以改造鞭毛蛋白为基础的广谱HPVL2多表位预防性疫苗的研究

The immunogenicity of HPV minor capsid protein L2 peptides or cross-neutralizing epitopes are quite weak. To develop an inexpensive, universal prophylactic vaccine against Chinese prevalent oncogenic and non-oncogenic HPVs, three concatenated multiple L2 peptides consisting all the possible cross-neutralizing epitopes from HPV type 16, -18, -58, -52, -31, -45, -33 and -6 will be constructed. By fusing 2 pan HLADR-binding epitopes (PADRE) at the N terminal and deletion of its hypervariable region, a modified Salmonella enterica fliC flagellin with enhanced CD4+T cell stimulating property will also be constructed. Then a panel of fusion proteins containing multitype L2 multiple epitope-based peptide and a modified flagellin will be expressed in bacteria, and properties of these fusion proteins will be evaluated both in vitro and in vivo. The fusion protein which could elicit the highest cross-protective immunity will be selected for further investigation to confirm if it could be more immunogenic when co-administrated with other TLR agonists. The resulting fusion protein vaccine could combine the highly adjuvant activity of the modified flagellin with the broadly protective immunity of multitype L2 peptide together efficiently. The immunity elicited by the innovative modified flagellin-L2 peptide fusion protein vaccine would provide protection against infections of most Chinese prevalent oncogenic and non-oncogenic HPVs at a low cost.

人乳头瘤病毒（HPV）次要外壳蛋白L2诱发的保护性抗体水平低，交叉保护范围远不能满足我国HPV预防的要求。本项目首先筛选出HPV18、-58、-52、-33、-45、-31及-6型L2所含的多种保守中和抗体表位，与HPV16 L2的多种保守中和抗体表位联合应用，获得整合有上述8型L2多种表位的L2多肽抗原；同时通过N端融合两个通用型Th表位及删除其超变区的方法，获得CD4+T细胞刺激活性增强的改造鞭毛蛋白，以此为载体构建鞭毛-L2多肽融合基因，在大肠杆菌内表达纯化后进行活性检测，优选兼具诱发中和抗体滴度高且中和病毒型别多、体内保护范围广且稳定性好的鞭毛-L2多肽融合蛋白，再与其它种类的TLR刺激剂协同免疫，确定疫苗的最佳免疫方案，明确其免疫机制。该新型L2疫苗特色是将改造鞭毛蛋白的高效佐剂活性与多型L2多肽的广谱保护活性有机结合起来，具有免疫活性好、保护范围广及成本低的特点，尚未见有报道。

人乳头瘤病毒（HPV）次要外壳蛋白L2诱发的保护性抗体水平低，交叉保护范围远不能满足我国HPV 预防的要求。本项目通过N端融合两个通用型Th表位及删除其超变区的方法获得3种改造鞭毛蛋白，并分别以此为载体构建各种鞭毛-HPV18 L2 aa.13-154多肽融合基因，在大肠杆菌内表达纯化后进行活性检测，结果表明删除鞭毛蛋白D3及D2超变区的FLIC△D2D3/18 L2N融合蛋白或删除鞭毛蛋白D3和CD2a超变区的FLIC△D3CD2a/18 L2N融合蛋白可有效诱发小鼠产生高滴度中和抗体及交叉中和抗体，且可保护免疫小鼠对抗同源及异源假病毒的攻击，而18 L2N蛋白单独免疫仅诱发低滴度的中和抗体和诱发针对HPV18的假病毒的免疫保护活性。研究结果表明鞭毛蛋白的突变体可作为分子内佐剂用于以L2为基础的广谱HPV疫苗的研究。