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Methods in
Molecular Biology 2183

Blaine A. Pfeifer
Andrew Hill Editors

Vaccine
Delivery
Technology
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
 Vaccine Delivery Technology

Methods and Protocols

Edited by

Blaine A. Pfeifer and Andrew Hill

Department of Chemical and Biological Engineering, University at Buffalo, The State University
of New York, Buffalo, NY, USA
Editors
Blaine A. Pfeifer Andrew Hill
Department of Chemical and Biological Department of Chemical and Biological
Engineering Engineering
University at Buffalo, The State University University at Buffalo, The State University
of New York of New York
Buffalo, NY, USA Buffalo, NY, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-0794-7 ISBN 978-1-0716-0795-4 (eBook)
https://doi.org/10.1007/978-1-0716-0795-4
© Springer Science+Business Media, LLC, part of Springer Nature 2021
Chapter 7 is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/). For further details see license information in the chapters.
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The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface

In this book for Methods in Molecular Biology, titled Vaccine Delivery Technology, we make a
holistic effort to cover the vaccine development process and the role delivery concepts
contribute to a global goal of effective final health outcomes. In so doing, readers are
presented with topics that span a broad array of ongoing research in vaccine production
intended to be educational and useful to those both within and outside of the vaccine fields.
Of note, we include chapters from both industry and academia with contributions coming
from different countries, continents, and perspectives.
This diversity in content and contributions adds to the educational breadth of the book
but also flavors applications by the regions of the globe contributing associated chapters. As
such, the general reader will also gain appreciation for the development of vaccination
research programs associated with regional disease concerns. An additional advantage to
this breadth of topics is the knowledge regarding diseases that rely upon vaccine research and
development to provide much needed treatment options.
In terms of content flow, the book begins with vaccine basics, including several early
chapters devoted to antigen identification and selection. Chapters include computational
approaches, provided by the Ellis and Daura groups, to antigen identification. These
chapters build on the immense level of data generated through various next generation
sequencing and associated ‘omics approaches. In related chapters, the Bidmos and McKay
groups discuss the identification of functional monoclonal antibodies resulting from antigen
exposure.
The following and related chapters next turn to antigen preparation and established
forms of antigen administration. Chapters devoted to genetic and protein antigen prepara-
tion are provided by the Lundstrom, Nichita, and Czermak groups. These preparation
methods overlap with the earlier introduced chapters on monoclonal antibody capture
from said antigens, particularly in the case for protein-based antigens. Included with these
chapters is a contribution from the Bracewell group on rapid and high-throughput methods
of antigen purification.
Subsequent chapters (contributed by the Smith, Keys, Wolff, Shukla, Micoli, Guo, and
Fiebig groups) cover various formats for vaccine formulations. These include the use of
viral-like particles (VLPs), whole cell vaccines, and glycoconjugate vaccines. Included in
these chapters are unique methodologies for combining emerging approaches for in vivo
glycoconjugation with VLP carriers. In addition, the Chakravortty group provides a chapter
on attenuation methods used in particular for live whole cell vaccines.
The next several chapters, contributed by the Ramsey, Czermak, Rak, Chakravortty,
Mancha-Agresti, and Pfeifer groups, focus on the central theme of this book, that is, vaccine
delivery. As such, topics span viral and nonviral gene delivery technology, the use of bacterial
and hybrid bacterial-biomaterial delivery devices, dual antigen delivery liposomal carriers,
and needle-less noninvasive delivery technology. Intermixed with these chapters are impor-
tant contributions by the Petrovsky, Zeng, and Cui groups that present methodologies
associated with vaccine adjuvant selection and long-term vaccine storage preparation.
The final series of chapters are devoted to vaccine delivery effectiveness assessment. The
McCluskie, Herbert, and Bou Ghanem groups provide chapters that cover confirmation of
vaccine delivery carriers, common assessment methodologies (ELISpot, ELISA), methods

v
vi Preface

that profile the in vivo progression of the immune response, novel monitoring of viral
infectivity quantification, and disease-specific antibody functionality. The emphasis on final
vaccine effectiveness thus concludes the range of topics covered by overall book
contributions.
In summary, this volume was designed broadly and generally enough to engage the
nonexpert interested in the vaccine development field. Alternatively, we highlight the central
theme of vaccine delivery technology through a series of chapters that cover delivery
methods designed to enable and/or enhance vaccine effectiveness across specific disease
applications. Combined, we expect this book to serve as a valuable resource, to those within
and outside of the field, in the ongoing research pursuits to expand and improve upon the
already significant impact made by vaccines.

Buffalo, NY, USA Blaine A. Pfeifer

Andrew Hill
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

1 Vaccine Delivery and Immune Response Basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Andrew Hill, Marie Beitelshees, and Blaine A. Pfeifer
2 Isolating Pathogen-Specific Human Monoclonal Antibodies (hmAbs)
Using Bacterial Whole Cells as Molecular Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Sara Siris, Camilla A. Gladstone, Yanping Guo,
Christopher L. Pinder, Robin J. Shattock, Paul F. McKay,
Paul R. Langford, and Fadil A. Bidmos
3 Use of Chlamydial Elementary Bodies as Probes to Isolate
Pathogen-Specific Human Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Christopher L. Pinder, Paul F. McKay, and Robin J. Shattock
4 Computational Antigen Discovery for Eukaryotic
Pathogens Using Vacceed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Stephen J. Goodswen, Paul J. Kennedy, and John T. Ellis
5 Antigen Discovery in Bacterial Panproteomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Daniel Yero, Oscar Conchillo-Solé, and Xavier Daura
6 Alphavirus-Based Antigen Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Kenneth Lundstrom
7 Production of Chimeric Hepatitis B Virus Surface Antigens
in Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Mihaela-Olivia Dobrica, Catalin Lazar,
and Norica Branza-Nichita
8 Bioreactor-Based Antigen Production Process Using the Baculovirus
Expression Vector System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Julie Harnischfeger, Lukas K€ a ßer, Jan Zitzmann,
Denise Salzig, and Peter Czermak
9 High-Throughput Process Development for the Chromatographic Purification
of Viral Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Shaleem I. Jacob, Spyridon Konstantinidis,
and Daniel G. Bracewell
10 Production of Zika Virus Virus-Like Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Atichat Kuadkitkan, Suwipa Ramphan, Suchin Worawichawong,
Wannapa Sornjai, Nitwara Wikan, and Duncan R. Smith
11 Biosynthesis of Glycoconjugate Virus-like Particles (VLPs). . . . . . . . . . . . . . . . . . . 205
Kathryn K. Oi, Tom A. Kloter, and Timothy G. Keys
12 Upstream and Downstream Processes for Viral Nanoplexes as Vaccines . . . . . . . . 217
Keven Lothert, Gregor Dekevic, Daniel Loewe, Denise Salzig,
Peter Czermak, and Michael W. Wolff

