(19)
(11)EP 2 376 525 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
03.05.2017 Bulletin 2017/18

(21)Application number: 09795419.2

(22)Date of filing:  17.12.2009
(51)Int. Cl.: 
C07K 14/025  (2006.01)
(86)International application number:
PCT/EP2009/067422
(87)International publication number:
WO 2010/070052 (24.06.2010 Gazette  2010/25)

(54)

IMMUNOGENIC POLYPEPTIDES COMPRISING A SCAFFOLD POLYPEPTIDE AND A L2 POLYPEPTIDE OR A FRAGMENT THEREOF

EIN GERÜST-POLYPEPTID UND EIN L2-POLYPEPTID ODER EIN FRAGMENT DAVON UMFASSENDE IMMUNOGENE POLYPEPTIDE

POLYPEPTIDES IMMUNOGÉNÉTIQUES COMPORTANT UN POLYPEPTIDE RECOMBINANT ET POLYPEPTIDE L2 OU SON FRAGMENT


(84)Designated Contracting States:
AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR

(30)Priority: 19.12.2008 EP 08172349

(43)Date of publication of application:
19.10.2011 Bulletin 2011/42

(73)Proprietor: Deutsches Krebsforschungszentrum
69120 Heidelberg (DE)

(72)Inventors:
  • MUELLER, Martin
    69151 Neckargemuend (DE)
  • RUBIO, Ivonne
    69117 Heidelberg (DE)
  • TOMMASINO, Massimo
    I-69390 Charly-Lyon (IT)
  • OTTONELLO, Simone
    I-43100 Parma (IT)
  • BOLCHI, Angelo
    I-43037 Lesignano de Bagni Parma (IT)

(74)Representative: Herzog, Fiesser & Partner Patentanwälte PartG mbB 
Dudenstrasse 46
68167 Mannheim
68167 Mannheim (DE)


(56)References cited: : 
WO-A-02/070004
WO-A-2009/001867
WO-A-2008/140474
  
  • GAMBHIRA RATISH ET AL: "A protective and broadly cross-neutralizing epitope of human papillomavirus L2" JOURNAL OF VIROLOGY, vol. 81, no. 24, December 2007 (2007-12), pages 13927-13931, XP002519253 ISSN: 0022-538X
  • KONDO ET AL: "Neutralization of HPV16, 18, 31, and 58 pseudovirions with antisera induced by immunizing rabbits with synthetic peptides representing segments of the HPV16 minor capsid protein L2 surface region" VIROLOGY, ACADEMIC PRESS,ORLANDO, US, vol. 358, no. 2, 25 January 2007 (2007-01-25), pages 266-272, XP005853978 ISSN: 0042-6822
  • DE JONG A ET AL: "Enhancement of human papillomavirus (HPV) type 16 E6 and E7-specific T-cell immunity in healthy volunteers through vaccination with TA-CIN, an HPV16 L2E7E6 fusion protein vaccine" VACCINE, BUTTERWORTH SCIENTIFIC. GUILDFORD, GB, vol. 20, no. 29-30, 4 October 2002 (2002-10-04), pages 3456-3464, XP004381809 ISSN: 0264-410X
  • SKERRA ET AL: "Alternative non-antibody scaffolds for molecular recognition" CURRENT OPINION IN BIOTECHNOLOGY, LONDON, GB, vol. 18, no. 4, 14 September 2007 (2007-09-14), pages 295-304, XP022244962 ISSN: 0958-1669
  • MAMBETISAEVA E T ET AL: "Expression of Three Functional Domains of Connexin 32 as Thioredoxin Fusion Proteins inEscherichia coliand Generation of Antibodies" PROTEIN EXPRESSION AND PURIFICATION, ACADEMIC PRESS, SAN DIEGO, CA, vol. 11, no. 1, 1 October 1997 (1997-10-01), pages 26-34, XP004451747 ISSN: 1046-5928
  • MORETTO NADIA ET AL: "Conformation-sensitive antibodies against Alzheimer amyloid-beta by immunization with a thioredoxin-constrained B-cell epitope peptide" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,; US, vol. 282, no. 15, 1 April 2007 (2007-04-01), pages 11436-11445, XP002451806 ISSN: 0021-9258
  • WOODMAN ET AL: "Design and Validation of a Neutral Protein Scaffold for the Presentation of Peptide Aptamers" JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 352, no. 5, 7 October 2005 (2005-10-07), pages 1118-1133, XP005074554 ISSN: 0022-2836
  • RUBIO ET AL.: "Potent anti-HPV immune responses induced by tandem repeats of the HPV16 L2 (20-38) peptide displayed on bacterial thioredoxin" VACCINE, vol. 27, 31 January 2009 (2009-01-31), pages 1949-1956, XP002519254
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description


[0001] The present disclosure relates to an immunogenic polypeptide comprising a) a scaffold polypeptide, and b) a L2 polypeptide or a fragment of said L2 polypeptide, wherein said scaffold polypeptide constrains the structure of said L2 polypeptide, or of the fragment of said L2 polypeptide. Moreover, the present disclosure relates to a vaccine comprising said immunogenic polypeptide. The present disclosure is also concerned with a method for producing an antibody against human papillomavirus. Also encompassed by the present disclosure is an antibody obtained by carrying out the said method.

[0002] Cervical cancer is women's second most frequent cancer worldwide. Clinical and molecular studies have shown that certain types of human papillomavirus (HPV), referred to as high-risk types, are the etiological agents of this disease. Two anti-HPV vaccines for the prophylaxis of cervical cancer have been licensed recently by Merck (Gardasil) and GlaxoSmithKline (Cervarix) (Schmiedeskamp et al, (2006) Human papillomavirus vaccines. Ann Pharmacother, 40, 1344-1352). Both vaccines rely on the major capsid protein L1 in the form of virus-like particles (VLPs) as antigen (Roden et al., (2006) How will HPV vaccines affect cervical cancer? Nat Rev Cancer, 6, 753-763); they protect against the HPV types from which the L1-VLPs were derived, yet are largely ineffective against all but the most closely related HPV types. The two most prominent high-risk HPV types, 16 and 18, are the major targets of both vaccines, although there is evidence for partial cross-protection against HPV types 31 and 45 (reviewed by Muller and Gissmann, (2007) A long way: history of the prophylactic papillomavirus vaccine. Dis Markers, 23, 331-336; Huh and Roden, (2008) The future of vaccines for cervical cancer. Gynecol Oncol, 109, S48-56). The limited cross-protective capacity of L1-based vaccines, which is the main reason for the continuing effort toward the development of improved vaccination strategies, likely reflects the HPV type specificity of L1 neutralizing epitopes (Giroglou et al., (2001) Immunological analyses of human papillomavirus capsids. Vaccine, 19, 1783-1793).

[0003] Antibodies against the minor capsid protein L2 also neutralize HPV infection and are often capable to cross-neutralize various non-cognate virions, although with varying efficiencies (Kondo et al. 2007, Neutralization of HPV16, 18, 31, and 58 pseudovirions with antisera induced by immunizing rabbits with synthetic peptides representing segments of the HPV16 minor capsid protein L2 surface region. Virology, 358, 266-272; Gambhira, R., (2007) A protective and broadly cross-neutralizing epitope of human papillomavirus L2. J Virol, 81, 13927-13931). The N-terminal region of L2 interacts with an as yet unidentified secondary receptor on the surface of target cells (Yang et al. (2003) Cell surface-binding motifs of L2 that facilitate papillomavirus infection. J Virol, 77, 3531-3541) and this interaction can be blocked by anti-L2 antibodies. The precise identity of the L2 region involved in HPV-cell surface interaction is still a matter of debate. This was initially proposed as the region comprised of amino acids (aa) 108-120, and antibodies targeting this particular L2 region were indeed shown to block viral infection in vitro albeit at low titers (Kawana et al. (2001) Nasal immunization of mice with peptide having a cross-neutralization epitope on minor capsid protein L2 of human papillomavirus type 16 elicit systemic and mucosal antibodies. Vaccine, 19, 1496-1502; Kawana et al. (2001b) Human papillomavirus type 16 minor capsid protein L2 N-terminal region containing a common neutralization epitope binds to the cell surface and enters the cytoplasm. J Virol, 75, 2331-2336). Subsequent experiments identified additional neutralizing epitopes in the aa 1-88 region (Pastrana et al. (2005) Cross-neutralization of cutaneous and mucosal Papillomavirus types with anti-sera to the amino terminus of L2. Virology, 337, 365-372) as well as in more extended N-terminal regions comprised of aa 11-200 and aa 18-144 (Kondo loc. cit). Perhaps the most prominent of these N-terminal epitopes is the one located between aa 17-36. This was identified as the target of an HPV16 neutralizing and protective monoclonal antibody (RG-1) as well as the major determinant of the neutralizing activity found in sera from rabbits and humans immunized with extended versions of L2 (aa 1-88, 11-200 or the full-length protein) (Gambhira, 2007, loc cit. ). Since it had been found that mutation of L2 amino acids 18 and 19 or of amino acids 20 and 21 disrupted both L2 binding to the cell surface and viral infection (Yang, R., et al. (2003). Cell surface-binding motifs of L2 that facilitate papillomavirus infection. J. Virol. 77:3531-3541), it was concluded that the epitope recognized by the RG-1 antibody overlaps the surface-binding motif of HPV16 L2.

[0004] Besides the lack of precise knowledge on the most relevant (cross) neutralizing epitope(s), a major problem with the use of L2 as a tool for HPV prophylaxis is the poor immunogenicity of the L2 protein and peptides thereof, as compared to L1-VLPs. A substantial increase in immunogenicity has been reported lately via chemical coupling of the HPV16 L2 peptide (17-36) to a broadly recognized T helper epitope and to the Toll-like receptor ligand dipalmitoyl S-glyceryl cysteine (Alphs et al. (2008) Protection against heterologous human papillomavirus challenge by a synthetic lipopeptide vaccine containing a broadly cross-neutralizing epitope of L2. Proc Natl Acad Sci USA, 105, 5850-5855). Alternatively, L2 peptides have been fused to Adenovirus surface proteins (WO 2008/140474) or to other HPV proteins to increase immunogenicity (WO 2002/070004, de Jong et al. (2002), Enhancement of human papillomavirus (HPV) type 16 E6 and E7-specific T-cell immunity in healthy volunteers through vaccination with TA-CIN, an HPV16 L2E7E6 fusion protein vaccine, Vaccine, 20(29-30):3456-3464).

[0005] A recently developed alternative strategy for increasing peptide immunogenicity relies on the use of thioredoxin (Trx) as a scaffold protein with the ability to constrain the structure of single-copy as well as multimeric (tandemly repeated) peptide epitopes inserted within its surface-exposed active site loop (Moretto et al. (2007) Conformation-sensitive antibodies against Alzheimer amyloid-beta by immunization with a thioredoxin-constrained B-cell epitope peptide. J Biol Chem, 282, 11436-11445).

[0006] Also, the authors of Mambetisaeva et al. (1997), Protein Expression and Purification 11(1):26, used expression of connexin 32 domains as thioredoxin fusion proteins for immunization. Moreover, WO2008/140474 used recombinant adenovirus constructs comprising a HPV L2 peptide as an immunogen in mice. WO 02/070004 and De Jong (2002), Vaccine 20(29-30):3456 disclosed a vaccine comprising HPV L2, E6, and E7 as a single fusion protein inducing an antibody response in man. Furthermore, the idea of using scaffolds in immunization was summarized in Skerra et al. (2007), Curr Opinion Biotechnol 18(4):295.

[0007] Thus, the L1 polypeptide is highly immunogenic and antibodies against it show only a limited cross-protective capacity, whereas antibodies against the L2 polypeptide are capable of cross-neutralizing various HPV genotypes. The L2 polypeptide, however has only limited immunogenicity.

[0008] Therefore, immunogenic polypeptides that are highly immunogenic and allow for a cross-neutralization of various HPV genotypes without the drawbacks as referred to above are highly required.

[0009] The technical problem underlying the present invention can be seen as the provision of means and methods for complying with the aforementioned needs.

[0010] The technical problem is solved by the embodiments characterized in the claims and herein below.

[0011] Accordingly, the present disclosure relates to an immunogenic polypeptide comprising
  1. a) a scaffold polypeptide, and
  2. b) a L2 polypeptide having an amino acid sequence as shown in SEQ ID NO:1, or a fragment of said L2 polypeptide,
wherein said scaffold polypeptide constrains the structure of said L2 polypeptide, or of the fragment of said L2 polypeptide.

[0012] The term "polypeptide" as used herein relates to a polymer comprising amino acids linked together by peptide bonds. The term "immunogenic polypeptide" is understood by the skilled person. Immunogenic polypeptides, preferably, elicit protective immune response in a host, preferably, in a human. The immunogenic polypeptide in the context of the present invention, preferably, shall allow for establishing or improving immunity to infection with various HPV genotypes. Preferably, the immunogenic polypeptide according to the present invention allows for establishing or improving immunity to infection with human papillomavirus genotypes 16, 18, 31, 45 and 58. Preferably, the said polypeptide also allows for establishing or improving immunity to infection with human papillomavirus genotypes 6, 52, 2, 27, 57 and/or 11. Immunogenic polypeptides are preferred reagents for vaccine compositions.

[0013] The term "L2 polypeptide", preferably, refers to the N-terminal region of the full-length L2 polypeptide of HPV16 (human papillomavirus 16). The full-length L2 is one of the two capsid proteins of HPV16 and is frequently also referred to as minor capsid protein. Together with the major capsid protein, L1, the full-length L2 polypeptide forms viral capsids. The L2 polypeptide in the context of the present invention, preferably, comprises the N-terminal amino acids 1 to 120 of the HPV16 L2 polypeptide as shown in SEQ ID NO:1.

[0014] The term "fragment" as used herein, preferably, refers to a sub-polypeptide of the L2 polypeptide (as shown in SEQ ID NO:1). Said fragment comprises at least 7, preferably, at least 10, at least 12, at least 15, or at least 20 consecutive amino acid residues of said L2 polypeptide. Preferred fragments of the L2 polypeptide have an amino acid sequence as shown in SEQ ID NO:2 (KTCKQAGTCPPDIIPKVEG), as shown in SEQ ID NO:3 (KTCKQAGTCPPD), as shown in SEQ ID NO:4 (TCKQAGTCPPD), as shown in SEQ ID NO:5 (CKQAGTCPPD), as shown in SEQ ID NO:6 (TCKQAGTCPP), as shown in SEQ ID NO:7 (CKQAGTCPP), as shown in SEQ ID NO:8 (DIIPKVEGKT), as shown in SEQ ID NO:9 (TGYIPLGTR).

[0015] The most preferred fragments in the context of the present invention are fragments having a sequence as shown in SEQ ID NO:2 (KTCKQAGTCPPDIIPKVEG, amino acids 20 to 38 of the L2 polypeptide as shown in SEQ ID NO:1)), or as shown in SEQ ID NO:5 (CKQAGTCPPD, amino acids 22 to 31 of the L2 polypeptide as shown in SEQ ID NO:1).

