Diese Studie wurde von den National Institutes of Health (NIH) der Vereinigten Staaten (RO1 AI68002) unterstützt.

1. Rekombinante SARS-CoV RBD Protein Preparation <ol><li> Bereiten Calciumphosphat Transfektionsreagenz <ol><li> 2X HBS-Puffer Zubereitung: Mischen Sie 16 g NaCl, 0,4 g Na 2 HPO 4 * 7H 2 O und 13,0 g HEPES. Der pH-Wert auf 7,00 und bringen Gesamtvolumen von 1000 ml in destilliertem Wasser. Nach Filtrieren der Lösung für die Sterilisation, Aliquot und speichern Sie es bei -20 ° C. Hinweis: Jede Veränderung des pH-Wertes würde die Transfektion Ergebnisse beeinflussen. So ist es ratsam, mehrere pH-Werten um 7,00 (z. B. 6.99, 7.00, bzw. 7,01) Test und finden Sie das beste für die Transfektion mit der Methode eingeführt unten. </li><li> 2,5 M CaCl 2 Vorbereitung: In 73,5 g CaCl 2 · 2H 2 O in destilliertem Wasser in einem Endvolumen von 200 mL. Filtern Sie die Lösung und bei -20 ° C. </li></ol></li><li> Das rekombinante Plasmid-Transfektion und Proteinreinigung <ol><li> Split 293T-Zellen bei 50-70% Konfluenz 24 h vor der Transfektion. Wachsen Zellen in T-175 cm 2 Zellkulturflaschen in 40 ml DMEM mit 10% Hitze-inaktiviertes (HALLO) FBS und 1% Penicillin / Streptomycin (P / S) bei 37 ° C in 5% CO 2. </li><li> Alle Transfektionsreagenzien sollten auf Raumtemperatur gebracht werden, bevor der Transfektion. Diese Reagenzien sind 2X HBS, 2,5 M CaCl 2, DIH 2 O, und rRBD Plasmid 6. Hinweis: Die endgültige rekombinante Plasmid verwendet für die Proteinexpression ein Signalpeptid enthalten sollte, um die Sekretion des exprimierten rekombinanten Proteine ​​im Kulturüberstand gewährleisten zu konstruieren. A 6x His-Tag kann der C-Terminus des exprimierten Proteins für eine einfache Reinigung hinzugefügt werden. </li><li> Bereiten Sie eine 50 ml (A) und 15 ml (B) BD Falcon-Röhrchen, und fügen Sie Reagenzien, die Rohre, wie in Tabelle 1 angegeben. Hinzufügen weiterer 2X HBS Puffer Rohr A. In Rohr B, fügen 2.5M CaCl 2 und rRBD Plasmid, und bringen Sie die Lautstärke auf den Bedarf in DIH 2 O. <table border="1"><tbody><tr><td colspan="4"> A. Ein T-175 cm 2 Zellkulturflasche (4000 ul / Kolben): Vorbereitung auf rRBD Ausdruck </td></tr><tr><td colspan="2"> Rohr A </td><td colspan="2"> Rohr B </td></tr><tr><td> 2X HBS </td><td> 2000 ul </td><td> 2.5M CaCl 2 </td><td> 200 ul </td></tr><tr><td></td><td></td><td> rRBD Plasmid-DNA </td><td> 40 ug </td></tr><tr><td></td><td></td><td> DIH 2 O zu </td><td> 2000 ul </td></tr><tr><td colspan="4"> B. Ein 100-mm-Petrischale (1000 ul / Schale): Vorbereitung auf SARS pseudovirus Produktion </td></tr><tr><td colspan="2"> Rohr A </td><td colspan="2"> Rohr B </td></tr><tr><td> 2X HBS </td><td> 500 ul </td><td> 2.5M CaCl 2 </td><td> 50 ul </td></tr><tr><td></td><td></td><td> SARS-CoV S Plasmid-DNA </td><td> 5 ug </td></tr><tr><td></td><td></td><td> HIV-1-Plasmid (pNL4-3.luc.RE) </td><td> 5 ug </td></tr><tr><td></td><td></td><td> DIH 2 O zu </td><td> 500 ul </td></tr></tbody></table> Tabelle 1. Transfektion Gemischaufbereitung und Volumina Die Volumina in Tabelle 