AU8091694A

AU8091694A - Structured synthetic antigen libraries as diagnostics, vaccines and therapeutics

Info

Publication number: AU8091694A
Application number: AU80916/94A
Authority: AU
Inventors: Barbara Hosein; Stephen M Kaminsky; C. Wayne Koff; Jacek Kowalski; Douglas F Nixon; Alan M. Walfield; Chang Yi Wang; John Ye; Timothy J Zamb
Original assignee: United Biomedical Inc
Current assignee: United Biomedical Inc
Priority date: 1993-10-26
Filing date: 1994-10-26
Publication date: 1995-05-22
Also published as: EP0725838A1; IL111417A0; EP0725838A4; WO1995011998A1; CA2175579A1

Description

STRUCTURED SYNTHETIC ANTIGEN LIBRARIES AS DIAGNOSTICS, VACCINES AND THERAPEUTICS

FIELD OF THE INVENTION

The present invention relates to "structured synthetic antigen libraries" (SSAL) composed of related peptides synthesized simultaneously in a single peptide synthesis. This "structured" library contrasts to those libraries previously described as "random peptide libraries" in that the order or structure within a synthetic antigen is provided by invariant amino acid residues that define the framework sequence of the synthetic antigen. The specific amino acids and their frequency of appearance at a variant locus within aligned peptide sequences is defined by the primary sequences of the several variants that make up the alignment used to construct the antigen peptide library. A method of constructing an open diagnostic, vaccine or therapeutic for a mutational infectious agent is also provided. The invention further provides the SSAL in diagnostic methods, kits, vaccination methods, vaccine compositions and pharmaceutical compositions. The libraries are prepared from variable domains in proteins and provide improved vaccines, diagnostics and therapeutics for infectious agents, etc., from such proteins.

BACKGROUND OF THE INVENTION

Synthetic peptides have been used increasingly to map antigenic or immunogenic sites on the surface of proteins. These approaches have used: (1) nested sets of peptides as in "site-directed serology" to identify diagnostically useful epitopes; (2) overlapping sets of peptides to scan the length of a protein antigen to locate antigenic sites (a technique colloquially known as pepscan) , and (3) totally random peptide libraries which can be systematically analyzed to characterize particular antigenic sites. In this regard, synthetic peptides provide a powerful tool for the diagnostic field, particularly in the areas of diagnostic virology, blood screening and others. For example, using site-directed serology, sensitive and specific tests have been developed for the envelope and core proteins of human immunodeficiency virus (HIV) [see, e.g., U.S. Pat. Nos. 4,735,896 and 4,879,212], for the gag protein of human T-lymphotropic viruses I and II (HTLV I/II) [see, e.g., U.S. Pat. No. 4,833,071], and the structural and non-structural proteins of the hepatitis C virus (HCV) [see, e.g., U.S. Pat. No. 5,106,726].

In certain cases, peptide-based assays are unmatched in their ability to differentiate between or among closely related viruses, a problem often associated with other common diagnostic tests based on either viral lysates or recombinantly-produced proteins. While having many advantages, there are still pitfalls to peptide-based immunodiagnostics, as well as the other formats for immunodiagnostic tests, when dealing with infectious agents that vary significantly over time or may have many serotypes. Peptide-based and other immunodiagnostics are static by design and can not accommodate such variation.

As is well-known, many proteins from infectious agents, or sites on those proteins, can vary in sequence by strain. At a given time or in a particular geographic locale, a particular antigen may contain one or multiple point mutations relative to an arbitrary prototype strain. Dynamic variation thereby creates an extremely complex antigenic profile for a given site for which sensitive or specific detection may be increasingly difficult as the site "drifts" further from the prototype. Nonetheless, each antigen maintains a "fundamental motif" or "structural framework" that is representative of the prototype sequence from which the variants arise. The structural framework consists of generally invariant residues which can be exploited in conjunction with the inherently variable residues to design improved diagnostic tests that overcome the limitations of the present tests. Accordingly, the present invention provides a means to construct a synthetic peptide library termed a "structured synthetic antigen library" (SSAL) which addresses the need for "open" diagnostics, i.e. diagnostic kits which can expressly accommodate antigenic variation. Site-directed synthetic peptides also find use as vaccines. Vaccine development and delivery has been highly successful as a public health strategy in the control of acute viral and bacterial infectious diseases. However, several major globally important infectious diseases has proven elusive to attempts at designing effective vaccines. A recurrent theme among certain pathogens, contributing to unsuccessful or limited success of vaccines, is antigenic variation. Extensive antigenic variation, for example, is a hallmark for example of HIV (AIDS) , rhinovirus (the common cold) , influenza virus (flu) , Borrelia burcrdorferi (Lyme disease) , Chla vdia (pelvic inflammatory disease) , Plasmodium falciparum (malaria), Trypanosoma cruzi (Chagas' disease) . While biotechnology may enable production of large quantities of purified antigens containing protective epitopes, antigenic variation has thus far proved to be a significant obstacle for vaccine development in certain pathogens.

Effective vaccines generally must induce long term memory responses so that a subsequent protective response is elicited immediately upon exposure to a pathogen. A single recombinant protein or a single peptide confers only limited protective immunity to pathogens that exhibit extensive antigenic variation because of their limited capacity to induce heterotypic immune responses. Synthetic peptide technology using SSALs offers a powerful tool to develop the next generation vaccines because they encompass a broad range of sequences required to overcome problems associated with antigenic variation as previously discussed for diagnostic markers.

Therefore, a novel strategy for vaccine-induced protection against pathogens having multiple strains, forms, serovars, serotypes, etc., is required. The "structured synthetic antigen library" (SSAL) provides a solution to the problem of developing vaccines for such pathogens for the following reasons. First, SSALs can be synthesized to address serological variants of each major neutralizing determinant for a pathogen, then combined for effective multicomponent vaccines. For example, current strategies for HIV may focus on the use of a mixture of 5-10 synthetic peptides representing variant sequences within a clade for a single principal neutralizing domain (V3 loop) . An antigenic peptide library can be constructed to address not only the 5-10 major representative sequences, but also the thousands of variants within each clade that the virus is capable of producing escape from neutralizing antibodies. Second, SSALs can be synthesized to provide T-helper epitopes for a pathogen, thus overcoming the problem of immunogenetic population diversity (i.e. responders vs. non-responders) which has thus far limited the efficacy of experimental synthetic peptide vaccines. Third, SSALs can also be synthesized to include cytotoxic T cell (CTL) epitopes, again to overcome the problem of immunogenetic population diversity. Such SSALs can thereby stimulate CTL responses in broad populations, which in conjunction with a complementary serological response, is required for successful vaccines.

The "structured synthetic antigen library" differs from the random peptide libraries known in the art (see, e.g., Jung et al . , 1992, Angew. Chem. Int. Ed. Enql. 31;367) . Fully random peptide libraries, produced both recombinantly and synthetically, have been used to identify receptor ligands, to determine substrate specificity, to identify epitopes. Moreover, recombinantly-produced antibody libraries, typically containing millions of randomly generated antibody Fab chains, have been described to select for high affinity antibodies.

.Among the previous reports was the construction and screening of vast libraries of peptides and proteins for the ability to bind a variety of receptor molecules. This was conducted by genetic techniques fusing DNA encoding a peptide to a filamentous bacteriophage phage coat protein, to another protein for display on E. coli cells, or to a DNA binding protein such as a lac repressor. In those approaches, peptides with the desired binding properties were isolated by affinity selection (panning) on an immobilized receptor. After several rounds of panning, individual clones from an enriched clonal population were analyzed to determine the binding properties and sequences of the peptides that they contained.

Further, there are reports of synthetic peptide combinatorial libraries composed of mixtures of free peptides in quantities sufficient for use in ligand-binding assays. In one instance, the screening of a positional scanning synthetic library combined with an iterative selection and synthesis process, permitted the systematic identification of optimal peptide ligands from a "random peptide library" composed of 34 million hexa-peptides (Dooley et al . , 1993, Life Sci. 52:1509) .

Rohren et al . (1993, J. Exp. Med. 177: 1713) reported a site-specific positional scanning synthetic peptide library (certain residues were fixed (anchors) and other positions had a random assortment of residues) . This peptide library was used to identify critical residues in peptide binding in the "pockets of MHC class I K^b molecules. Blake et al . (1992, Bioconiug Chem. 3_.:510) described random tetrapeptides (a set of 50,625 peptides) and hexapeptides (a set of 16,777,216 peptides) to identify residues important in the reactivity of the peptides with antibodies. The library was highly variable at each position.

Shumacher et al . (1992, Eur. J. Immunol. 22: 1405) reported a "limited complexity" peptide library to analyze the peptide preference of MHC class I molecules. This study taught that a singly represented T cell epitope could be detected and further suggested using the library to identify new T cell epitopes. The peptide library consisted of 8-mer or 9-mer libraries/mixtures with equimolar amounts of the variable amino acids at the multiply substituted positions and was used for epitope mapping. The peptides were based on the 9-mer Sendai virus NP peptide and the VSV-N peptide (specific for the mouse K^b locus) with the variable amino acids from correspondingly known K^b epitopes. Hortin et al . (1992, Biochem. Int. 26: 731) reported a series of 5 synthetic peptide libraries of 7 amino acids in length wherein the last two carboxyl residues were totally random, i.e. included all twenty amino acids in equal proportion. One of these mixtures inhibited platelet adhesion.

Gras-Masse et al . (1992, Peptide Res. 5_: 211) described a "mixotope" synthetic peptide library from the hypervariable gpl20 V3 loop of HIV-1 for use as a vaccine. The mixotope was 22-25 amino acids in length with invariant and variant positions. The variant-position residues (also termed degenerate positions) consisted only of those amino acids which exceeded 7% abundance at a particular position and were incorporated in equimolar amounts. The peptide library approaches described above with libraries of randomized complexity generally provide a research tool for the selection of a particular ligand or antibody to facilitate further drug design. The present invention provides "structured synthetic antigen libraries" SSAL as the key bioactive ingredient in vaccines, diagnostics and therapeutics. Such libraries provide the broad range of sequences necessitated by the strain or antigenic variation by anticipating temporal and geographic variation in antigens. The resulting SSALs are collections of peptides that maintain the character of the antigen and can simultaneously provide cross-reactivity to multiple strains of an infectious agent.

SUMMARY OF THE INVENTION

The present invention provides a library of peptides comprising a known antigenic site, epitope or ligand on a protein molecule, wherein (1) said library is optionally linked to a substantially invariant peptide domain or a core branched sequence; (2) said library consists of an ensemble domain; (3) the overall peptide length is about 8 to about 100 amino acids; (4) the sequence of said ensemble domain is represented by a consensus formula; (5) said ensemble is immunogenic, diagnostic for the epitope or is a therapeutic; and (6) the consensus formula provides that each sequence position in the ensemble contain either a single amino acid or multiple amino acids, and that when a position contains multiple amino acids, (a) the identity and ratio of those amino acids being determined by the relative prevalence of amino acids in a consensus of known variant sequences for that epitope or ligand or (b) the identity of those amino acids being determined by the amino acids present in a consensus of known variant sequences for that epitope or ligand and the ratio of amino acids is equimolar. Furthermore the antigenic sites, epitopes or ligands of the library are from a virus, bacterium, parasite, tumor antigen, allergen or other protein antigen, from a diagnostic marker site of an infectious agent or disease, from a therapeutically valuable ligand or from a helper T cell epitope or a cytotoxic T lymphocyte (CTL) epitope. More particularly, these antigenic sites, epitopes or ligands can be from an envelope, core, NS1, NS3, NS4 or NS5 proteins of HCV; a gpl20 V3 loop, gp41 envelope protein or the gp40 envelope protein of HIV; an envelope protein of

HTLV I/II; an HA protein or a mutant HA domain of influenza A virus; an major outer membrane protein of Chlamydia trachomtis; a neuraminidase, SAPA or CRA site of T. cruzi; an envelope B cell site or helper T cell site of type 2 Dengue virus; an M protein of streptococcus; a carbohydrate recognition site of selectin; a promiscuous helper T cell sites; a CTL epitope from HIV gag specific for HLA-B27; or an OspA, OsaB, OspC or flagellin protein of Lyme disease. SSAL libraries of the present invention are also shown in Figs. 2-11, 13, and 15-34.

Another aspect of the invention is directed to peptide compositions containing the above libraries, including immunogenic peptide compositions.

Still another aspect of the invention provides a method of detecting antibodies associated with an infectious agent or a disease state which comprises using an effective amount of the subject peptide compositions in an immunoassay procedure. Also provided is a method of detecting infection or a disease state which comprises contacting an effective amount of the subject peptide compositions with a body fluid, tissue or tissue extract in an immunoassay procedure for a time sufficient to form a complex between said peptide composition and any antibody in said fluid, said tissue, or said tissue extract, and subjecting said complex to a detecting means. Preferably, the immunoassay procedure is an ELISA or a PHA procedure. Similarly, the invention provides a kit for detection or diagnosis of an infectious agent or a disease state comprising a first container adapted to contain the subject peptide composition. In one embodiment, the kit is an ELISA or PHA test kit.

A still further aspect of the invention relates to a method of treating a disease which comprises administering an effective amount of the subject peptide compositions to a patient for a time sufficient to elicit an efficacious result. In this respect, the invention provides a pharmaceutical composition comprising the subject library, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Yet a further aspect of the invention is directed to a method of vaccination which comprises administering to a mammal an amount of the subject library effective to produce a protective immune response against an infectious agent which provided the basis of the library as well as a vaccine composition therefor. Such vaccine composition contains the subject library, or an acceptable salt thereof, formulated with immunologically acceptable carrier or adjuvant.

Another aspect of the invention is directed to a method of making an open diagnostic, vaccine or therapeutic composition for a mutatable infectious agent or for a protein site of known diversity which comprises (1) aligning a collection of primary amino acid sequences for a related family of antigens, epitopes, diagnostic markers or therapeutic sites from said mutatable infectious agent or said protein site; (2) identifying invariant and variant amino acid positions in said alignment; (3) calculating a consensus formula for the sequence of a structured synthetic antigen library (SSAL) ; (4) preparing the SSAL from the consensus formula. The consensus formula is determined by from examining each sequence position in the alignment to ascertain the presence of a single amino acid or multiple amino acids. When a position contains multiple amino acids, then (a) the identity and ratio of the multiple amino acids at that position in the SSAL is determined by the relative prevalence of amino acids in the alignment, (b) the identity of the multiple amino acids is determined by the amino acids present in the alignment and the ratio of the multiple amino acids is equimolar, or (c) the identity of the multiple amino acids is determined by the amino acids present in the alignment and the ratio of the multiple amino acids is weighted towards the predominant amino acid at said position. The process is repeated for each position of the sequence which can be represented by multiple amino acids. This method for making a vaccine, diagnostic or therapeutic SSAL can be used with mutatable infectious agents from a virus, bacterium or parasite or with a protein site from a tumor antigen, allergen or other protein antigen, from a helper T cell epitope or a CTL epitope. Similarly, the family of antigens, epitopes, diagnostic markers or therapeutic sites can be selected from an envelope, core, NS1, NS3, NS4 or NS5 proteins of HCV; a gpl20 V3 loop, gp41 envelope protein or the gp40 envelope protein of HIV; an envelope protein of HTLV I/II; an HA protein or a mutant HA domain of influenza A virus; an major outer membrane protein of Chlamydia trachomtis; a neuraminidase, SAPA or CRA site of T. cruzi; an envelope B cell site or helper T cell site of type 2 Dengue virus; an M protein of streptococcus; a carbohydrate recognition site of selectin; a promiscuous helper T cell site; a CTL epitope from HIV gag specific for HLA-B27; or an OspA, OspB, OspC or flagellin protein of Lyme disease. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates a generalized mathematical and chemical representation of a structured synthetic antigen library (SSAL) . Fig. 2 illustrates a primary amino acid sequence alignment (SEQ ID NO:l) of an antigenic NS4 peptide (47-mer) from eight representative strains of HCV. More weight (i.e. three times the listing frequency) is given to the prototype sequence used in the construction of an SSAL HCV NS4 to allow easy calculation of the ratio for each amino acid at the specified position.

Fig. 3 illustrates a primary amino acid sequence alignment (SEQ ID NO:2) of an antigenic core peptide (61- mer) from eight representative strains of HCV. An SS.AL HCV core is similarly constructed based on the given antigen structure and its source sequences.

Fig. 4 illustrates a primary amino acid sequence alignment (SEQ ID NO:3) of an antigenic NS3 peptide (80-mer) from eight representative strains of HCV. An SSAL HCV NS3 is similarly constructed based on the given antigen structure and its source sequences.

Fig. 5 illustrates a primary amino acid sequence alignment (SEQ ID NO:4) of an antigenic NS5 peptide (44-mer, Sequence #1) from eight representative strains of HCV. An SSAL HCV NS5 (#1) is similarly constructed based on the given antigen structure and its source sequences.

Fig. 6 illustrates a primary amino acid sequence alignment (SEQ ID NO:5) of an antigenic NS5 peptide (41-mer, Sequence #2) from eight representative strains of HCV. An SSAL HCV NS5 (#2) is similarly constructed based on the given antigen structure and its source sequences.

Fig. 7 illustrates a primary amino acid sequence alignment (SEQ ID NO:6) of an antigenic Env peptide (55-mer, Sequence #1) from sixteen representative strains of HCV. An SSAL HCV ENV (#1) is similarly constructed based on the given antigen structure and its source sequences.

Fig. 8 illustrates a primary amino acid sequence alignment (SEQ ID NO:7) of an antigenic Env peptide (44-mer, Sequence #2) from sixteen representative strains of HCV. An

SSAL HCV ENV (#2) is constructed based on the given antigen structure and its source sequences.

Fig. 9 illustrates a primary amino acid sequence alignment (SEQ ID NO:8) of an antigenic Env peptide (40-mer, Sequence #3) from sixteen representative strains of HCV. An

SSAL HCV ENV (#3) is constructed based on the given antigen structure and its source sequences.

Fig. 10 illustrates a primary amino acid sequence alignment (SEQ ID NO:9) of an antigenic Env peptide (35 mer, Sequence #4) from ten representative strains of HCV. An

SSAL HCV ENV (#4) is constructed based on the given antigen structure and its source sequences.

Fig. 11 illustrates a primary amino acid sequence alignment (SEQ ID NO:10) of an antigenic NS1 peptide from eight representative strains of HCV. An SSAL HCV NS1 is constructed based on the given antigen structure and its source sequences.

Fig. 12 illustrates four types of heteromeric synthetic carriers used in the production of branched SSALs: dendritic (A) , linear (B) , tree-like (C) , and tetrameric (D) .

Attached onto the core carriers is the SSAL synthesized according to a specified mathematical and chemical representation. Each dash shown within a peptide represents a particular amino acid corresponding to a conserved position in the original protein. Each 0 represents a position where peptides in the library contain any one of a fixed number of amino acids determined from the composition of the amino acid pool used in that cycle of synthesis. The variable positions can be at any position in the peptide except branch points.

Fig. 13A illustrates a primary amino acid sequence alignment (SEQ ID NO:11, top Fig. 13B) of an antigenic gpl20 Env peptide (33 mer) , representing the principal neutralizing determinant (PND), the V3 domain, of the HIV-1. An SSAL HIV-1 gpl20 V3 is constructed according to a previously optimized V3 framework (Fig. 13B; SEQ ID NO:11). Sixteen V3 sequences representative of geographically distinct HIV field isolates were used with more weight given to the Consensus S sequence to allow easier calculation of ratios for the various amino acids. An SSAL is constructed based on the given antigen structure and its source sequences. The SSAL HIV-1 gpl20 V3 of 10⁹ complexity is shown in Fig. 13C (SEQ ID NO:41). Fig. 13D depicts the neutralization titers found in the serum derived from the guinea pigs immunized with the SSALs of the indicated complexities and neutralization titers evoked by monovalent octameric V3 immunogens, •73_tm and V3₉₁_₃₃₀. Fig. 14 illustrates a primary amino acid sequence alignment (SEQ ID NO:12) of a highly antigenic gp41 Env peptide (35 mer) from 50 representative strains of HIV-1. The source of the sequences is derived from Human Retroviruses and AIDS, 1993, ed. by G. Myers et al. published by Los Alamos National Laboratory, USA. An SSAL is constructed based on the given antigen structure and its source sequences.

