Mechanism of action and therapeutic utility of immunostimulatory CpG oligonucleo

细菌DNA中未甲基化的CpG基序与toll样受体9相互作用，引发促炎免疫反应。CpG DNA还可以改善抗原提呈细胞的功能，从而促进适应性免疫的发展。在过去的几年里，我的实验室确定了表达免疫刺激CpG基序（CpG ODN）的合成寡核苷酸可以降低宿主对感染和过敏性炎症的易感性，是有效的疫苗佐剂，并在癌症治疗中发挥作用。最近，我们证明了它们也可以加速小鼠和非人类灵长类动物的伤口修复。这些临床前研究表明，CpG ODN既可用于治疗已建立的疾病，也可用于加速创伤或（肿瘤）手术后的伤口愈合。部分基于这些临床前数据，一些获得我们技术许可的团体已经启动了探索CpG ODN安全性和有效性的I - III期临床试验。我的实验室正在进行的临床前研究旨在确定CpG ODN递送的最佳治疗窗口，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配体和小分子激动性免疫增强剂）联合来加速和/或放大。我的实验室用来检测CpG ODN佐剂活性的模型系统之一涉及AVA，一种获得许可的炭疽疫苗。我们最近的研究结果表明，在AVA中添加CpG ODN通过两种不同的机制显著延长了保护性免疫的持续时间。首先，cpg佐剂AVA引起更快和更强的初始抗体反应，单次接种后抗炭疽抗体水平在保护范围内持续一年以上（明显长于单独接种AVA）。其次，cpg佐剂AVA诱导产生长期存在的高亲和力记忆B细胞群。这些B细胞对炭疽感染的反应如此之快，以至于即使在血清Ab水平下降后，它们也能对宿主产生抗药性。这代表了一种针对生物恐怖病原体提供长期保护的新机制。优化CpG ODN的治疗效用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，以及介导这些反应的调节途径。为了澄清这些问题，我们正在使用微阵列技术来识别CpG ODN引发的免疫刺激的核心基因和网络。这些实验是在体外高度纯化的细胞亚群和体内进行的，以监测生理条件下的基因表达。结果表明，在服用ODN后15分钟内可检测到基因表达的显著变化，并持续至少9天。研究发现，TNFa、IL-1b和IFNg在CpG介导的基因激活的初始上调中发挥了关键作用（最终扩散到涉及近700个基因的表达）。单个基因上调的幅度受到额外的共调节因子的影响，这些共调节因子主要在CpG ODN刺激后24小时内被激活。出乎意料的是，我们观察到CpG ODN递送到小鼠或人类细胞后，基因激活出现了两个高峰。第3小时的初始峰值主要涉及与免疫调节/激活相关的基因，而第2个峰值（第5天）的基因主要与细胞分裂相关。这第二个峰有助于解释CpG ODN在体内的活性延长。CpG ODN给药3天后，上调基因数量减少了85%。这种作用主要是由一组下调蛋白（包括MYC、FOS和SOCS）介导的，它们积极抑制cpg诱导的基因表达。这些抑制因子针对上述关键的上调基因，从而关闭整个刺激网络。该项目的另一个主要治疗目标是改善肿瘤特异性免疫的诱导。我们最近证明，通过将我们的CpG ODN偶联到被杀死的肿瘤细胞上，我们可以给正常小鼠接种疫苗，并显著降低它们对肿瘤攻击的易感性。这种效果不仅在预先接种疫苗然后再攻毒的小鼠中观察到，而且在用慢生长的TRAMP肿瘤细胞系攻毒后5周接种cpg杀伤肿瘤疫苗的动物中也观察到。

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 synthetic oligonucleotides expressing immunostimulatory CpG motifs (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. Most recently, we demonstrated that they can also accelerate wound repair in mice and non-human primates. 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 by groups who have licensed our technology. 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 AVA, the licensed anthrax vaccine. Our recent results show that adding CpG ODN to AVA significantly prolongs the duration of protective immunity via two distinct mechanisms. First, CpG-adjuvanted AVA elicits a faster and stronger initial Ab response, with anti-anthrax Ab levels persisting in the protective range for more than one year after a single vaccination (significantly longer than AVA alone). Second, CpG-adjuvanted AVA induces the generation of a high affinity memory B cell population that persists long-term. These B cells respond to anthrax infection so rapidly that they confer resistance to the host even after serum Ab levels have waned. This represents a novel mechanism for providing long-term protection against bioterror pathogens. 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. TNFa, IL-1b, and IFNg were identified as playing key roles in the initial up-regulation of CpG mediated gene activation (which eventually spreads to involve the expression of nearly 700 genes). The magnitude with which individual genes are up-regulated is influenced by additional co-regulators that are predominantly activated within 24 hr of CpG ODN stimulation. Unexpectedly, we observed two peaks of gene activation following CpG ODN delivery to mouse or human cells. An initial peak at 3 hr primarily involves genes associated with immune regulation/activation while genese in the second peak (at day 5) a largely associated with cell division. This second peaks help explain the prolonged activity of CpG ODN in vivo. By three days post CpG ODN administration, the number of up-regulated genes had decreased by >85%. This effect is largely mediated by a group of down-regulators (including MYC, FOS, and SOCS) that actively suppress CpG-induced gene expression. These suppressors target the critical up-regulatory genes described above, thereby shutting down entire stimulatory networks. The other major therapeutic goal of this project is to improve the induction of tumor-specific immunity. We recently demonstrated by by conjugating our CpG ODN to killed tumor cells, we can vaccinate normal mice and significantly reduce their susceptibility to tumor challenge. This effect is not only observed in mice pre-vaccinated and then challenged, but in animals immunized with our CpG-killed tumor vaccine up to 5 weeks post challenge with the slow-growing TRAMP tumor cell line.