Name: generation of recombinant arenavirus for vaccine development in fda-approved vero cells
Uploaded: 2013-08-01T13:00:00.000+00:00
Duration: 10 min 3 s
Description: Watch this Scientific Journal Video about Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells at JoVE.com

我们感谢过去和现在的成员JCT和LM-S实验室的沙粒病毒反向遗传学技术和质粒的​​发展和完善。我们也感谢Snezhana的季米特洛娃技术支持。单克隆抗体到JUNV NP（SA02-BG12）获得从BEI资源研究所（NIAID生物防御和新兴传染病研究资源库）。 BYHC支持由格兰特GM068411机构露丝属Kirschstein国家研究服务奖。 EO-R是一种的富布赖特CONICYT（BIO 2008）和罗切斯特疫苗院士（2012年）收件人。 EO-R目前支持从Office在美国罗切斯特大学教师发展与多样性的多样性与学术卓越的一个博士后奖学金。 LM-S实验室研究资助由NIH资助RO1 AI077719，R21NS075611-01，R03AI099681-01A1，NIAID的卓越中心的研究和监测流感（HHSN266200700008C），中心罗切斯特大学生物防御免疫建模（HHSN272201000055C）的。 研究在JCT实验室的补助RO1 RO1 AI077719，从NIH RO1 AI079665 AI047140支持。

