Mechanism of action and therapeutic utility of immunostimulatory CpG oligonucleo

细菌DNA中存在的未甲基化的CpG基序与Toll样受体9相互作用，触发促炎免疫反应。CpG DNA还可改善抗原提呈细胞功能，从而促进获得性免疫的发展。在过去的几年里，我的实验室证实CpG ODN可以降低宿主对感染和过敏性炎症的易感性，作为疫苗佐剂是有效的，并在癌症治疗中发挥作用。我们最近证明，它们还可以加速伤口修复。这些临床前研究表明，CpG ODN既可用于治疗已有的疾病，又可加速创伤或(肿瘤)手术后的伤口愈合。部分基于这些临床前数据，一些探索CpG ODN安全性和有效性的I-III期临床试验已经启动。我的实验室正在进行的临床前研究旨在确定CpG ODN传递的最佳治疗窗口，并检查它们引发的保护性免疫反应是否可以通过与其他免疫调节剂(如额外的TLR配体和小分子激动型免疫增强剂)相结合来加速和/或放大。我的实验室用来检查CpG ODN佐剂活性的模型系统之一涉及炭疽疫苗。相关小鼠模型的最新结果表明，将CpG ODN添加到目前获得许可的炭疽疫苗(AVA)中，可将保护性抗体效价在体内保持的持续时间延长四倍以上，并产生更高亲和力的记忆B细胞群，即使在抗体产生减少后仍能提供对攻击的保护。其他结果表明，CpG ODN的佐剂活性可以通过使用新的制剂或通过将它们与额外的免疫激活剂相结合来进一步提高。例如，当CpG ODN与Poly I：C结合使用时，可以观察到协同作用。要优化CpG ODN的治疗效果，需要详细了解它们激活的细胞(直接和间接)、它们的作用持续时间，以及参与调节这些反应的调节通路。为了阐明这些问题，我们正在使用微阵列技术来识别CpG ODN诱导的免疫刺激的核心基因和网络。这些实验在体外对高纯度的细胞亚群进行，并在体内进行，以监测生理条件下的基因表达。结果表明，基因表达的显著变化可以在给药后15分钟内检测到，并持续至少9天。肿瘤坏死因子α、IL-1β、干扰素-γ和IL-6在CpG介导的基因激活(至少涉及700个基因的表达)的初始上调中起着关键作用。单个基因被激活的程度受到另外一组共调控基因的影响。在注射ODN三天后，上调基因的数量下降了85%。这一作用部分是由一组下调调控因子(包括MYC、FOS和SOCS)介导的，它们主动抑制CpG诱导的基因表达。通过在体内将特定基因表达的变化与CpG ODN的疗效相关联，我们希望这些研究将识别预测临床有益结果的遗传标记。

The unmethylated CpG motifs present in bacterial DNA interact with toll-like receptor 9 to trigger a pro-inflammatory immune response. CpG DNA also improves antigen presenting cell function, thereby facilitating the development of adaptive immunity. Over the past several years, my laboratory established that CpG ODN could reduce host susceptibility to infection and allergic inflammation, were effective as vaccine adjuvants, and had a role in the treatment of cancer. We recently demonstrated that they could also accelerate wound repair. These pre-clinical studies suggest that CpG ODN may be used to both treat established diseases and speed wound healing after trauma or (oncologic) surgery. Based in part on this pre-clinical data, a number of phase I through III clinical trials exploring the safety and efficacy of CpG ODN have been initiated. Ongoing pre-clinical research in my lab is designed to identify the optimal therapeutic window for CpG ODN delivery, and examine whether the protective immune responses they elicit can be accelerated and/or magnified by combining them with other immunomodulatory agents (such as additional TLR ligands and small molecule agonistic immune potentiators). One of the model systems used by my lab to examine the adjuvant activity of CpG ODN involves the anthrax vaccine. Recent results in a relevant mouse model indicate that adding CpG ODN to the currently licensed anthrax vaccine (AVA) prolongs the duration over which protective Ab titers are maintained in vivo by more than four-fold, and generates a higher affinity memory B cell population capable of providing protecting against challenge even after Ab production wanes. Additional results suggest that the adjuvant activity of CpG ODN may be further improved through the use of novel formulations or by combining them with additional immune activators. For example, synergistic interactions were observed when CpG ODN were used in combination with poly I:C. Efforts to optimize the therapeutic utility of CpG ODN require a detailed understanding of the cells they activate (both directly and indirectly), their duration of action, and the regulatory pathways involved in mediating these responses. To clarify these issues, we are using microarray technology to identify the genes and networks central to the immune stimulation elicited by CpG ODN. Such experiments are conducted in vitro on highly purified cell subpopulations and in vivo to monitor gene expression under physiologic conditions. Results indicate that significant changes in gene expression are detectable within 15 minutes of ODN administration and persist for at least 9 days. TNFalpha, IL-1beta, IFNgamma and IL-6 were identified as playing key roles in the initial up-regulation of CpG-mediated gene activation (which involves the expression of at least 700 genes). The degree to which individual genes were activated was influenced by an additional set of co-regulatory genes. Three days post ODN administration, the number of up-regulated genes had fallen by >85%. This effect was mediated in part by a group of down-regulators (including MYC, FOS, and SOCS) that actively suppressed CpG-induced gene expression. By correlating changes in the expression of specific genes with the therapeutic efficacy of CpG ODN in vivo, we hope these studies will identify genetic markers predictive of clinically beneficial outcomes.