Effective Mucosal Vaccination through Engineered Outer Membrane Vesicles

粘膜是病原体传播的主要场所，但注射的疫苗导致粘膜免疫力低下。 口腔黏膜组织中的树突状细胞亚群可以诱导对抗原的免疫和耐受。这个 拟议的研究重点是通过激活粘膜树突状细胞来诱导粘膜免疫 重组外膜囊泡(ROMvs)。在过去的十年里，我们的研究团队设计和 开发rOMV以诱导对肽抗原的强大Th1/Th2免疫反应。在最近，但还没有 在未发表的工作中，我们的团队发现了一种激活和推动DC成熟的方法。也许最多的 令人兴奋的是，我们量化了rOMVS在猪口腔粘膜组织中的渗透深度，并显示了 ~100 nm的囊泡穿透组织的深度与幼稚髓系树突状细胞的数量一致 电池(&gt；100微米)。因此，这种基于rOMV的技术具有与粘膜递送相一致的特征。 疫苗，特别是通过口腔组织。研究的目的是阐明和确立其独特性 将rOMV方法作为口腔疫苗接种的一种方法。通过将亚基表位的使用与 ROMV的组织渗透性，我们将评估这样一个假设，即这些rOMV对 口腔黏膜可诱导幼稚树突状细胞活化，诱导强大而平衡的Th1/Th2免疫 回应。这一假设将通过完成以下目标来进行评估。目标1：量化 对“弱”抗原的免疫反应，以决定rOMvs的效力和效力。我们的 以前的研究使用绿色荧光蛋白(GFP)作为一种“弱”抗原的有效模型，证明了 皮下注射rOMvs的效力。我们将使用相同的方法来量化这两种幽默 以及rOMvs将GFP传递到猪的口腔粘膜的细胞反应(最接近于 人的口腔粘膜)。结果将与皮下注射的相同配方进行比较 (即，基准)，以便将口腔给药途径的有效性与传统的 行政管理。目的2：量化流感病毒血凝素(HA)抗原的免疫应答。 确定rOMV赋予的保护性免疫。在之前的工作中，我们使用了一系列HA 通过皮下注射rOMvs在小鼠皮下注射以量化测量的保护性免疫水平 通过血凝素抑制试验和抗体中和分析。我们将使用相同的方法 目的：量化重组OMVS对猪口腔黏膜HA的体液和细胞免疫反应。 结果将与皮下注射的相同配方(即基准)进行比较，以便 将口腔给药途径的疗效与传统给药途径联系起来。康奈尔大学- Putnam(rOMV疫苗设计)和Chang(猪免疫)的基础研究团队结合了两个实验室 专家们共同拥有创造一个有效的口腔注射疫苗平台的技能， 可能适用于广泛的粘膜传播病原体。

The mucosa is a primary site for pathogen transmission, but injected vaccines induce poor mucosal immunity. Mucosal dendritic cell subsets in the buccal tissue can induce both immunity and tolerance to antigens. The proposed research focuses on inducing mucosal immunity by activating mucosal dendritic cells using recombinant outer membrane vesicles (rOMVs). Over the past decade, our research team has designed and developed rOMVs to induce robust Th1/Th2 immune responses to peptide antigens. In more recent and yet unpublished work, our team has discovered a way to activate and drive the maturation of DCs. Perhaps most exciting is that we quantified the penetration depth of rOMVs in the buccal mucosal tissue of the pig and show that the ~100 nm vesicles penetrate the tissue to depths consistent with the population of naïve myeloid dendritic cells (>100 µm). This rOMV-based technology, therefore, has a profile consistent with a mucosally-deliverable vaccine, specifically via buccal tissue. The objective of the research is to illustrate and establish the uniqueness of the rOMV approach as a method for buccal vaccine delivery. By combining the use of subunit epitopes with the tissue penetration of rOMVs, we will evaluate the hypothesis that the administration of these rOMVs to the buccal mucosa will induce the activation of naïve DCs and induce a strong and balanced Th1/Th2 immune response. The hypothesis will be evaluated through the completion of the following Aims. Aim 1: to quantify the immune response to a “weak” antigen to determine the efficacy and potency of the rOMVs. Our previous work used green fluorescent protein (GFP) as an effective model of a “weak” antigen that demonstrated the potency of rOMVs administered subcutaneously. We will use the same approach to quantify both the humoral and cellular responses to GFP delivered by rOMVs to the buccal mucosa of pigs (the closest animal model to the human buccal mucosa). The results will be compared to the same formulation administered subcutaneously (i.e., benchmark) in order to correlate the efficacy of the buccal route of administration to a traditional route of administration. Aim 2: to quantify the immune response to the hemagglutinin (HA) antigen of influenza to determine the protective immunity conferred by the rOMVs. In previous work we used a sequence of HA delivered by subcutaneous injection of rOMVs in mice to quantify the level of protective immunity, as measured by the hemagglutinin inhibition assay and the antibody neutralization analysis. We will use the same approach to quantify both the humoral and cellular responses to HA delivered by rOMVs to the buccal mucosa of pigs. The results will be compared to the same formulation administered subcutaneously (i.e., benchmark) in order to correlate the efficacy of the buccal route of administration to a traditional route of administration. The Cornell- based research team of Putnam (rOMV vaccine design) and Chang (swine immunity) combines the labs of two experts who collectively have the skill to create an effective, buccally administered vaccine delivery platform that is potentially applicable to a wide range of mucosally-transmitted pathogens.