High-throughput mapping of antigen specificity to B-cell-receptor sequence for characterizing antibody responses in HIV-vaccinated and infected individuals

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Project Summary. The search for an effective HIV-1 vaccine remains a top priority, and a deeper understanding of how the immune system recognizes HIV-1 can help inform vaccine design. Lately, much effort has focused on understanding antibody responses to HIV-1 infection and vaccination, since antibodies have proven useful in therapy and prevention, and as templates for antibody-specific vaccine design. While antibody responses to HIV-1 are polyclonal and complex, advances in next-generation sequencing (NGS) technologies enable us to see such polyclonal responses at an unprecedented resolution, as a collection of individual monoclonal antibody sequences. Sequence identification is typically followed by functional antibody characterization, a primary component of which is the mapping of antigen/epitope specificity. A major challenge with the standard antibody analysis pipeline is that the sequence identification and functional characterization processes for antibodies are generally decoupled. This prevents truly high- throughput mapping of antibody-antigen specificity, providing only limited information for a small subset of selected antibody sequences from any given sample. To address this challenge, here we propose to develop a technology that, for a given sample, will enable the mapping of antibody sequence to antigen specificity from a single high-throughput experiment. The technology, LIBRA-seq (LInking B-cell Receptor to Antigen specificity through sequencing), involves physically mixing a B-cell sample with a (theoretically unlimited) pool of barcoded antigens, thus enabling the simultaneous recovery of: (i) paired heavy-light chain BCR sequences and (ii) antigen specificity for a given B cell. In particular, this technology development project will broadly focus on two specific aims: In Specific Aim 1, we will evaluate the effect of different antigen barcoding strategies and other assay variables on LIBRA-seq accuracy and performance. The goal in this aim is to optimize the LIBRA- seq ability to accurately detect BCR sequence and antigen specificity from a sequencing experiment. In Specific Aim 2, we will aim to simultaneously map the target epitope of a given HIV-specific B cell, by screening a cocktail of antigens with epitope-knockout mutations along with the wildtype antigens. These efforts will not only lead to the identification of HIV-specific B cells, but will also provide residue-level information about the specific epitope target on the antigen from the same high-throughput experiment. Ultimately, for a given infection or vaccination sample, the LIBRA-seq technology will provide the ability to recover antibody sequence and antigen specificity for tens to hundreds of thousands of B cells at the single- cell level. To demonstrate the utility of LIBRA-seq, we will characterize samples from HIV-1 infection and vaccination cohorts. More generally, LIBRA-seq will be an integral tool for efficient and accurate B-cell analysis, with the potential for broad impact on the fields of vaccine and antibody discovery not only for HIV-1 but also for a wide range of other pathogens of biomedical significance.