新型冠状病毒(Severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)是严重影响人类健康并造成全球性公共危机的新型冠状病毒肺炎（Corona Virus Disease 2019，COVID-19）的主要病原体，具有极强的传染性。为了抑制SARS-CoV-2的传播，已开发出多种不同类型的疫苗。这些疫苗的主要设计靶点是刺突蛋白（spike protein）S，然而通过对COVID-19感染恢复人群，以及未感染人群的免疫学比较分析，发现针对病毒核衣壳蛋白（Nucleocapsid protein）N的特异性细胞免疫具有显著差别，这提示我们该病毒的其他结构蛋白在机体免疫中可能发挥一定的作用。有研究观察表明，在COVID-19患者群体中，对N抗原表现出较强的的特异性细胞毒性T淋巴细胞（Cytotoxic T lymphocyte，CTL）的个体相对具有较好的临床预后。同时，在前期疫苗相关实验中我们发现，含有多种结构蛋白的灭活疫苗似乎具有更持久的保护效果。其中由于N蛋白相较于其他的如S、M蛋白更保守，并且可以诱导细胞免疫应答。因此，探索COVID-19预防性疫苗中有效抗原组分，尤其是N抗原的免疫原性及其与能够诱导中和抗体的S抗原之间的关系，为新冠疫苗的研发提供了一个新的方向。

基于上述基础，本研究利用重组蛋白疫苗背景简单、评价容易，加之安全性高、生物安全等级要求低、纯度高的特点，使用体外真核表达的S1和N抗原多肽，采用皮内和肌肉不同途径、单独和混合免疫实验用蛋白疫苗，以间隔14天两针免疫的方式对SARS-CoV-2较为敏感的叙利亚黄金地鼠模型进行免疫，分别观察了不同抗原诱导机体体液免疫反应的能力。为评价上述免疫反应对叙利亚黄金地鼠的保护作用，在加强免疫14天后（即首针免疫28天）感染原始型SARS-CoV-2活病毒。每日观察临床症状、排毒情况，定期观察各组织病毒载量以及各器官的病理损伤程度、检测血清抗体滴度。结果表明，叙利亚黄金地鼠单独接种SARS-CoV-2结构蛋白S或接种混合的S+N蛋白均能诱导抗体反应，皮内注射和肌肉注射诱导产生的抗体反应没有明显的差异。虽然两种疫苗都不能有效抑制病毒的增殖和复制，但免疫后的叙利亚黄金地鼠在受到病毒攻击时表现出部分缓解的症状。有趣的是，这两种蛋白（N、S1）的单独或同时存在并不能使叙利亚黄金地鼠抵抗病毒的感染，仅使其感染后体重减轻状况有所缓解，而灭活病毒疫苗所诱导相似水平的NAb则却能保护该动物抵御同等剂量病毒的感染。

正是由于两种类型的实验疫苗免疫相同动物模型，诱导相似的中和抗体水平，但却在抵御病毒感染时产生了天壤之别的现象，我们继续探究其产生的可能原因。但是因为商业化的叙利亚黄金地鼠检测试剂非常有限，除体液免疫外的很多免疫学指标检测困难，从而导致难以综合分析评价实验用疫苗的各种特性及相关原因，所以后续实验在Balb/c小鼠模型中进行。通过肌肉注射的免疫方法，采用与叙利亚黄金地鼠相同的免疫程序，免疫相同剂量的蛋白。在第2次免疫后的14天进行第3针刺激，并分别在3针刺激后的12h、24h、36h，检测其各种细胞免疫在内的一系列的免疫指标。结果表明， Balb/c与叙利亚黄金地鼠一样，单独接种SARS-CoV-2结构蛋白S或接种混合的S+N蛋白均能诱导抗体反应，在3针刺激后Balb/c小鼠还能够引起特异性T细胞免疫应答，激活特异性CD4+和CD8+ T细胞，并且N蛋白和S蛋白的联合免疫可能在一定程度上能够协同增强天然免疫细胞信号分子的作用。综上所述，提示我们进一步了解N蛋白的功能及在天然免疫中发挥的相关机制问题，为开发更有效的SARS-CoV-2疫苗提供理论基础，对防治COVID-19具有重要意义。

