HUMAN M2E PEPTIDE IMMUNOGENS

The present invention provides novel peptide immunogens comprising influenza virus matrix 2 protein epitopes and related compositions and methods. The present invention relates to a composition comprising a peptide immunogen useful for the prevention and treatment of an influenza virus-mediated disease. The invention also relates to vaccines, immunogenic products and immunogenic compositions containing the peptide immunogens.

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Description

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 13/523,710, filed Jun. 14, 2012 and issued as U.S. Pat. No. 8,932,597 on Jan. 13, 2015, which is a continuation of U.S. application Ser. No. 12/617,243, filed Nov. 12, 2009 and issued as U.S. Pat. No. 8,329,188 on Dec. 11, 2012, which claims the benefit of and priority to U.S. Provisional Application No. 61/113,880, filed Nov. 12, 2008, the contents of each of which are herein incorporated by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “THER006C02US SeqList.txt,” which was created on Jan. 12, 2015 and is 570 KB in size, are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to vaccines and therapeutics for influenza virus infection. The invention specifically relates to peptide immunogens suitable for generating influenza matrix 2 protein-specific antibodies and their manufacture and use.

BACKGROUND OF THE INVENTION

Influenza virus infects 5-20% of the population and results in 30,000-50,000 deaths each year in the U.S. Although the influenza vaccine is the primary method of infection prevention, four antiviral drugs are also available in the U.S.: amantadine, rimantadine, oseltamivir and zanamivir. As of December 2005, only oseltamivir (TAMIFLU™) is recommended for treatment of influenza A due to the increasing resistance of the virus to amantadine and rimantidine resulting from an amino acid substitution in the M2 protein of the virus. Recently, a drug resistant avian virus was found in a 14-year-girl in Vietnam. Resistance to Tamiflu has also been found in human influenza as well (Mai Le et al., Nature 437:1108 (2005)).

Influenza vaccines have been demonstrated to have a protective effect against influenza infection. However, yearly emerging antigenic variants of influenza viruses necessitate surveillance to contemporary of circulating virus strains. In some cases, difficulty in the prediction of new variant strains has prevented the timely production of the vaccine (Frace et al., Vaccine 17:2237 (1999)). Recently, pandemic avian influenza has become a serious threat due to the emergence of avian influenza viruses such as H5N1 in southern Asia. The currently available vaccines would be ineffective against avian viruses (Lipatov et al., J. Virology 78:8951 (2004); Osterholm et al., N Engl. Med. 352:1839 (2005)). A third problem with the current vaccine is the ineffectiveness in certain populations with compromised immune systems, for instance premature infants, the elderly, AIDS and transplant patients.

Disease caused by influenza A viral infections is typified by its cyclical nature. Antigenic drift and shift allow for different A strains to emerge every year. Added to that, the threat of highly pathogenic strains entering into the general population has stressed the need for novel therapies for flu infections.

SUMMARY OF THE INVENTION

The present invention relates to a synthetic peptide immunogen capable of inducing antibodies against a M2e target peptide of influenza A virus. In particular, the peptide immunogen of this invention comprises one or more epitopes. Optionally, the peptide immunogen further comprises a general immune stimulator. These peptide immunogens of the present invention are effective, capable of inducing antibodies against influenza A virus to prevent infection by the virus.

The peptide immunogen of this invention is represented by the following formula: [Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-[Xaa7]q-[Xaa8-[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-[Xaa7]q]n; wherein, m, p and q are independently 0 or 1, n is any number between 0 and 4, Xaa0 is any amino acid, preferably C; Xaa6 is any amino acid, preferably V or C; Xaa7 is any amino acid, preferably E; Xaa8 is any amino acid not including proline, preferably G or A; Xaa1-Xaa2-Xaa3-Xaa4-Xaa5- is S-L-L-T-E or, a peptide having a single substitution to the sequence S-L-L-T-E (SEQ ID NO: 47), the substitution selected from the group consisting of: Xaa1 is C or T; Xaa2 is A, C, F or K, Xaa3 is A, C, E, F, I, K, M, Q, S, T or V, and Xaa5 is D or C.

In another aspect the peptide immunogen of this invention is represented by the following formula: [Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-[Xaa7]q-[Xaa8-[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p[Xaa7]q]n; wherein, m, p and q are independently 0 or 1, n is any number between 0 and 4, Xaa0 is any amino acid, preferably C; Xaa6 is any amino acid, preferably V or C; Xaa7 is any amino acid, preferably E; Xaa8 is any amino acid not including proline, preferably G or A; Xaa1-Xaa2-Xaa3-Xaa4-Xaa5- is S-L-L-T-E or, a peptide having a single substitution to the sequence S-L-L-T-E (SEQ ID NO: 47), the substitution selected from the group consisting of: Xaa1 is A, C, D, L, T or V, Xaa2 is A, C, F, H, I, K, M, N, Q, R, T, W, or Y, Xaa3 is any amino acid, Xaa4 is M, N, Q, S, or W, and Xaa5 is A, D, F, H, I, K, M, N, Q, S, W, Y, or C.

In some aspects of the inventions, one or more amino acids are D-amino acids.

In some aspects of the inventions, the peptide immunogens are cycic. Cyclization of peptide immunogens can be performed by cross-linking cysteine residues present in the peptide or by chemical means.

In some aspects of the inventions, the peptide immunogens are conjugated to carrier proteins such as KLH through intermolecular crosslinking.

The invention relates to compositions comprising the peptide immunogen and a pharmaceutically acceptable adjuvant and/or carrier selected from the group consisting of alum, liposyn, saponin, squalene, L121, emulsigen monophosphyryl lipid A (MPL), polysorbate 80, QS21, Montanide ISA51, ISA35, ISA206 and ISA 720.

The invention relates to preventing or treating a disease associated with influenza virus infection by administering compositions comprising the peptide immunogens of the invention.

The invention relates to methods for generating antibodies reactive to influenza matrix 2 (M2) protein by administering compositions comprising the peptide immunogens of the invention. It is an object of the invention to develop an immunogen that will enable the generation of high levels of high affinity antibodies against M2 protein.

One aspect of this invention provides a vaccine comprising an immunologically effective amount of a peptide immunogen composition in accordance with this invention and one or more pharmaceutically acceptable carriers. The vaccine when administered at an appropriate dosage will generate immunotherapeutic antibodies directed against influenza A virus.

The present invention provides a vaccine delivery vehicle that is suitable for human or veterinary use for the prophylaxis and treatment of influenza.

The present invention and other objects, features, and advantages of the present invention will become further apparent in the following Detailed Description of the invention and the accompanying figures and embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the binding of three anti-M2 antibodies and control hu14C2 antibody to 293-HEK cells transfected with an M2 expression construct or control vector, in the presence or absence of free M2 peptide.

FIGS. 2A and 2B are graphs showing human monoclonal antibody binding to influenza A/PR/32.

FIG. 3A is a chart showing amino acid sequences of extracellular domains of M2 variants.

FIGS. 3B and 3C are bar charts showing binding of human monoclonal anti-influenza antibody binding to M2 variants shown in FIG. 3A.

FIG. 4 is a series of bar charts showing binding of MAbs 8I10 and 23K12 to M2 protein representing influenza strain A/HK/483/1997 sequence that was stably expressed in the CHO cell line DG44.

FIG. 5 is an illustration showing the anti-M2 antibodies bind a highly conserved region in the N-Terminus of M2e (SEQ ID NO: 66).

FIGS. 6A and 6B are schematic diagrams that depict a core peptide immunogen and amino acid variants that are effective in binding anti-M2e huMAbs 8I10 and 23K12 under high (6A) and low (6B) stringency conditions.

FIG. 6C is a schematic diagram that depicts variants of the core sequence.

FIGS. 6D, 6E and 6F are schematic diagrams that depict specific linear and cyclized peptide immunogens containing the core sequences of the invention.

DETAILED DESCRIPTION

The present invention relates to an immunogenic composition comprising synthetic peptide immunogens capable of inducing antibodies against the extracellular domain of the matrix 2 (M2) polypeptide of influenza A virus. The present invention provides peptides that bind human monoclonal antibodies specific against the extracellular domain of the matrix 2 (M2) polypeptide of influenza A virus.

The predominant fraction of neutralizing antibodies is directed to the polymorphic regions of the hemagglutinin and neuraminidase proteins. A third transmembrane protein of type A influenza virus, matrix protein 2 (M2), is abundantly expressed by virus-infected cells, where it is believed to provide an obligatory transmembrane proton flux for viral replication (Ciampor et al., Virus Research 22:247 (1992); Grambas and Hay, Virology 190:11 (1992); Sugrue et al., EMBO J. 9:3469 (1990)). Unlike HA and NA, M2 is conserved and may represent a target for the development of antibody-based passive immunotherapies for influenza patients (Ito et al., J. Virology 65:5491 (1991); Slepushkin et al., Vaccine 13:1399 (1995); Neirynck et al., Nature Med. 5:1157 (1999)). Thus, such a neutralizing MAb would presumably target only one or a few strains. A recent focus has been on the relatively invariant matrix 2 (M2) protein. Potentially, a neutralizing MAb to M2 would be an adequate therapy for all influenza A strains.

The M2 protein is found in a homotetramer that forms an ion channel and is thought to aid in the uncoating of the virus upon entering the cell. After infection, M2 can be found in abundance at the cell surface. It is subsequently incorporated into the virion coat, where it only comprises about 2% of total coat protein. The M2 extracellular domain (M2e) is short, with the amino terminal 2-24 amino acids displayed outside of the cell.

Anti-M2 monoclonal antibodies to date have been directed towards this linear sequence. Thus, they may not exhibit desired binding properties to cellularly expressed M2, including conformational determinants on native M2.

Recent vaccine development has used immunogenic peptides conjugated to carrier proteins. However, carrier proteins are too complex for use in driving antibody responses to site-specific targets. The mass of the carrier molecule is much greater than that of the functionally important target peptide site. Consequently, the major immune response is directed to the carrier protein rather than to the target site of the peptide immunogen. Moreover, immunization with hapten-carrier conjugates frequently leads to carrier-induced immune suppression (Schutze et al., J Immunol, 1985, 135:2319). A disadvantage with the peptide-carrier protein conjugates is that these molecules are highly complex and are difficult to characterize and it is difficult to develop effective quality control procedures for the manufacturing process.

To be effective, a peptide immunogen must do more than merely evoke an anti-peptide response. An effective peptide immunogen must also evoke a functional immune response, i.e., the antibody produced must have immunological cross-reactivity to the authentic target. It is known that peptide immunogens generally do not retain a preferred structure. Therefore, it is important in designing a peptide target site to introduce structural constraints. However, the imposed structural constraint must be able to mimic the conformation of the targeted epitope so that antibodies evoked will be cross-reactivities to that site on the authentic molecule (Moore, Chapter 2 in Synthetic Peptides A User's guide, ed Grant, WH Freeman and Company: New York, 1992, pp 63-67). Peptide immunogens have been designed employing promiscuous Th epitopes, the invasin domain, and with imposed structural constraint for a peptide-based vaccine for HIV (U.S. Pat. No. 6,090,388).

A long-felt need exists in the art for new antibodies that bind to the cell-expressed M2 and conformational determinants on the native M2. Accordingly, a suitable peptide-based immunogen that mimic M2 is needed for generating vaccines and therapeutics against influenza virus. It would be desirable to provide a synthetic peptide immunogen that generates a site-specific immune response without epitopic suppression by undesirable T cell responses. The peptide-based anti-M2e immunogen should provoke an early and strong immune response in humans for protective immunity without the adverse carrier-induced immune suppression. The peptide immunogen should also be stable and well defined chemically with no need of elaborate downstream processing for ease of manufacture and quality control to avoid the need of an elaborate production plant.

M2 is a 96 amino acid transmembrane protein present as a homotetramer on the surface of influenza virus and virally infected cells. M2 contains a 23 amino acid ectodomain (M2e) that is highly conserved across influenza A strains. Few amino acid changes have occurred since the 1918 pandemic strain thus M2e is an attractive target for influenza therapies. Peptides that incorporate immunogenic epitopes of M2e form a preferred aspect of the present invention.

Mimotopes which have the same characteristics as these epitopes, and immunogens comprising such mimotopes which generate an immune response which cross-react with the IgE epitope in the context of the IgE molecule, also form part of the present invention.

The present invention, therefore, includes isolated peptides encompassing these IgE epitopes themselves, and any mimotope thereof. The meaning of mimotope is defined as an entity which is sufficiently similar to the native M2e epitope so as to be capable of being recognized by antibodies which recognize the native M2e epitope; (Gheysen, H. M., et al., 1986, Synthetic peptides as antigens. Wiley, Chichester, Ciba foundation symposium 119, p 130-149; Gheysen, H. M., 1986, Molecular Immunology, 23, 7, 709-715); or are capable of raising antibodies, when coupled to a suitable carrier, which antibodies cross-react with the native M2e epitope.

Monoclonal Antibodies for Screening M2e Peptide Immunogens

The antibodies used for screening the peptide immunogens are referred to herein as huM2e antibodies. Monoclonal antibodies used are specific to the M2 ectodomain (M2e) and derived from full-length M2 is expressed in cell lines. The huM2e antibodies bind conformational determinants on the M2-transfected cells, as well as native M2, either on influenza infected cells, or on the virus itself. The huM2e antibodies do not bind the linear M2e peptide, but they do bind several natural M2 variants expressed upon cDNA transfection into cell lines. The human monoclonal antibodies exhibit specificity for a very broad range of influenza A virus strains.

The huM2e antibodies have one or more of the following characteristics: the huM2e antibody binds a) to an epitope in the extracellular domain of the matrix 2 (M2) polypeptide of an influenza virus; b) binds to influenza A infected cells; and/or c) binds to influenza A virus (i.e., virons). The huM2e antibodies of the invention eliminate influenza infected cells through immune effector mechanisms such as ADCC and promotes direct viral clearance by binding to influenza virons. The huM2e antibodies of the invention bind to the amino-terminal region of the M2e polypeptide. Preferably, the huM2e antibodies of the invention bind to the amino-terminal region of the M2e polypeptide wherein the N-terminal methionine residue is absent. Exemplary M2e sequences include those sequences listed on Table 1 below.

TABLE 1 Exemplary M2e sequences SEQ Type Name Subtype M2E Sequence ID NO: A BREVIG MISSION.1.1918 H1N1 MSLLTEVETPTRNEWGCRCNDSSD 48 A FORT MONMOUTH.1.1947 H1N1 MSLLTEVETPTKNEWECRCNDSSD 49 A .SINGAPORE.02.2005 H3N2 MSLLTEVETPIRNEWECRCNDSSD 50 A WISCONSIN.10.98 H1N1 MSLLTEVETPIRNGWECKCNDSSD 51 A WISCONSIN.301.1976 H1N1 MSLLTEVETPIRSEWGCRCNDSSD 52 A PANAMA.1.66 H2N2 MSFLPEVETPIRNEWGCRCNDSSD 53 A NEW YORK.321.1999 H3N2 MSLLTEVETPIRNEWGCRCNDSSN 54 A CARACAS.1.71 H3N2 MSLLTEVETPIRKEWGCRCNDSSD 55 A TAIWAN.3.71 H3N2 MSFLTEVETPIRNEWGCRCNDSSD 56 A WUHAN.359.95 H3N2 MSLPTEVETPIRSEWGCRCNDSSD 57 A HONG KONG.1144.99 H3N2 MSLLPEVETPIRNEWGCRCNDSSD 58 A HONG KONG.1180.99 H3N2 MSLLPEVETPIRNGWGCRCNDSSD 59 A HONG KONG.1774.99 H3N2 MSLLTEVETPTRNGWECRCSGSSD 60 A NEW YORK.217.02 H1N2 MSLLTEVETPIRNEWEYRCNDSSD 61 A NEW YORK.300.2003 H1N2 MSLLTEVETPIRNEWEYRCSDSSD 62 A SWINE.SPAIN.54008.2004 H3N2 MSLLTEVETPTRNGWECRYSDSSD 63 A GUANGZHOU.333.99 H9N2 MSFLTEVETLTRNGWECRCSDSSD 64 A HONG KONG.1073.99 H9N2 MSLLTEVETLTRNGWECKCRDSSD 65 A HONG KONG.1.68 H3N2 MSLLTEVETPIRNEWGCRCNDSSD 66 A SWINE.HONG KONG.126.1982 H3N2 MSLLTEVETPIRSEWGCRCNDSGD 67 A NEW YORK.703.1995 H3N2 MSLLTEVETPIRNEWECRCNGSSD 68 A SWINE.QUEBEC.192.81 H1N1 MSLPTEVETPIRNEWGCRCNDSSD 69 A PUERTO RIC0.8.34 H1N1 MSLLTEVETPIRNEWGCRCNGSSD 70 A HONG KONG.485.97 H5N1 MSLLTEVDTLTRNGWGCRCSDSSD 71 A HONG KONG.542.97 H5N1 MSLLTEVETLTKNGWGCRCSDSSD 72 A SILKY H9N2 MSLLTEVETPTRNGWECKCSDSSD 73 CHICKEN.SHANTOU.1826.2004 A CHICKEN.TAIWAN.0305.04 H6N1 MSLLTEVETHTRNGWECKCSDSSD 74 A QUAIL.ARKANSAS.16309-7.94 H7N3NSA MSLLTEVKTPTRNGWECKCSDSSD 75 A HONG KONG.486.97 H5N1 MSLLTEVETLTRNGWGCRCSDSSD 76 A CHICKEN.PENNSYLVANIA. H7N2NSB MSLLTEVETPTRDGWECKCSDSSD 77 13552-1.98 A CHICKEN.HEILONGJIANG.48.01 H9N2 MSLLTEVETPTRNGWGCRCSDSSD 78 A SWINE.KOREA.S5.2005 H1N2 MSLLTEVETPTRNGWECKCNDSSD 79 A HONG KONG.1073.99 H9N2 MSLLTEVETLTRNGWECKCSDSSD 80 A WISCONSIN.3523.88 H1N1 MSLLTEVETPIRNEWGCKCNDSSD 81 A X-31 VACCINE STRAIN H3N2 MSFLTEVETPIRNEWGCRCNGSSD 82 A CHICKEN.ROSTOCK.8.1934 H7N1 MSLLTEVETPTRNGWECRCNDSSD 83 A ENVIRONMENT.NEW YORK. H7N2 MSLLTEVETPIRKGWECNCSDSSD 84 16326-1.2005 A INDONESIA.560H.2006 H5N1 MSLLTEVETPTRNEWECRCSDSSD 85 A CHICKEN.HONG KONG.SF1.03 H9N2 MSLLTGVETHTRNGWGCKCSDSSD 86 A CHICKEN.HONGKONG.YU427.03 H9N2 MSLLPEVETHTRNGWGCRCSDSSD 87

In one embodiment, the peptide immunogens of the invention comprise a M2e peptide that wholly or partially includes the amino acid residues from position 2 to position 7 of M2e (SLLTEV). The huM2e antibodies bind wholly or partially to the amino acid sequence SLLTE (SEQ ID NO: 47) comprising the peptide immunogens of the invention.

Exemplary huM2e monoclonal antibodies that bind to the peptide immunogens are the 8I10, 21B15 and 23K12 antibodies described herein.

The 8I10 antibody includes a heavy chain variable region (SEQ ID NO: 88) encoded by the nucleic acid sequence shown below in SEQ ID NO: 89, and a light chain variable region (SEQ ID NO: 90) encoded by the nucleic acid sequence shown in SEQ ID NO: 91.

The amino acids encompassing the CDRs as defined by Chothia, C. et al. (1989, Nature, 342: 877-883) are underlined and those defined by Kabat E. A. et al. (1991, Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Heath and Human Services.) are highlighted in bold in the sequences below.

The heavy chain CDRs of the 8I10 antibody have the following sequences per Kabat definition: NYYWS (SEQ ID NO: 92), FIYYGGNTKYNPSLKS (SEQ ID NO: 93) and ASCSGGYCILD (SEQ ID NO: 94). The light chain CDRs of the 8I10 antibody have the following sequences per Kabat definition: RASQNIYKYLN (SEQ ID NO: 95), AA SGLQS (SEQ ID NO: 96) and QQSYSPPLT (SEQ ID NO: 97).

The heavy chain CDRs of the 8I10 antibody have the following sequences per Chothia definition: GSSISN (SEQ ID NO: 98), FIYYGGNTK (SEQ ID NO: 99) and ASCSGGYCILD (SEQ ID NO: 94). The light chain CDRs of the 8I10 antibody have the following sequences per Chothia definition: RASQNIYKYLN (SEQ ID NO: 95), AASGLQS (SEQ ID NO: 96) and QQSYSPPLT (SEQ ID NO: 97).

>8I10 VH nucleotide sequence: (SEQ ID NO: 89) CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACC CTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGG AGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTTATC TATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGTCACC ATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCTCTGTG ACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGTAGTGGT GGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCG >8I10 VH amino acid sequence: Kabat Bold, Chothia underlined (SEQ ID NO: 88) Q V Q L Q E S G P G L V K P S E T L S L T C T V S G S S I S N Y Y W S W I R Q S P G K G L E W I G F I Y Y G G N T KY N P S L K S R V T I S Q D T S K S Q V S L T M S S V T A A E S A V Y F C A R A S C S G G Y C I L D Y W G Q G T L V T V S >8I10 VL nucleotide sequence: (SEQ ID NO: 91) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC AGAGTCACCATCACTTGCCGGGCGAGTCAGAACATTTACAAGTATTTAAAT TGGTATCAGCAGAGACCAGGGAAAGCCCCTAAGGGCCTGATCTCTGCTGCA TCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGG ACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACT TACTACTGTCAACAGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACC AGGGTGGAGATCAAAC >8I10 VL amino acid sequence: Kabat Bold, Chothia underlined (SEQ ID NO: 90) D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q N I Y K Y L N W Y Q Q R P G K A K G L I S A A A S G L Q S G V P S R F S G S G S G T F T T L T I T S L Q P E D F A T Y Y C Q Q S Y S P P L T F G G G T R V E I K

The 21B15 antibody includes antibody includes a heavy chain variable region (SEQ ID NO: 100) encoded by the nucleic acid sequence shown below in SEQ ID NO: 101, and a light chain variable region (SEQ ID NO: 102) encoded by the nucleic acid sequence shown in SEQ ID NO: 103.

The amino acids encompassing the CDRs as defined by Chothia et al. 1989, are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.

The heavy chain CDRs of the 21B15 antibody have the following sequences per Kabat definition: NYYWS (SEQ ID NO: 92), FIYYGGNTKYNPSLKS (SEQ ID NO: 93) and ASCSGGYCILD (SEQ ID NO: 94). The light chain CDRs of the 21B15 antibody have the following sequences per Kabat definition: RASQNIYKYLN (SEQ ID NO: 95), AASGLQS (SEQ ID NO: 96) and QQSYSPPLT (SEQ ID NO: 97).

The heavy chain CDRs of the 21B15 antibody have the following sequences per Chothia definition: GSSISN (SEQ ID NO: 98), FIYYGGNTK (SEQ ID NO: 93) and ASCSGGYCILD (SEQ ID NO: 94). The light chain CDRs of the 21B15 antibody have the following sequences per Chothia definition: RASQNIYKYLN (SEQ ID NO: 95), AASGLQS (SEQ ID NO: 96) and QQSYSPPLT (SEQ ID NO: 97).

>21B15 VH nucleotide sequence: (SEQ ID NO: 101) CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACC CTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGG AGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTTATC TATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGTCACC ATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCTCTGTG ACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGTAGTGGT GGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCG >21B15 VH amino acid sequence: Kabat Bold, Chothia underlined (SEQ ID NO: 100) Q V Q L Q E S G P G L V K P S E T L S L T C T V S G S S I S N Y Y W S W I R Q S P G K G L E W I G F I Y Y G G N T KY N P S L K S R V T I S Q D T S K S Q V S L T M S S V T A A E S A V Y F C A R A S C S G G Y C I L D Y W G Q G T L V T V S >21B15 VL nucleotide sequence: (SEQ ID NO: 103) GACATCCAGGTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC AGAGTCACCATCACTTGCCGCGCGAGTCAGAACATTTACAAGTATTTAAAT TGGTATCAGCAGAGACCAGGGAAAGCCCCTAAGGGCCTGATCTCTGCTGCA TCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGG ACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACT TACTACTGTCAACAGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACC AGGGTGGATATCAAAC >21B15 VL amino acid sequence: Kabat Bold, Chothia underlined (SEQ ID NO: 102) D I Q V T Q S P S S L S A S V G D R V T I T C R A S Q N I Y K Y L N W Y Q Q R P G K A K G L I S A A A S G L Q S G V P S R F S G S G S G T D F T L T I T S L Q P E D F A T Y Y C Q Q S Y S P P L T F G G G T R V D I K

The 23K12 antibody includes antibody includes a heavy chain variable region (SEQ ID NO: 104) encoded by the nucleic acid sequence shown below in SEQ ID NO: 105, and a light chain variable region (SEQ ID NO: 106) encoded by the nucleic acid sequence shown in SEQ ID NO: 107.

The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.

The heavy chain CDRs of the 23K12 antibody have the following sequences per Kabat definition: SNYMS (SEQ ID NO: 108), VIYSGGSTYYADSVK (SEQ ID NO: 109) and CLSRMRGYGLDV (SEQ ID NO: 110). The light chain CDRs of the 23K12 antibody have the following sequences per Kabat definition: RTSQSISSYLN (SEQ ID NO: 111), AASSLQSGVPSRF (SEQ ID NO: 112) and QQSYSMPA (SEQ ID NO: 113).

The heavy chain CDRs of the 23K12 antibody have the following sequences per Chothia definition: GFTVSSN (SEQ ID NO: 114), VIYSGGSTY (SEQ ID NO: 115) and CLSRMRGYGLDV (SEQ ID NO: 110). The light chain CDRs of the 23K12 antibody have the following sequences per Chothia definition: RTSQSISSYLN (SEQ ID NO: 111), AASSLQSGVPSRF (SEQ ID NO: 112) and QQSYSMPA (SEQ ID NO: 113).

>23K12 VH nucleotide sequence: (SEQ ID NO: 105) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCC CTGAGAATCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACATG AGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATT TATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCAGATTCTCC TTCTCCAGAGACAACTCCAAGAACACAGTGTTTCTTCAAATGAACAGCCTG AGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATGTCTGAGCAGGATG CGGGGTTACGGTTTAGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCG >23K12 VH amino acid sequence:  Kabat Bold, Chothia underlined (SEQ ID NO: 104) E V Q L V E S G G G L V Q P G G S R I S C C A A S G F T V S S N Y M S W V R Q A P G K G L E W V S V I Y S G G S T YY A D S V K G R F S F S R D N S K N T V F L Q M N S L R A E D T A V Y Y C A R C L S R M R G Y G L D V W G Q G T T V T V S >23K12 VL nucleotide sequence:  (SEQ ID NO: 107) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC AGAGTCACCATCACTTGCCGGACAAGTCAGAGCATTAGCAGCTATTTAAAT TGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGCTGCA TCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGG ACAGATTTCACTCTCACCATCAGCGGTCTGCAACCTGAAGATTTTGCAACC TACTACTGTCAACAGAGTTACAGTATGCCTGCCTTTGGCCAGGGGACCAAG CTGGAGATCAAA >23K12 VL amino acid sequence:  Kabat Bold, Chothia underlined (SEQ ID NO: 106) D I Q M T Q S P S S L S A S V G D R V T I T C R T S Q S I S S Y L N W Y Q Q K P G K A P K L L I Y A A S S L Q S G V P S R F S G S G S G T F T T L T I S G L Q P E D F A T Y Y C Q Q S Y S M P A F G Q G T K L E I K

Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).

The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

DEFINITIONS

The following definitions are useful in understanding the present invention:

The term “antibody” (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

An “isolated antibody” is one that has been separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains and four CH domains for μ, and ε isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Ten and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71, and Chapter 6.

The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains (CL). Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), respectively. The γ and α classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term “variable” refers to the fact that certain segments of the V domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and around about 1-35 (H1), 50-65 (H2) and 95-102 (H3) in the VH; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop” (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the VL, and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the VH; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.

The monoclonal antibodies herein include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The present invention provides variable domainantigen-binding dequences derived from human antibodies. Accordingly, chimeric antibodies of primary interest herein include antibodies having one or more human antigen binding sequences (e.g., CDRs) and containing one or more sequences derived from a non-human antibody, e.g., an FR or C region sequence. In addition, chimeric antibodies of primary interest herein include those comprising a human variable domain antigen binding sequence of one antibody class or subclass and another sequence, e.g., FR or C region sequence, derived from another antibody class or subclass. Chimeric antibodies of interest herein also include those containing variable domain antigen-binding sequences related to those described herein or derived from a different species, such as a non-human primate (e.g., Old World Monkey, Ape, etc). Chimeric antibodies also include primatized and humanized antibodies.

Furthermore, chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

A “humanized antibody” is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization is traditionally performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting import hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.

A “human antibody” is an antibody containing only sequences present in an antibody naturally produced by a human. However, as used herein, human antibodies may comprise residues or modifications not found in a naturally occurring human antibody, including those modifications and variant sequences described herein. These are typically made to further refine or enhance antibody performance.

