CHAPTER-X

HUMAN
IMMUNODEFICIENCY VIRUS
(HIV)

R.KAVITHA, M.PHARM,
LECTURER, DEPARTMENT OF
PHARMACEUTICS,
SRM COLLEGE OF PHARMACY,
SRM UNIVERSITY, KATTANKULATHUR.
INTRODUCTION
| Etiologic agent of Acquired Immunodeficiency
Syndrome (AIDS).
| Discovered independently by Luc Montagnier of
France and Robert Gallo of the US in 1983-84.
| Former names of the virus include:
y Human T cell lymphotrophic virus (HTLV-III)
y Lymphadenopathy associated virus (LAV)
y AIDS associated retrovirus (ARV)
INTRODUCTION
| HIV-2 discovered in 1986, antigenically distinct
virus endemic in West Africa.
| One million people infected in US, 30 million
worldwide are infected.
| Leading cause of death of men aged 25-44 and
4th leading cause of death of women in this age
group in the US.
| http://www.cnn.com/2005/HEALTH/conditions/11/17/blacks
.hiv.ap/
CHARACTERISTICS OF THE VIRUS
| Icosahedral (20 sided), enveloped virus of
the lentivirus subfamily of retroviruses.
| Retroviruses transcribe RNA to DNA.

| Two viral strands of RNA found in core

surrounded by protein outer coat.
y Outer envelope contains a lipid matrix within
which specific viral glycoproteins are
imbedded.
y These knob-like structures responsible for
binding to target cell.
CHARACTERISTICS OF THE VIRUS
HIV
| The outer shell of the virus
is known as the Viral
enevlope. Embedded in the
viral envelope is a complex
protein known as env which
consists of an outer
protruding cap glycoprotein
(gp) 120, and a stem gp14.
Within the viral envelope is
an HIV protein called
p17(matrix), and within this
is the viral core or capsid,
which is made of another
viral protein p24(core
antigen).
STRUCTURAL GENES
| Three main structural genes:
y Group Specific Antigen (Gag)
y Envelope (Env)
y Polymerase (Pol)
GROUP SPECIFIC ANTIGEN (GAG)
| Located in nucelocapsid of virus.
| Icosahedryl capsid surrounds the internal nucleic
acids made up of p24 andp15.
| p17 lies between protein core and envelope and is
embedded in the internal portion of the envelope.
| Two additional p55 products, p7 and p9, are
nucleic acid binding proteins closely associated
with the RNA.
ENVELOPE (ENV)
| Envelope (Env) gene codes for envelope
proteins gp160, gp120 and gp41.
y These polyproteins will eventually be cleaved
by proteases to become HIV envelope
glycoproteins gp120 and gp41.
y gp160 cleaved to form gp120 and gp41.
y gp120 forms the 72 knobs which protrude from
outer envelope.
y gp41 is a transmembrane glycoprotein antigen
that spans the inner and outer membranes
and attaches to gp120.
y gp120 and gp41 both involved with fusion and
attachment of HIV to CD4 antigen on host
cells.
POLYMERASE (POL)
| Polymerase (Pol) codes for p66 and p51
subunits of reverse transcriptase and p31 an
endonuclease.
y Located in the core, close to nucleic acids.
y Responsible for conversion of viral RNA into DNA,
integration of DNA into host cell DNA and cleavage
of protein precursors.
VIRAL REPLICATION
| Firststep, HIV attaches to susceptible
host cell.
y Site of attachment is the CD4 antigen found
on a variety of cells
| helper T cells
| macrophages

| monocytes

| B cells

| microglial brain cells

| intestinal cells

y T cells infected later on.

