Dengue MTase mutants are attenuated and immunogenic.

<p>(A) Viremia kinetics of WT DENV-1 (strain West Pacific 74), DENV-1 K61A, and DENV-1-E216A or a combination of DENV-1 E216A and DENV-2 E217A <i>in vivo</i>. Mice were infected i.p. with 2.75×10⁵ PFU of the indicated virus. Viral titers in the plasma were measured at indicated time points by real-time RT-PCR. (B) Viral titers in plasma of mice immunized i.p. with 2.75×10⁵ PFU DENV-2 WT, DENV-2 E217A (strain TSV01) alone or in combination with DENV-1 E216A (2.75×10⁵ PFU DENV-1 E216A plus 2.75×10⁵ PFU DENV-2 E217A). Blood was taken at indicated time points and viral titers were measured by real-time PCR. (C) Viral titers in the plasma of mice immunized with 2′-<i>O</i>-MTase mutant and challenged as indicated. Numbers in gray boxes indicate WT virus, whereas numbers in white boxes indicate 2′-<i>O</i>-MTase mutant virus. Mice were immunized i.p. with 2.75×10⁵ PFU of the indicated 2′-<i>O</i>-MTase mutant serotype and challenged 30 days later with 5×10⁵ PFU WT DENV-1 strain (strain 05K3126 used for challenge due to its high virulence in mice, unpublished data) or 3×10⁶ PFU WT DENV-2. Blood was taken at indicated time points and viral titers were measured by plaque assay. ND: not detected. (D, E) IgG titers of mice immunized and challenged as described above. Blood was taken at indicated time points post challenge and IgG antibody titers against DENV-1 (D) and DENV-2 (E) were measured by ELISA. Data are representative of two experiments with three to four mice per group in each experiment (A, B) or two pooled experiments (C–E) with a total of 9 mice per group. Bars represent means with SD (A) or means with SEM (B–E).