Abstract After years of infection, a small subset of people with HIV can develop broadly neutralizing antibodies (bnAbs), defined as antibodies that neutralize a diverse range of HIV isolates. While eliciting bnAbs is a central focus of HIV-1 vaccine research, bnAbs may have additional roles as long-acting biologic antiretroviral therapy or as an immune effector arm in virus eradication studies. Broadly neutralizing antibodies target the HIV-1 envelope glycoproteins, heterotrimers of surface gp120 and transmembrane gp41 molecules, that are 50% glycan by mass. The cloning of antibody genes from individuals with HIV identified new bnAbs with increasing potency and breadth of neutralization that have been studied clinically as HIV-1 treatment. Whereas single infusions of bnAbs can reduce plasma virus loads in people with HIV, virus variants resistant to the individual bnAb emerge quickly and limit the activity and therapeutic potential of bnAb monotherapy. Classically, an antibody molecule contains an Fc region linked to two Fab regions with identical antigen binding sites. Recently, an antibody was engineered that combined three distinct Fab regions into a single molecule. SAR441236 is a tri-specific bnAb that combines the CD4bs specificity of VRC01-LS, the V1/V2 glycan-directed binding of PGDM1400, and the gp41 MPER binding of 10E8v4 into one antibody molecule. ACTG A5377 is a phase I first-in-human study of SAR441236 that investigates the safety, pharmacokinetics (PK), and anti-HIV-1 activity of this novel trispecific bnAb. A maximum of thirty viremic participants will be studied in a single infusion dose de-escalation trial design with 24 weeks of follow-up. The goal of this proposal is to leverage samples from A5377 to determine if HIV-1 decay in response to “triple” biologic ART differs from conventional combination ART and to define the mechanisms of virus escape from a trispecific bnAb. The purpose of this proposal is to combine innovative experimental and mathematical approaches with classic molecular virology to characterize the decay of viremia and define the mechanisms of HIV-1 escape from this first-in-class trispecific bnAb. We hypothesize that the trispecific bnAb, SAR441236, clears cell-free and cell-associated virus from blood, induces large and dynamic population shifts in the HIV-1 env quasispecies, and selects for virus escape variants at the protein and glycan level to maintain infectivity in the presence of bnAb. Specific Aims of this proposal are to determine the kinetics of SAR441236-induced HIV- 1 decay, understand the effects of SAR441236 on HIV-1 env quasispecies, and define the HIV-1 env and glycan shield determinants of SAR441236 resistance. Our approaches test hypotheses that are central to understand the dynamics and evolution of HIV-1 under a trispecific bnAb selection pressure.

Narrative SAR441236 is a novel synthetic antibody that combines three different anti-HIV-1 specificities into a single molecule. Our study leverages samples from the phase I ACTG study of SAR441236 and investigates the mechanisms of its antiviral effects and the virus adaptation to this neutralization. We believe our approaches test previously unexplored hypotheses that are central to understand how, for the first time, trispecific bnAbs may be used as HIV therapeutics.