Ringraziamo i membri passati e presenti in JCT e laboratori LM-S per lo sviluppo e il miglioramento delle tecniche di Arenavirus inversa e plasmidi genetica. Ringraziamo anche Snezhana Dimitrova per il supporto tecnico. L&#39;anticorpo monoclonale diretto a JUNV NP (SA02-BG12) è stato ottenuto da BEI Risorse (NIAID Biodefense ed Emerging Infections Research Resources Repository). BYHC è stato sostenuto da Grant Numero GM068411 da Institutional Ruth L. Kirschstein Servizio Nazionale Research Award. EO-R è un Fullbright-CONICYT (BIO 2008) e di un Rochester Vaccino Fellowship (2012) del destinatario. Supporto corrente EO-R è fornita da una borsa di studio postdottorato per la diversità e l&#39;eccellenza accademica presso l&#39;Ufficio per lo sviluppo delle capacità e diversità presso l&#39;Università di Rochester. La ricerca in laboratorio LM-S è finanziato dal NIH sovvenzioni RO1 AI077719, R21NS075611-01, R03AI099681-01A1, i Centri NIAID di Eccellenza per l&#39;influenza di ricerca e di sorveglianza (HHSN266200700008C), eL&#39;Università di Rochester Centro per Biodefense Modeling Immune (HHSN272201000055C). La ricerca presso il laboratorio JCT è stato sostenuto da sovvenzioni RO1 AI047140, RO1 AI077719, e RO1 AI079665 dal NIH.

