Nous remercions SIGA Technologies (Corvallis, OR) pour fournir ST-246. DKR a été soutenu par une subvention de formation NIH en immunologie à l&#39;Université de Boston (5T32AI 7309). Ce travail a été financé en partie par P41 086180, NIH RO1AI1096159-01, et SR3 (à JHC).

1. Cellules de la plaque <ol><li> Dissocier les cellules HeLa de la plaque et diluer dans un milieu de croissance (DMEM, 2 mM de L-surabondance, 10% de FBS) à environ 2,0 x 10 5 cellules / ml. Distribuer 100 pl / puits dans une plaque à fond plat noir clair paroi de 96 puits (20 000 cellules / puits). </li><li> Incuber les cellules pendant 24 heures jusqu&#39;à la confluence dans un incubateur à 37 ° C + 5% CO 2. </li></ol> 2. Infecter les cellules <ol><li> Diluer virus et infecter les cellules. <ol><li> Dégel TRPV (virus Triple; début Vénus, intermédiaire mCherry, fin TagBFP) et PLV (promoteur-moins Vénus) virus fluorescent et ventiler l&#39;aide ultrasons pendant 5 minutes. En variante, les stocks de virus brut peuvent être mélangés à 1:1 avec 0,25 mg / ml de trypsine dans un milieu d&#39;infection et incubées à 37 ° C pendant 30 min. </li><li> Diluer stocks de virus brut dans 37 ° C médias d&#39;infection (DMEM, 2 mM de L-surabondance, 2% de FBS). Pour les infections à MOI élevé (10 PFU / cellule), diluer stock de virus à 1,0 x 10 7 UFP / ml en supposant 5 x 10 4cellules / puits et le volume de l&#39;inoculum de 50 ul. Plan sur infecter 3 puits réplicats pour chaque traitement avec TRPV et 3 autres puits répétés pour le même traitement avec PLV pour tenir compte de la fluorescence de fond propres à chaque traitement. </li><li> Infecter puits en ajoutant 50 virus de pl / puits dilué dans les médias d&#39;infection. Elle est définie comme une infection temps = 0 h post (0 hpi). </li></ol></li><li> Diluer les composés de traitement souhaités dans les médias d&#39;infection. Pour tous les traitements d&#39;un seul master mix 2x dilution doit être établie et appliquée pour reproduire puits (par exemple 350 pi volume total de six à 96 puits avec 50 pi de chacun). <ol><li> Diluer composés expérimentaux dans les médias d&#39;infection. </li><li> Diluer le contrôle-véhicule solvant (par exemple PBS, DMSO) dans le milieu de l&#39;infection. Remarque: les concentrations finales similaires de solvants véhicule de contrôle devraient être utilisées comme appliquée pour les composés expérimentaux (par exemple si votre IBT stock est fait avec du DMSO et utilisé à une concentratio finalens de 1 pl / ml, puis utilisez DMSO seul à 1 pl / ml que le contrôle du véhicule). </li><li> Diluer composés de contrôle dans les médias d&#39;infection. Composés de commande recommandées comprennent: 3,6 uM (1 pg / ml) de 1-β-D-arabinofuranosylcytosine (AraC), 50 uM (11,7 ug / ml) isatine β-thiosemicarbazone (IBT), 60 uM (50 pg / ml) rifampicine, ou 5 uM (1,9 pg / ml) ST-246 8,15,16. Voir résultats représentatifs pour les effets escomptés. Remarque: rendre cette dilution à deux fois (2x) à la concentration finale désirée puisque ceci est ajouté à un rapport de 1:1 avec le volume déjà dans le puits. </li></ol></li><li> Immédiatement après l&#39;ajout de virus, ajouter 50 médias d&#39;infection ul contenant les traitements et les contrôles souhaités comme dilué dans l&#39;étape 2.2. Remarque: Pour doser l&#39;inhibition de l&#39;expression avant de stade précoce, le composé (s) peut soit être ajouté directement à l&#39;inoculum de virus dilué (étape 2.1.2), ou aux cellules hôtes avant l&#39;infection (avant l&#39;étape 2.1.3). </li><li> Incuber 6-24 h dans un 37 &amp; #176; incubateur C + 5% CO 2. </li></ol> 3. Fixer les cellules <ol><li> Fixer les cellules en ajoutant 100 pi 8% de paraformaldéhyde aux médias de l&#39;infection déjà dans chaque puits. Incuber à température ambiante pendant 15 min à l&#39;abri de la lumière. Note: Il est recommandé d&#39;ajouter 2x PFA (8%) directement sur un support d&#39;infection pour empêcher aérosol de la vaccine infectieuse qui peut se produire si les médias à titre élevé est inversée directement dans le plat des déchets avant la fixation. </li><li> Retirer fixateur en retournant la plaque dans le plat de déchets. </li><li> Ajouter 100 ul de PBS température ambiante. </li><li> Sceller la plaque avec un film adhésif optiquement clair. Remarque: Les plaques peuvent être conservés à 4 ° C si pas lire immédiatement. Soyez sûr de revenir plaques scellées à la température ambiante avant de le lire pour éviter la condensation de fausser les mesures du spectrophotomètre. </li></ol> 4. Quantifier la croissance du virus <ol><li> Mesurer critère fluorescence à 515:530 pour Vénus, 587:610 pour mCherry, et 415:457 fou TagBFP (excitation: longueur d&#39;onde d&#39;émission en nm). Quatre mesures devraient être en moyenne par puits en utilisant les réglages de gain optimisés pour chaque canal. Remarque: La longueur d&#39;onde d&#39;émission approprié peut être différente en fonction des caractéristiques des modèles et de filtrage spécifiques de votre lecteur de plaques. Ceci peut être déterminé de façon empirique en réalisant un balayage de longueur d&#39;onde d&#39;émission afin de déterminer le point où se trouve la plus grande différence entre TRPV et PLV pour chaque canal. </li><li> Normaliser les données brutes pour faciliter la comparaison entre les expériences. <ol><li> Pour chaque canal, déterminer la moyenne de répétition TRPV et puits de PLV. </li><li> Soustraire les moyennes des mesures de fond PLV-infectés à partir des mesures expérimentales TRPV infectés moyennes pour chaque traitement. </li><li> Normaliser les données en divisant chaque valeur soustraite fond par la valeur d&#39;un véhicule uniquement bien. </li><li> Une fois une expérience a été répétée plusieurs fois, une analyse unidirectionnelle de variance (ANOVA) et test de multiples comparaison post-test peut être effectué afin de déterminer si l&#39;un quelconque des traitements reflète une différence statistiquement significative par rapport au traitement avec le véhicule seulement pour chaque canal. </li></ol></li></ol> . 5 Alternative Protocole: essais sur plaque cinétiques <ol><li> Effectuez toutes les étapes par l&#39;ajout de traitements (étape 2.3). </li><li> Sceller la plaque avec un film adhésif et le lieu dans la chambre de lecteur de plaque équilibré à 37 ° C. Remarque: Une attention particulière doit être prise pour garder plaques constamment à 37 ° C pour prévenir le stress cellulaire excessive et la condensation. Pour les essais cinétiques, il est particulièrement important d&#39;utiliser un milieu tamponné (bicarbonate de sodium et de l&#39;HEPES) pour maintenir le pH correct, sans addition de 5% de CO 2. </li><li> Ensemble lecteur de plaque obtenir manuellement pour empêcher la saturation de points de temps ultérieurs. Remarque: l&#39;optimisation de gain précis peut être obtenue en réalisant un essais finaux dans des conditions similaires. </li><li> Acquérir des relevés horaires pour 8-24 hpi et normaliser au moyen de simiprocédure de lar comme dans l&#39;essai de point de terminaison (étape 4.2). </li><li> Points dans le temps individuel peuvent être analysées pour la signification statistique en utilisant des méthodes similaires que l&#39;essai du point de terminaison décrit précédemment. </li></ol>

