尿苷二磷酸与尿苷三磷酸两种佐剂对HAV抗原及HBsAg诱导小鼠体液免疫应答的影响

目的探讨尿苷二磷酸(Uridine diphosphate,UDP)与尿苷三磷酸(Uridine triphosphate,UTP)两种佐剂对HAV抗原和HBsAg诱导小鼠体液免疫应答的影响。方法分别在HBsAg(1μg)和HAV抗原(18 EU)中加入不同浓度的UDP和UTP(HBsAg组:UDP和UTP均500μg及1、2、5、10 mg,HAV抗原组:UDP和UTP均500μg及1、2 mg),均经腹部皮下多点注射免疫ICR小鼠(HBsAg组:2针,间隔2周,0.1 ml/只;HAV抗原组:单针免疫,0.2 ml/只),并设空白对照组(生理盐水100μl)、抗原组对照组(HBsAg 1μg;HAV抗原18 EU)、铝佐剂组对照组(铝佐剂300μg)、UDP+铝佐剂复合组(HBsAg 1μg+铝佐剂300μg+UDP 2 mg;HAV 18 EU+铝佐剂300μg+UDP 2 mg)和UTP+铝佐剂复合组(HBsAg 1μg+铝佐剂300μg+UTP 2 mg;HAV 18 EU+铝佐剂300μg+UTP 2 mg),分别于末次免疫后(HBsAg组:第4、8、12和16周;HAV抗原组:第4、8、12周)采血,分离血清,ELISA法检测小鼠血清中抗-HBsAg IgG和抗-HAV IgG水平。免疫18周后,分别取HBsAg组中UDP和UTP最佳浓度组及空白对照组小鼠心脏、肝脏、脾脏、肾脏及肺组织进行病理观察。结果除空白对照组小鼠血清检测不到抗-HBsAg IgG外,其余各组小鼠血清抗-HBsAg IgG及抗-HAV IgG水平均在第8周达到峰值,以后逐渐下降。末次免疫后,UDP及UTP各组抗-HBsAg IgG及抗-HAV IgG水平均高于抗原对照组,差异均有统计学意义(P均<0.05)。HBsAg组UDP+铝佐剂复合组产生的IgG水平明显高于抗原对照组、铝佐剂对照组及UDP和UTP各浓度组(P<0.05);HAV抗原组UTP+铝佐剂复合组产生的IgG水平明显高于抗原对照组、铝佐剂对照组及UDP和UTP各浓度组(P<0.05)。在两种抗原中,UDP和UTP的最佳浓度均为2 mg/只。在设置的浓度范围内,UDP和UTP佐剂均未观察到毒性反应。结论UDP和UTP均能明显增强HBsAg及HAV抗原诱导小鼠的体液免疫应答。

Effects of uridine diphosphate and uridine triphosphate adjuvant on humoral immune response induced by hepatitis A virus antigen and hepatitis B surface antigen in mice

Objective To investigate the effects of uridine diphosphate(UDP)and uridine triphosphate(UTP)on humoral immune response induced by hepatitis A virus(HAV)antigen and hepatitis B surface antigen(HBsAg) in mice. Methods The UDP and UTP at concentrations of 500 μg and 1,2,5 and 10 mg were added into 1 μg HBsAg,while those at concentrations of 500 μg and 1 and 2 mg into 18 EU HAV antigen,separately.ICR mice were immunized with the mixture by s.c.injection in several sites.The mixtures containing HBsAg were injected twice at an interval of 2 weeks,0.1 ml for each,while those containing HAV antigen were injected once,at a volume of 0.2 ml,using physiological saline(100 μl),adjuvant-free antigens(HBsAg 1 μg,HAV antigen 18 EU),aluminum adjuvant(300 μg), UDP + aluminum adjuvant(HBsAg 1 μg + aluminum adjuvant 300 μg + UDP 2 mg,HAV 18 EU + aluminum adjuvant 300 μg + UDP 2 mg)and UTP + aluminium adjuvant(HBsAg 1 μg + aluminum adjuvant 300 μg + UTP 2 mg,HAV 18 EU + aluminum adjuvant 300 μg + UTP 2 mg)as controls.Serum samples were collected at weeks 4,8,12 and 16 after the last immunization with mixtures containing HBsAg and at weeks 4,8 and 12 after the last immunization with mixtures containing HAV antigen,and determined for anti-HBsAg and anti-HAV IgG levels by ELISA.Eighteen weeks after the last immunization,the heart,liver,spleen,kidney and lung tissues of mice immunized with HBsAg containing UDP or UTP at optimal concentrations as well as those in blank control group(physiological saline)were collected for pathological examination.Results No anti-HBsAg IgG was detected in blank control group.However,all the IgG levels in sera of mice in other groups reached peak values at weeks 8 after the last immunization then decreased gradually.The anti-HBsAg and anti-HAV IgG levels of mice after the last immunization with the mixtures containing UDP and UTP adjuvant were significantly higher than those in adjuvant-free antigen control groups(P < 0.05).The IgG level in sera of mice immunized with HBsAg 1 μg + aluminum adjuvant 300 μg + UDP 2 mg were significantly higher than those in adjuvant-free antigen and aluminum adjuvant control groups and those with HBsAg containing various concentrations of UDP and UTP(P < 0.05).The IgG level in sera of mice immunized with HAV 18 EU + aluminum adjuvant 300 μg + UTP 2 mg were significantly higher than those in adjuvant-free antigen and aluminum adjuvant control groups and those with HAV antigen containing various concentrations of UDP and UTP(P < 0.05).Both the optimal concentrations of UDP and UTP for HBsAg and HAV antigen were 2 mg/mouse.No toxic reactions were observed in UDP or UTP within the range of designed concentrations.Conclusion Both UDP and UTP enhanced the humoral immune responses induced by HBsAg and HAV antigen in mice.