1. Arenavirus Rescue Transfezione Il nostro sistema di salvataggio si basa sull&#39;utilizzo di due polimerasi II proteiche plasmidi di espressione codificanti la nucleoproteina (NP) e RNA-dipendente-RNA-polimerasi (L), i fattori trans-agenti virali necessarie per la replicazione dell&#39;RNA e l&#39;espressione genica del genoma arenavirus 7, 19, e plasmidi capaci di dirigere la sintesi intracellulare, attraverso l&#39;RNA cellulare polimerasi I (pol-I), del S e L antigenome specie di RNA 7. Nei nostri studi usiamo l&#39;espressione proteica pCAGGS plasmide, che utilizza il pollo promotore β-actina e polyadenylanation (pA) sequenze di segnale, e il pol-I umana plasmide, che utilizza la polimerasi I umano promotore e sequenze di terminazione murini (Figura 2) . La L segmenti di RNA S e sono clonati nel plasmide HPOL-I in un orientamento antigenomic per consentire la generazione di segmenti di RNA genomici upon trascrizione da HPOL-I. Per il salvataggio di wild-type LCMV ricombinante (rLCMV) e Candid # 1 (rCandid # 1) la pCAGGS NP e L da ciascun virus sono state co-trasfettate con le rispettive L segmenti RNA (Figura 3) pol-I e S umana. Generazione di ricombinante trisegmented LCMV (r3LCMV) e Candid # 1 (r3Candid # 1) ha seguito un protocollo simile, ma il segmento S è stato diviso in due plasmidi pol-I S diversi, ognuno di codifica un gene distintivo giornalista: in una, la lettura NP aperto telaio (ORF) è stata sostituita con la proteina fluorescente verde (GFP) gene reporter (HPOL-I S GFP / GP) e, nell&#39;altro, il precursore glicoproteina virale (GPC) ORF viene sostituita con la Gaussia luciferasi (Gluc) gene reporter (HPOL-I S NP / Gluc). Una rappresentazione schematica del protocollo è illustrato in Figura 4. Il seguente protocollo di trasfezione e l&#39;infezione è stata stabilita per il 6-piastre a pozzetti. <ol><li> OptiMEM-Lipofectamine 2000 (LPF2000) miscela: Preparare 250 ml di mezzi OptiMEM e, a secondasul salvataggio virus, 10 - 12 mg di LPF2000 (1 mg / mL) per trasfezione (Tabella 1). Si consiglia un rapporto di 2,5 mcg LPF2000 per mg di DNA. Incubare per 5 - 10 minuti a temperatura ambiente. Nel frattempo, preparare la miscela di trasfezione plasmide in una provetta da microcentrifuga (passo 1.2). </li><li> Plasmide miscela di trasfezione del DNA: Preparare il cocktail trasfezione plasmide in una provetta con le quantità raccomandate per ogni salvataggio virus (vedi Tabella 1). Portare il volume finale di 50 microlitri con OptiMEM. </li><li> OptiMEM-LPF2000-DNA plasmidico miscela: Aggiungere 250 microlitri della miscela OptiMEM-LPF2000 (passo 1.1) nel plasmide DNA miscela di trasfezione (passo 1.2). Incubare questa miscela per 20 - 30 min a TA. Nel frattempo preparare e contare 1 x 10 6 cellule Vero per reazione trasfezione. Cellule Vero saranno transfettate in sospensione. </li><li> Preparazione delle cellule Vero: Vero celLS sono coltivate in 100 millimetri piatti. Prima di iniziare, portare l&#39;PBS 1x, DMEM 10% FBS 1% PS media, e miscela di tripsina-EDTA a 37 ° C. <ol><li> Lavare le cellule due volte con 5 ml di PBS 1x. </li><li> Trypsinize cellule con 1 ml di tripsina-EDTA e attendere cellule scollegamento (circa 5 min). Gentle intercettazioni forse necessario per distaccare completamente le cellule dai piatti. </li><li> Attentamente, risospendere le cellule in 10 ml di DMEM 10% FBS 1% PS in una provetta da centrifuga da 15 ml. </li><li> Centrifugare le cellule per 5 min a 1000 rpm in una centrifuga. </li><li> Risospendere le cellule in 10 ml di DMEM 10% FBS 1% PS e contare le cellule utilizzando un emocitometro e regolare la concentrazione di 1 x 10 6 cellule / ml. È stato osservato che 0,5-1 x 10 6 cellule Vero per trasfezione prodotto i migliori risultati. </li></ol></li><li> Mescolare LPF2000/DNA e cellule: dopo 20 - 30 minuti a temperatura ambiente di incubazione (passo 1.3), aggiungere 1 ml di cellule Vero (1 x 10 6) (passo 1,4) ala miscela plasmide OptiMEM-LPF2000-DNA e incubare per 5 min a temperatura ambiente. </li><li> Seme LPF2000/DNA-cell mix in 6-piastre a pozzetti: Aggiungere la miscela di DNA-LPF2000-Vero (passo 1.5) nei pozzetti della piastra da 6 pozzetti. Agitare delicatamente il 6-ben piatto e lasciare che la trasfezione incubare una notte in termostato a 37 ° C e 5% di CO 2. </li><li> Cambia medie trasfezione: circa 16 - 24 ore dopo la trasfezione, rimuovere il supporto di trasfezione, aggiungere 2 ml di mezzi di infezione e incubare cellule trasfettate per ulteriori 48 ore. </li><li> Passaggio Cell: Dopo 48 ore di incubazione nei media di infezione, rimuovere il supernatante di coltura tissutale (TCS) e passare le cellule trasfettate in un piatto di 100 mm. Il TCS raramente contiene virus infettivo, perché le cellule transfettate bisogno ulteriore incubazione per generare particelle virali. Per il passaggio delle cellule: <ol><li> Lavare le cellule due volte con 2 ml di PBS 1x. </li><li> Trypsinize cellule w<html> esimo 0,5 ml di tripsina-EDTA e attendere che le cellule si staccano. </li><li> Risospendere le cellule in 1 ml di DMEM 10% FBS 1% PS in una provetta da microcentrifuga da 1,5 ml. </li><li> Centrifugare le cellule per 5 min a 5000 rpm, a 4 ° C in una microcentrifuga. </li><li> Risospendere delicatamente le cellule in 1 ml di mezzo di infezione e trasferire le cellule a un piatto di 100 mm. Portare il volume totale nella piastra, con mezzi infettive, 8 ml, un volume sufficiente per evitare l&#39;essiccazione delle cellule così come per concentrare il virus. </li></ol></li><li> Agitare delicatamente il piatto 100 mm e continuare l&#39;incubazione di cellule Vero in incubatore a 37 ° C e 5% di CO 2 per 72 ore. </li><li> Collezione del TCS: Dopo 72 ore di incubazione, raccogliere il TCS in una provetta da centrifuga da 15 ml. Rimuovere