Novel Recombinant Vaccines to Protect Against Burkholderia Infections

描述（由申请人提供）：导致人类疾病的伯克氏菌包括洋葱伯克氏菌复合体（Bcc），可引起囊性纤维化患者的急性和慢性感染，以及马氏伯克氏菌和假马利氏伯克氏菌，这是B类选择的生物威胁剂，分别负责腺体病和meliodiosis。Bcc被认为是影响免疫功能低下个体的机会性病原体，而mallei B.和pseudomallei B.是剧毒生物，如果故意释放，将构成严重的健康威胁；目前还没有针对这些细菌的批准疫苗。我们的长期目标是开发针对这些传染性细菌病原体的有效疫苗。在这项为期2年的探索性资助中，将作为模型疫苗靶点的抗原是由一种流行的Bcc菌株合成的脂多糖的O抗原部分，以及测序的B. mallei和B. pseudomallei菌株。我们的基本原理是，这些碳水化合物抗原是细菌细胞的最外层成分，可能是免疫优势保护性抗原。我们将在沙门氏菌减毒株的表面表达这些抗原，并将这些疫苗传递给小鼠。该系统应允许这些O抗原处于更“原生确认”状态，并排除将它们与蛋白质载体结合以引起功能性免疫反应的需要。这种方法的另一个优点是，这些疫苗易于口服或鼻内注射，以非侵入性，无针的方式。我们将编码这些O抗原合成酶的基因转移并表达到沙门氏菌的减毒菌株中。一旦O抗原在这个异源宿主（Specific Aim 1）中被证实表达，这些重组菌株将通过口服或鼻内途径接种小鼠。在产生免疫应答后（特异性目的2），这些小鼠将使用每种病原体的标准感染模型进行挑战，以确定这些疫苗的保护功效。对于Bcc，我们将使用琼脂球模型，对于mallei B.和pseudomallei B.，我们将通过鼻内途径感染小鼠（Specific Aim 3）。我们假设免疫小鼠将在细菌负担方面受到保护，当这种情况发生时，显示出死亡时间增加，与未免疫小鼠相比，总体死亡率更低。如果一种免疫途径更有效，未来的实验将确定如何最好地优化这种疫苗方法，以引发保护性免疫反应，并产生被动免疫治疗试剂。我们的长期目标是利用这些知识和技术生产供人类使用的有效疫苗。这项为期两年的探索性拨款的重点是开发针对引起人类感染的伯克霍尔德菌的疫苗。特别地，我们建议在减毒沙门氏菌上表达一种流行的囊性纤维化伯克氏菌O抗原，这是一种新出现在囊性纤维化患者中的病原体，以及mallei伯克氏菌和假马利氏伯克氏菌的生物威胁因子。这些结构将被用于小鼠，并在相关的小鼠感染模型中测试它们的功效。我们的长期目标是开发人类使用的类似试剂。

DESCRIPTION (provided by applicant): Members of the Burkholderia species that cause diseases in humans include the Burkholderia cepacia complex (Bcc), which can cause acute and chronic infections in cystic fibrosis patients, and Burkholderia mallei and Burkholderia pseudomallei, which are category B select biothreat agents that are responsible for glanders and meliodiosis, respectively. Bcc are considered opportunistic pathogens affecting immunocompromised individuals, while B. mallei and B. pseudomallei are highly virulent organisms, and would pose serious health threats if intentionally released; there are currently no approved vaccines available for any of these bacteria. Our long-term objective is to develop effective vaccines for these infectious bacterial pathogens. The antigens that will serve as model vaccine targets in this 2-year exploratory grant are the O antigen portion of lipopolysaccharide synthesized by an epidemic strain of Bcc, and the sequenced B. mallei and B. pseudomallei strains. Our rationale is that these carbohydrate antigens are the outermost components of bacterial cells and may be immunodominant protective antigens. We will express these antigens on the surface of attenuated strains of Salmonella and deliver these vaccines to mice. This system should allow these O antigens to be in a more "native confirmation" as well as preclude the need to conjugate them to protein carriers in order to elicit a functional immune response. An additional advantage of this approach is that these vaccines are easy to administer, either orally or intranasally, in a non-invasive, needle-free manner. We will transfer and express the genes encoding the enzymes for the synthesis of these O antigens to attenuated Salmonella strains. Once O antigen expression is confirmed in this heterologous host (Specific Aim 1), these recombinant strains will be used to vaccinate mice by either the oral or intranasal route. After an immune response is generated (Specific Aim 2), these mice will be challenged using standard infection models for each pathogen to determine the protective efficacy of these vaccinations. For Bcc, we will use the agar bead model, and for B. mallei and B. pseudomallei we will infect the mice via the intranasal route (Specific Aim 3). We hypothesize that immunized mice will be protected with regard to bacterial burden, show increased time to death when this occurs, and less overall mortality compared to non-immune mice. If one route of immunization is more efficacious, future experiments will define how best to optimize this vaccine approach to elicit a protective immune response as well as to generate passive immunotherapeutic reagents. Our long- term goal is to exploit this knowledge and technology to produce effective vaccines for human use. The focus of this two-year exploratory grant is on the development of vaccines against the species of Burkholderia that cause infections in humans. In particular, we propose to express O antigens from an epidemic strain of Burkholderia cenocepacia, which is an emerging pathogen in cystic fibrosis patients, and the biothreat agents Burkholderia mallei and Burkholderia pseudomallei, on attenuated Salmonella strains. These constructs will be administered to mice and their efficacy tested in relevant mouse models of infection. Our long-term goal is to develop similar reagents for human use.