1 aufgelistet sind für eine Transfektion Einheit. Wenn mehr Flaschen oder Geschirr für die Transfektion eingesetzt werden, stellen Sie Volumen entsprechend. </li><li> Fügen Sie die DNA-Calcium-Lösung in Rohr B in das Rohr A in einem tropfenweise Weise, während eine konstante und schonende Durchmischung in einen Strudel. Lassen Sie die Mischung bei Raumtemperatur stehen lassen für 20-30 min. Der Schlüssel für eine erfolgreiche Calciumphosphat-Transfektion hängt von der Größe und Form der Niederschlag gebildet. So sollte die Mischung kontinuierlich und langsam verwirbelt werden, um diese Anforderung zu erfüllen und, als Folge, zu verbessern Transfektion Wirksamkeit. </li><li> Add-Gemisch in einer tropfenweise und sogar Weise in 293T-Zellen (4000 ul / Kolben). Kultur-Zellen im Brutschrank bei 37 ° C in 5% CO 2. </li><li> Ersetzen Sie das Kulturmedium mit frischem serumfreien OPTI-MEM I Reduced-Serum Medium (50 ml / Flasche) 8-10 h nach der Transfektion. Weiter zur Kultivierung von Zellen für zwei Tage in dem gleichen Zustand. </li><li> Sammeln enthaltende Überstand ausgedrückt rRBD Protein 72 h nach der Transfektion. Zentrifugation bei 6000 rpm für 15 min, um Zelltrümmer zu entfernen. Add-Protease-Inhibitor-Cocktail, um die gesammelten Überstand und lagern bei 4 ° C über Nacht. </li><li> Am nächsten Tag, zu reinigen rRBD rekombinanten Proteins aus dem Kulturüberstand mittels Ni-NTA Superflow folgenden Anweisungen des Herstellers. </li><li> Konzentrieren Sie sich gereinigtes Protein mit Amicon Ultra -15 Konzentration Röhren. Nach Proteinkonzentration,add PBS, um die Konzentration-und Zentrifugenröhrchen wieder Imidazol in Elutionspuffer zu entfernen. Berechnen Proteinkonzentration und speichern gereinigtes Protein bei -80 ° C bis zur Verwendung. </li></ol></li></ol> 2. Maus Immunisierung und Probennahme <ol><li> Pre-warm Sigma Adjuvans-System (SAS) auf 40-45 ° C nach den Anweisungen des Herstellers. 1 ml PBS pro Fläschchen geben und gut mischen. </li><li> Bereiten Protein-Adjuvans-Emulsion nach dem Protokoll in Tabelle 2. Pipette berechnet rRBD Protein in ein 1,5 ml-Röhrchen. Add gleichen Volumen SAS adjuvant mit dem Rohr und kräftig vortexen für 2-3 min, um Emulsion zu bilden. Hinweis: Die Volumina in Tabelle 2 aufgelistet sind für eine Maus. Lautstärkeanpassung entsprechend der tatsächlichen Maus Zahlen verwendet. Für jede Gruppe, mischen die Proteine ​​und Adjuvans für jeden Impfstoff erforderlich. Immer bereiten eine zusätzliche Probe für die Impfung, um die Genauigkeit zu gewährleisten. <table border="1"><tbody><tr><td> Gruppe </td><td> 1 M. Impfstoff (200 ul / Maus) </td><td> 2. Impfung (200 ul / Maus) </td><td> 3 rd-Impfstoff (200 ul / Maus) </td></tr><tr><td> rRBD Protein </td><td> 20 ug Protein in PBS (100 ul) + 100 ul SAS </td><td> 10 ug Protein in PBS (100 ul) + 100 ul SAS </td><td> 10 ug Protein in PBS (100 ul) + 100 ul SAS </td></tr><tr><td> PBS-Kontrolle </td><td> 100 ul PBS + 100 ul SAS </td><td> 100 ul PBS + 100 ul SAS </td><td> 100 ul PBS + 100 ul SAS </td></tr></tbody></table> Tabelle 