基于TLR2激活作用的新型分子内佐剂的设计、合成及其用于构建幽门螺杆菌脂肽疫苗的研究

Lipid molecules, such as Pam2CS, elicited epitope-specific Th, CTL and B- cell responses when conjugated with epitopes. Moreover, lipid molecules can be used in the mucosal route which resulted in the stimulation of local mucosal immune responses. Compared with conventional adjuvants for human beings, lipid molecules have obvious advantages. Nevertheless, intensity of the Pam2CS-elicited immune response was still not sufficient. The adjuvant activity of Pam2CS was attributed to the target-binding of Toll-like receptor 2. Our preliminary study suggests that enhanced interaction between lipid chain and hydrophobic pocket of TLR2, and raise rigidity of Cys-Ser skeleton were the right direction for the Pam2CS optimization. In this study, we intend to design and synthesize a series of compounds based on the above strategy , and then evaluate the TLR2 activity of the designed compounds in vitro. The structure-activity relationship will be carried out to determine the key structural factors, which provide theoretical guidance to research on novel adjuvants with TLR2 agonist. In addition, we intend to construct the Helicobacter pylori lipopeptide vaccine, in which Helicobacter pylori epitopes wad conjugated with the optimized lipids. The adjuvant activity of the optimized lipids was evaluated by analysis of the immune response after vaccination. The prophylactic potential of the lipopeptide vaccine was examined, which may lay the foundation of the development of novel vaccine against Helicobacter pylori.

Pam2CS等脂质分子与表位进行偶联，可以同时激发表位特异性Th、CTL和B细胞应答，且通过粘膜途径可以有效激发粘膜免疫应答，与现有人用佐剂相比具有明显优势。尽管如此，Pam2CS所诱导的免疫应答强度仍不够理想。Pam2CS的佐剂作用与其靶向激活TLR2密切相关，我们的前期研究提示增强脂链部分与TLR2疏水口袋的作用力以及提高Cys-Ser骨架的刚性是Pam2CS优化设计的正确方向。本项目拟基于以上策略设计与合成一系列化合物，体外评价所设计化合物的TLR2激活活性，并通过构效关系研究阐明影响活性的主要结构因素，为后续基于TLR2激活作用的新型佐剂的研究提供理论指导。对于TLR2激活活性和安全性兼优的化合物，本项目拟将其与幽门螺杆菌的优势表位进行偶联构建脂肽疫苗，通过免疫应答分析来考察目标化合物的体内佐剂活性，并评价幽门螺杆菌脂肽疫苗的免疫保护作用，为该类疫苗的进一步研究奠定基础。

Pam2CS等脂质分子可以辅助抗原激发特异性Th、CTL和B细胞应答，且可有效激发粘膜免疫应答，但所诱导的应答强度还不够理想。本研究采取增强脂链部分与TLR2疏水口袋的作用力以及提高Cys-Ser骨架的刚性等策略，合成了22个全新的化合物。通过体外TLR2激活活性评价，筛选到LP1（化合物4）和 LP2（化合物17）具有优于Pam2CS的体外TLR2激活活性，并呈剂量依赖关系。透射电镜下可见合成脂肽可以形成直径约200 nm的胶束，体外溶血实验和细胞毒性实验表明其体外安全性良好。进一步探索合成脂肽对提高重组HpaA对幽门螺旋杆菌感染的保护作用，合成脂肽与重组HpaA物理混合后免疫小鼠结果表明，LP2滴鼻组小鼠胃内幽门螺杆菌定植量显著低于单用HpaA滴鼻组，并且该组血清特异性IgG2a表达水平以及脾淋巴细胞培养上清IFN-γ和IL-17细胞因子的表达水平明显提高。综上，合成脂肽LP2具有显著的粘膜佐剂活性，通过增强机体对重组HpaA的特异性Th1和Th17细胞免疫应答水平，显著提高了重组HpaA对幽门螺杆菌感染的免疫保护作用。

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