Resistance Testing – Where Do I Start?

1. Resistance Testing – Where Do I Start? Iván Meléndez-Rivera, MD Fellow, American Academy of Family Physicians Assistant Professor of Family Medicine Ponce School of Medicine, Ponce PR, USA Board Member, American Academy of HIV Medicine Faculty, Florida/Caribbean AETC Medical Director, Centro Ararat, Inc Ponce PR, USA

2. Disclosures of Financial Relationships This speaker has significant financial relationships with the following commercial entities to disclose: • Advisory Board or Panel: Gilead, Merck • Consultant: Bristol-Myers Squibb • Grants/Research Support: Abbott, Boehringer, GlaxoSmithKline, Napo, Pfizer • Speakers Bureau: Abbott, Boehringer, Bristol-Myers Squibb, GlaxoSmithKline, Merck, Pfizer, Tibotec This speaker will not discuss any off-label use or investigational product during the program. This slide set has been peer-reviewed to ensure that there are no conflicts of interest represented in the presentation.

3. Objectives • Introduce major resistance mutations for each class of HIV therapy • Order currently available resistance tests • Present illustrative case(s) of diagnosing resistance pattern resulting in treatment plan adjustment

4. Definition of Antiviral Drug’s Resistance • Presence of viral mutations that reduce drug susceptibility compared with the susceptibility of wild-type viruses. • Can directly or indirectly interfere with a drug's activity. • Should be distinguished from other causes of drug failure such as: • Non-adherence • Insufficient drug levels • Drug regimens with intrinsically weak antiviral activity.

5. Why does Resistance Develop? • HIV can develop resistance quickly because: • HIV reverse transcriptase makes errors in matching bases when converting HIV RNA to DNA • HIV replicates at a rapid rate: 1 to 10 billion viral particles produced daily • In an untreated infected host, every possible mutation occurs at least once every 24 hours, some of which may impart drug resistance.

6. If you have developed resistance to a drug, does that mean that you are resistant to ALL the drugs in the same class? • True • False

7. When does resistance occur? • Resistance occurs when the virus has an opportunity to replicate in the presence of sub-inhibitory concentrations of drugs. • Treatment with <3 active drugs • Inappropriate selection of drugs • Pharmacokinetics or drug-drug interactions lead to inadequate drug exposures • Non-adherence to the treatment regimen • Interruption of treatment (even for a few days) • Adding one drug to a failing regimen • Prolonging a failing regimen

8. Selective Pressures of Therapy Drug-susceptible quasispecies Drug-resistant quasispecies Treatment begins Selection of resistant quasispecies Viral load • Incomplete suppression • Inadequate potency • Inadequate drug levels • Inadequate adherence • Pre-existing resistance Time

9. What is viral resistance? • A continuum of reduced susceptibility of HIV to the inhibitory effects of drugs MoreSusceptible More Resistant The terms "drug resistance" and "reduced drug susceptibility" have similar meanings, provided that each term is viewed as representing a continuum between susceptible and highly resistant. Because antiretroviral drugs differ in their potencies, reductions in susceptibility must be related to the activity of the drug against wild-type viruses.

10. Why is it important to measure drug resistance? Wild Virus vs Mutant virus

11. What is wild-type virus? • HIV virus that has not been exposed to drug therapy • The predominant sequence of nucleotide bases in a heterogeneous mixture of virions • It is the most fit form of HIV in the absence of drug

12. Identifying Mutations • How do we identify a resistance mutation? M 184 M “184 is the codon position” M is the “wild-type” amino acid (AA)

13. Identifying Mutations • How do we identify a resistance mutation? M 184 V “184 is the codon position” V is the mutant AA M is the “wild-type” amino acid

14. What is the Impact of HIV Mutations? • To the virus, mutations can be: • Changing the structure of the virus to evade antiretroviral treatment. These mutations may or may not affect viral fitness No effect on virus function Decreasing the virus’ ability to survive and/or replicate (viral fitness) or may make the virus hypersusceptible to certain antiretrovirals (ARVs) Johnson VA, et al. Topics in HIV Medicine. 2009;17:138-145.