detriti cellulari mediante centrifugazione per 5 min a 2500 rpm in una centrifuga. Arenavirus infettiva è trovato nella TCS, quindi rimuovere dalla cella detriti pellet e memorizzare il TCS a -80 ° C. </li></ol>Le &quot;&gt; 2. Conferma della ricombinante Arenavirus Rescue Rilevamento di un salvataggio di successo di rLCMV e rCandid # 1 è ottenuta tramite una unità di formazione di messa a fuoco (FFU) immunofluorescenza saggio (IFA). Per tipo selvatico rescue virus usiamo anticorpi specifici per NP virale. Il r3LCMV e r3Candid # 1 encode due geni reporter (GFP e Gluc), permettendo così di rilevamento virale e titolazione senza la necessità di anticorpi mediante microscopia a fluorescenza (GFP) e / o luminescenza (Gluc). <ol><li> Confermare la wild-type arenavirus salvataggio ricombinante: Il giorno prima della titolazione, lavare le cellule Vero due volte con PBS, trypsinize e preparare piastre da 96 pozzetti per raggiungere l&#39;80 - 90% confluenza giorno successivo (4 x 10 4 cellule / pozzetto). Scuotere piano le piastre a mano per ottenere una distribuzione uniforme delle cellule. Cultura delle cellule, durante la notte, nel 37 ° C incubatore con il 5% di CO 2. Controllare le cellule sotto il microscopio per confermare un monostrato, prima di procedere con l&#39;infezione: <ol><li> Diluire serialmente (10 diluizioni) contenente il virus-TCS recuperato dalle cellule trasfettate nel piatto 100 mm (passo 1.10) in OptiMEM. </li><li> Rimuovere i supporti dalle cellule Vero teste di serie, lavare due volte con 50 ml di PBS 1x, e infettare le cellule con 50 ml di virus diluito in serie (passo 2,1). Infettare le cellule per 1,5 ore a 37 ° C e 5% di CO 2. </li><li> Dopo l&#39;infezione, rimuovere l&#39;inoculo del virus, aggiungere 100 ml di supporti infezione / pozzetto ed incubare le cellule per 16 - 18 hr. Viral re-infezione può verificarsi dopo 18 ore di incubazione, che potrebbe comportare sovrastima dei titoli virali. </li><li> A 16 - 18 ore post-infezione, rimuovere il TCS da ciascun pozzetto e fissare le cellule con formaldeide al 4% diluito in PBS 1x, per 15 minuti a RT. </li><li> Poi, rimuovere la soluzione di fissaggio e permeabilize le cellule con 0,1% Triton X-100 diluito in PBS 1x, per 10 min a temperatura ambiente. </li><li> Aspirare la soluzione di permeabilizzazione, poi lavare le cellule 3 volte wiesimo 1x PBS. </li><li> Bloccare le cellule con 2,5% di albumina di siero bovino (BSA) in 1x PBS (soluzione bloccante) per 1 ora a RT. In alternativa, le cellule possono essere bloccati notte a 4 ° C e l&#39;incubazione proseguita con anticorpo il giorno seguente. </li><li> Nel frattempo preparare l&#39;anticorpo primario. Diluire gli anticorpi primari # 1-specifici LCMV-e Candid in soluzione di blocco, e quindi centrifugare l&#39;anticorpo / soluzione di blocco per 15 min a 3.500 giri al minuto. L&#39;anti-NP LCMV clone anticorpo monoclonale 1.1.3 (diluizione 1:30) 16 e l&#39;anticorpo monoclonale anti-anticorpo JUNV NP-SA02 BG12 con BEI Risorse (diluizione 1:500) può essere utilizzato per la rivelazione di rLCMV e rCandid # 1 , rispettivamente. </li><li> Dopo 1 ora di incubazione, aspirare la soluzione di saturazione e aggiungere 50 microlitri di anticorpo primario alle cellule e incubare per 1 ora a 37 ° C. </li><li> In questo momento, diluire l&#39;anticorpo secondario in una soluzione bloccante, quindi si centrifuga il / soluzione di anticorpo bloccante per 15 min a 3500 rpm. Il policlonale coniglio anti-topo IgG FITC anticorpo secondario per la rivelazione di anticorpi monoclonali primari è stato usato per ottenere immagini osservate in figura 5. </li><li> Dopo 1 h di incubazione, aspirare l&#39;anticorpo primario, lavare 3 volte con PBS 1x, e aggiungere l&#39;anticorpo secondario per 30 min a 37 ° C. </li><li> Dopo 30 minuti di incubazione, aspirare l&#39;anticorpo secondario e lavare 3 volte con PBS 1x. Le cellule sono ora pronti per essere osservato mediante microscopia a fluorescenza sia determinare il successo di salvataggio titolazione virale e contando il fuoco formando unità per ml (FFU / ml). </li></ol></li><li> r3LCMV e r3Candid # 1 di salvataggio virale: Dal momento che questi virus esprimono due geni reporter, il loro salvataggi possono essere monitorati mediante microscopia a fluorescenza per rilevare l&#39;espressione della GFP o espressione Gluc in TCS. Rescue può essere confermata anche dalla espressione di Gluc in TCS. Per titolare i virus trisegmented, seguiamo passi da 2.1 a 2.1.4, ma le cellule non hanno bisogno di be fissato per determinare un salvataggio virale di successo a causa di espressione GFP. La titolazione del virus sarà determinata contando il FFU / ml. Per determinare il salvataggio di successo virale di espressione Gluc, espressione Gluc dal TCS può essere misurata con un kit di test Gluc (Biolux Gaussia kit Assay luciferasi, New England Biolabs). A tal fine: <ol><li> Dispensare 100 ul di TCS in una piastra a 96 pozzetti bianco (micropiastre fondo piatto, Fisher Scientific). </li><li> Impostare il luminometro (LumiCount, Packard Biosciences). </li><li> Aggiungere 50 - 100 microlitri soluzione Assay Gluc (come consigliato dal produttore) per ogni campione e misurare la Gluc espressione del gene reporter con il luminometro. Come controllo negativo, è stata misurata l&#39;espressione Gluc del TCS dai virus wild-type cellule infettate. </li></ol></li></ol> 3. Passaggio del tessuto surnatanti di coltura Salvataggio virale dipende l&#39;efficienza di trasfezione. Cellule Vero sono state indicateavere efficienze di trasfezione inferiori rispetto altre linee cellulari 14. Se titoli di virus nel TCS sono bassi, infettare cellule Vero fresche in una molteplicità di infezione (MOI) di 0,01 (LCMV) o 0,1 (Candid # 1) per 72 ore per amplificare il virus.

