Buccal injection of synthetic HPV long peptide vaccine induces local and systemic antigen-specific CD8+ T-cell immune responses and antitumor effects without adjuvant

Abstract

Background: Human Papillomavirus is responsible for over 99 % of cervical cancers and is associated with cancers of the head and neck. The currently available prophylactic vaccines against HPV do not generate therapeutic effects against established HPV infections and associated lesions and disease. Thus, the need for a therapeutic vaccine capable of treating HPV-induced malignancies persists. Synthetic long peptides vaccination is a popular antigen delivery method because of its safety, stability, production feasibility, and its need to be processed by professional antigen presenting cells before it can be presented to cytotoxic CD8+ T lymphocytes. Cancers in the buccal mucosa have been shown to elicit cancer-related inflammations that are capable of recruiting inflammatory and immune cells to generate antitumor effects. In the current study, we evaluated the therapeutic potential of synthetic HPV long peptide vaccination in the absence of adjuvant in the TC-1 buccal tumor model.

Result: We show that intratumoral vaccination with E7 long peptide alone effectively controls buccal TC-1 tumors in mice. Furthermore, we observed an increase in systemic as well as local E7-specific CD8+ T cells in buccal tumor-bearing mice following the vaccination. Finally, we show that induction of immune responses against buccal tumors by intratumoral E7 long peptide vaccination is independent of CD4+ T cells, and that the phenomenon may be related to the unique environment associated with mucosal tissues.

Conclusion: Our results suggest the possibility for clinical translation of the administration of adjuvant free therapeutic long peptide vaccine as a potentially effective and safe strategy for mucosal HPV-associated tumor treatment.

Keywords: Adjuvant free; Buccal tumor; E7 long peptide; Immunotherapy.

Fig. 1

Characterization of antitumor effect in tumor bearing mice treated with intratumoral synthetic HPV-16 E7aa 43-62 long peptide vaccination in buccal mucosal region. 3 × 10⁴ TC-1-Luc cells were submucosally injected into the right buccal area of C57BL/6 mice (five per group). Three days after tumor injection, mice were vaccinated intratumorally with or without 50 μg of synthetic HPV-16 E7aa 43-62 peptide for four times in a 4-day intervals. a Schematic diagram of treatment regimens. b Line graph depicting the change in mean luminescence intensity of tumor bearing mice after tumor injection (mean ± SD). c Kaplan–Meier survival analysis of mice

Fig. 2

Characterization of systemic E7-specific CD8+ T cells and local E7-specific activated CD8+ T cells in tumor bearing mice. 3 × 10⁴ TC-1-Luc cells were submucosally injected into the right buccal area of C57BL/6 mice (five per group). Three days after tumor injection, mice were vaccinated intratumorally with or without 50 μg of synthetic HPV-16 E7aa 43-62 peptide for four times in a 4-day intervals. 21 days after tumor injection, peripheral blood was collected and the spleen and tumor were harvested. Cells obtained from the peripheral blood and tumor were stained with PE-conjugated HPV16 H-2D-RAHYNIVTF tetramer and APC-conjugated CD8 monoclonal antibody followed by flow cytometry analysis. Spleenocytes were stimulated with HPV16 E7aa49-57 peptide in the presence of GolgiPlug and IFN-γ-secreting CD8+ T cells were detected by intracellular cytokine staining followed by a flow cytometry analysis. a Representative flow cytometry images showing the amount of E7-specific CD8+ T cells per 1 × 10⁵ lymphocytes in the peripheral blood of various groups. b Bar graph depicting the amount of E7-specific CD8+ T cells per 1 × 10⁵ lymphocytes in the peripheral blood of various groups (mean ± SD). c Bar graph depicting the amount of IFN-γ positive lymphocytes per 1 × 10⁵ splenocytes (mean ± SD). d Bar graph depicting the percentage of E7-specific CD8+ T cells in all lymphocytes in the buccal tumor (mean ± SD)

Fig. 3

Effect of CD4+ or CD8+ T cell depletion on the antitumor response of synthetic HPV-16 E7aa 43-62 long peptide vaccine. 100 μg of purified rat monoclonal antibody GK1.5 (anti-CD4) or mAB 2.43 (anti-CD8) were injected intraperitoneally 2 days before tumor injection. The injections were repeated once per day for 2 days until the day of tumor challenge. On the day of tumor challenge, 3 × 10⁴ TC-1-Luc cells were submucosally injected into the right buccal area of C57BL/6 mice (five per group). Three days after tumor injection, mice were vaccinated intratumorally with or without 50 μg of synthetic HPV-16 E7aa 43-62 peptide for four times in a 4-day intervals. Mice also continued to receive anti-CD4 or anti-CD8 antibody injections once every week after tumor injection. a Schematic diagram of treatment regimens. b Line graph depicting the change in mean luminescence intensity of tumor bearing mice after tumor injection with or without CD8 depletion (mean ± SD). c Kaplan–Meier survival analysis of mice in CD8 depletion experiment. d Line graph depicting the change in mean luminescence intensity of tumor bearing mice after tumor injection with or without CD4 depletion (mean ± SD). e Kaplan–Meier survival analysis of mice in CD4 depletion experiment. NS indicates not significant

Fig. 4

Comparison of HPV-16 E7 specific CD8+ T cell responses induced by synthetic HPV-16 E7aa 43-62 long peptide vaccine in various tumor model. C57BL/6 mice (five per group) received either 3 × 10⁴ TC-1-Luc cells injection submucosally into the right buccal area or 1 × 10⁵ TC-1-Luc cells injection subcutaneously into the abdomen. Three days after tumor injection, mice were vaccinated intratumorally with or without 50 μg of synthetic HPV-16 E7aa 43-62 peptide for four times in a 4-day intervals. 21 days after tumor injection, spleenocytes were harvested and stimulated with HPV16 E7aa49-57 peptide in the presence of GolgiPlug and IFN-γ-secreting CD8+ T cells were detected by intracellular cytokine staining followed by a flow cytometry analysis. a Bar graph depicting the amount of IFN-γ positive lymphocytes per 3 × 10⁵ splenocytes for buccal treatments (mean ± SD). b Bar graph depicting the amount of IFN-γ positive lymphocytes per 3 × 10⁵ splenocytes for subcutaneous treatments (mean ± SD). NS indicates not significant

Fig. 5

Comparison of antitumor effects induced by synthetic HPV-16 E7aa 43-62 long peptide vaccine in various tumor model. C57BL/6 mice (five per group) received either 3 × 10⁴ TC-1-Luc cells injection submucosally into the right buccal area or 1 × 10⁵ TC-1-Luc cells injection subcutaneously into the abdomen. Three days after tumor injection, mice were vaccinated intratumorally with or without 50 μg of synthetic HPV-16 E7aa 43-62 peptide for four times in a 4-day intervals. a Line graph depicting the change in mean luminescence intensity of tumor bearing mice after buccal tumor injection (mean ± SD). b Line graph depicting the change in mean luminescence intensity of tumor bearing mice after subcutaneous tumor injection (mean ± SD)

Fig. 6

Effect of TLR4 Knockout on the antitumor response of synthetic HPV-16 E7aa 43-62 long peptide vaccine. 3 × 10⁴ TC-1-Luc cells were submucosally injected into the right buccal area of C57BL/6 or C57BL/10ScNJ (TLR4−/−) mice (five per group). Three days after tumor injection, mice were vaccinated intratumorally with or without 50 μg of synthetic HPV-16 E7aa 43-62 peptide for four times in a 4-day intervals. Figure represents the Kaplan–Meier survival analysis of mice in TLR4 knockout experiment