Limits...
Induction of a protective response in mice by the dengue virus NS3 protein using DNA vaccines.

Costa SM, Yorio AP, Gonçalves AJ, Vidale MM, Costa EC, Mohana-Borges R, Motta MA, Freire MS, Alves AM - PLoS ONE (2011)

Bottom Line: The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion.The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide.Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biotecnologia e Fisiologia de Infecções Virais, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brasil.

ABSTRACT
The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection.

Show MeSH

Related in: MedlinePlus

Production of INF-γ by T cells of animals immunized with the DNA vaccines encoding the full-length NS3 protein.Splenocytes collected from groups of mice (n = 6) inoculated with pcTPANS3, pcNS3, pcDNA3 or pcTPA plasmids were stimulated with a T-cell specific peptide and the number of spot-forming cells (SFC) were quantified in a 24h ELISPOT assay. Asterisks indicate statistically significant differences (**, p<0.003).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198735&req=5

pone-0025685-g006: Production of INF-γ by T cells of animals immunized with the DNA vaccines encoding the full-length NS3 protein.Splenocytes collected from groups of mice (n = 6) inoculated with pcTPANS3, pcNS3, pcDNA3 or pcTPA plasmids were stimulated with a T-cell specific peptide and the number of spot-forming cells (SFC) were quantified in a 24h ELISPOT assay. Asterisks indicate statistically significant differences (**, p<0.003).

Mentions: An IFN-γ ELISPOT assay was performed in order to investigate the T-cell response induced with plasmids encoding the full-length NS3 protein, using a previously identified NS3 immunodominant epitope in Balb/c mice. All animals immunized with either the pcTPANS3 or pcNS3 DNA vaccines presented higher and statistically different SFC values when compared to the control groups, inoculated with pcDNA3 or pcTPA plasmids (Fig. 6). Overall, animals vaccinated with the pcNS3 presented higher frequencies of NS3-specific IFN-γ-secreting cells comparing to pcTPANS3-immunized mice, although such differences were not statistically significant (Fig. 6). Positive control using Con A as stimulating antigen confirmed the cell viability (data not shown).


Induction of a protective response in mice by the dengue virus NS3 protein using DNA vaccines.

Costa SM, Yorio AP, Gonçalves AJ, Vidale MM, Costa EC, Mohana-Borges R, Motta MA, Freire MS, Alves AM - PLoS ONE (2011)

Production of INF-γ by T cells of animals immunized with the DNA vaccines encoding the full-length NS3 protein.Splenocytes collected from groups of mice (n = 6) inoculated with pcTPANS3, pcNS3, pcDNA3 or pcTPA plasmids were stimulated with a T-cell specific peptide and the number of spot-forming cells (SFC) were quantified in a 24h ELISPOT assay. Asterisks indicate statistically significant differences (**, p<0.003).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198735&req=5

pone-0025685-g006: Production of INF-γ by T cells of animals immunized with the DNA vaccines encoding the full-length NS3 protein.Splenocytes collected from groups of mice (n = 6) inoculated with pcTPANS3, pcNS3, pcDNA3 or pcTPA plasmids were stimulated with a T-cell specific peptide and the number of spot-forming cells (SFC) were quantified in a 24h ELISPOT assay. Asterisks indicate statistically significant differences (**, p<0.003).
Mentions: An IFN-γ ELISPOT assay was performed in order to investigate the T-cell response induced with plasmids encoding the full-length NS3 protein, using a previously identified NS3 immunodominant epitope in Balb/c mice. All animals immunized with either the pcTPANS3 or pcNS3 DNA vaccines presented higher and statistically different SFC values when compared to the control groups, inoculated with pcDNA3 or pcTPA plasmids (Fig. 6). Overall, animals vaccinated with the pcNS3 presented higher frequencies of NS3-specific IFN-γ-secreting cells comparing to pcTPANS3-immunized mice, although such differences were not statistically significant (Fig. 6). Positive control using Con A as stimulating antigen confirmed the cell viability (data not shown).

Bottom Line: The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion.The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide.Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biotecnologia e Fisiologia de Infecções Virais, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brasil.

ABSTRACT
The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection.

Show MeSH
Related in: MedlinePlus