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a Corona Virus Disease that seriously affects human health and causes a global public crisis 2019, COVID-19) is a major pathogen and is highly contagious. A number of different types of vaccines have been developed to suppress the spread of SARS-CoV-2. The primary design target of these vaccines is spike protein S. However, a comparative analysis of immunology between recovered and uninfected populations showed significant differences in cell-specific immunity against viral Nucleocapsid protein N. This suggests that other structural proteins of the virus may play a role in the body's immunity. Studies have shown that individuals with strong Cytotoxic T lymphocytes (CTL) for N antigen have a relatively good clinical prognosis in COVID-19 patients. Meanwhile, in previous vaccine-related experiments, we found that inactivated vaccines containing a variety of structural proteins seem to have a more durable protective effect. Because N protein is more conserved than other proteins such as S and M, it can induce cellular immune response. Therefore, exploring the immunogenicity of the active antigenic components in COVID-19 preventive vaccines, especially the N antigen and its relationship with the S antigen, which can induce neutralizing antibodies, provides a new direction for the development of COVID-19 vaccines. 

Based on the above basis, the recombinant protein vaccine is characterized by simple background, easy evaluation, high safety, low biosafety level requirements, and high purity. In this study, the in vitro eukaryotic expression of S1 and N antigen polypeptides is used, and the intradermal and muscular pathways, individual and mixed immunization protein vaccines are used. The Syrian golden ground mouse model sensitive to SARS-CoV-2 was immunized with two injections at an interval of 14 days, and the ability of different antigens to induce humoral immune response was observed. In order to evaluate the protective effect of the above immune response on Syrian golden ground squirrels, they were infected with the original SARS-CoV-2 live virus after 14 days of enhanced immunization (i.e., 28 days of initial immunization). Clinical symptoms and detoxification were observed daily, viral load of each tissue and pathological damage of each organ were observed regularly, and serum antibody titers were detected. The results showed that antibody response could be induced by inoculation of SARS-CoV-2 structural protein S alone or mixed with S+N protein, and there was no significant difference between intradermal and intradermal injection. Although neither vaccine was effective in inhibiting the proliferation and replication of the virus, immunized Syrian golden Gophers showed partial relief when attacked by the virus. Interestingly, the presence of the two proteins (N, S1) alone or in combination did not protect the Syrian Golden Ground squirrels from infection with the virus, only reducing their weight loss after infection, whereas similar levels of NAb induced by inactivated virus vaccine protected the animals from infection with the same dose of virus. 

We continue to explore the possible reasons why the two types of experimental vaccines immunize the same animal models and induce similar levels of neutralizing antibodies, but produce vastly different effects against viral infection. However, due to the limited commercial detection reagents for Syrian golden Gophers, it is difficult to detect many immunological indicators except humoral immunity, which makes it difficult to comprehensively analyze and evaluate various characteristics and related causes of experimental vaccines. Therefore, follow-up experiments were conducted in Balb/c mouse model. The same dose of protein is immunized by intramuscular injection, using the same immunization procedure as the Syrian golden Gophers. 14 days after the second immunization, the third dose of stimulation was administered, and 12h, 24h, 36h after the third dose of stimulation, a series of immune indicators including various cellular immunity were detected. The results showed that Balb/c mice, like Syrian golden ground mouse, were inoculated with SARS-CoV-2 structural protein S alone or mixed with S+N protein to induce antibody response. Balb/c mice could also induce specific T cell immune response and activate specific CD4+ and CD8+ T cells after 3 injections of stimulation. In addition, the combined immunity of N protein and S protein may synergistically enhance the role of natural immune cell signaling molecules to a certain extent. In conclusion, it has great significance for us to further understand the function of N protein and its related mechanism in natural immunity, and provide a theoretical basis for the development of more effective SARS-CoV-2 vaccine, which is of great significance for the prevention and treatment of COVID-19.