Donor Specific Antibody Libraries

Abstract

The present application concerns donor-specific antibody libraries derived from a patient donor who has suffered from, or is suffering from one or more diseases discussed herein. The present application also concerns the method of making and using the donor-specific antibodies. The present application further concerns the neutralizing antibodies obtained from the donor-specific antibody libraries and the methods of using these antibodies for the prevention/treatment of human disease.

Description

FIELD OF THE INVENTION

[0001] The present invention concerns donor-specific antibody libraries and methods of making and using thereof. The present invention also concerns neutralizing antibodies obtained from such donor-specific antibody libraries and methods of using the antibodies obtained for the prevention and/or treatment of various human diseases and conditions.

BACKGROUND OF THE INVENTION

[0002] The generation and identification of specific agents for the diagnosis, prevention, and treatment of human diseases requires access to vast collections of useful chemistries. With the advent and rapid development of a variety of techniques for the creation and screening of antibody libraries, monoclonal antibodies against disease targets have become one of the major categories of new drug candidates. Since for human use, in addition to specificity and efficacy, safety is of primary concern, libraries of human monoclonal antibodies have become of particular importance.

[0003] At present, the development of human antibody-based drug candidates are typically identified by screening of human antibody libraries comprising a random collection of antibody sequences from human repertoires that are typically unrelated to their intended application or applications. Each antibody library created from a specific human donor potentially contains antibodies to every component, physiology, and metabolic alteration stemming from, or creating, every unique challenge that the donor has encountered, challenged, and surmounted over the course of that individual's lifetime. As typical human antibody libraries made with the current approaches are constructed without the knowledge of the health history of donors, little is known of what would be expected in the resulting immunoglobulin repertoires.

[0004] Thus, it is of great interest to create antibody libraries from individuals who have successfully survived or are surviving an encounter with specific diseases because their resulting repertoires include antibodies that were used by the donor to defend specifically against a relevant disease. It is also important to provide methods for the efficient screening and handling of such libraries, including the ability to remove or isolate negative or positive elements, eliminate undesirable content, and produce human antibodies with improved properties.

[0005] The present invention addresses these needs by providing methods and means for the creation, screening and handling of donor-specific antibody libraries from individuals who have been exposed to and survived or are surviving an encounter with a specific target disease.

SUMMARY OF THE INVENTION

[0006] In one aspect, the invention concerns a vector collection comprising a repertoire of nucleic acid molecules encoding antibody light or heavy chains or fragments thereof, derived from a human patient donor who has suffered from, or is suffering from, a disease evoking antibody production to a target antigen, wherein the collection is identified with a unique barcode.

[0007] In one embodiment, the vector collection comprises a repertoire of nucleic acid molecules encoding antibody light chains or fragments thereof, such as antibody .lamda. light chains, or antibody .kappa. light chains, or fragments thereof.

[0008] In another embodiment, the vector collection comprises a repertoire of nucleic acid molecules encoding antibody heavy chains or fragments thereof.

[0009] In yet another embodiment, the barcode is a nucleotide sequence linked to or incorporated in the vectors present in the collection, and/or linked to or incorporated in the nucleic acid molecules encoding the antibody light or heavy chains or fragments thereof such that it does not interfere with the expression of the nucleic acid molecules.

[0010] Thus, the barcode may be contiguous non-coding nucleotide sequence of one to about 24 nucleotides, which may, for example, be linked to the 3' or 5' non-coding region of the nucleic acid molecules.

[0011] In a further embodiment, the barcode is a nucleotide sequence that is a coding sequence of one or more silent mutations incorporated into the nucleic acid molecules encoding the antibody light or heavy chains or fragments thereof.

[0012] In a still further embodiment, the barcode is a non-contiguous nucleotide sequence. At least part of the non-contiguous nucleotide sequence may be linked to or incorporated in the vectors present in the collection. Alternatively, at least part of the non-contiguous nucleotide sequence may be incorporated into the nucleic acid molecules encoding the antibody light or heavy chains or fragments thereof such that it does not interfere with the expression of said nucleic acid molecules.

[0013] In another embodiment, the barcode is a peptide or polypeptide sequence.

[0014] In a different embodiment, the vectors present in the vector collection are phagemid vectors, which may, for example, contain a bacteriophage gene III and a stop codon between the nucleic acid molecules encoding antibody light or heavy chains or fragments thereof and the bacteriophage III gene, and may have a barcode, such as a non-coding contiguous nucleotide sequence inserted in the untranslated region following the stop codon.

[0015] In another aspect, the invention concerns host cells comprising the vector collection of the present invention. The host cells may by eukaryotic or prokaryotic host cells, such as, for example, E. coli cells.

[0016] In a further aspect, the invention concerns a donor-specific antibody library comprising library members expressing a collection of antibodies or antibody fragments to a target antigen wherein the antibodies or antibody fragments are derived from a human donor who has suffered from, or is suffering from, a disease evoking antibody production to said target antigen, wherein said antibody library is identified with at least one unique barcode.

[0017] In one embodiment, the antibody heavy and light chains are separately identified each with a barcode unique to the human donor from whom it derived.

[0018] In another embodiment, the donor-specific antibody library is identified with one unique barcode.

[0019] In yet another embodiment, the antibodies or antibody fragments are composed of antibody heavy and light chains or fragments thereof encoded by nucleic acid molecules present in a vector.

[0020] In a further embodiment, the barcode is a nucleotide sequence linked to or incorporated in the vectors present in the library, and/or linked to or incorporated in the nucleic acid molecules encoding the antibody light or heavy chains or fragments thereof such that it does not interfere with the expression of the nucleic acid molecules.

[0021] In a still further embodiment, the barcode is a contiguous non-coding nucleotide sequence of one to about 24 nucleotides, which may, for example, be linked to the 3' or 5' non-coding region of the nucleic acid molecules.

[0022] In a different embodiment, the barcode is encoded by a coding sequence of one or more silent mutations incorporated into the nucleic acid molecules encoding the antibody light or heavy chains or fragments thereof.

[0023] In another embodiment, the barcode is encoded by a non-contiguous nucleotide sequence.

[0024] In a further embodiment, a least part of the non-contiguous sequence encoding the barcode is linked to or incorporated in the vectors present in the library.

[0025] In a still further embodiment, at least part of the non-contiguous sequence encoding the barcode is incorporated into the nucleic acid molecules encoding the antibody light or heavy chains or fragments thereof such that it does not interfere with the expression of such nucleic acid molecules.

[0026] In different embodiment, the barcode is a peptide or polypeptide sequence.

[0027] In another embodiment, vectors are phagemid vectors, may, for example, contain a bacteriophage gene III and a stop codon between the nucleic acid molecules encoding antibody light or heavy chains or fragments thereof and the bacteriophage II gene.

[0028] In yet another embodiment, the medical history of the human patient donor shows that the donor has suffered from, or is suffering from said disease. In some embodiments, it is independently confirmed that the human donor suffered from, or is suffering from the disease.

[0029] In an additional embodiment, the donor-specific antibody library is substantially devoid of antibodies and antibody fragments specifically binding antigens different from said target antigen.

[0030] In one embodiment, the target antigen is an influenza A virus, such as an isolate of influenza A virus H1, H2, H3, H5, H7, or H9 subtype.

[0031] In another embodiment, the library expresses at least one antibody or antibody fragment specifically binding to more than one influenza A virus subtype.

[0032] In yet another embodiment, the library expresses at least one antibody or antibody fragment binding to and neutralizing the H5N1 subtype of influenza virus A.

[0033] In a further embodiment, the human donor has suffered from, or is suffering from a disease selected from the group consisting of the diseases listed in Table 1 below.

[0034] In a still further embodiment, the antibody library expresses at least one antibody or antibody fragment binding to an antigen associated with the target disease.

[0035] In an additional embodiment, the antibody library expresses at least one antibody or antibody fragment binding to and neutralizing an antigen associated with the target disease.

[0036] The donor-specific antibody library may, for example, be a phage library, in an embodiment, it may contain sequences encoding more than 10.sup.6 different members of antibodies or antibody fragments, or more than 10.sup.9 different members of antibodies or antibody fragments.

[0037] In other embodiments, the donor-specific antibody library is, without limitation, a spore-display library, a ribosome display library, a mRNA display library, a microbial cell display library, a yeast display library, or a mammalian display library.

[0038] In an embodiment, the nucleic acid encoding the antibodies or antibody fragments present in the library is reverse-transcribed from mRNA extracted from lymphocytes of the human patient donor, where the lymphocytes may, for example, originate from bone marrow, blood, spleen, or lymph node.

[0039] If desired, a serological profile of said human donor may be generated prior to extraction of said mRNA.

[0040] Alternatively, or in addition, the medical history of the human donor, and optionally the donor's family, is examined prior to or following extraction of the mRNA.

[0041] In another aspect, the invention concerns a method of making a donor-specific library expressing a collection of antibodies or antibody fragments to a target antigen, comprising the steps of:

[0042] a) obtaining mRNA from lymphocytes of a human patient donor who has suffered from, or who is suffering from a disease evoking antibody production to said target antigen;

[0043] b) generating a collection of nucleic acids comprising sequences encoding an immunoglobulin repertoire of the patient by reverse transcription of said obtained mRNA; and

[0044] c) identifying the donor-specific library with an unique barcode labeling said nucleic acids.

[0045] The method may further comprise steps of generating a serological profile of said patient and/or examining medical history of the patient prior or subsequent to step a).

[0046] The method may further comprise the steps of d) inserting said nucleic acids into expression vector: e) expressing the immunoglobulin repertoire; and f) displaying the immunoglobulin repertoire in a display system.

[0047] In another embodiment, the method further comprises the step of selecting members of the library based their ability to neutralize or activate the target antigen.

[0048] In a still further embodiment, the method yields at least one neutralizing antibody.

[0049] In another embodiment, the method further comprises the step of creating one or more sub-libraries comprising library members that were found to neutralize or activate the target antigen.

[0050] In yet another embodiment, the method comprises the step of sequencing at least one library member identified.

[0051] In a further aspect, the invention concerns a method of treating or preventing a disease associated with a target antigen neutralized or activated by an antibody selected by the method described above, comprising administering to a human patient in need an effective amount of the antibody selected.

[0052] The antibody may, for example, be a neutralizing antibody, such as a neutralizing antibody to at least one influenza A virus subtype.

[0053] In another embodiment, the disease is selected from the group consisting of the diseases listed in Table 1 below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG. 1 is a flow chart schematically illustrating a typical method for the creatin of the human antibody libraries of the present invention.

[0055] FIG. 2 illustrates a typical panning enrichment scheme for increasing the reactive strength towards two different targets, A and B. Each round of enrichment increases the reactive strength of the pool towards the individual target(s).

[0056] FIG. 3 illustrates a strategy for the selection of clones cross-reactive with targets A and B, in which each successive round reinforces the reactive strength of the resulting pool towards both targets.

[0057] FIG. 4 illustrates a strategy for increasing the reactive strengths towards two different targets (targets A and B), by recombining parallel discovery pools to generate/increase cross-reactivity. Each round of selection of the recombined antibody library increases the reactive strength of the resulting pool towards both targets.

[0058] FIG. 5 illustrates a strategy for increasing cross-reactivity to a target B while maintaining reactivity to a target A. First, a clone reactive with target A is selected, then a mutagenic library of the clones reactive with target A is prepared, and selection is performed as shown, yielding one or more antibody clones that show strong reactivity with both target A and target B.

[0059] FIG. 6 illustrates a representative mutagenesis method for generating a diverse multifunctional antibody collection by the "destinational mutagenesis" method.

[0060] FIG. 7 shows the amino acid sequences of 15 known hemagglutinin (H) protein subtypes.

[0061] FIG. 8 shows the H5 hemagglutinin (HA) serology results for blood samples obtained from six human survivors of a Turkish H5N1 bird flu outbreak. The data demonstrate the presence of antibodies to the HA antigen.

[0062] FIG. 9 shows serology results obtained with serum samples of twelve local donors, tested on H5 antigen (A/Vietnam/1203/2004) and H1N1 (A/New Caledonia/20/99) and H3N2 (A/Panama/2007/99) viruses.

[0063] FIG. 10 illustrates the unique barcoding approach used in the construction of antibody phage libraries.

[0064] FIG. 11 shows the analysis of antibody binding to hemagglutinins from different influenza A subtypes.

[0065] FIG. 12 shows the positions of H5 hemagglutinin binding Group 1 required and dominant mutations on the crystal structure at Fab 47e.

[0066] FIGS. 13 and 14 illustrate the use of destinational mutagenesis to create diverse antibody heavy and light chain libraries using the antibody heavy and light chain sequences identified by analysis of sera and bone marrow of Turkish bird flu survivors.

DETAILED DESCRIPTION

A. Definitions

[0067] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), provides one skilled in the art with a general guide to many of the terms used in the present application.

[0068] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0069] The phrase "conserved amino acid residues" is used to refer to amino acid residues that are identical between two or more amino acid sequences aligned with each other.

[0070] The terms "disease," "disorder" and "condition" are used interchangeably herein, and refer to any disruption of normal body function, or the appearance of any type of pathology. The etiological agent causing the disruption of normal physiology may or may not be known. Furthermore, although two patients may be diagnosed with the same disorder, the particular symptoms displayed by those individuals may or may not be identical.

[0071] An "effective amount" is an amount sufficient to effect beneficial or desired therapeutic (including preventative) results. An effective amount can be administered in one or more administrations.

[0072] A "composition," as used herein, is defined as comprising an active ingredient, such as a neutralizing antibody generated from the present invention, and at least one additive, such as a pharmaceutically acceptable carrier, including, without limitation, water, minerals, proteins, and/or other excipients known to one skilled in the art.

[0073] As used herein, the term "treating" or "treatment" is intended to mean an amelioration of a clinical symptom indicative of a disease.

[0074] As used herein, the term "preventing" or "prevention" is intended to mean a forestalling of a clinical symptom indicative of a disease

[0075] The terms "subject" and "patient," as used herein, are used interchangeably, and can refer to any to animal, and preferably a mammal, that is the subject of an examination, treatment, analysis, test or diagnosis. Thus, subjects or patients include humans, non-human primates and other mammals, who may or may not have a disease or other pathological condition.

[0076] The term "amino acid" or "amino acid residue" typically refers to an amino acid having its art recognized definition such as an amino acid selected from the group consisting of: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile): leucine (Leu): lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val) although modified, synthetic, or rare amino acids may be used as desired. Thus, modified and unusual amino acids listed in 37 CFR 1.822(b)(4) are included within this definition and expressly incorporated herein by reference. Amino acids can be subdivided into various sub-groups. Thus, amino acids can be grouped as having a nonpolar side chain (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a positively charged side chain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr). Amino acids can also be grouped as small amino acids (Gly, Ala), nucleophilic amino acids (Ser, His, Thr, Cys), hydrophobic amino acids (Val, Leu. Ile. Met, Pro), aromatic amino acids (Phe, Tyr, Trp, Asp, Glu), amides (Asp, Glu), and basic amino acids (Lys, Arg).

[0077] The term "variant" with respect to a reference polypeptide refers to a polypeptide that possesses at least one amino acid mutation or modification (i.e., alteration) as compared to a native polypeptide. Variants generated by "amino acid modifications" can be produced, for example, by substituting, deleting, inserting and/or chemically modifying at least one amino acid in the native amino acid sequence.

[0078] An "amino acid modification" refers to a change in the amino acid sequence of a predetermined amino acid sequence. Exemplary modifications include an amino acid substitution, insertion and/or deletion.

[0079] An "amino acid modification at" a specified position, refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent to the specified residue. By insertion "adjacent" to a specified residue is meant insertion within one to two residues thereof. The insertion may be N-terminal or C-terminal to the specified residue.

[0080] An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence with another different "replacement" amino acid residue. The replacement residue or residues may be "naturally occurring amino acid residues" (i.e. encoded by the genetic code) and selected from the group consisting of: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His): isoleucine (Ile): leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). Substitution with one or more non-naturally occurring amino acid residues is also encompassed by the definition of an amino acid substitution herein.

[0081] A "non-naturally occurring amino acid residue" refers to a residue, other than those naturally occurring amino acid residues listed above, which is able to covalently bind adjacent amino acid residues(s) in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine and other amino acid residue analogues such as those described in Ellman et al. Meth. Enzym. 202:301 336 (1991). To generate such non-naturally occurring amino acid residues, the procedures of Noren et al. Science 244:182 (1989) and Ellman et al., supra, can be used. Briefly, these procedures involve chemically activating a suppressor tRNA with a non-naturally occurring amino acid residue followed by in vitro transcription and translation of the RNA.

[0082] An "amino acid insertion" refers to the incorporation of at least one amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, the present application contemplates larger "peptide insertions", e.g. insertion of about three to about live or even up to about ten amino acid residues. The inserted residue(s) may be naturally occurring or non-naturally occurring as disclosed above.

[0083] An "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0084] The term "polynucleotide(s)" refers to nucleic acids such as DNA molecules and RNA molecules and analogs thereof (e.g., DNA or RNA generated using nucleotide analogs or using nucleic acid chemistry). As desired, the polynucleotides may be made synthetically, e.g. using art-recognized nucleic acid chemistry or enzymatically using, e.g., a polymerase, and, if desired, be modified. Typical modifications include methylation, biotinylation, and other art-known modifications. In addition, the nucleic acid molecule can be single-stranded or double-stranded and, where desired, linked to a detectable moiety.

[0085] The term "mutagenesis" refers to unless otherwise specified, any art recognized technique for altering a polynucleotide or polypeptide sequence. Preferred types of mutagenesis include error prone PCR mutagenesis, saturation mutagenesis, or other site directed mutagenesis.

[0086] "Site-directed mutagenesis" is a technique standard in the art, and is conducted using a synthetic oligonucleotide primer complementary to a single-stranded phage DNA to be mutagenized except for limited mismatching, representing the desired mutation. Briefly, the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the single-stranded phage DNA, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells that harbor the phage. Theoretically, 50% of the new plaques will contain the phage having, as a single strand, the mutated form; 50% will have the original sequence. Plaques of interest are selected by hybridizing with kinased synthetic primer at a temperature that permits hybridization of an exact match, but at which the mismatches with the original strand are sufficient to prevent hybridization. Plaques that hybridize with the probe are then selected, sequenced and cultured, and the DNA is recovered.

[0087] The term "vector" is used to refer to a rDNA molecule capable of autonomous replication in a cell and to which a DNA segment, e.g., gene or polynucleotide, can be operatively linked so as to bring about replication of the attached segment. Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to herein as "expression vectors. The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0088] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0089] Percent amino acid sequence identity may be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtained from the National Institute of Health. Bethesda, Md. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask=yes, strand=all, expected occurrences=10, minimum low complexity length=15/5, multi-pass e-value=0.01, constant for multi-pass=25, dropoff for final gapped alignment=25 and scoring matrix=BLOSUM62.

[0090] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" are used herein to refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express FcRs (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. Various immune cells express different Fc receptors (FcRs). Thus, the primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII.

[0091] The terms "influenza A subtype" or "influenza A virus subtype" are used interchangeably, and refer to influenza A virus variants that are characterized by various combinations of the hemagglutinin (H) and neuraminidase (N) viral surface proteins, and thus are labeled by a combination of an H number and an N number, such as, for example, H1N1 and H3N2. The terms specifically include all strains (including extinct strains) within each subtype, which usually result from mutations and show different pathogenic profiles. Such strains will also be referred to as various "isolates" of a viral subtype, including all past, present and future isolates. Accordingly, in this context, the terms "strain" and "isolate" are used interchangeably.

[0092] The term "influenza" is used to refer to a contagious disease caused by an influenza virus.

B. General Techniques

[0093] Techniques for performing the methods of the present invention are well known in the art and described in standard laboratory textbooks, including, for example, Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997); Molecular Cloning: A Laboratory Manual. Third Edition, J. Sambrook and D. W. Russell, eds., Cold Spring Harbor, N.Y., USA, Cold Spring Harbor Laboratory Press, 2001; O'Brian et al., Analytical Chemistry of Bacillus Thuringiensis, Hickle and Fitch, eds., Am. Chem. Soc. 1990; Bacillus thuringiensis: biology, ecology and safety, T. R. Glare and M. O'Callaghan, eds., John Wiley, 2000; Antibody Phage Display, Methods and Protocols, Humana Press, 2001; and Antibodies, G. Subramanian, ed., Kluwer Academic, 2004. Mutagenesis can, for example, be performed using site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488-492 (1985)). PCR amplification methods are described in U.S. Pat. Nos. 4,683,192, 4,683,202, 4,800,159, and 4,965,188, and in several textbooks including "PCR Technology: Principles and Applications for DNA Amplification", H. Erlich, ed., Stockton Press, New York (1989); and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, San Diego, Calif. (1990).

[0094] The methods of the present invention are not limited by any particular technology used for the display of antibodies. Although the invention is illustrated with reference to phage display, antibodies of the present invention can also be identified by other display and enrichment technologies, such as, for example, ribosome or mRNA display (Mattheakis et al., Proc. Natl. Acad. Sci. USA 91:9022-9026 (1994); H-lanes and Pluckthun, Proc. Natl. Acad. Sci. USA 94:4937-4942 (1997)), microbial cell display, such as bacterial display (Georgiou et al., Nature Biotech. 15:29-34 (1997)), or yeast cell display (Kieke et al., Protein Eng. 10:1303-1310 (1997)), display on mammalian cells, spore display (Isticato et al., J. Bacteriol. 183:6294-6301 (2001); Cheng et al., Appl. Environ. Microbiol. 71:3337-3341 (2005) and co-pending provisional application Ser. No. 60/955,592, filed Aug. 13, 2007) viral display, such as retroviral display (Urban et al. Nucleic Acids Res. 33:e35 (2005), display based on protein-DNA linkage (Odegrip et al., Proc. Acad. Natl. Sci. USA 101:2806-2810 (2004); Reiersen et al., Nucleic Acids Res. 33:e10 (2005)), and microbead display (Sepp et al. FEBS Lett. 532:455-458 (2002)).

