HIV Infection- A summary

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)
30-Mar-18 3Biochemistry for medics

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

Incidence
• HIV (human immunodeficiency
virus) infection has now spread
to every country in the world.
• Approximately 40 million
people are currently living with
HIV infection, and
• An estimated 25 million have
died from this disease.

History of AIDS
• The first indication of this new syndrome
came in 1981 in homosexual drug addict
males;
• two things were common-
Pneumocystis pneumonia and Kaposi’s
sarcoma.
• Both these are markers of collapsed
immune system,

AIDS Markers
Pneumocystis pneumonia and Kaposi’s
sarcoma are considered AIDS markers,
since they reflect the underlying collapsed
immune system30-Mar-18 Biochemistry for medics 7

AIDS
Immune
incompetence
Infections and
malignancies
Collection of
symptoms

Etiological agent of AIDS
Retrovirus
(retroviridae family)
Latency
Persistent viremia
Infection of nervous
system
Weak host immune
responses
Lentivirus

Nomenclature of virus
• In 1986, The International
Committee on virus
Nomenclature decided on the
generic name of the causative
virus as the :
• Human Immunodeficiency
Virus.

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.

Immune deficiency in HIV Infection
Decreased
Number of
T-Helper
cells
Decreased
activity of
monocytes and
macrophages
Decreased
activity of B
lymphocytes
Decreased cell
mediated
immune
response
Decreased
activity of NK
cells

Structural Characteristics of HIV
(Envelop)
• Shape- Spherical
• Size- 90-120 nm in
diameter
• Envelop- Host lipoprotein
and viral glycoproteins
• Viral glycoproteins consist
of:
o Surface projecting knob like
spikes (gp 120)
o Anchoring transmembrane
pedicles(gp 41)

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)

Components of nucleocapsid core
• The enzymes
integrase p32,
• protease p10,
• reverse
transcriptase p55/66
and
• 2 copies of single
stranded genomic RNA
are present inside the
core.30-Mar-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

HIV Genome

HIV Genome
• gag
• pol
• envStructural
genes
• LTR
• Vif
• Vpr
• Vpu/vpx
• tat
• nef
• rev
Regulatory
genes

HIV Genome

HIV Genome
There are two types of genes analyzed-
a) Structural genes encode for products which
participate in formation of functional structure of
virus
1) gag gene
• encodes for core and shell of virus.
• The gene product is a precursor protein p55, which
is cleaved into p17, p24 and p15.
• The p24 antigen (major core antigen) can be
detected in serum during the early stages of
infection till the appearance of the antibodies.

Genome of HIV (Structural genes)
2) pol gene
• Encodes for the polymerase reverse
transcriptase and other viral enzymes such as
protease and integrase.
• It is expressed as a precursor protein, which is
cleaved in to components like p64 which has
reverse transcriptase and RNAse
activity: p51 which has only reverse
transcriptase activity: p10 is a protease that
cleaves gag precursor and p32 is an integrase.

b) Non structural and Regulatory
genes
1) vif - (Viral infectivity factor gene) influences
infectivity of viral particles.
2) vpr-stimulates promoter region of the virus
3) vpu (in HIV-1 ) and vpx (in HIV-2) enhance
maturation and release of progeny virus from
cells.
Detection of the type specific
sequences vpu and vpx is useful in distinguishing
between infection by HIV type 1 and 2.

HIV Types
HIV1
Original
isolates of
HIV
Present all
over the
world
HIV2
First isolated
from West
Africa
40 % genetic
similarity

Antigenic variations in HIV
Antigenic
variations
Different
persons
Same
person
sequential
isolates
Same
person-
different
sites
Different
races

Modes of transmission
Modes of
Transmission
Sexual contact
Sharing of
needles
Needle prick
injury
Organ
transplantation
Mother to child
Blood
transfusion

Modes of transmission

Age for HIV Infection
Age for
HIV
Infection
Adults
(25-49)
70%
Adolescent
(13-24)
25%
Young children
(<13 years)
Less than
5%%

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.

CD4 presenting cells
CD 4 cells
T Helper
cells
Monocytes
B
Lymphocytes
Macrophages

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.

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.

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.

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.

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.

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

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 DNA30-Mar-18 39Biochemistry for medics

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.

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

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.

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.

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)

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.

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.

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.

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.

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”.

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.

3. Persistent generalized
lymphadenopathy (PGL)
These are diagnostic of HIV when blood tests are positive for antibodies.
PGL
Enlarged lymph
nodes (>1cm)
Two or more
non -contiguous
extra- inguinal
sites
Persistence for
at least three
months
Absence of any
current illness
or medication

4. AIDS related complex
AIDS
related
complex
Weight
loss
Persistent
fever
Diarrhea
Generalize
d fatigue
Signs of
other
opportuni
stic
infections
The common
opportunistic
infections are oral
candidiasis, herpes
zoster, salmonellosis
or Tuberculosis and
hairy cell leucoplakia.

Opportunistic infections in AIDS

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.

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.

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.

Laboratory diagnosis of HIV
infection
Non Specific
Tests
Total Leukocyte
and lymphocyte
count-
T cell subset
Assays
IgA and Ig G
levels
Platelet count-
Specific
Tests
HIV antigen
Antibodies
Isolation of virus
Viral nucleic
acids

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.

2.Specific Tests for HIV infection- These
include demonstration of -
• HIV antigen,
• Antibodies,
• Viral nucleic acids or other components and
• Isolation of virus

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.

• 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.

Disease progression through different
phases in HIV infected cases
CD4 count comes down while the viral count
goes high with the passage of time

• 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.

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

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

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.

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

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.

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.

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

Treatment of HIV Infection
• All treatment for HIV infection and AIDS
today focuses on arresting the progression
of the disease within the body as measured
by T cell counts and tests for viral load.
• There are two principal approaches to
treatment: immunotherapy and anti-HIV
drug treatments

Immunotherapy
Immunotherapy
Immunoglobulins
Interleukins
Altered immune
cells

b) Anti-HIV drug treatment
• Treatment with anti-HIV drugs attempts to
reduce viral load by blocking new infection
in the host cell.
• The drugs used target two major enzymes of
HIV which are needed for the infection
cycle: reverse transcriptase and protease.

Anti HIV treatment
Reverse transcriptase
inhibitors
Protease inhibitors
Combination therapy
(HAART)

Prevention
Lifestyle
modifications

Development of vaccine
Development of vaccine is fraught with several problems
unique to this virus. These include-
1) HIV can mutate rapidly, thus, it is not possible to design
antibodies against all antigens.
2) Antibody alone is not sufficient, cell mediated immunity
may also be necessary.
3) Virus enters the body not as free virions but also as
infected cells, in which the virus or the provirus is
protected against antibody or cell mediated lysis.
4) Virus readily establishes life long latent infection hiding
from antibodies.

Failure to develop Vaccine
HIV can
mutate rapidly
Antibody
alone is not
sufficient
Virus readily
establishes life
long latent
infection
Virus enters
the body not
as free virions
but also as
infected cells

Summary
• Acquired immunodeficiency syndrome, is a
disease caused by Human Immunodeficiency Virus
and is characterized by marked
Immunosuppression.
• The mode of transmission is by sexual contact,
blood transfusions, needle prick injury or from
mother to child.
• 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”.

Summary
• ELISA is the method most commonly
employed for the screening purpose and
western Blotting is used for the
confirmation of the diagnosis.
• Treatment is imparted simply to check the
progression of the disease, there is no cure
of AIDS and there is no vaccine available for
prophylaxis of this disease.

HIV Infection- A summary

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HIV Infection- A summary