Criteria-Directed Vaccine Generation Via Ag Mimicry, Adjuvancy, And APC-Targeting

描述(由申请人提供)：建立一个有效和统一的疫苗开发战略，如我们在这里提出的，是征服当前和新出现的传染病的关键。尽管针对一系列细菌剂取得了成功，但目前疫苗开发的方法与它们所针对的微生物一样多样，需要的佐剂往往效果有限和/或毒副作用。因此，在许多高优先级病原体的情况下，疫苗发现通常是缓慢、低效和不成功的。我们提出/假设细菌病原体的疫苗生成可以通过一种高度创新的以标准为导向的方法进行统一和分流，该方法最大限度地利用了疫苗成功的三个最重要的标准：抗原(Ag)拟态、佐剂和有效靶向抗原提呈细胞(APC)的免疫原。我们将在一个非常严格的模型(感染人类毒力的图拉氏弗朗西斯杆菌)的背景下测试我们的假设 [FT])，我们在无佐剂粘膜疫苗(对C57BL/6小鼠的保护率高达75%)方面取得了非常令人鼓舞的结果。事实上，这是迄今为止证明灭活的Ft鼻腔给药能保护C57BL/6小鼠免受高毒力A类Ft SchuS4病原体黏膜攻击的唯一实例。因此，我们将使用Ft SchuS4作为模式病原体来建立一种以标准为导向的疫苗开发方法，我们坚信这将导致针对Ft SchuS4的完全保护性、佐剂无关的黏膜疫苗。在目标1中，我们将利用Ft SchuS4的差异培养和遗传操作来调节：1)体外培养的Ft免疫原与复制的感染性病原体之间的抗原相似性(宿主适应)，2)通过过表达TLR2激动剂来调节免疫原的细胞刺激能力，以及3)通过APC(B细胞和滤泡树突状细胞)上CR2的配体C3d的细菌表面表达来处理/递送免疫原。在目标2中，我们将分析：i)生长介质诱导的宿主适应，以及ii)TLR2配体过度表达对TLR2信号的影响，反映在树突状细胞(DC)成熟和细胞因子反应中。还将评估抗原宿主适应和TLR2信号改变对树突状细胞处理/呈递免疫原至Ft特异性T细胞的影响。在目标3中，我们将：i)分别和联合测定和优化AIMS 1-2中产生的免疫原的保护活性，以及ii)通过将免疫原靶向APC上的CR2和/或Fc受体来优化Ft免疫原处理/呈递的效率。然后将通过气雾剂攻击在野生型和人源化小鼠身上评估最具保护性的免疫原(S)，并确定保护的相关性/机制。该项目不仅将生产一种针对A类生物制剂的无佐剂粘膜疫苗，而且将建立一种适用于其他传染病病原体的创新和独特的以标准为导向的疫苗开发方法/平台，从而深刻影响预防医学/公共卫生，并改变疫苗开发的范式。

DESCRIPTION (provided by applicant): Establishment of an effective and uniform vaccine development strategy such as we propose here is key to conquering current and emerging infectious diseases. Despite successes against an array of bacterial agents, current approaches to vaccine development are as diverse as the microbes they target and require adjuvants that often have limited efficacy and/or toxic side effects. As a consequence, vaccine discovery is often slow, inefficient, and unsuccessful in the case of many high priority pathogens. We propose/hypothesize that vaccine generation for bacterial pathogens can be unified and stream-lined through a highly innovative criteria-directed approach that maximizes three of the most important criteria for vaccine success: Antigen (Ag)-mimicry, Adjuvanticity, and Efficient targeting of immunogens to Antigen Presenting Cells (APCs). We will test our hypothesis in the context of a very stringent model (respiratory infection with human-virulent Francisella tularensis [Ft]) in which we have obtained highly encouraging results with an adjuvant-free, mucosal vaccine (up to 75% protection of C57BL/6 mice). In fact, this is the only instance thus far in which it has been demonstrated that inactivated Ft administered intranasally protects C57BL/6 mice against mucosal challenge with the highly virulent Category A Ft SchuS4 pathogen. Thus, we will use Ft SchuS4 as a model pathogen to establish a criteria-directed approach to vaccine development, which we strongly believe will result in a fully protective adjuvant-independent mucosal vaccine against Ft SchuS4. In Aim 1, we will use differential cultivation and genetic manipulation of Ft SchuS4 to modulate: i) the antigenic similarity between in vitro- cultivated Ft immunogen and the replicating infectious pathogen (host-adaptation), ii) the cell-stimulatory capacity of the immunogen via over-expression of TLR2 agonist, and iii) processing/presentation of immunogen via bacterial surface expression of C3d, a ligand for CR2 on APCs (B cells and follicular dendritic cells). In Aim 2, we will analyze the impact of: i) growt medium-induced host-adaptation, and ii) over- expression of TLR2 ligands on TLR2 signaling, as reflected in dendritic cell (DC) maturation and cytokine responses. The impact of antigenic host-adaptation and altered TLR2 signaling on the processing/presentation of immunogen to Ft-specific T cells by DCs will also be evaluated. In Aim 3, we will: i) determine and optimize the protective activity of immunogens generated in Aims 1-2, separately and in combination, and ii) optimize the efficiency of Ft immunogen processing/presentation via the targeting of immunogen to CR2 and/or Fc receptors on APCs. The most protective immunogen(s) will then be evaluated in wildtype and humanized mice via aerosol challenge and correlates/mechanisms of protection identified. This project will not only yield an adjuvant-free mucosal vaccine against a Category A biothreat agent, but will establish an innovative and unique criteria-directed approach/platform for vaccine development applicable to other infectious agents, thus profoundly impacting preventive medicine/public health and changing the paradigm for vaccine development.