Fig 1.
Schematic diagrams of SFTSV DNA vaccines used in this study.
pSFTSV was constructed by inserting genes encoding ectodomains of Gn and Gc, and NP-NS fusion protein into the pGX2 vector. The viral antigens are preceded by the secretory signal sequence of tPA and the extracellular domain of Flt3L. pSFTSV-IL-12 includes additional genes encoding murine IL-12α and β to promote cellular immunity. ColE1, ColE1-type bacterial origin of replication; gIVS, rabbit β-globin intervening sequence; KanR, kanamycin resistance gene; IRES, internal ribosome entry site.
Fig 2.
Characterization of gene expression after SFTSV DNA vaccine transfection.
(A) Expression and secretion of the viral antigens and IL-12 in HEK293 cells transfected with the DNAs was confirmed by measuring the concentration of Flt3L and IL-12 in the cell culture supernatants by ELISA. Data are presented as mean + S.D. from triplicated experiments. (B) Expression of the viral antigens, Gn (~ 74 kDa), Gc (~ 71 kDa), and NP-NS fusion (~ 80 kDa) proteins, in cell lysates (left panels) and culture supernatants (right panels) of transfected HEK293 cells was assessed by immunoblot analysis using anti-Gn, Gc, or NP antibodies, respectively. The specific bands of antigens corresponding to their expected sizes are indicated with arrow heads. β-actin was used as loading control.
Fig 3.
Generation of antigen-specific antibodies and T cell responses in IFNAR KO mice vaccinated with SFTSV DNA vaccines.
(A) Anti-NP IgG response was measured by ELISA at two weeks after the third vaccination. The antibody titers of anti-NP IgG in sera of mice (n = 3) immunized with different DNA vaccines are presented. Cut-off titers (dashed line, mean O.D. + 3 x S.D. at 1:100 diluents) was determined using sera from vector-immunized mice. Error bar: mean ± S.D. (B and C) Splenocytes were collected from mice at two weeks after the third immunization with the indicated DNA vaccines. Production of IFN-γ by CD4+ T or CD8+ T cells were analyzed by flow cytometry after stimulation with the indicated antigens. Representative flow cytometric results are presented (B) and the percentile of cytokine positive cells among CD4+ or CD8+ T cell subsets are summarized (C). Data shown as mean + S.D. from duplicate assays with three mice per group. *, p < 0.05; **, p < 0.01.
Fig 4.
Complete protection of IFNAR KO mice after vaccination with pSFTSV-IL-12.
(A) Survival rates (left panel) and body weight changes (right panel) of mice (n = 5/group) immunized three times with the indicated DNAs and challenged s.c. with 105 FFU of SFTSV at two weeks after vaccination. (B) Platelet counts (left panel) and viral loads in plasma (right panel) of mice vaccinated with the indicated DNA vaccines are presented. Blood samples were collected at 2 (D2) and 4 (D4) days after viral challenge. Uninf, uninfected; n = 3 ~ 4 mice/group. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fig 5.
Partial protection of IFNAR KO mice after vaccination with Gn-Fc antigen.
(A) Anti-Gc (blue dots) or Gn (red dots) IgG response was measured by ELISA at two weeks after the third immunization with Gn-Fc or Gc-Fc in IFNAR KO mice (n = 4/group). Cut-off titers (dashed line, mean O.D. + 3 x S.D. at 1:100 diluents) was determined using Fc-immune sera (black dots). The antibody titers of anti-Gc or Gn IgG in sera of immunized mice are summarized in the right panel. Error bar, mean + S.D.; black line, mean. (B) Neutralizing antibody response to SFTSV generated by SFTSV DNA vaccines in IFNAR KO mice. The amount of neutralizing antibody against SFTSV was determined based on FRNT50 (left panel) and summarized (right panel). (C) Survival rates (left panel) and body weight changes (right panel) of mice immunized three times with Fc (black dots, n = 6), Gc-Fc (blue dots, n = 4), or Gn-Fc (red dots, n = 6) antigen and challenged s.c. with 105 FFU of SFTSV at two weeks after vaccination. *, p < 0.05; **, p < 0.01.