(425h) High-Throughput Production of HIV-Specific Antibodies Identified Via Microengraving in the Yeast Pichia Pastoris

Efforts to develop a vaccine that confers sterilizing immunity against the human immunodeficiency virus type 1 (HIV-1) have thus far been unsuccessful, due in part to the virus’ ability to rapidly mutate and evolve to evade the host immune response.  However, it is known that a subset of HIV-infected individuals can develop broadly-reactive antibodies over time that are capable of neutralizing the virus, despite its attempts to escape the immune response.  Identifying and studying such antibodies in large numbers from a broad range of individuals would provide greater details regarding their characteristics and the evolution of these responses within individuals.  Such information could ultimately inform more effective vaccine designs.  Direct identification of B cells producing HIV-specific antibodies by flow cytometry has made it possible to profile the antibody responses of small numbers of patients, but the secondary validation of the antibodies is slow and tedious.  Molecular cloning of each antibody by traditional methods of plasmid construction and propagation in bacteria limits the throughput and scale of evaluating recombinant antibodies from a subject.  Here we present an integrated method for identifying, producing, and characterizing such broadly-neutralizing antibodies from HIV+ patient samples in high-throughput.  Our approach combines a microengraving method established by our lab with overlap-extension PCR (OE-PCR) to rapidly assemble antibody-expression constructs for expression in the yeast Pichia pastoris.  Validation of the approach and scalability will be presented as well as feasibility of automation for production of manifold antibodies from subjects for secondary characterization such as binding affinities and neutralizing activity.