The overall objective of Core D: Functional Antibody Study Core is to provide a consistent, experimentally controlled and validated platform for evaluating functional antibody responses after vaccination with Neisseria gonorrhoeae antigens that are identified through the projects of the Gonorrhea Vaccine Cooperative Research Center (GV CRC). Serum bactericidal activity and opsonophagocytosis have been implicated in the protective immune response against pathogenic Neisseria, and both depend upon antibody binding to the bacterial surface. Evaluation of these three parameters of the functional antibody response is critical to understanding if a vaccine has the potential to elicit protective immunity. Therefore, Core D is critical to the success of the GV CRC. Core D will provide services to all four research projects and will interact routinely with the other Cores of this CRC, which offer distinct yet complementary expertises. For Core D to enable successful completion of the projects in the GV CRC, we propose three Specific Aims: 1) Bacterial Antibody Surface Binding: We will use imaging flow cytometry to quantify the ability of antibodies in sera from immunized mice to bind to the surface of intact N. gonorrhoeae. Bacteria of diverse strain backgrounds will be evaluated. 2) Serum Bactericidal Activity (SBA): We will measure the ability of antibodies in sera from immunized mice or humans immunized with N. meningitidis serogroup B vaccine to elicit SBA against a panel of N. gonorrhoeae strains, using antibody-depleted pooled normal human serum as the source of active complement. In addition to conventional colony count, we will pilot and optimize a high-throughput quantitative assay using a fluorometric metabolic dye as a surrogate measure of SBA. 3) Opsonphagocytic Activity (OPA): We will measure the ability of the antibodies in sera from immunized mice and N. meningitidis serogroup B-immunized humans to enhance OPA-dependent killing of N. gonorrhoeae, using HL-60 human promyelocytes as the phagocyte along with complement factor 6-depleted pooled normal human serum. In addition, we will use flow cytometry platforms employed in our laboratory in order to develop a high-throughput quantitative assay to measure OPA-dependent binding and internalization of N. gonorrhoeae. Overall, results from Core D will help the GV CRC and the field in general to establish the correlate(s) of protection for vaccines against gonorrhea, which to date are poorly understood. When integrated with results from other Cores in the GV CRC, the findings from Core D will contribute to selection of the most promising antigen(s) and platform(s) for the advancement of a novel vaccine for gonorrhea towards licensure.