MERS-CoV重组蛋白及病毒样颗粒疫苗的研究
发布时间：2019-06-13 06:34
【摘要】：中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus, MERS-CoV)是2012年在中东地区发现的一种新型冠状病毒,随后该病毒从中东地区向外传播。据世界卫生组织(World Health Organization, WHO)报道,截止到2016年4月26日,全球共有27个国家1728例实验室确诊病例,其中死亡624例,病死率高达36.1%。然而,目前尚无特异性药物治疗该病毒引起的感染。因此,丞待开发一种安全有效的疫苗预防或治疗该病毒引起的疾病。MERS-CoV为包膜病毒,其包膜上的棘突蛋白(Spike, S)在介导病毒与宿主细胞膜结合以及病毒进入细胞过程中发挥重要作用。S分为S1和S2两个亚单位,前者介导病毒和易感细胞上的受体结合,后者在病毒包膜和宿主细胞膜融合中发挥作用。S1进一步分为N端结构域(N terminal domain, NTD)和C端的受体结合结构域(Receptor binding domain, RBD)。RBD蛋白介导MERS-CoV与宿主细胞膜上的二肽基肽酶4(Dipeptidyl peptidase-4, DPP4)分子结合,富集中和性抗原表位,是MERS-CoV疫苗研发最重要的靶抗原。亚单位疫苗是预防病毒感染的重要手段,本文应用重组RBD或NTD蛋白作为亚单位疫苗,先后在小鼠或恒河猴体内检测了其诱导的免疫应答反应和免疫保护效果。同时构建了基于S蛋白的MERS-CoV嵌合型病毒样颗粒(Virus like particles, VLPs)疫苗,并在小鼠体内检测了该VLPs的免疫原性。具体研究内容及结果如下：1、重组RBD蛋白疫苗在小鼠体内免疫效果研究重组RBD蛋白与不同佐剂(单独铝佐剂、铝佐剂和CpG联合、铝佐剂和poly(I:C)联合)经肌肉注射,或与CpG和不完全弗氏佐剂(Incomplete Freund's adjuvant, IFA)联合应用经皮下注射,可在BALB/c小鼠体内诱导不同水平RBD特异性IgG抗体、中和抗体以及全身或局部细胞免疫应答反应。其中,重组RBD蛋白在铝佐剂和CpG联合作用下、经肌肉注射可在小鼠体内诱导最高水平的体液免疫应答,且该免疫策略可诱导多种效应性细胞因子的分泌。病毒攻击实验表明,该策略可在小鼠体内诱导有效的免疫保护反应,减轻MERS-CoV攻击后小鼠肺脏和气管组织的炎症反应。2、重组RBD蛋白疫苗在恒河猴体内免疫效果研究高(200μg)、低剂量(50μg)重组RBD蛋白疫苗肌肉注射三次免疫恒河猴后,在其体内诱导了强大而持久的体液免疫应答(IgG抗体和中和抗体)。高剂量组重组RBD蛋白疫苗还诱发了有效的细胞免疫应答。末次免疫后2w,恒河猴气管内接种6.5×107TCID50 MERS-CoV。结果发现,与对照组相比免疫恒河猴肺部炎症反应较轻、肺脏和气管病理改变轻微、肺组织MERS-CoV病毒载量和病毒滴度均较低。表明该重组RBD蛋白疫苗在恒河猴体内诱导了有效的免疫保护反应。3、重组NTD蛋白疫苗在小鼠体内免疫效果研究重组NTD蛋白在铝佐剂和CpG联合作用下、经肌肉注射三次免疫BALB/c小鼠。结果发现,该重组NTD蛋白疫苗在小鼠体内除诱导了中和抗体(水平略低于重组RBD蛋白疫苗)外,还诱导了高水平的细胞免疫应答(强度显著高于重组RBD蛋白疫苗)。该体液和细胞免疫应答在小鼠体内至少持续14w而无明显下降。MERS-CoV攻击后,病理结果示,虽然攻毒后的小鼠肺部出现炎症改变,但较对照组明显减轻,且该免疫保护水平与重组RBD蛋白疫苗相当。4、VLPs疫苗的研制及免疫原性分析以禽流感病毒H5N1的基质蛋白M1为骨架,在其表面嵌合MERS-CoV的S蛋白,应用杆状病毒表达系统在昆虫细胞中包装了MERS-CoV嵌合型VLPs。该VLPs在电镜下与MERS-CoV相似,呈球形,直径约100nm左右,具有典型的包膜以及“皇冠”样外观。该VLPs疫苗与铝佐剂和CpG联合应用、经肌肉注射免疫BALB/c小鼠后,可在其体内诱导S蛋白特异性IgG抗体,且抗体滴度与灭活MERS-CoV疫苗相当。此时血清亦检测到中和抗体,但抗体滴度低于灭活MERS-CoV疫苗免疫组。以上研究表明,本研究成功构建了有免疫原性的MERS-CoV嵌合型VLPs。综上所述,本研究在小鼠或恒河猴体内评价了重组RBD和NTD亚单位疫苗预防MERS-CoV感染的效果,结果表明该两种亚单位疫苗均在动物体内诱导了有效的免疫应答和明显的免疫保护效果。应用流感病毒M1为骨架,表面嵌合MERS-CoV S蛋白,成功构建了嵌合型VLPs疫苗。该VLPs疫苗在小鼠体内诱导了高水平的体液免疫应答反应。这些研究为MERS-CoV疫苗的研发以及人体应用奠定了理论基础。
[Abstract]:Middle East response syndrome coronavir (MERS-CoV) is a new coronavirus found in the Middle East in 2012, and the virus then spread out of the Middle East. According to the World Health Organization (WHO), as of 26 April 2016, there were 1728 laboratory-confirmed cases in 27 countries around the world, of which 624 died and the case fatality rate was as high as 36.1%. However, there is no specific drug currently being used to treat the infection caused by the virus. Therefore, a safe and effective vaccine is to be developed to prevent or treat the disease caused by the virus. MERS-CoV is a enveloped virus, and the spinous process protein (Spike, S) on the envelope plays an important role in mediating the binding of the virus to the host cell membrane and the entry of the virus into the cell. S is divided into S1 and S2 subunits, which mediate the binding of the receptors on the virus and the susceptible cells, which play a role in the fusion of the viral envelope and the host cell membrane. S1 is further divided into N-terminal domain (NTD) and C-terminal receptor binding domain (RBD). The RBD protein-mediated MERS-CoV binds to the dipeptidyl peptidase 4 (DPP4) on the host cell membrane to enrich the neutralizing antigen epitope and is the most important target antigen for the development of the MERS-CoV vaccine. Subunit vaccine is an important means for the prevention of viral infection. In this paper, the recombinant RBD or NTD protein is used as a subunit vaccine, and the induced immune response and the immune protective effect are detected in the mice or rhesus monkeys. The vaccine of the S-protein-based MERS-CoV chimeric