Long peptides of 22-45 amino acid residues that induce and/or enhance antigen specific immune responses
09650423 ยท 2017-05-16
Assignee
Inventors
- Sjoerd Hendrikus Van Der Burg (Waddinxveen, NL)
- Tom H. M. Ottenhoff (Oegstgeest, NL)
- Annemieke Geluk (Woubrugge, NL)
- Maria Johanna Philomena Schoenmaekers-Welters (Leiden, NL)
- Annemieke M. De Jong (Amsterdam, NL)
- Rienk Offringa (Leiden, NL)
- Cornelis Johannes Maria Melief (Haarlem, NL)
- Reinaldus Everardus Maria Toes (Leiden, NL)
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
A61K39/39
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
A61K40/11
HUMAN NECESSITIES
A61K2039/55555
HUMAN NECESSITIES
A61K2039/55561
HUMAN NECESSITIES
C12N2710/20022
CHEMISTRY; METALLURGY
C12N2710/20034
CHEMISTRY; METALLURGY
A61K2039/57
HUMAN NECESSITIES
A61K2039/55572
HUMAN NECESSITIES
International classification
A61K39/39
HUMAN NECESSITIES
Abstract
Epitopes derived from human papilloma virus and peptides having a size of about 22-45 amino acid residues comprising minimal T cell epitopes are disclosed. Also disclosed are clinically relevant approaches for immunizing subjects against (Myco) bacterially and/or virally infected cells or tumor cells. Peptide sequences of 22-35 amino acid residues in length can induce both peptide-specific CD8+ cytolytic cells and CD4+ T-helper cells. Moreover, vaccination with 22-35 residue long peptides results in a more vigorous CD8+ cytolytic T-cell response than vaccination with peptides of the exact minimal CTL epitope length. The intrinsic capacity of certain minimal CTL epitopes which instead of activating cytolytic effector cells tolerize these cytolytic cells, can be overcome by use of these 22-35 amino acid long peptides. Also disclosed are clinically relevant approaches for vaccination and/or treatment of subjects against HPV and methods and uses suited to treat subjects suffering from progressive lesions and/or cervical cancer.
Claims
1. An immunogenic composition comprising a peptide having a length of 22 to 45 amino acids that comprises a T helper cell epitope consisting of residues 127-142 of the E6 protein of a type 16 human papilloma virus (HPV), and an adjuvant.
2. The immunogenic composition of claim 1, wherein the peptide has a length of 22-40 amino acids.
3. The immunogenic composition of claim 1, wherein the peptide has a length of 22-35 amino acids.
4. The immunogenic composition of claim 1, wherein the peptide has a length of 32-35 amino acids.
5. The immunogenic composition of claim 1, wherein the peptide consists of the HPV16 E6 protein segment consisting of residues 109-143, 121-142, 124-158 or 127-158 of HPV 16 E6.
6. The immunogenic composition of claim 1, wherein the peptide consists of the HPV16 E6 protein segment consisting of residues 127-158 of HPV 16 E6.
7. The immunogenic composition according claim 1, further comprising a peptide comprising a fragment of HPV 16 E2 protein comprising residues 46-75, 51-70, 61-76, 311-325, 316-330, 346-355 or 351-365 of HPV16 E2.
8. The immunogenic composition according claim 1, further comprising a peptide comprising a fragment of HPV 16 E7 protein comprising residues 35-77, 35-50, 50-62 or 43-77 of HPV 16 E7.
9. The immunogenic composition according to claim 1, wherein the adjuvant is selected from the group consisting of (a) an exosome, (b) poly L:C, (c) poly l:poly C12U (d) monophosphoryl lipid A, (e) a CpG-containing nucleic acid, (f) a CD40ligand, and (g) a mixture of any of (a)-(f).
10. The immunogenic composition of claim 9, wherein the CD40 ligand is an anti-CD40 antibody.
Description
EXAMPLES
Example 1
(1) Material and Methods
(2) Subjects and Controls.
(3) Samples of umbilical cord blood mononuclear cells (CBC) were used as immunologically naive controls for influenza matrix-specific responses. PBMC of HLA-typed, anonymous healthy blood bank donors (D) obtained after informed consent, served as control PBMC for HPV16 E7 and influenza matrix-specific responses. Since these donors are anonymous, no additional data is available. However, donors with a known recent history of infection, including abnormal pap-smear, were, as part of normal regulations, discouraged to donate blood. The study of subjects (S; Table I) with CIN or cervical carcinoma in this paper was nested in the CIRCLE study that investigates cellular immunity against HPV16 infected cervical lesions. Women presenting with histologically proven CIN III or cervical carcinoma at the department of gynecology of the Leiden University Medical Center (LUMC) were, after informed consent, enrolled in this study. The study design was approved by the ethics committee of the LUMC. Blood was drawn at day of treatment. Subjects with CIN III were treated by LEEP or cold knife conisation. In case of stage IB-IIA a radical hysterectomy was performed. All individuals for whom enough PBMC were available were typed for HLA MHC class II (Naipal et al., 1984). Subjects were typed for HPV16 using HPV16-specific primers on DNA isolated from paraffin-embedded sections of biopsies or surgical resection specimens (Claas et al., 1989). Since HPV-specific T-helper response were expected to be found in subjects with progressive disease (de Gruijl et al., 1998), we chose to analyse three subjects presenting with CIN III, 4 subjects with stage IB cervical cancer and 4 subjects with recurrent cervical cancer.
(4) Antigens
(5) The peptides used spanning the E7 protein consisted of nine overlapping 22-mer peptides and are indicated by their first and last amino acid in the protein (1-22, 11-32, 21-42, 31-52, 41-62, 51-72, 61-82, 71-92 and 77-98) or 4 long peptides defined by amino acids 1-35, 22-56, 43-77 and 64-98. The peptides spanning the influenza matrix 1 protein of A/PR/8/43 that were used as control peptides in the ELISPOT assay consisted of sixteen 30-mer peptides overlapping by 15 amino acids. Peptides were synthesized and dissolved as described previously (van der Burg et al., 1999).
(6) Recombinant HPV16-E7 protein, HPV16-E6 protein and HIV-1 RT protein (the latter two proteins served as control proteins in proliferation assays) were produced in recombinant E. coli transformed with Pet-19b-HPV16-E7, Pet-19b-HPV16-E6 (De Bruijn et al., 1998) or Pet-19b-HIV-1 reverse transcriptase (RT) and purified as described previously (van der Burg et al., 1999).
(7) Memory Response Mix (MRM): A mixture of tetanus toxoid (1 LF/ml; National Institute of Public Health and the Environment, Bilthoven, The Netherlands) and Mycobacterium tuberculosis sonicate (2.5 g/ml; generously donated by Dr. P. Klatser, Royal Tropical Institute, The Netherlands) was used to control the capacity of PBMC to proliferate in response to typical recall antigens.
(8) HLA-DR-peptide Binding Assay.
(9) Binding of peptides was measured as reported previously (van der Burg et al., 1999). Briefly, as a source of HLA-DR molecules B-lymphoblastic cell lines homozygous for HLA-DR were used: LG2.1 (DRB*0101, DR1), IWB (DRB1*0201, DR2), HAR (DRB*0301, DR3), BSM (DRB*0401, DR4) and Pitout (DRB1*0701, DR7). DR molecules were purified by affinity chromatography and the purity confirmed by SDS-PAGE. The analysis of peptide binding to purified HLA-DR molecules was performed using N-terminally fluorescence-labeled standard peptides. As standard fluorescent peptides in the binding assays, either HA 308-319 (PKYVKQNTLKLAT, DR1 and DR2), (SEQ. ID. NO. 4) hsp65 3-13 (KTIAYDEEARR, DR3) (SEQ. ID. NO. 5), HA.sub.308-319 Y.fwdarw.F (PKFVKQNTLKLAT, DR4) (SEQ. ID. NO. 6) or Ii 80-103 (LPKPPKPVSKMRMATPLLMQA LPM, DR7) (SEQ. ID. NO. 7) were used.
(10) Immunogenicity Assay.
(11) HPV16-E7 derived peptide induced proliferation of PBMC isolated from blood obtained from healthy donors was measured as described previously (van der Burg et al., 1999).
(12) Briefly, PBMC were seeded at a density of 1.510.sup.5 cells/well of a 96 well U-bottom plate (Costar, Cambridge, Mass.) in 200, l of ISCOVE's medium (Gibco) enriched with 10% autologous serum, in the presence or absence of 10 g/ml of indicated 22 mer E7 peptide. As positive control, PBMC were cultured in the presence of a Memory Response Mix. Peptide specific proliferation was measured at day 6 by tritiumthymidine incorporation. Peptides were scored as immunogenic, i. e., able to stimulate T-cells, when the proliferation of >25% of the 8 test wells exceeded the mean proliferation+2 times the standard deviation of medium control wells.
(13) Proliferation Assays
(14) Cultures were pulsed with 0.5 Ci [3H] thymidine (5 Ci/mM, Amersham, UK) per well for 18 hours. Plates were harvested with a Micro cell Harvester (Skatron, Norway).
(15) Filters were packed in plastic bags containing 10 ml of scintillation fluid and subsequently counted in a 1205 Betaplate counter (Wallac, Turku, Finland).
(16) MHC class I1 blocking experiments were carried out as described before using murine monoclonal antibodies against HLA-DQ SPV. L3, against HLA-DR B8.11.2 and against HLA-DP B7/21 (van der Burg et al., 1999). Antibodies were added to APC 1 h prior to protein-APC incubation.