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Generazione di arenavirus ricombinanti utilizzando tecniche di genetica inversa plasmide-based è diventata un metodo ampiamente utilizzato per lo studio di molti e diversi aspetti della biologia arenavirus. Qui documentiamo un miglioramento fondamentale per l&#39;attuale sistema eseguendo arenavirus salvataggi su cellule Vero, consentendo la potenziale generazione di candidati vaccini approvati dalla FDA contro arenavirus e vettori di vaccini contro altre malattie infettive. Le procedure sp...

Arenavirus sono virus con involucro con bisegmented, negativo-stranded RNA del genoma 3 che appartengono alla famiglia Arenaviridae. Il genoma codifica arenavirus quattro proteine ​​in modo ambisense da due segmenti virali separati 3. Il grande segmento (L) codifica per la RNA polimerasi RNA-dipendente (L) e il piccolo anello (davvero interessante nuovo gene) finger proteina (Z), che serve come il principale motore di virus in erba. Il piccolo (S) segmento codifica la nucleoproteina virale (NP) e la glicoproteina di superficie (GP) (Figura 1). Diversi membri della famiglia Arenaviridae sono responsabili della febbre emorragica letale (HF) in esseri umani 3. Di principio preoccupazioni sono virus Lassa (LASV) e virus Junin (JUNV), che sono noti per causare un&#39;elevata mortalità nei pazienti ospedalizzati 8, 9. Anche se questi virus sono endemiche in Africa occidentale e rurale Argentina, rispettivamente,vi sono preoccupazioni crescenti di importazione di LASV e JUNV ad aree non endemiche a causa di un aumento di viaggio 10. Inoltre, anche se normalmente non associata a malattia grave nell&#39;uomo, il arenavirus prototipo virus coriomeningite linfocitaria (LCMV) è considerato un agente patogeno trascurato in quanto non vi sono stati casi di infezione letale in pazienti immunocompromessi 6, 15 ed è responsabile per i difetti congeniti alla nascita e aborti spontanei in donne in gravidanza 2, 13. Attualmente non ci sono US FDA ha approvato i vaccini contro arenavirus e il trattamento è limitato al nucleoside analogo ribavirina, che è solo parzialmente efficace e spesso associata a significativi effetti collaterali. L&#39;introduzione di inversione plasmide a base genetica 4 e la generazione di ricombinanti arenavirus 7, 19 hanno notevolmente migliorato il campo di ricerca arenavirus. Attualmente, cellule di roditore (come BHK-21) vengono utilizzati per la generzione di arenaviruses ricombinanti dovute alla specie-specifico murino RNA polimerasi I (pol-I) promotore dirigere la trascrizione iniziale della S e L segmenti. Tuttavia virus salvataggio in cellule BHK-21 non sono un metodo approvato per la generazione di ricombinanti arenavirus come potenziali candidati di semi vaccino. Qui documentiamo l&#39;uso del umano pol-I promoter di soccorso efficiente del prototipo Vecchio Mondo (OW) LCMV e il Nuovo Mondo (NW) JUNV Candid # 1 ceppo di cellule Vero. Utilizzando una metodologia simile, abbiamo generato ricombinante trisegmented LCMV (r3LCMV) e Candid # 1 (r3Candid # 1) arenavirus che contengono due geni estranei aggiuntivi codificati in due diversi segmenti di RNA S 5. Questo nuovo sistema segue non solo un protocollo altamente riproducibile e semplice ma può essere immediatamente attuata la generazione di arenaviruses ricombinanti come potenziale vaccino o semi vettore vaccino.

