1. HIV-1 Resistance - Implications For Clinicians Joseph J. Eron Jr., MD Professor of Medicine University of North Carolina

2. Overview of Presentation HIV-1 resistance to ARV; a consequence of viral replication and HIV biology Suppression of HIV replication limits resistance Clinical indications for resistance testing –Genotype vs. phenotype Clinical Importance of transmitted resistance Multiple causes of virologic failure of ART Resistance testing is one key to successful therapy in the treatment experienced patient Novel agents – novel resistance

3. Resistance Is a Matter of Degree High-level resistanceLittle or no virologic response Low-level / intermediate resistance Suboptimal virologic response SusceptibleNo reduced drug susceptibility

4. Suppression of Replication Limits Resistance Incidence of Resistance Decreasing with use of HAART

5. Types of Resistance Tests Genotype and Phenotype

6. Types of Resistance Tests Genotype –HIV gene sequencing of the patient’s virus to detect mutations known to confer drug resistance Phenotype –Measures ability of a recombinant virus derived from the patient sample to grow in different concentrations of antiretroviral drugs Hirsch et al. Clin Infect Dis. 2003;37:113-28.

7. Genotypic and Phenotypic Assays GenotypePhenotype Patient’s virus PR-RT-DNA Resistance test vector Recombinant virus Patient’s virus PR-RT-DNA Protein sequence Resistance mutations RT-PCR sequence selection interpretation RT-PCR vector transfection infection Measure of susceptibilityPrediction of susceptibility

8. Interpretation of Genotypic Assays Is an indirect measure of resistance –Requires knowledge of which mutation are associated with a change in susceptibility Expert advice –May not be available –Experts’ views may be inconsistent Rules-based algorithms –Provided by most labs, third-party sites –Need regular updating Virtual Phenotype (VircoType) –Phenotypic information using genotype –Database of matched genotypes and phenotypes

9. Provider Knowledge of Specific Resistance Mutations Mutations % Providers Recognizing Specific Mutations Salama et al. Clin Infect Dis. 2003;36:101-4. N = 100

10. Phenotypic Susceptibility Relationship Between Drug Concentration and Viral Inhibition Inhibition of Virus Replication (%) 50 0 100 Fold resistance Wild-type IC 50 Resistant IC 50 Wild-type Resistant Drug Concentration

11. Interpretation of Phenotypic Assays Results reported as IC50 or fold-change (FC) compared with IC50 of wild-type virus Individual results provided for each drug Thresholds to define reduced susceptibility –Biologic cutoff: based on biologic variations in treatment- naive patients (usually 2 SD > median) –Clinical cutoff: As good as clinical data used to estimate cut points Resistance is a continuum – precise breakpoints unlikely Two relevant breaks? –Decreased response and –Minimal response

12. Clinical Indications for Resistance Testing

13. Current Guidelines for Resistance Testing 1. DHHS. Guidelines. Oct 10, 2006. 2. Hirsch et al. Clin Infect Dis. 2003;37:113-28. 3. Vandamme et al. Antivir Ther. 2004;9:829-48. DHHS [1] IAS-USA [2] EuroGuidelines [3] Primary Infection Recommend PEP (Source Pt)——Recommend ChronicRecommend Recommend/ Consider Treatment Failure Recommend Pregnancy— Recommend * Pediatric—— Recommend ** * Only if mother is viremic ** Only if mother was viremic and on treatment at time of birth

14. Transmitted ARV Resistance Clinically Important in the Developed World Surveillance Essential in the Developing World

15. How Common is Drug Resistance at Diagnosis? US Variant, Atypical and Resistant HIV Surveillance System (VARHS) –Estimate prevalence of transmitted resistant mutations –Determine distribution of HIV subtypes March 2003 to October 2006; 11 states, 409 sites, n=3130 10% 5% AnyNRTINNRTIPI MDR M41L 45.1% of NRTI K103N 70.1% of NNRTI L90M 40.0% of PI 95% Subtype B Wheeler et al. 14 th CROI. Los Angeles, 2007. Abs 648 6.9% 3.6% 2.4% 1.9%

16. FTC-301A: Impact of Baseline Resistance on Treatment Outcome Borroto-Esoda et al. 11th CROI, 2004. Abs 672. Mutation type: Naive pts, baseline VL > 5000 copies/mL FTC + ddI + EFV (n=270) d4T + ddI + EFV (n=276) Incidence (%) of Virologic Failure

17. How Important Are Minority Variants?

18. Standard Genotype 181C 103N 190A 108I

19. Antiretroviral Response Following sdNVP Lockman et al. NEJM 2007;356:135-47.

20. Current Guidelines for Resistance Testing 1. DHHS. Guidelines. Oct 10, 2006. 2. Hirsch et al. Clin Infect Dis. 2003;37:113-28. 3. Vandamme et al. Antivir Ther. 2004;9:829-48. DHHS [1] IAS-USA [2] EuroGuidelines [3] Primary InfectionRecommend PEP (Source Pt)——Recommend Chronic (< 2 years)Recommend Recommend/ Consider Treatment Failure Recommend Pregnancy— Recommend * Pediatric—— Recommend ** * Only if mother is viremic ** Only if mother was viremic and on treatment at time of birth

21. Resistance and Virologic Failure Multiple Causes of Virologic Failure Replication in the Presence of Antiretrovirals Results in Resistance

22. Insufficient drug level Resistant virus Social/personal issues Regimen issues Toxicities Poor potency Wrong dose Host genetics Poor absorption Rapid clearance Poor activation Drug interactions Poor adherence Viral replication in the presence of drug Resistant virus Treatment-Experienced Patients: ARV Treatment Failure ART resistance testing. National resource center. Available at: http://www.aidsetc.org/aidsetc?page=et-01-00. Accessed November 29, 2006. Pre-existing resistance

23. Partially effective regimen Highly effective regimen Adherence Resistance Impact of Suboptimal Adherence Bangsberg et al. AIDS 2003;17:1925-32.

