WHO advises stratification of participants according to pre-vaccina-
tion serostatus during data analysis, as well as long-term follow-up of all vaccine
recipients.
The occurrence of breakthrough infections in the CYD-TDV vaccinees despite
having high titers of neutralizing antibodies has raised concerns regarding the value
of the currently used in vitro neutralization tests as correlates of protection. The
neutralizing potential of antibodies is commonly evaluated using the plaque reduc-
tion neutralization test (PRNT) in LLC-MK2, Vero, or BHK-21 cell lines. However,
neutralization of DENV infection in these FcγR-negative cells may not reflect the
ability of the antibodies to prevent infection of myeloid cells, which are primary
targets of DENV, and capable of internalizing antibody-opsonized DENV through
FcγR-mediated phagocytosis. The use of FcγR-bearing cell lines or primary myeloid
cells for in vitro neutralization has thus been proposed for measuring the neutraliz-
ing versus enhancing ability of anti-DENV antibodies (Mahalingam et al. 2013;
Gan et al. 2017). Furthermore, identification of stronger immune correlates for dis-
ease risk and protection is urgently needed (Katzelnick and Harris 2017).
While the results of Phase III trials of TV003/TV005 and TAK-003 vaccines are
awaited, the concern of vaccine-related ADE has promoted development of vac-
cines specifically designed to minimize ADE. Monoclonal antibody-based studies
have helped identify neutralizing and enhancing viral epitopes, making design of
such tailored vaccines possible. DENV EDIII induces predominantly neutralizing
antibodies, thus making recombinant EDIII an attractive subunit vaccine candidate
(Gil et al. 2017; Frei et al. 2018). DENV NS1 has also been considered as a vaccine
candidate as it avoids ADE (Hertz et al. 2017). Chimeric DENV constructs with pr
gene replaced with Japanese Encephalitis virus pr and DNA vaccines with substitu-
tions or removal of enhancing epitopes have demonstrated reduction in ADE (Crill
et al. 2012; Tang et al. 2015; Wang et al. 2015). Another strategy for evasion of ADE
is the development of vaccine candidates inducing protective CD4+ and CD8+
T-cell responses. DENV C protein is an immunodominant T-cell epitope, and
C-based vaccines have shown induction of protective T-cell-mediated immunity in
monkeys (Gil et al. 2014). Such next-generation vaccines do face the disadvantage
of not retaining the intact virion structure and the quaternary epitopes, which are
important for potent neutralization. However, these vaccines have shown encourag-
ing results in preclinical studies, and whether they are capable of conferring in vivo
protection remains to be seen.
2 Antibody-Dependent Enhancement of Viral Infections 23

2.4.4 Implications for Dengue Therapeutics

Humanized monoclonal antibodies (mAbs) are attractive therapeutic options against

DENV infection. Therapeutic mAbs must neutralize DENV, without increasing the
risk of ADE, in order to offer protection against dengue. Several mAbs targeting the
DENV E and/or prM proteins are being developed as therapeutic candidates, with
reduction of ADE as the major focus of development (Chan et al. 2013; Sun et al.
2018). Use of serotype-specific mAbs or high dose administration of cross-reactive
mAbs has demonstrated reduced risk of ADE in vitro and in animal models. Further,
antibodies with modifications in the Fc region such as deletion of nine amino acids
(at positions 231–239) in the N terminus, mutation of asparagine to glutamine at
position 297 (N297Q), or mutation of leucine to alanine at positions 234 and 235
(LALA mutants) have also shown reduced risk of ADE and exhibited prophylactic
and therapeutic efficacy in animal models (Balsitis et al. 2010; Shi et al. 2016; Xu
et al. 2017; Injampa et al. 2017; Lu et al. 2018). Fc region modifications that extend
the antibody half-life also help minimize ADE by maintaining the mAbs at high
levels.

2.4.5 ADE in Other Flaviviruses

ADE has also been reported in other viruses of family Flaviviridae including
Murray Valley Encephalitis virus, Japanese encephalitis virus (JEV), West Nile
virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV) of genus Flavivirus,
as well as hepatitis C virus (HCV), of genus Hepacivirus (Hawkes and Lafferty
1967; Cardosa et al. 1983; Gould and Buckley 1989; Meyer et al. 2008; Bardina
et al. 2017). Further, infection with one Flavivirus has also been demonstrated to
enhance subsequent infection with another virus of the same genus, as observed for
DENV-ZIKV in in vitro studies, mice models, and rhesus macaques (Bardina et al.
2017; George et al. 2017). Such DENV-ZIKV cross-enhancement has also been
observed in primary human PBMCs (Li et al. 2018); however, in the absence of
clinical or epidemiological evidence, the clinical relevance of these findings is
unknown. Interestingly, a beneficial effect of cross-enhancement among flaviviruses
has been reported recently in a clinical trial, with preexisting JEV vaccine-induced
antibodies enhancing immunogenicity of subsequently administered YFV vaccine
(Chan et al. 2016).

2.5 Human Immunodeficiency Virus

Human immunodeficiency virus (HIV), a member of family Retroviridae, genus

Lentivirus, is primarily a sexually transmitted virus, but nonsexual transmission
through intravenous inoculation or mother-to-child exposure can also occur. HIV
infects the CD4+ T cells leading to their progressive depletion and thus hampers the
host adaptive immune response. The final stage of HIV infection is acquired
24 R. Kulkarni

immunodeficiency syndrome (AIDS), characterized by immune system failure and

occurrence of life-threatening opportunistic infections. As of 2016, ~37 million
people worldwide have been estimated to be living with HIV (Ghosn et al. 2018).
While a protective vaccine or a complete cure of HIV infection remains elusive,
implementation of combination antiretroviral therapy (cART) has been successful
in prolonging the duration between HIV infection and AIDS and reducing AIDS-­
related deaths.

2.5.1  DE of HIV Infection: Experimental Evidence

A
and Molecular Mechanism

ADE of HIV infection was first reported in the 1980s, shortly after the identification
and isolation of HIV, and two different mechanisms, namely, complement-mediated
ADE (C-ADE) (Robinson Jr. et al. 1987, 1988) and FcR-mediated ADE (FcR-ADE)
(Takeda et al. 1988; Homsy et al. 1989), were described. Through either of these
mechanisms, virus-specific antibodies present in sera of HIV-infected individuals
were shown to enhance viral entry and in some cases replication within T cells,
monocytes/macrophages, and granulocytic cells (Beck et al. 2008). Further, both
these mechanisms have also been shown to enhance HIV infection of human syncy-
tiotrophoblast cells, thus suggesting contribution of ADE to materno-fetal transmis-
sion (Tóth et al. 1994). Both these forms of ADE are mediated by antibodies
targeting the viral surface glycoproteins gp41 and gp120 (Trischmann et al. 1995;
Takada and Kawaoka 2003). Such enhancing antibodies have been detected in 72%
of HIV patients (Subbramanian et al. 2002).
C-ADE of HIV infection has been well characterized through in vitro studies in
T-cell lines such as MT-2 and SupT1/R5, showing enhancement of cell line-adapted
HIV strains or primary virus isolates by human monoclonal antibodies or sera from
HIV-infected individuals, and has been associated with increased synthesis of viral
RNA and protein and enhanced release of infectious virions (Robinson Jr. et al.
1989, 1990a, b; Robinson Jr. 2006; Willey et al. 2011). Such C-ADE is primarily
mediated by antibodies targeting the N-terminal immunodominant domain of viral
gp41. Antibody binding to gp41 initiates the complement cascade leading to deposi-
tion of complement component C3d,g on the virion. Interaction of this opsonized
virus with the cell surface complement receptor type 2 (CR2) facilitates virus
attachment and internalization. Engagement of the HIV receptor CD4 and co-­
receptor CXCR4 is also required for this mode of virus uptake. Increased attach-
ment of the virus to target cell, rather than CR2-mediated signaling, has been
suggested to be responsible for enhancement of infection through C-ADE. An alter-
native route of complement component C1q binding followed by interaction with
C1q receptor has also been suggested for C-ADE (Prohaszka et al. 1997).
FcR-ADE has been demonstrated in vitro mainly in monocyte/macrophage cell
lines and primary cultures, with all three classes of FcγR, namely, FcγRI, FcγRII,
and FcγRIII, reported to support ADE (Homsy et al. 1989; Takeda et al. 1988, 1990;
Laurence et al. 1990; Connor et al. 1991; Trischmann et al. 1995). ADE by FcγRI
2 Antibody-Dependent Enhancement of Viral Infections 25

and FcγRII requires viral interaction with CD4, indicating that the FcRs facilitate
virus entry by potentiating attachment to CD4 receptors. Alternatively, FcR-­
mediated endocytosis of virus–antibody complexes followed by intracellular fusion
with endosomal membrane occurring as a result of binding of virus with CD4 recep-
tors on the endosomal membranes has also been suggested. FcγRIII-mediated ADE
has been shown to be CD4-independent. FcR-ADE assumes particular importance
in the viral life cycle as it mediates infection of macrophages, which are important
for maintaining a viral reservoir during persistent infections.
Another mechanism of complement-independent, FcR-independent ADE,
mediated by antibodies specific to viral gp120, has been described (Sullivan et al.
1998; Guillon et al. 2002). Antibody binding has been shown to modulate interac-
tion of gp120 with HIV co-receptor CCR5 or induce conformational changes in
gp120, leading to its activation and subsequent promotion of membrane fusion.
Further, FcαR-mediated ADE has also been reported, with human serum IgA
showing low-­level enhancement of HIV replication in vitro (Janoff et al. 1995;
Kozlowski et al. 1995).
An important feature of HIV is the rapid viral evolution during chronic infection.
While neutralizing antibodies are known to exert a selection pressure facilitating
emergence of neutralization escape mutants, enhancing antibodies have been sug-
gested to favor emergence of ADE-susceptible variants. Indeed, at later stages of
infection, patient sera have demonstrated increased proportion of enhancing anti-
bodies and potential for ADE, while virus strains isolated at such later stage have
exhibited increased susceptibility to ADE in vitro (Davis et al. 2001; Subbramanian
et al. 2002; Willey et al. 2011).

2.5.2 Clinical Significance of HIV-ADE

The HIV disease outcome is suggested to be influenced by the balance of neutral-

izing and enhancing antibodies in HIV patients, with studies showing lower neu-
tralizing and higher enhancing antibody levels among AIDS patients (Homsy et al.
1990; Toth et al. 1991; Fust et al. 1994; Szabo et al. 1999; Subbramanian et al.
2002). These studies have observed strong negative correlation between the extent
of ADE and CD4 cell count and positive correlation between ADE and plasma
viral load in HIV-positive individuals. As CD4 cell count and HIV plasma viral
load are considered the most potent predictors of HIV disease progression, such
observations indicate contribution of ADE to accelerated immunosuppression and
disease progression. Further support for this association was provided by reports of
decline in enhancing antibody levels and improvement in disease prognosis in
patients receiving highly active antiretroviral therapy (HAART) (Banhegyi et al.
2003). Contradictory observations of absence of any clinical correlation between
ADE and disease have also been reported (Montefiori et al. 1991); however, it has
been suggested to be the result of conduction of experiments in conditions which
do not sufficiently resemble in vivo conditions (Takada and Kawaoka 2003; Beck
et al. 2008).
26 R. Kulkarni

2.5.3 Implications for HIV Vaccines

The impact of ADE on HIV vaccine safety is not clearly understood. Studies in
Rhesus monkeys have demonstrated the association of active and passive immuni-
zation with enhanced disease progression on subsequent infection (Staprans et al.
2004; Sholukh et al. 2014). In humans, ADE has been indicated in two vaccine
clinical trials. In the AIDSVAX trial, the rate of HIV infection was observed to be
higher among vaccinees with low (non-protective) antibody responses (Gilbert et al.
2005). In the RV144 trial, positive correlation of vaccine-elicited plasma IgA
responses with risk for HIV acquisition was documented (Shmelkov et al. 2014).
Further, a study investigating the effect of passive immunization of HIV-positive
pregnant women on mother-to-infant transmission suggested the possibility of
antibody-­enhanced in utero transmission (Onyango-Makumbi et al. 2011). These
findings are suggestive of vaccine-induced ADE, but cannot confirm the same.
Assays capable of estimating neutralizing and enhancing potential of immune
serum, such as those involving use of complement and complement receptor carry-
ing target cells for C-ADE, have been proposed for a better understanding of the
clinical relevance of vaccine-related ADE. Such assays would also provide stronger
immune correlates during preclinical and clinical studies. Further, design of vaccine
immunogens that will elicit protective and not enhancing antibodies, by avoiding
inclusion of known enhancing epitopes, has been put forth as a mitigation strategy
for HIV-ADE (Beck et al. 2008).

2.6 Influenza Virus

Influenza is a highly communicable acute respiratory disease caused by infection

with influenza virus of family Orthomyxoviridae. The disease is a serious health
threat of epidemic and pandemic proportions, with severe cases associated with
pneumonia and lung tissue damage. Influenza A viruses are the most common in
human infections and are further classified into subtypes on the basis of the two
surface proteins, hemagglutinin (H) and neuraminidase (N), with 18 H and 11 N
subtypes identified (Shao et al. 2017). Influenza A virus undergoes rapid evolution
due to mechanisms such as antigenic drift, shift, and genetic reassortment, resulting
in periodic emergence of novel viral strains. Annual vaccination against the circulat-
ing viral strain is thus implemented for protection against the disease.
Epithelial cells of the respiratory tract are the primary targets of influenza virus
infection; however, in vitro ADE of this virus has been reported in primary macro-
phages and macrophage-like cell lines (P388D1), under the presence of monoclonal
antibodies, mice, rabbit, or human immune sera (Ochiai et al. 1988, 1990, 1992;
Tamura et al. 1991, 1994; Gotoff et al. 1994). Engagement of FcγRs by antibodies
specific to viral HA and NA has been shown to mediate virus uptake, with subtype
cross-reactive non-neutralizing antibodies being particularly involved in infection
enhancement of heterotypic viruses (different HA/NA type). While such internaliza-
tion usually results in abortive infection of macrophages, productive replication in
2 Antibody-Dependent Enhancement of Viral Infections 27

these cells has been shown to be possible in the presence of appropriate protease for
HA cleavage. Increased presentation of viral antigens by the infected macrophages
leading to augmented virus-specific T-cell activation is also suggested to contribute
to viral pathogenesis. Enhancement of virus fusion by anti-HA2 subunit antibodies
is another mechanism of influenza-ADE and has been shown to be responsible for
the occurrence of vaccine-associated enhanced respiratory disease (VAERD) in pigs
immunized with inactivated H1N2 vaccine and challenged with pandemic 2009
H1N1 virus (Gauger et al. 2011; Khurana et al. 2013). Influenza-­ADE is also sug-
gested by epidemiologic observations in humans. Retrospective analysis of medical
records from the 1918 influenza pandemic revealed association of prior influenza-
like illness with increased risk of medically attended illness during the pandemic
period (Shanks et al. 2016). More recently, prior receipt of seasonal influenza vac-
cine was found to increase the risk of medically attended pandemic H1N1 illness
during 2009 in Canada (Skowronski et al. 2010). Furthermore, presence of high titer
non-protective serum antibodies was associated with immune complex-mediated
severe H1N1 illness during this pandemic (Monsalvo et al. 2011; To et al. 2012).
Observation of VAERD in pigs and ferrets immunized with inactivated or HA
subunit vaccines raises concern regarding the safety of vaccines, particularly uni-
versal influenza vaccines (Gauger et al. 2011; Khurana et al. 2013; Rajao et al.
2014; Skowronski et al. 2014). However, vaccine-mediated protection and absence
of VAERD is also reported in few studies in pigs, suggesting that vaccine-induced
ADE occurs only in certain conditions, which are not yet clearly understood (Reeth
et al. 2004; Kyriakis et al. 2010; Ricklin et al. 2016). Regarding therapeutic candi-
dates, Phase 2 studies of anti-HA stalk mAbs have indicated possibility of enhanced
viral shedding in some treated human subjects. Screening for high potency protec-
tive antibodies, defining optimal dose range for effective neutralization, using cock-
tails of mAbs with different antigen specificities, has been suggested for mitigation
of ADE (Chan-Hui and Swiderek 2016).

2.7 Respiratory Syncytial Virus

Respiratory syncytial virus (RSV), a member of family Paramyxoviridae, genus

Pneumovirus, is the leading cause of lower respiratory tract infections among
infants and young children worldwide, with severe disease requiring hospitalization
occurring most frequently between 6 weeks and 6 months of life (Glezen et al.
1986; Hall et al. 2009). RSV disease is also a major health problem in adults and the
elderly. Severe bronchiolitis is one of the major outcomes of RSV infection.
Observation of enhanced disease among children receiving the formalin-­
inactivated RSV (FI-RSV) vaccine in the 1960s (Kim et al. 1969), as well as the
frequent occurrence of severe disease among infants <6 months of age, when mater-
nal antibodies are present, prompted investigations on ADE of RSV infections.
Subsequently, enhancement of RSV infection by RSV-specific monoclonal antibod-
ies and human immune sera was demonstrated in vitro in monocytic (U937, THP-1)
and macrophage-like (P388D1) cell lines (Gimenez et al. 1989, 1996; Krilov et al.
28 R. Kulkarni

1989; Osiowy et al. 1994). The phenomenon has also been reported in Bonnet mon-
keys developing enhanced disease following FI-RSV immunization (Ponnuraj et al.
2001, 2003). FcγR-mediated internalization of immune complexes is the suggested
mechanism, with antibodies against the viral surface glycoproteins, the attachment
glycoprotein (G) and fusion glycoprotein (F), playing an important role. Although
epithelial cells of the respiratory tract are primary targets of RSV infection, it has
been suggested that alveolar macrophages are infected through ADE, resulting in
activation of Th2 response and increased expression of TNF-α, IL-6, thus causing
enhanced disease (Gimenez et al., 1996). Further, ADE-mediated infection of lung
dendritic cells (DCs) has been demonstrated to negatively modulate DC cell func-
tion, resulting in impaired T-cell activation, and has been suggested to contribute to
RSV pathogenesis (Gomez et al. 2016). However, the clinical effects of RSV-ADE
still remain unclear with different studies reporting contradictory observations
regarding the association of maternal antibody-induced ADE and disease severity in
infants (Chanock et al. 1970; van Erp et al. 2017).
Enhanced RSV disease in FI-RSV vaccine recipients was initially attributed to
vaccine-induced ADE; however, recent studies have suggested a role of other mech-
anisms involving T cells (Huisman et al. 2009). Whether ADE would affect the
efficacy of other RSV vaccines in development remains to be seen. As far as immu-
notherapeutics are concerned, the F protein-specific humanized monoclonal anti-
body, Palivizumab, is the only clinically approved intervention. This mAb has
demonstrated ADE at sub-neutralizing concentrations in vitro, yet has been found to
have a protective effect in animal models and humans (TI-RS Group 1998; Mejıas
et al. 2004; van Mechelen et al. 2016).

2.8 Ebola Virus

Ebola virus is a member of family Filoviridae. Of the four Ebola virus species
infecting humans, Zaire ebolavirus (EBOV) is the most virulent and is the causative
agent of a severe hemorrhagic fever disease, Ebola virus disease (EVD), with a
fatality rate of ~90% (Feldmann and Geisbert 2011). The disease is zoonotic, with
initial cases occurring due to contact with infected fruit bats (reservoir) or their
contaminated material, followed by human-to-human transmission. The virus is
mainly endemic in Africa; however, due to its increased incidence and fast spread,
as observed during the 2014–2016 African outbreak, the virus is considered a pan-
demic threat.
ADE of Ebola virus infection has been demonstrated in vitro in a variety of cell
lines and is shown to be mediated by antibodies specific to the viral envelope glyco-
protein (GP) (Takada et al. 2001, 2003, 2007). Such enhancing antibodies have been
identified in the sera of EVD patients (Takada et al. 2003; Furuyama et al. 2016).
Two different mechanisms have been proposed for ADE of EBOV infection: (a)
FcγR-mediated ADE and (b) C1q-mediated ADE. In FcγR-mediated ADE, anti-
body–virus complexes bind cell surface FcγRIIA triggering phosphorylation of Src
family protein tyrosine kinases (PTKs) and activation of Src signaling pathways
2 Antibody-Dependent Enhancement of Viral Infections 29

leading to virus uptake through phagocytosis and/or micropinocytosis (Furuyama

et al. 2016). C1q-mediated ADE involves interaction of virus-bound antibodies with
complement component C1q and subsequent attachment to cell surface C1q recep-
tors, leading to virus internalization by C1q receptor-mediated endocytosis or via
virus-specific receptor (Takada et al. 2003, 2007). C1q receptors are present on
monocytes/macrophages, which are primary targets of EBOV, as well as most other
mammalian cells, including endothelial cells, epithelial cells, and hepatocytes. C1q-­
ADE mediated dissemination of EBOV infection to these “secondary target cells”
has been suggested to exacerbate disease.
Although clinical relevance of EBOV-ADE is not yet understood, repeated
in vitro demonstrations raise concerns regarding safety of vaccines and immuno-
therapeutics. Several candidate vaccines under development have demonstrated
protective efficacy in preclinical studies and immunogenicity in clinical trials
(Dhama et al. 2018). The protective effect of these vaccines seems to depend on
cytotoxic T-cell responses. Avoiding induction of known infectivity-enhancing anti-
bodies while retaining T-cell epitopes should thus be considered during vaccine
design. For immunotherapy, the use of well-characterized mAbs instead of poly-
clonal convalescent human serum has been suggested to reduce ADE.

2.9 Severe Acute Respiratory Syndrome: Coronavirus

Severe acute respiratory syndrome (SARS) is a respiratory disease caused by

SARS-coronavirus (SARS-CoV) of family Coronaviridae, which caused outbreaks
mainly in China, during 2002–2003. Although public health measures successfully
contained further spread and human outbreaks, future reemergence of SARS-CoV
or other related viruses remains a credible threat due to the zoonotic origin of this
virus.
ADE of SARS-CoV infection was first demonstrated in vitro in human B cells
using sera from immunized animals as well as convalescent patients (Kam et al.
2007). Further studies showed that antibodies specific to the viral surface spike
glycoprotein (S) are capable of enhancing viral infection of immune cells, particu-
larly monocytes and macrophages, thus allowing the virus to broaden its cellular
tropism (Jaume et al. 2011; Wang et al. 2014; Yip et al. 2014). Internalization of the
immune complexes by cellular FcγRs, mainly FcγRIIA, is the suggested mecha-
nism, with intracellular signaling by the FcγR cytosolic domain playing an impor-
tant role. This FcγR-mediated internalization follows a pH- and cysteine
protease-independent pathway and differs from the endosomal/lysosomal pathway
utilized by angiotensin I-converting enzyme 2 (ACE2), the accepted receptor for
SARS-CoV. Such antibody-mediated infection of macrophages, however, was not
found to support productive virus replication or modify expression of cytokines/
chemokines (Yip et al. 2016). Although demonstrated recently in rhesus monkeys
(Wang et al. 2016), the occurrence of SARS-ADE and its association with disease
severity in humans are still debated, with different clinical studies reporting both
30 R. Kulkarni

protective and disease-enhancing effects of anti-SARS-CoV antibodies (Lee et al.

2006; Zhang et al. 2006).

2.10 Chikungunya Virus

Chikungunya virus (CHIKV) is an arbovirus belonging to family Togaviridae,

genus Alphavirus. The virus is recognized as a significant global health threat since
the last decade. It is primarily transmitted through the bite of infected Aedes mos-
quitoes, Aedes aegypti and Aedes albopictus. CHIKV infection is associated with
a febrile illness accompanied by myalgia, arthralgia, and cutaneous rash (Kam
et al. 2009). Debilitating persistent polyarthralgia is the characteristic feature of
Chikungunya disease.
ADE of CHIKV infection was suggested by the association of low titers of anti-­
CHIKV IgG in immunized mice with the early onset of disease upon subsequent
CHIKV challenge (Hallengard et al. 2014). Another recent study observed enhanced
attachment of CHIKV to primary human monocytes and B cells and increased viral
replication in the murine macrophage cell line, RAW264.7, in the presence of anti-­
CHIKV antibodies (Lum et al. 2018). Further, this study also detected higher viral
RNA load and enhancement of disease severity in mice infected with CHIKV in the
presence of sub-neutralizing concentrations of antibody, thus providing the first evi-
dence for ADE of CHIKV infection and disease. The enhancement was found to be
FcγR-mediated, with FcγRII playing a predominant role. However, ADE of CHIKV
infection has not been reported to date in humans, and thus, the clinical significance
of these findings remains unknown and warrants further investigation.

2.11 Concluding Remarks

Virus-specific antibodies function to neutralize virus infection and are an important

component of the host antiviral immune response. However, under sub-neutralizing
or non-neutralizing conditions, the antibodies are capable of enhancing viral infec-
tion by providing an alternative route of virus entry into host cells. This phenome-
non of antibody-dependent enhancement facilitates viral infection of a larger
number of primary target cells and also allows viral entry into otherwise non-­
susceptible secondary target cells, thus broadening the virus tropism. Moreover,
ADE also modulates the host intracellular signaling pathways, thereby turning
down the antiviral immune response. Together these mechanisms serve to augment
viral replication and pathogenesis, with the balance between neutralizing and
enhancing antibodies influencing the outcome of viral infection and disease.
ADE has been demonstrated in vitro and in animal models for a number of
viruses including DENV, HIV, influenza, RSV, Ebola, SARS-CoV, and
CHIKV. While the occurrence of DENV-ADE in humans and its association with
severe disease have been clearly demonstrated, the clinical relevance of ADE in
other viruses is not completely understood. Nevertheless, ADE remains a concern in
2 Antibody-Dependent Enhancement of Viral Infections 31

development and use of vaccines and immunotherapeutics against these viruses.

Recent advances in identification of neutralizing and enhancing viral epitopes are
expected to guide future design of non-enhancing, protective viral vaccines and
therapeutic mAbs. Comprehensive evaluation of humoral and cell-mediated immune
response to vaccines in preclinical and clinical studies, with the help of robust
assays that can detect ADE, is suggested to be crucial for the assessment of vaccine
safety and efficacy. Such evaluations would require a better understanding of the
immune correlates of protection. Lastly, investigation of long-term effects of vac-
cine implementation is advisable while considering approval for future vaccines.

Acknowledgments The author is grateful to Dr. A.C. Mishra, Director, IRSHA, and Dr.
V.A. Arankalle, Head, Department of Communicable Diseases, IRSHA, for their constant
support.

Conflict of Interest The authors declare that they have no competing interests.

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Inflammation During Virus Infection:
Swings and Roundabouts 3
Sankar Bhattacharyya

Abstract
Inflammation constitutes a concerted series of cellular and molecular responses
that follow disturbance of systemic homeostasis, by either toxins or infectious
organisms. Leukocytes modulate inflammation through production of secretory
mediators, like cytokines and chemokines, which work in an autocrine and/or
paracrine manner. These mediators can either promote or attenuate the inflam-
matory response and depending on differential temporal and spatial expression
play a crucial role in the outcome of infection. Even though the objective is clear-
ance of the pathogen with minimum damage to host, the pathogenesis of multi-
ple human pathogenic viruses has been suggested to emanate from a dysregulation
of the inflammatory response, sometimes with fatal consequences. This review
discusses the nature and the outcome of inflammatory response, which is trig-
gered in the human host subsequent to infection by single-sense plus-strand
RNA viruses. In view of such harmful effects of a dysregulated inflammatory
response, an exogenous regulation of these reactions by either interference or
supplementation of critical regulators has been suggested. Currently multiple
such factors are being tested for their beneficial and adverse effects. A successful
use of such an approach in diseases of viral etiology can potentially protect the
affected individual without directly affecting the virus life cycle. Further, such
approaches whenever applicable would be useful in mitigating death and/or
debility that is caused by the infection of those viruses which have proven par-
ticularly difficult to control by either prophylactic vaccines and/or therapeutic
strategies using specific antiviral drugs.

S. Bhattacharyya (*)
Vaccine and Infectious Disease Research Centre, Translational Health Science and
Technology Institute, Faridabad, Haryana, India
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2020 43

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_3
44 S. Bhattacharyya

Keywords
Inflammation · RNA virus · Cytokine therapy

3.1 Introduction

The mammalian immune system has evolved arsenal and strategies to make a dis-
tinction between microbes that are either beneficial or benign or bad, an integral part
of which is differential treatment of “self” and “non-self.” Whereas recognition of
“self” as “non-self” can cause autoimmunity, the converse results in microbial colo-
nization. In fact the human gut does harbor multiple variety of microbes as natural
part of the biological ecosystem (Scarpellini et al. 2015). The recognized non-self
are counteracted by adaptive and innate effectors of the immune system, using dedi-
cated cells and biochemicals, which attempt to restrict the growth and impede colo-
nization by the pathogen. The innate response is nonspecific, while the secondary
adaptive response is specific for the pathogen or closely related species. The cellular
component includes innate immune cells like the monocytes/macrophages, neutro-
phils, and natural killer (NK) cells and adaptive immune cells like B- and
T-lymphocytes, which coordinate for an effective response. Cytokines are a dedi-
cated group of biochemicals involved in this coordination and include interferons
(IFNs), interleukins (ILs), and chemokines that are responsible for synchronizing
the initiation, regulation, and termination of an immune response. A group (~100)
of small polypeptides (<20 kDa) produced predominantly although not exclusively
by immune cells like macrophages and lymphocytes, cytokines are secreted out
exerting their function by engaging respective cell-surface receptors and depending
on biological function are labeled as either pro-inflammatory (PIC) or anti-­
inflammatory (AIC) cytokines (Turner et al. 2014). On the one hand, several cyto-
kines are functionally redundant, and on the other hand, some cells can express
receptors for multiple cytokines.

3.2 The Positive-Sense Single-Stranded RNA Viruses

Viruses with positive-sense single-stranded RNA as genome can either be envel-

oped (Togaviridae, Flaviviridae, and Coronaviridae) or non-enveloped (Astroviridae,
Caliciviridae, and Picornaviridae), and several from either group cause severe
human pathology (Fields et al. 2013). Entry into human host can be by diverse
means including mucosal contact (gut in enteroviruses) or vectorial inoculation
(e.g., in dengue and JEV) or parenteral blood transfer (e.g., hepatitis C virus).
Immobilization by interaction with extracellular matrix components like glycos-
aminoglycan is followed by tropism determinant cognate receptor-mediated entry
(Chen et al. 1997; Olenina et al. 2005; Tan et al. 2013). In enveloped viruses, the
envelope fuses with the endosomal membrane, while non-enveloped viruses breach
the membrane of either the cell or the endosome using specific cofactors, ultimately
3 Inflammation During Virus Infection: Swings and Roundabouts 45

releasing viral genome into the host cytosol (Kumar et al. 2018; Plemper 2011). A
culmination of the following steps results in direct translation of the genomic RNA
to produce a polyprotein, which is cleaved by virus-derived and host-origin prote-
ases to yield the multiple structural and nonstructural proteins (Fields et al. 2013).
The structural features of the genomic RNA facilitating translation can be, e.g., a
5’cap and a poly-A tail (Alphavirus, Togaviridae; Coronavirus, Coronaviridae) or a
5’cap without a poly-A tail (Flavivirus, Flaviviridae) or an internal-ribosome entry
site (IRES) serving for ribosome recruitment without a poly-A tail (hepatitis C
virus) or an IRES with a poly-A tail (Picornaviridae, Astroviridae, Caliciviridae).
After multiple rounds of translation, ribosome loading stops and the genomic RNA
is replicated by virus-encoded RNA-dependent RNA polymerase (RdRp), in endo-
plasmic reticulum (ER) membrane-associated replication complexes (RCs) during
which a double-stranded RNA intermediate is produced followed by its asymmetric
transcription to produce multiple copies of plus-sense genomic RNA. The new
genomic RNAs are packaged into virion particles that exit the cell by either secre-
tory pathway or plasma-membrane budding (for enveloped viruses) or by cell lysis
(for non-enveloped viruses) (Bird and Kirkegaard 2015; Pornillos et al. 2002).

3.3 Infection, Intimation, and Initiation of Inflammation

Although viruses can replicate in multiple types of cells, the pathological outcome
manifests in only one or a few specific cell/tissue types. Independent of organismal
entry site, the likeliest primary encounter of a virus is with mononuclear-phagocytic
cells like monocytes, macrophages (Mϕ), and dendritic cells (DCs). Mϕ and DCs
are tissue-localized cells constituting the first line of cellular defense, which when
infected can undertake antiviral steps in addition to “informing” the other effectors
of the innate and adaptive immune system (Pohl et al. 2007; Ginhoux and Jung
2014). Activated DCs shift to lymph nodes, process viral antigen, and “present” or
display it for clonal expansion of cognate lymphocytes population. Mϕ, which can
be either tissue-resident or differentiated from afferent monocytes postinfection,
play a more regulatory role and are important determinants of the outcome of the
inflammatory response (Ginhoux and Jung 2014; Mercer and Greber 2013; Hou
et al. 2012; Schulz et al. 2012). Tissue-resident Mϕ , which are a distinct population
from monocyte-derived ones, include microglial cells in CNS, liver Kupffer cells,
skin Langerhans cells, etc. (Davies et al. 2013)
Monocytes/Mϕ (and many other cell types) express molecular detector proteins
called pattern recognition receptors (PRRs), specialized for interacting with signa-
ture motifs on microbe-derived molecules, termed as pathogen-associated molecu-
lar pattern (PAMP). Viral PAMPs include double-stranded (dsRNA) RNA
(replication-intermediate formed during replication) and 5′-ppp (uncapped genomic
RNA polymerized by de novo replication). Cellular PRRs specific for these include
toll-like receptors (TLRs) like TLR3 (dsRNA) and RIG-I-like receptors (RLRs) like
RIG-I, MDA5 (dsRNA, 5′-ppp end on RNA) (Jensen and Thomsen 2012). Nod-like
receptors or NLRs form another class of cytosolic PRRs that can detect virus
46 S. Bhattacharyya

infection, albeit in an indirect manner (Takeuchi and Akira 2010; Ichinohe et al.
2013). Physical engagement with PAMPs activates the respective PRRs, stimulating
alterations in conformation of these sensors that allow them to interact with adapter
molecules mediating the assembly of multi-protein complexes called inflammo-
some, in parallel to activating the expression of cytokine genes coding for type-1
interferons (IFNs) and NFκB target genes (Kawai et al. 2005; Pichlmair and Reis e
Sousa 2007; Chen and Ichinohe 2015; Seth et al. 2005). Secreted type-I IFNs attach
specific receptors, in a paracrine or autocrine manner, thereby activating the expres-
sion of many interferon-sensitive genes (ISGs) with diverse functions that confer
antiviral property to their activity (Schneider et al. 2014; Schoggins and Rice 2011).
ISGs include PRR-coding genes producing a feed-forward loop and aggravating
inflammation. In parallel, NFκB enhances expression of pro-inflammatory genes
like TNF-α, IL-1β, COX2, IL6, IL-12p40, or IL-12 besides components of NLRP3
(Tak and Firestein 2001; Bauernfeind et al. 2009). Upon assembly the NLRP3
inflammosome catalyzes caspase-1 activation, a protease which slices the precursor
form of pleiotropic pro-inflammatory cytokines like IL-1β and IL-18 generating
their active secreted forms (Garlanda et al. 2013; Biet et al. 2002). IL-1β potentiates
the antiviral response by multiple ways in addition to inducing expression of type-I
IFNs and ISGs in DCs (Ben-Sasson et al. 2011; Aarreberg et al. 2018). Chemokines
(chemotactic cytokines) flag/point to the site of infection by a concentration gradi-
ent, attracting leukocytes like neutrophils, monocytes, and lymphocytes, subse-
quently activating them to release more cytokines thereby amplifying the
inflammatory response (Sokol and Luster 2015; Ley 2014). Among these IL-12 and
IL-2 (produced predominantly by DCs) have crucial immunomodulatory functions.
IL-12 attracts CD4+ T-helper (Th) cells influencing their differentiation into IFN-γ
secreting Th1 cells in addition to augmenting the cytotoxic activity of CD8+ T cells
and NK cells (Athie-Morales et al. 2004; Henry et al. 2008). IL-2 on the other hand
increases NK-cell sensitivity to IL-12 by receptor upregulation (Wang et al. 2000).
IFN-γ which in contrast to type-I IFNs is produced exclusively by immune cells (T
and NK cells) has pleiotropic antiviral effect including the capacity to polarize
existing or newly recruited Mϕ to M1 phenotype (Hu and Ivashkiv 2009; Verreck
et al. 2004). Mϕ either resident or monocyte-derived can acquire either an M1 or an
M2 phenotype differing in ontology, phenotype, and secretome, with unidirectional
plasticity from M1 to M2 (Halstead et al. 2010; Guiducci et al. 2005; Smith et al.
2016). M1-Mϕ promotes a Th1 immune response which is necessary for resolution
of infection, while the M2-Mϕ endorses tissue repair following inflammation, sug-
gesting that a premature skew in abundance of M2-Mϕ at the expense of M1-Mϕ
would limit viral clearance leading to chronic infection and prolonged inflammatory
response (Klenerman and Hill 2005). An emerging concept in modulation of inflam-
mation involves the role of bacterial surface components like lipopolysaccharide on
concurrent viral infection (Smith et al. 2016; Wilks and Golovkina 2012). Alterations
in gut microbiome have been reported and potential influences this might have on
disease outcome have been suggested (Preveden et al. 2017; Banks et al. 2015).
Though it is difficult to ascertain the number of asymptomatic infections for any
given virus, the percentage of symptomatic infection vis-à-vis asymptomatic ones is
3 Inflammation During Virus Infection: Swings and Roundabouts 47

often a multivariate variable, being known for only a few. For example, only 1
among 4 individuals infected with DENV shows febrile symptoms. This suggests a
success for the antiviral immune mechanisms in the majority of individuals. Animal
studies using gene knockout models have given evidence of this efficacy for many
viruses (Suthar et al. 2010; Samuel and Diamond 2005; Lazear et al. 2011; Deonarain
et al. 2004; Burdeinick-Kerr et al. 2007). In case of humans, these information are
complicated by differential efficacy of these pathways, protecting or predisposing
individuals under the influence of genotype, environment, etc. (Paalani et al. 2011;
Mitchell and Aneshensel 2016; Liu and Taioli 2015) Besides, there are few studies
that indicate potential influence of medication or noninfectious ailments or societal
stress on the outcome of infection through an influence on the immune system
(Mehrbod et al. 2014; Gilbert et al. 2005; Htun et al. 2015; Jean et al. 2007).

3.3.1  iver Damage Due to Hepatitis C Virus and Dengue Virus

L
Infection

HCV and DENV infection can cause liver damage through a chronic and acute
infection regime, respectively (Samanta and Sharma 2015; Axley et al. 2018). Liver
as an organ is characterized by a high capacity to regenerate; however, chronic
injury/scarring can lead to fibrosis, steatosis, or even hepatocellular carcinoma
resulting in liver failure (Forbes and Newsome 2016). Hepatocytes constitute two-­
thirds of all liver cells and are associated with all major liver functions besides play-
ing a crucial role in innate immune signaling (Kmiec 2001; Zhou et al. 2016).
Hepatocytes are permissible to both HCV and DENV, the latter being reported to
additionally infect Kupffer cells (Chang et al. 2003; Zehender et al. 1997; Boisvert
et al. 2001; Caussin-Schwemling et al. 2001; Goutagny et al. 2003; Marianneau
et al. 1999; de Macedo et al. 2006; Huerre et al. 2001). In acute infection, the major
damage is through apoptosis following direct infection of these cells, whereas
establishment of a chronic infection usually causes a sustained inflammation lead-
ing to infiltration of polymorphonuclear cells and lymphocytes (Huerre et al. 2001;
Lim et al. 2014; Masalova et al. 2017; Deng et al. 2008; Bala et al. 2012; Sung et al.
2012). Irrespective of the virus, these infections augment PIC levels in the liver with
drastic consequences. For example, hepatocyte apoptosis caused by either direct
infection or effect of PICs like TNF-α generates apoptotic bodies which when
engulfed by Kupffer cells induce the latter to release more PIC providing a positive
loop toward inflammation (Canbay et al. 2003a; Burdette et al. 2012; Negash et al.
2013; Shimizu et al. 2005). Cytokines like TGFβ and PDGF thus released can “acti-
vate” hepatic stellate cells initiating a metabolic transformation in them to secrete
more extracellular matrix that deposits as fibrotic tissue in addition to converting
them into smooth muscle fibers (Canbay et al. 2003b; Hernandez-Gea and Friedman
2011). In addition to virus infection-induced changes, bacterial LPS can also poten-
tially “activate” hepatic stellate cells (Brun et al. 2005). HCV infection skews mac-
rophage population to M2 phenotype restraining virus clearance while promoting
hepatic stellate cell activation mediated by TGFβ (Saha et al. 2016). Additionally, in
48 S. Bhattacharyya

case of infection by both of these viruses, immune suppression mediated by AIC

like IL10 is implicated for virus persistence and augmented pathology (MacDonald
et al. 2002; Sugimoto et al. 2003). In fact higher levels of cytokines like IL10 and
IL17 have shown positive correlation with liver damage (Fernando et al. 2016).
Liver steatosis, a clinical feature common among HCV patients, is the result of
intracellular ROS in hepatocytes (Okuda et al. 2002; Perlemuter et al. 2002).
Irrespective of the stimulus, a continuous cycle of injury and repair involving hepa-
tocytes strongly prognoses the growth of hepatocellular carcinoma, DNA damage
by augmented levels of ROS and RNS level playing a critical role (Bishayee 2014;
Capone et al. 2010).

3.3.2  NS Damage Due to JEV, WNV, Zika Virus Infection,

C
and Sometimes DENV Too

The central nervous system (CNS) is physiologically isolated from the rest of the
body by a specialized selectively permeable barricade called as the blood–brain bar-
rier (BBB), which allows passage to selected metabolites, respiratory gases, and an
extremely limited repertoire of circulatory tissue cells. This isolation is necessary
for protection of low regeneration capacity neuronal cells from systemic inflamma-
tion, which can also upset the structural and functional plasticity of neurons that is
dependent on cytokine signaling (Arnett et al. 2001; Gougeon et al. 2013; Mason
et al. 2001; Fischer et al. 2011; Brissoni et al. 2006). The CNS can have either neu-
ronal or non-neuronal glial cells; the latter provide vital functional support and
include microglia (macrophage-like immune cells), oligodendrocytes (which pro-
vide insulation for neurons), and astrocytes (responsible for repair of damaged neu-
ronal tissue). Microglial cells have immunomodulatory function in suppressing a
pathogenic inflammation (Seitz et al. 2018). Multiple viruses in the +ve-ssRNA
genome group, including Coronavirus, Picornavirus, Flaviviridae, and Togaviridae,
cause opportunistic infection of CNS (Bergmann et al. 2006; Koyuncu et al. 2013;
Fletcher and McKeating 2012).
In the absence of a direct admission route, these viruses undergo limited repli-
cation in peripheral tissue, before entering through either peripheral nerves or
BBB microvasculature or CNS infiltrating leukocytes (functioning as the prover-
bial “Trojan horse”) (Koyuncu et al. 2013; Jeha et al. 2003). A feature common
here is a breach of the vascular endothelial barrier at varying locations, e.g., BBB
for JEV/WNV, blood retinal barrier for ZIKV, and endothelial barriers in lungs/
peritoneum for DENV. Breach in BBB is more common for some viruses (e.g.,
WNV, JEV, ZIKAV, poliovirus) correlating with fatality. Interestingly, WNV and
JEV have been suggested to cause BBB disruption from inside the CNS (Li et al.
2015; Verma et al. 2009). Still other reports suggest infected endothelial cells to
secrete PICs that disrupt the BBB (Chen et al. 2014; Chang et al. 2017; Roach and
Alcendor 2017). The tissue damage is caused from a combination of either direct
neuronal infection which activates intrinsic apoptosis or a hyperactive inflamma-
tory response mediated by PICs or CD8+ cytotoxic T cells (CTLs) (Wang et al.
3 Inflammation During Virus Infection: Swings and Roundabouts 49

2003; Samuel et al. 2007). Infected neurons secrete chemokines that attract leuko-
cytes like monocytes and lymphocytes (Klein et al. 2005; Shrestha and Diamond
2004; Glass et al. 2005; Kelley et al. 2003; Lim et al. 2011; Bardina et al. 2015;
Durrant et al. 2015; Shrestha et al. 2008). The relation between a “good” and
“bad” immune response is, however, very tricky when it comes to the
CNS. Migration of CTLs expressing receptors for chemokines like CCL2, CCL3,
CCL4, CCL5, CXCL9–11, as well as its timing with respect to establishment of
infection, seems to play a crucial role in virus eradication and survival (Wang
et al. 2003; Shrestha and Diamond 2004; Diamond et al. 2003; Sussman et al.
1989; Getts et al. 2010; Nansen et al. 2000; Chen et al. 2004; Liu et al. 2001; Zink
et al. 2001; Winter et al. 2004). The CTLs exert their antiviral role by inducing
cell death through either a perforin-dependent or Fas-FasL-­mediated mechanism
(Rossi et al. 1998; Shrestha and Diamond 2007). In addition to CTLs, other PICs
might also induce direct cell death in neurons (Dhanwani et al. 2012; Olmo et al.
2017; Baer et al. 2016; Kumar et al. 2010).

3.3.3 Dengue Infection-Associated Vascular Leakage

Dengue virus causes a febrile illness with can turn fatal after a subsidence of the
fever. The severity emanates from leakage of fluid from the blood vessels by a
breach of the vascular endothelium. Circulating in four serotypes, severe disease is
mostly associated with secondary infection by a serotype different from the one
causing primary infection. Neutralizing antibodies generated during primary infec-
tion incompletely neutralize the secondary infection virus and instead promote their
uptake by monocytes, by a phenomenon called antibody-dependent enhancement or
ADE (Katzelnick et al. 2017; Dejnirattisai et al. 2016). Notwithstanding a primary
or secondary infection, the pathological symptoms are considered to be the result of
an unbridled host immune response (Basu and Chaturvedi 2008; Rothman 2011).
DENV infects a variety of cells including monocytes, dendritic cells (skin
Langerhans cells), macrophages (Kupffer cells), and vascular endothelial cells,
expectedly leading to PIC secretion (Wu et al. 2000; Jessie et al. 2004; Tolfvenstam
et al. 2011). Different studies have reported a positive association of DHF/DSS
development with extraordinarily augmented levels of different PICs that include
macrophage migration inhibitory factor (MIF), IFN-α, TNF-α (Green et al. 1999;
Kurane et al. 1993; Huang et al. 2000; Chen et al. 2006). Although multiple reports
have suggested correlation between specific PIC level and plasma leakage, the
mechanism is still elusive and limited to association studies (Priyadarshini et al.
2010; Her et al. 2017; Sehrawat et al. 2018; Malavige et al. 2012). Interestingly
however, multiple similar association studies have suggested a positive association
between levels of IL10 (an AIC) and severe/critical symptoms related to dengue
infection (Malavige et al. 2013; Tsai et al. 2013; Flores-Mendoza et al. 2017). IL10,
produced by multiple immune cells, suppresses immune response through upregu-
lation of SOCS (suppressor of cytokine signaling) function and downregulation of
IFN activity, the result being decreased T-cell cytotoxicity (Halstead et al. 2010;
50 S. Bhattacharyya

Katzelnick et al. 2017; Tsai et al. 2013; Azeredo et al. 2001; Brasier et al. 2012).
The augmentation of IL10 level has been suggested to emanate from monocytes
infected by the ADE route with additional influence from high viremia (Tsai et al.
2014). IL10 is a dominant regulator of the immune system that can prolong patho-
gen clearance through a subversion of the immune response (Couper et al. 2008).

3.3.4 Lung Infection and Pathology by Coronaviruses

Coronavirus infections are usually benign causing self-limiting mild flu-like symp-
toms. However, recent outbreaks involving, e.g., severe acute respiratory syndrome
coronavirus (SARS-CoV), which jumped species barrier through acquisition of
minor genome mutations, have projected them as potentially severe human patho-
gens (Guan et al. 2004). Spread through aerosols, SARS-CoV primarily infect lung
cells triggering an often fatal inflammatory response clinically called acute respira-
tory distress syndrome (ARDS) that starts with severe hypoxia, pulmonary edema
progressing to systemic inflammation, and failure of multiple organs, culminating
in high rate of mortality (Peiris et al. 2003; Lew et al. 2003; Tsushima et al. 2009;
Farcas et al. 2005). Although evidence suggests that SARS-CoV can infect multiple
cell types, lung type-II pneumocytes and ciliated epithelial cells constitute primary
sites of virus replication, consequent to which these cells undergo apoptotic and/or
necrotic death attracting innate immune cells and activating them to secrete PICs
(Sims et al. 2005; Chow et al. 2004; Nicholls et al. 2003). The nature of inflamma-
tion following SARS-CoV infection is characterized by a prompt production of
PICs through immediate NFκB activation and a delayed expression of type-I IFN
genes (Shi et al. 2014; Kong et al. 2009; Wong et al. 2004). Severity of symptoms
correlates positively with IL-6 levels while exhibiting negative correlation with that
of IL-8 and TGFβ (Zhang et al. 2004). As observed with many other viral pathogen-
esis models, macrophage polarization culminating in preferential enrichment of
M2-macrophages has been suggested to be responsible for SARS-CoV pathogene-
sis (Page et al. 2012). SARS-CoV infection is also associated with hemophagocyto-
sis or engulfment of different types of blood cells by histiocytes (a class within
macrophages), which is a clinical marker of immune system hyper-activation
(Usmani et al. 2013).

3.4 Therapeutic Approaches Using Cytokine

Traditionally prophylactic or therapeutic strategies for combating viral pathogene-

sis are designed using vaccines or directly acting antivirals (DAA), respectively. But
for many viruses there is no clinically approved product to serve in either approach.
Since the etiology of critical pathogenic symptoms is often associated with an
unbridled host inflammatory response, there have been suggestions and attempts to
control the harmful effects through modulation of key inflammatory signaling
(D’Elia et al. 2013). However, a holistic approach to complete “cure” should
3 Inflammation During Virus Infection: Swings and Roundabouts 51

probably involve investigations to provide support to both approaches simultane-

ously. Only ribavirin or the same combined with pegylated IFN-α was the therapeu-
tic strategy for controlling HCV infection, before the advent of high efficacy DAAs.
Similarly IFN-λ and glucocorticoids, both of which can consolidate the BBB, have
been suggested as therapeutics for combating viral diseases that disrupt this barrier
(Rhen and Cidlowski 2005; Daniels et al. 2014; Lazear et al. 2015; Wang et al.
2004; Blecharz et al. 2010; Fabene et al. 2008). Likewise, administration of PICs
like CCL7 and IL17A has shown efficacy in increasing survival of mice experimen-
tally infected with WNV (Bardina et al. 2015; Acharya et al. 2016). In dengue
patients, however, meddling with either promoter or inhibitor of inflammation has
been suggested as possible approaches (Tsai et al. 2013; Goh et al. 2014; Callaway
et al. 2015; Ji et al. 2005; Dinarello 2011). Small molecules that can influence the
function of the NLRP3 inflammosome have also been projected as potential thera-
pies for CHIKV and can be tested against dengue as well (Chen et al. 2017; Coll
et al. 2015; Hottz et al. 2013). Alternative approaches using pharmaceuticals that
indirectly mitigate the pathological effect without interfering with inflammation
have also been discussed (Olmo et al. 2017; Grip and Janciauskiene 2009; Reynolds
and Miller 1989; Thomas and Grossberg 2009; Giguere and Tremblay 2004; Raemer
et al. 2009).

3.5 Concluding Remarks

An ability to suppress innate immunity pathways is common among viruses that

cause severe human diseases. Nonetheless modulating inflammation needs extreme
caution, in order to reduce potential cytotoxicity of the administered therapeutic.
Therefore, there is a need to go beyond association studies to generate a clearer
picture of the exact role that inflammation plays in viral pathology, which can then
assist in developing therapeutic strategies that tinker with inflammation.

Conflict of Interest The author declares that they have no competing interests.

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Interferons and Their Role in Viral
Infection 4
Suji George, Gururaj Rao Deshpande,
and Gajanan N. Sapkal

Abstract
Interferons (IFNs) are a large family of naturally occurring cytokines, discovered
in 1957. They are secreted by host immune cells in response to virus infections,
tumors, and other biological inducers as diverse countermeasures against stimu-
lation. They elicit and regulate inter- and intracellular networks of immune sys-
tem by producing interferon-induced proteins. These proteins play a vital role in
inhibiting any vulnerable step in viral replication cycle starting from viral entry/
uncoating to maturation and release of the virus. Presently many interferons are
approved as therapeutic agents, and tremendous progress has been made in
understanding their role and usage as antiviral agents. This chapter explores the
essential link of IFNs with the innate and adaptive immune system during viral
infections. Further it discusses in detail the types of IFNs, genes induced by IFNs
in the host, IFN-induced proteins and their antiviral roles, and the strategies used
by viruses to counter the effects of antiviral action by IFNs. Finally, the chapter
concludes by summarizing the therapeutic application of IFNs to fight viral
diseases.

Keywords
Interferons · Antiviral activity · Immune system

4.1 Introduction

Viruses cause infectious diseases by invading living cells to multiply and produce
progeny viruses. The host immune system plays a key role in defending against the
infection and this defense barrier consists of innate immune cells such as dendritic

S. George · G. R. Deshpande · G. N. Sapkal (*)

National Institute of Virology, Pune, Maharashtra, India

© Springer Nature Singapore Pte Ltd. 2020 61

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_4
62 S. George et al.

cells, macrophages, and natural killer cells. These cells are capable of mounting an
immune response by recognizing a wide range of viral products. Activation of these
cells can result in rapid viral sensing and release of key antiviral cytokines, which
can inhibit viral replication and can further activate other components of innate and
adaptive immunity. Interferons (IFNs) are a group of highly specialized cytokines
capable of mediating antiviral, antigrowth, and immune modulation responses. It
was discovered by Isaacs and Lindenmann in 1957 as a molecule secreted by virus-­
infected cells and was able to prevent further infection of cells that were exposed to
it (Isaacs and Lindenmann 1957). IFN activity includes the induction of the tran-
scription of IFN-stimulated gene (ISG) that actively participates in the inhibition of
crucial steps in viral replication.

4.2 Classification of Interferons

IFNs are classified into three types (I, II, and III) based on the structural homology,
chromosomal location, and cell surface receptors to which they bind to induce intra-
cellular signaling pathways (de Weerd and Nguyen 2012). There are more than 10
IFN I subtypes of IFN-α, named IFN-α1, IFN-α2, etc., IFN-β, IFN-κ, IFN-ω, IFN-ε,
and limitin. Type I IFNs signal by binding to heterodimeric receptor composed of
low- (IFNAR1) and high-affinity (IFNAR2). While IFN II signals through trans-
membrane tetrameric IFN-γ receptor (IFNGR), comprising two units of IFNGR1
and IFNGR2. Type III IFNs (IFN-λ1, -λ1, -λ2, -λ3, and -λ4) signal through IFN-λ
receptor (IFNLR), a heterodimer of IFNLR1 and IL10RB (Fensterl et al. 2015).
The functions of three IFNs in some instances overlap, for example, their ability
to inhibit virus replication; at the same time many effects of signaling by type I, II,
III IFNs are distinct. These differences may be attributed to two properties. First,
IFN and its cognate receptors show differential expression as there are cell- and
tissue-specific differences. Although most of the virally infected cells can produce
IFN-α/β, IFN-γ is synthesized by certain cells of the immune system such as natural
killer T cells, B cells, and antigen-presenting cells (Samuel 2001). Type I, type II,
and IL10RB receptors are expressed in almost all cell types, except that IFNGR2 is
not found on the surface of Th1 cells and low expression of IFNAR2 is seen in the
human brain (de Weerd and Nguyen 2012). Unlike type I and II IFN receptors, the
distribution of type III IFN receptor (IFNLR1) is more restricted. IFNAR is abun-
dantly expressed only in cells of epithelial origin and hepatocytes and responds to
type III IFNs (IFN-λ) (Mordstein et al. 2010; Sheahan et al. 2014; Sommereyns
et al. 2008) Second, downstream targets of IFNs are different and this leads to
induction of different IFN-stimulated genes (Der et al. 1998), and there is only par-
tial overlap between IFNAR, IFNLR, and IFNGR. The set of genes induced by
IFNAR and IFNLR is similar and they do so by activating intracellular signaling
pathway through transcription factor complex ISG factor 3 (ISGF3) (Doyle et al.
2006). In contrast, activation of the transcription factor gamma-activated factor
(GAF) by IFNGR induces different set of genes (Decker et al. 1997; Fensterl et al.
2015) (Fig. 4.1).
4 Interferons and Their Role in Viral Infection 63

Fig. 4.1 Summary of Type I, Type II, and Type III interferon signaling: Different cell surface
receptors are involved in signaling of Type I, Type II, and Type III interferons. Binding of IFNs to
cognate receptor triggers phosphorylation of STAT through kinases (JAK-1, JAK-2, and TYK-2).
Further, phosphorylated STAT undergoes oligomerization and interacts with IRF-9 in case of Type
I and III IFNs to form a complex of ISGF3. Similarly, in the case of Type II IFNs STAT undergoes
dimerization and associates with GAF to form a complex which then triggers the transcriptional
activity

Type I and type III IFNs through IFNAR1/2 or IL-10RB/IFNLR1complexes

mediate phosphorylation of pre-associated Janus kinase 1 (JAK1) and tyrosine
kinase2 (TYK2), which then phosphorylate the receptors at specific intracellular
tyrosine residues. The phosphorylated JAK1 and TYK2 then recruit and phosphory-
late signal transducers and activators of transcription 1 and 2 (STAT1and 2) which
64 S. George et al.

further associate to form a heterodimer. The STAT heterodimer then interacts with
IFN-regulatory factor 9 (IRF9) to form the IFN-stimulated gene factor 3 (ISGF3).
Type II IFN dimers binding to the IFNGR1/2complex activate the cascade by phos-
phorylation of pre-associated JAK1 and JAK2 tyrosine kinases and transphosphory-
lation of the receptor chains leading to the recruitment and phosphorylation of
STAT1. Phosphorylated STAT1 homodimers form IFN-γ activation factor (GAF)
and translocate to the nucleus and bind to the gamma-activated sequence (GAS)
promoter elements, respectively, resulting in expression of antiviral genes. Similarly
the ISGF3 translocates to the nucleus to induce genes regulated by IFN-stimulated
response elements (ISRE), resulting in expression of antiviral genes.

