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].

Tutti i topi sono stati tenuti in condizioni controllate e osservati per qualsiasi segno di malattia. Il lavoro sperimentale è stato condotto in accordo con le direttive europee e nazionali per la protezione degli animali da esperimento e con l&#39;approvazione del competenti comitati etici locali. 1. Preparazione di quantificata glicerolo scorte di BCG danese e Mycobacterium tuberculosis H37Rv Nota: Tutti i protocolli descritti sono stati eseguiti in BSL3. <ol><li> Scongelare scorte glicerolo congelati di BCG danese ceppi H37Rv e inoculare 100 ml in 10 ml di 7H9 media 10 integrato con Tween-80 allo 0,05% (v / v) e ADC (albumina, destrosio, catalasi) 10% (v / v). </li><li> Incubare in condizioni statiche per una settimana a 37 ° C fino a quando la coltura è in fase di crescita logaritmica. </li><li> Scalare fino coltura batterica trasferendo la cultura da 10 ml a 200 ml di terreno fresco. Incubare per 20 aggiuntivogiorni in condizioni statiche. </li><li> Trasferimento a provette da centrifuga da 50 ml e centrifugare per 15 min a 2500 x g. </li><li> Eliminare sovranatante e risospendere pellet batterico nel volume residuo. Aggiungere ad ogni provetta perle di vetro sterili a 3 mm di diametro (10 - 15 per provetta). </li><li> Vortex vigorosamente per 1 minuto al fine di dissociare grumi batteriche. Risospendere ogni pellet in 10 ml di PBS con Tween-80 allo 0,05% (v / v). </li><li> Lasciare i tubi per 10 min permettendo i più grandi aggregati batterici e perle di vetro di stabilirsi. </li><li> Recupero 9,5 ml di surnatante da ciascuna delle 4 provette contenenti piccoli grumi e batteri singoli e volumi di trasferimento ad un singolo tubo da centrifuga da 50 ml fresca (volume finale recuperato 38 ml). Centrifugare per 10 min a 400 x g. </li><li> Recupero 35 ml di surnatante (contenenti batteri principalmente singoli) in una singola provetta da centrifuga da 50 ml fresca e aggiungere 15 ml di PBS con glicerolo 50% (v / v), ottenendo una concentrazione di glicerolo finale del 15% (v / v). </li><li> mescolare delicatamentee distribuire in aliquote da 1 ml in tubi appropriati per il congelamento. Conservare a -80 ° C (un lotto fornisce circa il 50 aliquote stock di glicerolo). </li><li> Per quantificare le scorte glicerolo, scongelare un glicerolo il giorno dopo il congelamento e di eseguire sette diluizioni seriali di dieci volte separati tubi da 1,5 ml contenente 900 ml di PBS. </li><li> Piatto 100 microlitri di ogni diluizione in 7H10 mezzo solido-agar addizionato con 10 ADC 10% (v / v) preparata in diametro piatti 55 mm Petri. </li><li> Aggiungere con cautela 3 mm di diametro perle di vetro (circa. 10 per piastra) e scuotere delicatamente piastra per distribuire equamente il volume su piastra di agar. Eliminare perline e sigillare piastra di agar con parafilm. </li><li> Incubare per 21 giorni a 37 ° C e determinare la concentrazione batterica contando unità formanti colonia (CFU) nelle diluizioni in cui singole colonie possono essere accuratamente distinti. </li></ol> 2. mouse Vaccinazione <ol><li> Scongelare un glicerolo pre-quantificato BCG e diluirlo in PBS a prepsono due sospensioni. Calcolare il volume finale considerando la dose per animale è di 100 ml per somministrazione sottocutanea (10 7 CFU per ml per la vaccinazione per via sottocutanea) e 40 ml per somministrazione intranasale (2,5 x 10 7 CFU per la vaccinazione intranasale). </li><li> Utilizzare una stazione di anestesia specializzato per roditori per anestetizzare topi con una miscela di isoflurano e ossigeno. Utilizzare isoflurano 5% per indurre anestesia e 2% per mantenere i topi nella camera di anestesia. (Altro metodo di anestesia accettato può essere utilizzato). </li><li> Per la somministrazione del vaccino sottocutanea riempire una siringa da 1 ml con un ago 26 G con 1 ml di sospensione batterica (10 7 CFU / ml). Rimuovere le bolle d&#39;aria. </li><li> Inserire un mouse anestetizzato su una superficie piana in posizione prona all&#39;interno di una cappa a flusso laminare e inoculare sottocutanea 100 microlitri (10 6 CFU / dose) di vaccino in un fianco della parte posteriore