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 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.
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.
2. Mouse Vaccination
3. Mouse Intranasal Challenge with H37Rv Strain
4. Analysis of Induced Immune Response in Lungs
5. Analysis of Immunoglobulins in Bronchoalveolar Lavage (BAL)
6. Bacterial load determination in lungs
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.
Figure 1 describes the workflow followed. Eight to ten week-old female DBA/2 mice are vaccinated with 106 CFU of BCG Danish by the subcutaneous or intranasal route of administration. Eight weeks later, a group of mice is sacrificed to analyze lung immune response induced by vaccination. BAL samples are first obtained and then we harvest lungs. In order to study vaccine-conferred protective efficacy, we inoculate a different group of mice with a low-dose intranasal challenge of M. tuberculosis H37Rv strain. One month later, we sacrifice animals and harvest lungs. In this case, for each animal we use the left lung to determine bacterial load and the right lung to assess vaccine-induced immune response post-challenge. The objective is to generate bacterial load and immune response data in lungs for each animal in order to study potential correlates of protection.
As shown in Figure 2, lungs were dissociated to obtain either an organ homogenate or a cellular suspension. Homogenized lungs were plated on solid agar medium to determine bacterial load four weeks post-challenge. Cellular suspension to study vaccine-induced immune response was obtained following lung enzymatic digestion with collagenase D and DNaseI.
Our results clearly indicate that, when compared to the subcutaneous route, the intranasal route of vaccination confers a much greater protective efficacy in lungs four weeks after challenge (Figure 3A). In addition, we confirmed that bacterial load in lungs from the intranasal BCG-vaccine group correspond to H37Rv and not to BCG vaccine. To this end, we analyzed a representative number of colonies from this group by specific PCR for the RD9 genome region, which amplifies different length fragments in BCG and M. tuberculosis (Figure 3B). Figure clearly showed that all the colonies analyzed provided a fragment of 0.4 kbp, which corresponded to H37Rv.
Our data revealed correlation between protective efficacy conferred by intranasal BCG vaccination and vaccine-induced immune response in lungs prior to challenge. Intranasal BCG clearly triggered higher IL17 and IFNγ production in lungs, measured by ELISA (Figure 4A). These data were confirmed by intracellular staining (ICS) and flow cytometry (data not shown). In addition, we also found a higher concentration of both total and PPD-specific IgA concentration in BAL samples (Figure 4B), indicating that pulmonary BCG vaccination induces production of IgA and translocation to respiratory airways.
Finally, we studied BCG-induced immune response in lungs after challenge (Figure 5). Our data revealed differences between IL17 and IFNγ; IL17A-producing CD4+ cells were only detected in the intranasal BCG group, whereas IFNγ-producing cells were found in all groups infected with H37Rv regardless of vaccination. Representation of data of each animal corresponding to IL17-producing cells and bacterial load showed a significant correlation between presence of IL17 and lung bacterial load reduction, which was not observed in the case of IFNγ.
Figure 1. Workflow to Compare Intranasal and Subcutaneous Route of BCG Vaccination. Eight weeks post BCG immunization, a set of mice (6 per experimental group) is used to harvest lungs and perform a BAL, and analyze pulmonary immune response induced by vaccination. Another set of mice (6/group), is inoculated with a low-dose intranasal challenge of M. tuberculosis H37Rv strain (100 CFU). One month later, animals are sacrificed and bacterial load is analyzed in the left lung and PPD-specific immune response in the right lung. Please click here to view a larger version of this figure.
Figure 2. Processing of Lung Samples. A tissue dissociator was used to process lungs. In the experiments to determine bacterial load, lungs were homogenised prior to plate them in solid agar medium. In experiments that require a lung cellular suspension, this was generated following enzymatic digestion with collagenase D and DNaseI. Please click here to view a larger version of this figure.
