双功能分子修饰脂质体构建疫苗脱冷链靶向载体-佐剂系统

Vaccination is an effective strategy used widely to keep disease under control. However, nowadays vaccines are generally labile bioproducts whose absolute dependence on cold chains for maintaining their immunity-inducing capacity makes mass inoculation more costly and inconvenient. Moreover, vaccines are mostly unpleasant injections unable to activate the mucosal immune systems which form a basal barrier to pathogen invasion. In an effort to offset these inadequacies, this project aims at developing a novel nasal vaccine carrier-adjuvant system. First, a dual function molecule of mannose-polyethylene glycol (PEG)-lipid A (MPL) is synthesized, and then it is incorporated along with specific pathogen antigens into liposomes by an emulsification-lyophilization procedure described as follows: preparation of emulsions containing antigens of interest and lyoprotectants in inner water phase and MPL and phospholipids in oil phase; lyophilization of emulsions resulting in a dry product (proMLML) which can be rehydrated to form mannose-PEG-lipid A modified liposomes (MLML) encapsulating the specific antigens. The MLML thus obtained can function as a targeting carrier and, simultaneously, an adjuvant agent due to the binding of MPL mannose group to mannose receptor and lipid A group to toll-like receptor 4 (TLR4), both of which are highly expressed on the nasal mucosal professional immune cells. Consequently, receptor-mediated endocytosis of MLML by antigen presenting cells (APC) will occur to promote antigen cross presentation and subsequently to facilitate cytotoxic T lymphocyte (CTL) response and B cell production of specific antibody; on the other hand, lipid A group-binding TLR4 will activate the body innate immune system, playing vigorously the role of an adjuvant. Notably, the big hydrophilic group of mannose-PEG- in MPL will inhibit lipid A from entering brain tissues avoiding its severe damage on central nervous system (CNS). The MLML, which can immediately form just prior to inoculation from a stable easy-producing dry product proMLML, possess a biological safety feature and are able to activate both mucosal and systemic immune systems and are, therefore, expected to be a promising vaccine outside-the-could-chain targeting carrier-adjuvant system (VOTCAS). The construction of VOTCAS and the related immune response mechanism exploration may lay a foundation in the field for the development of novel outside-the-cold-chain non-injection vaccines,underlining this hoped NSFC project fundamental scientific significance as well as wide clinical use value.

目前疫苗主要不足为依附冷链，多注射接种，无法诱导黏膜免疫应答。现发展一种新型鼻腔疫苗：先合成功能分子甘露糖-聚乙二醇-脂质A (MPL)；再将MPL、磷脂溶于油相，目标抗原、冻干保护剂溶于内水相，乳化-冻干，得冻干品（proMLML），接种前再水化形成包封抗原脂质体（MLML）。MLML具有载体、靶向、佐剂多重功能：MPL靶向免疫细胞表面甘露糖受体，实现受体介导内吞MLML，增强抗原交叉提呈，促进细胞毒性T细胞应答及抗体产生；MPL与TLR4结合激活固有免疫系统发挥佐剂功能。此外MPL能阻止lipid A由鼻腔向脑组织扩散，避免损伤中枢神经系统（CNS）。proMLML稳定,易制；MLML能诱导机体产生系统及粘膜免疫应答，生物安全性高，期望形成一种疫苗脱冷链靶向载体-佐剂系统（VOTCAS）。VOTCAS构建及相关机理探讨能为发展新型脱冷链非注射疫苗奠定基础，具有重要的科学意义及应用价值。

目前疫苗主要不足为依附冷链，多注射接种，无法诱导黏膜免疫应答。研究主要发展了一种新型鼻腔粘膜疫苗：先合成功能分子甘露糖-聚乙二醇-胆固醇结合物（mannose-PEG1000-cholesterol conjugate, MPC）；通过乳化-冻干制得冻干品（proMLLs），接种前再水化形成MPC-脂质A双重修饰阳离子脂质体（MPC and lipid A dually decorated liposomes, MLLs），抗原结合率38%，ζ电位5mV, 粒径180 nm。proMLL在室温储存2周仍保持免疫活性。体外细胞培养实验表明，与普通脂质体相比，MLLs能够促进抗原提呈细胞（APC）摄取疫苗，因为MLLs靶向免疫细胞表面甘露糖受体，并以受体介导内吞方式进入APC。体内实验发现，小鼠口腔粘膜接种MLLs没有表现出明显毒性，接种三周后外周血中IgG及腔道粘膜（口腔粘膜、产道粘膜、小肠粘膜）IgA等抗原特异性抗体水平显著增高，表明机体已经建立了体液及粘膜免疫应答；对于进一步分析表明IgG亚型IgG1及IgG2a均增高，但IgG1高于IgG2a，同时外周血及脾细胞培养液IFN-γ表达也显著增高，说明MLLs诱导机体产生了Th1/Th2混合型免疫应答。其原因可能为MLLs促进APC实现抗原交叉提呈，产生细胞免疫应答；而脂质A与TLR4 结合激活固有免疫系统发挥佐剂功能，并能够诱导机体产生Th1/Th2混合型免疫应答。可见，proMLLs 具有载体、靶向、佐剂多重功能，期望成为一种脱冷链疫苗佐剂传递系统（VADS）。