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。

<table><tbody><tr><td>Dulbecco&amp;acute;s Modified Eagle Medium (DMEM 1x)</td><td>Gibco RL-Life Technologies</td><td>41965-039</td><td></td></tr><tr><td>OptiMEM</td><td>Gibco RL-Life Technologies</td><td>31985-047</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen-Life Technologies</td><td>11668-027</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>PAA</td><td>p11-010</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>Embryonated eggs</td><td>Valo biomedia Gmbh</td><td></td><td></td></tr><tr><td>PBS (1x)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>70 &amp;mu;M Nylon Filters</td><td>Greiner-Biorad</td><td>542-070</td><td></td></tr><tr><td>Red Blood Cell Lysing buffer (RBCL) 10x</td><td>BD Bioscience</td><td>555899</td><td></td></tr><tr><td>CD16/CD32 Mouse BD Fc Block (2.4G2)</td><td>BD Pharmigen</td><td>553142</td><td></td></tr><tr><td>APC-Anti-mouse SIINFEKL-H2kb (25 D1.16)</td><td>Biolegend</td><td>141605</td><td></td></tr><tr><td>PE-Anti-mouse CD11c (HLA3)</td><td>BD Biosciences</td><td>553802</td><td></td></tr><tr><td>eFluor 450-Anti-mouse MHCII (Md/114.15.2)</td><td>eBioscience</td><td>48-5321-82</td><td></td></tr><tr><td>Pe-Cy7-Anti-mouse CD11b (M1/70)</td><td>Biolegend</td><td>101216</td><td></td></tr><tr><td>PerCp/Cy5.5-Anti-mouse CD103 (2E7)</td><td>Biolegend</td><td>121416</td><td></td></tr><tr><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><td></td></tr><tr><td>APC-Cy7-Anti-mouse CD3&amp;epsilon; (145-2611)</td><td>Biolegend</td><td>100325</td><td></td></tr><tr><td>eFluor450-Anti-mouse CD4 (GK 1.5)</td><td>eBioscience</td><td>48-0041-80</td><td></td></tr><tr><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></tr><tr><td>Dymethil-Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Ovalbumin&amp;nbsp;</td><td>Molecular probes&amp;nbsp;</td><td>O23020</td><td></td></tr><tr><td>Diphteria Toxin (DT)</td><td>Sigma-Aldrich</td><td>D0564</td><td></td></tr><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%)&amp;nbsp;</td><td>Life technologies</td><td>T10282</td><td></td></tr><tr><td>Countess Automatic Cell Counter</td><td>Invitrogen-Life Technologies</td><td>C10227</td><td></td></tr></tbody></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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

12.5K 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

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

插管介导气管内（IMIT）灌输:一种无创，龙专用输送系统

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

科研

医学

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 ...

免疫与感染

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 ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

二次细菌性肺炎的鼠种模型的精确病原体输送与恢复系统

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

病毒合并感染后 肺炎链球菌 从定植者转变为病原体的小鼠模型概括了年龄加重的疾病

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

Intranasal Immunization with BCG in a Mouse Model

Source: Uranga, S. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54440">Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice.</a>&#160;J. Vis. Exp. (2016)
This video demonstrates intranasal BCG immunization in mice, activating both innate and adaptive immune responses, leading to the development of lung-resident memory T cells and effector B cells, thus enhancing protection against Mycobacterium infections.

小鼠模型中的 BCG 鼻内免疫

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

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插管介导气管内（IMIT）灌输:一种无创，龙专用输送系统

的气管内管理

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

流感病毒在小鼠感染模型中的研究

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

宿主病原学反应及疫苗效果评价

二次细菌性肺炎的鼠种模型的精确病原体输送与恢复系统

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

病毒合并感染后肺炎链球菌从定植者转变为病原体的小鼠模型概括了年龄加重的疾病

小鼠模型中的 BCG 鼻内免疫

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

摘要

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插管介导气管内（IMIT）灌输:一种无创，龙专用输送系统

的气管内管理

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

流感病毒在小鼠感染模型中的研究

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

宿主病原学反应及疫苗效果评价

二次细菌性肺炎的鼠种模型的精确病原体输送与恢复系统

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

病毒合并感染后 肺炎链球菌 从定植者转变为病原体的小鼠模型概括了年龄加重的疾病

小鼠模型中的 BCG 鼻内免疫

病毒合并感染后肺炎链球菌从定植者转变为病原体的小鼠模型概括了年龄加重的疾病