(17) Isolation and Expansion of HPV16-E7-Specific T-helper Cells.
(18) Peptide-specific T-cell bulk cultures were generated as described previously (van der Burg et al., 1999) using either the E7 22-mer or E7 35-mer peptides. Specific proliferation was measured by incubation of 50,000 responders with an equal amount of irradiated (30Gy) APC (autologous PBMC unless indicated otherwise) and peptide or protein as indicated. E7-peptide and -protein-specific bulk T-cells were cloned by limiting dilution as described previously (van der Burg et al., 1999).
(19) Cytokine Assays
(20) To determine specific excretion of cytokines, T-cell clones were stimulated by incubation of 50.000 T-cells with an equal amount of APC (30Gy) together with 10 g/ml peptide, control peptide, E7 protein or control protein as indicated. After 24 hours of incubation, supernatant was harvested and replicate wells were pooled. Cytokine production was measured by Enzyme-Linked Immunosorbent Assay (ELISA) as described previously (van der Burg et al., 1999).
(21) Analysis of Antigen-specific T-cells by ELISPOT
(22) PBMC or cord blood cells (CBC) were seeded at a density of 210.sup.6 cells/well of a 24 well plate (Costar, Cambridge, Mass.) in 1 ml of ISCOVE's medium (Gibco) enriched with 10% FCS, in the presence or absence of 5 g/ml of indicated E7-derived 22-mer peptide. As positive control PBMC were cultured in the presence of indicated pools of influenza A/PR/8/34 M1 protein derived peptides consisting of 4 overlapping 30 amino acid long peptides in each pool. Based on our observations, we used a 4-day stimulation before PBMC were transferred to the ELISPOT plates. This resulted in a pronounced IFN-production towards influenza M1-derived peptides in the CD45RO+ (memory) subset of T-cells but not in naive T-cells obtained from adult PBMC (unpublished observations).
(23) Following 4 days of incubation at 37 C., PBMC were harvested, washed and seeded in six replicate wells at a density of 10.sup.5 cells/well of a Multiscreen 96-well plate (Millipore, Etten-Leur, The Netherlands) coated with a IFN catching antibody (Mabtech AB, Nacha, Sweden). The ELISPOT was further performed according to the instructions of the manufacturer (Mabtech). The number of spots were analyzed with a fully automated computer assisted video imaging analysis system (Carl Zeiss Vision).
(24) Specific spots were calculated by subtracting the mean number of spots+2SD of the medium only control from the mean number of spots of experimental wells. Antigenspecific T-cell frequencies were considered to be increased compared to non-responders when T-cell frequencies were >1/10, 000 PBMC.
(25) Results
(26) Identification of the Inmunogenic Sequences within HPV16 E7.
(27) We set out to identify the sequences within the HPV16 E7 protein that function as major immunogenic determinants in the context of MHC class II. A set of HPV16 E7 derived overlapping peptides was tested for binding to HLA-DR 1, 2, 3, 4 and 7 in a quantitative peptide/MHC binding assay (Geluk et al., 1995). Together, these HLA-DR molecules cover at least 50-60% of the caucasian, oriental and negroid populations (Baur et al., 1984). Four peptides, E7.sub.1-22, E7.sub.41-62, E7.sub.51-72 and E7.sub.77-98, bound to three or more different HLA-DR molecules (Table II): peptide E7.sub.1-22 bound to HLA DR2, 3, 4 and 7, peptide E7.sub.41-62 bound to DR1, 2, 3 and 4 whereas peptide E7.sub.51-72 bound to DR1, 3 and 7 and peptide E7.sub.77-98 bound to DR1, 2 and DR7. This is in accordance with the fact that the peptide binding restrictions for MHC class II molecules were found to be less strict than those for MHC class I (Rammensee et al., 1995). Of note, these peptides failed to bind to one or two other DR molecules tested indicating that their binding, although rather ubiquitous in character, was specific.
(28) Our subsequent experiments focused on the four peptides binding to multiple HLA-DR molecules, as these are the most likely to comprise naturally presented T-helper (Th) epitopes (Geluk et al., 1998). We analyzed whether the peptides could stimulate T-cells to proliferate by adding these to PBMC from 13 HLA-typed healthy blood donors. This assay does not discriminate between the reactivity of memory T-cells and naive, in vitro, primed T-cells but can be readily employed for identification of immunogenic peptides (van der Burg et al., 1999). All four peptides were able to stimulate proliferative responses in PBMC from multiple donors (Table II). Most PBMC cultures reacted to the peptides E7.sub.41-62 and E7.sub.51-72 indicating that the central region of the E7 protein harbors the most immunogenic sequences and is likely to be targeted by the immune system in HPV16 infected individuals.
(29) TABLE-US-00001 TABLE I Subject characteristics Patient HPV Stage of number Age type MHC class II type Disease 1 38 16 HLA DR4, 11(5); DQ3 CIN III 2 41 16 HLA DR15(2), 3; DQ6(1), 2 CIN III 3 32 16 HLA DR15(2), 4; DQ3, 6 CIN III 4 67 16 HLA DR15(2), 4; DQ6(1), 8(3) FIGO IB 5 59 16 HLA DR11(5); DQ3 FIGO IB 6 36 16 Non-Available FIGO IB 7 28 16 HLA DR1, 7; DQ5(1), 2 FIGO IB 8 48 16 HLA DR15(2), 7; DQ6(1) Recurrent cervical carcinoma 9 46 16 HLA DR15(2), 7; DQ6(1), 2 Recurrent cervical carcinoma 10 47 16 HLA DR6, 7; DQ1, 2 Recurrent cervical carcinoma 11 46 16 HLA DR3, DQ2 Recurrent cervical carcinoma
(30) TABLE-US-00002 TABLE II Binding capacity and immunogenicity of HPV16-E7 derived peptides Immunogenicity D #1 D #2 D #3 D #4 D #5 E7 Binding DR1,3 DR1,4 DR2,4 DR3,6 DR2,3 aa DR1 DR2 DR3 DR4 DR7 DQ1,2 DQ5,8 DQ6,8 DQ6,2 DQ1,2 1-22.sup.a >70.sup.b 10 0.8 30 7 .sup.c + 11-32 >70 >70 50 >70 >70 21-42 >70 12 >70 >70 >70 31-52 >70 >70 >70 >70 NT 41-62 7.4 50 55 38 >70 + + 51-72 70 >70 50 >70 11 + 61-82 >70 15 65 >70 >70 71-92 12 2 >70 >70 >70 77-98 46 22 >70 >70 0.9 + Memory Response Mix + + + + + Immunogenicity D #6 D #7 D #8 D #9 D #10 D #11 D #12 D #13 E7 DR2,3 DR1,3 DR2,13 DR4,7 DR1,3 DR7 DR3,13 DR7,13 aa DQ6,2 DQ5,2 DQ6 DQ3 DQ5,2 DQ2 DQ6,2 DQ6,3 1-22.sup.a + + 11-32 21-42 31-52 41-62 + + + + + + 51-72 + + + + + + 61-82 71-92 77-98 + + + + Memory + + + + + + + + Response Mix .sup.aFirst and last amino acid number of 22-mer overlapping peptides derived from the HPV16-E7 protein. .sup.bBinding capacity of each peptide is expressed as the IC50 value: this is the test peptide concentration (M) at which 50% of the maximal binding of the standard fluorescence labeled peptide is inhibited. >70, indicates undetectable binding. .sup.cFresh PBMC derived from HLA-DR and -DQ typed healthy blood donors (D) were stimulated with peptides that bound to 3 different HLA-purified DR molecules. Donors were considered to react specifically to a peptide when >25% of all wells tested displayed a proliferation that exceeded the mean + 2 standard deviation of the medium only control. More then 85% of all wells stimulated with the Memory Response Mix were found positive.