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccinia+virus+transcription.">Vaccinia virus transcription.</a> Journal of General Virology. 84 (9), 2293-2303 (2003).</li><li>Condit, R. C., Niles, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+viral+transcription+elongation+and+termination+during+vaccinia+virus+infection.">Regulation of viral transcription elongation and termination during vaccinia virus infection.</a> Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression. 1577 (2), 325-336 (2002).</li><li>D&#237;az-Guerra, M., Rivas, C., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+expression+of+the+2-5A+synthetase/RNase+L+system+results+in+inhibition+of+vaccinia+virus+replication.">Inducible expression of the 2-5A synthetase/RNase L system results in inhibition of vaccinia virus replication.</a> Virology. 227 (1), 220-228 (1997).</li><li>Cohrs, R. J., Condit, R. C., Pacha, R. F., Thompson, C. L., Sharma, O. 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Les auteurs n&#39;ont rien à révéler et pas de brevets sont en instance pour des virus ou des méthodes de dépistage.

Ici, nous avons décrit l&#39;utilisation pratique d&#39;un virus de la vaccine reporter multi-étape (TRPV), qui fournit reproductibles, en temps réel des mesures de rétroaction de la réplication du virus. En utilisant des inhibiteurs de poxvirus bien définis, nous avons pu montrer que TRPV répond d&#39;une manière compatible avec le mécanisme d&#39;action compris pour chaque inhibiteur. Alors que le virus TRPV fournit l&#39;information la plus complète à propos de la progression de la phase de virus, en deux ...

Traditionnellement, l&#39;expression de virus est étudiée en utilisant des techniques de biologie moléculaire (par exemple de transfert de Northern, Western blot, hybridation microarray, etc) 1. Bien que ces méthodes sont capables de fournir des informations détaillées en ce qui concerne la catégorisation des changements de l&#39;expression des ARNm ou des protéines individuelles, ils ne sont généralement pas prêter à des processus en temps réel et à haut débit. Des approches alternatives à l&#39;aide de journalistes basés sur la fluorescence ont été appliquées auparavant lorsque vous travaillez avec des poxvirus; Cependant, leur développement et leur utilisation a été motivé par des objectifs variés. Plusieurs de ces méthodes ont été conçues pour la sélection de virus recombinants 2,3. Ces techniques exprimer EGFP lors de la constitution et de l&#39;expression de l&#39;ADN exogène à partir de clones de la vaccine recombinants bon. De même, plusieurs souches de la vaccine exprimant de manière stable soluble ou EGFP protéines GFP-étiqueté exprimées sous un promoteur de la vaccine natif ont été largement utilisés. Ce sont typquement utilisé pour quantifier l&#39;entrée et la replication du virus au cours de dosages de neutralisation à base d&#39;anticorps, l&#39;inhibition chimique, ou la comparaison de l&#39;efficacité antivirale 6.4. Bien que ces virus se sont révélés utiles, ils sont limités dans leur capacité à fournir des informations détaillées sur le point d&#39;inhibition en raison de leur utilisation d&#39;un seul promoteur précoce / tardif virale ambiguë. Les méthodes précédentes ont également fait usage de la protéine EGFP avec des caractéristiques sous-optimaux signal-bruit. En raison de la complexité de la réplication du virus de la variole et le manque d&#39;outils existants disponibles pour analyser les changements en temps réel de l&#39;expression des gènes viraux, nous avons développé une série de virus rapporteurs unique et multi-étape 7,8. Comme décrit dans les publications précédentes, ces virus expriment un, deux, ou trois protéines fluorescentes spectralement distinctes de promoteurs de la vaccine indigènes au début, des étapes intermédiaires, ou à la fin de l&#39;infection. Ces virus peuvent nous êtreed comme indicateurs de progrès réplication du virus en utilisant un microscope à fluorescence et ils conviennent aussi bien pour la plaque et-lecteur basé tests à haut débit. Ces virus sont faciles à utiliser à la place de virus de type sauvage, de plus en plus avec une cinétique similaire pour atteindre titres équivalents 7. Lors de la planification et de la création de ces virus, on a pris soin dans le choix des fluorophores ayant des caractéristiques élevées signal à fond avec une efficacité supérieure de pliage pour faciliter la quantification fiable avec retour rapide à des changements dans l&#39;expression (Vénus, mCherry et TagBFP). En outre, des combinaisons de promoteurs viraux ont été choisis qui produisent de haute fidélité, l&#39;expression spécifique de stade sans ambiguïté, fournissant des informations sur la gamme complète de début (promoteur C11R), intermédiaire (promoteur G8R) et la fin (promoteur F17R) l&#39;expression des