H5N1亚型禽流感病毒样颗粒构建、优化与实验免疫研究
发布时间：2019-06-16 19:26
【摘要】：自2003年起,高致病性H5N1亚型流感病毒感染人的事件不断发生,并且衍生出新的基因谱系,其中,2.3.2.1和2.3.4谱系流行最为广泛。高致病性H5N1病毒存在大规模流行的可能性是被大家所公认的,需要对存在流行风险的多个谱系禽流感病毒进行疫苗的研究。目前广泛应用的灭活疫苗只能针对同谱系病毒感染提供免疫保护,利用杆状病毒表达系统生产的无感染性的病毒样颗粒疫苗,因生产成本低、无生物安全风险等优势受到广泛关注,特别是因其具有诱导细胞免疫能力可能针对同型的异源病毒提供免疫保护,而可能用于变异频繁的流感病毒疫苗研究。本研究的目的是应用昆虫杆状病毒表达系统,通过表达模式筛选、佐剂效应蛋白偶联等系列优化研究,研制H5N1病毒样颗粒,并评估对异源病毒感染的免疫保护作用。首先,利用昆虫杆状病毒表达系统研制了包含H5N1 A/meerkat/Shanghai/SH-1 2012 (clade 2.3.2.1) HA、NA和M1蛋白的病毒样颗粒。通过系列试验鉴定证明,包含HA、NA和M1蛋白的H5N1病毒样颗粒与天然流感病毒具有相似的形态结构和大小,并且具有血凝活性。与全病毒灭活疫苗相比,小鼠通过免疫病毒样颗粒疫苗能够诱导产生更强的体液免疫和细胞免疫,在加强免疫后,可以产生针对A/meerkat/Shanghai/SH-1/2012特异性IgG,其滴度为全病毒灭活组的20倍。接下来的攻毒结果表明,全病毒灭活组表现出对同源病毒80%的保护率,对异源病毒(A/duck/Jilin/JL-SIV/2013, clade 2.3.4)的保护率只有40%,而病毒样颗粒免疫组小鼠则完全存活,保护率均达到100%。结果显示,流感病毒样颗粒疫苗可以作为一种具有交叉保护活性的流感疫苗能够控制高致病H5N1流感病毒的爆发。为进一步提高病毒样颗粒的免疫原性,我们在疫苗中增加具有免疫佐剂活性的蛋白以期增强疫苗诱导的适应性免疫反应水平。以膜锚定形式构建了包含大肠杆菌不耐热肠毒素B亚基(LTB),鞭毛蛋白(Toll样受体5的配体,Flic)和粒细胞-巨噬细胞集落刺激因子(GM-CSF)的嵌合病毒样颗粒。鉴定表明,LTB-、Flic-和GM-CSF-VLPs病毒样颗粒包含所特有的蛋白,与天然流感病毒具有相似的形态结构和大小,并且具有血凝活性。与未偶联免疫刺激蛋白病毒样颗粒疫苗相比,LTB-、 Flic-和GM-CSF-VLPs病毒样颗粒疫苗能够诱导产生更强的体液免疫和细胞免疫。所有肌肉注射和滴鼻免疫组小鼠均能够保护同源和异源H5N1流感病毒的攻击,然而在口服免疫组中LTB-VLPs、Flic-VLPs或GM-CSF-VLPs能够对同源和异源H5N1流感病毒的攻击提供部分的保护活性,而单独VLPs组则不能保护流感病毒的攻击。LTB-或Flic-VLPs口服免疫组诱导产生比单独VLPs组高10倍的病毒特异性IgG抗体。说明具有免疫刺激活性的LTB-、Flic-和GM-CSF嵌合病毒样颗粒能够诱导产生更强的免疫反应。我们的研究结果表明,基于昆虫杆状病毒表达系统的流感病毒样颗粒可为同源甚至是抗原性存在差异的流感病毒感染提供免疫保护,偶联了具有免疫刺激活性的LTB-VLPs、Flic-VLPs或GM-CSF-VLPs可进一步提高病毒样颗粒的免疫原性。本研究为研发能够控制高致病H5N1亚型禽流感流感病毒流行具有交叉保护活性的疫苗奠定了基础,同时也为新型疫苗研究提供了可借鉴的数据与经验。
[Abstract]:Since 2003, the events of highly pathogenic H5N1 subtype influenza virus infections have continued and new genetic lineages have been derived, where 2.3. 2.1 and 2.3.4 are among the most popular. The possibility of a large-scale epidemic of highly pathogenic H5N1 viruses is recognized by all, and there is a need for a study of the vaccine of a number of lineages of avian influenza that have an epidemic risk. the inactivated vaccine which is widely used at present can only provide the immune protection against the virus infection of the same lineage, and the non-infectious virus-like particle vaccine produced by the baculovirus expression system is widely concerned by the advantages of low production cost, no biological safety risk and the like, In particular, because of its ability to induce cellular immunity, it is possible to provide an immune protection against a homotype of heterologous virus, which may be used in that study of influenza virus vaccine with frequent variation. The purpose of this study is to use the insect baculovirus expression system to study and develop the H5N1 virus-like particles by means of a series of optimization studies such as the expression pattern selection and the coupling of adjuvant effect protein, and to evaluate the immune protection effect on the heterologous virus infection. First, the virus-like particles containing the HA, NA and M1 proteins of the HA, NA and M1 proteins of the H5N1 A/ meerkat/ Shanhai/ SH-1 2012 (clade 2.3. 2.1) were developed using the insect baculovirus expression system. The identification of the series of tests demonstrated that the H5N1 virus-like particles containing the HA, NA and M1 proteins have similar morphological structure and size to the natural influenza virus and have a blood-clotting activity. Compared with the full-virus inactivated vaccine, the mice can induce stronger humoral and cellular immunity through the immune virus-like particle vaccine, and after the booster immunization, the specific IgG of the A/ meerkat/ Shanghai/ SH-1/2012 can be generated, and the titer is 20 times of that of the whole virus inactivation group. The next challenge results showed that the whole-virus inactivated group exhibited a protection rate of 80% against the homologous virus, with a