Dissecting Polyclonal Sera to Reveal Correlates of Productive Immune Responses to HIV

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Project Summary/Abstract Rational, structure-based immunogen design for HIV vaccine development has began to deliver promising results in both, pre-clinical non-human primate (NHP) and clinical studies such as the IAVI G001 phase I trial [NCT03547245]. Moreover, advances in single cell RNAseq and electron microscopy-based polyclonal antibody mapping (EMPEM) have enabled analysis of both vaccine and pathogen-induced antibody responses at unprecedented resolution, further enabling rational vaccine design. EMPEM and single cell B cell analysis deliver fundamentally different data formats that if properly integrated, can illuminate immune responses at unprecedented detail. We recently extended the original negative stain EMPEM method, capable of distinguishing polyclonal antibody epitope specificities and angle of approach, to high resolution cryoEM-based EMPEM to yield molecular details of epitope-paratope interfaces. In the best cases we can derive some sequence information for the complementarity determining regions (CDRs) from structural data, identify Vh/Vl gene usage, and query databases of antibody sequences to directly generate monoclonal antibodies. As such, we can short cut the laborious efforts to generate epitope specific monoclonal antibodies using conventional methods. These molecular details are critical for analyzing shepherding or polishing immunization strategies that are now being attempted for HIV vaccine development, wherein immunogens are designed to elicit and mature antibodies with specific features in CDRs that resemble known broadly neutralizing antibodies (bnAbs). In this renewal application we propose to extend the resolution, throughput, and utility of our EMPEM approach. Moreover, since most pathogens will enter the body through mucosal surfaces, analysis of (vaccine-induced) mucosal antibodies will be explored. The data generated will provide new correlates of productive immune responses to the portfolio of exciting HIV immunogens with epitope focusing or germline targeting properties that will help down select and prioritize amongst the various approaches being pursued.