gènes, contrairement aux ambiguë promoteurs début / fin. Ces virus peuvent être utilisés comme un outil pour investigating le cycle de vie du virus de la variole, le virus de la vaccine prototypique. Beaucoup ne sait toujours pas sur l&#39;interaction hôte-virus malgré sa longue histoire de l&#39;étude. Vaccine est complexe, produisant plus de 200 protéines uniques, dont beaucoup sont des immunomodulateurs et hébergent antagoniste. Lors de l&#39;infection, le virus de la vaccine commence immédiatement tôt transcription de l&#39;ARNm. Ceci est facilité par une ARN polymérase et des facteurs de transcription qui ont été chargés sur le génome viral lors de l&#39;emballage du virion et détenus dans un état de pause jusqu&#39;à ce que l&#39;infection ultérieure. Cette expression précoce produit principalement des protéines nécessaires à la suppression du système immunitaire de l&#39;hôte (ARNm de décapsulage, l&#39;ARN double brin séquestration, et des protéines réceptrices leurres ainsi que des inhibiteurs de l&#39;apoptose, la réponse au stress, et Toll, IL, et la signalisation NF-kB) et la réplication du génome. Expression précoce produit également des facteurs nécessaires à l&#39;expression intermédiaire de transcription. Intermédiaire expression inclut l&#39;expression de facteurs de transcription de fin d&#39;étape. Ceexpression cascade conduit à la production de protéines structurales et enzymatiques virales au cours des stades tardifs de l&#39;infection, qui sont nécessaires pour l&#39;assemblage du virion complet de la vaccine mature. Notre série de virus rapporteurs fluorescents permet de progresser plus rapidement réalisés dans la compréhension de la biologie poxvirus. L&#39;une des méthodes les plus courantes et fastidieuses dans le domaine de la virologie est le test de croissance. Cela implique généralement l&#39;infection des cellules, l&#39;adoption d&#39;une série de traitements, le virus de la récolte par la lyse des cellules infectées, et de quantifier le titre viral obtenue par dosage de plaque. Utilisation des virus rapporteurs décrits ici permet la mesure en temps réel de la croissance du virus qui peut être facilement testée et comparée entre les nombreux traitements effectués en parallèle. Nous prévoyons que cet ensemble de réactifs est utilisé dans divers protocoles pour identifier des altérations de l&#39;expression des gènes viraux en réponse à un traitement médicamenteux, l&#39;ARNi knockdown, ou restriction de la gamme d&#39;hôtes. Cette méthode permet également l&#39;analyse à haut contenu sur une plus grande échelle qu&#39;auparavant, permettant à haut débit de dépistage de drogues anti-viraux pour les étapes cibles spécifiquement définis d&#39;intérêt. Alors que de nombreux traitements possibles pour lutter contre l&#39;infection par le virus de la variole ont été identifiés, le seul approuvé par la FDA des thérapies efficaces pour le traitement de l&#39;infection par le virus de la variole sont nucléoside acyclique phosphonate, cidofovir, et le traitement par la vaccine immunoglobuline 9,10. Malgré l&#39;éradication de la variole en 1977 11, poxvirus restent une menace importante pour la santé humaine 12. Arrêt de la vaccination généralisée contre les virus de la variole a conduit à une sensibilité accrue à d&#39;autres poxvirus 13. Par exemple, les régions de l&#39;Afrique centrale qui étaient auparavant protégés par la vaccination connaissent une forte augmentation des infections par le virus monkeypox 14. Préoccupation importante a également étésoulevées au sujet de la sensibilité latente à la libération intentionnelle du virus de la variole. En raison des thérapies limitées actuellement disponibles, il ya un besoin urgent de développer de nouveaux traitements. Ces virus rapporteurs permettent le dépistage d&#39;inhibiteur de petite molécule rapide et à haut débit pour l&#39;inhibition d&#39;une étape spécifique de la réplication virale. Identification d&#39;inhibiteurs qui ciblent les stades d&#39;expression viraux actuellement pas la cible d&#39;inhibition facilitera le développement de thérapies de combinaison avec une puissance accrue. Beaucoup de choses peuvent être tirées à propos de la vaccine biologie cellulaire en observant les changements dans l&#39;expression des gènes de chaque étape. Atténuation par manipulation chimique ou génétique d&#39;une cellule hôte infectée est généralement exprimée en termes de titres viraux réduits. Toutefois, en comparant les changements dans chaque étage de la cascade d&#39;expression viral, on peut obtenir une compréhension plus complète de la façon de fitness viral est l&#39;impacted par un traitement particulier. Ces données ont été montré une bonne corrélation avec la sortie traditionnelle du titre de virus, mais de fournir des informations plus détaillées mécanique ainsi que des capacités à haut débit 7.

