We thank Sergio G&#243;mez-Medina for excellent technical support with mouse experiments. This work was supported by funds from the Leibniz Association and the Leibniz Center of Infection. A.L. is a recipient of a pre-doctoral fellowship from the Leibniz Graduate School.

动物实验按照批准的方案和下列德国动物保护法的准则进行的。所有开展动物实验的工作人员根据B类或欧洲实验动物科学协会联合会了C通过培训计划。 1.重组产生流感减毒活疫苗通过反向遗传学注：对于重组流感病毒通过反向遗传学产生的具体协议已经由先前的研究5和超出本报告的范围。简单地说，冷适应流感疫苗（CAV）的救助是在生物安全II类下柜生物安全水平进行2（BSL2）围堵，并涉及以下详细步骤。转染和感染协议得出的 马丁内斯Sobrido 等 5。 <ol><li>产生重配冷适应流感使用作为背景的​​冷适应毒株A /安阿伯/ 6/60（H2N2），以及血细胞凝集素（HA）和经修饰的神经氨酸酶（NA）的A / PR / 8/34（H1N1）病毒株的（疫苗图1A）。在NA基因的修改包括在蛋白质茎（残基65至72）基于PCR的置换的保守序列的一个短cDNA序列编码的鸡卵白蛋白（OVA）衍生的肽SIINFEKL（ 图1B）的。在SIINFEKL肽是在H-2b的MHC I类限制的C57BL / 6小鼠12的响应的情况下的高免疫肽。 </li><li>板10 6 293T每孔细胞中使用的DMEM 10％胎牛血清（FBS），补充有1％青霉素/链霉素（P / E）的6孔平板上。 </li><li>制备质粒转染混合物：添加1微克编码和的cDNA PB2，PB1，PA，NP，M NS从流感A /安阿伯/六十○分之六菌株​​的1μg质粒为NA编码的质粒中的每一个的-SIINFEKL和HA流感A / PR / 8/34。添加预热的OptiMEM（15微升100微升终体积/孔）。 </li><li>孵育转染混合物30分钟，在室温。 </li><li>添加100μl的转染混合物/孔孵育16小时，在37℃和5％的CO 2。 </li><li>由DMEM 0.3％BSA的1％的P / E在33℃下进行5小时改变细胞培养基和5％的CO 2。 </li><li>添加10 6流感容许MDCK细胞在含有从用L-1-Tosylamide -2-苯乙基氯甲基酮（TPCK胰蛋白酶）处理牛胰腺胰蛋白酶的1μg/ ml的DMSO中。保持293T-MDCK共培养2 - 1天在33℃和5％的CO 2。 </li><li>在260×g离心5分钟收集的MDCK-293T细胞共培养物的上清液并明确通过离心。 </li><li>接种9 - 10天，老鸡鸡胚在无菌条件下。要做到这一点，做一个洞蛋壳顶部由马丁内斯，Sobrido 等指示。5。 </li><li>覆盖蛋壳机智用棉签，孵育接种鸡蛋3天在33°] C H融化的蜡。 </li><li>收获来自感染胚卵的尿囊液：用70％的乙醇的蛋壳。具有非常平缓裂纹在上部和使用无菌镊子去除尿囊膜打开鸡蛋。用10毫升吸管收集尽可能多的流体尽可能（一般为6 - 10ml）中。离心机在260×g离心10分钟，在4℃，将清除尿囊液转移到新的管中。 </li></ol> 2.跟踪疫苗特异性免疫<ol><li>跟踪肽轴承树突细胞（DC）的<ol><li>对于小鼠免疫接种，麻醉使用精密汽化器小鼠用5％异氟烷在氧气中。使用200μl的移液管施用50微升PBS中含有10 3噬斑形成单位直接表达通过液滴的SIINFEKL肽与小鼠鼻孔疫苗（PFU）。 </li><li>三天后接种，euthaniz通过异氟烷麻醉小鼠ê其次是颈椎脱位。 </li><li>提起小鼠皮肤略低于钳胸腔中线，并通过用剪刀剪皮肤小切口。从切口的点，使3 - 5厘米长的通过朝头部和尾部的皮肤切开，则需要两个2厘米切口从中线横向从各端。剥离背部皮肤，露出腹腔和胸腔。通过围绕腹膜以暴露胸腔膜小心切开。 </li><li>访问纵隔切diafragma和笼肋的底部。找到并移除2纵隔淋巴结（MLNS），稍背和横向相邻胸腺，内侧相邻的头臂动脉，气管的腹侧近。 </li><li>为了收获MLNS使用弯钳。将镊子淋巴结下方，轻轻一拉起来。放置淋巴结在一个6孔板含有1ml DMEM中，并除去任何connecti已经或脂肪组织周围的节点。 <ol><li>附加至1ml注射器2台26摹针透逗每个样品。梳理淋巴结，按住一个针头，而砸开淋巴结与其他针。若这是正确完成，浓缩细胞从淋巴结爆破在媒体容易观察。通过所有的组织材料通过70微米的尼龙过滤器，以获得单细胞悬浮液。 </li></ol></li><li>离心机单细胞悬浮液在260×g离心在冷冻离心机10分钟。重悬在2ml的红细胞溶胞（RBCL）缓冲液将细胞沉淀以裂解红血细胞。允许裂解进行3分钟在室温下。 </li><li>通过离心分离与细胞再悬浮于2ml冰冷的PBS中和RBCL缓冲器。 </li><li>板的细胞在100微升最终体积的U形96孔培养板以10 7个细胞/ ml的浓度。 </li><li>流式细胞仪分析，阻断细胞的Fc受体使用1，每10 6细胞抗鼠CD16 / CD32抗体在冰上15分钟的微克。 </li><li>离心机板和丢弃上清液板轻弹。孵育单井与选择的表面抗体结合。典型地，对于检测的树突状细胞在淋巴结中，抗体鸡尾酒应包括：0.25微克抗CD11c的，0.25微克的CD11b，0.5微克的MHC II类，0.25微克CD103的和在100的终体积0.15微克CD8α的微升。 </li><li>为负门控，添加0.25微克的抗CD3，CD19，NK1.1或谱系混合物（ 图2A）的。备选策略门，以及对多个荧光染料适当的补偿协议，可以在免疫基因组计划的网站上找到。除了 ​​所选择的表面标记，针对H-2K b势必SIINFEKL到鸡尾酒抗体添加1微克。这市售抗体将允许DCS I可视化ñ其中SIINFEKL肽结合表面的MHC I类</li><li>孵育细胞表面的抗体，在冰上30分钟。避光板。离心机板在260×g离心5分钟，并洗涤用2ml的PBS两次。转移细胞到流式细胞仪管用于分析。 </li></ol></li><li>疫苗特异性T细胞增殖评估<ol><li>从市售的TCR转基因小鼠（OT-I小鼠）得到供体T细胞（C57BL / 6-Tg的（TCR aTcrb）1100Mjb / J），其中所有生成CD8 T细胞是特异于SIINFEKL肽。通过异氟烷麻醉颈椎脱位安乐死的小鼠。收获脾通过在腹腔进行切口。将脾/秒2毫升的DMEM /脾脏和消除结缔组织和脂肪组织。 </li><li>产生从脾脏以下在2.1.2和2.1.3中所述的方法单细胞悬浮液。 </li><li>进一步丰富对T细胞的单细胞悬浮液，适用于磁珠分离协议采用或正或负的甄选程序12。区分受体细胞供体，使用同类系小鼠使得例如受体小鼠表达白细胞标记CD45.1而供体的T细胞表达CD45.2 12。 </li><li>标记的T细胞，培养他们在1ml的PBS中含有5μM的羧基琥珀酰亚胺酯（CFSE）中的5×10 6个细胞/ ml的最佳细胞密度。孵育细胞20分钟，在37℃下从曝光保护。培养后，离心细胞，在260×g离心5分钟，弃去上清，重悬细胞在3ml胎牛血清（FBS）的中和的CFSE。 </li><li>在10分钟，重悬在PBS中的适当体积离心机细胞在260×g离心使100微升的溶液含有2×10 6个细胞。注入接受者同类系小鼠用2×10 6个细胞/经由眶后窦注射13鼠标。 </li><li>安乐死受体小鼠在天3，4和5后疫苗接种使用异氟烷麻醉颈椎脱位。收获纵隔淋巴结和制备单细胞悬浮液用于流式细胞术如2.1.2-2.1.5指示。 </li><li>确定供体细胞通过染色在100μl的抗体混合物含有的单细胞悬浮液：0.25微克抗小鼠CD3，CD4和CD8的抗体; 0.5抗小鼠CD45.1或CD45.2抗体（取决于供体细胞的表型）的微克。离开了FL-1通道血细胞计数打开（FITC）的流量，以确定CFSE 12（ 图2B）的稀释曲线。 </li></ol></li></ol> 3.嵌合体小鼠解剖基因特异性疫苗反应注意：使用DTR基于小鼠模型的竞争骨髓嵌合体的产生允许的小区特定功能在疫苗的免疫反应的判别。