4.3 Synthesis of Interferons

Although IFNs are not expressed in resting cells, in virus-infected cells there is a
need to accelerate the synthesis of IFNs as they are effective in limiting the virus
replication and spread. As all the viruses replicate inside the cells, detecting viral
products can be an effective strategy for inducing an immune response through
production of IFNs. Viral nucleic acid (RNA or DNA) can trigger the synthesis of
IFNs through the pattern recognition receptors (PRRs) by engaging pathogen-­
associated molecular patterns (PAMPs) (Thompson et al. 2011). Nucleic acid PRRs
can be endosomal membrane or cytosolic proteins. Recognition and binding of
PAMPs activates PRRs by conformational change, often by dimerization or oligo-
merization, and initiates intracellular signaling pathways resulting in the formation
of IFNs. In uninfected normal cells, the PRRs remain in an inactive conformation
unable to evoke an immune response. Toll-like receptors (TLRs) are receptors local-
ized within endosomes and plasma membrane and are efficient sensors of PAMPs
such as single-stranded RNA (ssRNA), lipopolysaccharides, lipoproteins, and fla-
gella. TLRs (TLR3, TLR7, TLR8, and TLR9) are nucleic acid sensing receptor
(Gay et al. 2014). TLR3 is a dsRNA sensor widely expressed in innate immune cells
and involved in the recognition of RNA viruses such as influenza A virus, picorna-
viruses, and DNA viruses such as herpes simplex virus-1 (Alexopoulou et al. 2001;
Le Goffic et al. 2006; Hardarson et al. 2007; Zhang et al. 2007). TLR-7 and TLR-8
are activated by ssRNA from many viruses such as vesicular stomatitis virus and
influenza A virus (Lee et al. 2007). DNA viruses are recognized by endosomal
TLR9 and several other cytoplasmic DNA sensors such as IFN-induced 16-kDA
protein (IFI16), cyclic GMP-AMP synthase (cGAS), DEAD-Box RNA Helicase
(DDX41), and DNA-dependent activator of IRFs (DAI) (Pandey et al. 2015; Wu and
Chen 2014). TLR9 can recognize unmethylated deoxycytidylate-phosphate-­
deoxyguanylate (CpG) motif in viral DNA in plasmacytoid dendritic cells
(Krug et al. 2004). RIG-I-like receptor family (RLRs) is another closely related
family consisting of retinoic acid-inducible gene (RIG-I), melanoma differentia-
tion-associated gene 5(MDA-5), and laboratory of genetics and physiology-2
(LGP2). RLRs are cytosolic RNA helicases involved in recognition of RNA viruses
(Yoneyama et al. 2015). RLRs play a crucial role in antiviral defense pathway in cell
types such as fibroblasts, epithelial cells, and dendrite cells (Thompson et al. 2011).
4 Interferons and Their Role in Viral Infection 65

RIG-I uses 5′ end of the ssRNA as a strategy to distinguish between viral and host
RNA. In addition, RIG-I can also recognize short dsRNA product produced during
viral replication (Goubau et al. 2014; Weber et al. 2013; Yoneyama et al. 2015).
MDA-5 differentiates viral and host RNA based on the length of the sequences as
long as RNA is not common in host cells (Kato et al. 2008; Züst et al. 2011).
RIG-I recognizes Vesicular Stomatitis virus (VSV), rabies virus, Newcastle dis-
ease virus, measles virus, influenza A and B, hepatitis C virus, Japanese encephalitis
virus, and Ebola virus (Fensterl et al. 2015; Thompson et al. 2011). LGP-2 can act
via two different pathways during cytoplasmic recognition. LGP2 binds to dsRNA
in tandem with MDA5 and thereby activates MDA5 (Bruns et al. 2014; Childs et al.
2013). Second, it can inhibit RIG-I activation by competing for dsRNA and by
direct interaction with RIG I (Saito et al. 2007).
Detection of PAMPs results in the activation of downstream signaling pathways
leading to the induction of IFNs. IFN-α subtypes use transcription factor IRF-7 and
IFN-λs utilize IRF-3, IRF-7, and NF-κB (Génin et al. 2009; Iversen and Paludan
2010; Onoguchi et al. 2007; Osterlund et al. 2007). IFN-β is induced by the syner-
gistic promoter binding of transcription factors IRF-3, NF-κB, and API (Ford and
Thanos 2010; Panne et al. 2007). Once activated by the PAMP, PRRs interact with
respective adapter proteins; TLR3 uses the TRIF and TLR7, TLR8, and TLR9 use
My88, while RLRs use mitochondrial and peroxisome-bound MAVS (Fensterl et al.
2015). Cytoplasmic DNA sensors use STING (stimulator of interferon genes) for
their signaling. Further the TRIF and MAVS interact with signaling proteins of the
TRAF family of ubiquitin ligases, as well as IKKα/β and TBK1 kinases which
phosphorylate and activate downstream transcription factors (Fensterl et al. 2015).
Phosphorylation of IRF3 at specific serine and threonine residues results in opening
of its conformation and subsequent dimerization and translocation to nucleus where
it binds to IFN-stimulated response elements (ISREs) in the promoter region of
IFN-β and IFN-γ (Pandey et al. 2015). NF-κB another transcription factor upon
phosphorylation is released from its inhibitor IκB to induce IFN genes (Fig 4.2).

4.4 Interferon-Mediated Induction of Gene Expression

Evidence suggests that multiple signaling pathways are activated and cooperation as
well as coordinated response by these pathways is required for the generation of
response to IFNs. Some of the major pathways involved in IFN signaling are dealt
in sufficient detail below. In addition to the signaling pathway described here, there
are many other pathways whose involvement in IFN signaling is documented; in
fact the list of newly identified IFN signaling pathway is growing and it seems prob-
able that more than one signaling pathway is involved in IFN-mediated response.

4.4.1 Jak-Stat Pathway in Interferon Signaling

IFNs are synthesized and secreted by virus-infected cells and it can elicit biological
responses by binding to specific receptors on the surface of the target cells. Binding
66 S. George et al.

Fig. 4.2 Signaling pathways for Type I and III interferons. Receptor proteins (toll-like receptors
(TLRs), RIG-I-like receptors (RLRs), and cyclic GMP-AMP synthase (cGAS) in endosomes or
cytoplasm recognize viral RNA or DNA and trigger signaling via adapter proteins such as TRIF,
My88, MAVS, and STING, which further activate the transcription factors of the IRF family and
NF-κB resulting in the transcription of Type I and Type III interferons

of IFNs to the respective receptors results in change in conformation of the receptor

intracellular domain and activates a cascade of intracellular signaling events that
culminates in the induction of target genes. The effect of IFNs is mediated by the
Jak/STAT signaling cascade. The Jak/STAT pathway has three components: (1) cell
receptor, (2) JAK proteins, and (3) STAT proteins. There are four members in the
JAK family [JAK 1, 2, 3 and tyrosine kinase 2 (TYK2)] and seven STATs (1–4, 5a,
5b, and 6) (Aaronson 2002). The subunit for receptor for type I IFNs and type II
IFNs interacts with member of the Janus-activated kinase (JAK) family. The
IFNAR1 subunit is constitutively associated with tyrosine kinase 2 (TYK2), whereas
IFNAR2 is associated with JAK1 unit of type I IFNs. While in type II IFN receptor
the IFNGR1 subunit associates with JAK1, whereas IFNGR2 is constitutively asso-
ciated with JAK2 (Raftery and Stevenson 2017). The first step in type I and type II
associated signaling is the activation of these receptor-associated JAKs. This hap-
pens in response to ligand binding and dimerization of the receptor subunits, subse-
quent autophosphorylation, and activation of the associated JAKs, TYK2, and JAK1
which in turn regulate the phosphorylation and activation of STATs (Raftery and
Stevenson 2017). The type of STATs activated by IFNs depends on the cell type,
while Type I activate STAT1, STAT2 and STAT3 in most cell types, in certain other
cell types acts via activation of STAT4, STAT5, STAT6 (Wen and Darnell 1997;
Wen et al. 1995; Platanias 2005). IFN-α activates STAT4 and STAT6; however, such
activation is seen only in certain cell types such as endothelial cells or cells of lym-
phoid origin (Platanias 2005). After activation by phosphorylation STATs can asso-
ciate to form homodimers or heterodimers. Then cytoplasmic IRF9 associates with
the STAT dimer to form ISG factor 3 complex, which subsequently translocates to
4 Interferons and Their Role in Viral Infection 67

the nucleus and initiates transcription by binding specific sites in the promoters
(IFN-stimulated response elements) of IFN-stimulated genes (ISGs) (Ivashkiv and
Donlin 2014; Platanias 2005). In addition, IFN-induced complex can also bind to
another site known as IFN-γ-activated site (GAS) element (Platanias 2005). IFNs
induce the expression of hundreds of genes, of these some have only ISREs or only
GAS elements in their promoter, whereas others have both elements. Thus, for opti-
mal transcriptional activation a combination of different STATS complex is required
to produce functionally distinct genes (Platanias 2005). STAT1 is activated by JAK1
and JAK2 in response to IFN-γ engagement of type II IFN receptors. Phosphorylation
of STAT1 on the tyrosine residue at position 701 (Tyr701) results in the formation
of STAT1-STAT1 homodimers which translocate to the nucleus to bring about tran-
scription by binding to GAS elements (Aaronson 2002; Boehm et al. 1997; Stark
et al. 1998). Type II IFNs are different from type I as they do not form ISGF3 com-
plexes and thereby cannot induce the formation of genes that have only ISREs in
their promoter.

4.4.2 Crk-Signaling Pathway in Interferon Signaling

In addition to the Jak STAT pathway, IFNs activates many other signaling pathways.
Such a pathway includes the Crk family of adaptor proteins which includes three
members—CrkL, CrkI, and CrkII, all of which are cellular homologues of the v-Crk
gene (Platanias and Fish 1999; Sattler and Salgia 1998). CrkL is present in the latent
form in cytoplasm and is known to constitutively associate with the guanine nucleo-
tide exchange factor (GEF) C3G. Binding of the IFN to the respective receptor
results in the association of CrkL-C3G with TYK2 and is activated by phosphoryla-
tion of the tyrosine. The active Crkl now forms a complex with STAT-5 to form
Crkl-STAT5 complex, which subsequently translocates to the nucleus and binds to
GAS elements (TTCTAGGAA) located at the promoters of ISGs. Activation of Crkl
can also result in the GEF activity of the C3G, thereby resulting in the activation of
GTPase activity of RAPI, a member of the Ras family of small GTPases which
mediate induction of the growth suppressive effects by IFNs (Platanias 2005;
Schmitt and Stork 2001). Both type I and type II IFNs activate RAPI in a Crkl-­
dependent manner (Lekmine et al. 2002). RAPI has also been shown to promote
cellular proliferation; thus, the effects on cell growth depend on the stimuli and cell
type that are involved (Hattori 2003).

4.4.3 PI-3-Kinase Pathway in Interferon Signaling

Phosphatidyl-inositol 3 kinases (PI3K) are enzymes known to regulate an array of

protein kinase signaling cascades involved in cellular processes like cell survival,
metabolism, proliferation, and inflammation/immunity. The class Ia PI3Ks are
dimeric enzymes composed of p110 catalytic subunit attached to a smaller, noncata-
lytic, regulatory subunit (p85). The p85 subunit contains the SRC homology 2
68 S. George et al.

(SH2) and SH3 domains which interact with other signaling proteins, while the
p110 regulates the phosphorylation of the D3 position of the phosphatidylinositol
lipids of the inositol ring. Several type I IFNs (IFNs-α, IFNs-β, IFNs-ω) have been
shown to induce tyrosine phosphorylation of IRS1 and the p85 subunit of PI3K
associates with IRS1 through its SH2 domains in an IFN-dependent manner result-
ing in activation of catalytic subunit of PI3K (p110) (Uddin et al. 1995). Similar to
IRS1, the IRS2 belonging to the same family was subsequently shown to undergo
phosphorylation in type I IFN-dependent manner (Burfoot et al. 1997; Platanias
et al. 1996). Thus, type I IFN can activate the PI3K signaling pathway in an IRS-­
dependent and STAT independent manner (Ivashkiv and Donlin 2014).
Similar to type I IFNs, type II IFNs activate PI3K pathway but in an IRS indepen-
dent manner. The protein that seems to play a role in type II IFN signaling is CBL
which has binding sites for the SH3 domain of p85 and is shown to be phosphory-
lated in an IFN-γ-dependent manner (Alsayed et al. 2000). As mentioned above
JAK1 and JAK2 are activated in response to IFN-γ engagement of type II IFN
receptors. The activated JAK through an intermediate can also activate the p110 of
PI3K, which in turn activates protein kinase C-δ (PKC-δ). Finally, the PKC-δ regu-
lates the phosphorylation of STAT1 on the Ser727 (Nguyen et al. 2001). Although
the modification of STAT1 on the Ser727 is not required for its translocation to
nucleus, it is essential for transcriptional activation. Many downstream effectors of
PI3K signaling pathway have been identified such as 3-phosphatidylinositol-­
dependent protein kinase 1 (PDK1), serine/threonine kinases (AKT), various iso-
forms of protein kinase C (PKC), and members of the TEC family of tyrosine
kinases (Platanias 2005).

4.4.4 MAPKs in Interferon Signaling

Mitogen-activated protein (MAP) kinases are serine-threonine kinases that play

crucial roles in many signal transduction pathways including IFN signaling in
mammalian cells (Uddin et al. 1995). There are three types of MAPK in cells: (1)
p38 Map kinases, (2) the extracellular signal-regulated kinase family, and (3) the
c-Jun NH2-terminal kinase phosphatidylinositol 3′ kinase family. The p38-kinase
pathway has been shown to be involved in type I, type II, and type III IFN recep-
tors and acts as an auxiliary cascade to the Jak-Stat pathways and is required for
IFN-­inducible expression. There are several isoforms of p38 (p38α, p38β, p38γ,
and p38δ) encoded by distinct genes, but show structural homology (Fensterl
et al. 2015).
Activation of Janus-activated kinase through type I IFN receptor regulates the
phosphorylation of VAV proto-oncogene and/or other guanine nucleotide exchange
factors (GEFs). This subsequently activates the RAC1 and the small G-protein
(SGPs). MAPKKK a MAPK kinase downstream to RAC1 and SGPs is activated,
which then regulates MAPK kinases MAPKK3 and MAPKK6, which phosphory-
late p38 and activate it. Several downstream effector kinases of p38 including
MAPK-activated protein kinase 2 (MAPKAPK2) (MAPKAPK3), mitogen and
4 Interferons and Their Role in Viral Infection 69

stress activated kinase 1 (MSK1), and MAPK interacting protein kinase 1 (MNK1)
then mediate gene transcription of ISG protein products. p38 MAPK pathway is
also activated by the type II IFN and type III receptor and plays important roles in
the generation of type II IFN-biological responses in several different cell types
(Fensterl et al. 2015).

4.5 Antiviral Action of Interferons

Binding of IFNs to its cognate receptors activates signaling cascades which culmi-
nates into the transcriptional induction of a number of antiviral genes called
interferon-­stimulated genes (ISGs). These genes encode direct antiviral molecules
with potential to regulate IFNs signaling positively or negatively. Hundreds of ISGs
have been identified by genome-wide transcriptional profiling and most of the ISGs
mainly target conserved aspects of viral infection. Specific mechanisms of the ISGs
are summarized below (Schneider et al. 2014; Schoggins and Rice 2011).

4.5.1 Classical ISGs

The best characterized IFN-induced ISGs are the “classical ISGs,” interferon-­induced,
double-stranded RNA-activated protein kinase encoded by Eif2ak2 (PKR), MX1 (the
myxovirus (influenza virus) resistance 1), and 2′-5′ oligoadenylate synthetase/RNase L
system (OAS1, OAS2, OAS3, encoded by Oas1, Oas2, Oas3). PKR is a serine/threo-
nine protein kinase and has been shown to be an inhibitor of cellular and viral mRNA
translation by phosphorylation of the protein synthesis initiation factor eIF2. OAS
enzyme system catalyzes the formation of 2′-5′ oligoadenylates, which then activate
RNase L to degrade viral genomes. The MX1 protein is a dynamin-like large guanosine
triphosphatase (GTPase) and it acts by trapping the viral components such as the nucleo-
capsids and prevents them from reaching their destination by limiting the viral growth.

4.5.2 Cholesterol-25-Hydroxylase (CH25H)

CH25H is an IFN-induced enzyme that converts cholesterol into

25-­hydroxycholesterol (25HC), an oxysterol. Recent reports suggest that a change
in the physical properties of cell membrane as a result of the formation of high lev-
els of 25HC prevents virus–host membrane fusion. As an enzyme, CH25H modu-
lates the cellular lipid composition and interferes in virus–host membrane fusion,
especially among enveloped viruses (e.g., hepatitis C virus, herpes simplex virus).
Besides this direct action, the antiviral activity of 25 HC partly may be due to its
ability to regulate sterol biosynthesis pathway. 25 HC can freely permeate mem-
branes and can inhibit sterol biosynthesis in both an autocrine and paracrine manner
(Liu et al. 2013).
70 S. George et al.

4.5.3 The Promyelocytic Leukemia (PML) Protein

Around 20 years ago, the expression of PML was detected in 98% of acute promy-
elocytic leukemia (APL) cases due to chromosomal translocation; later it was iden-
tified as INF-induced protein. The PML is known for inhibition of replication of
RNA and DNA viruses. The induction of PML enhances the expression of distinct
PML isoforms and nuclear bodies (NBs). Several PML isoforms have a key role in
transcription regulation and posttranslational modifications such as ubiquitinyl-
ation, SUMOylation, acetylation, and phosphorylation, which leads to consolidated
antiviral activity against specific viruses (Geoffroy and Chelbi-Alix 2011).

4.5.4 Interferon-Stimulated Gene 15 (ISG15)

Interferon-stimulated gene 15 is a 15 kDa ubiquitin-like protein induced by type 1

interferon. These proteins are responsible for ISGylation, a type of covalent addi-
tion to cytoplasmic and nuclear proteins, similar to ubiquitination. ISGylation
involves three main enzymes: UBE1L; E1 (ubiquitin-activating enzyme-E1-like
protein), E2 (ubiquitin-conjugating enzyme 8), and E3 (estrogen-responsive finger
protein and UBP43 or USP18 (ubiquitin-specific protease 18). ISG15 has numerous
antiviral functions including inhibition of virus release (e.g., retroviruses HIV-1,
ASLV, and Ebola virus) (Morales and Lenschow 2013).

4.5.5  he Interferon-Induced Protein with Tetratricopeptide

T
Repeats (IFIT) Family and IFN-­Induced Transmembrane
Protein Family (IFITM Families)

Type-1 and type III interferons chiefly induce IFIT family proteins. The genes for
these proteins are mainly conserved in mammals, amphibians, and fish but not in
lower animals like yeasts, fruit flies, etc. Presence of tetratricopeptide (TPR) mul-
tiple repeats is a unique property of these proteins. Some members of the IFIT fam-
ily act by binding to the subunits of the eIF3 translation initiation complex and
thereby reduce the efficiency of cellular cap-dependent protein translation. Besides,
IFIT can also function as viral RNA sensor by binding to uncapped 5′-ppp RNA and
sequestering it from the actively replicating pool. Studies have also shown that IFIT
can bind other proteins such as viral E1 helicase, a protein required for replication,
and prevent virus infection.
Humans consist of four IFITM proteins (IFITM1, IFITM2, IFITM3, and
IFITM5). Besides antiviral function, IFTM has been shown to play a role in many
biological processes such as cell signaling, germ cell homing and maturation, and
bone mineralization. IFN-induced transmembrane (IFITM) protein members inhibit
endocytic fusion events of a broad spectrum of viruses. However, further work has
to be done to establish how IFITM differentially restricts different viruses (Diamond
and Farzan 2013; Zhou et al. 2013).
4 Interferons and Their Role in Viral Infection 71

4.5.6 Radical SAM Domain-Containing Molecule/Viperin

Radical SAM domain-containing molecule (RSAD2) known as viperin is

induced by JAK-STAT signaling or by direct activation of IRF1/3. Viperin interacts
with mitochondrial trifunctional protein after relocalization from endoplasmic retic-
ulum or in the form of ER-derived lipid droplets. Viperin inhibits many viruses
which are enveloped, with diverse mechanisms. One of the mechanism identified
for influenza A virus is by decreasing the farnesyl diphosphate synthase activity (an
enzyme involved in isoprenoid biosynthesis) which alters membrane fluidity and
hence interferes in viral budding (e.g., HIV-1). In HCV, viperin has been shown to
inhibit viral RNA replication (Duschene and Broderick 2012).

4.5.7 Retroviral Restriction Factors: APOBEC3

Hosts of higher animals including mammals have evolved a mechanism to syn-

thesize virus restriction factors to restrict the infection. Among them apolipoprotein
B editing complex (APOBEC3) family members are well characterized for induc-
ing hypermutations in cDNA by interfering in the process of reverse transcription
among retroviruses (HIV-1). Overexpression of APOBEC3 leads to cytidine deami-
nation during reverse transcription and that results in lethal mutations. APOBEC3
gene family among humans has A, B, C, D, E, F, G, and H polymorphic forms. The
polymorphism in coding and regulatory regions of these genes has deleterious effect
on survival of Parvo, Herpes, Papilloma, and hepatitis B viruses in infected host
cells. Also APOBEC3 has a key role in boosting innate immunity against many
viruses (Harris and Dudley 2015).

4.5.8 SAMHD1

Sterile α motif and histidine-aspartic acid (HD) domain-containing protein 1

(SAMHD1) is another well-characterized retroviral restriction factor. SAMHD1 is
a deoxynucleoside triphosphate (dTNP) hydrolase which restricts viral replication
(HIV-1) by hydrolyzing deoxynucleotide triphosphates (dNTPs) present in virus-­
infected cells (CD+T and myeloid cells) (Li et al. 2017).

4.5.9 Bone Marrow Stromal Cell Antigen-2 (BST2)/Tetherin

Tetherin (bone marrow stromal cell antigen 2—BST-2, CD317) is a membrane-­

bound protein, which inhibits viral budding, release of viral particles from cell sur-
face in retrovirus (all classes), and many other viruses such as filoviruses (Ebola
virus and Marburg virus) and paramyxovirus (Nipah virus). Evidence suggests a
direct tethering mechanism of virus restriction where the tetherin dimers physically
crosslink virion and cellular/other virion membranes, thereby leading to the
72 S. George et al.

retention of mature virions in protease-sensitive layers on the plasma membrane.

Many viral proteins (glycoproteins) subvert tetherin function (e.g., HIV-1 VPU,
HIV-2 ENV, Ebola virus surface glycoprotein-VP-40) by ubiquitination of tetherin
resulting in tetherin degradation (Le Tortorec et al. 2011).

4.6 miRNA and IFNs

IFN is known to regulate the expression of a number of noncoding RNA genes,

especially micro RNA (miRNA). These miRNAs along with IFN-induced proteins
form the innate response system against the invading virus. Each type of IFN (I, II,
III) is capable of inducing the expression of miRNA capable of restricting the repli-
cation of virus by different mechanisms. Of the several miRNA induced by IFNβ,
eight miRNAs show sequence-predicted targets within the HCV genomic RNA and
three among these miR-351, miR-431, and miR-488 can inhibit HCV replication.
For a more comprehensive review, refer to Pedersen et al. (2007).

4.7 Evasion of the IFN System by Viruses

The IFN system is one of the first lines of defense mechanism mounted by the host
against the virus. Virus in due course of evolution has acquired mechanisms to
antagonize the IFN-mediated responses by inhibiting the IFN-activated signaling
cascade or else by suppressing the antiviral pathways. In the following section of
this article, a brief account of how viruses evade the IFN system is described. For a
more detailed review, refer to García-Sastre (2017).
Viruses such as flaviviruses form replication complex inside membrane vesicles
also termed as viroplasm-like structures (VLS) that protect the viral RNA from
interacting with RLRs during RNA synthesis (den Boon and Ahlquist 2010). While
in case of influenza virus, the replication occurs in nucleus far away from cytosolic
RLRs and TLRs, it has been shown that RIGI can recognize the influenza virus
RNA genome during the transit from cytoplasm to the nucleus (Jackson et al. 1982;
Weber et al. 2015). The viral RNA with unmethylated 5′ tri- or diphosphate ends is
recognized by the RLRs RIG-I and MDA 5, and this in turn activates and induces
IFNs. In order to mimic the host, some RNA viruses post-translationally modify
RNA by adding 5′ cap structures similar to eukaryotic using viral coded phospha-
tases and methyltransferases. For some viruses that lack enzymes for posttransla-
tional addition of their own RNA cap, they utilize the host cellular mRNA expressing
viral endonucleases and utilize these caps to mask viral RNA (Reguera et al. 2010).
Many DNA viruses such as herpes virus due to their large coding sequences
encode viral protein capable of interacting with key mediators of IFN signaling and
thereby suppress IFN response. For example, inhibition by herpesviruses of the
STING, a signaling molecule which is downstream of cGAS, has been reported
(Chen et al. 2016). Similar to DNA virus, some RNA viruses such as arenavirus Z
protein (Fan et al. 2010) and paramyxovirus V proteins (Andrejeva et al. 2004) bind
4 Interferons and Their Role in Viral Infection 73

to and inhibit members of RIG-I like receptors. The influenza virus PB1-F2 protein
is shown to interact with the downstream adaptor mitochondrial protein MAVS
(Varga et al. 2012). Viral proteins are also shown to interact with a number of tran-
scription factors involved in IFN induction. The binding of ORF36 protein of murine
gamma-herpesvirus to activated IRF3 in the nucleus prevents induction of IFN
mRNA synthesis (Hwang et al. 2009) The NS5 protein of yellow fever virus binds
to STAT2 and this affects its antiviral action by inhibiting binding of this transcrip-
tion factor to the IFN responsive promoter elements of the ISGs (Laurent-Rolle
et al. 2014). Similarly a number of viral encoded proteins have been shown to bind
to STAT1 and STAT2 such as the P proteins of rabies (Vidy et al. 2005), the C6
protein of vaccinia virus (Stuart et al. 2016), and the V and W proteins of paramyxo-
viruses (Rodriguez et al. 2002; Shaw et al. 2004).
Direct regulation of phosphorylation events involved in IFN induction is another
attractive strategy used by virus. These kinases are target of many viral encoded
proteins, for example, the IKKε kinase inhibition by the N protein of arenaviruses
(Pythoud et al. 2012). The NS4B protein of flaviviruses binds to and inhibits tank
binding kinase 1(TBK1) (Dalrymple et al. 2015), VP40 of Ebola virus inhibits the
JAK1 (Valmas et al. 2010), and TYK2 inhibition by LMP1 of Epstein-Barr virus
(Geiger and Martin 2006). IFN signaling is yet another pathway targeted by the
viruses to evade the host. The IFNAR1 chain of the IFN receptor is the preferred
target for degradation by phosphorylation-dependent ubiquitination, and this affects
type I signaling and antiviral defense (Liu et al. 2009).
Many viral proteases that are involved in the processing of the viral polyprotein
are also shown to cleave and degrade proteins necessary for IFN response. This is
most commonly seen in positive-strand RNA viruses. For example, the hepatitis C
virus protease cleaves MAVS (Li et al. 2005; Meylan et al. 2005), while the dengue
virus protease cleaves STING (Aguirre et al. 2012; Yu et al. 2012). Another mecha-
nism by which virus degrades host proteins is by marking the protein by addition of
K48 polyubiquitin chains makes it susceptible to proteosome degradation. This is
exemplified by NS5 of Zika virus that targets human STAT2 for proteosomal degra-
dation in infected cells (Grant et al. 2016).
Viruses have been shown to prevent gene expression by epigenetic regulation,
thereby interfering with transcription mechanism of the host. Adenovirus E1A pro-
tein has been shown to bind to and dissociate the host nuclear complex that is
required for histone monoubiquitination at H2B lysine 120 (Fonseca et al. 2012).
This modification prevents the opening of the chromatin to allow transcription, and
therefore IFN is unable to activate transcription of the ISGs. In addition to transcrip-
tional shutoff, viruses can suppress host gene expression by other mechanisms such
as the posttranscriptional inhibition of cellular RNA processing, trafficking, and
translational shutoff. The NS1 protein of influenza virus has been shown to prevent
the termination as well as polyadenylation of cellular mRNA (Nemeroff et al. 1998).
The M protein of VSV targets the nuclear pore components and thereby inhibits the
RNA export from the nucleus (von Kobbe et al. 2000). Similarly the ORF10 of the
Kaposi’s sarcoma-associated herpesvirus inhibits the translocation of mRNA of
genes coding for mitosis, gene silencing, DNA metabolic process, chromosome
74 S. George et al.

organization, cell cycle, and transcription regulation (Gong et al. 2016). These
genes although not involved in direct IFN response, may be detrimental for survival
of virus. Translational shutdown of host gene expression is another strategy used by
many viruses. This is best exemplified by the hepatitis C virus infection during
which the PKR mediated phosphorylation of the translation factor eiF2α results in
the inhibition of cellular translation, while the hepatitis mRNA translation continues
as it depends on a different mechanism of 5′ ribosomal entry site that is insensitive
to PKR mediated translation inhibition (Garaigorta and Chisari 2009). Many viruses
recruit decoy proteins to sequester the host proteins and prevent its binding to spe-
cific targets. The B18R protein of vaccinia virus, which is a poxvirus-encoded sol-
uble IFN receptor, can sequester IFN before it can bind to the IFN receptor (Symons
et al. 1995). Similarly the K3L protein of the poxvirus acts as a decoy for PKR
substrates (Beattie et al. 1991). Thus, a virus may use any of the above mechanism
or multiple mechanisms as a strategy to suppress the effect of IFN-mediated host
response.

4.8  herapeutic Intervention of Viral Infection Using

T
Interferons

As the virus hijacks the host immune response and causes chronic infection, for
elimination and controlling the virus replication a therapeutic intervention is needed.
The strong antiviral state conferred by the IFNs to the host cells combined with its
ability to modulate the immune system has made it an ideal candidate to be used in
antiviral therapy. IFN-α was initially used for the treatment of hepatitis C virus
(HCV) infection, but due to its short half-life and rapid elimination from the body
was unable to achieve the desired effect (Lin and Young 2014). In order to maintain
stable level in the blood, pegylated IFN was used for the treatment of chronic
HCV. IFN-α is also found to be effective against hepatitis B virus infection. Besides
the antiviral activity IFN-α is a powerful immune modulator and the studies related
to its efficiency as an adjuvant have met with limited success (Rizza et al. 2008).
IFN-α treatment is accompanied by systemic side effects such as neutropenia and
thrombocytopenia and as a remedy other IFN molecules such as albumin-IFN-αand
consensus IFN (CIFN) have been developed to replace PEG-IFN-α. CIFN with
10–100 times higher antiviral activity compared to standard IFN-α is a recombinant
protein and is found to be effective at a lower dose with limited side effects (Sjogren
et al. 2007). CIFN is a recombinant IFN with most commonly observed amino acids
from several type I IFN subtypes. Similar to type I IFNs, other classes of IFNs have
been shown to be useful in therapeutic intervention during viral infections. IFN-γ
has been shown to be protective against HIV-associated opportunistic infection with
or without highly active antiretroviral therapy (Jarvis et al. 2012). A comparison of
treatment with PEG-IFN-α and PEG-IFN-λ along with ribavirin showed higher
rates of rapid virologic response regardless of the HCV genotypes. Unlike IFN-α,
treatment of HCV with IFN-λ is much safer and less toxic as the restricted tissue
expression of the IFN-λR1 chain ensures a localized reaction (Lin and Young 2014).
4 Interferons and Their Role in Viral Infection 75

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ni.1979
Role of Cytokines in Infectious Viral
Disease 5
Pavani Sanapala and Sudhakar Pola

Abstract
Cytokines are cell signaling polypeptides. They regulate and promote immune
response, mostly related to autoimmunity. Role of cytokines in viral disease as
cytokine biology in recent studies shows multifaceted interactions that are
directly involved in the advancement of disease pathogenesis. Viruses induce the
expression of numerous cytokine protein genes that are activated from the dor-
mant state, and can also stimulate the expression or replication of extremely
identical cytokines. Cytokines are also involved in treating viral diseases, espe-
cially human interferons. The ongoing developmental swift in the area of cyto-
kines has led to the eventual growth of new antiviral therapeutic approaches
rooted in mimicking the cytokine network. Some cytokines, especially IL-12 in
combination with interferon γ, decrease the pathogenicity of viruses. Likewise,
cytokines induce or increase the viral pathogenicity, for instance, Leishmania
major, where IL-4 has a deleterious effect. This chapter details the salient fea-
tures of cytokines, classification, inter-relation of cytokines- viral expression and
signaling pathways of viruses inducing cytokines production.

Keywords
Cytokines · Chemokines · Viruses · NF-κβ activity · Signal transduction · viral
infections

P. Sanapala · S. Pola (*)

Department of Biotechnology, Andhra University, Visakhapatnam, Andhra Pradesh, India
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2020 81

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_5
82 P. Sanapala and S. Pola

5.1 Introduction

Cytokines, also called immunocytokines, are a broad group of polypeptides pro-

duced by both immune and non-immune cells (Ferreira et al. 2018). Cytokines
modulate a wide range of biological function together with hematopoiesis, inflam-
mation, proliferation, repair, differentiation, and innate and adaptive immunity even
at a lower concentration (Chung 2009; Balkwill and Burke 1989; Whicher and
Evans 1990). Cytokines act as a de facto host defense and amalgamate host meta-
bolic function. They play a crucial role in guarding against viral and bacterial infec-
tions. Indeed, cytokines are also concerned with developing disease symptoms and
pathogenesis (Balkwill and Burke 1989).
Viruses as small obligate parasites cannot replicate by themselves. For their rep-
lication and continued existence, they depend upon the cellular mechanism used
during growth and differentiation of the susceptible cell they invade (Ramshaw
et al. 1997). Viral infection characterizes an insightful challenge to host endurance,
where the ability of the virus to duplicate or persevere in the host is intended in
opposition to host antiviral defense mechanisms, i.e., both immune and non-immune
responses. Certainly, the point of contact of a virus with its host is complex and
active (Ramshaw et al. 1997; Campbell 1991), which rather seems not startling of
the function played by cytokines in viral infections. Cytokines uphold viral replica-
tion and dodge host defense in the course of regulating humoral and cellular immune
response (Campbell 1991).
The direct expression of multiple cytokines distinguishes many viral diseases.
Both hemopoietic and non-hemopoietic together constitute a direct change in cyto-
kine gene product expression by viruses. These approaches suggest that the law of
complete and local antiviral responses may possibly be mediated with cell exterior
to the hemopoietic cubicle. In this chapter, we review in brief general features of
cytokines and its importance in biological functions and mainly focus on its role in
infectious and viral diseases.

5.2 Cytokines

Cytokines are low molecular mass proteins habitually below 30 kDa in size. These
polypeptides are active on signaling molecules and cells invigorating them toward
sites of inflammation, infections, and traumas (Ferreira et al. 2018). Cytokines are
emanated by various cells at confined higher concentrations, and entailed in the cell
to cell communications either at the site they originated (autocrine effect) or at the
adjacent cells (paracrine fashion), or on distant cells (endocrine effect/systemic
effect) (Stadnyk 1994). As a result, the frontier between hormones and cytokines is
quite unclear. Indeed, standard hormones, for instance, prolactin (PRL), growth hor-
mone (GH), leptin, and erythropoietin (EPO), are all evidently cytokines confirmed
by their receptor structure and their manner of signaling. Perhaps it is just simplest
to accept that cell–cell communication and host defense went hand in hand during
evolution, and so functional and structural resemblance exists among families of
5 Role of Cytokines in Infectious Viral Disease 83

molecules that act on the immune, hematopoietic, endocrine, and nervous systems
(O’Shea et al. 2019).
Earlier many attempts for a suitable nomenclature were made, but Cohen et al.
emphasized that no single cell as a source is responsible for origination, as cyto-
kines are made by one or more cell types (Cohen et al. 1974). The first defined
cytokines were lymphokines a soluble product originated from lymphocytes in
stimuli to the polyclonal antigen. Other such previous attempts were monokines for
monocytes products. However, this nomenclature or classification did not seem apt
since few cytokines, namely, interleukin 6 and tumor necrosis factor α, were made
up of both cell types (Feldmann 1998). Cytokine may act interactively or antipa-
thetically (Zhang and An 2007).

5.3 Classification and Source of Cytokines

Cytokines are recognized by extensive pleiotropism and factor of redundancy,

with each cytokine comprising numerous overlie functions, and each role pro-
spectively transmitted by more than one cytokine. This, by reasons made the clas-
sification of cytokines a difficult chore. Classifying cytokines in the best way was
a challenge; hence they are grouped in different ways. One such categorization is
based on the type of receptor that they bind. This includes the following: type I
and type II receptors, i.e., hematopoietin and interferon family, tumor necrosis
factor family receptors, interleukin-­1 receptor, interleukin 17 receptor, toll-like
receptors, tyrosine kinase receptors, serine kinases, a receptor of transforming
growth factor-β family, and chemokine that binds to transmembrane domain
receptors (Vilcek 2003). Other categorizations were by numerical cataloging
based on discovery (IL-1 to IL-35), by their functional activity, by their kinetic
role in the inflammatory response, by the origin of the primary cell, and as a
recently added feature by structural homologies common with interrelated mole-
cules (McInnes 2017) (Table 5.1).
Many cells are a source for the production of cytokines. As illustrated above,
the most basic exemplary for cytokine action was resultant of T-lymphocytes that
stimulated the production of immature T cells (Hamblin 1988). By analogy with
cytokines derived from lymphocytes, two types of Helper T cells were known to
secrete and generate cytokine products with varied functional properties (Barrett
1996). Later in line, a second cellular family of monocytes or macrophages has
been an important source for cytokine production (Nathan 1987; Nicod 1993).
Another immune and non-immune cell synthesizing cytokine within and by sec-
ondary nerve tissues at the time of physiological and pathological practices is by
tissue-resident macrophages, recruited macrophages, mast cells, Schwann cells,
and endothelial cells. Likely, herniated nucleus pulposus can produce cytokines in
the spinal cord (DeLeo et al. 1996) and swollen dermis (Heijmans-Antonissen
et al. 2006).
84 P. Sanapala and S. Pola

Table 5.1 Different classification of cytokines and cytokine receptor

Classification based on receptor binding
Receptor family Cytokines
Type I GH, Prl, Epo, Leptin, IL-6, IL-11, Tpo, IL-27, IL-31, CNTF, LIF, Osm,
(hematopoetin) CT-1, GM-CSF, IL-3, IL-5, IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IL-13,
12, 23, IL-35, TSLP
Type II (interferon) IFN-α/β, IFN-γ, IL-10, IL-19, 20, 22, 24, 26, 28, 29, 30
Interleukin-1 family IL-1 α/β, IL-1F, IL-18, 33
Interleukin-17 Il-17A, B, C, D, E, F
family
TGF-β receptor TGF-β 1,2,3
serine kinase
Receptor tyrosine Stem cell factor, CSF-1, Flt-3 ligand, IL-32, 16, 34
kinase
Other classifications
Type of class Cytokines
Pro-inflammatory IL-1 α/β, TNF-α/β, IL-6, 11, 18, IFN-γ
cytokines
Anti-inflammatory IL-10, TGF-β, IL-1
cytokines
Neutrophils CXCL8/IL-8, IL-1 α/β, TNF-α/β, G-CSF, IL-17A, IL-17F
Eosinophil IL-2, 3, 4, 5, GM-CSF, CCL5, CCL11, CCL7, CCL13
T-helper (Th) cells Th1: IL-2, IFN-γ, IL-12, GM-CSF, TNF-α, TNF-β, Il-18
Th2: IL-4, 5, 6, 9, 10, 13, 25
T-regulatory (Tregs) Il-10 and TGF-β
cells
T cells IL-16, CCL5, CCL3, CCL4, TSLP, CCL17, CCL22
Growth hormones PDGF, VEGF, TGF-β, FGF, EGF, SCF
IL interleukin, IFN interferon, TGF transforming growth factor, CSF colony-stimulating factor,
G-CSF granulocyte CSF, CCL chemokine, TNF tumor necrosis factor, PDGF platelet-derived
growth factor, VEGF vascular endothelial growth factor, FGF fibroblast growth factor, SCF stem
cell factor, EGF epidermal growth factor, GM-CSF granulocyte macrophage colony-stimulating
factor

5.4 Viral Infection and Cytokines

Nearly all varied cytokines have a key role in many viral infections. Different
approaches such as viruses modulating cytokine expression, cytokines promoting
viral expression and replication, and a virus with cytokines or their receptors mim-
icking host immune response result in the cure of viral diseases.

5.4.1 Expression of Cytokine in Viral Disease

In general, cytokines were identified by its bioactivity and quantification by bioas-

say during its initial discovery. However, nowadays, tools such as sequence
homology in gene database and homologous receptor binding sequence are in use.
At present, knowledge on expression of cytokines in a diversity of viral infections
5 Role of Cytokines in Infectious Viral Disease 85

is gathered as a result of tissues and cells analysis through in situ and ex vivo
techniques.
The resistant reaction to viral diseases is a mix of numerous actions: viral antigen
introduction to the immune framework, multiplication of particular lymphocytes,
and cytokine mediation (Cavallo et al. 1994). A sign of viral contamination is an
intense response by the affected cell. This incorporates enactment of a previous
antiviral resistant mechanism, obligation headed for programmed cell death and
generation of explicit cytokines. These actions add toward the decrease of viral
duplication and the confinement of viral spread. Cytokines and chemokines have a
vital role in the host reaction to viral contagion just like in the immunopathology
related to numerous viral ailments. Numerous viral glycoproteins in association
with specific cell receptors mediate the direct synthesis of cytokines. Furthermore,
viral RNA and viral proteins endorse viral replication and pro-inflammatory protein
expression when meddling with cell signaling factor activity such as transduction
and transcription (Mogensen and Paludan 2001).

5.5  ignal Transduction in Viruses-Induced Cytokine

S
Production

Subsequent to viral infection, viral surface proteins in many viruses principally act
on the cellular surface protein initiating a cell response where in many cases directs
the primary signal of cytokine production. As well, some viruses in the absence of
viral proteins during initial virus infection can generate cytokines as proteins are
produced at some stage of the viral life cycle. However, the increase of viral RNA
and excess synthesis of the cellular macromolecule induce signs that activate early
host response to the infection.

5.5.1 Transcriptional Factors

Signal transduction pathways stimulated by viruses comprise many transcriptional

factors concerning cytokine induction. Recent studies revealed two factors, inter-
feron regulatory factor 3 and interferon regulatory factor 7 (IRF-3 and IRF-7,
respectively) play a significant role in IFN-α/β activation (Marié et al. 1998).
Activation of transcription factors is elicited by serine/threonine (S/T) phosphoryla-
tion. The MAP (mitogen-activated protein) kinases p38 and JNK (Jun N-terminal
Kinase) are stimulated in reaction to viruses. Downstream targets particularly acti-
vating transcription factor 2 (ATF-2) and Jun are phosphorylated by S/T kinases
after activation of MAP kinases p38 (Arora and Houde 1992). The protein Jun
forms homodimers with ATF-2 and heterodimers with Fos. The dimer ATF-2/Jun
joins to cyclic AMP response element. Likewise, the Jun and Jun/Fos homodimer
and heterodimer spot the TPA responsive element (Whitmarsh and Davis 1996). An
additional transcriptional factor, nuclear factor of activated T cell (NF-AT) present
in a latent form in the cytoplasm, is activated by viruses. The factor NF-AT is trig-
gered by calmodulin-dependent phosphatase calcineurin by dephosphorylation
86 P. Sanapala and S. Pola

process on increasing the cytoplasmic levels (Crabtree 1999). NF-κβ protein is gen-
erally originated in the cytoplasm in composite with Iκβ, an inhibitory protein.

5.5.2 Events in Signaling

Initiation of signal transduction is generated by activation of MAP 3 kinases that

uphold the stimulation of kinase complex capable of phorphorylating Iκβ at two
sites of N-terminal serine residues. The cofactors liable for Iκβ phosphorylation are
Iκβ kinase α (IKKα) and Iκβ kinase β (IKKβ). Phosphorylated Iκβ is degraded by
ubiquitin-dependent 26S proteasome pathway. Breakdown of Iκβ uncovers the tar-
get nuclear localization signal NF-κβ where it wanders toward the central cell
nucleus and thereby initiates the transcription process. NF-κβ has a significant role
in virus-independent cytokine expression and pathogenesis (Fig. 5.1). Viruses
inducing the production of a range of cytokines and chemokines profile are exem-
plified below.

5.5.2.1 Cytokines in Influenza Virus Infection

The influenza virus is a part of the Orthomyxoviridae family. It is an encased
virus with negative single-stranded RNA as a genome. Namely, four types of

Fig. 5.1 Activation of NF-κβ by viruses. The picture depicts different viruses, their proteins, and
pathways stimulating cellular response through cell membrane receptors
5 Role of Cytokines in Infectious Viral Disease 87

genera are distinguished: types A, B, C and Thogotovirus. The genera influenza

A and B are the only two that are clinically related to a human being. The virus
holds a pair of macromolecules: hemagglutinin (HA) and neuraminidase (NA)
dependable for viral cell attachment via exchanges with sialic acids on top of the
cell shell and breakdown of sialic acid that prop up viral disease respectively
(Scholtissek 1991). The main source for transmission of the virus is through
aerosol, i.e., via talking, coughing, or sneezing where the large droplets enter the
mucosa (Blut 2009).
In influenza viral infection, cytokines come into sight in acute phase within
1–3 days of contagion. A wide array of cytokines and chemokines were stimulated
in an influenza virus infection in various cell types. These comprise IL-1, 2, 6, 8,
10, 15, 18, TGF-β, TNF-α, IFN-α/β, IFN-γ, GM-CSF, and RANTES (Fawaz et al.
1999; Hayden et al. 1998; Hennet et al. 1992; Hofmann et al. 1997; Kallas et al.
1999). Studies showed a difference in the ability to influence virus inducing the
cytokine INF-α/β (Chomik 1981). Of all the cytokines, interferon (IFN) plays a
vital role in defense against influenza infection. IFN-α synthesized in virally
infected lymphocytes, IFN-β processed in virally infected fibroblast, and IFN-γ
produced in Th-1 cells are the three types of interferons studied for influenza virus.
In a study to detect the role of interferons protecting from the influenza virus, the
three subtypes were administrated as anti-interferons into mice model. When a
mouse infected with the influenza virus is treated with anti-IFN-α and β, all the
treated mice died within 7 days of postinfection. Similarly, treatment with anti-
IFN-γ showed 60% death while controls in both the cases survived to indicate that
IFN-α/β was active only in initial days of host-virus infection (Hoshino et al. 1983)
and IFN-γ is important in guarding against influenza virus infection. Likewise,
IL-6 which is produced in infected mice mast cells showed effective defense
against this infection by preventing uncontrolled amassing of neutrophils to swol-
len tissues and also restricting rigorous pathogenesis of the lung (Graham et al.
2015; Hurst et al. 2001; Lauder et al. 2013; McLoughlin et al. 2004; Modur et al.
1997; Skoner et al. 1999). Houde and Arora illustrated that the protein NA pro-
moted the production of IL-1 and TNF-α in murine peritoneal macrophages (Houde
and Arora 1989, 1990).
Cell signaling in influenza virus infections is instigated by the activation of
MAP kinases p38 and JNK and also by the downstream factors NF-κβ, AP-1, and
CRE-­binding protein (Fig. 5.1) (Hofmann et al. 1997; Bussfeld et al. 1997; Kujime
et al. 2000). Studies have shown cellular response on exposure to HA triggering
NF-κβ in excess in the endoplasmic reticulum (ER) (Baumann et al. 1998; Flory
et al. 2000; Pahl and Baeuerle 1995). Two additional proteins of influenza virus
(matrix protein and nucleoprotein) are stated to induce NF-κβ activation. For both
the mechanical systems, the activation pathway is mediated by IKKβ (Pahl and
Baeuerle 1997). Also, the double-stranded RNA of the virus is sensed to alter cell
signal by dsRNA-­activated protein kinase (PKR) through two mechanisms: one
with PKR inhibitor p58 (Melville et al. 1999) and the other with nonstructural
protein (Hatada et al. 1999).
88 P. Sanapala and S. Pola

5.5.2.2 Cytokine in Viral Myocarditis

Myocarditis is an inflammatory heart muscle disease, the main cause of dilated
cardiomyopathy globally (Schultz et al. 2009). Cytokines IL-1a, IL-10, TNF-α, and
G-CSF prominent in the serum of acute viral myocarditis probably play a role in
myocardial injury (Matsumori et al. 1994).
In an earlier study to examine the effect of cytokines within viral myocarditis, a
clinical trial in mice induced with virus encephalomyocarditis (EMCV) in the heart
was carried out by RT-PCR, ELISA, and bioassay methods. The results confirmed
that the expression of IL-15, 6, 10, TNF-α, IFN-α/β mRNA was enhanced in the
heart for the period of acute stage 2–3 days after EMCV infection. The cytokines
IL-6, TNF-α, IFN-γ, and IFN-β concentration were shown to be high in infected
mice in comparison with uninfected mice. Later anti-cytokine antibody in vivo
effects were observed, where anti-IL-6 and anti-IFN-γ resulted in considerably
decreased survival and raised myocardial damage in mice. An anti-TNF-α antibody
in EMCV-injected mice showed an improved survival rate and lesions. The study
signifies that IL-6 and TNF-α have a key role in protecting and execrating of acute
myocarditis correspondingly (Imanishi 2000).

5.5.2.3 Cytokines in the Hepatitis Virus

The hepatitis B virus (HBV) is a small DNA enveloped virus. It is a model virus of
the Hepadnaviridae family. It is a common cause liver cancer and liver disease
(Liang and Hepatitis 2009). The HBV genome is integrated into host DNA, leaving
the infection in the dormant stage. Later, accrual of HBV surface antigen (HBsAg)
leads to ground-glass hepatocyte pathology a feature of HBV infection (Chisari and
Ferrari 1995). Studies reported showed production of a large number of pro-­
inflammatory cytokines and chemokines such as IL-1β, IL-2, 6, 8, TNF-α, and
IFN-γ (Al-Wabel et al. 1993; Geneva-Popova and Murdjeva 1999; Mahe et al. 1991)
and also anti-inflammatory cytokine IL-10 (Oquendo et al. 1997; Hsu et al. 1990;
Vingerhoets et al. 1998). In another study, Penna et al. (1997) reported T helper 1
cytokine in acute self-stimulating HBV infection.
The viral component HBV x protein (HBx) is responsible for inducing cytokines
and chemokines (IL-6, IL-8, and TNF-α) production. HBx functions as a transcrip-
tional activator and is essential for viral infection. It is competent in upregulation of
cellular and viral genes. Besides HBx, HBsAg and core antigens (HBc Ag and HBe
Ag) trigger the production of cytokine (Hsu et al. 1990; Vingerhoets et al. 1998).
HBx protein stimulates the pro-inflammatory signaling cascade on activation of
MAP kinase network via MEKK1, GTPase Ras, and S/T kinases Raf (Benn and
Schneider 1994; Benn et al. 1996). This results in downstreaming of JNK and ERKs
which stimulate transcriptional factors AP-1, ATF-2, and NF-κβ. AP-1 triggers Ras
stimulation and NF-κβ (Cross et al. 1993; Natoli et al. 1994). Activation of NF-κβ
is stimulated by two diverge cytoplasmic pathways. The first is phosphorylation fol-
lowed by degradation of Iκβα and the latter is a decrease of p105 cytoplasmic levels
(Haviv et al. 1996). Another such means entailed is the interaction of Hbx and tran-
scriptional factor IIB (TFIIB), TFIIIB, RNA polymerase II (Su and Schneider 1996;
Lin et al. 1997), and raised levels of TATA-binding proteins (Wang et al. 1995a).
5 Role of Cytokines in Infectious Viral Disease 89

5.5.2.4 Cytokines in Herpes Virus

Herpes simplex virus (HSV) a herpes virus is a member of Alphaherpesvirinae.
HSV is reported to infect humans with disease like cold sores and eye and genital
infections. HSV infection is categorized into primary and secondary, where the ini-
tial bearing more severity than later. Primary HSV infection in host produces a
series of cytokines of interleukin family (IL-1β, IL-2, IL-6, IL-10, IL-12, and
IL-13), TNF-α, interferon α/β and γ, and GM-CSF (Ellermann-Eriksen 1993;
Ghanekar et al. 1996; He et al. 1999; Paludan et al. 2000). However, IL-8, macro-
phages, and inflammatory protein 1α and monocyte chemoattractant protein 1 and
RANTES are produced additionally (Rösler et al. 1998; Thomas et al. 1998).
Another related herpes virus cytomegalovirus (CMV), a member of
Betaherpesvirinae subgroup, is reported to induce the production of pro-­
inflammatory cytokines IL-1β, IL-6, 12, TNF-α, IFN α/β, and γ (Carlquist et al.
1999; Orange and Biron 1996; Peterson et al. 1992). CMV communicates as a dis-
ease in many cell types and institutes latency in leukocytes. Glycoprotein gB is the
main protein of the CMV virion. The anti-gBcan blocks IL-1β in monocytes of the
infected host. Likely, the ability to retain the induction of IL-6 is proliferated by
UV-inactivated virus in human CMV (Carlquist et al. 1999; Baumforth et al. 1999).
Also, Epstein-Barr virus (EBV), a lymphotropic herpes virus, belongs to the
Gammaherpesvirinae family. During its primary contagion, the virus ensures lytic
duplication in epithelial and B lymphocyte cells, where it begins latency. EBV
causes Burkitt’s lymphoma, Hodgkin’s disease, and nasopharyngeal malignancy
(Hsu and Glaser 2000; D’addario et al. 1999). The initial gesture bearing cytokines
and chemokines during EBV disease comprises IL-1β, IL-1 receptor antagonist,
IL-6, 8, 18, TNF-α, IFN α/β and γ, GM-CSF, and inducible protein-10 (Lay et al.
1997; Setsuda et al. 1999; Tanner et al. 1999; D’Addario et al. 2000). Similar to the
above-discussed herpesviruses, EBV surface protein activates early features of the
infection. For instance, EBV gp350 a glycoprotein induces the products IL-1β and
TNF-α (D’addario et al. 1999; McColl et al. 1997). Similar to CMV, EMV treated
with UV induced IL-6, GM-CSF, MIP-1α, IL-8 (Roberge et al. 1996; Eliopoulos
et al. 1999; Nakagomi et al. 1994). Moreover, a latent membrane protein 1 of EBV
is proficient at producing cytokines interleukins 6, 8, and 10 (Roberge et al. 1996).
Cellular signaling response is almost similar in all the herpes virus infections.
HSV infection is triggered by either UV insensible and reliant on gD or UV suscep-
tible and dependent on an efficient viral genome. The actually gD-dependent signal-
ing is not completely demonstrated, but HVS-2 inflammation of murine macrophages
provokes improved DNA binding activity of NF-κβ and ATF-2/c-Jun an hour after
the disease onset. The virus HSV uses Hve A as a receptor for signaling (Marsters
et al. 1997). For CMV, signals are activated for cytokine induction (IL-1β and IL-6)
by NF-κβ (Yurochko and Huang 1999). Part of signaling for EMV is similar to HSV
and CMV, while the unique property of the pathway is the use of gp350 protein via
cellular receptor complement receptor 2 (CD21) which acts as a signaling receptor.
These receptors induce tyrosine phosphorylation, PKC, activation, NF-κβ, and
phosphatidylinositol 3-kinase (Sugano et al. 1997). On the other side, proteins
BZLF-1 and BRLF-1 trigger signaling cellular trauma (Adamson et al. 2000).
90 P. Sanapala and S. Pola

5.5.2.5 Cytokines in Human Immunodeficiency Virus (HIV)

HIV belongs to the family Retroviridae of subfamily Orthoretroviridae, grouped to
the genus Lentivirus (Luciw 1996). Based on genetic features and variation in viral
genes the virus is differentiated into type 1 and type 2 (HIV-1 and HIV-2). The
virus infects CD4 T-lymphocytes and macrophages via CD 4 receptors and co-
receptors CXCR4 and CCR5 (Oberlin et al. 1996; Virelizier 1999). The severe host
reaction to initial HIV infection is differentiated by a T-helper cytokine profile of
both pro -and anti-inflammatory cytokines. At an advanced stage, the cytokine pro-
duction is triggered partially by Th-2 (Paludan et al. 1998; Rinaldo et al. 1990;
Toso et al. 1990).
The virus glycoprotein gp120 links with CD 4 and the co-receptors to induce the
production of IL-1α, IL-1β, IL 6, and 8, TNF-α, and interferon α/β/γ (Ameglio et al.
1994; Ankel et al. 1994). Cytokines IL-4 and -13 in basophils and IL-10 in mono-
nuclear cells are also produced by gp120 signifying the role of gp120 with cell
receptors. Also, the proteins transactivator (Trf), negative regulating factor (Nef),
and virus protein r (Vpr) are likely able to trigger the secretion of cytokines (Chen
et al. 1997).
Cellular activity of HIV infection in signal transduction is detailed profoundly.
The protein gp120 induces signaling when in contact with CD4 and CXCR4 and
CCR5 chemokines. The binding of CD4 with gp120 activates tyrosine kinase Lck
and Serine/threonine kinase Raf-1 receptors (Popik and Pitha 1996). Subsequently,
MAP kinases, ERK1, ERK2, and JNK, have also been activated which in turn acti-
vate NF-κβ, AP-1, and C/EBPβ (Lannuzel et al. 1997). The signaling mechanism
for chemokines was by phosphorylation of protein tyrosine kinase. Tat, a protein
inducer of many pro-inflammatory cytokines, activates viral replication by regulat-
ing transcriptional factors NF-κβ, NF-AT, Sp1, and C/EBPβ families (Ruocco et al.
1996; Vlach et al. 1995). Activation of NF-κβ by Tat protein is a prerequisite for
PKR and the process occurs via IKK is active in the cells infected by HIV. Tat pro-
tein initially aims Ikβ-α for degradation (Demarchi et al. 1996). This mechanism of
synthesis and degradation is sited at the promoter region in the presence of NF-κβ.
However, JNK and ATF-2 are also controlled by Tat in HIV infections. Another
feature regulated by Tat is the Sp1, a major source meant for basal and inducible
expressions of many genes. On phosphorylation, Sp1 enhances the promoter activ-
ity. No such study is reported regarding Nef protein and its signal transduction. Vpr
adapts the function of numeral DNA-binding proteins to stimulate HIV long-term
repeats via direct protein–protein interaction. This is duly regulated by physical
binding of Vpr and Sp1 factors (Wang et al. 1995b). Succeeding studies have
revealed that Vpr acts together with p53 antagonizing Vpr/Sp-1-driven transcription
(Schafer et al. 1996).