1。沙粒病毒救援转我们的救援体系的基础上使用两个聚合酶II蛋白表达质粒编码的核蛋白（NP）和RNA依赖的RNA聚合酶（L），病毒所需的沙粒病毒基因组RNA的复制和基因表达的反式作用因子7，19，和能够直接细胞内合成，通过细胞的RNA聚合酶I（聚合酶-I）中，将S和L反基因组RNA的物种7的质粒。在我们的研究中，我们使用pCAGGS中的蛋白表达载体，使用的鸡β-肌动蛋白启动子和polyadenylanation（PA）的信号序列，和人的聚合酶我的质粒，使用人的聚合酶I启动子和小鼠的终止子序列（ 图2） 。的S和L RNA片段被克隆到HPOL-I方向反基因的质粒HPOL-I的转录产生基因组RNA片段时允许。为抢救野生-TYPE重组LCMV（rLCMV）和坦诚的＃1（rCandid＃1）将pCAGGS NP和L每个病毒共转染连同各自的人力POL-I S和L RNA片段（ 图3）。重组trisegmented LCMV（r3LCMV）的和坦诚的＃1（r3Candid＃1）的产生遵循了类似的协议，但在S段被分成两个不同的POL-I S质粒，每个编码鲜明的报告基因于一体，NP开放阅读框（ORF）的绿色荧光蛋白（GFP）的报告基因（HPOL-I S GFP / GP）和病毒糖蛋白前体（GPC），在另一方面，Gaussia荧光素酶（GLUC）报告基因的ORF被替换为替换（HPOL-I S NP / GLUC的）。 图4中所示的协议的示意图。在6孔板中已建立以下的转染和感染协议。 <ol><li> 的OptiMEM脂质体2000（LPF2000）混合物：准备250微升的OptiMEM媒体，，并根据关于病毒的救援，10 - 12微克每转染LPF2000（1微克/微升）（ 表1）。建议2.5微克LPF2000每微克DNA的比率。 5 - 10分钟，在RT下孵育。同时，准备在一个单独的离心管（1.2）的质粒转染混合物。 </li><li> 质粒DNA转染混合物：准备质粒转染鸡尾酒离心管中，使用每种病毒救援的建议金额（ 见表1）。使终体积为50微升的OptiMEM。 </li><li> 的OptiMEM LPF2000-DNA质粒混合物：加入250μl的OptiMEM LPF2000混合成的质粒DNA转染混合物（1.2）（1.1）。孵育该混合物在RT为20 - 30分钟。同时准备和计数1×10 6每股Vero细胞转染反应。将被转染Vero细胞在悬浮液中。 </li><li> Vero细胞制备：维罗CELLS 100 mm培养皿中培养。在开始之前，将1倍PBS，DMEM培养液10％FBS的1％PS媒体，和胰蛋白酶-EDTA混合物到37℃。 <ol><li>洗涤细胞两次，用5ml的1×PBS。 </li><li>胰蛋白酶消化细胞用1毫升的胰蛋白酶-EDTA，并等待，直到细胞分离（约23分钟）。轻轻拍打可能需要完全分离的细胞从菜肴。 </li><li>小心地重悬细胞，在10毫升的DMEM 10％FBS的1％PS在一个15毫升的离心管中。 </li><li>在离心机中以1,000 rpm的5分钟，离心细胞。 </li><li>在10毫升的DMEM 10％FBS的1％PS和重悬细胞，用血细胞计数器计数细胞，并调整至1×10 6细胞/ ml的浓度。据观察，0.5 - 1×10 6个每转染Vero细胞中产生最好的结果。 </li></ol></li><li> 混合LPF2000/DNA细胞后20 - 30分钟，室温孵育（ 步骤1.3），加1毫升Vero细胞（1×10 6）（ 步骤1.4）LPF2000的OptiMEM质粒DNA混合物在室温下孵育5分钟。 </li><li> 种子LPF2000/DNA-cell到6孔板的组合：（ 步骤1.5）的DNA LPF2000维罗混合物加入到6孔板的孔中。轻轻摇动6孔板，让转染孵育过夜孵化器在37°C和5％的CO 2。 </li><li> 更改转染培养基：约16 - 24小时转染后，除去转染介质，加2毫升感染媒体，培育转染细胞增设48个小时。 </li><li> 细胞通过：感染媒体孵化48小时后，取出组织培养上清（TCS）和转染细胞传递到100毫米的菜。 TCS很少含有转染细胞感染病毒，因为需要额外的孵化产生的病毒颗粒。对于小区通道： <ol><li>洗涤细胞两次，用2毫升的1X PBS中。 </li><li>胰蛋白酶消化细胞<html>第i个0.5毫升的胰蛋白酶-EDTA和，等到细胞分离。 </li><li>在1毫升的DMEM 10％FBS的1％PS 1.5 ml离心管中重悬细胞。 </li><li>离心5分钟，在5,000 rpm，4℃下用微量细胞。 </li><li>小心地重悬细胞，在1毫升的感染媒体，将细胞转移到100毫米的菜。带来的总体积中板，8毫升，有足够的体积，以防止干燥的细胞，以及浓缩病毒，传染性媒体。 </li></ol></li><li>轻轻摇动100毫米盘，并继续培养Vero细胞在孵化器在37℃和5％CO 2 72小时。 </li><li> 收集的TCS：培养72小时后，收集TCS在15毫升离心管。在离心机中以2,500 rpm的转速下离心分离5分钟，以除去细胞碎片。发现传染性沙粒在TCS，所以去除细胞碎片颗粒和存储TCS在-80°C。 </li></ol>乐“&gt; 2。确认重组沙粒病毒救援实现通过一个焦点形成单元（FFU）免疫荧光法（IFA）检测抢救成功rLCMV rCandid＃1。对于野生型病毒拯救，我们使用病毒的NP特异性抗体。的r3LCMV和r3Candid的第1编码的两个报告基因（GFP和GLUC），从而使病毒的抗体，而不需要通过荧光显微镜（GFP）和/或发光（GLUC）的检测和滴定。 <ol><li> 确认野生型重组沙粒病毒救援：滴定前一天，Vero细胞洗两次，用PBS，胰蛋白酶消化并准备96孔板中，达到80 - 90％汇合第二天（4×10 4个细胞/孔）。轻轻摇动板手细胞获得一个穿制服的分布。过夜培养细胞，在37℃培养箱中，用5％的CO 2。在显微镜下检查细胞单层确认之前，继续感染： <ol><li>串行稀释（10倍稀释）的含病毒的回收的TCS从转染的细胞在100mm皿（ 步骤1.10）的OptiMEM。 </li><li>从种子的Vero细胞中取出介质，用50μl的1×PBS中洗两次，并用50μl的系列稀释的病毒（ 步骤2.1）感染细胞。感染细胞1.5小时，在37℃和5％CO 2。 </li><li>感染后，删除病毒接种，加100微升感染媒体/室温孵育16 - 18小时。病毒再感染后可能会出现18小时孵化，这可能会导致高估病毒滴度。 </li><li>于16 - 18小时，感染后，从各孔中取出的TCS，在1x PBS稀释，在RT下15分钟，用4％甲醛固定细胞。 </li><li>接着，去除固定溶液和通透细胞用0.1％Triton X-100在1x PBS稀释，在室温下10分钟。 </li><li>吸出通透的解决方案，然后洗细胞3次无线日1X PBS。 </li><li>座的细胞用2.5％牛血清白蛋白（BSA）在1x PBS（封闭溶液），在RT下进行1小时。另外，细胞可在4℃下封闭过夜，并继续与抗体孵育翌日。 </li><li>同时准备主抗体。用封闭液稀释LCMV和偷拍＃1特异性第一抗体，然后再离心抗体/封闭溶液在3,500 rpm转速下持续15分钟。可用于检测的rLCMV和rCandid＃1的单克隆抗LCMV NP抗体克隆BEI的资源（1:500稀释），1.1.3（1:30稀释）16和单克隆抗JUNV的NP抗体SA02-BG12元。 </li><li>孵育1小时后，吸去封闭液，加入​​50μl的第一抗体的细胞，并温育1小时，在37℃下</li><li>在这个时候，用封闭液稀释的二级抗体，抗体/封闭溶液，在3,500 rpm转速的15分钟，然后离心。的聚克隆的兔抗小鼠IgG抗体FITC标记的第二抗体的初级单克隆抗体，用于检测用于取得在图5中观察到的图像。 </li><li>孵育1小时后，吸出一次抗体，用1×PBS洗涤3次，添加二次抗体30分钟，在37℃下</li><li>孵育30 min后，吸二次抗体和1X PBS洗3次。现在已经准备好待观察细胞用荧光显微镜，同时确定成功的病毒救援和滴定形成单位每毫升（FFU /毫升）计数的焦点。 </li></ol></li><li> r3LCMV和r3Candid＃1病毒救援：由于这些病毒表达基因的两个记者，他们的救助，可以监视通过荧光显微镜检测GFP的表达或TCS GLUC表达。救援也可以证实表达GLUC的TCS。要滴定trisegmented的病毒，我们按照步骤2.1至2.1.4，但细胞并不需要到bE固定，以确定救援，因为一个成功的病毒GFP的表达。病毒滴定将由计数FFU /毫升。要确定病毒救援成功的的GLUC表达，从TCS GLUC表达可以使用GLUC检测试剂盒（碧路Gaussia荧光素酶的检测试剂盒，New England Biolabs公司）测量。为此： <ol><li>到一个96孔白色板（平底微量滴定板中，Fisher Scientific公司）的移液管将100μl的TCS。 </li><li>设置的的光度计（LumiCount，惠普公司）。 </li><li>添加50 - 100微升GLUC测定溶液（制造商建议的）每个样品测量的的记者GLUC基因表达与光度计。作为阴性对照，血糖测定从野生型病毒感染的细胞表达的TCS。 </li></ol></li></ol> 3。组织培养上清液通道病毒救援取决于转染效率。 Vero细胞已被证明具有较低的转染效率比其它细胞系14。如果在TCS的病毒滴度低，0.01（LCMV）或0.1（坦率＃1）的感染复数（MOI）感染新鲜的Vero细胞72小时，以扩增病毒。

