A Novel Influenza Vaccine Based On Bacillus Subtilis Spore

Objective: Our study aimed to prepare recombinant spore displayingconservative antigen of influenza virus M2protein extracellular area (M2e) based onthe Bacillus subtilis spore surface-displaying technique. Immunogenicity andimmunoprotective efficacy were analyzed and evaluated by performing animalexperiment using the recombinant spore as a new type of influenza virus candidatevaccine. Our study lays a foundation for development of a new type of influenza virusvaccine in Bacillus subtilis spore-displaying system.Methods:（1）The CotB gene from Bacillus subtilis was inserted into pDG1664integrational vector followed by ligation with three-repetitive M2e gene in thedownstream to construct recombinant plasmid pDG1664-CotB-M2e3, followed byintegration to genome of Bacillus subtilis PY79by homologous recombination. Therecombinant strain was screened by erythromycin resistance and recombinant singlecolony was selected to culture. The connection of M2e gene was identified by PCR andDNA sequencing followed by SDS-PAGE and Western Blot detecting the expression ofinfluenza virus M2e antigen protein on spore coat of the recombinant strain. Flowcytometry and immunofluorescence method were used to further determine theexpression of the M2e antigen on spore coat. The growth conditions and biochemicalcharacteristics of Recombinant Bacillus subtilis and spore was identified by analysis ofmorphology,biochemical events and16s rRNA.（2） To further analyze theimmunogenicity and immune protection of recombinant Bacillus subtilis expressiinginfluenza virus M2e, we apply recombinant Bacillus subtilis expressiing influenza virus M2e to immunization of animals and animal challenge experiments. Therecombinant spore expressing the M2e antigen protein was used as influenza viruscandidate vaccine to directly vaccinate mice orally without adjuvant on threeconsecutive days. Four weeks after first immunization the second vaccination wasperformed and the last was performed at the fifth week. At5th,7th,9th,11th,13th,15th,17th week after the first immunization the caudal vein blood was separated andhumoral immune response was detected and analyzed. Cytoimmunity was analyzed byflow cytometry. The immune protection effect of recombinant spore against influenzavirus as a new style influenza virus vaccine was evaluated by challenge protective test.Result：（1） Construction and identification of the recombinant Bacillussubtilis spore expressing influenza virus M2e. After optimized conditions,pDG1664-CotB-M2e3recombinant plasmid was transformed into PY79competentbacteria using the electrotransformation method. PCR and DNA sequencing resultsshowed that M2e antigen gene was integrated into the recombinant Bacillus subtilisgenome correctly. The M2e influenza virus antigen was successfully expressed inrecombinant Bacillus subtilis that conformed by SDS-PAGE and Western blot; Flowcytometry and immunofluorescence was used to further determine the formation ofthe M2e antigen on the coat.The molecular weight of recombinant Bacillus subtilis was(CotB-M2e fusion protein) about37kDa.Experimental studies showed that wesuccessfully constucted recombinant strains of Bacillus subtilis (named RSM2e31)which was integrated into M2e gene. The morphological observation and analysis ofrecombinant Bacillus subtilis by electron microscopy detection technology andrestructuring of Bacillus subtilis subculture showed that recombinant strain RSM2e31can form spores like the wild-type strain and the foreign gene can exist stably inrecombinant strains of Bacillus subtilis. Biochemical reaction (API CH50and API20E)and16s rRNA sequencing identified that the recombinant spore still belongs to Bacillus subtilis. The growth state of the recombinant Bacillus subtilis such as theformation of colony and biochemical reactions changes slightly, but the basicbiochemical characteristics of the recombinant strain was the same with the wild-typestrain.（2）The analysis of immunogenicity and immune response characteristics ofrecombinant Bacillus subtilis spore expressiing influenza virus M2e. To furtheranalyze the immunogenicity and immune response characteristics of recombinantBacillus subtilis expressiing influenza virus M2e, we apply immunization of animalswhich vaccinate mice orally. Immune results show that the oral immunization ofRSM2e31recombinant Bacillus subtilis spore without adjuvants for can induce goodhumoral immune response. At the17th week after immunization the antibody titercould still maintain at a high level (1:11200). Antibody subclass analysis showed thatIgG1take a major composition in IgG antibodies induced by the recombinant BacillusIgG1Results of intracellular cytokine staining of vaccinated mice splenocytes showedthat the recombinant RSM2e31could effectively induce Th2cellular immune responsein vivo. Detecting sIgA of Mouse lung and intestinal lavage showed that therecombinant Bacillus subtilis can induce antigen-specific mucosal immune respons（3） Challenge protective test of recombinant Bacillus subtilis spore vaccineexpressiing influenza virus M2e. After17weeks, the immunized mice werechallenged with A/PR/8/34(H1N1) influenza virus at the dose of103TCID50.Theresults showed that the survival rate was only10%in two control groups which wereimmunized with WT spore or PBS respectively; however, mice vaccinated withrecombinant spore RSM2e31were all survival. Virus titer in lung tissue of miceimmunized with RSM2e31significantly reduced which showed the effectivelyinhibition of virus replication in vivo. In conclusion, there is a good immunoprotectiveefficacy of the recombinant spore. Conclusion: In this study, we successfully prepared the recombinant Bacillus subtilis,which could stably express the exogenous protein M2e, a kind of conserved antigen oninfluenza virus surface. Mice that immunized recombinant Bacillus subtilis couldinduce effective humoral immune response, cellular immune response and mucosalimmune response. The vaccine of the recombinant Bacillus subtilis expressinginfluenza virus M2can effectively protect the mice against virus infection. Our study isan innovative exploration for development of a new type of influenza virus vaccine inBacillus subtilis spore-displaying system and lays a foundation for its in-depth study...