vii
viii Contents

13 Whole-Cell Vaccine Preparation: Options and Perspectives. . . . . . . . . . . . . . . . . . . 249

Punit Kumar, Sunita, Kashyap Kumar Dubey,
and Pratyoosh Shukla
14 O-Antigen Extraction, Purification, and Chemical Conjugation
to a Carrier Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Francesca Micoli, Carlo Giannelli,
and Roberta Di Benedetto
15 Oligosaccharide Antigen Conjugation to Carrier Proteins
to Formulate Glycoconjugate Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
Brittany R. Smith and Zhongwu Guo
16 Exploitation of Capsule Polymerases for Enzymatic Synthesis
of Polysaccharide Antigens Used in Glycoconjugate Vaccines. . . . . . . . . . . . . . . . . 313
Christa Litschko, Insa Budde, Monika Berger,
and Timm Fiebig
17 Attenuation Methods for Live Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
Dipasree Hajra, Akshay Datey,
and Dipshikha Chakravortty
18 Surface Modification of Adenovirus Vector to Improve Immunogenicity
and Tropism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
Yasmine Gabal and Joshua D. Ramsey
19 Production of Baculovirus and Stem Cells for Baculovirus-Mediated
Gene Transfer into Human Mesenchymal Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . 367
Friederike Eilts, Julie Harnischfeger, Daniel Loewe,
Michael W. Wolff, Denise Salzig, and Peter Czermak
20 Lipofection-Based Delivery of DNA Vaccines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
Monika Rak, Anna Go ra-Sochacka, and Zbigniew Madeja
21 Flavivirus DNA Vaccine Design and Adjuvant Selection. . . . . . . . . . . . . . . . . . . . . . 405
Lei Li, Yoshikazu Honda-Okubo, and Nikolai Petrovsky
22 Vaccine Delivery with a Detoxified Bacterial Toxin . . . . . . . . . . . . . . . . . . . . . . . . . . 423
Diana Diaz-Arévalo, Yanping Chen, and Mingtao Zeng
23 Needleless or Noninvasive Delivery Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437
Akshay Datey, Jagadeesh Gopalan, and Dipshikha Chakravortty
24 Lactic Acid Bacteria as Delivery Vehicle for Therapeutics Applications . . . . . . . . . 447
Viviane Lima Batista, Tales Fernando da Silva,
Luis Cláudio Lima de Jesus, Ana Paula Tapia-Costa,
Mariana Martins Drumond, Vasco Azevedo,
and Pamela Mancha-Agresti
25 A Hybrid Biological–Biomaterial Vector for Antigen Delivery . . . . . . . . . . . . . . . . 461
Ruiquan Qi, Andrew Hill, and Blaine A. Pfeifer
26 Liposomal Dual Delivery of Both Polysaccharide and Protein Antigens . . . . . . . . 477
Roozbeh Nayerhoda, Andrew Hill, and Blaine A. Pfeifer
27 Thin-Film Freeze-Drying Is a Viable Method to Convert Vaccines Containing Alumi-
num Salts from Liquid to Dry Powder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
Riyad F. Alzhrani, Haiyue Xu, Chaeho Moon,
Laura J. Suggs, Robert O. Williams III,
and Zhengrong Cui
 Contents ix

28 Application of Cryogenic Transmission Electron Microscopy for Evaluation

of Vaccine Delivery Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
Hui Qian, Yimei Jia, and Michael J. McCluskie
29 Methods to Evaluate Immune Cell Recruitment and Cellular Uptake
and Distribution of Antigen Following Intramuscular Administration
of Vaccine to Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
Gerard Agbayani, Felicity C. Stark, Bassel Akache,
and Michael J. McCluskie
30 The Quantification of Antigen-Specific T Cells by IFN-γ ELISpot . . . . . . . . . . . . 525
Bassel Akache and Michael J. McCluskie
31 Measurement of Antigen-Specific IgG Titers by Direct ELISA . . . . . . . . . . . . . . . 537
Bassel Akache, Felicity C. Stark, and Michael J. McCluskie
32 A Method to Evaluate In Vivo CD8+ T Cell Cytotoxicity
in a Murine Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549
Felicity C. Stark, Renu Dudani, Gerard Agbayani,
and Michael J. McCluskie
33 Testing Anti-Pneumococcal Antibody Function Using Bacteria
and Primary Neutrophils. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559
Manmeet Bhalla, Shaunna R. Simmons, Essi Y. I. Tchalla,
and Elsa N. Bou Ghanem
34 Viral Infectivity Quantification and Neutralization Assays
Using Laser Force Cytology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
Colin G. Hebert, Keya L. Rodrigues, Nicole DiNardo,
and Anna-Barbara Hachmann

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
Contributors

GERARD AGBAYANI • Human Health Therapeutics, National Research Council Canada,

Ottawa, ON, Canada
BASSEL AKACHE • Human Health Therapeutics, National Research Council Canada,
Ottawa, ON, Canada
RIYAD F. ALZHRANI • Division of Molecular Pharmaceutics and Drug Delivery, College of
Pharmacy, The University of Texas at Austin, Austin, TX, USA
VASCO AZEVEDO • Laboratory of Cellular and Molecular Genetics (LGCM), Department of
Genetics, Ecology and Evolution, Institute of Biological Sciences, Federal University of
Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil
VIVIANE LIMA BATISTA • Laboratory of Cellular and Molecular Genetics (LGCM),
Department of Genetics, Ecology and Evolution, Institute of Biological Sciences, Federal
University of Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil
MARIE BEITELSHEES • Department of Chemical and Biological Engineering, The State
University of New York at Buffalo, Buffalo, NY, USA
MONIKA BERGER • Institute of Clinical Biochemistry, Hannover Medical School, Hannover,
Germany
MANMEET BHALLA • Department of Microbiology and Immunology, University at Buffalo
School of Medicine, Buffalo, NY, USA
FADIL A. BIDMOS • Section of Paediatric Infectious Disease, Department of Infectious Disease,
Imperial College London, London, UK
ELSA N. BOU GHANEM • Department of Microbiology and Immunology, University at
Buffalo School of Medicine, Buffalo, NY, USA
DANIEL G. BRACEWELL • Department of Biochemical Engineering, University College
London, London, UK
NORICA BRANZA-NICHITA • Institute of Biochemistry of the Romanian Academy, Bucharest,
Romania
INSA BUDDE • Institute of Clinical Biochemistry, Hannover Medical School, Hannover,
Germany
DIPSHIKHA CHAKRAVORTTY • Department of Microbiology and Cell Biology, Indian Institute
of Science, Bengaluru, India; Centre for Biosystems Science and Engineering, Indian
Institute of Science, Bengaluru, India
YANPING CHEN • Center of Emphasis in Infectious Diseases, Department of Molecular and
Translational Medicine, Paul L. Foster School of Medicine, Texas Tech University Health
Sciences Center El Paso, El Paso, TX, USA
OSCAR CONCHILLO-SOLÉ • Institute of Biotechnology and Biomedicine, Universitat
Autònoma de Barcelona, Cerdanyola del Vallès, Spain; Department of Genetics and
Microbiology, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain
ZHENGRONG CUI • Division of Molecular Pharmaceutics and Drug Delivery, College of
Pharmacy, The University of Texas at Austin, Austin, TX, USA
PETER CZERMAK • Institute of Bioprocess Engineering and Pharmaceutical Technology
(IBPT), Technische Hochschule Mittelhessen (THM) - University of Applied Sciences,
Giessen, Germany; Faculty of Biology and Chemistry, Justus-Liebig-University Giessen,

