Abstract
BackgroundThe dengue non-structural 3 (NS3) is a multifunctional protein, containing a serine-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 evaluated the potential of the NS3 (protease domain) as a protective antigen by comparing the administration of a recombinant protein versus a DNA vaccine in the mouse model.ResultsBALB/c mice were immunized with the recombinant protein NS3-DEN3 via intraperitoneal and with plasmid pcDNA3/NS3-DEN3 intramuscularly and the immune response was evaluated. The activity of T lymphocytes was analyzed by the MTT assay, and cells of mice immunized with the recombinant protein showed no activity when stimulated with the homologous protein. However, cells from mice immunized with DNA, responded to stimulation with the recombinant protein. When the expression (RT-PCR) and cytokine production (ELISA) was evaluated in the splenocytes, different behavior depending on the type of immunization was observed, splenocytes of mice immunized with the recombinant protein expressed cytokines such as IL-4, IL-10 and produced high concentrations of IL-1, IL-6 and TNFα. Splenocytes from mice immunized with DNA expressed IL-2 and IFNγ and did not produce IL-6. In addition, immunization with the recombinant protein induced the production of antibodies that are detected up to a dilution 1:3200 by ELISA and Western blot assays, however, the serum of mice immunized with DNA presented no detectable antibody titers.ConclusionThe results obtained in this study show that administration of pcDNA3/NS3-DEN3 induces a favorable response in the activation of T lymphocytes with low production of specific antibodies against NS3-DEN3.
Highlights
The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serine-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region
DNA plasmids pcDNA3 and pcDNA3/NS3-DEN3 were isolated from bacteria by alkaline lysis procedure, and DNA bands were visualized by ethidium bromide staining (Fig. 2d)
The results showed that the antibodies obtained by immunization with the Glutation -STransferase (GST)-NS3-DEN3 recombinant protein recognize their homologous protein at a dilution of 1:1600 by Enzyme-Linked ImmunoSorbent Assay (ELISA) assays (Fig. 5a) and up to a dilution of 1:3200 by WB (Fig. 5b)
Summary
The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serine-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. The etiological agent of dengue disease is the Dengue virus (DENV) being the principal arthropod-borne viral pathogen affecting human populations It is an enveloped virus member of the Flaviviridae family, containing a ~11 kb genome of positive single-stranded RNA which encodes three structural proteins (C, pr-M, E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) [2]. The infection caused by dengue virus is classified into dengue with/without warning signs and severe dengue [4]
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