An “intact” antibody is one that comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

An “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The phrase “functional fragment or analog” of an antibody is a compound having qualitative biological activity in common with a full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody is one that can bind to an IgE immunoglobulin in such a manner so as to prevent or substantially reduce the ability of such molecule from having the ability to bind to the high affinity receptor, FcεRI.

Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab′)2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab′ fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The “Fc” fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term “diabodies” refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

As used herein, an antibody that “internalizes” is one that is taken up by (i.e., enters) the cell upon binding to an antigen on a mammalian cell (e.g., a cell surface polypeptide or receptor). The internalizing antibody will of course include antibody fragments, human or chimeric antibody, and antibody conjugates. For certain therapeutic applications, internalization in vivo is contemplated. The number of antibody molecules internalized will be sufficient or adequate to kill a cell or inhibit its growth, especially an infected cell. Depending on the potency of the antibody or antibody conjugate, in some instances, the uptake of a single antibody molecule into the cell is sufficient to kill the target cell to which the antibody binds. For example, certain toxins are highly potent in killing such that internalization of one molecule of the toxin conjugated to the antibody is sufficient to kill the infected cell.

As used herein, an antibody is said to be “immunospecific,” “specific for” or to “specifically bind” an antigen if it reacts at a detectable level with the antigen, preferably with an affinity constant, Ka, of greater than or equal to about 104 M−1, or greater than or equal to about 105 M−1, greater than or equal to about 106 M−1, greater than or equal to about 107 M−1, or greater than or equal to 108 M−1. Affinity of an antibody for its cognate antigen is also commonly expressed as a dissociation constant KD, and in certain embodiments, HuM2e antibody specifically binds to M2e if it binds with a KD of less than or equal to 10−4 M, less than or equal to about 10−5 M, less than or equal to about 10−6 M, less than or equal to 10−7 M, or less than or equal to 10−8 M. Affinities of antibodies can be readily determined using conventional techniques, for example, those described by Scatchard et al. (Ann. N.Y. Acad. Sci. USA 51:660 (1949)).

Binding properties of an antibody to antigens, cells or tissues thereof may generally be determined and assessed using immunodetection methods including, for example, immunofluorescence-based assays, such as immuno-histochemistry (IHC) and/or fluorescence-activated cell sorting (FACS).

An antibody having a “biological characteristic” of a designated antibody is one that possesses one or more of the biological characteristics of that antibody which distinguish it from other antibodies. For example, in certain embodiments, an antibody with a biological characteristic of a designated antibody will bind the same epitope as that bound by the designated antibody and/or have a common effector function as the designated antibody.

The term “antagonist” antibody is used in the broadest sense, and includes an antibody that partially or fully blocks, inhibits, or neutralizes a biological activity of an epitope, polypeptide, or cell that it specifically binds. Methods for identifying antagonist antibodies may comprise contacting a polypeptide or cell specifically bound by a candidate antagonist antibody with the candidate antagonist antibody and measuring a detectable change in one or more biological activities normally associated with the polypeptide or cell.

An “antibody that inhibits the growth of infected cells” or a “growth inhibitory” antibody is one that binds to and results in measurable growth inhibition of infected cells expressing or capable of expressing an M2e epitope bound by an antibody. Preferred growth inhibitory antibodies inhibit growth of infected cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g., from about 50% to about 100%) as compared to the appropriate control, the control typically being infected cells not treated with the antibody being tested. Growth inhibition can be measured at an antibody concentration of about 0.1 to 30 μg/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the infected cells to the antibody. Growth inhibition of infected cells in vivo can be determined in various ways known in the art. The antibody is growth inhibitory in vivo if administration of the antibody at about 1 μg/kg to about 100 mg/kg body weight results in reduction the percent of infected cells or total number of infected cells within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.

An antibody that “induces apoptosis” is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). Preferably the cell is an infected cell. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells. Preferably, the antibody that induces apoptosis is one that results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexin binding relative to untreated cell in an annexin binding assay.

Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. In certain embodiments, the FcR is a native sequence human FcR. Moreover, a preferred FcR is one that binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FCγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (see review M. in Daeron, Annu Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

“Human effector cells” are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include PBMC, NK cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source, e.g., from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

The terms “influenza A” and “Influenzavirus A” refer to a genus of the Orthomyxoviridae family of viruses. Influenzavirus A includes only one species: influenza A virus which cause influenza in birds, humans, pigs, and horses. Strains of all subtypes of influenza A virus have been isolated from wild birds, although disease is uncommon. Some isolates of influenza A virus cause severe disease both in domestic poultry and, rarely, in humans.

A “mammal” for purposes of treating n infection, refers to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.

“Treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully “treated” for an infection if, after receiving a therapeutic amount of an antibody according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of infected cells or absence of the infected cells; reduction in the percent of total cells that are infected; and/or relief to some extent, one or more of the symptoms associated with the specific infection; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

The term “therapeutically effective amount” refers to an amount of an antibody or a drug effective to “treat” a disease or disorder in a subject or mammal. See preceding definition of “treating.”

“Chronic” administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

“Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™ polyethylene glycol (PEG), and PLURONICS™.

“Label” as used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody so as to generate a “labeled” antibody. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

The term “epitope tagged” as used herein refers to a chimeric polypeptide comprising a polypeptide fused to a “tag polypeptide.” The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused. The tag polypeptide is also preferably fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).

A “small molecule” is defined herein to have a molecular weight below about 500 Daltons.

The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to single- or double-stranded RNA, DNA, or mixed polymers. Polynucleotides may include genomic sequences, extra-genomic and plasmid sequences, and smaller engineered gene segments that express, or may be adapted to express polypeptides.

An “isolated nucleic acid” is a nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. The term embraces a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure nucleic acid includes isolated forms of the nucleic acid. Of course, this refers to the nucleic acid as originally isolated and does not exclude genes or sequences later added to the isolated nucleic acid by the hand of man.

The term “polypeptide” is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product. Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence thereof. Particular polypeptides of interest in the context of this invention are amino acid subsequences comprising CDRs and being capable of binding an antigen or Influenza A-infected cell.

An “isolated polypeptide” is one that has been identified and separated and/or recovered from a component of its natural environment. In preferred embodiments, the isolated polypeptide will be purified (1) to greater than 95% by weight of polypeptide as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

A “native sequence” polynucleotide is one that has the same nucleotide sequence as a polynucleotide derived from nature. A “native sequence” polypeptide is one that has the same amino acid sequence as a polypeptide (e.g., antibody) derived from nature (e.g., from any species). Such native sequence polynucleotides and polypeptides can be isolated from nature or can be produced by recombinant or synthetic means.

A polynucleotide “variant,” as the term is used herein, is a polynucleotide that typically differs from a polynucleotide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the polynucleotide sequences of the invention and evaluating one or more biological activities of the encoded polypeptide as described herein and/or using any of a number of techniques well known in the art.

A polypeptide “variant,” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of techniques well known in the art.

Modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant or portion of a polypeptide of the invention, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence.

For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of its ability to bind other polypeptides (e.g., antigens) or cells. Since it is the binding capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences that encode said peptides without appreciable loss of their biological utility or activity.

In many instances, a polypeptide variant will contain one or more conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.

Polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.

When comparing polynucleotide and polypeptide sequences, two sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theon 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparison of both strands.

For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.

In one approach, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

“Homology” refers to the percentage of residues in the polynucleotide or polypeptide sequence variant that are identical to the non-variant sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. In particular embodiments, polynucleotide and polypeptide variants have at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% polynucleotide or polypeptide homology with a polynucleotide or polypeptide described herein.

“Vector” includes shuttle and expression vectors. Typically, the plasmid construct will also include an origin of replication (e.g., the ColE1 origin of replication) and a selectable marker (e.g., ampicillin or tetracycline resistance), for replication and selection, respectively, of the plasmids in bacteria. An “expression vector” refers to a vector that contains the necessary control sequences or regulatory elements for expression of the antibodies including antibody fragment of the invention, in bacterial or eukaryotic cells. Suitable vectors are disclosed below.

As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.

Preferred methods for determining mAb specificity and affinity by competitive inhibition can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference.

The techniques to raise antibodies of the present invention to small peptide sequences that recognize and bind to those sequences in the free or conjugated form or when presented as a native sequence in the context of a large protein are well known in the art. Such antibodies include murine, murine-human and human-human antibodies produced by hybridoma or recombinant techniques known in the art.

Screening Peptide Immunogens for Specific Binding to 8i10, or 23K12.

The present invention relates to peptide immunogens that bind HuM2e antibodies. In one embodiment, the antibody is an antibody designated herein as 8i10, 21B15, or 23K12. These antibodies are known to display preferential or specific binding to influenza A infected cells as compared to uninfected control cells of the same cell type.

In particular embodiments, the HuM2e antibodies bind to epitopes within M2e that are only present in the native conformation, i.e., as expressed in cells. In particular embodiments, these antibodies fail to specifically bind to an isolated M2e polypeptide, e.g., the 23 amino acid residue M2e fragment. It is understood that these antibodies recognize non-linear (i.e. conformational) epitope(s) of the M2 peptide. M2 ectodomain (M2e) includes or consists of the amino acid sequence SLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO: 326) and variants thereof (The Center for Disease Control (CDC) influenza A database at www.flu.lanl.gov/).

The specific conformational epitopes within the M2 protein, and particularly within M2e, were identified as peptide immunogens which can be used as vaccines to prevent the development of influenza infection within a subject.

The peptide immunogen sequences were identified by discontinuous epitope mapping using CLIPS™ (Chemically Linked Immunogenic Peptides on Scaffolds; PepScan) technology, which has been developed to improve the biological function of synthetic peptides. CLIPS uses small, chemical “scaffolds” onto which one or more peptides can be attached. In vaccine development, these are ideal for mapping conformational (or ‘discontinuous’) epitopes because they closely resemble the native structure of proteins.

Binding activity of anti-M2 antibodies to mutant M2 peptides was analyzed with an ELISA assay using different M2 peptides. Human anti-M2 antibody nos. Z3G1, 8I10 and 23K12 were used in the study. Peptides comprising M2e sequences were screened for the ability to specifically bind HuM2e antibodies 8I10 and 23K12 and distinguished from the ability to bind the anti-M2 human monoclonal antibody Z3G1 (ATCC Deposit No. PTA-5967) which has a broad M2 binding spectrum. 8I10 and 23K12 have been characterized for the ability to bind M2 and M2e under conditions resembling native conformation. The assays were performed at both 0.01 μg/mL and 0.001 μg/mL concentrations of peptides. At 0.01 μg/mL, signal levels of 1000 or greater were selected and at 0.001 μg/mL, signal levels of 300 or greater were selected as significant. Most peptides that bind strongly to 8I10 and 23K12 also bind Z3G1.