xi
xii Contributors

Giessen, Germany; Division Bioresources, Fraunhofer Institute for Molecular Biology and
Applied Ecology (IME), Giessen, Germany
TALES FERNANDO DA SILVA • Laboratory of Cellular and Molecular Genetics (LGCM),
Department of Genetics, Ecology and Evolution, Institute of Biological Sciences, Federal
University of Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil
AKSHAY DATEY • Centre for Biosystems Science and Engineering, Indian Institute of Science,
Bengaluru, India
XAVIER DAURA • Institute of Biotechnology and Biomedicine, Universitat Autònoma de
Barcelona, Cerdanyola del Vallès, Spain; Catalan Institution for Research and Advanced
Studies (ICREA), Barcelona, Spain
LUIS CLÁUDIO LIMA DE JESUS • Laboratory of Cellular and Molecular Genetics (LGCM),
Department of Genetics, Ecology and Evolution, Institute of Biological Sciences, Federal
University of Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil
GREGOR DEKEVIC • Institute of Bioprocess Engineering and Pharmaceutical Technology
(IBPT), Technische Hochschule Mittelhessen (THM) - University of Applied Sciences,
Giessen, Germany
ROBERTA DI BENEDETTO • GSK Vaccines Institute for Global Health, Siena, Italy
DIANA DIAZ-ARÉVALO • Grupo Funcional de Inmunologı́a, Fundacion Instituto de
Inmunologı́a de Colombia-FIDIC, School of Medicine and Health Sciences, Universidad
del Rosario, Bogotá, D.C., Colombia
NICOLE DINARDO • Thermo Fisher Scientific Inc., Grand Island, NY, USA
MIHAELA-OLIVIA DOBRICA • Institute of Biochemistry of the Romanian Academy, Bucharest,
Romania
MARIANA MARTINS DRUMOND • Laboratory of Cellular and Molecular Genetics (LGCM),
Department of Genetics, Ecology and Evolution, Institute of Biological Sciences, Federal
University of Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil; Department of
Biological Sciences, Federal Center for Technological Education of Minas Gerais (CEFET/
MG), Belo Horizonte, Minas Gerais, Brazil
KASHYAP KUMAR DUBEY • Department of Biotechnology, Central University of Haryana,
Mahendergarh, Haryana, India
RENU DUDANI • Human Health Therapeutics, National Research Council Canada,
Ottawa, ON, Canada
FRIEDERIKE EILTS • Institute of Bioprocess Engineering and Pharmaceutical Technology
(IBPT), Technische Hochschule Mittelhessen (THM) - University of Applied Sciences,
Giessen, Germany
JOHN T. ELLIS • School of Life Sciences, University of Technology Sydney (UTS), Ultimo, NSW,
Australia
TIMM FIEBIG • Institute of Clinical Biochemistry, Hannover Medical School, Hannover,
Germany
YASMINE GABAL • School of Chemical Engineering, Oklahoma State University, Stillwater,
OK, USA
CARLO GIANNELLI • GSK Vaccines Institute for Global Health, Siena, Italy
CAMILLA A. GLADSTONE • Section of Paediatric Infectious Disease, Department of Infectious
Disease, Imperial College London, London, UK
STEPHEN J. GOODSWEN • School of Life Sciences, University of Technology Sydney (UTS),
Ultimo, NSW, Australia
 Contributors xiii

JAGADEESH GOPALAN • Centre for Biosystems Science & Engineering, Indian Institute of
Science, Bangalore, Karnataka, India; Department of Aerospace Engineering, Indian
Institute of Science, Bangalore, Karnataka, India
ANNA GÓRA-SOCHACKA • Institute of Biochemistry and Biophysics, Polish Academy of Sciences,
Warsaw, Poland
YANPING GUO • Flow Cytometry Core Facility, National Heart and Lung Institute, Imperial
College London, London, UK
ZHONGWU GUO • Department of Chemistry, University of Florida, Gainesville, FL, USA
ANNA-BARBARA HACHMANN • Thermo Fisher Scientific Inc., Grand Island, NY, USA
DIPASREE HAJRA • Department of Microbiology and Cell Biology, Indian Institute of Science,
Bengaluru, India
JULIE HARNISCHFEGER • Institute of Bioprocess Engineering and Pharmaceutical Technology
(IBPT), Technische Hochschule Mittelhessen (THM) - University of Applied Sciences,
Giessen, Germany
COLIN G. HEBERT • LumaCyte, LLC, Charlottesville, VA, USA
ANDREW HILL • Department of Chemical and Biological Engineering, University at Buffalo,
The State University of New York, Buffalo, NY, USA
YOSHIKAZU HONDA-OKUBO • Vaxine Pty Ltd, Warradale, SA, Australia; College of Medicine
and Public Health, Flinders University, Adelaide, SA, Australia
SHALEEM I. JACOB • Department of Biochemical Engineering, University College London,
London, UK
YIMEI JIA • Human Health Therapeutics, National Research Council Canada, Ottawa, ON,
Canada
LUKAS KA߀ ER • Institute of Bioprocess Engineering and Pharmaceutical Technology (IBPT),
Technische Hochschule Mittelhessen (THM) - University of Applied Sciences, Giessen,
Germany
PAUL J. KENNEDY • School of Computer Science, Faculty of Engineering and Information
Technology and the Centre for Artificial Intelligence, University of Technology Sydney
(UTS), Ultimo, NSW, Australia
TIMOTHY G. KEYS • Institute of Microbiology, ETH Zurich, Zurich, Switzerland
TOM A. KLOTER • Institute of Microbiology, ETH Zurich, Zurich, Switzerland
SPYRIDON KONSTANTINIDIS • Department of Biochemical Engineering, University College
London, London, UK
ATICHAT KUADKITKAN • Institute of Molecular Biosciences, Mahidol University, Nakhon
Pathom, Thailand
PUNIT KUMAR • Department of Biotechnology, University Institute of Engineering and
Technology, Maharshi Dayanand University Rohtak, Rohtak, Haryana, India;
Department of Clinical Immunology, Allergology and Microbiology, Karaganda Medical
University, Karaganda, Kazakhstan
PAUL R. LANGFORD • Section of Paediatric Infectious Disease, Department of Infectious
Disease, Imperial College London, London, UK
CATALIN LAZAR • Institute of Biochemistry of the Romanian Academy, Bucharest, Romania
LEI LI • Vaxine Pty Ltd, Warradale, SA, Australia; College of Medicine and Public Health,
Flinders University, Adelaide, SA, Australia
CHRISTA LITSCHKO • Institute of Clinical Biochemistry, Hannover Medical School,
Hannover, Germany
xiv Contributors

