TgAMA1和TgRON2抗原表位合成肽鼻内免疫抗小鼠弓形虫病的作用

Intranasal immunization with synthetic peptides of TgAMA1 and TgRON2 containing epitopes protects mice against toxoplasmosis

目的观察含弓形虫顶膜抗原1(TgAMA1)和棒状体颈部蛋白2(TgRON2)T、B细胞抗原表位的多肽鼻内免疫小鼠诱导的抗弓形虫病免疫保护作用。方法化学方法合成含有经生物信息学预测的TgAMA1和TgRON2T细胞和B细胞表位多肽。5周龄BALB/c小鼠48只随机分为AMA1组、RON2组、A1+R2组和对照组(n=12),分别用AMA1 30μg、RON2 30μg或(A1+R2)各15μg鼻内免疫,对照组用20μl生理盐水滴鼻,共免疫3次,间隔2周。末次免疫后2周,采用ELISA法检测血清抗体水平;处死小鼠,取鼻、膀胱和肠道洗液,ELISA法检测SIgA水平;无菌取脾,制备脾细胞悬液,CCK-8法检测脾淋巴细胞增殖反应,ELISA法检测淋巴细胞培养上清细胞因子水平;取肝、脑组织,计数速殖子虫荷,以评价免疫诱导的黏膜及系统免疫应答和抗弓形虫感染保护作用。结果用AMA1和RON2多肽免疫小鼠可诱导产生高水平的特异性抗体反应和淋巴增殖反应。肽免疫小鼠血清特异性IgA、总IgG、IgG1和IgG2a水平均高于对照组(P<0.05或P<0.01),其中A1+R2组最高。小鼠脾淋巴细胞刺激指数(SI)显示,AMA1组(1.171±0.116)、RON2组(1.362±0.110)和A1+R2组(1.941±0.162)高于PBS组(0.743±0.081)(P<0.05或P<0.01),A1+R2组亦高于单肽组(P<0.05)。肽免疫小鼠脾细胞培养上清中IFN-γ、IL-2和IL-4水平显著高于对照组(P<0.05或P<0.01)。肽免疫诱导了小鼠高水平的黏膜免疫应答,肽免疫小鼠鼻腔、膀胱和小肠冲洗液SIgA水平均显著高于对照组(P<0.05或P<0.01)。肽免疫小鼠获得部分抗弓形虫感染保护作用,与对照组比较,肽免疫小鼠肝和脑中速殖子负荷显著降低,减虫率分别为A1+R2组55.79%和55.68%、AMA1组52.36%和46.46%、RON2组40.02%和47.33%(P<0.05或P<0.01)。结论含有T和B细胞表位的AMA1和RON2肽是一种有效的抗弓形虫病候选抗原,鼻内免疫小鼠可诱导强烈的黏膜和系统免疫应答,两种抗原表位肽联合使用的免疫效应比单用AMA1或RON2更强,抗弓形虫病作用更显著。

Objective To study the protective immunity induced by peptides of Toxoplasma gondii apical membrane antigen 1(TgAMA1)and rhoptry neck protein 2(TgRON2)containing T-and B-cell epitopes against toxoplasmosis in mice. Methods Chemically synthesized peptides of TgAMA1 and TgRON2 that contained T-and B-cell epitopes were predicted using bioinformatic analysis.Forty-eight five-week-old BALB/c mice were randomly divided into an AMA1 group,a RON2 group,an A1+R2 group,and a control group(n=12).Mice were immunized with 30μg of AMA1,30μg of RON2,or 15μg each of A1+R2.Mice in the control group received an intranasal drip with 20μl of normal saline.Mice were immunized 3 times at a 2-week interval.Two weeks after the final immunization,the serum antibody titer was determined using ELISA.SIgA levels in nasal,vesical,and intestinal washes were determined using ELISA.The spleen was removed,and a suspension of spleen cells was prepared.Lymphocytes were cultured,and the level of cytokines was determined.Samples of liver and brain tissues were collected,and tachyzoites were counted to evaluate mucosal and systemic immune responses and the protective effects against Toxoplasma gondii infection. Results Results indicated that mice immunized with AMA1 and RON2 peptides had high levels of a specific antibody response and a lymphoproliferative response.The levels of serum-specific IgA,total IgG,IgG1,and IgG2 ain mice immunized with peptides were higher than those in the control group(P<0.05 or P<0.01).Levels were highest in the A1+ R2 group.The spleen lymphocyte stimulation index(SI)indicated that levels in the AMA1 group(1.171±0.116),RON2 group(1.362±0.110),and A1+ R2 group(1.941± 0.162)were higher than those in the PBS group(0.743 ± 0.081)(P<0.05 or P<0.01).Levels in the A1+ R2 group were also higher than those in groups receiving a single peptide(P<0.05).The levels of IFN-γ,IL-2,and IL-4 in the spleen cell culture supernatant from mice immunized with peptides were significantly higher than those in the control group(P<0.05 or P<0.01).Peptide immunization induced a high level of mucosal immune response in mice.The SIgA levels in nasal,vesical,and intestinal washes from mice immunized with peptides were significantly higher than those in the control group(P<0.05 or P<0.01).The mice immunized with peptides also had partial protection against chronic Toxoplasma gondii infection.The tachyzoite load in the liver and brain of mice immunized with peptides decreased significantly in comparison to that the control group,and the rate of reduction was 55.79%and 55.68%in the A1 + R2 group,52.36% and 46.46%in the AMA1 group,40.02% and 47.33% in the RON2 group,respectively(P<0.05 or P<0.01). Conclusion Results indicated that AMA1 and RON2 peptides containing T-and B-cell epitopes are a potential antigen with which to combat toxoplasmosis,and intranasal immunization induced strong mucosal and systemic immune responses.A combination of the two peptides provided greater immune protection than that of AMA1 or RON2 alone,and action against toxoplasmosis was more marked.