DNA Vaccine Encoding HPV16 Oncogenes E6 and E7 Induces Potent Cell-mediated and Humoral Immunity Which Protects in Tumor Challenge and Drives E7-expressing Skin Graft Rejection

Abstract

We have previously shown that a novel DNA vaccine technology of codon optimization and the addition of ubiquitin sequences enhanced immunogenicity of a herpes simplex virus 2 polynucleotide vaccine in mice, and induced cell-mediated immunity when administered in humans at relatively low doses of naked DNA. We here show that a new polynucleotide vaccine using the same technology and encoding a fusion protein of the E6 and E7 oncogenes of high-risk human papillomavirus type 16 (HPV16) is immunogenic in mice. This vaccine induces long-lasting humoral and cell-mediated immunity and protects mice from establishment of HPV16-E7-expressing tumors. In addition, it suppresses growth of readily established tumors and shows enhanced efficacy when combined with immune checkpoint blockade targeted at PD-L1. This vaccine also facilitates rejection of HPV16-E7-expressing skin grafts that demonstrate epidermal hyperplasia with characteristics of cervical and vulvar intraepithelial neoplasia. Clinical studies evaluating the efficacy of this vaccine in patients with HPV16 premalignancies are planned.

FIGURE 1

Immunization with codon-optimized polynucleotides encoding HPV16-E6/E7 induce acute and long-lasting humoral and cytotoxic immune responses. Mice were immunized intradermally into the ear twice 3 weeks apart with HPV16-E6/E7 DNA vaccine (NTC-HPV16-E6/E7) or empty vector (NTC-empty). A and B, E7-specific IgG antibody responses of NTC-HPV16-E6/E7 immunized mice (grey circles) were determined in serum at week 0 and 5 by ELISA (A). E6-specific and E7-specific cytotoxic T-cell responses were measured by ELISPOT in splenocytes at week 5 after immunization (B) (n=10). C and D, E7-specific IgG antibody responses of NTC-HPV16-E6/E7 immunized mice (grey circles, n=5) or NTC-empty immunized mice (black squares, n=3) were determined in serum at week 0 and 5, 13, and 21 by ELISA (C). E6-specific and E7-specific T-cell responses were measured in splenocytes at week 21 by IFNγ ELISPOT (D). Each data point represents means of technical replicates of 1 individual animal. Mean values of averaged individuals and SDs are indicated. *P≤0.05, ***P≤0.001, ****P≤0.0001.

FIGURE 2

NTC-HPV16-E6/E7 immunization prevents TC-1 tumor growth and suppresses progression of established tumors. A and B, Mice were immunized intradermally twice 3 weeks apart with NTC-HPV16-E6/E7 or a vector expressing an irrelevant protein of herpes simplex virus 2 (NTC-gD2). One week later, mice were injected subcutaneously with 5×10⁵ HPV16-E7-expressing TC-1 tumor cells. Unimmunized mice were used as positive control for tumor growth. Mice were killed when tumor size reached 1 cm³. Shown are mean values of individual tumor sizes (n=10) with SE of mean) (A) and percentage of survival (B) over a period of 20 days. C and D, Mice were injected subcutaneously with 5×10⁴ HPV16-E7-expressing TC-1 tumor cells. Three days after tumor inoculation, mice were immunized 3 times in weekly intervals with NTC-HPV16-E6/E7 or NTC-gD2. One additional group was immunized 3 times in weekly intervals with NTC-HPV16-E6/E7 starting 7 days after tumor inoculation. Tumor size was measured and mice were killed when tumor size reached 1 cm³. Shown are mean values of individual tumor sizes (n=8) with SE of mean indicated (C) and percentage of survival (D) over a period of 50 days. *P≤0.01, **P≤0.01, ***P<0.001, ****P<0.0001.

FIGURE 3

Combination immunotherapy of HPV16-E6/E7 DNA vaccination adjacent to targeting PD-L1 further increases antitumor immunity. A and B, Mice were injected subcutaneously with 1×10⁵ HPV16-E7-expressing TC-1 tumor cells. Seven days after tumor inoculation, mice were immunized 3 times in weekly intervals with NTC-HPV16-E6/E7. Mice received monoclonal antibodies targeting PD-L1 twice weekly after each vaccination, for 3 constitutive weeks. Tumor size was measured and mice were killed when tumor size reached 1 cm³. Shown are mean values of individual tumor sizes (n=8) with SE of mean indicated (A) and percentage of survival (B) over a period of 50 days. C, TC-1 cells were analyzed by flow cytometry for the expression of PD-L1. Shown are histogram and median fluorescent intensity (MFI) of unstained, isotype stained, and α-PD-L1 stained TC-1 cells. *P<0.05, ***P<0.001.

FIGURE 4

E7TCR269 mice immunized with HPV16-E6/E7 DNA vaccine reject HPV16-E7-expressing skin grafts. E7TCR269 mice were immunized with HPV16-E6/E7 DNA vaccine or vector expressing an irrelevant protein (HSV gD2) intradermally into the ear twice 3 weeks apart. One week after the second immunization, mice received skin transplants from C57BL/6 and K14E7 animals. Grafts were measured weekly (n=4–6). A, Photographs taken 28 days after grafting. Shown is 1 representative per group. B, Graft size reduction was calculated based on graft size on day 7 after grafting, when bandages were removed. C, E6-specific and E7-specific T-cell responses were measured in splenocytes at the end of the grafting study by IFNγ ELISPOT. Each data point represents means of technical replicates of 1 individual animal. Mean values of averaged individuals and SDs are indicated. D, E7-specific IgG antibody responses were determined in serum at the end of the grafting study by ELISA. Each data point represents means of technical replicates of 1 individual animal. Mean values of averaged individuals and SDs are indicated. *P≤0.05, **P≤0.01.