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

Datta, R and Chowdhury, RR and Manjunath, K and Hanna, LE and Varadarajan, R (2020) A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing. In: Proceedings of the National Academy of Sciences of the United States of America, 117 (47). pp. 29584-29594.

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Official URL: https://doi.org/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. © 2020 National Academy of Sciences. All rights reserved.

Item Type: Journal Article
Publication: Proceedings of the National Academy of Sciences of the United States of America
Publisher: National Academy of Sciences
Additional Information: The copyright for this article belongs to the Author(s).
Keywords: cysteine; epitope; neutralizing antibody; cysteine; epitope; Human immunodeficiency virus antibody; Human immunodeficiency virus vaccine; neutralizing antibody; virus envelope protein, antibody response; Article; controlled study; gene sequence; human; human cell; Human immunodeficiency virus 1; Human immunodeficiency virus infection; mammal cell; matrix assisted laser desorption ionization time of flight mass spectrometry; nonhuman; priority journal; real time polymerase chain reaction; reverse genetics; sequence analysis; site directed mutagenesis; virion; virus envelope; virus infectivity; virus load; virus neutralization; virus strain; antibody production; antibody specificity; cell line; epitope mapping; HEK293 cell line; high throughput sequencing; Human immunodeficiency virus 1; immunization; immunology; procedures, AIDS Vaccines; Antibodies, Neutralizing; Antibody Formation; Antibody Specificity; Cell Line; Cysteine; env Gene Products, Human Immunodeficiency Virus; Epitope Mapping; Epitopes; HEK293 Cells; High-Throughput Nucleotide Sequencing; HIV Antibodies; HIV Infections; HIV Seropositivity; HIV-1; Humans; Immunization
Department/Centre: Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited: 10 Jan 2023 06:54
Last Modified: 10 Jan 2023 06:54
URI: https://eprints.iisc.ac.in/id/eprint/79003

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