Novel Promiscuous HPV16-Derived T Helper Epitopes for Immunotherapy

Abstract

The present invention relates to novel amino acid sequences of peptides derived from HPV16 that are able to promiscuously bind to MHC complexes of class II and shorter peptides contained in the promiscuously binding peptides that bind to MHC complexes of class I, and elicit an immune response. The present invention further relates to pharmaceutical products, such as vaccines and T-cells, based on said epitopes.

Claims

1. A peptide comprising a sequence selected from the group of SEQ ID NO: 7, and SEQ ID NOs: 32 to 42, or a variant of SEQ ID NO: 7, or any of SEQ ID NOs: 32 to 42, wherein said variant is 80% homologous to SEQ ID NO: 7, or any of SEQ ID NOs: 32 to 42, and wherein said peptide or parts thereof have the ability to bind to a molecule of the human major histocompatibility complex (MHC) class-II and/or class-I, and wherein said peptide has an overall length of between 8 and 50 amino acids.

2. The peptide or variant thereof according to claim 1, wherein said peptide or variant thereof has a length of between 15 and 30 amino acids for a peptide or variant thereof comprising a sequence according to SEQ ID NO: 7, or wherein said peptide or variant thereof has a length of between 8 and 30 amino acids for a peptide comprising a sequence according to any of SEQ ID NOs: 32 to 42.

3. The peptide according to claim 1, wherein said peptide consists essentially of an amino acid sequence selected from the group of SEQ ID NO: 7, and SEQ ID NOs: 32 to 42.

4. The peptide according to claim 1, wherein said peptide has a binding affinity of IC.sub.50?60 nM to a HLA molecule.

5. A pharmaceutical composition comprising the peptide according to claim 1, and optionally at least one additional peptide having an amino acid sequence selected from the group of SEQ ID NOs: 1 to 42.

6. The peptide according to claim 1, wherein said peptide is part of a fusion molecule.

7. A nucleic acid encoding the peptide according to claim 1.

8. An expression vector capable of expressing the nucleic acid according to claim 7.

9. A host cell comprising the nucleic acid according to claim 7.

10. A method for producing the peptide according to claim 1, said method comprising synthesizing said peptide; or culturing a host cell and isolating said peptide from said host cell or the culture medium thereof.

11. A method for producing activated T cells, the method comprising contacting T.sub.H cells or CTLs with antigen loaded human class II or I MHC molecules expressed on the surface of antigen-presenting cells for a period of time sufficient to activate said T cells in an antigen specific manner, wherein said antigen is a peptide according to claim 1.

12. Activated T cells, produced by the method according to claim 11, which cells selectively recognize cells that express an HPV polypeptide comprising a sequence selected from the group of SEQ ID NO: 7, and SEQ ID NOs: 32 to 42, or a variant of SEQ ID NO: 7, or any of SEQ ID NOs: 32 to 42, wherein said variant is 80% homologous to SEQ ID NO: 7, or any of SEQ ID NOs: 32 to 42, and wherein said peptide or parts thereof have the ability to bind to a molecule of the human major histocompatibility complex (MHC) class-II and/or class-I.

13. A pharmaceutical preparation, comprising at least one of: a) a nucleic acid encoding a sequence selected from the group of SEQ ID NO: 7, and SEQ ID NOs: 32 to 42, or a variant of SEQ ID NO: 7, or any of SEQ ID NOs: 32 to 42, wherein said variant is 80% homologous to SEQ ID NO: 7, or any of SEQ ID NOs: 32 to 42, and wherein said peptide or parts thereof have the ability to bind to a molecule of the human major histocompatibility complex (MHC) class-II and/or class-I, and wherein said peptide has an overall length of between 8 and 50 amino acids; b) an expression vector or a host cell comprising the nucleic acid of part a); and c) the activated T cells according to claim 12; and a pharmaceutically acceptable excipient.

14. A method for treating HPV infection, HPV-related premalignancies and/or malignancies comprising administering the pharmaceutical composition according to claim 5 to a patient in need of said treatment.

15. The method for treating HPV infection, HPV-related premalignancies and/or malignancies according to claim 14, wherein said treatment is MHC-I and/or MHC-II peptide presentation dependent.

16. A method for treating HPV infection, HPV-related premalignancies and/or malignancies comprising administering the pharmaceutical preparation according to claim 13 to a patient in need of said treatment.

17. The method for treating HPV infection, HPV-related premalignancies and/or malignancies according to claim 16, wherein said treatment is MHC-I and/or MHC-II peptide presentation dependent.

18. The host cell, according to claim 9, wherein the cell is an antigen presenting cell.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0070] The invention will now be described in more detail in the following examples with reference to the accompanying Figures and the sequence listing, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures,

[0071] FIG. 1 shows the detection of HPV16 peptide-specific T cell immunity in healthy subjects. The PBMC reactivity of 13 healthy donors was evaluated by stimulating with selected HPV16-derived peptides for 12 days. IFN-? peptide-specific T cells were analyzed by ELISPOT. Each dot represents the number of positive cells in one individual. Data represent the mean from triplicate wells after background subtraction. Mean responses (?SE) across donors are shown for each peptide. Responses are considered positive (?) if they were at least twofold higher than the background. An open circle (o) represents a non-responder. Percentages represent response rate of tested healthy subjects to the indicated HPV16-derived peptide.

