Mechanisms Involved in, and Development of, FcR-Enhanced Mucosal Vaccination

描述（由申请人提供）：F。土拉热是革兰氏阴性A类细胞内粘膜病原体。细胞免疫对于保护免受这种微生物的侵害至关重要，而抗体（Abs）则可以延缓感染的进展。将抗原（Ag）靶向抗原呈递细胞（APC）上的Fc受体（FcR）可以增强体液免疫和细胞免疫。我们假设靶向传染病抗原，如灭活F。土拉热菌（iFt）对粘膜部位的FcR的免疫应答将增强针对粘膜攻击的保护。在目的1中，我们将研究预先形成的mAb-iFt复合物和mAb加iFt混合物增强APC对Ag特异性T细胞的iFt结合、内化和呈递的能力，这是启动保护性免疫应答的关键事件。我们将：1）检查mAb：iFt比率对小鼠APC的mAb-iFt结合、内化和Ag呈递的影响; 2）使用小鼠APC确定mAb加iFt混合物是否可以用于代替预先形成的mAb-iFt; 3）确定上述FcR靶向策略是否也增强人APC的IFt结合、内化和呈递。在目的2中，我们将在小鼠中确定i.n. I.D.，i. m.，或s.c.以增强对I.N.或者用活的F.土拉菌，并确定体液和/或细胞的关键组成部分，所观察到的保护。我们将：1）验证在目标1中观察到的最佳FcR介导的结合、内化和呈递与由i.n.施用的FcR靶向免疫原产生的最佳保护相关; 2)确定CD 8和CD 4 T细胞、B细胞、FcR、Ab和IFN-γ在FcR依赖性保护中的作用，使用缺乏这些免疫组分的小鼠; 3）确定FcR靶向的iFt是否相对于非靶向的iFt优先定位于淋巴组织; 4）确定针对i.n.和i.d.可以在外围设备（i.d.，i. m.，或s.c.）免疫; 5）确定是否包含CTB（i.n.）和明矾（i.d.，i. m.，或s.c.）进一步增强由FcR靶向免疫原产生的保护。在目标3中，我们将验证该疫苗平台的灵活性和多病原体潜力。具体而言，我们将产生针对S的FcR靶向亚单位疫苗（Fc-PspA）。肺炎，一种引起重大公共卫生关注的细胞外粘膜病原体。PspA是S. pneumoniae，其在佐剂存在下产生Ab依赖性保护。我们将研究含有可变量的PspA并通过粘膜和外周途径施用的单价和多价Fc-PspA缀合物保护免受i.n.挑战S。肺炎。上述研究的意义是实质性的：1）需要新的和更安全的疫苗平台，其产生体液和细胞免疫; 2）后者在粘膜疫苗的情况下尤其明显; 3）迫切需要针对F. tularensis，以及针对S. 4）缺乏关于FcR在粘膜免疫中的作用以及产生针对粘膜病原体的保护的知识。拟议的研究将填补上述所有领域的重大空白。公共卫生相关性：拟议研究对公共卫生的意义是多方面的。需要新的和更安全的疫苗平台，特别是当其应用于粘膜免疫时。此外，还研制了一种有效的粘膜疫苗。tularensis（A类生物威胁剂），以及针对S.肺炎（一种重要的公共卫生问题的病原体）。我们提供了强有力的证据，拟议的研究将满足这些需求，并填补了我们的知识的重要差距，在粘膜和外周免疫的作用和使用Fc受体对细胞内和细胞外病原体。

DESCRIPTION (provided by applicant): F. tularensis is a gram-negative Category A intracellular mucosal pathogen. Cellular immunity is critical for protection against this organism, while antibodies (Abs) delay the progression of infection. Targeting antigen (Ag) to Fc receptors (FcR) on Ag presenting cells (APC) can enhance humoral and cellular immunity. We hypothesize targeting infectious disease Ag, such as inactivated F. tularensis (iFt), to FcR at mucosal sites will enhance protection against mucosal challenge. In Aim 1, we will investigate the ability of preformed mAb-iFt complexes and mAb plus iFt mixtures, to enhance binding, internalization, and presentation of iFt by APC to Ag-specific T cells, key events in initiating a protective immune response. We will: 1) Examine the impact of mAb:iFt ratio on mAb-iFt-binding, internalization, and Ag presentation by mouse APC; 2) Determine, using mouse APC, if mAb plus iFt mixtures can be used in place of preformed mAb-iFt; 3) Determine if the above FcR-targeting strategies also enhance IFt-binding, internalization, and presentation by human APC. In Aim 2, we will determine in mice, the ability of mAb-iFt complexes and/or mAb plus iFt mixtures administered i.n., i.d., i.m., or s.c. to enhance protection against i.n. or i.d challenge with live F. tularensis, and identify the humoral and/or cellular components critical to the observed protection. We will: 1) Verify optimal FcR-mediated binding, internalization, and presentation observed in Aim 1, correlates with optimal protection generated by FcR-targeted immunogens administered i.n.; 2) Determine the role of CD8 and CD4 T cells, B cells, FcR, Ab, and IFN-gamma in FcR-dependent protection, using mice lacking these immune components; 3) Determine if FcR-targeted iFt preferentially localizes to lymphoid tissues, versus non-targeted iFt; 4) Determine if protection against i.n. and i.d. challenge can be generated following peripheral (i.d., i.m., or s.c.) immunization; 5) Determine if inclusion of CTB (i.n.) and Alum (i.d., i.m., or s.c.) further enhances protection generated by FcR- targeted immunogens. In Aim 3, we will validate the flexibility and the multi-pathogen potential of this vaccine platform. Specifically, we will generate an FcR-targeted subunit vaccine (Fc-PspA) against S. pneumoniae, an extracellular mucosal pathogen of significant public health concern. PspA is a surface component of S. pneumoniae, which generates Ab-dependent protection in the presence of adjuvant. We will investigate the ability of mono- and multivalent Fc-PspA conjugates containing variable amounts of PspA, and administered via mucosal and peripheral routes, to protect against i.n. challenge with S. pneumoniae. The significance of the above studies is substantial: 1) New and safer vaccine platforms, which generate both humoral and cellular immunity are needed; 2) The latter is particularly evident in the case of mucosal vaccines; 3) There is an urgent need for an effective mucosal vaccine against F. tularensis, and a more efficacious vaccine against S. pneumoniae; 4) Knowledge regarding the role of FcR in mucosal immunity, and the generation of protection against mucosal pathogens, is lacking. The proposed studies will fill significant gaps in all the above areas. PUBLIC HEALTH RELEVANCE: The significance of the proposed studies to public health is multi-fold. There is a need for new and safer vaccine platforms, in particular as it applies to mucosal immunity. In addition, an effective mucosal vaccine against F. tularensis (a Category A biothreat agent), and a more efficacious vaccine against S. pneumoniae (a pathogen of significant public health concern), are also needed. We provide strong evidence the proposed studies will satisfy these needs, and fill significant gaps in our knowledge regarding the role and use of Fc receptors in mucosal and peripheral immunity against intracellular and extracellular pathogens.