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M., Kunz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+entry+by+human+pathogenic+arenaviruses.">Cell entry by human pathogenic arenaviruses.</a> Cellular Microbiology. 10, 828-835 (2008).</li><li>Rojek, J. M., Sanchez, A. B., Nguyen, N. T., de la Torre, J. C., Kunz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+mechanisms+of+cell+entry+by+human-pathogenic+Old+World+and+New+World+arenaviruses.">Different mechanisms of cell entry by human-pathogenic Old World and New World arenaviruses.</a> Journal of Virology. 82, 7677-7687 (2008).</li><li>Sanchez, A. B., de la Torre, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+the+prototypic+Arenavirus+LCMV+entirely+from+plasmid.">Rescue of the prototypic Arenavirus LCMV entirely from plasmid.</a> Virology. 350, 370-380 (2006).</li></ol>

代重组砂粒病毒使用基于质粒反向遗传学技术已经成为一种广泛使用的方法调查砂粒病毒生物学的许多不同的方面。在这里，我们记录到当前系统中的一个关键的改进，通过执行沙粒救援在Vero细胞中，允许对砂粒病毒和疫苗载体对其他传染病的潜在代FDA批准的候选疫苗。 实验过程涉及的一般完善，应该不会构成重大障碍。然而，有几个因素，应仔细监测，以确保一致的成功。 Vero细胞的正确维护是一个成功的病毒救援的关键。此外，它是必须有良好的载体制剂，产生重组的沙粒（见适当的细胞维护和质粒制备的协议 ）。对于救援的rLCMV rCandi的- ＃1野生型或trisegmented的版本建议三个独立的各重组病毒转染增加抢救成功的可能性，因为Vero细胞不容易转染14。如果有一个以上的重组病毒拯救尝试，相应地缩放以下步骤获救病毒的数量。作为阴性对照，我们包括在没有将pCAGGS（NP-pCAGGS中NP）转染。 由于LCMV和坦诚的＃1感染的细胞不显示典型的细胞病变效应（CPE），成功的病毒救援野生型rLCMV的和rCandid的＃1病毒通过使用经典传染性后代，可以做检测，必须进行评估斑块检测或通过IFA。我们推荐使用的IFA经典斑块的检测，因为前者可以完成24小时内，而不是5 - 6天斑块的发展。该r3LCMV或r3Candid的＃1被救出两个记者基因编码（GFP和GLUC），从而使病毒检测中和滴定法，而不需要的抗体，通过荧光显微镜（GFP）和/或发光（GLUC）。在重组LCMV偷拍＃1野生型的救援抢救，不成功的trisegmented的的病毒救援将导致荧光Vero细胞缺乏与TCS从转染细胞接种后。 由于S和L段由人类polyermase I启动子驱动，该救援系统可以应用在其他的人体细胞。事实上，我们还成功地产生两种重组的野生型trisegmented rLCMV和rCandid的＃1病毒在293T人胚肾细胞高效率。此外，转染细胞单层也导致病毒救援，虽然效率不高时，转染Vero细胞和293T细胞悬浮液中。在检测LCMV偷拍＃1野生型病毒救援方面，主要对其他病毒蛋白的抗体也可以使用。在R3病毒的情况下，我们可以检测GLUC GFP的表达，而不是发光。另外，其他报告基因可以用来代替GLUC和GFP 5。 我们的病毒拯救系统已被证明是高效的在我们手中，在这里，我们提供最佳转染条件。强烈建议使用指定金额的质粒。如果在其他人的细胞系，细胞的量的DNA，和/或LPF2000进行病毒拯救应该进行测试。 将允许代野生型和trisegmented的沙粒病毒在Vero细胞中的质粒为基础的反向遗传学研究沙粒病毒的生物学多个方面，以及为未来的发展FDA批准的疫苗和疫苗载体。

