Development of Cross Protective H5 Hemagglutinin Antigens

描述(申请人提供)：在东南亚家禽中流行的高致病性H5N1禽流感病毒最近通过野生候鸟在全球传播。这些禽流感病毒也能够感染人类，在过去的十年里已经导致了几次严重疾病的爆发，死亡率很高。有人担心，H5N1禽流感病毒可能在未来演变成容易传播给人和在人之间传播的形式。还有人担心，由于不可避免的抗原漂移和转变，针对过去和目前正在传播的H5流感病毒的疫苗可能不会对未来的病毒株有效。该项目的总体目标是开发血凝素抗原，以诱导针对广泛进化的H5N1流感毒株的交叉保护性而不是类型特异性的体液抗体反应。我们的工作将基于这样的假设，即去除H5血凝素上活跃进化的、特征良好的主要抗原决定簇将把免疫反应重定向到分子中保守的、通常不具抗原性的区域。与传统疫苗所针对的积极进化的抗原漂移表位相比，这些保守的“二级”表位更有可能被保存在未来的流行(可能是大流行)毒株中。因此，我们开发的重组血凝素可能是有用的“通用”免疫原，并在疫苗接种和免疫治疗方案中提供广泛的H5保护。具体目标1是设计、表达和纯化毫克量的几种血凝素。这些x-Has的设计将基于来自H5和相关流感病毒的自然漂移和逃逸突变体的大量测绘信息，以及越南/1203/04 H5 HA的晶体结构。为了中和主要抗原决定簇，逃逸突变体确定的主要抗原决定簇残基将被Discotope抗原-抗体界面数据库中出现频率较低的氨基酸取代。表达/纯化策略将遵循生产适当折叠的血凝素分子的既定方案。主要抗原决定簇敲除将通过一组映射到A/越南/1203/04 HA的单抗进行筛选来验证。具体目的2是制备抗对照和x-HA血凝素的小鼠抗血清，并用流感血凝素假型慢病毒载体报告试验检测它们的病毒中和能力。我们预测，所有主要抗原决定簇都被敲除的x-HA将诱导针对进化上保守的“次要”抗原决定簇的抗体，并将能够中和代表不同H5流感分支的报告病毒。含有中和抗体的抗血清将有助于发展针对H5流感病毒的被动免疫疗法，而用于产生这些抗体的x-HA抗原将成为开发通用疫苗的候选对象，以刺激对H5流感病毒产生广泛保护性体液免疫。与公共卫生相关：由于常规流感疫苗激发对部分病毒的免疫反应，这些病毒随着时间的推移稳步演变，因此有必要每年生产新的季节性流感疫苗。这项工作的目标是开发不针对病毒血凝素变化部分的禽流感疫苗，而是针对随时间保持不变的血凝素结构。在这项工作中开发的抗原分子可能有助于保护人们免受禽流感大流行毒株的侵袭，用于开发这些分子的策略也可能适用于生产具有广泛保护性的、针对季节性流感和其他通过改变表面结构逃避免疫系统的病原体的“通用”疫苗。

DESCRIPTION (provided by applicant): Highly pathogenic avian H5N1 influenza viruses circulating endemically in poultry of southeast Asia have recently been disseminated across the globe by wild migratory birds. These avian influenzas are also able to infect humans, and during the past decade have caused several outbreaks of severe illness with high mortality. There is concern that H5N1 bird flu viruses may in the future evolve to forms that are easily transmitted to, and among, people. There is also fear that vaccines directed against past and currently circulating H5 influenzas may not be effective against future strains of the virus due to inevitable antigenic drift and shift. The overall goal of this project is to develop hemagglutinin antigens that elicit cross protective, rather than type-specific, humoral antibody responses against a broad spectrum of evolving H5N1 influenza strains. Our work will be based on the hypothesis that ablating actively evolving, well-characterized primary antigenic determinants on the H5 hemagglutinin will redirect immune responses to regions of the molecule that are conserved and normally not antigenic. These conserved "secondary" epitopes are more likely to be preserved in future epidemic (and possibly pandemic) strains, than are the actively evolving, antigenic drift epitopes targeted by conventional vaccines. Therefore, the recombinant hemagglutinins we develop may be useful as "universal" immunogens and provide broad H5 protection in vaccination and immunotherapy protocols. Specific Aim 1 is to design, express and purify milligram amounts of several hemagglutinins. Design of these x-HAs will be based on a wealth of mapping information from natural drift and escape mutants of H5 and related influenzas, and the crystal structure of the Viet/1203/04 H5 HA. To neutralize the primary antigenic determinants, primary antigenic determinant residues identified by escape mutants will be replaced with amino acids that are present at low frequency in the Discotope database of antigen-antibody interfaces. Expression / purification strategies will follow established protocols for producing properly folded hemagglutinin molecules. Primary antigenic determinant site knockout will be verified by screening with a panel of mapped mAbs to the A/Viet/1203/04 HA. Specific Aim 2 is to prepare mouse antisera against control and x-HA hemagglutinins and assay their virus neutralization capacity using an influenza hemagglutinin pseudotyped lentiviral vector reporter assay. We predict that x-HA.s on which all primary antigenic determinants have been knocked out will elicit antibodies to evolutionarily conserved "secondary" antigenic determinants, and will be able to neutralize reporter viruses representing different H5 influenza clades. Antisera containing neutralizing antibodies will be useful for development of passive immunotherapeutics against H5 influenzas, and the x-HA antigens used to generate them will be candidates for the development of universal vaccines to stimulate the production of broadly protective humoral immunity to H5 influenzas. PUBLIC HEALTH RELEVANCE: Because conventional influenza vaccines stimulate immune responses against parts of the virus that evolve steadily over time, it is necessary to produce new seasonal flu vaccines each year. The goal of this work is to develop avian influenza vaccines that are not aimed at the parts of the viral hemagglutinin that change, and that instead target hemagglutinin structures which remain constant over time. The antigen molecules developed in this work may be useful for protecting people from pandemic strains of avian influenza, and the strategy used to develop them may also be applicable to generating broadly protective "universal" vaccines against seasonal flus and other pathogens that evade the immune system by changing their surface structures.