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].

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.

<ol>
	<li>Thaw frozen glycerol stocks of BCG Danish or H37Rv strains and inoculate 100 &#956;l in 10 ml of 7H9 medium10 supplemented with Tween-80 0.05% (v/v) and ADC (Albumin, Dextrose, Catalase) 10% (v/v).</li>
	<li>Incubate under static conditions for one week at 37 &#176;C until the culture is in log-phase growth.</li>
	<li>Scale up bacterial culture by transferring the 10-ml culture to 200 ml of fresh medium. Incubate for 20 additional days under static conditions.</li>
	<li>Transfer to 50-ml centrifuge tubes and centrifuge for 15 min at 2500 x g.</li>
	<li>Discard supernatants and resuspend bacterial pellet in the residual volume. Add to each tube sterile 3-mm-diameter glass beads (10 - 15 per tube).</li>
	<li>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).</li>
	<li>Leave tubes for 10 min allowing the greatest bacterial aggregates and glass beads to settle down.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Mix gently and distribute in 1-ml aliquots in appropriate tubes for freezing. Store at -80 &#176;C (one lot provides approximately 50 aliquots glycerol stock).</li>
	<li>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 &#956;l of PBS.</li>
	<li>Plate 100 &#956;l of each dilution in 7H10 solid-agar medium10 supplemented with ADC 10% (v/v) prepared in 55-mm diameter Petri dishes.</li>
	<li>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.</li>
	<li>Incubate for 21 days at 37 &#176;C and determine bacterial concentration by counting colony-forming units (CFU) in the dilutions where single colonies can be accurately distinguished.</li>
</ol>
2. Mouse Vaccination
<ol>
	<li>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 &#956;l for subcutaneous administration (107 CFU per ml for subcutaneous vaccination) and 40 &#956;l for intranasal administration (2.5 x 107 CFU for intranasal vaccination).</li>
	<li>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.&#160;(Other accepted anesthesia method can be used).</li>
	<li>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.</li>
	<li>Place an anesthetized mouse on a flat surface in prone position inside a laminar flow hood and inoculate subcutaneously 100 &#956;l (106 CFU/dose) of vaccine in a flank of the mouse back.&#160;Return the mouse to the cage and monitor it to ensure that it properly recovers from anesthesia.</li>
	<li>Change the syringe needle between each mouse administration. Removed air bubbles as described above.</li>
	<li>For intranasal administration, place an anesthetized mouse in supine position inside the hood.</li>
	<li>With a micropipette take 20 &#956;l 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.&#160;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.</li>
	<li>Re-fill the micropipette with another 20 &#956;l 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.&#160;Return the mouse to the cage and ensure that it properly recovers from anesthesia.</li>
</ol>
3. Mouse Intranasal Challenge with H37Rv Strain
<ol>
	<li>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 &#956;l.</li>
	<li>H37Rv is inoculated intranasally as described in 2.5 - 2.7. Final challenge dose per mouse is 102 CFU/40 &#181;l.</li>
</ol>
4. Analysis of Induced Immune Response in Lungs
<ol>
	<li>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 .
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>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.</li>
		<li>Add 100 &#956;l of Collagenase D 100 mg/ml (2 mg/ml final concentration), and 40 &#956;l 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.</li>
		<li>Place the tube in the tissue dissociator and use the predefined program m_lung_01 for lung fragmentation. Incubate the tube at 37 &#176;C in a water bath for 30 min.</li>
		<li>Place the tube in the dissociator and use the predefined program m_lung_02 for complete lung homogenization into single cellular suspension.</li>
		<li>Pass the cellular suspension through a 70-&#956;m nylon cell strainer fitted to a 50-ml centrifuge tube. Wash the strainer with 10 ml of RPMI 1640 medium.</li>
		<li>Centrifuge cells for 5 minutes at 400 x g and discard supernatant.</li>
		<li>Add 1 ml of erythrocytes lysing buffer, mix by vortexing and incubate for 1 min at room temperature.</li>
		<li>Add 10 ml of RPMI 1640 medium and centrifuge cells for 5 minutes at 400 x g.</li>
		<li>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 &#956;g/ml Streptomycin, 100 IU/ml Penicillin and 50 &#956;M 2-mercaptoethanol. 
		NOTE: FCS is inactivated by incubation at 56&#176;C for 30 minutes.</li>
		<li>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.</li>
		<li>Distribute 100 &#956;l of cellular suspension in U-bottom 96-well sterile plate (106 cells/well).</li>
		<li>Add 50 &#956;l/well of complete RPMI 1640 medium with 15 &#956;g/ml of commercial H37Rv Purified Protein Derivative (PPD) (5 &#956;g/ml final concentration).</li>
	</ol>
	</li>
	<li>Supernatant collection for cytokine determination by ELISA
	<ol>
		<li>Incubate plate from 4.1.14 for 48 hr at 37 &#176;C and 5% CO2.</li>
		<li>Centrifuge plate for 5 min at 400 x g and recover 100 &#956;l of supernatant.</li>
		<li>Store at -80 &#176;C.</li>
		<li>Dilute supernatants 1/10 and 1/2 in assay buffer for IFN&#947; and IL17A determination, respectively. Determine cytokine concentrations with commercial ELISA kits, according to manufacturer instructions (ELISA Development kits).</li>
	</ol>
	</li>
	<li>Intracellular staining for determination of cytokine-producing CD4+ cells by flow cytometry
	<ol>
		<li>Incubate plate from 4.1.14 for 18 hr at 37 &#176;C and 5% CO2.</li>
		<li>Add 1.5 &#956;l of Brefeldin A 1 mg/ml (stock dissolved in DMSO) to each well (10 &#956;g/ml final concentration) and incubate for six additional hours.</li>
		<li>Centrifuge the plate from 4.3.2 for 1 min at 3200 x g and discard supernatant.</li>
		<li>Resuspend cells in 50 &#956;l of anti-mouse CD4-FITC diluted in RPMI 1640 with 10% FCS (1/500) and incubate for 10 min at 4 &#176;C.</li>
		<li>Centrifuge the plate from 4.3.4 for 1 min at 3,200 x g, discard supernatant and add 100 &#956;l/well of fixation solution. Incubate at 4 &#176;C for 20 min.</li>
		<li>Wash cells twice with 150 &#956;l/wash of permeabilization solution. 
		NOTE: 10x concentrated stock is diluted in deionized water to prepare 1x work solution.</li>
		<li>Resuspend cells in 50 &#956;l of anti mouse IFN&#947;-APC and anti-mouse IL17A-APC.Cy7 diluted in permeabilization solution (1/250 both antibodies).</li>
		<li>Incubate for 1 hr at RT.</li>
		<li>Wash cells twice with 150 &#956;l/wash of permeabilization solution.</li>
		<li>Resuspend cells in 200 &#956;l of PBS for flow cytometry acquisition.</li>
	</ol>
	</li>
</ol>
5. Analysis of Immunoglobulins in Bronchoalveolar Lavage (BAL)
<ol>
	<li>Obtainment of BAL samples
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Introduce into the trachea a cannula connected to a sterile 1-ml syringe with 800 &#956;l of ice-cold PBS.</li>
		<li>Inoculate slowly the PBS into the lungs and then recover BAL volume pulling back the syringe&#39;s piston very slowly in order to avoid lung collapse. 
		NOTE: Recovery of 500 to 600 &#956;l is satisfactory.</li>
		<li>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).</li>
		<li>Centrifuge BAL samples for 5 min at 400 x g, and transfer supernatant to a new tube.</li>
		<li>Store supernatants at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Determination of IgA in BAL supernatants 
	NOTE: All reagents used are at RT.
	<ol>
		<li>Coat high-protein binding polystyrene flat-bottom 96-well plates with 100 &#956;l of PPD diluted in PBS (10 &#956;g/ml) for M. tuberculosis-specific IgA determination, or with 100 &#956;l of BAL supernatant diluted 10 times in PBS for total IgA determination.</li>
		<li>Incubate overnight at 4 &#176;C.</li>
		<li>Discard supernatant and tap the plate on paper towel to dry. Wash with 200 &#956;l/well of PBS.</li>
		<li>Block with 200 &#956;l/well of a bovine serum albumin (BSA) 1% (w/v) in PBS-Tween-80 0.05% (v/v) (Blocking buffer).</li>
		<li>Incubate 1 hr at RT.</li>
		<li>Wash once with 200 &#956;l/well of PBS-Tween-80 0.05% (v/v) (Washing buffer).</li>
		<li>Add 100 &#956;l 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 &#956;l washing buffer during this incubation.</li>
		<li>Wash three times with 200 &#956;l/well of washing buffer.0170004</li>
		<li>Add 100 &#956;l of horseradish peroxidase (HRP)-conjugated anti-mouse IgA diluted in blocking buffer (1/10,000).</li>
		<li>Incubate 1 hr RT.</li>
		<li>Wash five times with 200 &#956;l/well of washing buffer.</li>
		<li>Add 100 &#956;l/well of 3,3&#8242;,5,5&#8242;-Tetramethylbenzidine (TMB). Incubate 20 min at room temperature in the dark.</li>
		<li>Add 100 &#956;l/well of H2SO4 0.5N. Read the plate in a spectrophotometer at a wavelength of 450 nm.</li>
	</ol>
	</li>
</ol>
6. Bacterial load determination in lungs
<ol>
	<li>Lung homogenization and plating
	<ol>
		<li>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.</li>
		<li>Place the mouse in supine position fixed on a flat and disinfected surface inside a laminar flow hood.</li>
		<li>Make an incision and remove the skin from chest region to leave visible the thoracic area.</li>
		<li>Cut out the ribs and harvest the lungs and heart using sterile scissors and forceps. Place organs on a clean surface.</li>
		<li>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).</li>
		<li>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.</li>
		<li>Place the tube in the tissue dissociator and use the predefined program RNA_1 to homogenise the lungs.</li>
		<li>Make five ten-fold serial dilutions in 1.5-ml tubes, starting with 100 &#956;l of lung homogenate diluted in 900 &#956;l.</li>
		<li>Plate dilutions as described in 1.11-1.14.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Prepare a master mix stock for all samples with volumes per sample as indicated, and then distribute in PCR tubes (9 &#956;l/sample). 
		NOTE: Buffer 5x: 2 &#956;l. Forward primer 25 &#956;M (GTGTAGGTCAGCCCCATCC): 0.32 &#956;l, Reverse primer 25 &#956;M (GCCCAACAGCTCGACATC): 0.32 &#956;l, Taq polymerase 5 units/&#956;l: 0.04 &#956;l, Deionized H2O: 6.32 &#956;l.</li>
		<li>Touch a colony with a sterile toothpick and immerse the toothpick with the sample in a PCR tube containing the master mix.</li>
		<li>Run the PCR with the following program: 10 min 95 &#176;C (this step is to trigger bacterial disruption), 10 min 95 &#176;C (this step is to trigger bacterial disruption), 35 cycles ( 30 seconds at 95 &#176;C, 1 min at 58 &#176;C and 4 min at 72 &#176;C).</li>
		<li>Load the PCR samples in a 1% agarose gel with ethidium bromide and run samples to visualize the fragments.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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.
<p c...

