Characterization of human monoclonal antibodies against Neisseria gonorrhoeae and cognate antigen discovery

Gonorrhea, caused by the Gram negative bacterium Neisseria gonorrhoeae, affects millions of people globally. The lack of a vaccine and the increasing rates of antibiotic resistance make infection control difficult and underline the need for novel therapeutics. In this framework, monoclonal antibodies (mAbs) endowed with bactericidal activity have potential for treating bacterial infections resistant to currently available medications. This research project sought to isolate human mAbs and their cognate antigens as possible new tools for prevention and treatment of gonorrhea. The technology exploited here is named Reverse Vaccinology 2.0 and consisted in isolating plasma cells (PCs) and memory B cells (MBCs) from volunteers vaccinated with the anti-meningococcal vaccine Bexsero. The rationale for this approach relies on a retrospective study that demonstrated 31% cross-protection against gonorrhoea in people who had received Bexsero. Antibodies produced by PCs and MBCs were analysed to determine their binding profile to N. gonorrhoeae and functionality in vitro. The targets of the mAbs were then searched by combining genetic and biochemical studies. Although mAbs produced by PCs bound to a wide variety of N. gonorrhoeae strains, they were unable to kill the pathogen in complement-dependent assays. On the other hand, mAbs derived from MBCs displayed more interesting features. For instance, mAb_1 was bactericidal against the FA1090 strain. The anti-lipooligosaccharide (LOS) mAb_3 recognized distinct strains of N. gonorrhoeae (FA1090, BG27, F62, MS11) with different LOS structures thereby suggesting the existence of a common epitope. Monoclonal antibody 4 (mAb_4) bound specifically to an antigen named BamG (NGO_1985) involved in outer membrane biogenesis and stability. We chose to concentrate on characterizing this monoclonal and its target because this antigen is included in the approved meningococcal vaccination and provides protection in the animal model. Investigations of the interaction between mAb_4 and BamG were carried out in silico and predicted the epitope contacted by mAb_4. Crystallography trials were initiated with the purpose of determining the crystal structure of the complex. Of note, an effective immunoprecipitation/mass spectrometry (IP/MS) protocol for pinpointing mAb targets was established by including BamG/mAb_4 interactors as positive controls. Overall, this study generated a significant contribution to understanding the molecular bases of the cross-protection afforded by Bexsero and may provide guidance for future developments of antibody cocktail therapeutics, protein-based vaccines, and in vivo research.