Novel Chimeric Virus-like Particles Displaying Neisseria Surface Protein A Confer Protection Against Virulent Neisseria Meningitidis Serogroup B In Balb/c Mice

Objective: To investigate the specific humoral immune response and cellular immune response generated in immunized mice by the truncated hepatitis B core protein-like particles(HBc-N144)that fused with the Neisseria surface protein A(NspA),especially the evaluation on the mucosal immunity as well as the effect of the immunity protection.In order to explore the effective mucosal adjuvant,meanwhile,offer the theoretical basis for the development of vaccine against Neisseria meningitidis of serogroup B.Methods: 1.In this study,we searched the sequence of the NspA and the truncated HBc that was termed as HBc-N144,and the NspA was inserted into the major immunodominant region(MIR)between 79-80 amino acids of virus like particle(HBc-N144),and then the prokaryotic plasmid pet28a-HBc-N144-NspA was constructed by the overlap PCR.The recombinant protein was termed as HBc-N144-NspA.After thepurification of the fused plasmid,analysis by the transmission electron microscopy(TEM).Next,we observed the immunological properties of HBc-N144-NspA by animal immunization.The serum specific IgG,IgG1,IgG2 a and secreted IgA were evaluated by ELISA.The contents of cytokines IL-17 A,IL-4 and IFN-? in the supernatant of spleen lymphocyte culture were determined by ELISA.Meanwhile,the flow cytometry was used to detect Th1 and Th2 subsets of spleen lymphocytes.Serum bactericidal Activity(SBA)mediated by human serum complement was conducted using the specific antibody to evaluate the bactericidal Activity level in vitro.Two weeks after the last immunization,the lethal dose of Neisseria meningitidis MC58 was used to challenge the immunized mice to evaluate the immune protection effect of the vaccine.Finally,in order to investigate the safety of the HBc-N144-NspA,we collected the muscle tissue at the injection site of mice for pathological sections to analyze the inflammatory infiltration.Result: 1.TEM observation showed that HBc-N144 and HBc-N144-NspA presented granular structure with uniform size and particle sizes about 30 nm and 120 nm,respectively.2.The specific immune antibodies of HBc-N144-NspA+F and HBc-N144-NspA reach to a peak in the second week after last immunization,the immunization HBc-N144-NspA mixed Freund's adjuvant compared with the group ofHBc-N144-NspA without adjuvant had higher specific serum antibody as well as the secreted specific antibody but without evident difference(p>0.05).And the specific serum antibody and the secreted specific antibody levels of HBc-N144-NspA without adjuvant was evidently higher than NspA/F,with a statistical difference(p<0.05).The specific IgG1/IgG2 a ratio of HBc-N144-NspA+F,HBc-N144-NspA,NspA+F are all greater than 1.The Flow cytometry results showed that t HBc-N144-NspA,HBc-N144-NspA+F and rNspA+F can effectively induce Th2 type immune response.And the cytokine levels,according to the results of quantitative detection of IL-4,IL-17 A and INF-? produced by the immunization of the HBc-N144-NspA+F showed the greatest generation,but no obvious difference with the immunization of HBc-N144-NspA.The results of the attack experiment showed that the immune protection effect of HBc-N144-NspA+F and HBc-N144-NspA group enabled the mouse model to obtain the survival rate of 90%respectively,which was significantly higher than the 75% survival rate of the rNspA+F group.The vitro bactericidal test results showed that the bactericidal activity titer of HBc-N144-NspA+F group reached 1:16,higher than that of HBc-N144-NspA and rNspA+F group with 1:8.The results of pathological sections showed that both the HBc-N144-NspA+F group and the NspA+F group showed significant infiltration of inflammatory cells,while the HBc-N144-NspA groupshowed no significant infiltration of inflammatory cells,which suggesting that virus-like particle plays a relatively safe role in antigen transport as well as having good biocompatibility.Conclusion:1.The HBc-N144-NspA virus-like particles have good immunogenicity,and can induce mice to produce a higher level of specific humoral immunity,especially mucosal immunity2.The vaccine of the HBc-N144-NspA recombinant protein can elicit higher serum bactericidal titer and stronger protective effect on experimental mice.The immune protective effect was significantly higher than that of the rNspA+F immunization.