This work was supported by "Spanish Ministry of Economy and Competitiveness" [grant number BIO2014-5258P], "European Commission" by the H2020 programs [grant numbers TBVAC2020 643381].

所有小鼠保持在受控条件下和对疾病的任何迹象观察。试点工作是在与欧洲和国家指令实验动物保护，并与主管地方伦理委员会批准同意进行。 1. BCG丹麦的量化甘油股票和结核 分枝杆菌的制备注:所有所述的协议进行BSL3条件下进行。 <ol><li> BCG丹麦或分枝杆菌H37Rv菌株的解冻冷冻甘油股票和在10ml 7H9介质10补充有吐温80 0.05％（体积/体积）和ADC（白蛋白，葡萄糖，过氧化氢 ​​酶）的10％（V / V）接种100微升。 </li><li>静态条件下孵育一周，在37℃，直到培养是在对数生长期。 </li><li>由10毫升的文化转移到200毫升的新鲜培养基扩大细菌培养。孵育另外20静态条件下天。 </li><li>转移至50毫升离心管中并离心15分钟，在2500×克</li><li>丢弃上清液，剩余体积重悬细菌沉淀。添加到每个管无菌3-毫米直径的玻璃珠（10 - 15每管）。 </li><li>涡大力以1分钟内要分离的细菌团块。重悬在10毫升PBS中的各粒料用Tween-80 0.05％（体积/体积）。 </li><li>离开管10分钟允许的最大细菌聚集和玻璃珠定居下来。 </li><li>从每片含小团块和单细菌和转让卷一个新鲜的50毫升离心管（最终恢复卷38ml）中的4管的恢复9.5毫升上清液。离心机在400×g下10分钟。 </li><li>在一个单一的新鲜的50毫升离心管中回收35毫升上清液（主要含有单一细菌），并添加15毫升的PBS与甘油50％（体积/体积），得到15％的最终甘油浓度（体积/体积）。 </li><li>轻轻混匀并在1毫升的等分试样散布在适当的管中冷冻。存储在-80°C（一阄提供大约50等分甘油）。 </li><li>量化甘油股票，解冻甘油天冷冻后，在含有900微升的PBS单独1.5毫升管执行七点十分倍连续稀释。 </li><li>板在7H10固体琼脂培养基10 100微升每种稀释液的补充有55毫米直径的皮氏培养皿中制备的ADC 10％（体积/体积）。 </li><li>小心加入3毫米直径的玻璃珠（约10每盘）并轻轻摇动板均等地分布体积随琼脂平板上。丢弃珠和密封琼脂平板用封口膜。 </li><li>孵育21天，在37℃，并通过在稀释那里单个菌落可以准确分辨计数菌落形成单位（CFU）测定细菌的浓度。 </li></ol> 2.鼠标疫苗接种<ol><li>解冻预先定量卡介苗甘油并在PBS中稀释至准备两个悬浮液。计算最终体积考虑每只动物的剂量是用于皮下施用100微升（每毫升10 7 CFU用于皮下接种）和40微升用于鼻内给药（2.5×10 7 CFU鼻内疫苗）。 </li><li>使用专门麻醉工作站啮齿动物用异氟烷和氧气的混合物麻醉小鼠。使用异氟醚的5％以诱导麻醉和2％，以保持麻醉室中的小鼠。 （其他公认的麻醉方法都可以使用）。 </li><li>用于皮下施用疫苗填充1毫升注射器用＆G针用1ml细菌悬浮液（10 7 CFU /毫升）。去除气泡。 </li><li>放置在层流罩内俯卧的平坦表面麻醉小鼠并接种皮下100μl的疫苗（10 6 CFU /剂量）在小鼠的一侧面背面。返回鼠标笼并监视它，以确保它propeRLY从麻醉中恢复。 </li><li>改变各小鼠施用之间的注射器针头。如上所述除去气泡。 </li><li>用于鼻内给药，将麻醉小鼠在引擎盖内仰卧位。 </li><li>用微量取20微升悬浮的2.5×10 7 CFU /毫升。将接种下降逐滴两鼻孔直到整个卷已沉积之间，留下滴之间的时间来让鼠标呼吸音量注:鼠标可以在此过程中因呼吸窘迫这么辛苦，他们应该留给同化管理下一个，以防止它尽可能事先每下降。 </li><li>重新填写与另一个20微升微量并重复该操作。如果鼠标开始从第一接种后麻醉醒来，将其放置在麻醉室中的第二个前。返回鼠标笼子，并确保其恢复正常从麻醉。 </li></ol> 3.鼠标鼻内挑战与应变分枝杆菌<ol><li>解冻预先定量分枝杆菌甘油和稀释在PBS准备每毫升2500 CFU的菌悬液。计算最终体积考虑每只动物的剂量是40微升。 </li><li>在2.5中所述分枝杆菌是鼻内接种 - 2.7。每只小鼠最后的挑战剂量为10 2 CFU / 40微升。 </li></ol> 4.肺引起的免疫响应分析<ol><li> 肺细胞悬浮液 注:在肺中疫苗诱导的免疫应答的评估需要单细胞悬浮液的生成。为了这个目的，我们使用的组织离解，如GentleMACS。 <ol><li>牺牲颈椎脱位（或其他公认的人性化的方法）鼠标和鼠标确保是死触摸眼球，以确认没有瞬目反射。 </li><li>内部层流罩，提取第lungs用无菌剪刀和镊子，将其放在一个干净的表面。干净的肺移除气管和结缔组织，以及肺用PBS转移到1.5毫升试管（保持在冰上，直到处理）。 </li><li>以产生细胞悬浮液，将肺在含有5ml的10mM HEPES-NaOH中pH为7.4，150 mM氯化钠，5mM的氯化钾，1mM的MgCl 2的1.8毫米氯化钙2器官离解（C-离解器管）的适当管缓冲。 </li><li>添加的胶原酶D 100毫克/毫升100微升（2毫克/毫升终浓度），和DNA酶I 20000 IU / ml的40微升（160单位/毫升的最终浓度。 注:胶原酶D或DNA酶I的储备溶液制备分别溶解在PBS中冻干酶或甘油50％（V / V），20毫摩尔Tris -盐酸pH为7.5ý的MgCl 2为1mM，。 </li><li>将管在组织离解和使用肺碎片预定义的程序m_lung_01。孵育在37℃下在管的水浴中30分钟。 </li><li>第广场在离解ë管，并使用完全同质化的肺​​成单细胞悬浮液预定义的程序m_lung_02。 </li><li>通过装配到一个50毫升的离心管70微米尼龙细胞过滤器通过细胞悬浮液。用10ml的RPMI 1640培养基中洗涤过滤器。 </li><li>离心细胞在400 XG 5分钟，弃上清。 </li><li>加入1ml红细胞裂解缓冲液中，涡旋混合并孵育在室温下1分钟。 </li><li>加入10 mL的RPMI 1640培养基中，离心细胞在400×g下5分钟。 </li><li>在0.5的完全RPMI 1640培养基中重悬细胞在补充有10％热灭活胎牛血清（FCS），2mM的L-谷氨酰胺，100微克/毫升链霉素，100国际单位/毫升青霉素和50μM2-巯基乙醇。 注:FCS的温育在56℃灭活30分钟。 </li><li>计数细胞并添加完全RPMI 1640培养基中，达到10 7个细胞/ ml的终细胞密度。 注:对于细胞计数，我们使用自动细胞计数器。 </li><li>分发100微升细胞悬浮液在U型底96孔无菌板（10 6个细胞/孔）。 </li><li>添加50μl/孔的完全RPMI 1640培养基中的15微克/毫升的商业H37Rv的纯蛋白衍生物（PPD）（5微克/毫升的最终浓度）。 </li></ol></li><li> 上清液收集通过ELISA的细胞因子测定 <ol><li>孵育板从4.1.14为在37℃下48小时和5％ 的 CO 2。 </li><li>离心机板在400 xg离心5分钟，回收100μl的上清液。 </li><li>存储在-80℃。 </li><li>分别上清液稀释1/10和1/2在分析缓冲液的IFNγ和IL17A的决心。确定与商业ELISA试剂盒细胞因子浓度，根据制造商的说明（ELISA开发工具包）。 </li></ol></li><li> 细胞内染色测定细胞因子-producing CD4 +细胞通过流式细胞仪<ol><li>孵育板从4.1.14为在37℃下18小时和5％ 的 CO 2。 </li><li>添加1.5微升的布雷菲德菌素A 1毫克/毫升（股票溶于DMSO）至每孔（10微克/毫升终浓度）孵育额外六个小时。 </li><li>从离心4.3.2板1分钟在3200 XG弃上清。 </li><li>在抗小鼠CD4-FITC的50微升的RPMI 1640稀释，用10％的FCS（1/500）和悬浮细胞孵育在4℃下10分钟。 </li><li>离心从4.3.4板1分钟3,200 xg离心，弃去上清液，加入100μl/孔的固定溶液。孵育在4℃下20分钟。 </li><li>用通透的解决方案150微升/洗洗细胞两次。 注:10倍浓缩储液稀释于去离子水中以制备1×工作溶液。 </li><li>在50μl抗小鼠IFNγ-APC和抗小鼠IL17A-APC.Cy7的悬浮细胞稀释在permeabil化溶液（1/250两种抗体）。 </li><li>在室温下孵育1小时。 </li><li>用通透的解决方案150微升/洗洗细胞两次。 </li><li>悬浮细胞在200μl的PBS中进行流式细胞采集。 </li></ol></li></ol> 5.免疫球蛋白支气管肺泡灌洗液分析（BAL） <ol><li> BAL样品的获得 <ol><li>牺牲颈椎脱位（或其他公认的人性化的方法）鼠标和鼠标确保是死触摸眼球，以确认没有瞬目反射。 </li><li>揭露使用适当的精度无菌镊子和剪刀气管并执行气管一小截，确保不完全切除它。小心不要引起出血可能污染BAL样本。 </li><li>引入进气管连接到一个无菌的1-ml注射器用800微升冰冷的PBS的套管。 </li><li>慢慢地接种PBS进入肺部，则RECOVER BAL体积，以避免肺塌陷拉回注射器的活塞速度非常缓慢。 注:在500到600微升的恢复是令人满意的。 </li><li>转移BAL卷成1.5毫升管中并确认BAL保持无色，表明没有被血液污染的（保持在冰上，直到处理）。 </li><li>离心机BAL样品在400 xg离心5分钟，并转移上清液至新管。 </li><li>商店在上清-80℃。 </li></ol></li><li> 的IgA在BAL上清测定 注意:使用的所有试剂是在室温下。 <ol><li>用100μl的PPD在PBS（10微克/毫升）稀释分枝涂层高蛋白结合聚苯乙烯平底96孔板结核病特异性IgA检测，或用100微升BAL上清液稀释10倍的PBS总IgA决心。 </li><li>在4℃孵育过夜。 </li><li>弃上清并点击纸巾到D盘RY。用200微升/ PBS的好洗。 </li><li>用200μl/孔的牛血清白蛋白（BSA）的1％（重量/体积）在PBS吐温80 0.05％（体积/体积）（封闭缓冲液）阻断。 </li><li>孵育在RT 1小时。 </li><li>用200μl/ PBS吐温80 0.05％（体积/体积）（洗涤缓冲液）的孔洗一次。 </li><li>加入100μl未稀释的BAL上清液中的PPD-包被的孔。在室温下孵育两小时。 注意:如果总IgA判定与PPD特异性的IgA并行完成，离开总IgA孔用100μl洗涤缓冲液这个温育过程。 </li><li>用洗涤buffer.0170004的200μl/孔洗涤三次</li><li>添加100μl的辣根过氧化物酶（HRP）的缀合抗小鼠IgA稀释在封闭缓冲液（1 / 10,000）。 </li><li>孵育1小时RT。 </li><li>用洗涤缓冲液200μl/孔洗涤五次。 </li><li>添加的3,3100μl/孔&#39;，5,5&#39;-四甲基联苯胺（TMB）。孵育20分钟，在T室温他暗。 </li><li>添加100μl/孔的H 2 SO 4 0.5N。读在分光光度计板在450nm的波长。 </li></ol></li></ol> 6.细菌量测定肺<ol><li> 龙同质化和电镀 <ol><li>在攻击后四个星期，牺牲通过颈脱位（或其它公认的人道方法）的小鼠，并确保小鼠死触摸眼球确认不存在眨眼反射的。 </li><li>鼠标放置在固定在层流罩内平坦并消毒表面仰卧位。 </li><li>做一个切口，并从胸部区域去除皮肤留下明显的胸区域。 </li><li>剪下的肋骨和收获用无菌剪刀和镊子的肺和心脏。将机关在清洁的表面。 </li><li>通过移除心脏，气管和结缔组织清理肺部和肺部用1毫升去离子水转移到1.5毫升管（冰上保持，直至处理）。 </li><li>使用无菌镊子对各小鼠的肺转移到用于器官均质的适当管（M - 离解器管），含有1毫升去离子水。 </li><li>放置管在组织离解器和使用预定义的程序RNA_1至均质化肺部。 </li><li>使万分之五倍连续稀释在1.5毫升管，开始用100μl肺匀浆在900微升稀释。 </li><li>板稀释为1.11-1.14描述。 </li></ol></li><li> BCG和分枝杆菌之间的差异，通过PCR 注:在从6.1.6对应于鼻内途径接种BCG组的板块，我们分析殖民地的代表人数由BCG和分枝杆菌之间的挑剔的PCR，以确保CFU认为，评估疫苗赋予的保护效果相当以独特的分枝杆菌。 RD9的这种PCR扩增目标，基因组区域中BCG和M.涂不同berculosis，得到2618碱基对杆菌H37Rv，和465碱基对的BCG 11的片段。 <ol><li>制备主混合物的库存为每个样品体积所指示所有的样品，然后在PCR管分配（9微升/样品）。 注:缓冲区5X:2微升。正向引物25μM（GTGTAGGTCAGCCCCATCC）:0.32微升，反向引物25μM（GCCCAACAGCTCGACATC）:0.32微升，Taq DNA聚合酶5单位/微升:0.04微升，去离子水2 O:6.32微升。 </li><li>触摸菌落用无菌牙签在含有预混PCR管样品浸泡牙签。 </li><li>用下面的程序运行的PCR:10分钟95℃（此步骤是触发细菌破坏），10分钟95℃（此步骤是触发细菌破坏），35个循环（在95℃下30秒，1分钟在58℃，并在72℃4分钟）。 </li><li>加载的PCR样品用溴化乙锭和运行的样品在1％琼脂糖凝胶，以可视化的片段。 </l我&gt; </ol></li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriology+of+tuberculosis:+laboratory+methods.">Bacteriology of tuberculosis: laboratory methods.</a> Am J Public Health Nations Health. 48 (7), 844-853 (1958).</li><li>Brosch, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+evolutionary+scenario+for+the+Mycobacterium+tuberculosis+complex.">A new evolutionary scenario for the Mycobacterium tuberculosis complex.