RESUMEN
Effective Foot and Mouth Disease Virus (FMDV) peptide vaccines for cattle have two major constraints: resemblance of one or more of the multiple conformations of the major VP1 antigenic sites to induce neutralizing antibodies, and stimulation of T cells despite the variable bovine-MHC polymorphism. To overcome these limitations, a chimeric antigen was developed, using Vesicular Stomatitis Virus glycoprotein (VSV-G) as carrier protein of an in tandem-dimer of FMDV antigenic site A (ASA), the major epitope on the VP1 capsid protein (aa 139-149, FMDV-C3 serotype). The G-ASA construct was expressed in the Baculovirus system to produce a recombinant protein (DEL BAC) (cloned in pCDNA 3.1 plasmid) (Invitrogen Corporation, Carlsbad, CA) and was also prepared as a DNA vaccine (pC DEL). Calves vaccinated with both immunogens elicited antibodies that recognized the ASA in whole virion and were able to neutralize FMDV infectivity in vitro. After two vaccine doses, DEL BAC induced serum neutralizing titers compatible with an "expected percentage of protection" above 90%. Plasmid pC DEL stimulated FMDV specific humoral responses earlier than DEL BAC, though IgG1 to IgG2 ratios were lower than those induced by both DEL BAC and inactivated FMDV-C3 after the second dose. DEL BAC induced FMDV-specific secretion of IFN-γ in peripheral blood mononuclear cells of outbred cattle immunized with commercial FMDV vaccine, suggesting its capacity to recall anamnestic responses mediated by functional T cell epitopes. The results show that exposing FMDV-VP1 major neutralizing antigenic site in the context of N-terminal sequences of the VSV G protein can overcome the immunological limitations of FMDV-VP1 peptides as effective protein and DNA vaccines for cattle.
Asunto(s)
Epítopos/inmunología , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Glicoproteínas/inmunología , Vesiculovirus/inmunología , Proteínas Estructurales Virales/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Bovinos , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/prevención & control , Epítopos/genética , Fiebre Aftosa/inmunología , Virus de la Fiebre Aftosa/genética , Glicoproteínas/genética , Inmunoglobulina G/sangre , Memoria Inmunológica , Interferón gamma/metabolismo , Leucocitos Mononucleares/inmunología , Secuencias Repetidas en Tándem , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vesiculovirus/genética , Proteínas Estructurales Virales/genética , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
Transgenic plants are gaining increasing attention from the industry as a natural bioreactor for the production of industrial and chemical products. Optimization of transgene expression in plant cells holds the key to maximizing the potential of plants for producing proteins of commercial interest. This chapter is devoted to the description of the methods utilized for the generation of transgenic plants expressing a canine parvovirus vaccine peptide or virus-like particles from a rabbit calicivirus.
Asunto(s)
Antígenos/genética , Plantas Modificadas Genéticamente/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Datos de Secuencia Molecular , PlásmidosRESUMEN
Vaccines produced in transgenic plants constitute a promising alternative to conventional immunogens, presenting the possibility of stimulating secretory and systemic immunity against enteric pathogens when administered orally. Protection against enteric pathogens affecting newborn animals requires, in most cases, the stimulation of lactogenic immunity. Here, the group presents the development of an experimental immunogen based on expression of an immunorelevant peptide, eBRV4, of the VP4 protein of bovine rotavirus (BRV), which has been described as harbouring at least one neutralizing epitope as well as being responsible for the adsorption of the virus to epithelial cells. The eBRV4 epitope was efficiently expressed in transgenic alfalfa as a translational fusion protein with the highly stable reporter enzyme beta-glucuronidase (betaGUS), which served as a carrier, stabilized the synthesized peptide and facilitated screening for the higher expression levels in plants. Correlation of expression of the eBRV4 epitope in plants with those presenting the highest betaGUS activities was confirmed by a Western blot assay specific for the BRV peptide. The eBRV4 epitope expressed in plants was effective in inducing an anti-rotavirus antibody response in adult female mice when administered either intraperitoneally or orally and, more importantly, suckling mice born from immunized female mice were protected against oral challenge with virulent rotavirus. These results demonstrate the feasibility of inducing lactogenic immunity against an enteric pathogen using an edible vaccine produced in transgenic plants.