甲型流感病毒样颗粒疫苗的初步研究
发布时间：2018-08-14 10:49
【摘要】：流感是世界范围内广泛流行的呼吸系统传染病,每年都造成大量的住院及死亡病例。截至2010年7月18日,爆发于墨西哥的甲型H1N1流感已在全球214个国家造成18,336人死亡;自2003年以来,H5N1高致病性禽流感病毒已在世界范围内造成310人死亡,且死亡率高达59%。疫苗是防控流感最为有效的手段。流感疫苗的发展经历了灭活疫苗、裂解疫苗、减毒活疫苗、亚单位疫苗等阶段,季节性流感裂解疫苗虽然能在人群中产生有效保护作用,但是依然存在许多问题。首先,传统流感疫苗的生产工艺依赖于鸡胚,难以应对流感大规模爆发对疫苗的需求,且鸡胚卵清蛋白易引起过敏,至今还没有针对老年人、儿童及特殊人群理想的流感疫苗,因此现有流感疫苗的生产工艺亟待更新。其次,现有流感疫苗的有效成分是流感病毒HA和NA抗原。流感病毒具有易错倾向的RNA依赖性RNA聚合酶,使得HA和NA抗原不断发生抗原漂移;流感病毒的宿主极为广泛,可以感染鸟类、马、猪等多种动物并在这些动物体内发生基因节段的重组。因此,现有的季节性流感疫苗每年都需要更新。这不但需要对每年的病毒流行株进行预测,而且,每年接种流感疫苗,除了加重国家公共卫生经费的负担造成大量不必要的浪费之外,还不可避免的在人体中引入大量外源核酸,对人体健康造成潜在的威胁。因此,更新现有流感疫苗生产工艺,研发新类型的流感疫苗成为流感疫苗研究领域的发展方向。 病毒样颗粒(Virus-like particle, VLP)作为一种候选疫苗具有免疫原性好,没有感染性,稳定性好,不易失活等特点,因此在病毒感染性疾病的疫苗研发中极具研究价值。国内外已有大量关于病毒样颗粒的研究报道,包括人类乳头瘤病毒样颗粒、人类免疫缺陷病毒样颗粒、轮状病毒样颗粒等。其中,HPV-VLP疫苗已经在欧洲和美国上市,展现出了VLP疫苗良好的应用前景。由昆虫细胞衍生的流感VLP是最有希望替代现有流感疫苗的候选疫苗。目前已经成功重组的流感VLP包括H9N2、H3N2、H1N1、H5N1等多种亚型,这些VLP都具有免疫原性强,保护效果好,易于构建表达等优点。因此,研制流感病毒样颗粒疫苗具有极为重要的科学价值和社会效益。但是国内关于流感病毒样颗粒的文献还比较少,国外也未见关于将流感病毒样颗粒与现行裂解疫苗进行比较的报道。 现有的大部分通用疫苗研究以人来源的流感病毒M2蛋白外功能区(M2e)序列作为靶点。然而,M2e免疫原性低,M2e氨基酸序列在人来源和禽来源的A型流感病毒之间存在5-6个氨基酸残基的差异,且M2e诱导机体产生细胞免疫的能力较差。NP418-426表位是NP蛋白的免疫优势表位,与IFN-γ的产生高度相关并且能够有效的诱导特异的CTL发生免疫应答;禽流感病毒来源的NP418-426表位可以被人流感病毒特异的CTL交叉识别。因此,NP418-426表位有希望成为基于人来源M2e序列的流感病毒通用疫苗的重要组成成分,用以提高流感通用疫苗的种间交叉免疫保护效果,进一步提高通用疫苗的广谱性。乙肝核心蛋白(HBc)是使用最为广泛的外源表位载体。HBc可在体内或体外自组装成病毒样颗粒(VLP),将外源表位与HBc构建融合蛋白可以有效的诱导T细胞和B细胞免疫反应。基于以上研究背景,本研究分为两部分: 第一部分甲型H1N1病毒样颗粒疫苗的初步研究 1.通过昆虫-杆状病毒表达系统表达A/California/07/2009(H1N1)毒株的VLP。使用DEAE阴离子交换介质、Sepharose 4FF凝胶介质纯化甲型H1N1 VLP。电镜观察可知,纯化的甲型H1N1 VLP结构完整,直径在100nm左右。通过Western blot、血凝、单向免疫扩散等实验进一步证实,获得了重组甲型H1N1 VLP蛋白抗原并具有良好的免疫活性。 2.以10ug HA为免疫剂量,腹腔免疫4-6周龄雌性Balb/c小鼠,ELISA结果证实,研制的甲型H1N1 VLP能够诱导机体产生高滴度的特异性抗体,且甲型H1N1 VLP较甲型H1N1裂解疫苗能够诱导机体产生更高滴度的IgG抗体。 3.选用A/Beijing/501/2009 (H1N1)甲型H1N1流行株进行攻毒,结果显示,甲型H1N1 VLP候选疫苗能够保护小鼠抵抗50LD_(50)剂量的致死性攻击,保护率达到100%。 结论:研制的甲型H1N1 VLP候选疫苗具有良好的免疫原性,能够刺激机体产生体液免疫应答和细胞免疫应答,且生产较为便利。昆虫细胞生产的流感病毒VLP是值得深入研究,以期有希望替代现有流感疫苗的候选疫苗之一。 第二部分甲型流感通用病毒样颗粒疫苗的初步研究 1.使用大肠杆菌表达融合蛋白3M2e-NP-HBc、3M2e-HBc以及HBc N端149个氨基酸HBc-N149。采用His-trap纯化介质对重组蛋白进行纯化。电镜观察可知,纯化的重组蛋白可形成大小均一,结构完整的病毒样颗粒。通过M2e单克隆抗体进行Western blot实验可知,3M2e-NP-HBc及3M2e-HBc VLP抗原具有良好的M2e特异的免疫活性。 2.以20ug重组蛋白为免疫剂量,选取4-6周龄雌性Balb/c小鼠进行动物实验可知,重组3M2e-NP-HBc和3M2e-HBc VLP抗原均能够诱导机体产生高滴度的M2e特异性抗体。3M2e-NP-HBc较3M2e-HBc能够诱导机体产生更高滴度的IgG2a抗体且3M2e-NP-HBc较3M2e-HBc能够刺激机体产生更多的IFN-γ分泌型淋巴细胞。 