Immune Effects Of The Recombinant Protein NMB0315 With Combinative Mucosal Adjuvant Against Neisseria Meningitidis Serogroup B In Mice

Objective: To explore preliminarily the physicochemical and related characteristics of combinative mucosal adjuvant(Chi-CpG NP and Chi-C48/80 NP).Next,BALB/c mice were intranasal immunized with recombinant Neisseria meningitides serogroup B 0315 protein(r NMB0315)by using Chi-CpG NP or Chi-C48/80 NP as combinative mucosal adjuvant,the specific humoral and cellular immune responses,mucosal immune responses and the immunoprotection were detected.So as to provide some experimental basis for development of mucosal adjuvant and an effective B group epidemic cerebrospinal meningitis vaccine.Methods: 1.Immunopotentiators(C48/80 or CpG-ODN)were loaded into Chitosan nanoparticle(Chi NP)to form the combinative mucosal adjuvants(Chi-CpG NP and Chi-C48/80 NP).Transmission electron microscopy(TEM)was used to observe the morphology,the particles' s zate potential and polydispersity index by Zetasizer Nano-ZS.The loading efficacy(LE)of immunostimulator(CpG-ODN or C48/80)was obtained through determination of the amount of remaining free immunostimulator in the supernatant after centrifugation of the nanoparticle solution.The adsorption of r NMB0315 was measured by the method of BCA.2.Preparation of r NMB0315 vaccine.The recombinant E.coli BL21 strain,which successfully constructed by our group,were induced to express the r NMB0315 protein.The recombinant proteins were purified by Ni2+-charged column chromatography and identified by Western blot.3.Evaluation of the immunocompetence and immunoprotection of r NMB0315 vaccine.The Chi-CpG NP and Chi-C48/80 NP were mixed with the r NMB0315 protein,respectively.Following,the female BALB/c mice were intranasally immunized with r NMB0315+Chi-C48/80 NP,r NMB0315+Chi-CpG NP.The serum,nasal washes and vaginal washes samples were collected to detect the r NMB0315 specific serum antibodies Ig G,Ig G1 and Ig G2 a,as well as s Ig A antibody levels in vaginal wash and nasal wash by indirect ELISA.At day 42,spleen lymphocyte stimulation index(SI)was tested by CCK8 assay.Splenic lymphocyte cultured supernate was used to detect the levels of cytokine IFN-?,IL-4 and IL-17 A by using ELISA kits according to the manufacturer's instructions.The bactericidal titer of the immune serum was detected by serum bactericidal assay(SBA)and the immune prottective effect was investigated with the recombinant protein vaccine in vivo.Result: 1.TEM analysis showed that the Chi-CpG NP and Chi-C48/80 NP were spherical,with an average mean diameter of 150~200 nm;zeta potential were 11.25±0.052 mv and 19.54±0.069 mv;polydispersity index were 0.286 and 0.415,respectivily.In addition,For r NMB0315 loading efficacy on nanoparticles,Chi-C48/80 NP(85.5?)was higher than Chi-CpG NP(71.2?).2.The r NMB0315+Chi-C48/80 NP group,and r NMB0315+Chi-CpG NP group induced a stronger specific humoral immune response.The titer of specific Ig G,Ig G1,Ig G2 a antibody in r NMB0315+Chi-CpG NP group and r NMB0315+Chi-CpG NP group were 1:150000,1:40000,1:50000;1:170000,1:60000,1:50000,respectivily.The titer of r NMB0315-specific s Ig A antibodies from the nasal washes and vaginal washes in r NMB0315+Chi-CpG NP group and r NMB0315+Chi-CpG NP group were 1:40000,1:40000;1:60000 ?1:40000,respectivily.The level of IL-4 in r NMB0315+Chi-CpG NP group and r NMB0315+Chi-CpG NP group were 176.48±19.79 pg/m L;185.16±21.06pg/m L,respectivily.The experimental results indicate that both Chi-CpG NP and Chi-C48/80 NP are effective mucosal adjuvants for the induction of significantly higher r NMB0315-specific Ig G,Ig G1,Ig G2 a and s Ig A antibodies than r NMB0315 alone.Additionally,Chi-CpG NP and Chi-C48/80 NP changed the ratio of Ig G2a/Ig G1,the Ig G2a/Ig G1 ratios were close to an 1 at day 42.3.The rNMB0315+Chi-C48/80 NP group and r NMB0315+Chi-CpG NP group induced a stronger specific cellular immune response.A stimulation index(1.569±0.153,1.799±0.188)and the levels of IFN-?(126.09±11.77 pg/m L,182.48±16.25 pg/m L)detected in r NMB0315+Chi-C48/80 NP group and r NMB0315+Chi-CpG NP group were significantly higher than r NMB0315 group(P<0.001).The r NMB0315+Chi-CpG NP group induced a stronger specific cellular immune response than r NMB0315+Chi-C48/80 NP group(P<0.05,P<0.01).4.Chi-CpG NP and Chi-C48/80 NP significantly boosted r NMB0315-specific nterleukin-17A(IL-17A)production by spleen cells,showing that Th17 cells were induced in vivo.5.At day 42,the serum bactericidal titers of r NMB0315 group,r NMB0315+Chi-CpG NP group and r NMB0315+Chi-CpG NP group reached 1:2,1:4 and 1: 8respcetively,the protection rates of the three groups were 60%,75% and85% respectively against the N.meningitidis strain MC58.Conclusion: 1.The Chi-CpG NP and Chi-C48/80 NP mucosal adjuvant were developed by our group.2.Nasal immunization of mice with recombinant protein NMB0315(r NMB0315)adsorbed on combination mucosal adjuvant(Chi-CpG NP or Chi-C48/80 NP)could effectively induce specific humoral immunity and cellular immunity in mice.The r NMB0315+Chi-CpG NP and r NMB0315+Chi-CpG NP also promote specific mucosal immunity.2.The r NMB0315+Chi-CpG NP group had stronger bactericidal activity and a stronger protective effect against the N.meningitidis strain MC58 than r NMB0315+Chi-CpG NP group.