TABLE 2 Peptides that specifically bind for 23K21 and 8I10 (A numeral “1” in the peptide sequence indicates a differentially protected cystein allowing for selective CLIP attachment at certain residues) SEQ Peptides that bind ID NO: to 23K21 and 8I10 Signal 116 CLTEVETPIRNEWGSRCSLLTEVETPIRNEWGC 1922 117 CLLTEVETPIRNEWGSCSLLTEVETPIRC 1865 118 CSLLTEVETPIRNECSLLTEVETC 1829 119 CLTEVETPIRNEWGSRCSLLTEVETPIRNC 1825 120 CSLLTEVETPIRNCSLLTEVETPIRC 1781 121 CSLLTEVETPIRCSLLTEVETPIRC 1767 122 CSLLTEVETPIRNEWCSLLTEVETC 1752 123 CSLLTEVETPIRNECLTEVETPIRNEWGSRC 1702 124 CSLLTEVETPCSLLTEVETPIRC 1693 125 CTEVETPIRNEWGSRSCSLLTEVETPIRNEWGC 1659 126 CETPIRNEWGSRSNDSCSLLTEVETPIRNC 1650 127 CSLLTEVETCSLLTEVETC 1647 128 CSLLTEVETCSLLTEVETPIC 1638 129 CSLLTEVETPIRNCSLLTEVETPIRNC 1602 130 CSLLTEVETPIRNECSLLTEVETPIRC 1588 131 CSLLTEVETPIRCSLLTEVETPIRNC 1549 132 CVETPIRNEWGSRSNDCSLLTEVETPIC 1548 133 CSLLTEVETPIRNECSLLTEVETPIRNC 1534 134 CSLLTEVETCSLLTEVETPIRNC 1480 135 CSLLTEVETPIRNCSLLTEVETPIRNEWC 1476 136 CLTEVETPIRNEWGSRCSLLTEVETC 1470 137 CSLLTEVETPCSLLTEVETPIC 1458 138 CSLLTEVETPIRNEWGCLLTEVETPIRNEWGSC 1456 139 CSLLTEVETPIRNCSLLTEVETPIC 1449 140 CSLLTEVETPIRNEWGCSLLTEVETPIRNEWGC 1434 141 CSLLTEVETPIRNEWCSLLTEVETPIRNEWGC 1427 142 CSLLTEVETCSLLTEVETPC 1424 143 CSLLTEVETPIRNEWGCSLLTEVETPIRNC 1418 144 CSLLTEVETPIRNCSLLTEVETPC 1405 145 CSLLTEVETPIRNECSLLTEVETPIC 1395 146 CSLLTEVETPIRNECSLLTEVETPIRNEWC 1394 147 CSLLTEVETPIRCSLLTEVETC 1388 148 CSLLTEVETPIRNCSLLTEVETPIRNEWGC 1356 149 CTEVETPIRNEWGSRSCLTEVETPIRNEWGSRC 1348 150 CSLLTEVETCSLLTEVETPIRNEWC 1345 151 CSLLTEVETPIRCSLLTEVETPIC 1340 152 CSLLTEVETPIRNECSLLTEVETPIRNEWGC 1331 153 CSLLTEVETPCSLLTEVETPIRNC 1320 154 CETPIRNEWGSRSNDSCSLLTEVETPIRNEC 1313 155 CSLLTEVETPIRNEWCSLLTEVETPIRC 1293 156 CSLLTEVETPCSLLTEVETPC 1286 157 CSLLTEVETPIRNEWGCSLLTEVETPIRC 1278 158 CSLLTEVETPCSLLTEVETPIRNEWC 1269 159 CSLLTEVETPIRNEWGCVETPIRNEWGSRSNDC 1241 160 CSLLTEVETPIRCLLTEVETPIRNEWGSC 1211 161 CPIRNEWGSRSNDSSDCSLLTEVETC 1206 162 CLTEVETPIRNEWGSRCSLLTEVETPIRC 1203 163 CSLLTEVETCSLLTEVETPIRC 1200 164 CSLLTEVETPIRNECSLLTEVETPC 1194 165 CSLLTEVETPIRCSLLTEVETPIRNEC 1194 166 CSLLTEVETPIRNCSLLTEVETC 1192 167 CSLLTEVETPIRCSLLTEVETPIRNEWGC 1188 168 CVETPIRNEWGSRSNDCSLLTEVETPIRC 1186 169 CTPIRNEWGSRSNDSSCSLLTEVETPIRC 1180 170 CSLLTEVETPIRCLTEVETPIRNEWGSRC 1175 171 CSLLTEVETCSLLTEVETPIRNEC 1172 172 CEVETPIRNEWGSRSNCSLLTEVETPIRC 1168 173 CSLLTEVETPIRCSLLTEVETPIRNEWC 1167 174 CSLLTEVETPIRNEWCSLLTEVETPIRNEWC 1165 175 CSLLTEVETPIRNEWGCLTEVETPIRNEWGSRC 1148 176 CPIRNEWGSRSNDSSDCSLLTEVETPIRC 1146 177 CSLLTEVETPIRNEWCSLLTEVETPIC 1144 178 CLLTEVETPIRNEWGSCSLLTEVETPIRNC 1141 179 CSLLTEVETCLLTEVETPIRNEWGSC 1141 180 CSLLTEVETPIRNEWCSLLTEVETPIRNC 1138 181 CSLLTEVETPIRCSLLTEVETPC 1115 182 CSLLTEVETPCLLTEVETPIRNEWGSC 1111 183 CSLLTEVETPIRNCSLLTEVETPIRNEC 1110 184 CTEVETPIRNEWGSRSCSLLTEVETPIRC 1104 185 CTPIRNEWGSRSNDSSCSLLTEVETPIRNC 1103 186 CSLLTEVETPCSLLTEVETPIRNEWGC 1103 187 CSLLTEVETPICSLLTEVETC 1089 188 CLTEVETPIRNEWGSRCSLLTEVETPIRNEWC 1079 189 CSLLTEVETPIRNEWCSLLTEVETPC 1074 190 CETPIRNEWGSRSNDSCSLLTEVETPIRC 1069 191 CLTEVETPIRNEWGSRCSLLTEVETPIC 1040 192 CSLLTEVETPIRNCLLTEVETPIRNEWGSC 1036 193 CSLLTEVETPCSLLTEVETPIRNEC 1032 194 CVETPIRNEWGSRSNDCSLLTEVETPIRNC 1028 195 CSLLTEVETPIRNCLTEVETPIRNEWGSRC 1027 196 CEVETPIRNEWGSRSNCSLLTEVETPIRNC 1026 197 CSLLTEVETPIRNEWGCSLLTEVETPIC 1023 198 CSLLTEVETPCSLLTEVETC 1018 199 CSLLTEVETPIRCTEVETPIRNEWGSRSC 1017 200 CSLLTEVETPIRNEWGCSLLTEVETC 1014 201 CEVETPIRNEWGSRSNCSLLTEVETC 1007 202 CPIRNEWGSRSNDSSDCSLLTEVETPIRNC 1004 203 CSLLTEVETPICSLLTEVETPIRC 1001 204 CTEVETPIRNEWGSRSCSLLTEVETPIRNC 1000 205 CLLTEVETPIRNEWGSCSLLTEVETPIRNEWGC 1000 206 CSLLTEVETPIRNEWGCSLLTEVETPIRNEC 994 207 CSLLTEVETPICSLLTEVETPC 986 208 CSLLTEVETPICSLLTEVETPIRNC 981 209 CSLLTEVETPIRCEVETPIRNEWGSRSNC 980 210 CSLLTEVETPIRNCTEVETPIRNEWGSRSC 977 211 CSLLTEVETPICSLLTEVETPIRNEWC 975 212 CSLLTEVETPIRNEWGCSLLTEVETPIRNEWC 972 213 CETPIRNEWGSRSNDSCSLLTEVETC 971 214 CSLLTEVETPIRNEWCLLTEVETPIRNEWGSC 961 215 CSLLTEVETCLTEVETPIRNEWGSRC 958 216 CTEVETPIRNEWGSRSCSLLTEVETPIC 957 217 CSLLTEVETPIRNEWGCSLLTEVETPC 955 218 CLLTEVETPIRNEWGSCSLLTEVETPIRNEWC 922 219 CEVETPIRNEWGSRSNCSLLTEVETPIC 912 220 CSLLTEVETPIRNECSLLTEVETPIRNEC 910 221 CSLLTEVETPCLTEVETPIRNEWGSRC 907 222 CSLLTEVETPCTEVETPIRNEWGSRSC 907 223 CVETPIRNEWGSRSNDCSLLTEVETC 905 224 CSLLTEVETPICLLTEVETPIRNEWGSC 901 225 CTEVETPIRNEWGSRSCSLLTEVETPIRNEWC 900 226 CTEVETPIRNEWGSRSCSLLTEVETC 891 227 CSLLTEVETPIRNECLLTEVETPIRNEWGSC 870 228 CLTEVETPIRNEWGSRCSLLTEVETPC 867 229 CEVETPIRNEWGSRSNCSLLTEVETPC 862 230 CTPIRNEWGSRSNDSSCSLLTEVETC 861 231 CTPIRNEWGSRSNDSSCSLLTEVETPIC 854 232 CETPIRNEWGSRSNDSCSLLTEVETPC 842 233 CEVETPIRNEWGSRSNCSLLTEVETPIRNEWGC 838 234 CVETPIRNEWGSRSNDCSLLTEVETPIRNEWC 837 235 CSLLTEVETPICLTEVETPIRNEWGSRC 835 236 CSLLTEVETPICSLLTEVETPIRNEWGC 826 237 CETPIRNEWGSRSNDSCSLLTEVETPIC 825 238 CEVETPIRNEWGSRSNCSLLTEVETPIRNEWC 825 239 CSLLTEVETCEVETPIRNEWGSRSNC 821 240 CLTEVETPIRNEWGSRCSLLTEVETPIRNEC 817 241 CSLLTEVETPIRNEWCSLLTEVETPIRNEC 815 242 CVETPIRNEWGSRSNDCSLLTEVETPC 814 243 CTPIRNEWGSRSNDSSCSLLTEVETPIRNEWGC 812 244 CLLTEVETPIRNEWGSCSLLTEVETPIRNEC 808 245 CSLLTEVETPICSLLTEVETPIC 797 246 CTEVETPIRNEWGSRSCSLLTEVETPC 796 247 CTPIRNEWGSRSNDSSCSLLTEVETPC 794 248 CPIRNEWGSRSNDSSDCSLLTEVETPIRNEC 787 249 CPIRNEWGSRSNDSSDCSLLTEVETPIRNEWC 786 250 CSLLTEVETCTEVETPIRNEWGSRSC 786 251 CETPIRNEWGSRSNDSCSLLTEVETPIRNEWC 784 252 CLLTEVETPIRNEWGSCSLLTEVETPIC 762 253 CSLLTEVETPIRNCEVETPIRNEWGSRSNC 761 254 CLLTEVETPIRNEWGSCSLLTEVETC 752 255 CETPIRNEWGSRSNDSCSLLTEVETPIRNEWGC 749 256 CPIRNEWGSRSNDSSDCSLLTEVETPIRNEWGC 742 257 CVETPIRNEWGSRSNDCSLLTEVETPIRNEWGC 737 258 CSLLTEVETPIRNECTEVETPIRNEWGSRSC 736 259 CPIRNEWGSRSNDSSDCSLLTEVETPC 733 260 CSLLTEVETPICEVETPIRNEWGSRSNC 731 261 CTEVETPIRNEWGSRSCSLLTEVETPIRNEC 721 262 CSLLTEVETPIRNCVETPIRNEWGSRSNDC 721 263 CSLLTEVETPIRNEWGCTEVETPIRNEWGSRSC 715 264 CSLLTEVETCVETPIRNEWGSRSNDC 707 265 CSLLTEVETPCVETPIRNEWGSRSNDC 704 266 CTPIRNEWGSRSNDSSCSLLTEVETPIRNEC 699 267 CPIRNEWGSRSNDSSDCSLLTEVETPIC 691 268 SLLTEVETPIRNECGCRCNDSSD 682 269 CVETPIRNEWGSRSNDCSLLTEVETPIRNEC 679 270 CLLTEVETPIRNEWGSCSLLTEVETPC 678 271 CSLLTEVETPIRCVETPIRNEWGSRSNDC 678 272 CTPIRNEWGSRSNDSSCSLLTEVETPIRNEWC 677 273 CEVETPIRNEWGSRSNCSLLTEVETPIRNEC 669 274 CSLLTEVETPCEVETPIRNEWGSRSNC 650 275 CSLLTEVETPIRNEWGCEVETPIRNEWGSRSNC 607 276 CSLLTEVETPIRNECEVETPIRNEWGSRSNC 591 277 CSLLTEVETPIRNEWCTEVETPIRNEWGSRSC 575 278 CSLLTEVETPIRNEWCLTEVETPIRNEWGSRC 561 279 CSLLTEVETPIRNECVETPIRNEWGSRSNDC 538 280 CSLLTEVETPICTEVETPIRNEWGSRSC 526 281 CSLLTEVETCSLLTEVETPIRNEWGC 328 282 CSCLCEVGMSCLCEC 2705 283 CSLLTEVGSLLTEV 2494 284 ASLLTEVGSLLTCV 2443 285 MSLLTEVGMSLLTCV 2389 286 CSLLTEVGMSLLTCV 2311 287 MSLLTEVGMSLLTEV 2267 288 CSCLCEVGMSLLTEV 2154 289 MSLCTEVGMSLCTEV 2153 290 CSLLTEVGSLLTCV 2142 291 MSLLTEVGMCLCTCV 2110 292 CSLLTEVGSLLTEC 2094 293 CSLLTEVGMSLLTEV 2039 294 MSCLCECGMSLLTEV 2039 295 MSLCTEVGMSCLTEV 2026 296 MSLLTEVGMCLLTEV 2023 297 CSLLTEVGMCLCTCV 1972 298 SLCTEVGSCLCEC 1967 299 SLLTEVGCLCTCV 1963 300 SLLTEVETKIRNEWGCRCNDSSD 1960 301 SLLCEVGCSLLTEC 1959 302 ASLLTEVGSCLTEV 1944 303 CSLLTEVGMCLLTEV 1941 304 MSLCTEVGMSLLCEV 1935 305 CSCLCEVGMSLLTEC 1925 306 CSLLTECGMSLLTCV 1918 307 SLCTEVGCLCTCV 1907 308 MSLCTEVGMCLCTCV 1902 309 CSLLTEVGMSLLCEV 1891 310 MSCLCECGMSLLTEC 1887 311 SLLTEVGCLLTEV 1859 312 TLLTEVETPIRNEWGCRCNDSSD 1855 313 SLLTEVETPIRNEWGCRCNDSGD 1852 314 SLLTEVGSLLCEV 1834 315 MCLCTCVGCSLLTEC 1823 316 MSLLTEVGCSLLTEV 1809 317 MSLLTEVGMSLCTEV 1804 318 SLLTEVETPIRNEWGCRCKDSSD 1802 319 SLLTECGSLLTCV 1800 320 SLLTEVGSLLTEV 1794 321 ALLTEVETPIRNEWGCRCNDSSD 1794 322 SKLTEVETPIRNEWGCRCNDSSD 1784 323 SLCTEVGSLLTCV 1781 324 SLCTEVGSLLTEV 1772 325 SLLTECGSLLTEV 1770 326 SLLTEVETPIRNEWGCRCNDSSD 1749 327 SLMTEVETPIRNEWGCRCNDSSD 1729 328 SLETEVETPIRNEWGCRCNDSSD 1720 329 SLLTEVGSCLCEC 1708 330 SLLTEVETPIRNEWGCRCNDYSD 1694 331 MSLLTECGMSLLTCV 1690 332 SALTEVETPIRNEWGCRCNDSSD 1689 333 SLLTCVGSLLTEC 1683 334 CSLLTEVGMSLCTEV 1682 335 CSCLCEVGCSLLTEC 1677 336 SLLCEVGSLLTEV 1674 337 SLLTEVETPIRNEWGCRCNYSSD 1666 338 SLLTEVETPIRNEWGCRCNDGSD 1666 339 MSLLTECGMSLLTEV 1658 340 MSCLCECGCSLLTEC 1652 341 CSLLTEVCSLLTEC 1644 342 SLLTEVETPIRNEWGCRCNSSSD 1637 343 MSLLTEVGMSLLTEC 1630 344 SMLTEVETPIRNEWGCRCNDSSD 1622 345 CSLLTEVGSCLTEV 1616 346 SLLMEVETPIRNEWGCRCNDSSD 1616 347 SLLTEVETPIRNEWICRCNDSSD 1599 348 SLLTECGCSLLTEV 1597 349 SLATEVETPIRNEWGCRCNDSSD 1583 350 SLLTEVETPIRNEWGCRCNDSND 1577 351 SLLCEVGSLLTEC 1564 352 SLQTEVETPIRNEWGCRCNDSSD 1561 353 SLLTEVGCSLLTEC 1556 354 SLLTEVGCSLLTEV 1555 355 SCLCECGCSLLTEV 1555 356 SLLTEVETPIRNEWGCRCNDSDD 1553 357 SLLTEVETPIPNEWGCRCNDSSD 1548 358 SFLTEVETPIRNEWGCRCNDSSD 1546 359 MSLLTEVGMSCLTEV 1535 360 CSLLTECGCSLLTEV 1534 361 SCLCECGCSLLTEC 1520 362 CSLLTECGMCLLTEV 1517 363 MSLLTECGCSLLTEV 1500 364 CSLLTEVGCSLLTEV 1499 365 CSLLTEVCMSLLTEC 1498 366 SLLTEVETPIRNEWWCRCNDSSD 1498 367 SLCTEVGSLLTEC 1495 368 SLLTEVETPIRNEWG 1486 369 SLLTEAETPIRNEWGCRCNDSSD 1481 370 SLLTEVETPIRNEWGERCNDSSD 1474 371 MSLCTEVGMSLLTEV 1468 372 CSLLTEVGMSCLTEV 1466 373 CSLLTECGMSLLTEV 1458 374 SLLTECGCSLLTEC 1451 375 SLLTEVETPIRNEWGCRVNDSSD 1446 376 CSLLTECGMSLLTEC 1445 377 SLKTEVETPIRNEWGCRCNDSSD 1434 378 SLLTECGSLLTEC 1426 379 SLCTEVGCSCLCEV 1421 380 SLLTEVETPIRNTWGCRCNDSSD 1409 381 SLLQEVETPIRNEWGCRCNDSSD 1399 382 CSLLTECGSLLTEV 1393 383 CSLLTEVGCSLLTEC 1380 384 SLLTEVETPIRNEYGCRCNDSSD 1377 385 CSLLTEVGCLCTCV 1375 386 SLLTEVETPIRNEVGCRCNDSSD 1371 387 SLLTEVETPIRTEWGCRCNDSSD 1365 388 CSLLTEVGCLLTEV 1351 389 SLLTEVETPYRNEWGCRCNDSSD 1350 390 CLLTEVGSLLTEV 1349 391 SNLTEVETPIRNEWGCRCNDSSD 1345 392 SLLTEVETPKRNEWGCRCNDSSD 1342 393 SLLTEVETPIRNEWGCRCNLSSD 1334 449 SLNTEVETPIRNEWGCRCNDSSD 1333 450 SLLTEVEHPIRNEWGCRCNDSSD 1331 451 CSLLTEVGMSLLTEC 1326 452 SLLTEVGSLLTEC 1326 453 SLLTEVETPIRNEAGCRCNDSSD 1311 454 SLLTEVETPIMNEWGCRCNDSSD 1308 455 CLCTCVGSLLTEC 1303 456 CSLLTECGMSLLCEV 1294 457 SLLTEVETPMRNEWGCRCNDSSD 1281 458 CSLLTECGSCLTEV 1278 459 SLITEVETPIRNEWGCRCNDSSD 1277 460 MSLLTECGMSLLCEV 1268 461 SLLTEVETPLRNEWGCRCNDSSD 1268 462 SLLTECGCLLTEV 1259 463 CSLLTECGSLLTCV 1258 464 SLLTEVGSLCTEV 1256 465 SLLTEVETPIRNEWGCRCNKSSD 1254 466 SCLTEVGCSLLTEV 1250 467 SLLTEVENPIRNEWGCRCNDSSD 1245 468 SCLCECGSLLTEC 1244 469 MSLLTECGMSLLTEC 1236 470 MSLLTEVGCSLLTEC 1227 471 SLLTEEETPIRNEWGCRCNDSSD 1220 472 SLLTECGSLCTEV 1218 473 SLSTEVETPIRNEWGCRCNDSSD 1217 474 SLLTECETPIRNEWGCRCNDSSD 1214 475 MSLLTECGMSLCTEV 1212 476 CSLLTEVGCSCLCEV 1206 477 SLLTECGSLLCEV 1206 478 SLLTEVETPIRNEWGCRCNDHSD 1205 407 SLLTEVETPIRNEWGCRCNDHSD 1200 479 SLLTEDETPIRNEWGCRCNDSSD 1196 480 SLLTECGCLCTCV 1193 481 CSLLTECGMSLCTEV 1187 482 CLCTCVGSLLTEV 1178 483 CSLLTECGCSLLTEC 1177 484 SLLTEVETPIRNEWGCRCNDHSD 1175 485 CSLLTECGMCLCTCV 1168 486 SLLTEVETPIRNEWGCRCNDHSD 1156 487 SLLTNVETPIRNEWGCRCNDSSD 1142 394 SLLTEVETPIRNEWGCRCNMSSD 1133 488 SLLTEVETPIHNEWGCRCNDSSD 1112 489 MSLLTECGMCLLTEV 1111 490 SLLTEVETPIRNEWGCRCNDHSD 1109 491 SLLTEVETPIRNEWGCRCNDHSD 1109 492 MSLLTECGMCLCTCV 1107 493 SLLTEVETMIRNEWGCRCNDSSD 1104 494 SLLTEVETPIRNEWGCRCNDHSD 1102 495 SLLTEVETPIRNEWGCRCNDHSD 1097 496 SCLTEVGSLLCEV 1095 436 SLLTEVETPI 1084 497 SLLTECGSCLCEC 1077 498 SLLTEMETPIRNEWGCRCNDSSD 1076 499 CSLLTEVGMSCLCEC 1066 500 SCLTEVGSLLTEV 1066 501 SLLTEVETPIRNEWGCRCNDHSD 1065 502 CSCLCEVGSLLTEC 1064 503 SLLTEVETPIRNEWGCWCNDSSD 1061 504 SLLTEVETPIRNEWGCRCNDHSD 1057 505 SLLTEKETPIRNEWGCRCNDSSD 1055 506 SLCTEVGSLLCEV 1050 507 SLLTEVETPIRNEWGCRCNDSWD 1050 508 MSLCTEVGCSLLTEV 1049 509 SLLTEVETPIYNEWGCRCNDSSD 1049 510 SLRTEVETPIRNEWGCRCNDSSD 1047 511 SLLCEVGCSLLTEV 1044 512 SLLTEVETPIRNEWGCRCNDSWD 1039 513 MCLLTEVGMSLLTEV 1032 514 SLLTEVETPIRNEWGCRCNDSWD 1029 515 SLLTEWETPIRNEWGCRCNDSSD 1025 516 SLLTEVETPIRNEWGCRCPDSSD 1024 517 SLLTEVETPIRNEMGCRCNDSSD 1020 518 SYLTEVETPIRNEWGCRCNDSSD 1019 519 SLLTEVETPIRNEWGCRCNDSWD 1016 520 VLLTEVETPIRNEWGCRCNDSSD 1014 437 SLLTEVETPIR 1012 521 SLLTEVETPIRNEWGCRCNDSWD 1012 522 SLLTMVETPIRNEWGCRCNDSSD 1011 523 SLLTEVETPIRNEWGCRCNDSWD 1009 524 SLLTEVETPIDNEWGCRCNDSSD 1001 401 SLLTEVETPIRNEWGCRCNWSSD 999 525 SLLTEVGCSCLCEV 996 526 MSLLTECGMSCLCEC 992 527 SLLTEVETPIRNEWGCRCNDSWD 986 528 MSLLTCVGCSLLTEV 978 529 SLLTEVETNIRNEWGCRCNDSSD 978 445 SLLTEVETPIRNEWGCRCN 977 530 SLLTEVETPIRNEWGCRCNDSWD 974 433 SLLTEVETPIRNEWGCRCNDSWD 971 531 MSLCTEVGMSLLTEC 970 532 MSCLTEVGMSLLTEV 968 533 CSLLTEVGSCLCEC 963 534 SLLTEVEKPIRNEWGCRCNDSSD 963 535 SLLTEVETPIRNEWGCRCNDSFD 963 408 SLLTEVETPIRNEWGCRCNDSFD 962 536 MCLCTCVGCSLLTEV 954 537 SLLTEVEPPIRNEWGCRCNDSSD 953 538 LLLTEVETPIRNEWGCRCNDSSD 950 539 SLLTEYETPIRNEWGCRCNDSSD 950 422 SLLTEVETPIRNEWGCRCNDSFD 947 421 SLLTEVETPIRNEWGCRCNDSED 944 540 SLLTEHETPIRNEWGCRCNDSSD 942 541 SLLTEVETPIRNEWKCRCNDSSD 932 418 SLLTEVETPIRNEWGCRCNDWSD 929 542 MSLCTEVGMSLLTCV 927 395 SLLTEVETPIRNEWGCRCNNSSD 926 543 MSLLTCVGMSLLTEV 925 544 SLLTEVETPIRNEWGCRCRDSSD 924 545 SLLTEVETPIRNEWGCRCNDSFD 923 546 SLLTEVETPIRNRWGCRCNDSSD 921 547 SLLTEVETPIRNEWGCRCNDSFD 919 548 CSLLTECGSLCTEV 917 549 SLLTEVETPIRNEWGCRCNDSFD 916 550 SLLTENETPIRNEWGCRCNDSSD 914 551 SLLTEVETPIRNEWGCRCNDSFD 914 552 SLLTEVETPIRNEWGCYCNDSSD 913 553 SLLTEVETPIRNEWGCRCNDSFD 909 554 CLLTEVGSLLTCV 902 555 SLLTEVETPIRNEWGCRCWDSSD 901 556 SLLTEVETPIRNEWGCRCNDSFD 900 557 MSCLTEVGCSLLTEV 899 435 SLLTEVETP 899 409 SLLTEVETPIRNEWGCRCNDKSD 898 444 SLLTEVETPIRNEWGCRC 898 558 CSLLTECGMSCLCEC 897 559 SLLTEVETPIRNEWGCRCNDSYD 895 560 SLLTEVETPIRNEWGCRCNDSYD 894 561 SLLTEVETPIRNEWGCRCNDSYD 892 562 SLLTEVETPIRWEWGCRCNDSSD 890 398 SLLTEVETPIRNEWGCRCNRSSD 890 563 SLLTEVETPIRNEWGCRCNDSYD 887 564 SLLTEVETPIRNEWGCRCNDSYD 886 565 SLLTEVETPIRNEWGCRCNDSYD 884 566 SLLTEVETPIRNEWGCRCNDSYD 881 567 SLLTECGCSCLCEV 879 568 SLLTEVETPIRNEWGCRCNDSYD 877 569 SLLTEVETPIRNEWGCRCNDSYD 876 570 MCLLTEVGCSLLTEV 875 571 SLLTEVETPIRNEWGCRCNDSYD 870 572 SLLTEVETPIRNEKGCRCNDSSD 868 573 SLLTEVETPIRNEWGCRCNDSYD 867 574 CLLTEVGSLLTEC 866 575 SLLTEVRTPIRNEWGCRCNDSSD 866 400 SLLTEVETPIRNEWGCRCNDSYD 865 443 SLLTEVETPIRNEWGCR 864 576 SLLTEVETPIRNEWGCRCNDSYD 862 577 SLLTEVETPIRNEWGCRCNDSYD 862 434 SLLTEVETPIRNEWGCRCNDSYD 862 415 SLLTEVETPIRNEWGCRCNDRSD 860 578 SLLTERETPIRNEWGCRCNDSSD 859 579 SLLTEVETPIRNEWGCRWNDSSD 859 580 SLCTEVGCSLLTEV 858 581 SLLTEVETYIRNEWGCRCNDSSD 857 582 SLLTEVETPIRNEWGCRCNDSYD 856 583 SLLTEVWTPIRNEWGCRCNDSSD 855 584 SLLTEVETPIRNEWGCRRNDSSD 855 585 SLLTEVETPIRLEWGCRCNDSSD 853 586 SLLTEVETPIRNEWGCRCNISSD 851 587 SLLTEVETPIRCEWGCRCNDSSD 850 396 SLLTEVETPIRNEWGCRCNPSSD 849 588 SLLTEVETPIRNEWGCRCNCSSD 848 589 CSLLTECGMSCLTEV 846 590 SLLTEVEMPIRNEWGCRCNDSSD 845 591 SLLTEVETPHRNEWGCRCNDSSD 844 397 SLLTEVETPIRNEWGCRCNQSSD 842 592 MSLCTEVGCSLLTEC 840 593 SLLTEVEIPIRNEWGCRCNDSSD 839 594 SLLTEVQTPIRNEWGCRCNDSSD 838 595 SLLTEVETPIRNEWGWRCNDSSD 838 596 SLLTEVETPIRNEWFCRCNDSSD 837 597 SLLTEVETGIRNEWGCRCNDSSD 835 598 SLLTEVETPIRNEWGCRKNDSSD 835 599 SLLTEVERPIRNEWGCRCNDSSD 834 430 SLLTEVETPIRNEWGCRCNDSRD 833 448 SLLTEVETPIRNEWGCRCNDSS 833 600 SLLTEVETPIRNEWGLRCNDSSD 831 601 SLLTECGSCLTEV 830 602 SLLTEVETEIRNEWGCRCNDSSD 828 603 SLLTEVETPIRNEWGCRCNGSSD 828 604 SLCTEVGSCLTEV 824 605 SLLTEVETPIRNMWGCRCNDSSD 820 606 SLLTEVETQIRNEWGCRCNDSSD 819 607 SLLTEVESPIRNEWGCRCNDSSD 817 447 SLLTEVETPIRNEWGCRCNDS 817 608 SLLTEIETPIRNEWGCRCNDSSD 816 609 SLLTEVETHIRNEWGCRCNDSSD 816 610 SLLTEVETDIRNEWGCRCNDSSD 813 611 SLLTEVETRIRNEWGCRCNDSSD 813 612 SLLTEVETPIRNEWGCRCIDSSD 812 613 SLLTEVETPIRNEWGCRCNHSSD 812 614 SLLTEVETVIRNEWGCRCNDSSD 810 615 SLLTEVETPIRNEWGCDCNDSSD 810 616 SLLTEVETPIRNPWGCRCNDSSD 808 617 SLLTEVETPIRNSWGCRCNDSSD 807 618 SLLTEVETPIRNEWGCRCLDSSD 807 619 SLLTEVETPIRNEWGCRCNASSD 807 620 SLLTEVETPIRFEWGCRCNDSSD 806 621 SLLTEVEFPIRNEWGCRCNDSSD 805 622 SLLTEVETPIRMEWGCRCNDSSD 805 623 SLLTEVETCIRNEWGCRCNDSSD 800 624 SLLTEVETPIRNEWVCRCNDSSD 799 413 SLLTEVETPIRNEWGCRCNDPSD 797 399 SLLTEVETPIRNEWGCRCNTSSD 795 625 SLLTEVETPIRNEWYCRCNDSSD 794 416 SLLTEVETPIRNEWGCRCNDTSD 794 626 SLLTEVETPIRNEWGCRCYDSSD 791 627 SLLTEVETPIRIEWGCRCNDSSD 789 406 SLLTEVETPIRNEWGCRCNDFSD 788 628 SLLTEVETPIRNEWGCRCMDSSD 786 629 SLLTEVMTPIRNEWGCRCNDSSD 783 630 SLLTEVETPERNEWGCRCNDSSD 783 631 SLLTEVETPIRKEWGCRCNDSSD 782 423 SLLTEVETPIRNEWGCRCNDSHD 782 426 SLLTEVETPIRNEWGCRCNDSLD 779 632 SLLTEVETPIRNEWGCRCVDSSD 775 633 SLLTEVETPIRNEWHCRCNDSSD 772 403 SLLTEVETPIRNEWGCRCNDCSD 772 417 SLLTEVETPIRNEWGCRCNDVSD 772 428 SLLTEVETPIRNEWGCRCNDSPD 772 634 SLLTEVETPIRNEWGCRCNDSKD 770 425 SLLTEVETPIRNEWGCRCNDSKD 770 635 SLLTEVETPIRNEWGCRCNDSKD 769 410 SLLTEVETPIRNEWGCRCNDLSD 769 636 SLLTEVETPIRNEWGCRCNDSKD 768 637 SLLTEVETPIRNENGCRCNDSSD 768 638 SLLTEVETPIRNEWGCRCNDSKD 767 639 SLLTEVETPIRNEWGCRCNDSKD 767 640 SLLTEVETPIRNKWGCRCNDSSD 765 641 SLLTEVETPIRNEWGCRCNDSKD 765 642 SLLTEVETPIRNEWMCRCNDSSD 765 643 SLLTEVETPIRNEWGCRCNDSKD 764 644 SLLTEVETPIRNEWGCRCNDSKD 764 645 SLLTEVETPIRNEWGCRCNDSKD 763 646 SLLTEVETPIRNEWGCRCNDSKD 763 647 SLLTEVETPIRNEWGHRCNDSSD 763 648 SLLTEVETPIRNEWGKRCNDSSD 762 649 SLLTEVETPIRNEWGCRCNDSKD 760 650 SLLTEVETPIRNEWGCRCNDSKD 758 651 SLLTEVETPIRNEWGCRCHDSSD 758 652 SLLTEVEEPIRNEWGCRCNDSSD 757 653 SLLTEVETPIRNEWGCRCNDSKD 756 654 SLLTEVETPIRYEWGCRCNDSSD 755 655 SLYTEVETPIRNEWGCRCNDSSD 753 656 SLLTEVETPIWNEWGCRCNDSSD 753 657 SLLTEVDTPIRNEWGCRCNDSSD 751 658 SLLTEVYTPIRNEWGCRCNDSSD 751 438 SLLTEVETPIRN 751 659 SLLTEVETPIRNEWGCRCNDSKD 750 440 SLLTEVETPIRNEW 750 660 SLLTEVETPDRNEWGCRCNDSSD 749 661 SLLTEVETPIRNEWGCRCNDSKD 748 662 SLLTEVETPIRNEWGCRCNDMSD 747 663 MCLCTCVGMSLLTEV 746 664 SLLTEVETPIRNEWGNRCNDSSD 746 665 SLLTEVETPIRNEWGCRCNDMSD 745 411 SLLTEVETPIRNEWGCRCNDMSD 744 666 SLLTEVETPIRNEWGCRCNDMSD 743 667 SLLTEVETPIRNEWGCRCNDMSD 739 668 SLLTEVETPIRNEWGYRCNDSSD 739 414 SLLTEVETPIRNEWGCRCNDQSD 739 669 SLLTEVETPIRNEWGCRCNDMSD 738 446 SLLTEVETPIRNEWGCRCND 738 670 SLLTEVETWIRNEWGCRCNDSSD 736 671 SLLTEVETPIRNEPGCRCNDSSD 735 672 SLLTEVETPIRNEWGRRCNDSSD 735 673 SLLTEVETPIRNEWGCRCNDMSD 733 674 SLLTEVETPIRNEWGCRCNDMSD 733 675 SLLTEVNTPIRNEWGCRCNDSSD 732 676 