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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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Survival percentage of Balb/c mice immunized with pcTPANS3, pcTPANS3H and pcTPANS3P (a) or pcNS3, pcNS3H and pcNS3P (b) and challenged with the NGC DENV2.Mice were immunized with two DNA doses and challenged 4 weeks after the first plasmid inoculation. Non-immunized (DENV2) and pcTPA or pcDNA3-injected mice followed the same dengue virus infection procedure. Mice were daily monitored and death was recorded. Data represent compilation of two (a) or one (b) independent experiments, with groups of 10 animals in each test (n = 20 and n = 10, respectively).
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pone-0025685-g004: Survival percentage of Balb/c mice immunized with pcTPANS3, pcTPANS3H and pcTPANS3P (a) or pcNS3, pcNS3H and pcNS3P (b) and challenged with the NGC DENV2.Mice were immunized with two DNA doses and challenged 4 weeks after the first plasmid inoculation. Non-immunized (DENV2) and pcTPA or pcDNA3-injected mice followed the same dengue virus infection procedure. Mice were daily monitored and death was recorded. Data represent compilation of two (a) or one (b) independent experiments, with groups of 10 animals in each test (n = 20 and n = 10, respectively).

Mentions: The protective efficacy of plasmids encoding the full-length NS3 protein or its functional domains was evaluated in mice immunized with such DNA vaccines and challenged with a mouse brain-adapted DENV2. Results indicated that the tested vaccines induced different levels of protection depending on the codified NS3 region, regardless the presence or not of the signal peptide. Animals vaccinated with pcTPANS3 presented 90% of survival rate, while 70% of pcTPANS3H-immunized mice survived virus challenge, although this difference was not statistically significant (Fig. 4a). However, both group showed statistically significant differences of survival rates when compared to control groups, in which only 25% and 30% of non-immunized and pcTPA-inoculated animals, respectively, survived virus inoculation (p<0.0001) (Fig. 4a). Similar results were observed in the mouse groups inoculated with pcNS3 and pcNS3H plasmids, both presenting survival rates of 80%, which was statistically different (p<0.0001) from control animals (20% and 30%, in non-immunized and pcDNA3-inoculated mice, respectively) (Fig. 4b). Furthermore, the presence of the t-PA signal sequence in the constructions encoding either the entire NS3 or the helicase domain did not induce statistical differences in survival rates, when compared to results observed with similar plasmids without this signal peptide. In contrast, none of the DNA vaccines encoding only the NS3 protease domain induced protection against the DENV2 challenge, with survival rates of 25% and 30% for pcTPANS3P- and pcNS3P-immunized mice, respectively, which was similar to those observed in control groups (Figs. 4a and 4b).


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)

Survival percentage of Balb/c mice immunized with pcTPANS3, pcTPANS3H and pcTPANS3P (a) or pcNS3, pcNS3H and pcNS3P (b) and challenged with the NGC DENV2.Mice were immunized with two DNA doses and challenged 4 weeks after the first plasmid inoculation. Non-immunized (DENV2) and pcTPA or pcDNA3-injected mice followed the same dengue virus infection procedure. Mice were daily monitored and death was recorded. Data represent compilation of two (a) or one (b) independent experiments, with groups of 10 animals in each test (n = 20 and n = 10, respectively).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198735&req=5

pone-0025685-g004: Survival percentage of Balb/c mice immunized with pcTPANS3, pcTPANS3H and pcTPANS3P (a) or pcNS3, pcNS3H and pcNS3P (b) and challenged with the NGC DENV2.Mice were immunized with two DNA doses and challenged 4 weeks after the first plasmid inoculation. Non-immunized (DENV2) and pcTPA or pcDNA3-injected mice followed the same dengue virus infection procedure. Mice were daily monitored and death was recorded. Data represent compilation of two (a) or one (b) independent experiments, with groups of 10 animals in each test (n = 20 and n = 10, respectively).
Mentions: The protective efficacy of plasmids encoding the full-length NS3 protein or its functional domains was evaluated in mice immunized with such DNA vaccines and challenged with a mouse brain-adapted DENV2. Results indicated that the tested vaccines induced different levels of protection depending on the codified NS3 region, regardless the presence or not of the signal peptide. Animals vaccinated with pcTPANS3 presented 90% of survival rate, while 70% of pcTPANS3H-immunized mice survived virus challenge, although this difference was not statistically significant (Fig. 4a). However, both group showed statistically significant differences of survival rates when compared to control groups, in which only 25% and 30% of non-immunized and pcTPA-inoculated animals, respectively, survived virus inoculation (p<0.0001) (Fig. 4a). Similar results were observed in the mouse groups inoculated with pcNS3 and pcNS3H plasmids, both presenting survival rates of 80%, which was statistically different (p<0.0001) from control animals (20% and 30%, in non-immunized and pcDNA3-inoculated mice, respectively) (Fig. 4b). Furthermore, the presence of the t-PA signal sequence in the constructions encoding either the entire NS3 or the helicase domain did not induce statistical differences in survival rates, when compared to results observed with similar plasmids without this signal peptide. In contrast, none of the DNA vaccines encoding only the NS3 protease domain induced protection against the DENV2 challenge, with survival rates of 25% and 30% for pcTPANS3P- and pcNS3P-immunized mice, respectively, which was similar to those observed in control groups (Figs. 4a and 4b).

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