A Conserved E7-derived Cytotoxic T Lymphocyte Epitope Expressed on Human Papillomavirus 16-transformed HLA-A2+ Epithelial Cancers

Abstract

Human Papillomavirus 16 (HPV-16) has been identified as the causative agent of 50% of cervical cancers and many other HPV-associated tumors. The transforming potential/tumor maintenance capacity of this high risk HPV is mediated by two viral oncoproteins, E6 and E7, making them attractive targets for therapeutic vaccines. Of 21 E6 and E7 peptides computed to bind HLA-A*0201, 10 were confirmed through TAP-deficient T2 cell HLA stabilization assay. Those scoring positive were investigated to ascertain which were naturally processed and presented by surface HLA molecules for CTL recognition. Because IFN␥ ELISpot frequencies from healthy HPV-exposed blood donors against HLA-A*0201-binding peptides were unable to identify specificities for tumor targeting, their physical presence among peptides eluted from HPV-16-transformed epithelial tumor HLA-A*0201 immunoprecipitates was analyzed by MS 3 Poisson detection mass spectrometry. Only one epitope (E7 11-19 ) highly conserved among HPV-16 strains was detected. This 9-mer serves to direct cytolysis by T cell lines, whereas a related 10-mer (E7 11-20 ), previously used as a vaccine candidate, was neither detected by MS 3 on HPV-transformed tumor cells nor effectively recognized by 9-mer specific CTL. These data underscore the importance of precisely defining CTL epitopes on tumor cells and offer a paradigm for T cellbased vaccine design.The transforming potential of human Papillomavirus (HPV), 4 first suspected in the 1970s, has now been firmly established both biologically and epidemiologically (1-3). The single most important variable linked to malignant transformation is persistent infection with one of the high-risk HPV types. The E6 and E7 proteins encoded by high-risk HPVs have transforming activities and functionally inactivate the p53 and retinoblastoma (Rb) tumor suppressor proteins, respectively (3, 4). HPV-16 is the most abundant high risk HPV and has been detected in Ͼ50% of cervical cancer cases and in most other HPV-induced tumors, such as carcinomas of the vagina, anus, vulva, penis, and oropharynx (3, 5, 6). Worldwide, high risk HPVs are thought to be responsible for Ͼ500,000 malignancies per year, representing more than 5% of human cancers (7).A major breakthrough in combating HPV-induced disease was the development of prophylactic vaccines to prevent HPV infection in previously unexposed individuals. These vaccines are based on virus-like particles consisting of the L1 capsid protein (8, 9). Virus-like particles resemble natural virions and are able to induce high titers of L1-neutralizing antibodies. Two vaccines, one against HPV-16, -18, -6, and -11 and another against HPV-16 and -18, were approved for clinical use in 2006 (10 -12). Although the impact of prophylactic HPV vaccination on the incidence of vaccine type HPV-associated disease and cancer is unquestionable over time, these vaccines have no therapeutic efficacy for established HPV infections. Antibodies neutralize virus particles only before infection. Moreover, as HPV capsid proteins are exc...

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

“…By employing predictions as well as binding assays, and analyzing only non‐cysteine‐containing peptides, we reduced the number of possible HPV16 E6/E7‐derived HLA‐A2‐binding peptides to 17 actual binders, of which 9 had never been described before (Figure 2). The binding affinity results for already reported peptides were in accordance with our results, although they were tested with different in vitro assays 12, 13…”

“…Out of 14 tested donors, 8 showed reactivity against any of the tested peptides, indicating prior exposure to HPV16. Interestingly, the highest and most frequent responses were observed against E7 11–19 , which is the only peptide already detected to be presented on the cell surface of HPV16 + cells in a previous study 13. The overlapping peptide E7 12–19 also showed responses in four donors, albeit slightly weaker than the ones against E7 11–19 .…”

“…The reason for this rather high ratio may be our strategy of only looking for peptides whose HLA‐A2 binding capacity had been experimentally confirmed, which is in contrast to most other studies, which search for predicted HLA‐binding peptides. Only one identified peptide, E7 11–19 , was detected by MS before 13, 14. The major difference of our strategy compared to the approach described in ref.…”

“…The major difference of our strategy compared to the approach described in ref. 13, 14 is a faster and more sensitive MS instrumentation. This allowed for online LC‐MS 3 analysis of IP samples and measurement of more peptides in one analysis, whereas the authors of the earlier studies measured only one peptide at a time via static nanospray.…”

“…To date, several HLA‐A2‐restricted HPV16 target epitopes were identified with indirect biological assays 12. However, only one HLA‐A2‐restricted HPV16 peptide, E7 11–19 , was directly detected on the surface of HPV16 + cell lines13 and primary tumors 14. This is probably due to the fact that the amounts of HPV epitopes presented on the cell surface are extremely low, because of low viral protein expression and known HPV immune evasion mechanisms 15, 16…”