Application of mucosal vaccine against upper respiratory tract infections

1. 抗流感嗜血杆菌（Hi）粘膜疫苗的生产。已知外膜蛋白P6是Hi最常见的抗原。从急性中耳炎患者的Hi培养中纯化P6作为粘膜疫苗，并根据P6的生化分析制备重组P6 （rP6）。此外，为了检验mCT.2的佐剂性，制备了与rP6融合的重组突变霍乱毒素（mCT-rP6）。重组疫苗的黏膜免疫应答。对重组疫苗进行小鼠鼻内注射，并比较其粘膜免疫反应。结果表明，单独接种rP6的小鼠未观察到IgA应答。虽然mCT-rP6在小鼠鼻洗液中诱导了IgA抗体，但其反应比rP6与CT联合使用的小鼠要小。由于CT具有一定的毒性，不能用于人体，因此有必要开发一种新的给药系统。卵清蛋白联合纳米球的免疫应答。纳米球已被应用于抗HIV感染的粘膜疫苗。在我们的实验室中，纳米球与卵清蛋白结合，并给予小鼠鼻内注射。三次免疫后，观察免疫应答。结果表明：纳米球诱导粘膜免疫应答；但辅助作用较CT差。这些发现表明，为了使用纳米球诱导有效的粘膜免疫，需要更浓缩的疫苗。免疫能态细胞对内毒素的免疫反应。尽管在粘膜积液如中耳积液和鼻窦积液中存在内毒素，但这些粘膜的炎症反应被抑制。为了阐明其机制，我们分离扁桃体和鼻腔的单个核细胞，用内毒素刺激。结果表明，这些具有免疫能力的细胞可能对内毒素具有较高的耐受性。

1. Production of mucosal vaccines against Haemophilus influenzae (Hi).Outer membrane protein P6 is known to be a most common antigen of Hi. P6 was purified from Hi cultured from a patient with acute otitis media as a mucosal vaccine and recombinant P6 (rP6) was also produced based on the biochemical analysis of P6. Further, recombinant mutant cholera toxin (mCT) fused to rP6 (mCT-rP6) was produced in order to examine the adjuvancity of mCT.2. Mucosal immune responses against recombinant vaccines.Those recombinant vaccines were administered to mice intra-nasally and the mucosal immune responses were compared. In results, IgA responses were not observed in mice immunized with rP6 alone. Although mCT-rP6 induced IgA antibodies in nasal washes of mice, the responses were less than that in mice administered rP6 together with CT. Since CT cannot be used in human because of the toxicity, the results indicate the necessity of developing a new drug-delivery system.3. Immune responses against ovalbumin combined with nanosphere.Nanosphere has been already applied to mucosal vaccine against HIV infection. Nanospheres were combined with ovalbumin in our laboratory and were administered to mice intra-nasally. After three times immunization, the immune responses were investigated. The results showed that nanosphere induced mucosal immune responses ; however, the adjuvancity was less that CT. Those findings indicate that much more concentrated vaccines were needed in order to induce an effective mucosal immunity using nanosphere.4. Immune responses of immune competent cells against endotoxin.Despite the presence of endotoxin in mucosal effusions such as middle ear effusion and paranasal sinus effusion, inflammatory responses were suppressed in those mucosa. In order to clarify the mechanism, tonsillar and nasal mononuclear cells were isolated and stimulated with endotoxin. The results suggest that those immune competent cells might be hi tolerance to endotoxin.