Download Human Immunodeficiency Virus (HIV)

Unit 4
Part 6 Human Immunodeficiency Virus
Terry Kotrla, MS, MT(ASCP)BB
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
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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.
Reduced mortality resulting from the use of highly active
antiretroviral therapies is a major factor contributing to the
number of persons in the United States living with HIV disease.
Additionally, more than 56,000 new HIV infections are estimated
to occur annually
HIV in the US
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
 http://tinyurl.com/3425m69
 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
Viral Replication
 In late phase HIV infection,
most of the viruses are Ttropic, having gp120
capable of binding to CD4
and CXCR4 found on T4lymphocytes.
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.
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 mother’s CD 4 count.
Transmission
Primary HIV Syndrome
 Mononucleosis-like, cold or flu-like symptoms may
occur 6 to 12 weeks after infection.
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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:
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persistent herpes-zoster infection (shingles)
oral candidiasis (thrush)
oral hairy leukoplakia
Kaposi’s 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 EpsteinBarr virus under immunosuppressed conditions
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 ovalshaped 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
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Respiratory system
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Gastro-intestinal system
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Cryptosporidiosis
Candida
Cytomegolavirus (CMV)
Isosporiasis
Kaposi's Sarcoma
Central/peripheral Nervous system
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Pneumocystis Carinii Pneumonia (PCP)
Tuberculosis (TB)
Kaposi's Sarcoma (KS)
Cytomegolavirus
Toxoplasmosis
Cryptococcosis
Non Hodgkin's lymphoma
Varicella Zoster
Herpes simplex
Skin
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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.
‘typical’ primary HIV-1 infection
symptoms
symptoms
HIV proviral DNA
HIV antibodies
‘window’
period
HIV viral load
HIV-1 p24 antigen
0
1
1° infection
2
3
4
5
6
/
2
weeks
4
6
years
Time following infection
8
10
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.
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.
gp160
gp120
p68
p55
p53
gp41-45
Spectrum
of anti-HIV
testing
p40
p34
p24
p18
p12
early
DNA PCR
RNA PCR
p24 Ag
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, 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.
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 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.
Treatment - 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 or 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.”
 NRTIs block reverse transcriptase, a protein that HIV needs
to make more copies of itself.
 Prevents HIV from altering the genetic material of
healthy CD4 cells.
 Prevents the cells from producing new virus and
decreases the amount of virus in the body.
 May slow down HIV disease
References

http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/hivlc.html#translat
 http://pathmicro.med.sc.edu/lecture/HIV3.htm
 http://www.avert.org/hivstages.htm
 http://www.aidsinfo.nih.gov/guidelines/
 http://www.hopkins-aids.edu/publications/pocketguide/pocketgd0105.pdf
 http://www.modares.ac.ir/sci/saman_h/Pages/applications.htm
 http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-02-02-02
 http://www.hivandhepatitis.com/recent/test/realtime/061604_f.html