2. Maus Immunisierungsprotokoll </li><li> Subkutan prime-Immunisierung weibliche BALB / c-Mäuse (4-6 Wochen alt, 5 Mäuse / Gruppe), und steigern Sie zweimal mit rRBD und SAS als in Tabelle 2 angegebenen. Verwenden Sie PBS-Gruppe als Kontrolle. Der Standort für die subkutane Injektionen in der Regel gewählt wird, die lose Haut zwischen den Schulterblättern. Alternativ ist der ventralen Bauch häufig verwendet, weil es einfacher ist zu injizieren, und kann jede Leckage aus der Injektionsstelle zu beobachten. Bei der wiederholten Dosen von Impfstoffen verwendet werden, ist es einfach, verschiedene Injektionsstellen wählen, verhindern, dass potenzielle lokale Hautreaktionen. </li><li> Bleed Mäusen über retroorbitalen mit Betäubung vor der Immunisierung und 10 Tagen nach jeder Impfung und Wärme Seren bei 56 ° C für 30 min auf Komplement zu inaktivieren. Shop Mausseren bei -20 ° C bis zur Verwendung. </li></ol> 3. Neutralisation Erkennung anhand Pseudovirus Neutralisationstest <ol><li> Bereiten SARS pseudovirus <ol><li> Split 293T-Zellen in 100 mm Gewebekultur-Petrischalen (2x10 6 Zellen / Schale) 16 h vor der Transfektion und wachsen Zellen wie oben beschrieben. </li><li> Bereiten Transfektionsreagenzien wie in Tabelle 1 angegeben. Co-Transfektion ein Plasmid kodiert SARS-CoV S-Protein und ein Plasmid kodiert Env-defekt, Luciferase-exprimierende HIV-1-Genoms (pNL4-3.luc.RE) mit Calciumphosphat Transfektionsreagenz. </li><li> Ersetzen-Medium mit 10 ml frischem DMEM mit 10% FBS und 1% P / S 8-10 h nach der Transfektion. Sammeln enthaltende Überstand SARS pseudovirus 72 h nach der Transfektion. </li><li> Filter pseudovirus durch ein 0,45 um-Filter. Aliquotieren und bei -80 ° C bis zur Verwendung. </li></ol></li><li> SARS pseudovirus Neutralisationstest <ol><li> Split 293T-Zellen, die SARS-CoV-Rezeptor ACE2 (ACE2/293T) bei 10 4 Zellen/100 ul / well in 96-well-Zellkulturplatten 16 h vor der Infektion. </li><li> Verdünnen SARS pseudovirus durch 2-fach auf die Virustiter in ACE2/293T Zellen zu erkennen. </li><li> Serienmäßig verdünnen Mausseren in 96-well-Zellkulturplatten, und fügen Sie gleichen Volumen titriert SARS pseudovirus. Vorinkubieren die Platten bei 37 ° C für 1 h. </li><li> Nach der Inkubation mit 100 ul der sera-pseudovirus Mischung ACE2/293T Zellen und weiterhin Zellen bei 37 ° C wachsen in 5% CO 2. Fügen Sie frisches DMEM 24 h später. </li><li> Vollständig zu entfernen Kulturüberständen von den Platten 72 h nach der Infektion. Add 1X Luciferase Zellkultur Lysereagens (60 ul / well) und die Förderung der Zelllyse unter ständigem Schütteln der Platten für 1-2 h bei Raumtemperatur. </li><li> Transfer-Zelllysaten (50 ul / well) in Luminometer Platten (Microfluor 96-Well-Platten). Add Luciferase Substrat (50 ul / well) in Luciferase-Assay-Systems, sowie erkennen relativen Luciferaseaktivität in Ultra 384 Luminometer. </li><li> Berechnen SARS pseudovirus Neutralisation Titer und Gegenwart als 50% der Patienten neutralisierende Antikörper-Titer (NT 50) 6. </li></ol></li></ol>