(31) TABLE-US-00003 TABLE III Evaluation of HPV16 E7-specific T cell responses by IFN ELISPOT CBC #1 CBC #2 CBC #3 D #20 D #21 D #22 D #23 D #24 D #25 D #26 S #1 HLA- NA NA NA 3.sup.b 15 (2) 1 2 4 1 11 (5) 4 DR 13 (6) 13 (6) 4 8 7 11 (5) HLA- NA NA NA 1, 2 6 (1) 3, 5 1 4, 8 5 NA 3 DQ E7 1-22.sup.a <1 1 <1 <1 <1 <1 <1 <1 11-32 <1 1 <1 15 <1 <1 <1 <1 21-42 <1 <1 2 <1 <1 <1 <1 <1 31-52 <1 <1 <1 <1 <1 <1 1 <1 41-62 <1 2 2 <1 <1 <1 1 <1 51-72 <1 <1 4 <1 <1 <1 1 32 61-82 <1 <1 <1 <1 <1 <1 1 <1 71-92 <1 <1 <1 <1 <1 <1 2 <1 77-98 <1 <1 <1 <1 <1 <1 1 <1 M1 pool 1 <1.sup.c <1 <1 8 9 46 <1 7 5 13 <1 pool 2 <1 <1 <1 12 36 41 <1 28 <1 65 8 pool 3 <1 <1 <1 <1 47 42 <1 <1 9 17 92 pool 4 <1 <1 <1 <1 47 38 27 <1 15 2 <1 S #2 S #3 S #4 S #5 S #6 S #7 S #8 S #9 S #10 S #11 HLA- 15 (2) 15 (2) 15 (2) 11 (5) NA 1 15 (2) 15 (2) 6 3 DR 3 4 4 7 13 (6) 7 7 HLA- 6 (1), 2 6 (1), 3 6 (1), 8 (3) 3 NA 5 (1), 2 6 (1) (1), 2 1, 2 2 DQ E7 1-22.sup.a <1 <1 <1 16 <1 <1 <1 <1 <1 <1 11-32 <1 <1 <1 <1 <1 <1 <1 <1 <1 8 21-42 <1 <1 <1 4 <1 <1 1 <1 <1 2 31-52 8 <1 <1 <1 <1 <1 23 <1 <1 14 41-62 14 <1 <1 <1 <1 <1 19 <1 <1 5 51-72 76 <1 <1 98 <1 <1 <1 <1 <1 <1 61-82 <1 <1 <1 <1 <1 <1 <1 <1 <1 13 71-92 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 77-98 <1 <1 <1 12 <1 <1 6 <1 <1 6 M1 pool 1 <1 27 <1 <1 1 113 <1 <1 <1 15 pool 2 <1 29 47 2 8 66 11 <1 <1 19 pool 3 6 <1 59 100 <1 41 39 <1 <1 20 pool 4 22 15 <1 10 <1 34 17 <1 21 28 .sup.aFirst and last amino acid number of 22-amino acid long peptides derived from the HPV16-E7 protein that were used to stimulate T-cells of umbilical cord blood cells (CBC), PBMC from donors (D) or PBMC from HPV16+ subjects (S). For the influenza M1 protein only the number of the pool is indicated. Each pool consists of four 30 amino acid long peptides that overlap by 15. .sup.bHLA class II typing of each individual. NA, non available .sup.cThe number of antigen-specific cells per 100,000 cord blood cells or PBMC. Frequencies 1/10,000 are depicted in bold. Underscored are the responses that match in specificity to that of Th-clones described in FIGS. 1, 2, 3.
Mapping of Naturally Processed T-helper Epitopes in HPV1 6 E7.
(32) As a next step in the identification of Th-epitopes in HPV16 E7, peptide specific bulk T-cell cultures were generated from PBMC of HLA-typed healthy blood donors by repeated stimulation with several long peptides covering the central region of E7. The responding T-cells were subsequently tested for recognition of antigen presenting cells (APC) pulsed with either peptide or whole E7 protein. In the latter setting, presentation of the relevant peptide-epitopes requires antigen-uptake and -processing.
(33) Approximately 30% of the Th-cell cultures generated specifically responded to the peptides against which they were raised. Of these, three cultures also showed modest but specific activity against E7 protein pulsed APC, indicating the presence of Th-cells with the capacity to respond against naturally processed antigen (
(34)
(35) Furthermore, this Th-clone produced the Th type 1 cytokine IFN (
(36) Memory T-helper Responses in Subjects with HPV16+CIN III Lesions or Cervical Carcinoma.
(37) In an approach parallel to the mapping of Th-epitopes using PBMC cultures of healthy individuals, the response against HPV16 E7 in subjects with HPV16+ lesions was analysed (see Table I for subject characteristics). Primary, in vitro, stimulation of naive T-cells from newborns or adults can result in the production of IL-2 and proliferation of T-cells. However, at this stage such T-cells fail to secrete IFN. The production of IFN follows when the antigen is encountered again (Early and Reen, 1999; Pittet et al., 1999; Sallusto et al., 1999). This feature allowed us to discriminate between in vitro primed and memory T-cell responses by ELISPOT. Th-responses against the influenza A matrix (M1) protein, which are readily detectable in PBMC of most donors of diverse HLA-types (Table III and our unpublished observations), were measured in parallel as a positive control for the detection of memory T-cell immunity in the assay. Stimulation with MI-peptides of MACS-separated CD45RA+ (naive) T-cells and CD45RO+ (memory) T-cells resulted in the production of IFN in the CD45RO+ subset only, confirming that our ELISPOT setup specifically detects memory responses (
(38) Analysis of HPV16 E7-immunity revealed responses against one or more peptides in subjects diagnosed with CIN III and in subjects with cervical carcinoma. In addition, one of the seven donors tested displayed immunity against a peptide. The central region of HPV16 E7 that is covered by the peptides E7.sub.31-52, E7.sub.41-62 and E7.sub.51-72 was targeted by the immune system of all five HPV16+ individuals (Table III), not only confirming that this region is highly immunogenic (Table II) but also demonstrating that this region harbors epitopes that are targeted by natural immune responses against HPV16. Interestingly, the specificity of IFN responses detected in three HPV16+ subjects, in combination with their HLA-type, matched that of the established Th-clones for which we examined specificity and HLA-restriction in detail (
(39) In particular (Table III, underscored frequencies), subject 2 exhibited significantly increased immunity matching the specificity of the two HLA-DR restricted clones as well as weak immunity matching the HLA-DQ2/E7.sub.35-50 clone. Subject 8 displayed a response matching the DR15/E7.sub.50-62 clone, whereas subject 11 showed reactivity matching the HLA-DQ2/E7.sub.35-50 clone. It should be noted that in other subjects expressing the relevant MHC class II molecules such responses could not be detected (subjects 3, 4, 7 and 10).
Example 2
(40) Materials and Methods
(41) Lymphocytes
(42) Peripheral blood mononuclear cells (PBMC) and serum of HLA-typed anonymous healthy blood donors were obtained after informed consent. Because these donors are anonymous, no data on medical history are available. Importantly, donors with a known recent history of infection, including abnormal pap-smear were, as part of normal regulations, discouraged to donate blood.
(43) Antigens
(44) A set of peptides spanning the whole HPV16 E2 protein consisting of 23 overlapping peptides, 22 of which have a length of 30 amino acids and one of which (E2.sub.331-365) has a length of 35 amino acids, was used. These peptides share an overlap of 15 amino acids.
(45) For epitope fine-mapping and the determination of cross-reactivity of HPV16-E2 specific Th-clones, peptides with a length of 15 and 20 amino acids were used. The peptides spanning the Influenza Matrix 1 protein of A/PR/8/34, which were used as control peptides in ELISPOT assays, consisted of 16 30-mer peptides overlapping by 15 amino acids. Peptides were synthesized by solid phase technique on an automated multiple peptide synthesizer (Abimed AMS 422, Langenfeld, Germany), and analysed by reverse phase HPLC. The lyophilized peptides were dissolved in 50 l of DMSO, diluted in PBS to a final concentration of 2.5 mg/ml. The HPV16 E2 C terminal (E2.sub.280-365) protein and HPV16 E7 protein were produced according to previously described procedures (Franken et al., 2000) Memory Response Mix (MRM), consisting of a mixture of tetanus toxoid (0.75 LF/ml final concentration; National Institute of Public Health and Environment, Bilthoven, The Netherlands), Mycobacterium tuberculosis sonicate (2.5 g/ml; generously donated by Dr. P. Klatser, Royal Tropical institute, Amsterdam, The Netherlands) and Candida albicans (0.005%, HAL Allergenen Lab, Haarlem, The Netherlands), was used to confirm the capacity of PBMC to proliferate and produce cytokine in response to common recall antigens.
(46) HLA-DR-peptide Binding Assay
(47) Binding of peptides to HLA-DR was measured as reported previously (van der Burg et al., 1999). Briefly, as a source of DR molecules B-LCL homozygous for DR were used: LG2.1 (DRB*0101, DR1), IWB (DRB1*0201, DR2), HAR (DRB*0301, DR3), and BSM (DRB*0401, DR4). DR molecules were purified by affinity chromatography and the purity confirmed by SDS-PAGE. The analysis of peptide binding to purified DR molecules was performed using N-terminally fluorescence-labelled standard peptides. As standard peptide in the binding assays HA.sub.308-319 (PKYVKQNTLKLAT, DR1 and DR2), (SEQ. ID. NO. 4), hsp65 3-13 (KTIAYDEEARR, DR3) (SEQ. ID. NO. 5) or HA.sub.308-319 Y.fwdarw.F (PKFVKQNTLKLAT) (SEQ. ID. NO. 6) was used.
(48) Short-term T Cell Proliferation Assay
(49) Immunogenicity of individual HPV16 E2 peptides was determined by short term proliferation assays of healthy donor PBMC with HPV16-E2 peptides, according to previously described procedures (van der Burg et al. 1999). Briefly, freshly isolated PBMC were seeded at a density of 1.510.sup.5 cells/well in a 96-well U-bottom plate (Costar, Cambridge, Mass.) in 200 l of IMDM (Iscove's Modified Dulbecco's Medium, Bio Whittaker, Verviers, Belgium) supplemented with 10% autologous serum (10% FCS was used for CBC cultures). HPV16-E2 peptides were added at a concentration of 10 g/ml.
(50) Medium alone was taken along as negative control; phytohemagglutinine (PHA, 0.5 g/ml) served as a positive control. For each peptide 8 parallel micro-cultures were initiated; each donor was tested twice. Peptide-specific proliferation was measured at day 6 by .sup.3H-thymidine incorporation. Peptides were scored positive, whenin both assaysthe proliferation of >50% of the test wells exceeded the mean proliferation+3SD of the control wells, and the stimulation index (SI) of all test wells over medium control wells was higher than 3.
