Designing fixed divalent antigens for targeting VRC01 germline BCR 3393

Abstract Description A major goal for HIV vaccine design is immune focusing- the ability to target specific B cells for activation and proliferation in germinal centers to generate broadly neutralizing antibodies (bnAbs). We hypothesize that by presenting the HIV gp120 core antigen as a fixed-divalent antigen we can selectively target BCRs with high avidity, divalent binding and impart a survival advantage to these B cells. We have chosen the bnAb VRC01 as our model system. To fix two copies of the antigen, covalent immune complexes (ICs) were designed using 48d, a human antibody that targets the coreceptor binding site of gp120. Circularly permutated gp120 core was fused to the 48d heavy chain by a disulfide bond and light chains were crosslinked by mutating the loop containing Lys126, with K126C, to form Fab-Fab-crosslinked IC (FF-IC). Gel shift assays demonstrate 1:1 binding between both non-FF-IC and FF-IC whereas A32, which targets a different site on gp120, binds with 1:1 stoichiometry for non-FF-IC and 2:1 stoichiometry with FF-IC. Germline revertant of VRC01 binds to FF-IC with high avidity whereas a mutant of A32 having similar monovalent affinity binds weakly. Collectively, these results indicate that the IC design preferably binds to VRC01 forming a high avidity, 1:1 ring dimer and A32 with low avidity, 2:1 stoichiometry. Immunogenicity studies in mice may demonstrate the utility of this strategy in generating a favorable immune response. Funding Sources Supported by NIH/NIAID 1R21AI181698-01A1, and the Univ. of Hawaii, Office of Innovation and Commericalization with funding from the Office of Naval Research Topic Categories Vaccines and Immunotherapy (VAC)