Hiv

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Hiv

  1. 1. Human Immunodeficiency Virus(HIV)
  2. 2. Introduction• Etiologic agent of AcquiredImmunodeficiency Syndrome (AIDS).• Discovered independently by LucMontagnier of France and Robert Gallo ofthe 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. 3. Introduction• HIV-2 discovered in 1986, antigenicallydistinct virus endemic in West Africa.• One million people infected in US, 30million worldwide are infected.• Leading cause of death of men aged 25-44 and 4th leading cause of death ofwomen in this age group in the US.• http://www.cnn.com/2005/HEALTH/conditions/11/17/blacks.hiv.ap/
  4. 4. Characteristics of the virus• Icosahedral (20 sided), enveloped virus of thelentivirus subfamily of retroviruses.• Retroviruses transcribe RNA to DNA.• Two viral strands of RNA found in coresurrounded by protein outer coat.– Outer envelope contains a lipid matrix within whichspecific viral glycoproteins are imbedded.– These knob-like structures responsible for binding totarget cell.
  5. 5. Characteristics of the virus
  6. 6. HIV• The outer shell of the virus isknown as the Viral enevlope.Embedded in the viralenvelope is a complex proteinknown as env which consistsof an outer protruding capglycoprotein (gp) 120, and astem gp14. Within the viralenvelope is an HIV proteincalled p17(matrix), and withinthis is the viral core or capsid,which is made of another viralprotein p24(core antigen).
  7. 7. Structural Genes• Three main structural genes:– Group Specific Antigen (Gag)– Envelope (Env)– Polymerase (Pol)
  8. 8. Group Specific Antigen (Gag)• Located in nucelocapsid of virus.• Icosahedryl capsid surrounds the internalnucleic acids made up of p24 andp15.• p17 lies between protein core andenvelope and is embedded in the internalportion of the envelope.• Two additional p55 products, p7 and p9,are nucleic acid binding proteins closelyassociated with the RNA.
  9. 9. Envelope (Env)• Envelope (Env) gene codes for envelopeproteins gp160, gp120 and gp41.– These polyproteins will eventually be cleaved byproteases to become HIV envelope glycoproteinsgp120 and gp41.– gp160 cleaved to form gp120 and gp41.– gp120 forms the 72 knobs which protrude from outerenvelope.– gp41 is a transmembrane glycoprotein antigen thatspans the inner and outer membranes and attachesto gp120.– gp120 and gp41 both involved with fusion andattachment of HIV to CD4 antigen on host cells.
  10. 10. Polymerase (Pol)• Polymerase (Pol) codes for p66 and p51subunits of reverse transcriptase and p31an endonuclease.– Located in the core, close to nucleic acids.– Responsible for conversion of viral RNA intoDNA, integration of DNA into host cell DNAand cleavage of protein precursors.
  11. 11. Viral Replication• First step, HIV attaches to susceptible host cell.– Site of attachment is the CD4 antigen found on avariety of cells• helper T cells• macrophages• monocytes• B cells• microglial brain cells• intestinal cells– T cells infected later on.
  12. 12. Early Phase HIV Infection• In early phase HIVinfection, initialviruses are M-tropic.Their envelopeglycoprotein gp120 isable to bind to CD4molecules andchemokine receptorscalled CCR5 found onmacrophages
  13. 13. http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/hivad.html• In late phase HIVinfection, most of theviruses are T-tropic,having gp120 capableof binding to CD4 andCXCR4 found on T4-lymphocytes.
  14. 14. Viral Replication• The gp120 protein on virus bindsspecifically to CD4 receptor on host cellwith high affinity.• Gp41 causes fusion of the virus to the cellmembrane.– After fusion virus particle enters cell.– Viral genome exposed by uncoating particle.
  15. 15. Viral Replication• Reverse transcriptase produces viral DNAfrom RNA.– Becomes a provirus which integrates into hostDNA.– Period of latency occurs.• http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/hivdsdna.html
  16. 16. Viral Replication• After a period of latency lasting up to 10 yearsviral replication is triggered and occurs at highrate.• CD4 cell may be destroyed in the process, bodyattempts to replace lost CD4 cells, but over thecourse of many years body is unable to keep thecount at a safe level.• Destruction of large numbers of CD4 causesymptoms of HIV to appear with increasedsusceptibility to opportunistic infections, diseaseand malignancy.
