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

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

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....

Protocol

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.

1. Thaw frozen glycerol stocks of BCG Danish or H37Rv strains and inoculate 100 μl in 10 ml of 7H9 medium¹⁰ supplemented with Tween-....

Results

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.

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.

Materials

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