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

Sweety Samal,Manish Bansal,Supratik Das

doi:10.1016/j.mex.2019.04.013

Sweety Samal, Manish Bansal + Show 1 more

Open Access

https://doi.org/10.1016/j.mex.2019.04.013

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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.

Highlights

The HIV-1 Env is a trimer of a heterodimer of the gp120 soluble and gp41 transmembrane subunits that is formed by proteolytic cleavage of the precursor gp160 polypeptide
It has been shown that efficient cleavage of the Env gp160 precursor into its constituent subunits co-relate with specific binding to bNAbs and weak binding to non-NAbs [1,2], properties essential in functional Envs suitable for vaccine immunogen design
Efficient cleavage of HIV-1 Env gp160 precursor polypeptide into its constituent subunits leads to formation of functional Envs which is co-related with their efficient binding to bNAbs and weak binding to non-NAbs

Summary

Method summary

The HIV-1 envelope (Env) glycoprotein is the sole target of broadly neutralizing antibodies (bNAbs). It has been shown that efficient cleavage of the Env gp160 precursor into its constituent subunits co-relate with specific binding to bNAbs and weak binding to non-NAbs [1,2], properties essential in functional Envs suitable for vaccine immunogen design Based on these studies a six step strategy (see below) was devised to identify naturally occurring, membrane-bound, efficiently cleaved Envs which show antigenic properties suitable for immunogen design. The plasma membrane protein immunoprecipitation assay has been developed based on the manufacturer’s protocol for isolation of the plasma membrane fraction (Abcam, Cat# ab65400) Using this six step process we have identified the naturally occurring, membrane-bound efficiently cleaved Envs A5 and BG505 (clade A), JRCSF (clade B), 4-2.J41 (clade C) and LT5.J4b12C (clade B/C) [6,7,8,9]

Method details

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Discussion