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

改良安卡拉牛痘病毒（Modified Vaccinia virus Ankara，MVA）是一种DNA编码的痘病毒，是一种很有前途的候选减毒活疫苗载体，用于治疗HIV感染/AIDS、结核病和某些类型的癌症。它诱导有效的抗原特异性T细胞反应并在人类细胞中复制，但不会产生病毒后代。包括TLR、胞质DNA和RNA传感器以及炎性小体在内的多种先天性传感器已经涉及骨髓细胞的MVA传感。在骨髓细胞中，树突状细胞（DC）对于诱导保护性免疫应答至关重要。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。DCs如何感知MVA感染并对MVA感染作出反应仍然不完全清楚。我们正在研究人单核细胞衍生的DC（MDDC）对MVA的免疫应答，将其与HIV-1和Vpx（MDDC中cGAS依赖性传感的一个完善模型）进行比较，并研究未感染的旁观者DC在免疫激活中的作用。为此，从健康供体产生MDDC。我们使用含有慢病毒载体的shRNA过表达显性阴性IRF 3（IRF 3DN）和沉默cGAS。用MVAGFP或HIVGFP感染DC，并通过FACS分析⑶ 86、Siglec-1以及GFP和细胞因子表达。我们发现CD 86在未感染的旁观者DC上被诱导，而在MVA感染的DC上被下调。IRF 3DN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的应答，而它消除了DC对HIV的应答。MVA感染的DC和旁观者DC之间的相互作用是由可溶性因子介导的。使用针对IFN I和TNF的阻断抗体混合物，我们表明反式激活部分需要I型IFN、TNF以及另外的JAK/STAT途径。当在MVA感染前预活化DC时，我们注意到DC对MVA的易感性增强，而细胞死亡没有增加。出乎意料的是，我们发现MVA在WT DC中不复制，而在对照HeLa细胞中复制。我们确定SAMHD 1为负责阻断DC中MVA复制的限制因子。减轻这种限制增加了DCs中AraC敏感的MVA抗原表达。我们的研究结果强调了感染和旁观者的人树突状细胞之间的相互作用，以响应MVA。 然而，使用TLR配体预活化DC诱导高频率的DC表达靶抗原和CD 86。有趣的是，我们的研究结果表明，MVA抗原的生产在很大程度上受到限制因子SAMHD 1的活性。总之，这些见解可能有助于改善MVA作为推定的减毒疫苗载体。

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.