Microbiota-Derived Proteins Inducing Il-10 Release From Human Cells And Uses Thereof

Abstract

The present invention relates to microbiota-derived proteins, which are capable of inducing and/or enhancing secretion of IL-10 from human cells. Accordingly, the present invention provides microbial proteins, and fragments and sequence variants thereof, which stimulate IL-10 release from human immune cells. The present invention also relates to nucleic acids encoding such proteins, cells expressing such proteins, respective pharmaceutical compositions and uses thereof.

Claims

1. An isolated protein of non-pathogenic microbiota, or a fragment or sequence variant thereof, which is capable of inducing and/or enhancing IL-10 secretion from human cells.

2. A protein comprising or consisting of an amino acid sequence sharing at least 80% sequence identity with any one of SEQ ID NOs 1 to 10.

3. The protein according to claim 2, wherein the protein is capable of inducing and/or enhancing IL-10 secretion from human cells.

4. The protein according to claim 2 or 3, wherein the protein is a microbiota protein or a fragment or sequence variant thereof.

5. The protein, or a fragment or sequence variant thereof, according to any one of claims 1 to 4, wherein the protein is a protein of the metasecretome of non-pathogenic microbiota.

6. The protein, or a fragment or sequence variant thereof, according to any one of claims 1, 4 and 5, wherein the microbiota is selected from the group consisting of gastrointestinal tract microbiota, lung microbiota, saliva microbiota, seminal fluid microbiota, skin microbiota and vagina microbiota.

7. The protein, or a fragment or sequence variant thereof, according to any one of claims 1 and 4 to 6, wherein the microbiota is gastrointestinal tract microbiota selected from gut microbiota and oral cavity microbiota.

8. The protein, or a fragment or sequence variant thereof, according to any one of claims 1 to 7, wherein the protein is a bacterial protein, archaea protein, protist protein, fungi protein, virus protein and/or phage protein.

9. The protein, or a fragment or sequence variant thereof, according to any one of claims 1 to 8, wherein the protein is a protein of human non-pathogenic microbiota.

10. The protein, or a fragment or sequence variant thereof, according to any one of claims 1 to 9, wherein the protein is a bacterial protein, preferably of the human gastrointestinal tract microbiota metasecretome.

11. The protein, or a fragment or sequence variant thereof, according to any one of claims 1 to 10, wherein the protein comprises at least two cysteine residues.

12. The protein, or a fragment or sequence variant thereof, according to any one of claims 1 to 11, wherein the protein has a length of no more than 400 amino acids.

13. The protein, or a fragment or sequence variant thereof, according to any one of claims 1 and 3 to 12, wherein the human cells are human immune cells, preferably PBMCs.

14. The protein, or a fragment or sequence variant thereof, according to any one of claims 1 and 3 to 13, wherein the human cells are lymphocytes, preferably T lymphocytes.

15. The protein, or a fragment or sequence variant thereof, according to any one of claims 1 and 3 to 14, wherein IL-10 secretion from human cells stimulated with said protein, or the fragment or sequence variant thereof, is higher than IL-10 secretion from human cells stimulated with an E. coli lysate.

16. The protein, or a fragment or sequence variant thereof, according to any one of claims 1 and 3 to 15, wherein IL-10 secretion from human cells stimulated with said protein, or the fragment or sequence variant thereof, is higher than IL-10 secretion from human cells stimulated with PHA.

17. The protein, or a fragment or sequence variant thereof, according to any one of claims 1 and 3 to 16, wherein IL-10 secretion from human cells stimulated with 0.1 μM of said protein, or the fragment or sequence variant thereof, is higher than IL-10 secretion from human cells stimulated with 10 μg/ml PHA.

18. The protein, or a fragment or sequence variant thereof, according to claim 17, wherein IL-10 secretion from human cells stimulated with 0.025 μM of said protein, or the fragment or sequence variant thereof, is higher than IL-10 secretion from human cells stimulated with 10 μg/ml PHA.

19. The protein, or a fragment or sequence variant thereof, according to any one of claims 1 and 5 to 18, wherein the protein, or the fragment or sequence variant thereof, comprises or consists of an amino acid sequence sharing at least 80% sequence identity with any one of SEQ ID NOs 1 to 10.

20. The protein, or a fragment or sequence variant thereof, according to any one of claims 1 to 19, wherein the protein, or the fragment or sequence variant thereof, comprises or consists of an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 1.

