HIV Diagnosis and Pathogenesis

1. HIV Diagnosis and Pathogenesis Scott M. Hammer, M.D.

2. HIV Diagnosis • Consider in anyone presenting with symptoms and signs compatible with an HIV-related syndrome or in an asymptomatic person with a risk factor for acquisition • Full sexual and behavioral history should be taken in all patients • Assumptions of risk (or lack thereof) by clinicians are unreliable

3. Laboratory Diagnosis of Established HIV Infection: Antibody Detection • Screening • Serum ELISA • Rapid blood or salivary Ab tests • Confirmation • Western blot • Written consent for HIV Ab testing must be obtained and be accompanied by pre- and post-test counselling

4. Laboratory Diagnosis of Acute HIV-1 Infection • Patients with acute HIV infection may present to a health care facility before full antibody seroconversion • ELISA may be negative • ELISA may be positive with negative or indeterminant Western blot • Plasma HIV-1 RNA level should be done if acute HIV infection is suspected • Follow-up antibody testing should be performed to document full seroconversion (positive ELISA and WB)

5. HIV-1 Virion

6. HIV LifeCycle Tat = transcriptional activator Rev = regulator of mRNA nuclear export

7. HIV-1: Genetic Organization

8. Established HIV Infection:Pathogenesis • Active viral replication present throughout course of disease • Major reservoirs of infection exist outside of blood compartment • Lymphoreticular tissues • Central nervous system • Genital tract • Virus exists as multiple quasispecies • Mixtures of viruses with differential phenotypic and genotypic characteristics may coexist • At least 10 X 109 virions produced and destroyed each day • T1/2 of HIV in plasma is <6 h and may be as short as 30 minutes • Immune response, chemokine receptor status and HLA type are important codeterminants of outcome

9. Determinants of Outcome:Selected Viral Factors • Escape from immune response • Under immune selective pressure (cellular and humoral), mutations in gag, pol and env may arise • Attenuation • nef deleted viruses associated with slow or long-term nonprogression in case reports and small cohorts • Tropism • R5 to X4 virus conversion associated with increased viral pathogenicity and disease progression • Subtypes • Potential for varied subtypes to exhibit differential transmissibility and virulence • Potential for greater heterosexual spread of some subtypes

10. Host Factors in HIV Infection (I) • Cell-mediated immunity • Cytotoxic T cells • Eliminate virus infected cells • Play prominent role in control of viremia, slowing of disease progression and perhaps prevention of infection • T-helper response • Vital for preservation of CTL response • Humoral immunity • Role in prevention of transmission and disease progression unclear

11. Role of CTL’s in Control of Viremia Letvin N & Walker B: Nature Med 2003;9:861-866

12. Host Factors in HIV Infection (II) • Chemokine receptors • CCR5-Δ32 deletion • Homozygosity associated with decreased susceptibility to R5 virus infection • Heterozygosity associated with delayed disease progression • CCR2-V64I mutation • Heterozygosity associated with delayed disease progression • CCR5 promoter polymorphisms • 59029-G homozygosity associated with slower disease progression • 59356-T homozygosity associated with increased perinatal transmission

13. Host Factors in HIV Infection (III) • Other genetic factors • Class I alleles B35 and Cω4 • Associated with accelerated disease progression • Heterozygosity at all HLA class I loci • Appear to be protective • HLA-B57, HLA-B27, HLA-Bω4, HLA-B*5701 • Associated with long-term non-progression • HLA-B14 and HLA-C8 • ?Associated with long-term nonprogression

14. Mechanisms of CD4+ Cell Death in HIV Infection • HIV-infected cells • Direct cytolytic effect of HIV • Lysis by CTL’s • Apoptosis • Potentiated by viral gp120, Tat, Nef, Vpu • HIV-uninfected cells • Apoptosis • Release of gp120, Tat, Nef, Vpu by neighboring, infected cells • Activation induced cell death

15. The Variable Course of HIV-1 Infection Typical Progressor Rapid Progressor Primary HIVInfection Primary HIVInfection Clinical Latency AIDS AIDS CD4 Level CD4 Level Viral Replication Viral Replication A B months years months years Nonprogressor Primary HIVInfection Clinical Latency CD4 Level Viral Replication ? C months years Reprinted with permission from Haynes. In: DeVita et al, eds. AIDS: Etiology, Treatment and Prevention. 4th ed. Lippincott-Raven Publishers; 1997:89-99.