  17. 17. HIV (arrows) Infecting a T-lymphocyte
  18. 18. Viral Replication• Methods of transmission:– Sexual transmission, presence of STD increaseslikelihood 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.
  19. 19. Transmission
  20. 20. Primary HIV Syndrome• Mononucleosis-like, cold or flu-like symptomsmay occur 6 to 12 weeks after infection.– lymphadenopathy– fever– rash– headache– Fatigue– diarrhea– sore throat– neurologic manifestations.– no symptoms may be present
  21. 21. Primary HIV Syndrome• Symptoms are relatively nonspecific.• HIV antibody test often negative but becomespositive within 3 to 6 months, this process isknown as seroconversion.• Large amount of HIV in the peripheral blood.• Primary HIV can be diagnosed using viral loadtiter assay or other tests.• Primary HIV syndrome resolves itself and HIVinfected person remains asymptomatic for aprolonged period of time, often years.
  22. 22. Clinical Latency Period• HIV continues to reproduce, CD4 countgradually declines from its normal value of 500-1200.• Once CD4 count drops below 500, HIV infectedperson at risk for opportunistic infections.• The following diseases are predictive of theprogression to AIDS:– persistent herpes-zoster infection (shingles)– oral candidiasis (thrush)– oral hairy leukoplakia– Kaposi’s sarcoma (KS)
  23. 23. Oral Candidiasis (thrush)
  24. 24. Oral Hairy Leukoplakia• Being that HIV reduces immunologic activity, theintraoral environment is a prime target for chronicsecondary infections and inflammatory processes,including OHL, which is due to the Epstein-Barr virusunder immunosuppressed conditions
  25. 25. Kaposi’s sarcoma (KS)• Kaposi’s sarcoma(shown) is a rare cancerof the blood vessels thatis associated with HIV. Itmanifests as bluish-redoval-shaped patches thatmay eventually becomethickened. Lesions mayappear singly or inclusters.
  26. 26. AIDS• CD4 count drops below 200 person is considered tohave advanced HIV disease• If preventative medications not started the HIV infectedperson 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.
  27. 27. Other Opportunistic Infections• Respiratory system– Pneumocystis Carinii Pneumonia (PCP)– Tuberculosis (TB)– Kaposis Sarcoma (KS)• Gastro-intestinal system– Cryptosporidiosis– Candida– Cytomegolavirus (CMV)– Isosporiasis– Kaposis Sarcoma• Central/peripheral Nervous system– Cytomegolavirus– Toxoplasmosis– Cryptococcosis– Non Hodgkins lymphoma– Varicella Zoster– Herpes simplex• Skin– Herpes simple– Kaposis sarcoma– Varicella Zoster
  28. 28. Infants with HIV• Failure to thrive• Persistent oral candidiasis• Hepatosplenomegaly• Lymphadenopathy• Recurrent diarrhea• Recurrent bacterial infections• Abnormal neurologic findings.
  29. 29. Immunologic Manifestations• Early stage slight depression of CD4count, few symptoms, temporary.• Window of up to 6 weeks before antibodyis detected, by 6 months 95% positive.• During window p24 antigen present, acuteviremia and antigenemia.
  30. 30. Immunologic Manifestations• Antibodies produced to all major antigens.– First antibodies detected produced againstgag proteins p24 and p55.– Followed by antibody to p51, p120 and gp41– As disease progresses antibody levelsdecrease.
  31. 31. Immunologic Manifestations• Immune abnormalities associated with increasedviral replication.– Decrease in CD4 cells due to virus budding fromcells, fusion of uninfected cells with virally infectedcells and apoptosis.– B cells have decreased response to antigens possiblydue to blockage of T cell/B cell interaction by bindingof viral proteins to CD4 site.– CD8 cells initially increase and may remain elevated.– As HIV infection progresses, CD4 T cells dropresulting in immunosuppression and susceptibility ofpatient to opportunistic infections.– Death comes due to immuno-incompetence.
