Mechanisms of non-neutralizing antibody-mediated protection from influenza virus

描述(申请人提供)：在美国，甲型流感病毒感染每年导致约100,000人住院和36,000人死亡。人类可以通过接种季节性流感病毒株疫苗来保护自己。然而，目前的流感疫苗大多诱导针对病毒外部糖蛋白的抗体。这些抗体中和病毒，并防止感染，当给NAOVE小鼠。由于流感病毒株之间的糖蛋白高度不同，中和抗体主要对疫苗中使用的相同病毒有效。相比之下，针对变异性较小的病毒蛋白的疫苗可以预防多种毒株。这样的疫苗接种战略将有助于为新适应病毒的全球(大流行)传播做好准备，例如H5N1禽流感。用高度保守的甲型流感核蛋白(NP)免疫小鼠，可诱导对包括H5N1人类分离株在内的多种血清型病毒的免疫。尽管NP特异性细胞毒性T细胞可能介导这种保护，但不能排除NP的非中和抗体的作用。我们的初步结果表明，在重组NP免疫所产生的保护中需要抗体。然而，目前尚不清楚针对内部病毒蛋白的抗体如何在保护性免疫中发挥作用。这项研究的长期目标是了解这种非中和抗体对流感的保护潜力。然后，这种理解可用于加强人类疫苗的交叉保护，这可以：1)减轻季节性流感的影响，2)为意外的毒株和潜在的大流行疫情做好准备，3)提供长期保护，减少每年重新免疫的需要，从而产生经济效益。我们假设，抗NP抗体通过与感染细胞释放的NP结合形成复合体，激活固有的抗病毒机制，然后增强NP特异性的T细胞反应，从而促进免疫，最终导致病毒加速清除。因此，特定的AIM 1将决定保护幼小鼠所需的NP特异性抗体所需的效应分子(FCR、TLR、IFN12R和补体)。由于这些分子可能通过T细胞介导的机制促进病毒清除，特定的AIM 2将研究抗NP抗体如何影响T细胞反应，以及是否需要T细胞来进行RNP免疫抗体介导的保护。这些目标将通过用流感NP免疫小鼠和纯化血清中产生的抗体来实现。这种NP免疫抗体将被转移到抗体受体、其他效应分子或T细胞中缺乏的小鼠身上。在受体感染流感病毒后，将监测发病率(体重减轻)和肺部病毒水平。结果将在免疫抗体接受者和非免疫抗体接受者之间进行比较，并在正常接受者和在被测试的特定因素中缺陷的接受者之间进行比较。由于非中和抗体在流感免疫中的作用在很大程度上被低估，这些实验的结果将共同展示对交叉保护免疫机制的新理解。公共卫生相关性虽然目前的流感疫苗可以保护人类免受季节性疫情的影响，但它们仅限于对疫苗中使用的相同病毒产生反应，而不是对意想不到的毒株，如禽流感H5N1流感产生反应。我们的研究将考察疫苗接种可以诱导对一种在所有甲型流感病毒株中高度相似的蛋白质的免疫反应的独特和未被认识的方法。了解这些反应可以增强人类疫苗1)对多种病毒株的反应，而不考虑小的(季节性的)或大的(可能的大流行)变化，2)提供长期保护，减少每年重新免疫的需要。

DESCRIPTION (provided by applicant): Influenza A virus infection results in ~100,000 hospitalizations and 36,000 deaths in the U.S. per year. Humans can be protected by vaccination against seasonal influenza strains. However, current influenza vaccines mostly induce antibodies to the virus' external glycoproteins. These antibodies neutralize the virus and prevent infection when given to naove mice. Because the glycoproteins are highly variable among influenza strains, neutralizing antibodies are primarily effective against the same viruses used in the vaccine. By contrast, vaccines against less variable viral proteins could protect against multiple strains. Such a vaccination strategy would facilitate preparation for worldwide (pandemic) spread of newly adapted viruses, such as H5N1 avian influenza. Immunizing mice with the highly conserved influenza A nucleoprotein (NP) elicits immunity against viruses of multiple serotypes, including H5N1 human isolates. Although NP-specific cytotoxic T cells might mediate this protection, a role for non-neutralizing antibody against NP cannot be excluded. Our preliminary results show a requirement for antibody in protection elicited by recombinant NP immunization. However, it is unknown how antibodies against an internal viral protein might function in protective immunity. The long-term objective of this study is to understand the protective potential of such non-neutralizing antibodies to influenza. This understanding could then be used to enhance cross-protection of human vaccines that could: 1) lessen the impact of seasonal flu, 2) prepare for unexpected strains and potentially pandemic outbreaks, and 3) provide long-term protection that would lessen the need for annual re-immunization, and thus have an economic benefit. We hypothesize that anti-NP antibodies promote immunity by binding to NP released from infected cells to form complexes that activate innate anti-viral mechanisms, and then enhance NP-specific T cell responses - ultimately leading to accelerated viral clearance. Therefore, SPECIFIC AIM 1 will determine what effector molecules (FcR, TLR, IFN12R, and complement) are required for NP-specific antibodies to protect naive mice. Because these molecules may promote viral clearance via T cell-mediated mechanisms, SPECIFIC AIM 2 will examine how anti-NP antibodies influence T cell responses and whether T cells are required for rNP-immune antibody-mediated protection. These aims will be addressed by immunizing mice with influenza NP and purifying the antibody generated in the serum. This NP-immune antibody will be transferred to mice deficient in antibody receptors, in other effector molecules, or in T cells. After infection of the recipients with influenza virus, morbidity (weight loss), and the levels of virus in the lung will be monitored. The results will be compared between recipients of immune and non-immune antibody, and between normal recipients and those deficient in the given factor being tested. Because the role of non-neutralizing antibody in influenza immunity is largely underappreciated, the results from these experiments will collectively demonstrate a novel understanding of the mechanisms for cross-protective immunity. PUBLIC HEALTH RELEVANCE Although current influenza vaccines can protect humans from seasonal outbreaks, they are limited to reacting to the same viruses used in the vaccine, but not to unexpected strains, such as avian H5N1 influenza. Our research will examine unique and unappreciated ways in which vaccination can induce immune reactions to a protein that is highly similar among all influenza A strains. Understanding these reactions could then enhance human vaccines to 1) react against multiple virus strains regardless of small (seasonal) or large (potentially pandemic) changes, and 2) provide long-term protection that would lessen the need for annual re- immunization.