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 Learning objectives
To understand:
• Etiology
• Pathogenesis
• Laboratory diagnosis
• Treatment, and
• Prevention of HIV infection

30-Mar-18 Biochemistry for medics 2

 What is AIDS ?
o Acquired (acquired during lifetime, not
inherited)
o Immuno- (related with immune
system)
o Deficiency (related with deficient immune
response)
o Syndrome (collection of symptoms)

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 Syndrome ?
 A collection of symptoms
due to underlying infections and
malignancies
resulting from specific damage to immune
system
caused by human immunodeficiency virus
(HIV).

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 AIDS

Immune Infections and Collection of

incompetence malignancies symptoms

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 Etiological agent of AIDS

Latency

Lentivirus Persistent viremia

Retrovirus
(retroviridae family)
Infection of nervous
system

Weak host immune

responses

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 Human Immunodeficiency Virus
• HIV has high affinity for CD4 T
lymphocytes and monocytes.
• HIV binds to CD4 cells
and becomes internalized.
The virus replicates itself
by generating a DNA copy
by reverse transcriptase.
• Viral DNA becomes
incorporated into the host DNA,
enabling further replication.

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 Structural Characteristics of HIV
(Nucleocapsid core)
• Interior to the
envelope is an
outer icosahedral
nuclear capsid
shell(p17) and
• an inner cone-shaped
core containing
ribonucleoproteins
(p24). (The proteins and glycoproteins are
indicated by their mass expressed
as kilo Daltons)
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 Components of nucleocapsid core
• The enzymes
integrase p32,
• protease p10,
• reverse
transcriptase
p55/66
and
• 2 copies of single
stranded genomic RNA
care
30-Ma o present inside the
r- 18 Biochemistry for medics 15
 Functions of structural
components of HIV
Structural component Function
gp 120 Constitute the major surface component
of the virus which binds to the cell CD4
receptors on susceptible host cells.

gp41 causes cell to cell fusion

p17 Protects nucleocapsid core
p24 First diagnostic structural component
p10 (protease) Cleaves viral precursor proteins
P32(integrase) Integrates viral DNA to host genome
P55/66(reverse transcriptase) Catalyzes conversion of viral RNA to
DNA
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 HIV Types

HIV1 HIV2
Original First isolated
isolates of from West
HIV Africa

Present all
40 % genetic
over the
similarity
world

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 Antigenic variations in HIV
Different
persons

Same
Different Antigenic person
races sequential
isolates
variations

Same
person-
different
sites

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 Modes of transmission
Sexual contact

Blood Sharing of
transfusion needles

Modes of
Transmission

Needle prick
Mother to child
injury

Organ
transplantation

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 Modes of transmission

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 Age for HIV Infection
Adults
(25-49)
70%

Age for
HIV
Infectio
n
Young children Adolescent
(<13 years) (13-24)
Less than 25%
5%%

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 Pathogenesis
• Infection is transmitted when virus enters
the blood or tissues of a person and comes
in to contact with a suitable host cell,
principally the CD4 lymphocytes.
• The virus may infect any cell bearing the
CD4 antigen on the surface.
• Primarily these are the CD4 + helper T
lymphocytes.

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 CD4 presenting cells
T Helper
cells

Macrophages CD 4 cells Monocytes

B
Lymphocytes

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 CD 4 presenting cells
• Some other immune cells possessing CD4
antigens are also susceptible to infection,
like B lymphocytes, monocytes and
macrophages including specialized
macrophages such as Alveolar
macrophages in the lungs and
Langerhans cells in the dermis.
• Glial cells and microglia cells are also
susceptible.
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 Steps of viral entry in to the host
cell
1) Attachment of
virus to the host
cell –Specific
binding of the
virus to the CD4
receptors is by the
envelop
glycoprotein
gp120.
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 Steps of viral entry in to the host cell

2) Cell to cell
fusion – For infection
to take place the cell
fusion is essential.
o This is brought
about by the
transmembrane
glycoprotein gp 41.

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 Steps of viral entry in to the host
cell
• HIV-1 utilizes two major
co-receptors along with
CD4 to bind to, fuse with,
and enter target cells;
• these co-receptors are
CCR5 and CXCR4, which
are also receptors for
certain endogenous
chemokines.

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 Steps of viral entry in to the host
cell
o Strains of HIV that utilize
CCR5 as a co-receptor are
referred to as macrophage
tropic viruses (M –tropic
viruses)
o Strains of HIV that
utilize CXCR4 are referred
to as T - tropic viruses.
o Many virus strains are dual
tropic in that they utilize both
CCR5 and CXCR4.

