背景:由严重急性呼吸综合征冠状病毒 2(SARS-CoV-2)感染导致的疾病严重威胁着人类健康，研制制备简单、价格低廉的新冠疫苗可以降低中低收入国家的疫苗接种经济负担，同时便于针对突变毒株开展疫苗的快速研发。SARS-CoV-2 的 S 蛋白由 S1 和 S2 两个亚基组成，其中 S1 可以与血管紧张素转换酶 II (angiotensin-converting enzyme 2，ACE2)结合促进病毒对靶细胞的入侵，而 S1 上的受体结合域(receptor binding domain，RBD)是病毒与其受体结合的直接识别区域，因此是疫苗研制的关键靶点。利用细菌、昆虫或哺乳动物细胞表达免疫原性病毒蛋白通常可以制成有效且安全的亚单位疫苗，可以利用佐剂增强免疫反应，同时也可利用抗原的多聚化以及将抗原缀合在纳米颗粒上来提高 疫苗的免疫原性。由于原核表达系统中缺乏蛋白质糖基化以及可能形成变性蛋白包涵体，大多数针对 SARS-CoV-2 的疫苗开发工作都集中在价格昂贵且效率较低的哺乳动物表达系统上，而不是高效低廉的大肠杆菌系统。然而，大量的临床和临床前研究显示了使用大肠杆菌表达系统开发抗病毒疫苗的成功案例和有益探索，例如人乳头瘤病(human papillomavirus，HPV)和戊型肝炎病毒 (hepatitis E virus ，HEV)的重组病毒样颗粒(virus-like particle，VLP)在临床上获得应用，以及使用细菌外膜囊泡(bacterial outer-membrane vesicle， OMV)传递登革热病毒、流感病毒和中东呼吸综合征(Middle East respiratory syndrome ，MERS)冠状病毒的病毒抗原在动物模型中证明有效。SARS-CoV-2 的 RBD 在自然构象显示出三聚体结构，这种三聚体结构引起的宿主免疫反应可能与单体引起的宿主免疫反应不同，很多研究表明 RBD 的二聚体和多聚体形式比RBD 单体形式具有更好的免疫保护能力。因此，RBD 的多聚化可能是设计 SARS-CoV-2 疫苗的关键步骤。

目的:在大肠杆菌表达系统中，实现 SARS-CoV-2 RBD 重组蛋白的正确结构折叠以及多聚化纳米装配，探讨有效的亚单位纳米疫苗递送策略，为制备高免疫原性、高免疫保护力而价格低廉的 SARS-CoV-2 等重组疫苗提供平台。

方法:在第一部分“基于新型细菌仿生囊泡平台呈递 SARS-CoV-2 RBD 蛋白”的 研究中，利用膜孔蛋白细胞溶素 A (Cytolysin A, ClyA) 搭载全长的 RBD 形成融合蛋白 ClyA-RBD，基于 ClyA 的膜定位及自组装特性实现 RBD 在菌膜上高度规则的聚合化呈现。过表达 ClyA-RBD 的大肠杆菌细胞通过高压均质技术驱动细菌生物膜通过狭窄的缝隙后形成一种新型细菌仿生囊泡(bacterial biomimetic vesicle，BBV)。首先，考察囊泡的理化性质，以蛋白质组学分析 RBD-BBV 蛋白组成，通过与 hACE2 及中和抗体的结合实验考察 RBD 的构象折 叠;在体外细胞实验中，分析 RBD-BBV 促树突状细胞(dendritic cell，DC)摄取、溶酶体逃逸及促 DC 表型成熟;然后，在体内实验中，以活体及组织离体荧光成像分析 RBD-BBV 在注射部位及淋巴结的分布，以流式细胞术分析淋巴结中抗原摄取的细胞类型，以微阵列分析淋巴结基因表达情况;最后，进行小鼠免疫，ELISA 分析抗体应答水平及其对 SARS-CoV-2 S1 与 hACE2 结合的阻断作用，以流式细胞术与 ELISA 分别分析脾脏免疫细胞及其细胞因子水平，以 ELISpot 分析抗原特异的效应细胞应答。

