Early in vivo Expressed Antigens and their Role in Virulence, Immune Response, and Vaccines for Coccidioidomycosis

整个截面 早期体内表达的抗原及其在毒力、免疫应答和疫苗中的作用 球孢子菌病 总结 球孢子菌病，也被称为山谷热（VF），是一种重要的真菌疾病，由两种不同的真菌引起。 球孢子菌属的物种，导致区域重要的死亡率和更高的发病率。我们有 组建了一个团队来定义人类和动物对VF的免疫反应中发生的变化，并使用 这些知识用于设计新的疫苗和诊断测试。这项工作将利用我们详细的 在感染的最早阶段观察球孢子菌属的基因表达模式。我们假设 一些球孢子菌早期基因是致病因子，对致病至关重要。研究项目1 将通过使用CRISPR-Cas9技术的基因敲除和在蜡虫中的毒力测试来测试它们的作用 （Galleria）和小鼠VF模型。关键的毒力因子将成为诊断和疫苗的目标。此外 对于蜡虫和小鼠模型，我们开发了一种非人类灵长类动物（猪尾猕猴）， 与人类的VF非常相似在人类和脊椎动物模型中，T细胞的作用不能被解释。 研究项目2将使用集中的深度DNA测序来识别T细胞的类别， 受体（TCR），其响应于早期表达的球孢子菌属基因而发展。我们将产生TCR 来自跨越病原体流行区的三个临床地点的患者的序列。的TCR 将使用来自患者的库来鉴定新的诊断标记（例如，公共TCR），并帮助 确定对可用于疫苗开发的关键抗原的免疫应答。因此，TCR和 早期毒力基因是疫苗设计的最佳候选基因，将在研究中进行探索。 项目3使用基于核酸（NA）的疫苗（RNA和DNA），可以快速检测大量抗原 通过免疫小鼠来抵抗感染。DNA疫苗将以黄金为载体 纳米颗粒和基因枪，而mRNA采用自我复制的RNA分子（repRNA）和脂质 InOrganic Nanoparticle（LION）.这两种技术都是经过验证的技术，正在进入临床试验， 疾病我们在NA疫苗小鼠研究中的目标是确定用于免疫的最佳抗原和递送方式。 在NHP模型中进行疫苗测试并定义其免疫保护机制。这项工作只是 通过七个不同机构的研究者的综合努力，包括三个临床研究中心， 因为没有任何一个机构拥有所需的广泛专门知识和基础设施。当我们将生成 关于球孢子菌和VF的基本知识，我们也将在翻译方面取得进展， 预防和诊断疾病。

OVERALL Section Title: Early in vivo expressed antigens and their role in virulence, immune response, and vaccines for coccidioidomycosis. SUMMARY Coccidioidomycosis, also known as Valley Fever (VF), is an important fungal disease caused by two different Coccidioides species that results in regionally important mortality and even greater morbidity. We have assembled a team to define the changes that occur in human and animal immune responses to VF, and to use this knowledge for designing new vaccines and diagnostic tests. This work will capitalize upon our detailed observations of Coccidioides gene expression patterns during the earliest stages of infections. We hypothesize that some Coccidioides early genes are virulence factors and critical for causing disease. Research Project 1 will test their role through gene knockouts using CRISPR-Cas9 technology and virulence testing in wax worm (Galleria) and mouse VF models. Critical virulence factors will become diagnostic and vaccine targets. In addition to the wax worm and mouse models, we develop a non-human primate (pig-tailed macaques) that will more closely resemble VF in humans. In humans and vertebrate animal models, the role of T cells cannot be overemphasized and Research Project 2 will use focused deep DNA sequencing to identify classes of T cell receptors (TCR) that develop in response to early expressed Coccidioides genes. We will generate TCR sequences from patients at three clinical locations that span the endemic zones for the pathogen. The TCR repertoire from patients will be used to identify novel diagnostic signatures (e.g., public TCRs) and, also, help identify immune responses to key antigens that can be targeted for vaccine development. Hence, both TCR and early virulence genes represent excellent candidates for vaccine design that will be explored in Research Project 3 using nucleic acid (NA) based vaccines (RNA and DNA) that can rapidly test a large panel of antigens through the immunization of mice against infection. The DNA vaccine will be based upon delivery on gold nanoparticles and Gene Gun, while the mRNA employs self-replicating RNA molecules (repRNA) and a Lipid InOrganic Nanoparticle (LION). Both are proven technologies that are moving forward into clinical trials for other diseases. Our goal in the NA vaccine mouse studies is to identify the best antigens and delivery modality for vaccine testing in the NHP model and to define their immune mechanisms of protection. This work is only possible through the integrated efforts of investigators at seven different institutions, including three clinical sites, as no single institution has the requisite breadth of expertise and infrastructure. While we will generate fundamental knowledge about Coccidioides and VF, we will also make translational advances towards preventing and diagnosing the disease.