24. Resistance Testing in Treatment Experienced Patients Detectable plasma HIV RNA on therapy First Question ‘Is it time to switch therapy?’ –Obtain resistance testing The greater the resistance the more useful phenotype or virtual phenotype –Assess clinical situation Adherence Previous treatment history – ARV tolerability and toxicity Balance clinical urgency with availability of active agents including new drugs and expanded access New agents – especially those in a new class are likely to have the most activity

25. With New Agents in Existing Classes (PI and NNRTI) Resistance Patterns Are More Complex Darunavir (DRV) Tipranavir (TPV) Etravirine (ETV)

26. New Agents in Existing Classes Mutations to older agents are likely to be present –Majority variants –Minority variants Some degree of cross resistance can be anticipated –Cross resistance increases with the number and type of mutations The activity of a new agent from a new class is likely to be more predictable even with state of the art resistance testing

27. TORO Studies - Enfuvirtide Miralles et al. AIDS 2005;19:2178-9. Lalezari et al. NEJM 2003;348:2185-85. Lazzarin et al. NEJM 2003;348:2186-95. DC+Switch=Failure *p<0.05 * * * ** ENF+OB: 98 179 152 83 27 OB:49 88 84 46 16

28. TORO Studies - Enfuvirtide Miralles et al. AIDS 2005;19:2178-9. Lalezari et al. NEJM 2003;348:2185-85. Lazzarin et al. NEJM 2003;348:2186-95. DC+Switch=Failure *p<0.05 * * * ** ENF+OB: 98 179 152 83 27 OB:49 88 84 46 16

29. TORO Studies - Enfuvirtide Miralles et al. AIDS 2005;19:2178-9. Lalezari et al. NEJM 2003;348:2185-85. Lazzarin et al. NEJM 2003;348:2186-95. DC+Switch=Failure *p<0.05 * * * ** ENF+OB: 98 179 152 83 27 OB:49 88 84 46 16

30. Resistance Defines an Active Antiretroviral Agent CCR5 Inhibitors Integrase Inhibitor

31. CCR5 Inhibitors HIV-1 entry into CD4 cells is dependent on a second receptor: CCR5 (R5 viruses) or CXCR4 (X4 viruses) Early in HIV disease course most individuals have only R5 virus detectable In more advanced disease and highly treatment experienced patients dual tropic virus or mixtures of R5 and X4 viruses are more common CCR5 Inhibitors bind to host receptor (CCR5) blocking entry –Maraviroc and vicriviroc –Both have shown substantial activity in patients

32. Association Between Emergence of SI Virus and CD4+ Cell Count NSI virus predominates early in disease Dual/mixed virus detected in approximately 50% of patients over time CD4+ cell count decline accelerates following detection of SI in patients in whom NSI- only virus was previously detected Will emergence of X4 variants on R5 inhibitor therapy lead to CD4 cell decline? -36 Mean (SE) CD4+ Cell Count (cells/mm 3 ) Koot et al. Ann Intern Med. 1993;118:681-688. NSI NSI → SI 800 600 400 200 -12-240122436 Time (Months) 0 -48 SI

33. HIV-1 Resistance to CCR5 Antagonists Two mechanisms –Selection for (emergence of) viral variants that use CXCR4 (dual tropic viruses or mixed populations) Occurs about 2/3 of the time As yet NOT associated with rapid fall in CD4 cell count –Alterations (mutations) in HIV gp 120 No Change in viral tropism Allows virus to use CCR5 with inhibitor bound Plateau in antiviral effect as opposed to change in IC50 –The drug is unable to fully inhibit virus regardless of concentrations achieved

34. Integrase Inhibitors Integrase mechanism has 3 steps –Association with dsHIV DNA – pre-integration complex –3’ processing by integrase enzyme –Strand transfer of HIV DNA into host chromosome Potent inhibitors of stand transfer now in development –Raltegravir – Phase III studies recently presented –Elvitegravir – Phase II studies

35. Integrase Inhibitor Resistance Primary mutations surround the catalytic site Raltegravir –Resistance emerged in 3/4 patients with virologic failure who had genotype results –Two major pathways with apparent primary mutations but one or more additional mutations Likely compensatory for fitness –Cross resistance between integrase inhibitors is not yet known

36. Summary Antiretroviral resistance is a consequence of HIV replication and defines an active antiretroviral Resistance Impacts all stages of antiretroviral treatment typically with increasing complexity with increasing antiretroviral treatment experience In treatment experienced patients resistance information guides regimen selection but also influences the timing of therapy switch New agents with new mechanisms present challenges for the identification, quantification and clinical impact of resistance