Fig 1.
Schematic diagram of the DENV-2 genome (A) and the regions coded by the different recombinant plasmids (B). Dengue virus is a single-stranded, positive-sense RNA genome of about 11 kb in length. The open reading frame encodes the structural proteins capsid (C), membrane (M), envelope (E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). The region linking the protease domain and helicase domain of NS3 (residues169 to 179) are shown in yellow. The last 40 residues of NS2b (90 to 130) were added to the NS3 protease (NS3Pro) fragment (1 to 196). The NS3 helicase (NS3Hel) fragment contains residues 174 to 554.
Fig 2.
Purification and immunodetection of the recombinant NS3Pro and NS3Hel.
Conditions for growth of E. coli transformed by the His-tagged expression plasmids and purification by metal affinity are described under Materials and Methods. The purified proteins were fractionated by SDS-PAGE (4–12%) and the gel was stained by Coomassie blue (A), and anti-DENV-2 HMAF was used for the Western blot (B). Lane 1, molecular size marker; lane 2, NS3Pro; lane 3, NS3Hel. Pro = protease, Hel = helicase.
Fig 3.
Anti-DENV-2 antibody responses in immunized mice measured by ELISA using purified virions. Bars indicate reciprocal geometric mean endpoint titers (GMT) ≥ 0.1 OD405. For calculation purposes titers <100 were assigned a value of 5. PIV = purified inactivated vaccine, Pro = protease, Hel = helicase.
Fig 4.
Neutralizing antibody responses.
Anti-DENV-2 neutralizing antibody responses in immunized mice on Day 35 after first inoculation. Neutralizing antibody titers were determined for each animal by plaque reduction neutralization test (PRNT). The Y-axis indicates reciprocal 50% PRNT (PRNT50) titers (Log10). For calculation purposes, titers <20 were given a value of 5. PIV = purified inactivated vaccine, Pro = protease, Hel = helicase.
Fig 5.
T-cell responses induced by vaccination with recombinant protein.
Groups of vaccinated mice were tested for IFN-γ responses using a standard ELISPOT assay. Mouse spleen cells collected on Day 35 after first immunization, were stimulated with overlapping peptide pools spanning the entire M, E and NS3 protein. Responses are presented as number of spots per one million cells. Results indicate the mean of 3 mice with error bars equal to one standard deviation. PIV = purified inactivated vaccine, Pro = protease, Hel = helicase, E = envelope, M = membrane.
Fig 6.
Depletion of CD4 and CD8 T cell subsets followed by IFN- γ ELISPOT.
Whole spleen cells (a) or spleen cells depleted with either CD4 or CD8 subset (b, c), were stained with anti-mouse CD4 or CD8 monoclonal antibodies. The % of each subset is shown. IFN-γ response from whole or subset- depleted spleen cells was measured by ELISPOT assay (d). Results represent number of spots per million cells. Control = media without peptides.