Title
Bacterially expressed HIV-1 gp120 outer-domain fragment immunogens with improved stability and affinity for CD4 binding site neutralizing antibodies
Author
Ujjwal Rathore, Mansi Purwar, Venkada Subramanian Vignesh, Raksha Das, Aditya Arun Kumar, Sanchari Bhattacharyya, Heather E. Arendt, Joanne DeStefano, Aaron Wilson, Christopher Parks, Celia C. LaBranche, David C. Montefiori, Raghavan Varadarajan
Year
2018
Journal
Journal of Biological Chemistry
Abstract
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 (ODEC) 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 ODEC 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 ODEC, 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.
Instrument
FP-6300
Keywords
Fluorescence, Protein structure, Protein folding, Protein denaturation, Chemical stability, Biochemistry