del mouse. Ritorna il mouse per la gabbia e monitorarlo per assicurare che properly recupera da anestesia. </li><li> Cambiare l&#39;ago della siringa tra ogni amministrazione mouse. Rimosso bolle d&#39;aria come descritto sopra. </li><li> Per la somministrazione intranasale, posizionare un mouse anestetizzato in posizione supina all&#39;interno della cappa. </li><li> Con una micropipetta prendere 20 ml di sospensione con 2,5 x 10 7 CFU / ml. Posizionare l&#39;inoculo goccia a goccia tra le due narici fino a quando l&#39;intero volume è stato depositato, lasciando tempo tra gocce per consentire il mouse respirare il volume. Nota: i topi possono soffrire di distress respiratorio durante questa procedura in modo che dovrebbe essere lasciato a assimilare ogni goccia prima amministrare quello successivo per evitare il più possibile. </li><li> Ri-riempire la micropipetta con un altro 20 ml e ripetere l&#39;operazione. Se il mouse comincia a svegliare dall&#39;anestesia dopo il primo inoculo, posizionarlo nella camera di anestesia prima alla seconda. Ritorna il mouse per la gabbia e garantire che recupera correttamentedall&#39;anestesia. </li></ol> 3. mouse intranasale sfida con H37Rv Strain <ol><li> Scongelare un glicerolo pre-quantificato H37Rv e diluire in PBS per preparare una sospensione batterica di 2.500 CFU per ml. Calcolare il volume finale considerando la dose per animale è di 40 ml. </li><li> H37Rv viene inoculato per via intranasale, come descritto nella 2,5-2,7. Dose sfida finale per il mouse è di 10 CFU 2/40 ml. </li></ol> 4. Analisi dei indotta risposta immunitaria nei polmoni <ol><li> Sospensione cellulare Lung NOTA: Valutazione della risposta immunitaria adattativa indotta dal vaccino nei polmoni richiede la generazione di sospensione cellulare singolo. A questo scopo, usiamo un dissociatore tessuto come gentleMACS. <ol><li> Sacrificare il mouse per dislocazione cervicale (o altra metodologia umana accettato) e garantire il mouse è morto di toccare il bulbo oculare per confermare assenza di riflesso corneale. </li><li> All&#39;interno di una cappa a flusso laminare, estrarre lungs usando forbici e pinze sterili e collocarli su una superficie pulita. polmoni puliti rimozione della trachea e del tessuto connettivo, e trasferire i polmoni in una provetta da 1,5 ml con PBS (mantenere in ghiaccio fino al trattamento). </li><li> Per generare una sospensione cellulare, posizionare polmoni in un tubo adeguato per la dissociazione organo (tubo C-dissociatore) contenente 5 ml di 10 mM HEPES-NaOH pH 7,4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl 2 e 1.8 mM CaCl 2 buffer. </li><li> Aggiungere 100 ml di collagenasi D 100 mg / ml (2 mg / ml concentrazione finale) e 40 ml di DNAsi I 20.000 UI / ml (160 UI / ml concentrazione finale. NOTA: Soluzioni madre della collagenasi D o DNasiI sono preparati dissolvendo enzimi liofilizzati in PBS o glicerolo 50% (v / v), 20 mM Tris-HCl pH 7.5 y MgCl 2 1 mM, rispettivamente. </li><li> Posizionare il tubo nella dissociatore tessuti e utilizzare il programma di m_lung_01 predefinito per la frammentazione del polmone. Incubare la provetta a 37 ° C a bagnomaria per 30 minuti. </li><li> ° postoe tubo nella dissociatore e utilizzare il m_lung_02 programma predefinito per l&#39;omogeneizzazione del polmone completo in un&#39;unica sospensione cellulare. </li><li> Passare la sospensione cellulare attraverso un colino cella nylon 70 micron montato su un tubo da centrifuga da 50 ml. Lavare il filtro con 10 ml di RPMI 1640 medium. </li><li> celle di centrifugazione per 5 minuti a 400 xg ed eliminare il surnatante. </li><li> Aggiungere 1 ml di eritrociti lisi tampone, mescolare nel vortex e incubare per 1 minuto a temperatura ambiente. </li><li> Aggiungere 10 ml di RPMI 1640 cellule medie e centrifugare per 5 minuti a 400 x g. </li><li> Risospendere le cellule in 0,5 ml di RPMI completo 1640 supplementato con 10% inattivato al calore fetale Calf Serum (FCS), 2 mM L-Glutamin, 100 ug / ml streptomicina, 100 IU / ml penicillina e 50 mM 2-mercaptoetanolo. NOTA: FCS è inattivata tramite incubazione a 56 ° C per 30 minuti. </li><li> Contare le cellule e aggiungere