[0072] FIGS. 2A-2B show that T cell immunity induced by the selected HPV16-derived peptides is dependent on HLA class II peptide presentation. PBMC obtained from subject HD54 (2A) and HD55 (2B) were incubated in the presence or absence of anti-HLA-DR or panHLA-I W6/32 antibodies prior the indicated peptide exposure, and responses analyzed by IFN-? ELISPOT. Data represent the mean result from triplicate wells after background subtraction. Significant inhibition in presence of HLA-DR antibodies compared to non-treated PBMC is indicated by *(P?0.05, two-tailed Student's t-test). The HLA genotypes of tested subjects are indicated above the panels.

[0073] FIGS. 3A-3C show that the induced HPV16 peptide-specific T cell immunity is mediated by CD4+ T cells. PBMC of 7 healthy donors after stimulation with the indicated HPV16-derived peptides were tested by flow cytometry-based IFN-? intracellular staining (ICS) (3A). Each dot represents an individual. Results are presented as percentage of IFN-?-positive CD4+ (?) or CD8+ (o) cells after background subtraction. (3B) PBMC (?) obtained from 12 healthy individuals were depleted of CD4+ T cells (o), stimulated with selected peptides, and analyzed by IFN-? ELISPOT. Each dot represents an individual. Data represent the mean result from triplicate wells after background subtraction. (3C) PBMCs of two subjects (HD23 and HD24) were depleted of either CD4+ or CD8+ T cells prior to peptide exposure as indicated and analyzed by IFN-? ELISPOT. Data represent the mean result from triplicate wells after background subtraction. The HLA genotypes of tested subjects are indicated above the panels. Mean responses (?SE) are indicated for each peptide. Significant differences are indicated by *(P?0.05), **(P?0.01) or ***(P?0.001, two-tailed Student's t-test).

[0074] FIGS. 4A-4B show that the induced HPV16 peptide-specific immunity is associated with Th1-type cells but not with CD4+ regulatory T cells. (4A) The supernatants of T cell cultures from HD23 were tested 3 days after in vitro stimulation with indicated peptides for the indicated cytokines. Data represent the mean result from duplicate wells after background subtraction. The cut-off for positivity is indicated by a dashed line. (4B) PBMC were also investigated for presence of CD4+/CD25+/FoxP3+ regulatory T cells after 8 day in vitro stimulation with indicated peptides or without stimulation (DMSO) in a flow cytometry-based assay. Results are presented as percentage of CD25+/FoxP3+ T cells on the counter plot gated on CD4+ T cells. Representative results of one donor (HD 23) are shown. The HLA genotype of the tested subject is indicated above the panel.

[0075] FIG. 5 shows that PBMC of five healthy donors were investigated for presence of CD4+/CD25+/FoxP3+ regulatory T cells after 12 days in vitro stimulation with indicated peptides or without stimulation (DMSO) in a flow cytometry-based assay. Results are presented as percentage of CD25+/FoxP3+ T cells. Each dot represents an individual. Mean responses (?SE) across donors are shown for each peptide.

BRIEF DESCRIPTION OF THE SEQUENCES

[0076] SEQ ID NO: 1 to 42 show peptide sequences of HPV-related antigens and epitopes according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

Examples

Materials and Methods

Prediction of Promiscuous HLA Class II-Binding HPV-Derived Epitopes

[0077] An epitope prediction server, SYFPEITHI (http://www.syfpeithi.com) (39) was used for predicting 15-mer Th epitopes in E7 (GenBank: AAD33253.1), E6 (GenBank: AAD33252.1), E5 (GenBank: AA085413.1), and E2 (GenBank: AAD33255.1) proteins of HPV16. The in silico predictions were performed for multiple HLA-DR molecules encoded by DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101 and DRB1*1501 alleles. Higher binding score values indicate higher predicted binding affinity of candidate peptides to the selected HLA-DR allele. To identify promiscuous CD4+ T cell epitopes, for each protein, candidate peptides were ranked according to their predicted binding affinity among the indicated HLA-DR alleles, excluding epitopes harboring more than two cystein residues. Peptides predicted to bind at least five HLA-DR molecules were selected. In addition, HLA-DR binding epitopes were analyzed for dataset cross-comparison by the recently developed NetMHCII (NN-align) prediction method (Nielsen M, et al. 2010. NetMHCIIpan-2.0Improved pan-specific HLA-DR predictions using a novel concurrent alignment and weight optimization training procedure. Immunome Res 6: 9, Nielsen M, Lund O. 2009. NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction. BMC Bioinformatics 10: 296) (http://www.cbs.dtu.dk/services/NetMHCII/), which also allows to perform predictions for molecules encoded by DRB1*0901, DRB1*1302, DRB3*0101, DRB4*0101 and DRB5*0101 alleles. In this case, lower IC.sub.50 nM values indicate higher binding affinity of peptides to a HLA-DR molecule.

Peptides

[0078] The 15-mer peptides were synthesized using 9-fluorenylmethoxycarbonyl chemistry, and purified by high-performance liquid chromatography (>95% purity) and validated by mass spectrometry (Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ), Heidelberg, Germany). Synthetic peptides were reconstituted in DMSO (Sigma, Taufkirchen, Germany) at a concentration of 10 mg/ml, and stored at ?80? C. A MHC class II peptide pool (PANATecs, T?bingen, Germany) was included in experiments as a positive control.