在这里，我们显示，通过该组合DTR表达供体，c额外策略ELLS与来自敲除小鼠许可证细胞免疫在特定的细胞区室中来研究基因的特定功能。在这个例子中，我们生成小鼠特异性缺失在Langerin + CD103 +树突toll样受体3（TLR3）的。 <ol><li>骨髓供体细胞的制备<ol><li>下II生物安全柜级别工作。通过使用专门的仪器高压灭菌，避免污染。 </li><li>为了产生骨髓嵌合体小鼠，使用同类CD45.1 + C57BL / 6雌性小鼠为受体，并利用TLR3 - / - ，Langerin-DTR / EGFP，或重量小鼠表达CD45.2 +捐助者。使用同类系供体和受体小鼠允许供体和受体细胞通过流式细胞仪的分化，这使植入进行评估。 </li><li>以得到供体骨髓细胞，安乐死供体小鼠用异氟烷麻醉颈椎脱位。用锋利的剪刀进行绕脚踝切口，使得并购乌斯肌肉暴露。 </li><li>使用钝钳拔鼠皮和毛皮轻轻向臀部脚踝，使小腿的肌肉是可见的。 </li><li>用锋利的剪刀剪腿鼠标在臀部的高度和股骨的头部上方。 </li><li>放置腿培养皿并置于冰上。工作速度快，并删除所有的肌肉，露出骨头。 </li><li>独立的股骨胫骨和。放置骨头在70％乙醇中的溶液5分钟，以除去可能的细菌，并立即将它们放置在含有5ml无血清DMEM的另一个培养皿。切骨端用锋利的剪刀和冲洗骨髓成DMEM。通过插入连接于5ml的注射器＆G针为此，冲洗1毫升DMEM中成骨。确保骨骼冲洗后呈现白色（空）。 </li><li>收集在10毫升的DMEM从供体小鼠所有的骨髓细胞。通过使收获通过一个70微米的细胞过滤生成从骨髓细胞的单细胞悬浮液。 </li><li> CL骨髓细胞离心，在260×g离心10分钟，在4℃的冷冻离心机耳单细胞悬浮液。重悬细胞沉淀在10ml PBS中。耗尽使用RBCL溶液如在2.1.3和2.1.4中所述的红血细胞。 </li><li>使用一个血球或自动细胞计数器计数骨髓细胞。通过台盼蓝染色评估13细胞活力通过测定死细胞。最佳地，一个单一的动物产量约3×10 7个白血细胞/小鼠具有至少80％的生存力。 </li><li>在总的骨髓嵌合体的情况下，准备TLR-3 - / -或重量的供体细胞。最优细胞浓度为2×10 7个细胞/ ml，因为2×10 6个细胞在100μl的PBS将用于移植。如果不立即使用，使用慢速冷冻技术和FBS，用10％DMSO中保留的供体细胞在-80℃下。对于混合嵌合模型，混合Langerin-DTR-供体细胞和TLR3 - / - </sup&gt;供体细胞在75:25比率。制备75的混合：25 Langerin-DTR：重量骨髓细胞生成的对照小鼠。 </li></ol></li><li>小鼠照射和移植<ol><li>照射6之间雌性小鼠 - 8周龄的铯-137源照射。给小鼠两种剂量的550弧度4小时分开。分割照射最小化急性肝毒性，因此减少小鼠致死由于照射协议。 </li><li>照射后，养老鼠在用抗生素治疗两周。给予抗生素的饮用水在2.5 - 的拜有利/千克5毫克。 </li><li> 2 - 第二照射后4小时，移植小鼠供体骨髓细胞静脉注射。经由眶后窦注射13和异氟烷麻醉下使用的含2×10 6个细胞加入100μl的PBS最大容积进行注射的供体细胞。 </li><li>移植后4周，评价移植的1​​00的样品中微升外周血通过流式细胞术使用机器能够读取至少两种颜色。捐助者和广播性受体细胞之间的区别，用市售的荧光标记的CD45.1和使用1/100 1/200和稀释之间CD45.2抗鼠抗体。供体细胞的最佳植入应至少80％的总的造血细胞在外周血中。 </li></ol></li><li>疫苗接种和有针对性的枯竭<ol><li>通过鼻内接种小鼠重组减毒活流感疫苗。对于小鼠免疫接种，麻醉使用精密汽化器小鼠用5％异氟烷在氧气中。使用200μl的移液管施用50微升PBS中含有10 3噬斑形成单位疫苗（PFU）。 </li><li>为了产生最终的小鼠模型中，治疗的小鼠用50ng DT的稀释在50μlPBS中的鼻内给药。管理DT一滴一滴直接向麻醉小鼠的鼻孔中3.3.1说明。治疗心肌梗塞CE每日剂量开始治疗在-2天接种前一天，直到2接种后（ 图3B）。 </li></ol></li></ol>

