Pneumococcal immunization through a novel mechanism

说明(由申请人提供)：胶囊多糖-蛋白质结合疫苗在预防包括在内的血清型的肺炎链球菌(肺炎球菌)侵袭性疾病方面有效，但制造和管理成本很高，对中耳炎的效果最低，并且需要进行血清型替换(其中非纳入的血清型变得更加普遍)。因此，需要新的免疫方法。我们意外地发现，用肺炎球菌细胞壁多糖(CWPS，所有血清型的共同抗原)鼻腔免疫的小鼠对不同血清型的肺炎球菌的鼻咽定植和中耳感染产生了长期的抵抗力。值得注意的是，疫苗(即CWPS+粘膜佐剂)的保护作用不依赖于抗体，依赖于CD4+T细胞的存在。据我们所知，这种由多糖诱导的、细胞介导性的粘膜免疫，可以抵抗被“细胞外”包裹的细菌的定植，以前还没有得到证实，因此代表了一种新的疫苗接种方法。我们假设CWPS的两性离子性质在诱导这种T细胞依赖的反应中是关键的。我们的第一个目标是通过进一步纯化、聚合物合成、化学修饰改变两性离子基序、自动偶联或偶联到蛋白质载体来确定CWPS保护的结构基础。因此，两性离子假说将被证实和/或最小保护结构将被定义。其次，我们将更详细地研究CD4+T细胞对肺炎球菌定植的保护机制。过继转移实验将描述保护性T细胞反应的性质。进一步的方法将包括利用基因敲除小鼠或给予细胞因子来极化T细胞反应，中性粒细胞耗尽实验，以及共聚焦显微镜下的组织病理学。在第三个目标中，我们将通过使用Toll样受体(TLR)基因敲除小鼠和在免疫时联合给予TLR配体作为佐剂来评估先天免疫反应在调节获得性免疫定植中的作用。我们的研究将增加对肺炎球菌定植免疫的基本了解，并可能导致一种简单、明确且可能合成的疫苗，在针对这种儿童高度流行的病原体的疫苗接种中补充或取代多价胶囊结合物。

DESCRIPTION (provided by applicant): The capsular polysaccharide-protein conjugate vaccine is effective in prevention of invasive disease by Streptococcus pneumoniae (pneumococci) of the serotypes included but is costly to make and administer, minimally effective against otitis media, and subject to serotype replacement (in which non-included serotypes become more prevalent). Therefore, novel approaches to immunization are needed. We found unexpectedly that mice immunized intranasally with the pneumococcal cell wall polysaccharide (CWPS, an antigen common to all serotypes) develop long-lasting resistance to nasopharyngeal colonization and middle ear infection with pneumococci of different serotypes. Strikingly, protection by the vaccine (i.e. CWPS + mucosal adjuvant) is independent of antibody and dependent on the presence of CD4+ T cells. This polysaccharide-induced, cell-mediated mucosal immunity against colonization by an "extracellular" encapsulated bacterium has not, to our knowledge, been previously demonstrated and therefore represents a novel approach to vaccination. We hypothesize that the zwitterionic property of CWPS is critical in eliciting this T-cell-dependent response. Our first goal is to determine the structural basis of protection by CWPS by further purification, polymer synthesis, chemical modifications to alter the zwitterionic motif, auto-coupling, or coupling to a protein carrier. Thus either the zwitterion hypothesis will be confirmed and/or the minimal protective structure will be defined. Secondly, we will examine in more detail the mechanisms whereby CD4+ T cells confer protection against pneumococcal colonization. Adoptive transfer experiments will characterize the nature of the protective T cell responses. Further approaches will include polarization of T cell responses by use of knockout mice or administration of cytokines, neutrophil depletion experiments, and histopathology with confocal microscopy. In a third aim, we will evaluate the role of innate immune responses in modulating acquired immunity to colonization, by use of Toll-like receptor (TLR) knockout mice and co-administration of TLR ligands as adjuvants at the time of immunization. Our studies will increase basic understanding of immunity to pneumococcal colonization and could lead to a simple, defined, and possibly synthetic vaccine that would complement or replace the multivalent capsular conjugates in vaccination against this highly prevalent pathogen of children.