z

Funding for aspects of this research was provided by grants from the CanadianInstitutes for Health Research (CIHR), Réseau sida et maladies infectieuses,Fonds de Recherche du Québec FRQ–Santé (FRQ-S), FRQ-Nature et Technologie(FRQ-S/NT). We thank all patients, physicians and research staff from theMontreal PHI cohort including coordinators Mario Legault and Anne Vassal, aswell as participating clinicians from Actuel: Réjean Thomas, Benoit Trottier,Sylvie Vézina, Louise Charest, Catherine Milne, Jason Friedman, EmmanuelleHuchet; Clinique Quartier Latin: Jean-Guy Baril, Pierre Côté, Bernard Lessard,Serge Dufresne, Marc-André Charron; Clinique OPUS: Roger LeBlanc; HôpitalSt-Luc: Julie Bruneau; Centre hospitalier de l’Université de Montréal: CécileTremblay, Louise Labrecque, Claude Fortin, Marie Munoz; McGill UniversityHealth Centre : Jean-Pierre Routy, Norbert Gilmore, Richard Lalonde, MartinPotter, Marina Klein, Alexandra de Pokomandy, Jason Szabo. We also thank allMSM participants and members of the SPOT rapid testing site and preventioninitiative including research team: Joanne Otis, Karine Bertrand, Martin Blais,Bluma Brenner, Gilbert Émond, Ken Monteith, Michel Roger, Robert Rousseau,Mark Wainberg research staff: Thomas Haig, Jessica Caruso, Ludivine Veillette-Bourbeau, Michel Martel, Michel Martel, Carl Rodriguez, Chloé Rondeau,Patrice Bécotte and intervention team: Jean Boulanger, Rodolphe Coulon,Laurence Delisle, Dominique Harvey, Pierrette Héon, Ernesto Hernandez,Marc-André Primeau.

Bluma G. BrennerLady Davis Institute, McGill AIDS Centre3755 Cote Ste. Catherine Rd.. Montreal, Quebec CANADA H3T 1E2bluma.brenner@mcgill.ca515-340-8260

Acknowledgements

Contact Information

Large Cluster Viral Lineages Fueling the MSM Epidemic show Facilitated Escape

from Integrase InhibitorsBluma G. Brenner1, Maureen Oliveira1, Ruxandra-Ilinca Ibanescu1, Olga Golubkov1, Isabelle Hardy2, Michel Roger2, and Mark A. Wainberg1

1McGill AIDS Centre, Lady Davis Institute for Medical Research and Centre de Recherche du Centre hospitalier de l’Université de Montréal (CHUM), Montreal, Québec, Canada

Abstract

Background

The 25-50% decline in HIV transmissions with antiretroviral therapyhas raised optimism that Treatment-as-Prevention (TasP) maycontrol the global pandemic by 2030. Paradoxically, transmissionsamong MSM have not declined, raising concerns that early-stageinfection, frequently undiagnosed, may offset the benefit of TasP.Phylogenetics ascribe the growth of the Montreal MSM epidemic tolarge cluster outbreaks, averaging 43 linked transmissions/cluster.This study characterized the distinct genotypic and phenotypicfeatures of large cluster viral variants favoring their selectionadvantage.

Materials & Methods

Phylogenetic analysis was performed on RT/protease sequencesfrom all newly infected MSM (n=4319, 2002-2014), assessingclustering dynamics (frequency, size, periodicity). Nucleotide mixedbase calls estimated recency of infection, using a 0.44% cut-off forprimary HIV infection (PHI). PHI cohort data evaluated the naturalhistory (viral load, risk behaviour) for subjects belonging to largeclusters vs. solitary transmission groups.

Viral stocks uses were amplified from representative large clusters(n=7, 20+ linked transmissions/cluster) and solitary transmissions(n=6) through co-culture of patient CD-8 depleted peripheral bloodmononuclear cells (CBMCs). Viruses from both groups werecompared for tropism, drug susceptibility and emergent resistanceto integrase inhibitors. Amplified viruses were serially passaged inCBMCs in the presence of increasing concentrations of (DTG),elvitegravir (EVG), and DTG +3TC dual pressure. Genotyping wasperformed at select passages to evaluate time to the developmentof drug resistance.