Fig. 15 illustrates a primary amino acid sequence alignment (SEQ ID NO:13) of an antigenic gp40 Env peptide (36 mer) from 20 representative strains of Type 2 Human

Immunodeficiency Virus. The source of the sequences is from Human Retroviruses and AIDS, 1993, ed. by G. Myers et al. published by Los Alamos National Laboratory, USA. An SSAL is constructed based on the given antigen structure and its source sequences.

Fig. 16 illustrates a primary amino acid sequence alignment of three antigenic Env peptides from HTLV-I/II (SEQ ID NO:14, Fig. 16A; SEQ ID NO:15, Fig. 16B; SEQ ID

NO:16, Fig. 16C) . An SSAL is constructed based on the given antigen structure and its source sequences.

Fig. 17 illustrates a primary amino acid sequence (SEQ

ID NO:17) of the critical domain from Influenza A virus hemagglutinin (HA) , a surface glycoprotein capable of eliciting virus-neutralizing antibodies.

Alignment of the complete amino acid sequence of this domain, from residues 91-164, is shown for the H3N2 strains of Influenza virus A. The numbering of the amino acids begins with the first amino acid of the mature HA sequence

(i.e. following cleavage of the signal sequence) as the number one. The amino acid sequences were extracted from the EMBL and the SWISSPROT Databases.

Fig. 18A illustrates a primary amino acid sequence alignment (SEQ ID NO:18) of the critical HA domain from the

HlNl strains of Influenza A virus capable of eliciting virus neutralizing antibodies.

The structural organization of the antigenic sites and the sequence sources are the same as described in Fig. 17. WIL and FPR834 strains each harbor a single amino acid deletion within this domain. The location of the deletion is marked by X. Therefore, a separate library was constructed to represent these sequences (SEQ ID NO:40; Fig.

18B) . Fig. 19 illustrates a primary amino acid sequence alignment (SEQ ID NO:19) of the critical HA domain from two

H2N2 strains of Influenza A virus capable of eliciting virus neutralizing antibodies. Fig. 20 illustrates an SSAL for a Mutant Form of the Critical Influenza Virus HA Neutralizing Domain. Synthetic peptides were made from the sequences that immediately flank the loop structure present in the HA neutralizing domain which is capable of eliciting antibody that partially neutralizes virus infectivity. Therefore, these sequences are likely to form a conformationally-dependent epitope important for eliciting a protective immune response. Antigen libraries have been constructed to mimic this antigenic structure by linking the amino acid sequences present on either side of the loop. These libraries represent deletions of the amino acid residues present between Cys residues at positions 97 and 139 in the original HA domain structure for the H3N2 type virus. The sequence of the deletion libraries was extended at the amino-terminus to include all amino acids represented between the first Cysteine residue of the loop structure and the next Cys residue on the amino-terminal side of the loop. Structured libraries representing this mutant form of the HA neutralizing domain are presented for H3N2, HlNl and H2N2 influenza A viruses (SEQ ID NO:20, Fig. 20A; SEQ ID NO:21, Fig. 20B; SEQ ID NO:22, Fig. 20C; respectively).

Fig. 21 illustrates the amino acid sequence of the first variable domain from the major outer membrane protein (MOMP) of Chlamydia trachomatis . The primary amino acid sequence for the structured synthetic antigen library for VDI_a and VDI_b is shown in Figs. 21A and 2IB respectively (SEQ ID NO:23 and SEQ ID NO:24, respectively). The chlamydia VDI domain sequences were extracted from the EMBL database and from Kaltenboeck, et al. (1993, J. Bacteriol. 175:487) .

Fig. 22 illustrates the amino acid sequence of the fourth variable domain (VDIV) from the major outer membrane protein (MOMP) of Chlamydia trachomatis . The primary amino acid sequence for the structured synthetic antigen library for VDIV_a and VDIV_b is shown in Figs. 22A and 22B respectively (SEQ ID NO:25 and SEQ ID NO:26, respectively). The chlamydia VDIV domain sequences were extracted from the EMBL database and from Kaltenboeck, et al. (1993) .

Fig. 23 illustrates a primary amino acid sequence alignment (SEQ ID NO:27) derived from a series of 43 repeats present as a contiguous sequence of amino acid residues (596-1110) in the T. cruzi neuraminidase protein (NA) and a series of 14 repeats present as a contiguous sequence in the T. cruzi SAPA protein.

Fig. 24 illustrates a primary amino acid sequence alignment (SEQ ID NO:28) of a series of 21 repeats present as a contiguous sequence in T. cruzi cytoplasmic repetitive antigen (CRA) .

Fig. 25A illustrates a primary amino acid sequence alignment (SEQ ID NO:29) of a 65 mer peptide representative of the critical neutralizing determinant of the Env protein from 33 antigenic variants of type 2 Dengue virus. Fig. 25B illustrates a primary amino acid sequence (SEQ ID NO:30) of a 17-mer peptide representative of a helper T cell epitope present in the Env protein from 33 antigenic variants of type 2 Dengue virus.

Fig. 26 illustrates a primary amino acid sequence alignment (SEQ ID NO:31) of three type-specific sequences from the N-terminus of the streptococcal M protein. This N- terminus portion of the protein has been shown to be the site that elicits opsonic, or anti-phagocytic, antibodies. Fig. 27 illustrates a primary amino acid sequence alignment (SEQ ID NO:32) of a T cell helper epitope from the collagen type I/II proteins.

Fig. 28 illustrates a primary amino acid sequence alignment (SEQ ID NO:33) of a carbohydrate recognition site from a group of selectin molecules. Figs. 29A and B illustrates SSALs from two helper T cell determinants (T_h) , SEQ ID NO:34 and SEQ ID NO:35, respectively. The general feature of such SSALs is that the degenerate T_h sequence is separated from the LHRH sequence, EHWSYGLRPG, by two Gly residues. The Gly residues act as a spacer between the T_h epitope and the LHRH sequence.

Fig. 30 illustrates a primary amino acid sequence alignment (SEQ ID NO:36) of an HLA-B27 restricted self peptide antigen. The antigen sequences are derived from histone H3, HSP89α, HSP89B, HEF 2, Helicase, ribosomal protein and L28 (ribosomal protein) .

Fig. 31 illustrates a primary amino acid sequence alignment (SEQ ID NO:37) of a HLA B-27 restricted HIV GAG peptide CTL antigen. The GAG CTL antigen sequences represent those derived from HIV-1 MN, HIV-1 ELI, HIV-2 ROD and multiple HIV patient isolates.

Fig. 32 illustrates two SSAL_ospA antigens by chemical and mathematical representations #1 and #2 (SEQ ID NO:38 and SEQ ID NO:39, respectively). The libraries were designed according to the primary sequence alignment provided for 12 isolates of European and North American origin by Wallich et al; 1992 (ref. cited in Example 16). These included five isolates of genospecies B . burgdorferi sensu stricto (ospA serotype 1), five isolates of B . garinii (one of ospA serotype 4, three of ospA serotype 6, and one of an unexpressed ospA) , and sequences from two isolates of the ^v _46l genospecies (ospA serotype 2) . The amino acid ratios for variable positions were adjusted to give the B . burgdorferi sensu stricto sequences a weight of 50%, so as to account for the greater than 90% prevalence of this genospecies in North America. B . garinii and V₄₆₁ isolates were weighted to approximately 25%, to account for the distributions of these genospecies in Europe. SSAL_ospA #1 includes ospA amino acids 175-234 and #2 is an overlapping sequence of residues 210-273. The overlap in the two libraries is to ensure that each contains B and T cell antigenic determinants, both being required for protective immunity.

Fig. 33 illustrates an SSAL OspC antigen by chemical and mathematical representations (SEQ ID NO:42).

Fig. 34 illustrates an SSAL flagellin antigen by chemical and mathematical representations (SEQ ID NO:43).

DETAILED DESCRIPTION OF THE INVENTION

In this invention, a "structured synthetic antigen library" or SSAL corresponds to the antigenic region of a protein from an infectious agent or from another biologically important system, a diagnostic marker or a variable therapeutic domain of a protein. The design of an SSAL covers the broad range of sequences embraced by geographical or population differences and temporal variation in these molecules. A "structured synthetic antigen library" or SSAL is composed of an ordered set of from at least 3 to several million different but related peptides having sequences imposed upon an invariant structural framework capable of maintaining the antigenicity, diagnostic value or therapeutic bioactivity of that site. Fig. 1 provides a mathematical and chemical formula for the SSAL, which formulation describes the relative ratio of amino acids at each position in the SSAL. AA± to AA represent the amino acid sequence from N- to C-terminus of the library, j varies from 1 to n where n represents the number of possible different amino acids known at the ith amino acid position. Following this formula, a SSAL can be prepared in a single synthesis. The sequence of the SSAL is determined by aligning the primary amino acid sequences of a related family of antigens, markers or diagnostics and identifying the invariant and variant loci within the alignment. The invariant loci generally represent the structural framework of the SSAL. The degeneracy within the SSAL is determined by the loci within the alignment that harbor different amino acid residues relative to an arbitrary prototype sequence. After determining which amino acids are to be at each position, the degree of degeneracy for the multiresidue containing positions in the SSAL library is determined from the number of variants each individual amino acid represents. The SSAL is then synthesized with single amino acids at the invariant positions and with the requisite degeneracies at variant positions. Thus in a simple manner, the specific amino acids and their frequency of appearance at each position within the SSAL is defined by the primary sequences of the different antigens or molecules that make up the alignment. In another embodiment, amino acids not known to exist at a variant locus within aligned epitopes can also be incorporated into the library. For example, all residues of a specific class of amino acids (e.g. hydrophobic, charged, neutral or polar) can be incorporated at a variable locus. The class of amino acids incorporated at a specific locus can be determined by the predominant type of amino acid found at that variant locus within the antigen alignment.

In broad application, the sequence of any SSAL can be defined by homologous portions of existing primary amino acid sequences from a family of proteins or peptides of known biological significance. The sequences encoding the family of proteins or peptides encoding the target antigenic site, diagnostic marker or variable therapeutic domain of a protein or peptide may represent: 1) alternate alleles of the same gene; 2) genes that are evolutionarily related as deduced from sequence homology, but isolated from different strains or species of similar organisms; 3) genes encoding structurally similar proteins with similar function but isolated from very different organisms, or; 4) repeated sequences within the same gene, or repeats from different genes within the same organism or repeats within homologous genes from different, but related organisms. The order within any SSAL is provided by invariant amino acid residues that are defined as the framework sequence of the selected protein or peptide.

The SSAL can be from about 8 to about 100 amino acids in length depending on the particular site and preferably from about 10 to about 100. The overall length of the SSAL can be minimized to provide the fewest residues necessary to elicit the desired biological response. Similarly, extra residues can be added to the ends of the SSAL. For example, KKK can be added at the amino terminus to increase peptide solubility, cysteine can be added to facilitate directed coupling to carrier molecules, and methionine can be added for cyanogen bromide cleavage if necessary.

Moreover, the SSAL can be a domain within a peptide or can have other antigenic, diagnostic or therapeutic sites attached to it. The SSAL can be attached to a core sequence for facile delivery. These core sequences include dendritically branched cores, linear array type branched cores or randomly branched cores (e.g. poly-L-lysine) . The branched cores can be composed of an amino acid or an amino acid analog having two amino groups and one carboxyl group, each group capable of forming a peptide bond linkage.

Preferably such amino acids are lysine or a lysine analog such as ornithine. The amino acid analog can be an α-amino acid, a β-amino acid, or any other either natural or non- natural amino acid with two amino groups and one carboxyl group available for forming peptide bonds. Preferred branched peptides of the invention are dimers, tetramers and octamers, especially those having a branching core structure composed of lysine. Similarly, the branched cores can contain other residues interspersed among the branching residues as depicted, for example, in Fig. 12.

If the variant sequences comprise insertions and/or deletions of amino acids relative to the prototype sequence, separate SSALs may need to be prepared to fully accommodate the various strains. Such SSALs preserve the immunoreactivity or bioactivity of the desired antigen, marker or therapeutic.

The subject SSALs can also be used to form conjugates, i.e., the SSAL, either in branched or linear form can be coupled directly or indirectly, by methods known in the art, to carrier proteins such as bovine serum albumin (BSA) , human serum albumin (HSA) , or to red blood cells or latex particles.

As used herein, natural amino acids are the 20 amino acids commonly found in proteins (i.e. alanine, aspartic acid, asparagine, arginine, cysteine, glycine, gluta ine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan and valine) . The natural amino acids also include the D- and L- forms of such amino acids.

As used herein "unnatural amino acids" include both D- and L- forms of any other amino acids whether found in a protein, whether found in nature or whether synthetically produced. Unnatural amino acids can include, but are not limited to, β-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, gamma-amino butyric acid, homoserine, citrulline and the like.

The SSALs of the present invention are thus from antigenic sites of viruses, bacteria, parasites, tumor antigens, allergens or other protein antigens as well as the relevant diagnostic marker sites of these infectious agents or diseases. Likewise the SSALs include helper T cell epitopes, CTL epitopes or other variable immune epitopes. By way of example, SSALs can be prepared from the envelope, core, NS1, NS3, NS4 or NS5 proteins of HCV; the gpl20 V3 loop, gp41 envelope protein or the gp40 envelope protein of HIV; the envelope protein of HTLV I/II; the HA protein and a mutant HA domain of influenza A virus (including the H3N2, H2N2 and HlNl strains) ; the major outer membrane protein of Chlamydia tracho itis (including the VDI and VDIV sites) ; the neuraminidase (NA) , SAPA and CRA sites of T. cruzi; the envelope B cell site and helper T cell site of type 2 Dengue virus (or other types) ; the M protein of streptococcus; the carbohydrate recognition site of selectin; promiscuous helper T cell sites; the CTL epitope from HIV gag specific for HLA B-27; and the OspA, OspB, OspC and flagellin proteins of Lyme disease (from Borrelia burgdorferi and B. garinii. The SSALs of this invention further include but, are not limited to, those specifically provided in the Examples, s and Sequence Listings.

The SSALs are prepared by chemical synthesis using standard techniques well known in the art such as the solid- phase synthetic route pioneered by Merrifield. The coupling of multiple amino acids at a given position is accomplished by providing a mixture of the desired amino acids at the appropriate ratios. If necessary the ratio of amino acids in the mixture can be varied to account for different coupling efficiency of those amino acids. The SSALs can also be produced by standard recombinant DNA technology, pooled in batch, isolated if necessary and used as provided in accordance with the invention. Preferably, SSALs are prepared by chemical synthesis. The peptide compositions of the present invention can be composed of one or more SSAL. Preferably such compositions contain from one to about 30 SSALs depending on the application. SSALs in accordance with this invention are synthesized and tested to determine immunoreactivity, elicitation of neutralizing antibodies, diagnostic utility, therapeutic value and the like of the SSAL as described in the Examples, by ELISA, by PHA, by bioassay or any other technique appropriate to the source of the SSAL. For example, the efficacy of the HCV SSALs in detecting and diagnosing NANBH and HCV infection is determined by testing it for reactivity with serum specimens with known immunoreactivity for HCV. Such serum panels are commercially available. The strategy for serological validation, naturally, depends on the expected characteristics of the target epitopes. For example, universal immunodominant epitopes, such as the gp41 transmembrane peptide of HIV-1, can be screened by a single representative serum sample from a patient known to be infected with the virus. Epitopes which are not recognized by all infected individuals, or those for which antibody is produced late or only transiently, and especially epitopes which give rise to neutralizing antibodies, may need to be screened by large panels of sera. Both methods of screening can be employed in the present invention to assess the SSALs for selectivity and sensitivity.

Based on the immunoreactivities of the SSALs, they are useful in a vaccine composition to treat or prevent the infection caused by the infectious agent from which they are derived. These vaccine compositions containing one or more SSALs, alone or when coupled to a carrier or polymerized to homo- or hetero-dimers or higher oligomers by cysteine oxidation, by induced disulfide cross-linking, or by use of homo- or hetero-functional multivalent cross-linking reagents, can be introduced into normal subjects to stimulate production of antibodies. Similarly the subject SSALs can be formulated in a vaccine composition using adjuvants, pharmaceutically-acceptable carriers or other ingredients routinely provided in vaccine compositions.

Such formulations are readily determined by one of ordinary skill in the art and include formulations for immediate release and for sustained release, e.g., microencapsulation. The present vaccines can be administered by any convenient route including subcutaneous, oral, intramuscular, intravenous, or other parenteral or enteral route. Similarly the vaccines can be administered as a single dose or divided into multiple doses for administration.

The vaccine compositions of the instant invention contain an immunoeffective amount of the SSAL to treat or prevent the target infection. Such compositions in dosage unit form can contain about 0.1 μg to about 1 mg of the peptide (or mixture of peptides) per kg body weight. When delivered in multiple doses, the dosage unit form is conveniently divided into the appropriate amounts per dosage.

The SSAL peptide compositions prepared in accordance with the present invention can be used to detect or diagnose a target infection by using them as the test reagent in an enzyme-linked immunoadsorbent assay (ELISA) , an enzyme immunodot assay, a passive hemagglutination assay (e.g., PHA test) or other well-known immunoassays. In accordance with the present invention, any suitable immunoassay can be used with the SSALs. Such techniques are well known to the ordinarily skilled artisan and have been described in many standard immunology manuals and texts, see for example, by Harlow et al. (1988, Antibodies: A Laboratory Manual,, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 726 pp. In a preferred embodiment, the immunoassay is an ELISA using a solid phase coated with the peptide compositions of the present invention. ELISA techniques are well known in the art. In another preferred embodiment the immunoassay is a PHA assay. The immunoassays of the present invention are used to screen body fluids and tissues for the presence of the target infectious agent or diagnostic marker. The body fluids which can be subjected to such screening include blood and blood fractions (e.g. serum) , saliva, or any other fluid which contains antibodies specific for the target infectious agent or pathogen.

The examples serve to illustrate the present invention and are not to be used to limit the scope of the invention.

Example 1

EFFECTIVE DETECTION OF HCV ANTIBODIES BY NS-4 SSAL IN PATIENTS FROM GEOGRAPHICALLY DISTINCT REGIONS

An HCV NS4 antigen library was designed according to the sequence listing shown in Fig. 2. The HCV NS4 SSAL was synthesized as linear peptides using the solid phase peptide synthesis strategy employing Fmoc chemistry with standard side chain protecting groups. Protected amino acids were added sequentially during the synthesis process from C- to N-terminus according to the mathematical and chemical representation of the library as shown in Fig. 2B. The protected amino acid reagent added at each of these cycles consists of a collection of amino acid types, where the total concentration of amino acids is identical to that of non-variable positions, but the ratio of each type was set by an algorithm specific to the application of the individual SSAL. The deprotection and cleavage procedures were carried out by standard procedures applicable to the Fmoc chemistry of the synthesis. As controls, two NS4 peptides with corresponding HCV-1 and HCV J-8 sequences as listed in Fig. 2 were also synthesized as linear peptides for comparison of their relative HCV immunoreactivity. Wells of a microtiter plate were coated with NS-4 HCV-1 peptide, the J-8 variant analogue, or the NS-4 SSAL at 5 μg/mL. The NS-4 SSAL peptide was selectively substituted in 27/47 (57%) of its amino acids. In five of these substituted positions, the substituted amino acid corresponding to the J-8 sequence represents only 10% of the total. Specimens from two groups were tested: Japanese hemodialysis patients, a group at high risk for hepatitis C (HCV) infection, for whom recombinant C-100 assay data was available, and plasma donors confirmed seropositive for HCV by recombinant immunoblot (RIBA-II) . Two samples that were non-reactive on the HCV-1 peptide reacted with both the J-8 peptide and the SSAL peptide (Table la) , despite the minority representation of the J-8 sequence in the SSAL peptide. Both samples were non-reactive with the HCV-1 recombinant C-100 protein, indicating that additional antigenic regions that may exist in the C-100 protein do not contribute toward recognition of the Japanese patient antibody. The SSAL NS-4 peptide also detected samples that reacted with the HCV-1 peptide and recombinant C-100 protein but not with the J-8 peptide (Table lb) . In five samples in which both HCV-1 and J-8 peptides were reactive (Table lc) , the response to the SSAL peptide was greater than to either of the single strain peptides derived from HCV-1 or J-8. In the case of Japanese dialysis patient No. 97, the absorbance on the SSAL peptide was twice as high as on either HCV-1 or J-8 peptide.