(51) Generation and Analysis of Long-term HPV16-E2 Specific Th-Cultures
(52) Long-term HPV16 E2-specific T-cell cultures and Th-clones were established according to previously described procedures (van der Burg et al. 1999). Briefly, PBMC from healthy HLA-typed donors were stimulated in vitro with the following HPV16 E2 peptides (E2.sub.271-300+E2.sub.286-315, E2.sub.301-330+E2.sub.316-345, E2.sub.331-365). 1510.sup.6 PBMC were seeded in 25 cm.sup.2 culture flasks (Nalge Nunc, USA) in 6 ml IMDM supplemented with 10% autologous serum. Peptides were added at a concentration of 5 g/ml. At day 7.1510.sup.6 PBMC were added, together with fresh medium and peptides. At day 14 and 21 viable T cells were harvested from the cultures, counted and restimulated with an equal amount of autologous irradiated PBMC and peptide (5 g/ml). T-cell growth factor (Biotest, Dreieich, Germany) was added 2 days after restimulation at a final concentration of 10%. The T-cell cultures were tested for peptide recognition by proliferation assay at day 28. Peptide-specific T-cell cultures were cloned by limiting dilution and T-cell clones were subsequently tested for the recognition of E2-peptide and -protein-pulsed APC.
(53) Specificity of the Th clones was analysed as described previously (Van der Burg et al. 1999). Notably, in proliferation assays in which Th-clones were tested for protein recognition, autologous monocytes were used as APC. For measurement of proliferation, cultures were pulsed with 0.5 Ci [.sup.3H] thymidine (5 Ci/mM, Amersham, UK) per well for 18 hours. Plates were harvested with a Micro cell Harvester (Skatron, Norway). Filters were packed in plastic covers containing 10 ml of scintillation fluid and subsequently counted on a 1205 Betaplate counter (Wallac, Turku, Finland). HLA-class II blocking experiments were performed using murine monoclonal antibodies: anti-DQ SPV. L3, anti-DR B8. 11.2, and anti-DP B7/21. Supernatants of the proliferation assays were harvested 24 hrs after incubation and analysed for the presence of IFN- by ELISA (van der Burg et al., 1999).
(54) Detection of Memory Th-cells by ELISPOT
(55) Memory cells (CD45RO.sup.+) were isolated freshly from buffycoats by MACS after incubation with CD45RO microbeads (cat. no. 460-01, Miltenyi Biotec, Germany). The purity of the obtained CD45RO.sup.+ fraction was >95% as determined by floweytometry after surface staining for CD45RO and CD45RA (CD45RA-FITC, cat. no. 347723, CD45RO-PE, cat. no. 347967, Becton Dickinson Biosciences, USA). CD45RO+ cells were seeded at a density of 10.sup.6 cells/well in a 24-wells plate (Costar) in 1 ml of IMDM supplemented with 10% FCS. 10.sup.6 irradiated autologous cells were added to each well as APC. The responder cells were incubated with either medium alone, pools of HPV16-E2 peptides at 5 g/ml/peptide, MRM 1:50 dilution or pools of Influenza Matrix peptides (positive controls) and cultured for 11 days in order to improve the detection of antigenspecific cells (McCutcheon et al. 1997). The cells were then harvested, washed and seeded in 4 replicate wells at a density of 510.sup.4 cells/well of a Multiscreen 96-well plate (Millipore, Etten-Leur, The Netherlands) coated with a IFN- catching antibody.
(56) Per well, 10.sup.5 irradiated autologous PBMC were added as APC together with 5 g/ml peptide. ELISPOT analysis was further performed according to the instructions of the manufacturer (Mabtech AB, Natcha, Sweden). Analysis of the number of spots was done with a fully automated computer-assisted-video-imaging analysis system (Carl Zeiss Vision).
(57) Intracellular Cytokine Staining (ICS) of Memory T-cells
(58) Autologous monocytes were isolated from PBMC by adherence to a flat-bottom 48-wells plate during 2 hours in X-vivo 15 medium (Bio Whittaker, Verviers, Belgium) at 37 C., and then used as APC. CD45RO+ cells were stimulated for 11 days with peptide, then harvested, washed and suspended in IMDM+0.1% BSA at a concentration of 1.510.sup.6 cells/ml. 200 l of cell suspension was added to the monocytes+200 l of 10 g/ml HPV16-E2 peptide (stimulated) or 200 l of medium (non-stimulated control). After 1 hour of incubation at 37 C., 800 l of IMDM+10% FCS+12.5 g/mlg Brefeldin A (Sigma) was added and cells were incubated for another 5 hours. The cells were then harvested, transferred into a V-bottom 96-wells plate, washed twice with ice-cold PBS and fixed with 50 gel paraformaldehyde 4% for 4 minutes on ice. Following fixation, the cells were washed once with cold PBS and once with PBS/NaAz 0.2%/BSA 0.5%/Saponin 0.1%.
(59) This was followed by an incubation in 50 l PBS/NaAz 0.2%/BSA 0.5%/Saponin 0.1%/FCS 10% for 10 minutes on ice. Cells were washed twice with PBS/NaAz 0.2%/BSA 0.5%/Saponin 0.1% and supernatant was removed before 25 l of PBS/NaAz 0.2%/BSA 0.5%/Saponin 0.1% containing 1 l FITC-labelled mouse-anti-human IFN- (0.5 g/ml, BD Pharmingen, cat. no. 554551) 2 l PE-labelled anti-CD4 (BD Bioscience, cat. no. 345769), and 2 l PerCP-labelled anti-CD8 (BD Bioscience cat. no. 347314) was added. Following 30 minutes of incubation at 4 C., the cells were washed, suspended in 100 l paraformaldehyde and analysed by flowcytometry.
(60) Homology Search in Protein Database
(61) The search for sequence homology of the overlapping HPV16 E2 peptides in a protein database (SwissProt) was performed using standard Basic Local Alignment Tool (BLAST: www.ncbi.nlm.gov/blast/blastcgi). Statistical significance threshold (EXPECT) was 10 (Altschul et al., 1997). Reported matches with >60% aminoacid homology with HPV16 E2 peptides were included.
(62) Results
(63) High Reactivity of Healthy Donor PBMC Against HPV16 E2-Derived Peptides.
(64) We examined the proliferative responses of healthy donor PBMC against HPV16 E2 protein by using an array of overlapping 30-mer peptides covering the entire E2 sequence. Incubation of freshly isolated PBMC of 8 HLA-typed donors with each of the 23 E2-derived 30-mer peptides showed that 4 out of 8 donors reacted to 2 or more of the peptides. The observed E2 peptide-specific proliferative responses were remarkably strong (Table IV). In all cases more than 75% of the parallel micro cultures reacted against the stimulating peptide. For instance, in two independent experiments we found the peptide-specific proliferation of donor #8 against peptides E2.sub.31-60, E2.sub.46-75, E2.sub.91-120, E2.sub.151-180, E2.sub.271-300 and E2.sub.286-315 to exceeded background proliferation in 75-94% of all eight parallel micro cultures tested. This points at the presence of a very high frequency of E2-specific T-cells in the PBMC isolates. In particular, PBMC of donors #3, 5 and 8 displayed strong responses with a broad specificity. Please note that the 30-mer peptides have a 15 amino acid overlap with their neighboring peptides. Consequently, responses against adjacent peptides (e. g. donor #3, E2.sub.31-60 and E2.sub.46-75) most likely involve the same epitope, whereas responses against non-adjacent peptides are directed against distinct epitopes.
(65) Not only the frequency of responding cultures but also the magnitude of the proliferative responses were remarkably high. The peptide-specific proliferation of several cultures from donors #3, 5 and 8 exceeded background with mean stimulation indices ranging from 9.2-16.5 and, as such, are comparable to responses found against the tetanus toxoid antigen in several of the donors (#3, 4, 5 and 7; SI ranging from 13.3-25; see Table IV). These stimulation indices clearly exceed the threshold (SI3) that is commonly used for the detection of memory T cell responses (Bermas et al., 1997).
(66) Please note that responses against tetanus toxoid are considerably higher in some of the other donors (SI100 in donors #3 and 6), but that these strong values most likely represent very broad responses against multiple epitopes comprised by an entire antigen, rather than against a single 30-mer E2-peptide. Taken together, our data indicate that the T cell repertoire of healthy donors can contain particularly high frequencies of T-cells specific for the HPV16 E2 antigen resulting in vigorous proliferative responses, and suggest that these responses may reflect T-cell memory.
(67) TABLE-US-00004 TABLE IV Immunogenicity of HPV16 E2 derived peptides. EMI32.1 Table IV Immunogenicity of HPV16 E2 derived peptides. D1.sup.1 D2 D3 D4 D5 D6 D7 D8 E2.sub.1-30.sup.2 E2.sub.16-45 E2.sub.31-60 81% (8, 8).sup.3 88% (8, 6) E2.sub.46-75 88% (5, 5) 81% (6, 8) 94% (6, 5) E2.sub.61-90 E2.sub.76-105 E2.sub.91-120 94% (4, 8) 75% (4, 0) E2.sub.106-135 E2.sub.121-150 E2.sub.136-165 E2.sub.151-180 81% (6, 4) 81% (4, 1) E2.sub.166-195 94% (16, 5) 81% (10, 6) E2.sub.181-210 E2.sub.196-225 E2.sub.211-240 E2.sub.226-255 E2.sub.241-270 E2.sub.256-285 E2.sub.271-300 88% (4, 8) E2.sub.286-315 88% (16.2) 94% (9, 2) E2.sub.301-330 E2.sub.316-345 94% (4, 7) E2.sub.331-365 100% (11, 4) 75% (4, 1) TT 100% (29, 1) 100% (56, 5) 100% (25) 100% (16, 4) 100% (23, 5) 100% (139) 100% (13, 3) 100% (59) .sup.1D: Donor, Eight different healthy blood donors were tested. .sup.2HPV16 E2 peptides are indicated by the first and last amino acid. TT is the common recall antigen tetanus toxoid. .sup.3Peptides were scored positive, when - in both assays - the proliferation of >50% of the test wells exceeded the mean proliferation + 3 * SD of the control wells, and the stimulation index (SI) of the positive test wells over medium control wells was higher than 3. Only positive scores are depicted as the mean percentage of wells exceeding medium control + 3 * SD from the percentages found in both assays. Between brackets the mean stimulation index of all test wells of both assays.
