Microbiota Sequence Variants Of Tumor-Related Antigenic Epitopes
20200256877 ยท 2020-08-13
Inventors
- Laurent CHENE (Neuville aux Bois, FR)
- Francesco STROZZI (Paris, FR)
- Christophe BONNY (Paris, FR)
- Alessandra Cervino (Bois-le-Roi, FR)
- Celia Mendez (Charlestown, MA, US)
Cpc classification
G01N33/57484
PHYSICS
G16B45/00
PHYSICS
A61K39/39
HUMAN NECESSITIES
G16B20/20
PHYSICS
G16B50/00
PHYSICS
International classification
A61K39/39
HUMAN NECESSITIES
G16B45/00
PHYSICS
A61K39/00
HUMAN NECESSITIES
Abstract
The present invention relates to cancer immunotherapy, in particular to sequence variants of tumor-related antigenic epitope sequences. Namely, the present invention provides a method for identification of microbiota sequence variants of tumor-related antigenic epitope sequences. Such microbiota sequence variants are useful for the preparation of anticancer medicaments, since they differ from self-antigens and, thus, they may elicit a strong immune response. Accordingly, medicaments comprising microbiota sequence variants, methods of preparing such medicaments and uses of such medicaments are provided.
Claims
1. Method for identification of a microbiota sequence variant of a tumor-related antigenic epitope sequence, the method comprising the following steps: (i) selection of a tumor-related antigen of interest, (ii) identification of at least one epitope comprised in the tumor-related antigen selected in step (i) and determination of its sequence, and (iii) identification of at least one microbiota sequence variant of the epitope sequence identified in step (ii).
2. The method according to claim 1, wherein step (iii) comprises comparing the epitope sequence selected in step (ii) to one or more microbiota sequence(s), and identifying whether the one or more microbiota sequence(s) contain one or more microbiota sequence variant(s) of the epitope sequence.
3. The method according to claim 1 or 2, wherein the microbiota sequence variant shares at least 50% sequence identity with the tumor-related antigenic epitope sequence.
4. The method according to any one of claims 1-3, wherein the microbiota sequence variant is a human microbiota sequence variant and wherein the tumor-related antigen is a human tumor-related antigen.
5. The method according to any one of claims 1-4, wherein the microbiota sequence variant is selected from the group consisting of bacterial sequence variants, archaea sequence variants, protist sequence variants, fungi sequence variants and viral sequence variants.
6. The method according to claim 5, wherein the microbiota sequence variant is a bacterial sequence variant or an archaea sequence variant.
7. The method according to any one of claims 1-6, wherein the microbiota sequence variant is a sequence variant of microbiota of the gut.
8. The method according to claim 7, wherein the microbiota sequence variant is a gut bacterial sequence variant.
9. The method according to any one of claims 1-8, wherein the microbiota sequence variant is a peptide.
10. The method according to claim 9, wherein the peptide has a length of 8-12 amino acids, preferably of 8-10 amino acids, most preferably of 9 or 10 amino acids.
11. The method according to any one of claims 1-10, wherein the microbiota sequence variant shares at least 70%, preferably at least 75%, sequence identity with the tumor-related antigenic epitope sequence.
12. The method according to any one of claims 9-11, wherein the core sequence of the microbiota sequence variant is identical with the core sequence of the tumor-related antigenic epitope sequence, wherein the core sequence consists of all amino acids except the three most N-terminal and the three most C-terminal amino acids.
13. The method according to any one of claims 1-12, wherein the tumor-related antigenic epitope identified in step (ii) can bind to MHC I.
14. The method according to any one of claims 1-13, wherein the microbiota sequence variant in step (iii) is identified on basis of a microbiota database.
15. The method according to claim 14, wherein the microbiota database comprises microbiota data of multiple individuals.
16. The method according to claim 14, wherein the microbiota database comprises microbiota data of a single individual, but not of multiple individuals.
17. The method according to any one of claims 14-16, wherein step (iii) comprises the following sub-steps: (iii-a) optionally, identifying microbiota protein sequences or nucleic acid sequences from (a) sample(s) of a single or multiple individual(s), (iii-b) compiling a database containing microbiota protein sequences or nucleic acid sequences of a single or multiple individual(s), and (iii-c) identifying in the database compiled in step (iii-b) at least one microbiota sequence variant of the epitope sequence identified in step (ii).
18. The method according to claim 17, wherein the sample in step (iii-a) is a stool sample.
19. The method according to any one of claims 1-18, wherein the method further comprises the following step: (iv) testing binding of the at least one microbiota sequence variant to MHC molecules, in particular MHC I molecules, and obtaining a binding affinity.
20. The method according to claim 19, wherein step (iv) further comprises testing binding of the (respective reference) epitope to MHC molecules, in particular MHC I molecules, and obtaining a binding affinity.
21. The method according to claim 20, wherein step (iv) further comprises comparing of the binding affinities obtained for the microbiota sequence variant and for the respective reference epitope and selecting microbiota sequence variants having a higher binding affinity to MHC than their respective reference epitopes.
22. The method according to any one of claims 1-21, wherein the method further comprises the following step: (v) determining cellular localization of a microbiota protein containing the microbiota sequence variant.
23. The method according to claim 22, wherein step (v) further comprises identifying the sequence of a microbiota protein containing the microbiota sequence variant, preferably before determining cellular localization.
24. The method according to any one of claims 19-23, wherein the method comprises step (iv) and step (v).
25. The method according to claim 24, wherein step (v) follows step (iv) or wherein step (iv) follows step (v).
26. The method according to any one of claims 1-25, wherein the method further comprises the following step: (vi) testing immunogenicity of the microbiota sequence variant.
27. The method according to any one of claims 1-26, wherein the method further comprises the following step: (vii) testing cytotoxicity of the microbiota sequence variant.
28. The method according to any one of claims 1-28, wherein the tumor-related antigenic epitope sequence is the sequence as set forth in any one of SEQ ID NOs: 1-5, 55-65, and 126-131.
29. The method according to claim 29, wherein the tumor-related antigenic epitope sequence is the sequence as set forth in SEQ ID NO: 1.
30. Microbiota sequence variant of a tumor-related antigenic epitope sequence, preferably obtainable by the method according to claim 1-29.
31. The microbiota sequence variant according to claim 30, wherein the microbiota sequence variant is a (bacterial) peptide, preferably having a length of 8-12 amino acids, more preferably of 8-10 amino acids, most preferably 9 or 10 amino acids.
32. The microbiota sequence variant according to claim 31, wherein the microbiota sequence variant shares at least 70%, preferably at least 75%, sequence identity with the tumor-related antigenic epitope sequence, and/or wherein the core sequence of the microbiota sequence variant is identical with the core sequence of the tumor-related antigenic epitope sequence, wherein the core sequence consists of all amino acids except the three most N-terminal and the three most C-terminal amino acids.
33. The microbiota sequence variant according to claim 31 or 32, wherein the microbiota sequence variant comprises or consists of an amino acid sequence according to any one of SEQ ID NOs 6-18, preferably the microbiota sequence variant comprises or consists of an amino acid sequence according to SEQ ID NO: 6 or 18, more preferably the microbiota sequence variant comprises or consists of an amino acid sequence according to SEQ ID NO: 18.
34. The microbiota sequence variant according to claim 31 or 32, wherein the microbiota sequence variant comprises or consists of an amino acid sequence according to any one of SEQ ID NOs 66-84 and 126, preferably the microbiota sequence variant comprises or consists of an amino acid sequence according to SEQ ID NO: 75.
35. The microbiota sequence variant according to claim 31 or 32, wherein the microbiota sequence variant comprises or consists of an amino acid sequence according to any one of SEQ ID NOs 132-141 and 158, preferably the microbiota sequence variant comprises or consists of an amino acid sequence according to SEQ ID NO: 139.
36. Method for preparing a medicament, preferably for prevention and/or treatment of cancer, comprising the following steps: (a) identification of a microbiota sequence variant of a tumor-related antigenic epitope sequence according to the method according to any one of claims 1-29; (b) preparing a medicament comprising the microbiota sequence variant.
37. The method according to claim 36, wherein the medicament is a vaccine.
38. The method according to claim 36 or 37, wherein step (b) comprises loading a nanoparticle with the microbiota sequence variant.
39. The method according to claim 38, wherein step (b) further comprises loading the nanoparticle with an adjuvant.
40. The method according to claim 36 or 37, wherein step (b) comprises loading a bacterial cell with the microbiota sequence variant.
41. The method according to claim 40, wherein step (b) comprises a step of transformation of a bacterial cell with (a nucleic acid molecule comprising/encoding) the microbiota sequence variant.
42. The method according to any one of claims 36-41, wherein step (b) comprises the preparation of a pharmaceutical composition comprising (i) the microbiota sequence variant; (ii) a recombinant protein comprising the microbiota sequence variant; (iii) an immunogenic compound comprising the microbiota sequence variant; (iv) a nanoparticle loaded with the microbiota sequence variant; (v) an antigen-presenting cell loaded with the microbiota sequence variant; (vi) a host cell expressing the microbiota sequence variant; or (vii) a nucleic acid molecule encoding the microbiota sequence variant; and, optionally, a pharmaceutically acceptable carrier and/or an adjuvant.
43. Medicament comprising the microbiota sequence variant according to any one of claims 30-35, preferably obtainable by the method according to any one of claims 36-42.
44. The medicament according to claim 43 comprising a nanoparticle loaded with the microbiota sequence variant according to any one of claims 30-35.
45. The medicament according to claim 44, wherein the nanoparticle is further loaded with an adjuvant.
46. The medicament according to claim 43 comprising a bacterial cell expressing the microbiota sequence variant according to any one of claims 30-35.
47. The medicament according to claim 43 comprising (i) the microbiota sequence variant; (ii) a recombinant protein comprising the microbiota sequence variant; (iii) an immunogenic compound comprising the microbiota sequence variant; (iv) a nanoparticle loaded with the microbiota sequence variant; (v) an antigen-presenting cell loaded with the microbiota sequence variant; (vi) a host cell expressing the microbiota sequence variant; or (vii) a nucleic acid molecule encoding the microbiota sequence variant; and, optionally, a pharmaceutically acceptable carrier and/or an adjuvant.
48. The medicament according to any one of claims 43-47, wherein the medicament is a vaccine.
49. The medicament according to any one of claims 43-48, wherein the medicament is for use in the prevention and/or treatment of cancer.
50. The medicament according to claim 49, wherein the medicament is administered in combination with an anti-cancer agent, preferably with an immune checkpoint modulator.
51. A method for preventing and/or treating a cancer or initiating, enhancing or prolonging an anti-tumor response in a subject in need thereof comprising administering to the subject the medicament according to any one of claims 43-48.
52. The method according to claim 51, wherein the medicament is administered in combination with an anti-cancer agent, preferably with an immune checkpoint modulator.
53. A (in vitro) method for determining whether the microbiota sequence variant of a tumor-related antigenic epitope sequence according to any one of claims 30-35 is present in an individual comprising the step of determination whether the microbiota sequence variant of a tumor-related antigenic epitope sequence according to any one of claims 30-35 is present in an (isolated) sample of the individual.
54. The method according to claim 53, wherein the (isolated) sample is a stool sample or a blood sample.
55. The method according to claim 53 or claim 54, wherein the microbiota sequence variant of a tumor-related antigenic epitope sequence is obtained by a method according to any one of claims 1-29.
56. The method for preventing and/or treating a cancer or initiating, enhancing or prolonging an anti-tumor response according to claim 51 or 52 further comprising a step of determining whether the microbiota sequence variant of a tumor-related antigenic epitope sequence comprised by the medicament to be administered to the subject is present in the subject, preferably according to the method of any one of claims 53-55.
57. The method for preventing and/or treating a cancer or initiating, enhancing or prolonging an anti-tumor response according to claim 51 or 52, wherein the microbiota sequence variant of a tumor-related antigenic epitope sequence comprised by the medicament to be administered is present in the subject.
58. The method for preventing and/or treating a cancer or initiating, enhancing or prolonging an anti-tumor response according to claim 51 or 52, wherein the microbiota sequence variant of a tumor-related antigenic epitope sequence comprised by the medicament to be administered is not present in the subject.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0235] In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.
[0236]
[0237]
[0238]
[0239]
[0240]
[0241]
[0242]
[0243]
[0244]
EXAMPLES
[0245] In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.
Example 1
Identification of Bacterial Sequence Variants of Tumor-Related Epitopes in the Human Microbiome
[0246] 1. Selection of Tumor-Associated (TAA) and Tumor-Specific Antigens (TSA)
[0247] According to the classical definition, Tumor-Specific Antigens (TSA) are from antigens (proteins) present only on tumor cells, but not on any other cell type, while Tumor-Associated Antigens (TAA) are present on some tumor cells and also some normal (non-tumor) cells. The term tumor-related antigen, as used herein encompasses, tumor-associated (TAA) as well as tumor-specific antigens (TSA)
[0248] Selection of tumor-related proteins/antigens was performed based on literature, in particular based on well-known lists of TAAs and TSAs. For example, large numbers of potential TAA and TSA can be obtained from databases, such as Tumor T-cell Antigen Database (TANTIGEN; http://cvc.dfci.harvard.edu/tadb/), Peptide Database (https://www.cancerresearch.org/scientists/events-and-resources/peptide-database) or CTdatabase (http://www.cta.lncc.br/). Data from these database may be manually compared to recent literature in order to identify a feasible tumor-related antigen. For example, literature relating to specific expression of antigens in tumors, such as Xu et al., An integrated genome-wide approach to discover tumor-specific antigens as potential immunologic and clinical targets in cancer. Cancer Res. 2012 Dec. 15; 72(24):6351-61; Cheevers et al., The prioritization of cancer antigens: a national cancer institute pilot project for the acceleration of translational research. Clin Cancer Res. 2009 Sep. 1; 15(17):5323-37, may be useful to prioritize interesting antigens. A list of more than 600 candidate antigens was identified. All selected antigens were annotated regarding expression profile using available tools, such as Gent (http://medicalgenome.kribb.re.kr/GENT/), metabolic gene visualizer (http://meray.wi.mit.edu/), protein Atlas (https://www.proteinatlas.org/) or GEPIA (http://gepia.cancer-pku.cn). In addition, for each antigen the potential indication, relation to possible side effects, and driver vs passenger antigens were specified.
[0249] Among the 600 antigens, interleukin-13 receptor subunit alpha-2 (IL-13R2 or IL13RA2) was selected based on the facts that (i) it comprises an epitope identified as a CTL (cytotoxic T lymphocyte) epitope (Okano F, Storkus W J, Chambers W H, Pollack I F, Okada H. Identification of a novel HLA-A*0201-restricted, cytotoxic T lymphocyte epitope in a human glioma-associated antigen, interleukin 13 receptor alpha2 chain. Clin Cancer Res. 2002 September; 8(9): 2851-5); (ii) IL13RA2 is referenced in Tumor T-cell Antigen Database and CT database as an overexpressed gene in brain tumor; (iii) overexpression and selective expression of IL13RA2 was confirmed with tools as Gent, Metabolic gene visualizer and protein atlas, analyzing data from gene expression (microarrays studies); and (iv) overexpression was also reported in literature in brain tumors (Debinski et al., Molecular expression analysis of restrictive receptor for interleukin 13, a brain tumor-associated cancer/testis antigen. Mol Med. 2000 May; 6(5):440-9), in head and neck tumors (Kawakami et al., Interleukin-13 receptor alpha2 chain in human head and neck cancer serves as a unique diagnostic marker. Clin Cancer Res. 2003 Dec. 15; 9(17):6381-8) and in melanoma (Beard et al., Gene expression profiling using nanostring digital RNA counting to identify potential target antigens for melanoma immunotherapy. Clin Cancer Res. 2013 Sep. 15; 19(18):4941-50).
[0250] In particular, confirmation of overexpression and selective expression of IL13RA2 (point (iii)) was performed as follows: Analysis of mRNA data from the tissue atlas (RNA-seq data 37 normal tissues and 17 cancer types) generated by The Cancer Genome Atlas (TCGA; available at https://cancergenome.nih.gov/)) highlight the low basal level of IL13RA2 mRNA in normal tissue (with the exception of testis) and the high level of IL13RA2 mRNA expression in several tumor types with the highest expression observed in glioma samples. The same was observed when IL13RA2 mRNA expression was performed using Metabolic gEne RApid Visualizer (available at http://meray.wi.mit.edu/, analyzing data from the International Genomic Consortium, and NCBI GEO dataset) with a very low basal expression in most of the normal tissues tested, except for testis, and a strong expression in melanoma samples, glioblastoma and some samples of thyroid and pancreatic primary tumors.
[0251] IL13RA2 is a membrane bound protein that is encoded in humans by the IL13RA2 gene. In a non-exhaustive manner, IL13RA2 has been reported as a potential immunotherapy target (see Beard et al.; Clin Cancer Res; 72(11); 2012). The high expression of IL13RA2 has further been associated with invasion, liver metastasis and poor prognosis in colorectal cancer (Barderas et al.; Cancer Res; 72(11); 2012). Thus IL13RA2 could be considered as a driver tumor antigen.
[0252] 2. Selection of One or More Epitopes of Interest in the Selected Tumor-Related Antigen
[0253] In the next step, epitopes of the selected tumor-related antigen, which are presented specifically by MHC-I, were identified. To this end, the tumor-related antigen sequence (of IL13RA2) was analyzed by means of Immune epitope database and analysis resource (IEDB; http://www.iedb.org/; for MHC-I analysis in particular:
[0254] http://tools.immuneepitope.org/analyze/html/mhc_processing.htmlas used for IL13RA2 analysis, see also http://tools.immuneepitope.org/processing/) combining proteasomal cleavage, TAP transport, and MHC class I analysis tools for prediction of peptide presentation. Namely, the protein sequence of IL13RA2 was submitted to that IEDB analysis tool for identification of potential epitopes that could be presented by HLA.A2.1. Thereby, a list of 371 potential epitopes with HLA A2.1 binding properties was obtained. Two epitopes of that list were previously described as potential epitopes: WLPFGFILI (SEQ ID NO: 1) that was described and functionally validated by Okano et al. (Okano F, Storkus WJ, Chambers W H, Pollack I F, Okada H. Identification of a novel HLA-A*0201-restricted, cytotoxic T lymphocyte epitope in a human glioma-associated antigen, interleukin 13 receptor alpha2 chain. Clin Cancer Res. 2002 September; 8(9): 2851-5) and LLDTNYNLF (SEQ ID NO: 2) that was reported in IEDB database as found in a melanoma peptidome study (Gloger et al., Mass spectrometric analysis of the HLA class I peptidome of melanoma cell lines as a promising tool for the identification of putative tumor-associated HLA epitopes. Cancer Immunol Immunother. 2016 November; 65(11):1377-1393).
[0255] In order to identify epitopes, which have a good chance to be efficiently presented by MHC at the surface of tumor cells, in the list of the 371 potential epitopes with HLA A2.1 binding properties, in silico affinity of the 371 candidate epitopes to HLA A2.1 was calculated using the NetMHCpan 3.0 tool (http://www.cbs.dtu.dk/services/NetMHCpan/), with a maximum accepted affinity of 3000 nM (IC50). Thereby, a list of 54 IL13RA2 epitopes was obtained.
[0256] 3. Identification of Bacterial Sequence Variants of the Selected Epitopes in the Human Microbiome
[0257] Finally, the 54 selected ILI 3RA2-epitopes were compared to the Integrated reference catalog of the human gut microbiome (available at http://meta.genomics.cn/meta/home) in order to identify microbiota sequence variants of the 54 selected human IL13RA2-epitopes. To this end, a protein BLAST search (blastp) was performed using the PAM-30 protein substitution matrix, which describes the rate of amino acid changes per site over time, and is recommended for queries with lengths under 35 amino acids; with a word size of 2, also suggested for short queries; an Expect value (E) of 20000000, adjusted to maximize the number of possible matches; the composition-based-statistics set to 0, being the input sequences shorter than 30 amino acids, and allowing only un-gapped alignments. Thereafter, the blastp results were filtered to obtain exclusively microbial peptide sequences with a length of 9 amino acids (for binding to HLA-A2.1), admitting mismatches only at the beginning and/or end of the human peptide, with a maximum of two mismatches allowed per sequence. Thereby, a list of 514 bacterial sequences (nonapeptides, as a length of nine amino acid was used as a filter) was obtained, which consists of bacterial sequence variants of the selected IL13RA2 epitopes in the human microbiome.
