2. 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)
3. 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/
4. 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.
6. 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).
7. Structural Genes
• Three main structural genes:
– Group Specific Antigen (Gag)
– Envelope (Env)
– Polymerase (Pol)
8. 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.
9. 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.
10. 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.
11. 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.
12. 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
14. 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.
15. 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
16. 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.
19. 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 mother’s CD 4 count.
21. 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
22. 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.
23. 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
– Kaposi’s sarcoma (KS)
25. 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
26. Kaposi’s sarcoma (KS)
• Kaposi’s 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.
27. 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.
29. Infants with HIV
• Failure to thrive
• Persistent oral candidiasis
• Hepatosplenomegaly
• Lymphadenopathy
• Recurrent diarrhea
• Recurrent bacterial infections
• Abnormal neurologic findings.
30. 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.
31. 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.
32. 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.
33. 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.
35. 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.
36. 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
37. ‘typical’ primary HIV-1 infection
symptoms
HIV-1 p24 antigen
0 1 2 3 4 5 6 / 2 4 6 8 10
weeks years
HIV antibodies
Time following infection
HIV viral load
HIV proviral DNA
symptoms
‘window’
period
1° infection
38. Laboratory Diagnosis of HIV Infection
• Methods utilized to detect:
– Antibody
– Antigen
– Viral nucleic acid
– Virus in culture
39. 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
40. ELISA Testing
• ELISA tests useful for:
– Screening blood products.
– Diagnosing and monitoring patients.
– Determining prevalence of infection.
– Research investigations.
41. 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.
43. 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.
46. 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.
47. 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.
48. 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.
49. DNA PCR
RNA PCR
p24 Ag
3rd gen ELISA
1st gen ELISA
Detuned ELISA
1wk 2wk 3wk 2mo 6mo 1yr 2yr 3yr +8yr
gp160
gp120
p68
p55
p53
gp41-45
p40
p34
p24
p18
p12
gp160
gp120
p68
p55
p53
gp41-45
p40
p34
p24
p18
p12
gp160
gp120
p68
p55
p53
gp41-45
p40
p34
p24
p18
p12
early recent / established advanced
Spectrum
of anti-HIV
testing
50. Western Blot
• Expensive – $ 80 - 100
• technically more difficult
• visual interpretation
• lack standardisation
– - performance
– - interpretation
– - indeterminate reactions –
resolution of ??
• ‘Gold Standard’ for
confirmation
51. 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, Grave’s 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.
52. 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.
53.
54. 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.
55. 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.
56. Detection of p24 HIV antigen
• Most useful for the following:
– early infection suspected in seronegative
patient
– newborns
– CSF
– monitoring disease progress
57. 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.
58. 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.
59. 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.
60. 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.
61. 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.