[0016] Preferably, the terms "polypeptide" "L2 polypeptide" and "fragment of the L2 polypeptide", respectively, shall also encompass variants of said polypeptide, L2 polypeptide or variants of said fragment of said L2 polypeptide, respectively. Such variants have essentially the same immunological properties as the specific polypeptides, respectively. In particular, they share the same immunological properties if they are detectable by the same specific assays referred to in this specification, e.g., by ELISA assays using polyclonal or monoclonal antibodies specifically recognizing the said polypeptides, respectively. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and/or addition wherein the amino acid sequence of the variant is still at least 65%, preferably at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%, identical with the amino sequence of the specific polypeptide. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art. Preferably, the degree of identity is to be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad Sci. (USA) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by visual inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment and, thus, the degree of identity. Preferably, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. Variants referred to above may be allelic variants or any other species specific homologs, paralogs, or orthologs. Further included are variants which differ due to posttranslational modifications such as phosphorylation or myristoylation.

[0017] As mentioned above, in a preferred embodiment of the present invention the fragment of the L2 polypeptide comprised by the scaffold polypeptide of the immunogenic polypeptide has a sequence as shown in SEQ ID NO:2 (KTCKQAGTCPPDIIPKVEG), or as shown in SEQ ID NO:3 (KTCKQAGTCPPD), or as shown in SEQ ID NO:4 (TCKQAGTCPPD), or as shown in SEQ ID NO:5 (CKQAGTCPPD), or as shown in SEQ ID NO:6 (TCKQAGTCPP), or a sequence as shown in SEQ ID NO:7 (CKQAGTCPP), or a sequence as shown in SEQ ID NO:31 (IIPKVEGKT), or a sequence as shown in SEQ ID NO:32 (IPKVEGKT). Since it has been shown in the context of the present invention that the Alanine (A) residue comprised by the aforementioned fragments can be replaced with other amino acid residues (particularly, with a Glycine (G) residue) without significantly affecting the immunogenicity of the polypeptide according to the invention as well as the neutralizing capacity of the antibodies against the said immunogenic polypeptide (see Examples), variants of the aforementioned fragments preferably have the amino acid sequence as shown in SEQ ID NO:10 (KTCKQXGTCPPDIIPKVEG), or as shown in SEQ ID NO:11 (KTCKQXGTCPPD), or as shown in SEQ ID NO:12 (TCKQXGTCPPD), or as shown in SEQ ID NO:13 (CKQXGTCPPD), or as shown in SEQ ID NO:14 (TCKQXGTCPP), or a sequence as shown in SEQ ID NO: 15 (CKQXGTCPP). Preferably, X represents a Glycine (G) or an Alanine (A) residue. Moreover, experiments with the aforementioned fragments of the L2 polypeptide have shown that the most crucial amino acid residues for immunogenicity and for the generation of cross-neutralizing antibodies were amino acid residues 22 to 24 (CKQ) and 26 to 31 (GTCPPD) of the L2 polypeptide as shown in SEQ ID NO:1 (see Examples). Accordingly, the most preferred variant of a fragment of the L2 polypeptide has a sequence as shown in SEQ ID NO: 13), CKQXGTCPPD).

[0018] In one preferred embodiment of the present invention, the immunogenic polypeptide comprises a multimer of the L2 polypeptide or a fragment thereof (or a variant of said L2 polypeptide or a variant of any fragment thereof). Thus, the immunogenic polypeptide shall comprise more than one L2 polypeptide or more than one fragment of the L2 polypeptide. It is particularly envisaged that the immunogenic polypeptide comprises more than one fragment of the L2 polypeptide (or variants thereof). Preferably, the immunogenic polypeptide comprises multimers of 2 to 15 fragments of the L2 polypeptide, and more preferably multimers of 3 to 9 (and, thus, of 3, 4, 5 , 6, 7, 8 or 9) fragments of the L2 polypeptide. Most preferably, said immunogenic polypeptide comprises multimers of three or four fragments of the L2 polypeptide. Preferably, said fragments are directly linked together. More preferably, said fragments are linked via a linker peptide (for an explanation of the term "linker peptide", see herein below). Preferably, if the immunogenic polypeptide comprises more than one fragment of the L2 polypeptide, the fragments shall have the same or essentially the same sequence. However, it is also contemplated that the multimer comprises various fragments (or variants thereof) of the L2 polypeptide.

[0019] Other preferred L2 fragments (or variants thereof) are selected from the group consisting of SEQ ID NO: 33 to SEQ ID NO:479. SEQ ID NO:33 to SEQ ID NO:479 are also shown in Table 1. The sequences as shown in SEQ ID NO:33 to SEQ ID NO:79 and in SEQ ID NO:486 to SEQ ID NO:489 are variants of the L2 fragment KTCKQAGTCPPDIIPKVEG as shown in SEQ ID NO:2; the sequences as shown in SEQ ID NO:80 to SEQ ID NO:112 and in SEQ ID NO: 490 are variants of the L2 fragment KTCKQAGTCPPD as shown in SEQ ID NO:3; the sequences as shown in SEQ ID NO:113 to SEQ ID NO:139 are variants of the L2 fragment TCKQAGTCPPD as shown in SEQ ID NO:4; the sequences as shown in SEQ ID NO:140 to SEQ ID NO:161 are variants of the L2 fragment CKQAGTCPPD as shown in SEQ ID NO:5; the sequences as shown in SEQ ID NO:162 to SEQ ID NO:188 are variants of the L2 fragment TCKQAGTCPP as shown in SEQ ID NO:6; the sequences as shown in SEQ ID NO:189 to SEQ ID NO:210 are variants of the L2 fragment CKQAGTCPP as shown in SEQ ID NO:7; the sequences as shown in SEQ ID NO:211 to SEQ ID NO:238 are variants of the L2 fragment DIIPKVEGKT as shown in SEQ ID NO:8; the sequences as shown in SEQ ID NO:239 to SEQ ID NO:266 are variants of the L2 fragment IIPKVEGKT as shown in SEQ ID NO:31; the sequences as shown in SEQ ID NO:267 to SEQ ID NO:293 are variants of the L2 fragment IPKVEGKT as shown in SEQ ID NO:32; the sequences as shown in SEQ ID NO:294 to SEQ ID NO:301 are variants of the L2 fragment TGYIPLGTR as shown in SEQ ID NO:9; the sequences as shown in SEQ ID NO:302 to SEQ ID NO:348 are variants of the L2 fragment KTCKQXGTCPPDIIPKVEG as shown in SEQ ID NO:10; the sequences as shown in SEQ ID NO:349 to SEQ ID NO:381 are variants of the L2 fragment KTCKQXGTCPPD as shown in SEQ ID NO:11; the sequences as shown in SEQ ID NO: 382 to SEQ ID NO: 408 are variants of the L2 fragment TCKQXGTCPPD as shown in SEQ ID NO:12; the sequences as shown in SEQ ID NO: 409 to SEQ ID NO: 430 are variants of the L2 fragment CKQXGTCPPD as shown in SEQ ID NO:13; the sequences as shown in SEQ ID NO: 431 to SEQ ID NO: 457 are variants of the L2 fragment TCKQXGTCPP as shown in SEQ ID NO:14; the sequences as shown in SEQ ID NO:458 to SEQ ID NO:479 are variants of the L2 fragment CKQXGTCPP as shown in SEQ ID NO:15.

[0020] As mentioned above, the immunogenic polypeptide shall also comprise a linker peptide or more than one linker peptide. Said linker peptide, preferably, shall prevent the formation of junctional epitopes. Preferably, the linker peptide is positioned at the C- and/or N-Terminus of the L2 polypeptide, or of the fragment (or of the variant thereof). If the immunogenic polypeptide comprises more than one fragment of the L2 polypeptide (or more than one variant of said fragment), it is particularly contemplated that the immunogenic polypeptide comprises a linker peptide between the various fragments (or variants thereof). For example, SEQ ID NO:21 shows a multimer of L2 fragments with a GGP-linker (SEQ ID NO:16) inserted between any one of the L2 fragments.

[0021] Preferably, said linker has a length of 1 to 5 amino acids. The person skilled in the art knows how to select suitable linker peptides. Preferably, said 1 to 5 amino acids comprised by said linker peptide are selected from the group consisting of Glycine (G), Proline (P) or Serine (S). A particularly preferred linker peptide comprises the amino acid sequence GGP (SEQ ID NO: 16). However, also other linkers can be used such as GPGP (SEQ ID NO: 17), GPGPG (SEQ ID NO: 18), or SGSG (SEQ ID NO: 19). Preferably, said linker peptide is positioned at the junction of the scaffold polypeptide and the fragment of the L2 polypeptide and/or at the junction of two L2 fragments (or variants thereof). Thus, said linker peptide can be positioned either N-terminally or C-terminally from the L2 fragment (or variant thereof) or both.

[0022] A preferred multimer of a fragment of the L2 polypeptide comprised by the immunogenic polypeptide according to the invention has an amino acid sequence such as the one shown in SEQ ID NO: 20, or in SEQ ID NO: 21, or a sequence as shown in SEQ ID NO: 22.

[0023] Other preferred multimers are multimers comprising combinations of different homooligomers of fragments of the L2 polypeptide (e.g. a trimer of SEQ ID NO:2 linked to a trimer of SEQ ID NO:487 linked to a trimer of SEQ ID NO:487). More preferably, the L2 polypeptides comprised in said multimers are separated by linker sequences, see e.g. SEQ ID NO: 491. Also preferred are repeats of heterooligomers of fragments of the L2 polypeptide. A heterooligomer comprises e.g. SEQ ID NO:2 linked to SEQ ID NO:487 linked to SEQ ID NO:77, the corresponding multimer comprising e.g. said heterooligomer repeated three times. More preferably, the L2 polypeptides comprised in said multimers are separated by linker sequences, see e.g. SEQ ID NO:492.

[0024] The L2 polypeptide, or fragment thereof (or the variant of said L2 polypeptide or of the fragment thereof, or the corresponding multimers, see elsewhere herein) shall be comprised by a scaffold polypeptide which constrains the structure of the L2 polypeptide, or the fragment thereof (or the respective variants).

[0025] The term "constraining" as used herein, preferably, means that the L2 polypeptide, or the fragment thereof (or the respective variants) that is comprised by the scaffold protein is present in a conformation that mimics its natural conformation. Preferably, said L2 polypeptide, or the fragment thereof (or the respective variant) is kept by the scaffold polypeptide in a fixed conformation, when constrained.

[0026] Any scaffold polypeptide being capable of constraining the structure of said L2 polypeptide, or of the fragment of said L2 polypeptide, preferably, can be used for the production of the immunogenic polypeptide according to the present disclosure.

[0027] According to the present disclosure, the scaffold polypeptide is selected from the group consisting of thioredoxin, capsid polypeptides of adeno-associated viruses (e.g. AAV2, GenBank Accession No., NC_001401.2, GI:110645916; AAV8 GenBank Accession No., NC_006261.1, GI:51949963; AAV7 GenBank Accession No., NC_006260.1, GI:51949960), the tenth type III module of fibronectin (FN3, GenBank Accession No. 1TTF_A; GI:157834026, with insertion of the L2 polypeptide, fragment or variant thereof within the exposed PAVTVR (SEQ ID NO: 480) or GRGDSPASS (SEQ ID NO: 481) loop sites), lipocalins (particularly, the bilin-binding protein from Pieris brassicae, GenBank Accession No. CAA54063.1, GI:434995, with insertion of the L2 polypeptide, fragment or variant thereof within the PNSVEKY (SEQ ID NO: 482), IHGKE (SEQ ID NO: 483), TYGGVTK (SEQ ID NO: 484) and/or YDEDKKGH loop sites), a catalytically inactive version of Staphylococcus nuclease (e.g., GenBank Accession No. 2SNS_A, 2SNS_A, GI:157836360, with peptide insertion within the YKGQP (SEQ ID NO: 485) loop site); an alpha-amylase inhibitor, preferably tendamistat (GenBank Accession No. CAA00655.1, GI:413044, with peptide insertion within the EDD and/or IGSHG loop sites); or stefin A (GenBank Accession No. P01040.1, GI:118177, with insertion of the L2 polypeptide, fragment or variant thereof within the KSL loop site).

[0028] According to the present invention, the scaffold protein is a thioredoxin polypeptide.

[0029] Thioredoxin polypeptides are the major cellular disulfide redox components and serve as electron donors for enzymes such as ribonucleotide reductases, thioredoxin peroxidases and methionine sulfoxide reductases. Thioredoxins have an alpha/beta structure with two disulfide bondable cysteine residues. Thioredoxins are ubiquitous polypeptides and were shown to be present in most organisms (for a review see Arnér and Holmgren, Physiological functions of thioredoxin and thioredoxin reductase, European Journal of Biochemistry, Volume 267 Issue 20, Pages 6102 - 6109). The thioredoxin polypeptide in the context of the present invention may be derived from any organism. Preferably, the thioredoxin polypeptide comprises the so called thioredoxin display site CGPC (SEQ ID NO: 23). The thioredoxin display site, also known as thioredoxin motif or as dithiol/disulfide active site, is a highly conserved motif amongst thioredoxin polypeptides. Preferably, said thioredoxin polypeptide is selected from the group consisting of prokaryotic and eukaryotic thioredoxin polypeptides, or any other thioredoxin or thioredoxin-like protein, or proteins harbouring a thioredoxin (TRX) Pfam domain, bearing the conserved CGPC (SEQ ID NO: 23), or a CGXC, or a CXXC sequence motif (e.g., gi|40253454; gi|77456671; gi|31543902). More preferably, said thioredoxin polypeptide is selected from the group consisting of bacterial, animal and plant thioredoxin polypeptides Even more preferably, the thioredoxin polypeptide is a Escherichia coli thioredoxin as shown in SEQ ID NO: 24 (which shows 100% identity with the thioredoxin polypeptide of Salmonella typhi), or the homologous thioredoxin polypeptides from Salmonella enterica (SEQ ID NO: 25), mouse (SEQ ID NO: 26), rabbit (SEQ ID NO: 27), human (SEQ ID NO: 28), or any other thioredoxin or thioredoxin-like protein as shown in SEQ ID NO: 17. Also included are oligomers of said thioredoxin polypeptides, i.e. fusion polypeptides comprising at least two copies of thioredoxin polypeptides, e.g. dimers or trimers, wherein the C-terminus of one copy of a thioredoxin polypeptide is linked to the N-terminus of the following copy of a thioredoxin polypeptide. Preferably, at least one of the thioredoxin polypeptides comprises at least one L2 peptide inserted within the display site. More preferably, in said oligomers the thioredoxin polypeptides are separated by linker peptides, see e.g. SEQ ID NO:497 and SEQ ID NO: 498.

[0030] Preferably, the L2 polypeptide, or the fragment of said L2 polypeptide (or multimer or fragment thereof) is positioned within the so called "display site" of thioredoxin. Thus, the said L2 polypeptide or fragment thereof, preferably is positioned between the C and the G, or between the G and the P, or between the P and the C residues of the display site sequence CGPC (SEQ ID NO: 23) of the thioredoxin polypeptide. Also contemplated by the present invention is positioning the L2 polypeptide or fragment thereof adjacent to the display site, preferably, between any pair of amino acid residues located up to 20, up to 10, or up to 5 amino acid residues upstream or downstream from the display site.

