How human dendritic cells respond to the vaccine vector MVA: elucidating mechanisms at the intersection of viral replication and innate immune sensing

人类树突状细胞如何响应疫苗载体 MVA：阐明病毒复制和先天免疫感应交叉的机制

• 批准号: 390979210
• 负责人: Dr. Marius Döring, Ph.D.
• 金额: --
• 依托单位: Institut Curie
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/390979210?language=en
• 关键词: How; human; dendritic; cells; respond

Modified vaccinia virus Ankara (MVA) a DNA encoded poxvirus is a promising candidate live attenuated vaccine vector for HIV infection/AIDS, tuberculosis and some types of cancer. It induces potent antigen-specific T cell responses and replicates in human cells but does not produce viral progeny. Multiple innate sensors including TLRs, cytosolic DNA and RNA sensors as well as the inflammasome have been implicated in MVA sensing by myeloid cells. Among myeloid cells, dendritic cells (DCs) are critical for the induction of protective immune responses. In DCs, MVA infection leads to vaccine antigen expression, but paradoxically also to cell death and down regulation of costimulatory molecules. How DCs sense and respond to MVA infection remains incompletely understood. We are studying the immune response of human monocyte-derived DCs (MDDCs) to MVA, compared it to HIV-1 with Vpx a well-established model of cGAS-dependent sensing in MDDCs and investigate the role of uninfected bystander DCs in immune activation. To this end, MDDCs were generated from healthy donors. We overexpress a dominant-negative IRF3 (IRF3DN) and silenced cGAS using shRNA containing lentivectors. DCs were infected with MVAGFP or HIVGFP and CD86, Siglec-1, as well as GFP and cytokine expression were analyzed by FACS. We found that CD86 was induced on uninfected bystander DCs, while it was downregulated on MVA-infected DCs. IRF3DN inhibited DC activation by MVA and HIV. In contrast, cGAS depletion only modestly decreased DC response to MVA, while it abrogated the DC response to HIV. Cross-talk between MVA-infected and bystander DCs was mediated by soluble factors. Using blocking antibody cocktails against IFN I and TNF we showed that transactivation partially required type I IFN, TNF, and also additional JAK/STAT pathways. When pre-activating DCs before MVA-infection, we noticed an enhanced susceptibility of DCs to MVA without increased cell death. Unexpectedly, we found that MVA did not replicate in WT DCs, while it did in control HeLa cells. We identified SAMHD1 as restriction factor responsible for blocking MVA replication in DCs. Alleviating this restriction increased AraC-sensitive MVA antigen expression in DCs. Our results highlight the interplay between infected and bystander human DCs in response to MVA. However, the pre-activation of DCs using a TLR ligand induced high frequencies to DCs expressing both target antigen and CD86. Interestingly, our results show that MVA antigen production in DCs is largely compromised by the activity of the restriction factor SAMHD1. Altogether, these insights may help to improve MVA as a putative life attenuated vaccine vector.

修饰安卡拉痘苗病毒（MVA）是一种DNA编码的痘病毒，是HIV感染/艾滋病、结核病和某些类型癌症的一种有希望的候选减毒活疫苗载体。它诱导强效抗原特异性T细胞反应并在人细胞中复制，但不产生病毒后代。多种天然传感器包括tlr、细胞质DNA和RNA传感器以及炎性体都与髓细胞感知MVA有关。在髓细胞中，树突状细胞（dc）对诱导保护性免疫反应至关重要。在dc中，MVA感染导致疫苗抗原表达，但矛盾的是，也导致细胞死亡和共刺激分子的下调。dc如何感知和应对MVA感染仍不完全清楚。我们正在研究人类单核细胞来源的dc （MDDCs）对MVA的免疫应答，将其与HIV-1与Vpx进行比较，Vpx是MDDCs中cgas依赖性感知的成熟模型，并研究未感染的旁观者dc在免疫激活中的作用。为此目的，千年发展目标是由健康的捐助者产生的。我们使用含有慢载体的shRNA过表达显性阴性IRF3 （IRF3DN）和沉默cGAS。分别用MVAGFP、HIVGFP和CD86、siglec1感染dc，流式细胞仪检测GFP和细胞因子的表达。我们发现CD86在未感染的旁观者dc上被诱导，而在mva感染的dc上被下调。IRF3DN抑制MVA和HIV对DC的激活。相比之下，cGAS消耗仅适度降低了DC对MVA的反应，而消除了DC对HIV的反应。mva感染的细胞和周围dc之间的串扰是由可溶性因子介导的。使用阻断抗体鸡尾酒对抗IFN I和TNF，我们发现部分的转激活需要I型IFN、TNF和额外的JAK/STAT通路。当在MVA感染前预激活DCs时，我们注意到DCs对MVA的易感性增强，但细胞死亡却没有增加。出乎意料的是，我们发现MVA在WT dc中没有复制，而在对照HeLa细胞中却有。我们确定SAMHD1是负责阻断dc中MVA复制的限制因子。缓解这一限制可增加dc中arac敏感MVA抗原的表达。我们的结果强调了受感染的和旁观者的人类dc对MVA的反应之间的相互作用。然而，使用TLR配体对dc进行预激活可诱导高频率的dc同时表达靶抗原和CD86。有趣的是，我们的研究结果表明，dc中MVA抗原的产生在很大程度上受到限制因子SAMHD1活性的影响。总之，这些见解可能有助于改进MVA作为假定的生命减毒疫苗载体。