Figure 3. Protective Efficacy Conferred by BCG Immunization. Groups of 6 DBA/2 mice were vaccinated by the subcutaneous (BCG sc), intranasal (BCG in) route, or non-vaccinated (Unvacc) with BCG Danish vaccine 106 CFU. At two months post-vaccination, mice were inoculated intranasally with a low dose (100 CFU) H37Rv challenge, and one-month later bacterial burden in lungs was determined. A representative experiment of two independent is shown. (A) Data in the graphs are represented as mean+SD. One-way ANOVA test with Bonferroni post analysis was performed to calculate statistical significance. (B) A representative number of single colonies from the BCG intranasal group were analyzed by PCR specific for RD9 region (different in BCG and H37Rv genomes) to discern BCG and H37Rv colonies. (Previously published 9). Please click here to view a larger version of this figure.
Figure 4. Vaccine-specific Pulmonary Immune Response Analyzed Prior to Challenge with H37Rv. Groups of 6 DBA/2 mice were vaccinated by the subcutaneous (BCG sc) or intranasal (BCG in) route, or non-vaccinated (Unvacc) with BCG Danish vaccine 106 CFU. (A) At two months post-vaccination, a cellular suspension from harvested lungs was obtained. Cells were stimulated with PPD as described in methods section and IL17A (left panel) and IFNγ (right panel) production were analyzed by ELISA. (B) Total IgA, and M. tuberculosis (MTB)-specific IgA were analyzed from BAL samples by ELISA. Pooled data from two independent experiments are shown. Data in the graphs are represented as mean + SD. (Previously published 9). Please click here to view a larger version of this figure.
Figure 5. Vaccine-specific Pulmonary Immune Response Analyzed prior to Challenge with H37Rv. Groups of 6 DBA/2 mice were vaccinated by the subcutaneous (BCG sc) or intranasal (BCG in) route, or non-vaccinated (NV) with BCG Danish vaccine 106 CFU. A control group of non-vaccinated, non-infected mice was also included (NV/NI). At two months post-vaccination, mice were challenged intranasally with a low H37Rv dose (100 CFU), and one-month later animals were euthanized. Left and right lungs from the same animal were used to determine bacterial load and IL17A- (A) or IFNγ- (B) producing CD4+ cells, respectively. Data in left panels correspond to percentage of cytokine-producing cells measured by flow cytometry, and are represented as mean + SD. Right panels represent data from bacterial load and cytokine- producing CD4+ cells obtained for each mouse. Linear regression was calculated and the p-value obtained in each case is shown in the case of IL17A. Pooled data from two independent experiments are shown in the figure. (Previously published 9). Please click here to view a larger version of this figure.
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.
Our data clearly show that a change in the route of administration of BCG to mimic the natural route of infection could be a successful strategy to prevent pulmonary tuberculosis. Our results are in accordance with other authors showing equivalent data in different animal models, including guinea pigs and non-human primates (NHP) 6,9,12.
Remarkably, our unpublished data indicated that the volume of administration by the intranasal route is a critical step of the protocol. These results revealed that following intranasal delivery of 100 H37Rv bacteria resuspended in 10 μl (instead of 40 μl), we only recovered lung CFUs from around 20% of the animals (data not shown.
Importantly, one of the possible limitations of intranasal administration as a vaccine delivery route in clinic could be its proximity with the central nervous system13,14. In this regard, aerosol immunization might be safer as delivery route for pulmonary tuberculosis vaccines in humans.
This protocol describes a standardized methodology that can be adapted to other studies, as comparison of pulmonary vaccines, or different routes of pulmonary immunization, including aerosol.
Our data suggest that the analysis in parallel of protective efficacy and immune response, using lung samples from the same animal, could be a useful tool to identify biomarkers of protection. In this regard, our results reveal that lung IL17, but not IFNγ, seems to correlate with a better vaccine protective efficacy. These data highlight a possible role of IL17 in the protective response induced by intranasal BCG, which is in accordance with data reported by other authors using mucosal subunit TB vaccines 7,15. Our data also indicate that presence of IgA (both total and MTB-specific) in BAL samples correlates with protection conferred by intranasal BCG. Importantly, we described previously, in agreement with other works, that IL17 contributes to traslocation of IgA to respiratory airways and gut lumen.9,16,17
The authors have nothing to disclose.
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].
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 0.45×10 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 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 |