Evaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidates

Foot-and-mouth disease (FMD) is one of the most contagious veterinary viral diseases known, having economic, social and potentially devastating environmental impacts. The vaccines currently being marketed/sold around the world for disease control and prevention in bovines do not stimulate the produc...

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Autores:
Avendaño, Catalina
Celis Giraldo, Carmen Teresa
Ordóñez, Diego
Díaz Arévalo, Diana
Rodríguez Habibe, Ibett
Oviedo, Jairo
Curtidor, Hernando
García Castiblanco, Sebastián
Tipo de recurso:
Article of journal
Fecha de publicación:
2020
Institución:
Universidad de Ciencias Aplicadas y Ambientales U.D.C.A
Repositorio:
Repositorio Institucional UDCA
Idioma:
eng
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oai:repository.udca.edu.co:11158/3222
Acceso en línea:
https://www.scopus.com/search/form.uri?display=basic
Palabra clave:
A24 Cruzeiro serotype
Capsid proteins
Foot-and-mouth disease virus
O1 Campos serotype
Synthetic peptide
Péptidos sintéticos
Fiebre aftosa
Proteínas
Vacuna
Serotipos
Rights
openAccess
License
Derechos Reservados - Universidad de Ciencias Aplicadas y Ambientales
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dc.title.spa.fl_str_mv Evaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidates
title Evaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidates
spellingShingle Evaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidates
A24 Cruzeiro serotype
Capsid proteins
Foot-and-mouth disease virus
O1 Campos serotype
Synthetic peptide
Péptidos sintéticos
Fiebre aftosa
Proteínas
Vacuna
Serotipos
title_short Evaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidates
title_full Evaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidates
title_fullStr Evaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidates
title_full_unstemmed Evaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidates
title_sort Evaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidates
dc.creator.fl_str_mv Avendaño, Catalina
Celis Giraldo, Carmen Teresa
Ordóñez, Diego
Díaz Arévalo, Diana
Rodríguez Habibe, Ibett
Oviedo, Jairo
Curtidor, Hernando
García Castiblanco, Sebastián
dc.contributor.author.spa.fl_str_mv Avendaño, Catalina
Celis Giraldo, Carmen Teresa
Ordóñez, Diego
Díaz Arévalo, Diana
Rodríguez Habibe, Ibett
Oviedo, Jairo
Curtidor, Hernando
García Castiblanco, Sebastián
dc.subject.proposal.spa.fl_str_mv A24 Cruzeiro serotype
Capsid proteins
Foot-and-mouth disease virus
O1 Campos serotype
Synthetic peptide
topic A24 Cruzeiro serotype
Capsid proteins
Foot-and-mouth disease virus
O1 Campos serotype
Synthetic peptide
Péptidos sintéticos
Fiebre aftosa
Proteínas
Vacuna
Serotipos
dc.subject.agrovoc.spa.fl_str_mv Péptidos sintéticos
Fiebre aftosa
Proteínas
Vacuna
Serotipos
description Foot-and-mouth disease (FMD) is one of the most contagious veterinary viral diseases known, having economic, social and potentially devastating environmental impacts. The vaccines currently being marketed/sold around the world for disease control and prevention in bovines do not stimulate the production of antibodies having crossed reactions to different serotypes. This means that if an animal becomes infected by a serotype which has not been included in a vaccine then it will develop the disease. Synthetic peptide vaccines represent a safer option and (depending on the design) can stimulate antibodies protecting against different variants. Based on the forgoing, this work was aimed at evaluating FMDV VP1, VP2 and VP3 protein-derived, modified and chemically-synthesised peptides’ ability to induce an immune response for developing a vaccine contributing towards controlling the disease. VP1, VP2 and VP3 proteins’ conserved regions were selected for this. Peptides from these regions were chemically synthesised; binding assays were then carried out for ascertaining whether they were involved in BHK-21 cell binding. Selected peptides’ structure and location were studied. Peptides which did bind were modified and formulated with Montanide ISA 70 adjuvant; 17 animals were immunised twice with the formulation. The animals were genotyped by amplifying the BoLA-DRB3.2 gene. Blood samples were taken from 17 cattle on day 43 post-first immunisation for studying the formulation's immunogenicity. The sera were used in ELISA, immunofluorescence, flow cytometry, immunoadsorption and seroneutralisation assays. The A24 Cruzeiro and O1 Campos virus serotypes were used for these assays. The results revealed that even though protein exposure and 3D structure might be different amongst serotypes, the antibodies so produced could inhibit virus entry to cells, thereby showing the selected peptides’ in vitro protection-inducing ability.
