Developmental research of new generation DNA vaccine against microorganism using synthesized DNA

本研究对微生物感染性疾病的预防性DNA疫苗和病毒载体疫苗的重要性进行了研究。构建了含有OPR F/I、PCRV和Pill基因的三种假单胞菌DNA疫苗。DNA疫苗单独或联合免疫小鼠后，用D_1株攻击。当三种疫苗联合免疫时，90%以上的小鼠存活。预防效果依次为PCRV、OprF/I、PILI。未免疫的小鼠在攻击后2天死亡。另一方面，为了观察对HIV-1的预防效果，我们构建了将Gag-Pal和Env基因插入到CAG启动子中的新型DNA载体和用腺病毒5型纤维替换为腺病毒35型纤维的E1缺陷腺病毒载体。在使用这些新疫苗免疫后，我们用SHIV89.6挑战猴子。在DNA疫苗+Ad5/35免疫组，病毒载量一直保持在可检测到的水平以下，直到第25周。以往的5型腺病毒载体存在肝功能障碍等缺陷。然而，很明显Ad5/35载体对人类是最好的，根据这些结果，我们将开发更方便的假单胞菌疫苗免疫途径用于临床试验。在HIV-1方面，我们正计划与一些制药公司合作进行I期临床试验。此外，我们希望利用Ad5/35载体开发其他最好的针对O-157或Ave流感等严重传染病的DNA疫苗或病毒载体疫苗。

In this study, we studied the importance of preventive DNA vaccine and virus vector vaccine against microbe infectious diseases. We constructed three types of DNA vaccine of pseudomonas containing Opr F/I, Pcr V and Pill genes. After immunization to mice with each or the combination of DNA vaccine, the mice were challenged with D1 strain. When immunized with the combination of three vaccines, more than 90% of the mice were survived. The preventive effect was high in order of Pcr V, OprF/I and Pili. Non immunized mice died 2 days later after challenging. Therefore, the vaccines which we constructed have strong effects to prevent infection and we confirmed that Pcr V worked as strong preventive antigen.On the other hand, to observe preventive effects against HIV-1, we developed a new DNA vector which the genes of Gag-Pal and Env were inserted into CAG promoter and an E1 deficient adeno 5/35 virus vector which the fiber of adeno virus type 5 replaced into the fiber of adeno type35. After immunization using these novel vaccines, we challenged monkeys with SHIV89.6. At the group which were immunized with DNA vaccine+Ad5/35, the viral load kept under the detectable level up to 25th weeks. Former adeno virus vector of type 5 had some faults such as liver obstacle. However, it became clear that Ad5/35 vector was best for human.From these results, we are going to develop more convenient immunizing route of pseudomonas vaccine for clinical trial. On HIV-1, we are planning to join with some pharmaceutical companies to do phase I clinical trial. Furthermore, we hope to develop other best DNA vaccines or virus vector vaccines against severe infectious diseases such as O-157 or Ave influenza using Ad5/35 vector.