RESUMEN
Expression of Norwalk virus nonstructural polyprotein precursor in vitro resulted in rapid cotranslational cleavage at specific sites. The cleavage products were similar to those previously identified for Southampton virus, a highly related virus. We inactivated the virally encoded proteinase responsible for cleavage of the nonstructural polyprotein by mutation of the putative catalytic cysteine residue, which resulted in production of full-length polyprotein precursor. NV proteinase was expressed in Escherichia coli as a glutathione S-transferase fusion and purified by GST-affinity chromatography. Activity of the purified proteinase was demonstrated by incubation with the full-length precursor protein. trans cleavage of the nonstructural protein precursor resulted in cleavage products similar to those observed during cotranslational cleavage, however, at lesser efficiency. NV proteinase displayed sensitivities to cysteine and serine protease inhibitors similar to poliovirus 3C proteinase, suggesting that NV proteinase is a member of the viral cysteine proteinase family. We propose that the proteinase may play a regulatory role in viral replication.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Virus Norwalk/fisiología , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/metabolismo , Proteasas Virales 3C , Cinética , Inhibidores de Proteasas/farmacología , Replicación ViralRESUMEN
In this study, we evaluated the potential of a genetically modified cholera toxin, CT-E29H as an adjuvant for recombinant Norwalk virus like particle (NV-VLP) vaccine. This detoxified mutant, containing E to H substitution at amino acid 29 of the CT-A1 subunit, was administered with a recombinant Norwalk virus like particle vaccine to Balb/c mice by mucosal routes to monitor the induction of mucosal, humoral and cellular responses. We observed that a low dose of NV-VLP (5 microg) with the adjuvant delivered by the intranasal route (IN) was more effective than the highest dose (200 microg) delivered by oral route at inducing both cellular and NV-VLP specific IgG and IgA responses. Higher counts of antigen specific IgA secreting cells were observed in the Peyer's Patches (PP) following delivery of the vaccine with CT-E29H as compared to delivery of vaccine by mucosal routes without CT-E29H. Furthermore, there was an increase in antigen specific cells producing IL-4 from animals that received the vaccine with the adjuvant. Delivery of the vaccine by the oral route results in antigen specific CD4(+) and CD8(+) T cells in PP and spleen. Addition of CT-E29H results in an increase of antigen specific CD4(+) cell population in PP and both CD4(+) and CD8(+) populations in the spleen. These cellular and cytokine responses suggest that combining the vaccine with CT-E29H results in a stronger Th2 type response. Collectively, these results indicate that immune responses to NV-VLP vaccine are qualitatively and quantitatively improved when the vaccine is delivered along with CT-E29H, and thus merits its further consideration as a mucosal adjuvant.