1. 1 Novel HIV Suppressive Approaches with Integrase Inhibitors Mark A Wainberg McGill University AIDS Centre Montreal, Canada

2. Global distribution of HIV-1 subtypes 1.3 million 4.8 million 2 million B 10% A 12% URF 4.2% AG 6.7% AE 3.1% G 5% D 3.6%

3. Rapid Selection of K65R Resistance in Subtype C Isolates

6. 6

7. 7 Previous work in our lab showed that MK-2048, a Merck INSTI, selected G118R followed by E138K. The latter augmented levels of resistance against MK-2048 and also restored replicative fitness.

8. Major resistance pathways against INSTIs (clinical and tissue culture data) Resistance pathways Fold resistance RALEVGDTG Y143 pathway Y143C<10<2 Y143R<50<2 T97A/Y143C>100<2 T97A/Y143R>100<2 L74M/T97A/Y143G<50ND<2 L74M/T97A/E138A/Y143C<20ND<2 N155 pathway N155H<50 <2 E92Q/N155H<100>100<10 L74M/N155H<50 <2 Q148 pathway Q148H<20<10<2 Q148K<100 <2 Q148R<50<100<2 E138K/Q148H<10<20<2 E138K/Q148K>100 <20 E138K/Q148R>100 <10 G140S/Q148H>100 <20 G140S/Q148K<10<100<2 G140S/Q148R>100 <10 E138A/G140S/Y143H/Q148H>100ND<50 Quashie et al., Curr. Opin. Infect. Diseases, in press

9. Secondary INSTI-resistance mutations often restore HIV replication capacity Mbisa et al., Infect. and drug resistance, 2011--Canducci et al., JAC, 2010--Reigadas et al., Plos One, 2010--Delelis et al., AAC, 2009 Secondary Mutations (pathway) Effect on viral fitness in the presence of primary resistance mutations Y143 pathway- (often) L74M, T97A+ N155H pathway- Q95K, T97Q, G163R/K+ Q148 pathway- G140A/S/C, E138K/A+

10. Dolutegravir activity on RAL- resistant clinical isolates (n=39) (median IC 50 for wild-type=1.07 nM) GenotypeMedian fold change N155H1.37 Y143R/T97A1.05 Q148H/G140S3.75 Q148R/G140S13.3 Underwood et al., JAIDS, 2012

11. Resistance to INSTIs in clinical trials in treatment-naïve patients Treatment Major resistance mutations detected by genotyping in treatment-naïve patients failing therapy Minor resistance mutations Raltegravir Y143 N155H Q148 Multiple Elvitegravir T66I E92Q N155H Q148 Multiple DolutegravirNONE RALTEGRAVIR Cooper et al., NEJM, 2008 Sichtig et al, JAC, 2009 Canducci et al, AIDS, 2009 Hatano et al, JAIDS, 2010 ELVITEGRAVIR Sax et al, Lancet, 2012 DeJesus et al, Lancet, 2012 DOLUTEGRAVIR vanLunzen et al., Lancet Infect. Dis., 2012

12. Selection results with DTG Quashie, et al, Journal of Virology 2012

13. Selection results with DTG Quashie, et al, Journal of Virology 2012

14. Selection results with DTG Quashie, et al, Journal of Virology 2012

15. Subtype-specific mutations selected in vitro with dolutegravir HIV-1 subtype Most common mutations selected with dolutegravir BR263K, H51Y CG118R, H51Y Quashie, Mesplède et al., Journal of Virology, 2012

16. The R263K mutation confers low-level resistance to dolutegravir in cell culture GenotypeIC 50 fold change* R263K2.5 to 6 *Methodological differences (EC 50 for wild-type ≈1-6nM) Quashie, Mesplède et al., Journal of Virology, 2012

17. The R263K mutation decreases integrase activity in cell-free assays Quashie, Mesplède et al., Journal of Virology, 2012

18. The R263K mutation decreases dolutegravir residency time in an integrase-vDNA complex

19. The addition of H51Y to R263K further decreases IN strand transfer activity A B

20. The combination of H51Y and R263K negatively impacts viral fitness

21. Effects of G118R and H51Y on in vitro strand transfer activity IN protein Relative strand transfer activity (RFU/hr) Vmax± SEM WT7,751.2480.7 H51Y7,590.0265.3 G118R6,138.5621.5 H51Y/G118R3,223.0130.2

22. Effects of H51Y, G118R and R263K mutations on susceptibility to dolutegravir in cell culture GenotypeIC 50 fold change* R263K2.5 to 6 H51Y1 (no change) H51Y/R263K6 to 12 G118R3 to 7 H51Y/G118R6 to 10 *Methodological differences (EC 50 for wild-type ≈1-6nM)

23. Dolutegravir resistance associates with a decrease in viral replication capacity GenotypeResistance Effect on viral fitness R263K+- H51YNone H51Y/R263K++- G118R+- H51Y/G118R++-

24. Conclusions Resistance mutations selected in vitro with dolutegravir are: R263K or G118R plus H51Y R263K and G118R confer low-level resistance against dolutegravir, e.g. 2.5-6 fold The addition of H51Y to either R263K or G118R increases resistance against DTG but also further decreases viral fitness These findings help to explain why resistance against dolutegravir in INSTI-naïve patients has not been observed

25. No compensatory mutations in regard to DTG resistance and viral fitness have developed over more than one year in culture. Might there be other implications for a drug that selects for an unfit virus and can animal models be of use?

26. Bluma Brenner Hongtao Xu Dimitri Coutsinos Jerry Zaharatos Maureen Oliveira Thibault Mesplède Peter Quashie Acknowledgements

27. MERCI