DANIEL LOEWE • Institute of Bioprocess Engineering and Pharmaceutical Technology

(IBPT), Technische Hochschule Mittelhessen (THM) - University of Applied Sciences,
Giessen, Germany
KEVEN LOTHERT • Institute of Bioprocess Engineering and Pharmaceutical Technology
(IBPT), Technische Hochschule Mittelhessen (THM) - University of Applied Sciences,
Giessen, Germany
KENNETH LUNDSTROM • PanTherapeutics, Lutry, Switzerland
ZBIGNIEW MADEJA • Department of Cell Biology, Faculty of Biochemistry, Biophysics and
Biotechnology, Jagiellonian University, Krakow, Poland
PAMELA MANCHA-AGRESTI • Laboratory of Cellular and Molecular Genetics (LGCM),
Department of Genetics, Ecology and Evolution, Institute of Biological Sciences, Federal
University of Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil; Biomedical
Science Institute (ICBM), Catholic of Cuyo University, San Juan, CP, Argentina
MICHAEL J. MCCLUSKIE • Human Health Therapeutics, National Research Council
Canada, Ottawa, ON, Canada
PAUL F. MCKAY • Section of Immunology of Infection, Department of Infectious Disease,
Imperial College London, London, UK
FRANCESCA MICOLI • GSK Vaccines Institute for Global Health, Siena, Italy
CHAEHO MOON • Division of Molecular Pharmaceutics and Drug Delivery, College of
Pharmacy, The University of Texas at Austin, Austin, TX, USA
ROOZBEH NAYERHODA • Department of Biomedical Engineering, University at Buffalo, The
State University of New York at Buffalo, Buffalo, NY, USA
KATHRYN K. OI • Institute of Microbiology, ETH Zurich, Zurich, Switzerland
NIKOLAI PETROVSKY • Vaxine Pty Ltd, Warradale, SA, Australia; College of Medicine and
Public Health, Flinders University, Adelaide, SA, Australia
BLAINE A. PFEIFER • Department of Chemical and Biological Engineering, University at
Buffalo, The State University of New York, Buffalo, NY, USA
CHRISTOPHER L. PINDER • Section of Immunology of Infection, Department of Infectious
Disease, Imperial College London, London, UK
RUIQUAN QI • Department of Chemical and Biological Engineering, University at Buffalo,
The State University of New York at Buffalo, Buffalo, NY, USA
HUI QIAN • Nanotechnology Research Center, National Research Council Canada,
Edmonton, AB, Canada
MONIKA RAK • Department of Cell Biology, Faculty of Biochemistry, Biophysics and
Biotechnology, Jagiellonian University, Krakow, Poland
SUWIPA RAMPHAN • Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom,
Thailand
JOSHUA D. RAMSEY • School of Chemical Engineering, Oklahoma State University, Stillwater,
OK, USA
KEYA L. RODRIGUES • LumaCyte, LLC, Charlottesville, VA, USA
DENISE SALZIG • Institute of Bioprocess Engineering and Pharmaceutical Technology (IBPT),
Technische Hochschule Mittelhessen (THM) - University of Applied Sciences, Giessen,
Germany
ROBIN J. SHATTOCK • Section of Immunology of Infection, Department of Infectious Disease,
Imperial College London, London, UK
PRATYOOSH SHUKLA • Enzyme Technology and Protein Bioinformatics Laboratory,
Department of Microbiology, Maharshi Dayanand University Rohtak, Rohtak, Haryana,
India
 Contributors xv

SHAUNNA R. SIMMONS • Department of Microbiology and Immunology, University at Buffalo

School of Medicine, Buffalo, NY, USA
SARA SIRIS • Section of Paediatric Infectious Disease, Department of Infectious Disease,
Imperial College London, London, UK
BRITTANY R. SMITH • Department of Chemistry, University of Florida, Gainesville, FL, USA
DUNCAN R. SMITH • Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom,
Thailand
WANNAPA SORNJAI • Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom,
Thailand
FELICITY C. STARK • Human Health Therapeutics, National Research Council Canada,
Ottawa, ON, Canada
LAURA J. SUGGS • Department of Biomedical Engineering, Cockrell School of Engineering,
The University of Texas at Austin, Austin, TX, USA
SUNITA • Enzyme Technology and Protein Bioinformatics Laboratory, Department of
Microbiology, Maharshi Dayanand University Rohtak, Rohtak, Haryana, India
ANA PAULA TAPIA-COSTA • Biomedical Science Institute (ICBM), Catholic of Cuyo
University, San Juan, CP, Argentina
ESSI Y. I. TCHALLA • Department of Microbiology and Immunology, University at Buffalo
School of Medicine, Buffalo, NY, USA
NITWARA WIKAN • Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom,
Thailand
ROBERT O. WILLIAMS III • Division of Molecular Pharmaceutics and Drug Delivery,
College of Pharmacy, The University of Texas at Austin, Austin, TX, USA
MICHAEL W. WOLFF • Institute of Bioprocess Engineering and Pharmaceutical Technology
(IBPT), Technische Hochschule Mittelhessen (THM) - University of Applied Sciences,
Giessen, Germany
SUCHIN WORAWICHAWONG • Department of Pathology, Faculty of Medicine, Ramathibodi
Hospital, Mahidol University, Bangkok, Thailand
HAIYUE XU • Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy,
The University of Texas at Austin, Austin, TX, USA
DANIEL YERO • Institute of Biotechnology and Biomedicine, Universitat Autònoma de
Barcelona, Cerdanyola del Vallès, Spain; Department of Genetics and Microbiology,
Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain
MINGTAO ZENG • Center of Emphasis in Infectious Diseases, Department of Molecular and
Translational Medicine, Paul L. Foster School of Medicine, Texas Tech University Health
Sciences Center El Paso, El Paso, TX, USA
JAN ZITZMANN • Institute of Bioprocess Engineering and Pharmaceutical Technology (IBPT),
Technische Hochschule Mittelhessen (THM) - University of Applied Sciences, Giessen,
Germany
 Chapter 1

Vaccine Delivery and Immune Response Basics

Andrew Hill, Marie Beitelshees, and Blaine A. Pfeifer

Abstract
In this opening chapter, we outline the basics of vaccine delivery and subsequent immune reactivity. Vaccine
delivery is an augmentation to immunization more generally in that a delivery reagent is harnessed to
improve administration of the key ingredient (i.e., the antigen) needed to provoke an immune response. In
this chapter, we discuss the evolution of vaccine design and how such efforts evolved into targeted
administration/delivery of key antigens. We then provide overview descriptions of vaccine immune
responses and methods for assessment. More generally, the chapter sets the tone for the remainder of this
book, which will focus upon each step of the vaccine process with a special emphasis on how vaccine delivery
contributes to overall health outcomes.

Key words Vaccine, Antigen, Adjuvant, Antigen-presenting cell, Humoral response, Cellular
response

1 Introduction to Vaccination

Vaccines represent one of the greatest medical achievements in the

history of mankind due to the role they have played in preventing
disease. Since their introduction, vaccines have significantly cur-
tailed various diseases such as small pox, diphtheria, polio, measles,
pneumococcal disease, and so on in countries such as the USA (see
Table 1) [1]. The reduction of many of these disease cases have
been achieved through the implementation of pediatric regimens
against such pathogens [1]. Furthermore, vaccines are useful within
the broader population to reduce the burden associated with dis-
eases caused by pathogens such as the influenza virus, shingles, and
the human papillomavirus (HPV). In addition, vaccines against
diseases such as yellow fever are available to provide protection to
individuals during travel. The primary benefit that is realized from
vaccination is that it provides a prophylaxis against potentially
deadly diseases, rather than treating the diseases as they occur.
These benefits can be enhanced through the achievement of a
phenomena known as herd immunity, in which vaccination of a

Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021

1
2 Andrew Hill et al.

