Therapeutic vaccination using minimal HPV16 epitopes in a novel MHC-humanized murine HPV tumor model

Abstract

Therapeutic vaccination as a treatment option for HPV-induced cancers is actively pursued because the two HPV proteins E6 and E7 represent ideal targets for immunotherapy, as they are non-self and expressed in all tumor stages. MHC-humanized mice are valuable tools for the study of therapeutic cancer vaccines - given the availability of a suitable tumor model. Here, we present for the first time an HPV16 tumor model suitable for fully MHC-humanized A2.DR1 mice, PAP-A2 cells, which in contrast to existing HPV16 tumor models allows the exclusive study of HLA-A2- and DR1-mediated immune responses, without any interfering murine MHC-presented epitopes. We used several HPV16 epitopes that were shown to be presented on human cervical cancer cells by mass spectrometry for therapeutic anti-tumor vaccination in the new tumor model. All epitopes were immunogenic when rendered amphiphilic by incorporation into a molecule containing stearic acids. Prophylactic and therapeutic vaccination experiments with the epitope E7/11-19 demonstrated that effective immune responses could be induced with these vaccination approaches in A2.DR1 mice. Interestingly, the combination of E7/11-19 with other immunogenic HPV16 E6/E7 epitopes caused a reduction of vaccine efficacy, although all tested combinations resulted in a survival benefit. In summary, we present the first HPV16 tumor model for exclusive studies of HLA-A2-mediated anti-HPV tumor immune responses and show anti-tumor efficacy of minimal epitope vaccines.

Keywords: A2.DR1; Cancer immunotherapy; HLA-humanized mouse model; PAP-A2; human papillomavirus (HPV); therapeutic vaccination.

Figure 1.

Characterization of the novel HPV16 E6/E7-expressing A2.DR1 tumor cell line, PAP-A2. (A) PAP-A2 cells were lysed and analyzed for E6 (left) and E7 (right) expression by Western blot. CaSki: HPV16⁺ cervical cancer cells, positive control; 2277NS: untransduced parental cell line of PAP-A2, negative control; PAP-A2: cell line lysate; PAP-A2 tumor: lysate generated from a PAP-A2 tumor having grown in an A2.DR1 mouse. The Western blots shown are representative of three Western blots. (B) Tumor growth curves of PAP-A2 cell injection number titrations in A2.DR1 mice. Mean ± SD are shown. (C) Mouse survival curves for the experiment shown in B. n = 3 (5x10⁶) or n = 5 (other groups) mice per group.

Figure 2.

Induction of anti-HPV16 E6/E7 responses in A2.DR1 mice and specific killing of PAP-A2 tumor cells by vaccination-induced CD8⁺ T cells. (A) Structure and modular synthesis of amph-peptides, exemplified by amph-E7/11–19. (B) A2.DR1 mice (n = 6 per group) were injected with indicated amph-peptides + 50 µg pI:C according to the indicated schedule. Splenocytes were isolated and incubated with either an irrelevant HLA-A2-binding peptide or the cognate peptide. After 5 h of incubation, cells were stained for intracellular IFN-γ and analyzed by flow cytometry. Frequencies of IFN-γ⁺ cells among CD8⁺ T cells after incubation with the irrelevant or the cognate peptide are shown. Each dot represents one mouse, mean ± SD is indicated. Statistical analysis was performed with Student’s unpaired t test. (C) Splenocytes of amph-peptide-vaccinated A2.DR1 mice were isolated and cultured 7 days in the presence of the respective indicated cognate peptide. CD8⁺ T cells were isolated by untouched MACS isolation. CD8⁺ T cells (effector cells) were added to wells containing a 1:1 mixture of specific target cells (PAP-A2, CFSE labeled) and control target cells (parental 2277NS cells, FR labeled). 48 h after addition of CD8⁺ T cells, cells were analyzed via flow cytometry. “% of specific killing” was calculated from the ratio of specific to control target cell killing. The experiment was performed once in triplicates; error bars: SD.

Figure 3.

Prophylactic and therapeutic vaccination with amph-E7/11–19 reduces the growth of PAP-A2 tumors. (A, B) A2.DR1 mice (n = 15 per group for amph-E7/11–19 and vehicle, n = 14 for untreated) were treated with three injections of amph-E7/11–19 or controls and were challenged 7 days after the last vaccination with 1.5x10⁶ PAP-A2 cells. A: Cumulative survival curves of groups, B: Tumor growth curves of individual mice. (C, D) A2.DR1 mice (n = 8 per group) were injected with 1.5x10⁶ PAP-A2 cells and were vaccinated as indicated with amph-E7/11–19, vehicle control or left untreated. C: Cumulative survival curves of groups, D: Tumor growth curves of individual mice. Statistical analysis for differences in survival was performed with the Gehan-Breslow-Wilcoxon test.

Figure 4.

Therapeutic vaccination with the amphiphilic HPV16 E6/E7 epitopes E7/7–15, E7/82–90 and E6/25–33 only weakly reduces the growth of PAP-A2 tumors. (A, B) A2.DR1 mice (n = 10 per group) were injected with 1.5x10⁶ PAP-A2 cells and were vaccinated as indicated with amph-peptides or the vehicle control. A: Cumulative survival curves of groups, B: Tumor growth curves of individual mice. Statistical analysis for differences in median survival was performed with the Gehan-Breslow-Wilcoxon test. All differences were found to be non-significant.

Figure 5.

Vaccination with combinations of amphiphilic HPV16 E6/E7 epitopes leads to reduced frequencies of E7/7–15, E7/11–19 and E6/25–33-specific CD8⁺ cells compared to single vaccination. A2.DR1 mice (n = 5 per group) were vaccinated as indicated in Figure 2B with the combinations of amph-peptides shown in the respective graph’s title. Splenocytes were isolated and incubated with either an irrelevant HLA-A2-binding peptide, the cognate peptide or a combination of all cognate peptides. After 5 h of incubation, cells were stained for intracellular IFN-γ and analyzed by flow cytometry. Frequencies of IFN-γ⁺ cells among CD8⁺ T cells after incubation with the indicated peptide are shown. The last group in each graph shows the mathematical sum of the frequencies of all HPV16 E6/E7 epitopes used in this treatment group. Each dot represents one mouse, mean ± SD is indicated. Vaccination with amph-peptides is abbreviated as follows: 11 = amph-E7/11–19, 7 = amph-E7/7–15, 82 = amph-E7/82–90, 25 = amph-E6/25–33.

Figure 6.

Therapeutic vaccination with combinations of amphiphilic HPV16 E6/E7 epitopes leads to reduced anti-tumor effects compared to amph-E7/11–19 single vaccination. A2.DR1 mice (n = 10 per group) were injected with 1.5x10⁶ PAP-A2 cells and were vaccinated as indicated in the group’s title and in Figures 3C and 4A with amph-peptides or the vehicle control. Cumulative survival curves of groups are shown. Vaccination with amph-peptides is abbreviated as follows: 11 = amph-E7/11–19, 7 = amph-E7/7–15, 82 = amph-E7/82–90, 25 = amph-E6/25–33. Statistical analysis for differences in survival was performed with the Gehan-Breslow-Wilcoxon test.