Damos las gracias a los miembros pasados ​​y presentes en los laboratorios LM-S JCT y para el desarrollo y mejora de las técnicas de ida y plásmidos genética arenavirus. También agradecemos a Snezhana Dimitrova para soporte técnico. El anticuerpo monoclonal dirigido a JUNV NP (SA02-BG12) se obtuvo de Recursos BEI (NIAID Biodefensa y Enfermedades Infecciosas investigación Repositorio de Recursos). BYHC fue apoyada por número de concesión GM068411 del Institucional Ruth L. Kirschstein Premio Nacional de Servicio de Investigación. EO-R es un Fullbright-Conicyt (BIO 2008) y una beca Vacuna Rochester (2012) destinatario. Soporte actual EO-R es proporcionado por una beca postdoctoral de la Diversidad y Excelencia Académica de la Oficina de Desarrollo de la Facultad y Diversidad de la Universidad de Rochester. La investigación en LM-S laboratorio está financiado por el NIH subvenciones RO1 AI077719, R21NS075611-01, R03AI099681-01A1, los Centros de Excelencia del NIAID para la Investigación y Vigilancia de la Gripe (HHSN266200700008C) yLa Universidad de Rochester Centro de Biodefensa Modeling Inmune (HHSN272201000055C). La investigación en laboratorio JCT fue apoyado por becas SR1 AI047140, AI077719 SR1 y SR1 AI079665 de los NIH.

