Project #3:Active Vaccination and Immunotherapy Against Coccidioidomycosis

项目摘要 据估计，美国每年有35万人感染球虫。有一个 开发更好的化疗和主动免疫和/或免疫疗法疫苗的需求未得到满足 抗球孢子菌病(CM)。我们之前已经创造了一种重组嵌合多肽抗原 (RCpa1)由先前鉴定的3个球虫抗原和5个球虫抗原的免疫原性最强的片段组成 人类T细胞表位。细菌表达的rCpa1被装载到酵母葡聚糖-甲壳素颗粒(GCPs)中，以 研制GCP-rCpa1蛋白疫苗。后者刺激强大的Th1和Th17反应，并赋予 人MHC II基因表达的人类白细胞抗原-DR4转基因小鼠对致死性肺部感染的保护作用 球孢菌科的两个种。将蛋白质疫苗推进到临床准备就绪的挑战是生产 高数量和高质量的rCpa1抗原，并验证人类对疫苗的免疫反应。在这 项目，我们建议创建两种类型的表达方法，使用mRNA和基于载体的传递和 佐剂系统。疫苗接受者将在体内表达球虫抗原，为自己的疫苗做好准备 免疫系统。我们的中心假设是，使用mRNA或载体的优化的基于RCPA-1的疫苗 给药/佐剂平台将刺激针对CM的强大的Th1和Th17免疫。这两种送货方式 平台可以很容易地扩大规模，生产用于临床前和临床测试的安全疫苗。GCP-rCpa1 蛋白质疫苗将被用作比较的基准。有三个具体目标。目标1：我们将设计 用于编码球虫rCpa1抗原的mRNAs体外转录的DNA构建。IVT-mRNA将 将其装载到GCP中，以产生GCP-rCpa1-mRNA疫苗。第二种方法将创建基于向量的 使用新城疫杆菌突变株(FN-iglD)的疫苗。野生型FN很少引起图拉热症，而图拉氏丝虫 (FT)具有很强的毒性。FN-iglD是高度减弱的，它保护老鼠和猕猴免受致命的 肺部Ft感染。FN-IgD也能诱导Th1/Th17混合免疫，目前正在研制中 临床安全性试验。我们将创造一种FN-iglD-rCpa1疫苗。这些平台的抗原表达水平 以及它们刺激人单核细胞THP-1细胞和小鼠巨噬细胞产生细胞因子的能力 将会被确定。目标2：新发明的疫苗既有良好的抗原表达，又有能力 诱导细胞因子的产生将进一步评估它们的有效性、免疫相关性和保护性 机械论。我们将应用人类白细胞抗原-DR4的CM小鼠模型和人类血细胞来描绘免疫 使用全球免疫基因图谱方法的相关性和保护机制，并通过 我们实验室里不合逻辑的方法。目的：建立慢性粒细胞白血病小鼠免疫治疗模型。 评价最具保护性的疫苗单独或与唑类或新城疫联合使用的疗效 确定了项目1和项目2中的药物。

Project Summary An estimated 350,000 people in the United States become infected with Coccidioides annually. There is an unmet need to develop better chemotherapies and a vaccine for active immunization and/or immunotherapy against coccidioidomycosis (CM). We have previously created a recombinant chimeric polypeptide antigen (rCpa1) composed of the most immunogenic fragments of 3 previously identified Coccidioides antigens and 5 human T-cell epitopes. The bacterial-expressed rCpa1 is loaded into yeast glucan-chitin particles (GCPs) to create GCP-rCpa1 protein vaccine. The latter stimulates a robust Th1 and Th17 response and confers protection for human MHC II-expressing HLA-DR4 transgenic mice against a lethal pulmonary infection with both species of Coccidioides. The challenges to advance a protein vaccine to clinical readiness are to produce high quantity and quality of the rCpa1 antigen and to validate human immune response to the vaccine. In this project, we propose to create two types of formulation methods using mRNA and vector-based delivery and adjuvant systems. The vaccine recepients will express the coccidioidal antigen in vivo to prime their own immune system. Our central hypothesis is that an optimized rCpa-1-based vaccine using an mRNA or vector delivery/adjuvant platform will stimulate robust Th1 and Th17 immunity against CM. These two delivery platforms can be easily scaled up to produce safe vaccines for preclinical and clinical testing. The GCP-rCpa1 protein vaccine will be used as a benchmark for comparison. There are 3 specific aims. Aim 1: We will design DNA constructs for in vitro transcription of mRNAs that encode Coccidioides rCpa1 antigen. IVT-mRNAs will be loaded into GCPs to create GCP-rCpa1-mRNA vaccines. The second approach will create a vector-based vaccine using a Francisella novicida mutant (Fn-iglD). Wild typle Fn rarely causes tularemia, while F. tularensis (Ft) is highly virulent. Fn-iglD is highly attenuated and it protects both rats and macaques againt a lethal pulmonary Ft infection. Fn-iglD also elictis a mixed Th1/Th17 immunity and it is currently under formulation for clinical safety trial. We will create a Fn-iglD-rCpa1 vaccine. The antigen expression levels of these platforms and their capacity to stimulate cytokine production in human monoctyic THP-1 cells and murine macrophages will be determined. Aim 2: The newly created vaccines that show both good antigen expression and capable of eliciting cytokine production will be further evaluated for their efficacy, immune correlates and protective mecahnisms. We will apply the HLA-DR4 murine model of CM and human blood cells to delineate immune correlates and protective mechanisms using a global immune gene profiling method and confirmed with immiunlogical methods in our laboratories. Aim3: We will establish a murine immunotherapy model of CM for evaluating treatment efficacy of the most protective vaccine alone or in combination with an azole or the newly identified drugs in Project #1 and Project #2.