MERS-CoV重組蛋白及病毒樣顆粒疫苗的研究
發(fā)布時間：2019-06-13 06:34
【摘要】：中東呼吸綜合征冠狀病毒(Middle East respiratory syndrome coronavirus, MERS-CoV)是2012年在中東地區(qū)發(fā)現(xiàn)的一種新型冠狀病毒,隨后該病毒從中東地區(qū)向外傳播。據(jù)世界衛(wèi)生組織(World Health Organization, WHO)報道,截止到2016年4月26日,全球共有27個國家1728例實驗室確診病例,其中死亡624例,病死率高達36.1%。然而,目前尚無特異性藥物治療該病毒引起的感染。因此,丞待開發(fā)一種安全有效的疫苗預防或治療該病毒引起的疾病。MERS-CoV為包膜病毒,其包膜上的棘突蛋白(Spike, S)在介導病毒與宿主細胞膜結合以及病毒進入細胞過程中發(fā)揮重要作用。S分為S1和S2兩個亞單位,前者介導病毒和易感細胞上的受體結合,后者在病毒包膜和宿主細胞膜融合中發(fā)揮作用。S1進一步分為N端結構域(N terminal domain, NTD)和C端的受體結合結構域(Receptor binding domain, RBD)。RBD蛋白介導MERS-CoV與宿主細胞膜上的二肽基肽酶4(Dipeptidyl peptidase-4, DPP4)分子結合,富集中和性抗原表位,是MERS-CoV疫苗研發(fā)最重要的靶抗原。亞單位疫苗是預防病毒感染的重要手段,本文應用重組RBD或NTD蛋白作為亞單位疫苗,先后在小鼠或恒河猴體內檢測了其誘導的免疫應答反應和免疫保護效果。同時構建了基于S蛋白的MERS-CoV嵌合型病毒樣顆粒(Virus like particles, VLPs)疫苗,并在小鼠體內檢測了該VLPs的免疫原性。具體研究內容及結果如下：1、重組RBD蛋白疫苗在小鼠體內免疫效果研究重組RBD蛋白與不同佐劑(單獨鋁佐劑、鋁佐劑和CpG聯(lián)合、鋁佐劑和poly(I:C)聯(lián)合)經肌肉注射,或與CpG和不完全弗氏佐劑(Incomplete Freund's adjuvant, IFA)聯(lián)合應用經皮下注射,可在BALB/c小鼠體內誘導不同水平RBD特異性IgG抗體、中和抗體以及全身或局部細胞免疫應答反應。其中,重組RBD蛋白在鋁佐劑和CpG聯(lián)合作用下、經肌肉注射可在小鼠體內誘導最高水平的體液免疫應答,且該免疫策略可誘導多種效應性細胞因子的分泌。病毒攻擊實驗表明,該策略可在小鼠體內誘導有效的免疫保護反應,減輕MERS-CoV攻擊后小鼠肺臟和氣管組織的炎癥反應。2、重組RBD蛋白疫苗在恒河猴體內免疫效果研究高(200μg)、低劑量(50μg)重組RBD蛋白疫苗肌肉注射三次免疫恒河猴后,在其體內誘導了強大而持久的體液免疫應答(IgG抗體和中和抗體)。高劑量組重組RBD蛋白疫苗還誘發(fā)了有效的細胞免疫應答。末次免疫后2w,恒河猴氣管內接種6.5×107TCID50 MERS-CoV。結果發(fā)現(xiàn),與對照組相比免疫恒河猴肺部炎癥反應較輕、肺臟和氣管病理改變輕微、肺組織MERS-CoV病毒載量和病毒滴度均較低。表明該重組RBD蛋白疫苗在恒河猴體內誘導了有效的免疫保護反應。3、重組NTD蛋白疫苗在小鼠體內免疫效果研究重組NTD蛋白在鋁佐劑和CpG聯(lián)合作用下、經肌肉注射三次免疫BALB/c小鼠。結果發(fā)現(xiàn),該重組NTD蛋白疫苗在小鼠體內除誘導了中和抗體(水平略低于重組RBD蛋白疫苗)外,還誘導了高水平的細胞免疫應答(強度顯著高于重組RBD蛋白疫苗)。該體液和細胞免疫應答在小鼠體內至少持續(xù)14w而無明顯下降。MERS-CoV攻擊后,病理結果示,雖然攻毒后的小鼠肺部出現(xiàn)炎癥改變,但較對照組明顯減輕,且該免疫保護水平與重組RBD蛋白疫苗相當。4、VLPs疫苗的研制及免疫原性分析以禽流感病毒H5N1的基質蛋白M1為骨架,在其表面嵌合MERS-CoV的S蛋白,應用桿狀病毒表達系統(tǒng)在昆蟲細胞中包裝了MERS-CoV嵌合型VLPs。該VLPs在電鏡下與MERS-CoV相似,呈球形,直徑約100nm左右,具有典型的包膜以及“皇冠”樣外觀。該VLPs疫苗與鋁佐劑和CpG聯(lián)合應用、經肌肉注射免疫BALB/c小鼠后,可在其體內誘導S蛋白特異性IgG抗體,且抗體滴度與滅活MERS-CoV疫苗相當。此時血清亦檢測到中和抗體,但抗體滴度低于滅活MERS-CoV疫苗免疫組。以上研究表明,本研究成功構建了有免疫原性的MERS-CoV嵌合型VLPs。綜上所述,本研究在小鼠或恒河猴體內評價了重組RBD和NTD亞單位疫苗預防MERS-CoV感染的效果,結果表明該兩種亞單位疫苗均在動物體內誘導了有效的免疫應答和明顯的免疫保護效果。應用流感病毒M1為骨架,表面嵌合MERS-CoV S蛋白,成功構建了嵌合型VLPs疫苗。該VLPs疫苗在小鼠體內誘導了高水平的體液免疫應答反應。這些研究為MERS-CoV疫苗的研發(fā)以及人體應用奠定了理論基礎。
[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.
【學位授予單位】：中國疾病預防控制中心
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R392
，
本文編號：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.
【學位授予單位】：中國疾病預防控制中心
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R392
，
本文編號：2498314
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