Immunobiology Laboratory, Cancer Research UK, London Research Institute, London, UK.
Gene Ther. 2009 Mar;16(3):359-66. doi: 10.1038/gt.2008.164. Epub 2008 Dec 4.
Replicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines. We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells. However, we find that cytotoxic T-cell responses to a replicon plasmid intramuscular vaccine are not diminished in the absence of TLR3 in vivo. Our results underscore the potential role of TLR3 in mediating immune activation by dsRNA-bearing replicon plasmid-transfected cells and indicate that other innate sensing pathways can compensate for TLR3 absence in vivo.
复制子质粒编码的甲病毒 RNA 复制酶构成了替代传统 DNA 质粒的一种选择，有望用于人类的 DNA 疫苗接种。复制酶活性通过涉及双链 (ds) RNA 中间体的复制过程来扩增转基因 mRNA 的水平，dsRNA 中间体通过激活先天抗病毒反应有助于疫苗的免疫原性。Toll 样受体 3 (TLR3) 是一种 dsRNA 先天免疫受体，由抗原呈递树突状细胞 (DC) 表达。在这里，我们检验了 TLR3 是否是基于复制子质粒的 DNA 疫苗免疫原性所必需的假设。我们表明，体外鼠 CD8α+DC 吞噬死亡的复制子质粒转染细胞，并通过这些细胞内存在的 dsRNA 以 TLR3 依赖的方式被激活。然而，我们发现，体内缺乏 TLR3 并不会降低对肌肉内注射复制子质粒疫苗的细胞毒性 T 细胞反应。我们的结果强调了 TLR3 在介导带有 dsRNA 的复制子质粒转染细胞的免疫激活中的潜在作用，并表明其他先天感应途径可以在体内弥补 TLR3 的缺失。
