Human Immunodeficiency Virus

(HIV)

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:
Human T cell lymphotrophic virus (HTLV-III)
Lymphadenopathy associated virus (LAV)
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.
Outer envelope contains a lipid matrix within which
specific viral glycoproteins are imbedded.
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:
Group Specific Antigen (Gag)
Envelope (Env)
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.
These polyproteins will eventually be cleaved by
proteases to become HIV envelope glycoproteins
gp120 and gp41.
gp160 cleaved to form gp120 and gp41.
gp120 forms the 72 knobs which protrude from outer
envelope.
gp41 is a transmembrane glycoprotein antigen that
spans the inner and outer membranes and attaches
to gp120.
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.
Located in the core, close to nucleic acids.
Responsible for conversion of viral RNA into
DNA, integration of DNA into host cell DNA
and cleavage of protein precursors.

Viral Replication
First step, HIV attaches to susceptible host cell.
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

T cells infected later on.

Early Phase HIV Infection

In early 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

http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/hivad.html

In late phase HIV

infection, most of the
viruses are T-tropic,
having gp120 capable
of binding to CD4 and
CXCR4 found on T4lymphocytes.

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 doublestranded 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
The gp120 protein on virus binds
specifically to CD4 receptor on host cell
with high affinity.
Gp41 causes fusion of the virus to the cell
membrane.
After fusion virus particle enters cell.
Viral genome exposed by uncoating particle.

Viral Replication
Reverse transcriptase produces viral DNA
from RNA.
Becomes a provirus which integrates into host
DNA.
Period of latency occurs.

http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/hivdsdna.html

Viral Replication
After a period of latency lasting up to 10 years
viral replication is triggered and occurs at high
rate.
CD4 cell may be destroyed in the process, body
attempts to replace lost CD4 cells, but over the
course of many years body is unable to keep the
count at a safe level.
Destruction of large numbers of CD4 cause
symptoms of HIV to appear with increased
susceptibility to opportunistic infections, disease
and malignancy.

HIV (arrows) Infecting a T-lymphocyte

Viral Replication
Methods of transmission:
Sexual transmission, presence of STD increases
likelihood of transmission.
Exposure to infected blood or blood products.
Use of contaminated clotting factors by hemophiliacs.
Sharing contaminated needles (IV drug users).
Transplantation of infected tissues or organs.
Mother to fetus, perinatal transmission variable,
dependent on viral load and mothers CD 4 count.

Transmission

Primary HIV Syndrome

Mononucleosis-like, cold or flu-like symptoms
may occur 6 to 12 weeks after infection.
lymphadenopathy
fever
rash
headache
Fatigue
diarrhea
sore throat
neurologic manifestations.
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:
persistent herpes-zoster infection (shingles)
oral candidiasis (thrush)
oral hairy leukoplakia
Kaposis sarcoma (KS)

Oral Candidiasis (thrush)

Oral Hairy Leukoplakia

Being that HIV reduces immunologic activity, the

intraoral environment is a prime target for chronic
secondary infections and inflammatory processes,
including OHL, which is due to the Epstein-Barr virus
under immunosuppressed conditions

Kaposis sarcoma (KS)

Kaposis sarcoma
(shown) is a rare cancer
of the blood vessels that
is associated with HIV. It
manifests as bluish-red
oval-shaped patches that
may eventually become
thickened. Lesions may
appear singly or in
clusters.

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:
Pneumocystis carinii pneumonia (PCP)
cryptococcal meningitis
toxoplasmosis

If CD4 count drops below 50:

Mycobacterium avium
Cytomegalovirus infections
lymphoma
dementia
Most deaths occur with CD4 counts below 50.

Other Opportunistic Infections

Respiratory system

Gastro-intestinal system

Cryptosporidiosis
Candida
Cytomegolavirus (CMV)
Isosporiasis
Kaposi's Sarcoma

Central/peripheral Nervous system

Pneumocystis Carinii Pneumonia (PCP)

Tuberculosis (TB)
Kaposi's Sarcoma (KS)

Cytomegolavirus
Toxoplasmosis
Cryptococcosis
Non Hodgkin's lymphoma
Varicella Zoster
Herpes simplex

Skin

Herpes simple
Kaposi's sarcoma
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.
First antibodies detected produced against
gag proteins p24 and p55.
Followed by antibody to p51, p120 and gp41
As disease progresses antibody levels
decrease.

Immunologic Manifestations
Immune abnormalities associated with increased
viral replication.
Decrease in CD4 cells due to virus budding from
cells, fusion of uninfected cells with virally infected
cells and apoptosis.
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.
CD8 cells initially increase and may remain elevated.
As HIV infection progresses, CD4 T cells drop
resulting in immunosuppression and susceptibility of
patient to opportunistic infections.
Death comes due to immuno-incompetence.

Immunologic Manifestations
Immune abnormalities associated with increased
viral replication.
Decrease in CD4 cells due to virus budding from
cells, fusion of uninfected cells with virally infected
cells and apoptosis.
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.
CD8 cells initially increase and may remain elevated.
As HIV infection progresses, CD4 T cells drop
resulting in immunosuppression and susceptibility of
patient to opportunistic infections.
Death comes due to immuno-incompetence.

The Move Toward Lower Pill Burdens

Regimen

Dosing

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

Daily pill burden

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
10
1 infection

3
weeks

Time following infection

6
years

Laboratory Diagnosis of HIV Infection

Methods utilized to detect:
Antibody
Antigen
Viral nucleic acid
Virus in culture

ELISA Testing
First serological test developed to detect
HIV infection.
Easy to perform.
Easily adapted to batch testing.
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:
Screening blood products.
Diagnosing and monitoring patients.
Determining prevalence of infection.
Research investigations.

ELISA Testing
Different types of ELISA techniques used:
indirect
competitive
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.
A rapid, cost-effective and may become an alternative
to standard ELISA and Western blot testing.

Particle Agglutination

Western Blot
Most popular confirmatory test.
Utilizes a lysate prepared from HIV virus.
The lysate is electrophoresed to separate out the HIV
proteins (antigens).
The paper is cut into strips and reacted with test sera.
After incubation and washing anti-antibody tagged
with radioisotope or enzyme is added.
Specific bands form where antibody has reacted with
different antigens.
Most critical reagent of test is purest quality HIV
antigen.
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.
No bands, negative.
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.

early

3rd gen ELISA

1st gen ELISA
Detuned ELISA

1wk

2wk

recent / established advanced

3wk

2mo 6mo

1yr 2yr 3yr

+8yr

Western Blot

Expensive $ 80 - 100
technically more difficult
visual interpretation
lack standardisation
- performance
- interpretation
- 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:

prior blood transfusions, even with non-HIV-1 infected blood

prior or current infection with syphilis
prior or current infection with malaria
autoimmune diseases (e.g., diabetes, Graves disease, etc)
infection with other human retroviruses
second or subsequent pregnancies in women.
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:
early infection suspected in seronegative
patient
newborns
CSF
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 patients 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.