RESUMEN
Amphotericin B (AmB) is an antifungal antibiotic the activity of which has been associated with modulation of pro-inflammatory cytokines expression in cultured cells. Herein we reveal that co-administration with AmB enhances the immunogenicity of oral Lip-JENS1 vaccine which derived from liposomes functionalized with DSPC (distearoylphosphatidylcholine) and cholesterol (2:1, molar ratio)-bearing JE virus NS1 protein (600 microg ml(-1)). Oral single dose of Lip-JENS1 elicited a detectable serum NS1-specific IgG antibody response from a mouse model. Remarkably, the addition of AmB (125 microg per mouse), particularly, 2 h prior to, but not simultaneously with, the administration of Lip-JENS1 significantly enhanced the systemic antigen-specific antibody response, providing superior protection against lethal JEV challenges. Further, we observed AmB-induced the transcription of cytokine expression and translocation of transcriptional factor NF-kappaB from the cytoplasm to the nucleus for the murine macrophage J774A.1. Moreover, Peyer's-patch lymphocytes (PPL) from AmB-treated mice produced high levels of IL-1beta, IL-6 and TNF-alpha expression compared to the corresponding control of cells from non-treated mice. Taken together, the results suggest that AmB exerts a profound influence upon mucosal vaccination with Lip-JENS1, possibly playing an adjuvant-augmented role to "fine-tune" humoral as well as cellular immune response, thus conferring enhanced protective immunity for immunising individuals against JE infection.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Anfotericina B/administración & dosificación , Vacunas contra la Encefalitis Japonesa/administración & dosificación , Vacunas contra la Encefalitis Japonesa/inmunología , Liposomas/administración & dosificación , Proteínas no Estructurales Virales/inmunología , Administración Oral , Animales , Anticuerpos Antivirales/sangre , Núcleo Celular/química , Citocinas/metabolismo , Citoplasma/química , Modelos Animales de Enfermedad , Virus de la Encefalitis Japonesa (Especie)/inmunología , Encefalitis Japonesa/prevención & control , Femenino , Inmunoglobulina G/sangre , Linfocitos/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Ganglios Linfáticos Agregados/inmunología , Análisis de SupervivenciaRESUMEN
BACKGROUND AND PURPOSE: The Caf1 secretion pathway of Yersinia pestis is one of the most well-characterized export machineries. To facilitate the secretion of human epidermal growth factor (hEGF) in Escherichia coli, a DNA fragment containing the synthetic gene for hEGF was joined to a sequence encoding the signal peptide of Yersinia pestis Caf1 protein. METHODS: The gene for hEGF was synthesized by overlapping polymerase chain reaction technique and was placed under the control of the caf1 gene promoter in the recombinant plasmid pHL401 which was used to transfect E. coli BL-21 for production of hEGF. The biological function of recombinant hEGF was measured by estimating its ability to stimulate the proliferation of human embryonic kidney-293 cells. RESULTS: The results indicated that the expressed hybrid protein was processed during the secretion process. The majority of the mature hEGF was recovered from the periplasm and medium fractions, with a small amount of the expressed hEGF deposited in the cytoplasm. Furthermore, it was found that the cell proliferation was enhanced by the recombinant hEGF. CONCLUSION: These results suggested that the recombinant hEGF was successfully secreted through the inner membrane of cells into the periplasm and then through the outer membrane into the medium via the action of the signal peptide of Y. pestis Caf1 in E. coli. The mitogenic activity of hEGF in cells was demonstrated.