Development of microanalysis tools for characterization of the humoral response to infections diseases

Abstract

Antibodies are higher order protein structures produced by a subset of lymphocytes (B cells) in the immune system for protection against pathogenic species. These homodimers of heterodimers form highly specific interactions with their cognate antigens and hence have become very important for the development of prophylactic or therapeutic agents against different disease pathogens. A key step in the development of human monoclonal antibodies as therapeutics is identification of candidate antibodies either by direct screening of human antibody repertoires or by filtering through combinatorial libraries of human variable genes using display technologies. Combinatorial libraries of human variable genes afford the flexibility to pursue many targets of interest, but often result in the selection of low affinity antibodies or unnatural heavy and light chain pairings that would have been selected against in vivo. With direct screening of the human B cell repertoire, the challenge is how to efficiently isolate clones of interest. Presented in this thesis is a high-throughput, integrated, single-cell methodology based on microengraving that allows the rapid recovery of antigen-specific human B cells. Microengraving is an analytical technique wherein secreted molecules from individual cells seeded into a dense array of subnanoliter wells are captured on the surface of a glass slide, generating a microarray from which desirable cells can be identified and recovered. Combined with high resolution epifluorescence microscopy and single-cell RT-PCR, we have developed assays for the simultaneous profiling of surface-expressed phenotypes of primary antibodyproducing cells, as well as functional characteristics of their secreted antibodies and germline variable gene usage. Using clinical samples from HIV- and West Nile virus-infected subjects, we demonstrate that the method can identify antigen-specific neutralizing antibodies from both plasmablast/ plasma cell and memory B cell populations, and is ideal for the detailed characterization of cells from anatomical sites where sample sizes are often limited and disease pathophysiology is poorly understood (e,g. gut tissue, bone marrow).