EARLY PHASE HIV INFECTION
| Inearly phase HIV
infection, initial
viruses are M-tropic.
Their envelope
glycoprotein gp120 is
able to bind to CD4
molecules and
chemokine receptors
called CCR5 found on
macrophages
LIFE CYCLE
| (a) HIV (red) attaches to two cell-surface receptors
(the CD4 antigen and a specific chemokine
receptor).
| (b) The virus and cell membrane fuse, and the
virion core enters the cell.
| (c) The viral RNA and core proteins are released
from the virion core and are then actively
transported to the nucleus.
| (d) The viral RNA genome is converted into
double-stranded DNA through an enzyme unique
to viruses, reverse transcriptase (red dot).
| (e) The double-stranded viral DNA moves into the
cell nucleus.
| (f) Using a unique viral enzyme called integrase,
the viral DNA is integrated into the cellular DNA.
| (g) Viral RNA is synthesized by the cellular
enzyme RNA polymerase II using integrated viral
DNA as a template. Two types of RNA transcripts
shorter spliced RNA (h) and full-length genomic
RNA (j) are produced.
| (h) Shorter spliced RNAs are transported to the
cytoplasm and used for the production of several
viral proteins that are then modified in the Golgi
apparatus of the cell (i).
| (j) Full-length genomic RNAs are transported to
the cytoplasm (k).
| (l) New virion is assembled and then buds off.
| (m) Mature virus is released.
VIRAL REPLICATION
| Methods of transmission:
y Sexual transmission, presence of STD
increases likelihood of transmission.
y Exposure to infected blood or blood products.
y Use of contaminated clotting factors by
hemophiliacs.
y Sharing contaminated needles (IV drug users).
y Transplantation of infected tissues or organs.
y Mother to fetus, perinatal transmission
variable, dependent on viral load and mother’s
CD 4 count.
PRIMARY HIV SYNDROME
| Mononucleosis-like,
cold or flu-like
symptoms may occur 6 to 12 weeks after
infection.
y lymphadenopathy
y fever
y rash
y headache
y Fatigue
y diarrhea
y sore throat
y neurologic manifestations.
y no symptoms may be present
PRIMARY HIV SYNDROME
| Symptoms are relatively nonspecific.
| HIV antibody test often negative but
becomes positive within 3 to 6 months,
this process is known as seroconversion.
| Large amount of HIV in the peripheral
blood.
| Primary HIV can be diagnosed using viral
load titer assay or other tests.
| Primary HIV syndrome resolves itself and
HIV infected person remains
asymptomatic for a prolonged period of
time, often years.
CLINICAL LATENCY PERIOD
| HIV continues to reproduce, CD4 count
gradually declines from its normal value
of 500-1200.
| Once CD4 count drops below 500, HIV
infected person at risk for opportunistic
infections.
| The following diseases are predictive of
the progression to AIDS:
y persistent herpes-zoster infection (shingles)
y oral candidiasis (thrush)
y oral hairy leukoplakia
y Kaposi’s sarcoma (KS)
AIDS
| CD4 count drops below 200 person is considered
to have advanced HIV disease
| If preventative medications not started the HIV
infected person is now at risk for:
y Pneumocystis carinii pneumonia (PCP)
y cryptococcal meningitis
y toxoplasmosis
| If CD4 count drops below 50:
y Mycobacterium avium
y Cytomegalovirus infections
y lymphoma
y dementia
y Most deaths occur with CD4 counts below 50.
OTHER OPPORTUNISTIC INFECTIONS
| Respiratory system
y Pneumocystis Carinii Pneumonia (PCP)
y Tuberculosis (TB)
y Kaposi's Sarcoma (KS)
| Gastro-intestinal system
y Cryptosporidiosis
y Candida
y Cytomegolavirus (CMV)
y Isosporiasis
y Kaposi's Sarcoma
| Central/peripheral Nervous system
y Cytomegolavirus
y Toxoplasmosis
y Cryptococcosis
y Non Hodgkin's lymphoma
y Varicella Zoster
y Herpes simplex
| Skin
y Herpes simple
y Kaposi's sarcoma
y Varicella Zoster
INFANTS WITH HIV
| Failure to thrive
| Persistent oral candidiasis

| Hepatosplenomegaly

| Lymphadenopathy

| Recurrent diarrhea

| Recurrent bacterial infections

| Abnormal neurologic findings.

IMMUNOLOGIC MANIFESTATIONS
| Early stage slight depression of CD4 count, few
symptoms, temporary.
| Window of up to 6 weeks before antibody is
detected, by 6 months 95% positive.
| During window p24 antigen present, acute
viremia and antigenemia.
IMMUNOLOGIC MANIFESTATIONS
| Antibodies produced to all major antigens.
y First antibodies detected produced against gag
proteins p24 and p55.
y Followed by antibody to p51, p120 and gp41
y As disease progresses antibody levels decrease.
IMMUNOLOGIC MANIFESTATIONS
| Immune abnormalities associated with
increased viral replication.
y Decrease in CD4 cells due to virus budding
from cells, fusion of uninfected cells with
virally infected cells and apoptosis.
y B cells have decreased response to antigens
possibly due to blockage of T cell/B cell
interaction by binding of viral proteins to CD4
site.
y CD8 cells initially increase and may remain
elevated.
y As HIV infection progresses, CD4 T cells drop
resulting in immunosuppression and
susceptibility of patient to opportunistic
infections.
y Death comes due to immuno-incompetence.
IMMUNOLOGIC MANIFESTATIONS
| Immune abnormalities associated with
increased viral replication.
y Decrease in CD4 cells due to virus budding
from cells, fusion of uninfected cells with
virally infected cells and apoptosis.
y B cells have decreased response to antigens
possibly due to blockage of T cell/B cell
interaction by binding of viral proteins to CD4
site.
y CD8 cells initially increase and may remain
elevated.
y As HIV infection progresses, CD4 T cells drop
resulting in immunosuppression and
susceptibility of patient to opportunistic
infections.
y Death comes due to immuno-incompetence.
The Move Toward Lower Pill Burdens
Regimen Dosing Daily pill burden

