两种佐剂系统对重组疟疾蛋白疫苗免疫效果的影响

Effect of two adjuvant systems on immune effect of recombinant malaria protein vaccine

目的比较两种佐剂系统对重组疟疾蛋白疫苗免疫效果的影响。方法将BALB/c小鼠随机分为02、03佐剂组、Pr组和NS组,每组24只。02、03佐剂组、Pr组分别经大腿内侧肌肉注射15μg恶性疟原虫融合蛋白-2.9(combine protein 2.9 of Plasmodium falciparum,PfCP-2.9)+15μg恶性疟原虫环孢子蛋白-2(circumsporozoite protein of Plasmodium falciparum 2,PfCSP-2)+02佐剂系统[75μg BCG-CpG-DNA+0.2 mg A(lOH)3)]、15μg PfCP-2.9+15μg PfCSP-2+03佐剂系统[(50μg PolyI:C+0.2 mg A(lOH)3]和15μg PfCP-2.9+15μg PfCSP-2,NS组注射生理盐水。隔周免疫1次,共5次,于初次免疫2周后每周内眦采血,分离血清,采用ELISA法检测血清IgG值及抗体效价;于2、3、4、5次免疫后2周,无菌取小鼠脾脏,制备脾细胞悬液,采用ELISPOT法,对分泌IFNγ、IL-2、IL-4的特异性淋巴细胞数量进行检测。结果 02佐剂组小鼠血清IgG抗体效价在2次免疫后2周即可达到1∶105,03佐剂组在3次免疫后1周达到1∶105,而Pr组在5次免疫后2周达到1∶105,4次免疫后02、03佐剂组达到最高值。分泌IL-4的特异性淋巴细胞数,03佐剂组均明显高于02佐剂组、Pr组及NS组,02佐剂组3、4、5次免疫后才明显高于NS组,5次免疫后才明显高于Pr组(P均<0.05);分泌IL-2的特异性淋巴细胞数,02佐剂组与03佐剂组比较差异无统计学意义(P>0.05),3次免疫后02、03佐剂组均明显高于NS组,且03佐剂组也明显高于Pr组,4次免疫后02、03佐剂组、Pr组均明显高于NS组(P均<0.05),但三者之间差异无统计学意义(P>0.05);分泌IFNγ的特异性淋巴细胞数,02、03佐剂组、Pr组间差异无统计学意义(P>0.05),且03佐剂组在3次免疫后明显高于NS组,而02佐剂组、Pr组在5次免疫后才明显高于NS组(P均<0.05);02、03佐剂组分别在3、5次免疫后分泌IL-4的特异性淋巴细胞数量达到最高,且分别在5、3次免疫后,分泌IL-2的特异性淋巴细胞数量达到最高,二者均需5次免疫后分泌IFNγ的特异性淋巴细胞数量才可达到最高。结论 03佐剂系统可更加有效地增强重组疟疾蛋白疫苗的免疫原性,诱导机体产生较强的细胞免疫和体液免疫反应,5次免疫后可达到最佳免疫效果。

Objective To compare the effects of two adjuvant systems on immune effect of recombinant malaria protein vaccine. Methods BALB/c mice were randomly divided into four groups,24 for each. The mice in 02 adjuvant,03 adjuvant and Pr groups were injected i. m. with 15 μg combine protein 2. 9 of Plasmodium falciparum（PfCP-2. 9）＋ 15 μg（circumsporozoite protein of Plasmodium falciparum 2（PfCSP-2）＋ 02 adjuvant system[75 μg BCG-CpG-DNA ＋ 0. 2 mg Al（OH）3 ],15 μg PfCP-2. 9 ＋ 15 μg PfCSP-2 ＋ 03 adjuvant system[50 μg Poly I ： C ＋ 0. 2 mg Al（OH）3 ]and 15 μg PfCP-2. 9 ＋ 15 μg PfCSP-2 respectively,every other week for 5 times,while these in control group with normal saline（NS）. Serum samples were collected every weeks 2 weeks after the first immunization and determined for IgG level and antibody titer by ELISA. The mouse spleens were collected 2 weeks after the 2nd,3rd,4th and 5th immunization and prepared into splenocyte suspension,and lymphocytes secreting specific IFNγ,IL-2 and IL-4 were counted by ELISPOT. Results The serum IgG titer of mice in 02 adjuvant group reached 1 ∶ 1052 weeks after the 2nd immunization,while that in 03 adjuvant group one week after the 3rd immunization,and that in Pr group 2 weeks after the 5th immunization. However,the IgG titers in 02 and 03 adjavant groups reached the maximum after the 4th immunization. The count of specific lymphocytes secreting IL-4 was significantly higher in 03 adjuvant group than in 02 adjuvant,Pr and NS groups. However,the count in 02 adjuvant group was significantly higher than that in NS group after the 3rd,4th and 6th immunization,and higher than that in Pr group after the 5th immunization（each P〈 0. 05）. The count of specific lymphocytes secreting IL-2 showed no significant difference in 02 and 03 adjuvant groups（P〉0. 05）,which was significantly higher in 02 and 03 adjuvant groups than in NS group after the 3rd immunization,while in 03 adjuvant group than in Pr group（each P〈 0. 05）. However,the counts after the 4th immunization were significantly higher in 02 and 03 adjuvant and Pr groups than in NS group（each P〈 0. 05）,while showed no significant difference in 02 and 03 adjuvant and Pr groups（P〉 0. 05）. The count of specific lymphocytes secreting IFNγ showed no significant difference in 02 and 03 adjuvant and Pr groups（P〉 0. 05）,which was significantly higher in 03 adjuvant group than in NS group after the 3rd immunization, and in 02 adjuvant and Pr groups than in NS group after the 5th immunization（each P〈 0. 05）. The counts of specific lymphocytes secreting IL-4 in 02 and 03 groups reached the maximum after the 3rd and 5th immunization,while those secreting IL-2 after the 5th and 3rd immunization,respectively,and those secreting IFNγ after the 5th immunization. Conclusion Compared with 02 adjuvant system,03 adjuvant system enhanced the immunogenicity of recombinant malaria protein vaccine effectively,and induced strong cellular and humoral immune responses. The immune effect was satisfactory after the 5th immunization.