Development of Cross Protective H5 Hemagglutinin Antigens

描述（由申请人提供）：在东南亚家禽中流行的高致病性H5N1流感病毒最近通过野生候鸟在全球传播。这些禽流感也能够感染人类，并且在过去十年中造成了几次严重疾病的暴发，死亡率很高。令人担忧的是，H5N1禽流感病毒将来可能演变成易于在人与人之间传播的形式。人们还担心，由于不可避免的抗原漂移和转移，针对过去和目前流行的H5流感的疫苗可能对未来的病毒株无效。该项目的总体目标是开发血凝素抗原，该抗原可引起针对广泛的不断演变的H5N1流感毒株的交叉保护性而非类型特异性体液抗体反应。我们的工作将基于这样的假设，即去除H5血凝素上积极进化的、特征良好的初级抗原决定因子，将免疫反应重新定向到分子中保守的、通常不具有抗原性的区域。这些保守的“二级”表位比传统疫苗靶向的主动进化的抗原漂移表位更有可能在未来的流行病（和可能的大流行）毒株中保存下来。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。特异性目的1是设计、表达和纯化数毫克量的几种血凝素。这些x-HA的设计将基于来自H5和相关流感的自然漂移和逃逸突变体的丰富图谱信息，以及Viet/1203/04 H5 HA的晶体结构。为了中和初级抗原决定因子，由逃逸突变体识别的初级抗原决定因子残基将被存在于distope抗原-抗体界面数据库中的低频率氨基酸所取代。表达/纯化策略将遵循既定的方案，以产生适当折叠的血凝素分子。初级抗原决定位点敲除将通过筛选a /Viet/1203/04 HA的一组定位单克隆抗体来验证。特异性目的2是制备小鼠抗对照血凝素和x-HA血凝素的抗血清，并使用流感血凝素假型慢病毒载体报告试验测定它们的病毒中和能力。我们预测x-HA。所有初级抗原决定因子都被敲除的抗体将引发针对进化上保守的“二级”抗原决定因子的抗体，并将能够中和代表不同H5流感进化支的报告病毒。含有中和抗体的抗血清将有助于开发针对H5流感的被动免疫疗法，用于生成它们的x-HA抗原将成为开发通用疫苗的候选物，以刺激产生针对H5流感的广泛保护性体液免疫。公共卫生相关性：由于传统流感疫苗会刺激针对部分病毒的免疫反应，这些病毒会随着时间的推移而稳定进化，因此每年有必要生产新的季节性流感疫苗。这项工作的目标是开发禽流感疫苗，这种疫苗不是针对病毒血凝素发生变化的部分，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害，用于开发它们的策略也可能适用于生产具有广泛保护性的“通用”疫苗，以预防季节性流感和其他通过改变其表面结构逃避免疫系统的病原体。

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.