[0031] In a preferred embodiment the thioredoxin polypeptide is selected from the group consisting of
  1. a) a polypeptide having a sequence as shown in SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, or SEQ ID No: 28 (or any other thioredoxin polypeptide as recited herein); and
  2. b) a variant polypeptide having a sequence at least 70% identical to the sequence shown in SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, or SEQ ID No: 28 (or any other thioredoxin polypeptide as recited herein),
wherein said polypeptide constrains the structure of the L2 polypeptide, or which constrains the structure of a fragment of said L2 polypeptide (or of a variant thereof).

[0032] As set forth above the thioredoxin polypeptide in the context of the present invention, preferably, shall comprise the thioredoxin display site.

[0033] In another preferred embodiment the thioredoxin polypeptide is derived from a thermophile bacterium. The use of a thioredoxin polypeptide from a thermophile bacterium allows for storage of the immunogenic polypeptides, e.g., at room temperature (instead of storing said polypeptide, e.g., at 4°C or at even lower temperatures). Storing the immunogenic polypeptide, e.g., at 20°C is, particularly, advantageous if said polypeptide is used as a vaccine since it allows the distribution of the polypeptide even in regions where cooling systems are not available.

[0034] Thermophile bacteria are known to grow at elevated temperatures (> 50°C), particularly in and/or around geothermal vents in marine or aquatic environments. A variety of termophile bacteria is known in the art. Preferred thermophile bacteria in the context of the present invention are Archaebacteria, particularly Methanosaeta thermophila, Archaeoglobus fulgidus, Metallosphaera sedula, Sulfolobus solfataricus, Sulfolobus tokodaii, Sulfolobus acidocaldarius, Metallosphaera sedula, Thermofilum pendens, Picrophilus torridus, Caldivirga maquilingensis. The amino acid sequence of thioredoxin polypeptides of a variety of thermophile bacteria is well known in the art. Preferred thiorexodin polypeptides derived from thermophile bacteria have an amino acid sequence as shown in GenBank-Accession Numbers: Methanosaeta thermophila (gi|116754023, YP_843141; gi|116754438, YP_843556); Archaeoglobus fulgidus (gi|11498883, NP_070112; gi|11499727, NP_070969); Metallosphaera sedula (gi|146304377, YP_001191693; gi|146303559, YP_001190875); Sulfolobus solfataricus (gi|15897303, NP_341908; gi|15899007, NP_343612); Sulfolobus tokodaii (gi|15922449, NP_378118; gi|15921676, NP_377345); Sulfolobus acidocaldarius (gi|70605894, YP_254764.1; gi|70607552, YP_256422.1; gi|70607229, YP_256099); Thermofilum pendens (gi|119720035, YP_920530); Picrophilus torridus (gi|48477193, YP_022899); Caldivirga maquilingensis (gi|159040636, YP_001539888). Also included are thioredoxin polypeptides from Pyrococcus furiosus (SEQ ID NO: 493), Thermococcus kodakarensis (SEQ ID NO: 494), Thermococcus onnurineus (SEQ ID NO: 495), and Thermococcus sibiricus (SEQ ID NO: 496).

[0035] In a preferred embodiment the immunogenic polypeptide further comprises a polypeptide that further stimulates (enhances) immunogenicity of said immunogenic polypeptides. Such polypeptides stimulating immunogenicity are well known in the art. Preferred stimulating polypeptides are C4bp (Complement component 4 binding protein) and MDC/CCL22 (Macrophage-Derived Chemokine_CC motif_ligand 22. It is to be understood that the immunogenic polypeptide and the stimulating polypeptide are fused in frame. Preferably, the stimulating polypeptide is fused to the N- or C-terminus of to the immunogenic polypeptide

[0036] Preferably, the immunogenic polypeptide according to the invention is a polypeptide having an amino acid sequence as shown in SEQ ID NO: 29, or SEQ ID NO: 30.

[0037] The immunogenic polypeptide as shown in SEQ ID NO:29 comprises a multimer of 3 of the L2 fragment having a sequence as shown in SEQ ID NO:2, said fragments being connected by a linker peptide having a sequence as shown in SEQ ID NO: 16.

[0038] The immunogenic polypeptide as shown in SEQ ID NO:30 comprises a multimer of 9 of the L2 fragment having a sequence as shown in SEQ ID NO:2, said fragments being connected by a linker peptide having a sequence as shown in SEQ ID NO:16.

[0039] The sequences as shown in SEQ ID NO: 29, and SEQ ID NO: 30 comprise two hexahistidine-tags for purification of said polypeptides. It is to be understood that these tag do not contribute to the immunogenicity of said polypeptide and, thus, can be omitted.

[0040] Advantageously, it was shown in the studies underlying the present disclosure that an immunogenic polypeptide comprising a scaffold polypeptide and a L2 polypeptide or a fragment thereof, wherein said scaffold protein constrains the structure of said polypeptide or of said fragment, confers strong immunogenicity and induces strong neutralizing responses against HPV16 as well as strong cross-neutralizing responses against other HPV genotypes such as HPV18, HPV31, HPV45 and HPV58. Particularly, it was shown that a thioredoxin polypeptide that comprises within its display site the L2 polypeptide or a fragment has a strong immunogenicity and allows for strong neutralizing as well as cross-neutralizing responses (see Examples). The immunogenicity and (cross-)neutralizing response was further enhanced when multimers of the L2 polypeptides or fragments thereof were inserted within the display site of the thioredoxin polypeptide (see Examples).

[0041] The immunogenic polypeptide according to the present invention is of advantage over prior art polypeptides, since the polypeptides as disclosed in prior art have a low immunogenicity or only induce strong but not cross-neutralizing responses. For example, L2 based peptides that are disclosed in the art are poorly immunogenic whereas L1 based peptides have a limited cross-protective capacity. Thus, the immunogenic polypeptide according to the present invention allows for the production of vaccines against a broad range of HPV genotypes, particularly high-risk HPV genotypes.

[0042] Moreover, the present invention relates to a polynucleotide encoding the immunogenic polypeptide according to the present invention.

[0043] The polynucleotides of the present invention may contain further nucleic acid sequences as well. Specifically, the polynucleotides of the present invention may encode fusion proteins wherein one partner of the fusion protein is a polypeptide being encoded by a nucleic acid sequence recited above. Such fusion proteins may comprise as additional part peptide sequences for monitoring expression (e.g., green, yellow, blue or red fluorescent proteins, alkaline phosphatase and the like) or so called "tags" which may serve as a detectable marker or as an auxiliary measure for purification purposes. Tags for the different purposes are well known in the art and comprise FLAG-tags, 6-histidine-tags, MYC-tags and the like.

[0044] The term "polynucleotide" as used herein refers to a linear or circular nucleic acid molecule. It encompasses DNA as well as RNA molecules. The polynucleotide of the present invention shall be provided, preferably, either as an isolated polynucleotide (i.e. isolated from its natural context) or in genetically modified form. The term encompasses single as well as double stranded polynucleotides. Moreover, comprised are also chemically modified polynucleotides including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides or artificially modified derivatives such as biotinylated polynucleotides. The polynucleotide of the present invention is characterized in that it shall encode a polypeptide as referred to above. The polynucleotide, preferably, has a specific nucleotide sequence as mentioned above. Moreover, due to the degeneracy of the genetic code, polynucleotides are encompassed which encode a specific amino acid sequence as recited above.

[0045] Moreover, the term "polynucleotide" as used in accordance with the present invention further encompasses variants of the aforementioned specific polynucleotides. Said variants may represent orthologs, paralogs or other homologs of the polynucleotide of the present invention. The polynucleotide variants, preferably, comprise a nucleic acid sequence characterized in that the sequence can be derived from the aforementioned specific nucleic acid sequences by at least one nucleotide substitution, addition and/or deletion whereby the variant nucleic acid sequence shall still encode a polypeptide having a structure as specified in the claims. Variants also
encompass polynucleotides comprising a nucleic acid sequence which is capable of hybridizing to the aforementioned specific nucleic acid sequences, preferably, under stringent hybridization conditions. These stringent conditions are known to the skilled worker and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. A preferred example for stringent hybridization conditions are hybridization conditions in 6 × sodium chloride/sodium citrate (= SSC) at approximately 45°C, followed by one or more wash steps in 0.2 × SSC, 0.1% SDS at 50 to 65°C. The skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example when organic solvents are present, with regard to the temperature and concentration of the buffer. For example, under "standard hybridization conditions" the temperature differs depending on the type of nucleic acid between 42°C and 58°C in aqueous buffer with a concentration of 0.1 to 5 × SSC (pH 7.2). If an organic solvent is present in the above mentioned buffer, for example 50% formamide, the temperature under standard conditions is approximately 42°C. The hybridization conditions for DNA:DNA hybrids are preferably for example 0.1 × SSC and 20°C to 45°C, preferably between 30°C and 45°C. The hybridization conditions for DNA:RNA hybrids are preferably, for example, 0.1 × SSC and 30°C to 55°C, preferably between 45°C and 55°C. The above mentioned hybridization temperatures are determined for example for a nucleic acid with approximately 100 bp (= base pairs) in length and a G + C content of 50% in the absence of formamide. The skilled worker knows how to determine the hybridization conditions required by referring to textbooks such as the textbook mentioned above, or the following textbooks: Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, "Nucleic Acids Hybridization: A Practical Approach", IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential Molecular Biology: A Practical Approach", IRL Press at Oxford University Press, Oxford. Alternatively, polynucleotide variants are obtainable by PCR-based techniques such as mixed oligonucleotide primer- based amplification of DNA, i.e. using degenerated primers against conserved domains of the polypeptides of the present invention. Conserved domains of the polypeptide of the present invention may be identified by a sequence comparison of the nucleic acid sequence of the polynucleotide or the amino acid sequence of the polypeptide of the present invention with sequences of other members of the enzyme families referred to in accordance with this invention.

[0046] Oligonucleotides suitable as PCR primers as well as suitable PCR conditions are described in the accompanying Examples. As a template, DNA or cDNA from bacteria, fungi, plants or animals may be used. Further, variants include polynucleotides comprising nucleic acid sequences which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the specific nucleic acid sequences. Moreover, also encompassed are polynucleotides which comprise nucleic acid sequences encoding amino acid sequences which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the specific amino acid sequences referred to herein. The percent identity values are, preferably, calculated over the entire amino acid or nucleotide sequence region. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To carry out the sequence alignments, the program PileUp (Higgins 1989, CABIOS, 5 1989: 151-153) or the programs Gap and BestFit (Needleman 1970, J. Mol. Biol. 48; 443-453 and Smith 198, Adv. Appl. Math. 2: 482-489), which are part of the GCG software packet from Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711, version 1991, are to be used. The sequence identity values recited above in percent (%) are to be determined, preferably, using the program GAP over the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which, unless otherwise specified, shall always be used as standard settings for sequence alignments.

[0047] Moreover, the present invention relates to a vaccine comprising the immunogenic polypeptide according to the invention.

[0048] The term "vaccine" as used herein, preferably, relates to a composition which -when administered to an animal, preferably a human- elicits an immune response against various HPV genotypes. Thus, administering said vaccine would stimulate the immune system and establish or improve immunity to infection with various HPV genotypes. Preferably, the vaccine according to the present invention allows for establishing or improving immunity to infection with human papillomavirus genotypes 16, 18, 31, 45 and 58. Preferably, the vaccine according to the present invention also allows for establishing or improving immunity to infection with human papillomavirus genotypes 6, 52, 2, 27, 57 and/or 11. It is to be understood that the vaccine according to the present invention may comprise further components.

[0049] A preferred further component is an adjuvant. Adjuvants are compounds which may not elicit an immune response when administered to the host alone but which may further enhance the immune response of the host when administered together with the immunogenic polypeptides. It is known in the art that adjuvants may act as surfactants which promote concentration of immunogenic polypeptides over a large surface area, or may have immunostimulatory properties.

[0050] Preferred adjuvants in the context of the present invention are muramyl dipeptide, saponins such as QS21 and Quil A, monophosphoryl lipid A, mineral oil/surfactant mixtures (e.g., Montanide), aluminum hydroxide, aluminum phosphate, hydroxyapatite, complete and/or incomplete Freund's adjuvant, or cytokines such as interleukins, macrophage derived chemokines, complement binding proteins and tumor necrosis factor (either free or fused to the scaffold protein), and human use-approved live microbial carriers such as the live attenuated Salmonella enterica serovar Typhimurium strain.

[0051] Moreover the present disclosure relates to the use of the immunogenic polypeptide according the invention for the preparation of a vaccine for immunization of a subject against infection with HPV.

[0052] Preferably, said subject is an animal, more preferably, said subject is a vertebrate, even more preferably, said subject is a mammal and, most preferably, said subject is a human. Preferably, the immunization of said subject, establishes or improves immunity of said subject to various HPV genotypes as referred to elsewhere herein. It is to be understood that the immunogenic polypeptide according to the invention or vaccine according to the invention has to be administered to said subject for immunization. Said administration can be done by any method deemed appropriate such as oral or parenteral administration.

[0053] Moreover, the present disclosure relates to a method for producing an antibody against the immunogenic polypeptide according to the invention, comprising the following steps:
  1. a) providing the immunogenic polypeptide according to the invention;
  2. b) immunizing a host with said immunogenic polypeptide, and
  3. c) harvesting the antibody against said immunogenic polypeptide.


[0054] Preferably, the host will be sacrificed after the method has been carried out. It is to be understood that such a method is not deemed to be a method of treatment of the human or animal body.

[0055] The "host" in the context may be any host deemed appropriate. Preferably, the host is a non-human host. Preferred host for the production of monoclonal antibodies is a mouse or a rabbit. A host for the production of polyclonal antibodies is preferably selected from the group consisting of rabbits, mice, chickens, goats, guinea pigs, hamsters, horses, rats, and sheep.

[0056] Antibodies against the immunogenic polypeptide according to the present invention can be prepared by well known methods using said immunogenic polypeptide as an antigen. Preferably, the produced antibody is a polyclonal antibody. More preferably, said antibody is a monoclonal antibody.

[0057] In a further disclosure, said monoclonal antibody is produced by the hybridoma cell line which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany on November 27, 2008 under deposit number DSM ACC2983 according to the Budapest Treaty, or a fragment thereof (preferably, F(ab)2, F(ab')2, Fab, F (ab'), Dab, Fv, sFv,scFv, or Fc fragments), or said monoclonal antibody produced by the hybridoma cell line which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany on November 27, 2008 under deposit number DSM ACC2984 according to the Budapest Treaty, or a fragment thereof (preferably, F(ab)2, F(ab')2, Fab, F (ab'), Dab, Fv, sFv,scFv, or Fc fragments). The monoclonal antibody produced by the hybridoma cell line which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany on November 27, 2008 under deposit number DSM ACC2983 is herein also referred to as K4L2(20-38)4.1B. The monoclonal antibody produced by the hybridoma cell line which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany on November 27, 2008 under deposit number DSM ACC2984 is herein also referred to as K18L2(20-38)XIII.5G.

