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Human Immunodeficiency Virus
(HIV) & AIDS
Dr. Md. Abdulla-hel Kafi&
Md. Mesbah Uddin
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.
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.
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.
• (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.
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
Late Phase HIV Infection
• In late phase HIV infection, most of the viruses are T-tropic, having gp120 capable of binding to CD4 and CXCR4 found on T4-lymphocytes.
HIV (arrows) Infecting a T-lymphocyte
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– 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 Epstein-Barr 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 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
– Pneumocystis Carinii Pneumonia (PCP) – Tuberculosis (TB)– Kaposi's Sarcoma (KS)
• Gastro-intestinal system– Cryptosporidiosis– Candida– Cytomegolavirus (CMV)– Isosporiasis – Kaposi's Sarcoma
Other Opportunistic Infections• Central/peripheral Nervous system
– 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.
TreatmentANTIRETROVIRAL THERAPYART that is capable of suppressing viral replication has been
available since 1996. ART has transformed HIV from a progressive illness with a fatal outcome into a chronic manageable disease (Box 14.18). The goals of ART are to:
• The specific goals of treatment are to restore immunologic function that reduces the incidence of both opportunistic infections and certain malignancies as well as to reduce viral load, which reduces the chance of transmission to others.
• Reduce the viral load to an undetectable level for as long as possible
Treatment• Improve the CD4 count to over 200 cells/mm3 so
that severe HIV-related disease is unlikely• Improve the quantity and quality of life without
unacceptable drug toxicity• Reduce HIV transmission.
Many of the antiretroviral drugs which were used initially have been largely abandoned because of toxicity or poor efficacy. The drugs that are currently most commonly used are shown in Box
Drugs Used for the Treatment of HIV Infection
Class of Drug Reverse transcriptase inhibitors
Nucleosides (NRTI)
• Abacavir (ABC) (Ziagen) • Didanosine (ddI) (Videx)• Emtricitabine (FTC) (Emtriva)• Lamivudine (3TC) (Epivir)• Stavudine (d4T) (Zerit)• Zidovudine (AZT, ZDV) (Retrovir)
Class of Drug Name of DrugReverse transcriptase inhibitorsNucleosides (NRTI)
Abacavir (ABC) (Ziagen)Didanosine (ddI) (Videx)Emtricitabine (FTC) (Emtriva)Lamivudine (3TC) (Epivir)Stavudine (d4T) (Zerit)Zidovudine (AZT, ZDV) (Retrovir)
Nucleotides Tenofovir (Viread)
Non-nucleosides (NNRTI) Delavirdine (Rescriptor)Efavirenz (Sustiva)Etravirine (Intelence)Nevirapine (Viramune)Rilpivirine (Endurant)
Class of Drug Name of DrugProtease inhibitors1 Amprenavir (Agenerase)
Atazanavir (Reyataz)Darunavir (Prezista)Fosamprenavir (Lexiva)Indinavir (Crixivan)Lopinavir/ritonavir (Kaletra)Nelfinavir (Viracept)Ritonavir (Norvir)Saquinavir (Invirase andFortovase)Tipranavir (Aptivus)
Entry inhibitorsFusion inhibitor
Enfuvirtide (Fuzeon)
Coreceptor antagonist Maraviroc (Selzentry)Integrase inhibitor Raltegravir (Isentress)
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.
DNA PCRDNA PCRRNA PCRRNA PCR
p24 Agp24 Ag3rd gen ELISA1st gen ELISA
Detuned ELISA1wk 2wk 3wk 2mo 6mo 1yr 2yr 3yr +8yr
gp160gp120
p68p55p53
gp41-45
p40
p34
p24
p18
p12
gp160gp120
p68p55p53
gp41-45
p40
p34
p24
p18
p12
gp160gp120
p68p55p53
gp41-45
p40
p34
p24
p18
p12
early recent / established advanced
Spectrum Spectrum of anti-HIV of anti-HIV
testing testing
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.
The End…