מגן היעילות ריאתי החיסונית בתגובה תת עורי ואת אפי BCG מינהל עכברים
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
שחפת (TB) היא אחת המחלות הזיהומיות המובילים בגרימת מותם מזוהה יותר HIV בעולם בשילוב עם העלייה הגואה של זנים עמידים multidrug גורם שחפת בעיה בריאותית עולמית מדאיגה 1. כלי אבחון חדשים, תרופות יעילות יותר ופחות רעילות, וחיסוני TB בטוחים ויעילים חדשים הם צורך דחוף, במיוחד במדינות המתפתחות.
לחיות Bacille Calmette-גרן מוחלשים (BCG) היא כיום חיסון מורשה רק נגד שחפת, אשר כבר מנוהל intradermally בלידה מאז 1970 ברחבי העולם. BCG נחשב יעיל במניעת צורות חמורות של המחלה (דלקת קרום המוח שחפת miliary) בילדים, אך הראה יעילות עולה בקנה אחד נגד שחפת ריאתית אחראית של העברת מחלות 2.
חיסון ריאתי, אשר מחק מסלול טבעי של זיהום שחפת, מייצג גישה אטרקטיבית עבור תחול תגובה מקומי חיסון של פונדקאיים. בהקשר זה, יצירות פרה-קליניים שונים במודלים של בעלי חיים TB רלוונטיים שונים הוכיחו יעילות החיסון יותר בעקבות חיסון ריאתי כפי בהשוואה לתוואי תת עורית או תוך-3-6. עם זאת, מנגנוני ההגנה מופעלים על ידי חיסון ריאתי אינם מובנים היטב. בשנים האחרונות, כמה יצירות הצביעו לעבר בתגובה בתיווך IL17 כגורם חשוב של תגובה חיסונית ברירית ספציפית TB, כמו במודלים של עכברים לוקים עבור יעילות מגן עקב חיסון רירי IL17 נפגע 7,8.
לאחרונה הראינו לראשונה כי אפי ממשל BCG מוגן DBA / 2 עכברים, זן עכבר מאופיין בהיעדר הגנה לאחר תת עורית BCG חיסון 9. תוצאות אלו הראו כי חיסון TB נשימה יכול להיות יעיל יותר בהפחתת שיעור השחפת במדינות אנדמיות, שבו BCG תוך-עורית נחשב יעיל נגד pulmonTB האר"י.
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…Declarações
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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