与一个bisegmented，负链RNA基因组的3属于沙粒病毒科的的砂粒病毒是有包膜的病毒。沙粒病毒基因组编码的蛋白质在一个的时尚ambisense从两个不同的病毒分部3。大（L）段编码RNA依赖的RNA聚合酶（L）和小环（非常有趣的新基因）指蛋白（Z）作为病毒出芽的主要推动力。小段（S）编码病毒的核蛋白（NP）的表面糖蛋白（GP）（ 图1）。 沙粒病毒科的几位成员负责致命的出血热（HF）在人类3。原则问题是拉沙病毒（LASV）和胡宁病毒（JUNV），这是众所周知的导致高死亡率住院患者8，9。尽管这些病毒的西非和阿根廷农村地区，分别是地方性的，有越来越多的关注进口LASV JUNV的非流行地区由于旅游增加10。此外，尽管通常不伴有严重的人类疾病，被认为是原型沙粒淋巴细胞性脉络丛脑膜炎病毒（LCMV）一个被忽视的病原体已经有致死性感染的情况下，在免疫功能低下的患者6，15，先天性出生缺陷和自然流产负责孕妇中，13。目前，还没有美国FDA批准的疫苗的砂粒病毒和治疗是有限的，以三氮唑核苷核苷类似物，这是只是部分有效，并经常伴有显着的副作用。 基于质粒重组砂粒病毒7,19反向遗传学4代的推出，极大地推进了砂粒病毒研究领域。目前，啮齿动物细胞（如BHK-21）是用于根儿振动性的重组的砂粒病毒由于种属特异性的小鼠RNA聚合酶I（聚合酶-I）启动子指导的S和L段的初始转录。然而病毒拯救BHK-21细胞的代重组砂粒病毒作为潜在疫苗种子候选人未获批准的方法。在这里，我们记录使用人POL-I启动旧世界的原型（OW）LCMV和新世界（NW）JUNV偷拍＃1株在Vero细胞有效的救援。使用类似的方法，我们产生重组trisegmented LCMV（r3LCMV）和坦率＃1（＃1 r3Candid）的砂粒病毒，包含两个额外的编码的外源基因在两个不同的S RNA片段5。这个新的系统，不仅按照高度的重现性和简单的协议，但在生成的重组砂粒病毒作为的潜在疫苗或疫苗载体的种子，可以立即实施。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Material and methods&lt;/strong&gt;</td></tr><tr><td colspan="4"> &lt;strong&gt;Cell lines&lt;br/&gt; &lt;/strong&gt;Vero E6 (African green monkey kidney epithelial cells) are maintained in a 37 &amp;deg;C incubator with 5 % CO&lt;sub&gt;2&lt;/sub&gt; in DMEM 10 % FBS 1 % PS. Cells are available from the American Type Culture Collection (ATCC, catalogue number CRL-1586).&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;Plasmids&lt;br/&gt; &lt;/strong&gt;All plasmids, with the exception of hpol-I L, can be grown at 37 &amp;deg;C, for 16-18 hr. We recommend that cultures of the hpol-I L be grown at 30 &amp;deg;C for 24 hr. Plasmids are prepared using a plasmid maxi kit (EZNA Fastfilter Plasmid Maxi Kit, Omega Bio-tek) following the manufacturer&#039;s recommendations and stored at -20 &amp;deg;C. The concentration of the purified DNA plasmid is determined by spectrophotometry at 260 nm, with purity being estimated using the 260:280 nm ratio. Preparations with 1.8-2.0 260:280 nm ratios are considered appropriate for virus rescue purposes. Additionally, plasmid concentration and purity should be confirmed with agarose gel electrophoresis.&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;Viruses&lt;br/&gt; &lt;/strong&gt;The described protocol for rescuing rLCMV (Armstrong 53b) and rCandid#1 can be executed under biosafety level (BSL) 2 conditions. Contaminated material, including TCS and cells should be sterilized before disposal. Rescue of other arenavirus may require higher BSL facilities so proper safety/security measures must be followed.&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;Tissue culture media and solutions&lt;br/&gt; &lt;/strong&gt;&lt;strong&gt;DMEM 10 %FBS 1 %PS:&lt;/strong&gt; 445 ml Dulbecco&#039;s modified Eagle&#039;s medium (DMEM), 50 ml of Fetal Bovine Serum (FBS), and 5 ml of 100X Penicillin/Streptomycin (PS). Store at 4 &amp;deg;C. This media will be used for maintenance of Vero cells.&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;Infectious Media:&lt;/strong&gt; 2-to-1 mixture of OptiMEM and DMEM 10 %FBS 1 %PS. Store at 4 &amp;deg;C. This media will be used during viral infections.&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;10X Phosphate buffered saline (PBS):&lt;/strong&gt; 80 g of NaCl, 2 g of KCl, 11.5 g of Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O, 2 g of KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;. Add ddH&lt;sub&gt;2&lt;/sub&gt;O up to 1 liter. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature.&lt;br/&gt; &lt;strong&gt;1X PBS:&lt;/strong&gt; Dilute 10X PBS 1:10 with ddH&lt;sub&gt;2&lt;/sub&gt;O. Sterilize by autoclave and store at room temperature.&lt;br/&gt; &lt;strong&gt;2.5 % BSA&lt;/strong&gt;: 2.5 g of BSA in 97.5 ml of 1X PBS. Store at 4 &amp;deg;C. This is used as a blocking solution for IFA. </td></tr></tbody></table>

generation of recombinant arenavirus for vaccine development in fda-approved vero cells

