Study Of The Immunoprotective Effect Of Neisseria Gonorrhoeae Vaccine Candidate Antigen NGO2105

Objective:NGO2105 protein is a serine protease self-transport protein identified by our group,which plays a role in the pathogenesis of Neisseria gonorrhoeae,involving in adhesion and invasion.Antiserum against NGO2105 protein inhibited the adhesion and colonization of Neisseria gonorrhoeae,suggesting that it may be a potential protein vaccine target.Therefore,this study investigated the immune protective effects of NGO2105 protein through the intranasal mucosal immune pathway by using the Passenger and Translocator domains of NGO2105 protein as targets,and provided an experimental basis for the subsequent development of a vaccine against Neisseria gonorrhoeae using NGO2105 as a target.Methods:The recombinant proteins of the Passenger and Translocator domains of the NGO2105 protein were expressed in the Prokaryotic and purified.The obtained recombinant proteins were mixed with CTB adjuvant and immunized via the intranasal route in BALB/c mice.Serum and vaginal wash were collected 7 days after each immunization for antibody titer testing.The ability of the antibodies to bind to Neisseria gonorrhoeae was measured using flow cytometry.Antisera were collected for serum bactericidal assay,opsonophagocytic assay and adhesion inhibition assay.To initially assess the effect of the purified recombinant protein on the induction of cellular immunity,we examined the secretion of cytokines in splenocytes of immunized mice after immunization with serum subtypes and protein stimulation.A model of Neisseria gonorrhoeae infection was constructed by treating immunized mice with estradiol,and the protective effect of each immunized group against Neisseria gonorrhoeae infection was assessed by serial observation of colony-forming units of bacterial.Results:Passenger,Translocator and pColdTF recombinant proteins were successfully induced to be expressed,the purity of recombinant proteins was above90%.Mice immunized 3 times with the recombinant protein via the intranasal mucosal route obtained higher titer of antiserum;after the 4th intraperitoneal shock booster immunization,the antibody titers in the sera of the different immunized groups further increased,all reaching 1 x 10~7（P<0.0001）.Anti-Passenger and anti-Translocator IgG and IgA antibody titer in vaginal wash also increased significantly after the 4th intraperitoneal shock.The above results suggest that Passenger domain and Translocator domain proteins can effectively induce IgG and IgA antibodies in serum and vaginal mucosa.The results of antibody subtyping showed that both the recombinant Passenger and Translocator domain proteins induced high titers of IgG1,IgG2a and IgG2b antibodies,while IgG3 antibody titers were weak compared to the above three subtypes.The results of the flow cytometry analysis showed that both anti-Passenger and anti-Translocator sera bind well to Neisseria gonorrhoeae FA1090,and the anti-serum in the Passenger and Passenger+Translocator groups were effective against Neisseria gonorrhoeae（P<0.05）,The Passenger+Translocator group was the most effective and the Passenger group was the second most effective.The results of the opsonophagocytic assay showed that the opsonophagocytic assay titer of the Passenger and Passenger+Translocator groups was 80,whereas the serum bactericidal assay and the opsonophagocytic assay of the Translocator immunization group alone were both lower.The antibody adhesion inhibition assay showed an adhesion inhibition titer of1:80 for both Passenger antiserum and anti-Passenger+Translocator serum against Neisseria gonorrhoeae,while the Translocator group had an adhesion inhibition titer of 1:40（P<0.05）.Compared to PBS and pColdTF control groups,the secretion of cytokines IL-17A,IFN-γwas significantly higher in Passenger,Passenger+Translocator,Translocator protein-induced splenocytes（P<0.001）,and the order is Translocator group>Passenger+Translocator group>Passenger group.In addition,in vivo colonization assays suggested that immunization with Passenger,Translocator and Passenger+Translocator proteins were effective in inhibiting colonization of the lower genital tract of mice by Neisseria gonorrhoeae（P<0.0001）.Conclusion:Intranasal mucosal immunization with the Passenger and Translocator domains of NGO2105 protein induced high titer of IgG and IgA antibodies in mouse serum and vaginal wash,Antisera from both the Passenger and Passenger+Translocator groups showed good bactericidal activity,opsonophagocytic assay and adhesion inhibition.In vivo experiments also showed that both the Passenger and Translocator domains of the intranasal mucosal immune NGO2105 protein were protective against Neisseria gonorrhoeae infection.These results suggest that NGO2105 may be a promising target for Neisseria gonorrhoeae vaccines.