virus-like particles (VLPs) was constructed, and the immunogenicity of the VLPs was also detected in the mice. The specific research contents and results are as follows:1. The recombinant RBD protein vaccine is used to study the immune effect of the recombinant RBD protein in the mouse, and the recombinant RBD protein and the different adjuvants (the separate aluminum adjuvant, the aluminum adjuvant and the CpG combination, the aluminum adjuvant and the poly (I: C)) are administered by intramuscular injection, Or in combination with CpG and incomplete Freund's aduviant, IFA), and can induce different levels of RBD-specific IgG antibodies, neutralizing antibodies, and systemic or local cellular immune response reactions in BALB/ c mice. In which, the recombinant RBD protein can induce the highest level of humoral immune response in the mouse body by intramuscular injection under the combination of the aluminum adjuvant and the CpG, and the immune strategy can induce the secretion of a plurality of effector cytokines. The virus attack experiment shows that the strategy can induce effective immune protection reaction in the mouse body, and the inflammation reaction of the lung and the trachea tissue of the mouse after the MERS-CoV attack is reduced. A strong and persistent humoral immune response (IgG antibody and neutralizing antibody) was induced in the body of the rhesus after the low-dose (50. mu.g) recombinant RBD protein vaccine was injected intramuscularly three times with the rhesus monkey. The high-dose recombinant RBD protein vaccine also induces an effective cellular immune response. 5-107 TCID50 MERS-CoV was inoculated in the trachea of the rhesus monkey after the last immunization. The results showed that, compared with the control group, the pulmonary inflammatory response of the rhesus monkey was light, the pathological changes of the lung and the trachea were mild, the lung tissue MERS-CoV viral load and the virus titer were both low. The results showed that the recombinant RBD protein vaccine induced an effective immune protective response in the rhesus monkey.3. The recombinant NTD protein vaccine was used to study the effect of the recombinant NTD protein in the mice. The BALB/ c mice were immunized with the three-time intramuscular injection of the recombinant NTD protein in the combination of aluminum adjuvant and CpG. As a result, the recombinant NTD protein vaccine induced a high level of cellular immune response (significantly higher in intensity than the recombinant RBD protein vaccine), in addition to the induction of neutralizing antibodies (slightly lower than the recombinant RBD protein vaccine) in the mice. The humoral and cellular immune response was at least 14 w in the body of the mouse without significant decrease. After the attack of MERS-CoV, the pathological results show that, although the inflammation of the lung of the mice after challenge is changed, the control group is obviously relieved, and the immune protection level is comparable to that of the recombinant RBD protein vaccine.4, the development and the immunogenicity analysis of the VLPs vaccine are based on the base protein M1 of the avian influenza virus H5N1, MERS-CoV chimeric VLPs were packaged in insect cells using a baculovirus expression system. The VLPs are similar to MERS-CoV under the electron microscope, have a spherical shape with a diameter of about 100 nm, and have a typical envelope and a "crown"-like appearance. The VLPs vaccine is used in combination with the aluminum adjuvant and the CpG. After the BALB/ c mice are immunized by intramuscular injection, the S-protein-specific IgG antibody can be induced in the BALB/ c mice, and the antibody