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.

13Identification of specific epitopes targeted by neutralizing antibodies is essential to advance 14 epitope-based vaccine design strategies. We report a methodology for rapid epitope mapping 15 of neutralizing antibodies against HIV-1 Env at single residue resolution, using viral 16 neutralization assays and deep sequencing. We extended our methodology to map multiple 17 specificities of epitopes targeted in polyclonal sera, elicited in immunized animals as well as 18 in HIV-We recently described a methodology to decipher epitopes of monoclonal antibody panels 35 using yeast surface display, Cys labeling and deep sequencing 1, 2 . Here we built on this to map 36 epitopes of neutralizing monoclonal antibodies and polyclonal sera against the HIV-1 virus. 37 The design of an effective vaccine against HIV-1 has met with limited success so far, because 38 of the inability of immunogens to elicit broadly neutralizing antibodies (bNAbs) targeted to the 39 trimeric envelope (Env) glycoprotein, the sole viral antigen on the surface of the virus. Most 40 bNAbs isolated from infected individuals are directed to one of the major sites of vulnerability 41 on Env: V1/V2 loop apex, V3 loop, CD4-binding site, centre of the gp120 silent face, gp120-42 gp41 subunit interface, gp41 fusion peptide and membrane proximal external region (MPER) 43 of gp41. 44 We developed a facile methodology for mapping neutralizing HIV-1 epitopes at single residue 45 resolution. To this end, a library of single-site Cys mutations were engineered at selected 46 solvent-exposed sites in the Env glycoprotein on the surface of the virus. Cys mutants were 47 covalently labeled using a Cys reactive reagent (Maleimide-PEG2-Biotin) that masks epitope 48 residues, followed by infection of the labeled mutant viruses in mammalian cells in the 49 presence of bNAbs. Subsequently, deep sequencing of the env gene from bNAb-resistant 50 viruses was used to delineate epitopes ( Figure 1). We focused our epitope mapping efforts to 51 the Env ectodomain of the clade B, CCR5-tropic primary HIV-1 isolate JRFL, which has been 52 extensively characterized both biochemically and structurally, and is used as a model primary 53 virus. 54A total of 81 solvent-exposed residues (Table S1) were selected from the X-ray crystal structure 55 of the pre-fusion HIV-1 Env ectodomain using a combined criterion of >30% solvent 56 accessibility and sidechain-sidechain centroid distance >8 Å ( Figure S1). This led to the 57 identification of a set of solvent-exposed residues which collectively exhibit uniform surface 58 coverage of the Env trimer ( Figure S2). These Env residues were mutated to Cys in the pLAI-59 JRFL molecular clone, pooled in equimolar quantities to create a plasmid DNA library, and 60 transfected in HEK293T cells to produce the Exposed Cys Library, a library of single-site Cys 61 mutant viruses. Wt Env lacks free Cys residues. In single-cycle and multi-cycle infectivity 62 assays, the Exposed Cys Library retained significant viral infectivity ( Figure S3) and ...