21. The protein according to any one of claims 1 to 20, wherein the protein has an amino acid sequence according to SEQ ID NO: 1.

22. A nucleic acid comprising a polynucleotide encoding the protein, or the fragment or sequence variant thereof, according to any one of claims 1 to 21.

23. The nucleic acid according to claim 22, wherein the nucleic acid is a DNA molecule or an RNA molecule; preferably selected from genomic DNA; cDNA; siRNA; rRNA; mRNA; antisense DNA; antisense RNA; ribozyme; complementary RNA and/or DNA sequences; RNA and/or DNA sequences with or without expression elements, regulatory elements, and/or promoters; a vector; and combinations thereof.

24. The nucleic acid according to claim 22 or 23, wherein the nucleic acid sequence of the polynucleotide encoding the protein, or the fragment or sequence variant thereof, shares at least 80% sequence identity with any one of SEQ ID NOs 11 to 20.

25. The nucleic acid according to any one of claims 22 to 24, wherein the polynucleotide encoding the protein, or the fragment or sequence variant thereof, is codon-optimized for expression by prokaryotic cells, preferably bacteria.

26. An expression cassette comprising a polynucleotide encoding the protein, or the fragment or sequence variant thereof, according to any one of claims 1 to 21 and, operably linked thereto, a regulatory element, preferably for expression in a prokaryotic cell, such as a bacterium.

27. The expression cassette according to claim 26 comprising a regulatory element for heterologous expression and/or overexpression of the encoded protein, or the fragment or sequence variant thereof.

28. A vector comprising the nucleic acid according to any one of claims 21 to 25 or the expression cassette according to claim 26 or 27.

29. A (host) cell expressing the microbiota protein, or the fragment or sequence variant thereof, according to any one of claims 1 to 21; or comprising the nucleic acid according to any one of claims 22 to 25, the expression cassette according to claim 26 or 27, or the vector according to claim 28.

30. The (host) cell according to claim 28, wherein the (host) cell is a bacterium, preferably a genetically engineered bacterium.

31. A genetically engineered bacterium capable of inducing and/or enhancing IL-10 secretion from a human cell, the bacterium comprising the nucleic acid according to any one of claims 22 to 25, the expression cassette according to claim 26 or 27, or the vector according to claim 28.

32. A genetically engineered bacterium (over)expressing the protein, or the fragment or sequence variant thereof, according to any one of claims 1 to 21.

33. A culture medium comprising the protein, or the fragment or sequence variant thereof, according to any one of claims 1 to 21, the (host) cell according to claim 29 or 30, or the bacterium according to claim 31 or 32.

34. The culture medium according to claim 33 further comprising a (self) antigen.

35. An isolated human cell cultured with the culture medium according to claim 33 or 34.

36. The cell according to claim 35, wherein the cell is a human immune cell, preferably a PBMC.

37. The cell according to claim 35 or 36, wherein the cell is a monocyte, a macrophage, a dendritic cell or a lymphocyte, preferably a T lymphocyte.

38. A pharmaceutical composition comprising the protein, or the fragment or sequence variant thereof, according to any one of claims 1 to 21, the nucleic acid according to any one of claims 22 to 25, the vector according to claim 28, the cell according to claim 29 or 30, the bacterium according to claim 31 or 32, or the human cell according to any one of claims 35 to 37 and, optionally, a pharmaceutically acceptable excipient or carrier.

39. The protein, or the fragment or sequence variant thereof, according to any one of claims 1 to 21, the nucleic acid according to any one of claims 22 to 25, the vector according to claim 28, the cell according to claim 29 or 30, the bacterium according to claim 31 or 32, the human cell according to any one of claims 35 to 37, or the pharmaceutical composition according to claim 38 for use in medicine.

40. The protein, or the fragment or sequence variant thereof, the nucleic acid, the vector, the cell, the bacterium, the human cell or the pharmaceutical composition for use according to claim 39 in treating an inflammatory disease or an autoimmune disorder.

41. A method for reducing, treating, alleviating symptoms of or ameliorating an inflammatory disease or an autoimmune disorder in a subject, comprising the step of administering to said subject the protein, or the fragment or sequence variant thereof, according to any one of claims 1 to 21, the nucleic acid according to any one of claims 22 to 25, the vector according to claim 28, the cell according to claim 29 or 30, the bacterium according to claim 31 or 32, the human cell according to any one of claims 35 to 37, or the pharmaceutical composition according to claim 38.