5.5.2.6 Cytokines in Human T-Lymphocyte Virus Type-1

The retrovirus HTLV-1 belongs to Oncovirinae subfamily. The viral disease is
habitually asymptomatic. In its latency period, it causes mature T-cell lymphocytic
leukemia (ATLL) or non-cancerous HTLV-1. HTLV 1 virus modulated the expres-
sion of many cellular genes, even for cytokines through posttranscriptional and
5 Role of Cytokines in Infectious Viral Disease 91

transcriptional pathways. During the infectious period, the production of cytokines

IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-15, TGF-β, TNF-α, interferon
α/β/γ and GM-CSF is triggered by the host (Azimi et al. 1998). The proteins Tax
and Rex enhance the secretion of cytokine, chemokines, and the receptors (Baba
et al. 1996). However, Tax protein intercedes transactivation of TNF-β and TGF-β
genes, where cellular transcriptional factor NF-κβ (Kim et al. 1990) and AP-1 (Paul
et al. 1990) are essential. Posttranscriptional regulation through protein Rex adds to
the stimulation of cellular genes cytokines. The product assists the transfer and
expression of nucleus and cytoplasm of HTLV-1 gaga and env mRNAs and post-
transcriptionally alleviates the IL-2 receptor protein mRNA, which considerably
accumulates mRNA followed by the viral infection (Kanamori et al. 1990).
The protein Tax is certainly the major immune stimulator, not just regulating
duplication but as well taking part in the expression of cell signaling. Signaling via
Tax is a complex mechanism. Initially, Tax stimulates NF-κβ via IKK pathway that
results in the release of active NF-κβ (Geleziunas et al. 1998). IKK-γ and MEKK-1
are involved in stimulating IKK by Tax protein. This process of specific interaction
linking Tax and IKK-γ has a requirement of leucine zipper domains (Harhaj and
Sun 1999; Xiao et al. 2000). Several discrete NF-κβ pathways are activated by Tax
protein. One such pathway is the use of PKC isoforms (α, δ, and η) with phosphory-
lation and autophosphorylation of Tax and PKC correspondingly. A second factor
ATF-2 in group with CREB transcription factor family activates viral transcription
along CRE-like sites in HTLV-1 LTR (Franklin et al. 1993; Gachon et al. 2000).
Next, Tax activates NF-AT that binds to CD28 responsive element in the promoter
region of the genes triggering cell expression.

5.5.2.7 Cytokines in Filovirus Infection

Filovirus is nearly 80 nm in diameter recognized by its filamentous shape catego-
rized under the family Filoviridae. The virus is divided into three genera: Ebolavirus,
Marburgvirus, and Cuevavirus (Kuhn et al. 2010). The genome of the virus consti-
tutes 19 kb single linear non-segmented negative-sense RNA. The virus codes for
the proteins: nucleoprotein, polymerase factor VP35, the matrix proteins VP40 and
VP24, transcriptional protein VP30, glycoprotein and the RNA-dependent RNA
polymerase (Bixler and Goff 2015). This viral infection is differentiated by elevated
levels of pro-inflammatory cytokines and chemokines that are produced by con-
taminated macrophages and monocytes. The cyto- and chemokines include IL-1β,
IL-8, IL-15, IL-18, MIP-1α, MCP-1, IP-10, Gro-α, and eotaxin. However, at lower
levels, IL-2 was observed in association with T cell (Geisbert et al. 2007).

5.6 Cytokines and Viral Replication

Cytokines besides their role as mediators in local and systematic host antiviral
response are able to endorse viral replication and multiply some of viruses, espe-
cially by IFNs and TNF. The well-known suitable example explained is
HIV. Activation of HIV expression from inactive state of replication is reliant partly
92 P. Sanapala and S. Pola

on the stimulation of the host cell (Rosenberg and Fauci 1990). Activation of CD4+
lymphocytes and monocytes is linked with the production of a number of cytokines
that induce the expression of HIV from chronic infected T lymphocyte and mono-
cyte cell lines. Specifically, the cytokines TNF-α and IL-6 upbeat transcriptional
and posttranscriptional expression levels (Poli et al. 1990).

Acknowledgements The authors gratefully acknowledge Andhra University, Visakhapatnam, for

the support extended.

Conflict of Interest The authors declare that they have no competing interests.

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B Cells and Their Role in Combating Viral
Diseases 6
Devanabanda Mallaiah and Pallaval Veera Bramhachari

Abstract
Humoral immunity mediated by B cells plays an important role in combating
different types of viral diseases. Two types of B cells (B-1 and B-2) originate in
the primary lymphoid organ called bone marrow from hematopoietic stem cells
(HSCs). Both B-1and B-2 cells develop separately through sequential steps
(pro-B cells—pre-B cells—immature B cells) in bone marrow from distinct
common lymphoid progenitors before their release as immature B cells into cir-
culation. In the serous cavities, the immature B-1 cells differentiate into mature
B-1 cells through transitional B cells. The immature B-2 cells differentiate into
transitional B cells, which mature finally into marginal zone (MZ) and follicular
(FO) B cells in the secondary lymphoid organs. The B-1 cells produce poly-­
specific natural antibodies (antibodies produced before infection), which provide
first line of defense. The B-1 cells mainly defend mucosal and blood-borne
pathogens in a T-cell independent manner. The MZ B cells produce immune
response against blood-borne pathogens and undergo both T-independent and
T-dependent activation. In addition, both B1 and MZ B cells behave like innate
immune cells by expressing toll-like receptors (TLR) and produce immune
response without or with their membrane-bound poly-reactive B-cell receptors
(BCR). Finally, FO B cells are the conventional B cells of adaptive immunity and
primarily responsible for T-dependent immune response by their membrane-­
bound mono-reactive BCR. The antigen-activated B-2 cells differentiate into
antibodies secreting plasma cells and memory B cells. Along with natural anti-
bodies, the non-neutralizing, neutralizing, and broadly neutralizing specific

D. Mallaiah (*)
Department of Biotechnology and Bioinformatics, Yogi Vemana University,
Kadapa, Andhra Pradesh, India
P. V. Bramhachari
Department of Biotechnology, Krishna University,
Machilipatnam, Andhra Pradesh, India

© Springer Nature Singapore Pte Ltd. 2020 99

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_6
100 D. Mallaiah and P. V. Bramhachari

a­ ntibodies are important in combating various viral diseases. Additionally, B

cells modulate T-cell functions by presenting antigens, providing co-stimulation,
and secreting cytokines. This chapter describes different types of B cells, anti-
bodies, and their role in combating viral diseases (HIV, influenza and hepatitis).

Keywords
B cells · Humoral immunity · Antibodies · Viral diseases

6.1 Introduction

Serum therapy by Von Behring and Kitasato provided the first evidence that humoral
response played an important role in the host resistance against infectious agents
such as diphtheria and tetanus toxins. They explained that serum therapy gives dis-
ease resistance due to the presence of antibodies in the serum. Paul Ehrlich demon-
strated the basis for serum therapy and established the field of humoral immunity.
Today, the importance of antibodies as immunotherapeutic is well characterized and
is used to treat a wide range of infectious diseases. Antibodies are main components
in many effective vaccines and have been used to prevent many human diseases
caused by viruses (Yamada 2011; Burton 2002).
The success of immune system in combating different types of viruses depends
on both non-specific innate immune system and specific-adaptive immune system.
Humoral immunity is a branch of adaptive immunity mediated by antibodies
secreted from B cells, which plays an important role in combating viruses along
with cell-mediated immunity (Bonilla and Oettgen 2010). Humoral immune
response to antigens is categorized as primary and secondary response. In the pri-
mary response, T helper cells are of primary importance whereas in the secondary
immune response, the specific-neutralizing antibodies play a primary role against
reinfection and T cells are of secondary importance. The primary immune response
can take 1–2 weeks and IgM is the predominant type of antibody elicited. The sec-
ondary immune response is more rapid than primary immune response and pre-
dominant type of antibody formed is IgG (Lefevre et al. 2009).
The T helper cells, typically follicular T helper cells (TFH cells), were activated
by the same antigen and cooperate with antigen-activated mature B cells to differ-
entiate into plasma cells and memory B cells. The activated B cells remain in the
margins of T-cell zone and differentiate into short-lived plasma cells. The activated
B cells which initiate germinal centers differentiate into long-lived plasma cells and
memory B cells. The high-affinity and antigen-specific antibodies are secreted and
maintained by both short-lived and long-lived plasma cells. The long-lived plasma
cells migrate to survival niches in the bone marrow and persist for several years
(Gourley et al. 2004). The protective natural antibodies are produced before the
foreign antigen exposure or pathogens by B-1 cells and marginal zone B cells in a
T-cell independent manner. About 80% of all circulating natural antibodies are IgM
type and remaining are IgG and IgA (Palma et al. 2018). B cells provide several
lines of protection against viruses. In the first line, natural antibodies provide
6 B Cells and Their Role in Combating Viral Diseases 101

defense against initial viral infections. The long-lived plasma cells generate high
amounts of neutralizing IgG and provide second line of defense, and reactive long-­
lived memory B cells provide third line of defense against viruses (Dörner and
Radbruch 2007).
Natural antibodies are encoded by germ line variable genes and not shaped by
post-recombination processes such as somatic hypermutation and class switching.
Natural antibodies recruit antigens into the spleen to prevent infection of vital
organs and also to induce early neutralizing antibodies without T-cell help. Natural
antibodies activate complement to enhance B- and T-cell-specific immune
responses. Therefore, natural antibodies are considered an important link between
innate and adaptive immunity (Matter and Ochsenbein 2008). The natural antibod-
ies are poly-­reactive that they can bind to different structurally unrelated antigens
and also auto-­reactive that they can bind to self-antigens. Along with natural anti-
bodies, poly-reactive antigen-binding B cells provide protection against multiple
pathogens. In addition to poly-reactive antibodies, the specific neutralizing and
cross-­reactive antibodies also provide effective antiviral immune response (Warter
et al. 2012).
The highly specific antibodies protect from viral infection by both neutralizing
and Fc-mediated effector mechanisms. Neutralizing antibodies inhibit the viruses
by binding through their antigen-binding sites or Fab region and also through their
constant or Fc region. The non-neutralizing antibodies bind to many epitopes
including neutralizing antibody targets and induce the destruction of microbe or
infected host cells by innate immune system (Hua and Ackerman 2017). Recently,
novel broadly neutralizing antibodies were characterized, which are able to neutral-
ize diverse isolates of viruses (Sok and Burton 2018). This chapter describes about
different types of B cells, antibodies, and their role in combating different types of
viruses (HIV, influenza, and hepatitis).

6.2 B Cells

The B lymphocytes are subset of lymphocytes, which express diverse specific sur-
face immunoglobulins and constitute 5–15% of total circulating lymphoid cells.
The antigen-independent development of B cells starts from the fetal liver and bone
marrow of an adult, where the hematopoietic stem cells differentiate into pro-B
cells, which in turn differentiate pre-B cells to immature B cells. Along with the
development of phenotypically distinct precursor cells, rearrangements of VDJ
genes occur parallelly, which contribute to formation of diverse specific immature
B cells with membrane-bound IgM. Immature B cells differentiate into mature B
cells with membrane-bound IgM and IgD as its BCR in the secondary lymphoid
organs. Two types of B lymphocytes (B-1 and B-2) have been identified. Mature B
lymphocytes are activated by different types of antigens. There are three types of
antigens activating the B lymphocytes. (1) T-independent antigen type 1, for exam-
ple, LPS (lipopolysaccharide), which is polyclonal activator of both mature and
immature B cells. LPS activates the B cells through TLR. (2) T-independent antigen
102 D. Mallaiah and P. V. Bramhachari

type 2, for example, bacterial polysaccharides, which have repeated epitopes,

require help from cytokines secreted by T helper cells but not their direct contact.
(3) T-dependent antigens, for example, soluble protein antigens, require T helper
cells’ direct contact and cytokines secreted by them to activate B lymphocytes
(Maddaly et al. 2010).
The origin and development of B-1 and B-2 cells are described by two compet-
ing models. The selection model proposes that the response to a particular antigen
decides the differentiation of B cell to become either B-1 or B-2. The other layered
immune system hypothesis of Herzenberg proposes that they emerge from separate
distinct progenitors. The B-1 cells present in serous cavities (peritoneal and pleural
cavities) further differentiates into B-1a and B-1b. The B-2 cells in secondary lym-
phoid organs also differentiate into two types of subsets such as follicular (FO) and
marginal zone (MZ) B cells after passing through T1, T2, and T3 transitional stages
(Montecino-Rodriguez and Dorshkind 2012).
The B-1a cells produce poly-specific IgM natural antibodies in the serum of
newborn or germ-free animals. The B-1b cells secrete antibodies after stimulation
with thymus-independent antigens (Matter and Ochsenbein 2008). In addition to
IgM, the B-1 cells also produce poly-reactive IgA antibodies for mucosal immunity
(Suzuki et al. 2010). The MZ B cells express poly-reactive BCR and produce poly-­
specific IgM antibodies against blood-borne microorganisms. Both B-1 and MZ B
cells express TLR and produce response to pathogen-associated or endogenous
TLR ligands with or without recognition through BCR. Unlike B-1 cells, the MZ B
cells generate response to T-dependent proteins and produce high-affinity isotype-­
switched antibodies. Although the FO B cells respond to T-independent antigens,
they are primarily responsible for high-affinity IgG antibodies production against
T-dependent antigens. The T-dependent antigens activate the FO B cells in the pres-
ence of T helper cells and their secreted cytokines. Antigen-activated FO B cells
involves in the formation of germinal center (GC) reaction. The clonal expansion,
isotype-switching, somatic hypermutation, and affinity maturation are characteris-
tics of GC reaction. The activated FO B cells undergo proliferation and differentia-
tion into antibody secreting long-lived plasma cells and memory B cells (Fig 6.1)
(Hoffman et al. 2016). The external antigens are captured through BCR of B cells
and they are degraded inside the B cells and the corresponding antigen fragments
presented by B cells on MHC-II molecule to helper T cells are an important step in
adaptive cellular immunity (Yuseff et al. 2013).
B cell surface not only consists of membrane-bound Ig, but also other comple-
ment component receptors and Fc receptors are expressed. In addition to that with
the advancement of monoclonal antibody technology, many B-cell-specific surface
molecules were identified. To bring common nomenclature, monoclonal antibodies
were designated as clusters of differentiation (CD). The CD designation is embraced
as a label for the target molecule rather than grouping of monoclonal antibodies
with common reactivity. The target molecules or CD are involved in B-cell develop-
ment, function and communication with extracellular environment. These CD mol-
ecules also provide cellular context which interprets the BCR signals; for example,
in BCR signaling and B-cell development, CD79a (Igα) and CD79b (Igβ) are
6 B Cells and Their Role in Combating Viral Diseases 103

Fig. 6.1 Schematic diagram of B-cell development and different humoral effector immune cells

non-­covalently attached with BCR and their cytoplasmic domains certainly con-
tains conserved motifs for tyrosine phosphorylation and Src family kinase dock-
ing respectively (LeBien and Tedder 2008). In addition to production of antibodies,
B cells also actively participate in cellular immune response mediated by T cells.
The B cells directly modulate effector, memory, and regulatory T-cell functions via
antigen-specific but antibody-independent mechanisms. B cells modulate T cells by
antigen presentation, by providing co-stimulation, and by secreting cytokines. B
cells are functionally subdivided into two types: (1) Be-1 cells and (2) Be-2 cells. In
the presence of TH1-type cytokines, the Be-1 cells do not secrete significant
amounts of IL-4, IL-13, or IL-2 but secrete IFN-γ and IL-12 including IL-10, TNF-­
α, and IL-6. In the presence of TH2-type cytokines, Be-2 cells do not secrete signifi-
cant amounts of IFN-γ and IL-12 but secrete IL-4, IL-13, or IL-2 including IL-10,
TNF-α, and IL-6. The regulatory B cells also called B-10 cells produce IL-10 cyto-
kines that suppress the CD4+ T-cell responses. In addition to suppression, cytokines
producing B cells enhance the T-cell-mediated immune responses (Lund and
Randall 2010).

6.3 Antibodies

The B lymphocytes differentiate finally into plasma cells, which are the source for
antibodies. The two competing natural selection and clonal selection theories for
antibody formation were proposed by Jerne and Burnet, respectively, but finally,
clonal selection theory was supported by experimental evidence (Burnet 1976).
Antibodies are chemically glycoprotein molecules. They are also called immu-
noglobulins and are present on the surface of B cells as BCR or free molecules in
blood, plasma, and extracellular fluids. The fluids formerly were called humors and
are part of humoral immune response. They have two principal functions in
humoral defense. The first function is that it recognizes and binds to antigenic
104 D. Mallaiah and P. V. Bramhachari

determinants or epitopes on the surface of foreign molecules, for example, enve-

lope spikes on the virus surface. The second function is that it triggers elimination
mechanisms, e.g., complement activation and phagocytosis by neutrophils and
macrophages. The first function requires huge diversified specific antibodies and
the second function requires common features in all different types of antibodies.
The Y-shaped antibodies are made up of four proteins chains: the upper and lower
parts of heavy and light chains are called variable and constant regions, respec-
tively. The two heavy and two light chains are covalently linked by disulfide bonds
at constant section. The two heavy chains again linked at their constant section
contain hinge region, which provide flexibility to the antibodies (Fig. 6.2). The
antibodies consist of three units, of which two units present at N-terminus of the
chains are identical and responsible for antigen binding—the Fab (fragment anti-
gen binding) arms. The variable domains of both heavy and light chains form the
antigen-binding site or paratope. The variable domain again contains special
hypervariable segments called complementary-­ determining regions, which are
specific to antibodies. The third unit is called Fc (fragment crystalline) arm involved
in effector functions (Hey 2015; Chan et al. 2009).
There are five classes or isotypes (IgG, IgM, IgA, IgD, and IgE), which are dif-
ferent in their C-terminus region of H chains termed γ, μ, α, δ, and ε, respectively.
There are four subclasses of IgG (IgG1, IgG2, IgG3, and IgG4. The effector mecha-
nisms are mainly dependent on the type of antibodies; for example, IgM and IgG 3
are complement activators, whereas IgG1 and IgGE activate macrophages and mast
cells, respectively (Hoffman et al. 2016).
Antibodies act on viruses directly by neutralization or indirectly via interaction
with complement or Fc receptors on innate immune cells (Fig. 6.3). The multiple
effector functions of antibodies include (1) neutralization; (2) antibody-dependent
cellular toxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) of
both viruses and infected cells through interaction with Fc receptors on innate

Fig. 6.2 Structure of antibody molecule

6 B Cells and Their Role in Combating Viral Diseases 105

Fig. 6.3 Antiviral mechanism of antibodies. (a) Neutralization, (b) antibody-bound virus or
infected cell interacts with Fc receptor on innate effector cell, (c) complement component-bound
antibody initiates MAC formation on infected cell or interacts with complement-component recep-
tor on innate effector cell, (d) infected cell bound by antibody interacts with dendritic cell to
release type 1 interferon which in turn stimulates NK cells and antiviral proteins in infected cells,
(e) antigen presentation of viral peptides to T cells. MAC membrane attack complex, APC antigen-­
presenting cell, MHC major histocompatibility complex, TCR T-cell receptor

immune cells; (3) complement-dependent cytotoxicity (CDC), where complement

component C1q mediates formation of membrane attack complex on infected cells
or opsonization-based phagocytosis mediated through complement receptors on
innate immune effector cells; (4) antibodies bound to infected cells interact with
dendritic cells to release type 1 interferons, which stimulate antiviral activity of NK
cells and antiviral functions in infected cells, or antigen-presenting cells phagocy-
tize the antibody–virus immune complex and present specific viral peptides to T
cells for cellular immunity (Forthal 2014; Hua and Ackerman 2017).
Antibodies neutralize the pathogen by multiple mechanisms such as (1) pre-­
attachment neutralization, where antibodies directly bind with pathogen to aggluti-
nate. For example, the neutralizing IgG antibodies aggregate the poliovirus to
decrease their infectivity. (2) Interference with pathogen attachment—antibodies
bind to pathogen ligands which are essential for pathogen attachment. For example,
antibodies against HIV-1 gp120 interfere with their attachment to CD4+ T cells. (3)
Post-attachment neutralization-inhibition of fusion or entry. For example, a mono-
clonal antibody 2F5 inhibits the fusion of viral and cellular membranes. (4)
Inhibition of various steps in pathogen life cycle—once the pathogen internalized,
in order to neutralize pathogen, antibodies must be internalized and interfere with
106 D. Mallaiah and P. V. Bramhachari

the replication, genetic material expression, and release of pathogen. (5) Inhibition
at later steps—antibodies inhibit the liberation of virus or budding (Forthal 2014).

6.4 Humoral Immune Response Against to Viruses

Viruses are intracellular pathogens and need host cells machinery for their survival.
After synthesizing many viral copies, viruses burst out and reinfect healthy new
host cells. These viruses directly kill the host cells and are called acutely cytopathic
viruses (polioviruses, rabies, and small pox viruses). Some viruses do not kill the
host cells and they are called non-cytopathic viruses (hepatitis B and C, herpes
virus, herpes simplex virus 1 and 2, cytomegalovirus, and Epstein-Barr viruses).
The role of immune system in antiviral defense is both protective and pathogenic,
where it eliminates the viruses by effective immune response and in addition also
causes more damage to host cells than viruses themselves. With regard to effective
immunity, the viruses have developed an array of strategies to escape elimination by
immune system through co-evolution (Dörner and Radbruch 2007).
Viruses are chemically nucleoprotein particles with a size of ~20–200 nm. Due
to their small size, viruses move freely in the lymphatic system and interact with B
cells in secondary lymphoid organs. The highly repetitive structures of viral parti-
cles allow the cross-linking of BCRs, which is an initial step in the activation of B
cells. Further, the repetitive viral structures also bind with natural antibodies, fix
complement, and also carry TLR ligands (DNA or RNA) for TLR 7/8 or 9 to acti-
vate B cells directly (Zabel et al. 2013).

6.4.1 Human Immunodeficiency Virus

AIDS (acquired immune deficiency syndrome) is caused by HIV (human immuno-

deficiency virus). HIV is genetically highly diverse virus and can be found in two
types: HIV-1 and HIV-2. HIV-1 is the most common and falls into three groups: M,
N, and O. Group M is the most common and divided into subtypes or clades (A–D,
F–H, J, and K), of which B is predominant subtype in the Western world and C in
India, China, and Africa. The CD4+ T cells, macrophages, and different subsets of
dendritic cells are the major targets of HIV-1 infection. HIV-1 belongs to Retroviridae
family and lentivirus genus. HIV-1 is an enveloped virus containing two positive
sense RNA strands. The enveloped glycoproteins of HIV-1 present as trimers of
gp120/gp41 heterodimers on the surface of virus (Phogat et al. 2007).
After few days of following HIV-1 postinfection, anti-gp41 antibodies and anti-
­gp120 antibodies are produced, but these antibodies are unable to neutralize the
infecting virus strain or autologous virus. The autologous neutralizing antibodies
are produced several months postinfection, which are not able to neutralize the het-
erologous virus. The cross-reacting antibodies, which can neutralize many heterolo-
gous viruses, develop 2–4 years after seroconversion, but in some patients develop
earlier. Some HIV patients like “Elite neutralizers” develop highly cross-­reacting
6 B Cells and Their Role in Combating Viral Diseases 107

antibodies or broadly neutralizing antibodies (bNAbs) 20-months postinfection.

Diversity of HIV strains and molecular peculiarities of HIV envelope protein are
some obstacles in recognition and neutralization of HIV strains. However, some
patients produce bNAbs that neutralize many HIV strains. The discovery and well
characterization of bNAbs has given new hope in the treatment of HIV, so that vac-
cines should be prepared that induce broadly neutralizing antibodies and antibodies
with particular functional activity. Not only systemic immune response but also
mucosal immune response is produced against HIV virus. Even though, very little
is known about mucosal immune response, some studies have shown that both sero-
negative and positive patients develop HIV-specific mucosal IgA, which inhibits
HIV transcytosis and replication in epithelial cells (Mouquet 2014; Baum 2010).
Non-neutralizing antibody plays an important role in the prevention and control
of HIV in humans. In addition, limited protection was observed with RV144 vaccine
in the absence of neutralizing antibodies, which indicates the role of non-­neutralizing
functions by antibodies (Zolla-Pazner 2016; Mayr et al. 2017). Some studies
reported that they found anti-HIV antibodies are part of innate immune system.
They defend HIV by binding to the HIV protein Tat. Natural antibodies for host
receptor CCR5 prevent HIV infection effectively at major sites of virus entry such
as mucosal tissues (Ward 2001; Lopalco 2010).

6.4.2 Influenza Virus

Influenza or flu viruses cause respiratory diseases and continuously threaten human
health. Genome high mutational rates of influenza viruses cause emergences of new
strains by genetic drifts. Influenza affects 5–30% of global population and causes
hospitalization and death of many people. They are enveloped viruses and contain
single-stranded, segmented, and negative sense 7–8 RNA strands. Influenza viruses
are categorized into four types: A, B, C, and D. Influenza A virus is again classified
based on the antigenic properties of two surface viral glycol proteins such as hem-
agglutinin (HA) and neuraminidase (NA) with 18 (H1-H18) and 11 (N1-N11) anti-
genic subtypes, respectively. Influenza A (H1N1 and H3N2) virus causes seasonal
influenza epidemics in humans along with other members of influenza viruses such
as influenza B virus (Sautto et al. 2018).
Two important hypotheses such as original antigenic sin (OAS) and immune
imprinting or antigenic seniority explain how humoral immunity gets affected by
influenza viruses. Both are dependent on humoral immune memory response rather
than on de novo humoral immune response to drifted or altered influenza viruses
(Guthmiller and Wilson 2018). The original antigenic sin concept refers that the first
exposure of influenza variant in early life dictates lifelong immunity to all variants
of influenza viruses in subsequent encounters. The immune memory produced by
the first influenza variant influences the immune response to subsequently expose
influenza distinct variants, but how this sequential exposure shapes the immune
response remains obscure. The antigenic seniority concept better explains the hier-
archical nature of immune response to previously exposed variants of influenza
108 D. Mallaiah and P. V. Bramhachari

virus. According to the antigenic seniority concept, the first exposed influenza vari-
ant in childhood takes the senior antigenic position in immune repertoire and subse-
quent exposed strains take the junior positions. The immune response to the first
exposure is larger than the responses to subsequent exposures. Understanding of
how previous exposure shapes the antibody responses to vaccination and infection
is critical for the development of universal influenza vaccine (Henry et al. 2018).
Neutralizing antibodies produced against HA head region of influenza are strain-­
specific and bind to highly variable regions. Antibodies neutralize more strains of
influenza and are called broadly neutralizing or cross-reactive antibodies. The
broadly neutralizing antibodies produced against influenza virus mainly target the
conserved regions of HA stem domain. The existence of non-neutralizing antibod-
ies also clears the influenza virus infection by exploiting non-neutralizing effector
mechanisms (Sicca et al. 2018).
The natural antibodies against influenza virus are produced by B-1 cells and
therefore reported to suggest that innate and acquired humoral immunity comes
from separate effector arms of immune system (Baumgarth et al. 1999).

6.4.3 Hepatitis C Virus

Hepatitis C (HCV) is non-cytopathic virus which infects millions of people around

the world, and it is transmitted mainly by unsafe injections and transfusions. During
acute infections few people (20%) only clear the virus spontaneously and remaining
70–80% people suffer from chronic infection. The acute hepatitis C infections are
asymptomatic. The chronic viral hepatitis by HCV leads to cirrhosis, hepatocellular
carcinoma, and end-stage liver disease (Webster et al. 2015).
Hepatitis C virus is small enveloped virus and has positive single-stranded RNA
genome encoding a single polyprotein which undergoes posttranslational modifica-
tion by cellular and viral-encoded proteases into structural (core proteins and enve-
lope proteins) and nonstructural proteins. The HCV RNA genome interacts with
core proteins to form the nucleocapsid that is surrounded by a lipid membrane,
called the viral envelope, in which envelope glycoproteins are anchored (Irshad
et al. 2008).
The clearance of HCV is associated with induction of cellular immune response.
In addition, evidence is accumulating that neutralizing antibodies also contribute to
HCV clearance. Acutely HCV-infected individuals produce antibodies against epit-
opes present on the structural and nonstructural proteins of HCV virus. A small
fraction of antibodies called neutralizing antibodies are able to inhibit virus binding,
entry, and uncoating. The glycoproteins E1 and E2 are major targets for the neutral-
izing antibodies. Neutralizing antibodies are generated against HVR1 region of
envelope glycoprotein E2. The generation of neutralizing antibodies in the early
phase of infection correlates with the resolution of HCV infection in some acutely
infected people. In contrast, chronic infected people showed delayed induction of
neutralizing antibodies (Lapa et al. 2019).
6 B Cells and Their Role in Combating Viral Diseases 109

The HVR1 antibodies are type-specific but some evidence shows that anti-
HVR1 antibodies are cross-reactive. Some neutralizing antibodies against E1 and
E2 envelope proteins show cross-neutralizing potential (Drummer 2014). However,
there is strong evidence on the production of broadly neutralizing antibodies
against HCV infections (Kinchen et al. 2018). The HCV virus also has immuno-
logic regions for virus escape or non-neutralizing antibodies in the envelope glyco-
proteins (Fuerst et al. 2018). Similar to HIV virus, the humoral immune response
also evolves with time in HCV. The evolution of Ab specificity (non-neutralizing—
autologous neutralizing—heterologous neutralizing—broadly neutralizing) during
the course of infection produces increased breadth in the neutralizing activity
(Murira et al. 2016). There are also reports on natural antibodies which recognize
the linear epitopes on hepatitis C envelope glycoprotein E2 to confer additional
neutralization (Tarr et al. 2012).

6.5 Future Perspectives and Conclusions

B cells are the main players of humoral immunity against viruses. B cells also act as
antigen-presenting cells, contributing to cell-mediated immunity. Both B-1 and B-2
cells secrete natural and highly specific antibodies for the prevention and control of
viral infections, respectively. B-2 cells capture the viral antigens through BCR and
differentiate into memory B cells and plasma cells, which secrete different types of
specific adaptive antibodies. Recently, many studies reported the generation of
broadly neutralizing antibodies, which can neutralize different strains of viruses.
Understanding the production of antibodies and their antiviral mechanisms will pro-
vide new ways to develop novel, efficient prophylactic and therapeutic vaccines.

Acknowledgements The authors are grateful to YVU Kadapa and Krishna University,
Machilipatnam, for the support extended.

Conflict of Interest The authors declare that they have no competing interests.

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 Part II
Role of Adaptive Immunity in Combating Viral
Diseases
Significant Role of Cellular Activation
in Viral Infections 7
Bojjibabu Chidipi, Samuel Ignatious Bolleddu,
Ganugula Mohana Sheela, and Alavala Matta Reddy

Abstract
Immune response during viral infection is the result of pathogen recognition and
antibody production by the memory cells residing in the host target tissue. These
cells by origin are differentiated from the cytotoxic CD8+ T cells and play a
major role in developing adaptive immunity. Early activation of these T cells by
ribosomal binding proteins during various cell death pathways can induce a criti-
cal autoimmune response suitable to develop enhanced strategic immune thera-
pies for many chronic viral infections. In addition, there are various genes which
get expressed in good amount in the process of T-cell activation during infections
like Epstein-Barr virus. Focusing on these changes can help in determining the
pathogenic process and orientation of the viral infection. Since we can only con-
trol but not cure most of the viral infections using present medications, following
these novel strategies can help in focusing on the host cell death pathways,
thereby developing the drugs that can give complete immunity against virus
invasion by facilitated viral cell elimination from the humoral environment.

Keywords
Cytotoxic CD8+ T cells · Epstein-Barr virus · Humoral environment · Cell death
pathways · Memory cells

B. Chidipi · S. I. Bolleddu
Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
G. M. Sheela
Department of Biotechnology, Krishna University,
Machilipatnam, Andhra Pradesh, India
A. Matta Reddy (*)
School of Life and Health Sciences, Adikavi Nannaya University,
Rajamahendravaram, Andhra Pradesh, India

© Springer Nature Singapore Pte Ltd. 2020 115

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_7
116 B. Chidipi et al.

7.1 Introduction

The main role of the immune system is to identify the presence of any foreign pro-
tein complexes or antigens and initiate an immune response to eliminate them with-
out harming the metabolism of the host by implementing the pathogen-specific
defense response. Immune response during viral infection is the result of pathogen
recognition and antibody production by the memory cells residing in the host target
tissue. B lymphocytes produce the antibodies during the humoral immune response
while T cells initiate cell-mediated immunity (Finotti et al. 2018).
During viral infections, the cellular activation of various lymphocytes leads to
degranulation of various inflammatory mediators like histamines, TNFα, and vari-
ous amino acids within the first few seconds. The host inborn resistant reaction is a
profoundly strong boundary of barrier containing numerous intrinsic safe cells, for
example, dendritic cells (DCs), macrophages, and common executioner (NK) cells.
These cells are fit for perceiving a broad scope of viral items through a complex and
covering atomic system of pattern recognition receptors (PRRs), including toll-like
receptors (TLRs) just as cytosolic sensors, for example, the helicases, retinoic acid-­
inducible gene 1 (RIG-I) and MDA5. Actuation of intrinsic cells through these
PRRs advances fast popular detecting and the arrival of critical antiviral cytokines
with the possibility of quickly controlling viral replication and driving further natu-
ral and versatile invulnerable reactions. Specifically, the sort I interferon (IFN-I) is
a pleiotropic cytokine family comprising 13 IFNα isoforms and one IFNβ isoform
that move through a common pervasively communicated receptor (IFNαβR).
Eicosanoids like leukotrienes and prostaglandins are released as a secondary
mediator after few minutes. In a prolonged immune response, cytokines and chemo-
kines along with growth factors are released (Bull and Plummer 2014; Frossi et al.
2018; Yu et al. 2016; Brown et al. 2018; Gieseck et al. 2018).
The viral reproduction process in the host cells after the viral invasion starts with
the synthesis of new viral proteins and nucleic acids intracellularly. The new viral
particles or virions are then self-assembled by the aggregation of the macromole-
cules. These virions are seen in the blood circulation invading the healthy cells after
the rupture of the infected host cells. The envelope or capsids of the virus particles
offer protection and platform for inducing an immune response. Motioning through
IFNαβR prompts both autocrine and paracrine actuation, bringing about phosphory-
lation of flag transducer and activator of interpretation 1 and 2 (STAT1 and STAT2).
This actuates several IFN-I-animated qualities (ISGs) that advance an antiviral state
and initiate various safe cells (Brown et al. 2018). Despite this complex inborn reac-
tion, numerous infections, including those that accomplish interminable disease,
can stifle and additionally avoid these early natural host reactions to support their
replication and transmission. In this segment, we centered on IFN-I to show the
dynamic changes that the natural resistant framework experiences and the critical
and expansive results that such changes can have amid perpetual viral diseases
(Ning et al. 2018).
This new wave of viral invasion directly stimulates glycoprotein inducing cells
for the production of the antiviral proteins called interferons. Vital cells of the
7 Significant Role of Cellular Activation in Viral Infections 117

immune system like leucocytes, fibroblasts, and natural killer cells play a vital role
in the production of interferons and lysosomes that lyse a wide variety of virus par-
ticles, thereby recognizing the antigen and developing a suitable antibody.

7.1.1 Type I Interferon Induction by Persisting Viruses

Within days after HIV discovery in plasma from tainted patients, and inside hours
after contamination with LCMV Cl13 in mice, raised dimensions of fundamental
IFN-I are distinguished. Moreover, upgraded ISG articulation is visible in tissues as
well as T cells after LCMV Cl13, HCV, and SIV diseases (Fig. 7.1). Interestingly,
qualities with intrinsic insusceptible capacity are imperceptible right on time after
in vivo HBV contamination in chimpanzees (Haas and Trumpp 2018).

7.2 Role in Adaptive Immunity

Early activation of these T cells by ribosomal binding proteins can induce a critical
immune response suitable to develop enhanced strategic immune therapies for
many chronic infections. Several studies confirm T lymphocytes functional modifi-
cations as the key pathological change responsible for adaptive immunity. A few
non-hematopoietic and hematopoietic cells, including macrophages, ordinary DCs
(cDCs), and plasmacytoid DCs (pDCs), can add to the first vigorous IFNI reaction

MMP-9

CD4+CD28- Th17 CD31 CD31 Treg

truncated intact

Pro-inflammatory Anti-inflammatory

T lymphocyte subsets altered in acute coronary syndromes

Fig. 7.1 depicts several subtypes of T lymphocytes implicated in dysregulated immune responses
during acute coronary syndromes. Regulatory T cells counterbalance the pro-inflammatory proper-
ties of CD4+ CD 28− and Th17 T cells. IL-17A produced by Th17 cells may not only promote
inflammation but also favor fibrosis in atheromata. The rainbow hue of the Th17 cell portrays its
potential dual roles. T lymphocytes that express intact CD31 on their surface interfere with adap-
tive immune responses. Proteolytic cleavage of CD31 by the enzyme matrix metalloproteinase-9
removes T cells from inhibition and licenses them to contribute to the stimulation of adaptive
immunity. As patients with acute coronary syndromes have higher circulating concentrations of
matrix metalloproteinase-9, this mechanism for boosting T-cell responses may operate particularly
during acute ischemic episodes (Libby and Hansson 2018)
118 B. Chidipi et al.

by perceiving and reacting to viral disease through PRR-intervened flagging path-

ways. For instance, in LCMV Cl13 disease, cDCs and macrophages produce IFNI
through MDA5/MAVS-subordinate pathways. Even though the phone source and
the kind of IFN (e.g., IFN-I as well as IFN-III) that prompts raised ISGs in the liver
upon HCV disease are as yet hazy, demonstrating that hepatocytes are fit for creat-
ing IFN-I through RIG-I motioning in light of HCV contamination (Libby and
Hansson 2018; Shuai et al. 2016).
For instance, the CD4 type of cells in the lymphocyte population plays a key role
in the process of adaptive immunity during cellular response by initiating the effec-
tive antibodies against the foreign pathogens. This is usually done by secreting the
cytokines that are capable of the effector mechanisms that combat with antigen
interactions with the host system. At long last, monocyte determined DCs can detect
HIV-2 (and HIV-1 when the host catalyst SAMHD1 does not limit its replication)
using cyclic guanosine monophosphate–adenosine monophosphate synthase
(cGAS) pathways, initiating expert incendiary cytokines and IFN-I creation.
Regardless of the way that practically all phone types can create IFN-I upon viral
experience, pDCs are very particular for discharging these cytokines, delivering
multiple times more IFN-I than some other platelet type, and integrating a more
extensive scope of IFN-I isoforms upon TLR incitement. pDCs can straightfor-
wardly perceive few steady infections, incorporating HIV and HCV in people and
LCMV in mice, through TLR7. pDCs were determined to be basic for T-cell reac-
tions and viral control amid ceaseless LCMV disease, although another examina-
tion utilizing an alternate LCMV variation and an inducible pDC cancelation
framework showed just insignificant impacts (Gao et al. 2018).
The microbes present in the host body also play a vital role in the process of
adaptive immunity by interacting with the antigen–host responses. The macromol-
ecules present in the microbes interact with the immune mediators and alter the
specificity of the autoimmune response, thereby leading to adaptive immunity in
natural process. Remarkably, rather than IFN-I creation in most cell types, PDC
IFN-I generation does not require viral replication, and most of DC IFN-I originates
from uninfected pDCs right on time after LCMV Cl13 contamination.
Correspondingly, uninfected pDCs can detect neighboring tainted hepatocytes by
perceiving HCV RNA-containing exosomes, and neither viral combination nor rep-
lication is required for PDC acknowledgment of HIV. All in all, these investigations
demonstrate that most of industriously recreating infections can be detected by
inborn cells using PRRs, bringing about a vigorous IFN-I reaction in the beginning
of in vivo unending disease (Ost and Round 2018).

1. Antibodies play a vital role in the treatment of viral infections, but a new class of
human monoclonal antibodies called super-antibodies are drawing attention in
immunotherapy. These antibodies are rarely seen in human infections. They are
majorly generated during screening using the B-cell approach. This strategy is
mainly implemented to increase the half-life and immune functions in the pro-
phylaxis and treatment of a wide range of viral infections. This strategy can be a
promising approach in the future (Walker and Burton 2018).
7 Significant Role of Cellular Activation in Viral Infections 119

2. The current antiviral therapies for HBV are effective but need to be administered
for a long period without allowing the virus to rebound or replicate. There are
some immunotherapies which focus on the response of the host to the virus and
maintaining the viral load under control. One of the best strategies is to develop
the antiviral drug based on the toxic outcome and therapeutic efficiency to reduce
the viral levels by immunotherapy studies in the varied populations (Bertoletti
and Le Bert 2018).
3. The new approach in the targeting of the immunodeficiency diseases like HIV,
HBV, and HCV is to target the T regulatory cells which play a major role in
protecting important organs like the liver and kidneys from injury due to immu-
nosuppression. This approach will help protect a vast majority of the white blood
cells that are destroyed by viral infections whose primary goal is to eliminate the
large populations of the macrophages (Karkhah et al. 2018).
4. Another very successful strategy in the treatment of viral infections soon after
transplantation of the stem cells of the blood is to utilize the T lymphocytes taken
from the infected donors who are specific to virus. This can treat patients suffer-
ing from primary immunodeficiency disorders like HIV. This type of therapy is
also known as adoptive immunotherapy (Keller et al. 2018).
5. In one approach DNA was collected from different patients with immunodefi-
ciency disorders and transcriptional analysis was done on the genome to study
the genetic sequences coding for viral replication by nuclear protein binding.
The outcome is studied through mass spectroscopy and the allele or non-allele
dependent messenger RNA transcription is done in possible ways (Lummus
et al. 2018).
6. Both the lymphocytes aggregate and get compromised in the lung injuries like
asthma induced by dust particles. To study the extent of the exposure and the
injury, various fluids from the bronchiolar tissues and the alveoli are collected
and antibodies are confirmed by ELISA or western blotting techniques to study
autoimmunity associated with it. Even the lung tissues can be stained for the
identification of particular proteins (Poole et al. 2018).

7.3 T-Cell Activation During Infections

There are various genes which get expressed in good amount during T-cell activa-
tion during infections like Epstein-Barr virus. The natural regulatory T cells are
usually involved in the activation of the receptors on the cell surface like the CD25.
The T lymphocytes develop in the thymus and are differentiated into regulatory and
non-regulatory cells when they migrate to the peripheral region of the thymus gland.
These regulatory cells are further divided into natural regulatory and the induc-
ible regulatory T cells and include various other subtypes. All these cells are classi-
fied based on their role in the process of eliciting the immune response according to
the invading mechanism of the pathogen.
120 B. Chidipi et al.

7.3.1 Quick Attenuation and Persistence of Type I Interferon

Underlying IFN-I reaction after contamination, the foundational IFN-I winds up

imperceptible at later stages, regardless of kept up viral burdens. This quick atten-
tuation is collated by a significant decrease in pDC IFN-I creation limit amid LCMV,
HIV, and HCV diseases that frequently harmonize with changed natural reactions to
auxiliary, disconnected pathogens. Notwithstanding, it is vital to take note of that in
LCMV, HCV, HIV, and SIV contaminations; IFN-I or ISG transcripts are tena-
ciously recognized in all-out DCs, CD4+, and CD8+ T cells, or potentially tissues,
but at lower sums than those distinguished amid the pinnacle IFN-I reaction. A few
components may add to the weakening of IFN-I in the post-acute phases of intermi-
nable viral contaminations, including (an) immediate concealment of intrinsic path-
ways by viral items, and have immunomodulatory systems. Instances of direct
systems of IFN-I restraint incorporate HCV NS3–4A protein hindrance of TRIF
(the TLR3 connector), RIG-I, and MAVS viral detecting pathways upstream of IFN
creation just as LCMV nucleoprotein (NP) barricade of IRF3 actuation (Gasteiger
et al. 2017).

7.4 Viral Pathogenesis

The pathophysiology of viral infection is very complex unless we can identify the
type of virus species more specifically under careful correlation with the clinical
symptoms of the infection. This can help in determining the pathogenic process and
orientation of the disease. All the cells in the immune system rely on the energy
pathways to survive and produce the response related to their metabolism. Amino
acids like arginine, tryptophan, and alanine play a major role in the creation of the
immune response during pathological conditions and inhibition of the unwanted
immune response during viral attack. Also, the HIV proteins Vpr and Vif initiate
IRF3 debasement (31), and direct HIV gp120 communication with pDCs restrains
TLR9 (however not TLR7) reactions, including PDC actuation and IFNα discharge.
Additionally the HIV capsid connects with chosen have cofactors to avoid PRR
detecting in macrophages. The previously mentioned hidden dimensions of natural
qualities ahead of schedule after in vivo HBV contamination might be mostly clari-
fied by HBV polymerase impedance with IRF3 phosphorylation or potentially by
hindrance of MAVS motioning by the viral HBx protein. Also, by the diminished
TLR2 articulation in hepatocytes, monocytes, and Kupffer cells (liver-inhabitant
macrophages) from ceaseless HBV patients, HBV dissolvable antigen can decrease
articulation of TLR2 in hepatic cell lines. Thus, HBV dissolvable antigen likewise
stifles TLR9 (however not TLR7) motioning in pDCs, and TLR9 articulation is
diminished in blood pDCs from incessantly contaminated patients. Notwithstanding
the direct IFN-I inhibitory exercises of viral proteins, infections that build up unend-
ing diseases additionally advance host immunomodulatory reactions that may cause
deviant working of natural cells (Coe et al. 2014).
7 Significant Role of Cellular Activation in Viral Infections 121

In chronic immunosuppressing diseases like HIV, there is a huge loss of T-cell

CD4 population along with the increased blood levels of the mediators like cyto-
kines. Initially they compromise the host immune system alarmingly leading to the
clinical symptoms of opportunistic infections in HIV patients. Combinational ther-
apy with both antiretroviral therapy and antibiotics will help reduce the levels of
immunosuppression (Moreno-Fernandez et al. 2012).
However, in hepatitis virus infection the number of regulatory T lymphocytes
increases drastically in chronic conditions to induce a good suppression effect on
the virus particles. This is mainly due to the hepatic-cellular invasion of the carcino-
genic virus particles. This mechanism will also help in protecting the morphology
of the liver in the severe inflammatory conditions (Li et al. 2016).

7.5 Host Cell Death Pathways

Since we can only control but not cure most of the viral infections, these strategies
can help in focusing on the host cell death pathways, thereby developing drugs that
can give complete immunity against the virus by facilitated viral cell elimination.
Some of the virus will escape the host immune system and become persistent in the
human body causing infection in the future immunocompromised pathological
conditions.

7.5.1  dvantageous and Deleterious Effects of Type I

A
Interferon on Antiviral Immunity

IFN-I antiviral impacts are interceded by acceptance of a few ISGs that have the
capacity to impede viral replication through different systems [e.g., protein interpre-
tation restraint, viral RNA debasement (investigated by Brown et al. 2018). IFN-I
likewise coordinates both inborn and versatile resistant cells, for example, DCs,
macrophages, NK cells, and T cells following viral diseases, giving enactment and
survival signals. It reliably examines persevering LCMV contamination, in which
the infection is cleared from the blood and most tissues by 2–3 months postinfec-
tion, showing that mice lacking IFNαβR build up long-lasting viremia. Besides,
Sandler et al. exhibited that treatment of rhesus macaques with IFN αβR killing
counteracting agent amid serious SIV contamination brought about improved CD4+
T-cell consumption, diminished antiviral quality articulation, decreased extents of
cytotoxic NK cells, and expanded SIV repositories, all associated with quicker
movement to simian AIDS. What is more, treatment with recombinant IFN-I can
regularly support the host barrier and upgrade viral control (Cooney et al. 2018).
A plethora of examples of such infections are human immunodeficiency virus,
Epstein-Barr virus, hepatitis B and C virus, and so on. These viruses can be preva-
lent irrespective of the advancement in the treatment and vaccination. For instance,
amid incessant HCV disease, PDC IFN-I creation limit is disabled, in any event to
a limited extent, by monocyte-determined tumor rot factor α (TNFα) and interleukin
122 B. Chidipi et al.

(IL)-10. It ought to be noticed that not all parts of natural reactions are weakened
amid unending viral diseases; in fact, select pathways are improved. For example,
though LCMV Cl13-tainted mice react ineffectively to TLR9 ligand (CpG) chal-
lenge, they produce unusually high IFN-I levels upon TLR4 ligand (LPS) incite-
ment, prompting quick passing. By these perceptions, unending HCV contamination
likewise advances hyperresponsive macrophages, including Kupffer cells, adding to
the constant liver irritation that is normal for endless HCV disease. At long last,
macrophages treated with HIV and antigen-displaying cells detached from HIV-­
contaminated patients show hyperresponsiveness to random TLR ligands and com-
mensal microbes, individually. Together, these investigations feature that although
natural resistant cells are equipped for IFN-I reaction in the beginning of most con-
stant viral contaminations, blocking of intrinsic pathways by viral proteins associ-
ated with host immunomodulatory atoms prompts a significant (yet inadequate)
constriction of IFN-I at later phases of the disease (Alvarado-Mora and Pinho 2013).
There are various novel treatment strategies focusing on the host cell death path-
ways to cure these persistent diseases. One involves clearing the latent infected cells
first which have the potential to cause infection; in the future, this can be done by
screening and eluting these cells. But these latent cells are very hard to differentiate
from the healthy cells in the absence of the infection. This strategy is very success-
ful in targeting the HIV virus with antiretroviral therapy. The very high rate of muta-
tions in the virus genome can also be a problem to cure the infection when they
acquire resistance to the antiretroviral drugs administered after the diagnosis and
treatment of HIV (Liang et al. 2015).
Wang and colleagues demonstrated that treatment with recombinant IFNα5 and
IFNβ between days 2 and 5 after LCMV Cl13 contamination (maybe identical to
deferring the IFN-I lessening mentioned above) brought about early popular regula-
tion joined by improved infection explicit CD8+ T-cell reactions, despite the fact
that a similar treatment in the unending period of disease had no impact.
Also, prophylactic treatment with pegylated IFNα2a (pIFNα) preceding SIV
mucosal test prompted expanded protection from contamination interceded to some
degree by an expansion in CD56+ NK cells; in any case, persistent pIFNα treatment
brought about an IFN-I desensitization state with decreased ISG levels and expanded
cell-related viral burdens. Outstandingly, pIFNα additionally improves articulation
of a few ISGs known to be direct enemy of HIV movement. In any case, treatment
with pIFNα in HIV-tainted patients has yielded mixed outcomes; only few investi-
gations have appeared.
In some infections like hepatitis, there are few transcriptionally very active frag-
ments which can code for the viral DNA or RNA and produce new viral fragments.
They invade the host cells and remain attached to the host genome. These fragments
may remain in the body for long periods of time and relapse during favorable condi-
tions producing the antigens. These infective active fragments may get inserted in
the host genome and remain inactive for a period of time which is known as the
latent phase during which screening for the infection is very tiresome (Battivelli
et al. 2018).
7 Significant Role of Cellular Activation in Viral Infections 123

Acknowledgments The authors are grateful for the support extended from Adikavi Nannaya
University, Osmania University, Krishna University, and the University of South Florida, USA.

Conflict of Interest The authors declare that they have no competing interests.

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Macrophages/Microvesicles and Their
Task in Viral Diseases 8
Bojjibabu Chidipi, Samuel Ignatius, Madhavi Maddala,
Pallaval Veera Bramhachari, and Alavala Mattareddy

Abstract
Viral infectious diseases are mainly associated with immunological responses trig-
gered by immunocytes present in the circulation like macrophages. A better under-
standing of these immunity mechanisms will help us to control the progression of
the disease to chronic conditions. Microvesicles are an extracellular group of
plasma membrane secretions, which play a significant role in the spread of infec-
tion or evasion of viral entities to surrounding healthy cells acting as a viral vehicle.
Their involvement in the viral cycle can be utilized productively in hampering the
period by developing the antibodies that shield the evasion. These extracellular
vesicles will facilitate the communication between the cells in the body humoral
environment leading to host and viral interactions in the immune system. Thereby
attempting to study the role of macrophages in the immunity process will help us
to eliminate the viral pathogens by either delaying or damaging their spread to
healthy cells. Focusing on these mechanisms of the microvesicles and macro-
phages will help lead to the early cure of many life-threatening infectious diseases
like HIV, Hepatitis, and Dengue by developing a single specific antibiotic.

Keywords
Macrophages · Viral cycle · HIV · Hepatitis · Dengue · Humoral environment ·
Extracellular vesicles

B. Chidipi (*) · S. Ignatius

Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
M. Maddala
Department of Zoology, Nizam College, Osmania University, Hyderabad, Telangana, India
P. V. Bramhachari
Department of Biotechnology, Krishna University, Machilipatnam, Andhra Pradesh, India
A. Mattareddy
School of Life and Health Science, Adikavi Nannaya University, Rajahmundry, India

© Springer Nature Singapore Pte Ltd. 2020 125

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_8
126 B. Chidipi et al.