HPV16 E2-Specific Th Cultures Recognize Naturally Processed Epitopes
(68) The proliferation data pointed at the existence of multiple immunogenic Th-epitopes within HPV16 E2. We performed a more detailed analysis of the nature and specificity of such responses, thereby focussing on the N-terminal and C-terminal region which our data (Table IV) revealed to comprise several highly immunogenic peptides. The overlapping peptides comprised in this C-terminal region (E2.sub.271-300; E2.sub.286-315, E2.sub.301-330, E2.sub.316-345, E2.sub.331-365) were tested for their capacity to bind to HLA-DR molecules. Each of the five peptides showed intermediate to strong binding to two or more of the common HLA-DR molecules tested (Table V), which supports the notion that these peptides can indeed represent class II MHC-restricted Th-epitopes.
(69) TABLE-US-00005 TABLEV BindingaffinityofHPV16-E2derivedpeptidesto differentHLA-DRtypes Aminoacidsequence HPV16-E2 DR1 DR2 DR3 DR4 FNSSHKGRINCNSNTTPIVHLKGDANTLKC 271-300 22 65 37 >70 (SEQ.ID.NO.8) TPIVHLKGDANTLKCLRYRFKKHCTLYTAV 286-315 8 68 20 >70 (SEQ.ID.NO.9) LRYRFKKHCTLYTAVSSTWHWTGHNVKHKS 301-330 6 8 >70 10 (SEQ.ID.NO.10) SSTWHWTGHNVKHKSAIVTLTYDSEWQRDQ 316-345 6 55 30 >70 (SEQ.ID.NO.11) AIVTLTYDSEWQRDQFLSQVKIPKTITVSTGFMSI 331-365 8 20 10 15 (SEQ.ID.NO.12) Binding affinity of each peptide is expressed as the IC.sub.50 value: this is the peptide concentration (M) at which binding of the standard fluorescence labeled peptide is reduced to 50% of its maximal value >70 represents undetectable binding.
(70) Subsequently, long-term E2-specific Th cultures were generated through stimulation of PBMC from HLA-typed healthy blood donors at weekly intervals with either peptides E2.sub.271-300 and E2.sub.286-315, peptides E2.sub.301-330 and E23.sub.316-345, or with peptide E2.sub.331-365 or with peptide E2.sub.46-75. PBMC from 2 donors showed strong peptide-specific proliferative responses against one or more of the stimulating peptides (data not shown). Through cloning via limiting dilution of these cultures, we succeeded in the isolation of stable T cell clones, uniformly displaying a CD4.sup.+CD8.sup. phenotype, against six distinct peptideepitopes. Two of these Th clones were established from HLA-DR15 (2)-, -DQ6 (1)-PBMC stimulated with E2.sub.301-330 and E2.sub.316-345. Although both Th-clones recognized E2.sub.301-830, in depth analysis of the specificity of these Th clones revealed that they recognized distinct, yet overlapping, sequences, restricted by different class II HLA molecules. One of the clones recognized peptide E2.sub.316-330 in the context of HLA-DR15 (2), whereas the other clone was specific for peptide E2.sub.311-325 in the context of HLA-DQ6 (1) (
(71) Detection of HPV16 E2 Specific Memory Th-cells in Healthy Individuals
(72) The strikingly frequent detection of HPV16 E2-specific Th immunity in healthy individuals, as described in the first paragraph, prompted us to analyse whether the underlying T cell repertoire would represent immunological memory as a the result of previous encounter with antigen, or whether it would primarily consist of particularly abundant naive T cell precursors specific for this antigen. In view of the high incidence of, generally transient, genital HPV infections in young sexually active individuals (Karlsson et al., 1995; Koutsky et al., 1997; Ho et al., 1998), as well as the prominent expression of E2 during HPV infection, it seems conceivable that T cell memory against E2 is to be found in healthy subjects. We examined the nature of the HPV16 E2-specific immunity detected by us through analysis of the E2-specific reactivity of the CD45RO.sup.+ fraction of healthy donor PBMC, which contains antigen-experienced T-cells but is devoid of their naive counterparts (Young et al., 1997). Because we found the T cell repertoire of healthy donors to contain IFN-producing Th cells (
(73) Interestingly, these CD45RO.sup.+ PBMC were found to respond against multiple E2 peptides (
(74) Cross-reactivity of HPV16 E2 Specific Th-clones with Peptide Sequences of Other HPV Types
(75) Due to the common nature of HPV infections, a majority of the human population is likely to encounter multiple HPV types (Thomas et al. 2000; Koutsky et al. 1997). Furthermore, the protein sequences of the viral gene products are conserved to considerable extend between HPV types. It is therefore possible that at least a fraction of the T cell repertoire induced by a previous encounter with a given type of HPV could cross-react, and therefore cross-protect, during subsequent infection with other HPV types. Alignment of the sequence of the HPV16 E2 protein with that of various other HPV types revealed that it is most prominently conserved with that of other high risk types. Although this conservation is somewhat less conspicuous when the HPV16 E2 sequence is compared to that of low risk or common types it is evident that in all cases maximal conservation is confined to certain regions within the E2 sequence. In particular, three areas of HPV16E2 share homology with E2 of other HPV types: the N-terminal portions E2.sub.31-120 and E2.sub.151-195 as well as the C-terminal portion E2.sub.271-365. These regions co-localise with the major functional domains of E2, in that the N-terminal domain harbours the transcriptional activation functions of this protein whereas the C-terminal portion mediates its sequence-specific DNA-binding properties. The intervening sequences ranging from residues 210 to 270 constitute the so-called hingeregion connecting the two key functional domains, which is poorly conserved between HPV types. Interestingly, our analyses of E2-specific responses in short-term proliferation assays have revealed that the most immunogenic peptides are clustered in the two conserved domains of the HPV16 E2 sequence (see Table IV). In view of these considerations, we tested whether our established Th clones, raised against epitopes derived from the C-terminal part of the HPV16 E2 sequence (
(76) These data indicate that part of the HPV16 E2-reactive Th memory detected in our assays may relate to encounter of HPV types other than HPV16, but also suggest that the majority of this immune repertoire was most likely established through encounter with HPV16 itself.
Example 3
(77) Material and Methods
(78) Subjects and Controls
(79) PBMC of anonymous healthy blood bank donors (D) were obtained. Since these donors are anonymous, no additional data is available. However, donors with a known recent history of infection, including abnormal pap-smear, were, as part of normal regulations, discouraged to donate blood.
(80) The study of subjects (S; Table II) with CIN or cervical carcinoma in this paper was nested in the CIRCLE study that investigates cellular immunity against HPV16 infected cervical lesions. Women presenting with histologically proven CIN III or cervical carcinoma at the department of gynecology of the Leiden University Medical Center (LUMC) were, after informed consent, enrolled in this study. The study design was approved by the ethics committee of the LUMC. Blood was drawn at day of treatment. Subjects with CIN III were treated by LEEP or cold knife conization. In case of stage IB-IIA a radical hysterectomy was performed. Subjects were typed for HPV16 using HPV16-specific primers on DNA isolated from paraffin-embedded sections of biopsies or surgical resection specimens (Claas et al., 1989). Since HPV-specific Thelper response were expected to be found in subjects with progressive disease (de Gruijl et al., 1998), we chose to analyse subjects with stage IB cervical cancer.
(81) Antigens.
(82) The peptides used spanning the E6 protein consisted of 15 overlapping 22-mer peptides and are indicated by their first and last amino acid in the protein (e. g. 1 22, 11-32, 2142, 31-52, 41-62, 51-72, 61-82, 71-92 and so forth, the last peptide consists of amino acid 137-158). Peptides were synthesized and dissolved as described previously (van der Burg et al., 1999).
(83) Memory Response Mix (MRM): A mixture of tetanus toxoid (1 LF/ml; National Institute of Public Health and the Environment, Bilthoven, The Netherlands) and Mycobacterium tuberculosis sonicate (2.5 g/ml; generously donated by Dr. P. Klatser, Royal Tropical Institute, The Netherlands) was used to control the capacity of PBMC to proliferate in response to typical recall antigens.
(84) Analysis of Antigen-specific T-cells by ELISPOT.
(85) PBMC were seeded at a density of 210.sup.6 cells/well of a 24 well plate (Costar, Cambridge, Mass.) in 1 ml of ISCOVE's medium (Gibco) enriched with 10% FCS, in the presence or absence of 5) g/ml of indicated E6-derived 22-mer peptide. As positive control PBMC were cultured in the presence of indicated pools of influenza A/PR/8/34 MI protein derived peptides consisting of 4 overlapping 30 amino acid long peptides in each pool.
(86) In case CD45RO+ cells were used the CD45RO+ cells were seeded at a density of 10.sup.6 cells/well in a 24-wells plate (Costar) in 1 ml of IMDM supplemented with 10% FCS together with indicated single or pools of peptides at a concentration of 10 g/ml/peptide.
