Cette étude a été soutenue par les National Institutes of Health (NIH) des États-Unis (RO1 AI68002).

1. Recombinante Préparation du SARS-CoV protéines RBD <ol><li> Préparer réactif de transfection de phosphate de calcium <ol><li> Préparation de 2X tampon HBS: Mélanger 16 g de NaCl, 0,4 g de Na 2 HPO 4 * 7H 2 O, et 13,0 g d&#39;HEPES. Ajuster le pH à 7,00 et amener le volume total à 1000 ml d&#39;eau distillée. Après filtration de la solution pour la stérilisation, aliquote et le stocker à -20 ° C. Astuce: Toute variation de la valeur du pH aurait une incidence sur les résultats de la transfection. Ainsi, il est conseillé de tester plusieurs valeurs de pH autour de 7,00 (par exemple, 6,99, 7,00, ou 7,01) et de trouver le meilleur pour la transfection en utilisant la méthode présentée ci-dessous. </li><li> 2,5 M CaCl 2 préparation: Ajouter 73,5 g de CaCl 2 * 2H 2 O à l&#39;eau distillée dans un volume final de 200 ml. Filtrer la solution et conserver à -20 ° C. </li></ol></li><li> Recombinant transfection plasmidique et purification des protéines recombinantes <ol><li> Fractionner les cellules 293T à 50-70% de confluence 24 h avant la transfection. Cultiver des cellules de T-175 cm 2 flacons de culture tissulaire dans 40 ml de DMEM contenant 10% inactivés par la chaleur (HI) FBS et 1% de pénicilline / streptomycine (P / S) à 37 ° C dans 5% de CO 2. </li><li> Tous les réactifs de transfection devrait être porté à la température ambiante avant la transfection. Ces réactifs comprennent 2X HBS, 2.5M CaCl 2, Dih 2 O, et rRBD 6 plasmide. Astuce: Le plasmide final recombinant de construction utilisé pour l&#39;expression protéique doit contenir un peptide signal pour assurer la sécrétion de protéines recombinantes exprimées dans le surnageant de culture. Un 6x Son étiquette peut être ajoutée à la C-terminale de la protéine exprimée pour la purification facile. </li><li> Préparer un 50 ml (A) et un 15 ml (B) BD Falcon tube, et ajouter des réactifs dans les tubes comme indiqué dans le tableau 1. Ajouter 2X tampon HBS à A. Dans le tube tube B, ajouter 2,5 M CaCl 2 et rRBD plasmide, et de porter le volume à l&#39;exigence de Dih 2 O. <table border="1"><tbody><tr><td colspan="4"> A. Un T-175 cm 2 flacon de culture tissulaire (4000 ul / flacon): se préparer pour l&#39;expression rRBD </td></tr><tr><td colspan="2"> Un tube </td><td colspan="2"> Le tube B </td></tr><tr><td> 2X HBS </td><td> 2000 uL </td><td> 2,5 M CaCl 2 </td><td> 200 uL </td></tr><tr><td></td><td></td><td> rRBD ADN plasmidique </td><td> 40 ug </td></tr><tr><td></td><td></td><td> Dih 2 O </td><td> 2000 uL </td></tr><tr><td colspan="4"> B. Une de 100 mm boîte de Petri (1000 ul / plat): se préparer pour la production pseudovirus SRAS </td></tr><tr><td colspan="2"> Un tube </td><td colspan="2"> Le tube B </td></tr><tr><td> 2X HBS </td><td> 500 ul </td><td> 2,5 M CaCl 2 </td><td> 50 ul </td></tr><tr><td></td><td></td><td> ADN plasmidique S SRAS-CoV </td><td> 5 ug </td></tr><tr><td></td><td></td><td> VIH-1 plasmide (pNL4-3.luc.RE) </td><td> 5 ug </td></tr><tr><td></td><td></td><td> Dih 2 O </td><td> 500 ul </td></tr></tbody></table> Tableau 1. Préparation du mélange de transfection et les volumes Les volumes indiqués dans le tableau 1 sont pour une unité de transfection. Si plus des flacons ou des plats sont utilisés pour la transfection, ajuster les volumes en conséquence. </li><li> Ajouter la solution d&#39;ADN et de calcium dans le tube B dans le tube A dans un goutte à goutte, tout en maintenant constante et douce de mélange dans un tourbillon. Laissez reposer le mélange à la température ambiante pendant 20-30 min. La clé pour la transfection de phosphate de calcium succès dépend de la taille et la forme du précipité formé. Ainsi, le mélange doit être constamment vortexé et lentement pour répondre à cette exigence et, par conséquent, d&#39;améliorer l&#39;efficacité de la transfection. </li><li> Ajouter le mélange goutte à goutte, et même dans les cellules 293T (4000 ul / flacon). Cellules en culture dans un incubateur à 37 ° C dans 5% de CO 2. </li><li> Remplacez le milieu de culture avec des produits frais sans sérum OPTI-MEM-je réduite Sérum Medium (50 ml / flacon) 8-10 h post-transfection. Continuer à cultiver des cellules pour deux jours de plus dans le même état. </li><li> Recueillir le surnageant contenant la protéine exprimée rRBD 72 h post-transfection. Centrifuger à 6000 rpm pendant 15 min pour éliminer les débris cellulaires. Ajouter un cocktail inhibiteur de protéase pour le surnageant collecté et stocker à 4 ° C pendant la nuit. </li><li> Le lendemain, purifier rRBD protéine recombinante à partir du surnageant de culture en utilisant les instructions du fabricant Superflow Ni-NTA suivante. </li><li> Concentré protéine purifiée en utilisant des tubes ultra concentration Amicon -15. Après la concentration de protéines,Ajouter PBS à la concentration des tubes et centrifuger à nouveau pour enlever l&#39;imidazole dans le tampon d&#39;élution. Calculer la concentration de protéines et de stocker des protéines purifiées à -80 ° C jusqu&#39;à utilisation. </li></ol></li></ol> 2. Immunisation de souris et de prélèvement d&#39;échantillons <ol><li> Préchauffer Sigma adjuvant System (SAS) à 40-45 ° C selon les instructions du fabricant. Ajouter 1 ml de PBS par flacon et bien mélanger. </li><li> Préparer protéines adjuvant émulsion selon le protocole dans le tableau 2. Pipeter calculée protéines rRBD dans un tube de 1,5 ml. Ajouter un volume égal de SAS adjuvant dans le tube et vortexer vigoureusement pendant 2-3 min pour former l&#39;émulsion. Astuce: Les volumes indiqués dans le tableau 2 sont pour une souris. Ajustez le volume en fonction du nombre de souris utilisées réelle. Pour chaque groupe, mélanger les protéines et de l&#39;adjuvant nécessaire pour chaque vaccin. Toujours préparer un échantillon supplémentaire pour la vaccination pour assurer l&#39;exactitude. <table border="1"><tbody><tr><td> Groupe </td><td> 1 er vaccins (200 ul / souris) </td><td> 2 ème vaccin (200 ul / souris) </td><td> 3 ème vaccin (200 ul / souris) </td></tr><tr><td> protéines rRBD </td><td> 20 g de protéine dans du PBS (100 ul) + 100 uL SAS </td><td> 10 g de protéine dans du PBS (100 ul) + 100 uL SAS </td><td> 10 g de protéine dans du PBS (100 ul) + 100 uL SAS </td></tr><tr><td> PBS de contrôle </td><td> 100 ul de PBS + 100 uL SAS </td><td> 100 ul de PBS + 100 uL SAS </td><td> 100 ul de PBS + 100 uL SAS </td></tr></tbody></table> Tableau 2. Protocole d&#39;immunisation de la souris </li><li> Sous-cutanée en prime-vacciner les femmes souris Balb / c (4-6 semaines, 5 souris / groupe), et de stimuler deux fois avec rRBD et SAS comme indiqué au tableau 2. Utilisez PBS groupe comme le contrôle. Le site d&#39;injections sous-cutanées généralement choisi est la peau lâche entre les omoplates. Alternativement, l&#39;abdomen ventral est couramment utilisé, car il est plus facile à injecter, et peut observer des fuites provenant des sites d&#39;injection. Lorsque des doses répétées de vaccins sont utilisés, il est facile de sélectionner des sites d&#39;injection différents, empêchant le potentiel des réactions cutanées locales. </li><li> Purger souris par rétro-orbitale avec anesthésie avant l&#39;immunisation et 10 jours après chaque vaccination, et la chaleur des sérums à 56 ° C pendant 30 min pour inactiver le complément. Magasin de sérums de souris à -20 ° C jusqu&#39;à utilisation. </li></ol> 3. Détection de neutralisation utilisant test de neutralisation pseudovirus <ol><li> Préparer pseudovirus SRAS <ol><li> Fractionner les cellules 293T en 100 mm plats de Pétri de culture tissulaire (2x10 6 cellules / boîte) 16 h avant la transfection, et cultiver des cellules comme ci-dessus. </li><li> Préparer des réactifs de transfection, comme indiqué dans le tableau 1. Co-transfection un plasmide codant pour le SRAS-CoV protéine S et d&#39;un plasmide codant pour Env-défectueux, la luciférase exprimant génome VIH-1 (pNL4-3.luc.RE) en utilisant le réactif de transfection de phosphate de calcium. </li><li> Remplacer moyenne avec 10 ml DMEM frais contenant 10% de FBS et 1% P / S 8-10 h post-transfection. Recueillir le surnageant contenant pseudovirus SRAS 72 h post-transfection. </li><li> Pseudovirus Filtrer à travers un filtre de 0,45 um. Aliquoter et conserver à -80 ° C jusqu&#39;à utilisation. </li></ol></li><li> Dosage de SRAS neutralisation pseudovirus <ol><li> Fractionner les cellules 293T exprimant des récepteurs du SARS-CoV ACE2 (ACE2/293T) à 10 4 cellules/100 ul / puits de plaques de 96 puits de culture de tissus 16 h avant l&#39;infection. </li><li> Diluer pseudovirus SRAS en deux volets afin de détecter le titre du virus dans les cellules ACE2/293T. </li><li> Série diluer sérums de souris dans les plaques de 96 puits de culture tissulaire, et ajouter un volume égal de pseudovirus SRAS titré. Préincuber les plaques à 37 ° C pendant 1 h. </li><li> Après incubation, ajouter 100 ul du mélange de sérums-pseudovirus aux cellules ACE2/293T, et de continuer à cultiver des cellules à 37 ° C dans 5% de CO 2. Ajouter DMEM frais 24 h plus tard. </li><li> Supprimer complètement les surnageants de culture à partir des plaques de 72 h post-infection. Ajouter 1X réactif luciférase lyse cellulaire de la culture (60 ul / puits), et de promouvoir la lyse cellulaire avec agitation constante de plaques pendant 1-2 heures à température ambiante. </li><li> Lysats de cellules de transfert (50 ul / puits) en plaques luminomètre (Microfluor plaques de 96 puits). Ajouter un substrat luciférase (50 ul / puits) inclus dans le système de dosage de la luciférase, et de détecter relativement activité luciférase dans les Ultra 384 luminomètre. </li><li> Calculer titre SRAS neutralisation pseudovirus et présents sous le titre d&#39;anticorps neutralisant 50% (NT 50) 6. </li></ol></li></ol>