The results obtained from the above evaluation of critical serological samples with HCV immunoassays demonstrated a clear advantage in using the SSAL antigen design approach for the detection of antibodies to infectious agents, particularly those with high variability such as HCV.

Table 1 Detection of HCV Antibodies by NS-4 SSAL in HCV-infected Japanese Dialysis

Patients and US Plasma Donors

RIBA- ^■II¹ NS-4 Peptide (A₄₉₂)

10 Specimen Sample rClOO Source ID EIA 511 clOO c33 C22 HCV-1 J-8 SSAL

a. Japan 97 Neg ND ND ND ND 0.022 1.155 0.635 US 9 Neg 0 0 4 4 0.051 0.313 0.233 b. US 1 Pos 3 4 4 4 1.264 0.043 1.206

15 US 3 Pos 2 2 4 4 1.073 0.066 1.026 US 21 Pos 4 4 4 0 2.273 0.089 2.398 CD c. Japan 57 Pos ND ND ND ND 0.520 0.521 1.096 US 5 Pos 4 3 4 4 2.009 1.084 2.507 US 16 Pos 4 4 4 2 1.583 1.301 2.120

20 ^xIn a recombinant immunoblot assay (RIBA) , antibody reactivity to each of the HCV recombinant proteins 511, clOO, c33 and c22 is scored from 0 to 4 with 4 being the strongest.

25

ND: not done

2008Z-WO 10/25/94

Example 2

SSAL HCV PEPTIDES CAN BE EFFECTIVELY FORMULATED INTO AN HCV IMMUNOASSAY WITH IMPROVED SENSITIVITY AND SPECIFICITY OVER ASSAYS USING INDIVIDUAL HCV-1 PEPTIDES

Five HCV antigen [NS4, core, NS3, NS5(#1) and NS5(#2)] libraries were designed according to the sequence listing shown in Figs. 2, 3, 4, 5 and 6 respectively. All five HCV antigen SSALs were synthesized as linear peptides using the solid phase peptide synthesis strategy employing Fmoc chemistry with standard side chain protecting groups according to the mathematical and chemical representation of each of the libraries as illustrated in Figs. 2, 3, 4, 5 and 6. All deprotection and cleavage methods were identical to standard procedures applicable to the Fmoc chemistry of the synthesis.

SSAL peptides NS4, core, NS5(#1), NS5(#2), and NS3 were coated onto wells of a microtiter plate at a concentration of 2, 1, 0.5, 0.5 and 5 μg/Ml, respectively. The NS3 SSAL peptide was employed as a conjugate to bovine serum albumin (BSA) . The reactivity of the mixed SSAL peptides was compared to the standard HCV peptides under the same formulation. Sensitivity was evaluated using commercially available low titer and mixed titer HCV panels from Boston Biomedica, Inc. In five samples from panels 102 and 202, the SSAL mixture of peptides provided greater sensitivity than did the mixture of individual HCV-1 peptides (Table 2) . According to RIBA-II analysis, the reactivity of the five samples that had higher absorbance on the SSAL mixture of peptides varied: patterns of core only, NS-3 only and mixtures of NS-3/NS-4 or NS-3/core were all represented. Specificity of the two formats tested over 264 random plasma samples was equivalent. One well- characterized false positive sample, detected by the HCV-1 mixture but not by RIBA-II, was negative on the SSAL peptide mixture. The results demonstrated improved HCV immunoassay performance using SSAL-based HCV peptides.

Example 3

BRANCHED SSALS REPRESENTING THE HIGHLY VARIABLE YET ANTIGENIC

REGIONS OF THE HCV ENV AND NSl PROTEINS ARE USED AS THE KEY INGREDIENTS IN A POLYVALENT VACCINE FOR THE PREVENTION OF INFECTION BY HIGHLY DIVERGENT GLOBAL STRAINS OF HCV

HCV has been associated with liver disease, both acute and chronic, and with the development of liver cancer. Chronic liver disease occurs in at least 50% of infected individuals, and up to 20% of these go on to develop cirrhosis. Variation in immune responsiveness to HCV may account for the variation in outcome associated with HCV infection.

In this HCV vaccine development approach, branched SSALs derived from highly variable yet antigenic regions of the HCV Env and NSl proteins are used in a polyvalent vaccine. More specifically, five HCV SSALs derived from both the Env and NSl protein regions are designed according to the sequence listing shown in Figs. 7, 8, 9, 10 and 11. Analogues of the four Env peptides and one NSl peptide with sequences corresponding to the HCV J-l strain were previously found to be antigenic, in that they reacted with sera from HCV- infected individuals. In this HCV vaccine design, all five HCV Env and NSl SSALs are synthesized as branched peptides onto a heteromeric dendritic branched carrier as shown in Fig. 12a, using the solid phase peptide synthesis strategy employing Fmoc chemistry with Table 2 Improved HCV Assay Sensitivity with Low Titer HCV Antibodies by Employing

HCV SSAL as the Solid Phase Antigens

10 HCV HCV SSAL Abbott Ortho Peptide Antigen² 2.0 2.0 Ortho RIBA 2.0

Sample Antigen¹ HCV HCV Number Abs Ratio Ratio Ratio 5-1-5 ClOO-3 C33C C22-3 Result

Abs Ratio

PHV-102-05 0.220 1.41 0.836 4.26 1.1 2.7 — _ — 1+ IND

15 PHV-202-09 0.587 3.76 1.413 7.21 4.7 4.4 3+ 1+ 4+ - OS

PHV-202-10 0.260 1.67 0.396 2.02 2.7 2.8 - - 3+ - IND

PHV-202-14 1.172 7.51 2.285 11.66 4.7 4.4 - - 3+ 4+ POS

PHV-202-22 0.853 5.46 1.543 7.87 4.7 4.4 - - 4+ - IND to

20 *Five HCV peptides (1 from core, 1 from NS4 , 2 from NS5 and 1 from NS3 regions) were used to formulate an HCV enzyme immunoassay. ²Five HCV antigen libraries corresponding to each of the five antigenic peptides were used to formulate an SSAL HCV enzyme immunoassay.

25

30

standard side chain protecting groups. Protected amino acids are added sequentially during the synthesis process from C- toN-terminus according to the mathematical and chemical representation of each of the libraries. All deprotection and cleavage methods are carried by standard procedures applicable to the Fmoc chemistry of the synthesis.

After the synthesis of five HCV Env/NSl SSALs as branched peptides, they are formulated with appropriate vaccine vehicles and adjuvants and tested for their capacity to induce HCV neutralizing antibodies in laboratory animals which will block the growth of HCV isolates in vitro. Following demonstration of neutralizing antibody production in vivo, the SSALs can be formulated into microparticles or other appropriate vaccine delivery systems for efficacy testing in human volunteers at risk for HCV infection.

Example 4

SYNTHETIC AIDS VACCINE FOR THE PREVENTION OF HIV-1 INFECTION USING SSAL REPRESENTATIVE OF UNIQUE AMINO ACID SEQUENCE FRAME DERIVED FROM THE GP120 V3 REGION

The third hypervariable region (V3) of the human immunodeficiency virus-1 (HIV-1) external envelope glycoprotein (gpl20) contains a cysteine loop, the V3 loop, that constitutes the principal neutralizing determinant (PND) . To enhance the immunogenicity of PND peptides, the V3-derived peptides have been presented as radial eight-branched octamers based on an optimized amino acid sequence frame. A heptalysyl core bearing eight reactive NH₂-termini served as a carrier onto which eight identical V3 peptides were attached by solid-phase synthesis. Significant titers of V3 reactive neutralizing antibodies were observed after immunization with this branched peptide vaccine (Wang et al. 1991, Science 254: 285) . This V3 peptide immunogen thus serves as a promising approach for an AIDS vaccine. However, two major problems were encountered subsequently with immunogenicity during the development of such a vaccine. The sources of the problems, the hypervariability of HIV-1 V3 and the genetic restriction of peptide vaccines by the human hosts, served to render a simple peptide insufficiently immunogenic for application in a peptide-based vaccine.

First, the hypervariability of the HIV-1 gpl20 V3 PND presented a major challenge. Although immunizations with a mixture of the branched octameric V3 peptides carefully selected from the HIV-1 V3 sequences shown in Figure 13A resulted in broadly crossreactive anti-V3 neutralizing antibodies capable of reacting with and neutralizing isolates from each major clade of HIV-1 (e.g. Clades A, B, C, D, E) , intra-clade variation, e.g., variation within clade A (e.g. A- 1. A-2... , A-n) still presented a significant obstacle to an effective vaccine.

The second major problem, the genetic restriction of the cellular immune response to a peptide immunogen, became evident during a phase I clinical trial using the monovalent branched octameric ■72_1W peptide in a prototype vaccine. Only a subpopulation of the vaccinees responded to the V3 immunogen after a six-month three-dose protocol. This narrow range in the responsiveness of humans to the peptide-based vaccine was a manifestation of the simplicity of the immunogen design: The monovalent peptide, comprised of eight identical V-l_m peptides attached to heptalysine core, had an insufficient number of T cell epitopes for broad responsiveness in the genetically diverse human population.

More diversity associated with the V3 sequences is needed to allow the synthetic immunogen to simultaneously induce neutralizing antibodies to the globally diverse HIV-l V3 spectrum (e.g. inter-clade and intra-clade variation) and to elicit responses in the majority of genetically variable human subjects. Because of the large numbers of different viruses expressing different V3 loop primary sequences, the synthesis of individual sequences, either by synthetic or by recombinant DNA means becomes impractical, if not impossible. Clearly, the production of an array of V3 immunogens as an SSAL, by a single peptide synthesis, provides a useful solution to this problem. Furthermore, a V3 SSAL can impart an additional advantage to a peptide-based vaccine. By the insertion of amino acid mixtures at the variable loci within the V3 epitope array, a V3 SSAL immunogen can elicit protective immune responses specific to HIV-1 types that have not yet been produced by the readily mutable virus, thereby anticipating viral evolution and preventing evasion of the immune response through escape mutation. A branched SSAL with sequences derived from the previously characterized V3 region of the HIV-1 gpl20 protein can be synthesized onto a standard heptalysyl core or any one of the four types of heteromeric branched core resins shown in Fig. 12 using the solid phase Fmoc chemistry with standard side chain protecting groups. For example, a V3 SSAL was synthesized onto a heptalysyl core resin according to the mathematical and chemical representation of the library as shown in Figs. 13A and B. The protected amino acid reagent added at each of these cycles (representing a variant position) , consists of a collection of amino acid types, where the total concentration of amino acids at the variant position is identical to that of non-variable positions, but the ratio of each type is set by an algorithm specific to the application of the individual SSAL. All deprotection and cleavage methods were carried out identically to standard procedures for the Fmoc chemistry of the synthesis.

The SSAL HIV-1 V3 immunogen was evaluated by immunizations followed by assays for serological and cellular immune activities; and, in demonstration of the value and flexibility for the complexity of the V3 SSAL, complexity was varied and its influence on immunogenicity was observed.

a) Serology of response to the SSAL HIV-crpl20 V3 immunogen. Following the synthesis of the SSAL HIV-gpl20 V3, the peptide library was formulated in 0.5% alum and injected into four guinea pigs at 100 μg per dose. Three and six weeks after the first injection, sera from the animals were tested by a panel of anti-peptide ELISAs (enzyme-linked immunosorbent assays) for their immune reactivities against the immunizing V3 peptide SSAL as well as for reactivities to other individual V3 peptides whose sequences (Figure 13A) contributed to the construction of the V3 SSAL. As shown in Table 3, the SSAL HIV-gp 120 V3 immunogen induced significant antibody responses (Log₁₀ titer > 4.0) to itself, the "V3 library", and more significantly, demonstrated broad reactivity to monovalent V3 peptides with sequences derived from geographically distinct worldwide isolates. In this experiment, the branched SSAL HIV-1 gpl20 V3 demonstrated potent immunogenicity and elicited highly crossreactive V3 antibodies. Another aspect of the serological reactivity evoked by the V3 SSAL immunogen is neutralization activity, i.e., the capability of the anti-sera to neutralize or inactivate infectious virus. The ability to elicit viral neutralization by antibodies is an important component of a protective vaccine against HIV infection. Neutralization activity was determined by the Rapid Microplaque Assay (MT-2 assay) decribed by Hanson et al. (1990, J. Clin. Microbiol. 28:2030) in which the infectivity of HIV-1 (preincubated with neutralizing serum) is determined on HIV-sensitive MT-2 cells. The particular MT-2 assay was a stringent assay in which the endpoint was the antibody dilution at which 90% of input virus, North American HIV-1 isolate MN of Clade B, is neutralized. This endpoint is expressed as the "MT-2MN_go". The pooled antiserum from the V3 SSAL-immunized guinea pigs had an MT-2MN₉₀ of 1:2430. This titer was comparable to the MT-2mn₉₀ of >1:2430 achieved by guinea pigs administered comparable doses of an analogous monovalent peptide immunogen representing the V3 sequence for HIV-1 MN only. These titers were comparable dispite the minute proportion of V-._m sequence present in the SSAL immunogen (10^_11%) compared to the proportion of VS^ in the monovalent formulation (100%) .

Neutralization activity for the anti-SSAL V3 antibodies in the pooled guinea pig anti-sera was also determined by a stringent assay, the infectivity reduction assay (IRA) , described in White-Scharf et al. (1993, Virology 192:197), which measures neutralization activity against primary field isolates. IRA neutralization activity, expressed as infectious units (IU) blocked 100% by 1:10 dilution of serum, was 10 IU against the Zambian HIV-1 isolate Zam200370, a field isolate of Clade C, which was not contained in the V3 SSAL. This cross-neutralization activity illustrates the library's capacity for broad immunogenicity.

b) CTL response to the SSAL HIV-gpl20 V3 immunogen. Cell-mediated immunity, including cytotoxic T lymphocyte response (CTL) is an important component of the protective immune response to HIV-1. CTL response results in the destruction of HIV-infected cells. The CTL response to a V3 SSAL immunogen is useful both in a prophylactic vaccine by preventing the transmission of infected cells, and in a therapeutic vaccine by reducing the viral load in HIV-infected individuals. CTL responsiveness to a peptide immunogen in humans is determined by how well the immunogen is recognized by the Class I HLA antigens of the genetically diverse human population. Antigens of greater complexity are more likely to be recognized by the Class I antigens of a greater part of the population. The capability of the V3 SSAL to be recognized by a Class I major histocompatibility complex and evoke CTL was demonstrated in a mouse model system.

The immunizations were accomplished by the following protocol: The V3 SSAL branched peptide was formulated into polylactide-co-glycolide microparticles that are suspended in phosphate-buffered saline (O'Hagan et al. 1991, Vaccine, 9:768) and injected intraperitoneally into a group of female Balb/c mice (6-8 weeks old) , three mice per group, at 100 μg of peptide in 0.5 ml of microparticulate vaccine per dose. Booster immunizations were administered on days 12 and 21. Animals were sacrificed on day 27 and splenocytes collected and restimulated with a monovalent linear peptide of sequence I-G-P-G-R-A-F-Y-T-T, corresponding to a portion of the V3 domain of HIV-1 MN that was present in the SSAL as an individual sequence. The same peptide was also used to sensitize target cells. The cultured splenocytes were restimulated on days 7 and 14 after sacrifice and assayed for CTL 6 days later by ⁵¹Cr-release assay (Hioe et al. 1990, J. Virol. 64:6246). Target cells were ⁵¹Cr-labeled A20.1-11 (H- 2^d) cells pulsed with the above monovalent linear peptide or irrelevant peptide and no peptide as controls. A highly significant level of CTL immune reactivity specific for the ^V3MN peptide was observed, as represented by per cent ⁵¹Cr released of (% specific lysis) 51%, 42%, and 21% for respective Effector:Target ratios of 150, 50, and 17. By comparison, CTL results with unsensitized target cells and for target cells coated with an irrelevant peptide were less than 3%. Splenocytes cultured from placebo-immunized mice displayed only background levels of ⁵¹Cr release.

c) Optimization of SSAL complexity. The V3 SSAL evaluated above in parts a and b, with a complexity of 10¹³, was evaluated for immunogenicity relative to that of a series of V3 libraries of diminishing complexities. Complexity is a highly significant parameter for immunogenicity as it determines not only the number of different peptides that is delivered by an SSAL vaccine, but also the dose of any individual peptide in the SSAL. Four V3 SSAL immunogens of increasingly simpler compositions were synthesized, of complexities 10¹², 10⁹, 10⁶, and 10³. These were used to immunize groups of five guinea pigs, as described in part a of this Example. The composition of the V3 SSAL of 10⁹ complexity is illustrated in, Figure 13c. Neutralization activities (against HIV-1 MN) elicited by those libraries were determined for the pooled anti-sera of each group at week 6 as MT-2MN₉₀ values and results were compared to the neutralization titer of the anti-sera to the original V3 SSAL (Figure 13B) and to neutralization titers evoked by monovalent branched V3 peptide immunogens. Neutralization activities are plotted in Figure 13D and show a strongly significant neutralization titer for the SSAL of 10⁹ complexity (MT-2MN₉₀ 1:9081) and the other SSALs elicited titers comparable to the results obtained from immunization with monovalent V2_m immunogen. Immunization with a monovalent V3 immunogen other than MN that was also present in the libraries evoked poorer cross-neutralizing activity against HIV-1 MN than was evoked by any of the SSAL immunogens regardless of library complexity. The V3 SSAL immunogens of complexities 10¹³ and 10³ were compared for immunogenicity by CTL assays against VS^-pulsed cells. Mice were immunized with these two libraries and evaluated for CTL by the protocols described in part b. At Effector:Target cell ratio of 150, results were 53% specific release for the CTL elicited by SSAL of 10¹³ sequences and 79% specific release for the SSAL of 10³ sequences, strongly suggestive of the influence of complexity on the Table 3

Branched SSAL HIV gp 120 V3 Demonstrated Potent Immunogenicity and Elicited Highly Crossreactive V3 Antibodies Required of a Synthetic AIDS Vaccine

Clades V3 Library B S D B B E B A C B B D

10 V3 Sequence UB1 BAL-1 Lab. Thai, MN SC Thai Thai lvoCot Di jibo Indi, Ruanda Haiti UMJ2 Uganda Source Uganda d Zar

Cφde of 620 369M 468 471 200M 281M 481 482 485 469 470 473 478 282M 486 Octamer

Cross

15 Reactivity 4.015 3.500 4.165 3.529 3.670 3.461 3.512 3.995 3.490 3.630 3.130 3.039 2.568 3.675 2.990

Log,- Titer

(3 wks)

Cross

Reactivity 4.749* 4.200 4.628 4.077 4.346 4.229 4.339 4.514 4.204 4.087 4.012 3.795 3.653 4.467 4.088

20 Log,- Titer

(6 wks)

Sera obtained from 6 wks G .P . bleeds also reacted strongly wi th V3- l ibrary based branched pept i des presented on a tetrameri c l i near array as shown in F i g. 12(d) .

immunogenicity of HIV-1 gpl20 V3 SSAL immunogens, and a clear illustration of the potent immunogenicity of a V3 SSAL.

