A Conserved E7-derived Cytotoxic T Lymphocyte Epitope Expressed on Human Papillomavirus 16-transformed HLA-A2+ Epithelial Cancers
2010; Elsevier BV; Volume: 285; Issue: 38 Linguagem: Inglês
10.1074/jbc.m110.126722
ISSN1083-351X
AutoresAngelika B. Riemer, Derin B. Keskin, G. Zhang, Maris Handley, Karen S. Anderson, Vladimir Brusić, Bruce B. Reinhold, Ellis L. Reinherz,
Tópico(s)vaccines and immunoinformatics approaches
ResumoHuman 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 IFNγ 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 MS3 Poisson detection mass spectrometry. Only one epitope (E711–19) highly conserved among HPV-16 strains was detected. This 9-mer serves to direct cytolysis by T cell lines, whereas a related 10-mer (E711–20), previously used as a vaccine candidate, was neither detected by MS3 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 cell-based vaccine design. 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 IFNγ 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 MS3 Poisson detection mass spectrometry. Only one epitope (E711–19) highly conserved among HPV-16 strains was detected. This 9-mer serves to direct cytolysis by T cell lines, whereas a related 10-mer (E711–20), previously used as a vaccine candidate, was neither detected by MS3 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 cell-based vaccine design. IntroductionThe transforming potential of human Papillomavirus (HPV),4Moscicki A.B. Schiffman M. Kjaer S. Villa L.L. Vaccine. 2006; 24: 42-51Crossref Scopus (393) Google Scholar first suspected in the 1970s, has now been firmly established both biologically and epidemiologically (1zur Hausen H. Curr. Top. Microbiol. Immunol. 1977; 78: 1-30Crossref PubMed Scopus (530) Google Scholar, 2Kreimer A.R. Clifford G.M. Boyle P. Franceschi S. Cancer Epidemiol. Biomarkers Prev. 2005; 14: 467-475Crossref PubMed Scopus (1644) Google Scholar, 3Munoz N. Castellsague X. de Gonzalez A.B. Gissmann L. Vaccine. 2006; 24: 1-10Crossref PubMed Scopus (1067) Google Scholar). 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 (3Munoz N. 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Vaccine. 2006; 24: S3/11-25Google Scholar).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 (8Berzofsky J.A. Ahlers J.D. Janik J. Morris J. Oh S. Terabe M. Belyakov I.M. J. Clin. Invest. 2004; 114: 450-462Crossref PubMed Scopus (117) Google Scholar, 9Lowy D.R. Schiller J.T. J. Clin. Invest. 2006; 116: 1167-1173Crossref PubMed Scopus (311) Google Scholar). 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 (10Printz C. Cancer. 2009; 115: 5130Crossref PubMed Scopus (1) Google Scholar, 11Ramqvist T. Andreasson K. Dalianis T. Expert Opin. Biol. Ther. 2007; 7: 997-1007Crossref PubMed Scopus (82) Google Scholar, 12Vetter K.M. Geller S.E. J. Womens Health. 2007; 16: 1258-1268Crossref Scopus (20) Google Scholar). 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 exclusively expressed late in the viral replication cycle within the upper layers of the epithelium, immune responses against capsid proteins do not affect persistently infected basal cells and, thus, fail to clear the infection (13Frazer I.H. Nat. Rev. Immunol. 2004; 4: 46-54Crossref PubMed Scopus (306) Google Scholar). Moreover, high risk HPV-associated cancers generally represent nonproductive infections, and the capsid proteins are not expressed (5Longworth M.S. Laimins L.A. Microbiol. Mol. Biol. 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Therapeutic vaccines aimed to induce targeted T cell-mediated immune responses against dysplastic and neoplastic cells, therefore, seem a logical extension for achieving beneficial clinical results. Given that E6 and E7 are consistently expressed in HPV-associated cancers, these proteins themselves represent promising targets for vaccine design. Although most tumor-specific antigens are derived from normal or mutated endogenous self-proteins (15Boon T. Coulie P.G. Van den Eynde B. Immunol Today. 1997; 18: 267-268Abstract Full Text PDF PubMed Scopus (496) Google Scholar) (TANTIGEN: Tumor T cell Antigen Database), E6 and E7 are foreign viral antigens. These two proteins are required for the induction and maintenance of the malignant phenotype of high-risk HPV-associated cancer cells (5Longworth M.S. Laimins L.A. Microbiol. Mol. Biol. Rev. 2004; 68: 362-372Crossref PubMed Scopus (483) Google Scholar, 6Minger K. Baldwin A. Edwards K.M. Hayakawa H. Nguyen C.L. Owens M. Grace M. Huh K. J. Virol. 