<table border="0" cellpadding="0" cellspacing="0" width="1249">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:747px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:104px;">11995-065</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fetal Bovine Serum - Optima, Heat Inactivated (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S12450H</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">200 mM L-Glutamine, (L-glut)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">96 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3603</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">384 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3712</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Trypsin, 2X, Sterile, Irradiated</td>
			<td>Worthington Biochemical Corp.</td>
			<td>TRLVMF</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dimethyl Sulfoxide, Cell Culture (DMSO)</td>
			<td>American Bioanalytical</td>
			<td>AB03091</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1-&#946;-D-Arabinofuranosylcytosine&#160; (AraC), MW= 280 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>C6645</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">isatin &#946; -thiosemicarbazone (IBT), MW= 234 g/mol</td>
			<td>Fisher Scientific</td>
			<td>NC9075202</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Rifampicin, MW= 823 g/mol</td>
			<td>SigmaAldrich Co</td>
			<td>R3501</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">ST-246, MW= 376 g/mol</td>
			<td>SIGA Labs, Corvallis, OR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">8% Paraformaldehyde (formaldehyde) aqueous solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-8</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TempPlate RT optically clear film</td>
			<td>USA Scientific</td>
			<td>2978-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
	</tbody>
</table>

vaccinia reporter viruses for quantifying viral function at all stages of gene expression

