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，该策略对抗原漂移变异比目前的疫苗病毒更新更有效，目前的疫苗病毒更新通常比流行病毒晚一年。本资助期内拟议工作的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，并确定人群中多克隆抗体如何选择抗原变体。下一个授权期的特定目的基于以下假设：表位的免疫原性不同，大部分抗体应答可能针对抗原位点的一个小子集。抗原漂移期间突变的积累可导致特定抗原位点变得更少或更多免疫显性。具体目标1是获得并表征从2002年到5年资助的与H3 N2流感分离株的HA和NA反应的单克隆抗体（mAb）组。人mAb将与生成的小鼠mAb组一起使用，以填补任何空白。具体目标2是定位HA和NA上的表位。中和表位是构象的，不被肽模拟。构象表位将通过逃逸突变体的选择和表征、竞争测定、定向诱变和在少数情况下抗原-抗体复合物的X射线晶体学来绘制。具体目标3是在与人血清的竞争测定中使用来自目标1的mAb确定个体表位的体内相对免疫显性，以确定某些抗原位点在接种疫苗或感染后的人中是否占优势，以及在老年或免疫抑制受试者中是否存在差异。具体目标4是确定免疫优势是否随着病毒在资助的5年内经历抗原漂移而发生变化。每年将从接种疫苗的受试者中测量人血清中针对漂移病毒单个抗原位点的抗体。具体目标5是确定是否可以通过制备突变病毒或抗原来改变免疫原性，所述突变病毒或抗原被设计为抑制特定表位的免疫原性。那么对其他表位的反应是否会增强？这些实验的结果将显示某些抗原位点是否存在免疫显性，在抗原漂移期间免疫显性是否改变，以及免疫显性是否可以系统地改变。这些知识最终可以用于操纵疫苗抗原，从而使抗体能够针对更广泛的中和表位，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而具有不同的有效性。流感疫苗每年都会更新新的病毒，但由于确定新菌株正在传播并使其适应疫苗使用所需的时间，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流感变异的机制，并利用这些知识来修改疫苗，使其对更广泛的变异病毒有效。如果新的大流行开始，所获得的知识将适用于新的流感亚型。

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.