Designing Potent Anthrax Vaccine with Engineered Protective Antigen

描述（由申请人提供）：我们研究的长期目标是开发有效和安全的炭疽疫苗，并了解炭疽毒性的基本机制和免疫系统对炭疽感染的反应。本研究的目的是探索提高现有炭疽疫苗的主要抗原--保护性抗原（PA）的免疫原性。PA是三重炭疽毒素复合物的中心成分，负责结合细胞并将致死因子（LF）和水肿因子（EF）转运到发生中毒的细胞溶质中。该途径可能已经进化为最佳的，用于通过PA靶向递送LF/EF以杀死宿主，同时最小化宿主免疫的诱导。基于这一假设，我们假设PA的免疫原性可以通过工程改造蛋白质来增强，这样它不仅可以阻断中毒步骤，而且还可以改变其细胞运输。预期这种修饰的PA具有大大改善的免疫原性，并且可以诱导显著量的保护性抗体。为了支持这一假设，我们最近证明了显性负抑制（DNI）突变体在诱导保护性抗体方面比PA更有效。在本研究中，我们将比较天然PA和四种不同类型的PA突变体（目的1）的免疫原性。这些突变体各自在PA介导的中毒的不同步骤中受损，为探测可能导致突变体相对于天然PA的免疫原性增强的细胞和生物化学途径的序列提供了有价值的分子工具。为了剖析PA免疫原性的基本机制，并解释增强的抗体反应DNI，我们最近提出，DNI，这是有缺陷的插入到内体膜，并不移位LF/EF，可能仍然被困内体，因此，更有效地处理比原生PA的抗原呈递细胞。为了实验性地测试这种“内体捕获”假设，我们将研究DNI是否增强共递送的抗原如LF、LFn-GFP和LFn-PGA的免疫原性，因为DNI预期也将这些抗原捕获在内体内（目的2）。此外，我们将更详细地检查PA和DNI的细胞命运（目的3）。本研究不仅有助于阐明PA免疫原性的基本机制和炭疽中毒的生物学基础，而且有助于获得更有效、更安全的炭疽疫苗候选物。

DESCRIPTION (provided by applicant): The long-term goal of our research is to develop potent and safe anthrax vaccines and to understand the fundamental mechanisms of anthrax toxicity and the response of the immune system to anthrax infection. The aim of this proposal is to explore enhancement of the immunogenicity of protective antigen (PA), the major antigen of currently available anthrax vaccine. PA is the central component of the tripartite anthrax toxin complex, responsible for binding to cells and transporting lethal factor (LF) and edema factor (EF) into the cytosol where intoxication occurs. This pathway has presumably evolved to be optimal for the targeted delivery of LF/EF by PA to kill the host while minimizing the induction of host immunity. Based on this assumption, we hypothesize that the immunogenicity of PA can be augmented by engineering the protein such that it will not only block intoxication steps but also change its cellular trafficking. Such modified PA would be expected to possess much improved immunogenicity and may induce significant amounts of protective antibodies. In support of this hypothesis, we recently demonstrated that a dominant-negative inhibitory (DNI) mutant is more potent than PA in inducing protective antibodies. In the present study, we will compare the immunogenicities of native PA and four distinct classes of PA mutants (Aim 1). These mutants, each being impaired in a different step of PA-mediated intoxication, provide valuable molecular tools for probing the sequence of cellular and biochemical pathways that may lead to enhanced immunogenicity of mutant vs. native PA. To dissect the basic mechanisms of PA immunogenicity and to explain the enhanced antibody response to DNI, we recently proposed that DNI, which is defective in inserting into endosomal membranes and does not translocated LF/EF, may remain trapped endosomally and is, therefore, processed more efficiently by antigen-presenting cells than native PA. To experimentally test this "endosomal trapping" hypothesis, we will investigate whether DNI enhances the immunogenicity of co-delivered antigens such as LF, LFn-GFP, and LFn-PGA since DNI is expected to also trap these antigens within endosomes (Aim 2). Furthermore, we will examine the cellular fates of PA and DNI in greater detail (Aim 3). This study is highly significant in that it will not only shed light on the basic mechanisms of PA immunogenicity and the basic biology of anthrax intoxication but it will also yield more potent and safer anthrax vaccine candidates.