Human & mouse antibodies against influenza virus

描述（由申请人提供）：我们工作的长期目标是开发一种流感疫苗策略，这种疫苗策略比目前更新的疫苗病毒更有效，后者通常比流行病毒晚一年。本资助期提出的工作总体目标是表征由疫苗接种或感染诱导或召回的人血清中抗体识别的抗原位点，并确定人群中的多克隆抗体如何选择抗原变体。下一个批准期的具体目标是基于一个假设，即表位不具有相同的免疫原性，大部分抗体反应可能针对一小部分抗原位点。抗原漂移过程中突变的积累可能导致特定抗原位点的免疫优势减弱或增强。具体目标1是从2002年到5年拨款期间获得与H3N2流感毒株HA和NA反应的单克隆抗体（mab）并对其进行鉴定。人类单克隆抗体将与生成的小鼠单克隆抗体面板一起使用，以填补任何空白。特异性目标2是绘制HA和NA上的表位。中和表位是构象的，不被肽模仿。构象表位将通过逃逸突变体的选择和表征、竞争测定、定向诱变和抗原-抗体复合物的x射线晶体学在少数情况下绘制。特异性Aim 3是利用来自Aim 1的单克隆抗体在与人血清的竞争试验中确定个体表位在体内的相对免疫优势，以确定某些抗原位点在接种疫苗或感染后是否在人群中占主导地位，以及在老年人或免疫抑制受试者中是否存在差异。具体目标4是确定在5年的资助期内，免疫优势是否会随着病毒抗原漂移而改变。每年将从接种疫苗的受试者中测量人血清中针对漂流病毒单个抗原位点的抗体。特异性目的5是确定是否可以改变免疫原性，通过制造突变病毒或抗原来抑制特定表位的免疫原性。那么对其他表位的反应会得到加强吗？这些实验的结果将显示某些抗原位点是否存在免疫优势，在抗原漂移过程中免疫优势是否发生变化，以及免疫优势是否可以被系统地改变。这一知识最终可用于操纵疫苗抗原，以便针对更广泛的中和性表位制造抗体，并更有效地对抗抗原漂移。公共卫生相关性：流感疫苗是安全的，但由于人群中流行的流感病毒的抗原变异，其有效性存在差异。流感疫苗每年都有新病毒的更新，但由于确定一种新毒株正在传播并使其适应疫苗使用需要时间，疫苗通常比病毒晚一年。这项研究的目的是确定在人群中选择新的流感变种的机制，并利用这一知识修改疫苗，使其对更广泛的变种病毒有效。如果新的流感大流行开始，所获得的知识将适用于一种新的流感亚型。

DESCRIPTION (provided by applicant): The long term goal of our work is to develop a vaccine strategy for influenza that is more effective against antigenic drift variants than the current updating of vaccine viruses, which is typically a year behind the circulating viruses. The overall goal of the work proposed for this funding period is to characterize the antigenic sites recognized by antibodies in human sera that are induced or recalled by vaccination or infection and to determine how antigenic variants are selected by polyclonal antibodies in the human population. The Specific Aims for the next granting period are based on a hypothesis that epitopes are not equally immunogenic and the bulk of the antibody response may be to a small subset of antigenic sites. Accumulation of mutations during antigenic drift may cause particular antigenic sites to become less or more immunodominant. Specific Aim 1 is to obtain and characterize panels of monoclonal antibodies (mAbs) that react with HA and NA of influenza H3N2 isolates from 2002 through the 5 year grant. Human mAbs will be used with panels of mouse mAbs generated to fill in any gaps. Specific Aim 2 is to map the epitopes on the HA and NA. Neutralizing epitopes are conformational and not mimicked by peptides. Conformational epitopes will be mapped by selection and characterization of escape mutants, competition assays, directed mutagenesis and X-ray crystallography of antigen-antibody complexes in a few cases. Specific Aim 3 is to determine the relative immunodominance in vivo of individual epitopes using mAbs from Aim 1 in competition assays with human sera to determine if certain antigenic sites are dominant in people after vaccination or infection, and if there are differences in elderly or immunosuppressed subjects. Specific Aim 4 is to determine if immunodominance changes as the virus undergoes antigenic drift over the 5 years of the grant. Antibodies in human sera against individual antigenic sites of drifted viruses will be measured from subjects vaccinated each year. Specific Aim 5 is to determine if immunogenicity can be altered, by making mutant viruses or antigens that are designed to suppress immunogenicity of particular epitopes. Will response to other epitopes then be enhanced? The results of these experiments will show if there is immunodominance of certain antigenic sites, if the immunodominance changes during antigenic drift, and if immunodominance can be systematically altered. This knowledge could ultimately be used to manipulate vaccine antigens so that antibodies would be made against a wider spectrum of neutralizing epitopes and be more effective against antigenic drift. PUBLIC HEALTH RELEVANCE: Influenza vaccines are safe but have varying effectiveness due to antigenic variation of circulating influenza viruses in the human population. Influenza vaccine is updated every year with new viruses but due to the time required to determine that a new strain is spreading and to adapt it for vaccine use, the vaccine is usually a year behind the virus. The goal of this research is to determine the mechanisms by which new influenza variants are selected in the human population and to use this knowledge to modify the vaccine so that it is effective against a broader range of variant viruses. The knowledge gained will be applicable to a new subtype of influenza if and when a new pandemic begins.