Viral Genetic Traits of Durable Control in Dual Immunotherapy-treated SHIV-infected Rhesus Macaques

The interruption of antiretroviral therapy (ART) in HIV-1 infected individuals leads to rapid viral rebound, highlighting the persistent challenge of eradicating HIV-1 reservoirs. Our previous study demonstrated that combined administration of N-803 (Anktiva) combined with the broadly neutralizing antibodies (bNAbs), 10-1074 and 3BNC117 results in durable virologic remission following ART cessation in SHIV-infected, ART-suppressed rhesus macaques (RM). Identifying viral genetic features associated with therapeutically induced viral remission is of great importance to the development of effective HIV cure strategies. Here, we investigate the impact of this curative intervention on viral sequence diversification to identify SHIV genetic features associated with durable remission. We performed single genome amplification (SGA) of plasma-derived SHIV genomes and comprehensively analyzed the genetic features of envelope (env) sequences during the post-ART viral rebound phase. Our data show that the rebounding SHIV variants after analytical treatment interruption (ATI) in dual immunotherapy-treated macaques exhibited significantly elevated viral divergence and increased purifying selection as compared to viruses from control animals. Env sequences from treated animals harbored distinct mutational patterns in the signal peptide, V1/V2 region, V3 region, fusion peptide, and contact site. Moreover, the loss of specific N-glycosylation sites in the V1 region was observed, likely reflecting changes in humoral immune pressure resulting from the intervention. Consistently, CD8 depletion in treated animals resulted in viral rebound, increased Env diversification, and emergence of additional mutations in CD8+ CTL and CD4+ helper T cell epitopes, compatible with the concept that CD8+ T cells are critical mediators of viral containment following dual immunotherapy. The observed viral control in N-803+bNAb-treated RMs correlates with elevated frequencies of CD8+ T cells, demonstrating that reprogrammed cell-mediated immune pressure maintains durable virologic control. Therefore, the viral genetic signatures revealed by our analyses may inform the development and optimization of immune-based HIV cure strategies that minimize the likelihood of viral recrudescence following ART cessation.