L'efficacia e la protezione polmonare risposta immunitaria seguito sottocutanea e intranasale BCG Amministrazione nei topi
Summary
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.
Abstract
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.
Introduction
La tubercolosi (TB) è una delle principali malattie infettive che causano più morti associate di HIV nel mondo e combinato con l'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'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'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.
Protocol
Representative Results
Discussion
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.
<p c…Disclosures
The authors have nothing to disclose.
Acknowledgements
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].
Materials
| Middlebrook 7H9 broth | BD | 271310 | |
| Middlebrook ADC Enrichment | BD | 211887 | |
| Tween 80 | Scharlau | TW00800250 | |
| 3-mm diameter Glass Beads | Scharlau | 038-138003 | |
| Middlebrook 7H10 Agar | BD | 262710 | |
| 1-ml syringe 26GA 0.45×10 mm | BD | 301358 | |
| GentleMACS dissociator | Miltenyi Biotec | 130-093-235 | |
| C tubes | Miltenyi Biotec | 130-093-237 | |
| M tubes | Miltenyi Biotec | 130-093-236 | |
| Collagenase D | Roche | 11088882001 | |
| DNaseI | Applichem | A3778,0100 | |
| Falcon 70µm Cell Strainer | Corning | 352350 | |
| RPMI 1640 | Sigma | R0883 | |
| Red Blood Cell Lysing Buffer | Sigma | R7757 | |
| GlutaMAX Supplement | Gibco | 35050-061 | 100X concentrated |
| Penicillin-Streptomycin Solution | Sigma | P4333 | 100X concentrated |
| Fetal Calf Serum | Biological Industries | 04-001-1A | |
| 2-Mercaptoethanol | Sigma | M3148-25ML | |
| Scepter 2.0 Handheld Automated Cell Counter | Millipore | PHCC20040 | |
| Scepter Cell Counter Sensors, 40 µm | Millipore | PHCC40050 | |
| Mycobacterium Tuberculosis – Tuberculin PPD | Statens Serum Institut (SSI) | 2390 | |
| Mouse IFN-γ ELISA development kit | Mabtech | 3321-1H | |
| Mouse IL17A ELISA development kit | Mabtech | 3521-1H | |
| Brefeldin A | Sigma | B7651 | |
| FITC Rat Anti-Mouse CD4 | BD | 553047 | |
| BD Cytofix/Cytoperm Kit | BD | 555028 | |
| APC-Cy7 Rat Anti-mouse IL-17A | BD | 560821 | |
| APC Mouse Anti-mouse IFNg | BD | 554413 | |
| LACHRYMAL OLIVE LUER LOCK 0.60 x 30 mm. 23G x 1 1/4” | UNIMED | 27.134 | Used as trachea cannula for BAL |
| high-protein binding polystyrene flat-bottom 96-well plates MAXISORP | NUNC | 430341 | |
| Albumin, from bovine serum | Sigma | A4503 | |
| Goat Anti-Mouse IgA (α-chain specific)−Peroxidase antibody | Sigma | A4789 | |
| 3,3′,5,5′-Tetramethylbenzidine (TMB) | Sigma | T0440 | |
| MyTaq DNA Polymerase | Bioline | BIO-21107 | The kit Includes Buffer 5x |
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