Example 5

DETECTION OF HIV-1 ANTIBODIES WITH AN SSAL DERIVED FROM A

HIGHLY ANTIGENIC REGION OF THE GP41 TRANSMEMBRANE

ENVELOPE PROTEIN OF HIV-1

The gp41 transmembrane envelope protein of the human immunodeficiency virus, Type 1 (HIV-1) contains an immunodominant antigenic region useful for detection of antibodies to HIV-1. Although this antigenic region is highly conserved, variant amino acid substitutions have been observed in field isolates.

An HIV-1 gp41 antigen (35 mer) library was designed according to the sequence listing of 50 field isolates shown in Fig. 14. The HIV-1 gp41 SSAL was synthesized as linear peptides using solid phase Fmoc chemistry. Wells of a microtiter plate were coated with the SSAL HIV-1 gp41 at 1 μg/mL. Confirmed HIV-1 positive specimens from geographical diverse areas were tested with the HIV-1 gp41 SSAL and found to have strong seroreactivities.

Due to the inherent design advantages associated with the SSAL, such a gp41 antigen preparation is useful for comprehensive HIV-1 antibody detection.

Example 6

DETECTION OF HIV-2 ANTIBODIES WITH AN SSAL DERIVED FROM A HIGHLY ANTIGENIC REGION OF THE GP40 TRANSMEMBRANE ENVELOPE PROTEIN OF HIV-2

The gp40 transmembrane envelope protein of the human immunodeficiency virus, Type 2 (HIV-2) contains an immunodominant antigenic region useful for detection of antibodies to HIV-2. Although this antigenic region is highly conserved, variant amino acid substitutions have been observed in field isolates.

An HIV-2 gp40 antigen (36mer) library is designed according to the sequence listing of 20 field isolates shown in Fig. 15. The HIV-2 gp40 SSAL is synthesized as linear peptides using the solid phase Fmoc chemistry. Wells of a icrotiter plate can be coated with the SSAL HIV-2 gp40 at lμg/ L. Confirmed HIV-2 positive specimens, mostly from West Africa are tested against these SSALs. Due to the inherent design advantages associated with the SSAL, such an HIV-2 gp40 SSAL antigen preparation is thus useful for comprehensive HIV-2 antibody detection in the event of antigenic drift.

Example 7

SIMULTANEOUS DETECTION OF HTLV I/II ANTIBODIES WITH SSALS DERIVED FROM THE HIGHLY ANTIGENIC REGIONS OF THE EXTERNAL PORTION (GP46) AND TRANSMEMBRANE

PORTION (GP21) OF THE HTLV ENVELOPE PROTEIN

HTLV-I and HTLV-II are closely related viruses with extensive serologic cross-reactivity. HTLV-I is the causative agent of adult T-cell leukemia and a degenerative neurological disorder, HTLV-I-associated myelopathy (HAM) or tropical spastic paraparesis. Disease association with HTLV-II is less well-established, but HTLV-II virus has been identified in patients with a neurological disease similar to HAM.

HTLV-I and HTLV-II can be transmitted by blood transfusion. Immunodominant epitopes have been identified in the gp46 external portion and gp21 transmembrane portion of the HTLV-I envelope that are useful for detection of antibodies to HTLV-I.

Three HTLV I/II antigen (gp46, gp21) libraries are designed according to the sequence listing shown in 16A, B and C. All three HTLV-I/II antigen SSALs are synthesized as linear peptides using the solid phase peptide synthesis strategy employing Fmoc chemistry. SSAL peptides 16A (gp46) , 16B (gp46) and 16C (gp21) can be individually coated onto wells of a microplate at a concentration of 2 μg/mL. These SSAL peptides are tested with sera from patients infected with either HTLV-I or HTLV-II. SSAL HTLV-I/II peptides thus demonstrated their cost-effective feature in screening for HTLV-I/II antibodies when compared with the use of individual HTLV-I or HTLV-II peptide antigens.

Example 8

SSAL BASED UNIVERSAL FLU VACCINE FOR THE PROTECTION AGAINST DOMINANT HUMAN FLU VIRUS TYPES (e.g. H3N2, HlNl AND H2N2)

Influenza A viruses belong to the Orthomyxoviridae family and cause febrile respiratory disease on a frequent and worldwide basis. High morbidity and mortality are typically associated with epidemics caused by influenza A viruses. Young children, the elderly and patients with chronic illnesses are especially susceptible to pneumonia resulting from infection. Antigenic variation occurs with exceptionally high frequency in influenza A virus. Mutations in the genes encoding the viral surface glycoproteins, namely the hemagglutinin (HA) and neuraminidase proteins (NA) are rapidly selected for by immune responses mounted against the virus. These mutations accumulate around the regions containing antigenic sites such that the mutants are capable of escaping immunity, thereby causing recurrent epidemics of respiratory disease on nearly an annual basis. Because the existence of large numbers of different virus strains and their capacity to escape immune surveillance through mutation, new vaccines structured to block infection by the current circulating virus strain(s) must be formulated on an annual basis. Herein is described a realistic alternative to this approach by using structured synthetic antigen libraries (SSALs) . The libraries are designed to mimic the major neutralizing sites present on the HA molecule, which is the dominant immunogen of this virus. Therefore, a single vaccine composed of a simple mixture of structured libraries, based upon the dominant HA antigenic sites which are capable of eliciting virus neutralizing antibodies, should eliminate and replace the need to prepare "customized" Flu virus vaccines in the conventional manner.

The first step in the design of an effective Flu vaccine is the identification of the primary amino acid sequence of the critical domain from Influenza A virus hemagglutinin (HA) surface glycoprotein capable of eliciting virus neutralizing antibodies.

A synthetic peptide representing the amino-terminus of this domain (residues 91-108) , when used as a vaccine immunogen was found capable of protecting mice from virulent virus challenge (Muller, et al. 1982, Proc Natl Acad Sci USA 7_9: 569) . Its carboxyl-terminus (residues 138-164) , was also found to confer partial protection to mice (Shapira et al, 1984, Proc Natl Acad Sci USA 81; 2461). Alignment of the complete amino acid sequence of this domain, from residue 91-164, is hereby described for the H3N2 strains of Influenza virus A. The major structural feature of this domain is a loop formed by a disulfide bond between the Cysteine (Cys) residues at positions 97 and 139. The numbering of the amino acids begins with the first amino acid of the mature HA sequence (i.e. following cleavage of the signal sequence) as the first residue. The primary sequence of the structured synthetic library for the H3N2 viruses (i.e. SSAL1 Inf A-HA-H3N2) is described in Fig. 17.

Extending the amino acid sequence framework information obtained from H3N2 strains of influenza virus A to the HlNl strains, a separate SSAL (i.e. SSAL2 Inf A-HA-H1N1) for HlNl was similarly constructed as shown in Fig. 18A. However, there were two strains in the HlNl family, namely WIL and FPR 834 that were found to harbor a single amino acid deletion within this domain. A third library was therefore constructed (SSAL3 Inf A-HA-H1N1) to represent the two sequences (Fig. 18B) .

The "framework" information of the amino acid sequences representing the HA critical neutralizing site of two H2N2 strains was further extended, and used to construct a fourth SSAL (i.e. SSAL 4 Inf A-HA-H2N2) (Fig. 19).

Since synthetic peptides made based upon the sequences that immediately flank the loop structure present in the HA neutralizing domain were found capable of eliciting antibody that partially neutralizes virus infectivity, these flanking sequences are likely to form a conformationally dependent epitope important for eliciting a protective immune response. Three more SSALs representing a mutant form of the HA critical neutralizing domain based on the sequences of H3N2, HlNl and H2N2 were thus constructed to mimic this "Base" structure by simply linking the amino acid sequences present on either side of the loop as shown in Fig. 20. These libraries represent deletions of the amino acid residues present between Cys residues at positions 97 and 139 in the original critical HA domain structure described above. The sequence of the deletion libraries (i.e. SSAL5 Inf A-HA-H3N2, SSAL6 Inf A-HA-H1N1, SSAL7 Inf A-HA-H2N2) was extended at the amino-terminus to include all residues represented between the first Cysteine residue of the loop structure and the next Cys residue on the amino-terminal side of the loop.

Based on the above structure design, seven SSALs are synthesized as branched peptides. These seven SSALs are tested alone and in combinations for the induction of virus- specific antibody responses and virus-neutralizing antibody responses in laboratory animals. A response capable of neutralizing the broad-spectrum of human influenza viruses is anticipated. Following demonstration of virus neutralizing responses in vitro, mixtures of the these SSALs are formulated with appropriate vaccine vehicles and adjuvants and used to immunize mice. Following demonstration of an immune response to the influenza A HA SSALs in mice, the animals are challenged with the different types of human viruses to determine the breadth of protection conferred by this novel immunogen. Following mouse protection studies and demonstration of safety in laboratory animals, the SSAL-based vaccine is tested for efficacy in human volunteers.

Example 9

SSAL-BASED SYNTHETIC CHLAMYDIA VACCINE DERIVED FROM THE NEUTRALIZING DOMAIN OF THE MAJOR OUTER MEMBRANE PROTEIN (MOMP) FOR PROTECTION FROM CHLAMYDIA INFECTION

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that is the leading cause of sexually transmitted disease (including cervicitis, epididymitis, urethritis and pelvic inflammatory disease) in industrialized countries, and the dominant pathogen associated with preventable blindness (i.e. trachoma) in developing countries. The many different isolates of this pathogen have been divided into three biovars based upon their nucleic acid composition, antigenicity and pathogenicity. Isolates that produce disease in humans come from the lymphogranuloma venereum and trachoma biovars. Within these two biovars, the different bacterial strains are further subdivided into at least 15 different serovars based upon serologic responses. These serovars are collected into three serogroups based largely upon antigenic responses to the major outer membrane protein (MOMP) : the B serogroup contains the B, Ba, D, E, LI and L2 serovars; the intermediate group is composed of the F, G, K and L3 serovars; while, the C serogroup covers the A, C, H, I, and J serovars. The MOMP protein contains major antigenic structures that cause the production of serovar-, serogroup- , and species-specific antibody responses. Since it is the serovar- and serogroup-specific antibody responses, particularly the anti-MOMP responses that are capable of neutralizing chlamydial infectivity, MOMP has been the focus of subunit vaccine research and development. MOMP structure is conserved between serovars and consists of four variable domains separated by constant regions. The variable domains I and IV (i.e. VDI and VDIV) contain antigenic sites which elicit protective neutralizing antibody (Zhang et al. 1989, Infect. Immun. 57:636; Yuan et al. 1989, Infect. Immun. 57:1040)

Alignment of the amino acid sequence of the first variable domain (VDI) from the 15 known serovars of C. trachomatis has established two primary amino acid sequence patterns, designated VDI_a and VDI_b. The prototype VDI_a pattern is defined by the VDI domain from C. trachomatis serovar A (two alternate sequences for the VDI domain of serovar A exist, designated A_λ and A above) and represents MOMP residues 81-106. Serovars A, C, H, I, J, K and L3 are representatives of the VDI_a pattern. The C. trachomatis serovar B VDI domain is the prototype VDI_b pattern and represents MOMP residues 81-104. Serovars B, Ba, D, E, LI and L2 are representatives of the VDI_b pattern. The primary amino acid sequences for the SSALs for VDI_a and VDI_b are described in Figs. 21A and 2IB.

Alignment of the amino acid sequences of the fourth variable domain (VDIV) from the 15 known C. trachomatis serovars has established two primary amino acid sequence patterns, designated VDIV_a and VDIV_b. The prototype VDIV_a pattern is defined by the VDIV domain from C. trachomatis serovar A and represents MOMP residues 312-341. Serovars A, B, Ba, D, E, I, LI and L2 are representatives of the VDIV_a pattern. The C. trachomatis serovar C VDIV domain is the prototype VDIV_b pattern and represents MOMP residues 312- 342. Serovars C, F, G, H, J, K and L3 are representatives of the VDIV_b pattern. The primary amino acid sequence for the structured synthetic epitope library for VDIV_a and VDIV_b is described in Figs. 22A and 22B.

Based on the above vaccine design, four SSALs were synthesized as branched peptides using Fmoc chemistry. Since the VDI and VDIV domains do not contain potent helper T cell epitopes one can be provided to both libraries. Addition of the helper epitope is required to potentiate antibody-producing B cell responses to the chlamydial SSALs. A T cell helper epitope, named A8, has been located between MOMP variable domains I and II (Su et al. 1990, J Exp Med 172:203) . This helper epitope sequence was included at the amino-ter inus of the VDI and VDIV libraries in an unmodified and non-degenerate form to elicit the T cell responses required for the production of neutralizing antibodies to the degenerate B cell domains present within the SSALs. Following synthesis of the VDI and VDIV SSALs, they were formulated into experimental vaccines with Freund's Complete (FCA) and Incomplete Adjuvants (IFA) and used to immunize groups of five guinea pigs, at 100 μg of peptide per dose with FCA on week 0 and with IFA for booster immunizations on weeks 3 and 10, intraperitoneally. Blood was collected from the guinea pigs on weeks 0, 5, 8, and 12, processed into serum, then stored until evaluated by anti- peptide ELISA on homologous peptides. Every group of five animals that had received Chlamydia MOMP SSAL displayed strong anti-peptide reactivity at weeks 5, 8, and 12, with ELISA reactivities of >0.5 A_492nm at serum dilution of 1:100,000. Serum samples from week 5 or 8 were pooled for each group, serially diluted and tested by dot immunoassay blot assay (Zhang et al. 1989) for cross-reactivity to the elementary bodies (EB) of a selection of Chlamydia trachomatis serovars representing each of the three serogroups (EBs of serovars B,Ba,D,E,F,G,A,C,H,I,J,and K) . The anti-serum to the VDI_a, VDI_b, VDIV_a, and VDIV_b SSAL vaccines were cross-reactive to all of the EBs at a 1:200 dilution. The reactivity of the VDI_a SSAL anti-serum was compared to guinea pig anti-serum made against an analogous but monovalent VDI peptide immunogen representing serovar A. The antibodies in the SSAL anti-serum bound to the EBs of each serovar approximately equally while the antibodies in the anti-VDl A monovalent antiserum bound preferentially to the serovar A EBs with reduced reactivities for the heterogeneous EBs, in a clear demonstration of the improved immunogenicity offered by the SSAL. The SSAL guinea pig anti-sera can be further tested to demonstrate the capacity of the SSAL immunogens to induce antibodies that block C. trachomatis infections to all relevant serovars in vitro; prevent vaginal infections in mice; and prevent infections of the eye and sexually transmitted disease in primates. The VDI and VDIV libraries were initially administered by parenteral immunization. Following demonstration of antibody production specific to C. trachomatis elementary bodies that is capable of blocking infection in vitro, the SSALs can be incorporated into microparticles, or other appropriate biodegradable delivery systems, for oral, vaginal or ocular delivery. Since chlamydial infections occur at mucosal surfaces, antigen delivery to these surfaces is considered critical for successful immunization to prevent these infections.

Example 10

SSALs FROM Trypanosoma cruzi ANTIGENS AS EXAMPLES OF PEPTIDE LIBRARIES DERIVED FROM REPEATS WITHIN THE SAME GENE AND USED AS DIAGNOSTIC REAGENTS FOR THE DETECTION OF T. cruzi INFECTION

Chagas' disease, caused by the parasite T. cruzi, is a chronic disease affecting about 20 million people in South and Central America. Transmission occurs through the bite . of a hematophagous insect carrying infective trypomastigotes in its excreta. The acute phase of the infection is symptomless, with 90% of infected individuals progressing to the chronic phase. This phase is often characterized by myocarditis, and since no infective organisms are detectible at this stage, it is speculated that an autoimmune process is involved. An effective diagnostic test for Chagas' disease is urgently needed, both for early detection of acutely infected individuals, and for prevention of transmission by blood transfusion.

Several T. cruzi antigens have been found to be proteins characterized by regions of highly repeated sequence. In the few cases that have been investigated, the repeats constitute antigenic sites. For example, shed acute- phase antigen (SAPA) is a lipid-anchored family of proteins shed from T. cruzi during the acute phase of the infection. Some of the members of this family possess neuraminidase activity but all members contain variable numbers of a specific repeated sequence. The sequence of SEQ ID NO:27 has been found to be an immunodominant B-cell epitope

(Prioli et al., 1992, Mol. Biochem. Parasitol .52_.:85) which is found in as many as 43 imperfect tandem repeats in SAPA proteins. This is a very good candidate for the design of a diagnostic test because it is potentially reactive with all members of the family; in addition it offers the possibility of early detection during the acute phase of Chagas* disease. The contiguous sequence of residues 596-1110 of one member of this family (lined up in rows of twelve residues) is shown in Fig. 23. An analogous region from another member of the SAPA family is shown in Fig. 23. The two proteins combined contain 57 imperfect repeats. An SSAL designed to consist of tandem repeats of several such partially degenerate dodecamers is also shown in Fig. 23.

Another T. cruzi antigen useful for design of a diagnostic test is known as cytoplasmic repetitive antigen (CRA) . It has been demonstrated that the CRA antigen is recognized by all of a panel of 6 chagasic sera, and none of a panel of 9 sera from patients with infections that produce cross-reacting antibodies (Krieger et al., 1992, Am J Trop Med Hvσ 4_.6:427). By analogy with other T. cruzi antigens, repeated sequences are likely to constitute antigenic sites in this protein as well. Computer analysis of the sequence of the CRA gene with a program that detects potential epitopes in proteins shows that 21 imperfect repeats of a 14 residue sequence are highly likely to be antigenic. The 21 repeats are shown in Fig. 24. A diagnostic SSAL consists of numerous tandem repeats of this sequence. An SSAL based on tandem repeats of CRA sequences is also represented in Fig. 24.

By analogy with the previous example, it can also contain equal amounts of the two residues at any given variant position.

Diagnostic reagents based on the SSALs as designed used either individually or in combination would constitute a significant improvement over peptide reagents derived from a single sequence because they have the potential to react with a broader spectrum of antibodies.

Based on the above design, two SSALs are synthesized as linear peptides using Fmoc chemistry. Both SSALs are employed as solid phase antigens and formulated into an immunoassay, both individually and in combination, for the detection of antibodies to T. cruzi .

Example 11

SSAI^eu_{g VI RUS}-BASED VACCINE FOR IMMUNIZATION AGAINST VARIANTS OF DENGUE VIRUSES

Dengue viruses are members of the Flavivirus family and cause hemorrhagic fever and shock syndrome which result in significant morbidity/mortality in Asia, and Central/South

America. Dengue viruses are divided principally into four major antigenic groups (Dengue subtypes 1-4) , with each group subdivided into several antigenic variants. Vaccine development for Dengue virus has thus far proven difficult, since enhancing epitopes from one or more subtypes primes individuals for enhanced disease complications. Structured Synthetic Antigen Libraries (SSAL) provide significant advantages for the design of Dengue vaccines, since variable protective neutralizing B cell antibody epitopes can be covered through this approach, along with T cell helper epitopes. As an example, an SSAL derived from the critical neutralizing domain (aa 121-185) of the Env protein of the type-2 Dengue virus is constructed as shown in Fig. 25A, based on the primary amino acid sequence alignment from 33 antigenic variants. Three other SSALs derived from the corresponding Env regions, on the primary amino acid sequence alignments, from antigenic variants of Dengue subtypes 1, 3 and 4 can be constructed similarly.

These four SSALs (i.e. Dengue Env 1, 2, 3 and 4) can be combined with a library derived from amino acids 352-368 of the Dengue 2 Env protein representing a T helper epitope capable of conferring T helper effects to heterologous flaviviruses, thus completing the requisite T-B requirements for immunization against variants of four subtypes of Dengue viruses.

The combined four SSALs can be formulated with appropriate vaccine vehicles and adjuvants and tested for their capacity to induce neutralizing antibodies in laboratory animals to block the replication of all relevant antigenic variants of Dengue virus in vitro. Following demonstration of neutralizing antibody production in vivo , the SSALs are then incorporated into microparticles or other appropriate vaccine delivery systems for efficacy testing in human volunteers at risk for infection by Dengue viruses.

