HPV subtype 16 (HPV16) causes a persistent viral infection resulting in the majority of cervical cancers and oropharyngeal cancers (HPVOPC). Despite effective prophylactic vaccines, the high incidence of persistent HPV infections provides a strong rationale for the development of novel targeted immunotherapies. Immune therapies such as PD-1 checkpoint inhibitors have the potential to enhance tumor infiltrating CD8+ T-cells (CTLs) in HPV-lesions targeting human leukocyte antigen (HLA) class I-restricted HPV16 epitopes. CTL-epitopes derived from HPV16 are ideal candidates for therapeutic vaccine development as well as immune monitoring of HPV16 associated-cancers. However, the CTL epitope landscape of HPV-16 during viral clearance and tumor progression is largely unknown. We sought to identify epitopes from HPV16 E2, E6, and E7 due to the sustained expression of these proteins in pre-cancerous and in cancerous lesions. To identify potential epitopes with >90% global population coverage, we performed a comprehensive bioinformatics analysis to predict potential HPV16 epitopes from 10 HLA-alleles representing common HLA class I-supertypes (A*0101, A*0201, A*0301, A*1101, A*2402, B*0702, B*0801, B*2705, B*3501, B*5701) using 5 different HLA-binding and antigen processing prediction servers. Over 2000 possible candidate peptides from each HPV16 antigen spanning the 10 HLAs were evaluated. Incorporating stringent selection criteria to control for inter-algorithmic variations and normalization, we selected the 58 top-ranked candidate peptides (24 from E2, 20 from E6 and 14 from E7) as potential HPV16-CTL epitope candidates. We confirmed our prediction strategy with well-described HLA-A2 restricted E6 (KLPQLCTEL, TIHDIILECV, FAFRDLCIV) and E7 epitopes (LLMGTLGIV, YMLDLQPET). The immunogenicity of the 58 candidate HPV16 epitopes were evaluated by IFN-gamma Elispot assays using short term ex vivo T-cell lines stimulated in the presence of PD-1 blockade from healthy donors and patients with HPVOPC. HPV-16 E2 and E6-reactive T-cells have been detected at varying specificity and frequencies in PBMCs isolated from healthy blood donors (ranging from 45 to 175 SFUs/10^6 PBMCs) indicating the high prevalence of HPV in the general population. We have confirmed the immunogenicity of previously identified HLA-A2 restricted E2 epitopes TLQDVSLEV and YICEEASVTV. We have also identified novel HLA-A11 and B07-restricted-E6 epitopes (130 and 70 SFUs/10^6 PBMCs respectively) and novel HLA-A11 and B07 restricted-E2 epitopes (445 and 251 SFUs/10^6 PBMCs respectively) from healthy donors. The role of the identified epitopes in HPV clearance is currently unknown and is under investigation. E2 and E6-reactive T-cells were also found in HPVOPC patients indicating the possible presence of viral episomes in HPVOPC lesions. Additional CTL screens for epitope discovery are ongoing. The identified epitopes may be targets for globally relevant immunotherapies and monitoring of HPV related disease.
Citation Format: Sri Krishna, Jacqueline Nelson, Falguni Parikh, Amelia Clark, Seunghee Kim-Schulze, Marshall Posner, Andrew Sikora, Karen S. Anderson. Discovering immunotherapeutic T-cell epitopes for HPV16-associated cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B098.
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