Early in vivo expressed antigens and their role in virulence, immune response, and vaccines for coccidioidomycosis

早期体内表达的抗原及其在球孢子菌病毒力、免疫反应和疫苗中的作用

• 批准号: 10689675
• 负责人: Bridget Marie Barker
• 金额: $ 27.65万
• 依托单位: NORTHERN ARIZONA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-24 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10689675
• 关键词: Adjuvant; Algorithms; Animal Model; Animals; Antibodies; Antigens; Applied Genetics; Attenuated; Bioinformatics; Biology; CRISPR/Cas technology; Candidate Disease Gene; Cellular Immunity; Centers for Disease Control and Prevention (U.S.); Coccidioides; Coccidioides immitis; Coccidioides posadasii; Coccidioidomycosis; Collaborations; Data; Data Analyses; Development; Diagnostic; Diagnostic tests; Disease; Disease Outcome; Fungal Drug Resistance; Gene Deletion; Gene Expression Profiling; Gene Proteins; Gene Silencing; Gene Targeting; Generations; Genes; Genetic Transcription; Genomics; Goals; Growth; Health; Human; Hypothetical Protein; Immune response; Immunity; Immunization; In Vitro; Incidence; Infection; Investigation; Knock-out; Knowledge; Length of Stay; Life; Lung; Marketing; Methods; Mus; Mycoses; Nucleic Acid Vaccines; Parents; Pathogenesis; Pathogenicity; Patients; Play; Population; Production; Protein Secretion; Proteins; Reaction; Recombinant Proteins; Recombinants; Reporting; Research; Research Project Grants; Research Proposals; Role; Safety; Serology test; Sister; Structure; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Epitopes; Testing; Therapeutic; Transcript; Vaccination; Vaccine Antigen; Vaccine Design; Vaccines; Virulence; Virulence Factors; adaptive immune response; cost; desert fever; diagnostic tool; disorder prevention; gene function; genetic analysis; genetic approach; genome analysis; genome sequencing; genomic data; immunogenic; immunogenicity; improved; in silico; in vivo; insight; nano-string; novel; pathogen; pathogenic fungus; protein complex; response; reverse genetics; side effect; targeted biomarker; tool; transcriptome sequencing; treatment strategy; vaccine candidate; vaccine development; whole genome

Project Summary/Abstract (Research Project 1) Emerging and endemic mycoses have a growing impact on human health on a global scale and little is known about virulence mechanisms for most emerging fungal pathogens. Coccidioides immitis and C. posadasii cause coccidioidomycosis (aka Valley Fever), which is of specific concern due to recent rapid increases in US disease incidence with a more than a 10-fold increase of reports over the last 20 years. The Centers for Dis- ease Control and Prevention (CDC) report that the average cost for treating Valley fever is $50,000 per patient, including extensive hospital stays and treatment regimes. Advanced genomic and genetic analyses of these fungal pathogens can help improve our understanding of mechanisms of pathogenicity, support the identifica- tion of new biomarker targets, and represent the foundational framework that is necessary for development of diagnostic tools, treatment strategies and prevention of disease via vaccination. Currently, few specific viru- lence mechanisms have been investigated in Coccidioides. Complicating these investigations is the lack of in vivo data, as the biology and gene expression analysis of in vitro cultures does not replicate the infective se- quence. We have remedied this gap by detailed transcriptional analysis of both Coccidioides species in mouse lungs five days after infection. Based on our transcriptional analyses and existing genomic data, plus in silico functional predictions, we have identified 26 putative antigens/virulence factors. These Coccidioides spp. tran- scripts are highly expressed under in vivo conditions and are predicted to be small secreted proteins. Our working hypothesis is that these proteins will be antigenic and potentially virulence factors. We will test this hy- pothesis by creating CRISPR-Cas9 gene deletion strains. These strains will be tested for virulence in two ani- mal models in collaboration with the Animal Core. We will further investigate these genes’ functions in both Coccidioides species, specifically the effects on development of the parasitic structure (spherule), virulence, and host and pathogen response in vivo using nanoString. RNAseq analysis of gene deletion strains compared to parent strain will be used to compare transcriptional networks. Analysis of these data will provide new in- sights into gene functions, and effectively double the number of gene deletion strains investigated for this path- ogen, which will increase our understanding of fungal pathogenesis mechanisms. These data will be used by the other two Research Projects to identify T cell epitopes and the cognate T cell receptors for understanding the host immune response and possible advanced diagnostic tests, as well as to identify targets for nucleic acid vaccines. Programming the immune response toward early infection virulence factors has the great poten- tial to provide protective immunity to Valley Fever. In total, this proposal represents a unique pairing of ad- vanced genomic data analysis and functional assessments in an important, yet understudied, fungal pathogen.

项目概要/摘要（研究项目1） 新发和地方性真菌病在全球范围内对人类健康的影响越来越大，但人们对此知之甚少 关于大多数新兴真菌病原体的毒力机制。粗球孢子菌（Coccidioides immitis）和C.波萨达西 引起球孢子菌病（又名山谷热），这是特别关注，由于最近迅速增加，在美国 在过去20年中，报告的发病率增加了10倍以上。该中心为Dis- 疾病控制和预防中心（CDC）报告说，治疗山谷热的平均费用是每名患者5万美元， 包括长期住院和治疗方案。这些基因的先进的基因组和遗传分析 真菌病原体可以帮助我们提高致病机制的理解，支持鉴定， 新的生物标志物靶点，并代表了发展生物标志物所必需的基础框架。 诊断工具、治疗策略和通过接种疫苗预防疾病。目前，很少有特定的维鲁- 在球孢子菌属中已经研究了种子机制。使这些调查复杂化的是， 体内数据，因为体外培养物的生物学和基因表达分析不能复制感染性SE， 顺序。我们通过对小鼠中两种球孢子菌的详细转录分析弥补了这一空白 感染后五天肺部基于我们的转录分析和现有的基因组数据，加上计算机模拟 功能预测，我们已经确定了26个推定的抗原/毒力因子。这些球孢子菌属（Coccidioides spp.）反式 脚本在体内条件下高度表达，并被预测为小的分泌蛋白。我们 工作假设是，这些蛋白质将是抗原性的和潜在的毒力因子。我们会测试这个- 通过创建CRISPR-Cas9基因缺失菌株来进行假设。这些菌株将在两种动物中进行毒力测试。 与Animal Core合作建立的模型。我们将进一步研究这些基因在这两种细胞中的功能， 球孢子菌属物种，特别是对寄生结构（小球）发育的影响，毒力， 以及使用nanoString的体内宿主和病原体响应。基因缺失菌株的RNAseq分析比较 将用于比较转录网络。对这些数据的分析将提供新的... 着眼于基因功能，并有效地增加了基因缺失菌株的数量， 原，这将增加我们对真菌致病机制的理解。这些数据将用于 其他两个研究项目，以确定T细胞表位和同源T细胞受体，以了解 宿主免疫反应和可能的先进诊断测试，以及确定核酸的靶点， 酸性疫苗规划针对早期感染毒力因子的免疫应答具有巨大的潜力， 提供对山谷热的保护性免疫。总的来说，这个提案代表了广告的独特配对- 先进的基因组数据分析和功能评估，在一个重要的，但研究不足，真菌病原体。