A eficácia de protecção e Pulmonar resposta imune Seguindo subcutânea e intranasal BCG Administração em Ratinhos
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
A tuberculose (TB) é uma das doenças infecciosas, causador de mortes associadas mais do que o HIV no mundo e combinada com o aumento do crescimento de estirpes resistentes a múltiplos fármacos torna a TB um problema de saúde mundial alarmante 1. Novas ferramentas de diagnóstico, drogas mais eficazes e menos tóxicos, e novas vacinas contra a tuberculose seguros e eficazes são uma necessidade urgente, especialmente nos países em desenvolvimento.
Vivo atenuado Bacilo de Calmette-Guerin (BCG) é atualmente a única vacina licenciada contra a tuberculose, que foi administrado por via intradérmica no nascimento desde 1970 em todo o mundo. BCG é considerada eficaz na prevenção de formas graves da doença (meningite e TB miliar) em crianças, mas tem demonstrado eficácia inconsistente contra a TB pulmonar responsável da transmissão da doença 2.
vacinação pulmonar, que imita via natural de infecção por tuberculose, representa uma abordagem atraente para o priming resposta imune host locals. A este respeito, vários trabalhos pré-clínicos em diferentes modelos animais relevantes de TB demonstram maior eficácia da vacina após imunização pulmonar, em comparação com a via subcutânea ou intradérmica rota 3-6. No entanto, os mecanismos de protecção activadas por vacinação pulmonar não são bem compreendidos. Nos últimos anos, vários trabalhos têm apontado para resposta mediada por IL17 como um fator importante da resposta imune da mucosa específicos de TB, como em modelos de ratos deficientes em IL17 eficácia protetora induzida pela vacina da mucosa é prejudicada 7,8.
Recentemente, demonstrou pela primeira vez que intranasal administração BCG protegido ratinhos DBA / 2, uma cepa de rato caracteriza-se pela falta de proteção após a BCG subcutânea imunização 9. Estes resultados sugerem que a vacinação TB respiratória poderia ser mais eficaz na redução da taxa de TB em países endêmicos, onde BCG intradérmica é considerado ineficaz contra 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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