Expression of a functional B cell receptor with the specificity of VRC01, a broadly neutralizing antibody against HIV (159.8)

Abstract

Abstract VRC01, a CD4-binding site antibody that neutralizes over 90% of HIV-1 isolates has recently been identified. One of its unusual features is that the variable domain goes through extensive somatic hypermutation (30%) from its germline precursors, which leads to greatly enhanced binding affinity and neutralization capacity. To understand the somatic hypermutation process of VRC01 during B cell development and to identify immunogens that drive the affinity maturation of VRC01, the B cell receptor (BCR) form of VRC01 and its germline precursor 02*02 was expressed and examined for antigen-binding, BCR microclustering and BCR signaling. Co-expression of surface IgM and kappa chain transgenes was detected on 293T and Ramos cells by either transfection or lentivirus-mediated transduction. The transgenic VRC01 BCR bound to different gp120 variants as determined by flow cytometry. The VRC01 BCR also exhibited higher binding affinity to the gp120 Resurfaced Stabilized Cores (RSC3) presented on lipid bilayers than to the mutant form ΔRSC3 as indicated by more BCR microclusters upon binding to RSC3. Stimulation of VRC01-BCR-expressing B cells with RSC3 or anti-IgM induced phosphorylation of the B cell signaling tyrosine kinases Fyn and Syk. In conclusion, a functional VRC01-BCR was successfully expressed on 293T and B cells. This transgenic system allows us to test the feasibility of guiding the maturation of VRC01 with appropriate antigens.