Este estudio fue financiado por los Institutos Nacionales de Salud (NIH) de los Estados Unidos (RO1 AI68002).

1. Recombinante SARS-CoV RBD preparación de proteínas <ol><li> Prepare fosfato de calcio reactivo de transfección <ol><li> 2X preparación de buffer HBS: Mezcle 16 g de NaCl, 0,4 g de Na 2 HPO 4 * 7H 2 O, y 13.0 g de HEPES. Ajustar el pH a 7,00 y criar a un volumen total de 1.000 ml de agua destilada. Después de filtrar la solución para la esterilización, alícuota y almacenar a -20 ° C. Sugerencia: Cualquier variación del valor de pH podría afectar los resultados de la transfección. Por lo tanto, es aconsejable probar varios valores de pH alrededor de 7,00 (por ejemplo, 6,99, 7,00 o 7,01) y encontrar el mejor para la transfección utilizando el método se presentan a continuación. </li><li> 2,5 M CaCl 2 Preparación: Añadir 73,5 g de CaCl 2 * 2H 2 O en agua destilada en un volumen final de 200 ml. Filtrar la solución y almacenar a -20 ° C. </li></ol></li><li> Transfección del plásmido recombinante y purificación de proteínas <ol><li> Dividir las células 293T en 50-70% de confluencia de 24 horas antes de la transfección. Se cultivan las células en la T-175 cm 2 frascos de cultivo de tejidos de 40 ml de DMEM con 10% inactivado por calor (HI), FBS y 1% de penicilina / estreptomicina (P / S) a 37 ° C en 5% de CO 2. </li><li> Todos los reactivos de transfección deben ser llevados a la temperatura ambiente antes de la transfección. Estos reactivos son 2X HBS, 2,5 M CaCl 2, DIH 2 O, y rRBD plásmido 6. Sugerencia: El plásmido recombinante final de construcción utilizada para la expresión de la proteína debe contener un péptido señal para garantizar la secreción de proteínas recombinantes expresadas en el sobrenadante del cultivo. A 6x Su etiqueta se puede agregar a la C-terminal de la proteína expresada para la purificación fácil. </li><li> Prepare un 50 mL (A) y uno de 15 ml (B) BD tubo Falcon, y añadir los reactivos a los tubos como se indica en la Tabla 1. Añadir 2X tampón HBS para A. tubo en tubo B, añadir 2,5 M CaCl 2 y rRBD plásmido, y llevar el volumen a la exigencia en el DIH 2 O. <table border="1"><tbody><tr><td colspan="4"> A. Una T-175 cm 2 frasco de cultivo de tejidos (4000 l / frasco): prepararse para la expresión rRBD </td></tr><tr><td colspan="2"> Un tubo </td><td colspan="2"> Tubo B </td></tr><tr><td> 2X EPF </td><td> 2000 l </td><td> 2,5 M de CaCl2 </td><td> 200 l </td></tr><tr><td></td><td></td><td> ADN plásmido rRBD </td><td> 40 g </td></tr><tr><td></td><td></td><td> DIH 2 O a </td><td> 2000 l </td></tr><tr><td colspan="4"> B. Una de 100 mm de una caja de petri (1000 l / placa): prepararse para la producción pseudovirus SRAS </td></tr><tr><td colspan="2"> Un tubo </td><td colspan="2"> Tubo B </td></tr><tr><td> 2X EPF </td><td> 500 l </td><td> 2,5 M de CaCl2 </td><td> 50 L </td></tr><tr><td></td><td></td><td> ADN plásmido SARS-CoV S </td><td> 5 mg </td></tr><tr><td></td><td></td><td> VIH-1 plásmido (pNL4-3.luc.RE) </td><td> 5 mg </td></tr><tr><td></td><td></td><td> DIH 2 O a </td><td> 500 l </td></tr></tbody></table> Tabla 1. Transfección preparación de la mezcla y los volúmenes Los volúmenes que figuran en el Cuadro 1 se para la unidad de la transfección