Table 1
Disease reduction associated vaccine implementation in the USA [16]

Type Prevaccine Year vaccine Postvaccine

Disease of vaccine annual cases introduced annual casesa
Smallpox Similar virus 29,000 1798 0 (100%)
Diphtheria Toxoid 21,053 1923 0 (100%)
Pertussis Inactivated 200,752 1926 15,632 (92.2%)
Tetanus Toxoid 580 1927 41 (92.9%)
Polio Inactivated 36,110 1955 0 (100%)
Measles Attenuated 530,217 1963 56 (99.9%)
Mumps Attenuated 162,344 1967 6584 (95.9%)
Rubella Attenuated 47,897 1969 12 (99.9%)
Hepatitis B Subunit 66,232 1981 13,169 (80.1%)
b
Hib Glycoconjugate 20,000 1987 50 (99.8%)
Hepatitis A Inactivated 117,333 1995 15,298 (87.0%)
Varicella Attenuated 4,085,120 1995 612,768 (85.0%)
Pneumococcal Glycoconjugate 63,067 2000 41,550 (34.1%)
a
Percentages refer to percentage of disease reduction observed in 2006 following the introduction of the vaccine
Haemophilus influenzae type b
b

portion of the population is able to extend protection to the

population that is unvaccinated. This occurs because the pathogens
do not have available hosts through which they can transmit them-
selves to infect the vulnerable population. The result enables vac-
cines to provide substantial economic benefits that are typically
presented as quality-adjusted life years (QALYs) or disability-
adjusted life years (DALYs) saved compared to nonvaccination
[2, 3].
The isolation of pathogens subsequently provided an opportu-
nity for the development of vaccines targeting specific targets. Early
vaccine development involved the isolation of various human
pathogens and subjecting them to various procedures to weaken
their virulence. One of the initial studies to achieve this was the
development of a vaccine against anthrax in animals using bacteria
that had been attenuated using carbolic acid [4]. A similar approach
utilized formaldehyde to attenuate the rabies virus that had been
isolated from cerebral tissue [5]. In subsequent vaccines, additional
strategies for the attenuation or inactivation of pathogens were
developed, which include heat, chemical methods (e.g., hydroxyl-
amine, β-propiolactone, and methylene blue), and irradiation (e.g.,
ultraviolet) [6]. These studies have facilitated the development and
manufacturing of whole-pathogen vaccines (WPVs). This laid the
 Immunology Overview for Vaccine Development 3

framework for future work which has provided a demonstration

that immunization with a weakened or inactivated pathogen could
be used to provide immunity towards the live version.
Despite the utility of various WPVs, advanced fermentation and
culturing techniques for growing the pathogens were not initially
developed. As such technology became more advanced, vaccines
that targeted toxic factors produced by the pathogens were increas-
ingly developed. This approach facilitated the development of vac-
cines against the bacteria that cause diphtheria and tetanus. As the
appreciation for using components of pathogens in vaccines in
place of the whole pathogen grew, subunit vaccines containing
polysaccharides (e.g., Pneumovax 23®), proteins (e.g., Flublok®),
and glycoconjugates (e.g., Prevnar) became increasingly developed.
However, one distinct disadvantage associated with subunit vac-
cines is that they invoke a considerably weaker immune response
compared to killed or attenuated vaccines [7]. This observation led
to the development of vaccine components known as adjuvants,
which boost the immunogenicity of subunit vaccines. Although
aluminum salt (alum-based) adjuvants are the most commonly
used adjuvants, alternatives containing attenuated endotoxin (i.e.,
monophosphoryl lipid A) [8] and an emulsion-based system [9]
have been developed.
As increasingly diverse systems were developed for the assembly
of vaccines, increasing the scientific understanding of the immune
system became important for optimizing vaccine efficacy. For
example, understanding the mechanism by which various portions
of bacteria or viruses (i.e., antigens) illicit an immune response is an
important factor in vaccine design. Protein antigens are generally
recognized as thymus cell (T cell)-dependent antigens, whereas
polysaccharides are T cell-independent antigens [10]. Conse-
quently, solutions of bacterial polysaccharides are poorly immuno-
genic in infants and do not create a long-lasting immunological
memory [10]. This led to the development of glycoconjugate
vaccines, which conjugate bacterial polysaccharides to carrier pro-
teins to promote a T cell-dependent response against the polysac-
charides and provide potent immunity in infants [10]. Such
vaccines have been utilized for vaccination of infants against Strep-
tococcus pneumoniae to provide serotype-specific immunization that
has reduced the incidence of invasive pneumococcal disease by over
90% [11, 12]. Taken together, these examples have motivated a
higher understanding of how the immune response vaccines illicit
translates into clinical efficacy.
4 Andrew Hill et al.

2 Overview of Immune Response

Following the injection of the vaccine into a patient, the antigens

contained within the vaccines are processed to develop an immune
response. Upon initial introduction of the antigens, the body must
recognize the antigens introduced by the vaccine and mount an
immune response toward them. Although the body’s innate
immune response can mount an initial response to the antigens,
the goal of a vaccine is to develop a longer-lasting response from the
body’s adaptive immune system.
The initial stages of this process occur when the cells of the
body’s immune system recognize epitopes or moieties associated
with the vaccine antigen. Antigen-presenting cells (APCs) such as
dendritic cells will recognize and phagocytose the vaccine particles
and process them internally. As a result of this process, fragments of
the digested antigen are displayed on the APC’s surface in associa-
tion with the major histocompatibility (MHC) II protein (Fig. 1).
The APC can then travel to the body’s lymph nodes to help develop
a stronger and longer-lasting humoral immune response against the
vaccine antigen. Alternative B cells can recognize antigens that have
not been processed by an APC and mount an immune response.
These antigens, also known as T cell-independent antigens, include
molecules such as bacterial capsular polysaccharides. When this type
of immune response is generated, it is typically immunoglobulin
(Ig)M-biased and demonstrates a weaker and shorter-lasting
immune response compared to that obtained through the involve-
ment of T cells.
Once the APC has reached the lymph nodes, it is capable of
participating in T cell-dependent maturation of B cells. Both of
these cells travel to lymph nodes to play a role in the adaptive
immune response. During the initial stage of the process, receptors
on the surface of CD4+ Helper T cells interact with the MHCII
receptor of the APC and the processed vaccine antigen that is
associated with it. This interaction activates the CD4+ T cell and
can enable it to stimulate reproduction and activation of B cells.
When the activated T cell interacts with a B cell displaying the
appropriate antigen, it releases cytokines that promote proliferation
of the B cell population and their subsequent maturation into
antibody (typically IgG)-secreting plasma cells. In addition, this
interaction also promotes a process known as somatic hypermuta-
tion in which a great degree of mutation of the variable region of
the B cell antibody is achieved. As a consequence of this process,
antibody variants processing greater degrees of affinity for the
antigen can be generated to develop a highly potent and specific
immune response. Once these immune cells have been generated,
they can either disseminate to become effector cells or remain in
lymph nodes as memory cells to enable the body to mount a rapid
response if a similar infection occurs.
 Immunology Overview for Vaccine Development 5

Fig. 1 Overview of humoral and cellular immunity. In the humoral immune response (shown on left), an APC
such as a dendritic cell phagocytoses a pathogen or vaccine antigen and processes it internally. After
processing, an antigen epitope is displayed on the cell’s surface attached to the MHC II receptor. When the
APC is a B cell, it can become a plasma cell and secrete IgM antibodies. When a CD4+ T cell interacts with the
APC, it becomes activated and promotes proliferation and somatic hypermutation within B cells. These B cells
can then either be stored as memory cells or disseminate into the blood as plasma cells secreting IgG
antibodies. In the cellular immune response (shown on the right), a virus-infected or tumor cell containing a
nucleus processes an intracellular protein and presents an epitope on its surface attached to the MHC I
receptor. A CD8+ T cell can then bind to the MHC I receptor and become activated. Upon activation, the CD8+
T cell secretes various cytokines as well as cytotoxic chemicals that form pores within the bound cell and
promote apoptosis

