PROJECT SUMMARY/ABSTRACT There is as yet no effective vaccine to HIV despite concerted efforts by numerous groups worldwide to produce one. The target for vaccines is the envelope spike protein (gp120/gp41), which mediates binding to receptors on the surface of CD4 T cells. The problem from a vaccine perspective is that the spike protein is heavily glycosylated, with up to 25 or more N-linked glycans, creating a `glycan shield' that prevents an immune response to the underlying protein. Yet, many HIV infected individuals develop broadly neutralizing antibodies (bnAbs) that utilize glycans as part of their binding epitopes or accommodate glycans for binding to conserved features of the underlying protein. In the past project period, we have developed methods for analyzing site specific glycosylation of the N-linked glycans of the envelope protein produced recombinantly and isolated from virus grown in peripheral blood lymphocytes. We have observed that site specific glycan processing of native virus envelope trimer differs significantly from the corresponding SOSIP trimers being evaluated as vaccine candidates. In this project we will increase the sensitivity of the methods to analyze glycosylation of native virus from blood of HIV patients. For representative strains, we will develop soluble trimers that have glycosylation similar to the envelope of native virus and compare them with SOSIP stabilized trimers for their ability to elicit neutralizing antibodies in animal models. The results will help assess the importance of establishing native-like glycosylation for optimally eliciting neutralizing antibodies of HIV.