uno. Si hay más botellas o platos se utilizan para la transfección, ajustar el volumen en consecuencia. </li><li> Añadir la solución de ADN-B del calcio en el tubo en el tubo de una manera gota a gota, mientras se mantiene constante y suave mezcla en un torbellino. Deja reposar la mezcla a temperatura ambiente durante 20-30 min. La clave del éxito para la transfección de fosfato de calcio depende del tamaño y la forma del precipitado formado. Por lo tanto, la mezcla en un vórtex constantemente y poco a poco para cumplir con este requisito y, en consecuencia, mejorar la eficacia de transfección. </li><li> Agregue la mezcla de una manera gota a gota, e incluso en las células 293T (4000 l / frasco). Las células de cultivo en la incubadora a 37 ° C en 5% de CO 2. </li><li> Vuelva a colocar el medio de cultivo con suero fresco libre de OPTI-MEM I reducido Serum Medium (50 ml / frasco) 80 a 10 h después de la transfección. Continuar con las células de cultivo para dos días más en las mismas condiciones. </li><li> Recoger el líquido sobrenadante que contiene la proteína expresada rRBD 72 h después de la transfección. Centrifugar a 6000 rpm durante 15 minutos para eliminar los desechos celulares. Añadir un cóctel de inhibidores de proteasa para el sobrenadante se recogió y se almacenan a 4 ° C durante la noche. </li><li> Al día siguiente, purificar la proteína recombinante rRBD del sobrenadante del cultivo siguiendo las instrucciones del fabricante de Ni-NTA SuperFlow siguiente. </li><li> Concentrado de proteína purificada usando Amicon Ultra tubos de concentración de -15. Después de la concentración de proteínas,añadir PBS a la concentración de los tubos y centrifugar de nuevo para eliminar imidazol en tampón de elución. Calcular la concentración de proteínas y almacenar la proteína purificada a -80 ° C hasta su uso. </li></ol></li></ol> 2. La inmunización del ratón y Toma de Muestras <ol><li> Pre-caliente el sistema Sigma adyuvante (SAS) a 40-45 ° C de acuerdo con las instrucciones del fabricante. Añadir 1 ml de PBS por vial y mezclar bien. </li><li> Prepare la proteína-adyuvante emulsión de acuerdo con el protocolo en la Tabla 2. Pipeta calcula rRBD proteína en un tubo de 1,5 ml. Añadir un volumen igual de adyuvante SAS en el tubo y agitar vigorosamente durante 2-3 minutos para formar la emulsión. Sugerencia: Los volúmenes que figuran en el Cuadro 2 son para un ratón. Ajustar los volúmenes de acuerdo con los números del ratón real utilizado. Para cada grupo, mezclar las proteínas y adyuvante para cada vacuna. Siempre prepare una muestra adicional de la vacuna para asegurar la exactitud. <table border="1"><tbody><tr><td> Grupo </td><td> 1 de la vacuna (200 l / ratón) </td><td> 2 de la vacuna (200 l / ratón) </td><td> 3 de la vacuna (200 l / ratón) </td></tr><tr><td> rRBD proteínas </td><td> 20 mg de proteína en PBS (100 l) + 100 l SAS </td><td> 10 g de proteína en PBS (100 l) + 100 l SAS </td><td> 10 g de proteína en PBS (100 l) + 100 l SAS </td></tr><tr><td> PBS de control </td><td> 100 L de PBS + 100 L SAS </td><td> 100 L de PBS + 100 L SAS </td><td> 100 L de PBS + 100 L SAS </td></tr></tbody></table> Tabla 2. Ratón protocolo de inmunización </li><li> Por vía subcutánea estelar