Example 2
Testing Binding of Selected Bacterial Sequence Variants to MHC
[0258] As binding of microbial mimics to MHC molecules is essential for antigen presentation to cytotoxic T-cells, affinity of the 514 bacterial sequences to MHC class I HLA.A2.01 was calculated using the NetMHCpan 3.0 tool (http://www.cbs.dtu.dk/services/NetMHCpan/). This tool is trained on more than 180000 quantitative binding data covering 172 MHC molecules from human (HLA-A, B, C, E) and other species. The 514 bacterial sequences (blastp result of Example 1) were used as input, and the affinity was predicted by setting default thresholds for strong and weak binders. The rank of the predicted affinity compared to a set of 400000 random natural peptides was used as a measure of the binding affinity. This value is not affected by inherent bias of certain molecules towards higher or lower mean predicted affinities. Very strong binders are defined as having % rank <0.5, strong binders are defined as having % rank 0.5 and <1.0, moderate binders are defined as having % rank of 1.0 and 2.0 (in particular, moderate binders include moderate to strong binders, which are defined as having % rank 1.0 and <1.5) and weak binders are defined as having % rank of <2.0. Namely, from the 514 bacterial sequences, only those were selected, which show a very strong affinity (% rank <0.5), and where the human reference epitope shows at least moderate to strong affinity (for human peptide) (% rank <1.5), preferably where the human reference epitope shows at least strong affinity (for human peptide) (% rank <1).
[0259] Thereby, the following 13 bacterial sequence variants (Peptide 1-Peptide 13 were identified (Table 3):
TABLE-US-00003 Human Affinity Affinity Affinity Affinity Bacterial reference human human bacterial bacterial peptide, epitope, peptide peptide peptide peptide SEQ ID # SEQ ID # % rank [nM] % rank [nM] 6 3 1.3 143.467 0.18 13.5048 7 3 1.3 143.467 0.06 6.6623 8 3 1.3 143.467 0.20 16.0441 9 4 0.5 35.5261 0.01 2.8783 10 4 0.5 35.5261 0.02 3.6789 11 4 0.5 35.5261 0.04 5.0586 12 4 0.5 35.5261 0.05 5.8467 13 4 0.5 35.5261 0.18 13.3325 14 4 0.5 35.5261 0.40 25.3124 15 5 0.09 8.0315 0.04 5.5211 16 5 0.09 8.0315 0.40 26.9535 17 5 0.09 8.0315 0.40 26.9535 18 1 0.8 66.1889 0.08 7.4445
Example 3
Determining Annotation and Cellular Localization of the Bacterial Proteins Comprising the Selected Bacterial Sequence Variants
[0260] Next, the annotation of the bacterial proteins containing the selected bacterial epitope sequence variants was performed. To this end, a blast-based comparison against both the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/) and the National Center for Biotechnology Information (NCBI) Reference Sequence Database (RefSeq) (https://www.ncbi.nlm.nih.gov/refseq/). RefSeq provides an integrated, non-redundant set of sequences, including genomic DNA, transcripts, and proteins. In KEGG, the molecular-level functions stored in the KO (KEGG Orthology) database were used. These functions are categorized in groups of orthologues, which contain proteins encoded by genes from different species that evolved from a common ancestor.
[0261] In a next step, a prediction of the cellular localization of the bacterial proteins containing the selected bacterial epitope sequence variants was performed using two different procedures, after which a list of the peptide-containing proteins with the consensus prediction is delivered. First, a dichotomic search strategy to identify intracellular or extracellular proteins based on the prediction of the presence of a signal peptide was carried out. Signal peptides are ubiquitous protein-sorting signals that target their passenger protein for translocation across the cytoplasmic membrane in prokaryotes. In this context both, the SignalP 4.7. (www.cbs.dtu.dk/services/SignalP) and the Phobius server (phobius.sbc.su.se) were used to deliver the consensus prediction. If the presence of a signal peptide was detected by the two approaches, it was interpreted that the protein is likely to be extracellular or periplasmic. If not, the protein probably belongs to the outer/inner membrane, or is cytoplasmic. Second, a prediction of the transmembrane topology is performed. Both signal peptides and transmembrane domains are hydrophobic, but transmembrane helices typically have longer hydrophobic regions. SignalP 4.1. and Phobius have the capacity to differentiate signal peptides from transmembrane domains. A minimum number of 2 predicted transmembrane helices is set to differentiate between membrane and cytoplasmic proteins to deliver the final consensus list. Data regarding potential cellular localization of the bacterial protein is of interest for selection of immunogenic peptides, assuming that secreted components or proteins contained in secreted exosomes are more prone to be presented by APCs.
[0262] Table 4 shows the SEQ ID NOs of the bacterial proteins containing the 13 bacterial peptides shown in Table 4, their annotation and cellular localization:
TABLE-US-00004 Bacterial Bacterial Consensus peptide, protein Kegg cellular SEQ ID # SEQ ID # Phylum Genus Species orthology localization 6 19 Firmicutes Lachno- Lachno- K01190 No transmembrane clostridium clostridium phyto- fermentans 7 20 unknown unknown unknown unknown No transmembrane 8 21 Firmicutes Lacto- unknown unknown Transmembrane bacillus 9 22 unknown unknown unknown unknown No transmembrane 10 23 Firmicutes Rumino- Rumino- K07315 No transmembrane coccus coccus sp. 5_1_39BFAA 11 24 unknown unknown unknown unknown No transmembrane 12 25 Firmicutes unknown unknown K19002 No transmembrane 13 26 Bacteroidetes Bacteroides Bacteroides unknown No transmembrane fragilis 14 27 unknown unknown unknown K01992 Transmembrane 15 28 Firmicutes Copro- Copro- K07636 No transmembrane bacillus bacillus sp. 8_1_38FAA 16 29 unknown unknown unknown unknown No transmembrane 17 30 unknown unknown unknown unknown No transmembrane 18 31 unknown unknown unknown K19427 Transmembrane
[0263] Based on the data shown in Tables 3 and 4, the bacterial peptide according to SEQ ID NO: 18 (amino acid sequence: FLPFGFILV; also referred herein as IL13RA2-B), which is a sequence variant of the human IL13RA2 reference epitope according to SEQ ID NO: 1
[0264] (WLPFGFILI, see Table 2; also referred herein as IL13RA2-H), was selected for further studies. Effectively, the human reference epitope has intermediate affinity, and is presented at the surface of tumor cells. This MHC presentation was confirmed in several published studies (Okano et al., Identification of a novel HLA-A*0201-restricted, cytotoxic T lymphocyte epitope in a human glioma-associated antigen, interleukin 13 receptor alpha2 chain. Clin Cancer Res. 2002 September; 8(9):2851-5).
[0265] The bacterial sequence variant (SEQ ID NO: 18) has a very strong binding affinity for HLA.A2.01. Furthermore, this bacterial peptide sequence variant is comprised in a bacterial protein, which is predicted to be expressed at the transmembrane level, thereby increasing the probability of being part of exosome that will be trapped by antigen-presenting cells (APC) for MHC presentation.
Example 4
Bacterial Peptide IL13RA2-B (SEQ ID NO: 18) has Superior Affinity to the HLA-A*0201 Allele In Vitro than the Human Epitope IL13RA2-H (SEQ ID NO: 1)
[0266] This Example provides evidence that the bacterial peptide of sequence SEQ ID NO: 18 (FLPFGFILV; also referred herein as IL13RA2-B) has high affinity to the HLA-A*0201 allele in vitro, whereas the corresponding reference human peptide derived from IL13RA2 (WLPFGFILI, SEQ ID NO: 1, also referred herein as IL13RA2-H) has low affinity.
[0267] A. Materials and Methods
[0268] A1. Measuring the Affinity of the Peptide to T2 Cell Line.
[0269] The experimental protocol is similar to the one that was validated for peptides presented by the HLA-A*0201 (Tourdot et al., A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides: implication in the identification of cryptic tumor epitopes. Eur J Immunol. 2000 December; 30(12):3411-21). Affinity measurement of the peptides is achieved with the human tumoral cell T2 which expresses the HLA-A*0201 molecule, but which is TAP1/2 negative and incapable of presenting endogenous peptides.
[0270] T2 cells (2.10.sup.5 cells per well) were incubated with decreasing concentrations of peptides from 100 M to 0.1 M in a AIMV medium supplemented with 100 ng/l of human 2m at 37 C. for 16 hours. Cells were then washed two times and marked with the anti-HLA-A2 antibody coupled to PE (clone BB7.2, BD Pharmagen).
[0271] The analysis was performed by FACS (Guava Easy Cyte). For each peptide concentration, the geometric mean of the labeling associated with the peptide of interest was subtracted from background noise and reported as a percentage of the geometric mean of the HLA-A*0202 labeling obtained for the reference peptide HIV pol 589-597 at a concentration of 100 M. The relative affinity is then determined as follows:
relative affinity=concentration of each peptide inducing 20% of expression of HLA-A*0201/concentration of the reference peptide inducing 20% of expression of HLA-A*0201.
[0272] A2. Solubilisation of Peptides
[0273] Each peptide was solubilized by taking into account the amino acid composition. For peptides which do not include any cysteine, methionine, or tryptophan, the addition of DMSO is possible to up to 10% of the total volume. Other peptides are re-suspended in water or PBS pH7.4.
[0274] B. Results
[0275] For T2 Cells: Mean fluorescence intensity for variable peptidic concentrations: Regarding the couple IL13RA2 peptides (IL13RA2-H and IL13RA2-B), the human peptide does not bind to HLA-A*0201, whereas the bacterial peptide IL13RA2-B binds strongly to HLA-A*0201: 112.03 vs 18.64 at 100 M; 40.77 vs 11.61 at 10 M; 12.18 vs 9.41 at 1 M; 9.9 vs 7.46 at 0.1 M. Also, IL13RA2-B at 4.4 M induces 20% of expression of the HLA-A*0201 (vs 100 M for IL13RA2-H).
[0276] Similar results were obtained from a second distinct T2 cell clone.
Example 5
Bacterial Peptide 1L13RA2-B (SEQ ID NO: 18) has Superior Affinity to the HLA-A*0201 Allele In Vitro
[0277] This Example provides evidence that the bacterial peptide of sequence SEQ ID NO: 18 (FLPFGFILV; also referred herein as IL13RA2-B) has higher affinity to the HLA-A*0201 allele than other sequence variants of the corresponding reference human peptide derived from IL13RA2 (WLPFGFILI, SEQ ID NO: 1, also referred herein as IL13RA2-H). In this experiment, the bacterial peptide of sequence SEQ ID NO: 18 (FLPFGFILV; also referred herein as IL13RA2-B) was compared to [0278] the peptide 1A9V, as described by Eguchi Junichi et al., 2006, Identification of interleukin-13 receptor alpha 2 peptide analogues capable of inducing improved antiglioma CTL responses. Cancer Research 66(11): 5883-5891, in which the tryptophan at position 1 of SEQ ID NO: 1 was substituted by alanine (1A) and the isoleucine at position 9 of SEQ ID NO: 1 was substituted by valine (9V); [0279] peptide 1I9A, wherein the tryptophan at position 1 of SEQ ID NO: 1 was substituted by isoleucine (11) and the isoleucine at position 9 of SEQ ID NO: 1 was substituted by alanine (9A); and [0280] peptide 1F9M, wherein the tryptophan at position 1 of SEQ ID NO: 1 was substituted by phenylalanine (1F) and the isoleucine at position 9 of SEQ ID NO: 1 was substituted by methionine (9M).
[0281] A. Materials and Methods
[0282] The experimental protocol, materials and methods correspond to those outlined in Example 4, with the only difference that the above mentioned antigenic peptides were used.
[0283] B. Results
[0284] The following in vitro binding affinities were obtained (Table 5):
TABLE-US-00005 In vitro Peptide binding affinity IL13RA2-B (SEQ ID No18) 0.49 1A9V 3.06 1I9A 2.22 1F9M 2.62
[0285] Accordingly, the antigenic peptide according to the present invention (IL13RA2-B (SEQ ID N 31)) showed considerably higher binding affinity to HLA-A*0201 than all other peptides tested, whereas the peptide 1A9V, as described by Eguchi Junichi et al., 2006, Identification of interleukin-13 receptor alpha 2 peptide analogues capable of inducing improved antiglioma CTL responses. Cancer Research 66(11): 5883-5891, showed the lowest affinity of the peptides tested.
Example 6
Vaccination of Mice with the Bacterial Peptide IL13RA2-B (SEQ ID NO: 18) Induces Improved T Cell Responses in a ELISPOT-IFN Assay
[0286] A. Materials and Methods
[0287] A. 1 Mouse Model
[0288] The features of the model used are outlined in Table 6:
TABLE-US-00006 Mouse Model C57BL/6J B2m .sup.tm1UncIAb.sup./Tg(HLA-DRA HLA-DRB1*0301).sup.#Gih Tg(HLA-A/H2-D/B2M).sup.1Bpe Acronym /A2/DR3 Description Immunocompetent, no mouse class I and class II MHC Housing SOPF conditions (ABSL3) Number of mice 24 adults (>8 weeks of age)
[0289] These mice have been described in several reports (Koller et al., Normal development of mice deficient in beta 2M, MHC class I proteins, and CD8+ T cells. Science. 1990 Jun. 8; 248(4960):1227-30. Cosgrove et al., Mice lacking MHC class II molecules. Cell. 1991 Sep. 6; 66(5):1051-66; Pascolo et al., HLA-A2.1-restricted education and cytolytic activity of CD8(+) T lymphocytes from beta2 microglobulin (beta2m) HLA-A2.1 monochain transgenic H-2Db beta2m double knockout mice. J Exp Med. 1997 Jun. 16; 185(12):2043-51).
[0290] A.2. Immunization Scheme.
[0291] The immunization scheme is shown in
TABLE-US-00007 TABLE 7 Experimental group composition. h-pAg: helper peptide; vacc-pAg: vaccination peptide. The number of boost injections is indicated into brackets. Peptide Helper Animal Group (vacc-pAg) (h-pAg) Prime Boost number 1 IL13RA2-B HHD-DR3 + + (1X) 6 (100 g) (150 g) SEQ ID No 18 SEQ ID No32 2 IL13RA2-H HHD-DR3 + + (1X) 6 (100 g) (150 g) SEQ ID No 1 SEQ ID No32
[0292] The peptides were provided as follows: [0293] couples of vacc-pAg: IL13RA2-H and IL13RA2-B; all produced and provided at a 4 mg/ml (4 mM) concentration; [0294] h-pAg: HHD-DR3 peptide (SEQ ID NO: 32); provided lyophilized (50.6 mg; Eurogentec batch 1611166) and re-suspended in pure distilled water at a 10 mg/mL concentration.
[0295] The animals were immunized on day 0 (d0) with a prime injection, and on d14 with a boost injection. Each mouse was injected s.c. at tail base with 100 L of an oil-based emulsion that contained: [0296] 100 g of vacc-pAg (25 L of 4 mg/mL stock per mouse); [0297] 150 g of h-pAg (15 L of 10 mg/mL stock per mouse); [0298] 10 L of PBS to reach a total volume of 50 L (per mouse); [0299] Incomplete Freund's Adjuvant (IFA) added at 1:1 (v:v) ratio (50 L per mouse).
[0300] A separate emulsion was prepared for each vacc-pAg, as follows: IFA reagent was added to the vacc-pAg/h-pAg/PBS mixture in a 15 mL tube and mixed on vortex for repeated cycles of 1 min until forming a thick emulsion.
[0301] A.3. Mouse Analysis
[0302] Seven days after the boost injection (i.e. on d21), the animals were euthanized and the spleen was harvested. Splenocytes were prepared by mechanical disruption of the organ followed by 70 m-filtering and Ficoll density gradient purification.
[0303] The splenocytes were immediately used in an ELISPOT-IFN assay (Table 8). Experimental conditions were repeated in quadruplets, using 2*10.sup.5 total splenocytes per well, and were cultured in presence of vacc-pAg (10 M), Concanavalin A (ConA, 2.5 g/mL) or medium-only to assess for their capacity to secrete IFN. The commercial ELISPOT-IFN kit (Diaclone Kit Mujrine IFN ELISpot) was used following the manufacturer's instructions, and the assay was performed after about 16 h of incubation.
TABLE-US-00008 TABLE 8 Setup of the ELISPOT-IFN assay. Group Stimulus Wells Animal Total 1 IL13RA2-B (10 M) SEQ ID No 18 4 6 24 IL13RA2-H (10 M) SEQ ID No 1 4 6 24 ConA (2.5 g/ml) 4 6 24 Medium 4 6 24 2 IL13RA2-B (10 M) SEQ ID No 18 4 6 24 IL13RA2-H (10 M) SEQ ID No 1 4 6 24 ConA (2.5 g/ml) 4 6 24 Medium 4 6 24
[0304] Spots were counted on a Grand ImmunoSpot S6 Ultimate UV Image Analyzer interfaced to the ImmunoSpot 5.4 software (CTL-Europe). Data plotting and statistical analysis were performed with the Prism-5 software (GraphPad Software Inc.).
[0305] The cell suspensions were also analyzed by flow cytometry, for T cell counts normalization. The monoclonal antibody cocktail (data not shown) was applied on the purified leucocytes in presence of Fc-block reagents targeting murine (1:10 diluted anti-mCD16/CD32 CF11 cloneinternal source) Fc receptors. Incubations were performed in 96-well plates, in the dark and at 4 C. for 15-20 minutes. The cells were washed by centrifugation after staining to remove the excess of monoclonal antibody cocktail, and were re-suspended in PBS for data acquisition.
[0306] All data acquisitions were performed with an LSR-II Fortessa flow cytometer interfaced with the FACS-Diva software (BD Bioscience). The analysis of the data was performed using the FlowJo-9 software (TreeStar Inc.) using a gating strategy (not shown).
TABLE-US-00009 TABLE 9 FACS panel EXP-1. Target Label Clone Provider Dilution mCD3 FITC 145-2C11 Biolegend 1/100 mCD4 PE RM4-5 Biolegend 1/100 mCD8 APC 53-6,7 Biolegend 1/100
[0307] B. Results
[0308] A total of 14 /A2/DR3 mice were used for this experiment (see Table 8). At time of sacrifice, the spleen T cell population was analysed by flow cytometry, showing that the large majority belonged to the CD4+ T cell subset.
TABLE-US-00010 TABLE 10 Individual mouse features (groups 1 & 2). Each mouse is identified by a unique ear tag ID number. T Mouse Age.sup.a Group cells.sup.b T4.sup.c T8.sup.c ID Sex (wks) (pAg) (%) (%) (%) Note.sup.d 826 M 14 1 (IL13RA2-B) 18.6 72.0 13.7 P1/2 827 M 14 1 (IL13RA2-B) 21.1 82.5 8.7 P1/2 828 M 14 1 (IL13RA2-B) 20.9 78.4 8.6 P1/2 829 F 15 1 (IL13RA2-B) 23.8 67.0 17.5 P1/2 830 F 15 1 (IL13RA2-B) 29.2 73.3 12.5 P1/2 831 F 15 1 (IL13RA2-B) N.A. N.A. N.A. ID tag lost (excluded) 17 M 9 1 (IL13RA2-B) 8.3 83.7 10.4 P5 832 F 15 2 (IL13RA2-H) 28.3 83.4 5.7 P1/2 833 F 15 2 (IL13RA2-H) N.A. N.A. N.A. ID tag lost (excluded) 834 F 15 2 (IL13RA2-H) 27.5 79.7 7.2 P1/2 835 M 13 2 (IL13RA2-H) 33.8 84.2 8.5 P1/2 836 M 13 2 (IL13RA2-H) 31.4 84.7 6.3 P1/2 837 M 15 2 (IL13RA2-H) 30.8 83.4 5.4 P1/2 18 M 9 2 (IL13RA2-H) 11.2 85.9 9.2 P5 .sup.aage at onset of the vaccination protocol (in weeks); .sup.bpercentage of T cells in total leukocytes; .sup.cpercentage of CD4+ or CD8+ T cells in total T cells; .sup.dplate (P) number.
[0309] After plating and incubation with the appropriate stimuli, the IFN-producing cells were revealed and counted. The data were then normalized as a number of specific spots (the average counts obtained in the medium only condition being subtracted) per 10.sup.6 total T cells.
[0310] The individual average values (obtained from the quadruplicates) were next used to plot the group average values (see
[0311] Overall, vaccination with the IL13RA2-B pAg bacterial peptide induced improved T cell responses in the ELISPOT-IFN assay, as compared to IL13RA2-H pA (reference human)-vaccinated animals (group 2). For group 1 (IL13RA2-B), ex vivo re-stimulation with the IL13RA2-B pAg promoted higher response than with the IL13RA2-H pAg. It was not the case for group 2 (IL13RA2-H). The percentage of ConA-induced response (mean+/SEM) for each condition was as follows: [0312] Group 1 (IL13RA2-B)/IL13RA2-B pAg: 56.3%+/18.1 [0313] Group 1 (IL13RA2-B)/IL13RA2-H pAg: 32.3%+/11.8 [0314] Group 2 (IL13RA2-H)/IL13RA2-B pAg: 2.0%+/0.8 [0315] Group 2 (IL13RA2-H)/IL13RA2-H pAg: 1.1%+/0.8
[0316] Accordingly, those results provide experimental evidence that tumor-antigen immunotherapy targeting IL13RA2 is able to improve T cell response in vivo and that the IL13RA2-B bacterial peptide (SEQ ID NO: 18), which was identified as outlined in Examples 1-3, is particularly efficient for that purpose.
