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

描述（由申请人提供）：土拉菌是一种革兰氏阴性的a类细胞内粘膜病原体。细胞免疫对抵抗这种生物至关重要，而抗体（Abs）可以延缓感染的进展。Ag呈递细胞（APC）上靶向抗原（Ag）到Fc受体（FcR）可以增强体液免疫和细胞免疫。我们假设将感染性疾病Ag，如灭活的土拉菌（iFt）靶向于粘膜部位的FcR将增强对粘膜攻击的保护。在Aim 1中，我们将研究预形成的单克隆抗体-iFt复合物和单克隆抗体加iFt混合物增强APC对ag特异性T细胞的结合、内化和呈递iFt的能力，这是启动保护性免疫反应的关键事件。我们将：1)研究mAb:iFt比例对小鼠APC中mAb-iFt结合、内化和Ag呈递的影响；2)使用小鼠APC，确定单抗+ iFt混合物是否可以代替预制的单抗-iFt；3)确定上述fcr靶向策略是否也能增强人类APC的ift结合、内化和呈递。在Aim 2中，我们将在小鼠中确定单抗-iFt复合物和/或单抗+ iFt混合物在体内、体外、体外或体外给予的能力，以增强对活土拉菌感染的保护作用，并确定对观察到的保护作用至关重要的体液和/或细胞成分。我们将：1)验证在目的1中观察到的最佳的fcr介导的结合、内化和呈递，与使用fcr靶向免疫原产生的最佳保护相关；2)利用缺乏这些免疫成分的小鼠，确定CD8和CD4 T细胞、B细胞、FcR、Ab和ifn - γ在FcR依赖性保护中的作用；3)确定fcr靶向的iFt是否优先定位于淋巴组织，而非靶向的iFt；4)确定是否可以在外围（id、i.m.或s.c）免疫后产生针对i.n.和i.d挑战的保护；5)确定CTB （i.n）和Alum （i.d, i.m或s.c）的加入是否能进一步增强FcR靶向免疫原产生的保护作用。在Aim 3中，我们将验证该疫苗平台的灵活性和多病原体潜力。具体来说，我们将生产一种针对肺炎链球菌的fcr靶向亚单位疫苗（Fc-PspA），肺炎链球菌是一种具有重大公共卫生关注的细胞外粘膜病原体。PspA是肺炎链球菌的表面成分，在佐剂存在下产生抗体依赖性保护。我们将研究含有可变量PspA的单价和多价Fc-PspA偶联物的能力，并通过粘膜和外周途径给药，以防止肺炎链球菌的侵袭。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，既能产生体液免疫，也能产生细胞免疫；2)后者在粘膜疫苗中尤为明显；3)迫切需要一种有效的土拉菌黏膜疫苗和一种更有效的肺炎链球菌疫苗；4)缺乏关于FcR在粘膜免疫中的作用以及对粘膜病原体产生保护作用的知识。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：拟议的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，特别是适用于粘膜免疫的疫苗平台。此外，还需要一种有效的土拉菌（a类生物威胁剂）粘膜疫苗，以及一种更有效的肺炎链球菌（一种具有重大公共卫生关注的病原体）疫苗。我们提供了强有力的证据，证明拟议的研究将满足这些需求，并填补了我们关于Fc受体在对抗细胞内和细胞外病原体的粘膜和外周免疫中的作用和使用方面的重大知识空白。

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.