结核多组分重组蛋白疫苗EPRHP014的免疫原性和保护效果的初步评价

Preliminary evaluation of immunogenicity and protective effect of multicomponent recombinant protein vaccine EPRHP014 against tuberculosis

目的初步评价自主构建的结核多组分重组蛋白疫苗EPRHP014免疫原性和保护效果, 为研制结核新疫苗、有效防控结核病提供科学基础。方法选择结核分枝杆菌全长蛋白抗原3种(EsxH、Rv2628和HspX)和经表位预测和优化的表位优势蛋白抗原2种(nPPE18和nPstS1), 共5种组分构建蛋白抗原组合物EPRHP014, 包括融合表达纯化的多组分蛋白抗原(EPRHP014f)和分别表达纯化单个蛋白构成多组分混合蛋白抗原(EPRHP014m)。EPRHP014f和EPRHP014m分别辅以铝佐剂制备多组分蛋白疫苗, 采用卡介苗(BCG)作对照。皮下注射免疫BALB/c小鼠后, 采用ELISA法检测血清特异性抗体效价, 采用ELISpot和Luminex技术检测多种细胞因子分泌情况, 采用结核分枝杆菌体外生长抑制试验观察其免疫保护作用。结果采用t检验或秩和检验进行统计分析。结果 EPRHP014m和EPRHP014f免疫小鼠后均能诱导产生高效价的IgG抗体及其亚型IgG1和IgG2a, 抗体效价与BCG免疫组小鼠差异无统计学意义(P>0.05)。EPRHP014f组诱导产生的分泌IFN-γ和IL-4的斑点形成细胞(SFC)数量均高于EPRHP014m组和BCG组(P<0.05), 而EPRHP014m组和BCG组的IFN-γ和IL-4的SFC数量差异无统计学意义(P>0.05)。EPRHP014m组诱导分泌的GM-CSF、IL-12p70均高于BCG组(P<0.05), 而IL-6和IL-10分泌水平与BCG组的差异无统计学意义(P>0.05);EPRHP014f组和BCG组的IL-6、IL-10、IL-12和GM-CSF分泌水平差异无统计学意义(P>0.05)。EPRHP014m组、EPRHP014f组和BCG组具有明显的体外抑菌作用, 差异无统计学意义(P>0.05)。结论 EPRHP014f和EPRHP014m免疫后均能诱导小鼠产生较强的体液免疫和细胞免疫应答, 且有较强的体外抑制结核分枝杆菌生长的能力, 显示抗原组合物EPRHP014具有良好的结核疫苗研发和应用价值。

Objective To evaluate the immunogenicity and protective effect of a multicomponent recombinant protein vaccine EPRHP014 constructed independently and provide a scientific basis for developing new tuberculosis(TB)vaccine and effective prevention and control of TB.Methods Three full-length Mycobacterium(M.)tuberculosis protein antigens(EsxH,Rv2628,and HspX)and two epitope-predicted and optimized epitope-dominant protein antigens(nPPE18 and nPstS1)were selected,from which five protein antigens were used to construct a protein antigen composition EPRHP014,including a fusion expression multi-component protein antigen(EPRHP014f)and a multi-component mixed protein antigen(EPRHP014m)formed with the five single protein using clone,purification,and purification respectively.Multicomponent protein vaccines EPRHP014f and EPRHP014m were prepared with aluminum adjuvant,and the BCG vaccine was used as a control.ELISA detected the titer of serum-specific antibodies,the secretion of various cytokines was detected by ELISpot and Luminex,and immune protection was observed by the M.tuberculosis growth inhibition test in vitro.The results were statistically analyzed by t-test or rank sum test,and P<0.05 was considered a statistically significant difference.Results Mice Immunized with EPRHP014m and EPRHP014f could produce highly effective IgG antibodies and their subtypes IgG1 and IgG2a,and the antibody titers were similar to those of mice immunized with BCG,with no statistical significance(P>0.05).The number of spot-forming cells(SFC)secreting IFN-γand IL-4 induced by EPRHP014f group was significantly higher than those by EPRHP014m group and BCG group(P<0.05),but there was no significant difference in the number of SFC for IFN-γand IL-4 induced between EPRHP014m group and BCG group(P>0.05).The secretion levels of GM-CSF and IL-12p70 induced by the EPRHP014m group were higher than those of the BCG group(P<0.05),but there was no significant difference in the levels of IL-6 and IL-10 induced between EPRHP014m group and BCG group(P>0.05).There was no significant difference in the secretions of IL-6,IL-10,IL-12,and GM-CSF between the EPRHP014f and BCG groups(P>0.05).EPRHP014m group,EPRHP014f group,and BCG group had obvious antibacterial effects in vitro,and the difference was insignificant(P>0.05).Conclusion Both EPRHP014f and EPRHP014m can induce strong humoral and cellular immune responses in mice after immunization,and have a strong ability to inhibit the growth of M.tuberculosis in vitro,indicating that the antigen composition EPRHP014 has good potential in the development and application of TB vaccine.