Healthy Volunteers and Peripheral Blood T Cell Sample Acquisition

[0079] Peripheral blood samples or buffy coats of whole blood from anonymous healthy individuals were obtained from the IKTZ Heidelberg Blood Bank. As these donors are anonymous, no data are available to determine HPV infection status. Peripheral blood mononuclear cells (PBMC) were isolated within 12 h using a standard Ficoll-Hypaque density gradient procedure. PBMC were cultured directly or cryopreserved in RPMI-1640 medium (PAA, Coelbe, Germany) containing 20% fetal bovine serum (FBS) (Bio West, Nuaille, France) and 10% DMSO and stored in the gas phase of liquid nitrogen for later use. The HLA class I and class II haplotypes of the blood donors were typed by the Transplantation and Immunology Laboratory at the Institute of Immunology and Serology, Heidelberg, Germany. Sampling and use of PBMC were in accordance with the Institutional Review Board at DKFZ and the University of Heidelberg, Heidelberg, Germany.

Depletion of CD4+ T Cells from PBMC

[0080] PBMC were depleted of CD4+ T cells according to the manufacturer's instruction using anti-CD4 MicroBeads (Miltenyi Biotec, Bergisch Gladbach. Germany). Purity was assessed by flow cytometry. PBMC were effectively (>98%) depleted of CD4+ cells.

T-Cell Culture and IFN-? ELISPOT Assay

[0081] PBMC were used for CD4+ T cell depletion or cultured directly in RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin and 100 ?g/ml streptomycin (R10 medium). The immunogenicity of candidate HPV16-derived peptides was evaluated by ELISPOT analysis of short-term PBMC cultures. Briefly, PBMC were seeded at a density of 10.sup.6 cells/well in 24-well plates (Becton Dickinson (BD), Heidelberg, Germany) in 1 ml of R10 medium, in the presence or absence of individual HPV16-derived peptides with a final concentration of 20 ?g/ml. As a positive control, PBMC were cultured in the presence of the positive peptide pool (4 ?g/ml) or 5 ?g/ml of phytohaemagglutinin (PHA; Sigma). On day 5, PBMC were harvested, and seeded in triplicate wells at a density of 10.sup.5 cells/well on Multiscreen-HA ELISPOT plates (Millipore, Darmstadt, Germany) coated with 2 ?g/ml of anti-human IFN-? antibody 1-D1K (Mabtech AB, Nacka Strand, Sweden). Cells were incubated for another 20 h at 37? C. The ELISPOT assay was developed according to the manufacturer's instructions (Mabtech AB). The number of spots was analyzed with an AID ELISPOT reader (AID Diagnostika, Strassberg, Germany). Specific spots were calculated by subtracting the mean number of spots of the DMSO background control from the mean number of spots of test wells. A peptide-specific T cell response was considered positive when the mean spot number for a given peptide was at least twofold higher than mean background spot number.

[0082] In experiments in which selected HPV-derived peptides were further analyzed, the PBMC or depleted PBMC were cultured for 12 days. Briefly, PBMC or depleted PBMC were seeded at a density of 5?10.sup.5 to 1?10.sup.6 cells/well in 24-well plates in 1 ml of R10 medium supplemented with recombinant human interleukin-7 (rhIL-7, R&D Systems, Wiesbaden, Germany) at a final concentration of 330 U/ml per well. Cells were incubated in the presence of either DMSO (background control), selected HPV16-derived peptides (20 ?g/ml) or the positive peptide pool (4 ?g/ml). On days 3 and 7, cell culture was supplemented with rhIL-2 (PeproTech, Hamburg, Germany) at a final concentration 20 U/ml per well and the medium was replaced (no rhIL-2) on day 10. Cells were restimulated on day 11 with the indicated peptides (20 ?g/ml). On day 12, cultures were analyzed for the presence of peptide-specific T cells by IFN-? ELISPOT assay as described above.

[0083] To block peptide-specific IFN-? production, anti-HLA-DR antibody L243 (BioLegend, Fell, Germany) or anti-HLA class I antibody W6/32 were added to the cell cultures at a final concentration of 10 ?g/ml for 30 min before peptide exposure.

IFN-? Intracellular Cytokine Staining

[0084] PBMC stimulated for 11 days with peptide as described above, were harvested, washed and incubated in the presence of either DMSO or selected HPV16-derived peptide (20 ?l/ml). GolgiPlug (1/1000; BD) was added after 1 h for a further 12 h incubation period. The cells were washed and blocked with PBS/10% FBS and next stained for the surface markers CD3-APC, CD4-PE, CD8-FITC (BD). For intracellular staining, cells were fixed and permeabilized using Cytofix/Cytoperm solution according to the manufacturer's instructions (BD). The cells were washed with Perm/Wash buffer (BD) before staining with anti-human IFN-? eFluor450-conjugated antibody (eBioscience, Frankfurt, Germany). After fixation with 1% formaldehyde, stained cells were analyzed on a FACS Canto II cytometer (BD). Respective isotype controls were used in all experiments. Data analysis was performed with FlowJo (TreeStar, Ashland, Oreg., USA). Identical gates were used for all samples. A response was considered positive for peptide-specific T cells if the response was greater than 0.04% after subtraction of the mean DMSO background.

Cytokine Assays

[0085] The supernatants collected on day 3 of the PBMC cultured with or without selected HPV-derived peptides were subjected to cytokine magnetic bead assay Milliplex MAP (Millipore) according to the manufacturer's instructions. The read-out of the assay was performed on a Luminex MAGPIX instrument with xPONENT software (Luminex, Austin, Tex., USA). In this assay, the levels of IFN-?, TNF-?, GM-CSF, IL-4, IL-5 and IL-2 were determined. A peptide-specific cytokine production was considered positive if (after subtraction of the mean DMSO control) the cytokine concentration was over the cutoff value of 20 ?g/ml (50 ?g/ml for IFN-?).