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The authors have nothing to disclose.

在这项研究中，我们描述了如何反向遗传学和嵌合小鼠模型可用于阐明疫苗诱导的免疫的生理和分子机制。流感反向遗传学是建立在许多实验室，并发挥在了解发病的流感，复制和传输17主要作用。在我们的协议的一个关键点是表达外源抗原决定冷适应流感疫苗抢救。同时引入短的cDNA进神经氨酸苷酶的茎的策略已经由许多组，研究者需要确保没有另外的突变过程中的疫苗生产蛋和该疫苗?...

免疫记忆在没有疾病的产生是有效的疫苗接种1的生理基础。最近，系统生物学为基础的方法已经表明，成功的疫苗，如黄热病疫苗，诱导强的诱导先天免疫反应和活化的树突细胞（DC），几种亚群这反过来，导致多向活化的抗原特异性T细胞2,3。由于区议会是唯一的免疫细胞群，激活抗原特异性幼稚T细胞4的能力，其功能的疫苗接种过程中的研究是至关重要的理解免疫反应的疫苗，并设计未来战略打击有挑战性的病原体。 一种系统，允许在免疫应答的疫苗不同的DC子集的跟踪是需要的，以便建立直流偏移的精确动力学到淋巴组织，并因此提供洞察负责疫苗的特异性适应性免疫启动的生理机制。反向遗传学为基础的方法提供以产生修改的可能性，减毒活疫苗，可通过实验用于此目的。因为它的实现对流感的研究，基于质粒的反向遗传学已被广泛采用，以产生重组流感病毒株包括LAIVs。标准协议来拯救重组流感病毒所需要的多转染的含有八个流感病毒节段，以及扩增在一个宽松的系统，如的Madin-Darby犬肾高度转染的细胞系的双义质粒（产生正的和负义RNA）（ MDCK）细胞和/或鸡胚5。然而，反向遗传学中的应用，以产生为了研究疫苗的免疫机制的分子工具仍然未开发。 发电新的小鼠模型允许免疫细胞亚群，包括DCS具体枯竭，开辟了新的可能性，以了解潜在的疫苗引发保护的基本免疫机制。在小鼠和人类DC亚群功能之间的比较表明，在很大的程度上，小鼠和人DCs是功能性同源6,7-，这些发现，有力地表明，小鼠模型的发展允许DC的在稳定状态下的特定耗尽并且在炎性病症，可能有助于理解直流反应在人体中的生理。近年来已经产生了许多小鼠模型携带转基因的表达猿猴白喉毒素（DT）的受体（DTR）的兴趣8,9的基因的启动子区的控制之下。由于小鼠组织中不自然地表达DTR，这些模型允许携带鼠标时接种DT感兴趣的靶基因细胞亚群的条件枯竭。因此，我们的abiliTY过程中的生理过程耗尽特定DC和白细胞等在体内 ，已经大大的DTR-RO为主的发展增强。然而，虽然这些转基因小鼠模型已被广泛使用，以了解免疫系统的个体发育，其应用到疫苗开发已几乎没有测试。这里，通过结合流感反向遗传学和DTR基于竞争的骨髓嵌合体中，我们提出在疫苗的免疫应答的体内研究疫苗免疫以及个别基因功能的动力学的方法。这种技术的对具有挑战性的传染病新型疫苗临床前评价中的应用可以帮助理顺疫苗设计和体内测试候选疫苗。

