Structure of the Recombinant Neisseria gonorrhoeae Adhesin Complex Protein (rNg-ACP) and Generation of Murine Antibodies with Bactericidal Activity against Gonococci

Abstract

Neisseria gonorrhoeae (gonococcus [Ng]) is the causative organism of the sexually transmitted disease gonorrhoea, and the organism is listed by the World Health Organization as a high-priority pathogen for research and development of new control measures, including vaccines. In this study, we demonstrated that the N. gonorrhoeae adhesin complex protein (Ng-ACP) was conserved and expressed by 50 gonococcal strains and that recombinant proteins induced antibodies in mice that killed the bacteria in vitro. We determined the structure of Ng-ACP by X-ray crystallography and investigated structural conservation with Neisseria meningitidis ACP and MliC/PliC proteins from other bacteria which act as inhibitors of the human innate defense molecule lysozyme. These findings are important and suggest that Ng-ACP could provide a potential dual target for tackling gonococcal infections.

Feasibility of Using a Luminescence-Based Method to Determine Serum Bactericidal Activity against Neisseria gonorrhoeae

“…Determination of bacterial survival by the ATP method was sufficient to show development of higher SBA titres to the P9-17 strain with anti-sera raised to homologous OMVs, compared to an intermediate or no detectable cross-reactive SBA with anti-sera against MS11 or FA1090 OMVs, respectively. The homologous anti-P9-17 SBA response reported here is similar to a previous study, which indicated that vaccination of mice with sodium deoxycholate-extracted OMVs from P9-17 stimulated a SBA titre of 256 when delivered in alum adjuvant, compared to a titre of >1000 with OMVs prepared without detergent [35].…”

“…The N. gonorrhoeae strains used in this study were chosen because they are frequently used for identification or testing of novel vaccine candidate antigens [35,41,42], for understanding gonococcal virulence [43] and for use as challenge strains for murine [44][45][46] or human models of infection [9,10].…”

“…This was partially because a portion of the re-suspended bacteria had to be transferred into another plate and combined with an equal volume of substrate for detection of ATP by luminescence. Results obtained with this higher number of bacteria were compared to a well-established CFU-based SBA that used~1 × 10 3 CFU [35]. The reduced serum sensitivity and antibody-mediated bacterial killing detected with the ATP assay was likely due to the addition of approximately five-fold more bacteria.…”

“…Measurement of SBA by CFU was determined as previously described [35], with minor modifications for quantification of bacterial ATP by luminescence. N. gonorrhoeae was grown to mid-log on GCA and suspended in Dulbecco's modification of PBS (PBSB).…”

Integrated Bioinformatic Analyses and Immune Characterization of New Neisseria gonorrhoeae Vaccine Antigens Expressed during Natural Mucosal Infection

“…Some protection was shown by antibodies to the gonococcal lipoligosaccharides (LOS) and a porin-based vaccine was also explored [2] but, like pilin, porin is subject to immunological pressure [18][19][20] and also affects complement-dependent bacterial killing [21]. Pre-clinical vaccine studies have been carried out in mouse models of immunization and infection [22,23] using classical surface-exposed antigens (for example, the highly conserved LOS epitope 2C7 [24], transferrin binding proteins (TbpA and TbpB) [25,26]), conserved and/or variable proteins (reviewed in Reference [2]) identified by conventional screenings, reverse vaccinology (initially developed for the Neisseria meningitidis serogroup B vaccine 4CMenB [27,28]), "omics" and bioinformatics [29][30][31][32][33][34][35]. Interest in outer membrane vesicles (OMVs) has been recently renewed by evidence of cross-reactive protection against N. gonorrhoeae by a meningococcal OMV-based vaccine [36][37][38][39].…”