Bacterially expressed HIV-1 gp120 outer-domain fragment immunogens with improved stability and affinity for CD4 binding site neutralizing antibodies.

Protein-minimization is an attractive approach for designing vaccines against rapidly evolving pathogens such as HIV-1 since it can help in focussing the immune response towards conserved conformational epitopes present on complex targets. The outer domain (OD) of HIV-1 gp120 contains epitopes for a large number of neutralizing antibodies and therefore is a primary target for structure-based vaccine design. We have previously designed a bacterially expressed outer domain immunogen (OD) that bound CD4 binding site (CD4bs) ligands with 3-12µM affinity and elicited a modest neutralizing antibody response in rabbits. In this study， we have optimized OD using consensus sequence design， cyclic permutation and structure-guided mutations to generate a number of variants with improved yields， biophysical properties， stabilities and affinities (KD of 10-50 nM) for various CD4bs targeting， broadly neutralizing antibodies， including the germline reverted version of the broadly neutralizing antibody， VRC01. In contrast to OD， the optimized immunogens elicited high anti-gp120 titers in rabbits as early as 6 weeks post-immunization， before any gp120 boost was given. Following two gp120 boosts， sera collected at week 22 showed cross-clade neutralization of Tier 1 HIV-1 viruses. Using a number of different prime: boost combinations， we have identified a cyclically permuted OD fragment as the best priming immunogen， and a trimeric， cyclically permuted gp120 as the most suitable boosting molecule amongst the tested immunogens. This study also provides insights into some of the biophysical correlates of improved immunogenicity.