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.

<table><tbody><tr><td>Middlebrook 7H9 broth</td><td>BD</td><td>271310</td><td></td></tr><tr><td>Middlebrook ADC Enrichment</td><td>BD</td><td>211887</td><td></td></tr><tr><td>Tween 80</td><td>Scharlau</td><td>TW00800250</td><td></td></tr><tr><td>3-mm diameter Glass Beads</td><td>Scharlau</td><td>038-138003</td><td></td></tr><tr><td>Middlebrook 7H10 Agar</td><td>BD</td><td>262710</td><td></td></tr><tr><td>1-ml syringe 26GA 0.45 x 10 mm</td><td>BD</td><td>301358</td><td></td></tr><tr><td>GentleMACS dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td></td></tr><tr><td>C tubes</td><td>Miltenyi Biotec</td><td>130-093-237</td><td></td></tr><tr><td>M tubes</td><td>Miltenyi Biotec</td><td>130-093-236</td><td></td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088882001</td><td></td></tr><tr><td>DNaseI</td><td>Applichem</td><td>A3778,0100</td><td></td></tr><tr><td>Falcon 70 &amp;micro;m Cell Strainer</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>RPMI 1640</td><td>Sigma</td><td>R0883</td><td></td></tr><tr><td>Red Blood Cell Lysing Buffer</td><td>Sigma</td><td>R7757</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco</td><td>35050-061</td><td>100x concentrated</td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Sigma</td><td>P4333</td><td>100x concentrated</td></tr><tr><td>Fetal Calf Serum</td><td>Biological Industries</td><td>04-001-1A</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M3148-25ML</td><td></td></tr><tr><td>Scepter 2.0 Handheld Automated Cell Counter</td><td>Millipore</td><td>PHCC20040</td><td></td></tr><tr><td>Scepter Cell Counter Sensors, 40 &amp;micro;m</td><td>Millipore</td><td>PHCC40050</td><td></td></tr><tr><td>Mycobacterium Tuberculosis - Tuberculin PPD</td><td>Statens Serum Institut (SSI)</td><td>2390</td><td></td></tr><tr><td>Mouse IFN-&amp;gamma; ELISA development kit&amp;nbsp;</td><td>Mabtech</td><td>3321-1H</td><td></td></tr><tr><td>Mouse IL17A ELISA development kit&amp;nbsp;</td><td>Mabtech</td><td>3521-1H</td><td></td></tr><tr><td>Brefeldin A</td><td>Sigma</td><td>B7651</td><td></td></tr><tr><td>FITC Rat Anti-Mouse CD4</td><td>BD</td><td>553047</td><td></td></tr><tr><td>BD Cytofix/Cytoperm Kit</td><td>BD</td><td>555028</td><td></td></tr><tr><td>APC-Cy7 Rat Anti-mouse IL-17A</td><td>BD</td><td>560821</td><td></td></tr><tr><td>APC Mouse Anti-mouse IFNg</td><td>BD</td><td>554413</td><td></td></tr><tr><td>LACHRYMAL OLIVE LUER LOCK 0.60 x 30 mm. 23G x 1 1/4&amp;rdquo;</td><td>UNIMED</td><td>27.134</td><td>Used as trachea cannula for BAL</td></tr><tr><td>high-protein binding polystyrene flat-bottom 96-well plates MAXISORP</td><td>NUNC</td><td>430341</td><td></td></tr><tr><td>Albumin, from bovine serum</td><td>Sigma</td><td>A4503</td><td></td></tr><tr><td>Goat Anti-Mouse IgA (&amp;alpha;-chain specific)&amp;minus;Peroxidase antibody</td><td>Sigma</td><td>A4789</td><td></td></tr><tr><td>3,3&amp;prime;,5,5&amp;prime;-Tetramethylbenzidine (TMB)&amp;nbsp;</td><td>Sigma</td><td>T0440</td><td></td></tr><tr><td>MyTaq DNA Polymerase</td><td>Bioline</td><td>BIO-21107</td><td>The kit Includes Buffer 5x</td></tr></tbody></table>

