보호 효능 및 폐 면역 반응은 마우스에 피하 및 비강 BCG 관리에 따라
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
결핵 (TB)는 TB 놀라운 세계 보건 문제 일 수 있습니다 세계에서 HIV보다 더 많은 관련 사망의 원인이되는 주요 감염 질환 중 하나 다 약제 내성 균주의 증가 증가와 결합이다. 새로운 진단 도구,보다 효과적이고 독성이 적은 약물, 새로운 안전하고 효과적인 결핵 백신은 특히 개발 도상국에서, 긴급한 필요하다.
감쇠 Bacille 칼 메트 – GUERIN은 (BCG)은 현재 전 세계적으로 1970 년대 이후 출생시 피내 투여 된 TB, 대한 전용 허가 백신 살고있다. BCG는 아이들의 질환 (수막염 및 속 립성 결핵)의 심한 형태의 예방에 효과적인 것으로 간주되지만, 질병 전파 (2)의 책임 폐결핵에 대한 일관성 효과를 보여 주었다.
TB 감염의 자연 경로를 모방 폐 예방 접종은 로컬 호스트 면역 반응을 프라이밍위한 매력적인 접근 방식을 나타냅니다에스. 피하 또는 피내 경로 3-6에 비해 이와 관련하여, 다른 관련 TB의 동물 모델에서 다양한 전임상 작품 폐 면역화 다음 큰 백신 효능을 보여 주었다. 그럼에도 불구하고, 폐 예방 접종에 의해 유발 보호 메커니즘은 잘 이해되지 않습니다. 지난 몇 년 동안, 여러 작품 IL17 점막 백신에 의한 예방 효과에 대한 결핍 된 마우스 모델에서와 같이, TB-특정 점막 면역 반응의 중요한 요소로 IL17 중재 응답으로 지적은 7,8를 손상된다.
최근에 우리는 BCG 관리는 DBA / 2 마우스, 피하 BCG 예방 접종 9 후 보호의 부족을 특징으로하는 마우스 변형을 보호 비강 처음으로 보여 주었다. 이러한 결과는 호흡기 결핵 예방 접종은 피내 BCG가 pulmon에 대한 효과로 간주됩니다 발병 국가에서 TB의 비율을 감소 시키는데 더 효과적 일 수 있다고 제안진 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…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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