3.分别使用A/Beijing/501/2009 (H1N1)甲型H1N1毒株、A/PR/8/34(H1N1)及A/Ostrich/SuZhou/097/2003 (H5N1)高致病性禽流感毒株进行攻毒实验。结果显示,3M2e-NP-HBc和3 2e HBc均能够保护小鼠抵抗10LD_(50)的A/Beiji g/501/2 09 (H1N1)、A/PR/8/34(H1N1)毒株的致死性攻击,保护率达到100%。3M2e-NP-HBc较3M2e-HBc能够更好的保护小鼠抵抗10LD_(50)的A/Ostrich/SuZhou/097/2003 (H5N1)毒株的致死性攻击。 结论:NP418-426表位的嵌入能够有效的诱导机体产生细胞免疫应答,有效的提高基于M2e的甲型流感通用候选疫苗的种间交叉保护效果。流感病毒内部蛋白作为潜在的通用疫苗候选抗原应当进行深入的研究。
[Abstract]:As of July 18, 2010, influenza A (H1N1) outbreak in Mexico has killed 18,336 people in 214 countries worldwide; since 2003, the highly pathogenic H5N1 avian influenza virus has caused 310 deaths worldwide. Vaccines are the most effective means to prevent and control influenza. The development of influenza vaccines has gone through inactivated vaccines, lysed vaccines, live attenuated vaccines, subunit vaccines and so on. Although seasonal split influenza vaccines can produce effective protection in the population, there are still many problems. First, the traditional influenza vaccines The production process is dependent on chicken embryos, which is difficult to meet the demand for vaccines in large-scale influenza outbreaks, and egg albumin is susceptible to allergies. So far, there is no ideal influenza vaccine for the elderly, children and special populations. Therefore, the existing influenza vaccine production process needs to be updated urgently. Secondly, the effective ingredient of the existing influenza vaccine is influenza virus H. A and NA antigens. Influenza viruses have a RNA dependent RNA polymerase which is prone to be misaligned, which makes the antigen shift of HA and NA antigens. The host of influenza virus is very extensive, * it can infect many kinds of animals such as birds, horses, pigs and so on, and reorganize gene segments in these animals. New. This requires not only the annual prediction of the virus epidemic strains, but also the annual vaccination of influenza vaccines, in addition to increasing the burden of national public health expenditure caused by a large number of unnecessary waste, but also the inevitable introduction of a large number of exogenous nucleic acids in the human body, a potential threat to human health. The development of new types of influenza vaccine has become the development direction of influenza vaccine research.
