Isolation of neutralizing monoclonal antibodies against HIV-1 with predetermined conformational epitope specificity

Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs) from B cells. However, using the whole antigen for B-cell sorting implies a need for extensive screening of newly isolated mAbs, followed by epitope mapping. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes) that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs) that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs). To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. Using our strategy we isolated mAbs targeting the conformational V3 loop crown of HIV Env. The new mAbs showed strong dependence on epitope conformation and cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents.