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Representative Results





Immunology and Infection

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Published: September 19th, 2016



1Department of Microbiology, University of Zaragoza

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.

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.

Tuberculosis (TB) is one of the leading infectious diseases causing more associated deaths than HIV in the world and combined with rising increase of multidrug resistant strains makes TB an alarming global health problem1. New diagnostic tools, more effective and less toxic drugs, and new safe and effective TB vaccines are an urgent need, especially in the developing world.

Live attenuated Bacille Calmette-Guerin (BCG) is currently the only licenced vaccine against TB, which has been administered intradermally at birth since the 1970s worldwide. BCG is considered effective in preventing severe forms of the disease (meningitis and....

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All mice were kept under controlled conditions and observed for any sign of disease. Experimental work was conducted in agreement with European and national directives for protection of experimental animals and with approval from the competent local ethics committees.

1. Preparation of Quantified Glycerol Stocks of BCG Danish and Mycobacterium tuberculosis H37Rv

NOTE: All the protocols described were performed under BSL3 conditions.


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This work describes the comparison of two routes of administration of BCG: subcutaneous and intranasal. Subcutaneous route is comparable to the intradermal, which is the current clinical route for BCG worldwide. Intranasal route of vaccination aims to mimic the natural route of infection of M. tuberculosis, with the objective to induce immune response directly in the lungs, the primary target organ of this pathogen.

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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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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].


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Name Company Catalog Number Comments
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 0.45x10 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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