表达HPV16 E7的重组减毒李斯特菌诱导的小鼠抗肿瘤作用

Protective immune responses induced by a recombinant Listeria monocytogenes delivering HPV16 E7

【目的】研究运送HPV16 E7抗原的重组李斯特菌(LM4△hly::E7)诱导的特异性免疫应答,并进行免疫保护性效力评价。【方法】将重组减毒李斯特菌LM4△hly::E7腹腔注射免疫C57BL/6小鼠,2次免疫后,通过ELISPOT方法、细胞毒性杀伤实验及脾脏中效应性T细胞比例分析来研究重组菌激发的细胞免疫应答,同时,用ELISA方法检测小鼠血清中的E7抗体效价。最后,将TC-1肿瘤细胞皮下注射免疫小鼠进行保护性效力评价。【结果】ELISPOT结果表明,重组菌LM4△hly::E7以诱发Th1型免疫为主;同时LM4△hly::E7能够诱导强烈的特异性CTL杀伤活性,杀伤率可达到72%,与对照组相比,差异极显著(P<0.01);并且研究结果显示重组李斯特菌诱导小鼠脾脏内产生较多数量的CD4+T细胞和CD8+T细胞(P<0.05);ELISA检测LM4△hly::E7免疫小鼠血清中的E7抗体效价为1∶400;保护性效力评价结果证实,LM4△hly::E7能够100%保护小鼠抵御肿瘤细胞的攻击。【结论】重组减毒菌LM4△hly::E7能够诱导机体产生E7特异性的细胞免疫应答和体液免疫应答,具有较好的免疫保护效力,显示出减毒李斯特菌在肿瘤疫苗研发中的良好应用前景。

[Objective]To investigate specific immune responses elicited by a recombinant Listeria monocytogenes strain LM4△hly：E7 and assess protective effect in C57BL/6 mice.[Methods]C57BL/6 mice were intraperitoneally immunized with LM4△hly：E7 at 1-week intervals.After the second immunization,cellular immunity elicited by this recombinant L.monocytogenes strain was analyzed via an ELISPOT assay,Cytotoxic T lymphocytes（CTL） measurement assay and analysis of effector T cells proportion in the splenocytes.Also,the serum antibodies against HPV16 E7 protein were determined in an ELISA assay.Finally,protective effect was assessed against the challenge with TC-1 tumor cells.[Results]The immune responses elicited by LM4△hly：E7 were biased towards Th1 type in the ELISPOT assay.Also,LM4△hly：E7 was able to induce E7-specific CTL activity,with average specific lysis of 72%,which was highly significant difference compared with the controls（P0.01）.Moreover,the proportion of effector T cells in the spleens were increased in mice immunized with recombinant L.monocytogenes strain（P0.05）.The titer of E7-specific antibodies in mice immunized with LM4△hly：E7 was 1：400 in the ELISA assay.Furthermore,immunization with LM△hly：E7 protected all mice against the lethal tumor cell challenge.[Conclusion]The data suggest that attenuated Listeria monocytogenes delivering HPV16 E7 antigen could induce both E7-specific cell mediated immunity and humoral immunity,and had a protective effect against challenge with TC-1 tumor cells.