1. Arenavirus transfección Rescate Nuestro sistema de rescate se basa en el uso de ambos II plásmidos de expresión de proteínas de polimerasa que codifican la nucleoproteína (NP) y dependiente de ARN-ARN-polimerasa (L), los factores que actúan en trans virales necesarias para la replicación de ARN y la expresión de genes del genoma arenavirus 7, 19, y los plásmidos capaces de dirigir la síntesis intracelular, celular a través de la ARN polimerasa I (pol-I), de la S y L ​​especies de ARN antigenoma 7. En nuestros estudios usamos el pCAGGS plásmido de expresión de la proteína, que utiliza el pollo promotor de β-actina y polyadenylanation (pA) secuencias de señal, y el pol-I humano plásmido, que utiliza la polimerasa I humana secuencias promotoras y terminadoras murinos (Figura 2) . La S y segmentos de ARN L se clonaron en el plásmido HPOL-I en una orientación antigenómico para permitir la generación de segmentos de ARN genómico en la transcripción por HPOL-I. Para el rescate de wild-tyPE LCMV recombinante (rLCMV) y Candid # 1 (rCandid # 1) el pCAGGS NP y L a partir de cada virus fueron co-transfectadas junto con sus respectivos pol-I humano S y L ​​segmentos de ARN (Figura 3). Generación de LCMV trisegmented recombinante (r3LCMV) y Candid # 1 (r3Candid # 1) siguió un protocolo similar pero el segmento S se dividió en dos plásmidos pol I-S diferentes, cada uno que codifica un gen reportero distintivo: en uno, la lectura NP abierto marco (ORF) se reemplazó con la proteína (GFP) gen verde fluorescente reportero (HPOL-I S GFP / GP) y, en el otro, el precursor de la glicoproteína viral (GPC) ORF se sustituye con la luciferasa Gaussia (Gluc) gen reportero (HPOL-I S NP / Gluc). Una representación esquemática del protocolo se ilustra en la Figura 4. El siguiente protocolo de transfección y la infección se ha establecido para los de 6 pocillos y placas. <ol><li> OptiMEM-Lipofectamine 2000 (LPF2000) mezcla de: preparar 250 l de los medios de comunicación Optimem y, dependiendoen el virus de rescate, 10 - 12 g de LPF2000 (1 g / l) por transfección (Tabla 1). Se recomienda una relación de 2,5 g LPF2000 por g de ADN. Incubar durante 5 - 10 min a RT. Mientras tanto, preparar la mezcla de transfección del plásmido en un tubo de microcentrífuga separado (paso 1.2). </li><li> Mezcla de transfección de ADN de plásmido: Preparar el cóctel de transfección del plásmido en un tubo de microcentrífuga usando las cantidades recomendadas para cada virus de rescate (véase la Tabla 1). Llevar el volumen final a 50 l con OptiMEM. </li><li> Plásmido mezcla OptiMEM-LPF2000-ADN: Añadir 250 l de la mezcla-LPF2000 OptiMEM (paso 1.1) en la mezcla de transfección de ADN plásmido (paso 1.2). Se incuba esta mezcla durante 20 - 30 min a TA. Mientras tanto preparar y contar 1 x 10 6 células Vero por reacción de transfección. Células Vero se transfectaron en suspensión. </li><li> Preparación de células Vero: Vero cells se cultivan en placas de 100 mm. Antes de comenzar, llevar el 1x PBS, DMEM FBS al 10% PS 1%, y la mezcla de tripsina-EDTA a 37 ° C. <ol><li> Lavar las células dos veces con 5 ml de PBS 1x. </li><li> Tripsinizar las células con 1 ml de tripsina-EDTA y esperar hasta que las células de desconexión (aproximadamente 5 min). Golpecitos suaves quizá necesaria para retirar completamente las células de los platos. </li><li> Con cuidado, resuspender las células en 10 ml de DMEM 10% de FBS 1% PS en un tubo de centrífuga de 15 ml. </li><li> Centrifugar las células durante 5 min a 1000 rpm en una centrífuga. </li><li> Resuspender las células en 10 ml de DMEM 10% de FBS 1% PS y contar las células usando un hemocitómetro y ajustar la concentración de 1 x 10 6 células / ml. Se observó que el 0,5 - 1 x 10 6 células Vero por transfección produce los mejores resultados. </li></ol></li><li> Mezclar LPF2000/DNA y células: Después de 20 - 30 min sala de incubación a temperatura ambiente (paso 1.3), añadir 1 ml de células Vero (1 x 10 6) (paso 1.4) ael plásmido mezcla OptiMEM-LPF2000-ADN y se incuba durante 5 min a temperatura ambiente. </li><li> Semilla LPF2000/DNA-cell mezcla en 6-así placas: Añadir la mezcla de ADN-LPF2000-Vero (paso 1.5) en los pocillos de la placa de 6 pocillos. Agitar suavemente la placa de pocillos 6 y dejar que la transfección se incuba durante la noche en la incubadora a 37 ° C y 5% de CO2. </li><li> Cambiar medio de transfección: Aproximadamente 16 - 24 horas después de la transfección, eliminar el medio de transfección, añadir 2 ml de medio de infección y se incuban las células transfectadas durante 48 h adicionales. </li><li> Paso de la célula: Después de 48 h de incubación en los medios de comunicación de infección, eliminar el sobrenadante de cultivo de tejido (TCS) y pasar las células transfectadas en una placa de 100 mm. El TCS rara vez contiene virus infeccioso debido a que las células transfectadas necesitan incubación adicional para generar partículas de virus. Para el paso de células: <ol><li> Lavar las células dos veces con 2 ml de PBS 1x. </li><li> Tripsinizar células w<html> ith 0,5 ml de tripsina-EDTA y esperar hasta que las células se desprenden. </li><li> Resuspender las células en 1 ml de DMEM 10% de FBS 1% PS en un tubo de microcentrífuga de 1,5 ml. </li><li> Centrifugar las células durante 5 min a 5000 rpm, 4 ° C en una microcentrífuga. </li><li> Cuidadosamente resuspender las células en 1 ml de medio de infección y transferir las células a un plato de 100 mm. Llevar el volumen total en la placa, con los medios de comunicación infecciosas, a 8 ml, un volumen suficiente para prevenir el secado de las células, así como para concentrar el virus. </li></ol></li><li> Agite suavemente el plato de 100 mm y continuar la incubación de células Vero en la incubadora a 37 ° C y 5% de CO 2 durante 72 horas. </li><li> Colección de TCS: Después de 72 h de incubación, recoger