  32. 32. Immunologic Manifestations• Immune abnormalities associated with increasedviral replication.– Decrease in CD4 cells due to virus budding fromcells, fusion of uninfected cells with virally infectedcells and apoptosis.– B cells have decreased response to antigens possiblydue to blockage of T cell/B cell interaction by bindingof viral proteins to CD4 site.– CD8 cells initially increase and may remain elevated.– As HIV infection progresses, CD4 T cells dropresulting in immunosuppression and susceptibility ofpatient to opportunistic infections.– Death comes due to immuno-incompetence.
  33. 33. The Move Toward Lower Pill BurdensDosing Daily pill burdenRegimen1996Zerit/Epivir/Crixivan 10 pills, Q8H20023 pills, BIDCombivir (AZT/3TC)/EFV1998Retrovir/Epivir/Sustiva 5 pills, BID20033 pills, QDViread/ Emtriva/Sustiva20042 pills, QDTruvada/Sustiva
  34. 34. Sustiva + Truvada Treatment• Sustiva + Truvada (FTC + tenofovor) is one of the mostpopular and effective starting HIV regimens.• Many patients will have dream/sleep/central nervoussystem effects particularly in the first month (due to theSustiva).• Upset stomach/bloating/gas/loose stools is also fairlycommon during the first month and for most patients isfairly mild.• HIV levels in the blood will often drop by > 99% in thefirst month and the CD4 count (marker of immunesystem function) will often increase providing protectionagainst AIDS related diseases within weeks/months ofstarting the medication.
  35. 35. Truvada• Truvada is made up of HIV drugs from aclass called nucleoside/nucleotide reversetranscriptase inhibitors (NRTIs), alsoknown as “nukes.”• The NRTIs block reverse transcriptase, aprotein that HIV needs to make morecopies of itself. This may slow down HIVdisease
  36. 36. ‘typical’ primary HIV-1 infectionsymptomsHIV-1 p24 antigen0 1 2 3 4 5 6 / 2 4 6 8 10weeks yearsHIV antibodiesTime following infectionHIV viral loadHIV proviral DNAsymptoms‘window’period1° infection
  37. 37. Laboratory Diagnosis of HIV Infection• Methods utilized to detect:– Antibody– Antigen– Viral nucleic acid– Virus in culture
  38. 38. ELISA Testing• First serological test developed to detectHIV infection.– Easy to perform.– Easily adapted to batch testing.– Highly sensitive and specific.• Antibodies detected in ELISA includethose directed against: p24, gp120, gp160and gp41, detected first in infection andappear in most individuals
  39. 39. ELISA Testing• ELISA tests useful for:– Screening blood products.– Diagnosing and monitoring patients.– Determining prevalence of infection.– Research investigations.
  40. 40. ELISA Testing• Different types of ELISA techniques used:– indirect– competitive– sandwich• ELISAs are for screening only, falsepositives do occur and may be due to AIdisease, alcoholism, syphilis, andimmunoproliferative diseases.
  41. 41. ELISA Sandwich
  42. 42. Other Screening Tests• Agglutination tests using latex particles, gelatinparticles or microbeads are coated with HIVantigen and will agglutinate in the presence ofantibody.• Dot-Blot Testing utilizes paper or nitrocelluloseimpregnated with antigen, patient serum isfiltered through, and anti-antibody is added withenzyme label, color change is positive.– A rapid, cost-effective and may become an alternativeto standard ELISA and Western blot testing.
  43. 43. Particle Agglutination
  44. 44. Western Blot• Most popular confirmatory test.– Utilizes a lysate prepared from HIV virus.– The lysate is electrophoresed to separate out the HIVproteins (antigens).– The paper is cut into strips and reacted with test sera.– After incubation and washing anti-antibody taggedwith radioisotope or enzyme is added.– Specific bands form where antibody has reacted withdifferent antigens.– Most critical reagent of test is purest quality HIVantigen.– The following antigens must be present: p17, p24,p31, gp41, p51, p55, p66, gp120 and gp160.
