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 miliary TB) in children, but has shown inconsistent efficacy against pulmonary TB responsible of disease transmission ².

Pulmonary vaccination, which mimics natural route of TB infection, represents an attractive approach for priming local host immune responses. In this regard, various preclinical works in different relevant TB animal models have demonstrated greater vaccine efficacy following pulmonary immunization as compared to the subcutaneous or intradermal route ^3-6. Nevertheless, the protective mechanisms triggered by pulmonary vaccination are not well understood. In the last years, several works have pointed towards IL17-mediated response as an important factor of TB-specific mucosal immune response, as in mouse models deficient for IL17 mucosal vaccine-induced protective efficacy is impaired ^7,8.

Recently we demonstrated for the first time that intranasal BCG administration protected DBA/2 mice, a mouse strain characterized by the lack of protection after subcutaneous BCG immunization ⁹. These results suggested that respiratory TB vaccination could be more effective in reducing rate of TB in endemic countries, where intradermal BCG is considered ineffective against pulmonary TB.

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-80 0.05% (v/v) and ADC (Albumin, Dextrose, Catalase) 10% (v/v).
2. Incubate under static conditions for one week at 37 °C until the culture is in log-phase growth.
3. Scale up bacterial culture by transferring the 10-ml culture to 200 ml of fresh medium. Incubate for 20 additional days under static conditions.
4. Transfer to 50-ml centrifuge tubes and centrifuge for 15 min at 2500 x g.
5. Discard supernatants and resuspend bacterial pellet in the residual volume. Add to each tube sterile 3-mm-diameter glass beads (10 - 15 per tube).
6. Vortex vigorously during 1 min in order to dissociate bacterial clumps. Resuspend each pellet in 10 ml of PBS with Tween-80 0.05% (v/v).
7. Leave tubes for 10 min allowing the greatest bacterial aggregates and glass beads to settle down.
8. Recover 9.5 ml of supernatant from each of the 4 tubes containing small clumps and single bacteria and transfer volumes to a single fresh 50-ml centrifuge tube (final recovered volume 38 ml). Centrifuge for 10 min at 400 x g.
9. Recover 35 ml of supernatant (containing mainly single bacteria) in a single fresh 50-ml centrifuge tube and add 15 ml of PBS with glycerol 50% (v/v), obtaining a final glycerol concentration of 15% (v/v).
10. Mix gently and distribute in 1-ml aliquots in appropriate tubes for freezing. Store at -80 °C (one lot provides approximately 50 aliquots glycerol stock).
11. To quantify glycerol stocks, thaw a glycerol the day after freezing and perform seven ten-fold serial dilutions in separate 1.5-ml tubes containing 900 μl of PBS.
12. Plate 100 μl of each dilution in 7H10 solid-agar medium¹⁰ supplemented with ADC 10% (v/v) prepared in 55-mm diameter Petri dishes.
13. Carefully add 3 mm-diameter glass beads (approx. 10 per plate) and gently shake plate to equally distribute volume over agar plate. Discard beads and seal agar plate with parafilm.
14. Incubate for 21 days at 37 °C and determine bacterial concentration by counting colony-forming units (CFU) in the dilutions where single colonies can be accurately distinguished.