Virus-like particles (VLP), as a candidate vaccine, have good immunogenicity, no infectivity, good stability, and are not easy to inactivate. Therefore, it is of great value in the research and development of vaccine for viral infectious diseases. Among them, HPV-VLP vaccines have been marketed in Europe and the United States, showing a promising future for VLP vaccines. Influenza VLP derived from insect cells is the most promising alternative to existing influenza vaccines. H5N1 and other subtypes, these VLPs have the advantages of strong immunogenicity, good protective effect, easy construction and expression. Therefore, the development of influenza-like granule vaccine has extremely important scientific value and social benefits. Comparison of split vaccine was reported.
However, M2e immunogenicity is low. There are 5-6 amino acid residues in the M2e amino acid sequence between human and avian influenza A viruses, and the ability of M2e to induce cellular immunity is poor. NP epitope is an immunodominant epitope of NP protein, which is highly correlated with the production of IFN-gamma and can effectively induce specific CTL immune response. NP418-426 epitope derived from avian influenza virus can be cross-identified by human influenza virus-specific CTL. Therefore, NP418-426 epitope is hopeful to become a universal influenza virus based on human M2e sequence. Hepatitis B core protein (HBc) is the most widely used exogenous epitope vector. HBc can self-assemble into virus-like particles (VLP) in vivo or in vitro, and construct fusion proteins between exogenous epitopes and HBc. In order to effectively induce T cell and B cell immune response, based on the above research background, the study is divided into two parts:
The first part is a preliminary study of a H1N1 virus like particle vaccine.
1. Expressing the VLP of A/California/07/2009 (H1N1) strain by insect-baculovirus expression system. Using DEAE anion exchange medium and Sepharose 4FF gel medium to purify A/H1N1 VLP. The structure of purified A/H1N1 VLP was intact and its diameter was about 100 nm. It was confirmed that the recombinant H1N1 VLP protein antigen was obtained and had good immunological activity.
2. Female Balb/c mice aged 4-6 weeks were immunized by intraperitoneal injection of 10ug HA. ELISA results showed that the developed A H1N1 VLP could induce high titer of specific antibodies, and A H1N1 VLP could induce higher titer of IgG antibodies than A H1N1 lysis vaccine.
3. A/Beijing/501/2009 (H1N1) influenza A H1N1 strain was selected to attack the virus. The results showed that the candidate vaccine of A H1N1 VLP could protect mice against lethal attack of 50 LD_ (50) dose, and the protection rate reached 100%.
CONCLUSION: The candidate vaccine for influenza A H1N1 VLP has good immunogenicity, can stimulate humoral and cellular immune responses, and is convenient for production.
The second part is a preliminary study of influenza A virus like particle vaccine.
