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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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Related in: MedlinePlus

Schematic representation of the DENV2 genome (a) and the regions coded by the different recombinant plasmids (b): pcTPANS3, pcTPANS3P, pcTPANS3H, pcNS3, pcNS3P, pcNS3H.Boxes represent the NS3 region (gray areas), the protease domain (streaked areas) and the helicase/RTPase/NTPase (dotted areas) domain; black box symbolizes the signal peptide derived from the human tissue plasminogen activator (t-PA).
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pone-0025685-g001: Schematic representation of the DENV2 genome (a) and the regions coded by the different recombinant plasmids (b): pcTPANS3, pcTPANS3P, pcTPANS3H, pcNS3, pcNS3P, pcNS3H.Boxes represent the NS3 region (gray areas), the protease domain (streaked areas) and the helicase/RTPase/NTPase (dotted areas) domain; black box symbolizes the signal peptide derived from the human tissue plasminogen activator (t-PA).

Mentions: Six DNA vaccines, pcNS3, pcNS3H, pcNS3P, pcTPANS3, pcTPANS3H and pcTPANS3P, were constructed as described in materials and methods and represented in figure 1. The pcNS3 and pcTPANS3 vaccines encode the full-length NS3 protein. The pcNS3P and pcTPANS3P plasmids contain the sequence that codes the first 185 NS3 N-terminal amino acids, which enclose the protease domain, while constructs pcNS3H and pcTPANS3H encode the 433 NS3 C-terminal amino acids (from residues 186 to 618), which correspond to the RTPase/NTPase/helicase domains. Fragments were cloned without any signal sequence (pcNS3P, pcNS3H and pcNS3), for intracellular expression, or in frame with the t-PA signal peptide (pcTPANS3P, pcTPAND3H and pcTPANS3), for the secretion of recombinant proteins (Fig. 1).


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)

Schematic representation of the DENV2 genome (a) and the regions coded by the different recombinant plasmids (b): pcTPANS3, pcTPANS3P, pcTPANS3H, pcNS3, pcNS3P, pcNS3H.Boxes represent the NS3 region (gray areas), the protease domain (streaked areas) and the helicase/RTPase/NTPase (dotted areas) domain; black box symbolizes the signal peptide derived from the human tissue plasminogen activator (t-PA).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0025685-g001: Schematic representation of the DENV2 genome (a) and the regions coded by the different recombinant plasmids (b): pcTPANS3, pcTPANS3P, pcTPANS3H, pcNS3, pcNS3P, pcNS3H.Boxes represent the NS3 region (gray areas), the protease domain (streaked areas) and the helicase/RTPase/NTPase (dotted areas) domain; black box symbolizes the signal peptide derived from the human tissue plasminogen activator (t-PA).
Mentions: Six DNA vaccines, pcNS3, pcNS3H, pcNS3P, pcTPANS3, pcTPANS3H and pcTPANS3P, were constructed as described in materials and methods and represented in figure 1. The pcNS3 and pcTPANS3 vaccines encode the full-length NS3 protein. The pcNS3P and pcTPANS3P plasmids contain the sequence that codes the first 185 NS3 N-terminal amino acids, which enclose the protease domain, while constructs pcNS3H and pcTPANS3H encode the 433 NS3 C-terminal amino acids (from residues 186 to 618), which correspond to the RTPase/NTPase/helicase domains. Fragments were cloned without any signal sequence (pcNS3P, pcNS3H and pcNS3), for intracellular expression, or in frame with the t-PA signal peptide (pcTPANS3P, pcTPAND3H and pcTPANS3), for the secretion of recombinant proteins (Fig. 1).

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