RESUMEN
Vaccination is the best strategy to control Paratuberculosis (PTB), which is a significant disease in cattle and sheep. Previously we showed the humoral and cellular immune response induced by a novel vaccine candidate against PTB based on the Argentinian Mycobacterium avium subspecies paratuberculosis (Map) 6611 strain. To improve 6611 immunogenicity and efficacy, we evaluated this vaccine candidate in mice with two different adjuvants and a heterologous boost with a recombinant modified vaccinia Ankara virus (MVA) expressing the antigen 85A (MVA85A). We observed that boosting with MVA85A did not improve total IgG or specific isotypes in serum induced by one or two doses of 6611 formulated with incomplete Freund's adjuvant (IFA). However, when 6611 was formulated with ISA201 adjuvant, MVA85A boost enhanced the production of IFNγ, Th1/Th17 cytokines (IL-2, TNF, IL-17A) and IL-6, IL-4 and IL-10. Also, this group showed the highest levels of IgG2b and IgG3 isotypes, both important for better protection against Map infection in the murine model. Finally, the heterologous scheme elicited the highest levels of protection after Map challenge (lowest CFU count and liver lesion score). In conclusion, our results encourage further evaluation of 6611 strain + ISA201 prime and MVA85A boost in bovines.
Asunto(s)
Adyuvantes Inmunológicos , Anticuerpos Antibacterianos , Citocinas , Modelos Animales de Enfermedad , Inmunización Secundaria , Inmunoglobulina G , Mycobacterium avium subsp. paratuberculosis , Paratuberculosis , Animales , Mycobacterium avium subsp. paratuberculosis/inmunología , Inmunización Secundaria/métodos , Ratones , Paratuberculosis/prevención & control , Paratuberculosis/inmunología , Inmunoglobulina G/sangre , Citocinas/metabolismo , Femenino , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/administración & dosificación , Ratones Endogámicos BALB C , Virus Vaccinia/inmunología , Virus Vaccinia/genética , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/genética , Inmunidad Celular/inmunología , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/administración & dosificación , Adyuvante de Freund/administración & dosificación , Adyuvante de Freund/inmunologíaRESUMEN
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.
Asunto(s)
Administración Intranasal , Ratones Endogámicos BALB C , Virus Sincitial Respiratorio Bovino , Animales , Virus Sincitial Respiratorio Bovino/inmunología , Ratones , Femenino , Bovinos , Proteínas Virales de Fusión/inmunología , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/administración & dosificación , Vacunas contra Virus Sincitial Respiratorio/inmunología , Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Vectores Genéticos , Infecciones por Virus Sincitial Respiratorio/prevención & control , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/veterinaria , Virus Vaccinia/inmunología , Virus Vaccinia/genética , Anticuerpos Antivirales/sangre , Inmunidad Mucosa , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Inmunización/métodos , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/administración & dosificaciónRESUMEN
Mycobacterium tuberculosis (Mtb) infection is one of the leading causes of death worldwide. The Modified Vaccinia Ankara (MVA) vaccine vector expressing the mycobacterial antigen 85A (MVA85A) was demonstrated to be safe, although it did not improve BCG efficacy, denoting the need to search for improved tuberculosis vaccines. In this work, we investigated the effect of IL-12 DNA -as an adjuvant- on an Ag85A DNA prime/MVA85A boost vaccination regimen. We evaluated the immune response profile elicited in mice and the protection conferred against intratracheal Mtb H37Rv challenge. We observed that the immunization scheme including DNA-A85A+DNA-IL-12/MVA85A induced a strong IFN-γ production to Ag85A in vitro, with a significant expansion of IFN-γ+CD4+ and IFN-γ+CD8+ anti-Ag85A lymphocytes. Furthermore, we also detected a significant increase in the proportion of specific CD8+CD107+ T cells against Ag85A. Additionally, inclusion of IL-12 DNA in the DNA-A85A/MVA85A vaccine scheme induced a marked augment in anti-Ag85A IgG levels. Interestingly, after 30 days of infection with Mtb H37Rv, DNA-A85A+DNA-IL-12/MVA85A vaccinated mice displayed a significant reduction in lung bacterial burden. Together, our findings suggest that IL-12 DNA might be useful as a molecular adjuvant in an Ag85A DNA/MVA prime-boost vaccine against Mtb infection.