8.1 Introduction

Viral infectious diseases are mainly associated with immunological responses trig-
gered by immunocytes like Macrophages. A better understanding of these immunity
mechanisms and the knowledge related to the prevalence of the infection will help
us to control the progression of the disease to chronic conditions by actual estima-
tion of the number of infected individuals by screening methods (Birdwell et al.
2018). The primary route of entry of the virus into the human body is through the
epithelial lining, every time epithelial cells will be the first cells to get infected or
encounter the virus particles. When infected, one of the surface of the epithelial
cells will get polarized since both the inner and outer surfaces of the epithelial cells
are morphologically distinct (Hoebe et al. 2018). With or without finishing the life
cycle inside the epithelium these viruses may exit the cells to invade one or other
surfaces (Baraldo et al. 2018).
The process by which a viral infection leads to disease is known as viral patho-
genesis, the consequences and the period of the viral infection depend on various
host and viral-related interplaying factors. They can develop as an acute infection
but may last for months to years depending on the favorable viral factors. If the
disease becomes chronic it may easily get transmitted to others and also recovery
from such conditions is sporadic (Jaimes and Whittaker 2018). The cell damage
created by the infection depends on the type of virus because not all viruses can
damage cells even though they replicate and widely spread throughout the body.
Some viruses belonging to the class known as retrovirus can cause persistent infec-
tion but cannot cause cell death as they are released from the cells without any lysis
by a unique phenomenon called budding (Shaheen 2018).
Whereas picornavirus can cause lysis of cell and cause cell damage and rhinovi-
rus cause mucus secretion and poliovirus paralysis the tissues causing tremendous
cell damage. The progressive clinical symptoms are noticed with these infections
until the viral population minimizes to noninfectious levels (Carrasco et al. 2018).

8.2 Viral Infection Cycle

An infection must utilize cell procedures to repeat. The viral replication cycle can
create dramatic biochemical and fundamental changes in the host cell, which may
cause cell harm. These changes, called cytopathic (causing cell harm) impacts, can
change cell function. Some infected cells, for example, those contaminated by the
regular cold infection known as rhinovirus, kick the bucket through lysis (bursting)
or apoptosis (customized cell demise or “cell suicide”), discharging all offspring
virions without a moment’s delay. The side effects of viral maladies result from the
insusceptible reaction to the infection, which endeavors to control and dispense
with the virus from the body, and from cell harm brought about by the virus
(McDougall et al. 2019).
Numerous viral infections, for example, HIV (human immunodeficiency infec-
tion), leave the contaminated cells of the safe framework by a procedure known as
8 Macrophages/Microvesicles and Their Task in Viral Diseases 127

sprouting, where virions go to the cell separately. Amid the growing process, the
cell does not experience lysis and is not promptly slaughtered. In any case, the harm
to the cells that the infection taints may make it outlandish for the cells to work
ordinarily, even though the cells stay alive for a timeframe. Most beneficial viral
diseases pursue comparative strides in the infection replication cycle: connection,
entrance, un-coating, replication, gathering, and discharge. In Fig. 8.1, the host cell
is annihilated toward the finish of the replication cycle—recollect this does not gen-
erally occur: here and there the host cell lives on and keeps on reproducing the
infection (Khalili and Malcolm 2019).

Connection An infection joins to a particular receptor site on the host cell film
through connection proteins in the capsid or using glycoproteins inserted in the viral
envelope. The explicitness of this communication decides the host—and the cells
inside the host—that can be tainted by a specific infection. This can be outlined by
thinking about a few keys and a few locks, where each key will fit just a single
explicit lock (Zhao et al. 2019).

Entry Phase The nucleic acid of bacteriophages enters the host cell exposed, leaving the
capsid outside the cell. Plant and creature infections can enter through endocytosis, in
which the cell film encompasses and overwhelms the whole virus. Some wrapped infec-
tions come to the cell when the viral envelope melds legitimately with the cell layer. Once

Fig. 8.1 Microvesicle formation mechanism. (Top panel) In resting cells, both phosphatidylserine
and phosphatidylethanolamine are negatively charged and segregated in the inner side of the
plasma membrane, on the other hand sphingomyelin and phosphatidylcholine, both are positively
charged head group, are in the outer side of the plasma membrane (Seigneuret et al. 1984). (Lower
panel) The “flippase” and “floppase are an ATP-dependent transmembrane enzyme. Flippase
reacts with phosphatidylserine and help to inward folding, meanwhile floppase intermediate the
both anionic and cationic phospholipids to outward fold (Daleke 2003; Laberge et al. 2018)
128 B. Chidipi et al.

inside the cell, the viral capsid is debased, and the viral nucleic acid is discharged, which
at that point ends up accessible for replication and translation (Appaiah et al. 2019).

Replication Phase The replication instrument relies upon the viral genome. The
viral infections generally use cell proteins and compounds to make extra DNA that
is translated to ambassador RNA (mRNA), which is then used to coordinate protein
union. RNA infections, for the most part, utilize the RNA center as a layout for the
union of viral genomic RNA and mRNA. The viral mRNA guides the host cell to
orchestrate viral catalysts and capsid proteins, and gather new virions (Ilan 2019).

There are individual cases to this example. On the off chance that a host cell does
not give the compounds essential to viral replication, viral qualities supply the data
to coordinate the union of the missing proteins. Retroviruses, for example, HIV,
have an RNA genome that must be turned around translated into DNA, which at that
point is consolidated into the host cell genome (Solomon and Geretti 2019).

Assembly Phase They are inside gathering VI of the Baltimore characterization

plot. To change over RNA into DNA, retroviruses must contain qualities that encode
the infection explicit protein switch transcriptase that deciphers an RNA format to
DNA. Invert translation never happens in uninfected host cells—the required com-
pound switch transcriptase is just gotten from the outflow of viral qualities inside
the infected host cells (Jin and Musier-Forsyth 2019).

The way that HIV creates its very own portion compounds not found in the host
has enabled scientists to develop drugs that hinder these chemicals. These medica-
tions, including the turnaround transcriptase inhibitor AZT, inhibit HIV replication
by diminishing the movement of the protein without influencing the host's diges-
tion. This methodology has prompted the advancement of an assortment of medica-
tions used to treat HIV and has been successful at decreasing the number of
irresistible virions (duplicates of viral RNA) in the blood to non-discernible dimen-
sions in numerous HIV-contaminated people (Roder et al. 2019).

Budding Phase The last phase of viral replication is the arrival of the new virions
delivered in the host living being, the place they can taint neighboring cells and rehash
the replication cycle. As you have adapted, some infections are discharged when the
host cell bites the dust, and different infections can leave contaminated cells by sprout-
ing through the layer without legitimately slaughtering the phone (May 2019).

8.3 Microvesicles

Microvesicles are the extracellular group of plasma membrane secretions, which

play a significant role in the spread or evasion of viral entities to surrounding healthy
cells acting as a viral vehicle. Microvesicles formed from the lipid rafts also called
8 Macrophages/Microvesicles and Their Task in Viral Diseases 129

cholesterol- and sphingolipid-rich membrane microdomains (Fig. 8.1). These

microdomains involved in many cellular processes including microvesicles release.
These being the cell-derived vesicles encapsulated by a bilayer of lipids are pres-
ent throughout the body in the amniotic fluids, breast milk, nasal secretions, cere-
brospinal fluid, blood, and serum. Higher concentrations of these cysts will be
noticed during the pregnancy and tumorous conditions in a diameter of about
30–100 nm (Chen et al. 2018) (Fig. 8.2).
The primary biological function of microvesicles in the human body is to elimi-
nate unwanted molecules like proteins from the healthy cells during the process like
blood coagulation. Their secondary purpose is intercellular communication between
the cells during the exchange of materials between them. During the dangerous
conditions, their role is to propagate the pathogens for the immune response by
inhibitory or regulatory mechanisms and also in the presentation of antigens
(Laberge et al. 2018).
The formation of microvesicles is by a unique process known as the membrane
remodeling in which the integrity of the cell membrane is changed due to increased
levels of calcium deposition leading to the formation of small vesicles on the sur-
face and are released by the disruption of the cell membrane. These vesicles are
released into the blood circulation and reach their target cells. These vesicles bind
to the surface of the host cell and undergo ligand-specific interaction to enter the
host cell or may undergo fusion or endocytosis to gain entry into the cell cytoplasm
(Bello-Morales et al. 2018a).
The main constituents of the microvesicles are proteins, lipids, messenger RNA,
or Micro RNA. These are spontaneously released or shed by many types of cells like
microvesicles during cell activation or apoptosis. They help in communication
between cells with their surface receptors and play a potential role in the exchange
of genetic material (Yao et al. 2018).

8.4 Role of Microvesicles in the Viral Cycle

Microvesicles have many common characteristics with that of the enveloped viruses.
During viral infections, many infected cells secrete a vast number of microvesicles
that may or may not be similar to the biogenic and biophysical characteristics of
viral counterparts. For instance, HIV virions will shed from the plasma membrane
to but not from the cytoplasm in a similar budding mechanism like microvesicles
spread from cell to cell. Moreover, it is tough to separate HIV particles from exo-
some vesicles during centrifugation as they both have identical biological properties
(Bello-Morales et al. 2018a).
Microvesicles as discussed above will play a vital role in assisting the virally
infected cells in promoting the intercellular microvesicle-mediated communication.
These vesicles, when fused to the surface of the other cells, will eventually transfer the
cellular components exerting a biological function to the recipient cells. During infec-
tion with herpes simplex virus, microvesicles promote the viral pathogenesis and
infectivity by using the exosome components and endosome makers like tetraspanin.
130 B. Chidipi et al.

Fig. 8.2 Size and number of microvesicles. (a) Cryo-electron micrograph of extracellular vesicles
secreted by MLP-29 cells (Conde-Vancells et al. 2008). The percentage of vesicles that belonged
to each category and their size distribution within each category are shown in cultured HMC-1
cells (b, c) (Zabeo et al. 2017)

These particles are similar to the endosome vesicles in size making them use the exo-
some cellular pathways for their invasion and evasion (Sharma et al. 2018).
The primary functions of the microvesicles in the viral infection are to modulate
the cellular processes like angiogenesis, cell proliferation, cell invasion, gene regu-
lation, and immune regulation. These can also generate the essential ligands for
receptor cell surfaces to initiate the signaling pathways and release their contents
into the plasma membrane by endocytosis. Their involvement in the viral cycle can
be utilized in hampering the period by developing antibodies that shield the evasion.
Virally infected cells will sometimes secrete microvesicles to remove unwanted cel-
lular proteins from the cytoplasm thereby regulating the signaling complexes and
pathological process (Meckes and Raab-Traub 2011).
8 Macrophages/Microvesicles and Their Task in Viral Diseases 131

8.5 The Communication Between the Cells

Extracellular vesicles facilitate the connection between the cells in the body humoral
environment leading to host and viral interactions in the immune system. These
extracellular vesicles are usually the exosome or microvesicles containing RNA’s,
proteins, and lipids (Birdwell et al. 2018). These involve in the extracellular com-
munication by a unique mechanism in which the cells selectively discharge mem-
brane and elute the internal components. This mechanism is identified using the
exosome secreted by antigen-presenting cells major histocompatibility complex 2
using the immunogold labels (Paolicelli et al. 2018).
Even in the tumor environment, extracellular vesicles (EVs) play a vital role in
the mediation of communication between the tumor cells. EVs contribute to regulat-
ing the bystander effects in which the stressed tumor cells will cause DNA damage
to the neighboring healthy cells (Birdwell et al. 2018). Evidence shows that exo-
somes help in the cross-communication between cells from which they were derived
from, for example, between macrophages, dendrocytes, both T and B lymphocytes,
and natural killer cells in the cancer environment. These exosomes will influence
the functions of the cells by modifying their genotype due to mutations in the cancer
cells. These are playing a vital role in the generation of the vaccines for various
diseases involved with macrovesicle mediation (Raposo et al. 1996).
The significant contribution of these vesicles in their release and function by
biogenesis will be the novel tool for the detection and treatment of the viral dis-
eases. It has been revealed that they contribute to homeostasis in the central nervous
system trauma conditions (Baraldo et al. 2018). During the therapeutic purpose, the
same exosomes play a vital role in targeting and modifying specific tissue cells and
combating the significant barriers in immunological diseases. They also help in the
development of various vaccines against those infectious diseases that mimic the
microvesicles for exploiting the host immune system (Samuel et al. 2017).

8.6 Macrophages in the Immunity Process

Macrophages play a vital role in the generation of the immune response in the
human body (Baraldo et al. 2018). Thereby attempting to study the role of macro-
phages in the immunity process will help us to eliminate the viral pathogens by
either delaying or damaging their spread to healthy cells. Macrophages also contrib-
ute a vital role in increasing the genome load for viral infections like infectious
bronchitis virus as they accumulate in the respiratory tract (Shen and Ren 2018).
During viral infections, a large number of infected cells will undergo a pro-
grammed cell death process called apoptosis during which the macrophages elimi-
nate the cells preventing further necrosis (Shaheen 2018). They also play a supportive
role in the activation of the tissue repair process by activating the inflammatory
cascade by recruiting inflammatory mediators thereby initiating the wound healing
process (Pegtel et al. 2014).
132 B. Chidipi et al.

8.7 Role of Macrophages in Infectious Diseases

In infections like hepatitis, the microvesicles budding from the infected cells will
participate in the viral cycle along with the virus. This was the first time discovered
under the electron microscope when observing the infected liver cells for the herpes
simplex virus virions. In hepatitis infection, microvesicles harbor the virions as part
of the viral cycle, and when these are ingested by the macrophages they are infect-
ing the naïve cells as part of the productive infection. In hepatitis, virus particles are
depending on the microvesicles to promote the disease effectively in the host, which
can be used therapeutically by shielding the microvesicles with antibodies (Huang-­
Doran et al. 2017).
In dengue infection, the virus uses the arthropod vesicles to promote the disease
from the mosquito to humans. These extracellular vesicles contain the viral RNA
and proteins that are infectious. In the treatment of the dengue infection effectively
scientists are using the glycoprotein present in the tetraspanin domain of the
microvesicle as a marker to identify the pathophysiology and blocking the spread of
disease by targeting the microvesicle-mediated transmission by DNA silencing and
gene isolation studies. These changes are confirmed by immunoprecipitation stud-
ies (Vora et al. 2018).
In HIV infection microvesicles play a vital role in the remodeling of the pulmo-
nary vasculature. The actual reason for the cardiac regeneration in the humans is
anonymous for recent times and later discovered to be due to the extracellular vesi-
cles released from the human monocyte-derived macrophages. The main constitu-
ent of these vesicles is miRNA-130a that plays a significant role in the decrease in
the expression of the phosphatase and tensin molecules that are responsible for the
perivascular inflammation in the HIV patients (Sharma et al. 2018).
With respect to entry and departure of virus, four noteworthy stages have been
proposed to portray these procedures: capsid get together and DNA bundling in the
core; essential envelopment and de-envelopment at the atomic envelope; tegumen-
tation and optional envelopment in the cytoplasm; and, at long last, exocytosis of
viral particles at the plasma layer and additionally cell-to-cell transmission at cell
intersections (Lannes et al. 2019). A unique method of viral communication in
human tissues is cell-to-cell spread, that is, the immediate entry of descendant’s
infection from a contaminated cell to a nearby one (Bracq et al. 2018) (Fig. 8.3).
It is broadly acknowledged that this system of dissemination speaks to a resistant
avoidance technique since it shields the infection from insusceptible observation.
Be that as it may, as referenced above, HSV-1 may utilize a few methods of spread
to go from tainted to uninfected cells (You et al. 2018). Numerous viewpoints con-
cerning the procedure of viral spread, for example, the instruments of viral depar-
ture from epithelial cells and spread to neurons and the other way around, are not
seen yet (Bayliss and Piguet 2018).
Elucidating the devices of viral scattering and the ensuing passage into neighbor-
ing cells remains a vital advance to comprehend the viral cycle in the host. In this
unique situation, discharged vesicles have risen as another object of consideration
given their capacity to take part in the intercellular correspondence process amid
8 Macrophages/Microvesicles and Their Task in Viral Diseases 133

Fig. 8.3 Intercellular structures and processes involved in cell-to-cell transmission of HIV-1. (a–
g) Schemes represent the different pathways for HIV-1 cell-to-cell transfer between donor cells (in
green) and target cells (in pink) (Bracq et al. 2018)

viral diseases. Extracellular vehicles (EVs) are a very different gathering of emitted
layer vesicles, which have been separated from most cell types and natural liquids.
Three noteworthy subgroups of EVs have been recognized: apoptotic bodies;
microvesicles (MVs), which get from the shedding of the plasma film; and exo-
somes, which are intraluminal vesicles discharged to the endless extracellular supply
of multivesicular bodies (MVBs) with the plasma layer (Fig. 8.4) (Roth et al. 2019).
Exosome usually is 30–100 nm in the distance across, while MVs have a differ-
ent size running from 100 nm to 1 m in breadth. EVs have been appeared to be
associated with various physiological and neurotic procedures, for example, aggra-
vation and the insusceptible reaction, cell bond, coagulation, squander the board,
tumor movement, and viral spread. Oligodendrocytes (OLs) are the myelin-shaping
cells of the focal sensory system (CNS). The myelin sheath is an electrically pro-
tecting layer that encompasses axons in both the focal and fringe sensory systems,
permitting salutatory conduction of activity potential (Domingues et al. 2018).
All neural cell types discharge EVs, which have a focal job in procedures, for
example, myelination or guideline of synaptic movement and may be associated with
the pathogenesis of a few neurodegenerative maladies or, despite what might be
expected, in neuroprotection. To be sure, OLs discharge exosomes conveying myelin
proteins, for example, proteolipid protein (PLP), the real myelin protein in the CNS;
134 B. Chidipi et al.

Fig. 8.4 Multiple stages of the retroviral life cycle, however, no current antiretroviral drug blocks
the expression of HIV proteins and RNA from integrated viral DNA, or their subsequent sorting
into exosomes and secretion from infected cells (Patters and Kumar 2018)

2=3=-cyclic nucleotide phosphodiesterase (CNPase); myelin essential protein

(MBP); and myelin oligodendrocyte glycoprotein (MOG), just as various chemicals,
for example, glycolytic or oxidative pressure easing compounds (Cinelli et al. 2019).
Past works completed by our gathering have demonstrated that OLs are profoundly
vulnerable to HSV-1 disease. Consequent actions uncovered that oligodendrocyte
antecedent cells (OPCs) and cells of the human oligodendroglia HOG cell line
refined under separation conditions become progressively vulnerable to HSV-1, a
reality that was steady with the later finding that PLP, a myelin protein upregulated
all through separation, is engaged with the viral passage (Ueda et al. 2018).
EVs got from OLs have likewise been associated with such procedures, and there
is expanding proof for the job of EVs in myelination and neuron–glia correspon-
dence. EVs have additionally been engaged with viral contamination as a method
for infections to enter have cells, improve spread, or avoid the host resistant reaction
(Charlton et al. 2018). Two noteworthy elements of EVs amid viral diseases are the
exchange of viral genomes into target cells and the strengthening of contamination
by changing the physiology of target cells. Generation of discharged vesicles by
HSV-1-contaminated cells has been known for quite a while. The first to be found,
8 Macrophages/Microvesicles and Their Task in Viral Diseases 135

named L-particles, are like the virions in appearance and do not have the viral
nucleocapsid and the genome and along these lines are not irresistible (Bello-­
Morales et al. 2018a). In any case, L-particles have been appeared to encourage
HSV-1 disease by conveying viral proteins and the cellular factors required for
infection replication and invulnerable avoidance (Bello-Morales and López-­
Guerrero 2018).
Hence, it has been confirmed that the comparative capacities watched for vhs and
TIF among virions and L-particles recommend that viral connection, combination,
and the arrival of covering proteins are the equivalent for both. Likewise, L-particles
share comparative gathering and departure pathways with virions, recommending
that the covering and glycoproteins are adequate to provoke auxiliary envelopment.
It has been exhibited that useful viral proteins can be exchanged to uninfected
onlooker cells by means of L-particles, a procedure that may show a methodology
for viral invulnerable break. Different particles, the previral DNA replication-­
encompassed particles (PREPs), are morphologically like L-particles; however,
they vary in their relative protein arrangements (Sun et al. 1999). Nonetheless, to
date, there is no proof of virions being bundled inside EVs. Here, we propose a
unique job for MVs in spread of infection. Recent discoveries show out of the blue
that virions might be exchanged from tainted to uninfected cells through MVs. By
methods of transmission electron microscopy (TEM), we identified miniaturized
scale vesicles containing HSV-1 virions. What is more, we found that the no lenient
Chinese hamster ovary (CHO) cell line was powerless to HSV disease directly after
immunization with infection containing MVs recently detached from supernatant of
infected HOG cells. These outcomes recommend that MVs emitted by HOG cells
tainted with HSV-1 may be associated with viral spread and may add to evading
resistant reconnaissance (Bello-Morales et al. 2018b).

Acknowledgments The authors are grateful for the support extended from Adikavi Nannaya
University, Krishna University, and University of South Florida, USA.

Conflict of Interest The authors declare that they have no competing interests.

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T Cells in Viral Infections: The Myriad
Flavours of Antiviral Immunity 9
Achanta Jagadeesh, A. M. V. N. Prathyusha,
Ganugula Mohana Sheela, and Pallaval Veera Bramhachari

Abstract
Viral diseases are a major cause of morbidity and mortality and result in a signifi-
cant public health burden. T lymphocytes first identified in the chordate lineage
and constitute a highly sophisticated branch of adaptive immune system. Apart
from B cells, it is the only cell type that exhibits antigenic specificities; achieved
by gene rearrangement. T cells are unique with respect to diversity of their sub-
sets, which have distinct effector specificities, proliferative abilities, memory
generation, and life span. T cells are impactful in viral infections by virtue of
their capability to combat intracellular pathogens. The effector functions of T
cells are mediated through cytokines/chemokines and by direct cytotoxicity of
infected cells. T cell response can be beneficial or detrimental to host; prognosis
depending on qualitative and quantitative differences in the response. Persistent
viral infections are associated with functionally suboptimal, exhausted T cell
responses, which are unable to clear virus. Specific subsets such as regulatory T
cells (Tregs) dampen antiviral responses; thereby favouring viral persistence.
However, Tregs protect the host from immunopathology by limiting perpetual
inflammation. Certain other subsets such as Th17 cells may contribute to autoim-
mune component of viral infections. The importance of T cells is highlighted by
the fact that modern vaccination and therapeutic approaches focus on modulating
T cell frequencies and effector functions. This chapter emphasises the under-
standing how T cells influence outcomes of viral infections, modern vaccination
and therapeutic strategies with thrust on T cell biology.

Achanta Jagadeesh, A. M. V. N. Prathyusha, G. Mohana Sheela and Pallaval Veera Bramhachari

contributed equally with all other contributors.

A. Jagadeesh
Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul
National University, Seoul, South Korea
A. M. V. N. Prathyusha · G. M. Sheela · P. V. Bramhachari (*)
Department of Biotechnology, Krishna University, Machilipatnam, Andhra Pradesh, India

© Springer Nature Singapore Pte Ltd. 2020 139

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_9
140 A. Jagadeesh et al.

Keywords
Adaptive immune system · T cell responses · Viral infections · Therapeutic
strategies

9.1 Introduction

Viruses are infectious agents consists of nucleic acids coated in a simple protein
casing, infects, replicates in host cells and causes acute, chronic infections.
Mammals established a refined immune system to cope with several viral and bacte-
rial infections (García-Sastre and Biron 2006). Remarkably, adaptive arm of
immune responses is significant in action against virus particles and infected cells.
T Lymphocytes are the key players in adaptive, cell-mediated immune responses
and also in elimination of foreign pathogens by activating various immune responses.
There exist many viruses for which both CD8+ and CD4+ T cells, reported to play
key role in viral control viz. measles virus (Nelson et al. 2017), cytomegalovirus
(CMV) (Wehrens et al. 2016), hepatitis C virus (HCV) (Sheiko et al. 2016) and HIV
(Jones and Walker 2016). Typically, TH1 cells are assumed to be efficient in antiviral
T cell response. Nonetheless, many viruses can inhibit TH1 response by downregu-
lating interferons release from infected cells, which greatly influence outcome of
virus infection (Laidlaw et al. 2016). The humoral immune response involves anti-
bodies specific for virus to block host–virus interactions, neutralize virus and recog-
nise viral antigens on infected cells and activates antibody-dependent cytotoxic
cells (ADCC) or complement-mediated lysis to kill infected cells. However, if these
antibodies are ineffective, viruses are able to infect host cells where adaptive arm
acts to control viral pathogenesis (Rosendahl Huber et al. 2014).
Virus infects host cells if humoral immunity fails, viruses use protein synthesis
and replication machinery of host cells for their replication and synthesis of their
own proteins. Some newly synthesized proteins may degrade into peptide frag-
ments. If these peptides have sufficient binding affinity, to class I MHC molecules
they appear on cell surface of an infected cell as class I MHC–peptide complex.
This complex activates CD8+ T cells and induces infected cell apoptosis by releas-
ing cytotoxic granules and production of TNF-α and IFN-γ. Activation of CD8+ T
cells also occurs in draining lymph nodes, where antigen-presenting cells (APCs)
encounter naïve T cells. Priming of naïve T cells will not only occur through classi-
cal pathway via infection of cell, but also through cross-presentation of viral pep-
tides on MHC class I molecules, taken up from extracellular sources. Priming of T
cells triggers a massive expansion of antigen-specific T cells. Their progeny usually
accumulate in large numbers of armed effector T cells and these normally contribute
to the eradication of viral pathogens.
T cells in chronic viral infections typically exhibit strong impairments in the
production of cytokines (IFN-g, TNF and IL-2) and express high levels of inhibitory
receptors viz. PD-1 (programmed cell death-1) and Lymphocyte-activation gene 3
(Lag-3). These phenotypic changes along with failure of immune system to clear
9 T Cells in Viral Infections: The Myriad Flavours of Antiviral Immunity 141

pathogens in chronic infection exhaust functional T cell response. These primarily

promote terminally differentiated T cells and inhibiting formation of CD8+ T cell
memory. Furthermore, antiviral activity and CD8+ T cell response drastically
increase when signalling through PD-1 is prevented. These observations signify that
T cell immune response has ceased reversibly. However, molecular mechanisms
that conserve terminally differentiated T cells in chronic infections and improve-
ment in T cell response after checkpoint inhibition remain poorly understood of
time, which still mediates certain level of virus control.
The pathogenesis of T cell also depends on processing pathogen components
by Antigen Presentation Cells (APCs), and their presentation via Major
Histocompatibility Complex (MHC). Antigenic diversity of peptides enhances
viral pathogenesis, where diversity of MHC and TCR repertoire in viral patho-
genesis is yet unexplored. The different methods are administered likewise
in vivo and ex vivo to elucidate T cell responses and their mechanism in viral
pathogenesis. Therefore, to explore the diversity of the viral proteins and its
pathogenesis in effector T cell immune reaction and their mechanisms are
important. As a result, there is a potential for designing new therapeutics to
combat the viral pathogenicity. Additionally, viruses also exploit complement
system for cellular entry as well as their spread.

9.2 T Cell Responses in SARS Virus

There are more than 8000 cases of respiratory diseases among which the Severe
acute Respiratory Syndrome (SARS) is caused by novel coronavirus (SARS-CoV),
and contributed to 10% of mortality in 2002–2003. Pro-inflammatory responses
enhance disease progression. The mechanism of immune evasion is mainly charac-
terised by poor antigen presentation by antigen presentating cells (Legge and
Braciale 2003). The antigen presentation is key for activation of T cell and produces
chemokines and cytokines that regulate disease progression (Seder et al. 2008). The
immune evasion of novel coronavirus targets APC and suppresses T cell activation.
Dendritic cell immunisation activates T cells. As a result, the production of IFN-­
gamma, IL-2 and TNF-alfa are released (Zhao et al. 2010). Therefore, the viral titre
gets reduced as immunisation with Dendritic cells (DC) by T cell activation suc-
cessfully suppressed viral pathogenesis. It can be characterised as a potent immuno-
gen to activate immune response.

9.3 T Cell Responses in West Nile Virus

West Nile Virus is a positive sense single-stranded RNA belongs to Flaviviridae and
transmitted by mosquito vectors and originated in the USA in 1999, it causes mos-
quito borne encephalitis. It is asymptomatic in majority of individuals and symptoms
usually are arthralgia, myalgia and cephalea. The minor part of pathogenesis can be
neurologic deficits and neuroinvasive in elderly people. Viral pathogenesis mainly
142 A. Jagadeesh et al.

favours by efferocytosis activator TIM-3, which inhibits CD8+ T cell activation

(Lanteri et al. 2014). The viral RNA plays major role in dampening the CD8+ T cell
activation and it activates CD4+ T cell expresses Th1 and Th17 cytokines that favours
neuroinvasion (James et al. 2016). viral RNA mainly responsible in regulating T cell
pathogenesis and responsible for neuroinvasion. As per study, CD8+ T cell activation
controls viral replication, tissue tropism and infection (Aguilar-Valenzuela et al.
2018). Despite the fact that CD8+ T cell activation reaches peak expansion in the
periphery of west nile virus by 7-day post infection followed by chemokine activa-
tion of CXCR3. As a result, viral clearance activated by membrane-­mediated apop-
tosis (Shrestha and Diamond 2007) was recently evidenced to combat viral
replication, novel recombinant TCR can enhance immune response (Aguilar-
Valenzuela et al. 2018).

9.4 T Cell Responses in Japanese Encephalitis Virus

Japanese encephalitis virus (JEV) is an arthropod borne member belongs to family

Flaviviridae, which is endemic to rural parts of South and Southeast Asia. The virus
mainly infects children and it causes death. The JEV possess a single-stranded,
positive sensed RNA genome and its 10 kb open reading frame (ORF) encodes four
structural proteins, envelope (E), premembrane (prM), core (C) and seven non-­
structural (NS). The JEV viral pathogenesis mainly inhibits infiltration of T cells
due to production of low levels of IFN-gamma and regulates production of IL-2
(Kumar et al. 2004). As a result, JEV is successful in disease progression. The IFN-­
gamma plays important role in the activation of T cells. In recent study, modes of
activation of TCR by various viral epitopes and maintaining levels of IFN-gamma
and increasing infiltration of activated T cells (Turtle et al. 2016).

9.5 T Cell Responses in Acute Dengue Virus

The immune evasion strategy of viruses mainly by producing diversity of viral

proteins and enhances viral pathogenesis. The HLA alleles on CD8+ T cell and
diversity of viral protein presentation are not uniform in viral pathogenic environ-
ment makes the strong presentation of viral pathogenesis (Weiskopf et al. 2013).
As a result, T cell activation is suppressed due to less polymorphism of HLA. The
T cell cross-reactivity plays an important role in disease pathogenesis. Likewise,
disease serotypes fail to provide immune response against later stage secondary
infection of dengue virus and unresponsiveness to T memory cells to secondary
infection, which are produced in primary infection and it also produces Types 1
and 2 cytokines and responsible for other pathologies (Duangchinda et al. 2011).
Therefore, cytotoxic memory T cell enhances viral replication and it is considered
as original antigenic sin (Mongkolsapaya et al. 2003). Therefore inorder to combat
the strategy was applied to activate CD8+ T cell, as a result IFN-gamma enhances
immune response against DENV peptides.
9 T Cells in Viral Infections: The Myriad Flavours of Antiviral Immunity 143

9.6 T Cell Responses in Viral Hemorrhage Fever

The viral hemorrhage fever (VHF) majorly regulates T cell priming, effector T
cell response and T cell activation (Dahlke et al. 2017). Early during VHF, the
antigen-­presenting cells produce high levels of IFN-gamma and TNF-alfa and
IL-6. As a result, activation of T cells increases viremia and overproduction of
IFN-gamma and TNF-alfa this results in over activation of T cells (Perdomo-Celis
and Salvato 2019) This condition is termed as cytokine storm. During VHF the
proportions of regulatory T lymphocytes decreases and therefore, this enhances
disease pathogenicity.

9.7 T Cell Responses in Chronic and Acute Viral Infections

The chronic and viral infections effect a plethora of T cell populations drastically.
The CD4+ T cells and CD8+ T cells are major players in regulating viral pathogen-
esis. The other differentiated T cell called regulatory T cell, which regulates immune
responses and inflammatory responses. During chronic and viral infections, T reg
plays important role in regulating immune responses and various cytokines (Keynan
et al. 2008). As a result, it regulates effector T cells population. In chronic viral
infections, depletion of Treg cells, CD8+ T cell proliferation (Boettler et al. 2005),
IFN-gamma production and increase cytolytic activity are predominant (Haeryfar
et al. 2005). In HIV infection, due to decrease in Treg population, hyper activation
of CD4+ and CD8+ T cells is predominant (Oswald-Richter et al. 2004) As a result,
it favours viral replication.

9.8 T Cell Responses in Human Papilloma Virus (HPV)

Human Papilloma Virus (HPV), a small DNA virus majorly infects birds, reptiles
and mammals, there are 300 viral genotypes that have been discovered till now
(Vande and Klingelhutz 2013). The HPV infects mucosal and/or cutaneous skin and
causes benign or malignant tumours. HPV associates with cervical cancer, oral
squamous carcinoma and Head and Neck cancers (Forman et al. 2012). The HPV
oncoproteins, E6 and E7, majorly regulates the host immune responses and plays
vital role in tumorigenesis (den Boon et al. 2015). The HPV16 viral particle involves
in the host immune dysregulation by epigenetic mechanisms. The viral pathogene-
sis regulates the synthesis of chemokines that required for T cell activation, i.e.
CXCL14 (Cicchini et al. 2016). The CXCL14 synthesis regulated epigenetically by
HPV viral protein, i.e. E7. Likewise, the HPV viral protein interacts with the host
DNMT1 and stimulates the methylase activation (Burgers et al. 2007) and as a
result, CXCL14 is repressed. Therefore, evasion of immune responses against virus,
by inhibiting the T cell activation. The HPV16 E7 suppresses the production of pro-­
inflammatory responses like IL-8, IL18, CCL2, CCL20 (Cho et al. 2001; Guess and
McCance 2005; Huang and McCance 2002; Kleine-Lowinski et al. 2003)
144 A. Jagadeesh et al.

Additionally, HPV16 E7 upregulates the immunosuppressive genes like IDO1 and

it triggers the activation of Treg cells (Mittal et al. 2013). Strikingly, the HPA18 E6
and E7 proteins jointy dysregulate the T cell activation by binding directly to the
proteins involved in non-receptor tyrosine kinase signalling pathway (Li et al.
1999). As result, it downregulates the IFN-alfa and other cytokines like IL-6 and
IL12. The HPV proteins regulate the IFN signalling for its major mechanism in
evading the host T cell responses. The MHC restriction is a typical immune escape
used by the HPV protein E5 by downregulating the MHC-I complex (Ashrafi et al.
2005, 2006). Hence HPV hinders the activation of CD8+ T cell and evasion of host
responses are dysregulated by viral proteins and favours for the tumour
progression.

9.9 T Cell Responses in Hepatitis B Virus

There are more than 350 million people infected with the Hepatitis B virus. The
virus mainly infects liver and causes chronic and acute pathology. The infection
carried commonly by mother to child during birth. The one in all among the viruses,
which is a non-retroviruses that uses the host RNA polymerase for its transcription
and as a result, the closed circular DNA gets transcribed and involved in the host
disease pathogenesis. The adaptive immunopathogenesis plays important role in the
disease progression. The liver residing APC plays an important role in the activation
of naïve CD4 T and CD8 T cells via cross-priming and facilitates the persistence of
the virus (Lan et al. 2016). The downregulation of the TLR-7 and TLR-9 in plasma-
cytoid dendritic cell and as a result, the IFN signalling is inactivated and cytokines
are inhibited (Seeger and Mason 2000). The NK cells are also major cells providing
immune to the HBV by expressing death ligand and inactivating the CD4 T cells
Bertoletti and Ferrari 2016). The CD8 T cells exhaustion is the major episode in the
disease pathology by expressing death receptors such as TIM-3, PD-1 and 2B4,
poor proliferative signal, IL-2 and IFN-gamma (Raziorrouh et al. 2010). The CTL
apoptosis of CTL by upregulation of various apoptotic genes likewise, Bim and
TRAIL-R2. Due to downregulation of T-bet results in exhaustion of the T cells.

9.10 T Cell Responses in Zika Virus

Zika virus is mainly infected by mosquito borne flavivirus and it has unexpected
links with microcephaly and Guillain–Barre syndrome. It is hypothesised as the
infection resultant to the testis damage. The Zika viral infection results in the expres-
sion of TIM-4, which is one of the phagocytic markers and facilitates viral replica-
tion (Osuna et al. 2016; Zhang et al. 2018). The population of lymphocyte subsets
are reduced in the period of infection. The virus getting successfully evading the
host immune responses. The current study in combatting the viral immune evasion
9 T Cells in Viral Infections: The Myriad Flavours of Antiviral Immunity 145

mainly on designing the RNA vaccines and T cell epitope tetramer for activation of
T cells, recombinant vector-based vectors (Zang et al. 2018).

9.11 Future Perspectives and Conclusions

Acquired immunity plays an important role to eliminate the pathogen by activating

APC, CD4 T and CD8 T cells. The APC presentation by MHC and the presented
processed peptide activates the TCR and CD4 and CD8 associated with MHC stabi-
lises the TCR activation. The TCR activation supported by the co-stimulatory mol-
ecules like CD28/B7 activates the T cell proliferation. As a result, the IL-2 and other
cytokines and chemokines are released. The TLR’s are the receptors which activates
the innate immunity and as a result, the activation of chemokines and cytokines are
upregulated and various proliferative signals are activated with the response to the
PAMP’s. The naïve T cells, as aresult differentiates into CD4 and further has differ-
ent subsets like, Th1, Th2, Th17 and T reg. The other CD8 T and memory T cells
are prominent cells in elimination of disease. The virus infection evades the acquired
immunity and immune responses by various mechanisms. The viral antigen manip-
ulates the host immune responses by inhibiting the proliferative signals, expressing
exhausting receptors, MHC restriction and various anti-inflammatory cytokines.
The viral proteins upregulate apoptotic genes and inhibits immune responses of T
cells. Due to high diversity of viral proteins, the HLA polymorphism is restricted,
hence the MHC restriction and MHC cross presentation makes more significant in
the viral persistence. The TLR-7 and TLR-9 are majorly downregulated in the HBV
and provide immune to the viral propagation. The differentiation of the CD4 Th
cells into Regulatory T lymphocytes hinders the immune activation and provide
immune-resistance to the virus. In few viral serotypes, the T memory cells fail to
respond to the secondary infection. In VHF, the overexpression of cytokines like
TNF-alfa, IFN-gamma and IL-6 are responsible for ‘cytokine storm’ and increase
viremia and favour in disease progression. The NK cell-mediated immune evasion
in cancer causing viruses is unique by causing exhaustion of T cell and therefore,
inhibiting immune responses.
There are vast potential targets to design the therapeutic and diagnosing markers
for the virus. Recently, immunotherapy is a novel application for treating various
virus pathogenicity by PD-1/PD-L1, this infers the overcoming the exhaustion of T
cells (Pauken and Wherry 2015). The active T cell induces immune responses
against viral titre and resolving the immune evasion of viruses. The tcf1 can be a
potential biomarker for the chronic viral infections. The acquired immunity is a
potential target to combat against the viral infection. The RNA-based Next-­
generation sequencing play an imperative role in transplanting the γδ T cell antigen
receptors from the human cohort (Utzschneider et al. 2016). Ex vivo application of
IFN-gamma is also a potential therapeutic activation of viral immune responses
(Turtle et al. 2016). Immunisation of acquired immune responses by activating the
DC cells viral peptide and evade the anergic condition (Zhao et al. 2010). The main-
tenance of antiviral peptides from the healthy infected individuals is the potential
strategy for combatting the immune evasion.
146 A. Jagadeesh et al.

Acknowledgments The authors gratefully acknowledge Krishna University, Machilipatnam and

Seoul National University, Seoul, South Korea for the support extended.

Conflict of Interest The authors declare that they have no competing interests.

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Potentiality of Toll-Like Receptors (TLRS)
in Viral Infections 10
A. M. V. N. Prathyusha, Prudhvi Lal Bhukya,
and Pallaval Veera Bramhachari

Abstract
Living organisms exist in nature usually perceptible to infectious agents like
viruses, bacteria, and many other pathogens. Immune system of host organism
typically elicits both nonspecific innate and “specific” adaptive immune
responses against foreign pathogens. Cells of innate immunity expresses a diver-
sity of pattern recognition receptors (PRRs), which were developed during evo-
lutionary process, recognizes conserved structures of distinct pathogens known
as pathogen-associated molecular patterns (PAMPs). There are several classes of
PRRs like Toll-like receptors (TLRS), RLRs, NLRs, and many DNA and RNA
sensors. Among these PRRs, Toll-like receptors plays an important role in elicit-
ing innate immunity, development of B and T cell responses and as well as
pathogen-specific adaptive immune response. Immune responses in any viral
infections are elicited by the recognition viral PAMPs like nucleic acids such as
DNA and RNA, viral capsid proteins by the host PRR. TLRs sense these viral
PAMPs and induce the antiviral response by inducing the immune active chemo-
kines and cytokines. This chapter focuses on the responses of different TLRs
with viral PAMPS, immune responses mediated by TLRS in viral infections,
agonists of TLRs for treatment of viral infections.

Keywords
Viral Infections · PAMP · PRR · TLRs · Innate immune responses

A. M. V. N. Prathyusha · P. Veera Bramhachari (*)

Department of Biotechnology, Krishna University, Machilipatnam,
Andhra Pradesh, India
P. L. Bhukya
National Institute of Virology, Pune, Maharashtra, India

© Springer Nature Singapore Pte Ltd. 2020 149

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_10
150 A. M. V. N. Prathyusha et al.

10.1 Introduction

Innate immune system expresses diverse pattern recognition receptors (PRRs)

developed during the evolutionary process, which recognizes the distinct pathogens
conserved region known as pathogen-associated molecular patterns (PAMPs).
Eventually the cells of innate immune system detects the virus or viral components
like viral nucleic acid (DNA or RNA), carbohydrate moiety and even viral proteins
through germ line encoded receptors equipped on innate immune cells or in intra-
cellular compartments of cells these receptors generally known as PRRs (Thompson
et al. 2011). Several PRRs like Toll-like receptors (TLRs), RIG-I-like receptors
(RIG-1 and MDA5), nucleotide-binding oligomerization domain (NLRs), and
cyclic GMP-AMP synthase (cGAS) involve in regulation of viral infection. Among
these, TLRs are most studied, well characterized, and also plays critical role in
innate responses against complex viral pathogens, viz. herpes simplex virus (HSV),
Herpes simplex encephalitis (HSE), influenza A virus, respiratory syncytial virus
(RSV), coxsackie B virus, yellow fever virus, West Nile virus (WNV), Epstein Barr
virus (EBV), human cytomegalovirus, Lymphocytic choriomeningitis virus
(LCMV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), enterovirus 71 (EV71),
and coxsackievirus A16 (CA16).
Different TLRs recognize some viral components and modulates viral replica-
tion, cytokine production in vitro. For instance, TLR2, and TLR3 were reported to
participate in cellular activation and cytokine production against Flavivirus.
Furthermore, TLRs 2, 3, 4, 7, 8, and 9 are well characterized reported to be involved
in activating inflammatory response against several viruses viz influenza A virus (Le
Goffic et al. 2007), HSV (Sato et al. 2006), coxsackie B virus (Triantafilou et al.
2005), or RSV (Rudd et al. 2005). Multiple TLRs including TLR3, 7, 8, and 9 are
significantly associated with yellow fever virus, moreover, TLR3 signaling was
highlighted during WNV infection merely not for mounting cellular antiviral
response nonetheless for shaping detrimental innate and adaptive immune response
in vivo (Wang et al. 2004).
TLRs generate innate immune signals that orchestrate innate and also adaptive
immune response. TLRs has the ability to employ diverse downstream signaling
molecules furthermore cross talk with other immune pathways is an essential factor
in determining type, magnitude, and duration of inflammatory response. Several
single nucleotide polymorphisms (SNPs) in TLR genes are associated with altered
susceptibility in viral infections and immune response which promotes viral replica-
tion (Medvedev 2017). This chapter emphasizes the role of multiple TLRs and their
agonists in viral infections.

10.2 Toll-Like Receptors

TLRs are membrane sentinels belong to a class of type I transmembrane proteins

helps in detection of microbial infection through conserved molecular pattern of
pathogen proteins derived from DNA and RNA viruses, Gram-positive and -nega-
tive bacteria, fungi, and protozoan (Pasare and Medzhitov 2005; Dowling and
10 Potentiality of Toll-Like Receptors (TLRS) in Viral Infections 151

Dellacasagrande 2016). In human, there are 11 types of TLRs (TLR1–TLR11)

whereas in mouse 12 TLRs (TLR1–TLR9 and TLR11–TLR13) have been identified
(Gay and Gangloff 2007). Each TLR has specific location on innate immune cells
either intra- or extracellular. Subgroup of TLRs like TLR1, TLR2, TLR4, and TLR5
are generally expressed on cells surface and recognizes PAMPS of viral proteins,
extracellular fungal and bacterial cell wall portions. Another subgroup of TLRs like
TLR3, TLR7, TLR8, and TLR9 localized in cellular compartments such as endo-
somes and senses nucleic acids, DNA and RNA (Gay and Gangloff 2007; Takeda
and Akira 2004). Not all the TLRs function independently. For instance, TLR4
involves MD2, however, TLR2 requires TLR1 or TLR6, while ds RNA-mediated
activation of TLR3 is enhanced by CD14 (Lee et al. 2006). Responses of TLR class
proteins in combating viral PAMPs have observed both in vivo and in vitro
studies.

10.3 TLR 1/TLR2 in Viral Infection

TLR1 and TLR2 regulate innate immune response by recognizing PAMPs-like gly-
coproteins and lipoproteins to activate immune cells (Ma et al. 2015). TLR2-­
dependent secretion of pro-inflammatory cytokines and cell surface molecules
which activate promotion of T cells function in non-parenchymal liver cells like
Kupffer cells (Lin et al. 2010). Preiss et al. (2008) reported that stimulation of TLR2
leads to activation of NF-κB and other cytokines like tumor necrosis factor alpha
(TNF-α) and interleukin 8 (IL-8) in MyD88-dependent manner to suppress invading
pathogens in both hepatoma cell lines and human primary hepatocytes. In an in vitro
study carried out by Thompson et al. (2009) reported that when Hepatoma cell lines
stimulated with IlL-1 receptor and TLR2, replication and capsids formation of HBV
is inhibited. Secretions of pro-inflammatory cytokines viz. IL6, IL12p40, and
TNF-α were observed in macrophages in a TLR2-dependent fashion when triggered
with full-length HBV-HBc antigen (Cooper et al. 2005). Zhang et al. (2012) reported
that TLR2 activates antiviral signalling pathways like NFKB, PI3/Akt, and MAPK
producing various pro-inflammatory cytokines in hepatoma cell lines. In a similar
study, it is observed that in HepG2.2.15 cells and primary woodchuck hepatocytes
TLR2-mediated innate immune response that leads to decreased replication and
gene expression of HBV and woodchuck hepatitis virus. Even though TLR2 inhib-
its replication and nucleocapsid formation of HBV while virus has evolved a clever
strategy to counteract antiviral function of TLR2 by using its antigens like HBeAg,
HBsAg, and by whole HBV virions to decrease expression level of TLR2, which
ultimately affects amount of inflammatory cytokines. In many other DNA and RNA
viruses, like EBV, HSV, human cytomegalovirus, HCV, RSV, and LCMV antiviral
action mediated by TLR2 was observed. The antiviral response of TLR2 was
observed in many cell types like glial cells of nervous system in LCMV virus infec-
tion, monocytes in EBV infection, microglial cells in HSV infection and leukocytes
in HSV infection (Zhou et al. 2009; Gaudreault et al. 2007; Aravalli et al. 2008;
Murawski et al. 2009). Transmembrane domain of HIV-1 envelope interacts with
152 A. M. V. N. Prathyusha et al.

transmembrane domain of TLR2 and hinders secretion of TNF-α and MCP-1 in

mouse macrophages.

10.4 TLR3 in Viral Infection

Toll-like receptor 3 (TLR3) is one of the important intracellular receptors present on

intracellular membrane organelles like endosomes, lysosome, and endoplasmic
reticulum (ER). Apart from these organelles other immune cells like natural killer
cells (NK cells), mast cells, dendritic cells (DCs), monocytes, epithelial cells, and
variety of antigen processing cells also express TLR3 on their cell surface (Latz
et al. 2004; Nicodemus and Berek 2010). TLR3 is well known for detection of viral
intracellular double-stranded RNA (ds RNA) and stimulates NF-κB activation
through antiviral TRIF pathway (Karimi-Googheri and Arababadi 2014). When
TLR3 interacts with its ligand ds RNA, the cytoplasmic domain of TLR3 known as
Toll/interleukin 1 receptor (TIR) interacts with TRIF and further activates the down-
stream signalling molecules viz. TRAF6, RIP-1, and TBK1 which finally activates
several antiviral transcription factors like NF-κB, AP-1, and IRF-3 which tran-
scribes the genes of cytokines, chemokines, and several co stimulatory molecules
(Karimi-Googheri and Arababadi 2014; Chang and Toledo-Pereyra 2012).
TLR3-dependent inflammatory responses permit WNV to affect CNS (central
nervous system) prompting reversible breakdown of blood–brain barrier. TLR3
enhances WNV’s ability to replicate in CNS triggering lethal encephalitis (Wang
et al. 2004). TLR3 also initiates inflammatory response against HBV and HCV and
affects chronicity of virus infection, and thus subsequent pathological changes.
Several studies reported the correlation of genetic polymorphisms in TLR3 gene
with susceptibility or resistance to copious infectious and immune diseases. SNPs
in TLR3 gene may associate with changes in protein or gene expression, which
influences function and efficacy of signal transduction. These changes in TLR3
cause an altered immune response. Previous reports, TLR3 polymorphisms
rs1879026, rs3775296, rs3775291, and rs5743305 have been associated with out-
come of HCV and HBV infection, and development of consequential liver cirrhosis
and HCC (hepatocellular carcinoma) primarily in Asian populations (Fischer et al.
2018). TLR3 gene represents a good candidate approach for predicting progression
of HCV and immune control over HBV infections (Al-Anazi et al. 2017).

10.5 TLR4 in Viral Infection

TLR4 is a receptor for sensing various microbial PAMPs such as lipopolysaccha-

rides (LPS) and activates intracellular antimicrobial and antiviral signalling path-
ways. Apart from LPS, it also senses hyaluronan, heat shock protein (hsp60), free
fatty acids, and adjuvant monophosphoyl lipid A (MPLA). Structure of TLR4 con-
sists of three main domains such as extracellular domain that is rich in leucine
repeats, hydrophobic transmembrane domain, and cytoplasmic TIR domains
10 Potentiality of Toll-Like Receptors (TLRS) in Viral Infections 153

(Yamamoto et al. 2004). In comparison with other TLRs, TLR4 has a unique prop-
erty as it combines with two different signalling adaptors molecules like MyD88
and TRIF activates transcription of several pro-inflammatory cytokines like IL6,
IL12, TNFα, and type 1 interferon (Yamamoto et al. 2004; Evans et al. 2003). In
case of MyD88-dependent pathway, TLR4 which is present on cells’ surface dimer-
izes with myeloid differentiation 2 proteins (MD-2), which further results in recruit-
ment of two adaptor proteins known as TIRAP and MyD88. This series of events
results in activation of pro-inflammatory transcription factors NF-κB, AP-1, IRF5,
and production of pro-inflammatory cytokines (Rosadini et al. 2015). TLR4 is
known to internalize into endosomes and recruits TRAM and TRIF adaptor proteins
and activates transcription factor like IRF3 which induces production of type I inter-
feron (Kawai and Akira 2011).

10.6 TLR5 in Viral Infection

TLR5 recognizes flagellin protein as a ligand from invading motile bacterial patho-
gens and mediates immune responses by the production of cytokines. When TLR5
receptor is activated by bacterial flagellin, TLR5 activates NF-κB and further stimu-
lates TNFα production to elicit efficient immunity against invading pathogens
(Srivastava et al. 2013). Increasing numbers of recent studies have demonstrated the
efficacy of flagellin in adjuvant activity and also about bridging innate and adaptive
immune responses via TLR5. Isogawa in 2005 reported that intravenous injection of
TLR5-specific ligand flagellin from Salmonella muenchen in HBV transgenic mice
exhibited complete inhibition of HBV replication in liver non-cytopathically within
24 h in an enhanced α/β interferon dependent way (Isogawa et al. 2005). This study
states that ligand specific for TLR5 can be used as therapeutic targets for inhibiting
the replication of HBV. Zhao et al. developed a new vaccine with the adjuvant activ-
ity of fusion protein Flagellin (FliC) from Salmonella abortus equi and gD protein
from EHV-1 virus. This novel vaccine significantly induced specific antibody
responses and increased IFN-γ and IL-4 levels in host (Zhao et al. 2019).

10.7 TLR7 in Viral Infection

TLR7 plays a significant role in immunopathogenesis of HBV, influenza A virus,

HCV, EV71, and CA16 viral infections. Single-stranded RNA (ssRNA) that is
derived from invading pathogens viz. viruses, bacterium, fungi, and parasites act as
a ligand for TLR7 receptors (Krieg 2007; Hayashi et al. 2012). TLR7 is expressed
on intracellular membrane organelles like endosomes, endoplasmic reticulum (ER),
and lysosome of different immune cells like NK cells (Hackstein et al. 2012;
Bourquin et al. 2009), common dendritic cells (cDC), plasmacytoid dendritic cells
(pDC) (Kader et al. 2013), and cytotoxic T lymphocytes and macrophages (Budimir
et al. 2013; Gantier et al. 2008). Structurally N-terminal part of TLR7 contains
leucine-rich repeats (LRRs), which is located on inner surface of endosomes.
154 A. M. V. N. Prathyusha et al.

Following this LRR domain, there are transmembrane region and cytoplasmic TIR
receptor domain. Interaction of TLR7 with its ligand-like pathogens ssRNAs results
in activation of MyD88-IRF7-dependent innate immune signalling pathways to
elicit cytokine production (Sajadi et al. 2013), promote formation of autophago-
somes which directly combat pathogen invasion (Song et al. 2018). Viruses evolved
several strategies to combat TLR7 pathway-dependent autophagy and enhance their
replication in host cells. EV71 and CA16 virus evade immune pathways by lower-
ing production of INF-1, downstream signaling molecules in TLR7 pathway this
results in reduced autophagosomes production and in turn autophagy (Song et al.
2018). This ultimately leads to successful replication of viruses in host cells.

10.8 TLR8 in Viral Infection

Despite other TLRs in viral infections, TLR8 recognizes the PAMPs expressed by
infectious agents and stimulates innate immune responses for production of cyto-
kines to combat invading pathogens (Sarvestani et al. 2012). TLR8 is present on
endosomes which specifically recognizes GU and G-rich nucleotides in ssRNAs of
pathogens. TLR8 recognizes many viral genomes like HCV and HIV. TLR8 medi-
ates its antiviral action through MyD88-dependent manner (Heil et al. 2004; Zhang
et al. 2016).

10.9 TLR9 in Viral Infection

TLR9 is a receptor that is generally present on extracellular surfaces and on mem-

branes of intracellular organelles like endosomes and ER. TLR9 identifies unmeth-
ylated CpG DNA of invading pathogens like bacteria, viruses, and parasites. TLR9
belongs to type 1 transmembrane proteins and structurally TLR9 consists of LRRs
repeats to its N terminal domain, followed by LRRs domain there are transmem-
brane regions and cytoplasmic TOLL/TIR domains are present. Expression of TLR9
receptor is constrained only to pDCs and B lymphocytes. Once TLR9 binds to its
ligand, activates MyD88, forms complex with IRAK family protein employs signal-
ing cascade that eventually triggers NF-κB and AP-1 transcription factors to elicit
immune responses against invading pathogens (Hemmi et al. 2000; Latz et al. 2004).
Interestingly, few viruses viz. HIV, HCV, EBV, and HPV thought to inhibit TLR9
signaling to escape from immune response. HBV virus might escape TLR9 pathway
either directly through activity of surface antigen (HBsAg), or indirectly through
increased secretion of IL-10 by monocytes. HBV virus directly escapes immune
system by binding HBsAg to C-type lectin receptor BDCA-2 upregulates SOCS-1,
which inhibits TLR9–IRF7–IFN pathway thus reducing the secretion of IFN from
pDC cells. HSV virus modulates NF-κB signaling via TLR pathway to directly
benefit virus replication which simultaneously endows suppression of INF secre-
tion. Nonetheless, omnipresent NF-κB signals trigger transcription of key modules
of innate feedbacks to viral infection such as cytokines, chemokines, adhesion, as
well as antiapoptotic proteins. Remarkably, HSV modulates the expression of
10 Potentiality of Toll-Like Receptors (TLRS) in Viral Infections 155

NF-κB through its numerous gene products. Therefore, HSV impairs both TLR-­
facilitated NF-κB pathway but also inhibits/activates NF-κB signaling through its
own gene proteins in TLR independent manner to ensure viral replication and
immune escape. Studies on HSV discovered that TLR2 and NF-κB-dependent path-
ways are harnessed for viral replication.