completo RPMI 1640 medium per raggiungere densità finale cellulare di 10 7 cellule / ml. NOTA: Per il conteggio delle cellule, usiamo il contatore delle cellule automatizzato. </li><li> Distribuire 100 ml di sospensione cellulare in U-bottom piastra a 96 pozzetti sterili (10 6 cellule / pozzetto). </li><li> Aggiungere 50 ml / pozzetto di completo RPMI 1640 medium con 15 ug / ml di H37Rv commerciale purificata proteine ​​derivati ​​(PPD) (5 mg / ml concentrazione finale). </li></ol></li><li> Raccolta surnatante per la determinazione di citochine da parte ELISA <ol><li> Incubare la piastra da 4.1.14 per 48 ore a 37 ° C e 5% CO 2. </li><li> Piastra centrifugare per 5 minuti a 400 xg e recuperare 100 ml di surnatante. </li><li> Conservare a -80 ° C. </li><li> Diluire surnatanti 1/10 e 1/2 nel tampone per IFNγ e determinazione IL17A, rispettivamente. Determinare le concentrazioni di citochine con kit commerciali ELISA, secondo le istruzioni del produttore (kit ELISA Sviluppo). </li></ol></li><li> Colorazione intracellulare per la determinazione delle citochine-produzione di cellule CD4 + mediante citometria di flusso <ol><li> Incubare la piastra da 4.1.14 per 18 ore a 37 ° C e 5% CO 2. </li><li> Aggiungere 1,5 ml di brefeldina a 1 mg / ml (archivio disciolto in DMSO) a ciascuna (concentrazione finale 10 ug / ml) e incubare per sei ore aggiuntive. </li><li> Centrifugare la piastra da 4.3.2 per 1 min a 3200 xg e scartare il surnatante. </li><li> Risospendere le cellule in 50 ml di anti-topo CD4-FITC diluito in RPMI 1640 con 10% FCS (1/500) e incubare per 10 minuti a 4 ° C. </li><li> Centrifugare la piastra da 4.3.4 per 1 min a 3.200 xg, scartare il surnatante e aggiungere 100 ml / pozzetto di soluzione di fissaggio. Incubare a 4 ° C per 20 min. </li><li> Lavare le cellule due volte con 150 ml / lavaggio di soluzione di permeabilizzazione. NOTA: 10x magazzino concentrato viene diluito in acqua deionizzata per preparare la soluzione di lavoro 1x. </li><li> Risospendere le cellule in 50 ml di anti- topo IFNγ-APC e anti-topo-IL17A APC.Cy7 diluiti in permeabilsoluzione zione (1/250 entrambi gli anticorpi). </li><li> Incubare per 1 ora a RT. </li><li> Lavare le cellule due volte con 150 ml / lavaggio di soluzione di permeabilizzazione. </li><li> Risospendere le cellule in 200 ml di PBS per l&#39;acquisizione di citometria a flusso. </li></ol></li></ol> 5. Analisi delle immunoglobuline nel lavaggio broncoalveolare (BAL) <ol><li> Ottenimento di campioni BAL <ol><li> Sacrificare il mouse per dislocazione cervicale (o altra metodologia umana accettato) e garantire il mouse è morto di toccare il bulbo oculare per confermare assenza di riflesso corneale. </li><li> Esporre la trachea utilizzando adeguata precisione pinza sterile e forbici ed eseguire un piccolo taglio nella trachea, facendo attenzione a non asportare completamente. Fare attenzione a non causare sanguinamento che potrebbe contaminare campione BAL. </li><li> Introdurre nella trachea una cannula collegata ad una siringa sterile da 1 ml con 800 ml di PBS ghiacciato. </li><li> Seminare il PBS lentamente nei polmoni e poi recover volume di BAL tirando indietro il pistone della siringa molto lentamente per evitare il collasso del polmone. NOTA: Il recupero di 500 a 600 microlitri è soddisfacente. </li><li> Trasferire il volume BAL in un tubo da 1,5 ml e confermare BAL rimane incolore, che indica che non è contaminato con il sangue (mantenere in ghiaccio fino al trattamento). </li><li> centrifugare i campioni BAL per 5 minuti a 400 xg, e il trasferimento surnatante in una nuova provetta. </li><li> surnatanti conservare a -80 ° C. </li></ol></li><li> Determinazione delle IgA nel BAL surnatanti NOTA: Tutti i reagenti utilizzati sono a temperatura ambiente. <ol><li> Coat-alto contenuto di proteine ​​di polistirolo legame a fondo piatto piastre a 96 pozzetti con 100 ml di PPD diluito in PBS (10 mg / ml) per M. tubercolosi determinazione IgA SPECIFICI, o con 100 ml di BAL surnatante diluiti 10 volte in PBS per la determinazione totale di IgA. </li><li> Incubare per una notte a 4 ° C. </li><li> Gettare il surnatante e toccare la piastra su carta assorbente per