[0095] In ribosome display, the antibody and the encoding mRNA are linked by the ribosome, which at the end of translating the mRNA is made to stop without releasing the polypeptide. Selection is based on the ternary complex as a whole.

[0096] In a mRNA display library, a covalent bond between an antibody and the encoding mRNA is established via puromycin, used as an adaptor molecule (Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750-3755 (2001)). For use of this technique to display antibodies, see, e.g. Lipovsek and Pluckthun, J. Immunol. Methods. 290:51-67 (2004).

[0097] Microbial cell display techniques include surface display on a yeast, such as Saccharomyces cerevisiae (Boder and Wittrup. Nat. Biotechnol. 15:553-557 (1997)). Thus, for example antibodies can be displayed on the surface of S. cerevisiae via fusion to the .alpha.-agglutinin yeast adhesion receptor, which is located on the yeast cell wall. This method provides the possibility of selecting repertoires by flow cytometry. By staining the cells by fluorescently labeled antigen and an anti-epitope tag reagent, the yeast cells can be sorted according to the level of antigen binding and antibody expression on the cell surface. Yeast display platforms can also be combined with phage (see, e.g., Van den Beucken et al., FEBS Leit. 546:288-294 (2003)).

[0098] For a review of techniques for selecting and screening antibody libraries see, e.g. Hoogenboom, Nature Biotechnol. 23(9): 1105-1116 (2005).

C. Detailed Description of Preferred Embodiments

[0099] I. Preparation of Donor-Specific Antibody Libraries

[0100] The present invention concerns donor-specific antibody libraries from individuals who have successfully survived or are surviving an encounter with a specific disease. The resulting antibody repertoires will include antibodies that were used by the donor to defend specifically against a relevant disease and thus are important tools, for example, for developing neutralizing antibodies for the prevention and/or treatment of a target disease.

[0101] While the present invention is applicable to any target disease that evokes antibody production in a human subject, representative, non-limiting, examples of such diseases are listed in Table 1.

TABLE-US-00001 TABLE 1 Type of disorder Representative examples infectious disorder Influenza viral infection, hepatitis C virus (HCV) infection, herpes simplex virus (HSV) infection, human immunodeficiency virus (HIV) infection, Methicillin-resistant Staphylococcus aureus (MRSA) infection, Epstein-Barr virus (EBV) infection, respiratory syncytial virus (RSV) infection, Pseudomonas. Candida infections Respiratory disorder Asthma, Allergies, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), adult respiratory distress syndrome (ARDS) metabolic disorder Frailty, cachexia, sarcopenia, Obesity, type II diabedyslipidemia, metabolic syndrome- associated myocardial infarction (MI), chronic renal failure (CRF), osteoporosis digestive disorder irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), Chron's disease, fatty liver disease, fibrosis, drug-induced liver disease Neurological disorder Alzheimer's disease, multiple sclerosis (MS), Parkinson's disease, bovine spongiform encephalopathy (BSE, mad cow disease) Cancer e.g., breast, renal, stomach, melanoma, lung, colon, glioma, lymphoma

[0102] A method of creating the donor-specific libraries of the present invention is schematically illustrated in FIG. 1. As a first step, potential donors are identified. The patient donor may currently suffer from or may have recovered from and survived a target disease. Thus, for example, as illustrated in the Examples, the donor-specific libraries herein may be created from the bone marrow of convalescent patients of prior influenza infections, including seasonal influenza outbreaks, epidemics, and pandemics.

[0103] When selecting or identifying a patient donor, it is important to confirm that the patient indeed had or is having the target disease. Part of the confirmation is the examination of the medical history of the patient donor. In addition to the medical history, various other factors, such as the medical history of the patient's family, the patient's sex, weight, health state, etc., should be taken into consideration. If the patient history is not available or unreliable, or for any other reason such as a further confirmation measure the serological profile of the patient may be determined. Serological assays are well known in the art and can be performed in various formats, such as in the form of various ELISA assay formats. Thus, for example, the presence of antibodies to an influenza virus can be detected by the well-known hemagglutinin inhibition (HAI) assay (i, A. P., M. S. Pereira, and J. J. Skehel. 1982. Concepts and procedures for laboratory-based influenza surveillance. U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control, Atlanta. Ga.), or the microneutralization assay (Harmon et al., J. Clin. Microbiol. 26:333-337 (1988)). This step might not be necessary if the serum sample has already been confirmed to contain influenza neutralizing antibodies.

[0104] In order to prepare donor-specific human antibody libraries, samples containing lymphocytes are collected from individuals (patient donors) known to have developed a target disease such as at least one disease from those listed in Table 1. The sample may for example, derive from bone marrow blood, spleen, lymph nodes, tonsils, thymus and the like. Bone marrow is a preferred source of the antibody libraries herein since it represents the complete "fossil archive" of individual donor's mature antibody repertoire.

[0105] Samples containing lymphocytes can be collected from the patient donor at various time points. In one embodiment, lymphocytes are collected from a patient who has recovered from the targeted disease(s) at least for 1, 5, 10, 15, 20, 25 days, at least for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months, or at least for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years. In another embodiment, lymphocytes are collected from a patient who is having the targeted disease(s) at the time of collection, and has been diagnosed as having the disease(s) at least 1, 5, 10, 15, 20, 25 days or at least 1, 2, 3, 4, 5, 6, 8, 9, 10 months, or 1, 2, 3, 4, or 5 years prior to collection.

[0106] Peripheral blood samples, especially from geographically distant sources, may need to be stabilized prior to transportation and use. Kits for this purpose are well known and commercially available, such as, for example, BD Vacutainer.RTM. CPT.TM. cell preparation tubes can be used for centrifugal purification of lymphocytes, and guanidium. Trizol, or RNAlater used to stabilize the samples. Methods and kits for isolating lymphocytes from other sources, such as lymphoid organs are also well known and commercially available.

[0107] Upon receipt of the stabilized lymphocytes or whole bone marrow, RNA is extracted and RT-PCR is performed to rescue antibody heavy and light chain repertoires, using immunoglobulin oligo primers known in the art.

[0108] Methods for preparation of RNA from bone marrow lymphocytes, or lymphocytes from any other source are well known in the art. General methods for mRNA extraction are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). RNA purification kits are available from commercial manufacturers, such as Qiagen, and can be used according to the manufacturer's instructions.

[0109] Since RNA cannot serve as a template for PCR, it is first reverse transcribed into cDNA, which is subjected to PCR amplification. The two most commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling. For example, extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer. CA, USA), following the manufacturer's instructions. The derived cDNA can then be used as a template in the subsequent PCR reaction.

[0110] In order to create phage-display libraries, the PCR repertoire products may be combined with linker oligos to generate scFv libraries to clone directly in frame with m13 pIII protein, following procedures known in the art. Libraries using other display techniques, such as those discussed above, can be prepared by methods well known in the art.

[0111] In a typical protocol, whole RNA is extracted by Tri BD reagent (Sigma) from fresh or RNAlater stabilized tissue. Subsequently, the isolated donor total RNA is further purified to mRNA using Oligotex purification (Qiagen). Next first strand cDNA synthesis, is generated by using random nonamer oligonucleotides and or oligo (dT).sub.18 primers according to the protocol of AccuScript reverse transcriptase (Stratagene). Briefly, 100 ng mRNA, 0.5 mM dNTPs and 300 ng random nonamers and or 500 ng oligo (dT).sub.18 primers in Accuscript RT buffer (Stratagene) are incubated at 65.degree. C. for 5 min, followed by rapid cooling to 4.degree. C. Then, 100 mM DTT, Accuscript RT, and RNAse Block are added to each reaction and incubated at 42.degree. C. for 1 h, and the reverse transcriptase is inactivated by heating at 70.degree. C. for 15 minutes. The cDNA obtained can be used as a template for RT-PCR amplification of the antibody heavy and light chain V genes, which can then be cloned into a vector, or, if phage display library is intended, into a phagemid vector. This procedure generates a repertoire of antibody heavy and light chain variable region clones (V.sub.H and V.sub.L libraries), which can be kept separate or combined for screening purposes. The vector, such as a phagemid vector, can then be introduced into a host cell, such as an E coli host, to generate a vector collection comprising a repertoire of nucleic acid molecules encoding antibody light chains or heavy chains or fragments thereof. In each case, the vector collection may comprise a single or more than one antibody light chain or heavy chain subtype. Thus, the vector collection may comprise sequences encoding antibody .kappa. and/or .lamda. light chains.

[0112] In the methods of the present invention, typically antibody light chains and antibody heavy chains are at first cloned separately, as discussed above, also separating the .kappa. and .lamda. light chain libraries. The libraries can be archived, and, when needed, the heavy chain library can be combined with the segregated .kappa. and .lamda. light chain libraries and heavy and light chain pairings can be identified, e.g. by panning, in the case of phage display. It is possible to repeat these steps multiple times with various libraries or sub-libraries, depending on the goal to be attained. The methods of the present invention provide great flexibility in including or excluding libraries, sub-libraries or clones, as needed during panning in order to maximize success.

[0113] In particular, because the sequences present in the vector collection harbor the coding sequences of the antibody heavy and light chains (or fragments) separately, the sequences may be excised and inserted into one or more expression vectors for expression of the antibody heavy and light chains, or fragments thereof. Preferably, the coding sequences of the antibody heavy and light chains, or fragments thereof, are inserted into the same expression vector for coexpression of the heavy and light chains to produce the library of the antibodies or antibody fragments.

[0114] The expression vectors of the present invention contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2.mu. plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.

[0115] Expression vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

[0116] An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibodies-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al. Proc. Natl. Acad. Sci. USA. 77:4216 (1980). A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].

[0117] Expression and cloning vectors usually contain a promoter operably linked to the antibody-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the .beta.-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding antibodies

[0118] Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

[0119] Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73.657.

[0120] Transcription of the heavy chain or light chain genes in the expression vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.

[0121] Transcription of a DNA encoding the antibody genes by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, .alpha.-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the antibody coding sequence, but is preferably located at a site 5' from the promoter.

[0122] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding antibody heavy and light chains.

[0123] Still other methods, vectors, and host cells suitable for adaptation to the synthesis of polypeptide, in recombinant vertebrate cell culture are described in Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

[0124] In a particular embodiment, the antibody library is produced in the form of a phage library, where the coding sequences of the antibody heavy and light chains, or fragments thereof, are cloned into a phagemid vector, such as a vector comprising the bacteriophage gene III. Phagemid vectors are well known and commercially available, including, for example, the pBluescript vector SKII+(Stratagene, Genbank Accession X52328), and other pBluscript vectors. Phage display technology enables the generation of large repertoires of human antibodies, and the biopanning procedure allows the selection of individual antibodies with any desired specificity or other properties.

[0125] For example, immunoglobulin repertoires from peripheral lymphocytes of survivors of earlier epidemics and pandemics, such as the 1918 Spanish Flu, can be retrieved, stabilized, rescued and expressed in a manner similar to that described above. For additional H1 and H3 libraries, repertoires can be recovered from properly timed vaccinated locally-sourced donors. As an additional option, commercially available bone marrow total RNA or mRNA can be purchased from commercial sources to produce libraries suitable for H1 and H3, and, depending upon the background of donor, also suitable for H2 antibody screening. In general, for target diseases where vaccination is an available treatment option, antibodies can be isolated from biological samples obtained from immunized human donors as well. From immunized patients that have developed titers of antibody recognizing the particular antigen, bone marrow, blood, or another source of lymphocytes, is collected, and antibodies produced are isolated, amplified and expressed as described above.

[0126] As discussed above, for each donor, antibody light and heavy chain libraries can be cloned separately. Thus, for each donor, various .kappa. and .lamda.light chain families can be separately pooled and cloned in equimolar amounts. Similarly, for each donor, various heavy chain families can be pooled and cloned in equimolar amounts. By enabling gene family specific rescue of antibodies, the methods of the present invention yield libraries more completely representing the antibody repertoire of the donor, including antibodies that are less abundant and, in the case of pooled antibody libraries, guaranteeing immunoglobulin contributions from any and all individuals. For example, as illustrated in Example 1 by the Examples, in preparing the influenza heavy and light chain libraries herein, 6 .kappa. light chain families, 11.lamda. light chain families and 4 heavy chain families were rescued.

[0127] A typical screen can yield zero, one, or more than one target specific positive clone(s). If a particular combinatorial antibody library or libraries have been exhaustively screened and no further solutions seem attainable this may not be a failure of the heavy and light chain repertoire(s) ability to bind target, but rather the collection may have failed to bring together the necessary heavy and light chain pair required to bind target. A typical rescued repertoire of light chains from any individual may contain between about 10.sup.5-10.sup.6 unique light chains and between about 10.sup.6-10.sup.8 unique heavy chains. The possible combinatorial products of such pairings range from 10.sup.11 to 10.sup.14. Such a collection exceeds the practical limits of most display systems, such as phage display, by several orders of magnitude. Consequently, with current display technologies (such as phage display), only a fraction of the combinatorial possibilities are captured and assessed in any single phage antibody library. Therefore, recloning the original set of heavy chains with the original collection of light chains will generate an entirely new set of shuffled heavy and light chain combinations with likely novel antibodies to a particular target. Such newly reshuffled collections were found to transform previously existing poorly performing donor specific libraries into highly productive collections. Specifically, for a collection from a single donor previously only 0.3.times.-fold enrichment could be achieved compared to background after three rounds of selection. However, when this collection was recloned and reshuffled it became capable of 15-fold enrichment following 3 rounds of panning resulting in 55 novel sequences from 92 selected clones.

[0128] As mentioned previously, a typical screen can yield any number of target specific positive clones. The present invention enables the identification of the origins of any clone by their embedded barcode. As a typical antibody screen may combine phage antibodies from numerous donor specific libraries it is possible that some of the libraries and their combinatorial clones are not completely represented as antibody bearing phage particles. In which case a positive clone may have resulted from only a limited physical set of all the possible cloned solutions present in the sub-library phage population being screened. In such an instance it is of considerable interest to more fully interrogate the collection of donor specific phage. In this case the barcode from a positive clone guides one to the specific library responsible for the clone and allows to exclusively and more deeply screen the collection of interest.

[0129] In instances where desired antibodies must have functional capabilities beyond those initially used as the basis for the initial library construction, such as neutralization, or activation, we can prospectively profile individual donor sera for evidence of such activities. If the desired activities are present at reasonable titers in any particular donor sera, one can select those corresponding libraries are selected to screen against the target of interest. In other instances the relevant selection criteria may be unrelated to serology, but related to donor characteristics such as age, gender, or medical histories. In any event, donor profiles are logical guides for library selection and possible only in donor specific and segregated antibody libraries.

[0130] It is not unusual to complete a phage panning screen and discover the presence of immunodominant clones. Furthermore, it is also not unusual to rediscover such clone upon repeated panning screening regimens. In the case of a dominant clone or clones, where either more or different clones are desirable, it is important to avoid the library material responsible for the presence of this clone. In typical phage antibody libraries the specific library or materials responsible for the clones origin are not separable from the collection, however in donor specific libraries it is possible to rescreen the libraries and simply omit the undesirable donor sublibrary or sublibraries, thereby forcing positive selection away from previously identified dominant clones.

[0131] Although, for simplicity, the libraries are described as heavy or light chain libraries, it will be apparent to those of ordinary skill in the art that the same description applies to the libraries of antibody fragments, fragments of antibody heavy and/or light chains, and libraries of antibody-like molecules.

[0132] In a particular embodiment, antibodies with dual specificities, such as, for example, showing reactivity with two different influenza A subtypes and/or with two strains (isolates) of the same subtype, and/or with human and non-human isolates, can be discovered and optimized through controlled cross-reactive selection and/or directed combinatorial and/or mutagenic engineering.

[0133] In a typical enrichment scheme, illustrated in FIG. 2, a library including antibodies showing cross-reactivity to two targets, designated as targets A and B, are subjected to multiple rounds of enrichment. If enrichment is based on reactivity with target A, each round of enrichment will increase the reactive strength of the pool towards target A. Similarly, if enrichment is based on reactivity with target B, each round of enrichment will increase the reactive strength of the pool towards target B. Although FIG. 2 refers to panning, which is the selection method used when screening phage display libraries (see below), the approach is equally applicable to any type of library discussed above, or otherwise known in the art, and to any type of display technique. Targets A and B include any targets to which antibodies bind, including but not limited to various isolates, types and sub-types of influenza viruses.

[0134] If the goal is to identify neutralizing antibodies with multiple specificities, a cross-reactive discovery selection scheme can be used. In the interest of simplicity, this scheme is illustrated in FIG. 3 showing the selection of antibodies with dual specificities. In this case, an antibody library including antibodies showing reactivity with two targets, targets A and B, is first selected for reactivity with one of the targets, e.g., target A, followed by selection for reactivity with the other target, e.g., target B. Each successive selection round reinforces the reactive strength of the resulting pool towards both targets. Accordingly, this method is particularly useful for identifying antibodies with dual specificity. Of course, the method can be extended to identifying antibodies showing reactivity towards further targets, by including additional rounds of enrichment towards the additional target(s). Again, if the library screened is a phage display library, selection is performed by cross-reactive panning, but other libraries and other selection methods can also be used.

[0135] A combination of the two methods discussed above includes two separate enrichment rounds for reactivity towards target A and target B, respectively, recombining the two pools obtained, and subsequent cross-reactive selection rounds, as described above. This approach is illustrated in FIG. 4. Just as in the pure cross-reactive selection, each round of selection of the recombined library increases the reactive strength of the resulting pool towards both targets.

[0136] In a further embodiment, illustrated in FIG. 5, first a clone showing strong reactivity with a target A, and having detectable cross-reactivity with target B is identified. Based on this clone, a mutagenic library is prepared, which is then selected, in alternating rounds, for reactivity with target B and target A respectively. This scheme will result in antibodies that maintain strong reactivity with target A, and have increased reactivity with target B. Just as before, selection is performed by panning, if the libraries screened are phage display libraries, but other libraries, other display techniques, and other selection methods can also be used, following the same strategy.

[0137] As discussed above, targets A and B can, for example, be two different subtypes of the influenza A virus, two different strains (isolates) of the same influenza A virus, subtypes or isolates from two different species, where one species is preferably human. Thus, for example, target A may be an isolate of the 2004 Vietnam isolate of the H5N1 virus, and target B may be a 1997 Hong Kong isolate of the H5N1 virus. It is emphasized that these examples are merely illustrative, and antibodies with dual and multiple specificities to any two or multiple targets can be identified, selected and optimized in an analogous manner.

[0138] Once neutralizing antibodies with the desired properties have been identified, it might be desirable to identify the dominant epitope or epitopes recognized by the majority of such antibodies. Methods for epitope mapping are well known in the art and are disclosed, for example, in Morris, Glenn E., Epitope Mapping Protocols, Totowa, N. J. ed., Humana Press, 1996: and Epitope Mapping: A Practical Approach, Westwood and Hay. eds. Oxford University Press, 2001.

[0139] II. Identifying Donor-Specific Antibody Library with Unique Barcoding

[0140] According to the present invention, following amplification of the antibody heavy and light chain repertoires from cDNA, such as bone marrow cDNA, prepared as described above, preferably antibody heavy and light chain libraries are cloned separately for each patient donor, where the individual libraries can be distinguished using unique barcodes.

[0141] The barcodes preferably are selected such that they are capable of propagating along with the clone(s) labeled, without interfering with the expression of the desired antibody chain or fragment thereof. In an exemplary embodiment of the present invention, the barcode is inserted into the sequence of the expression vector, preferably, the 3' untranslated region following the terminal pIII stop codon when a phagemaid vector is used. Upon clonal isolation, a vector's unique sequence is determined and subsequently dedicated to a single defined library. This defined library can be derived not only from a single donor, but also from discrete pools of donors, or a synthetic repertoire or a semi-synthetic collection. In another embodiment, the barcode is inserted into the coding sequence of the antibody heavy and/or light chain or fragment thereof, at a position or in a form that does not interfere with the expression of the respective chains.

[0142] Thus the barcodes can be non-coding DNA sequences of about 1-24 contiguous non-coding nucleotides in length that can be deconvoluted by sequencing or specific PCR primers. This way, a collection of nucleic acids, such as an antibody repertoire, can be linked at the cloning step. In a exemplary embodiment of the present invention, the barcode is 3 or bases of randomly generated sequence.

[0143] In another example, the barcodes are coding sequences of silent mutations. If the libraries utilize restriction enzymes that recognize interrupted palidromes (e.g. Sfi GGCCNNNNNGGCC), distinct nucleotides can be incorporated in place of the "N's" to distinguish various collections of clones, such as antibody libraries. This barcoding approach has the advantage that the repertoire is linked at the amplification step.

[0144] In a further embodiment, the barcodes are non-contiguous nucleotide sequences, which may be present in the vector sequence and/or the coding sequence of the desired antibody chain. Thus, a barcode with a non-contiguous sequence provides a great degree of flexibility in identifying the origins of the various individual sequences, and monitoring their subsequent handling.

[0145] In a different example, the barcodes are coding sequences that encode immunologically distinct peptide or protein sequences fused to phage particles. Examples include, for example, epitope (e.g. Myc, HA, FLAG) fusions to pIII, pVIII, pVII, or pIX phages. The epitopes can be used singly or in various combinations, and can be provided in cis (on the library-encoding plasmid) or in trans (specifically modified helper phage) configuration.

[0146] Other examples of possible barcodes include, without limitation, chemical and enzymatic phage modifications (for phage libraries) with haptens or fluorescent chromophores. Such tags are preferred for a single round of selection.

[0147] The individual heavy and light chain libraries obtained from individual donors, or other barcoded clone or collections, can be pooled, without losing the ability to distinguish the source of individual sequences.

[0148] III. Optimizing Neutralizing Antibodies from the Donor-Specific Antibody Libraries

[0149] If desired, cross-reactivity of the neutralizing antibodies with dual or multiple specificity can be further improved by methods known in the art, such as, for example, by Look Through Mutagenesis (LTM), as described in US. Patent Application Publication No. 20050136428, published Jun. 23, 2005, the entire disclosure of which is hereby expressly incorporated by reference.