15. Single-base Mutations May Confer Antiretroviral Resistance 3 nucleotides specify an amino acid HIV RNA mutant HIV RNA wild type Single-base mutation Drug resistant 123 X Y Wild-type AA: X Position AA: 123 Mutant AA: Y AA=amino acid Hoffman C et al. HIV Medicine 2007. 15th ed. Paris, France: Flying Publisher. 2007.

16. Resistance Profile and Potential for Cross Resistance Region of HIV RNA associated with antiretroviral drug class specific resistance HIV RNA Mutant Drug A Drug B B cross resistance Drug C A & B & C cross resistance Drug D Adapted from: ParedesR, et al. Antiviral Res. 2010 Jan;85(1):245-65.

17. How is resistance measured? By Genotype • Amino acid substitution on chain By phenotype (IC50) • IC50 • The minimum concentration of a drug needed to inhibit the growth of the virus by 50% in vitro • IC50 is analogous to MIC90 • IC90 is not sufficiently reproducible for routine clinical use • The lower the IC50, the more potent the drug

18. Resistance mutations per HIV class therapy

19. Available Antiretroviral Agents • Protease Inhibitors • Saquinavir (SQV) • Ritonavir (RTV) • Indinavir (IDV) • Nelfinavir (NFV) • Amprenavir (APV) • Lopinavir/r (LPV/r) • Atazanavir (ATV) • Fosamprenavir (Fos-APV) • Tipranavir (TPV) • Darunavir (DRV) • Nonnucleos(t)ide RTIs • Nevirapine (NVP) • Delavirdine (DLV) • Efavirenz (EFV) • Etravirine (ETR) • Rilpivirine (RPV) • Nucleoside Reverse • Transcriptase • Inhibitors (RTIs) • Zidovudine (ZDV) • Didanosine (ddI) • Zalcitabine (ddC) • Stavudine (d4T) • Lamivudine (3TC) • Abacavir (ABC) • Emtricitabine (FTC) • Tenofovir DF (TDF) • Integrase Inhibitors • Raltegravir (RAL) • Elvitegravir(ELV)* • Dolutegravir (DTG)* • Boosters • Ritonavir (RTV) • Cobicistat(cobi) • Fusion Inhibitor • Enfuvirtide (T-20) • CCR5 Antagonist • Maraviroc (MVC) * In expanded access August 28, 2012

20. Nucleoside Reverse Transcriptase Inhibitors (NRTI’s) • Zidovudine (ZDV) • Didanosine(ddI) • Stavudine (d4T) • Lamivudine (3TC) • Abacavir (ABC) • Tenofovir (TDF) • Emtricitabine (FTC) Structure of a covalently trapped catalytic complex of HIV-1 RT published by Huang H et al, Science 1998. http://hivdb.stanford.edu/pages/3DStructures/rt.html Accessed Sept 3, 2012

21. NRTI’s Resistance Mutations Red: Primary Mutations to confer resistanceGray: Secondary Mutations ***: Increase susceptibility Adapted from: http://hivdb.stanford.edu/DR/NRTIResiNote.html. Accessed Sept 3, 2012

22. NRTI’s Signature Mutations

23. Not ALL mutations are bad • M184V/I cause high-level resistance to 3TC and FTC and low-level resistance to ddI and ABC. • M184V/I are not contraindications to continued treatment with 3TC or FTC because they increase susceptibility to AZT, TDF, and d4T and are associated with clinically significant decreased HIV-1 replication. http://hivdb.stanford.edu/DR/cgi-bin/rules_comments_hivdb.cgi?class=NRTI

24. Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTI’s) • Nevirapine (NVP) • Efavirenz (EFV) • Etravirine (ETR) • Rilpivirine (RPV) Non-nucleoside RT inhibitor (NNRTI)-resistance mutations http://hivdb.stanford.edu/pages/3DStructures/rt.html Accessed Sept 3, 2012