42. A method for inducing and/or enhancing IL-10 secretion in a subject, comprising the step of administering to said subject the protein, or the fragment or sequence variant thereof, according to any one of claims 1 to 21, the nucleic acid according to any one of claims 22 to 25, the vector according to claim 28, the cell according to claim 29 or 30, the bacterium according to claim 31 or 32, the human cell according to any one of claims 35 to 37, or the pharmaceutical composition according to claim 38.

43. A method for inducing tolerance in a subject, comprising the step of administering to said subject the protein, or the fragment or sequence variant thereof, according to any one of claims 1 to 21, the nucleic acid according to any one of claims 22 to 25, the vector according to claim 28, the cell according to claim 29 or 30, the bacterium according to claim 31 or 32, the human cell according to any one of claims 35 to 37, or the pharmaceutical composition according to claim 38.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0184] 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.

[0185] FIG. 1 shows for Example 1 the results of AlphaLISA for IL-10 secretion from human PBMCs by stimulation with the 10 exemplified microbiota proteins in the second round of cell-free synthesis.

[0186] FIG. 2: shows for Example 2 the results of AlphaLISA for IL-10 secretion from human PBMCs by stimulation with the 5 selected microbiota proteins at doses of 0.5, 0.25, 0.1 and 0.025 μM (final concentration).

[0187] FIG. 3: shows exemplified DNA sequences encoding the 10 exemplified proteins inducing IL-10, as identified in Example 1.

[0188] FIG. 4: shows amino acid sequences of the 10 exemplified proteins inducing IL-10, as identified in Example 1.

EXAMPLES

[0189] 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 Human Microbiota-Derived Proteins Inducing IL-10 Release from Human Cells

[0190] The aim of this study was to identify proteins expressed by human microbiota, which are able to induce secretion of IL-10 from human immune cells. To this end, a library of proteins expressed by human microbiota was screened to identify proteins inducing secretion of IL-10 from human immune cells.

Experimental Procedures:

Library: In Silico Method

[0191] A compound library of secreted proteins from gut commensal bacteria was generated by an in silico-based approach. The library included more than 12,000 proteins predicted from human gut microbiome catalogues and from bacterial species known for their role in immune modulation. To obtain the library, bacterial proteins having a length from 50 to 350 amino acids were screened for the presence of secretory signal peptides using the bioinformatic tool Phobius and were annotated using HMMSCAN and the PFAM database. A cut-off at 75% was applied to reduce sequence redundancy.

[0192] In view of the relevance of small cysteine-rich proteins in immune modulation an additional selection criterion was applied to identify cysteine-rich proteins: at least two cysteines were required to be present to form a disulphide bond. To ensure correct synthesis and folding in vitro, the amino acid sequences corresponding to the signal peptide were removed.

Library: Cell Free Proteins Synthesis and Quantification

[0193] The protein library was generated using an Escherichia co/i Cell Free kit suitable for generation of disulphide bonds (RTS 100 E. coli Disulfide Kit; Biotechrabbit, Hennigsdorf, Germany) according to the supplier's protocol. The Cell-Free system is based on the continuous exchange between the reaction compartment, containing components for transcription and translation, and the feeding chamber, containing amino acids and other energy components, through a semipermeable membrane.

[0194] Heterologous protein expression using the transcriptional machinery of E. coli was improved through a codon optimization algorithm (Twist Bioscience, San Francisco, USA) applied to all the selected sequences. All synthetized ORFs were subcloned into pIVEX 2.4 vector (Biotechrabbit, Hennigsdorf, Germany) specifically designed for high-yield Cell-Free expression of His-tagged proteins.

[0195] For the detection of His-tagged proteins, the 6His Check kit Gold using the HTRF® technology (Cisbio, Codolet, France) was used according to the supplier's protocol. Proteins, previously diluted at 1:20 in 1×PBS, were quantified in 384 well plates against a standard curve of 6×His GFP at 0.1 μg/mL (ThermoFisher, Waltham, USA) diluted in serial dilution in the lysate used for the Cell-Free synthesis (lysate was also diluted at 1:20 in 1×PBS).

