Viral variants that initiate and drive maturation of V1V2-directed HIV-1 broadly neutralizing antibodies.

CAPRISA, 2015.Abstract available in pdf

Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies.

CAPRISA, 2014.Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development

Tracking B-cell repertoires and clonal histories in normal and malignant lymphocytes

Methods for tracking B-cell repertoires and clonal history in normal and malignant B-cells based on immunoglobulin variable region (IGV) gene analysis have developed rapidly with the advent of massive parallel next-generation sequencing (mpNGS) protocols. mpNGS permits a depth of analysis of IGV genes not hitherto feasible, and presents challenges of bioinformatics analysis, which can be readily met by current pipelines. This strategy offers a potential resolution of B-cell usage at a depth that may capture fully the natural state, in a given biological setting. Conventional methods based on RT-PCR amplification and Sanger sequencing are also available where mpNGS is not accessible. Each method offers distinct advantages. Conventional methods for IGV gene sequencing are readily adaptable to most laboratories and provide an ease of analysis to capture salient features of B-cell use. This chapter describes two methods in detail for analysis of IGV genes, mpNGS and conventional RT-PCR with Sanger sequencing

The Effects of Somatic Hypermutation on Neutralization and Binding in the PGT121 Family of Broadly Neutralizing HIV Antibodies

Broadly neutralizing HIV antibodies (bnAbs) are typically highly somatically mutated, raising doubts as to whether they can be elicited by vaccination. We used 454 sequencing and designed a novel phylogenetic method to model lineage evolution of the bnAbs PGT121–134 and found a positive correlation between the level of somatic hypermutation (SHM) and the development of neutralization breadth and potency. Strikingly, putative intermediates were characterized that show approximately half the mutation level of PGT121–134 but were still capable of neutralizing roughly 40–80% of PGT121–134 sensitive viruses in a 74-virus panel at median titers between 15- and 3-fold higher than PGT121–134. Such antibodies with lower levels of SHM may be more amenable to elicitation through vaccination while still providing noteworthy coverage. Binding characterization indicated a preference of inferred intermediates for native Env binding over monomeric gp120, suggesting that the PGT121–134 lineage may have been selected for binding to native Env at some point during maturation. Analysis of glycan-dependent neutralization for inferred intermediates identified additional adjacent glycans that comprise the epitope and suggests changes in glycan dependency or recognition over the course of affinity maturation for this lineage. Finally, patterns of neutralization of inferred bnAb intermediates suggest hypotheses as to how SHM may lead to potent and broad HIV neutralization and provide important clues for immunogen design.National Institutes of Health (U.S.) (Bioinformatics and Integrative Genomics Fellowship)National Institutes of Health (U.S.) (Center for Excellence in Genomic Science