[0150] Look-through mutagenesis (LTM) is a multidimensional mutagenesis method that simultaneously assesses and optimizes combinatorial mutations of selected amino acids. The process focuses on a precise distribution within one or more complementarity determining region (CDR) domains and explores the synergistic contribution of amino acid side-chain chemistry. LTM generates a positional series of single mutations within a CDR where each wild type residue is systematically substituted by one of a number of selected amino acids. Mutated CDRs are combined to generate combinatorial single-chain variable fragment (scFv) libraries of increasing complexity and size without becoming prohibitive to the quantitative display of all variants. After positive selection, clones with improved properties are sequenced, and those beneficial mutations are mapped. To identify synergistic mutations for improved binding properties, combinatorial libraries (combinatorial beneficial mutations, CBMs) expressing all beneficial permutations can be produced by mixed DNA probes, positively selected, and analyzed to identify a panel of optimized scFv candidates. The procedure can be performed in a similar manner with Fv and other antibody libraries.

[0151] Mutagenesis can also be performed by walk-through mutagenesis (WTM), as described above.

[0152] Another useful mutagenic method to intentionally design cross-reactivity of the antibodies herein with more than one influenza A subtype and/or more than one isolate of the same subtype, is referred herein as "destinational" mutagenesis. Destinational mutagenesis can be used to rationally engineer a collection of antibodies based upon one or more antibody clones, preferably of differing reactivities. In the context of the present invention, destinational mutagenesis is used to encode single or multiple residues defined by analogous positions on like sequences such as those in the individual CDRs of antibodies. In this case, these collections are generated using oligo degeneracy to capture the range of residues found in the comparable positions. It is expected that within this collection a continuum of specificities will exist between or even beyond those of the parental clones. The objective of destinational mutagenesis is to generate diverse multifunctional antibody collections, or libraries, between two or more discrete entities or collections. To create a destinational mutagenesis library, the CDR sequences for both antibodies are first attained and aligned. Next all positions of conserved identity are fixed with a single codon to the matched residue. At non-conserved positions a degenerate codon is incorporated to encode both residues. In some instances the degenerate codon will only encode the two parental residues at this position. However, in some instances additional co-products are produced. The level of co-product production can be dialed in to force co-product production or eliminate this production dependent upon size limits or goals.

[0153] Thus, for example, if the first position of the two antibodies respectively are threonine and alanine, the degenerate codon with A/G-C- in the first two positions would only encode threonine or alanine, irrespective of the base in the third position. If, for example, the next position residues are lysine and arginine the degenerate codon A-A/G-A/G will only encode lysine or arginine. However, if the degenerate codon A/C-A/G-A/G/C/T were used then asparagine, histidine, glutamine, and serine coproducts will be generated as well.

[0154] As a convenience it is simpler to use only antibodies with matched CDR lengths. One way to force this is to screen a size restricted library for the second antigen, based on the CDR length and potentially even framework restrictions imparted by the initially discovered antibody. It is noted, however, that using CDRs of equal length is only a convenience and not a requirement. It is easy to see that, while this method will be useful to create large functionally diverse libraries of influenza A virus neutralizing antibodies, its applicability is much broader. This mutagenesis technique can be used to produce functionally diverse libraries or collections of any antibody. Thus, FIG. 6 is included herein to illustrate the use of the destinational mutagenesis method using CDRs of a TNF-.alpha. antibody and a CD11a antibody as the parental sequences mutagenized.

[0155] Other exemplary mutagenesis methods include saturation mutagenesis and error prone PCR.

[0156] Saturation mutagenesis (Hayashi et al., Biotechniques 17:310-315 (1994)) is a technique in which all 20 amino acids are substituted in a particular position in a protein and clones corresponding to each variant are assayed for a particular phenotype. (See, also U.S. Pat. Nos. 6,171,820; 6,358,709 and 6,361,974.)

[0157] Error prone PCR (Leung et al., Technique 1:11-15 (1989); Cadwell and Joyce, PCR Method Applic. 2:28-33 (1992)) is a modified polymerase chain reaction (PCR) technique introducing random point mutations into cloned genes. The resulting PCR products can be cloned to produce random mutant libraries or transcribed directly if a T7 promoter is incorporated within the appropriate PCR primer.

[0158] Other mutagenesis techniques are also well known and described, for example, in In Vitro Mutagenesis Protocols. J. Braman, Ed. Humana Press. 2001.

[0159] Optimization can be based on any of the libraries discussed above, or any other types of libraries known in the art, alone or in any combination.

[0160] IV. Production of Neutralizing Antibodies

[0161] Once antibodies with the desired neutralizing properties are identified, such antibodies, including antibody fragments can be produced by methods well known in the art, including, for example, hybridoma techniques or recombinant DNA technology.

[0162] In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0163] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[0164] Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor. J. Immunol. 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0165] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

[0166] Recombinant monoclonal antibodies can, for example, be produced by isolating the DNA encoding the required antibody chains and co-transfecting a recombinant host cell with the coding sequences for co-expression, using well known recombinant expression vectors. Recombinant host cells can be prokaryotic and eukaryotic cells, such as those described above.

[0167] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987)). It is important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.

[0168] In addition, human antibodies can be generated following methods known in the art. For example, transgenic animals (e.g., mice) can be made that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al. Nature 362:255-258 (1993); Bruggermann et al., Year in Immuno. 7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807.

[0169] V. Use of Neutralizing Antibodies

[0170] The neutralizing antibodies of the present invention can be used for the prevention and/or treatment of the targeted diseases. For therapeutic applications, the antibodies or other molecules, the delivery of which is facilitated by using the antibodies or antibody-based transport sequences, are usually used in the form of pharmaceutical compositions.

[0171] Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, 18th Edition. Mack Publishing Co. (Easton, Pa. 1990). See also, Wang and Hanson "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers," Journal of Parenteral Science and Technology. Technical Report No. 10, Supp. 42-2S (1988).

[0172] Antibodies are typically formulated in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride: hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben: catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol): low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium: metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).

[0173] The antibodies also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

[0174] The neutralizing antibodies disclosed herein may also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

[0175] Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al. J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange reaction. A chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al. J. National Cancer Inst. 81(19)1484 (1989).

[0176] For the prevention or treatment of disease, the appropriate dosage of antibody will depend on the type of infection to be treated, the severity and course of the disease, and whether the antibody is administered for preventive or therapeutic purposes. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 .mu.g/kg to about 15 mg/kg of antibody is a typical initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.

[0177] Further details of the invention are illustrated by the following non-limiting Examples.

Example 1

Antibody Libraries from Survivors of Prior Bird Flu Outbreaks and Preparation of Neutralizing Antibodies

[0178] Materials and Bone Marrow Protocol and Sera Preparation

[0179] Blood was obtained by standard venopuncture, allowed to clot, and processed to recover serum. The serum was stored at -20.degree. C. for 3-4 days until they were shipped on dry ice. Donors were anaesthetized with an injection of a local anesthetic and 5 ml of bone marrow was removed from the pelvic bone of each H5N1 survivor. Next the 5 ml of bone marrow was placed into a sterile 50-ml tube containing 45 ml RNAlater (Ambion). The mixture was gently inverted approximately 8-20 times, until there were no visible clumps and the marrow and RNAlater were mixed well. Next the specimen was refrigerated the between 2-10.degree. C. overnight. Following the overnight refrigeration, the specimens were stored at -20.degree. C. for 3-4 days until they were shipped on dry ice. Upon receipt the RNAlater/marrow and sera containing tubes were stored at -80.degree. C. until processed.

[0180] Serology: H/A ELISA

[0181] ELISA plates (Thermo, Immulon 4HBX 96W) were coated with 100 .mu.l of 100 ng/mL H5 hemagglutinin (Protein Sciences, A/Vietnam/1203/2004) in IX ELISA Plate Coating Solution (BiolFX) by overnight incubation at room temperature. The next day plates were washed three times with 300 .mu.l PBS/0.05% Tween-20 (PBST). Following the wash, 300 .mu.l of a blocking solution (4% Non-Fat dry Milk in PBS/0.05% Tween-20) was added and incubated for 1 hour at RTf. Following the blocking step, the plates were washed three times with 300 .mu.l PBS/0.05% Tween-20. Next, 100 .mu.l serum samples diluted 1:20,000 in PBS/0.05% Tween were incubated for 1-2 hours at RT and then washed three times with 300 .mu.l PBS/0.05% Tween-20. 100 .mu.l of an anti-human Fc-HRP conjugate diluted 1:5,000 in PBS/0.05% Tween was incubated for 1-2 hours at RT and then washed three times with 300 .mu.l PBS/0.05% Tween-20. Following this final wash, 100 .mu.l of chromogenic substrate solution was added (TMB1 Substrate, BioFx) and after sufficient amount of time terminated by the addition of 100 .mu.l of STOP Solution (BioFx). Absorbances at 450 nm were read on a plate reader (Molecular Devices Thermomax microplate reader with Softmax Pro software), data recorded, and subsequently plotted using Excel (Microsoft).

[0182] Bone Marrow: RNA Extraction and mRNA Purification

[0183] Bone marrow (.about.2.5 ml in 20 ml RNA later), previously stored at -80.degree. C., was recovered by centrifugation to remove RNA later and then resuspended in 11.25 ml TRI BD reagent (Sigma) containing 300 .mu.l Acetic Acid. The pellet was then vortexed vigorously. Next 1.5 ml BCP (1-bromo-3-chloropropane, Sigma) was added, mixed by vortexing, incubated at RT for 5 min, and then centrifuged at 12000.times.g for 15 min at 4.degree. C. The aqueous phase was carefully removed to not disturb the interface. Total RNA from the aqueous phase was next precipitated by addition of 25 ml isopropanol, incubation at RT for 10 minutes, and centrifugation at 12000.times.g for 10 min at 4.degree. C. Following the addition of isopropanol, two phases were formed due to residual RNAlater, resulting in the precipitated RNA settling at the interface. To eliminate the residual RNAlater and allow maximal recovery of RNA, 5 ml aliquots of 50% isopropanol in H.sub.20 were added and mixed until no phase separation was noticeable, at which point the RNA was pelleted by centrifugation at 12000.times.g for 10 min at 4.degree. C. The RNA pellet was washed with 75% EtOH, transferred to an RNAse-free 1.6 ml microcentrifuge tube, and again recovered by centrifugation. Finally the RNA pellet was resuspended in 100 .mu.l 1 mM Na-phosphate, pH 8.2 and the A.sub.260 and A.sub.280 were read to assess RNA purity.

[0184] Prior to reverse transcription mRNA was purified from total RNA according to Qiagen Oligotex mRNA purification kit. Briefly, 50-200 .mu.g bone marrow RNA was brought to 250 .mu.l with RNasc-free water and mixed with 250 .mu.l of OBB buffer and Oligotex suspension followed by incubation for 3 min at 70.degree. C. Hybridization between the oligo dT.sub.30 of the Oligotex particle and the mRNA poly-A-tail was carried out at room temperature for min. The hybridized suspensions were then transferred to a spin column and centrifuged for 1 min. The spin column was washed twice with 400 .mu.l Buffer OW2. Purified mRNA was then eluted twice by centrifugation with 20 .mu.l hot (70.degree. C.) Buffer OEB. Typical yields were 500 ng to 1.5 .mu.g total RNA.

[0185] Reverse Transcription Using N9 and Oligo dT on Bone Marrow mRNA

[0186] Reverse transcription (RT) reactions were accomplished by mixing together 75-100 ng mRNA with 2 .mu.l 10.times. Accuscript RT Buffer (Stratagene), 0.8 .mu.l 100 mM dNTPs, and either N9 (300 ng) or oligo dT primer (100 ng) and then brought to a final volume of 17 .mu.l with water. The mixtures were heated at 65.degree. C. for 5 min, and then allowed to cool to room temperature. Next 2 .mu.l DTT, 0.5 .mu.l RNase Block (Stratagene), 0.5 .mu.l AccuScript RT (Stratagene) were added to each reaction. Next, the N9 primed reactions were incubated for 10 minutes at room temperature and the oligo-dT primed reactions were incubated on ice for 10 minutes. Finally, both reactions were incubated at 42.degree. C. for 60 minutes followed by 70.degree. C. for 15 minutes to kill the enzyme.

[0187] PCR from bone marrow-derived cDNA

[0188] Antibody heavy and light chain repertoires were amplified from bone marrow cDNA essentially using previously described methods and degenerate primers (O'Brien, P. M., Aitken R. Standard protocols for the construction of scFv Libraries. Antibody Phage Display Methods and Protocols, vol. 178, 59-71, 2001, Humana Press) based upon human germline V and J regions.

[0189] Briefly, PCR reactions using Oligo dT primed cDNA (from 75 ng mRNA) for lambda light chains and N9 primed cDNA (from 75 ng mRNA for kappa light chains, from 100 ng mRNA for heavy chains) were mixed together with 5 .mu.l 10.times. amplification buffer (Invitrogen), 1.5 .mu.l dNTPs (10 mM), 1 .mu.l MgSO4 (50 mM), 2.5 .mu.l V.sub.region primers (10 uM) and 2.5 .mu.l J.sub.region primers (10 uM)-10 uM for V.sub.H, 0.5 .mu.l Platinum Pfx Polymerase (Invitrogen), and sterile dH.sub.2O to final volume of 50 .mu.l. PCR parameters were as follows: step 1-95.degree. C. 5 minutes, step 2-95.degree. C. 30 seconds, step 3-58.degree. C. 30 seconds, step 4-68.degree. C. 1 minute, step 5-cycle step 2-4 40 times, step 6-68.degree. C. 5 minutes. Light chain PCR products were cleaned up using Qiagen PCR Cleanup kit. Heavy chains PCR products were gel purified from 1.5% agarose gel using Qiagen Gel Extraction Kit and then reamplified. Heavy chain reamplification was carried out as follows: Mixed 10 .mu.l 10.times. amplification buffer (Invitrogen), 3 .mu.l dNTPs (10 mM), 2 .mu.l MgSO4 (50 mM), 5 .mu.l each V.sub.H primers (10 uM) and J.sub.H primers (10 uM), 5 .mu.l Heavy chain Primary PCR product, 1 .mu.l Platinum Pfx, volume adjusted to 100 .mu.l with water. Cycling parameters were as follows: step 1-95.degree. C. 5 minutes, step 2-95.degree. C. 30 seconds, step 3-58.degree. C. 30 seconds, step 4-68.degree. C. 1 minute, step 5-cycle step 2-4 20 times, step 6-68.degree. C. 5 minutes. Re-amplified heavy chain PCR products were cleaned up from a 1.5% agarose-TAE gel using Qiagen Extraction Kit.

[0190] Antibody Phage Library Construction

[0191] Separate antibody libraries for each individual bird flu survivor were constructed using unique identifying 3-nucleotide barcodes inserted in the untranslated region following the stop codon of the pIII gene of filamentous phage.

[0192] Light Chain Cloning:

[0193] 1 .mu.g each of pooled kappa light chain and pooled lambda light chain per donor were digested with NotI and BamHI and gel purified from a 1.5% agarose-TAE gel using Qiagen Gel Extraction Kit. 5 .mu.g of each vector (pAMPFab) was digested with NotI and BamHI and gel purified from a 1% agarose-TAE gel using Qiagen Gel Extraction Kit. Library ligations were performed with 200 ng of gel purified Kappa or Lambda inserts and 1 .mu.g of gel purified vector in 60 .mu.l for 1 hour at RT or overnight at 14.degree. C. Ligations were desalted using Edge BioSystem Perfroma spin columns. The library was transformed in five electroporations in 80 .mu.l TG-1 or XL-1 Blue aliquots, each recovered in 1 ml SOC, pooled and outgrown for one hour at 37.degree. C. Total number of transformants was determined following this outgrowth by plating an aliquot from each of the transformations. The remaining electroporation was amplified by growing overnight at 37.degree. C. in 200 ml 2YT+50 .mu.g/ml Ampicillin+2% glucose. The subsequent light chain library was recovered by plasmid purification from these overnight cultures using a Qiagen High Speed Maxiprep Kit.

[0194] Heavy Chain Cloning:

[0195] 1.5-2 .mu.g each of the donor-specific heavy chains (V.sub.H1, V.sub.H2, 5, 6 pool. V.sub.H3, and V.sub.H4) were digested with a 40 Unit excess/.mu.g DNA with Sfil and Xhol and gel purified from a 1.5% agarose-TAE gel using Qiagen Gel Extraction Kit. 15 .mu.g of each light chain library vector was digested with 40 Unit/.mu.g DNA with Sfil and Xhol and gel purified from a 1% agarose-TAE gel using Qiagen Gel Extraction Kit. Library ligations were set up by combining 1.2 .mu.g Sfil/Xhol digested, gel purified heavy chain donor collections and 5 .mu.g of each light chain library (kappa and lambda) overnight at 14.degree. C. The library ligations were then desalted with Edge BioSystem Pefroma spin columns and then transformed through 20 electroporations per library in 80 .mu.l TG-1 aliquots, each recovered in 1 ml SOC, pooled and outgrown for one hour at 37.degree. C. Again following this outgrowth an aliquot of each was used to determine the total number of transformants with the remainder transferred to 1L 2YT+50 .mu.g/ml Ampicillin +2% glucose and grown at 37 C with vigorous aeration to an OD.sub.600 of .about.0.3. Next M13K07 helper phage was then added at a multiplicity of infection (MOI) of 5:1 and incubated for 1 hour at 37.degree. C., with no agitation. Next the cells were harvested by centrifugation and resuspended in 1L 2YT+50 .mu.g/ml Ampicillin, 70 .mu.g/ml Kanamycin and grown overnight at 37.degree. C. with vigorous aeration to allow for scFv phagemid production. The next morning the cells were collected by centrifugation and supernatant containing phagemid was collected. The phagemids were precipitated from the supernatant by the addition of 0.2 volumes 20% PEG/5 M NaCl solution and incubation for 1 hour on ice. The phagemid library stocks were then harvested by centrifugation and resuspended in 20 ml sterile PBS. Residual bacteria were removed by an additional centrifugation and the final phagemid libraries were stored at -20.degree. C. in PBS+50% glycerol.

[0196] Phagemid Panning and Amplification

[0197] ELISA plates (Immulon 4HBX flat bottom, Nunc) were coated with 100 .mu.l of 100 ng/ml, H5 hemagglutinin protein(Protein Sciences, A/Vietnam/1203/2004) in ELISA Coating Solution (BioFX) by overnight incubation at room temperature. The next day plates were washed three times with 300 .mu.l PBST. Following the wash, 300 .mu.l of a blocking solution (4% Non-Fat dry Milk in PBS/0.05% Tween-20) was added and incubated for 30 mins on ice. Following the blocking step, the plates were washed three times with 300 .mu.l PBST. Just prior to phage panning, the glycerol was removed from the frozen phagemid stocks using Millipore Amicon Ultra columns and then blocked in 4% nonfat dry milk for 15 minutes. Next, 100 .mu.l aliquots of phagemid were distributed into 8 wells (total phage .about.1.times.10.sup.12 CFU) and incubated for 2 hours at 4.degree. C. followed by washing 6-8 times with 300 .mu.l PBST. Phagemid were collected following a 10 min at room temperature in 100 .mu.l/well Elution buffer (0.2M glycine-HCl, pH 2.2, 1 mg/ml BSA). The eluate was then neutralized by the addition of 56.25 .mu.l 2M Tris base per ml eluate. Following neutralization. 5 ml TGI cells (OD.sub.6oo .about.0.3) were infected with 0.5 ml neutralized phage at 37.degree. C. for 30 minutes in 2-YT with no shaking. Following this step some cells were plated onto LB AMP Glucose plates to determine total phagemid recovery. The remaining inoculum was placed into 10 ml 2-YTAG (final concentration 2% glucose and 50 .mu.g/ml ampicillin) and grown at 37.degree. C. with vigorous aeration to OD.sub.600--0.3. Next the cultures were infected with M13K07 helper phage at an MOI of 5:1 and incubated at 37.degree. C. for 30-60 minutes with no shaking. The cells were collected by centrifugation and resuspended in 25 ml 2-YTAK (Ampicillin 50 .mu.g/ml, Kanamycin 70 .mu.g/ml), transferred to a fresh culture flask, and grown ON at 37.degree. C. with shaking. Subsequent rounds were similarly recovered and amplified.

[0198] scFv ELISA

[0199] Individual colonies of E. coli HB2151 transformed cells from biopanned phage were grown overnight at 37.degree. C. in 1 ml of 2YT+100 .mu.g/ml AMP. The following morning the cells were harvested by centrifugation and resuspended in 1.5 ml periplasmic lysis buffer (1 ml BBS (Teknova)+0.5 ml 10 mg/ml lysozyme+EDTA to 10 mM final concentration). The cells were again pelleted by centrifugation and the scFv containing periplasmic lysates were collected. The scFv lysates were combined 1:1 with dilution buffer (PBS/0.05% BSA) and 100 .mu.l was added to wells that had been previously antigen coated with and blocked with dilution buffer. The samples were incubated for 2 hours at room temperature and then washed three times with PBS/0.05% Tween. Next 100 .mu.l of 1:5000 diluted Biotin Anti-Histidine mouse (Serotec) in dilution buffer was added to each well and incubated for 1 hr at room temperature. Following this incubation the wells were washed three times with PBS/0.05% Tween and then to each well 100 .mu.l of 1:2500 Streptavidin: HRP (Serotec) was added and incubated for 1 hr at room temperature and then washed three times with PBS/0.05% Tween. Following this final wash, 100 .mu.l of chromogenic substrate solution was added (TMB1 Substrate, BioFx) and after sufficient amount of time terminated by the addition of 100 .mu.l of STOP Solution (BioFx). Absorbances at 450 nm were read on a plate reader (Molecular Devices Thermomax microplate reader with Softmax Pro software), data recorded, and subsequently plotted using Excel (Microsoft).