2. Mouse Vaccination

1. Thaw a pre-quantified BCG glycerol and dilute it in PBS to prepare two suspensions. Calculate final volume considering the dose per animal is 100 μl for subcutaneous administration (10⁷ CFU per ml for subcutaneous vaccination) and 40 μl for intranasal administration (2.5 x 10⁷ CFU for intranasal vaccination).
2. Use a specialized anesthesia workstation for rodents to anesthetize mice with a mixture of isoflurane and oxygen. Use isoflurane 5% to induce anesthesia and 2% to maintain the mice in the anesthesia chamber. (Other accepted anesthesia method can be used).
3. For subcutaneous vaccine administration fill a 1-ml syringe with a 26 G needle with 1 ml of bacterial suspension (10⁷ CFU/ml). Remove air bubbles.
4. Place an anesthetized mouse on a flat surface in prone position inside a laminar flow hood and inoculate subcutaneously 100 μl (10⁶ CFU/dose) of vaccine in a flank of the mouse back. Return the mouse to the cage and monitor it to ensure that it properly recovers from anesthesia.
5. Change the syringe needle between each mouse administration. Removed air bubbles as described above.
6. For intranasal administration, place an anesthetized mouse in supine position inside the hood.
7. With a micropipette take 20 μl of the suspension with 2.5 x 10⁷ CFU/ml. Place the inoculum drop-by-drop between the two nostrils until the whole volume has been deposited, leaving time between drops to allow the mouse breathe the volume in. Note: mice can suffer from respiratory distress during this procedure so they should be left to assimilate each drop prior to administrate the next one to prevent it as much as possible.
8. Re-fill the micropipette with another 20 μl and repeat the operation. If the mouse starts to wake up from the anesthesia after the first inoculation, place it in the anesthesia chamber prior to the second one. Return the mouse to the cage and ensure that it properly recovers from anesthesia.

3. Mouse Intranasal Challenge with H37Rv Strain

1. Thaw a pre-quantified H37Rv glycerol and dilute it in PBS to prepare a bacterial suspension of 2,500 CFU per ml. Calculate final volume considering the dose per animal is 40 μl.
2. H37Rv is inoculated intranasally as described in 2.5 - 2.7. Final challenge dose per mouse is 10² CFU/40 µl.

4. Analysis of Induced Immune Response in Lungs

1. Lung cellular suspension
   NOTE: Assessment of vaccine-induced adaptive immune response in lungs requires the generation of single cell suspension. For this purpose, we use a tissue dissociator such as GentleMACS .
   1. Sacrifice the mouse by cervical dislocation (or other accepted humane methodology) and ensure mouse is dead touching the eyeball to confirm absent of blink reflex.
   2. Inside a laminar flow hood, extract the lungs using sterile scissors and forceps and place them on a clean surface. Clean lungs removing trachea and connective tissue, and transfer the lungs to a 1.5-ml tube with PBS (maintain on ice until processing).
   3. To generate a cellular suspension, place lungs in a proper tube for organ dissociation (C-dissociator tube) containing 5 ml of 10 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂ and 1.8 mM CaCl₂ buffer.
   4. Add 100 μl of Collagenase D 100 mg/ml (2 mg/ml final concentration), and 40 μl of DNAse I 20,000 IU/ml (160 IU/ml final concentration.
      NOTE: Stock solutions of Collagenase D or DNaseI are prepared dissolving lyophilized enzymes in PBS or glycerol 50% (v/v), 20 mM Tris-HCl pH 7.5 y MgCl₂ 1 mM, respectively.
   5. Place the tube in the tissue dissociator and use the predefined program m_lung_01 for lung fragmentation. Incubate the tube at 37 °C in a water bath for 30 min.
   6. Place the tube in the dissociator and use the predefined program m_lung_02 for complete lung homogenization into single cellular suspension.
   7. Pass the cellular suspension through a 70-μm nylon cell strainer fitted to a 50-ml centrifuge tube. Wash the strainer with 10 ml of RPMI 1640 medium.
   8. Centrifuge cells for 5 minutes at 400 x g and discard supernatant.
   9. Add 1 ml of erythrocytes lysing buffer, mix by vortexing and incubate for 1 min at room temperature.
   10. Add 10 ml of RPMI 1640 medium and centrifuge cells for 5 minutes at 400 x g.
   11. Resuspend cells in 0.5 ml of complete RPMI 1640 medium supplemented with 10% heat-inactivated Foetal Calf Serum (FCS), 2 mM L-Glutamin, 100 μg/ml Streptomycin, 100 IU/ml Penicillin and 50 μM 2-mercaptoethanol.
       NOTE: FCS is inactivated by incubation at 56°C for 30 minutes.
   12. Count cells and add complete RPMI 1640 medium to reach final cellular density of 10⁷ cells/ml.
       NOTE: For cell count, we use the automated cell counter.
   13. Distribute 100 μl of cellular suspension in U-bottom 96-well sterile plate (10⁶ cells/well).
   14. Add 50 μl/well of complete RPMI 1640 medium with 15 μg/ml of commercial H37Rv Purified Protein Derivative (PPD) (5 μg/ml final concentration).
2. Supernatant collection for cytokine determination by ELISA
   1. Incubate plate from 4.1.14 for 48 hr at 37 °C and 5% CO₂.
   2. Centrifuge plate for 5 min at 400 x g and recover 100 μl of supernatant.
   3. Store at -80 °C.
   4. Dilute supernatants 1/10 and 1/2 in assay buffer for IFNγ and IL17A determination, respectively. Determine cytokine concentrations with commercial ELISA kits, according to manufacturer instructions (ELISA Development kits).
3. Intracellular staining for determination of cytokine-producing CD4+ cells by flow cytometry
   1. Incubate plate from 4.1.14 for 18 hr at 37 °C and 5% CO_2.
   2. Add 1.5 μl of Brefeldin A 1 mg/ml (stock dissolved in DMSO) to each well (10 μg/ml final concentration) and incubate for six additional hours.
   3. Centrifuge the plate from 4.3.2 for 1 min at 3200 x g and discard supernatant.
   4. Resuspend cells in 50 μl of anti-mouse CD4-FITC diluted in RPMI 1640 with 10% FCS (1/500) and incubate for 10 min at 4 °C.
   5. Centrifuge the plate from 4.3.4 for 1 min at 3,200 x g, discard supernatant and add 100 μl/well of fixation solution. Incubate at 4 °C for 20 min.
   6. Wash cells twice with 150 μl/wash of permeabilization solution.
      NOTE: 10x concentrated stock is diluted in deionized water to prepare 1x work solution.
   7. Resuspend cells in 50 μl of anti mouse IFNγ-APC and anti-mouse IL17A-APC.Cy7 diluted in permeabilization solution (1/250 both antibodies).
   8. Incubate for 1 hr at RT.
   9. Wash cells twice with 150 μl/wash of permeabilization solution.
   10. Resuspend cells in 200 μl of PBS for flow cytometry acquisition.