protection rate of only 40% for the heterologous virus (A/ duck/ Jilin/ JL-SIV/2013, clade 2.3.4), while the virus-like particle-like mice were fully alive and the protection rate was 100%. The results show that influenza virus-like particle vaccine can be used as a kind of influenza vaccine with cross-protection activity to control the outbreak of highly pathogenic H5N1 influenza virus. To further improve the immunogenicity of the virus-like particles, we increase the immune-adjuvant-active protein in the vaccine with a view to enhancing the adaptive immune response level induced by the vaccine. The chimeric virus-like particles of the non-heat-labile enterotoxin B subunit (LTB), the flagellin (Toll-like receptor 5, and the granulocyte-macrophage colony-stimulating factor (GM-CSF) were constructed in the form of membrane-anchoring. The identification shows that LTB-, Flic-and GM-CSF-VLPs virus-like particles contain the specific proteins and have a similar morphological structure and size to the natural influenza virus and have a blood-clotting activity. LTB-, public-and GM-CSF-VLPs viral-like particle vaccines can induce stronger humoral and cellular immunity as compared to unconjugated immunostimulatory protein-like particle vaccines. All of the intramuscular and nasal immune group mice are capable of protecting both homologous and heterologous H5N1 influenza viruses, whereas LTB-VLPs, public-VLPs, or GM-CSF-VLPs in the oral immunization group can provide a partial protection activity against the attack of homologous and heterologous H5N1 influenza viruses, And the individual VLPs group cannot protect the attack of the influenza virus. The LTB-or the Flic-VLPs oral immune group induced a 10-fold higher virus-specific IgG antibody than the individual VLPs group. The LTB-, Flic-and GM-CSF chimeric virus-like particles with immunostimulatory activity can induce a stronger immune response. Our research results show that the influenza virus-like particles based on the insect baculovirus expression system can provide the immune protection for influenza virus infection which is homologous or even antigenic, and the LTB-VLPs with immunostimulating activity are coupled, The public-VLPs or GM-CSF-VLPs can further improve the immunogenicity of the virus-like particles. The research has laid a foundation for the development of a vaccine which can control the epidemic of highly pathogenic H5N1 subtype avian influenza virus and has cross-protection activity, and also provides useful data and experience for new vaccine research.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392
[Abstract]:Since 2003, the events of highly pathogenic H5N1 subtype influenza virus infections have continued and new genetic lineages have been derived, where 2.3. 2.1 and 2.3.4 are among the most popular. The possibility of a large-scale epidemic of highly pathogenic H5N1 viruses is recognized by all, and there is a need for a study of the vaccine of a number of lineages of avian influenza that have an epidemic risk. the inactivated vaccine which is widely used at present can only provide the immune protection against the virus infection of the same lineage, and the non-infectious virus-like particle vaccine produced by the baculovirus expression system is widely concerned by the advantages of low production cost, no biological safety risk and the like, In particular, because of its ability to induce cellular immunity, it is possible to provide an immune protection against a homotype of heterologous virus, which may be used in that study of influenza virus vaccine with frequent variation. The purpose of this study is to use the insect baculovirus expression system to study and develop the H5N1 virus-like particles by means of a series of optimization studies such as the expression pattern selection and the coupling of adjuvant effect protein, and to evaluate the immune protection effect on the heterologous virus infection. First, the virus-like particles containing the HA, NA and M1 proteins of the HA, NA and M1 proteins of the H5N1 A/ meerkat/ Shanhai/ SH-1 2012 (clade 2.3. 