Intranasal administration af rekombinant influenzavacciner i kimære musemodeller at studere slimhindeimmunitet
Summary
There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.
Abstract
Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies.
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.
Introduction
Dannelsen af immunologisk hukommelse i fravær af sygdom er den fysiologiske grundlag af en effektiv vaccination 1. For nylig har systemer biologi tilgange afsløret, at vellykkede vacciner såsom gul feber-vaccine inducerer en stærk induktion af medfødte immunrespons og aktivering af flere undergrupper af dendritiske celler (DC'er), som igen, at bly multilineage aktivering af antigen specifikke T-celler 2,3. Da DC'er er de eneste immune cellepopulation med evnen til at aktivere antigenspecifikke naive T-celler 4, undersøgelse af deres funktion under vaccination er vigtigt at forstå immunresponser på vacciner og udforme fremtidige strategier mod udfordrende patogener.
Et system, der giver sporing af forskellige DC'er delmængder under immunreaktioner på vacciner ville være ønskeligt for at etablere en nøjagtig kinetik for DC migration til lymfoide væv, og dermed til at giveindsigt i de fysiologiske mekanismer, der er ansvarlige for at indlede vaccine-specifikke adaptiv immunitet. Reverse genetik tilgange giver mulighed for at generere modificeret, levende svækkede vacciner, der kan bruges eksperimentelt med dette formål. Siden dens gennemførelse på influenza forskning har plasmid-baserede revers genetik været almindeligt anvendt til at generere rekombinante influenzastammer herunder LAIVs. Standardprotokoller at redde rekombinante influenzavirus må flere transfektion af højt transficerbare cellelinjer med ambisense plasmider (der producerer både positiv og negativ sense-RNA) indeholdende otte influenza virale segmenter samt amplifikation i en permissiv system som Madin-Darby hunde nyre ( MDCK) celler og / eller kylling befrugtede æg 5. Men anvendelsen af revers genetik at generere molekylære værktøjer for at studere immun mekanismer vaccination forbliver uudforsket.
Genereringenaf nye musemodeller tillader specifik udtømning af immuncelleundergrupper, herunder udviklingslandene, har åbnet nye muligheder for at forstå de grundlæggende immun mekanismer bag vaccine-fremkaldte beskyttelse. Sammenligningen mellem DC subset funktioner i mus og mennesker har vist, at for en stor del, muse og humane DC'er er funktionelt homologe 6,7, disse resultater tyder på, at udviklingen af musemodeller muliggør specifik udtømning af DC'er i steady state og under inflammatoriske tilstande, kan tjene til at forstå fysiologi DC responser hos mennesker. I de senere år er blevet genereret et antal musemodeller transporterer transgener udtrykker simian difteritoksin (DT) receptor (DTR) under kontrol af promotorregionen af et gen af interesse 8,9. Da musevæv ikke naturligt udtrykker DTR, disse modeller tillader betinget udtømning af celledelmængder bærer den målrettede gen af interesse ved mus podning med DT. Således er vores ability at nedbryder specifikke DC'er og andre leukocytter in vivo under fysiologiske processer, er blevet stærkt forbedret gennem udviklingen af DTR-baserede ro. Men mens disse transgene musemodeller er blevet anvendt i vid udstrækning til at forstå ontogenese af immunsystemet, deres anvendelse til vaccineudvikling er næppe testet. Her, ved at kombinere influenza revers genetik og DTR-baserede konkurrencedygtige knoglemarvsceller kimærer, foreslår vi en metode til at studere kinetikken af vaccinen immunitet samt individuelle genfunktion under immunresponser på vacciner in vivo. Anvendelsen af denne teknologi til præklinisk evaluering af nye vacciner mod udfordrende infektionssygdomme kunne bidrage til at rationalisere vaccine design og teste vaccinekandidater in vivo.
Protocol
Representative Results
Discussion
I denne undersøgelse beskriver vi, hvordan revers genetik og kimære mus-modeller kan anvendes til at belyse de fysiologiske og molekylære mekanismer for vaccine-inducerede immunitet. Influenza revers genetik er etableret i mange laboratorier og har spillet en ledende rolle i forståelsen influenza patogenese, replikation, og transmission 17. Et centralt punkt i vores protokol er redning af koldadapterede influenzavacciner udtrykker fremmede epitoper. Mens strategien om at indføre korte cDNAer i stilken af…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We thank Sergio Gómez-Medina for excellent technical support with mouse experiments. This work was supported by funds from the Leibniz Association and the Leibniz Center of Infection. A.L. is a recipient of a pre-doctoral fellowship from the Leibniz Graduate School.
Materials
| Dulbecco´s Modified Eagle Medium (DMEM 1X) | Gibco RL-Life Technologies | 41965-039 | |
| Opti MEM | Gibco RL-Life Technologies | 31985-047 | |
| Lipofectamine 2000 | Invitrogen-Life Technologies | 11668-027 | |
| Penicillin-Streptomycin (10.000 U/ml) | PAA | p11-010 | |
| Bovine Serum Albumin | Sigma-Aldrich | A2153 | |
| Embryonated eggs | Valo biomedia Gmbh | ||
| PBS (1X) | Sigma-Aldrich | D8537 | |
| 70 μM Nylon Filters | Greiner-Biorad | 542-070 | |
| Red Blood Cell Lysing buffer (RBCL) 10X | BD Bioscience | 555899 | |
| CD16/CD32 Mouse BD Fc Block (2.4G2) | BD Pharmigen | 553142 | |
| APC-Anti-mouse SIINFEKL-H2kb (25 D1.16) | Biolegend | 141605 | |
| PE-Anti-mouse CD11c (HLA3) | BD Biosciences | 553802 | |
| eFluor 450-Anti-mouse MHCII (Md/114.15.2) | eBioscience | 48-5321-82 | |
| Pe-Cy7-Anti-mouse CD11b (M1/70) | Biolegend | 101216 | |
| PerCp/Cy5.5-Anti-mouse CD103 (2E7) | Biolegend | 121416 | |
| PE-Anti-mouse CD45.1 (A20) | eBioscience | 12-0453-82 | |
| V500-Anti-mouse CD45.2 (1O4) | BD Bioscience | 562130 | |
| PerCp-eFluor710 -Anti-mouse CD8a (53-6.7) | eBioscience | 46-0081-80 | |
| APC-Cy7-Anti-mouse CD3ε (145-2611) | Biolegend | 100325 | |
| eFluor450-Anti-mouse CD4 (GK 1.5) | eBioscience | 48-0041-80 | |
| CFSE Proliferation dye | eBioscience | 65-0850-85 | |
| Baytril 2.5% | Bayer | 65-0850-85 | |
| Dymethil-Sulfoxide (DMSO) | Sigma-Aldrich | D2650 | |
| Ovalbumin | Molecular probes | O23020 | |
| Diphteria Toxin (DT) | Sigma-Aldrich | D0564 | |
| Trypsin-TPCK | Sigma-Aldrich | T1426 | |
| BD FACsCanto II Flow cytometer | BD Biosciences | ||
| FlowJo cell analysis software 9.5 | Flowjo inc. | ||
| Trypan Blue Stain (0.4%) | Life technologies | T10282 | |
| Countess Automatic Cell Counter | Invitrogen-Life Technologies | C10227 |
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