Example 12 STREPTOCOCCAL M PROTEIN BASED VACCINE USING THE SSAL

APPROACH

The efforts to date in producing an effective vaccine against Group A streptococcal infection have been thwarted by the serological diversity of the antiphagocytic and major protective antigen designated streptococcal M protein. Only type specific responses have been elicited using M-protein based vaccines. M protein sequences are characterized by a seven amino acid periodicity (Hosein, B., Mccarty, M. , and Fischetti, V.A. , Proc. Natl. Acad. Sci. USA, 76, 3765, 1979) that allows them to assume an alpha-helical coli structure that is rare among bacterial surface molecules but is common in mammalian proteins. The extreme variability at the amino terminus has formed the basis for serological typing of M proteins, with over 100 distinct M types being identified to date. The N-terminus of M proteins from across a number of types has been shown to produce opsonic, or antiphagocytic, antibodies. In humans and experimental animals, systemic immunity to group A streptococcal infections is due primarily to these type-specific opsonic antibodies directed against the variable amino-terminal half of M proteins. An SSAL based on an antigenic peptide structure derived from the N-terminus of the streptococcal M protein was constructed. The N-terminus of Type 5 M protein was aligned as a prototype with corresponding regions of M proteins from Types 1 and 6 in such a way as to preserve the register across types of the seven-amino acid periodicity. Despite the wide diversity of sequences across types, Leucine is completely conserved in positions 28 and 35, thus providing an anchor structure for the SSAL. Corresponding regions of M protein from Types 19 and 24 are also aligned for sequence comparison, although the extreme diversity of these two sequences has prevented us from including them into the construction of an SSAL streptococcal M. A 35 mer SSAL beginning at the invariant C-terminal

Leucine position designed according to the mathematical and chemical representation of the library (Fig. 26) , is synthesized onto a heteromeric (Lysine and Alanine copolymer core) octa-branched carrier employing Fmoc chemistry. Following synthesis of the SSAL_{streptococcal M}, this SSAL is formulated with appropriate vaccine vehicles and adjuvants and tested for its capacity to elicit protective opsonic, or anti-phagocytic, antibodies in laboratory animals.

Following demonstration of opsonic antibody production directed against multiple strains of streptococcus pyogenes, the SSAL is then incorporated into microparticles or other appropriate vaccine delivery systems for efficacy testing in human volunteers at risk for streptococcal infection. Example 13

SSALCOLLAGEN Type I/II (Th)-BASED TREATMENT OF RHEUMATOID

ARTHRITIS

Rheumatoid arthritis (RA) is a common autoimmune disease in which the joints become painful and swollen under immune system attack. The symptoms of RA can be relieved somewhat by treatment with steroids or anti-cell proliferation drugs, but these therapies have serious side effects. Recently, Trentham et al. (1993, Science 261:1727) have described a treatment of RA in human volunteers by oral tolerization with collagen.

In a related study in a mouse model of collagen-induced arthritis (Myers et al. 1993, J. Immunol. 150:4652) . a peptide from collagen that was an effective competitor with collagen for MHC Class II molecules was identified. When mice were co-immunized with collagen and this MHC class II binding collagen peptide, the incidence and severity of arthritis were reduced. In another related study (Ku et al. 1993, Eur. J. Immunol. 2J3:591), a collagen peptide was found to prevent the development of arthritis in the rat model. Resistance to collagen-induced arthritis in rats and rhesus monkeys was found following an immunization protocol with attenuated type-II collagen aimed at maintaining T cell epitopes, thus implying that collagen-related T cell epitopes are important in inducing a tolerized T cell state, thus preventing the development or induction of the autoimmune disease (Hart et al. 1993, Eur. J. Immunol. 3:1588).

From the above observations, we therefore adopted the use of a collagen derived T cell epitope SSAL, an approach to broaden the responder population for the treatment of patients with RA. More specifically, a collagen T cell epitope SSAL (12mer in size) as shown in Fig. 27 can be used to immunize patients with RA with a total of from 100 to 500 μg per dose, emulsified in incomplete Freund's adjuvant at multiple sites and administered intradermally. The patients are monitored at entry, 1 month, 2 months and 3 months after immunization for various clinical parameters and symptoms. It is predicted that SSALCOLLAGEN Type I/II (Th)-based immunization will induce T cell tolerization to the collagen, thus relieving the symptoms of RA.

Example 14 SSAL_selectins-BASED TREATMENT OF INFLAMMATION

Developing effective anti-inflammatory compounds is a major goal for medical sciences. In a recent report, pertussis toxin was found to bind target cells through the carbohydrate recognition properties of two subunits, S2 and S3, which share amino acid sequence similarity with the lectin domains of the eukaryotic "selectin" family. Selectins are a group of proteins that play an important role in promoting the reversible rolling of leukocytes at sites of tissue inflammation. To date, three selectins have been characterized and all shared a terminal lectin domain. P-selectin and E-selectin appear on activated endothelial cells and mediate adherence of leukocytes by recognition of sialylated and non-sialylated determinants. L-selectin (human leukocyte homing receptor, hLHR) is present on lymphocytes and mediates homing to lymphoid tissue that bears sialyated glycoconjugates. More importantly, this report found that pertussis toxin S2 and S3 subunits, and synthetic peptides representing their carbohydrate recognition domains, competitively inhibit adherence of neutrophils to selectin-coated surfaces and to endothelial cells. Based on the above observation, an SSAL representing the carbohydrate recognition domains is useful to treat inflammation. A careful examination of peptide sequences derived from multiple selectins and pertussis toxin subunits that block neutrophil adherence has allowed construction of an SSAL as shown in Fig. 28, based on selectins or peptides from prokaryotic organisms with related sequences, for the treatment of inflammation.

More specifically, a selectin SSAL is synthesized as linear peptides according to the mathematical and chemical representation shown in Fig. 28. Following the synthesis, the SSAL selectins can be formulated with an appropriate delivery system for application at site of inflammation.

Example 15

HELPER T CELL (Th) EPITOPE BASED SSALs ENHANCE ANTIBODY RESPONSE TO LHRH IN A GENETICALLY DIVERSE POPULATION

B cell responses to a foreign antigen, resulting in the production of antibodies to that antigen, are modulated by T cell responses. During antigen processing, domains or epitopes that are composed of specific linear segments of the antigen are recognized by and are presented in the context of MHC class II molecules on the surfaces of antigen presenting cells. The peptide segments presented in this fashion are recognized by CD4⁺ T cells. These activated T cells stimulate [termed helper T cells (Th) ] or suppress [termed suppressor T cell (Ts) ] antibody production to B cell epitopes present on the foreign molecule in an antigen specific manner. Often, synthetic peptide antigens are poor immunogens because they do not effectively stimulate Th responses. This lack of immune reactivity can be overcome by the addition of known Th epitopes to the synthetic antigen.

A number of Th epitopes have been characterized to date. They range in size from approximately 15-30 amino acid residues in length and often share common structural features and may contain specific landmark sequences. For example, a common feature is amphipathic helices, which are alpha-helical structures with hydrophobic amino acid residues dominating one face of the helix and with charged and polar residues dominating the surrounding faces. Th epitopes frequently contain additional primary amino acid patterns such as a Gly or charged residue followed by two to three hydrophobic residues, followed in turn by a charged or polar residue. This pattern defines what are called Rothbard sequences. Also, Th epitopes often obey the 1, 4, 5, 8 rule, where a positively charged residue is followed by hydrophobic residues at the fourth, fifth and eighth positions after the charged residue. Since all of these structures are composed of common hydrophobic, charged and polar amino acids each structure can exist simultaneously within a single Th epitope.

A further class of Th epitopes have been defined as being universal, in that they are capable of stimulating B cell responses in most members of a population that expresses diverse HLA haplotypes. A representative of this class of epitope is more appropriately described as promiscuous, because a majority, but not all, individuals expressing different HLA antigens are capable of responding to it. Most, if not all, of the promiscuous T cell epitopes contain at least one and usually more than one of the Th epitope features described above.

The design and synthesis of two SSALs that act as artificial Th epitopes and can elicit immune responses to luteinizing hormone releasing hormone (LHRH) in all members of a genetically diverse population are described below.

The general feature of such SSALs is that the degenerate Th sequence is separated from the LHRH sequence, positions 19-28 of SEQ ID NO:34, by two Gly residues. The Gly residues act as a spacer between the T_h epitope and the LHRH sequence. The Gly spacer and the LHRH sequence are invariant in the library sequences.

The degenerate helper T cell epitope present in SSAL1 THILHRH is modeled after a universal epitope identified from the F protein of measles virus (Partidos et al. 1990, J Gen Virol 71: 2099) . This epitope is represented by residues 288-302 of the F protein. Charged residues Glu/Asp are added at position 1 to increase the charge surrounding the hydrophobic face of the epitope. This face is made up of residues at positions 2, 5, 8, 9, 10, 13 and 16. The hydrophobic residues commonly associated with promiscuous epitopes are added at these positions. A Rothbard sequence is indicated by the residues in bold type in the prototype sequence. Sequences obeying the 1, 4, 5, 8 rule are underlined in the prototype sequence.

The degenerate helper epitope present in SSAL2 TH2LHRH is modeled after the universal epitope identified from the hepatitis B virus surface antigen (Greenstein et al. 1992, J Immunol 48: 3970) . This epitope is represented by residues 19-33 of HBsAg. Positively charged Lys and Arg residues are added at positions 1, 2, 3 and 5 to increase the charge surrounding the hydrophobic face of the helical structure. The charged amino acids at position 3 also contributes the required residue to generate a sequence obeying the 1, 4, 5, 8 rule (residues underlined in the prototype sequence) . Hydrophobic residues at positions 4, 6, 7, 10, 11, 13, 15 and 17 make up the hydrophobic face of an amphipathic helix. A Rothbard sequence is indicated by the residues in bold type in the prototype sequence.

The SSALs described above and as shown in Figs. 29 A and B are therefore the first artificial members of the promiscuous class of helper epitopes. The synthetic Th:

LHRH library can be used as a therapy for the treatment of androgen driven diseases including prostate cancer, prostatic hyperplasia, breast cancer and endometriosis. In addition, this same synthetic antigen can be used as a contraceptive in both males and females, with a focus on companion animals. It will also be used to prevent the syndrome commonly known as "boar taint", the offensive aroma and flavor of meat caused by the presence of testosterone in intact male food animals. The synthetic peptide libraries can be formulated in an appropriate carrier, which may include but is not limited to saline, alum, mineral or vegetable oil emulsions, microparticles, other biodegradable vehicles, and the like. Additional compounds can also be added as adjuvants to further potentiate the immune response to the SSAL formulations including, but not limited to, pluronic polymers, lipid amines, saponin, muramyl dipeptides or their analogues, cytokines, derivatives of cytokines, and lipids which may be either mixed with or covalently linked to the synthetic peptide libraries. The final formulation can be administered by immunization, oral delivery or delivery to other mucosal surfaces.

The production of structured synthetic T_h: LHRH libraries provides an ideal model for testing the efficacy of the synthetic universal helper epitopes, since LHRH is a self molecule and therefore not antigenic, i.e. antibody is not produced to unmodified LHRH when exogenously administered. Example 16

^SSAL _HLA-B27 self-eep_ti_des BASED TREATMENT OF H.LA-B 21 RELATED ARTHRITIS

The MHC molecule HLA-B27 is very strongly linked with ankylosing spondylitis and related arthropathies, including reactive arthritis. It is thought that these autoimmune diseases are caused by cytotoxic T cells reacting against an arthritogenic peptide of cellular or microbial origin, which is selectively presented by HLA-B27. The stability of HLA molecules on the surface of a cell is dependent on the binding of a peptide, and in an uninfected cell HLA molecules are stabilized by self-derived peptides. When an individual with HLA-B27 becomes infected with a microorganism which contains an arthritogenic peptide, cytotoxic T cells recognize the HLA-B27 peptide complex and attack the cell leading to autoimmune disease. An SSAL based on the sequences of naturally produced self peptides can be used to competitively occupy the peptide binding sites and prevent the presentation of the arthritogenic peptide by HLA-B27.

SSAL peptides based on the sequences of naturally processed self-peptides from HLA-B27 molecules can be used as competitive blockers to prevent presentation of arthritogenic peptides. An SSAL HLA-B27 self-peptides is synthesized as linear peptides according to the mathematical and chemical representation shown in Fig. 30. Following the synthesis, the SSAL HLA-B27 self-peptides can be formulated in an appropriate delivery system for application at the site of inflammation. Example 17

SSAL_{CTL it} -BASED VACCINE THERAPY . FOR PREVENTION AND TREATMENT OF DISEASE

5

- Cytotoxic T lymphocytes (CTL) are a vital part of the immune response in many diseases, including infectious diseases such as influenza and AIDS, and in some malignant

10 diseases such as malignant melanoma. A strong cellular response is an important component of a prophylactic vaccine and, in addition stimulation of the CTL response, can be used therapeutically to improve selected CTL responses. The stimulation of this cellular response can be achieved by the

15 use of peptides representing target epitopes within a protein, such as the gag protein of HIV. The advantage of the SSAL_{CTL ep}i_t0pe based vaccine therapy is that sequences from a large number of related but not identical sequences in a virus can be covered. CTL epitopes that are restricted

20 by the HLA-B27 molecule, from virus variants of HIV-1, HIV-2 and variants found from sequencing patients viruses can be incorporated into a SSAL-based vaccine. This gives a set of peptides which can stimulate a broadly-reactive CTL response and overcome possible escape mutant sequences. The SSAL_CTL

25 _epi_to_pe vaccine can be encapsulated with a microparticle to be administered parenterally. In this example (see Fig. 31) , the SSAL_{CTL ep}i_tope vaccine therapy is designed on an epitope within the gag protein of HIV that has been shown to be restricted by HLA-B27. The SSAL can be administered to

30 HIV-infected patients with HLA-B27 to stimulate an HIV specific CTL response. Patients can be monitored at entry and each month following administration for improvement in immune function and clinical status. Example 18 DIAGNOSTIC MARKERS AND VACCINE FOR LYME DISEASE

Lyme disease or Ly e borreliosis is an inflammatory illness in humans and other mammals with varied involvements of the skin, heart, liver, kidneys, musculoskeletal system, and the central and peripheral nervous systems (Steere. 1989, N Engl J Med 321:586) . The etiological agents are a group of spirochetes which are collectively known as Borrelia burgdorferi , and which are transmitted by ticks of the genus Ixodes . The disease is of global distribution and is the most prevalent tick-borne disease in the U.S. Prevention is by tick-control methods, since there is no effective vaccine available. The organisms responsible for Lyme disease have been subdivided by recent phylogenetic analysis into at least three genospecies referred to as B . burgdorferi sensu strictu, Borrelia garinii , and Borrelia afzelii (formerly known as VS461) . B . burgdorferi sensu strictu is common in Europe and North America. It accounts for over 90% of isolates in the United States. It tends to be correlated with arthritis. Borrelia garinii is found in Europe and tends to be associated with neuroborreliosis. B . afzelii isolates have been found in Europe and Japan and are linked to acrodermatitis chronica atrophicans (Cann et al, 1992

Lancet 339:1598) . Antigenic variation can be correlated to these subgroups by serotypic and sequence differences in major outer surface proteins OspA and OspB. This variation occurs at both inter- and intra-species levels. The influence of antigenic variation and strain specificity can be observed in the limited cross-protection between isolates in animal vaccine trials with Osp subunit antigens (Marconi et al. 1993 Infect Immun 62.:2611) and in a lack of natural cross-immunity in humans.

Recombinant proteins corresponding to variable major outer surface proteins (Osp) of B. burgdorferii, OspA and OspB have been proven in mouse models to elicit protective neutralizing antibodies and to have potential as protective vaccines (Telford et al. 1993 J Exp Med 178:755) . OspA is the more conserved of the two among North American isolates but was found to display considerable variability (Wallich et al. 1992 Infect Immun 60: 4856) . OspA and OspB vaccines formulated to correspond to single strains do not elicit strongly cross-protective immunity. SSALs can provide significant advantages for the design of Lyme disease vaccine, since variable protective epitopes can be effectively included for wide efficacy.

OspA antigenicity has been extensively analyzed. Studies with monoclonal antibodies and patient antibodies have shown that the C-terminal half, residues 108 to 273, is exposed to the human immune system (Schubach et al. 1991 Infect Immun 5_9:1911-1915) . This half of OspA is highly variable between strains and bears one or more crucial non¬ contiguous antigenic determinants between residues 133 to 273 which are responsible for strain-specific protective immunity. Class II T cell determinants have been identified at residues 221-235, 258-273, and 248 to 263 and B cell epitopes are recognized by human serum samples on the terminal 60 residues, 211-273 (Shanafelt et al. 1992 J Immunol 148:218) .

Two useful SSALs depicted in Figs. 32A and B can be constructed as OspA SSAL immunogens, based on the primary amino acid sequence alignment from 12 antigenically variant isolates and antigenic mapping. They are useful for the detection of antibodies to Borrelia in sera and body fluids when formulated into immunoassays, and they are key components of a protective vaccine for humans and animals when incorporated into microparticles or other appropriate vaccine delivery system.

OspC is another heterogeous outer surface protein common to the three genospecies of the Borrelia burgdorferi group. OspC elicits a prominent antibody response early in infection making it a useful diagnostic marker and studies with animal models have shown it to be a candidate for an effective vaccine (Jauris-Heipke et al, 1993) . It is somewhat more variable in sequence and antigenicity than OspA, a heterogeneity that suggests that it can be effectively applied to diagnostics and vaccines as an SSAL antigen. An SSAL (Fig. 33) useful for those applications can be constructed based on an alignment of primary amino acid sequences from OspC of 15 Borrelia isolates. These sequences were published: 13 in the NCBI CD/ROM database "Entrez" as NCBI seq ID nos. 311392, 311394, 313272, 313274, 313276, 313278, 313280, 434658, 434662, 434664, 434666, 443725, and 495736; and two in GenBank as accession nos. U08284 and X62162. The SSAL immunogen as shown in Fig. 33 is incorporated into vaccine and diagnostic compositions.

A central region of the primary sequence for the flagellin protein of B . burgdorferi comprises a cluster of antigenic determinants that cause the production of antibodies specifically associated with Lyme disease. Serological detection and measurement of these antibodies is useful for the diagnosis of Lyme disease and for the prognosis of clinical outcome (Schneider et al, 1992 Infect Immun j50_:316). This segment is encompassed by a region spanning flagellin residues 175-235 that is described by the SSAL of Fig. 34. This SSAL was designed in accordance with amino acid sequences deduced for 12 isolates from North America and Europe (Collins and Peltz, 1991 Infect Immun 59.:514; Picken, 1992 J Clin Microbiol 20:99; Jauris-Heipke et al, 1993 Med Microbiol Immunol 182:37) . It includes a conserved framework sequence that is species-specific and common to the isolates of B . burgdorf ri , and ten positions within the framework sequence that vary among the isolates. Though flagellin sequence is more conserved than OspA sequence for the relevant genospecies of Borrelia , the pattern of isolate to isolate variation for flagellin does display some correlation with the heterogeneity of OspA (Jauris-Heipke et al, 1993; Picken, 1992).

Antibodies to regions of flagellin outside of the segment described by the SSAL of Fig. 34 share homologies and cross-reactivities to the flagellins of other bacteria. Thereby, a reduced specificity for the detection of Lyme disease is imparted to immunoassays which are sensitized with flagellin antigens that include the cross-reactive domains (Schneider et al, 1992) . Conversely, the isolate- specific variation of the species-specific central domain suggests reduced antigenicity for immunoassays which are sensitized to flagellin by single-strain central region peptides. Thus, a specific and comprehensive SSAL such as that of Fig. 34 is advantageously employed in immunoassays for the diagnosis and prognosis of Lyme disease. A peptide composition comprised of the SSAL depicted in Fig. 34 is useful for the detection of antibodies to the Borrelia burgdorferi group of bacteria in sera and body fluids in North America and Europe, and contributes to the diagnosis and prognosis of Lyme disease.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: United Biomedical, Inc.

