Project #3:Active Vaccination and Immunotherapy Against Coccidioidomycosis

项目摘要 据估计，美国每年有35万人感染球孢子菌。有一个 开发更好的化学疗法和主动免疫和/或免疫疗法疫苗的需求未得到满足 抗球孢子菌病（CM）。我们先前已经创建了一个重组嵌合多肽抗原 （rCpa 1）由3种先前鉴定的球孢子菌属抗原的最具免疫原性的片段和5种 人T细胞表位。将细菌表达的rCpa 1加载到酵母葡聚糖-几丁质颗粒（GCPs）中， 构建GCP-rCpa 1蛋白疫苗。后者刺激强有力的Th 1和Th 17应答，并赋予 保护表达人MHCII的HLA-DR 4转基因小鼠免受致命的肺部感染 这两种球孢子菌。将蛋白质疫苗推向临床准备阶段的挑战是生产 rCpa 1抗原的高数量和质量，并验证人对疫苗的免疫应答。在这 项目，我们建议创建两种类型的配方方法，使用mRNA和基于载体的交付， 辅助系统疫苗受体将在体内表达球孢子虫抗原以引发其自身免疫 免疫系统我们的中心假设是，使用mRNA或载体的优化的rCpa-1疫苗 递送/佐剂平台将刺激针对CM的稳健的Thl和Thl 7免疫。这两个交付 平台可以很容易地扩大规模，以生产用于临床前和临床测试的安全疫苗。GCP-rCpa1 蛋白疫苗将用作比较的基准。有三个具体目标。目标1：设计 用于编码球孢子菌rCpa 1抗原的mRNA的体外转录的DNA构建体。IVT-mRNA将 将其装载到GCP中以产生GCP-rCpa 1-mRNA疫苗。第二种方法将创建一个基于矢量的 使用新杀弗朗西丝氏菌突变体（Fn-iglD）的疫苗。野生型Fn很少引起兔热病，而F. tularensis (Ft)是剧毒的Fn-iglD是高度减毒的，它保护大鼠和猕猴免受致命的 肺部感染Fn-iglD还引发混合的Th 1/Th 17免疫，并且其目前正在配制用于 临床安全性试验。我们将制造Fn-iglD-rCpa 1疫苗。这些平台的抗原表达水平 以及它们在人单核THP-1细胞和鼠巨噬细胞中刺激细胞因子产生的能力 将被确定。目的2：新创建的疫苗既显示良好的抗原表达，又能够 将进一步评估诱导细胞因子产生的功效、免疫相关性和保护性。 机械我们将应用HLA-DR 4小鼠CM模型和人血细胞来描述免疫反应。 相关和保护机制，使用全球免疫基因分析方法，并证实与 我们实验室的免疫学方法。目的3：建立CM小鼠免疫治疗模型， 评估最具保护性的疫苗单独或与唑类或新的抗病毒药物联合使用的治疗效果， 在项目#1和项目#2中确定了药物。

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.