HIV Resistance Basics

1. HIV Resistance Basics Michael J. Harbour, MDClinical Assistant ProfessorStanford University School of MedicineStanford Positive Care Clinic

2. 19 Drugs – How to Pick the Best Regimen?

3. 2002-03 increase 180-280K 30-40k 34-54K 150-270K 43-67k 45-80K 610-1.1M 120-180k 3-3.4M 700-1K The AIDS pandemicAdults and children living with HIV/AIDS, end 2003 Eastern Europe & Central Asia 1,2-1.8M North America 790,000-1.2M Western Europe 520-680K East Asia & Pacific 700k-1.3M North Africa & Middle East 470-730K Caribbean 350-590,000 S & SE Asia 4.6-8.2M Latin America 1.3-1.9 M Sub-Saharan Africa 25-28.2 M Australia & New Zealand 12-18K • 5 Million new infections in 2003 • 3 Million deaths due to HIV/AIDS in 2003 • 40 Million living with HIV/AIDS; 50% Female Source: USAIDS

4. Leading Causes of Death 1987-2000:Persons 25-44 Years of Age Unintentional injury Cancer Heart disease Suicide HIV infection Homicide Chronic liver disease Stroke Diabetes Deaths/100,000 Population Year CDC: Preliminary Mortality data for 2000.

5. High-risk sex in HIV+ adults with known drug-resistant HIV • SCOPE (Study of the Consequences Of the Protease inhibitor Era) cohort in San Francisco • 168 patients on treatment but viremic and with genotypically proven drug-resistant HIV Chin-Hong PV, et al. 11thCROI, San Francisco 2004, #845

6. 4510 MSM participating in the VaxGen 004 Phase III study1 Seroconversion for MSM in the VaxGen trial • AIDSVAX had no influence on pretreatment HIV RNA set-point in those who seroconverted2 All p<0.0001 except *p<0.01 1. Ackers M, et al. 11thCROI, San Francisco 2004, #857; 2. Shepherd B, et al. ibid, #284

7. Mean change in HIV RNA and CD4+ after superinfection +1.6 log10 c/mL –132 cells/mm3 ΔCD4+ (cells/mm3) ΔRNA (log10 c/mL) p=0.05 vs controls without superinfection HIV superinfection • Superinfection recently described in the literature1−4 • 3 of 78 (4.1%) patients in the first 6 to 20 months of infection in San Diego and Los Angeles5 • 1 of 32 (3.1%) newly infected subjects from the MACS6 • CD4+ progressed to <200 cells/mm3 2.4 years postinfection • Implications: • Counseling of HIV-infected partners • Concern regarding vaccine strategies 1. Altfeld M, et al. Nature 2002;420:434; 2. Jost S, et al.NEJM 2002;347:731; 3. Koelsch K, et al.AIDS 2003;17:F11; 4. Ramos A, et al. J Virol 2002;76:7444; 5. Smith D, et al. 11th CROI, San Francisco 2004, #21; 6. Gottlieb G, et al. ibid, #454

8. Evolving high-risk groups Men on the Down Low (“DL”)1 • Heterosexually identified black men who have sex with men but do not tell their female partners • Don’t identify with gay subculture • Usually unaware or non-disclosing of their HIV status San Francisco4 • Rising unprotected anal sex in Asian MSM Screening and Tracing Active Transmission (“STAT”)2,3 • Cases of HIV among 18−30 year old men (n=998) in North Carolina attending college rose from 4% (late 2001) to 15% (2003) • More likely to be African-American, have acute/recent infection, have sex with men and women, use ecstasy, travel outside of NC • 73% of HIV+ felt they were at low risk for HIV acquisition 1. Millett G. 11thCROI, San Francisco 2004, #83; 2. Hightow LB, et al.ibid, #84; 3. Fitzpatrick L, et al.ibid, #85LB; 4. Troung HM, et al. ibid, #844

9. GENESEQ™ AND PHENOSENSE™ COMPLEMENTARY TECHNOLOGIES PhenoSense™ HIV GeneSeq™ Patient virus Patient virus RT-PCR RT-PCR PR-RT DNA PR-RT DNA Vector Assembly Sequencing Protein Sequence Resistance Test Vector Transfection Selection Resistance Mutations Recombinant Virus Interpretation Infection Measure of Drug Susceptibility Prediction of DrugSusceptibility ?