<ol>
	<li>Pitcovski, J., Gutter, B., Gallili, G., Goldway, M., Perelman, B., Gross, G., Krispel, S., Barbakov, M., Michael, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+large-scale+use+of+recombinant+VP2+vaccine+for+the+prevention+of+infectious+bursal+disease+of+chickens.">Development and large-scale use of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens.</a> Vaccine. 21, 4736-4743 (2003).</li><li>Tait, A., Hall, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theileria+annulata:+control+measures,+diagnosis+and+the+potential+use+of+subunit+vaccines.">Theileria annulata: control measures, diagnosis and the potential use of subunit vaccines.</a> Rev. Sci. Tech. 9, 387-403 (1990).</li><li>Qi, Z., Zhou, L., Zhang, Q., Ren, L., Dai, R., Wu, B., Wang, T., Zhu, Z., Yang, Y., Cui, B., Wang, Z., Wang, H., Qiu, Y., Guo, Z., Yang, R., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+mouse,+guinea+pig+and+rabbit+models+for+evaluation+of+plague+subunit+vaccine+F1+rV270.">Comparison of mouse, guinea pig and rabbit models for evaluation of plague subunit vaccine F1+rV270.</a> Vaccine. 28, 1655-1660 (2010).</li><li>Rosser, M. P., Xia, W., Hartsell, S., McCaman, M., Zhu, Y., Wang, S., Harvey, S., Bringmann, P., Cobb, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transfection+of+CHO-K1-S+using+serum-free+medium+in+suspension:+a+rapid+mammalian+protein+expression+system.">Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system.</a> Protein Expr. Purif. 40, 237-243 (2005).</li><li>Du, L., Zhao, G., Li, L., He, Y., Zhou, Y., Zheng, B. J., Jiang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenicity+and+immunogenicity+of+SARS-CoV+S+protein+receptor-binding+domain+stably+expressed+in+CHO+cells.">Antigenicity and immunogenicity of SARS-CoV S protein receptor-binding domain stably expressed in CHO cells.</a> Biochem. Biophys. Res. Commun. 384, 486-490 (2009).</li><li>Du, L., Zhao, G., Chan, C. C., Sun, S., Chen, M., Liu, Z., Guo, H., He, Y., Zhou, Y., Zheng, B. J., Jiang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+receptor-binding+domain+of+SARS-CoV+spike+protein+expressed+in+mammalian,+insect+and+E.+coli+cells+elicits+potent+neutralizing+antibody+and+protective+immunity.">Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity.</a> Virology. 393, 144-150 (2009).</li><li>Ambrosino, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acids+270+to+510+of+the+severe+acute+respiratory+syndrome+coronavirus+spike+protein+are+required+for+interaction+with+receptor.">Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor.</a> J. Virol. 78, 4552-4560 (2004).</li><li>Li, W., Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., Somasundaran, M., Sullivan, J. L., Luzuriaga, K., Greenough, T. C., Choe, H., Farzan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin-converting+enzyme+2+is+a+functional+receptor+for+the+SARS+coronavirus.">Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus.</a> Nature. 426, 450-454 (2003).</li><li>He, Y., Zhou, Y., Liu, S., Kou, Z., Li, W., Farzan, M., Jiang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-binding+domain+of+SARS-CoV+spike+protein+induces+highly+potent+neutralizing+antibodies:+implication+for+developing+subunit+vaccine.">Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine.</a> Biochem. Biophys. Res. Commun. 324, 773-781 (2004).</li><li>Batard, P., Jordan, M., Wurm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+high+copy+number+plasmid+into+mammalian+cells+by+calcium+phosphate+transfection.">Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection.</a> Gene. 270, 61-68 (2001).</li><li>Pramfalk, C., Lanner, J., Andersson, M., Danielsson, E., Kaiser, C., Renstrom, I. M., Warolen, M., James, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+receptor+activation+and+down-regulation+by+cationic+lipid+transfection+reagents.">Insulin receptor activation and down-regulation by cationic lipid transfection reagents.</a> BMC. Cell Biol. 5, 7-7 (2004).</li><li>Rosser, M. P., Xia, W., Hartsell, S., McCaman, M., Zhu, Y., Wang, S., Harvey, S., Bringmann, P., Cobb, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transfection+of+CHO-K1-S+using+serum-free+medium+in+suspension:+a+rapid+mammalian+protein+expression+system.">Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system.</a> Protein Expr. Purif. 40, 237-243 (2005).</li><li>Jiang, M., Chen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Ca2+-phosphate+transfection+efficiency+in+low-density+neuronal+cultures.">High Ca2+-phosphate transfection efficiency in low-density neuronal cultures.</a> Nat. Protoc. 1, 695-700 (2006).</li><li>Jiang, M., Deng, L., Chen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Ca(2+)-phosphate+transfection+efficiency+enables+single+neuron+gene+analysis.">High Ca(2+)-phosphate transfection efficiency enables single neuron gene analysis.</a> Gene Ther. 11, 1303-1311 (2004).</li><li>Han, D. P., Kim, H. G., Kim, Y. B., Poon, L. L., Cho, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+safe+neutralization+assay+for+SARS-CoV+and+characterization+of+S-glycoprotein.">Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein.</a> Virology. 326, 140-149 (2004).</li><li>Toledo, J. R., Prieto, Y., Oramas, N., Sanchez, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine-based+transfection+method+as+a+simple+and+effective+way+to+produce+recombinant+lentiviral+vectors.">Polyethylenimine-based transfection method as a simple and effective way to produce recombinant lentiviral vectors.</a> Appl. Biochem. Biotechnol. 157, 538-544 (2009).</li></ol>

