HIV DNA疫苗与重组腺病毒伴随病毒联合免疫效果的研究

Immune response induced by HIV DNA vaccine combined with recombinant adeno-associated virus

目的 构建含HIV 1B亚型中国株gagV3基因的DNA疫苗及重组腺病毒伴随病毒(rAAV)疫苗 ,并研究DNA疫苗和rAAV联合免疫的免疫效果。方法 将HIV 1B亚型中国株gagV3基因克隆入真核表达载体pCI neo上 ,构建了含HIV 1gagV3基因的DNA疫苗pCI gagV3。采用电击法将pCI gagV3质粒转染p815细胞 ,用G4 18压力筛选 ,得到转入重组质粒的细胞系p815 gagV3,用免疫酶法检测细胞系中HIV 1基因的表达。用该DNA疫苗进行小鼠免疫实验 ,检测免疫效果 ;用该DNA疫苗初次免疫 ,含同样gagV3基因的重组腺病毒伴随病毒rAAV gagV3加强免疫 ,采用免疫酶法检测免疫小鼠血清中HIV 1特异性的抗体水平 ,用乳酸脱氢酶法检测免疫小鼠的HIV 1特异性CTL水平。结果 pCI gagV3可以在p815细胞中表达HIV 1的基因 ,免疫BALB c小鼠后可以在小鼠体内诱发HIV 1特异性的细胞和体液免疫反应。HIV 1特异性抗体滴度为 1∶2 0 ;效靶比为 5 0∶1时 ,CTL平均杀伤率为4 1 7%。pCI gagV3与rAAV gagV3联合免疫并不能明显提高抗体水平 ,但可以提高CTL反应 ,效靶比为 5 0∶1时 ,CTL平均杀伤率为 6 1 3% ,高于单独用DNA疫苗或重组AAV疫苗免疫后产生的CTL活性。结论 DNA疫苗与重组腺病毒伴随病毒联合免疫可以提高免疫小鼠产生的HIV 1特异性CTL反应。

Objective HIV-1 DNA vaccine and recombinant adeno-associated virus (rAAV) expressing gagV3 gene of HIV-1 subtype B were constructed and BALB/c mice were immunized by vaccination regimen consisting of consecutive priming with DNA vaccine and boosting with rAAV vaccine; the CTL and antibody response were detected and compared with those induced by DNA vaccine or rAAV vaccine separately Methods ⑴ HIV-1 subtype B gagV3 gene was inserted into the polyclonal site of plasmid pCI-neo, DNA vaccine pCI-gagV3 was thereby constructed; ⑵ pCI-gagV3 was transfected into p815 cells, G418-resistant cells were obtained through screening transfected-cells with G418, the expression of HIV-1 antigen in G-418-resistant cells was detected by EIA; ⑶BALB/c mice were immunized with pCI-gagV3 and the immune response was tested; BALB/c mouse immunized with pCI-gagV3 and combined with rAAV expressing the same gagV3 genes were tested for antibody level in sera by EIA method and cytotoxicity response by LDH method Results ⑴pCI-gagV3 could express HIV-1 gene in p815 cells; ⑵ pCI-gagV3 could induce HIV-1 specific humoral and cell-mediated immune response in BALB/c mice The HIV-1 specific antibody level was 1∶20; when the ratio of effector cells: target cells was 50∶1, the average specific cytotoxicity was 417%; ⑶ there was no evident increase in the antibody level induced by pCI-gagV3 combined with rAAV, but there was increase in CTL response, the average specific cytotoxicity was 613% when effector cells: target cells ratio was 50∶1Conclusion HIV-1 specific cytotoxicity in BALB/c mice can be increased by immunization of BALB/c mice with DNA vaccine combined with rAAV vaccine