沙粒病毒反向遗传学的发展和实施代表的一个重大突破在沙粒领域4。沙粒病毒的生命周期的不同步骤的调查病毒的决定因素的贡献，以及病毒 - 宿主细胞为基础的沙粒一起能够产生预定在他们的基因组突变重组传染性砂粒病毒微型基因组系统的使用提供了便利相互作用和机制沙粒病毒发病机制1，3，11。此外，已经允许发展trisegmented砂粒病毒沙粒病毒基因组的表达还额外的外源基因的使用，从而打开的可能性的沙粒病毒为基础的疫苗载体的应用5。同样，单周期能够表达报告基因的传染性砂粒病毒的发展提供了一种新的实验工具，提高了安全性的研究，涉及极力Ÿ人类致病的砂粒病毒16。代重组砂粒病毒质粒为基础的反向遗传学技术至今依赖于啮齿动物细胞线使用7,19，这带来了一些障碍，发展食品和药物管理局（FDA）许可的疫苗或疫苗载体。为了克服这个障碍，我们在这里描述的高效生成重组砂粒病毒在FDA批准的Vero细胞。

抢救成功重组野生型沙粒将被确认使用IFA（ 图5）病毒抗原的存在。成功的病毒拯救的重组trisegmented病毒的情况下，可以评估通过观察GFP的表达，通过荧光显微镜观察（ 图6A）。抢救成功，将进一步证实通过评估GLUC的表达（ 图6B）。代表性的成果，说明抢救成功野生型和trisegmented的沙粒使用上述表示的协议。 <img alt="图1" src="/files/ftp_upload/50662/50662fig1.jpg" /> 图1。沙粒病毒基因组结构和病毒粒子的结构。的砂粒病毒是包膜的负链RNA病毒与的bisegmented基因组3。每个段使用一个ambisense编码策略，可直接进行合成的两种病毒proteiNS在相反的方向3。大（L）段（7.2 kb的）编码RNA依赖性RNA聚合酶（L）和小环指蛋白（Z）具有类似矩阵的功能，包括指挥萌芽过程3。小（S）段（3.5 KB）编码的糖蛋白前体（GPC）和核蛋白（NP）。 GPC是翻译后加工以生产GP-1和GP-2，关联形成的糖蛋白复合物（GP）的病毒粒子的结构的表面上负责受体识别单元格条目17，18构成的尖峰。 NP encapsidates的病毒RNA基因组（病毒RNA），连同L蛋白形式病毒核糖（vRNPs）的，是最小的病毒的复制和转录的组件12。 IGR：间隔区。 <img alt="图2" src="/files/ftp_upload/50662/50662fig2.jpg" /> 图2。沙粒RESCU的ë键质粒。沙粒病毒在Vero细胞中产生重组质粒的示意图。蛋白表达质粒pCAGGS中使用的鸡β-肌动蛋白启动子和兔β-珠蛋白多聚腺苷酸化信号序列（pA的）直接合成病毒L和NP 4，7。 CMV-IE的增强剂：巨细胞病毒立即早期增强子。内含：鸡β-肌动蛋白内含子序列。 HPOL-I质粒使用人类聚合酶I启动子和终止子序列小鼠聚合酶，直接病毒RNA片段的合成。 pCAGGS中HPOL-I质粒氨苄青霉素抗性（AMPR）。 <img alt="图3" src="/files/ftp_upload/50662/50662fig3.jpg" /> 图3。的野生型和trisegmented的沙粒病毒救援在黑色椭圆形的重组野生型的沙粒病毒的产生所需的质粒pCAGGS中NP，L，和HPOL-I S和L NP和L的要求d来启动病毒的转录和复制。 HPOL-I，S和L直接细胞内合成，通过POL-I，S和L基因组RNA物种。在红色椭圆形所需的代trisegmented砂粒病毒质粒pCAGGS中NP和L，以及HPOL I L质粒，其中所描述的野生型沙粒病毒救援是相同的。 HPOL-I S被分离成两个质粒：质粒，NP与绿色荧光蛋白（HPOL-I S GFP / GP）取代，在其他质粒，GP取代GLUC基因（HPOL-I S NP / GLUC ）。 <img alt="图4" src="/files/ftp_upload/50662/50662fig4.jpg" /> 图4。沙粒沙粒病毒救援的救援协议。在Vero细胞使用6孔板格式的。简言之，Vero细胞悬浮在第1天使用脂质体2000转。在转染后24小时，用新鲜的传染性介质的介质被替换。转染的细胞培养<html>增设48个小时，然后按比例增加为100毫米菜（4天）。 Vero细胞在100mm平皿孵育72小时。