SLLTEVETPIENEWGCRCNDSSD 731 677 SLLTEVETPIRNEWGCRCNDMSD 731 678 SLLTEVETPIRNEWGCRCNDMSD 730 679 SLLTEVETPIRNEWGCECNDSSD 730 680 SLLTEVETPIRNEWGCRCNDMSD 729 681 SLLTEVETPIRNEWGCRCNDMSD 729 682 SLLTEVETPIRNEWGCRCNDMSD 729 683 SLLTEVETPIRNEWGCRCNDMSD 729 684 SLLTEVETPIRNHWGCRCNDSSD 728 685 SLLTEVETPIRNEWGCRCNDMSD 728 686 SLLTEVETPIRNEWGCRCNDMSD 728 432 SLLTEVETPIRNEWGCRCNDSVD 728 420 SLLTEVETPIRNEWGCRCNDSCD 725 687 SLLTEVETPIRNEWGCRCNDMSD 724 688 SLLTEVETPIRNEWGCRCNDMSD 724 689 SLLTEVETPIRNEWGCRCGDSSD 724 690 SLLTEVHTPIRNEWGCRCNDSSD 722 691 SLLTEVETPIRNEWGCRMNDSSD 722 692 SLLTEVETPILNEWGCRCNDSSD 721 693 SLLTEVETPWRNEWGCRCNDSSD 720 694 SLLTEVETPIRNEWGCRCNESSD 720 695 SLLTEVEVPIRNEWGCRCNDSSD 719 696 SLLTEVETPIRNEHGCRCNDSSD 719 697 SLLTEVETPIRNEWGCRINDSSD 718 698 SLLTEVETPIRDEWGCRCNDSSD 716 699 SLLTEVETPIRNEWGCRYNDSSD 716 700 SLLTEVETSIRNEWGCRCNDSSD 715 701 SLLTEVETPIRNEWGCKCNDSSD 715 702 SLLTEVETPIRNYWGCRCNDSSD 714 703 SLLTEVETPIRNWWGCRCNDSSD 713 704 SLLTEVETPIRNEWRCRCNDSSD 713 705 SLLTEVKTPIRNEWGCRCNDSSD 712 706 SLLTEVETPIRNEWGCRCADSSD 712 707 SLLTEVETPIANEWGCRCNDSSD 708 708 SLLTEVETPIRNEGGCRCNDSSD 708 412 SLLTEVETPIRNEWGCRCNDNSD 708 709 SLLTEVLTPIRNEWGCRCNDSSD 707 710 SLLTEVETPIRREWGCRCNDSSD 707 711 CSLLTEVGSLCTEV 707 712 SLLTEVETPIRNDWGCRCNDSSD 706 713 SLLTEVETPIRNEWGQRCNDSSD 705 714 SLLTEVETPIRNEWCCRCNDSSD 704 715 SLLTEVETPARNEWGCRCNDSSD 702 716 SLLTEVETPIRNEFGCRCNDSSD 702 717 SLLTEVETPIRNEWDCRCNDSSD 701 431 SLLTEVETPIRNEWGCRCNDSTD 701 718 SLLTEVECPIRNEWGCRCNDSSD 700 719 SLLTEVETPIRNEWGCRCNDSQD 700 720 SLLTEVETPIRNEWGCRCNDSQD 699 721 SLLTEVETPIRHEWGCRCNDSSD 699 722 SLLTEVETPIRNEWGCRCNDSQD 699 723 SLLTEVETPIRNEWGCRCNDSQD 698 724 MCLLTEVGMSLLTEC 697 725 SLLTEVETPIRNEWGCRCNDSQD 697 726 SLLTEVETPIRNEWECRCNDSSD 697 727 SLLTEVETPIRNEWGPRCNDSSD 696 728 SLLTEVETPIRNEWGCRCNDSQD 696 729 SLLTEVETPIRNEWGCRCNDSQD 696 730 SLLTEVETPIRNEWGCMCNDSSD 695 429 SLLTEVETPIRNEWGCRCNDSQD 695 731 SLLTEVETPIRNEWGCRCNDSQD 694 732 SLLTEVETPIRNEWGMRCNDSSD 693 733 SLLTEVETPIRNEWGCRCNDSQD 692 734 SLLTEVETPIRNEWGCRHNDSSD 692 424 SLLTEVETPIRNEWGCRCNDSQD 692 735 SLLTEVETPIRNEWGCRCNDSQD 691 736 SLLTEVETPIRNEWGCRCNDSQD 691 737 SLLTEVETPIRNERGCRCNDSSD 690 738 SLLTEVETPPRNEWGCRCNDSSD 689 739 SLLTEVETPIRNEWGCRCEDSSD 687 740 SHLTEVETPIRNEWGCRCNDSSD 686 741 SLLTEVETPIRNNWGCRCNDSSD 686 742 SLLTEVETPIRNEWGCRCNDSQD 684 404 SLLTEVETPIRNEWGCRCNDDSD 684 743 SLLTEVETPIRNEWGCNCNDSSD 683 402 SLLTEVETPIRNEWGCRCNDASD 683 744 SLLTEVETPIRNEWGCRCNDSQD 681 745 SLLTEVETPIRNEWGCRCNDSQD 681 746 SLLTEVETPIRNEWGCRNNDSSD 681 747 SLCTEVGCSLLTEC 680 748 SLLTEVETPIRNEWGCRCNDESD 680 442 SLLTEVETPIRNEWGC 680 749 MSLLCEVGMSLLTEV 677 750 SLLTEVETPRRNEWGCRCNDSSD 677 405 SLLTEVETPIRNEWGCRCNDESD 676 751 SLLTEVETPIRNEDGCRCNDSSD 675 752 SLLTEVETPIRNEWGCRCNDESD 674 753 SLLTEVETPIRPEWGCRCNDSSD 673 754 SLLTEVETPIRNEWGCRCNDESD 672 755 SLLTEVETPIRNEWGCRCNDESD 671 756 SLLTEVETPIRNEWGCRCNDESD 671 757 SLLTEVETPIRNEWGCRPNDSSD 669 758 SLLTEVETPIRNEWGCRCNDESD 668 427 SLLTEVETPIRNEWGCRCNDSMD 668 759 SLHTEVETPIRNEWGCRCNDSSD 667 760 SLLTEVETPIRNEWGCRCNDESD 667 761 MSLLTEVGCSCLCEV 663 762 SLLTEVETPIRNEWGCRCDDSSD 662 763 SLLTEVEWPIRNEWGCRCNDSSD 660 764 SLLTEPETPIRNEWGCRCNDSSD 659 765 SLLTEVETPIRNEWGCRCNDESD 657 766 SLLTEVETPIRNEWGCRCNDESD 653 767 SLLTEVETPIRNEWGCPCNDSSD 653 768 MSCLTEVGMSLLTEC 650 769 MCLLTEVGCSLLTEC 650 770 SLLTEVETPNRNEWGCRCNDSSD 649 771 SLLTEVETPIREEWGCRCNDSSD 648 772 SLLTEVETPIRNEWGCRCNDESD 646 773 SLLTEVETPIRNEWGCRCNDESD 644 774 SLLTEVETPIRNEWPCRCNDSSD 644 775 SLLTEVETPIRNEWGCRCNDESD 644 807 CLLTEVGCSLLTEV 520 808 SCLTEVGCSLLTEC 514 809 CSLLTECGCLLTEV 507 810 SLLTCVGCSLLTEV 504 811 CSLLTECGCLCTCV 458 812 SLLTCVGCSLLTEC 447 813 CSLLTECGSLLTEC 441 814 CSLLTECGSLLCEV 430 815 CSLLTECGSCLCEC 419 816 CLLTEVGCSLLTEC 409 817 CSLLTECGCSCLCEV 404 818 MSLLTECGCSLLTEC 386 819 CLCTCVGCSLLTEC 340 820 MSLLTCVGCSLLTEC 330 821 CSCLCEVGCSLLTEV 325 822 SLLTDVETPIRNEWGCRCNDSSD 324 823 SCLTEVGSLLTEC 321 824 SLLTCVGSLLTEV 320 825 MSLLCEVGMSLLTEC 314 826 MSLLCEVGCSLLTEC 312 827 MSCLCECGCSLLTEV 306 828 MCLCTCVGMSLLTEC 306 829 MSLLTEVGMSCLCEC 304 830 1CSLLTEVETP1SLLTEVETPCLLTEVETPI1 2303 831 1CSLLTEVETP1SLLTEVETPCSLLTEVETP1 2147 832 1CLTEVETPIR1SLLTEVETPCLLTEVETPI1 2145 833 SLLTEVETCSLLTEVETATPIRNEWGCRC 2133 834 SLLTEVET1CSLLTEVET1SLLTEVETCEWG1R 2107 835 SLLTEVET1CSLLTEVET1SLLTEVETAWGCR1 2103 836 1CLTEVETPIR1SLLTEVETPCSLLTEVETP1 1936 837 1CETPIRNEWG1SLLTEVETPCLLTEVETPI1 1912 838 1CTEVETPIRN1SLLTEVETPCLLTEVETPI1 1887 839 1CTEVETPIRN1SLLTEVETPCSLLTEVETP1 1796 840 SLLTEVET1CSLLTEVET1SLLTEVETCEWGSR1 1765 841 1CSLLTEVETP1LTEVETPIRCSLLTEVETP1 1732 842 1CSLLTEVETP1TEVETPIRNCSLLTEVETP1 1717 843 1CVETPIRNEW1SLLTEVETPCLTEVETPIR1 1715 844 SLLTEVET1CSLLTEVET1SLLTEVETAGCR1N 1712 845 1CSLLTEVETP1SLLTEVETPCETPIRNEWG1 1704 846 SLLTEVETCSLLTEVETCNEWGSRSNDSSC 1697 847 1CLLTEVETPI1SLLTEVETPCLLTEVETPI1 1665 848 1CEVETPIRNE1SLLTEVETPCSLLTEVETP1 1655 849 1CLLTEVETPI1LTEVETPIRCSLLTEVETP1 1651 850 1CSLLTEVETP1SLLTEVETPCVETPIRNEW1 1649 851 1CSLLTEVETP1VETPIRNEWCSLLTEVETP1 1634 852 SLLTEVET1CSLLTEVET1SLLTEVETCIRNEW1 1598 853 SLLTEVET1CSLLTEVET1SLLTEVETCLLTEV1 1592 854 1CTEVETPIRN1LTEVETPIRCSLLTEVETP1 1590 855 1CEVETPIRNE1SLLTEVETPCLLTEVETPI1 1584 856 SLLTEVETCSLLTEVETCVETPIRNEWGC 1584 857 1CLTEVETPIR1SLLTEVETPCLTEVETPIR1 1576 858 1CETPIRNEWG1SLLTEVETPCSLLTEVETP1 1559 859 1CSLLTEVETP1SLLTEVETPCLTEVETPIR1 1552 860 1CVETPIRNEW1SLLTEVETPCSLLTEVETP1 1531 861 SLLTEVET1CSLLTEVET1SLLTEVETCSLLTE1 1490 862 SLLTEVET1CSLLTEVET1SLLTEVETCTPIRN1 1481 863 1CLTEVETPIR1LTEVETPIRCSLLTEVETP1 1458 864 1CTEVETPIRN1SLLTEVETPCLTEVETPIR1 1452 865 SLLTEVETCSLLTEVETAEWGCRCNDSSD 1451 866 1CVETPIRNEW1SLLTEVETPCETPIRNEWG1 1446 867 1CLLTEVETPI1SLLTEVETPCSLLTEVETP1 1438 868 1CEVETPIRNE1SLLTEVETPCETPIRNEWG1 1425 869 1CSLLTEVETP1EVETPIRNECSLLTEVETP1 1405 870 1CEVETPIRNE1EVETPIRNECSLLTEVETP1 1400 871 1CVETPIRNEW1SLLTEVETPCLLTEVETPI1 1396 872 1CEVETPIRNE1LTEVETPIRCSLLTEVETP1 1382 873 1CSLLTEVETP1VETPIRNEWCLLTEVETPI1 1382 874 SLLTEVETCSLLTEVETCEWGSRSNDSSDC 1375 875 SLLTEVETCSLLTEVETCSLLTEVETPIRC 1369 876 1CVETPIRNEW1LTEVETPIRCSLLTEVETP1 1327 877 1CVETPIRNEW1ETPIRNEWGCSLLTEVETP1 1317 878 1CSLLTEVETP1TEVETPIRNCLLTEVETPI1 1313 879 SLLTEVETCSLLTEVETCETPIRNEWGCR 1306 880 1CLTEVETPIR1TEVETPIRNCSLLTEVETP1 1290 881 CSLLTEVETPIRNEWGCETPIRNEWGSRSNDSC 1289 882 1CETPIRNEWG1LTEVETPIRCSLLTEVETP1 1286 883 1CLLTEVETPI1TEVETPIRNCSLLTEVETP1 1270 884 SLLTEVETCSLLTEVETCRNEWGSRSNDSC 1255 885 1CSLLTEVETP1ETPIRNEWGCSLLTEVETP1 1250 886 1CSLLTEVETP1LTEVETPIRCLTEVETPIR1 1241 887 1CTEVETPIRN1TEVETPIRNCSLLTEVETP1 1229 888 CSLLTEVETCPIRNEWGSRSNDSSDC 1224 889 1CSLLTEVETP1EVETPIRNECLLTEVETPI1 1221 890 SLLTEVET1CSLLTEVET1SLLTEVETCETPIR1 1208 891 CSLLTEVETPIRNCETPIRNEWGSRSNDSC 1204 892 1CSLLTEVETP1LLTEVETPICLTEVETPIR1 1203 893 SLLTEVET1CSLLTEVET1SLLTEVETCVETPI1 1200 894 CSLLTEVETCTPIRNEWGSRSNDSSC 1196 895 SLLTEVET1CSLLTEVET1SLLTEVETCRNEWG1 1195 896 1CETPIRNEWG1ETPIRNEWGCSLLTEVETP1 1180 897 1CEVETPIRNE1SLLTEVETPCLTEVETPIR1 1169 898 SLLTEVET1CSLLTEVET1SLLTEVETACR1ND 1169 899 SLLTEVETCSLLTEVETAPIRNEWGCRCN 1165 900 1CSLLTEVETP1LLTEVETPICSLLTEVETP1 1157 901 SLLTEVET1CSLLTEVET1SLLTEVETCLTEVE1 1144 902 1CLTEVETPIR1ETPIRNEWGCSLLTEVETP1 1125 903 1CEVETPIRNE1ETPIRNEWGCSLLTEVETP1 1124 904 SLLTEVETCSLLTEVETAIRNEWGCRCND 1120 905 CSLLTEVETPICETPIRNEWGSRSNDSC 1120 906 CSLLTEVETPIRNEWGCPIRNEWGSRSNDSSDC 1118 907 SLLTEVET1CSLLTEVET1SLLTEVETCPIRNE1 1104 908 SLLTEVET1CSLLTEVET1SLLTEVETCNEWG1 1103 909 SLLTEVETCSLLTEVETCLLTEVETPIRNC 1101 910 1CETPIRNEWG1SLLTEVETPCLTEVETPIR1 1099 911 CSLLTEVETPIRCETPIRNEWGSRSNDSC 1087 912 SLLTEVETCSLLTEVETCTPIRNEWGSRSC 1081 913 1CTEVETPIRN1SLLTEVETPCEVETPIRNE1 1079 914 SLLTEVET1CSLLTEVET1SLLTEVETCEVETP1 1062 915 SLLTEVET1CSLLTEVET1SLLTEVETCTEVET1 1053 916 1CSLLTEVETP1LTEVETPIRCLLTEVETPI1 1048 917 CSLLTEVETPIRNECETPIRNEWGSRSNDSC 1037 918 1CSLLTEVETP1SLLTEVETPCTEVETPIRN1 1022 919 1CETPIRNEWG1TEVETPIRNCSLLTEVETP1 1021 920 CSLLTEVETPIRNECTPIRNEWGSRSNDSSC 1019 921 1CEVETPIRNE1TEVETPIRNCSLLTEVETP1 1014 922 CSLLTEVETPCETPIRNEWGSRSNDSC 1010 923 1CSLLTEVETP1LLTEVETPICLLTEVETPI1 1009 924 CSLLTEVETPIRCTPIRNEWGSRSNDSSC 1000 925 SLLTEVETCSLLTEVETCVETPIRNEWGSC 979 926 SLLTEVETCSLLTEVETARNEWGCRCNDS 963 927 SLLTEVETCSLLTEVETCETPIRNEWGSRC 935 928 SLLTEVETCSLLTEVETANEWGCRCNDSS 897 929 1CETPIRNEWG1VETPIRNEWCSLLTEVETP1 890 930 SLLTEVETCSLLTEVETCEVETPIRNEWGC 867 931 CSLLTEVETPCPIRNEWGSRSNDSSDC 853 932 1CLTEVETPIR1VETPIRNEWCSLLTEVETP1 852 933 SLLTEVETCSLLTEVETCPIRNEWGSRSNC 840 934 1CEVETPIRNE1VETPIRNEWCSLLTEVETP1 834 935 1CVETPIRNEW1SLLTEVETPCVETPIRNEW1 815 936 1CETPIRNEWG1EVETPIRNECSLLTEVETP1 809 937 1CSLLTEVETP1ETPIRNEWGCLLTEVETPI1 805 938 CSLLTEVETCETPIRNEWGSRSNDSC 796 939 1CSLLTEVETP1LLTEVETPICETPIRNEWG1 777 940 SLLTEVETCSLLTEVETCTEVETPIRNEWC 775 941 1CEVETPIRNE1SLLTEVETPCTEVETPIRN1 772 942 1CETPIRNEWG1SLLTEVETPCTEVETPIRN1 749 943 1CLLTEVETPI1VETPIRNEWCSLLTEVETP1 743 944 1CSLLTEVETP1TEVETPIRNCLTEVETPIR1 740 945 1CLTEVETPIR1SLLTEVETPCVETPIRNEW1 733 946 1CLTEVETPIR1SLLTEVETPCTEVETPIRN1 729 947 1CVETPIRNEW1SLLTEVETPCTEVETPIRN1 724 948 SLLTEVETCSLLTEVETCIRNEWGSRSNDC 721 949 1CLLTEVETPI1SLLTEVETPCLTEVETPIR1 717 950 1CETPIRNEWG1SLLTEVETPCVETPIRNEW1 715 951 1CTEVETPIRN1SLLTEVETPCVETPIRNEW1 711 952 1CTEVETPIRN1VETPIRNEWCSLLTEVETP1 710 953 1CVETPIRNEW1TEVETPIRNCSLLTEVETP1 708 954 CSLLTEVETPICPIRNEWGSRSNDSSDC 700 955 1CVETPIRNEW1VETPIRNEWCSLLTEVETP1 699 956 1CTEVETPIRN1SLLTEVETPCETPIRNEWG1 691 957 1CTEVETPIRN1SLLTEVETPCTEVETPIRN1 684 958 1CSLLTEVETP1SLLTEVETPCEVETPIRNE1 684 959 1CEVETPIRNE1SLLTEVETPCVETPIRNEW1 674 960 SLLTEVETCSLLTEVETCLTEVETPIRNEC 666 961 1CLTEVETPIR1EVETPIRNECSLLTEVETP1 665 962 1CLTEVETPIR1SLLTEVETPCETPIRNEWG1 657 963 1CVETPIRNEW1EVETPIRNECSLLTEVETP1 652 964 1CETPIRNEWG1SLLTEVETPCETPIRNEWG1 645 965 CSLLTEVETPCTPIRNEWGSRSNDSSC 645 966 CSLLTEVETPICTPIRNEWGSRSNDSSC 622 967 1CLLTEVETPI1EVETPIRNECSLLTEVETP1 617 968 1CLLTEVETPI1ETPIRNEWGCSLLTEVETP1 608 969 CSLLTEVETPIRCPIRNEWGSRSNDSSDC 606 970 CSLLTEVETPIRNCPIRNEWGSRSNDSSDC 589 971 1CSLLTEVETP1LTEVETPIRCTEVETPIRN1 571 972 1CSLLTEVETP1LTEVETPIRCETPIRNEWG1 564 973 CSLLTEVETPIRNECPIRNEWGSRSNDSSDC 553 974 1CSLLTEVETP1LLTEVETPICVETPIRNEW1 551 975 1CLLTEVETPI1SLLTEVETPCVETPIRNEW1 539 976 1CLTEVETPIR1SLLTEVETPCEVETPIRNE1 538 977 1CVETPIRNEW1LLTEVETPICSLLTEVETP1 537 978 1CTEVETPIRN1EVETPIRNECSLLTEVETP1 535 979 CSLLTEVETPIRNCTPIRNEWGSRSNDSSC 532 980 CSLLTEVETPIRNEWGCTPIRNEWGSRSNDSSC 508 981 1CSLLTEVETP1TEVETPIRNCTEVETPIRN1 505 982 1CTEVETPIRN1ETPIRNEWGCSLLTEVETP1 504 983 SLLTEVET1CSLLTEVET1SLLTEVETCNEWGS1 346 984 CSLLTEVETPIRNC 2154 985 SLLTEVCTCIRNEWG 2047 986 SLLTEVETCSLLTEVETPIRNEWGSRC 1883 987 SLLTEVCTPICNEWG 1857 988 SLLTEVCTPIRCEWG 1853 989 SLLTEVECPIRCEWG 1849 990 CSLLTEVCTPIRNE 1721 991 SLLTEVETCIRNEWGC 1707 992 SLLTEVETCIRNCWG 1691 993 SLLTEVETCCRNEWG 1675 994 SLLTEVETPIRCCWG 1674 995 SLLTEVETSLLTEVETPIRNEWGCRCND 1663 996 SLLTEVCTPCRNEWG 1551 997 CSLLTEVETPIRCE 1512 998 SLLTEVETCIRCEWG 1495 999 SLLTEVETCSLLTEVETPIRNEWGSRSC 1488 1000 SLLTEVCTPIRNCWG 1440 1001 SLLTEVECPIRNCWG 1438 1002 SLCTEVETCIRNEWG 1385 1003 SLLTECETCIRNEWG 1384 1004 SLLTEVECCIRNEWG 1377 1005 SLLTEVETPCRNCWG 1377 1006 SLLTEVETCICNEWG 1360 1007 SLLTEVECPIRNC 1341 1008 SLLTECETPIRNCWG 1298 1009 SLLTEVETCIRNEWG 1286 1010 SLLTEVETPICNCWG 1255 1011 SLLTEVETCSLLTEVETPIRNEWGSRSNC 1239 1012 CSLLTEVETPICNE 1232 1013 CSLLTEVETCIRNE 1220 1014 SLLTEVCCPIRNEWG 1195 1015 SLLTEVETPIRNCWGC 1177 1016 SLLTEVCTPIRNC 1163 1017 SLLTEVECPIRNEWC 1161 1018 CSLLTEVETPIRNE 1159 1019 SLLTEVCTPIRNEWG 1156 1020 CSLLTEVETPCRNE 1148 1021 SLLTEVETPIRCEWGC 1135 1022 SLLTEVETCIRNC 1134 1023 SLLTEVETPIRCECG 1121 1024 CSLLTEVECPIRNE 1116 1025 SLLTEVECPCRNEWG 1110 1026 SCLTEVETPCRNEWG 1083 1027 CSLLTECETPIRNE 1082 1028 SLLTEVECPICNEWG 1079 1029 SLLTECETPIRCEWG 1052 1030 SLLTEVETCIRNEWC 1049 1031 SLCTEVETPIRNC 1035 1032 SLLTEVETCSLLTEVETPIRNEWGCR 1032 1033 SLLTECECPIRNEWG 1031 1034 SLLTEVETPICCEWG 1029 1035 SLLTEVECPIRCE 1027 1036 SLLTEVETPIRNCWG 1021 1037 SCLTEVCTPIRNE 1019 1038 SCLTEVETCIRNEWG 1015 1039 SLLTEVETPIRNCWC 1002 1040 SLLTEVETCSLLTEVETPIRC 995 1041 SLLTEVECPIRNEWG 980 1042 SLLTEVETSLLTEVETPIRNEWGCRC 979 1043 SLLTEVETSLLTEVETPIRNEWGCRCN 970 1044 SLLTEVCTCIRNE 949 1045 SLCTEVETPIRCE 945 1046 SLLTEVETCIRNECG 941 1047 SCLTEVETPIRNC 940 1048 SLLTEVETCSLLTEVETPIRNEWGC 938 1049 SLLTEVCTPIRNEWC 937 1050 SLLTEVECPIRNE 927 1051 SLLTEVETPICNEWGC 923 1052 SLLTEVETCSLLTEVETPIRNEWGSRSNDSSC 916 1053 SLLTEVETPIRCEWG 902 1054 SLCTEVETPIRNCWG 899 1055 SLLTEVETCIRCE 898 1056 SLLTECETPCRNEWG 897 1057 SLLTEVETPICNEWG 890 1058 SLLTEVETCSLLTEVETPIRNC 890 1059 SLLTECETPICNEWG 885 1060 SLLTECETPIRNEWG 880 1061 SLLTEVETPCRCEWG 875 1062 SLLTEVETCSLLTEVETPIRNEWGSRSNDC 874 1063 SLLTECCTPIRNEWG 866 1064 SLLTEVETCSLLTEVETPIRNEWGSRSNDSC 861 1065 SLLTEVCTPCRNE 853 1066 SCLTECETPIRNEWG 847 1067 SLLTEVETCSLLTEVETPC 844 995 SLLTEVETSLLTEVETPIRNEWGCRCNDS 836 1068 SLLTEVETCSLLTEVETPIRNEWGSRSNDSSDC 831 1069 SLLTEVETCSLLTEVETPIC 830 1070 SLLTEVETCSLLTEVETC 820 1071 SLLTEVETPCRNEWG 813 1072 CSLLTEVETPIRNEWGC 798 1073 SLLTEVCTPIRCE 789 1074 SLLTECETCIRNE 785 1075 SCLTECETPIRNE 780 1076 SLLTEVETCSLLTEVETPIRNEWC 768 1077 SLLTEVETPCCNEWG 766 1078 SLCTEVECPIRNEWG 750 1079 SLCTEVETPIRCEWG 750 1080 SLLTECETPIRNEWC 741 1081 SLLTEVCTPIRNEWGC 739 1082 SLLTEVETPIRCEWC 732 1083 SCLTEVETCIRNE 727 1084 SLLTEVETCICNE 727 1085 SLLTEVETPICNEWC 724 1086 SLLTEVECPIRNEWGC 715 1087 SLCTEVETCIRNE 712 1088 SLLTEVETCSLLTEVETPIRNEWGSC 712 1089 SLLTEVETSLLTEVETPIRNEWGCRCNDSS 711 1090 SCLTEVECPIRNE 705 1091 SLLTEVCTPICNE 699 1092 SLLTEVETPCRNEWGC 696 1093 SLLTEVETCIRNE 675 1094 SLLTEVCTPIRNECG 674 1095 SLLTEVETSLLTEVETPIRNEWGCRCNDSSD 669 1096 SLLTEVETPICNC 665 1097 SCLTEVETPIRNCWG 662 1098 SLLTEVETPICNECG 660 1099 SLLTEVECPIRNECG 656 1100 SLCTEVCTPIRNE 653 1101 SLLTECETPIRCE 648 1102 SLCTECETPIRNEWG 648 1103 SLCTEVECPIRNE 645 1104 SLLTECETPIRNC 643 1105 SLCTEVCTPIRNEWG 641 1106 SLLTEVETPIRNCCG 638 1107 SLLTEVETCSLLTEVETPIRNEC 629 1108 SLLTECETPIRNEWGC 607 1109 SLCTEVETPCRNEWG 607 1110 SLLTEVETPCRNEWC 589 1111 SLLTEVETPCRNECG 587 1112 SLLTEVECPICNE 582 1113 SLLTECETPIRNE 578 1114 SLCTEVETPICNEWG 578 1115 SLLTEVETPCRNC 571 1116 SLLTEVCTPIRNE 568 1117 SLLTEVETPIRNEWC 558 1118 SCLTEVECPIRNEWG 542 1119 SLLTEVECPCRNE 538 1120 SLCTEVETPCRNE 535 1121 SLCTEVETPIRNEWC 535 1122 SLLTECETPICNE 531 1123 SLLTECETPIRNECG 528 1124 SLCTEVETPIRNEWG 510 1125 SLLTECECPIRNE 507 1126 CMSLLTEVETPIRNC 1864 1127 MSLLTEVEKPIRNEWGCRCN 1854 1128 MSLLTEVETPCRNC 1834 1129 MSLLTEVECPIRNC 1752 1130 MSLLTEVCTPIRNC 1738 1131 DSLLTEVETPIRNEWGCRCN 1713 1132 MSLLTEVETPIRNEAGCRCN 1700 1133 MSLLTEVETPCRCE 1651 1134 MSLLTEVETPCRNE 1638 1135 MSLLTEVEMPIRNEWGCRCN 1580 1136 MSLLTEVEAPIRNEWGCRCN 1544 1137 MSLLTEVELPIRNEWGCRCN 1521 1138 MSLLTELETPIRNEWGCRCN 1497 1139 MSLLTEVETPIRNAWGCRCN 1480 1140 MSLTTEVETPIRNEWGCRCN 1474 1141 MTLLTEVETPIRNEWGCRCN 1470 1142 MSLLTEVETPICNC 1451 1143 MSLLTEVCTPIRCE 1428 1144 MSLLTEVETAIRNEWGCRCN 1398 1145 MSLCTEVETPIRNC 1395 1146 MSLLTEVETCIRNE 1387 1147 MSLLTEVETCICNE 1378 1148 MSLLTEVEEPIRNEWGCRCN 1361 1149 MSLLTEVETPIRNEWGCRCN 1360 1150 MSLLTEVEQPIRNEWGCRCN 1345 1151 MSLLTEVECPIRCE 1336 1152 MSLLTEKETPIRNEWGCRCN 1324 1153 ESLLTEVETPIRNEWGCRCN 1310 1154 MSLCTEVETCIRNE 1305 1155 MSLLTETETPIRNEWGCRCN 1279 1156 MSLLTEIETPIRNEWGCRCN 1255 1157 MSLLTEVETPIRNEWGCRAN 1252 1158 MSLLTEVCTPCRNE 1244 1159 TSLLTEVETPIRNEWGCRCN 1243 1160 MSLCTEVCTPIRNE 1243 1161 MSLLTEVETPIRNEWGCRCA 1233 1162 MSLVTEVETPIRNEWGCRCN 1202 1163 MSCLTEVETPIRCE 1192 1164 MSLATEVETPIRNEWGCRCN 1188 1165 MSLLTEVVTPIRNEWGCRCN 1183 1166 MSLLTEVQTPIRNEWGCRCN 1172 1167 MSLITEVETPIRNEWGCRCN 1167 1168 MSLLTEVECPIRNE 1158 1169 MSLLTEVCTPICNE 1157 1170 MSLLTEPETPIRNEWGCRCN 1153 1171 MSLLTEVEFPIRNEWGCRCN 1149 1172 MSLLTEVETPARNEWGCRCN 1149 1173 MSLLTEVETPIRNEWGARCN 1149 1174 MSLLTEVMTPIRNEWGCRCN 1133 1175 MSLLTECETPIRCE 1131 1176 CMSLLTEVECPIRNE 1122 1177 ASLLTEVETPIRNEWGCRCN 1111 1178 MSLLTEVETCIRCE 1105 1179 MSCLTEVETPIRNC 1102 1180 MSLLTEVEGPIRNEWGCRCN 1099 1181 MSLLTEVETPIRNEWACRCN 1087 1182 MSLLTEMETPIRNEWGCRCN 1075 1183 KSLLTEVETPIRNEWGCRCN 1068 1184 MSLLTEVEVPIRNEWGCRCN 1064 1185 MSLCTEVETPCRNE 1064 1186 MSCLTEVCTPIRNE 1055 1187 CMSLLTEVCTPIRNE 1054 1188 VSLLTEVETPIRNEWGCRCN 1054 1189 MSLLTEVESPIRNEWGCRCN 1051 1190 MSLLTEVSTPIRNEWGCRCN 1045 1191 MSLLTEFETPIRNEWGCRCN 1044 1192 CMSLLTEVETPIRCE 1034 1193 MSLCTEVECPIRNE 1023 1194 MSLLTECETPICNE 1016 1195 MSLLTEVTTPIRNEWGCRCN 1013 1196 RSLLTEVETPIRNEWGCRCN 1012 1197 MSLLTEVEDPIRNEWGCRCN 1004 1198 MSLLTEAETPIRNEWGCRCN 1003 1199 CMSLLTEVETPCRNE 992 1200 MSLLTEVETPIRAEWGCRCN 978 1201 MSLLTEVCTCIRNE 959 1202 MSLLTECECPIRNE 958 1203 HSLLTEVETPIRNEWGCRCN 956 1204 CMSLLTECETPIRNE 937 1205 MSLLTEVENPIRNEWGCRCN 936 1206 MSLLTECETPCRNE 932 1207 MSLLTEVERPIRNEWGCRCN 926 1208 MSLLTEQETPIRNEWGCRCN 913 1209 MSLLTEVETCIRNC 908 1210 MSFLTEVETPIRNEWGCRCN 907 1211 CMSLLTEVETCIRNE 896 1212 PSLLTEVETPIRNEWGCRCN 841 1213 MSLETEVETPIRNEWGCRCN 838 1214 MSLLTEVEHPIRNEWGCRCN 838 1215 YSLLTEVETPIRNEWGCRCN 835 1216 MSLLTEVCTPIRNE 824 1217 MSLLTEVECPICNE 821 1218 MSLLTEVETPIANEWGCRCN 814 1219 LSLLTEVETPIRNEWGCRCN 801 1220 WSLLTEVETPIRNEWGCRCN 742 1221 MSLLTEVETPIRNEWGCACN 739 1222 MSLLTEVECPCRNE 736 1223 MSCLTEVECPIRNE 730 1224 FSLLTEVETPIRNEWGCRCN 707 1225 NSLLTEVETPIRNEWGCRCN 706 1226 MSLLTEVEYPIRNEWGCRCN 685 1227 MSLLTDVETPIRNEWGCRCN 679 1228 MSKLTEVETPIRNEWGCRCN 672 1229 MSCLTEVETCIRNE 649 1230 MSLSTEVETPIRNEWGCRCN 648 1231 MSLLTECETPIRNE 630 1232 ISLLTEVETPIRNEWGCRCN 629 1233 MSCLTEVETPCRNE 625 1234 MSLCTEVETPIRNE 618 1235 CMSLLTEVETPICNE 615 1236 MSLLTEVEPPIRNEWGCRCN 610 1237 MSCLTEVETPIRNE 602 1238 GSLLTEVETPIRNEWGCRCN 584 1239 MSLCTEVETPIRCE 568 1240 MSLKTEVETPIRNEWGCRCN 567 1241 MSLLTEEETPIRNEWGCRCN 545 1242 CSLCTEVETPIRNE 518 1243 QSLLTEVETPIRNEWGCRCN 440 1244 MSLLTEVEIPIRNEWGCRCN 409 1245 MSLLTESETPIRNEWGCRCN 369 1246 MSLLTEGETPIRNEWGCRCN 357 1247 MSLLTECETPIRNC 344 1248 MSLLTECETCIRNE 312 1249 SSLLTEVETPIRNEWGCRCN 303

