Figure 1.
Construction of bicistronic DNA vaccines encoding HPV, HIV and HSV antigens for expression in mammalian cells.
(A) Schematic linear representation of the trivalent DNA vaccines. pIRES I and pIRES II contain gDp24 and gDE7 chimeric gene fusions, which are inverted with regard to the CMV promoter and IRES sequence. The empty vector pIRES Ø was used as a control. The nucleotide numbers corresponding to the IRES sequence and the cloned chimeric genes are indicated. (B) In vitro expression of the chimeric proteins encoded by pIRES I (left panels) and pIRES II (right panels). Non-permeabilized pIRES I- or pIRES II-transfected COS-7 cells were labeled with antigen-specific antibodies for the simultaneous detection of the HSV-1 protein gD and the HIV-1 protein p24 or the HPV-16 oncoprotein E7. Green, gD; red, p24 or E7; yellow, co-localization of gD with p24 or E7; blue, DAPI nuclear staining.
Figure 2.
Activation of antigen-specific IFN-γ-producing CD8+ T cell precursors in mice immunized with pIRES I or pIRES II.
(A-B) Spleen cells from BALB/c mice spleen cells were stimulated with the MHC-I-restricted p24-specific peptide, and the p24-specific IFN-γ-producing CD8+ T cells were detected by intracellular cytokine staining (A) or ELISPOT assay (B). (C–D) Spleen cells from C57BL/6 mice were stimulated with the MHC-I-restricted E7-specific peptide, and the E7-specific IFN-γ-producing CD8+ T cells were detected by IFN-γ intracellular staining (C) or ELISPOT assay (D). Mice were i.m. immunized with three doses of the DNA vaccines with one week intervals between doses (100 µg/dose). The CD8+ T-cell responses were analyzed two weeks after the last dose. *p<0.05. Data represent the compilation of two independent experiments with four mice per immunization group (n = 8) and results expressed by each animal analyzed. pIRES is the empty vector used as immunization control.
Figure 3.
Induction of in vivo E7- and p24-specific cytolytic CD8+ T cell responses in immunized mice.
(A–B) In vivo antigen-specific CD8+ T cell-dependent cytotoxic responses in the vaccinated mice was measured two weeks after the last immunization dose. Spleen cells from BALB/c or C57BL/6 mice were labeled with CFSE and pulsed with synthetic peptides representing the immunodominant MHC-I-restricted epitopes of p24 (A) or E7 (B). Data shown in A and B represent the compilation of two independent experiments, encompassing four and five mice per group (n = 9) with results based on the response of each animal. (C) The protective immunity elicited in BALB/c mice immunized with pIRES I or pIRES II was measured after challenge with a recombinant vaccinia virus expressing the HIV-1 protein Gag. Female BALB/c mice were challenged with 2×106 P.F.U. of rVV-Gag, and 5 days later, the level of viable vaccinia virus in the ovaries was determined after titration in Vero cells. Data shown in C represent the compilation of two independent experiments carried out with pooled samples from five mice per group. *p<0.05. (D) Prophylactic and (E) therapeutic anti-tumor immunity in C57BL/6 mice immunized with pIRES I or PIRES II. The prophylactic anti-tumor effects were determined in five vaccinated female mice after the s.c. transplantation of 7.5×104 TC-1 cells two weeks after the last vaccination. The therapeutic anti-tumor effects induced by the vaccines were determined after transplantation of 7.5×104 TC-1 cells one day before the administration of the first vaccine dose. Data shown in D and E represent the compilation of two independent experiments, with five mice per group. The survival curves D and E raised p values of 0.0001 and 0.0006, respectively, in the Logrank test for trend. pIRES is the empty vector used as immunization control.
Figure 4.
Induction of gD-specific functional T cell responses in mice immunized with pIRES I or pIRES II.
(A) Detection of gD-specific IFN-γ-secreting cells in vaccinated BALB/c mice was performed two weeks after the last immunization dose. Spleen cells from individual mice (n = 8) were cultured in the presence of full-length recombinant purified gD for 72 h. The frequencies of the gD-specific IFN-γ-secreting cells were measured by ELISPOT. (B) Detection of gD-specific IFN-γ-secreting CD8+ and CD4+ T cells in those mice described in (A) with spleen cells previously incubated for 72 h with the recombinant gD analyzed by flow cytometry. Data shown in A and B represent the compilation of two independent experiments, with four mice per group (n = 8). *p<0.05. (C) Protective anti-HSV immunity elicited in mice immunized with pIRES I or pIRES II. Vaccinated male BALB/c mice were challenged intranasal with the HSV-1 strain EK (5×104 P.F.U./mouse) two weeks after last vaccine dose and mice survival was monitored for 40 days. Data shown in C represent the compilation of two independent experiments, with five mice per group (n = 10). p = 0.0009 (Logrank test for trend). pIRES is the empty vector used as immunization control.