Protocol to identify and monitor key mutations of broadly neutralizing antibody lineages following sequential immunization of Ig-humanized mice

Xuejun Chen; Stephen D Schmidt; Hongying Duan; Nicole A Doria-Rose; John R Mascola

doi:10.1016/j.xpro.2022.101180

Protocol to identify and monitor key mutations of broadly neutralizing antibody lineages following sequential immunization of Ig-humanized mice

STAR Protoc. 2022 Feb 18;3(1):101180. doi: 10.1016/j.xpro.2022.101180. eCollection 2022 Mar 18.

Authors

Xuejun Chen¹, Stephen D Schmidt¹, Hongying Duan¹, Nicole A Doria-Rose¹, John R Mascola¹

Affiliation

¹ Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Abstract

Using the VRC01-class of anti-HIV-1 broadly neutralizing antibodies (bnAbs) elicited in sequentially immunized Ig-humanized mice as an example, we describe a protocol to identify key mutations for bnAb function by point mutagenesis and antibody binding and neutralization assays. We also describe steps to monitor how the key mutations arise in response to specific immunogens, which is critical for vaccine evaluation and design, via longitudinal antibody mutation profiling. This protocol can be customized for other V-gene-specific bnAbs and animal models. For complete details on the use and execution of this profile, please refer to Chen et al. (2021).

Keywords: Antibody; Bioinformatics; Flow Cytometry/Mass Cytometry; Immunology; Model Organisms; Molecular Biology; Protein expression and purification; Sequence analysis.

Publication types

Research Support, N.I.H., Intramural

MeSH terms

Animals
Antibodies, Neutralizing
Broadly Neutralizing Antibodies
HIV Antibodies
HIV Infections*
HIV-1* / genetics
Immunization
Immunoglobulins / immunology
Mice
Mutation

Substances

Antibodies, Neutralizing
Broadly Neutralizing Antibodies
HIV Antibodies
Immunoglobulins