1. 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

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

3. 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

4. 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

5. 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

6. Generation of Barcoded SIVmac239M
CGCGGGCTACCNNNNNNNNNNTTGCAAG
CCGATGGNNNNNNNNNNAACGTTCGCGC or

7. Sivmac239 contains ~10,000 unique barcoded viral variants

8. 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

9. 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

10. 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
Days Post Infection

11. 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

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

13. 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
SIVmac239 Variant
100
-Δ-
-Δ-
-Δ-
10-1
Reactivation rate = 1 every 1.5 days
10-2
10-3
Days Post Release

14. 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
Days Post Release
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
Days Post Release
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
Days Post Release
Days Post Release
Days Post Release
Days Post Release

15. 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

16. 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

17. 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 HHSN E