2004; 78: 11451-11460Crossref PubMed Scopus (723) Google Scholar), and because HPV uses the cellular DNA replication machinery for genome synthesis, the mutation rate of HPV proteins is low. Thus, it is unlikely that HPV will evade immune attack through loss or mutation of the E6 and/or E7 gene products (16Devaraj K. Gillison M.L. Wu T.C. Crit. Rev. Oral. Biol. Med. 2003; 14: 345-362Crossref PubMed Scopus (67) Google Scholar).Studies on therapeutic vaccines, therefore, have mostly focused on E6 and E7 as target antigens. To date these targets have been delivered as naked DNA vaccines, with recombinant viral or bacterial vectors, as protein or peptide vaccines, and as fusion constructs with toll-like receptor agonists or proteins that enhance antigen delivery or presentation (for review, see Refs. 13Frazer I.H. Nat. Rev. Immunol. 2004; 4: 46-54Crossref PubMed Scopus (306) Google Scholar, 17Leggatt G.R. Frazer I.H. Curr. Opin. 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Recently, multiple long synthetic peptide fragments of E6 and E7 have been used to create a polyepitope vaccine, which when tested in patients with HPV-16-positive vulvar intraepithelial neoplasia, exhibited promising clinical efficacy (35Kenter G.G. Welters M.J. Valentijn A.R. Lowik M.J. Berends-van der Meer D.M. Vloon A.P. Essahsah F. Fathers L.M. Offringa R. Drijfhout J.W. Wafelman A.R. Oostendorp J. Fleuren G.J. van der Burg S.H. Melief C.J. N. Engl. J. Med. 2009; 361: 1838-1847Crossref PubMed Scopus (818) Google Scholar). This type of vaccination-induced clinical response has been the most efficacious to date and argues that a robust outcome can be engendered by peptides in conventional adjuvants.One of the key practical challenges to specific epitope-based vaccines to stimulate cytotoxic T lymphocytes stems from the fundamental nature of T cell receptor (TCR)-based recognition. TCR recognition is referred to as MHC-restricted as, unlike antibody-based recognition, a TCR physiologically interacts with a peptide in complex with an MHC molecule (pMHC) (for review, see Refs. 36Rudolph M.G. Stanfield R.L. Wilson I.A. Annu. Rev. Immunol. 2006; 24: 419-466Crossref PubMed Scopus (901) Google Scholar and 37Wang J.H. Reinherz E.L. Mol. Immunol. 2002; 38: 1039-1049Crossref PubMed Scopus (91) Google Scholar). Further complexity to TCR-based recognition is that a given peptide binds to some but not all MHC molecules. Each human being expresses 3–6 MHC class I molecules (so-called HLA molecules) and at least as many MHC class II molecules. More than 3000 variants of human MHC class I and 1000 variants of MHC class II have been characterized throughout the world to date (38Robinson J. Waller M.J. Fail S.C. McWilliam H. Lopez R. Parham P. Marsh S.G. Nucleic Acids Res. 2009; 37: D1013-D1017Crossref PubMed Scopus (138) Google Scholar). Cytotoxic T cell recognition of foreign protein antigens occurs via short (generally 9–10 amino acids long) peptides produced through proteolytic cleavage in the cytoplasmic proteasome complex. These are subsequently transported into the endoplasmic reticulum, bound to MHC class I molecules and ultimately displayed on the cell surface as a pMHC. The viral pMHC serves as a flag to target an infected or transformed cell for destruction by a CTL.Bioinformatic approaches are important tools for peptide-based vaccines and immunotherapy. Computational methods now offer accuracies that are useful in reducing the number of potential candidate peptides that must be tested experimentally for binding to a given MHC allele (39Brusic V. August J.T. Petrovsky N. Expert Rev. Vaccines. 2005; 4: 407-417Crossref PubMed Scopus (27) Google Scholar, 40De Groot A.S. Berzofsky J.A. Methods. 2004; 34: 425-428Crossref PubMed Scopus (80) Google Scholar, 41Purcell A.W. McCluskey J. Rossjohn J. Nat. Rev. Drug Discov. 2007; 6: 404-414Crossref PubMed Scopus (577) Google Scholar). In silico methods cannot predict, however, which MHC class I-binding peptides are actually processed and displayed on a cell surface. We have developed an MS3 Poisson detection mass spectrometry approach to directly assess the physical presence of predicted CTL target epitopes on tumors and infected cells. Our “predict/detect” method achieves sensitivities comparable with that of a T cell with a dynamic range of one peptide among 100,000 pMHCs displayed per cell.Here for the first time we have interrogated the MHC class I peptide array of several HLA-A*0201 HPV-16-transformed epithelial tumor cells for the presence of any and all predicted HLA-A*0201-binding E6- and E7-derived peptides. Among E6 and E7 proteins, only a single 9-mer epitope was found on all HPV-16 transformants tested. This conserved peptide, termed E711–19, is predicted to have the capacity to bind to the vast majority of globally distributed A2 alleles (100 of 116 HLA-A2 alleles). We suggest that the lack of prior clinical effectiveness of targeted CTL epitope vaccination (32van Driel W.J. Ressing M.E. Kenter G.G. Brandt R.M. Krul E.J. van Rossum A.B. Schuuring E. Offringa R. Bauknecht T. Tamm-Hermelink A. van Dam P.A. Fleuren G.J. Kast W.M. Melief C.J. Trimbos J.B. Eur. J. Cancer. 