Assessing individual viral reactivations of the latent reservoir using a novel barcoded virus Christine M. Fennessey, Ph.D AIDS and Cancer Virus Program Frederick National Laboratory for Cancer Research 24 July 2017

All contributors to this work have no conflicts of interest to declare

Viral reservoirS: A major obstacle to Definitive treatment of HIV HIV establishes viral reservoirs early in infection; interruption of cART almost invariably leads to viral recrudescence Eradicating integrated virus from latently infected cells or establishing cART-free remission is a significant challenge In vitro and ex vivo based assays are useful, but suffer from numerous limitations Time to measurable rebound viremia following cART interruption is a clinically relevant gold standard, but provides limited information

Assessing individual rebound events Chronic phase HIV swarms provide ample genetic diversity to potentially discriminate variants BUT, sequencing limitations preclude sensitive, deep analysis of full length viral genomes (or even just Env) Robust discrimination of lesser diversity during acute infection not feasible Fitness effects of genetic variation across viral genome

Studying Viral recrudescence in nonhuman primates Robust viral suppression now achievable with cART in SIV/SHIV infected NHP, providing animal models for reservoir/reactivation studies To maximize NHP model value, we developed an isogenic, molecularly barcoded “synthetic swarm” of phenotypically equivalent variants of SIVmac239 to allow detailed tracking of distinct chains of events in establishment, maintenance, and reactivation of infection by different viral variants contributing to viral reservoirs Potential advantages: smaller cohort size, shorter cART duration, increased accuracy, and greater statistical power

Generation of Barcoded SIVmac239M CGCGGGCTACCNNNNNNNNNNTTGCAAG CCGATGGNNNNNNNNNNAACGTTCGCGC or

Sivmac239 contains ~10,000 unique barcoded viral variants

Barcoded SIVmac239M Replicates comparably to wild type SIVmac239 in vitro and in vivo In Rhesus ng RT/mL vRNA copies/mL Days Post Infection Days Post Infection

Assessment of reactivation events After Treatment Interruption in SIVmac239M Infected Macaques IV inoculation of 6 rhesus macaques with high dose SIVmac239M – intent to initially seed reservoir with many variants Samples collected on day 4 post-inoculation, just prior to cART initiation cART interrupted on day 300, 380, or 480 post-inoculation Animals monitored for rebound viremia Barcodes of rebounding variants sequenced

Barcode Variants at pre-ART Peak Viremia TFV/FTC/RAL/IND/RTV DEJX, DFGV H090, DEJW DEPI, H105 Number of Barcodes Relative proportion vRNA Copies/mL Range 250-2900 Days Post Infection

Early art suppression: Variants at Peak rebound viremia TFV/FTC/RAL/IND/RTV DEJX, DFGV H090, DEJW DEPI, H105 vRNA Copies/mL Days Post Infection

Abundance and proportion of rebounding barcodes Provides insights into Reactivation dynamics Rebound barcodes Pre-therapy barcodes

Proportion of Rebounders can be correlated with reactivation timing 107 106 1 0.1 0.001 0.0001 105 104 103 102 vRNA Copies/mL 101 2220 3019 5603 4754 3820 546 SIVmac239 Variant 100 -Δ- -Δ- -Δ- 10-1 Reactivation rate = 1 every 1.5 days 10-2 10-3 0 5 10 15 20 25 Days Post Release

Estimation of Reactivation Rate DEJX DFGV 107 107 106 106 105 105 104 104 103 103 Average reactivation rate: 1 activation every 1.6 days 300 days of cART vRNA Copies/mL 102 vRNA Copies/mL 102 101 101 100 100 10-1 10-1 10-2 10-2 10-3 10-3 0 5 10 15 20 25 Days Post Release 0 5 10 15 20 25 Days Post Release Days Post Release Days Post Release DEJW H090 107 107 106 106 105 105 104 104 Average reactivation rate: 1 activation every 1.7 days 103 103 380 days of cART vRNA Copies/mL 102 vRNA Copies/mL 102 101 101 100 100 10-1 10-1 10-2 10-2 10-3 10-3 0 5 10 15 20 25 Days Post Release 0 5 10 15 20 25 Days Post Release Days Post Release Days Post Release DEPI H105 107 107 106 106 105 105 104 104 103 Average reactivation rate: 1 activation every 2.4 days 480 days of cART 103 vRNA Copies/mL 102 vRNA Copies/mL 102 101 101 100 100 10-1 10-1 10-2 10-2 10-3 10-3 0 5 10 15 20 25 Days Post Release 0 5 10 15 20 25 Days Post Release Days Post Release Days Post Release

Conclusions I.V. inoculation with SIVmac239M can establish disseminated systemic infection/viral reservoirs with numerous phenotypically similar but distinct barcoded viruses and allows monitoring of persistence and reactivation events by individual viral variants upon cART interruption Modeling based on rebound of SIVmac239M barcode variants allows estimation of latent virus reactivation rates SIVmac239M should allow efficacy assessment of reservoir targeting interventions, based on number and dynamics of rebounding variants, even if time to measurable rebound viremia is not affected

Future Directions Prepare barcoded SHIV and minimally chimeric HIV stocks for addtl NHP studies Test in vivo efficacy of novel reservoir depleting strategies Characterize origins and viral dynamics of immune escape

COLLABORATORS AND FUNDING ACVP/FNLCR U. New South Wales Brandon Keele Taina Immonen Carolyn Reid Laura Newman Leslie Lipkey Jacob Estes Claire Deleage Greg Del Prete Jeff Lifson Miles Davenport Mykola Pinkevych Arnold Reynaldi Kelli Oswald Claes Ohlen Dave Ott BJ Boche Matt Trivett AIDS and Cancer Virus Program Contract HHSN261200800001E