IN VITRO SELECTED RESISTANCE TO NEW INTEGRASE INHIBITORS BY B & NON-B SUBTYPE VIRUSES

Contact Information 1 1 1 1 1 1 Bluma G. Brenner Bluma G. Brenner , Maureen Oliveira , Ruxandra-Ilinca Ibanescu , Bonnie Spira , Thibault Mesplede , Jean-Pierre Routy Lady Davis Institute, McGill AIDS Centre 1 3755 Cote Ste. Catherine Rd. McGill University, Montreal , QC, Canada Montreal, Quebec CANADA H3T 1E2 [email protected] (514-340-8260)

Background Table 1. Selection of drug resistance to dolutegravir (DTG), bictegravir (BIC), cabotegravir (CAB) and elvitegravir(EVG) at the final week of passage Acquired mutations at final passage (week 46) of selective drug pressure Virus isolate Subtype The integrase strand transfer inhibitors (INSTIs) bictegravir DTG BIC CAB EVG (BIC), dolutegravir (DTG), and cabotegravir (CAB) have 14514 B R263K None None T66I improved resistance profiles when compared to raltegravir 10387 B None None None T66I and elvitegravir (EVG), with few reported cases of 10249 B R263K None None E92Q resistance in the clinic. This study used in vitro drug 14624 B none None H51HY T66I selections with primary HIV-1 isolates to examine potential 14637 B R263K R263K R263K T66I, E157Q, R263K pathways for resistance to EVG and DTG and the 14947 B R263K R263K R263K, S153A T66I, E138EK, S147G, Q148R investigational drugs BIC and CAB. 5326 B H51H/Y S153Y L74M, G140S, S147G, Q148K R263K, S153A 4742 C None None R263K E92EG, R263KR 10947 C R263K R263K S147G E92V, R263K Methods 6343 AE R263K S153Y S153Y, G163R T66I, R263K 14515 AG None None None T66I, H51HY Subtype B (n=7) and non-B subtypes (n=5) strains were 96USSN20 AG R263K S153FS, E157EK L74M, E138K, Q148R, R263K amplified from PBMCs of primary HIV infections through pNL4.3 B R263K, M50I R263K, M50I S153F T66I, T97A, S147G, S119R, S153A co-culture in cord blood mononuclear cells (CBMCs). The acquisition of R263K, H51Y, S153Y/F conferred low-level resistance with 1-10 nM final drug concentrations as compared to the high-level resistance highlighted in bold CBMCs were serially passaged in escalating where the acquisition of complex resistance mutational motifs with CAB or EVG allowed for viral breakthrough at final drug concentrations of 0.1 to 2.5 µM. concentrations of DTG, EVG, BIC, and/or CAB for 36-46 Table 2. Phenotypic drug susceptibility of viral strains to integrase strand transfer inhibitors (INSTIs) harvested at the designated week of selection with weeks. Sanger and ultradeep sequencing ascertained the cabotegravir (CAB) or elvitegravir (EVG). acquisition of resistance mutations under selective drug Viral variant-Drug EC (nM) in CBMCs (fold-resistance relative to WT control) Acquired resistance mutations 50 pressure at weeks 8-9, 16, 24-30, and 43-46. Selection Week DTG BIC CAB EVG RAL 5326 -No Drug WT 0.8 0.4 0.2 0.3 1.1 5326-CAB wk17 Q148K 1.6 (2x) 0.9 (2x) 0.6 (3x) 3.0 (10x) 3.6 (3x) Delay in the acquisition of resistance to DTG and BIC, 5326-CAB wk28 G140S, S147GS, Q148K 3.60 (5x) 0.72 (2x) 8.07 (40x) 180 (720x) 675 (60x) compared to CAB and EVG 5326 CAB wk45 L74M. G140S, S147G, Q148K 125 (162x) 49.01 (120x) 139.7 (700x) 3429 (>10000x) 1007 (900x) 5326-EVG wk45 R263K, S153A 0.52 (0.7x) 0.53 (1.3x) 0.19 (1.0x) 53 (212x) 1.12 (1x)

1 0 96USSN20-No Drug WT (CRF02_AG) 0.68 0.8 0.3 0.2 1.6 96USSN20-CAB wk17 E138EK, Q148R, R263KR 8.1 (14x) 5.83(8x) 2.4 (8x) 22.4 (112x) 12.0 (8x) E V G 96USSN20-CAB wk27 L74LM, E138K, Q148R, R263K 10.0 (17x) 9.0 (12x) >30 (>100x) >300 (>1500x) 300 (188x) 1 96USSN20-CAB wk45 L74M, E138K, Q148R, R263K 14.0 (24x) 13.4 (17x) 47.8 (165x) 1612 (8060x) 568 (355x)