E. coli Production of Recombinant Proteins

[0196] DNA from positive hits was subcloned in pET-28a vector carrying an N-terminal 6×His-Tag (Twist Bioscience, San Francisco, USA) and then transformed in E. coli BL21(DE3) thermo competent cells (New England Biolabs, Ipswich, Mass., USA). For the expression of recombinant proteins, pre-cultures of BL21(DE3) clones were performed in LB-medium at 30° C. under shaking (180 rpm) conditions. Cultures were made under the same conditions and the induction was started when an OD600 of 0.4-0.8 was reached by using 0.5 mM IPTG. Depending on the properties of each protein, induction was performed for 2 hours to overnight.

[0197] Cultures were centrifuged for 15 min at 4° C., 4500 rpm. Supernatants were removed and the pellets were frozen at −80° C. to break the cells. Pellets were then thawed and resuspended in 1× BugBuster® (Novagen®, Merck KGaA, Darmstadt, Germany) supplemented with Benzonase® Nuclease (Sigma-Aldrich, St. Louis, USA) and Lysozyme (Sigma-Aldrich, St. Louis, USA). Samples were incubated at room temperature by gentle shaking and centrifuged at 4° C., 15,000 g for 30 minutes. Soluble proteins were purified from supernatants onto Nickel packed columns (Protino®, Macherey-Nagel, Duren, Germany) according to the supplier's protocol. Proteins produced in inclusion bodies were solubilized from pellets using 8M urea. Imidazole (250 mM), used for proteins elution, was removed by buffer exchange using 3 kDa cutoff filters (Amicon®, Merck Millipore Ltd., Burlington, USA). Proteins were visualized on 12% Bis-Tris acrylamide gels (ThermoFisher, Waltham, USA) stained with Coomassie Blue (Imperial protein Stain; ThermoFisher, Waltham, USA) and detected by Western Blot using the 6×-His Tag monoclonal antibody HIS.H8 (ThermoFisher, Waltham, USA) diluted at 1:3000 and revealed using the secondary antibody Goat anti-Mouse IgG H+L WesternDot625 (ThermoFisher, Waltham, USA). Purified proteins were quantified by Bradford protein assay (Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254).

IL10 Screening

[0198] IL-10 screening of the microbiota protein library was performed on CD14 depleted human peripheral blood mononuclear cells (PBMCs). This cellular model was chosen to reduce the background due to cell wall components and other bacterial contaminants possibly present in the lysate of the Cell Free synthesis kit on the synthesised (but not purified) proteins.

CD14-Depleted PBMCs:

[0199] PBMCs were isolated from buffy coats as follows: 80 ml PBS were added to 50 ml blood; 4 SepMate™-50 IVD tubes (Stemcell Technologies, Vancouver, Canada) were filled with 15 mL of Ficoll® (Ficoll® Paque Plus; Sigma-Aldrich, St. Louis, USA) per donor, then 30 ml of PBS-diluted blood were gently added. Samples were centrifuged for 20 min at 1200 g at room temperature and washed three times with PBS. To lyse the red blood cells, pellets were resuspended in Red Blood Cells Lysis buffer 1× (Miltenyi Biotec, Bergisch Gladbach, Germany) and incubated for 10 min at room temperature. Cells were then washed with MACS buffer and counted.

[0200] PBMC depletion was performed using the CD14 Microbeads kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the supplier's protocol. Depleted monocytes were resuspended in Iscove's Modified Dulbecco's Medium (IMDM; GIBCO™, Life Technologies, Carlsbad, USA) supplemented with 1% L-Glutamine (Sigma-Aldrich, St. Louis, USA), 1% Penicillin-Streptomycin (Sigma-Aldrich, St. Louis, USA) and 10% of heat-inactivated FBS (Sigma-Aldrich, St. Louis, USA).

IL-10 Screening:

[0201] Screening was performed in 384 well plates in a final volume of 60 μl. PBMCs were seeded at 72,000 cells/well by multidrop (Hamilton Robotics, Martinsried, Germany) and stimulated for 72 hours with 10% (vol/vol) of the library (proteins) previously diluted at 1:10 with PBS in a humidified 5% CO.sub.2 atmosphere at 37° C. The E. coli lysate included in the cell free kit was used (at the same dilution as the library) as negative control. Phytohaemagglutinin (PHA) at 10 μg/ml (0.087 μM) was used as positive control. To ensure technical robustness, the screening was performed on CD14 depleted PBMCs from at least two different donors.

