重组弓形虫肌动蛋白解聚因子滴鼻免疫小鼠诱导的免疫应答及保护作用

目的观察重组弓形虫肌动蛋白解聚因子(Recombinant Toxoplasma gondii actin depolymerizing factor,rTgADF)滴鼻免疫小鼠诱导的免疫应答及抗弓形虫攻击的保护作用。方法 40只雌性BALB/c小鼠随机分为2组,分别用30μg rTgADF(溶于20μl PBS)和20μl PBS于第0、14和21d各滴鼻免疫1次。末次免疫后14d,每组处死小鼠5只,ELISA法测定血清IgA、IgG和IgG1/IgG2a及鼻咽、阴道和小肠冲洗液sIgA以及脾淋巴细胞培养上清中IFN-γ、IL-2、IL-4和IL-10水平。每组另取5只小鼠,用4×104 RH株弓形虫速殖子/只灌胃攻击(致死性),观察小鼠健康状况并计算存活率。其余小鼠用1×104速殖子/只灌胃攻击(慢性),攻击后30d处死,计数肝、脑组织速殖子。结果 rTgADF诱发小鼠产生较强的系统免疫和黏膜免疫应答,免疫组小鼠血清IgG、IgA和IgG1/IgG2a水平与对照组比较差异有统计学意义(P<0.05或P<0.01),鼻咽、阴道和小肠冲洗液sIgA水平与对照组比较差异有统计学意义(P<0.01),脾淋巴细胞培养液上清中IFN-γ和IL-2、IL-4水平与对照组比较差异有统计学意义(P<0.05或P<0.01),IL-10水平组间比较差异无统计学意义(P>0.05)。致死性攻击后30d,对照组小鼠全部死亡,免疫组小鼠存活率为20%,差异有统计学意义(P<0.01);慢性感染后30d,免疫组小鼠肝和脑虫荷分别比对照组减少63.87%(P<0.01)和50.14%(P<0.05)。结论 rTgADF滴鼻免疫小鼠诱导产生较强的黏膜及系统免疫应答,并可部分抵抗弓形虫致死性和慢性攻击,可作为弓形虫候选蛋白疫苗。

Intranasal immunization with recombinant Toxoplasma gondii actin depolymerizing factor （rTgADF） in- duces immune responses and protection in mice

Objective To observe the immunogenicity and immunoprotection against acute infection and chronic infection induced by intranasai immunization with recombinant T.gondii actin depolymerizing factor （rTgADF）. Methods For- ty BALB/c mice were randomly divided into two groups that were intranasally vaccinated with 30 μg（20 μl）rTgADF or 20 μl PBS. Both groups were vaccinated three times （on days 0, 14, and 21）. Two weeks after the final inoculation, 5 mice from each group were randomly selected and sacrificed. The levels of IgA, IgG, IgG1/IgG2a in sera, slgA in nasopharyn geal, vaginal, and intestinal washes, and IFN-γ, IL-2, IL- 4 and IL-10 in the supernatant of cultured splenic lymphocytes were determined using EI.ISA. Five more mice were randomly selected from each group and were used in an acute chal lenge. Each mouse was challenged intragastrically with 4 × 104 tachyzoites （fatal）, and the health and survival time of mice was observed for 30 days. The remaining mice were challenged intragastrically with 1 × 104 tachyzoites per mouse. Thirty days after the challenge, the number of tachyzoites in the brain and middle lobe of the liver was observed micro scopically. Results rTgADF effectively induced stronger systemic and mucosal immune responses in mice. Compared to the control group, the immunized group had significantly higher （P〈0.05 or P〈0.01） levels of serum IgG, IgA, and IgG1/IgG2a. The immunized group had significantly higher （P〈0.01） levels of slgA antibodies in nasopharyngeal, vagi- nal, and intestinal washes compared to the control group. The levels of IFN-γ, IL-2, and IL-4 in the supernatant of cul- tured spleen lymphocytes were higher than those in the control group （P〈0.05 or P〈0.01）. In the acute infection test,30 days after the challenge immunized mice had a higher survival rate （20%, P〈0.01） than the control group （no mice survived）. In the chronic infection test, immunized mice had significantly fewer tachyzoites in the liver and brain than did the control group. In immunized mice, the tachyzoite load in the liver and brain decreased by 63.87% （P〈0.01） and 50. 14% （P〈0.05）, respectively. Conclusion Intranasal immunization with rTgADF can effectively induce systemic and mucosal immune responses to partially protect mice against fatal and chronic infection with T. gondii, rTgADF could be used to create a potential vaccine against T.gondii.