TABLE 3 Peptides that show specific binding to Z3G1 (A numeral “1” in the peptide sequence indicates a differentially protected cystein allowing for selective CLIP  attachment at certain residues) SEQ ID NO: Peptides that bind to Z3G1 Signal 1177 ASLLTEVETPIRNEWGCRCN 2816 1131 DSLLTEVETPIRNEWGCRCN 2814 1153 ESLLTEVETPIRNEWGCRCN 2816 1224 FSLLTEVETPIRNEWGCRCN 2665 1238 GSLLTEVETPIRNEWGCRCN 2635 1203 HSLLTEVETPIRNEWGCRCN 2490 1232 ISLLTEVETPIRNEWGCRCN 2438 1183 KSLLTEVETPIRNEWGCRCN 2618 1219 LSLLTEVETPIRNEWGCRCN 2506 1149 MSLLTEVETPIRNEWGCRCN 2711 1225 NSLLTEVETPIRNEWGCRCN 2485 1212 PSLLTEVETPIRNEWGCRCN 2694 1243 QSLLTEVETPIRNEWGCRCN 2619 1196 RSLLTEVETPIRNEWGCRCN 2550 1249 SSLLTEVETPIRNEWGCRCN 2404 1159 TSLLTEVETPIRNEWGCRCN 2834 1188 VSLLTEVETPIRNEWGCRCN 2816 1220 WSLLTEVETPIRNEWGCRCN 2693 1215 YSLLTEVETPIRNEWGCRCN 2650 1250 MALLTEVETPIRNEWGCRCN 2604 1251 MDLLTEVETPIRNEWGCRCN 2726 1252 MELLTEVETPIRNEWGCRCN 2679 1253 MFLLTEVETPIRNEWGCRCN 1707 1254 MGLLTEVETPIRNEWGCRCN 2635 1255 MHLLTEVETPIRNEWGCRCN 2449 1256 MILLTEVETPIRNEWGCRCN 1634 1257 MKLLTEVETPIRNEWGCRCN 2629 1258 MLLLTEVETPIRNEWGCRCN 2091 1259 MMLLTEVETPIRNEWGCRCN 2666 1260 MNLLTEVETPIRNEWGCRCN 2733 1261 MPLLTEVETPIRNEWGCRCN 2521 1262 MQLLTEVETPIRNEWGCRCN 2526 1263 MRLLTEVETPIRNEWGCRCN 2519 1141 MTLLTEVETPIRNEWGCRCN 2809 1264 MVLLTEVETPIRNEWGCRCN 2465 1265 MWLLTEVETPIRNEWGCRCN 2615 1266 MYLLTEVETPIRNEWGCRCN 1960 1210 MSFLTEVETPIRNEWGCRCN 1087 1267 MSILTEVETPIRNEWGCRCN 2582 1268 MSMLTEVETPIRNEWGCRCN 1329 1269 MSVLTEVETPIRNEWGCRCN 2111 1164 MSLATEVETPIRNEWGCRCN 2747 1270 MSLDTEVETPIRNEWGCRCN 2594 1213 MSLETEVETPIRNEWGCRCN 2768 1271 MSLFTEVETPIRNEWGCRCN 2115 1272 MSLHTEVETPIRNEWGCRCN 2058 1167 MSLITEVETPIRNEWGCRCN 2684 1240 MSLKTEVETPIRNEWGCRCN 2691 1273 MSLMTEVETPIRNEWGCRCN 2804 1274 MSLNTEVETPIRNEWGCRCN 2787 1275 MSLPTEVETPIRNEWGCRCN 2007 1276 MSLQTEVETPIRNEWGCRCN 2792 1277 MSLRTEVETPIRNEWGCRCN 2554 1230 MSLSTEVETPIRNEWGCRCN 2768 1140 MSLTTEVETPIRNEWGCRCN 2742 1162 MSLVTEVETPIRNEWGCRCN 2776 1278 MSLYTEVETPIRNEWGCRCN 1313 1279 MSLLAEVETPIRNEWGCRCN 2807 1280 MSLLDEVETPIRNEWGCRCN 2763 1281 MSLLEEVETPIRNEWGCRCN 2805 1282 MSLLFEVETPIRNEWGCRCN 2581 1283 MSLLGEVETPIRNEWGCRCN 2717 1284 MSLLHEVETPIRNEWGCRCN 2691 1285 MSLLIEVETPIRNEWGCRCN 2371 1286 MSLLKEVETPIRNEWGCRCN 2825 1287 MSLLLEVETPIRNEWGCRCN 2804 1288 MSLLMEVETPIRNEWGCRCN 2847 1289 MSLLNEVETPIRNEWGCRCN 2824 1290 MSLLPEVETPIRNEWGCRCN 2728 1291 MSLLQEVETPIRNEWGCRCN 2792 1292 MSLLREVETPIRNEWGCRCN 2699 1293 MSLLSEVETPIRNEWGCRCN 2790 1294 MSLLVEVETPIRNEWGCRCN 2732 1295 MSLLWEVETPIRNEWGCRCN 2531 1296 MSLLYEVETPIRNEWGCRCN 2464 1227 MSLLTDVETPIRNEWGCRCN 2476 1297 MSLLTQVETPIRNEWGCRCN 1697 1298 MSLLTTVETPIRNEWGCRCN 2699 1156 MSLLTEIETPIRNEWGCRCN 2799 1138 MSLLTELETPIRNEWGCRCN 2589 1155 MSLLTETETPIRNEWGCRCN 2641 1299 MSLLTEVATPIRNEWGCRCN 2633 1300 MSLLTEVDTPIRNEWGCRCN 2856 1301 MSLLTEVFTPIRNEWGCRCN 1858 1302 MSLLTEVGTPIRNEWGCRCN 2789 1303 MSLLTEVHTPIRNEWGCRCN 2751 1304 MSLLTEVITPIRNEWGCRCN 2668 1305 MSLLTEVKTPIRNEWGCRCN 2708 1306 MSLLTEVLTPIRNEWGCRCN 2714 1174 MSLLTEVMTPIRNEWGCRCN 2813 1307 MSLLTEVNTPIRNEWGCRCN 2807 1308 MSLLTEVPTPIRNEWGCRCN 2380 1166 MSLLTEVQTPIRNEWGCRCN 2813 1309 MSLLTEVRTPIRNEWGCRCN 2668 1190 MSLLTEVSTPIRNEWGCRCN 2783 1195 MSLLTEVTTPIRNEWGCRCN 2791 1165 MSLLTEVRTPIRNEWGCRCN 2732 1310 MSLLTEVWTPIRNEWGCRCN 2546 1311 MSLLTEVYTPIRNEWGCRCN 2181 1136 MSLLTEVEAPIRNEWGCRCN 2833 1197 MSLLTEVEDPIRNEWGCRCN 2826 1148 MSLLTEVEEPIRNEWGCRCN 2835 1171 MSLLTEVEFPIRNEWGCRCN 2728 1180 MSLLTEVEGPIRNEWGCRCN 2802 1214 MSLLTEVEHPIRNEWGCRCN 2753 1244 MSLLTEVEIPIRNEWGCRCN 2742 1127 MSLLTEVEKPIRNEWGCRCN 2837 1137 MSLLTEVELPIRNEWGCRCN 2797 1135 MSLLTEVEMPIRNEWGCRCN 2839 1205 MSLLTEVENPIRNEWGCRCN 2805 1236 MSLLTEVEPPIRNEWGCRCN 2797 1150 MSLLTEVEQPIRNEWGCRCN 2826 1207 MSLLTEVERPIRNEWGCRCN 2753 1189 MSLLTEVESPIRNEWGCRCN 2712 1184 MSLLTEVEVPIRNEWGCRCN 2768 1312 MSLLTEVEWPIRNEWGCRCN 2541 1226 MSLLTEVEYPIRNEWGCRCN 2571 1144 MSLLTEVETAIRNEWGCRCN 2722 1172 MSLLTEVETPARNEWGCRCN 2774 1218 MSLLTEVETPIANEWGCRCN 2683 1200 MSLLTEVETPIRAEWGCRCN 2742 1139 MSLLTEVETPIRNAWGCRCN 2803 1132 MSLLTEVETPIRNEAGCRCN 2831 1181 MSLLTEVETPIRNEWACRCN 2732 1173 MSLLTEVETPIRNEWGARCN 2760 1221 MSLLTEVETPIRNEWGCACN 2662 1157 MSLLTEVETPIRNEWGCRAN 2769 1161 MSLLTEVETPIRNEWGCRCA 2770 1126 CMSLLTEVETPIRNC 2767 1192 CMSLLTEVETPIRCE 2453 1235 CMSLLTEVETPICNE 2331 1199 CMSLLTEVETPCRNE 2789 1211 CMSLLTEVETCIRNE 2713 1176 CMSLLTEVECPIRNE 2730 1187 CMSLLTEVCTPIRNE 2633 1313 CMSLLCEVETPIRNE 2582 1314 CMSLCTEVETPIRNE 1705 1315 CMCLLTEVETPIRNE 2347 984 CSLLTEVETPIRNC 2782 997 CSLLTEVETPIRCE 2516 1012 CSLLTEVETPICNE 2444 1020 CSLLTEVETPCRNE 2470 1013 CSLLTEVETCIRNE 2301 1024 CSLLTEVECPIRNE 2226 990 CSLLTEVCTPIRNE 1942 1316 CSLLCEVETPIRNE 2527 1242 CSLCTEVETPIRNE 1832 1317 MCLLTEVETPIRNC 2743 1318 MCLLTEVETPIRCE 2307 1319 MCLLTEVETPICNE 2379 1320 MCLLTEVETPCRNE 2404 1321 MCLLTEVETCIRNE 1948 1322 MCLLTEVECPIRNE 2421 1323 MCLLTEVCTPIRNE 1407 1324 MCLLCEVETPIRNE 2038 1145 MSLCTEVETPIRNC 2601 1239 MSLCTEVETPIRCE 1555 1185 MSLCTEVETPCRNE 1673 1193 MSLCTEVECPIRNE 1538 1160 MSLCTEVCTPIRNE 1906 1325 MSLLCEVETPIRNC 2823 1326 MSLLCEVETPIRCE 2772 1327 MSLLCEVETPICNE 2704 1328 MSLLCEVETPCRNE 2652 1329 MSLLCEVETCIRNE 2589 1330 MSLLCEVECPIRNE 2606 1331 MSLLCEVCTPIRNE 2601 1130 MSLLTEVCTPIRNC 2787 1143 MSLLTEVCTPIRCE 2667 1169 MSLLTEVCTPICNE 2526 1158 MSLLTEVCTPCRNE 2572 1201 MSLLTEVCTCIRNE 2143 1168 MSLLTEVECPIRNE 2613 1129 MSLLTEVECPIRNC 2755 1151 MSLLTEVECPIRCE 2675 1217 MSLLTEVECPICNE 2440 1222 MSLLTEVECPCRNE 2361 1146 MSLLTEVETCIRNE 2129 1209 MSLLTEVETCIRNC 2309 1178 MSLLTEVETCIRCE 2739 1147 MSLLTEVETCICNE 2721 1134 MSLLTEVETPCRNE 2695 1128 MSLLTEVETPCRNC 2810 1133 MSLLTEVETPCRCE 2597 1142 MSLLTEVETPICNC 2758 1018 CSLLTEVETPIRNE 2211 1332 MCLLTEVETPIRNE 2364 1234 MSLCTEVETPIRNE 2054 1333 MSLLCEVETPIRNE 2454 1216 MSLLTEVCTPIRNE 2529 287 MSLLTEVGMSLLTEV 2878 293 CSLLTEVGMSLLTEV 2827 513 MCLLTEVGMSLLTEV 2697 532 MSCLTEVGMSLLTEV 2336 371 MSLCTEVGMSLLTEV 2691 749 MSLLCEVGMSLLTEV 2786 543 MSLLTCVGMSLLTEV 2627 339 MSLLTECGMSLLTEV 2170 288 CSCLCEVGMSLLTEV 1812 663 MCLCTCVGMSLLTEV 2509 294 MSCLCECGMSLLTEV 1006 373 CSLLTECGMSLLTEV 1235 316 MSLLTEVGCSLLTEV 2650 364 CSLLTEVGCSLLTEV 2503 570 MCLLTEVGCSLLTEV 2717 557 MSCLTEVGCSLLTEV 2506 508 MSLCTEVGCSLLTEV 2832 781 MSLLCEVGCSLLTEV 2761 528 MSLLTCVGCSLLTEV 2467 363 MSLLTECGCSLLTEV 1235 536 MCLCTCVGCSLLTEV 2241 360 CSLLTECGCSLLTEV 1427 296 MSLLTEVGMCLLTEV 2809 303 CSLLTEVGMCLLTEV 2618 1334 MSLLCEVGMCLLTEV 2676 359 MSLLTEVGMSCLTEV 2793 372 CSLLTEVGMSCLTEV 2554 1335 MCLLTEVGMSCLTEV 1869 1336 MSLLCEVGMSCLTEV 2846 317 MSLLTEVGMSLCTEV 2895 334 CSLLTEVGMSLCTEV 2857 1337 MCLLTEVGMSLCTEV 2513 289 MSLCTEVGMSLCTEV 1093 1338 MSLLCEVGMSLCTEV 2595 793 MSLLTEVGMSLLCEV 2819 309 CSLLTEVGMSLLCEV 2734 1339 MCLLTEVGMSLLCEV 2234 1340 MSCLTEVGMSLLCEV 2341 304 MSLCTEVGMSLLCEV 2616 1341 MSLLCEVGMSLLCEV 2851 1342 MSLLTCVGMSLLCEV 2712 460 MSLLTECGMSLLCEV 2373 1343 CSCLCEVGMSLLCEV 1945 1344 MCLCTCVGMSLLCEV 2431 1345 MSCLCECGMSLLCEV 1307 456 CSLLTECGMSLLCEV 1640 285 MSLLTEVGMSLLTCV 2872 286 CSLLTEVGMSLLTCV 2768 1346 MCLLTEVGMSLLTCV 1098 542 MSLCTEVGMSLLTCV 2722 1347 MSLLCEVGMSLLTCV 2784 343 MSLLTEVGMSLLTEC 2831 451 CSLLTEVGMSLLTEC 2613 825 MSLLCEVGMSLLTEC 2617 761 MSLLTEVGCSCLCEV 2053 476 CSLLTEVGCSCLCEV 1680 1348 MSLCTEVGCSCLCEV 2001 1349 MSLLCEVGCSCLCEV 2586 291 MSLLTEVGMCLCTCV 2842 297 CSLLTEVGMCLCTCV 2649 1350 MSLLCEVGMCLCTCV 2664 829 MSLLTEVGMSCLCEC 2444 499 CSLLTEVGMSCLCEC 2101 1351 MSLLCEVGMSCLCEC 2492 470 MSLLTEVGCSLLTEC 2349 383 CSLLTEVGCSLLTEC 2486 592 MSLCTEVGCSLLTEC 1971 826 MSLLCEVGCSLLTEC 2573 365 CSLLTEVCMSLLTEC 1891 1352 CCLLTEVETPIRNE 2155 1353 CLLTEVETPIRNC 1996 1354 CLLTEVETPIRCE 1813 1355 CLLTEVETPICNE 1647 1356 CLLTEVETPCRNE 1991 1357 CLLTEVETCIRNE 1661 1358 CLLTEVECPIRNE 2326 1359 CLLTEVCTPIRNE 1585 1360 CLLCEVETPIRNE 2314 1100 SLCTEVCTPIRNE 1674 1361 SLLCEVETPIRNC 2460 1362 SLLCEVETPIRCE 2307 1363 SLLCEVETPICNE 1921 1364 SLLCEVETPCRNE 2035 1365 SLLCEVETCIRNE 2305 1366 SLLCEVECPIRNE 2377 1367 SLLCEVCTPIRNE 2283 1016 SLLTEVCTPIRNC 2308 1073 SLLTEVCTPIRCE 1873 1091 SLLTEVCTPICNE 1659 1065 SLLTEVCTPCRNE 2016 1044 SLLTEVCTCIRNE 1695 1050 SLLTEVECPIRNE 2469 1007 SLLTEVECPIRNC 2635 1035 SLLTEVECPIRCE 2426 1112 SLLTEVECPICNE 2022 1119 SLLTEVECPCRNE 1840 1093 SLLTEVETCIRNE 1792 1022 SLLTEVETCIRNC 2289 1055 SLLTEVETCIRCE 2161 1084 SLLTEVETCICNE 1953 1368 SLLTEVETPCRNE 1665 1115 SLLTEVETPCRNC 1816 1369 SLLTEVETPCRCE 1546 1096 SLLTEVETPCRNC 2075 1370 CLLTEVETPIRNE 1524 1371 SLLCEVETPIRNE 2511 1116 SLLTEVCTPIRNE 1854 1072 CSLLTEVETPIRNEWGC 2232 1372 CLLTEVETPIRNEWGC 1866 1373 SLLCEVETPIRNEWGC 2281 1081 SLLTEVCTPIRNEWGC 1866 1086 SLLTEVECPIRNEWGC 2142 991 SLLTEVETCIRNEWGC 2284 1092 SLLTEVETPCRNEWGC 1873 1051 SLLTEVETPICNEWGC 2101 1021 SLLTEVETPIRCEWGC 2435 1015 SLLTEVETPIRNCWGC 2513 1374 SLLTEVETPIRNECGC 1660 1375 SLLTEVETPIRNEWCC 1813 1376 CLLTEVETPIRNEWC 1743 1377 CLLTEVETPIRNECG 1501 1378 CLLTEVETPIRNCWG 2388 1379 CLLTEVETPIRCEWG 2169 1380 CLLTEVETPICNEWG 2120 1381 CLLTEVETPCRNEWG 2074 1382 CLLTEVETCIRNEWG 2296 1383 CLLTEVECPIRNEWG 2157 1384 CLLCEVETPIRNEWG 2388 1385 CLLTEVETPIRNEWG 2142 1105 SLCTEVCTPIRNEWG 1477 1386 SLCCEVETPIRNEWG 1603 1387 SLLCEVETPIRNEWC 2406 1388 SLLCEVETPIRNECG 2107 1389 SLLCEVETPIRNCWG 2789 1390 SLLCEVETPIRCEWG 2601 1391 SLLCEVETPIRNEWG 2536 1392 SLLCEVETPCRNEWG 2465 1393 SLLCEVETCIRNEWG 2531 1394 SLLCEVECPIRNEWG 2529 1395 SLLCEVCTPIRNEWG 2384 1396 SLLCEVETPIRNEWG 2384 1049 SLLTEVCTPIRNEWC 1883 1094 SLLTEVCTPIRNECG 1782 1000 SLLTEVCTPIRNCWG 2420 988 SLLTEVCTPIRCEWG 2090 987 SLLTEVCTPIRNEWG 1883 996 SLLTEVCTPCRNEWG 1841 985 SLLTEVCTCIRNEWG 1546 1014 SLLTEVCCPIRNEWG 2253 1019 SLLTEVCTPIRNEWG 2692 1397 CLLTEVCTPIRNEWG 1634 1017 SLLTEVECPIRNEWC 2141 1099 SLLTEVECPIRNECG 2001 1001 SLLTEVECPIRNCWG 2589 989 SLLTEVECPIRCEWG 2509 1028 SLLTEVECPICNEWG 2449 1025 SLLTEVECPCRNEWG 2303 1004 SLLTEVECCIRNEWG 2507 1041 SLLTEVECPIRNEWG 2267 1030 SLLTEVETCIRNEWC 2337 1046 SLLTEVETCIRNECG 2018 992 SLLTEVETCIRNCWG 2616 998 SLLTEVETCIRCEWG 2566 1006 SLLTEVETCICNEWG 2498 993 SLLTEVETCCRNEWG 2389 1009 SLLTEVETCIRNEWG 2462 1110 SLLTEVETPCRNEWC 1867 1111 SLLTEVETPCRNECG 1470 1005 SLLTEVETPCRNCWG 2602 1061 SLLTEVETPCRCEWG 2155 1077 SLLTEVETPCCNEWG 2208 1071 SLLTEVETPCRNEWG 2167 1085 SLLTEVETPICNEWC 2488 1098 SLLTEVETPICNECG 2041 1010 SLLTEVETPICNCWG 2624 1034 SLLTEVETPICCEWG 2525 1057 SLLTEVETPICNEWG 2395 1082 SLLTEVETPIRCEWC 2255 1023 SLLTEVETPIRCECG 2464 994 SLLTEVETPIRCCWG 2755 1053 SLLTEVETPIRCEWG 2365 1039 SLLTEVETPIRNCWC 2321 1106 SLLTEVETPIRNCCG 1923 1036 SLLTEVETPIRNCWG 2350 1398 SLLTEVETPIRNECC 1493 1399 SLLTEVETPIRNECG 1545 1117 SLLTEVETPIRNEWC 1856 1070 SLLTEVETCSLLTEVETC 2104 1067 SLLTEVETCSLLTEVETPC 2082 1069 SLLTEVETCSLLTEVETPIC 2169 1040 SLLTEVETCSLLTEVETPIRC 2402 1058 SLLTEVETCSLLTEVETPIRNC 2295 1107 SLLTEVETCSLLTEVETPIRNEC 2025 1076 SLLTEVETCSLLTEVETPIRNEWC 2169 1048 SLLTEVETCSLLTEVETPIRNEWGC 2010 1088 SLLTEVETCSLLTEVETPIRNEWGSC 2180 986 SLLTEVETCSLLTEVETPIRNEWGSRC 2757 999 SLLTEVETCSLLTEVETPIRNEWGSRSC 2792 1011 SLLTEVETCSLLTEVETPIRNEWGSRSNC 2673 1062 SLLTEVETCSLLTEVETPIRNEWGSRSNDC 2483 1064 SLLTEVETCSLLTEVETPIRNEWGSRSNDSC 2335 1052 SLLTEVETCSLLTEVETPIRNEWGSRSNDSSC 2404 1068 SLLTEVETCSLLTEVETPIRNEWGSRSNDSSDC 2294 1032 SLLTEVETCSLLTEVETPIRNEWGCR 2395 1042 SLLTEVETSLLTEVETPIRNEWGCRC 2324 1043 SLLTEVETSLLTEVETPIRNEWGCRCN 2390 995 SLLTEVETSLLTEVETPIRNEWGCRCND 2393 995 SLLTEVETSLLTEVETPIRNEWGCRCNDS 2260 1089 SLLTEVETSLLTEVETPIRNEWGCRCNDSS 2212 1095 SLLTEVETSLLTEVETPIRNEWGCRCNDSSD 1980 831 1CSLLTEVETP1SLLTEVETPCSLLTEVETP1 2714 867 1CLLTEVETPI1SLLTEVETPCSLLTEVETP1 2593 836 1CLTEVETPIR1SLLTEVETPCSLLTEVETP1 2645 839 1CTEVETPIRN1SLLTEVETPCSLLTEVETP1 2420 848 1CEVETPIRNE1SLLTEVETPCSLLTEVETP1 2460 860 1CVETPIRNEW1SLLTEVETPCSLLTEVETP1 2474 858 1CETPIRNEWG1SLLTEVETPCSLLTEVETP1 2566 900 1CSLLTEVETP1LLTEVETPICSLLTEVETP1 2437 1400 1CLLTEVETPI1LLTEVETPICSLLTEVETP1 2220 1401 1CLTEVETPIR1LLTEVETPICSLLTEVETP1 2316 1402 1CTEVETPIRN1LLTEVETPICSLLTEVETP1 2258 1403 1CEVETPIRNE1LLTEVETPICSLLTEVETP1 2133 977 1CVETPIRNEW1LLTEVETPICSLLTEVETP1 1946 1404 1CETPIRNEWG1LLTEVETPICSLLTEVETP1 2288 841 1CSLLTEVETP1LTEVETPIRCSLLTEVETP1 2521 849 1CLLTEVETPI1LTEVETPIRCSLLTEVETP1 2601 863 1CLTEVETPIR1LTEVETPIRCSLLTEVETP1 2476 854 1CTEVETPIRN1LTEVETPIRCSLLTEVETP1 2469 872 1CEVETPIRNE1LTEVETPIRCSLLTEVETP1 2304 876 1CVETPIRNEW1LTEVETPIRCSLLTEVETP1 2350 882 1CETPIRNEWG1LTEVETPIRCSLLTEVETP1 2282 842 1CSLLTEVETP1TEVETPIRNCSLLTEVETP1 2591 883 1CLLTEVETPI1TEVETPIRNCSLLTEVETP1 2492 880 1CLTEVETPIR1TEVETPIRNCSLLTEVETP1 2396 887 1CTEVETPIRN1TEVETPIRNCSLLTEVETP1 2405 921 1CEVETPIRNE1TEVETPIRNCSLLTEVETP1 2272 953 1CVETPIRNEW1TEVETPIRNCSLLTEVETP1 2118 919 1CETPIRNEWG1TEVETPIRNCSLLTEVETP1 2223 869 1CSLLTEVETP1EVETPIRNECSLLTEVETP1 2582 967 1CLLTEVETPI1EVETPIRNECSLLTEVETP1 2220 961 1CLTEVETPIR1EVETPIRNECSLLTEVETP1 2331 978 1CTEVETPIRN1EVETPIRNECSLLTEVETP1 2295 870 1CEVETPIRNE1EVETPIRNECSLLTEVETP1 2618 963 1CVETPIRNEW1EVETPIRNECSLLTEVETP1 2365 936 1CETPIRNEWG1EVETPIRNECSLLTEVETP1 2368 851 1CSLLTEVETP1VETPIRNEWCSLLTEVETP1 2708 943 1CLLTEVETPI1VETPIRNEWCSLLTEVETP1 2422 932 1CLTEVETPIR1VETPIRNEWCSLLTEVETP1 2344 952 1CTEVETPIRN1VETPIRNEWCSLLTEVETP1 2380 934 1CEVETPIRNE1VETPIRNEWCSLLTEVETP1 2448 955 1CVETPIRNEW1VETPIRNEWCSLLTEVETP1 2307 929 1CETPIRNEWG1VETPIRNEWCSLLTEVETP1 2419 885 1CSLLTEVETP1ETPIRNEWGCSLLTEVETP1 2591 968 1CLLTEVETPI1ETPIRNEWGCSLLTEVETP1 2377 902 1CLTEVETPIR1ETPIRNEWGCSLLTEVETP1 2296 1405 1CTEVETPIRN1ETPIRNEWGCSLLTEVETP1 2268 903 1CEVETPIRNE1ETPIRNEWGCSLLTEVETP1 2140 877 1CVETPIRNEW1ETPIRNEWGCSLLTEVETP1 2349 896 1CETPIRNEWG1ETPIRNEWGCSLLTEVETP1 2425 830 1CSLLTEVETP1SLLTEVETPCLLTEVETPI1 2786 847 1CLLTEVETPI1SLLTEVETPCLLTEVETPI1 2722 832 1CLTEVETPIR1SLLTEVETPCLLTEVETPI1 2711 838 1CTEVETPIRN1SLLTEVETPCLLTEVETPI1 2616 855 1CEVETPIRNE1SLLTEVETPCLLTEVETPI1 2576 871 1CVETPIRNEW1SLLTEVETPCLLTEVETPI1 2544 837 1CETPIRNEWG1SLLTEVETPCLLTEVETPI1 2653 923 1CSLLTEVETP1LLTEVETPICLLTEVETPI1 2534 1406 1CLLTEVETPI1LLTEVETPICLLTEVETPI1 2350 1407 1CLTEVETPIR1LLTEVETPICLLTEVETPI1 2316 1408 1CTEVETPIRN1LLTEVETPICLLTEVETPI1 2446 1409 1CEVETPIRNE1LLTEVETPICLLTEVETPI1 2194 1410 1CVETPIRNEW1LLTEVETPICLLTEVETPI1 2331 1411 1CETPIRNEWG1LLTEVETPICLLTEVETPI1 2405 916 1CSLLTEVETP1LTEVETPIRCLLTEVETPI1 2722 1412 1CLLTEVETPI1LTEVETPIRCLLTEVETPI1 2436 1413 1CLTEVETPIR1LTEVETPIRCLLTEVETPI1 1445 1414 1CTEVETPIRN1LTEVETPIRCLLTEVETPI1 1793 1415 1CEVETPIRNE1LTEVETPIRCLLTEVETPI1 1407 1416 1CVETPIRNEW1LTEVETPIRCLLTEVETPI1 1369 1417 1CETPIRNEWG1LTEVETPIRCLLTEVETPI1 1474 878 1CSLLTEVETP1TEVETPIRNCLLTEVETPI1 2664 1418 1CLLTEVETPI1TEVETPIRNCLLTEVETPI1 2371 1419 1CLTEVETPIR1TEVETPIRNCLLTEVETPI1 1907 1420 1CTEVETPIRN1TEVETPIRNCLLTEVETPI1 1905 1421 1CEVETPIRNE1TEVETPIRNCLLTEVETPI1 1469 1422 1CVETPIRNEW1TEVETPIRNCLLTEVETPI1 1452 1423 1CETPIRNEWG1TEVETPIRNCLLTEVETPI1 1735 889 1CSLLTEVETP1EVETPIRNECLLTEVETPI1 2595 1424 1CLLTEVETPI1EVETPIRNECLLTEVETPI1 2331 1425 1CLTEVETPIR1EVETPIRNECLLTEVETPI1 1049 1426 1CTEVETPIRN1EVETPIRNECLLTEVETPI1 1107 873 1CSLLTEVETP1VETPIRNEWCLLTEVETPI1 2767 1427 1CLLTEVETPI1VETPIRNEWCLLTEVETPI1 2410 1428 1CLTEVETPIR1VETPIRNEWCLLTEVETPI1 1465 1429 1CTEVETPIRN1VETPIRNEWCLLTEVETPI1 1559 1430 1CEVETPIRNE1VETPIRNEWCLLTEVETPI1 1477 1431 1CVETPIRNEW1VETPIRNEWCLLTEVETPI1 1588 1432 1CETPIRNEWG1VETPIRNEWCLLTEVETPI1 1453 937 1CSLLTEVETP1ETPIRNEWGCLLTEVETPI1 2646 1433 1CLLTEVETPI1ETPIRNEWGCLLTEVETPI1 2361 1434 1CLTEVETPIR1ETPIRNEWGCLLTEVETPI1 1699 1435 1CTEVETPIRN1ETPIRNEWGCLLTEVETPI1 1573 1436 1CEVETPIRNE1ETPIRNEWGCLLTEVETPI1 1434 1437 1CVETPIRNEW1ETPIRNEWGCLLTEVETPI1 1582 1438 1CETPIRNEWG1ETPIRNEWGCLLTEVETPI1 1968 1439 1CSLLTEVETP1SLLTEVETPCLTEVETPIR1 2738 949 1CLLTEVETPI1SLLTEVETPCLTEVETPIR1 2403 857 1CLTEVETPIR1SLLTEVETPCLTEVETPIR1 2755 1440 1CTEVETPIRN1SLLTEVETPCLTEVETPIR1 2705 897 1CEVETPIRNE1SLLTEVETPCLTEVETPIR1 2565 843 1CVETPIRNEW1SLLTEVETPCLTEVETPIR1 2498 910 1CETPIRNEWG1SLLTEVETPCLTEVETPIR1 2473 892 1CSLLTEVETP1LLTEVETPICLTEVETPIR1 2546 1441 1CLLTEVETPI1LLTEVETPICLTEVETPIR1 2288 1442 1CLTEVETPIR1LLTEVETPICLTEVETPIR1 2057 1443 1CTEVETPIRN1LLTEVETPICLTEVETPIR1 2213 1444 1CEVETPIRNE1LLTEVETPICLTEVETPIR1 2000 1445 1CVETPIRNEW1LLTEVETPICLTEVETPIR1 2280 1446 1CETPIRNEWG1LLTEVETPICLTEVETPIR1 2367 886 1CSLLTEVETP1LTEVETPIRCLTEVETPIR1 2623 1447 1CLLTEVETPI1LTEVETPIRCLTEVETPIR1 2283 944 1CSLLTEVETP1TEVETPIRNCLTEVETPIR1 2646 1448 1CLLTEVETPI1TEVETPIRNCLTEVETPIR1 2440 1449 1CSLLTEVETP1EVETPIRNECLTEVETPIR1 2404 1450 1CLLTEVETPI1EVETPIRNECLTEVETPIR1 2188 1451 1CSLLTEVETP1VETPIRNEWCLTEVETPIR1 2549 1452 1CLLTEVETPI1VETPIRNEWCLTEVETPIR1 2607 1453 1CSLLTEVETP1ETPIRNEWGCLTEVETPIR1 2465 1454 1CLLTEVETPI1ETPIRNEWGCLTEVETPIR1 2218 918 1CSLLTEVETP1SLLTEVETPCTEVETPIRN1 2618 1455 1CLLTEVETPI1SLLTEVETPCTEVETPIRN1 2283 946 1CLTEVETPIR1SLLTEVETPCTEVETPIRN1 2434 957 1CTEVETPIRN1SLLTEVETPCTEVETPIRN1 2402 941 1CEVETPIRNE1SLLTEVETPCTEVETPIRN1 2692 947 1CVETPIRNEW1SLLTEVETPCTEVETPIRN1 2733 942 1CETPIRNEWG1SLLTEVETPCTEVETPIRN1 2601 1456 1CSLLTEVETP1LLTEVETPICTEVETPIRN1 2103 1457 1CLLTEVETPI1LLTEVETPICTEVETPIRN1 1884 1458 1CLTEVETPIR1LLTEVETPICTEVETPIRN1 1756 1459 1CTEVETPIRN1LLTEVETPICTEVETPIRN1 1749 1460 1CEVETPIRNE1LLTEVETPICTEVETPIRN1 1620 1461 1CVETPIRNEW1LLTEVETPICTEVETPIRN1 1835 1462 1CETPIRNEWG1LLTEVETPICTEVETPIRN1 1710 971 1CSLLTEVETP1LTEVETPIRCTEVETPIRN1 2360 1463 1CLLTEVETPI1LTEVETPIRCTEVETPIRN1 2188 1464 1CSLLTEVETP1TEVETPIRNCTEVETPIRN1 2755 1465 1CLLTEVETPI1TEVETPIRNCTEVETPIRN1 2488 1466 1CSLLTEVETP1EVETPIRNECTEVETPIRN1 2134 1467 1CLLTEVETPI1EVETPIRNECTEVETPIRN1 1851 1468 1CSLLTEVETP1VETPIRNEWCTEVETPIRN1 2301 1469 1CLLTEVETPI1VETPIRNEWCTEVETPIRN1 1713 1470 1CSLLTEVETP1ETPIRNEWGCTEVETPIRN1 2331 1471 1CLLTEVETPI1ETPIRNEWGCTEVETPIRN1 1983 958 1CSLLTEVETP1SLLTEVETPCEVETPIRNE1 2203 1472 1CLLTEVETPI1SLLTEVETPCEVETPIRNE1 2057 976 1CLTEVETPIR1SLLTEVETPCEVETPIRNE1 2216 913 1CTEVETPIRN1SLLTEVETPCEVETPIRNE1 2139 1473 1CEVETPIRNE1SLLTEVETPCEVETPIRNE1 2006 1474 1CVETPIRNEW1SLLTEVETPCEVETPIRNE1 1986 1475 1CETPIRNEWG1SLLTEVETPCEVETPIRNE1 2642 1476 1CSLLTEVETP1LLTEVETPICEVETPIRNE1 2472 1477 1CLLTEVETPI1LLTEVETPICEVETPIRNE1 2022 1478 1CLTEVETPIR1LLTEVETPICEVETPIRNE1 1681 1479 1CTEVETPIRN1LLTEVETPICEVETPIRNE1 1627 1480 1CEVETPIRNE1LLTEVETPICEVETPIRNE1 1394 1481 1CVETPIRNEW1LLTEVETPICEVETPIRNE1 1592 1482 1CETPIRNEWG1LLTEVETPICEVETPIRNE1 1648 1483 1CSLLTEVETP1LTEVETPIRCEVETPIRNE1 2040 1484 1CLLTEVETPI1LTEVETPIRCEVETPIRNE1 1777 1485 1CSLLTEVETP1TEVETPIRNCEVETPIRNE1 2036 1486 1CLLTEVETPI1TEVETPIRNCEVETPIRNE1 1798 1487 1CSLLTEVETP1EVETPIRNECEVETPIRNE1 1917 1488 1CLLTEVETPI1EVETPIRNECEVETPIRNE1 1631 1489 1CSLLTEVETP1VETPIRNEWCEVETPIRNE1 2359 1490 1CLLTEVETPI1VETPIRNEWCEVETPIRNE1 1730 1491 1CSLLTEVETP1ETPIRNEWGCEVETPIRNE1 2338 1492 1CLLTEVETPI1ETPIRNEWGCEVETPIRNE1 1781 850 1CSLLTEVETP1SLLTEVETPCVETPIRNEW1 2491 975 1CLLTEVETPI1SLLTEVETPCVETPIRNEW1 2316 945 1CLTEVETPIR1SLLTEVETPCVETPIRNEW1 2366 951 1CTEVETPIRN1SLLTEVETPCVETPIRNEW1 2460 959 1CEVETPIRNE1SLLTEVETPCVETPIRNEW1 2441 935 1CVETPIRNEW1SLLTEVETPCVETPIRNEW1 2470 950 1CETPIRNEWG1SLLTEVETPCVETPIRNEW1 2465 974 1CSLLTEVETP1LLTEVETPICVETPIRNEW1 2285 1493 1CLLTEVETPI1LLTEVETPICVETPIRNEW1 2661 1494 1CLTEVETPIR1LLTEVETPICVETPIRNEW1 2443 1495 1CTEVETPIRN1LLTEVETPICVETPIRNEW1 2088 1496 1CEVETPIRNE1LLTEVETPICVETPIRNEW1 1625 1497 1CVETPIRNEW1LLTEVETPICVETPIRNEW1 1817 1498 1CETPIRNEWG1LLTEVETPICVETPIRNEW1 1817 1499 1CSLLTEVETP1LTEVETPIRCVETPIRNEW1 2319 1500 1CLLTEVETPI1LTEVETPIRCVETPIRNEW1 2171 1501 1CSLLTEVETP1TEVETPIRNCVETPIRNEW1 2497 1502 1CLLTEVETPI1TEVETPIRNCVETPIRNEW1 2229 1503 1CSLLTEVETP1EVETPIRNECVETPIRNEW1 2469 1504 1CLLTEVETPI1EVETPIRNECVETPIRNEW1 2081 1505 1CSLLTEVETP1VETPIRNEWCVETPIRNEW1 2398 1506 1CLLTEVETPI1VETPIRNEWCVETPIRNEW1 2074 1507 1CSLLTEVETP1ETPIRNEWGCVETPIRNEW1 2778 1508 1CLLTEVETPI1ETPIRNEWGCVETPIRNEW1 2495 845 1CSLLTEVETP1SLLTEVETPCETPIRNEWG1 2417 1509 1CLLTEVETPI1SLLTEVETPCETPIRNEWG1 2057 962 1CLTEVETPIR1SLLTEVETPCETPIRNEWG1 2230 956 1CTEVETPIRN1SLLTEVETPCETPIRNEWG1 2352 868 1CEVETPIRNE1SLLTEVETPCETPIRNEWG1 2326 866 1CVETPIRNEW1SLLTEVETPCETPIRNEWG1 2328 964 1CETPIRNEWG1SLLTEVETPCETPIRNEWG1 2357 939 1CSLLTEVETP1LLTEVETPICETPIRNEWG1 2446 1510 1CLLTEVETPI1LLTEVETPICETPIRNEWG1 1999 1511 1CLTEVETPIR1LLTEVETPICETPIRNEWG1 2175 1512 1CTEVETPIRN1LLTEVETPICETPIRNEWG1 2645 1513 1CEVETPIRNE1LLTEVETPICETPIRNEWG1 2466 1514 1CVETPIRNEW1LLTEVETPICETPIRNEWG1 2575 1515 1CETPIRNEWG1LLTEVETPICETPIRNEWG1 2427 972 1CSLLTEVETP1LTEVETPIRCETPIRNEWG1 2583 1516 1CLLTEVETPI1LTEVETPIRCETPIRNEWG1 2320 1517 1CSLLTEVETP1TEVETPIRNCETPIRNEWG1 2299 1518 1CLLTEVETPI1TEVETPIRNCETPIRNEWG1 1972 1519 1CSLLTEVETP1EVETPIRNECETPIRNEWG1 2692 1520 1CLLTEVETPI1EVETPIRNECETPIRNEWG1 2433 1521 1CSLLTEVETP1VETPIRNEWCETPIRNEWG1 2569 1522 1CLLTEVETPI1VETPIRNEWCETPIRNEWG1 1931 1523 1CSLLTEVETP1ETPIRNEWGCETPIRNEWG1 2340 1524 1CLLTEVETPI1ETPIRNEWGCETPIRNEWG1 2007 861 SLLTEVET1CSLLTEVET1SLLTEVETCSLLTE1 2691 853 SLLTEVET1CSLLTEVET1SLLTEVETCLLTEV1 2690 901 SLLTEVET1CSLLTEVET1SLLTEVETCLTEVE1 2396 915 SLLTEVET1CSLLTEVET1SLLTEVETCTEVET1 2218 914 SLLTEVET1CSLLTEVET1SLLTEVETCEVETP1 2268 893 SLLTEVET1CSLLTEVET1SLLTEVETCVETPI1 2422 890 SLLTEVET1CSLLTEVET1SLLTEVETCETPIR1 2507 862 SLLTEVET1CSLLTEVET1SLLTEVETCTPIRN1 2690 907 SLLTEVET1CSLLTEVET1SLLTEVETCPIRNE1 2435 852 SLLTEVET1CSLLTEVET1SLLTEVETCIRNEW1 2663 895 SLLTEVET1CSLLTEVET1SLLTEVETCRNEWG1 2476 908 SLLTEVET1CSLLTEVET1SLLTEVETCNEWG1 2461 834 SLLTEVET1CSLLTEVET1SLLTEVETCEWG1R 2855 835 SLLTEVET1CSLLTEVET1SLLTEVETAWGCR1 2870 844 SLLTEVET1CSLLTEVET1SLLTEVETAGCR1N 2814 898 SLLTEVET1CSLLTEVET1SLLTEVETACR1ND 2686 908 SLLTEVET1CSLLTEVET1SLLTEVETCNEWGS1 2726 840 SLLTEVET1CSLLTEVET1SLLTEVETCEWGSR1 2730 1525 CLLTEVETPIRNEWGSCVETPIRNEWGSRSNDC 2278 938 CSLLTEVETCETPIRNEWGSRSNDSC 1916 922 CSLLTEVETPCETPIRNEWGSRSNDSC 2170 905 CSLLTEVETPICETPIRNEWGSRSNDSC 2041 911 CSLLTEVETPIRCETPIRNEWGSRSNDSC 2024 891 CSLLTEVETPIRNCETPIRNEWGSRSNDSC 1931 917 CSLLTEVETPIRNECETPIRNEWGSRSNDSC 1808 1526 CSLLTEVETPIRNEWCETPIRNEWGSRSNDSC 2012 881 CSLLTEVETPIRNEWGCETPIRNEWGSRSNDSC 2501 1527 CLLTEVETPIRNEWGSCETPIRNEWGSRSNDSC 2414 894 CSLLTEVETCTPIRNEWGSRSNDSSC 2068 965 CSLLTEVETPCTPIRNEWGSRSNDSSC 2351 966 CSLLTEVETPICTPIRNEWGSRSNDSSC 2125 924 CSLLTEVETPIRCTPIRNEWGSRSNDSSC 