RESUMO
Bovine respiratory syncytial virus (BRSV) affects both beef and dairy cattle, reaching morbidity and mortality rates of 60-80% and 20%, respectively. The aim of this study was to obtain a recombinant MVA expressing the BRSV F protein (MVA-F) as a vaccine against BRSV and to evaluate the immune response induced by MVA-F after systemic immunization in homologous and heterologous vaccination (MVA-F alone or combined with a subunit vaccine), and after intranasal immunization of mice. MVA-F administered by intraperitoneal route in a homologous scheme elicited levels of neutralizing antibodies similar to those obtained with inactivated BRSV as well as better levels of IFN-γ secretion. In addition, nasal administration of MVA-F elicited local and systemic immunity with a Th1 profile. This study suggests that MVA-F is a good candidate for further evaluations combining intranasal and intramuscular routes, in order to induce local and systemic immune responses, to improve the vaccine efficacy against BRSV infection.
Assuntos
Administração Intranasal , Camundongos Endogâmicos BALB C , Vírus Sincicial Respiratório Bovino , Animais , Vírus Sincicial Respiratório Bovino/imunologia , Camundongos , Feminino , Bovinos , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vetores Genéticos , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/veterinária , Vaccinia virus/imunologia , Vaccinia virus/genética , Anticorpos Antivirais/sangue , Imunidade nas Mucosas , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Imunização/métodos , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagemRESUMO
Modified vaccinia Ankara virus (MVA) is extensively used as a vaccine vector. We have previously observed that MVAΔ008, an MVA lacking the gene that codes for interleukin-18 binding protein, significantly increases CD8+ and CD4+ T-cell responses to vaccinia virus (VACV) epitopes and recombinant HIV antigens. However, the efficacy of this vector against pathogens or tumor cells remains unclear. Thus, the aim of this study was to evaluate the cellular immune response and the protection induced by recombinant MVAs encoding the model antigen ovalbumin (OVA). We used the MO5 melanoma tumor model (OVA-expressing tumor) as an approach for evaluating the vector-induced efficacy. Our results show that MVAΔ008-OVA (optimized vector) induced higher in vivo specific cytotoxicity and ex vivo T-cell IFN-γ responses against OVA than the conventional MVA vector. Importantly, the recombinant vectors were capable of controlling MO5 tumor growth. Indeed, the administration of MVAΔ008-OVA or MVA-OVA in prophylactic and therapeutic schemes provided total protection and longer survival of mice, respectively. Overall, our results demonstrate the improved immunogenicity and the protective capacity of MVAΔ008 against a heterologous model antigen. These findings suggest that MVAΔ008 constitutes an excellent candidate for vaccine development against pathogens or cancer therapy.
Assuntos
Melanoma Experimental/imunologia , Ovalbumina/imunologia , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Animais , Vetores Genéticos , Camundongos , Vacinas de DNARESUMO
Different immunogens such as subunit, DNA or live viral-vectored vaccines against Infectious Bursal Disease virus (IBDV) have been evaluated in the last years. However, the heterologous prime-boost approach using recombinant modified vaccinia Ankara virus (rMVA), which has shown promising results in both mammals and chickens, has not been tried against this pathogen yet. IBD is a highly contagious and immunosuppressive disease of poultry that affects mainly young chicks. It is caused by IBDV, a double-stranded RNA virus carrying its main antigenic epitopes on the capsid protein VP2. Our objective was to evaluate the immune response elicited by two heterologous prime-boost schemes combining an rMVA carrying the VP2 mature gene (rVP2) and a recombinant VP2 protein produced in Nicotiana benthamiana (pVP2), and to compare them with the performance of the homologous pVP2-pVP2 scheme usually used in our laboratory. The SPF chickens immunized with the three evaluated schemes elicited significantly higher anti-VP2 antibody titers (p<0.001) and seroneutralizing titers (p<0.05) and had less T-cell infiltration (p<0.001), histological damage (p<0.001) and IBDV particles (p<0.001) in their bursae of Fabricius when compared with control groups. No significant differences were found between both heterologous schemes and the homologous one. However, the rVP2-pVP2 scheme showed significantly higher anti-VP2 antibody titers than pVP2-rVP2 and a similar tendency was found in the seroneutralization assay. Conversely, pVP2-rVP2 had the best performance when evaluated through bursal parameters despite having a less potent humoral immune response. These findings suggest that the order in which rVP2 and pVP2 are combined can influence the immune response obtained. Besides, the lack of a strong humoral immune response did not lessen the ability to protect from IBDV challenge. Therefore, further research is needed to evaluate the mechanisms by which these immunogens are working in order to define the combination that performs better against IBDV.