10.10 TLR Antagonists in Antiviral Therapy

Association of viral outbreaks with public health concern globally, there is a dire
need for the development of safe and effective innate immune strategies to combat
viral infections. Innate immunity delivers the first line of defense against viruses,
especially TLRs, constitute critical components of innate immune pathways.
Therefore, copious viruses evolved strategies to deploy TLR signaling to escape
defensive responses of host. Agonist therapy might control viral infections during
very early stage as they block virus attachment to cell surface via TLRs or by pre-
venting TLR-mediated immune activation. Agonists are molecules that mimic the
viral pattern recognition molecules and bind to TLRs to elicit both innate and adap-
tive immune response. Furthermore, agonist viral therapy may be useful for prophy-
lactic or therapeutic treatment for viruses. Several agonists were successfully
employed in preclinical and clinical settings against different viruses. Each TLR
responds to diverse agonists, viz. bacterial and fungal components as well as viral
RNA, except for TLR10, whose agonist is unknown (Lee et al. 2006). TLR3, 4, 7,
8, and 9 agonists were used in nonhuman primate models for DENV and hepatitis
B virus (Chihab et al. 2019) and in murine models for HSV types-1 and -2. These
agonists were discovered to reduce viral symptoms and replication. Conversely,
TLR7 and TLR8 agonists were also reported to be used in treating viral infections
along with inflammatory disorders. However, the first identified TLR guanosine
analog Isatoribine has comparable chemical structural features in common with
ribonucleoside ribavirin, component of current standard for HCV care therapy.
TLR7 agonist Isatoribine may signify an important and unique approach for chronic
HCV therapy (Xiang et al. 2007). Interestingly, both TLR7 (Imidazoquinoline) and
TLR8 (R848 (resiquimod) agonists were used to treat HSV-2 induced psoriasis and
genital lesions (Mark et al. 2007; Gotovtseva et al. 2008) and related imiquimod is
used in treatment of genital warts caused by human papillomavirus.
Nonetheless, carboxy-terminal domain and amino-terminal amphipathic helix
are indispensable for antiviral activity against HCV (Helbig et al. 2011) while SAM
domain (Upadhyay et al. 2014) is obligatory for activity against TBEV and carboxy-­
terminal domain for DENV, respectively (Helbig et al. 2013). Notably, Viperin is
another agonist that comprises carboxy-terminal domain, central domain with con-
served CXXCXXC motif typical for radical SAM family of enzymes (SAM domain)
and amino-terminal domain that mimics TLR ligands that acts as antiviral agents
(Vanwalscappel et al. 2018). TLR7/8 agonist R848 is deemed as the most potent
inhibitor and might be for prophylactic or therapeutic treatment of ZIKA. RNA-seq
analysis recognized several genes that were strongly induced by R848 in
156 A. M. V. N. Prathyusha et al.

monocytes. Viperin, an interferon-induced gene was identified to be active against

several viruses, which also has the ability to inhibit ZIKV replication. Notably, the
transduction of viperin lentiviral expression vector to microglial CHME3 cells ren-
dered them resistant to ZIKA, preventing viral replication (synthesis of viral protein
and RNA). CRISPR/Cas9 knockout of viperin in macrophages relieved block to
infection, demonstrate the role of viperin. TLR agonists may be useful for prophy-
lactic or therapeutic treatment for ZIKV (Vanwalscappel et al. 2018).
TLR9 agonists have been used for cancer and chronic infections for their ability
to enhance both innate and adaptive antitumoral/antiviral responses. TLR9 Ligands
agonists PF-3512676 and ISS-1018 have been tested in clinical trials as immune
cancer therapeutics (Aillot et al. 2018). Low-molecular-weight mannogalactofu-
cans (LMMGFs), a potent TLR2 agonist, block the interaction of viral components
with TLR and suppress deleterious effects of TLR2 in response to HSV as seen in
HSE cases (Jahanban-Esfahlan et al. 2019). GS-9688 is a potent and selective small
molecule agonist of human TLR8, currently in Phase 2 trials for treatment of CHB
(Chronic Hepatitis B infection) (Amin et al. 2019).

10.11 Conclusion and Future Perspectives

TLRs represent as primary sensors of innate immune response against viral patho-
gens. Priming of TLR results in the activation of cellular signaling pathways
involved in both innate and adaptive immune responses. TLRs in mount protective
immune system against infection and their cross talk among other PRRs in associa-
tion with pathogen recognition. Triggering of TLRs 7, 8, and 9 prompts secretion of
pro-inflammatory cytokines (IFN-α, IL-12, and TNF-α) that ensues in TH1-like
immune responses, important in antiviral and tumor immunity. TLR activation by
topical administration of TLR7/8/9 agonists represents a powerful treatment modal-
ity for epithelial viral and neoplastic lesions. Currently, imiquimod represents the
only TLR agonist licensed for the treatment of humans (e.g., CA, actinic keratosis,
superficial basal cell carcinoma). Several other agonists of TLR7, 8, and 9 were
shown to be effective therapeutic agents against infections, cancer, and allergic dis-
orders, but are not yet approved for treatment in humans. Agonists of TLRs 4, 7, 8,
and 9 were also used as adjuvants for prophylactic and therapeutic vaccination.

Acknowledgments The authors are grateful to NIV Pune and Krishna University, Machilipatnam
for the support extended.

Conflict of Interest The authors declare that they have no competing interests.
10 Potentiality of Toll-Like Receptors (TLRS) in Viral Infections 157

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Activation of Complement System
During Viral Infections: Prospects 11
and Future Challenges

Prudhvi Lal Bhukya and Pallaval Veera Bramhachari

Abstract
The complement system is homeostatic system evolved to remain constant check
on pathogen but as per the recent knowledge it is involved in many biological
processes including complementing adaptive immunity. It gets activated in most
of the viral infections and leads to neutralization of virus via opsonization of
C3b, aggregation, phagocytosis, membrane attack complex (MAC) mediated
lysis of virus or virus-infected cells. Primary work of complement in viral dis-
eases clears the virus by MAC or by opsonization nonetheless it offers favorable
milieu locally during localized infection. It increases vascular permeability, gen-
erates edema, recruits phagocytes by chemotaxis, mediates release of cytokines
depending on the cell type. This acute inflammation generated due to local acti-
vation of complement in initial stage of infection is crucial. C3a and C5a ana-
phylatoxins modulate adaptive immunity generation via modulating priming of
T cells and enhancing Th1 immunity. In the absence of certain complement com-
ponents and its receptors, some viruses become more pathogenic than in general,
denote complement functions at more than one step in different viruses of their
pathogenesis.

Keywords
Complement components · Complement activation · Viral infection · Pathogenesis

P. L. Bhukya (*)
Hepatitis Division, National Institute of Virology, Pune, Maharashtra, India
P. V. Bramhachari
Department of Biotechnology, Krishna University,
Machilipatnam, Andhra Pradesh, India

© Springer Nature Singapore Pte Ltd. 2020 161

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_11
162 P. L. Bhukya and P. V. Bramhachari

11.1 Introduction

Complement system is the first line of defense system presumed to be evolved in

invertebrates by gene duplication and diversions (Smith et al. 1999). Conventionally
it is considered as an innate immune system as it keeps constant vigilance on patho-
gens and killing of pathogens in body fluids. However, recent research studies high-
lighted its function as a link between innate and adaptive immune systems
(Dunkelberger and Song 2010). It also acts as critical mediator for clearance of
immune complexes and injured cells. Complement system is versatile and works
apart from immune functions like maturation of neuronal synapse, angiogenesis,
tissue repair and regeneration, immune complex clearance, and in lipid metabolism.
This versatile function might be because it coevolved with the immunity and other
cellular pathways (Ricklin et al. 2010).
Indeed complement contributes to pathogen clearance, moreover aiding adap-
tive immunity against viruses, especially C3d fragment linked with antigen
increases antibody response basically by mounting enhanced germinal center
assembly. Complement also augments a good CD4+ and CD8+ T cell response
against viruses through signaling of complement receptors on these cells and
enhanced antigen presentation function by Antigen Presenting cells (APCs) (Kolev
et al. 2014). However, on the other side viruses either cleared by complement or
viruses use complement for invading inside cells through complement receptors
moreover some viruses suppress synthesis of complement or code/acquire host
complement regulatory proteins (Agrawal et al. 2017). This chapter emphasizes on
the protective role of complement during viral infections.

11.2 Complement System

Complement system is a group of soluble and cell surface proteins that starts the
series of activation cascade of proteins against the inflammatory signal. These pro-
teins are present in all vertebrates in inactive form zymogen while activated form is
serine proteases. However, a host cell does not activate complement due to presence
of complement regulatory proteins present on their surface. There are mainly three
major pathways by which complement system gets activated. These are classical
pathway, alternate pathway, lectin pathway; converge at single step of C3 conver-
tase formation, and cleavage of C3 component.
Classical pathway (CP) is antibody dependent since it needs antibody for its
activation, upon binding of IgG or IgM to the antigen C1 complex can bind to anti-
body. C1q multimeric protein possesses globular heads that bind to Fc portion of
antibody while C1r and C1s subsequently get activated and further cleaves C4 and
C2. The activation product C4bC2a forms classical pathway C3 convertase leaving
away C4a and C2b. The alternate pathway (AP) activated by tick over mechanism
of C3, that is, spontaneous hydrolysis of C3 into C3H2O further binds to foreign
surface. Factor B binds to bound C3H2O followed by factor D that cleaves factor B
into Ba and Bb, this generates alternate pathway C3 convertase C3bBb. Properdin
11 Activation of Complement System During Viral Infections: Prospects and Future… 163

Fig. 11.1 The complement system activation during viral infection, classical alternate, and lectin
pathway

binding stabilizes alternate pathway convertase. Alternate pathway loop continues

C3b formation for coating pathogen leading either neutralization or phagocytosis by
phagocytic cells (Fig. 11.1).
The lectin pathway (LP) activated in the presence of carbohydrate moieties
such as mannose, complement proteins mannose-binding lectin and ficolin.
Although there are four types of MASPs (MBL-associated proteins) MASP1, 2,
3, and MAP 19.
Only MASP2 is capable to cleave C4 and C2when it is associated with MBL. C3
convertase is the same for classical and lectin pathway C4bC2a (Fig. 11.1). Research
reports on the role of MASP1, MASP3, and MAP 19 are not clearly documented.
However only MASP1 can cleave C2 for the matter of fact that, this step increases
activation of lectin pathway in presence of pathogen carbohydrate moieties.
The C3 convertase formed by CP, LP, and AP cleaves C3 into C3a and C3b. C3b
generated can bind on pathogen surface for clearance while few C3b molecules bind
to C3 convertase to form C5 convertase (C3bBbC3b) AP or (C4bC2aC3b) CP and
LP. C5 convertase cleaves C5 into C5a and C5b, which binds away from C5 conver-
tase. C3a and C5a generated during complement activation act as anaphylatoxins.
C6, C7, and C8 bind to C5b to form C6C7C8 complex also referred as a nascent
MAC (Membrane Attack Complex), binding of poly C9 completes the MAC. Around
22 C9 molecules bound to C5bC6C7C8 forms pore of approx. 120-A0 size on the
cell membrane of pathogen leads to lysis of pathogen (Dudkina et al. 2016).
164 P. L. Bhukya and P. V. Bramhachari

11.3 Downstream Effects of Complement Activation

Activation of complement increases vascular permeability helps the recruitment of

immune cells, as well as chemotactic cells by secretion of anaphylatoxins during
virus infection in a local milieu. Infected cells and immune cells generate a number
of different cytokines also called a cytokine storm. This acute inflammation gener-
ated due to local activation of complement in the initial stage of infection is
crucial.
Reciprocal interactions exist between complement system and proinflammatory
cytokines (Markiewski and Lambris 2007). Plerthora of studies have reported that,
proinflammatory cytokines increase the expression of complement receptors in
inflammatory conditions (Mäck et al. 2001). In different pathophysiological sce-
narios, the effect of generation of anaphylatoxins does not lead to similar outcome,
for example, in hepatitis B virus infection generation of C5a eventually leads to
fulminant hepatitis condition while in the case of influenza virus infection, C3 is
necessary for recovery from infection, whereas c5a does not seem to have a protec-
tive role in this case (Xu et al. 2014).

11.4  ross Talk Between Complement and Adaptive

C
Immunity

C3b when cleaved into iC3b and C3dg by factor I (Atkinson et al. 2018), C3dg
binding to antigen interacts with cr2 receptor on B cells which is B cell co-receptor
and it has very strong costimulatory effect on B cells (Roozendaal and Carroll 2007)
CR2-mediated signaling helps B cells to survive in germinal center (81) it helps DC
for long retention of antigen thereby it contributes to enhanced B cell memory
(Fischer et al. 1998) C3a and C5a has a regulatory effects on B cells such as C3a
inhibits polyclonal response and C5a promotes migration of B cells to the site of
complement activation (Burg et al. 1995; Fischer and Hugli 1997; Ottonello et al.
1999).
In the absence of C3a-C3aR signaling, DCs fail to induce potent CD4 T cell
response against antigen (Sacks 2010) anaphylatoxins provide a co-stimulatory and
survival signal to T cells (Strainic et al. 2008). Anaphylatoxins’ interaction with
their respective receptors has a very important role which decides the consequence
of APC and T cell interaction, induction of Th1, Th2, Th17, and T reg cells (Strainic
et al. 2013). Especially it has been shown that complement enhances CD4 and CD8
T cell response against virus infection through anaphylatoxin receptors (Kolev et al.
2014). In addition, complement also enhances CD8 T cell immunity in lymphocytic
choriomeningitis virus infection. (Fang et al. 2007). These mechanisms composed
of direct engagement between complement proteins with CRs on T cells, while
indirect regulation via APC engagement, and alteration of cytokine profiles through
CR–TLR cross talk.
11 Activation of Complement System During Viral Infections: Prospects and Future… 165

11.5 Conclusions and Future Perspectives

Complement pathway is reported and accepted to a mediator/connecting link among

adaptive and innate immunity. However, C3d–CR2 role in monitoring Innate immu-
nity is long known, while recent studies have established complex approach that
complement drives T cell responses. To date, it was expected that complement path-
way occurs only extracellular milieu, therefore, it is able for opsonization of viruses
only outside cell. However, it is now clear that major complement activation pro-
teins like C3 and C5 can be cleaved inside cell in a noncanonical way, which sug-
gests viruses opsonization with C3b may occur even inside the cell. Recent studies
elegantly documented that cleavage of C3 and C5 proteins inside T cell, subse-
quently engages respective receptors for their cleaved products in an autocrine fash-
ion control TH1 induction. Therefore, intracellularly generated C3a and C5a
fragments are anticipated to play predominant role in producing effective defensive
response against viruses. However, numerous subversion mechanisms of virus
evade complement-mediated adaptive immunity have been depicted. Conversely,
many evasion strategies of viruses remain undiscovered. This raises the question—
how do T cell tropic viruses manipulate the noncanonical complement activation
inside the cell for their survival? And more importantly, in general, how viruses
regulate intracellular complement activation? Better knowledge on unknown eva-
sion mechanisms devised by viruses would not only add to our knowledge yet pre-
requisite for capitalizing rational vaccine design and therapeutic potential.

Acknowledgments The authors are grateful to NIV, Pune and Krishna University, Machilipatnam
for the support extended.

Conflict of Interest The authors declare that they have no competing interests.

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MicroRNAs and Their Role in Viral
Infection 12
Divya Tiraki

Abstract
MicroRNAs (miRNAs) are small noncoding RNA molecules that act as central
regulators of gene expression. miRNAs have diverse functions in physiology. As
regulatory molecules, miRNAs are involved in the manifestation of various viral
diseases in humans. Viruses can take advantage of host miRNAs to facilitate their
survival and replication while, for host cells, miRNAs can serve as direct antivi-
ral entities. miRNAs modulate viral pathogenesis either by directly binding to
viral genomes or by modulating cellular antiviral responses. Circulating miR-
NAs are increasingly being recognized as an emerging class of disease biomark-
ers. These miRNAs exhibit a consistent expression profile among healthy
individuals. Another level of complexity is added by the miRNAs encoded by the
viral genomes using the host RNAi machinery. These vir-miRNAs help in the
viral infection establishment. In this chapter, we discuss in brief about what are
miRNAs, their functional role, different viral miRNAs, and cellular miRNAs
involved in the pathogenesis of classical viral infections.

Keywords
MicroRNAs · Virus · Replication · Cellular miRNA · Viral miRNA

12.1 What Are miRNAs?

MicroRNAs (miRNAs) are 21–23 nucleotide small noncoding RNA molecules that
post-transcriptionally regulate gene expression by binding to complementary
sequences on target mRNA transcripts (mRNAs) resulting in translational repression

D. Tiraki (*)
Department of Communicable Diseases, Interactive Research School for Health Affairs
(IRSHA), Bharati Vidyapeeth Deemed University, Pune, Maharashtra, India

© Springer Nature Singapore Pte Ltd. 2020 167

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_12
168 D. Tiraki

or target degradation (Girard et al. 2008; Bartel 2009). miRNAs have attracted
immense attention during the last decade as tiny regulators with profound impact on
eukaryotic gene expression (Scaria et al. 2007). During the last decade, miRNA
research has gone from discovering the existence of miRNAs in mammals to explor-
ing their therapeutic applications in numerous diseases.
miRNAs are well conserved in eukaryotic organisms and are vital and evolution-
arily ancient component of genetic regulation (Tanzer and Stadler 2004). miRNAs
can specifically bind to 3′UTRs (Grimson et al. 2007), 5′UTR (Lytle et al. 2007), or
the coding region of mRNA transcripts (Friedman et al. 2008). miRNAs have
diverse functions in physiology; ranging from cell differentiation, proliferation,
apoptosis to the endocrine system, hematopoiesis, limb morphogenesis, fat metabo-
lism, etc. (Girard et al. 2008). They display different expression profiles from tissue
to tissue, reflecting diversity in cellular phenotypes. A direct role of miRNAs in
immune response also has been demonstrated (Gantier et al. 2007). miRNAs are
generally tissue specific and cell associated, however, they have also been found in
the extracellular milieu (circulating miRNAs). Circulating miRNAs in serum and
plasma are increasingly being recognized as an emerging class of potentially useful
disease biomarkers readily detectable in the blood (Wang et al. 2009; Zhang et al.
2010a, b).

12.2 Biogenesis of miRNAs in Animals

The biogenesis of miRNA is a complex multistep process, which begins in the

nucleus, goes through several post-transcriptional modifications, and terminates in
the cytoplasm. A majority of miRNAs are derived from long double-stranded RNAs
(dsRNAs), which are sequentially cleaved into shorter intermediates by specialized
ribonuclease III (RNase III) enzymes that partner with dsRNA-binding proteins
(Axtell et al. 2011; Kim et al. 2009). Mature miRNAs are found as one arm of a
hairpin structure within RNA polymerase II (Pol II) transcripts called pri-miRNAs,
which are characteristically longer, capped, and polyadenylated (Cai et al. 2004;
Lee et al. 2004).
RNA Pol II generates long pri-miRNAs either from introns of protein-coding
genes or independent genomic transcription units (Cai et al. 2004; Corcoran et al.
2009; Lee et al. 2004). Drosha, a nuclear RNase III enzyme, together with its
dsRNA-binding domain (dsRBD) partner DGCR8 further processes the pri-­miRNAs
into pre-miRNAs (~70 nt stem–loop structures) (Denli et al. 2004; Han et al. 2004;
Lee et al. 2002, 2003). A RanGTP-dependent Exportin5 transporter then exports
this pre-miRNA hairpin to the cytoplasm (Bohnsack et al. 2004; Yi et al. 2003; Zeng
and Cullen 2004). In the cytoplasm, second enzymatic processing is brought about
by Dicer (RNase III enzyme) in association with its dsRBD partner TRBP, liberat-
ing a ~22 nt dsRNA duplex (miRNA–miRNA∗) from the pre-miRNA (Bernstein
et al. 2001; Hutvágner et al. 2001; Ketting et al. 2001).
These duplexes contain the mature miRNA bound to its complement, miRNA∗.
The dsRNA duplex is subsequently loaded onto an Argonaute (Ago 1–4) protein,
12 MicroRNAs and Their Role in Viral Infection 169

forming an effector complex known as the RNA-induced silencing complex (RISC)

(Hammond et al. 2000; Mourelatos et al. 2002). The orientation of loading onto the
RISC is determined by the thermodynamic stability present at the 5′ end of the
mature miRNA (Khvorova et al. 2003). The strand which is relatively more unstable
base pairs at the 5′end. This helps in the preferential usage of the mature miRNA
strand whereas the passenger strand miRNA∗, which remains unused is degraded. In
the effector complex, miRNAs bind the host mRNAs through the sequence comple-
mentarity at the 3′ UTR.

12.3 Interplay of miRNAs with Their Targets

A single miRNA may control the expression of more than one target mRNAs. The
specificity required for the binding interactions is attributed to the 5′ region of
miRNA. The target interactions are restricted to a region confined to ~6–8 nt
sequence at the 5′ end of the miRNA, known as the “seed sequence” (Wee et al.
2012). This seed sequence is highly conserved among different species and change
in the sequence alters the target binding (Ameres et al. 2007). The loops formed in
the miRNA:mRNA duplexes may also have a profound effect on the efficiency of
miRNA-mediated gene regulation (Ye et al. 2008). The properties associated with
the target:miRNA interactions such as sequence, structure-associated free energy,
and evolutionary conservation are used in the current target prediction algorithms
(Doench and Sharp 2004; Martin et al. 2007).

12.4 Effector Mechanisms of miRNAs

miRNAs direct the RISC to downregulate gene expression by one of the two post-
transcriptional mechanisms; mRNA cleavage or translational repression. The degree
of miRNA: mRNA complementarity determines which mechanism is followed.
Higher degree of complementarity directs toward the Ago-catalyzed degradation of
target miRNA sequences through the mRNA cleavage mechanism. Conversely, a
central mismatch bypasses degradation and facilitates the translational repression
process.

12.5 Types of miRNAs

miRNAs are often tissue specific and cell-associated, however, they are also found
in the extracellular milieu. Hence, based on their location in the body, they can be
classified as (a) cell-associated miRNAs and (b) circulating miRNAs (cell-free
miRNAs). Based on their origin, miRNAs are also classified as (a) host-encoded
miRNAs and (b) virus-encoded miRNAs.
170 D. Tiraki

12.5.1 Cell-Associated miRNAs

All miRNAs originate in particular cells/tissues and play a vital role in the regula-
tion of cellular processes there. For example, miR-122, a liver-specific miRNA is
abundantly present in the liver and constitutes ~70% of total miRNA pool. Cell-­
associated miRNAs play a role in the pathogenesis of diseases, which is attributed
to their dysregulation in the tissue of their origin. Thus, these miRNAs can act as
biomarkers or indicators of diseases. For example, miR-122 has been reported as an
indicator of liver disease progression (Girard et al. 2008), miR-208 acts as a predic-
tive marker for myocardial injury (Ji et al. 2009).

12.5.2 Circulating miRNAs

Cell-free miRNAs that are found circulating in the extracellular milieu are called as
circulating miRNAs. In 2008, Lawrie et al. first reported the presence of miRNAs in
serum (Lawrie et al. 2008). Cell-free miRNAs are not only detected in plasma and
serum, but also in body fluids such as urine, tears, breast milk, amniotic fluid, pleu-
ral fluid, cerebrospinal fluid, and saliva (Chen et al. 2008; Weber et al. 2010). Due
to their remarkable stability and ease of access, circulating miRNAs serve as an
important tool as a biomarker of various diseases (Mitchell et al. 2008). These cell-­
free miRNAs act as mediators for cell–cell communication and immune regulation
(Valadi et al. 2007). Altered levels of circulating miRNAs have been reported in
different pathological conditions (Laterza et al. 2009; Mitchell et al. 2008; Mo et al.
2012; Nogales-Gadea et al. 2014; Zhang et al. 2010a, b). The origin of miRNAs is
diverse and microRNAs are released in circulation by blood cells (Hunter et al.
2008); organs of the body (Laterza et al. 2009) and tumors (Mo et al. 2012).
Circulating miRNAs are widely used as biomarkers in a variety of pathological
conditions and few of them have been summarized in Table 12.1. As biomarkers,
these miRNAs help in various ways like tracking the progression of the disease, as
a prognostic tool, and as a diagnostic tool.

Table 12.1 Circulating miRNAs as biomarkers

miRNA Pathological condition References
miR-122 and Inflammatory biomarkers for (Wang et al. 2009; Bala et al. 2012)
miR-155 different liver injuries
miR-25 and Lung cancer (Chen et al. 2008)
miR-223
miR-184 Squamous cell carcinoma (Wong et al. 2008)
miR-92a Leukemia (Tanaka et al. 2009)
miR-20 and Predictive biomarkers in HCV liver (Shrivastava et al. 2013)
miR-92a disease
miR-21 B lymphoma (Lawrie et al. 2008)
miR-208 Myocardial injury (Ji et al. 2009)
12 MicroRNAs and Their Role in Viral Infection 171

12.5.3 Host-Encoded miRNAs

This class of miRNAs includes all the miRNAs that originate in the mammalian
system through the canonical miRNA biogenesis pathways. These include both the
cell-associated and circulating miRNAs discussed above.

12.5.4 Virus-Encoded miRNAs

Several studies have reported the existence of virus-encoded miRNAs (vmiRNAs)

in DNA and RNA viruses. The biogenesis of these miRNAs is mainly dependent on
host proteins and enzymes. Most of the viruses generate viral pri-miRNAs using
host RNA polII, which are further processed to pre-miRNAs by host microproces-
sor complex. Viral miRNAs sharing homology with host miRNAs are capable of
cheating host cells and directly takeover the regulatory pathways of host miRNAs.
The host–virus interactions directed by viral miRNAs have been studied extensively
in DNA viruses, like Human cytomegalovirus (HCMV) (Grey et al. 2007), Herpes
simplex virus-1 (HSV-1) (Flores et al. 2013; Umbach et al. 2008). HCMV-encoded
miR-UL112-1 targets the 3′UTR of MICB, vital for NK cell-mediated destruction
of virus infected cells. HSV-1-encoded miR-H2-3p and miR-H3 target ICP0 and
ICP34.5 genes, thereby maintaining the latency. The examples of RNA viruses that
encode miRNAs are West Nile virus (WNV): KUN-miR-1 (Hussain et al. 2012),
Dengue virus (DENV): DENV-vsRNA-5 (Hussain and Asgari 2014), enterovirus
71: vsRNA1 (Weng et al. 2014) and Hepatitis A virus (HAV): hav-miR-1-5p, hav-­
miR-­2-5p, and hav-miR-N1-3p (Shi et al. 2014a, b).

12.6 miRNAs in Viral Infections

A growing body of evidence suggests a significant role of cellular miRNA-mediated

RNAi in intricate networks of host–virus interactions. Lecellier et al. reported the
first antiviral miRNA in human cells capable of restricting accumulation of reovirus
primate foamy virus type 1 (PFV-1) (Lecellier et al. 2005). Cellular as well as viral
miRNAs have been reported to be involved in the pathogenesis of viruses such as
hepatitis viruses (Hepatitis A virus (HAV), Hepatitis B virus (HBV), Hepatitis C
virus (HCV), Hepatitis E virus (HEV)), West Nile Virus (WNV), Measles virus
(MeV), Influenza A virus (IAV), Human Immunodeficiency Virus-1 (HIV-1),
Dengue (DENV), and Herpes Simples virus-1 (HSV-1) (Bogerd et al. 2014).
Multiple lines of evidence have confirmed the ability of miRNAs to modulate
antiviral activity through two mechanisms:

1. miRNAs directly exert an antiviral function by interacting with the genome.

Computational and experimental approaches predict UTRs, particularly 3′UTR
to be a more common miRNA target. However, few reports have shown that
miRNAs can bind to 5′UTR as well. Few examples for virus–miRNA i­ nteractions
172 D. Tiraki

are 5 IFN-inducible miRNAs, miR-196,-296, -351, -431, and -448 target HCV
genome and inhibit replication (Pedersen et al. 2007), miR-23 inhibits PRRSV
replication by targeting the PRRSV RNAs (Zhang et al. 2014a, b), let 7c inhibits
H1N1 by targeting the 3′UTR of viral M1 gene (Ma et al. 2012).
2. miRNAs modulate host factors to provide a less conducive environment for virus
replication. Examples are miR-146 that is involved in the pathogenesis of
Dengue virus (Wu et al. 2013), Chikungunya (Selvamani et al. 2014), and VSV
(Hou et al. 2009), miR-23 that inhibits PRRSV replication by upregulating type
1 IFNs (Zhang et al. 2014a, b), miR-30e∗, which suppresses DENV replication
through NF-κB-dependent IFN production (Zhu et al. 2014a), miR-21 attenuates
human cytomegalovirus (HCMV) replication by targeting Cdc25a, a cell cycle
regulator (Fu et al. 2014), miR-23a facilitates HSV-1 replication by suppressing
IFN regulatory factor 1 (IRF1) (Ru et al. 2014), miR-15b modulates HBV repli-
cation by targeting HNF1α (Dai et al. 2014). Qian et al. have reported the role of
miR-122 in Borna virus-induced disease. miR-122 exerted a direct antiviral
function by translation and replication inhibition and also by indirectly acting
through IFN to increase the host immunity (Qian et al. 2010).

12.6.1 Regulation of Viral Infection by Viral miRNAs

Viral miRNAs have been reported in Human Immunodeficiency Syndrome (HIV),

Polyomaviruses, Herpesviruses, and Adenoviruses. Over 90% of these miRNAs are
found to be conserved between related viral species. Some miRNAs make use of the
cellular miRNA pathway to regulate the viral gene expression and few others also
have cellular targets.

12.6.1.1 HIV miRNAs

Owing to the presence of secondary structures in the viral genome, there is a high
chance that HIV-1 may encode miRNAs. Omoto et al. showed that HIV-1 encodes
miR-N367 in the Nef/LTR overlapping region, which targets the negative response
element of the LTR U3 region and inhibits the LTR’s promoter activity (Omoto
et al. 2004). Another miRNA, miR-H1 downregulates the expression of apoptosis
antagonizing transcription factor (AATF) (Kaul et al. 2009). Next, Ouellet et al.
reported miR-TAR-5p/3p in HIV-1 infected cell lines to be involved in the asym-
metrical processing of the HIV-1 TAR element by Dicer (Ouellet et al. 2008). They
also prevented apoptosis of infected cells thereby promoting survival and replica-
tion by targeting host genes nucleophosmin 1 (NPM), caspase 8 (CASP8), Ikaros
family Zinc Finger 1 (IKZF1), and Ikaros family Zinc Finger 3 (Aiolos). Further,
miR-H3 targeting TATA box in the viral 5′ LTR promoter was reported (Zhang et al.
2014a, b).

12.6.1.2 Polyomavirus miRNAs

These viruses are capable of establishing persistent infections in humans and
transforming the infected cells. SV40, a monkey polyomavirus encodes miRNAs
12 MicroRNAs and Their Role in Viral Infection 173

that regulate viral pathogenesis. SV40 encodes a single functional pre-miRNA

which is antisense to the SV40 early mRNA. It is involved in evasion of immune
responses by cleaving the large T-antigen mRNA (Sullivan et al. 2009). Several
similar miRNAs have been reported in other polyomaviruses like BK polyomavi-
rus (BKPyV), JC polyomavirus (JCPyV), simian agent 12 (SA12), Murine poly-
omavirus (MPyV), and Markel cell polyomavirus (MCPyV) (Seo et al. 2008,
2009; Sullivan et al. 2009).

12.6.1.3 Herpesvirus miRNAs

This is a DNA virus causing persistent infection. Few of these herpesviruses and
their encoded miRNAs are discussed in brief in below sections.

12.6.1.3.1 Epstein-Barr Virus (EBV)

It encodes five nonhomologous miRNAs as identified in the B cell line latently
infected with EBV (Pfeffer et al. 2004). These are located in two major regions:
BHFR1 (developmental stage specific) and BART (tissue specific) genes. These
may target chemokines, cytokines, apoptotic, and cell growth control genes.

12.6.1.3.2 Kaposi’s Sarcoma-Associated Herpesvirus (KSHV)

A cluster of 11 Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) miRNAs have
been identified using RACE-like cloning from a single 4.5-kb region of the genome
(Cai et al. 2006; Pfeffer et al. 2005; Samols et al. 2007). Out of these 11, miR-
K12-­10 codes for Kaposi’s family of proteins, which affect cell growth, enhance
cytokine production in infected cells. Computational predictions suggest that these
miRNA might play a pivotal role in regulating viral pathogenesis and replication.

12.6.1.3.3 Human Cytomegalovirus

Human cytomegalovirus (HCMV) encodes for nine miRNAs, which are dispersed
throughout the viral genome. Of these, three are from complementary strands of
known ORFs, five in intergenic regions and one within intron (Pfeffer et al. 2005).
These miRNAs are associated with the autoregulation of virus and are involved in
the latent stage of the life cycle.

12.6.1.4 Adenovirus miRNAs

Adenovirus (ADV), a DNA virus encodes a 160-nt noncoding RNA, VA1pre-­
miRNA during the late lytic replication. This miRNA could inhibit the RNAi
expression in vivo by inhibiting the nuclear transport of shRNA through its compe-
tition with exportin 5 (Cullen 2006).

12.6.1.5 Hepatitis A
Using computational analysis and experimental validation, Shi et al. reported the
production of HAV-encoded miRNA on the antigenome, hav-miR-N1-3p, in
KMB17 and HEK 293 T cells (Shi et al. 2014a). Parallelly in the same year, the
same group reported identification of two more HAV-encoded miRNAs, hav-miR-­
1-5p and hav-miR-2-5p using deep sequencing and in silico approaches in KMB17
174 D. Tiraki

cells (Shi et al. 2014b). Further in 2016, once again Shi et al. reported the mecha-
nism of these HAV vmiRNAs wherein they proposed that these vmiRNAs serve as
a self-mediated feedback regulator during HAV infection by attenuating the accu-
mulation of viral genomic RNAs (Shi et al. 2016).

12.6.2 Cellular miRNAs in Viral Infection

Several cellular miRNAs are known to regulate viral pathogenesis by altering the
host mRNA/gene expression. Several other cellular miRNAs involved in the host
immune system are modulated by the viruses thereby affecting the course of infec-
tion. In the section below, we shed light upon the effect of viruses on cellular miR-
NAs and cellular miRNAs that regulate the viral life cycle in several human viral
infections.

12.6.2.1 Hepatitis B
HBV infection can cause acute or chronic hepatitis B, liver cirrhosis, and hepatocel-
lular carcinoma (Kiyosawa et al. 1990). In an attempt to identify host miRNAs
involved in HBV replication, both miRNAs inhibiting and promoting HBV replica-
tion have been identified.

12.6.2.1.1 miRNAs Inhibiting HBV Replication

Wang et al. addressed the effect of miR-122 on HBV and found that miR-122 mim-
ics inhibit HBV replication whereas anti-miR-122 increased HBV replication in
hepatoma cells (Wang et al. 2012). Several other studies showed that HBV infection
reduced miR-122 levels and viral load inversely correlated with the miR-122 levels
in HBV-infected patients (Chen et al. 2011; Fan et al. 2011; Qiu et al. 2010; Wang
et al. 2012). Furthermore, it was also shown that HBx could bind PPARγ and inhibit
miR-122 transcription (Song et al. 2013b). miR-122 was involved in regulating
HBx protein. miR-122 induced downregulation of SOCS3 thereby enhancing IFN-­
mediated inhibition of HBV (Gao et al. 2015). Pederson et al. showed that upregu-
lated IFNs result in lowering of miR-122 both in vitro and in vivo (Pedersen et al.
2007). Wang et al. have reported a correlation of circulating miR-122 levels with the
prognosis of CHB related liver failure (Wang et al. 2016).
Zhang et al. observed inhibition of HBV replication by miR-199a-3p and miR-­
210 by binding to S protein and preS1 coding regions respectively (Zhang et al.
2010a, b). Similarly, miR-125a-5p inhibited translation of S transcript (Potenza
et al. 2011). Few additional miRNAs, let-7, miR-196b, -205, -345, -433, and -511
target HBV genome (Wu et al. 2011). miR-99a targets IGF-1R and induces cell
cycle arrest (Li et al. 2011). miR-141 inhibits HBV replication by downregulating
PPARα required for transcription of HBV genome (Hu et al. 2012). Downregulation
of miR-22, a tumor suppressor, was seen in HBV-related HCC (Shi and Xu 2013).
Kohno et al. observed suppression of HBV replication by overexpression of mi-1231
without affecting the interferon-stimulated genes (ISGs) (Kohno et al. 2014).
Recently, Wang et al. showed that HBV expression dramatically downregulates
12 MicroRNAs and Their Role in Viral Infection 175

miR-101 whereas overexpression of miR-101 suppresses HBV replication by tar-

geting FOXO1 (Wang and Tian 2017).

12.6.2.1.2 miRNAs Promoting HBV Replication

miR-1 upregulates farnesoid X receptor α (FXRα), a nuclear receptor required in
HBV transcription (Zhang et al. 2011). Serum miR-122 levels correlated well with
ALT levels as well as HBV and HBsAg (Bala et al. 2012). Jin et al. have shown
downregulation of HBX1P, an HBV inhibitor, by miR-501 (Jin et al. 2013). Few
other studies have shown upregulation of miR-99a and -125 in HBV patients
(Akamatsu et al. 2015; Giray et al. 2014; Hayes et al. 2012). In 2016, one more
study reported enhanced HBV replication by miR-449a, an epigenetically regulated
miRNA by targeting CREBP5 (Zhang et al. 2016). Lin et al. showed that miR-99
family promoted autophagy through mTOR/ULK1 signaling which in turn enhanced
HBV replication (Lin et al. 2017). Another study by Qiu et al. reported that miRNA
let-7a promotes HBV replication in HCC tissues (Qiu et al. 2017). And previously,
Deng et al. demonstrated sequestration of let-7a by HBV (Deng et al. 2016).
Recently, Deng et al. have reported that miR-125b-5p could target the LIN28B/let-7
axis and stimulate HBV replication (Deng et al. 2017b). This is the first evidence for
posttranscriptional miRNA regulation.

12.6.2.2 Hepatitis C
HCV infection disrupts multiple pathways regulated by miRNAs, like antigen pro-
cessing, cell cycle, immune response, proteasome, and lipid metabolism (Ura et al.
2009). miRNAs act either by controlling target gene expression or by directly tar-
geting the HCV genome (Kerr et al. 2011; Pedersen et al. 2007; Peng et al. 2009).

12.6.2.2.1 miRNAs Promoting HCV Replication

Jopling and colleagues, for first the time, reported a positive role of miR-122 in
HCV infection (Jopling et al. 2005). This study demonstrated a reduction of HCV
RNA levels by sequestration of miR-122 in subgenomic replicon (SGR) cells. It has
been observed that miR-122 expression facilitates an efficient replication in HepG2
(Narbus et al. 2011), as well as many non-hepatic cells upon infection with HCV
(Chang et al. 2008; Fukuhara et al. 2012). miR-122 enhances HCV replication by
binding directly to two closely spaced target sites in the 5′UTR of the viral genome,
resulting in increased accumulation and expression of the viral RNA (Jangra et al.
2010; Jopling et al. 2008; Machlin et al. 2011; Shimakami et al. 2012; Pang et al.
2012). These target sites are conserved across HCV genotypes and hence are a
potential therapeutic candidate.
The beneficial role of miR-122 seen in vitro does not translate into a correlation
with HCV viral load in patients. This comment is supported by the finding that
nonresponders to standard IFN-cased therapy have low miR-122 pre-treatment lev-
els (Kamo et al. 2014; Sarasin-Filipowicz et al. 2009; Su et al. 2013). In fact pre-­
treatment miR-122 levels might serve to be biomarker for predicting the therapeutic
outcome. Circulating miR-122 serves as a biomarker to predict therapeutic efficacy
in HCV-infected patients (Jiao et al. 2017). Koberle et al. showed that IFN therapy
176 D. Tiraki

did not lower miR-122 in HCV-infected patients (Köberle et al. 2013). Hepatic
miR-122 expression levels were significantly increased in HCV-3-infected patients
(Oliveira et al. 2016). Recently, van der Ree et al. demonstrated miravirsen dosing
decreased miR-­122 levels in CHC patients (van der Ree et al. 2016). Insufficient
clinical data is available and hence more studies are required to understand the regu-
latory role of miR-122 in various states of HCV infection. Also, the results for the
clinical trials for miravirsen are awaited.
miRNAs like miR-141, -192, -491, and -215 are reported to facilitate HCV rep-
lication (Banaudha et al. 2011; Ishida et al. 2011). miR-199a-5p facilitated HCV
replication by regulating pro-survival pathways through PI3K/Akt, Ras/ERK, and
Wnt/β-catenin (Wang et al. 2015). Recently, Bandiera et al. reported facilitation of
HCV infection by upregulating miR-146a-5p in hepatocytes (Bandiera et al. 2016).

12.6.2.2.2 miRNAs Inhibiting HCV Replication

Several studies have identified miRNAs with anti-HCV activity, miR-­
199, -196, -29, -130, -27, and let-7b (Bandyopadhyay et al. 2011; Bhanja Chowdhury
et al. 2012; Cheng et al. 2012; Hou et al. 2010; Murakami et al. 2009). Pederson
et al. have reported induction of antiviral miRNAs like miR-196, -296, -431, -351
and -441 by IFNβ (Pedersen et al. 2007). Mukherjee et al. have recently demon-
strated that miR-181c binds to E1 and NS5A regions of HCV genome, enhanced
HOXA1 expression (required for normal development and differentiation of mam-
malian tissue) (Mukherjee et al. 2014). Further, they showed that overexpression of
miR-181c inhibited viral replication indicating it to be a therapeutic target. Recently,
Chou et al. demonstrated that let-7g inhibits HCV replication by cooperating with
IFN/RBV through p38/AP-1 signaling (Chou et al. 2016).

12.6.2.3 Hepatitis A
A few studies have reported the involvement of cellular miRNAs in HAV infec-
tion. Profiling and characterization of cellular miRNAs were carried out in HAV
infected human fibroblasts (Shi et al. 2016). The alteration of miRNA profile was
suggested to be related to the establishment of persistent HAV infection (Shi et al.
2016). In another study, Weseslindtner et al. have shown upregulation of miR-
106a and -197 in patients with acute viral hepatitis (caused by HAV, HBV, HCV,
and HEV). This report, however, does not differentiate between these etiologies
(Weseslindtner et al. 2016).

12.6.2.4 Hepatitis E
Cheng et al. showed downregulation of miR-221 and -222 in swine HEV-4 ORF3
expressing HEK 293 cells by regulating the p27kip1 expression (Cheng et al. 2013).
Another study by Trehanpati et al. reported for the first time, the role of miRNA
signatures in predicting ALF in HE during pregnancy. They demonstrated that the
interaction of distinct miRNAs in particular immune pathways was responsible for
the diverse outcomes of HE infection including inflammatory responses, liver fail-
ure, or death (Trehanpati et al. 2017). As mentioned in the earlier section,
12 MicroRNAs and Their Role in Viral Infection 177

Weseslindtner et al. have reported the upregulation of miR-106a, -122, and -197 in
patients with acute viral hepatitis (caused by HAV, HBV, HCV, and HEV) with no
differentiation in the different etiologies (Weseslindtner et al. 2016). Recently, posi-
tive regulation of Hepatitis E virus replication by interacting with the RdRp region
of the viral genome has been reported by Haldipur et al. (2018).

12.6.2.5 Influenza A Virus

Several cellular miRNAs are involved in regulating the Influenza A virus (IAV) life
cycle. These miRNAs act either as diagnosis markers or by targeting IAV viral RNA
or by controlling the IAV inflammatory responses.

12.6.2.5.1 microRNAs as Diagnostic Markers

PBMCs from critically ill patients with swine-origin pandemic H1N1 had increased
levels of miR-148 and decreased levels of miR-29a and -31 (Song et al. 2013a).
Higher levels of miR-34c-3p and lower levels of miR-29a-3p, -181a-5p, and -30c-­
5p were reported in throat swabs of H1N1 patients (Peng et al. 2016). Sera showed
significantly high levels of miR-150 in critically ill patients as compared to healthy
controls and those with mild disease (Moran et al. 2015). miRNA microarray profil-
ing of PBMCs from H1N1/H3N2-infected patients revealed dysregulation of 14
miRNAs (increased miR-229-5p, -335, -1260, and -664 levels and decreased miR-­
26a, -18a, 185, -30a, -34b, -628-3p, -576-3p, -665, -765, and -1285 levels). H&N9-­
infected sera showed upregulation of miR-17, -20a, -106a, and -376c (Zhu et al.
2014b).

12.6.2.5.2 microRNAs Targeting IAV RNA

PB1 mRNA of (1) H1N1 is destabilized by miR-491, -323, and -654 (Song et al.
2010); (2) H5N1 is targeted by miR-485 (Ingle et al. 2015); and (3) H3N2 is tar-
geted by miR-3145 (Khongnomnan et al. 2015). Next, Ma et al. showed that let 7c
precursor binds to 3′UTR of M1 mRNA and inhibits viral replication (Ma et al.
2012). miR-33a indirectly diminishes replication by binding to 3′UTR of Archain
(ARCN1) thereby suppressing NP and M1 expression (Hu et al. 2016). Another
miRNA, miR-21 inhibits the virus by targeting NP, PB1, PB2, NA, and HA of H1N1
(Waring et al. 2018).

12.6.2.5.3 microRNAs Controlling IAV Inflammatory Response

Chen et al. have reported that increased expression of IRF5 gene correlated with
reduced levels of miR-302a in PBMCs and throat swabs of IAV patients as com-
pared to healthy individuals (Chen et al. 2017). miR-146a directly targets TRAF6 in
H3N2-infected human nasal epithelial cells (Deng et al. 2017a). miR-4776 binds to
3′UTR of NFkB and leads to its reduction thereby inhibiting viral replication
(Othumpangat et al. 2017). H3N2 infection decreases miR-302c expression which
targets NFkB inducing kinase (NIK) (Gui et al. 2015). miR-132, -46a, and -1275
reduced IRAK and MAPK3 expression in H1N1/H3N2-infected cells (Buggele
et al. 2012). Yet another study by Dong et al. reported that miR-9 promoted viral
replication by suppressing MCP1P1 (Dong et al. 2017).
178 D. Tiraki

Several other miRNAs like miR-302c, -449b, -125a/b, -136, -194, -483-3p, -132,
and -26a have been shown to target cytokine production (Chen et al. 2017; Gui et al.
2015; Buggele et al. 2013; Hsu et al. 2017; Zhao et al. 2015; Wang et al. 2017;
Maemura et al. 2018; Lagos et al. 2010; Gao et al. 2017).

12.6.2.6 Dengue (DENV)

Several studies have reported increased levels of let-7c and miR-30e∗ in DENV-­
infected HeLa; Huh7, and U937-DC-SIGN cells (Escalera-Cueto et al. 2015; Zhu
et al. 2014a). Let-7c inhibits viral replication by activating TLR2 via NS1 and leads
to IL6 and TNFα production (Chen et al. 2015). miR-30e∗ inhibits viral replication
by targeting NFkB thereby increasing NFkB-dependent IFNβ expression. miR-­
126-­5p is lowered during DENV infection (Kakumani et al. 2013) whereas miR-­
126-­ 5p mimics decreased both viral RNA levels and viremia in supernatants
(Kakumani et al. 2016). DENV induces miR-146a in THP-1 cells and primary
monocytes thereby facilitating replication through impairment of IFNβ production
(Wu et al. 2013). miR-147a (a negative regulator of pro-inflammation) was signifi-
cantly increased in PBMCs infected with DENV (Tolfvenstam et al. 2011).

12.6.2.7 Human Immunodeficiency Virus-1

Involvement of RNAi machinery in Human Immunodeficiency Virus-1 (HIV-1)
came into limelight when Dicer and Drosha were knockeddown in HIV-1-infected
PBMCs. Since then several miRNAs have been studied. miR-29a and -29b
(expressed in PBMCs) targeted sites in the HIV nef region and affected the HIV-1
life cycle (Ahluwalia et al. 2008). An experiment by Triboulet et al. using HIV-1-­
infected Jurkat cells showed downregulation of miR-17/92 cluster, which targeted
P300/CBP-associated factor (PCAF), required for HIV-1 Tat (Triboulet et al. 2007).
miR-28, -125b, -15o, -223, and -383 (anti-HIV) were under expressed activated
CD4+ cells. These miRNAs targeted 3′end of HIV-1 mRNA (Huang et al. 2007).
miR-29a regulates the viral replication by targeting both the 3′end of HIV-1 tran-
scripts and HIV-1 nef (Nathans et al. 2009; Ahluwalia et al. 2008; Hariharan et al.
2005). miRNAs also play a role in the HIV-1-induced encephalitis (HIVE). miR-­
129∗ and -130a (targeting caspase-6 mRNA) and miR-146a (targeting MCP-2) are
under expressed in the brains of HIVE patients (Noorbakhsh et al. 2010; Rom et al.
2010). On the other hand, miR-21 which targets MEF2C is overexpressed in the
brains of HIVE patients (Yelamanchili et al. 2010).

12.6.2.8 Herpes Simplex Virus-1 (HSV-1)

Unlike several other viral infections discussed above, cellular miRNAs are also
involved in Herpes Simplex Virus (HSV-1) life cycle. miR-101 targets 3′UTR of
mitochondrial ATP synthase subunit β (ATP5B) which is required for HSV-1 repli-
cation (Zheng et al. 2011), thereby contributing to viral latency. On the other hand,
miR-23a targets IRF-1 and inhibits interferon pathway, thus, facilitating HSV-1 rep-
lication (Ru et al. 2014). Further, HSV-1 induces miR-146a (a pro-inflammatory
miRNA) that targets complement factor H and induces arachidonic cascade, con-
tributing to Alzheimer-like neuropathological change (Hill et al. 2009).
12 MicroRNAs and Their Role in Viral Infection 179

12.7 Conclusion

As discussed in the sections above, it is increasingly becoming apparent that viruses

can make use of the host RNAi machinery in several ways so as to regulate the viral
life cycle and host response. Viral regulation of cellular miRNA profile seems to be
a complex yet widely used mechanism affecting host cell environment and viral life
cycle by variations in host miRNA targets. Another level of complexity is added by
the viral-encoded miRNAs. This is another strategy used by the viruses to establish-
ing themselves in the host by counterattacking the host response. Here we have
touched upon a few examples of viral infections as discussing all the viral infections
is beyond the scope of this chapter. Most of the miRNAs serve as potential diagnos-
tic and prognostic biomarkers for several viral infections. Others which act on either
the host or the viral components required for the replication may act as potential
therapeutic targets. A classical example is the anti-miR-122 (Miravirsen) against
HCV replication, which is being in clinical trial and results are promising. It would
be very interesting to utilize the potential therapeutic miRNAs reported against viral
infections and develop strategies for management of such viral infections.

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Evolution, Distribution, and Diversity
of Immunodeficiency Viruses 13
Harika Sai Vemuri, Surekha Challa,
and Nageswara Rao Reddy Neelapu

Abstract
Immunodeficiency viruses infect the host and primarily affect the immune sys-
tem of an organism, i.e., host. Till date Human Immunodeficiency Virus (HIV),
Simian Immunodeficiency Virus (SIV), Feline Immunodeficiency Virus (FIV),
Bovine Immunodeficiency Virus (BIV), and Dog Immunodeficiency Virus (DIV)
were reported in the literature. These viruses belong to phylum—Incertae sedis,
family—Retroviridae, genus—Lentivirus, and order—Ortervirales. The review
discusses about evolution, distribution, and diversity of immunodeficiency
viruses, which helps in understanding the biology of HIV and how to develop a
vaccine to the most harmful and dreadful diseases.

Keywords
Bovine immunodeficiency virus (BIV) · Dog immunodeficiency virus (DIV) ·
Feline immunodeficiency virus (FIV) · Human immunodeficiency virus (HIV) ·
Immunodeficiency virus · Simian immunodeficiency virus (SIV)

13.1 Immunodeficiency Viruses

Viruses that infect the host and affect the immune system of the host upon infec-
tion are generally known as immunodeficiency viruses (IV). The host then acquires
a disease known as Acquired Immunodeficiency Disease Syndrome (AIDS).
These immunodeficiency viruses belong to phylum—Incertae sedis, family—
Retroviridae, genus—Lentivirus, and order—Ortervirales. The different types of

H. S. Vemuri · S. Challa · N. R. R. Neelapu (*)

Department of Biochemistry and Bioinformatics, Institute of Science, Gandhi Institute of
Technology and Management (GITAM) (Deemed to be University), Rushikonda,
Visakhapatnam, Andhra Pradesh, India

© Springer Nature Singapore Pte Ltd. 2020 187

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_13
188 H. S. Vemuri et al.

immunodeficiency viruses are Human Immunodeficiency Virus (HIV) (Barré-­

Sinoussi et al. 1983; Clavel et al. 1986), Simian Immunodeficiency Virus (SIV)
(Daniel et al. 1985), Feline Immunodeficiency Virus (FIV) (Pedersen et al. 1987),
Bovine Immunodeficiency Virus (BIV) (Van Der Maaten et al. 1972a), and Dog
Immunodeficiency Virus (DIV) (Safran et al. 1992). Among these viruses, HIV
and SIV are widely studied and there are also reports on other immunodeficiency
viruses like FIV, BIV, and DIV.

13.2 Human Immunodeficiency Virus

HIV is a retrovirus that infects human and affects the immune system of the human
upon infection (Fig. 13.1). The host then acquires a disease known as
AIDS. Worldwide statistics as of 2017 for HIV and AIDS are mentioned in
Table 13.1. In 1983, HIV called HIV1 was isolated by “… researchers at the Pasteur
Institute in France …” which was known to cause AIDS (Barré-Sinoussi et al.
1983). In 1986 “… HIV-2, was isolated from AIDS patients in West Africa …”
(Clavel et al. 1986).

13.2.1 Evolution, Distribution, and Diversity of HIV

Two types of HIVs exist, HIV1 and HIV2; among them, HIV1 was discovered
first and HIV2 was identified later (Fig. 13.2). HIV1 is distributed worldwide
(Fig. 13.3), whereas HIV2 is observed mainly in western Africa (Vidal et al.
2000). The reason for widespread HIV1 can due to the ancestor of HIV1, which
might have mutated at a much faster rate and traveled along with Homo sapiens
population generating diversity in HIV1 (Vidal et al. 2000; Sharp and Hahn 2008).
HIV1 group might have stemmed from strain SIVcpz of SIV (Keele et al. 2006),
whereas HIV2 group might have stemmed from strain SIVsmm of SIV (Gao et al.
1992, 1994). HIV1 is further classified into group M (main), group O (outlier),
group N (non-M/non-O), and group P. Group M is further subdivided into ten
subtypes A–K, CRF’s (Vidal et al. 2000; Sharp and Hahn 2008). Lemey et al.
(2004) classified HIV2 into groups A and B. Further, HIV2 was classified

Table 13.1 Worldwide statistics as of 2017 for HIV and AIDS (Global Statistics 2017)
S. no. Parameter Statistics
1 People living with HIV 36.9 million (31.1–43.9 million)
2 People on antiretroviral therapy 21.7 million (19.1−22.6 million)
3 People newly infected with HIV 1.8 million (1.4–2.4 million)
4 People died with HIV infection 940,000 (670,000–1.3 million)
5 People living with HIV since epidemic 77.3 million (59.9–100 million)
6 People died with HIV infection since 35.4 million (25.0–49.9 million)
epidemic
13 Evolution, Distribution, and Diversity of Immunodeficiency Viruses 189

Fig. 13.1 (a) HIV infects humans and causes disease known as AIDS, (b) SIV infects nonhuman
primates and causes disease known as SAIDS, (c) FIV infects cats and causes disease known as
FAIDS, and (d) BIV infects cattle and causes disease known as BAIDS

phylogenetically into hypothetical groups such as group C–G, and 96FR12034

(Damond et al. 2004). Epidemiological, physiological, clinical, and phylogenetic
evidence favored that HIV1 and HIV2 are due to “… several cross-species trans-
mission of HIV from chimpanzee to humans …” (Castro-­Nallar et al. 2012; Huet
et al. 1990; Hahn et al. 2000; Plantier et al. 2009; Van Heuverswyn and Peeters
2007). Intensive studies were carried out on the evolution and divergence of HIV1
and HIV2 using phylogeny. The divergence time of HIV1, subtype A of HIV2 and
subtype B of HIV2 dated to 1920s (Worobey et al. 2008), 1940 ± 16 (Lemey et al.
2003) and 1945 ± 14 (Gilbert et al. 2007), respectively. HIV1 was introduced into
North America in 1968 (1966–1970) (Pérez-Losada et al. 2010; Surekha and
Neelapu 2019).
190 H. S. Vemuri et al.

