Chitosan-DNA基因免疫诱导粘膜特异性免疫应答及其抗CVB3感染作用

Intranasal gene immunization with Chitosan-DNA vaccine generates mucosal IgA and protects BALB/C mice against CVB3 challenge

目的 :构建新型粘膜基因疫苗 ,诱生CVB3VP1特异性粘膜免疫应答 ,探讨粘膜免疫抗CVB3感染的作用 ,为病毒性心肌炎的特异性防治奠定基础。方法 :抽提CVB3RNA ,以RT PCR扩增得VP1基因 ,插入真核表达载体pcDNA3中 ,构建质粒pcDNA3 VP1,以Chitosan多糖包裹形成Chitosan DNA基因疫苗。将该质粒转染Hela细胞 ,观察其体外表达情况。以 5 0 μgDNA剂量的Chitosan DNA疫苗滴鼻免疫BALB C小鼠 3次 ,检测CVB3特异性体液和细胞免疫应答 ;隔 4周以 5LD50 活CVB3致死攻击小鼠 ,观察攻击后存活情况。结果 :制备了直径为 80～ 10 0nm的Chitosan DNA复合物颗粒 ,体外转染证实Chitosan DNA复合物中VP1的表达高于pcDNA3 VP1质粒 Lipofectamine的表达水平。该疫苗滴鼻 3次免疫后 ,不仅诱生了高水平的IgG ,而且诱生了高水平的粘膜IgA抗体 ,第 6周抗体P N值分别达 3.5和 3.2。特异性细胞免疫应答研究发现 ,该疫苗诱生了较强的VP1特异性CTL杀伤作用 ,并显著高于pcDNA3 VP1和pcDNA3组。CVB3攻击后 ,可保护 33.3%小鼠长期存活 ,而pcDNA3和pcDNA3 VP1质粒滴鼻免疫对照组的平均存活天数分别为 8.5和 10 .8天。病理学研究显示 :Chitosan DNA免疫小鼠心肌组织基本正常 ,而对照小鼠死亡前心肌显示大量的灶性坏死和炎性浸润。结论

Objective:To construct a novel Chitosan-embedded gene vaccine harboring VP1 encoding sequences of Coxsackievirus B type 3 for mucosal immunization.Methods:The gene coding for VP1 was obtained by RT-PCR and was then cloned into pcDNA3 to construct pcDNA3-VP1 plasmid.Chitosan-DNA complexes were prepared by embedding plasmid DNA pcDNA3-VP1 with chitosan.The morphology of the complex was checked by transmission electron microscopy(TEM).In vitro expression of VP1 was performed by transfection of Hela cells with the plasmid and detection of the transgene products by ELISA.BALB/C mice were immunized intranasally three times with 50 μg DNA each time,and challenged with 5LD 50CVB3 four weeks after the last immunization.Results:Chitosan-pcDNA3-VP1 complexes were obtained after embedding pcDNA3-VP1 with Chitosan and proved to be spherical with uniform diameter of about 80～100 nm by TEM.Intranasal immunization with Chitosan-pcDNA3-VP1 generated high levels of both IgG and IgA,the P/N value of which went up to 3.5 and 3.2 at week 6.CVB3 specific cellular immune responses in terms of CTL activity was successfully induced. 33.3% mice immunized which Chitosan-pcDNA3-VP1 were protected from CVB3 lethal challenge to live up to more than 28 days while control mice only lived up to 8.6 and 10.8 days.Infiltrating immune cells or unusual high levels of connective tissue,indicating ongoing myocarditis or fibrosis,were not observed in Chitosan-pcDNA3-VP1 immunized mice but did exist in control mice.Conclusion:Intranasal delivery of Chitosan-pcDNA3-VP1 vaccine could successfully induce CVB3-specific immune responses and protect from lethal challenge by CVB3.Chitosan-pcDNA3-VP1 gene vaccine might be a promising vaccine against CVB3 infection.