Phenotypic CD4+ T Cell Analysis by Flow Cytometry

[0086] PBMC stimulated for 11 days with peptide as described above, were harvested, washed and incubated in the presence of either DMSO or relevant HPV-derived peptide (20 ?l/ml). GolgiPlug (1/1000; BD) was added after 1 h for a further 12 h incubation period. The cells were washed and blocked with PBS/10% FBS and next stained for the surface markers CD4-FITC, CD25-APC (BD). For intracellular staining, cells were fixed and permeabilized using Transcription Factor FIX/Perm Buffer according to the manufacturer's instructions (BD). The cells were washed with Perm/Wash buffer (BD) before staining with anti-human FoxP3 PE-conjugated antibody (BD). After fixation with 1% formaldehyde, stained cells were analyzed on a FACS Canto II cytometer (BD).

Statistical Analysis

[0087] Statistical analysis was performed using GraphPad Prism 5 Software Inc. Two-tailed Student's t-test with Welch's correction was used to analyze the data. P values ?0.05 were considered significant.

Results

[0088] Prediction and Selection of Candidate Peptides Derived from the HPV16 E2, E5, E6 and E7 Proteins with High Binding Potency for Multiple HLA-DR Molecules

[0089] The inventors' study was designed to identify epitopes binding to multiple HLA-DR alleles, derived from HPV16 early proteins E2, E5, E6 and E7. Therefore the inventors first analyzed the amino acid sequence of the indicated HPV16 proteins using two epitope prediction algorithms (Rammensee H, et al. 1999. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50: 213-9; Nielsen M, et al. 2010. NetMHCIIpan-2.0Improved pan-specific HLA-DR predictions using a novel concurrent alignment and weight optimization training procedure. Immunome Res 6: 9; Nielsen M, Lund O. 2009. NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction. BMC Bioinformatics 10: 296). 144 peptides for E6, 84 peptides for E7, 351 peptides for E2 and 69 peptides for E5 binding to eleven different HLA-DR molecules were identified. To select promiscuous CD4+ T cell epitopes, for each protein, the 15-mer candidate peptides were ranked according to their binding affinity to multiple HLA-DR alleles, excluding those harboring more than two cystein residues to minimize complexities resulting from intramolecular or intermolecular disulfide bond formation. Six candidate peptides of E6 and E7 as well as eleven and eight candidate peptides from E2 and E5, respectively, which were predicted as binders by any one of the prediction algorithms and bound to at least 5 different HLA-DR molecules (Table 1) were synthesized and used for subsequent assays. As shown in Table 1, predictions were not completely consistent between the two algorithms.

Evaluation of HPV16 Peptide-Specific T Cell Immunity in Healthy Subjects

[0090] To investigate whether the predicted candidate peptides were able to stimulate T cells from a cohort of healthy donors, the inventors used a 6-day IFN-? ELISPOT assay. Frequencies of reactive T cells to each candidate peptide and the number of positive responders are reported for E6- and E7-derived peptides in Table 2, and for E2- and E5-derived peptides in Table 3. T cells isolated from the majority of healthy subjects showed reactivity against one or more of the HPV16-derived candidate peptides, after a single round of in vitro stimulation. The peptides E6/54-68 (30% response rate of tested healthy subjects), E6/74-88 (45%), E7/12-26 (40%), E7/64-78 (40%), E2/76-90 (30%), E2/99-113 (35%), E2/156-170 (40%), E5/33-48 (50%), E5/54-69 (55%), and E5/67-81 (60%) were found to be the most potent epitopes in this cohort of individuals expressing a wide variety of HLA-DR molecules. To further characterize the immune potency of these ten selected peptides, PBMC from an independent set of 13 healthy subjects were analyzed in a 12 day culture expansion of preexisting memory T cells. Using this approach, even higher frequencies of IFN-? producing T cells against the selected HPV16-derived peptides could be observed (FIG. 1). For each peptide a high response rate was observed, with 92% cumulative response rate (against all selected peptides) in the whole cohort of tested healthy donors. The five peptides showing the highest response rates and average spot numbers (E2/156-170, E5/33-48, E6/74-88, E7/12-26 and E7/64-78) were chosen for further analysis.

Peptide Reactivity is Abrogated in the Presence of Anti-HLA-DR Antibodies

[0091] To determine whether the selected HPV16-derived peptides were HLA-DR-restricted, PBMC were incubated with the HLA-DR blocking antibody L243 before peptide exposure and then subjected to IFN-? ELISPOT assay. Similarly, PBMC cultures were incubated with the pan-specific HLA class I blocking antibody W6/32. Two subjects were analyzed in this assay. As shown in FIG. 2, peptide-specific T cell responses were abrogated in all cases under anti-HLA-DR antibody treatment, indicating that the epitopes are truly HLA-DR-restricted. However, the reactivity of the E5/33-48 peptide was partially reduced when PBMC from subject HD54 were cultured with the anti-panHLA-I antibody W6/32 (FIG. 2A). This was not observed with this peptide with PBMC from HD55 (FIG. 2B). Further, blocking activity of the anti-panHLA-I antibody was found in PBMC cultures of both subjects stimulated with E7/64-78 peptide, and with E6/74-88-stimulated PBMC from subject HD55. These results indicate that the 15-mer peptides E5/33-48, E6/74-88 and E7/64-78 are harboring epitopes restricted by some HLA class I molecules.

