人乳头瘤病毒18型L1-E6、E7嵌合基因DNA疫苗的体内免疫效应

目的：检测HPV18 L1-E6、E7嵌合基因DNA疫苗在小鼠体内的体液和细胞免疫效应。方法：将实验动物BALB/c小鼠54只随机分为9组，按不同免疫方式(肌肉接种或鼻内滴注)分别给予不同的重组质粒(pVAX1-L1-E6M3或pVAX1-L1-E7M3)和免疫佐剂(pLXHDmB7-2或LTB)。用免疫原免疫3次，末次免疫后取眼球后血进行ELISA抗体检测。末次免疫后进行小鼠足垫迟发型超敏反应(DTH)试验。断足进行小鼠足垫HE染色。取小鼠脾脏制成单细胞悬液，进行脾细胞增殖试验，并进行CD4^ /CD8^ T细胞中IFN-γ^ 或IL-4^ 细胞的FACS分析。结果：与对照组相比，各实验组均获得明显的免疫效果。实验组免疫后血清抗体4值均高于相应组别免疫前；肌肉注射组每次免疫后抗体水平较前次明显升高。实验组小鼠注射VLP抗原的左后足垫局部有红肿硬结，镜下观察可见大量单核细胞侵润。肌肉接种组的DTH反应强度、脾细胞增碹刺激指数(SI)和CD8^ IFN-γ^ 细胞数均高于鼻内滴注组；而鼻内滴注组CD4^ IL-4^ 细胞数高于单纯质粒肌肉接种组；加入pLXHDmB7-2的联合免疫组各项指标均高于单纯质粒组。结论-证实了重组pVAX1-HPV18L1/E6、E7嵌和基因DNA疫苗能诱导小鼠的体液免疫和细胞免疫效应：同时，证实B7-2分子能显著提高该质粒在小鼠体内的免疫反应效果.

Study of immune responses induced by human papillomavirus type 18 L1-E6 and L1-E7 chimeric gene DNA vaccines in mice

AIM: To examine the humoral and cellular immunoresponses induced by HPV18 L1-E6 and L1-E7 chimeric gene DNA vaccines in mice. METHODS: 54 BALB/c mice were divided into 9 groups randomly, and then vaccinated with various recombinant plasmids(pVAX1-L1-E6M3 or pVAX1-L1-E7M3) and immune adjuvants (pLXHDmB7-2 or LTB) through different administration routes (intramuscular or intranasal). After the third inoculation, blood samples were taken to measure specific antibody, and footpad swelling test was used to detect delayed-type hypersensitivity(DTH).Mice were killed and spleens were taken to prepare single spleen cell suspension for lymphocyte proliferation assay and CD4(+)/CD8(+), IFN-gamma(+) or IL-4(+) T cells double staining FACS assay. And then the pathological changes of the swollen footpad were observed. RESULTS: Compared with control, significant immunoresponses were observed in different experimental groups. The level of specific serum IgG against HPV in experiment groups was much higher than that of control group, and intramuscular immunization group had the highest antibody level. The footpad from immunized mice injected with virus-like particles was swollen and harden, and a large amounts of mononuclear cells can be seen in the footpad tissues. Intramuscular immunization groups were superior to intranasal immunization groups in DTH response, splenocyte proliferation and CD8(+) IFN-gamma(+) cell number, while CD4(+) IL-4(+) cell number was higher in intranasal immunization groups. The immunization groups using pLXHDmB7-2 as adjuvant were superior to other groups in immune responses. CONCLUSION: The recombinant plasmids could induce strong humoral and cellular immunoresponses in mice. Costimulatory molecule B7-2 could enhance the immune responses against HPV18 induced by the L1-E6 and L1-E7 DNA vaccines when used as an adjuvant.