Designing Potent Anthrax Vaccine with Engineered Protective Antigen

描述(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。这项建议的目的是探索增强保护性抗原(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体的中心成分，负责与细胞结合，并将致死因子(LF)和水肿因子(EF)运输到发生中毒的细胞质中。这条途径可能已经进化为PA靶向传递LF/EF以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对蛋白质进行改造来增强，从而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和四种不同类型的PA突变体的免疫原性(目标1)。这些突变体分别在PA介导的中毒过程中的不同步骤受到损害，为探索可能导致突变体与天然PA免疫原性增强的细胞和生化途径序列提供了有价值的分子工具。为了分析PA免疫原性的基本机制和解释对DNI的增强抗体反应，我们最近提出了DNI可能仍然被困在内吞体内，因此被抗原提呈细胞比天然PA更有效地处理。为了从实验上验证这一“内体捕获”假说，我们将研究DNI是否能增强联合传递的抗原，如LF、LFN-GFP和LFN-PGA的免疫原性，因为DNI预计也会将这些抗原捕获在内体内(目标2)。此外，我们将更详细地研究PA和DNI的细胞命运(目标3)。这项研究具有非常重要的意义，因为它不仅将阐明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疫苗候选。

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.