Serum-Saving Systems Serology Screen for Functional Antibody Profiling

Abstract

Vaccine-induced antibodies confer protection in part through neutralization, whereby antibodies bind to viruses, bacteria, or fungi and block pathogen attachment to host cell receptors or entry into host cells. Increasingly, studies have shown the importance of non-neutralizing functions of antibodies, such as monocyte and neutrophil phagocytosis, complement deposition, and cytotoxicity. Identifying the capacity of vaccine candidates to elicit such antibody-dependent immune effector functions against target pathogens is thus necessary for complete evaluation of vaccine responses. Because there is a substantial number of antigen targets against which vaccine recipients might have antigen-specific antibodies, and clinical serum sample volumes are limited, high-throughput antibody-profiling tools linking many specificities to functions are needed. This thesis describes the development of an approach to interrogate the functional activity of antibodies in serum against 3 to 5 antigen targets simultaneously. Existing single-bead based assays used to measure the capacity of antibodies to induce phagocytosis were adapted to be compatible with 3 to 5 different fluorescently colored beads. This allows for concurrent investigation of antibody responses directed against multiple antigens for a single sample using as little serum as possible. This high-throughput assay was used to measure Fc-mediated antibody responses against an array of pathogens, including antigens derived from HIV, influenza, herpes-simplex virus 1 and 2, and Ebola. To demonstrate the robustness of the multiplexed assays and their wide-reaching applications, multiplexed phagocytosis assays were applied to probe differences in antibody-mediated effector functions between HIV controllers and progressors against HIV antigens gp120, gp41, and p24. Multiplexed phagocytosis assays accurately detected known differences in antibody function between distinct clinical sample groups, while simultaneously showing consistent trends across individuals for antibody-dependent effector functions directed against control antigens, ¬influenza and Ebola. Together, the findings show that multiplexing allows for accurate and more efficient evaluation of antibody-dependent monocyte and neutrophil phagocytosis.