A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing

Abstract

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.

Mutational scan inferred binding energetics and structure in intrinsically disordered protein CcdA

“…DMS methods simultaneously evaluate functional features of large libraries, by exploiting variant enrichment following selection, or distribution of the variants amongst different bins based on a functional attribute. Several groups have successfully studied mutational effects on protein–protein interactions (PPIs) (DeBartolo et al, 2012; Ernst et al, 2010; Fowler et al, 2010; Fujino et al, 2012; McLaughlin et al, 2012; Starita et al, 2013; Whitehead et al, 2012) using DMS and attempted epitope mapping (Datta et al, 2020; Doolan & Colby, 2015; Kowalsky et al, 2015; Mata‐Fink et al, 2013; Najar et al, 2017; Van Blarcom et al, 2015) as well as quantifying interactions for large numbers of variants (Adams et al, 2016; Jenson et al, 2018).…”

Mutational scan inferred binding energetics and structure in intrinsically disordered protein CcdA

“…DMS methods simultaneously evaluate functional features of large libraries, by exploiting variant enrichment following selection, or distribution of the variants amongst different bins based on a functional attribute. Several groups have successfully studied mutational effects on protein-protein interactions (PPIs)(DeBartolo et al, 2012; Ernst et al, 2010; Fowler et al, 2010; Fujino et al, 2012; McLaughlin et al, 2012; Starita et al, 2013; Whitehead et al, 2012) using DMS and attempted epitope mapping(Van Blarcom et al, 2015; Datta et al, 2020; Doolan and Colby, 2015; Kowalsky et al, 2015; Mata-Fink et al, 2013; Najar et al, 2017) as well as quantifying interactions for large numbers of variants(Adams et al, 2016; Jenson et al, 2018).…”