</a> Proc Natl Acad Sci U S A. 99 (6), 3684-3689 (2002).</li><li>Kaushal, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+vaccination+with+attenuated+Mycobacterium+tuberculosis+induces+strong+central+memory+responses+and+protects+against+tuberculosis.">Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis.</a> Nat Commun. 6, 8533 (2015).</li><li>Lochhead, J. J., Thorne, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+of+biologics+to+the+central+nervous+system.">Intranasal delivery of biologics to the central nervous system.</a> Adv Drug Deliv Rev. 64 (7), 614-628 (2012).</li><li>Lochhead, J. J., Wolak, D. J., Pizzo, M. E., Thorne, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+transport+within+cerebral+perivascular+spaces+underlies+widespread+tracer+distribution+in+the+brain+after+intranasal+administration.">Rapid transport within cerebral perivascular spaces underlies widespread tracer distribution in the brain after intranasal administration.</a> J Cereb Blood Flow Metab. 35 (3), 371-381 (2015).</li><li>Griffiths, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholera+toxin+enhances+vaccine-induced+protection+against+Mycobacterium+tuberculosis+challenge+in+mice.">Cholera toxin enhances vaccine-induced protection against Mycobacterium tuberculosis challenge in mice.</a> PLoS One. 8 (10), e78312 (2013).</li><li>Hirota, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+Th17+cells+in+Peyer's+patches+is+responsible+for+the+induction+of+T+cell-dependent+IgA+responses.">Plasticity of Th17 cells in Peyer's patches is responsible for the induction of T cell-dependent IgA responses.</a> Nat Immunol. 14 (4), 372-379 (2013).</li><li>Jaffar, Z., Ferrini, M. E., Herritt, L. A., Roberts, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+lung+mucosal+Th17-mediated+responses+induce+polymeric+Ig+receptor+expression+by+the+airway+epithelium+and+elevate+secretory+IgA+levels.">Cutting edge: lung mucosal Th17-mediated responses induce polymeric Ig receptor expression by the airway epithelium and elevate secretory IgA levels.</a> J Immunol. 182 (8), 4507-4511 (2009).</li></ol>

