Maurice Sarah, Nussinovitch Amos, Jaffe Nicole, Shoseyov Oded, Gertler Arieh
Institute of Biochemistry, The Hebrew University of Jerusalem, P.O. Box 12, Rechovot 76100, Israel.
Vaccine. 2004 Dec 9;23(4):450-9. doi: 10.1016/j.vaccine.2004.06.022.
This study was focused on the utilization of a recombinant expression system to produce a unique modified subunit vaccine possessing a self-contained delivery system which could potentially improve the uptake and delivery of vaccine products as well their immunogenic potential. For this purpose the A-layer protein (At-R) associated with the fish pathogen atypical Aeromonas salmonicida was cloned and modified by the genetic fusion of the protein transduction domain (MTS) derived from Kaposi fibroblast growth factor (At-MTS). The potential for these proteins to be employed as antigens for oral immunization of goldfish was examined by encapsulation of At-R, At-MTS and the control, BSA, into biodegradable alginate gel macrospheres which were fed to goldfish in place of standard pellet fish feed. The bead physical properties were modified only in the presence of At-R and the temporal release of proteins was significantly less when At-MTS was employed. Western blot analysis of serum samples collected from fish following intubation with the recombinant proteins determined that the rate of protein uptake from the digestive tract into the blood system improved considerably when MTS was fused to At-R. Experimental fish were fed one of three protein-alginate formulae on a schedule of 3 days/week or 5 days/month for a period of 2 months. After 1 month, animals fed on the 5-day protocol demonstrated increased serum antibody titers while following an additional month of feeding this level decreased and titers were found to be higher in fish maintained on the 3-day regime. Fish fed At-MTS maintained the highest titer at the end of 2-month period. To determine whether the diminished antibody titers were a result of oral tolerance fish were injected intraperitoneally with the At-R antigen. Only experimental groups which had been fed At-R or At-MTS demonstrated increased antibody titers which paralleled a typical secondary humoral response. In spite of the presence of an increased titer to A-protein, vaccinated fish did not demonstrate resistance to infection with atypical A. salmonicida.
本研究聚焦于利用重组表达系统生产一种独特的改良亚单位疫苗，该疫苗拥有一个自成一体的递送系统，这可能会改善疫苗产品的摄取和递送及其免疫原性潜力。为此，将与鱼类病原体非典型杀鲑气单胞菌相关的A层蛋白（At-R）进行克隆，并通过源自卡波西成纤维细胞生长因子的蛋白质转导结构域（MTS）的基因融合进行修饰（At-MTS）。通过将At-R、At-MTS和对照牛血清白蛋白（BSA）封装到可生物降解的海藻酸盐凝胶大球体中，并将其代替标准颗粒鱼饲料喂给金鱼，来检测这些蛋白质作为金鱼口服免疫抗原的潜力。仅在存在At-R的情况下珠子的物理性质发生了改变，并且当使用At-MTS时蛋白质的时间释放明显减少。对用重组蛋白插管后的鱼所采集的血清样本进行的蛋白质印迹分析确定，当MTS与At-R融合时，蛋白质从消化道吸收到血液系统中的速率有了显著提高。实验鱼按照每周3天或每月5天的时间表喂食三种蛋白质-海藻酸盐配方之一，持续2个月。1个月后，按照每月5天方案喂食的动物血清抗体滴度升高，而在额外喂食1个月后该水平下降，并且发现按照每周3天方案饲养的鱼的滴度更高。喂食At-MTS的鱼在2个月期末保持最高滴度。为了确定抗体滴度降低是否是口服耐受的结果，对鱼进行腹腔注射At-R抗原。只有喂食At-R或At-MTS的实验组显示抗体滴度升高，这与典型的二次体液反应相似。尽管对A蛋白的滴度有所增加，但接种疫苗的鱼并未表现出对非典型杀鲑气单胞菌感染的抵抗力。