Asunto(s)
Mycobacterium tuberculosis , Vacunas contra la Tuberculosis , Tuberculosis , Vacunas de ADN , Aciltransferasas/genética , Animales , Antígenos Bacterianos/genética , Vacuna BCG , ADN , Inmunización Secundaria , Interleucina-12/genética , Ratones , Mycobacterium tuberculosis/genética , Tuberculosis/prevención & control , Vacunas de ADN/genéticaRESUMEN
Canarypox viruses (CNPV) are excellent candidates to develop recombinant vector vaccines due to both their capability to induce protective immune responses and their incompetence to replicate in mammalian cells (safety profile). In addition, CNPV and the derived recombinants can be manipulated under biosafety level 1 conditions. There is no commercially available system to obtain recombinant CNPV; however, the methodology and tools required to develop recombinant vaccinia virus (VV), prototype of the Poxviridae family, can be easily adapted. This chapter provides protocols for the generation, plaque isolation, molecular characterization, amplification and purification of recombinant CNPV.
Asunto(s)
Virus de la Viruela de los Canarios/crecimiento & desarrollo , Fibroblastos/virología , Vacunas Sintéticas/inmunología , Animales , Virus de la Viruela de los Canarios/genética , Virus de la Viruela de los Canarios/inmunología , Línea Celular , Pollos , Fibroblastos/inmunología , Vacunas Virales/inmunologíaRESUMEN
In this study, we evaluated the immunogenicity and efficacy of mucosal delivery of a recombinant modified vaccinia Ankara virus (MVA) expressing the secreted version of bovine herpesvirus type 1 (BoHV-1) glycoprotein D (MVA-gDs) without addition of adjuvant in two animal models. First, we demonstrated the capability of MVA-gDs of inducing both local and systemic anti-gD humoral immune response after intranasal immunization of mice. Then, we confirmed that two doses of MVA-gDs administered intranasally to rabbits induced systemic anti-gD antibodies and conferred protection against BoHV-1 challenge. Our results show the potential of using MVA as a vector for the rational design of veterinary vaccines capable of inducing specific and protective immune responses both at local and systemic level.
Asunto(s)
Portadores de Fármacos , Infecciones por Herpesviridae/prevención & control , Herpesvirus Bovino 1/inmunología , Vacunas contra Herpesvirus/inmunología , Virus Vaccinia/genética , Proteínas Virales/inmunología , Administración Intranasal , Animales , Anticuerpos Antivirales/sangre , Modelos Animales de Enfermedad , Femenino , Infecciones por Herpesviridae/inmunología , Vacunas contra Herpesvirus/administración & dosificación , Vacunas contra Herpesvirus/genética , Ratones Endogámicos BALB C , Conejos , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas Virales/genéticaRESUMEN
MVA is an attenuated vector that still retains immunomodulatory genes. We have previously reported its optimization after deleting the C12L gene, coding for the IL-18 binding-protein. Here, we analyzed the immunogenicity of MVA vectors harboring the simultaneous deletion of A44L, related to steroid synthesis and A46R, a TLR-signaling inhibitor (MVAΔA44L-A46R); or also including a deletion of C12L (MVAΔC12L/ΔA44L-A46R). The absence of biological activities of the deleted genes in the MVA vectors was demonstrated. Adaptive T-cell responses against VACV epitopes, evaluated in spleen and draining lymph-nodes of C57Bl/6 mice at acute/memory phases, were of higher magnitude in those animals that received deleted MVAs compared to MVAwt. MVAΔC12L/ΔA44L-A46R generated cellular specific memory responses of higher quality characterized by bifunctionality (CD107a/bâº/IFN-γâº) and proliferation capacity. Deletion of selected genes from MVA generated innate immune responses with higher levels of determining cytokines related to T-cell response generation, such as IL-12, IFN-γ, as well as IL-1ß and IFN-ß. This study describes for the first time that simultaneous deletion of the A44L, A46R and C12L genes from MVA improved its immunogenicity by enhancing the host adaptive and innate immune responses, suggesting that this approach comprises an appropriate strategy to increase the MVA vaccine potential.
