Use of Enzyme-digested Virus-like Particles as Probes for Flow Cytometric Sorting of HIV-specific Neutralizing Ab-producing B-cells

Abstract

OA06.04 Background: Isolating new broadly neutralizing antibodies (bnAbs) provides fresh insights for rational HIV-1 vaccine design. Several sites of vulnerability to bnAbs are now well defined, and it is likely that additional targets exist. Virus-like particles (VLPs) that express Env trimers display all of the natural epitopes and are therefore attractive candidates for use as probes. However, VLP surfaces are also decorated with nonfunctional forms of HIV Env. Exposing VLPs to proteases clears nonfunctional Env, leaving native trimeric Env intact. The resulting “trimer-VLPs” preferentially bind to nAbs compared to non-neutralizing antibodies and therefore may have gained sufficient selective power for use as B cell probes to identify new bnAbs. Methods: PBMC from HIV-infected patients were stained with the enzyme-digested trimer-VLPs, and VLP-positive memory B cells were sorted by flow cytometry. Multiplex RT-PCR was performed on cell lysates to amplify kappa, lambda, and heavy chain genes. Cloned antibodies were assessed for neutralization breadth and potency against a panel of Env-pseudoviruses using the TZM-bl neutralization assay. Results: VLPs were used to sort B cells from the donor of the broad CD4bs antibody VRC13. The sort yielded relatives of VRC13, as well as unrelated sequences that exhibited high sequence divergence from germline VH genes and/or long CDR3 regions, which are characteristics of known bnAbs. Cloning and characterization of these Abs is ongoing. Conclusions: Unlike many previously used B cell probes, VLPs express multiple epitopes including those for trimer specific antibodies which allows for simultaneous sorting of B-cells of different specificities. Additionally, these probes are devoid of non-functional forms of Env and therefore preferentially bind to neutralizing Ab-producing B cells. VLPs therefore hold great promise for isolating novel HIV-specific bnAbs that can better inform vaccine design.