1999; 35: 946-952Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) is a consequence of misidentification of peptides displayed on tumor cells because of the use of indirect immunological surrogates (killing, proliferation, cytokine production, etc.) to judge T cell epitope expression. Our results offer a direct path to select allele-specific targets that should afford tumor protection to a broad population of patients.DISCUSSIONHPV-induced dysplasia and cancer cause significant morbidity worldwide (7Parkin D.M. Bray F. Vaccine. 2006; 24: S3/11-25Google Scholar). Although prophylactic vaccines are now available, immunization does not reach everyone at risk. Given that HPV-associated cancers develop years and often decades after initial infection, it was predicted that no measurable decline of HPV-associated cancers in women may occur before 2040. This prediction was based upon higher acceptance rates for the vaccines than is currently achieved in the United States (for review, see Ref. 13Frazer I.H. Nat. Rev. Immunol. 2004; 4: 46-54Crossref PubMed Scopus (306) Google Scholar). Furthermore, the approved prophylactic vaccines have no therapeutic effects (54Schiller J.T. Castellsague X. Villa L.L. Hildesheim A. 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A vaccine could be offered to patients who do not clear HPV infection spontaneously during a finite observation period as well as to patients with established lesions.HPV-related diseases represent an ideal set of clinical disorders to test development of a therapeutic cancer vaccine as the viral oncoproteins E6 and E7 are consistently expressed in HPV-associated cancers (16Devaraj K. Gillison M.L. Wu T.C. Crit. Rev. Oral. Biol. Med. 2003; 14: 345-362Crossref PubMed Scopus (67) Google Scholar), and they represent “non-self” cancer antigens. Given their key role in cancerous transformation, a large body of research focusing on E6 and E7 as therapeutic vaccine targets has been conducted. Multiple MHC class I- and class II-restricted epitopes, mostly of HPV-16 and HPV-18, have been reported (19Peng S. Trimble C. Wu L. Pardoll D. Roden R. Hung C.F. Wu T.C. Clin. Cancer Res. 2007; 13: 2479-2487Crossref PubMed Scopus (56) Google Scholar, 30Ressing M.E. Sette A. Brandt R.M. Ruppert J. 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However, because the success of a therapeutic vaccine is dependent on accurate identification of HPV epitopes displayed as pMHC on HPV-infected target cells or HPV-transformed tumor cells, it is essential to define HPV-16 E6 and E7 T cell epitopes that are naturally processed and presented on the surface of virally altered cells. Only those HPV peptide/MHC class I complexes are capable of being recognized by cytolytic T lymphocytes to target destruction of transformed cells.To this end, we have developed a new methodology, nanospray MS3 Poisson detection mass spectrometry. This methodology works by filtering the ion beam through two stages of mass selection and fragmentation (generating MS3 spectra) and detecting a target molecule by a probabilistic measure of the target's known dissociation patterns in the MS3 spectra. The methodology combines instrumental and ionization optimizations in a detection mode format to provide a high dynamic range from limited sample amounts. An instrumental geometry in which a quadrupole filter is placed in front of an ion trap (QTrap 4000) achieves a high duty cycle for MS3 spectra. Static nanospray avoids losses from surface exposure associated with chromatography and in its low (a few nanoliters per minute) flow, an optimal conversion of molecules in the condensed phase into gas phase ions.Our findings are that none of the clinically targeted A2 peptides employed in epitope-based T cell vaccines to date could be detected on HPV-16-transformed cell lines tested herein. Two examples of this discordance are of particular note. The E786–93 peptide has been previously reported to be by far the best HLA-A*0201-binding peptide derived from HPV-16 (46Kast W.M. Brandt R.M. Sidney J. Drijfhout J.W. Kubo R.T. Grey H.M. Melief C.J. Sette A. J. Immunol. 1994; 152: 3904-3912PubMed Google Scholar). It is among the top predicted binders in the present study and the strongest binder in the HLA-A*0201 T2 binding assay. Nonetheless, E786–93 could not be detected by mass spectro
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