2162 979 CSLLTEVETPIRNCTPIRNEWGSRSNDSSC 1927 920 CSLLTEVETPIRNECTPIRNEWGSRSNDSSC 1988 1528 CSLLTEVETPIRNEWCTPIRNEWGSRSNDSSC 1959 980 CSLLTEVETPIRNEWGCTPIRNEWGSRSNDSSC 2116 1529 CLLTEVETPIRNEWGSCTPIRNEWGSRSNDSSC 2268 888 CSLLTEVETCPIRNEWGSRSNDSSDC 2246 931 CSLLTEVETPCPIRNEWGSRSNDSSDC 2377 954 CSLLTEVETPICPIRNEWGSRSNDSSDC 2267 969 CSLLTEVETPIRCPIRNEWGSRSNDSSDC 2228 970 CSLLTEVETPIRNCPIRNEWGSRSNDSSDC 2089 973 CSLLTEVETPIRNECPIRNEWGSRSNDSSDC 1955 1530 CSLLTEVETPIRNEWCPIRNEWGSRSNDSSDC 1983 906 CSLLTEVETPIRNEWGCPIRNEWGSRSNDSSDC 1954 1531 CLLTEVETPIRNEWGSCPIRNEWGSRSNDSSDC 1420 875 SLLTEVETCSLLTEVETCSLLTEVETPIRC 2643 909 SLLTEVETCSLLTEVETCLLTEVETPIRNC 2618 960 SLLTEVETCSLLTEVETCLTEVETPIRNEC 2157 940 SLLTEVETCSLLTEVETCTEVETPIRNEWC 2302 930 SLLTEVETCSLLTEVETCEVETPIRNEWGC 2300 925 SLLTEVETCSLLTEVETCVETPIRNEWGSC 2347 927 SLLTEVETCSLLTEVETCETPIRNEWGSRC 2311 912 SLLTEVETCSLLTEVETCTPIRNEWGSRSC 2434 933 SLLTEVETCSLLTEVETCPIRNEWGSRSNC 2192 948 SLLTEVETCSLLTEVETCIRNEWGSRSNDC 1961 884 SLLTEVETCSLLTEVETCRNEWGSRSNDSC 2620 846 SLLTEVETCSLLTEVETCNEWGSRSNDSSC 2640 874 SLLTEVETCSLLTEVETCEWGSRSNDSSDC 2509 856 SLLTEVETCSLLTEVETCVETPIRNEWGC 2671 879 SLLTEVETCSLLTEVETCETPIRNEWGCR 2631 833 SLLTEVETCSLLTEVETATPIRNEWGCRC 2640 899 SLLTEVETCSLLTEVETAPIRNEWGCRCN 2576 904 SLLTEVETCSLLTEVETAIRNEWGCRCND 2538 926 SLLTEVETCSLLTEVETARNEWGCRCNDS 2463 928 SLLTEVETCSLLTEVETANEWGCRCNDSS 2358 865 SLLTEVETCSLLTEVETAEWGCRCNDSSD 2254 1532 CSLLTEVETPIRNEWGCRCNDSSD 2284 1533 CLLTEVETPIRNEWGCRCNDSSD 1979 1534 SLLCEVETPIRNEWGCRCNDSSD 2161 566 SLLTEVCTPIRNEWGCRCNDSSD 1210 718 SLLTEVECPIRNEWGCRCNDSSD 1974 623 SLLTEVETCIRNEWGCRCNDSSD 1992 687 SLLTEVETPCRNEWGCRCNDSSD 2050 725 SLLTEVETPICNEWGCRCNDSSD 2005 587 SLLTEVETPIRCEWGCRCNDSSD 1956 733 SLLTEVETPIRNCWGCRCNDSSD 2006 268 SLLTEVETPIRNECGCRCNDSSD 2554 714 SLLTEVETPIRNEWCCRCNDSSD 2388 127 CSLLTEVETCSLLTEVETC 2555 198 CSLLTEVETPCSLLTEVETC 2341 187 CSLLTEVETPICSLLTEVETC 2554 147 CSLLTEVETPIRCSLLTEVETC 2662 166 CSLLTEVETPIRNCSLLTEVETC 2538 118 CSLLTEVETPIRNECSLLTEVETC 2489 122 CSLLTEVETPIRNEWCSLLTEVETC 2454 200 CSLLTEVETPIRNEWGCSLLTEVETC 2371 254 CLLTEVETPIRNEWGSCSLLTEVETC 2177 136 CLTEVETPIRNEWGSRCSLLTEVETC 2026 226 CTEVETPIRNEWGSRSCSLLTEVETC 2140 201 CEVETPIRNEWGSRSNCSLLTEVETC 2288 223 CVETPIRNEWGSRSNDCSLLTEVETC 2233 213 CETPIRNEWGSRSNDSCSLLTEVETC 1997 230 CTPIRNEWGSRSNDSSCSLLTEVETC 2168 161 CPIRNEWGSRSNDSSDCSLLTEVETC 2759 142 CSLLTEVETCSLLTEVETPC 2716 156 CSLLTEVETPCSLLTEVETPC 2673 207 CSLLTEVETPICSLLTEVETPC 2440 181 CSLLTEVETPIRCSLLTEVETPC 2448 144 CSLLTEVETPIRNCSLLTEVETPC 2673 164 CSLLTEVETPIRNECSLLTEVETPC 2582 189 CSLLTEVETPIRNEWCSLLTEVETPC 2505 217 CSLLTEVETPIRNEWGCSLLTEVETPC 2445 270 CLLTEVETPIRNEWGSCSLLTEVETPC 2307 228 CLTEVETPIRNEWGSRCSLLTEVETPC 2237 246 CTEVETPIRNEWGSRSCSLLTEVETPC 2182 229 CEVETPIRNEWGSRSNCSLLTEVETPC 2290 242 CVETPIRNEWGSRSNDCSLLTEVETPC 2379 232 CETPIRNEWGSRSNDSCSLLTEVETPC 2425 247 CTPIRNEWGSRSNDSSCSLLTEVETPC 2249 259 CPIRNEWGSRSNDSSDCSLLTEVETPC 2441 128 CSLLTEVETCSLLTEVETPIC 2876 137 CSLLTEVETPCSLLTEVETPIC 2815 245 CSLLTEVETPICSLLTEVETPIC 2492 151 CSLLTEVETPIRCSLLTEVETPIC 2561 139 CSLLTEVETPIRNCSLLTEVETPIC 2666 145 CSLLTEVETPIRNECSLLTEVETPIC 2684 177 CSLLTEVETPIRNEWCSLLTEVETPIC 2548 197 CSLLTEVETPIRNEWGCSLLTEVETPIC 2481 252 CLLTEVETPIRNEWGSCSLLTEVETPIC 2333 191 CLTEVETPIRNEWGSRCSLLTEVETPIC 2405 216 CTEVETPIRNEWGSRSCSLLTEVETPIC 2308 219 CEVETPIRNEWGSRSNCSLLTEVETPIC 2363 132 CVETPIRNEWGSRSNDCSLLTEVETPIC 2372 237 CETPIRNEWGSRSNDSCSLLTEVETPIC 2436 231 CTPIRNEWGSRSNDSSCSLLTEVETPIC 2460 267 CPIRNEWGSRSNDSSDCSLLTEVETPIC 2227 163 CSLLTEVETCSLLTEVETPIRC 2531 124 CSLLTEVETPCSLLTEVETPIRC 2839 203 CSLLTEVETPICSLLTEVETPIRC 2628 121 CSLLTEVETPIRCSLLTEVETPIRC 2767 120 CSLLTEVETPIRNCSLLTEVETPIRC 2751 130 CSLLTEVETPIRNECSLLTEVETPIRC 2751 155 CSLLTEVETPIRNEWCSLLTEVETPIRC 2646 157 CSLLTEVETPIRNEWGCSLLTEVETPIRC 2619 117 CLLTEVETPIRNEWGSCSLLTEVETPIRC 2622 162 CLTEVETPIRNEWGSRCSLLTEVETPIRC 2476 184 CTEVETPIRNEWGSRSCSLLTEVETPIRC 2419 172 CEVETPIRNEWGSRSNCSLLTEVETPIRC 2467 168 CVETPIRNEWGSRSNDCSLLTEVETPIRC 2484 190 CETPIRNEWGSRSNDSCSLLTEVETPIRC 2514 169 CTPIRNEWGSRSNDSSCSLLTEVETPIRC 2518 176 CPIRNEWGSRSNDSSDCSLLTEVETPIRC 2586 134 CSLLTEVETCSLLTEVETPIRNC 2633 153 CSLLTEVETPCSLLTEVETPIRNC 2691 208 CSLLTEVETPICSLLTEVETPIRNC 2548 131 CSLLTEVETPIRCSLLTEVETPIRNC 2753 129 CSLLTEVETPIRNCSLLTEVETPIRNC 2748 133 CSLLTEVETPIRNECSLLTEVETPIRNC 2724 180 CSLLTEVETPIRNEWCSLLTEVETPIRNC 2538 143 CSLLTEVETPIRNEWGCSLLTEVETPIRNC 2657 178 CLLTEVETPIRNEWGSCSLLTEVETPIRNC 2574 119 CLTEVETPIRNEWGSRCSLLTEVETPIRNC 2459 204 CTEVETPIRNEWGSRSCSLLTEVETPIRNC 2358 196 CEVETPIRNEWGSRSNCSLLTEVETPIRNC 2429 194 CVETPIRNEWGSRSNDCSLLTEVETPIRNC 2440 126 CETPIRNEWGSRSNDSCSLLTEVETPIRNC 2440 185 CTPIRNEWGSRSNDSSCSLLTEVETPIRNC 2492 202 CPIRNEWGSRSNDSSDCSLLTEVETPIRNC 2500 171 CSLLTEVETCSLLTEVETPIRNEC 2424 193 CSLLTEVETPCSLLTEVETPIRNEC 2387 1535 CSLLTEVETPICSLLTEVETPIRNEC 1851 165 CSLLTEVETPIRCSLLTEVETPIRNEC 2648 183 CSLLTEVETPIRNCSLLTEVETPIRNEC 2580 220 CSLLTEVETPIRNECSLLTEVETPIRNEC 2522 241 CSLLTEVETPIRNEWCSLLTEVETPIRNEC 2349 206 CSLLTEVETPIRNEWGCSLLTEVETPIRNEC 2419 244 CLLTEVETPIRNEWGSCSLLTEVETPIRNEC 2327 240 CLTEVETPIRNEWGSRCSLLTEVETPIRNEC 2221 261 CTEVETPIRNEWGSRSCSLLTEVETPIRNEC 2074 273 CEVETPIRNEWGSRSNCSLLTEVETPIRNEC 2008 269 CVETPIRNEWGSRSNDCSLLTEVETPIRNEC 2140 154 CETPIRNEWGSRSNDSCSLLTEVETPIRNEC 2122 266 CTPIRNEWGSRSNDSSCSLLTEVETPIRNEC 2080 248 CPIRNEWGSRSNDSSDCSLLTEVETPIRNEC 2249 150 CSLLTEVETCSLLTEVETPIRNEWC 2577 158 CSLLTEVETPCSLLTEVETPIRNEWC 2567 211 CSLLTEVETPICSLLTEVETPIRNEWC 2423 173 CSLLTEVETPIRCSLLTEVETPIRNEWC 2566 135 CSLLTEVETPIRNCSLLTEVETPIRNEWC 2820 146 CSLLTEVETPIRNECSLLTEVETPIRNEWC 2720 174 CSLLTEVETPIRNEWCSLLTEVETPIRNEWC 2647 212 CSLLTEVETPIRNEWGCSLLTEVETPIRNEWC 2545 218 CLLTEVETPIRNEWGSCSLLTEVETPIRNEWC 2541 188 CLTEVETPIRNEWGSRCSLLTEVETPIRNEWC 2522 225 CTEVETPIRNEWGSRSCSLLTEVETPIRNEWC 2482 238 CEVETPIRNEWGSRSNCSLLTEVETPIRNEWC 2391 234 CVETPIRNEWGSRSNDCSLLTEVETPIRNEWC 2412 251 CETPIRNEWGSRSNDSCSLLTEVETPIRNEWC 2374 272 CTPIRNEWGSRSNDSSCSLLTEVETPIRNEWC 2313 249 CPIRNEWGSRSNDSSDCSLLTEVETPIRNEWC 2397 281 CSLLTEVETCSLLTEVETPIRNEWGC 2479 186 CSLLTEVETPCSLLTEVETPIRNEWGC 2523 236 CSLLTEVETPICSLLTEVETPIRNEWGC 2345 167 CSLLTEVETPIRCSLLTEVETPIRNEWGC 2600 148 CSLLTEVETPIRNCSLLTEVETPIRNEWGC 2553 152 CSLLTEVETPIRNECSLLTEVETPIRNEWGC 2732 141 CSLLTEVETPIRNEWCSLLTEVETPIRNEWGC 2669 140 CSLLTEVETPIRNEWGCSLLTEVETPIRNEWGC 2678 205 CLLTEVETPIRNEWGSCSLLTEVETPIRNEWGC 2593 116 CLTEVETPIRNEWGSRCSLLTEVETPIRNEWGC 2492 125 CTEVETPIRNEWGSRSCSLLTEVETPIRNEWGC 2449 233 CEVETPIRNEWGSRSNCSLLTEVETPIRNEWGC 2442 257 CVETPIRNEWGSRSNDCSLLTEVETPIRNEWGC 2400 255 CETPIRNEWGSRSNDSCSLLTEVETPIRNEWGC 2414 243 CTPIRNEWGSRSNDSSCSLLTEVETPIRNEWGC 2466 256 CPIRNEWGSRSNDSSDCSLLTEVETPIRNEWGC 2460 179 CSLLTEVETCLLTEVETPIRNEWGSC 2416 182 CSLLTEVETPCLLTEVETPIRNEWGSC 2540 224 CSLLTEVETPICLLTEVETPIRNEWGSC 2388 160 CSLLTEVETPIRCLLTEVETPIRNEWGSC 2520 192 CSLLTEVETPIRNCLLTEVETPIRNEWGSC 2519 227 CSLLTEVETPIRNECLLTEVETPIRNEWGSC 2484 214 CSLLTEVETPIRNEWCLLTEVETPIRNEWGSC 2661 138 CSLLTEVETPIRNEWGCLLTEVETPIRNEWGSC 2624 1536 CLLTEVETPIRNEWGSCLLTEVETPIRNEWGSC 2616 1537 CLTEVETPIRNEWGSRCLLTEVETPIRNEWGSC 2667 1538 CTEVETPIRNEWGSRSCLLTEVETPIRNEWGSC 2425 1539 CEVETPIRNEWGSRSNCLLTEVETPIRNEWGSC 2461 1540 CVETPIRNEWGSRSNDCLLTEVETPIRNEWGSC 2218 1541 CETPIRNEWGSRSNDSCLLTEVETPIRNEWGSC 2255 1542 CTPIRNEWGSRSNDSSCLLTEVETPIRNEWGSC 2363 1543 CPIRNEWGSRSNDSSDCLLTEVETPIRNEWGSC 2260 215 CSLLTEVETCLTEVETPIRNEWGSRC 2328 221 CSLLTEVETPCLTEVETPIRNEWGSRC 2513 235 CSLLTEVETPICLTEVETPIRNEWGSRC 2351 170 CSLLTEVETPIRCLTEVETPIRNEWGSRC 2587 195 CSLLTEVETPIRNCLTEVETPIRNEWGSRC 2585 123 CSLLTEVETPIRNECLTEVETPIRNEWGSRC 2496 278 CSLLTEVETPIRNEWCLTEVETPIRNEWGSRC 2429 175 CSLLTEVETPIRNEWGCLTEVETPIRNEWGSRC 2641 1544 CLLTEVETPIRNEWGSCLTEVETPIRNEWGSRC 2500 250 CSLLTEVETCTEVETPIRNEWGSRSC 2474 222 CSLLTEVETPCTEVETPIRNEWGSRSC 2703 280 CSLLTEVETPICTEVETPIRNEWGSRSC 2241 199 CSLLTEVETPIRCTEVETPIRNEWGSRSC 2623 210 CSLLTEVETPIRNCTEVETPIRNEWGSRSC 2643 258 CSLLTEVETPIRNECTEVETPIRNEWGSRSC 2553 277 CSLLTEVETPIRNEWCTEVETPIRNEWGSRSC 2388 263 CSLLTEVETPIRNEWGCTEVETPIRNEWGSRSC 2496 1545 CLLTEVETPIRNEWGSCTEVETPIRNEWGSRSC 2592 239 CSLLTEVETCEVETPIRNEWGSRSNC 2549 274 CSLLTEVETPCEVETPIRNEWGSRSNC 2701 260 CSLLTEVETPICEVETPIRNEWGSRSNC 2680 209 CSLLTEVETPIRCEVETPIRNEWGSRSNC 2699 253 CSLLTEVETPIRNCEVETPIRNEWGSRSNC 2699 276 CSLLTEVETPIRNECEVETPIRNEWGSRSNC 2620 1546 CSLLTEVETPIRNEWCEVETPIRNEWGSRSNC 2519 275 CSLLTEVETPIRNEWGCEVETPIRNEWGSRSNC 2397 1547 CLLTEVETPIRNEWGSCEVETPIRNEWGSRSNC 2074 264 CSLLTEVETCVETPIRNEWGSRSNDC 2374 265 CSLLTEVETPCVETPIRNEWGSRSNDC 2484 1548 CSLLTEVETPICVETPIRNEWGSRSNDC 2174 271 CSLLTEVETPIRCVETPIRNEWGSRSNDC 2480 262 CSLLTEVETPIRNCVETPIRNEWGSRSNDC 2566 279 CSLLTEVETPIRNECVETPIRNEWGSRSNDC 2465 1549 CSLLTEVETPIRNEWCVETPIRNEWGSRSNDC 2433 159 CSLLTEVETPIRNEWGCVETPIRNEWGSRSNDC 2415 320 SLLTEVGSLLTEV 2530 283 CSLLTEVGSLLTEV 2903 390 CLLTEVGSLLTEV 2494 500 SCLTEVGSLLTEV 1610 324 SLCTEVGSLLTEV 1780 336 SLLCEVGSLLTEV 2475 824 SLLTCVGSLLTEV 1772 482 CLCTCVGSLLTEV 1953 354 SLLTEVGCSLLTEV 2511 807 CLLTEVGCSLLTEV 1558 580 SLCTEVGCSLLTEV 2382 511 SLLCEVGCSLLTEV 2562 810 SLLTCVGCSLLTEV 1992 798 CLCTCVGCSLLTEV 1489 311 SLLTEVGCLLTEV 1924 388 CSLLTEVGCLLTEV 2427 1550 SLLCEVGCLLTEV 1997 302 ASLLTEVGSCLTEV 2770 345 CSLLTEVGSCLTEV 2703 1551 SLLCEVGSCLTEV 2499 464 SLLTEVGSLCTEV 2219 711 CSLLTEVGSLCTEV 2409 1552 SLLCEVGSLCTEV 2722 314 SLLTEVGSLLCEV 2392 792 CSLLTEVGSLLCEV 2594 1553 CLLTEVGSLLCEV 2168 496 SCLTEVGSLLCEV 1896 506 SLCTEVGSLLCEV 1863 1554 SLLCEVGSLLCEV 2399 1555 SLLTCVGSLLCEV 2264 477 SLLTECGSLLCEV 2403 1556 CSCLCEVGSLLCEV 1583 1557 CLCTCVGSLLCEV 2246 1558 SCLCECGSLLCEV 1747 814 CSLLTECGSLLCEV 2256 284 ASLLTEVGSLLTCV 2358 290 CSLLTEVGSLLTCV 2503 1559 SLLCEVGSLLTCV 1972 452 SLLTEVGSLLTEC 1862 292 CSLLTEVGSLLTEC 2213 351 SLLCEVGSLLTEC 2438 525 SLLTEVGCSCLCEV 1829 379 SLCTEVGCSCLCEV 1500 1560 SLLCEVGCSCLCEV 2112 299 SLLTEVGCLCTCV 2346 385 CSLLTEVGCLCTCV 2474 1561 SLLCEVGCLCTCV 2331 329 SLLTEVGSCLCEC 2558 533 CSLLTEVGSCLCEC 2625 1562 SLLCEVGSCLCEC 2614 353 SLLTEVGCSLLTEC 2380 747 SLCTEVGCSLLTEC 2050 301 SLLCEVGCSLLTEC 2637 341 CSLLTEVCSLLTEC 1122 321 ALLTEVETPIRNEWGCRCNDSSD 2225 1563 DLLTEVETPIRNEWGCRCNDSSD 2320 1564 ELLTEVETPIRNEWGCRCNDSSD 2201 1565 FLLTEVETPIRNEWGCRCNDSSD 1018 1566 GLLTEVETPIRNEWGCRCNDSSD 2321 1567 HLLTEVETPIRNEWGCRCNDSSD 1946 1568 ILLTEVETPIRNEWGCRCNDSSD 1044 1569 KLLTEVETPIRNEWGCRCNDSSD 1826 538 LLLTEVETPIRNEWGCRCNDSSD 1399 1570 MLLTEVETPIRNEWGCRCNDSSD 2126 1571 NLLTEVETPIRNEWGCRCNDSSD 2135 1572 PLLTEVETPIRNEWGCRCNDSSD 1641 1573 QLLTEVETPIRNEWGCRCNDSSD 1627 1574 RLLTEVETPIRNEWGCRCNDSSD 1338 326 SLLTEVETPIRNEWGCRCNDSSD 2476 312 TLLTEVETPIRNEWGCRCNDSSD 2510 520 VLLTEVETPIRNEWGCRCNDSSD 1758 1575 WLLTEVETPIRNEWGCRCNDSSD 2180 1576 YLLTEVETPIRNEWGCRCNDSSD 1882 349 SLATEVETPIRNEWGCRCNDSSD 2145 1577 SLDTEVETPIRNEWGCRCNDSSD 1505 328 SLETEVETPIRNEWGCRCNDSSD 1963 459 SLITEVETPIRNEWGCRCNDSSD 1996 377 SLKTEVETPIRNEWGCRCNDSSD 1453 327 SLMTEVETPIRNEWGCRCNDSSD 2149 449 SLNTEVETPIRNEWGCRCNDSSD 1908 352 SLQTEVETPIRNEWGCRCNDSSD 2025 510 SLRTEVETPIRNEWGCRCNDSSD 1034 473 SLSTEVETPIRNEWGCRCNDSSD 2052 478 SLTTEVETPIRNEWGCRCNDSSD 1804 484 SLVTEVETPIRNEWGCRCNDSSD 2029 1578 SLLAEVETPIRNEWGCRCNDSSD 2356 1579 SLLDEVETPIRNEWGCRCNDSSD 2276 1580 SLLEEVETPIRNEWGCRCNDSSD 2420 1581 SLLFEVETPIRNEWGCRCNDSSD 2137 1582 SLLGEVETPIRNEWGCRCNDSSD 2301 1583 SLLHEVETPIRNEWGCRCNDSSD 2462 1584 SLLIEVETPIRNEWGCRCNDSSD 1899 1585 SLLKEVETPIRNEWGCRCNDSSD 2611 1586 SLLLEVETPIRNEWGCRCNDSSD 2344 346 SLLMEVETPIRNEWGCRCNDSSD 2477 1587 SLLNEVETPIRNEWGCRCNDSSD 2425 381 SLLQEVETPIRNEWGCRCNDSSD 2432 1588 SLLREVETPIRNEWGCRCNDSSD 2286 803 SLLSEVETPIRNEWGCRCNDSSD 2305 1589 SLLVEVETPIRNEWGCRCNDSSD 1932 1590 SLLWEVETPIRNEWGCRCNDSSD 2221 1591 SLLYEVETPIRNEWGCRCNDSSD 2181 822 SLLTDVETPIRNEWGCRCNDSSD 1113 522 SLLTMVETPIRNEWGCRCNDSSD 1244 608 SLLTEIETPIRNEWGCRCNDSSD 2016 556 SLLTELETPIRNEWGCRCNDSSD 1380 547 SLLTETETPIRNEWGCRCNDSSD 1594 551 SLLTEVATPIRNEWGCRCNDSSD 2093 657 SLLTEVDTPIRNEWGCRCNDSSD 2095 731 SLLTEVFTPIRNEWGCRCNDSSD 1708 784 SLLTEVGTPIRNEWGCRCNDSSD 2005 690 SLLTEVHTPIRNEWGCRCNDSSD 2057 795 SLLTEVITPIRNEWGCRCNDSSD 1438 705 SLLTEVKTPIRNEWGCRCNDSSD 1905 709 SLLTEVLTPIRNEWGCRCNDSSD 1743 629 SLLTEVMTPIRNEWGCRCNDSSD 2331 675 SLLTEVNTPIRNEWGCRCNDSSD 2150 594 SLLTEVQTPIRNEWGCRCNDSSD 2289 575 SLLTEVRTPIRNEWGCRCNDSSD 1024 545 SLLTEVSTPIRNEWGCRCNDSSD 2288 553 SLLTEVTTPIRNEWGCRCNDSSD 2102 645 SLLTEVVTPIRNEWGCRCNDSSD 1492 583 SLLTEVWTPIRNEWGCRCNDSSD 1889 658 SLLTEVYTPIRNEWGCRCNDSSD 1092 635 SLLTEVEAPIRNEWGCRCNDSSD 2325 779 SLLTEVEDPIRNEWGCRCNDSSD 2216 652 SLLTEVEEPIRNEWGCRCNDSSD 2362 621 SLLTEVEFPIRNEWGCRCNDSSD 2297 646 SLLTEVEGPIRNEWGCRCNDSSD 2341 450 SLLTEVEHPIRNEWGCRCNDSSD 2241 593 SLLTEVEIPIRNEWGCRCNDSSD 2365 534 SLLTEVEKPIRNEWGCRCNDSSD 2505 564 SLLTEVELPIRNEWGCRCNDSSD 2407 590 SLLTEVEMPIRNEWGCRCNDSSD 2516 467 SLLTEVENPIRNEWGCRCNDSSD 2549 537 SLLTEVEPPIRNEWGCRCNDSSD 2522 527 SLLTEVEQPIRNEWGCRCNDSSD 2344 599 SLLTEVERPIRNEWGCRCNDSSD 2131 607 SLLTEVESPIRNEWGCRCNDSSD 2156 695 SLLTEVEVPIRNEWGCRCNDSSD 2286 763 SLLTEVEWPIRNEWGCRCNDSSD 2178 783 SLLTEVEYPIRNEWGCRCNDSSD 2113 559 SLLTEVETAIRNEWGCRCNDSSD 2415 610 SLLTEVETDIRNEWGCRCNDSSD 2392 602 SLLTEVETEIRNEWGCRCNDSSD 2368 549 SLLTEVETFIRNEWGCRCNDSSD 2301 597 SLLTEVETGIRNEWGCRCNDSSD 2366 609 SLLTEVETHIRNEWGCRCNDSSD 2288 653 SLLTEVETKIRNEWGCRCNDSSD 2096 300 SLLTEVETKIRNEWGCRCNDSSD 2485 491 SLLTEVETLIRNEWGCRCNDSSD 2225 493 SLLTEVETMIRNEWGCRCNDSSD 2320 529 SLLTEVETNIRNEWGCRCNDSSD 2253 606 SLLTEVETQIRNEWGCRCNDSSD 2189 611 SLLTEVETRIRNEWGCRCNDSSD 2159 700 SLLTEVETSIRNEWGCRCNDSSD 2151 649 SLLTEVETTIRNEWGCRCNDSSD 2295 614 SLLTEVETVIRNEWGCRCNDSSD 2264 670 SLLTEVETWIRNEWGCRCNDSSD 2146 581 SLLTEVETYIRNEWGCRCNDSSD 2290 715 SLLTEVETPARNEWGCRCNDSSD 2244 660 SLLTEVETPDRNEWGCRCNDSSD 2390 630 SLLTEVETPERNEWGCRCNDSSD 2480 665 SLLTEVETPFRNEWGCRCNDSSD 2229 495 SLLTEVETPGRNEWGCRCNDSSD 2676 591 SLLTEVETPHRNEWGCRCNDSSD 2425 392 SLLTEVETPKRNEWGCRCNDSSD 2300 461 SLLTEVETPLRNEWGCRCNDSSD 2239 457 SLLTEVETPMRNEWGCRCNDSSD 2297 770 SLLTEVETPNRNEWGCRCNDSSD 2299 738 SLLTEVETPPRNEWGCRCNDSSD 2302 782 SLLTEVETPQRNEWGCRCNDSSD 2341 750 SLLTEVETPRRNEWGCRCNDSSD 2376 673 SLLTEVETPSRNEWGCRCNDSSD 2396 758 SLLTEVETPTRNEWGCRCNDSSD 2417 650 SLLTEVETPVRNEWGCRCNDSSD 2331 693 SLLTEVETPWRNEWGCRCNDSSD 2190 389 SLLTEVETPYRNEWGCRCNDSSD 2303 707 SLLTEVETPIANEWGCRCNDSSD 2302 524 SLLTEVETPIDNEWGCRCNDSSD 2337 676 SLLTEVETPIENEWGCRCNDSSD 2252 576 SLLTEVETPIFNEWGCRCNDSSD 2176 744 SLLTEVETPIGNEWGCRCNDSSD 2073 488 SLLTEVETPIHNEWGCRCNDSSD 2121 720 SLLTEVETPIINEWGCRCNDSSD 2262 778 SLLTEVETPIKNEWGCRCNDSSD 2322 692 SLLTEVETPILNEWGCRCNDSSD 2363 454 SLLTEVETPIMNEWGCRCNDSSD 2369 780 SLLTEVETPINNEWGCRCNDSSD 2277 357 SLLTEVETPIPNEWGCRCNDSSD 2515 677 SLLTEVETPIQNEWGCRCNDSSD 2372 754 SLLTEVETPISNEWGCRCNDSSD 2292 766 SLLTEVETPITNEWGCRCNDSSD 2358 680 SLLTEVETPIVNEWGCRCNDSSD 2366 656 SLLTEVETPIWNEWGCRCNDSSD 2250 509 SLLTEVETPIYNEWGCRCNDSSD 2296 582 SLLTEVETPIRAEWGCRCNDSSD 2306 698 SLLTEVETPIRDEWGCRCNDSSD 2187 771 SLLTEVETPIREEWGCRCNDSSD 2156 620 SLLTEVETPIRFEWGCRCNDSSD 2364 773 SLLTEVETPIRGEWGCRCNDSSD 2228 721 SLLTEVETPIRHEWGCRCNDSSD 2296 627 SLLTEVETPIRIEWGCRCNDSSD 2444 631 SLLTEVETPIRKEWGCRCNDSSD 2437 585 SLLTEVETPIRLEWGCRCNDSSD 2468 622 SLLTEVETPIRMEWGCRCNDSSD 2385 753 SLLTEVETPIRPEWGCRCNDSSD 2301 661 SLLTEVETPIRQEWGCRCNDSSD 2367 710 SLLTEVETPIRREWGCRCNDSSD 2278 678 SLLTEVETPIRSEWGCRCNDSSD 2322 387 SLLTEVETPIRTEWGCRCNDSSD 2360 565 SLLTEVETPIRVEWGCRCNDSSD 2300 562 SLLTEVETPIRWEWGCRCNDSSD 2302 654 SLLTEVETPIRYEWGCRCNDSSD 2202 666 SLLTEVETPIRNAWGCRCNDSSD 2334 712 SLLTEVETPIRNDWGCRCNDSSD 2324 662 SLLTEVETPIRNFWGCRCNDSSD 2417 643 SLLTEVETPIRNGWGCRCNDSSD 2408 684 SLLTEVETPIRNHWGCRCNDSSD 2377 577 SLLTEVETPIRNIWGCRCNDSSD 2490 640 SLLTEVETPIRNKWGCRCNDSSD 2402 571 SLLTEVETPIRNLWGCRCNDSSD 2415 605 SLLTEVETPIRNMWGCRCNDSSD 2455 741 SLLTEVETPIRNNWGCRCNDSSD 2319 616 SLLTEVETPIRNPWGCRCNDSSD 2288 512 SLLTEVETPIRNQWGCRCNDSSD 2402 546 SLLTEVETPIRNRWGCRCNDSSD 2257 617 SLLTEVETPIRNSWGCRCNDSSD 2300 380 SLLTEVETPIRNTWGCRCNDSSD 2314 685 SLLTEVETPIRNVWGCRCNDSSD 2281 703 SLLTEVETPIRNWWGCRCNDSSD 2293 702 SLLTEVETPIRNYWGCRCNDSSD 2311 453 SLLTEVETPIRNEAGCRCNDSSD 2383 751 SLLTEVETPIRNEDGCRCNDSSD 2359 787 SLLTEVETPIRNEEGCRCNDSSD 2330 716 SLLTEVETPIRNEFGCRCNDSSD 2444 708 SLLTEVETPIRNEGGCRCNDSSD 2486 696 SLLTEVETPIRNEHGCRCNDSSD 2398 636 SLLTEVETPIRNEIGCRCNDSSD 2402 572 SLLTEVETPIRNEKGCRCNDSSD 2580 641 SLLTEVETPIRNELGCRCNDSSD 2436 517 SLLTEVETPIRNEMGCRCNDSSD 2398 637 SLLTEVETPIRNENGCRCNDSSD 2265 671 SLLTEVETPIRNEPGCRCNDSSD 2279 765 SLLTEVETPIRNEQGCRCNDSSD 2264 737 SLLTEVETPIRNERGCRCNDSSD 2328 745 SLLTEVETPIRNESGCRCNDSSD 2346 688 SLLTEVETPIRNETGCRCNDSSD 2401 386 SLLTEVETPIRNEVGCRCNDSSD 2411 384 SLLTEVETPIRNEYGCRCNDSSD 2426 681 SLLTEVETPIRNEWACRCNDSSD 2431 717 SLLTEVETPIRNEWDCRCNDSSD 2322 726 SLLTEVETPIRNEWECRCNDSSD 2364 596 SLLTEVETPIRNEWFCRCNDSSD 2493 633 SLLTEVETPIRNEWHCRCNDSSD 2327 347 SLLTEVETPIRNEWICRCNDSSD 2359 541 SLLTEVETPIRNEWKCRCNDSSD 2328 569 SLLTEVETPIRNEWLCRCNDSSD 2379 642 SLLTEVETPIRNEWMCRCNDSSD 2378 785 SLLTEVETPIRNEWNCRCNDSSD 2236 774 SLLTEVETPIRNEWPCRCNDSSD 2328 760 SLLTEVETPIRNEWQCRCNDSSD 2338 704 SLLTEVETPIRNEWRCRCNDSSD 2371 735 SLLTEVETPIRNEWSCRCNDSSD 2374 669 SLLTEVETPIRNEWTCRCNDSSD 2401 624 SLLTEVETPIRNEWVCRCNDSSD 2418 366 SLLTEVETPIRNEWWCRCNDSSD 2406 625 SLLTEVETPIRNEWYCRCNDSSD 2362 659 SLLTEVETPIRNEWGARCNDSSD 2396 790 SLLTEVETPIRNEWGDRCNDSSD 2267 370 SLLTEVETPIRNEWGERCNDSSD 2286 504 SLLTEVETPIRNEWGFRCNDSSD 2349 563 SLLTEVETPIRNEWGGRCNDSSD 2270 647 SLLTEVETPIRNEWGHRCNDSSD 2113 667 SLLTEVETPIRNEWGIRCNDSSD 2211 648 SLLTEVETPIRNEWGKRCNDSSD 2295 600 SLLTEVETPIRNEWGLRCNDSSD 2389 732 SLLTEVETPIRNEWGMRCNDSSD 2269 664 SLLTEVETPIRNEWGNRCNDSSD 2328 727 SLLTEVETPIRNEWGPRCNDSSD 2334 713 SLLTEVETPIRNEWGQRCNDSSD 2369 672 SLLTEVETPIRNEWGRRCNDSSD 2372 682 SLLTEVETPIRNEWGSRCNDSSD 2346 638 SLLTEVETPIRNEWGTRCNDSSD 2404 644 SLLTEVETPIRNEWGVRCNDSSD 2444 595 SLLTEVETPIRNEWGWRCNDSSD 2439 668 SLLTEVETPIRNEWGYRCNDSSD 2410 683 SLLTEVETPIRNEWGCACNDSSD 2301 501 SLLTEVETPIRNEWGCCCNDSSD 2515 615 SLLTEVETPIRNEWGCDCNDSSD 2207 679 SLLTEVETPIRNEWGCECNDSSD 2148 639 SLLTEVETPIRNEWGCFCNDSSD 2312 775 SLLTEVETPIRNEWGCGCNDSSD 2186 776 SLLTEVETPIRNEWGCHCNDSSD 2134 722 SLLTEVETPIRNEWGCICNDSSD 2269 701 SLLTEVETPIRNEWGCKCNDSSD 2290 748 SLLTEVETPIRNEWGCLCNDSSD 2292 730 SLLTEVETPIRNEWGCMCNDSSD 2413 743 SLLTEVETPIRNEWGCNCNDSSD 2340 767 SLLTEVETPIRNEWGCPCNDSSD 2295 756 SLLTEVETPIRNEWGCQCNDSSD 2294 736 SLLTEVETPIRNEWGCSCNDSSD 2348 719 SLLTEVETPIRNEWGCTCNDSSD 2347 742 SLLTEVETPIRNEWGCVCNDSSD 2334 503 SLLTEVETPIRNEWGCWCNDSSD 2437 552 SLLTEVETPIRNEWGCYCNDSSD 2397 634 SLLTEVETPIRNEWGCRANDSSD 2265 794 SLLTEVETPIRNEWGCRDNDSSD 2033 796 SLLTEVETPIRNEWGCRENDSSD 2097 723 SLLTEVETPIRNEWGCRFNDSSD 2303 752 SLLTEVETPIRNEWGCRGNDSSD 2345 734 SLLTEVETPIRNEWGCRHNDSSD 2353 697 SLLTEVETPIRNEWGCRINDSSD 2409 598 SLLTEVETPIRNEWGCRKNDSSD 2435 686 SLLTEVETPIRNEWGCRLNDSSD 2363 691 SLLTEVETPIRNEWGCRMNDSSD 2356 746 SLLTEVETPIRNEWGCRNNDSSD 2384 757 SLLTEVETPIRNEWGCRPNDSSD 2326 772 SLLTEVETPIRNEWGCRQNDSSD 2327 584 SLLTEVETPIRNEWGCRRNDSSD 2383 519 SLLTEVETPIRNEWGCRSNDSSD 2344 573 SLLTEVETPIRNEWGCRTNDSSD 2374 375 SLLTEVETPIRNEWGCRVNDSSD 2316 579 SLLTEVETPIRNEWGCRWNDSSD 2364 699 SLLTEVETPIRNEWGCRYNDSSD 2169 706 SLLTEVETPIRNEWGCRCADSSD 2329 728 SLLTEVETPIRNEWGCRCCDSSD 2377 762 SLLTEVETPIRNEWGCRCDDSSD 2289 739 SLLTEVETPIRNEWGCRCEDSSD 2273 560 SLLTEVETPIRNEWGCRCFDSSD 2538 689 SLLTEVETPIRNEWGCRCGDSSD 2343 651 SLLTEVETPIRNEWGCRCHDSSD 2350 612 SLLTEVETPIRNEWGCRCIDSSD 2450 318 SLLTEVETPIRNEWGCRCKDSSD 2549 618 SLLTEVETPIRNEWGCRCLDSSD 2480 628 SLLTEVETPIRNEWGCRCMDSSD 2470 516 SLLTEVETPIRNEWGCRCPDSSD 2449 568 SLLTEVETPIRNEWGCRCQDSSD 2438 544 SLLTEVETPIRNEWGCRCRDSSD 2447 729 SLLTEVETPIRNEWGCRCSDSSD 2141 674 SLLTEVETPIRNEWGCRCTDSSD 2253 632 SLLTEVETPIRNEWGCRCVDSSD 2366 555 SLLTEVETPIRNEWGCRCWDSSD 2462 626 SLLTEVETPIRNEWGCRCYDSSD 2385 619 SLLTEVETPIRNEWGCRCNASSD 2454 588 SLLTEVETPIRNEWGCRCNCSSD 2505 694 SLLTEVETPIRNEWGCRCNESSD 2282 535 SLLTEVETPIRNEWGCRCNFSSD 2509 603 SLLTEVETPIRNEWGCRCNGSSD 2418 613 SLLTEVETPIRNEWGCRCNHSSD 2439 586 SLLTEVETPIRNEWGCRCNASSD 2517 465 SLLTEVETPIRNEWGCRCNKSSD 2597 393 SLLTEVETPIRNEWGCRCNLSSD 2634 394 SLLTEVETPIRNEWGCRCNMSSD 2574 395 SLLTEVETPIRNEWGCRCNNSSD 2502 396 SLLTEVETPIRNEWGCRCNPSSD 2445 397 SLLTEVETPIRNEWGCRCNQSSD 2376 398 SLLTEVETPIRNEWGCRCNRSSD 2514 342 SLLTEVETPIRNEWGCRCNASSD 2440 399 SLLTEVETPIRNEWGCRCNTSSD 2483 400 SLLTEVETPIRNEWGCRCNVSSD 2476 401 SLLTEVETPIRNEWGCRCNWSSD 2556 337 SLLTEVETPIRNEWGCRCNYSSD 2475 402 SLLTEVETPIRNEWGCRCNDASD 2267 403 SLLTEVETPIRNEWGCRCNDCSD 2318 404 SLLTEVETPIRNEWGCRCNDDSD 2237 405 SLLTEVETPIRNEWGCRCNDESD 2203 406 SLLTEVETPIRNEWGCRCNDFSD 2395 338 SLLTEVETPIRNEWGCRCNDGSD 2226 407 SLLTEVETPIRNEWGCRCNDHSD 2537 408 SLLTEVETPIRNEWGCRCNDISD 2527 409 SLLTEVETPIRNEWGCRCNDKSD 2484 410 SLLTEVETPIRNEWGCRCNDLSD 2494 411 SLLTEVETPIRNEWGCRCNDMSD 2405 412 SLLTEVETPIRNEWGCRCNDNSD 2389 413 SLLTEVETPIRNEWGCRCNDPSD 2450 414 SLLTEVETPIRNEWGCRCNDQSD 2349 415 SLLTEVETPIRNEWGCRCNDRSD 2502 416 SLLTEVETPIRNEWGCRCNDTSD 2394 417 SLLTEVETPIRNEWGCRCNDVSD 2389 418 SLLTEVETPIRNEWGCRCNDWSD 2491 330 SLLTEVETPIRNEWGCRCNDYSD 2445 419 SLLTEVETPIRNEWGCRCNDSAD 2219 420 SLLTEVETPIRNEWGCRCNDSCD 2269 356 SLLTEVETPIRNEWGCRCNDSDD 2181 421 SLLTEVETPIRNEWGCRCNDSED 2527 422 SLLTEVETPIRNEWGCRCNDSFD 2492 313 SLLTEVETPIRNEWGCRCNDSGD 2485 423 SLLTEVETPIRNEWGCRCNDSHD 2353 424 SLLTEVETPIRNEWGCRCNDSID 2320 425 SLLTEVETPIRNEWGCRCNDSKD 2361 426 SLLTEVETPIRNEWGCRCNDSLD 2400 427 SLLTEVETPIRNEWGCRCNDSMD 2304 350 SLLTEVETPIRNEWGCRCNDSND 2299 428 SLLTEVETPIRNEWGCRCNDSPD 2344 429 SLLTEVETPIRNEWGCRCNDSQD 2175 430 SLLTEVETPIRNEWGCRCNDSRD 2373 431 SLLTEVETPIRNEWGCRCNDSTD 2205 432 SLLTEVETPIRNEWGCRCNDSVD 2232 433 SLLTEVETPIRNEWGCRCNDSWD 2477 434 SLLTEVETPIRNEWGCRCNDSYD 2479 435 SLLTEVETP 2380 436 SLLTEVETPI 2465 437 SLLTEVETPIR 2297 438 SLLTEVETPIRN 2130 439 SLLTEVETPIRNE 1819 440 SLLTEVETPIRNEW 2334 368 SLLTEVETPIRNEWG 2429 442 SLLTEVETPIRNEWGC 2369 443 SLLTEVETPIRNEWGCR 2444 444 SLLTEVETPIRNEWGCRC 2478 445 SLLTEVETPIRNEWGCRCN 2506 446 SLLTEVETPIRNEWGCRCND 2276 447 SLLTEVETPIRNEWGCRCNDS 2348 448 SLLTEVETPIRNEWGCRCNDSS 2325