[0200] Sequencing

[0201] To deduce the heavy and light chain sequences, individual clones were grown and plasmid DNA extracted (Qiagen). The plasmid DNA was subjected to standard DNA sequencing.

[0202] Hemagglutinin Inhibition (HAI) Assays

[0203] Hemagglutination Inhibition was performed essentially following the method of Rogers et al. Virology 131:394-408 (1983), in round bottom microliter plates (Corning) using 4 HAU (hemagglutinating units) of virus or protein/well. For HAI determinations 25 .mu.l samples of purified single chain variable fragments (scFv) were mixed with 25 .mu.l of PBS containing 4 HAU of the test virus in each microtiter well. Following a preincubation of 15 minutes at room temperature, 25 .mu.l of 0.75% human erythrocytes were added, and mixed. HAI antibody activity was determined by visual inspection following a 60 min incubation at room temperature.

[0204] In particular, the following protocols were used:

[0205] Antibodies, Proteins and Viruses.

[0206] IgG.sub.1 proteins were generated from either scFv or Fabs by subcloning their coding regions into a pCI-based (Promega) full length mammalian protein expression and then transfecting them into 293 Freestyle cells (Invitrogen) according to manufacturers guidelines. Briefly 20 .mu.g of light chain and 10 .mu.g heavy chain encoding plasmid were combined with 1.0 ml 293fectin and incubated for 60 minutes. Following this preincubation the DNA mixture was combined with 3.times.10.sup.7 cells in 30 ml media and the resulting cell suspension was grown according to manufacturers suggestion for 7 days. After seven days the secreted immunoglobulins were purified from the culture supernatants using protein A chromatography (Calbiochem). The resulting purified antibodies were buffer exchanged into sterile PBS using centrifugal size filtration (Centricon Plus-20) and their protein concentrations determined by colorimetric BCA assay (Pierce).

[0207] Cross-reaction IgG ELISA.

[0208] Microtiter plates were coated with 0.1 ml of the following antigens diluted in coating buffer and incubated overnight at room temperature: 100 ng/ml H5N1 Vietnam 1203/04, 250 ng/ml H5N1 Turkey/65596/06, 1 .mu.g/ml H5N1 Indonesia/5/05, 700 ng/ml H1N1 New Caledonia/20/99, 1 .mu.g/ml H1N1 North Carolina/1/18, 100 ng/ml and H3N2 Wisconsin/67/05. Blocking was done with 0.3 ml of blocking buffer (4% Non-fat dry milk in PBS/0.05% Tween-20). Following blocking antibodies diluted to 0.5 .mu.g/ml in 2% non-fat dry milk blocking buffer were incubated for two hours at 4 C, washed, and later detected using a 1:3000 dilution of peroxidase conjugated anti-human F.sub.c antibody (Jackson ImmunoResearch) in 2% non-fat dry milk blocking buffer and standard TMB substrate detection (BioFX). Absorbance at 450 nm was read, data recorded, and reported herein

[0209] Viral Microneutralization.

[0210] Indonesia and Turkey hemagglutinin genes were synthetically assembled using human codon optimized sequences (DNA 2.0) and then used to generate recombinant engineered viruses. Recombinant influenza viruses were generated using reverse genetics as previously described (Fodor, E. et al. J Virol. 1999; 73(11): 9679-82). Briefly, 1 ug each of 10 plasmids was transfected into 293 T cells in monolayer. Each transfection contained ambisense plasmids (for the expression of both vRNAs and mRNAs) for the A/Puerto Rico/8/34/PA, PB1, PB2, NP, M, and NS segments, in addition to vRNA (pPOL1 type) and protein expression plasmids (pCAGGS type) for A/Vietnam/1203/04 HA and NA (pCAGGS expression plasmid was kindly provided by J. Miyazaki, Osaka University, Osaka, Japan) (Miyazaki, J. et al. Gene 1989; 79(2):269-77). Twenty hours following transfection, 293T cells were resuspended in cell culture supernatant, and used to inoculate 10-day-old embryonated eggs.

[0211] Antibodies were screened for neutralizing activity against viruses as follows. Two fold serial dilutions of each Mab were incubated with 100 TCIDs.sub.50 of virus in PBS at 37.degree. for 1 h. Madin-Darby Canine Kidney cell monolayers in 24 well plates were washed once with PBS and inoculated with virus-antibody mixtures. Following incubation for 1 h at 37.degree. C. in 5% CO.sub.2 the inoculum was removed and monolayers were again washed once with PBS. Opti-MEM supplemented with 0.3% BSA, 0.01% FBS and 1 ug/ml TPCK-treated trypsin was added and cells were incubated for 72 h at 37.degree. C. The presence of virus in cell culture supernatants was assessed by HA assays using 0.5% chicken red blood cells.

Results

[0212] Bone marrow and blood samples were collected from six survivors of the H5N1 bird flu outbreak that had taken place in Turkey in January 2006, approximately four months after the outbreak. For all six survivors the initial diagnosis of bird flu was made following by physical examination, clinical laboratory testing, and molecular diagnostic determination, sanctioned by the Turkish Ministry of Health. Four of these survivors were additionally confirmed by the World Health Organization (WHO). Serum samples were analyzed to confirm the presence of antibodies to H5 hemagglutinin (A/Vietnam/1203/2004) using the serology protocol described above. As shown in FIG. 8, the blood samples of all six patients (designated SLB H1-H6, respectively) demonstrated the presence of antibodies to the H5 antigen. Following this confirmation, RNA was extracted from the bone marrow samples of these individuals, and bone marrow mRNA was purified and reverse transcribed using the protocols described above. The antibody heavy and light chain repertoires were then amplified from the bone marrow cDNA as described above, and individual antibody heavy and light chain phage libraries were cloned separately for each survivor, using the above-described three-nucleotide bar coding to distinguish the individual libraries.

[0213] Using this vector with its coding system, we successfully cloned repertoires from the bone marrow of five of the six survivors in both single chain (scFv) and Fab phagemid formats. Each collection from an individual survivor has a diversity of greater than 1.0.times.10.sup.8 members. Furthermore, we created additional bar coded libraries comprised of mixed survivor light and heavy chains with a final diversity of 1.1.times.10.sup.9. Collectively the 5 donor-specific collections and the pooled libraries from all donors have a total diversity of 1.0.times.10.sup.9 as a scFv collection and 4.2.times.10.sup.9 as a Fab-displayed collection (Table 4).

Table 2 shows the light chain and full library total transformants in both scFv and Fab formats. Total diversity represented by all libraries is 5.6.times.10.sup.9

TABLE-US-00002 TABLE 2 Light Chains Completed Libraries Kappa Lambda Kappa Lambda scFV H5-1 3.00E+06 4.00E+06 1.50E+08 1.20E+08 H5-2 3.00E+06 3.00E+06 4.00E+07 1.60E+07 H5-3 8.20E+05 1.70E+06 5.30E+07 1.50E+08 H5-5 7.00E+06 5.60E+06 6.50E+08 5.60E+07 H5-6 1.50E+06 5.00E+06 ND 1.00E+07 H5Pool Not Determined 1.80E+08 5.70E+08 Totals 1.10E+09 9.20E+08 scFv Total 2.00E+09 Fab H5-1 1.50E+06 2.90E+06 2.90E+08 4.60E+08 H5-2 3.10E+06 9.40E+05 4.40E+08 4.30E+08 H5-3 2.80E+06 2.30E+06 3.90E+08 3.90E+08 H5-5 7.00E+06 5.60E+06 7.20E+08 1.70E+08 H5-6 1.50E+06 5.00E+06 ND ND H5Pool 1.90E+07 2.60E+08 Totals 2.10E+09 1.45E+09 Fab Total 3.60E+09

[0214] Bone marrow and blood samples were also collected from twelve local donors who were treated for flu symptoms in the year of 2006. Serology was performed as described above to confirm the presence of antibodies to H1, H3 and H5 hemagglutinin, respectively. As shown in FIG. 8, all serum samples tested positive for antibodies to H1 and/or H3 hemagglutinins, where the dominance of a certain subtype depended on the influenza A virus subtype to which the particular donor was exposed most throughout his or her lifetime. Interestingly, there were donors whose serum contained a significant level of antibodies of H5 hemagglutinin as well (donors SLB1 and SLB5 in FIG. 9). Following this confirmation, RNA was extracted from the bone marrow samples of the donors, and bone marrow mRNA was purified and reverse transcribed using the protocols described above. The antibody heavy and light chain repertoires were then amplified from the bone marrow cDNA as described above, and individual antibody heavy and light chain phage libraries were cloned separately for each donor, using the above-described three-nucleotide bar coding to distinguish the individual libraries.

[0215] Selecting Binding Antibodies

[0216] The H5N1 survivor libraries summarized in Table 2, were panned against inactivated virus containing the Vietnam/1203/04 virus HA and NA proteins or recombinant purified hemagglutinin (Barbas, C. et al. (2001) Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press)). Following three to four rounds of phage panning, individual clones from enriched phage pools were analyzed by ELISA against H5N1 virus or purified hemagglutinin and the positive clones were sequenced to determine their heavy and light chain sequences and to read their survivor bar code (D. W. Coomber, Methods Mol Biol 178, 133 (2002)). From this, we isolated specific H5 hemagglutinin binding clones from all five of the individual survivor libraries. In total, more than 300 hundred different antiviral antibodies were recovered, of which 146 specifically bind the H5 hemagglutinin protein.

[0217] General Features of the Selected Clones

[0218] Overall, the individual donors use different germ lines for both heavy and light chains, demonstrating that individual patients have found different solutions to the same potentially lethal immunological challenge. The major features of combinatorial antibody libraries that can be used both to give confidence as to the quality of the obtained repertoire and to provide information as to the chemistry of antibody binding and/or neutralization are seen in these clones. These clones contain all the hallmarks of the previously described repeated clones ("jackpot solution") to antigen binding that is found in the natural progression of affinity maturation, as well as in selected synthetic antibody libraries (Lerner, R. A. Angew Chem Int Ed Engl 45, 8106 (Dec. 11, 2006); A. Rajpal et al., Proc Natl Acad Sci USA 102, 8466 (Jun. 14, 2005)). The presence of "jackpots" in these large collections validates the screening procedure because, unless the phage were selected on the basis of activity, the chance of obtaining the same clone multiple times is highly improbable. Moreover, when one analyzes the heavy chain differences within groups, it was observed that many of the amino acid substitutions were chemically and structurally conservative (Table 4). As with repeated clones, the appearance of multiple amino acid substitutions that are chemically reasonable is unlikely to be a random event.

[0219] Binding Specificity of Recovered Antibodies

[0220] Six clones were selected from three survivors that recognized two different epitopes for conversion into full IgG.sub.1 proteins. The binding of four of these antibodies was mapped to the HA1 subunit of the hemagglutinin protein, while the remaining two did not. As the HA1 subunit has tremendous relevance in infection we further analyzed the activities of those four antibodies as described below.

[0221] One goal of these studies was to recover those rare antibodies that broadly neutralize divergent viral strains. There was a suggestion that some of our antibodies might be broadly reactive because the serum from the donors had high titer antibodies against a divergent subfamily of H5N1 viruses that extended beyond the virus with which they were infected. To determine the degree of cross reactivity at the level of individual antibodies, we first analyzed binding of our clones to different influenza hemagglutinin antigens (FIG. 11). Not surprisingly, these antibodies recognize hemagglutinin from the corresponding infecting Turkey/65596/06 strain, and in addition recognize the heterologous hemagglutinin from the Vietnam/1203/04 strain used for selection. Moreover, they recognize the antigenically divergent Indonesian/5/05 H5 hemagglutinin. Furthermore, we found that four prototype antibodies bound hemagglutinin from the closely related subtype H1N1 contemporary reference strain New Caledonia/20/99. Notably, the three neutralizing antibodies belonging to survivor 5 also bound hemagglutinin from the H1N1 South Carolina/1/18 isolate that emerged during the 1918 Spanish Flu pandemic. Conversely, none of these four antibodies bound hemagglutinin from the contemporary H3N2 Wisconsin/67/05 reference strain, indicating that even though the antibodies display broad spectrum binding amongst and between Influenza subtypes, the reactivity did not extend to all influenza subtypes. FIG. 11 shows the cross-reactivity of H5N1 antibodies from two survivors with hemagglutinins from H1N1 viruses. (A) Bars are H5N1 Vietnam 1203/04(dark grey), H5N1 Turkey/65596/06 (white), H5N1 Indonesia/5/05 (diagonal stripes), H1N1 New Caledonia/20/99 (vertical stripes), H1N1 South Carolina/1/18 (crosshatch stripes), and H3N2 Wisconsin/67/05 (light gray).

[0222] Neutralization Studies

[0223] Initially the antibodies were assayed for their ability to neutralize an H5 HA (Vietnam/1203/04) containing influenza virus. One antibody derived from survivor 2 and 3 from survivor 5 that recognized a common epitope (epitope "A") were all neutralizing whereas the two antibodies derived from survivor 1 that recognized a second epitope (epitope "13") were not.

[0224] Based on the striking sequence similarity of clones separately isolated from survivor 5 against either H5N1 or H1N1 hemagglutinin, we predicted that their cross reactivity would extend beyond simple binding and they would also have the highly unusual property of neutralizing both H5N1 and H1N1 virus. To test the cross neutralizing activity of the IgGs, we tested representative antibodies from the H5N1 screen in a neutralization assay to see if they would also neutralize H1N1 or H3N2 virus (Table 3). We studied the H1 bearing virus A/New Cal/20/99 and the H3 bearing virus A/Hong Kong/68. A collection of viruses bearing H5 subtype hemagglutinin was also tested (A/Vn/1203/04; A/Indo/5/05; A/Turkey/65596/06; A/Egypt/06). The antibodies showed no activity against H3 subtype influenza. However, three of the monoclonal antibodies (1-3) that neutralized H5 containing viruses also strongly neutralized all viruses bearing HA from subtypes H1 (Table 5).

TABLE-US-00003 TABLE 3 Virus* H5 H1 H3 A/Vietnam A/Vietnam A/Indonesia A/Turkey A/Egypt A/New A/Hong 1203/04 .dagger-dbl. 1203/04 .dagger-dbl. 5/05 65596/06 14725/06 Cal 20/99 Kong/68 Ab 1.dagger. 32-64 7-28 28 28 3.5-7 28 >1000 Ab 2.dagger. 188 162-650 80 325 20-40 162-325 >1000 Ab 3.dagger. 175 53 47 94 12-23 23-47 >1000 Ab 4.dagger. 5-19 1.6-6.6 >1000 Not done Not done >1000 >1000

[0225] Immunochemical Basis of Neutralization

[0226] One advantage of antibody libraries is that when one obtains large numbers of antibodies, they can be grouped as to their relatedness. Thus, when a function for a given antibody in the collection is observed one can predict that other members of the group to which it belongs will have similar activity.

[0227] Table 4 shows example sequences displaying the immunochemical basis of neutralization discovered from Survivor 5 libraries following H5N1 Vietnam panning. The 61 unique heavy chain sequences aligned with their germline variable regions from the 114 unique heavy and light chain combinations. Requisite mutations are shown in bolded, underlined text (column 5--PI to GM and A to T; column 6--KS to EL or EM or XL) and predominant mutations are shown in italicized, underlined text (column 2--A to T; column 3--IS to VT; column 5--G to A; column 8--K to Q or R). Heavy chains sequences also discovered in H1N1 New Caledonia panning are highlighted in gray. Antibody regions and Kabat numbering ranges are listed at the top of each sequence column. The heavy chain/light chain pairing is indicated in the first column as follows: *--paired with 2 unique light chains, .dagger.--paired with 3 unique light chains, .dagger-dbl.--paired with 4 unique light chains, .sctn.--paired with 5 unique light chains, and --paired with 12 unique light chains.

TABLE-US-00004 TABLE 4 ##STR00001## ##STR00002##

[0228] Members of the group that contained the neutralizing antibody collection against epitope "A" from survivor 5, are shown in Table 4. The group is comprised of 61 unique members that most closely resemble the V.sub.H1e germ line heavy chain. Some heavy chains are paired with more than one light chain. In total these heavy chains have 114 unique pairings to both kappa and lambda light chains. Comparing these heavy chains to the highly related VH1e germline, we observe three types of point substitutions. Some changes appear to be required, others are dominant, and some residues have only been changed sporadically. The changes that are required occur in every clone in the group within CDR2 at position 52A (Pro>Gly), 53 (Ile>Met), and 57 (Ala>Thr), as well as in the framework 3 region at position 73 (Lys>Glu) and 74 (Ser>Leu or Met), all of which vary from the germline side chain chemistries, suggesting that these mutations are critical to antigen binding and neutralization. The second set of mutations is dominant and found in most clones. The first, in framework 1 at position 24 (Ala>Thr), represents a significant chemical change. The next three are conservative changes in CDR1 at positions 34(Ile>Val) and 35 (Ser>Thr) and also in CDR2 at position 50 (Gly>Ala). All four of these dominant substitutions, however, are dispensable, suggesting that, while beneficial, they are not essential. The sporadic changes found throughout framework regions 1, 3, and 4, as well as CDR3, are all conservative and likely represent minor optimization events.

[0229] FIG. 12 shows the positions of the required mutations in the structure of the antibody superimposed on the crystal structure of a highly related anti-HIV Fab called 47e (lrzi.pdb) (Huang, C. C. et al. (2004)Proc. Nat. Acad. Sci. 101, 2706-2711). FIG. 12 shows the positions of H5 hemagglutinin binding Group 1 required and dominant mutations on the crystal structure of Fab 47e. The required mutations are shown as G52 (52A (Pro>Gly)), M53 (Ile>Met), T57 (Ala>Thr), E73 (Lys>Glu) and LM74 (Ser>Leu or Met). The dominant mutations are shown as T24 (Ala>Thr), V34 (Ile>Val), T35 (Ser>Thr), and A50 (Gly>Ala). The required and dominant Group 1 heavy chain sequences identified in H5 Vietnam/1203/2004H A biopanning are superimposed on the crystal structure of the highly related anti-HIV Fab 47e. Mutations are shown in both backbone (top) and space-filling (bottom) models. A tight cluster is formed by four of the required mutations in and adjacent to CDR2. The required mutations 52A (Pro>Gly), 53 (Ile>Met), 73 (Lys>Glu) and 74 (Ser>Leu or Met) form a remarkably tight cluster on the exposed surface of the heavy chain variable domain where they form a ridge that prominently protrudes from the protein surface (FIG. 12). The remaining required mutation 57 (Ala>Thr) is partially buried at the base of the CDR2 loop. The surface exposed changes in CDR 2 and framework 3 are likely to have a direct role in antigen binding while the less exposed required mutation and the non-essential dominant mutations may have indirect effects through stabilizing and/or positioning the CDR2 loop.

[0230] The antibodies from survivor 2 are comprised of 2 unique heavy chains that most closely resemble the V.sub.H4-4b germ line heavy chain (Table 5). The first heavy chain has been found paired with 5 unique lambda light chains, four of which are from the infrequently used lambda 6 light chain family and the other is paired with a single kappa light chain. Antibody 4 whose neutralization profile was more restricted came from this group.

TABLE-US-00005 TABLE 5 Group FR1 CDR1 FR2 CDR2 2 (1-29) (30-35) (36-46) (47-58) Heavy QVQLQESGPGLVKPSETLSLTCTVSGYSF DSGYYWG WLRQPPGKGLE WIGSIYHSRNTY chain Lambda FMLTQPHSVSESPGKTVTISCTGSGGN IARNYVQWY QQRPGSAPV TVILEDDKRP light FMLTQPHSVSESPGKTVTISCTGSSGS IASNYVQWY QQRPGSAPT TVIYEDYQRP chains SVLTQPPSASGTPGQRVTISCSGSSSN IGSNTVNWY KQLPGTAPR LLIYSNDQRP SVLTQPPSASGTPGQRVTLSCSGSSSN IGGNSVNWY QHVPGTAPK LLMHSDDQRP PELTQPHSVSESPGKTVTISCTGSGGR IATNHVQWY QQRPGSAPT IVIYENNQRP PELTQPPSASGTPGQRVTISCSGSSSN IGSNTVNWY QQLPGTAPK LLIYSNNQRP Kappa DIQMTQSPSSLSAFVGDRVTITCQASQDI SNYLNWY QQKPGKAPK LLIYDATNLE light chain Group FR3 CDR3 FR4 2 (59-92) (93-101) (102-113) Heavy YNPSLKSRVTISVDTSKNQFSLQLSSVTAADTAVYYC ARGTWYSSNLRYWFD PWGKGTLVRVSS chain Lambda SGIPDRFSGSIDRSSNSASLTISGLRTEDEALYYC QSYDDSDLV VFGGGTKLT light SGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC QSYDDSDHL IFGGGTKLTVL chains SGVPDRFSGSKSGTSASLAISGLQSEDEANYYC AAWDDSLSGW VFGGGTKLTVL SGVPDRFSGSKSGTSXSLAISGLQSEDEADYYC AXWDDSLNAW VFGGXTKVTVL SGVPNRFSGSIDDSSNSASLTISALRTEDEADYYC QSADATNV FFGGGTKVTVL SGVPDRFSGSKSGTSASLAISGLQSEDEADYYC AAWDDSLNGW VFGGXTKLTVL Kappa TGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLPL TFGGGTKVDIKR light chain

[0231] The probability that a given mutation is important to the activity of an antibody increases as a function of the number of times it was independently selected. To determine if the required mutations were selected during somatic mutation from independent clones or were from the progeny of a single clone that further mutated during subsequent replications, the codon usage of the dominant mutations were analyzed (Table 6A-6B). The data reveal that although different codons were used they resulted in the same amino acid changes, demonstrating that these mutations arose independently in different clones and were, thus, selected multiple times. This convergent outcome for independently selected events is strong evidence that these dominant mutations play a critical role in the binding to the virus and/or its neutralization.