To describe an example of this process, we will highlight recent

work to formulate a vaccine against the bacterial pathogen Strepto-
coccus pneumoniae. This vaccine was comprised of 20–24 pneumo-
coccal capsular polysaccharides encapsulated within a liposome
[13, 14]. The liposome was also surface-decorated with pneumo-
coccal virulence factors using either a His-tag or streptavidin-biotin
system [13]. Through this approach, the vaccine was able to gener-
ate a CD4+ T cell-dependent immune response resulting in signifi-
cant IgG production against both the capsular polysaccharide and
protein antigens [13, 14]. Moreover, this work also demonstrated
that encapsulation of the polysaccharides, normally a T cell-
independent antigen, within a protein decorated liposome was
able to generate a T cell-dependent response. This was driven by
the observation of IgM to IgG class switching that is a hallmark of
the T cell-dependent response. Interestingly, this provided evidence
that physical colocalization of the polysaccharides with a T cell-
6 Andrew Hill et al.

dependent antigen, the surface protein, was able to provide an

immune response similar to that observed for direct chemical con-
jugation of polysaccharides to carrier proteins (i.e., glycoconjugate
vaccines). Furthermore, the researchers demonstrated that the
resulting immune response provides protection against pneumo-
coccal disease using various animal models and an in vitro correlate
assay [13, 14]. This work highlights how a vaccine was designed to
invoke a specific immune response to provide protection against
pneumococcal infections.
As an alternative to the aforementioned immune response, the
immune system can direct a response towards infected bodily cells.
This occurs through the action of CD8+ T cells that recognize
peptides (processed antigen fragments produced intracellularly)
attached to the surface of cells via the MHC I receptor (Fig. 1).
Unlike MHC II, which is present on APCs, MHC I is expressed on
the surface of nucleated bodily cells. If a cell becomes infected with
a virus or becomes a tumor, intracellular proteins can be processed
for presentation on the cellular surface via attachment to MHC I
and then be recognized by CD8+ T cells. When the T cells recog-
nize and bind to the epitope-bound MHC I receptor, the T cell
becomes activated and can initiate a cellular immune response. In
this response, the T cell secretes cytokines, such as tumor necrosis
factor alpha (TNFα) or interferon gamma (IFN-γ), that demon-
strate antitumor and antiviral effects. The T cells also secrete cyto-
toxic chemicals that serve to create pores within the membrane of
the bound cells through which proteases can enter. Once the
proteases have entered the targeted cell, they are able to degrade
any viral proteins and promote cellular apoptosis. Once this has
been achieved, the CD8+ T cell is able to continue its response as
needed against additional infected cells.

3 Development of Assays to Evaluate Vaccine Efficacy

In order to determine whether a vaccine has the potential to

provide protective immunity within a patient, various assays have
been developed to evaluate the immune response and determine
whether it is sufficiently protective. One significant challenge facing
various vaccines is the fact that the incidence rate of disease is
frequently very low. As shown in Table 1, the incidence and fatality
rates of bacterial pathogens frequently occurs on the rate of a few
cases per hundred thousand people. Consequently, performance of
clinical trials to demonstrate efficacy of vaccines against these
organisms represents a substantial challenge since a massive number
of patients would be necessary. For example, if a clinical trial is
seeking to demonstrate the efficacy of the vaccine through the
reduction of disease incidence or associated death, a sufficient
number of patients would be required to capture differences of
 Immunology Overview for Vaccine Development 7

groups when cases of disease occurs around 1 in 10,000 people.

Furthermore, multiple trial arms would be required to cover a
placebo control as well as other potential groups that would be
necessary depending on the vaccine being evaluated. This is further
complicated for vaccines in which a current standard of care exists
and cannot be ethically denied to patients. Consequently, the tested
vaccine needs a mechanism for demonstrating both superiority to
the current vaccine in certain tests while demonstrating
non-inferiority in areas of overlap. This situation is made more
complex by bacteria for which there are multiple strains or sero-
types (e.g., Streptococcus pneumoniae) in which vaccines provide
protection against only a subset of bacterial diversity. If reduction
of disease were the only mechanism for evaluating efficacy, then not
only would new vaccines need to include sufficient patients to
statistically evaluate reduction of disease, this number would need
follow-up assays to demonstrate the vaccine’s efficacy in a serotype-
dependent manner.
Thus, numerous assays have been developed to link in vitro
assay results to clinical efficacy of vaccines. The quantitative power
of these assays, called “correlates of protection” (COPs), have been
demonstrated in a clinical setting to predict clinical utility of a
vaccine through in vitro testing. As a result, a vaccine’s efficacy
can be potentially determined using a number of patients that is
far lower than required using reduction in disease incidence.
One such COP for protein antigens against bacterial pathogens
is the development of assays that measure the production of anti-
bodies against the bacteria. These may include enzyme-linked
immunosorbent assays (ELISAs) or neutralization assays such as
opsonophagocytic activity (OPA) assays in which antibody titers
and their ability to promote opsonization of bacteria is determined.
For example, through clinical trials, such assays have been devel-
oped for pneumococcal disease that include ELISAs which can
show IgG concentrations >0.35 μg/mL and OPA assays showing
50% bacteriocidal activity when dilution is less than 1 in
8 [15]. Since each of these values have been associated with the
efficacy of pneumococcal glycoconjugate vaccines, subsequent vac-
cines against the bacteria are able to demonstrate efficacy by achiev-
ing each of these titers for both previous and new serotypes
included in vaccines. Similar assays have also been developed
against various other bacterial pathogens to measure vaccine effi-
cacy without requiring additional massive clinical trials.
8 Andrew Hill et al.

References
1. Centers for Disease Control and Prevention Dramé M, Roman F, Gillard P (2011) Immu-
(CDC) (1999) Impact of vaccines universally nogenicity profile of a 3.75-μg hemagglutinin
recommended for children--United States, pandemic rH5N1 split virion AS03(A)-
1990-1998. MMWR Morb Mortal Wkly Rep adjuvanted vaccine in elderly persons: a rando-
48(12):243 mized trial. J Infect Dis 203(8):1054–1062.
2. Whitney CG, Zhou F, Singleton J, Schuchat A https://doi.org/10.1093/infdis/jiq174
(2014) Benefits from immunization during the 10. Weintraub A (2003) Immunology of bacterial
vaccines for children program era-United polysaccharide antigens. Carbohydr Res 338
States, 1994-2013. MMWR Morb Mortal (23):2539–2547. https://doi.org/10.1016/j.
Wkly Rep 63(16):352–355 carres.2003.07.008
3. Szucs T (2000) Cost-benefits of vaccination 11. CDC (2000) Active bacterial core surveillance
programmes. Vaccine 18(Suppl 1):S49–S51. report, emerging infections program network,
https://doi.org/10.1016/S0264-410X(99) Streptococcus pneumoniae, 1999. Centers for
00464-8 Disease Control and Prevention, Paris
4. Scorpio A, Blank TE, Day WA, Chabot DJ 12. CDC (2015) Active bacterial core surveillance
(2006) Anthrax vaccines: pasteur to the pres- report, emerging infections program network,
ent. Cell Mol 63(19-20):2237–2248. https:// Streptococcus pneumoniae, 2015. Centers for
doi.org/10.1007/s00018-006-6312-3 Disease Control and Prevention, Paris
5. Smith KA (2012) Louis Pasteur, the father of 13. Hill AB, Beitelshees M, Nayerhoda R, Pfeifer
immunology? Front Immunol 3:68. https:// BA, Jones CH (2018) Engineering a next-
doi.org/10.3389/fimmu.2012.00068 generation glycoconjugate-like streptococcus
6. Turner GS, Squires EJ, Murray HGS (1970) pneumoniae vaccine. ACS Infect Dis 4:1553.
Inactivated smallpox vaccine. A comparison of https://doi.org/10.1021/acsinfecdis.
inactivation methods. J Hyg 68(2):197–210. 8b00100
https://doi.org/10.1017/ 14. Jones CH, Zhang G, Nayerhoda R,
s0022172400028679 Beitelshees M, Hill A, Rostami P, Li Y, David-
7. Moyle PM, Toth I (2013) Modern subunit son BA, Knight P, Pfeifer BA (2017) Compre-
vaccines: development, components, and hensive vaccine design for commensal disease
research opportunities. ChemMedChem 8 progression. Sci Adv 3(10):e1701797. https://
(3):360–376. https://doi.org/10.1002/ doi.org/10.1126/sciadv.1701797
cmdc.201200487 15. Andrews NJ, Waight PA, Burbidge P, Pearce E,
8. Didierlaurent AM, Morel S, Lockman L, Gian- Roalfe L, Zancolli M, Slack M, Ladhani SN,
nini SL, Bisteau M, Carlsen H, Kielland A, Miller E, Goldblatt D (2014) Serotype-specific
Vosters O, Vanderheyde N, Schiavetti F, effectiveness and correlates of protection for
Larocque D, Van Mechelen M, Garcon N the 13-valent pneumococcal conjugate vaccine:
(2009) AS04, an aluminum salt- and TLR4 a postlicensure indirect cohort study. Lancet
agonist-based adjuvant system, induces a tran- Infect Dis 14(9):839–846. https://doi.org/
sient localized innate immune response leading 10.1016/S1473-3099(14)70822-9
to enhanced adaptive immunity. J Immunol 16. Roush SW, Murphy TV (2007) Historical com-
183(10):6186–6197. https://doi.org/10. parisons of morbidity and mortality for
4049/jimmunol.0901474 vaccine-preventable diseases in the United
9. Heijmans S, De Meulemeester M, Reynders P, States. JAMA 298(18):2155–2163. https://
Giet D, Demanet E, Devresse P-Y, Icardi G, doi.org/10.1001/jama.298.18.2155
 Chapter 2

