Delivery of self-amplifying RNA vaccines in in vitro reconstituted virus-like particles
Delivery of self-amplifying RNA vaccines in in vitro reconstituted virus-like particles
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DOI：
10.1371/journal.pone.0215031
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发表时间：
2019-06-04
期刊：
影响因子：
3.7
通讯作者：
Gelbart, William M.
中科院分区：
文献类型：
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作者：
Biddlecome, Adam;Habte, Habtom H.;Gelbart, William M.
许多基于mRNA的疫苗因其激活树突状细胞(DC)的特殊潜力而被研究，树突状细胞是免疫系统中高度专门化的抗原提呈细胞，在诱导有效的CD4(+)和CD8(+)T细胞反应中发挥关键作用。在这篇文章中，我们报告了一个新的疫苗/基因传递平台，它展示了使用在病毒蛋白衣壳中保护的自我放大(“复制子”)mRNAs的好处。从植物病毒豌豆褪绿斑驳病毒(CCMV)中提纯的衣壳蛋白被用来在体外组装含有报告蛋白(如荧光素酶或EYFP)或RNA基因形式的串联重复模式抗原SIINFEKL的单分散类病毒颗粒(VLP)，并将其偶联到诺达村昆虫病毒的依赖于RNA的RNA聚合酶上。与未包装的复制子mRNA孵育相比，未成熟DC与这些VLP孵育后，成熟标志物CD80、CD86和MHC-II的激活程度和RNA复制水平都有所提高。CCMV-VLP预先与抗CCMV抗体共同孵育后，RNA摄取/复制增加，DC活化增强，且呈剂量依赖关系。在所有实验中，成熟标志物的表达与DC的RNA水平相关。总体而言，这些研究表明：VLP保护增强了树突状细胞的mRNA摄取；将复制子与目标基因偶联增加了细胞中的RNA和蛋白质水平；抗VLP抗体的存在提高了体外培养的树突状细胞的mRNA水平和激活。最后，用SIIN-FEKL-REPLICON VLP接种小鼠的初步体内实验表明，对干扰素和转铁蛋白诱导呈双重阳性的抗原特异性T细胞略有增加，但显著增加。
Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4(+) and CD8(+) T-cell responses. In this paper we report a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying ("replicon") mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins (e.g., Luciferase or eYFP) or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers - CD80, CD86 and MHC-II - and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIIN-FEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction.