Novel Recombinant Vaccines to Protect Against Burkholderia Infections

描述(申请人提供)：在人类中引起疾病的伯克霍尔德氏菌的成员包括洋葱伯克霍尔德氏菌复合体(BCC)，它可以导致囊性纤维化患者的急性和慢性感染，以及伯克霍尔德氏菌和假腮腺伯克霍尔德氏菌，它们是B类精选生物硫尿剂，分别导致腺体和甲状腺疾病。BCC被认为是影响免疫受损个体的机会性病原体，而马来杆菌和假鼻疽杆菌是高度毒力的有机体，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。在这项为期两年的探索性拨款中，将作为模式疫苗靶标的抗原是BCC流行株合成的脂多糖的O抗原部分，以及测序的马来和假鼻炎杆菌株。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。我们将在减毒沙门氏菌的表面表达这些抗原，并将这些疫苗传递给小鼠。这个系统应该允许这些O抗原处于更“天然的确认”状态，并排除了将它们连接到蛋白质载体以引起功能性免疫反应的需要。这种方法的另一个优点是，这些疫苗很容易以非侵入性、无针头的方式经口或鼻腔给药。我们将编码合成这些O抗原的酶的基因转移并表达到减毒沙门氏菌菌株中。一旦证实O抗原在这个异源宿主中表达(特异性目标1)，这些重组菌株将被用来通过口服或鼻腔途径接种小鼠。在产生免疫反应(特定目标2)后，这些小鼠将接受针对每种病原体的标准感染模型的挑战，以确定这些疫苗的保护效果。对于基底细胞癌，我们将使用琼脂微珠模型，而对于马来杆菌和假鼻疽杆菌，我们将通过鼻腔途径感染小鼠(特定目标3)。我们假设，与非免疫小鼠相比，免疫小鼠将在细菌负担方面受到保护，表现出更长的死亡时间，总体死亡率更低。如果有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，以诱导保护性免疫反应，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出有效的人类使用的疫苗。这笔为期两年的探索性赠款的重点是开发针对导致人类感染的伯克霍尔德氏菌的疫苗。特别是，我们建议在减毒沙门氏菌菌株上表达来自囊性纤维化患者的新病原体-盲肠伯克霍尔德氏菌流行株的O抗原，以及生物调节剂伯克霍尔德氏菌和假鼻疽伯克霍尔德氏菌的O抗原。这些构建物将被应用于小鼠，并在相关的小鼠感染模型中测试它们的有效性。我们的长期目标是开发类似的试剂供人类使用。

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.