Fig. 13.2 Classification of HIV into two major types HIV1 and HIV2

13.2.2 Pathogenesis of HIV

HIV1 advances into the host cells using two molecules of HIV envelope (glycopro-
tein—gp120 and gp41). This ingress of glycoproteins stimulates intracellular signal
cascades and facilitates replication of virus. The glycoproteins gp120 and gp41
form spikes on the surface of virion’s. The protein gp120 binds to the CD4+ receptor
and attaches to the host cell membrane. A marked decrease in CD4+ T cells (both
activated and memory) is the characteristic feature of infection and disease. The
virus then interacts with receptors (CCR5, CXCR4) leading to structural changes of
the protein. The viral and the cellular membranes of the host are fused and form a
viral pore. This allows the transfer of the viral core into the cytoplasm of the host.
After the core disassembles, the RNA is reverse transcribed into DNA by the virus’
with the help of enzyme reverse transcriptase. The DNA is then integrated into the
13 Evolution, Distribution, and Diversity of Immunodeficiency Viruses 191

Fig. 13.3 Worldwide representation of HIV1 and its subtypes across the continents (Source:
Castro-Nallar et al. (2012))

gene rich and transcriptionally active domains of the host’s genome with the help of
the viral protein integrase, DNA repair enzymes of the host and LEDGF/p75 (lens
epithelium-derived growth factor—integrase binding host factor). Once the host
cell is transformed into a potential virus producer, transcription of viral genetic
material occurs. Later viral proteins are transported and assembled at the cell mem-
brane with the help of vesicular sorting pathway (ESCRT-I, -II, and -III). TSG101
domain, a short sequence motif in p6 of Gag I is used for protein sorting. Cleavage
of the Gag-Pol polyprotein by the viral protease produces mature infectious virions
…” (Naif 2013).

13.3 Simian Immunodeficiency Virus

Simian Immunodeficiency Virus (SIV) infects “non-human primates” and affects

the immune system of the “non-human primates” upon infection (Fig. 13.1). The
host then acquires a disease known as Simian Acquired Immunodeficiency Disease
Syndrome (SAIDS). SIV was isolated in 1985 from captive rhesus macaques suffer-
ing from SAIDS (Daniel et al. 1985).

13.3.1 Evolution, Distribution, and Diversity of SIV

SIVs infect nearly 45 different species of “non-human primates.” Different types of

SIV infecting “non-human primates” are mentioned in Table 13.2 (Gordon et al.
2005). SIV may be present at least 32,000 years ago in monkeys and apes (Worobey
192 H. S. Vemuri et al.

Table 13.2 Major types of SIVs and their corresponding nonhuman primate hosts infected with
SIV
S. Nonhuman primate S. Nonhuman primate
no. SIV type infected with SIV no. SIV type infected with SIV
1 SIVcpz Chimpanzee 19 SIVsun Sun-tailed monkey
2 SIVgor Gorillas 20 SIVprg Preuss’s guenon
3 SIVagm African green 21 SIVwrc Western red
monkeys colobus
4 SIVsmm Sooty mangabeys 22 SIVolc Olive colobus
5 SIVrcm Red-capped 23 SIVkrc Kibale red colobus
mangabeys
6 SIVgsn Greater spot-nosed 24 SIVtrc Tshuapa red
monkeys colobus
7 SIVmus Mustached guenons 25 SIVsyk Sykes’ monkey
8 SIVmon Mona monkeys 26 SIVdeb De Brazza’s
monkey
9 SIVagi Agile mangabey 27 SIVden Dent’s mona
monkey
10 SIVmnd 2 Mandrill 28 SIVwol Wolf’s mona
monkey
11 SIVdrl Drill monkey 29 SIVgsn/SIVmon/ Northern talapoin
SIVmus 1/SIVmus 2
clade, SIVtal
12 SIVmac Rhesus macaque 30 SIVasc Red-tailed guenon
13 SIVmne Pig-tailed macaque 31 SIVbkm Black mangabey
14 SIVstm Stump-tailed 32 SIVreg [formerly Red-eared guenon
macaque SIVery]
15 SIVsab, Grivet monkey 33 SIVblu Blue monkey
SIVver,
SIVgri
16 SIVtan Tantalus monkey 34 SIVcol Colobus guereza
17 SIVmnd 1 Mandrill 35 SIVkcol 1 Black-and-white
colobus
18 SIVlho L’hoest’s monkey 36 SIVkcol 2, SIVblc Black colobus
(formerly SIVbcm)

et al. 2010). Primate’s infection with SIV dated back to 14 Ma, if the virus and host
were coevolved then it dates to 85 Ma (Compton et al. 2013). The “non-human
primates” include species of gorilla, chimpanzees, and monkeys. The species of
gorilla are Gorilla gorilla (western lowland gorillas), and Gorilla beringei (eastern
Grauer’s gorillas). The species of western gorilla are subdivided into Gorilla gorilla
diehli (Cross River gorilla) and Gorilla gorilla gorilla (western lowland gorilla).
The eastern species are classified into Gorilla beringei graueri (Grauer’s gorilla),
Gorilla beringei beringei (mountain gorilla), and Gorilla beringei subspecies
(Bwindi gorilla). The chimpanzee species include Pan troglodytes verus (western
Africa), Pan troglodytes ellioti (Nigerian), Pan troglodytes troglodytes (central), and
Pan troglodytes schweinfurthii (eastern). The monkey species include African green
monkeys, sooty mangabeys, Cercocebus torquatus (red-capped mangabeys),
13 Evolution, Distribution, and Diversity of Immunodeficiency Viruses 193

Cercopithecus nictitans (greater spot-nosed monkeys), Cercopithecus cephus (mus-

tached guenons), and Cercopithecus mona (mona monkeys). The association of SIV
with apes, gorillas, and monkeys can be approximately 32,000 years or even much
longer (Worobey et al. 2010). It is hypothesized that “… several cross-species trans-
mission events …” of SIVcpz and SIVsmm might have resulted in evolution of
HIV1 and HIV2 (Huet et al. 1990; Hahn et al. 2000; Plantier et al. 2009; Van
Heuverswyn and Peeters 2007). SIVcpz from chimpanzees “… crossed the species
barrier …” and migrated to humans giving rise to HIV1. Similarity, SIVsmm from
sooty mangabeys “… crossed the species barrier …” and migrated to humans giving
rise to HIV-2.

13.3.2 Pathogenesis of SIV

SIV infects CD4+ T cells and the SIV-infected cells undergo apoptosis within 1 day
of infection. The immune system of simians is not able to replace the cells at the
same pace leading to the deterioration of immune function. Subsequently, the host
acquires immunodeficiency syndrome leaving the animal susceptible to fatigue and
life-threatening coinfections (Fig. 13.4a). “… The interaction of SIV with the host’s
immune system triggers innate immune responses followed by virus-specific adap-
tive cellular and humoral immune responses. Rapidly occurring mutations lead to
immune evasion. Chronic immune activation contributes to the functional impair-
ment of the immune system. As the disease progresses, adaptive immune responses
are unsuccessful in containing the virus replication, and overt signs of a chronic
immune suppression become evident …” (Schmitz and Korioth-Schmitz 2013).
SIV infection leads to changes in mucosal tissues (cervicovaginal, gastrointestinal,
and penile tissues) and all organ systems (brain, lung, and heart) (Haase 2011). The
severity of the infection has an impact and functionally impairs the organs (Alammar
et al. 2011; Kelly et al. 2012). Successful infection of SIV also results in loss of
CD4+ T cells. SIV uses glycoproteins to bind CD4+ receptors of T cells and interacts
with co-receptor CXCR4 leading to conformational changes of the protein. “… The
viral and the cellular membranes of the host are fused allowing the transfer of the
SIV genome into the cytoplasm of the host. The RNA is reverse transcribed into
DNA by the virus with the help of enzyme reverse transcriptase. Then this DNA is
integrated into the host cell’s genome, transforming the host cell into a potential
virus producer. The virus then uses host cell’s replication machinery to transcribe its
DNA back into RNA. The copies of RNA are packed into virus particles of about
80–100 nm in diameter and bud off or free from the host cell infecting more cells.
SIV infections are non-pathogenic in their natural African simian primates.
However, if the virus infects an Asian or Indian rhesus macaque, these non-African
simian primates will develop simian AIDS (SAIDS). SIVs nef gene down-regulates
expression of CD3, CD4, and MHC class I and therefore do not induce immunode-
ficiency. Whereas, nef gene in HIV-1 lost its ability to down-regulate CD3, which
results in the immune activation and apoptosis …” (Swigut et al. 2004).
194 H. S. Vemuri et al.

Fig. 13.4 (a) Consequences and immunopathogenesis in simian when infected with a simian
immunodeficiency virus (SIV) (Source: Schmitz and Korioth-Schmitz (2013)). (b) Replication of
SIV in the host simian (Source: Encyclopaedia Britannica Inc. https://www.britannica.com/sci-
ence/SIV)
13 Evolution, Distribution, and Diversity of Immunodeficiency Viruses 195

13.4 Feline Immunodeficiency Virus

Feline Immunodeficiency Virus (FIV) infects cats and affects the immune system of
the cats upon infection (Fig. 13.1). The cat then acquires a disease known as Feline
Acquired Immunodeficiency Disease Syndrome (FAIDS). FIV was first isolated
from a cat exhibiting an immunodeficiency-like syndrome (Pedersen et al. 1987).

13.4.1 Evolution, Distribution, and Diversity of FIV

FIV is endemic in Felidae species (Biek et al. 2003, 2006; Brown et al. 1994;
Carpenter et al. 1996, 1998; Carpenter and O’Brien 1995; Driciru et al. 2006;
Hofmann-Lehmann et al. 1996; Munson et al. 2004; Olmsted et al. 1992; Troyer
et al. 2004; Troyer et al., 2005), from free-ranging lions (Pecon-Slattery et al. 2004)
to domestic cats (Olmsted et al. 1989). FIV occurs in Felis catus (domestic cats),
Puma concolor (pumas) (Langley et al. 1994), Panthera leo (lions), Otocolobus
manul (pallas cat) (Barr et al. 1997), Panthera pardus (puma leopard), Acinonyx
jubatus (cheetah), Leopardus pardalis (ocelot), Panthera onca (jaguar), Leopardus
weidii (margay), Leopardus tigrina (tigrina), large African lions, and Crocuta cro-
cuta (spotted hyaena) (Troyer et al. 2005). The different types of FIVs like FIVPle,
FIVFca, FIVPco, FIVOma, FIVLpa, FIVAju, FIVCcr, FIVPon, FIVLwe, and FIVLti were
reported in literature (Fig. 13.5). The major types of FIVs and their corresponding
felines infected with FIV are listed in Table 13.3.
Currently, isolates of FIV are classified into six subtypes (A, B, C, D, E, and F)
(Weaver 2010). Weaver (2010) performed a detailed phylogenetic analysis of FIV
and classified FIV into six subtypes (A, B, C, D, E, and F) (Fig. 13.4). Sodora et al.
(1994) initially classified FIV into three subtypes (A, B, and C). Sodora et al. (1994)
used phylogenetic analysis to classify FIV isolates based on FIV env (envelope
sequence). The geographical location of the subtype A is California and Europe;
subtype B is Japan and the central and eastern USA; and Subtype C is southwestern
Canada. Nishimura et al. (1998), later classified FIV into five subtypes (A, B, C, D,

Fig. 13.5 Classification of FIV and their subtypes

196 H. S. Vemuri et al.

Table 13.3 Major types of S. no. FIV type Felines infected with FIV
FIVs and their corresponding 1 FIVPle Panthera leo (lions)
felines infected with FIV
2 FIVFca Felis catus (domestic cats)
3 FIVPco Puma concolor (pumas)
4 FIVOma Otocolobus manul (pallas cat)
5 FIVPpa Panthera pardus (puma leopard)
6 FIVAju Acinonyx jubatus (cheetah)
7 FIVCcr Crocuta crocuta (spotted hyaena)
8 FIVLpa Leopardus pardalis (ocelot)
9 FIVPon Panthera onca (jaguar)
10 FIVLwe Leopardus weidii (margay)
11 FIVLti Leopardus tigrina (tigrina)

Fig. 13.6 Worldwide distribution of FIV and its subtypes across the continents (Source: Teixeira
et al. (2012))

and E) based on FIV env (envelope sequence). The geographical location of the
subtype A is California and Northern Europe; subtype B is central and eastern USA
and southern European countries; subtype C is California and British Columbia;
subtype D is Japan; and subtype E is Argentina. FIVPle has diverged into six sub-
types A–F, each with distinct geographic areas of endemicity (Brown et al. 1994;
Troyer et al. 2004; O’Brien et al. 2006). FIVFca has diverged into five subtypes, A–E
(Sodora et al. 1994; Kakinuma et al. 1995; Pecoraro et al. 1996), where subtypes A,
B, and C are widespread worldwide. Subtype D is found in Japan and Vietnam
(Kakinuma et al. 1995; Nakamura et al. 2003), whereas subtype E is only found in
Argentina (Pecon-Slattery et al. 2008). The four subtypes A–D are found in cat
populations from Japan (Nishimura et al. 1998; Kakinuma et al. 1995). FIVPco has
diverged into three subtypes A, B, and C (Pecon-Slattery et al. 2008) (Fig. 13.6).
13 Evolution, Distribution, and Diversity of Immunodeficiency Viruses 197

13.4.2 Pathogenesis of FIV

FIV uses receptor CD9 (Poeschla and Looney 1998) for entry and then infects CD4+
T (Brown et al. 1991), FIV can also infect astroglial cells, CD8+ T lymphocytes,
macrophages, and kidney cells (Pedersen et al. 1989; Brunner and Pedersen 1989;
Phillips et al. 1990; Zenger et al. 1995). “… This leads to a significant drop in cells
which have critical roles in the immune system. Low levels of CD4+ and other
affected immune system cells cause the cat to be susceptible to opportunistic dis-
eases once the disease progresses to feline acquired immune deficiency syndrome
(FAIDS) …” (Bendinelli et al. 1995). Symptoms of immunodeficiency associated
with FIV are chronic lesions, opportunistic infections, neurological abnormalities,
and weight loss (Yamamoto et al. 2010).
CD134 is mostly present on T cells which are activated and binds to OX40
ligand, causing T cell activation, stimulation, proliferation, and apoptosis. The virus
enters the host’s cells using the glycoprotein env and interacts with surface recep-
tors. The glycoprotein SU binds to receptor CD134 of the host cell leading to con-
formational changes of protein SU. These changes facilitate interaction between SU
and the chemokine receptor CXCR4; and fuses viral membrane and cellular mem-
brane of the host (Hu 2012). “… This allows the transfer of the viral RNA into the
cytoplasm of the host. Then RNA is reverse transcribed and integrated into the
genome of the host through non-homologous recombination. Once viral RNA is
integrated into the host genome, the virus can be dormant in the asymptomatic stage
without being detected by the immune system of the host or can cause lysis of the
host cell …” (Lecollinet and Richardson 2008; Hartmann 2011).

13.5 Bovine Immunodeficiency Virus

Bovine Immunodeficiency Virus (BIV) infects cattle and affects the immune system
of the cattle upon infection (Fig. 13.1). The cattle then acquires a disease known as
Bovine Acquired Immunodeficiency Disease Syndrome (BAIDS). BIV strain “…
R-29 was originally isolated from an 8-year-old dairy cow suspected of having
bovine lymphosarcoma …” (Van Der Maaten et al. 1972a).

13.5.1 Evolution, Distribution, and Diversity of BIV

BIV is widespread in dairy and beef cattle in Australia (Forman et al. 1992), Canada
(McNab et al. 1994), France (Polack et al. 1996), Germany (Muluneh 1994), Italy
(Cavirani et al. 1998), Japan (Hirai et al. 1996; Meas et al. 1998), Korea (Cho et al.
1999), Netherlands (Horzinek et al. 1991a), New Zealand (Horner 1991), the UK
(Clayton 1994), the USA (Amborski et al. 1989; Cockerell et al. 1992; St. Cyr Coats
et al. 1994), and buffaloes in Pakistan (Meas et al. 2000). The prevalence of BIV in
dairy cattle is higher than beef cattle (Amborski et al. 1989; Cho et al. 1999). The
different BIV strains reported in literature are BIV R-29 (isolated from cow) (Van
198 H. S. Vemuri et al.

Der Maaten et al. 1972b), BIV-106, and BIV-127 (variants of BIV R-29) (Braun
et al. 1988; Garvey et al. 1990), BIV CR1 (BIV strain from Costa Rica (Hidalgo
et al. 1995), BIV FL112 (Hirari et al. 1996), and FL491 (Suarez et al. 1993) (strain
from Florida, USA).

13.5.2 Pathogenesis of BIV

BIV infects nondividing host cells and uses two phases—entry phase (first phase)
and replication phase (second phase) to replicate itself (Berkowitz et al. 2001). In
the first phase, glycoprotein of the virus envelope interacts and binds with the spe-
cific receptor of the cell. “… Then virus uses either of the ways, receptor mediated
endocytosis or viral envelope-cell membrane fusion to enter into the host cell. The
virus then dissembles in the cell and reverse-transcribes the RNA genome into DNA
with the help of enzyme reverse transcriptase (Gonda, 1992). The activity of reverse
transcriptase is higher at lower concentrations of Mn2+ ions when compared to Mg2+
ions (Horzinek et al. 1991b). The DNA is then transported to nucleus and is inte-
grated into the genome of the host cell. In the second phase, the integrated viral
DNA is transcribed and the transcript (viral mRNA) is transported and translated in
the cytoplasm ….” The translated viral structural proteins are then assembled into
virus particles and form a viral complex (virus buds) along with the viral RNA at the
plasma membrane. Then viral proteases process the viral buds and are released from
the cell in the mature stages, which are ready to infect another cell (Gonda 1992).

13.6 Dog Immunodeficiency Viruses

Dog Immunodeficiency Virus (DIV) is a retrovirus which infects dog and affects the
immune system of the dogs upon infection. The dog then acquires a disease known
as DAIDS. Safran et al. (1992) isolated canine immunodeficiency virus, (CIV)—
(canine lentivirus) from a leukemic dog. The ultrastructure and morphogenesis of
CIV is strikingly similar to that displayed by other lentiviruses, while the immuno-
logical relatedness close to any other lentivirus or oncovirus was not established.
These findings suggest that this canine retrovirus, fits in the subfamily lentivirus and
not related to other known members. Additional, investigations on DIV (CIV) are
essential to establish the biology, immunopathogenesis of virus; and how immuno-
deficiency is acquired by dogs.

13.7 Conclusions and Future Perspectives

Immunodeficiency viruses infect the host and primarily affect the immune system
of an organism i.e. host. HIV, SIV, FIV, BIV, and DIV were known to infect humans,
simians, cats, cattle, and dogs respectively. The ultrastructure and morphogenesis of
IVs like HIV, SIV, FIV, BIV, and DIV are strikingly similar. At the same time
13 Evolution, Distribution, and Diversity of Immunodeficiency Viruses 199

immunological relatedness of the lentiviruses like HIV, SIV, FIV and BIV are close
with exception in DIV. Therefore, there is a need to establish the immunological
relatedness of the DIV. The similarities in the ultrastructure, morphogenesis and
immunological relatedness of lentivirus provide us with an opportunity for better
understanding of the immunodeficiency in different hosts. This information further
can be used to develop a vaccine to the most harmful and dreadful disease.

Acknowledgements HSV, SC, and NRRN are grateful to GITAM (Deemed to be University) for
providing necessary facilities to carry out the research work and for extending constant support.

Authors Contribution HSV, CS and NNRR initiated the review, participated in

writing and revised the manuscript.

Conflict of Interest Statement The authors declare that there is no potential con-
flict of interest.

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Role of Immune Cells in Hepatitis B
Infection 14
Prakriti Sinha and Parul Sahu

Abstract
Hepatitis B virus infection is a common cause of liver cirrhosis and hepatocel-
lular carcinoma leading to about one million deaths annually. The disease patho-
genesis is complex and incompletely understood, especially when it turns
chronic. Among various factors known to be responsible for the disease progres-
sion, the immune system has the most important role to play. Viral clearance is
largely T cells dependent, and progression to a chronic state is due to insufficient
T cells response, partly due to T-regulatory cells. In chronic infections, functions
of important immune cells like natural killer cells and dendritic cells are impaired,
which enables the viral persistence and promotes the pathogenesis in the host
body. Similarly, the virus is reported to modulate functions of monocytes, mac-
rophages, and Kupffer cells to promote an immune-tolerant local environment in
the liver. Though B cells are expected to be central to the humoral immune
response that clears the virus, its role in chronic HBV is still obscure. This chap-
ter would elaborate on the significant roles of immune cells in pathogenesis and
in clearance of the hepatitis B virus.

Keywords
Hepatitis B virus · Innate immunity · Adaptive immunity · T cells ·
Immunopathogenesis

P. Sinha (*) · P. Sahu

National Institute of Immunology, New Delhi, India

© Springer Nature Singapore Pte Ltd. 2020 205

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_14
206 P. Sinha and P. Sahu

14.1 Hepatitis B: The Disease

Hepatitis B virus infection is the most common cause of liver pathologies like cir-
rhosis and hepatocellular carcinoma leading to about one million deaths annu-
ally and around 257 million people are chronically infected globally (WHO report
2017). Most adults clear an acute viral infection without any symptoms, but 5–10%
of them become chronic patients due to persisting presence of virus, while most
neonates born to mothers with an active infection develop chronic hepatitis B infec-
tion. The infection could lead to a range of pathologies depending on the virus and
host immune system interplay, from acute self-contained infection to chronic hepa-
titis, leading to cirrhosis, hepatocellular carcinoma (HCC), and death of the patient.
Most acute infections are mild and asymptomatic with only a small proportion
developing fatigue, nausea, jaundice, and rarely, acute liver failure. In chronic infec-
tions, the disease manifests itself to variable degree in each patient depending on
various factors. Some patients remain asymptomatic or have nonspecific symptoms;
some show symptoms similar to that in acute infection, while more severe cases
have a more morbid prognosis with a 50% 5-year survival rate (Liang 2009).
Current treatment includes pegylated-interferon (IFN)-α and five nucleos(t)ide
analogs (NAs). Though each of these drugs show some benefit in controlling the
virus and symptoms, they do not completely clear the virus. Antiviral resistance and
side effects limit usage of these drugs. Hence, therapy is advised for only those who
are likely to develop serious complications (Koumbi 2015).

14.2 The Virus

The hepatitis B virus belongs to the Hepadnaviridae family of hepatotropic DNA

viruses. Similar viruses are found to infect other apes, woodchucks, squirrels, ducks,
and herons (Liang 2009). These viruses majorly infect the liver, but trace amounts
of their DNA have been reported to be found in pancreas, kidney, and peripheral
blood mononuclear cells (Murakami et al. 2004).

Fig. 14.1 Schematic

depiction of a hepatitis B
virion (Liang 2009)
14 Role of Immune Cells in Hepatitis B Infection 207

The hepatitis B virus is 42 nm in diameter and consists of an outer host-derived

lipid membrane, in which the hepatitis B surface antigen (HBsAg) is embedded.
The inner nucleocapsid is composed of hepatitis B core antigen (HBcAg) which
encapsulates the virally encoded polymerase and a partially double-stranded viral
DNA genome (Fig. 14.1) (Liang 2009). An important feature of this virus is its
replication strategy employing an RNA intermediate and the error-prone reverse
transcriptase enzyme resulting in high mutation rate (Trepo et al. 2014).
The virus binds to host hepatocytes through its surface antigen (HBsAg) and
Sodium taurocholate cotransporting polypeptide (NTCP) receptor, found on the
surface of hepatocytes (Yan et al. 2012). After fusion with the host membrane, the
core enters the cytosol and is transported to the host nucleus. With the help from
host molecular machineries, the viral DNA is replicated, via an RNA intermediate,
the viral proteins are expressed and new virions are assembled and released
(Fig. 14.2) (Mohd-Ismail et al. 2019). Some of the viral proteins like HbsAg and
HBeAg are synthesized and released in excess and are known to have immuno-
modulatory roles (Liang 2009).

Fig. 14.2 Schematic representation of the Hepatitis B virus life cyce (Mohd-Ismail et al. 2019)
208 P. Sinha and P. Sahu

14.3 Natural History and Pathogenesis

HBV reaches high titers in the blood and this is the major route of transmission. On
infection there is minimal immune response and hence the virus spreads rapidly in
the hepatocytes. Immune activation within the first few weeks leads to inflammation
in the liver and antiviral response. Cytotoxic T cells are the major cells responsible
for killing infected hepatocytes as well as arresting viral replication by secretion of
antiviral cytokines like INF-γ and TNF-α. Extensive cell death in the liver may
result in some symptoms, indicative of liver inflammation and damage. Appearance
of antibodies against the viral surface antigen (HBsAb) in serum is a major indicator
of clearance of the virus from the circulation.
As mentioned earlier, 5–10% of adults and most HBV-infected infants are unable
to fully clear the virus and develop a chronic HBV infection. Insufficient or
exhausted T cell response is believed to be the primary reason for this persistent
infection. Low levels of viral DNA and particles are detected beyond 6 months of
infection and this is accompanied by occasional flares of viral–immune activity.
This continued cycle of inflammation and cell damage, over the years, leads to
development of liver cirrhosis and hepatocellular carcinoma in many patients. The
status of the disease in a chronic patient can be categorized as immune-tolerant,
immune-active, or immune-inactive carrier phase, depending upon the viral activity
and host immune response (Trepo et al. 2014).
The course of the disease and its outcome is variable in chronic HBV patients
making it tricky to manage. Hence, extensive studies have been done to fully under-
stand the immune pathogenesis of HBV infection and identify possible targets to
help manage the infection. The narrow host range of HBV makes it challenging to
study. Most of the understanding of the HBV life cycle comes from animal models,
i.e., chimpanzees and Tupaia Belangeri (Tree shrew) which can be infected by HBV
as well as closely related viruses like Duck-HBV and Woodchuck-HBV in their
respective host. Many studies are also done on transgenic and humanized mice.
Viral biology is also studied in vitro, on either primary human hepatocytes or NTCP
expressing transgenic cell lines. Major gaps still exist in the understanding of the
pathogenesis and involvement of immune cells in HBV infection (Lamontagne
et al. 2016).

14.4 T Cells

T lymphocytes are the major effector cells involved in adaptive immune response to
viruses. A robust T cell response clears the infection in acutely infected HBV
patients, while weak and inadequate T cell response is believed to result in chronic
HBV infection. In a mouse model of HBV infection, the results strongly show that
CD4+ T cells (T helper cells) serve as major regulators of the adaptive immune
response and the CD8+ T cells (cytotoxic T cells or CTLs) being the main effector
cells for HBV clearance along with supporting functions of NK cells and interfer-
ons (Yang et al. 2010).
14 Role of Immune Cells in Hepatitis B Infection 209

14.4.1 CD4+ Cells or T Helper Cells

CD4+ T helper cells are involved in early priming and development of CD8+ cyto-
toxic T cell function as well as B cell function of production of antibodies to clear
free viral particles (Sant and McMichael 2012). Th1 CD4+ cells have been found to
increase during hepatic inflammatory activity and in active phases of chronic HBV
infection (Jiang et al. 2002), but not during peak viremia; rather they are required
early in the infection for priming of CD8+ T cells for successfully clearing the infec-
tion. This was supported by another report (Fisicaro et al. 2009) where they found
that CD4+ and CD8+ T cell responses are found to peak, early in the infection, with
IFN-γ producing CD8+ cells found at higher frequencies at the height of immune
response. Hence, even though CD4+ T cells may not be direct effector cells for viral
clearance, they play an important role by early induction of CD8+ T cells. The CD4+
T cells also exert their immunological function by producing cytokines like
Interferon (IFN)-γ and Tumor Necrosis Factor (TNF)-α to control the infection and
regulate other immune cells (Li et al. 2016a). Chronic HBV infection is character-
ized by T cell exhaustion and inadequacy of CD4+ T cells function may be another
contributing factor to the effector CD8+ T cell exhaustion in chronic viral infections
(Ye et al. 2015). Indeed, inhibitory molecule PD-1 (programmed cell death protein
1) has been found to be upregulated in CD4+ T cells in chronic HBV infection
(Raziorrouh et al. 2014), hence this could be a contributing factor for the dysfunc-
tion of the T cell compartment.

14.4.2 CD8+ T Cells or Cytotoxic T Cells

Early reports show that strong HBV-specific CD8+ T cell response correlates with
viral clearance during acute infection. The highest frequency of peripheral HBV-­
specific CD8+ T cells are found in the acute phase of HBV infection; however, a
transient attenuation of the CD8+ proliferation (Maini et al. 1999) and functions,
coinciding with increase in the immunosuppressive cytokine, IL-10 during peak
viremia is noted (Dunn et al. 2009). An effective CD8+ response is robust, poly-
clonal and multi-antigen specific, while such a response is undetectable in chronic
patients (Rehermann et al. 1995; Stelma et al. 2017). These are the main effector
cells that clear the HBV infection by both cytolytic and non-cytolytic mechanisms,
as observed, in chimpanzees (Thimme et al. 2003).
The non-cytolytic mechanism of viral inhibition, mediated by IFN-γ and TNF-α,
inhibits viral replication and clears viral intermediates from infected cells (Guidotti
et al. 1996) without liver damage (Maini et al. 2000) and is the predominant mode
of viral clearance in an effective response, while significant cytolytic activity is
observed by nonspecific but activated lymphoid cells (Phillips et al. 2010).
The cytotoxic T cell response (CTL) is weaker and less specific in chronic HBV
infections with dampening of not only HBV-specific but global T cells functions
(Park et al. 2015). The HBV-specific CD8+ T cells show dysfunction correlating
with viral levels and they can be detected in patients with lower viremia while rarely
210 P. Sinha and P. Sahu

found in patients with higher viral load. HBV-specific CTLs, specific for the core
antigen (HBcAg), is associated with viral control, while those specific for envelop
and polymerase epitopes are more frequent in higher viremia patients, implying that
the core specific CD8+ cells are therapeutically more relevant (Webster et al. 2004).
Also, HBV mutational escape and evolution is observed during progression of the
chronic infection and is believed to be due to the selection pressure from HBV core
specific CTLs (Zhang et al. 2018); however, complete understanding of viral escape
from CD8+ immune response is still incomplete (Lumley et al. 2018).
The virus-specific CD8+ cells found in circulation of chronic patients show an
exhausted phenotype with impaired proliferation, cytokine secretion, expression of
inhibitory molecules like PD-1 (Boni et al. 2007), cytotoxic T lymphocyte antigen-4
(CTLA-4) (Schurich et al. 2011), T cell immunoglobulin domain and mucin domain-
containing molecule-3 (Tim-3) (Nebbia et al. 2012; Wu et al. 2012) and CD244
(Raziorrouh et al. 2010), which lead to their deletion on interacting with their respec-
tive ligands. This exhausted phenotype is a stable form of dysfunction and cannot be
reversed by changing the immune environment (Wang et al. 2018). However, their
functionality could be improved in vitro, by blocking these inhibitory molecules and
receptors highlighting important immunotherapeutic targets in chronic HBV infec-
tion. Blocking of PD-1 with antibodies has especially shown promise by increasing
IFN-γ producing CTLs in the liver in mice (Maier et al. 2007), and improved viral
control in combination with nucleoside analog and DNA vaccine therapy in wood-
chuks models (Liu et al. 2014b). Therapeutic efficacy in woodchuck model showed
no significant liver injury in acute models and good viral control in infected animals
expressing PD-1 on CD8+ T cells; however, combination treatment with additional
antiviral agents was recommended to achieve a robust and more consistent response
for complete immune clearance (Balsitis et al. 2018). Nivolumab, a PD-1 blocking
monoclonal antibody has been approved for therapeutic use in many forms of cancer
and has recently been approved in the USA for hepatocellular carcinoma caused by
various afflictions including HBV infection (Highleyman 2017).
Successful antiviral treatment with pegylated-IFN-α-2b is accompanied with
increase in number of circulating HBV-core or envelop-specific CD8+ T cells, along
with increased Th1-type cytokines (IL-12, TNF-α, IFN-γ) (Chen et al. 2010). T
cells with TCRs (T cell Receptor) which have high avidity for HBV antigens hold
lot of potential for adoptive transfer and treatment, and such TCRs have also been
cloned and studied in vitro for possible applications in therapy (Wisskirchen et al.
2017). Adoptive transfer of HBV-specific CD4+ and CD8+ T cells along with immu-
notherapies to prevent T cell exhaustion has been proposed for treatment of chronic
viral infection as well as cancer (Kamphorst and Ahmed 2013).

14.4.3 Regulatory T Cells (T regs)

Regulatory T cells or Tregs are a set of naturally occurring and inducible CD4+
CD25+ and FoxP3 expressing T cells with a basic role of controlling hyperactivity
of effector T cells. Even though their main role is to prevent autoimmune reactions
14 Role of Immune Cells in Hepatitis B Infection 211

and reduce collateral tissue damage during infections, an undesirable consequence

is immune suppression and pathogen persistence (Belkaid 2007). Probable mecha-
nisms by which they exert the suppressive function is by secretion of immunosup-
pressive cytokines IL-10, IL-35, TGF-β, regulating maturation of dendritic cells and
production of metabolites that inhibit effector T cells with or without direct-cell
contact (Li et al. 2016b).
A higher percentage of CD4+ CD25+ Tregs were detected in circulation (Stoop
et al. 2005) as well as in the liver of chronic HBV patients, and the increase in pro-
portion was more pronounced in the convalescent phase of infection and correlated
with the serum viral load in the patients (Xu et al. 2006). These cells were capable
of suppressing activation of CD8+ T cells, while their depletion resulted in increase
in HBV-specific CD8+ response, as observed ex vivo (Franzese et al. 2005; Stoop
et al. 2005). Tregs in the liver correlate strongly with the viral load and are function-
ally distinct from peripheral blood Tregs, i.e., these were found to not express CD25,
had lower expression of HLA-DR and CTLA-4 (Stoop et al. 2008). HBcAg-specific
Tregs are found to decline during acute exacerbations in chronic HBV patients
accompanied by increase in HBcAg-specific CD8+ T cells (Feng et al. 2007). It has
been suggested that the immunosuppressive function of Tregs is important to mini-
mize extensive histological damage as concluded by Stross et al. (2012) who found
that Tregs downregulated the cytokine production and cytotoxicity of the effector T
cells, but did not affect the development of HBV-specific effector and memory T
cells. Also, Tregs reduced recruitment of macrophages and dendritic cells to the
liver, which inadvertently delayed clearance of the virus, in transgenic mouse model
(Stross et al. 2012).
In cases of vertical transfer of infection, as seen in the peripheral and cord
blood of HBsAg positive newly borns, only the frequency of FoxP3 expressing
Tregs was significantly high while the frequency of CD4+ and CD8+ T cells
remained unchanged, though the CD8+ T cells were found to be dysfunctional,
explaining the development of chronic infections in infected neonates (Shrivastava
et al. 2013). Levels of circulating Tregs is also significantly elevated in acute-on-
chronic liver failure (ACLF) patients compared to healthy donors and chronic
patients, and is proposed to be indicative of the disease severity (Yang et al. 2012).
Increased Tregs levels have also been found to increase in frequency as well as
impair CD8+ T cell function, promoting disease progression in chronic HBV
patients with HCC and proposed to have prognostic value as well as serve as a
potential therapeutic target (Fu et al. 2007; Han et al. 2011; Kalathil et al. 2013;
Li et al. 2016b). During treatment for HCC with anti-angiogenic agent, Sorafenib,
decreased Tregs numbers correlated with overall survival of the HCC patients
(Kalathil et al. 2016). Hence, even though Tregs are important for controlling
excessive immune-mediated damage, their suppressive function often leads to
disease progression in chronic infection setting, making Tregs an important prog-
nostic marker and therapeutic target for immunotherapy in HBV infection (Aalaei-
Andabili and Alavian 2012).
212 P. Sinha and P. Sahu

14.4.4 T Helper: 17 (Th17) Cells

The T-helper-17 cells are a newly identified lineage of the CD4+ T helper cells that
predominantly produce IL-17, IL-21, IL-22 and play an important role in protection
against extracellular pathogens as well as in inflammation and autoimmunity
(Harrington et al. 2005; Park et al. 2005; Bettelli et al. 2007; Ouyang et al. 2008).
The Th17 cells in circulation, as well as intrahepatic, increase with disease progres-
sion and correlate with viral load, serum ALT, and histopathology index and this
may be through activation of mDCs, monocytes, and increased production of pro-­
inflammatory cytokines in chronic patients (Zhang et al. 2010a). IL-17(a) activates
hepatic stellate cells to express fibrogenic factors thereby leading to fibrosis and
cirrhosis in an inflammatory environment in the liver (Tan et al. 2013), and this is
suggested to be a major mechanism of cirrhosis in severe cases of chronic HBV
infection (Sun et al. 2012). TH17 and IL-17 levels are found to be higher in non-­
survivors and decrease in circulation of recovering patients (Yang et al. 2013).
Th17 and Treg cells play opposite roles of mediating and restraining inflamma-
tion and a lower Treg/Th17 ratio indicates extensive liver injury and fibrosis in
chronic HBV patients (Li et al. 2012). The serum IL-17 levels and frequency of
Th17 cells correlate with ALT levels while nTregs correlate positively with HBV
DNA load and inversely with Th17 frequencies (Yang et al. 2016). TGF-β/IL-17
ratio have also been found to markedly increase in decompensated cirrhosis patients
and non-survivors, and hence both these ratios can help in monitoring and prognosis
of HBV-associated cirrhotic patients (Yu et al. 2014). On administration of NA anti-
viral therapy, the Treg/Th17 ratio declined in complete responders due to increased
Th17 cells and IL-17 and decreased Treg and their cytokines, correlating with
decrease in HBV DNA and ALT levels (Yu et al. 2013). Cell-based therapy using
autologous bone marrow mesenchymal stem cells, post antiviral therapy in HBV-­
associated cirrhotic patients, showed improved liver function and an increase in the
Treg/Th17 ratio (Xu et al. 2014a).

14.5 B Cells and Bregs

The neutralizing antibodies against HBsAg produced by B cells is essential for

clearance of free viral particles and prevention of reinfection (Paul et al. 2017).
Chronic HBV patients mostly lack neutralizing antibodies against the viral particle
and this explains why the virus persists in these cases (Alberti et al. 1978). B cells
have been found to actively present HBsAg to T helper cells more efficiently than
classical APCs like macrophages and dendritic cells, in a mice model (Milich et al.
1997). B cells can bind HBcAg and give an IgM response, in a T cell independent
fashion; however, these could not class switch to IgG, highlighting the need of T
cell’s help for this. The same response was not found for HbeAg (Cao et al. 2001).
This antigen presentation potential of B cells was also employed to activate HBV-­
specific cytolytic T lymphocytes especially for chronic conditions, where in B cells
from healthy donor was first activated with a soluble CD40 ligand and then pulsed
14 Role of Immune Cells in Hepatitis B Infection 213

with a peptide from HBcAg and coculture with autologous T cells, showed increase
in percentage of induced CTLs (Wu et al. 2010).
The B cells do not seem to be activated in chronic HBV patients as seen by the
proportion of CD86+ B cells in their blood (Zhao et al. 2015). However, effective
treatment can activate the B cell subsets. The B cell subsets remodeling, from naïve
and post-germinal to transitional and plasmablasts strongly correlated with
decrease in serum HBsAg, as observed in PEG-IFNα treated patients (Aspord et al.
2016). In chronic HBV patients, an exhausted or defective subset of B cells has
also been recently characterized. It has been found that HBsAg-specific B cells
persist in circulation; however, these show an atypical Memory B cell phenotype
(atMBC) which is a functionally defective subset enhanced in conditions of con-
tinuous antigen exposure. These cells had enhanced expression of inhibitory mol-
ecules like PD-1, were prone to apoptosis, and perhaps diminished ability to
differentiate to antibody producing plasma cells and attenuated signaling and cyto-
kine production. These at MBC were found to preferentially accumulate in the
infected liver and it is possible that the highly inflammatory microenvironment
promotes the development of the defective phenotype. PD-1 blockade was found to
partially rescue the premature apoptosis and impaired antibody and cytokine pro-
duction (Burton et al. 2018). Similar subset of impaired B cells was also defined in
another study, where these cells from chronic patients were found to express inhib-
itory receptors of FcRL family and downregulate genes responsible for antigen
presentation. Treatment of B cells from healthy subjects, with HBV core particles,
resulted in expression of the inhibitory molecules and this altered phenotype, high-
lighting another immunosuppressive effect of HBV particles on immune cells
(Poonia et al. 2018).
A regulatory subset of B cells which predominantly produces IL-10 was also
identified. These were increased in patients, and their frequency and IL-10 levels
correlated temporally with liver inflammation. These cells were found to suppress
HBV-specific CD8+ T cell response in a IL-10 dependent manner and hence play an
important role in controlling liver inflammation or in establishment of a tolerogenic
environment (Das et al. 2012). This subset was further defined as Bregs and found
to be elevated in immune-active chronic HBV patients, which correlated with the
serum IL-10, ALT, and AST levels. Also, Bregs and IL-10 levels were higher in
inactive carrier group than healthy controls. The expression of TLR2 is elevated and
TLR9 is reduced on Bregs of chronic HBV patients compared to healthy controls;
however, its significance is yet to be elucidated (Wang et al. 2017).

14.6 Monocytes and Macrophages

Macrophages, Kupffer cells, dendritic cells, and hepatocytes would be few of the
first cells to recognize and respond to HBV, through their Toll-like receptors (Ma
et al. 2018). Monocytes mediate host antimicrobial defense by migrating to breached
tissues in response to chemokines and depending upon interaction with the
214 P. Sinha and P. Sahu

pathogen, differentiate into macrophages and dendritic cells, along with secretion of
immune modulators. Many viruses are known to preferentially infect monocytes,
express viral proteins in the cell, and trigger their rapid differentiation to dendritic
cells (Hou et al. 2012). Early reports claim that HBV DNA is found in monocytes
of chronically infected patients (Yoffe et al. 1986; Guang 1992). As found from
in vitro experiments, recombinant HBsAg binds preferentially to B cells and mono-
cytes (Pontisso et al. 1991) especially by interacting and binding to CD14 receptor
(Vanlandschoot et al. 2002). Monocytes from chronic HBV patients have been
found to be the only antigen presenting cells to contain intracellular HBsAg, which
when differentiated ex vivo, was able to cross present and stimulate expansion of
HBV-specific T cells (Gehring et al. 2013). However, literature regarding the effect
of such interaction of monocytes with different subviral particles of HBV has been
ambiguous.
HBcAg induces production of pro-inflammatory cytokines IL-6, IL-12, and
TNF-α in monocytic cell line THP-1, by signaling through TLR2 (Cooper et al.
2005). However, there is a reduction of surface expression TLR2 accompanied by
reduction of secretion of TNF-α on interaction with HBeAg (Riordan et al. 2006;
Visvanathan et al. 2007) and IL-12 on interaction with HBsAg with monocytes
(Wang et al. 2013). TLR8 is another important virus-sensing receptor expressed
strongly on monocytes but found to be reduced in monocytes from chronic HBV
patients with a concomitant reduction in production of antiviral cytokines, also cor-
relating with reduced response to interferon therapy (Deng et al. 2017). Also, while
some in vitro studies report an immunosuppressive response of the monocytes on
interaction with HbsAg, where in they secrete IL-10 and TNF-α and suppress func-
tions of pDCs (Shi et al. 2012), other similar study observe a robust secretion of
inflammatory cytokines IL-6 and TNF by monocytes on treatment with HBsAg,
though the serum level of these cytokines in chronic HBV patients did not correlate
serum HBV and HBsAg levels (Boltjes et al. 2014). Hence, more studies are
required to delineate the exact effect of the monocyte–virus interaction and role of
these cells in the disease pathogenesis.
Pro-inflammatory CD16+ subset of monocytes have also been implicated to con-
tribute to liver injury in immune-active patients since these increase in circulation
and in liver of these patients, correlating with their serum ALT and extent of liver
damage (Zhang et al. 2011). Kupffer cells (KCs), the liver tissue resident macro-
phages are also expected to have a significant role in the liver microenvironment
even though there is sparse information about their role in HBV immunity or patho-
genesis. Intrahepatic KCs from chronic HBV patients have been found to be HBsAg
positive and more activated than controls (Boltjes et al. 2015). In a mouse model of
HBV infection, KCs were found to secrete immunosuppressive IL-10 which could
be induced by viral particles to favor viral persistence (Xu et al. 2014a). On the
other hand, KCs and lymphoid cells from chronic patients are found to express
Fas-L (Tang et al. 2003) while HBsAg sensitizes liver cells to Fas mediated apopto-
sis. Hence, KCs may play in important role in clearance of infected cells, but at the
same time, cause extensive cell death and injury in an infected liver.
14 Role of Immune Cells in Hepatitis B Infection 215

14.7 Dendritic Cells

The dendritic cells (DCs) are classical antigen presenting cells and their role is
important for antigen presentation and activation of T cells. Two major subtypes of
DCs are plasmacytoid DC (pDC) and myeloid DC (mDC) based on their lineage of
origin. These differ by expression of certain cell surface markers, with pDCs having
a predominant role in antiviral response by producing type I interferons (Kadowaki
2009).
HBV DNA and RNA has been reported to be found in DCs enriched and cultured
from chronic HBV patient’s blood and these DCs showed impaired functioning like
reduced ability to stimulate T cells and reduced secretion of IL-12 (Arima et al.
2003). mDCs show reduction in maturation and secretion of TNF-α while pDCs
produce less interferon-α in in vitro experiments (van der Molen et al. 2004). DCs
generated from patient monocytes in vitro (moDCs) show impaired T cell stimula-
tory function and lower IL-12 secretion, which could be restored with antiviral
treatment (Beckebaum et al. 2003). Similar functional impairment along with the
presence of viral DNA and viral particles within the cells was observed in pDCs
from chronic patients (Beckebaum et al. 2002). Even though DCs take up the viral
particles, they do not support the viral replication or subsequent steps within the
cells (Untergasser et al. 2006). These cells reduce in circulation and enrich in the
liver of immune-active chronic HBV patients (Zhang et al. 2007) while antiviral
treatment (with adefovir) that reduces the viral load shows some restoration of func-
tion as well as number of circulating DCs in the circulation, concomitant with
decreased inflammation in the liver (Van der Molen et al. 2006).
The functional impairment of DCs on interaction with viral particles is expected
to contribute to the inadequate immune response and persistence of virus in chronic
HBV infection. Reviving DC function in chronic infections would be one of the
ways to overcome the tolerogenic environment in chronic infections. It has been
observed in one study that monocytes retained a depot of HBsAg and on differenti-
ating them into DCs in vitro, they were able to crosspresent the antigen to T cells
(Gehring et al. 2013). Hence an idea of using in vitro activated autologous DCs as
vaccine in chronic patients has been explored with some success in achieving
immune clearance in these patients (Chen et al. 2005; Luo et al. 2010). On treating
MoDCs from patient’s PBMCs, with HBV sub-viral particles like HBsAg and
HBcAg, they were found to have strong capacity to stimulate T cell proliferation
and produce IL-12, showing some potential for autologous therapy for chronic
HBV patients (Shi et al. 2007). Hence, MoDCs have been considered to help boost
HBV-specific T cell response in chronic HBV patients with hepatocellular carci-
noma (HCC).
Studies have also been done to understand the interaction of the virus with DCs
and mechanism of the dysfunction. Dendritic cells would be one of the first cells to
sense the virus through its Toll-like receptor (Ma et al. 2018). It has been found that
HBV can downregulate the expression of TL9 and the subsequent IFN-α production
from pDCs (Vincent et al. 2011) which is the case in chronic HBV patients (Xu
et al. 2012). Targeting activation of Toll-like receptors, specially use of TLR7 ligand
216 P. Sinha and P. Sahu

GS-9620, to elicit an antiviral response is an attractive idea and has been tested with
some success in Woodchuk HBV model (Menne et al. 2011) and chimpanzee model
(Lanford et al. 2013); however, it was not as effective in patients (Janssen et al.
2018). Another approach was the use of nanoparticles containing HBV-CpG and
HBsAg which would activate TLR9, and was used with success in mouse in elicit-
ing an enhanced CD4+ T cell response (Lv et al. 2014). TLR 4 and 9 agonists are
already used as adjuvants for HBV vaccines while agonists for TLR 3, 7, and 8
show potential and need to be further investigated for use in therapeutics (Ma et al.
2018).

14.8 Natural Killer Cells

Natural killer (NK) cells are from the lymphoid lineage, and are involved mainly in
innate immune response against intracellular pathogens and abnormal cells. They
exert their antiviral activity by recognition and killing of infected cells and secretion
of antiviral cytokines like IFN-γ and TNF-α. They express CD56 and can be catego-
rized as two subsets, i.e., CD56bright, which have a predominant function of antiviral
cytokine secretion and CD56dim, which are more involved in killing target cells (Wu
et al. 2015). NK cells also promote release of antiviral cytokines like IFN-α and
IL-6 from pDCs. IFN-α in turn upregulates cyotoxic activity of NK cells and IL-6
promotes B cell differentiation (Della Chiesa et al. 2006). NK cells are the most
abundant innate immune cells in the liver (Faure-Dupuy et al. 2017) and are one of
the first cells to respond to HBV infection in patients which would allow a timely
adaptive immune response to be induced (Fisicaro et al. 2009).
Natural killer cells have been found to increase in number post-peak viremia in
acute HBV infection, where in a transient reduction of NK cells and T cell responses
coincided with a surge in IL-10 immunosuppressive cytokine (Dunn et al. 2009).
After peak viremia, an increase in TRAIL (TNF-related apoptosis-inducing
ligand)-expressing CD56bright NK cells is observed and is believed to be responsible
for clearance of the infected cells (Stelma et al. 2017) while these are also respon-
sible for elimination of HBV-specific CD8+ T cells (Peppa et al. 2013) and CD4+ T
cells (Ghosh et al. 2016) which show an exhausted phenotype and express death
receptors in chronic infections. Patients who clear an acute infection have fully
functional and activated NK cells which correlate with serum ALT levels and
inversely with serum viral load, while NK cells from chronic HBV patients pro-
duce IL-10 elicited by HBV-induced monocytes (Li et al. 2017), have diminished
IFN-γ secretion but enhanced cytolytic activity (Zhao et al. 2012), correlating with
liver histopathology and serum ALT levels especially in patients in the immune-
active phase (Zhang et al. 2010a, b). This skew in the NK cell function, correlated
with serum viral load and antiviral therapy partially restored normal NK cell func-
tioning and interferon production by these cells (Tjwa et al. 2011; Stelma et al.
2015).
Major liver damage is caused by the increased cytolytic activity of NK cells in
chronic HBV infection. Hence, it has been proposed to block NKG2D receptor of
14 Role of Immune Cells in Hepatitis B Infection 217

NK cells to alleviate the severe liver damage caused by these cells (Huang et al.
2013). PD-1/PD-L1 blockade is another popular potential strategy which would
restore IFN-γ production of NK cells, along with alleviating T cell exhaustion.
Further study to understand NK cell function in HBV infection will reveal more
therapeutic targets for treatment of HBV infection.

14.9 Neutrophils

Neutrophils are the most abundant immune cells which actively eliminate patho-
gens and target cells. However, over-activation can lead to extensive tissue damage,
as happens in hepatitis (Xu et al. 2014b). Reports of direct interaction of the virus
and neutrophils is scant, but these cells have a more significant role in the secondary
effects of the viral infection. Though one study noted the immunosuppressive effect
of recombinant HBeAg by suppression of respiratory burst in neutrophils, as well as
their chemotactic functions in vitro (Leu et al. 2014), most studies are focused
toward its role in the pathogenesis of injury in hepatitis.
In a mouse model of HBV infection, it was observed that transferring HBV-­
specific cytotoxic T cells resulted in recruitment of nonspecific inflammatory cells
that caused liver pathology. In this setting, depletion of neutrophils reduced the
recruitment of antigen nonspecific inflammatory cells, hence ameliorating the
severity of liver damage, but did not affect antiviral activity of CTLs. It can be
inferred that the neutrophils have a role in the pathogenesis of the disease and may
serve as a target for immunotherapeutic approaches (Sitia et al. 2002). Concurrently,
blocking a serine protease like neutrophil elastase with an inhibitor, reduced serum
ALT levels, and the migration of inflammatory cells in the liver, post injection of
antigen-specific CTLs in a HBV mouse model, and hence administration of neutro-
phil elastase inhibitors may hold therapeutic potential against severe hepatitis (Takai
et al. 2005). The recruitment of neutrophils can be due to the expression of CXCL-8
by CTLs which recruits and elicits cytotoxic activity of neutrophils (Gehring et al.
2011) by interacting with the CXCR1 and CXCR2 on neutrophils (Nasser et al.
2009). Hence another approach could be to block CXCR1/CXCR2, which was
found to be increased in neutrophils in HBV-related acute-on-chronic liver failure
(ACLF) patients. Blocking CXCR1/CXCR2 had significantly reduced cell death in
in vitro experiments with hepatic cell lines (Khanam et al. 2017).
The neutrophil-to-lymphocyte ratio (NLR) has been widely accepted as reflec-
tion of inflammatory status of the patient before treatment and has been used in the
context of hepatitis B infection to predict acute-on-chronic liver failure in patients.
Low NLR indicates better prognosis, while higher NLR indicates a high short-term
mortality rate (Liu et al. 2014a). The NLR has been found to be a useful predictive
marker in patients with HBV-related decompensated cirrhosis (Zhang et al. 2016),
ACLF patients treated with artificial liver support system (Fan et al. 2017), in
patients receiving PEG interferon treatment (Le et al. 2017), and may be applied to
many other similar conditions.
218 P. Sinha and P. Sahu

14.10 Conclusions and Future Perspectives

Hepatitis B virus infection is causative of significant number of deaths each year.

While a robust immune system manages to clear the infection effectively, a chronic
infection is a result of a combination of viral and host factors, of which the immune
system plays an integral role. While much of the mechanism of viral clearance or
persistence and development of pathology is understood, many aspects still remain
obscure. Ambiguity in several aspects exists in the available literature, which may
be due to observations from different experimental settings and patient cohorts. The
narrow host range and cell specificity of HBV also makes experimental work chal-
lenging. However, the undisputed roles of the major immune cells in the pathogen-
esis, along with the significant observations, have been summarized in this report.
The key players involved in the process of immune clearance and immune-­pathology
would be the logical targets for developing novel therapeutic approaches.
CD8+ T cells are the most important cells involved in clearance of the virus
through production of antiviral cytokines like IFN-γ and TNF-α which arrest viral
replication, as well as by cytolysis of infected cells and requires help from CD4+ T
cells and dendritic cells for its activation. However, in a setting where the viral load
far exceeds the CD8+ response, this subset becomes exhausted and express exhaus-
tion markers like PD-1, CTLA-4, Tim-3, and CD244, which in turn marks these
cells for removal by natural killer cells. The role of Tregs and Bregs is to prevent
over-activation of the immune cells and control extensive histological damage by
secretion of immunosuppressive cytokines, during phases of peak T cell activation.
However, their sustained activity is believed to contribute to immune tolerance in
chronic infections. Th17 is another subset of CD4+ T cells, which secretes pro-­
inflammatory IL-17 and involved in development of liver fibrosis and Treg/h17 ratio
has been found to be of value in prognosis of the disease.
There is scarce literature about role of B cells in chronic infections, while they
certainly play a major role in immune clearance in acute, self-resolved infections by
secretion of anti-HBs antibodies. The effect of the virus on monocytes and macro-
phages is also ambiguous since there are reports of these cells interacting with the
virus; however, it is not clear if these cells respond with an immunostimulatory or
immunosuppressive effect. Dendritic cells have been found to be mostly impaired in
chronic infection setting and many research efforts have been to activate them
in vitro so that they may activate the T cells on being administered in vivo. Activated
pro-inflammatory CD16+ monocytes, tissue resident Kupffer cells, natural killer
cells, and neutrophils are believed contribute to liver injury by inducing cell death
in the liver, in an attempt to clear infected hepatocytes. The neutrophil-to-­lymphocyte
ratio is another predictive value used to gauge the inflammatory status of the patient
and make a prognosis.
The limitations of the current therapy in treatment of HBV infection, i.e., inter-
feron therapy or nucleos(t)ide analogs, warrants the development of more effective
therapies. Advancement in the understanding of the disease pathogenesis re-empha-
sizes the role of the immune system in clearance of the infection, which when mal-
functions, leads to chronic infection, thereby also serving as potential targets for
14 Role of Immune Cells in Hepatitis B Infection 219

treatment. Most immunotherapeutic efforts is toward enhancing CD8+ T cells func-

tion in chronic infections by either reactivating impaired T cells by blocking inhibi-
tory molecules (PD-1, CTLA-4, etc.) or by adoptive transfer of HBV-specific T
cells. Antibody response may also be boosted by vaccination and in addition to the
prevalent HBsAg recombinant vaccine, the potential of DNA-based vaccines is
being evaluated in clinical trials (Fontaine et al. 2015). Some of the significant
potential therapeutic strategies targeting the innate immune branch focus on use of
TLR agonists and antiviral and immunostimulatory cytokines (Bertoletti and Le
Bert 2018). Minimizing liver damage caused due to over-activation of some of the
immune cells like NK cells and neutrophils, leading to fibrosis and cirrhosis, may
also prove beneficial for recovery of the patient. A single therapeutic strategy may
not be sufficient for an effective treatment, depending on the degree of pathology in
a patient. Hence a combination of therapies targeting various viral and immune fac-
tors promises to be the most effective therapeutic approach for treatment of HBV
infection.