1996
Zerit/Epivir/Crixivan 10 pills, Q8H

1998
Retrovir/Epivir/Sustiva 5 pills, BID

2002
Combivir (AZT/3TC)/EFV 3 pills, BID

2003
Viread/ Emtriva/Sustiva 3 pills, QD
2004
Truvada/Sustiva 2 pills, QD
SUSTIVA + TRUVADA TREATMENT
| Sustiva + Truvada (FTC + tenofovor) is one of the
most popular and effective starting HIV
regimens.
| Many patients will have dream/sleep/central
nervous system effects particularly in the first
month (due to the Sustiva).
| Upset stomach/bloating/gas/loose stools is also
fairly common during the first month and for
most patients is fairly mild.
| HIV levels in the blood will often drop by > 99%
in the first month and the CD4 count (marker of
immune system function) will often increase
providing protection against AIDS related
diseases within weeks/months of starting the
medication.
TRUVADA
| Truvada is made up of HIV drugs from a class
called nucleoside/nucleotide reverse transcriptase
inhibitors (NRTIs), also known as “nukes.”
| The NRTIs block reverse transcriptase, a protein
that HIV needs to make more copies of itself.
This may slow down HIV disease
 ‘TYPICAL’ PRIMARY HIV-1 INFECTION
symptoms symptoms
HIV proviral DNA

HIV antibodies
‘window’
period

HIV viral load

HIV-1 p24 antigen

0 1 2 3 4 5 6 / 2 4 6 8 10
1° infection weeks years
Time following infection
LABORATORY DIAGNOSIS OF HIV
INFECTION
| Methods utilized to detect:
y Antibody
y Antigen
y Viral nucleic acid
y Virus in culture
ELISA TESTING
| First serological test developed to detect HIV
infection.
y Easy to perform.
y Easily adapted to batch testing.
y Highly sensitive and specific.
| Antibodies detected in ELISA include those
directed against: p24, gp120, gp160 and gp41,
detected first in infection and appear in most
individuals
ELISA TESTING
| ELISA tests useful for:
y Screening blood products.
y Diagnosing and monitoring patients.
y Determining prevalence of infection.
y Research investigations.
ELISA TESTING
| Different types of ELISA techniques used:
y indirect
y competitive
y sandwich

| ELISAs are for screening only, false positives do

occur and may be due to AI disease, alcoholism,
syphilis, and immunoproliferative diseases.
ELISA SANDWICH
OTHER SCREENING TESTS
| Agglutination tests using latex particles,
gelatin particles or microbeads are coated
with HIV antigen and will agglutinate in
the presence of antibody.
| Dot-Blot Testing utilizes paper or
nitrocellulose impregnated with antigen,
patient serum is filtered through, and
anti-antibody is added with enzyme label,
color change is positive.
y A rapid, cost-effective and may become an
alternative to standard ELISA and Western
blot testing.
PARTICLE AGGLUTINATION
WESTERN BLOT
| Most popular confirmatory test.
y Utilizes a lysate prepared from HIV virus.
y The lysate is electrophoresed to separate out
the HIV proteins (antigens).
y The paper is cut into strips and reacted with
test sera.
y After incubation and washing anti-antibody
tagged with radioisotope or enzyme is added.
y Specific bands form where antibody has
reacted with different antigens.
y Most critical reagent of test is purest quality
HIV antigen.
y The following antigens must be present: p17,
p24, p31, gp41, p51, p55, p66, gp120 and
gp160.
WESTERN BLOT
| Antibodies to p24 and p55 appear earliest but
decrease or become undetectable.
| Antibodies to gp31, gp41, gp 120, and gp160
appear later but are present throughout all
stages of the disease.
WESTERN BLOT
| Interpretation of results.
y No bands, negative.
y In order to be interpreted as positive a minimum of 3
bands directed against the following antigens must
be present: p24, p31, gp41 or gp120/160.
| CDC criteria require 2 bands of the following:
p24, gp41 or gp120/160.
 gp160
gp120

