Development of a Spontaneous HPV16 E6/E7-Expressing Head and Neck Squamous Cell Carcinoma in HLA-A2 Transgenic Mice

Human papillomavirus (HPV)-associated head and neck squamous cell carci-noma (HNSCC) is a growing global health problem. HPV16 has been attributed to a ma-jority of HPV-associated HNSCCs. In order to test candidate immunotherapies, we devel-oped a spontaneous HPV16-driven HNSCC model in HLA-A2 (AAD) transgenic mice. We sought to eliminate the confounding effects of dominant HPV antigen presentation through murine major histocompatibility complex class I (MHC-I) via epitope mutagenesis (without compromising tumorigenicity). We generated HPV16 E6(R55K)(delK75) and E7 (N53S) expression constructs with mutations in known dominant H-2Db epitopes and characterized their presentation through murine and human MHC-I molecules using in vitro and in vivo activation of HPV16 E6/E7 antigen-specific CD8+ T cells. In addition, we tested the ability of E6(R55K)(delK75) and E7(N53S) for oncogenicity. The mutated E7 (N53S) abolished the presentation of murine H-2Db-restricted HPV16 E7 peptide (i.e., amino acids [aa] 49 to 57) cytotoxic T lymphocyte (CTL) epitope and resulted in HLA-A2-restricted presentation of the HPV16 E7 (aa 11 to 20)-specific CTL epitope. The mutated E6(R55K)(delK75) abolished the activation of murine MHC-I-restricted E6-specific CD8(+) T cell-mediated immune responses in C57BL/6 mice. In addition, the vaccination led to the activation of human HLA-A2-restricted E6-specific CD8(+) T cell-mediated immune responses in HLA-A2 (AAD) transgenic mice. Injection of DNA plasmids encoding LucE7(N53S)E6 (R55K)(delK75), AKT, c-Myc, and SB100 followed by electroporation results in development of squamous cell carcinoma in the oral/pharyngeal cavity of all of the HLA-A2 (AAD) trans-genic mice (5/5), with 2/5 tumor-bearing mice developing metastatic carcinoma in the neck lymph nodes. IMPORTANCE Our data indicate that mutated HPV16 E6(R55K)(delK75) and mutated HPV16 E7(N53S) DNA abolishes the presentation of HPV16 E6 and E7 through murine MHC-I and results in their presentation through human HLA-A2 molecules. Additionally, the mutated HPV16 E6 and E7 remain oncogenic. Our approach is potentially applicable to different human MHC-I transgenic mice for the identification of human MHC-I re-stricted HPV16 E6/E7-specific CTL epitopes as well as the generation of spontaneous HPV E6/E7-expressing oral/pharyngeal carcinoma.

Automated summary

The study developed a spontaneous HPV16-driven HNSCC model in HLA-A2 transgenic mice to test candidate immunotherapies. They eliminated the confounding effects of dominant HPV antigen presentation through epitope mutagenesis and characterized their presentation in murine and human MHC-I molecules.