Immune responses and therapeutic antitumor effects of an experimental DNA vaccine encoding human papillomavirus type 16 oncoproteins genetically fused to herpesvirus glycoprotein D

Abstract

Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8(+) T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8(+) T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4(+) T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8(+) T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8(+) T-cell responses, measured by intracellular gamma interferon (IFN-γ) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2D(b)-restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 × 10(5) TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors.

FIG. 1.

Activation of E7-specific CD8⁺ T-cell precursors and preventive anticancer responses in mice immunized with pgD-E7E6E5 administered via the i.m. route. Individual (A) and pooled (B) CD8⁺ T-cell responses in mice immunized with one or two doses of pgD, pE7E6E5, or pgD-E7E6E5 delivered via the i.m. route (100 μg/dose). The level of E7-specific CD8⁺ T cells was determined 2 weeks after the last vaccine dose. Detection of IFN-γ-producing E7-specific CD8⁺ T cells was carried out with PBMC stimulated with the MHC-I-restricted E7 peptide (⁴⁹RAHYNIVTF⁵⁷) and stained for CD8 (with FITC) and intracellular IFN-γ (with PE).

FIG. 2.

E7-specific CD8⁺ T-cell response and preventive antitumor effects in pgD-E7E6E5-vaccinated mice. Mice were immunized with one dose of pgD-E7E6E5, pE7E6E5, or pgD and challenged with 5 × 10⁵ TC-1 tumor cells 100 days after vaccine administration. Long-term (A) and time point-specific (day 121) (B) detection of IFN-γ-producing E7-specific CD8⁺ T cells in pooled PBMC. E7-specific CD8⁺ T cells were stimulated with the E7 peptide (⁴⁹RAHYNIVTF⁵⁷) and stained for CD8 (with FITC) and intracellular IFN-γ (with PE). (C) Long-term preventive antitumor responses in vaccinated mice. Tumor growth was monitored up to 60 days after challenge. The arrow indicates when the TC-1 cell challenge occurred.

FIG. 3.

Priming/boosting effects on E7-specific CD8⁺ T-cell responses in mice submitted to a heterologous prime-boost immunization regimen using pgD-E7E6E5 and a recombinant adenovirus vector encoding the same antigen. Groups of mice (n = 5) were primed with pgD-E7E6E5 and subsequently boosted with the recombinant adenovirus vector (AdC68gD-E7E6E5) 3 months later (▴). The same procedure was repeated in mice immunized with AdC68gD-E7E6E5 and subsequently boosted with pgD-E7E6E5 (▪). Detection of IFN-γ-producing E7-specific CD8⁺ T cells was carried out with pooled PBMC stimulated with the MHC-I-restricted E7 peptide (⁴⁹RAHYNIVTF⁵⁷) and stained for CD8 (with FITC) and intracellular IFN-γ (with PE).

FIG. 4.

Activation of antigen-specific cytotoxic CD8⁺ T cells in mice immunized with pgD-E7E6E5 after challenge with TC-1 cells. (A) Frequencies of IFN-γ-producing CD8⁺ T cells following in vitro stimulation of splenocytes harvested from mice therapeutically treated with three doses of pgD, pE7E6E5, or pgD-E7E6E5. Fourteen days after the last vaccine dose, splenocytes were stimulated with the E5, E6, or E7 peptide and stained for CD8 (with FITC) and intracellular IFN-γ (with PE). Mice were challenged with 5 × 10⁵ TC-1 tumor cells. (B) Frequencies of CD107a-producing CD8⁺ T cells following in vitro stimulation of splenocytes harvested from mice therapeutically treated with three doses of pgD, pE7E6E5, or pgD-E7E6E5. Stimulation with the specific E5, E6, or E7 peptide was performed in the presence of anti-CD107a (with PE), and after the incubation period, spleen cells were stained for CD8 (with PE Cy5) and intracellular IFN-γ (with FITC). Mice were challenged with 5 × 10⁵ TC-1 tumor cells. (C) In vivo cytotoxic activities of antigen-specific CD8⁺ T cells induced in mice immunized with different DNA vectors. Ten days after the last vaccine dose, mice received target spleen cells pulsed with E5, E6, or E7 CD8⁺ T-cell-specific peptide and stained with 5 mM CSFE (right-side curves) or 0.5 mM CSFE (left-side curves) but not pulsed with the target peptides. One day later, total spleen cells were harvested and monitored by flow cytometry. Results from representative mice immunized with one of the tested DNA vectors. Reduction of the peak area corresponding to the cell population labeled with antigen-specific derived peptides (right-side curve), with regard to CFSE-labeled cells not exposed to the peptides (left-side curve), reflects the in vivo cytotoxic activity of antigen-specific CD8⁺ T cells. (D) Individual in vivo cytotoxic activities detected in mice immunized with different DNA vaccine vectors. CFSE-labeled spleen cells were pulsed with MCH-I-restricted peptides derived from E5 (•), E6 (▴), and E7 (⧫) oncoproteins. Values detected only in mice immunized with pgD-E7E6E5 after stimulation with E7- or E6-specific peptides showed statistically significant differences (P < 0.0001) with regard to mice immunized with pgD or pE7E6E5.

FIG. 5.

Therapeutic antitumor effects and induced E7-specific CD8⁺ T-cell responses in mice immunized with pgD-E7E6E5. (A) Dose-dependent antitumor protection in mice immunized with one, two, or three i.m. doses of pgD-E7E6E5. (B) Frequencies of E7-specific IFN-γ-producing CD8⁺ T cells in mice challenged with TC-1 cells and i.m immunized 8 h later with one, two, or three doses of pgD-E7E6E5 delivered at weekly intervals. (C) Therapeutic antitumor effects of pgD-E7E6E5 in mice challenged with TC-1 cells and subsequently immunized with three doses of the DNA vaccine on the same day (day 0) of the TC-1 challenge or 3, 5, 7, or 10 days after challenge.

FIG. 6.

Coadministration of pgD-E7E6E5 and IL-12- or GM-CSF-encoding plasmid confers full therapeutic protection to the TC-1 cell challenge. Mice were i.m. immunized with three doses (100 μg) of pE7E6E5 or pgD-E7E6E5 admixed with 100 μg of pcDNA3.1 (empty vector), pGM-CSF, or pIL-12 vector. The vaccines were administered 8 h after challenge with 5 × 10⁵ TC-1 cells. Tumor growth was monitored up to 60 days after challenge. Statistically significant differences were noted (P = 0.0023) using the log rank test with regard to mice immunized with pgD-E7E6E5 plus pcDNA3.1 and pgD-E7E6E5 plus pIL-12 or pgD-E7E6E5 plus pGM-CSF vectors.