el TCS en un tubo de centrífuga de 15 ml. Eliminar los restos celulares por centrifugación durante 5 min a 2500 rpm en una centrífuga. Arenavirus infecciosa se encuentra en el TCS, por lo que eliminar de la sedimento de restos celulares y almacenar el TCS a -80 ° C. </li></ol>le &quot;&gt; 2. Confirmación de rescate Arenavirus recombinante La detección de un rescate exitoso de rLCMV y rCandid # 1 se logra a través de una unidad de formación de enfoque (FFU) ensayo de inmunofluorescencia (IFA). Para rescatar virus natural que utilizamos anticuerpos específicos para el NP viral. El r3LCMV y r3Candid # 1 codifican dos genes informadores (GFP y Gluc), permitiendo así la detección viral y valoración sin la necesidad de anticuerpos por microscopía de fluorescencia (GFP) y / o luminiscencia (Gluc). <ol><li> Confirmación de tipo salvaje rescate arenavirus recombinante: El día antes de la titulación, lavar las células Vero dos veces con PBS, trypsinize y preparar placas de 96 pocillos para llegar al 80 - 90% de confluencia día siguiente (4 x 10 4 células / pocillo). Agite suavemente las placas con la mano para conseguir una distribución uniforme de las células. Cultura de las células, durante la noche, en el 37 ° C incubadora con 5% de CO 2. Compruebe las células bajo el microscopio para confirmar una monocapa, antes de proceder con la infección: <ol><li> Diluir en serie (diluciones de 10 veces) que contiene el virus-TCS se recuperó de las células transfectadas en la placa de 100 mm (paso 1,10) en OptiMEM. </li><li> Retire el medio de las células sembradas Vero, lavar dos veces con 50 l de 1x PBS, e infectar las células con 50 l del virus diluido en serie (paso 2.1). Infectar a las células durante 1,5 horas a 37 ° C y 5% de CO 2. </li><li> Después de la infección, eliminar el inóculo del virus, añadir 100 ml de los medios de comunicación de infección / pocillo e incubar las células durante 16 - 18 h. Re-infección viral puede ocurrir después de 18 h de incubación, lo que podría dar lugar a una estimación de los títulos virales. </li><li> En 16 - 18 horas después de la infección, eliminar el TCS de cada pocillo y fijar las células con formaldehído al 4% diluido en 1x PBS, durante 15 min a TA. </li><li> A continuación, eliminar la solución de fijación y permeabilizar las células con 0,1% de Triton X-100 diluido en 1x PBS, durante 10 min a temperatura ambiente. </li><li> Aspirar la solución de permeabilización, lavar las células 3 veces wiª 1x PBS. </li><li> Bloquear las células con albúmina de suero bovino 2,5% (BSA) en 1x PBS (solución de bloqueo) durante 1 hora a TA. Alternativamente, las células pueden ser bloqueados durante la noche a 4 ° C y se continuó con la incubación del anticuerpo el día siguiente. </li><li> Mientras tanto preparar el anticuerpo primario. Diluir los anticuerpos primarios # 1-específicos LCMV-y Candid en solución de bloqueo, y luego centrifugar la solución de anticuerpo / bloqueo durante 15 min a 3500 rpm. El anticuerpo monoclonal anti-LCMV NP clon del anticuerpo 1.1.3 (dilución 1:30) 16 y el anticuerpo anti-NP JUNV monoclonal SA02-BG12 de Recursos BEI (dilución 1:500) se puede utilizar para la detección de rLCMV y rCandid # 1 , respectivamente. </li><li> Después de 1 hora de incubación, aspirar la solución de bloqueo y añadir 50 l de anticuerpo primario a las células e incubar durante 1 hora a 37 ° C. </li><li> En este momento, diluir el anticuerpo secundario en solución de bloqueo, y luego centrifugar el / solución de bloqueo de anticuerpos durante 15 min a 3500 rpm. El polide conejo anti-IgG de ratón FITC anticuerpo secundario clonal para la detección de anticuerpos monoclonales primarios se utilizó para obtener imágenes observadas en la Figura 5. </li><li> Después de 1 hora de incubación, aspirar el anticuerpo primario, lavar 3 veces con 1 x PBS, y añadir el anticuerpo secundario durante 30 min a 37 ° C. </li><li> Después de 30 min de incubación, aspirar el anticuerpo secundario y lavar 3 veces con 1 x PBS. Las células están ahora listos para ser observados mediante microscopía de fluorescencia para determinar tanto el éxito del rescate viral y titulación contando el foco formando unidades por ml (FFU / ml). </li></ol></li><li> r3LCMV y r3Candid # 1 rescate viral: Puesto que estos virus expresan dos genes indicadores, su rescates puede ser monitoreada por microscopía de fluorescencia para detectar la expresión de GFP o expresión Gluc en TCS. Rescate puede ser también confirmado por la expresión de Gluc en TCS. Para valorar los virus trisegmented, seguimos los pasos 2.1 a 2.1.4, pero las células no necesitan be fija para determinar un rescate viral éxito debido a la expresión de GFP. La titulación del virus se determinó contando el FFU / ml. Para determinar rescate viral con éxito por la expresión Gluc, expresión Gluc de TCS se puede medir usando un kit de ensayo de Gluc (Biolux Gaussia kit de ensayo de luciferasa, New England Biolabs). Para ello: <ol><li> Pipetear 100 l de TCS en una placa de 96 pozos (placas de microtitulación de fondo plano, Fisher Scientific). </li><li> Configure el luminómetro (LumiCount, Packard Biosciences). </li><li> Añadir 50 - 100 l solución de ensayo Gluc (según lo recomendado por el fabricante) a cada muestra y medir la expresión del gen informador Gluc con el luminómetro. Como control negativo, se midió la expresión de Gluc el TCS de los virus de tipo salvaje células infectadas. </li></ol></li></ol> 3. Paso de sobrenadantes de cultivo de tejidos Rescate viral depende de las eficacias de transfección. Células Vero se ha demostradoa tener menores eficiencias de transfección que otras líneas de células 14. Si los títulos de virus en el TCS son bajos, infectar células Vero frescas a una multiplicidad de infección (MOI) de 0,01 (LCMV) o 0.1 (Candid # 1) durante 72 horas para amplificar el virus.