The Induced HPV16 Peptide-Specific T Cell Immunity is CD4+ T Cell-Mediated

[0092] To verify whether the observed peptide-specific T cell responses are indeed mediated by CD4+ T cells, the inventors tested the five selected peptides in an IFN-? intracellular cytokine secretion (ICS) assay in 7 healthy subjects. Even if HPV16 peptide-specific T cell reactivities detected by this assay were generally low, a predominance of CD4+ T cells compared to CD8+ T cells could be observed (FIG. 3A). This difference was found to be significant for E2/156-170 (P=0.013), E6/74-88 (P=0.01), E7/64-78 (P=0.05), but not for E5/33-48 (P=0.14) and E7/12-26 (P=0.10). This approach allowed us to assess T cell subsets taking part in immune response against the selected HPV16 peptides. However, the inventors conclude that the ICS method is not sensitive enough to assess preexisting HPV responses in healthy subjects.

[0093] To independently verify these findings with another method, PBMC were depleted of CD4+ T cells (and, if sufficient PBMC were available from a donor, also of CD8+ T cells) prior to in vitro peptide stimulation and subjected to ELISPOT analysis. The same five selected peptides were analyzed in an independent set of twelve subjects for this assay. CD4+ T cell-depletion resulted in significant loss of peptide reactivity (P?0.05) in the majority of tested subjects (FIG. 3B), again showing that the peptide-specific immune responses are mediated by CD4+ T cells. CD8+ T cell-depletion did not affect peptide-specific IFN-? production (FIG. 3C). However, reactivity of the E7/64-78 peptide was not abolished in CD4+ T cell-depleted PBMC from subject HD24, indicating some CD8+ T cell reactivity to this peptide in this donor. Taken together, these data provide strong evidence that the selected HPV16-derived peptides are able to induce CD4+ T cell-mediated immunity.

Peptide-Specific CD4+ T Cells Have A Th1 Phenotype

[0094] In order to investigate the functional properties of peptide-specific CD4+ T cells, the inventors assessed the Th1/Th2 cytokine profile. Analysis of supernatants of T cell cultures from HD23 for presence of IFN-?, TNF-?, GM-CSF, IL-4, IL-5 and IL-2 showed predominant secretion of Th1 effector cytokines in response to the selected HPV16-derived peptides (FIG. 4A). High levels of IFN-?, TNF-? (except for peptide E5/33-48) and GM-CSF were detected, whereas only weak or no production of IL-4 and IL-5 was observed. Interestingly, IL-2 was not detected in these T cell cultures, which is probably due to its consumption by the proliferating T cells (this cytokine was not provided during the PBMC culture). Furthermore, the inventors tested in five subjects whether the observed CD4+ T cell-mediated immunity in the peptide-stimulated T cell cultures contains regulatory T cells (Treg). To do so, the inventors determined the frequency of peptide-specific CD4+/CD25+/FoxP3+ T cells after 12 days of peptide stimulation. Only minor populations of peptide-specific CD4+/CD25+/FoxP3+ T cells were found in PBMC cultures after stimulation with the selected HPV16-derived peptides (FIG. 4B, representative results of one donorHD23are shown).

