Name: intranasal administration of recombinant influenza vaccines in chimeric mouse models to study mucosal immunity
Uploaded: 2015-06-25T13:00:00
Description: Watch this Scientific Journal Video about Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity at JoVE.com

We thank Sergio G&#243;mez-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.

Animal experimenten werden uitgevoerd volgens de goedgekeurde protocollen en volgens de richtlijnen van de Duitse dierenbescherming wet. Alle medewerkers uitvoeren van dierproeven doorgegeven opleidingen volgens categorie B of C van de Federation of European Laboratory Animal Science Associations. 1. Generatie van recombinant levend verzwakt Influenza Vaccines door Reverse Genetics OPMERKING: De gedetailleerd protocol voor het genereren van recombinant influenzavir...

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In deze studie beschrijven we hoe reverse genetics chimerische muismodellen kan worden gebruikt om de fysiologische en moleculaire mechanismen van door vaccin geïnduceerde immuniteit te helderen. Influenza reverse genetics is opgericht in veel laboratoria en heeft een hoofdrol in het begrijpen van influenza pathogenese, replicatie en transmissie 17 gespeeld. Een belangrijk punt in ons protocol is redding van koude aangepaste influenzavaccins uiting buitenlandse epitopen. Hoewel de strategie van integratie va...

Het genereren van immunologische geheugen in de afwezigheid van ziekte is de fysiologische basis van efficiënte vaccinatie 1. Onlangs zijn systemen-biologie gebaseerde benaderingen bleek dat succesvolle vaccins zoals gele koorts vaccin, induceert een sterke inductie van de aangeboren immuunrespons en activatie van verschillende subsets van dendritische cellen (DCs), die op hun beurt leiden tot activatie van antigeen multilineage specifieke T-cellen 2,3. Aangezien DCs de enige immuuncel populatie met de mogelijkheid om antigenspecifieke naïeve T-cellen activeren 4, de studie van hun functie tijdens de vaccinatie essentieel voor de immuunrespons op vaccins begrijpen en toekomstige strategieën te ontwikkelen tegen pathogenen uitdagend. Een systeem dat opsporing van verschillende DC subsets tijdens de immuunrespons op vaccins zou wenselijk zijn om een ​​nauwkeurige kinetiek van DC migratie naar lymfoïde weefsel vast te stellen en derhalve te voorzieninzicht in de fysiologische mechanismen die verantwoordelijk zijn voor de initiatie van de vaccin-specifieke adaptieve immuniteit. Reverse genetica gebaseerde aanpak bieden de mogelijkheid genereren gemodificeerd levend-verzwakte vaccins die experimenteel kan worden met dit doel. Sinds de uitvoering ervan op influenza onderzoek, heeft-plasmide gebaseerde reverse genetics op grote schaal toegepast om recombinante influenzastammen inclusief LAIVs genereren. Standaardprotocollen te redden recombinante influenzavirussen vereist multi-transfectie van sterk transfectable cellijnen ambisense plasmiden (die zowel positieve als negatieve sense RNA) die de acht segmenten van influenza virus en amplificatie in een tolerante systeem zoals Madin-Darby Canine Kidney ( MDCK) cellen en / of geëmbryoneerde kippeneieren 5. De toepassing van reverse genetica moleculaire technieken genereren om de immune mechanismen van vaccinatiestudie blijft onontdekt. The generationvan nieuwe muismodellen waardoor specifieke uitputting van immuuncelsubklassen, inclusief DC, heeft geopend nieuwe mogelijkheden om de fundamentele immuun onderliggende mechanismen-vaccin ontlokte bescherming begrijpen. De vergelijking tussen DC subgroep functies in muis en mens is gebleken dat, voor een groot deel, muis en humane DCs functioneel homoloog 6,7 Deze bevindingen suggereren sterk dat de ontwikkeling van muismodellen om specifieke depletie van DCs in de steady state en bij ontstekingsaandoeningen, kan dienen om de fysiologie van DC responsen in mensen begrijpen. In de afgelopen jaren een aantal muismodellen zijn gegenereerd dragende transgenen tot expressie het simian difterietoxine (DT) receptor (DTR) onder de controle van het promotorgebied van een gen van interesse 8,9. Sinds muisweefsels nature niet uitdrukken DTR, deze modellen maken voorwaardelijke uitputting van de cel subsets die het doelwit gen van belang bij de muis inenting met DT. Aldus onze ability specifieke DCs en andere leukocyten in vivo afbreken tijdens fysiologische processen, is sterk verbeterd door de ontwikkeling van DTR-gebaseerde ro. Hoewel transgene muismodellen zijn uitgebreid toegepast om de ontogenie van het immuunsysteem begrijpen ervan op vaccinontwikkeling is nauwelijks onderzocht. Hier, door het combineren van influenza reverse genetics en DTR-gebaseerde competitieve beenmerg chimeren, stellen we een methode om de kinetiek van vaccinimmuniteit evenals individuele bestudering van genfunctie tijdens de immuunrespons op vaccins in vivo. De toepassing van deze technologie voor preklinische evaluatie van nieuwe vaccins tegen uitdagende infectieziekten kan bijdragen tot vaccinontwerp rationaliseren en vaccin gegadigden in vivo.

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intranasal administration of recombinant influenza vaccines in chimeric mouse models to study mucosal immunity

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.

Generering van recombinant levend geattenueerde influenza vaccins kunnen worden bereikt door transfectie van plasmiden die coderen voor de acht segmenten van influenza virus onder controle van bidirectionele promoters 5. Een koude aangepast influenzavaccin bevat gewoonlijk zes segmenten van een aan koude aangepaste stam en de HA en NA van influenza-stam gekozen (bijvoorbeeld H1N1) (Figuur 1A). Het principe van aanpassing aan koude is gebaseerd op virus beperkte replicatie bij 33 ° C...

Watch this Scientific Journal Video about Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity at JoVE.com

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

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.

Intranasale toediening van recombinante Influenza Vaccines in Chimerisch muismodellen om mucosale immuniteit bestuderen

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 ...

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