الحماية فعالية والرئوي الاستجابة المناعية بعد تحت الجلد والطريق الأنفي BCG الإدارة في الفئران
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
السل (TB) هو واحد من الأمراض المعدية الرائدة تسبب المزيد من الوفيات المرتبطة من فيروس نقص المناعة البشرية في العالم، وجنبا إلى جنب مع ارتفاع الزيادة من أدوية المتعددة السلالات المقاومة يجعل السل مشكلة صحية عالمية تنذر بالخطر 1. أدوات جديدة للتشخيص والمخدرات أكثر فعالية وأقل سمية، واللقاحات السل آمنة وفعالة جديدة هي حاجة ملحة، خاصة في العالم النامي.
تعيش الموهن عصيات كالميت غيران (BCG) حاليا لقاح مرخص الوحيد ضد السل، الذي تديره الأدمة عند الولادة منذ 1970s في جميع أنحاء العالم. ويعتبر السل فعالة في الوقاية من أشكال حادة من المرض (التهاب السحايا والسل الدخني) في الأطفال، ولكن أظهرت فعالية تتعارض ضد السل الرئوي المسؤول عن انتقال المرض 2.
التطعيم الرئوي، الذي يحاكي الطريق الطبيعي لعدوى السل، يمثل نهجا جذابا للفتيلة المضيف المحلي الاستجابة المناعيةالصورة. وفي هذا الصدد، أظهرت أعمال مختلفة قبل السريرية في نماذج السل حيوانية مختلفة ذات الصلة أكبر فعالية لقاح التالية التحصين الرئوي مقارنة تحت الجلد أو داخل الأدمة الطريق 3-6. ومع ذلك، فإن آليات الحماية الناجمة عن التطعيم الرئوي ليست مفهومة جيدا. في السنوات الأخيرة، وقد أشار العديد من الأعمال من أجل الاستجابة بوساطة IL17 باعتبارها عاملا مهما من الاستجابة المناعية المخاطية-TB معين، كما في نماذج الفئران ناقصة لIL17 المخاطية فعالية الحماية التي يسببها اللقاح تضعف 7،8.
نحن في الآونة الأخيرة أظهرت لأول مرة أن الأنف إدارة BCG باي DBA / 2 الفئران، سلالة الماوس تتميز انعدام الحماية بعد التطعيم ضد مرض السل تحت الجلد 9. وتشير هذه النتائج إلى أن تطعيم السل التنفسي يمكن أن تكون أكثر فعالية في الحد من نسبة السل في البلدان الموبوءة، حيث يعتبر BCG داخل الأدمة غير فعالة ضد pulmonالسل آرى.
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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