Conflict of Interest The authors declare that they have no competing interests.

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Significance of RNA Sensors
in Activating Immune System 15
in Emerging Viral Diseases

Preethika Nair and Siddhesh U. Sapre

Abstract
Viruses are known for their utilization of the host machinery to aid their replica-
tion within the infected cells. The viral nucleic acid, their intermittent forms con-
tain pathogen-associated molecular patterns (PAMP) which enable the pattern
recognition receptors (PRR) to distinguish them from self and mount a response
against them. Highly robust targets for the PRRs are the cell entry points and
phases of viral replication. RNA-sensing PRR can be classified as endosomal
PRR and cytosolic PRR. Both enveloped and non-enveloped viruses utilize endo-
somal compartments to undergo proteolytic cleavage or pH-­dependent conforma-
tional changes to ensure membrane fusion. Thus, monitoring the endosomal
compartments in order to restrict the virus from penetrating into the cytoplasm is
a key antiviral strategy. Endosomal compartments are guarded by Toll-like recep-
tors (TLRs) which are the earliest discovered PRRs. TLR3, 7, and 8 specifically
cater to viral RNA sensing. They can detect dsRNA and stable stem structures of
ssRNA. Cytosolic PRRs include RIG-I like receptors (RLR) and nucleotide-bind-
ing oligomerization domain-containing (NOD)—like receptors (NLR). RLRs are
cytosolic helicases which detect viral RNA, ds RNA, short 5′ppp RNA, and RNase
L cleaved RNA. Retinoic acid-inducible gene I product (RIG-I), melanoma differ-
entiation-associated antigen 5 (MDA5), and laboratory of genetics and physiology
2 (LGP2) are aspartate-glutamate-any amino acid-aspartate/histidine (DExD/H)-
box helicases belonging to the SF2 superfamily. Other RNA helicases like
SNRNP200 which belongs to the Ski-2 superfamily, or DDX60 belonging to the
Ski-2-like helicase family are also involved in viral RNA sensing. NLRs like
NLRP3 and NOD2 are cytosolic RNA-sensing PRRs. In addition to these, RNA-
binding proteins like RNase L, protein kinase R (PKR), and interferon-inducible

P. Nair (*)
NMIMS School of Science, Mumbai, India
S. U. Sapre
National Institute of Virology, Pune, India

© Springer Nature Singapore Pte Ltd. 2020 229

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_15
230 P. Nair and S. U. Sapre

transmembrane protein (IFIT) are also present which play pertinent roles to
enhance the antiviral immunity during a viral infection. Recognition of PAMPs or
DAMPs by PRRs activate various signaling cascades which ultimately triggers the
transcription of type I/III IFN, production of various pro-inflammatory cytokines,
and various other genes that can ensure an intracellular antiviral state which will
aid in containing the viral infection. Furthermore, these interferons can lead to the
expression of multiple interferon-stimulated genes (ISGs) which induce antiviral
activities controlling the life cycle of the virus, restricting its replication, and
transmission to the surrounding cells. This chapter will elaborate on these RNA-
sensing PRRs, their structures, agonists, mechanism of activation, and response
mounted against viral infection.

Keywords
Host machinery RNA sensors · Viral diseases · Immune activation

15.1 Introduction

Viruses are known for their utilization of the host machinery to aid their replication
within the infected cells. The viral nucleic acid, its intermittent forms contain
pathogen-­associated molecular patterns (PAMP) which enable the pattern recogni-
tion receptors (PRR) to distinguish them from self and mount a response against
them. Highly robust targets for the PRRs are the cell entry points and phases of viral
replication. RNA-sensing PRR can be classified as endosomal PRR and cytosolic
PRR. Both enveloped and non-enveloped viruses utilize endosomal compartments
to undergo proteolytic cleavage or pH-dependent conformational changes to ensure
membrane fusion. Thus, monitoring the endosomal compartments in order to restrict
the virus from penetrating into the cytoplasm is a key antiviral strategy. Endosomal
compartments are guarded by Toll-like receptors (TLRs) which are the earliest dis-
covered PRRs. TLR3, 7, and 8 specifically cater to viral RNA sensing. They can
detect dsRNA and stable stem structures of ssRNA.
Cytosolic PRRs include RIG-I like receptors (RLR) and nucleotide-binding
oligomerization domain-containing (NOD)—like receptors (NLR). RLRs are cyto-
solic helicases which detect viral RNA, ds RNA, short 5′ppp RNA and RNase L
cleaved RNA. Retinoic acid-inducible gene I product (RIG-I), melanoma
differentiation-­associated antigen 5 (MDA5), and laboratory of genetics and physi-
ology 2 (LGP2) are aspartate-glutamate-any amino acid-aspartate/histidine (DExD/
H)-box helicases belonging to the SF2 superfamily. Other RNA helicases like
SNRNP200 which belongs to the Ski-2 superfamily, or DDX60 belonging to the
Ski-2-like helicase family are also involved in viral RNA sensing. NLRs like NLRP3
and NOD2 are cytosolic RNA-sensing PRRs. In addition to these, RNA-binding
proteins like RNase L, protein kinase R (PKR), and interferon-inducible transmem-
brane protein (IFIT) are also present which play pertinent roles to enhance the anti-
viral immunity during a viral infection.
15 Significance of RNA Sensors in Activating Immune System in Emerging Viral… 231

Recognition of PAMPs or DAMPs by PRRs activate various signaling cascades

which ultimately triggers the transcription of type I/III IFN, production of various
pro-inflammatory cytokines, and various other genes that can ensure an intracellular
antiviral state which will aid in containing the viral infection. Furthermore, these
interferons can lead to the expression of multiple interferon-stimulated genes (ISGs)
which induce antiviral activities controlling the life cycle of the virus, restricting its
replication and transmission to the surrounding cells. This chapter will elaborate on
these RNA-sensing PRRs, their structures, agonists, mechanism of activation, and
response mounted against viral infection.

15.2 Innate Immunity: Soldiers Against Viruses

Innate immunity provides the first line of defence against the invading pathogens. It
exerts its role by distinguishing between self and non-self, subsequently initiating a
host response against the invading pathogen. Innate immunity is triggered by the
identification of PAMPs (pathogen-associated molecular patterns) via the PRRs
(pattern recognition receptors). PRRs are germline-encoded proteins expressed by a
variety of cells which can carry out surveillance to detect any pathogenic invasions.
PRRs have been classified into various families based on protein domain homology;
Toll-like receptors (TLRs), RIG-I like receptors (RLRs), NOD-like receptors
(NLRs), C-type lectin receptors (CLRs), AIM2-like receptors (ALRs), and cytosol
DNA sensing PRR cyclic GMP-AMP synthase (cGAS). PRRs not only survey the
PAMPS but also the DAMPS (danger-associated molecular patterns), which are
typically the cellular products produced as a response to cell stress (Anafu et al.
2013; Chen et al. 2017; Chow et al. 2018; Said et al. 2018).
Recognition of PAMPs or DAMPs by PRRs leads to transcription of genes involved
in pro-inflammatory responses. The responses mounted against a viral infection,
upregulates type I and III interferons, pro-inflammatory chemokines, and various
other genes that can ensure an intracellular antiviral state in order to contain the infec-
tion. Furthermore, these interferons can lead to the expression of multiple interferon-
stimulated genes (ISGs) which induce antiviral activities controlling the life cycle of
the virus, restricting its replication and transmission to the surrounding cells.
Viruses are known for their utilization of the host machinery to aid their replica-
tion within the infected cells. The viral nucleic acid, their intermittent forms contain
PAMPs which enable the PRRs to distinguish them from self and mount a response
against them. Highly robust targets for the PRRs are the cell entry points and phases
of viral replication. Both enveloped and non-enveloped viruses utilize endosomal
compartments to undergo proteolytic cleavage or pH-dependent conformational
changes to ensure membrane fusion (Said et al. 2018). Thus, monitoring the endo-
somal compartments in order to restrict the virus from penetrating into the cyto-
plasm is a key antiviral strategy. Endosomal compartments are guarded by Toll-like
receptors (TLRs) which are the earliest discovered PRRs. TLR3, 7, and 8 specifi-
cally cater to viral RNA sensing. Cytosolic PRRs include RIG-I, MDA5, LGP2,
NLRP3, and NOD2. RNA sensing is also carried out by other RNA helicases like
232 P. Nair and S. U. Sapre

SNRNP200 and DDX60 which will be discussed in details. In addition to these,

RNA-binding proteins like RNase L, PKR, and IFITs are also present which play
pertinent roles to enhance the antiviral immunity during a viral infection.

15.3 RNA-Sensing Endosomal PRRs

15.3.1 Toll-Like Receptors (TLRs)

TLRs are a very important and earliest detected type of PRRs. They comprise of 11
genes on the human genome. Majority of the TLRs are associated with the plasma
membrane. However, the TLRs associated with RNA sensing namely, TLR3, 7, and
8 are present in the endosomal compartment. TLRs are type I transmembrane pro-
teins which consists of N-terminal ectodomain or extracellular leucine-rich domain
(ECD), middle transmembrane domain (TM), and C-terminal cytoplasmic Toll/IL-1
receptor (TIR) domain. Leucine-rich regions (LRRs) in the ECD are attached into a
horseshoe-shaped solenoid structure. α-helices of each LRR forms into the convex
structure of the solenoid and the β sheets assemble into the concave surfaces of the
solenoid structure. It is a unique property of the TLRs to bind their agonists to the
lateral convex surface instead of concave surface.
The formation of M-shaped dimer or multimer is needed for all TLR activation,
so that the C-terminal regions of the two TLR ECDs are brought into proximity. It
in turn causes the multimerization of cytoplasmic TIR domains, which will recruit
downstream adaptors TRIF or MyD88 through homotypic interaction, further form-
ing signaling complex called signalosome and activating downstream transcription
factors like NF-κB which induces pro-inflammatory cytokines and interferon regu-
latory factor (IRF) which in turn induces type I interferon.

15.3.1.1 TLR3: Expression, Structure, and Signaling Pathway

Immune cells like monocytes, macrophages, conventional dendritic cells, NK cells,
T and B lymphocytes, mast cells, eosinophils, and basophiles demonstrate the
expression of TLR3 in their endosomal compartments (Chen et al. 2017; Said et al.
2018; Gantier and Williams 2011; Hewson et al. 2005; Thomas et al. 2007). They
are also expressed in nonimmune cells like keratinocytes, fibroblasts, hepatocytes,
astrocytes, and microglia. TLR3 senses dsRNA; however, stable stem structures of
ssRNA are also recognized by TLR3. Its structure consists of N-terminal ectodo-
main or extracellular 23 leucine-rich domain (ECD), middle transmembrane domain
(TM), and C-terminal cytoplasmic Toll/IL-1 receptor (TIR) domain. The 23 LRR
ECD is responsible for the viral dsRNA binding. Dimerization of the ECD initiates
the signaling cascade wherein TIR domain-containing adaptor protein-inducing
IFN-β (TRIF) is then recruited and undergoes slight conformational changes to
form a signaling complex together with TNF receptor-associated factor 6 (TRAF6),
TRAF3, TBK1, IKKε, and IKK. This leads to the activation of IRF3/IRF7 and
NF-κB, which results in the production of type 1 IFNs and inflammatory cytokines,
respectively.
15 Significance of RNA Sensors in Activating Immune System in Emerging Viral… 233

Further, there are positive regulators to this pathway like S100A9 which acts dur-
ing the early stages of TLR3 activation by easing the maturation of TLR3-containing
early endosomes into late endosomes, etc. Similarly, there are negative regulators
such as Rho proteins that decrease the production of pro-inflammatory cytokines
upon TLR3 triggering. The pathway is also regulated via post-translational modifi-
cations.TLR3 has been implicated in pathogenesis of HCV, infection, HSV-1, HBV,
etc. This makes TLR3 an interesting candidate to prod into antiviral therapies.

15.3.1.2 TLR7 and 8: Expression, Structure, and Signaling Pathway

TLR7/8/9 subfamily members are localized at endosomes of the cells. They are
present on the endosomes of monocytes, macrophages, plasmocystoid dendritic
cells, NK cells, and T and B cells. TLR8 is also present on mast cells and Tregs.
Both have very similar ligand recognition and intracellular signaling. TLR7 and 8
are activated by small molecular agonists and GU or U rich single-stranded RNA
(ssRNA) (Chen et al. 2017; Gantier and Williams 2011; Crozat and Beutler 2004;
Jensen and Thomsen 2012; Jurk et al. 2002; Patel et al. 2014). In viral genomes,
untranslated terminal regions (UTR) are GU or U rich. They are highly conserved
sequences as they are involved in viral protein translation and RNA replication.
Further, they can also recognize phagocytosed vRNA, by the adenosine-to-­inosine
(A-to-I) editing, which is an important arm of the antiviral response. But
2′-O-methylation within an RNA sequence leads to the triggering of TLR8 but not
TLR7.
The ECDs of both TLR7 and 8 have 26 LRR motifs in their extracellular domain,
which contain multiple insertions such as the Z-loop or undefined region situated
between LRRs 14 and 15. Cathepsins and arginine endopeptidase cleave the TLR7
and 8 in the endosomes, at this z-loop. Once cleaved, the fragments are still associ-
ated to each other by the intermolecular interactions as they both are crucial for the
receptor activation. Recent crystal structures have demonstrated the presence of two
binding sites: one for a small chemical stimuli or degradation product of ssRNA and
the other which recognizes the viral ssRNA. At steady state they both exist as dimers.
However, on association with the agonists, the conformation changes causing multi-
merization of the cytoplasmic TIR domains. A downstream adaptor MyD88 is
recruited through homotypic interaction; a signaling complex called myddosome is
formed involving IRAK4, IRAK1, TRAF6, and TRAF3 and downstream transcrip-
tion factors NF-κB and IRF7 are activated to induce pro-­inflammatory cytokines and
IFNs, respectively (Fig. 15.1). Furthermore, TLR7 recognizes Streptococcus Group
B (SGB) RNA and TLR8 recognize the RNAs from Escherichia coli, Mycobacteria
bovis, Helicobacter pylori, and Borrelia burgdorferi.

15.4 RNA-Sensing Cytosolic PRRs

The first line of cytosolic RNA sensors include the PRR families RLRs and
NLRs. RLRs consists of cytosolic helicases: RIG-I, MDA5, and LGP2; other
DEXD/H-­box helicases like DDX3, DDX60, and SNRP20 (Chow et al. 2018;
Dang et al. 2018).
234 P. Nair and S. U. Sapre

Fig. 15.1 Structure and mechanism of action of endosomal PRRs—TLR3, 7/8

15.4.1 RIG-I Like Receptors (RLRs)

Cytosolic helicases are ubiquitously expressed in low levels in most cell types.
They belong to a family of aspartate-glutamate-any amino acid-aspartate/histi-
dine (DExD/H)-box helicases (SF2 superfamily). RLRs are also known as double-­
stranded RNA (dsRNA)-dependent ATPases. Their function includes sensing the
viral RNA in the cytosol, binding to the non-self RNA stably, and imposing the
innate immune response against the target RNA. Three members widely studied
are the retinoic acid-inducible gene I product (RIG-I), melanoma differentiation-­
associated antigen 5 (MDA5), and laboratory of genetics and physiology 2
(LGP2).
15 Significance of RNA Sensors in Activating Immune System in Emerging Viral… 235

Overall structure of the RLRs are as follows:

1. Zinc-binding C-terminal domain (CTD), otherwise known as the repressor

domain (RD)
2. Attached to the CTD is a central RNA helicase core consisting of two RecA-like
helicase domains promotes dsRNA recognition.
3. Further, RIG-I and MDA5 contain at the N-terminus tandem caspase activation
and recruitment domains (CARDs) that enables signaling capabilities. LGP2
uniquely lacks this domain.

15.4.1.1 RIG-I
When disengaged, the RIG-I CARDs and RD are bound to the helicase region and
this leads to autoinhibition (Chow et al. 2018; Dang et al. 2018; Pichlmair et al.
2006). During viral replication the CTD/RD detects and associates with the viral
RNA along with the helicase, exposing the CARDS. These CARDs are no longer
autorepressed; the RLRs oligomerize and further associate with the mitochondrial
antiviral signaling adaptor called MAVS (also referred to as IPS-1/VISA/cardif).
MAVS are located at the cytosolic face of the outer mitochondrial membrane, and
this mitochondrial association is necessary for initiating further signaling events.
On association the MAVS undergo a prion-like aggregation. This leads to the
recruitment of E3 ubiquitin ligases and the downstream effector proteins TNF
receptor-associated factor 2 (TRAF2), TRAF3, and TRAF6 which assembles into
an active “signalosome.” The resulting cascade leads to the phosphorylation and
nuclear translocation of key innate immune transcription factors IRF3 and IRF7.
Further, it leads to the activation of NF-κB to drive the expression of type I and III
interferons, innate immune genes, pro-inflammatory cytokines, and chemokines
that will aid in containing the virus infection.
The RD domain is not only critical for RNA sensing but also confers it with the
displayed selectivity. RIG-I can recognize 5′ppp dsRNA, 5′pp dsRNA, pU/UC
genomic RNA, AU-rich 3 UTR, RNase L cleavage products and circular viral RNA
(Chow et al. 2018; Said et al. 2018; Dang et al. 2018; Liu and Gale Jr 2011;
Yoneyama and Fujita 2007; Zou et al. 2009). The distinguishing between self and
non-self RNA is based on the 5′-7-methylguanosine cap, which is subject to
2′-O-methylation.

15.4.1.2 MDA5
RIG-I and MDA5 being homologues showcase the same overall domain structures.
The RD domain however recognizes long dsRNA which under normal circum-
stances is absent in an uninfected cell. The presence of this PAMP signifies vial
invasion which activates MDA5 and stimulates a cytokine response similar to the
RIG-I. MDA5 binds to the long dsRNA replicative intermediates generated by
picornavirus, consisting of its positive-sense genome annealed to the negative-sense
antigenome, AU-rich motifs and RNase L cleavage products (Said et al. 2018; Dang
et al. 2018; Bruns et al. 2014; Rodriguez et al. 2014).
236 P. Nair and S. U. Sapre

On binding with the target RNA it undergoes a conformational change and the 2
CARD domains are exposed. The 2 CARDS assemble into a stable tetramer due to
its binding with the dsRNA and they form a filament structure. They therefore are
independent on the polyubiquitin chain binding; the tetramer itself nucleates the
MAVS, which in turn activates TRAF3/TBK1/IKKe/IRF3 and TRAF6/IKK/NF-jB,
which drive IFN and pro-inflammatory cytokine expression, respectively.

15.4.1.3 LGP2
Although LGP2 is homologues to RIG-I and MDA5, it lacks the CARD domain
which restricts it from binding to the MAVS. However, LGP2 is shown to bind to
the termini of blunt-ended dsRNA of different lengths with high affinity, forming
complexes with 2:1 stoichiometry. LGP2 has been both implicated as a positive and
negative effector of RIG-I and MDA5 response (Said et al. 2018; Jensen and
Thomsen 2012; Bruns et al. 2014; Rodriguez et al. 2014). Studies have shown that
CTDs of LGP2 and RIG-I are analogous because of which LGP2 CTD interacts
with RIG-I to abolish its ability to initiate antiviral signaling. RIG-I also undergoes
attenuation of viral sensing. LGP2 has also been believed to utilize its ATP depen-
dent/RNA helicase activity to increase the interaction of nucleic acid PAMPS for
improved antiviral RLR signaling. LGP2 has also been implicated for its activity
against DICER protein to maintain the cytosolic PAMPs in intact condition for
detection by the cytosolic RNA sensors. Therefore, LGP2 overall is a modulator of
the innate immune response to a viral infection and not a sensor of PAMPs. LGP2
does not initiate antiviral gene expression.

15.4.1.4 Other DEXD/H-Box Helicases

Certain helicases which are a member of the DEXD/H-box helicases family have
been anticipated to function in the RLR-based RNA sensing. DDX3 is one such heli-
cases which associates itself with MAVS, and when it encounters transfected dsRNA,
it induces interferon production. DDX3 has also been suggested to aid as a scaffold
for the assembly of an RLR signaling complex, promoting innate immunity signaling
(Chow et al. 2018; Said et al. 2018; Jensen and Thomsen 2012). DDX3 is being stud-
ied further to understand the function and mechanism of action in RNA sensing.
Another DEXD/H-box Helicase DDX60 (Ski-2-like helicase) is implicated in
the cytosolic antiviral response. DDX60 maintains the quality of host RNA by act-
ing as an exosome complex to degrade other foreign RNAs. Exosome-mediated
degradation also enables the degraded vRNA to be recognized by RIG-I/MDA5.
DDX60 also functions as an ISG to suppress viral infection in an infected cell.
vRNA and RIG-I/MDA5 interactions are brought together by DDX60 in order to
enhance the type 1 interferon production. Thus, DDX60 functions on two levels
augmenting the antiviral response by detecting the vRNA and subject them to the
RIG-I/MDA5 based RLR pathway; further, they aid in revealing the molecular sig-
nature (PAMP) of the vRNA via the exosome-mediated degradation, making
DDX60 a immune-stimulatory molecule.
SNRNP200 is another member of the Ski-2 RNA helicase family. It has been
recently discovered for promoting vRNA sensing and IRAF3 activation by interact-
ing with TBK-1, found in the RIG-I/MAVS signaling pathway. SNRP200 is a mem-
ber of the spliceosome complex for removal of introns. During viral infection, the
15 Significance of RNA Sensors in Activating Immune System in Emerging Viral… 237

amino terminal of SNRP200 binds to the vRNA, perinuclear relocation occurs and
it aids as an adaptor o trigger IRF3 signaling.

15.4.2 NLRs

Nucleotide-binding oligomerization domain-containing (NOD)-like receptors

(NLR) are divided into five subfamilies (based on the N terminal effector domains):
NLRA (acid activation domain), NLRB (baculovirus inhibitor of apoptosis repeats),
NLRC (caspase activation and recruitment domain CARD), NLRP (pyrin domain
PYD), and NLRX (unknown domain). NLR is another family of cytosolic RNA-­
sensing PRRs. All NLRs upon activation lead to inflammasome formation except
NOD1 and NOD2. General structure of NLRs are as follows:

1. N-terminal effector domain

2. Followed by nucleotide-binding and oligomerization domain (NOD)
3. C-terminal leucine-rich repeats (LRRs).

NLRC2 (NOD2) recognizes ssRNA viruses. Recognition leads to association

with MAVS. This interaction is LRR-dependent and nucleotide binding domains.
This triggers the MAVS-dependent pathway which leads to type I IFN and pro-­
inflammatory cytokine release.
NLRC5 is another NLR which has a distinguished longer LRR domain which
thereby forms a helical structure instead of the horseshoe shape. Upon interaction
with the viral DNA it induces IFN-mediated JAK-STAT (Janus kinase-signal trans-
ducer and activator of transcription) pathway. NLRC5 is an amplifier of the antiviral
responses.
NLRP3 on the other hand has a pyrin domain in its N-terminal which enables it
to interact with the PYD in the N terminal of apoptosis-associated speck-like pro-
tein containing a CARD (ASC). The CARD domain in the C-terminal of ASC fur-
ther activates caspase-1, leading to the formation of fully functional IL-1 and IL-18.
NLPR3 can be activated by lysosomal disintegration, membrane disruption, or gen-
eration of ROS caused due to viral PAMPS and DAMPS; therefore, it serves as an
indirect sensor of viral invasion (Fig. 15.2).

15.4.3 RNA-Binding Proteins

Multiple RNA-binding proteins are also involved directly or indirectly in the pro-
cess of vRNA sensing.

15.4.3.1 RNase L/Oligoadenylate Synthase (OAS)

RNase L is an antiviral endoribonuclease. Oligoadenylate synthetase (OAS) cata-
lyzes the conversion of ATP into 2-5-linked oligoadenylates (2-5A) that in turn
become the second messengers that bind to and activate RNase L (Chakrabarti et al.
2015; Malathi et al. 2007; Silverman 2007). vRNAs, cellular self-RNA are
238 P. Nair and S. U. Sapre

Fig. 15.2 Structure, activation, and mechanism of action of cytosolic PRRs—RIG-I, MDA5, and
LGP2. Overview of endosomal and cytosolic RNA sensors

substrates to RNase L. The cleavage products are utilized for increasing the activa-
tion of RIG-I/MDA5. These are being studied in order to use them as targets for
antiviral therapies (Sarkar 2014).

15.4.3.2 Protein Kinase R (PKR)

PKR functions by inhibiting translation of the viral infected cell (Dauber and Wolff
2009; Zhu et al. 2008). Being a serine/threonine kinase it gets activated on binding
to a viral dsRNA/short dsRNA with 5′ppp ends and limited secondary structures
(Dauber and Wolff 2009; Gil and Esteban 2000), ultimately inhibiting the eukary-
otic translation initiation factor 2A (eIF2A). Antiviral response by PKR is also
mediated by stabilizing MDA5.

15.4.3.3 Interferon-Inducible Transmembrane Protein (IFIT)

Multiple viruses induce the interferon response via both innate and adaptive immune
responses. This leads to the stimulation of ISGs like IFIT which triggers antiviral
cell-intrinsic restriction factors. Though the exact molecular mechanism is yet to be
elucidated, they have showcased inhibition of viral translation or the sequestration
of viral RNA to inhibit either virus replication or its packaging into new virions
(Anafu et al. 2013; Diamond 2014; Vladimer et al. 2014). Furthermore, IFIT acts on
the late endosomes blocking the endosomal viral entry. IFIT1 and IFIT5 recognize
5′ppp ssRNA. IFIT2 recognizes U-rich RNAs in vitro, independent of a 5′ppp.
 15

Endosomal RNA sensors Cytosolic RNA sensors RNA-binding proteins

TLRs Rlrs-SF2 family cytosolic helicases NLRs
TLR3 TLR7 TLR8 RIG-I MDA5 NLRC2 (NOD2) NLRP3 RNase L PKR IFIT
Cellular Endosomes Endosomes of Endosomes of Cytoplasm of Cytoplasm of Cytoplasm. Cytoplasm Nucleoplasm Activated PKR Mitochondria
localization, of immune monocytes, monocytes, all cell types all cell types Macrophages, Ubiquitously is fund in both and
distribution cells except, macrophages, macrophages, monocytes, expressed cytoplasm and cytoplasm
non-immune plasmocystoid dendritic cells, DCs nucleus
cells like dendritic cells, NK cells, T
keratinocytes, NK cells, T and B cells
fibroblasts, and B cells also in mast
hepatocytes, cells and Tregs
astrocytes
and microglia
Agonists dsRNA, GU or U rich GU or U rich 5′ppp-dsRNA, Long dsRNA Bacterial and Pathogen vRNAs, Viral dsRNA, 5′ppp
stable stem single-­stranded single-­stranded short dsRNA virus RNA ssRNA/ cellular short dsRNA ssRNA,
structures of RNA (ssRNA) RNA (ssRNA) dsRNA and self-RNA with 5′ppp ends U-rich RNAs
ssRNA other distinct and limited in vitro
set of ligands secondary
structures
Mechanism dsRNA Z-loop Z-loop Polyubiquitin Filament Tetramerisation Potassium Binding to Dimersiation on Interferon
of activation induced proteolytic proteolytic chain binding formation efflux-­NEK7 the agonist, encountering dependent
dimerization cleavage, and cleavage, and mediated mediated involved apoptosis the agonists pathways
receptor dimer receptor dimer receptor receptor NLRP3 ad interferon leads to
conformational conformational tetramerization tetramerization inflammas- dependent auto-
change change ome complex pathways phosphorylation
Cell TRIF-­ MyD88-­ MyD88-­ MAVS-­TRAF3-­ MAVS-­ RIP2-IKKs-­ ASC-­
signaling TRAF3-­ IRAK4/ IRAK4/ TBK1/ TRAF3-­TBK1/ NF-κB inflammasome
pathways TBK1/ IRAK1-­IRF7. IRAK1-­IRF7. IKKe-IRF3. IKKe-­IRF3. caspase-
IKKe-IRF3 MyD88-­ MyD88-­ MAVS-­FADD/ MAVS-­FADD/ 1-IL-1/IL-18
TRIF-­ IRAF6-­IKKs- IRAF6-­IKKs-­ TRAF6-IKKs-­ TRAF6-­IKKs-­
TRAF6-­ NF-κB NF-κB NF-κB NF-κB
IKKs-­NF-κB
Significance of RNA Sensors in Activating Immune System in Emerging Viral…

(continued)
239
 Endosomal RNA sensors Cytosolic RNA sensors RNA-binding proteins
240

TLRs Rlrs-SF2 family cytosolic helicases NLRs

TLR3 TLR7 TLR8 RIG-I MDA5 NLRC2 (NOD2) NLRP3 RNase L PKR IFIT
Recognized MCMV, SeV, flu virus, SeV, flu virus, BOV, MV, SeV, EMCV, RSV, IAV, and Flu virus, SeV Influenza A,
pathogens HSV-1, coxsackie coxsackie NDV, RSV, flu poliovirus and HCMV and bacteria H5N1,
EMCV, WNV virus, vaccinia virus, vaccinia virus, coxasackie Rotavirus
and virus, MV, virus, MV, hantavirus, virus.
enteroviruses RSV, RSV, VSV, RV, HCV, Rotavirus,
retrovirus, retrovirus, E. JEV, dengue virus,
SGB coli, M. bovis, adenovirus, WNV, murine
H. pylori, B. vaccinia virus, hepatitis virus
burgdorferi HSV, L.
monocytogenes,
H. pylori, S.
flexneri.
Rotavirus,
dengue virus,
WNV, murine
hepatitis virus
Crosstalk Positive Positive Positive Negative Negative Negative Positive Positive Positive
regulation by regulation by regulation by regulation by regulation by regulation by regulation by regulation of regulation by
TLR8 NOD2 TLR3, NOD2 TLR3, NOD2 TLR3 RIG-I MDA5 RIG-I/ interferon
Negative MDA5 inducing
regulation by mechanisms
TLR7
P. Nair and S. U. Sapre
15 Significance of RNA Sensors in Activating Immune System in Emerging Viral… 241

Acknowledgments Authors are grateful to NIV Pune and NMIMS Pune for the support extended.

Conflict of Interest The authors declare that they have no competing interests.

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Potentiality of DNA Sensors
in Activating Immune System 16
in Emerging Viral Infectious Diseases

Siddhesh U. Sapre and Preethika Nair

Abstract
Viruses are obligatory intracellular parasites and hijack the host cell machinery
to make more identical copies of it and continue self-propagation. They attach
and replicate in the susceptible and permissive hosts and host derived cell lines.
They enter the cells either through direct attachment, receptor-mediated endocy-
tosis, or phagocytosis. Hence, to thwart the invasion by viruses, hosts have devel-
oped immunity in ascending stages—intrinsic, innate and adaptive immunity. A
robust intrinsic and innate immune response governs an effective adaptive
immune response, should that be needed. Both enveloped as well as non-­
enveloped viruses are subject to distinct types of DNA sensors, subject to their
site of replication. DNA sensors of viral PAMPs can be classified into three
types, based on the location of their PAMPs in the host cellular compartment viz.
cell surface, cytoplasmic and nuclear. The host cell membrane both, surface as
well as intra cellular, is continuously monitored for the non-host, pathogenic
components or PAMPs. Among the intracellular sensors of the viral genome,
there are two types—essentially due to the two types of major viral genomes i.e.
RNA and DNA sensors. The cytosolic DNA sensors include AIM2, IFI16, cGAS,
RNA Pol III, DNA-PK, DDX9, DHX36, DDX41, DDX60, DAI, LRRFIP1,
HMGB, ABCF1 and MRE11. PYHIN family of sensors include AIM2, IFI16,
IFIX and MNDA. Another recently discovered family of sensor called stimulator
of interferon (IFN) genes (STING), specifically houses on the endoplasmic retic-
ulum (ER) and functions in association with its upstream sensor, cGAS. Some
DNA sensors shuttle between the cytosol and nucleus pre- and post-extraneous
DNA binding. These include IFI16, IFIX, RNA Pol III, etc. There is no exclusive
nuclear DNA sensor. Many enzymes known to be present in the cells for their

S. U. Sapre
National Institute of Virology, Pune, India
P. Nair (*)
NMIMS School of Science, Mumbai, India

© Springer Nature Singapore Pte Ltd. 2020 243

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_16
244 S. U. Sapre and P. Nair

obvious primary functions also additionally function as DNA sensors. The

DNAse family of sensors include DNAse II and TREX1, which are ubiquitously
present in the cell for their housekeeping functions. The RNAse family of sensor
includes one member—RNA Pol III. Additionally, DNA-PK also functions to
cater to viral DNA sensing. The endosomal DNA sensors include TLR7 and
TLR9, which belong to the Toll-like receptor (TLR) family. The DExD/H-box
helicase family include the putative DNA sensors recently discovered including
DDX9, DHX36, DDX41 and DDX60. Several other sensors remain to be char-
acterised or are less classified viz. DAI, LRRFIP1, HMGB, ABCF1, MRE11. In
general, response to a viral RNA or DNA produces three types of responses,
namely, production of antiviral cytokines including Types I and III IFNs, release
of pro-inflammatory and inflammatory cytokines and chemotactic factors. This
chapter discusses the structure, function and mechanism of action of the viral
DNA sensors explored till date.

Keywords
Viral DNA sensors · Immune responses · Immune activation

16.1 Introduction

Defence mechanisms employed by the innate branch of immunity include myriad

types of cells and soluble molecules in tissues and circulatory system. Such pre-­
existing deployments constantly thwart the attacks on the organism by the patho-
genic microorganisms ubiquitously present throughout the biosphere, thus averting
the invasion, establishment and multiplication of such agents. In fact, if microbes do
institute a niche, the innate immune responses equip early defence, before the
involvement of adaptive branch of the immune system.
The peculiarities of the recognition by players of innate immune system have
emerged to contest microbes which have outstanding common features. The
innate immunity has evolved to perceive the molecular structures which are pres-
ent on the surface or inside the microorganism in question, as well as the induced
pathogenic microbial products. Such biomolecules which spur the innate immune
system are often shared by classes of microbes are called pathogen-associated
molecular patterns (PAMPS), or even more appropriately, microbial-associated
molecular patterns (MAMPs). Such moieties include nucleic acids (single-
stranded RNA/DNA), characteristic feature(s) on protein, carbohydrate and lipid
products restricted to microorganisms, or even a combination of the biochemical
elements. Such building blocks are often indispensable for the survival of micro-
organisms. Not only is it capable of recognising the foreign biomolecular struc-
tures, but also some, which are derived from self: in aberrant conditions like
damaged or dying cells. These substances are known as death- or damage-associ-
ated molecular patterns (DAMPs).
16 Potentiality of DNA Sensors in Activating Immune System in Emerging Viral… 245

The cell-associated recognition counterpart that assists in perceiving the molecu-

lar patterns are called pattern recognition receptors (PRRs). PRRs constitute various
types of cell receptors, present in distinct premises of the cell (surface as well as
intracellular), as soluble factors in the circulation and other body secretions.
PRRs (based on the location)

Viral PAMPS
Cellular

Soluble (blood)
ssRNA of dsRNA of dsDNA of
Mucosal surfaces viruses viruses viruses
(sectretions)

Cell-associated molecules are mostly restricted to the cells of the immune sys-
tem like phagocytes—Macrophages and Neutrophils; antigen presenting cells
(APCs)—Dendritic cells; cells that form the obstacle between the internal milieu
and the external environment—epithelial cells; as well as other cells like mast cells
and tissue resident cells.
Pathogens like viruses, bacteria, fungi and protozoa can establish themselves
within any compartment of the cell viz. cytosol, nucleus, endosomes or on the sur-
face of the cell, tissue, etc. The innate immune system efficiently tackles the micro-
bial invasion in all these compartments by activating the signal transduction
pathways downstream of the recognition molecules which ultimately promote the
pro-inflammatory and anti-microbe activity.
Broadly though, the pattern recognition receptors can be either cell-associated or
soluble.
The cell-associated receptors include:

1. Toll-like receptors (TLRs)

2. NOD-like receptors (NLRs)
3. RIG-I-like receptors (RLRs)
4. Cytosolic DNA sensors (CDSs)
5. C-type lectin-like receptors (CLRs)
6. Scavenger receptors
7. N-formyl met-leu-phe receptors

The soluble receptors include:

1. Pentraxins
2. Collectins
3. Ficolins
4. Complement
246 S. U. Sapre and P. Nair

The scope of discussion on each of the above PRRs is wide. However, since this
chapter specifically deals with the DNA-sensing molecules, further discussion
would be restricted to the viral DNA sensors.

16.2 Sources of Cytosolic DNA

There are several ways by which DNA can be present in the cell cytoplasm.
These routes include (but are not limited to):

1. Intracellular pathogen infection

2. Impaired ability of clearing exogenous DNA innately metabolised in the endo-­
lysosomal compartment
3. An asymmetric management of endogenous DNA products and turnover.

Modes of entry of pathogen DNA in the cytoplasm are depicted below.

The following table summarises the currently known DNA sensors in brief—
proposed DNA sensor families and the examples:

Site of DNA
PRR Cell types sensing Response References
Toll-like receptor (TLR) family
TLR9 pDCs Endosomes Type I IFN (Hemmi et al. 2000;
Latz et al. 2004, 2007)
PYHIN family
AIM2 Macrophages, Cytoplasm IL-1β, IL-18 (Hornung et al. 2009;
DCs Fernandes-Alnemri
et al. 2009;
Burckstummer et al.
2009; Roberts et al.
2009)
IFI16 Macrophages, Cytoplasm, IFN-β, CXCL10, (Unterholzner et al.
endothelial cells nucleus IL-6, IL-1β 2010; Horan et al. 2013;
Kerur et al. 2011)
IFIX Macrophages Nucleus IL-1β, IL-18 (Diner et al. 2015a)
MNDA Less explored
STING activator family
cGAS L929, THP-1, Cytoplasm IFN-β (Sun et al. 2013)
HEK293
DNAse family
DNAse II Ubiquitous Lysosomes DNA degradation (Okabe et al. 2005)
TREX1 Ubiquitous Cytoplasm-ER Degradation of (Stetson et al. 2008;
associated DNA elements Yang et al. 2007)
derived from
endogenous
retroviruses
RNAse family
16 Potentiality of DNA Sensors in Activating Immune System in Emerging Viral… 247

Site of DNA
PRR Cell types sensing Response References
RNA Pol EBV+ B cell, Cytoplasm, IFN-β (Ablasser et al. 2009;
III macrophage, nucleus Chiu et al. 2009)
cell line
Protein kinase (PK) family
DNA-PK 293T, MEFs Cytoplasm IFN-λ1, IFN-β, (Zhang et al. 2011a;
IL-6 Ferguson et al. 2012)
DExD/H-box helicase family
DDX9 pDCs Cytoplasm TNF-α (Kim et al. 2010)
DHX36 pDCs Cytoplasm IFN-α (Kim et al. 2010)
DDX41 DCs Cytoplasm IFN-α, β (Zhang et al. 2011b)
DDX60 HeLa cells Cytoplasm IFN-β, CXCL10 (Miyashita et al. 2011)
Others/less characterised family
DAI Fibroblasts Cytoplasm IFN-β, necrosis (Takaoka et al. 2007;
Upton et al. 2012)
LRRFIP1 Less studied Cytoplasm Type I IFN (Sabbah et al. 2009;
HMGB Cytoplasm induction Yanai et al. 2009; Yang
et al. 2010)
ABCF1 Cytoplasm Less known
MRE11 MEFs, DCs Cytoplasm IFN-β, CXCL10, (Kondo et al. 2013)
IL-6

16.3 TLR Family

16.3.1 Structure

TLRs belong to the type I integral membrane glycoproteins embedded in the surface
membrane or in the endosomal membrane. They possess an extracellular (or intra-­
endosomal) region—characteristic leucine-rich repeats (LRRs) which are sur-
rounded by cysteine-rich motifs which essentially bind ligand(s), a transmembrane
region, and an intracellular (or cytosolic) region—known as the Toll/IL-1 receptor
(TIR) involved as a part of the cytoplasmic tail. There are about 18–26 copies of
LRRs which vary in different TLRs. Each LRR of TLR protein is composed of
around 20–25 amino acids and multiple such LRRs make up a typical question mark
hook shaped protein scaffold which adapts for ligand binding. Both, the concave as
well as convex surfaces, are involved in ligand binding. The N-terminus of a TLR is
at its LRR end, whereas the C-terminus is at its TIR end.

16.3.2 Function

There are nine different functional TLRs in humans (TLR1-9). The function of
TLR-10 is unknown. TLRs always act as dimers. Some of them form heterodimers
like TLR-1 and TLR-2, whereas others form homodimers like TLR-3, TLR-4,
248 S. U. Sapre and P. Nair

TLR-5, TLR-5, TLR-6, TLR-7, TLR-8, TLR-9 and TLR-10. TLR-1, 2, 4, 5 and 6
are expressed on the surface of the membrane, whereas TLR-3, 7, 8 and 9 are
expressed inside the endosomal membrane. TLR-4 utilises accessory proteins MD2,
LPS-binding protein (LBP) and CD14.

PAMPs

TLR activation

Inflammator Chemotactic Antimicrobia Antiviral

y cytokines factors l peptides cytokines

Amongst all, TLR-9 recognises unmethylated CpG dinucleotides. In the human

genome, the DNA methyltransferases heavily methylate cytosine residues. However,
in the genome of bacteria and many viruses, CpG dinucleotides remain unmethyl-
ated and thus serve as PAMP. Delivery of TLR-9 (and TLR-3, 7, 8) from the endo-
plasmic reticulum to the endosome relies on UNC-93B, a protein composed of 12
transmembrane domains. Deficiency of UNC-93B1 due to rare human mutations
increases the susceptibility to herpes simplex encephalitis.

16.3.3 Mechanism of Action

Ligand binding to the TLR brings about dimerization of the respective TLRs
involved. This brings about the cytoplasmic tails to come in proximity. TIR domain
containing adaptor proteins are now recruited, which bind the TIR domains in the
cytoplasmic tails. Further, this brings about recruitment and activation of distinct
protein kinases, which activate the transcription factors. Nuclear factor κ-B (NF-­
κB), activation protein-1 (AP-1), interferon response factor 3 (IRF3) and IRF7 are
the major transcription factors which are activated by TLR signalling pathways.
NF-κB and AP-1 are responsible for the expression of genes encoding inflammatory
response molecules, including (but not limited to) inflammatory cytokines, endothe-
lial adhesion molecules and chemokines. IRF3 and IRF7 stimulate the production
type I interferons (IFNs), which are central to antiviral innate immune responses.
Different TLRs use different combination of adaptors and signalling intermediates
and thus mediate unique downstream effects. TLR9 uses the MyD88-dependent,
TRIF-independent pathway and activates both NF-κB and IRFs. Hence, they induce
both inflammatory and antiviral responses.
Two protein domains of MyD88 allow it to function as an adaptor protein: a TIR
domain at its carboxy terminus that associates with the TIR domains of the TLR
16 Potentiality of DNA Sensors in Activating Immune System in Emerging Viral… 249

cytoplasmic tails and a death domain at its amino terminus, which associates with
the death domains present in other intracellular signalling proteins. It is worth-
while to note that both the domains of MyD88 are essential for signalling. The
MyD88 death domain recruits and activates IL-1 receptor associated kinase
(IRAK4) and IRAK1, which are both serine-threonine protein kinases. The com-
plex involving IRAK, TIR of cytoplasmic tails and MyD88 executes two func-
tions—executing the enzymes that produce a signalling scaffold, employing the
scaffold to recruit other molecules which are then phosphorylated by the IRAKs.
The formation of signalling scaffold is a multi-step process: The IRAK complex
brings in the enzyme tumour necrosis factor receptor-associated factor 6 (TRAF6),
which functions as an E3 ubiquitin ligase in association with TRIKA1 (composed
of UBC13, which is an E2 ubiquitin ligase with a cofactor for Uve1A). TRAF6 and
UBC13 together have the function of polyubiquitination. This polyubiquitin has
linkages between lysine63 of pervious subunit and C terminus of the next, leading
to K63 linkages. The same process can be initiated on vivid proteins, including
TRAF6 or the multi-ubiquitin chains can exist independently as free linear chains
which can be extended to form polyubiquitin chains, which can bind to other sig-
nalling proteins. Further, the scaffold brings in signalling complex that is made up
of TAB1, TAB2-ubiquitin binding adaptor molecules, and TAK1-a serine-threo-
nine kinase. IRAK complex phosphorylates TAK1, which propagates signalling by
activation of certain MAPKs like c-Jun terminal kinase (JNK) and MAPK14 (p38
MAPK). This brings about downstream activation of AP-1 family of transcription
factors which transcribe cytokine genes.
TAK1 additionally phosphorylates and activates the IκB kinase (IKK) complex.
IKKα, IKKβ and IKKγ constitute the IKK complex (the IKKγ is also known as
NF-Κb essential modifier or NEMO). NEMO binds to polyubiquitin chains, which
leads to IKK complex close to TAK1. TAK1 then activates IKKβ by its phosphory-
lation. IKKβ then phosphorylates inhibitor of κB (IκB), which is a cytoplasmic
protein that natively binds to the transcription factor NF-κB. IκB is made up of two
subunits—p50 and p65. The binding of IκB prevents the movement of NF-κB to the
nucleus from cytoplasm. Post phosphorylation of IKK, the IκB is released from the
functional subunit of NF-κB, which leads to translocation of NF-κB to nucleus,
where it leads to transcription of genes for pro-inflammatory cytokines like TNF-α,
IL-1β and IL-6. It is noteworthy that the effect of TLR activation varies depending
on the cell type in which it occurs.
The nucleic acid sensing TLRs, including TLR9 activate IRF family of proteins.
Natively present in the cytoplasm, they only get activated upon phosphorylation of
serine and threonine residues in their C terminal. Upon activation, they move to the
nucleus to act as transcription factors. Among all the IRFs, IRF3 and IRF7 are par-
ticularly important for TLR signalling and expression of antiviral type I IFNs. For
TLR9 signalling in plasmacytoid dendritic cells, MyD88 exclusively is used as an
adaptor protein. The TIR domain of MyD88 employs IRAK1/IRAK4 complex as
described earlier. However, the IRAK complex carries out a different function
beyond recruiting TRAFs which generates a signalling scaffold. In these cells,
IRAK1 can also interact with IRF7, which is highly expressed by plasmacytoid
250 S. U. Sapre and P. Nair

Table 16.1 Containing location of different TLRs, their ligand (Janeway)

Cellular or
Toll-like Functional subcellular Adaptor
receptor association location Ligand proteins
TLR1 TLR1:TLR2 Monocytes, Lipomannans (mycobacteria), MyD88/
heterodimer dendritic cells, Lipoproteins (diacyl MAL
TLR2 TLR2-TLR6 mast cells, lipopeptides; triacyl
heterodimer eosinophils, lipopeptides); Lipoteichoic
basophils acids (gram-positive bacteria);
cell-wall β-glucans (bacteria
and fungi)
TLR3 TLR3-TLR3 Macrophages, Double-stranded RNA TRIF
homodimer dendritic cells, (viruses), poly I:C
intestinal
epithelium
TLR4 TLR4-TLR4 Macrophages, LPS (gram-negative bacteria) MyD88/
homodimer dendritic cells, MAL;
mast cells, TRIF/
eosinophils TRAM
TLR5 TLR5-TLR5 Intestinal Flagellin (bacteria) MyD88
homodimer epithelium,
macrophages,
dendritic cells
TLR7 TLR7-TLR7 Plasmacytoic Single-stranded RNA (viruses) MyD88
homodimer dendritic cells,
macrophages,
eosinophils, B
cells
TLR8 TLR8-TLR8 Macrophages, Single-stranded RNA (viruses) MyD88
homodimer neutrophils
TLR9 TLR9-TLR9 Plasmacytoid DNA with unmethylated CpG MyD88
homodimer dendritic cells, (bacteria and herpesviruses)
eosinophils, B
cells, basophils
TLR10 TLR3-TLR3 Plasmacytoid Unknown Unknown
homodimer dendritic cells,
eosinophils, B
cells, basophils

dendritic cells. This enables IRAK1 to phosphorylate IRF7 which leads to induction
of type I IFNs (Table 16.1).

16.4 PYHIN Family

There are 4 PYHIN proteins in humans, and 13 in mice. Two human proteins in this
class, namely AIM2 and IFN-γ inducible (IFI16) have been predicted based on the
studies so far, to be necessary for different DNA-modulated immune responses and
perhaps, also function as DNA sensors (Hornung et al. 2009; Roberts et al. 2009;
16 Potentiality of DNA Sensors in Activating Immune System in Emerging Viral… 251

Unterholzner et al. 2010). PYHIN family of proteins can also activate the formation
of inflammasome.

Inflammasome Inflammasomes is a complex structure which consists of many

proteins which form in the cytosol upon stimulation with cytosolic PAMPs and
DAMPs, the outcome of which is production of active forms of IL-1β and IL-18.
IL1-β and IL-18 are actually produced as inactive precursors and their activation is
dependent on their proteolytic cleavage by the enzyme caspase-1. These cytokines
are then released from the cell, which then promote inflammatory responses.
Inflammasomes are made up of oligomers of a sensor, caspase-1, and an adaptor
which links the interaction between the rest of the two components. The oligomeric
complexes only form upon stimulation with DNA detected by sensors.

16.4.1 Structure of PYHIN

The PYHIN family proteins contain an N-terminal pyrin (PY) domain and an H
inversion (HIN) domain

16.4.1.1 AIM2
Absent in melanoma (AIM2) is one such example of a PYHIN family of proteins.
The HIN region of AIM2 recognises dsDNA genome and triggers caspase 1 activa-
tion. This occurs through the simultaneous interaction of pyrin domain with
ASC. Primarily AIM2 is involved in the inflammasome formation (Schattgen and
Fitzgerald 2011). Microbial DNA is one such PAMP. It is usually located in the
cytosol and is key to responses in vitro to vaccinia virus (Janeway). However, it
ubiquitously oligomerises upon stimulation. The downstream aggregates so formed
recruit and activate a protease-caspase-1 that leads to the maturation of pro-­
inflammatory cytokines IL-1β and IL-18 which ultimately culminates into a pro-
grammed cell death, termed ‘pyroptosis’ (Bergsbaken et al. 2009; Miao et al. 2011).

16.4.1.2 IFI16
Interferon-inducible protein 16 (IFI16) was recently characterised as the first viral
DNA sensor to function within the nucleus. It is a member of PYHIN protein fam-
ily (Diner et al. 2015a; Schattgen and Fitzgerald 2011). IFI16 has two HIN
domains. At its C terminus, it has two HIN200 domains which bind to DNA. At its
N-terminal, is a pyrin (PY) domain which mediates homotypic interactions within
the molecules as well as cooperative assembly of small subunits of IFI16 (Li et al.
2013; Morrone et al. 2014). This binding is sequence-independent manner (Jin
et al. 2012). It primarily functions in the nucleus, where it is located. It recognises
viral dsDNA.
The function of IFI16 depends on the type of cell in which it functions. In the
immune cells, IFI16 binds to cytosolic viral DNA, engaging sting and induces IFN
production (Unterholzner et al. 2010; Horan et al. 2013; Jakobsen et al. 2013).
However, in non-immune cells, IFI16 majorly functions in the nucleus by localising
252 S. U. Sapre and P. Nair

there (Diner et al. 2015a; Li et al. 2013, 2012; Orzalli et al. 2012). A plausible expla-
nation for this is that the there exists a multi-partite nuclear localisation signal on
IFI16 and is necessary for its transit between nucleus and cytoplasm (Li et al. 2012).
Especially in case of herpesvirus infections, both IFI16 and STING are required for
inducing the expression of IFN and IFN-stimulated genes (Orzalli et al. 2012). The
differences in IFI16 DNA-sensing are possibly due to the cell type-­dependent pro-
cess (Diner et al. 2015b). Prompt responses are required in case of immune cells,
and thus, DNA-sensing components localised in the cytoplasm makes more sense,
whereas, in non-immune cells, the IFI16 might play some housekeeping functions
but additionally may respond to the successful viral infections in the nucleus.
Additionally, IFI16 also mounts inflammatory and apoptotic responses to foreign
DNA through inflammasome—a multiprotein assembly (Kerur et al. 2011; Johnson
et al. 2013; Ansari et al. 2013; Singh et al. 2013; Monroe et al. 2014). In reality, the
evidence that IFI16 elicits both, type I IFN response as well as inflammation is con-
tradictory, since type I IFNs are known to exhibit anti-inflammatory effect
(Theofilopoulos et al. 2005; Billiau 2006; Guarda et al. 2011). Recently, IFI16 was
shown to play a direct role in inhibiting the formation of both, AIM2 and NLR fam-
ily inflammasomes (Veeranki et al. 2011). Hence, it is quite possible that the role of
IFI16 in inflammasome responses is cell type dependent and other yet unknown
factors do play a role too. Recent evidences suggest that IFI16 and cGAS may func-
tion in harmony to execute immune signalling to nuclear foreign DNA (Orzalli et al.
2015). This also suggests that IFI16 is a dominant nuclear DNA sensor, whereas
cGAS has auxiliary functions like stabilising the IFI16 to enable or prolong signal
efficiency.

16.4.1.3 IFIX
Some of the PYHIN family members, namely, IFIX and MNDA also majorly local-
ise in the nucleus. Yet, their functions were not known in the early phase and were
suspected to have some immunological function. IFIX associates with antiviral fac-
tors and its expression is inversely associated with the capacity of herpesvirus rep-
lication (Diner et al. 2015a) as it binds to the DNA of the virus and remains localised
in the nucleus. It binds DNA substrates in a sequence-dependent fashion and leads
to type I IFN response (Diner et al. 2015a). IFIX expression, like IFI16, is depen-
dent on the type of cell and tissue, thus making it likely that its function varies from
one cell type to other (Ding et al. 2004; Haque et al. 2014).

16.5 STING Activator Family

16.5.1 STING (a.k.a. MITA, ERIS and TMEM173)

STING is an ER localised transmembrane adaptor protein that is anchored by an

amino-terminal tetraspan transmembrane domain. Its carboxy-terminal domain
extends into the cytoplasm and interacts to form an inactive form of STING—the
STING homodimer. Type I IFN response is crucial for successful defence against
16 Potentiality of DNA Sensors in Activating Immune System in Emerging Viral… 253

viral pathogens. Stimulator of IFN genes (STING) is one such pathway vital to the
mechanism of dsDNA-induced stimulation of type I IFN responses. If a viral
dsDNA happens to exist in the cytosol, post entry and uncoating, it activates the
enzyme cGAS (cyclic GMP-AMP synthase) that generates cyclic guanosine
monophosphate-­adenosine monophosphate (cGAMP), a signalling molecule. cGAS
contains a protein motif as a part of the nucelotidyltransferase (NTase) family of
enzymes, including adenylate cyclase and distinct DNA polymerases (Janeway).
cGAS has an affinity for DNA and readily attaches to the cytosolic DNA. This inter-
action stimulates the cGAS enzymatic activity that leads to generation of cGAMP
from GTP and ATP in the cytoplasm. cGAMP binds to both the subunits of STING
dimer and activates STING signalling.
It activates TANK binding kinase 1 (TBK1) through interaction with
cGAMP. TBK1 phosphorylates and activates a transcription factor IRF3 which
induces expression of type I IFN genes. Besides these, the STING is also known to
respond to other cytosolic DNA sensors like DAI and IFI16. Additionally, STING
induces autophagy. In the innate immune system, autophagy is a potential mecha-
nism of delivering cytosolic microbes to lysosome where they are acted upon by the
proteolytic enzymes.
It is quite interesting to know that STING, MAVS and TRIF, all have similar
amino acid sequence motif at their carboxy termini

16.6 DNAse Family

16.6.1 DNAse II

Cells possess DNAses, which degrade the unwanted DNA in the compartment it is
not expected to be present. DNAse II is restricted to lysosomes which digests patho-
genic and by-products of dead cells which enter this compartment (Okabe et al.
2005). Originally, this mechanism has evolved for the degradation of the cells
undergoing programmed cell death, i.e. apoptosis, which are usually taken up by
macrophages. In cells which lack DNAse II activity, DNA may stimulate aberrant
responses by entering cytoplasm, consequently stimulating the cytosolic DNA sen-
sors (Okabe et al. 2005).

