基于工程铝盐纳米粒子和Toll样受体激动剂的复合疫苗佐剂作用机制研究

Aluminum-containing vaccine adjuvants, e.g., aluminum hydroxide, aluminum phosphate, and aluminum hydroxyphosphate sulfate, are prevailingly used in human vaccines. Despite of their safe applications, the detailed mechanism of how aluminum salts exert their adjuvant immune effect remains unclear. The aluminum salts lacks specific physicochemical characteristics that affect the interactions at the nano-bio interface and immunogenicity. Additionally, aluminum salts have been found to induce Th2-biased humoral immune responses, but not Th1 and/or CD8+ T cell mediated immune responses needed for vaccines targeting intracellular pathogens. There is already some evidence that the simultaneous stimulation of various immune targets by combination adjuvants could produce a more effective and longer lasting immune response, and rigorously controlled structure-based adjuvants may allow us to address these adjuvant effects. In this project, we aim to understand the molecular mechanisms of combination adjuvants containing engineered aluminum-based nanoparticles, i.e., aluminum oxyhydroxide or aluminum phosphate, and Toll-like receptor agonists, i.e., monophosphoryl lipid A (MPL), imiquimod, and CpG-ODN. We assess their effects on the stimulation of immune responses in vitro and potentiation of immunogenicity in vivo. The understanding of immunological functions and the molecular mechanisms that are responsible for the activity of combination adjuvants could lead to future development of adjuvants that target multiple immune pathways. These pathways, likely affecting multiple immune cell types within both innate and/or adaptive immunity, could improve the duration of the immune response, as well as the quality and magnitude of the response. This project will benefit the design and development of effective vaccine adjuvants with improved immunogenicity.

疫苗佐剂是添加在疫苗配方中用以增强并延长免疫应答的物质。其中，铝盐是人用疫苗中使用最广泛的一种佐剂。铝盐诱导Th2型体液免疫，在长期使用过程中已被证明具有良好的安全性和有效性。然而，铝盐的免疫增强机制不是十分清楚并存有争议，导致了铝盐佐剂设计缺乏理论指导。不同结构的铝盐佐剂应用在疫苗配方中，同时，铝盐不能诱导Th1型免疫应答和毒性T淋巴细胞反应，限制了其在治疗型疫苗中的应用。为此，本项目构建基于工程铝盐纳米粒子（包括羟基氧化铝、磷酸铝）和Toll样受体激动剂（包括单磷酸类脂A、咪喹莫特、胞嘧啶-磷酸-鸟嘌呤寡脱氧核苷酸）的复合疫苗佐剂，通过精确调控铝盐纳米粒子的物理化学特性，从工程和免疫学角度研究复合佐剂的结构对体液免疫和细胞免疫增强的影响，揭示佐剂的作用机制，并建立佐剂的结构与免疫响应的构效关系。本项目采用免疫工程的策略，强化刺激多种免疫途径，为新型疫苗佐剂的设计提供理论基础和依据。

项目构建了物理化学特性可控的羟基氧化铝佐剂、磷酸铝佐剂、羟基氧化铝-CpG ODN复合佐剂、羟基磷灰石佐剂。通过使用乙肝表面抗原（HBsAg）、人乳头瘤病毒病毒样颗粒（HPV VLP）及金黄色葡萄球菌mSEB或MntC抗原进行了佐剂作用机制研究，并构建了佐剂结构-免疫响应的构效关系。研究表明，高长径比的羟基氧化铝佐剂、羟基磷灰石佐剂及表面带正电的磷酸铝佐剂具有优异的佐剂效果，而通过共价结合的羟基氧化铝-CpG ODN复合佐剂能够诱导平衡和细胞及体液免疫。 构建了具有不同长径比的基于鞭毛蛋白纤维的黏膜佐剂，在卵清蛋白 (OVA) 黏膜免疫模型中，较短的鞭毛纳米纤维在粘膜部位和血清中表现出增强的免疫反应。构建了以VLP为生物模板的二氧化硅佐剂疫苗递送系统（VLP@Silica），以乙肝表面病毒样颗粒（HBsAg VLP）或人乳头瘤病毒18型病毒样颗粒（HPV 18 VLP）为生物模板的VLP@Silica疫苗，能促进抗原特异性抗体（包括IgG, IgG1和IgG2c）的高水平释放以及CD4+ 和CD8+ T细胞的激活。同时，基于工程佐剂材料的构建及调控，拓展进行了疫苗制剂研究。通过选择物化特性可控的羟基氧化铝纳米佐剂，使用HBsAg、SARS-COV-2刺突蛋白受体结合域（RBD）、牛血清白蛋白（BSA）和OVA作为模型抗原，通过构建佐剂与抗原的吸附等温线，揭示了抗原的吸附能力、吸附强度与纳米佐剂的比表面积、表面羟基含量的相关性。使用OVA作为模型抗原，通过生物物理方法对吸附后抗原结构和稳定性进行评估。本项目采用多学科交叉的研究方法，为新型疫苗佐剂的设计提供了理论基础和依据。