[0058] It is also contemplated by the present disclosure that the antibody is a single chain antibody, a recombinant, human or humanized antibody or primatized, chimerized or a fragment of the antibody according to the present invention.

[0059] Also comprised by the aforementioned method of the present disclosure is the production of a synthetic antibody, an antibody fragment, such as F(ab)2, F(ab')2, Fab, F (ab'), Dab, Fv, sFv, scFv, or Fc fragments etc., or a chemically modified derivative of any of these. The antibody may belong to any immunoglobulin class, including IgM, IgG, IgD, IgE, IgA, or subclasses of IgG (such as IgG1, IgG2, IgG2, IgG2a, IgG2b, IgG3 or IgGM).

[0060] How to produce and harvest the aforementioned antibodies and fragments is well known in the art. Antibodies or fragments thereof can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies can be prepared by the techniques originally described in Köhler and Milstein, Nature 256 (1975), 495, and Galfré, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. It is also contemplated that monoclonal antibodies are produced by fusing myeloma cells with the B-cells from rabbits that have been immunized with the desired antigen.

[0061] It is to be understood that the antibody produced by the aforementioned method shall specifically bind the immunogenic polypeptide according to the invention. Specific binding can be tested by various well known techniques. Preferably, the antibody produced by the aforementioned method shall specifically bind the L2 polypeptide or fragment thereof. More preferably, said antibody shall specifically bind the L2 polypeptide or fragment thereof, when comprised by the immunogenic polypeptide according to the present invention (linked to the scaffold polypeptide), and thus when being present in a constrained structure. Thus, the antibody according to the present disclosure shall not specifically bind the parts of the immunogenic polypeptide that are derived from the scaffold polypeptide.

[0062] The aforementioned method disclosed herein, preferably, allows for the production of an antibody against human papillomavirus. Preferably, said antibody binds the L2 polypeptide or fragments thereof of various HPV genotypes. Preferably, said antibody binds the L2 polypeptide or fragment thereof of HPV genotypes 16, 18, 31, 45 and 58. Preferably, the said antibody also binds the L2 polypeptide or fragments thereof of HPV genotypes 52, 2, 27, 57 and/or 11.

[0063] The present disclosure relates also to an antibody obtainable/produced by the aforementioned method of the present invention.

[0064] Said antibody of the present disclosure, preferably is a polyclonal antibody and, more preferably, a monoclonal antibody.

[0065] Most preferably, the antibody according to the present disclosure is the monoclonal antibody K4L2(20-38)4.1B (see Examples) produced by the hybridoma cell line which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany on November 27, 2008 under deposit number DSM ACC2983 according to the Budapest Treaty, or a fragment thereof (preferably, F(ab)2, F(ab')2, Fab, F (ab'), Dab, Fv, sFv,scFv, or Fc fragments), or the antibody according to the present invention is the monoclonal antibody K18L2(20-38)XIII.5G (see Examples), which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany on November 27, 2008 under deposit number DSM ACC2984 according to the Budapest Treaty, or a fragment thereof (preferably, F(ab)2, F(ab')2, Fab, F (ab'), Dab, Fv, sFv,scFv, or Fc fragments).

[0066] The antibodies according to the present disclosure can be used, for example, for the immunoprecipitation and immunolocalization of the immunogenic polypeptides of the present invention as well as for monitoring the presence of said variant polypeptides; for example, for the diagnosis of HPV infection, particularly for diagnosing infection with HPV genotypes 16, 18, 31, 45 and/or 58. Preferably, said diagnosis is done by determining the amount (or presence) of the L2 polypeptide in a biological sample from a subject suspected to be infected with HPV genotype 16, 18, 31, 45 and/or 58 (e.g. in a Pap smear). The presence of the L2 polypeptide (or increased amounts of the L2 polypeptide compared with a reference amount, e.g. the amount of said polypeptide in a sample from a subject not infected with HPV) indicates infection with HPV, whereas the absence of the L2 polypeptide (or decreased amounts of the L2 polypeptide compared with a reference amount, e.g. the amount of said polypeptide in a sample of a subject not infected with HPV) indicates that said subject is not infected with HPV.

[0067] Moreover, the antibodies according to the present disclosure can be used for the preparation of a pharmaceutical composition for passive immunization against various HPV genotypes, particularly against HPV genotypes 16, 18, 31, 45 and/or 58. For passive immunization, the antibody according to the present disclosure is administered to a subject in order to protect said subject against infection with various HPV genotypes and/or to treat an existing HPV infection, particularly infection with HPV genotypes 16, 18, 31, 45 or 58.
Also, the antibody of the present disclosure can be used for the production of anti-idiotypic antibodies. An "anti-idiotypic antibody" in the context of the present disclosure is an antibody that specifically binds to the idiotypic region of the antibody according to the present disclosure, or a fragment thereof. The idiotypic region of the antibody according to the present disclosure (or a fragment thereof) is, preferably, the unique part of its variable region that specifically binds to the immunogenic polypeptide according to the present disclosure. Preferably, the anti-idiotypic antibody is a monoclonal antibody.

[0068] Anti-idiotypic antibodies as well as methods for their production are well known in the art, see, e.g., US20080127359, or US Patent 5792455; Dalgleish: An anti-idiotype vaccine for AIDS based on the HIV receptor. Ann Ist Super Sanita. 1991;27(1):27-31, or Attanasio, Int Rev Immunol. 1990;7(1):109-19.

[0069] Preferably, said anti-idiotypic antibodies are produced by a) providing an antibody according to the present disclosure (preferably, a monoclonal antibody according to the invention, more preferably, K4L2(20-38)4.1B, or a fragment thereof, or K18L2(20-38)XIII.5G, or a fragment thereof), b) immunizing a host with said antibody, and c) harvesting the resulting anti-idiotypic antibody.

[0070] Accordingly, the present disclosure also relates to a method for producing anti-idiotypic antibodies by carrying out the aforementioned steps a) and b).

[0071] Moreover, the present disclosure relates to the use of the hybridoma cell line which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany on November 27, 2008 under deposit number DSM ACC2983, and to the use of the hybridoma cell line which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany on November 27, 2008 under deposit number DSM ACC2984 for the production of a monoclonal antibody that specifically binds the L2 peptide (as described herein).

[0072] Finally, the present disclosure also relates to the hybridoma cell line which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany on November 27, 2008 under deposit number DSM ACC2983 or under deposit number DSM ACC2984 according to the Budapest Treaty.

[0073] The Figures show:

Figure 1. Trx-L2 peptides. (A) Schematic representation of the HPV16 L2 peptides examined in this study. L2 aa 1-120 1x (1x SEQ ID NO:1 + 1x SEQ ID NO:24, thus the L2 polypeptide having a sequence as shown in SEQ ID NO1 inserted within the display site of the thioredoxin polypeptide having a sequence as shown in SEQ ID NO:24), 2x (SEQ ID NO:1 (2x) + SEQ ID NO:16(1x) + SEQ ID NO:24, thus two fragments of the L2 polypeptide, said fragments having a sequence as shown in SEQ ID NO:2, said fragments linked via one linker peptide having a sequence as shown in SEQ ID NO:16, inserted within the display site of the thioredoxin polypeptide having a sequence as shown in SEQ ID NO:24), 3x (SEQ ID NO:1 (3x)+ SEQ ID NO:16(2x) + SEQ ID NO:24); L2 aa 20-38 1x (SEQ IS NO:2 + SEQ ID NO:24), 3x(SEQ ID NO:29), 9x(SEQ ID NO:30), 15X (SEQ IS NO:2 (15x) + SEQ ID NO:16 (14x)+ SEQ ID NO:24); L2 aa 28-42 1x, 4x, 8x; L2 aa 56-75 1x, 4x; L2 aa 64-81 1x, 4x, 8x; L2 aa 95-115 1x, 4x, 8x. (B) Representative example of the expression analysis of pTrx-L2(20-38)n constructs with varying L2 peptide insert multiplicity (n). SDS-PAGE of total bacterial lysates from different clones ordered according to the multiplicity of their L2(20-38) peptide inserts; the peptide insert multiplicity (n) of the various fusion proteins and the migration positions of molecular mass markers are indicated on the right hand side and on the left hand side, respectively. (C) Representative examples of purified Trx-L2(20-38)n fusion proteins used for mice immunization (1) L2 aa 20-38 1x (SEQ IS NO:2 + SEQ ID NO:24), 3x(SEQ ID NO:29), 9x(SEQ ID NO:30), 15X (SEQ IS NO:2 (15x) + SEQ ID NO:16 (14x)+ SEQ ID NO:24). (D) Comparison of the immune responses elicited by the HPV16 L2(20-38) peptide chemically conjugated to KLH and by the same peptide grafted to Trx, both administered (100 µg/dose) with the same CFA/IFA immunization protocol described in 'Materials and methods'. ELISA data, obtained using GST-L2 as target antigen and expressed as A405 values, are presented as dot plots. The L2 binding activity of individual sera as well as the mean binding activity of each group (horizontal bars) are shown; KLH and Trx indicate the unconjugated carrier proteins utilized as negative controls. Please note that the numbers correspond to the amino acid positions of the L2 polypeptide as shown in SEQ ID NO:1. E.g., "L2(20-38)" stands for a fragment of the L2 polypeptide as shown in SEQ ID NO:1 comprising amino acids 20 to 28 of said polypeptide.

Figure 2. Antibody titers of mice vaccinated with the Trx-L2 peptide fusions. GST-L2 ELISA was used to determine the antibody titers of sera from mice immunized three times with the indicated Trx-L2(peptide)n fusions, for group L2 aa 20-38 1x (SEQ IS NO:2 + SEQ ID NO:24), 3x(SEQ ID NO:29), 9x(SEQ ID NO:30), 15X (SEQ IS NO:2 (15x) + SEQ ID NO:16 (14x)+ SEQ ID NO:24) and group Trx-L2 L2 aa 1-120 1x (1x SEQ ID NO:1 +1x SEQ ID NO:24), 2x (SEQ ID NO:1 (2x)+ SEQ ID NO:16(1x)+ SEQ ID NO:24), 3x (SEQ ID NO:1 (3x)+ SEQ ID NO:16(2x)+ SEQ ID NO:24); L2 aa 28-42 1x, 4x, 8x; L2 aa 56-75 1x, 4x; L2 aa 64-81 1x, 4x, 8x; L2 aa 95-115 1x, 4x, 8x (100 µg each, corresponding to 1.7-4.3 nmol of protein, depending on the size of the peptide insert; n values are shown on the x-axis) administered with the CFA/IFA immunization protocol described in 'Materials and methods'. Sera from mice immunized with the Trx protein scaffold only (not shown) were used as negative controls and were assayed in parallel in each set of ELISAs. Binding titers are given as the reciprocal of the maximum antisera dilutions that yielded A405 values higher than the mean absorbance plus four standard deviations of sera from mice immunized with the Trx scaffold only. Data are presented as log10 dot-plots of the titers; horizontal bars represent the geometric mean of the titers for each of the indicated subgroups of Trx-L2 antisera. The P values in each panel indicate the statistical significance of the differences between the immune responses induced by monopeptide and multipeptide Trx-L2 fusions in each group.

Figure 3. Neutralization of HPV 16 infection by Trx-L2 peptide antisera. Serial dilutions of antisera raised against the monopeptide and multipeptide Trx-L2 fusions shown in Fig. 2 were analyzed for their capacity to block infection of 293TT cells by HPV16 pseudovirions using secreted alkaline phosphatase (SEAP) activity as readout. Neutralization efficiency was determined relative to that of mock-treated (0% neutralization) and HPV16 L1-specific mAb-treated (100% neutralization) controls, which were run in parallel in each assay (see 'Materials and methods' for details). Data for the monopeptide and the aggregated multipeptide forms of each Trx-L2 immunogen L2 aa 20-38 1x (SEQ IS NO:2 + SEQ ID NO:24), 3x(SEQ ID NO:29), 9x(SEQ ID NO:30), 15X (SEQ IS NO:2 (15x) + SEQ ID NO:16 (14x)+ SEQ ID NO:24); L2 aa 28-42 1x, 4x, 8x; L2 aa 56-75 1x, 4x; L2 aa 64-81 1x, 4x, 8x and L2 aa 95-115 1x, 4x, 8x are expressed as the reciprocal of the maximum dilution causing ≥70% neutralization. The geometric means of the titers and the 95% confidence intervals for each group of anti-Trx-L2 peptide antisera plus the anti-Trx-L2(1-120) 1x (1x SEQ ID NO:1 +1x SEQ ID NO:24), 2x (SEQ ID NO:1 (2x)+ SEQ ID NO:16(1x)+ SEQ ID NO:24), 3x (SEQ ID NO:1 (3x)+ SEQ ID NO:16(2x)+ SEQ ID NO:24) reference are represented on a log scale.

Figure 4. Cross-neutralization of HPV 31, 45, and 58 pseudovirions. The crossneutralization activities of the indicated subset of Trx-L2 peptide antisera were assayed at a fixed 1:200 dilution against three heterologous pseudovirions (HPV 31, 45 and 58) plus the cognate HPV16 type. Mock-treated 293TT cells and cells treated with type-specific neutralizing antibodies served as negative and positive controls, respectively (see Fig. 3) legend and 'Materials and methods' for details). Cumulative monopeptide and multipeptide data are presented for each immunogen except Trx-L2 aa 20-38 1x (SEQ IS NO:2 + SEQ ID NO:24), 3x(SEQ ID NO:29), 9x(SEQ ID NO:30), 15X (SEQ IS NO:2 (15x) + SEQ ID NO:16 (14x)+ SEQ ID NO:24), the only one for which a trend toward a peptide multiplicity-dependent increase in cross-neutralization activity was observed; they are represented as the mean plus SD of the neutralization values for the various Trx-L2 peptide antisera relative to those obtained with HPV type-specific antibodies.

Figure 5. Neutralization of homologous and heterologous pseudovirions by Trx-L2(20-38)n antisera. The strongest HPV 16 neutralizing antisera from each group of Trx-L2(20-38)n antigens (n = 1, 3, 9, 15) 1x (SEQ IS NO:2 + SEQ ID NO:24), 3x(SEQ ID NO:29), 9x(SEQ ID NO:30), 15X (SEQ IS NO:2 (15x) + SEQ ID NO:16 (14x)+ SEQ ID NO:24) were titrated against homologous (HPV16) and heterologous (HPV18, 31, 45, 58) pseudovirions (see 'Materials and methods' and Fig. 3 legend for details).

Figure 6. Sequence comparison of the L2(20-38) region of the examined HPV types. Multiple sequence alignment was performed with CLUSTAL W [30]; amino acids identical to those of the cognate HPV16 type are indicated with dots. Conservative and non-conservative substitutions are shown in standard and in bold characters, respectively; non-conservative substitutions occurring in only one of the five examined HPV types are boxed.

