非甲基化CpG寡聚脱氧核苷酸对人乳头瘤病毒治疗性蛋白疫苗的免疫佐剂作用

Adjuvant effects of CpG-ODN on therapeutic hmnan papillomavirus protein vaccines

目的研究非甲基化CpG寡聚脱氧核苷酸（CpG-ODN）联合宫颈癌治疗性融合蛋白疫苗HPV16L2ESET（以下简称蛋白疫苗）诱导小鼠产生的免疫应答及其在肿瘤免疫治疗中的免疫佐剂作用。方法免疫学试验：108只雌性C57BL／6小鼠随机分成CpG组、蛋白疫苗组、CpG＋蛋白疫苗组，每组36只，分别肌肉注射CpG-ODN（10μg／只）、蛋白疫苗（120μg／只）、蛋白疫苗（120μg/只）和CpG-ODN（10μg／只）的混合溶液，免疫程序为0,3、7d；ELISPOT法检测IFN-T，间接ELISA法检测IgG抗体水平，分别评价小鼠产生的细胞及体液免疫效果。肿瘤抑制试验：将40只雌性C57BL／6小鼠皮下接种TC．1肿瘤细胞（1×104／只），24h后随机分成4组接种疫苗，每组10只，CpG组、蛋白疫苗组和CpG＋蛋白疫苗组分别肌肉注射CpG-ODN（10μg/只）、蛋白疫苗（120μg／只）、蛋白疫苗（120μg／只）和CpG-ODN（10μg／只）的混合溶液，免疫程序为0、3、7d；对照组不给药。通过成瘤率分析抗肿瘤效果。结果免疫学试验表明，免疫后14、21和28d,CpG＋蛋白疫苗组小鼠分泌IFN-y的T细胞高于蛋白疫苗组（Z=-2．36、-3．58、-3．58，P均〈0．05），在21d达到最高水平；免疫后5周，CpG＋蛋白疫苗组IgG抗体滴度维持在1：1×105左右，其中在10、14、21、42d与蛋白疫苗组比较差异显著（t=6．15、5．84、4．57、7．91，P均〈0．01）；抑瘤试验表明，蛋白疫苗组能延缓肿瘤生长，成瘤率为40％，添加CpG-ODN后，成瘤率降为0（x^2=5．00，P〈0．05）。结论将CpG-ODN与HPV16L2E6E7融合蛋白疫苗联合应用，可以诱导C57BL／6雌性小鼠产生更强的特异性体液和细胞免疫应答，对荷瘤鼠有更好的抗肿瘤效果，有望提高疫苗的免疫治疗效果。

Objective To investigate the immune responses and antitumor effects of therapeutic HPV fusion pro- tein vaccine HPV16L2E6E7 （termed protein vaccine） combined with unmethylated CpG-containing oligodeoxynucleotide （CpG-ODN） adjuvant. Methods A total of 108 female C57BL/6 mice were put in the immunologic tests, and were ran- domly divided into CpG （ n = 36）, protein vaccine （ n = 36） and CpG plus protein vaccine （ n = 36） group, CpG-ODN （10 μg for each mouse ）, protein vaccine （120μgfor each mouse） and the mixture of protein vaccine （ 120 ttg for each mouse） and CpG-ODN （ 10 μg for each mouse） were intramuscuarly injected respectively on 0, 3, 7 d; ELISA and ELISPOT were used to detect IgG and IFN-γlevels, so as to determine the cellular and humoral immune responses in mice. Forty female C57BL/6 mice subcutaneously inoculated with 1 × 104 TC-1 tumor cells were put in the tumor suppression tests, and were randomly divided into four groups after 24 h： CpG （ n = 10）, protein vaccine （ n = 10）, CpG-plus protein vaccine （ n = 10） and control group （ n = 10）, CpG-ODN （10 μg for each mouse）, protein vac- cine （ 120 μg for each mouse） and the mixture of protein vaccine （ 120μg for each mouse） and CpG-ODN （ 10 μg for each mouse） were intramuscularly injected on 0, 3, 7 d respectively; no drug was administered in the control group. The anti- tumor effects were detected through tumorigenic rates. Results Immunologic tests showed that vaccination of HPV16L2E6E7 protein vaccine combined CpG-ODN induced stronger E7-specific cellular immune response on 14, 21 and 28 d than protein vaccine group（ Z = -2.36,-3.58,-3.58, P all 〈 0.05）and reached peak value on 21 d. The titers of antigen-specific IgG kept about 1 ： 1 × 10^5 during 5 weeks after vaccination, and the titers had significant difference be- tween protein vaccine and CpG phis protein vaccine group on 10,14,21,42 d （ t = 6.15, 5.84, 4.57, 7.91; P all 〈 0.01）. Tumor suppression test showed that protein vaccine decreased the growth of tumor with a tumorigenic rate of 40%, while the rate dropped to 0 in CpG plus protein vaccine group （ Z2 = 5.00, P 〈 0.05 ）. Conclusions HPV1612E6E7 protein vaccine combined with CpG-ODN adjuvant can induce stronger specific cellular and humoral im- mune responses in female C57BIZ6 mice and has potent antitumor effects, which provide a new way to improve the im- munngenicity of HPV16L2E6E7 protein vaccine.