Immunology and Infection
Published: September 19th, 2016
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....
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.
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.
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.
|Middlebrook 7H9 broth
|Middlebrook ADC Enrichment
|3-mm diameter Glass Beads
|Middlebrook 7H10 Agar
|1-ml syringe 26GA 0.45x10 mm
|Falcon 70µm Cell Strainer
|Red Blood Cell Lysing Buffer
|Fetal Calf Serum
|Scepter 2.0 Handheld Automated Cell Counter
|Scepter Cell Counter Sensors, 40 µm
|Mycobacterium Tuberculosis - Tuberculin PPD
|Statens Serum Institut (SSI)
|Mouse IFN-γ ELISA development kit
|Mouse IL17A ELISA development kit
|FITC Rat Anti-Mouse CD4
|BD Cytofix/Cytoperm Kit
|APC-Cy7 Rat Anti-mouse IL-17A
|APC Mouse Anti-mouse IFNg
|LACHRYMAL OLIVE LUER LOCK 0.60 x 30 mm. 23G x 1 1/4”
|Used as trachea cannula for BAL
|high-protein binding polystyrene flat-bottom 96-well plates MAXISORP
|Albumin, from bovine serum
|Goat Anti-Mouse IgA (α-chain specific)−Peroxidase antibody
|MyTaq DNA Polymerase
|The kit Includes Buffer 5x
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