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

描述（由申请人提供）：甲型流感病毒感染导致美国每年约10万人住院，3.6万人死亡。人类可以通过接种季节性流感毒株疫苗得到保护。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。这些抗体可以中和病毒，防止幼鼠感染。由于糖蛋白在流感毒株之间变化很大，中和抗体主要针对疫苗中使用的相同病毒有效。相比之下，针对变异较少的病毒蛋白的疫苗可以预防多种毒株。这种疫苗接种战略将有助于为H5N1禽流感等新适应病毒的全球（大流行）传播做好准备。用高度保守的甲型流感核蛋白（NP）免疫小鼠可引起对多种血清型病毒的免疫，包括H5N1人类分离株。尽管NP特异性细胞毒性T细胞可能介导这种保护，但不能排除非中和抗体对NP的作用。我们的初步结果表明重组NP免疫引起的保护需要抗体。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起作用。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。这一认识可用于加强人用疫苗的交叉保护，从而可以：1)减轻季节性流感的影响，2)为意外毒株和潜在的大流行疫情做好准备，3)提供长期保护，减少每年重新接种疫苗的需要，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结合形成复合物，激活先天抗病毒机制，然后增强NP特异性T细胞反应，最终导致加速病毒清除，从而促进免疫。因此，SPECIFIC AIM 1将确定np特异性抗体需要哪些效应分子（FcR、TLR、IFN12R和补体）来保护幼稚小鼠。由于这些分子可能通过T细胞介导的机制促进病毒清除，因此SPECIFIC AIM 2将研究抗np抗体如何影响T细胞反应，以及rnp免疫抗体介导的保护是否需要T细胞。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解决。这种np免疫抗体将被转移到缺乏抗体受体、其他效应分子或T细胞的小鼠体内。在受者感染流感病毒后，将监测发病率（体重减轻）和肺部病毒水平。结果将在免疫抗体受体和非免疫抗体受体之间，以及在正常受体和缺乏被检测因子的受体之间进行比较。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证明对交叉保护性免疫机制的新理解。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作出反应，而不是对意想不到的毒株作出反应，例如禽流感H5N1。我们的研究将检查独特的和未被认识到的方式，其中疫苗接种可以诱导对所有甲型流感毒株中高度相似的蛋白质的免疫反应。了解这些反应可以增强人类疫苗：1)无论小（季节性）或大（潜在的大流行）变化，都能对多种病毒株作出反应；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.