[0232] As illustrated in Tables 6A-6B, codon usage of individual clones shows independent origin of selected H5 HA binding clones. DNA alignment and encoded amino acids for 6 representative Group 1 antibodies against VH1-e germline. The use of different codons for the same amino acids demonstrates that each unique sequence is of a distinct origin. Table 6A corresponds to CDR2 and Table 6B corresponds to Framework 3. Germ line codons are shown as bolded codons. A change from a germ line codon to the same amino acid is shown as a plain text codon. A first change from a germ line amino acid is shown as a bolded, underlined codon. A second change from a germ line amino acid is shown as an italicized, underlined codon. A third change from a germ line amino acid is shown as an underlined,

TABLE-US-00006 TABLE 6A Clone Kabat CDR2 Number Sequence 47 48 49 50 51 52 52A 52B 52C 53 54 55 56 57 58 VH1-e TGG ATG GGA AGG ATC ATC CCT ATC CTT GGT ATA GCA AAC TAC GCA germline W M G R I I P I L G I A N Y A 27 TGG ATG GGC GCG ATC ATC GGT ATG TTT GGT ACA ACA AAC TAC GCA W M G A I I G M F G T T N Y A 30 TGG ATG GGA GGG ATC ATC GGT ATG TTT GGA ACA ACC AAC TAT GCA W M G G I I G M F G T T N Y A 33 TGG ATG GGA GCG ATC ATC GGT ATG TTT GGT ACA ACA AAC TAC GCA W M G A I I G M F G T T N Y A 41 TGG ATG GGC GCG ATC ATC GGT ATG TTT GGT ACA ACA AAC TAC GCA W M G A I I G M F G T T N Y A 50 TGG ATG GGA GGG ATC ATC GGT ATG TTT GGT ACA ACG AAC TAT GCA W M G G I I G M F G T T N Y A 17 TGG ATG GGA GGG ATC ATC GGT ATG TTT GGT ACA ACA AAC TAC GCA W M G G I I G M F G T T N Y A # Codons 1 1 2 2 1 1 1 1 1 2 1 3 1 2 1 used

TABLE-US-00007 TABLE 6B Clone Kabat Framework 3 Number Sequence 67 68 69 70 71 72 73 74 75 76 77 78 VH1-e GTC ACG ATT ACC GCG GAC AAA TCC ACG AGC ACA GCC germline V T I T A D K S T S T A 27 GTC ACG CTT ACC GCG GAC GAA TTA ACG TCC ACA GCC V T L T A D E L T S T A 30 CTC ACA ATC ACC GCG GAC GAG ATG ACG TCC ACA GCC L T I T A D E M T S T A 33 GTC ACA ATC ACC GCG GAC GAA TTA ACG TCC ACA GCC V T I T A D E L T S T A 41 GTC ACG CTT ACC GCG GAC GAA TTA ACG TCC ACA GCC V T L T A D E L T S T A 50 GTC ACG ATT ACC GCG GAC GAG ATG ACG TCC ACA GCC V T I T A D E M T S T A 17 GTC ACG ATT ACC GCG GAC GAA TTA ACG TCC ACA GCC V T I T A D E L T S T A # Codons 2 2 3 1 1 1 2 2 1 1 1 1 used

Example 2

Constructing Donor-Specific Antibody Library for Patients Infected with HIV

[0233] Bone Marrow Protocol and Sera Preparation

[0234] Blood is obtained by standard venopuncture, allowed to clot, and processed to recover serum. The serum is stored at -20.degree. C. for 3-4 days until they are shipped on dry ice. Donors are anaesthetized with an injection of a local anesthetic and 5 ml of bone marrow is removed from the pelvic bone of each patient donor. Next the 5 ml of bone marrow is placed into a sterile 50-ml tube containing 45 ml RNAlater (Ambion). The mixture is gently inverted approximately 8-20 times, until there are no visible clumps and the marrow and RNAlater are mixed well. Next the specimen is refrigerated the between 2-10.degree. C. overnight. Following the overnight refrigeration, the specimens are stored at -20.degree. C. for 3-4 days until they are shipped on dry ice. Upon receipt the RNAlater/marrow and sera containing tubes are stored at -80.degree. C. until processed. Candidate patient should be tested HIV positive prior to be selected as donors.

[0235] Bone marrow extraction and mRNA purification, reverse transcription, PCR, antibody light and heavy chain construction, phagemid panning and amplification, ELISA and sequencing are performed essentially as described in Example 1.

[0236] Although in the foregoing description the invention is illustrated with reference to certain embodiments, it is not so limited. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0237] All references cited throughout the specification are hereby expressly incorporated by reference.

Sequence CWU 1

1

128118DNAArtificial sequenceDescription of Artificial Sequence Synthetic oligonucleotide 1tttttttttt tttttttt 18213DNAArtificial sequenceDescription of Artificial Sequence Synthetic oligonucleotide 2ggccnnnnng gcc 133120PRTHomo sapiens 3Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 1204120PRTHomo sapiens 4Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115 1205120PRTHomo sapiens 5Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 1206120PRTHomo sapiens 6Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 1207120PRTHomo sapiens 7Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 1208120PRTHomo sapiens 8Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 1209120PRTHomo sapiens 9Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12010120PRTHomo sapiens 10Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Arg Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12011120PRTHomo sapiens 11Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12012120PRTHomo sapiens 12Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12013120PRTHomo sapiens 13Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12014120PRTHomo sapiens 14Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12015120PRTHomo sapiens 15Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12016120PRTHomo sapiens 16Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12017120PRTHomo sapiens 17Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12018120PRTHomo sapiensMOD_RES(89)..(89)Any amino acid 18Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Xaa Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12019120PRTHomo sapiens 19Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12020120PRTHomo sapiens 20Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12021120PRTHomo sapiens 21Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Ser Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12022120PRTHomo sapiens 22Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser

Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12023120PRTHomo sapiens 23Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12024120PRTHomo sapiens 24Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12025120PRTHomo sapiens 25Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12026120PRTHomo sapiensMOD_RES(24)..(24)Any amino acid 26Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Xaa Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12027120PRTHomo sapiens 27Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12028120PRTHomo sapiens 28Glu Val Gln Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12029120PRTHomo sapiens 29Glu Val Gln Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Val Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gly 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115 12030120PRTHomo sapiens 30Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Gln Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12031120PRTHomo sapiens 31Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Ala Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12032120PRTHomo sapiens 32Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12033120PRTHomo sapiens 33Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12034120PRTHomo sapiens 34Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12035119PRTHomo sapiens 35Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser 11536120PRTHomo sapiens 36Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12037120PRTHomo sapiens 37Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12038120PRTHomo sapiens 38Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12039120PRTHomo sapiens 39Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12040120PRTHomo sapiens 40Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115 12041120PRTHomo sapiens 41Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12042120PRTHomo sapiens 42Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12043120PRTHomo sapiens 43Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12044120PRTHomo sapiens 44Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12045120PRTHomo sapiens 45Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12046120PRTHomo sapiensMOD_RES(74)..(74)Any amino acid 46Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Xaa Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12047120PRTHomo sapiens 47Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12048120PRTHomo sapiens 48Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12049120PRTHomo sapiens 49Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12050120PRTHomo sapiens 50Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12051120PRTHomo sapiens 51Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12052120PRTHomo sapiens 52Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12053120PRTHomo sapiens 53Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12054120PRTHomo sapiens 54Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12055120PRTHomo sapiens 55Gly Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12056120PRTHomo sapiens 56Gly Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Arg 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12057120PRTHomo sapiens 57Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12058120PRTHomo sapiens 58Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12059120PRTHomo sapiens 59Gln Val Gln Leu Leu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Thr Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12060120PRTHomo sapiens 60Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12061120PRTHomo sapiens 61Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Ser Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12062120PRTHomo sapiens 62Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85

90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Asn Ser Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12063120PRTHomo sapiens 63Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Gln 100 105 110Gly Thr Met Val Thr Val Ser Ser 115 12064120PRTHomo sapiens 64Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Arg Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Val Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Leu Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Ser Tyr Tyr Tyr Glu Thr Thr Leu Asp Tyr Trp Gly Lys 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 12065123PRTHomo sapiens 65Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Phe Asp Ser Gly 20 25 30Tyr Tyr Trp Gly Trp Leu Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly Ser Ile Tyr His Ser Arg Asn Thr Tyr Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Gln Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Thr Trp Tyr Ser Ser Asn Leu Arg Tyr Trp Phe Asp Pro 100 105 110Trp Gly Lys Gly Thr Leu Val Arg Val Ser Ser 115 12066108PRTHomo sapiens 66Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys Thr1 5 10 15Val Thr Ile Ser Cys Thr Gly Ser Gly Gly Asn Ile Ala Arg Asn Tyr 20 25 30Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Val Thr Val Ile 35 40 45Leu Glu Asp Asp Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Ile Asp Arg Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80Arg Thr Glu Asp Glu Ala Leu Tyr Tyr Cys Gln Ser Tyr Asp Asp Ser 85 90 95Asp Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr 100 10567110PRTHomo sapiens 67Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys Thr1 5 10 15Val Thr Ile Ser Cys Thr Gly Ser Ser Gly Ser Ile Ala Ser Asn Tyr 20 25 30Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val Ile 35 40 45Tyr Glu Asp Tyr Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asp Ser 85 90 95Asp His Leu Ile Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 11068109PRTHomo sapiens 68Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg1 5 10 15Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr 20 25 30Val Asn Trp Tyr Lys Gln Leu Pro Gly Thr Ala Pro Arg Leu Leu Ile 35 40 45Tyr Ser Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser65 70 75 80Glu Asp Glu Ala Asn Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Ser 85 90 95Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 10569109PRTHomo sapiensMOD_RES(71)..(71)Any amino acid 69Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg1 5 10 15Val Thr Leu Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn Ser 20 25 30Val Asn Trp Tyr Gln His Val Pro Gly Thr Ala Pro Lys Leu Leu Met 35 40 45His Ser Asp Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Lys Ser Gly Thr Ser Xaa Ser Leu Ala Ile Ser Gly Leu Gln Ser65 70 75 80Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Xaa Trp Asp Asp Ser Leu Asn 85 90 95Ala Trp Val Phe Gly Gly Xaa Thr Lys Val Thr Val Leu 100 10570109PRTHomo sapiens 70Pro Glu Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys Thr1 5 10 15Val Thr Ile Ser Cys Thr Gly Ser Gly Gly Arg Ile Ala Thr Asn His 20 25 30Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val Ile 35 40 45Tyr Glu Asn Asn Gln Arg Pro Ser Gly Val Pro Asn Arg Phe Ser Gly 50 55 60Ser Ile Asp Asp Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Ala Leu65 70 75 80Arg Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ala Thr 85 90 95Asn Val Phe Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 10571109PRTHomo sapiensMOD_RES(103)..(103)Any amino acid 71Pro Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg1 5 10 15Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr 20 25 30Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser65 70 75 80Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn 85 90 95Gly Trp Val Phe Gly Gly Xaa Thr Lys Leu Thr Val Leu 100 10572108PRTHomo sapiens 72Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Phe Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Thr Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg 100 1057345DNAHomo sapiensCDS(1)..(45) 73tgg atg gga agg atc atc cct atc ctt ggt ata gca aac tac gca 45Trp Met Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala1 5 10 157415PRTHomo sapiens 74Trp Met Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala1 5 10 157545DNAArtificial sequencesource/note="Description of artificial sequence Synthetic oligonucleotide" 75tgg atg ggc gcg atc atc ggt atg ttt ggt aca aca aac tac gca 45Trp Met Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala1 5 10 157615PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 76Trp Met Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala1 5 10 157745DNAArtificial sequencesource/note="Description of artificial sequence Synthetic oligonucleotide" 77tgg atg gga ggg atc atc ggt atg ttt gga aca acc aac tat gca 45Trp Met Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala1 5 10 157815PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 78Trp Met Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala1 5 10 157945DNAArtificial sequencesource/note="Description of artificial sequence Synthetic oligonucleotide" 79tgg atg gga gcg atc atc ggt atg ttt ggt aca aca aac tac gca 45Trp Met Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala1 5 10 158045DNAArtificial sequencesource/note="Description of artificial sequence Synthetic oligonucleotide" 80tgg atg ggc gcg atc atc ggt atg ttt ggt aca aca aac tac gca 45Trp Met Gly Ala Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala1 5 10 158145DNAArtificial sequencesource/note="Description of artificial sequence Synthetic oligonucleotide" 81tgg atg gga ggg atc atc ggt atg ttt ggt aca acg aac tat gca 45Trp Met Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala1 5 10 158245DNAArtificial sequencesource/note="Description of artificial sequence Synthetic oligonucleotide" 82tgg atg gga ggg atc atc ggt atg ttt ggt aca aca aac tac gca 45Trp Met Gly Gly Ile Ile Gly Met Phe Gly Thr Thr Asn Tyr Ala1 5 10 158336DNAHomo sapiensCDS(1)..(36) 83gtc acg att acc gcg gac aaa tcc acg agc aca gcc 36Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala1 5 108412PRTHomo sapiens 84Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala1 5 108536DNAArtificial sequencesource/note="Description of artificial sequence Synthetic oligonucleotide" 85gtc acg ctt acc gcg gac gaa tta acg tcc aca gcc 36Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala1 5 108612PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 86Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala1 5 108736DNAArtificial sequencesource/note="Description of artificial sequence Synthetic oligonucleotide" 87ctc aca atc acc gcg gac gag atg acg tcc aca gcc 36Leu Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala1 5 108812PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 88Leu Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala1 5 108936DNAArtificial sequencesource/note="Description of artificial sequence Synthetic oligonucleotide" 89gtc aca atc acc gcg gac gaa tta acg tcc aca gcc 36Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala1 5 109012PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 90Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala1 5 109136DNAArtificial sequencesource/note="Description of artificial sequence Synthetic oligonucleotide" 91gtc acg ctt acc gcg gac gaa tta acg tcc aca gcc 36Val Thr Leu Thr Ala Asp Glu Leu Thr Ser Thr Ala1 5 109236DNAArtificial sequencesource/note="Description of artificial sequence Synthetic oligonucleotide" 92gtc acg att acc gcg gac gag atg acg tcc aca gcc 36Val Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala1 5 109312PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 93Val Thr Ile Thr Ala Asp Glu Met Thr Ser Thr Ala1 5 109436DNAArtificial sequencesource/note="Description of artificial sequence Synthetic oligonucleotide" 94gtc acg att acc gcg gac gaa tta acg tcc aca gcc 36Val Thr Ile Thr Ala Asp Glu Leu Thr Ser Thr Ala1 5 109539DNAArtificial sequencesource/note="Description of artificial sequence Synthetic oligonucleotide" 95acn aar gcn tcn tay ctn agy acn agy agy agy ctn gay 39Thr Lys Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp1 5 109613PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 96Thr Lys Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp1 5 109739DNAArtificial sequencesource/note="Description of artificial sequence Synthetic oligonucleotide" 97gcn cgn ggn ath tay tty tay ggn acn acn tay tty gay 39Ala Arg Gly Ile Tyr Phe Tyr Gly Thr Thr Tyr Phe Asp1 5 109813PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 98Ala Arg Gly Ile Tyr Phe Tyr Gly Thr Thr Tyr Phe Asp1 5 109939DNAArtificial sequencesource/note="Description of artificial sequence Synthetic consensus sequence" 99rcn mrn gsn wyn tay ytn wry rsn asn asn wry ytn gay 39Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp1 5 1010013PRTArtificial sequencesource/note="Description of artificial sequence Synthetic consensus sequence" 100Thr Lys Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp1 5 10101595PRTArtificial sequencesource/note="Description of artificial sequence Synthetic consensus sequence" 101Met Glu Lys Ile Ile Ile Ile Leu Leu Leu Leu Ala Leu Ala Ala Cys1 5 10 15Ser Gly Ala Leu Pro Gly Asn Asp Asn Ser Thr Asp Lys Ile Cys Ile 20 25 30Gly Tyr His Ala Asn Asn Ser Thr Glu Thr Val Asp Thr Leu Thr Glu 35 40 45Lys Asn Val Glu Val Thr His Ala Thr Glu Leu Val Glu Thr Thr His 50 55 60Asn Gly Lys Leu Cys Ser Leu Asn Gly Lys Ser Pro Leu Asp Leu Gly65 70 75 80Asp Cys Ser Ile Glu Gly Trp Ile Leu Gly Asn Pro Gln Cys Asp Leu 85 90 95Leu Leu Gly Gly Arg Glu Trp Ser Tyr Ile Val Glu Arg Pro Asn Ala 100 105 110Pro Asn Gly Leu Cys Tyr Pro Gly Asp Phe Glu Asn Tyr Glu Glu Leu 115 120 125Arg His Leu Phe Ser Ser Ser Gly Ser Phe Glu Lys Ile Glu Ile Phe 130 135 140Pro Lys Thr Phe Thr Trp Gly Asn Val Val Thr Thr Asn Gly Thr Thr145 150 155 160Lys Ala Cys Lys Asp Arg Ser Gly Gly Ser Ser Phe Tyr Arg Asn Leu 165 170 175Val Trp Leu Thr Ser Lys Lys Lys Gly Ser Ala Tyr Pro Val Ile Lys 180 185 190Gly Thr Tyr Asn Asn Thr Arg Gly Glu Asp Ile Leu Ile Ile Trp Gly 195 200 205Ile His His Pro Pro Thr Thr Thr Glu Gln Thr Lys Leu Tyr Gly Asn 210 215 220Ala Asp Thr Tyr Val Ser Val Gly Thr Ser Thr Tyr Asn Arg Arg Phe225 230 235 240Val Pro Glu Ile Gly Ala Arg Pro Lys Val Asn Gly Gln Ser Gly Arg 245 250 255Met Asp Phe Tyr Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile Thr Phe 260 265 270Glu Ser Asn Gly Asn Leu Ile Ala Pro Arg Tyr Ala Tyr Lys Leu Ile 275 280 285Lys Gly Gly Pro Ser Gly Ile Glu Tyr Asn Gly Lys Gly Arg Ile Ile 290 295 300Gln Ser Glu Asp Leu Pro Ile Gly Ala Asn Cys Asn Thr Lys Cys Gln305 310 315 320Thr Pro Gly Gly Ala Ile Asn Thr Ser Lys Pro Phe Gln Asn Ile Ser 325