dry. Lavare con 200 pl / pozzetto di PBS. </li><li> Blocco con 200 pl / pozzetto di una albumina sierica bovina (BSA) 1% (w / v) in PBS-Tween-80 allo 0,05% (v / v) (tampone di bloccaggio). </li><li> Incubare 1 ora a temperatura ambiente. </li><li> Risciacquare una volta con 200 ml / pozzetto di PBS-Tween-80 0,05% (v / v) (tampone di lavaggio). </li><li> Aggiungere 100 ml di diluito surnatante BAL nei pozzetti PPD-rivestiti. Incubare due ore a temperatura ambiente. NOTA: Se la determinazione totale IgA viene fatto in parallelo con il PPD-specifica IgA, lasciare pozzi totale di IgA con tampone di lavaggio 100 microlitri Durante questa incubazione. </li><li> Lavare tre volte con 200 pl / pozzetto di lavaggio buffer.0170004 </li><li> Aggiungere 100 ml di perossidasi di rafano (HRP) coniugata anti-topo IgA diluito in tampone di bloccaggio (1 / 10.000). </li><li> Incubare 1 ora RT. </li><li> Lavare cinque volte con 200 pl / pozzetto di tampone di lavaggio. </li><li> Aggiungere 100 ml / pozzetto di 3,3 &#39;, 5,5&#39;-Tetrametilbenzidina (TMB). Incubare 20 minuti a temperatura ambiente in tegli scuro. </li><li> Aggiungere 100 pl / pozzetto di H 2 SO 4 0.5N. Leggere la piastra in uno spettrofotometro ad una lunghezza d&#39;onda di 450 nm. </li></ol></li></ol> 6. determinazione carica batterica nei polmoni <ol><li> Omogeneizzazione del polmone e placcatura <ol><li> Quattro settimane dopo la sfida, sacrificare il mouse per dislocazione cervicale (o altra metodologia umana accettato) e garantire il mouse è morto di toccare il bulbo oculare per confermare assenza di riflesso corneale. </li><li> Posizionare il mouse in posizione supina fissa su una superficie piana e disinfettati all&#39;interno di una cappa a flusso laminare. </li><li> Fare un&#39;incisione e rimuovere la pelle da regione toracica lasciare visibile la zona toracica. </li><li> Tagliare le costole e raccogliere i polmoni e il cuore con le forbici e pinze sterili. Mettere organi su una superficie pulita. </li><li> Pulire polmoni rimuovendo cuore, trachea e tessuto connettivo e trasferire i polmoni in una provetta da 1,5 ml con 1 ml di acqua deionizzata(Mantenere in ghiaccio fino lavorazione). </li><li> Utilizzare pinze sterili per trasferire i polmoni di ciascun topo in una provetta adeguata per omogeneizzazione organo (M - tubo dissociatore), contenente 1 ml di acqua deionizzata. </li><li> Posizionare il tubo nella dissociatore tessuti e utilizzare il programma RNA_1 predefinito per omogeneizzare i polmoni. </li><li> Fare cinque diluizioni seriali dieci volte in tubi da 1,5 ml, a partire da 100 ml di omogenato polmone diluito in 900 ml. </li><li> diluizioni piastra come descritto in 1,11-1,14. </li></ol></li><li> La differenziazione tra BCG e H37Rv mediante PCR NOTA: In piastre dalla 6.1.6 corrispondenti al gruppo vaccinato con BCG per via intranasale, abbiamo analizzato un numero rappresentativo di colonie da una PCR discernere tra BCG e H37Rv per garantire che il CFU considerato per valutare l&#39;efficacia protettiva del vaccino-conferito corrispondeva unica per H37Rv. Questo target di amplificazione PCR di RD9, una regione genomica diversa in BCG e M. tutuberculosis, dando un frammento di 2618 bp per H37Rv, e 465 bp per BCG 11. <ol><li> Preparare uno stock master mix per tutti i campioni con volumi per campione come indicato, e quindi distribuire in provette da PCR (9 ml / campione). NOTA: Buffer 5x: 2 ml. Avanti di primer 25 micron (GTGTAGGTCAGCCCCATCC): 0,32 ml, Reverse Primer 25 micron (GCCCAACAGCTCGACATC): 0,32 microlitri, Taq polimerasi 5 unità / mL: 0,04 ml, deionizzata H 2 O: 6.32 ml. </li><li> Toccare una colonia con uno stuzzicadenti sterile e immergere lo stuzzicadenti con il campione in un tubo di PCR contenente la master mix. </li><li> Eseguire il PCR con il seguente programma: 10 min 95 ° C (questo passaggio è per innescare interruzione batterica), 10 min a 95 ° C (questo passaggio è per innescare interruzione batterica), 35 cicli (30 secondi a 95 ° C, 1 min a 58 ° C e 4 minuti a 72 ° C). </li><li> Caricare i campioni di PCR in un 1% gel con i campioni di etidio bromuro e correre a visualizzare i frammenti. </li&gt; </ol></li></ol>