La densité cellulaire est un facteur important affectant l&#39;efficacité du phosphate de calcium à base de transfection. Dans notre expérience, moins de 70% de confluence des cellules apporte les meilleurs résultats. Ainsi, afin d&#39;améliorer l&#39;efficacité de transfection, il est recommandé que la densité cellulaire sera maintenu à environ 50-70% de confluence. La méthode de transfection de phosphate de calcium est généralement considéré comme moins efficace par rapport aux méthodes de transfection d&#39;autres, tels que 16 transfection des lipides. Toutefois, dans ce protocole, nous avons utilisé une méthode de calcium de phosphate modifiés transfection lequel constante et lente de mélange de la solution de transfection dans un vortex assure la formation d&#39;un précipité avec la taille et de forme appropriées, améliorant ainsi l&#39;efficacité de la transfection.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>Na2HPO4*7H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S2429</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2*2H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C5080</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12430</td>
<td>&nbsp;</td>
<tr>
<td>HI FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10438</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>OPTI-MEM I Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31985</td>
<td>&nbsp;</td>
<tr>
<td>Protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11836170001</td>
<td>&nbsp;</td>
<tr>
<td>Ni-NTA Superflow</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>30450</td>
<td>&nbsp;</td>
<tr>
<td>Sigma adjuvant system</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6322</td>
<td>&nbsp;</td>
<tr>
<td>Luciferase assay system</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>E1501</td>
<td>&nbsp;</td>
<tr>
<td>Luciferase cell culture lysis reagent (5X)</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>E1531</td>
<td>&nbsp;</td>
<tr>
<td>Amicon Ultra - 15</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC901024</td>
<td>&nbsp;</td>
<tr>
<td>Microfluor 96-well plates</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>7905</td>
<td>&nbsp;</td>
<tr>
<td>Ultra 384 luminometer</td>
<td>&nbsp;</td>
<td>Tecan Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

