论文题名(中文): | SARS-CoV-2结构蛋白在抗病毒免疫中的作用研究 |
姓名: | |
论文语种: | chi |
学位: | 硕士 |
学位类型: | 学术学位 |
学校: | 北京协和医学院 |
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专业: | |
指导教师姓名: | |
校内导师组成员姓名(逗号分隔): | |
论文完成日期: | 2023-05-07 |
论文题名(外文): | The effects of SARS-CoV-2 structural proteins on antiviral immunity |
关键词(中文): | |
关键词(外文): | SARS-CoV-2 structural protein innate immune specific T-cell response humoral immunity |
论文文摘(中文): |
2019年,严重急性呼吸综合征冠状病毒2型(Severe Acute Respiratory Syndrome Coronavirus 2,SARS-CoV-2)作为一种新型病原体出现,导致2019年冠状病毒病(2019 Coronavirus Disease,COVID-19)大流行。SARS-CoV-2可引起上呼吸道和下呼吸道感染,通常与发烧、咳嗽、嗅觉和味觉丧失有关,大多数感染症状轻微,约20-40% 的患者无症状。然而,也有一些患者会产生更严重的症状,包括全身炎症、组织损伤、急性呼吸窘迫综合征、血栓栓塞并发症、心脏损伤、细胞因子风暴等。SARS-CoV-2 刺突蛋白(Spike Protein,S)是一种关键的病毒抗原蛋白,能够产生高中和抗体,而其他结构蛋白,包括膜蛋白(Membrane Protein,M)、核衣壳蛋白(Nucleocapsid Protein,N)和包膜蛋白(Envelope Protein,E)在抗病毒免疫中的作用研究得还不够透彻。 近年来,对SARS-CoV-2病毒感染具有较强保护作用的疫苗快速发展,除了中和抗体以外,T细胞免疫反应在疫苗介导的保护中也发挥着巨大的作用。在关于免疫持久性的研究中,有报道认为病毒的结构蛋白在维持患者或疫苗接种者的抗体水平方面具有重要意义,同时针对病毒抗原的特异性T细胞反应对促进机体免疫系统清除病毒具有积极效应。那么是不是更多的、更系统的特异性T细胞反应更能有效清除病毒呢?在本研究中,我们构建了五个关于结构蛋白S1、S2、M、N和E的真核表达重组质粒,然后质粒在人支气管上皮细胞(16HBE)中表达,检测16HBE细胞中与固有免疫反应以及炎症相关的细胞因子转录水平,以探讨SARS-CoV-2不同结构蛋白对固有免疫反应以及炎症反应产生的影响。然后,分别使用两剂SARS-CoV-2灭活疫苗或两剂mRNA疫苗免疫Balb/c小鼠,体外分离出免疫小鼠的外周血单个核细胞(Peripheral Bood Mononuclear Cells,PBMCs),并用S1、S2、M、N和E 5种结构蛋白刺激PBMCs,评估疫苗接种后相应的特异性T细胞免疫反应。此外,我们还比较了注射两剂灭活疫苗后使用一剂mRNA疫苗加强、注射两剂同源SARS-CoV-2灭活疫苗、注射两剂同源mRNA疫苗诱导的小鼠的体液免疫水平。实验结果表明,在SARS-CoV-2病毒结构蛋白S1、S2、M、N、E中,S1蛋白上调16HBE细胞中部分与固有免疫/炎症相关的细胞因子的转录水平,S2、M、N、E蛋白可以下调16HBE细胞中部分与固有免疫/炎症相关的细胞因子的转录水平。SARS-CoV-2病毒结构蛋白能显著诱导特异性T细胞反应和特异性结合抗体反应,然而,针对SARS-CoV-2病毒结构蛋白M、N、E的免疫反应并不是协调/增强以S蛋白为靶点的体液免疫以及中和抗体的关键。但是,相比于其他疫苗,灭活疫苗完整的结构蛋白免疫机体后激起的免疫反应应该是更为系统的,病毒结构蛋白诱导的特异性T细胞反应究竟在宿主应对病毒感染的过程当中发挥什么样的具体作用,依然值得我们深入探讨。 |
论文文摘(外文): |
In 2019, the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) emerged as a novel pathogen, leading to the 2019 coronavirus disease (COVID-19) pandemic. SARS-CoV-2 can cause upper and lower respiratory tract infections and is often associated with fever, cough, and loss of smell and taste. Most infections are mild and about 20-40% of patients are asymptomatic. However, some patients develop more severe symptoms, including systemic inflammation, tissue damage, acute respiratory distress syndrome (ARDS), thromboembolic complications, heart damage, cytokine storms, and more. The SARS-CoV-2 Spike (S) protein is a critical viral antigenic protein that enables the production of neutralizing antibodies, while other structural proteins, including the membrane (M), nucleocapsid (N) and envelope (E) proteins, have unclear roles in antiviral immunity. In recent years, vaccines with strong protective effect against SARS-CoV-2 virus infection have been developed rapidly. In addition to the neutralizing antibodies, T cell immune responses also play an important role in vaccine-mediated protection. In the study of immune persistence, it has been reported that the structural proteins of viruses are important to maintain the antibody level of patients or immunized persons. Meanwhile, specific T cells targeting virus antigens have a positive effect of promoting the body's immune system to clear the virus. Therefore, is more systematic T-cell response more effective in eliminating the virus? In this study, five eukaryotic expression recombinant plasmids related to structural proteins S1, S2, M, N, and E were constructed and then expressed in human bronchial epithelial cells (16HBE) to detect transcription levels of cytokines associated with innate immune response and inflammation response. Then,Balb/c mice were immunized with two doses of SARS-CoV-2 inactivated vaccine and mRNA vaccine respectively. Peripheral blood mononuclear cells (PBMCs) of the immunized mice were isolated in vitro and stimulated with S1, S2, M, N, and E structural proteins to evaluate the corresponding specific T-cell immune response after vaccination. In addition, the levels of humoral immunity induced by two-dose inactivated vaccine priming followed by mRNA vaccine boosting, two homologous inactivated vaccine doses and two homologous mRNA vaccine doses in immunized mice were compared. The results showed that among SARS-CoV-2 structural proteins, S1 protein up-regulated the transcription levels of some cytokines related to innate immunity/inflammation in 16HBE cells, while S2, M, N, and E proteins down-regulated the transcription levels of some cytokines related to innate immunity/inflammation in 16HBE cells. Viral structural proteins can significantly induce specific T cell responses and specific binding antibody responses. However, immune responses against viral structural proteins M, N, and E are not the key to coordinate/enhance humoral immunity and neutralizing antibodies targeting S protein. However, compared with other vaccines, the immune response stimulated by the complete structural proteins of inactivated vaccines should be more systematic. What specific role the specific T-cell response induced by viral structural proteins plays in the host's response to viral infection is still worth further investigation. |
开放日期: | 2023-05-24 |