(87) Following 4 days of incubation at 37 C., PBMC were harvested, washed and seeded in four to six replicate wells at a density of 10.sup.5 cells/well of a Multiscreen 96-well plate (Millipore, Etten-Leur, The Netherlands) coated with a IFN catching antibody (Mabtech AB, Nacha, Sweden). The ELISPOT was further performed according to the instructions of the manufacturer (Mabtech). The number of spots were analysed with a fully automated computer assisted video imaging analysis system (Carl Zeiss Vision).
(88) Specific spots were calculated by subtracting the mean number of spots+2SD of the medium only control from the mean number of spots of experimental wells. Antigenspecific T-cell frequencies were considered to be increased compared to non-responders when T-cell frequencies were=1/10,000 PBMC.
(89) Isolation of the CD45RO+ Memory Fraction of PBMC
(90) Memory cells (CD45RO.sup.+) were isolated freshly from buffycoats by MACS after incubation with CD45RO microbeads (cat. no. 460-01, Miltenyi Biotec, Germany) according to the instructions of the manufacturer. The purity of the obtained CD45RO+ fraction was >95% as determined by floweytometry after surface staining for CD45RO and CD45RA (CD45RA-FITC, cat. no. 347723, CD45RO-PE, cat. no. 347967, Becton Dickinson Biosciences, USA).
(91) Results
(92) In view of the high incidence of, generally transient, genital HPV infections in young sexually active individuals (Karlsson et al., 1995; Koutsky et al., 1997; Ho et al., 1998), as well as the prominent expression of HPV16 E2-specific immunity (see example 2) in about half of the healthy subjects tested, we studied the natural, in vivo induced, HPV16 E6-specific response in the human population by means of the 4-day IFN-ELISPOT assay. This assay detects only memory T helper-cells, which upon stimulation will secrete IFN whereas their naive counterparts will not (see example 1 and 2 and references therein). A survey of E6-specific reactivity against the full array of peptides present in PBMC from 18 healthy blood donors, revealed that 11 of these 18 (>60%) PBMC isolates responded against one or more of the HPV16 E6 peptides (Table VIa).
(93) The majority of the responses were found at the C-terminal end of E6 (E6.sub.81-158).
(94) Furthermore, all healthy donors reacted against the common recall antigen influenza matrix 1 protein. The frequency of both influenza MI and HPV16 E6-specific T-helper cells were of comparable magnitude. Interestingly, a similar survey in HPV16+ patients revealed that only 3/12 (25%) reacted against HPV16 E6 peptides indicating that the E6 response found in healthy subjects is protective against disease.
(95) TABLE-US-00006 TABLE VI Detection of HPV16 E6-specific T-helper reactivity in healthy donor PBMC. Donor.sup.1 1-22.sup.2 11-32 21-42 31-52 41-62 51-72 61-82 71-92 81-102 91-112 101-122 1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 2 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 3 <1 4 <1 3 <1 <1 <1 <1 <1 <1 <1 4 <1 <1 <1 10 <1 <1 <1 <1 <1 <1 <1 5 <1 5 1 1 2 9 5 7 6 10 2 6 3 34 <1 9 <1 <1 2 <1 11 14 6 7 <1 2 1 <1 <1 <1 <1 <1 2 14 <1 8 4 4 <1 <1 9 <1 <1 <1 12 14 3 9 2 <1 7 3 <1 <1 4 <1 <1 3 <1 10 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 11 2 <1 <1 2 <1 <1 <1 7 3 4 2 12 <1 <1 <1 <1 <1 <1 <1 <1 1 <1 <1 13 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 14 5 5 <1 10 5 <1 2 20 12 5 28 15 18 16 4 15 2 1 <1 3 12 21 8 16 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 17 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 18 <1 <1 <1 <1 4 <1 <1 <1 2 <1 <1 Donor.sup.1 111-132 121-142 131-152 137-158 M1P1 M1P2 M1P3 M1P4 1 <1 <1 <1 <1 <1 16 <1 <1 2 <1 <1 <1 4 2 26 12 7 3 3 8 18 4 30 7 19 <1 4 <1 <1 <1 <1 22 15 14 10 5 8 5 8 1 7 10 12 37 6 12 10 <1 6 <1 33 41 5 7 <1 1 3 14 6 21 57 28 8 6 7 3 <1 27 38 34 16 9 <1 3 <1 <1 22 10 14 4 10 <1 7 <1 <1 13 23 19 11 11 15 10 27 17 33 28 24 52 12 <1 14 15 <1 5 10 12 <1 13 1 <1 <1 <1 <1 <1 1 21 14 18 37 40 2 12 42 28 45 15 11 8 <1 <1 52 68 64 5 16 <1 <1 <1 <1 39 66 49 48 17 <1 <1 <1 <1 4 3 <1 66 18 11 1 1 <1 <1 1 10 <1 .sup.118 different healthy blood donors were tested. .sup.2First and last amino acid number of HPV16 E6-derived peptides are indicated. M1P1-M1P4 are the four different pools of 4, by 15 residues overlapping, 30-amino acid long peptides derived from influenza A matrix 1 protein (inf A/PR/8/34). .sup.3Indicated are the number of specific spots per 100,000 PBMC. Specific spots are calculated by subtracting the mean number of spots + 2 SD of the medium control from the mean number of spots after stimulation with peptide. Numbers in bold indicate T-cell frequencies 1/10,000
(96) TABLE-US-00007 TABLE VIb Detection of HPV16 E6-specific T-helper reactivity in HPV16+ patients. patient 1-22 11-32 21-42 31-52 41-62 51-72 61-82 71-92 81-102 91-112 101-122 C40 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 C41 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 2 C43 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 C47 <1 <1 <1 28 <1 <1 <1 <1 <1 <1 <1 C57 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 2 C58 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 C63 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 C66 <1 <1 <1 <1 <1 11 <1 <1 <1 <1 <1 C73 <1 <1 <1 <1 2 1 <1 3 4 5 <1 C75 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 C80 <1 <1 <1 <1 <1 <1 1 1 2 <1 1 C91 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 patient 111-132 121-142 131-152 137-158 M1P1 M1P2 M1P3 M1P4 C40 1 <1 <1 <1 <1 <1 <1 2 C41 <1 <1 <1 <1 <1 <1 10 <1 C43 <1 <1 <1 <1 <1 <1 4 <1 C47 <1 <1 13 <1 <1 <1 <1 <1 C57 <1 <1 22 <1 <1 2 62 30 C58 <1 <1 <1 <1 <1 <1 <1 <1 C63 <1 <1 <1 <1 <1 1 <1 <1 C66 <1 <1 <1 <1 31 <1 13 <1 C73 8 7 7 9 5 2 4 2 C75 <1 <1 <1 <1 <1 <1 15 <1 C80 1 <1 2 <1 7 2 7 1 C91 <1 <1 <1 <1 3 2 <1 <1
(97) To confirm our observation that HPV16-specific memory T-cells were present in a large fraction of the human population we isolated the specific CD45RO+, memory fraction of PBMC before they were subjected to stimulation with HPV16 E6 peptides. After a 4 days of stimulation a clear response to HPV16 E6 could be detected. Taken together, our data reveal the presence of CD45RO.sup.+ memory-type, IFN secreting T helper cells reactive against both HPV16 E6 peptides in approximately half of the healthy donors tested.
(98) Furthermore, PBMC of two different HLA-DR1 positive blood donors were stimulated with a pool of two long 35-mer peptides that together covered E6 109-158. PBMC of these donors proliferated specifically against peptide E6 121-142 and peptide E6 127158 as well as E6 protein. A CD4+ T-helper clone derived from one of these bulks was analysed further and specifically proliferated and produced IFN when stimulated with HPV16 E6 peptide and HPV16 E6 protein in a HLA-DR1 restricted manner (
(99) Conclusions
(100) Through analysis of the IFN-responses in PBMC cultures from healthy subjects against the HPV16 E6 antigen we have demonstrated that this protein contains highly immunogenic peptide sequences to which strong T-cell reactivity is detected in approximately half of these donors. Subsequent testing of the CD45RO.sup.+ memory fraction of healthy donor PBMC revealed that these HPV16 E6-specific IFN-secreting CD4.sup.+ T cells were present in the T-cell memory fraction of PBMC and thus have been primed in vivo upon encounter with HPV. A similar survey demonstrated that HPV16 E6-specific IFN secreting T-cells were almost absent in HPV16+ patients. Taken together, our data demonstrate that the T-cell repertoire of a majority of the healthy subjects contains particularly high frequencies of memory type 1 cytokine producing T helper cells reactive against the HPV16 E6 antigen and indicates that these are protective against disease.
(101) Importantly, we have not found any previous report concerning the presence of memory T-cell responses against E2 and E6 or any of the other non-structural HPV16 proteins in healthy individuals. In fact, healthy individuals served in many cases as negative control for the culture conditions used. We have now shown, in two independent studies (example 2 and example 3), that healthy donor derived PBMC contain HPV16-specific T-helper cells. The majority of the T-helper responses are directed against specific regions in E2 and E6. The presence of these memory type T-helper cells show that these individuals have encountered HPV16 infection, responded to it via the cellular T-cell arm of the immune system and cleared the infection. This implies that the T-cell response to these antigens is a part of the protective response to HPV infection.
(102) Induction or boosting of these type of responses can therefore be a powerful tool in the protection against HPV induced diseases.
