霍乱毒素A、B亚单位增强DNA疫苗免疫效果的佐剂活性和机制研究

Both cholera toxin subunit A and B were widely used as adjuvants for protein based vaccines. However,in a pilot experiment,we found that cholera toxin subunit A and B also showed potent adjuvant activity towards DNA vaccine, when their genes were fused with antigen coding gene. And the fusion gene vaccine showed no obvious cytotoxicity. To elucidate the underlying mechanisms, in this study, we will first investigate the influences of cholera toxin subunit A and B on the efficacy of DNA vaccine and fucntion of specific CD8+ T cells elicited by DNA vaccine.Then, by using the methods of RNA chip and mouse cytokine Th1/Th2/Th17 cytometric beads array, we will investigate the gene transciption and cytokine secretion profile of antigen presenting cells after being stimulated both in vitro and in vivo with DNA vaccines encoding fusion immunogens containing CTA or CTB. Finally, by using gene knockout mice or in vivo RNAi technique, we shall clarify the relationship between key differential genes or cytokines and the adjuvant activities of CTA and CTB. This study will not only help to indentify new adjuvant mechanisms of CTA and CTB,but also will provide a new kind of adjuvant for DNA vaccine.

霍乱毒素A、B亚单位通常被用作蛋白类疫苗佐剂，而我们在前期工作中发现：在与目标免疫原融合表达时，霍乱毒素A、B亚单位同样可以显著增强DNA疫苗免疫原性，并且未发现明显的细胞毒性作用。为进一步阐明其佐剂作用特征和机制，本研究将首先以BALB/c鼠为模型，运用多色流式技术观察霍乱毒素A、B亚单位对DNA疫苗所活化的特异性CD8+ T细胞功能和疫苗保护效果的影响。 然后，采用表达谱芯片和多重细胞因子检测技术，在细胞模型和小鼠模型上观察霍乱毒素A、B亚单位对抗原提呈细胞基因转录和细胞因子分泌的影响，并探讨其分子机制。最后，运用基因敲除鼠或体内RNA干扰技术，解析差异基因或细胞因子对霍乱毒素A、B亚单位发挥佐剂效应的影响。本研究的开展不仅有助于补充、深化对CTA、CTB佐剂效应特征及机制的认识，亦可为DNA疫苗寻找到一类新的有效佐剂。

免疫原性弱是制约DNA疫苗向临床应用转化的主要瓶颈，通过添加细胞因子佐剂以及使用电穿孔等新型疫苗接种技术虽可提高DNA疫苗在小鼠体内的免疫原性，但临床试验表明，这些方法并不能有效提高DNA疫苗在人体内的免疫原性。为了寻求增强DNA疫苗免疫原性的新手段，本课题首先探讨了以CTA、CTB为佐剂来提高DNA疫苗免疫原性的可行性及其作用机制。我们发现，将CTA或CTB基因与抗原基因融合后，DNA疫苗的免疫原性均可获得提高，其中尤以CTA的佐剂效果更为显著：经过三次DNA疫苗免疫之后，CTA可显著提高抗原特异性的T细胞应答和抗体应答；而CTB的佐剂效果则需要通过重组痘病毒疫苗加强免疫之后方能得以充分体现。经过进一步探索，我们发现CTA 发挥佐剂效应的主要机制是通过其ADP-核糖基转移酶活性升高细胞内cAMP浓度，藉此激活下游PKA信号通路，继而促进IL-6和IL-1β的表达。在此基础上，为了进一步提高DNA疫苗的免疫原性，我们首次提出并验证了换位免疫策略，实验数据表明：将CTA佐剂与换位免疫策略联合使用可使DNA疫苗的免疫原性得到进一步提升。综上所述，本课题不仅阐明了CTA、CTB对DNA疫苗的佐剂效应，还为进一步提高DNA疫苗免疫原性提供了一条新途径。