p68
p55
p53

gp41-45

Spectrum p40

p34
of anti-HIV p24

testing p18

p12

early recent / established advanced

DNA PCR
RNA PCR
p24 Ag
3rd gen ELISA
1st gen ELISA
Detuned ELISA
1wk 2wk 3wk 2mo 6mo 1yr 2yr 3yr +8yr
WESTERN BLOT
| Expensive – $ 80 - 100
| technically more difficult

| visual interpretation

| lack standardisation
y - performance
y - interpretation
y - indeterminate reactions –
resolution of ??
| ‘Gold Standard’ for
confirmation
WESTERN BLOT
| Indeterminate results are those samples that
produce bands but not enough to be positive, may
be due to the following:
y prior blood transfusions, even with non-HIV-1 infected
blood
y prior or current infection with syphilis
y prior or current infection with malaria
y autoimmune diseases (e.g., diabetes, Grave’s disease,
etc)
y infection with other human retroviruses
y second or subsequent pregnancies in women.
y run an alternate HIV confirmatory assay.
| Quality control of Western Blot is critical and
requires testing with strongly positive, weakly
positive and negative controls.
INDIRECT IMMUNOFLUORESCENCE
| Can be used to detect both virus and antibody to
it.
| Antibody detected by testing patient serum
against antigen applied to a slide, incubated,
washed and a fluorescent antibody added.
| Virus is detected by fixing patient cells to slide,
incubating with antibody.
DETECTION OF P24 HIV ANTIGEN
| The p24-antigen screening assay is an
EIA performed on serum or plasma.
| P24 antigen only present for short time,
disappears when antibody to p24 appears.
| Anti-HIV-1 bound to membrane,
incubated with patient serum, second
anti-HIV-1 antibody attached to enzyme
label is added (sandwich technique), color
change occurs.
| Optical density measured, standard curve
prepared to quantitate results.
DETECTION OF P24 HIV ANTIGEN
| Positive confirmed by neutralizing reaction,
preincubate patient sample with anti- HIV,
retest, if p24 present immune complexes form
preventing binding to HIV antibody on
membrane when added.
| Test not recommended for routine screening as
appearance and rate of rise are unpredictable.
| Sensitivity lower than ELISA.
DETECTION OF P24 HIV ANTIGEN
| Most useful for the following:
y early infection suspected in seronegative patient
y newborns
y CSF
y monitoring disease progress
POLYMERASE CHAIN REACTION
(PCR)
| Looks for HIV DNA in the WBCs of a person.
| PCR amplifies tiny quantities of the HIV DNA
present, each cycle of PCR results in doubling of
the DNA sequences present.
| The DNA is detected by using radioactive or
biotinylated probes.
| Once DNA is amplified it is placed on
nitrocellulose paper and allowed to react with a
radiolabeled probe, a single stranded DNA
fragment unique to HIV, which will hybridize
with the patient’s HIV DNA if present.
| Radioactivity is determined.
VIRUS ISOLATION
| Virus isolation can be used to definitively
diagnose HIV.
| Best sample is peripheral blood, but can
use CSF, saliva, cervical secretions,
semen, tears or material from organ
biopsy.
| Cell growth in culture is stimulated,
amplifies number of cells releasing virus.
| Cultures incubated one month, infection
confirmed by detecting reverse
transcriptase or p24 antigen in
supernatant.
VIRAL LOAD TESTS
| Viral load or viral burden is the quantity of HIV-
RNA that is in the blood.
| RNA is the genetic material of HIV that contains
information to make more virus.
VIRAL LOAD TESTS
| Viral load tests measure the amount of
HIV-RNA in one milliliter of blood.
| Take 2 measurements 2-3 weeks apart to
determine baseline.
| Repeat every 3-6 months in conjunction
with CD4 counts to monitor viral load ant
T-cell count.
| Repeat 4-6 weeks after starting or
changing antiretroviral therapy to
determine effect on viral load.
TESTING OF NEONATES
| Difficult due to presence of maternal IgG
antibodies.
| Use tests to detect IgM or IgA antibodies, IgM
lacks sensitivity, IgA more promising.
| Measurement of p24 antigen.

| PCR testing may be helpful but still not detecting

antigen soon enough: 38 days to 6 months to be
positive.