inmunizar a las mujeres ratones Balb / c (4-6 semanas de edad, 5 ratones / grupo), y aumentar dos veces con rRBD SAS y como se indica en la Tabla 2. Use grupo PBS como control. El sitio de las inyecciones subcutáneas generalmente elegido es la piel suelta entre los omoplatos. Por otra parte, la parte ventral del abdomen es de uso común, porque es más fácil de inyectar, y puede observar el líquido de la inyección. Cuando las dosis repetidas de las vacunas se utilizan, es fácil para seleccionar varios sitios de inyección, evitando posibles reacciones cutáneas locales. </li><li> Sangrar ratones a través de retro-orbital con anestesia antes de la inmunización y 10 días después de cada vacunación, y el calor los sueros a 56 ° C durante 30 min para inactivar el complemento. Almacenar sueros de ratón a -20 ° C hasta su uso. </li></ol> 3. La neutralización de detección mediante ensayo de neutralización Pseudovirus <ol><li> Prepare pseudovirus SRAS <ol><li> Dividir las células 293T en 100 mm de cultivo de tejidos placas de Petri (2x10 6 células / placa) 16 h antes de la transfección, las células crecen y que el anterior. </li><li> Preparar los reactivos de transfección, como se indica en la Tabla 1. Proteínas co-transfectar un plásmido que codifica el SARS-CoV S y un plásmido que codifica Env defectuoso, la luciferasa que expresan el VIH-1 del genoma (pNL4-3.luc.RE) con fosfato de calcio reactivo de transfección. </li><li> Vuelva a colocar medio con 10 ml DMEM con 10% de SFB y 1% P / S 08.10 h post-transfección. Recoger el líquido sobrenadante pseudovirus para contener el SRAS 72 h después de la transfección. </li><li> Pseudovirus filtro a través de un filtro de 0,45 micras. Alícuota y almacenar a -80 ° C hasta su uso. </li></ol></li><li> Pseudovirus SRAS ensayo de neutralización <ol><li> Dividir las células 293T expresando el SARS-CoV receptor ACE2 (ACE2/293T) a 10 4 células/100 l / pocillo en placas de 96 pocillos de cultivo de tejidos de 16 horas antes de la infección. </li><li> Diluir pseudovirus SRAS por dos veces para detectar el título del virus en las células ACE2/293T. </li><li> Serie diluir sueros de ratón en placas de 96 pocillos de cultivo de tejidos, y añadir un volumen igual de pseudovirus titulada SRAS. Preincubación de las placas a 37 ° C durante 1 h. </li><li> Después de la incubación, añadir 100 ml de la mezcla de suero-pseudovirus a las células ACE2/293T, y continúan creciendo las células a 37 ° C en 5% de CO 2. Añadir DMEM 24 horas más tarde. </li><li> Eliminar por completo la cultura sobrenadantes de las placas 72 horas después de la infección. Añadir 1X cultura luciferasa de células reactivo de lisis (60 l / pocillo), y promover la lisis celular con agitación constante de las placas durante 1-2 horas a temperatura ambiente. </li><li> Lisados ​​de la transferencia de células (50 l / pocillo) en placas de luminómetro (Microfluor de 96 pocillos). Añadir el sustrato de luciferasa (50 l / pocillo) incluidos en el sistema de ensayo de luciferasa, y detectar la actividad luciferasa relativa en Ultra 384 luminómetro. </li><li> Calcular pseudovirus SRAS título de neutralización y se presentan como título de anticuerpos neutralizantes del 50% (NT 50) 6. </li></ol></li></ol>

