In this work, we report the evaluation of two DNA vaccines against dengue-3 virus (DENV-3). The first construction, called pVAC3DEN3, was engineered inserting the pre-membrane (prM) and envelope (E) gene of DENV-3 tru...In this work, we report the evaluation of two DNA vaccines against dengue-3 virus (DENV-3). The first construction, called pVAC3DEN3, was engineered inserting the pre-membrane (prM) and envelope (E) gene of DENV-3 truncated with a restriction site between them, as previously described. The second construction was developed cloning the full gene sequence of prM and E from DENV-3 virus in pCI plasmid for mammalian expression and was denominated pVAC1WDEN3. The results showed that both constructions were capable of expressing the prM and E proteins, as demonstrated by ELISA and immunoblotting detection in cell culture transfected with the plasmids. After positive “in vitro” results, the vaccine candidates were used to immunize BALB/c mice and the elicited response was investigated. After immunization by intramuscular inoculation with three doses of each vaccinal clone the animals were sacrificed, the cytokine levels and T cell response were analyzed in the spleens, after three days of culture with stimulus, our analysis showed that the two constructions elicited T cell responses mea- sured by BrdU incorporation assay and high levels of IFN-γ, detected in the supernatant of the cultures. Moreover, both constructions induced detectable titers of neutralizing antibodies in mice. And finally the survival rate of the immunized animals after intracerebral challenge was analyzed, showing a better result in the pVAC3DEN3 group with an 80% survival compared with a 50% survival of the pVAC1 WDEN3. Thus, these data showed that our two constructions were able to induce specific immune response and protects mice against a lethal challenge with DENV-3, and these vaccine candidates can be employed to develop a viable dengue vaccine.展开更多
基金Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP)
文摘In this work, we report the evaluation of two DNA vaccines against dengue-3 virus (DENV-3). The first construction, called pVAC3DEN3, was engineered inserting the pre-membrane (prM) and envelope (E) gene of DENV-3 truncated with a restriction site between them, as previously described. The second construction was developed cloning the full gene sequence of prM and E from DENV-3 virus in pCI plasmid for mammalian expression and was denominated pVAC1WDEN3. The results showed that both constructions were capable of expressing the prM and E proteins, as demonstrated by ELISA and immunoblotting detection in cell culture transfected with the plasmids. After positive “in vitro” results, the vaccine candidates were used to immunize BALB/c mice and the elicited response was investigated. After immunization by intramuscular inoculation with three doses of each vaccinal clone the animals were sacrificed, the cytokine levels and T cell response were analyzed in the spleens, after three days of culture with stimulus, our analysis showed that the two constructions elicited T cell responses mea- sured by BrdU incorporation assay and high levels of IFN-γ, detected in the supernatant of the cultures. Moreover, both constructions induced detectable titers of neutralizing antibodies in mice. And finally the survival rate of the immunized animals after intracerebral challenge was analyzed, showing a better result in the pVAC3DEN3 group with an 80% survival compared with a 50% survival of the pVAC1 WDEN3. Thus, these data showed that our two constructions were able to induce specific immune response and protects mice against a lethal challenge with DENV-3, and these vaccine candidates can be employed to develop a viable dengue vaccine.
