ABSTRACT Gonorrhea is a major public health problem globally. About 87 million new cases of gonorrhea occur worldwide annually. In 2018, 583,405 cases were reported in the U.S, an 82.6% increase in disease incidence since the historic low in 2009. Concomitant infection with HIV and gonorrhea can increase rates of HIV transmission 5- fold. Dysbiosis of the vaginal microbiome (i.e., bacterial vaginosis (BV)) increases the risk of gonorrhea and HIV acquisition and transmission. Serious sequelae of gonorrhea in women include infertility, ectopic pregnancy and chronic pelvic pain. Neisseria gonorrhoeae (Ng) has become resistant to almost every antibiotic in clinical use. Reports of resistance to ceftriaxone and azithromycin from almost every continent portends an era of untreatable gonorrhea. Development of novel therapies against Ng is a global public health priority. Gonococci deploy a unique immune evasion strategy wherein it caps its lipooligosaccharide (LOS) by a surface LOS sialyltransferase (Lst) using host-derived CMP-Neu5Ac. LOS sialylation enables Ng to evade several aspects of host immunity, including complement and cationic antimicrobial peptides (CAMPs). We discovered that gonococci fed with CMP-nonulosonates (CMP-NulOs), such as CMP-legionaminic acid (CMP- Leg5,7Ac2) and CMP-ketodeoxynonulosonate (CMP-Kdn) cap their LOS with these NulOs. Incorporation of NulOs into Ng LOS renders bacteria susceptible to complement and CAMPs. Importantly, CMP-NulOs significantly shorten the duration and burden of Ng vaginal colonization in mice. Cathelicidin (a member of the CAMP family) played a key role in the MOA of CMP-NulOs. Our efficacy, safety and stability studies have established CMP-Leg5,7Ac2 and CMP-Kdn as promising candidates for intravaginal delivery to prevent Ng acquisition. In years 1-2 of Aim 1, the NRC will produce CMP-NulOs for all work in this proposal, perform a pharmacoeconomic analysis and assess scale-up. Process development, quality release assays, development of potency assays and stability in simulated genital tract fluids of the identified lead will follow in years 3-5. Aim 2 will elucidate factors that may affect safety efficacy of CMP-NulOs. These include effects of CMP-NulOs on growth and viability of favorable or unfavorable vaginal bacteria, the effects of sialidases elaborated by BV- associated pathogens and naturally-occurring anti-NulO antibodies on the efficacy of CMP-NulOs in vitro and in vivo, examining cervicovaginal secretions for CMP-NulO hydrolase activity and testing the specificity of CAMPs to bind NulO-containing glycans to refine the MOA of CMP-NulO against Ng. Aim 3 will formulate the CMP-NulOs into intravaginal rings (IVRs), perform PK studies in vitro and in a sheep model, and examine biofilm formation by vaginal microbiota on IVRs. Work in years 1-2 of the Aims 1-3 will identify a single lead; works in years 3-5 will focus on further product development and IND-enabling activities. Aim 4 will verify activ...