10. PhenoSense HIV • Allows determination of drug susceptibility against patient’s virus compared to wild-type virus • Measurement of single drug, not the combination • Can be performed on samples with viral loads  500 copies/mL • Results based on direct measurement --not inference from genotype (Virtual Phenotype) • Able to detect majority and (depending on percentage) some minority species

11. Genotype Test • High degree of accuracy and reproducibility • Results are specific to HIV RNA sequence • Detects many minority populations of drug-resistant virus • Can be performed on samples with viral loads  500 copies/mL • Software analyzes every position of the nucleotide sequence, therefore may detect mutations other tests may miss • Reports mixtures • Reports all mutations (including polymorphisms) • Reports subtype

12. WHEN DOES GENOTYPING HELP?

13. ADVANTAGES OF ACCESSING BOTH GENOTYPIC AND PHENOTYPIC RESISTANCE DATA Combining genotypic data with phenotypic data provides a comprehensive picture of the resistance profile of the patient’s virus • Allows clinician maximum efficiency in antiretroviralmanagement • Enhances ability to preserve future treatment options • Facilitates individualization of antiretroviral therapymanagement for optimal clinical outcomes • Phenotypic and genotypic results can be provided from a single patient sample

14. PHENOSENSE GTTM TEST REPORT • Combination phenotype/genotype test report form • Page 1 provides all info necessary for clinical interpretation • Page 2 provides more detailed assay data

15. PhenoSense GT Test Report G E F B A C D Fold-change bar graph Phenotypic cutoff indication Resistance mutations by drug class Side-by-side phenotype/genotype interpretations Net assessment of susceptibility (based on proprietary algorithm; helpful in cases of discordance) References for detailed comments found on Page 2 HIV-1 subtype info

16. Glossary of Phenotype Terminology • Fold change: The change in susceptibility above or below wild type reference strain • Cut point (Cut off): The fold change value above which drug susceptibility declines. There are different means of assessing a cut point • Hypersusceptibility: Increased drug susceptibility compared to wild type

17. Highest Clinical Relevance Moderate HIV drug resistance cutoffs • Clinical cutoffs: • based on outcome data from clinical trials involving patients • Biologic cutoffs: • based on natural variability of wild-type viruses from patients • Reproducibility cutoffs: • based on assay variability with repeated testing of patient samples

18. % Patient: Control: % Inhibition FC=1 IC50(patient) IC50(control) Fold Change = Nelfinavir PhenoSense Inhibition Curves Patient’s virus is sensitive to the drug

19. Patient: Control: High-Level Drug Resistance Patient’s virus is highly resistant to the drug % FC=200 % Inhibition Nelfinavir

20. Patient: Control: Hypersusceptibility

21. Structure and Function in Biology:What’s the practical difference between a genotype and a phenotype? Why may they not tell you the same thing?

22. Genotype Depicts Structural Changes HIV RNA Translation Linear sequence of amino acids Processing and Folding

23. Genotype Depicts Structural Changes HIV RNA Translation Linear sequence of amino acids Processing and Folding Genotype sees this… And needs an algorithm To predict this

24. HIV RNA RTV Phenotype Assesses Functional Aspects PhenoSense tests the ability of each drug to interfere with the FUNCTION of the viral enzymes that are the actual targets of the drugs.

25. Interpreting Resistance Test Reports

26. How We Identify a Mutation • How do we identify a resistance mutation? M 184 V “M”is the “wild type” amino acid “184”is the codon position “V”is the mutant amino acid

27. A = alanine C = cysteine D = Aspartate E = glutamate F = phenylalanine G = glycine H = histidine I = Isoleucine K = lysineL L = leucine M = methionine N = asparagine P = proline Q = glutamine R = arginine S = serine T = threonine V = valine W = tryptophan Y = tyrosine 20 Amino Acid Symbols

28. What Are TAMs? • Thymidine Analog (Resistance) Mutations • Previously known as ZDV resistance mutations • Selected by ZDV and/or d4T • 41L • 67N • 70R • 210W • 215Y/F • 219Q/E • Other ZDV-selected mutations include • 44D/A, 118I, 207D/E, 208Y

29. TAMs Confer Cross-resistance to NRTIs • ZDV resistance mutations now recognized as multinucleoside resistance mutations • Cross-resistance with d4T, ddI, ddC, 3TC • Presence of 2 TAMs + 184V significantly reduces potency of ABC • Presence of 3 TAMs including 41L + 210W significantly reduces activity of TDF

30. Gentotype and Phenotype Disconcordance

31. What do we mean by discordance? Discordance refers to disagreement between the results of the phenotypic measurement of susceptibility and the genotypic interpretation of susceptibility based on mutational patterns

32. Discordance Review • Observed differences between results from phenotypic/genotypic resistance testing is more common than generally thought • Discordance occurs because the interpretation of the results may be different and the tests “see” different aspects of the virus • Genotypic and phenotypic tests provide complimentary information that gives the most complete picture of resistance • In many cases, interpretation of these results can be facilitated by learning several patterns and rules

