Novel Strategies to Improve Production of a Full Length Native HIV Env Spike

Project Summary Production of HIV envelope protein Env trimeric gp cleaved to gp gp for clinical studies is a critical objective for vaccine development Its conformational plasticity which allows it to adopt multiple conformational states important for function often makes its expression and purification very difficult Our recent work shows a route for overcoming this problem First we have identified two full length gp s from difficult to neutralize HIV primary isolates that are conformationally homogenous when expressed on the surface of human cells Their antigenic properties correlate closely with antibody neutralization Second truncation of the gp cytoplasmic tail CT has minimal impact on the activity of Env as a fusogen but has an unexpected role in constraining the conformational variability of the ectodomain Third we have determined an atomic structure of the HIV Env transmembrane domain TMD which is a well ordered helical trimer Introduction into Env of a set of mutations that disrupts the TMD trimer alters antibody sensitivity Thus both the TMD and the CT contribute to Env stability and antigenicity In Phase I of this project we will build on the three findings to identify specific structural determinants of HIV Env stability and expression consistent with its native antigenic properties We will combine these structural determinants along with structure based modifications in the TMD to develop a general strategy for high yield production in mammalian cells of essentially any HIV Env regardless of isolate or clade We will also explore various expression vectors and cell lines to establish a robust production pipeline In a projected Phase II we will develop a purification scheme suitable for producing clinical grade materials We will also evaluate immunogenicity of those Envs in animal models The Phase I Specific Aims include to identify structural determinants in HIV Env that have significant impact on its stability and expression to develop a general strategy for mammalian cell expression of full length HIV Env trimers with native antigenic properties optimize expression conditions to maximize the yield of full length native HIV Env By the end of Phase I of this proposal we expect to establish an expression system with high yield andgt fold increase in protein levels over current well expressed variants of full length HIV Env trimers derived from many HIV isolates Such a system will provide a solid foundation for future Phase II studies to produce HIV Env trimer immunogens suitable for clinical trials Project Narrative Production of a recombinant form of HIV envelope protein Env that corresponds closely to its native structure found on the surface of virion in large quantities suitable for clinical studies remains a major technical hurdle for vaccine development In this proposal we will build on our recent unexpected discoveries to establish a robust pipeline for production of full length HIV Env trimers derived from many HIV isolates