Mechanisms of vaccine immunity against coccidioidomycosis

摘要 球孢子菌病是一种重新出现的感染，NIH已将其列为疫苗预防的优先事项。试验性的 减毒孢子活疫苗鼻腔注射后具有很强的保护性。我们建议研究这种疫苗 明确肺上皮细胞调节持久粘膜T细胞免疫的机制。抵抗 吸入的微生物被认为存在于组织驻留记忆(TRM)细胞中，但人们对此知之甚少 鼻腔疫苗诱导肺TRM。我们的初步数据显示，疫苗会引起保护性球虫 内切葡聚糖酶2(C-Eng2)特异的CD4T细胞及细支气管球细胞和钙钙调神经磷酸酶信号转导通路 这些细胞需要动员炎症细胞和T细胞来应对疫苗。我们还发现微折(M) 细胞从细支气管杆细胞中下来，促进T细胞对疫苗的反应。 根据这些初步数据，我们假设细支气管球细胞和M细胞调节粘膜 鼻腔疫苗的细胞免疫应答。为了验证这一假设，我们创造了创新的工具：(I) 转基因小鼠消耗上皮细胞亚群或其产物，以进一步确定它们在诱导免疫中的作用； (Ii)C-Eng2特异性四聚体，用于跟踪和分析C57BL6小鼠保护性的CD4T细胞和TRM； 方法分离培养人肺上皮细胞，将小鼠肺上皮细胞结果转化为人肺上皮细胞。我们建议 三个目的是验证我们的假设。在目标1中，我们将阐明炎症反应的早期阶段 由细支气管球细胞和M细胞调节的鼻腔疫苗；在目标2中，我们将鉴定肺上皮细胞 受体-Dectin-1，DUOX1和DUOXA1-及其下游的PLCG2可能感知鼻腔疫苗和 通过钙和钙调神经磷酸酶信号来动员粘膜免疫；在目标3中，我们将定义 通过研究肺TRM和肺上皮的调节作用，疫苗诱导持久的粘膜免疫。 总之，我们解决了球孢子菌病疫苗接种的未得到满足的需求。我们的工作意义重大，因为 它将确定一种很有前途的疫苗在肺粘膜建立T细胞免疫的机制。结果 将确定对其他疫苗免疫原鼻腔注射有用的策略，包括亚单位疫苗。这项工作 将确定推进这种减毒疫苗或下一代亚单位疫苗所需的免疫相关因素 对抗这种高度优先的病原体。这项工作将使用最先进的尖端工具来完成。我们的团队 PI和Co-I‘s将允许我们利用工具和试剂将结果从小鼠转化为人类，用于人类肺上皮细胞。

ABSTRACT Coccidioidomycosis is a re-emerging infection that NIH has prioritized for vaccine prevention. An experimental vaccine of live attenuated spores is highly protective after intranasal delivery. We propose to study this vaccine to define mechanisms by which lung epithelium regulates durable mucosal T cell immunity. Resistance against inhaled microbes is thought to reside within tissue-resident memory (TRM) cells, but little is known about how this intranasal vaccine induces lung TRM. Our preliminary data reveal that the vaccine elicits protective, Coccidioides endoglucanase 2 (C-Eng2) specific CD4+ T cells and that bronchiolar club cells and Ca++ calcineurin signaling in the cells are needed to mobilize inflammatory and T cells in response to vaccine. We also find that Microfold (M) cells descend from the bronchiolar club cells and facilitate T cell priming in response to the vaccine. From these preliminary data, we hypothesize that bronchiolar club cells and M cells regulate mucosal cellular immunity in response to intranasal vaccine. To test this hypothesis, we have created innovative tools: (i) transgenic mice to deplete epithelial cell subsets or their products to further define their role in inducing immunity; (ii) C-Eng2 specific tetramers to track and analyze protective CD4+ T cells and TRM in C57BL6 mice; and (iii) methods to isolate and culture human lung epithelial cells to translate results from mice to humans. We propose three aims to test our hypothesis. In Aim 1, we will elucidate early stages of the inflammatory response to intranasal vaccine regulated by bronchiolar club cells and M cells; in Aim 2, we will identify lung epithelial cell receptors - dectin-1, DUOX1 and DUOXA1 - and downstream PLCG2 that may sense intranasal vaccine and signal via Ca++ and calcineurin to mobilize mucosal immunity; and in Aim 3, we will define mechanisms of vaccine-induced durable mucosal immunity by studying lung TRM and the regulatory role of lung epithelium. In sum, we address the unmet need of vaccination against coccidioidomycosis. Our work is significant as it will define mechanisms by which a promising vaccine establishes T cell immunity at the lung mucosa. Results will identify tactics useful for other vaccine immunogens given intranasally, including subunit vaccines. The work will define correlates of immunity needed to advance this attenuated vaccine or next generation subunit vaccines against this high priority pathogen. The work will be done with state-of-the-art, cutting-edge tools. Our team of PI and Co-I’s will let us translate results from mouse to human, with tools and reagents for human lung epithelium.