TABLE-US-00007 TABLE1 Candidatepan-HLA-DRbindingpeptidesderivedfromtheHPV16E2,E5,E6andE7proteins. NetMHCII Peptide SYFPEITHI Highaffinity (protein/ Sequence Highscoring Otherscoring binding Otherbinding position) (SEQIDNo.) HLA-DRalleles.sup.a HLA-DRalleles.sup.b HLA-DRalleles.sup.c HLA-DRalleles.sup.d E6/16-30 PRKLPQLCTELQTTI DRB1*01,*04 DRB1*03,*07, DRB1*01,*04, (SEQIDNo.1) *11,*15 DRB4 E6/42-56 QQLLRREVYDFAFRD DRB1*04 DRB1*01,*03, DRB1*01,*03, (SEQIDNo.2) *07,*11,*15 *04,*15,DRB3, DRB4,DRB5 E6/54-68 FRDLCIVYRDGNPYA DRB1*04,*11 DRB1*01,*03, DRB1*01,*04, (SEQIDNo.3) *15 *11,*13,*15, DRB3,DRB5 E6/74-88 LKFYSKISEYRHYCY DRB1*07 DRB1*01,*04, DRB1*01,*15, DRB1*04,*07, (SEQIDNo.4) *11 DRB5 *09,*11 E6/92-106 GTTLEQQYNKPLCDL DRB1*01,*03, DRB1*15 DRB1*01,*13, (SEQIDNo.5) *04,*07 DRB4 E6/129-143 KQRFHNIRGRWTGRC DRB1*01,*04 DRB1*07,*11, DRB1*01,*04, (SEQIDNo.6) *15 *07,*09,*11, *15,DRB5 E7/12-26 MLDLQPETTDLYCYE DRB1*03,*04, DRB1*01,*15 DRB1*01,*03 (SEQIDNo.7) *07 E7/63-77 STLRLCVQSTHVDIR DRB1*01 DRB1*04,*07 DRB1*01,*07, DRB1*03,*04, (SEQIDNo.8) DRB4 *09,*11,*13, *15,DRB5 E7/64-78 TLRLCVQSTHVDIRT DRB1*01 DRB1*03,*04, DRB1*01,*07, DRB1*03,*04, (SEQIDNo.9) *07,*15 DRB4 *09,*11,*13, *15,DRB5 E7/71-85 STHVDIRTLEDLLMG DRB1*01,*03, DRB1*01,*03, (SEQIDNo.10) *07,*15 *04,*07,DRB4 E7/73-87 HVDIRTLEDLLMGTL DRB1*01,*04, DRB1*03,*11 DRB1*01 DRB1*03,*04, (SEQIDNo.11) *07,*15 DRB4 E7/76-90 IRTLEDLLMGTLGIV DRB1*01,*04 DRB1*03,*07, DRB1*01 DRB1*04,*07, (SEQIDNo.12) *11,*15 DRB4 E2/27-42 RDHIDYWKHMRLECA DRB1*04,*15 DRB1*01,*03, DRB1*11 DRB1*01,*07, (SEQIDNo.13) *07,*11 *15,DRB4 E2/51-65 FKHINHQVVPTLAVS DRB1*01 DRB1*03,*04, DRB1*01,*07 DRB1*04,*09, (SEQIDNo.14) *07,*15 *11,*13,*15, DRB4,DRB5 E2/59-73 VPTLAVSKNKALQAI DRB1*03,*04, DRBI*01,*15 DRB1*01,*13 DRB1*03,*07, (SEQIDNo.15) *07 *09,*11, DRB4,DRB5 E2/76-90 QLTLETIYNSQYSNE DRB1*03,*04, DRB1*01,*11, DRB1*01,*04, (SEQIDNo.16) *07 *15 *15 E2/99-113 LEVYLTAPTGCIKKH DRB1*01,*04, DRB1*11 DRB1*01,*07, DRB1*04,*09, (SEQIDNo.17) *07 DRB5 *11,*15,DRB3 E2/156-170 GLYYVHEGIRTYFVQ DRB1*01,*03, DRB1*01,*07, DRB1*03,*04, (SEQIDNo.18) *04,*07,*11 *15,DRB3, *09,*11,DRB4 DRB5 E2/208-223 PEIIRQHLANHPAAT DRB1*01 DRB1*03,*04, DRB1*01,DRB4 DRB1*04,*07, (SEQIDNo.19) *11,*15 *09,*11,*13, *15,DRB5 E2/267-282 ILTAFNSSHKGRINC DRB1*07,*09, DRB1*01,*04, (SEQIDNo.20) DRB5 *11,*13,*15 E2/288-302 IVHLKGDANTLKCLR DRB1*03,*04 DRB1*01,*07, DRB1*03 DRB1*01,*04, (SEQIDNo.21) *15 DRB3,DRB5 E2/300-314 CLRYRFKKHCTLYTA DRB1*01 DRB1*03,*04, DRB1*01,*07, DRB1*04,*09, (SEQIDNo.22) *07,*11 *11,*15 DRB3,DRB5 E2/344-359 DQFLSQVKIPKTITV DRB1*04 DRB1*01,*03, DRB1*01,*11, DRB1*04,*07, (SEQIDNo.23) *11,*15 DRB5 *09,*13,*15, DRB4 E5/1-15 MTNLDTASTTLLACF DRB1*01,*03, DRB1*15 DRB1*01,*07 DRB1*03,*04, (SEQIDNo.24) *04,*07 *09,*13 E5/30-45 RPLLLSVSTYTSLIL DRB1*01,*04 DRB1*03,*07, DRB1*01,*04, DRB1*03,*09, (SEQIDNo.25) *11,*15 *07,*15 *11,*13, DRB4,DRB5 E5/33-48 LLSVSTYTSLILLVL DRB1*01,*04, DRB1*03 DRB1*01,*07, DRB1*04,*09, (SEQIDNo.26) *07,*15 *15 *11, E5/45-60 LVLVLWITAASAFRC DRB1*01,*04 DRB1*03,*07, DRB1*01,*07, DRB1*04,*11, (SEQIDNo.27) *11,*15 *09,DRB5 *13,*15,DRB3 E5/54-69 ASAFRCFIVYIVFVY DRB1*01,*04, DRB1*07,*11 DRB1*01,*11, (SEQIDNo.28) *15 *15 E5/60-74 FIVYIVFVYIPLFLI DRB1*04,*15 DRB1*01,*07, DRB1*01,*07, (SEQIDNo.29) *11 *15,DRB3 E5/67-81 VYIPLFLIHTHARFL DRB1*11 DRB1*01,*04 DRB1*01,*04, DRB1*03,*09, (SEQIDNo.30) *07,*11,*13, DRB3,DRB4 *15,DRB5 E5/69-84 IPLFLIHTHARFLIT DRB1*01,*11 DRB1*03,*04, DRB1*01,*03, DRB1*09,DRB3, (SEQIDNo.31) *07 *04,*07,*11, DRB4 *13,*15,DRB5 .sup.aThreshold for good binding peptides: score ? 20. .sup.bThreshold for other binding peptides: score 19 ? 10. .sup.cThreshold for good binding peptides: IC.sub.50 ? 60 nM. .sup.dThreshold for other binding peptides IC.sub.50: 60 ? 1000 nM.

