Development of DNA Vaccine Targeting E6 and E7 Proteins of Human Papillomavirus 16 (HPV16) and HPV18 for Immunotherapy in Combination with Recombinant Vaccinia Boost and PD-1 Antibody

Abstract

Immunotherapy for cervical cancer should target high-risk human papillomavirus types 16 and 18, which cause 50% and 20% of cervical cancers, respectively. Here, we describe the construction and characterization of the pBI-11 DNA vaccine via the addition of codon-optimized human papillomavirus 18 (HPV18) E7 and HPV16 and 18 E6 genes to the HPV16 E7-targeted DNA vaccine pNGVL4a-SigE7(detox)HSP70 (DNA vaccine pBI-1). Codon optimization of the HPV16/18 E6/E7 genes in pBI-11 improved fusion protein expression compared to that in DNA vaccine pBI-10.1 that utilized the native viral sequences fused 3' to a signal sequence and 5' to the HSP70 gene of Mycobacterium tuberculosis Intramuscular vaccination of mice with pBI-11 DNA better induced HPV antigen-specific CD8+ T cell immune responses than pBI-10.1 DNA. Furthermore, intramuscular vaccination with pBI-11 DNA generated stronger therapeutic responses for C57BL/6 mice bearing HPV16 E6/E7-expressing TC-1 tumors. The HPV16/18 antigen-specific T cell-mediated immune responses generated by pBI-11 DNA vaccination were further enhanced by boosting with tissue-antigen HPV vaccine (TA-HPV). Combination of the pBI-11 DNA and TA-HPV boost vaccination with PD-1 antibody blockade significantly improved the control of TC-1 tumors and extended the survival of the mice. Finally, repeat vaccination with clinical-grade pBI-11 with or without clinical-grade TA-HPV was well tolerated in vaccinated mice. These preclinical studies suggest that the pBI-11 DNA vaccine may be used with TA-HPV in a heterologous prime-boost strategy to enhance HPV 16/18 E6/E7-specific CD8+ T cell responses, either alone or in combination with immune checkpoint blockade, to control HPV16/18-associated tumors. Our data serve as an important foundation for future clinical translation.IMPORTANCE Persistent expression of high-risk human papillomavirus (HPV) E6 and E7 is an obligate driver for several human malignancies, including cervical cancer, wherein HPV16 and HPV18 are the most common types. PD-1 antibody immunotherapy helps a subset of cervical cancer patients, and its efficacy might be improved by combination with active vaccination against E6 and/or E7. For patients with HPV16+ cervical intraepithelial neoplasia grade 2/3 (CIN2/3), the precursor of cervical cancer, intramuscular vaccination with a DNA vaccine targeting HPV16 E7 and then a recombinant vaccinia virus expressing HPV16/18 E6-E7 fusion proteins (TA-HPV) was safe, and half of the patients cleared their lesions in a small study (NCT00788164). Here, we sought to improve upon this therapeutic approach by developing a new DNA vaccine that targets E6 and E7 of HPV16 and HPV18 for administration prior to a TA-HPV booster vaccination and for application against cervical cancer in combination with a PD-1-blocking antibody.

Keywords: DNA vaccine; E6; E7; HPV-associated cancer; HPV16; HPV18; PD-1; TA-HPV; human papillomavirus.

Copyright © 2021 Peng et al.

Figures

FIG 1

Schematic of the DNA constructs and map of pBI-11. (A) Schematic of the DNA constructs that are cloned into the pNGVL4a plasmid. The genes in the plasmids pBI-1, pBI-10.1, pBI-11, and pBI-12 are depicted. pBI-1 is in use for clinical trials under the name pNGVL4a-Sig/E7(detox)/HSP70. Blue indicates the native sequence of the target antigen. HSP70 is derived from Mycobacterium tuberculosis. Red indicates the gene was codon optimized prior to cloning it into the vector. (B) DNA construct map of pBI-11.

FIG 2

Western blot analysis to characterize the expression of the encoded HPV16/18 E6/E7 fusion protein in transfected cells. 293 expi cells were transfected with the designated plasmids and analyzed by Western blotting to assess the levels of target antigens. HPV16 E7-specific (A) and HPV18 and HPV16 E6-specific (B) MAbs were used as probes for the Western blot analysis of the antigen expression of the DNA vaccine constructs. 293 cells were transfected with the vaccines. (C) GAPDH was used as the loading control.

FIG 3

Characterization of HPV16 E7 and HPV18 E6 antigen presentation by cells transfected with the various DNA vaccines. 293-Db or 293 Kb cells were transfected with either pBI-10.1, pBI-11, or pBI-12 DNA vaccine or mock transfected using Lipofectamine 2000. Twenty-four hours later, these cells were harvested and cocultured with either murine H-2Db-restricted HPV16 E7 (aa 49 to 57) peptide-specific CD8+ T cells or murine H-2Kb-restricted HPV 18 E6 (aa 67 to 75) peptide-specific CD8+ T cells in the presence of GolgiPlug. The cells were then harvested, and IFN-γ intracellular staining was performed to determine the activation of HPV16 E7 or HPV18 E6 antigen-specific CD8+ T cells. (A) Bar graph to summarize flow cytometry data for activation of HPV16 E7-specific CD8+ T cells. (B) Bar graph to summarize the flow cytometry data for the activation of HPV18 E6-specific CD8+ T cells.

