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1.
《Vaccine》2022,40(6):934-944
Respiratory Syncytial Virus (RSV) remains a leading cause of severe respiratory disease for which no licensed vaccine is available. We have previously described the derivation of an RSV Fusion protein (F) stabilized in its prefusion conformation (preF) as vaccine immunogen and demonstrated superior immunogenicity in naive mice of preF versus wild type RSV F protein, both as protein and when expressed from an Ad26 vaccine vector. Here we address the question if there are qualitative differences between the two vaccine platforms for induction of protective immunity. In naïve mice, both Ad26.RSV.preF and preF protein induced humoral responses, whereas cellular responses were only elicited by Ad26.RSV.preF. In RSV pre-exposed mice, a single dose of either vaccine induced cellular responses and strong humoral responses. Ad26-induced RSV-specific cellular immune responses were detected systemically and locally in the lungs. Both vaccines showed protective efficacy in the cotton rat model, but Ad26.RSV.preF conferred protection at lower virus neutralizing titers in comparison to RSV preF protein. Factors that may contribute to the protective capacity of Ad26.RSV.preF elicited immunity are the induced IgG2a antibodies that are able to engage Fcγ receptors mediating Antibody Dependent Cellular Cytotoxicity (ADCC), and the induction of systemic and lung resident RSV specific CD8 + T cells. These data demonstrate qualitative improvement of immune responses elicited by an adenoviral vector based vaccine encoding the RSV preF antigen compared to the subunit vaccine in small animal models which may inform RSV vaccine development.  相似文献   
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赵丹华  李玉华 《中国药事》2019,33(11):1264-1269
目的:构建带有荧光素酶基因的乙型脑炎(简称乙脑)病毒感染性克隆,并通过体外拯救获得恢复病毒,用于乙脑病毒致病机理研究、病毒感染在小鼠体内的动态分布以及乙脑疫苗免疫效果评价的高通量方法建立。方法:应用反向遗传学、融合PCR以及无缝拼接等体外重组技术,将荧光素酶报告基因(F-LUC)插入到乙脑病毒SA14-14-2全长感染性克隆的5''非编码区与C蛋白编码区之间,线性化后体外转录为RNA并转染BHK21细胞,在细胞感染和动物试验分别检测恢复病毒的拯救和F-LUC的表达情况。结果:成功构建了带有F-LUC报告基因的SA14-14-2乙脑病毒全长感染性克隆,并拯救获得了重组病毒。基因序列分析及细胞与动物水平均证明拯救的病毒可较高水平表达荧光素酶基因。结论:本研究构建出带有荧光素酶报告基因F-LUC的SA14-14-2感染性克隆,并拯救获得带有荧光素酶的重组乙脑病毒,为乙脑血清中和抗体的高通量筛选、病毒感染在小鼠体内的动态分布和致病机理研究奠定了基础。  相似文献   
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花瞻  安静  王培刚 《中国热带医学》2019,19(11):1009-1013
目的 拟将荧光素酶基因整合入登革病毒(DENV)亚病毒颗粒(RSPs)中,构建能用于抗体介导的感染增强(ADE)高通量筛选的研究工具。方法 将荧光素酶基因luciferase插入DENV包膜蛋白基因prME的3'端,取代prME第二个跨膜区,构建prME-luc融合基因。用prME-luc融合基因转染293T细胞,在上清中检测携带荧光素酶的登革亚病毒颗粒(RSP-luc)。结果 转染prME-luc的293T细胞可以将RSP-luc释放至培养上清中,共转染野生型prME能够提高RSP-luc的产量。Western-blot技术确认了RSP-luc中含有DENV包膜蛋白和荧光素酶形成的融合蛋白。RSP-luc易于制备并可通过超速离心分离纯化,且可通过测定荧光素酶活性快速定量。体外实验显示RSP-luc能够与Vero细胞、U937细胞以及小鼠腹腔巨噬细胞等DENV靶细胞结合,结合过程可被受体类似物硫酸肝素竞争性抑制,也可被DENV特异性抗体4E11增强。结论 RSP-luc 能够模拟DENV 感染过程,有望成为深入研究DENV 与宿主相互作用及ADE 发生机制的有效工具。  相似文献   
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目的建立孕烷X受体(pregnane X receptor,PXR)应答元件萤光素酶报告基因,并检测其活性。方法利用化学合成方法得到五聚体PXR应答元件(everted repeat elelment-6,ER-6元件)5和(direct repeat element-3,DR-3)5序列;将五聚体PXR应答元件序列(ER6或DR3)克隆至p GL3-Promoter载体上;利用萤光素酶报告基因系统检测PXR应答元件报告基因的活性。结果 PXR应答元件萤光素酶报告基因具有明确的活性。PXR激动剂茴香霉素能够剂量依赖地诱导ER6-Luc(R2=0.95;P=0.002 2)和DR3-Luc(R2=0.96;P=0.000 91)报告基因的活性,其EC50值分别为(0.11±0.04)μmol/L和(0.13±0.06)μmol/L;PXR拮抗剂酮康唑能够剂量依赖地降低茴香霉素诱导的ER6-Luc(R2=0.97;P=0.000 85)和DR3-Luc(R2=0.98;P=0.000 11)的活性,IC50值分别为(0.71±0.11)μmol/L和(1.73±0.15)μmol/L。结论成功构建了PXR应答元件报告基因,建立了PXR转录活性的检测方法。  相似文献   
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《Vaccine》2021,39(26):3498-3508
Adenovirus infections are a major cause of epidemic keratoconjunctivitis (EKC), which can lead to corneal subepithelial infiltrates and multifocal corneal opacity. In the current study, we investigated the use of an E1/E3-deleted adenovirus serotype 5 (Ad5) vector as a vaccine administered intramuscularly (IM) or intranasally (IN) against subsequent challenges with a luciferase-expressing Ad5 (Ad5-Luci) vector via eyedrop. We evaluated the adaptive immune response to Ad5 vector vaccination and confirmed a robust polyfunctional CD8 T cell response in splenic cells. Neutralizing Ad5 antibodies were also measured in the sera of vaccinated mice as well as Ad5 antibody in the eye wash solutions. Upon challenge with Ad5-Luci vector 8 weeks post the primary immunization, transduction was significantly reduced by > 70% in the vaccinated mice, which was slightly better in IM- vs. that in IN-vaccinated animals. Resistance to subsequent challenge was observed 10 months post primary IM vaccination, with sustained reduction up to 60% in the Ad5-Luci vector transduction. Passive immunization of naive mice with antisera from IM to vaccinated mice subsequently challenged with the Ad5-Luci vector resulted in approximately 40% loss in transduction efficiency. Furthermore, the mice that received IM immunization with or without CD8 T cell depletion showed > 40% and 70% reductions, respectively, in Ad8 genomic copies after Ad8 topical challenge. We conclude that Ad-vector vaccination successfully induced an adaptive immune response that prevented subsequent Ad transduction in the cornea and conjunctiva-associated tissues in a mouse model of adenovirus keratoconjunctivitis, and that both cellular and humoral immunity play an important role in preventing Ad transduction.  相似文献   
