抗原蛋白可控组装：“自运载”纳米疫苗探索

Adjuvants have made great success in prophylactic vaccine, however it is not the same true if applied for therapeutic vaccine due to inadequate ability to initiate T cell response. Nanovaccine raises the hope to address the above issue for the mimic feature of microbes that enables to dense, multicopy displays and followed cross-presentation of antigen. It is therefore predicted as the next-generation vaccine. Nonetheless, two critical challenges not favorable to elicit T cell response remain to be addressed. It includes: difficulty antigen display for inner entrapment and low encapsulation efficiency of antigen (low antigen density). In this project, we proposed a “self-carrier” nanovaccine, in which antigens act as the carrier to form nanovaccine, driven by intermolecular disulfide bond cross-linking between antigens. To make the process proceed, the hydrophobic interaction as well as hydrogen bond of antigen protein structure should be carefully regulated, so as to expose the free cysteine groups and make them intermolecularly accessible for heat-mediated cross-linking. CpG agonist, terminated with thiol group, can be further incorporated by integration into the matrix disulfide network as “danger signal” necessary for activating innate immune system. The most intriguing feature of “self-carrier” nanovaccine is the high antigen content (more than 95%), which enables to maximize the merits of dense antigen display similar to microbes. The synthetic feasibility as well as enhanced immune efficacy have preliminarily verified. This project aims to not only develop novel strategy for protein nano-assembly, but also to pave the new route for vaccine development and hold the clinic impact.

预防性疫苗取得巨大成功，佐剂的“载体功能”功不可没，但传统佐剂载体诱导T细胞免疫乏力，制约了疫苗免疫治疗潜力。纳米载体兼具传统佐剂特性及类病毒尺寸属性，被誉为新一代佐剂的希望。然而，传统纳米载体用于疫苗设计难以充分发挥“类病毒”免疫优势：抗原表面展示困难（被包封）及抗原密度小（负载率低）。因此限制了T细胞免疫效应的提高。针对以上瓶颈，本项目拟不借助传统纳米载体，而通过挖掘抗原自组装潜力，构建一种“自运载”纳米疫苗，其中抗原同时承担“抗原与载体”双重功能。组装策略是利用小分子协同调控抗原蛋白疏水、氢键网络，以暴露巯基，介导抗原分子间二硫键交联，实现抗原自身为单元的可控组装。组装过程中可借助巯基引入寡核苷酸CpG作为危险信号。整个纳米疫苗抗原占比将>95wt%。“自运载”设计增强T细胞免疫行为在预实验中得到确证。本项目不仅有望发展一种蛋白组装新方法，更将为纳米疫苗提供新思路，推动领域发展。

增强疫苗的免疫效应是实现疫苗临床效果的关键。纳米载体兼具传统佐剂特性及类病毒尺寸属性，被誉为新一代佐剂的希望。传统纳米载体用于疫苗设计难以充分发挥“类病毒”免疫优势：抗原表面展示困难（被包封）及抗原密度小（负载率低）。针对以上瓶颈，本项目发展了一种新技术：不借助传统纳米载体，而通过挖掘抗原自组装潜力，构建一种“自运载”纳米疫苗，其中抗原同时承担“抗原与载体”双重功能。我们成功设计并制备出了合成方法简单、过程绿色、方便快捷的一种新型纳米疫苗。该纳米疫苗是分别由占组分97%wt为抗原蛋白OVA和3%wt为免疫佐剂CpG构成，该疫苗不仅完美契合了疫苗的成分最简化原则，更保证了纳米疫苗的抗原高密度。通过十二烷基硫酸钠的精确调控，抗原OVA的结构在反应过程中在分子水平上得以精准的调控，随后可与巯基化的CpG通过二硫键反应组装形成稳定的纳米颗粒。经静态光散射测算，一个50nm的OVA蛋白颗粒含有约500个OVA 抗原分子。该疫苗近乎由100%的抗原组成，因此赋予其高效刺激机体产生高水平的细胞杀伤效应，在B1-OVA黑色素瘤预免疫治疗模型上，约70%的小鼠处于完全的免疫保护状态。除此之外，该纳米疫苗还具有控制释放、易转运，保护CpG免于酶解的能力，在疫苗注射部位，该疫苗表现出明显的储库效应，纳米疫苗的缓释性能促进了肿瘤疫苗的转运，提升了对机体的长效刺激的能力。这种疫苗设计上的新突破为成分最简化这一原则提供了一个新思路，有望在临床免疫治疗上得到应用。