JY佐剂对人乳头瘤病毒18型L1蛋白病毒样颗粒黏膜免疫效果的影响

目的探讨JY佐剂对人乳头瘤病毒(Human papillomavirus,HPV)18型L1蛋白病毒样颗粒(Virus-like parti-cle,VLP)黏膜免疫效果的影响,并观察鼻腔反复免疫后与HPV58、11和6型的免疫交叉反应。方法用含或不含JY佐剂的HPV18 L1 VLP分别经肌肉和鼻腔免疫BALB/c小鼠,并设PBS对照组,于第0、3、6周各免疫1次,免疫后第2、5、8周采血,ELISA法检测血清IgG抗体滴度;于第8周分离脾淋巴细胞并收集呼吸道灌洗液,采用IFNγELISPOT试剂盒检测细胞免疫效果,ELISA法检测黏膜免疫效果;鼻腔免疫各组取5只小鼠,继续免疫10次,每次间隔3周,收集第14、20、26、32和38周的血清,ELISA法检测HPV18 L1蛋白与HPV58、11和6型的免疫交叉反应。结果免疫3次后,不含或含有佐剂的HPV18 L1蛋白经肌肉免疫后的血清IgG抗体滴度(均为104.6)高于经鼻腔免疫后的血清IgG抗体滴度(分别为101.9和102.8)。含有佐剂的HPV18 L1蛋白经鼻腔免疫后呼吸道灌洗液中sIgA抗体浓度(30.06μg/ml)明显高于不含佐剂的HPV18 L1蛋白鼻腔免疫组(23.47μg/ml)(P<0.01),但肌肉免疫组未检测到sIgA抗体。含有佐剂的HPV18 L1蛋白经鼻腔免疫诱导的细胞斑点数明显高于不含佐剂的HPV18 L1蛋白鼻腔免疫组(P<0.01),不含佐剂与含佐剂的HPV18 L1蛋白肌肉免疫组诱导的细胞斑点数差异无统计学意义(P>0.05)。HPV18 L1免疫小鼠后的血清与HPV58、11和6型之间均有交叉反应,在第32周血清特异性IgG抗体滴度最高。结论 HPV18 L1 VLP经肌肉免疫后,其血清抗体水平高于鼻腔免疫组,但该蛋白鼻腔免疫后细胞免疫反应和黏膜sIgA抗体水平均高于肌肉免疫组;JY佐剂能增强HPV18 L1蛋白经鼻腔免疫后的小鼠细胞反应及黏膜sIgA抗体应答,但肌肉免疫组佐剂作用不明显;HPV18 L1蛋白反复鼻腔免疫后,可拓宽抗原保护谱。

Effect of JY adjuvant on mucosal immunity of virus-like particles of human papillomavirus type 18 L1 protein

Objective To investigate the effect of JY adjuvant on mucosal immunity of virus-like particles(VLPs)of human papillomavirus(HPV)type 18 L1 protein,and observe the immune cross reactions among HPV types 58,11 and 6 after repeat intranasal immunization.Methods BALB/c mice were immunized with HPV type 18 L1 protein with or without JY adjuvant for 3 times by intramuscular and intranasal routes,at weeks 0,3 and 6,respectively,using PBS as control.Serum samples were collected at weeks 2,5 and 8 respectively and determined for IgG titer by ELISA.Splenic lymphocytes and washes of respiratory tract were collected at week 8 and determined for cellular and mucosal immunities by IFNγ ELISPOT Kit and ELISA respectively.Half of the mice immunized by intranasal route were further immunized for 10 times,each at an interval of 3 weeks,of which the serum samples were collected at weeks 14,20,26,32 and 38,and determined for cellular immunity as well as cross reactions among HPV18 L1 protein and HPV types 58,11 an 6 by IFNγ ELISPOT.Results The serum IgG titers induced by HPV18 L1 protein with and without adjuvant by intramus cular route(both 10 4.6)were higher than those by in tranasal route(10 1.9 and 10 2.8 respectively).The sIgA concentration in washes of respiratory tract of mice immunized with adjuvant-containing HPV18 L1 protein(30.06 μg/ml)was significantly higher than that with adjuvant-free HPV18 L1 protein(23.47 μg/ml)(P < 0.01).However,no sIgA was detected in the washes of respiratory tract of mice immunized by intramuscular route.The number of spot forming cells was significantly higher in mice immunized with adjuvant-containing HPV18 L1 protein by intranasal route than in those with adjuvant-free L1 protein(P < 0.01),while showed no significant difference in the mice immunized with adjuvant-containing and adjuvant-free HPV18 L1 proteins by intramuscular route(P > 0.05).The sera of mice immunized with HPV18 L1 protein showed cross-reaction with HPV types 58,11 and 6,and the serum specific IgG titer reached the peak value at week 32.Conclusion The serum antibody level induced by HPV18 L1 VLPs by intramuscular route was higher than that by intranasal route.However,the cellular immunity and sIgA level induced with HPV18 L1 VLP by intranasal route was higher than those by intramuscular route.JY adjuvant enhanced the cellular immunity and sIgA level induced with HPV18 L1 protein by intranasal route,while showed no significant influence on those by intramuscular route.Repeat intranasal immunization with HPV18 L1 protein broadened the antigen protective spectrum.