Figure 7. Neutralizing titers of supernatants of monoclonal antibodies against the aa 20-31 from HPV16 L2 protein
IgG concentration in supernatants was adjusted to 0,6 µg/ml, titer was defined as the last dilution that can protect 70% of pseudovirions infection. There are not big differences in the neutralization capacity of antibodies #4 (K4L2(20-38)4.1B) and #18 (K18L2(20-38)XIII.5G) except for the neutralization of HPV31. Antibody #18 can neutralize the infection although with low titer, but, antibody #4 is unable to neutralize the infection even at low dilution factor. Antibody #8 and #1 can neutralize only HPV16. Antibody #1 can neutralize HPV16 with high titer.

Figure 8. Identification of epitopes recognized by neutralizing (boxed) and non-neutralizing antibodies
The different monoclonal antibodies raised against different regions of the HPV 16 N-terminus were tested for their reactivity with a set of overlapping peptides (amino acids 1-15, 5-19, 106-120) in ELISA. All four neutralizing antibodies show a distinct pattern in binding the peptides, different to the pattern of the non-neutralizing antibodies. The two cross-neutralizing antibodies (K4L2(20-38)4.1B) and #18 (K18L2(20-38)XIII.5G) are directed against region 20-38. Antibody #15, which shows a similar binding pattern compared to #18, has an about 30 fold lower affinity to its target which explains its failure to neutralize HPV pseudovirions.

Figure 9. Epitopes for non-neutralizing, neutralizing and cross-neutralizing antibodies (K4L2(20-38)4.1B and K18L2(20-38)XIII.5G) within region 20-42 of HPV 16 L2. Scheme with the recognition patron of all mAbs isolated against the region 20-42. Cross-neutralizing antibodies Mab K4L2(20-38)4.1B recognize the sequence aa 21-30 SEQ ID NO:4 and K18L2(20-38)XIII.5G recognize the sequence aa 22-30 SEQ ID NO:5. Neutralizing antibody anti HPV16 K8L2(28-42)12.4B recognize the sequence aa 32-39 SEQ ID NO:31.

Figure 10. Epitope mapping for the two neutralizing antibodies (K4L2(20-38)4.1B and #18 (K18L2(20-38)XIII.5G). To determine the amino acids required for binding of the two antibodies K4 (A9 and K18 (B) an peptide-alanine scan was performed. Antibody #4 the five amino acids xTCKxxxxCPxx are essential for binding while for K8 only the two cysteine residues are crucial for binding, although the remainder residues might contribute to the binding.

Figure 11. Neutralization assay of HPV 16 and HPV 31 pseudovirions with modified L2 proteins. To determine why antibodies #4 (K4L2(20-38)4.1B) and #18 (K18L2(20-38)XIII.5G) have different abilities to neutralize HPV 31 we tested hybrid particles composed of HPV 16 L1 HPV 31 L2 and vice versa. In addition, the corresponding epitope in HPV 31 recognized by K4 and K18 was modified. Results indicate that the ability of (K4L2(20-38)4.1B) and #18 (K18L2(20-38)XIII.5G) antibodies to neutralize depends on the epitope sequence as HPV 31 L1/16L2 pseudovirions can be neutralized by both antibodies. Altering serine at position 30 into proline restores the ability to neutralize HPV 31 pseudovirions indicating that this residue is important in binding the antibodies.


Examples


Example 1



[0074] Monopeptide (1 x SEQ ID NO:2) and multipeptide ((SEQ ID NO:2 (3x)+ SEQ ID NO:16(2x)) or 3x (SEQ ID NO:2 (3x)+ SEQ ID NO:16(2x)) immunogenic peptides are inserted within the display site of the thioredoxin polypeptide having a sequence as shown in SEQ ID NO: 493 to SEQ ID NO: 496. Fusion proteins are produced in E. coli cells, purified from cell extracts and used for immunization.
Table 1 List of L2 peptide immunogens (and variants thereof)_
SEQ ID NO: 33 RGCKQAGTCPPDVINKVEQ
SEQ ID NO: 34 RGCKASNTCPPDVINKVEQ
SEQ ID NO: 35 RGCKAAGTCPPDVINKVEQ
SEQ ID NO: 36 QSCKAAGTCPPDVLNKVEQ
SEQ ID NO: 37 QSCKAAGTCPPDVVNKVEQ
SEQ ID NO: 38 QTCKQAGTCPPDVINKVEQ
SEQ ID NO: 39 QTCKQAGTCPPDVVNKVEQ
SEQ ID NO: 40 RTCKQAGTCPPDVINKVES
SEQ ID NO: 41 RTCKQAGTCPPDVINKVEQ
SEQ ID NO: 42 KGCKASGTCPPDVINKVEQ
SEQ ID NO: 43 RTCKQSGTCPPDVVPKVEG
SEQ ID NO: 44 RTCKQAGTCPPDVIPKVEG
SEQ ID NO: 45 RTCKVTGTCPADVVPKVEG
SEQ ID NO: 46 RTCKATGTRPADVIPKVEG
SEQ ID NO: 47 RTCKQSGTCPPDIIPRVEQ
SEQ ID NO: 48 RTCKQAGTCPPDIIPRLEQ
SEQ ID NO: 49 RTCKQAGTCPPDIIPRVEQ
SEQ ID NO: 50 KTCKVAGTCPPDVIPKVEG
SEQ ID NO: 51 KTCKAAGTCPPDVIPKVEG
SEQ ID NO: 52 RTCKAAGTCPPDVIPKVEG
SEQ ID NO: 53 RTCKASGTCPPDVIPKVEG
SEQ ID NO: 54 STCKAAGTCPADVIPKVEG
SEQ ID NO: 55 KTCKLSGTCPEDVINKVEQ
SEQ ID NO: 56 KTCKQSGTCPPDIIPKVEG
SEQ ID NO: 57 KTCKQAGTCPPDIVPKVEG
SEQ ID NO: 58 QTCKASGTCPPDVIPKVEG
SEQ ID NO: 59 KTCKQAGTCPPDVIPKVEG
SEQ ID NO: 60 QTCKAAGTCPSDIIPKVEH
SEQ ID NO: 61 QTCKASGTCPPDVIPKVEQ
SEQ ID NO: 62 QTCKLTGTCPPDVIPKVEH
SEQ ID NO: 63 QTCKAAGTCPSDVINKVEH
SEQ ID NO: 64 KQCQLGADCPPDVRNKVEG
SEQ ID NO: 65 AKCQLSGNCLPDVKNKVEA
SEQ ID NO: 66 AKCQLSGDCLPDVKNKVEA
SEQ ID NO: 67 RHCALSGTCPDDVKNKVEN
SEQ ID NO: 68 KHCAGSGTCPEDVKNKVEQ
SEQ ID NO: 69 KTCLQGGDCIPDVKNKFEN
SEQ ID NO: 70 RSCLQGGDCIPDVQNKFEG
SEQ ID NO: 71 QTCKATGTCPPDVIPKVEG
SEQ ID NO: 72 KTCKQSGTCPPDVVPKVEG
SEQ ID NO: 73 RTCKQSGTCPPDVINKVEG
SEQ ID NO: 74 KTCKQAGTCPSDVINKVEG
SEQ ID NO: 75 KTCKLSGTCPEDVVNKIEQ
SEQ ID NO: 76 RTCKQSGTCPPDVVDKVEG
SEQ ID NO: 77 STCKAAGTCPPDVVNKVEG
SEQ ID NO: 78 PTCKIAGNCPADIQNKFEN
SEQ ID NO: 79 PACKISNTCPPDIINKYEN
   
SEQ ID NO: 80 RGCKQAGTCPPD
SEQ ID NO: 81 RGCKASNTCPPD
SEQ ID NO: 82 RGCKAAGTCPPD
SEQ ID NO: 83 QSCKAAGTCPPD
SEQ ID NO: 84 QTCKQAGTCPPD
SEQ ID NO: 85 RTCKQAGTCPPD
SEQ ID NO: 86 KGCKASGTCPPD
SEQ ID NO: 87 RTCKQSGTCPPD
SEQ ID NO: 88 RTCKVTGTCPAD
SEQ ID NO: 89 RTCKATGTRPAD
SEQ ID NO: 90 KTCKVAGTCPPD
SEQ ID NO: 91 KTCKAAGTCPPD
SEQ ID NO: 92 RTCKAAGTCPPD
SEQ ID NO: 93 RTCKASGTCPPD
SEQ ID NO: 94 STCKAAGTCPAD
SEQ ID NO: 95 KTCKLSGTCPED
SEQ ID NO: 96 KTCKQAGTCPED
SEQ ID NO: 97 QTCKASGTCPPD
SEQ ID NO: 98 QTCKAAGTCPSD
SEQ ID NO: 99 QTCKLTGTCPPD
SEQ ID NO: 100 KQCQLGADCPPD
SEQ ID NO: 101 AKCQLSGNCLPD
SEQ ID NO: 102 AKCQLSGDCLPD
SEQ ID NO: 103 RHCALSGTCPDD
SEQ ID NO: 104 KHCAGSGTCPED
SEQ ID NO: 105 KTCLQGGDCIPD
SEQ ID NO: 106 RSCLQGGDCIPD
SEQ ID NO: 107 QTCKATGTCPPD
SEQ ID NO: 108 KTCKQSGTCPPD
SEQ ID NO: 109 KTCKQAGTCPSD
SEQ ID NO: 110 STCKAAGTCPPD
SEQ ID NO: 111 PTCKIAGNCPAD
SEQ ID NO: 112 PACKISNTCPPD
   
SEQ ID NO: 113 GCKQAGTCPPD
SEQ ID NO: 114 GCKASNTCPPD
SEQ ID NO: 115 GCKAAGTCPPD
SEQ ID NO: 116 SCKAAGTCPPD
SEQ ID NO: 117 TCKQSGTCPSD
SEQ ID NO: 118 GCKASGTCPPD
SEQ ID NO: 119 TCKQSGTCPPD
SEQ ID NO: 120 TCKVTGTCPAD
SEQ ID NO: 121 TCKATGTRPAD
SEQ ID NO: 122 TCKVAGTCPPD
SEQ ID NO: 123 TCKAAGTCPPD
SEQ ID NO: 124 TCKASGTCPPD
SEQ ID NO: 125 TCKAAGTCPAD
SEQ ID NO: 126 TCKLSGTCPED
SEQ ID NO: 127 TCKAAGTCPSD
SEQ ID NO: 128 TCKLTGTCPPD
SEQ ID NO: 129 QCQLGADCPPD
SEQ ID NO: 130 KCQLSGNCLPD
SEQ ID NO: 131 KCQLSGDCLPD
SEQ ID NO: 132 HCALSGTCPDD
SEQ ID NO: 133 HCAGSGTCPED
SEQ ID NO: 134 TCLQGGDCIPD
SEQ ID NO: 135 SCLQGGDCIPD
SEQ ID NO: 136 TCKATGTCPPD
SEQ ID NO: 137 TCKQAGTCPSD
SEQ ID NO: 138 TCKIAGNCPAD
SEQ ID NO: 139 ACKISNTCPPD
   
SEQ ID NO: 140 CKQSGTCPDD
SEQ ID NO: 141 CKASNTCPPD
SEQ ID NO: 142 CKAAGTCPPD
SEQ ID NO: 143 CKASGTCPPD
SEQ ID NO: 144 CKQSGTCPPD
SEQ ID NO: 145 CKVTGTCPAD
SEQ ID NO: 146 CKATGTRPAD
SEQ ID NO: 147 CKVAGTCPPD
SEQ ID NO: 148 CKAAGTCPAD
SEQ ID NO: 149 CKLSGTCPED
SEQ ID NO: 150 CKAAGTCPSD
SEQ ID NO: 151 CKLTGTCPPD
SEQ ID NO: 152 CQLGADCPPD
SEQ ID NO: 153 CQLSGNCLPD
SEQ ID NO: 154 CQLSGDCLPD
SEQ ID NO: 155 CALSGTCPDD
SEQ ID NO: 156 CAGSGTCPED
SEQ ID NO: 157 CLQGGDCIPD
SEQ ID NO: 158 CKATGTCPPD
SEQ ID NO: 159 CKQAGTCPSD
SEQ ID NO: 160 CKIAGNCPAD
SEQ ID NO: 161 CKISNTCPPD
   
SEQ ID NO: 162 GCKQAGTCPP
SEQ ID NO: 163 GCKASNTCPP
SEQ ID NO: 164 GCKAAGTCPP
SEQ ID NO: 165 SCKAAGTCPP
SEQ ID NO: 166 TCKLAGTCPP
SEQ ID NO: 167 GCKASGTCPP
SEQ ID NO: 168 TCKQSGTCPP
SEQ ID NO: 169 TCKVTGTCPA
SEQ ID NO: 170 TCKATGTRPA
SEQ ID NO: 171 TCKVAGTCPP
SEQ ID NO: 172 TCKAAGTCPP
SEQ ID NO: 173 TCKASGTCPP
SEQ ID NO: 174 TCKAAGTCPA
SEQ ID NO: 175 TCKLSGTCPE
SEQ ID NO: 176 TCKAAGTCPS
SEQ ID NO: 177 TCKLTGTCPP
SEQ ID NO: 178 QCQLGADCPP
SEQ ID NO: 179 KCQLSGNCLP
SEQ ID NO: 180 KCQLSGDCLP
SEQ ID NO: 181 HCALSGTCPD
SEQ ID NO: 182 HCAGSGTCPE
SEQ ID NO: 183 TCLQGGDCIP
SEQ ID NO: 184 SCLQGGDCIP
SEQ ID NO: 185 TCKATGTCPP
SEQ ID NO: 186 TCKQAGTCPS
SEQ ID NO: 187 TCKIAGNCPA
SEQ ID NO: 188 ACKISNTCPP
   
SEQ ID NO: 189 CKLAGTCPP
SEQ ID NO: 190 CKASNTCPP
SEQ ID NO: 191 CKAAGTCPP
SEQ ID NO: 192 CKASGTCPP
SEQ ID NO: 193 CKQSGTCPP
SEQ ID NO: 194 CKVTGTCPA
SEQ ID NO: 195 CKATGTRPA
SEQ ID NO: 196 CKVAGTCPP
SEQ ID NO: 197 CKAAGTCPA
SEQ ID NO: 198 CKLSGTCPE
SEQ ID NO: 199 CKAAGTCPS
SEQ ID NO: 200 CKLTGTCPP
SEQ ID NO: 201 CQLGADCPP
SEQ ID NO: 202 CQLSGNCLP
SEQ ID NO: 203 CQLSGDCLP
SEQ ID NO: 204 CALSGTCPD
SEQ ID NO: 205 CAGSGTCPE
SEQ ID NO: 206 CLQGGDCIP
SEQ ID NO: 207 CKATGTCPP
SEQ ID NO: 208 CKQAGTCPS
SEQ ID NO: 209 CKIAGNCPA
SEQ ID NO: 210 CKISNTCPP
   
