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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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Morbidity degree of Balb/c mice inoculated with pcTPANS3, pcTPANS3H, pcTPANS3P and pcTPA (a) or pcNS3, pcNS3H, pcNS3P and pcDNA3 (b) and challenged with the NGC DENV2.Control groups of non-immunized animals (DENV2) were also challenge with the dengue virus. Clinical signs of infection, mainly hind leg paralysis, alterations in spinal column and deaths, were monitored during 21 days post challenge. The semi-quantitative analysis of morbidity degrees after virus challenge were performed using a subjective scale ranging from 0 to 3 (0 =  none, 1 =  mild paralyses in one hind leg or alteration of the spinal column with small bump, 2 =  one severe hind leg paralyses and alteration of the spinal column with small bump or two severe hind leg paralyses, 3 =  two severe hind leg paralyses and bump deformed spinal column or death). Asterisks indicate statistically significant differences (***, p<0.0001; **, p<0.01; *, p<0.04).
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pone-0025685-g005: Morbidity degree of Balb/c mice inoculated with pcTPANS3, pcTPANS3H, pcTPANS3P and pcTPA (a) or pcNS3, pcNS3H, pcNS3P and pcDNA3 (b) and challenged with the NGC DENV2.Control groups of non-immunized animals (DENV2) were also challenge with the dengue virus. Clinical signs of infection, mainly hind leg paralysis, alterations in spinal column and deaths, were monitored during 21 days post challenge. The semi-quantitative analysis of morbidity degrees after virus challenge were performed using a subjective scale ranging from 0 to 3 (0 =  none, 1 =  mild paralyses in one hind leg or alteration of the spinal column with small bump, 2 =  one severe hind leg paralyses and alteration of the spinal column with small bump or two severe hind leg paralyses, 3 =  two severe hind leg paralyses and bump deformed spinal column or death). Asterisks indicate statistically significant differences (***, p<0.0001; **, p<0.01; *, p<0.04).

Mentions: Although most of animals immunized with the DNA vaccines based in full-length NS3 survived virus challenge, 40% and 60% of pcTPANS3- and pcNS3-immunized mice, respectively, showed clinical signs of infection (Fig. 5). In fact, 30% of these animals manifested severe neurological signs (grades 2 and 3). However, the morbidity rate in these groups were significant different comparing to non-immunized mice, in which 95% to 100% of animals presented several clinical signs of infection (p<0,0001 for pcTPANS3 and p<0,01 for pcNS3) (Figs. 5a and 5b). In contrast, 75% to 80% of mice inoculated with the vaccines containing the helicase domain and 90% of animals injected with plasmids based on the protease sequence presented clinical signs after challenge (Fig. 5), and these morbidity degrees were not statistically different from that observed in non-immunized groups. Furthermore, results showed that pcTPANS3H-immunized animals presented statistically higher morbidity degrees when compared to mice vaccinated with the pcTPANS3 (p<0.04). Thus, data indicated that only immunization with plasmids based on the full-length NS3 protein confered some protection, when mortality and morbidity parameters were taken together.


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)

Morbidity degree of Balb/c mice inoculated with pcTPANS3, pcTPANS3H, pcTPANS3P and pcTPA (a) or pcNS3, pcNS3H, pcNS3P and pcDNA3 (b) and challenged with the NGC DENV2.Control groups of non-immunized animals (DENV2) were also challenge with the dengue virus. Clinical signs of infection, mainly hind leg paralysis, alterations in spinal column and deaths, were monitored during 21 days post challenge. The semi-quantitative analysis of morbidity degrees after virus challenge were performed using a subjective scale ranging from 0 to 3 (0 =  none, 1 =  mild paralyses in one hind leg or alteration of the spinal column with small bump, 2 =  one severe hind leg paralyses and alteration of the spinal column with small bump or two severe hind leg paralyses, 3 =  two severe hind leg paralyses and bump deformed spinal column or death). Asterisks indicate statistically significant differences (***, p<0.0001; **, p<0.01; *, p<0.04).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198735&req=5

pone-0025685-g005: Morbidity degree of Balb/c mice inoculated with pcTPANS3, pcTPANS3H, pcTPANS3P and pcTPA (a) or pcNS3, pcNS3H, pcNS3P and pcDNA3 (b) and challenged with the NGC DENV2.Control groups of non-immunized animals (DENV2) were also challenge with the dengue virus. Clinical signs of infection, mainly hind leg paralysis, alterations in spinal column and deaths, were monitored during 21 days post challenge. The semi-quantitative analysis of morbidity degrees after virus challenge were performed using a subjective scale ranging from 0 to 3 (0 =  none, 1 =  mild paralyses in one hind leg or alteration of the spinal column with small bump, 2 =  one severe hind leg paralyses and alteration of the spinal column with small bump or two severe hind leg paralyses, 3 =  two severe hind leg paralyses and bump deformed spinal column or death). Asterisks indicate statistically significant differences (***, p<0.0001; **, p<0.01; *, p<0.04).
Mentions: Although most of animals immunized with the DNA vaccines based in full-length NS3 survived virus challenge, 40% and 60% of pcTPANS3- and pcNS3-immunized mice, respectively, showed clinical signs of infection (Fig. 5). In fact, 30% of these animals manifested severe neurological signs (grades 2 and 3). However, the morbidity rate in these groups were significant different comparing to non-immunized mice, in which 95% to 100% of animals presented several clinical signs of infection (p<0,0001 for pcTPANS3 and p<0,01 for pcNS3) (Figs. 5a and 5b). In contrast, 75% to 80% of mice inoculated with the vaccines containing the helicase domain and 90% of animals injected with plasmids based on the protease sequence presented clinical signs after challenge (Fig. 5), and these morbidity degrees were not statistically different from that observed in non-immunized groups. Furthermore, results showed that pcTPANS3H-immunized animals presented statistically higher morbidity degrees when compared to mice vaccinated with the pcTPANS3 (p<0.04). Thus, data indicated that only immunization with plasmids based on the full-length NS3 protein confered some protection, when mortality and morbidity parameters were taken together.

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