[0202] IL-10 secretion was measured by AlphaLISA® (IL10 (human) AlphaLISA Detection Kit; PerkinElmer, Waltham Mass., USA) on the undiluted supernatants according to the supplier's protocol. Results were expressed as AlphaLISA signal (Counts). Results were considered as positive hits, either when at least a single raw data signal (of the two signals of the two PBMC donors) was higher than the corresponding plate mean+3 SD (Standard Deviation) or when both raw data signals (of the two signals of the two PBMC donors) were higher than the corresponding plate means+2SD (Standard Deviation). In order to avoid false positives, the concentration of the potential hits obtained by the primary screening was compared to that of the corresponding plate mean.

[0203] Potential hits were then validated by a new round of Cell-Free synthesis and test on CD14 depleted PBMCs from several donors (as described above). Further characterization was performed on recombinant proteins produced in the E. coli BL21 (DE3) strain transformed with a pET-28a vector containing the target sequence (as described above, see paragraph “E. coli production of recombinant proteins”). Alternatively, a cell-free production of some proteins was performed by a commercial supplier (Synthelis, La Tronche, France).

Results

Screening

[0204] A total of 11904 proteins of the library was screened on CD14 depleted PBMCs in order to identify proteins able to stimulate IL-10 secretion from human PBMCs. From various potential hits obtained in the primary screening, so far ten proteins were confirmed in the second round of cell-free synthesis. These microbiota proteins, which are able to stimulate IL-10 secretion from human PBMCs, include ID3166 (SEQ ID NO: 1); ID6359 (SEQ ID NO: 2); ID1888 (SEQ ID NO: 3); ID1889 (SEQ ID NO: 4); ID2661 (SEQ ID NO: 5); ID5682 (SEQ ID NO: 6); ID5138 (SEQ ID NO: 7); ID6077 (SEQ ID NO: 8); ID6274 (SEQ ID NO: 9); and ID6298 (SEQ ID NO: 10).

[0205] Results of the AlphaLISA for IL-10 secretion from human PBMCs of the second round of cell-free synthesis are shown in FIG. 1. The data show that ten IL-10 secretagogue proteins were identified by screening of a human microbiome metasecretome protein library on stimulation of IL-10 release from human PBMCs. These microbiota proteins induce IL-10 release from human immune cells.

Example 2: Dose-Response of Selected IL-10 Inducing Microbiota Proteins

[0206] Microbiota proteins ID3166 (SEQ ID NO: 1); ID2661 (SEQ ID NO: 5); ID5682 (SEQ ID NO: 6); and ID5138 (SEQ ID NO: 7) were selected. These proteins contain 121 (ID3166), 144 (ID2661), 58 (ID5682) and 44 (ID5138) amino acids, respectively, corresponding to a size of 14, 15.5, 6 and 4 kDa respectively.

[0207] Before testing, the proteins were purified. In view thereof, CD14 depletion of PBMCs was not required, since the background induced by the residual contaminants present in the purified proteins is very low. Therefore, the cellular assays of Example 2 to characterize the purified proteins were performed on total PBMCs.

[0208] The selected proteins, were tested at concentrations of 0.5, 0.25, 0.1 and 0.025 μM (final concentration) on PBMCs. Cells were seeded in 384 well plates at 72,000 cells/well in a final volume of 60 μl and stimulated with either the purified proteins (10% vol/vol) or the relative controls (positive and negative) for 24 hours in a humidified 5% CO.sub.2 atmosphere at 37° C. Samples were tested in duplicate on at least two different PBMCs donors. All the dilutions were made in PBS.

[0209] Secretion of IL-10 was measured by AlphaLISA and compared to those of PBS (negative control) and PHA at 10 μg/ml (positive control), essentially as described above.

[0210] Results of the AlphaLISA for IL-10 secretion from human PBMCs at different doses of the selected microbiota proteins are shown in FIG. 2. The data show that all the tested proteins are as effective (ID5138) as PHA (positive control) or even more effective (ID 3166; ID2661; and ID5682) than PHA in inducing IL-10 secretion from PBMCs. Moreover, microbiota proteins ID3166 and ID5682 show higher effectivity than PHA even at the lowest doses tested.