protocol for recombinant rbd-based sars vaccines: protein preparation, animal vaccination and neutralization detection

Basé sur leur profil d&#39;innocuité et sa capacité à induire des réponses immunitaires contre les infections, vaccins sous-unitaires ont été utilisés comme des candidats pour une grande variété de pathogènes 1-3. Puisque le système de cellules de mammifères est capable de modifications post-traductionnelles, formant ainsi des protéines correctement repliées et glycosylée, protéines recombinantes exprimées dans les cellules de mammifères ont montré le plus grand potentiel pour maintenir l&#39;antigénicité et l&#39;immunogénicité élevée 4-6. Bien qu&#39;aucun nouveau cas de SRAS ont été rapportés depuis 2004, de futures éclosions sont une menace constante, par conséquent, le développement de vaccins contre le SRAS-CoV est une étape prudente et préventive doit être effectuée. Les RBD de la protéine S SRAS-CoV joue un rôle important dans la liaison aux récepteurs spécifiques et l&#39;induction d&#39;anticorps neutralisants contre 7-9 infection par le virus. Par conséquent, dans ce protocole, nous décrivons de nouvelles méthodes pour développer un vaccin sous-unité RBD-Unis contre le SRAS. En bref, la protéine recombinante RBD (rRBD) a été exprimée dans le surnageant de culture de cellules de mammifères 293T d&#39;obtenir une protéine correctement repliée avec une conformation correcte et l&#39;immunogénicité élevée 6. La transfection du recombinant plasmide codant pour RBD aux cellules a été ensuite effectuée en utilisant une méthode de transfection de phosphate de calcium 6,10 avec quelques modifications. Comparativement à la méthode de transfection des lipides 11,12, cette méthode de calcium de phosphate modifiés transfection est moins cher, plus facile à manipuler, et a le potentiel pour atteindre une grande efficacité une fois complexes de transfection avec une taille convenable et la forme est formée 13,14. Enfin, un test de neutralisation du SRAS pseudovirus été introduite dans le protocole et utilisé pour détecter l&#39;activité neutralisante des sérums de souris vaccinées avec la protéine rRBD. Ce dosage est relativement sûr, n&#39;implique pas une maladie infectieuse du SRAS-CoV, et peut être effectuée sans l&#39;exigence d&#39;un laboratoire de biosécurité 3 15. Le protocole décrit ici peut également être utilisé pour la conception et l&#39;étude des vaccins sous-unitaires recombinants contre d&#39;autres virus avec des protéines de fusion de classe I, par exemple, le VIH, le virus respiratoire syncytial (VRS), virus Ebola, virus de la grippe, ainsi que les virus Nipah et Handra. En outre, les méthodes pour générer une pseudovirus et ensuite établir un test de neutralisation pseudovirus peut être appliquée à tous ces virus.

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

Ce protocole décrit une procédure générale pour l&#39;étude de liaison au récepteur recombinant domaine (RBD) basé vaccins sous-unitaires contre le SRAS. Il inclut des méthodes pour la transfection et l&#39;expression des protéines dans les cellules 293T RBD, l&#39;immunisation de souris avec RBD et la détection de l&#39;activité de neutralisation de sérums de souris en utilisant un dosage établie SRAS neutralisation pseudovirus.

