Beschermende werkzaamheid en Pulmonale immuunrespons na subcutane en intranasale BCG Administration in Muizen
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
Tuberculose (tbc) is een van de toonaangevende infectieziekten veroorzaken meer geassocieerd doden dan HIV in de wereld en in combinatie met de stijgende toename van multidrug-resistente stammen maakt TB een alarmerend wereldwijd gezondheidsprobleem 1. Nieuwe diagnostische hulpmiddelen, effectiever en minder giftige medicijnen, en de nieuwe veilige en effectieve tbc-vaccins zijn dringend noodzakelijk, vooral in de ontwikkelingslanden.
Leef verzwakt Bacille Calmette-Guerin (BCG) is momenteel de enige in licentie vaccin tegen tbc, die intradermaal werd toegediend bij de geboorte sinds 1970 wereldwijd. BCG is effectief in het voorkomen van ernstige vormen van de ziekte (meningitis en miliaire TB) bij kinderen beschouwd, maar inconsistente werkzaamheid tegen longtuberculose verantwoordelijk ziekteoverdracht 2 getoond.
Pulmonale vaccinatie, die de natuurlijke route van tuberculose-infectie nabootst, vertegenwoordigt een aantrekkelijke benadering voor het vullen van lokale host immuunresponss. In dit verband hebben verschillende preklinische werken in verschillende relevante TB diermodellen groter vaccineffectiviteit na pulmonaire immunisatie aangetoond in vergelijking met de subcutane of intradermale route 3-6. Toch is de beschermingsmechanismen veroorzaakt door pulmonale vaccinatie zijn niet goed begrepen. In de afgelopen jaren hebben een aantal werken wees naar-IL17 gemedieerde respons als een belangrijke factor van TB-specifieke mucosale immuunrespons, zoals in muismodellen deficiënte voor IL17 mucosale vaccingeïnduceerde beschermende werking in het gedrang 7,8.
Onlangs toonden we voor de eerste keer dat intranasale toediening BCG beschermd DBA / 2 muizen, een muizenstam gekenmerkt door het ontbreken van bescherming na subcutane BCG immunisering 9. Deze resultaten suggereren dat de luchtwegen TB vaccinatie meer effectief in het verminderen tarief van tuberculose in endemische landen, waar de intradermale BCG ineffectief tegen pulmón wordt beschouwd zou kunnen zijnAry TB.
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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