A Therapeutic DNA Vaccine Targeting HPV16 E7 in Combination with Anti-PD-1/PD-L1 Enhanced Tumor Regression and Cytotoxic Immune Responses

Abstract

Persistent infection of high-risk human papillomavirus (HPV) and the expression of E6 and E7 oncoproteins are the main causes of cervical cancer. Several prophylactic HPV vaccines are used in the clinic, but these vaccines have limited efficacy in patients already infected with HPV. Since HPV E7 is vital for tumor-specific immunity, developing a vaccine against HPV E7 is an attractive strategy for cervical cancer treatment. Here, we constructed an HPV16 E7 mutant that loses the ability to bind pRb while still eliciting a robust immune response. In order to build a therapeutic DNA vaccine, the E7 mutant was packaged in an adenovirus vector (Ad-E7) for efficient expression and enhanced immunogenicity of the vaccine. Our results showed that the Ad-E7 vaccine effectively inhibited tumor growth and increased the proportion of interferon-gamma (IFN-γ)-secreting CD8⁺ T cells in the spleen, and tumor-infiltrating lymphocytes in a mouse cervical cancer model was achieved by injecting with HPV16-E6/E7-expressing TC-1 cells subcutaneously. Combining the Ad-E7 vaccine with the PD-1/PD-L1 antibody blockade significantly improved the control of TC-1 tumors. Combination therapy elicited stronger cytotoxic T lymphocyte (CTL) responses, and IFN-γ secretion downregulated the proportion of Tregs and MDSCs significantly. The expressions of cancer-promoting factors, such as TNF-α, were also significantly down-regulated in the case of combination therapy. In addition, combination therapy inhibited the number of capillaries in tumor tissues and increased the thickness of the tumor capsule. Thus, Ad-E7 vaccination, in combination with an immune checkpoint blockade, may benefit patients with HPV16-associated cervical cancer.

Keywords: E7; PD-1; cervical cancer; human papillomavirus; therapeutic vaccine.

Figure 1

Construction and identification of the Ad-E7 vaccine. (A) The amplified E7 (delD21-C24) gene was identified using agarose gel electrophoresis. Lane 1 is the DNA marker, which is 5 Kb, 3 Kb, 2 Kb, 1.5 Kb, 1 Kb, 750 bp, 500 bp, 250 bp and 100 bp from top to bottom, and lane 2 is E7 (delD21-C24). (B) E7 (delD21-C24) mRNA level was detected using qRT-PCR following transfection of E7 (delD21-C24) plasmid into 293T cells. (C) Western blot analysis of E7 (delD21-C24) protein level in the 293T and PA317 cells infected by Ad-E7 adenovirus in vitro. (D) qRT-PCR was employed to detect mRNA level of E7 (deld21-c24) in the 293T and PA317 cells infected by Ad-E7 adenovirus in vitro. *** p < 0.001.

Figure 2

Effects of different treatments on tumor growth in tumor models. (A) Schematic diagram of treatment time in tumor-bearing mice. Purple arrow indicates the time points for injection of TC-1 cells; green arrows indicate the time points for measurement of tumors every three days; blue arrows represent the time points for injection of related antibodies using the indicated dosage; brown arrows represent the time points for vaccination at day 6 and day 15 (for booster immunization). (B) Average tumor growth curve of mice in each group. (C) Tumor growth curve of each mouse in each group with different treatments (n = 6/group). (D) The tumors of the mice were isolated and photographed. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not statistically significant (p > 0.05).

Figure 3

Expression of related immune cells in spleens of mice. (A) CD8⁺ T lymphocyte populations were analyzed using flow cytometry. (B) The frequencies of CD8⁺ T lymphocytes in spleen lymphocytes in each group with different treatments. (C) The positive proportion of IFN-γ in CD8⁺ T lymphocytes in each group with different treatments. (D) The positive proportion of PD-1 in CD8⁺ T lymphocytes in each group with different treatments. (E) MDSC populations were analyzed using flow cytometry. (F) The proportion of MDSCs in spleen lymphocytes in each group with different treatments. (G) Treg populations were detected using flow cytometry. (H) The proportion of Tregs in spleen lymphocytes in each group with different treatments. (I) The positive ratio of PD-L1 in Tregs in each group with different treatments. Bar graph is used to summarize the flow cytometry data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Analysis of tumor-infiltrating lymphocytes (TILs). (A) Schematic diagram of treatment time in tumor-bearing mice. Purple arrow indicates the time points for injection of TC-1cells; green arrows indicate the time points for measurement of tumors every three days; blue arrows represent the time points for injection of related antibodies using the indicated dosage; brown arrow represents the time points for vaccination at day 6. (B) The proportion of CD8⁺ T lymphocytes in TILs. The white bars represent the first part being the Ad-E7 vaccine combined with the PD-1 antibody, and the gray bars represent the second being the Ad-E7 vaccine combined with the PD-L1 antibody. (C) The positive proportion of IFN-γ in CD8⁺ T lymphocytes. (D) The positive proportion of PD-1 in CD8⁺ T lymphocytes. (E) The positive proportion of MDSCs in TILs. (F) The positive proportion of Tregs in TILs. (G) The positive proportion of PD-L1 in Tregs. Bar graph is used to summarize the flow cytometry data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Expression of related cytokines in tumors. (A,B) qRT-PCR analysis of CCL2, CCL3, CCL5, CCL12, CCL19, CCL20, CCL21, CXCL10, IL-2, IL-4, IL-10, MMP2, MMP3, TNF-α, and TGF-β mRNA levels in four groups (TC-1 + Ad-control + IgG 2a, TC-1 + Ad-control + PD-1, TC-1 + Ad-E7 + IgG 2a, TC-1 + Ad-E7 + PD-1). (C,D) qRT-PCR analysis of CCL2, CCL3, CCL5, CCL12, CCL19, CCL20, CCL21, CXCL10, IL-2, IL-4, IL-10, MMP2, MMP3, TNF-α, and TGF-β mRNA levels in four groups (TC-1 + Ad-control + IgG 2b, TC-1 + Ad-control + PD-L1, TC-1 + Ad-E7 + IgG 2b, TC-1 + Ad-E7 + PD-L1). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

The H&E staining of tumor tissues. (A) The H&E staining of tumor tissues in four groups (TC-1 + Ad-control + IgG 2a, TC-1 + Ad-control + PD-1, TC-1 + Ad-E7 + IgG 2a, TC-1 + Ad-E7 + PD-1). Scale bars, 125 μm. (B) The H&E staining of tumor tissues in four groups (TC-1 + Ad-control + IgG 2b, TC-1 + Ad-control + PD-L1, TC-1 + Ad-E7 + IgG 2b, TC-1 + Ad-E7 + PD-L1). Red arrows indicate capillary, and black arrows indicate tumor capsule. Scale bars, 125 μm.