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.

Gli esperimenti sugli animali sono stati condotti secondo protocolli approvati e seguendo le linee guida della legge sulla protezione degli animali tedesca. Tutto il personale che effettuano esperimenti sugli animali passati programmi di formazione a seconda della categoria B o C della Federazione delle Associazioni scientifiche europee animali da laboratorio. 1. Generazione di ricombinante vivo attenuato Influenza vaccini da Reverse Genetics NOTA: Il protocollo de...

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In questo studio viene descritto come la genetica e modelli di mouse chimerici inversa può essere utilizzata per chiarire i meccanismi fisiologici e molecolari dell&#39;immunità indotta dal vaccino. Influenza genetica inversa è stabilito in molti laboratori e ha svolto un ruolo principale nella comprensione patogenesi dell&#39;influenza, la replica, e la trasmissione 17. Un punto chiave nel nostro protocollo è il salvataggio di vaccini antinfluenzali adattate freddo esprimono epitopi stranieri. Mentre la ...

La generazione di memoria immunologica in assenza della malattia è la base fisiologica di vaccinazione efficace 1. Recentemente, sistemi di approcci basati biologia, hanno rivelato che i vaccini di successo come il vaccino contro la febbre gialla, inducono una forte induzione di risposte immunitarie innate e l&#39;attivazione di diversi sottoinsiemi di cellule dendritiche (DC), che a loro volta, portano a multilineare attivazione di antigene specifiche cellule T 2,3. Dal momento che le DC sono l&#39;unica popolazione di cellule immunitarie con la possibilità di attivare le cellule T naïve antigene-specifiche 4, lo studio della loro funzione durante la vaccinazione è fondamentale per capire le risposte immunitarie ai vaccini e per progettare strategie future contro gli agenti patogeni difficili. Un sistema che consente di risalire diverse sottopopolazioni PVS durante la risposta immunitaria ai vaccini sarebbe auspicabile, al fine di stabilire un accurato cinetica della migrazione DC a tessuti linfoidi, e quindi quello di fornirecomprensione dei meccanismi fisiologici responsabili per l&#39;avvio di specifici vaccini immunità adattativa. Genetica inversa a base di approcci offrono la possibilità di generare modificato, vaccini vivi attenuati che possono essere utilizzati in via sperimentale con questo scopo. Dal momento della sua attuazione in materia di ricerca di influenza, genetica inversa basata plasmide-è stato ampiamente impiegato per generare ceppi influenzali ricombinanti compreso LAIVs. Protocolli standard per salvare virus influenzali ricombinanti richiedono multi-trasfezione di linee altamente transfectable cellulari con plasmidi ambisense (che producono sia positivi che negativi senso RNA) che contengono i segmenti virali otto influenzali, nonché di amplificazione in un sistema permissiva come Madin-Darby rene canino ( MDCK) cellule e / o di gallina embrionate uova 5. Tuttavia, l&#39;applicazione di genetica inversa per generare strumenti molecolari per studiare i meccanismi immunitari della vaccinazione rimane inesplorato. La generazionedi nuovi modelli di mouse che permettono specifico esaurimento delle sottopopolazioni di cellule del sistema immunitario, tra cui DC, ha aperto nuove possibilità per comprendere i meccanismi immunitari fondamentali alla base di protezione del vaccino-suscitato. Il confronto tra le funzioni sottoinsieme DC nei topi e nell&#39;uomo ha rivelato che, in larga misura, mouse e DCS umani sono funzionalmente omologhe 6,7, questi risultati, suggeriscono fortemente che lo sviluppo di modelli murini consentendo specifica esaurimento delle DC nello stato stazionario e in condizioni infiammatorie, può servire per capire la fisiologia delle risposte DC nell&#39;uomo. Negli ultimi anni una serie di modelli di topo sono state generate portando transgeni esprimenti il recettore simian tossina difterica (DT) (DTR) sotto il controllo della regione del promotore di un gene di interesse 8,9. Dal momento che i tessuti di topo non naturalmente esprimono DTR, questi modelli consentono esaurimento condizionale di sottoinsiemi di cellule che trasportano il gene targeting di interesse sul inoculazione mouse con DT. Così, il nostro ability ad esaurire DC specifici e altri leucociti in vivo durante i processi fisiologici, è stata notevolmente rafforzata dallo sviluppo di ro-based DTR. Tuttavia, mentre questi modelli di topi transgenici sono stati ampiamente utilizzati per comprendere l&#39;ontogenesi del sistema immunitario, la loro applicazione per lo sviluppo del vaccino è stato appena testato. Qui, combinando l&#39;influenza genetica inversa e competitivi chimere del midollo osseo a base di DTR, proponiamo un metodo per studiare la cinetica di vaccino immunità così come la funzione del gene individuo durante la risposta immunitaria ai vaccini in vivo. L&#39;applicazione di questa tecnologia per la valutazione preclinica di nuovi vaccini contro le malattie infettive difficili potrebbe contribuire a razionalizzare la progettazione di vaccini e per testare vaccini candidati in vivo.