330 335Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys Ser Gly Ser Leu 340 345 350Lys Leu Ala Thr Gly Leu Arg Asn Val Pro Glu Ile Ile Glu Arg Arg 355 360 365Arg Lys Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly 370 375 380Gly Trp Pro Gly Leu Ile Asp Gly Trp Tyr Gly Phe His His Gln Asn385 390 395 400Ala Gln Gly Thr Gly Ile Ala Ala Asp Lys Lys Ser Thr Gln Lys Ala 405 410 415Ile Asp Gln Ile Thr Asn Lys Val Asn Asn Ile Ile Glu Lys Met Asn 420 425 430Thr Gln Phe Glu Ala Ile Asp His Glu Phe Ser Glu Val Glu Lys Arg 435 440 445Ile Asn Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Thr Asp Ile Trp 450 455 460Ser Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Gln His Thr Leu465 470 475 480Asp Leu His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Arg 485 490 495Gln Leu Arg Asp Asn Ala Glu Asp Asp Gly Asn Gly Cys Phe Glu Ile 500 505 510Tyr His Lys Cys Asp Asp Glu Cys Met Glu Ser Ile Arg Asn Gly Thr 515 520 525Tyr Asp His Pro Glu Tyr Arg Glu Glu Ser Lys Leu Asn Arg Gln Glu 530 535 540Ile Asp Gly Val Lys Leu Glu Ser Gly Gly Asn Val Tyr Lys Ile Leu545 550 555 560Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Ala Ala Leu Ile 565 570 575Ala Gly Phe Ile Phe Trp Ala Cys Ser Asn Gly Asn Cys Arg Cys Thr 580 585 590Ile Cys Ile 595102561PRTInfluenza virus A 102Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Asp1 5 10 15Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val Asp 20 25 30Thr Ile Leu Glu Arg Asn Val Thr Val Thr His Ala Lys Asp Ile Leu 35 40 45Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn Gly Ile Pro Pro Leu 50 55 60Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro Glu65 70 75 80Cys Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile Met Glu Lys 85 90 95Glu Asn Pro Arg Asp Gly Leu Cys Tyr Pro Gly Ser Phe Asn Asp Tyr 100 105 110Glu Glu Leu Lys His Leu Leu Ser Ser Val Lys His Phe Glu Lys Val 115 120 125Lys Ile Leu Pro Lys Asp Arg Trp Thr Gln His Thr Thr Thr Gly Gly 130 135 140Ser Arg Ala Cys Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn Met145 150 155 160Val Trp Leu Thr Glu Lys Gly Ser Asn Tyr Pro Val Ala Lys Gly Ser 165 170 175Tyr Asn Asn Thr Ser Gly Glu Gln Met Leu Ile Ile Trp Gly Val His 180 185 190His Pro Asn Asp Glu Lys Glu Gln Arg Thr Leu Tyr Gln Asn Val Gly 195 200 205Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys Arg Ser Thr Pro 210 215 220Asp Ile Ala Thr Arg Pro Lys Val Asn Gly Leu Gly Ser Arg Met Glu225 230 235 240Phe Ser Trp Thr Leu Leu Asp Met Trp Asp Thr Ile Asn Phe Glu Ser 245 250 255Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys Arg 260 265 270Gly Ser Ser Gly Ile Met Lys Thr Glu Gly Thr Leu Glu Asn Cys Glu 275 280 285Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro Phe 290 295 300His Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys305 310 315 320Ser Glu Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val Pro Gln Ile 325 330 335Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly 340 345 350Trp Gln Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Ser Asn Asp 355 360 365Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala Phe 370 375 380Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr385 390 395 400Gln Phe Glu Ala Val Gly Lys Glu Phe Ser Asn Leu Glu Arg Arg Leu 405 410 415Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr 420 425 430Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp 435 440 445Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Met Gln 450 455 460Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe Tyr465 470 475 480His Lys Cys Asp Asp Glu Cys Met Asn Ser Val Lys Asn Gly Thr Tyr 485 490 495Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu Ile 500 505 510Lys Gly Val Lys Leu Ser Ser Met Gly Val Tyr Gln Ile Leu Ala Ile 515 520 525Tyr Ala Thr Val Ala Gly Ser Leu Ser Leu Ala Ile Met Met Ala Gly 530 535 540Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys545 550 555 560Ile103561PRTInfluenza virus A 103Met Tyr Lys Val Val Val Ile Ile Ala Leu Leu Gly Ala Val Lys Gly1 5 10 15Leu Asp Arg Ile Cys Leu Gly His His Ala Val Ala Asn Gly Thr Ile 20 25 30Val Lys Thr Leu Thr Asn Glu Gln Glu Glu Val Thr Asn Ala Thr Glu 35 40 45Thr Val Glu Ser Thr Asn Leu Asn Lys Leu Cys Met Lys Gly Arg Ser 50 55 60Tyr Lys Asp Leu Gly Asn Cys His Pro Val Gly Met Leu Ile Gly Thr65 70 75 80Pro Val Cys Asp Pro His Leu Thr Gly Thr Trp Asp Thr Leu Ile Glu 85 90 95Arg Glu Asn Ala Ile Ala His Cys Tyr Pro Gly Ala Thr Ile Asn Glu 100 105 110Glu Ala Leu Arg Gln Lys Ile Met Glu Ser Gly Gly Ile Ser Lys Met 115 120 125Ser Thr Gly Phe Thr Tyr Gly Ser Ser Ile Thr Ser Ala Gly Thr Thr 130 135 140Lys Ala Cys Met Arg Asn Gly Gly Asp Ser Phe Tyr Ala Glu Leu Lys145 150 155 160Trp Leu Val Ser Lys Thr Lys Gly Gln Asn Phe Pro Gln Thr Thr Asn 165 170 175Thr Tyr Arg Asn Thr Asp Thr Ala Glu His Leu Ile Ile Trp Gly Ile 180 185 190His His Pro Ser Ser Thr Gln Glu Lys Asn Asp Leu Tyr Gly Thr Gln 195 200 205Ser Leu Ser Ile Ser Val Glu Ser Ser Thr Tyr Gln Asn Asn Phe Val 210 215 220Pro Val Val Gly Ala Arg Pro Gln Val Asn Gly Gln Ser Gly Arg Ile225 230 235 240Asp Phe His Trp Thr Leu Val Gln Pro Gly Asp Asn Ile Thr Phe Ser 245 250 255Asp Asn Gly Gly Leu Ile Ala Pro Ser Arg Val Ser Lys Leu Thr Gly 260 265 270Arg Asp Leu Gly Ile Gln Ser Glu Ala Leu Ile Asp Asn Ser Cys Glu 275 280 285Ser Lys Cys Phe Trp Arg Gly Gly Ser Ile Asn Thr Lys Leu Pro Phe 290 295 300Gln Asn Leu Ser Pro Arg Thr Val Gly Gln Cys Pro Lys Tyr Val Asn305 310 315 320Gln Arg Ser Leu Leu Leu Ala Thr Gly Met Arg Asn Val Pro Glu Val 325 330 335Val Gln Gly Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn 340 345 350Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe Arg His Gln Asn 355 360 365Ala Gln Gly Thr Gly Gln Ala Ala Asp Tyr Lys Ser Thr Gln Ala Ala 370 375 380Ile Asp Gln Ile Thr Gly Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn385 390 395 400Thr Glu Phe Glu Ser Ile Glu Ser Glu Phe Ser Glu Thr Glu His Gln 405 410 415Ile Gly Asn Val Ile Asn Trp Thr Lys Asp Ser Ile Thr Asp Ile Trp 420 425 430Thr Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn Gln His Thr Ile 435 440 445Asp Met Ala Asp Ser Glu Met Leu Asn Leu Tyr Glu Arg Val Arg Lys 450 455 460Gln Leu Arg Gln Asn Ala Glu Glu Asp Gly Lys Gly Cys Phe Glu Ile465 470 475 480Tyr His Thr Cys Asp Asp Ser Cys Met Glu Ser Ile Arg Asn Asn Thr 485 490 495Tyr Asp His Ser Gln Tyr Arg Glu Glu Ala Leu Leu Asn Arg Leu Asn 500 505 510Ile Asn Pro Val Lys Leu Ser Ser Gly Tyr Lys Asp Ile Ile Leu Trp 515 520 525Phe Ser Phe Gly Glu Ser Cys Phe Val Leu Leu Ala Val Val Met Gly 530 535 540Leu Val Phe Phe Cys Leu Lys Asn Gly Asn Met Arg Cys Thr Ile Cys545 550 555 560Ile104570PRTInfluenza virus A 104Met Asn Thr Gln Ile Ile Val Ile Leu Val Leu Gly Leu Ser Met Val1 5 10 15Lys Ser Asp Lys Ile Cys Leu Gly His His Ala Val Ala Asn Gly Thr 20 25 30Lys Val Asn Thr Leu Thr Glu Arg Gly Val Glu Val Val Asn Ala Thr 35 40 45Glu Thr Val Glu Ile Thr Gly Ile Asp Lys Val Cys Thr Lys Gly Lys 50 55 60Lys Ala Val Asp Leu Gly Ser Cys Gly Ile Leu Gly Thr Ile Ile Gly65 70 75 80Pro Pro Gln Cys Asp Leu His Leu Glu Phe Lys Ala Asp Leu Ile Ile 85 90 95Glu Arg Arg Asn Ser Ser Asp Ile Cys Tyr Pro Gly Arg Phe Thr Asn 100 105 110Glu Glu Ala Leu Arg Gln Ile Ile Arg Glu Ser Gly Gly Ile Asp Lys 115 120 125Glu Ser Met Gly Phe Arg Tyr Ser Gly Ile Arg Thr Asp Gly Ala Thr 130 135 140Ser Ala Cys Lys Arg Thr Val Ser Ser Phe Tyr Ser Glu Met Lys Trp145 150 155 160Leu Ser Ser Ser Met Asn Asn Gln Val Phe Pro Gln Leu Asn Gln Thr 165 170 175Tyr Arg Asn Thr Arg Lys Glu Pro Ala Leu Ile Val Trp Gly Val His 180 185 190His Ser Ser Ser Leu Asp Glu Gln Asn Lys Leu Tyr Gly Thr Gly Asn 195 200 205Lys Leu Ile Thr Val Gly Ser Ser Lys Tyr Gln Gln Ser Phe Ser Pro 210 215 220Ser Pro Gly Ala Arg Pro Lys Val Asn Gly Gln Ala Gly Arg Ile Asp225 230 235 240Phe His Trp Met Leu Leu Asp Pro Gly Asp Thr Val Thr Phe Thr Phe 245 250 255Asn Gly Ala Phe Ile Ala Pro Asp Arg Ala Thr Phe Leu Arg Ser Asn 260 265 270Ala Pro Ser Gly Ile Glu Tyr Asn Gly Lys Ser Leu Gly Ile Gln Ser 275 280 285Asp Ala Gln Ile Asp Glu Ser Cys Glu Gly Glu Cys Phe Tyr Ser Gly 290 295 300Gly Thr Ile Asn Ser Pro Leu Pro Phe Gln Asn Ile Asp Ser Arg Ala305 310 315 320Val Gly Lys Cys Pro Arg Tyr Val Lys Gln Ser Ser Leu Pro Leu Ala 325 330 335Leu Gly Met Lys Asn Val Pro Glu Lys Ile Arg Thr Arg Gly Leu Phe 340 345 350Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Leu Ile Asp 355 360 365Gly Trp Tyr Gly Phe Arg His Gln Asn Ala Gln Gly Gln Gly Thr Ala 370 375 380Ala Asp Tyr Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile Thr Gly Lys385 390 395 400Leu Asn Arg Leu Ile Glu Lys Thr Asn Lys Gln Phe Glu Leu Ile Asp 405 410 415Asn Glu Phe Thr Glu Val Glu Gln Gln Ile Gly Asn Val Ile Asn Trp 420 425 430Thr Arg Asp Ser Leu Thr Glu Ile Trp Ser Tyr Asn Ala Glu Leu Leu 435 440 445Val Ala Met Glu Asn Gln His Thr Ile Asp Leu Ala Asp Ser Glu Met 450 455 460Asn Lys Leu Tyr Glu Arg Val Arg Arg Gln Leu Arg Glu Asn Ala Glu465 470 475 480Glu Asp Gly Thr Gly Cys Phe Glu Ile Phe His Arg Cys Asp Asp Gln 485 490 495Cys Met Glu Ser Ile Arg Asn Asn Thr Tyr Asn His Thr Glu Tyr Arg 500 505 510Gln Glu Ala Leu Gln Asn Arg Ile Met Ile Asn Pro Val Lys Leu Ser 515 520 525Ser Gly Tyr Lys Asp Val Ile Leu Trp Phe Ser Phe Gly Ala Ser Cys 530 535 540Val Met Leu Leu Ala Ile Ala Met Gly Leu Ile Phe Met Cys Val Lys545 550 555 560Asn Gly Asn Leu Arg Cys Thr Ile Cys Ile 565 570105560PRTInfluenza virus A 105Met Asn Thr Gln Ile Leu Ile Leu Ala Leu Val Ala Ile Ile Pro Thr1 5 10 15Asn Ala Asp Lys Ile Cys Leu Gly His His Ala Val Ser Asn Gly Ala 20 25 30Lys Val Asn Thr Leu Thr Glu Arg Gly Val Glu Val Val Asn Ala Thr 35 40 45Glu Thr Val Glu Arg Thr Asn Val Pro Arg Ile Cys Ser Lys Gly Lys 50 55 60Arg Thr Val Asp Leu Gly Gln Cys Gly Leu Leu Gly Thr Ile Thr Gly65 70 75 80Phe Pro Gln Cys Asp Gln Phe Leu Glu Phe Ser Ala Asp Leu Ile Ile 85 90 95Glu Arg Arg Glu Gly Asn Asp Val Cys Tyr Pro Gly Lys Phe Val Asn 100 105 110Glu Glu Ala Leu Arg Gln Ile Leu Arg Lys Ser Gly Gly Ile Asp Lys 115 120 125Glu Thr Met Gly Phe Thr Tyr Ser Gly Ile Arg Thr Asn Gly Ala Thr 130 135 140Ser Ala Cys Arg Arg Ser Gly Ser Ser Phe Tyr Ala Glu Met Lys Trp145 150 155 160Leu Leu Ser Asn Thr Asp Asn Ala Ala Phe Pro Gln Met Thr Lys Ser 165 170 175Tyr Lys Asn Ile Arg Lys Asp Pro Ala Leu Ile Ile Trp Gly Ile His 180 185 190His Ser Gly Ser Thr Ala Glu Gln Thr Lys Leu Tyr Gly Ser Gly Asn 195 200 205Lys Leu Ile Thr Val Gly Ser Ser Asn Tyr Gln Gln Ser Phe Val Pro 210 215 220Ser Pro Gly Ala Arg Pro Gln Val Asn Gly Gln Ser Gly Arg Ile Asp225 230 235 240Phe His Trp Leu Met Leu Asn Pro Asn Asp Thr Val Thr Phe Ser Phe 245 250 255Asn Gly Ala Phe Ile Ala Pro Asp Arg Ala Ser Phe Leu Arg Gly Lys 260 265 270Ser Met Gly Ile Gln Ser Glu Val Gln Val Asp Ala Asn Cys Glu Gly 275 280 285Asp Cys Tyr His Asp Gly Gly Thr Ile Leu Ser Ser Leu Pro Phe Gln 290 295 300Asn Ile Asn Ser Arg Thr Val Gly Glu Cys Pro Arg Tyr Val Lys Gln305 310 315 320Glu Ser Leu Leu Leu Ala Thr Gly Met Lys Asn Val Pro Glu Ile Pro 325 330 335Lys Gly Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly 340 345 350Trp Glu Gly Leu Val Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ala 355 360 365Gln Gly Glu Gly Thr Ala Ala Asp Tyr Lys Ser Thr Gln Ser Ala Ile 370 375 380Asp Gln Ile Thr Gly Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn Gln385 390 395 400Gln Phe Glu Leu Ile Asp Asn Glu Phe Thr Glu Val Glu Lys Gln Ile 405 410 415Gly Asn Val Ile Asn Trp Thr Arg Asp Ser Leu Thr Glu Met Trp Ser 420 425 430Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn Gln His Thr Ile Asp 435 440 445Leu Ala Asp Ser Glu Met Asn Lys Leu Tyr Glu Arg Val Arg Arg Gln 450 455 460Leu Arg Glu Asn Ala Glu Glu Asp Gly Thr Gly Cys Phe Glu Ile Phe465 470 475 480His Lys Cys Asp Asp Asp Cys Met Ala Ser Ile Arg Asn Asn Thr Tyr 485 490 495Asp His Ser Lys Tyr Arg Glu Glu Ala Ile Gln Asn Arg Ile Gln Ile

500 505 510Asp Pro Val Lys Leu Ser Ser Gly Tyr Lys Asp Val Ile Leu Trp Phe 515 520 525Ser Phe Gly Ala Ser Cys Phe Ile Leu Leu Ala Ile Ala Met Gly Leu 530 535 540Val Phe Ile Cys Val Lys Asn Gly Asn Met Arg Cys Thr Ile Cys Ile545 550 555 560106566PRTInfluenza virus A 106Met Glu Ala Arg Leu Leu Val Leu Leu Cys Ala Phe Ala Ala Thr Asn1 5 10 15Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Lys Leu Lys Gly Ile 50 55 60Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly65 70 75 80Asn Pro Glu Cys Asp Leu Leu Leu Thr Ala Ser Ser Trp Ser Tyr Ile 85 90 95Val Glu Thr Ser Asn Ser Glu Asn Gly Thr Cys Tyr Pro Gly Asp Phe 100 105 110Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125Glu Lys Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Glu 130 135 140Thr Thr Lys Gly Val Thr Ala Ala Cys Ser Tyr Ala Gly Ala Ser Ser145 150 155 160Phe Tyr Arg Asn Leu Leu Trp Leu Thr Lys Lys Gly Ser Ser Tyr Pro 165 170 175Lys Leu Ser Lys Ser Tyr Val Asn Asn Lys Gly Lys Glu Val Leu Val 180 185 190Leu Trp Gly Val His His Pro Pro Thr Gly Thr Asp Gln Gln Ser Leu 195 200 205Tyr Gln Asn Ala Asp Ala Tyr Val Ser Val Gly Ser Ser Lys Tyr Asn 210 215 220Arg Arg Phe Thr Pro Glu Ile Ala Ala Arg Pro Lys Val Arg Asp Gln225 230 235 240Ala Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr 245 250 255Ile Thr Phe Glu Ala Thr Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe 260 265 270Ala Leu Asn Arg Gly Ser Gly Ser Gly Ile Ile Thr Ser Asp Ala Pro 275 280 285Val His Asp Cys Asn Thr Lys Cys Gln Thr Pro His Gly Ala Ile Asn 290 295 300Ser Ser Leu Pro Phe Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys305 310 315 320Pro Lys Tyr Val Arg Ser Thr Lys Leu Arg Met Ala Thr Gly Leu Arg 325 330 335Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly 340 345 350Phe Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr 355 360 365His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser 370 375 380Thr Gln Asn Ala Ile Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile385 390 395 400Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Asn 405 410 415Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe 420 425 430Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn 435 440 445Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Arg Asn Leu Tyr Glu 450 455 460Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly465 470 475 480Cys Phe Glu Phe Tyr His Lys Cys Asp Asp Ala Cys Met Glu Ser Val 485 490 495Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu 500 505 510Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Met Gly Val Tyr 515 520 525Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu 530 535 540Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu545 550 555 560Gln Cys Arg Ile Cys Ile 565107552PRTInfluenza virus A 107Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val1 5 10 15Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 20 25 30Leu Glu Arg Thr His Asn Gly Lys Leu Cys Asp Leu Asn Gly Val Lys 35 40 45Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 50 55 60Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val65 70 75 80Glu Lys Ala Ser Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn 85 90 95Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 100 105 110Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asn His Asp Ala Ser 115 120 125Ser Gly Val Ser Ser Ala Cys Pro Tyr Leu Gly Arg Ser Ser Phe Phe 130 135 140Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Ser Tyr Pro Thr Ile145 150 155 160Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 165 170 175Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln 180 185 190Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg 195 200 205Leu Val Pro Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Gln Ser Gly 210 215 220Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn225 230 235 240Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile 245 250 255Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly 260 265 270Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser 275 280 285Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 290 295 300Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Thr305 310 315 320Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile 325 330 335Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr 340 345 350Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln 355 360 365Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser 370 375 380Ile Ile Asn Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe385 390 395 400Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp 405 410 415Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met 420 425 430Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu 435 440 445Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly 450 455 460Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu465 470 475 480Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala 485 490 495Arg Leu Asn Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Met Gly 500 505 510Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala 515 520 525Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly 530 535 540Ser Leu Gln Cys Arg Ile Cys Ile545 550108564PRTInfluenza virus A 108Met Leu Ser Ile Val Ile Leu Phe Leu Leu Ile Ala Glu Asn Ser Ser1 5 10 15Gln Asn Thr Tyr Gly Asn Pro Val Ile Cys Met Gly His His Ala Val 20 25 30Ala Asn Gly Thr Met Val Lys Thr Leu Ala Asp Asp Gln Val Glu Val 35 40 45Val Thr Ala Gln Glu Leu Val Glu Ser Gln Asn Leu Pro Glu Leu Cys 50 55 60Pro Ser Pro Leu Arg Leu Val Asp Gly Gln Thr Cys Asp Ile Ile Asn65 70 75 80Gly Ala Leu Gly Ser Pro Gly Cys Asp His Leu Asn Gly Ala Glu Trp 85 90 95Asp Val Phe Ile Glu Arg Pro Asn Ala Val Asp Thr Cys Tyr Pro Phe 100 105 110Asp Val Pro Glu Tyr Gln Ser Leu Arg Ser Ile Leu Ala Asn Asn Gly 115 120 125Lys Phe Glu Phe Ile Ala Glu Glu Phe Gln Trp Asn Thr Val Lys Gln 130 135 140Asn Gly Lys Ser Gly Ala Cys Lys Arg Ala Asn Val Asp Asp Phe Phe145 150 155 160Asn Arg Leu Asn Trp Leu Val Lys Ser Asp Gly Asn Ala Tyr Pro Phe 165 170 175Gln Asn Leu Thr Lys Ile Asn Asn Gly Asp Tyr Ala Arg Leu Tyr Ile 180 185 190Trp Gly Val His His Pro Ser Thr Ser Thr Glu Gln Ile Asn Leu Tyr 195 200 205Lys Asn Asn Pro Gly Arg Val Thr Val Ser Thr Lys Thr Ser Gln Thr 210 215 220Ser Val Val Pro Asp Ile Gly Ser Arg Pro Leu Val Arg Gly Gln Ser225 230 235 240Gly Arg Val Ser Phe Tyr Trp Thr Ile Val Glu Pro Gly Asp Leu Ile 245 250 255Val Phe Asn Thr Ile Gly Asn Leu Ile Ala Pro Arg Gly His Tyr Lys 260 265 270Leu Asn Asn Gln Lys Lys Ser Thr Ile Leu Asn Thr Ala Ile Pro Ile 275 280 285Gly Ser Cys Val Ser Lys Cys His Thr Asp Lys Gly Ser Leu Ser Thr 290 295 300Thr Lys Pro Phe Gln Asn Ile Ser Arg Ile Ala Val Gly Asp Cys Pro305 310 315 320Arg Tyr Val Lys Gln Gly Ser Leu Lys Leu Ala Thr Gly Met Arg Asn 325 330 335Ile Pro Glu Lys Ala Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340 345 350Ile Glu Asn Gly Trp Gln Gly Leu Ile Asp Gly Trp Tyr Gly Phe Arg 355 360 365His Gln Asn Ala Glu Gly Thr Gly Thr Ala Ala Asp Leu Lys Ser Thr 370 375 380Gln Ala Ala Ile Asn Gln Ile Asn Gly Lys Leu Asn Arg Leu Ile Glu385 390 395 400Lys Thr Asn Asp Lys Tyr His Gln Ile Glu Lys Glu Phe Glu Gln Val 405 410 415Glu Gly Arg Ile Gln Asp Leu Glu Asn Tyr Val Glu Asp Thr Lys Ile 420 425 430Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln 435 440 445His Thr Ile Asp Val Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Arg 450 455 460Val Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Lys Gly Asn Gly Cys465 470 475 480Phe Glu Ile Phe His Lys Cys Asp Asn Asn Cys Ile Glu Ser Ile Arg 485 490 495Asn Gly Thr Tyr Asp His Asp Ile Tyr Arg Asp Glu Ala Ile Asp Asn 500 505 510Arg Phe Gln Ile Gln Gly Val Lys Leu Thr Gln Gly Tyr Lys Asp Ile 515 520 525Ile Leu Trp Ile Ser Phe Ser Ile Ser Cys Phe Leu Leu Val Ala Leu 530 535 540Leu Leu Ala Phe Ile Leu Trp Ala Cys Gln Asn Gly Asn Ile Arg Cys545 550 555 560Gln Ile Cys Ile109566PRTInfluenza virus A 109Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Ala Leu Gly1 5 10 15Gln Asp Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly 20 25 30His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile Thr Asp Asp 35 40 45Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr 50 55 60Gly Lys Ile Cys Asn Asn Pro His Arg Ile Leu Asp Gly Ile Asp Cys65 70 75 80Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Val Phe Gln 85 90 95Asn Glu Thr Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Phe Ser Asn 100 105 110Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val 115 120 125Ala Ser Ser Gly Thr Leu Glu Phe Ile Thr Glu Gly Phe Thr Trp Thr 130 135 140Gly Val Thr Gln Asn Gly Gly Ser Asn Ala Cys Lys Arg Gly Pro Gly145 150 155 160Ser Gly Phe Phe Ser Arg Leu Asn Trp Leu Thr Lys Ser Gly Ser Thr 165 170 175Tyr Pro Val Leu Asn Val Thr Met Pro Asn Asn Asp Asn Phe Asp Lys 180 185 190Leu Tyr Ile Trp Gly Val His His Pro Ser Thr Asn Gln Glu Gln Thr 195 200 205Ser Leu Tyr Val Gln Ala Ser Gly Arg Val Thr Val Ser Thr Arg Arg 210 215 220Ser Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg225 230 235 240Gly Leu Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly 245 250 255Asp Val Leu Val Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro Arg Gly 260 265 270Tyr Phe Lys Met Arg Thr Gly Lys Ser Ser Ile Met Arg Ser Asp Ala 275 280 285Pro Ile Asp Thr Cys Ile Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile 290 295 300Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Ile Thr Tyr Gly Ala305 310 315 320Cys Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met 325 330 335Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Leu Phe Gly Ala Ile Ala 340 345 350Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly 355 360 365Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys 370 375 380Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val385 390 395 400Ile Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser 405 410 415Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr 420 425 430Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu 435 440 445Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe 450 455 460Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn465 470 475 480Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Glu Ser 485 490 495Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu 500 505 510Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys 515 520 525Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys 530 535 540Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Arg Gly Asn Ile545 550 555 560Arg Cys Asn Ile Cys Ile 565110565PRTInfluenza virus A 110Met Ile Ala Ile Ile Ile Leu Ala Ile Val Ala Ser Thr Ser Lys Ser1 5 10 15Lys Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Thr Gln Asp 20 25 30Thr Leu Ile Leu Lys Asn Val Thr Val Thr His Ser Val Glu Leu Leu 35 40 45Glu Ser Gln Lys Glu Glu Arg Phe Cys Arg Val Leu Asn Lys Ala Pro 50 55 60Leu Asp Leu Lys Gly Cys Thr Ile Glu Gly Trp Ile Leu Gly Asn Pro65 70 75 80Gln Cys Asp Ile Leu Leu Gly Asp Gln Arg Trp Ser Tyr Ile Val Glu 85 90 95Arg Pro Gly Ala Gln Asn Gly Ile Cys Tyr Pro Gly Ile Leu Asn Glu 100 105 110Leu Glu Glu Leu Lys Ala Leu Ile Gly Ser Gly Glu Arg Val Gln Arg 115 120 125Phe Glu Met Phe Pro Lys Ser Thr Trp Ala Gly Val Asp Thr Ser Arg 130 135 140Gly Val Thr Lys Ala Cys Pro Tyr Ile Ser Gly Ser Ser Phe Tyr Gly145 150 155 160Asn Leu Leu Trp Ile Ile Lys

