Scanning B. pseudomallei proteome for vaccine antigens

描述(申请人提供)：由于潜在的生物恐怖主义威胁，开发安全有效的假鼻疽杆菌疫苗是一个全国性和世界性的目标。加州大学欧文分校的应用蛋白质组学实验室开发了一种称为PCR Express的高通量蛋白质表达系统，该系统可用于从任何已测序的微生物中快速生成完整的蛋白质组，包括A、B和C类生物恐怖分子。这项技术允许数百种不同的基因以每周数百种不同蛋白质的速度从他们的PCR产物中直接表达出来。在这里，将产生假鼻疽伯克霍尔德氏菌蛋白质组，并将其应用于识别B细胞和T细胞识别的抗原的问题，这将在针对该病毒的亚单位疫苗中有用。第一种形式的蛋白质组将在微阵列芯片上，该芯片将用于定量BALB/c小鼠(2D2)和泰国东北部流行区自然暴露于假鼻疽杆菌的人对每一种细菌蛋白的抗体滴度。对于第二种蛋白质组形式，每个单独的蛋白质都将被提纯，并以一种形式呈现，使它们能够用于体外T细胞再刺激试验，使用来自2D2疫苗接种的小鼠的脾细胞或来自暴露的人类人群的全血。用ELISA法测定T细胞的活化，以检测LFN？的分泌。通过疫苗抗原扫描确定的反应抗原将在假鼻疽杆菌感染的小鼠模型中进行有效性测试，并且由于假鼻疽杆菌和马来杆菌之间广泛的遗传相似性，将在腺体动物模型中测试它们的有效性。这一定量体液和细胞免疫反应扫描将在人类和实验动物中产生第一个针对假鼻疽杆菌的完整免疫反应谱，并将确定最有效的候选抗原，用于开发针对人类类鼻疽病和腺体的DNA或亚单位疫苗。

DESCRIPTION (provided by applicant): Because of the potential bioterrorism threat, the development of a safe and effective B. pseudomallei vaccine is a national and worldwide goal. The Applied Proteomics Laboratory at UC Irvine has developed a high throughput protein expression system called PCR Express which can be used to rapidly generate complete proteomes from any sequenced microorganism, including the Class A, B & C Bioterrorism Agents. The technology allows hundreds of different genes to be expressed directly from their PCR products at the rate of hundreds of different proteins per week. Here the Burkholderia pseudomallei proteome will be generated and applied to the problem of identifying antigens recognized by B-cells and T-cells that will be useful in a subunit vaccine against the agent. The first form of the proteome will be on microarray chips which will be used to quantify serum antibody titers from BALB/c mice vaccinated with a rationally attenuated auxotroph of B. pseudomallei (2D2) and humans naturally exposed to B. pseudomallei in endemic regions of NE Thailand against each of the individual bacterial proteins. For the second proteome format, each individual protein will be purified and presented in a form that will enable them to be used in T cell restimulation assays in vitro using either spleen cells from 2D2 vaccinated mice or whole blood from exposed human populations. T cell activation will be determined by ELISA assay for secretion of lFN?. The responsive antigens identified by this Vaccine Antigen Scan will be tested for their efficacy in a murine model of infection with B. pseudomallei and, because of the extensive genetic similarity between B. pseudomallei and B. mallei, in animal models of glanders. This quantitative humoral and cellular immune response scan will produce the first complete profile of the immune response against B. pseudomallei in humans and experimental animals and will identify the most effective candidate antigens for development of a DNA or subunit vaccine against human melioidosis and glanders.