) C A B M

( 96USSN20-EVG wk27 T66I, T97A, Q147G 0.4 (0.7x) 0.8 (1x) 1.2 (4x) >100 (>500x) 1.7 (1x)



n

o 0 .1 i

t 6343-CAB wk45 WT 0.010 0.17 0.23 0.21

a r

t 6343-CAB wk46 S153Y, G163R 0.060 (6x) 1.33 (8x) 0.87 (3.2) 0.53 (2.5x) n

e B IC

c 0 .0 1 D T G

n pNL4.3 WT 0.5 0.4 0.3 0.5 <0.3 o

C pNL4.3 R263K 0.8 (2x) 0.9 (2x) 0.4 (2x) 1.7 (3x) <0.3

0 .0 0 1 pNL4.3 S153F 1.5 (3x) 0.0 (2x) 0.6 (2x) 1.0 (2x) 75 (>250x) Samples in bold represent greater than 5-fold reduction in drug susceptibility. Table 3. Viral outgrowth of elvitegravir (EVG) resistant viruses switched to dolutegravir (DTG), bictegravir (BIC) or cabotegravir (CAB). 0 .0 0 0 1 Viral Switch Week 17 polymorphisms Lost mutations Acquired mutations 0 8 1 6 2 4 3 2 4 0 4 8 isolate Drug [Drug] (µM) following switch 6343 Pre-switch 0.25 T66I, R263K Figure 1. Growth of 96USSN20 cells in escalating concentrations of 6343 DTG 0.01 R263K, M50MI (T66I) M50IM dolutegravir (DTG), bictegravir (BIC), cabotegravir (CAB) and elvitegravir 6343 BIC 0.05 R263K (T66I) (EVG). The rise in drug concentrations is related to the acquisition of resistance mutations. 6343 CAB 0.05 R263K (T66I) 14637 Pre-switch 1.0 T66I, E157Q, R263K 14637 DTG 0.01 E157Q, R263K (T66I) Results 14637 BIC 0.025 E157Q, R263K (T66I) 14637 CAB 0.05 E157Q, R263K (T66I) Parallel in vitro selections found resistance mutations in 5326 Pre-switch 1 S153A, R263K more strains for EVG (12/12), followed by CAB (8/12), and 5326 DTG 0.01 S153A, R263K 5326 BIC 0.01 S153A, R263K BIC and DTG (6/12 each). For EVG, T66I (n=8), E92G/V/Q 5326 CAB 0.05 S153A, R263K (n=3) or R263K (n=1) were followed by the accumulation of 14624 Pre-switch 1.0 T66I mutations leading to viral escape and high-level resistance 14624 DTG 0.005 T66I, L74M, E138K, S147G, M154IM L74M, E138K, S147G, M154IM (bold). With CAB, at the final passages, 8/12 selections 14624 BIC 0.01 H51HY (T66I) H51HY resulted in R263K (n=3), Q148K, S153Y, or S147G with 14624 CAB 0.1 T66I, L74M, G140GS, S147GS L74M, G140GS, S147GS acquisition of Q148R/K in two strains leading to viral 14947 Pre-switch 5 T66I, E138EK, S147G, Q148R escape. For DTG and BIC at the final passages, 6/12 14947 DTG 0.005 T66I, E138EK, S147G, Q148R, S230N S230N strains had singleton mutations R263K, S153Y or H51Y 14947 BIC 0.025 T66I, E138EK, S147G, Q148R, S230N S230N which conferred low-level (<3-fold) resistance and reduced 14947 CAB 0.1 T66I, L74M, E138EK, S147G, Q148R, S230N L74M, S230N, L74LM 96USSN20 Pre-switch 2.5 T66I, Q146R, S147G replicative fitness, precluding escalations in DTG and BIC 96USSN20 DTG 0.1 T66I, Q146R, S147G, E138E1AEKT, Q148R (Q146R) E138AEKT, Q148R beyond 5-25nM. 96USSN20 BIC 0.05 T66I, Q146R, S147G, E138A, T97A (Q146R) T97A, E138A 96USSN20 CAB 0.25 T66I, Q146QR, S147G, E138A, Q148QR (Q146QR) E138A, Q148QR pNL4.3 Pre-switch 2.5 T66I, T97A, S147G, V151I, S153A Conclusion pNL4.3 DTG 0.025 T66I, T97A, S147G, V151I, S153A pNL4.3 BIC 0.025 T66I, T97A, S147G, V151I, S153A pNL4.3 CAB 0.25 T66I, T97A, S147G, V151I, S153A There is a high genetic barrier to resistance to BIC and Patient-derived viral strains, subjected to EVG selective pressure for 46 weeks, were switched to serially increasing concentrations of DTG, BIC or CAB for 17 weeks. DTG compared to CAB and EVG. Emergent resistance by Genotyping was performed to monitor the loss and acquisition of mutations. singleton mutations, R263K or S153Y confers <1.5-3 Acknowledgments resistance retaining antiviral activity to DTG and BIC, whereas more complicated patterns of high-level This study was sponsored in part, by grants from Gilead Sciences, the Canadian Institutes for Health Research (CIHR), and the Fonds de Recherche du Québec (FRQ, 202685). resistance were selected by CAB and EVG. Ongoing studies will deduce resistance to INSTIs in larger panel of viral strains. The authors thank all participants of in the Montreal PHI cohort, Mario Legault, coordinator of the cohort and all participating physicians.