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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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La tubercolosi (TB) è una delle principali malattie infettive che causano più morti associate di HIV nel mondo e combinato con l&#39;aumento della crescita di ceppi resistenti multiresistenti rende TB un allarmante problema di salute globale 1. I nuovi strumenti diagnostici, farmaci più efficaci e meno tossici, e nuovi vaccini TB sicuri ed efficaci sono una necessità urgente, soprattutto nel mondo in via di sviluppo. Vivo attenuato Bacille Calmette-Guerin (BCG) è attualmente l&#39;unico vaccino autorizzato contro la tubercolosi, che è stato somministrato per via intradermica alla nascita dal 1970 in tutto il mondo. BCG è considerata efficace nel prevenire le forme gravi della malattia (meningite e la tubercolosi miliare) nei bambini, ma ha dimostrato efficacia in contrasto contro la tubercolosi polmonare responsabile della trasmissione della malattia 2. la vaccinazione polmonare, che imita percorso naturale di infezione da TBC, rappresenta un approccio interessante per l&#39;innesco della risposta immunitaria host localeS. A questo proposito, varie opere preclinici in modelli animali TB pertinenti hanno dimostrato una maggiore efficacia del vaccino a seguito di immunizzazione polmonare rispetto alla via sottocutanea o intradermica 3-6. Tuttavia, i meccanismi di protezione innescati dalla vaccinazione polmonare non sono ben compresi. Negli ultimi anni, diversi lavori hanno puntato verso di risposta IL17-mediata come un importante fattore di mucosa risposta immunitaria specifica-TB, come nei modelli murini carenti per IL17 mucosa efficacia protettiva del vaccino-indotta è compromessa 7,8. Recentemente abbiamo dimostrato per la prima volta che intranasale amministrazione BCG protetto / 2 topi DBA, un ceppo di topi caratterizzata dalla mancanza di protezione dopo BCG sottocutaneo vaccinazione 9. Questi risultati suggeriscono che la vaccinazione respiratorio TB potrebbe essere più efficace nel ridurre il tasso di tubercolosi nei paesi endemici, in cui intradermica di BCG è considerato inefficace contro pulmonTB ary.

<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.

Questo lavoro descrive il confronto delle due vie di somministrazione di BCG: sottocutaneo e intranasale. Via di somministrazione sottocutanea è paragonabile al intradermica, che è il percorso clinica corrente BCG mondo. Via intranasale della vaccinazione si propone di imitare la via naturale di infezione di M. tuberculosis, con l&#39;obiettivo di indurre risposta immunitaria direttamente nei polmoni, l&#39;organo bersaglio primario di questo patogeno. <p class="jove_conte...

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.

L&#39;efficacia e la protezione polmonare risposta immunitaria seguito sottocutanea e intranasale BCG Amministrazione nei topi

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.5K Views.  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.

Video: 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.

Un test funzionale di sangue intero per misurare la vitalità dei micobatteri, utilizzando Reporter-Gene Tagged BCG o 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 ...

Ricerca

Immunologia e infezioni

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.

Endolinfatica immunoterapia e di vaccinazione nei topi

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.

L&#39;immunoterapia sublinguale come alternativa di indurre una protezione contro le infezioni respiratorie acute

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 ...

Medicina

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.

Un test CFSE-based per studiare la migrazione delle cellule dendritiche murini della pelle in drenanti linfonodi durante l&#39;infezione con<em&gt; Mycobacterium bovis</em&gt; Bacille Calmette-Guérin

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.

Espressione di antigeni esogeni nel Mycobacterium bovis BCG vaccino via genetica decorazione superficiale con il sistema avidina-biotina

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.

Un protocollo di aspirazione Blister per studiare T-cellula umana richiamo 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 ...

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.

Valutazione delle risposte ospite-patogeno e l'efficacia del vaccino nei topi

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.

Arricchimento di vescicole extracellulari native e ricombinanti di micobatteri

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

Accelerazione della determinazione della tubercolosi con il metodo Micro-CFU

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.

Autore in primo piano: Ottimizzazione della determinazione delle CFU per una valutazione efficiente dell'efficacia del vaccino contro la tubercolosi e dell'analisi della presentazione dell'antigene 

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.

Saggio dell'unità formante microcolonie per la valutazione dell'efficacia dei vaccini contro la tubercolosi

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 ...

L'efficacia e la protezione polmonare risposta immunitaria seguito sottocutanea e intranasale BCG Amministrazione nei topi

Riepilogo

Esplora Altri Video

Capitoli in questo video

Un test funzionale di sangue intero per misurare la vitalità dei micobatteri, utilizzando Reporter-Gene Tagged BCG o M. Tb (BCG Lux / M. Tb Lux)

Endolinfatica immunoterapia e di vaccinazione nei topi

L'immunoterapia sublinguale come alternativa di indurre una protezione contro le infezioni respiratorie acute

Un test CFSE-based per studiare la migrazione delle cellule dendritiche murini della pelle in drenanti linfonodi durante l'infezione con

Espressione di antigeni esogeni nel Mycobacterium bovis BCG vaccino via genetica decorazione superficiale con il sistema avidina-biotina

Un protocollo di aspirazione Blister per studiare T-cellula umana richiamo Responses In Vivo

Valutazione delle risposte ospite-patogeno e l'efficacia del vaccino nei topi

Arricchimento di vescicole extracellulari native e ricombinanti di micobatteri

Saggio dell'unità formante microcolonie per la valutazione dell'efficacia dei vaccini contro la tubercolosi

Saggio dell'unità formante microcolonie per la valutazione dell'efficacia dei vaccini contro la tubercolosi