protective efficacy and pulmonary immune response following subcutaneous and intranasal bcg administration in mice

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.

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

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 ...

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Research

JoVE Journal

Immunology and Infection

10.6K Views.  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.

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

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

Medicine

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites, lipids, proteins, and nucleic acids, several groups have evaluated either the native or recombinant versions of bEVs for their protective potency as subunit vaccine candidates. Unlike native EVs, recombinant EVs are molecularly engineered to contain one or more immunogens of interest. Over the last decade, different groups have explored diverse approaches for generating recombinant bEVs. However, here, we report the design, construction, and enrichment of recombinant mycobacterial EVs (mEVs) in mycobacteria. Towards that, we use Mycobacterium smegmatis (Msm), an avirulent soil mycobacterium as the model system. We first describe the generation and enrichment of native EVs of Msm. Then, we describe the design and construction of recombinant mEVs that contain either mCherry, a red fluorescent reporter protein, or EsxA (Esat-6), a prominent immunogen of Mycobacterium tuberculosis. We achieve this by separately fusing mCherry and EsxA N-termini with the C-terminus of a small Msm protein Cfp-29. Cfp-29 is one of the few abundantly present proteins of MsmEVs. The protocol to generate and enrich recombinant mEVs from Msm remains identical to the generation and enrichment of native EVs of Msm.

This protocol details the enrichment of native mycobacterial extracellular vesicles (mEVs) from axenic cultures of Mycobacterium smegmatis (Msm) and how mCherry (a red fluorescent reporter)-containing recombinant MsmEVs can be designed and enriched. Lastly, it verifies the novel approach with the enrichment of MsmEVs containing the EsxA protein of Mycobacterium tuberculosis.

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, ...

Accelerating Tuberculosis Determination Using the Micro-CFU Method

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during the 2019-2021 pandemic. The disease is caused by the bacterium Mycobacterium tuberculosis (M.tb). The Mycobacterium bovis strain Bacillus Calmette-Gu&#233;rin (BCG), the only TB vaccine, is the oldest licensed vaccine in the world, still in use. Currently, there are 12 vaccines in clinical trials and dozens of vaccines under pre-clinical development. The method of choice used to assess the efficacy of TB vaccines in pre-clinical studies is the enumeration of bacterial colonies by the colony-forming units (CFU) assay. This time-consuming assay takes 4 to 6 weeks to conclude, requires substantial laboratory and incubator space, has high reagent costs, and is prone to contamination. Here we describe an optimized method for colony enumeration, the micro-CFU (mCFU), that offers a simple and rapid solution to analyze M.tb vaccine efficacy results. The mCFU assay requires tenfold fewer reagents, reduces the incubation period threefold, taking 1 to 2 weeks to conclude, reduces lab space and reagent cost, and minimizes the health and safety risks associated with working with large numbers of M.tb. Moreover, to evaluate the efficacy of a TB vaccine, samples may be obtained from a variety of sources, including tissues from vaccinated animals infected with Mycobacteria. We also describe an optimized method to produce a unicellular, uniform, and high-quality mycobacterial culture for infection studies. Finally, we propose that these methods should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB.

Author Spotlight: Optimizing CFU Determination for Efficient Assessment of TB Vaccine Efficacy and Antigen Presentation Analysis 

The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...

Intranasal Immunization with BCG in a Mouse Model

Source: Uranga, S. et al., <a href="https://app.jove.com/v/54440">Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice.</a>&#160;J. Vis. Exp. (2016)
This video demonstrates intranasal BCG immunization in mice, activating both innate and adaptive immune responses, leading to the development of lung-resident memory T cells and effector B cells, thus enhancing protection against Mycobacterium infections.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

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

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