Development of Cross Protective H5 Hemagglutinin Antigens

交叉保护性 H5 血凝素抗原的开发

• 批准号: 8072917
• 负责人: SUSAN C BOCK
• 金额: $ 1.14万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-24 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8072917
• 关键词: Amino Acids; Antibodies; Antibody Formation; Antigens; Avian Influenza; Avian Influenza A Virus; Biological Assay; Bird Flu vaccine; Birds; Databases; Development; Disease Outbreaks; Domestic Fowls; Epidemic; Epitopes; Escape Mutant; Frequencies; Fright; Future; Goals; H5 hemagglutinin; Hemagglutinin; Human; Humoral Immunities; Immune Sera; Immune response; Immune system; Immunotherapeutic agent; Immunotherapy; Influenza; Influenza A Virus, H5N1 Subtype; Influenza Hemagglutinin; Knock-out; Lentivirus Vector; Maps; Mus; Production; Protocols documentation; Recombinants; Reporter; Screening procedure; Site; Southeastern Asia; Structure; Surface; Time; Vaccination; Vaccines; Viral Hemagglutinins; Virus; Work; base; design; influenza virus vaccine; migratory bird; milligram; mortality; neutralizing antibody; pandemic disease; pathogen; public health relevance; seasonal influenza

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.

描述（由申请方提供）：在东南亚家禽中流行的高致病性H5 N1禽流感病毒最近已通过野生候鸟传播到地球仪。这些禽流感也能够感染人类，并在过去十年中引发了多次严重疾病的爆发，死亡率很高。人们担心，H5 N1禽流感病毒将来可能会演变成容易传染给人和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，其引起针对广谱进化的H5 N1流感毒株的交叉保护性而非类型特异性体液抗体应答。我们的工作将基于这样的假设，即消融H5血凝素上的积极进化的、充分表征的初级抗原决定簇将使免疫应答重定向到分子的保守且通常不具有抗原性的区域。这些保守的“第二”表位更有可能在未来的流行（可能是大流行）菌株中保留，而不是由常规疫苗靶向的积极进化的抗原漂移表位。因此，我们开发的重组血凝素可用作“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-HA的设计将基于来自H5和相关流感病毒的天然漂移和逃逸突变体的大量作图信息，以及Viet/1203/04 H5 HA的晶体结构。为了中和一级抗原决定簇，由逃逸突变体鉴定的一级抗原决定簇残基将被抗原-抗体界面的Discotope数据库中以低频率存在的氨基酸取代。表达/纯化策略将遵循用于产生正确折叠的血凝素分子的既定方案。将通过使用一组定位于A/Viet/1203/04 HA的mAb进行筛选，验证一级抗原决定簇位点敲除。具体目标2是制备抗对照和x-HA血凝素的小鼠抗血清，并使用流感血凝素假型慢病毒载体报告基因试验测定其病毒中和能力。我们预测，x-HA.s上的所有主要抗原决定簇已被敲除，将引发抗体进化保守的“二级”抗原决定簇，并将能够中和报告病毒代表不同的H5流感进化枝。含有中和抗体的抗血清将有助于开发针对H5流感的被动免疫治疗剂，并且用于产生它们的x-HA抗原将是开发通用疫苗以刺激产生针对H5流感的广泛保护性体液免疫的候选物。公共卫生关系：由于传统的流感疫苗会刺激免疫反应，对抗随着时间推移而稳定进化的病毒部分，因此每年都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，不针对病毒血凝素的变化部分，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株的侵害，并且用于开发它们的策略也可能适用于产生广泛保护性的“通用”疫苗，以对抗季节性流感和其他通过改变其表面结构来逃避免疫系统的病原体。