Mapping the mutational landscape of CD4bs broadly neutralizing HIV antibodies

About This Grant

PROJECT SUMMARY Broadly neutralizing antibodies (bnAbs) that target the CD4 binding site (CD4bs) on the HIV envelope glycoprotein (Env) are an attractive target for an HIV vaccine and antibody-based therapy for HIV protection. However, eliciting CD4bs bnAbs in a vaccine is challenging, and current CD4bs bnAbs are insufficient to provide long-term HIV suppression. This difficulty in CD4bs bnAb-mediated treatment stems from Env mutations near the CD4bs referred to as “loop D,” including the gain of a glycan at N276 that has yet to be dependably overcome in vaccine regimens. Although improvements in antibody isolation have provided mechanistic and evolutionary insight into some CD4bs bnAbs, it remains poorly understood how CD4bs bnAbs evolve breadth and potency from their naïve precursors, complicating attempts to elicit them via vaccination. To address this issue, high-throughput deep mutational scanning (DMS) techniques will be used to characterize the effect of all possible single-amino acid mutations at each position in the variable domains of the VRC01 and IOMA CD4bs bnAbs that represent two stereotyped mechanisms of Env CD4bs recognition. VRC01 and IOMA variant libraries will be expressed on the cell surface of yeast. Using this yeast-surface display (YSD) platform, both VRC01 and IOMA variant binding affinities will be measured against various Env proteins using fluorescence-activated cell sorting and high-throughput sequencing (FACS-seq), facilitating the measurement of the binding properties of these bnAb libraries in parallel. Aim 1 will determine the mutations in CD4bs bnAbs that enable HIV Env N276 glycan accommodation. DMS, YSD, and FACS-Seq will be performed on libraries of mature VRC01 and its naïve precursor to determine their binding affinities to two closely related donor-autologous Env proteins that contain and lack the N276 glycan. These experiments will reveal routes to overcome common barriers in CD4bs bnAb development and illuminate how the next generation of immunogens will better elicit broad and potent bnAbs. Aim 2 will map the mutational landscape that confers CD4bs bnAbs breadth and potency. DMS, YSD, and FACS-seq will be performed on mature VRC01 and IOMA to measure the impact of all single mutations on binding to a previously established panel of 12 heterologous Env strains that comprise a reference of global Env diversity. These results will illustrate how mutations in these antibody scaffolds confer binding to divergent Env strains versus promoting strain-specific binding, fully defining the functional constraints and flexibilities of these CD4bs bnAb classes. The results of this proposal will illustrate a new approach to investigate the evolution and engineering of HIV CD4bs bnAbs, setting the stage for the development of improved immunogens and therapeutic antibodies against HIV. More broadly, the methodological innovation herein will provide a new platform for antibody engineering for the treatment and prevention of various infectious diseases.