1. 16 th Conference on Retroviruses and Opportunistic Infections February 8-11, 2009 Palais des Congrès de Montréal Montréal, Canada Javier Martinez-Picado

2. HIV Drug Resistance Drug Resistance 61 abstracts (6,1%) 1003 Abstracts

3. HIV Drug Resistance Oral Sessions: 18. Mechanisms of Drug Resistance and Optimal Timing of ART (9) 32. HIV Drug Resistance and Treatment Response (4) Poster Sessions: 114. Evolution of RT and PI Resistance in Patients (9) 115. Drug Resistance and Viral Fitness (4) 116. Outcomes of HIV Drug Resistance (11) 117. Circulating Drug Resistance in HIV-infected Populations (6) 118. Transmission of Drug-resistant HIV (7) 119. Use of Novel Technologies to Understand HIV Drug Resistance (7) 120. Resistance Assays and Their Imterpretation (4)

4. Mechanisms of Drug Resistance

5. N348I in HIV-1 RT counteracts the antagonism between TAMs and Y181C (Abstract 68) Mechanisms of Resistance to RT inhibitors AZT + NVP  antagonistic interaction between TAMs and Y181C  Treatment efficacy  AZT + NVP  These patients were 2.6 times more likely to acquire N348I in the connection domain of HIV-1 RT  N348I is highly associated with TAMs and Y181C polconnectionRNase H N348I 1 250 1 318-9426-7560 Current genotyping N348I Nikolenko, PNAS 2007; Yap, PLoS Med. 2007; Delviks-Frankenberry, PNAS 2008; Ehteshami, J Biol Chem. 2008; Hachiya, J Virol. 2008

6. Mechanisms of Resistance to RT inhibitors  Biochemical and cell-based assays demonstrate that N348I restores AZT-resistance phenotype N348I (AZT/NVP) Y181C (NVP) AZT resentization TAM (AZT) AZT-resistance N348I in HIV-1 RT counteracts the antagonism between TAMs and Y181C (Abstract 68)

7. Evolution of RT and PI Resistance in Patients

8. Polymorphism A376S in the Connection Domain (Abst. 646) polconnectionRNase H N348I 1 250 1 318-9426-7560 Current genotyping A376S  Present in approx. 5% of the naïve patients  Increases up to 15% in ARV-experienced patients  Associated with NVP failure  No association with other Drug Resistance Mutations

10. Use of Novel Technologies to Understand HIV Drug Resistance variants

11. Use of Novel Technologies Ultra-deep Sequencing (Abst. 124, 125, 679, 680, 682, 683) System throughput/8h = 1Gb Total clonal reads > 2,000,000  Read length = 400 bp  Cost = $10,000

12. Mix DNA Library & capture beads (limited dilution) “Break micro-reactors” Isolate DNA containing beads Generation of millions of clonally amplified sequencing templates on each bead No cloning and colony picking Create “Water-in-oil” emulsion + PCR Reagents + Emulsion Oil Perform emulsion PCR Adapter carrying library DNA A B Micro-reactors 1. Emulsion Based Clonal Amplification

13. Centrifuge Step Load Enzyme Beads 44 μm Load beads into PicoTiter™Plate 2. Depositing DNA Beads into the PicoTiter™Plate 1.6 million wells

14. 3. Sequencing by Synthesis (PyroSequencing)

15. 4. Biocomputational analysis

16. Use of Novel Technologies Ultra-deep Sequencing  Quantification of tropism (Abst. 124, 679, 680)  Detecting minority virus: Broad distribution of prevalence of X4 variants in clinical isolates that are not detected by standard sequencing techniques  Implications for ART: Initially minority variants can increase in frequency in a matter of days due to drug pressure A low prevalence of X4 was associated with improved virological response to Maraviroc, even when standard Trofile indicated DM or X4 virus

17. HIV Drug Resistance variants and Treatment Response

18. HIV Drug Resistance and Treatment Response Emerging Patterns of Resistance to Integrase Inhibitors (Abst. 125)  Resistance data from 5 clinical studies are generally consistent:  3 studies in treatment-experienced patients and 2 in treatment-naïve patients  Three pathways  primary mut Y143C/H/R, Q148H/K/R, N155H  Secondary mutations lead to higher resistance  If not present at VF, evolves at later time  Q148 pathway is preferred  Virus population can switch N155 to Q148  When combined with secondary mut, Q148 mutants lead to greater resistance

