La eficacia protectora y la respuesta inmune pulmonar tras la administración subcutánea e intranasal BCG en ratones
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 tuberculosis (TB) es una de las principales enfermedades infecciosas que causan más muertes asociadas que el VIH en el mundo y unida a la creciente aumento de cepas resistentes a múltiples fármacos TB hace un problema de salud mundial alarmante 1. Las nuevas herramientas de diagnóstico, medicamentos más eficaces y menos tóxicos, y nuevas vacunas contra la tuberculosis seguros y eficaces son una necesidad urgente, especialmente en el mundo en desarrollo.
En vivo atenuado bacilo de Calmette-Guerin (BCG) es actualmente la única vacuna autorizada contra la tuberculosis, que ha sido administrada por vía intradérmica en el nacimiento desde 1970 en todo el mundo. BCG se considera eficaz en la prevención de las formas graves de la enfermedad (meningitis y tuberculosis miliar) en niños, pero ha demostrado eficacia contra la TB pulmonar incompatible responsable de la transmisión de la enfermedad 2.
la vacunación pulmonar, que imita ruta natural de la infección tuberculosa, representa un enfoque atractivo para la imprimación de la respuesta inmune anfitrión locals. En este sentido, varios trabajos preclínicos en diferentes modelos animales pertinentes TB han demostrado una mayor eficacia de la vacuna después de la inmunización pulmonar en comparación con la vía subcutánea o intradérmica 3-6. Sin embargo, los mecanismos de protección provocados por la vacunación pulmonar no se conocen bien. En los últimos años, varios trabajos han señalado hacia respuesta mediada por IL17 como un factor importante de la respuesta inmune de la mucosa-TB específica, como en modelos de ratones deficientes para IL17 eficacia protectora inducida por la vacuna de la mucosa se vea obstaculizada 7,8.
Recientemente hemos demostrado por primera vez que la administración intranasal BCG protege ratones DBA / 2, una cepa de ratones que se caracteriza por la falta de protección después de la vacunación con BCG subcutáneo 9. Estos resultados sugieren que la vacunación contra la tuberculosis respiratoria podría ser más eficaz en la reducción de la tasa de tuberculosis en los países endémicos, donde se considera ineficaz contra pulmon BCG intradérmicaTB aria.
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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