TABLE-US-00008 TABLE 2 HPV16 E6- and E7-specific T-cell responses in healthy donors as measured by IFN-? ELISPOT HLA- HPV16 E6 peptides HPV16 E7 peptides DRB1 E6/ E6/ E6/ E6/ E7/ E7/ E7/ Donor genotype 16-30 42-56 54-68 74-88 E6/92-106 E6/129-143 E7/12-26 E7/63-77 E7/64-78 71-85 73-87 76-90 HD1 *04, .sup.55.sup.a 12 25 35 88 128 58 98 88 82 192 HD2 *03, *11 27 13 13 HD3 *03, *04 187 260 233 183 63 113 80 80 133 177 180 323 HD4 *04, *16 17 30 7 7 27 HD5 *01, *16 2 288 392 528 122 28 205 185 HD6 *03, *13 50 130 220 7 97 567 3 63 13 63 20 HD7 *08, *11 7 3 3 60 7 HD8 *13, 63 HD9 *01, *07 2 2 2 35 18 2 18 5 HD10 *04, *13 85 15 2 142 18 45 68 78 245 58 115 48 HD11 *01, *04 250 163 77 73 70 207 347 113 133 43 117 HD12 *03, *11 263 73 250 393 243 83 223 447 270 350 190 223 HD13 *11, *14 125 105 108 222 168 135 258 68 265 15 72 38 HD14 *07, *13 143 33 213 87 37 190 140 227 127 40 113 HD15 *01, *08 20 37 63 40 13 10 63 87 130 237 67 73 HD16 *07, *13 45 195 18 22 45 25 192 172 98 328 72 22 HD17 *01, *15 142 282 132 332 235 458 342 242 812 438 298 278 HD18 *08, *13 5 162 85 5 258 228 215 48 98 HD19 *01, *15 518 162 362 168 108 355 315 418 215 202 82 HD20 *07, *15 175 85 75 95 115 118 318 128 108 192 165 95 Positive 5 (25%) 5 (25%) 6 (30%) 9 (45%) 2 (10%) 5 (25%) 8 (40%) 5 (25%) 8 (40%) 6 (30%) 4 (20%) 3 (15%) responders (%) .sup.aNumber of peptide-specific memory T cells per 10.sup.6 PBMC. Data represent the mean result from triplicate wells after background subtraction. A dash indicates that no specific T cell reactivity was detected. Responses are considered positive (bold) if they were at least twofold higher than the background.

TABLE-US-00009 TABLE 3 HPV16 E2- and E5-specific T-cell responses in healthy donors as measured by IFN-? ELISPOT HLA- HPV16 E2 peptides DRB1 E2/ E2/ E2/ E2/ E2/ E2/ E2/ E2/ E2/ Donor genotype 27-42 51-65 59-73 76-90 99-113 156-170 208-223 267-282 288-302 HD21 *07, 123.sup.a 7 47 50 147 13 13 7 13 *13 HD22 *04, 3 3 7 27 7 10 3 *15 HD23 *11, 10 13 20 *13 HD24 *09, 3 7 7 20 10 17 10 3 *12 HD25 *03, 170 3 110 *15 HD26 *04, 10 23 17 *16 HD27 *01, 10 57 87 60 133 53 40 87 3 *11 HD28 *11, 27 27 223 493 73 17 57 *15 HD29 *04, 17 23 57 33 87 3 13 *16 HD30 *07, 10 3 3 3 *11 HD31 *07, 3 50 77 47 23 27 *11 HD32 *01, 3 7 40 40 7 3 3 *16 HD33 *04, 150 40 173 120 73 617 93 270 *07 HD34 *04, 13 3 7 10 HD35 *11, 3 17 33 20 7 7 HD36 *04, 47 47 403 13 HD37 *04, 47 10 167 307 83 167 17 40 77 *07 HD38 *13, 100 10 30 33 350 47 53 23 7 *15 HD39 *04, 10 17 27 33 13 13 3 *13 HD40 *03, 10 37 17 3 23 3 *04 Positive 3 2 4 6 7 8 3 2 2 responders (15%) (10%) (20%) (30%) (35)% (40%) (15%) (10%) (10%) (%) HPV16 E2 peptides HPV16 E5 peptides E2/ E2/ E5/ E5/ E5/ E5/ E5/ E5/ Donor 300-314 344-359 1-15 30-45 33-48 45-60 54-69 60-74 E5/67-81 E5/69-84 HD21 7 40 7 67 20 153 97 40 3 HD22 3 3 3 17 13 7 220 7 10 HD23 13 23 3 43 193 200 3 HD24 10 7 23 17 433 3 3 HD25 13 70 207 43 7 17 27 23 HD26 30 13 7 77 50 33 100 HD27 77 73 223 247 73 97 140 200 133 13 HD28 7 10 270 60 37 250 143 147 180 63 HD29 143 7 3 13 10 40 60 113 HD30 7 13 3 7 27 13 HD31 20 3 13 73 7 3 80 107 HD32 10 30 47 33 13 3 HD33 123 103 283 130 500 407 367 27 73 110 HD34 13 30 30 7 40 43 HD35 17 30 10 10 170 20 157 207 50 20 HD36 127 123 80 77 27 40 17 HD37 123 43 183 50 297 80 127 93 17 HD38 27 37 53 173 160 247 280 183 227 50 HD39 83 107 3 10 27 10 73 47 23 HD40 20 3 30 17 20 20 53 27 20 23 5 3 (15%) 4 5 10 6 11 12 7 3 (25%) (20%) (25%) (50%) (30%) (55%) (60%) (35%) (15%) .sup.aNumber of peptide-specific memory T cells per 10.sup.6 PBMC. Data represent the mean result from triplicate wells after background subtraction. A dash indicates that no specific T cell reactivity was detected. Responses are considered positive (bold) if they were at least twofold higher than the background.

