Immune Effects Of A Vaccination Regimen Combining A DNA Vaccine Encoding NMB0315 And A Recombinant Protein NMB0315 Boosting Against Neisseria Meningitidis Serogroup B In Mice

Objective: To evaluate preliminarily immunocompetence and immunoprotection of NMB0315 nucleic acid vaccine, recombinant protein vaccine and nucleic acid vaccine plus recombinant protein vaccine against Neisseria meningitidis serogroup B in mice, and to provide reliable experimental basis for an effective B group epidemic cerebrospinal meningitis vaccine. Methods: 1.The recombinant plasmid pc DNA3.1(+)/NMB0315 was identified by Polymerase Chain Reaction(PCR), enzyme digestion and then prepared.The female BALB/c mice were inoculated with the eukaryotic expression recombinant through intramuscular immunization at the week 0, 2,4, 6. Next, specific Ig G in serum, s Ig A in genital tract mucosa secretion,IFN-?, IL-4 in spleen lymphocytes supernatant fluid were detected by indirect ELISA, and spleen lymphocyte stimulation index(SI) was detected by CCK8 assay. 2.The recombinant plasmid p ET-30a/NMB0315 was expressed efficiently in Escherichia coli BL21. The female BALB/c mice were inoculated with the purified recombinant protein NMB0315 through intraperitoneal immunization at the week 0, 2, 4, 6 and humoral immunologicresponse and cellullar immunologic response were detected. 3.The NMB0315 nucleic acid vaccine and recombinant protein vaccine were prepared. Female BALB/c mice were inoculated with a NMB0315 DNA vaccine followed by boosting with recombinant protein NMB0315 through intramuscular and intraperitoneal immunization respectively. Next, humoral immunologic response and cellullar immunologic response were detected in female BALB/c mice by ELISA. The survival rate of BALB/c mice was used to evaluate immunoprotection of the NMB0315 vaccines in mice. Results: 1.The immunogenicity of NMB0315 nucleic acid vaccine was strong, the levels of specific s Ig A in genital tract and Ig G in serum shown upward trend along with the days post innoculation in pc DNA3.1(+)/NMB0315, the titers were up to1:45,000 and 1:30,000 respectively at the week 8, which was significantly higher than controls(P<0.05); Stimulation index(2.112±0.342) of the splenic lymphocyte and the levels of IFN-?(142.31±19.45 pg/m L) ? IL-4(178.04±18.38 pg/m L)induced by splenic lymphocyte were significantly higher than PBS?pc DNA3.1(+) and Cp G controls(P<0.05). 2.High levels of humoral immune responses were induced by the NMB0315 protein vaccine,the levels of specific s Ig A and Ig G shown upward trend along with the days post innoculation in the recombinant protein, the titers of Ig G and s Ig A were up to 1:180,000 and 1:40,000 respectively at the week 8;The levels of IL-4(248.47±23.37 pg/m L),IFN-?(198.12±20.02pg/m L) and stimulation index(1.631±0.062) at the week 8 were significantly higher than PBS and FAcontrols(P<0.05). 3.Specific Ig G,Ig G1,Ig G2 a,Ig G2 b,Ig G3 and s Ig A were induced by the NMB0315 DNA vaccine prime-protein boost at week 8, the titers were up to 1:150 000,1:70 000,1:50 000,1:22 000,1:45 000,1:30 000 respectively,level of IL-4(219.58±20.01 pg/m L)was lower than the single r NMB0315 group; Stimulation index(2.185±0.086) and the level of IFN-?(226.60±22.76 pg/ml) were significantly higher than the single r NMB0315 group(P<0.05); The bactericidal titer of nucleic acid vaccine,protein vaccine group and combined immunization group reached 1:64,1:128and 1:128 respectively,the protection rates of the three vaccine groups were70%,80% and 95% respectively against the N. meningitidis strain MC58.4.The Ig G2a/Ig G1 ratios of the nucleic acid vaccine group, the recombinant protein vaccine group and the combined immunization vaccine group were all less than 1 at week 2, 4, 6, 8.Conclusion:1.The humoral immunity effects(including mucosal immune) induced by the NMB0315 vaccines were as follows: the recombinant protein vaccine group > the combined immunization vaccine group > the nucleic acid vaccine; and the cellular immune effects were as follows: the combined immunization vaccine group > the nucleic acid vaccine group > the recombinant protein vaccine group; 2. The protection effects induced by the NMB0315 vaccines in BALB/c mice within 72 hours were as follows: the recombinant protein vaccine group > the combined immunization vaccine group > the nucleic acid vaccine group.