Neisserial Porins and Antigen Presenting Cells

描述（由申请人提供）：在过去的二十年里，我们的实验室一直在研究奈瑟氏菌porin PorB的免疫生物学。我们首先研究了这种蛋白质作为潜在的抗奈斯球菌候选物的用途，但通过这项工作，我们发现这种蛋白质具有独特的免疫刺激能力，超出了它作为疫苗的潜在用途。我们发现PorB可以作为疫苗的免疫佐剂，这种能力与其刺激抗原呈递细胞（APC）和增加共刺激分子CD86和II类MHC的表达的能力直接相关。此外，我们发现PorB刺激和激活抗原提呈细胞的能力直接归因于与模式识别受体toll样受体2 （TLR2）的相互作用，并且需要TLR适配器蛋白MyD88。如初步数据部分所述，我们已经进一步表明，为了使PorB刺激这些apc， TLR2需要与TLR1（而不是TLR6）异二聚化。利用这些信息，研究PorB免疫刺激佐剂活性的下一个合乎逻辑的步骤是将其活性与其他已知的疫苗佐剂进行比较。这将允许更好地使用PorB作为佐剂，通过了解与其他佐剂相比，它诱导的免疫反应类型。此外，我们认为有必要继续研究PorB的佐剂潜力，因为它是目前唯一被研究作为疫苗佐剂的TLR2配体。我们将把PorB的免疫刺激与其他佐剂进行比较，包括唯一许可的佐剂明胶（炎症小体介导的免疫激活）、其他TLR配体，包括CpG DNA （TLR9）、单磷酰脂质A (MPL)（TLR4）和QS-21。我们将检查这些佐剂诱导对测试抗原卵清蛋白的免疫反应的能力，我们之前在初步数据部分中描述了卵清蛋白。我们将测量诱导的抗体水平、诱导的同型和IgG亚型、抗体亲和力、CD4 T细胞反应以及诱导APC迁移和淋巴结特异性T细胞刺激的能力。此外，我们将比较这些不同的佐剂保护小鼠免受分泌卵泡蛋白的李斯特菌感染的能力。这是一个具有良好特征的模型，先前已被用来证明，当这种病原体被构建为分泌OVA时，卵细胞特异性CD4和CD8 T细胞的存在对于保护小鼠免受感染是必要的。我们将从哈佛医学院微生物系的达伦·希金斯博士处获得该菌株，他在该模型方面具有重要的专业知识，并将担任本提案的顾问。尽管大多数获得许可的疫苗所诱导的保护作用是由于诱导了中和抗体或功能性抗体，但我们认为，开始明确比较这些佐剂（包括PorB）的能力是非常重要的，它们不仅能诱导功能性抗体，还能潜在地诱导保护性CD8 CTL反应。

DESCRIPTION (provided by applicant): Our laboratory has been investigating the immunobiology of the Neisserial porin PorB for the last two decades. We first began by investigating the use of this protein as a potential anti-Neisserial candidate, but through this work we found that this protein had unique immune stimulatory abilities above and beyond its own potential use as a vaccine. We found that PorB can be used as an immune adjuvant for vaccines and that this ability was directly related to its ability to stimulate antigen presenting cells (APC) and increase expression of the costimulatory molecule CD86 and class II MHC. Moreover, we found that the ability of PorB to stimulate and activate antigen presenting cells was directly due to an interaction with the pattern recognition receptor, TOLL-like Receptor 2 (TLR2) and requires the TLR adaptor protein MyD88. We have further shown, as described in the preliminary data section and in that TLR2 needs to heterodimerize with TLR1 (and not TLR6) in order for PorB to stimulate these APCs. Utilizing this body of information, the next logical step for studying the immune stimulating adjuvant activity of PorB is to compare this activity to other known vaccine adjuvants. This will allow for the better use of PorB as adjuvant, by understanding what type of immune response it induces as compared to these other adjuvants. Moreover, we believe it is essential to continue to investigate the adjuvant potential of PorB, as it is currently the only TLR2 ligand being investigated as a vaccine adjuvant. We shall compare the immune stimulation by PorB to panel of other adjuvants, including the only licensed adjuvant, Alum (inflammasomes mediated immune activation, other TLR ligands, including CpG DNA (TLR9), and monophosphoryl lipid A (MPL)(TLR4), and QS-21. We shall examine the ability of these adjuvants to induce immune responses to a test antigen, ovalbumin, which we have used previously as described in the Preliminary Data section. We shall measure antibody levels induced, isotypes and IgG subtypes induced, antibody affinity, CD4 T cell response and ability to induce APC migration and lymph node specific T cell stimulation. Moreover, we shall compare the ability of these different adjuvants to protect mice from infection with Listeria which secretes OVA. This is a well characterized model and has been used previously to demonstrate that the presence of OVA specific CD4 and CD8 T cells are necessary for protection of mice from infection with this pathogen when it is constructed to secrete OVA. We shall obtain this strain from Dr. Darren Higgins in the Dept. of Microbiology at the Harvard Medical School, who has significant expertise in this model and will act as a consultant on this proposal. Even though the protection induced by most licensed vaccines are due to the induction of neutralizing or functional antibodies, we feel it would extremely important to begin to definitively compare the ability of these adjuvants, including PorB, to not only induce functional antibodies, but to potentially induce a protective CD8 CTL response. PUBLIC HEALTH RELEVANCE: We have shown that the Neisserial porin, PorB, is a potent immune adjuvant and works via recognition by TLR2 and induction of antigen presenting cell activation. This proposal will examine the adjuvant activity of PorB and compare this activity to other currently investigated adjuvants (including Alum, CpG DNA, MPL and QS-21). We shall examine induction of both humoral responses and CD8 T cell responses and the ability of the vaccines to prevent Listeria infection in mice. Moreover, we shall further examine the mechanism of PorB adjuvant activity via the use of MyD88 floxed mice bred with mice expressing cell specific cre recombinase.