Name: protective efficacy and pulmonary immune response following subcutaneous and intranasal bcg administration in mice
Uploaded: 2016-09-19T15:00:00
Description: Watch this Scientific Journal Video about Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice at JoVE.com

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.

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	<li>Zumla, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+WHO+2014+global+tuberculosis+report--further+to+go.">The WHO 2014 global tuberculosis report--further to go.</a> Lancet Glob Health. 3 (1), e10-e12 (2015).</li><li>Mangtani, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+by+BCG+vaccine+against+tuberculosis:+a+systematic+review+of+randomized+controlled+trials.">Protection by BCG vaccine against tuberculosis: a systematic review of randomized controlled trials.</a> Clin Infect Dis. 58 (4), 470-480 (2014).</li><li>Aguilo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+Mycobacterium+bovis+BCG+vaccination+confers+dose-dependent+superior+protection+compared+to+that+of+subcutaneous+vaccination.">Pulmonary Mycobacterium bovis BCG vaccination confers dose-dependent superior protection compared to that of subcutaneous vaccination.</a> Clin Vaccine Immunol. 21 (4), 594-597 (2014).</li><li>Chen, L., Wang, J., Zganiacz, A., Xing, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+intranasal+mucosal+Mycobacterium+bovis+BCG+vaccination+confers+improved+protection+compared+to+subcutaneous+vaccination+against+pulmonary+tuberculosis.">Single intranasal mucosal Mycobacterium bovis BCG vaccination confers improved protection compared to subcutaneous vaccination against pulmonary tuberculosis.</a> Infect Immun. 72 (1), 238-246 (2004).</li><li>Giri, P. K., Verma, I., Khuller, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+efficacy+of+intranasal+vaccination+with+Mycobacterium+bovis+BCG+against+airway+Mycobacterium+tuberculosis+challenge+in+mice.">Protective efficacy of intranasal vaccination with Mycobacterium bovis BCG against airway Mycobacterium tuberculosis challenge in mice.</a> J Infect. 53 (5), 350-356 (2006).</li><li>Lagranderie, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BCG-induced+protection+in+guinea+pigs+vaccinated+and+challenged+via+the+respiratory+route.">BCG-induced protection in guinea pigs vaccinated and challenged via the respiratory route.</a> Tuber Lung Dis. 74 (1), 38-46 (1993).</li><li>Gopal, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-17-dependent+CXCL13+mediates+mucosal+vaccine-induced+immunity+against+tuberculosis.">Interleukin-17-dependent CXCL13 mediates mucosal vaccine-induced immunity against tuberculosis.</a> Mucosal Immunol. 6 (5), 972-984 (2013).</li><li>Khader, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-23+and+IL-17+in+the+establishment+of+protective+pulmonary+CD4++T+cell+responses+after+vaccination+and+during+Mycobacterium+tuberculosis+challenge.">IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during Mycobacterium tuberculosis challenge.</a> Nat Immunol. 8 (4), 369-377 (2007).</li><li>Aguilo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+but+Not+Subcutaneous+Delivery+of+BCG+Vaccine+Confers+Protection+to+Tuberculosis-Susceptible+Mice+by+an+Interleukin+17-Dependent+Mechanism.">Pulmonary but Not Subcutaneous Delivery of BCG Vaccine Confers Protection to Tuberculosis-Susceptible Mice by an Interleukin 17-Dependent Mechanism.</a> J Infect Dis. , (2015).</li><li>Middlebrook, G., Cohn, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriology+of+tuberculosis:+laboratory+methods.">Bacteriology of tuberculosis: laboratory methods.</a> Am J Public Health Nations Health. 48 (7), 844-853 (1958).</li><li>Brosch, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+evolutionary+scenario+for+the+Mycobacterium+tuberculosis+complex.">A new evolutionary scenario for the Mycobacterium tuberculosis complex.</a> Proc Natl Acad Sci U S A. 99 (6), 3684-3689 (2002).</li><li>Kaushal, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+vaccination+with+attenuated+Mycobacterium+tuberculosis+induces+strong+central+memory+responses+and+protects+against+tuberculosis.">Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis.</a> Nat Commun. 6, 8533 (2015).</li><li>Lochhead, J. J., Thorne, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+of+biologics+to+the+central+nervous+system.">Intranasal delivery of biologics to the central nervous system.</a> Adv Drug Deliv Rev. 64 (7), 614-628 (2012).</li><li>Lochhead, J. J., Wolak, D. J., Pizzo, M. E., Thorne, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+transport+within+cerebral+perivascular+spaces+underlies+widespread+tracer+distribution+in+the+brain+after+intranasal+administration.">Rapid transport within cerebral perivascular spaces underlies widespread tracer distribution in the brain after intranasal administration.</a> J Cereb Blood Flow Metab. 35 (3), 371-381 (2015).</li><li>Griffiths, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholera+toxin+enhances+vaccine-induced+protection+against+Mycobacterium+tuberculosis+challenge+in+mice.">Cholera toxin enhances vaccine-induced protection against Mycobacterium tuberculosis challenge in mice.</a> PLoS One. 8 (10), e78312 (2013).</li><li>Hirota, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+Th17+cells+in+Peyer's+patches+is+responsible+for+the+induction+of+T+cell-dependent+IgA+responses.">Plasticity of Th17 cells in Peyer's patches is responsible for the induction of T cell-dependent IgA responses.</a> Nat Immunol. 14 (4), 372-379 (2013).</li><li>Jaffar, Z., Ferrini, M. E., Herritt, L. A., Roberts, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+lung+mucosal+Th17-mediated+responses+induce+polymeric+Ig+receptor+expression+by+the+airway+epithelium+and+elevate+secretory+IgA+levels.">Cutting edge: lung mucosal Th17-mediated responses induce polymeric Ig receptor expression by the airway epithelium and elevate secretory IgA levels.</a> J Immunol. 182 (8), 4507-4511 (2009).</li></ol>