所收集的第7天转染后TCS要么IFA（rLCMV和rCandid＃1）或荧光显微镜（r3LCMV和r3Candid＃1）感染后在新鲜的Vero细胞病毒检测。如果病毒滴度低于预期，TCS可用于感染新鲜的Vero细胞以扩增病毒。 <img alt="图5" src="/files/ftp_upload/50662/50662fig5.jpg" /> 图5。通过一个成功的野生型病毒拯救。TCS从细胞传代（100mm平皿）用于感染新鲜的Vero细胞在96孔板中。在感染后24小时，细胞被固定，透化，阻止前LCMV-或偷拍第1特异性抗体染色。用荧光显微镜观察到病毒抗原的存在。比例尺，100微米。 帐篷“FO：保持together.within”=“总是”&gt; <img alt="图6" src="/files/ftp_upload/50662/50662fig6.jpg" /> 图6。成功抢救的trisegmented病毒重组trisegmented LCMV（r3LCMV）的和坦诚的＃1（r3Candid＃1）编码GFP和GLUC。成功的病毒，可以迅速确定救援用荧光显微镜（A）。 GLUC报告基因的表达，可以作为病毒拯救的二次确认。折感应正常化的发光GLUC值由野生型病毒感染（B）。比例尺，100微米。 <img alt="表1" src="/files/ftp_upload/50662/50662table1.jpg" fo:content-width="4.5in" fo:src="/files/ftp_upload/50662/50662table1highres.jpg" /> 表1中。脂质体/质粒DNA浓度为沙粒救援。推荐的DNA和LPF2000浓度产生重组WIL的d型和trisegmented的砂粒病毒在Vero细胞中表示。

Watch this Scientific Journal Video about Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells at JoVE.com

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

救援重组砂粒病毒克隆的cDNA，称为反向遗传学的方法，使研究人员能够调查特定的病毒基因产物的作用，以及其不同的具体领域和残留物的贡献，砂粒病毒的生物学的许多不同方面。同样，反向遗传学技术在FDA批准的细胞系（Vero）疫苗研制的新一代安全有效的疫苗，打击人类致病的砂粒病毒提供了新的可能性。

在FDA批准的Vero细胞疫苗开发产生重组沙粒

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

generation-recombinant-arenavirus-for-vaccine-development-fda

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

16.8K 次观看.  University of Rochester School of Medicine and Dentistry. 救援重组砂粒病毒克隆的cDNA，称为反向遗传学的方法，使研究人员能够调查特定的病毒基因产物的作用，以及其不同的具体领域和残留物的贡献，砂粒病毒的生物学的许多不同方面。同样，反向遗传学技术在FDA批准的细胞系（Vero）疫苗研制的新一代安全有效的疫苗，打击人类致病的砂粒病毒提供了新的可能性。

视频: Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

重组细小病毒与腺病毒派生系统的帮助下高效生产

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

科研

免疫与感染

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

从救援的cDNA重组新城疫病毒

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 1010 infectious particles per ml and can be directly administrated in vivo.