(ii) TITLE OF INVENTION: Structured Synthetic Antigen Libraries (SSAL) for Diagnostics, Vaccines and Therapeutics

(iii) NUMBER OF SEQUENCES: 43

(iv) CORRESPONDENCE .ADDRESS:

(A) ADDRESSEE: Maria CH. Lin, Esq.

(B) STREET: 345 Park Avenue

(C) CITY: New York

(D) STATE: New York

(E) COUNTRY: U.S.A.

(F) ZIP: 10154

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: WordPerfect 5.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 26-OCT-1994

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/143,412

(B) FILING DATE: 26-OCT-1993

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Lin, Maria CH.

(B) REGISTRATION NUMBER: 29,323

(C) REFERENCE/DOCKET NUMBER: 1151-4120PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 212-758-4800

(B) TELEFAX: 212-751-6849

(2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

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(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "S8;N2"

(ix) FEATURE:

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(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "G8;D1;Q1"

(ix) FEATURE:

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(ix) FEATURE:

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(ix) FEATURE:

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(D) OTHER INFORMATION: /note= "I6;V4"

(ix) FEATURE:

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(D) OTHER INFORMATION: /note= "I6;V2;A2"

(ix) FEATURE:

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(D) OTHER INFORMATION: /note= "R8;K2"

(ix) FEATURE:

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(B) LOCATION: 12 (D) OTHER INFORMATION: /note= "V9;I1"

(ix) FEATURE:

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(D) OTHER INFORMATION: /note= "R3;Q5;E2"

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(ix) FEATURE:

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(D) OTHER INFORMATION: /note= "S4;A6"

(ix) FEATURE:

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(ix) FEATURE:

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(D) OTHER INFORMATION: /note= "H8;K1;R1"

(ix) FEATURE:

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(ix) FEATURE:

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(ix) FEATURE:

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(ix) FEATURE:

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Ser Gly Lys Pro Ala lie lie Pro Asp Arg Glu Val Leu Tyr Arg Glu 1 5 10 15

Phe Asp Glu Met Glu Glu Cys Ser Gin His Leu Pro Tyr lie Glu Gin 20 25 30

Gly Met Met Leu Ala Glu Gin Phe Lys Gin Lys Ala Leu Gly Leu 35 40 45 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 61 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "P9;G1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

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(ix) FEATURE:

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(D) OTHER INFORMATION: /note= "N3;T7"

(ix) FEATURE:

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(D) OTHER INFORMATION: /note= "V9;I1"

(ix) FEATURE:

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(B) LOCATION: 48

(D) OTHER INFORMATION: /note= "T9;P1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 52

(D) OTHER INFORMATION: /note= "S9;W1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Ser Thr Asn Pro Lys Pro Gin Lys Lys Asn Lys Arg Asn Thr Asn Arg 1 5 10 15

Arg Pro Gin Asp Val Lys Phe Pro Gly Gly Gly Gin lie Val Gly Gly 20 25 30

Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr 35 40 45 Arg Lys Thr Ser Glu Arg Ser Gin Pro Arg Gly Arg Arg 50 55 60

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 80 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "K9;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "L6;I4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "E8;A1;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "V4;A3;T1;F1;Yl"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /note= "K9;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "K8;A2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 31

(D) OTHER INFORMATION: /note= "V4;S3;T1;R2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 32 (D) OTHER INFORMATION: /note= "A5;G5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 33

(D) OTHER INFORMATION: /note= "L8;M2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 35

(D) OTHER INFORMATION: /note= "I6;L3;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 36

(D) OTHER INFORMATION: /note= "N9;H1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 51

(D) OTHER INFORMATION: /note= "T9;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 52

(D) OTHER INFORMATION: /note= "S7;I1;Q2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 54

(D) OTHER INFORMATION: /note= "D9;N1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 59

(D) OTHER INFORMATION: /note= "A9;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 67

(D) OTHER INFORMATION: /note= "Y5;F5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 79

(D) OTHER INFORMATION: /note= "T8;V2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 80

(D) OTHER INFORMATION: /note= "C8;A2" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Lys Ala lie Pro Leu Glu Val lie Lys Gly Gly Arg His Leu lie Phe 1 5 10 15

Cys His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala 20 25 30

Leu Gly lie Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val 35 40 45 lie Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met 50 55 60

Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val lie Asp Cys Asn Thr Cys 65 70 75 80

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "R9;P1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "P8;L1;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "V5;L4;I1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "T5;S4;P1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 15

(D) OTHER INFORMATION: /note= "K4;D4;R2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /note= "D9;G1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 19

(D) OTHER INFORMATION: /note= "E5;V4;Q1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 21

(D) OTHER INFORMATION: /note= "P9;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 22

(D) OTHER INFORMATION: /note= "V8;T2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 24

(D) OTHER INFORMATION: /note= "H8;L1;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

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(D) OTHER INFORMATION: /note= "P8;A2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 30

(D) OTHER INFORMATION: /note= "P9;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 31

(D) OTHER INFORMATION: /note= "P5;T3;V1;I1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 32

(D) OTHER INFORMATION: /note= "K8;P1;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 33 (D) OTHER INFORMATION: /note= "S4;A4;K1;Q1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 34

(D) OTHER INFORMATION: /note= "P8;T2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 36

(D) OTHER INFORMATION: /note= "V5;I4;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 42

(D) OTHER INFORMATION: /note= ^,IK7;R3"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 44

(D) OTHER INFORMATION: /note= "T9;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 41

(D) OTHER INFORMATION: /note= "K4;R6"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu Thr Trp Lys Lys Pro 1 5 10 15

Asp Tyr Glu Pro Pro Val Val His Gly Cys Pro Leu Pro Pro Pro Lys 20 25 30

Ser Pro Pro Val Pro Pro Pro Arg Lys Lys Arg Thr 35 40

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 2

(D) OTHER INFORMATION: /note= "E8;D2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "V8;T2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "A8;E1;D1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

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(D) OTHER INFORMATION: /note= "T9;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 24

(D) OTHER INFORMATION: /note= "A6;T4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 26

(D) OTHER INFORMATION: /note= "G5;K3;A2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Cys Glu Pro Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp 1 5 10 15

Pro Ser His lie Thr Ala Glu Ala Ala Gly Arg Arg Leu Ala Arg Gly 20 25 30

Ser Pro Pro Ser 35

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 55 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "L12;I2;V2"

(ix) FEATURE:

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(D) OTHER INFORMATION: /note= "V7;T4;I5"

(ix) FEATURE:

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(ix) FEATURE:

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(B) LOCATION: 9

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(ix) FEATURE:

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(D) OTHER INFORMATION: /note= "Q5;E11"

(ix) FEATURE:

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(D) OTHER INFORMATION: /note= "R11;H3;K2"

(ix) FEATURE:

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(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "S5;V7;I4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "T3;S13"

(ix) FEATURE:

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(B) LOCATION: 16

(D) OTHER INFORMATION: /note= "G12;S2;T2^M

(ix) FEATURE:

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(B) LOCATION: 17

(D) OTHER INFORMATION: /note= "L5;I7;S2;G2"

(ix) FEATURE:

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(D) OTHER INFORMATION: /note= "H12;Y2;M2"

(ix) FEATURE:

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(B) LOCATION: 20

(D) OTHER INFORMATION: /note= "V14;A2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 25

(D) OTHER INFORMATION: /note= "P5;S9;T2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 27

(D) OTHER INFORMATION: /note= "S10;A2;N2;D2"

(ix) FEATURE:

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(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "115;VI"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 30

(D) OTHER INFORMATION: /note= "V12;T4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 31

(D) OTHER INFORMATION: /note= "Y12;W4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 32

(D) OTHER INFORMATION: /note= "E12;Q4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 33

(D) OTHER INFORMATION: /note= "A12;L4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 34

(D) OTHER INFORMATION: /note= "A11;H1;T2;Q2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 35 (D) OTHER INFORMATION: /note= "D13;A2;N1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 36

(D) OTHER INFORMATION: /note= "A9;L2;M4;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 37

(D) OTHER INFORMATION: /note= "112;V4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 38

(D) OTHER INFORMATION: /note= •^lL9;M7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 40

(D) OTHER INFORMATION: /note= "T11;L2;V2;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 47

(D) OTHER INFORMATION: /note= "V12;E4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 48

(D) OTHER INFORMATION: /note= "R12;N2;K2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 49

(D) OTHER INFORMATION: /note= "E12;D2;V2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 50

(D) OTHER INFORMATION: /note= "G9;S2;N4;D1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 51

(D) OTHER INFORMATION: /note= "N14;G2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 53

(D) OTHER INFORMATION: /note= "S14;L2" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 52

(D) OTHER INFORMATION: /note= "A4;S5;F2;T4;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 54

(D) OTHER INFORMATION: /note= "R15;H1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Cys Leu Thr Val Pro Ala Ser Ala Tyr Gin Val Arg Asn Ser Thr Gly 1 5 10 15

Leu Tyr His Val Thr Asn Asp Cys Pro Asn Ser Ser lie Val Tyr Glu 20 25 30

Ala Ala Asp Ala lie Leu His Thr Pro Gly Cys Val Pro Cys Val Arg 35 40 45

Glu Gly Asn Ala Ser Arg Cys 50 55

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "V12;E4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "R12;K2;N2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "E12;V2;D2"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "G9;S2;N4;D1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "N14;G2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "A4;S5;F2;T4;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "S14;L2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /note= "R15;H1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "V12;I4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "A12;Q2;P2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "M3;V6;L7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "T14;S2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /note= "T12;N4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 18

(D) OTHER INFORMATION: /note= "V9;L7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 20

(D) OTHER INFORMATION: /note= "T5;A7;V4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 21

(D) OTHER INFORMATION: /note= "R12;Q2;K2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 22

(D) OTHER INFORMATION: /note= "D5;N7;Q2;H2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 23

(D) OTHER INFORMATION: /note= "G5;A2;S2;V2;P2;R2;N1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 24

(D) OTHER INFORMATION: /note= "K5;S5;T2;G4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 25

(D) OTHER INFORMATION: /note= "L5;V3;I4;A4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 26

(D) OTHER INFORMATION: /note= "P12;L4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 27

(D) OTHER INFORMATION: /note= "A3;T13"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 28

(D) OTHER INFORMATION: /note= "T11;Q2;H1;A1;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29 (D) OTHER INFORMATION: /note= "Q5;T7;G2;N1;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 30

(D) OTHER INFORMATION: /note= "L10;I6"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 32

(D) OTHER INFORMATION: /note= "R12;T4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 34

(D) OTHER INFORMATION: /note= "I7;V9"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 36

(D) OTHER INFORMATION: /note= "L12;M4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 37

(D) OTHER INFORMATION: /note= "L12;I2;V2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 39

(D) OTHER INFORMATION: /note= "G12;M4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 40

(D) OTHER INFORMATION: /note= "S7;A8;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 42

(D) OTHER INFORMATION: /note= "T9;A7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 43

(D) OTHER INFORMATION: /note= "L9;F5;V1;A1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Cys Val Arg Glu Gly Asn Ala Ser Arg Cys Trp Val Ala Met Thr Pro 1 5 10 15

Thr Val Ala Thr Arg Asp Gly Lys Leu Pro Ala Thr Gin Leu Arg Arg 20 25 30

His lie Asp Leu Leu Val Gly Ser Ala Thr Leu Cys 35 40

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "T12;I3;M1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "F12;V3;I1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "R12;Q3;E1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "R13;H3"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "H13;Y3"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /note= "W7;E5;V2;N2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10 (D) OTHER INFORMATION: /note= "T12;F4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "T7;V7;L2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "G3;D11;E2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 20

(D) OTHER INFORMATION: /note= "P14;Q2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 22

(D) OTHER INFORMATION: /note= "H15;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 23

(D) OTHER INFORMATION: /note= "I9;L1;V6"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 24

(D) OTHER INFORMATION: /note= "T10;S6"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 31

(D) OTHER INFORMATION: /note= "D15;N1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 34

(D) OTHER INFORMATION: /note= "M14;L2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 35

(D) OTHER INFORMATION: /note= "N15;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 40

(D) OTHER INFORMATION: /note= "T8;A6;L2" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Phe Thr Phe Ser Pro Arg Arg His Trp Thr Thr Gin Gly Cys Asn Cys 1 5 10 15

Ser lie Tyr Pro Gly His lie Thr Gly His Arg Met Ala Trp Asp Met 20 25 30

Met Met Asn Trp Ser Pro Thr Thr 35 40

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "A5;G5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "E3;H2;D2;S1;T1;Q1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "H7;I2;Y1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "V7;S1;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "T6;S3;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "G9;Q1" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "S2;Q1;R2;A2;N1;T1;E1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "H2;R4;S2 ;K2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "T6;S2;N1;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 16

(D) OTHER INFORMATION: /note= "V2;T6;M1;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /note= "S2;A2;N2;Q2;H1;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 18

(D) OTHER INFORMATION: /note= "G4;S3;R2;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 19

(D) OTHER INFORMATION: /note= "F2;L8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 20

(D) OTHER INFORMATION: /note= "V7;A2;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 21

(D) OTHER INFORMATION: /note= "S7;G3"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 22

(D) OTHER INFORMATION: /note= "L5;M3;W2"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 24

(D) OTHER INFORMATION: /note= "A3;S3;T4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 25

(D) OTHER INFORMATION: /note= "P3;Q3;S2;T1;L1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 27

(D) OTHER INFORMATION: /note= "A6;P4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 28

(D) OTHER INFORMATION: /note= "K4;S5;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 30

(D) OTHER INFORMATION: /note= "N4;K6"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 32

(D) OTHER INFORMATION: /note= "Q9;Y1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 34

(D) OTHER INFORMATION: /note= "I8;V2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "A4;Q2;V3;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "G4;A6"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 23

(D) OTHER INFORMATION: /note= "L5;F5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 31

(D) OTHER INFORMATION: /note= "V4;I5;L1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Val Asp Ala Glu Thr His Val Thr Gly Gly Ser Ala Gly His Thr Val 1 5 10 15

Ser Gly Phe Val Ser Leu Leu Ala Pro Gly Ala Lys Gin Asn Val Gin 20 25 30

Leu lie Asn 35

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "Q8;I1;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "P8;T1;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "I8;L2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "S5;T3;E1;Q1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "Y9;H1"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /note= "A6;D1;T1;E2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "N5;E2;M1;T1;D1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "*8;N2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "*8;V2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "G5;S1;P1;A1;T2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "S6;E1;D1;N2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "G5;R1;S1;I1;D1;P1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 16

(D) OTHER INFORMATION: /note= "P4;L1;S2;Q1;G1;E1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 18

(D) OTHER INFORMATION: /note= "Q7;E1;M2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 26

(D) OTHER INFORMATION: /note= "P8;A2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 28

(D) OTHER INFORMATION: /note= "K4;R5;P1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "P8;Q2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 31

(D) OTHER INFORMATION: /note= "G9;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 32

(D) OTHER INFORMATION: /note= "I9;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 34

(D) OTHER INFORMATION: /note= "P9;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 36

(D) OTHER INFORMATION: /note= "K5;S4;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 37

(D) OTHER INFORMATION: /note= "S6;E1;Q2;T1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Gin Gly Trp Gly Pro lie Ser Tyr Ala Asn Xaa Xaa Gly Ser Gly Pro 1 5 10 15

Asp Gin Arg Pro Tyr Cys Trp His Tyr Pro Pro Lys Pro Cys Gly lie 20 25 30

Val Pro Ala Lys Ser Val Cys Gly Pro Val Tyr Cys 35 40

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "E12;K4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "S11;P2;A2;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "E9;R2;K2;Q1;T1;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "N15;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "T15;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "P14;L1;H1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "N12;S1;Y3"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "N13;Y1;K1;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "N15;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 14 (D) OTHER INFORMATION: /note= "T13;K1;V1;I1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "R14;K1;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 16

(D) OTHER INFORMATION: /note= "K9;R3;T1;E1;Q2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /note= "S11;R2;G3"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 18

(D) OTHER INFORMATION: /note= "110;V3;L1;T2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 19

(D) OTHER INFORMATION: /note= "H8;P4;R2;G1;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 20

(D) OTHER INFORMATION: /note= "I12;L2;V2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 22

(D) OTHER INFORMATION: /note= "P14;W1;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 24

(D) OTHER INFORMATION: /note= "Q9;R6;K1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 25

(D) OTHER INFORMATION: /note= "A11;T3;V1;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 26

(D) OTHER INFORMATION: /note= "F10;W2;L3;Y1" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 27

(D) OTHER INFORMATION: /note= "Y12;F2;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 28

(D) OTHER INFORMATION: /note= "A6;T7;R2;K1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "T14;R1;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 30

(D) OTHER INFORMATION: /note= "G13;K1;R2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 31

(D) OTHER INFORMATION: /note= "D7;E3;N3;Q1;Y1;R1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Glu Ser Val Glu lie Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys 1 5 10 15

Ser lie His lie Gly Pro Gly Gin Ala Phe Tyr Ala Thr Gly Asp Met 20 25 30

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "I9;V38;L3"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "L49;Q1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "V41;L5;I4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "R47;S1;T2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "Y47;F1;L2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /note= "L49;I1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "K27;Q6;G1;R16"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "D47;E1;N2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "Q49;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "L48;R1;I1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "L49;M1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 17

(D) OTHER INFORMATION: /note= "142;F2;M2;G1;L3"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 18

(D) OTHER INFORMATION: /note= "W49;L1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 19

(D) OTHER INFORMATION: /note= "G49;W1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 20

(D) OTHER INFORMATION: /note= "C49;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 21

(D) OTHER INFORMATION: /note= "S48;K2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 23

(D) OTHER INFORMATION: /note= "K49;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 24

(D) OTHER INFORMATION: /note= "L43;H4;I2;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 25

(D) OTHER INFORMATION: /note= "147;V3"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 27

(D) OTHER INFORMATION: /note= "T46;Y3;P1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "A22;T20;N5;F1;S2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 31 (D) OTHER INFORMATION: /note= "P48;K2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 34

(D) OTHER INFORMATION: /note= "A26;T8;S14;R1;N1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 35

(D) OTHER INFORMATION: /note= "S48;T2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 16

(D) OTHER INFORMATION: /note= "G46;E1;R1;N1;S1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Arg lie Leu Ala Val Glu Arg Tyr Leu Lys Asp Gin Gin Leu Leu Gly 1 5 10 15 lie Trp Gly Cys Ser Gly Lys Leu lie Cys Thr Thr Ala Val Pro Trp 20 25 30

Asn Ala Ser 35

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "T19;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "Q1;K18;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 11

(D) OTHER INFORMATION: /note= "D19;H1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "R2;Q16;L1;X1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "LI9;XI"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /note= "S14;A6"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "T18;S1;X1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 32

(D) OTHER INFORMATION: /note= "W19;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 33

(D) OTHER INFORMATION: /note= "V7;A1;P12"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 35

(D) OTHER INFORMATION: /note= "D10;E4;A6"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 36

(D) OTHER INFORMATION: /note= "S11;T8;N1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Arg Val Thr Ala lie Glu Lys Tyr Leu Gin Asp Gin Ala Arg Leu Asn 1 5 10 15

Ser Trp Gly Cys Ala Phe Arg Gin Val Cys His Thr Thr Val Pro Trp 20 25 30 Val Asn Asp Ser 35

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "LI;II"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "N1;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "T1;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "S1;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "L1;P1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "A1;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 16

(D) OTHER INFORMATION: /note= "L1;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17 (D) OTHER INFORMATION: /note= "P1;H1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 18

(D) OTHER INFORMATION: /note= "H1;D1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 20

(D) OTHER INFORMATION: /note= "W1;D1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 22

(D) OTHER INFORMATION: /note= "D1;E1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 24

(D) OTHER INFORMATION: /note= "II;VI"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 26

(D) OTHER INFORMATION: /note= "E1;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "I1;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 30

(D) OTHER INFORMATION: /note= "PI;SI"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 32

(D) OTHER INFORMATION: /note= "K1;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 33

(D) OTHER INFORMATION: /note= "S1;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 35

(D) OTHER INFORMATION: /note= "LI;II" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 37

(D) OTHER INFORMATION: /note= "T1;K1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 38