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			<td>Dulbecco&#180;s Modified Eagle Medium (DMEM 1X)</td>
			<td>Gibco RL-Life Technologies</td>
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			<td>Embryonated eggs</td>
			<td>Valo biomedia Gmbh</td>
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			<td>PBS (1X)</td>
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			<td>D8537</td>
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			<td>70 &#956;M Nylon Filters</td>
			<td>Greiner-Biorad</td>
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			<td>Red Blood Cell Lysing buffer (RBCL) 10X</td>
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			<td>CD16/CD32 Mouse BD Fc Block (2.4G2)</td>
			<td>BD Pharmigen</td>
			<td>553142</td>
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			<td>APC-Anti-mouse SIINFEKL-H2kb (25 D1.16)</td>
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			<td>PE-Anti-mouse CD11c (HLA3)</td>
			<td>BD Biosciences</td>
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			<td>eFluor 450-Anti-mouse MHCII (Md/114.15.2)</td>
			<td>eBioscience</td>
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			<td>PerCp/Cy5.5-Anti-mouse CD103 (2E7)</td>
			<td>Biolegend</td>
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			<td>PE-Anti-mouse CD45.1 (A20)</td>
			<td>eBioscience</td>
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			<td>V500-Anti-mouse CD45.2 (1O4)</td>
			<td>BD Bioscience</td>
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			<td>PerCp-eFluor710 -Anti-mouse CD8a (53-6.7)</td>
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			<td>eFluor450-Anti-mouse CD4 (GK 1.5)</td>
			<td>eBioscience</td>
			<td>48-0041-80</td>
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			<td>CFSE Proliferation dye</td>
			<td>eBioscience</td>
			<td>65-0850-85</td>
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			<td>Baytril 2.5%</td>
			<td>Bayer</td>
			<td>65-0850-85</td>
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			<td>Dymethil-Sulfoxide (DMSO)</td>
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			<td>Ovalbumin&#160;</td>
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			<td>Diphteria Toxin (DT)</td>
			<td>Sigma-Aldrich</td>
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			<td>Trypsin-TPCK</td>
			<td>Sigma-Aldrich</td>
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			<td>BD FACsCanto II Flow cytometer</td>
			<td>BD Biosciences</td>
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			<td>FlowJo cell analysis software 9.5</td>
			<td>Flowjo inc.</td>
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			<td>Trypan Blue Stain (0.4%)&#160;</td>
			<td>Life technologies</td>
			<td>T10282</td>
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			<td>Countess Automatic Cell Counter</td>
			<td>Invitrogen-Life Technologies</td>
			<td>C10227</td>
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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.

Generazione di ricombinanti vaccini influenzali vivo attenuato può essere ottenuto mediante trasfezione di plasmidi che codificano le otto segmenti del virus dell&#39;influenza sotto il controllo di promotori bidirezionali 5. Un vaccino influenzale adattato fredda solito contiene sei segmenti di un ceppo adattato fredda, nonché l&#39;HA e NA del ceppo influenzale di scelta (ad esempio, H1N1) (Figura 1A). Il principio di freddo-adattamento si basa sulla replicazione virale limitato ...

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 somministrazione di ricombinante Influenza vaccini nei modelli chimerico topo per studiare immunità delle mucose

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 ...

Research

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Generation of Recombinant Influenza Virus from Plasmid DNA

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Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

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Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Protocollo per ricombinante RBD-based Vaccines SARS: Preparazione di proteine, vaccinazione degli animali e la rivelazione di neutralizzazione

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

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Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

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Esaminando il ruolo della rinofaringeo associata Tissue linforeticolare (NALT) nelle risposte mouse ai vaccini

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

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Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

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Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

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Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

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Misurare Influenza neuraminidasi Inibizione anticorpo I titoli da Enzyme-linked lectina Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

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Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...