肠炎沙门氏菌基因工程减毒株的免疫效果及安全性评价

Evaluation of immunogenicity and safety of genetic engineering attenuated strains for Salmonella enteritidis

摘要:

[背景] 相较于灭活疫苗和弱毒疫苗，沙门氏菌（Salmonella）基因工程减毒活疫苗具有的优越性逐渐显现，研究也不断深入。[目的] 探究肠炎沙门氏菌G9菌株的4株基因缺失株G9（ΔhilA）、G9（ΔhilD）、G9（ΔssrABhilA）和G9（ΔssrABhilAhilD）的免疫效果及生物安全性。[方法] 以肠炎沙门氏菌基因缺失菌株G9（ΔhilA）、G9（ΔhilD）、G9（ΔssrABhilA）、G9（ΔssrABhilAhilD）及其亲本菌株G9的最佳免疫剂量接种小鼠后，利用间接ELISA法、流式细胞术、MTT法、小鼠攻毒试验及倾注平板法等对各缺失株的免疫效果和安全性进行评价。[结果] G9（ΔhilD）诱导血清IgG抗体效价最高，G9（ΔhilD）和G9（ΔssrABhilAhilD）产生肠黏膜IgA抗体效价最高，4株缺失菌诱导IL-4、IL-10、IFN-γ、TNF-β、MCP-1细胞因子的能力与亲本株G9差异不显著（P>0.05），产生的CD4⁺/CD3⁺、CD8⁺/CD3⁺ T细胞比率呈上升趋势，而且G9（ΔssrABhilA）和G9（ΔssrABhilAhilD）最高；G9（ΔssrABhilAhilD）诱发的脾淋巴细胞增殖指数最高；免疫小鼠后4株缺失株对G9攻毒提供的保护率为80%-100%，而且小鼠肝脏、脾脏及小肠绒毛无明显病理变化，对鼠伤寒沙门氏菌攻毒提供的保护率为50%-80%；接种后12 d，小鼠肝脏、脾脏及小肠中定殖的菌株能基本清除，而且在体外能连续稳定传代30代。[结论] G9（ΔhilA）、G9（ΔhilD）、G9（ΔssrABhilA）和G9（ΔssrABhilAhilD）对小鼠的免疫效果和生物安全性均良好，有成为肠炎沙门氏菌基因工程减毒活疫苗的可能。

Abstract:

[Background] Compared with inactivated vaccines and attenuated vaccines, the superiority of Salmonella genetically engineered live attenuated vaccines has gradually emerged, and research has continued to deepen. [Objective] To explore the immunogenicity and biological safety of four gene-deleted strains of Salmonella enteritidis G9 strain:G9(ΔhilA), G9(ΔhilD), G9(ΔssrABhilA) and G9(ΔssrABhilAhilD).[Methods] Salmonella enteritidis strain G9 and gene deletion strains G9(ΔhilA), G9(ΔhilD), G9(ΔssrABhilA) and G9(ΔssrABhilAhilD) were inoculated with the optimal immunization doses to mice, and then used indirect ELISA and flow cytometry, MTT method, mouse challenge test and pouring plate method to evaluate the immunogenicity and safety of each deletion strain.[Results] G9(ΔhilD) induced the highest titer of serum IgG antibody, G9(ΔhilD) and G9(ΔssrABhilAhilD) produced the highest titer of intestinal mucosal IgA antibody, and 4 strains of deletion bacteria induced IL-4, IL-10, IFN-γ, TNF-β, MCP-1 cytokine capacity was not significantly different from that of the parent strain G9 (P>0.05), the ratio of CD4⁺/CD3⁺, CD8⁺/CD3⁺ T cells showed an upward trend, and G9(ΔssrABhilA) and G9(ΔssrABhilAhilD) were the highest; G9(ΔssrABhilAhilD) induced the highest proliferation index of splenic lymphocytes; after immunization of mice, the protection rate of the four deletion strains against G9 challenge was 80%-100%. The structure of the spleen and small intestine villi was relatively complete without obvious pathological changes. The protection rate against Salmonella typhimurium challenge is 50% to 80%; 12 days after inoculation, the colonizing strains in the liver, spleen and small intestine of mice could be basically eliminated. And it could be continuously and stably passaged for 30 generations in vitro.[Conclusion] G9(ΔhilA), G9(ΔhilD), G9(ΔssrABhilA) and G9(ΔssrABhilAhilD) had good immune effects and biological safety in mice, and they had the possibility of becoming genetically engineered live attenuated vaccines of Salmonella enteritidis.