(D) OTHER INFORMATION: /note= "L1;F1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 39

(D) OTHER INFORMATION: /note= "VI;II"

( i) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Leu Asn Thr Glu Pro Ser Gin Leu Pro Pro Thr Ala Pro Pro Leu Leu 1 5 10 15

Pro His Ser Trp Leu Asp His lie Leu Glu Pro Ser lie Pro Trp Lys 20 25 30

Ser Lys Leu Leu Thr Leu Val Gin Leu Thr Leu Gin Ser 35 40 45

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "F1;Y1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "D1;Q1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "Q1;R1" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "II;LI"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "V1;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "S1;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "S1;D1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "P1;N1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 18

(D) OTHER INFORMATION: /note= "LI;II"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 25

(D) OTHER INFORMATION: /note= "S1;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "T1;P1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 31

(D) OTHER INFORMATION: /note= "G1;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 32

(D) OTHER INFORMATION: /note= "S1;T1"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 34

(D) OTHER INFORMATION: /note= "S1;R1"

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Cys Phe Asp Pro Gin lie Gin Ala lie Val Ser Ser Pro Cys His Asn 1 5 10 15

Ser Leu lie Leu Pro Pro Ser Leu Ser Pro Val Pro Thr Leu Gly Ser 20 25 30

Arg Ser Arg Arg Ala 35

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 71 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "Q1;H1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /note= "K1;Q1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 19

(D) OTHER INFORMATION: /note= "LI;II"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 21

(D) OTHER INFORMATION: /note= "K1;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 22

(D) OTHER INFORMATION: /note= "II;VI" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 24

(D) OTHER INFORMATION: /note= "N1;Q1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 47

(D) OTHER INFORMATION: /note= "LI;II"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 52

(D) OTHER INFORMATION: /note= "R1;C1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 54

(D) OTHER INFORMATION: /note= "PI;LI"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 57

(D) OTHER INFORMATION: /note= "T1;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 59

(D) OTHER INFORMATION: /note= "S1;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 62

(D) OTHER INFORMATION: /note= "PI;SI"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 63

(D) OTHER INFORMATION: /note= "II;VI"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Val Asp Lys Asp lie Ser Gin Leu Thr Gin Ala lie Val Lys His His 1 5 10 15

Lys His Leu Leu Lys lie Ala Asn Tyr Ala Ala Gin His Arg Arg Gly 20 25 30

Leu Asp Leu Leu Phe Trp Glu Gin Gly Gly Leu Cys Lys Ala Leu Gin 35 40 45 Glu Gin Cys Arg Phe Pro His lie Thr His Ser His Val Pro lie Leu 50 55 60

Gin Glu Arg Pro Pro Leu Glu 65 70

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 74 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "S95;G5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "K82;N18"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "Y86;F14"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 20

(D) OTHER INFORMATION: /note= "S95;Y5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 30

(D) OTHER INFORMATION: /note= "F95;C5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 32

(D) OTHER INFORMATION: /note= "N50;T45;S5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 34

(D) OTHER INFORMATION: /note= "G90;N5;D5" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 36

(D) OTHER INFORMATION: /note= "T60;N40"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 37

(D) OTHER INFORMATION: /note= "W90;L10"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 39

(D) OTHER INFORMATION: /note= "G95;E5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 43

(D) OTHER INFORMATION: /note= "N77;S23"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 47

(D) OTHER INFORMATION: /note= "S60;Y30;S10"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 52

(D) OTHER INFORMATION: /note= "G90;E10"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 53

(D) OTHER INFORMATION: /note= "P72;S27"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 54

(D) OTHER INFORMATION: /note= "D46;V18;A18;G18"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 55

(D) OTHER INFORMATION: /note= "N82;S14;K4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 56

(D) OTHER INFORMATION: /note= "G68;S32"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 65

(D) OTHER INFORMATION: /note= "Y55;T45"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 66

(D) OTHER INFORMATION: /note= "K82;E18"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 68

(D) OTHER INFORMATION: /note= "G64;E36"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 69

(D) OTHER INFORMATION: /note= "S86;Y14"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 70

(D) OTHER INFORMATION: /note= "T72;K27"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 73

(D) OTHER INFORMATION: /note= "V86;A14"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 74

(D) OTHER INFORMATION: /note= "L91;Q9"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "S91;N9"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Ser Lys Ala Tyr Ser Ser Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 10 15

Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe lie Asn 20 25 30

Glu Gly Phe Thr Trp Thr Gly Val Thr Gin Asn Gly Gly Ser Ser Ala 35 40 45 Cys Lys Arg Gly Pro Asp Asn Gly Phe Phe Ser Arg Leu Asn Trp Leu 50 55 60

Tyr Lys Ser Gly Ser Thr Tyr Pro Val Leu 65 70

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 78 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "T85;I15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "Y85;D15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "A85;I15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 39

(D) OTHER INFORMATION: /note= "S85;F15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 40

(D) OTHER INFORMATION: /note= "W84;N8;T8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 41

(D) OTHER INFORMATION: /note= "P84;W8;L8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 42

(D) OTHER INFORMATION: /note= "N62;K23;T15" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 43

(D) OTHER INFORMATION: /note= "H85;G15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 44

(D) OTHER INFORMATION: /note= "T69;N31"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 45

(D) OTHER INFORMATION: /note= "V70;T15;Q15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 47

(D) OTHER INFORMATION: /note= "K62;R23;N15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 49

(D) OTHER INFORMATION: /note= "V85;G15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 50

(D) OTHER INFORMATION: /note= "T85;S15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 51

(D) OTHER INFORMATION: /note= "A84;S8;N50"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 52

(D) OTHER INFORMATION: /note= "S69;A31"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 54

(D) OTHER INFORMATION: /note= "S85;K15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 55

(D) OTHER INFORMATION: /note= "H85;R15"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 56

(D) OTHER INFORMATION: /note= "K55;A15;N15;G15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 57

(D) OTHER INFORMATION: /note= "G84;P8;R8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 58

(D) OTHER INFORMATION: /note= "K70;D15;R15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 60

(D) OTHER INFORMATION: /note= "S85;G15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 62

(D) OTHER INFORMATION: /note= "Y85;F15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 63

(D) OTHER INFORMATION: /note= "R85;S15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 64

(D) OTHER INFORMATION: /note= "N85;R15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 66

(D) OTHER INFORMATION: /note= "L85;N15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 69

(D) OTHER INFORMATION: /note= "T85;Y15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 70

(D) OTHER INFORMATION: /note= "E70;G15;K15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 71

(D) OTHER INFORMATION: /note= "K85;S15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 72

(D) OTHER INFORMATION: /note= "N77;G15;E8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 73

(D) OTHER INFORMATION: /note= "G77;S15;E8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 74

(D) OTHER INFORMATION: /note= "L62;S23;T15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 77

(D) OTHER INFORMATION: /note= "N70;K15;V15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 78

(D) OTHER INFORMATION: /note= "L92;Q8"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Pro Asn Ser Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe Ala Asp Tyr 1 5 10 15

Glu Glu Leu Arg Glu Gin Leu Ser Ser Val Ser Ser Phe Glu Arg Phe 20 25 30

Glu lie Phe Pro Lys Ser Ser Trp Pro Asn His Thr Val Thr Lys Gly 35 40 45

Val Thr Ala Ser Cys Ser His Lys Gly Lys Ser Ser Phe Tyr Arg Asn 50 55 60

Leu Leu Trp Leu Thr Glu Lys Asn Gly Leu Tyr Pro Asn Leu

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 77 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "D1;Y1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "G1;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "S1;N1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 50

(D) OTHER INFORMATION: /note= "R1;K1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 57

(D) OTHER INFORMATION: /note= "N1;K1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 70

(D) OTHER INFORMATION: /note= "E1;K1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 72

(D) OTHER INFORMATION: /note= "SI;PI"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 73

(D) OTHER INFORMATION: /note= "D1;N1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

Glu Asn Pro Arg Asp Gly Leu Cys Tyr Pro Gly Ser Phe Asn Asp Tyr 1 5 10 15

Glu Glu Leu Lys His Leu Leu Ser Ser Val Lys His Phe Glu Lys Val 20 25 30 Lys lie Leu Pro Lys Asp Arg Trp Thr Gin His Thr Thr Thr Gly Gly 35 40 45

Ser Arg Ala Cys Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn Met 50 55 60

Val Trp Leu Thr Lys Glu Gly Ser Asp Tyr Pro Val Ala 65 70 75

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "G64;V36"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "N77;D23"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "T59;K41"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "V95;I5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "S95;G5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 16

(D) OTHER INFORMATION: /note= "K82;N18"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 18

(D) OTHER INFORMATION: /note= "Y86;F14"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 20

(D) OTHER INFORMATION: /note= "S90;N10"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 25

(D) OTHER INFORMATION: /note= "G90;E10"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 26

(D) OTHER INFORMATION: /note= "P73;S27"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 27

(D) OTHER INFORMATION: /note= "D46;V18;A18;G18"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 28

(D) OTHER INFORMATION: /note= "N82;S14;K4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "G68;S32"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 38

(D) OTHER INFORMATION: /note= "Y55;T45"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 39

(D) OTHER INFORMATION: /note= "K82;E18"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 41

(D) OTHER INFORMATION: /note= "G64;E36"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 42 (D) OTHER INFORMATION: /note= "S86;Y14"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 43

(D) OTHER INFORMATION: /note= "T73;K27"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 46

(D) OTHER INFORMATION: /note= "V86;A14"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 47

(D) OTHER INFORMATION: /note= "L90;Q10"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Asp Gly Phe Gin Asn Glu Thr Trp Asp Leu Phe Val Glu Arg Ser Lys 1 5 10 15

Ala Tyr Ser Ser Cys Cys Lys Arg Gly Pro Asp Asn Gly Phe Phe Ser 20 25 30

Arg Leu Asn Trp Leu Tyr Lys Ser Gly Ser Thr Tyr Pro Val Leu 35 40 45

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "E85;D15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "S85;P15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 4

(D) OTHER INFORMATION: /note= "F77;L15;V8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "S85;P15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "K70;V15;Q15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "K70;E15;R15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "S92;E8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "A85;V15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 22

(D) OTHER INFORMATION: /note= "T85;I15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 25

(D) OTHER INFORMATION: /note= "S85;K15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 26

(D) OTHER INFORMATION: /note= "S85;K15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 27

(D) OTHER INFORMATION: /note= "H85;R15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 28 (D) OTHER INFORMATION: /note= "K55;A15;N15;G15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "G84;P8;R8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 30

(D) OTHER INFORMATION: /note= "K70;D15;R15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 32

(D) OTHER INFORMATION: /note= "S85;G15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 34

(D) OTHER INFORMATION: /note= "Y85;F15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 35

(D) OTHER INFORMATION: /note= "R85;S15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 36

(D) OTHER INFORMATION: /note= "N85;R15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 38

(D) OTHER INFORMATION: /note= "L85;N15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 41

(D) OTHER INFORMATION: /note= "T85;Y15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 42

(D) OTHER INFORMATION: /note= "E70;G15;K15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 43

(D) OTHER INFORMATION: /note= "K85;S15" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 44

(D) OTHER INFORMATION: /note= "N77;G15;E8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 45

(D) OTHER INFORMATION: /note= "G77;S15;E8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 46

(D) OTHER INFORMATION: /note= "L62;S23;T15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 49

(D) OTHER INFORMATION: /note= "N70;K15;V15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 50

(D) OTHER INFORMATION: /note= "L92;Q8"

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Glu Ser Leu Phe Ser Lys Lys Ser Trp Ser Tyr lie Ala Glu Thr Pro 1 5 10 15

Asn Ser Glu Asn Gly Thr Cys Cys Ser Ser His Lys Gly Lys Ser Ser 20 25 30

Phe Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Asn Gly Leu Tyr Pro 35 40 45

Asn Leu 50

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 49 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "S1;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 20

(D) OTHER INFORMATION: /note= "D1;Y1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 21

(D) OTHER INFORMATION: /note= "G1;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "N1;K1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 42

(D) OTHER INFORMATION: /note= "E1;K1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 44

(D) OTHER INFORMATION: /note= "S1;P1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 45

(D) OTHER INFORMATION: /note= "D1;N1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr lie Met Glu Lys Glu 1 5 10 15

Asn Pro Arg Asp Gly Leu Cys Cys Ala Val Ser Gly Asn Pro Ser Phe 20 25 30

Phe Arg Asn Met Val Trp Leu Thr Lys Glu Gly Ser Asp Tyr Pro Val 35 40 45

Ala

(2) INFORMATION FOR SEQ ID NO:23 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "A7;E1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "S5;N1;K1;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "V6;A1;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "A7;E1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "G7;D1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /note= "E3;Q4;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 18

(D) OTHER INFORMATION: /note= "K2;N6"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 21

(D) OTHER INFORMATION: /note= "V2;T5;K1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 22 (D) OTHER INFORMATION: /note= "A1;T6;V1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Glu Phe Gin Met Gly Ala Ala Pro Thr Thr Ser Asp Val Ala Gly Leu 1 5 10 15

Glu Lys Asp Pro Val Ala Asn Val Ala Arg 20 25

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "A5;D1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "T3;A2;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "T4;D1;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "A5;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 16

(D) OTHER INFORMATION: /note= "V1;A3;T2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 19

(D) OTHER INFORMATION: /note= "S5;T1" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 21

(D) OTHER INFORMATION: /note= "L4;C2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

Glu Phe Gin Met Gly Ala Lys Pro Thr Thr Thr Thr Gly Asn Ala Val 1 5 10 15

Ala Pro Ser Thr Leu Thr Ala Arg 20

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "L2;S6"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "K1;T4;E3"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "P1;A4;T3"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "V2;I6"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "L2;F6"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 8

(D) OTHER INFORMATION: /note= "T4;V4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 18

(D) OTHER INFORMATION: /note= "K2;A6"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 20

(D) OTHER INFORMATION: /note= "T2;D5;E1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 22

(D) OTHER INFORMATION: /note= "V2;K6"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 23

(D) OTHER INFORMATION: /note= "S2;T3;A3"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 24

(D) OTHER INFORMATION: /note= "S6;G1;N1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 27

(D) OTHER INFORMATION: /note= "N2;G6"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 28

(D) OTHER INFORMATION: /note= "E3;Q5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 30

(D) OTHER INFORMATION: /note= "A2;G6"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Leu Ala Lys Pro Val Leu Asp Thr Thr Thr Leu Asn Pro Thr lie Ala 1 5 10 15

Gly Lys Gly Thr Val Val Ser Ser Ala Glu Asn Glu Leu Ala 20 25 30 (2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "A6;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "E5;T1;K1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "A6;P1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "I5;V2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "L6;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "V6;I1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 16

(D) OTHER INFORMATION: /note= "A6;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 18

(D) OTHER INFORMATION: /note= "K5;C2"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 20

(D) OTHER INFORMATION: /note= "S5;T1;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 22

(D) OTHER INFORMATION: /note= "V6;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 23

(D) OTHER INFORMATION: /note= "S2;A4;G1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 24

(D) OTHER INFORMATION: /note= "A3;S4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 25

(D) OTHER INFORMATION: /note= "G5;N2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 26

(D) OTHER INFORMATION: /note= "T2;S5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 27

(D) OTHER INFORMATION: /note= "D3;E4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 28

(D) OTHER INFORMATION: /note= "N5;G2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "E3;D2;Q2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 30

(D) OTHER INFORMATION: /note= "L5;I2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 31

(D) OTHER INFORMATION: /note= "A5;S2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

Leu Ala Glu Ala lie Leu Asp Val Thr Thr Leu Asn Pro Thr lie Ala 1 5 10 15

Gly Lys Gly Ser Val Val Ser Ala Gly Thr Asp Asn Glu Leu Ala 20 25 30

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "S55;T2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "S37;G20"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "T56;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "A33;V24"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

Asp Ser Ser Ala His Ser Thr Pro Ser Thr Pro Ala 1 5 10

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "V19;A2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "A19;V2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "A19;T2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "R3;K18"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "T15;M4;A2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

Val Ala Glu Ala Glu Lys Gin Arg Ala Ala Glu Ala Thr Lys 1 5 10

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 65 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2 (D) OTHER INFORMATION: /note= "K32;Q1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "N31;S2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "K3;E30"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /note= "V16;I16;F1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "Q30;L2;H1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 19

(D) OTHER INFORMATION: /note= "129;V4"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 21

(D) OTHER INFORMATION: /note= "128;V5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "H22;N10;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 40

(D) OTHER INFORMATION: /note= "K32;E1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 42

(D) OTHER INFORMATION: /note= "I27;L1;V5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 43

(D) OTHER INFORMATION: /note= "K30;V3" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 44

(D) OTHER INFORMATION: /note= "124;V9"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 50

(D) OTHER INFORMATION: /note= "I30;M1;T1;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 61

(D) OTHER INFORMATION: /note= "V32;I1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 64

(D) OTHER INFORMATION: /note= "E31;D1;Q1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

Cys Lys Lys Asn Met Lys Gly Lys Val Val Gin Pro Glu Asn Leu Glu 1 5 10 15

Tyr Thr lie Val lie Thr Pro His Ser Gly Glu Glu His Ala Val Gly 20 25 30

Asn Asp Thr Gly Lys His Gly Lys Glu lie Lys lie Thr Pro Gin Ser 35 40 45

Ser lie Thr Glu Ala Glu Leu Thr Gly Tyr Gly Thr Val Thr Met Glu 50 55 60

Cys 65

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1 (D) OTHER INFORMATION: /note= "132;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "N32;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "V31;A1;G1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "S32;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "N32;K1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 16

(D) OTHER INFORMATION: /note= "I31;V2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /note= "E32;D1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: lie Thr Val Asn Pro lie Val Thr Glu Lys Asp Ser Pro Val Asn lie 1 5 10 15

Glu

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "T1;V1;*"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "V1;F1;*"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "T1;N1;P1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "R2;G1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "G2;D1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "T2;G1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "I1;N1;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /note= "SI;PI;El"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "D1;R1;N1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "P2;E1"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "Q1;V1;D1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "R1;I1;K1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "A2;E1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "K1;D1;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 16

(D) OTHER INFORMATION: /note= "E2;L1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /note= "A2;L1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 18

(D) OTHER INFORMATION: /note= "L2;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 19

(D) OTHER INFORMATION: /note= "D1;N2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 20

(D) OTHER INFORMATION: /note= "K2;N1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 21

(D) OTHER INFORMATION: /note= "Y2;P1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 22

(D) OTHER INFORMATION: /note= "E1;A1;D1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 23

(D) OTHER INFORMATION: /note= "LI;II;VI"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 24

(D) OTHER INFORMATION: /note= "E2;Q1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 26

(D) OTHER INFORMATION: /note= "HI;II;SI"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 27

(D) OTHER INFORMATION: /note= "D1;R1;M1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "K1;R1;Q1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 30

(D) OTHER INFORMATION: /note= "T1;H1;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 31

(D) OTHER INFORMATION: /note= "K1;E1;N1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 33

(D) OTHER INFORMATION: /note= "E1;K1;D1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 34

(D) OTHER INFORMATION: /note= "G1;D1;K1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 36 (D) OTHER INFORMATION: /note= "K2;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 37

(D) OTHER INFORMATION: /note= "T2;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 38

(D) OTHER INFORMATION: /note= "E2;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 39

(D) OTHER INFORMATION: /note= "N2;L1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 40

(D) OTHER INFORMATION: /note= "E2;N1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 41

(D) OTHER INFORMATION: /note= "G1;N1;L1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 42

(D) OTHER INFORMATION: /note= "L2;A1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

Arg Thr Val Thr Arg Gly Thr lie Ser Asp Pro Gin Arg Ala Lys Glu 1 5 10 15

Ala Leu Asp Lys Tyr Glu Leu Glu Asn His Asp Leu Lys Thr Lys Asn 20 25 30

Glu Gly Leu Lys Thr Glu Asn Glu Gly Leu 35 40

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "E3;I1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "A3;P1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "Q3;P1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "P3;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "E3;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "P3;S1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Pro Glu Gly Ala Gin Gly Pro Arg Gly Glu Pro Gly 1 5 10

