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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.

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Western blots of whole-cell extract (a) and culture supernatants (b) of BHK-21 cells transfected with the different DNA vaccines.The recombinant proteins were detected in SDS-PAGE, using a mouse polyclonal antibody against the DENV2 NS3, harvested from transfections with pcTPA (lane 1), pcTPANS3P (lane 2), pcTPANS3H (lane 3), pcTPANS3 (lane 4), pcNS3P (lane 5), pcNS3H (lane 6) and pcNS3 (lane 7). Arrows indicate bands corresponding to the protease, helicase and full-length NS3 recombinant proteins.
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pone-0025685-g003: Western blots of whole-cell extract (a) and culture supernatants (b) of BHK-21 cells transfected with the different DNA vaccines.The recombinant proteins were detected in SDS-PAGE, using a mouse polyclonal antibody against the DENV2 NS3, harvested from transfections with pcTPA (lane 1), pcTPANS3P (lane 2), pcTPANS3H (lane 3), pcTPANS3 (lane 4), pcNS3P (lane 5), pcNS3H (lane 6) and pcNS3 (lane 7). Arrows indicate bands corresponding to the protease, helicase and full-length NS3 recombinant proteins.

Mentions: The expression of recombinant proteins was evaluated in BHK-21 cells transiently transfected with the different plasmids. Immunofluorescence assays revealed positive reaction in cells transfected with all recombinant plasmids, indicating that constructs were able to promote expression of the full-length NS3 protein or its functional domains in eukaryotic systems, regardless the presence of the t-PA signal peptide (Fig. 2). Cells transfected with control vectors (pcTPA or pcDNA3) did not react with NS3-specific antibodies (data not shown). Western blot analysis of transfected cell extracts confirmed the identity of the recombinant proteins, showing predictable molecular weights. A protein of approximately 20 kDa, corresponding to the NS3 protease domain, was detected in pcTPANS3P- and pcNS3P-transfected cell lysates, while extracts from cells transfected with pcTPANS3H and pcNS3H revealed a band around 50 kDa, according to the 433 amino acids of the helicase domain (Fig. 3a). Furthermore, a band of approximately 70 kDa was observed in pcTPANS3- and pcNS3-transfected cell extracts, which corresponds to the full-length NS3 protein (Fig. 3a). Other specific bands, with different molecular weights, were also observed in the extract of cells transfected with plasmids encoding the full-length NS3 or the helicase domain, probably resulting from spontaneous cleavage and/or degradation of the NS3 protein. As expected, NS3-specific recombinant proteins were detected only in the supernatants of cells transfected with plasmids containing the t-PA signal sequence (pcTPANS3P, pcTPANS3H and pcTPANS3), which demonstrated that this signal peptide indeed targeted the proteins to secretion (Fig. 3b). No NS3-specific band was observed in extracts or supernatants of cells transfected with control vectors (Fig. 3).


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)

Western blots of whole-cell extract (a) and culture supernatants (b) of BHK-21 cells transfected with the different DNA vaccines.The recombinant proteins were detected in SDS-PAGE, using a mouse polyclonal antibody against the DENV2 NS3, harvested from transfections with pcTPA (lane 1), pcTPANS3P (lane 2), pcTPANS3H (lane 3), pcTPANS3 (lane 4), pcNS3P (lane 5), pcNS3H (lane 6) and pcNS3 (lane 7). Arrows indicate bands corresponding to the protease, helicase and full-length NS3 recombinant proteins.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0025685-g003: Western blots of whole-cell extract (a) and culture supernatants (b) of BHK-21 cells transfected with the different DNA vaccines.The recombinant proteins were detected in SDS-PAGE, using a mouse polyclonal antibody against the DENV2 NS3, harvested from transfections with pcTPA (lane 1), pcTPANS3P (lane 2), pcTPANS3H (lane 3), pcTPANS3 (lane 4), pcNS3P (lane 5), pcNS3H (lane 6) and pcNS3 (lane 7). Arrows indicate bands corresponding to the protease, helicase and full-length NS3 recombinant proteins.
Mentions: The expression of recombinant proteins was evaluated in BHK-21 cells transiently transfected with the different plasmids. Immunofluorescence assays revealed positive reaction in cells transfected with all recombinant plasmids, indicating that constructs were able to promote expression of the full-length NS3 protein or its functional domains in eukaryotic systems, regardless the presence of the t-PA signal peptide (Fig. 2). Cells transfected with control vectors (pcTPA or pcDNA3) did not react with NS3-specific antibodies (data not shown). Western blot analysis of transfected cell extracts confirmed the identity of the recombinant proteins, showing predictable molecular weights. A protein of approximately 20 kDa, corresponding to the NS3 protease domain, was detected in pcTPANS3P- and pcNS3P-transfected cell lysates, while extracts from cells transfected with pcTPANS3H and pcNS3H revealed a band around 50 kDa, according to the 433 amino acids of the helicase domain (Fig. 3a). Furthermore, a band of approximately 70 kDa was observed in pcTPANS3- and pcNS3-transfected cell extracts, which corresponds to the full-length NS3 protein (Fig. 3a). Other specific bands, with different molecular weights, were also observed in the extract of cells transfected with plasmids encoding the full-length NS3 or the helicase domain, probably resulting from spontaneous cleavage and/or degradation of the NS3 protein. As expected, NS3-specific recombinant proteins were detected only in the supernatants of cells transfected with plasmids containing the t-PA signal sequence (pcTPANS3P, pcTPANS3H and pcTPANS3), which demonstrated that this signal peptide indeed targeted the proteins to secretion (Fig. 3b). No NS3-specific band was observed in extracts or supernatants of cells transfected with control vectors (Fig. 3).

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