16. 0 T1/2 = 1 d (productively infected CD4’s) -1 T1/2 = 2-4 wks (macrophages, latently infected CD4’s, release of trapped virions) Change in HIV RNA (log10) T1/2 = 6-44 mos (resting, memory CD4’s) -2 2-4 16-24 Time (weeks) Phases of Decay Under the Influence of Potent Antiretroviral Therapy

17. Therapeutic Implications of First and Second Phase HIV RNA Declines • Antiviral potency can be assessed in first 7-14 days • Should see 1-2 log declines after initiation of therapy in persons with drug susceptible virus who are adherent • HIV RNA trajectory in first 1-8 weeks can be predictive of subsequent response • Durability of response translates into clinical benefit

18. 0 T1/2 = 1 d (productively infected CD4’s) -1 T1/2 = 2-4 wks (macrophages, latently infected CD4’s, release of trapped virions) Change in HIV RNA (log10) T1/2 = 6-44 mos (resting, memory CD4’s) -2 2-4 16-24 Time (weeks) Phases of Decay Under the Influence of Potent Antiretroviral Therapy

19. Model of Post-Integration Latency Resting naïve CD4+ T cell +Ag Activated CD4+ T cell Preintegration Latency Postintegration Latency -Ag -Ag Resting memory CD4+ T cell +Ag +Ag +Ag Activated CD4+ T cell Siliciano R et al

20. Therapeutic Implications of Third Phase of HIV RNA Decay: Latent Cell Reservoir • Viral eradication not possible with current drugs • Archive of replication competent virus history is established • Drug susceptible and resistant • Despite the presence of reservoir(s), minimal degree of viral evolution observed in patients with plasma HIV RNA levels <50 c/ml suggests that current approach designed to achieve maximum virus suppression is appropriate

21. Initiation of Therapy in Established HIV Infection: Considerations • Patient’s disease stage • Symptomatic status • CD4 cell count • Plasma HIV-1 RNA level • Patient’s commitment to therapy • Philosophy of treatment • Pros and cons of ‘early’ intervention

22. Initiation of Therapy in Asymptomatic Persons: Population Based Studies • Clinical outcome compromised if Rx begun when CD4 <200 • Miller et al (EuroSIDA), Ann Intern Med 1999;130:570-577 • Hogg et al (British Columbia), JAMA 2001;286:2568 • Sterling et al (JHU), AIDS 2001;15:2251-2257 • Pallela et al (HOPS), Ann Intern Med 2003;138:620-626 • Sterling et al (JHU), J Infect Dis 2003;188:1659-1665 • No virologic or immunologic advantage to starting at CD4 >350 vs. 200-350; increased rate of virologic failure when starting at CD4 <200 • Cozzi-Lepri et al (ICONA), AIDS 2001;15:983-990 • Virologic responses comparable among groups with CD4 >200; slower decline to RNA <500 in those with RNA’s >100,000 at baseline • Phillips et al (SHCS, EuroSIDA, Frankfurt), JAMA 2001;286:2560-2567 • Clinical outcome compromised if Rx begun when CD4 <200 or RNA >100,000 • Egger et al (13 cohorts, >12,000 persons), Lancet 2002;360:119-129

23. Prognosis According to CD4 and RNA:ART Cohort Collaboration Egger M et al: Lancet 2002;360:119-129

24. 1000 800 600 400 200 0 Early Opportunistic Infections CD4Cells + Late Opportunistic Infections 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Infection Time in Years Natural History of Untreated HIV-1 Infection

25. MACS: CD4 Cell Decline by HIV RNA Stratum Mellors et al: Ann Intern Med 1997;126:946-954

26. CD4 and HIV-1 RNA (I) • Independent predictors of outcome in most studies • Near-term risk defined by CD4 • Longer-term risk defined by both CD4 and HIV-1 RNA • Rate of CD4 decline linked to HIV RNA level in untreated persons

27. CD4 and HIV-1 RNA (II) • Good but incomplete surrogate markers • For both natural history and treatment effect • Thresholds are arbitrary • Disease process is a biologic continuum • Gender specificity of HIV RNA in early-mid stage disease needs to be considered • Treatment decisions should be individualized • Baseline should be established • Trajectory determined

28. HIV Resistance: Underlying Concepts • Genetic variants are continuously produced as a result of high viral turnover and inherent error rate of RT • Mutations at each codon site occur daily • Survival depends on replication competence and presence of drug or immune selective pressure • Double mutations in same genome also occur but 3 or more mutations in same genome is a rare event • Numerous natural polymorphisms exist

