重组狂犬病病毒糖蛋白活酵母疫苗经小鼠口服给药后的免疫效果

Protection Effect of Recombinant Oral Rabies Vaccine for Human Use on Immunity of Mice

目的：研究以活酵母为输送载体的狂犬病疫苗对小鼠的免疫保护能力和免疫疗程。方法：小鼠首先灌食高浓度空白活酵母INVSI，并于灌胃后8h和12h分别采集小鼠空肠和回肠组织并提取小肠浸出液培养，计算活酵母经肠胃环境后的存活率；分别取狂犬病糖蛋白（glycoprotein，G）分泌型表达茵株pYes-InG和胞内表达型菌株pYes-G灌胃小鼠，灌胃结束后12h采集小鼠血清和小肠组织，采用免疫组织化学方法检测抗原物质G在小肠上皮细胞的分布，采用ELISA检测小鼠血清中和性抗体的滴度。结果：活酵母经灌食消化8h后在小肠中的存活率最高达36．11％，12h后降至0．59％；口服分泌型pYes．InG重组酵母的小鼠小肠组织和血清中能检测到抗原物质G和低量的中和性抗体，ELISA分析显示，小鼠经过3-4次免疫接种，免疫效果基本恒定，而口服胞内表达型pYes-G重组酵母的小鼠小肠组织和血清中均未检测到目标物。结论：分泌型重组酵母pYes-InG经多次口服可对狂犬病起到一定的预防作用，但它诱导产生的中和性抗体浓度低，免疫应答慢，虽不适合用于控制突发性狂犬病的传染以及治疗狂犬病患者，但从免疫机制、免疫方式、安全性以及生产成本等因素考虑，仍具有良好的研究价值。

Purpose To evaluation of the immune protection effect of a oral rabies vaccine and on the immune program in mice for potential human use delivered by live recombinant Saccharomyces cerevisiae. Methods Two groups of Kunming mice （ each containing ten mice ） were fed by oral gavage with high density live cells of Saccharomyces cerevisiae （S. cerevisiae） INVSCI. At 8 and 12 hours after being fed, the jejunum and ileum of the mice were collected and extracted, respectively, and then those extracts was cultured for 2 -3 days at 30°C for calculation of the survival rates of yeast cells passed through digestive tract. Then the other two groups were fed with two kinds of recombinant yeast expressing rabies virus glycoprotein （G） in different forms including secretory expression pYes-InG and intracellular expression pYes-G. Blood serum and intestine in mice were isolated at 12 hours of fed time. Antibodies against rabies virus in their sera were analyzed by ELISA and the distribution of G antigen in intestinal slices was measured by immunohistochemistry methods. Results The survival rate reached the greatest value of about 36.11% （n -- 10） at 8 hours after being fed with empty yeast cells and the survival rate after 12 hours was about 0.59% （ n = 12）. The G antigen and low neutralizing antibody titer were detected in tissue samples from the mice fed with yeast cells of secretory expression pYes-InG. The concentration of neutralizing antibody was maintained constant after oral gavage three to four times. However, no antigen or antibody was found in tissue samples from the mice fed with yeast cells of pYes-G. Conclusions Although the new kind of recombinant S. cerevisiae vaccine secreting G antigen （pYes-InG） was unsuitable for preventing infection of rabies virus and treating those patients exposed to rabies virus due to the slow immune response and low antibody titer, they showed potential application for human use considering their immune effect, immune mechanism of oral vaccine, the low production costs and the occupational safety regulations.