Development of a DNA vaccine targeting human papillomavirus type 16 oncoprotein E6

Abstract

Human papillomavirus (HPV), particularly type 16 (HPV-16), is present in more than 99% of cervical cancers. The HPV oncoproteins E6 and E7 are constantly expressed and therefore represent ideal targets for HPV vaccine development. We previously developed DNA vaccines encoding calreticulin (CRT) linked to HPV-16 E7 and generated potent E7-specific CD8(+) T-cell immune responses and antitumor effects against an E7-expressing tumor. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a DNA vaccine encoding CRT linked to E6 (CRT/E6). Our results indicated that the CRT/E6 DNA vaccine, but not a wild-type E6 DNA vaccine, generated significant E6-specific CD8(+) T-cell immune responses in vaccinated mice. Mapping of the immunodominant epitope of E6 revealed that an E6 peptide comprising amino acids (aa) 48 to 57 (E6 aa48-57), presented by H-2K(b), is the optimal peptide and that the region of E6 comprising aa 50 to 57 represents the minimal core sequence required for activating E6-specific CD8(+) T lymphocytes. We also demonstrated that E6 aa48-57 contains cytotoxic T-lymphocyte epitopes naturally presented by E6-expressing TC-1 cells. Vaccination with a CRT/E6 but not a CRT/mtE6 (lacking aa 50 to 57 of E6) DNA vaccine could protect vaccinated mice from challenge with E6-expressing TC-1 tumors. Thus, our data indicate that E6 aa48-57 contains the immunodominant epitope and that a CRT/E6 DNA vaccine may be useful for control of HPV infection and HPV-associated lesions.

FIG. 1.

Intracellular cytokine staining with flow cytometry analysis to determine the proportion of IFN-γ-expressing, E6-specific CD8⁺ T-cell precursors. Mice were immunized, and splenocytes were collected and cultured, as described in Materials and Methods. Pooled splenocytes from vaccinated mice (5 mice/group) were cultured in vitro with various overlapping E6 peptides overnight and were stained for both CD8 and intracellular IFN-γ. (A) Representative flow cytometry data for splenocytes harvested from mice vaccinated with pcDNA3-E6 and either left unstimulated or stimulated overnight with the E6 peptide aa1-45, aa36-80, aa71-115, aa106-138, or aa134-158. (B) Representative flow cytometry data for splenocytes harvested from mice vaccinated with pcDNA3-CRT/E6 and stimulated overnight with one of the E6 peptides listed above. (C) Bar graph depicting the number of E6-specific IFN-γ-expressing CD8⁺ T-cell precursors/3 × 10⁵ splenocytes (mean ± SD) from mice vaccinated with pcDNA3-E6 or pcDNA3-CRT/E6. Data presented in this figure are from one experiment representative of two performed. Note that the E6 peptide aa36-80 was the only peptide to activate E6-specific CD8⁺ T cells.

FIG. 2.

Intracellular cytokine staining with flow cytometry analysis of IFN-γ-expressing, E6-specific CD8⁺ T-cell precursors generated by splenocytes stimulated with various E6 peptides. Mice were immunized with pcDNA3-CRT/E6, and splenocytes were collected and cultured. (A) Representative flow cytometry data for splenocytes harvested from mice and either left unstimulated or stimulated overnight with the E6 peptide aa36-80, aa48-57, or aa50-57. (B) Bar graph showing the number of E6-specific IFN-γ-expressing CD8⁺ T-cell precursors per 3 × 10⁵ splenocytes (mean ± SD) generated by in vitro stimulation. Data presented in this figure are from one experiment representative of two performed.

FIG. 3.

Intracellular cytokine staining and flow cytometry analysis to determine the MHC class I H-2^b binding restriction of the E6 aa50-57 epitope. An E6-specific CD8⁺ T-cell line was used to determine the MHC class I binding restriction. Two E6 peptides (aa48-57 and aa50-57) were used. A peptide from ovalbumin (OVA) was used as a negative control. (A) Representative flow cytometry data showing percentages of IFN-γ-expressing E6-specific CD8⁺ T cells in cultures of E6-specific CD8⁺ T cells coincubated with peptide-pulsed C1R, C1R/D^b, or C1R/K^b cells. (B) Bar graph showing percentages of IFN-γ⁺ CD8⁺ T cells per total CD8⁺ T cells in cultures described above. Note that the data show that the E6 aa48-57 and aa50-57 epitopes are K^b restricted.

FIG. 4.

Determination of the percentage of activated E6-specific CD8⁺ T cells within the total number of E6-specific CD8⁺ T cells at decreasing peptide concentrations. An E6-specific CD8⁺ T-cell line was used to determine the optimal and essential components of the E6 immunodominant epitope. T2-K^b cells were pulsed with various E6 peptides (aa48-56, aa48-57, aa49-57, aa50-57, or aa51-58) or a negative-control peptide from ovalbumin (SIINFEKL) at sequentially decreasing concentrations from 10⁻⁴ to 10⁻⁹ M. The peptide-pulsed T2-K^b cells were then coincubated with an HPV-16 E6-specific CTL line at an E:T ratio of 1:10. Intracellular cytokine staining followed by flow cytometry analysis was used to determine the percentage of IFN-γ-expressing E6-specific CD8⁺ T cells within the total E6-specific CD8⁺ T-cell population.

FIG. 5.

Intracellular cytokine staining with flow cytometry analysis and chromium release assay to demonstrate that TC-1 cells naturally process the E6 epitope containing aa 50 to 57. (A) Mice were immunized with pcDNA3-CRT/E6 or pcDNA3-CRT/mtE6. Flow cytometry data indicate the number of IFN-γ-expressing CD8⁺ T-cell precursors generated from splenocytes harvested from vaccinated mice and pulsed with either E6 aa48-57 or E6-expressing TC-1 cells. (B) Bar graph shows the number of E6-specific IFN-γ⁺ CD8⁺ T cells/3 × 10⁵ splenocytes (mean ± SD). (C) The lytic activity of T cells was assessed by using a standard chromium release assay. TC-1 and EL-4 cells were used as target cells. EL-4 cells pulsed with the E6 peptide aa48-57 were used as a positive control. Splenocytes stimulated in vitro with E6 peptide served as effector cells. Percent cytotoxicity (specific lysis) was calculated. Data were collected from cultures measured at E:T ratios of 1:10, 1:1, 10:1, and 25:1. Results are expressed as percent cytotoxicity ± SD.

FIG. 6.

In vivo tumor protection experiment to demonstrate the antitumor effect generated by pcDNA3-CRT/E6 against E6-expressing TC-1 tumors and the effects of lymphocyte subsets on tumor protection. (A) Mice were immunized with pcDNA3-CRT, pcDNA3-E6, pcDNA3-CRT/E6, or pcDNA3-CRT/mtE6. One week after vaccination, mice were challenged subcutaneously with 5 × 10⁴ TC-1 cells/mouse; they were then monitored for evidence of tumor growth by palpation and inspection twice a week. (B) In vivo antibody depletion experiments to determine the effects of lymphocyte subsets on the tumor protection of the CRT/E6 DNA vaccine. CD4, CD8, and NK1.1 depletions were initiated 1 week before tumor challenge. Data shown in this figure are from one experiment representative of two performed.