仿生矿化优化口蹄疫病毒样颗粒耐热抗逆性研究

How to improve the thermal stability and reduce the cost of the vaccine cold-chain is a strategic issue in current Vaccinology.Virus-like particles (VLPs) based-vaccine is considered as the best potential candidate for a safe and effective alternative to inactivated Foot-and-Mouth disease (FMD) vaccine. VLPs vaccine will be great significance in effective prevention and control of FMD, protection of livestock healthy and sustainable development of animal husbandry. Therefore, this study will use the combination of biomimetic mineralization principle and genetic engineering technology to introduce nucleation factor in FMD VLPs. The surface of VLPs will be enriched mineral layer of calcium phosphate or silica by using in situ mineralization technology. The study will: 1) to analyze the relationship between mineralization sites, crystal form and physical morphology of VLPs with its stability, and clarify VLPs thermal protection mechanism in liquid environment; 2) to investigate the recognition，process and presentation of mineralized VLPs process by dendritic cells, and clarify the relationship between stability and immune mechanism of mineralized VLPs in vitro; 3) to determine the characteristics of bio-distribution and the regular of immune response against mineralized VLPs in vivo, and validate the immune mechanism in which biomineralization enhances the stability of VLPs. Results from the proposed research will not only illuminate the mechanism and advantage of biomineralization VLPs from the aspect in vitro and in vivo, but will also provide scientific knowledge and new thoughts to thermostable vaccinology research.

如何提高疫苗的热稳定性、降低冷链成本是当前疫苗学的战略性问题。病毒样颗粒（VLPs）被认为是目前能够替代口蹄疫灭活疫苗的最佳候选疫苗形式，对有效防控并实现口蹄疫净化、保障畜牧业健康可持续发展具有重要社会经济意义。本研究将根据生物矿化原理，结合基因改造引入矿物成核因子，以原位矿化技术使口蹄疫病毒VLPs表面构成磷酸钙或氧化硅矿物层，通过 1）分析矿化VLPs形貌理化特性、矿化位点和结晶形式与VLPs稳定性的关系，阐明体外液态环境中矿化层对VLPs的热保护机理；2）探讨树突状细胞摄入、递呈矿化VLPs的过程，从细胞水平阐明矿化VLPs的稳定性与免疫机制的关系；3）追踪矿化VLPs在体内生物分布特性及诱导免疫反应规律，从活体水平验证生物矿化提高VLPs稳定性的免疫机制。本研究从体外、细胞及活体层面证实生物矿化赋予VLPs稳定性的机制，为VLPs疫苗及抗逆疫苗学的研究提供新的思路和理论依据。

本研究旨在利用生物矿化策略提高口蹄疫病毒样颗粒疫苗的热稳定性，同时从细胞递呈层面阐述矿化所赋予病毒样颗粒诱发免疫反应的优势。通过基因工程的方法将N6、NW及W6三种矿化肽插入到口蹄疫病毒结构蛋白VP1的139位点，构建嵌合的病毒样颗粒。蛋白定量、抗原定量ELISA及蔗糖密度离心等方法表明插入的矿化肽并不影响目标蛋白的表达及VLPs的组装，其中以W6肽嵌合体表达量最高；利用磷酸钙对VLPs、VLPs-W6、VLPs-NW及VLPs-N6进行矿化，结果显示W6和NW肽对VLPs的矿化有明显的增强作用，而N6肽作用不明显。TEM、SEM及Element Mapping等方法表明在VLPs的表面形成一层无定形磷酸钙组成的无极壳，且矿化壳并不影响VLPs对BHK- 21细胞的感染和巨噬细胞对其的摄取。获得无极壳的VLPs-W6-CaP的耐酸性可降至pH5.5，在常温和37℃可分别放置13天和11天。37℃处理的VLPs-W6-CaP疫苗诱发的特异性抗体、中和抗体及攻毒保护率仍和正常的VLPs相当。研究对以上嵌合VLPs还进行了硅矿化，结果显示SiO2与矿化肽的结合更具特异性，VLPs-W6的硅化率可达99%。DLS、TEM及SEM检测发现硅化VLPs-W6的粒径为70~100nm,小于VLPs-W6-CaP，同样在外表面形成一层硅化壳。耐热性试验表明硅化VLPs-W6的耐热性可达20天，强于VLPs-W6-CaP。.研究进一步在DC细胞上对VLPs-W6-CaP的加工递呈进行了验证，结果显示，VLPs-W6-CaP能更高效的活化DC细胞并刺激表达高水平的MHC-Π分子、共刺激分子及炎症因子，并能活化天然的T细胞。为VLPs-W6-CaP诱发高水平的免疫反应提供了理论支持。.综合以上研究表明：⑴ 磷酸钙矿化及SiO2硅化均可提高FMD VLPs的耐热性，无极壳增强细胞对VLPs对内吞；⑵ 矿化VLPs能更高效的活化DC细胞和激活天然的T细胞。