霍乱病毒B亚单位作为基因佐剂对单纯疱疹病毒2型DNA疫苗免疫效果的影响

Influence of cholera toxin B subunit as a genetic adjuvant on immune effect of herpes simplex virus type 2 DNA vaccine

目的研究霍乱病毒B亚单位(cholera toxin subunit B,CTB)作为基因佐剂对单纯疱疹病毒2型(herpes simplex virus type 2,HSV-2)DNA疫苗pg D免疫效果的影响。方法将CTB片段克隆至真核表达质粒pc DNA3Kan(p Kan)中,构建基因佐剂pc DNA3Kan-CTB(p CTB)。将BALB/c小鼠随机分为p CTB无疫苗组、pg D/p CTB佐剂组、pg D/p Kan空载体组及PBS对照组,经后腿肌肉多点注射,共免疫3次,每次间隔2周。于末次免疫2周后,经小鼠眼球采血,分离血清,ELISA法检测HSV-2特异性Ig G水平及Ig G1、Ig G2a亚型抗体滴度;制备小鼠脾细胞,MTS法检测小鼠脾脏T细胞增殖能力;将病毒稀释后经腹腔接种小鼠,于免疫2周后,进行HSV-2致死剂量攻毒试验。结果p CTB经双酶切及测序鉴定证明构建正确;pg D/p CTB佐剂组小鼠血清中HSV-2特异性Ig G水平明显高于其他3组(P<0.001),Ig G1和Ig G2a滴度均明显高于pg D/p Kan空载体组(P<0.001),其中Ig G2a增幅更明显,Ig G2a/Ig G1比值与pg D/p Kan空载体组相比,由(1.176±0.11)增加至(1.316±0.07)(P<0.05);pg D/p CTB佐剂组小鼠脾细胞在HSV-2刺激下诱导的特异性T细胞增殖反应明显强于PBS对照组及pg D/p Kan空载体组(P<0.05);攻毒14 d内,pg D/p CTB佐剂组小鼠存活率高达100%。结论 p CTB作为基因佐剂能够显著提高pg D诱导小鼠产生抗原特异性Ig G抗体水平,增强HSV-2特异性T细胞增殖反应,并介导以细胞免疫为主的Th1/Th2混合型免疫反应。

Objective To investigate the influence of cholera toxin B subunit（CTB）as a genetic adjuvant on immune effect of herpes simplex virus type 2（HSV-2）DNA vaccine pg D.Methods CTB gene was cloned into eukaryotic expression vector pc DNA3Kan（p Kan）to construct recombinant plasmid pc DNA3Kan-CTB（p CTB）.BALB / c mice were randomly divided into four groups and immunized with p CTB without vaccine,pg D / p CTB,pg D / p Kan PBS as control respectively by intramuscular injection in several sites for 3 times each at an interval of 2 weeks.Serum samples were collected 2 weeks after the last injection and determined for specific Ig G level against HSV-2 as well as Ig G1 and Ig G2 titers by ELISA.Murine splenocytes were prepared and determined for proliferation of T lymphocytes by MTS colorimetric assay.Mice were inoculated i.p.with diluted HSV-2,and challenged with HSV-2 at a lethal dosage 2 weeks later.Results Restriction analysis and sequencing proved that recombinant plasmid p CTB was constructed correctly.The specific Ig G level against HSV-2 in sera of mice in pg D / p CTB group was significantly higher than those in other three groups（P〈.001）,while the Ig G1 and Ig G2 a titers were significantly higher than that in pg D / p Kan group（P〈.001）,of which Ig G2 a titers increased significantly.Compared with that in pg D / p Kan group,the ratio of Ig G2 a to Ig G1 in pg D /p CTB group increased from（1.176 ± 0.11）to（1.316 ± 0.07）（P〈.05）.The proliferation level of HSV-2 specific splenic T lymphocytes in pg D / p CTB group was significantly higher than those in PBS and pg D / p Kan groups（P〈.05）.The survival rate of mice in pg D / p CTB group within 14 d after the lethal challenge was 100%.Conclusion As a genetic adjuvant,p CTB increased the specific Ig G antibody level induced by pg D,enhanced the T HSV-2 specific lymphocyte proliferation response,and mediated a mixed Th1 / Th2 with Th1-biased immune response.