在第二部分“基于聚合孔纳米颗粒平台递送 SARS-CoV-2 RBD 蛋白”研究中，利用表面活性剂的方法将 ClyA-RBD 从菌膜上提取后自组装成为聚合孔纳米颗粒，体外对该颗粒进行了形态、折叠及促 DC 作用分析，体内考察主要组织器官分布、淋巴结生发中心免疫细胞的激活，并进一步在免疫小鼠中考察体液及细胞免疫应答。

结果:在第一部分研究中，成功制备了 ClyA 介导呈递 RBD 的细菌仿生囊泡 RBD-BBV，与细菌自然分泌的 OMV 相比，囊泡产量得到极大提升，并显著提高了囊泡表面外源蛋白荷载量，解决了两个限制细菌囊泡应用发展的重要问题。基于 ClyA 的膜孔组装特性，BBV 表面观察到大量由 RBD 聚合形成的环状结构，且以纳米孔展示在囊泡表面的 RBD 与单体相比具有与 ACE2 更高的、与真核表达的 S1-Fc 相当的亲和力，此外，RBD-BBV 能与多种具有中和作用的单克隆抗体结合，提示 RBD-BBV 正确折叠，具有与真核系统中表达的 S1 相似的空间构象，提示 RBD 可提供正确的免疫原性。进一步的研究发现， BBV 促进 DC 的抗原摄取及成熟，同时可发生溶酶体逃逸，有利于细胞免疫诱导;此外， 在注射部位缓释、淋巴结靶向、免疫反应激活等方面展示了纳米蛋白脂质囊泡的特点。在小鼠免疫应答评估中，RBD-BBV 诱导了特异性体液免疫，抗血清在野生型 SARS-CoV-2 病毒细胞攻击中和实验中表现出了中和保护作用，同时，RBD-BBV 免疫激发了抗原特异的细胞免疫应答，显著刺激效应及记忆性的 CD4+和 CD8+ T 细胞产生。

为探讨更简化的疫苗形式，论文基于表面活性剂可以模拟细菌膜对 ClyA 构象变化的触发并能促进孔结构自组装，进一步制备了重组 ClyA-RBD 构成的聚合孔(polymerized porin ，PP)纳米颗粒 RBD-PP。与 ClyA-RBD 相比，RBD-PP 表现出更强的中和抗体及 ACE2 识别能力，提示其更为正确的空间构象折叠，推测是由于 RBD 在 PP 上的高度规则排列与重复排列暴露以及在伴随 ClyA 聚合过程中的空间构象折叠。RBD-PP 可以高效地靶向淋巴结，并促进抗原提呈细胞的高效摄取与成熟，以及 T 滤泡辅助 (T follicular helper，Tfh)细 胞和生发中心(germinal center，GC) B 细胞的生成。在小鼠免疫中，RBD-PP 较之 RBD 单体，诱导出更为显著的中和抗体应答，以及效应及记忆性 CD4+与 CD8+ T 细胞的应答，并表现出平衡的 Th1 与 Th2 应答特征。

结论:利用 ClyA 作为载体携带 RBD 实现了大肠杆菌中 RBD 的高效表达，并开发了一种细菌仿生囊泡系统以及一种聚合孔纳米颗粒作为新型的疫苗抗原递送平台，成功实现了正确构像的 RBD 聚合化纳米递送，在体外细胞水平及小鼠免疫模型中展示了促进抗原加工递呈，刺激机体产生有效免疫应答的能力。论文为 SARS-CoV-2 亚单位疫苗研发，尤其是应对不断突变的病毒带来的挑战奠定了重要基础，同时也为其他病毒疫苗的研发提供了新思路，并可扩展到到细菌疫苗、肿瘤疫苗以及药物递送等研究应用领域。

