B Cell Repertoire Analysis

The Laboratory of B cell Repertoire Analysis is directed by Mattia Bonsignori, MD and begun its operations in 2009 with the scope of establishing technologies for the high-throughput screening of large numbers of B cells for human antibodyome analyses. The lab has focused their activity on identifying memory B cells from HIV-1-infected subjects to support the aims of the Duke Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID). In addition, the lab is identifying memory B cells from vaccine recipients who were enrolled in the RV144 Thai trial to support the efforts of the Collaboration for AIDS Vaccine Discovery (CAVD).

High-throughput screening of the memory B cell repertoire

The lab has developed an in vitro culture system to induce the rapid proliferation and differentiation of memory B cells cultured at clonal dilution into antibody-secreting cells. This system allows for the simultaneous screening of multiple specificities with functional tests using a single sample. The most relevant advantages of this system over pre-existing methods are that the VH + VL immunoglobulin (Ig) chain pairs can be isolated directly from cell cultures without the need for single-cell sorting or manual pairing and that memory B cells are preserved and remain available for further studies. By implementing semi-automated operations with a Biomek FXP, a BenchCel Microplate Handler, and an EL406 washer dispenser in its portfolio, the laboratory can culture ~60,000 memory B cells and screen 8 binding specificities from a single subject in 3 weeks.

Broadly neutralizing antibodies from HIV-1 chronically infected subjects

From the memory B cells of a Tanzanian HIV-1-infected elite neutralizer who was enrolled in the CHAVI001 protocol, we identified a clonal lineage of four quaternary structure-preferring V1/V2 broadly neutralizing antibodies (bNAbs), CH01 through CH04, and their unmutated common ancestor (UCA) antibodies. Envelope glycorproteins and scaffolded V1/V2 peptides were identified to bind to the mature and ancestral antibodies with affinity that was predicted to trigger B cell development. The identification of another clonal lineage of bNAbs, CH30 through CH34, in the same subject that were directed against the CD4bs represents the first evidence that multiple specificities of bNAbs can be naturally produced in vivo. We demonstrated that two bNAbs (CH01 and CH31) were sufficient to vastly recapitulate this subject’s plasma neutralizing activity and to achieve near pan-HIV-1 neutralization in vitro. Autologous neutralizing antibodies from HIV-1 acutely infected subjets From an HIV-1 acutely infected subject, we isolated two clonally related mAbs (CH83 and CH84) with strain-specific neutralization of the transmitted/founder virus. Binding to autologous Env was blocked by the bNAb PGT121. The two NAbs nearly recapitulated the polyclonal neutralizing reactivity in plasma as measured by site-directed mutagenesis neutralization studies. Identification of correlates of protection in vaccinated individuals The ALVAC-prime AIDSVax-boost RV144 HIV-1 vaccine efficacy trial induced a partial short-lived protection from HIV-1 infection. However, the antibodies induced by the vaccine did not demonstrate potent or broad neutralization and other mechanisms of protection have been hypothesized. In collaboration with the Laboratory of B cell Immunotechnology, we isolated 23 ADCC-mediating mAbs from 6 vaccine recipients (21 IgG and 2 IgA) directed against the C1, the V1/V2 and the V3 regions of the gp120 Env. One mAb, CH23, also mediated neutralization of tier-1 pseudoviruses, similar to two additional antibodies that were isolated from the same subjects (CH21 and CH22). The ADCC-mediating mAbs displayed a preferential usage of the VH1 gene family. The C1-directed mAbs could be blocked by A32, a mAb directed against a common target of ADCC-mediating mAbs during acute infection. The binding of two mAbs that were directed against the V1/V2 region (CH58 and CH59) was abrogated by the K169A substitution, the site of immune pressure identified by sieve analysis, and despite displaying only limited neutralizing activity, recognized an epitope overlapping that of V1/V2 bNAbs, such as PG9 and CH01. Crystal structures of CH58 and PG9 in complex with V2 peptides revealed that the two mAbs recognize different conformations of the same region. These observations raise the hypothesis that antibodies that are targeted at K169 in V2 may have played a role in providing vaccine-induced protection.