Evaluations of Immunogenicity and Efficacy of A Novel HPV16 E6 and E7 Multi-epitope DNA Vaccine
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摘要:
目的 构建和评价HPV16 E6、E7多表位DNA疫苗诱导的特异性CTL细胞应答及其对肿瘤生长的干预作用，从而揭示其作为候选HPV治疗性疫苗的潜能。 方法 首先通过IEDB网站中的MHC I Processing Predictions和MHC I Binding Predictions方法，分别预测人类HLA-A*02:01、HLA-A*11:01、HLA-A*24:02和C57BL/6小鼠H-2b的限制性CTL表位，然后根据评分以及ELISPOT实验筛选出2者共同呈递的CTL表位，并将其构建成多表位DNA疫苗（pVAX1-10P）。从预防性和治疗性2个方面研究pVAX1-10P对小鼠移植TC-1异位癌的免疫干预作用，流式细胞术检测特异性CTL应答。 结果 获得10条可被人与鼠MHC分子共呈递的CTL表位，ELISPOT结果表明这10条CTL表位均能诱导小鼠淋巴细胞产生特异性免疫应答；由此构建的多表位DNA疫苗pVAX1-10P无论在预防性实验还是治疗性实验中，均能诱导特异性的细胞免疫并抑制肿瘤的生长。 结论 构建的HPV16 E6、E7多表位DNA疫苗pVAX1-10P能够诱导特异性CTL应答，显著抑制肿瘤生长，有望作为候选HPV治疗性DNA疫苗。 Abstract:Objective To construct and evaluate the specific CTL cell response induced by HPV16 E6 and E7 multi epitope DNA vaccines and their intervention effects on tumor growth so as to reveal their potential as candidate HPV therapeutic vaccines. Methods The CTL epitopes of human HLA-A*02:01, HLA-A*11:01, HLA-A*24:02 restriction and C57BL/6 mouse H-2b restriction were predicted by the MHC-I processing and MHC-I binding methods in the IEDB website, and then screened for co-presentation of both based on scoring as well as ELISPOT experiment. The predicted CTL epitopes obtained were constructed into a multi-epitope DNA vaccine （pVAX1-10P）, and the immunological interventions were performed in the mice transplanted with TC-1 tumour cells from prophylactic and therapeutic strategies respectively, and the ability of pVAX1-10P to induce specific CTL responses was assessed by flow cytometry. Results Prediction and screening yielded 10 CTL antigenic epitopes co-presented by human and murine MHC molecules. ELISPOT results showed that each peptide in the experimental group induced the specific immune responses in mouse lymphocytes. The constructed multi-epitope DNA vaccine, pVAX1-10P, induced specific cellular immunity and significantly inhibited tumour growth in both prophylactic and therapeutic experiments. Conclusion The constructed HPV16 E6 and E7 multi-epitope DNA vaccine, pVAX1-10P can induce specific CTL response and inhibit tumour growth, which is expected as a promising candidate of HPV therapeutic DNA vaccine. -
Key words:
- Cervical cancer /
- Human papillomavirus 16 /
- E6 protein /
- E7 protein /
- multi-epitope DNA vaccine
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图 1 小鼠免疫程序
A：疫苗的预防性抗肿瘤评价中的小鼠免疫程序；B：疫苗的治疗性抗肿瘤评价中的小鼠免疫程序。
Figure 1. Immunisation program in mice
图 2 ELISPOT检测结果
A：实验组及对照组代表性酶联免疫斑点法图；B：ELISPOT检测IFN-γ斑点数统计图，条形图以$ \bar x \pm s $表示。
Figure 2. ELISPOT results
图 4 多表位DNA疫苗诱导的预防性抗肿瘤免疫
A：肿瘤解剖图；B：流式细胞术检测的CD8+T IFN-γ占比；与对照组比较，*P < 0.05。
Figure 4. Prophylactic anti-tumor immunity induced by multi-epitope vaccine
图 5 多表位DNA疫苗诱导的治疗性抗肿瘤免疫
A：肿瘤解剖图；B：流式细胞术检测的CD8+T IFN-γ占比；与对照组比较，*P < 0.05，nsP ˃ 0.05。
Figure 5. Therapeutic anti-tumor immunity induced by multi-epitope vaccine
表 1 预测得到的人鼠共呈递的CTL表位
Table 1. Predicted human and mouse co-presented CTL epitopes
蛋白 肽名称 起始位点 结束位点 序列 总分（鼠） 总分（人） 等级排名（鼠） 等级排名（人） E6 E6p1 38 45 VYCKQQLL −0.81 −1.39 1.4 0.45 E6p2 49 57 VYDFAFRDL −1.39 −1.23 1.3 0.28 E6p3 75 83 KFYSKISEY −0.72 −0.74或−0.75 1.5 1.6 E6p4 124 132 RHLDKKQRF −1.3 −1.06 1.4 0.46 E6p5 81 90 SEYRHYCYSL −0.29 1.7 82 90 EYRHYCYSL −0.68 0.57 E6p6 86 95 YCYSLYGTTL −0.52 −0.37 1.6 0.41 E7 E7p1 49 57 RAHYNIVTF 0.83或−0.59 −0.3 0.01或0.6 0.54 E7p2 7 15 TLHEYMLDL −1.08 −0.28 1.3 0.21 E7p3 50 57 AHYNIVTF −0.04或−1.26 −1.12 0.23或1.2 1.2 E7p4 77 87 RTLEDLLMGTL −1.37 −1.36 1.3 1.2 
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