Example 4
(103) Material & Methods
(104) Male C57/B16 mice (n=8 per group) were vaccinated with either 10 g of the exact E1A-derived CTL epitope (SGPSNTPPEI) (SEQ. ID. NO. 1) or with 30 g of the HPLC-purified 32-amino acid long peptide RECNSSTDSCDSGPSNTPPEIHPVVRLCPIKP (SEQ. NO. ID. 2) containing this CTL epitope. Peptides were dissolved in PBS and mixed in a 1:1 ratio with IFA. Mice were vaccinated subcutaneously with peptide in a volume of 200 l at day 1. Fourteen days later mice were challenged with 0.510.sup.6 AR5 tumor cells (E1A+Ras transformed mouse embryo cells). Survival of mice was monitored during a 100 day follow-up.
(105) Results
(106) Vaccination with synthetic peptides representing cytotoxic T lymphocyte (CTL) epitopes can lead to a protective CTL-mediated immunity against tumors or viruses. B6 tumor cells transformed by the human adenovirus early region 1 (Ad5E1) present an Ad5E1A- and an Ad5E1B-encoded CTL epitope to the immune system. CTL clones directed against either of these epitopes are able to eradicate established Ad5E1-induced tumors, showing that these CTL epitopes are targets of CTL that can mediate tumor regression. Protective immunity against Ad5E1-expressing tumor cells can be established by immunization with Ad5E1-transformed cells and with an adenovirus vector containing the Ad5E1 region. Protective immunity, in either case, is associated with specific CTL memory. When, however, mice were vaccinated with either the minimal peptide-epitope sequence of the E1A-derived CTL epitope or the minimal peptide-epitope sequence of the E1B-derived CTL epitope protection against Ad5A1-expressing tumors was lost.
(107) Vaccination with a CTL epitope derived from the human adenovirus type 5 ER-region (Ad5E1A234-243), enhances rather than inhibits the growth of Ad5E1A-expressing tumors. In contrast to peptide vaccination, immunization with adenovirus, expressing Ad5E1A, induced Ad5E1A-specific immunity and prevented the outgrowth of Ad5E1A expressing tumors. These results show that immunization with synthetic peptides can lead to the elimination of anti-tumor CTL responses (Toes et al., 1996a). Furthermore, vaccinated mice s. c. with a low dose of the Ad5E1B peptide also showed this adverse reaction. The Ad5E1B peptide was chosen because the CTL response against the Ad5E1B-encoded CTL epitope contributes most to the antitumor response in B6 mice after vaccination with Ad5E1-transformed cells. Ad5E1B peptide-vaccinated mice were not protected against the outgrowth of Ad5E1-expressing tumor cells, but instead were no longer able to reject a tumor inoculum that was rejected by nonvaccinated mice.
(108) Moreover, the protection induced by tumor cell vaccination against Ad5E1B-expressing tumors was gone when the Ad5E1B-encoded CTL epitope was injected a few days before tumor challenge. This is associated with peptide-induced tolerance of Ad5E1B-specific CTL activity (Toes et al. 1996b).
(109) In conclusion, immunization with synthetic peptides of the exact CTL epitope length, can also lead to CTL tolerance associated with the inability to reject tumors. The issue of tolerance or functional deletion of CTL by immunization with minimal epitopes is circumvented by the use of long peptide (22-35 amino acid residues). To proof this statement we vaccinated C57/B16 mice with a 32 amino long E1A derived peptide that contains the 10 amino acid long CTL E1A-derived epitope, which when given as the exact peptide-epitope is involved in loss of tumor-protection. As shown in
(110) Conclusion
(111) For a clinically relevant approach of immunizing subjects against virally infected cells or tumor cells both specific T-helper cells and CTL should be induced. We have already shown that immunization with minimal CTL epitopes results in protection against tumors in some models (Kast et al. 1991, Feltkamp et al. 1993) whereas it can also lead to tolerance or functional deletion of virus- and tumor-specific CTL that when otherwise induced are protective (Toes et al. 1996ab). Processing of exogenous antigens for presentation by MHC class I molecules by cross-priming as well as by other mechanisms is now widely recognized second pathway of processing for presentation by MHC class I, next to the well known endogenous route (Jondal et al., 1996, Reimann et al. 1997). We have now shown that in contrast to vaccination of mice with the exact peptide-epitope, vaccination with long peptide sequences containing CTL epitopes does not result in the loss of CTL that are involved in the protection of mice against tumors but instead result in a CTL mediated immune response that protects mice against a subsequent tumor-challenge.
Example 5
(112) Mice and cell lines. C57BL/6 (B6, H-2.sup.b) mice were obtained from IFFA Credo (Paris, France). MHC class II.sup./ B6 mice were purchased from Taconic (USA) and CD40.sup./ B6 mice were obtained from The Jackson Laboratory (Maine, USA). Tumor cell line TC-1 was generated by transfection of Mouse Embryo Cells (MEC) of C57BL/6 origin by HPV 16 E6/E7 and c-H-ras oncogenes. Tumor cell line 13.2 was derived from MEC (B6) transformed with adenovirus type 5 derived E1 protein in which the H-2Db E1A epitope was replaced with the HPV 16 E7.sup.49-57 CTL epitope. D1 cells are long-term growth factor dependent immature splenic dendritic cells (DC) derived from C57BL/6 mice.
(113) Peptides. The HPV16-E7-derived peptides E7.sub.49-57: RAHYNIVTF (SEQ. ID. NO. 3) and E7.sub.43-77: GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR (SEQ. ID. NO. 13) were synthesized by solid phase strategies on an automated multiple peptide synthesizer (Abimed AMS 422, Langenfeld, Germany). The peptides were analysed by reverse phase HPLC for contaminants and stored at 20 C.
(114) Tetramers and antibodies. PE-labelled H-2D.sup.b epitope E7.sup.49-57 (RAHYNIVTF) (SEQ. ID. NO. 3)containing tetramers were constructed and used for the analysis of peptide-specific CTL-immunity. FITC labelled anti-CD8b.2 Ab (Ly-3.2) (clone 53-5.8), APC labeled anti-CD4 Ab (L3T4) (clone RM4-5) and PE labeled anti-IFN- Ab (clone XMG1.2) (BD PharMingen, San Diego, USA) were used in the various FACS procedures.
(115) Adjuvantia. IFA (incomplete Freund's adjuvant) was obtained from Difco Laboratories (Michigan, USA). Montanide ISA 51 was purchased from SEPPIC (Paris, France). CpGoligodeoxynucleotides (ODN) 1826 were kindly provided by Dr. G. B. Lipford, Technical University of Munich (Munich, Germany). GM-CSF was obtained from PeproTech (Rocky Hill, USA). The FGK-45 hybridoma cells producing stimulatory anti-CD40 Ab were provided by A. Rolink. MPL was kindly provided by Dr. M. Johnson, Ribi Immunochem. Research (Hamilton, USA).
(116) Immunization strategies. C57BL/6 mice were injected subcutaneously with either 50 g E7.sup.49-57 peptide or 150 g E7.sup.43-77 35-mer dissolved in PBS in order to achieve similar molar levels of the E7.sup.49-57 CTL epitope in both cases. Combinations with various adjuvantia were tested. In the case of IFA and Montanide, the dissolved peptides were emulsified in 50% of these respective substances. ODN-CpG (50 g/mouse), MPL (10 g/mouse) and GM-CSF (4 g/mouse) were all dissolved in PBS and mixed with the peptides before subcutaneous vaccination. The total injected volume was 200 l/mouse.
(117) Anti-CD40 Ab was dissolved in PBS and injected separately from the peptides intravenously on day 0, 1 and 2 at an amount of 100 g per injection (total volume 200, l/mouse). Either spleens were harvested after 10 days or, when indicated, mice were boosted with identical vaccines 50 days after priming and spleens were harvested 10 days after booster immunization. In the case of the 35-mer E7.sup.43-77, the latter strategy might allow the formation of memory CTL- and T helper cells after the first vaccination and activation of DC by HPV16 E7.sup.43-77 specific T helper cells when the booster immunization was given. In the therapeutic anti-tumor experiments, tumor-bearing mice were vaccinated twice: at the time that tumors were palpable in all mice and 14 days later.
(118) T cell cultures. T cells were obtained from immunized mice by culturing spleen cells (510.sup.6 cells/well of a 24-wells plate) in complete medium in the presence of 0.510.sup.6 E7.sup.49-57-expressing cells (tumor cell line 13.2) or, when indicated, in the presence of D1 cells.
(119) Before use, the D1 cells were incubated for 16 hours with the E7.sup.43-77 35-mer and subsequently activated by adding LPS (10 g/ml) for 6 hours and then thoroughly washed. Complete medium consists of Iscove's Modified Dulbecco's Medium (IMDM; BioWhittaker, Walkersville, Md., USA) supplemented with 8% FCS, 100 IU/ml penicillin, 2 mM glutamine (ICN, Aurora, Ohio, USA) and 30 M 2-ME (Merck, Darmstadt, Germany). Cultures were maintained at 37 C. in humidified air containing 5% CO.sub.2. No exogenous IL-2 was added. On day six, dead cells were removed from the culture by centrifugation over a Ficoll density gradient and remaining cells were seeded in 24-wells plates at 110.sup.6 cells/well. On day seven tetramer staining or intracellular cytokine staining was performed.