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 2

(D) OTHER INFORMATION: /note= "E1;A1;N1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "E2;D1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "N2;E1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /note= "S1;K2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "I1;T1;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "S1;P2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "Y2;F1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "S2;Y1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

Lys Glu Glu lie Glu Tyr Leu Asn Ser lie Leu Ser Tyr Ser 1 5 10

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "DO.50;E0.50"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "LO.25;10.25;V0.25;F0.25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "EO.50;DO.50"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "LO.25;I0.25;V0.25;F0.25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "KO.50;R0.50"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "L25;I25;V25;F25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "L25;I25;V25;F25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /note= "L25;I25;V25;F25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "K50;R50"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12 (D) OTHER INFORMATION: /note= "L25;I25;V25;F25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "D50;E50"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "L25;I25;V25;F25"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

Asp Leu Glu Leu Lys Gly Leu Leu Leu His Lys Leu Asp Gly Leu Gly 1 5 10 15

Gly Glu His Trp Ser Tyr Gly Leu Arg Pro Gly 20 25

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "K50;R50"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "K50;R50"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "K50;R50"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "L25;I25;V25;F25" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "F34;K33;R33"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "L25;I25;V25;F25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "L25;I25;V25;F25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /note= "K50;R50"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "L25;I25;V25;F25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "L25;I25;V25;F25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "L25;I25;V25;F25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "Q20;L20;I20;F20;V20"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /note= "L25;I25;V25;F25"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

Lys Lys Lys Leu Phe Leu Leu Thr Lys Leu Leu Thr Leu Pro Gin Ser 1 5 10 15 Leu Gly Gly Glu His Trp Ser Tyr Gly Leu Arg Pro Gly 20 25

(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "R5;G1;F1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "Y2;I2;V1;W1;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "Q1;K3;L1;D1;N1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "K2;E3;P1;G1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "S1;I2;V1;A1;P1;L1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "T2;V2;I2;G1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "E1;K2;D1;V1;L1;H1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9 (D) OTHER INFORMATION: /note= "L1;K3;R2;A1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

Arg Arg Tyr Gin Lys Ser Thr Glu Leu 1 5

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "K8;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "W8;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "I8;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "I5;V3;Q1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "L5;I2;M2"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "G8;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9 (D) OTHER INFORMATION: /note= "N8;Q1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "K8;R1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

Lys Arg Trp lie lie Leu Gly Leu Asn Lys 1 5 10

(2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 58 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "T76;K16;N8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "L84;V16"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "A58;T42"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "A67;N25;S8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "D50;E42;G8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9 (D) OTHER INFORMATION: /note= "T67;V25;A8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "T92;A8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "L92;V8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "V34;E33;K33"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "V92;I8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "K75;T25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 19

(D) OTHER INFORMATION: /note= "T75;V25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 21

(D) OTHER INFORMATION: /note= "S92;K8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 22

(D) OTHER INFORMATION: /note= "K92;R8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 23

(D) OTHER INFORMATION: /note= "N50;E34;H16"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 25

(D) OTHER INFORMATION: /note= "S42;A25;L17;P8;D8" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 26

(D) OTHER INFORMATION: /note= "K92;N8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 27

(D) OTHER INFORMATION: /note= "S92;N8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "E92;K8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 30

(D) OTHER INFORMATION: /note= "V75;I25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 31

(D) OTHER INFORMATION: /note= "T67;S33"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 32

(D) OTHER INFORMATION: /note= "V92;A8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 33

(D) OTHER INFORMATION: /note= "E50;A42;S8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 35

(D) OTHER INFORMATION: /note= "N75;D25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 37

(D) OTHER INFORMATION: /note= "T73;S27"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 39

(D) OTHER INFORMATION: /note= "T58;S42"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 40

(D) OTHER INFORMATION: /note= "T58;S34;A8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 41

(D) OTHER INFORMATION: /note= "Q58;A34;S8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 42

(D) OTHER INFORMATION: /note= "A92;G8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 43

(D) OTHER INFORMATION: /note= "T92;S8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 47

(D) OTHER INFORMATION: /note= "G58;A42"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 48

(D) OTHER INFORMATION: /note= "A58;K26;S8;N8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 50

(D) OTHER INFORMATION: /note= "D58;N34;Q8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 51

(D) OTHER INFORMATION: /note= "S84;A8;E8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 52

(D) OTHER INFORMATION: /note= "K42;G42;S8;N8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 53

(D) OTHER INFORMATION: /note= "T92;S8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 38

(D) OTHER INFORMATION: /note= "D73;N27"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

Gly Thr Leu Ala Ala Asp Gly Lys Thr Thr Leu Val Val Lys Glu Gly 1 5 10 15

Thr Val Thr Leu Ser Lys Asn lie Ser Lys Ser Gly Glu Val Thr Val 20 25 30

Glu Leu Asn Asp Thr Asp Thr Thr Gin Ala Thr Lys Lys Thr Gly Ala 35 40 45

Trp Asp Ser Lys Thr Ser Thr Leu Thr lie 50 55

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 71 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "N75;D25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "T73;S27"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "D73;N23"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5

(D) OTHER INFORMATION: /note= "T58;S42"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 6

(D) OTHER INFORMATION: /note= "T58;S33;A9"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "Q58;A33;S9"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "A92;G8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /note= "T92;S8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "G58;A42"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "A58;K25;S8;N9"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 16

(D) OTHER INFORMATION: /note= "D58;N33;Q9"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /note= "S83;A8;E9"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 18

(D) OTHER INFORMATION: /note= "K42;G42;S8;N8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 19

(D) OTHER INFORMATION: /note= "T92;S8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 24 (D) OTHER INFORMATION: /note= "S58;T42"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 25

(D) OTHER INFORMATION: /note= "V92;A8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 27

(D) OTHER INFORMATION: /note= "S92;N8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 28

(D) OTHER INFORMATION: /note= "K75;Q17;R8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 31

(D) OTHER INFORMATION: /note= "K75;T25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 32

(D) OTHER INFORMATION: /note= "D42;N25;Q25;A8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 33

(D) OTHER INFORMATION: /note= "L92;I8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 36

(D) OTHER INFORMATION: /note= "T92;L8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 37

(D) OTHER INFORMATION: /note= "K92;T8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 38

(D) OTHER INFORMATION: /note= "E58;Q33;N9"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 39

(D) OTHER INFORMATION: /note= "D58;N33;G9" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 43

(D) OTHER INFORMATION: /note= "V92;S8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 45

(D) OTHER INFORMATION: /note= "K58;Q33;N9"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 49

(D) OTHER INFORMATION: /note= "A75;N25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 52

(D) OTHER INFORMATION: /note= "N58;K42"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 56

(D) OTHER INFORMATION: /note= "S42;T33;K17;N8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 58

(D) OTHER INFORMATION: /note= "V92;A8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 61

(D) OTHER INFORMATION: /note= "K58;T42"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 62

(D) OTHER INFORMATION: /note= "T58;K42"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 64

(D) OTHER INFORMATION: /note= "D83;K17"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 66

(D) OTHER INFORMATION: /note= "L67;I33"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 68

(D) OTHER INFORMATION: /note= "N75;D17;A8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 71

(D) OTHER INFORMATION: /note= "R25;K75"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

Asn Asp Thr Asp Thr Thr Gin Ala Thr Lys Lys Thr Gly Ala Trp Asp 1 5 10 15

Ser Lys Thr Ser Thr Leu lie Ser Val Asn Ser Lys Lys Thr Lys Asp 20 25 30

Leu Val Phe Thr Lys Glu Asp Thr lie Thr Val Gin Lys Tyr Asp Ser 35 40 45

Ala Gly Thr Asn Leu Glu Gly Ser Ala Val Glu lie Lys Thr Leu Asp 50 55 60

Glu Leu Lys Asn Ala Leu Arg 65 70

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 78 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "A1;I1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 46

(D) OTHER INFORMATION: /note= "F1;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 51

(D) OTHER INFORMATION: /note= "V1;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 52

(D) OTHER INFORMATION: /note= "S1;A1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 56

(D) OTHER INFORMATION: /note= "R1;E1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 70

(D) OTHER INFORMATION: /note= "K1;E1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 72

(D) OTHER INFORMATION: /note= "G1;E1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 73

(D) OTHER INFORMATION: /note= "D1;G1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 74

(D) OTHER INFORMATION: /note= "LI;SI"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

Pro Asn Ser Glu Asn Gly Ala Cys Tyr Tyr Gly Asp Phe lie Asp Tyr 1 5 10 15

Glu Glu Leu Arg Glu Gin Leu Ser Ser Val Ser Ser Phe Glu Arg Phe

20 25 30

Glu lie Phe Pro Lys Glu Ser Ser Trp Pro Asn His Thr Phe Asn Gly 35 40 45

Val Thr Val Ser Cys Ser His Arg Gly Lys Ser Ser Phe Tyr Arg Asn 50 55 60

Leu Leu Trp Leu Thr Lys Lys Gly Asp Leu Tyr Pro Lys Leu 65 70 75 (2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "E6;K3;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "S6;P3;T1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "E7;L1;Q1;V1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "T5.5;12.5;S1;P1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "N9;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "N9;K1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "N9;Y1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "-8;I1;R1"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "T9;Q1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 16

(D) OTHER INFORMATION: /note= "R8;K1;S1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /note= "K9;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 18

(D) OTHER INFORMATION: /note= "S5.5;G2.5;I1;R1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 19

(D) OTHER INFORMATION: /note= "19;VI"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 21

(D) OTHER INFORMATION: /note= "I6;L3;M1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 23

(D) OTHER INFORMATION: /note= "P9;W1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 25

(D) OTHER INFORMATION: /note= "R6.5;Q3.5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 27

(D) OTHER INFORMATION: /note= "F6.5;W3.5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 28

(D) OTHER INFORMATION: /note= "Y8;V1;H1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 29

(D) OTHER INFORMATION: /note= "A6.5;T3.5"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 30

(D) OTHER INFORMATION: /note= "T9;I1"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 32

(D) OTHER INFORMATION: /note= "D3;K2.5;Q2.5;E1;G1"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

Glu Ser Val Glu lie Asn Cys Thr Arg Pro Asn Asn Asn Xaa Thr Arg 1 5 10 15

Lys Ser lie His lie Gly Pro Gly Arg Ala Phe Tyr Ala Thr Gly Asp 20 25 30

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "T60;K26;D7;A7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /note= "193;N7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 4

(D) OTHER INFORMATION: /note= "T67;K20;A13"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 5 (D) OTHER INFORMATION: /note= "Q46;E40;T7;K7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 7

(D) OTHER INFORMATION: /note= "L86;T7;I7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 8

(D) OTHER INFORMATION: /note= "S53;D33;N7;G7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 9

(D) OTHER INFORMATION: /note= "G40;K26;V20;A7;S7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 10

(D) OTHER INFORMATION: /note= "L87;I13"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 11

(D) OTHER INFORMATION: /note= "K85;N15"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 12

(D) OTHER INFORMATION: /note= "N60;G20;D20"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 13

(D) OTHER INFORMATION: /note= "S67;L33"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 14

(D) OTHER INFORMATION: /note= "E86;G14"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "E66;G27;K7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /note= "K93;S7" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 18

(D) OTHER INFORMATION: /note= "E67;T13;A13;K7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 19

(D) OTHER INFORMATION: /note= "K67;E33"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 21

(D) OTHER INFORMATION: /note= "E40;A34;D13;K13"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 22

(D) OTHER INFORMATION: /note= "K40;A26;E13;N7;D7;T7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 23

(D) OTHER INFORMATION: /note= "A87;V13"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 25

(D) OTHER INFORMATION: /note= "K66;D13;E7;N7;Q7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 26

(D) OTHER INFORMATION: /note= "C93;A7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 27

(D) OTHER INFORMATION: /note= "S93;N7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 28

(D) OTHER INFORMATION: /note= "E60;D12;K7;Q7;A7;T7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "E40;K20;A13;D13;T7;S7"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 31

(D) OTHER INFORMATION: /note= "T93;S7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 32

(D) OTHER INFORMATION: /note= "N60;K26;T20;D7;A7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 35

(D) OTHER INFORMATION: /note= "K72;E7;T7;Q7;S7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 36

(D) OTHER INFORMATION: /note= "S40;D33;G13;E7;N7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 37

(D) OTHER INFORMATION: /note= "S47;E20;G13;N13;K7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 38

(D) OTHER INFORMATION: /note= "H93;Q7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 39

(D) OTHER INFORMATION: /note= "A80;T20"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 40

(D) OTHER INFORMATION: /note= "E46;D27;Q20;V7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 43

(D) OTHER INFORMATION: /note= "I40;K33;V13;A7;L7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 44

(D) OTHER INFORMATION: /note= "Q40;E26;A20;V7;D7"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 45

(D) OTHER INFORMATION: /note= "G40;N34;D13;S13"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

Thr Leu lie Thr Gin Lys Leu Ser Gly Leu Lys Asn Ser Glu Glu Leu 1 5 10 15

Lys Glu Lys lie Glu Lys Ala Lys Lys Cys Ser Glu Glu Phe Thr Asn 20 25 30

Lys Leu Lys Ser Ser His Ala Glu Leu Gly lie Gin Gly 35 40 45

(2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 61 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /note= "N83;T17"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /note= "A75;S25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 25

(D) OTHER INFORMATION: /note= "S83;A17"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 29

(D) OTHER INFORMATION: /note= "A92;S8"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 31

(D) OTHER INFORMATION: /note= "A50;T50"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 34

(D) OTHER INFORMATION: /note= "A75;T25"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 41

(D) OTHER INFORMATION: /note= "V67;A33"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 43

(D) OTHER INFORMATION: /note= "Q83;E17"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 50

(D) OTHER INFORMATION: /note= "A67;T33"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 56

(D) OTHER INFORMATION: /note= "S83;T17"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

Val His Val Gly Ala Asn Gin Asp Glu Ala lie Ala Val Asn lie Tyr 1 5 10 15

Ala Ala Asn Val Ala Asn Leu Phe Ser Gly Glu Gly Ala Gin Ala Ala 20 25 30

Gin Ala Ala Pro Val Gin Glu Gly Val Gin Gin Glu Gly Ala Gin Gin 35 40 45

Pro Ala Pro Ala Thr Ala Pro Ser Gin Gly Gly Val Asn 50 55 60

Claims

We Claim:

1. A library of peptides comprising a known antigenic site, epitope or ligand on a protein molecule, wherein

(1) said library is optionally linked to a substantially invariant peptide domain or a core branched sequence;

(2) said library consists of an ensemble domain;

(3) the overall peptide length is about 8 to about 100 amino acids;

(4) the sequence of said ensemble domain is represented by a consensus formula;

(5) said ensemble is immunogenic, diagnostic for the epitope or is a therapeutic; and

(6) the consensus formula provides that each sequence position in the ensemble contain either a single amino acid or multiple amino acids, and that when a position contains multiple amino acids, (a) the identity and ratio of those amino acids being determined by the relative prevalence of amino acids in a consensus of known variant sequences for that epitope or ligand or (b) the identity of those amino acids being determined by the amino acids present in a consensus of known variant sequences for that epitope or ligand and the ratio of amino acids is equimolar.

2. The library of Claim 1, wherein said antigenic site, epitope or ligand is from a virus, bacterium, parasite, tumor antigen, allergen or other protein antigen, from a diagnostic marker site of an infectious agent or disease, from a therapeutically valuable ligand or from a helper T cell epitope or a cytotoxic T lymphocyte (CTL) epitope.

3. The library of Claim 1, wherein said antigenic site, epitope or ligand is from an envelope, core, NSl, NS3, NS4 or

NS5 proteins of HCV; a gpl20 V3 loop, gp41 envelope protein or the gp40 envelope protein of HIV; an envelope protein of HTLV I/II; an HA protein or a mutant HA domain of influenza A virus; an major outer membrane protein of Chlamydia trachomtis; a neuraminidase, SAPA or CRA site of T. cruzi; an envelope B cell site or helper T cell site of type 2 Dengue virus; an M protein of streptococcus; a carbohydrate recognition site of selectin; a promiscuous helper T cell 5 sites; a CTL epitope from HIV gag specific for HLA-B27; or an OspA, OspB, OspC or flagellin protein of Lyme disease.

4. The library of Claim 1 wherein said ensemble domain is represented by an SSAL library sequence provided in any one of Figs. 2-11, 13-34 (SEQ ID NO:l-43). 10

5. A peptide composition comprising the library of Claim 1.

6. The peptide composition of Claim 5 which is immunogenic.

7. A method of detecting antibodies associated with an 15 infectious agent or a disease state which comprises using an effective amount of a peptide composition according to Claim 5 in an immunoassay procedure.

8. A method of detecting infection or a disease state which comprises contacting an effective amount of a peptide

20 composition of Claim 5 with a body fluid, tissue or tissue extract in an immunoassay procedure for a time sufficient to form a complex between said peptide composition and any antibody in said fluid, said tissue, or said tissue extract, and subjecting said complex to a detecting means.

25 9. The method of Claim 7 wherein said immunoassay procedure is an ELISA or a PHA procedure.

10. The method of Claim 8 wherein said immunoassay procedure is an ELISA or a PHA procedure.

11. A kit for detection or diagnosis of an infectious 30 agent or a disease state comprising a first container adapted to contain the peptide composition of Claim 5.

I

12. The kit of Claim 11 wherein said kit is an ELISA or

PHA test kit.

13. A method of treating a disease which comprises administering an effective amount of a composition of Claim 5 to a patient for a time sufficient to elicit an efficacious result.

14. A pharmaceutical composition comprising the library of Claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

15. A method of vaccination which comprises administering an amount of the library of Claim 1 to a mammal effective to produce a protective immune response against an infectious agent which provided the basis of the library.

16. A vaccine composition comprising a library of Claim 1, or a salt thereof, and immunologically acceptable carrier.

17. A method of making an open diagnostic, vaccine or therapeutic composition for a mutatable infectious agent or for a protein site of known diversity which comprises

(1) aligning a collection of primary amino acid sequences for a related family of antigens, epitopes, diagnostic markers or therapeutic sites from said mutatable infectious agent or said protein site;

(2) identifying invariant and variant amino acid positions in said alignment;

(3) calculating a consensus formula for the sequence of a structured synthetic antigen library (SSAL) ; (4) preparing said SSAL from said consensus formula.

18. The method of Claim 17 wherein said consensus formula provides that each sequence position in the alignment contains either a single amino acid or multiple amino acids, and that when a position contains multiple amino acids, (a) the identity and ratio of said multiple amino acids being determined by the relative prevalence of amino acids in said alignment, (b) the identity of said multiple amino acids being determined by the amino acids present in said alignment and the ratio of said multiple amino acids is equimolar, or (c) the identity of said multiple amino acids being determined by the amino acids present in said alignment and the ratio of said multiple amino acids is weighted towards the predominant amino acid at said position.

19. The method of Claim 17 wherein said mutatable infectious agent is from a virus, bacterium or parasite.

20. The method of Claim 17 wherein said protein site is from a tumor antigen, allergen or other protein antigen, from a helper T cell epitope or a cytotoxic T lymphocyte (CTL) epitope.

21. The method of Claim 17, wherein said family of antigens, epitopes, diagnostic markers or therapeutic sites is from an envelope, core, NSl, NS3, NS4 or NS5 proteins of HCV; a gpl20 V3 loop, gp41 envelope protein or the gp40 envelope protein of HIV; an envelope protein of HTLV I/II; an HA protein or a mutant HA domain of influenza A virus; an major outer membrane protein of Chlamydia trachomtis; a neuraminidase, SAPA or CRA site of T. cruzi; an envelope B cell site or helper T cell site of type 2 Dengue virus; an M protein of streptococcus; a carbohydrate recognition site of selectin; a promiscuous helper T cell sites; a CTL epitope from HIV gag specific for HLA-B27; or an OspA, OspB, OspC or flagellin protein of Lyme disease.