publishDate 2020
dc.date.accessioned.spa.fl_str_mv 2020-05-04T23:20:42Z
dc.date.available.spa.fl_str_mv 2020-05-04T23:20:42Z
dc.date.issued.spa.fl_str_mv 2020
dc.type.spa.fl_str_mv Artículo de revista
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dc.identifier.citation.spa.fl_str_mv Avendaño, C., Celis-Giraldo, C., Ordoñez, D., Díaz-Arévalo, D., Rodríguez-Habibe, I., Oviedo, J., Curtidor, H., García-Castiblanco, S., Martínez-Panqueva, F., Camargo-Castañeda, A., Reyes, C., Bohórquez, M.D., Vanegas, M., Cantor, D., Patarroyo, M.E., Patarroyo, M.A. Evaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidates (2020) Vaccine, 38 (23), pp. 3942-3951.
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dc.identifier.doi.spa.fl_str_mv 10.1016/j.vaccine.2020.04.006
identifier_str_mv Avendaño, C., Celis-Giraldo, C., Ordoñez, D., Díaz-Arévalo, D., Rodríguez-Habibe, I., Oviedo, J., Curtidor, H., García-Castiblanco, S., Martínez-Panqueva, F., Camargo-Castañeda, A., Reyes, C., Bohórquez, M.D., Vanegas, M., Cantor, D., Patarroyo, M.E., Patarroyo, M.A. Evaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidates (2020) Vaccine, 38 (23), pp. 3942-3951.
10.1016/j.vaccine.2020.04.006
url https://www.scopus.com/search/form.uri?display=basic
dc.language.iso.spa.fl_str_mv eng
language eng
dc.relation.ispartofseries.spa.fl_str_mv Vaccine;Vol.38, No.23, May 13, 2020, páginas 3942-3951
dc.relation.indexed.spa.fl_str_mv Agricultura
dc.rights.spa.fl_str_mv Derechos Reservados - Universidad de Ciencias Aplicadas y Ambientales
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spelling Avendaño, CatalinaCelis Giraldo, Carmen TeresaOrdóñez, DiegoDíaz Arévalo, DianaRodríguez Habibe, IbettOviedo, JairoCurtidor, HernandoGarcía Castiblanco, Sebastián2020-05-04T23:20:42Z2020-05-04T23:20:42Z2020Avendaño, C., Celis-Giraldo, C., Ordoñez, D., Díaz-Arévalo, D., Rodríguez-Habibe, I., Oviedo, J., Curtidor, H., García-Castiblanco, S., Martínez-Panqueva, F., Camargo-Castañeda, A., Reyes, C., Bohórquez, M.D., Vanegas, M., Cantor, D., Patarroyo, M.E., Patarroyo, M.A. Evaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidates (2020) Vaccine, 38 (23), pp. 3942-3951.https://www.scopus.com/search/form.uri?display=basic10.1016/j.vaccine.2020.04.006Foot-and-mouth disease (FMD) is one of the most contagious veterinary viral diseases known, having economic, social and potentially devastating environmental impacts. The vaccines currently being marketed/sold around the world for disease control and prevention in bovines do not stimulate the production of antibodies having crossed reactions to different serotypes. This means that if an animal becomes infected by a serotype which has not been included in a vaccine then it will develop the disease. Synthetic peptide vaccines represent a safer option and (depending on the design) can stimulate antibodies protecting against different variants. Based on the forgoing, this work was aimed at evaluating FMDV VP1, VP2 and VP3 protein-derived, modified and chemically-synthesised peptides’ ability to induce an immune response for developing a vaccine contributing towards controlling the disease. VP1, VP2 and VP3 proteins’ conserved regions were selected for this. Peptides from these regions were chemically synthesised; binding assays were then carried out for ascertaining whether they were involved in BHK-21 cell binding. Selected peptides’ structure and location were studied. Peptides which did bind were modified and formulated with Montanide ISA 70 adjuvant; 17 animals were immunised twice with the formulation. The animals were genotyped by amplifying the BoLA-DRB3.2 gene. Blood samples were taken from 17 cattle on day 43 post-first immunisation for studying the formulation's immunogenicity. The sera were used in ELISA, immunofluorescence, flow cytometry, immunoadsorption and seroneutralisation assays. The A24 Cruzeiro and O1 Campos virus serotypes were used for these assays. The