titer is equivalent to the inactivated MERS-CoV vaccine. The serum also detected neutralizing antibodies, but the antibody titer was lower than that of the inactivated MERS-CoV vaccine. The above studies have shown that this study successfully constructed the immunogenic MERS-CoV chimeric VLPs. To sum up, the effect of recombinant RBD and NTD subunit vaccine on the prevention of MERS-CoV infection was evaluated in mice or rhesus monkeys, and the results showed that the two subunit vaccines induced an effective immune response and a clear immune protective effect in the animal. The chimeric VLPs vaccine was successfully constructed by using the influenza virus M1 as a skeleton and the surface-chimeric MERS-CoV S protein. The VLPs vaccine induced a high level of humoral immune response in mice. These studies have laid a theoretical foundation for the development of MERS-CoV vaccine and the application of human body.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392
，
本文编号：2498314
[Abstract]:Middle East response syndrome coronavir (MERS-CoV) is a new coronavirus found in the Middle East in 2012, and the virus then spread out of the Middle East. According to the World Health Organization (WHO), as of 26 April 2016, there were 1728 laboratory-confirmed cases in 27 countries around the world, of which 624 died and the case fatality rate was as high as 36.1%. However, there is no specific drug currently being used to treat the infection caused by the virus. Therefore, a safe and effective vaccine is to be developed to prevent or treat the disease caused by the virus. MERS-CoV is a enveloped virus, and the spinous process protein (Spike, S) on the envelope plays an important role in mediating the binding of the virus to the host cell membrane and the entry of the virus into the cell. S is divided into S1 and S2 subunits, which mediate the binding of the receptors on the virus and the susceptible cells, which play a role in the fusion of the viral envelope and the host cell membrane. S1 is further divided into N-terminal domain (NTD) and C-terminal receptor binding domain (RBD). The RBD protein-mediated MERS-CoV binds to the dipeptidyl peptidase 4 (DPP4) on the host cell membrane to enrich the neutralizing antigen epitope and is the most important target antigen for the development of the MERS-CoV vaccine. Subunit vaccine is an important means for the prevention of viral infection. In this paper, the recombinant RBD or NTD protein is used as a subunit vaccine, and the induced immune response and the immune protective effect are detected in the mice or rhesus monkeys. The vaccine of the S-protein-based MERS-CoV chimeric virus-like particles (VLPs) was constructed, and the immunogenicity of the VLPs was also detected in the mice. The specific research contents and results are as follows:1. The recombinant RBD protein vaccine is used to study the immune effect of the recombinant RBD protein in the mouse, and the recombinant RBD protein and the different adjuvants (the separate aluminum adjuvant, the aluminum adjuvant and the CpG combination, the aluminum adjuvant and the poly (I: C)) are administered by intramuscular injection, Or in combination with CpG and incomplete Freund's aduviant, IFA), and can induce different levels of RBD-specific IgG antibodies, neutralizing antibodies, and systemic or local cellular immune response reactions in BALB/ c mice. In which, the recombinant RBD protein can induce the highest level of humoral immune response in the mouse body by intramuscular injection under the combination of the aluminum adjuvant and the CpG, and the immune strategy can induce the secretion of a plurality of effector cytokines. The virus attack