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 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.
Tuberkulose (TB) er en av de ledende smittsomme sykdommer som forårsaker mer assosiert dødsfall enn HIV i verden, og kombinert med økende økning av multiresistente stammer gjør TB en alarmerende global helseproblem en. Nye diagnostiske verktøy, mer effektive og mindre giftige stoffer, og nye trygge og effektive TB vaksiner er et presserende behov, spesielt i utviklingsland.
Levende svekkede Bacille Calmette-Guerin (BCG) er i dag den eneste lisensierte vaksine mot tuberkulose, som har blitt administrert intradermalt ved fødselen siden 1970-tallet på verdensbasis. BCG er ansett som effektiv for å hindre alvorlige former av sykdommen (meningitt og miliærtuberkulose TB) hos barn, men har vist inkonsekvent effekt mot lungetuberkulose ansvarlig for sykdomsoverføring 2.
Lunge vaksinasjon, som etterligner naturlig rute fra TB-infeksjon, representerer en attraktiv metode for grunning lokale vertens immunresponss. I denne forbindelse har forskjellige prekliniske arbeider på forskjellige relevante TB dyremodeller vist større vaksineeffektivitet følgende pulmonal immunisering, sammenlignet med subkutan eller intradermal rute 3-6. Likevel er de beskyttende mekanismer som utløses av lunge vaksinasjon ikke godt forstått. I de siste årene har flere verk peker mot IL17-mediert reaksjon som en viktig faktor for TB-spesifikke mucosal immunrespons, som i musemodeller mangelfull for IL17 slimhinne vaksineindusert beskyttende effekt er svekket 7,8.
Nylig viste vi for første gang at intranasal BCG administrasjon beskyttet DBA / 2 mus, en musestamme preget av mangel på beskyttelse etter subkutan BCG immunisering 9. Disse resultatene antydet at luft TB vaksinasjon kan være mer effektiv i å redusere frekvensen av TB i endemiske land, der intradermal BCG anses ineffektive mot pulmonær TB.
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…The authors have nothing to disclose.
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].
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 |