TABLE-US-00010 TABLE 4 Comparison of the predicted HLA-DRB1 alleles with the experimentally tested HLA-DRB1 molecules presenting selected HPV16-derived peptides. HLA-DRB1 Peptide molecules predicted HLA-DRB1 genotype (protein/position) to bind a given peptide of responders E6/54-68 *01, *03, *04, *11, *03/*04; *01/*16; *03/*13; *13, *15 *03/*11; *01/*08; *01/*15 E6/74-88 *01, *04, *07, *09, *03/*04; *01/*16; *03/*13; *11, *15 *01/*07; *04/*13; *03/*11; *07/*13; *01/*08; *01/*15 E7/12-26 *01, *03, *04, *07, *03/*04; *03/*13; *01/*04; *15 *03/*11; *01/*08; *01/*15; *07/*15 E7/64-78 *01, *03, *04, *07, *03/*04; *03/*13; *04/*13; *09, *11, *13, *15 *03/*11; *07/*13; *01/*08; *01/*15 E7/71-85 *01, *03, *04, *07, *03/*04; *08/*11; *03/*11; *15 *01/*08; *07/*13; *01/*15 E2/76-90 *01, *03, *04, *07, *07/*13; *01/*11; *11/*15; *11, *15 *04/*16; *04/*07; *04/*13 E2/99-113 *01, *04, *07, *09, *07/*13; *09/*12; *03/*15; *11, *15 *01/*11; *04/*07; *13/*15 E2/156-170 *01, *03, *04, *07, *09/*12; *01/*11; *11/*15; *09, *11, *15 *04/*16; *04/*07; *04/; *13/*15 E5/33-48 *01, *03, *04, *07, *07/*13; *09/*12; *03/*15; *09, *11, *15 *01/*11; *04/*07; *13/*15; *04/*13 E5/45-60 *01, *03, *04, *07, *11/*13; *01/*11; *11/*15; *09, *11, *13, *15 *04/*07; *13/*15 E5/54-69 *01, *04, *07, *11, *07/*13; *11/*13; *09/*12; *15 *01/*11; *11/*15; *04/*07; *11/; *13/*15; *04/*13; *03/*04 E5/60-74 *01, *04, *07, *11, *07/*13; *04/*15; *11/*13; *15 *09/*12; *01/*11; *11/*15; *07/*11; *04/; *11/; *04/*07; *13/*15; *04/*13 E5/67-81 *01, *03, *04, *07, *04/*16; *01/*11; *11/*15; *09, *11, *13, *15 *04/*07; *13/*15; *04/*13 Peptides in bold: selected for detailed analysis. HLA-DRB1 molecules in bold: predicted as high affinity binding or scoring.

TABLE-US-00011 TABLE5 Experimentalbindingaffinity ofselectedHPV16-derivedpeptides Peptide Binding (protein/ affinity HLA position) Sequence (?M) type E7/15-24 LQPETTDLYC 38.45 HLA-B15 (SEQIDNo.32) E7/15-25 LQPETTDLYCY 7.3 HLA-B15 (SEQIDNo.33) E7/12-19 MLDLQPET 53.66 HLA-A2 (SEQIDNo.34) E7/18-25 ETTDLYCY 11.5 HLA-A1 (SEQIDNo.35) E7/14-23 DLQPETTDLY 1.62 HLA-A1 (SEQIDNo.36) E7/15-23 LQPETTDLY 4.68 HLA-B15 (SEQIDNo.37) E7/12-20 MLDLQPETT 3.6 HLA-A2 (SEQIDNo.38) E7/11-18 YMLDLQPE 8.07 HLA-A2 (SEQIDNo.39) E7/11-19 YMLDLQPET 1.4 HLA-A2 (SEQIDNo.40) E7/11-20 YMLDLQPETT 2.19 HLA-A2 (SEQIDNo.41) E7/11-21 YMLDLQPETTD 2.37 HLA-A2 (SEQIDNo.42)

TABLE-US-00012 TABLE6 Surfacepresentation, Tcellreactivity,andexperimental cytotoxicityofselectedHPV16-derivedpeptides MS- detection Tcell ofsurface reac- Cy- HLApresen- tivity to- Peptide tation inIFN- tox- (protein/ inHPV16+ gamma ic- position) Sequence cellline ELISpot ity E7/15-24 LQPETTDLYC N/A N/A N/A (SEQIDNo.32) E7/15-25 LQPETTDLYCY N/A yes N/A (SEQIDNo.33) E7/12-19 MLDLQPET yes yes N/A (SEQIDNo.34) E7/18-25 ETTDLYCY N/A N/A N/A (SEQIDNo.35) E7/14-23 DLQPETTDLY N/A N/A N/A (SEQIDNo.36) E7/15-23 LQPETTDLY N/A no N/A (SEQIDNo.37) E7/12-20 MLDLQPETT no yes yes (SEQIDNo.38) E7/11-18 YMLDLQPE no yes N/A (SEQIDNo.39) E7/11-19 YMLDLQPET yes yes yes (SEQIDNo.40) E7/11-20 YMLDLQPETT yesat yes N/A (SEQIDNo.41) limitof detection E7/11-21 YMLDLQPETTD yesat yes yes (SEQIDNo.42) limitof detection N/A: analysis not performed