SEQ ID NO: 211 DVINKVEQTT
SEQ ID NO: 212 DVINKVEQST
SEQ ID NO: 213 DVINKVEQKT
SEQ ID NO: 214 DVLNKVEQTT
SEQ ID NO: 215 DVVNKVEQTT
SEQ ID NO: 216 DVINKVESTT
SEQ ID NO: 217 DVINKVEQNT
SEQ ID NO: 218 DVVPKVEGDT
SEQ ID NO: 219 DVIPKVEGDT
SEQ ID NO: 220 DIIPRVEQNT
SEQ ID NO: 221 DIIPRLEQNT
SEQ ID NO: 222 DIIPRVEQDT
SEQ ID NO: 223 DVIPKVEGTT
SEQ ID NO: 224 DIIPKVEQKT
SEQ ID NO: 225 DVIPKVEGST
SEQ ID NO: 226 DIIPKVEHNT
SEQ ID NO: 227 DVIPKVEQNT
SEQ ID NO: 228 DVIPKVEHNT
SEQ ID NO: 229 DVINKVEHTT
SEQ ID NO: 230 DVRNKVEGTT
SEQ ID NO: 231 DVKNKVEADT
SEQ ID NO: 232 DVKNKVEANT
SEQ ID NO: 233 DVKNKVENNT
SEQ ID NO: 234 DVKNKVEQTT
SEQ ID NO: 235 DVKNKFENST
SEQ ID NO: 236 DVQNKFEGNT
SEQ ID NO: 237 DIQNKIEQTT
SEQ ID NO: 238 DVIKRYEQTT
   
SEQ ID NO: 239 VINKVEQTT
SEQ ID NO: 240 VINKVEQST
SEQ ID NO: 241 VINKVEQKT
SEQ ID NO: 242 VLNKVEQTT
SEQ ID NO: 243 VVNKVEQTT
SEQ ID NO: 244 VINKVESTT
SEQ ID NO: 245 VINKVEQNT
SEQ ID NO: 246 VVPKVEGDT
SEQ ID NO: 247 VIPKVEGDT
SEQ ID NO: 248 IIPRVEQNT
SEQ ID NO: 249 IIPRLEQNT
SEQ ID NO: 250 IIPRVEQDT
SEQ ID NO: 251 VIPKVEGTT
SEQ ID NO: 252 IIPKVEQKT
SEQ ID NO: 253 VIPKVEGST
SEQ ID NO: 254 IIPKVEHNT
SEQ ID NO: 255 VIPKVEQNT
SEQ ID NO: 256 VIPKVEHNT
SEQ ID NO: 257 VINKVEHTT
SEQ ID NO: 258 VRNKVEGTT
SEQ ID NO: 259 VKNKVEADT
SEQ ID NO: 260 VKNKVEANT
SEQ ID NO: 261 VKNKVENNT
SEQ ID NO: 262 VKNKVEQTT
SEQ ID NO: 263 VKNKFENST
SEQ ID NO: 264 VQNKFEGNT
SEQ ID NO: 265 IQNKIEQTT
SEQ ID NO: 266 VIKRYEQTT
   
SEQ ID NO: 267 INKVEQTT
SEQ ID NO: 268 INKVEQST
SEQ ID NO: 269 INKVEQKT
SEQ ID NO: 270 LNKVEQTT
SEQ ID NO: 271 VNKVEQTT
SEQ ID NO: 272 INKVESTT
SEQ ID NO: 273 INKVEQNT
SEQ ID NO: 274 VPKVEGDT
SEQ ID NO: 275 IPKVEGDT
SEQ ID NO: 276 IPRVEQNT
SEQ ID NO: 277 IPRLEQNT
SEQ ID NO: 278 IPRVEQDT
SEQ ID NO: 279 IPKVEGTT
SEQ ID NO: 280 IPKVEHKT
SEQ ID NO: 281 IPKVEGST
SEQ ID NO: 282 IPKVEHNT
SEQ ID NO: 283 IPKVEQNT
SEQ ID NO: 284 INKVEHTT
SEQ ID NO: 285 RNKVEGTT
SEQ ID NO: 286 KNKVEADT
SEQ ID NO: 287 KNKVEANT
SEQ ID NO: 288 KNKVENNT
SEQ ID NO: 289 KNKVEQTT
SEQ ID NO: 290 KNKFENST
SEQ ID NO: 291 QNKFEGNT
SEQ ID NO: 292 QNKIEQTT
SEQ ID NO: 293 IKRYEQTT
   
SEQ ID NO: 294 TGYIPLQTR
SEQ ID NO: 295 TGYVPLGST
SEQ ID NO: 296 TGYVPLGNT
SEQ ID NO: 297 TGYVPLSTG
SEQ ID NO: 298 TGYIPLQST
SEQ ID NO: 299 TGYVPVGST
SEQ ID NO: 300 TGYVPLQTS
SEQ ID NO: 301 TGYVPLTTG
   
SEQ ID NO: 302 RGCKQXGTCPPDVINKVEQ
SEQ ID NO: 303 RGCKAXNTCPPDVINKVEQ
SEQ ID NO: 304 RGCKAXGTCPPDVINKVEQ
SEQ ID NO: 305 QSCKAXGTCPPDVLNKVEQ
SEQ ID NO: 306 QSCKAXGTCPPDVVNKVEQ
SEQ ID NO: 307 QTCKQXGTCPPDVINKVEQ
SEQ ID NO: 308 QTCKQXGTCPPDVVNKVEQ
SEQ ID NO: 309 RTCKQXGTCPPDVINKVES
SEQ ID NO: 310 RTCKQXGTCPPDVINKVEQ
SEQ ID NO: 311 KGCKAXGTCPPDVINKVEQ
SEQ ID NO: 312 RTCKQXGTCPPDVVPKVEG
SEQ ID NO: 313 RTCKQXGTCPPDVIPKVEG
SEQ ID NO: 314 RTCKVXGTCPADVVPKVEG
SEQ ID NO: 315 RTCKAXGTRPADVIPKVEG
SEQ ID NO: 316 STCKAXGTCPPDVIPKLEG
SEQ ID NO: 317 RTCKQXGTCPPDIIPRLEQ
SEQ ID NO: 318 RTCKQXGTCPPDIIPRVEQ
SEQ ID NO: 319 KTCKVXGTCPPDVIPKVEG
SEQ ID NO: 320 KTCKAXGTCPPDVIPKVEG
SEQ ID NO: 321 STCKAXGTCPPDVIPKVEG
SEQ ID NO: 322 RTCKAXGTCPPDVIPKVEG
SEQ ID NO: 323 STCKAXGTCPADVIPKVEG
SEQ ID NO: 324 KTCKLXGTCPEDVINKVEQ
SEQ ID NO: 325 KTCKQXGTCPPDIIPKIEG
SEQ ID NO: 326 KTCKQXGTCPPDIVPKVEG
SEQ ID NO: 327 STCKQXGTCPPDIIPRVEQ
SEQ ID NO: 328 KTCKQXGTCPPDVIPKVEG
SEQ ID NO: 329 QTCKAXGTCPSDIIPKVEH
SEQ ID NO: 330 QTCKAXGTCPPDVIPKVEQ
SEQ ID NO: 331 QTCKLXGTCPPDVIPKVEH
SEQ ID NO: 332 QTCKAXGTCPSDVINKVEH
SEQ ID NO: 333 KQCQLXADCPPDVRNKVEG
SEQ ID NO: 334 AKCQLXGNCLPDVKNKVEA
SEQ ID NO: 335 AKCQLXGDCLPDVKNKVEA
SEQ ID NO: 336 RHCALXGTCPDDVKNKVEN
SEQ ID NO: 337 KHCAGXGTCPEDVKNKVEQ
SEQ ID NO: 338 KTCLQXGDCIPDVKNKFEN
SEQ ID NO: 339 RSCLQXGDCIPDVQNKFEG
SEQ ID NO: 340 QTCKAXGTCPPDVIPKVEG
SEQ ID NO: 341 KTCKQXGTCPPDVVPKVEG
SEQ ID NO: 342 RTCKQXGTCPPDVINKVEG
SEQ ID NO: 343 KTCKQXGTCPSDVINKVEG
SEQ ID NO: 344 KTCKLXGTCPEDVVNKIEQ
SEQ ID NO: 345 RTCKQXGTCPPDVVDKVEG
SEQ ID NO: 346 STCKAXGTCPPDVVNKVEG
SEQ ID NO: 347 PTCKIXGNCPADIQNKFEN
SEQ ID NO: 348 PACKIXNTCPPDIINKYEN
   
***X = Gly (G) or Ala (A)
   
SEQ ID NO: 349 RGCKQXGTCPPD
SEQ ID NO: 350 RGCKAXNTCPPD
SEQ ID NO: 351 RGCKAXGTCPPD
SEQ ID NO: 352 QSCKAXGTCPPD
SEQ ID NO: 353 QTCKQXGTCPPD
SEQ ID NO: 354 RTCKQXGTCPPD
SEQ ID NO: 355 KGCKAXGTCPPD
SEQ ID NO: 356 PTCKAXGTCPPD
SEQ ID NO: 357 RTCKVXGTCPAD
SEQ ID NO: 358 RTCKAXGTRPAD
SEQ ID NO: 359 KTCKVXGTCPPD
SEQ ID NO: 360 KTCKAXGTCPPD
SEQ ID NO: 361 RTCKAXGTCPPD
SEQ ID NO: 362 STCKAXGTRPPD
SEQ ID NO: 363 STCKAXGTCPAD
SEQ ID NO: 364 KTCKLXGTCPED
SEQ ID NO: 365 ATCKQXGTCPPD
SEQ ID NO: 366 STCKQXGTCPPD
SEQ ID NO: 367 QTCKAXGTCPSD
SEQ ID NO: 368 QTCKLXGTCPPD
SEQ ID NO: 369 KQCQLXADCPPD
SEQ ID NO: 370 AKCQLXGNCLPD
SEQ ID NO: 371 AKCQLXGDCLPD
SEQ ID NO: 372 RHCALXGTCPDD
SEQ ID NO: 373 KHCAGXGTCPED
SEQ ID NO: 374 KTCLQXGDCIPD
SEQ ID NO: 375 RSCLQXGDCIPD
SEQ ID NO: 376 QTCKAXGTCPPD
SEQ ID NO: 377 KTCKQXGTCPED
SEQ ID NO: 378 KTCKQXGTCPSD
SEQ ID NO: 379 STCKAXGTCPPD
SEQ ID NO: 380 PTCKIXGNCPAD
SEQ ID NO: 381 PACKIXNTCPPD
   
***X = Gly (G) or Ala (A)
   
SEQ ID NO: 382 GCKQXGTCPPD
SEQ ID NO: 383 GCKAXNTCPPD
SEQ ID NO: 384 ACKAXGTCPPD
SEQ ID NO: 385 SCKAXGTCPPD
SEQ ID NO: 386 KCKAXGTCIPD
SEQ ID NO: 387 GCKAXGTCPPD
SEQ ID NO: 388 KCKAXGTCPPD
SEQ ID NO: 389 TCKVXGTCPAD
SEQ ID NO: 390 TCKAXGTRPAD
SEQ ID NO: 391 TCKVXGTCPPD
SEQ ID NO: 392 SCKLXGTCPPD
SEQ ID NO: 393 SCKQXGTCPSD
SEQ ID NO: 394 TCKAXGTCPAD
SEQ ID NO: 395 TCKLXGTCPED
SEQ ID NO: 396 TCKAXGTCPSD
SEQ ID NO: 397 TCKLXGTCPPD
SEQ ID NO: 398 QCQLXADCPPD
SEQ ID NO: 399 KCQLXGNCLPD
SEQ ID NO: 400 KCQLXGDCLPD
SEQ ID NO: 401 HCALXGTCPDD
SEQ ID NO: 402 HCAGXGTCPED
SEQ ID NO: 403 TCLQXGDCIPD
SEQ ID NO: 404 SCLQXGDCIPD
SEQ ID NO: 405 TCKAXGTCPPD
SEQ ID NO: 406 TCKQXGTCPSD
SEQ ID NO: 407 TCKIXGNCPAD
SEQ ID NO: 408 ACKIXNTCPPD
   
***X = Gly (G) or Ala (A)
   
SEQ ID NO: 409 CKQXGTCPDD
SEQ ID NO: 410 CKAXNTCPPD
SEQ ID NO: 411 CLAXGTCPAD
SEQ ID NO: 412 CLAXGTCPPD
SEQ ID NO: 413 CKLXGTCPAD
SEQ ID NO: 414 CKVXGTCPAD
SEQ ID NO: 415 CKAXGTRPAD
SEQ ID NO: 416 CKVXGTCPPD
SEQ ID NO: 417 CKAXGTCPAD
SEQ ID NO: 418 CKLXGTCPED
SEQ ID NO: 419 CKAXGTCPSD
SEQ ID NO: 420 CKLXGTCPPD
SEQ ID NO: 421 CQLXADCPPD
SEQ ID NO: 422 CQLXGNCLPD
SEQ ID NO: 423 CQLXGDCLPD
SEQ ID NO: 424 CALXGTCPDD
SEQ ID NO: 425 CAGXGTCPED
SEQ ID NO: 426 CLQXGDCIPD
SEQ ID NO: 427 CKAXGTCPPD
SEQ ID NO: 428 CKQXGTCPSD
SEQ ID NO: 429 CKIXGNCPAD
SEQ ID NO: 430 CKIXNTCPPD
   
***X = Gly (G) or Ala (A)
   
SEQ ID NO: 486 KTCKQSGTCPSDVVNKVEG
SEQ ID NO: 487 QTCKAAGTCPSDVIPKIEH
SEQ ID NO: 488 KTCKQSGTCPPDVIDKVEG
SEQ ID NO: 489 STCKAAGTCPPDVIPKVKG
SEQ ID NO: 490 KTCKQSGTCPSD
   
SEQ ID NO: 491 ((SEQ ID NO:2)x3+ (SEQ ID NO: 487)x3 +(SEQ ID NO:77)x3 with a tripeptide (GGP) linker):


 
   
SEQ ID NO: 492 ((SEQ ID NO:2)+ (SEQ ID NO:487) +(SEQ ID NO:77))x3 with a tripeptide (GGP) linker


 
Table 2: List of thioredoxin variants
SEQ ID NO: 493 (variant thiorexodin polypeptide from hyperthermophile archaebacterium Pyrococcus furiosus)


 
SEQ ID NO: 494 (variant thiorexodin polypeptide from hyperthermophile archaebacterium Thermococcus kodakarensis)


 
 
SEQ ID NO: 495 (variant thiorexodin polypeptide from hyperthermophile archaebacterium Thermococcus onnurineus)


 
 
SEQ ID NO: 496 (variant of thiorexodin polypeptide from hyperthermophile archaebacterium Thermococcus sibiricus)


 
 
SEQ ID NO: 497 (Dimer of Escherichia coli thioredoxin variants)


 
 
SEQ ID NO: 498 (Trimer of Escherichia coli thioredoxin variants)


 

SEQUENCE LISTING



[0075] 

<110> DKFZ Deutsches Krebsforschungszentrum, Stiftung des öffentlichen Rechts

<120> Immunogenic polypeptides comprising a scaffold polypeptide and a L2 polypeptide or fragment

