埃博拉和马尔堡病毒囊膜糖蛋白嵌合型重组水泡性口炎病毒的构建与免疫原性研究

发布时间：2019-06-24 16:15

【摘要】：埃博拉出血热(Ebola hemorrhagic fever,EHF)和马尔堡出血热(Marburg hemorrhagic fever,MHF)分别是由埃博拉病毒(Ebola virus,EBOV)和马尔堡病毒(Marburg virus,MARV)引起的以急性,发热并伴有严重出血为症状的人兽共患烈性传染病。EBOV和MARV同属丝状病毒科,它们是目前人类所知最致命的病原之一,其致死率可高达90%,严重威胁着公众健康和国家生物安全,同时也是重要的生物武器及生物反恐防范对象。虽然EHF和MHF主要流行于非洲地区,我国尚未发现该病,但随着日益密切的国际贸易和人员流动,该病传入我国的风险日益增高。研究证明使用疫苗可有效预防和控制该病的传播和危害,因此开展EBOV和MARV的储备性疫苗研究对我国具有十分重要的公共卫生和生物安全意义。本研究在水泡性口炎病毒(Vesicular stomatitis virus,VSV)反向遗传操作平台的基础上,通过限制性酶切位点改造,成功建立了嵌合型VSV反向遗传操作平台,将VSV基因组中自身囊膜糖蛋白G替换为ZEBOV,SEBOV或MARV的囊膜糖蛋白GP并成功拯救出重组病毒,分别命名为rVSV?G/ZEBOVGP,rVSV?G/SEBOVGP和rVSV?G/MARVGP。Western Blot和间接免疫荧光试验表明三种重组病毒的GP蛋白均能够正确表达且定位于细胞膜表面,免疫电镜结果表明GP嵌合在重组病毒粒子表面,重组病毒呈现典型的子弹状形态特征。进一步在小鼠上进行三种病毒的安全性评价,在观察期内小鼠未表现明显的体重下降和任何临床症状,初步表明三种重组病毒对小鼠具有良好的安全性。为了评估重组病毒对小鼠的免疫原性,将三种病毒以单独(1×106pfu)或三重联合(每种病毒1×106 pfu)经肌肉注射途径接种小鼠,初次免疫3周后用相同剂量加强免疫。利用ELISA和中和试验测定小鼠血清特异性抗体水平。结果显示,一次免疫后小鼠即能产生较高水平的GP特异性IgG和中和抗体,加强免疫后抗体水平显著上升。表明三种重组病毒在小鼠上具有良好的免疫原性。本研究在国内首次成功构建了以嵌合型VSV为载体的几种重要烈性外来出血热病毒储备性疫苗,并证明这几种疫苗无论单独或联合应用均对小鼠安全,且能诱导产生高水平中和抗体。本研究结果表明rVSV?G/ZEBOVGP,rVSV?G/SEBOVGP和rVSV?G/MARVGP作为防控EBOV和MARV的储备性疫苗具有潜在应用价值。重组病毒在非人灵长类动物(Nonhuman primates,NHPs)上的安全性和有效性需要进一步评估。
[Abstract]:Ebola hemorrhagic fever (Ebola hemorrhagic fever,EHF) and Marburg hemorrhagic fever (Marburg hemorrhagic fever,MHF) are human and animal infectious diseases characterized by acute, febrile and severe bleeding caused by Ebola virus (Ebola virus,EBOV) and Marburg virus (Marburg virus,MARV), respectively. EBOV and MARV belong to the filamentous virus family, which are one of the deadliest pathogens known to human beings, and their fatality rate can be as high as 90%. It is a serious threat to public health and national biosafety, but also an important biological weapons and biological counter-terrorism prevention target. Although EHF and MHF are mainly popular in Africa, the disease has not yet been found in China, but with the increasing international trade and mobility of people, the risk of the disease entering China is increasing day by day. Studies have proved that the use of vaccines can effectively prevent and control the spread and harm of the disease, so the study of EBOV and MARV reserve vaccines is of great significance to public health and biosafety in China. In this study, based on the reverse genetic operation platform of vesicular stomatitis virus (Vesicular stomatitis virus,VSV), a chimeric VSV reverse genetic operating platform was successfully established by restriction endonuclease modification. The envelope glycoprotein G in VSV genome was replaced with the envelope glycoprotein GP of ZEBOV,SEBOV or MARV, and the recombinant virus was successfully saved, named rVSV?G/ZEBOVGP, respectively. RVSV?G/SEBOVGP, rVSV?G/MARVGP.Western Blot and indirect immunofluorescence assay showed that the GP proteins of the three recombinant viruses could be correctly expressed and located on the surface of cell membrane. The results of immunoelectron microscope showed that GP chimerized on the surface of recombinant virus particles, and the recombinant virus showed typical bullet morphology. The safety of the three viruses was further evaluated in mice. During the observation period, the mice did not show obvious weight loss and any clinical symptoms, which preliminarily showed that the three recombinant viruses were safe in mice. In order to evaluate the immunogenicity of the recombinant virus to mice, the three viruses were inoculated by intramuscular injection (1脳106pfu) or triple combination (1 脳 106pfu per virus). After the first immunization for 3 weeks, the mice were immunized with the same dose. The serum specific antibody level of mice was measured by ELISA and neutralization test. The results showed that mice could produce high levels of GP specific IgG and neutralizing antibody after one immunization, and the antibody level increased significantly after enhanced immunization. The results showed that the three recombinant viruses had good immunogenicity in mice. In this study, several important virulent hemorrhagic fever virus storage vaccines with chimeric VSV as vector were successfully constructed in China for the first time, and it was proved that these vaccines were safe for mice and could induce the production of high levels of neutralizing antibodies. The results of this study show that rVSV?G/ZEBOVGP,rVSV?G/SEBOVGP and rVSV?G/MARVGP have potential application value as reserve vaccines for prevention and control of EBOV and MARV. The safety and effectiveness of recombinant viruses in non-human primates (Nonhuman primates,NHPs) need to be further evaluated.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.65

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