Results

Phylogenetic analysis revealed a significant rise in the relativecontribution of large clusters (10+ linked transmissions/cluster) intransmission dynamics, accounted for 35%, 40%, and 49% of newinfections over the 2002-2006, 2007-2010 and 2011-2014 periods.Overall, 40 viral lineages contributed to 1235 transmissions ascompared to 1375 solitary “dead-end” transmissions. PHI/EHI (0-0.44% genetic diversity) accounted for 57% and 26% oftransmissions in large cluster and solitary transmission groups,respectively. Viruses from seven representative large clusters oftenharbored X4/R5 dual tropic viruses (n=5/7) as compared todominant R5 tropism in the unique transmission group (0/6).Overall, 10% of transmitted viruses associated with large clustersharbored drug resistance mutations (G190A, K103N, T215revertants). Tissue culture studies showed accelerated developmentof resistance under DTG, (R263K, H51Y or S153Y), elvitegravir (EVG),3TC (M184I/V), and DTG/3TC dual pressure (R263K +M184V).Resistance mutations for DTG arose within 6-8 weeks in large clusterlineages while solitary transmission variants retained wild-typegenotype at 26 weeks.

Conclusion

Failure to control early stage transmissions is leading to worrisometrends towards the selection of super-viruses showing prolongedhigh viremia, dual tropism, rapid tropism shift, and/or facilitatedescape from drug pressure.

All MSM infections (n=6135) Subtype B, men, non-IDU/HET

Untreated MSMPHI (0-6 months, n=1464)

Untreated MSMCUN (> 6 months, n=2565)

Non-B subtype HET (n=1094)*MSM clusters (n=71)

Population-level phylogenetic surveillance (RT/protease region); Integrase (post-2009)

IDU/HET (n=1517)Female/ IDU clusters

Treated MSM (n=2106)

Surveillance of the Quebec HIV-1 epidemic (2002-2014)

Cluster group association of individual infections monitored annually and cumulatively. Bootstrap >95% and shared natural polymorphisms

Sequences evaluated for transmitted resistance & molecular recency (% mixed base calls).

First RT/protease sequences of all genotyped persons in Quebec (n =8746, 2002-2014)

New infections

Phylogenetic surveillance of MSM epidemic

0

5

10

15

20

25

30

35

40

45

Singleton 2-4 5-9 10-19 20+

2002-2006 2007-2010 2011-2014

40%

Evolutionary shift towards large cluster outbreakssustain the MSM epidemic

21%

29%

40%

34%

26%

Episodic genesis and expansion of large cluster outbreaks fuel new MSM infections in the era of optimized ART

Cluster 45 (n=38), cluster 118 (n=43) and cluster (n=44) share a common integrase.Cluster 27, 53, and 185 show accelerated selection of resistance to INIs in cell culture.

26 1 59 44 68 89 18 83 62 86 19 51 7512015

612

5 72 94159 50 99 67 27 53 4511

818

516

3

0

20

40

60

80

100

120

Clu

ste

r siz

e 2002-2006: 21%

2007-2010: 29%

2011-2014: 40%

Clusters 20+Post-2011

Cluster 50 (n=132, G190A) and Cluster 99 (n=34/69, K103N) harbor transmitted resistanceto first generation NNRTIs (EFV) and hypersensitivity to rilpivirine and etravirine.

0.01 Transmitted Drug Resistance (RT/protease)10%

NNRTI, (n=247, 6.2%) NNRTI+NRTI, (n=30, 0.8%)NNRTI+PI. (n=4, 0.1%)NNRTI+NRTI+PI (n=9, 0.2%)

TDR(n=394/3957)

10.0%

NRTI, (n=65, 1.6%) NNRTI+NRTI (0.8%)

NRTI+PI (n=5, 0.1%)NNRTI+NRTI+PI (0.2%)

NNRTIs 7.6%

NRTIs 2.8%

PIs 0.9% (n=23, 0.5%)

0 4 8 12 16 20 24

0.001

0.01

0.1

Dolutegravir selection

Weeks in culture

[DT

G]