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.
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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.

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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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Watch this Scientific Journal Video about Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice at JoVE.com

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 ...

The overall goal of this procedure is to study immune responses in the lungs following tuberculosis immunization, including the protective efficacy of the vaccine against an intranasal challenge and the local vaccine-induced lung immune response. This methodology can help answer our key questions in the tuberculosis vaccine felt, including identification of the defensive lung immuno responses tuckered by pulmonary vaccines for optimal lung immune protection. One benefit of this methodology is that the lung immune response from its animal is individually assessed in parallel with protective efficacy.allowing the identification of the specific immune parameters that correlate with protection. For subcutaneous administration of the BCG vaccine, first, fill a one milliliter syringe equipped with a 26 gauge needle with one milliliter of the BCG vaccine suspension, and remove the air bubbles. Then place an anesthetized mouse in the prone position inside a Laminar flow hood, and subcutaneously inoculate the animal with 100 microliters of bacteria in a rear flank.For intranasal administration, load a micro-pipette with 20 microliters of the BCG suspension, and place the anesthetized mouse in the supine position inside the hood. Administer the inoculum dropwise between the two nostrils until the entire 20 microliter volume has been deposited, leaving time between the drops to allow the mouse to inhale the suspension. Then, reload the micro-pipette with another 20 microliters of bacteria and repeat the administration.For intranasal challenge with the H37Rv microbacterium tuberculosis strain, at the appropriate experimental time point post-vaccination, inoculate the animals with intranasal administration of 40 microliters of the H37Rv bacterial suspension as just demonstrated. To acquire the bronchoalveolar lavage or ball sample, first use sterile forceps and scissors to expose the trachea. Then make a small incision in the trachea, taking care not to excise the tissue completely, and insert a cannula connected to a sterile 1 mL syringe, containing 800 microliters of ice-cold PBS.When the cannula is in place, carefully fill the lungs with the PBS, then slowly pull back on the plunger to recover at least 500 to 600 microliters of lavage fluid. Dispense the ball into a 1.5 mL tube on ice, and confirm that the suspension is colorless, indicating a lack of blood contamination. Store samples on ice until ball processing.To determine the bacterial load and immune response in the lungs, fix the mouse with needles in the supine position on a flat and disinfected surface inside the Laminar flow hood, and make an incision in the upper skin over the chest. Then, peel back the skin to expose the thoracic area, and cut open the lungs. Use sterile scissors and forceps to harvest the lungs and heart, and place the organs on a clean surface.Then, remove the heart, trachea, and connective tissue. Place each lung in a different 1.5 mL tube with 1 mL of de-ionized water on ice for downstream determination of bacterial load and cellular immune responses. When compared to the subcutaneous route, the intranasal route of vaccination confers a much greater protective efficacy in the lungs.Analysis of a representative number of colonies from the intranasal BCG vaccine group by PCR specific for the RD9 genome region demonstrates that all of the colonies provide a fragment of 0.4 kilo-base-pairs that corresponded to H37Rv. Intranasal BCG triggers higher IL17 and interfere on gamma production in the lungs, revealing a correlation between the protective efficacy conferred by the intranasal BCG vaccination and the vaccine induced immune response in the lungs prior to challenge with a higher concentration of both the total and the H37Rv purified protein derivate-specific IgA concentration in the lavage samples observed for the intranasal vaccine recipients. Further analysis of cytokine producing cells in the lungs following the tuberculosis challenge revealed that whereas interfere on gamma producing cells were detected in all infected groups, IL17A producing CD4+cells were found only in the intranasal BCG group, demonstrating a significant correlation between the presence of IL17 and lung-bacterial load reduction.Following this procedure, it is possible to compare the local lung immuno-responses in the use of biodiverse tuberculosis vaccines as well as different routes of administration and time points after challenge. In addition, this methodology can be used to evaluate the immunicity in the used by vaccination after challenge. Which could allowed for the identification of the immuno responses that correlate with protection.Importantly, working with Mycobacterium Tuberculosis requires BCL-free lab facilities, and the appropriate precautions should always be taken when performing this procedure.

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An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

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 ...