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 Steps of viral entry in to the host cell
3) Uncoating of the viral
envelope and entry of nuclear
capsid core into the cell
o After fusion of virus with the
host cell membrane, HIV
genome is uncoated and
internalized in to cell.
o Viral RNA is released into
the core cytoplasm

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 Steps of viral entry in to the host cell
4) Viral transcription
o viral reverse
transcriptase mediates
transcription of its RNA;
o RNA-DNA hybrid is formed.
o Original RNA strand is
degraded by ribonuclease H,
followed by
o synthesis of second strand
of DNA to yield double
strand HIV DNA Biochemistry for medics
30-Mar-18 39
 Steps of viral entry in to the host cell

4) Integration into the host DNA as

provirus
o The double stranded DNA is integrated in to
the genome of the infected host cell through
the action of the viral integrase enzyme,
causing a latent infection.

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 Fate of provirus
o From time to time, lytic infection is initiated
releasing progeny virions, which infect
other cells.
o The long and variable incubation period of
HIV is because of the latency.
o In an infected individual the virus can be
isolated from the blood, lymphocytes, cell
free plasma, semen, cervical secretions,
saliva, urine and breast milk
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 Steps of viral exit from host cell
• Transcription back into RNA
o The viral DNA is transcribed into RNA and
multiple copies of viral RNA are
produced.
o There are only nine genes in HIV RNA, and
these code for the production of structural
proteins, accessory proteins, and enzymes
essential for the virus's replicative cycle.

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 Steps of viral exit from host cell
• Virion assembly - With the help of viral
protease, the new virions are assembled
into the polypeptide sequences needed for
HIV virion formation and infectivity.
• Cell lysis. The infected cell is made to burst
open, presumably by the action of cellular
proteins.

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 Clinical Manifestations
• The center for disease control (USA) has
classified the clinical course of HIV infection
under various groups.
• Acute HIV infection
• Asymptomatic or Latent infection
• Persistent generalized lymphadenopathy
(PGL)
• AIDS related complex
• Full blown AIDS (Last stage)

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 1. Acute HIV infection
• Symptoms- A flu-like illness with fever,
sore throat, headache, tiredness, skin
rashes and enlarged lymph nodes in the
neck within several days to weeks after
exposure to virus.
• These symptoms usually disappear of their
own within a few weeks.
• Some patients do not develop symptoms
after they first get infected with HIV.

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 1. Acute HIV infection
• This phase is also
called window period or
phase of Seroconversion.
• The test for HIV antibodies
appears negative while HIV
antigenemia (p24
antigen) and viral nucleic
acids can be demonstrated
at the beginning of the
phase.
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 2. Asymptomatic or Latent infection
• All persons infected with HIV, pass through
a phase of symptomless infection
(Clinical latency), which may last up to
several years.
• Even though the person has no symptoms,
he or she is contagious and can pass HIV to
others.
• This state may last from a few months to
more than 10 years.

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 2. Asymptomatic or Latent infection
(contd.)
• The virus continues to multiply actively and
infects and kills the cells of the immune
system
• The virus destroys the CD4 cells that are the
primary infection fighters.
• The patients show positive antibody tests
during this phase.

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 2. Asymptomatic or Latent
infection (contd.)
• The median time between primary HIV
infection and development of AIDS has been
stated as approximately 10 years.
• About 5-10 % percent of the infected appear
to escape clinical AIDS for 15 years or more.
• They have been ‘long term survivors” or
“long term non progressors”.

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 3. Persistent generalized lymphadenopathy
(PGL)
• This has been defined by presence of:
• enlarged lymph nodes,
• at least I cm in diameter,
• in two or more non contiguous extra inguinal
sites,
• that persist for at least three months,
• in the absence of any current illness or
medication that may cause lymphadenopathy.

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 3. Persistent generalized
lymphadenopathy
(PGL)
Two or more
non -contiguous Persistence for
extra- inguinal at least three
sites months

Absence of any
Enlarged lymph
current illness
nodes (>1cm)
or medication

PGL
These are diagnostic of HIV when blood tests are positive for antibodies.
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 4. AIDS related complex

Weigh
t
loss
The common
opportunistic Signs of
infections are oral other
opportuni Persistent
candidiasis, herpes fever
zoster, salmonellosis
stic
infections
AIDS
or Tuberculosis and related
hairy cell leucoplakia.
complex

Generalize
Diarrhea
d fatigue

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 Opportunistic infections in AIDS

