Alternative approaches for NALT-based immunity to respiratory pathogens

描述(申请人提供)：鼻咽道是包括炭疽杆菌和流感病毒在内的使人虚弱和潜在致命的病原体的主要入口。能够促进黏膜组织中抗体和细胞毒性T细胞反应的黏膜疫苗，除了一般的血流外，比经典的注射疫苗更有效地保护呼吸道病原体。因此，需要安全的粘膜佐剂和疫苗递送系统。这笔赠款之前的工作重点是将小鼠鼻部相关淋巴网状组织(NALT)定义为针对鼻咽道的免疫反应的诱导部位。我们使用霍乱毒素并开发了缺乏ADP核糖基转移酶活性的新型衍生物来规避这种肠毒素佐剂的反应性。此外，我们还评估了炭疽毒素对小鼠NALT的作用。这些研究还包括炭疽保护性抗原抗体(Abs)的特征，特别强调保护粘膜组织的可能性。总共成功地实现了五个最初的具体目标。在我们的研究过程中，一个重要的和意想不到的发现是，鼻腔注射炭疽杆菌保护性抗原和cAMP诱导的炭疽杆菌水肿因子突变体可以增强粘膜和系统对这两种分子的免疫力。此外，我们还发现，水肿性毒素(EdTx或保护性抗原+水肿性因子)衍生物可增强黏膜和系统对重组鼠疫菌F1-V抗原等无关抗原的免疫。与神经节苷脂结合的霍乱肠毒素不同，EdTx及其衍生物在鼻腔应用后都不会针对中枢神经系统组织。在这一续期拨款中，我们将解决总体假设，即舌下应用EdTx衍生物将诱导基于NALT的免疫并预防呼吸道病原体，而不会出现通常与鼻用肠毒素相关的不良反应。具体目的一是通过舌下注射途径，建立与保护性抗原(PA)共同作用的水肿型因子衍生物的佐剂活性，以增强对炭疽毒素成分的免疫力。具体目标二将表征以EdTx衍生物为佐剂的舌下免疫对呼吸道病毒感染的保护性免疫。具体目标三的研究将确定EdTx衍生物舌下免疫后产生分泌性IgA(SIgA)抗体和粘膜免疫的诱导部位。最后，具体目标四将确定作为舌下佐剂的EdTx-衍生物诱导SIgA反应的潜在分子信号。这笔赠款将揭开EdTx衍生物作为舌下疫苗佐剂并诱导基于NALT的免疫的机制。我们还将验证一种新的粘膜疫苗递送途径，以及用于诱导对呼吸道病原体免疫的新型PA基黏膜佐剂(S)。公共卫生相关性：到目前为止，疫苗鼻腔注射被认为是最有效的手段，可以触发免疫反应的诱导部位，以发展对呼吸道病原体的最佳免疫力。然而，鼻用疫苗中使用的肠毒素佐剂可能会引起严重的副作用，这是因为它们能够靶向中枢神经系统并维持炎症反应。这笔赠款将探索水肿性毒素衍生物作为舌下疫苗佐剂的有效性。完成后，我们将验证一种新的疫苗递送系统，以及用于诱导对呼吸道病原体免疫的新粘膜佐剂(S)。

DESCRIPTION (provided by applicant): The nasopharyngeal tract is a major portal entry of debilitating and potentially lethal pathogens including Bacillus anthracis and influenza virus. Mucosal vaccines capable of promoting antibody and cytotoxic T cell responses in mucosal tissues, in addition to the general bloodstream, protect more effectively against respiratory pathogens than classical injected vaccines. Therefore, there is a need for safe mucosal adjuvants and vaccine delivery systems. Previous work on this grant focused at defining murine nasal-associated lymphoreticular tissues (NALT) as inductive sites for immune responses targeting the nasopharyngeal tract. We used cholera toxin and developed novel derivatives lacking ADP ribosyl transferase activity to circumvent the reactogenicity of this enterotoxin adjuvant. In addition, we have assessed the action of anthrax toxins on murine NALT. These studies have also included the characterization of antibodies (Abs) to anthrax protective antigens with special emphasis on the potential to protect mucosal tissues. A total of five original Specific Aims were successfully addressed. A major and unexpected finding during the course of our studies was the fact the nasal co-administration of Bacillus anthracis protective antigen together with a mutant of the cAMP-inducing Bacillus anthracis edema factor enhanced mucosal and systemic immunity against these two molecules. Furthermore, we found that the edema toxin (EdTx or protective antigen plus edema factor) derivative enhanced mucosal and systemic immunity to co-administered unrelated antigens such as recombinant Yersinia pestis F1-V antigen. Unlike the ganglioside-binding enterotoxin cholera toxin, neither EdTx nor its derivatives target central nervous system tissues after nasal application. In this renewal grant, we will address the overall hypothesis that sublingual application of EdTx derivatives will induce NALT-based immunity and protect against respiratory pathogens, without the adverse effects often associated with nasal application of enterotoxins. Specific aim one will establish the adjuvant activity of edema factor derivatives co-administered with protective antigen (PA) via the sublingual route for enhanced immunity to anthrax toxin components. Specific aim two will characterize protective immunity to respiratory viral infection afforded by sublingual immunization with EdTx derivatives as adjuvant. Studies in specific aim three will identify inductive sites for the generation of secretory IgA (SIgA) Abs and mucosal immunity after sublingual immunization with EdTx-derivatives. Finally, specific aim four will determine molecular signals underlying the induction of SIgA responses by EdTx-derivatives as sublingual adjuvant. This grant will unravel the mechanisms by which EdTx derivatives act as adjuvant for sublingual vaccines and induce NALT-based immunity. We will also validate a new route for mucosal vaccine delivery, as well as new PA-based mucosal adjuvant(s) for the induction of immunity against respiratory pathogens. PUBLIC HEALTH RELEVANCE: Thus far, nasal delivery of vaccines was believed to be the most effective mean to trigger inductive sites of immune responses for the development of optimal immunity against respiratory pathogens. However, enterotoxin adjuvants employed in nasal vaccines could induce severe side effects resulting from their ability to target the central nervous system and sustain inflammatory responses. This grant will explore the efficacy of edema toxin derivatives as adjuvant for sublingual vaccines. Upon completion, we will have validated a new vaccine delivery system, as well as new mucosal adjuvant(s) for the induction of immunity against respiratory pathogens.