The authors have nothing to disclose.

Although current vaccine against tuberculosis, BCG, is the most widely administered vaccine in history, tuberculosis remains one of the leading causes of death and morbidity from infectious diseases worldwide. This paradox is explained by the lack of protection of this vaccine against pulmonary tuberculosis, the responsible form of transmission. New vaccination approaches effective against pulmonary forms of the disease are urgently needed, as they would have the greatest impact on disease transmission globally.
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结核病（TB）是全球领先的传染性疾病导致性比HIV相关的死亡在世界之一，耐多药菌株的增加上升相结合，使得TB惊人的全球性健康问题1。新的诊断工具，更有效和毒性较小的药物，以及新的安全和有效的结核病疫苗的迫切需要，特别是在发展中世界。 减毒活卡介苗（BCG）是目前预防结核病，已在皮内诞生以来全球20世纪70年代给予唯一的持牌疫苗。卡介苗被认为是有效地防止儿童疾病（脑膜炎和粟粒TB）的严重形式，但已经显示出对负责疾病变速 ​​器2的肺结核功效不一致。 肺接种疫苗，它模仿结核感染的自然路线，代表了当地引发宿主的免疫反应有吸引力的方法秒。在这点上，相比于皮下或皮内途径3-6在不同的有关结核的动物模型的各种临床前工作已经证明以下肺免疫更大疫苗效力。尽管如此，通过肺接种引发的保护机制尚不十分清楚。在过去的几年中，一些作品指着具体TB-粘膜免疫应答的重要因子IL17介导的反应，如在缺陷的IL17粘膜疫苗诱导的保护效力小鼠模型受损7,8。 最近，我们展示的第一次鼻内BCG管理保护的DBA / 2鼠标，鼠标应变特点是皮下BCG免疫后9缺乏保护的。这些结果表明，呼吸道结核疫苗可以减少在流行国家，皮内BCG被认为是对pulmon无效结核病率更有效元TB。