Thr Glu Ser Ala Ala Tyr Pro Val Ile 165 170 175Lys Gly Thr Tyr Asn Asn Thr Gly Ser Gln Pro Ile Leu Tyr Phe Trp 180 185 190Gly Val His His Pro Pro Asp Thr Asn Glu Gln Asn Thr Leu Tyr Gly 195 200 205Ser Gly Asp Arg Tyr Val Arg Met Gly Thr Glu Ser Met Arg Phe Ala 210 215 220Lys Ser Pro Glu Ile Ala Ala Arg Pro Ala Val Asn Gly Gln Arg Gly225 230 235 240Arg Ile Asp Tyr Tyr Trp Ser Val Leu Lys Pro Gly Glu Thr Leu Asn 245 250 255Val Glu Ser Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Tyr Lys Phe 260 265 270Thr Ser Ser Asn Asn Lys Gly Ala Val Phe Lys Ser Asn Leu Pro Ile 275 280 285Glu Asn Cys Asp Ala Val Cys Gln Thr Val Ala Gly Ala Leu Arg Thr 290 295 300Asn Lys Thr Phe Gln Asn Val Ser Pro Leu Trp Ile Gly Glu Cys Pro305 310 315 320Lys Tyr Val Lys Ser Asp Ser Leu Arg Leu Ala Thr Gly Leu Arg Asn 325 330 335Val Pro Gln Ala Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340 345 350Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His 355 360 365His Glu Asn Ser Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr 370 375 380Gln Lys Ala Ile Asp Gly Ile Thr Asn Lys Val Asn Ser Ile Ile Asp385 390 395 400Lys Met Asn Thr Gln Phe Glu Ala Val Asp His Glu Phe Ser Asn Leu 405 410 415Glu Arg Arg Val Asp Asn Leu Asn Lys Arg Met Glu Asp Gly Phe Leu 420 425 430Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 435 440 445Arg Thr Leu Asp Leu His Asp Ala Asn Val Lys Asn Leu Tyr Glu Lys 450 455 460Val Lys Ser Gln Leu Arg Asp Asn Ala Lys Asp Leu Gly Asn Gly Cys465 470 475 480Phe Glu Phe Trp His Lys Cys Asp Asp Glu Cys Ile Asn Ser Val Lys 485 490 495Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Gln Asp Glu Ser Lys Leu Asn 500 505 510Arg Gln Glu Ile Asp Ser Val Lys Leu Glu Asn Leu Gly Val Tyr Gln 515 520 525Ile Leu Ala Ile Tyr Ser Thr Val Ser Ser Gly Leu Val Leu Val Gly 530 535 540Leu Ile Ile Ala Met Gly Leu Trp Met Cys Ser Asn Gly Ser Met Pro545 550 555 560Cys Lys Ile Cys Ile 565111565PRTInfluenza virus A 111Met Ala Val Lys Val Leu His Leu Leu Ile Ile Val Leu Gly Arg Tyr1 5 10 15Ser Ile Ala Asp Lys Ile Cys Ile Gly Tyr Leu Ser Asn Asn Ser Ser 20 25 30Asp Thr Val Asp Thr Leu Thr Glu Asn Gly Val Pro Val Thr Ser Ser 35 40 45Ile Asp Leu Val Glu Thr Asn His Thr Gly Thr Tyr Cys Ser Leu Asn 50 55 60Gly Ile Ser Pro Ile His Leu Gly Asp Cys Ser Phe Glu Gly Trp Ile65 70 75 80Val Gly Asn Pro Ser Cys Ala Thr Asn Ile Asn Ile Arg Glu Trp Ser 85 90 95Tyr Leu Ile Glu Asp Pro Asn Ala Pro Asn Lys Leu Cys Phe Pro Gly 100 105 110Glu Leu Asp Asn Asn Gly Glu Leu Arg His Leu Phe Ser Gly Val Asn 115 120 125Ser Phe Ser Arg Thr Glu Leu Ile Ser Pro Ser Lys Trp Gly Asp Val 130 135 140Leu Asp Gly Val Thr Ala Ser Cys Leu Asp Lys Gly Ala Ser Ser Phe145 150 155 160Tyr Arg Asn Leu Val Trp Leu Val Lys Gln Asn Asp Arg Tyr Pro Val 165 170 175Val Arg Gly Asp Tyr Asn Asn Thr Thr Gly Arg Asp Val Leu Val Leu 180 185 190Trp Gly Ile His His Pro Asp Thr Glu Thr Thr Ala Thr Lys Leu Tyr 195 200 205Val Asn Lys Asn Pro Tyr Thr Leu Val Ser Thr Lys Glu Trp Ser Lys 210 215 220Arg Tyr Glu Leu Glu Ile Gly Thr Arg Ile Gly Asp Gly Gln Arg Ser225 230 235 240Trp Met Lys Ile Tyr Trp His Leu Met His Pro Gly Glu Arg Ile Met 245 250 255Phe Glu Ser Asn Gly Gly Leu Leu Ala Pro Arg Tyr Gly Tyr Ile Ile 260 265 270Glu Lys Tyr Gly Thr Gly Arg Ile Phe Gln Ser Gly Ile Arg Met Ala 275 280 285Lys Cys Asn Thr Lys Cys Gln Thr Ser Met Gly Gly Val Asn Thr Asn 290 295 300Lys Thr Phe Gln Asn Ile Glu Arg Asn Ala Leu Gly Asp Cys Pro Lys305 310 315 320Tyr Ile Lys Ser Gly Gln Leu Lys Leu Ala Thr Gly Leu Arg Asn Val 325 330 335Pro Ser Ile Gly Glu Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile 340 345 350Glu Gly Gly Trp Pro Gly Leu Ile Asn Gly Trp Tyr Gly Phe Gln His 355 360 365Gln Asn Glu Gln Gly Thr Gly Ile Ala Ala Asp Lys Ala Ser Thr Gln 370 375 380Lys Ala Ile Asn Glu Ile Thr Thr Lys Ile Asn Asn Ile Ile Glu Lys385 390 395 400Met Asn Gly Asn Thr Asp Ser Ile Arg Gly Glu Phe Asn Gln Val Glu 405 410 415Lys Arg Ile Asn Met Leu Ala Asp Arg Val Asp Asp Ala Val Thr Asp 420 425 430Ile Trp Ser Tyr Asn Ala Lys Leu Leu Val Leu Ile Glu Asn Asp Arg 435 440 445Thr Leu Asp Leu His Asp Ala Asn Val Lys Asn Leu His Glu Gln Val 450 455 460Lys Arg Ala Leu Lys Asn Asn Ala Ile Asp Glu Gly Asp Gly Cys Phe465 470 475 480Asn Leu Leu His Lys Cys Asn Asp Ser Cys Met Glu Thr Ile Arg Asn 485 490 495Gly Thr Tyr Asn His Glu Asp Tyr Arg Glu Glu Ser Gln Leu Lys Arg 500 505 510Gln Glu Ile Glu Gly Ile Lys Leu Lys Thr Glu Asp Asn Val Tyr Lys 515 520 525Val Leu Ser Ile Tyr Ser Cys Ile Ala Ser Ser Ile Val Met Val Gly 530 535 540Leu Ile Leu Ala Phe Ile Met Trp Ala Cys Ser Ser Gly Asn Cys Arg545 550 555 560Phe Asn Val Cys Ile 565112565PRTInfluenza virus A 112Met Glu Lys Phe Ile Ala Ile Ala Thr Leu Ala Ser Thr Asn Ala Tyr1 5 10 15Asp Arg Ile Cys Ile Gly Tyr Gln Ser Asn Asn Ser Thr Asp Thr Val 20 25 30Asn Thr Leu Ile Glu Gln Asn Val Pro Val Thr Gln Thr Met Glu Leu 35 40 45Val Glu Thr Glu Lys His Pro Ala Tyr Cys Asn Thr Asp Leu Gly Ala 50 55 60Pro Leu Glu Leu Arg Asp Cys Lys Ile Glu Ala Val Ile Tyr Gly Asn65 70 75 80Pro Lys Cys Asp Ile His Leu Lys Asp Gln Gly Trp Ser Tyr Ile Val 85 90 95Glu Arg Pro Ser Ala Pro Glu Gly Met Cys Tyr Pro Gly Ser Val Glu 100 105 110Asn Leu Glu Glu Leu Arg Phe Val Phe Ser Ser Ala Ala Ser Tyr Lys 115 120 125Arg Ile Arg Leu Phe Asp Tyr Ser Arg Trp Asn Val Thr Arg Ser Gly 130 135 140Thr Ser Lys Ala Cys Asn Ala Ser Thr Gly Gly Gln Ser Phe Tyr Arg145 150 155 160Ser Ile Asn Trp Leu Thr Lys Lys Glu Pro Asp Thr Tyr Asp Phe Asn 165 170 175Glu Gly Ala Tyr Val Asn Asn Glu Asp Gly Asp Ile Ile Phe Leu Trp 180 185 190Gly Ile His His Pro Pro Asp Thr Lys Glu Gln Thr Thr Leu Tyr Lys 195 200 205Asn Ala Asn Thr Leu Ser Ser Val Thr Thr Asn Thr Ile Asn Arg Ser 210 215 220Phe Gln Pro Asn Ile Gly Pro Arg Pro Leu Val Arg Gly Gln Gln Gly225 230 235 240Arg Met Asp Tyr Tyr Trp Gly Ile Leu Lys Arg Gly Glu Thr Leu Lys 245 250 255Ile Arg Thr Asn Gly Asn Leu Ile Ala Pro Glu Phe Gly Tyr Leu Leu 260 265 270Lys Gly Glu Ser Tyr Gly Arg Ile Ile Gln Asn Glu Asp Ile Pro Ile 275 280 285Gly Asn Cys Asn Thr Lys Cys Gln Thr Tyr Ala Gly Ala Ile Asn Ser 290 295 300Ser Lys Pro Phe Gln Asn Ala Ser Arg His Tyr Met Gly Glu Cys Pro305 310 315 320Lys Tyr Val Lys Lys Ala Ser Leu Arg Leu Ala Val Gly Leu Arg Asn 325 330 335Thr Pro Ser Val Glu Pro Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340 345 350Ile Glu Gly Gly Trp Ser Gly Met Ile Asp Gly Trp Tyr Gly Phe His 355 360 365His Ser Asn Ser Glu Gly Thr Gly Met Ala Ala Asp Gln Lys Ser Thr 370 375 380Gln Glu Ala Ile Asp Lys Ile Thr Asn Lys Val Asn Asn Ile Val Asp385 390 395 400Lys Met Asn Arg Glu Phe Glu Val Val Asn His Glu Phe Ser Glu Val 405 410 415Glu Lys Arg Ile Asn Met Ile Asn Asp Lys Ile Asp Asp Gln Ile Glu 420 425 430Asp Leu Trp Ala Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Gln 435 440 445Lys Thr Leu Asp Glu His Asp Ser Asn Val Lys Asn Leu Phe Asp Glu 450 455 460Val Lys Arg Arg Leu Ser Ala Asn Ala Ile Asp Ala Gly Asn Gly Cys465 470 475 480Phe Asp Ile Leu His Lys Cys Asp Asn Glu Cys Met Glu Thr Ile Lys 485 490 495Asn Gly Thr Tyr Asp His Lys Glu Tyr Glu Glu Glu Ala Lys Leu Glu 500 505 510Arg Ser Lys Ile Asn Gly Val Lys Leu Glu Glu Asn Thr Thr Tyr Lys 515 520 525Ile Leu Ser Ile Tyr Ser Thr Val Ala Ala Ser Leu Cys Leu Ala Ile 530 535 540Leu Ile Ala Gly Gly Leu Ile Leu Gly Met Gln Asn Gly Ser Cys Arg545 550 555 560Cys Met Phe Cys Ile 565113565PRTInfluenza virus A 113Met Glu Lys Thr Leu Leu Phe Ala Ala Ile Phe Leu Cys Val Lys Ala1 5 10 15Asp Glu Ile Cys Ile Gly Tyr Leu Ser Asn Asn Ser Thr Asp Lys Val 20 25 30Asp Thr Ile Ile Glu Asn Asn Val Thr Val Thr Ser Ser Val Glu Leu 35 40 45Val Glu Thr Glu His Thr Gly Ser Phe Cys Ser Ile Asn Gly Lys Gln 50 55 60Pro Ile Ser Leu Gly Asp Cys Ser Phe Ala Gly Trp Ile Leu Gly Asn65 70 75 80Pro Met Cys Asp Glu Leu Ile Gly Lys Thr Ser Trp Ser Tyr Ile Val 85 90 95Glu Lys Pro Asn Pro Thr Asn Gly Ile Cys Tyr Pro Gly Thr Leu Glu 100 105 110Ser Glu Glu Glu Leu Arg Leu Lys Phe Ser Gly Val Leu Glu Phe Asn 115 120 125Lys Phe Glu Val Phe Thr Ser Asn Gly Trp Gly Ala Val Asn Ser Gly 130 135 140Val Gly Val Thr Ala Ala Cys Lys Phe Gly Gly Ser Asn Ser Phe Phe145 150 155 160Arg Asn Met Val Trp Leu Ile His Gln Ser Gln Thr Tyr Pro Val Ile 165 170 175Lys Arg Thr Phe Asn Asn Thr Lys Gly Arg Asp Val Leu Ile Val Trp 180 185 190Gly Ile His His Pro Ala Thr Leu Thr Glu His Gln Asp Leu Tyr Lys 195 200 205Lys Asp Ser Ser Tyr Val Ala Val Gly Ser Glu Thr Tyr Asn Arg Arg 210 215 220Phe Thr Pro Glu Ile Asn Thr Arg Pro Arg Val Asn Gly Gln Ala Gly225 230 235 240Arg Met Thr Phe Tyr Trp Lys Ile Val Lys Pro Gly Glu Ser Ile Thr 245 250 255Phe Glu Ser Asn Gly Ala Phe Leu Ala Pro Arg Tyr Ala Phe Glu Ile 260 265 270Val Ser Val Gly Asn Gly Lys Leu Phe Arg Ser Glu Leu Asn Ile Glu 275 280 285Ser Cys Ser Thr Lys Cys Gln Thr Glu Ile Gly Gly Ile Asn Thr Asn 290 295 300Lys Ser Phe His Asn Val His Arg Asn Thr Ile Gly Asp Cys Pro Lys305 310 315 320Tyr Val Asn Val Lys Ser Leu Lys Leu Ala Thr Gly Pro Arg Asn Val 325 330 335Pro Ala Ile Ala Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile 340 345 350Glu Gly Gly Trp Pro Gly Leu Ile Asn Gly Trp Tyr Gly Phe Gln His 355 360 365Arg Asp Glu Glu Gly Thr Gly Ile Ala Ala Asp Lys Glu Ser Thr Gln 370 375 380Lys Ala Ile Asp Gln Ile Thr Ser Lys Val Asn Asn Ile Val Asp Arg385 390 395 400Met Asn Thr Asn Phe Glu Ser Val Gln His Glu Phe Ser Glu Ile Glu 405 410 415Glu Arg Ile Asn Gln Leu Ser Lys His Val Asp Asp Ser Val Val Asp 420 425 430Ile Trp Ser Tyr Asn Ala Gln Leu Leu Val Leu Leu Glu Asn Glu Lys 435 440 445Thr Leu Asp Leu His Asp Ser Asn Val Arg Asn Leu His Glu Lys Val 450 455 460Arg Arg Met Leu Lys Asp Asn Ala Lys Asp Glu Gly Asn Gly Cys Phe465 470 475 480Thr Phe Tyr His Lys Cys Asp Asn Lys Cys Ile Glu Arg Val Arg Asn 485 490 495Gly Thr Tyr Asp His Lys Glu Phe Glu Glu Glu Ser Lys Ile Asn Arg 500 505 510Gln Glu Ile Glu Gly Val Lys Leu Asp Ser Ser Gly Asn Val Tyr Lys 515 520 525Ile Leu Ser Ile Tyr Ser Cys Ile Ala Ser Ser Leu Val Leu Ala Ala 530 535 540Leu Ile Met Gly Phe Met Phe Trp Ala Cys Ser Asn Gly Ser Cys Arg545 550 555 560Cys Thr Ile Cys Ile 565114564PRTInfluenza virus A 114Met Glu Lys Phe Ile Ile Leu Ser Thr Val Leu Ala Ala Ser Phe Ala1 5 10 15Tyr Asp Lys Ile Cys Ile Gly Tyr Gln Thr Asn Asn Ser Thr Glu Thr 20 25 30Val Asn Thr Leu Ser Glu Gln Asn Val Pro Val Thr Gln Val Glu Glu 35 40 45Leu Val His Arg Gly Ile Asp Pro Ile Leu Cys Gly Thr Glu Leu Gly 50 55 60Ser Pro Leu Val Leu Asp Asp Cys Ser Leu Glu Gly Leu Ile Leu Gly65 70 75 80Asn Pro Lys Cys Asp Leu Tyr Leu Asn Gly Arg Glu Trp Ser Tyr Ile 85 90 95Val Glu Arg Pro Lys Glu Met Glu Gly Val Cys Tyr Pro Gly Ser Ile 100 105 110Glu Asn Gln Glu Glu Leu Arg Ser Leu Phe Ser Ser Ile Lys Lys Tyr 115 120 125Glu Arg Val Lys Met Phe Asp Phe Thr Lys Trp Asn Val Thr Tyr Thr 130 135 140Gly Thr Ser Lys Ala Cys Asn Asn Thr Ser Asn Gln Gly Ser Phe Tyr145 150 155 160Arg Ser Met Arg Trp Leu Thr Leu Lys Ser Gly Gln Phe Pro Val Gln 165 170 175Thr Asp Glu Tyr Lys Asn Thr Arg Asp Ser Asp Ile Val Phe Thr Trp 180 185 190Ala Ile His His Pro Pro Thr Ser Asp Glu Gln Val Lys Leu Tyr Lys 195 200 205Asn Pro Asp Thr Leu Ser Ser Val Thr Thr Val Glu Ile Asn Arg Ser 210 215 220Phe Lys Pro Asn Ile Gly Pro Arg Pro Leu Val Arg Gly Gln Gln Gly225 230 235 240Arg Met Asp Tyr Tyr Trp Ala Val Leu Lys Pro Gly Gln Thr Val Lys 245 250 255Ile Gln Thr Asn Gly Asn Leu Ile Ala Pro Glu Tyr Gly His Leu Ile 260 265 270Thr Gly Lys Ser His Gly Arg Ile Leu Lys Asn Asn Leu Pro Met Gly 275 280 285Gln Cys Val Thr Glu Cys Gln Leu Asn Glu Gly Val Met Asn Thr Ser 290 295 300Lys Pro Phe Gln Asn Thr Ser Lys His Tyr Ile Gly Lys Cys Pro Lys305 310 315 320Tyr Ile Pro Ser Gly Ser Leu Lys Leu Ala Ile Gly Leu Arg Asn Val 325 330 335Pro Gln Val Gln Asp Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile 340 345 350Glu Gly Gly Trp Pro Gly Leu Val Ala Gly Trp Tyr Gly Phe