5. Analysis of Immunoglobulins in Bronchoalveolar Lavage (BAL)

1. Obtainment of BAL samples
   1. Sacrifice the mouse by cervical dislocation (or other accepted humane methodology) and ensure mouse is dead touching the eyeball to confirm absent of blink reflex.
   2. Expose the trachea using proper precision sterile forceps and scissors and perform a small cut in the trachea, making sure not to excise it completely. Take care not to cause bleeding that could contaminate BAL sample.
   3. Introduce into the trachea a cannula connected to a sterile 1-ml syringe with 800 μl of ice-cold PBS.
   4. Inoculate slowly the PBS into the lungs and then recover BAL volume pulling back the syringe's piston very slowly in order to avoid lung collapse.
      NOTE: Recovery of 500 to 600 μl is satisfactory.
   5. Transfer BAL volume into a 1.5-ml tube and confirm BAL remains uncoloured, indicating that is not contaminated with blood (maintain on ice until processing).
   6. Centrifuge BAL samples for 5 min at 400 x g, and transfer supernatant to a new tube.
   7. Store supernatants at -80 °C.
2. Determination of IgA in BAL supernatants
   NOTE: All reagents used are at RT.
   1. Coat high-protein binding polystyrene flat-bottom 96-well plates with 100 μl of PPD diluted in PBS (10 μg/ml) for M. tuberculosis-specific IgA determination, or with 100 μl of BAL supernatant diluted 10 times in PBS for total IgA determination.
   2. Incubate overnight at 4 °C.
   3. Discard supernatant and tap the plate on paper towel to dry. Wash with 200 μl/well of PBS.
   4. Block with 200 μl/well of a bovine serum albumin (BSA) 1% (w/v) in PBS-Tween-80 0.05% (v/v) (Blocking buffer).
   5. Incubate 1 hr at RT.
   6. Wash once with 200 μl/well of PBS-Tween-80 0.05% (v/v) (Washing buffer).
   7. Add 100 μl of undiluted BAL supernatant in the PPD-coated wells. Incubate two hours at room temperature.
      NOTE: If the total IgA determination is done in parallel with the PPD-specific IgA, leave total IgA wells with 100 μl washing buffer during this incubation.
   8. Wash three times with 200 μl/well of washing buffer.0170004
   9. Add 100 μl of horseradish peroxidase (HRP)-conjugated anti-mouse IgA diluted in blocking buffer (1/10,000).
   10. Incubate 1 hr RT.
   11. Wash five times with 200 μl/well of washing buffer.
   12. Add 100 μl/well of 3,3′,5,5′-Tetramethylbenzidine (TMB). Incubate 20 min at room temperature in the dark.
   13. Add 100 μl/well of H₂SO₄ 0.5N. Read the plate in a spectrophotometer at a wavelength of 450 nm.