33. Types of Pheno/Geno Discordance • PT-Resistant, GT-Susceptible 25% • PT-S, GT-R, mixtures* absent 41% • PT-S, GT-R, mixtures* present 34% *Mixtures = patient sample has mixture of drug resistant and drug sensitive virus, usually observed during transitionbetween completely drug resistant and completely drug sensitive virus, such as shortly after interrupting therapy or during the brief period when drug resistant virus first emerges Parkin et al, JAIDS 2002; 31: 128-136

34. Major explanations of discordance • Incomplete genotypic algorithms (rules) • Novel mutations • Improper weighting of mutations in algorithms (both over- and under-weighted) • Non-B subtype resistance patterns • Immaturity of interpretive algorithms • Mixtures present • Suppressive mutations or “re-sensitization” caused by specific mutations (e.g. 184V)

35. Case Study (1):35 yr. old Gay Hispanic Male • HIV+ since 1996 • Presents for care November, 2000

36. Past Medical History • Treated with IDV/AZT/3TC for 2 mos • Stopped HAART due to side effects • Last CD4=230 HIV RNA=unknown • No Prior OI’s • Pancreatitis 1999 • No Prior Surgery • NKDA • No current Medications

37. Social History • Alcohol Abuse (6-12 beers daily) • No drug use • No tobacco • Living with male partner for 2 yrs who is also active alcoholic • Works as shipping clerk at the Gap

38. Review of Systems • 33 lb weight loss over 6 mos • Chills, sweats • Diarrhea • Vomiting on occasion • Allergic rhinitis • Visual changes • Depressive Symptoms

39. Wt=68.6 kg BP=122/84 T=98.3 Pulse=74 Resp=18 Nervous Appearing Smelled of ETOH No swollen nodes No thrush Chest Clear No HSM Skin clear Normal genitals Guiac Neg Physical Exam

40. Data • CBC Normal • CD4=169 HIV RNA=230,000 • ALT=90 AST=120 Amylase=96 • RPR Negative • Hep A non immune • Hep B immune • Hep C Negative • Normal UA • Testosterone=355 (400-1080) • CXR Normal

41. HIV Genotype • PR Mutations • L63P • RT Mutations • None

42. Initial Treatment • HIV therapy held until ETOH abuse was under better control • AA meetings encouraged, but pt did not connect • Short course of Antabuse • PCP Prophylaxis begun • Testosterone Replacement • Paxil 20 mg/d • Referred for counseling

43. Follow-up Care • Tcell count=200 HIV RNA=58,000 six weeks later • Patient able to substantially reduce ETOH intake • Started on Sustiva 600 qhs and Combivir bid on March 2, 2001

44. Patient Complaints • Called office complaining of nausea and vomiting • Feels as if he has a hangover in the morning • Headache • Just “can’t function” • Feels like “shit” • Nightmares • Partner complains of somniloquy

45. Should Sustiva Be Withheld in Certain Patients? • Patients with Depression or Anxiety? • Patients with Active Substance use? • Patients with Schitzophrenia or other severe mental illness?

46. How I Treated Patient • Telephone Reassurance • Follow-up Office Visit to check compliance • Ativan 0.5-1.0mg qhs • Compazine 10 mg q 8 hrs prn

47. Case Study 2 32 Year old White male Dx: HIV + 1990 Transferred Care: 6/1/00 on CBV BID only HIV PCR= 1,000 Tcell=138 (23%) PMH: No hx OI’s Depression (severe) HIV Risk Factor: “Prolific” Sex (mostly hetero, some homo) SH: Works for technology start up company

48. Case 2 Contd • HIV Med History • AZT monotherapy followed by AZT/ddC dual therapy • SQV/RTV dual therapy in late 1990’s • Transferred on Combivir and Septra • Genotype Performed 7/00 • PR Mutations: None • RT Mutations: D67N, T69N, K70R, M184V, T215, K219Q • What would you have done in 7/2000?

49. Case 2 Contd • 7/00 Meds Changed to: • Viramune 200 BID • ddI 400 QD • d4T 40 BID • LAB DATA: • HIV PCR = BDL • T cell = 398 (25%) • Regimen Maintained until 9/02 when • HIV PCR = 2900 • T cell = 387 (22%)

50. Case 2 Contd • Gentotype Done 7/02 • PR—None • RT—D67N, T69N, K103N, V106A, V118I, T215F, K219Q, • New Medication Started 9/02 • Viread 300 QD • Abacavir 300 BID • ddIEC 250 QD • Lab Data • HIV PCR = 1500 • T cell = 358 (20%)