RESUMO
Classical swine fever virus causes significant mortality and morbidity in commercial piggeries in many countries in Europe and Asia. The protective antigen, gp55, is highly conformation-dependent and thus killed virus or bacterially produced proteins are not protective. This report demonstrates that DNA vaccination with the gene encoding gp55 can provide protective immunity with inoculation of two doses of 25 microg DNA or a single shot of 200 microg. Furthermore, the DNA can be delivered intramuscularly or by a simple spring-loaded needleless inoculator. In addition it is shown that inoculation of the DNA at a single site conveys the same level of immunity as division of the dose between two sites.
Assuntos
Vírus da Febre Suína Clássica/imunologia , Peste Suína Clássica/prevenção & controle , Vacinas de DNA/administração & dosagem , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/genética , Animais , Anticorpos Antivirais/biossíntese , Peste Suína Clássica/imunologia , DNA Viral/administração & dosagem , DNA Viral/genética , DNA Viral/imunologia , DNA Viral/uso terapêutico , Injeções Intramusculares , Testes de Neutralização , Suínos , Vacinas de DNA/uso terapêutico , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/uso terapêutico , Vacinas Virais/administração & dosagemRESUMO
The expression of antigens or other molecules from recombinant vaccinia viruses requires the insertion of coding sequence at specific sites in the viral genome. Here we investigate the influence of two different sites on the level of protein expressed during a viral infection. The level of immune response in mice to vaccinia virus-expressed murine interleukin 2 (IL-2) or IL-4 varied depending on whether the coding sequence was inserted into the vaccinia virus thymidine kinase (tk) gene or into the HindIII F fragment of the viral genome where herpes simplex virus (HSV) tk was used as a selectable marker. In each case the intensity of the response was greater when the relevant gene was expressed from the HindIII F insertion site. In order to quantify these differences a series of recombinant viruses expressing luciferase was constructed. Luciferase activity from coding sequence inserted into the HindIII F fragment was significantly higher than that from the tk gene insertion, provided HSV tk(+) constructs were compared. Insertion of a marker gene (HSV tk) into the HindIII F site with disruption of the F7L open reading frame led to a reduced level of luciferase expressed from the tk insert, despite more than 45 kb of intervening sequence. In mice, luciferase expression was higher from the HindIII F inserted gene than from the tk insert in both lungs and ovaries.
Assuntos
Vaccinia virus/genética , Animais , Feminino , Expressão Gênica , Vetores Genéticos , Interleucina-2/genética , Interleucina-2/farmacologia , Interleucina-4/genética , Interleucina-4/farmacologia , Luciferases/genética , Pulmão/virologia , Camundongos , Camundongos Endogâmicos CBA , Camundongos Nus , Ovário/virologia , Proteínas Recombinantes/genética , Recombinação Genética , Vaccinia virus/patogenicidade , Virulência/genéticaRESUMO
A synthetic vaccinia virus promoter (Psel) was constructed based upon sequences which increase activity of the P7.5 early/late promoter. Comparison of luciferase activity in lysates from cells infected with recombinant vaccinia viruses expressing the luciferase gene either under the control of the P7.5 promoter or Psel, demonstrated significantly enhanced activity mediated by Psel at both early and late times post infection. This promoter may be of considerable benefit in the construction of recombinant poxviruses where early foreign gene expression is important for generating a protective immune response in vaccinated animals, or in reporter/target gene expression in vitro.