Asunto(s)
Inmunidad Innata , Eliminación de Secuencia , Linfocitos T/inmunología , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Proteínas Virales/genética , Animales , Antígenos Virales/inmunología , Citocinas/metabolismo , Epítopos/inmunología , Ganglios Linfáticos/inmunología , Ratones Endogámicos C57BL , Bazo/inmunologíaAsunto(s)
Infecciones por Birnaviridae/veterinaria , Virus de la Viruela de los Canarios/inmunología , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Enfermedades de las Aves de Corral/inmunología , Proteínas Estructurales Virales/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Antígenos Virales/inmunología , Infecciones por Birnaviridae/inmunología , Infecciones por Birnaviridae/prevención & control , Bolsa de Fabricio/diagnóstico por imagen , Bolsa de Fabricio/patología , Virus de la Viruela de los Canarios/genética , Virus de la Viruela de los Canarios/metabolismo , Pollos , Vectores Genéticos , Enfermedades de las Aves de Corral/prevención & control , Enfermedades de las Aves de Corral/virología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas Estructurales Virales/biosíntesis , Proteínas Estructurales Virales/genética , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
Orf virus (ORFV) is the etiological agent of contagious ecthyma (CE), a pustular dermatitis of sheep and goats. Outbreaks of ORFV have been observed in all geographical regions of the world, including Argentina. The origin and identity of Argentinian ORFVs are unknown, and no comparative or phylogenetic studies of these viruses have been performed. In this study, we described the sequencing and analysis of five ORFV molecular markers: a partial B2L gene (ORF011), VIR (ORF020), an envelope mature protein (ORF109), vIL10 (ORF127), and GIF (ORF117) from two particular Argentinian outbreaks of CE.
Asunto(s)
ADN Viral/genética , Ectima Contagioso/virología , Virus del Orf/clasificación , Virus del Orf/aislamiento & purificación , Animales , Argentina , ADN Viral/química , Cabras , Datos de Secuencia Molecular , Virus del Orf/genética , Análisis de Secuencia de ADN , OvinosRESUMEN
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.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Virus de la Viruela de los Canarios/genética , Portadores de Fármacos , Vectores Genéticos , Enfermedades de las Aves de Corral/prevención & control , Proteínas Estructurales Virales/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Infecciones por Birnaviridae/inmunología , Infecciones por Birnaviridae/prevención & control , Pollos , Enfermedades de las Aves de Corral/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas Estructurales Virales/genética , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
Punta Lara is located in the Río de la Plata estuary near industrial areas contaminated mainly by organic pollutants. In this work, the responses and status of hepatic biomarkers were studied in juvenile carp (Cyprinus carpio) by means of a 21-day field exposure in cages and collection of juvenile native fish (Leporinus obtusidens) at Punta Lara. The analyzed hepatic biomarkers were: enzymatic activity of glutathione-S-transferase (GST), catalase (CAT) and superoxide dismutase (SOD), lipid peroxidation level using the thiobarbituric acid reaction (TBARS), and CYP1A protein expression, condition factor (CF) and liver somatic (LSI) index. Taking into account oxidative stress responses, SOD activity was increased in both species, while CAT was increased in C. carpio and decreased in L. obtusidens; TBARS levels indicated that oxidative damage was possibly exerted only in L. obtusidens. Biotransformation responses mediated by CYP1A were observed in both species, while GST activity was induced mainly in carps. Considering morphometric indices, CF and LSI were significantly increased in carps while CF decreased in native species. The anthropogenic pollution detected in this study in Punta Lara was associated with differences in biomarkers on both fish species, although a different pattern of response was observed.
