Method to identify efficiently cleaved, membrane-bound, functional HIV-1 (Human Immunodeficiency Virus-1) envelopes

Abstract 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.