Novel Nanoparticle Respiratory Tract Mucosal Vaccine

摘要 细菌和病毒呼吸道病原体与粘膜表面密切相互作用。特化的先天和适应性粘膜 免疫系统保护这些表面，是身体的第一道防线。更重要的原因之一是 粘膜疫苗的发展是激发和准备局部粘膜免疫的越来越多的证据 应对措施对于预防结核病等传染病具有重要意义。尽管整个免疫系统 预防结核分枝杆菌(Mtb)感染所需的反应谱尚不清楚，最近的数据在 动物模型表明，疫苗诱导的T辅助17(Th17)细胞亚型的CD4+细胞，自然地向 在呼吸道，可以加速保护性Th1细胞的募集和干扰素、IL-17等细胞因子的产生。 细胞毒性CD8+和CD4+T细胞也很重要，可以通过抗原特异性的穿孔素诱导水平来评估。 颗粒酶B和颗粒溶素。此外，免疫球蛋白A和免疫球蛋白免疫球蛋白的粘膜抗体已被证明可以干扰病变的进展。 对于其他呼吸道病原体的疾病，可能对结核分枝杆菌起类似的作用。呼吸道上皮细胞是一种 黏膜疫苗的主要目标。粘膜的主要部分由能够提供屏障功能的细胞组成。 作为传感器，通过模式识别受体检测危险微生物成分并传递信号 以触发先天免疫反应并促进适应性免疫反应。涉及多个结核分枝杆菌的研究 包括HBHA、Rv3351c和ESAT6在内的分泌蛋白已经确定了吸入者最初的相互作用之间的联系 病原体与肺泡上皮细胞和随后的微生物从肺部传播。这些Mtb蛋白 也已被证明在皮下或鼻腔给药的小鼠中能产生重要的免疫反应。 纳米粒是一种极具吸引力的粘膜疫苗/免疫治疗载体，因为它可以通过 抗原提呈细胞，NPs优先流向淋巴管而不是血流，并依赖于 大小和组成，NPs通过粘液扩散和跨越粘膜屏障的能力。交付以NP为基础的疫苗 对呼吸道的作用可能是增强先天免疫反应的一种手段，也将是本研究的重点。通过 将所有这三种上皮细胞靶向Mtb蛋白结合在NP载体上，结合VacSIM®免疫- 刺激基质加佐剂，并作为黏膜增强疫苗部署到皮下卡介苗，我们预计 动物肺的保护性细胞和体液反应和显著提高对结核分枝杆菌感染的保护力 与单独接种卡介苗相比。在目标1和目标2中，我们将评估疗效和免疫反应 用于多种疫苗制剂，并确定两种最具保护性的疫苗，将在目标3中进行安全性、稳定性、 然后将在豚鼠身上证实保护效果。我们的假设是通过刺激体液和细胞 利用我们的结核分枝杆菌多抗原粘膜纳米颗粒基质疫苗的免疫应答，随后的结核分枝杆菌气雾剂暴露将 导致肺部细菌复制减少，并增强卡介苗引发的全身免疫反应 疫苗可以更有效地清除残留细菌，减少或防止传播。我们对这种方法充满信心 将在两种不同的动物模型上成功对抗结核分枝杆菌，从而为后续的非人类奠定基础 灵长类动物试验。

Summary Bacterial and viral respiratory pathogens interact closely with mucosal surfaces. Specialized innate and adaptive mucosal immune systems protect these surfaces and are the first line of defense for the body. One of the more important reasons for the development of mucosal vaccines is the increasing evidence that stimulating and preparing local mucosal immune responses is important for protection against infectious against diseases, such as tuberculosis. Although the entire immune response repertoire needed for protection against Mycobacterium tuberculosis (Mtb) infection is not known, recent data in animal models suggest that vaccine-induced CD4+ cells of the T helper 17 (Th17) cell subtype, which naturally traffic to the airways, can accelerate the recruitment of protective Th1 cells and production of IFN, IL-17 and other cytokines. Cytotoxic CD8+ and CD4+ T cells also are important and can be assessed via levels of antigen-specific induction of perforin, granzyme B, and granulysin. In addition, IgA and IgG mucosal antibodies have been shown to interfere with the progression towards disease with other respiratory pathogens and may act similarly against Mtb. The respiratory epithelial cell is a primary target for mucosal vaccines. A major portion of the mucosa is comprised of cells that can provide a barrier function and serve as sensors to detect dangerous microbial components through pattern-recognition receptors and transmit signals to underlying mucosal cells to trigger innate and promote adaptive immune responses. Studies involving several Mtb secreted proteins including HBHA, Rv3351c and ESAT6 have identified links between the initial interaction of the inhaled pathogen with alveolar epithelial cells and the subsequent dissemination of the microbes from the lung. These Mtb proteins also have been shown to generate important immune responses in mice given subcutaneous or intranasal doses. Nanoparticles (NPs) are attractive mucosal vaccine/immunotherapy delivery vehicles due to the enhanced uptake by antigen-presenting cells, the preferential draining of NPs to lymphatics rather than to the bloodstream, and depending on size and composition, the ability of NPs to diffuse through mucus and cross mucosal barriers. Delivery of NP-based vaccines to the respiratory tract may be a means of enhancing innate immune responses and will be the emphasis of this study. By combining all three of these epithelial cell-targeting Mtb proteins on a NP vehicle combined with VacSIM® immune- stimulating matrix plus adjuvant, and deploying as a mucosal booster vaccine to the subcutaneous BCG prime, we expect protective cellular and humoral responses in animal lungs and significantly-elevated protection from Mtb infection compared to that conferred by BCG vaccination alone. In Aims 1 and 2, we will evaluate the efficacy and immune responses for multiple vaccine preparations and identify the two most protective which will be assessed in Aim 3 for safety, stability, and then protective efficacy will be confirmed in guinea pigs. Our hypothesis is that by stimulating humoral and cellular immune responses with our Mtb multi-antigen mucosal nanoparticle matrix vaccine, subsequent Mtb aerosol exposure will result in reduced bacterial replication in the lungs and augment systemic immune responses generated by the BCG-priming vaccine to more-effectively clear residual bacteria and decrease or prevent dissemination. We are confident this approach will be successful against Mtb in two different animal models, and thus, lay the groundwork for subsequent non-human primate trials.