Les poxvirus sont une famille de virus à ADN à double brin qui comprennent des agents pathogènes humains actifs tels que la variole du singe, le molluscum contagiousum et Contagalo virus. La famille comprend également le virus de la variole, de la variole. En raison de la complexité de la réplication du virus de la variole, de nombreuses questions restent encore en ce qui concerne leur stratégie de l&#39;expression des gènes. Dans cet article, on décrit la conception et l&#39;utilisation des virus de la vaccine recombinants qui permettent de mesurer en temps réel des étapes uniques et multiples de l&#39;expression du gène viral dans un format à haut débit. Cela est rendu possible grâce à l&#39;utilisation de protéines fluorescentes spectralement distinctes comme reporters pour chacune des trois étapes de la réplication virale. Ces virus constituent un rapport signal-bruit tout en conservant les modes de scène spécifiques d&#39;expression, permettant des analyses basées sur plaque et observations microscopiques de la propagation du virus et la réplication. Ces outils ont des utilisations pour la découverte d&#39;antiviraux, les études de l&#39;interaction virus-hôte, et bio évolutivelogie.

A titre d&#39;exemple de l&#39;utilisation normale de ces virus, le virus triple fluorescence reporter a été utilisé pour comparer le point d&#39;inhibition de plusieurs inhibiteurs de poxvirus bien définies. Des cellules HeLa ont été étalées dans une culture tissulaire traitée claires des plaques à 96 puits à fond plat à parois noires et incubées pendant une nuit. Des monocouches confluentes ont été infectées à une multiplicité d&#39;infection (MOI) de 10 en utilisant soit...

Watch this Scientific Journal Video about Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression at JoVE.com

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Nous décrivons l&#39;utilisation d&#39;un virus de la vaccine rapporteur fluorescent qui permet la mesure en temps réel de l&#39;infectivité virale et l&#39;expression des gènes par le biais de l&#39;expression spécifique au stade de fluorophores spectralement distincts rapporteurs. Nous détaillons une méthode basée plaque pour identifier avec précision l&#39;étape à laquelle la réplication du virus est affecté en réponse à petite molécule inhibition.

Reporter vaccine virus pour quantifier la fonction virale à toutes les étapes de l&#39;expression des gènes

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

vaccinia-reporter-viruses-for-quantifying-viral-function-at-all

Research

JoVE Journal

Immunology and Infection

11.2K vues.  Boston University School of Medicine.  Nous décrivons l'utilisation d'un virus de la vaccine rapporteur fluorescent qui permet la mesure en temps réel de l'infectivité virale et l'expression des gènes par le biais de l'expression spécifique au stade de fluorophores spectralement distincts rapporteurs. Nous détaillons une méthode basée plaque pour identifier avec précision l'étape à laquelle la réplication du virus est affecté en réponse à petite molécule inhibition. 

Vidéo: Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Génération de cellules T spécifiques Multivirus pour prévenir / traiter les infections virales, après allogreffe de cellules souches hématopoïétiques

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Recherche

Immunologie et infection

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Phage »Bioluminescent&quot; Reporter pour la détection des bactéries pathogènes de la catégorie A

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

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

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Analyses quantitatives de tous influenza de type A hémagglutinines virales et les neuraminidases utilisant des anticorps Universal en simples essais slot blot

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

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Rentable méthode de suivi des sources microbiennes spécifiques utilisant des virus humains et animaux

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

Isolement des variantes des virus à ARN de Fidelity et caractérisation de la fréquence de mutation de virus

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

L&#39;établissement d&#39;une culture liquide recouvert de Airway humaines épithéliales polarisées cellules Calu-3 pour étudier la réponse cellulaire aux agents pathogènes respiratoires hôte<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

Souris foie fœtal Système de culture à disséquer les fonctions du gène cible dans les stades précoces et tardifs de l&#39;aérogare de l&#39;érythropoïèse

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

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.

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.

Administration intranasale de vaccins contre la grippe recombinant chimérique dans les modèles de souris pour étudier l&#39;immunité muqueuse

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 ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Une approche simple et efficace de construire Mutant Vaccinia vecteurs de virus

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Développement d&#39;un système de virus de l&#39;hépatite B Reporter pour surveiller les premiers stades du cycle de réplication

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...