RESUMEN
Immunogenicity of recombinant vaccinia virus strain (VR26) expressing Venezuelan equine encephalomyelitis (VEE) virus structural protein genes was studied by oral immunization. Sera of animals immunized with VR26 contained antibodies specific to VEE virus, among which antibodies with virus-neutralizing activity were present. Evaluation of the protective efficiency of oral immunization with VR26 demonstrated a high level of animal protection from lethal doses of VEE virus. Rabbits immunized orally were highly resistant (protection index 142.9) to intranasal infection, which is of priority importance for antiVEE vaccine. Comparative analysis of the results of scarification and oral immunization with VR26 indicates that the type of immune response depends on the method of immunization. These results demonstrate good prospects of oral vaccination with recombinant VR26 strain for immunoprophylaxis of VEE.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/genética , Genes Virales , Virus Vaccinia/inmunología , Proteínas Estructurales Virales/genética , Vacunas Virales/inmunología , Administración Oral , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Encefalomielitis Equina Venezolana/prevención & control , Pruebas de Neutralización , Conejos , Recombinación Genética , Virus Vaccinia/genética , Vacunas Virales/administración & dosificaciónRESUMEN
Venezuelan equine encephalomyelitis (VEE) virus-based vectors for expression of heterologous genes have been constructed using full-length cDNA copy of the interstrain recombinant virus (Trinidad Donkey and 230 strain). Gene cassettes carrying the subgenomic mRNA promoter of VEE virus and the preS2-S gene of hepatitis B virus (HBV) were inserted before or after the genes of structural viral proteins. Live virus stocks were obtained by transfection of chick embryo fibroblasts with in vitro transcribed full-length RNA. Insertions of gene cassettes before the structural region resulted in expression of HBsAg (VEHB-25 and VEHB-361 viruses), whereas insertions in the 3' region did not. Recombinant virus VEHB-25 expressed HBsAg during 5 passages in Vero cells. VEHB-25 stimulated immune response to HBsAg and was less virulent than the parental virus.
Asunto(s)
Encefalomielitis Equina Venezolana/genética , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Precursores de Proteínas/genética , Proteínas del Envoltorio Viral/genética , Animales , Línea Celular , Embrión de Pollo , Chlorocebus aethiops , Clonación Molecular , Encefalomielitis Equina Venezolana/inmunología , Antígenos de Superficie de la Hepatitis B/ultraestructura , Microscopía Electrónica , Plásmidos , Regiones Promotoras Genéticas , Precursores de Proteínas/ultraestructura , ARN Mensajero/genética , ARN Viral/genética , Conejos , Recombinación Genética , Pase Seriado , Células VeroRESUMEN
A recombinant strain of vaccinia virus (VR26) containing a DNA-copy of the subgenomic 26S RNA of Venezuelan equine encephalomyelitis virus (VEE) inserted into the coding region of thymidine kinase (TK) gene was produced. This subgenomic RNA contained the genes for all structural proteins of the VEE virus, the strain Trinidad donkey (TRD). VR26 effectively expressed VEE virus glycoproteins on the membranes of the infected cells. Blood sera of VR26-immunized animals were found to contain VEE virus-specific antibodies. VR26-immunized mice and rabbits showed a high level of resistance to subcutaneous inoculation with the pathogenic TRD strain of VEE virus. VR26 also provided a high level of protection in animals against aerogenic infection. The absence of virus-neutralizing antibodies in most VR26-immunized animals resistant to inoculation with high doses of VEE suggests the dominant role of the cell component in the immune response. The immune response induced by the recombinant VR26 strain was stable as demonstrated by the resistance of the animals to a challenge with VEE virus 7 months after immunization. The experimental results suggest that this recombinant strain may be considered as a candidate for vaccine preparation.
Asunto(s)
ADN Complementario/genética , ADN Viral/genética , Virus de la Encefalitis Equina Venezolana/genética , Regulación Viral de la Expresión Génica/inmunología , ARN Mensajero/genética , ARN Ribosómico/genética , ARN Viral/genética , Recombinación Genética/inmunología , Virus Vaccinia/inmunología , Animales , Encefalomielitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/prevención & control , Regulación Viral de la Expresión Génica/genética , Sueros Inmunes/inmunología , Inmunización/métodos , Ratones , Conejos , Recombinación Genética/genética , Factores de Tiempo , Vaccinia/inmunología , Vaccinia/prevención & control , Virus Vaccinia/genéticaRESUMEN
Nine peptides were synthesized for detailed mapping of VEE virus E2-2 and E2-6 sites responsible for the formation of protective antibodies that neutralize the virus and block hemagglutination. The sequence of the peptides over-lapped the regions of amino acid residues 30-75 and 202-250 of VEE virus E2 protein in which antigenic mutations caused by monoclonal antibodies to E2-2 and E2-6 sites had been mapped. None of the synthesized peptides reacted in the enzyme immunoassay with a panel of 17 Mabs to VEE virus E2 protein. However, eight peptides reacted with polyclonal antiviral serum and two of them elicited antiviral antibody production. The E2-2 site might be associated with amino acid residues 30-45, and the region of E2 residues 57-62 in which antigenic mutations are observed is not a linear type antigenic determinant, but participates in the formation of antigenic determinants of the conformational type. The mapping of residues 202-250 demonstrated that all the peptides in this region were well recognized by polyclonal antiviral serum. The residues 235-240 were shown to form a linear epitope which provided a crossover between VEE and EEE viruses and was not recognized by 19 types of monoclonal antibodies cross-reacting with VEE and EEE viruses.