1. The fusion protein 3M2e-NP-HBc, 3M2e-HBc and 149 amino acids HBc-N149 were expressed in E. coli. The recombinant protein was purified by his-trap purification medium. The purified recombinant protein could form virus-like particles with uniform size and complete structure. The Western blot test with M2e monoclonal antibody showed that the recombinant protein was 3. M2e-NP-HBc and 3M2e-HBc VLP antigen have good M2e specific immune activity.
2. Using 20ug recombinant protein as the immune dose, female Balb/c mice aged 4-6 weeks were selected for animal experiment. The results showed that recombinant 3M2e-NP-HBc and 3M2e-HBc VLP antigens could induce high titer of M2e-specific antibody. 3M2e-NP-HBc could induce higher titer of IgG2a antibody than 3M2e-HBc and 3M2e-NP-HBc could prick higher titer of IgG2a antibody than 3M2e-HBc. The stimulated organism produces more IFN- - ray secreting lymphocytes.
3. Attack experiments were carried out using A/Beijing/501/2009 (H1N1) A/H1N1 strain, A/PR/8/34 (H1N1) and A/Ostrich/SuZhou/097/2003 (H5N1) highly pathogenic avian influenza strains. The results showed that 3M2e-NP-HBc and 32e-HBc could protect mice against lethal attack by 10LD_ (50) A/Beiji g/501/209 (H1N1), A/PR/8/34 (H1N1) strains. Compared with 3M2e-HBc, 3M2e-NP-HBc could protect mice against lethal attack of 10LD_ (50) A/Ostrich/SuZhou/097/2003 (H5N1).
CONCLUSION: NP418-426 epitope insertion can effectively induce cellular immune response and improve the cross-protection effect of M2e-based universal vaccine candidates for influenza A. As a potential candidate antigen for universal vaccine, influenza virus internal protein should be further studied.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
本文编号：2182618
[Abstract]:As of July 18, 2010, influenza A (H1N1) outbreak in Mexico has killed 18,336 people in 214 countries worldwide; since 2003, the highly pathogenic H5N1 avian influenza virus has caused 310 deaths worldwide. Vaccines are the most effective means to prevent and control influenza. The development of influenza vaccines has gone through inactivated vaccines, lysed vaccines, live attenuated vaccines, subunit vaccines and so on. Although seasonal split influenza vaccines can produce effective protection in the population, there are still many problems. First, the traditional influenza vaccines The production process is dependent on chicken embryos, which is difficult to meet the demand for vaccines in large-scale influenza outbreaks, and egg albumin is susceptible to allergies. So far, there is no ideal influenza vaccine for the elderly, children and special populations. Therefore, the existing influenza vaccine production process needs to be updated urgently. Secondly, the effective ingredient of the existing influenza vaccine is influenza virus H. A and NA antigens. Influenza viruses have a RNA dependent RNA polymerase which is prone to be misaligned, which makes the antigen shift of HA and NA antigens. The host of influenza virus is very extensive, * it can infect many kinds of animals such as birds, horses, pigs and so on, and reorganize gene segments in these animals. New. This requires not only the annual prediction of the virus epidemic strains, but also the annual vaccination of influenza vaccines, in addition to increasing the burden of national public health expenditure caused by a large number of unnecessary waste, but also the inevitable introduction of a large number of exogenous nucleic acids in the human body, a potential threat to human health. The development of new types of influenza vaccine has become the development direction of influenza vaccine research.
Virus-like particles (VLP), as a candidate vaccine, have good immunogenicity, no infectivity, good stability, and are not easy to inactivate. Therefore, it is of great value in the research and development of vaccine for viral infectious diseases. Among them, HPV-VLP vaccines have been marketed in Europe and the United States, showing a promising future for VLP vaccines. Influenza VLP derived from insect cells is the most promising alternative to existing influenza vaccines. H5N1 and other subtypes, these VLPs have the advantages of strong immunogenicity, good protective effect, easy construction and expression. Therefore, the development of influenza-like granule vaccine has extremely important scientific value and social benefits. Comparison of split vaccine was reported.
However, M2e immunogenicity is low. There are 5-6 amino acid residues in the M2e amino acid sequence between human and avian influenza A viruses, and the ability of M2e to induce cellular immunity is poor. NP epitope is an immunodominant epitope of NP protein, which is highly correlated with the production of IFN-gamma and can effectively induce specific CTL immune response. NP418-426 epitope derived from avian influenza virus can be cross-identified by human influenza virus-specific CTL. Therefore, NP418-426 epitope is hopeful to become a universal influenza virus based on human M2e sequence. Hepatitis B core protein (HBc) is the most widely used exogenous epitope vector. HBc can self-assemble into virus-like particles (VLP) in vivo or in vitro, and construct fusion proteins between exogenous epitopes and HBc. In order to effectively induce T cell and B cell immune response, based on the above research background, the study is divided into two parts:
The first part is a preliminary study of a H1N1 virus like particle vaccine.
1. Expressing the VLP of A/California/07/2009 (H1N1) strain by insect-baculovirus expression system. Using DEAE anion exchange medium and Sepharose 4FF gel medium to purify A/H1N1 VLP. The structure of purified A/H1N1 VLP was intact and its diameter was about 100 nm. It was confirmed that the recombinant H1N1 VLP protein antigen was obtained and had good immunological activity.
2. Female Balb/c mice aged 4-6 weeks were immunized by intraperitoneal injection of 10ug HA. ELISA results showed that the developed A H1N1 VLP could induce high titer of specific antibodies, and A H1N1 VLP could induce higher titer of IgG antibodies than A H1N1 lysis vaccine.
3. A/Beijing/501/2009 (H1N1) influenza A H1N1 strain was selected to attack the virus. The results showed that the candidate vaccine of A H1N1 VLP could protect mice against lethal attack of 50 LD_ (50) dose, and the protection rate reached 100%.
CONCLUSION: The candidate vaccine for influenza A H1N1 VLP has good immunogenicity, can stimulate humoral and cellular immune responses, and is convenient for production.
The second part is a preliminary study of influenza A virus like particle vaccine.
1. The fusion protein 3M2e-NP-HBc, 3M2e-HBc and 149 amino acids HBc-N149 were expressed in E. coli. The recombinant protein was purified by his-trap purification medium. The purified recombinant protein could form virus-like particles with uniform size and complete structure. The Western blot test with M2e monoclonal antibody showed that the recombinant protein was 3. M2e-NP-HBc and 3M2e-HBc VLP antigen have good M2e specific immune activity.
2. Using 20ug recombinant protein as the immune dose, female Balb/c mice aged 4-6 weeks were selected for animal experiment. The results showed that recombinant 3M2e-NP-HBc and 3M2e-HBc VLP antigens could induce high titer of M2e-specific antibody. 3M2e-NP-HBc could induce higher titer of IgG2a antibody than 3M2e-HBc and 3M2e-NP-HBc could prick higher titer of IgG2a antibody than 3M2e-HBc. The stimulated organism produces more IFN- - ray secreting lymphocytes.
3. Attack experiments were carried out using A/Beijing/501/2009 (H1N1) A/H1N1 strain, A/PR/8/34 (H1N1) and A/Ostrich/SuZhou/097/2003 (H5N1) highly pathogenic avian influenza strains. The results showed that 3M2e-NP-HBc and 32e-HBc could protect mice against lethal attack by 10LD_ (50) A/Beiji g/501/209 (H1N1), A/PR/8/34 (H1N1) strains. Compared with 3M2e-HBc, 3M2e-NP-HBc could protect mice against lethal attack of 10LD_ (50) A/Ostrich/SuZhou/097/2003 (H5N1).
CONCLUSION: NP418-426 epitope insertion can effectively induce cellular immune response and improve the cross-protection effect of M2e-based universal vaccine candidates for influenza A. As a potential candidate antigen for universal vaccine, influenza virus internal protein should be further studied.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
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