25. NRTI’s Resistance Mutations Red: Highest Levels of resistance Gray: Intermediate level of resistance Adapted from: http://hivdb.stanford.edu/DR/NRTIResiNote.html. Accessed Sept 3, 2012

26. NNRTI’s SignatureMutations

27. Protease Inhibitors (PI’s) Saquinavir (SQV) Ritonavir (RTV) Indinavir (IDV) Nelfinavir (NFV) Lopinavir/r (LPV/r) Atazanavir (ATV) Fosamprenavir (FPV) Tipranavir (TPV) Darunavir (DRV) Major protease inhibitor (PI)-resistance mutations http://hivdb.stanford.edu/pages/3DStructures/rt.html, Accessed Sept 3, 2012

28. PI’s Resistance Mutations Red: Highest Levels of resistance Gray: Intermediate level of resistance Adapted from: http://hivdb.stanford.edu/DR/NRTIResiNote.html. Accessed Sept 3, 2012

29. PI’s Signature Mutations

30. Integrase Inhibitors (INI) Raltegravir (RAL) Elvitegravir(ELV) Dolutegravir(DTG)* * Investigational http://www.scientistlive.com/European-Science-News/Pharmacology/HIV_integrase_inhibitor_effective_when_beginning_treatment/23101/ Accessed 06/OCT/12

31. INI Resistance Mutations Red: Highest Levels of resistance Gray: Intermediate level of resistance Adapted from: http://hivdb.stanford.edu/DR/NRTIResiNote.html. Accessed Sept 3, 2012

32. INI Signature Mutations * In expanded access

33. Fusion Inhibitors (FI) Enfuvirtide (T-20) www.fuzeon.com Accessed Sept 3, 2012

34. Fusion Inhibitor Resistance Mutations Gray: Intermediate level of resistance Adapted from: http://hivdb.stanford.edu/DR/NRTIResiNote.html. Accessed Sept 3, 2012

35. CCR5 Antagonist Maraviroc (MVC) Moore J, et al. ProcNatlAcadSci USA. 2003;100:10598-10602; Yost R, et al. Am J Health-Syst Pharm. 2009;66:715-726 Accessed Sept 3, 2012

36. CCR5 Antagonist Resistance is Associated withAmino Acid Changes in the V3 Loop of gp120* • Variable pattern of Maraviroc resistance-associated amino acid substitutions • No signature mutations have been identified • Currently, there is no assay available to assess Maraviroc resistance G/A Tip Tip Stem Stem Base Base • *Substitutions outside the V3 loop of gp120 may also contribute to reduced susceptibility to Maraviroc Change Maraviroc-resistant Insertion Deletion Adapted from Data on file. ViiV Healthcare, RTP, NC.

37. MVCres Virus Mutated gp120 recognizes CCR5 differently MVCres gp120 Binding siteNOT disruptedby maraviroc Entry CCR5-Resistant Virus Recognizes Drug-Bound Receptors Mori J, et al. 16thIHIVDRW. Barbados, 2007. Abstract 10.

38. Recommendations for drug resistance testing

39. In which one of the following situations would HIV resistance testing NOT usually be recommended? • Acute HIV infection, regardless of whether treatment is to be started • Chronic HIV infection, at entry into care • After discontinuation (>4 weeks) of ARVs • Suboptimal suppression of viral load after starting antiretroviral therapy (ART)

40. Trends of Phenotypic 1-, 2-, and 3-Class Resistance 1-Class Resistance 2-Class Resistance 3-Class Resistance 60 60 60 PI PI and NRTI NNRTI PI and NNRTI NRTI NRTI and NNRTI 50 50 50 40 40 40 30 30 30 Resistant Samples (%) 20 20 20 10 10 10 0 0 0 2003 2004 2005 2006 2007 2008 2009 2010 2003 2004 2005 2006 2007 2008 2009 2010 2003 2004 2005 2006 2007 2008 2009 2010 NNRTI=non-nucleoside reverse transcriptase inhibitor; NRTI=nucleoside reverse transcriptase inhibitor; PI=protease inhibitor. Adapted from Paquet A et al. 51st Interscience Conference on Antimicrobial Agents and Chemotherapy; September 17-20, 2011; Chicago, Illinois. Abstract H2-800.

