Oral vaccine from daily lactic acid bacteria! -Development of new vaccine adjuvants-

家畜对食物生产非常重要。然而，他们患有慢性肠道疾病，导致粮食生产损失和抗生素过量使用，这是一个非常严重的问题。在这项研究中，最终的目标是利用乳制品和细菌DNA开发一种新型的口服疫苗佐剂。为了达到这一最终目标，我们首先试图从乳酸菌(LAB)中寻找具有活性的DNA基序，并建立一种新的猪TLR9转基因免疫检测系统，最后阐明LAB DNA在肠道中的免疫刺激机制。我们发现了几个免疫刺激DNA基序，其中包括一个来自实验室的新基序。我们从猪的Peyer‘s斑块中克隆了Toll样受体9(TLR9)，并成功地构建了猪TLR9的转染体。该转染体可用于实验室活性DNA的鉴定。在Peyer‘s斑块，DNA基序增强了Th1型细胞因子(IL-6、IL-12、干扰素γ)的表达。猪TLR9基因在猪的Peyer‘s Patches(PPS)和肠系膜淋巴结(MLN)中优先表达，其表达程度约为脾的3倍，而在其他组织中表达较弱。我们首次发现在PPS和MLN中形成高表达的(TLR9^+)细胞。此外，TLR9~+细胞不仅存在于树突状细胞、B细胞等免疫细胞中，也存在于膜细胞(M细胞)中，属于滤泡相关上皮细胞(FAE)。这些结果表明，PPS和MLN为宿主防御提供了在初始免疫反应的传导部位对来自细菌的各种生物活性寡核苷酸(ODN)做出反应的能力。这些结果表明，来自LAB的免疫刺激DNA基序通过TLR9在肠道中发挥了细胞因子的诱导作用，提示LAB DNA是一种很好的口服疫苗佐剂。

Domestic animals are very important for food production. However, it becomes very severe problem that they have chronic intestinal diseases resulted in loss of food production and overadministration of antibiotics. In this study, the final goal is development of a novel oral vaccine adjuvant by using dairy lactic and bacterial DNA. In order to go ahead to the final goal, we first tried to find the active DNA motifs from lactic and bacteria (LAB) and develop a new immunoassay system with swine TLR9 transfectant and finally elucidate the mechanisms of the immunostimulation by the LAB DNA in the gut. We found several immunostimulatory DNA motifs including a novel one from the LAB. We cloned Toll-like receptor 9 (TLR9) from swine peyer's patch, which is expected for human model, and successfully developed a swine TLR9 transfectant. The transfectant was useful for the evaluation of active DNA from the LAB. The DNA motifs augmented an induction of Th1 type cytokine (IL-6, IL-12, IFNγ) mRNA in peyer's patches. The swine TLR9 gene was preferentially expressed peyer's patches (Pps) and mesenteric lymph nodes (MLNs), of which their degrees were approximately three times higher than that of spleens but weak in the other tissues. We firstly showed that high express (TLR9^+) cells were formed in Pps and MLNs. In addition, TLR9^+ cells existed not only in immune cells such as dendritic cells and B cells but also in membranous cells (M cells), belonging to follicle-associated epithelia (FAE) in Pps. These results suggest that Pps and MLNs provide the host defense with the ability to respond to a variety of bioactive oligonucleotides (ODNs) from bacteria at a conductive site of initial immune responses. From these results, immunostimulatory DNA motif from the LAB exerted the cytokine induction in the gut via TLR9 suggesting that the LAB DNA is a good candidate of an oral vaccine adjuvant.