Purification and immunodetection of the recombinant NS3Pro and NS3Hel.

<p>Conditions for growth of <i>E</i>. <i>co</i>li 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.</p

T-cell responses induced by vaccination with recombinant protein.

<p>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.</p

Depletion of CD4 and CD8 T cell subsets followed by IFN- γ ELISPOT.

<p>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.</p

Serum IgG antibody responses.

<p>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.</p

Neutralizing antibody responses.

<p>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.</p