(120) Tetramer staining. Spleen cultures, stimulated with tumor cell line 13.2 for seven days, were transferred at an amount of 4010.sup.4 per well to 96-well V-bottom microtiter plates and washed twice with PBS/BSA 0.5%. Subsequently, PE-labelled E749-57-containing tetramer was added. After 30 minutes of incubation at room temperature, cells were washed twice with PBS/BSA 0.5% and incubated with FITC-labelled anti CD8b Ab for 30 minutes. Subsequently cells were washed twice in PBS/BSA 0.5%, suspended in PBS/BSA 0.5% containing Propidium Iodide (PI) (0.5) g/ml) and transferred to tubes. Cell samples were analysed in a FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif., USA) using CellQuest software. A total of 2010.sup.4 events was analysed for fluorescence intensity. Debris was gated out using the PI staining and subsequently the CD8.sup.+ fraction was gated. Mean background tetramerstaining of similarly cultured and stained cells from non-immunized control mice was found to be below 1 percent of CD8+ cells (0.94%, standard deviation 0.36%). As a positive control the HPV16 E7 specific CTL clone 9.5 was used.
(121) Intracellular cytokine staining. Spleen cultures were stimulated with D1 cells pulsed with E7.sup.43-77 35-mer for seven days. Subsequently, the percentage of CD8.sup.+ and CD4.sup.+ IFN- producing T cells was measured as described before (van der Burg et al., 2001). Notably, LPS-activated non-pulsed D1 cells or D1 cells pulsed with E7.sup.49-57 or E7.sup.43-77 35-mer (5 g/ml) were used as stimulator cells.
(122) Results
(123) Prime-Boost Vaccinations with the E7.sup.43-77 35 Amino Acid Long HPV16 E7 T Helper- and CTL Epitope Containing Peptide Result in a Vigorous CTL Response.
(124) Mice (B6) were vaccinated once with either the minimal CTL epitope HPV16 E7.sup.49-57 or the HPV16 E7.sup.43-77 35 amino acid residue long peptide, admixed with IFA. Ten days following vaccination spleens were harvested and stimulated in vitro for 1 week. Subsequently, the percentage of E7.sup.49-57 peptide-specific CTL was determined by H2-D.sup.b E7.sup.49-57 (RAHYNIVTF) (SEQ. ID. NO. 3)tetramer staining (van der Burg et al., 2001). In both groups 3 out of 9 mice responded to the vaccine while generally 5 percent of the CD8.sup.+ T cells stained with tetramers (
(125) To obtain a vigorous E7.sup.49-57-specific CTL response, both vaccines were used in a primeboost regimen, which allowed primed T cells to form memory T cells before the response was boosted by a second vaccination 50 days later. Mice that received twice the minimal CTL epitope, showed CD8.sup.+ T cell responses that were comparable to that after one vaccination (
(126) The Vigorous E7.sup.49-57-Specific CTL Response is Dependent on MHC Class II-Restricted T Helper Cells and CD40-CD40L Interactions.
(127) In order to demonstrate that the impressive CTL responses, detected following primeboost vaccinations with the long E7.sup.43-77 peptide, were in fact enhanced by MHC class II-restricted E7-specific T helper cells, MHC class II.sup./ mice were prime-boosted with the long peptide vaccine. The number of E7.sup.49-57-specific CTL detected in the MHC class II.sup./ mice was far lower than found in B6 mice after two vaccinations, and comparable to that found after one is vaccination (
(128) Subsequently the percentage of E7.sup.43-77-specific IFN?-producing CD4.sup.+ T cells was measured by intracellular cytokine staining Whereas no responses over background were observed in naive mice (data not shown), 5 percent or more of the CD4.sup.+ T cells from vaccinated mice specifically responded upon stimulation with the long E7.sup.43-77 peptide (
(129) Professional APC, that process and present the long peptide upon the booster immunization, are activated through CD40-CD40L interactions by E7.sup.43-77 peptide-specific T helper cells. This will subsequently lead to enhanced CTL activation.
(130) Single Vaccinations with the E7.sup.43-77 Peptide and DC-Activating Agents Result in Vigorous T Helper- and CTL Responses Towards HPV16 E7.
(131) If the T helper-mediated activation of professional APC is important for the observed vigorous CTL response, direct activation of DC should bypass a lack of T cell help and a single vaccination should be sufficient to raise adequate CTL responses. To address this subject, B6 mice were vaccinated once with the long peptide or the minimal E7.sup.49-57 peptide in combination with various DC-activating agents. IFA and Montanidea human grade IFAwere used as controls. As with IFA (
(132) Furthermore, mice vaccinated with either the minimal CTL epitope or the long peptide combined with anti-CD40 antibody or GM-CSF displayed no or minimal CTL responses (
(133) Mice receiving the minimal peptide mixed with MPL or ODN-CpG showed clear-cut CTL responses in several mice (
(134) Vaccination with the 35-Mer and the DC-Activating Adjuvant ODN-CpG can Effectively Eradicate HPV16 Expressing Tumors.
(135) In order to test the efficacy of the long peptide vaccine, tumor bearing mice were immunized with the E7.sup.49-57 peptide or the E7.sup.43-77 long peptide admixed with ODN-CpG.
(136) Notably, the vaccination was given at the time when tumors were palpable (day 10-14) in all mice. A second vaccination was administered 14 days later in order to sustain E7 specific T helper- and CTL immunity. Mice treated with ODN-CpG alone did not display anti-tumor activity (
(137) Although complete eradication was observed in 3 out of 9 mice treated with the E7.sup.49-57 peptide and ODN-CpG (30% of the animals), others were only capable of stabilizing tumor growth temporarily and then died of their tumor. In contrast, 8 out of 10 mice treated with the long peptide and ODN-CpG eradicated their tumors, some of them sizing up from 200-500 mm.sup.3.
(138) Conclusion
(139) We show that the HPV16 E7-specific CTL response is far more vigorous after vaccination with a HPV16 E7 derived 35-residue long peptide than following vaccination with the minimal CTL epitope. Our data demonstrate that at least one of two independent mechanisms account for this. Firstly, the long 35-residue peptide used in this example contains both a CTL epitope and a T helper epitope. Vaccination of MHC class II/mice demonstrated that the interaction between APC and E7-specific T-helper cells contributed considerably to the level of the CTL response. Furthermore, administration of said long peptide mixed with strong DC-activating agents was able to bypass the requirement for T cell help. Secondly, a direct comparison of the CTL response induced by the minimal CTL epitope (9 residues) of this example or said long peptide vaccine combined with DC activating agents in both wild type and MHC class II/mice showed that vaccination with the long peptide resulted in a far better CTL response, showing that in contrast to the minimal CTL epitope, the long peptide is preferably presented by professional APC. Moreover, we show that vaccination with the long peptide and strong DC activating agents result in the complete eradication of established tumors. These data provide a scientific basis for the use of long overlapping peptides, alone or in combination with strong DC activating agents in future human trials.
(140) The high efficacy of our long peptide-vaccine is among other things due to the fact that the T helper- and CTL epitope are physically linked to each other. The potential advantage of epitope-linkage lies in the increased chance for simultaneous presentation of both the MHC class I and class II restricted epitopes on the surface of a single APC, thereby facilitating the delivery of cognate T cell help to CTL priming. A direct comparison of vaccines that used a mix of CTL and Th-epitopes with vaccines that used physically linked, but identical, CTL and Th-epitopes demonstrated that the latter resulted in more vigorous CTL responses (Shirai, 1994; Hiranuma, 1999; Bristol, 2000).
(141) Our vaccinations of normal B6 and MHC class II/mice confirm that the use of a physically linked T-helper epitope is advantageous for the development of a strong CTL response. In addition, these experiments demonstrated that the interaction between E7 specific Th-cells and APC is responsible for this boost of the CTL response.
(142) Comparison of the CTL response induced by the minimal CTL epitope and the long peptide in normal and MHC class II/mice revealed another interesting property of our long peptide vaccine. In these experiments the contribution of E7-specific Th-cells was eliminated and only differences in physical properties or kinetics of the peptides are likely to play a role. Because of its size, which excluded direct binding of peptide to MHC class I, said long peptide needed to be taken up by professional APC that are able to process exogenously derived antigens and present peptides in MHC class I. We demonstrated that DC activation only marginally affected the outcome of the E7 specific CTL response when the minimal peptide was used whereas DC activation was a prerequisite for the induction of a superior E7-specific CTL response by the long peptide vaccine. Together these data show that the CTL and Th-epitope present in said long peptide are preferably presented at the surface of professional APC.
(143) Vaccination with the minimal HPV16-derived CTL epitope results in a detectable CTL response that is not improved when DC activating agents are co-injected. This indicates that said peptide is not only presented at the surface of activated professional APC but also in MHC class I molecules of other nucleated cells. This is not surprising because said peptide can directly bind to MHC class I at the cell surface (Feltkamp et al., 1993).
(144) Presentation of said CTL peptide by cells that cannot deliver co-stimulation (e. g. nonimmune cells) results in a dampening of CTL responses and the presentation of said E7 CTL epitope by these non-professional APC are, therefore, deleterious to the induction of E7-specific CTL. Notably, we have shown previously that vaccination with the minimal E1A-derived CTL epitope resulted in tolerance of E1A-specific CTL that, otherwise, were protective against E1A induced tumors (Toes 1996). Although, the possible presentation of the HPV16 E7-derived CTL epitope at the surface of nonprofessional APC does not hinder the induction of E7-specific CTL as seen in the adenovirus tumor model, the anti-tumor efficacy of these responses is hampered.
(145) Treatment of established tumors by therapeutic vaccination with the minimal CTL epitope and the DC activating agent CpG is far less effective when compared to the long peptide vaccine mixed with DC activating agents.
(146) These results provide a scientific basis for the evaluation of this vaccine in human trials for both prophylactic and therapeutic intervention against HPV16 induced disease.
BRIEF DESCRIPTION OF THE FIGURES
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