Isolating Pathogen-Specific Human Monoclonal Antibodies

(hmAbs) Using Bacterial Whole Cells as Molecular Probes
Sara Siris, Camilla A. Gladstone, Yanping Guo, Christopher L. Pinder,
Robin J. Shattock, Paul F. McKay, Paul R. Langford, and Fadil A. Bidmos

Abstract
The immunoglobulin capture assay (ICA) enables the enrichment for pathogen-specific plasmablasts from
individuals with a confirmed adaptive immune response to vaccination or disseminated infection. Only
single recombinant antigens have been used previously as probes in this ICA and it was unclear whether the
method was applicable to complex probes such as whole bacterial cells. Here, we describe the enrichment of
plasmablasts specific for polysaccharide and protein antigens of both Streptococcus pneumoniae and Neisseria
meningitidis using whole formalin-fixed bacterial cells as probes. The modified ICA protocol described here
allowed for a pathogen-specific hmAb cloning efficiency of >80%.

Key words Immunoglobulin capture assay, Pathogen-specific plasmablasts, Bacteria, Whole cells,
Vaccine antigen discovery

1 Introduction

Methods for analysis of the human adaptive immune response to

vaccination or disseminated infection include the cloning and
in vitro expression of human monoclonal antibodies (hmAbs)
from antibody-producing cells (APCs: memory B-cells, plasma-
blasts and plasma cells). Targeting plasmablasts, especially, for
hmAb cloning is useful as rapid expansion of a plasmablast popula-
tion occurs immediately following antigen encounter—this expan-
sion is characterized by differentiation leading to increased
specificity for the presenting antigen [1]. Expression cloning of
hmAbs from plasmablasts has been achieved by sorting of individ-
ual cells into multiwell plates followed by a sequential PCR that
generates amplicons of the variable regions of heavy (VH) and light

Sara Siris and Camilla Gladstone contributed equally to this work.

Paul Langford and Fadil Bidmos contributed equally to this work.

Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021

9
10 Sara Siris et al.

(VL ¼ κ or λ) chains of individual hmAbs. These VH and VL

amplicons are subsequently incorporated into expression plasmids
using molecular cloning strategies (restriction endonuclease or
ligation-independent) [2]. In vitro production of individual recom-
binant hmAbs occurs in human embryonic kidney cells (HEK-293)
transfected with cognate VH and VL plasmid pairs; hmAbs secreted
into culture supernatants are subsequently screened for pathogen/
antigen specificity and functional activity.
Low hmAb cloning efficiencies are obtained in studies where
rare antigens are sought, blood sample volume is low or when the
magnitude/timing of the plasmablast component of the immune
response to the pathogen/antigen of interest has not been suffi-
ciently quantified. To counter low hmAb cloning efficiencies,
enrichment for plasmablasts of interest is performed prior to
fluorescence-activated cell sorting (FACS). In vivo enrichment of
human plasmablasts has been achieved by transplantation of irra-
diated SCID/beige mice with human peripheral blood mononu-
clear cells (PBMCs) premixed with antigens of interest [3]. In vitro
enrichment has also been achieved by separation of individual cells
into droplets, analyses of the secretome of each cell within the
droplet, and immediate sorting of cells producing the desired anti-
bodies [4]. However, the use of these enrichment methods has not
been widely reported in the literature for a variety of reasons
including the complexity of the techniques and unavailability of
specialist equipment in standard research laboratories.
Recently, a simplified enrichment protocol, known as the
immunoglobulin capture assay (ICA), was described for the
in vitro identification of plasmablasts of interest. In the ICA, a
streptavidin anti-CD45 and biotin anti-human IgG scaffold is
assembled on the surface of plasmablasts to prevent diffusion of
secreted IgG away from the secreting plasmablast. Interactions
between these “captured” IgG molecules and antigens of interest
are subsequently analyzed during FACS. Positive interaction
events, that is, plasmablasts whose IgG have bound to the antigen
of interest, are sorted, while “nonbinders” (non-IgG and nonspe-
cific IgG) are excluded [5]. Production of hmAbs from individual
plasmablasts is subsequently performed as described above. Clon-
ing into expression vectors and expression of hmAbs have been
comprehensively described in published protocols [2, 6].
Only single recombinant vaccine antigens have been utilized in
this assay previously. Here, we demonstrate that a complex probe
such as whole bacterial cells can be efficiently utilized for the
enrichment of pathogen-specific plasmablasts (Fig. 1). Using
formalin-fixed cells representing four capsular variants (6A, 7F,
14, and 19F) of the Gram-positive pneumococcus, we were able
to achieve a hmAb cloning efficiency of ~82%. Our panel of cloned
hmAbs targeted either the 6A, 7F, or 14 capsules—no cross-
reactivity between structurally dissimilar capsules was discerned.
The modified ICA protocol was also readily applicable to
 Isolating Bacterial Pathogen-Specific Monoclonal Antibodies 11

Fig. 1 Schematic illustration of the experimental procedure. Quasi-presentation of secreted IgG is achieved by
exploitation of the surface expression of CD45 on plasmablasts. An anti-CD45 streptavidin conjugate antibody
is then used as a link between the CD45-expressing plasmablast and the anti-human IgG (conjugated to biotin)
that will capture IgG secreted into the immediate milieu of the plasmablast. This captured IgG can then bind to
a cognate antigen, if present, on the bacterial surface

Gram-negative bacteria yielding similar results following cloning of

capsular and protein vaccine-induced antimeningococcal hmAbs
from ICA-enriched plasmablasts.