41. Testing for Drug Resistance • Before initiation of ART: • Transmitted resistance in 6-16% of HIV-infected patients • In absence of therapy, resistance mutations may decline over time and become undetectable by current assays, but may persist and cause treatment failure when ART is started • Identification of resistance mutations may optimize treatment outcomes • Resistance testing (genotype) recommended for all at entry to care • Recommended for all pregnant women • Patients with virologic failure: • Perform while patient is taking ART, or ≤4 weeks afterdiscontinuing therapy • Interpret in combination with history of ARV exposureand ARV adherence • Coffey S. Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents: Initiation of Therapy [PowerPoint]. AIDS Education and Training Centers, National Resource Center; March 2012. Available at http://aidsetc.org/aidsetc?page=etres-display&resource=etres-6. Accessed Sept 03, 2012.

42. Drug Resistance Testing: Recommendations • Coffey S. Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents: Initiation of Therapy [PowerPoint]. AIDS Education and Training Centers, National Resource Center; March 2012. Available at http://aidsetc.org/aidsetc?page=etres-display&resource=etres-6. Accessed Sept 03, 2012.

43. Drug Resistance Testing: Recommendations(2) • Coffey S. Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents: Initiation of Therapy [PowerPoint]. AIDS Education and Training Centers, National Resource Center; March 2012. Available at http://aidsetc.org/aidsetc?page=etres-display&resource=etres-6. Accessed Sept 03, 2012.

44. Drug Resistance Testing: Recommendations(3) • Coffey S. Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents: Initiation of Therapy [PowerPoint]. AIDS Education and Training Centers, National Resource Center; March 2012. Available at http://aidsetc.org/aidsetc?page=etres-display&resource=etres-6. Accessed Sept 03, 2012.

45. Drug Resistance Testing: Recommendations(4) • Coffey S. Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents: Initiation of Therapy [PowerPoint]. AIDS Education and Training Centers, National Resource Center; March 2012. Available at http://aidsetc.org/aidsetc?page=etres-display&resource=etres-6. Accessed Sept 03, 2012.

46. Available Resistance test http://www.tumblr.com/tagged/genotype Accessed 06/OCT/12

47. HIV RNA Translation into linear string of amino acids Folding into functional protein Genotypic assays test this Phenotypic assays test this Genotypic and Phenotypic Resistance Tests

48. Available Tests • NRTI, NNRTI AND PI • GENOTYPE • Virtual Phenotype • PHENOTYPE • ENTRY INHIBITORS • PHENOTYPE • INTEGRASE INHIBITORS • GENOTYPE • PHENOTYPE • Co-Receptor Tropism Assay • RNA • DNA

49. General Limitations of Resistance Testing • Lack of uniform quality controls across different laboratories • High cost compared with other tests routinely done in HIV care • Cannot be reliably performed with HIV RNA < 500-1000 copies/mL • May not be able to detect minority populations of resistant virus if they account for < 20% of the sample—especially common after drug discontinuation • Resistant strains that are in viral reservoirs also not detected

50. Reversion to Predominant Wild-Type Virus After Discontinuing ART 0 Behrens C, Kindrick A, Harrington R. Antiretroviral Resistance Testing in the Management of HIV-Infected Patients [PowerPoint]. Northwest AIDS Education and Training Center; July 2006. Available at http://aidsetc.org/aidsetc?page=etres-display&resource=etres-9. Accessed SEP 3, 2012. Illustration by David Spach, MD