Preliminary Study On Novel Vaccine Against Hand, Foot And Mouth Disease

Hand,foot and mouth disease (HFMD) is a viral disease,caused by intestinal infection. The main pathogens are Enterovirus 71 (EV71) and Coxsackie A 16 (CA16), central nervous system complications often were caused by EV71, myocarditis and pericarditis were caused by CA16. There were no drugs to treat HFMD,vaccines were the most effective means of prevention and control, It was imperative to develop safe and effective vaccines.In this study, EV71 (Anhui Fuyang strain,China,Genbank----FJ607337) VP1 and CA16 (Shandong strain,China,Genbank----GQ253390) VP1 genes were amplified by reverse transcription polymerase chain reaction (RT-PCR), were constructed recombinant plasmid pFastBac HT A-EV71 VP1,pFastBac HT A-CA16 VP1 and pFastBac HT B-Mel-EV71 VP1-CA16 VP1,all recombinant plasmids were transformed to E.coli DH10. Each recombinant Bacmid DNA was obtained and then transfected into insect cells (Sf9), the recombinant protein were expressed in insect baculovirus expression system (Bac-to-Bac).Optimization codon of EV71 P1 and EV71 3CD in insect cells were cloned into pFastBac Dual polyhedrin promoter pPh and Pp10, pFastBac Dual EV71 P1-3CD and Bacmid were constructed by homologous recombination, Bacmid DNA was transfected into Sf9. Structural protein capsid P1 was cut by virus-encoded protease 3CD in Bac-to-Bac, EV71 virus like particles (VLPs) were assembled and purified by sucrose density gradient centrifugation. The expressed protein was detected by immunocytochemistry. mice were immunized by intraperitoneal and intranasal immunization, recombinant protein vaccine were assessed. The results showed as follows:1.The cloned DNA genes were consistent with DNA sequence in Genbank. EV71 VP1 and CA16 VP1 protein were expressed in Sf9, their concentration were 0.318mg/mLand 0.412 mg/mL,respectively, and their purity>90%.2.The best antigen doses of recombinant EV71 VP1 and CA16 VP1 protein were 20μg. Their specific IgG antibody titers were 875.43 and 1588.95, neutralizing antibody titers were 282.84 and 250.00, antibody subtype distribution were mainly IgG1 and IgG2b,CD4+/CD8+ were significantly different from control groups(P<0.05), T lymphocyte proliferation were enhanced, the number of cytokines IL-4 were more than IFN-γin spleen cells (p <0.05),sIgA antibodies in nasal and intestinal lavage with PBS were no significant differences (p>0.05) in intraperitoneal injection groups.They were induced humoral and cellular, no mucosal immune response, so mainly caused humoral immune response.3.Intranasal immuned groups , their serum IgG titers were 278.54 and 211.66,respectively. sIgA antibodies in nasal lavage were 64.31 and 54.22,respectively. sIgA antibodies in intestinal lavage were 15.98 and 13.55,respectively. sIgA-ASCs numbers in spleen cells were increased, recombinant protein vaccines induced humoral and mucosal immune response.4.EV71 VP1-CA16 VP1 fusion protein was expressed in Bac-to-Bac, the concentration of purified protein was 0.516mg/mL, its purity > 90%.5.EV71 3CD and EV71 P1 were optimized by insect preferred codon, and EV71 VLPs was assembled successfully in Bac-to-Bac, the concentration of purified EV71 VLPs was 0.817mg/mL, its purity>90%.Conclusion: EV71 VP1 and CA16 VP1 protein vaccines were preparated, they were able to induce better humoral immune response, also induced certain cellular and mucosal immune response. The recombinant EV71 VP1-CA16 VP1 protein and EV71 VLPs were constructed,expressed and purified.