Example 7
Bacterial Peptide IL13RA2-B (SEQ ID NO: 18) Provides In Vitro Cytotoxicity Against Tumor Cells
[0317] This Example provides evidence that the bacterial peptide of sequence SEQ ID NO: 18(FLPFGFILV; also referred herein as IL13RA2-B) provides in vitro cytotoxicity against U87 cells, which are tumor cells expressing IL13RA2. In contrast, the corresponding reference human peptide derived from IL13RA2 (WLPFGFILI, SEQ ID NO: 1, also referred herein as IL13RA2-H) does not provide in vitro cytotoxicity against U87 cells.
[0318] Methods:
[0319] Briefly, CD8 T cells from mice immunized with IL13RA2-H or IL13RA2-H were used. These cells were obtained after sorting of splenocyte from immunized mice and were placed on top of U87 cells (tumor cells expressing IL13RA2).
[0320] In more detail, CD3.sup.+ T cells were purified from splenocytes of HHD mice immunized with IL13RA2-H (WLPFGFILI, SEQ ID NO: 1) or IL13RA2-B (FLPFGFILV, SEQ ID NO: 18). To this end, B6 2m.sup.ko HHD/DR3 mice were injected s.c. at tail base with 100 L of an oil-based emulsion containing vaccination peptide plus helper peptide plus CFA (complete Freund's adjuvant), at day 0 and day 14 as described in Example 6. On d21, i.e. seven days after the boost injection, the animals were euthanized and the spleen was harvested. Splenocytes were prepared by mechanical disruption of the organ. CD3+ purification was performed using the mouse total T cells isolation kit from Miltenyi biotec using the recommended procedure. Efficient purification of cells and viability was validated by cytometry using appropriate marker for viability, CD8, CD4, CD3, and CD45.
[0321] U87-MG cells were seeded at 610.sup.5 cells/well in flat-bottomed 24-well culture plates and incubated for 24 h at 37 C. in DMEM (Dulbecco's Modified Eagle Medium) containing 10% of FCS (fetal calf serum) and antibiotics. After 24 hours, culture media were removed and replaced with media containing purified T CD3+ cells. The following ratios of T cells vs. U87-MG cells were used: 1/0.5, 1/1 and 1/5.
[0322] 72 hours after co-culture of U87-MG cells and CD3+ T cells, all cells from the wells were harvested and specific U87-MG cell death was evaluated after immunostaining of CD45 negative cells with DAPI and fluorescent annexin V followed by cytometry analysis.
[0323] Results:
[0324] Results are shown in
Example 8
Identification of Bacterial Sequence Variants of an Epitope of Tumor-Related Antigen FOXM1 in the Human Microbiome
[0325] In the present example, among the 600 antigens, forkhead box M1 (FOXM1) was selected based on the facts that (i) it comprises an epitope identified as a CTL (cytotoxic T lymphocyte) epitope (Yokomine K, Senju S, Nakatsura T, Irie A, Hayashida Y, Ikuta Y, Harao M, Imai K, Baba H, lwase H, Nomori H, Takahashi K, Daigo Y, Tsunoda T, Nakamura Y, Sasaki Y, Nishimura Y. The forkhead box M1 transcription factor as a candidate of target for anti-cancer immunotherapy. Int J Cancer. 2010 May 1; 126(9):2153-63. doi: 10.1002/ijc.24836); (ii) FOXM1 is found overexpressed in many tumors in several database, including GEPIA, Gent, Metabolic gene visualizer and protein atlas, analyzing data from gene expression (microarrays studies); and (iii) overexpression was also reported in brain tumors (Hodgson J G, Yeh R F, Ray A, Wang N J, Smirnov I, Yu M, Hariono S, Silber J, Feiler H S, Gray J W, Spellman P T, Vandenberg S R, Berger M S, James C D Comparative analyses of gene copy number and mRNA expression in glioblastoma multiforme tumors and xenografts. Neuro Oncol. 2009 October; 11(5):477-87. doi: 10.1215/15228517-2008-113), in pancreatic tumors (Xia J T, Wang H, Liang Li, Peng B G, Wu Z F, Chen L Z, Xue L, Li Z, Li W. Overexpression of FOXM1 is associated with poor prognosis and clinicopathologic stage of pancreatic ductal adenocarcinoma. Pancreas. 2012 May; 41(4):629-35. doi: 10.1097/MPA.0b013e31823bcef2), in ovarian cancer (Wen N, Wang Y, Wen L, Zhao S H, Ai Z H, Wang Y, Wu B, Lu H X, Yang H, Liu W C, Li Y. Overexpression of FOXM1 predicts poor prognosis and promotes cancer cell proliferation, migration and invasion in epithelial ovarian cancer. J Transl Med. 2014 May 20; 12:134. doi: 10.1186/1479-5876-12-134), in colorectal cancer (Zhang H G, Xu X W, Shi X P, Han B W, Li Z H, Ren W H, Chen P J, Lou Y F, Li B, Luo X Y. Overexpression of forkhead box protein M1 (FOXM1) plays a critical role in colorectal cancer. Clin Transl Oncol. 2016 May; 18(5):527-32. doi: 10.1007/s12094-015-1400-1), and many other cancers.
[0326] In particular, confirmation of overexpression and selective expression of FOXM1 in tumor/cancer as described above was performed as follows: Analysis of mRNA data from the tissue atlas (RNA-seq data 37 normal tissues and 17 cancer types) generated by The Cancer Genome Atlas (TCGA; available at https://cancergenome.nih.gov/)) highlight the low basal level of FOXM1 mRNA in normal tissue (with the exception of testis) and the high level of FOXM1 mRNA expression in several tumor types. The same was observed when FOXM1 mRNA expression was performed using Metabolic gEne RApid Visualizer (available at http://meray.wi.mit.edu/, analyzing data from the International Genomic Consortium, and NCBI GEO dataset) with a very low basal expression in most of the normal tissues tested, except for embryo) and a strong expression in many tumor samples including samples of breast cancer, oesophagal cancer, lung cancer, melanoma, colorectal samples and glioblastoma samples.
[0327] FOXM1 is a transcription factor involved in G1-S and G2-M progression that is encoded in humans by the FOXM1 gene. In a non-exhaustive manner, FOXM1 has been proposed as a potential immunotherapy target (Yokomine K, Senju S, Nakatsura T, Irie A, Hayashida Y, Ikuta Y, Harao M, Imai K, Baba H, Iwase H, Nomori H, Takahashi K, Daigo Y, Tsunoda T, Nakamura Y, Sasaki Y, Nishimura Y; The forkhead box M1 transcription factor as a candidate of target for anti-cancer immunotherapy. Int J Cancer. 2010 May 1; 126(9):2153-63. doi: 10.1002/ijc.24836). The high expression of FOXM1 has further been associated with oncogenic transformation participating for example in tumor growth, angiogenesis, migration, invasion, epithelial-mesenchymal transition, metastasis and chemotherapeutic drug resistance (Wierstra I.FOXM1 (Forkhead box M1) in tumorigenesis: overexpression in human cancer, implication in tumorigenesis, oncogenic functions, tumor-suppressive properties, and target of anticancer therapy. Adv Cancer Res. 2013; 119:191-419. doi: 10.1016/B978-0-12-407190-2.00016-2). Thus, FOXM1 could be considered as a driver tumor antigen.
[0328] In the next step, epitopes of the selected tumor-related antigen, which are presented specifically by MHC-I, were identified. To this end, the tumor-related antigen sequence (of FOXM1) was analyzed by means of Immune epitope database and analysis resource (IEDB; http://www.iedb.org/; for MHC-I analysis in particular: http://tools.immuneepitope.org/analyze/html/mhc_processing.htmlas used for FOXM1 analysis, see also http://tools.immuneepitope.org/processing/) combining proteasomal cleavage, TAP transport, and MHC class I analysis tools for prediction of peptide presentation. Namely, the protein sequence of FOXM1 was submitted to that IEDB analysis tool for identification of potential epitopes that could be presented by HLA.A2.1. Thereby, a list of 756 potential epitopes with HLA A2.1 binding properties was obtained. Three epitopes of that list were previously described as potential epitopes: YLVPIQFPV (SEQ ID NO: 55), SLVLQPSVKV (SEQ ID NO: 56)/LVLQPSVKV (SEQ ID NO: 57) and GLMDLSTTPL (SEQ ID NO: 58)/LMDLSTTPL (SEQ ID NO: 59) that was described and functionally validated by Yokomine et al. (Yokomine K, Senju S, Nakatsura T, Irie A, Hayashida Y, Ikuta Y, Harao M, Imai K, Baba H, lwase H, Nomori H, Takahashi K, Daigo Y, Tsunoda T, Nakamura Y, Sasaki Y, Nishimura Y. The forkhead box M1 transcription factor as a candidate of target for anti-cancer immunotherapy. Int Cancer. 2010 May 1; 126(9):2153-63. doi: 10.1002/ijc.24836).
[0329] In order to identify epitopes, which have a good chance to be efficiently presented by MHC at the surface of tumor cells, in the list of the 756 potential epitopes with HLA A2.1 binding properties, in silico affinity of the 756 candidate epitopes to HLA A2.1 was calculated using the NetMHCpan 4.0 tool (http://www.cbs.dtu.dk/services/NetMHCpan/), with a maximum accepted affinity of 3000 nM (IC50). Thereby, a list of 35 FOXM1 epitopes was obtained.
[0330] Finally, the 35 selected FOXM1-epitopes were compared to the Integrated reference catalog of the human gut microbiome (available at http://meta.genomics.cn/meta/home) in order to identify microbiota sequence variants of the 35 selected human FOXM1-epitopes. To this end, a protein BLAST search (blastp) was performed using the PAM-30 protein substitution matrix, which describes the rate of amino acid changes per site over time, and is recommended for queries with lengths under 35 amino acids; with a word size of 2, also suggested for short queries; an Expect value (E) of 20000000, adjusted to maximize the number of possible matches; the composition-based-statistics set to 0, being the input sequences shorter than 30 amino acids, and allowing only un-gapped alignments. Thereafter, the blastp results were filtered to obtain exclusively microbial peptide sequences with a length of 9 or 10 amino acids (for binding to HLA-A2.1), admitting mismatches only at the beginning and/or end of the human peptide, with a maximum of two mismatches allowed per sequence (in addition to the maximum two mismatches, a third mismatch was accepted for an amino acid with similar properties, i.e. a conservative amino acid substitution as described above. Thereby, a list of 573 bacterial sequences was obtained, which consists of bacterial sequence variants of the selected FOXM1 epitopes in the human microbiome.
Example 9
Testing Binding of Selected Bacterial Sequence Variants to MHC
[0331] As binding of microbial mimics to MHC molecules is essential for antigen presentation to cytotoxic T-cells, affinity of the 573 bacterial sequences to MHC class I HLA.A2.01 was calculated using the NetMHCpan 4.0 tool (http://www.cbs.dtu.dk/services/NetMHCpan/). The 573 bacterial sequences (blastp result of Example 8) were used as input, and the affinity was predicted by setting default thresholds for strong and weak binders. The rank of the predicted affinity compared to a set of 400000 random natural peptides was used as a measure of the binding affinity. This value is not affected by inherent bias of certain molecules towards higher or lower mean predicted affinities. Very strong binders are defined as having rank <0.5, strong binders are defined as having % rank 0.5 and <1.0, moderate binders are defined as having % rank of 1.0 and 2.0 and weak binders are defined as having % rank of <2.0. Namely, from the 573 bacterial sequences, only those were selected, which show a very strong affinity (% rank <0.5), and where the human reference epitope shows at least strong affinity (for human peptide) (% rank <1).
[0332] Thereby, the following 20 bacterial sequence variants were identified (Table 11):
TABLE-US-00011 Human Affinity Affinity Affinity Affinity reference Bacterial human human bacterial bacterial epitope, peptide, peptide peptide peptide peptide SEQ ID # SEQ ID # [nM] % rank [nM] % rank 60 66 33.8685 0.5 36.7574 0.5 61 67 35.0299 0.5 24.6073 0.4 61 68 35.0299 0.5 18.9641 0.25 62 69 22.1919 0.3 3.4324 0.015 62 70 22.1919 0.3 5.4835 0.04 62 71 22.1919 0.3 32.5867 0.5 55 72 2.0623 0.01 10.1452 0.125 55 73 2.0623 0.01 18.7154 0.25 59 74 36.1922 0.5 28.9885 0.4 59 75 36.1922 0.5 20.6064 0.3 63 76 58.7874 0.7 1.7952 0.01 63 77 58.7874 0.7 4.8682 0.04 63 78 58.7874 0.7 20.2275 0.3 63 79 58.7874 0.7 2.5715 0.01 63 80 58.7874 0.7 3.0709 0.01 63 81 58.7874 0.7 2.1973 0.01 64 82 39.9764 0.6 35.5715 0.5 65 83 4.1604 0.025 14.2518 0.175 62 84 22.1919 0.3 8.3115 0.09
Example 10
Determining Annotation and Cellular Localization of the Bacterial Proteins Comprising the Selected Bacterial Sequence Variants
[0333] Next, the annotation of the bacterial proteins containing the selected bacterial epitope sequence variants was performed. To this end, a blast-based comparison against both the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/) and the National Center for Biotechnology Information (NCBI) Reference Sequence Database (RefSeq) (https://www.ncbi.nlm.nih.gov/refseq/). RefSeq provides an integrated, non-redundant set of sequences, including genomic DNA, transcripts, and proteins. In KEGG, the molecular-level functions stored in the KO (KEGG Orthology) database were used. These functions are categorized in groups of orthologues, which contain proteins encoded by genes from different species that evolved from a common ancestor.
[0334] In a next step, a prediction of the cellular localization of the bacterial proteins containing the selected bacterial epitope sequence variants was performed using two different procedures, after which a list of the peptide-containing proteins with the consensus prediction is delivered. First, a dichotomic search strategy to identify intracellular or extracellular proteins based on the prediction of the presence of a signal peptide was carried out. Signal peptides are ubiquitous protein-sorting signals that target their passenger protein for translocation across the cytoplasmic membrane in prokaryotes. In this context both, the SignalP 4.1. (www.cbs.dtu.dk/services/SignalP) and the Phobius server (phobius.sbc.su.se) were used to deliver the consensus prediction. If the presence of a signal peptide was detected by the two approaches, it was interpreted that the protein is likely to be extracellular or periplasmic. If not, the protein probably belongs to the outer/inner membrane, or is cytoplasmic. Second, a prediction of the transmembrane topology is performed. Both signal peptides and transmembrane domains are hydrophobic, but transmembrane helices typically have longer hydrophobic regions. SignalP 4.1. and Phobius have the capacity to differentiate signal peptides from transmembrane domains. A minimum number of 2 predicted transmembrane helices is set to differentiate between membrane and cytoplasmic proteins to deliver the final consensus list. Data regarding potential cellular localization of the bacterial protein is of interest for selection of immunogenic peptides, assuming that secreted components or proteins contained in secreted exosomes are more prone to be presented by APCs.
[0335] Table 12 shows the SEQ ID NOs of the bacterial proteins containing the bacterial peptides shown in Table 11, their annotation and cellular localization:
TABLE-US-00012 Bacterial Bacterial Consensus peptide, protein Kegg cellular SEQ ID # SEQ ID # Phylum Genus Species orthology localization 66 85 Bacteroidetes Barnesiella unknown K00347 transmembrane 67 86 unknown unknown unknown unknown cytoplasmic 68 87 Firmicutes unknown Hungatella K02335 cytoplasmic hathewayi 68 88 Firmicutes unknown Hungatella K02335 cytoplasmic hathewayi 69 89 unknown unknown unknown unknown cytoplasmic 70 90 unknown unknown unknown unknown cytoplasmic 71 91 unknown unknown unknown K03310 transmembrane 72 92 unknown unknown unknown K02355 cytoplasmic 73 93 Bacteroidetes unknown unknown K02355 cytoplasmic 74 94 Firmicutes Coprococcus Coprococcus catus K10117 cytoplasmic 74 95 Firmicutes Blautia unknown K10117 cytoplasmic 74 96 Firmicutes Blautia unknown K10117 secreted 74 97 Firmicutes Blautia unknown K10117 secreted 74 98 Firmicutes Coprococcus unknown K10117 secreted 74 99 Firmicutes Eubacterium Eubacterium K10117 secreted hallii 74 100 Firmicutes Blautia Blautia obeum K10117 secreted 74 101 Firmicutes Blautia unknown K10117 cytoplasmic 74 102 Firmicutes Blautia unknown K10117 cytoplasmic 74 103 Firmicutes Eubacterium Eubacterium K10117 cytoplasmic ramulus 74 104 Firmicutes Dorea unknown K10117 cytoplasmic 74 105 Firmicutes Blautia unknown K10117 secreted 75 106 Firmicutes Faecalibacterium Faecalibacterium K10117 cytoplasmic prausnitzii 74 107 Firmicutes Blautia unknown K10117 secreted 74 108 Firmicutes Blautia unknown K10117 cytoplasmic 74 109 Firmicutes Coprococcus unknown K10117 cytoplasmic 74 110 Firmicutes Blautia unknown K10117 secreted 75 111 Firmicutes Faecalibacterium unknown K10117 cytoplasmic 75 112 Firmicutes Faecalibacterium unknown K10117 secreted 75 113 Firmicutes Faecalibacterium unknown K10117 secreted 75 114 Firmicutes Faecalibacterium Faecalibacterium K10117 secreted prausnitzii 75 115 Firmicutes Faecalibacterium unknown K10117 cytoplasmic 126 116 unknown unknown unknown unknown cytoplasmic 76 117 unknown unknown unknown unknown cytoplasmic 77 118 unknown unknown unknown K05569 transmembrane 78 119 unknown unknown unknown K01686 cytoplasmic 79 120 unknown unknown unknown unknown cytoplasmic 80 121 unknown unknown unknown K06147 transmembrane 81 122 unknown unknown unknown K07089 transmembrane 82 123 unknown unknown unknown K03654 cytoplasmic 83 124 unknown unknown unknown unknown cytoplasmic 84 125 Firmicutes Oscillibacter Oscillibacter sp K03324 cytoplasmic
[0336] Based on the data shown in Tables 11 and 12, the bacterial peptide according to SEQ ID NO: 75 (amino acid sequence: LMDLSTTEV; also referred to as FOXM1-B2), which is a sequence variant of the human FOXM1 reference epitope according to SEQ ID NO: 59 (LMDLSTTPL; also referred to as FOXM1-H2), was selected for further studies. Effectively, the human reference epitope has medium/high affinity, and is presented at the surface of tumor cells. This MHC presentation was confirmed in published studies (Yokomine K, Senju S, Nakatsura T, He A, Hayashida Y, Ikuta Y, Harao M, Imai K, Baba H, Iwase H, Nomori H, Takahashi K, Daigo Y, Tsunoda T, Nakamura Y, Sasaki Y, Nishimura Y. The forkhead box M1 transcription factor as a candidate of target for anti-cancer immunotherapy. Int J Cancer. 2010 May 1; 126(9):2153-63. doi: 10.1002/ijc.24836).
[0337] The bacterial sequence variant of SEQ ID NO: 75 (LMDLSTTEV) has a strong binding affinity for HLA.A2.01. Furthermore, this bacterial peptide sequence variant is comprised in a bacterial protein, which is predicted to be secreted, thereby increasing the probability of being trapped by antigen-presenting cells (APC) for MHC presentation.
Example 11
Bacterial Peptide FOXM1 B2 (SEQ ID NO: 75) Binds to HLA-A*0201 Allele In Vitro and has Superior Affinity to the HLA-A*0201 Allele In Vitro than the Human Epitope
[0338] This Example provides evidence that the bacterial peptide of sequence SEQ ID NO: 75 (LMDLSTTEV; also referred herein as FOXM1-B2) binds to HLA-A*0201 allele in vitro and has high affinity to the HLA-A*0201 allele in vitro, whereas the corresponding reference human peptide derived from FOXM1-H2 (LMDLSTTPL, SEQ ID NO: 59, also referred herein as FOXM1-H2) has slightly lower affinity.
[0339] A. Materials and Methods
[0340] A 1. Measuring the Affinity of the Peptide to T2 Cell Line
[0341] The experimental protocol is similar to the one that was validated for peptides presented by the HLA-A*0201 (Tourdot et al., A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides: implication in the identification of cryptic tumor epitopes. Eur J Immunol. 2000 December; 30(12):3411-21). Affinity measurement of the peptides is achieved with the human tumoral cell T2 which expresses the HLA-A*0201 molecule, but which is TAP1/2 negative and incapable of presenting endogenous peptides.