results revealed that even though protein exposure and 3D structure might be different amongst serotypes, the antibodies so produced could inhibit virus entry to cells, thereby showing the selected peptides’ in vitro protection-inducing ability.application/pdfengVaccine;Vol.38, No.23, May 13, 2020, páginas 3942-3951AgriculturaDerechos Reservados - Universidad de Ciencias Aplicadas y Ambientaleshttps://creativecommons.org/licenses/by-nc-sa/4.0/info:eu-repo/semantics/openAccessAtribución-NoComercial-CompartirIgual 4.0 Internacional (CC BY-NC-SA 4.0)http://purl.org/coar/access_right/c_abf2https://www.scopus.com/search/form.uri?display=basichttps://udca.elogim.com:2119/science/article/pii/S0264410X20304643?via%3DihubEvaluating the immunogenicity of chemically-synthesised peptides derived from foot-and-mouth disease VP1, VP2 and VP3 proteins as vaccine candidatesArtículo de 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 nombre de la parte o las partes que el Autor Original y/o el Licenciante hubieren designado para la atribución (v.g., un instituto patrocinador, editorial, publicación) en la información de los derechos de autor del Licenciante, términos de servicios o de otras formas razonables; el título de la Obra si está provisto; en la medida de lo razonablemente factible y, si está provisto, el Identificador Uniforme de Recursos (Uniform Resource Identifier) que el Licenciante especifica para ser asociado con la Obra, salvo que tal URI no se refiera a la nota sobre los derechos de autor o a la información sobre el licenciamiento de la Obra; y en el caso de una Obra Derivada, atribuir el crédito identificando el uso de la Obra en la Obra Derivada (v.g., "Traducción Francesa de la Obra del Autor Original," o "Guión Cinematográfico basado en la Obra original del Autor Original"). Tal crédito puede ser implementado de cualquier forma razonable; en el caso, sin embargo, de Obras Derivadas u Obras Colectivas, tal crédito aparecerá, como mínimo, donde aparece el crédito de cualquier otro autor comparable y de una manera, al menos, tan destacada como el crédito de otro autor comparable.
d.	Para evitar toda confusión, el Licenciante aclara que, cuando la obra es una composición musical:
i.	Regalías por interpretación y ejecución bajo licencias generales. El Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública o la ejecución pública digital de la obra y de recolectar, sea individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, SAYCO), las regalías por la ejecución pública o por la ejecución pública digital de la obra (por ejemplo Webcast) licenciada bajo licencias generales, si la interpretación o ejecución de la obra está primordialmente orientada por o dirigida a la obtención de una ventaja comercial o una compensación monetaria privada.
ii.	Regalías por Fonogramas. El Licenciante se reserva el derecho exclusivo de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, SAYCO), una agencia de derechos musicales o algún agente designado, las regalías por cualquier fonograma que Usted cree a partir de la obra (“versión cover”) y distribuya, en los términos del régimen de derechos de autor, si la creación o distribución de esa versión cover está primordialmente destinada o dirigida a obtener una ventaja comercial o una compensación monetaria privada.
e.	Gestión de Derechos de Autor sobre Interpretaciones y Ejecuciones Digitales (WebCasting). Para evitar toda confusión, el Licenciante aclara que, cuando la obra sea un fonograma, el Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública digital de la obra (por ejemplo, webcast) y de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, Acinpro), las regalías por la ejecución pública digital de la obra (por ejemplo, webcast), sujeta a las disposiciones aplicables del régimen de Derecho de Autor, si esta ejecución pública digital está primordialmente dirigida a obtener una ventaja comercial o una compensación monetaria privada.