<table><tbody><tr><td>Middlebrook 7H9 broth</td><td>BD</td><td>271310</td><td></td></tr><tr><td>Middlebrook ADC Enrichment</td><td>BD</td><td>211887</td><td></td></tr><tr><td>Tween 80</td><td>Scharlau</td><td>TW00800250</td><td></td></tr><tr><td>3-mm diameter Glass Beads</td><td>Scharlau</td><td>038-138003</td><td></td></tr><tr><td>Middlebrook 7H10 Agar</td><td>BD</td><td>262710</td><td></td></tr><tr><td>1-ml syringe 26GA 0.45 x 10 mm</td><td>BD</td><td>301358</td><td></td></tr><tr><td>GentleMACS dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td></td></tr><tr><td>C tubes</td><td>Miltenyi Biotec</td><td>130-093-237</td><td></td></tr><tr><td>M tubes</td><td>Miltenyi Biotec</td><td>130-093-236</td><td></td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088882001</td><td></td></tr><tr><td>DNaseI</td><td>Applichem</td><td>A3778,0100</td><td></td></tr><tr><td>Falcon 70 &amp;micro;m Cell Strainer</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>RPMI 1640</td><td>Sigma</td><td>R0883</td><td></td></tr><tr><td>Red Blood Cell Lysing Buffer</td><td>Sigma</td><td>R7757</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco</td><td>35050-061</td><td>100x concentrated</td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Sigma</td><td>P4333</td><td>100x concentrated</td></tr><tr><td>Fetal Calf Serum</td><td>Biological Industries</td><td>04-001-1A</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M3148-25ML</td><td></td></tr><tr><td>Scepter 2.0 Handheld Automated Cell Counter</td><td>Millipore</td><td>PHCC20040</td><td></td></tr><tr><td>Scepter Cell Counter Sensors, 40 &amp;micro;m</td><td>Millipore</td><td>PHCC40050</td><td></td></tr><tr><td>Mycobacterium Tuberculosis - Tuberculin PPD</td><td>Statens Serum Institut (SSI)</td><td>2390</td><td></td></tr><tr><td>Mouse IFN-&amp;gamma; ELISA development kit&amp;nbsp;</td><td>Mabtech</td><td>3321-1H</td><td></td></tr><tr><td>Mouse IL17A ELISA development kit&amp;nbsp;</td><td>Mabtech</td><td>3521-1H</td><td></td></tr><tr><td>Brefeldin A</td><td>Sigma</td><td>B7651</td><td></td></tr><tr><td>FITC Rat Anti-Mouse CD4</td><td>BD</td><td>553047</td><td></td></tr><tr><td>BD Cytofix/Cytoperm Kit</td><td>BD</td><td>555028</td><td></td></tr><tr><td>APC-Cy7 Rat Anti-mouse IL-17A</td><td>BD</td><td>560821</td><td></td></tr><tr><td>APC Mouse Anti-mouse IFNg</td><td>BD</td><td>554413</td><td></td></tr><tr><td>LACHRYMAL OLIVE LUER LOCK 0.60 x 30 mm. 23G x 1 1/4&amp;rdquo;</td><td>UNIMED</td><td>27.134</td><td>Used as trachea cannula for BAL</td></tr><tr><td>high-protein binding polystyrene flat-bottom 96-well plates MAXISORP</td><td>NUNC</td><td>430341</td><td></td></tr><tr><td>Albumin, from bovine serum</td><td>Sigma</td><td>A4503</td><td></td></tr><tr><td>Goat Anti-Mouse IgA (&amp;alpha;-chain specific)&amp;minus;Peroxidase antibody</td><td>Sigma</td><td>A4789</td><td></td></tr><tr><td>3,3&amp;prime;,5,5&amp;prime;-Tetramethylbenzidine (TMB)&amp;nbsp;</td><td>Sigma</td><td>T0440</td><td></td></tr><tr><td>MyTaq DNA Polymerase</td><td>Bioline</td><td>BIO-21107</td><td>The kit Includes Buffer 5x</td></tr></tbody></table>