Further, the peptides were screened for the ability to specifically bind 23K21 and 8I10 and NOT Z3G. In one set, peptides with high binding values for 23K21 and 8I10 that are NOT recognized by Z3G1 were identified. In a second set, peptides with high binding values for Z3G1 that are NOT recognized by 23K21 and 8I10 were identified.

Peptide sequences with high binding values for 23K12 and BI10 with no binding to Z3G1 were identified for low stringency (0.01 μg/mL) conditions (Table 4A) and high stringency (0.001 μg/mL) conditions (Table 4B).

TABLE 4A

TABLE 4B

Properties of the peptides that bind specifically to HuM2e antibodies 23K12/BI10 at different stringency conditions were analyzed. Antibodies 8i10 and 23k12 bind a conformational epitope with SLLTE as its core sequence. The best 23k12 binder is SLLTEVGSLLTEV (SEQ ID NO: 320), which is also recognized by Z3G1. The best 8i10 binder is CSLLTEVGSLLTEV (SEQ ID NO: 283), which is also recognized by Z3G1. The very best specific binder is CSLLTECGSLLTCV (SEQ ID NO: 463). The binding sequences contain a remarkably high number of cysteines.

Peptide sequences with high binding values for Z3G1 with no binding to 23K12 and BI10 were identified for low stringency (0.01 μg/mL) conditions (Table 5A) and high stringency (0.001 μg/mL) conditions (Table 5B).

TABLE 5A

TABLE 5B

Now Dominant Motif Occurs!

There is a clear difference between binding specificity between antibodies 8i10/23k12 versus Z3G1. The very best specific binder is a longer sequence than with 23k12/8i10, has no dimer topology and also no CLIPS. Among the very best specific binder is mostly the whole native sequence, MSLLTEVETPIRNEWGCRCN (SEQ ID NO: 1149). The result with 0.01 μg/ml shows that LLXEVEXPIRN (SEQ ID NO: 1594) is the core of the Z3G1 binding epitope.

Specific binding motifs can derived from 0.001 μg/ml screening. Thus, mAbs 23k12/8i10 recognize M2e peptides with SLLTE as the core of the epitope. The T residue in S-L-L-T-E differentiates binding between 23k12/8i10 and Z3G1. In contrast, mAb Z3G1 recognize M2e with LLXEVEXPIRN (SEQ ID NO: 1594) as the core of the epitope. (Tables 6A and 6B).

TABLE 6A

TABLE 6B

Peptide Immunogens

In particular, a peptide immunogen of this invention that binds the HuM2e 23k12/8i10 comprises a core sequence of S-L-L-T-E as well as variants, modifications and multimers thereof. Core sequences for low stringency (0.01 μg/mL) conditions (FIG. 6A) and high stringency (0.001 μg/mL) conditions (FIG. 6B) and variants thereof derived from the binding data are shown in FIGS. 6A and 6B.

In some embodiments, the core sequence comprises a first additional amino acid at the C terminal end of S-L-L-T-E-Xaa6, wherein Xaa6 is any amino acid, preferably V or C. (FIG. 6C).

Further, in some embodiments, the core sequence comprises a first additional amino acid at the C terminal end of S-L-L-T-E-Xaa6-Xaa7, wherein Xaa7 is any amino acid, but preferably E. (FIG. 6C).

In some embodiments, the peptide immunogens comprise a plurality of core sequences linked by an amino acid, preferably a small amino acid such as G or A. However, amino acids such as proline, which can act as a structural disruptor in the middle of regular secondary structure elements such as alpha helices and beta sheets, are less desirable. The number of core sequences in peptide immunogens of the invention can be 1, 2, 3, 4, 5 or more. (FIG. 6D).

In some embodiments, an N-terminal amino acid Xaa0 is present in the peptide immunogens. Xaa0 can be any amino acid, but preferably is a cysteine. In some embodiments a N-terminal cysteine is used to cyclize the peptide immunogen. (FIG. 6E)

Peptide immunogens (linear or cyclized) with particularly strong affinity for 23K12/8I10 binding are shown in FIG. 6F.

The peptide immunogens of this invention that bind the huM2e monoclonal antibodies under high stringency conditions are represented by the following formula:

[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-[Xaa7]q- [Xaa8-[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p- [Xaa7]q]n

wherein, m, p and q are independently 0 or 1,
n is any number between 0 and 4,
Xaa0 is any amino acid, preferably C;
Xaa6 is any amino acid, preferably V or C;
Xaa7 is any amino acid, preferably E;
Xaa8 is any amino acid not including proline, preferably G or A;

Xaa1-Xaa2-Xaa3-Xaa4-Xaa5 is S-L-L-T-E,

or a peptide having a single substitution to the sequence S-L-L-T-E, the substitution selected from the group consisting of:

Xaa1 is C or T;

Xaa2 is A, C, F or K,

Xaa3 is A, C, E, F, I, K, M, Q, S, T or V, and

Xaa5 is D or C.

The peptide immunogens of this invention that bind the huM2e monoclonal antibodies under low stringency conditions are represented by the following formula:

[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-[Xaa7]q- [Xaa8-[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p- [Xaa7]q]n

wherein, m, p and q are independently 0 or 1,
n is any number between 0 and 4,
Xaa0 is any amino acid, preferably C;
Xaa6 is any amino acid, preferably V or C;
Xaa7 is any amino acid, preferably E;
Xaa8 is any amino acid not including proline, preferably G or A;

Xaa1-Xaa2-Xaa3-Xaa4-Xaa5 is S-L-L-T-E or,

a peptide having a single substitution to the sequence S-L-L-T-E, the substitution selected from the group consisting of:

Xaa1 is A, C, D, L, T or V,

Xaa2 is A, C, F, H, I, K, M, N, Q, R, T, W, or Y,

Xaa3 is any amino acid,

Xaa4 is M, N, Q, S, or W, and

Xaa5 is A, D, F, H, I, K, M, N, Q, S, W, Y, or C.

Preparation of Linear and Cyclic Peptide Immunogens

Linear peptide immunogens can be prepared synthetically and then screened for a particular characteristic in various biological assays. E.g., Scott, J. K. and G. P. Smith, Science 249:386, 1990; Devlin, J. J., et al., Science 24:404, 1990; Furka, A. et al., Int. J. Pept. Protein Res. 37:487, 1991; Lam, K. S., et al., Nature 354:82, 1991.

