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

• Published in: Proceedings of the National Academy of Sciences - PNAS 2020-11, Vol.117 (47), p.29584-29594
• Main Authors: Datta, Rohini, Chowdhury, Rohan Roy, Manjunath, Kavyashree, Hanna, Luke Elizabeth, Varadarajan, Raghavan
• Format: Article
• Language: English
• Subjects: AIDS Vaccines - immunology; Antibodies; Antibodies, Neutralizing - immunology; Antibody Formation - immunology; Antibody Specificity - immunology; Biological Sciences; Cell Line; Cysteine - immunology; Display devices; Env gene; env Gene Products, Human Immunodeficiency Virus - immunology; Epitope mapping; Epitope Mapping - methods; Epitopes - immunology; Gene sequencing; Genetics; Glycoproteins; HEK293 Cells; High-Throughput Nucleotide Sequencing - methods; HIV; HIV Antibodies - immunology; HIV Infections - immunology; HIV Seropositivity - immunology; HIV-1 - immunology; Human immunodeficiency virus; Humans; Immunization; Immunization - methods; Infections; Labeling; Mammalian cells; Mapping; Mutation; Neutralization; Neutralizing; Polyclonal antibodies; Residues; Vaccines; Virions; Viruses
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2010256117

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.