Immune Effects Of Aluminum Hydroxide Nanoparticles Adsorbed Recombinant Protein Nmb0315 Against Neisseria Meningitidis Serogroup B In Mice

Objective:To explore preliminarily the specific humoral and cellular immune responses induced by aluminum hydroxide nanoparticles adsorbed recombinant Neisseria meningitides serogroup B0315 protein?rNMB0315?in BALB/c mice and evaluate the immunoprotection of the vaccine against Neisseria meningitidis serogroup B in mice,so as to provide some experimental basis for development of a suitable rNMB0315 vaccine adjuvant and an effective B group epidemic cerebrospinal meningitis vaccine.Methods:1.Characterization of aluminum hydroxide particles.Scanning electron microscopy?SEM?was used to observe the morphology and size of the particles,and the particles's zate potential was measured by zate potential analyzer.The property of aluminum hydroxide particles adsorption of rNMB0315 was measured by the method of BCA.2.Preparation of rNMB0315 vaccine.The recombinant E.coli BL21 strain,which successfully constructed by our group,were induced to express rNMB0315 protein.The recombinant proteins were purified by affinity chromatography and identified by Western blot.3.Evaluation of the immunocompetence and immunoprotection of r NMB0315 vaccine.The aluminum hydroxide nanoparticles?NPs?and microparticles?MPs?were mixed with the rNMB0315 protein at a 2:1ratio,respectively.The female BALB/c mice were intramuscularly immunizedwithr NMB0315-adsorbedaluminumhydroxide nanoparticles?rNMB0315+NPs?.The inoculations were performed four times at week 0,1,2,3 and serum and vaginal secretions samples were collected before each immunization.The levels of specific antibody IgG,Ig G1,IgG2a,IgG2b,IgG3 in serum and specific sIgA in genital tract were detected by indirect ELISA.At week 5,IL-4 and IFN-?in spleen lymphocytes supernatant fluid were detected by indirect ELISA and spleen lymphocyte stimulation index?SI?was detected by CCK8 assay.The bactericidal titer of the immune serum was determined by serum bactericidal assay?SBA?in vitro and the immune prottective effect was investigated with the recombinant protein vaccine in vivo.Result:1.The aluminum hydroxide nanoparticles prepared by hydrothermal method had excellent physical and chemical properties:SEM analysis showed that the nanoparticles were rod-shaped,with an average mean diameter of 100²00 nm;the surface charge of nanoparticles in ddH₂O or rNMB0315 were 48±1 mv and 26±6 mv respectivily,which suggested the nanoparticles had excellent stability in the dispersion system.In addition,aluminum hydroxide nanoparticles showed excellent adsorption ability.When the nanoparticles were mixed with rNMB0315 at a 1:2 ratio,the rNMB0315 protein was fully adsorbed by the nanoparticles.2.rNMB0315+NPs group induced a stronger specific humoral immune response.The titers of specific IgG,Ig G1,IgG2a,IgG2b,IgG3 and sIgA antibody in rNMB0315+NPs group were 1:131072,1:65536,1:32768,1:16384,1:32768 and 1:32768.The Ig G2a/IgG1 ratios were less than 1 at week 4 and week 5 in rNMB0315+NPs group.3.rNMB0315+NPs group induced a stronger specific cellular immune response.A stimulation index?1.849±0.144?detected in r NMB0315+NPs group was significantly higher than rNMB0315+MPs group?P<0.05?.4.The levels of IFN-??132.54±15.32 pg/mL?of r NMB0315+NPs group in culture supernatant of the spleen lymphocytes was significantly higher than r NMB0315+MPs group?97.79±14.37pg/mL,P<0.05?.Howerve,The levels of IL-4 in culture supernatant of the spleen lymphocytes had no significant differences between r NMB0315+NPs group?185.36±31.37 pg/mL?and r NMB0315+MPs group?174.23±24.84 pg/mL?,P>0.05.5.At week 5,the serum bactericidal titers of rNMB0315 group,rNMB0315+MPs group and r NMB0315+NPs group reached 1:64,1:128 and 1:128 respcetively in vitro,the protection rates of the three groups were 65%,75%and 80%respectively against the N.meningitidis strain MC58 in vivo.Conclusion:1.The aluminum hydroxide nanoparticles adsorbed recombinant protein NMB0315?rNMB0315+NPs?could effectively induce specific humoral immunity and cellular immunity in mice.2.The immune serum induced by rNMB0315+NPs vaccine had strong bactericidal activity in vitro and the vaccine had a strong protective effect against the N.meningitidis strain MC58 in vivo.