Multiplexed Epitope Mapping of HIV-1 Neutralizing Antibodies by Coupling Chemical Labeling of Cysteines to Deep Sequencing.

Identification of neutralizing antibody (NAb) binding sites or epitopes on an antigen is a prerequisite for epitope-focused vaccine design strategies. HIV-1 infection is associated with polyclonal antibody responses comprising NAbs that target multiple epitopes on the envelope glycoprotein (Env), the primary target of the immune response. Current epitope mapping methods, such as X-ray crystallography and cryo-EM microscopy that rely on purified antigen-antibody complexes, fail to reliably deconvolute epitope specificities of polyclonal HIV-1 antibodies. We describe a method to map antigen-antibody binding sites at single residue resolution, using chemically masked cysteines coupled to deep sequencing. This was achieved by generating a panel of cysteine mutants of the HIV-1 Env antigen on the viral surface, followed by chemical labeling of cysteines using Cys-reactive probes that block antibody binding. Epitopes are inferred using assays that monitor viral infectivity in the absence and presence of NAbs, followed by deep sequencing. We successfully applied this technique to distinguish residues at the epitope from residues lying outside the epitope for several NAbs. The methodology is also able to accurately map epitopes of polyclonal NAbs from HIV-1-infected individuals and HIV-1-immunized animals. Here, we describe protocols to facilitate adoption of this methodology. Knowledge of polyclonal epitope specificities can provide useful insight into the nature of the antibody response elicited by the virus, which can be used to design better immunogens against HIV-1 and emerging viral pathogens.