RESUMEN
Particulate material is more efficient in eliciting immune responses. Here we describe the production of micro- and nanospheres formed by protein muNS-Mi from avian reoviruses, loaded with foreign epitopes for their use as vaccines.
Asunto(s)
Orthoreovirus Aviar , Vacunas , Adyuvantes Inmunológicos , Epítopos/metabolismo , Orthoreovirus Aviar/metabolismo , Vacunas/metabolismo , Vacunas de Subunidad , Proteínas no Estructurales Virales/metabolismoRESUMEN
We have previously developed a methodology to produce protein microspheres (MS) that can be loaded with proteins of interest in living cells through their C or N-terminal tagging with the so-called IC-Tag. The IC-Tagging method has many applications ranging from the production of immobilized enzymes for industrial use to the production of subunit vaccines due to its intrinsic adjuvancy. Here we show the adaptation of the IC-Tagging to work inside the endoplasmic reticulum and bacteria, allowing us to produce properly modified viral glycoproteins. Additionally, we were able to express the Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), whose expression remained elusive to date possibly due to its toxicity when over-expressed. IGRP is an antigen of enormous pharmaceutical interest as it is specifically targeted during the autoimmune response taking place in both the Non-Obese Diabetic (NOD) mice and type 1 diabetes (T1D) patients leading to the destruction of insulin-producing beta cells.
Asunto(s)
Autoantígenos/aislamiento & purificación , Glucosa-6-Fosfatasa/aislamiento & purificación , Cuerpos de Inclusión Viral/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Línea Celular , Embrión de Pollo , Vectores Genéticos/genética , Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/metabolismo , Glicoproteínas/genética , Orthoreovirus Aviar/genética , Plásmidos/genética , Dominios Proteicos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/aislamiento & purificación , Proteínas no Estructurales Virales/metabolismoRESUMEN
Particulate material is more efficient in eliciting immune responses. Here we describe the production of microspheres formed by protein muNS-Mi from avian reoviruses, loaded with foreign epitopes by means of IC-Tagging, for their use as vaccines.
Asunto(s)
Epítopos/inmunología , Orthoreovirus Aviar/efectos de los fármacos , Vacunación/métodos , Proteínas no Estructurales Virales/inmunología , Animales , Aves/inmunología , Aves/virología , Fluorescencia , Microesferas , Orthoreovirus Aviar/patogenicidadRESUMEN
Vaccination is critical for controlling the spread of bluetongue virus (BTV). The inactivated BTV vaccines that are now being used in Europe are effective in preventing outbreaks of BTV but secondary effects associated to repetitive inoculation of aluminum-containing adjuvants and the need to develop safer, cross-reactive, and more efficacious vaccines with differential diagnostic capability have re-stimulated the interest in developing improved vaccination strategies against BTV. We have engineered a subunit BTV vaccine candidate based on proteins VP2, VP7, and NS1 of BTV-4 incorporated into avian reovirus (ARV) muNS-Mi microspheres (MS-VP2/MS-VP7/MS-NS1). IFNAR(-/-) mice immunized with MS-VP2/MS-VP7/MS-NS1 without adjuvant generated significant levels of neutralizing antibodies specific to BTV-4. In addition, vaccination stimulated specific T cell responses, predominantly CD4+, against the virus. Immunized mice were fully protected against a homologous challenge with a lethal dose of BTV-4 and partially cross-protected against a heterologous challenge with a lethal dose of BTV-1. These results support MS-VP2/MS-VP7/MS-NS1 as a promising subunit vaccine candidate against multiple serotypes of BTV as well as the use of microspheres as an alternative delivery method with potent intrinsic adjuvant activity.
Asunto(s)
Virus de la Lengua Azul/inmunología , Proteínas de la Cápside/inmunología , Orthoreovirus Aviar/inmunología , Proteínas no Estructurales Virales/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Baculoviridae/genética , Lengua Azul/inmunología , Lengua Azul/prevención & control , Linfocitos T CD4-Positivos/inmunología , Proteínas de la Cápside/genética , Línea Celular , Embrión de Pollo , Chlorocebus aethiops , Cricetinae , Portadores de Fármacos/uso terapéutico , Masculino , Ratones , Ratones Noqueados , Microesferas , Orthoreovirus Aviar/genética , Células Sf9 , Ovinos/virología , Spodoptera , Vacunación , Vacunas de Subunidad/inmunología , Células Vero , Proteínas no Estructurales Virales/genéticaRESUMEN
We have recently developed a versatile tagging system (IC-tagging) that causes relocation of the tagged proteins to ARV muNS-derived intracellular globular inclusions. In the present study we demonstrate (i) that the IC-tag can be successfully fused either to the amino or carboxyl terminus of the protein to be tagged and (ii) that IC-tagged proteins are able to interact between them and perform complex reactions that require such interactions while integrated into muNS inclusions, increasing the versatility of the IC-tagging system. Also, our studies with the DsRed protein add some light on the structure/function relationship of the evolution of DsRed chromophore.