Development of nanobody immunotherapeutics that prevent and treat gonorrhea

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PROJECT SUMMARY/ABSTRACT About 87 million new cases of gonorrhea occur worldwide annually. In 2020, 677,769 cases were reported to the CDC, a 111% increase in annual incidence since the historic low in 2009. Neisseria gonorrhoeae (Ng) has become resistant to almost every antibiotic in clinical use. Reports of resistance to ceftriaxone – the only currently recommended first-line of treatment – from almost every continent portends an era of untreatable gonorrhea. Development of novel treatments and preventives against Ng is a global public health priority. Our group, in collaboration with Evaxion Biotech, has identified two putative cell-division proteins (NGO0265 and NGO1549 (FtsN)) as lead vaccine candidates. NGO0265 and FtsN are expressed by all Ng isolates and FtsN is essential for bacterial viability. Immunization of mice with NGO0265 and FtsN elicits bactericidal antibodies (Abs) and significantly reduces the duration and burden of gonococcal colonization of mouse vaginas. Ng deploy a unique immune evasion strategy wherein it caps its lipooligosaccharide (LOS) with sialic acid using its surface LOS sialyltransferase (Lst) and host- derived CMP-sialic acid. LOS sialylation enables Ng to evade complement, cationic antimicrobial peptides (CAMPs) and down-regulate the inflammatory response by engaging host Siglec receptors. Gonococcal lst deletion mutants are attenuated in mice. Supporting its key role in pathogenesis, all gonococcal disease isolates sequenced thus far possess a functional Lst gene. Camelid single-domain antibodies called VHHs or nanobodies, have been found to possess unique properties that offer enormous versatility to facilitate the rapid development of simple and economical antibody-based therapeutics. VHH- based products can exploit the small size and high stability of VHH components, their propensity to bind conformation- dependent neutralizing epitopes, and their tractability for functional expression as heteromultimers. These characteristics make possible commercially favorable antibody therapeutic agents having ultrahigh target affinities, broad natural variant specificities and the ability to bind multiple different targets. Here, we aim to develop nanobody-based therapeutics against NGO0265, FtsN and Lst for use as an adjunctive treatment and as a preventive against multidrug-resistant gonorrhea. In Aim 1, we will immunize alpacas with purified recombinant chimeric NGO0265-NGO1549 and Lst, create a nanobody-display phage library from immune B cells and identify nanobodies that recognize non-overlapping epitopes on their targets with high-affinity. The ability of the nanobodies to block enzyme function or to activate complement and kill Ng when linked to IgG Fc will be evaluated in Aim 2. In Aim 3, we will test the ability of the lead anti-Ng nanobodies against each of the targets to clear gonococcal colonization in a well-established mouse vaginal colonization model. Successful completion of the work will identify and characterize nanobodies with activity against multidrug-resistant Ng, which can then be adapted to a variety of formats and drug delivery platforms.