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.

这部作品描述了BCG给药两种途径的比较:皮下和鼻内。皮下途径是可比的皮内，这是全世界的卡介苗目前临床路径。疫苗接种的鼻内途径旨在模仿M的感染的天然途径肺结核 ，目标是直接诱导免疫反应在肺，这种病原体的主要靶器官。 图1描述了工作流程遵循。八到十周龄的雌性DBA / 2小鼠用10 6...

Watch this Scientific Journal Video about Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice at JoVE.com

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

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 ...

protective-efficacy-pulmonary-immune-response-following-subcutaneous

Research

JoVE Journal

Immunology and Infection

10.5K 次观看.  University of Zaragoza. 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.

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

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

科研

免疫与感染

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

淋巴免疫治疗和疫苗接种小鼠

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

舌下免疫治疗作为一种替代起到保护作用对急性呼吸道感染

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

医学

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

基于CFSE-试验鼠皮肤树突状细胞的迁移习进引流淋巴结在与感染<em&gt;牛分枝杆菌</em&gt;卡介苗

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

非遗传表面修饰的素-生物素系统在牛分枝杆菌卡介苗中的外源抗原表达

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

一种研究人体 T 细胞召回反应的吸水泡协议

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

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 ...

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites, lipids, proteins, and nucleic acids, several groups have evaluated either the native or recombinant versions of bEVs for their protective potency as subunit vaccine candidates. Unlike native EVs, recombinant EVs are molecularly engineered to contain one or more immunogens of interest. Over the last decade, different groups have explored diverse approaches for generating recombinant bEVs. However, here, we report the design, construction, and enrichment of recombinant mycobacterial EVs (mEVs) in mycobacteria. Towards that, we use Mycobacterium smegmatis (Msm), an avirulent soil mycobacterium as the model system. We first describe the generation and enrichment of native EVs of Msm. Then, we describe the design and construction of recombinant mEVs that contain either mCherry, a red fluorescent reporter protein, or EsxA (Esat-6), a prominent immunogen of Mycobacterium tuberculosis. We achieve this by separately fusing mCherry and EsxA N-termini with the C-terminus of a small Msm protein Cfp-29. Cfp-29 is one of the few abundantly present proteins of MsmEVs. The protocol to generate and enrich recombinant mEVs from Msm remains identical to the generation and enrichment of native EVs of Msm.

This protocol details the enrichment of native mycobacterial extracellular vesicles (mEVs) from axenic cultures of Mycobacterium smegmatis (Msm) and how mCherry (a red fluorescent reporter)-containing recombinant MsmEVs can be designed and enriched. Lastly, it verifies the novel approach with the enrichment of MsmEVs containing the EsxA protein of Mycobacterium tuberculosis.

富集分枝杆菌的天然和重组细胞外囊泡

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, ...

使用 Micro-CFU 方法加速结核病测定

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during the 2019-2021 pandemic. The disease is caused by the bacterium Mycobacterium tuberculosis (M.tb). The Mycobacterium bovis strain Bacillus Calmette-Gu&#233;rin (BCG), the only TB vaccine, is the oldest licensed vaccine in the world, still in use. Currently, there are 12 vaccines in clinical trials and dozens of vaccines under pre-clinical development. The method of choice used to assess the efficacy of TB vaccines in pre-clinical studies is the enumeration of bacterial colonies by the colony-forming units (CFU) assay. This time-consuming assay takes 4 to 6 weeks to conclude, requires substantial laboratory and incubator space, has high reagent costs, and is prone to contamination. Here we describe an optimized method for colony enumeration, the micro-CFU (mCFU), that offers a simple and rapid solution to analyze M.tb vaccine efficacy results. The mCFU assay requires tenfold fewer reagents, reduces the incubation period threefold, taking 1 to 2 weeks to conclude, reduces lab space and reagent cost, and minimizes the health and safety risks associated with working with large numbers of M.tb. Moreover, to evaluate the efficacy of a TB vaccine, samples may be obtained from a variety of sources, including tissues from vaccinated animals infected with Mycobacteria. We also describe an optimized method to produce a unicellular, uniform, and high-quality mycobacterial culture for infection studies. Finally, we propose that these methods should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB.

作者聚焦:优化 CFU 测定以有效评估结核病疫苗功效和抗原呈报分析 

The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

用于结核病疫苗功效评估的微集落形成单位测定

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

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一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

淋巴免疫治疗和疫苗接种小鼠

舌下免疫治疗作为一种替代起到保护作用对急性呼吸道感染

基于CFSE-试验鼠皮肤树突状细胞的迁移习进引流淋巴结在与感染

非遗传表面修饰的素-生物素系统在牛分枝杆菌卡介苗中的外源抗原表达

一种研究人体 T 细胞召回反应的吸水泡协议

宿主病原学反应及疫苗效果评价

富集分枝杆菌的天然和重组细胞外囊泡

用于结核病疫苗功效评估的微集落形成单位测定

用于结核病疫苗功效评估的微集落形成单位测定