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+the+prototypic+Arenavirus+LCMV+entirely+from+plasmid.">Rescue of the prototypic Arenavirus LCMV entirely from plasmid.</a> Virology. 350, 370-380 (2006).</li></ol>

Generación de arenavirus recombinantes utilizando técnicas de genética inversa basadas en plásmidos ha convertido en un método ampliamente utilizado para la investigación de muchos aspectos diferentes de la biología arenavirus. Aquí se documenta una mejora fundamental en el sistema actual mediante la realización de arenavirus rescata en células Vero, lo que permite la generación potencial de las vacunas aprobadas por la FDA contra el arenavirus y vectores de vacunas contra otras enfermedades infecciosas. ...

Los arenavirus son virus envueltos con un bisegmented, genoma de ARN de cadena negativa 3 que pertenecen a la familia Arenaviridae. El genoma arenavirus codifica cuatro proteínas de una manera ambisense a partir de dos segmentos virales separadas 3. El segmento grande (L) codifica la ARN polimerasa dependiente de ARN (L) y el anillo pequeño (realmente interesante nuevo gen) de proteínas dedo (Z), que sirve como la principal fuerza impulsora del virus en ciernes. El pequeño segmento (S) codifica la nucleoproteína viral (NP) y la glicoproteína de superficie (GP) (Figura 1). Varios miembros de la familia Arenaviridae son responsables de la fiebre hemorrágica letal (HF) en los seres humanos 3. De las preocupaciones principales son el virus de Lassa (LASV) y el virus Junín (JUNV), que se sabe que causa una alta mortalidad en los pacientes hospitalizados, 8, 9. A pesar de estos virus son endémicos de África occidental y rural Argentina, respectivamente,hay cada vez más preocupación de importación de LASV y JUNV a zonas no endémicas debido al aumento de los viajes 10. Además, a pesar de que normalmente no se asocia con una enfermedad grave en el hombre, el arenavirus prototípico virus de la coriomeningitis linfocítica (LCMV) se considera un patógeno descuidado ya que ha habido casos de infección letal en pacientes inmunodeprimidos 6, 15 y es responsable de los defectos congénitos y abortos espontáneos en mujeres embarazadas 2, 13. Actualmente no hay EE.UU. FDA aprobó las vacunas contra arenavirus y el tratamiento se limita al análogo de nucleósido ribavirina, que es sólo parcialmente efectiva ya menudo asociado con efectos secundarios significativos. La introducción de la marcha atrás plásmido basado en la genética 4 y la generación de arenavirus recombinantes 7, 19 han avanzado en gran medida el campo de la investigación arenavirus. Actualmente, las células de roedores (tales como células BHK-21) se utilizan para la Generación de arenavirus recombinantes debido a la ARN polimerasa I murino específico de la especie (pol-I) promotor que dirige la transcripción inicial de la S y L segmentos. Sin embargo rescate del virus en las células BHK-21 no son un método aprobado para la generación de arenavirus recombinantes como posibles candidatos vacunales de siembra. Aquí se documenta el uso de la pol-I promotor humano de rescate eficiente del Viejo Mundo prototípico (OW) LCMV y el Nuevo Mundo (NW) JUNV Candid # 1 cepa de células Vero. Usando una metodología similar, hemos generado recombinante LCMV trisegmented (r3LCMV) y Candid # 1 (r3Candid # 1) arenavirus que contienen dos genes extraños adicionales codificados en dos segmentos de ARN diferentes S 5. Este nuevo sistema no sólo sigue un protocolo simple y altamente reproducible, pero se puede implementar inmediatamente en la generación de arenavirus recombinantes como vacuna potencial o semillas de vector de vacuna.

