Construct novel influenza A (H1N1) virus chimeric HA genes for development of bro

描述（由申请方提供）：甲型流感病毒在多种禽类和哺乳动物宿主（包括人和猪）中引起高度传染性呼吸道疾病。新型猪源性甲型H1N1流感病毒2009 A（H1N1）的出现和流行分类对公共卫生构成了重大威胁。有人担心该病毒可能与季节性人类流感病毒重组，从而增加传播性或致病性。特别是，这些病毒似乎保留了传播回猪的可能性，因此与猪病毒的持续重配可能产生更具毒性的病毒。需要确定和实施有效的战略，以防止当前和未来的疫情。控制流感病毒流行的主要手段是接种疫苗，其中针对表面糖蛋白HA的中和抗体是大多数目前许可的流感病毒疫苗的靶标。然而，针对流感病毒的疫苗接种的功效受到HA基因内的快速抗原变异（抗原漂移）的严重限制。雪貂感染后抗血清针对目前流行的季节性人类A（H1N1）病毒没有与2009年A（H1N1）猪源性菌株反应。从历史上看，每种流感亚型包含一种以上的分离株受到抗原性限制，包括刺激免疫所需的HA量、总HA含量耐受性以及一种疫苗株相对于另一种疫苗株的免疫优势潜力。为了开发一种疫苗，可以广泛有效地对抗各种病毒株，并限制新的猪源H1N1流感病毒的传播，这项拟议的研究将测试分子育种（DNA改组）的方法来创建新的HA基因的H1N1流感病毒的广泛保护疫苗的开发的可行性。这种方法将通过组合来自猪和人源H1N1流感病毒的代表五个不同系统发育簇的HA基因来创建新的HA基因。将使用猪攻击模型评估改组的嵌合HA抗原诱导的保护的功效。具体目标是：1）通过从甲型H1N1流感病毒的五个主要致突变基因簇的流感HA的分子育种（DNA改组）来产生嵌合HA基因; 2）评估在猪攻击模型中使用改组的嵌合HA抗原作为亚单位疫苗的潜力。这项研究将产生新的HA抗原，并测试它们诱导针对猪和人源甲型H1N1流感病毒的广泛免疫应答的能力。以这种方式开发疫苗将作为诱导针对人类和动物病毒分离株的免疫应答的疫苗将提供广泛免疫力并防止将来从动物来源进入人群的新型HA的出现的原理的证明。研究团队将招募积极性高的本科生参与该项目，该项目在研究方法，疾病机制和疾病预防方面提供了有价值的培训（参见附件：本科生培训）。 公共卫生相关性：在工业化国家，季节性流感病毒感染与每年300 - 500万人住院和250- 500，000人死亡有关。最近出现的猪源甲型H1N1流感病毒与人类季节性流感疫苗株在基因上不同，这表明需要在短时间内开发有效的疫苗。这项拟议的研究将使用分子育种方法，通过DNA改组和筛选来产生嵌合流感HA抗原，这些抗原具有诱导针对猪和人源甲型H1N1流感病毒的广泛保护性免疫应答的能力。本研究建立的技术可应用于流感病毒其他亚型内（或之间）的嵌合HA基因。我们研究的长期目标是开发通用疫苗，可以引起广泛的免疫力，以防止未来的流行病。

DESCRIPTION (provided by applicant): Influenza A virus causes highly contagious respiratory diseases in a variety of avian and mammalian hosts, including humans and pigs. The recent emergence and pandemic classification of novel swine-origin influenza A H1N1 virus, 2009 A (H1N1) poses a significant threat to public health. There are concerns that the virus may reassort with seasonal human influenza viruses giving rise to either increased transmissibility or pathogenicity. In particular, these viruses appear to retain the potential to transmit back to swine and thus continued reassortment with swine viruses may generate more virulent viruses. Identification and implementation of effective strategies to prevent current and future outbreaks are needed. The primary means for controlling influenza virus epidemics is vaccination, with neutralizing antibody against the surface glycoprotein HA being the target of most of the currently licensed influenza virus vaccines. However, the efficacy of vaccination towards influenza virus is severely limited by rapid antigenic variations within the HA gene (antigenic drift). Ferret postinfection antisera raised against the currently circulating seasonal human A (H1N1) viruses did not react with the 2009 A (H1N1) swine-origin stains. Historically, inclusion of more than one isolate per subtype of influenza has been limited by antigenic constraints, including the amount of HA required to stimulate immunity, the total HA content tolerance, and the potential for immunodominance by one vaccine strain over the other. In order to develop a vaccine that can be broadly effective against various strains of virus, and limit the spread of novel swine-origin H1N1 influenza viruses, this proposed study will test the feasibility of a molecular breeding (DNA shuffling) approach to create novel HA genes of influenza H1N1 viruses for development of broadly protective vaccines. This approach will create the novel HA genes by combination of HA genes from swine and human- origin influenza H1N1 viruses representing five distinct phylogenetic clusters. The efficacy of the shuffled chimeric HA antigen-induced protection will be assessed using a pig challenge model. Specific aims are: 1) To create chimeric HA genes by molecular breeding (DNA shuffling) of influenza HAs from five major phylogenic clusters of influenza A H1N1 viruses; 2) To evaluate the potential of using the shuffled chimeric HA antigen as a subunit vaccine in a pig challenge model. This study will generate novel HA antigens and test their ability to induce broad immune responses against swine and human-origin influenza A H1N1 viruses. Development of a vaccine in this manner will serve as a proof of principle that vaccines inducing immune response toward both human and animal viral isolates will provide broad immunity, and prevent future emergence of novel HAs from animal sources into the human population. The research team will recruit highly motivated undergraduate students to participate in the project, which provides valuable training in research methods, disease mechanisms and disease prevention (refer to attached document: Undergraduate student training). PUBLIC HEALTH RELEVANCE: Seasonal influenza virus infections are associated with 3-5 million hospitalizations and 250-500,000 deaths on an annual basis in the industrialized world. The recent emergence of the swine-origin influenza A (H1N1) virus that is genetically divergent from human seasonal influenza vaccine strain exemplifies the need for development of an effective vaccine in a short period of time. This proposed study will use molecular breeding approach by DNA shuffling and screening to generate chimeric influenza HA antigens that have the capacity to induce a broadly protective immune response against swine and human-origin influenza A H1N1 viruses. The technology established in this study can be applied to chimeric HA genes within (or between) other subtypes of influenza virus. The long term goal of our study is to develop universal vaccines that can elicit broad immunity to prevent future pandemics.