TABLE-US-00004 TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING): SEQ ID NO Sequence Remarks SEQ ID NO: 1 AFLFTSTGVPKKAAEAAFFLYLNKGTKKGSRSCFFIY ID3166 LDRGTKKGSRSCFFYLPRQGYQKGSRGCFFIYLDR GTKKGSRGCFFIYLDCEKRAGNVCIRKCRGRYLHK KTPRRYRNAEATCS SEQ ID NO: 2 QTRKQREDAKREAWKKERKEKKALEAQQDSVSF ID6359 MKDTESCCASKAFFSLRSFFHASRLASSLCFLVCAFV TPLKQTSNNAAKNNTFFIIKAVLLINISFR SEQ ID NO: 3 ARNYTCDVCGNGTIQIVSSHIIHNVHCGFIPCNKI D1888 NGVMDEVVYKTVTENNEACNNCGVSYTYKVYG DMEIICKAKAN SEQ ID NO: 4 AEPADTAISERRVELCGNCGGRMVTSTTWGSWYT ID1889 VAQIKCTHHNYGTDLRQQRDGTATTKCQGCGQ GYTTSKSQTRIVCHGYDS SEQ ID NO: 5 AAFVFSNSLKPANASSAESSRLLIHVNSFFSQLGLKP ID2661 ISENLLRKTAHFCEFGMLGILASSACAMFSGAYSAA SLPSLRRRGFFISFGVSVACAVCDETIQYFVPGRAC RVTDMLIDSAGALCGLAAVLAFCAAIRVRRRRRRN SEQ ID NO: 6 LAGPGSGCRFTPSCSTYFIQAVEIHGALKGSLMGI ID5682 WRILRCNPWGGCGYDPVPPRKPR SEQ ID NO: 7 AKLGMAAGAMLVLGLLAAGASGGTLILAALALCA ID5138 VTLLCGRKKQ SEQ ID NO: 8 VEKKTVITKCAITVNEYREKVVPSMRKIHAIVIFVSYS ID6077 INHLYKNCEPEQLFSPGRKTKKPPPATCRKRLNLQ YF SEQ ID NO: 9 EITQPCNHVKSDWIIDKEATCIGSYAFYNCTSLTSIE ID6274 ISTSVTKIKYRAFASCRALNNIYYTGTLTQWNEISK DTNWNWAAPLNCKVICLNGTCYL SEQ ID NO: 10 LLVSVCTAAGLLAVAMRQIEPLLAWLRTLEVYFQG ID6298 QSPAVLLRALGIALVAQFAADTCREAGLCAASTAIE LCGRVLVLLQALPLLRSLLGSFADYLQ SEQ ID NO: 11 GCTTTTTTATTTACCTCGACAGGGGTACCAAAA ID3166 AAGGCAGCAGAGGCTGCTTTTTTTCTTTACCTC AACAAGGGTACCAAAAAAGGCAGCAGAAGCT GCTTTTTTATTTACCTCGACAGGGGTACCAAAA AAGGCAGCAGAAGCTGCTTTTTTTATTTACCTC GACAGGGGTACCAAAAAGGCAGCAGAGGCTG CTTTTTTATTTACCTCGACAGGGGTACCAAAAA AGGTAGCAGAGGCTGCTTTTTTATTTACCTCGA TTGCGAAAAGCGCGCAGGCAATGTCTGCATAC GCAAATGCCGCGGGCGGTATTTGCACAAAAAA ACGCCGCGGCGGTATCGAAACGCCGAGGCGA CGTGCTCCTAA SEQ ID NO: 12 CAAACCAGAAAACAAAGAGAAGATGCCAAAC GCGAAGCATGGAAAAAAGAACGTAAGGAAAA GAAGGCCTTGGAAGCACAGCAAGATTCCGTGT CTTTCATGAAAGACACGGAATCTTGCTGTGCTT CCAAGGCCTTCTTTTCCTTACGTTCTTTTTTCCAT GCTTCGCGTTTGGCATCTTCTCTTTGTTTTCTGG TTTGTGCATTTGTTACTCCGCTGAAGCAAACAA GTAACAATGCTGCTAAGAATAATACTTTTTTCAT AATTAAAGCTGTTTTGTTAATCAATATATCTTTTA GATAA SEQ ID NO: 13 GCAAGGAACTATACATGTGATGTATGTGGAAA D1888 TGGAACAATACAAATCGTTTCTTCTCATATAATC CATAATGTACACTGTGGATTCATTCCATGTAAT AAAATTAACGGAGTTATGGACGAAGTTGTTTAT