<130> DK65846PC

<160> 498

<170> PatentIn version 3.5

<210> 1
<211> 120
<212> PRT
<213> Human papillomavirus type 16

<400> 1

<210> 2
<211> 19
<212> PRT
<213> Human papillomavirus type 16

<400> 2



<210> 3
<211> 12
<212> PRT
<213> Human papillomavirus type 16

<400> 3

<210> 4
<211> 11
<212> PRT
<213> Human papillomavirus type 16

<400> 4

<210> 5
<211> 10
<212> PRT
<213> Human papillomavirus type 16

<400> 5

<210> 6
<211> 10
<212> PRT
<213> Human papillomavirus type 16

<400> 6

<210> 7
<211> 9
<212> PRT
<213> Human papillomavirus type 16

<400> 7

<210> 8
<211> 10
<212> PRT
<213> Human papillomavirus type 16

<400> 8

<210> 9
<211> 9
<212> PRT
<213> Human papillomavirus type 16

<400> 9

<210> 10
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 10

<210> 11
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 11

<210> 12
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 12

<210> 13
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 13

<210> 14
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 14

<210> 15
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 15

<210> 16
<211> 3
<212> PRT
<213> artificial

<220>
<223> linker peptide

<400> 16

<210> 17
<211> 4
<212> PRT
<213> artificial

<220>
<223> linker peptide

<400> 17

<210> 18
<211> 5
<212> PRT
<213> artificial

<220>
<223> linker peptide

<400> 18

<210> 19
<211> 4
<212> PRT
<213> artificial

<220>
<223> linker peptide

<400> 19

<210> 20
<211> 63
<212> PRT
<213> artificial

<220>
<223> Multimers with (GGP) linker

<400> 20

<210> 21
<211> 195
<212> PRT
<213> artificial

<220>
<223> Multimers with (GGP) linker

<400> 21



<210> 22
<211> 72
<212> PRT
<213> artificial

<220>
<223> Multimers with (GGP) linker

<400> 22

<210> 23
<211> 4
<212> PRT
<213> artificial

<220>
<223> Thioredoxin display site

<400> 23

<210> 24
<211> 109
<212> PRT
<213> Escherichia coli

<400> 24

<210> 25
<211> 109
<212> PRT
<213> Salmonella enterica

<400> 25



<210> 26
<211> 105
<212> PRT
<213> Mus musculus

<400> 26

<210> 27
<211> 105
<212> PRT
<213> Rattus norvegicus

<400> 27

<210> 28
<211> 105
<212> PRT
<213> Homo sapiens

<400> 28

<210> 29
<211> 216
<212> PRT
<213> artificial

<220>
<223> immunogenic polypeptide

<400> 29



<210> 30
<211> 348
<212> PRT
<213> artificial

<220>
<223> immunogenic polypeptide

<400> 30



<210> 31
<211> 9
<212> PRT
<213> Human papillomavirus type 16

<400> 31

<210> 32
<211> 8
<212> PRT
<213> Human papillomavirus type 16

<400> 32

<210> 33
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 33

<210> 34
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 34

<210> 35
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 35

<210> 36
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 36

<210> 37
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 37

<210> 38
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 38

<210> 39
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 39

<210> 40
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 40

<210> 41
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 41

<210> 42
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 42

<210> 43
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 43

<210> 44
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 44

<210> 45
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 45

<210> 46
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 46

<210> 47
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 47

<210> 48
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 48

<210> 49
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 49

<210> 50
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 50

<210> 51
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 51

<210> 52
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 52

<210> 53
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 53

<210> 54
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 54

<210> 55
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 55

<210> 56
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 56

<210> 57
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 57

<210> 58
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 58

<210> 59
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 59

<210> 60
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 60

<210> 61
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 61

<210> 62
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 62

<210> 63
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 63

<210> 64
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 64

<210> 65
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 65

<210> 66
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 66

<210> 67
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 67

<210> 68
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 68

<210> 69
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 69

<210> 70
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 70

<210> 71
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 71

<210> 72
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 72

<210> 73
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 73

<210> 74
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 74

<210> 75
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 75

<210> 76
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 76

<210> 77
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 77

<210> 78
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 78

<210> 79
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 79

<210> 80
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 80

<210> 81
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 81

<210> 82
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 82

<210> 83
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 83

<210> 84
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 84

<210> 85
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 85

<210> 86
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 86

<210> 87
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 87

<210> 88
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 88

<210> 89
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 89

<210> 90
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 90

<210> 91
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 91

<210> 92
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 92

<210> 93
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 93

<210> 94
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 94

<210> 95
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 95

<210> 96
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 96

<210> 97
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 97

<210> 98
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 98

<210> 99
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 99

<210> 100
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 100

<210> 101
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 101

<210> 102
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 102

<210> 103
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 103

<210> 104
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 104

<210> 105
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 105

<210> 106
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 106

<210> 107
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 107

<210> 108
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 108

<210> 109
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 109

<210> 110
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 110

<210> 111
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 111

<210> 112
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 112

<210> 113
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 113

<210> 114
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 114

<210> 115
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 115

<210> 116
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 116

<210> 117
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 117

<210> 118
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 118

<210> 119
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 119

<210> 120
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 120

<210> 121
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 121

<210> 122
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 122

<210> 123
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 123

<210> 124
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 124

<210> 125
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 125

<210> 126
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 126

<210> 127
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 127

<210> 128
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 128

<210> 129
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 129

<210> 130
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 130

<210> 131
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 131

<210> 132
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 132

<210> 133
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 133

<210> 134
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 134

<210> 135
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 135

<210> 136
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 136

<210> 137
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 137

<210> 138
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 138

<210> 139
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 139

<210> 140
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 140

<210> 141
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 141

<210> 142
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 142

<210> 143
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 143

<210> 144
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 144

<210> 145
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 145

<210> 146
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 146

<210> 147
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 147

<210> 148
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 148

<210> 149
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 149

<210> 150
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 150

<210> 151
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 151

<210> 152
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 152

<210> 153
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 153

<210> 154
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 154

<210> 155
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 155

<210> 156
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 156

<210> 157
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 157

<210> 158
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 158

<210> 159
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 159

<210> 160
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 160

<210> 161
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 161

<210> 162
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 162

<210> 163
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 163

<210> 164
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 164

<210> 165
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 165

<210> 166
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 166

<210> 167
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 167

<210> 168
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 168

<210> 169
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 169

<210> 170
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 170

<210> 171
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 171

<210> 172
<211> 10
<212> PRT
<213> artifiicial

<400> 172

<210> 173
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 173

<210> 174
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 174

<210> 175
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 175

<210> 176
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 176

<210> 177
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 177

<210> 178
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 178

<210> 179
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 179

<210> 180
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 180

<210> 181
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 181

<210> 182
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 182

<210> 183
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 183

<210> 184
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 184

<210> 185
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 185

<210> 186
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 186

<210> 187
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 187

<210> 188
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 188

<210> 189
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 189

<210> 190
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 190

<210> 191
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 191

<210> 192
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 192

<210> 193
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 193

<210> 194
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 194

<210> 195
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 195

<210> 196
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 196

<210> 197
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 197

<210> 198
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 198

<210> 199
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 199

<210> 200
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 200

<210> 201
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 201

<210> 202
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 202

<210> 203
<211> 9
<212> PRT
<213> artifical

<400> 203

<210> 204
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 204

<210> 205
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 205

<210> 206
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 206

<210> 207
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 207

<210> 208
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 208

<210> 209
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 209

<210> 210
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 210

<210> 211
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 211

<210> 212
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 212

<210> 213
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 213

<210> 214
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 214

<210> 215
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 215

<210> 216
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 216

<210> 217
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 217

<210> 218
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 218

<210> 219
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 219

<210> 220
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 220

<210> 221
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 221

<210> 222
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 222

<210> 223
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 223

<210> 224
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 224

<210> 225
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 225

<210> 226
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 226

<210> 227
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 227

<210> 228
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 228

<210> 229
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 229

<210> 230
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 230

<210> 231
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 231

<210> 232
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 232

<210> 233
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 233

<210> 234
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 234

<210> 235
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 235

<210> 236
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 236

<210> 237
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 237

<210> 238
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 238

<210> 239
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 239

<210> 240
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 240

<210> 241
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 241

<210> 242
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 242

<210> 243
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 243

<210> 244
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 244

<210> 245
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 245

<210> 246
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 246

<210> 247
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 247

<210> 248
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 248

<210> 249
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 249

<210> 250
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 250

<210> 251
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 251

<210> 252
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 252

<210> 253
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 253

<210> 254
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 254

<210> 255
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 255

<210> 256
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 256

<210> 257
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 257

<210> 258
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 258

<210> 259
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 259

<210> 260
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 260

<210> 261
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 261

<210> 262
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 262

<210> 263
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 263

<210> 264
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 264

<210> 265
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 265

<210> 266
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 266

<210> 267
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 267

<210> 268
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 268

<210> 269
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 269

<210> 270
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 270

<210> 271
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 271

<210> 272
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 272

<210> 273
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 273

<210> 274
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 274

<210> 275
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 275

<210> 276
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 276

<210> 277
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 277

<210> 278
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 278

<210> 279
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 279

<210> 280
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 280

<210> 281
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 281

<210> 282
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 282

<210> 283
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 283

<210> 284
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 284

<210> 285
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 285

<210> 286
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 286

<210> 287
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 287

<210> 288
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 288

<210> 289
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 289

<210> 290
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 290

<210> 291
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 291

<210> 292
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 292

<210> 293
<211> 8
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 293

<210> 294
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 294

<210> 295
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 295

<210> 296
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 296

<210> 297
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 297

<210> 298
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 298

<210> 299
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 299

<210> 300
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 300

<210> 301
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<400> 301

<210> 302
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 302

<210> 303
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 303

<210> 304
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represent Gly or Ala

<400> 304

<210> 305
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 305

<210> 306
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 306

<210> 307
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 307

<210> 308
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 308

<210> 309
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 309

<210> 310
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 310



<210> 311
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 311

<210> 312
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 312

<210> 313
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 313

<210> 314
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 314

<210> 315
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 315

<210> 316
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 316

<210> 317
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 317

<210> 318
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 318

<210> 319
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 319

<210> 320
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 320

<210> 321
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 321

<210> 322
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 322

<210> 323
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 323

<210> 324
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 324

<210> 325
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 325

<210> 326
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 326

<210> 327
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 327

<210> 328
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 328

<210> 329
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 329

<210> 330
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 330

<210> 331
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 331



<210> 332
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 332

<210> 333
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represenst Gly or Ala

<400> 333

<210> 334
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 334

<210> 335
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 335

<210> 336
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 336

<210> 337
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 337

<210> 338
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 338

<210> 339
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 339

<210> 340
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 340

<210> 341
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 341

<210> 342
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 342

<210> 343
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 343

<210> 344
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 344

<210> 345
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 345

<210> 346
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 346

<210> 347
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 347

<210> 348
<211> 19
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 348

<210> 349
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 349

<210> 350
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 350

<210> 351
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represent Gly or Ala

<400> 351

<210> 352
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 352

<210> 353
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 353

<210> 354
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represents Gly or Ala

<400> 354

<210> 355
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa represent Gly or Ala

<400> 355

<210> 356
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 356

<210> 357
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 357

<210> 358
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 358

<210> 359
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 359



<210> 360
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 360

<210> 361
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 361

<210> 362
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 362

<210> 363
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 363

<210> 364
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 364

<210> 365
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 365

<210> 366
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 366

<210> 367
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 367

<210> 368
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 368

<210> 369
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 369

<210> 370
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 370

<210> 371
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 371

<210> 372
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 372

<210> 373
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 373

<210> 374
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 374

<210> 375
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 375

<210> 376
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 376

<210> 377
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 377

<210> 378
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 378

<210> 379
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 379

<210> 380
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 380

<210> 381
<211> 12
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (6) .. (6)
<223> Xaa represents Gly or Ala

<400> 381

<210> 382
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 382

<210> 383
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 383

<210> 384
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 384

<210> 385
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 385

<210> 386
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 386

<210> 387
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 387

<210> 388
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 388

<210> 389
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 389

<210> 390
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 390

<210> 391
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 391

<210> 392
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 392

<210> 393
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 393

<210> 394
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 394

<210> 395
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 395

<210> 396
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 396

<210> 397
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 397

<210> 398
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 398

<210> 399
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 399

<210> 400
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 400

<210> 401
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 401

<210> 402
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 402

<210> 403
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 403

<210> 404
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 404

<210> 405
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 405

<210> 406
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 406

<210> 407
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 407

<210> 408
<211> 11
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 408

<210> 409
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represent Gly or Ala

<400> 409

<210> 410
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 410

<210> 411
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 411

<210> 412
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 412

<210> 413
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 413

<210> 414
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 414

<210> 415
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 415

<210> 416
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 416

<210> 417
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 417

<210> 418
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 418

<210> 419
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> L2 fragment

<400> 419

<210> 420
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 420

<210> 421
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 421

<210> 422
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 422



<210> 423
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 423

<210> 424
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 424

<210> 425
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 425

<210> 426
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 426

<210> 427
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 427

<210> 428
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 428

<210> 429
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 429

<210> 430
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 430

<210> 431
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 431

<210> 432
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 432

<210> 433
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 433

<210> 434
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 434

<210> 435
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 435

<210> 436
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 436

<210> 437
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 437

<210> 438
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 438

<210> 439
<211> 10
<212> PRT
<213> artificial

<220>
<223> Xaa represents Gly or Ala

<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa can be any naturally occurring amino acid

<400> 439

<210> 440
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 440

<210> 441
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 441

<210> 442
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 442

<210> 443
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 443

<210> 444
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 444

<210> 445
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 445

<210> 446
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 446

<210> 447
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 447

<210> 448
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 448

<210> 449
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 449

<210> 450
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 450

<210> 451
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 451

<210> 452
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 452

<210> 453
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 453

<210> 454
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 454

<210> 455
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 455

<210> 456
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 456

<210> 457
<211> 10
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa represents Gly or Ala

<400> 457

<210> 458
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 458

<210> 459
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 459

<210> 460
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 460

<210> 461
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 461

<210> 462
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 462

<210> 463
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 463

<210> 464
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 464

<210> 465
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 465

<210> 466
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 466

<210> 467
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 467

<210> 468
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 468

<210> 469
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 469



<210> 470
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 470

<210> 471
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 471

<210> 472
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 472

<210> 473
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 473

<210> 474
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 474

<210> 475
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 475

<210> 476
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 476

<210> 477
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 477

<210> 478
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 478

<210> 479
<211> 9
<212> PRT
<213> artificial

<220>
<223> L2 fragment

<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa represents Gly or Ala

<400> 479

<210> 480
<211> 6
<212> PRT
<213> artificial

<220>
<223> loop site of FN3

<400> 480

<210> 481
<211> 9
<212> PRT
<213> artificial

<220>
<223> loop site of FN3

<400> 481

<210> 482
<211> 7
<212> PRT
<213> artificial

<220>
<223> loop site of bilin-binding protein

<400> 482

<210> 483
<211> 5
<212> PRT
<213> artificial

<220>
<223> loop site of bilin-binding protein

<400> 483

<210> 484
<211> 7
<212> PRT
<213> artificial

<220>
<223> loop-site of bilin-binding protein

<400> 484

<210> 485
<211> 5
<212> PRT
<213> artificial

<220>
<223> loop-site

<400> 485

<210> 486
<211> 19
<212> PRT
<213> Human papillomavirus type 18

<400> 486

<210> 487
<211> 19
<212> PRT
<213> Human papillomavirus type 31

<400> 487

<210> 488
<211> 19
<212> PRT
<213> Human papillomavirus type 68

<400> 488

<210> 489
<211> 19
<212> PRT
<213> Human Paillomavirus Type 82

<400> 489

<210> 490
<211> 12
<212> PRT
<213> Human papillomavirus type 18

<400> 490

<210> 491
<211> 195
<212> PRT
<213> Artificial Sequence

<220>
<223> (SEQ ID NO:2)x3+ (SEQ ID NO: 487)x3 +(SEQ ID NO:77)x3 with tripeptide (GGP) linkers

