Maturation Pathways for the Broad and Potent Antibody 10E8 Using a Combination of Next Generation Sequencing, Bioinformatics and Functional Analysis

Abstract

OA06.06 Background: Antibodies that target the membrane-proximal external region (MPER) of the HIV-1 gp41 subunit represent one of the few categories of antibodies that effectively neutralize HIV-1. Such antibodies provide potential templates to guide vaccine strategies, but details of their generation and maturation have been unclear. Methods: From the PBMCs of a single time point of donor N152, we used 454 Pyrosequencing to obtain the sequences of the variable domains of heavy and light antibodies from B cell transcripts. Bioinformatics sieving of gene families and structure-based sequence signatures delineated potential transcripts from the maturation pathway of the effective MPER-directed antibody 10E8. We calculated maturation intermediates along these pathways, synthesized heavy and light chains, and used transient transfection to express antibodies, which we assessed for neutralization and for MPER-peptide affinity. Results: We identified 235 heavy chain sequences with V, D and J segments matching the mature 10E8 antibody and 147,691 light chain sequences with V and J segments matching the mature 10E8 antibody. These sequences were further sieved with structural signatures. Pathways - based on maximal intermediates, minimal sequence reversions, and minimal N-nucleotide additions - for heavy and light chain were identified, and we paired heavy and light chain sequences for the unmutated common ancestor (UCA) along with three inferred heavy and light chain sequences. Unexpectedly, the CDR H3 of the UCA differed substantially from the mature sequence. Nonetheless, all of the paired intermediates, including the UCA, showed binding to MPER peptide, while only the two most mature intermediates showed neutralization. Binding to MPER in the context of lipid micelles, meanwhile, mirrored that of neutralization. Conclusions: The combination of cross-sectional NGS data, bioinformatics, and functional analysis can be used to define the maturation pathways for an antibody, even with cross-sectional data.