These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro&#160;and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

生产的非复制型犬2型腺病毒衍生载体与纯化

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

有效生成荧光标记疫苗病毒蛋白的方法

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both as an oncolytic virus and a viral-vectored vaccine. NDV is an attractive therapeutic and prophylactic agent due to its well-established reverse genetics system, potent immunostimulatory properties, and excellent safety profile. When administered as an oncolytic virus or a viral-vectored vaccine, NDV elicits a robust antitumor or antigen-specific immune response, activating both the innate and adaptive arms of the immune system. 
Given these desirable characteristics, NDV has been evaluated in numerous clinical trials and is one of the most well-studied oncolytic viruses. Currently, there are two registered clinical trials involving NDV: one evaluating a recombinant NDV-vectored vaccine for SARS-CoV-2 (NCT04871737), and a second evaluating a recombinant NDV encoding Interleukin-12 in combination with Durvalumab, an antiPD-L1 antibody (NCT04613492). To facilitate further advancement of this highly promising viral vector, simplified methods for generating high-titer, in vivo-grade, recombinant NDV (rNDV) are needed. 
This paper describes a detailed procedure for amplifying rNDV in specified pathogen-free (SPF) embryonated chicken eggs and purifying rNDV from allantoic fluid, with improvements to reduce loss during purification. Also included are descriptions of the recommended quality control assays, which should be performed to confirm lack of contaminants and virus integrity. Overall, this detailed procedure enables the synthesis, purification, and storage of high-titer, in vivo-grade, recombinant, lentogenic, and mesogenic NDV for use in preclinical studies.

Here we provide a detailed procedure for production, purification, and quantification of high-titer recombinant Newcastle disease virus. This protocol consistently yields &#62; 6 &#215; 109 plaque-forming units/mL, providing virus quantities appropriate for in vivo animal studies. Additional quality control assays to ensure safety in vivo are described.

从尿囊液中生产高滴度重组新城疫病毒

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both ...

癌症研究

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

用 crispr/cas9 基因编辑生成重组禽流感病毒载体

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian and avian host species, including humans. With the recent advent of rotavirus reverse genetics systems, it has become possible to use directed mutagenesis to explore rotavirus biology, modify and optimize existing rotavirus vaccines, and develop rotavirus multitarget vaccine vectors. In this report, we describe a simplified reverse genetics system that allows the efficient and reliable recovery of recombinant rotaviruses. The system is based on co-transfection of T7 transcription vectors expressing full-length rotavirus (+)RNAs and a CMV vector encoding an RNA capping enzyme into BHK cells constitutively producing T7 RNA polymerase (BHK-T7). Recombinant rotaviruses are amplified by overseeding the transfected BHK-T7 cells with MA104 cells, a monkey kidney cell line that is highly permissive for virus growth. In this report, we also describe an approach for generating recombinant rotaviruses that express a separate fluorescent reporter protein through the introduction of a 2A translational stop-restart element into genome segment 7 (NSP3). This approach avoids deleting or modifying any of the viral open reading frames, thus allowing the production of recombinant rotaviruses that retain fully functional viral proteins while expressing a fluorescent protein.

Generation of recombinant rotaviruses from plasmid DNA provides an essential tool for the study of rotavirus replication and pathogenesis, and the development of rotavirus expression vectors and vaccines. Herein, we describe a simplified reverse genetics approach for generating recombinant rotaviruses, including strains expressing fluorescent reporter proteins.

简化反向遗传学方法，恢复重组性轮状病毒表达报告蛋白

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian ...

Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV&#8217;s easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3&#8217; arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.

This is a method to generate &#8220;scarless&#8221; recombinant vaccinia viruses using host-range selection and visual identification of recombinant viruses.

使用主机范围和视觉选择快速、无缝地生成重组痘病毒

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a ...

Efficient Adeno-Associated Virus Vector Production Using a Cell Factory Platform

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

As the field of gene therapy continues to evolve, there is a growing need for innovative methods that can address these challenges. Here, a unique method is presented, which streamlines the process of generating high-yield and high-purity AAV vectors using a cell factory platform, meeting the quality standards for in vivo studies.

利用 Cell Factory 平台生成高滴度、高质量腺相关病毒载体的简化和标准化程序

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. ...

生物工程

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.