[0342] T2 cells (2.10.sup.5 cells per well) were incubated with decreasing concentrations of peptides from 100 M to 0.1 M in a AIMV medium supplemented with 100 ng/l of human 2m at 37 C. for 16 hours. Cells were then washed two times and marked with the anti-HLA-A2 antibody coupled to PE (clone BB7.2, BD Pharmagen).
[0343] The analysis was performed by FACS (Guava Easy Cyte). For each peptide concentration, the geometric mean of the labeling associated with the peptide of interest was subtracted from background noise and reported as a percentage of the geometric mean of the HLA-A*0202 labeling obtained for the reference peptide HIV pol 589-597 at a concentration of 100 M. The relative affinity is then determined as follows:
relative affinity=concentration of each peptide inducing 20% of expression of HLA-A*0201/concentration of the reference peptide inducing 20% of expression of HLA-A*0201.
[0344] A2. Solubilisation of Peptides
[0345] Each peptide was solubilized by taking into account the amino acid composition. For peptides which do not include any cysteine, methionine, or tryptophan, the addition of DMSO is possible to up to 10% of the total volume. Other peptides are re-suspended in water or PBS pH7.4.
[0346] B. Results
[0347] For T2 Cells: Mean fluorescence intensity for variable peptidic concentrations: Both, bacterial peptide FOXM1-B2 (SEQ ID NO: 75) and human peptide FOXM1-H2 (SEQ ID NO: 59) bind to HLA-A*0201. However, the bacterial peptide FOXM1-B2 (SEQ ID NO: 75) has a better binding affinity to HLA-A*0201 than the human peptide FOXM1-H2 (SEQ ID NO: 59), namely, 105 vs 77.6 at 100 M; 98.2 vs 65.4 at 25 M; and 12.7 vs 0.9 at 3 M. Also, the bacterial peptide FOXM1-B2 induces at 6.7 M 20% of expression of the HLA-A*0201, while for the same expression a higher concentration of the human peptide FOXM1-H2 is required, namely 12.6 M.
[0348] Similar results were obtained from a second experiment. These data show that the bacterial peptide FOXM1-B2 is clearly superior to the corresponding human peptide FOXM1-H2.
Example 12
Vaccination of Mice with the bacterial peptide FOXM1-B2 (SEQ ID NO: 75) Induces Improved T Cell Responses in a ELISPOT-IFNs Assay
[0349] A. Materials and Methods A.1 Mouse Model
[0350] The features of the model used are outlined in Table 13:
TABLE-US-00013 Mouse Model C57BL/6J B2m .sup.tm1UncIAb.sup./Tg(HLA-DRA HLA-DRBl*0301).sup.#Gjh Tg(HLA-A/H2-D/B2M).sup.1Bpe Acronym /A2/DR3 Description Immunocompetent, no mouse class I and class II MHC Housing SOPF conditions (ABSL3) Number of mice 15 adults (>8 weeks of age)
[0351] These mice have been described in several reports (Koller et al., Normal development of mice deficient in beta 2M, MHC class I proteins, and CD8+ T cells. Science. 1990 Jun. 8; 248(4960):1227-30. Cosgrove et al., Mice lacking MHC class II molecules. Cell. 1991 Sep. 6; 66(5):1051-66; Pascolo et al., HLA-A2.1-restricted education and cytolytic activity of CD8(+) T lymphocytes from beta2 microglobulin (beta2m) HLA-A2.1 monochain transgenic H-2Db beta2m double knockout mice. J Exp Med. 1997 Jun. 16; 185(12):2043-51).
[0352] A.2. Immunization Scheme.
[0353] The immunization scheme is shown in
TABLE-US-00014 TABLE 14 Experimental group composition. h-pAg: helper peptide; vacc-pAg: vaccination peptide. The number of boost injections is indicated into brackets. Peptide Helper Animal Group (vacc-pAg) (h-pAg) Prime Boost number 1 FOXM1-B2 HHD-DR3 + + (1X) 15 (100 g) (150 g)
[0354] The peptides were provided as follows:
[0355] couples of vacc-pAg: FOXM1-B2 and FOXM1-H2; all produced and provided at a 4 mg/ml (4 mM) concentration;
[0356] h-pAg: HHD-DR3 peptide (SEQ ID NO: 32); provided lyophilized (50.6 mg; Eurogentec batch 1611166) and re-suspended in pure distilled water at a 10 mg/mL concentration.
[0357] The animals were immunized on day 0 (d0) with a prime injection, and on d14 with a boost injection. Each mouse was injected s.c. at tail base with 100 L of an oil-based emulsion that contained:
[0358] 100 g of vacc-pAg (25 L of 4 mg/mL stock per mouse);
[0359] 150 g of h-pAg (15 L of 10 mg/mL stock per mouse);
[0360] 10 L of PBS to reach a total volume of 50 L (per mouse);
[0361] Incomplete Freund's Adjuvant (IFA) added at 1:1 (v:v) ratio (50 L per mouse).
[0362] A separate emulsion was prepared for each vacc-pAg, as follows: IFA reagent was added to the vacc-pAg/h-pAg/PBS mixture in a 15 mL tube and mixed on vortex for repeated cycles of 1 min until forming a thick emulsion.
[0363] A.3. Mouse Analysis
[0364] Seven days after the boost injection (i.e., on d21), the animals were euthanized and the spleen was harvested. Splenocytes were prepared by mechanical disruption of the organ followed by 70 m-filtering and Ficoll density gradient purification.
[0365] The splenocytes were immediately used in an ELISPOT-IFN assay (Table 15). Experimental conditions were repeated in duplicates, using 2*10.sup.5 total splenocytes per well, and were cultured in presence of vacc-pAg (10 M), Concanavalin A (ConA, 2.5 g/mL) or medium-only to assess for their capacity to secrete IFN. The commercial ELISPOT-IFN kit (Diaclone Kit Mujrine IFN ELISpot) was used following the manufacturer's instructions, and the assay was performed after about 16 h of incubation.
TABLE-US-00015 TABLE 15 Setup of the ELISPOT-IFN assay. Group Stimulus Wells Animal Total 1 FOXM1-H2 (10 M) 2 15 30 FOXM1-B2 (10 M) 2 15 30 ConA (2.5 g/ml) 2 15 30 Medium 2 15 30
[0366] Spots were counted on a Grand ImmunoSpot S6 Ultimate UV Image Analyzer interfaced to the ImmunoSpot 5.4 software (CTL-Europe). Data plotting and statistical analysis were performed with the Prism-5 software (GraphPad Software Inc.).
[0367] The cell suspensions were also analyzed by flow cytometry, for T cell counts normalization.
[0368] The monoclonal antibody cocktail (data not shown) was applied on the purified leucocytes in presence of Fc-block reagents targeting murine (1:10 diluted anti-mCD16/CD32 CF11 cloneinternal source) Fc receptors. Incubations were performed in 96-well plates, in the dark and at 4 C. for 15-20 minutes. The cells were washed by centrifugation after staining to remove the excess of monoclonal antibody cocktail, and were re-suspended in PBS for data acquisition.
[0369] All data acquisitions were performed with an LSR-I1 Fortessa flow cytometer interfaced with the FACS-Diva software (BD Bioscience). The analysis of the data was performed using the FlowJo-9 software (TreeStar Inc.) using a gating strategy (not shown).
TABLE-US-00016 TABLE 16 FACS panel EXP-1. Target Label Clone Provider Dilution mCD3 FITC 145-2C11 Biolegend 1/100 mCD4 PE RM4-5 Biolegend 1/100 mCD8 APC 53-6,7 Biolegend 1/100
[0370] B. Results
[0371] A total of 14 /A2/DR3 mice were used for this experiment (see Table 15). At time of sacrifice, the spleen T cell population was analysed by flow cytometry, showing that the large majority belonged to the CD4+ T cell subset.
TABLE-US-00017 TABLE 17 Individual mouse features (groups 1 & 2). Each mouse is identified by a unique ear tag ID number. Mouse Age T cells T4 T8 Nb Id Sex (weeks).sub.a (%).sub.b (%).sub.c (%).sub.d 1 731 M 22 16.9 80.6 9.58 2 736 M 27 19.9 70.8 15 3 744 F 24 24.1 71.9 12.3 4 753 F 24 19.2 63.2 17.9 5 758 F 24 23.2 68.3 17.7 11 733 M 22 25.4 71.2 12.6 12 738 M 24 30.9 74.9 12.2 13 746 F 22 25.7 70.9 10.8 14 755 F 24 20.5 68.4 14.8 15 756 F 26 15.8 70.7 14.1 21 740 M 24 22.1 77.6 13.7 22 742 F 22 25.6 70.3 16.5 23 748 F 22 17.1 55.1 16.3 24 749 F 23 14 65.5 17.5 25 752 F 24 15.4 60.3 20.1 .sub.aage at onset of the vaccination protocol (in weeks); .sub.bpercentage of T cells in total leukocytes; .sub.cpercentage of CD4+ or CD8+ T cells in total T cells; .sub.dplate (P) number.
[0372] After plating and incubation with the appropriate stimuli, the IFN-producing cells were revealed and counted. The data were then normalized as a number of specific spots (the average counts obtained in the medium only condition being subtracted) per 10.sup.6 total T cells.
[0373] The individual average values (obtained from the quadruplicates) were next used to plot the group average values (see
[0374] Accordingly, those results provide experimental evidence that tumor-antigen immunotherapy targeting FOXM1 is able to improve T cell response in vivo and that the FOXM1-B2 bacterial peptide (SEQ ID NO: 75), which was identified as outlined in Examples 8 and 9, is particularly efficient for that purpose.
Example 13
Validation of 10 aa Bacterial Sequence Variants of Tumor-Related Epitopes in the Human Microbiome
[0375] In the following, it is demonstrated that bacterial sequences having a length of 10 amino acids (10 aa) identified according to the present invention are able to induce immune activation against tumor associated epitopes.
[0376] Interleukin-13 receptor subunit alpha-2 (IL-13R2 or IL13RA2) was selected as tumor associated antigen essentially for the same reasons as described in Example 1. Briefly, IL13RA2 selection was based on the facts that (i) it comprises an epitope identified as a CTL (cytotoxic T lymphocyte) epitope (Okano F, Storkus W J, Chambers W H, Pollack I F, Okada H. Identification of a novel HLA-A*0201-restricted, cytotoxic T lymphocyte epitope in a human glioma-associated antigen, interleukin 13 receptor alpha2 chain. Clin Cancer Res. 2002 September; 8(9): 2851-5); (ii) IL13RA2 is referenced in Tumor T-cell Antigen Database and CT database as an overexpressed gene in brain tumor; (iii) overexpression and selective expression of IL13RA2 was confirmed with tools as Gent, Metabolic gene visualizer and protein atlas, analyzing data from gene expression (microarrays studies); (iv) overexpression was also reported in literature in brain tumors (Debinski et al., Molecular expression analysis of restrictive receptor for interleukin 13, a brain tumor-associated cancer/testis antigen. Mol Med. 2000 May; 6(5):440-9), in head and neck tumors (Kawakami et al., Interleukin-13 receptor alpha2 chain in human head and neck cancer serves as a unique diagnostic marker. Clin Cancer Res. 2003 Dec. 15; 9(17):6381-8) and in melanoma (Beard et al., Gene expression profiling using nanostring digital RNA counting to identify potential target antigens for melanoma immunotherapy. Clin Cancer Res. 2013 Sep. 15; 19(18):4941-50), and (v), a 9 aa bacterial sequence (SEQ ID NO: 18) able to induce T cell activation against an IL13RA2 epitope (SEQ ID NO: 1) was already identified (Examples 1-7).
[0377] Epitopes of IL13RA2, which have a length of 10 amino acids and which are presented specifically by MHC-I, were identified. To this end, the tumor-related antigen sequence (of IL13RA2) was analyzed by means of Immune epitope database and analysis resource (IEDB; http://www.iedb.org/; for MHC-I analysis in particular: http://tools.immuneepitope.org/analyze/html/mhc_processing.htmlas used for IL13RA2 analysis, see also http://tools.immuneepitope.org/processing/) combining proteasomal cleavage, TAP transport, and MHC class I analysis tools for prediction of peptide presentation. Namely, the protein sequence of IL13RA2 was submitted to that IEDB analysis tool for identification of potential epitopes that could be presented by HLA.A2.1. silico affinity of candidate epitopes to HLA A2.1 was calculated using NetMHCpan 3.0 tool (http://www.cbs.dtu.dk/services/NetMHCpan/) with a maximum accepted affinity of 3000 nM (IC50), to identify epitopes, which have a good chance to be efficiently presented by MHC Affinity. Thereby, a list of 19 potential IL13RA2 epitopes of 10 amino acids was obtained.
[0378] The 19 selected IL13RA2-epitopes were compared to the Integrated reference catalog of the human gut microbiome (available at http://meta.genomics.cn/meta/home) in order to identify microbiota sequence variants. To this end, a protein BLAST search (blastp) was performed using the PAM-30 protein substitution matrix, which describes the rate of amino acid changes per site over time, and is recommended for queries with lengths under 35 amino acids; with a word size of 2, also suggested for short queries; an Expect value (E) of 20000000, adjusted to maximize the number of possible matches; the composition-based-statistics set to 0, being the input sequences shorter than 30 amino acids, and allowing only un-gapped alignments. Thereafter, the blastp results were filtered to obtain exclusively microbial peptide sequences with a length of 10 amino acids (for binding to HLA-A2.1), admitting mismatches only at the beginning and/or end of the human peptide, with a maximum of 3 mismatches allowed per sequence. Furthermore, only bacterial sequences were selected, which show a very strong affinity (% rank <0.5), and where the human reference epitope shows at least strong affinity (for human peptide) (% rank <1.5). Thereby a list of 11 bacterial peptides having similarity with 5 IL13RA2 tumor associated peptides were identified.
TABLE-US-00018 TABLE 18 10aa bacterial peptides having similarity with epitopes of human IL13RA2 Human Affinity Affinity Affinity Affinity Bacterial reference human human bacterial bacterial peptide, epitope, peptide peptide peptide peptide SEQ ID # SEQ ID # % rank [nM] % rank [nM] 132 127 0.7 54.6434 0.4 24.6345 133 127 0.7 54.6434 0.06 6.4119 134 127 0.7 54.6434 0.4 23.1945 135 128 0.125 9.6997 0.25 17.3756 136 129 0.7 51.5016 0.05 5.5782 137 129 0.7 51.5016 0.05 5.5782 138 130 0.7 50.2853 0.4 25.6338 139 131 1.3 136.856 0.03 4.4932 140 131 1.3 136.856 0.06 6.4084 158 131 1.3 136.856 0.05 5.8225 141 130 0.7 50.2853 0.4 26.8938
[0379] Next, the bacterial proteins containing the bacterial peptides shown in Table 18 were identified. Moreover, the annotation of the bacterial proteins containing the selected bacterial epitope sequence variants was performed as described above. Results are shown in Table 19.
[0380] Table 19 shows the SEQ ID NOs of the bacterial proteins containing the bacterial peptides shown in Table 18, their annotation and cellular localization:
TABLE-US-00019 Bacterial Bacterial Consensus peptide, protein cellular SEQ ID # SEQ ID # Phylum Genus localization 132 22 Unknown Unknown cytoplasmic 133 142 Firmicutes Hungatella transmembrane 134 143 Unknown Unknown cytoplasmic 135 144 Firmicutes Unknown transmembrane 136 28 Firmicutes Coprobacillus transmembrane 137 145 Unknown Unknown transmembrane 138 146 Unknown Unknown cytoplasmic 139 147 Unknown Unknown cytoplasmic 139 148 Firmicutes Blautia transmembrane 139 149 Unknown Unknown transmembrane 139 150 Firmicutes Blautia transmembrane 139 151 Firmicutes Blautia transmembrane 140 152 Firmicutes Clostridium transmembrane 140 153 Firmicutes Clostridium transmembrane 140 154 Unknown Unknown transmembrane 158 155 Unknown Unknown transmembrane 140 156 Firmicutes Lachnoclostridium transmembrane 141 157 Unknown Unknown cytoplasmic
[0381] Table 19 shows that the bacterial peptide according to SEQ ID NO: 139 (FLPFGFILPV; also referred to herein as IL13RA2-BL) was identified in the most distinct bacterial proteins expressed in human microbiota, namely, in five distinct bacterial proteins. For this reason, the bacterial peptide according to SEQ ID NO: 139 (FLPFGFILPV) was selected for in vitro and in vivo experimental testing. The corresponding human IL13RA2 epitope WLPFGFILIL (IL13RA2-HL, SEQ ID NO: 131), encompasses the sequence of IL13RA2-H peptide (SEQ ID NO: 1).
Example 14
Bacterial Peptide IL13RA2-BL (SEQ ID NO: 139) Binds to HLA-A*0201 Allele In Vitro and has Superior Affinity to the HLA-A*0201 Allele In Vitro than the Corresponding Human Epitope
[0382] This Example provides evidence that the bacterial peptide of sequence SEQ ID NO: 139 (FLPFGFILPV; also referred herein as IL13RA2-BL) binds to HLA-A*0201 allele in vitro and has high affinity to the HLA-A*0201 allele in vitro, while the corresponding reference human peptide derived from IL13RA2 displays low affinity.
[0383] A. Materials and Methods
[0384] A 1. Measuring the Affinity of the Peptide to T2 Cell Line.
[0385] The experimental protocol is similar to the one that was validated for peptides presented by the HLA-A*0201 (Tourdot et al., A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides: implication in the identification of cryptic tumor epitopes. Eur J Immunol. 2000 December; 30(12):3411-21). Affinity measurement of the peptides is achieved with the human tumoral cell T2 which expresses the HLA-A*0201 molecule, but which is TAP1/2 negative and incapable of presenting endogenous peptides.
[0386] T2 cells (2.10.sup.5 cells per well) were incubated with decreasing concentrations of peptides from 100 M to 0.1 M in a AIMV medium supplemented with 100 ng/l of human 2 m at 37 C. for 16 hours. Cells were then washed two times and marked with the anti-HLA-A2 antibody coupled to PE (clone BB7.2, BD Pharmagen).
[0387] The analysis was performed by FACS (Guava Easy Cyte). For each peptide concentration, the geometric mean of the labeling associated with the peptide of interest was subtracted from background noise and reported as a percentage of the geometric mean of the HLA-A*0202 labeling obtained for the reference peptide HIV pol 589-597 at a concentration of 100 M. The relative affinity is then determined as follows:
relative affinity=concentration of each peptide inducing 20% of expression of HLA-A*0201/concentration of the reference peptide inducing 20% of expression of HLA-A*0201.
[0388] A2. Solubilisation of Peptides
[0389] Each peptide was solubilized by taking into account the amino acid composition. For peptides which do not include any cysteine, methionine, or tryptophan, the addition of DMSO is possible to up to 10% of the total volume. Other peptides are re-suspended in water or PBS pH7.4.
[0390] B. Results
[0391] For T2 Cells: Mean fluorescence intensity for variable peptidic concentrations: The bacterial peptide IL13RA2-BL (SEQ ID NO: 139) binds to HLA-A*0201, while the corresponding human peptide does not bind to HLA-A*0201. The bacterial peptide IL13RA2-BL (SEQ ID NO: 139) shows a strong binding affinity to HLA-A*0201, namely, 69% of maximum HIV pol 589-597 binding activity at 100 M; 96% at 25 M and 43% at 6.25 M. Results are also shown in
Example 15
Vaccination of Mice with the Bacterial Peptide IL13RA2-BL (SEQ ID NO: 139) Induces Improved T Cell Responses in a ELISPOT-IFN Assay
[0392] A. Materials and Methods
[0393] A. 7 Mouse model
[0394] Two different mice models were used for the study. The features of the model used are outlined in Table 20:
TABLE-US-00020 Model 1 C57BL/6J B2m .sup.tm1UncIAb.sup./Tg(HLA-DRA HLA-DRBl*0301).sup.#Gjh Tg(HLA-A/H2-D/B2M).sup.1Bpe Acronym /A2/DR3 HHDDR3 Description Immunocompetent, no mouse class I and class II MHC Model 2 C57BL/6JB2m.sup.tm1UncIAb.sup./Tg(HLA-DRA, HLA-DRB1*0101).sup.#GjhTg(HLA-A/H2-D/B2M)1Bpe Acronym /A2/DR1 HHDDR1 Description Immunocompetent, no mouse class I and class II MHC
[0395] These mice have been described in several reports (Koller et al., Normal development of mice deficient in beta 2M, MHC class I proteins, and CD8+ T cells. Science. 1990 Jun. 8; 248(4960):1227-30. Cosgrove et al., Mice lacking MHC class II molecules. Cell. 1991 Sep. 6; 66(5):1051-66; Pascolo et al., HLA-A2.1-restricted education and cytolytic activity of CD8(+) T lymphocytes from beta2 microglobulin (beta2m) HLA-A2.1 monochain transgenic H-2Db beta2m double knockout mice. J Exp Med. 1997 Jun. 16; 185(12):2043-51).