AAAACTGTTACAGAAAATAACGAAGCTTGTAAC AACTGTGGTGTTTCTTATACTTATAAAGTATATG GGGACATGGAAATAATATGTAAAGCTAAAGCA AATTAG SEQ ID NO: 14 GCCGAACCGGCGGACACCGCGATTTCTGAAC ID1889 GGCGCGTGGAACTTTGCGGCAACTGCGGCGG ACGAATGGTAACTTCCACTACATGGGGAAGCT GGTACACAGTTGCTCAAATCAAATGCACACAC CATAATTATGGTACCGATCTCCGTCAGCAGAG AGACGGTACGGCAACAACAAAGTGCCAGGGC TGCGGACAGGGTTACACCACTTCCAAATCTCA GACAAGAATTGTTTGCCACGGTTACGATTCCTA A SEQ ID NO: 15 GCAGCATTTGTTTTTTCCAACTCGCTCAAACCC ID2661 GCTAATGCCTCAAGCGCGGAAAGCAGCCGGC TCCTCATCCATGTGAACTCATTTTTTTCACAGCT TGGGCTTAAACCGATAAGCGAAAACCTGCTTC GCAAAACGGCGCATTTCTGCGAGTTCGGTATG CTCGGAATACTTGCATCCTCCGCCTGCGCAAT GTTTTCCGGAGCTTATTCTGCCGCCTCCCTGCC CTCTCTGCGCCGCCGTGGCTTTTTCATCTCGTTT GGCGTATCTGTCGCGTGCGCCGTATGTGACGA AACGATCCAGTATTTTGTTCCCGGCAGAGCCT GCCGCGTGACCGATATGCTCATCGACTCCGCC GGAGCACTCTGCGGGCTTGCTGCCGTACTTGC GTTCTGCGCGGCAATTCGCGTCCGGCGCCGG CGCCGCCGGAATTGA SEQ ID NO: 16 CTGGCCGGGCCGGGTTCCGGCTGTCGGTTCA ID5682 CCCCCAGCTGTTCGACCTACTTCATTCAGGCCG TGGAGATTCACGGCGCGCTCAAGGGCTCGCT GATGGGCATCTGGCGCATTCTGCGCTGCAATC CTTGGGGCGGCTGCGGCTATGATCCCGTGCC GCCGCGAAAGCCCCGATGA SEQ ID NO: 17 CCAAAGCTCGGCATGGCGGCGGGCGCAATGC ID5138 TGGTGCTCGGGCTGCTGGCGGCGGGTGCCTC CGGCGGCACGCTCATCCTTGCGGCGCTGGCA CTGTGTGCCGTGACCCTGTTGTGCGGGAGGA AAAAGCAATGA SEQ ID NO: 18 GTGGAAAAGAAAACAGTGATTACGAAATGTGC ID6077 AATCACAGTAAACGAATACCGCGAAAAAGTTG TCCCGTCAATGCGCAAAATTCACGCCATTGTCA TATTTGTGAGCTACTCAATAAATCATTTGTACAA AAATTGTGAGCCAGAACAGCTATTTTCACCGG GCAGAAAAACAAAAAAACCGCCCCCGGCGAC GTGCCGAAAGCGGTTAAATTTACAATATTTTTA A SEQ ID NO: 19 GAAATAACACAACCATGTAATCATGTTAAAAGT ID6274 GATTGGATAATAGATAAAGAAGCAACATGTAT AGGAAGTTATGCCTTTTACAATTGTACTAGTTTA ACAAGTATAGAAATTTCAACAAGTGTTACAAAA ATAAAATATCGTGCATTTGCTAGTTGTCGAGCT TTAAATAATATATACTACACAGGAACACTAACT CAATGGAATGAAATAAGTAAGGATACTAATTG GAATTGGGCTGCACCATTAAATTGTAAAGTTAT TTGTTTAAATGGTACATGTTACTTATAA SEQ ID NO: 20 CTTTTGGTCTCGGTATGCACCGCCGCCGGGCT ID6298 GCTTGCTGTGGCGATGCGCCAGATCGAGCCG CTGCTCGCTTGGCTGCGCACACTGGAGGTCTA CTTTCAGGGGCAGAGCCCGGCGGTGCTGCTG CGCGCTCTCGGCATTGCGCTGGTCGCGCAGTT TGCCGCCGACACCTGCCGCGAGGCCGGTCTTT GCGCGGCCTCCACCGCCATCGAGCTCTGCGG CCGTGTGCTGGTGCTTTTGCAGGCTCTGCCGC TGCTGCGGTCGCTGCTCGGCTCCTTTGCGGAT TATTTGCAGTAA