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La densidad celular es un factor importante que afecta la eficacia de fosfato de calcio basado en la transfección. En nuestra experiencia, menos del 70% de confluencia de las células trae los mejores resultados. Por lo tanto, con el fin de mejorar la eficiencia de la transfección, se recomienda que la densidad celular se mantuvo en torno al 50-70% de confluencia. El fosfato de calcio método de transfección se piensa generalmente para ser menos eficiente en comparación con otros métodos de transfección, tales como la transfección de lípidos 16. Sin embargo, en este protocolo, el fosfato de calcio modificados método de transfección mediante el cual constante y lento la mezcla de la solución de transfección en un vórtice se asegura la formación de un precipitado con el tamaño y forma apropiada, mejorando así la eficiencia de transfección.

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

En base a su perfil de seguridad y capacidad de inducir potentes respuestas inmunes contra las infecciones, las vacunas de subunidades se han utilizado como candidatos para una amplia variedad de patógenos 3.1. Dado que el sistema de células de mamíferos es capaz de modificaciones post-traduccionales, formando así proteínas correctamente plegadas y glicosilada, proteínas recombinantes expresadas en células de mamíferos han mostrado el mayor potencial de mantener alta antigenicidad y la inmunogenicidad de 4-6. Aunque no hay nuevos casos de SARS se han reportado desde el año 2004, los brotes futuros son una amenaza constante, por lo tanto, el desarrollo de vacunas contra el SARS-CoV es un paso prudente y preventiva debe llevarse a cabo. La RBD de proteína SARS-CoV S juega un papel importante en la unión al receptor y la inducción de anticuerpos neutralizantes específicos contra la infección por el virus de 7-9. Por lo tanto, en este protocolo, se describen nuevos métodos para el desarrollo de una vacuna subunidad RBD basada contra el SARS. En pocas palabras, la proteína recombinante RBD (rRBD) se expresó en el sobrenadante del cultivo de células de mamíferos 293T para obtener una proteína correctamente plegada con la conformación adecuada y alta inmunogenicidad 6. La transfección del plásmido recombinante que codifica RBD a las células se realizó mediante un método de transfección de fosfato de calcio 6,10, con algunas modificaciones. En comparación con el método de transfección de lípidos 11,12, este fosfato de calcio modificados método de transfección es más barato, más fácil de manejar, y tiene el potencial para llegar a una alta eficacia una vez que un complejo de transfección con el tamaño y forma adecuados está formado 13,14. Por último, un ensayo de neutralización pseudovirus SRAS fue introducido en el protocolo y se utiliza para detectar la actividad neutralizante de los sueros de los ratones vacunados con la proteína rRBD. Este ensayo es relativamente seguro, no implica una enfermedad infecciosa SARS-CoV, y se puede realizar sin necesidad de un laboratorio de bioseguridad 3 15. El protocolo descrito aquí también se puede utilizar para el diseño y estudio de las vacunas recombinantes contra la subunidad otros virus con las proteínas de fusión de clase I, por ejemplo, el VIH, el virus respiratorio sincitial (VRS), el virus del Ébola, el virus de la influenza, así como los virus Nipah y Handra. Además, los métodos para la generación de un pseudovirus y, posteriormente, el establecimiento de un ensayo de neutralización pseudovirus se puede aplicar a todos estos virus.

Watch this Scientific Journal Video about Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection at JoVE.com

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

Este protocolo describe un procedimiento general para el estudio de unión al receptor recombinante de dominio (RBD) basado en vacunas de subunidades contra el SARS. Que incluye los métodos de transfección y expresión de la proteína en las células 293T RBD, la inmunización de ratones con RBD y la detección de la actividad de neutralización de los sueros de ratón utilizando un pseudovirus SRAS establecida una prueba de neutralización.

Protocolo para las vacunas recombinantes para RBD basada en el SRAS: Preparación de la proteína, la vacunación de animales y la detección de neutralización

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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Video: 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).

Combinación de adhesivo basado en cinta de muestreo y de fluorescencia In situ La hibridación para la detección rápida de Salmonella En los productos frescos

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

Investigación

Inmunología e Infección

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.

Imaging Whole-animal y de citometría de flujo Las técnicas para T CD8 + respuestas celulares Análisis de antígeno-específicos después de nanopartículas Vacunación

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.

Administración intranasal de vacunas contra la influenza recombinante en modelos de ratón quimérico para el Estudio de la mucosa Inmunidad

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.

En Vivo Ensayo para la detección de la función citolítica de células T específicas de antígeno utilizando un modelo de vacunación

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.

Generación de vectores recombinantes Herpesvirus aviar con CRISPR/Cas9 Gene edición

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.

Análisis de análogos de ácido iophenoxic en pequeña mangosta india (Herpestes Auropunctatus) Sera para su uso como un marcador biológico de vacunación de rabia oral

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 ensayo de neutralización igG e IgM SARS-CoV-2 de doble multiplex multiplexing para un sistema de análisis de flujo multiplexado basado en perlas

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.

Detección de anticuerpos de dominio de unión al receptor SARS-CoV-2 mediante un bioinformador basado en 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.

Imágenes en vivo y cuantificación de la infección viral en ratones transgénicos K18 hACE2 utilizando SARS-CoV-2 recombinante que expresa el reportero

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.

Detección de epítopos sensibles a la neutralización en antígenos mostrados en vacunas basadas en partículas similares a virus (VLP) utilizando un ensayo de captura

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 ...