Induction of a Protective Response in Mice by the Dengue Virus NS3 Protein Using DNA Vaccines

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

Primers used for amplifications of different DENV2 NS3 sequences for construction of the different recombinant plasmids.

a<p>Vectors used for cloning.</p>b<p>Genome coordinates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025685#pone.0025685-Irie1" target="_blank">[64]</a>.</p>c<p>Forward primer (start codon is marked in bold type and <i>Eco</i>RV restriction site is underlined).</p>d<p>Reverse primer (stop codon is marked in bold type and <i>Xba</i> I restriction site is underlined).</p

Production of INF-γ by T cells of animals immunized with the DNA vaccines encoding the full-length NS3 protein.

<p>Splenocytes collected from groups of mice (n = 6) inoculated with pcTPANS3, pcNS3, pcDNA3 or pcTPA plasmids were stimulated with a T-cell specific peptide and the number of spot-forming cells (SFC) were quantified in a 24h ELISPOT assay. Asterisks indicate statistically significant differences (**, p<0.003).</p

Western blots of whole-cell extract (a) and culture supernatants (b) of BHK-21 cells transfected with the different DNA vaccines.

<p>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.</p

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.

<p>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).</p

Immunofluorescence of BHK-21 cells transfected with recombinant plasmids based on NS3 protein or its functional domains: pcTPANS3 (a), pcTPANS3H (b), pcTPANS3P (c), pcNS3 (d), pcNS3H (e) and pcNS3P (e).

<p>Cells were treated with a mouse polyclonal antibody against the DENV2 NS3 protein. Magnification 400x.</p

Schematic representation of the DENV2 genome (a) and the regions coded by the different recombinant plasmids (b): pcTPANS3, pcTPANS3P, pcTPANS3H, pcNS3, pcNS3P, pcNS3H.

<p>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).</p

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.

<p>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).</p