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

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).