CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

242 A BROAD POLYCLONAL RESPONSE IS MEDIATED BY SUPERINFECTION WITH 2 DISTINCT VIRUSES Bingjie Wang 1 , Katherine L. Williams 1 , Valerie Cortez 1 , James A. Williams 2 , Kelly K. Lee 2 , R. Scott McClelland 2 , Julie Overbaugh 1 1 Fred Hutchinson Cancer Rsr Cntr, Seattle, WA, USA, 2 Univ of Washington, Seattle, WA, USA

Background: While broadly neutralizing antibodies (bNAbs) develop in a subset of HIV-infected individuals, the contribution of viral diversity to this process remains poorly defined. Individuals who have been infected with two or more distinct viral variants develop a broader and more potent neutralizing antibody repertoire than singly-infected individuals. Thus, superinfection provides a unique setting to examine the dynamic relationship between bNAb development and viral evolution. Methods: We isolated and characterized neutralizing antibodies (NAb) from an individual initially infected with a subtype D variant and superinfected with a subtype A variant 11 months later. IgG-producing B cells were sorted and cultured from 2282 days post-initial infection (dpi) and HIV-specific B cells were identified using microneutralization assays. Antibody breadth was determined using a cross-clade panel of viruses. Epitope specificity of the broadest NAb was identified using negative stain electron microscopy and confirmed using viruses with mutations at the known target site. Autologous viral variants were identified using single genome PCR. Results: Six NAbs were isolated and together recapitulated 53% of plasma breadth against a cross-clade panel of nineteen Tier 1, 2, and 3 viruses. Five of six antibodies exhibited limited neutralizing activity while one antibody, QA013.2, neutralized 42% of viruses tested in a larger panel of 31 HIV variants that includes the NIH global reference panel. This NAb is dependent upon the N332 residue in the V3 loop of gp120 and has a predicted germline gene usage pattern unique from other N332-dependent NAbs with 21% divergence in the VH gene and no indels in either the VH or VL genes. Thirty-four autologous viral variants were cloned from five timepoints following initial infection to explore the development of these antibody lineages. Two of the six NAbs originated from the same clonal lineage and preliminary data indicate that they neutralized a single clade D variant isolated from 287 dpi. In contrast, the more potent NAb QA013.2 strongly neutralized the superinfecting clade A viruses isolated at 385, 765, and 987 dpi while the remaining three NAbs neutralized one virus isolated at 385 dpi and three viruses isolated at 765 dpi. Conclusion: These data provide strong evidence for development of a polyclonal antibody response resulting from superinfection with viruses from two different HIV clades. 243 HIGH-RESOLUTION MAPPING OF AN ENV EPITOPE BY COMPREHENSIVE ESCAPE MUTANT SELECTION Adam Dingens , Hugh Haddox, Julie Overbaugh, Jesse Bloom Fred Hutchinson Cancer Rsr Cntr, Seattle, WA, USA Background: A detailed understanding of broadly neutralizing antibody (bnAb) epitopes is vital to designing vaccines that target these sites of vulnerability, as well as using bnAbs as therapeutics and prophylactics. Current functional epitope mapping strategies are labor-intensive and derive from interrogation of single mutations at selected positions of Env. Methods: We have developed a deep mutational scanning approach, which we termmutational antigenic profiling, to comprehensively map the functional epitopes of anti-Env antibodies in an unbiased fashion. This approach involves generating libraries containing viruses with nearly all amino acid mutations in Env, incubating these libraries with an antibody, and then infecting target cells. Finally, we use deep sequencing to quantify the effect of each mutation on neutralization phenotype by comparing its frequency in the selected library to a control library not treated with antibody. Results: To validate this technique, we profiled a well-mapped bnAb, PGT151, with mutant libraries of a transmitted clade A Env, BF520. Antibody selection strongly enriched for escape mutants at a variety of sites known to be in or near the PGT151 epitope, including loss of key glycans at sites 611 and 637, sites in the fusion peptide, and sites near the gp120-gp41 interface. These precisely corresponded to epitope information known from cryo-EM and neutralization assays with panels of alanine scanning mutants. Further, we have better defined PGT151’s epitope by identifying escape mutants at sites that have not been tested previously, as well as at sites where alanine scanning has been shown to not affect neutralization phenotype, but more biochemically dissimilar amino acids were enriched after selection in our experiments. Comparing amino acids that are or are not enriched suggests potential mechanisms of escape. For example, many mutations that result in loss of glycosylation at site 611 are consistently enriched, but primarily positively charged mutants are enriched in a stretch of residues in the heptad repeat 2 (HR2) that neighbors the CDRH3 of bound PGT151. Conclusion: Mutational antigenic profiling not only identified known sites of the epitope with high accuracy, it also identified new positions that may contribute to PGT151’s functional epitope. This approach has the potential to enable rapid, comprehensive, and unbiased functional epitope mapping of HIV-1 Env antibodies, as well as complete identification of pathways of viral escape.

Poster and Themed Discussion Abstracts

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CROI 2017