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HIV Diagnosis and Pathogenesis Scott M. Hammer, M.D. 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 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 posttest counselling 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) HIV-1 Virion HIV Life Cycle Tat = transcriptional activator Rev = regulator of mRNA nuclear export HIV-1: Genetic Organization 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 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 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 Role of CTL’s in Control of Viremia Letvin N & Walker B: Nature Med 2003;9:861-866 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 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 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 The Variable Course of HIV-1 Infection Typical Progressor AIDS Viral Replication B months years Nonprogressor months years Clinical Latency CD4 Level C Viral Replication Primary HIV Infection AIDS CD4 Level CD4 Level A Primary HIV Infection Viral Replication Primary HIV Infection Clinical Latency Rapid Progressor ? 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. Change in HIV RNA (log10) Phases of Decay Under the Influence of Potent Antiretroviral Therapy 0 T1/2 = 1 d (productively infected CD4’s) -1 T1/2 = 2-4 wks (macrophages, latently infected CD4’s, release of trapped virions) -2 2-4 16-24 Time (weeks) T1/2 = 6-44 mos (resting, memory CD4’s) 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 Change in HIV RNA (log10) Phases of Decay Under the Influence of Potent Antiretroviral Therapy 0 T1/2 = 1 d (productively infected CD4’s) -1 T1/2 = 2-4 wks (macrophages, latently infected CD4’s, release of trapped virions) -2 2-4 16-24 Time (weeks) T1/2 = 6-44 mos (resting, memory CD4’s) Model of Post-Integration Latency Resting naïve CD4+ T cell +Ag Activated CD4+ T cell Preintegration Latency -Ag -Ag Resting memory CD4+ T cell +Ag Activated CD4+ T cell Siliciano R et al +Ag +Ag Postintegration Latency 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 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 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;286:25602567 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 Prognosis According to CD4 and RNA: ART Cohort Collaboration Egger M et al: Lancet 2002;360:119-129 Natural History of Untreated HIV-1 Infection 1000 800 + CD4 Cells 600 Early Opportunistic Infections Late Opportunistic Infections 400 200 0 1 Infection 2 3 4 5 6 7 8 9 Time in Years 10 11 12 13 14 MACS: CD4 Cell Decline by HIV RNA Stratum Mean D ecrease in CD 4+ Count per Year, cells/mm 3 0 -10 -20 -30.4 -30 -36.3 -40 -42.3 -39.1 -44.8 -50.7 -50 -50.5 -55.2 -60 -59.8 -59.6 -64.8 -70 -70.0 -80 -90 -70.5 -76.5 -82.9 (n=118) <500 (n=250) (n=386) (n=383) (n=394) 501-3000 3001-10 000 10 001-30 000 >30 000 Plasma HIV-1 RN A Concentration, copies/mL Mellors et al: Ann Intern Med 1997;126:946-954 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 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 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 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 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 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 HIV Drug Resistance: Methodologies • Genotyping - Different platforms » Dideoxy sequencing » Gene chip » Point mutation assays • Phenotyping - Recombinant virus assays • Virtual phenotyping - Informatics Mutations Associated with nRTIs/ntRTIs M Zidovudine E 41 44 L D Stavudine M E 41 44 L D Didanosine Abacavir Lamivudine 70 R D K 67 N 70 R 74 R V T L 69 D 74 V L K L Y M 65 R 74 V 115 F 184 V 65 R K T K 210 215 219 W YF QE M 184 V V 118 I T 210 215 219 W YF QE V 118 I 65 K www.iasusa.org 67 N L V 118 I L E 44 D Tenofovir K K K 65 R Zalcitabine D M 184 VI Mutations Associated with nRTIs/ntRTIs Multi-nRTI Resistance: 151 Complex Multi-nRTI Resistance: 69 Insertion Complex A V F 62 75 77 I L V Y Q 151 M L K M 41 A 62 67 69 70 L V N Inser R T K 210 215 219 W YF QE t M Multi-nRTI Resistance: NAMs D F 116 E 41 44 L D www.iasusa.org D K 67 N 70 R V 118 I L T K 210 215 219 W YF QE Nucleoside Analog Resistance TAM’s (M41L, D67N, K70R, L210W, T215F/Y, K219Q/E/N) M184V K65R Confer ZDV resistance thru ZDV-MP excision Confers 3TC resistance thru decreased 3TC-TP incorporation Confers non-ZDV NRTI resistance thru decreased analog incorporation Antagonize K65R Decreases ZDV resistance thru decreased ZDV-MP excision Decreases ZDV resistance thru decreased ZDV-MP excision Pyrophosphorolysis primer3'-terminal AZTMP regeneration of primer free 3'-OH 3' 5' O -O O P O -O P O O O O Mg2+ O- [R] O P O P A O U A U 3' HO O OO 3' 5' OH -O P O O O O T + A A 3' N N+ O -N [R] 5' O P O- O O P O- O O P OH O 5' O where R = AMP (ATP-dependent phosphorolysis) N or R=H +N -N (pyrophosphorolysis) Courtesy M. Parniak Mellors, 9th CROI, 2002 T Mutations Selected by NNRTIs Multi-NNRTI Resistance Multi-NNRTI Resistance: Accumulation of Mutations Nevirapine N M L V Y G M 100 106 181 190 230 I A CI SA L L K V V 100 103 106 108 I N AM I Y 181 CI Y G 188 190 CLH A K V 103106 Y Y P 181 188 236 N M C L L L K V V 100 103 106 108 Y Y I www.iasusa.org Y 188 L Delavirdine Efavirenz K V 103106 N M I 181 CI G 188 190 L SA P 225 H Mutations Selected by PIs Multi-PI Resistance: Accumulation of Mutations Indinavir M 46 54 FIRV IL VML L 10 K 20 IRV MR Ritonavir L 24 V M M I 32 36 46 I I I IL I Nelfinavir Amprenavir Lopinavir/ Ritonavir V I L 82 84 A G V 54 71 73 77 V VT SA 90 AFT S V M V I L 82 84 90 I AFT V M A V V I L 82 84 L K V L M M 10 20 32 33 36 46 54 71 77 I F I IL VL VT I I A G FIRV MR Saquinavir I L 10 AFT S V V 90 V M I L L G 10 48 54 71 73 77 82 84 90 IRV V VL VT S I A V M A V V I N L L D M M 10 30 36 46 71 77 82 84 88 90 FI N I IL VT I AFT S V DS M L V M I I 10 32 46 47 FIRV I IL V M I L K L 10 20 24 FIRV MR I V I V Atazanavir www.iasusa.org L 32 33 F 46 47 IL 32 M 46 I I V 50 I 54 G 73 84 L 90 V LVM S V M I F I L 50 53 54 63 V L VL P A G I V L 82 84 90 VT AFT V S M S A V 50 I 54 71 L L V I I 71 73 N 82 I 84 88 L 90 A V S M Mutations in the GP41 Envelope Gene Associated With Resistance to Entry Inhibitors Enfuvirtide Q N N 36 37 38 39 42 43 D S T G I V V A M R D HR1 Region Progress in HIV Disease HIV Pathogenesis Monitoring Therapy