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 4. AIDS related complex
• The patients are usually severely ill and many
of them progress to AIDS in few months.
• The CD4 cell count decreases steadily when
the count falls to 200, or less, clinical AIDS
usually sets in.
• For this reason the case definition by CDC
includes all HIV infected cases with CD4 + T
cell counts of 200 or less, irrespective of
clinical condition.
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 5. Full blown AIDS
• This is the end stage disease representing the
irreversible break down of immune defense
mechanisms.
• In addition to the opportunistic infections the
patient may develop primary CNS lymphomas
and progressive encephalopathy, dementia
and other neurological abnormalities.
• Kaposi sarcoma and Pneumocystis
pneumonia are almost always observed in a
majority of patients.
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 Kaposi’s sarcoma

• Kaposi’s sarcoma- is an indolent, multifocal

non metastasizing mucosal or cutaneous tumor
probably of endothelial origin, represented in
the form of purple spots in the skin.
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 Laboratory diagnosis of HIV
infection
Non Specific Specific
Tests Tests

Total Leukocyte
and lymphocyte HIV antigen
count-

T cell subset
Antibodies
Assays

Platelet count-
Isolation of virus

IgA and Ig G
levels Viral nucleic
acids

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 Laboratory Diagnosis of HIV infection
1) Non Specific Tests- The following tests help to
establish the immunodeficiency in HIV infection.
a) Total Leukocyte and lymphocyte count- to
demonstrate leucopenia and lymphopenia.
The lymphocytic count is usually below
2000/mm3
b) T cell subset Assays- Absolute CD4+ cell count is less
than 200 /L.T4 T8 ratio is reversed. The decrease in CD4
is the hall mark for AIDS.
c) Platelet count- shows Thrombocytopenia.
d) IgA and Ig G levels are raised
e) Diminished cell mediated Immunity as indicated
by skin tests
f) Lymph node biopsy shows profound abnormalities.
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 Laboratory Diagnosis of HIV infection

2.Specific Tests for HIV infection- These

include demonstration of -
• HIV antigen,
• Antibodies,
• Viral nucleic acids or other components
and
• Isolation of virus

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 Laboratory Diagnosis of HIV infection

i) Detection of antigen
o The major core antigen p24 is the earliest
virus marker to appear in blood.
o The p24 Capture ELISA assay, which uses
anti p24 antibody as the solid phase can
be used for this.

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 Laboratory Diagnosis of HIV infection
• i) Detection of antigen (contd.)
• Free p24 antigen disappears from circulation
and remains absent during the long
asymptomatic phase to reappear only when
severe clinical disease sets in.
• This test is positive in about 30% of the
infected persons.
• In the first few weeks after infection and in the
terminal phase, the test is uniformly positive.
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Disease progression through different
phases in HIV infected cases

CD4 count comes down while the viral count

goes high with the passage of time
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 Laboratory Diagnosis of HIV infection
• Detection of antibodies
o It takes 2-8 weeks to months for the
antibodies to appear in circulation
o IgM antibodies appear first, to be followed
by IgG antibodies
o Once antibodies appear they increase in
titer for the next several months
o IgM antibodies disappear in 8-10weeks
while IgG antibodies remain through out.
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 Laboratory Diagnosis of HIV infection

• Detection of
antibodies (contd.)
o ELISA- ELISA is the
most frequently used
method for
screening of blood
samples for HIV
antibody.

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 Laboratory Diagnosis of HIV
infection
Significance of ELISA
• Antibodies can be detected within 6-12 weeks
after infection using the earlier generation of
assays.
• Using the newer third generation ELISA,
Antibodies can be detected within 3-4 weeks

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 Laboratory Diagnosis of HIV
infection
Supplemental test
Western blotting-Confirmatory test
• Western blots are regarded as the
gold standard
• antibodies against both the env and the
gag
proteins are detected.

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 Laboratory Diagnosis of HIV
infection
3. Demonstration of viral Nucleic acid
• probes or
• PCR
• The latter may be useful because of its extremely high
sensitivity.

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 Laboratory Diagnosis of HIV
infection
• PCR -In this the target HIV
RNA or proviral DNA is
amplified enzymatically in
vitro by chemical
reaction.
• It is an extremely sensitive
assay because a single copy
of proviral DNA can be
amplified.
• Qualitative PCR is useful for
diagnostic purposes.
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 Laboratory Diagnosis of HIV
infection
4. Virus isolation
• Virus isolation is tedious and time-consuming (weeks) and
is successful in only 70 to 90% of cases.
• Therefore virus isolation is mainly used for the
characterization of the virus.

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 Laboratory Diagnosis of HIV
infection
5. Alternative to classical tests
a) Oral fluid (saliva) HIV tests
b) Urine tests

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