Asunto(s)
Carpas/metabolismo , Characiformes/metabolismo , Contaminantes Químicos del Agua/toxicidad , Animales , Argentina , Hidrocarburo de Aril Hidroxilasas/metabolismo , Biomarcadores/metabolismo , Catalasa/metabolismo , Estuarios , Sedimentos Geológicos/química , Glutatión Transferasa/metabolismo , Peroxidación de Lípido , Hígado/metabolismo , Estrés Oxidativo , Especificidad de la Especie , Superóxido Dismutasa/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
The Luján River basin, which is located in the northwest area of the province of Buenos Aires, Argentina, receives different anthropogenic inputs before reaching the Río de la Plata estuary. The aim of this study was to assess the adverse impact of the river in the middle part of the basin. To this end, an in situ cage assay was conducted in two sites of the river (S1 and S2) near Luján city, and the responses of hepatic biomarkers of both a standardized (Cyprinus carpio) and a native (Pimelodella laticeps) species were evaluated. The biomarkers studied were the condition factor and liver somatic indices (LSI), the enzymatic activities of catalase (CAT), superoxide dismutase (SOD), and glutathione-S-transferase (GST), lipid peroxidation levels (thiobarbituric acid reactive substances, TBARS) and the induction of hepatic cytochrome P450 1A (CYP1A) and vitellogenin (Vtg) proteins. After 14 days, LSI and GST activity increased, and TBARS levels decreased in both species exposed at S1 and S2. In addition, exposure at both sites promoted an increase in SOD activity and CYP1A induction in C. carpio, while Vtg expression was observed only at S1. A shorter exposure period (7 days) caused an initial response only at S2 mediated only by CAT in P. laticeps. Finally, our results demonstrate that a 14-day period of in situ exposure in Luján River could lead to antioxidant and biotransformation processes in C. carpio and to phase II biotransformation responses in P. laticeps.
Asunto(s)
Biomarcadores/metabolismo , Carpas , Bagres , Hígado/enzimología , Ríos/química , Contaminación del Agua/efectos adversos , Animales , Antioxidantes/metabolismo , Argentina , Biotransformación , Catalasa/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Glutatión Transferasa/metabolismo , Peroxidación de Lípido , Estrés Oxidativo , Superóxido Dismutasa/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Vitelogeninas/metabolismoRESUMEN
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.(AU)
Asunto(s)
Animales , Pollos/virología , Virus de la Viruela de los Canarios , Proteínas Virales , Virus de la Enfermedad Infecciosa de la BolsaRESUMEN
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.
Asunto(s)
Animales , Pollos/virología , Proteínas Virales , Virus de la Enfermedad Infecciosa de la Bolsa , Virus de la Viruela de los CanariosRESUMEN
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.
Asunto(s)
Antígenos Virales/inmunología , Virus de la Viruela de los Canarios/inmunología , Glicoproteínas/inmunología , Vacunas Antirrábicas , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Antígenos Virales/genética , Virus de la Viruela de los Canarios/genética , Virus de la Viruela de los Canarios/crecimiento & desarrollo , Virus de la Viruela de los Canarios/aislamiento & purificación , Línea Celular/virología , Embrión de Pollo , Chlorocebus aethiops , Cricetinae , Fibroblastos/virología , Glicoproteínas/genética , Riñón , Mesocricetus , Ratones , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Rabia/prevención & control , Vacunas Antirrábicas/inmunología , Organismos Libres de Patógenos Específicos , Vacunas Sintéticas/inmunología , Células Vero/virología , Proteínas del Envoltorio Viral/genética , Cultivo de VirusRESUMEN
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.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Virus de la Enfermedad Infecciosa de la Bolsa/inmunología , Enfermedades de las Aves de Corral/prevención & control , Virus Vaccinia/genética , Proteínas Estructurales Virales/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales , Infecciones por Birnaviridae/prevención & control , Células Cultivadas , Embrión de Pollo , Pollos , Fibroblastos/metabolismo , Enfermedades de las Aves de Corral/virología , Organismos Libres de Patógenos Específicos , Virus Vaccinia/inmunología , Virus Vaccinia/metabolismo , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismoRESUMEN
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.
