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。本建议的目的是探索增强保护抗原（PA）的免疫原性，PA是目前可用的炭疽疫苗的主要抗原。PA是三方炭疽毒素复合物的核心成分，负责与细胞结合并将致死因子（LF）和水肿因子（EF）运送到发生中毒的细胞质中。这一途径可能是PA靶向递送LF/EF杀死宿主的最佳途径，同时最大限度地减少对宿主免疫的诱导。基于这一假设，我们假设PA的免疫原性可以通过工程蛋白来增强，这样它不仅可以阻断中毒步骤，还可以改变其细胞运输。这种修饰的PA有望具有更好的免疫原性，并可能诱导大量的保护性抗体。为了支持这一假设，我们最近证明了显性阴性抑制（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PA突变体的免疫原性（目的1）。这些突变体，每个都在PA介导的中毒的不同步骤中受损，为探测细胞和生化途径的序列提供了有价值的分子工具，这些途径可能导致突变体与天然PA的免疫原性增强。为了剖析PA免疫原性的基本机制并解释对DNI增强的抗体反应，我们最近提出，DNI在插入内体膜时存在缺陷，不会使LF/EF易位，可能仍被困在内体中，因此抗原呈递细胞比天然PA更有效地处理。为了通过实验验证这种“内体捕获”假说，我们将研究DNI是否会增强共递送抗原（如LF、LFn-GFP和LFn-PGA）的免疫原性，因为DNI预计也会在内体中捕获这些抗原（目的2）。此外，我们将更详细地研究PA和DNI的细胞命运（目的3）。这项研究不仅揭示了PA免疫原性的基本机制和炭疽中毒的基本生物学，而且还将产生更有效和更安全的炭疽候选疫苗，具有重要的意义。

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.