IL-15基因佐剂对DNA初始-重组痘苗病毒加强免疫效果的影响

Enhancing effect of adjuvant IL-15 on the immune responses elicited by DNA prime-recombinant vaccinia virus boost in mice

目的比较IL-15在DNA疫苗初始和重组天坛痘苗病毒加强免疫阶段对免疫原性的影响。方法应用PCR方法构建重组片段I8R-pE/L-IL-15-GFP-G1L,通过同源重组将IL-15基因插入到表达HIV-1CN54Gag,Pol和Env基因的重组天坛艾滋病疫苗rTV的基因组中。HIV DNA疫苗在0、3、6周与质粒pIL-15共免疫BALB/c小鼠,或仅使用DNA疫苗,rTV和rTV-IL-15在12周分别进行加强免疫。末次免疫后1、4周应用ICS和ELISPOT方法检测小鼠脾淋巴细胞中HIV特异性细胞免疫。末次免疫后1、4周和12周ELISA方法检测小鼠血清中Gp120特异性抗体。结果 IL-15基因正确插入重组天坛艾滋病疫苗rTV基因组中,成功构建了重组病毒rTV-IL-15。各组免疫小鼠均诱导出HIV特异性细胞和体液免疫。尽管各组诱导的HIV特异性CD8+T细胞免疫反应差异无统计学意义,但加强免疫后4周,DNA-IL-15/rTV-IL-15组分泌IFN-γ、TNF-α以及IFN-γ/TNF-α双因子的CD8+T效应记忆细胞百分比均值显著高于DNA-IL15/rTV组(P<0.05),分别为0.86%,0.37%和0.31%。DNA/rTV-IL-15组的gp120抗体滴度在加强免疫后1周即达到1∶40 381,显著高于DNA-IL-15/rTV组和DNA-IL-15/rTV-IL-15组(P=0.000 1,P=0.020 5),且抗体滴度持续至12周未见明显下降。而DNA-IL-15/rTV组和DNA-IL-15/rTV-IL-15组的抗体滴度则从加强免疫1周后逐渐上升,在12周达到高峰(1∶11 143,1∶16 889),DNA-IL-15/rTV组4周和12周的抗体滴度显著高于1周(P=0.009,P=0.012)。结论在初始和加强免疫阶段使用IL-15佐剂可以提高CD8+T效应记忆细胞应答水平。

Objective To construct recombinant vaccinia Tiantan HIV vaccine that expresses murine IL-15 and compare the effect of IL-15 as an adjuvant when combined with HIV DNA vaccines during the prime and/or boost stages.Methods The recombinant fragment I8R-pE/L-IL-15-GFP-G1L was constructed by polymerase chain reaction（PCR）.Using homologous recombination techniques,the IL-15 gene was inserted in the genome of recombinant vaccinia Tiantan HIV vaccine rTV that express HIV-1 CN54 Gag,Pol and Env genes.Several groups of BALB/c female mice were primed with HIV DNA vaccines at weeks 0,3 and 6,with or without plasmid pIL-15,rTV or rTV-IL-15 boosted at week 12.At weeks 1 and 4 post the final vaccination,the HIV specific cellular immune responses were detected by ELISPOT and ICS;HIV-1 gp120 antibody was evaluated by ELISA at weeks 1,4 and 12 post the final vaccination.Results The recombinant virus rTV-IL-15 was constructed by inserting IL-15 gene into the genome of rTV.All groups of immunized mice elicited HIV-1 specific cellular and humoral immune responses.No significant difference was detected in the HIV-specific CD8＋ T cell response among mice with or without IL-15 co-immunization.The frequencies of effector CD8＋ memory T cells that secret IFN-γ（0.86%）,TNF-α（0.37%） and IFN-γ/TNF-α（0.31%） in the DNA-IL15/rTV-IL-15 group were significantly higher than that in DNA-IL15/rTV group at 4 weeks post the final immunization（P〈0.05）.The titer of gp120-specific antibody in mice immunized with DNA and rTV-IL-15 reached to 1∶40 381 at 1 week post boost,which is markedly higher than that in group DNA-IL-15/rTV and DNA-IL-15/rTV-IL-15（P=0.000 1,P=0.020 5,respectively）,and the high titer levels maintained for at least 3 months.The antibody titers in group DNA-IL-15/rTV and DNA-IL-15/rTV-IL-15 started to rise at week 1 post boost and reached to the peak level after 11 weeks at 1∶11 143 and 1∶16 889,respectively.Conclusions Application of adjuvant IL-15 in prime and boost stages could improve the HIV-specific CD8＋ memory T cell responses.