Imaging antigen and adjuvant uptake for enhancing response to mucosal vaccines

描述（由申请方提供）：由于大多数人类感染影响或通过口腔-胃肠道、呼吸道和泌尿生殖道的粘膜表面进入，未来在这些部位引发保护的疫苗可能通过粘膜途径给药，胃内（i.g.）或鼻内（i.n.）。在设计用于增强对粘膜疫苗的应答的众多策略中，细菌热不稳定肠毒素如霍乱毒素（CT）和大肠杆菌的不稳定毒素（LT-I、LT-IIa和LT-IIb）已被开发作为强有力的免疫刺激佐剂和偶联至疫苗抗原的载体分子。然而，目前还不清楚这些佐剂和免疫原靶向哪些组织和细胞，以及它们如何和在哪里被加工并呈递给T细胞以启动免疫应答。一些抗原在i.g.单独或与CT的B亚基偶联可抑制免疫反应，这种现象称为“口服耐受性”。诱导这两种不同应答的机制知之甚少，但在粘膜疫苗的设计和开发中至关重要，无论是用于产生抗感染保护，还是用于抑制不期望的免疫应答，如自身免疫性疾病或过敏。参与疫苗抗原和佐剂摄取的细胞的精确组织部位和特征，以及佐剂在活化抗原呈递细胞中的作用模式，在确定随后的免疫应答的性质方面是至关重要的。通过应用新兴的新型ImageStream（Amnis Corp.）技术.在粘膜免疫学和流式细胞术和成像细胞术专家之间的这个合作项目中，将在小鼠中研究基于抗原肠毒素嵌合蛋白的荧光染料标记的粘膜免疫原和肠毒素佐剂（或其无毒衍生物）的摄取。具体目的是：（1）确定经i.g.给药的抗原-肠毒素嵌合蛋白摄取的细胞途径。或I.N.，并表征所涉及的抗原呈递细胞的类型、位置和活化;和（2）与对照蛋白相比，当i.g.或I.N.预期结果将鉴定这些免疫原和佐剂摄取的精确位点，定义由它们刺激的细胞的特征，从而确定诱导的免疫应答的类型。这些信息对于未来粘膜应用疫苗的设计将是重要的。 公共卫生相关性：正在开发通过鼻内或胃内途径施用的新一代疫苗，以在大多数人类感染发生的粘膜表面引起保护，并且具有无针的额外优点。然而，摄取并响应于粘膜施用的疫苗抗原和佐剂的精确组织和细胞还没有被充分理解，然而这种知识对于设计这样的疫苗是必不可少的，无论是用于保护免受感染还是抑制自身免疫疾病或过敏中的不期望的应答。该提案将应用基于Amnis图像流的新型成像技术来识别粘膜疫苗和佐剂靶向的组织和细胞，并表征启动免疫应答的抗原呈递细胞。

DESCRIPTION (provided by applicant): Because most human infections affect or gain entry through the mucosal surfaces of the oro-gastrointestinal, respiratory, and genitourinary tracts, future vaccines to elicit protection at these sites will likely be administered by mucosal routes, intragastrically (i.g.) or intranasally (i.n.). Among numerous strategies designed to enhance responses to mucosal vaccines, bacterial heat-labile enterotoxins such as cholera toxin (CT) and the labile toxins of Escherichia coli (LT-I, LT-IIa and LT-IIb) have been exploited as both powerful immunostimulatory adjuvants and carrier molecules coupled to vaccine antigens. However, it is not known precisely which tissues and cells are targeted by these adjuvants and immunogens, and how and where they are processed and presented to T cells to initiate the immune response. Some antigens when given i.g. alone or coupled to the B subunit of CT suppress immune responses in the phenomenon known as "oral tolerance". The mechanisms by which these two divergent responses are induced are poorly understood, but are critical in the design and development of mucosal vaccines, whether intended for generating protection against infections, or for suppressing undesirable immune responses such as in autoimmune diseases or allergies. The precise tissue sites and characteristics of the cells involved in the uptake of vaccine antigens and adjuvants, as well as the mode of action of the adjuvants in activating antigen-presenting cells, are critical in determining the nature of the ensuing immune response. Limitations in previous technologies for tracking the uptake of antigens and adjuvants in vivo can be overcome by applying the emerging novel ImageStream (Amnis Corp.) technology. In this collaborative project between experts in mucosal immunology and flow and imaging cytometry, the uptake of fluorochrome-labeled mucosal immunogens based on antigen-enterotoxin chimeric proteins, and of enterotoxin adjuvants (or their nontoxic derivatives), will be studied in mice. The Specific Aims are: (1) to determine the cellular pathways of uptake of antigen-enterotoxin chimeric proteins administered i.g. or i.n., and to characterize the type, location, and activation of antigen-presenting cells involved; and (2) to compare the cellular pathways of uptake of the intact enterotoxin adjuvants, their binding- site mutants, and B subunits, in comparison with control proteins, when administered i.g. or i.n. The results are expected to identify the precise sites of uptake of these immunogens and adjuvants, define the characteristics of the cells stimulated by them, and thereby determine the type of immune responses that are induced. Such information will be important for the design of future mucosally applied vaccines. PUBLIC HEALTH RELEVANCE: New generations of vaccines administered by intranasal or intragastric routes are being developed to elicit protection at the mucosal surfaces where most human infections arise, and have the additional advantage of being needle-free. However, the precise tissues and cells that takes up and responds to mucosally administered vaccine antigens and adjuvants are not adequately understood, yet this knowledge is essential for the design of such vaccines whether intended for protection against infections or to suppress undesirable responses in autoimmune diseases or allergies. This proposal will apply novel imaging technology based on the Amnis Image Stream to identify the tissues and cells targeted by mucosal vaccines and adjuvants, and to characterize the antigen-presenting cells that initiate the immune responses to them.