2 Materials

Refer to storage instructions for all reagents. Ensure safety guide-

lines and proper waste disposal procedures are followed.

2.1 Bacterial Growth, 1. Frozen stocks of pneumococcal and meningococcal strains.

Fixing, and Staining 2. Brain-Heart Infusion (BHI) broth supplemented with 0.5%
yeast extract (BHI-YE): Suspend 14.8 g of BHI powder and
2 g of yeast extract in 383.2 mL of deionized water. Sterilize at
12 Sara Siris et al.

121  C for 15 min. Allow to cool to room temperature

(RT) before use. Prepare fresh each time.
3. Blood agar plates.
4. Cell culture flask with vented caps.
5. 10 μL sterile disposable inoculation loops.
6. Research CO2 incubator.
7. Benchtop centrifuge.
8. Dulbecco’s phosphate buffered saline (DPBS).
9. Bacterial fixing solution: 0.5% formaldehyde solution in DPBS.
10. 0.1 M sodium hydrogen carbonate (0.1 M NaHCO3): Dis-
solve 3.36 g of NaHCO3 crystals in 396.64 mL of deionized
water. Sterilize at 121  C for 15 min. Allow to cool to room
temperature (RT) before use.
11. Spectrophotometer.
12. Cell staining solution: Dissolve 100 mg of 5(6)-
carboxyfluorescein N-hydroxysuccinimide ester (FAM SE) in
10 mL of dimethyl sulfoxide (DMSO) to give a final concen-
tration of 10 mg/mL. Protect from light by wrapping in
aluminum foil.

2.2 ICA 1. Frozen stocks of PBMCs.

2. 5 mL round bottom polystyrene tubes.
3. Water bath.
4. Cell counter, slides, and trypan blue dye.
5. Single-color compensation beads.
6. RPMI-1640 medium.
7. R10 medium: RPMI-1640, 10% fetal bovine serum.
8. ICA buffer (DPBS, 1% bovine serum albumin, 50 U/mL
benzonase nuclease): Dissolve 5 g of bovine serum albumin
(BSA) flakes in 495 mL DPBS. Add 100 μL benzonase nuclease
to give 50 U/mL final concentration (see Note 1). Prepare on
day of experiment.
9. Zombie NIR™ Fixable Viability Kit: Follow manufacturer
instructions for preparation of the dye.
10. Anti-human CD45 streptavidin conjugate: Prepare using a
conjugation kit. Follow the manufacturer’s instructions (see
Note 2).
11. Biotin-SP-AffiniPure F(ab0 )2 fragment goat anti-human IgG,
Fcγ fragment specific (biotin anti-human IgG).
12. Fluorescent antibodies:
(a) APC anti-human CD3 antibody (APC).
 Isolating Bacterial Pathogen-Specific Monoclonal Antibodies 13

(b) APC anti-human CD14 antibody (APC).

(c) Brilliant Violet 421™ (BV421) anti-human CD19
antibody.
(d) Brilliant Violet 605™ (BV605) anti-human CD20
antibody.
(e) PE anti-human CD27 antibody (PE).
(f) PerCP/Cyanine5.5 anti-human CD38 antibody (PerCP/
Cy5.5).
(g) Alexa Fluor® 594 AffiniPure goat anti-human serum IgA,
α chain specific (AF594).
(h) Brilliant Violet 650™ anti-human IgM antibody
(BV650).
(i) Brilliant Violet 785™ anti-human IgD antibody
(BV785).
13. Catch buffer: Nuclease- and protease-free water, 10 mM Tris–
HCL 8.0, 1 U RNAsin.

3 Methods

3.1 Pneumococcal 1. Streak a loopful of pneumococcal frozen stocks on blood agar.

Growth, Fixing, Incubate for 14–16 h at 37  C, 5% CO2.
and Staining 2. From the blood agar plates, suspend 10–20 colonies of pneu-
mococcal growth in 10 mL of BHI-YE culture medium in a
50 mL centrifuge tube.
3. Transfer the pneumococcal suspension to a cell culture flask
with vented cap (see Note 3).
4. Incubate the flask overnight for 14–16 h at 37  C, 5% CO2
without shaking (see Note 4).
5. Pellet pneumococci from the overnight culture at 3000  g for
5 min, RT.
6. Resuspend pneumococcal cells in 10 mL bacterial fixing solu-
tion. Incubate for 4 h at RT.
7. Wash pneumococcal cells thrice in 10 mL 0.1 M NaHCO3.
Resuspend cells to OD600 of 0.2 (108 cells/mL).
8. Resuspend each aliquot of 108 cells in 495 μL of 0.1 M
NaHCO3. Add 5 μL of cell staining solution. Incubate for
1 h at 37  C.
9. Wash stained pneumococcal cells five times in 500 μL of 0.1 M
NaHCO3.
10. Resuspend stained pneumococcal cells in ICA buffer. Stained
cells can be stored overnight at 4  C, protected from light.
14 Sara Siris et al.

3.2 Meningococcal 1. Streak a loopful of meningococcal frozen stocks on blood agar.

Growth, Fixing, Incubate for 14–16 h at 37  C, 5% CO2.
and Staining 2. Harvest cells from the overnight growth into 10 mL bacterial
fixing solution using a sterile 10 μL disposable loop.
3. Incubate for 4 h at RT.
4. Wash meningococcal cells thrice in 10 mL 0.1 M NaHCO3.
Resuspend cells to OD600 of 0.1 (~107 cells/mL).
5. Resuspend each aliquot of 107 cells in 495 μL of 0.1 M
NaHCO3. Add 5 μL of cell staining solution. Incubate for
1 h at 37  C.
6. Wash stained meningococcal cells at 800  g for 10 min in
500 μL of 0.1 M NaHCO3.
7. Repeat step 6 above four more times.
8. Resuspend stained meningococcal cells in ICA buffer. Stained
cells can be stored overnight at 4  C, protected from light.

3.3 ICA 1. Warm the RPMI-1640 and R10 to RT. Transfer 9 mL of

RPMI-1640 per five million live PBMCs into a 50 mL
3.3.1 PBMC Thawing
centrifuge tube.
2. Transfer PBMC vials from 80  C to a 37  C water bath.
Check the vials periodically and remove following complete
thawing.
3. Immediately after complete thawing, add the PBMC sample
dropwise into the RPMI-1640 medium.
4. Pellet the PBMCs at 500  g, RT for 8 min. Discard the
supernatant.
5. Resuspend the PBMCs in 10 mL of ICA buffer.
6. Add 10 μL of the sample to 10 μL of trypan blue dye. Count
the number of cells in this mixture using an automated cell
counter.
7. Pellet the PBMCs (from step 5 above) at 500  g, RT for
8 min. Discard the supernatant.
8. Resuspend the PBMCs at a concentration of 3–4 million live
PBMCs per 100 μL in ICA buffer. Store on ice.

3.3.2 IgG Secretion 1. Label 2 mL microcentrifuge tubes as follows:

and Capture (a) “C” for unstained PBMCs.
(b) “CS” for stained PBMCs (no ICA).
(c) “CF-APC” for fluorescence minus APC control.
(d) “CF-BV421” for fluorescence minus BV421 control.
(e) “CF-BV605” for fluorescence minus BV605 control.
(f) “CF-PE” for fluorescence minus PE control.
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