16.6.2 TREX1

Yet another cellular DNAse is three primer repair endonuclease 1(TREX1) that is
present in association with the endoplasmic reticulum (ER) in the cytoplasm. Under
housekeeping conditions, basal amount of DNA tends to accumulate in the cytosol.
TREX1 regularly degrades such DNA in the cytosol.
254 S. U. Sapre and P. Nair

16.7 RNAse Family

Another putative cytosolic DNA sensor discovered is RNA Polymerase III (PolIII).
It uses AT-rich and herpesvirus DNA as a template to produce 5′triphosphate RNAs.
RNAs so produced get detected by the cytosolic RNA sensors like RIG-I (Ablasser
et al. 2009; Chiu et al. 2009). However, the broad mechanism being the Pol III as a
potential DNA senor remains to be completely defined.

16.8 Protein Kinase (PK) Family

Some proteins which are usually involved in DNA damage repair also serve as DNA
sensors. DNA-PK (DNA-dependent protein kinase which is composed of Ku70,
Ku80 and DNA-PKc).

16.9 DExD/H-Box Helicase Family

Both, RNA, as well as DNA helicases constitute the DExD/H-box (DDX) protein
family which has it characteristic DExD/H-box domain. DDXs are multi-functional
and control the gene induction at multiple points that includes signal transduction
pathways, gene promoters, mRNA splicing, and translation regulation. Several
DNA sensors have been identified in the DDX family (Kim et al. 2010; Zhang et al.
2011b; Yoneyama et al. 2004; Schroder et al. 2008).

16.9.1 DDX9 and DHX36

DHX9 and DHX36 are the other two DExD/H-box helicases which bind to CpG
DNA and interact with MyD88 (Kim et al. 2010). The inflammatory response and
type I interferon response depend partly on DHX9 and DHX36, respectively.

16.9.2 DDX41

DEAD box polypeptide 1 (DDX41) is closely related to RIG-I. It appears to signal

through the STING. It has been reported that DDX41 interacts with dsDNA, both,
in vitro and in vivo. In some cells, it has also been shown to be crucial to the DNA-­
dependent activation of type I IFN production involving STING and TBK1 (Zhang
et al. 2011b). In cells where the IFI16 expression is less or restricted, the DDX41
acts as the primary DNA sensor of cytoplasmic DNA which further induced the IFN
induction and IFI16 expression, the latter of which, amplifies the innate immune
responses (Zhang et al. 2011b). The pattern of DNA sensor expression across differ-
ent types of cells is vital to define the type of sensor which mediates the DNA
primed innate immune responses. Also, recently, DDX41 has also been shown to
16 Potentiality of DNA Sensors in Activating Immune System in Emerging Viral… 255

directly bind the cyclic di-nucleotides (CDNs) and thus, indirectly, the IFN response
induced was DDX41 dependent (Parvatiyar et al. 2012)

16.9.3 DDX60

It is a novice antiviral factor among the pre-existing list of DExD/H box helicases
and functions in association with RIG-I, MDA5 and LGP2 to induce the type I IFN
response (Miyashita et al. 2011).

16.10 Other/Less Characterised Family

There are many unknown and known but less characterised candidate DNA sensors.
They might play some vital role in other cellular processes, but their role as a DNA
sensor is yet to be confirmed. Very less is known regarding their mechanism of rec-
ognition and signalling, or their in vivo function.

16.10.1 DAI

Another protein is DAI (a.k.a. ZBP1) (Takaoka et al. 2007). The peculiarity of this
protein is that the response of DAI as a DNA sensor is decided by the cell type
(Unterholzner et al. 2010; Upton et al. 2012, 2010; DeFilippis et al. 2010; Ishii
et al. 2008). It interacts with the dsDNA and drives type I IFN response (DeFilippis
et al. 2010).

16.10.2 LRRFIPI

It senses the DNA present in the cytoplasm and phosphorylates β-catenin. Β-catenin
translocates to the nucleus to induce IFN-β production.

16.10.3 HMGB

Three subtypes of HMGB, viz. HMGB1, HMGB2 and HMGB3 are known to
respond to cytosolic DNA. Primarily, ABCF1 binds to the cytosolic DNA. This
complex then binds to HMGB2 and IFI16 to stimulate further innate immune
response (Yanai et al. 2009; Yang et al. 2010; Lee et al. 2013; Goubau et al. 2013)

16.10.4 ABCF1

They function in harmony with the HMGB group of sensors.

256 S. U. Sapre and P. Nair

16.10.5 MRE11

Meiotic recombination 11 homolog a (MRE11A) has the capability to sense dsDNA

in the cytosol and activates STING pathway.

16.11 Sensing of Viral Components by DNA Sensors

There are numerous mechanisms through which the DNA sensors in the host cells
can detect the viral genomic components and trigger a cascade of downstream reac-
tions. Some of them have been discussed below:

16.11.1 Herpesviridae

Till date, the role of DNA sensors has been best studied extensively using herpesvi-
ruses, HSV, in particular (Paludan et al. 2011). Quite evidently, currently known-­
well, established, as well as prospective DNA sensors have been shown to counteract
herpesvirus infections. The very primary DNA sensor was TLR9, followed by the
contemporary DNA sensors. To state a few, the HSV-1 infection in human origin
primary MDMs (monocyte-derived macrophages) induces the production of pro-­
inflammatory cytokines like IL-1β, which imitates the response post HSV-1 infec-
tion along IFI16 axis (Horan et al. 2013). However, this response is altered when the
cell type differs. HSV-1 infection of primary murine dendritic cells (DCs) invokes a
type I IFN response with the protein DDX41 involved (Zhang et al. 2011b). IRF3 is
known to drive the type I IFN response as well. DAI drives IRF3 activation as well.
However, contrastingly, DAI (or ZBP1) is shown to play a central role in inducing
necroptosis, post infection with MCMV (Upton et al. 2012).
As discussed earlier, STING is central to all the DNA induced antiviral responses.
This coincides with the study which demonstrated that STING deficiency in mice
causes increased susceptibility to HSV-1 infection (Ishikawa et al. 2009). While it is
not known if the cell-specific response applies to all the cell-lines, the above docu-
mented work irrefutably states that the response to herpesvirus infections in primary
cells is cell type specific.

16.11.2 Retroviridae

HIV is the most widely studied retrovirus for known reasons on its complex patho-
genesis, drug resistance and co-evolution with the host. As is known, HIV replica-
tion proceeds through and RNA–DNA double-stranded intermediate in the process
of its RNA being converted to a dsDNA form. Hence, both, RNA as well as DNA
sensors, play a crucial role in the detection of retroviral genome upon target cell
entry (Solis et al. 2011; Berg et al. 2012; Doitsh et al. 2010; Yan et al. 2010). A
recent study, intended to understand the type I IFN response against HIV proved
16 Potentiality of DNA Sensors in Activating Immune System in Emerging Viral… 257

that ssDNA, rather than dsDNA form of HIV, is more potent at inducing the type I
IFN response. TREX1 is a 3′5′-exonuclease that degrades the unintegrated cyto-
solic cDNA, making it unavailable for sensing to the DNA sensors. Eliminating the
expression of TREX1 leads to upregulation in type I IFN response and was more so
in case of ssRNA intermediate of HIV, rather than dsDNA form. Undoubtedly, thus,
cell does employ the intracellular sensors of DNA that culminates in type I IFN
response. Although the definite sensors involved remain to be defined to date, they
sure function through a STING-TBK1-IRF3 axis.
Other virus families and the respective DNA sensors have been summarised in
Table 16.2

16.11.3 M
 echanisms Employed By Viruses to Evade DNA
Sensors

Viral genomes are subject to recognition by DNA sensors only if they happen to
expose their characteristic features, leading to the consequent recognition as non-­
self genomic entity by the host DNA sensing mechanisms. However, myriad viruses
evade and subvert the host immune responses so as to render these mechanisms of
detecting the viral components ineffective.
One such strategy is to simply obscure their genome and replication intermedi-
ates involving potential ligand to the DNA sensors:
Viruses belonging to the Herpesviridae family inhibit DNA sensors like DAI,
DHX9 and IFI16 responses.

1. HSV-1 encodes a protein ICP0, an E3 ubiquitin ligase, engages proteasomal deg-

radation of IFI16 and also prevents its nuclear relocalisation. These further limits
the activation of IRF3 (Orzalli et al. 2012).
2. HCMV possesses pUL83/pp65, a matrix protein. It inhibits ISG induction. In
addition to this, it is known to interact with IFI16 (Cristea et al. 2010).

Table 16.2 Human nuclear replicating DNA viruses and implicated DNA sensors
Virus Implicated DNA sensors
Adenovirus C (types 1, 2, 5 and cGAS, TLR9
6)
Hepatitis B virus AIM2, cGAS
Cytomegalovirus AIM2, DAI/ZBP1, IFI16, TLR7, TLR9
Epstein–Barr virus RNA Pol III, IFI16, TLR9
Herpes simplex virus type I cGAS, DAI/ZBP1, DDX41, DDX60, DHX9, DHX36,
DNA-PK, RNA Pol III, IFI16, TLR9, IFIX
Herpes simplex virus type II DNA-PK, TLR9
Herpesvirus 8 (Kaposi sarcoma- IFI16
associated virus)
Varicella zoster virus NLRP3, TLR9
Papillomavirus (>170 types) TLR9
258 S. U. Sapre and P. Nair

3. Proteases of viral origin like the NS2B3 protease, coronavirus papain-like prote-
ases subvert STING in a direct manner (Aguirre et al. 2012; Sun et al. 2012; Yu
et al. 2012).

Acknowledgements Authors are grateful to NIV Pune and NMIMS Pune for the support
extended.

Conflict of Interest The authors declare that they have no competing interests.

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Advanced Immunotechnological
Methods for Detection and Diagnosis 17
of Viral Infections: Current Applications
and Future Challenges

Pallaval Veera Bramhachari, Ganugula Mohana Sheela,

A. M. V. N. Prathyusha, M. Madhavi, K. Satish Kumar,
Neelapu Nageswara Rao Reddy,
and Chanda Parulekar Berde

Abstract

Diagnosis and identification of viruses is an important component of diag-

nostic virology laboratory. Although various modes of diagnostic methods
are now available at disposal, a vast majority of the diseases across the globe
remain undiagnosed. This is largely due to the overlapping undifferentiated
set of symptoms across myriad set of RNA and DNA viral diseases. As such,
it becomes critical to take into consideration several factors for viral diagno-
sis ranging from the type and quality of specimen collected, time of speci-
men collection, mode of transport, accuracy, specificity, sensitivity, and the
type of diagnostic method used. This chapter broadly emphasizes various
methods on diagnostic virology ranging from the classical methods of diag-
nosis to the most recently developed molecular methods of detection of
virus.

P. V. Bramhachari · G. Mohana Sheela · A. M. V. N. Prathyusha (*)

Department of Biotechnology, Krishna University, Machilipatnam, India
M. Madhavi
Department of Zoology, Nizam College, Osmania University, Hyderabad, Telangana, India
K. Satish Kumar
Department of Zoology, Adikavi Nannaya University, Rajamundry, Andhra Pradesh, India
N. N. R. Reddy
Department of Biochemistry and Bioinformatics, GITAM Institute of Science, Gandhi
Institute of Technology and Management (GITAM) (Deemed-to-be-University),
Visakhapatnam, Andhra Pradesh, India
C. P. Berde
Gogate Jogalekar College, Ratnagiri, Maharashtra, India

© Springer Nature Singapore Pte Ltd. 2020 261

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_17
262 P. V. Bramhachari et al.

Keywords
RNA and DNA viral diseases · Diagnostics · Accuracy · Specificity · Sensitivity

17.1 Introduction

Viruses are obligate intracellular parasites and their detection and identification is
an imperative component clinically. Viral infectious signify a major portion in pub-
lic health perspectives with thousands of deaths annually. Notwithstanding from
highly contagious infections and serious pandemics to common influenza episodes,
clinical diagnosis of viral infection often relies on early detection. Therefore, effec-
tual detection of viruses is indispensable aid to avert transmission, initiate befitting
therapy, and scrutinize response to treatment which leads to effective disease con-
trol and management (Souf 2016).
Viral diagnosis is a dynamic process. Although prophylaxis is better than cure,
an accurate diagnosis of any virus fundamental to the infection is equally vital.
Generally, diagnostic tests are categorized into three groups: direct detection, iso-
lation of virus, and serology tests. Direct examination methods are merely faster
and examined directly for presence of virus particles, virus antigen, or viral
nucleic acids. Immunofluorescence assay is extensively used for rapid identifica-
tion of virus infections through detection of virus antigen or virus-specific anti-
bodies in clinical specimens. Viral diagnosis is a crucial revolution and renders
more accessible and makes it potential to standardize the recently developed diag-
nostic methods. Serology essentially comprises bulk of work of any virology
laboratory. A serological diagnosis can be identified by increasing titers of anti-
body among acute and convalescent stages of infection, or by the detection of
Immunoglobulin M (IgM).
The diagnosis of viral infections were enhanced noticeably all through 1990s,
with the onset of highly sensitive methods, viz. enzyme-linked immunosorbent
assay (ELISA) and PCR are superseded for this reason. Nevertheless, during the last
20 years this technique is being utilized, due to its unique nature, making diagnosis
probable through visualization of virus. Then after, introduction of ELISA then
revolutionized viral diagnosis by simplifying detection and shortening the time
(Torrance and Jones 1981). Molecular diagnostic tools for viral diagnosis has trialed
speedy advancements in last few years (Hayden and Persing 2001), and revolution-
ized diagnosis of infectious diseases, particularly viral diseases. The use of amplifi-
cation techniques, viz. PCR, RT-PCR, or NASBA (nucleic acid sequence-based
amplification) (Van Belkum and Niesters 1995) for detection of virus, genotyping,
and quantification have several advantages such as high reproducibility and sensi-
tivity, including broad dynamic range (Ebner et al. 2005, 2006). Several molecular
diagnostic techniques were recently swapped by fully automated devices that use
less time, maneuver smaller volumes of liquids, and provide quantified results with
improved accuracy. The current review emphasizes the advanced immunotech-
niques, how the specific characteristics of diagnostic methods revolutionized field
of viral diagnosis clinically from a decade.
17 Advanced Immunotechnological Methods for Detection and Diagnosis of Viral… 263

17.2 Recent Advances in Viral Disease Diagnosis

17.2.1 Advanced Methods in Diagnostic Virology

17.2.1.1 Microscopy
Transmission electron microscopy (TEM) is a merely imaging technique that per-
mits direct image of viruses attributable to its nanoscale resolution. This is a classi-
cal technique owing to its advantage of direct visualization of virus. Amid the 1960s
and 1990s, TEM contributed a breakthrough and doled out as a diagnostic tool for
recognizing numerous virus unswervingly in any biological samples. For that rea-
son, in modern years, role of TEM in diagnosis of anonymous infectious agents in
specific viral outbreaks or transmission clusters shifted from regular use to an initial
screening test. TEM is also considered an important method for assessment of viral
safety in biopharmaceutical products. Application of TEM understands the unknown
viruses for which there is an imminent risk of contamination, and TEM will undeni-
ably be functional for documenting the subsistence of viruses or virus-like particles
in these cells and the products derivative from them (Roingeard et al. 2019).

17.2.2 Advanced Serological Immunoassays

The immunoassays are primarily antibody/antigen dependent assays. This princi-

ple works on immunization with the antigens and activating, or infection. Therefore,
measuring of IgG immunoglobulin and quantifying the antibodies are preferen-
tially used as diagnostic markers. The antigens or antibodies are coated with con-
jugated labels like metals, radioactive isotopes, fluorescent tags, respectively. As a
part of modern research on immunotechniques, a diagnostic approach for chronic
hepatitis C infection (CHC), detects specific antibody to HCV (anti-HCV) (indi-
rect tests) and assays that can detect, quantify, or characterize components of HCV
viral particles, viz. HCV RNA and core antigen (direct tests). Quantification of
HCV core antigen (cAg) as a one-step procedure has been indispensable. Hence, in
order to evaluate the performance of cAg quantification in diagnosing CHC and
how it is predisposed by concomitant HIV or HBV infections, cross-sectional vali-
dation assays, i.e., HCV Ag quantification as a one-step procedure in diagnosing
CHC in Cameroon was designed to abridge the diagnostic process (Duchesne et al.
2017). It is pertinent to note that a multiplex microsphere immunoassay (MIA)
recently developed possess diagnostic power to incarcerate viral envelope protein,
which evokes to be strong cross-reactive antibodies to other flaviviruses and dif-
ferential power of viral nonstructural proteins NS1 and NS5 was. Interestingly this
serologic assay needs to be employed in rapid clinical diagnosis of ZIKV and/or
dengue virus infections for screening immune responses in vaccine trials (Wong
et al. 2017). It is noteworthy that oral fluid is a noninvasive biospecimen that can
harbor pathogen-­specific antibodies and reach potential to replace blood-based
testing protocols. Therefore, a saliva-based oral fluid immunoassay was developed
to assess past and recent hepatitis E virus (HEV) infections from noninvasive
264 P. V. Bramhachari et al.

sampling methods. The sensitivity and specificity of this assay was comparable to
serum-based ELISAs. This salivary assay could improve our understanding of the
ecology and natural history of HEV (Pisanic et al. 2017).
Diagnosing ZIKV remains a great challenge, as detection of viral RNA is only
possible merely few days after onset of symptoms. Conversely, novel high-­
throughput image-based fluorescent neutralization method for identification of
ZIKV was thoroughly evaluated and developed which reported higher sensitivity
than Plaque reduction neutralization test (PRNT) and MAC-ELISA, respectively.
This test might employ for clinical diagnosis, clinical trials, and confirmation and
seroprevalence studies of ZIKV infection (Koishi et al. 2018). In one of the recent
studies, detection of serum HEV antigen (Ag) is deemed to be sensitive and prom-
ising biomarker for HEV antigen diagnosis with HEV RNA in both acute and
chronic genotypes. Strikingly an antigen assay was recently evaluated for diagnos-
ing HEV genotypes with higher sensitivity than commercial anti-HEV IgM and
HEV RNA ELISA tests (Zhang et al. 2019). Nonetheless, recent studies on respira-
tory syncytial virus (RSV) developed Luciferase Immunoprecipitation Systems
(LIPS) assay to detect IgG Antibodies against Human RSV G-Glycoprotein.
Moreover, Human RSV G-Glycoprotein also acts as biomarker for natural expo-
sure or immunization. RSV genes encoding native and mutated G (mG) proteins
from subgroups A and B strains were cloned, expressed as luciferase-tagged pro-
teins, and experimented separately to spot anti-RSV-G specific IgG antibodies
employing a high-throughput luciferase immunoprecipitation system (LIPS-G). It
was pertinent to note that RSV monoclonal antibodies and polyclonal antisera
explicitly bound in LIPS-GA and/or -GB assays (Crim et al. 2019).
The diagnosis of (ZIKV) and dengue virus (DENV) infections against viral
envelope protein and nonstructural proteins (NS) was developed using flavivirus
multiplex microsphere immunoassay (MIA). However MIA could not differentiate
more recent from past infections, which represents a key diagnostic challenge;
therefore, in a most recent report an immunoglobulin G (IgG)-based avidity assay
was developed for its diagnostic performance to accurately differentiate between
recent ZIKA and past dengue virus infections. This assay was found useful in
patients with high risk of ZIKA complications, viz. pregnant women and monitor-
ing immune responses in vaccine trials (Furuya et al. 2019). Consecutively to
develop serological diagnosis of ZIKV-IgA and ZIKV-IgG, avidity assays were
evaluated to characterize ZIKA infections in want of viremia. These assay facili-
tated construed low avidity of IgG and IgA results, enhanced the serological diag-
nosis of ZIKV (Amaro et al. 2019). In another study, homologous proteins of diverse
flaviviruses exhibited high degrees of sequence uniqueness, mainly within sub-
groups. This led to prevalent immunological cross-reactivity. Therefore, a propor-
tional deconvolution of complex B cell responses against ZIKV and other flavivirus
were deliberated by screening with a microarray chip-based high-resolution sero-
logical analysis primed from overlapping peptides covering the whole amino acid
sequence of ZIKV genomic polyprotein was developed. Additionally with advent of
this assay several infections, viz. dengue, yellow fever, tick-borne encephalitis, and
West Nile viruses shall be diagnosed (Hansen et al. 2019).
17 Advanced Immunotechnological Methods for Detection and Diagnosis of Viral… 265

17.2.2.1 ELISA-Based Immunodetection

Enzymes are extensive tool for diagnosing virus which have various applications
like enzyme immune assay, ELISA. Enzyme immune assay has different applica-
tions like fluorescence polarization immune assay (FPIA), micro-particle immune
assay (MEIA), chemiluminescent (CLIA). Enzyme immune assays work with anti-
gen–antibody interaction with the conjugated tags like fluorescent tags, chemilumi-
nescent tags which are complemented with substrates like polarized light and
fluorescent substrates. As a part of most advanced immunotechniques, an ultrasensi-
tive colorimetric assay called magnetic nano(e)zyme-linked immunosorbent assay
(MagLISA) was developed, wherein silica-shelled magnetic nanobeads (MagNBs)
and gold nanoparticles were pooled to monitor influenza A virus up to femtogram
per milliliter concentration (Oh et al. 2018). Sensitive and specific detection of
Crimean–Congo hemorrhagic fever virus (CCHFV) was developed employing spe-
cific IgM and IgG antibodies in human sera using recombinant CCHFV nucleopro-
tein as antigen in μ-capture and IgG immune complex (IC) ELISA tests (Emmerich
et al. 2018). Recently, truncated forms of HeV and NiV (Hendra and Nipah virus)
G proteins as well as full-length NiV nucleocapsid (N) protein were used for detec-
tion of Hendra and Nipah virus specific antibodies in pigs. These recombinant pro-
teins were expressed through diverse expression systems and an indirect ELISA
was developed for detection (Fischer et al. 2018). A rapid diagnostic platform for
colorimetric differential detection of DENV and CHIKV viral infections was
recently developed with a possibility to alter clinical diagnosis of acute febrile ill-
nesses in resource-limited settings. This platform principally facilitates consistent
and accurate multiplexed detection of chikungunya and dengue IgM/IgG antibodies
in human clinical samples within short stint (Wang et al. 2019).

17.2.2.2 Immunofluorescence-Based Immunodetection

Immunofluorescence (IF) is extensively used for speedy detection of viral infections
clinically started in early 1970s. IF is used for diagnosis of virus antigen and virus-­
specific IgG/IgA/IgM antibody in clinical specimens. In this technique, fluorescein-­
labeled antibody to stain specimens containing specific virus antigens, were used
for UV illumination. As a part of modern research on immunotechniques, new
recombinant rabies virus expressing green fluorescent protein (rRV-GFP) is more
rapid, simpler, and less expensive detection and for quantification of virus neutral-
izing antibodies in blood sera. This technique simplified multistep Rapid Fluorescent
Focus Inhibition Test (RFFIT) procedure by purging immunostaining step (Qin
et al. 2019a, b).

17.2.2.3 PCR/RT-PCR-Based Immunodetection

Nucleic acid (DNA and RNA) amplification assays are conventionally known as
polymerase chain reactions (PCRs). Distinct PCRs exists based on type of nucleic
acid and information known a propos the sequences of genomes for immunodetec-
tion. Rous Sarcoma Virus (RSV) is one of the most significant causative agents of
respiratory tract infection in children and related with high morbidity and mortal-
ity. However, very little is known with reference to effects of respiratory viral
266 P. V. Bramhachari et al.

infections. A reverse transcription recombinase polymerase amplification assay

(RT-RPA) is nucleic acid probe based on novel isothermal amplification technique
which has been widely employed to detect human RSV. The results exemplified
that concurrence rates between RT-RPA assay and qRT-PCR assay for clinical
samples was 96%, demonstrating that RT-RPA assay holds better diagnostic pre-
sentation on clinical samples in remote rural areas in developing countries (Xi
et al. 2019). Using quantitative PCR (qPCR) for common respiratory viruses and
for two genes (CCL8/CXCL11) is recognized to be extremely upregulated in viral
infections. Notably, RNA-seq virus detection achieved 86% sensitivity compared
to qPCR-­based screening in asthmatic children which consequently drives immune
cell airway infiltration, cellular remodeling, and alteration of asthmogenic gene
expression (Wesolowska-Andersen et al. 2017). Nevertheless as a part of latest
advancements on immunotechniques, isothermal reverse transcription and recom-
binase polymerase amplification (RT-RPA) of synthetic RNA (Ebola virus)
employing paper microfluidics devices was developed initially. Later on based on
RNA detection and multiplexed analysis for Ebola virus diagnostics were opti-
mized and demonstrated with high sensitivity. Additionally, nine-spot multilayered
device achieving parallel detection of three distinct RNA sequences opens a route
in the direction for detection of multiple viral pathogens (Magro et al. 2017).
Rabies virus (RABV) is one of the most significant global zoonotic pathogens.
Two sensitive real-time quantitative RT-PCR assays were developed and validated
for large-spectrum detection of RABV, with a focus on African isolates. The primer
and probe sets were targeted for highly conserved regions of nucleoprotein (N) and
polymerase (L) genes. Effective detection and high sensitivity of these assays can
be effectively functional in general research and used in diagnostic process and
epizootic surveillance (Faye et al. 2017). However, in recent times a fluorescent
reverse transcription loop-mediated isothermal amplification (RT-LAMP) employ-
ing quenching probes for detection of Middle East respiratory syndrome coronavi-
rus was developed. Additionally, detection efficacy of QProbe RT-LAMP was
comparable to that of RT-PCR assay (Azhar 2018). Furthermore this assay can as
well be used as authoritative diagnostic tool for rapid detection and surveillance of
MERS-CoV infections (Shirato et al. 2018). Consecutively to assist detection of
ZIKV infections, and distinguish these infections from DENV and CHIKV, Trioplex
real-time RT-PCR assay was recently developed. However the performance of
Trioplex real-time RT-PCR assay was particularly employed for the detection of
ZIKV, DENV and CHIKV viruses. Simultaneous testing of more than one specimen
type from each patient affords a superior diagnostic sensitivity of this technique
(Santiago et al. 2018).
An in situ hybridization (RNA-ISH) assay was recently developed to detect viral
hemorrhagic septicemia virus (VHSV), an OIE listed piscine rhabdovirus, in
infected fish cells with fathead minnow (FHM) as model cell line. Two antisense
RNA probes targeting fragments of N and G genes were amplified by RT-PCR
employing VHSV-specific primers trailed by transcription reaction in presence of
digoxigenin dUTP has competently localized VHSV mRNAs in infected cells. The
diagnostic sensitivity of RNA-ISH assay was better than immunocytochemistry,
17 Advanced Immunotechnological Methods for Detection and Diagnosis of Viral… 267

qRT-PCR and TCID50 (Qadiri et al. 2019). Development and evaluation of a new
one-step, real-time RT-PCR assay was developed for detecting latest H9N2 influ-
enza viruses competent of causing human infection. The sensitivity of one-step,
real-time RT-PCR assay was generally determined to be used in vitro transcribed
RNA, devoid of any cross-reactivity against RNA from H1–15 subtypes of influ-
enza viruses and other viral respiratory pathogens with no nonspecific reactions
(Saito et al. 2019).

17.2.2.4 Spectroscopy-Based Immunodetection

In the current trends mass spectrometry (MS) is a benchmark for qualitative and
quantitative diagnosis of viruses clinically. In clinical laboratories, MALDI (matrix-­
assisted laser desorption ionization) and ES (electrospray) ionization methods are
most frequently used as they allow ionization of analyte in considerable amounts.
The combination (RT-PCR/ESI-MS) was able to detect viral pathogens (acute viral
respiratory infections and influenza A viruses) usually for those viruses which are
undetected by regular testing methods as well as provides rapid and detailed data
(about types and subtypes of virus) in short period (Deyde et al. 2010; Chen et al.
2011). Yet another study reported that near-infrared spectroscopy (NIRS) is a rapid,
reagent-free, and cost-effective tool used to detect ZIKV noninvasively in heads and
thoraces of intact Aedes aegypti mosquitoes with high prediction accuracies relative
to quantitative RT-qPCR reaction. Perhaps this technique could be extended upon
for identifying probable arbovirus hotspots to guide spatial prioritization of vector
control (Fernandes et al. 2018). Recently developed surface plasma resonance
(SPR) spectroscopy was developed to be a valuable optical biosensor and potential
method for diagnosis of dengue virus E-protein and also for identification of anti-
bodies to DENV antigen. The diagnosis limit, sensitivity, and selectivity of SPR
sensing in DENV antigen was amazingly high. This technique was introduced as a
novel 3D-PAMAM-SAM-Au multilayer thin film for future research on SPR sens-
ing applications (Omar and Fen 2018).

17.2.2.5 miRNA-Based Immunodetection

miRNA are conserved small noncoding RNA with 19–24 nucleotides which regu-
lates post-transcriptional modification. miRNAs are transcribed by RNA polymerase
II into pre-miRNA which is processed by Dorsa/DGCR-8 and Dicer and transported
to cytoplasm. RISC in cytoplasm processes pre-miRNA into mature miRNA and
regulates post-transcriptional activities. As a part of the most advanced immunotech-
niques, exosomal microRNAs were recently studied as potential diagnostic markers
for various malignancies, including hepatocellular carcinoma (HCC). Serum exo-
somal microRNAs combined with alpha-fetoprotein as diagnostic markers of hepa-
tocellular carcinoma (Wang et al. 2018). In another study, synthetic miRNA-based
approach was developed to express neutralizing antibodies directly in lung via aero-
sol, to prevent from human RSV and influenza infections. Engineered mRNA-
expressed antibodies prevented RSV infection. It is noteworthy that an expressing
membrane-anchored broadly neutralizing antibody in lungs could potentially be
promising pulmonary prophylaxis approach (Tiwari et al. 2018). In a most recent
268 P. V. Bramhachari et al.

Table 17.1 Advanced immunotechnological techniques for detection and diagnosis of viral
infections
Application to diagnosis of emerging
S. No. Techniques viral infections
1. Serological tests Diagnostic approach for chronic
hepatitis C infection, ZIKV, RSV,
DENV, tick-borne encephalitis, and
West Nile viruses
2. Enzyme assays (fluorescence polarization Identification of structural proteins of
immune assay (FPIA), microparticle immune Hendra and Nipah virus, Crimean–
assay (MEIA), chemiluminescent (CLIA) Congo hemorrhagic fever virus
(CCHFV), chikungunya
3. PCR-based immunodetection (RT-RPA, Detection of Ebola virus, Rabies virus,
RT-LAMP, RNA-ISH, RT-PCR, Trioplex Middle East respiratory syndrome
real-time PCR) coronavirus, hemorrhagic septicemia
virus, influenza viruses
4. Mass spectrometry (MS/MS) Prognosis of DENV antigen, ZIKV
5. miRNA-based immunodetection Circulating microRNA biomarkers in
detection of arboviruses; hepatitis
infection
6. Next-generation sequencing (NGS) Provides more exact and precise time
estimates of infection, crucial for
HIV-1 surveillance
7. Metagenomics (panpathogen metagenomic Diagnostic standard techniques used in
sequencing assay) diagnosis and genetic analysis of
influenza and other clinical respiratory
viruses
8. Immunosensors (sandwich-type Diagnostic test for HBV, swine flu
electrochemiluminescence (ECL) (H1N1) infection, H7N9 virus with
immunosensor) high sensitivity
9. Microfluidic technology Used to test the convenience of
neutralizing antibodies to explore
impact on virus–cell interactions
10. CRISPR/Cas system Prognosis of coxsackievirus, hepatitis
B virus, Zika virus
11. Nanoparticles-based immunodetection Nanoparticle-based lateral flow
immunoassay as point-of-care
diagnostic tool for infectious agents
and diseases

study, endogenous microRNAs (miRNA) are evolutionarily conserved and their

presence in biological fluids signifies regulatory role of circulating miRNAs in
pathogenesis, immune responses, and viral infections. On the other hand, noninva-
sive diagnostic approach, using biomarkers, currently plays a central role in early
diagnosis of viral infections. Given the fact, a recent report depicted numerous circu-
lating microRNA biomarkers viz. miR155 and miR1260 in influenza; miR12,
miRVP3p, and miR184 in arboviruses; and miR29b and miR125 in hepatitis infec-
tion for diagnostic function, respectively (Ojha et al. 2019) (Table 17.1).
17 Advanced Immunotechnological Methods for Detection and Diagnosis of Viral… 269

17.2.2.6  ext-Generation Sequencing (NGS)-Based

N
Immunodetection
Next-generation sequencing (NGS) is one of the noteworthy achievements recorded
in the current era. Ahead from genome sequencing of known organisms, permitted
breakthrough of new viruses dependable for unknown human diseases, for tracking
viral outbreaks and pandemics as influenza to comprehend their emergence and
transmission profiles. Viral diversity from next-generation sequencing of HIV-1
samples provides more exact and precise estimates of time since infection, conse-
quently, the infection regencies are also crucial for HIV-1 surveillance and under-
standing of viral pathogenesis. NGS-derived average pair-wise diversity exhibited
higher sensitivity and specificity compared to fraction of ambiguous nucleotides
(Carlisle et al. 2019).

17.2.2.7 Metagenomics-Based Immunodetection

It is noteworthy that metagenomic next-generation sequencing assay (mNGS) for
pan-pathogen detection has been effectively tested in patients with acute illness of
several viral etiologies. In this connection a customized bioinformatics pipeline,
SURPI+, was recently designed to quickly analyze mNGS data, generate an auto-
mated summary of detected pathogens, and provide a graphical user interface for
evaluating and interpreting results (Miller et al. 2019). Panpathogen Metagenomic
sequencing assay for SLEV (St. Louis encephalitis virus) infection in CSF (cerebro-
spinal fluid) is an unbiased approach to infectious disease testing, although several
challenges still remain relating to test availability, interpretation, and validation that
were reported. However, recently metagenomic next-generation sequencing was
employed to diagnose fatal case of meningoencephalitis caused by SLEV (Chiu
et al. 2017). In another study, metagenomic viral sequencing is the potential diag-
nostic test for influenza which also provides insights on transmission, drug resis-
tance, evolution, and simultaneously detects other viruses. It is pertinent that Oxford
Nanopore Technology was employed to metagenomic sequencing of respiratory
samples. This technology operated with very high sensitivity compared to current
diagnostic standard techniques and certainly this approach may show a great prom-
ise for nanopore platform to be used in diagnosis and genetic analysis of influenza
and other clinical respiratory viruses (Lewandowski et al. 2019) (Table 17.1).

17.2.2.8 Monoclonal Antibodies-Based Immunodetection

In the recent past designing diagnostic and therapeutic platforms based on aptamer
technology is undoubtedly a potential approach in viral infections. Nevertheless
the oligonucleotide aptamers which are potential alternatives for monoclonal anti-
bodies based detection could be aimed against any protein in infected cells and any
components of viral particles are deemed as probable novel diagnostic molecules
270 P. V. Bramhachari et al.

against viral hepatitis. It is noteworthy that these aptamer molecules could be a

favorable substitute for monoclonal antibody in near future (Mirian et al. 2017).

17.2.2.9 Immunosensors-Based Immunodetection

A sensitive and selective electrochemical immunosensor for label-free ZIKV pro-
tein detection was recently developed, employing functionalized interdigitated
microelectrode of gold (IDE-Au) array. This ZIKV immune-sensing chip can be
integrated with miniaturized potentiostat (MP)-interfaced with smart phone for
rapid ZIKV-infection detection is obligatory for early stage diagnostics at point-of-­
care application (Kaushik et al. 2018). However, a cost-effective and portable
graphene-­enabled biosensor to detect ZIKV with a highly specific immobilized
monoclonal antibody was recently developed. Field effect biosensing (FEB) with
monoclonal antibodies covalently linked to graphene enables the real-time, quanti-
tative detection of native ZIKV antigens. This assay is first-of-its-kind graphene-­
enabled ZIKA biosensor which makes it an ideal candidate for the development as
a medical diagnostic test (Afsahi et al. 2018).
An accurate and timely diagnosis of any new reassortment of avian influenza is
very crucial for controlling disease outbreaks. In view of this, a simple strategy for
rapid and sensitive detection of H7N9 virus was achieved by employing an intensity-­
modulated surface plasmon resonance (IM-SPR) biosensor technique integrated
with newly generated monoclonal antibody. This novel antibody demonstrates note-
worthy specificity to identify H7N9 virus compared to homemade target-captured
ELISA, qRT-PCR, and rapid influenza diagnostic test (RIDT) with high sensitivity
(Chang et al. 2018).
In a recent study, sandwich-type electrochemiluminescence (ECL) immunosen-
sor was developed for ultrasensitive determination of HBV surface antigen. The
primary antibody of HBs (Ab1) was immobilized on surface of the carboxyl-­
modified magnetic nanoparticles (MNPs). Then, the PAMAM dendrimer with many
amine functional groups was employed as carrier for immobilizing CdTe@CdS
quantum dots (QDs) and the secondary antibody (Ab2) amplified ECL signal of
QDs considerably improved sensitivity. Strikingly, this ECL sensor was designed
based on signal amplification with dendrimer–quantum dots structures (Babamiri
et al. 2018). In another study, hemagglutinin (HA), a glycoprotein present on the
surface of influenza A subtype virus H1N1, virus binds to human cells with sialic
acid on membrane of upper respiratory tract. For early detection of swine flu (H1N1)
infection in human, an impedimetric hemagglutinin gene-based biosensor was
developed by immobilizing amino-labeled single-stranded DNA probe onto cyste-
ine modified gold surface of the screen printed electrode for early and rapid detec-
tion of H1N1 (swine flu) in human (Mohan et al. 2019).

17.2.2.10 Microfluidic Technology-Based Immunodetection

Microfluidic technique permitted research of viral measurement of fusion kinetics,
viral infectivity, and screening of viral responses to neutralizing molecules. Besides
that, microfluidic platforms also signify promising and innovative clinical tools
with applications in clinical diagnostics including drug screening. Microfluidics
17 Advanced Immunotechnological Methods for Detection and Diagnosis of Viral… 271

specifically emphasizes an array of techniques concerned with the precise control

and manipulation of fluids, within microscopic channels (Whitesides 2006). This
moderately clear-cut perception underpins a range of biological research tech-
niques, from flow cytometric and DNA analyses to enzyme and immunoassays
(Duncombe et al. 2015). A recent study investigating the use of an integrated micro-
fluidic system for diagnostics utilized aptamers against IAV (H1N1) for viral detec-
tion (Tsang et al. 2016) was studied. Remarkably, two recent studies into the
infectivity of murine norovirus (MNV) also utilized droplet-based microfluidic plat-
forms to test the convenience of neutralizing antibodies and explored their impact
on virus–cell interactions (Fischer et al. 2018). Since viral infections are complex
and highly dynamic process, appreciably affected by the physical and chemical
environment, studies into infection biology should preferably occur in microfluidics
system

17.2.2.11 CRISPR/Cas System-Based Immunodetection

The use of CRISPR/Cas system for RNA-based gene therapy is currently raising
numerous potential therapeutic applications. CRISPR/cas9 is the latest and
unique RNA targeted gene therapy successfully applied in 2007 and observed in
Staphylococcus pneumonia. The CRISPR is RNA sequence repeats which targets
the foreign DNA cleavage by binding to the PAM flanking sequences which
mediates the endonuclease called Cas through guide RNA (g-RNA) and respon-
sible for the double strand breaks in the host or foreign DNA and silences the
gene expression by nonhomologous end joining (NHEJ). There are different
kinds which are mediated by different cas proteins. Notably like class 2, type II
has cas9 where g-RNA complements cas protein and other system, eventually
class II and type V complements with cas12a and the CRISPR codes crRNA acts
as guide RNA by complementing the type V cas 12 protein complex. There are
other 29 CRISPER/cas systems that were identified (Makarova et al. 2015). The
application of CRISPR/CAS in viral diagnostics makes a breakthrough and it
increased specificity. The type V CRISPR-CAS12a is used for the detection of
the viral DNA. The CAS12a is designed specific to the viral DNA. The ssDNA is
tagged with a Reporter which has fluorophore and quencher on both ends. The
cleavage activity of viral DNA by cas12a confers the cleavage of the ssDNA. As
a result flourophore reporter, the cleavage of ssDNA emits the fluorescence
which is detected by quenchers and further amplified by recombinase polymerase
PCR. The technique was characterized by DETECTR. There are other methods
like SHERLOCK (Gootenberg et al. 2018) which works consiently based on the
Cas13 system. This tool is specially applied in the ZIKA virus diagnosis
(Table 17.1).
A novel arrayed CRISPR screen is aptly based on the plasmid library expressing
single-guide RNA (sgRNA) and disrupted 1514 genes, encoding kinases, proteins
related to endocytosis, and Golgi-localized proteins, individually using 4542
sgRNAs. This CRISPR screen uncovered host factors indispensable for infection by
coxsackievirus B3 (CVB3) which comprises human pathogens causing diverse dis-
eases. This technique is more sensitive as compared to arrayed screens based on
272 P. V. Bramhachari et al.

siRNA-mediated knockdown (Kim et al. 2018).The lenti viral-based CRISPR

screen-based sg-RNA plasmid pool can be employed as potential diagnostic tool for
HPV and can target other viral diseases. An automated POC system for EBV detec-
tion with RNA-guided RNA endonuclease Cas13a, employing its collateral RNA
degradation subsequent to its activation was recently developed by Qin and col-
leagues. Followed by an automated microfluidic mixing and hybridization, nonspe-
cific cleavage products of Cas13a were allowed to quantify by a custom-integrated
fluorometer. This CRISPR-Cas13a based diagnostic method is rapid, amplification-­
free, simple, and sensitive, thus establishing a key technology toward a useful POC
diagnostic platform (Qin et al. 2019a, b).

17.2.2.12 Nanoparticles-Based Immunodetection

The prologue of a new class of nanoscale materials with manifold exceptional prop-
erties and functions has sparked series of breakthrough applications in biomedical
and diagnostic applications. It is pertinent to note that some recent advances in
nanoparticle-based lateral flow immunoassay as point-of-care diagnostic tool for
infectious agents and diseases has been recently developed for the detection of
infectious viral agents. Lateral flow immunoassay (LFIA) technology is a paper-­
based, point-of-care strip biosensor designed to detect a specific analyte in virus-­
infected samples (Banerjee and Jaiswal 2018). AuNP-based detection techniques
were reported by various groups of clinically relevant viruses with a unique focus
on applied types of bio-AuNP hybrid structures, virus detection targets, and assay
modalities and formats were recently developed (Draz and Shafiee 2018).

17.3 Future Perspectives and Conclusions

The most recently developed diagnostic viral techniques are redesigning the field
of clinical virology, which could contribute to reduction in incidence of serious
infectious viral diseases. The foremost advantages of molecular techniques are its
higher sensitivity and specificity matched up with other diagnostic methods, viz.
serological assays and culture methods, as well as its rapidity and possibility of
automation. Nevertheless, the technological capabilities alone are inadequate if
not sustained by health promotion policies to boost the consciousness a propos the
significance of early detection of infectious viral diseases outbreak and its spread.
In conclusion, good quality diagnosis has a cost that only developed countries can
afford in regular practice so far, and this is delaying the execution of new-fangled
methods in developing world and in disease endemic areas. Conversely, it is antic-
ipated that asserted efforts may persist toward developing new high-quality tests
inexpensive in low-­income countries, which would considerably reinforce disease
control strategies.

Acknowledgments The authors are grateful to their respective academic institutions for the sup-
port extended.
17 Advanced Immunotechnological Methods for Detection and Diagnosis of Viral… 273

Conflict of Interest The authors declare that they have no competing interests.

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Current Advances in Multi-Epitope Viral
Vaccines Development and Research 18
A. M. V. N. Prathyusha and Pallaval Veera Bramhachari

Abstract
Viral diseases are major public health concern and cause significant morbidity
and mortality globally. The following dreadful viruses viz. Influenza virus, Ebola
virus (EBOV), and Sudan virus are the most recent viruses to cause a global
health concern. Despite recent progress in reduced deaths by viral infection, need
for new molecular, immunoinformatic tools, together with safe plus effective
vaccines is prerequisite due to the pros and cons of prophylactic and therapeutic
vaccines in failure to provide optimal protection. The challenges for viral vaccine
advancements are not restricted for recognition of suitable antigens or adjuvants
and delivery methods, nonetheless cover technical and manufacturing hurdles in
transforming a vaccine to clinic. Research and process improvement is techno-
logical basis which prompts production of new vaccines, essential for commer-
cialization. In this review, we emphasize the present status and recent advances
in designing and developing viral vaccines.

Keywords
Viral vaccines · Multi-epitopes · Inluenza virus · Ebola virus (EBOV) · Hepatitis
E virus

18.1 Introduction

During the past three decades several notable zoonotic virus infections, viz. HIV,
Nipah (NiV) viruses, Hendra (HeV), Ebola, avian influenza, Marburg filoviruses,
Lassa virus (LASV), Crimean–Congo hemorrhagic fever (CCHF) viruses and many
more have emerged suddenly from anonymity and became serious health threats

A. M. V. N. Prathyusha · P. V. Bramhachari (*)

Department of Biotechnology, Krishna University, Machilipatnam, India

© Springer Nature Singapore Pte Ltd. 2020 277

P. V. Bramhachari (ed.), Dynamics of immune activation in viral diseases,
https://doi.org/10.1007/978-981-15-1045-8_18
278 A. M. V. N. Prathyusha and P. V. Bramhachari

globally, provoking concern regarding their sustained epidemic transmission in

immunologically naive human population. With each new threat rapid need for
development of effective and efficient vaccines have been significant part for public
health concern. Indeed, vaccines are considered powerful strategy for prevention of
emerging viral infections, since, in most of the cases, further options for treatment
or effectiveness of therapeutics are limited or nonexistent. Vaccines for number of
viral diseases with major health concern are not yet available. However, struggles
are still enduring to develop fully effective vaccine strategies for recurrent and
emerging viruses and other current threats need to be addressed. In addition, resur-
gent interest remains, in development of new vaccines strategies against pandemic
viruses (Kanekiyo et al. 2019).
Traditional vaccine development approaches are amenable yet for emerging
viruses, while application of molecular techniques in virology has profoundly influ-
enced our understanding of virus biology. New approaches and technologies hold
an essential role in modern vaccine development and in silico approaches, multi-­
epitope (B and T cell epitopes, peptide, subunit) vaccines using immunoinformatics
depicts a major role to address challenges in vaccine development. In silico
approaches have attained great acceptance with current advancements in genome
and protein sequence databases. With the advancements in immunoinformatics,
limitations related to prediction of accurate B cell epitopes and human class I and II
restricted T cell epitopes are surmounted. This strategy can assemble vaccines with
epitopes for optimal presentation by phagocytic processing machinery. Furthermore,
several promising approaches are heading from lab to clinic to address unresolved
challenges in viral vaccine development. It is notable that multi-epitope (ME) vac-
cines possesses the ability to stimulate ample repertoires of immune responses,
along with dealing to pathogens effectively with genetic variations. Several
approaches of vaccination relying to replicating, attenuated, and nonreplicating
virus vectors have become useful vaccine delivery platforms. This chapter empha-
sizes a few recent multi-epitope vaccine approaches for effective vaccines encom-
passing some current vaccine platform strategies.

18.2 Multi-Epitope Vaccines

Epitope-based vaccines (epitope vaccines) signify novel approach for generation of

specific immune response and prevention of unfavorable responses against other
epitopes in complete antigen. EVs have several advantages over other forms of vac-
cines, particularly with regard to safety, ease of production, storage and distribution,
without cold chain issues. They also offer opportunity to vaccinate against several
pathogens or multiple epitopes from same pathogen. Potential advantages of
epitope-­based vaccines also include increased safety, opportunity to rationally engi-
neer epitopes for increased potency and breadth, and ability to focus immune
responses on conserved epitopes.
18 Current Advances in Multi-Epitope Viral Vaccines Development and Research 279

18.3 Multi-Epitope Subunit Vaccines

Subunit vaccines comprise merely antigenic part of virus with potential to promote
immune response while overcoming problems associated with conventional vac-
cines. Exploitation of vaccination strategy in combating virus is a major concern
since synthesis of conventional vaccines often has several obstacles. Hasan et al.
developed novel reverse vaccinology approach that aims to link immunogenetics,
immunogenomics using bioinformatics to identify new vaccine targets. Detection of
T cell and B cell epitopes and HLA (human leukocyte antigen) using in silico
approaches reinforced a possibility in potent vaccine candidate discovery. Vaccine
candidates may be identified as foreign molecules after injection to body. Therefore,
prediction of antigenicity is a crucial part in synthesis of subunit vaccines.
Furthermore, assessment of hydrophilicity is a significant criterion for B cell epit-
ope prediction. Hasan and group reported a design to synthesize nonallergic, immu-
nogenic, and thermostable chimeric ME monovalent subunit vaccine against avian
influenza A (H7N9) virus, and Marburg virus using vaccinomics approach. Viral
proteome was assessed for epitopes with high antigenicity using TMHMM, VaxiJen
v2.0 server (transmembrane topology screening), AllerTOP, AllergenFP, PA3P,
Allermatch servers (allergenicity and toxicity analysis), IEDB’s epitope conser-
vancy analysis tool (population coverage assessment), and MGL Tools (molecular
docking). Two proteins, envelope glycoprotein (GP) and matrix protein (VP40),
generated potent T cell epitopes and are recognized to be most antigenic proteins in
Marburg virus. PEP-FOLD de novo approach was used in prediction of 3D-peptide
structures from amino acid sequences of top ranked epitopes. Finally, three vaccines
were designed most effectively using suitable adjuvants with PADRE sequence,
combined in sequential manner with highly immunogenic (CTL, HTL, and
BCL) respectively. Such constructed vaccines with predicted epitopes were vali-
dated experimentally using animal models for their nonallergic reactions and immu-
nogenic potential (Hasan et al. 2019a, b). Using similar approaches subunit vaccines
may be produced against Chikungunya virus (Narula et al. 2018), Kaposi’s sarcoma-­
associated herpesvirus (KSHV) (Chauhan et al. 2019) respectively.

18.4 Multi-Epitope DNA Vaccines

DNA vaccines imply a convenient method to design vaccines with tailored epitopes
being administered into plasmid vectors with desirable memory response.
Furthermore, to evade unfavorable immunodominant epitopes from pathogens, chi-
meric multi-epitope-based DNA vaccine was designed against subgroup J avian
leukosis virus in chickens (Xu et al. 2016). Bounds and his group using in silico
algorithms designed DNA construct entailing HLA class II-restricted T cell epit-
opes obtained from GP and NP (nucleocapsid protein) of EBOV (Ebola virus),
SUDV (Sudan virus), and structural proteins of VEEV (Venezuelan equine enceph-
alitis virus). Identified epitopes with high specificity were examined for binding
ability to soluble HLA molecules. Then vaccinated BALB/c mice with ME-DNA
280 A. M. V. N. Prathyusha and P. V. Bramhachari

vaccine developed and evaluated for immune response using interferon (IFN)-g
ELISpot analyses. These studies provide a concept for designing an ME immuno-
gen along with evaluation focusing on immune response concerning preferred T cell
epitopes (Bounds et al. 2017).

18.5 Multi-Epitope Peptide Vaccines

Peptide vaccination can induce both humoral- and cell-mediated immune response
by stimulating T cell immunity in both humans and animals with minimal side
effects. Peptide vaccines are short immunogenic peptide fragments which can elicit
targeted immune response avoiding the chance of allergenic responses. Several pep-
tide vaccines were under progress, viz. vaccine for hepatitis C virus (HCV), human
immunodeficiency virus (HIV), foot and mouth disease, malaria, influenza, swine
fever, human papilloma virus (HPV), and anthrax (Verma et al. 2018). With advance-
ments in computational biology and immunoinformatics approaches, designing
effective strategies for prediction of antigenic epitopes became easier. A peptide-­
based vaccine is majorly presented via class II MHC molecules and gets processed
by endocytic pathway. Immune response can also be induced by cytotoxic T cells
(CTL) by cross-presentation where exogenous antigens are processed and presented
onto class I MHC. To improve antigenic presentation, cell penetrating peptides
(CPPs) was one of the promising approach to penetrate peptides efficiently into
cells using cationic peptides (TAT). Commonly used methods for CPP designing
and tagging to antigen were: (1) chemical linkage through covalent bonds, and (2)
recombinant fusion constructs by bacterial expression vectors. Gross et al. tagged a
C-terminal viral protein R (Vpr55-91 and Vpr55-82) fragment of human papilloma-
virus to CPP which paved way for practical ME immunization for neoantigen vac-
cination in cancer patients and is effective now clinically (Gross et al. 2019).

18.6 Vector-Based ME Vaccines

Site-specific recombination with Cre-recombinase based multi-epitope (ME) vac-

cine was designed using relatively conserved immunogenic domains of antigeni-
cally distinct strains of the H5, H7, and H9 avian influenza viruses. Three domains
M2 ectodomain (M2e), hemagglutinin (HA) fusion domain (HFD), T cell epitope of
nucleoprotein (TNP) and HA α-helix domain (HαD) were separated by linkers and
inserted into Human Adenovirus (Ad) vector. BALB/c mice were vaccinated and
evaluated for immune responses and protection efficacy using hemagglutination
inhibition, viral neutralization, ELISA, and ELISpot assays. Such ME vaccine
approach provided broad protection against avian influenza virus (Hassan et al.
2017). Yasmin and group have designed an RNA-dependent RNA polymerase (L)
epitope-based ME vaccine using immunoinformatics. Two conserved envelope gly-
coproteins GP1 and GP2 of EBOV were recognized as targets for synthesis of epit-
ope vaccine using various softwares. Collected EBOV glycoproteins were sequenced
using further examination for proteins with good immunogenicity by in silico
18 Current Advances in Multi-Epitope Viral Vaccines Development and Research 281

approaches. Such predicted B and T cell epitopes can confer long-lasting immunity
against EBOV with better ability of protection (Yasmin and Nabi 2016).

18.7 Current Vaccine Platform Strategies

Strategies for growth of triumphant vaccines are due to design of an antigen delivery
system that optimizes antigen presentation and stimulates broad protective immune
responses. Recent advances in vector delivery technologies, immunology, basic
virology, genetics, and molecular cell biology have led to in-depth understanding of
cellular mechanisms by which vaccines should stimulate the adaptive immune
response, thus presenting novel strategies of vaccination. Classic approach to vac-
cine development is still acquiescent to emerging viruses, the application of molec-
ular techniques in virology has overwhelmingly predisposed our perception of virus
biology, and vaccination schemes based on replication strategies, attenuated and
nonreplicating virus vector approaches that have turned out to be valuable vaccine
platforms. Several virus-like particle (VLP)-based vaccines are currently commer-
cializes in global market, including vaccines against hepatitis B virus and human
papillomavirus (Roldao et al. 2010). Furthermore, through genetic fusion or chemi-
cal conjugation, VLPs are attractive carrier proteins of foreign antigens, since they
can efficiently display them within a host immune system (Tissot et al. 2010;
Plummer and Manchester 2011, Brune et al. 2016). An important advantage of
VLP-based vaccine platforms is that VLPs can present antigens in a dense, repeti-
tive manner, thus effectively enabling the cross-linking of B cell receptors (BCRs)
(Zabel et al. 2014).

18.8 Innovation Challenges and Opportunities

Uncertainty of the public health priority and demand for some targets may be
unclear, which increases uncertainty of potential return on investment (ROI).
Furthermore, new vaccine targets are scientifically more complex and challenging.
The challenges presented may require substantial investment in new tools, stan-
dards, analysis methods, other novel approaches to demonstrate safety and effec-
tiveness of vaccine and also limited knowledge in science to progress optimal
vaccines. However, there is a limited understanding of viral pathogenesis and
immune responses against some targeted infectious viral agents, lack of optimal
immune response to potential vaccine candidates, and a limited understanding for
protection mechanism against some vaccine candidates. Conducting clinical trials
to evaluate safety and efficacy of certain preventive vaccines may be particularly
challenging for several reasons, including: relatively low disease incidence (con-
genital cytomegalovirus disease; neonatal group B streptococcal disease); limited
infrastructure in affected geographic areas (EBV vaccine); ethical considerations
(assessing novel approaches to pertussis vaccination of pregnant women to prevent
neonatal pertussis which is recommended); or new settings (hospital acquired
infections).
282 A. M. V. N. Prathyusha and P. V. Bramhachari

18.9 Conclusion and Future Perspectives

Increasing incidence of viral infections development of effective and efficient vac-

cines has mandated a significant part of public health concern. Strikingly, bioinfor-
matics, immunogenetics, immunoinformatics, reduces time and covers the need,
efforts should be expended in designing of new viral vaccines. However, epitope
vaccines made progress in development of more effective vaccines. Interpreting vac-
cine design, despite of using immunoinformatics and in silico approaches remain at
development stage yet. Furthermore, moderate successes might lead to significant
progress on ME vaccine efficiency against human and animal pathogens, particularly
in terms of optimizing T cell epitope polypeptide construct in vaccine assembly. This
further improves cellular processing in epitope presentation and protection against
highly variable viral pathogens.

Acknowledgment The authors gratefully acknowledge Krishna University, Machilipatnam for

the support extended.

Conflict of Interest The authors declare that they have no competing interests.

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