Construction Of A Nucleic Acid Vaccine Of Serogroup B Neisseria Meningitis Surface Protein A And Investigation Of Its Immunocompetence And Immunoprotection In Mice

Objective: Amplified NspA (Neisseria meningitis surface Protein A) gene is used forbuilding the recombine eukaryotic vector pcDNA3.1(+)/NspA. The female BALB/c micewere immunized by the methods of intramuscular and intranasal. Immune protective rate ofrecombinant NspA was calculated by the level of humoral and cellular immune responses.To provide relative experimental evidences for the study of nucleic acid vaccines ofserogroup B Neisseria meningitis.Methods: the recombinant eukaryotic vector pcDNA3.1(+)/NspA were transfectedinto RAW264.7and COS-7cells by lpofectin-mediated gene transfer. The mRNA level ofNspA in cells was detected by Real-time fluorescent quantitative(RT-qPCR) and theexpression of NspA incells was assayed by immunohistochemistry.3~4weeks old BALB/cmice were immunized with the methoded of intramuscular and intranasal. The level of IgGanti-NapA antibody in the serum and sIgA antibody in the genital tract of immunized micewas detected by ELISA. The level of IFN-γ and IL-4in the mice splenocytes culturesupernatant was detected by ELISA. The proliferation of splenocytes was determined byCCK8. The animal infection model of meningococcus was established, and the immuneprotection rate of DNA vaccine in mice was observed.Results：（1）DNA sequence analysis and restriction enzyme identification showed that theeukaryotic expression vector pcDNA3.1(+)/NspA was successfully constructed.（2）The recombine eukaryotic vector pcDNA3.1(+)/NspA was transcribed andexpressed efficiently in eukaryotic cells.(3) After being immunized by the intramuscular and intranasal of DNA vaccine, thevaccine can stimulate the mice to produce specific antiNspA antibody,and the level of the antibody rised as time goes on,results were shown below: ①On week6, among groups which were immunized with the method of nasal drip,the NspA specific sIgA level in the genital tract of mice was significantly higher inpcDNA3.1(+)/NspA+CpG group than that of the groups pcDNA3.1(+) and PBS（P<0.01）.The sIgA level had no significant difference between pcDNA3.1(+)/NspA+CpG andpcDNA3.1(+)/NspA group. Antibody titer of pcDNA3.1(+)/NspA+CpG group was1:3000.Among groups which were immunized with the method of intramuscular injection, thelevel of sIgA in group pcDNA3.1(+)/NspA+CpG was significantly higher than that ofgroups pcDNA3.1(+) and PBS (p<0.01), while the results between the grouppcDNA3.1(+)/NspA+CpG and pcDNA3.1(+)/NspA were no significant difference (p>0.05),and the antibody titer of pcDNA3.1(+)/NspA+CpG group was1:1875.②On week6, among groups which were immunized with the method of nasal drip,the NspA specific IgG level in the serum of mice was significantly higher in pcDNA3.1(+)/NspA+CpG group than that of the groups pcDNA3.1(+) and PBS（P<0.01）. The sIgAlevel had no significant difference between pcDNA3.1(+)/NspA+CpG and pcDNA3.1(+)/NspA group. Antibody titer of pcDNA3.1(+)/NspA+CpG group was1:3750. Amonggroups which were immunized with the method of intramuscular injection, the level of IgGin group pcDNA3.1(+)/NspA+CpG was significantly higher than that of groupspcDNA3.1(+) and PBS (p<0.01), while the results between the grouppcDNA3.1(+)/NspA+CpG and pcDNA3.1(+)/NspA were no significant difference (p>0.05),and the antibody titer of pcDNA3.1(+)/NspA+CpG group was1:5000.③The level of IL-4or IFN-γ of the pcDNA3.1(+)/NspA+CpG group immunized withthe method of intramuscular injection and nasal drip was significantly higher than that ofthe groups pcDNA3.1(+) and PBS（P<0.01）in splenocyte supernatant, while the resultsbetween the group pcDNA3.1(+)/NspA+CpG and pcDNA3.1(+)/NspA were no significantdifference (p>0.05).④The stimulation index of the splenic lymphocyte of pcDNA3.1(+)/NspA+CpGgroup was significantly higher than those of the groups pcDNA3.1(+) and PBS(P<0.05) ineither intramuscular injection or nasal drip, while the results between the grouppcDNA3.1(+)/NspA+CpG and pcDNA3.1(+)/NspA were no significant difference(p>0.05). (4) The effect of different approaches was shown below:①Intramuscular injection: The72h survival rate of pcDNA3.1(+)/NspA+CpG andpcDNA3.1(+)/NspA groups was90%and80%, respectively; the24h survival rate ofpcDNA3.1(+) and PBS groups was30%and20%, respectively; the48h survival rate ofpcDNA3.1(+) and PBS groups was0%.②Nasal drip: The72h survival rate of pcDNA3.1(+)/NspA+CpG andpcDNA3.1(+)/NspA groups was80%and70%, respectively; the24h survival rate ofpcDNA3.1(+) and PBS groups was30%and20%, respectively; the48h survival rate ofpcDNA3.1(+) and PBS groups was0%..Conclusions：(1) Powerful and specific humoral immunity and cellular immune response of mice couldbe induced by pcDNA3.1(+)/NspA.(2) Nucleic acid vaccine had some immune protective effect in B meningococcal infectedmice. The immune protective effect of intramuscular injection was significantly better thanthat of nasal drip.(3) Vaccine adjuvants CpG had no obvious effect on immune activity and protective effectof NspA nucleic acid vaccine.