<400> 491



<210> 492
<211> 195
<212> PRT
<213> Artificial Sequence

<220>
<223> ((SEQ ID NO:2)+ (SEQ ID NO:487) +(SEQ ID NO:77))x3 with tripeptide (GGP) linkers

<400> 492



<210> 493
<211> 100
<212> PRT
<213> Pyrococcus furiosus

<400> 493



<210> 494
<211> 101
<212> PRT
<213> Thermococcus kodakarensis

<400> 494

<210> 495
<211> 105
<212> PRT
<213> Thermococcus onnurineus

<400> 495



<210> 496
<211> 100
<212> PRT
<213> Thermococcus sibiricus

<400> 496

<210> 497
<211> 253
<212> PRT
<213> Artificial Sequence

<220>
<223> Dimer of Escherichia coli thioredoxin variants

<400> 497



<210> 498
<211> 396
<212> PRT
<213> Artificial

<220>
<223> Trimer of Escherichia coli thioredoxin variants

<400> 498






Claims

1. An immunogenic polypeptide comprising

a) a multimer (i) of an L2 polypeptide comprising an amino acid sequence as shown in SEQ ID NO:1 or a variant thereof, or (ii) of a fragment or fragments of said L2 polypeptide or variants thereof,
wherein the amino acid sequence of said variants is at least 65% identical with said L2 polypeptide or fragment of said L2 polypeptide, and
wherein said fragments comprise at least 7 consecutive amino acid residues of said L2 polypeptide;
and

b) a scaffold polypeptide,

wherein said scaffold polypeptide constrains the structure of said L2 polypeptides or variants thereof, or of said fragments of said L2 polypeptide or variants thereof, and
wherein said scaffold polypeptide is selected from the group consisting of thioredoxin polypeptides and thioredoxin polypeptides derived from thermophile bacteria.
 
2. The immunogenic polypeptide of claim 1, wherein the scaffold polypeptide is a thioredoxin polypeptide selected from the group consisting of

a) a polypeptide having a sequence as shown in SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, or SEQ ID No: 28, and

b) a variant polypeptide having a sequence at least 70% identical to the sequence shown in SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, or SEQ ID No: 28,

wherein said polypeptide constrains the structure of the HPV L2 polypeptide, or which constrains the structure of a fragment of said HPV L2 polypeptide.
 
3. The immunogenic polypeptide according to claim 1 or 2, wherein the fragment or variant of the HPV L2 polypeptide is selected from the group consisting of SEQ ID NO: 2 to 15, 20 to 22, and 31 to 479.
 
4. The immunogenic polypeptide according to anyone of claims 1 to 3, wherein the fragment of the HPV L2 polypeptide is selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 31 and 32.
 
5. The immunogenic polypeptide according to any one of claims 1 to 5, wherein the fragment of the HPV L2 polypeptide has a sequence as shown in SEQ ID NO:2.
 
6. The immunogenic polypeptide according to any one of claims 1 to 5, comprising a multimer of 2 to 15 fragments of said L2 polypeptide or variants thereof.
 
7. The immunogenic polypeptide according to any one of claims 1 to 6, wherein the immunogenic polypeptide has a sequence as shown in SEQ ID NO: 29 or 30.
 
8. A polynucleotide encoding the immunogenic polypeptide according to anyone of claims 1 to 7.
 
9. A vaccine comprising the immunogenic polypeptide according to any one of claims 1 to 7.
 
10. Use of the immunogenic polypeptide according to any one of claims 1 to 7 for the preparation of a vaccine for immunization of a subject against infection with HPV.
 
11. A method for producing an antibody against the immunogenic polypeptide of any one of claims 1 to 7 comprising the steps:

a) providing said immunogenic polypeptide,

b) immunizing a non-human host with said immunogenic polypeptide, and

c) harvesting the antibody against said immunogenic
polypeptide.


 
12. The method of claim 11, wherein the antibody is against HPV genotypes 52, 2, 27, 57 and/or 11.
 
13. The method of claim 10 or 11, wherein the antibody is polyclonal or monoclonal.
 
14. An immunogenic polypeptide comprising

a) a scaffold polypeptide, and

b) an L2 polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 1, or a fragment of said L2 polypeptide,

wherein said scaffold polypeptide constrains the structure of said L2 polypeptide, or of said fragment of said L2 polypeptide,
wherein said scaffold polypeptide is a thioredoxin polypeptide derived from thermophile bacteria, and
wherein said fragment comprises at least 7 consecutive amino acid residues of said L2 polypeptide.
 


Ansprüche

1. Immunogenes Polypeptid, umfassend

a) ein Multimer (i) eines L2-Polypeptids, das eine Aminosäuresequenz gemäß SEQ ID NO:1 umfasst, oder einer Variante davon oder (ii) eines Fragments bzw. von Fragmenten des L2-Polypeptids oder von Varianten davon,
wobei die Aminosäuresequenz der Varianten zu wenigstens 65% mit dem L2-Polypeptid bzw. Fragment des L2-Polypeptids identisch ist und wobei die Fragmente wenigstens 7 aufeinander folgende Aminosäurereste des L2-Polypeptids umfassen;
und

b) ein Gerüstpolypeptid,

wobei das Gerüstpolypeptid die Struktur der L2-Polypeptide oder Varianten davon bzw. der Fragmente des L2-Polypeptids oder Varianten davon Beschränkungen unterwirft und
wobei das Gerüstpolypeptid aus der aus Thioredoxin-Polypeptiden und aus thermophilen Bakterien stammenden Thioredoxin-Polypeptiden bestehenden Gruppe ausgewählt ist.
 
2. Immunogenes Polypeptid nach Anspruch 1, wobei es sich bei dem Gerüstpolypeptid um ein Thioredoxin-Polypeptid handelt, das aus der aus

a) einem Polypeptid mit einer Sequenz gemäß SEQ ID Nr. 24, SEQ ID Nr. 25, SEQ ID Nr. 26, SEQ ID Nr. 27 oder SEQ ID Nr. 28 und

b) einer Polypeptidvariante mit einer Sequenz, die zu wenigstens 70% mit der Sequenz gemäß SEQ ID Nr. 24, SEQ ID Nr. 25, SEQ ID Nr. 26, SEQ ID Nr. 27 bzw. SEQ ID Nr. 28 identisch ist,

bestehenden Gruppe ausgewählt ist, wobei das Polypeptid die Struktur des HPV-L2-Polypeptids Beschränkungen unterwirft oder die Struktur eines Fragments des HPV-L2-Polypeptids Beschränkungen unterwirft.
 
3. Immunogenes Polypeptid gemäß Anspruch 1 oder 2, wobei das Fragment bzw. die Variante des HPV-L2-Polypeptids aus der aus SEQ ID NO: 2 bis 15, 20 bis 22 und 31 bis 479 bestehenden Gruppe ausgewählt ist.
 
4. Immunogenes Polypeptid gemäß einem der Ansprüche 1 bis 3, wobei das Fragment des HPV-L2-Polypeptids aus der aus SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 31 und 32 bestehenden Gruppe ausgewählt ist.
 
5. Immunogenes Polypeptid gemäß einem der Ansprüche 1 bis 5, wobei das Fragment des HPV-L2-Polypeptids eine Sequenz gemäß SEQ ID NO: 2 aufweist.
 
6. Immunogenes Polypeptid gemäß einem der Ansprüche 1 bis 5, umfassend ein Multimer von 2 bis 15 Fragmenten des L2-Polypeptids oder Varianten davon.
 
7. Immunogenes Polypeptid gemäß einem der Ansprüche 1 bis 6, wobei das immunogene Polypeptid eine Sequenz gemäß SEQ ID NO: 29 oder 30 aufweist.
 
8. Polynukleotid, codierend das immunogene Polypeptid gemäß einem der Ansprüche 1 bis 7.
 
9. Impfstoff, umfassend das immunogene Polypeptid gemäß einem der Ansprüche 1 bis 7.
 
10. Verwendung des immunogenen Polypeptids gemäß einem der Ansprüche 1 bis 7 zur Herstellung eines Impfstoffs für die Immunisierung eines Individuums gegen eine Infektion mit HPV.
 
11. Verfahren zur Erzeugung eines Antikörpers gegen das immunogene Polypeptid nach einem der Ansprüche 1 bis 7, umfassend die Schritte:

a) Bereitstellen des immunogenen Polypeptids,

b) Immunisieren eines nichtmenschlichen Wirts mit dem immunogenen Polypeptid und

c) Ernten des Antikörpers gegen das immunogene Polypeptid.


 
12. Verfahren nach Anspruch 11, wobei es sich bei dem Antikörper um einen Antikörper gegen HPV-Genotypen 52, 2, 27, 57 und/oder 11 handelt.
 
13. Verfahren nach Anspruch 10 oder 11, wobei der Antikörper polyklonal oder monoklonal ist.
 
14. Immunogenes Polypeptid, umfassend

a) ein Gerüstpolypeptid und

b) ein L2-Polypeptid, das eine Aminosäuresequenz gemäß SEQ ID NO:1 umfasst, oder ein Fragment des L2-Polypeptids,

wobei das Gerüstpolypeptid die Struktur des L2-Polypeptids bzw. des Fragments des L2-Polypeptids Beschränkungen unterwirft,
wobei es sich bei dem Gerüstpolypeptid um ein aus thermophilen Bakterien stammendes Thioredoxin-Polypeptid handelt und
wobei das Fragment wenigstens 7 aufeinander folgende Aminosäurereste des L2-Polypeptids umfasst.
 


Revendications

1. Polypeptide immunogène comprenant

a) un multimère (i) d'un polypeptide L2 comprenant une séquence d'acides aminés telle que présentée dans la SEQ ID n° : 1 ou un variant de celle-ci, ou (ii) d'un fragment ou de fragments dudit polypeptide L2 ou de variants de celui/ceux-ci,
dans lequel la séquence d'acides aminés desdits variants est identique, au moins à 65%, au dit polypeptide L2 ou à un fragment dudit polypeptide L2, et
dans lequel lesdits fragments comprennent au moins 7 résidus d'acides aminés consécutifs dudit polypeptide L2 ;
et

b) un polypeptide d'échafaudage,

dans lequel ledit polypeptide d'échafaudage contraint la structure desdits polypeptides L2 ou de variants de ceux-ci, ou desdits fragments dudit polypeptide L2 ou de variants de ceux-ci, et
dans lequel ledit polypeptide d'échafaudage est choisi dans le groupe constitué par des polypeptides de thiorédoxine et polypeptides de thiorédoxine dérivés d'une bactérie thermophile.
 
2. Polypeptide immunogène selon la revendication 1, dans lequel le polypeptide d'échafaudage est un polypeptide de thiorédoxine choisi dans le groupe constitué par

a) un polypeptide ayant une séquence telle que présentée dans la SEQ ID n° : 24, la SEQ ID n° : 25, la SEQ ID n° : 26, la SEQ ID n° : 27 ou la SEQ ID n° : 28, et

b) un polypeptide variant ayant une séquence étant identique, au moins à 70%, à la séquence présentée dans la SEQ ID n° : 24, la SEQ ID n° : 25, la SEQ ID n° : 26, la SEQ ID n° : 27 ou la SEQ ID n° : 28,

dans lequel ledit polypeptide contraint la structure du polypeptide L2 du HPV ou contraint la structure d'un fragment dudit polypeptide L2 du HPV.
 
3. Polypeptide immunogène selon la revendication 1 ou 2, dans lequel le fragment ou un variant du polypeptide L2 du HPV est choisi dans le groupe constitué par les SEQ ID n° : 2 à 15, 20 à 22 et 31 à 479.
 
4. Polypeptide immunogène selon l'une quelconque des revendications 1 à 3, dans lequel le fragment du polypeptide L2 du HPV est choisi dans le groupe constitué par les SEQ ID n° : 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 31 et 32.
 
5. Polypeptide immunogène selon l'une quelconque des revendications 1 à 5, dans lequel le fragment du polypeptide L2 du HPV a une séquence telle que présentée dans la SEQ ID n° : 2.
 
6. Polypeptide immunogène, selon l'une quelconque des revendications 1 à 5, comprenant un multimère de 2 à 15 fragments dudit polypeptide L2 ou de variants de celui-ci.
 
7. Polypeptide immunogène selon l'une quelconque des revendications 1 à 6, le polypeptide immunogène ayant une séquence telle que présentée dans la SEQ ID n° : 29 ou 30.
 
8. Polynucléotide codant pour le polypeptide immunogène selon l'une quelconque des revendications 1 à 7.
 
9. Vaccin comprenant le polypeptide immunogène selon l'une quelconque des revendications 1 à 7.
 
10. Utilisation du polypeptide immunogène, selon l'une quelconque des revendications 1 à 7, pour la préparation d'un vaccin destiné à l'immunisation d'un sujet contre une infection par HPV.
 
11. Procédé de production d'un anticorps contre le polypeptide immunogène, selon l'une quelconque des revendications 1 à 7, comprenant les étapes consistant à :

a) fournir ledit polypeptide immunogène,

b) immuniser un hôte non humain avec ledit polypeptide immunogène, et

c) recueillir l'anticorps contre ledit polypeptide immunogène.


 
12. Procédé selon la revendication 11, dans lequel l'anticorps est contre les génotypes 52, 2, 27, 57 et/ou 11 du HPV.
 
13. Procédé selon la revendication 10 ou 11, dans lequel l'anticorps est polyclonal ou monoclonal.
 
14. Polypeptide immunogène comprenant

a) un polypeptide d'échafaudage, et

b) un polypeptide L2 comprenant une séquence d'acides aminés telle que présentée dans la SEQ ID n° : 1, ou un fragment dudit polypeptide L2,

dans lequel ledit polypeptide d'échafaudage contraint la structure dudit polypeptide L2 ou dudit fragment dudit polypeptide L2,
dans lequel ledit polypeptide d'échafaudage est un polypeptide de thiorédoxine dérivé d'une bactérie thermophile, et
dans lequel ledit fragment comprend au moins 7 résidus d'acides aminés consécutifs dudit polypeptide L2.
 




Drawing
















































REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description




Non-patent literature cited in the description