( M

)

0 4 8 12 16 20 24

0.001

0.01

0.1

1

10

Elvitegravir selection

Weeks in culture

[EV

G]

( M

)

3TC selection

0 4 8 12 16 20 24

0.01

0.1

1

10

Weeks in culture

[3T

C]

( M

)

0 4 8 12 16 20 24

0.0001

0.001

0.01

0.1

Dual 3TC + DTG selection

Weeks in culture

[Dru

g]

( M

)

C185 (n=2)

C027 & C053 (n=2)

Singleton (n=4)

Large cluster variants show accelerated escape from drug pressure in cell culture selections

0.01 Transmitted NRTI Resistance(n=2.8%) Large Cluster (n=5-18) Small Cluster (n=2-4)Singleton

C149 (n=18)T215D/E

C161 (n=5))M41L, L210W, T215D

C144 (n=10) M41L, T215D

C129 (n=7)M41L, L120LW, T215C/S, N348ID30N, L33F, N88L

C122 (n=11) M41L, T215S

C238 – M184V (2/8), K103N (8/8)

C219 (n=6)M41L, T215C

C232 (n=6)M184V, T215

0.01

Transmitted NNRTI Resistance7.6% (302/3957)

Large Cluster (8-132)

(Large Cluster – wt)

Small Cluster (2-5)

Singleton

C050 (n=132)G190A

C099(n=34/69 K103N)

C072 – V179D

C068 – (n=14/41, K103R, V108I, V179D)

C042 – 5/13, PI ± K103NC189 –( n=8 ,108I, 179D, 181C, 221Y)

C129 – MDR, 215C, 348I

C105 – (n=9/11, 103N)

C166 (n=13, K103NC238 – (n=8, K103N)

C099 – wt

Quebec city

NGS

Cluster group(cluster size)

Lab number Drug First appearance of drug resistance Drug resistanceWeek 36Week Mutation

Singletons 4 isolates DTG - WT

Cluster 185 (44) 14947 DTG 6 R263K R263K

Cluster 185 (44) 14637 DTG 8 S153Y S153Y

Cluster 27 (40) 14977 DTG 8 12 R263K/R R263K R263K

Cluster 53 (24) 14969 DTG 23 H51Y H51Y

Singletons 14514 EVG 23 T66I T66I

Singletons 15366 EVG 23 T66I, L74M T66I, L74M

Cluster 185 14947 EVG 6 8 T66I T66I, R263K/R T66I, R263K, E157Q

Cluster 185 14637 EVG 12 N155H N155H, Q95K, S230R

Cluster 27 (40) 14997 EVG 8 T66IT, E92EG T66I, E92EG, L74LM, E157Q

Cluster 53 (24) 14969 EVG 12 S147G S147G, Q148R

Singletons 1 isolates 3TC M184I/V

Cluster 185 (44) 14947 3TC 6 M184V M184V M184V

Cluster 185 (44) 14637 3TC 8 15 M184I M184V M184V

Cluster 27 (40) 14977 3TC 8 M184I M184I

Cluster 53 (24) 14969 3TC 8 M184I M184I

Singletons 4 isolates DTG+3TC WR

Cluster 185 (44) 14947 DTG +3TC 8 12 R263K R263K, M184IM R263K M184V

Jewish General

Hospital

Lady Davis Institute

Impact of Clustering on Transmitted Resistance Large cluster variants show accelerated selection of resistance

Integrase inhibitors (INIs), including raltegravir (RAL), elvitegravir (EVG) anddolutegravir (DTG) are a drug class of choice in first-line therapy. Emergentresistance to EVG and RAL include the N155H and G140A/G148RHQpathways, conferring RGV/EVG cross-resistance, and Y143RHC (RGV) andT66I/E92Q/G (EVG) pathways. To date, DTG shows a higher barrier toresistance with no reported mutations in first-line therapy. “Super-transmissible” viral lineages showed facilitated development of resistance incell culture, revealing pathways to DTG resistance that could not beotherwise ascertained.

Clusters 118 (n=43), 45 (n=38) and 185 (n=44) share a common integrase

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