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The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.  相似文献   
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Luminopsins are fusion proteins of luciferase and opsin that allow interrogation of neuronal circuits at different temporal and spatial resolutions by choosing either extrinsic physical or intrinsic biological light for its activation. Building on previous development of fusions of wild-type Gaussia luciferase with channelrhodopsin, here we expanded the utility of luminopsins by fusing bright Gaussia luciferase variants with either channelrhodopsin to excite neurons (luminescent opsin, LMO) or a proton pump to inhibit neurons (inhibitory LMO, iLMO). These improved LMOs could reliably activate or silence neurons in vitro and in vivo. Expression of the improved LMO in hippocampal circuits not only enabled mapping of synaptic activation of CA1 neurons with fine spatiotemporal resolution but also could drive rhythmic circuit excitation over a large spatiotemporal scale. Furthermore, virus-mediated expression of either LMO or iLMO in the substantia nigra in vivo produced not only the expected bidirectional control of single unit activity but also opposing effects on circling behavior in response to systemic injection of a luciferase substrate. Thus, although preserving the ability to be activated by external light sources, LMOs expand the use of optogenetics by making the same opsins accessible to noninvasive, chemogenetic control, thereby allowing the same probe to manipulate neuronal activity over a range of spatial and temporal scales.Optogenetics, which offers precise temporal control of neuronal activity, has been used widely in experimental neuroscience. Although optogenetic probes are indispensable tools, conventionally their application in vivo requires invasive optical fiber implants and thus imposes significant limitations for clinical applications and for applications involving multiple brain regions (1). On the other hand, chemogenetics can modulate neuronal activity throughout the brain using a genetically targeted actuator when combined with a systemically administered small molecule. Although systemic injection of a small molecule is far less invasive than implantation of fiber optics, chemogenetics has its own limitations, such as slow response kinetics and dependence on G protein signaling, which potentially elicits unwanted secondary effects in target neurons (2).Combining the distinct advantages of opto- and chemogenetic approaches would create unprecedented opportunities for interrogation of neural circuits at a wide range of spatial scales. To allow manipulation of activity of dispersed neuronal populations using optogenetic probes without fiber-optic implants, we proposed a different approach where bioluminescence—biological light produced by enzymatic reaction between a protein, luciferase, and its diffusible substrate, luciferin—activates an opsin, which is tethered to the luciferase (3). After injection to the peripheral bloodstream, luciferin reaches a target in the brain because it crosses the blood–brain barrier (4). Light is generated by the luciferase and then activates the opsin, resulting in activation (in case of channelrhodopsins) or inhibition (in case of proton or chloride pumps) of the target neurons. Capitalizing on the major advantage of opsins as powerful generators of electrical current, our approach integrates opto- and chemogenetic methods by preserving conventional photoactivation of opsins where desired, while at the same time providing chemogenetic access to the same molecules, thus allowing manipulation of neuronal activity over a range of spatial and temporal scales in the same experimental animal.Initial proof-of-concept studies showed that Gaussia luciferase (GLuc)-emitted light is able to activate opsins when the two molecules are fused together (luminescent opsin or luminopsin, LMO) (3). Here we report a set of new LMOs, incorporating brighter versions of GLuc, with significantly improved performance. We found that the improved LMOs could modulate neuronal activity via bioluminescence in vitro, ex vivo, and in vivo and could elicit behaviors in freely moving mice.  相似文献   
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