Summary

A eficácia de protecção e Pulmonar resposta imune Seguindo subcutânea e intranasal BCG Administração em Ratinhos

Published: September 19, 2016
doi:

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

Todos os ratinhos foram mantidos sob condições controladas e observados quanto a quaisquer sinais de doença. O trabalho experimental foi realizado de acordo com as directivas europeias e nacionais para a protecção dos animais experimentais e com a aprovação dos comités de ética locais competentes. 1. Preparação de Stocks quantificada glicerol de BCG dinamarquesa e H37Rv Mycobacterium tuberculosis Nota: Todos os protocolos descritos fora…

Representative Results

Este trabalho descreve a comparação de duas vias de administração de BCG: subcutânea e intranasal. via subcutânea é comparável à intradérmica, que é a via clínica corrente de BCG em todo o mundo. Via intranasal de vacinação destina-se a imitar a via natural de infecção de M. tuberculose, com o objetivo de induzir resposta imune diretamente nos pulmões, o principal órgão alvo deste patógeno. <strong…

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.

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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

References

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Cite This Article
Uranga, S., Marinova, D., Martin, C., Aguilo, N. Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice. J. Vis. Exp. (115), e54440, doi:10.3791/54440 (2016).

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