[0396] A.2. Immunization Scheme.
[0397] The immunization scheme is shown in
[0398] The peptides were provided as follows: [0399] vacc-pAg: IL13RA2-BL; all produced and provided at a 4 mg/ml (4 mM) concentration; [0400] h-pAg: HHD-DR3 peptide (SEQ ID NO: 32); for immunization of /A2/DR3 HHDDR3 mice provided at a 4 mg/ml (4 mM) concentration [0401] h-pAg: UCP2 peptide (SEQ ID NO: 159); for immunization of /A2/DR1 HHDDR1 mice provided at a 4 mg/ml (4 mM) concentration
[0402] The animals were immunized on day 0 (d0) with a prime injection, and on d14 with a boost injection. Each mouse was injected s.c. at tail base with 100 L of an oil-based emulsion that contained: [0403] 100 g of vacc-pAg (25 L of 4 mg/mL stock per mouse); [0404] 150 g of h-pAg (15 L of 10 mg/mL stock per mouse); [0405] 10 L of PBS to reach a total volume of 50 L (per mouse); [0406] Incomplete Freund's Adjuvant (IFA) added at 1:1 (v:v) ratio (50 L per mouse).
[0407] A separate emulsion was prepared for each vacc-pAg, as follows: IFA reagent was added to the vacc-pAg/h-pAg/PBS mixture in a 15 mL tube and mixed on vortex for repeated cycles of 1 min until forming a thick emulsion.
[0408] A.3. Mouse Analysis
[0409] Seven days after the boost injection (i.e. on d21), the animals were euthanized and the spleen was harvested. Splenocytes were prepared by mechanical disruption of the organ followed by 70 m-filtering and Ficoll density gradient purification.
[0410] The splenocytes were immediately used in an ELISPOT-IFN assay (Table 21). Experimental conditions were repeated in quadruplets, using 2*10.sup.5 total splenocytes per well, and were cultured in presence of vacc-pAg (10 M), Concanavalin A (ConA, 2.5 g/mL) or medium-only to assess for their capacity to secrete IFN. The commercial ELISPOT-IFN kit (Diaclone Kit Mujrine IFN ELISpot) was used following the manufacturer's instructions, and the assay was performed after about 16 h of incubation.
TABLE-US-00021 TABLE 21 Setup of the ELISPOT-IFN assay. Vaccination Peptide Group (vacc-pAg) Stimulus Wells Animal Total 1 HHD DR3 IL13RA2 BL Medium 2 15 30 mice (vacc-pAg) plus ConA 2 15 30 (15 mice) HHDDR3 helper (2.5 g/ml) (h-pAg) plus IL13RA2-BL 2 15 30 IFA IL13RA2-L 2 15 30 2 HHD DR1 IL13RA2 BL Medium 3 5 15 mice (vacc-pAg) plus ConA 3 5 15 (5 mice) UCP2 helper (2.5 g/ml) (h-pAg) plus IL13RA2-BL 3 5 15 IFA IL13RA2-HL 3 5 15
[0411] Spots were counted on a Grand ImmunoSpot S6 Ultimate UV Image Analyzer interfaced to the ImmunoSpot 5.4 software (CTL-Europe). Data plotting and statistical analysis were performed with the Prism-5 software (GraphPad Software Inc.).
[0412] Results are shown in
Example 16
Validation of the Method for Identification of a Microbiota Sequence Variant in a Mouse Model
[0413] The present invention relates to identification of peptides expressed from microbiota, such as commensal bacteria, and able to promote immune response against tumor specific antigens of interest. In particular, the method enables identification of bacterial peptides, which are sequence variants of tumor associated peptides and which able to bind to human MHC (such as HLA.A2.01). The examples described herein provide evidence that the method according to the present invention enables identification of microbiota sequence variants of epitopes with strong binding affinity to MHC (for example, HLA.A2) and vaccination with microbiota sequence variants of epitopes is able to induce immunogenicity against the respective reference epitopes.
[0414] Without being bound to any theory, the present inventors assume that reference epitopes (from self) result in specific T cell clone exhaustion during thymic selection. Furthermore, without being bound to any theory, the present inventors also assume that immune system has been primed with the bacterial proteins/peptides of commensal bacteria and/or has the ability to better react to bacterial proteins/peptides of commensal bacteria.
[0415] The in vivo experiments described above were performed in HLA transgenic mice expressing class 1 and class 2 MHC (HHD DR3 mice) using bacterial peptides identified from human microbiota and epitopes of tumor associated antigens identified from human tumors. However, commensal bacterial species are different in human and in mice, and epitope sequences of human tumor specific antigens may not always have full homologs in the mice genome. Accordingly, epitopes of human tumor antigens may represent more immunogenic not self sequences in mice, while they represent less immunogenic self sequences in humans.
[0416] In view thereof, in the present example microbiota sequence variants of epitopes were identified in mice commensal bacterial proteins. Those mice microbiota sequence variants elicit immunogenicity against epitopes of mice antigens in wild-type mice.
[0417] 1. Identification of Bacterial Sequence Variants in the Murine Microbiome
[0418] To identify epitopes of murine proteins, mouse annotated proteins were used as reference sequences. Two mouse reference epitopes of interest were selected, namely, H2 Ld M5 (VSSVFLLTL; SEQ ID NO: 160) of mouse gene Phtf1 for BALB/c mice, and H2 Db M2 (INMLVGAIM; SEQ ID NO: 161) of mouse gene Stra6 for C57BU6 mice. Phtf1 encodes the putative homeodomain transcription factor 1, which is highly expressed in mice testis, but also expressed at low level in most of mouse tissues. Strati (stimulated by retinoic acid 6) encodes a receptor for retinol uptake, a protein highly expressed in mice placenta, but also expressed at medium level in in mice ovary, kidney, brain, mammary gland, intestine and fat pad.
[0419] In order to identify murine microbiota sequence variants thereof, stool samples from BALB/c and C57BL/6 mice were collected for mice commensal microbiota sequencing. After collection, microbial DNA was extracted using 1HMS procedure (International Human Microbiome Standards; URL: http://www.microbiome-standards.org/#SOPS). Sequencing was performed using Illumina (NextSeq500) technology and a mice gut gene catalogue was generated.
[0420] Murine microbiota sequence variants of the above described murine reference epitopes were identified using essentially the same identity criteria as in the above examples relating to the human gut microbiome. In particular, to reproduce the criteria used in the above examples in the context of human microbiota and human tumor-associated epitopes, peptides were further selected on the basis of molecular mimicry to the murine reference sequence, assuming that the selected murine reference peptide is expressed at low-medium level in different mice organs and has the ability to bind to mice MHC class 1 at a medium low level.
[0421] Table 22 shows the two bacterial peptides candidates were selected for in vivo studies:
TABLE-US-00022 Mouse strain BALB/c C57BL/6 Mouse gene/protein Phtf1 Stra6 Murine epitope VSSVFLLTL INMLVGAIM SEQ ID NO. 160 161 peptide name H2 Ld M5 H2 Db M2 Mice rank 2.5 3.5 Microbial sequence KPSVFLLTL GAMLVGAVL SEQ ID NO. 162 163 peptide name H2 Ld B5 H2 Db B2 Microbial rank 0.07 0.6
[0422] Bacterial peptide H2 Ld B5 (SEQ ID NO: 162) is a fragment of a protein found in the microbiota of BALB/c mice. H2 Ld B5 is a sequence variant of the Phtf1 peptide (H2 Ld M5; SEQ ID NO: 160).
[0423] Bacterial peptide H2 Db B2 (SEQ ID NO: 163) is a fragment of a protein found in the microbiota of C57BL/6 mice. H2 Db B2 is a sequence variant of the Stra6 peptide (H2 Db M2; SEQ ID NO: 161).
[0424] 2. Bacterial Peptides H2 Ld B5 (SEQ ID NO: 162) and H2 Db B2 (SEQ ID NO: 163) Induce Immunogenicity in Mice and Allow Activation of T Cells Reacting Against Mice Homolog Peptides
[0425] A. Materials and Methods
[0426] A.1 Mouse Model
[0427] Healthy female BALB/c mice (n=12) and healthy female C57BL/6J mice (n=11), 7 weeks old, were obtained from Charles River (France). Animals were individually identified and maintained in SPF health status according to the FELASA guidelines.
[0428] A.2. Immunization Scheme.
[0429] The immunization scheme is shown in
TABLE-US-00023 TABLE 23 experimental groups Peptide Helper Animal Group Mice (vacc-pAg) (h-pAg) Prime Boost number 1 BALB/c No OVA + + (1X) 6 323-339 2 BALB/c H2 Ld B 5 OVA + + (1X) 6 323-339 3 C57BL/6 No OVA + + (1X) 5 323-339 4 C57BL/6 H2 Db B 2 OVA + + (1X) 6 323-339
[0430] The peptides were provided as follows: [0431] couples of vacc-pAg: H2 Ld B5 and H2 Db B2; all produced and provided at a 4 mg/ml (4 mM) concentration; and [0432] h-pAg: OVA 323-339 (SEQ ID NO: 164); provided at a 4 mg/ml (4 mM) concentration.
[0433] The animals were immunized on day 0 (d0) with a prime injection, and on d14 with a boost injection. Each mouse was injected s.c. at tail base with 100 L of an oil-based emulsion that contained: [0434] 100 g of vacc-pAg (25 L of 4 mg/mL stock per mouse); [0435] 150 g of h-pAg (15 L of 10 mg/mL stock per mouse); [0436] 10 L of PBS to reach a total volume of 50 L (per mouse); [0437] Incomplete Freund's Adjuvant (IFA) added at 1:1 (v:v) ratio (50 L per mouse).
[0438] A separate emulsion was prepared for each vacc-pAg, as follows: IFA reagent was added to the vacc-pAg/h-pAg/PBS mixture in a 15 mL tube and mixed on vortex for repeated cycles of 1 min until forming a thick emulsion.
[0439] A.3. Mouse Analysis
[0440] Seven days after the boost injection (i.e. on d21), the animals were euthanized and the spleen was harvested. Splenocytes were prepared by mechanical disruption of the organ followed by 70 m-filtering and Ficoll density gradient purification. Spleen weight, splenocyte number and viability were immediately assessed (Table 24).
TABLE-US-00024 TABLE 24 Setup of the ELISPOT-IFN assay. Spleen Num Via- Mouse Animal weight (Mil- bility Group strain Vaccination No. (mg) lions) (%) 1 BALB/c OVA + IFA 6 126.0 101.8 97.1 7 125.1 135.4 96.9 8 137.9 132.8 97.0 9 144.2 79.2 96.7 10 111.2 69.5 97.3 11 111.6 74.5 97.8 2 BALB/c OVA + IFA + 42 135.0 95.9 98.4 H2 Ld B5 43 166.0 116.2 97.6 44 161.8 78.5 98.2 45 159.0 91.3 98.7 46 231.0 133.1 98.7 47 148.3 108.8 98.1 3 C57BL/6 OVA + IFA 54 93.8 129.1 98.4 55 91.6 89.0 98.2 56 125.1 123.1 97.9 57 97.6 81.3 98.4 58 110.6 90.2 98.2 11 C57BL/6 OVA + IFA + 59 101.5 85.6 98.9 H2 Db B2 60 103.9 75.5 98.9 61 97.5 82.0 99.1 62 134.3 88.0 98.1 63 105.7 96.6 99.0 64 90.7 90.5 99.1
[0441] The splenocytes were used in an ELISPOT-IFN assay (Table X). Experimental conditions were repeated in quadruplets, using 2*10.sup.5 total splenocytes per well, and were cultured in presence of vacc-pAg (10 M), mice peptide homolog, positive control (1 ng/ml of Phorbol 12-myristate 13-acetate (PMA) and 500 ng/ml of Ionomycin) or medium-only to assess for their capacity to secrete IFN.
[0442] The commercial ELISPOT-IFN kit (Diaclone Kit Mujrine IFN ELISpot) was used following the manufacturer's instructions, and the assay was performed after about 16 h of incubation.
TABLE-US-00025 TABLE 25 Setup of the ELISPOT-IFN assay. An- Group Mice Stimulus Wells imal Total 1 BALBc H2 Lb B5 (KPSVFLLTL) 3 6 18 PMA plus ionomycin 3 6 18 Medium 3 6 18 2 BALBc H2 Lb B5 (KPSVFLLTL) 3 6 18 H2 Ld M5 (VSSVFLLTL) 3 6 18 PMA plus ionomycin 3 6 18 Medium 3 6 18 3 C57BL6 H2 Db B2 (GAMLVGAVL) 3 5 15 PMA plus ionomycin 3 5 15 Medium 3 5 15 4 C57BL6 H2 Db B2 (GAMLVGAVL) 3 6 18 H2 Db M2 (INMLVGAIM) 3 6 18 PMA plus ionomycin 3 6 18 Medium 3 6 18
