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

总体科 标题：早期体内表达的抗原及其在毒力、免疫反应和疫苗中的作用 球孢子菌病。 摘要 球孢子菌病，又称谷热，是由两种不同的真菌引起的一种重要的真菌病 球孢子虫物种，导致区域重要的死亡和甚至更高的发病率。我们有 组建了一个团队，以确定人类和动物对VF的免疫反应发生的变化，并使用 这些知识有助于设计新的疫苗和诊断测试。这项工作将充分利用我们详细的 球虫感染早期阶段基因表达模式的观察。我们假设 一些球虫早期基因是毒力因子，对致病至关重要。研究项目1 将通过使用CRISPR-Cas9技术的基因敲除和对蜡虫的毒力测试来测试它们的作用 (Galleria)和鼠标VF模型。关键的毒力因子将成为诊断和疫苗的目标。此外 对于蜡虫和老鼠的模型，我们开发了一种非人类灵长类动物(猪尾猕猴)，它会比 与人类的室颤非常相似。在人类和脊椎动物模型中，T细胞的作用不能 过度强调和研究项目2将使用聚焦的深度DNA测序来识别T细胞的类别 对早期表达的球虫基因作出反应而产生的受体(TCR)。我们将生成TCR 来自三个临床地点的患者的序列，这些序列跨越了病原体的流行区。TCR 来自患者的曲目将被用来识别新的诊断特征(例如，公共TCR)，还将帮助 确定针对关键抗原的免疫反应，这些免疫反应可作为疫苗开发的靶点。因此，TCR和 早期毒力基因是疫苗设计的极佳候选者，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)，可以快速测试大量抗原 通过免疫使小鼠免受感染。DNA疫苗将以黄金为基础交付 纳米粒子和基因枪，而信使核糖核酸使用自我复制的RNA分子(EpRNA)和脂类 无机纳米粒子(LIME)。这两种技术都是经过验证的技术，正在进入其他 疾病。我们在NA疫苗小鼠研究中的目标是确定最好的抗原和注射方式 在NHP模型中进行疫苗测试，并确定其免疫保护机制。这项工作只是 通过七个不同机构的调查人员的综合努力，包括三个临床地点， 因为没有一家机构拥有必要的专业知识和基础设施。而我们将会产生 关于球虫和VF的基础知识，我们还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.