An ideal vaccine against HIV-1 will specifically elicit bNAbs (broadly neutralizing antibodies) which can cross-neutralize a wide spectrum of circulating viral strains belonging to different clades. The current paradigm for developing such a vaccine is to generate HIV-1 envelope (Env)-based immunogens which can specifically elicit bNAbs. For this purpose, it is necessary to identify Envs, belonging to different clades, suitable for immunogen design. Efficient cleavage of the HIV-1 Env precursor gp160 polypeptide into its constituent subunits determines its ability to selectively bind to bNAbs and poorly to non-NAbs (non-neutralizing antibodies), properties desirable in Env-based immunogens. Thus, efficiently cleaved HIV-1 Envs with desirable antigenic properties can be good candidates for developing immunogens. Here we describe in detail a six step method we have used in our laboratory to identify such efficiently cleaved Envs. Some of these protocols are optimizations of previously reported assays such as FACS-based cell surface antibody binding assay, pseudovirus neutralization assay and gp120 shedding assay. Other protocols like biotinylation-neutravidin-agarose pull-down assay and plasma membrane protein immunoprecipitation assay have been developed by taking inputs from reagent/kit manufacturer's protocols and previous studies. These protocols will help the field in identifying more such Envs which can be used for immunogen development. •Six step process to identify efficiently cleaved, membrane-bound, functional HIV-1 Envs with high degree of repeatability.•Method applicable for characterizing any HIV-1 envelope protein.•New method of immunoprecipitation of plasma membrane fraction to validate efficiently cleaved HIV-1 envelopes.
Keywords: Biochemistry; Biotinylation; Broadly neutralizing antibodies; FACS; Identifying cleaved, functional HIV-1 Envs; Immunoprecipitation; Neutravidin-agarose pull down; Non-neutralizing antibodies; Plasma membrane fraction; Pseudovirus neutralization; gp120 shedding.