Background: The disease caused by SARS-CoV-2 infection threatens human health. The development of vaccines that are simple to prepare and inexpensive can reduce the economic burden of vaccination in low- and middle-income countries, and at the same time facilitate the rapid development of vaccines against mutant strains. The S protein of SARS-CoV-2 consists of two subunits, S1 and S2. S1 can combine with angiotensin-converting enzyme II (ACE2) to promote virus invasion of target cells, and the receptor-binding domain (RBD) on S1 is the direct recognition region for the virus to bind to its receptor and is therefore a key target for vaccine development. The use of bacterial, insect or mammalian cells to express immunogenic viral proteins can often make effective and safe subunit vaccines. Adjuvants can be used to enhance the immune response, while multimeric antigen display and conjugation to nanoparticles can also be used to improve the immunogenicity of vaccines. Due to lack of protein glycosylation and possible formation of denatured protein inclusion bodies in prokaryotic expression systems, most vaccine development against SARS-CoV-2 has focused on the expensive and less efficient mammalian expression system rather than the highly efficient Escherichia coli system. However, a large number of clinical and preclinical studies have shown successful cases and beneficial explorations for the development of antiviral vaccines using E. coli expression systems, such as the recombinant virus-like particle (VLP) of human papillomavirus (HPV) and hepatitis E virus (HEV) has been applied clinically, and the use of bacterial outer-membrane vesicles (OMVs) to deliver viral antigens from dengue, influenza, and Middle East respiratory syndrome coronaviruses (MERS-CoV) has proven effective in animal models. The RBD of SARS-CoV-2 exhibits a trimeric structure in its natural conformation, and the host immune response elicited by this trimeric structure may be different from that elicited by the monomeric structure. Many studies have shown that the dimer and multimeric forms of RBD have better immune protection ability than RBD monomer. Therefore, multimerization of RBD may be a critical step in designing a SARS-CoV-2 vaccine.

Objective: In the E.coli expression system, realize the correct structural folding and multimerization nano-assembly of SARS-CoV-2 RBD recombinant protein, and explore the effective subunit nano-vaccine delivery strategy, for the preparation of highly immunogenic, high immune protection and inexpensive recombinant vaccines such as SARS-CoV-2 provide a platform.

Methods: In the first part of the study "Presentation of the SARS-CoV-2 RBD protein based on the novel bacterial biomimetic vesicle platform". The porin cytolysin A (ClyA) is used to carry the full-length RBD to form the fusion protein ClyA-RBD. Based on the membrane localization and self-assembly properties of ClyA, the highly regular polymerization presentation of RBD on bacterial membranes is realized. E.coli cells overexpressing ClyA-RBD form a new type of bacterial biomimetic vesicle (BBV) after the bacterial membrane passes through a narrow gap driven by high-pressure homogenization technology. First, the physicochemical properties of the vesicles were evaluated, the protein composition of RBD-BBV was analyzed by proteomics, and the conformational folding of RBD was investigated through binding experiments with hACE2 and neutralizing antibodies. Using in vitro cell experiments to analyze the effect of RBD-BBV on DC uptake, lysosome escape and DC phenotype maturation. Then, in the in vivo experiment, the distribution of RBD-BBV at the injection site and lymph nodes was analyzed by in vivo and tissue ex vivo fluorescence imaging, the cell types of antigen uptake in lymph nodes were analyzed by flow cytometry, and the gene expression of lymph nodes was analyzed by microarray. Finally, the mice were immunized, and the antibody response level and its blocking effect on the combination of SARS-CoV-2 S1 and hACE2 were analyzed by ELISA. Spleen immune cells and their cytokine levels were analyzed by flow cytometry and ELISA, and antigen-specific effector cell responses were analyzed by ELISpot.