[0443] Spots were counted on a Grand ImmunoSpor S6 Ultimate UV Image Analyzer interfaced to the ImmunoSpot 5.4 software (CTL-Europe). Data plotting and statistical analysis were performed with the Prism-5 software (GraphPad Software Inc.).
[0444] B. Results
[0445] Results are shown in
[0446] In summary, those results provide experimental evidence that the method for identification of microbiota sequence variants as described herein is efficient for identification of microbiota sequence variants inducing activation of T cells against host reference peptides.
TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING)
[0447]
TABLE-US-00026 SEQIDNO Sequence Remarks SEQIDNO:1 WLPFGFILI IL13RA2epitope, IL13RA2-H SEQIDNO:2 LLDTNYNLF IL13RA2epitope SEQIDNO:3 CLYTFLIST 1L13RA2epitope SEQIDNO:4 FLISTTFGC IL13RA2epitope SEQIDNO:5 VLLDTNYNL IL13RA2epitope SEQIDNO:6 YLYTFLIST Sequencevariant SEQIDNO:7 KLYTFLISI Sequencevariant SEQIDNO:8 CLYTFLIGV Sequencevariant SEQIDNO:9 FLISTTFTI Sequencevariant SEQIDNO:10 FLISTTFAA Sequencevariant SEQIDNO:11 TLISTTFGV Sequencevariant SEQIDNO:12 KLISTTEGI Sequencevariant SEQIDNO:13 NLISTTFGI Sequencevariant SEQIDNO:14 FLISTTFAS Sequencevariant SEQIDNO:15 VLLDTNYEI Sequencevariant SEQIDNO:16 ALLDTNYNA Sequencevariant SEQIDNO:17 ALLDTNYNA Sequencevariant SEQIDNO:18 FLPFGFILV Sequencevariant, IL13RA2-B SEQIDNO:19 QYTNVKYPFPYDPPYVPNENPTGLYHQKFHLSK Bacterialprotein EQKQYQQFLNFEGVDSCFYLYVNKTFVGYSQVS HSTSEFDITPFTVEGQNELHVIVLKWCDGSYLED QDKERMSGIERDVYLMFRPENYVWDYNIRTSLS NENSKAKIEVFIMNQGQLKNPHYQLLNSEGIVL WEQYTKDTSFQFEVSNPILWNAEAPYLYTFLISTE EEVIVQQLGIREVSISEGVLLINGKPIKLKGVNRH DMDPVTGFTISYEQAKKDMTLMKEHNINAIRTS HYPNAPWFPILCNEYGFYVIAEADLEAHGAVSFY GGGYDKTYGDIVQRPMFYEAILDRNERNLMRD KNNPSIFMWSMGNEAGYSKAFEDTGRYLKELDP TRLVHYEGSIHETGGHKNDTSMIDVFSRMYASV DEIRDYLSKPNKKPFVLCEFIHAMGNGPGDIEDY LSLEYEMDRIAGGFVWEWSDHGIYMGKTEEGIK KYYYGDDFDIYPNDSNFCVDGLTSPDRIPHQGL LEYKNAIRPIRAALKSAIYPYEVTLINCLDFTNAKD LVELNIELLKNGEVVANQRVECPDIPPRCSTNIKI DYPHFKGVEWQEGDYVHINLTYLQKVAKPLTPR NHSLGEDQLLVNEPSRKEEWSVGNEFDIQNRTPI DNNEEISIEDLGNKIQLHHTNEHYVYNKFTGLED SIVWNQKSRLTKPMEFNIWRALIDNDKKHADD WKAAGYDRALVRVYKTSLTKNPDTGGIAIVSEFS LTAVHIQRILEGSIEWNIDRDGVLTFHVDAKRNL SMPFLPREGIRCFLPSAYEEVSYLGEGPRESYIDKH RASYFGQFHNLVERMYEDNIKPQENSSHCGCRF VSLQNNAKDQIYVASKEAFSFQASRYTQEELEKK RHNYELVKDEDTILCLDYKMSGIGSAACGPELAE QYQLKEEEIKESLQIRFDRS SEQIDNO:20 MKTIRKLYTFLISIEVILSLCSCYNDTHIITWQNED Bacterialprotein GTILAVDEVANGQIPVFQGSTPTKDSSSQYEYSF SEQIDNO:21 MATLYCLYTFLIGVLYHSAWFLTQAFYYLLLFLIRL Bacterialprotein ILSHQIRTSCNSSPLTRLKTCLMIGWLLLLFTPILSG MTILIPHQESSTTHFSQNVLLVVALYTFINLGNVL RGFAKPRRATVLLKTDKNVVMVTMMTSLYNLQ TLMLAAYSHDKSYTQLMTMTTGLVIIVITIGLAL WMIIESRHKIKQLANNAG SEQIDNO:22 ICAKNNGNPNTSSTNYAFLISTTFTINKGFVDVYS Bacterialprotein ELNHALYSYDTVTFSGGTIIARTGSSASSSYRPIRL GLNSSNPIVINAPTFTLDLSKQSDGSAMTTYSDV SNDKVKTLLAASGSSANHYAKLTSEFPPTVSTSTT GSGVTVSVKTDGQQQYLFIARYDSTGHLLELQ QRLRGEEAILKAEFTFPTVSPT SEQIDNO:23 MEHKRKKQWILIIMLLLTVCSVFVVYAGREWMF Bacterialprotein TNPFKPYTFSSVSYASGDGDGCTYVIDDSNRKIL KISADGRLLWRACASDKSFLSAERVVADGDGNV YLHDVRIEQGVQIASEGIVKLSSKGKYISTVASVE AEKGSVRRNIVGMVPTEHGVVYMQKEKEGILVS NTEQGSSKVFSVADAQDRILCCAYDRDSDSLFY VTYDGKIYKYTDSGQDELLYDSDTVDGSIPQEIS YSDGVLYSADIGLRDIIRIPCDMENTGSTDRLTVE ESLKEREIAYHVSAPGTLVSSTNYSVILWDGEDYE QFWDVPLSGKLQVWNCLLWAACAVIVAAVLFF AVTLLKILVKKFSFYAKITMAVIGIIVGVAALFIGTL FPQFQSLLVDETYTREKFAASAVTNRLPADAFQR LEKPSDFMNEDYRQVRQVVRDVFFSDSDSSQDL YCVLYKVKDGTVTLVYTLEDICVAYPYDWEYEG TDLQEVMEQGATKTYATNSSAGGFVFIHSPIRDK SGDIIGIIEVGTDMNSLTEKSREIQVSLIINLIAIMV VFFMLTFEVIYFIKGRQELKRRKQEEDNSRLPVEIF RFIVFLVFFFTNLTCAILPIYAMKISEKMSVQGLSPA MLAAVPISAEVLSGAIFSALGGKVIHKLGAKRSVF VSSVLLTAGLGLRVVPNIWLLTLSALLLGAGWGV LLLLVNLMIVELPDEEKNRAYAYYSVSSLSGANCA VVFGGFLLQWMSYTALFAVTAVLSVLLFLVANK YMSKYTSDNEEENCETEDTHMNIVQFIFRPRIISFF LLMMIPLLICGYFLNYMFPIVGSEWGLSETYIGYT YLLNGIFVLILGTPLTEFFSNRGWKHLGLAVAAFI YAAAFLEVTMLQNIPSLLIALALIGVADSEGIPUTS YFTDLKDVERFGYDRGLGVYSLFENGAQSLGSF VFGYVLVLGVGRGLIFVLILVSVLSAAFLISTTFAA HRDKRRSKNMEKRRKLNVELIKFLIGSMLVVGVL MLLGSSLVNNRQYRKLYNDKALEIAKTVSDQVN GDFIEELCKEIDTEEFEQIQKEAVAADDEQPIIDW LKEKGMYQNYERINEYLHSIQADMNIEYLYIQMI QDHSSVYLFDPSSGYLTLGYKEELSERFDKLKGNE RLEPTVSRTEFGWLSSAGEPVLSSDGEKCAVAFV DIDMTEIVRNTIRFTVLMVCLCILIILAAGMDISRKI KKRISRPIELLTEATHKFGNGEEGYDENNIVDLDI HTRDEIEELYHATQSMQKSIINYMDNLTRVTAEK ERIGAELNVATQIQASMLPCIFPAFPDRDEMDIY ATMTPAKEVGGDFYDFFMVDDRHMAIVMADV SGKGVPAALFMVIGKTLIKDHTQPGRDLGEVETE VNNILCESNENGMFITAFEGVLDLVTGEFRYVNA GHEMPFVYRRETNTYEAYKIRAGFVLAGIEDIVYK EQKLQLNIGDKIFQYTDGVTEATDKDRQLYGM DRLDHVLNQQCLSSNPEETLKLVKADIDAFVGD NDQFDDITMLCLEYTKKMENQRLLNNC SEQIDNO:24 MAACAACRWLMNEKTLISTTFGVGQLTLNAVE Bacterialprotein HKAKQDCY SEQIDNO:25 MAKLNIGIFTDTYFPQLNGVATSVQTLRRELEKR Bacterialprotein GHQVYIFTPYDPRQQQETDDHIFRLPSMPFIFVK NYRACFVCPPHILRKIHQLKLDIIHTQTEFSLGFL GKLISTTEGIPMVHTYHTMYEDYVHYIAGGHLIS AEGAREFSRIFCNTAMAVIAPTQKTERLLLSYGVN KPISIIPTGIDTSHFRKSNYDPAEILELRHSLGLKAD TPVLISIGRIAKEKSIDVIIGALPKLLEKLPNTMMVI VGEGMEIENLKKYADSLGIGDHLLFTGGKPWSEI GKYYQLGDVFCSASLSETQGLTFAEAMAGGIPV VARRDDCIVNFMTHGETGMFFDDPAELPDLLYR VLTDKPLREHLSTTSQNTMESLSVETFGNHVEELY EKVVRAFQNAESIPLHSLPYIKGTRVVHRISKIPKK LAHRSRSYSSQIAERLPFLPRHRS SEQIDNO:26 MIILNAMKLINLISTTEGIGVQDLLLKESENEVEVC Bacterialprotein FRLPRPFCVIADDINLFYAQILDDCQFDFLYCGN SEITINSLHSITDVENFVSHISDKLASLDLNDPDDI EVVNSFSILVKIRKEIRERVLNIYDFIALCNYWNDL TWENRLFVLSKEELKRGIVFYLLEDDICSFKTEGFY FSHNREEKPHIVNCLEDIRENVYWGNLDVYKLTP LYFHITQRSNVENIFQETEDVLSAVESLCSILDIVSL NAKDGKLVYKLCGYKNINGELNIDNSFSLLKNTE NEYFKIFRWIYIGEGNKTDKIGIARNVLSLFIAND NIAIEDNVFISIQSSEKTYLKENLDKYVAIRNQIYQ ELDAIISLSSAVKKDFLEGFKHNLLACITFFFSTIVLE VLGGNSKSYFLFTKEVCILCYAVFFISFLYLLWMR GDIEVEKKNISNRYVVLKKRYSDLLIPKEIDIILRNG EELKEQMGYIDLVKKKYTALWICSLLTLCVIVTVLS PIGNMFAGMIFAFKSIIVIEGLLIFLLVRLGSFIL SEQIDNO:27 MNVFAGIQFGIRKGLRYKVNTYSWFLADLALYA Bacterialprotein SVILMYFLISTTFASFGAYTKTEMGLYISTYFIINNLF AVLFSEAVSEYGASILNGSFSYYQLTPVGPLRSLILL NENFAAMLSTPALLAMNIYFVVQLFTTPVQVILY YLGVLFACGTMLFVFQTISALLLFGVRSSAIASAM TQLFSIAEKPDMVFHPAFRKVEITVIPAFLFSAVPS KVMLGTAAVSEIAALFLSPLFFYALFRILEAAGCRK YQHAGF SEQIDNO:28 MNKALFKYFATVLIVTLLFSSSVSMVILSDQMMQ Bacterialprotein TTRKDMYYTVKLVENQIDYQKPLDNQVEKLND LAYTKDTRLTIIDKDGNVLADSDKEGIQENHSGR SEFKEALSDQFGYATRYSSTVKKNMMYVAYYHR GYVVRIAIPYNGIFDNIGPLLEPLFISAALSLCVALA LSYRFSRTLTKPLEEISEEVSKINDNRYLSFDHYQY DEFNVIATKLKEQADTIRKTLKTLKNERLKINSILD KMNEGFVLLDTNYEILMVNKKAKQLFGDKMEV NQPIQDFIFDHQIIDQLENIGVEPKIVTLKKDEEV YDCHLAKVEYGVTLLFVNITDSVNATKMRQEFFS NVSHELKTPMTSIRGYSELLQTGMIDDPKARKQA LDKIQKEVDQMSSLISDILMISRLENKDIEVIQHPV HLQPIVDDILESLKVEIEKKEIKVTCDLTPQTYLAN HQHVQQLMNNLINNAVKYNKQKGSLNIHSYL VDQDYIIEVSDTGRGISLIDQGRVFERFFRCDAG RDKETGGTGLGLAIVKHIVQYYKGTIHLESELGK GTTFKIVLPINKDSL SEQIDNO:29 MSISLAEAKVGMADKVDQQVVDEFRRASLLLD Bacterialprotein MLIFDDAVSPGTGGSTLTYGYTCLKTPSTVAVRE LNTEYTPNEAKREKKTADLKIFGGSYQIDRVIAQT SGAVNEVEFQMREKIKAAANYFHMLVINGTGA GSGAGYVTNTFDGLKKILSGSDTEYTAEDVDIST SALLDTNYNAFLDAVDTFISKLAEKPDILMMNTE MLTKVRSAARRAGYYDRSKDDFGRAVETYNGIK LLDAGYYYNGSTTEPVVAIETDGSTAIYGIKIGLN AFHGVSPKGDKIIAQHLPDFSQAGAVKEGDVE MVAATVLKNSKMAGVLKGIKIKPTE SEQIDNO:30 MPVTLAEAKVGMADKVDQQVIDEFRRSSLLLD Bacterialprotein MLTFDDSVSPGTGGSTLTYGYVRLKTPSTVAVRS INSEYTANEAKREKATANVIILGGSFEVDRVIANTS GAVDEIDFQLKEKTKAGANYFHNLVINGTSAAS GAGFVVNTFDGLKKILSGSDTEYTSESDISTSALL DTNYNAFLDELDAFISKLAEKPDILLMNNEMLTK TRAAARRAGFYERSVDGFGRTVEKYNGIPMMD AGQYYNGSATVDVIETSTPSTSAYGETDIYAVKL GLNAFHGISVDGSKM1HTYLPDLQAPGAVKKGK VELLAGAILKNSKMAGRLKGIKIKPKTTAGG SEQIDNO:31 MVFVFSLLFSPFFALFFLLLYLYRYKIKKIHVALSVFL Bacterialprotein VAFIGIYWYPWGDNQTHFAIYYLDIVNNYYSLA LSSSHWLYDYVIYHIASLTGQYIWGYYFWLFVPF LFFSLLVWQIVDEQEVPNKEKWLLLILLILFLGIREL LDLNRNTNAGLLLAIATLLWQKNKALSITCVIVSL LLHDSVRYFIPFLPFGFILVKQSQRKTDLIIITTIIISG FLIKVIAPLVVSERNAMYLEVGGGRGVGSGFMVL QGYVNILIGIIQYLIIRRNKSVIAKPLYVVYIVSILIA AALSSMWVGRERFLLVSNILATSIILTSWSKLRLVE GVIKVLRNEQUIGSYSMKIIINLLLVYSAHYVENSA TTDNQKEFSIVARSFYMPTEMLFDIENYGESDKKE MNLYDRVDSTIDGE SEQIDNO:32 MAKTIAYDEEARRGLERGLN HHD-DR3 SEQIDNO:33 IISAVVGIA peptide SEQIDNO:34 ISAVVGIV peptide SEQIDNO:35 LFYSLADLI peptide SEQIDNO:36 ISAVVGIAV peptide SEQIDNO:37 SAVVGIAVT peptide SEQIDNO:38 YIISAVVGI peptide SEQIDNO:39 AYIISAVVG peptide SEQIDNO:40 LAYIISAVV peptide SEQIDNO:41 ISAVVGIAA peptide SEQIDNO:42 SAVVGIAAG peptide SEQIDNO:43 RIISAVVGI peptide SEQIDNO:44 QRIISAVVG peptide SEQIDNO:45 AQRIISAVV peptide SEQIDNO:46 SAVVGIVV peptide SEQIDNO:47 AISAVVGI peptide SEQIDNO:48 GAISAVVG peptide SEQIDNO:49 AGAISAVV peptide SEQIDNO:50 LLFYSLADL peptide SEQIDNO:51 ISAVVG peptide SEQIDNO:52 SLADLI peptide SEQIDNO:53 IISAVVGIL peptide SEQIDNO:54 LLYKLADLI peptide SEQIDNO:55 YLVPIQFPV FOXM1epitope SEQIDNO:56 SLVLQPSVKV FOXM1epitope SEQIDNO:57 LVLQPSVKV FOXM1epitope SEQIDNO:58 GLMDLSTTPL FOXM1epitope SEQIDNO:59 LMDLSTTPL FOXM1epitope SEQIDNO:60 NLSLHDMFV FOXM1epitope SEQIDNO:61 KMKPLLPRV FOXM1epitope SEQIDNO:62 RVSSYLVPI FOXM1epitope SEQIDNO:63 ILLDISFPG FOXM1epitope SEQIDNO:64 LLDISFPGL FOXM1epitope SEQIDNO:65 YMAMIQFAI FOXM1epitope SEQIDNO:66 SLSLHDMFL Sequencevariant SEQIDNO:67 KLKPLLPWI Sequencevariant SEQIDNO:68 KLKPLLPFL Sequencevariant SEQIDNO:69 MLSSYLVPI Sequencevariant SEQIDNO:70 LLSSYLVPI Sequencevariant SEQIDNO:71 FVSSYLVPT Sequencevariant SEQIDNO:72 KVVPIQFPV Sequencevariant SEQIDNO:73 KIVPIQFPI Sequencevariant SEQIDNO:74 LMDLSTTNV Sequencevariant SEQIDNO:75 LMDLSTTEV Sequencevariant SEQIDNO:76 WLLDISFPL Sequencevariant SEQIDNO:77 HLLDISFPA Sequencevariant SEQIDNO:78 ELLDISFPA Sequencevariant SEQIDNO:79 VLLDISFEL Sequencevariant SEQIDNO:80 VLLDISFKV Sequencevariant SEQIDNO:81 IMLDISFLL Sequencevariant SEQIDNO:82 LLDISFPSL Sequencevariant SEQIDNO:83 YQAMIQFLI Sequencevariant SEQIDNO:84 RLSSYLVEI Sequencevariant SEQIDNO:85 MFQSVFEGFESFLEVPNTTSRSGVHIHDSIDSKRT Bacterialprotein MTVVIVALLPALLFGMYNVGYQHYLAIGELAQT SFWSLFLEGFLAVLPKIVVSYVVGLGIEFTAAQLR HHEIQEGFLVSGMLIPMIVPVDTPLWMIAVATAF AVIFAKEVEGGTGMNIFNIALVTRAFLFFAYPSKM SGDEVEVRTGDTEGLGAGQIVEGFSGATPLGQ AATHTGGGALHLTDILGNSLSLHDMFLGFIPGSI GETSTLAILIGAVILLVTGIASWRVMLSVFAGGIV MSLICNVVCANPDIYPAAQLSPLEQICLGGFAFA AVFMATDPVTGARTNTGKYIEGFLVGVLAILIRV FNSGYPEGAMLAVLLMNAFAPLIDYFVVEANIR HRLKRAKNLTK SEQIDNO:86 MEGLEGEDAITCFNDSENHLKDRPDWDGYITLK Bacterialprotein EANEWYRSGNGEPLEADINKIDEDNYVSWGEK YVGETYVINYLLHIGRNIQTHIGAKVAGQGTAF NINIYGKKKLKPLLPWIK SEQIDNO:87 MDKEKLVLIDGHSIMSRAFYGVPELTNSEGLHTN Bacterialprotein AVYGFLNIMFKILEEEQADHVAVAFDLKEPTFRH QMFEQYKGMRKPMPEELHEQVDLMKEVLGAM EVPILTMAGFEADDILGTVAKESQAKGVEVVVVS GDRDLLQLADEHIKIRIPKTSRGGTEIKDYYPEDV KNEYHVTPKEFIDMKALMGDSSDNIPGVPSIGEK TAAAIIEAYGSIENAYAHIEEIKPPRAKKSLEENYSL AQLSKELAAINTNCGIEFSYDDAKTDSLYTPAAY QYMKRLEFKSLLSRFSDTPVESPSAEAHFRMVTDF GEAEAVFASCRKGAKIGLELVIEDHELTAMALCT GEEATYCFVPQGFMRAEYLVEKARDLCRTCERVS VLKLKPLLPFLKAESDSPLFDAGVAGYLLNPLKDT YDYDDLARDYLGLTVPSRAGLIGKQSVKMALET DEKKAFTCVCYMGYIAFMSADRLTEELKRTEMYS LFTDIEMPLIYSLFHMEQVGIKAERVRLKEYGDRL KVQIAVLEQKIYEETGETFNINSPKQLGEVLFDH MKLPNGKKTKSGYSTAADVLDKLAPDYPVVQM ILDYRQLTKLNSTYAEGLAVYIGPDERIHGTFNQ TITATGRISSTEPNLQNIPVRMELGREIRKIFVPED GYVFIDADYSQIELRVLAHMSGDERLIGAYRHAE DIHAITASEVFHTPLDEVTPLQRRNAKAVNFGIV YGISSFGLSEGLSISRKEATEYINKYFETYPGVKEFL DRLVADAKETGYAVSMFGRRRPVPELKSANFM QRSFGERVAMNSPIQGTAADIMKIAMIRVDRAL KAKGLKSRIVLQVHDELLIETRKDEVEAVKALLVD EMKHAADLSVSLEVEANVGDSWFDAK SEQIDNO:88 MDKEKIVLIDGHSIMSRAFYGVPELTNSEGLHTN Bacterialprotein AVYGELNIMFKILEEEQADHVAVAFDRKEPTERH