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

El desarrollo e implementación de arenavirus genética inversa representa un avance significativo en el campo arenavirus 4. El uso de los sistemas de minigenoma arenavirus basados ​​en células, junto con la capacidad de generar recombinantes arenavirus infecciosas con mutaciones predeterminadas en sus genomas ha facilitado la investigación de la contribución de los determinantes virales para las diferentes etapas del ciclo de vida arenavirus, así como virus-huésped interacciones y mecanismos de patogénesis arenavirus 1, 3, 11. Además, el desarrollo de arenavirus trisegmented ha permitido el uso del genoma arenavirus para expresar genes extraños de interés adicionales, abriendo así la posibilidad de aplicaciones de vectores de vacunas basados ​​en arenavirus 5. Del mismo modo, el desarrollo de un solo ciclo de arenavirus infecciosos capaces de expresar genes indicadores proporciona una nueva herramienta experimental para mejorar la seguridad de la investigación con highlŸ arenavirus patógenos humanos 16. La generación de arenavirus recombinantes utilizando técnicas de genética inversa basadas en plásmidos ha basado hasta ahora en el uso de líneas celulares de roedores 7,19, lo que plantea algunas de las barreras para el desarrollo de la Administración de Alimentos y Medicamentos (FDA) con licencia de vacuna o vectores de vacuna. Para superar este obstáculo, como se describe aquí la generación eficiente de los arenavirus recombinantes en células Vero aprobados por la FDA.

Rescate exitoso de un arenavirus de tipo salvaje recombinante se confirmó por la presencia de antígenos virales mediante IFA (Figura 5). En el caso de los virus recombinantes trisegmented, rescate viral exitosa puede evaluarse mediante la observación de la expresión de GFP mediante microscopía de fluorescencia (Figura 6A). Rescate exitoso será confirmado por la evaluación de la expresión Gluc (Figura 6B). Los resultados representativos que ilustran el rescate co...

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

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

Rescate de arenavirus recombinantes a partir de ADNc clonado, un enfoque conocido como la genética inversa, permite a los investigadores estudiar la función de los productos específicos de genes virales, así como la contribución de los diferentes dominios y residuos específicos, a diferentes aspectos de la biología de los arenavirus . Del mismo modo, las técnicas de genética inversa en líneas celulares aprobados por la FDA (Vero) para el desarrollo de vacunas ofrece nuevas posibilidades para la generación de vacunas seguras y eficaces para combatir arenavirus patógenos humanos.

Generación de Arenavirus recombinante para el Desarrollo de Vacunas de la FDA aprobados células Vero

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

generation-recombinant-arenavirus-for-vaccine-development-fda

Research

JoVE Journal

Immunology and Infection

16.8K vistas.  University of Rochester School of Medicine and Dentistry. Rescate de arenavirus recombinantes a partir de ADNc clonado, un enfoque conocido como la genética inversa, permite a los investigadores estudiar la función de los productos específicos de genes virales, así como la contribución de los diferentes dominios y residuos específicos, a diferentes aspectos de la biología de los arenavirus . Del mismo modo, las técnicas de genética inversa en líneas celulares aprobados por la FDA (Vero) para el desarrollo de vacunas of...

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

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

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

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

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

Aislamiento de linfocitos T humanos, cultura y análisis de la migración In Vitro</em

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

Investigación

Inmunología e Infección

Generation of Recombinant Influenza Virus from Plasmid DNA

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

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

Generación de recombinantes del virus de influenza a partir del ADN plásmido

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

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

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

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

Generación de una nueva vacuna de células dendríticas usando melanoma y células madre escamosas del cáncer

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

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

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

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

Usando Click Química para medir el efecto de la infección viral en el Host-Cell ARN Síntesis

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

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

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

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

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

El análisis in vitro de la respuesta inmune celular Myd88 mediada por el Virus del Nilo Occidental cepa mutante Infección

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

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

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

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

La utilización de la Estrategia de la cápside-Incorporación Antigen para el desarrollo de enfoques adenovirus serotipo 5-Vectored de vacunas

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

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

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

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

Generación de recombinante humano IgG anticuerpos monoclonales a partir de células inmortalizadas B Ordenada

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

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

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

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

Ensayo muy sensible para la medición del Adjunto de células arenavirus

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

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

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

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

Generación, amplificación y titulación de virus sincitiales respiratorios recombinantes

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

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

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

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

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

Producción de escamas libres de células y adición adyuvante a una proteína principal recombinante de membrana externa de Chlamydia muridarum para el desarrollo de vacunas