突变型与野生型HPV16DNA疫苗的免疫原性

Immunogenicity of Mutant and Wild HPV16 DNA Vaccines

目的评价突变人乳头瘤病毒16(HPV16)E7锌指结合区后HPV16E7DNA疫苗的免疫原性。方法将构建的野生型穴pcDNA3.1/16E7雪和突变型穴pcDNA3.1/16ME7雪DNA疫苗经肌肉免疫C57BL/6小鼠,分离脾单个核细胞,用E7特异性多肽刺激后,测定E7特异性白介素-2(IL-2)和干扰素-γ(IFN-γ)的水平及细胞毒性T淋巴细胞(CTL)反应。同时以未加E7多肽组作对照组。结果经E7特异性多肽刺激后,pcDNA3.1/16ME7产生IL-2的斑点数明显高于pcDNA3.1/16E7、pcDNA3.1和空白对照组。pcDNA3.1/16ME7的斑点数是pcDNA3.1/16E7的5倍多,两组间差异有显著性(P<0.05);pcDNA3.1/16ME7产生的IFN-γ是pcDNA3.1/16E7的2倍,差异有显著性(P<0.05);LDH结果显示在E∶T为45∶1时,pcDNA16/E7、pcDNA16/ME7、pcDNA3.1及未经免疫的小鼠4组之间CTL细胞杀伤率分别为穴28.7±1.2雪%、穴55±2.2雪%、穴12.5±2.0雪%和穴11.5±1.2雪%熏前两组与后两组中任一组之间差异均有显著性,前2组之间差异亦有显著性(P<0.05)。而未加E7多肽组,各组间差异均无显著性(P>0.05)。结论突变HPV16E7锌指结合区能显著增强DNA疫苗的免疫原性,是提高DNA疫苗免疫原性的策略之一。

Objective To evaluate the efficacy of mutation of human papillomavirus 16?(HPV16?)E7 in two zinc-binding motifs on HPV16 E7 C terminus on antigen-specific immunity. Methods pcDNA3.1/16E7 and pcDNA3.1/ME7 were successfully constructed by inserting the E7?(ME7?)into pcDNA3.1 BamHⅠ? EcoRⅠ cut sites. After intramuscular injection with pcDNA3.1? pcDNA3.1/16E7? and pcDNA3.1/16ME7 on C57BL/6 mice? splenocytes from vaccinated mice was isolated. After have been stimulated with E7-specific peptide? interferon γ?(IFN-γ?)? interleukin 2?(IL-2?)? and cytotoxic T lymphocyte?(CTL?)were detected by ELISA,Eli-spot? and LDH assay respectively?鸦 splenocytes without E7 peptide stimulation were used as control group. Results Splenocytes from mice vaccinated with pcDNA/ME7? stimulated with E7 peptide? generated significantly larger number of E7-specific IL-2 compared with pcDNA3.1/16E7? pcDNA3.1? and control group. The E7-specific IL-2 generated in pcDNA-ME7 group was 5-fold of that of pcDNA3.1/16E7? and the difference was statistically significant?(P < 0.05?). Splenocytes from mice vaccinated with pcDNA/ME7 and stimulated with E7 peptide? generated significantly larger number of E7-specific IFN-γ compared with other vaccines. pcDNA-ME7 generated a 2-fold increase in the number of E7-specific IFN-γ compared with wild-type E7 and the difference was statistically significant?(P < 0.05?). The highest CTL activity in mice vaccinated with pcDNA/ME7 at an E∶T ratio of 45∶1 was achieved compared with mice vaccinated with other vaccines. The percents of specific lysis generated by pcDNA3.1/ME7? pcDNA3.1/E7? pcDNA3.1? and without vaccination were of?(28.7 ± 1.2?)%,?(55 ± 2.2?)%,?(12.5 ± 2.0?)%,and?(11.5 ± 1.2?)% respectively? and significant difference existed between the former and the latter two groups?(P < 0.05?). However? no significant difference was found among all the groups without specific E7 peptide stimulation?(P > 0.05?). Conclusions The mutation of zinc-binding motifs on HPV16 E7 C terminus may greatly enhance the immunogenicity.