29. Pre-existence of Resistant Mutants • Viral replication cycles: 109-1010/day • RT error rate: 10-4-10-5/base/cycle • HIV genome: 104 bp • Every point mutation occurs 104-105 times/day • In drug naïve individuals • Single and double mutants pre-exist • Triple and quadruple mutants would be predicted to be rare

30. HIV Resistance: Underlying Concepts • Implications • Resistance mutations may exist before drug exposure and may emerge quickly after it is introduced • Drugs which develop high level resistance with a single mutation are at greatest risk • e.g., 3TC, NNRTI’s (nevirapine, efavirenz) • Resistance to agents which require multiple mutations will evolve more slowly • Partially suppressive regimens will inevitably lead to emergence of resistance • A high ‘genetic barrier’ needs to be set to prevent resistance • Potent, combination regimens

31. HIV Drug Resistance: Definitions • Genotype • Determines phenotype • Major and minor mutations for PIs • Phenotype • Drug susceptibility • Virtual phenotype • Result of large relational genotype and phenotype database

32. HIV Drug Resistance: Methodologies • Genotyping • Different platforms • Dideoxy sequencing • Gene chip • Point mutation assays • Phenotyping • Recombinant virus assays • Virtual phenotyping • Informatics

33. M Zidovudine 41 L L T M K Didanosine 74 184 65 69 V R V D L T K D K Zalcitabine 210 215 219 67 70 W YF QE N R K M L Y Stavudine 184 65 74 115 R V F V M 41 E E E V V V M D K L T K Abacavir L 118 118 118 44 44 44 184 67 70 210 215 219 D D D I I I VI N R W YF QE L K K 65 Tenofovir 74 65 R V R Lamivudine Mutations Associated with nRTIs/ntRTIs www.iasusa.org

34. V A F Q 62 77 F 75 Multi-nRTIResistance: 151 Complex 116 L I 151 V Y M D  K A M 62 41 67 69 70 Multi-nRTIResistance:69 InsertionComplex V L N R Insert D K M Multi-nRTIResistance:NAMs 41 67 70 L N R E V L L T T K K 118 44 210 210 215 215 219 219 D I W W YF YF QE QE Mutations Associated with nRTIs/ntRTIs www.iasusa.org

36. Nucleoside Analog Resistance

37. Pyrophosphorolysis Courtesy M. Parniak Mellors, 9th CROI, 2002

38. K 103 Y V Multi-NNRTIResistance N 106 188 L M Multi-NNRTIResistance:Accumulationof Mutations L V Y G M 100 106 181 190 230 I A CI SA L K V V V V Y Y G L Nevirapine 100 103 106 106 106 108 181 188 190 I N AM M M I CI CLH A Y K Y P Delavirdine 188 103 181 236 N C L L L K V Y Y G P Efavirenz 100 103 108 181 188 190 225 H I N I CI L SA Mutations Selected by NNRTIs www.iasusa.org

39. Multi-PI Resistance: Accumulationof Mutations 10 46 54 82 84 90 Indinavir V M M M I I A G V V V I I L L L L K L 10 20 24 32 36 46 54 71 73 77 82 84 90 FIRV IRV MR I I I IL IL VML V VT SA I AFTS AFT V V M M Ritonavir L K V L M M I A V V I L 10 20 32 33 36 46 54 71 77 82 84 90 FIRV MR I F I IL VL VT I AFTS V M Saquinavir L G I A G V V I L 10 48 54 71 73 77 82 84 90 IRV V VL VT S I A V M Nelfinavir L D M M A V V I N L 10 30 36 46 71 77 82 84 88 90 FI N I IL VT I AFTS V DS M Amprenavir L K L V L M I I F I L A G V I L L V M I I I I G I I L Lopinavir/Ritonavir 10 20 24 32 33 46 47 50 53 54 63 71 73 82 84 90 50 50 54 73 84 84 90 10 32 46 47 FIRV I IL V L V LVM S V V M MR I I F IL V V L VL P VT S V M M I L 71 88 46 54 90 Atazanavir 82 I L M Mutations Selected by PIs FIRV AFTS A N V V 32 V S A I www.iasusa.org

40. Enfuvirtide N I Q V N G HR1 Region 36 37 38 39 42 43 R AM T V DS D Mutations in the GP41 Envelope Gene Associated With Resistance to Entry Inhibitors

41. Progress in HIV Disease HIV Pathogenesis Monitoring Therapy