PT - JOURNAL ARTICLE AU - Rohini Datta AU - Rohan Roy Chowdhury AU - Kavyashree Manjunath AU - Luke Elizabeth Hanna AU - Raghavan Varadarajan TI - A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing AID - 10.1101/2020.04.15.041996 DP - 2020 Jan 01 TA - bioRxiv PG - 2020.04.15.041996 4099 - http://biorxiv.org/content/early/2020/09/20/2020.04.15.041996.short 4100 - http://biorxiv.org/content/early/2020/09/20/2020.04.15.041996.full AB - Identification of specific epitopes targeted by neutralizing antibodies is essential to advance epitope-based vaccine design strategies. We report a facile methodology for rapid epitope mapping of neutralizing antibodies (NAbs) against HIV-1 Envelope (Env) at single residue resolution, using Cys labeling, viral neutralization assays and deep sequencing. This was achieved by the generation of a library of Cys mutations in Env glycoprotein on the viral surface, covalent labeling of the Cys residues using a Cys reactive label that masks epitope residues, followed by infection of the labeled mutant virions in mammalian cells in the presence of NAbs. Env gene sequencing from NAb-resistant viruses was used to accurately delineate epitopes for the NAbs VRC01, PGT128 and PGT151. These agreed well with corresponding experimentally determined structural epitopes previously inferred from NAb:Env structures. HIV-1 infection is associated with complex and polyclonal antibody responses, typically composed of multiple antibody specificities. Deconvoluting the epitope specificities in a polyclonal response is a challenging task. We therefore extended our methodology to map multiple specificities of epitopes targeted in polyclonal sera, elicited in immunized animals as well as in an HIV-1 infected elite neutralizer capable of neutralizing Tier-3 pseudoviruses with high titers. The method can be readily extended to other viruses for which convenient reverse genetics or lentiviral surface display systems are available.Competing Interest StatementRD and RV are inventors of a patent on mapping protein binding sites and conformational epitopes using Cys labeling (PCT WO/ 2018/104967).