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			<td>Dulbecco&#180;s Modified Eagle Medium (DMEM 1X)</td>
			<td>Gibco RL-Life Technologies</td>
			<td>41965-039</td>
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			<td>Opti MEM</td>
			<td>Gibco RL-Life Technologies</td>
			<td>31985-047</td>
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			<td>Lipofectamine 2000</td>
			<td>Invitrogen-Life Technologies</td>
			<td>11668-027</td>
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			<td>PAA</td>
			<td>p11-010</td>
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			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
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			<td>Embryonated eggs</td>
			<td>Valo biomedia Gmbh</td>
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			<td>PBS (1X)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
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			<td>70 &#956;M Nylon Filters</td>
			<td>Greiner-Biorad</td>
			<td>542-070</td>
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			<td>Red Blood Cell Lysing buffer (RBCL) 10X</td>
			<td>BD Bioscience</td>
			<td>555899</td>
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			<td>CD16/CD32 Mouse BD Fc Block (2.4G2)</td>
			<td>BD Pharmigen</td>
			<td>553142</td>
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			<td>APC-Anti-mouse SIINFEKL-H2kb (25 D1.16)</td>
			<td>Biolegend</td>
			<td>141605</td>
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			<td>PE-Anti-mouse CD11c (HLA3)</td>
			<td>BD Biosciences</td>
			<td>553802</td>
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			<td>eFluor 450-Anti-mouse MHCII (Md/114.15.2)</td>
			<td>eBioscience</td>
			<td>48-5321-82</td>
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			<td>Pe-Cy7-Anti-mouse CD11b (M1/70)</td>
			<td>Biolegend</td>
			<td>101216</td>
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			<td>PerCp/Cy5.5-Anti-mouse CD103 (2E7)</td>
			<td>Biolegend</td>
			<td>121416</td>
			<td></td>
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			<td>PE-Anti-mouse CD45.1 (A20)</td>
			<td>eBioscience</td>
			<td>12-0453-82</td>
			<td></td>
		</tr>
		<tr>
			<td>V500-Anti-mouse CD45.2 (1O4)</td>
			<td>BD Bioscience</td>
			<td>562130</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCp-eFluor710 -Anti-mouse CD8a (53-6.7)</td>
			<td>eBioscience</td>
			<td>46-0081-80</td>
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			<td>APC-Cy7-Anti-mouse CD3&#949; (145-2611)</td>
			<td>Biolegend</td>
			<td>100325</td>
			<td></td>
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		<tr>
			<td>eFluor450-Anti-mouse CD4 (GK 1.5)</td>
			<td>eBioscience</td>
			<td>48-0041-80</td>
			<td></td>
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			<td>CFSE Proliferation dye</td>
			<td>eBioscience</td>
			<td>65-0850-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Baytril 2.5%</td>
			<td>Bayer</td>
			<td>65-0850-85</td>
			<td></td>
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			<td>Dymethil-Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
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			<td>Ovalbumin&#160;</td>
			<td>Molecular probes&#160;</td>
			<td>O23020</td>
			<td></td>
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		<tr>
			<td>Diphteria Toxin (DT)</td>
			<td>Sigma-Aldrich</td>
			<td>D0564</td>
			<td></td>
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		<tr>
			<td>Trypsin-TPCK</td>
			<td>Sigma-Aldrich</td>
			<td>T1426</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACsCanto II Flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo cell analysis software 9.5</td>
			<td>Flowjo inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)&#160;</td>
			<td>Life technologies</td>
			<td>T10282</td>
			<td></td>
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		<tr>
			<td>Countess Automatic Cell Counter</td>
			<td>Invitrogen-Life Technologies</td>
			<td>C10227</td>
			<td></td>
		</tr>
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</table>

intranasal administration of recombinant influenza vaccines in chimeric mouse models to study mucosal immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

重组减毒活流感疫苗的产生可通过质粒双向启动子5的控制下编码流感病毒的八个段的转染来实现。冷适应流感疫苗通常含有冷适应株以及HA和NA选择的流感病毒株（ 如 H1N1）（ 图1A）的六个部分。冷适应的原则在33℃，在小鼠和人类14上层呼吸道（URT）的温度是基于病毒限制复制。因此，疫苗可以复制到在​​URT某种程度上但不能引起下呼吸道肺炎。在病毒神经?...

Watch this Scientific Journal Video about Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity at JoVE.com

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

intranasal-administration-recombinant-influenza-vaccines-chimeric

Research

JoVE Journal

Immunology and Infection

12.4K 次观看.  Heinrich Pette Institute, Leibniz Institute for Experimental Virology. There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

视频: Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

科研

免疫与感染

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

协议RBD - SARS疫苗的重组蛋白的制备，动物疫苗接种和中检测

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

鼻咽癌相关淋巴组织（NALT）在鼠标的响应中的作用研究疫苗

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

的气管内管理<em&gt;流感嗜血杆菌</em&gt;在小鼠模型来研究气道炎症

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...