RESUMO
A pre-designed plasmid containing the gene encoding the second subunit of Echinococcus granulosus AgB8 (EgAgB8/2) was used to study the effect of the immunization route on the immune response in BALB/c mice. Mice were immunized with pDRIVEEgAgB8/2 or pDRIVE empty cassette using the intramuscular (i.m.), intranasal (i.n.) or the epidermal gene gun (g.g.) routes. Analysis of the antibody response and cytokine data revealed that gene immunization by the i.m. route induced a marked bias towards a T helper type 1 (Th1) immune response as characterized by high IFN-γ gene expression and a low IgG1/IgG2a reactivity index (R.I.) ratio of 0.04. The i.n. route showed a moderate IFN-γ expression but a higher IgG1/IgG2a R.I. ratio of 0.25 indicating a moderate Th1 response. In contrast, epidermal g.g. immunization induced a Th2 response characterized by high IL-4 expression and the highest IgG1/IgG2a R.I. ratio of 0.58. In conclusion, this study showed the advantage of genetic immunization using the i.m. route and i.n. over the epidermal g.g. routes in the induction of Th1 immunity in response to E. granulosus AgB gene immunization.
Assuntos
Equinococose/prevenção & controle , Echinococcus granulosus/imunologia , Lipoproteínas/imunologia , Células Th1/imunologia , Vacinas de DNA/administração & dosagem , Administração Intranasal , Animais , Anticorpos Anti-Helmínticos/biossíntese , Anticorpos Anti-Helmínticos/sangue , Biolística/métodos , Biolística/normas , DNA de Helmintos/imunologia , Echinococcus granulosus/genética , Feminino , Expressão Gênica , Imunização Secundária , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Injeções Intramusculares , Interferon gama/biossíntese , Interferon gama/genética , Interleucina-4/biossíntese , Interleucina-4/genética , Lipoproteínas/administração & dosagem , Lipoproteínas/genética , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Ovinos , Doenças dos Ovinos/parasitologiaRESUMO
It has recently been demonstrated that a recombinant replication-deficient human adenovirus 5 (Ad5) vector expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) fused to the p31 invariant (Ii) chain confers broad, long-lasting T-cell immunity that completely protects C57BL/6 mice against lethal peripheral challenge. The current study questioned whether the same strategy, i.e. linkage of GP to an Ii chain, could be applied to a naked DNA vaccine. Following gene-gun immunization with the linked construct (DNA-IiGP), GP-specific CD4(+) T cells could not be detected by flow cytometry. However, inclusion of the Ii chain augmented the priming of GP-specific CD8(+) T cells directed towards both immunodominant (GP(33-41)) and subdominant (GP(276-286) and GP(92-101)) epitopes, and vaccination with DNA-IiGP conferred significantly improved protection against systemic LCMV infection compared with the unlinked construct. In contrast, substantial protection against peripheral challenge was not observed. Additional experiments with T-cell subset-depleted or perforin-deficient mice revealed that virus control in vaccinated mice depends critically on cytotoxic CD8(+) T cells. Finally, priming with the naked DNA vaccine was shown to augment the immune response raised by subsequent immunization with the Ad5 vector. In conclusion, this study showed that the immunoenhancing effect of Ii chain linkage is not limited to the Ad5 vector, but is also relevant with a DNA platform. Furthermore, given the fact that the Ii chain enhances the presentation of more than one epitope, this suggests that Ii-chain-based DNA vaccines may be promising candidates for various heterologous prime-boost regimes.
Assuntos
Antígenos Virais/imunologia , Biolística/métodos , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Linfócitos T/imunologia , Vacinas de DNA/administração & dosagem , Vacinas Virais/administração & dosagem , Animais , Biolística/normas , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Escherichia coli/imunologia , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/mortalidade , Coriomeningite Linfocítica/prevenção & controle , Vírus da Coriomeningite Linfocítica/genética , Camundongos , Camundongos Endogâmicos C57BL , Taxa de Sobrevida , Subpopulações de Linfócitos T/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
The transcriptional downregulation of the SERCA2 gene is studied using neonatal rat cardiomyocytes stimulated with endothelin-1 to induce hypertrophy. Liposome-based transfection of cells with a 1.9 kb SERCA2 promoter fragment directed expression of a reporter gene identical to the downregulation of genomic SERCA2 expression by endothelin-1. Results of a new gene gun technology for transient transfection of cardiomyocytes with a RSV-beta-galactosidase construct are reported. This new method for propelling DNA-coated gold beads into cardiomyocytes is extremely suitable for directly testing promoter/reporter gene DNA constructs since the transfection efficiency (approximately 10%) appears to be higher than traditional transfection methods.
Assuntos
Biolística/normas , ATPases Transportadoras de Cálcio/genética , Cardiomegalia/genética , Regulação da Expressão Gênica/fisiologia , Retículo Sarcoplasmático/enzimologia , Transfecção/fisiologia , Animais , Cardiomegalia/patologia , Células Cultivadas , Endotelina-1/farmacologia , Expressão Gênica/efeitos dos fármacos , Miocárdio/citologia , Ratos , Transcrição Gênica/fisiologiaRESUMO
This work examines the effect of delivering a DNA plasmid encoding murine erythropoietin (pVRmEpo) to BALB/c mice by gene gun. Whereas intramuscular injection elicits a rise in hematocrit persisting >8 months, intradermal delivery triggers the dose-dependent secretion of biologically active erythropoietin (Epo) for approximately 1 month. Repeated administration of pVRmEpo by gene gun elicits a stable increase in hematocrit. The source of the Epo produced following gene gun delivery was analyzed by periodically grafting the site of injection onto naive recipients. Results indicate that both stationary cells (presumably keratinocytes) and migratory (presumably dendritic) cells were transfected and secreted biologically active Epo in vivo. Gene gun administration of plasmid DNA appears to be safe, and provides an additional strategy for achieving the regulated secretion of an exogenous gene product.