Asunto(s)
Animales , Embrión de Pollo , Cricetinae , Ratones , Antígenos Virales/inmunología , Virus de la Viruela de los Canarios/inmunología , Glicoproteínas/inmunología , Vacunas Antirrábicas , Proteínas del Envoltorio Viral/inmunología , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Antígenos Virales/genética , Chlorocebus aethiops , Virus de la Viruela de los Canarios/genética , Virus de la Viruela de los Canarios/crecimiento & desarrollo , Virus de la Viruela de los Canarios/aislamiento & purificación , Línea Celular/virología , Fibroblastos/virología , Glicoproteínas/genética , Riñón , Mesocricetus , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Vacunas Antirrábicas/inmunología , Rabia/prevención & control , Organismos Libres de Patógenos Específicos , Cultivo de Virus , Vacunas Sintéticas/inmunología , Células Vero/virología , Proteínas del Envoltorio Viral/genéticaRESUMEN
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.(AU)
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.(AU)
Asunto(s)
Animales , Embrión de Pollo , Cricetinae , Ratones , Antígenos Virales/inmunología , Virus de la Viruela de los Canarios/inmunología , Glicoproteínas/inmunología , Vacunas Antirrábicas , Proteínas del Envoltorio Viral/inmunología , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Antígenos Virales/genética , Virus de la Viruela de los Canarios/genética , Virus de la Viruela de los Canarios/crecimiento & desarrollo , Virus de la Viruela de los Canarios/aislamiento & purificación , Línea Celular/virología , Chlorocebus aethiops , Fibroblastos/virología , Glicoproteínas/genética , Riñón , Mesocricetus , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Rabia/prevención & control , Vacunas Antirrábicas/inmunología , Organismos Libres de Patógenos Específicos , Vacunas Sintéticas/inmunología , Células Vero/virología , Proteínas del Envoltorio Viral/genética , Cultivo de VirusRESUMEN
BACKGROUND: Modified Vaccinia Ankara (MVA) is an attenuated strain of Vaccinia virus (VACV) currently employed in many clinical trials against HIV/AIDS and other diseases. MVA still retains genes involved in host immune response evasion, enabling its optimization by removing some of them. The aim of this study was to evaluate cellular immune responses (CIR) induced by an IL-18 binding protein gene (C12L) deleted vector (MVAΔC12L). METHODOLOGY/PRINCIPAL FINDINGS: BALB/c and C57BL/6 mice were immunized with different doses of MVAΔC12L or MVA wild type (MVAwt), then CIR to VACV epitopes in immunogenic proteins were evaluated in spleen and draining lymph nodes at acute and memory phases (7 and 40 days post-immunization respectively). Compared with parental MVAwt, MVAΔC12L immunization induced a significant increase of two to three-fold in CD8(+) and CD4(+) T-cell responses to different VACV epitopes, with increased percentage of anti-VACV cytotoxic CD8(+) T-cells (CD107a/b(+)) during the acute phase of the response. Importantly, the immunogenicity enhancement was also observed after MVAΔC12L inoculation with different viral doses and by distinct routes (systemic and mucosal). Potentiation of MVA's CIR was also observed during the memory phase, in correlation with a higher protection against an intranasal challenge with VACV WR. Of note, we could also show a significant increase in the CIR against HIV antigens such as Env, Gag, Pol and Nef from different subtypes expressed from two recombinants of MVAΔC12L during heterologous DNA prime/MVA boost vaccination regimens. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the relevance of IL-18 bp contribution in the immune response evasion during MVA infection. Our findings clearly show that the deletion of the viral IL-18 bp gene is an effective approach to increase MVA vaccine efficacy, as immunogenicity improvements were observed against vector antigens and more importantly to HIV antigens.
