防御効力および肺免疫応答は、マウスに皮下および鼻腔内BCG投与後の
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
結核（TB）は、結核驚くべき世界的な健康問題が1になり、世界のHIVよりも関連の死亡を引き起こし大手感染症の一つであり、多剤耐性株の上昇を高めることと組み合わせています。新しい診断ツール、より効果的かつ毒性の少ない薬、新たな安全かつ効果的な結核ワクチンは、特に発展途上国では、緊急の必要性です。
弱毒生カルメット・ゲラン桿菌（BCG）は、現在、全世界で1970年代以降、出生時に皮内投与された結核に対する唯一の認可ワクチンです。 BCGは、子供の病気（髄膜炎や粟粒結核）の重症型の予防に有効であると考えられるが、病気の伝染2の責任肺結核に対する矛盾した有効性を示しています。
結核感染の自然経路を模倣肺ワクチン接種は、ローカルホストの免疫応答をプライミングするための魅力的なアプローチを表し秒。この点において、異なる関連するTBの動物モデルにおいて、様々な前臨床作品は、皮下または皮内経路3-6と比較して、肺の免疫後の大きいワクチンの有効性を実証しています。それにもかかわらず、肺のワクチン接種によってトリガ保護メカニズムはよく理解されていません。 IL17粘膜ワクチンによって誘導される防御効力欠損マウスモデルで7,8を損なわれているように最後の年では、いくつかの作品は、TB-特有の粘膜免疫応答の重要な要因として、IL17が媒介する応答に向かって指摘しています。
最近では、BCGの投与は、DBA / 2マウス、皮下BCGの予防接種9後の保護の欠如によって特徴づけられるマウス系統を保護鼻腔内を初めて実証しました。これらの結果は、皮内BCGをpulmonに対して効果がないとみなされる場合、呼吸TBワクチンは流行国でTBの速度を低下させる、より効果的であることが示唆しました進TB。
Protocol
Representative Results
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.
<p c…Disclosures
The authors have nothing to disclose.
Acknowledgements
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].
Materials
| 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 |
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