5. Representaciones, Garantías y Limitaciones de Responsabilidad.
A MENOS QUE LAS PARTES LO ACORDARAN DE OTRA FORMA POR ESCRITO, EL LICENCIANTE OFRECE LA OBRA (EN EL ESTADO EN EL QUE SE ENCUENTRA) “TAL CUAL”, SIN BRINDAR GARANTÍAS DE CLASE ALGUNA RESPECTO DE LA OBRA, YA SEA EXPRESA, IMPLÍCITA, LEGAL O CUALQUIERA OTRA, INCLUYENDO, SIN LIMITARSE A ELLAS, GARANTÍAS DE TITULARIDAD, COMERCIABILIDAD, ADAPTABILIDAD O ADECUACIÓN A PROPÓSITO DETERMINADO, AUSENCIA DE INFRACCIÓN, DE AUSENCIA DE DEFECTOS LATENTES O DE OTRO TIPO, O LA PRESENCIA O AUSENCIA DE ERRORES, SEAN O NO DESCUBRIBLES (PUEDAN O NO SER ESTOS DESCUBIERTOS). ALGUNAS JURISDICCIONES NO PERMITEN LA EXCLUSIÓN DE GARANTÍAS IMPLÍCITAS, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.
6. Limitación de responsabilidad.
A MENOS QUE LO EXIJA EXPRESAMENTE LA LEY APLICABLE, EL LICENCIANTE NO SERÁ RESPONSABLE ANTE USTED POR DAÑO ALGUNO, SEA POR RESPONSABILIDAD EXTRACONTRACTUAL, PRECONTRACTUAL O CONTRACTUAL, OBJETIVA O SUBJETIVA, SE TRATE DE DAÑOS MORALES O PATRIMONIALES, DIRECTOS O INDIRECTOS, PREVISTOS O IMPREVISTOS PRODUCIDOS POR EL USO DE ESTA LICENCIA O DE LA OBRA, AUN CUANDO EL LICENCIANTE HAYA SIDO ADVERTIDO DE LA POSIBILIDAD DE DICHOS DAÑOS. ALGUNAS LEYES NO PERMITEN LA EXCLUSIÓN DE CIERTA RESPONSABILIDAD, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.
7. Término.
a.	Esta Licencia y los derechos otorgados en virtud de ella terminarán automáticamente si Usted infringe alguna condición establecida en ella. Sin embargo, los individuos o entidades que han recibido Obras Derivadas o Colectivas de Usted de conformidad con esta Licencia, no verán terminadas sus licencias, siempre que estos individuos o entidades sigan cumpliendo íntegramente las condiciones de estas licencias. Las Secciones 1, 2, 5, 6, 7, y 8 subsistirán a cualquier terminación de esta Licencia.
b.	Sujeta a las condiciones y términos anteriores, la licencia otorgada aquí es perpetua (durante el período de vigencia de los derechos de autor de la obra). No obstante lo anterior, el Licenciante se reserva el derecho a publicar y/o estrenar la Obra bajo condiciones de licencia diferentes o a dejar de distribuirla en los términos de esta Licencia en cualquier momento; en el entendido, sin embargo, que esa elección no servirá para revocar esta licencia o que deba ser otorgada , bajo los términos de esta licencia), y esta licencia continuará en pleno vigor y efecto a menos que sea terminada como se expresa atrás. La Licencia revocada continuará siendo plenamente vigente y efectiva si no se le da término en las condiciones indicadas anteriormente.
8. Varios.
a.	Cada vez que Usted distribuya o ponga a disposición pública la Obra o una Obra Colectiva, el Licenciante ofrecerá al destinatario una licencia en los mismos términos y condiciones que la licencia otorgada a Usted bajo esta Licencia.
b.	Si alguna disposición de esta Licencia resulta invalidada o no exigible, según la legislación vigente, esto no afectará ni la validez ni la aplicabilidad del resto de condiciones de esta Licencia y, sin acción adicional por parte de los sujetos de este acuerdo, aquélla se entenderá reformada lo mínimo necesario para hacer que dicha disposición sea válida y exigible.
c.	Ningún término o disposición de esta Licencia se estimará renunciada y ninguna violación de ella será consentida a menos que esa renuncia o consentimiento sea otorgado por escrito y firmado por la parte que renuncie o consienta.
d.	Esta Licencia refleja el acuerdo pleno entre las partes respecto a la Obra aquí licenciada. No hay arreglos, acuerdos o declaraciones respecto a la Obra que no estén especificados en este documento. El Licenciante no se verá limitado por ninguna disposición adicional que pueda surgir en alguna comunicación emanada de Usted. Esta Licencia no puede ser modificada sin el consentimiento mutuo por escrito del Licenciante y Usted.