Gln His 355 360 365Gln Asn Ala Glu Gly Thr Gly Ile Ala Ala Asp Arg Asp Ser Thr Gln 370 375 380Arg Ala Ile Asp Asn Met Gln Asn Lys Leu Asn Asn Val Ile Asp Lys385 390 395 400Met Asn Lys Gln Phe Glu Val Val Asn His Glu Phe Ser Glu Val Glu 405 410 415Ser Arg Ile Asn Met Ile Asn Ser Lys Ile Asp Asp Gln Ile Thr Asp 420 425 430Ile Trp Ala Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Gln Lys 435 440 445Thr Leu Asp Glu His Asp Ala Asn Val Arg Asn Leu His Asp Arg Val 450 455 460Arg Arg Val Leu Arg Glu Asn Ala Ile Asp Thr Gly Asp Gly Cys Phe465 470 475 480Glu Ile Leu His Lys Cys Asp Asn Asn Cys Met Asp Thr Ile Arg Asn 485 490 495Gly Thr Tyr Asn His Lys Glu Tyr Glu Glu Glu Ser Lys Ile Glu Arg 500 505 510Gln Lys Val Asn Gly Val Lys Leu Glu Glu Asn Ser Thr Tyr Lys Ile 515 520 525Leu Ser Ile Tyr Ser Ser Val Ala Ser Ser Leu Val Leu Leu Leu Met 530 535 540Ile Ile Gly Gly Phe Ile Phe Gly Cys Gln Asn Gly Asn Val Arg Cys545 550 555 560Thr Phe Cys Ile115566PRTInfluenza virus A 115Met Ala Leu Asn Val Ile Ala Thr Leu Thr Leu Ile Ser Val Cys Val1 5 10 15His Ala Asp Arg Ile Cys Val Gly Tyr Leu Ser Thr Asn Ser Ser Glu 20 25 30Arg Val Asp Thr Leu Leu Glu Asn Gly Val Pro Val Thr Ser Ser Ile 35 40 45Asp Leu Ile Glu Thr Asn His Thr Gly Thr Tyr Cys Ser Leu Asn Gly 50 55 60Val Ser Pro Val His Leu Gly Asp Cys Ser Phe Glu Gly Trp Ile Val65 70 75 80Gly Asn Pro Ala Cys Thr Ser Asn Phe Gly Ile Arg Glu Trp Ser Tyr 85 90 95Leu Ile Glu Asp Pro Ala Ala Pro His Gly Leu Cys Tyr Pro Gly Glu 100 105 110Leu Asn Asn Asn Gly Glu Leu Arg His Leu Phe Ser Gly Ile Arg Ser 115 120 125Phe Ser Arg Thr Glu Leu Ile Pro Pro Thr Ser Trp Gly Glu Val Leu 130 135 140Asp Gly Thr Thr Ser Ala Cys Arg Asp Asn Thr Gly Thr Asn Ser Phe145 150 155 160Tyr Arg Asn Leu Val Trp Phe Ile Lys Lys Asn Thr Arg Tyr Pro Val 165 170 175Ile Ser Lys Thr Tyr Asn Asn Thr Thr Gly Arg Asp Val Leu Val Leu 180 185 190Trp Gly Ile His His Pro Val Ser Val Asp Glu Thr Lys Thr Leu Tyr 195 200 205Val Asn Ser Asp Pro Tyr Thr Leu Val Ser Thr Lys Ser Trp Ser Glu 210 215 220Lys Tyr Lys Leu Glu Thr Gly Val Arg Pro Gly Tyr Asn Gly Gln Arg225 230 235 240Ser Trp Met Lys Ile Tyr Trp Ser Leu Ile His Pro Gly Glu Met Ile 245 250 255Thr Phe Glu Ser Asn Gly Gly Phe Leu Ala Pro Arg Tyr Gly Tyr Ile 260 265 270Ile Glu Glu Tyr Gly Lys Gly Arg Ile Phe Gln Ser Arg Ile Arg Met 275 280 285Ser Arg Cys Asn Thr Lys Cys Gln Thr Ser Val Gly Gly Ile Asn Thr 290 295 300Asn Arg Thr Phe Gln Asn Ile Asp Lys Asn Ala Leu Gly Asp Cys Pro305 310 315 320Lys Tyr Ile Lys Ser Gly Gln Leu Lys Leu Ala Thr Gly Leu Arg Asn 325 330 335Val Pro Ala Ile Ser Asn Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340 345 350Ile Glu Gly Gly Trp Pro Gly Leu Ile Asn Gly Trp Tyr Gly Phe Gln 355 360 365His Gln Asn Glu Gln Gly Thr Gly Ile Ala Ala Asp Lys Glu Ser Thr 370 375 380Gln Lys Ala Ile Asp Gln Ile Thr Thr Lys Ile Asn Asn Ile Ile Asp385 390 395 400Lys Met Asn Gly Asn Tyr Asp Ser Ile Arg Gly Glu Phe Asn Gln Val 405 410 415Glu Lys Arg Ile Asn Met Leu Ala Asp Arg Ile Asp Asp Ala Val Thr 420 425 430Asp Ile Trp Ser Tyr Asn Ala Lys Leu Leu Val Leu Leu Glu Asn Asp 435 440 445Lys Thr Leu Asp Met His Asp Ala Asn Val Lys Asn Leu His Glu Gln 450 455 460Val Arg Arg Glu Leu Lys Asp Asn Ala Ile Asp Glu Gly Asn Gly Cys465 470 475 480Phe Glu Leu Leu His Lys Cys Asn Asp Ser Cys Met Glu Thr Ile Arg 485 490 495Asn Gly Thr Tyr Asp His Thr Glu Tyr Ala Glu Glu Ser Lys Leu Lys 500 505 510Arg Gln Glu Ile Asp Gly Ile Lys Leu Lys Ser Glu Asp Asn Val Tyr 515 520 525Lys Ala Leu Ser Ile Tyr Ser Cys Ile Ala Ser Ser Val Val Leu Val 530 535 540Gly Leu Ile Leu Ser Phe Ile Met Trp Ala Cys Ser Ser Gly Asn Cys545 550 555 560Arg Phe Asn Val Cys Ile 565116559PRTInfluenza virus A 116Met Glu Thr Ile Ser Leu Ile Thr Ile Leu Leu Val Val Thr Ala Ser1 5 10 15Asn Ala Asp Lys Ile Cys Ile Gly His Gln Ser Thr Asn Ser Thr Glu 20 25 30Thr Val Asp Thr Leu Thr Glu Thr Asn Val Pro Val Thr His Ala Lys 35 40 45Glu Leu Leu His Thr Glu His Asn Gly Met Leu Cys Ala Thr Ser Leu 50 55 60Gly His Pro Leu Ile Leu Asp Thr Cys Thr Ile Glu Gly Leu Val Tyr65 70 75 80Gly Asn Pro Ser Cys Asp Leu Leu Leu Gly Gly Arg Glu Trp Ser Tyr 85 90 95Ile Val Glu Arg Ser Ser Ala Val Asn Gly Thr Cys Tyr Pro Gly Asn 100 105 110Val Glu Asn Leu Glu Glu Leu Arg Thr Leu Phe Ser Ser Ala Ser Ser 115 120 125Tyr Gln Arg Ile Gln Ile Phe Pro Asp Thr Thr Asn Val Val Tyr Thr 130 135 140Asn Gly Ser Arg Ala Cys Ser Gly Ser Phe Tyr Arg Ser Met Arg Trp145 150 155 160Leu Ile Gln Lys Ser Gly Phe Tyr Pro Val Gln Asp Ala Gln Tyr Thr 165 170 175Asn Asn Arg Gly Lys Ser Ile Leu Phe Val Trp Gly Ile His His Pro 180 185 190Pro Thr Tyr Thr Glu Gln Thr Asn Leu Tyr Ile Arg Asn Asp Thr Thr 195 200 205Thr Ser Val Thr Thr Glu Asp Leu Asn Arg Thr Phe Lys Pro Val Ile 210 215 220Gly Pro Arg Pro Leu Val Asn Gly Leu Gln Gly Arg Ile Asp Tyr Tyr225 230 235 240Trp Ser Val Leu Lys Pro Gly Gln Thr Leu Arg Val Arg Ser Asn Gly 245 250 255Asn Leu Ile Ala Pro Trp Tyr Gly His Val Leu Ser Gly Gly Ser His 260 265 270Gly Arg Ile Leu Lys Tyr Asp Leu Lys Gly Gly Asn Cys Val Val Gln 275 280 285Cys Gln Thr Glu Lys Gly Gly Leu Asn Ser Thr Leu Pro Phe His Asn 290 295 300Ile Ser Lys Tyr Ala Phe Gly Thr Cys Pro Lys Tyr Val Arg Val Asn305 310 315 320Ser Leu Lys Leu Ala Val Gly Leu Arg Asn Val Pro Ala Arg Ser Ser 325 330 335Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Pro 340 345 350Gly Leu Val Ala Gly Trp Tyr Gly Phe Gln His Ser Asn Asp Gln Gly 355 360 365Val Gly Met Ala Ala Asp Arg Asp Ser Thr Gln Lys Ala Ile Asp Lys 370 375 380Ile Thr Ser Lys Val Asn Asn Ile Val Asp Lys Met Asn Lys Gln Tyr385 390 395 400Glu Ile Ile Asp His Glu Phe Ser Glu Val Glu Thr Arg Leu Asn Met 405 410 415Ile Asn Asn Lys Ile Asp Asp Gln Ile Gln Asp Val Trp Ala Tyr Asn 420 425 430Ala Glu Leu Leu Val Leu Leu Glu Asn Gln Lys Thr Leu Asp Glu His 435 440 445Asp Ala Asn Val Asn Asn Leu Tyr Asn Lys Val Lys Arg Ala Leu Gly 450 455 460Ser Asn Ala Met Glu Asp Gly Lys Gly Cys Phe Glu Leu Tyr His Lys465 470 475 480Cys Asp Asp Gln Cys Met Glu Thr Ile Arg Asn Gly Thr Tyr Asn Arg 485 490 495Arg Lys Tyr Arg Glu Glu Ser Arg Leu Glu Arg Gln Lys Ile Glu Gly 500 505 510Val Lys Leu Glu Ser Glu Gly Thr Tyr Lys Ile Leu Thr Ile Tyr Ser 515 520 525Thr Val Ala Ser Ser Leu Val Leu Ala Met Gly Phe Ala Ala Phe Leu 530 535 540Phe Trp Ala Asn Ser Asn Gly Ser Cys Arg Cys Asn Ile Cys Ile545 550 55511734PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 117Ser Thr Tyr Gly Met His Trp Val Ala Val Ser Ser Tyr Asp Gly Arg1 5 10 15Asn Glu Tyr Ala Lys Glu Val Gly Met Arg Ser Tyr Asp Ser Tyr Gly 20 25 30Met Asp11834PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 118Thr Ser Tyr Gly Met His Trp Val Ala Val Ser Ser Tyr Asp Gly Arg1 5 10 15Lys Lys Tyr Ala Lys Asp Val Ser Leu Arg Ala Tyr Asp His Tyr Gly 20 25 30Met Asp11934PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 119Ser Ser Tyr Gly Met His Trp Val Ala His Ile Ser Tyr Asp Gly Thr1 5 10 15Glu Thr His Ala Lys Asp Val Ser Leu Arg Ala Tyr Asp His Tyr Gly 20 25 30Met Asp12021PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 120Ser Ser Tyr Gly Met His Trp Val Ala Val Ile Ser Tyr Asp Gly Ser1 5 10 15Asn Lys Tyr Ala Lys 201218PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 121Tyr Tyr Tyr Tyr Tyr Gly Met Asp1 512234PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 122Ser Ser Tyr Gly Met His Trp Val Ala Val Ile Ser Tyr Asp Gly Ser1 5 10 15Asn Lys Tyr Ala Lys Asp Val Gly Met Arg Tyr Tyr Tyr Tyr Tyr Gly 20 25 30Met Asp12325PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 123Asn Ser Trp Leu Ala Trp Tyr Val Leu Phe Gly Ala Ala Ser Ser Leu1 5 10 15Gln Gln Gln Ser Asn Asn Phe Pro Tyr 20 2512424PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 124Ser Ser Trp Leu Ala Trp Tyr Leu Leu Ile Tyr Ala Ala Ser Ser Leu1 5 10 15Gln Gln Gln Ala Asn Ser Phe Pro 2012525PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 125Ser Ser Trp Leu Ala Trp Tyr Leu Leu Ile Tyr Ala Ala Ser Ser Leu1 5 10 15Gln Gln Gln Ala Asn Ser Phe Pro Tyr 20 2512627PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 126Asn Tyr Tyr Ala Asn Trp Tyr Leu Val Ile Tyr Gly Gly Asn Ser Arg1 5 10 15Pro Asp Ser Arg Asp Ser Ser Asp Asn His Arg 20 2512726PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 127Ser Tyr Tyr Ala Ser Trp Tyr Leu Val Ile Tyr Gly Lys Asn Asn Arg1 5 10 15Pro Asn Ser Arg Asp Ser Ser Gly Asn His 20 2512827PRTArtificial sequencesource/note="Description of artificial sequence Synthetic peptide" 128Ser Tyr Tyr Ala Ser Trp Tyr Leu Val Ile Tyr Gly Lys Asn Asn Arg1 5 10 15Pro Asn Ser Arg Asp Ser Ser Gly Asn His Ala 20 25

Claims

1. A vector collection comprising a repertoire of nucleic acid molecules encoding antibody light or heavy chains or fragments thereof, derived from a human patient donor who has suffered from, or is suffering from, a disease evoking antibody production to a target antigen, wherein said collection is identified with a unique barcode.

2. The vector collection of claim 1 comprising a repertoire of nucleic acid molecules encoding antibody light chains or fragments thereof.

3. The vector collection of claim 2 wherein the antibody light chains are .lamda. chains.

4. The vector collection of claim 2 wherein the antibody light chains are .kappa. chains.

5. The vector collection of claim 1 comprising a repertoire of nucleic acid molecules encoding antibody heavy chains or fragments thereof.

6. The vector collection of claim 1 wherein the barcode is a nucleotide sequence linked to or incorporated in the vectors present in the collection, and/or linked to or incorporated in the nucleic acid molecules encoding the antibody light or heavy chains or fragments thereof such that it does not interfere with the expression of said nucleic acid molecules.

7. The vector collection of claim 6 wherein said barcode is a contiguous non-coding nucleotide sequence of one to about 24 nucleotides.

8. The vector collection of claim 7 wherein said nucleotide sequence is linked to the 3' or 5' non-coding region of said nucleic acid molecules.

9. The vector collection of claim 6 wherein said nucleotide sequence is a coding sequence of one or more silent mutations incorporated into the nucleic acid molecules encoding the antibody light or heavy chains or fragments thereof.

10. The vector collection of claim 6 wherein said nucleotide sequence in non-contiguous.

11. The vector collection of claim 10 wherein at least part of said non-contiguous nucleotide sequence is linked to or incorporated in the vectors present in the collection.

12. The vector collection of claim 10 wherein at least part of said non-contiguous sequence is incorporated into the nucleic acid molecules encoding the antibody light or heavy chains or fragments thereof such that it does not interfere with the expression of said nucleic acid molecules.

13. The vector collection of claim 1 wherein the barcode is a peptide or polypeptide sequence.

14. The vector collection of claim 1 wherein the vectors are phagemid vectors.

15. The vector collection of claim 15 wherein the phagemid vectors contain a bacteriophage gene III and a stop codon between the nucleic acid molecules encoding antibody light or heavy chains or fragments thereof and the bacteriophage III gene.

16. The vector collection of claim 15 wherein the barcode is a non-coding contiguous nucleotide sequence inserted in the untranslated region following said stop codon.

17. Host cells comprising the vector collection of claim 1.

18. The host cells of claim 16 which are E. coli host cells.

19. A donor-specific antibody library comprising library members expressing a collection of antibodies or antibody fragments to a target antigen wherein said antibodies or antibody fragments are derived from a human donor who has suffered from, or is suffering from, a disease evoking antibody production to said target antigen, wherein said antibody library is identified with at least one unique barcode.

20. The donor-specific antibody library of claim 19 wherein said antibody heavy and light chains are separately identified each with a barcode unique to the human donor from whom it derived.

21. The donor-specific antibody library of claim 19 which is identified with one unique barcode.

22. The donor-specific antibody library of claim 19 wherein said antibodies or antibody fragments are composed of antibody heavy and light chains or fragments thereof encoded by nucleic acid molecules present in a vector.

23. The donor-specific antibody library of claim 22 wherein the barcode is a nucleotide sequence linked to or incorporated in the vectors present in the library, and/or linked to or incorporated in the nucleic acid molecules encoding the antibody light or heavy chains or fragments thereof such that it does not interfere with the expression of said nucleic acid molecules.

24. The donor-specific antibody library of claim 23 wherein said barcode is a contiguous non-coding nucleotide sequence of one to about 24 nucleotides.

25. The donor-specific antibody library of claim 24 wherein said nucleotide sequence is linked to the 3' or 5' non-coding region of said nucleic acid molecules.

26. The donor-specific antibody library of claim 23 wherein said nucleotide sequence is a coding sequence of one or more silent mutations incorporated into the nucleic acid molecules encoding the antibody light or heavy chains or fragments thereof.

27. The donor-specific antibody library of claim 23 wherein said nucleotide sequence in non-contiguous.

28. The donor-specific antibody library of claim 27 wherein at least part of said non-contiguous sequence is linked to or incorporated in the vectors present in the library.

29. The donor-specific antibody library of claim 27 wherein at least part of said non-contiguous sequence is incorporated into the nucleic acid molecules encoding the antibody light or heavy chains or fragments thereof such that it does not interfere with the expression of said nucleic acid molecules.

30. The donor-specific antibody library of claim 19 wherein the barcode is a peptide or polypeptide sequence.

31. The donor-specific antibody library of claim 22 wherein the vectors are phagemid vectors.

32. The donor-specific antibody library of claim 31 wherein the phagemid vectors contain a bacteriophage gene III and a stop codon between the nucleic acid molecules encoding antibody light or heavy chains or fragments thereof and the bacteriophage III gene.

33. The donor-specific antibody library of claim 19 expressing a collection of antibody heavy chains or fragments thereof.

34. The donor specific antibody library of claim 33 comprising more than one antibody heavy chain family.

35. The donor-specific antibody library of claim 19 wherein the medical history of said human donor shows that the patient has suffered from, or is suffering from said disease.

36. The donor-specific antibody library of claim 35 wherein it is independently confirmed that said human donor suffered from, or is suffering from said disease.

37. The donor-specific antibody library of claim 19, which is substantially devoid of antibodies and antibody fragments specifically binding antigens different from said target antigen.

38. The donor-specific antibody library of claim 19 wherein said target antigen is an influenza A virus.

39. The donor-specific antibody library of claim 38 wherein said target antigen is an isolate of influenza A virus H1, H2 or H3 subtype.

40. The donor-specific antibody library of claim 38 wherein the target antigen is selected from the group comprising H5, H7 and H9 influenza A virus subtypes.

41. The donor-specific antibody library of claim 38 expressing at least one antibody or antibody fragment specifically binding to more than one influenza A virus subtype.

42. The donor-specific antibody library of claim 39 expressing at least one antibody or antibody fragment binding to and neutralizing the H5N1 subtype of influenza virus A.

43. The donor-specific antibody library of claim 19 wherein the human donor has suffered from, or is suffering from a disease selected from the group consisting of the diseases listed in Table 1.

44. The donor-specific antibody library of claim 43 expressing at least one antibody or antibody fragment binding to an antigen associated with said target disease.

45. The donor-specific antibody library of claim 43 expressing at least one antibody or antibody fragment binding to and neutralizing an antigen associated with said target disease.

46. The donor-specific antibody library of claim 19 expressing a least one neutralizing antibody.

47. The donor-specific antibody library of Claim 19, wherein said donor-specific antibody library is a phage library.

48. The donor-specific antibody library of Claim 47, wherein said collection contains sequences encoding more than 106 different members of antibodies or antibody fragments.

49. The donor-specific antibody library of Claim 47, wherein said collection contains sequences encoding more than 109 different members of antibodies or antibody fragments.

50. The donor-specific antibody library of claim 19, which is selected from the group consisting of a spore-display library, a ribosome display library, a mRNA display library, a microbial cell display library, a yeast display library, and a mammalian display library.

51. The donor-specific antibody library of Claim 50, wherein said nucleic acid is reverse-transcribed from mRNA extracted from lymphocytes of said human donor.

52. The donor-specific antibody library of Claim 51, wherein said lymphocytes are derived from bone marrow, blood, spleen, or lymph node.

53. The donor-specific antibody library of Claim 52, wherein a serological profile of said human donor is generated prior to extraction of said mRNA.

54. The donor-specific antibody library of Claim 53, wherein a medical history of said human donor is examined prior to or following extraction of said mRNA.

55. A method of making a donor-specific library expressing a collection of antibodies or antibody fragments to a target antigen, comprising the steps of: a) obtaining mRNA from lymphocytes of a human patient donor who has suffered from, or who is suffering from a disease evoking antibody production to said target antigen; b) generating a collection of nucleic acids comprising sequences encoding an immunoglobulin repertoire of said patient by reverse transcription of said obtained mRNA: and c) identifying said donor-specific library with an unique barcode labeling said nucleic acids.

56. The method of Claim 55, further comprising steps of generating a serological profile of said patient and/or examining medical history of said patient prior or subsequent to step a).

57. The method of Claim 56, further comprises steps of: d) inserting said nucleic acids into expression vector; e) expressing said immunoglobulin repertoire; and f) displaying said immunoglobulin repertoire in a display system.

58. The method of Claim 57, wherein the display system is a phagemid.

59. The method of claim 58 further comprising the step of selecting members of the library based their ability to neutralize or activate the target antigen.

60. The method of claim 59 that yields at least one neutralizing antibody.

61. The method of claim 59 that yields more than one neutralizing antibody.

62. The method of claim 61 further comprising the step of creating one or more sub-libraries comprising library members that were found to neutralize or activate the target antigen.

63. The method of claim 59 comprising the step of sequencing at least one library member identified.

64. A method of treating or preventing a disease associated with a target antigen neutralized or activated by an antibody selected by the method of claim 59, comprising administering to said human patient in need an effective amount of the antibody selected.

65. The method of claim 64 wherein the antibody is a neutralizing antibody.

66. The method of claim 65 wherein the disease is an influenza virus A infection.

67. The method of claim 64 wherein the disease is selected from the group consisting of the diseases listed in Table 1.