2.1) were developed using the insect baculovirus expression system. The identification of the series of tests demonstrated that the H5N1 virus-like particles containing the HA, NA and M1 proteins have similar morphological structure and size to the natural influenza virus and have a blood-clotting activity. Compared with the full-virus inactivated vaccine, the mice can induce stronger humoral and cellular immunity through the immune virus-like particle vaccine, and after the booster immunization, the specific IgG of the A/ meerkat/ Shanghai/ SH-1/2012 can be generated, and the titer is 20 times of that of the whole virus inactivation group. The next challenge results showed that the whole-virus inactivated group exhibited a protection rate of 80% against the homologous virus, with a protection rate of only 40% for the heterologous virus (A/ duck/ Jilin/ JL-SIV/2013, clade 2.3.4), while the virus-like particle-like mice were fully alive and the protection rate was 100%. The results show that influenza virus-like particle vaccine can be used as a kind of influenza vaccine with cross-protection activity to control the outbreak of highly pathogenic H5N1 influenza virus. To further improve the immunogenicity of the virus-like particles, we increase the immune-adjuvant-active protein in the vaccine with a view to enhancing the adaptive immune response level induced by the vaccine. The chimeric virus-like particles of the non-heat-labile enterotoxin B subunit (LTB), the flagellin (Toll-like receptor 5, and the granulocyte-macrophage colony-stimulating factor (GM-CSF) were constructed in the form of membrane-anchoring. The identification shows that LTB-, Flic-and GM-CSF-VLPs virus-like particles contain the specific proteins and have a similar morphological structure and size to the natural influenza virus and have a blood-clotting activity. LTB-, public-and GM-CSF-VLPs viral-like particle vaccines can induce stronger humoral and cellular immunity as compared to unconjugated immunostimulatory protein-like particle vaccines. All of the intramuscular and nasal immune group mice are capable of protecting both homologous and heterologous H5N1 influenza viruses, whereas LTB-VLPs, public-VLPs, or GM-CSF-VLPs in the oral immunization group can provide a partial protection activity against the attack of homologous and heterologous H5N1 influenza viruses, And the individual VLPs group cannot protect the attack of the influenza virus. The LTB-or the Flic-VLPs oral immune group induced a 10-fold higher virus-specific IgG antibody than the individual VLPs group. The LTB-, Flic-and GM-CSF chimeric virus-like particles with immunostimulatory activity can induce a stronger immune response. Our research results show that the influenza virus-like particles based on the insect baculovirus expression system can provide the immune protection for influenza virus infection which is homologous or even antigenic, and the LTB-VLPs with immunostimulating activity are coupled, The public-VLPs or GM-CSF-VLPs can further improve the immunogenicity of the virus-like particles. The research has laid a foundation for the development of a vaccine which can control the epidemic of highly pathogenic H5N1 subtype avian influenza virus and has cross-protection activity, and also provides useful data and experience for new vaccine research.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392
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