PROJECT SUMMARY / ABSTRACT Neisseria gonorrhoeae (Gc) is the Gram-negative bacterium that causes gonorrhea, resulting in an estimated 87 million cases globally each year. Gonococcal disease can result in pelvic inflammatory disease, ectopic pregnancies, and infertility. Rapidly emerging antibiotic resistance and detrimental effects on human health and fertility make gonorrhea an urgent threat. Additionally, adaptive humoral immunity to native Gc infections is weak and short-lived, making vaccines against gonorrhea a current public health priority. Immune factors recruited to Gc-infected tissues include neutrophils (PMNs) which combat Gc through phagocytosis/antimicrobial compounds, and the complement (C’) cascade. C’ enhances phagocyte activation and bactericidal capacity and promotes direct bacterial lysis via the pore-forming membrane attack complex (MAC). The efficiency of C’ effector functions is enhanced by pathogen-directed antibodies that activate the classical C’ cascade. Underscoring the importance of C’ to control Neisseria infections, individuals with impaired C’ have a more than 1,000-fold increased risk of serious Neisseria infections. Yet, despite the importance of C’ to Neisseria pathogenesis, we still have an incomplete understanding of how C’ contributes to control of Gc. Furthermore, the exact mechanism by which the MAC kills Gc and other Gram-negative pathogens is incompletely elucidated. Although the MAC only inserts within the outer membrane, inner membrane damage is required for MAC- mediated killing. It has been proposed that the MAC serves as a pore in the outer membrane barrier through which antibiotics and immunologic antimicrobials can transit, thereby enhancing antimicrobial access and activity. In Aim 1, I will determine how C’ destabilizes Gc to potentiate the activities of antimicrobials with distinct targets (periplasm, inner membrane, cytoplasm) and human PMNs. We will specifically test the contribution of multimeric C9 in facilitating C’ potentiation activities by using C9-depleted and -replete serum, and through vitronectin, a MAC-inhibiting protein in serum. Recent studies in humans show multivalent Serogroup B meningococcal vaccines, such as 4CMenB (BEXSERO), confer protection against gonorrhea and generate cross-reactive antibodies with bactericidal activity against Gc. In Aim 2, I will characterize the efficacy of human 4CMenB-elicited antibodies in combatting Gc in an integrated whole blood model system and identify the contributing complement effector functions. Together, the research proposed in this F30 application will define how C’ controls Gc and how this knowledge can be leveraged to advance the development of vaccines and therapeutics against gonococcal disease. This research plan, along with professional development activities, will provide me with the training and expertise to accomplish my overarching career goal of becoming a physician- scientist leading an independent research program at the in...