KMFEPYKGTRKPMPEELHEQVDLMKEVLGAME VPILTMAGYEADDILGTVAKESQAKGVEVVVVS GDRDLLQLADEHIKIRIPKTSRGGTEIKDYYPEDV KNEYHVTPTEFIDMKALMGDSSDNIPGVPSIGEK TAAAIIEAYGSIENAYAHIEEIKPPRAKKSLEENYSL AQLSKELATININCGIEFSYDDAKADNLYTPAAY QYMKRLEFKSLLSRFSDTPVESPSAEAHFQMVTD FGEAEAIFAACKAGAKIGLELVIEDHELTAMALCT GEEATYCFVPQGFMRAEYLVEKARDLCRSCERVS VLKLKPLLPFLKAESDSPLFDASVAGYLLNPLKDT YDYDDLARDYLGMTVPSRADLLGKQTIKKALES DEKKAFTCICYMGYIAFMSADRLTEELKKAEMYS LFTDIEMPLIYSLEHMEQVGIKAERERLKEYGDRL KVQIVALEQKIYEETGETENINSPKQLGEVLEDH MKLPNGKKTKSGYSTAADVLDKLAPDYPVVQM ILDYRQLTKLNSTYAEGLAVYIGPDERIHGTENQ TITATGRISSTEPNLQNIPVRMELGREIRKIFVPED GCVFIDADYSQIELRVLAHMSGDERLIGAYRHA DDIHAITASEVFHTPLNEVTPLQRRNAKAVNFGI VYGISSFGLSEGLSISRKEATEYINKYFETYPGVKEF LDRLVADAKETGYAVSMFGRRRPVPELKSTNFM QRSFGERVAMNSPIQGTAADIMKIAMIRVDRAL KAKGLKSRIVLQVHDELLIETQKDEVEAVKALLV DEMKHAADLSVSLEVEANVGDSWFDAK SEQIDNO:89 MHTDQFFKEPKRGGRESMLDNTQRIVSIADAN Bacterialprotein ASSSAMDTENADTLDDYEVITKLQKKKTVIVPRV QSMQDYILKHHKRMILAEINRQLDGGTLQEIAQ DAQHPVTLHVGDCRFGDMIFWRYDARVLLTD VIISAYIHTGEATQTYDLYCELWVDMSKGMTFT CGECGFLEDKPCRNLWMLSSYLVPILRKDEVEQ GAEELLLRYCPKALEDLREHDAYRLADRMACG WNVIRFTERKAPSACFSSVRVK SEQIDNO:90 MFRIDSDTQTYPNAFTSDNMEEDENPRLDRTQE Bacterialprotein KTVVVPRIQSMKNYILKHHKRMILSELNRQIDGG TLQEIQATAKGCVTLNAQNCTFPDMNFWRYDT YTLLAEVLVCVNIEIDGILQTYDLYCELIVDMRKS MKFGYGECGFLKDKPERDLWLLSSYLVPILRKDE VEQGAEELLLRYCPNALTDRKEHNAYVLAENMG LHVERYPLYRQSATLSVLFFCDGYVVAEEQDEEG RGLDTPYTVKVSAGTIIINTNAVHKDCCQLEIYH ECIHYDWHYMFFKLQDMHNSDIRNLKTKRIVLI RDKSVTNPTQWMEWQARRGSFGLMMPLCMM EPLVDTMRMERVNNGQHPGKEFDSIARTIARDY KLPKFRVKARLLQMGYIAAKGALNYVDGRYIEPF AFSAENGSGNNSEVIDRKSAFAIYQENEAFRKQI QSGRYVYADGHICMNDSKYVCETNNGLMLTS WANAHIDTCCLRFTSNYEPCGISDYCFGVMNS DEEYNRHYMAFANAKKELTEKEKLAAMTRILYSL PASFPEALSYLMKQAHITIEKLEEKACISSRTISRLRT EERRDYSLDQ SEQIDNO:91 RDALGKKKLGILFASLLTFCYMLAFNMLQANNM Bacterialprotein STAFEYFIPNYRSGIWPWVIGIVESGLVACVVEG GIYRISFVSSYLVPTMASVYLLVGLYIIITNITEMPRI LGIIFKDAFDFQSITGGFAGSVVLLGIKRGLLSNE AGMGSAPNSAATADTSHPAKQGVMQILSVGID TILICSTSAFIILLSKTPMDPKMEGIPLMQAAISSQV GVWGRYFVTVSIICFAFSAVIGNEGISEPNVLFIK DSKKVLNTLK SEQIDNO:92 MKVYKTNEIKNISLLGSKGSGKTTLAESMLYECG Bacterialprotein VINRRGSIANNNTVCDYFPVEKEYGYSVESTVEY AEFNNKKLNVIDCPGMDDEVGNAVTALNITDA GVIVVNSQYGVEVGTQNIYRTAAKINKPVIFALN KMDAENVDYDNLINQLKEAFGNKVVPIQFPVA TGPDFNSIVDVLIMKQLTWGPEGGAPTITDIAPE YQDRAAEMNQALVEMAAENDETLMDKFFEQG ALSEDEMREGIRKGLIDRSICPVFCVSALKDMGV RRMMEFLGNVVPFVNEVKAPVNTEGVEIKPDAN GPLSVFFEKTTVEPHIGEVSYFKVMSGTLKAGMD LNNVDRGSKERLAQISVVCGQIKTPVEALEAGDI GAAVKLKDVRTGNTLNDKGVEYRFDFIKYPAPK YQRAIRPVNESEIEKLGAILNRMHEEDPTWKIEQS KELKQTIVSGQGEFHLRTLKWRIENNEKVQIEYLE PKIPYRETITKVARADYRHKKQSGGSGQFGEVH LIVEAYKEGMEEPGTYKEGNQEFKMSVKDKQEIA LEWGGKIVIYNCIVGGAIDARFIPAIVKGIMDRM EQGPVTGSYARDVRVCIYDGKMHPVDSNEISFR LAARHAFSEAFNAASPKVLEPVYDAEVLMPADC MGDVMSDLQGRRAIIMGMEEANGLQKINAKV PLKEMASYSTALSSITGGRASFTMKFASYELVPTDI QEKLHKEYLEASKDDE SEQIDNO:93 MKVYETKEIKNIALLGSKGSGKTTLAEAMLLECG Bacterialprotein VIKRRGSVENKNTVSDYFPVEKEYGYSVESTVEYA EFLNKKLNVIDCPGSDDEVGSAITALNVTDTGVI LIDGQYGVEVGTQNIFRATEKLQKPVIFAMNQI DGEKADYDNVLQQMREIFGNKIVPIQFPISCGP GENSMIDVLLMKMYSWGPDGGTPTISDIPDEY MDKAKEMHQGLVEAAAENDESLMEKFFDQGTL SEDEMRSGIRKGLIGRQIFPVFCVSALKDMGVRR MMEFLGNVVPFVEDMPAPEDTNGDEVKPDSKG PLSLEVEKTTVEPHIGEVSYEKVMSGTLNVGEDLT NMNRGGKERIAQIYCVCGQIKTNV SEQIDNO:94 MKMKKWSRVLAVLLALVTAVLLLSACGGKRAEK Bacterialprotein EDAETITVYLWSTKLYDKYAPYIQEQLPDINVEFV VGNNDLDFYKFLKENGGLPDIITCCRFSLHDASP LKDSLMDLSTTNVAGAVYDTYLNNFMNEDGSV NWLPVCADAHGFVVNKDLFEKYDIPLPTDYKSF VSACQAFDKVGIRGFTADYYYDYTCMETLQGLS ASELSSVDGRKWRTTYSDPDNTKREGLDNTVW PKAFERMEQFIQDTGLSQDDLDMNYDDIVEMY QSGKLAMYFGSSSGVKMFQDQGINTTFLPFFQE NGEKWLMTTPYFQVALNRDLTQDETRLKKANK VLNIMLSEDAQTQILYEGQDLLSYSQDVDMQLT EYLKDVKPVIEENHMYIRIASNDFFSVSKDVVSK MISGEYDAEQAYESFNTQLLEEESHSESVVLDSQ KSYSNRFHSSGGNAAYSVMANTLRGIYGTDVLI ATGNSFTGNVLKAGYTEKMAGDMIMPNDLAA YSSTMNGAELKETVKNFVEGYEGGFIPFNRGSLP VFSGISVEVKETEDGYTLSKVTKDGKKVQDNDT FTVTCLAIPKHMETYLADENIVFDGGDTSVKDT WTGYTSDGEAILVEPEDYINVR SEQIDNO:95 MEKKKWNRVLSVLFVMVTALSLLSGCGGKRAEK Bacterialprotein EDKETITVYLWTTNLYEKYAPYIQKQLADINIEFV VGNNDLDFYKFLKENGGLPDIITCCRFSLHDASP LKDSLMDLSTTNVAGAVYDTYLNSFQNEDGSV NWLPVCADAHGFLVNKDLFEKYDIPLPTDYESF VSACEAFDKVGIRGFTSDYFYDYTCMETLQGLS ASELSSPDGRKWRTGYSDPDNTKIEGLDRTVWP EAFERMEQFIRDTGLSRDDLDMDYDAVRDMFK SGKLAMYFGSSADVKMMQEQGINTTFLPFFQE NGEKWIMTTPYFQVALNRDLSKDDTRRKKAMK ILSTMLSEDAQKRIISDGQDLLSYSQDVDFKLTKY LNDVKPMIQENHMYIRIASNDFFSVSKDVVSKMI SGEYDAGQAYQVFHSQLLEEESASENIVLDSQKS YSNRFHSSGGNEAYSVMVNTLRGIYGTDVLIAT GNSFTGNVLKAGYTEKMAGDMIMPNGLSAYSS KMSGTELKETLRNFVEGYEGGFIPFNRGSLPVVS GISVEIRETDEGYTLGKVTKDGKQVQDNDIVTV TCLALPKHMEAYPADDNIVEGGEDTSVKDTWLE YISEGDAILAEPEDYMTLR SEQIDNO:96 MKKKKWNKILAVLLAMVTAVSLLSGCGGKSAEK Bacterialprotein EDAETITVYLWSTNLYEKYAPYIQEQLPDINVEFV VGNNDLDFYKFLEENGGLPDIITCCRFSLHDASP MKDSLMDLSTTNVAGAVYDTYLRNFMNEDGS VNWLPVCADAHGFVVNKDLFEKYDIPLPTDYES FVSACQVFEEMGIRGFAADYYYDYTCMETLQGL SASELSSADGRRWRTTYSDPDSTKREGLDSTVW PEAFERMEQFIQDTGLSQDDLDMNYDDIVEMY QSGKLAMYEGSSEGVKMFQDQGINTTFLPFFQE NGEKWLMTTPYFQVALNRDLTKDETRRKKAME VLSTMLSEDAQNRIISEGQDMLSYSQDVDMQL TEYLKDVKSVIEENHMYIRIASNDFFSISKDVVSK MISGEYDAEQAYQSFNSQLLEEKATSENVVLNS QKSYSNRFFISSGGNAAYSVMANTLRGIYGTDV LIATGNSFTGSVLKAGYTEKMAGDMIMPNVLLA YNSKMSGAELKETVRNEVEGYQGGFIPENRGSL PVVSGISVEVKETADGYTLSKIIKDGKKIQDNDTF TVTCLMMPQHMEAYPADGNITFNGGDTSVKD TWTEYVSEDNAILAESEDYMTLK SEQIDNO:97 MKRKKWNKVFSILLVMVTAVSLLSGCGGKSAEK Bacterialprotein EDAEIITVYLWSTSLYEKYAPYIQEQLPDINVEFVV GNNDLDFYRFLEENGGLPDIITCCRFSLHDASPL KDSLMDLSTTNVAGAVYDTYFSNFMNEDGSVN WLPVCADAHGEVVNKDLFEKYDIPLPTDYESEV SACQAFDKVGIRGFTADYYYDYTCMETLQGLSA SKLSSVEGRKWRTIYSDPDNTKKEGLDSTVWPEA FERMEQFIKDTGLSRDDLDMNYDDIAKMYQSG RLAMYEGSSEGVKMFQDQGINTTFLPFFQENGE KWIMTTPYFQAALNRDLTKDETRRKKAIKVLSTM LSEDAQKRIISEGQDLLSYSQDVDIHLTEYLKDVK PVIEENHMYIRIASNDFFSVSKDVVSKMISGEYDA RQAYQSENSQLLKEESTLEAIVLDSQKSYSNREHS SGGNAAYSVMANTLRSIYGTDVLIATANSFTGN VLKAGYTEKMAGNMIMPNDLFAYSSKLSGAELK ETVKNEVEGYEGGFIPENRGSLPVVSGISVEVKET EDGYTLSKVTKEGKQIRDEDIFTVTCLATLKHME AYPTGDNIVFDGENTSVKDTWTGYISNGDAVL AEPEDYINVR SEQIDNO:98 MKKKKWSRVLAVLLAMVTAISLLSGCGGKSAEK Bacterialprotein EDAGTITVYLWSTKLYEKYAPYIQEQLPDINVEFV VGNNDLDFYKELDENGGLPDIITCCRFSLHDAS PLKESLMDLSTTNVAGAVYDTYLSNFMNEDGSV NWLPVCADAHGFVVNKDLFEKYDIPLPTDYESF VSACQAFDKVGIRGFTADYYYDYTCMETLQGLS ASELSSVDGRKWRTTYSDPDNTKREGLDSTVWP GAFERMEQFIRDTGLSRDDLDLNYDDIVEMYQS GKLAMYEGSSSGVKMFQDQGINTTFLPFFQEN GEKWLMTAPYFQVALNRDLTQDETRLKKANKV LNIMLSEDAQTQILYEGQDLLSYSQDVDMQLTE YLKDVKPVIEENHMYIRIASNDFFSVSKDVVSKMI SGEYDAEQAYASFNTQLLEEESASESVVLDSQKS YSNRFHSSGGNAAYSVMANTLRGIYGTDVLIAT GNSFTGNVLKAGYTEKMAGDMIMPNDLSAYSS KMSGVELKKTVKNEVEGYEGGFIPENRGSLPVFS GISLEVEETDNGYTLSKVIKDGKEVQDNDTFTVT CLAIPKHMEAYPADENTVFDRGDTTVKGTWTG YTSDGEAILAEPEDYINVR SEQIDNO:99 MRKKKWNRVLAVLLMMVMSISLLSGCGSKSAEK Bacterialprotein EDAETITVYLWSTNLYEKYAPYIQEQLPDINVEFI VGNNDLDFYKFLNENGGLPDIITCCRFSLHDAS PLKDNLMDLSTTNVAGAVYDTYLSNFMNEDGS VNWLPVCADAHGFVVNKDLFEKYDIPLPTDYES FVSACQTFDKVGIRGFTADYYYDYTCMETLQGL SASELSSVDGRKWRTTYSDPDNTKREGLDSTVW PKAFERMEQFIQDTGLSQDDLDMNYDDIVEMY QSGKLAMYFGTSAGVKMFQDQGINTTFLPFFQ ENGEKWIMTTPYFQVALNSNLTKDETRRKKAMK VLDTMLSADAQNRIVYDGQDLLSYSQDVDLQL TEYLKDVKPVIEENHMYIRIASNDFFSVSKDVVSK MISGEYDAGQAYQSFDSQLLEEKSTSEKVVLDS QKSYSNRFHSSGGNAAYSVMANTLRGIYGSDV LIATGNSFTGNVLKAGYTEKMAGDMIMPNELSA YSSKMSGAELKEAVKNFVEGYEGGFTPFNRGSLP VLSGISVEVKETDDDYTLSKVTKDGKQIQDNDT FTVTCLAIPKHMEAYPADDNIVEDGGNTSVDDT WTGYISDGDAVLAEPEDYMTLR SEQIDNO:100 FVMKKKKWNRVLAVLLMMVMSISLLSGCGGKS Bacterialprotein TEKEDAETITVYLWSTNLYEKYAPYIQEQLPDINV EFVVGNNDLDFYKFLKKNGGLPDIITCCRFSLHD ASPLKDSLMDLSTTNVAGAVYDTYLSNFMNED GSVNWLPVCADAHGFVVNKDLFEKYDIPLPTD YESEVSACQAFDKVGIRGETADYYYDYTCMETL QGLSASELSSVDGRKWRTAYSDPDNTKREGLDS TVWPKAFERMEQFIQDTGLSQDDLDMNYDDI VEMYQSGKLAMYFGTSAGVKMFQDQGINTTFL PFFQENGEKWLMTTPYFQVALNRDLTQDETRR KKAMKVLSTMLSEDAQERIISDGQDLLSYSQDV DMQLTEYLKDVKSVIEENHMYIRIASNDFFSVSK DVVSKMISGEYDAEQAYQSFNSQLLEEEAISENIV LDSQKSYSNRFHSSGGNAAYSVMANTLRGIYGS DVLIATGNSFTGNVLKAGYTEKMAGDMIMPNS LSAYSSKMSGAELKETVKNFVEGYEGGFIPFNRG SLPVFSGISVEIKETDDGYTLSNVTMDGKKVQD NDTFTVTCLAIPKHMEAYPTDENIVFDGGDISV DDTWTAYVSDGDAILAEPEDYMTLR SEQIDNO:101 MKRKLRGGFIMKKKKWNRVLAVLLAMVTAITLL Bacterialprotein SGCGGKSAEKEDAETITVYLWSTNLYEKYAPYIQ EQLPDINVEFVVGNNDLDFYRFLKENGGLPDIIT CCRFSLHDASPLKDSLMDLSTTNVAGAVYDTYL SSFMNEDGSVNWLPVCADAHGFVVNKDLFEKY DIPLPTDYESEVSACEAFEEVGIRGFTADYYYDYT CMETLQGLSASELSSVDGRKWRTAYSDPDNTKR EGLDSTVWPKAFERMEQFIQDTGLSQDDLDMN YDDIVEMYQSGKLAMYFGSSAGVKMFQDQGI NTTFLPFFQENGEKWIMTTPYFQVALNRDLTKD ETRRKKAMKVLNTMLSADAQNRIVYDGQDLLS YSQDVDLKLTEYLKDVKPVIEENHMYIRIASNDF FSVSQDVVSKMISGEYDAEQAYQSFNSQLLEEES ASEDIVLDSQKSYSNRFHSSGGNAAYSVMANTL RGIYGTDVLIATGNSFTGNVLKAGYTEKMAGD MIMPNGLSAYSSKMSGAELKETVKNFVEGYEGG FIPENCGSLPVFSGISVEIKKTDDGYTLSKVTKDG KQIQDDDTFTVTCLATPQHMEAYPTDDNIVED GGDTSVKDTWTGYISNGNAVLAEPEDYINVR SEQIDNO:102 MRTISEGGLLMKMKKRSRVLSALFVMAAVILLLA Bacterialprotein GCAGNSAEKEEKEDAETITVYLWSTKLYEKYAPYI QEQLPDINVEFVVGNNDLDFYKFLKENGGLPDII TCCRFSLHDASPLKDSLMDLSTTNVAGAVYDTY LNNFMNKDGSVNWIPVCADAHGVVVNKDLFE TYDIPLPTDYASEVSACQAFDKAGIRGETADYSY DYTCMETLQGLSAAELSSVEGRKWRTAYSDPDN TKKEGLDSTVWPEAFERMDQFIHDTGLSRDDLD MDYDAVMDMEKSGKLAMYEGSSAGVKMFRD QGIDTTFLPFFQQNGEKWLMTTPYFQVALNRD LTKDETRREKAMKVLNTMLSEDAQNRIISDGQD LLSYSQDVDMHLTKYLKDVKPVIEENHMYIRIAS SDFFSVSKDVVSKMISGEYDAGQAYQSFHSQLL NEKSTSEKVVLDSPKSYSNRFHSNGGNAAYSVM ANTLRGIYGTDVLIATGNSFTGNVLKAGYTEKM AGSMIMPNSLSAYSCKMTGAELKETVRNFVEGY EGGLTPFNRGSLPVVSGISVEIKETDDGYTLKEVK KDGKTVQDKDTFTVTCLATPQHMEAYPADEHV GFDAGNSFVKDTWTDYVSDGNAVLAKPEDYM TLR SEQIDNO:103 MITKSGKQVGRVVMKKKKWNKLLAVFLVMATV Bacterialprotein LSLLAGCGGKRAEKEDAETITVYLWSTSLYEAYAP YIQEQLPDINIEFVVGNNDLDFYRFLEKNGGLPD IITCCRFSLHDASPLKDSLMDLSTTNVAGAVYNT YLNNFMNEDGSVNWLPVCADAHGFVVNKDLF ETYDIPLPTDYESFVSACQAFDKAGIRGFTADYFY DYTCMETLQGLSASELSSVDGRKWRTSYSDPGN IIREGLDSTVWPEAFERMERFIRDTGLSRDDLEM NYDDIVELYQSGKLAMYFGTSAGVKMFQDQGI NTTFLPFFQENGEKWLMTTPYFQVALNRDLTQ DETRRTKAMKVLSTMLSEDAQNRIISDGQDLLSY SQDVDIHLTEYLKDVKSVIEENHMYIRIASNDFFS VSKDVVSKMISGEYDAGQAYQSFQTQLLDEKTT SEKVVLNSEKSYSNREHSSGGNEAYSVMANTLR GIYGTDVLIATGNSFTGNVLKAGYTEKMAGDMI MPNGLSAYSCKMNGAELKETVRNFVEGYPGGF LPFNRGSLPVFSGISVELMETEDGYTVRKVTKDG KKVQDNDTFTVTCLATPQHMEAYPADQNMVF AGGETSVKDTWTAYVSDGNAILAEPEDYINVR SEQIDNO:104 MENNFTRESILKKEKMEQLPNINVEFVVGNNDL Bacterialprotein DFYKFLKENGGLPDIITCCRFSLHDASPLKDSLM DLSTTNVAGAVYDTYLNNFMNEDGSVNWLPV CADAHGFVVNKDLFEQ SEQIDNO:105 MKKKKWNKILAVLLAMVTAISLLSGCGSKSAEKE Bacterialprotein DAETITVYLWSTNLYEKYAPYIQEQLPDINVEFVV GNNDLDFYKFLKENGGLPDIITCCRFSLHDASPL KDSLMDLSTTNVAGAVYDTY SEQIDNO:106 RFSLNDAAPLAEHLMDLSTTEVAGTFYSSYLNNN Bacterialprotein QEPDGAIRWLPMCAEVDGTAANVDLFAQHNIP LPTNYAEFVAAIDAFEAVGIKGYQADWRYDYTC LETMQGCAIPELMSLEGTTWRMNYESETEDSST GLDDVVWPKEGL SEQIDNO:107 MKKKAWNKLLAQLVVMVTAISLLSGCGGKSVE Bacterialprotein KEDAETITVYLWSTKLYEKYAPYIQEQLPDINIEFV VGNNDLDFYRFLDENGGLPDIITCCRFSLHDAS PLKDSLMDLSTTNVAGAVYDTYLNSFMNEDGS VNWLPVCADVHGFVVNRDLFEKYDIPLPTDYES FVSACRAFEEVGIR SEQIDNO:108 KDSLMDLSTTNVAGAVYDTYLSNFMNEDGSVN Bacterialprotein WLPVCADAHGFVVNKDLFEKYDIPLPTDYESFV SACQVFDEVGIRGFTADYYYDYTCMETLQGLSA SELSSVDGRKWRTAYSDPDNTKREGLDSTVWP AAFEHMEQFIRDTGLSRDDLDMNYDDIVEMYQ SGKLAMYEGSSSGVKMFQDQGINIIFLPFFQKD GEKWLMTTPYFQVALNSDLAK SEQIDNO:109 MQRKLRGGFVMEKKKWKKVLSVSFVMVTAISLL Bacterialprotein SGCGGKSAEKEDAETITVYLWSTNLNEKYAPYIQ EQLPDINVEFVVGNNDLDFYKFLNENGGLPDIIT CCRFSLHDASPLKDSLMDLSTTNVAGAVYDTYL NNFMNEDGSVNWLPVCADAHGFVVNKDLFEK YDIPLPTDYESFVSACQAFDQVGIRGFTADYYY DYTCMETLQGLSVSDLSSVDGRKWRTTYS SEQIDNO:110 MKKKKWNRVLAVLLMMVMSISLLSGCGGKSTE Bacterialprotein KEDAETITVYLWSTNLYEKYAPYIQEQLPDINVEF