Cyclized peptides are often found to possess superior immunogenic activity compared to linear peptide immunogens. Linear peptide immunogens comprising three or more core sequences are found to bind with the terminal sequences only, while cyclization allows binding by all core sequences present in the peptide immunogens. Various methods for producing cyclic peptides have been described. One involves solution or liquid phase peptide synthesis, where amino acid residues in solution are linked by peptide bonds, with reactive groups not involved in the peptide bond formation, such as the amino group of the N-terminal residue, the carboxy group of the C-terminal residue, sulfhydryl groups on cysteine residues and similar or other reactive groups in the amino acid side chains, protected by suitable protecting groups.

In one embodiment cyclic peptide immunogens are formed using terminal cysteine residues by reduction of thiol groups to form disulfide bridges.

Another approach involves solid phase peptide synthesis, in which synthesis is carried out on an insoluble solid matrix. Protecting groups are employed for reactive side chains. The general methodology of solid phase synthesis is well known in the art. Merrifield, R. B., Solid phase synthesis (Nobel lecture). Angew Chem 24:799-810 (1985) and Barany et al., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross, E. and Meienhofer, J., Eds. Academic Press 1-284 (1980). For example, chemical reaction protocols, such as those described in U.S. Pat. Nos. 4,033,940 and 4,102,877, have been devised to produce circularized peptides. In other techniques, biological and chemical methods are combined to produce cyclic peptides. These latter methods involve first expressing linear precursors of cyclic peptides in cells (e.g., bacteria) to produce linear precursors of cyclic peptides and then adding of an exogenous agent such as a protease or a nucleophilic reagent to chemically convert these linear precursors into cyclic peptides. See, e.g., Camerero, J. A., and Muir, T. W., J. Am. Chem. Society. 121:5597 (1999); Wu, H. et al, Proc. Natl. Acad. Sci. USA, 95:9226 (1998).

Head-to-tail (backbone) peptide cyclization has been used to rigidify structure and improve in vivo stability of small bioactive peptides (see Camarero and Muir, J. Am. Chem. Soc., 121:5597-5598 (1999)). An important consequence of peptide cyclization is retention of biological activity and/or the identification of new classes of pharmacological agents. A chemical cross-linking approach was used to prepare a backbone cyclized version of bovine pancreatic trypsin inhibitor (Goldenburg and Creighton, J. Mol. Biol., 165:407-413 (1983)). Other approaches include chemical (Camarero et al., Angew. Chem. Int. Ed., 37:347-349 (1998); Tam and Lu, Prot. Sci., 7:1583-1592 (1998); Camarero and Muir, Chem. Commun., 1997:1369-1370 (1997); and Zhang and Tam, J. Am. Chem. Soc. 119:2363-2370 (1997)) and enzymatic (Jackson et al., J. Am. Chem. Soc., 117:819-820 (1995)) intramolecular ligation methods which allow linear synthetic peptides to be efficiently cyclized under aqueous conditions.

A native chemical ligation approach utilizes inteins (internal proteins) to catalyze head-to-tail peptide and protein ligation in vivo (see, for example, Evans et al., J. Biol. Chem. 274:18359-18363 (1999); Iwai and Pluckthun, FEBS Lett. 459.166-172 (1999); Wood et al., Nature Biotechnology 17:889-892 (1999); Camarero and Muir, J. Am. Chem. Soc. 121:5597-5598 (1999); and Scott et al., Proc. Natl. Acad. Sci. USA 96:13638-13643 (1999)).

The invention also encompasses isolated nucleic acid molecules comprising a sequences that encode M2e peptide immunogens of the invention. Also provided by the present invention are nucleic acid expression constructs, and host cells containing such nucleic acids, which encode M2e peptides, and variants thereof, which have at least one epitope characteristic of M2e peptide immunogens. This aspect of the invention pertains to isolated nucleic sequences encoding an M2e sequence or M2e peptide immunogen sequence as described herein, as well as those sequences readily derived from isolated nucleic molecules such as, for example, complementary sequences, reverse sequences and complements of reverse of sequences.

A related embodiment includes a nucleic acid expression construct comprising a promoter operably linked to the isolated nucleic acid molecule such that a M2e peptide immunogen or fusion protein comprising a M2e peptide immunogen as described herein is expressed in a host cell. In another embodiment, the invention provides a host cell containing such a nucleic acid expression construct. In a related embodiment, the invention provides a method for producing a peptide immunogen, comprising growing the described host cells for a time sufficient to express the peptide immunogen encoded by the nucleic acid expression construct.

Conjugated Peptide Immunogens

The approach of increasing immunogenicity of small immunogenic molecules by conjugating these molecules to large “carrier” molecules has been utilized successfully for decades (see, e.g., Goebel et al. (1939) J. Exp. Med. 69: 53). For example, many immunogenic compositions have been described in which purified capsular polymers have been conjugated to carrier proteins to create more effective immunogenic compositions by exploiting this “carrier effect.” Schneerson et al. (1984) Infect. Immun. 45: 582-591).

In one aspect of the invention, method for conjugating a M2e peptide immunogen via a reactive group of an amino acid residue of the peptide immunogen to a protein/polypeptide carrier having one or more functional groups is provided. The protein/polypeptide carrier may be human serum albumin, keyhole limpet hemocyanin (KLH), immunoglobulin molecules, thyroglobulin, ovalbumin, influenza hemagglutinin, PAN-DR binding peptide (PADRE polypeptide), malaria circumsporozite (CS) protein, hepatitis B surface antigen (HBSAg19-28, Heat Shock Protein (HSP) 65, Bacillus Calmette-Guerin (BCG), cholera toxin, cholera toxin mutants with reduced toxicity, diphtheria toxin, CRM197 protein that is cross-reactive with diphtheria toxin, recombinant Streptococcal C5a peptidase, Streptococcus pyogenes ORF1224, Streptococcus pyogenes ORF1664, Streptococcus pyogenes ORF 2452, Chlamydia pneumoniae ORF T367, Chlamydia pneumoniae ORF T858, Tetanus toxoid, HIV gp120 T1, microbial surface components recognizing adhesive matrix molecules (MSCRAMMS), growth factor/hormone, cytokines or chemokines.

Methods for protecting a subject from infection or decreasing susceptibility of a subject to infection by one or more influenza strains/isolates or subtypes, i.e., prophylactic methods, are additionally provided. In one embodiment, a method includes administering to the subject an amount of M2e peptide immunogens that specifically bind influenza M2 effective to protect the subject from infection, or effective to decrease susceptibility of the subject to infection, by one or more influenza strains/isolates or subtypes.

Symptoms or complications of influenza infection that can be reduced or decreased include, for example, chills, fever, cough, sore throat, nasal congestion, sinus congestion, nasal infection, sinus infection, body ache, head ache, fatigue, pneumonia, bronchitis, ear infection, ear ache or death.

Peptide Immunogens as Vaccines

The peptide immunogens can be used as vaccines to generate an anti-influenza M2-mediated immune response in order to prevent influenza infections. Synthetic peptides require both stabilization and adjuvantation for the induction of an effective immune response in vivo. Various methods have been employed to protect synthetic peptide immunogens against degradation in vitro and in vivo, mediated by various processes including chemical and physical pathways. (Manning M C, et al. Pharmaceutical Research, 1989, 6:903-918).

Numerous adjuvants and/or depot-based parenteral, mucosal or transdermal delivery systems destined for use with human or veterinary vaccines have been developed to enhance the immune response. These include the use of mineral salts, water-in-oil (w/o)-emulsions, liposomes, polymeric microparticles, nanoparticles and gels/hydrogels. (Cox J C, et al. Vaccine, 1997, 15:248-256). Freund's complete adjuvant (FCA), a suspension of heat-killed M. tuberculosis mycobacteria in mineral oil containing a surfactant, has been recognized as one of the most powerful adjuvants. Adjuvants are well known in the art (Vaccine Design—The Subunit and Adjuvant Approach, 1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, M. F., and Newman, M. J., Plenum Press, New York and London, ISBN 0-306-44867-X). Preferred adjuvants for use with immunogens of the present invention include aluminium or calcium salts (hydroxide or phosphate). Adjuvants may be selected from GM-CSF, 529 SE, IL-12, aluminum phosphate, aluminum hydroxide, Mycobacterium tuberculosis, Bordetella pertussis, bacterial lipopolysaccharides, aminoalkyl glucosane phosphate compounds, MPL™ (3-O-deacylated monophosphoryl lipid A), a polypeptide, Quil A, STIMULON™ QS-21, a pertussis toxin (PT), an E. coli heat-labile toxin (LT), IL-1alpha, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon-alpha, interferon-β, interferon-gamma, G-CSF, TNF-alpha and TNF-β.

Still other adjuvants include mineral oil and water emulsions, calcium salts such as calcium phosphate, aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, etc., Amphigen, Avridine, L121/squalene, D-lactide-polylactide/glycoside, pluronic acids, polyols, muramyl dipeptide, killed Bordetella, saponins, such as Stimulon™ QS-21 (Antigenics, Framingham, Mass.), described in U.S. Pat. No. 5,057,540, which is hereby incorporated by reference 3, and particles generated therefrom such as ISCOMS (immunostimulating complexes), Mycobacterium tuberculosis, bacterial lipopolysaccharides, synthetic polynucleotides such as oligonucleotides containing a CpG motif (U.S. Pat. No. 6,207,646, which is hereby incorporated by reference), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularly LT-K63, LT-R72, PT-K9/G129; see, e.g., International Patent Publication Nos. WO 93/13302 and WO 92/19265, which are/incorporated herein by reference for all purposes.

Also useful as adjuvants are cholera toxins and mutants thereof, including those described in published International Patent Application No. WO 00/18434 (wherein the glutamic acid at amino acid position 29 is replaced by another amino acid (other than aspartic acid, preferably a histidine). Similar CT toxins or mutants are described in published International Patent Application number WO 02/098368 (wherein the isoleucine at amino acid position 16 is replaced by another amino acid, either alone or in combination with the replacement of the serine at amino acid position 68 by another amino acid; and/or wherein the valine at amino acid position 72 is replaced by another amino acid). Other CT toxins are described in published International Patent Application number WO 02/098369 (wherein the arginine at amino acid position 25 is replaced by another amino acid; and/or an amino acid is inserted at amino acid position 49; and/or two amino acids are inserted at amino acid position 35 and 36).

Various methods may be employed to adjuvant synthetic peptide-based immunogens, but normally a carrier or depot system is required for effective long-term immunogenic responses. Notable examples include adsorbing the immunogen onto a mineral salt or gel. For example, encapsulating a peptide immunogen within a polymeric matrix (monolithic matrix) or gel, or layering a polymeric material around a peptide immunogen (core-shell) may be an effective strategy. Or, an immunogen may be incorporated in a liposome or vesicular type of formulation, with the immunogen either embedded in the lipid matrix or physically entrapped in the internal aqueous phase. Another strategy may employ a mineral-based, vegetable-based or animal-based oil, with an aqueous solution of the immunogen in various proportions, to prepare a water-in-oil (w/o)-emulsion or a water-in-oil-in-water (w/o/w)-double emulsion. Powell M F, et al., Pharmaceutical Biotechnology, Vol. 6, Plenum Press, New York, 1995.

Kits

It is particularly useful to use antibody binding sequences in the kit, which correspond to defined epitope sequences known to be specific for the immunogen under investigation. This kit will lead to a more specific answer than those kits used today, and hence to a better selection of immunogen vaccine therapy for the individual patient.

In an extension of this approach, one could also characterize the patient's serum by identifying the corresponding antibody binding peptides among a random display library using the aforementioned methods. This again may lead to optimisation of the epitope information, and thus to a better diagnosis.

Further, one could use the individual antibody binding sequences as (immunogen) vaccines leading to more specific (immunogen) vaccines. These antibody binding sequences could be administered in an isolated form or fused to a membrane protein of the phage display system, or to another carrier protein, which may have beneficial effect for the immunoprotective effect of the antibody binding peptide (Dalum et al., Nature Biotechnology, Vol. 17, pp. 666-669 (1999)).

The present invention relates to a kit for predicting binding of a specific antibody to at least one potential immunogen. The kit of the invention would also be useful for other screening purposes where it is desirable to test for antibody binding to peptide sequences, such as epitope variant.

In one embodiment the peptide immunogen may be immobilized on a solid support. Suitable solid support could be any chemical support, including micro titer plates, beads, capillary tubing or membranes. Each of these supports could be activated, supporting covalent, ionic or hydrophobic binding, chelation or affinity binding, or inactivated, promoting ionic or hydrophobic binding. Immobillisation could take place by attachment through covalent binding, ionic or hydrophobic binding, chelation, affinity binding, or through van der Waal bonds. A solid support could also be biological in nature, such as phages, bacteria, red blood cells or any related system allowing display of heterologous proteins or peptides.

The kit also can be used for screening different antigenic peptide sequences corresponding to structural epitopes at the same time. The kit above also can be used in a high throughput screening method for screening many samples, obtained e.g. from humans or animals, at the same time and thereby predicting which humans or animals will display an immunogenic response towards particular immunogens. Any practical combination of the number of antigenic peptide sequences and the number of humans or animals would be possible.

The following examples illustrate embodiments of the invention. It will be appreciated by one of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLES

Example 1

Identification of M2-Specific Antibodies

Mononuclear or B cells expressing three of the MAbs identified in human serum were diluted into clonal populations and induced to produce antibodies. Antibody containing supernatants were screened for binding to 293 FT cells stably transfected with the full length M2E protein from influenza strain Influenza subtype H3N2. Supernatants which showed positive staining/binding were re-screened again on 293 FT cells stably transfected with the full length M2E protein from influenza strain Influenza subtype H3N2 and on vector alone transfected cells as a control.

The variable regions of the antibodies were then rescue cloned from the B cell wells whose supernatants showed positive binding. Transient transfections were performed in 293 FT cells to reconstitute and produce these antibodies. Reconstituted antibody supernatants were screened for binding to 293 FT cells stably transfected with the full length M2E protein as detailed above to identify the rescued anti-M2E antibodies. Three different antibodies were identified: 8i10, 21B15 and 23K12.

Antibodies 21B15, 23K12, and 8I10 bound to the surface of 293-HEK cells stably expressing the M2 protein, but not to vector transfected cells (see FIG. 1). In addition, binding of these antibodies was not competed by the presence of 5 mg/ml 24-mer M2 peptide, whereas the binding of the control chimeric mouse V-region/human IgG1 kappa 14C2 antibody (hu14C2) generated against the linear M2 peptide was completely inhibited by the M2 peptide (see FIG. 1). These data confirm that these antibodies bind to conformational epitopes present in M2e expressed on the cell or virus surface, as opposed to the linear M2e peptide.

Example 2

Viral Binding of Human Anti-Influenza Monoclonal Antibodies

UV-inactivated influenza A virus (A/PR/8/34) (Applied Biotechnologies) was plated in 384-well MaxiSorp plates (Nunc) at 1.2 μg/ml in PBS, with 25 μl/well, and was incubated at 4° C. overnight. The plates were then washed three times with PBS, and blocked with 1% Nonfat dry milk in PBS, 50 μl/well, and then were incubated at room temp for 1 hr. After a second wash with PBS, MAbs were added at the indicated concentrations in triplicate, and the plates were incubated at room temp for 1 hour. After another wash with PBS, to each well was added 25 μl of a 1/5000 dilution of horseradish peroxidase (HRP) conjugated goat anti-human IgG Fc (Pierce) in PBS/1% Milk, and the plates were left at room temp for 1 hr. After the final PBS wash, the HRP substrate 1-Step™ Ultra-TMB-ELISA (Pierce) was added at 25 μl/well, and the reaction proceeded in the dark at room temp. The assay was stopped with 25 μl/well 1N H2SO4, and light absorbance at 450 nm (A450) was read on a SpectroMax Plus plate reader. Data are normalized to the absorbance of MAb 8I10 binding at 10 μg/ml. Results are shown in FIGS. 2A and 2B.

Example 3

Binding of Human Anti-Influenza Monoclonal Antibodies to Full-Length M2 Variants

M2 variants (including those with a high pathology phenotype in vivo) were selected for analysis. See FIG. 3A for sequences.

M2 cDNA constructs were transiently transfected in HEK293 cells and analyzed as follows: To analyze the transient transfectants by FACS, cells on 10 cm tissue culture plates were treated with 0.5 ml Cell Dissociation Buffer (Invitrogen), and harvested. Cells were washed in PBS containing 1% FBS, 0.2% NaN3 (FACS buffer), and resuspended in 0.6 ml FACS buffer supplemented with 100 μg/ml rabbit IgG. Each transfectant was mixed with the indicated MAbs at 1 μg/ml in 0.2 ml FACS buffer, with 5×105 to 106 cells per sample. Cells were washed three times with FACS buffer, and each sample was resuspended in 0.1 ml containing 1 μg/ml alexafluor (AF) 647-anti human IgG H&L (Invitrogen). Cells were again washed and flow cytometry was performed on a FACSCanto device (Becton-Dickenson). The data is expressed as a percentage of the mean fluorescence of the M2-D20 transient transfectant. Data for variant binding are representative of 2 experiments. Data for alanine mutants are average readouts from 3 separate experiments with standard error. Results are shown in FIGS. 3B and 3C.

Example 4

Epitope Blocking

To determine whether the MAbs 8I10 and 23K12 bind to the same site, M2 protein representing influenza strain A/HK/483/1997 sequence was stably expressed in the CHO (Chinese Hamster Ovary) cell line DG44. Cells were treated with Cell Dissociation Buffer (Invitrogen), and harvested. Cells were washed in PBS containing 1% FBS, 0.2% NaN3 (FACS buffer), and resuspended at 107 cells/ml in FACS buffer supplemented with 100 μg/ml rabbit IgG. The cells were pre-bound by either MAb (or the 2N9 control) at 10 μg/ml for 1 hr at 4° C., and were then washed with FACS buffer. Directly conjugated AF647-8I10 or -23K12 (labeled with the AlexaFluor® 647 Protein Labeling kit (Invitrogen) was then used to stain the three pre-blocked cell samples at 1 μg/ml for 106 cells per sample. Flow cytometric analyses proceeded as before with the FACSCanto. Data are average readouts from 3 separate experiments with standard error. Results are shown in FIG. 4.

Example 5

Binding of Human Anti-Influenza Monoclonal Antibodies to M2 Variants and Truncated M2 Peptides

The cross reactivity of mAbs 8i10 and 23K12 to other M2 peptide variants was assessed by ELISA. Peptide sequences are shown in Tables 7A and 7B. Additionally, a similar ELISA assay was used to determine binding activity to M2 truncated peptides.

In brief, each peptide was coated at 2 μg/mL to a flat bottom 384 well plate (Nunc) in 25 μL/well of PBS buffer overnight at 4° C. Plates were washed three times and blocked with 1% Milk/PBS for one hour at room temperature. After washing three times, MAb titers were added and incubated for one hour at room temperature. Diluted HRP conjugated goat anti-human immunoglobulin FC specific (Pierce) was added to each well after washing three times. Plates were incubated for one hour at room temperature and washed three times. 1-Step™ Ultra-TMB-ELISA (Pierce) was added at 25 μl/well, and the reaction proceeded in the dark at room temp. The assay was stopped with 25 μl/well 1N H2SO4, and light absorbance at 450 nm (A450) was read on a SpectroMax Plus plate reader. Results are shown in Tables 7A and 7B.

TABLE 7A Cross reactivity binding of anti-M2 antibodies to variant M2 peptides. seqNo Name Size Description 14C2 8I10 23K12 2N9 1 M2 23 aa SLLTEVETPIRNEWGCRCNDSSD + 2 M2SG 23 aa SLLTEVETPIRSEWGCRCNDSGD + 3 M2EG 23 aa SLLTEVETPIRNEWECRCNGSSD + 4 M2P 23 aa SLPTEVETPIRNEWGCRCNDSSD + 5 M2G 23 aa SLLTEVETPIRNEWGCRCNGSSD + 6 M2DLTGS 23 aa SLLTEVDTLTRNGWGCRCSDSSD + 7 M2KNS 23 aa SLLTEVETPIRKEWGCNCSDSSD + 8 M2LGS 23 aa SLLTEVETLIRNGWGCRCSDSSD 9 M2LTKGS 23 aa SLLTEVETLTKNGWGCRCSDSSD 10 M2SY 23 aa SLLTEVETPIRSEWGCRYNDSSD + 11 M2TGEKS 23 aa SLLTEVETPTRNGWECKCSDSSD + 12 M2HTGEKS 23 aa SLLTEVETHTRNGWECKCSDSSD 13 M2KTGEKS 23 aa SLLTEVKTPTRNGWECKCSDSSD 14 M2LTGS 23 aa SLLTEVETLTRNGWGCRCSDSSD + 15 M2TDGEKS 23 aa SLLTEVETPTRDGWECKCSDSSD + 16 M2TGS 23 aa SLLTEVETPTRNGWGCRCSDSSD + W 17 M2TGEK 23 aa SLLTEVETPTRNGWECKCNDSSD + 18 M2LTGEKS 23 aa SLLTEVETLTRNGWECKCSDSSD W 19 M2K 23 aa SLLTEVETPIRNEWGCKCNDSSD + W + 20 M2FG 23 aa SPLTEVETPIRNEWGCRCNGSSD + W 21 M2TGE 23 aa SLLTEVETPTRNGWECRCNDSSD + 22 M2KGENS 23 aa SLLTEVETPIRKGWECNCSDSSD + 23 M2TES 23 aa SLLTEVETPTRNEWECRCSDSSD + 24 M2GHTGKS 23 aa SLLTGVETHTRNGWGCKCSDSSD 25 M2PHTGS 23 aa SLLPEVETHTRNGWGCRCSDSSD Percentage compared relative to binding to wild-type peptide (Seq 1) Note: mAhs were tested at 5 μg/mL >25% − no binding 25-40% W weak binding >40% + positive binding

TABLE 7B Binding activity of M2 antibodies to truncated M2 peptides. seqNo Name Size Description 14C2 8I10 23K12 2N9  1 M2 23 aa SLLTEVETPIRNEWGCRCNDSSD 3.85 0.11 0.22 0.06 26 M16 16 aa LLTEVETPIRNEWGCR 3.94 0.09 0.21 0.09 27 M15 15 aa LTEVETPIRNEWGCR 3.95 0.09 0.21 0.09 28 M12 12 aa VETPIRNEWGCR 0.15 0.09 0.20 0.09 29 CM17 17 aa ETPIRNEWGCRCNDSSD 0.19 0.11 0.34 0.11 30 CM16 16 aa TPIRNEWGCRCNDSSD 0.23 0.13 0.35 0.12 31 CM15 15 aa PIRNEWGCRCNDSSD 0.19 0.12 0.34 0.11 32 CM14 14 aa IRNEWGCRCNDSSD 0.23 0.14 0.36 0.13 33 CM13 13 aa RNEWGCRCNDSSD 0.22 0.14 0.34 0.13 34 CM12 12 aa NEWGCRCNDSSD 0.27 0.14 0.39 0.14 35 NM17 17 aa SLLTEVETPIRNEWGCR 3.99 0.26 0.58 0.10 36 NM16 16 aa SLLTEVETPIRNEWGC 3.90 0.29 0.62 0.09 37 NM15 15 aa SLLTEVETPIRNEWG 3.97 0.12 0.30 0.11 38 NM14 14 aa SLLTEVETPIRNEW 3.97 0.11 0.24 0.09 39 NM13 13 aa SLLTEVETPIRNE 0.18 0.11 0.25 0.10 40 NM12 12 aa SLLTEVETPIRN 0.20 0.10 0.24 0.09 41 NM11 11 aa SLLTEVETPIR 0.21 0.13 0.30 0.12 42 NM10 10 aa SLLTEVETPI 0.17 0.10 0.24 0.10 43 NM8  8 aa SLLTEVET 0.15 0.10 0.20 0.09 44 NM7  7 aa SLLTEVE 0.14 0.10 0.20 0.08 45 NM9  9 aa SLLTEVETP 0.21 0.12 0.30 0.19 46 M2e 24 aa MSLLTEVETPIRNEWGCRCNDSSD 3.98 0.13 0.43 0.10 CMV HVIR1 0.16 0.11 0.21 3.99 Note: mAbs were tested at 5 μg/mL

OTHER EMBODIMENTS

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

1. A peptide immunogen comprising a sequence of:

[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-[Xaa7]q-[Xaa8-[Xaa0]m-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-[Xaa6]p-[Xaa7]q]n
wherein, m, p and q are independently 0 or 1,
n is any number between 0 and 4,
Xaa0, Xaa6, Xaa7 and Xaa8 is independently any amino acid,
Xaa1-Xaa2-Xaa3-Xaa4-Xaa5 is S-L-L-T-E, or a peptide having a single substitution to the sequence S-L-L-T-E, wherein the substitution is selected from the group consisting of:
(i): Xaa1 is C or T; Xaa2 is A, C, F or K, Xaa3 is A, C, E, F, I, K, M, Q, S, T or V, and Xaa5 is D or C; and
(ii): Xaa1 is A, C, D, L, T or V, Xaa2 is A, C, F, H, I, K, M, N, Q, R, T, W, or Y, Xaa3 is any amino acid, Xaa4 is M, N, Q, S, or W, and Xaa5 is A, D, F, H, I, K, M, N, Q, S, W, Y, or C.

2. The peptide immunogen of claim 1, wherein Xaa0 is C.

3. The peptide immunogen of claim 1, wherein Xaa6 is V or C.

4. The peptide immunogen of claim 1, wherein Xaa7 is E.

5. The peptide immunogen of claim 1, wherein Xaa8 is G or A.

6. The peptide immunogen of claim 1, wherein said peptide is non-linear.

7. The peptide immunogen of claim 6, wherein said peptide is cyclic.

8. The peptide immunogen of claim 6, wherein said peptide is cyclic and n is any number between 1 and 4.

9. The peptide immunogen of claim 1, wherein said peptide binds specifically to HuMe2 antibodies 8I10 or 23K12.

10. The peptide immunogen of claim 1, wherein said peptide comprises D-amino acids.

11. The peptide immunogen of claim 1, wherein said peptide is a linear or cyclized peptide having a sequence selected from the group consisting of SLLTEVGSLLTEV (SEQ ID NO: 320); CSLLTEVGSLLTEV (SEQ ID NO: 283); and CSLLTECGSLLTCV (SEQ ID NO: 463).

12. The peptide immunogen of claim 1, wherein said peptide does not bind the antibody Z3G1.

13. The peptide immunogen of claim 1, wherein said peptide is conjugated to a carrier.

14. The peptide immunogen of claim 13, wherein the carrier is human serum albumin, keyhole limpet hemocyanin (KLH), immunoglobulin molecules, thyroglobulin, ovalbumin, influenza hemagglutinin, PAN-DR binding peptide (PADRE polypeptide), malaria circumsporozite (CS) protein, hepatitis B surface antigen (HBSAg19-28, Heat Shock Protein (HSP) 65, Bacillus Calmette-Guerin (BCG), cholera toxin, cholera toxin mutants with reduced toxicity, diphtheria toxin, CRM197 protein that is cross-reactive with diphtheria toxin, recombinant Streptococcal C5a peptidase, Streptococcus pyogenes ORF1224, Streptococcus pyogenes ORF1664, Streptococcus pyogenes ORF 2452, Chlamydia pneumoniae ORF T367, Chlamydia pneumoniae ORF T858, Tetanus toxoid, HIV gp120 T1, microbial surface components recognizing adhesive matrix molecules (MSCRAMMS), growth factor/hormone, cytokine or chemokine.

15. A vaccine composition comprising the peptide immunogen of claim 1.

16. The composition of claim 15, further comprising an adjuvant selected from the group consisting of mineral salts, GM-CSF, 529 SE, IL-12, aluminum phosphate, aluminum hydroxide, Mycobacterium tuberculosis, Bordetella pertussis, bacterial lipopolysaccharides, aminoalkyl glucosane phosphate compounds, MPL™ (3-O-deacylated monophosphoryl lipid A), a polypeptide, Quil A, STIMULON™ QS-21, a pertussis toxin (PT), an E. coli heat-labile toxin (LT), IL-1alpha, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon-alpha, interferon-β, interferon-gamma, G-CSF, TNF-alpha and TNF-β and Freund's complete adjuvant (FCA).

17. An immunogenic composition, comprising the peptide immunogen of claim 1, together with one or more pharmaceutically acceptable excipients, diluents, and/or adjuvants.

18. The immunogenic composition of claim 17, wherein the pharmaceutically acceptable adjuvant and/or carrier is selected from the group consisting of alum, liposyn, saponin, squalene, L121, emulsigen monophosphyryl lipid A (MPL), polysorbate 80, QS21, Montanide ISA51, ISA35, ISA206 and ISA 720.

19. A method of preventing or treating an influenza virus mediated disease by administrating to a mammal a peptide immunogen according to claim 1.

20. A kit comprising the peptide immunogen according to claim 1.

Patent History

Publication number: 20150125423
Type: Application
Filed: Jan 12, 2015
Publication Date: May 7, 2015
Inventors: Jennifer Mitcham (Redmond, WA), Matthew Moyle (Newtown, CT)
Application Number: 14/594,461