FIG 4

Comparison of HPV16/18 E6/E7-specific CD8+ T cell responses generated by the various HPV DNA vaccines. (A) Schematic illustration of the experimental design. Briefly, 5- to 8-week-old female C57BL/6 mice (5 mice/group) were vaccinated with either 25 μg/mouse of pBI-10.1, pBI-11, or pBI-12 DNA vaccine through intramuscular injection. The mice were boosted twice with the same dose and regimen with 1-week intervals. Seven days after the last vaccination, splenocytes were prepared from the vaccinated mice and stimulated with HPV16 E7 (aa 49 to 57) peptide, HPV16 E6 (aa 50 to 57) peptide, or HPV18 E6 (aa 67 to 75) peptide in the presence of GolgiPlug overnight. The splenocytes were stained with PE-conjugated anti-mouse CD8a. After permeabilization and fixation, the cells were further stained with FITC-conjugated anti-mouse IFN-γ. The cells were acquired with a FACSCalibur flow cytometer, and data were analyzed with CellQuest Pro software. (B) Bar graphs summarizing the data from flow cytometry analysis of HPV16 E6 (a), HPV16 E7 (b), and. HPV18 E6 (c) peptide-specific CD8+ T cell responses analyzed by IFN-γ intracellular staining. (d) Splenocytes pulsed without peptide as negative control. N.S., not significant.

FIG 5

Characterization of HPV antigen-specific CD8+ T cell-mediated immune responses and therapeutic antitumor effects of the various DNA vaccines in mice injected with HPV16 E6/E7-expressing tumors, model TC-1. Six- to eight-week-old female C57BL/6 mice (5 mice/group) were injected with 2 × 105 of TC-1 cells subcutaneously on day 0. On day 3, the mice were vaccinated with either pBI-10.1, pBI-11, or pBI-12 DNA (25 μg/50 μl/mouse) through i.m. injection and boosted twice as indicated or left untreated as control. (A) Schema of the experiment. (B) Detection of HPV16 E7-specific CD8+ T cells in peripheral blood using HPV16 E7 (aa 49 to 57) peptide-loaded tetramer staining. (C) Detection of HPV18 E6-specific CD8+ T cells in peripheral blood using HPV18 E6 (aa 67 to 75) peptide-loaded tetramer staining. (D) Summary of the TC-1 tumor volumes of the mice. (E) Kaplan-Meier analysis of the survival of TC-1 tumor-bearing mice.

FIG 6

Characterization of HPV antigen-specific CD8+ T cell-mediated immune responses in mice vaccinated with pBI-11 DNA alone or followed by TA-HPV. Six- to eight-week-old female C57BL/6 mice (5 mice/group) were vaccinated with pBI-11 DNA (25 μg/50 μl/mouse) through i.m. injection. The mice were boosted with the same regimen 7 days later. One week after the second vaccination, one group of the mice was vaccinated with pBI-11 DNA (25 μg/50 μl/mouse) through i.m. injection (DDD). Another group of mice was vaccinated with TA-HPV (1 × 106 PFU/50 μl/mouse) through i.m. injection (DDV). (A) Schema of the experiment. Six days after the last vaccination, peripheral blood was collected from the vaccinated or naive mice for HPV16 E7 tetramer staining. Fourteen days after the last vaccination, splenocytes were prepared from the vaccinated mice and stimulated with either HPV16 E6 (aa 50 to 57), HPV16 E7 (aa 49 to 57), or HPV18 E6 (aa 67 to 75) peptide in the presence of GolgiPlug. Intracellular IFN-γ cytokine staining assay was performed to detect antigen-specific CD8+ T cells. The cells were acquired with a FACSCalibur flow cytometer, and data were analyzed with CellQuest Pro software. (B) Summary of the HPV16 E7 tetramer flow cytometry analysis. (C) Intracellular IFN-γ cytokine staining assay after stimulation with either HPV16 E6 (aa 50 to 57) peptide (5 μg/ml), HPV16 E7 (aa 49 to 57) peptide (1 μg/ml), or HPV18 E6 (aa 67 to 75) peptide (1 μg/ml).

FIG 7

Characterization of the HPV antigen-specific immune response and antitumor effects in a TC-1 tumor-bearing mouse treated with DDV with or without anti-PD-1. Six- to eight-week-old female C57BL/6 mice (5 to 8 mice/group) were injected with 2 × 105 of TC-1 cells subcutaneously on day 0. On day 3, the mice were divided into 4 groups. The first group was used as the untreated control. The second group was injected with purified anti-mouse PD-1 monoclonal antibody (MAb; clone 29F.1A12, 200 μg/mouse) via intraperitoneal injection. The treatment was repeated every other day. The third group was vaccinated with pBI-11 DNA (25 μg/50 μl/mouse) through i.m. injection and boosted once 3 days later. The mice were further boosted with TA-HPV vaccinia virus 3 days later through skin scarification. The fourth group was treated with both anti-mouse PD-1 MAb and pBI-11 DNA vaccine prime followed by TA-HPV vaccinia virus boost as described in Materials and Methods. (A) Schema of the experiment. (B) Detection of HPV16 E7-specific CD8+ T cells in peripheral blood using HPV16 E7 tetramer staining. (C) Summary of the TC-1 tumor volumes in the mice. (D) Kaplan-Meier analysis of the survival of TC-1 tumor-bearing mice.