Modulation of immune responses mediated by porb from neisseria gonorrhoeae - antibiotic resistance in neisseria gonorrhoeae has been rising over the last decade, leading the who and the US CDC to…

Modulation of immune responses mediated by porb from neisseria gonorrhoeae - antibiotic resistance in neisseria gonorrhoeae has been rising over the last decade, leading the who and the US CDC to label the emergence of antimicrobial-resistant n. Gonorrhoeae a serious public health threat. Development of a vaccine against n. Gonorrhoeae is considered a critical step in prevention strategies to slow the spread of antibiotic-resistant n. Gonorrhoeae. However, individuals infected with n. Gonorrhoeae remain susceptible to repeat n. Gonorrhoeae infection because they fail to develop effective protective immune responses. Our research has found that the porb porin, the most abundant protein antigen in outer membrane vesicles (omvs), suppresses the capacity of antigen-presenting dendritic cells to stimulate t cell proliferation when treated prior to addition of t cells. This immunosuppressive effect likely contributes to immune evasion by the bacteria and the failure to develop protective immune responses. Recently, n. Gonorrhoeae infections have fallen in countries or regions that deployed mass vaccination campaigns with vaccines against n. Meningitidis serogroup b made from omvs, suggesting that these vaccines may offer partial cross-species protection against n. Gonorrhoeae. However, these omvs have very high levels of porb (and a related porin, pora). We hypothesize that because of the documented immunosuppressive effects of porb, IT is likely that these porins limit the immunogenicity of OMV-based vaccines. One possibility is that the immunosuppressive effects of porb are due to its channel activity. To test whether decreasing or ablating the channel activity reduces the immunosuppression mediated by porb, we propose in specific aim 1 to use a structural model of porb to introduce cysteine (CYS) mutations at locations within the pore that would have a high likelihood of decreasing or ablating channel permeation following modification by CYS-directed chemical reagents and crosslinkers. We will establish the effectiveness of these different reagents to inhibit permeation by assessing channel activity in lipid bilayers and in multilamellar vesicles using established protocols. Porin mutants that are readily inactivated by sulfhydryl-directed reagents will be examined for their capacity to inhibit dendritic cell-mediated t cell proliferation. To test whether reducing the level of porb in omvs impacts immunogenicity of vaccination with OMV, in specific aim 2 we will create a strain of n. Gonorrhoeae with regulated porb expression. We will test whether omvs containing decreasing levels of n. Gonorrhoeae porb exhibit reduced capacity to inhibit dendritic cell-mediated t cell proliferation and whether immunization of mice with these omvs results in increased cellular immune responses against n. Gonorrhoeae. These experiments will serve as an important step in the future development of more effective OMV-based vaccines.