VVGNNDLDFYKFLKENGGLPDIITCCRFSLHDAS PLKDSLMDLSTTNVAGAVYDTYLSSFMNEDGSV NWLPVCADAHGFVVNKDLFEKYDIPLPTDYESF VSACEAFEEVGIRGFTADYYYDYTCMETLQGLSA SELSSVDGRKWRTTYSAPDNTKREGLDSTVWPK AFERMEQFIQDTGLSQDDLDMNYDDI SEQIDNO:111 GGELCFANASCLQSTRFFALAMQKQLETLLLQW Bacterialprotein YNKIVFLWENQRKAQCGQAASAGIPMWCVRT ATAALRSAALRYCEEGIYMMKKISRRSFLQACGV AAATAALTACGGGKAESDKSSSQNGKIQITFYL WDRSMMKELTPWLEEKEPEYEFHFIQGENTMDY YRDLLNRAEQLPDIITCRRFSLNDAAPLAEHLMD LSTTEVAGTFYSSYLNNNQEPDGAIRWLPMCAE VDGTAANVDLFAQHNIPLPTNYAEFVAAIDAFE AVGIKGYQADWRYDYTCLETMQGSAIPELMSLE GTTWRMNYESETEDGSTGLDDVVWPKVFEK SEQIDNO:112 MMKKISRRSFLQVCGITAATAALTACGGGKADS Bacterialprotein GKGSQNGRIQITFYLWDRSMMKELTPWLEQKF PEYEENFIQGFNTMDYYRDLLNRAEQLPDIITCR RFSLNDAAPLAEHLMDLSTTEVAGTFYSSYLNNN QEPDGAIRWLPMCAEVDGTAANVDLFAQYNIP LPTNYAEFVAAINAFEAVGIKGYQADWRYDYTC LETMQGSAIPELMSLEGTTWRMNYESETEDGST GLDDVVWPKVFEKYEQFLRDVRVQPGDDRLEL NPIAKPFYARQTAMIRTTAGIADVMPDQYGFNA SILPYFGETANDSWLLTYPMCQAAVSNTVAQDE AKLAAVLKVLGAVYSAEGQSKLASGGAVLSYNK EVNITSSASLEHVEDVISANHLYMRLASTEFFRISE DVGHKMITGEYDARAGYDAFNEQLVTPKADPE AEILFTQNTAYSLDMTDHGSAAASSLMNALRAA YDASVAVGYSPLVSTSIYCGDYSKQQLLWVMA GNYAVSQGEYTGAELRQMMEWLVNVKDNGA NPIRHRNYMPVTSGMEYKVTEYEQGKERLEELTI NGTPLDDTAAYTVEVAGTDVWIENEVYCNCPM PENLKTKRTEYAIEKADSRSCLKDSLAVSKQFPAP SEYLTIVQGE SEQIDNO:113 MMNKISRRSFLQAAGVVAAAAALTACGGKTEA Bacterialprotein DKGSSQNGKIQITFYLWDRSMMKELTPWLEQK FPEYEENFIQGENTMDYYRDLLNRAEQLPDIITC RRFSLNDAAPLAEYLMDLSTTEVAGTFYSSYLNN NQEPDGAIRWLPMCAEVDGTAANVDLFAQYN IPLPTNYAEFVAAIDAFEAVGIKGYQADWRYDY TCLETMQGCAIPELMSLEGTTWRMNYESETEDG STGLDDVVWPKVFEKYEQFLKDVRVQPGDDRL ELNPIAKPFYARQTAMIRTTAGIADVMLDLHGF NASILPYFGETANDSWLLTYPMCQAAVSNTVA QDEAKLAAVLKVLGAVYSAEGQSKLAAGGAVLS YNKEVNITSSTSLEHVADVISANHLYMRLASTEIF RISEDVGHKMITGEYDAKAGYEAFNEQLVTPKA DPETEILFTQNTAYSIDMTDHGSAAASSLMTALR TTYDASIAIGYSPLVSTSIYCGDYSKQQLLWVMA GNYAVSQGEYTGAELRQMMEWLVNVKDNGA NPIRHRNYMPVTSGMEYKVTEYEQGKFRLEELTV NGAPLDDTATYTVEVAGTDVWIENEVYCSCPM PENLKTKRTEYAIEGADSRSCLKDSLAVSKQFPAP SEYLTIVQGE SEQIDNO:114 MMKKISRRSFLQACGIAAATAALTACGGGKAES Bacterialprotein GKGSSQNGKIQITFYLWDRSMMKALTPWLEEKF PEYEFTFIQGFNTMDYYRDLLNRAEQLPDIITCRR FSLNDAAPLAEHLMDLSTTEVAGTFYSSYLNNN QEPDGAIRWLPMCAEVDGTAANVDLFAQHNIP LPTNYAEFVAAIDAFEAVGIKGYQADWRYDYTC LETMQGCAIPELMSLEGTTWRMNYESETEDGST GLDDVVWPKVFKKYEQFLKDVRVQPGDARLEL NPIAEPFYARQTAMIRTTAGIADVMFDLHGENT SILPYFGETANDSWLLTYPMCQAAVSNTVAQDE AKLAAVLKVLESVYSAEGQNKMAVGAAVLSYNK EVNITSSTSLEHVADIISANHLYMRLASTEIFRISED VGHKMITGEYDAKAAYDAFNEQLVTPRVDPEA EVLFTQNTAYSLDMTDHGSAAASSLMNALRATY DASIAVGYSPLVSTSIYCGDYSKQQLLWVMAGN YAVSQGDYTGAELRQMMEWLVNVKDNGANPI RHRNYMPVTSGMEYKVTEYEQGKFRLEELTING APLDDTATYTVEVAGTDVWMEDKAYCNCPMP ENLKAKRTEYAIEGADSRSCLKDSLAVSKQFPAPS EYLTIVQGE SEQIDNO:115 MCHFSLFPVSEIQNLPDFSCKILQDVQNQLETLL Bacterialprotein LQWYNNTVILWENQRKAQCGQAASAGIPVGC VRIATAALRYCACAVLPSDTVRKYICMMKKISRRS FLQVCGITAATAALTACGSGKAEGDKSSSQNGK IQITFYLWDRSMMKALTPWLEEKFPEYEFNFIQG FNTMDYYRDLLNRAEQLPDIITCRRFSLNDAAPL AEHLMDLSTTEVAGTFYSSYLNNNQEPDGAIRW LPMCAEVDGTAANVDLFAQYNIPLPTNYAEFVA AINAFEAVGIKGYQADWRYDYTCLETMQGSAIP ELMSLEGTTWRRNYESETEDGSTGLDDVVWPK VFEKYEQFLKDVRVQPGDDRLELNPIAKPFYAR QTAMIRTTAGIADVMPDQYGFNASILPYFGETA NDSWLLTYPMCQAAVSNTVAQDEAKLAAVLKV LEAVYSAEGQSKMAGGAAVLSYNKEINITSSTSLE QVADIISANHLYMRLASTEIFRISEDVGHKMITGE YDAKAAYDAFNEQLVTPRADPEAEVLFTQNTAY SIDMTDHGSAAASSLMNALRATYDASIAVGYSP LVSTSIYCGEYSKQQILWVMAGNYAVSQGEYTG AELRQMMEWLVNVKDNGANPIRHRNYMPVTS GMEYKVTEYEQGKFRLEELTINGAPLDDTATYTV FVAGTDVWIENEVYCNCPMPENLKAKRTEYAIE GAESRSCLKDSLAVSKQFPAPSEYLTIVQGE SEQIDNO:116 MKLLAVTFVVASNFVSCSKGIAEADKLDLSTTPV Bacterialprotein QTVDDVFAVQTKNGEMGMRMEAVRLERYNK DGTKTDLFPAGVSVFGYNEEGLLESVIVADKAEH TVPSSGDEIWKAYGNVILHNVLKQETMETDTIF WDSSKKEIYTDCYVKMYSRDMFAQGYGMRSD DRMRNAKLNSPENGYVVTVRDTTAVIIDSVNYI GPFPKK SEQIDNO:117 GMTLMHSPPMLYSRAAAKTHRVPFWLLDISFPLS Bacterialprotein MKKALCPKNGQRA SEQIDNO:118 MLKQWFKLTCLLYILWLILSGHFEAKYLILGLLGS Bacterialprotein ALIGYFCLPALTITSSIGKRDFHLLDISFPAFCGYW LWLLKEIIKSSLSVSAAILSPKMKINPVIIEIDYIFNN PAAVTVFVNSIILTPGTVTIDVKDERYFYVHALTD SAALGLMDGERQRRISRVFER SEQIDNO:119 MKHITFSNGDKVCTIGQGTWNMGRNPLCEKSE Bacterialprotein ANALLTGIDLGMNMIDTAEMYGNEKFIGKVIKS CRDKVFLVSKVHPENADYQGTIKACEESLRRLGI EVLDLYLLHWKSRYPLSETVEAMCRLQRDGKIRL WGVSNLDVDDMELIDDIPNGCSCDANQVLYN LQERGVEYDLIPYAQQRDIPVIAYSPVGEGKLLR HPVLRTIAEKHNATPAQIALSWIIRNPGVMAIPK AGSAEHVKENEGSVSITLDTEDIELLDISFPAPQH KIQLAGW SEQIDNO:120 MMKPDEIAKAFLHEMNPTNWNGQGEMPAGF Bacterialprotein DTRTMEFITDMPDVLLDISFELCMEDDGTFQWE HYCELVQESSDTIVDCAHGYGINSVQNLTDTIS QLLEVNVK SEQIDNO:121 MRENLSGIRVVRAFNAEKYQEDKFEGINNRLTN Bacterialprotein QQMFNQRTFNFLSPIMYLVMYFLTLGIYFIGANL INGANMGDKIVLEGNMIVESSYAMQVIMSFLML AMIFMMLPRASVSARRINEVLDTPISVKEGNVTM NNSDIKGCVEFKNVSFKYPDADEYVLLDISFKVN KGETIAFIGSTGSGKSTLINLIPRFYDATSGEILIDGI NVRDYSFEYLNNIIGYV SEQIDNO:122 MILFRHWCWSFLGVVIESLPFIVIGAIISTIIQFYISE Bacterialprotein DIIKRIVPRRRGLAFLVAAFIGLVFPMCECAIVPVA RSLIKKGVPIGITITFMLSVPIVNPFVITSTYYAFEA NLTIVLIRVVGGILCSIIVGMLITYIFKDSTIESIISDG YLDLSCTCCSSNKKYYISKLDKLITIVCQASNEFLN ISVYVILGAFISSIFGSIINEEILNDYTENNILAVIIML DISFLLSLCSEADAFVGSKFLNNFGIPAVSAFMILG PMMDLKNAILTLGLFKRKFATILIITILLVVTAFSICL SFISL SEQIDNO:123 MMTAAQTLKEYWGYDGFRPMQEEIISSALEGRD Bacterialprotein TLAILPTGGGKSICFQVPAMMRDGIALVVTPLIAL MKDQVQNLEARGIRAIAVHAGMNRREVDTAL NNAAYGDYKFLYVSPERLGTSLEKSYLEVLDVNEI VVDEAHCISQWGYDFRPDYLRIGEMRKVLKAPL IALTATATPEVARDIMQKLVRPGTPSQVERNLEN FTLLRSGFERPNLSYIVRECEDKTGQLLNICGSVP GSGIVYMRNRRKCEEVAALLSGSGVSASFYHAG LGALTRTERQEAWKKGEIRVMVCTNAFGMGID KPDVRFVLHLGLPDSPEAYFQEAGRAGRDGQR SWAALLWNKTDIRRLRQLLDISFPSLEYIEDIYQKI HIFNKIPYEGGEGARLKFDLEAFARNYSLSRAAV HYAIRYLEMSDHLTYTEDADISTQVKILVDRQAL YEVSLPDPMMLRLLDALMRAYPGIFSYIVPVDEE RLAHLCGVSVPVLRQLLYNLSLEHVIRYVPCDKA TVIFLHHGRLMPGNLNLRKDKYAFLKESAEKRA GAMEEYVTQTEMCRSRYLLAYFGQTESRDCGC CDVCRSRAARERTEKLILGYASSHPGFTLKEFKA WCDDPGNALPSDVMEIYRDMLDKGKLLYLHP DES SEQIDNO:124 MPKPGSSLEDAREQKFSSAVTEYGDLNPSEGIQV Bacterialprotein MSIDWDGDFKEDDDGGMFFKDGFEYQAMIQF LIDPNGKYDTDYIIKNGEYILDGSRIKVTVNGKP AHVQNSTPYVIYMDIQFLIGSGGKGLDRELASG RAYQSSVNYALCNNLIDEELLGNDYTKSLNQLQ LRSLAVRLAEELVGKEIKVEKKVEGKYNDAITFSTI APGERVWVVGPRLGGMSEYLPVKEPVTGQTLY VKANCFRPVRKYVFKSEKTTLREGEFKNYVDGQ YIWYRWN SEQIDNO:125 MDIFSVFTLCGGLAFFLYGMTVMSKSLEKMAGG Bacterialprotein KLERMLKRMTSSPFKSLLLGAGITIAIQSSSAMTV MLVGLVNSGVMELRQTIGIIMGSNIGTTLTAWIL SLTGIESENVFVNLLKPENFSPLIALAGILLIMGSKR QRRRDVGRIMMGFAILMYGMELMSGAVSPLAE MPQFAGLLTAFENPLLGVLVGAVFTGIIQSSAAS VAILQALAMTGSITYGMAIPIIMGQNIGTCVTALI SSIGVNRNAKRVAVVHISFNVIGTAVCLILFYGG DMILHFITLNQAVGAVGIAFCHTAFNVETTILLL PFSRQLEKLARRLVRTEDTRESFAFLDPLLLRTPGA AVSESVAMAGRMGQAARENICLATDQLSQYSR ERETQILQNEDKLDIYEDRLSSYLVEISQHGLSMQ DMRTVSRLLHAIGDFERIGDHAVNIQESAQELH DKELRFSDSAREELQVLLSALDDILDLTIRSFQAA DVETARRVEPLEETIDQLIEEIRSRHIQRLQAGQC TIQLGFVLSDLLTNIERASDHCSNIAVSVIEECSG GPGRHAYLQEVKAGGAFGEDLRRDRKKYHLPE A SEQIDNO:126 KLDLSTTPV Sequencevariant SEQIDNO:127 FLISTTFGCT IL13RA2epitope SEQIDNO:128 YLYLQWQPPL IL13RA2epitope SEQIDNO:129 GVLLDTNYNL IL13RA2epitope SEQIDNO:130 FQLQNIVKPL IL13RA2epitope SEQIDNO:131 WLPFGFILIL IL13RA2epitope SEQIDNO:132 FLISTTFTIN Sequencevariant SEQIDNO:133 FMISTTFMRL Sequencevariant SEQIDNO:134 QMISTTFGNV Sequencevariant SEQIDNO:135 WLYLQWQPSV Sequencevariant SEQIDNO:136 FVLLDTNYEI Sequencevariant SEQIDNO:137 FILLDTNYEI Sequencevariant SEQIDNO:138 YELQNIVLPI Sequencevariant SEQIDNO:139 FLPFGFILPV Sequencevariant SEQIDNO:140 FMPFGFILPI Sequencevariant SEQIDNO:141 FMLQNIVKNL Sequencevariant SEQIDNO:142 MGGRWMGYILIGIYVLLVLYHLVKDINGDVKW Bacterialprotein AMVYITEGFLFYLCSHCEYLNTYDLSNYNAQYA YYNPMWDKSFTLYYLFLTMMRLGQIAEISFVNW WWITLAGAFLIIIIAVKIHRFNPHHFLVFFMMYYII NLYTGLKFFYGFCIYLLASGELLRGGRKNKLLYVF LTAVAGGMHVMYYAFILFALINTDMPASMEECS LNIYSHIRRHRIIAVLVIASLTLSEVLRLSGSANEFLS RVFSFIDSDKMDDYLSLSTNGGFYIPVIMQLLSLY LAFIIKKQSKRASLLNQQYTDVLYYFNLLQVIFYP LEMISTTFMRLITATSMVTIAAGGYNKFEIKQRKR FKIIGASFLIVAASLFRQLVLGHWWETAVVPLFHL SEQIDNO:143 MEKQKIIEDVDPGVDDCMALILSFYEPSIDVQMI Bacterialprotein STTFGNVSVEQTTKNALFIVQNFADKDYPVYKG AAQGLNSPIHDAEEVHGKNGLGNKIIAHDVTK QIANKPGYGAIEAMRDVILKNPNEIILVAVGPVT NVATLENTYPETIDKLKGLVLMVGSIDGKGSITPY ASFNAYCDPDAIQVVLDKAKKLPIILSTKENGTTC YFEDDQRERFAKCGRLGPLEYDLCDGYVDKIUP GQYALHDTCALFSILKDEEFFTREKVSMKINTTED EKRAQTKFRKCASSNITLLTGVDKQKVIKRIEKILK RT SEQIDNO:144 PGAQGRGSAAGGDDMIWELLVQLAAAFGATV Bacterialprotein GFAVLVNAPPREFVWAGVTGAVGWGCYWLYL QWQPSVAVASLLASLMLALLSRVFSVVRRCPAT VFLISGIFALVPGAGIYYTAYYFIMGDNAMAVAK GVETFKIAVALAVGIVLVLALPGRLFEAFAPCAGK KKGER SEQIDNO:145 MNKALFKYFATVLIITLLFSSSVSMVILSDQMMQT Bacterialprotein TRKDMYYTVKLVENQIDYQKPLEKQIDKLNDLA YTKDTRLTIIDKEGNVLADSDKEGIQENHSGRSE FKEALSDQFGYATRYSSTVKKNMMYVAYYHRG YVVRIAIPYNGIFDNIGPLLEPLFISAALSLCVALAL SYRFSRTLTKPLEEISEEVSKINDNRYLSFDHYQYD EFNVIATKLKEQADTIRKTLKTLKNERLKINSILDK MNEGFILLDTNYEILMVNKKAKQLFSDRMEVNQ PIQDFIFDHQIIDQLENIGVEPKIVTLKKDEEVYD CHLAKVEYGVTLLFVNVTESVNATKMRQEFFSN VSHELKTPMTSIRGYSELLQAGMIDDPKVRKQAL DKIQKEVDHMSQLIGDILMISRLENKDIEVIKHPV HLQPIVDDILESLKVEIEKREITVECDLTSQTYLAN HQHIQQLMNNLINNAVKYNKQKGSLNIHSYLV DQDYIIEVSDTGRGISLIDQGRVFERFFRCDAGR DKETGGTGLGLAIVKHIVQYYKGTIHLESELGKG TTFKVVLPIIKDSL SEQIDNO:146 MIKCTVHKLSPSKTLYLEDSNKKTIASTIKDSLYLY Bacterialprotein KIPTKLAEILEDDDIVYLDIDENYELQNIVLPIKKSS EVKASIYKTEYFEINWLNTKIEDLSSTVDKKEKAIIR VLGIIENKFKILHLWSTINTLWIIVLTIVILNLI SEQIDNO:147 MGILLFAVYVILLIYFLFFSEEYGRVAQAERVYRYN Bacterialprotein LVPFVEIRRFWVYREQLGAFAVFTNIFGNVIGFLP FGFILPVIFRRMNSGFLICISGFVLSLTVEVIQLVTK VGCFDVDDMILNTLGAALGYVLFLICNHIRRKF HYGKKI SEQIDNO:148 MKKETKHIIRTLGTILFILYVLALIYFLFFSEEYGRAA Bacterialprotein LEERQYRYNLIPFVEIRRFWVYRRQLGFMAVAAN LFGNVIGFLPFGFILPVILDRMRSGWLIILAGFGLS VTVEVIQLITKVGCFDVDDMILNTAGAALGYLLF FICDHLRRKIYGKKI SEQIDNO:149 YDDLRGEFLKKETKTLIRRMGILLFVIYIIFLVYFLFF Bacterialprotein SEEYGRAAEAQRVYRYNLIPFVEIRRFWIYREQLG TFAVFSNIFGNVIGFLPFGFILPVIFRRMNSGFLIC VSGFILSLTVEVIQLVTKVGCFDVDDMILNTLGA TLGYVLFFVCNHIVTVHW SEQIDNO:150 RLQKQEKTLKKETKHIIRTLGTILFILYVLALIYFLFF Bacterialprotein SEEYGRAAMEERQYRYNLIPFVEIRRFWVYRKQL GLMAVVTNLFGNVIGFLPFGFILPVILDKMRSG WLIVLAGFGLSVTVEVIQLITKVGCFDVDDMILN TAGAALGYLLFFICDHLRRKIYGKKI SEQIDNO:151 MWFFSQKQEKTLKKETKHIIRTLGTVLFILYVLALI Bacterialprotein YFLFFSEEYGRVAMEEREYRYNLIPFVEIRRFWVYR KQLGFLAVCTNLEGNVIGFLPFGFILPVILERMRS GWLIILAGFGLSVTVEVIQLITKVGCFDVDDMIL NTAGAALGYLLFFICNHLRRKIYGKKI SEQIDNO:152 AFLINTVGNVVCFMPFGFILPIITEFGKRWYNTFL Bacterialprotein LSFLMTFTIETIQLVFKVGSFDVDDMFLNTVGGV AGYILVVICKVIRRAFYDPET SEQIDNO:153 MWKRTKTHQKVCWVLFIGYLLMLTYFMFFSDG Bacterialprotein FSRSEYTEYHYNITLEKEIKREYTYRELLGMKAFLIN TVGNVVCFMPFGFILPIITELGKRWYNTFLLSFLM TFTIETIQLVFKVGSFDVDDMFLNTVGGIAGYILV IICKAMRRVFYDSET SEQIDNO:154 MWKKEKTHQKICWILFESYLLMLTYFMFFSDGF Bacterialprotein GRSEYTEYHYNLTLFKEIRRFYTYRELVGTKAFLLN IVGNVVCFMPFGFILPIITRLGERWLNTLLLSFLLT LSIETIQLVFRVGSFDVDDMFLNTVGGAAGYVS VTMLKWIRRAFHGSKNEKDFIH SEQIDNO:155 MAKHSTRNQRLGWVLFVLYLGALFYLMFFADM Bacterialprotein AERGLGVKENYTYNLKPFVEIRRYLFCASQIGFRG VELNLYGNILGEMPFGFILGVISSRCRKYWYDAVI CTYLLSYSIEMIQLFFRAGSCDVDDIILNTLGGTL GYIAFHIVQHERIRRYFLKHPKKKRPQQ SEQIDNO:156 MENSGAVLRDGCLLIDGENMIKKTRMHQKICW Bacterialprotein VLFISYLVVLTYFMFFSDGFGRSGHEEYAYNLILFK EIKREYKYRELLGMRSELLNTVGNVICFMPFGFILP IISRRGKKWYNTFLLSELMSEGIETIQLIFKVGSFD VDDMFLNTLGGIAGYICVCMAKGVRRMASGAS DR SEQIDNO:157 LCKIVASNFSSRIRFFMLQNIVKNLEKVKWLEDSS Bacterialprotein SRFSRLKM SEQIDNO:158 FMPFGFILGV Sequencevariant SEQIDNO:159 KSVWSKLQSIGIRQH UCP2peptide SEQIDNO:160 VSSVFLLTL Mouseepitope SEQIDNO:161 INMLVGAIM Mouseepitope SEQIDNO:162 KPSVFLLTL Sequencevariant SEQIDNO:163 GAMLVGAVL Sequencevariant SEQIDNO:164 ISQAVHAAHAEINEAGR OVA323-339 peptide