Strategies to Study HIV-1 Viral Escape from Broadly Neutralizing Antibodies In Vitro
Open Access DepositedBroadly neutralizing antibodies (bNAbs) targeting conserved regions of the HIV-1 envelope (Env) protein represent a promising avenue for HIV-1 cure strategies. However, the effectiveness of bNAb-based therapies is limited by the rapid emergence of viral escape. A comprehensive understanding of escape pathways across diverse viral species is critical to designing robust bNAb combinations. To address this need, we optimized a high-throughput in vitro viral escape assay capable of capturing the pathways of resistance across a range of HIV-1 isolates and bNAbs.Optimization of the in vitro assay was performed by testing different viral stocks, including donor-derived viral stocks and infectious molecular clones (IMCs), varying antibody concentrations and multiplicity of infection (MOI). An MOI of 1 was determined to be optimal for accumulation of escape mutations while preserving viral replication dynamics. A 56-day assay protocol was established, during which antibody concentrations were gradually increased to mimic selective pressure, enabling longitudinal observation of viral escape. In preliminary experiments, escape from CD4 binding site (CD4bs) bNAb VRC01 was studied with the loss of the N276 glycan conferring resistance. When tested against a panel of pseudoviruses, this mutation conferred differential resistance or sensitivity depending on the Env and specific bNAb used, illustrating the nuanced interplay between glycosylation patterns in viral resistance to CD4bs bNAbs. This methodology was then applied to study non-subtype B escape pathways. Viruses isolated from the RV329 AFRICOS cohort were sequenced, subtyped, and evaluated for sensitivity to a panel of nine clinically relevant bNAbs. Sensitive bNAb and virus pairings were used in our in vitro escape assay to study escape pathways. Resistance was observed by day 14 in some cultures, with complete resistance usually emerging by day 56. CD4bs-directed bNAbs demonstrated the longest time to resistance, averaging 49.4 days, with bNAb 1-18 showing the most resistance to viral escape. Sequencing of escape variants revealed mutations localized within known antibody binding epitopes and potential compensatory mutations to maintain fitness during escape. This work provides an optimized model for investigating HIV-1 escape from bNAbs in vitro. This method enables detailed analysis of escape kinetics, Env-specific resistance pathways, and the influence of viral diversity on HIV-1 escape from bNAbs. These findings will contribute to the design of bNAb combinations in downstream in vivo work, thereby informing future clinical strategies aimed at durable HIV-1 suppression or cure.
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