6. Bacterial load determination in lungs

1. Lung homogenization and plating
   1. Four weeks after challenge, sacrifice the mouse by cervical dislocation (or other accepted humane methodology) and ensure mouse is dead touching the eyeball to confirm absent of blink reflex.
   2. Place the mouse in supine position fixed on a flat and disinfected surface inside a laminar flow hood.
   3. Make an incision and remove the skin from chest region to leave visible the thoracic area.
   4. Cut out the ribs and harvest the lungs and heart using sterile scissors and forceps. Place organs on a clean surface.
   5. Clean out lungs by removing the heart, trachea and connective tissue and transfer the lungs to a 1.5-ml tube with 1 ml of deionized water (maintain on ice until processing).
   6. Use sterile forceps to transfer the lungs of each mouse into a proper tube for organ homogenization (M - dissociator tube), containing 1 ml of deionized water.
   7. Place the tube in the tissue dissociator and use the predefined program RNA_1 to homogenise the lungs.
   8. Make five ten-fold serial dilutions in 1.5-ml tubes, starting with 100 μl of lung homogenate diluted in 900 μl.
   9. Plate dilutions as described in 1.11-1.14.
2. Differentiation between BCG and H37Rv by PCR
   NOTE: In the plates from 6.1.6 corresponding to the group vaccinated with BCG by the intranasal route, we analyzed a representative number of colonies by a discerning PCR between BCG and H37Rv to ensure that the CFU considered to assess vaccine-conferred protective efficacy corresponded uniquely to H37Rv. This PCR targets amplification of RD9, a genomic region different in BCG and M. tuberculosis, giving a fragment of 2618 bp for H37Rv, and 465 bp for BCG ¹¹.
   1. Prepare a master mix stock for all samples with volumes per sample as indicated, and then distribute in PCR tubes (9 μl/sample).
      NOTE: Buffer 5x: 2 μl. Forward primer 25 μM (GTGTAGGTCAGCCCCATCC): 0.32 μl, Reverse primer 25 μM (GCCCAACAGCTCGACATC): 0.32 μl, Taq polymerase 5 units/μl: 0.04 μl, Deionized H₂O: 6.32 μl.
   2. Touch a colony with a sterile toothpick and immerse the toothpick with the sample in a PCR tube containing the master mix.
   3. Run the PCR with the following program: 10 min 95 °C (this step is to trigger bacterial disruption), 10 min 95 °C (this step is to trigger bacterial disruption), 35 cycles ( 30 seconds at 95 °C, 1 min at 58 °C and 4 min at 72 °C).
   4. Load the PCR samples in a 1% agarose gel with ethidium bromide and run samples to visualize the fragments.

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

List of materials used in this article

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 0.45 x 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 70 µm 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