Imaging antigen and adjuvant uptake for enhancing response to mucosal vaccines

描述（由申请人提供）：由于大多数人类感染通过口胃肠道、呼吸道和泌尿生殖道的粘膜表面感染或进入，未来在这些部位产生保护作用的疫苗可能会通过粘膜途径给药，如灌胃（例如）或鼻内（例如）。在众多旨在增强粘膜疫苗应答的策略中，细菌热不稳定肠毒素，如霍乱毒素（CT）和大肠杆菌不稳定毒素（LT-I， LT-IIa和LT-IIb）已被开发为强大的免疫刺激佐剂和与疫苗抗原偶联的载体分子。然而，目前尚不清楚哪些组织和细胞是这些佐剂和免疫原靶向的，以及它们如何和在何处被加工并呈现给T细胞以启动免疫反应。当某些抗原单独或与CT的B亚基结合时，会抑制免疫反应，这种现象被称为“口服耐受”。诱导这两种不同反应的机制尚不清楚，但对于粘膜疫苗的设计和开发至关重要，无论是用于产生抗感染的保护，还是用于抑制自身免疫性疾病或过敏等不良免疫反应。参与疫苗抗原和佐剂摄取的细胞的精确组织部位和特征，以及佐剂在激活抗原呈递细胞中的作用方式，对于确定随后的免疫反应的性质至关重要。通过应用新兴的ImageStream （Amnis公司）技术，可以克服以前在体内追踪抗原和佐剂摄取技术的局限性。在这个由粘膜免疫学、流式细胞术和成像细胞术专家合作的项目中，将在小鼠身上研究基于抗原-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)确定抗原-肠毒素嵌合蛋白的细胞摄取途径，并确定所涉及的抗原呈递细胞的类型、位置和活化；(2)比较完整肠毒素佐剂、其结合位点突变体和B亚基的细胞摄取途径，与对照蛋白相比，在i.g.或i.g.给药时。这些结果有望确定这些免疫原和佐剂摄取的精确位置，定义受其刺激的细胞的特征，从而确定诱导的免疫反应的类型。这些信息对于设计未来的粘膜应用疫苗将是重要的。

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.