Protocole pour vaccins recombinants contre le SRAS RBD-base: préparation de protéines, vaccination des animaux et de détection de neutralisation

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

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Research

JoVE Journal

Immunology and Infection

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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinaison de adhésif à base de bandes d&#39;échantillonnage et de fluorescence In situ</emHybridation> pour la détection rapide des Salmonella Sur les produits frais

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Recherche

Immunologie et infection

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Imagerie animal entier et cytométrie de flux techniques pour T CD8 + Analyse des réponses spécifiques de l&#39;antigène de cellule après nanoparticules vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

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 ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

In Vivo Test pour la détection de cellules T spécifiques de l’antigène fonction cytolytique en utilisant un modèle de Vaccination

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Génération des vecteurs recombinants Avian Herpesvirus avec CRISPR/Cas9 gène édition

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

Analyse des analogues de l'acide iophénoxique chez la petite mangouste indienne (Herpestes Auropunctatus) Sera pour utilisation comme marqueur biologique de vaccination contre la rage buccale

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

Un test de neutralisation rapide et multiplex à double rapporteur IgG et IgM SARS-CoV-2 pour un système d’analyse de débit à base de billes multiplexées

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The increasing number of SARS-CoV-2 infections raises the need for better antibody detection assays. Current antibody detection methods compromise sensitivity for speed or are sensitive but time-consuming. A large proportion of SARS-CoV-2-neutralizing antibodies target the receptor-binding domain (RBD), one of the primary immunogenic compartments of SARS-CoV-2. We have recently designed and developed a highly sensitive, bioluminescent-tagged RBD (NanoLuc HiBiT-RBD) to detect SARS-CoV-2 antibodies. The following text describes the procedure to produce the HiBiT-RBD complex and a fast assay to evaluate the presence of RBD-targeting antibodies using this tool. Due to the durability of the HiBiT-RBD protein product over a wide range of temperatures and the shorter experimental procedure that can be completed within 1 h, the protocol can be considered as a more efficient alternative to detect SARS-CoV-2 antibodies in patient serum samples.

The outlined protocol describes the procedure for producing the HiBiT-receptor-binding domain protein complex and its application for fast and sensitive detection of SARS-CoV-2 antibodies.

Détection de l’anticorps du domaine de liaison au récepteur du SARS-CoV-2 à l’aide d’un biorapporteur basé sur HiBiT

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

Imagerie vivante et quantification de l’infection virale chez des souris transgéniques K18 hACE2 à l’aide du SARS-CoV-2 recombinant exprimant le rapporteur

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs. This protocol describes the binding of antigen-specific antibodies to protein A- or G-conjugated magnetic beads. In our study, human immunodeficiency virus (HIV)-derived VLPs formed by the group-specific antigen (Gag) viral core precursor protein p55 Gag and displaying the envelope glycoproteins (Env) of HIV are examined. The VLPs are captured utilizing broadly neutralizing antibodies (bNAbs) directed against neutralization-sensitive epitopes in Env. The VLP capture assay outlined here represents a sensitive and easy-to-perform method to demonstrate that (i) the VLPs are decorated with the respective target antigen, (ii) the surface antigen retained its structural integrity as demonstrated by the epitope-specific binding of bNAbs used in the assay and (iii) the structural integrity of the VLPs revealed by the detection of Gag proteins in a subsequent Western blot-analysis. Consequently, the utilization of bNAbs for immunoprecipitation facilitates a prediction of whether VLP vaccines will be able to elicit a neutralizing B cell response in vaccinated humans. We anticipate that this protocol will furnish other researchers with a valuable and straightforward experimental approach to examine potential VLP-based vaccines.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle VLP-Based Vaccines Using a Capture Assay

Here, we present a protocol to detect neutralization epitopes on antigen-displaying virus-like particles (VLPs). Immunoprecipitation of the human immunodeficiency virus (HIV)-derived VLPs is performed using envelope glycoproteins-specific monoclonal antibodies coupled to protein G-conjugated magnetic beads. Captured VLPs are subsequently subjected to SDS-PAGE and Western blot-analysis employing viral core protein Gag-specific antibodies.

Détection d’épitopes sensibles à la neutralisation dans les antigènes affichés sur les vaccins à base de particules virales (VLP) à l’aide d’un test de capture

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate ...