【作者】 温永俊；

【导师】 杨福合；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2010， 博士

【摘要】 最近研究发现，先天性免疫反应的激活是狂犬病病毒（Rabies Virus,RV）减弱的机制之一。诱导先天性免疫的因素包括炎性趋化因子（RANTES, MIP-1α, IP-10等）和细胞因子（IL-6, IL-1β,和TNF-α），干扰素和干扰素相关基因（IFN-α, STAT1, Mx-1），Toll样受体(TLRs1-3)。这些先天性免疫分子具有诱导树突状细胞活化的能力而且能够引发很强的获得性免疫和对机体提供很好的保护能力。因此，我们把免疫刺激分子整合到病毒中用来增强免疫效率。树突状细胞的活化是非常重要的，特别是在加强针对狂犬病病毒的保护性免疫反应方面。我们利用树突状细胞活化分子作为狂犬病病毒疫苗的遗传佐剂，试图影响先天性免疫反应，诱导出更高的特异性的中和抗体，包括从淋巴结中成熟的和迁移的树突状细胞聚集到炎症部位。本研究中，证明树突状细胞活化分子能够诱导出针对RV的保护性免疫。粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophagecolony-stimulating factor GM-CSF）,巨噬细胞来源的趋化因子CCL22和巨噬细胞炎性蛋白1α（Macrophage Inflammatory Protein-1alpha MIP-1α）分别被克隆到RV基因组全长CDNA中，并且每一个重组的RV分子的抗原性和安全性都在小鼠模型上得到了确认。所有的重组的RV分子对成年鼠无致病性，即使用最高剂量攻击成年鼠也不产生临床症状。对重组狂犬病病毒感染小鼠3天和6天的时候利用RT-PCR技术，检测病毒的总RNA和小鼠腿部肌肉中所含的插入基因，在感染开始之后，RV和重组狂犬病病毒基因组几乎相同，然而插入激活因子的病毒，相应的在免疫后激活因子含量明显升高。通过流式细胞术对进入淋巴结和血液双阳性的B细胞和树突状细胞（Dendritic Cell DC）细胞的数量和活化状态进行量化比较。重组GM-CSF的RV rLBNSE-GM-CSF,重组MDC的RV rLBNSE-MDC和重组MIP-1α的RV rLBNSE-MIP-1α实验组与无外源基因插入的重组RV rLBNSE组感染比较，在淋巴结和血液中产生了更多的B细胞和DC细胞。说明我们插入的趋化因子和细胞因子可以吸引更多的B细胞到神经末梢免疫系统，可以对成熟的DC起到激发和活化的作用。用RV的强毒CVS-24攻击免疫的小鼠，针对重组RV分子的一个单一的免疫反应能够产生GM-CSF或者CCL22的保护率是100%，而针对重组RV分子产生MIP-1α的保护率是80%，总结我们的结果表明，重组的RV分子表达树突状细胞活化分子能够加强免疫保护反应并且具有发展成为下一代RV疫苗的潜力。更多还原

【Abstract】 Recently it was found that activation of the innate immune responses is one of the mechanisms by which rabies virus (RV) is attenuated. Induced innate response genes include inflammatory chemokines (RANTES, MIP-1 , IP-10, etc) and cytokines (IL-6, IL-1 , and TNF- ), IFN and IFN-related genes (IFN- , STAT1, Mx-1), and Toll-like receptors (TLRs1-3). These innate immune molecules are capable of inducing activation of dendritic cells triggered the strongest adaptive immune responses and provided the best protection, so we incorporate the immunostimulatory molecules into chimeric RVs to increase their efficacy. We use of dendritic cell-activation molecules as genetic adjuvants for rabies virus vaccines try to affect the very early events of an immune response and induce much higher specific neutralizing antibody responses, which includes maturation and migration of DCs from the site of inflammation to the draining lymph nodes.The recombinant rabies virus (rRV) carrying chemokines and cytokines were constructed from the SAD L16 strain which mutated the position 194 and 333 amino acids on the G protein. To determine the function of rRVs with expressing dendritic cell-activation molecules in innate and adaptive immunity, we generated rRVs that expressed murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), murine CCL22 (mCCL22) and Macrophage Inflammatory Protein 1 alpha(mMIP-1 ). The rRVs carrying dendritic cell-activation molecules induced much higher increases of RV-specific antibody titers, compared to those responses elicited by just backbone rRV. The rRVs expressing each of these molecules are avirulent since infection with the highest possible dose did not induce any diseases in adult mice but have limited pathogenicity in the baby mice. Peripheral immunization with each of the virus the viral total RNA and insert gene in the mouse muscle were examined by an RT-PCR analysis for rRVs infected mice at 3 and 6dpi. There was almost the same RV genome in rRVs infected mice at the onset of infection, of course, the insert gene products were increased after immunization with recombinant virus respectliy. Double positive numbers of B cells and DC cells were assessed by flow cytometry in the lymph nodes and blood. When cells were stimulated specifically by rLBNSE-GM-CSF, rLBNSE-MDC, and rLBNSE-MIP-1 double positive B cells and DC cells showed a higher signal than rLBNSE B cells in the lymph nodes and blood. These results s ggested that the B and DC cells were more activated by different rRVs with inserts than backbone virus.One single immunization can induce 100% protection in mice against the challenge infection with virulent rabies virus. Together our results indicate that these recombinant rabies viruses expressing dendritic cell-activation molecules enhance humoral and cellular immune responses and have the potential to be developed as the next generation of rabies virus vaccines.更多还原