  45. 45. Western Blot• Antibodies to p24 and p55 appear earliestbut decrease or become undetectable.• Antibodies to gp31, gp41, gp 120, andgp160 appear later but are presentthroughout all stages of the disease.
  46. 46. Western Blot• Interpretation of results.– No bands, negative.– In order to be interpreted as positive aminimum of 3 bands directed against thefollowing antigens must be present: p24, p31,gp41 or gp120/160.• CDC criteria require 2 bands of thefollowing: p24, gp41 or gp120/160.
  47. 47. DNA PCRRNA PCRp24 Ag3rd gen ELISA1st gen ELISADetuned ELISA1wk 2wk 3wk 2mo 6mo 1yr 2yr 3yr +8yrgp160gp120p68p55p53gp41-45p40p34p24p18p12gp160gp120p68p55p53gp41-45p40p34p24p18p12gp160gp120p68p55p53gp41-45p40p34p24p18p12early recent / established advancedSpectrumof anti-HIVtesting
  48. 48. Western Blot• Expensive – $ 80 - 100• technically more difficult• visual interpretation• lack standardisation– - performance– - interpretation– - indeterminate reactions –resolution of ??• ‘Gold Standard’ forconfirmation
  49. 49. Western Blot• Indeterminate results are those samples that producebands but not enough to be positive, may be due to thefollowing:– 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 requirestesting with strongly positive, weakly positive andnegative controls.
  50. 50. Indirect immunofluorescence• Can be used to detect both virus andantibody to it.• Antibody detected by testing patient serumagainst antigen applied to a slide,incubated, washed and a fluorescentantibody added.• Virus is detected by fixing patient cells toslide, incubating with antibody.
  51. 51. Detection of p24 HIV antigen• The p24-antigen screening assay is an EIAperformed on serum or plasma.• P24 antigen only present for short time,disappears when antibody to p24 appears.• Anti-HIV-1 bound to membrane, incubated withpatient serum, second anti-HIV-1 antibodyattached to enzyme label is added (sandwichtechnique), color change occurs.• Optical density measured, standard curveprepared to quantitate results.
  52. 52. Detection of p24 HIV antigen• Positive confirmed by neutralizingreaction, preincubate patient sample withanti- HIV, retest, if p24 present immunecomplexes form preventing binding to HIVantibody on membrane when added.• Test not recommended for routinescreening as appearance and rate of riseare unpredictable.• Sensitivity lower than ELISA.
  53. 53. Detection of p24 HIV antigen• Most useful for the following:– early infection suspected in seronegativepatient– newborns– CSF– monitoring disease progress
  54. 54. 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 DNAsequences present.• The DNA is detected by using radioactive or biotinylatedprobes.• Once DNA is amplified it is placed on nitrocellulosepaper and allowed to react with a radiolabeled probe, asingle stranded DNA fragment unique to HIV, which willhybridize with the patient’s HIV DNA if present.• Radioactivity is determined.
  55. 55. Virus isolation• Virus isolation can be used to definitivelydiagnose HIV.• Best sample is peripheral blood, but can useCSF, saliva, cervical secretions, semen, tears ormaterial from organ biopsy.• Cell growth in culture is stimulated, amplifiesnumber of cells releasing virus.• Cultures incubated one month, infectionconfirmed by detecting reverse transcriptase orp24 antigen in supernatant.
  56. 56. Viral Load Tests• Viral load or viral burden is the quantity ofHIV-RNA that is in the blood.• RNA is the genetic material of HIV thatcontains information to make more virus.
  57. 57. Viral Load Tests• Viral load tests measure the amount of HIV-RNAin one milliliter of blood.• Take 2 measurements 2-3 weeks apart todetermine baseline.• Repeat every 3-6 months in conjunction withCD4 counts to monitor viral load ant T-cell count.• Repeat 4-6 weeks after starting or changingantiretroviral therapy to determine effect on viralload.
  58. 58. Testing of Neonates• Difficult due to presence of maternal IgGantibodies.• 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 notdetecting antigen soon enough: 38 days to6 months to be positive.
  59. 59. 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

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