CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

178

MEASURING FITNESS OF IN VIVO ESCAPE MUTATIONS IN SIV ENV BY FITSEQ Sergio Ita 1 , Alison Hill 2 , Evan Lam 2 , Ismael B. Fofana 2 , Welkin E. Johnson 2 1 Univ of California San Diego, La Jolla, CA, USA, 2 Boston Coll, Chestnut Hill, MA, USA

Background: The target of antibodies is the viral Envelope glycoprotein (Env). SIV env sequence variation arising from evasion of antibody is well known. However, the impact of antibody escape Env adaptations on viral fitness, or relative replication capacity (RRC), of SIV is poorly defined. To investigate the impact of escape mutations on inherent replicative capacity, we developed a probe-free, multiplex fitness assay based on deep-sequencing technology, which we call FITSEQ. Methods: To measure RRC, we tested individual Env substitutions and deletions using a deep-sequencing-based viral competition assay, described here as FITseq. These changes were either reported previously or identified in a deep-sequencing longitudinal analysis of SIV env sequences isolated frommacaques during early SIV infection. RRC of each mutant was measured relative to SIVmac239 in two immortalized rhesus (Rh) T-cell lines. Viral RNA isolated from supernatants was subjected to Illumina (MiSeq) sequencing and assembled to the SIVmac239 reference genome (M33262), the wild type (WT) strain. The frequency of mutant reads, relative to WT, was plotted over time and linear regression analysis was used to determine the slope of the line as a direct measure of the RRC. An F-test was used to determine whether the slope of the line was significantly different than zero for each mutant. Results: 5 of 5 substitutions in V1 (left panel) and 2 of 4 substitutions in V4 incurred no viral fitness costs in either T-cell line (middle panel). V4 loop change P421Q incurred a minimal, but significant, reduction in RRC in Rh 221 cells (p=0.001) while P421S resulted in a reduced RRC in both cell lines (Rh 221: p<0.05, Rh 444: p<0.05) (middle panel). Of the two V4 loop deletion mutants tested, a four amino acid deletion (∆423-EQHK-426; ∆V1) had no effect on RRC in either cell line (right panel). An eight amino acid deletion in V4 (∆418-NQKPKEQH-425; ∆V2) had RRC values of -0.292 and -0.463 in the Rh 221 and 444 cell lines, respectively, with the reduction in RRC reaching significance in the Rh 444 cell line only (p<0.05) (right panel). Conclusion: The observation that several variable loop changes, including antibody escape adaptations, had little to no effect on viral fitness suggests SIV Env evolves along specific sequence pathways that confer escape to antibody without hindering viral replicative fitness. Further, FITseq provides a novel high-resolution and high-throughput approach towards investigation of viral fitness.

Poster and Themed Discussion Abstracts

179 SIV ESCAPE FROM A VACCINE TARGETING CLEAVAGE SITES IS ASSOCIATED WITH A FITNESS LOSS So-Yon Lim 1 , David Tang 2 , David La 2 , Shaun Tyler 2 , Ben Liang 2 , Francis Plummer 2 , Ma Luo 2 , James Whitney 1 1 Beth Israel Deaconess Med Cntr, Boston, MA, USA, 2 Natl MicroBio Lab, Winnipeg, Canada

Background: The classical approach to vaccine development against HIV has shown only limited efficacy in dealing with viral antigenic diversity. The present study was initiated to explore the efficacy of a novel HIV vaccine candidate targeting the highly conserved protease cleavage sites (PCS) in a cynomolgus macaque-SIV-challenge model. In that study, PCS specific antibody and T cell responses correlated with reduced acquisition and disease progression after SIVmac239 challenge. Methods: We sequenced SIV populations isolated after challenge from vaccinees (n=12) immunized with a VSV vector expressing a 20-amino acid peptides overlapping each of the SIVmac239 12 PCS regions, then boosted intra-nasally with nanopackaged peptides, or from control animals that received sham inoculations (n=5). Unique mutations were identified in vaccinees and associations were analyzed between the immune driven viral mutations surrounding the PCS and alterations in viral load and CD4 count. To evaluate whether the vaccine-elicited mutations were detrimental to the virus, we produced 11 transfection-derived viral stocks harboring each PCS mutation alone or in combination. Each of these full-length mutant viral stocks was evaluated for viral p27-CA content, SIV RNA levels, replication rate and proteolytic processing of Pr55Gag. Results: Vaccinees showed significantly higher frequencies of mutations in both PCS2 (p27/p2) and PCS12 (Nef) regions that correlated with reduced viremia post-challenge. All recombinant mutant clones containing only PCS2 preparations were impaired. Virus preparations of these mutants contained significantly reduced viral RNA levels and p27-CA content. These mutants also displayed impaired proteolyitic Gag processing. All PCS2 mutant clones were significantly impaired in their ability to replicate in CEMx174 cells. Interestingly, recombinant virus harboring only PCS12 mutations replicated at comparable rates to wild type SIV. Importantly, we observed compensation of the defects incurred by PCS2 by the addition of the PCS12 mutation. This compensating PCS12 mutation restored viral replication to PCS2 mutants, compared to those mutants harboring mutation in PCS2 region alone. Conclusion: These results show that focused immune responses targeting the PCS region result in mutations surrounding the protease cleavage region, impair the replication ability of virus and correlate significantly with reduction in viral load, and the maintenance of CD4+ T cells in vivo. 180 MUTATIONS IN HIV-1 ENV RESCUE REPLICATION DEFECTS DESPITE POOR CELL-FREE INFECTIVITY Rachel Van Duyne , Lillian Kuo, Ken Fujii, Eric O. Freed NCI, Frederick, MD, USA Background: The p6 domain of HIV-1 Gag contains a YPXnL motif, a “late” domain, which promotes the release of virions through a direct interaction with the ESCRT-associated protein Alix. We previously demonstrated a functional role for the binding between Gag p6 and Alix in HIV-1 particle production and replication by introducing mutations in the YPXnL motif. The most striking defect observed was a severe delay in virus replication. The goal of this study was to further characterize the role of Alix in HIV-1 replication by identifying compensatory mutations that correct the original replication defect.

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