In the second part of the study " Presentation of the SARS-CoV-2 RBD protein based on the polymeric pore nanoparticle platform", the ClyA-RBD was extracted from the bacterial membrane using surfactants and then self-assembled into polymeric pore nanoparticles. In vitro experiments were carried out to investigate the morphology, folding and DC-promoting effect of the particles. In vivo experiments were used to investigate the distribution of major tissues and organs, the activation of immune cells in lymph node germinal centers, and further immunization of mice to investigate humoral and cellular immune responses.

Results: In the first part of the study, a bacterial biomimetic vesicle RBD-BBV that presents RBD mediated by ClyA was successfully prepared. Compared with the OMV naturally secreted by bacteria, the yield of BBV has been greatly improved, and the loading of exogenous proteins on the vesicle surface has been significantly increased, which solves two important problems that limit the application and development of bacterial vesicles. Based on the membrane pore assembly properties of ClyA, a large number of ring structures formed by RBD polymerization were observed on the surface of BBV. Compared with the monomer, the RBD displayed on the surface of the vesicle in the form of nanopores has a higher affinity with ACE2, and the affinity is comparable to that of S1-Fc expressed in the eukaryotic system. In addition, RBD- BBV can bind to a variety of neutralizing monoclonal antibodies, suggesting that RBD-BBV is correctly folded and has a similar spatial conformation to S1 expressed in eukaryotic systems, suggesting that RBD can provide correct immunogenicity. Further studies have found that BBV promotes the antigen uptake and maturation of DCs, and at the same time, lysosomal escape that is conducive to the induction of cellular immunity can occur. In addition, the characteristics of nano-proteolipid vesicles were demonstrated in terms of sustained release at the injection site, lymph node targeting, and activation of immune responses. In the mouse immune model evaluation, RBD-BBV induced specific humoral immunity, and the antiserum showed neutralizing protection in the wild-type SARS-CoV-2 virus neutralization experiment. At the same time, RBD-BBV immunization stimulated antigen-specific cellular immune responses, significantly stimulating the production of effector and memory CD4+ and CD8+ T cells.

In order to explore a more simplified form of vaccine, based on the characteristics that the surfactant can simulate the bacterial membrane triggering the conformational change of ClyA and promote the self-assembly of the pore structure, the polymeric porin nanoparticle (RBD-PP) composed of recombinant ClyA-RBD was further prepared. Compared with ClyA-RBD, RBD-PP showed a stronger ability to recognize neutralizing antibodies and ACE2, suggesting that it was folded in a more correct spatial conformation. Compared with ClyA-RBD, RBD-PP showed a stronger ability to recognize neutralizing antibodies and ACE2, suggesting that it is folded in a more correct spatial conformation, presumably because RBD is highly exposed to PP in a regular repeat arrangement and the steric conformational folding that occurs during concomitant ClyA polymerization. RBD-PP efficiently targets lymph nodes and promotes efficient uptake and maturation of antigen-presenting cells, as well as the generation of T follicular helper (Tfh) cells and germinal center (GC) B cells. In mouse immunization, RBD-PP induced more significant neutralizing antibody responses, effector and memory CD4+ and CD8+ T cell responses than RBD monomers, and exhibited balanced Th1 and Th2 response characteristics.

Conclusion: Using ClyA as a carrier to carry RBD achieved high-efficiency expression of RBD in Escherichia coli, and developed a bacterial biomimetic vesicle system and a polymeric porin nanoparticle as the novel vaccine antigen delivery platform. The polymerized nano-delivery of RBD with the correct conformation has been successfully realized. The ability to promote antigen processing and presentation and stimulate the body to produce an effective immune response has been demonstrated at the cellular level in vitro and in mouse immune models. The paper lays an important foundation for the development of SARS-CoV-2 subunit vaccines, especially in response to the challenges posed by constantly mutating viruses. At the same time, it also provides new ideas for the research and development of other virus vaccines, and can be extended to research and application fields such as bacterial vaccines, tumor vaccines, and drug delivery.