Evaluation of hyperstable HIV-1 Env trimers as immungens

One of the major obstacles to the development of a prophylactic HIV vaccine is believed to be the scarcity of functional envelope spikes compared to the relative abundance of non-functional ones. Moreover, irrelevant structures seem to be intrinsically immunodominant over neutralizing epitopes, hence distracting the immune system from developing neutralizing antibodies (nAbs). To date only few nAbs have been isolated, such as 2F5, 2G12, 4E10, b12, and Fab Z13e. They all target the envelope spike (Env), supporting the notion that this glycoprotein is the only viral protein accessible by the immune system. The present project is based on the development of Env libraries that contain mutations in regions that have been shown to be involved in spike stability (i.e. gp41-gp120 interactions or (gp41-gp120)-(gp41-gp120) interactions). The proposed in vitro mutagenesis approach moreover circumvents the influence of sequence censorship that would otherwise limit the use of viruses carrying disadvantageous mutations (Aim 1). In the next step, these Env libraries will be subjected to different selection pressures such as heat, denaturants, and soluble CD4, eventually yielding hyperstable mutants. These mutants will be compared to the parental virus strain and further characterized employing infectivity and virus capture assays, as well as Blue Native PAGE (Aim 2). In the final phase, the most stable mutants will be used to immunize rabbits in a DNA-prime/VLP-boost vaccination regimen. Sera will be characterized with respect to binding specificity as well as neutralizing potential (Aim 3). The results are expected to yield important insights that should take vaccine design a step closer to the development of functional immunogens capable of eliciting a protective immune response to HIV-1.