experiment shows that the strategy can induce effective immune protection reaction in the mouse body, and the inflammation reaction of the lung and the trachea tissue of the mouse after the MERS-CoV attack is reduced. A strong and persistent humoral immune response (IgG antibody and neutralizing antibody) was induced in the body of the rhesus after the low-dose (50. mu.g) recombinant RBD protein vaccine was injected intramuscularly three times with the rhesus monkey. The high-dose recombinant RBD protein vaccine also induces an effective cellular immune response. 5-107 TCID50 MERS-CoV was inoculated in the trachea of the rhesus monkey after the last immunization. The results showed that, compared with the control group, the pulmonary inflammatory response of the rhesus monkey was light, the pathological changes of the lung and the trachea were mild, the lung tissue MERS-CoV viral load and the virus titer were both low. The results showed that the recombinant RBD protein vaccine induced an effective immune protective response in the rhesus monkey.3. The recombinant NTD protein vaccine was used to study the effect of the recombinant NTD protein in the mice. The BALB/ c mice were immunized with the three-time intramuscular injection of the recombinant NTD protein in the combination of aluminum adjuvant and CpG. As a result, the recombinant NTD protein vaccine induced a high level of cellular immune response (significantly higher in intensity than the recombinant RBD protein vaccine), in addition to the induction of neutralizing antibodies (slightly lower than the recombinant RBD protein vaccine) in the mice. The humoral and cellular immune response was at least 14 w in the body of the mouse without significant decrease. After the attack of MERS-CoV, the pathological results show that, although the inflammation of the lung of the mice after challenge is changed, the control group is obviously relieved, and the immune protection level is comparable to that of the recombinant RBD protein vaccine.4, the development and the immunogenicity analysis of the VLPs vaccine are based on the base protein M1 of the avian influenza virus H5N1, MERS-CoV chimeric VLPs were packaged in insect cells using a baculovirus expression system. The VLPs are similar to MERS-CoV under the electron microscope, have a spherical shape with a diameter of about 100 nm, and have a typical envelope and a "crown"-like appearance. The VLPs vaccine is used in combination with the aluminum adjuvant and the CpG. After the BALB/ c mice are immunized by intramuscular injection, the S-protein-specific IgG antibody can be induced in the BALB/ c mice, and the antibody titer is equivalent to the inactivated MERS-CoV vaccine. The serum also detected neutralizing antibodies, but the antibody titer was lower than that of the inactivated MERS-CoV vaccine. The above studies have shown that this study successfully constructed the immunogenic MERS-CoV chimeric VLPs. To sum up, the effect of recombinant RBD and NTD subunit vaccine on the prevention of MERS-CoV infection was evaluated in mice or rhesus monkeys, and the results showed that the two subunit vaccines induced an effective immune response and a clear immune protective effect in the animal. The chimeric VLPs vaccine was successfully constructed by using the influenza virus M1 as a skeleton and the surface-chimeric MERS-CoV S protein. The VLPs vaccine induced a high level of humoral immune response in mice. These studies have laid a theoretical foundation for the development of MERS-CoV vaccine and the application of human body.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392
，
本文编号：2498314
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