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 Cell lines 
Vero E6 (African green monkey kidney epithelial cells) are maintained in a 37 &deg;C incubator with 5 % CO2 in DMEM 10 % FBS 1 % PS. Cells are available from the American Type Culture Collection (ATCC, catalogue number CRL-1586). 
 
Plasmids 
All plasmids, with the exception of hpol-I L, can be grown at 37 &deg;C, for 16-18 hr. We recommend that cultures of the hpol-I L be grown at 30 &deg;C for 24 hr. Plasmids are prepared using a plasmid maxi kit (EZNA Fastfilter Plasmid Maxi Kit, Omega Bio-tek) following the manufacturer's recommendations and stored at -20 &deg;C. The concentration of the purified DNA plasmid is determined by spectrophotometry at 260 nm, with purity being estimated using the 260:280 nm ratio. Preparations with 1.8-2.0 260:280 nm ratios are considered appropriate for virus rescue purposes. Additionally, plasmid concentration and purity should be confirmed with agarose gel electrophoresis. 
 
Viruses 
The described protocol for rescuing rLCMV (Armstrong 53b) and rCandid#1 can be executed under biosafety level (BSL) 2 conditions. Contaminated material, including TCS and cells should be sterilized before disposal. Rescue of other arenavirus may require higher BSL facilities so proper safety/security measures must be followed. 
 
Tissue culture media and solutions 
DMEM 10 %FBS 1 %PS: 445 ml Dulbecco's modified Eagle's medium (DMEM), 50 ml of Fetal Bovine Serum (FBS), and 5 ml of 100X Penicillin/Streptomycin (PS). Store at 4 &deg;C. This media will be used for maintenance of Vero cells. 
 
Infectious Media: 2-to-1 mixture of OptiMEM and DMEM 10 %FBS 1 %PS. Store at 4 &deg;C. This media will be used during viral infections. 
 
10X Phosphate buffered saline (PBS): 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 liter. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature. 
1X PBS: Dilute 10X PBS 1:10 with ddH2O. Sterilize by autoclave and store at room temperature. 
2.5 % BSA: 2.5 g of BSA in 97.5 ml of 1X PBS. Store at 4 &deg;C. This is used as a blocking solution for IFA.
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generation of recombinant arenavirus for vaccine development in fda-approved vero cells