Assuntos
Vírus da Doença Infecciosa da Bursa/imunologia , Vacinação/métodos , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Bolsa de Fabricius/patologia , Galinhas , Portadores de Fármacos/administração & dosagem , Vírus da Doença Infecciosa da Bursa/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Linfócitos T/imunologia , Nicotiana , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/metabolismo , Vaccinia virus/genética , Proteínas Estruturais Virais/administração & dosagem , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/isolamento & purificação , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/metabolismoAssuntos
Infecções por Birnaviridae/veterinária , Vírus da Varíola dos Canários/imunologia , Vírus da Doença Infecciosa da Bursa/genética , Doenças das Aves Domésticas/imunologia , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Antígenos Virais/imunologia , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/prevenção & controle , Bolsa de Fabricius/diagnóstico por imagem , Bolsa de Fabricius/patologia , Vírus da Varíola dos Canários/genética , Vírus da Varíola dos Canários/metabolismo , Galinhas , Vetores Genéticos , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/virologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas Estruturais Virais/biossíntese , Proteínas Estruturais Virais/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens.
Assuntos
Infecções por Birnaviridae/veterinária , Vírus da Varíola dos Canários/genética , Portadores de Fármacos , Vetores Genéticos , Doenças das Aves Domésticas/prevenção & controle , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/prevenção & controle , Galinhas , Doenças das Aves Domésticas/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas Estruturais Virais/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens.
Assuntos
Animais , Galinhas/virologia , Proteínas Virais , Vírus da Doença Infecciosa da Bursa , Vírus da Varíola dos CanáriosRESUMO
Development and preliminary assessment of a recombinant canarypox virus as an antirabic vaccine candidate. In Argentina, rabies is limited to some northern provinces. Availability of new vaccines abolishing the handling of the rabies virus and allowing disease control has regional and national strategic importance. Vaccines based on recombinant poxviruses have been successfully used as antirabic vaccines worldwide. Although these systems are not commercially available, the platform to obtain recombinant canarypox viruses (CNPV) has been previously set up in our laboratory. The aim of this work was the development and evaluation of an antirabic vaccine candidate based on recombinant CNPV expressing the rabies virus (RV) glycoprotein G (RG). A recombinant virus (CNPV-RG) expressing the RG coding sequence was designed. Inoculation of mice with this virus induced high RV seroneutralizing antibodies (3.58 and 9.76 IU/ml after 1 or 2 immunizations, respectively) and protected 78% of intracerebrally RV-challenged animals. In addition, it was determined that CNPV-RG has a relative potency of 3.5 IU/ml. The obtained results constituted the first stage of CNPV-RG evaluation as antirabic vaccine candidate. Further assays will be necessary to confirm its utility in species of veterinary interest.
Assuntos
Antígenos Virais/imunologia , Vírus da Varíola dos Canários/imunologia , Glicoproteínas/imunologia , Vacina Antirrábica , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Vírus da Varíola dos Canários/genética , Vírus da Varíola dos Canários/crescimento & desenvolvimento , Vírus da Varíola dos Canários/isolamento & purificação , Linhagem Celular/virologia , Embrião de Galinha , Chlorocebus aethiops , Cricetinae , Fibroblastos/virologia , Glicoproteínas/genética , Rim , Mesocricetus , Camundongos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Raiva/prevenção & controle , Vacina Antirrábica/imunologia , Organismos Livres de Patógenos Específicos , Vacinas Sintéticas/imunologia , Células Vero/virologia , Proteínas do Envelope Viral/genética , Cultura de VírusRESUMO
A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry.
Assuntos
Infecções por Birnaviridae/veterinária , Vírus da Doença Infecciosa da Bursa/imunologia , Doenças das Aves Domésticas/prevenção & controle , Vaccinia virus/genética , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais , Infecções por Birnaviridae/prevenção & controle , Células Cultivadas , Embrião de Galinha , Galinhas , Fibroblastos/metabolismo , Doenças das Aves Domésticas/virologia , Organismos Livres de Patógenos Específicos , Vaccinia virus/imunologia , Vaccinia virus/metabolismo , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismoRESUMO
En la Argentina, la rabia está circunscripta a algunas provincias del norte. La disponibilidad de nuevas vacunas que eliminen la manipulación del virus rábico y que permitan el control de la enfermedad es de importancia estratégica nacional y regional. Las vacunas basadas en poxvirus recombinantes se han utilizado con éxito como vacunas antirrábicas a nivel mundial. SI bien estos sistemas no están disponibles comercialmente, la plataforma de obtención de virus canarypox (CNPV) recombinantes ya ha sido implementada en nuestro laboratorio. El objetivo de este trabajo fue obtener y evaluar un candidato a vacuna antirrábica basado en CNPV recombinantes que expresan la glicoproteína G (RG) del virus rábico (RV). Se construyó un virus recombinante que expresa la secuencia codificante de RG (CNPV-RG). La inoculación de ratones con este virus indujo altos títulos de anticuerpos seroneutralizantes de RV (3,58 y 9,76 Ul/ml después de una o dos inmunizaciones, respectivamente) y protegió al 78 % de los animales desafiados intracerebralmente con RV. Además, se determinó que el CNPV-RG posee una potencia relativa de 3,5 Ul/ml. Los resultados obtenidos constituyen la primera etapa en la evaluación del CNPV-RG como candidato a vacuna antirrábica. Se requerirán nuevos ensayos para confirmar su utilidad en especies de interés veterinario.