Asunto(s)
Vacunas contra el SIDA/uso terapéutico , Péptidos y Proteínas de Señalización Intercelular/química , Virus Vaccinia/metabolismo , Vacunas contra el SIDA/química , Animales , Linfocitos T CD8-positivos/metabolismo , Pollos , Femenino , Eliminación de Gen , Vectores Genéticos , Memoria Inmunológica , Péptidos y Proteínas de Señalización Intercelular/genética , Interferón gamma/metabolismo , Interleucina-18 , Interleucina-2/metabolismo , Ganglios Linfáticos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Bazo/metabolismoRESUMEN
Bovine herpesvirus-1 (BoHV-1) infection is distributed worldwide and the development of new tools to fight against this pathogen has become extremely important. In this work a recombinant modified vaccinia virus Ankara (MVA) vector expressing the secreted version of glycoprotein D, MVA-gDs, was obtained and evaluated as a candidate vaccine. First, the correct expression, antigenicity, and N-glycosylation of glycoprotein D were confirmed by molecular techniques. Then MVA-gDs was used as parenteral immunogen in BALB/C mice in which a specific anti-gD humoral immune response was induced and maintained for 7 mo. Two doses of MVA-gDs supplemented with cholera toxin delivered by intranasal immunization induced IgA anti-gD humoral immune responses in nasal and bronchopulmonary washes, as well as IgG anti-gD antibodies in serum samples. In order to evaluate the protection conferred by MVA-gDs immunization, a rabbit BoHV-1 challenge assay was performed. A shorter viral excretion period and a reduction in the number of animals shedding BoHV-1 was observed in the group immunized with recombinant MVA-gDs. In conclusion our data encourage further studies to evaluate MVA-gDs, alone or combined with other immunogens, as a candidate vaccine for BoHV-1.
Asunto(s)
Portadores de Fármacos , Vacunas contra Herpesvirus/inmunología , Virus Vaccinia/genética , Proteínas Virales/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/genética , Administración Intranasal , Animales , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/sangre , Líquido del Lavado Bronquioalveolar/inmunología , Toxina del Cólera/administración & dosificación , Toxina del Cólera/genética , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/prevención & control , Vacunas contra Herpesvirus/administración & dosificación , Vacunas contra Herpesvirus/genética , Inmunoglobulina A/análisis , Inmunoglobulina G/sangre , Masculino , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal/inmunología , Enfermedades de los Roedores/inmunología , Enfermedades de los Roedores/prevención & control , Factores de Tiempo , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas Virales/genética , Esparcimiento de VirusRESUMEN
The US3 protein is a unique protein kinase only present in the Alphaherpesvirinae subfamily of the herpesviruses. Studies performed with several alphaherpesviruses demonstrated that the US3 protein is involved in cytoskeleton modifications during viral infection and displays anti-apoptotic activity. However, the US3 protein of BoHV-5 has not been studied up to now. As reported for other alphaherpesviruses, our results showed that BoHV-5 US3 confers resistance against apoptosis and induces cytoskeletal reorganization leading to cell rounding, actin stress fiber breakdown and cell projections that interconnect cells. The expression of a kinase-dead version of BoHV-5 US3 showed that the anti-apoptotic activity and the induction of cell projections are kinase-dependent whereas kinase activity is not absolutely required for actin stress fiber breakdown. Besides, the kinase-dead version of US3, but not the wild type protein, was found excluded from the nucleus. These results constitute the first report on the BoHV-5 US3 functions, and highlight that there are functional differences and similarities among US3 proteins of different alphaherpesviruses.