Lo sviluppo e l&#39;attuazione di arenavirus genetica inversa rappresenta un significativo passo avanti nel campo 4 arenavirus. L&#39;uso di sistemi minigenome Arenavirus base di cellule insieme con la capacità di generare ricombinanti arenaviruses infettive con mutazioni predeterminate nei loro genomi ha facilitato l&#39;indagine del contributo dei determinanti virali nelle diverse fasi del ciclo di vita arenavirus, così come virus-ospite interazioni e meccanismi di patogenesi arenavirus 1, 3, 11. Inoltre, lo sviluppo di arenaviruses trisegmented ha permesso l&#39;utilizzo del genoma arenavirus di esprimere ulteriori geni estranei di interesse, aprendo così la possibilità di applicazioni vettore di vaccino arenavirus basati 5. Analogamente, lo sviluppo di ciclo unico arenaviruses infettivi capaci di esprimere geni reporter fornisce un nuovo strumento sperimentale per migliorare la sicurezza di ricerca che coinvolge highly patogeni arenavirus umani 16. La generazione di arenavirus ricombinanti utilizzando tecniche di genetica inversa plasmide-based ha finora invocato l&#39;uso di linee cellulari di roditori 7,19, il che pone alcune barriere per lo sviluppo di Food and Drug Administration (FDA) con licenza vaccino o di vettori vaccinali. Per superare questo ostacolo, si descrive qui la generazione efficiente di arenavirus ricombinanti in cellule Vero approvato dalla FDA.

Salvataggio di successo di un ricombinante arenavirus wild-type sarà confermata dalla presenza di antigeni virali mediante IFA (Figura 5). Nel caso dei virus ricombinanti trisegmented, salvataggio di successo virale può essere valutata osservando espressione GFP mediante microscopia a fluorescenza (Figura 6A). Salvataggio di successo sarà ulteriormente confermato dalla valutazione dell&#39;espressione Gluc (Figura 6B). Rappresentante risultati illustrano il salvatagg...

Watch this Scientific Journal Video about Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells at JoVE.com

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

Salvataggio di arenavirus ricombinanti da cDNA clonati, un approccio denominato invertire la genetica, permette ai ricercatori di indagare il ruolo di specifici prodotti genici virali, così come il contributo dei loro diversi domini e residui specifici, per diversi aspetti della biologia di arenavirus . Allo stesso modo, invertire tecniche di genetica in linee cellulari approvato dalla FDA (Vero) per lo sviluppo di vaccini fornisce nuove possibilità per la generazione di vaccini efficaci e sicuri per combattere arenavirus patogeni umani.

Generazione di Arenavirus ricombinante per lo sviluppo del vaccino nel approvati dalla FDA cellule Vero

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

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Research

JoVE Journal

Immunology and Infection

16.8K Views.  University of Rochester School of Medicine and Dentistry. Salvataggio di arenavirus ricombinanti da cDNA clonati, un approccio denominato invertire la genetica, permette ai ricercatori di indagare il ruolo di specifici prodotti genici virali, così come il contributo dei loro diversi domini e residui specifici, per diversi aspetti della biologia di arenavirus . Allo stesso modo, invertire tecniche di genetica in linee cellulari approvato dalla FDA (Vero) per lo sviluppo di vaccini fornisce nuove possibilità per la generazione d...

Video: Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Isolamento linfociti T umani, la cultura e l&#39;analisi delle migrazioni In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

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Immunologia e infezioni

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Generazione di influenza da virus ricombinante del DNA plasmidi

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

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

Generazione di un nuovo vaccino a cellule dendritiche con melanoma e cellule staminali tumorali squamose

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Utilizzando Clicca Chimica per misurare l&#39;effetto dell&#39;infezione virale sulla cellula ospite RNA Sintesi

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In Analisi Vitro di Myd88 mediata Cellular Immune Response to West Nile virus mutante Strain Infezione

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

Utilizzando la strategia di Antigen Capside-Incorporation per lo sviluppo di approcci Adenovirus sierotipo 5 Vectored vaccino

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Generazione di ricombinante umano IgG anticorpi monoclonali da immortalizzate cellule ordinati B

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Saggio altamente sensibile per la misura dell&#39;attaccamento Arenavirus-cell

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

Generazione, amplificazione e titolazione del virus sinciziale respiratorio ricombinante

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. 
This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (&lt;1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. 
Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

This protocol describes using commercial, cell-free protein expression kits to produce membrane proteins supported in nanodisc that can be used as antigens in subunit vaccines.

Produzione su scala libera da cellule e aggiunta adiuvante a una proteina principale ricombinante della membrana esterna da Chlamydia muridarum per lo sviluppo di vaccini