In Argentina, rabies is limited to some northern provinces. Availability of new vaccines abolishing the handling of the rabies virus and allowing disease control has regional and national strategic importance. Vaccines based on recombinant poxviruses have been successfully used as antirabic vaccines worldwide. Although these systems are not commercially available, the platform to obtain recombinant canarypox viruses (CNPV) has been previously set up in our laboratory. The aim of this work was the development and evaluation of an antirabic vaccine candidate based on recombinant CNPV expressing the rabies virus (RV) glycoprotein G (RG). A recombinant virus (CNPV-RG) expressing the RG coding sequence was designed. Inoculation of mice with this virus induced high RV seroneutralizing antibodies (3.58 and 9.76 lU/ml after 1 or 2 immunizations, respectively) and protected 78% of intracerebrally RV-challenged animals. In addition, it was determined that CNPV-RG has a relative potency of 3.5 lU/ml. The obtained results constituted the first stage of CNPV-RG evaluation as antirabic vaccine candidate. Further assays will be necessary to confirm its utility in species of veterinary Interest.
Assuntos
Animais , Embrião de Galinha , Cricetinae , Camundongos , Antígenos Virais/imunologia , Vírus da Varíola dos Canários/imunologia , Glicoproteínas/imunologia , Vacina Antirrábica , Proteínas do Envelope Viral/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Chlorocebus aethiops , Vírus da Varíola dos Canários/genética , Vírus da Varíola dos Canários/crescimento & desenvolvimento , Vírus da Varíola dos Canários/isolamento & purificação , Linhagem Celular/virologia , Fibroblastos/virologia , Glicoproteínas/genética , Rim , Mesocricetus , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Vacina Antirrábica/imunologia , Raiva/prevenção & controle , Organismos Livres de Patógenos Específicos , Cultura de Vírus , Vacinas Sintéticas/imunologia , Células Vero/virologia , Proteínas do Envelope Viral/genéticaRESUMO
This work describes the development of a novel protein expression system based on Rachiplusia nu larvae for the production of the recombinant E2 protein to be used as a vaccine candidate against bovine viral diarrhea virus (BVDV). A recombinant baculovirus (Ac-E2pol+) bearing the E2 glycoprotein coding sequence of BVDV was obtained. Fourth-instar R. nu larvae were infected orally with recombinant polyhedra and the expression of E2 protein was confirmed by immunoblot. In order to test the recombinant product as a vaccine candidate, an immunization assay was performed and the neutralizing humoral immune response against BVDV NADL strain was evaluated. Mice vaccinated with Ac-E2pol+ extracts of per os infected larvae developed a neutralizing antibody titer of 3.16 after the administration of three doses of the immunogen. This report demonstrates the efficacy of per os infected larval extracts as a BVDV recombinant immunogen, which constitutes an easier and economic approach for producing recombinant antigens.
Assuntos
Anticorpos Antivirais/biossíntese , Baculoviridae/genética , Vírus da Diarreia Viral Bovina/imunologia , Lepidópteros/virologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Vírus da Diarreia Viral Bovina/genética , Imunização , Larva/genética , Larva/metabolismo , Lepidópteros/genética , Lepidópteros/metabolismo , Camundongos , Testes de Neutralização , Proteínas Recombinantes/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
A good candidate antigen to create a therapeutic vaccine against TB is the ESAT-6 protein. Antigens produced in plants have already been successfully used as experimental vaccines, and small single-stranded RNA plant viruses have emerged as promising tools to rapidly express large amounts of foreign proteins in susceptible host plants. Here, we present the expression of ESAT-6 protein in Nicotiana tabacum using a vector based on potato virus X (PVX). The complete ESAT-6 open reading frame is expressed as a fusion protein with the 2A peptide of Foot and Mouth Disease Virus and the amino terminal of the PVX coat protein (CP) (PVXESAT-6). This strategy allows the production of free CP and ESAT-6 as well as fused ESAT-2A-CP to obtain recombinant chimaeric virions expressing ESAT-6 at the surface to be used as particulate antigen in vaccination. ESAT-6 expression was tested in agroinfiltrated tobacco leaves and products of the expected molecular masses corresponding to cleaved CP and ESAT-2A-CP fusion protein were observed, with ESAT-6 yields ranging from 0.5% to 1% of total soluble protein. Our study describes for the first time the expression of the ESAT-6 protein in tobacco plants using a PVX-derived vector. This strategy should serve as a convenient, rapid, low-cost expression system and can also be used for the assessment of ESAT-6 production and function prior to stable plant transformation.