19. HIV Drug Resistance and Treatment Response Emerging Patterns of Resistance to Integrase Inhibitors (Abst. 125)  RAL resistance-associated mutations (RAMs):  No primary RAL RAMs (i.e., 143, 148, 155) detected at baseline, consistent with previously published data by: Buzón et al. Raltegravir susceptibility and fitness progression of HIV type-1 integrase in patients on long- term antiretroviral therapy. Antivir Ther. 2008;13:881-93  Secondary RAL RAMs ( L74M, E92Q, T97A, E138A/K, G140A/S ) : only T97A observed at baseline; frequency not significantly different between patients with and without virologic failure  Polymorphisms  25 of 28 baseline polymorphisms analyzed to date had no significant difference in frequency between virologic failures and treatment successes (exceptions: S17N, M50I, and D256E)--Further data needed to confirm

20. HIV Drug Resistance and Treatment Response Emerging Patterns of Resistance to Integrase Inhibitors (Abst. 125, 685)  Minority variants  Primary RAL RAMs were detected at exceedingly low frequencies in baseline samples None detected by ultra-deep sequencing (LOQ = 0.4%) Frequencies of <0.2% were detected by PASS  Primary RAL RAMs detected by PASS at baseline did not emerge in any patients who experienced virologic failure  Primary RAL RAMs appearing during PN004 monotherapy were rare, occurred at low levels, and did not result in virologic failure during the combination therapy phase

21. Circulating Drug Resistance in HIV-infected Populations

22. Circulating Drug Resistance Lack of drug resistance monitoring is associated with an increased risk of mortality (Abst. 666)  Clinical and demographic factor associated with failing to receive HIV drug resistance testing when indicated  Association between access to resistance testing before and after starting HAART and mortality.  1820 participants that initiated HAART between Jan’00 and Jun’06 and were followed until Jun’07 in an environment with universal free access to health services.

23. Circulating Drug Resistance Lack of drug resistance monitoring is associated with an increased risk of mortality (Abst. 666)  Pre-Tx: Resistance testing was most likely ordered in patients:  with CD4 cell count <350 cells/mm 3 (0R 1.64; 95%CI 1.26 to 2.15)  initiating Tx in the past 3 years (0R 3.65; 95%CI 2.81 to 4.73)  During HAART: Resistance testing was most likely ordered in:  females, IVD users, patients with high pVL, patients with adherence <95%  Mortality:  No tested before Tx initiation  1.7-fold higher risk of mortality  Fewer resistance testing while on HAART  2.7-fold higher risk of mortality  These results do not allow to establish a causal relationship regarding the association between physician-ordered genotypic resistance testing and survival.

24. Outcomes of HIV Drug Resistance

25. Residual Viremia (Abst. 664) < 50 HVI-1 RNA copies / mL Clinical Implications ?

26. Transmission of Drug-resistant HIV

27. Pre-Exposure Proxylaxis - PrEP (Abst. 678)  PrEP could substantially reduce HIV in San Francisco, but could also increase levels of Transmitted Drug Resistance  Based on “complex” mathematical models …  Assumptions: PrEP used by a median of 70% of MSM with a median efficacy of 60% in a population with a 10% transmitted drug resistance Preventing 58% of new infections Increasing TDR up to 31% next 10 years San Francisco

28. A final and global view...

29. WHO Updated List of Mutations for Surveillance of TDR HIV (Abst. 689) PI 23I, 24I, 0N, 32I, 46I/L, 47V/A, 48M/V, 50V/L, 53L/Y, 54V/M/L/T/S/A, 58E, 73S/T/C/A, 76V, 82A/F/L/T/S/M/C, 83D, 84V/A/C, 85V, 88D/S, 90M NRTI 41L, K65R/N, 67N/G/E/del, 69D/G/N/ins, 70E/R/G, 74I/V, 75T/A/M/S, 77L, 115F, 116Y, 151M, 184V/I, 210W, 215C/D/E/F/V/Y/S/I, 219E/Q/N/R NNRTI 100I, 101E, 103N, V106A/M, 179F, 181C/I/V, 188L/C, 190A/S/E/Q/C, 225H, 230L, 236L new mutations italicized Examined expert lists: IAS–USA, Los Alamos, Stanford HIV Database, ANRS, and Rega Analyzed numbers of subtype B and non-B sequences: 7395 and 7710 from PI-naive persons; and 5539 and 5982 from RTI-naïve persons. Numbers of non-B sequences from untreated persons were: A, 1,524; AE, 890; AG, 1,415; C, 2,145; D, 512; F, 605; G, 619. Non-polymorphic mutations associated with ART were included in the list.