<ol>
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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. 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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. 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Die Zelldichte ist ein wichtiger Faktor für die Wirksamkeit von Calciumphosphat-basierte Transfektion. Nach unserer Erfahrung bringt weniger als 70% Konfluenz der Zellen die besten Ergebnisse. So, um die Transfektionseffizienz zu verbessern, wird empfohlen, dass die Zelldichte bei ca. 50-70% der Konfluenz gehalten werden. Die Calciumphosphat-Transfektion Methode wird allgemein angenommen, weniger effizient zu sein im Vergleich zu anderen Transfektion Methoden, wie Lipid-Transfektion 16. Allerdings ist in diesem Protokoll verwendeten wir eine modifizierte Calciumphosphat-Transfektion Methode, bei der konstanten und langsamen Vermischung der Transfektion Lösung in einen Wirbel sorgt die Bildung eines Niederschlags mit der entsprechenden Größe und Form und verbessert so die Transfektionseffizienz.

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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

Basierend auf ihr Sicherheitsprofil und die Fähigkeit, wirksame Immunantwort gegen Infektionen auslösen, haben Subunit-Impfstoffe als Kandidaten für eine Vielzahl von Krankheitserregern 1-3 verwendet worden. Da die Säugetierzellen ist in der Lage post-translationale Modifikation, wodurch richtig gefaltet und glykosylierte Proteine ​​sind rekombinante Proteine ​​in Säugetierzellen exprimiert das größte Potenzial, um hohe Antigenität und Immunogenität 4-6 pflegen gezeigt. Obwohl keine neuen Fälle von SARS seit 2004 berichtet wurden, sind zukünftige Ausbrüche eine ständige Bedrohung, daher ist die Entwicklung von Impfstoffen gegen die SARS-CoV einer vorsichtigen vorbeugenden Schritt und sollte durchgeführt werden. Die RBD des SARS-CoV S-Protein spielt eine wichtige Rolle in Rezeptor-Bindung und die Induktion von spezifischen neutralisierenden Antikörper gegen Virus-Infektion 7-9. Daher wird in diesem Protokoll, beschreiben wir neuartige Methoden für die Entwicklung eines RBD-basierte Subunit-Impfstoff gegen SARS. Kurz gesagt, wurde das rekombinante RBD-Protein (rRBD) im Kulturüberstand von Säugetier-293T-Zellen exprimiert, um ein korrekt gefaltetes Protein mit der richtigen Konformation und eine hohe Immunogenität 6 zu erhalten. Die Transfektion der rekombinanten Plasmid kodiert RBD zu den Zellen wurde dann mit Hilfe einer Calciumphosphat-Transfektion Methode 6,10 mit einigen Änderungen. Verglichen mit dem Lipid Transfektionsmethode 11,12, ist dieser modifizierten Calciumphosphat-Transfektion Methode billiger, einfacher zu handhaben und hat das Potenzial, eine hohe Wirksamkeit einmal Transfektion Komplex mit geeigneter Größe und Form ist 13,14 gebildet erreichen. Schließlich wurde eine SARS pseudovirus Neutralisationstest in das Protokoll eingeführt und verwendet werden, um die neutralisierende Aktivität der Seren von Mäusen mit rRBD Protein geimpft zu erkennen. Dieser Test ist relativ sicher, nicht um eine ansteckende SARS-CoV, und kann ohne das Erfordernis eines Biosafety-3-Labor 15 durchgeführt werden. Die hier beschriebene Protokoll kann auch zur Gestaltung und das Studium rekombinanter Subunit-Impfstoffe gegen andere Viren, die mit Klasse-I-Fusionsproteine, z. B. HIV, Respiratory Syncytial Virus (RSV), Ebola-Virus, Influenza-Virus, sowie Nipah und Handra Viren sein. Darüber hinaus können die Methoden zur Erzeugung eines pseudovirus und anschließend über eine pseudovirus Neutralisationstest auf alle diese Viren angewendet werden.

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

Dieses Protokoll beschreibt ein allgemeines Verfahren für die Untersuchung rekombinanten Rezeptor-bindende Domäne (RBD)-basierte Subunit-Impfstoffe gegen SARS. Es umfasst Methoden für die Transfektion und Expression von RBD-Protein in 293T-Zellen, Immunisierung von Mäusen mit RBD und den Nachweis von Neutralisation Aktivität Mausseren anhand eines etablierten SARS pseudovirus Neutralisationstest.

Protokoll für die rekombinante RBD-basierte Impfstoffe SARS: Protein Preparation, Animal Impfung und Neutralisation Erkennung

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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Immunology and Infection

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Serie: 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).

Kombination von Klebstoff-tape-basierten Sampling und Fluoreszenz In situ Hybridisierung zum schnellen Nachweis von Salmonellen 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 ...

Forschung

Immunologie und Infektion

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.

Whole-Tier Imaging und Fließzytometrieverfahren zur Analyse von Antigen-spezifischen CD8 + T-Zellantworten nach der Nanopartikel-Impfung

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.

Die intranasale Verabreichung von rekombinantem Influenza Vaccines in chimären Maus-Modelle zu Mucosale Immunität Studieren

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 zum Nachweis von Antigen-spezifischen T-Zell Zytolyse-Funktion mit einer Impfung-Modell

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.

Generierung von rekombinanten aviären Herpesvirus Vektoren mit CRISPR/Cas9 gen bearbeiten

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 von Iophenoxic Acid Analoga in Small Indian Mongoose (Herpestes Auropunctatus) Sera zur Verwendung als orale Tollwut-Impfung Biologischer 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 ...

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.

Ein schneller Multiplex-Dual-Reporter-IgG- und IgM-SARS-CoV-2-Neutralisationsassay für ein multiplexed Bead-basiertes Durchflussanalysesystem

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.

Nachweis eines SARS-CoV-2-Rezeptor-bindenden Domänenantikörpers mit einem HiBiT-basierten Bioreporter

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.

Live-Bildgebung und Quantifizierung von Virusinfektionen in transgenen K18-hACE2-Mäusen mit Reporter-exprimierendem rekombinantem 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 ...

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.

Nachweis neutralisationsempfindlicher Epitope in Antigenen, die auf VLP-basierten Impfstoffen (Virus Like Particle) mit einem Capture-Assay angezeigt werden

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 ...