Rhamnose polysaccharide

Abstract

The present invention relates to a method for synthesizing rhamnose polysaccharide. The present invention also relates to streptococcal polysaccharides, streptococcal glycoconjugates, immunogenic compositions or vaccines comprising streptococcal polysaccharides or glycoconjugates, and polysaccharides, glycoconjugates, immunogenic compositions or vaccines for enhancing the immune response of animals or for treating or preventing diseases, disorders or infections caused by streptococcal etiology.

Description

Technical Field

[0001] The present invention relates to a method for synthesizing rhamnose polysaccharide. The present invention also relates to streptococcal polysaccharides, streptococcal glycoconjugates, immunogenic compositions or vaccines comprising streptococcal polysaccharides or glycoconjugates, and polysaccharides, glycoconjugates, immunogenic compositions or vaccines for enhancing the immune response of animals or for treating or preventing diseases, disorders or infections caused by streptococcal etiology. Background Art

[0002] Streptococcus bacteria are a group of versatile Gram-positive bacteria that can infect a wide range of hosts and cause a large number of diseases.

[0003] Streptococcus pyogenes (Group A Streptococcus, GAS) is a human-specific pathogenic Gram-positive bacterium that can cause a variety of diseases. An assessment of the possible underestimated prevalence of this organism indicates that more than 700 million people are affected worldwide each year, leading to various diseases such as impetigo, pharyngitis, scarlet fever, necrotizing fasciitis, meningitis and toxic shock syndrome. In addition, post-infectious autoimmune sequelae such as acute rheumatic fever, acute glomerulonephritis or rheumatic heart disease can affect individuals previously infected with GAS, thus expanding the list of clinical manifestations caused by this pathogen. Group A carbohydrate (GAC) is a peptidoglycan-anchored rhamnose polysaccharide (RhaPS) from Streptococcus pyogenes, which is essential for bacterial survival and contributes to the ability of Streptococcus pyogenes to infect the human host.

[0004] Streptococcus agalactiae (Group B Streptococcus, GBS) is a (pathogenic) commensal bacterium carried by 20 - 40% of adults. 25% of women carry GBS in their vagina, where it usually exists asymptomatically. However, in pregnant women, GBS is a recognized cause of preterm birth, maternal infection, stillbirth and late miscarriage. Despite current preventive strategies, 1 in every 1000 babies born in the UK develops GBS infection. It is well known that preterm infants are particularly vulnerable to GBS infection because their immune systems are underdeveloped. This results in one baby dying from GBS infection and one baby surviving but with long-term disabilities every week in the UK.

[0005] Group C Streptococcus (GCS) can cause epidemic pharyngitis and cellulitis, which are clinically indistinguishable from human GAS diseases. It is also known to cause sepsis, endocarditis, septic arthritis and necrotizing infections in patients with susceptible diseases such as diabetes, cancer or in elderly patients. In horses, GCS is the cause of a highly contagious and severe upper respiratory tract infection (known as strangles), which is endemic worldwide.

[0006] Group G streptococcus (GGS) is an important human pathogen that causes skin infections, such as human skin infections. GGS also infects the oropharynx, gastrointestinal tract, and female genital tract. Other infections associated with GGS include several life-threatening infections, such as sepsis, endocarditis, meningitis, peritonitis, pneumonia, empyema, and septic arthritis.

[0007] The antimicrobial options for effectively controlling, treating, and preventing GAS infections are becoming increasingly limited. This is due to the emergence of antibiotic resistance, the development of pandemics, and the spread of hypervirulent strains. Therefore, there is an obvious need to develop a safe and effective candidate vaccine. For a vaccine capable of targeting most of the more than 120 different GAS serotypes, it needs to be based on a prevalent, conserved, and essential GAS target. One such target is GAC, which is not only an important structural component of the pathogen but also a virulence determinant.

[0008] Current forms of vaccine development are limited to methods of chemical and enzymatic extraction from natural bacteria and chemical conjugation with any receptor compound, such as a protein or peptide. This is highly labor-intensive and results in limited yields and quality of the product. There is an obvious need for a method of producing GAS polysaccharide that is less labor-intensive and results in homogeneous, pure, and high-yield polysaccharides. The present invention has been designed in view of these problems.

[0009] Description

[0010] The present disclosure relates, in its broadest sense, to a method for synthesizing a polysaccharide, particularly a rhamnose polysaccharide.

[0011] According to a first aspect, there is provided a method for synthesizing a rhamnose polysaccharide, the method comprising:

[0012] (i) transferring a rhamnose moiety to a hexose monosaccharide, disaccharide, or trisaccharide using a hexose-β-1,4-rhamnosyltransferase, hexose-α-1,2-rhamnosyltransferase, and / or hexose-α-1,3-rhamnosyltransferase or an enzymatically active fragment or variant thereof to form a disaccharide, trisaccharide, or tetrasaccharide comprising a rhamnose moiety at the non-reducing end of the disaccharide, trisaccharide, or tetrasaccharide; and

[0013] (ii) extending from the rhamnose moiety at the non-reducing end of the disaccharide, trisaccharide, or tetrasaccharide by using the heterologous bacterial enzyme Streptococcus pyogenes group A carbohydrate enzyme C (GacC) and / or Streptococcus pyogenes group A carbohydrate enzyme G (GacG) or an enzymatically active homolog, variant, or fragment thereof to generate a rhamnose polysaccharide.

[0014] The bacterial species from which the enzyme GacC and / or the enzyme GacG or an enzymatically active homolog, variant or fragment thereof is derived is heterologous to the bacterial species from which the hexose-β-1,4-rhamnosyltransferase, hexose-α-1,2-rhamnosyltransferase, hexose-α-1,3-rhamnosyltransferase or an enzymatically active fragment or variant thereof used in step (i) is derived.

[0015] The inventors have for the first time discovered that the Streptococcus pyogenes enzyme GacB that initiates GAC rhamnan synthesis is an α-D-GlcNAc-β-1,4-L-rhamnosyltransferase. Quite surprisingly, the inventors have found that these rhamnans can be synthesized using rhamnosyltransferases from bacterial species different from the bacterial species from which GacB is derived. In other words, the inventors have found that rhamnans can be synthesized using rhamnosyltransferases from bacterial species other than Streptococcus pyogenes. This was completely unexpected as the function of GacB was previously unknown. Equally surprisingly, enzymes from different species can co-synthesize rhamnans.

[0016] In some embodiments, step (ii) comprises elongating to generate a rhamnan from the rhamnose moiety at the non-reducing end of a disaccharide, trisaccharide or tetrasaccharide by using the heterologous bacterial enzyme GacC or an enzymatically active homolog, variant or fragment thereof.

[0017] Polysaccharide is a term known in the art and is used to denote a molecule containing multiple, usually more than four, identical or different monosaccharides. Thus, the term rhamnan as used herein will be understood to refer to a molecule containing multiple, usually more than four, rhamnose moieties optionally linked to one or more other monosaccharide moieties. Conveniently, the rhamnan can be a single straight chain containing repeating units of rhamnose, which are bound to each other by α1,3 or α1,2 bonds. Each repeating unit can consist only of rhamnose, or each repeating unit can contain rhamnose and one or more different monosaccharides. An exemplary repeating unit containing rhamnose is the rhamnose-galactose disaccharide repeating unit. Each / any repeating unit and / or rhamnose moiety can include or not include any side groups. In one embodiment, there are no side groups, while in another embodiment, there may be one or more side groups (e.g., sugars) with or without additional modifications (e.g., phosphoglycerol; or phosphate).

[0018] In an embodiment, the method is carried out in bacteria.

[0019] In such an embodiment, the method will be understood as a microbiological method. Embodiments other than those carried out in bacteria will be understood as in vitro methods. For "bacteria", this will be understood to refer to bacterial cells. It should be understood that the present invention also includes methods carried out in bacteria. Such microbiological methods are well suited for the production of large quantities and homogeneous specific products, in this case, rhamnan polysaccharide.

[0020] The rhamnan polysaccharide produced by this method will be understood as synthetic rhamnan polysaccharide. As will be understood by a person skilled in the art, synthetic rhamnan polysaccharide will be understood to refer to rhamnan polysaccharide that is not the result of a naturally occurring process. This is because the method of the first aspect uses enzymes, the combination of which is not naturally occurring. In one embodiment, the bacterium is a Streptococcus species other than Streptococcus pyogenes, an Escherichia species (such as E. coli), or a Shigella species (such as Shigella dysenteriae or Shigella flexneri).

[0021] Generally, the rhamnan polysaccharide produced by this method is a streptococcal polysaccharide. For example, the polysaccharide may include a polysaccharide or a fragment or variant thereof selected from the group consisting of group A, group B, group C, and group G carbohydrates.

[0022] For the rhamnose moiety, this will be understood to refer to rhamnose monosaccharide or its derivatives. It should be understood that a rhamnose derivative refers to a rhamnose monosaccharide that is modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide, provided that at least one carbon of the rhamnose monosaccharide is still capable of forming a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. Derivatives of rhamnose may include acetyl or methyl forms of rhamnose, amino rhamnose, carboxyethyl rhamnose, halogenated rhamnose, and phospho rhamnose. Unless the context otherwise indicates, the rhamnose moiety will generally be referred to hereinafter, but this should not be construed as limiting.

[0023] Halogenated rhamnose should be understood to refer to such a rhamnose monosaccharide in which one or more groups of rhamnose (such as one or more OH groups) are replaced by a halogen such as a fluoride ion or a chloride ion to form fluoro rhamnose or chloro rhamnose, respectively.

[0024] Amino rhamnose will be understood to refer to a rhamnose monosaccharide in which one or more groups of rhamnose are replaced by an amino group.

[0025] Exemplary acetyl rhamnose may include 2-O-acetyl-α-L-rhamnose, while exemplary methyl rhamnose may include 3-O-methyl-L-rhamnose. Another exemplary derivative of rhamnose may include carboxyethyl rhamnose, such as 4-O-(1-carboxyethyl)-L-rhamnose.

[0026] By an enzymatic active fragment or variant, we encompass that the sequence of the relevant enzyme can be different from the naturally occurring sequence, provided that the fragment or variant substantially retains the enzymatic activity of the enzyme. Retaining the enzymatic activity of the enzyme means that, compared with the native enzyme, the fragment and / or variant retains at least a part of the enzymatic activity. Generally, the fragment and / or variant retains at least 50%, such as 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% of the activity. In some cases, the fragment and / or variant may have a higher enzymatic activity than the native enzyme. In some embodiments, compared with the native enzyme, the fragment and / or variant can show an increase in another physiological characteristic. For example, compared with the native enzyme, the fragment and / or variant can have a longer half-life in vitro and / or in vivo. Tests for determining the half-life of an enzyme or its fragment or variant will be known to those skilled in the art. Briefly, in vitro tests may involve incubating the enzyme for different periods of time at a specific temperature and pH. At the end of each period, the activity of the enzyme or its fragment or variant can be measured using an enzyme assay well-known to those skilled in the art.

[0027] As used herein, the enzyme GacC will be understood to refer to Streptococcus pyogenes group A carbohydrate enzyme C (UniProtKB-Q9A0G4 (Q9A0G4_STRP1)). An exemplary amino acid sequence encoding GacC is provided by SEQ ID NO: 1.

[0028] As used herein, the enzyme GacG will be understood to refer to Streptococcus pyogenes group A carbohydrate enzyme G (UniProtKB-Q9A0G0 (Q9A0G0_STRP1)). In some embodiments, the enzyme GacG comprises SEQ ID NO: 2 or its enzymatic active fragment or variant or consists of SEQ ID NO: 2 or its enzymatic active fragment or variant.

[0029] In the method of the present invention, GacG (or its enzymatic active homolog, variant or fragment) is used in place of GacC, or GacG (or its enzymatic active homolog, variant or fragment) is used in addition to GacC. GacC is a rhamnose-1,3α rhamnosyltransferase, while GacG is a predicted bifunctional glycosyltransferase that can synthesize the repeating unit of GAC (alpha1,3-alpha1,2).

[0030] "Homolog" can include enzymes that exhibit at least about 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the GacC or GacG amino acid sequence.

[0031] In some embodiments, the enzymatic active homolog is a homolog of GacC.

[0032] The degree (or percentage) of "homology" between two or more amino acid sequences can be calculated by aligning the sequences and determining the number of identical residues in the alignment and adding to this the number of conservative amino acid substitutions. The combined total is then divided by the total number of residues compared, and the resulting number is multiplied by 100 - this gives the percentage of homology between the aligned sequences.

[0033] Typically, homologs of GacC or GacG include enzymes that substantially retain the enzymatic activity of GacC or GacG.

[0034] In some embodiments, a homolog of GacC comprises or consists of rfbG. RfbG is an α-1-3 rhamnosyltransferase derived from Shigella flexneri that has 30% identity with GacC. Thus, in the context of the present invention, rfbG is an enzymatic activity homolog of GacC. In some embodiments, rfbG comprises or consists of SEQ ID NO:3. RfbG can be identified using UniProtKB - A0A2D0WWB9 (A0A2D0WWB9_9ENTR).

[0035] A homolog of GacC or GacG can comprise or consist of rfbG, which is an enzyme sourced from Lancefield group species other than Streptococcus pyogenes and / or non-Lancefield group Streptococcus species other than Streptococcus pneumoniae.

[0036] In some embodiments, a homolog of GacC or GacG is an enzyme sourced from Lancefield group species other than Streptococcus pyogenes and / or non-Lancefield group Streptococcus species other than Streptococcus pneumoniae.

[0037] As will be appreciated by those skilled in the art, the Lancefield grouping of bacteria refers to a set of different bacterial species, mainly Streptococcus species, which are catalase-negative and coagulase-negative. This grouping is based on the carbohydrate composition of cell wall antigens.

[0038] Lancefield group bacteria include:

[0039] · Group A - Streptococcus pyogenes, Streptococcus dysgalactiae subsp. equisimilis

[0040] · Group B - Streptococcus agalactiae

[0041] · Group C - Streptococcus equisimilis, Streptococcus equi, Streptococcus zooepidemicus, Streptococcus dysgalactiae, Streptococcus dysgalactiae subsp. equisimilis

[0042] · Group D - Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, and Streptococcus bovis

[0043] · Group E - Enterococcus

[0044] · Groups F, G, and L - Streptococcus anginosus, Streptococcus dysgalactiae subsp. equisimilis.

[0045] · Group H - Streptococcus sanguis

[0046] · Group K - Streptococcus salivarius

[0047] · Group L - Streptococcus dysgalactiae

[0048] · Groups M&O - Streptococcus mitior

[0049] · Group N - Lactococcus lactis

[0050] · Groups R&S - Streptococcus suis

[0051] Non - Lancefield streptococcal species can include Streptococcus mutans or Streptococcus uberis. In some embodiments, the non - Lancefield streptococcal species can include Streptococcus mutans or consist of Streptococcus mutans.

[0052] The enzymatic activity homologs of GacC or GacG can be selected from homologs of Group B, C, G streptococci, Streptococcus mutans, Streptococcus uberis, or enzymatic activity fragments or variants thereof.

[0053] In some embodiments, the enzymatic activity homologs of GacC or GacG can be selected from homologs of Group B, C, G streptococci, Streptococcus mutans, or enzymatic activity fragments or variants thereof.

[0054] In some embodiments, the enzymatic activity homologs of GacC are selected from the GacC homologs of Group B, Group C, Group G streptococci, Streptococcus mutans, Streptococcus uberis, or enzymatic activity fragments or variants thereof. Those skilled in the art will know the streptococcal homologs of GacC. For example, the Group B homolog of GacC can be GbcC (UniProtKB-Q8DYQ2 (Q8DYQ2_STRA5)). The Group C homolog of GacC can be GccC (UniProtKB-M4YWQ3 (M4YWQ3_STREQ)). The Group G homolog of GacC can be GgcC (UniProtKB-C5WFT8 (C5WFT8_STRDG)), and the S. mutans homolog of GacC can be SccC (UniProtKB-A0A0E2EN43 (A0A0E2EN43_STRMG)). The S. uberis homolog of GacC can be SucC (UniProtKB-B9DU25 (B9DU25_STRU0)).

[0055] The amino acid sequence of GbcC can comprise SEQ ID NO: 4 or consist of SEQ ID NO: 4. The amino acid sequence of GccC can consist of SEQ ID NO: 5, and the amino acid sequence of GgcC can consist of SEQ ID NO: 6. In some embodiments, SccC comprises SEQ ID NO: 7 or consists of SEQ ID NO: 7. The amino acid sequence of SucC can comprise SEQ ID NO: 8 or consist of SEQ ID NO: 8.

[0056] In some embodiments, the enzymatic activity homologs of GacG are selected from the GacG homologs of Group C, Group G streptococci, Streptococcus mutans, Streptococcus uberis, or enzymatic activity fragments or variants thereof. Suitable enzymatic activity homologs of GacG include, but are not limited to, the Group C homolog GccG of GacG, the Group G homolog GgcG of GacG, the S. uberis homolog SucG of GacG, and the S. mutans homolog SccG of GacG.

[0057] In some embodiments, GccG comprises SEQ ID NO: 9 and consists of SEQ ID NO: 9. In some embodiments, GccG comprises two proteins or consists of two proteins. These two proteins can comprise SEQ ID No: 10 and 11 or consist of SEQ ID No: 10 and 11.

[0058] GgcG may comprise two proteins or consist of two proteins. These two proteins may have UniProtKBs C5WFU2 (C5WFU2_STRDG) and C5WFU3 (C5WFU3_STRDG), respectively. In some embodiments, GgcG may comprise SEQ ID No: 12 and 13 or consist of SEQ ID No: 12 and 13.

[0059] SucG may comprise the amino acid sequence identified by UniProtKB - B9DU29 (B9DU29_STRU0) or consist of the amino acid sequence. For example, SucG may comprise the amino acid sequence SEQ ID NO: 14 or consist of the amino acid sequence SEQ ID NO: 14.

[0060] SccG may comprise the amino acid sequence identified by UniProtKB - O82878 (O82878_STRMG) or consist of the amino acid sequence. In some embodiments, SccG comprises the amino acid sequence SEQ ID NO: 15 or consists of the amino acid sequence SEQ ID NO: 15.

[0061] The enzymatic activity homologs of GacC or GacG may be selected from homologs of Streptococcus mutans, Streptococcus uberis, or fragments or variants thereof.

[0062] In some embodiments, step (ii) comprises generating rhamnan by extending from the rhamnose moiety at the non - reducing end of a disaccharide, trisaccharide, or tetrasaccharide using an enzymatic activity homolog of GacC and / or GacG from Streptococcus mutans or an active variant or fragment thereof.

[0063] The present invention also includes nucleic acid sequences encoding the enzymes (and / or enzymatic activity fragments, variants, or homologs) of the present invention.

[0064] As used herein, when an enzyme "is derived from" a particular bacterial species, this means that the enzyme naturally occurs in the particular bacterial species. In the context of the present invention, an enzyme "derived from" a particular bacterial species may include an enzyme endogenous to the bacteria in which the method can be performed, an enzyme isolated from a particular bacterial species, or the nucleic acid encoding the enzyme, or variants or fragments thereof. In embodiments where the method is performed in bacteria, an enzyme isolated from a particular bacterial species or the nucleic acid encoding the enzyme can be transferred into the bacteria in which the method is performed.

[0065] In an embodiment of the method carried out in bacteria, the enzyme of step (i) and / or the enzyme of step (ii) can be overexpressed in bacteria. "Overexpression" will be understood to mean that the expression level of the enzyme is higher than the expression level of the naturally occurring enzyme observed when it is endogenously expressed in its natural bacteria. Various techniques for overexpression are known to those skilled in the art. More information on overexpression techniques can be found in Current Protocols in Molecular Biology (2019), which is incorporated herein by reference.

[0066] In the context of the present invention, heterologous is used to refer to different. A heterologous bacterial species will be understood to mean a bacterial species that is different from another bacterial species, or a bacterial genus that is different from another bacterial genus.

[0067] It should be understood that in the context of the present invention, heterologous does not include a bacterial strain that is different from another bacterial strain (i.e., for example, two strains of Streptococcus mutans).

[0068] For "variants" of an enzyme, we encompass insertions, deletions, and substitutions of the amino acid sequence, whether conservative or non-conservative, where the physicochemical properties of each amino acid are substantially unchanged (e.g., conservative substitutions such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr). Those skilled in the art will understand that such conservative substitutions should not affect the function of the corresponding enzyme. In addition, small deletions within non-functional regions of the enzyme are also tolerated and are thus considered "variants" for the purposes of the present invention. "Variants" also include recombinant enzyme proteins in which the amino acids have been post-translationally modified, such as by glycosylation or disulfide bond formation. Those skilled in the art can readily employ the experimental procedures described herein to determine whether a "variant" can still be used as an enzyme.

[0069] Preferred variants have an amino acid sequence that has at least 75%, more preferably at least 80%, further preferably at least 85%, still further preferably at least 90%, most preferably at least 95%, 97%, 98%, or 99% identity to the "naturally occurring" amino acid sequence of the enzyme.

[0070] It should be understood that variants also include variants of the nucleic acid sequence encoding the enzyme. In particular, we encompass nucleotide sequence variants in which these changes do not significantly alter the enzyme activity of the enzyme encoded by them. Those skilled in the art will know that such sequences can be altered without loss of enzyme activity. In particular, a single change in the nucleotide sequence may not result in a change in the amino acid sequence after sequence expression.

[0071] In some embodiments, the method is performed in a bacterial species that is heterologous to the bacterial species or genus from which the enzyme GacC and / or GacG or an enzymatically active homolog, variant, or fragment thereof is derived. In some embodiments, the method is performed in Gram-positive bacteria. The method can be performed in Gram-negative bacteria. For example, the method can be performed in Gram-negative bacteria such as Escherichia coli or Campylobacter species. Other suitable Gram-negative bacteria are known to those skilled in the art. In an embodiment, the bacterial species can be heterologous to the bacterial species or genus from which the hexose-β-1,4-rhamnosyltransferase, hexose-α-1,2-rhamnosyltransferase, or hexose-α-1,3-rhamnosyltransferase is derived.

[0072] In some embodiments, the method is performed in Escherichia coli.

[0073] Step ii) of the method can include using one or more additional enzymes from the bacterial enzyme Gac cluster, or one or more enzymatically active homologs, variants, or fragments thereof.

[0074] As will be understood by those skilled in the art, GacB is one of a plurality of enzymes encoded by a gene cluster in Streptococcus pyogenes. As defined by van Sorge et al. in 2014, this gene cluster (also referred to as the Gac gene cluster) (gacA-gacL, MGAS5005_Spy_0602-0613) is understood to encode 12 different enzymes. These 12 enzymes are GacA, GacB, GacC, GacD, GacE, GacF, GacG, GacH, GacI, GacJ, GacK, and GacL. Thus, step ii) of the method can further include using one or more additional enzymes from the bacterial enzyme Gac cluster, or one or more enzymatically active homologs, variants, or fragments thereof. Thus, in some embodiments, step ii) of the method includes using one or more additional enzymes, or one or more enzymatically active homologs, variants, or fragments thereof, the additional enzymes being selected from GacA, GacC, GacD, GacE, GacF, GacG, GacH, GacI, GacJ, GacK, GacL.

[0075] In some embodiments, step ii) of the method further includes using one or more enzymatically active homologs of one or more of GacA, GacC, GacD, GacE, GacF, GacG, GacH, GacI, GacJ, GacK, GacL, or an enzymatically active variant or fragment thereof.

[0076] One or more enzymatically active homologs can be derived from Streptococcus mutans and / or Streptococcus uberis.

[0077] In some embodiments, one or more enzymatically active homologs are derived from Streptococcus mutans.

[0078] Step ii) may further comprise using the enzyme GacA or an enzymatically active homolog, fragment or variant thereof. In some embodiments, step ii) may comprise using the enzymes GacC and GacG, or one or more enzymatically active homologs, variants or fragments thereof.

[0079] In some embodiments, step ii) comprises using the enzymes GacC, GacA and GacG, or one or more enzymatically active homologs, variants or fragments thereof. Step ii) may further comprise using the enzymes GacD, GacE and GacF, or one or more enzymatically active homologs, fragments or variants thereof.

[0080] Step ii) may comprise using the enzymes GacC, GacA, GacG, GacD, GacE and GacF, or one or more enzymatically active homologs, fragments or variants thereof.

[0081] In some embodiments, step ii) comprises using the enzymes GacA, GacC, GacD, GacE, GacF, GacG, GacH, GacI, GacJ, GacK and GacL, or one or more enzymatically active homologs, variants or fragments thereof.

[0082] Step ii) may comprise using enzymatically active homologs of GacA, GacC, GacD, GacE, GacF, GacG and GacH from Streptococcus mutans and / or Streptococcus uberis.

[0083] In some embodiments, step ii) comprises using enzymatically active homologs of GacA, GacC, GacD, GacE, GacF, GacG and GacH from Streptococcus mutans.

[0084] GacA may comprise SEQ ID NO: 16 or consist of SEQ ID NO: 16. Without wishing to be bound by theory, it is believed that the function of GacA is to synthesize the rhamnose moiety required for rhamnan production. GacG is thought to be involved in rhamnan production by extending from the rhamnose moiety at the reducing end.

[0085] GacD and GacE may play a role in forming an ATP-dependent ABC transporter. As will be understood by those skilled in the art, an ATP-dependent ABC transporter transports substrates across a membrane. Thus, without wishing to be bound by theory, GacD and GacE may help transport rhamnan across the bacterial membrane so that it can subsequently be presented on the bacterial cell wall.

[0086] GacH may comprise SEQ ID NO: 17 or consist of SEQ ID NO: 17. GacH can also be identified using UniProtKB-J7M7C2 (J7M7C2_STRP1).

[0087] In some embodiments, step ii) further comprises using the enzymes GacH, GacI, GacJ, GacK and GacL, or one or more enzyme activity homologs, variants or fragments thereof.

[0088] It is believed that GacI and / or GacJ can improve the catalytic efficiency of the method for synthesizing rhamnopolysaccharide.

[0089] Enzyme activity homologs of GacA can be selected from homologs of GacA from Group B, C, G streptococci, Streptococcus mutans, Streptococcus uberis, or enzyme activity fragments or variants thereof. For example, the Group B streptococcus homolog of GacA is RmID. The Group C streptococcus homolog of GacA is RmID, and the same is true for the Group G streptococcus homolog of GacA.

[0090] The Group B streptococcus homolog RmID of GacA may have UniProtKB-A0A0E1EP43 (A0A0E1EP43_STRAG). In some embodiments, the Group B streptococcus homolog RmID of GacA comprises SEQ ID NO: 18 or consists of SEQ ID NO: 18.

[0091] The Group C streptococcus homolog RmID of GacA may have UniProtKB-K4Q921 (K4Q921_STREQ). In some embodiments, the Group C streptococcus homolog RmID of GacA comprises SEQ ID NO: 19 or consists of SEQ ID NO: 19.

[0092] The Group G streptococcus homolog RmID of GacA may have UniProt-KB A0A2X3AIL5 (A0A2X3AIL5_STRDY). The Group G streptococcus homolog of GacA can comprise SEQ ID NO: 20 or consist of SEQ ID NO: 20.

[0093] The Streptococcus mutans homolog of GacA can be identified using UniProtKB-O33664 (O33664_STRMG). In some embodiments, the Streptococcus mutans homolog of GacA can comprise SEQ ID NO: 21 or consist of SEQ ID NO: 21.

[0094] The Streptococcus uberis homolog of GacA can be identified using UniProtKB - B9DU23 (B9DU23_STRU0). In some embodiments, the Streptococcus uberis homolog of GacA can comprise SEQ ID NO: 22 or consist of SEQ ID NO: 22.

[0095] Enzymatically active homologs of GacD, GacE, and / or GacF can be selected from homologs from Group C streptococci, Group G streptococci, Streptococcus mutans, Streptococcus uberis, or enzymatically active fragments or variants thereof. Suitable homologs of GacD include, but are not limited to, Group C streptococcal enzyme GccD, Group G streptococcal enzyme GgcD, and Streptococcus mutans enzyme SccD. Suitable homologs of GacE include, but are not limited to, Group C streptococcal enzyme GccE, Group G streptococcal enzyme GgcE, and Streptococcus mutans enzyme SccE. Suitable homologs of GacF include, but are not limited to, Group C streptococcal enzyme GccF, Group G streptococcal enzyme GgcF, Streptococcus mutans enzyme SccF, and Streptococcus uberis enzyme SucF.

[0096] In some embodiments, GccD comprises the amino acid sequence SEQ ID NO: 23 or consists of SEQ ID NO: 23. GccE can be identified using UniProtKB - A0A380KIL0 (A0A380KIL0_STREQ). In some embodiments, GccE comprises the amino acid sequence SEQ ID NO: 24 or consists of SEQ ID NO: 24. GccF can be identified using UniProtKB - A0A3S4QIR3 (A0A3S4QIR3_STREQ). Optionally, GccF comprises SEQ ID NO: 25 or consists of SEQ ID NO: 25.

[0097] In some embodiments, GgcD comprises the amino acid sequence SEQ ID NO: 26 or consists of the amino acid sequence SEQ ID NO: 26. GgcD can be identified using UniProtKB - C5WFT9 (C5WFT9_STRDG).

[0098] In some embodiments, GgcE is identified by UniProtKB - M4YXS7 (M4YXS7_STREQ). Optionally, GgcE comprises SEQ ID NO: 27 or consists of SEQ ID NO: 27. GgcF can be identified by UniProtKB - C5WFU1 (C5WFU1_STRDG). In some embodiments, GgcF comprises SEQ ID NO: 28 or consists of SEQ ID NO: 28.

[0099] SccD may comprise or consist of SEQ ID NO: 29. Optionally, SccD is identified using UniProtKB-I6L8Z4 (I6L8Z4_STRMU).

[0100] SccE may comprise or consist of SEQ ID NO: 30. Optionally, SccE is identified using UniProtKB-I6L8X8 (I6L8X8_STRMU).

[0101] SccF may be identified using UniProtKB-O82877 (O82877_STRMG). Optionally, SccF comprises or consists of SEQ ID NO: 31.

[0102] SucD may be identified using UniProtKB-B9DU26 (B9DU26_STRU0). In some embodiments, SucD comprises or consists of SEQ ID NO: 32.

[0103] SucE may be identified using UniProtKB-B9DU27 (B9DU27_STRU0). In some embodiments, SucE comprises or consists of SEQ ID NO: 33.

[0104] SucF may be identified using UniProtKB-B9DU28 (B9DU28_STRU0). In some embodiments, SucF comprises the amino acid sequence SEQ ID NO: 34 or consists of SEQ ID NO: 34.

[0105] An enzyme activity homolog of GacH may comprise or consist of the Streptococcus mutans enzyme SccH or an enzyme activity fragment or variant thereof. The enzyme SccH may be identified using UniProtKB-Q8DUS0 (Q8DUS0_STRMU).

[0106] In some embodiments, SccH comprises or consists of SEQ ID NO: 35.

[0107] In some embodiments, the hexose-β-1,4-rhamnosyltransferase is not an N-acetylglucosamine (GlcNAc)-β-1,4-rhamnosyltransferase. In some embodiments, the hexose-β-1,4-rhamnosyltransferase is not GacB.

[0108] "Hexose-β-1,4-rhamnosyltransferase" shall be understood to be an enzyme capable of transferring a rhamnose moiety to a hexose to form a β-1,4 linkage between the hexose and the rhamnose moiety. Once the rhamnose moiety is transferred, it shall be understood that the hexose is at the reducing end and the rhamnose moiety is at the non-reducing end, i.e., the end from which the rhamnose polysaccharide extends.

[0109] Hexose-β-1,4-rhamnosyltransferase may comprise or consist of the following: allose-β-1,4-rhamnosyltransferase, altrose-β-1,4-rhamnosyltransferase, glucose-β-1,4-rhamnosyltransferase, mannose-β-1,4-rhamnosyltransferase, xylose-β-1,4-rhamnosyltransferase, idose-β-1,4-rhamnosyltransferase, galactose-β-1,4-rhamnosyltransferase, talose-β-1,4-rhamnosyltransferase, diacetylbacillosamine-β-1,4-rhamnosyltransferase, or an enzymatically active fragment or variant thereof.

[0110] In some embodiments, hexose-β-1,4-rhamnosyltransferase comprises glucose (Glc)-β-1,4-rhamnosyltransferase or an enzymatically active fragment or variant thereof. As will be understood by those skilled in the art, glucose (Glc)-β-1,4-rhamnosyltransferase is an enzyme capable of transferring a rhamnose moiety to glucose to form a β-1,4 linkage between the glucose and the rhamnose moiety. Hexose-β-1,4-rhamnosyltransferase may comprise the WchF enzyme, or an enzymatically active fragment or variant thereof. The WchF enzyme shall be understood to be derived from Streptococcus pneumoniae and to be glucose (Glc)-β-1,4-rhamnosyltransferase.

[0111] In some embodiments, the WchF enzyme comprises SEQ ID NO: 36, or an enzymatically active fragment or variant thereof.

[0112] An enzymatically active fragment or variant of WchF may have at least 30% amino acid sequence identity with the WchF enzyme.

[0113] In some embodiments, an enzymatic fragment or variant of WchF has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid identity with the WchF enzyme. For example, the WchF homologs from Streptococcus mitis, Streptococcus oralis, Streptococcus pseudopneumoniae, and Streptococcus peroris have 87%, 93%, 87%, and 81% amino acid identity with WchF, respectively. In the context of the present invention, these specific homologs will thus be understood as enzymatic variants of WchF.

[0114] Hexose-α-1,2-rhamnosyltransferase may comprise or consist of: allose-α-1,2-rhamnosyltransferase, altrose-α-1,2-rhamnosyltransferase, glucose-α-1,2-rhamnosyltransferase, mannose-α-1,2-rhamnosyltransferase, xylose-α-1,2-rhamnosyltransferase, idose-α-1,2-rhamnosyltransferase, α-galactose α-1,2-rhamnosyltransferase, talose-α-1,2-rhamnosyltransferase, diacetylbacillosamine-α-1,2-rhamnosyltransferase, GlcNAc-α-1,2-rhamnosyltransferase, or an enzymatic fragment or variant thereof.

[0115] In some embodiments, hexose-α-1,2-rhamnosyltransferase comprises or consists of: galactose-α-1,2-rhamnosyltransferase, or an enzymatic fragment or variant thereof. Hexose-α-1,2-rhamnosyltransferase may comprise the WbbR enzyme, or an enzymatic fragment or variant thereof. As will be understood by those skilled in the art, the WbbR enzyme (WP_001045977.1-UniProtKB-Q32EG0 (Q32EG0_SHIDS)) is derived from Shigella dysenteriae and is galactose-α-1,2-rhamnosyltransferase.

[0116] The WbbR enzyme may comprise SEQ ID NO: 37 or consist of SEQ ID NO: 37.

[0117] The hexose-α-1,3-rhamnosyltransferase may comprise or consist of the following: allose-α-1,3-rhamnosyltransferase, altrose-α-1,3-rhamnosyltransferase, glucose-α-1,3-rhamnosyltransferase, mannose-α-1,3-rhamnosyltransferase, xylose-α-1,3-rhamnosyltransferase, idose-α-1,3-rhamnosyltransferase, galactose-α-1,3-rhamnosyltransferase, talose-α-1,3-rhamnosyltransferase, diacetylbacillosamine-α-1,3-rhamnosyltransferase, GlcNAc-α-1,3-rhamnosyltransferase, or an enzymatically active fragment or variant thereof.

[0118] In some embodiments, the hexose-α-1,3-rhamnosyltransferase comprises or consists of the following: GlcNAc-α-1,3-rhamnosyltransferase, diNAcBac-α-1,3-rhamnosyltransferase, Glc-α-1,3-rhamnosyltransferase, galactose-α-1,3-rhamnosyltransferase, or a fragment or variant thereof. The hexose-α-1,3-rhamnosyltransferase may comprise or consist of the following: GlcNAc-α-1,3-rhamnosyltransferase or galactose-α-1,3-rhamnosyltransferase, or an enzymatically active fragment or variant thereof.

[0119] The GlcNAc-α-1,3-rhamnosyltransferase may comprise the WbbL enzyme or an enzymatically active fragment or variant thereof. The WbbL enzyme is derived from Escherichia coli. The WbbL enzyme may comprise SEQ ID NO: 38 or an enzymatically active fragment or variant thereof or consist of SEQ ID NO: 38 or an enzymatically active fragment or variant thereof.

[0120] The enzymatically active fragment or variant of WbbL may have at least 20% or at least 25% amino acid sequence identity with the WchF enzyme. For example, a WbbL homolog with 27% amino acid identity to WbbL has been identified in Mycobacterium tuberculosis and is also referred to as WbbL. Thus, in the context of the present invention, this homolog will be understood as an enzymatically active variant of WbbL. This homolog of WbbL derived from Mycobacterium tuberculosis may comprise SEQ ID NO: 39 or consist of SEQ ID NO: 39. Another suitable homolog of WbbL comprises the enzyme rfbF or consists of the enzyme rfbF, which is derived from Shigella flexneri. RfbF may comprise SEQ ID NO: 40 or consist of SEQ ID NO: 40. RfbF can be identified using UniProtKB-A0A2Y2Z3I0 (A0A2Y2Z3I0_SHIFL).

[0121] The galactose-α-1,3-rhamnosyltransferase may comprise the WsaD enzyme or an enzymatically active fragment or variant thereof. The WsaD enzyme is derived from Geobacillus stearothermophilus. In some embodiments, the WsaD enzyme comprises SEQ ID NO: 41 or consists of SEQ ID NO: 41.

[0122] The enzymatically active fragment or variant of WsaD may be derived from other Bacillus strains, such as Brevibacillus species and Paenibacillus species. The enzymatically active fragment or variant of WsaD may have at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% amino acid identity with WsaD.

[0123] The inventors surprisingly found that chimeras of hexose-β-1,4-rhamnosyltransferase, hexose-α-1,2-rhamnosyltransferase, hexose-α-1,3-rhamnosyltransferase or enzymatically active variants, fragments and GacB or an enzymatically active variant, fragment or homolog thereof are capable of transferring the rhamnose moiety to hexose monosaccharides, disaccharides or trisaccharides. Thus, in some embodiments, GacB / hexose-β-1,4-rhamnosyltransferase, hexose-α-1,2-rhamnosyltransferase, hexose-α-1,3-rhamnosyltransferase or an enzymatically active fragment or variant chimera thereof is used to transfer the rhamnose moiety to hexose monosaccharides, disaccharides or trisaccharides. It should be understood that in such embodiments, hexose-β-1,4-rhamnosyltransferase is not GacB.

[0124] The chimera may comprise at least the C-terminal region of GacB linked to the N-terminal region of hexose-β-1,4-rhamnosyltransferase, hexose-α-1,2-rhamnosyltransferase, hexose-α-1,3-rhamnosyltransferase, or an enzymatically active fragment or variant thereof. In some embodiments, the chimera comprises the C-terminal region of GacB linked to the N-terminal region of WchF.

[0125] In some embodiments, the chimeric body comprises the complete amino acid sequence of GacB except for the first 50, 100, 150, 160, 170, 180, 190, or 200 amino acids, and the first 50, 100, 150, 160, 170, 180, 190, or 200 amino acids are replaced by the corresponding hexose-β-1,4-rhamnosyltransferase, hexose-α-1,2-rhamnosyltransferase, hexose-α-1,3-rhamnosyltransferase, or an enzymatically active fragment or variant of their amino acids. An exemplary chimeric body may comprise the amino acid sequence of GacB except for the first 178 amino acids of GacB, and the first 178 amino acids of GacB are replaced by the corresponding WchF amino acids (amino acids 1 to 186).

[0126] The hexose monosaccharide, disaccharide, or trisaccharide to which the rhamnose moiety is transferred can be any hexose. In an embodiment, the hexose monosaccharide is not the rhamnose moiety.

[0127] In embodiments in which the rhamnose moiety is transferred to a hexose disaccharide or trisaccharide, the monosaccharides of the disaccharide or trisaccharide can be the same as or different from each other. For example, the disaccharide can comprise two galactose monosaccharides. Alternatively, the disaccharide can comprise GlcNAc and galactose. GlcNAc can be at the reducing end of the disaccharide, while galactose is at the non-reducing end.

[0128] The disaccharide can comprise one rhamnose moiety. The trisaccharide can comprise one or two rhamnose moieties.

[0129] In some embodiments, the monosaccharide at the reducing end of the hexose monosaccharide, disaccharide, or trisaccharide to which the rhamnose moiety is transferred (i.e., the first monosaccharide of the hexose monosaccharide or disaccharide or trisaccharide) is glucose or a glucose derivative.

[0130] In the context of the present invention, the glucose derivative will be understood to refer to GlcNAc or diNAcBac. In some embodiments, the hexose monosaccharide, disaccharide, or trisaccharide does not include GlcNAc.

[0131] It should be understood that the monosaccharide at the non-reducing end of the hexose monosaccharide, disaccharide, or trisaccharide determines the specificity of the rhamnosyltransferase. This is because the rhamnosyltransferase transfers the rhamnose moiety to the monosaccharide at the non-reducing end of the hexose monosaccharide, disaccharide, or trisaccharide. Thus, when the monosaccharide at the non-reducing end is galactose, the rhamnosyltransferase is galactose rhamnosyltransferase.

[0132] The disaccharide or trisaccharide can comprise a rhamnose moiety at its non-reducing end.

[0133] Exemplary disaccharides can include a reducing-end glucose linked to a rhamnose moiety at the non-reducing end. Other exemplary disaccharides include, but are not limited to, a reducing-end diNAcBac linked to a rhamnose moiety at the non-reducing end, or a reducing-end galactose linked to a rhamnose moiety at the non-reducing end.

[0134] Exemplary trisaccharides include, but are not limited to, a reducing-end glucose linked to a hexose linked to a rhamnose moiety at the non-reducing end, a reducing-end diNAcBac linked to a rhamnose moiety at the non-reducing end, or a reducing-end GlcNAc linked to a hexose linked to a rhamnose moiety at the non-reducing end. Optionally, the hexose of the trisaccharide can be a rhamnose moiety or galactose.

[0135] When referring to a "linkage" between hexoses, this will be understood to refer to a glycosidic bond. In a disaccharide or trisaccharide, the glycosidic bond between the two hexoses in the disaccharide or trisaccharide can be an α or β glycosidic bond. The α bond can be an α1,3 or α1,2 bond. The β bond can be a β1,4 bond.

[0136] The characteristics of the hexose monosaccharides, disaccharides, and trisaccharides described herein also apply to the hexose monosaccharides, disaccharides, and trisaccharides of the Streptococcus polysaccharides of the present invention.

[0137] Example 2 provides other examples of monosaccharides, disaccharides, and trisaccharides to which the rhamnose moiety can be transferred in step (i) of the method and / or monosaccharides, disaccharides, and trisaccharides that include, consist of, or are composed of the hexose monosaccharides, disaccharides, or trisaccharides of the Streptococcus polysaccharides of the present invention.

[0138] In embodiments where step (i) includes transferring a rhamnose moiety to a hexose disaccharide or trisaccharide, the method can further include forming the hexose disaccharide or trisaccharide. The hexose disaccharide or trisaccharide can be formed using a hexosyltransferase (i.e., an enzyme capable of transferring a hexose to another hexose). For a trisaccharide hexose, if each monosaccharide of the trisaccharide is the same (e.g., the trisaccharide is composed of three glucoses), then one hexosyltransferase can be used to transfer each hexose onto another to form the trisaccharide. However, in embodiments where the hexose trisaccharide is formed from at least two different hexoses, two different hexosyltransferases are required to form the hexose trisaccharide.

[0139] When the method further includes forming a hexose disaccharide, a hexose-α-1,3-hexosyltransferase or an enzymatically active fragment or variant thereof can be used to form the hexose disaccharide. A hexose-α-1,3-hexosyltransferase is understood to refer to an enzyme capable of transferring a hexose to another hexose to form an α-1,3 bond. In the context of the present invention, a bond can alternatively be used to refer to a linkage. In some embodiments, a hexose-α-1,3-galactosyltransferase is used to form the hexose disaccharide. The hexose-α-1,3-galactosyltransferase can comprise or consist of: GlcNAc-α-1,3-galactosyltransferase, optionally enzyme WbbP, or an enzymatically active fragment or variant thereof. Enzyme WbbP can be identified using UniProt KB-Q53982 (Q53982_SHIDY). In some embodiments, WbbP can comprise the amino acid sequence SEQ ID NO: 42 or consist of the amino acid sequence SEQ ID NO: 42. Thus, in some embodiments, the disaccharide consists of GlcNAc at its reducing end and galactose at its non-reducing end, and the two hexoses are linked by an α-1,3 bond.

[0140] In some embodiments, the method includes using enzyme WbbP or an enzymatically active fragment or variant thereof to form a hexose disaccharide, and then using enzyme WbbR or an enzymatically active fragment or variant thereof to transfer a rhamnose moiety to the hexose disaccharide.

[0141] A hexose-α-1,3-rhamnosyltransferase or an enzymatically active fragment or variant thereof can be used to form the hexose disaccharide. For example, galactose-α-1,3-rhamnosyltransferase (such as WsaD or an enzymatically active fragment or variant thereof) can be used to form the hexose disaccharide. In such embodiments, it should be understood that the hexose disaccharide is formed from galactose at the reducing end and a rhamnose moiety at the non-reducing end. When using galactose-α-1,3-rhamnosyltransferase to form the hexose disaccharide, enzyme WsaP can also optionally be used for the formation of the disaccharide, such as to link a lipid to galactose. The WsaP enzyme is derived from Geobacillus stearothermophilus. WsaP can be identified using UniprotKB-Q7BG44 (Q7BG44_GEOSE). In some embodiments, the WsaP enzyme comprises SEQ ID NO: 43 or consists of SEQ ID NO: 43.

[0142] The enzymatically active fragments or variants of WsaP can be derived from other Bacillus strains, such as Brevibacillus species and Paenibacillus species. The enzymatically active fragments or variants of WsaP can have at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% amino acid identity with WsaP.

[0143] Hexose disaccharides can be extended using hexose-α-1,2-hexosyltransferases or enzymatic active fragments or variants thereof to form trisaccharides or tetrasaccharides, and then further extended from the rhamnose moiety at the non-reducing end of the trisaccharide or tetrasaccharide using the heterologous bacterial enzymes GacC and / or GacG or their enzymatically active homologs, variants or fragments. Exemplary hexose-α-1,2-hexosyltransferases can include but are not limited to WsaC and WsaE. WsaC can be identified by UniProtKB-Q7BG54 (Q7BG54_GEOSE). Optionally, WsaC comprises SEQ ID NO:44 or consists of SEQ ID NO:44. WsaE can be identified by UniProtKB-Q7BG51 (Q7BG51_GEOSE). Optionally, WsaE can comprise SEQ ID NO:45 or consists of SEQ ID NO:45.

[0144] When the method further includes forming a hexose trisaccharide, two monosaccharides can be linked together as described for the disaccharide, and then an additional hexose is transferred to the non-reducing end of the disaccharide using an additional hexosyltransferase. The additional hexosyltransferase can comprise a hexose-rhamnosyltransferase, thereby transferring the rhamnose moiety to the non-reducing end. Suitable hexose-rhamnosyltransferases can include any hexose-rhamnosyltransferase described herein. Suitable hexose-rhamnosyltransferases can include rhamnose-α-1,3-rhamnosyltransferases (such as the enzyme WbbQ or WsaC), or enzymatic active variants or fragments thereof. WbbQ can be identified using UniProtKB-A0A090NIC3 (A0A090NIC3_SHIDY). In some embodiments, WbbQ comprises SEQ ID NO:46 or consists of SEQ ID NO:46.

[0145] In some embodiments, a hexose trisaccharide is formed using a rhamnose-α-1,3-rhamnosyltransferase that is not GacC.

[0146] Further information regarding exemplary hexosyltransferases for use in the present invention is provided in the Examples.

[0147] The hexose monosaccharide, disaccharide or trisaccharide to which the rhamnose moiety is transferred can be linked to a lipid. Thus, step (i) can include transferring the rhamnose moiety to a lipid-linked hexose monosaccharide, disaccharide or trisaccharide. The linkage between the hexose monosaccharide, disaccharide or trisaccharide can include undecaprenyl diphosphate.

[0148] The method can further include the step of conjugating the rhamnose polysaccharide with a receptor molecule to form a rhamnose glycoconjugate using an oligosaccharyltransferase capable of recognizing the hexose monosaccharide at the reducing end of the rhamnose polysaccharide (step (iii)).

[0149] Oligosaccharyltransferase is an enzyme used to catalyze the transfer of a carbohydrate moiety to a target protein during a process called protein glycosylation. Protein glycosylation is the process of covalently linking a carbohydrate moiety (i.e., a polysaccharide) to a protein substrate. Oligosaccharyltransferase acts by cleaving a phospho-monosaccharide bond at the reducing end of the polysaccharide. In order to be able to interact with the substrate, the oligosaccharyltransferase must be able to recognize the first two monosaccharides after the phospho bond. The substrate can alternatively be referred to as the acceptor. Thus, the acceptor molecule can contain a peptide or a protein. This results in the formation of a glycoconjugate containing the rhamnose polysaccharide of the present invention. Such a glycoconjugate is particularly useful as an antigen and can be used in immunogenic compositions or vaccines. In addition, when the method is carried out in bacteria, the glycosylation process results in the presentation of the glycoconjugate on the bacterial surface. This allows the glycoconjugate to be isolated from the bacteria for further use, or the whole bacteria to be used as an antigen, which can be used in immunogenic compositions or vaccines.

[0150] In some embodiments, the oligosaccharyltransferase is able to recognize glucose or a glucose derivative. In such embodiments, the hexose monosaccharide at the reducing end of the rhamnose polysaccharide will be glucose or a glucose derivative, such as N-acetylglucosamine (GlcNAc).

[0151] The oligosaccharyltransferase can comprise PgIB, PgIL, PgIS or WsaB or an enzymatically active homolog, fragment or variant thereof.

[0152] The PgIB enzyme can be derived from a Campylobacter species, such as Campylobacter jejuni or Campylobacter lari. Without wishing to be bound by theory, it is believed that the PgIB enzyme is able to recognize any hexose other than glucose.

[0153] The PgIL enzyme can be derived from Neisseria meningitides. It is believed that the PgIL enzyme is able to recognize any hexose other than glucose.

[0154] The PgIS enzyme can be derived from an Acinetobacter species. It is believed that the PgIS enzyme is able to recognize glucose.

[0155] The WsaB enzyme is derived from Geobacillus stearothermophilus. Enzymatically active variants of the WsaB enzyme can be derived from other Geobacillus species.

[0156] In some embodiments, the oligosaccharyltransferase is derived from a bacterial species heterologous to the bacteria in which the method is carried out.

[0157] The method may further include an additional step of purifying the rhamnose glycoconjugate. Purification may include high performance liquid chromatography (HPLC) (such as recycling HPLC), affinity chromatography, or size exclusion chromatography. Other suitable purification methods will be known to the person skilled in the art.

[0158] It should be understood that the method can be carried out on an industrial scale. As the person skilled in the art will realize, the bacteria in which the method is carried out are grown in a liquid medium. This liquid medium containing bacteria can be used to fill a bioreactor on an industrial scale, such as a bioreactor with a volume of at least 50 liters, 100 liters, or 1000 liters. This advantageously results in the synthesis of a large amount of the polysaccharide product of the present invention. A commonly used liquid medium is Luria Broth, which can also be referred to as Lysogeny Broth. Other liquid media will be known to the person skilled in the art.

[0159] When the method is carried out in bacteria, the method can be a fed-batch method. "Fed-batch" is a term familiar to those skilled in the art. However, for the sake of clarity, "fed-batch" will be understood to refer to a synthesis method in which nutrients are provided to the bacteria through a liquid medium during cultivation.

[0160] Suitable nutrients will be known to the person skilled in the art. Some exemplary but non-limiting nutrients can include a rhamnose moiety, hexoses other than the rhamnose moiety, and / or divalent cations including but not limited to magnesium and / or manganese.

[0161] In some embodiments, the rhamnose moiety contains rhamnose. Rhamnose can be provided to the liquid medium in the D or L isotype, preferably in the L isotype.

[0162] Which hexose other than the rhamnose moiety is provided to the liquid medium depends on the composition of the rhamnose polysaccharide produced by the method. If the hexose monosaccharide, disaccharide, or trisaccharide to which the rhamnose moiety is transferred includes glucose, the person skilled in the art will understand that the suitable nutrient to be supplied to the liquid medium is glucose. If the hexose monosaccharide, disaccharide, or trisaccharide contains galactose, the person skilled in the art will understand that the suitable nutrient to be supplied to the liquid medium will be galactose. Thus, the hexose supplied to the liquid medium can be appropriately selected from one or more of allose, altrose, glucose, mannose, xylose, idose, galactose, talose, diacetylbacillosamine, GalNAc, or GlcNAc.

[0163] The rhamnose moiety and / or other hexoses can (each) be supplied to the liquid medium at a final concentration of 0.1 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L or 15 g / L in the liquid medium. In some embodiments, the rhamnose moiety and / or other hexoses (each) are supplied to the liquid medium at a final concentration of about 4 g / L in the liquid medium.

[0164] The rhamnose moiety and / or other hexoses can (each) be supplied to the liquid medium at a final concentration of 0.05 mg / ml, 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.25 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml or 1.0 mg / ml in the liquid medium.

[0165] In an embodiment, the rhamnose moiety is supplied to the liquid medium as L-rhamnose. L-rhamnose can be supplied to the liquid medium at a final concentration of 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL or 1.0 mg / mL in the liquid medium.

[0166] When magnesium is added to the liquid medium, it can be provided in the form of MgSO 4 or MgCl 2 MgSO 4 or MgCl 2 can be supplied to the liquid medium to form a final concentration between 0 mM and 10 mM in the medium.

[0167] Before step (i), when the method is carried out in bacteria, the method may further comprise introducing into the bacteria one or more nucleic acids encoding one or more of the enzymes described herein. For example, the method may further comprise introducing into the bacteria a nucleic acid encoding an oligosaccharyltransferase and / or a nucleic acid encoding a hexose-β-1,4-rhamnosyltransferase, hexose-α1,2-rhamnosyltransferase, hexose-α-1,3-rhamnosyltransferase or an enzymatic active fragment or variant thereof. In some embodiments, the method further comprises introducing into the bacteria a nucleic acid encoding the bacterial enzyme GacC and / or the bacterial enzyme GacG or one or more enzymatic active homologs, variants or fragments thereof. The enzymes can then be expressed from their respective nucleic acids. The nucleic acid encoding one or more enzymes may also comprise a nucleic acid sequence encoding an endogenous or constitutive promoter and / or an artificial ribosome binding site.

[0168] Methods for introducing one or more nucleic acids into bacteria are well known to those skilled in the art. A commonly used method is the transformation method. As used herein, transformation (transforming / transformation) (which may alternatively be referred to as transfection (transfecting / transfection)) refers to the process of introducing free nucleic acids into cells by allowing the nucleic acids to cross the plasma membrane of the cells. For free nucleic acids, this will be understood to refer to nucleic acids that are not contained within a virus, virus-like particle or other organism; that is, the nucleic acids are independent of an organism (although it should be understood that the nucleic acids may be derived from or isolated from the nucleic acid sequences of an organism).

[0169] Transfection methods generally include altering the plasma membrane so that free nucleic acids can cross the plasma membrane (e.g., electroporation methods) or complexing the free nucleic acids with a reagent capable of enabling the free nucleic acids to cross the plasma membrane.

[0170] It should be understood that the nucleic acid used for transfection can be in the form of a plasmid, which is a circular nucleic acid strand. Thus, the plasmid may contain one or more nucleic acids encoding one or more enzymes.

[0171] The nucleic acid is usually DNA, although RNA can also or alternatively be contemplated.

[0172] Transfection can include polyethylenimine, poly-L-lysine, calcium phosphate, electroporation or liposome-based methods. In an embodiment, transfection can include polyethylenimine, calcium phosphate or liposome-based methods.

[0173] It should be understood that a variety of liposome-based reagents are commercially available for liposome-based transfection methods. Liposome methods can include, but are not limited to, liposome-based transfection or transfection based on HD (Promega Corporation, Wisconsin, USA).

[0174] More information about transformation / transfection techniques can be found in Current Protocols in Molecular Biology (2019), which is incorporated herein by reference.

[0175] The plasmid may also contain appropriate regulatory sequences, including promoter sequences, terminator fragments, enhancer sequences, marker genes, and / or other sequences. See, for example, Sambrook & Russell, Molecular Cloning: A Laboratory Manual: 3rd edition, for more details.

[0176] The plasmid can be further engineered to contain regulatory sequences that act as enhancer and promoter regions and result in efficient transcription of the fusion protein sequence carried on the construct. Many parts of the regulatory unit are located upstream of the heterologous gene coding sequence and are operably linked to the heterologous gene coding sequence. The regulatory sequences can direct constitutive or inducible expression of the heterologous coding sequence. Such regulatory sequences are particularly applicable if expression is desired to occur in a time-specific manner. Expression can be induced by providing an inducer to the liquid medium. The inducer can include arabinose, IPTG, or rhamnose or consist of arabinose, IPTG, or rhamnose. Regulatory sequences that can direct inducible expression when exposed to arabinose, IPTG, or rhamnose will be known to the person skilled in the art.

[0177] Arabinose can be supplied to the liquid medium at a final concentration of 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, or 10 g / L in the liquid medium. Optionally, arabinose is supplied to the liquid medium at a concentration of about 2 g / L.

[0178] IPTG can be supplied to the liquid medium at a final concentration of 0.1 mM to 5 mM in the liquid medium. In some embodiments, IPTG is supplied to the liquid medium at a final concentration of 0.1 to 2 mM, preferably at a concentration of about 1 mM, in the liquid medium.

[0179] L-rhamnose can be supplied as an inducer to the liquid medium at a final concentration of 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg / mL.

[0180] Also provided is a product obtainable by the method according to the first aspect. The product obtainable by the method according to the first aspect is particularly pure and homogeneous due to its synthetic production method. Thus, the products of the present invention are very suitable for commercial use, for example, for large-scale production for use as antigens or for research applications.

[0181] According to a third aspect, there is provided a synthetic streptococcal polysaccharide having a non-reducing end comprising a linear chain of rhamnose moieties and a reducing end comprising a hexose monosaccharide, disaccharide or trisaccharide as described in the method aspect. The polysaccharide comprises an α-1,3 bond or an α-1,2 bond between the hexose monosaccharide, disaccharide or trisaccharide and the linear chain of rhamnose moieties, or the polysaccharide comprises a β-1,4 bond between the hexose monosaccharide, disaccharide or trisaccharide and the linear chain of rhamnose moieties, and the hexose monosaccharide, disaccharide or trisaccharide does not include N-acetylglucosamine.

[0182] As the inventors have discovered, the naturally occurring GAC from Streptococcus pyogenes contains GlcNAc (N-acetylglucosamine) monosaccharides linked to a linear chain of rhamnose monosaccharides by β-1,4 glycosidic bonds. By altering this natural composition of the reducing terminal sugar, the inventors have generated a synthetic polysaccharide that retains the chemical composition and antigenic capacity of the α-1,2-α-1,3 rhamnose disaccharide repeating unit of GAC while being capable of producing the polysaccharide on an industrial scale with high purity and tightly controlled size distribution to improve the uniformity of product length.

[0183] Thus, generally, the polysaccharide comprises a polysaccharide or a fragment or variant thereof selected from the group consisting of Group A, Group B, Group C and Group G carbohydrates.

[0184] In some embodiments, the polysaccharide comprises an α-1,3 bond between the hexose monosaccharide, disaccharide or trisaccharide and the linear chain of rhamnose moieties. The hexose monosaccharide disaccharide or trisaccharide may include N-acetylglucosamine, N,N'-diacetylbacillosamine, glucose or galactose.

[0185] In some embodiments, the polysaccharide comprises an α-1,2 bond between the hexose monosaccharide, disaccharide or trisaccharide and the linear chain of rhamnose moieties. The hexose may include galactose.

[0186] In some embodiments, the polysaccharide comprises a β-1,4 bond between the hexose monosaccharide, disaccharide or trisaccharide and the linear chain of rhamnose moieties, and the hexose includes glucose.

[0187] According to a fourth aspect, there is provided a streptococcal rhamnose glycoconjugate comprising a streptococcal polysaccharide according to the third aspect conjugated to a receptor. The glycoconjugate has strong antigenic potential and thus the rhamnose glycoconjugate of the present invention has particular utility in enhancing an immune response, such as as an immunogenic composition or vaccine, or as part of an immunogenic composition or vaccine.

[0188] In an embodiment, the polysaccharide is conjugated to the receptor at the reducing end of the polysaccharide. The receptor may include a peptide or a protein.

[0189] In some embodiments, the streptococcal rhamnose glycoconjugate is expressed on the surface of a bacterial host cell (optionally a Gram-negative bacterium such as Escherichia coli). Accordingly, the invention also encompasses a bacterial host cell that comprises on its cell surface the streptococcal rhamnose glycoconjugate of the fourth aspect. Conveniently, expression on the cell surface of the bacterial host cell facilitates the isolation of the glycoconjugate. Even more conveniently, this means that a bacterial host cell that comprises the streptococcal rhamnose glycoconjugate on its cell surface can be used as a component of an immunogenic composition or a vaccine without the need to isolate the glycoconjugate from the bacterial host cell. This reduces the time and cost required to produce the glycoconjugate for downstream use as an immunogenic composition or a vaccine.

[0190] Accordingly, in a fifth aspect, there is provided a bacterial host cell that comprises a hexose-β-1,4-rhamnosyltransferase, a hexose-α-1,2-rhamnosyltransferase or a hexose-α-1,3-rhamnosyltransferase, or an enzymatically active fragment or variant thereof, and a heterologous bacterial enzyme GacC and / or GacG, or an enzymatically active homolog, variant or fragment thereof, as described herein.

[0191] The bacterial host cell can be heterologous to the species from which the hexose-β-1,4-rhamnosyltransferase, the hexose-α-1,2-rhamnosyltransferase or the hexose-α-1,3-rhamnosyltransferase or the enzymatically active fragment or variant thereof is derived. Optionally, the bacterial host cell is a Gram-negative bacterium such as Escherichia coli. The bacterial host cell can comprise the enzymes described herein and / or nucleic acid sequences encoding these enzymes.

[0192] In a sixth aspect, there is provided an immunogenic composition or a vaccine that comprises the rhamnose polysaccharide of the second or third aspect or the streptococcal glycoconjugate of the fourth aspect. The immunogenic composition or vaccine can further comprise a pharmaceutically acceptable and / or sterile excipient, carrier and / or diluent.

[0193] In some embodiments, the immunogenic composition or vaccine further comprises an antigen, a polypeptide and / or an adjuvant.

[0194] The composition can further comprise a pharmaceutically acceptable carrier, diluent or excipient. As used herein, "pharmaceutically acceptable carrier" refers to any physiological medium known to those of ordinary skill in the art that can be used to formulate a pharmaceutical composition. As used herein, "diluent" refers to any substance known to those of ordinary skill in the art that can be used to dilute a medicament for use in a pharmaceutical composition. The medicament can be mixed with a carrier, diluent or excipient, or dissolved, suspended or dispersed in a carrier, diluent or excipient.

[0195] The composition can be in the form of a capsule, tablet, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle, transdermal patch, liposome, or any other suitable form that can be administered to an animal suffering from a disease, disorder or infection caused by a streptococcal etiology or at risk of developing a disease, disorder or infection caused by a streptococcal etiology.

[0196] The compositions and / or vaccines of the present invention can be formulated for oral, topical (including dermal and sublingual), intramammary, parenteral (including subcutaneous, intradermal, intramuscular and intravenous), transdermal and / or mucosal administration. In embodiments, the compositions and vaccines of the present invention can be formulated for parenteral administration, optionally subcutaneous, intradermal, intramuscular and / or intravenous administration.

[0197] Also provided are rhamnopolysaccharides of the second or third aspect, streptococcal glycoconjugates of the fourth aspect, or immunogenic compositions or vaccines of the sixth aspect for eliciting an immune response in an animal or for treating or preventing a disease, disorder or infection caused by a streptococcal etiology.

[0198] The animal can be any mammalian subject, such as a dog, cat, rat, mouse, human, sheep, goat, donkey, horse, cow, pig, and / or chicken.

[0199] In embodiments, the animal is an ovine animal, a caprine animal, an equine animal, a porcine animal, a bovine animal or a human. In embodiments, the animal is an ovine animal. For "ovine animal", this will be understood to include sheep.

[0200] One of ordinary skill in the art will understand that the term "caprine" includes goats, while "bovine" includes cows. Equine is a term that can be understood to include horses. As used herein, the term "porcine" includes pigs.

[0201] An immune response that contributes to an animal's ability to resolve an infection / infestation and / or helps to alleviate symptoms associated with the infection / infestation can be referred to as a "protective response". In the context of the present invention, an immune response elicited by utilizing the rhamnopolysaccharides described herein can be referred to as a "protective" immune response. The term "protective" immune response can include any immune response that: (i) promotes or affects a reduction in the host pathogen burden; (ii) alleviates one or more effects or symptoms of the infection / infestation; and / or (iii) prevents, reduces or limits the occurrence of further (subsequent / secondary) infections.

[0202] Thus, a protective immune response can prevent an animal from being infected / infested by a particular pathogen and / or from developing a particular disease or disorder.

[0203] "Immune response" can be regarded as any response that elicits an antibody response (such as IgA, IgM, and / or IgG or any other relevant isotype) and / or a cytokine or cell-mediated immune response. The immune response can target the rhamnopolysaccharide of the present invention. For example, the immune response can include antibodies that have an affinity for the epitope of the rhamnopolysaccharide or the entire rhamnopolysaccharide.

[0204] There is also provided a method for treating an animal suffering from a disease, disorder or infection caused by a streptococcal etiology, the method comprising administering to the animal a therapeutically effective amount of the rhamnopolysaccharide of the second or third aspect, the streptococcal glycoconjugate of the fourth aspect, or the immunogenic composition or vaccine according to the sixth aspect.

[0205] The therapeutically effective amount should be understood to mean an amount sufficient to eliminate, reduce or prevent a disease, disorder or infection caused by a streptococcal etiology.

[0206] The rhamnopolysaccharide, glycoconjugate or immunogenic composition or vaccine can be administered in a single dose or multiple doses. Multiple doses can be administered within a day (for example, 2, 3 or 4 doses can be administered at intervals of, for example, 3, 6 or 8 hours). The agent can be administered regularly (for example, daily, every other day or weekly) over a period of days, weeks or months as appropriate.

[0207] It should be understood that the optimal dose to be administered can be determined by those skilled in the art and will vary depending on the particular agent used, the strength of the formulation, the mode of administration, and the progression or severity of the disease, disorder or infection caused by the streptococcal etiology. Other factors depending on the particular subject being treated will result in a need to adjust the dose, including the subject's age, weight, gender, diet and time of administration. Known procedures, such as those commonly used in the pharmaceutical industry (such as in vivo experiments, clinical trials, etc.), can be used to establish the particular formulation and precise therapeutic dosing regimen to be used according to the present invention.

[0208] There is also provided a kit, which comprises:

[0209] (i) a nucleic acid sequence encoding hexose-β1,4-rhamnosyltransferase, hexose-α-1,2-rhamnosyltransferase or hexose-α1,3-rhamnosyltransferase or an enzymatically active fragment or variant thereof; and

[0210] (ii) a nucleic acid sequence encoding the heterologous bacterial enzymes GacC and / or GacG or an enzymatically active homolog, variant or fragment thereof. Suitable nucleic acid sequences for the kit are as described herein with respect to the methods of the present invention.

[0211] In some embodiments, the kit further comprises one or more nucleic acid sequences encoding an oligosaccharyltransferase as described herein.

[0212] Other nucleic acid sequences that the kit may contain may include one or more nucleic acid sequences encoding one or more of the following 12 enzymes: GacA, GacD, GacE, GacF, GacH, GacI, GacJ, GacK, and GacL, or one or more enzyme activity homologs, variants, or fragments thereof.

[0213] In some embodiments, the kit further contains a nucleic acid sequence encoding GacA or an enzyme activity homolog, variant, or fragment thereof. In some embodiments, the kit contains a nucleic acid sequence encoding GacG or an enzyme activity homolog, variant, or fragment thereof.

[0214] In some embodiments, the kit contains a nucleic acid sequence encoding GacG and GacC or one or more enzyme activity homologs, variants, or fragments thereof.

[0215] In some embodiments, the kit further contains a nucleic acid sequence encoding the enzymes GacA, GacD, GacE, and GacF, or one or more enzyme activity homologs, fragments, or variants thereof.

[0216] The kit may also contain one or more nucleic acid sequences encoding reporter genes. The reporter sequence may encode a gene or peptide / protein whose expression can be detected by certain means. Suitable reporter sequences can encode genes and / or proteins whose expression can be detected by, for example, optical, immunological, or molecular means. Exemplary reporter sequences can encode, for example, fluorescent and / or luminescent proteins. Examples may include sequences encoding firefly luciferase (Luc: including codon-optimized forms), green fluorescent protein (GFP), and red fluorescent protein (dsRed). One or both of the nucleic acid sequences described in (i) and (ii) of the kit may contain a reporter sequence.

[0217] The kit may optionally further contain bacteria, such as Gram-negative bacteria, such as Escherichia coli. The bacteria may be heterologous to the bacterial species from which the hexose-β-1,4-rhamnosyltransferase, hexose-α-1,2-rhamnosyltransferase, hexose-α-1,3-rhamnosyltransferase, or an enzyme activity fragment or variant thereof is derived.

[0218] It should be understood that multiple nucleic acid sequences may be provided in one or more plasmids.

[0219] Unless otherwise stated, all features described herein (including any appended claims, abstract, and drawings) may be combined in any combination with any of the above aspects.

[0220] Detailed Description

[0221] The present invention will now be described by way of example with reference to the following drawings, which show:

[0222] Figure 1 A) shows the gene complementation strategies and maps of Streptococcus pyogenes and Streptococcus mutans genes required for rhamnose chain production. Streptococcus mutans cluster: sccA (Smu0824), sccB (Smu0825), sccC (Smu0826), sccD (Smu0827), sccE (Smu0828), sccF (Smu0829), sccG (Smu0830). Streptococcus pyogenes cluster: gacA (M5005_Spy_0602), gacB (M5005_Spy_0603), gacC (M5005_Spy_0604), gacD (M5005_Spy_0605), gacE (M5005_Spy_0606), gacF (M5005_Spy_0607), gacG (M5005_Spy_0607). B) Bacterial complementation assay. Western blot of whole cell samples probed with anti-group A antibody. Legend on the figure;

[0223] Figure 2 shows the Western blot of whole cell samples probed with anti-GAC antibody, showing the complementation of sccB_TTG, sccB_ATG, and gacB for ΔsccB or ΔgacB;

[0224] Figure 3 shows the thin layer chromatography analysis of radiolabeled lipo-linked oligosaccharides extracted from E. coli cells expressing empty vector, Streptococcus mutans SccAB-DEFG, Streptococcus pyogenes GacB, or Streptococcus mutans SccB;

[0225] Figure 4 shows the in vitro assessment of GacB activity detected by MALDI-MS. Spectra were obtained from dTDP-Rha and the following enzymatic reaction products: A. acceptor 1 (C 13 -PP-GlcNAc) B. acceptor 1 + GacB-GFP C. acceptor 1 + GacB cleavage (without GFP) D. acceptor 2 (phenol-O-C11-PP-GlcNAc). E. acceptor 2 + GacB-GFP; F. acceptor 2 + GacB cleavage (without GFP); G. acceptor 2 + GacB-D160N-FGFP; H. acceptor 2 + GacB-Y182N-F-GFP;

[0226] Figure 5Shows an in vitro assessment of the specificity of GacB towards different activated nucleotide sugar donors using MALDI-MS. Spectra were obtained from the enzyme reaction products between GacB-GFP, acceptor 2 and: dTDP-Rha (A), UDP-Glc (B), UDP-GlcNAc (C), or UDP-Rha (D). Conversion to products (818 m / z and 840 m / z) was only observed when dTDP-Rha was used as the nucleotide sugar donor;

[0227] Figure 6 Shows an in vitro assessment of the metal ion dependence of GacB by MALDI MS. Spectra were obtained from the enzymatic reaction products between dTDP-Rha, acceptor 2 (A) and: GacB-GFP (B), 1 mM MgCl 2 (C), 1 mM MnCl 2 (D), or EDTA (E). Conversion to products (818 m / z and 840 m / z) was observed under all conditions with GacB-GFP, regardless of whether metal ions or a metal chelator were added;

[0228] Figure 7 Shows the 800 MHz 1 H NMR spectra of A) (a) acceptor substrate 1, (b) product 1, (c) acceptor substrate 2, (d) product 2; B) A partial 2D ROESY spectrum of product 1 shows correlations between H1 of β-L-Rha and the protons of rhamnose (R) and GlcNAc (G). The F2 cross-section through H1 of Rha is shown in red. C) Chemical structure with proton numbering.

[0229] Figure 8 Shows a schematic of the initiation of RhaPS in different streptococci compared to the capsular polysaccharide in Streptococcus pneumoniae. RhaPS biosynthesis is initiated by GacO (green background) on Und-P, followed by the action of GacB (teal) to generate the conserved core structure Und-PP-GlcNac-Rha. The percentage of amino acid sequence identity, positive amino acids, and gaps within the sequence are given below each homolog compared to GacO or GacB: Streptococcus mutans serotype c SccB, Streptococcus agalactiae (GBS) RfaB, Streptococcus dysgalactiae subsp. equisimilis 167 (GCS) RgpAc, Streptococcus dysgalactiae subsp. equisimilis ATCC 12394 (GGS) Rs03945. The specific carbohydrate composition that extends each group of lipid-linked core structures is depicted on the right. The repeating unit (RU) of the carbohydrate is highlighted (light pink background), and the symbols for the sugar residues are shown in the legend;

[0230] Figure 9Showing (top) anti-Lipid A and anti-GAC Western blots of total cell lysates of Escherichia coli. Complementation of the dgacB gene cluster by WchF restored RhaPS biosynthesis in 21548 cells (lacking Und-PP-GlcNAc with inactivated wecA gene), while no other GacB and homologous enzymes could initiate RhaPS biosynthesis. (Bottom) All gene combinations led to functional RhaPS biosynthesis in CS2775 cells (containing Und-PP-GlcNAc with functional wecA gene);

[0231] Figure 10 A) shows the phylogenetic relationships among 48 partially or fully sequenced streptococcal pathogens. The tree was constructed using the default neighbor-joining clustering method of Clustal Omega based on the multiple sequence alignment of GacB homologs. The tree was drawn using the iTOL online tool. Black squares on the branches indicate species with fully sequenced genomes. (B) The bar graphs associated with each node represent the percentage amino acid identity to the individual GacB (blue) or GacO (magenta) homologs;

[0232] Figure 11 Left) shows an anti-GAC Western blot of total cell lysates, a Western blot of Escherichia coli 21548 cells expressing the dgacB gene cluster and gacB, gacB mutants, or gacB-WchF chimeras. The GacB-WchF chimera complemented the dgacB RhaPS cluster, indicating that the N-terminal WchF domain was sufficient to change the acceptor substrate specificity of GacB from Und-PP-GlcNAc to Und-PP-Glc; Right) Coomassie-stained membrane after loading control - Western blot;

[0233] Figure 12 is a schematic diagram showing the composition of naturally occurring GAC; and

[0234] Figure 13 is a schematic diagram showing an embodiment of the present invention;

[0235] Figure 14 is a schematic diagram showing another embodiment of the present invention;

[0236] Figure 15 is a schematic diagram showing yet another embodiment of the present invention;

[0237] Figure 16 is a schematic diagram showing another embodiment of the present invention;

[0238] Figure 17 is a schematic diagram showing an embodiment of the present invention;

[0239] Figure 18is another schematic diagram further showing the present invention;

[0240] Figure 19 is an anti-GAC Western blot showing that WbbL can be used instead of GacB or SccB in the method according to the present invention. This figure shows an anti-GAC Western blot of total E. coli lysates from cells expressing the gene clusters RmlD-SccC-SccD-SccE-SccF-SccG (deltaSccB) and GacA-GacC-GacD-GacE-GacF-GacG (deltaGacB). Supplemented with an empty plasmid control or WbbL. The arabinose induction concentration is expressed as a percentage;

[0241] Figure 20 and 21 is an image of radioactively labeled lipooligosaccharides prepared in vivo;

[0242] Figure 22 shows the results of E. coli complementation studies;

[0243] Figure 23 shows the results of phylogenetic studies of GacO, GacB, and GacC enzymes from the genus Streptococcus;

[0244] Figure 24 shows the functional characterization of GacC and how GacC installs polymannose onto the linker / stem;

[0245] Figure 25 shows the assignment of proton and carbon sugar signals obtained from 2D TOCSY and NOESY spectra and how it is converted into a mannose polysaccharide molecule;

[0246] Figure 26 shows a Western blot image obtained by generating mannose polysaccharide with the WbbPQR linker / stem;

[0247] Figure 27 shows a schematic diagram of mannose polysaccharide generated from a Shigella spp. linker / stem and GAC repeat units; and

[0248] Figure 28 shows that the mannose polysaccharide prepared according to the present invention can act as a substrate for the E. coli glycoconjugate system.

[0249] Example 1 - GacB is an α-D-GlcNAcβ-1,4-L-mannosyltransferase

[0250] Introduce

[0251] Streptococcus pyogenes relies on different mechanisms to defend against host defenses (1-5). These mechanisms are supported by the synthesis of multiple virulence factors, including group A carbohydrate (GAC), which is a surface polysaccharide that accounts for 40% to 60% of the bacterial cell wall (6-9). GAC consists of a [→3)α-Rha(1→2)α-Rha(1→] rhamnose polysaccharide (RhaPS) backbone that is modified with β-d-GlcNAc(1→3) side chains on each α-1,2-linked rhamnose (9-11). Recent structural examinations and compositional analyses of GAC have also indicated the presence of glycerophosphate (GroP) (12), an observation that has gone unnoticed for over 50 years (13,14). Further, Edgar et al. demonstrated that approximately 25% of the GAC side chain GlcNAcs are modified with GroP, rendering this polymer negatively charged, which has implications for S. pyogenes biology and defense mechanisms (12,13,15). This feature, previously found in other surface glycans (16,17), provides new insights into the structural composition, biosynthesis, and function of GAC.

[0252] It has been proposed that GAC is synthesized by 12 proteins GacABCDEFGHIJKL, which are encoded in a gene cluster (i.e., MGAS5005_spy0602-0613), which has been found in all S. pyogenes species identified to date (1,18). By sequencing a transposon mutant library, Le Breton et al. found that 8 of these genes, gacABCDEFG and gacL, are essential for the survival of S. pyogenes (4,19). This information supports the observations of van Sorge et al., who determined by insertional mutagenesis that the first three genes of the cluster (gacABC) are essential (1).

[0253] It is currently hypothesized that GAC is formed in five consecutive steps: (i) lipid-linked acceptor initiation, (ii) [→3)α-Rha(1→2)α-Rha(1→] RhaPS backbone synthesis, (iii) membrane translocation, (iv) post-translocation chain modification in the extracellular environment, and (v) ligation to peptidoglycan (9). The cytoplasmic pool of dTDP-rhamnose is provided by enzymes encoded in two separate gene clusters, rmlABC and gacA / rmlD (16).

[0254] Despite these recent findings, some pressing questions regarding the biosynthesis of GAC remain unanswered. For example, the products of 6 of the 12 genes that make up the GAC cluster (gacBCDEFG) have not been characterized, and thus the GAC initiation, RhaPS backbone biosynthesis, and translocation steps are unknown.

[0255] As a means of obtaining more information on the initial steps of GAC, we performed an in-depth examination of the second enzyme encoded in the GAC gene cluster. Here, we demonstrate that GacB, which is inconsistent with its initial genetic annotation and the currently proposed role (8), is the first retaining rhamnosyltransferase that catalyzes the transfer of L-rhamnose from dTDP-β-L-rhamnose. GacB forms a β-1,4 glycosidic bond with lipid-linked GlcNAc-diphosphate through a metal-independent mechanism. More importantly, our study of phylogenetically related homologs from other important human pathogenic streptococci, particularly from Lancefield groups B, C, and G streptococci, reveals that the role of GacB is well conserved in the genus Streptococcus, suggesting a common first step in the production of RhaPS from all Lancefield groups.

[0256] Experimental procedures

[0257] Bioinformatics analysis

[0258] Protein sequence alignments were performed using NCBI Blast Global alignment (https: / / goo.gl / vB9zmD) and ClustalOmega (https: / / goo.gl / 8FbvYP) (49). Molecular weight predictions were obtained using the ProtParam tool on the Expasy server (http: / / www.expasy.org / ). Topology predictions were generated using SpOctopus (http: / / octopus.cbr.su.se / ) and the TMHMM algorithm (www.cbs.dtu.dk / services / TMHMM / ).

[0259] Secondary structure predictions were generated using the Phyre2 (https: / / goo.gl / zrGKJ7) or RaptorX (raptorx.uchicago.edu) homology recognition engines and viewed and analyzed using the PyMOL Molecular Graphics System (Educational Edition 1.8 LLC). The Carbohydrate-Active Enzyme database (CAZy) (http: / / www.cazy.org / ) (50) was examined to obtain information on the classification and characterization of carbohydrate-active enzymes. Phylogenetic relationships were established using Clustal Omega, Clustal X, and the interactive Tree of Life iTOL (22).

[0260] Bacterial strains and growth conditions

[0261] Escherichia coli strains DH5α and MC1061 were used indifferently as host strains for recombinant plasmid propagation and plasmid integration. Escherichia coli CS2775, a strain lacking Rha modification on lipopolysaccharide, was used as a host strain to evaluate the production of RhaPS. Escherichia coli 21548, an Und-PP-GlcNAc-deficient strain containing a wecA deletion, was used as a negative control for RhaPS production. Escherichia coli strain C43(DE3) was used for the production of recombinant proteins. All Escherichia coli strains were grown in LB medium. Unless otherwise specified, all bacterial cultures were incubated at 37 °C with a rotation speed of 200 rpm in an orbital shaker incubator. When necessary, one or more antibiotics were added to the medium at the following final concentrations: 100 μg / μL carbenicillin (Amp), 300 μg / μL erythromycin (Erm), or 50 μg / mL kanamycin (Kan).

[0262] Molecular genetics techniques

[0263] Table 1 shows the DNA sequences of the forward and reverse oligonucleotide primer pairs used for amplifying, deleting, or mutagenizing target genes. All primers were obtained from Integrated DNA Technologies (IDT). All PCR reactions were carried out using a SimpliAmp thermal cycler from ThermoFisher Scientific according to the standard procedure. Constructs were cloned using standard molecular biology procedures, including restriction enzyme digestion and ligation. All constructs were verified by DNA sequencing.

[0264]

[0265]

[0266]

[0267] Table 1

[0268] Determination of RhaPS production

[0269] 50 μL of OD 600 The standardized overnight culture was mixed with 50 μL of 6x SDS loading buffer at 37 °C and separated in a 20% Tricine-SDS gel (29). RhaPS production was evaluated by immunoblotting on a PVDF membrane according to traditional immunoblotting techniques. Primary antibody: Rabbit anti-group A Streptococcus pyogenes carbohydrate polyclonal antibody (Abcam, ab21034). Secondary antibody: Goat anti-rabbit IgG HRP conjugate (Biorad, 170-6515). After exposure to Clarity Western ECL (Biorad), GENESYS was used TMA 10S UV-Vis spectrophotometer (Thermo Scientific) captures the immune response signal.

[0270] Extraction and radiolabeling of lipo-oligosaccharides

[0271] Use 1:1 CHCl 3 / CH 3 OH and water-saturated butan-1-ol (1:1 v / v) solution to extract radiolabeled lipo-oligosaccharides (LLS) from induced Escherichia coli CS2775 cells carrying the selected plasmid, and determine the in vivo addition of sugar residues after supplementing with glucose D[6s3H](N) (PerkinElmer) (1 mCi / mL). Measure the incorporated radioactivity in a Beckman LS6000SE scintillation counter. Normalize the organic phase containing LLS to 0.05 μCi / μL. Use a C:M:AC:A:W mobile phase (180 mL chloroform + 140 mL methanol + 9 mL 1 M ammonium acetate + 9 mL 13 M ammonia solution, 23 mL distilled water), then dry and spray with En3Hance liquid (Perkin Elmer). Use XAR Film and MS Intensifying Screens to obtain autoradiographic images after 5 to 10 days.

[0272] Purification of recombinantly expressed membrane-associated proteins

[0273] Purification was carried out according to the protocol established by Waldo et al. (3) with the following modifications. Dilute an overnight culture of Escherichia coli C43(DE3) cells expressing a C-terminal GFP fusion protein 1:100, incubate for 3 hours until OD 600 = 0.6, induce with 0.5 mM IPTG and transfer to room temperature overnight, both with shaking at 200 rpm. Detect GPF expression by in-gel fluorescence using a Fuji FLA-5000 laser scanner. Cloning, expression, and purification of GacB-WT, GacB-D160N-GFP, and GacB-Y182-GFP: As described in Table 1, construct plasmids carrying GFP-His 8 tagged recombinant proteins into the vector pWaldo-E (30). For protein production and purification purposes, transform the vector into Escherichia coli C43(DE3) cells and express as described above. Fractionate the cells using an Avestin C3 high-pressure homogenizer (Biopharma, UK) and centrifuge at 4000 xg. Further centrifuge the supernatant at 200,000 xg for 2 hours to obtain 2 - 3 g of membranes containing the GacB-GFP protein. Dissolve the membranes in buffer 1 (500 mM NaCl, 10 mM Na 2 HPO4 , 1.8 mM KH 2 PO 4 (2.7 mM KCl, pH 7.4, 20 mM imidazole, 0.44 mM TCEP), 1% DDM (Anatrace) was added, kept at 4 °C for 2 h, and bound to a 1 mL Ni-Sepharose 6 Fast Flow (GE Healthcare) column, prewashed with Buffer 1 plus 0.03% DDM. Elution was performed using Buffer 1 supplemented with 250 mM imidazole and 0.03% DDM. Imidazole was removed using a HiPrep 26 / 10 desalting column (GE Healthcare) equilibrated with Buffer (PBS, 0.03% DDM, 0.4 mM TCEP). The GFP-His tag was removed by overnight cleavage at 4 °C with PreScission Protease at a ratio of 1:100. The cleaved GacB protein was collected after negative IMAC. Protein identity and purity were determined by tryptic peptide mass fingerprinting, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) (the ‘Fingerprint’ Proteomics Facility, University of Dundee).

[0274] Synthesis of Receptors 1 and 2

[0275] Receptor 2 (P 1 -(11-phenoxyundecyl)-P 2 -(2-acetamido-2-deoxy-α-d-glucopyranosyl) diphosphate) was synthesized as the sodium salt from phenoxyundecyl dihydrogen phosphate and 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-α-d-glucopyranosyl dihydrogen phosphate according to the procedure of T.N. Druzhinina et al. 2010 (94). Receptor 1 (P 1 -tridecyl-P 2 -(2-acetamido-2-deoxy-α-d-glucopyranosyl) diphosphate) was synthesized from tridecyl dihydrogen phosphate (obtained similarly to phenoxyundecyl dihydrogen phosphate) by the same procedure as described for Receptor 2.

[0276] In vitro enzymatic reaction of GacB

[0277] Purified GacB-WT-GFP, GacB-D160N-GFP, GacB-Y182F-GFP, and GacB (untagged) proteins (final concentration 0.15 mg / ml) were mixed in 100 μl of TBS buffer, and 1 mM TDP-Rha was added as the sugar donor and 1 mM Receptor 1 (C 13 -PP-GlcNAc) or 1 mM Receptor 2 (phenol-O-C 11 H22 -PP-GlcNAc) as the acceptor substrate. The reaction was incubated at 30 °C for 3 to 24 hours. The assay mixture was adjusted by exchanging the nucleotide sugar donor for UDP-Rha or UDP-GlcNAc and adding 1 mM MgCl 2 、1 mM MnCl 2 or 1 mM EDTA to determine the importance of metal dependence.

[0278] Mass spectrometry

[0279] Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) was used to analyze the acceptor and products of the GacB in vitro assay. 100 μl of the reaction sample was purified on a 100 μL Sep-Pak C18 cartridge (Waters, UK), pre-equilibrated with 5% EtOH. The bound sample was washed with 800 μl H 2 O and 800 μl 15% EtOH and eluted in two steps with a) 800 μl 30% and b) 800 μl 60% EtOH. The two elution fractions were dried in a high-speed vacuum and resuspended in 20 μl 50% MeOH. 1 μl of the sample was mixed with 1 μl of 2,5-dihydroxybenzoic acid (DHB) acid matrix (15 mg / mL dissolved in 30:70 acetonitrile:0.1% TFA), and then 1 μl was added to the MALDI grid. The sample was analyzed by MALDI in an Autoflex speed mass spectrometer (Bruker, Germany) set to reflectron positive ion mode.

[0280] NMR analysis

[0281] The purified GacB in vitro assay product (0.5 - 2 mg) was dissolved in D 2 O (550 μL) and measured at 300 K. The spectra were obtained on a 4-channel Avance III 800 MHz Bruker NMR spectrometer equipped with a 5 mm TCI CryoProbe TM with automatic matching and tuning capabilities. 1D spectra were obtained using relaxation and acquisition times of 5 and 1.8 s, respectively. 32 to 512 scans were performed using a spectral width of 11 ppm. J connectivity was established in a series of 1D and 2D TOCSY experiments with mixing times between 20 and 120 ms. Selective 1D TOCSY spectra (32) were obtained using a 40 ms Gaussian pulse and DIPSI-2 sequence (33) (γB 1 / 2π = 10 kHz) for spin locking between 20 and 120 ms. The following parameters were used to acquire 2D TOCSY and ROESY experiments: t 2 and t 1There are 2048 and 768 complex points in respectively, F 2 and F 1 The spectral widths in are 11 and 8 ppm respectively, t 2 and t 1 The acquisition times are 116 and 60 ms respectively. A relaxation time of 1.5 s is used for each t 1 increment for 16 scans. The total acquisition time for each experiment is 6 - 7 hours. In F 1 Forward linear prediction of 4096 points is applied. In F 2 Zero filling up to 4096 is applied. Before Fourier transform, apodization is performed using a cosine-squared window function in both dimensions. The ROESY mixing time is applied in the form of a 250 ms rectangular pulse with γB 1 / 2π = 4167 Hz. The DIPSI-2 sequence (γB 1 / 2π = 10 kHz) is applied for 20, 80, and 120 ms spin locking. 2D amplitude-mode HMBC experiment: t 2 and t 1 There are 2048 and 128 complex points in respectively, F 2 and F 1 The spectral widths in are 6 and 500 ppm respectively, t 2 and t 1 The acquisition times are 0.35 s and 0.6 ms respectively. A relaxation time of 1.2 s is used for each of the 128 t 1 increments for 2 scans. The total acquisition time is 8 minutes. In F 1 Forward linear prediction of 512 points is applied; in F 2 Zero filling up to 4096 is applied. Before Fourier transform, apodization is performed using a sine-squared window function in both dimensions.

[0282] GacC / Homologous Enzyme Protein Purification

[0283] For the production of recombinant proteins, the target genes (GacC, GbcC, Cps2F, SccC) are synthesized using IDT's gBlock gene fragment synthesis service. The wild-type sequences of GacC and its homologs are amplified by PCR, and the overhangs are designed for cloning into pOPINF 1 in which pOPINF1 contains an N-terminal 6x histidine tag for affinity purification. Using In-Fusion TMThe plasmid pOPINF was cloned using Clontech. The resulting plasmid was then transformed into DH5α competent cells for propagation and extraction (miniprep kit; Qiagen). Positive transformed plasmids were identified by gel electrophoresis by comparing their sizes with that of the untransformed control pOPINF plasmid, and were subsequently confirmed by DNA sequencing. To insert point mutants, the wild-type plasmid was used as a template and two overlapping fragments containing the desired point mutants were PCR amplified. The fragments were designed to contain at least 15 bp of overlap and were cloned into pOPINF, and the sequences were verified as for the wild-type plasmid. The complete list of primers used for wild-type and mutant cloning can be found in Table A.

[0284] The sequence-verified plasmids were then transformed into C43 cells for protein expression. For activity assays, 1 L of E. coli culture typically yields enough protein for >50 assays (1 mg L -1 ). The cultures were grown at 37 °C and shaken at 200 RPM until the OD reached 0.6 - 1, at which point they were transferred to 18 °C for 1 h and then induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG). The cultures were shaken overnight at 18 °C. After centrifuging the cultures at 3000 x g, the proteins were extracted using an Avestin C3 cell disruptor in buffer A0 (50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 2 mM TCEP) supplemented with protease inhibitors according to the manufacturer's instructions. The lysed cultures were then ultracentrifuged at 200,000 x g and the supernatant was collected. The supernatant containing the soluble protein of interest was then purified on a nickel affinity (Thermofisher) column using wash buffer A (50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 2 mM TCEP, 20 mM imidazole) and elution buffer B (50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 2 mM TCEP, 400 mM imidazole) according to the manufacturer's instructions. The eluted fraction containing the target protein was then passed through a desalting column pre-equilibrated with buffer A0 to remove imidazole. The protein samples were concentrated to 0.5 - 1 mg / ml and snap-frozen in liquid nitrogen until use.

[0285] Table A

[0286]

[0287]

[0288] 1. Berrow NS, Alderton D, Sainsbury S, Nettleship J, Assenberg R, Rahman N, Stuart DI, Owens RJ. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic acids research. 2007 Mar 1;35(6):e45.

[0289] HPLC determination

[0290] For in vitro enzyme assays, 50 μl reactions were set up to contain 2.5 mM synthetic lipid receptor PH-O-C 11 H 22 -PP-α-NAG, 12.5 mM TDP-L-rhamnose, 0.5 to 1.5 μM GacB-GFP and 1.25 to 2.5 μM GacC or homologues / mutants of interest, made up to 50 μl with TBS buffer supplemented with 2 mM MnCl 2 The reactions were incubated at 30 °C and when the desired time point was reached, quenched with 50 μl of acetonitrile and placed on ice for 15 minutes. The reactants were spun in a benchtop centrifuge at 14,000 RPM to filter out precipitated proteins and then injected onto an Xbridge BEH Amide OBS Prep column ( 5 μM, 10 x 250 mm) connected to an HPLC system equipped with a UV detector (Ultimate 3000, Thermo) set at 270 nm. Using running buffer A (95% acetonitrile, 10 mM ammonium acetate, pH 8) and running buffer B (50% acetonitrile, 10 mM ammonium acetate, pH 8), samples were loaded onto the column at a flow rate of 4 ml / min in a gradient of increasing B concentration. Increasingly polar products with additional sugar residues were then eluted into the gradient, with the triply rhamnosylated GacC product typically eluting at around 14 minutes in a 36-minute run time. Products purified by HPLC were dried in a rapid vacuum to remove excess acetonitrile and then lyophilized to remove residual water and ammonium acetate. Samples could be stored at -20 °C for structural analysis.

[0291] NMR analysis of GacC products

[0292] For NMR analysis at the University of Dundee, HPLC-purified products (0.5 to 2 mg) were resuspended in 600 μl of D2O, and NMR spectra were recorded at 293 K. Spectra were acquired on a Bruker AVANCE III HD 500 MHz NMR spectrometer equipped with a 5-mm QCPI cryoprobe. NMR spectra were recorded as described for the GacB reaction products. Spectra were analyzed using Bruker Topsin (4.0.7).

[0293] Results

[0294] GacB is essential for the biosynthesis of the GAC RhaPS chain

[0295] To study GacB function and identify potential catalytic residues, we used Escherichia coli as a heterologous expression system to investigate the biosynthesis of the GAC RhaPS backbone. We constructed two vectors that carried the homologous genes from Streptococcus pyogenes, gacACDEFG (gacA-G; ΔgacB) and gacB( Figure 1 A).

[0296] It was speculated that the RhaPS chain translocated to the outer membrane in E. coli, which naturally contains rhamnose attached to lipopolysaccharide. Therefore, to avoid non-specific binding of anti-GAC antibodies, all transformations were carried out using an rfaS-deficient strain (20). Disruption of the rfaS gene impeded the attachment of rhamnose to LPS on the bacterial outer membrane, resulting in strains lacking endogenous rhamnose on their surface (20). The role of GacB was investigated using the conventional complementation strategy described in Figure 1 .

[0297] We investigated the RhaPS produced by gacA-G from our complementation approach using immunoblotting of total cell lysates( Figure 1 B). If the expression of GacBCDEFG was sufficient to produce the RhaPS chain, then we should be able to detect the synthesized RhaPS using a specific anti-GAC antibody. The results showed that E. coli cells lacking the gacA-G gene cluster (empty vector) did not produce RhaPS( Figure 1 , lane 2). Similarly, transformants carrying the ΔgacB or ΔsccB plasmids lost reactivity with the GAC antibody( Figure 1 , lanes 3 and 5). In contrast, co-transformation of sccB+ΔsccB or gacB+ΔgacB restored RhaPS production, highlighting the importance of sccB and gacB for GAC backbone biosynthesis( Figure 1 , lanes 4 and 6).

[0298] To investigate whether GacB and SccB catalyze the same reaction, we tested the ability of GacB to functionally replace SccB and vice versa by co-transforming ΔsccB + gacB and ΔgacB + sccB. In all cases, SccB and GacB were interchangeable ( Figure 2 ). The predicted start codon of GacB differed from that of Streptococcus mutans SccB, which uses TTG instead of ATG ( Figure 2 ). We decided to test two versions of SccB; one with TTG as the start codon and the other with ATG as the start codon. Both versions provided active enzymes that could complement ΔsccB and ΔgacB ( Figure 2 ). Unless otherwise stated, all further work was performed using the sccB construct with the native TTG start codon.

[0299] GacB extended lipid-linked precursor

[0300] We investigated whether GacB is a GT that uses GlcNAc-PP-Und as the acceptor. We performed in vivo experiments to generate radiolabeled lipo-oligosaccharides (LLO), which were isolated from the bacterial membrane and separated by thin-layer chromatography (TLC). Based on the annotation as a rhamnosyltransferase, radiolabeled dTDP-β-L-rhamnose would be the preferred sugar donor for GacB. However, this compound was not commercially available, so tritiated glucose was chosen as a substitute. Inside bacterial cells, glucose is used as a substrate for the synthesis of various organic components, including dTDP-L-rhamnose (25).

[0301] We hypothesized that GacB transfers the activated sugar from the (radiolabeled) nucleotide sugar donor to the membrane-bound receptor monosaccharide-PP-Und, such as GlcNAc-PP-Und. Therefore, we expected a change in the size of the membrane-bound receptor compared to the signal of the monosaccharide lipid-linked receptor after running the samples on the TLC plate. As a negative control, we used Escherichia coli CS2775 (ΔrfaS) transformed with the empty vector. This transformant showed a signal consistent with the production of monosaccharide-PP-Und ( Figure 3 lane 1). After the expression of the gacB or sccB gene, we observed the accumulation of a radiolabeled signal that migrated slower on the TLC plate, indicating a higher molecular weight for these compounds ( Figure 3 , lanes 3 and 4). For the sccAB-DEFG (ΔsccC) construct ( Figure 3, the same change was observed in lane 2, indicating that sccB and gacB can glycosylate the lipid-linked precursor. According to the literature, we hypothesized that the upper radiolabeled band corresponds to GlcNAc-PP-Und, while the lower radiolabeled band corresponds to Rha-GlcNAc-PP-Und (8, 9).

[0302] GacB is a rhamnosyltransferase that can transfer rhamnose from TDP-β-l-Rha to the GlcNAc-PP-lipid acceptor.

[0303] The observed band shift indicates that GacB adds a monosaccharide to the lipid-linked precursor, most likely GlcNAc-PP-Und. We used recombinantly produced and purified GacB WT and amino acid mutants (mutants D 160 N and Y 182 F) to study this hypothesis. We established an in vitro assay using the predicted nucleotide sugar donor, TDP-β-l-rhamnose, and synthetic acceptor substrates. We tested two of these synthetic substrates designed to mimic the native lipid-linked acceptor: C 13 H 27 -PP-GlcNAc (receptor 1) or phenyl-O-C 11 H 22 -PP-GlcNAc (receptor 2) ( Figure 7 C). The reactions were purified and characterized using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in positive ion mode.

[0304] The MALDI-MS spectra of the enzymatic reactions ( Figure 4 ) confirmed that GacB catalyzes the addition of one rhamnose to both receptor substrates when incubated with TDP-β-l-rha ( Figure 4 B and E). The molecular weight of receptor 1 is 563 Da and was detected at m / z = 608 [M - 1H+2Na] + and m / z = 630 [M - 2H+3Na] + ( Figure 4 A). GacB-GFP and GacB lacking the GPF tag modified the receptor, resulting in a major peak at m / z = 776 [M - 2H+3Na] + ( Figure 4 B, C). In this spectrum, we also observed additional peaks of lower intensity at m / z = 754 [M - 1H+2Na] + , corresponding to the adduct with 2 Na + ions instead of 3 Na +Ion-coupled modified receptor 1. In both cases, compared with the unmodified receptor, the product shifted by m / z = 146, which is consistent with the addition of one rhamnose via a glycosidic linkage. For the second receptor, the same mass shift was observed; the peaks of the unmodified receptor 2 ( + and m / z = 694 [M-2H+3Na] + were detected at Figure 4 D), while the product peaks appeared at m / z = 818 [M-1H+2Na] + and m / z = 840 [M-2H+3Na] + ( Figure 4 E and 4F). We also tested the ability of GacB to catalyze the rhamnosylation of GlcNAc-α-1-P, but no detectable product was produced in this reaction (data not shown), indicating that the enzyme not only interacts with GlcNAc-P but may also require a second phosphate and lipid component to recognize the receptor substrate.

[0305] We further investigated the specificity of GacB for sugar nucleotide donors. In particular, we tested whether GacB is selective for thymidine-based nucleotides and tolerates uridine-based nucleotides such as UDP-Glc, UDP-GlcNAc, and UDP-Rha. As shown previously, in the presence of TDP-β-l-Rha, two products were observed in the spectrum, consistent with the incorporation of rhamnose and two or three sodium cations ( Figure 5 A). In contrast, no product peaks were observed when UDP-α-D-Glc or UDP-α-D-GlcNAc was used as the substrate ( Figure 5 B and C), while residual activity of UDP-β-l-Rha was detected ( Figure 5 D). This data indicates that GacB is intolerant to nucleotide sugars in the α-D configuration. In addition, GacB is specific for deoxyribose (TDP-rhamnose) and / or requires the binding of the thymine methyl.

[0306] Finally, we evaluated the metal ion dependence in vitro. Compared with the control reaction ( Figure 6 B), we noted that when GacB was added with MgCl 2 , MnCl 2 or EDTA as a metal chelator, there was no significant difference in the rhamnosylation activity of the enzyme ( Figure 6 C, D, E), indicating that the activity of GacB does not require divalent metal ions.

[0307] In summary, these data confirm our previous conclusion from the LLS radiolabeling assay ( Figure 3)。This is the first in vitro evidence that GacB is a metal-independent rhamnosyltransferase that catalyzes the initiating step in GAC RhaPS backbone biosynthesis by transferring a single rhamnose to GlcNAc-PP-Und using TDP-β-l-Rha as the sole activated nucleotide sugar donor.

[0308] Study of the catalytic residues of GacB

[0309] We were unable to obtain crystals of diffraction quality from the detergent-extracted protein that would ultimately reveal a detailed understanding of the catalytic region. We constructed a structural model of GacB based on two enzymes belonging to the GT-4GT family: BaBshA from Bacillus anthracis (PDB entry 3mbo) (72) and MshA from Corynebacterium glutamicum (PDB ID: 3c4v) (24). BaBshA shares 64 amino acids out of 424, with 15% identity. MshA is a "homologous" GT that shares a 71-amino acid sequence out of 446, with 16% identity. Based on the scarce information provided by the structural model and the multiple sequence alignments described in detail below, we mutated several residues that are highly conserved among more than 40 pathogenic streptococci.

[0310] Our in vitro Escherichia coli system is the first to be able to study GacB mutant proteins and identify mutants that abolish or reduce RhaPS backbone production. This is not possible in Streptococcus pyogenes because deletion of the gacB gene renders the cells non-viable (1, 20). We used the information available in the above GT models and sequence alignments of multiple streptococci to select residues that are likely to be involved in substrate binding, which tend to be conserved in GTs. By site-directed mutagenesis, we constructed nine recombinant versions of GacB containing the following amino acid substitutions: D126A, D126N, E222A, E222Q, D160A, D160N, Y182A, Y182F, and K131R. The last mutation was included as a negative control because it is a conserved predicted surface residue that may not be involved in catalytic activity or may inactivate the enzyme.

[0311] We found that the substitution of D160 with asparagine led to a sharp decrease in the production of the RhaPS chain, while the alanine residue did not have such a significant effect. This indicates that the carboxyl group of D160 may be essential for catalysis and that it may be replaced by a water molecule in the alanine mutant. A more severe effect was observed for the Y182 mutation. The alanine substitution of Y182 (Y182A) significantly hindered the biosynthesis of the RhaPS backbone, while Y182F completely inactivated GacB, indicating that the Y182 hydroxyl group plays an important role in the enzymatic activity of GacB.

[0312] We further investigated the mutants D160N and Y182F in in vitro assays using recombinantly expressed and purified GacB-GFP-fusions. MALDI-MS analysis of the reaction products from GacB-D160N-GFP and GacB-Y182F-GFP showed that both mutants lacked enzymatic activity in vitro ( Figure 4 Figs. G and H). These results support the hypothesis that residues D160 and Y182 play a role in substrate binding or catalysis.

[0313] Finally, we created three N-terminal truncated versions of GacB to attempt to determine whether the enzyme remained active in the absence of predicted membrane-associated residues. Our results showed that truncation of the first 22 (GacB 23-385 ), 75 (GacB 76-385 ), and 118 residues (GacB 119-385 ) led to enzyme inactivation. Their inability to complement ΔgacB indicates that the N-terminal domain is essential for activity and supports the hypothesis that GacB is a membrane-associated rhamnosyltransferase.

[0314] GacB is a retaining β-1,4-rhamnosyltransferase

[0315] Current gene annotations indicate that GacB is a reverse α-1,2-rhamnosyltransferase (1,8). This annotation is incompatible with the acceptor sugar GlcNAc, as the carbon at its C2 position is already modified with an N-acetyl group. Therefore, GacB can only transfer rhamnose to available hydroxyl groups at C3, C4, or C6. Additionally, the GAC backbone consists of rhamnose repeat units linked by α-1,3-1,2 bonds (9,12), indicating that GacB would be the only rhamnosyltransferase in the pathway that uses a retaining mechanism. According to the CAZy database, the GacB sequence is classified as a member of the GT-4 family, and the GT-4 family is classified as retaining GTs (27). If this classification is correct for GacB, the stereochemical configuration at the anomeric centre of the sugar donor TDP-β-l-rhamnose should be retained in the final product.

[0316] To elucidate whether GacB is a reverse or retained rhamnosyltransferase, we performed nuclear magnetic resonance (NMR) spectroscopy on the purified reaction products 1 and 2. The spectra were collected at 800 MHz. 1 H NMR spectroscopy to establish the structural integrity of receptors 1 and 2 ( Figure 7 A) and determine the chemical structures of the products after the enzymatic reaction (products 1 and 2). The NMR parameters were determined by one-dimensional and two-dimensional (1D and 2D) and 2D total correlation spectroscopy (TOCSY) experiments ( Figure 7 B); their chemical shifts are summarized in Table 2. For both receptors, the anomeric protons of α-d-GlcNAc appear as doublets with 3J(H1,H2) = 3.4 Hz and 3J(H1,P) = 7.2 Hz. The proton H2 of α-d-GlcNAc is also split by coupling with P at 3J(H2,P) = 2.4 Hz. The 2D 1H,31P HMQC spectra (data not shown) reveal the correlation of these two H-1' protons with P at -13.5 ppm. The 31P at -10.6 ppm and the adjacent CH 2 Another correlation between the protons of the groups emerged, confirming the integrity of the acceptor substrate. For acceptor 2, a typical signal pattern of monosubstituted benzene with an integrated intensity of 2:2:1 was observed.

[0317] The addition of rhamnose to both acceptor substrates is accompanied by the appearance of a characteristic signal in the anomeric region of the spectrum (4.88 ppm, H1) next to the water signal. The anomeric configuration of this monosaccharide is established in a number of ways. 3 The J(H1,H2) coupling constants indicate the β-l configuration of β-l and α-l-Rha (reported as 1.1 and 1.8 Hz, respectively). Rotating frame nuclear Overhauser effect (ROESY) spectroscopy ( Figure 4 B) shows that H1 of rhamnose is close in space to the other four protons. Among them are H2, H3, and H5 protons of rhamnose, the latter two of which confirm the 1,3 double axis arrangement between H1, H3, and H5, which indicates a β-1Rha configuration. Finally, 1 The H chemical shifts were compared with those of α-l and β-l-rhamnose ( Figure 7C), showed very good agreement with the chemical shifts of β-l-rhamnose (75), thus confirming the configuration of the ring. The fourth ROESY cross-peak of rhamnose H1 was accompanied by H4 of GlcNAc, indicating a (1→4) linkage between the two monosaccharides. Comparison of the GlcNAc 1H chemical shifts of the acceptor substrate and the product further supported this observation. Here, an increase in the chemical shift of H4 (+0.21 ppm) was observed after glycosylation, while the average of the absolute values of the differences in the chemical shifts of the other corresponding protons of GlcNAc was 0.03 ppm. As expected, the signals of the alkyl and aryl side chains were virtually unchanged in their respective acceptor-product pairs.

[0318] In summary, 1 The 1H NMR spectrum revealed the formation of the β-l-Rha(1→4)d-GlcNAc moiety and the integrity of the product.

[0319] Streptococcus groups A, B, C, and G share a common RhaPS initiation step

[0320] In addition to S. mutans SccB, GacB homologs with high sequence identity are also present in other clinically important streptococcal species, such as those from group B (GBS), group C (GCS), and group G (GGS) streptococci. All homologous enzymes are located in the respective gene clusters encoding the biosynthesis of the Lancefield antigens, i.e., the group B, C, and G carbohydrates (15). The homologous gene products share 67%, 89%, and 89% amino acid identity with GacB, respectively (Table 2, Figure 8 ). There is a general understanding of the chemical structure of the RhaPS of these streptococci based on varying degrees of evidence depending on the species (9). The currently accepted GAC, GBC, GCC, GGC, and SCC structures are shown as Figure 8 follows. Notably, none of the studies leading to the understanding of the surface carbohydrate structures included data describing the mechanism of action of the enzymes involved in the initiation step of each RhaPS biosynthesis.

[0321] Based on the high sequence identity with GacB, we hypothesized that the carbohydrate biosynthesis of Streptococcus groups A, B, C, and G has a conserved initiation step in which the first rhamnose residue is transferred to a lipid-linked acceptor to form Rha-β-1,4-GlcNAc-PP-Und. We tested the ability of the homologs from GBS, GCS, and GGS (GbsB, GcsB, and GgsB, respectively) to functionally replace GacB in RhaPS chain production ( Figure 9 ). Our results showed that all homologous proteins were able to restore the RhaPS backbone when their genes were co-expressed with the ΔgacB expression plasmid, indicating that these enzymes can carry out the same enzymatic reaction.

[0322] We found that GacB requires GlcNAc-PP-Und as a receptor, but the enzymes from GBS, GCS, and GGS may use different lipid-linked receptor substrates, such as Glc-PP-Und. Therefore, to determine whether GacB homologs require GlcNAc-PP-Und as a lipid receptor, we performed complementation assays using Escherichia coli ΔwecA cells lacking GlcNAc-PP-Und (23). As a positive control, we identified Streptococcus pneumoniae WchF, which is a Glc-1,4-β-rhamnosyltransferase that uses only Glc-PP-Und as a substrate (28). As expected, in the absence of GlcNAc-PP-Und, GacB was unable to restore the RhaPS chain when co-transformed with the ΔgacB vector ( Figure 9 A, lane 2). The GacB homologs from GBS, GCS, and GGS also failed to produce the RhaPS backbone ( Figure 9 A, lanes 4-6), but could replace the function of GacB in the ΔrfaS strain ( Figure 9 B). Only WchF, which transfers rhamnose residues using the Glc-PP-Und receptor, restored RhaPS biosynthesis in the absence of GlcNAc-PP-Und ( Figure 9 A, lane 3). Combining the data from our in vitro enzymatic reactions, these results indicate that the GacB homologs from GBS, GCS, and GGS are also GlcNAc-1,4-β-rhamnosyltransferases that require GlcNAc-PP-Und as a membrane-bound receptor.,

[0323] Most streptococcal pathogens are predicted to have GlcNAc-1,4-β-rhamnosyltransferase

[0324] Streptococcus pneumoniae wchF encodes a Glc-β-1,4-rhamnosyltransferase that requires Glc-PP-Und as a receptor (28). It has 51% amino acid identity with GacB, compared with 67-89% amino acid identity with the homologous enzymes from GBS, GCS, GGS, and Streptococcus mutans. To better understand the conservation of GacB in the genus Streptococcus, we extended our bioinformatics analysis to search for other strains containing the GacB homolog gene. We found that 48 human / veterinary pathogenic streptococcal species have a single GacB homolog with 50% to 94% sequence identity (Table 2, Figure 10)。Among the 48 species we identified, 5 had a percentage identity equal to or lower than 51% (Streptococcus mitis, Streptococcus pneumoniae, Streptococcus oralis subsp. tigurinus, Streptococcus oralis, and Streptococcus pseudopneumoniae), while all other encoded proteins had more than 65% homology to GacB. For simplicity, we refer to the five streptococcal strains with low amino acid identity as the "low identity" subgroup, and the remaining species as the "high identity" subgroup.

[0325] Sequence analysis paired with complementary analysis led us to hypothesize that all GacB homologs contained in the "high identity" subgroup have GlcNAc-β-1,4-rhamnosyltransferase activity. In contrast, the "low identity" subgroup contains Streptococcus pneumoniae WchF, a known Glc-1,4-β-rhamnosyltransferase (28). Compared to WchF, all five members of the "low identity subgroup" showed very high sequence identity (>90%).

[0326] GacO (a WecA homolog) from Streptococcus pyogenes was shown to be responsible for the biosynthesis of GlcNAc-PP-Und (the substrate of GacB) (8, 9). Therefore, we hypothesized that the "low" and "high identity" subgroups utilize different substrates, and thus investigated whether equivalent differences should be observed when comparing the sequence identities of GacO homologs. Among the 48 pathogenic streptococcal genomes (Table 2, Figure 10 ), we found that all strains from the "high identity" subgroup shared gacO homologs with 63 - 92% sequence identity. Importantly, any genome from the "low identity" subgroup contained gene products with a sequence identity to GacO equal to or less than 30%. This subgroup presented gene products with high homology to Streptococcus pneumoniae Cps2E, which transfers Glc-1-P to P-Und to generate Glc-PP-Und (28). The Streptococcus mitis, Streptococcus oralis subsp. tigurinus, Streptococcus pseudopneumoniae, and Streptococcus oralis homologs had 98% sequence identity to Cps2E.

[0327] The phylogenetic conservation of GacB in Streptococcus highlights the importance of this gene for the survival and pathogenesis of streptococcal pathogens. Overall, these results led us to propose that streptococcal species with highly identical (>65%) GacB homologs are GlcNAc-β-1,4-rhamnosyltransferases that catalyze the first key step in the biosynthesis of surface RhaPS by transferring rhamnose from TDP-β-l-rhamnose to membrane-bound GlcNAc-PP-Und. In contrast, we hypothesized that, based on the function of Streptococcus pneumoniae serotype 2 WchF, species in the "low identity" subgroup contain rhamnosyltransferases that act on lipid-linked Glc-PP-Und.

[0328]

[0329] Table 2 Percent sequence conservation of GacB and GacO homolog enzymes from 48 Streptococcus species.

[0330] The N-terminal domain of GacB encodes specificity for the GlcNAc receptor

[0331] We performed a multiple sequence alignment of GacB homologs from all 48 streptococcal pathogens to identify the most variable and conserved regions in the protein sequence. We observed a large difference in its N-terminal domain between the "high identity" and "low identity" subgroups (Table 2). More precisely, a low sequence conservation region was identified between GacB amino acid residues 40 and 80, suggesting that this part of the domain is involved in GlcNAc receptor sugar recognition or in essential protein-protein interactions.

[0332] We knew from previous experiments that GacB was unable to initiate RhaPS biosynthesis in the context of a wecA deletion ( Figure 9 A, lane 2). Based on this information and to identify the residues involved in sugar receptor recognition, we introduced mutations in the GacB amino acid sequence. The goal was to rescue the RhaPS initiation step in a WecA-deficient Escherichia coli strain that recognizes lipid-linked sugar receptors other than GlcNAc-PP-Und by the GacB mutant.

[0333] Therefore, we studied a structural model based on the GacB homolog BaBshA from Bacillus anthracis (PDB entry 3mbo), which suggested that residues L128, R131, GNT100 might be involved in sugar receptor recognition. We mutated these residues to mimic those found in WchF. A complementation assay using GacB L128H_R131L failed to complement ΔgacB in the ΔwecA background ( Figure 11, lane 2). Following the sequential approach, we modified the GacB primary sequence by introducing additional amino acids corresponding to the amino acid substitutions found in WchF: L128H_R131L_GNT100ARC and L128H_R131L_GNT100ARC_A105P. None of these mutants recognized glucose to initiate rhamnose chain and thus did not restore GacB activity. Finally, we replaced the first 178 residues of GacB with the corresponding WchF amino acids (1-186). When expressed in a wecA-deleted background, this WchF-GacB chimera was able to synthesize the RhaPS backbone on the exclusive acceptor substrate Glc-PP-Und ( Figure 11 , lane 5).

[0334] Discussion

[0335] This work reveals the first key step in GAC biosynthesis and provides insights into the function of GacB, the first reported metal-independent, retaining, and non-processive α-D-GlcNAcβ-1,4-L-rhamnosyltransferase. Figure 12 This insight is schematically depicted, showing the elucidated structure of GAC and the endogenous Streptococcus mutans enzymes involved in the synthesis of each part. Other enzymes involved in polysaccharide biosynthesis from Gram-negative and Gram-positive bacteria use lipid-linked GlcNAc as the acceptor and dTDP-L- or GDP-D-rhamnose nucleotides, however, their reactions result in α-1,3 or α-1,4 glycosidic bonds (29-31). Additionally, the fact that the GAC backbone consists of rhamnose repeat units linked by α-1,3-1,2 bonds (9,13) indicates that GacB is the only rhamnosyltransferase in this pathway that uses a retaining mechanism of action.

[0336] We also showed that using the enzyme WchF, Streptococcus RhaPS can be synthesized on a different acceptor Und-PP-Glu in a recombinant expression system, i.e., Escherichia coli. This is schematically depicted in Figure 13 . Specifically, Figure 13 shows how the enzyme WchF can be used to transfer the rhamnose moiety to a glucose monosaccharide to form a disaccharide that has glucose at the reducing end and a rhamnose moiety at the non-reducing end. The WchF enzyme facilitates the formation of a β-1,4 glycosidic bond between the two monosaccharides. The rhamnose polysaccharide is then generated by elongation from the rhamnose moiety at the non-reducing end of the disaccharide using the bacterial enzyme GacC or its enzymatically active homolog GbcC. WchF is derived from Streptococcus pneumoniae, which is heterologous to the bacteria from which GacC or GbcC are derived (Streptococcus mutans and Streptococcus agalactiae). In this particular embodiment, the method is carried out in Escherichia coli, which is also a different species from the bacteria from which WchF, GacC, and GbcC are derived.

[0337] This results in the formation of a Streptococcal polysaccharide that has a non-reducing end comprising a linear rhamnose moiety and a reducing end comprising a glucose monosaccharide, and the polysaccharide comprises a β-1,4 bond between the glucose and the linear rhamnose moiety. As will be understood by those skilled in the art, this is different from the naturally occurring GAC (which is shown in Figure 12 ) in that the monosaccharide at the reducing end is glucose rather than GlcNAc.

[0338] Example 2

[0339] To further illustrate the present invention, this example relates to further exemplary synthetic methods and the rhamnose polysaccharides of the present invention.

[0340] Figure 14 is another exemplary embodiment of the present invention. Figure 14 shows how the enzyme WbbL from Escherichia coli can be used to transfer a rhamnose moiety to a GlcNAc monosaccharide. This forms a disaccharide that has GlcNAc at the reducing end and a rhamnose moiety at the non-reducing end and an α-1,3 glycosidic bond between the rhamnose moiety and GlcNAc. Then the bacterial enzyme GacC or its enzymatically active homolog GbcC is used to extend from the rhamnose moiety at the reducing end of the disaccharide to generate the rhamnose polysaccharide. Since WbbL is from Escherichia coli, it is from a bacterial species heterologous to the bacterial species from which GacC and GbcC are derived.

[0341] In this particular example, the method is carried out in Escherichia coli, although other bacteria can be envisioned for this purpose. Thus, in this particular embodiment, WbbL can be endogenous to Escherichia coli or it can be overexpressed in Escherichia coli.

[0342] As Figure 14 shows, the method results in the production of a Streptococcal polysaccharide that has a non-reducing end comprising a linear rhamnose moiety and a reducing end comprising a GlcNAc monosaccharide, and the polysaccharide comprises an α-1,3 bond between the GlcNAc and the linear rhamnose moiety. This is different from the endogenous GAC (as Figure 12 shows) in that GAC contains a β-1,4 bond between GlcNAc and the rhamnose straight chain. Any other hexose-α-1,3-rhamnosyltransferase can be used in place of WbbL, as Figure 15 shows. Figure 15 Differs from Figure 14 in that the monosaccharide is glucose rather than GlcNAc. Thus, Figure 14The product is a synthetic streptococcal polysaccharide that has a non-reducing end with a linear rhamnose moiety and a reducing end with a glucose monosaccharide, and the polysaccharide contains an α-1,3 bond between the glucose and the linear rhamnose moiety. This is different from endogenous GAC (as Figure 12 shown) in that it contains glucose and an α-1,3 bond.

[0343] Other synthetic methods are also within the scope of the present invention. Figure 16 An exemplary method is shown. In this method, diNAcBac-α-1,3-rhamnosyltransferase is used to transfer the rhamnose moiety to diNAcBac monosaccharide. Thus, a disaccharide is formed that has diNAcBac at its reducing end and a rhamnose moiety at its non-reducing end. These two monosaccharides are linked by an α-1,3 glycosidic bond. Then, the bacterial enzyme GacC or its enzymatically active homolog GbcC is used to elongate from the rhamnose moiety at the non-reducing end of the disaccharide to generate a rhamnose polysaccharide. The diNAcBac-α-1,3-rhamnosyltransferase is derived from a bacterial species different from the bacterial species from which GacC or its enzymatically active homolog GbcC is derived.

[0344] Figure 16 The method results in the production of a synthetic streptococcal polysaccharide that has a non-reducing end with a linear rhamnose moiety and a reducing end with a diNAcBac monosaccharide, and the polysaccharide contains an α-1,3 bond between the diNAcBac and the linear rhamnose moiety. This is different from endogenous GAC (as Figure 12 shown) in that GAC contains a β-1,4 bond between GlcNAc and the linear rhamnose.

[0345] Figure 17 Another exemplary method and product are shown. In this method, a disaccharide, trisaccharide, or tetrasaccharide can be formed before elongation from the rhamnose moiety. For the disaccharide, galactose-α-1,2-rhamnosyltransferase WbbR is used to transfer the rhamnose moiety to a galactose monosaccharide. This forms a disaccharide that has galactose at its reducing end and a rhamnose moiety at its non-reducing end. Then, a rhamnose polysaccharide is generated by elongating a linear rhamnose moiety from this rhamnose moiety. In this example, the elongation uses the enzymes GacC, GacG, or GbcC (see the penultimate schematic and the top schematic of Figure 17 ). WbbR is derived from Shigella, which is a bacterial species different from the streptococcus from which each of GacC, GacG, or GbcC is derived. The method results in the production of a synthetic streptococcal polysaccharide that has a non-reducing end with a linear rhamnose moiety and a reducing end with a galactose monosaccharide, and the polysaccharide contains an α-1,2 bond between the diNAcBac and the linear rhamnose moiety.

[0346] Another embodiment is also asFigure 17 As shown in the top schematic and the penultimate schematic, the trisaccharide is formed before the extension from the rhamnose moiety. For the trisaccharide, the galactose monosaccharide is transferred to GlcNAc using the enzyme WbbP, thereby forming an α-1,3 glycosidic bond between the two monosaccharides. Then, as described above for the disaccharide, the enzyme WbbR is used to transfer the rhamnose moiety to galactose. Thereafter, the extension can occur as detailed above for the disaccharide.

[0347] Figure 17 On the left is a dot blot (positive antibody blot). Each blot represents a sample from an experiment; each row represents triplicates under the same conditions. For each experiment, the sample from the reaction was added as a dot, and an anti-GAC antibody was used to determine whether the reaction successfully formed rhamnopolysaccharide. The middle row shows the triplicate samples obtained from the reaction, where the enzyme WbbP was used to transfer the galactose monosaccharide to GlcNAc, followed by the enzyme WbbR, and then GacG. The dot plot on the left confirms that the reaction was able to produce the rhamnopolysaccharide of the present invention.

[0348] WbbP can alternatively be used to form a disaccharide (i.e., the galactose monosaccharide at its non-reducing end is linked to GlcNAc at its reducing end by an α-1,3 glycosidic bond, and subsequently the rhamnopolysaccharide is generated by the extension of the rhamnose moiety from the non-reducing end of the disaccharide (see Figure 17 the bottom schematic). The dot plot row on the left side of this schematic confirms that the reaction was also able to produce the rhamnopolysaccharide of the present invention.

[0349] Optionally, before the extension step detailed above, one or two additional rhamnose moieties can be transferred to the rhamnose moiety linked to galactose to form a tetrasaccharide or a pentasaccharide. One or two additional rhamnose moieties can be transferred using the enzyme WbbQ, and then further extension can be carried out using GacC with GbcC, as Figure 17 shown in the third schematic. The dot plot row on the left side of this figure confirms that the reaction containing WbbP, WbbR, WbbQ, and GacC successfully generated the rhamnopolysaccharide according to the present invention.

[0350] For the trisaccharide, tetrasaccharide, or pentasaccharide methods, these methods result in the production of a Streptococcus polysaccharide that has a reducing end containing a straight chain of rhamnose moieties and a non-reducing end containing GlcNac and galactose, and the polysaccharide contains an α-1,2 bond between the straight chain of rhamnose moieties and galactose and an α-1,3 bond between galactose and GlcNAc.

[0351] In embodiments where the rhamnose moiety is transferred to a disaccharide or a trisaccharide, it is contemplated that any hexose combination can be used to form the disaccharide or trisaccharide using the α or β bonds described herein. This is shown in Figure 18This is described in []. Similarly, for extending rhamnose polysaccharides from rhamnose moieties, it is envisioned that any GacC, GacG homologs with enzymatic activity, or fragments or variants thereof can be used, provided that α-1,2 and / or α-1,3 glycosidic bonds are formed between each pair of rhamnose moieties.

[0352] Figure 19 It was demonstrated that WbbL can be used in place of GacB or SccB in the method of the present invention to produce rhamnose polysaccharides. The figure shows an anti-GAC Western blot of total E. coli lysates from cells expressing the gene clusters RmlD-SccC-SccD-SccE-SccF-SccG (deltaSccB) and GacA-GacC-GacD-GacE-GacF-GacG (deltaGacB). Supplemented with an empty plasmid control or WbbL. The first column is a ladder. The second column demonstrates that GAC is not produced in E. coli cells with an RgpA deletion, while the third column demonstrates that the individual expression of WbbL in RgpA-deficient cells does not restore GAC synthesis. The third column shows the lysate from E. coli cells with an RgpA deletion but also expressing the gene cluster GacA-GacC-GacD-GacE-GacF-GacG (deltaGacB). No GAC was found in these cells. However, the fourth column shows that GAC is produced when WbbL is expressed in the cells of the third column. The same result was observed when rgpA-deficient cells were co-expressed with the gene cluster RmlD-SccC-SccD-SccE-SccF-SccG (deltaSccB) and WbbL (see duplicates in the last two columns). This data confirms that WbbL can be used with heterologous enzymes from other species to produce rhamnose polysaccharides according to the present invention.

[0353] Figure 20 It was confirmed that GacC incorporates up to five rhamnose sugars into the product generated by GacB. Figure 20 Shows the radiolabeling of lipo-linked oligosaccharides (LLOS) in vivo (E. coli). Film exposure of a TLC plate with radiolabeled LLOS from E. coli CS2775 with gacB (lane 1) or gacBC (lane 2).

[0354] Homologs of GacC can function in a similar manner. Figure 21 Shows results similar to those shown in Figure 20 but using GbcC, GccC, and GgcC, homologs of the enzymes from Streptococcus groups B, C, and G. Figure 21Shows the film exposure of a TLC plate with radiolabeled LLOS from Escherichia coli CS2775 with gacB and gacC (lane 1), gacB alone (lane 2), gacB and gbcC (lane 3), gacB and gccC (lane 4), and gacB and ggcC (lane 5). GacC, GbcC, GccC, GgcC are homologous enzymes from Streptococcus groups A, B, C, and G, and the figure shows that all 3 - 5 rhamnoses are transferred to the product of GacB.

[0355] Similarly, the inventors have demonstrated that GacC enzyme function is conserved in streptococci and can complement the SccC enzyme of Escherichia coli. Figure 22 Shows:

[0356] A) Gene complementation strategy. The sccC gene was replaced with homologous genes gacC, gbcC, gccC, ggcC.

[0357] B) Immunoblot of whole - cell lysates for bacterial complementation assays probed with anti - group A antibody.

[0358] Complementation studies confirmed that GacC enzyme function is conserved in streptococci from groups B, C, G, and Streptococcus mutans.

[0359] Phylogenetic analysis of GacO, GacB, and GacC enzymes shows high similarity, so the function in streptococci is conserved - it is expected that pathogenic strains will all produce RhaPS with the same linker / stem, and thus, all of these are suitable for use according to the present invention.

[0360] Figure 23 Shows A) Phylogenetic tree based on GacB orthologous protein sequences identified from 48 pathogenic streptococci. An asterisk after the species name indicates that the orthologous sequence was not retrieved from the whole - genome sequence. To study GacB function and identify potential catalytic residues, we used Escherichia coli as a heterologous expression system to study GAC RhaPS backbone biosynthesis. B) Bar graph showing the degree of homology to Streptococcus pyogenes GacO (red), GacB (blue), or GacC (green) as a percentage. The numbers next to the GacO, GacB, and GacC labels represent the steps catalyzed by Streptococcus pyogenes. The numbers in the indented center of the figure are based on our current understanding of the roles of Streptococcus pneumoniae Cps2E, Cps2T (WchF), and Cps2F (James 2013).

[0361] Figure 24 Shows in vitro GacC rhamnosylation of the synthetic LLO substrate (GacB product).

[0362] A) HPLC analysis showed that GacC extension uses chemoenzymatically lipid-linked disaccharides generated by GacB with 3 additional rhamnose residues. Chemical ligation was subsequently analyzed by NMR. B) Chemical mapping of GacB / C reaction with in vitro receptor substrates

[0363] Further studies by the inventors using NMR and mass spectrometry techniques (not showing all data) confirmed that GacC can add up to 4 rhamnoses and that GacC is a reverse α-1,3 rhamnosyltransferase. Figure 25 Complete assignments of proton and carbon sugar signals are shown. 1 H assignments are based on the analysis of several F1-band selective 2D TOCSY spectra. Using 2D 1H, 13 CHSQC assignments 13 C signals. Connectivities were assigned using 2D NOESY experiments. The chemical shifts of each sugar residue are in good agreement with the published data for pyranose 1H and 13C signals.

[0364] The present inventors further showed that the rhamnose polysaccharides according to the present invention can be generated using different enzyme combinations. Figure 26 Shows that the rhamnose polysaccharides according to the present invention can be produced using enzymes from Shigella dysenteriae in combination with Escherichia coli and Shigella dysenteriae and Streptococcus mutans. Figure 26 Shows a whole cell Western blot using anti-group A carbohydrate antibody. Total E. coli cell lysates were separated by SDS-PAGE. NewRhaPS was constructed by combining Shigella dysenteriae gene products with Streptococcus mutans / Group A Streptococcus gene products. RmlD_GacD_E_F_G plus WbbP_Q_R is sufficient to construct NewRhaPS. NewRhaPS can also be constructed using RmlD_SccC_D_E_F_G plus WbbP_Q_R.

[0365] Based on the above evidence, it is expected that Shigella can be further used to provide linker / stem and GAC repeat units, as Figure 27 shown. In the native system, GacB and GacC enzymes install the linker / stem region (red box) before GacG installs the immunogenic repeat units. This figure shows an example of 3 α-1,3-rhamnoses installed by GacC.

[0366] Replacing the GacB / C enzymes (replacing the GlcNAc-β1,4-rhamnose-α1,3-rhamnose linker / stem) to generate NewRhaPS provides an alternative method of maintaining the immunogenic repeat units (suggested to be introduced by GacG enzyme activity). Replacing the linker region with an O-Otase compatible polysaccharide / oligosaccharide (green box) is sufficient to construct an immunogenic polysaccharide (α1,2-α1,3 rhamnose).

[0367] As described herein, the rhamnan polysaccharide of the invention can be conjugated to a suitable protein and presented on the surface of bacteria. Figure 28 It was shown that the rhamnan polysaccharide prepared according to the invention is a suitable substrate for the Escherichia coli glycoconjugation system. A periplasmic expression test system was established according to the procedure described by Reglinski et al., npj Vaccines (2108) 3:53. Figure 28 It was shown that NewRhaPS is a compatible substrate for the O-Otase (PglB) / proteoglycan conjugation technology (PGCT).

[0368] The periplasmic expression of the test protein NanA (according to Reglinski) was tested in the + / - active / inactive NewRhaPS system (1-8).

[0369] Lanes 5 and 7 show that two different expression conditions of the NewRhaPS system are positive for NanA-NewRhaPS glycosylation.

[0370] Lane 9: GAC chemically extracted from Streptococcus pyogenes (positive control for GAC antibody).

[0371] This description should not be construed as limiting, and it should be understood that other variants and embodiments fall within the scope of the invention.

[0372] References

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[0398] 26. Parsonage, D., Newton, G. L., Holder, R. C., Wallace, B. D., Paige, C., Hamilton, C. J., Dos Santos, P. C., Redinbo, M. R., Reid, S. D., and Claiborne, A. (2010) Characterization of the N-acetyl-α-D-glucosaminyl l-malate synthase and deacetylase functions for bacillithiol biosynthesis in Bacillus anthracis. Biochemistry (Mosc.). 49, 8398–8414

[0399] 27. Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M., and Henrissat, B. (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, D490–495

[0400] 28. James, D. B. A., and Yother, J. (2012) Genetic and Biochemical Characterizations of Enzymes Involved in Streptococcus pneumoniae Serotype 2 Capsule Synthesis Demonstrate that Cps2T (WchF) Catalyzes the Committed Step by Addition of β1-4 Rhamnose, the Second Sugar Residue in the Repeat Unit. J. Bacteriol. 194, 6479–6489

[0401] 29. H. (2006) Tricine - SDS - PAGE. Nat. Protoc. 1, 16–22

[0402] 30. Waldo, G. S., Standish, B. M., Berendzen, J., and Terwilliger, T. C. (1999) Rapid protein - folding assay using green fluorescent protein. Nat. Biotechnol. 17, 691–695

[0403] 31. Druzhinina, T. N., Danilov, L. L., Torgov, V. I., Utkina, N. S., Balagurova, N. M., Veselovsky, V. V., and Chizhov, A. O. (2010) 11 - Phenoxyundecyl phosphate as a 2 - acetamido - 2 - deoxy - α - d - glucopyranosyl phosphate acceptor in O - antigen repeating unit assembly of Salmonella arizonae O:59. Carbohydr. Res. 345, 2636–2640

[0404] 32. Robinson, P. T., Pham, T. N., and Uhrín, D. (2004) In phase selective excitation of overlapping multiplets by gradient - enhanced chemical shift selective filters. J. Magn. Reson. San Diego Calif 1997. 170, 97–103

[0405] 33. Rucker, F. J., and Osorio, D. (2008) The effects of longitudinal chromatic aberration and a shift in the peak of the middle-wavelength sensitive cone fundamental on cone contrast. Vision Res. 48, 1929–1939

[0406] Sequence

[0407] SEQ ID NO:1 GacC

[0408] MNINILLSTYNGERFLAEQIQSIQRQTVNDWTLLIRDDGSTDGTQDIIRTFVKEDKRIQWINEGQTENLGVIKNFYTLLKHQKADVYFFSDQDDIWLDNKLEVTLLEAQKHEMTAPLLVYTDLKVVTQHLAVCHDSMIKTQSGHANTSLLQELTENTVTGGTMMITHALAEEWTTCDGLLMHDWYLALLASAIGKLVYLDIPTELYRQHDANVLGARTWSKRMKNWLTPHHLVNKYWWLITSSQKQAQLLLDLPLKPNDHELVTAYVSLLDMPFTKRLATLKRYGFRKNRIFHTFIFRSLVVTLFGYRRK

[0409] SEQ ID NO:2 GacG

[0410] MNRILLYVHFNKYNKISAHVYYQLEQMRSLFSKIVFISNSKVSHEDLKRLKNHCLIDEFLQRKNKGFDFSAWHDGLIIMGFDKLEEFDSLTIMNDTCFGPIWEMAPYFENFEEKETVDFWGITNNRGTKAFKEHVQSYFMTFKNQVIQNKVFQQFWQSIIEYENVQEVIQHYETQLTSILLNEGFSYQTVFDTRKAESSFMPHPDFSYYNPTAILKHHVPFIKVKAIDANQHIAPYLLNLIRETTNYPIDLIVSHMSQISLPDTKYLLSQKYLNCQRLAKQTCQKVAVHLHVFYVDLLDEFLTAFENWNFHYDLFITTDSDIKRKEIKEILQRKGKTADIRVTGNRGRDIYPMLLLKDKLSQYDYIGHFHTKKSKEADFWAGESWRKELIDMLVKPADSILSAFETDDIGIIIADIPSFFRFNKIVNAWNEHLIAQEMMSLWRKMDVKKQIDFQAMDTFVMSYGTFVWFKYDALKSLFDLELTQNDIPSEPLPQNSILHAIERLLVYIAWGDSYDFRIVKNPYELTPFIDNKLLNLREDEGAHTYVNFNQMGGIKGALKYIIVGPAKAMKYIFLRLMEKLK

[0411] SEQ ID NO:3 RfbG

[0412] MHSSDQKRVAVLMATYNGECWIEEQLKSIIEQKDVDISIFISDDLSTDNTLNICEEFQLSYPSIINILPSVNKFGGAGKNFYRLIKDVDLENYDYICFSDQDDIWYKDKIKNAIDCLVFNNANCYSSNVIAYYPSGRKNLVDKAQSQTQFDYFFEAAGPGCTYVIKKETLIEFKKFIINNKNAAQDICLHDWFLYSFARTRNYSWYIDRKPTMLYRQHENNQVGANISFKAKYKRLGLVRNKWYRKEVTKIANALADDSFVNNQLGKGYIGNLILALSFWKLRRKKADKIYILLMLILNIF

[0413] SEQ ID NO:4 GbcC

[0414] MKVNILMATYNGEKFLAQQIESIQKQTFKEWNLLIRDDGSSDKTCDIIRNFTAKDSRIRFINENEHHNLGVIKSFFTLVNYEVADFYFFSDQDDVWLPEKLSVSLEAAKHKASDVPLLVYTDLKVVNQELNILQDSMIRAQSHHANTTLLPELTENTVTGGTMMINHALAEKWFTPNDILMHDWFLALLAASLGEIIYLDLPTQLYRQHDNNVLGARTMDKRFKILREGPKSIFTRYWKLIHDSQKQASLIVDKYGDIMTANDLELIKCFIKIDKQPFMTRLRWLWKYGYSKNQFKHQVVFKWLIATNYYNKR

[0415] SEQ ID NO:5 GccC

[0416] MNINILLSTYNGERFLAEQIQSIQKQTIKDWTLLIRDDGSTDRTPDIIREFVKQDQRIQWINENQIENLGVIKNFYTLLKYQAADVYFFSDQDDIWLEDKLEVTLLEAQKHDLSKPLLVYTDLKVVNQQLEITHASMIKTQSAHANTTLLQELTENTVTGGTMMINQALAKEWNTCEGLLMHDWYLALVAAARGKLVCLDIPTELYRQHDANVLGARTWSKRMKHWLRPHQLIRKYWWLITSSQQQAQLLLDLPLQPKDRDMVEAYVSLLTMSLTKRLATLKTYGFRKNRAFHTLVFWSLVITLFGYRRK

[0417] SEQ ID NO:6 GgcC

[0418] MNINILLSTYNGERFLAEQIQSIQKQTIKDWTLLIRDDGSTDRTPDIIREFVKQDQRIQWINENQIENLGVIKNFYTLLKYQAADVYFFSDQDDIWLEDKLEVTLLEAQKHDLSKPLLVYTDLKVVNQQLEITHASMIKTQSAHANTTLLQELTENTVTGGTMMINQALAKEWNTCEGLLMHDWYLALVAAARGKLVYLDIPTELYRQHDANVLGARTWSKRMKHWLRPHQLIRKYWWLITSSQQQAQLLLDLPLQPKDRDMVEAYVSLLTMSLTKRLATLKTYGFRKNRAFHTLVFWSLVITLFGYRRK

[0419] SEQ ID NO:7 SccC

[0420] MKVNILMSTYNGQEFIAQQIQSIQKQTFENWNLLIRDDGSSDGTPKIIADFAKSDARIRFINADKRENFGVIKNFYTLLKYEKADYYFFSDQDDVWLPQKLELTLASVEKENNQIPLMVYTDLTVVDRDLQVLHDSMIKTQSHHANTSLLEELTENTVTGGTMMVNHCLAKQWKQCYDDLIMHDWYLALLAASLGKLIYLDETTELYRQHESNVLGARTWSKRLKNWLRPHRLVKKYWWLVTSSQQQASHLLELDLPAANKAIIRAYVTLLDQSFLNRIKWLKQYGFAKNRAFHTFVFKTLIITKFGYRRK

[0421] SEQ ID NO:8 SucC

[0422] MKINILMSTYNGEKFLAEQIESIQKQTVTDWTLLIRDDGSSDRTPEIIQDFVAKDSRIHFINADHRINFGVIKNFFTLLKYEEADYYFFSDQDDVWLPHKIETSLNKAKELEKNRPFLIYTDLTIVNQSLETIHESMISFQSDHANTTLLEELTENTVTGGTALINHALAELWTDDKDLLMHDWFLALLASAMGNLVYINEATELYRQHDRNVLGARTWSKRLKTWSKPHLMLNKYWWLIQSSQQQAQKLLDLPLSSDKRKLVEHYVTLLEKPLMTRLRDLKKYGYKKNRAFHTFVFRMLIITKIGYRRTVKNGIIQ

[0423] SEQ ID NO:9 GccG

[0424] MNRVLLYVHFNKYNKVSKHIYYQLEKLRPLFTTVVFISNSKVEQKELENLQKQRLIDSFIQRENKGFDFAAWHDGMMKIGFDDLTLCDSLTIMNDTCFGPLWGMAPYFEKFDNNQSVDFWGLTNNRKTSSFKEHIQSYFITFKQHVIQSDAFLNFWKTIKEYDDVQEVIQKYETQVTTTLLEAGFNYQTVFDTREADSSFMLHPDFSYYNPTAILQHRVPFIKVKAIDANQHITPYLLNMIEEETTYPVDLIISHMSQVGLPDAKYLLARKYLPFESLVTQNVPRIAVHLHVFYVDLLNEFLEGFASWEFQYDLYITTDTQEKKEAIEKLLVQSNRHAHLYVTGNVGRDVLPMLLLKDKLRDYDYIGHFHTKKSKEADFWAGESWRKELINMLIKPANEIVRSFENNDIGIVIADIPSFFRFNKIVDAWNEHLIAPEMMRLWKEMGLKKEIDFQSMDTFVMSYGTFVWFKFDALKPLFDLDLTVDDIPKEPLPQNSILHAIERLLVYIAWDRFYDFRIVKNPYNLSPFIDNKLLNLRESGGARTYVNFDHMGGIKGALKYIIIGPARAMKYIVKRVLKSKR

[0425] SEQ ID NO:10 GccG protein 1

[0426] MNRVLLYVHFNKYNKVSKHIYYQLEKLRPLFTTVVFISNSKVEQKELENLQKQRLIDSFIQRENKGFDFAAWHDGMMKIGFDDLTLCDSLTIMNDTCFGPLWGMAPYFEKFDNNQSVDFWGLTNNRKTSSFKEHIQSYFITFKQHVIQSDAFLNFWKTIKEYDDVQEVIQKYETQVTTTLLEAGFNYQTVFDTREADSSFMLHPDFSYYNPTAILQHRVPFIKVKAIDANQHITPYLLNMIEEETTYPVDLIISHMSQVGLPDAKYLLARKYLPFESLVTQNVPRIAVHLHVFYVDLLNEFLEGFASWEFQYDLYITTDTQEKRKQLKNY

[0427] SEQ ID NO:11 GccG protein 2

[0428] MGVSVRPLYYNRYSRKKEAIEKLLVQSNRHAHLYVTGNVGRDVLPMLLLKDKLRDYDYIGHFHTKKSKEADFWAGESWRKELINMLIKPANEIVRSFENNDIGIVIADIPSFFRFNKIVDAWNEHLIAPEMMRLWKEMGLKKEIDFQSMDTFVMSYGTFVWFKFDALKPLFDLDLTVDDIPKEPLPQNSILHAIERLLVYIAWDRFYDFRIVKNPYNLSPFIDNKLLNLRESGGARTYVNFDHMGGIKGALKYIIIGPARAMKYIVKRVLKSKR

[0429] SEQ ID NO:12 GgcG protein 1

[0430] MIGKIIRSYQDEGGRATLRKIRQRLQGGGHPQSAGKIDLNRIPIMPQLEDIAQADYINHPYQRPAKLDKKQLNIAWVSPPVGKGGGGHTTISRFVKYLQSQGHHITFYIYHNNTIEQSAKEAQEIFSKAYGIEVAVDDLKNFSNQDLVFATSWETAYAVFNLKSENLHKFYFVQDFEPIFYGVGSRYKLAEATYKFGFYGITAGKWLTHKLKDYHMDADYFNFGADTDIYKPKAPLQKKKKIAFYARAHTERRGFELGVMALKIFKDKHPEYDIEFFGQDMSHYDIPFDFIDRGILNKEELAAIYHESVACLVLSLTNVSLLPLELLVAGCIPVMNSGDNNTMVLGENDDIAYAEAYPVALAEELCKAVERSDIDTYANEMSQKYDGVSWENSYRKVEEIIRREVIND

[0431] SEQ ID NO:13 GgcG protein 2

[0432] MTDKIKATVFIPVYNGENDHLEETLTALYTQKTDFSWNVMITDSESKDRSVAIIETFAERYGNLQLIKLKKSDYSHGATRQMAAELSSAEYMVYLSQDAVPANEHWLAEMLKPFTIHHDIVAVLGKQKPRIGCFPAMKYDINAVFNEQGVAGAITLWTRQEESLKGKYTKESFYSDVCSAAPRDFLVNEIGYRSVPYSEDYEYGKDILDAGYMKAYNSDAIVEHSNDVLLSEYKQRIFDETYNVRRNSGVTTPISVSTVLIQFLKSSVKDAMKIVSDQDYSWKRKLYWLAVNPLFHFEKWRGMRLANSVDMTKDNSKHSLENSKSKG

[0433] SEQ ID NO:14 SucG

[0434] MKRLLLYVHFNKYNRLSPHVLYQLKKMRPLFSNLIFISNSSLNDSDRQELLSSGLVNEVIQRQNIGFDFAAWRDGMATVGFESLSEYDNVTIMNDTCFGPLWDMKPYFLTYEDDEEVDFWGLTNNRQTKEFDEHIQSYFISFKKTVLSNETFLHFWRTVQDFTDVQDVIKNYETQVTTGLLKEGFRYKCIFNTVTADASGMLHADFSYYNPTAILKHQVPFIKVKTIDANQSIAPYLLQVIKNQTDYPVDLIVSHMSDIHYPDAPYLLSQKYLEKQEESDLKVSEHSIAVHLHVFYVDLLEEFLHAFTSFKFPFDLYITTDKSEKESEIKAILDSFRVSAKIVVTGNIGRDVLPMLKLKDELSQYDYIGHFHTKKSKEADFWAGESWRNELIDMLIKPANTIINQFEDPAIGIIIADIPSFFRFNKIVTPLNEHLIAPEMNKLWEKMNLSKTIDFEQFDTFVMSYGTFVWFKYDALKPLFDLNLKDGDVPKEPLPQNSILHAVERLLIYIAWDSHFDFRIAKNNVELTPFLDNKLLNDKSNSLPNTYVDFTYMGGIKGALKYIFIGPARAIKYIYIRTKEKIFNG

[0435] SEQ ID NO:15 SccG

[0436] MKRLLLYVHFNKYNRVSSHVVYQLTQMRSLFSKVIFISNSQVADADVKMLREKHLIDDFIQRQNSGFDFAAWRDGMVFVGFDELVTYDSVTTMNDTCFGPLWEMYSIYQEFETKTTVDFWGLTNNRATKSFREHIQSYFISFKASVLRSTAFRDFWENIKEYQDVQKVIDQYETKVTTTLLDAGFQYDVVFDTTKEDASHMLHADFSYYNPTAILNHRVPFIKVKAIDNNQHITPYLLNDIQKNSTYPIDLIVSHMSEINYPDFSYLLGHKYVKKRERVDLKNQKVAVHLHVFYVDLLEEFLTAFKQFHFSYDLFITTDSDDKKAEIEEILSANGQEAQVFVTGNIGRDVLPMLKLKNYLSAYDFVGHFHTKKSKEADFWAGQSWREELIDMLVKPADNILAQLQQNPKIGLVIADMPTFFRYNKIVDAWNEHLIAPEMNTLWQKMGMTKKIDFNAFHTFVMSYGTFVWFKYDALKPLFDLNLTDDDVPEEPLPQNSILHAIERLLIYIAWNEHYDFRISKNPVDLTPFIDNKLLNERGNSAPNTFVDFNYMGGIKGAFKYIFIGPARAVKYILKRSLQKIKS

[0437] SEQ ID NO:16 GacA

[0438] MLENTKILRKVFYLWQKGELMILITGSNGQLGTELRYLLDERGVDYVAVDVAEMDITNEDKVEAVFAQVKPTLVYHCAAYTAVDAAEDEGKALNEAINVTGSENIAKACGKYGATLVYISTDYVFDGNKPVGQEWVETDHPDPKTEYGRTKRLGELAVERYAEHFYIIRTAWVFGNYGKNFVFTMEQLAENHSRLTVVNDQHGRPTWTRTLAEFMCYLTENQKAFGYYHLSNDAKEDTTWYDFAKEILKDKAVEVVPVDSSAFPAKAKRPLNSTMNLDKAKATGFVIPTWQEALKAFYQQGLKK

[0439] SEQ ID NO:17 GacH

[0440] MIKDTFLKTNWLNISHHIILLVFGFYFSFYSLAKELVSSTAQPVNYYAHLLNVSFVGYIISLIGLSYYLSRQVSRQLFLKTSFIVISYLIVSYWVQITQHLNDKRFDIWSLTKNQFYQFQALPSLLIILVMATLIKILVAYFAIEKDRFGLLGYQGNTFSVALILAVVPINDIHLLKLISSRFSELVTAGNSQIALLKISGLLIVLLVIFATIIYVVLNALKHLKSNKPSFSVAATTSLFLALVFNYTFQYGVKGDEALLGYYVFPGATLFQIVAITLVALLAYVITNRYWPTTFFLLILGTIISVVNDLKESMRSEPLLVTDFVWLQELGLVTSFVKKSVIVEMVVGLAICIVVAWYLHGRVLAGKLFMSPVKRASAVLGLFIVSCSMLIPFSYEKEGKILSGLPIISALNNDNDINWLGFSTNARYKSLAYVWTRQVTKKIMEKPTNYSQETIASIAQKYQKLAEDINKDRKNNIADQTVIYLLSESLSDPDRVSNVTVSHDVLPNIKAIKNSTTAGLMQSDSYGGGTANMEFQTLTSLPFYNFSSSVSVLYSEVFPKMAKPHTISEFYQGKNRIAMHPASANNFNRKTVYSNLGFSKFLALSGSKDKFKNIENVGLLTSDKTVYNNILSLINPSESQFFSVITMQNHIPWSSDYPEEIVAEGKNFTEEENHNLTSYARLLSFTDKETRAFLEKLTQINKPITVVFYGDHLPGLYPDSAFNKHIENKYLTDYFIWSNGTNEKKNHPLINSSDFTAALFEHTDSKVSPYYALLTEVLNKASVDKSPDSPEVKAIQNDLKNIQYDVTIGKGYLLKHKTFFKISR

[0441] SEQ ID NO:18 RMID of Group B

[0442] MILITGANGQLGSELRHLLDERTQEYVAVDVAEMDITNAEMVDKVFEEVKPSLVYHCAAYTAVDAAEDEGKELDFAINVTGTENVAKAAAKHDATLVYISTDYVFDGEKPVGQEWEVDDLPDPKTEYGRTKRMGEELVEKYASKFYTIRTAWVFGNYGKNFVFTMQNLAKTHKTLTVVNDQHGRPTWTRTLAEFMTYLAENQKDFGYYHLSNDAKEDTTWYDFAVEILKDTDVEVKPVDSSQFPAKAKRPLNSTMSLEKAKATGFVIPTWQDALKEFYKQEVKK

[0443] SEQ ID NO:19 RMID of Group C

[0444] MILITGSNGQLGTELRYLLDERHVDYVAVDVAEMDITDADKVEAVFAQVKPTLVYHCAAYTAVDAAEDEGKALNEAINVTGSENIAKACGKYGATLVYISTDYVFDGNKPVGQEWLETDVPDPQTEYGRTKRLGELAVEQYAEHFYIIRTAWVFGNYGKNFVFTMQQLAEKHPRLTVVNDQHGRPTWTRTLAEFMCYLAENQKAFGYYHLSNDAKEDTTWYDFAKEILKDKAVEVVPVDSSAFPAKAKRPLNSTMNLDKAKATGFVIPTWQEALKEFYQQDRHQ

[0445] SEQ ID NO:20 RMID of Group G

[0446] MILITGSNGQLGTELRYLLDERHVDYVAVDVAEMDITDADKVEAVFAQVKPTLVYHCAAYTAVDAAEDEGKALNEAINVTGSENIAKACGKYGATLVYISTDYVFDGNKPVGQEWLETDVPDPQTEYGRTKRLGELAVEQYAEHFYIIRTAWVFGNYGKNFVFTMQQLAEKHPRLTVVNDQHGRPTWTRTLAEFMCYLAENQKAFGYYHLSNDAKEDTTWYDFAKEILKDKAIEVVPVDSSAFPAKAKRPLNSTMNLDKAKATGFVIPTWQEALKEFYQQDRHQ

[0447] SEQ ID NO:21 RMID of Streptococcus mutans

[0448] MILITGSNGQLGTELRHLLNERNEDYVAVDVAEMDITKAEKVDEVFLQVKPSLVYHCAAYTAVDAAEDEGKELDYAINVTGTENIAKACEKYNATLVYISTDYVFDGEKPVGQEWEVDDKPDPKTEYGRTKRLGEEAVEKYVKNFYIIRTAWVFGNYGKNFVFTMQHLAKSHNSLTVVNDQHGRPTWTRTLAEFMTYLAENQKEYGYYHLSNDATEDTTWYDFALEILKDTDVVVKPVDSSQFPAKAKRPLNSTMSLTKAKATGFVIPTWQEALQEFYKQDVKK

[0449] SEQ ID NO:22 RMID of Streptococcus uberis

[0450] MILITGSNGQLGTELRYLLDERNVEYVAVDVAEMDITNPDMVDEVFAQVKPTLVYHCAAYTAVDAAEDEGKALNQAINVDGTVNIAKACQKYNATLVYISTDYVFDGTKTVGQEWLETDIPDPKTEYGRTKRLGEEAVEKYVDQFYIIRTAWVFGHYGKNFVFTMQNLAKTHPKLTVVNDQYGRPTWTRTLAEFMCHLTENQKDYGYYHLSNDSKEDTSWYDFAKEILKDTDVEVVPVDSSAFPAKAKRPLNSTMNLDKAKATGFVIPTWQEALNEFYKQEVKK

[0451] SEQ ID NO:23 GccD

[0452] MNFLTKKNRILLREMVKTDFKLRYQGSAIGYLWSILKPLMMFTIMYLVFIRFLRLGGNIPHFPVALLLANVIWSFFSEATSMGMVSIVSRGDLLRKLNFSKHIIVFSAILGALINFLINLVVVLIFALINGVTISNYAYFSFFLFIELVVFVVGIALLLSTVFVYYRDLAQVWEVLLQAGMYATPIIYPITFVLEGHPLAAKILMLNPIAQMIQDFRYLLIDRANVTIWQMSTNWFYIAIPYLIPFILLFIGITVFKKNATKFAEII

[0453] SEQ ID NO:24 GccE

[0454] MTNNKIAVKVEHVSKSFKLPTEATKSFRTTLVNRFRGIKGFTEQQVLKDINFEVHKGDFFGIVGRNGSGKSTLLKIISQIYVPEKGQVTVDGKMVSFIELGVGFNPELTGRENVYMNGAMLGFTKEEINAMYDDIVDFAELHDFMNQKLKNYSSGMQVRLAFSVAIKAQGDVLILDEVLAVGDEAFQRKCNDYFMERKDSGKTTILVTHDMGAVKKYCNRAVLIEDGLVKAYGEPFDVANQYSVDNTETKEELQDSEKVAISDIVQQLRVNLTSKQRITPKEIISFEVSYEVLRDEPTYIAFSLTDMDRNIWVYNDNSRDQLVEGIGKKTISYQCHLSHLNDIKLKLEVTVRDKDGQMLLFSTAEQSPKIIIQRDDITSDDFSALDSASGLYQRNGQWTFS

[0455] SEQ ID NO:25 GccF

[0456] MHKVSIICTNYNKAPWLGEALDSFLNQKTNFEVDIIVIDDASTDESKTILEDYQTRFPEKITLLFNDHNLGITKTWIKACLYAKGKYIARCDGDDYWTDDLKLQKQVDALEASKYSKWSNTDFDFVDNKGKVLHSNVFETGYIPFTDTYEKVLALKGMTMASTWVVDAELMRFVNQKINIETPDDTFDMQLELFQLTSLTYINDSTTVYRMTSNSDSRPADKKRMIHRIKQLLQTQVFYLAKYPQANIPQIANLLMEQDGKNELRIHELSCLINDLRQELNEKTEQQKEREFEIKEIIENQSRQICELTHQYNCVINSRRWKYMSKLIDFIRRKK

[0457] SEQ ID NO:26 GgcD

[0458] MNFLTKKNRILLREMVKTDFKLRYQGSFIGHLWSILKPMLLFTIMYLVFVRFLKFDDGTPHYAVSLLLGMVTWNFFTEATNMGMLSIVSRGDLLRKINFPKEIIVISSVVGATINYFINILVVFAFALINGVQPSFGVFILIPLFLELFLFATGVAFILATLFVKYRDMGPIWEVMLQAGMYGTPIIYSITYIIQRGHLGIAKVMMMNPLAQIIQELRHFIVYSGATINWDIFENKFFTLIPIILSLSAFVIGYVIFKRNAKKFAEIL

[0459] SEQ ID NO:27 GgcE

[0460] MSEKKVVLSVDSVSKSFKLPTEASNSLRTSLVNYFKGIKGYTEQHVLDDISFQVEEGDFFGIVGRNGSGKSTLLKIISKIYEPEKGTVTVDGKLVPFIELGVGFNPELTGRENVFMNGALLGFSRDEVAAMYDDIVSFAELHDFMDQKLKNYSSGMQVRLAFSIAIKAKGDILILDEVLAVGDEAFQRKCFDYFAQLKREHKTVILVTHSMEQVQRFCNKAMLIDKGHHMEVGTPLEISQIYKQLNGLNVAKESAKETENNGISLSSQFINHKDDTLTFTFDVHFEQTIEDPVLTFTIHKDTGELLYRWVSDEEVEGSIMIKNHKVSIDFAIQNIFPNGKFTTEFGVKSRDRSKEYAMFSGICNFELINRGKSGNNIYWKPETTVKLS

[0461] SEQ ID NO:28 GgcF

[0462] MRMYQGKRFLLTHIWLRGFSGAEINILELATYLKEAGAQVEVFTFLAKSPMLDEFQKNGIPVIDDSDYPFDVSQYDVVCSAQNIIPPAMIEALGKSQEKLPKFIFFHMAALPEHVLEQPYIYQLEKKISSATLAISEEIVNKNLKRFFKDIPNLHYYPNPAPESYAAMEHLKKQSPERILVISNHPPQEVIDMEPLLAKKGIHVDYFGVWSDHYELVTPELLASYDCVVGIGKNAQYCLVMGKPIYIYDHFKGPGYLTETNFEAAALNNFSGRGFEEQEKTAEELVDDLLEHYQSAQAFQHNHLYDYRSRYTISTIVDHIYKSINIIPKAIAPLEQVDVEYIKAITLFIRTRLVRLENDVANLWEAVHRYEQLDRKATAKREALEQLLTAKTTELNLIKTSRMFKLYQLLWRIKGFFFRKEHLKRAK

[0463] SEQ ID NO:29 SccD

[0464] MDFFSRKNRILLKELIKTDFKLRYQGSAIGYLWSILKPLMLFAIMYIVFVRFLPLGGDVPHWPVALLLGNVIWTFFQETTMMGMVSVVTRGDLLRKLNFSKQTIVFSAVSGAAINFGINVIVVLIFALLNGVTFTFRWNLFLLIPLFLELLLFSTGIAFILSTLYVRYRDIGPVWEVILQGGFYGTPIIYSLTYIATRSVVGAKLLLLSPIAQIIQDMRHILIDPANVTIWQMINHKSIAVIPYLVPIFVFIIGFLVFNYNAKKFAEII

[0465] SEQ ID NO:30 SccE

[0466] MTKNNIAVKVDHVSKYFKLPVESTQSLRTALVNRFKGIKGYKKQHVLRDIDFEVEKGDFFGIVGRNGSGKSTLLKIISQIYVPEQGKVTVDGKLVSFIELGVGFNPELTGRENVYMNGAMLGFTTEEVDTMYQDIVDFAELQDFMNQKLKNYSSGMQVRLAFSVAIKAQGDVLILDEVLAVGDEAFQRKCNDYFLERKNSGKTTILVTHDMAAVKKYCNKAVLIDDGLIKAIGEPFDVANQYSLDNTDQIVEDKQEEEAAVQEEEQIVVDNLEVKLLSANRMTPRDSIRFEISYNVLADVGTYIALSLTDVDRNIWIYNDNSLDYLSSGSGKKRVFYECHLKSLNDIKLKLEVTVRDKQGQMLAFSSATNTPIISINRDDLEGDDKSAMDSASGLIQRNGQWQFS

[0467] SEQ ID NO:31 SccF

[0468] MVKVSIICTNYNKGSWIGEAIDSFLKQETSFPYEIIIVDDASTDHSVHIIKTYQKQYPDLIRAFFNQENQGITKTWSDICKKARGQYIARCDGDDYWIDPFKLQKQIDLLETSPESKWSNTDFDMVDSKGNIIHKDVLKNNIIPFMDSYEKMLALKGMTMASTWLVETKLMLEINDRINKDAVDDTFNIQLELFKKTKLAFLRDSTTVYRMDAESDSRSKDSEKLAQRFDRLLETQLEYIEKYPDSDYKKVLEYLLPKHNDFEKVLAQDGKNVWDNQQITIYLAKGDDQEFSEENCFQFPLQHSGNIQLTFPENIRKIRIDLSEIPSYYRQVSLVNTTVNTELLPTWTNAKVFGYSYYFIAPDPQMIYDLTAQEGQDFKLTYEWFNVDQPSQPDFLANHLVKELDQKKVELKMLSPYKYQYQKAVAERDLYLEQLNEMVVRYNSVTHSRRWTIPTKIINLFRRKK

[0469] SEQ ID NO32 SucD

[0470] MELFSKKNRILLKELVKTDFKLRYQGSAIGYLWSILKPLLMFTIMYLVFIRFLRLGGSVPHFPVALLLANVIWSFFSEATGMGMVSIVTRGDLLRKLNFSKHTIVFSAVLGALINFSINLVVVLIFALINGVTISPFAYMAIPLFIELLILAVGVALLLSTLFVYYRDLAQVWEVLMQAAMYATPIIYPITFVSDKNPLAAKILMLNPLAQMIQDLRFLLIDRANATIWQMSNHWYYVMIPYLIPFLVLALGILVFNKNAKKFAEII

[0471] SEQ ID NO33 SucE

[0472] MSTRDIAVKVEHVSKSFKLPTEATKSFRTTLVNRFRGIKGYTEQKVLKDINFEVKKGDFFGIVGRNGSGKSTLLKIISQIYVPEKGTVTVEGKMVSFIELGVGFNPELTGRENVYMNGAMLGFTQEEVDAMYEDIVDFAELHDFMNQKLKNYSSGMQVRLAFSVAIKAQGDVLILDEVLAVGDEAFQRKCNDYFMERKESGKTTILVTHDMAAVKKYCNRAVLIEDGLVKALGDPDDVANQYSFDNAIASETVEKKEDGKSTEKKESQLISDFSAQLLTKPQISPDEDITISFSYNVLKNMETHVALSFIDIDTNLGLYNDNSMSLKTNGQGQKTVTMTCQMSYLNHAKLKLAATVRDKDKHPLAFLPVNEIPVILIDRKVDASNESEWDANTGILRRSSQWT*

[0473] SEQ ID NO34 SucF

[0474] MKKILFVSPTGTLDNGAEISITNLMVLLTQEGYDIINVIPKIKHSTHDAYLHKMRENQIKVYELDYTNWWWESAPGDKIGHLEDRSAYYQKYIYEIRKIIAEEAVDLVITSTANLFQGALAAACERIPHYWIIHEFPLDEFAYYKELIPFIEEYSDKIFTVEGKLTEFLRPLLKESQKLFPFVPFVNIKKNNNLKTGEETRLISISRINENKNQLELLKAYQSMAEPKPELLFVGDWDDSYKEKCDDFIQSHQLKTVRFLGHQSNPWNLMTDKDILVLNSKMETFGLVFVEALIQGIPVLASNNYGYSSVVDYFGCGKLYHLGDEKELVALLNEFVTNFSEEKKKSLTQSFMVEEKYTIEKSYCALLDAISNENSVKSDRPIWLSQFLGAYNPLSTFSPAGKESISIYYRDENGNWSENQKLVFSLFNRDSFTFSVPKGMTRIRLDMSERPSYYDKITLVDSDTMTQLLPTNVSGFEENNSFYFNHSDPQMEFNVSFSKNNVFQLSYQLANLENIFQDSFLPNQLVQKLLSFKEKQSDLEMLKIENHQLQEKNKLKQEQLEEMVVRYNSVIHSRRWSIPTKMINFLRRKK

[0475] SEQ ID NO:35 SccH

[0476] MKQLKKIWDMLGKQKLLIFIFIFALNVTLRNYDLLIGRRANSSLSFKVISKNFDIMIEHWEALPSHFKIIGGVCLVIYVLSILGLSFYLSKNLKKTFFIELLLGYGLYIVISYFLAVTRELNNESFKIWDLAKNHFFQPYFLPTLVLIIVCTLALNYLIRVKMKRSHLSRKMTLLLENFSETEFLLTGLIVSFILSDTLYVKLLQESLRAYYHKPLAYESLLFLYTLLTLILFSVIVEACFNAYRSIKLNRPNLSLAFVSSLLFATIFNYAFQYGLKNDADLLGKYIVPGATAYQILVLTAAGFFLYLIINRYLLVTFLIVILGSIITVVNVLKVGMRNEPLLVTDFAWVTNIRLLARSVNANIIFSTLLILAALILLYLFLRKRLLQGKITENHRLKVGLISSICLLGFSIFIIFRNEKGSKIVNGIPVISQVNNWVDIGYQGFYSNASYKSLMYVWTKQVTKSIMDKPSDYSKERILKLAKKYNNVANKINKVRTENISNQTVIYILSESFSDPDRVKGVNLSRDVIPNIKQIKEKTTSGLMHSDGYGGGTANMEFQSLTGLPYYNFNSSVSTLYTEVVPDMSVFPSISNQFKSKNRVVIHPSSASNYSRKYVYDKLKFPTFVASSGTSDKITHSEKVGLNVSDKTTYQNILDKINPSQSQFFSVMTMQNHVPWASDEPSDVVATGKGYTKDENGSLSSYARLLTYTDKETKDFLAQLSQLKHKVTVVFYGDHLPGLYPESAFKKDPDSQYQTDYFIWSNYNTKTLNHSYVNSSDFTAELLEHTNSKVSPYYALLTEVLDNTTVGHGKLTKEQKEIANDLKLIQYDITVGKGYIRNYKGFFDIR

[0477] SEQ ID NO:36 WchF_pHD0486

[0478] MKQSVYIIGSKGIPAKYGGFETFVEKLTEYQKDGNIQYYVACMRENSAKSGFTADTFEYNGAICYNIDVPNIGPARAIAYDIAAVNKAIELSKGNKDEAPIFYILACRIGPFISGLKKKIRSIGGRLLVNPDGHEWLRAKWSLPVRKYWKFSEQLMVKHADLLVCDSKNIEKYIREDYKQYQPKTTYIAYGTDTTPSSLKSEDAKVRNWYREKGVSENGYYLVVGRFVPENNYETMIREFIKSKSNKDFVLITNVEQNKFYDQLLKETGFDKDLRVKFVGTVYDQELLKYIRENAFAYFHGHEVGGTNPSLLEALASTKLNLLLDVGFNREVGEDGAIYWKKDELAHVIEEVERFDEGDITELDEKSSQRIADAFTWEKIVSDYEEVFTV

[0479] SEQ ID NO:37 WbbR

[0480] MNKYCILVLFNPDISVFIDNVKKILSLDVSLFVYDNSANKHAFLALSSQEQTKINYFSICENIGLSKAYNETLRHILEFNKNVKNKSINDSVLFLDQDSEVDLNSINILFETISAAESNVMIVAGNPIRRDGLPYIDYPHTVNNVKFVISSYAVYRLDAFRNIGLFQEDFFIDHIDSDFCSRLIKSNYQILLRKDAFFYQPIGIKPFNLCGRYLFPIPSQHRTYFQIRNAFLSYRRNGVTFNFLFREIVNRLIMSIFSGLNEKDLLKRLHLYLKGIKDGLKM

[0481] SEQ ID NO:38 WbbL_pHD0480

[0482] MVYIIIVSHGHEDYIKKLLENLNADDEHYKIIVRDNKDSLLLKQICQHYAGLDYISGGVYGFGHNNNIAVAYVKEKYRPADDDYILFLNPDIIMKHDDLLTYIKYVESKRYAFSTLCLFRDEAKSLHDYSVRKFPVLSDFIVSFMLGINKTKIPKESIYSDTVVDWCAGSFMLVRFSDFVRVNGFDQGYFMYCEDIDLCLRLSLAGVRLHYVPAFHAIHYAHHDNRSFFSKAFRWHLKSTFRYLARKRILSNRNFDRISSVFHP

[0483] SEQ ID NO:39 WbbL

[0484] MVAVTYSPGPHLERFLASLSLATERPVSVLLADNGSTDGTPQAAVQRYPNVRLLPTGANLGYGTAVNRTIAQLGEMAGDAGEPWGDDWVIVANPDVQWGPGSIDALLDAASRWPRAGALGPLIRDPDGSVYPSARQMPSLIRGGMHAVLGPFWPRNPWTTAYRQERLEPSERPVGWLSGSCLLVRRSAFGQVGGFDERYFMYMEDVDLGDRLGKAGWLSVYVPSAEVLHHKAHSTGRDPASHLAAHHKSTYIFLADRHSGWWRAPLRWTLRGSLALRSHLMVRSSLRRSRRRKLKLVEGRH

[0485] SEQ ID NO:40 RfbF

[0486] MNSNIYAVIVTYNPELKNLNALITELKEQNCYVVVVDNRTNFTLKDKLADIEKVHLICLGRNEGIAKAQNIGIRYSLEKGAEKIIFFDQDSRIRNEFIKKLSCYMDNENAKIAGPVFIDRDKSHYYPICNIKKNGLREKIHVTEGQTPFKSSVTISSGTMVSKEVFEIVGMMDEELFIDYVDTEWCLRCLNYGILVHIIPDIEMVHAIGDKSVKICGINIPIHSPVRRYYRVRNAFLLLRKNHVPLLLSIREVVFSLIHTTLIIATQKNKIEYMKKHILATLDGIRGITGGGRYNA

[0487] SEQ ID NO:41 WsaD

[0488] MDISIIIVNYNTPKLTVEAIESILKSKTKYSYEIIVVDNHSSDDSVRILKGKFPNIVVIENKQNVGFSKANNQAIKLSKGRYILLLNSDTIVKEDTIEKMIEFMDKSKKVGASGCEVVLPNGELDRACHRGFPTPEASFYYLVGLARLFPRSRRFNQYHLGYMNLNEPHPIDCLVGAFMMVRREVIEQVGLLDEEFFMYGEDIDWCYRIKQAGWEIYYCPFTSIIHYKGASSKKKPFKIVYEFHRAMFLFHRKHYARKYPFIVNCLVYTGIAAKFILSAIINTFRKIGG

[0489] SEQ ID NO:42 WbbP

[0490] MKISIIGNTANAMILFRLDLIKTLTKKGISVYAFATDYNDSSKEIIKKAGAIPVDYNLSRSGINLAGDLWNTYLLSKKLKKIKPDAILSFFSKPSIFGSLAGIFSGVKNNTAMLEGLGFLFTEQPHGTPLKTKLLKNIQVLLYKIIFPHINSLILLNKDDYHDLIDKYKIKLKSCHILGGIGLDMNNYCKSTPPTNEISFIFIARLLAEKGVNEFVLAAKKIKKTHPNVEFIILGAIDKENPGGLSESDVDTLIKSGVISYPGFVSNVADWIEKSSVFVLPSYYREGVPRSTQEAMAMGRPILTTNLPGCKETIIDGVNGYVVKKWSHEDLAEKMLKLINNPEKIISMGEESYKLARERFDANVNNVKLLKILGIPD

[0491] SEQ ID NO:43 WsaP

[0492] MVKVIRGRERFLTKLYAFVDFAMMQGAFFLAWVLKFKVFHNGVGGHLPLEDYLFWSFVYGAIAIVIGYLVELYAPKRKEKFSNELAKVLQVHTLSMFVLLSVLFTFKTVDVSRSFLLLYFAWNLILVSIYRYIVKQSLRTLRKKGYNKQFVLIIGAGSIGRKYFENLQMHPEFGLEVVGFLDDFRTKHAPEFAHYKPIIGQTADLEHVLSHQLIDEVIVALPLQAYPKYREIIAVCEKMGVRVSIIPDFYDILPAAPHFEIFGDLPIINVRDVPLDELRNRVLKRSFDIVFSLVAIIVTSPIMLLIAIGIKLTSPGPIIFKQERVGLNRRTFYMYKFRSMKPMPQSVSDTQWTVESDPRRTKFGAFLRKTSLDELPQFFNVLKGDMSIVGPRPERPFFVEKFKKEIPKYMIKHHVRPGITGWAQVCGLRGDTSIQERIEHDLFYIENWSLWLDIKIILLTITNGLVNKNAY

[0493] SEQ ID NO:44 WsaC

[0494] MEMPLVSIVVATYFPRTDFFEKQLQSLNNQTYENIEIIICDDSANDAEYEKVKKMVENIISRFPCKVIRNEKNVGSNKTFERLTQEANGDYICYCDQDDIWLSEKVERLVNHITKHHCTLVYSDLSLIDENDRIIHKSFKRSNFRLKHVHGDNTFAHLINRNSVTGCAMMIRADVAKSAIPFPDYDEFVHDHWLAIHAAVKGSLGYIKEPLVWYRIHLGNQIGNQRLVNITNINDYIRHRIEKQGNKYRLTLERLSLTLQQKQLVYFQIHLTEARKKFSQKPCLGNFFKIVPLIKYDIILFLFELMIFTVPFTCSIWIFKKLKY

[0495] SEQ ID NO:45 WsaE

[0496]

[0497] SEQ ID NO:46 WbbQ

[0498] MARSGGVVIKKKVAAIIITYNPDLTILRESYTSLYKQVDKIILIDNNSTNYQELKKLFEKKEKIKIVPLSDNIGLAAAQNLGLNLAIKNNYTYAILFDQDSVLQDNGINSFFFEFEKLVSEEKLNIVAIGPSFFDEKTGRRFRPTKFIGPFLYPFRKITTKNPLTEVDFLIASGCFIKLECIKSAGMMTESLFIDYIDVEWSYRMRSYGYKLYIHNDIHMSHLVGESRVNLGLKTISLHGPLRRYYLFRNYISILKVRYIPLGYKIREGFFNIGRFLVSMIITKNRKTLILYTIKAIKDGINNEMGKYKG Sequence Listing <110> University of Dundee <120> Rhamnan Polysaccharide <130> PE959537WO <150> GB 1908528.1 <151> 2019-06-13 <160> 128 <170> PatentIn version 3.5 <210> 1 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 1 Met Asn Ile Asn Ile Leu Leu Ser Thr Tyr Asn Gly Glu Arg Phe Leu 1 5 10 15 Ala Glu Gln Ile Gln Ser Ile Gln Arg Gln Thr Val Asn Asp Trp Thr 20 25 30 Leu Leu Ile Arg Asp Asp Gly Ser Thr Asp Gly Thr Gln Asp Ile Ile 35 40 45 Arg Thr Phe Val Lys Glu Asp Lys Arg Ile Gln Trp Ile Asn Glu Gly 50 55 60 Gln Thr Glu Asn Leu Gly Val Ile Lys Asn Phe Tyr Thr Leu Leu Lys 65 70 75 80 His Gln Lys Ala Asp Val Tyr Phe Phe Ser Asp Gln Asp Asp Ile Trp 85 90 95 Leu Asp Asn Lys Leu Glu Val Thr Leu Leu Glu Ala Gln Lys His Glu 100 105 110 Met Thr Ala Pro Leu Leu Val Tyr Thr Asp Leu Lys Val Val Thr Gln 115 120 125 His Leu Ala Val Cys His Asp Ser Met Ile Lys Thr Gln Ser Gly His 130 135 140 Ala Asn Thr Ser Leu Leu Gln Glu Leu Thr Glu Asn Thr Val Thr Gly 145 150 155 160 Gly Thr Met Met Ile Thr His Ala Leu Ala Glu Glu Trp Thr Thr Cys 165 170 175 Asp Gly Leu Leu Met His Asp Trp Tyr Leu Ala Leu Leu Ala Ser Ala 180 185 190 Ile Gly Lys Leu Val Tyr Leu Asp Ile Pro Thr Glu Leu Tyr Arg Gln 195 200 205 His Asp Ala Asn Val Leu Gly Ala Arg Thr Trp Ser Lys Arg Met Lys 210 215 220 Asn Trp Leu Thr Pro His His Leu Val Asn Lys Tyr Trp Trp Leu Ile 225 230 235 240 Thr Ser Ser Gln Lys Gln Ala Gln Leu Leu Leu Asp Leu Pro Leu Lys 245 250 255 Pro Asn Asp His Glu Leu Val Thr Ala Tyr Val Ser Leu Leu Asp Met 260 265 270 Pro Phe Thr Lys Arg Leu Ala Thr Leu Lys Arg Tyr Gly Phe Arg Lys 275 280 285 Asn Arg Ile Phe His Thr Phe Ile Phe Arg Ser Leu Val Val Thr Leu 290 295 300 Phe Gly Tyr Arg Arg Lys 305 310 <210> 2 <211> 581 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 2 Met Asn Arg Ile Leu Leu Tyr Val His Phe Asn Lys Tyr Asn Lys Ile 1 5 10 15 Ser Ala His Val Tyr Tyr Gln Leu Glu Gln Met Arg Ser Leu Phe Ser 20 25 30 Lys Ile Val Phe Ile Ser Asn Ser Lys Val Ser His Glu Asp Leu Lys 35 40 45 Arg Leu Lys Asn His Cys Leu Ile Asp Glu Phe Leu Gln Arg Lys Asn 50 55 60 Lys Gly Phe Asp Phe Ser Ala Trp His Asp Gly Leu Ile Ile Met Gly 65 70 75 80 Phe Asp Lys Leu Glu Glu Phe Asp Ser Leu Thr Ile Met Asn Asp Thr 85 90 95 Cys Phe Gly Pro Ile Trp Glu Met Ala Pro Tyr Phe Glu Asn Phe Glu 100 105 110 Glu Lys Glu Thr Val Asp Phe Trp Gly Ile Thr Asn Asn Arg Gly Thr 115 120 125 Lys Ala Phe Lys Glu His Val Gln Ser Tyr Phe Met Thr Phe Lys Asn 130 135 140 Gln Val Ile Gln Asn Lys Val Phe Gln Gln Phe Trp Gln Ser Ile Ile 145 150 155 160 Glu Tyr Glu Asn Val Gln Glu Val Ile Gln His Tyr Glu Thr Gln Leu 165 170 175 Thr Ser Ile Leu Leu Asn Glu Gly Phe Ser Tyr Gln Thr Val Phe Asp 180 185 190 Thr Arg Lys Ala Glu Ser Ser Phe Met Pro His Pro Asp Phe Ser Tyr 195 200 205 Tyr Asn Pro Thr Ala Ile Leu Lys His His Val Pro Phe Ile Lys Val 210 215 220 Lys Ala Ile Asp Ala Asn Gln His Ile Ala Pro Tyr Leu Leu Asn Leu 225 230 235 240 Ile Arg Glu Thr Thr Asn Tyr Pro Ile Asp Leu Ile Val Ser His Met 245 250 255 Ser Gln Ile Ser Leu Pro Asp Thr Lys Tyr Leu Leu Ser Gln Lys Tyr 260 265 270 Leu Asn Cys Gln Arg Leu Ala Lys Gln Thr Cys Gln Lys Val Ala Val 275 280 285 His Leu His Val Phe Tyr Val Asp Leu Leu Asp Glu Phe Leu Thr Ala 290 295 300 Phe Glu Asn Trp Asn Phe His Tyr Asp Leu Phe Ile Thr Thr Asp Ser 305 310 315 320 Asp Ile Lys Arg Lys Glu Ile Lys Glu Ile Leu Gln Arg Lys Gly Lys 325 330 335 Thr Ala Asp Ile Arg Val Thr Gly Asn Arg Gly Arg Asp Ile Tyr Pro 340 345 350 Met Leu Leu Leu Lys Asp Lys Leu Ser Gln Tyr Asp Tyr Ile Gly His 355 360 365 Phe His Thr Lys Lys Ser Lys Glu Ala Asp Phe Trp Ala Gly Glu Ser 370 375 380 Trp Arg Lys Glu Leu Ile Asp Met Leu Val Lys Pro Ala Asp Ser Ile 385 390 395 400 Leu Ser Ala Phe Glu Thr Asp Asp Ile Gly Ile Ile Ile Ala Asp Ile 405 410 415 Pro Ser Phe Phe Arg Phe Asn Lys Ile Val Asn Ala Trp Asn Glu His 420 425 430 Leu Ile Ala Gln Glu Met Met Ser Leu Trp Arg Lys Met Asp Val Lys 435 440 445 Lys Gln Ile Asp Phe Gln Ala Met Asp Thr Phe Val Met Ser Tyr Gly 450 455 460 Thr Phe Val Trp Phe Lys Tyr Asp Ala Leu Lys Ser Leu Phe Asp Leu 465 470 475 480 Glu Leu Thr Gln Asn Asp Ile Pro Ser Glu Pro Leu Pro Gln Asn Ser 485 490 495 Ile Leu His Ala Ile Glu Arg Leu Leu Val Tyr Ile Ala Trp Gly Asp 500 505 510 Ser Tyr Asp Phe Arg Ile Val Lys Asn Pro Tyr Glu Leu Thr Pro Phe 515 520 525 Ile Asp Asn Lys Leu Leu Asn Leu Arg Glu Asp Glu Gly Ala His Thr 530 535 540 Tyr Val Asn Phe Asn Gln Met Gly Gly Ile Lys Gly Ala Leu Lys Tyr 545 550 555 560 Ile Ile Val Gly Pro Ala Lys Ala Met Lys Tyr Ile Phe Leu Arg Leu 565 570 575 Met Glu Lys Leu Lys 580 <210> 3 <211> 301 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 3 Met His Ser Ser Asp Gln Lys Arg Val Ala Val Leu Met Ala Thr Tyr 1 5 10 15 Asn Gly Glu Cys Trp Ile Glu Glu Gln Leu Lys Ser Ile Ile Glu Gln 20 25 30 Lys Asp Val Asp Ile Ser Ile Phe Ile Ser Asp Asp Leu Ser Thr Asp 35 40 45 Asn Thr Leu Asn Ile Cys Glu Glu Phe Gln Leu Ser Tyr Pro Ser Ile 50 55 60 Ile Asn Ile Leu Pro Ser Val Asn Lys Phe Gly Gly Ala Gly Lys Asn 65 70 75 80 Phe Tyr Arg Leu Ile Lys Asp Val Asp Leu Glu Asn Tyr Asp Tyr Ile 85 90 95 Cys Phe Ser Asp Gln Asp Asp Ile Trp Tyr Lys Asp Lys Ile Lys Asn 100 105 110 Ala Ile Asp Cys Leu Val Phe Asn Asn Ala Asn Cys Tyr Ser Ser Asn 115 120 125 Val Ile Ala Tyr Tyr Pro Ser Gly Arg Lys Asn Leu Val Asp Lys Ala 130 135 140 Gln Ser Gln Thr Gln Phe Asp Tyr Phe Phe Glu Ala Ala Gly Pro Gly 145 150 155 160 Cys Thr Tyr Val Ile Lys Lys Glu Thr Leu Ile Glu Phe Lys Lys Phe 165 170 175 Ile Ile Asn Asn Lys Asn Ala Ala Gln Asp Ile Cys Leu His Asp Trp 180 185 190 Phe Leu Tyr Ser Phe Ala Arg Thr Arg Asn Tyr Ser Trp Tyr Ile Asp 195 200 205 Arg Lys Pro Thr Met Leu Tyr Arg Gln His Glu Asn Asn Gln Val Gly 210 215 220 Ala Asn Ile Ser Phe Lys Ala Lys Tyr Lys Arg Leu Gly Leu Val Arg 225 230 235 240 Asn Lys Trp Tyr Arg Lys Glu Val Thr Lys Ile Ala Asn Ala Leu Ala 245 250 255 Asp Asp Ser Phe Val Asn Asn Gln Leu Gly Lys Gly Tyr Ile Gly Asn 260 265 270 Leu Ile Leu Ala Leu Ser Phe Trp Lys Leu Arg Arg Lys Lys Ala Asp 275 280 285 Lys Ile Tyr Ile Leu Leu Met Leu Ile Leu Asn Ile Phe 290 295 300 <210> 4 <211> 313 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 4 Met Lys Val Asn Ile Leu Met Ala Thr Tyr Asn Gly Glu Lys Phe Leu 1 5 10 15 Ala Gln Gln Ile Glu Ser Ile Gln Lys Gln Thr Phe Lys Glu Trp Asn 20 25 30 Leu Leu Ile Arg Asp Asp Gly Ser Ser Asp Lys Thr Cys Asp Ile Ile 35 40 45 Arg Asn Phe Thr Ala Lys Asp Ser Arg Ile Arg Phe Ile Asn Glu Asn 50 55 60 Glu His His Asn Leu Gly Val Ile Lys Ser Phe Phe Thr Leu Val Asn 65 70 75 80 Tyr Glu Val Ala Asp Phe Tyr Phe Phe Ser Asp Gln Asp Asp Val Trp 85 90 95 Leu Pro Glu Lys Leu Ser Val Ser Leu Glu Ala Ala Lys His Lys Ala 100 105 110 Ser Asp Val Pro Leu Leu Val Tyr Thr Asp Leu Lys Val Val Asn Gln 115 120 125 Glu Leu Asn Ile Leu Gln Asp Ser Met Ile Arg Ala Gln Ser His His 130 135 140 Ala Asn Thr Thr Leu Leu Pro Glu Leu Thr Glu Asn Thr Val Thr Gly 145 150 155 160 Gly Thr Met Met Ile Asn His Ala Leu Ala Glu Lys Trp Phe Thr Pro 165 170 175 Asn Asp Ile Leu Met His Asp Trp Phe Leu Ala Leu Leu Ala Ala Ser 180 185 190 Leu Gly Glu Ile Ile Tyr Leu Asp Leu Pro Thr Gln Leu Tyr Arg Gln 195 200 205 His Asp Asn Asn Val Leu Gly Ala Arg Thr Met Asp Lys Arg Phe Lys 210 215 220 Ile Leu Arg Glu Gly Pro Lys Ser Ile Phe Thr Arg Tyr Trp Lys Leu 225 230 235 240 Ile His Asp Ser Gln Lys Gln Ala Ser Leu Ile Val Asp Lys Tyr Gly 245 250 255 Asp Ile Met Thr Ala Asn Asp Leu Glu Leu Ile Lys Cys Phe Ile Lys 260 265 270 Ile Asp Lys Gln Pro Phe Met Thr Arg Leu Arg Trp Leu Trp Lys Tyr 275 280 285 Gly Tyr Ser Lys Asn Gln Phe Lys His Gln Val Val Phe Lys Trp Leu 290 295 300 Ile Ala Thr Asn Tyr Tyr Asn Lys Arg 305 310 <210> 5 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 5 Met Asn Ile Asn Ile Leu Leu Ser Thr Tyr Asn Gly Glu Arg Phe Leu 1 5 10 15 Ala Glu Gln Ile Gln Ser Ile Gln Lys Gln Thr Ile Lys Asp Trp Thr 20 25 30 Leu Leu Ile Arg Asp Asp Gly Ser Thr Asp Arg Thr Pro Asp Ile Ile 35 40 45 Arg Glu Phe Val Lys Gln Asp Gln Arg Ile Gln Trp Ile Asn Glu Asn 50 55 60 Gln Ile Glu Asn Leu Gly Val Ile Lys Asn Phe Tyr Thr Leu Leu Lys 65 70 75 80 Tyr Gln Ala Ala Asp Val Tyr Phe Phe Ser Asp Gln Asp Asp Ile Trp 85 90 95 Leu Glu Asp Lys Leu Glu Val Thr Leu Leu Glu Ala Gln Lys His Asp 100 105 110 Leu Ser Lys Pro Leu Leu Val Tyr Thr Asp Leu Lys Val Val Asn Gln 115 120 125 Gln Leu Glu Ile Thr His Ala Ser Met Ile Lys Thr Gln Ser Ala His 130 135 140 Ala Asn Thr Thr Leu Leu Gln Glu Leu Thr Glu Asn Thr Val Thr Gly 145 150 155 160 Gly Thr Met Met Ile Asn Gln Ala Leu Ala Lys Glu Trp Asn Thr Cys 165 170 175 Glu Gly Leu Leu Met His Asp Trp Tyr Leu Ala Leu Val Ala Ala Ala 180 185 190 Arg Gly Lys Leu Val Cys Leu Asp Ile Pro Thr Glu Leu Tyr Arg Gln 195 200 205 His Asp Ala Asn Val Leu Gly Ala Arg Thr Trp Ser Lys Arg Met Lys 210 215 220 His Trp Leu Arg Pro His Gln Leu Ile Arg Lys Tyr Trp Trp Leu Ile 225 230 235 240 Thr Ser Ser Gln Gln Gln Ala Gln Leu Leu Leu Asp Leu Pro Leu Gln 245 250 255 Pro Lys Asp Arg Asp Met Val Glu Ala Tyr Val Ser Leu Leu Thr Met 260 265 270 Ser Leu Thr Lys Arg Leu Ala Thr Leu Lys Thr Tyr Gly Phe Arg Lys 275 280 285 Asn Arg Ala Phe His Thr Leu Val Phe Trp Ser Leu Val Ile Thr Leu 290 295 300 Phe Gly Tyr Arg Arg Lys 305 310 <210> 6 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 6 Met Asn Ile Asn Ile Leu Leu Ser Thr Tyr Asn Gly Glu Arg Phe Leu 1 5 10 15 Ala Glu Gln Ile Gln Ser Ile Gln Lys Gln Thr Ile Lys Asp Trp Thr 20 25 30 Leu Leu Ile Arg Asp Asp Gly Ser Thr Asp Arg Thr Pro Asp Ile Ile 35 40 45 Arg Glu Phe Val Lys Gln Asp Gln Arg Ile Gln Trp Ile Asn Glu Asn 50 55 60 Gln Ile Glu Asn Leu Gly Val Ile Lys Asn Phe Tyr Thr Leu Leu Lys 65 70 75 80 Tyr Gln Ala Ala Asp Val Tyr Phe Phe Ser Asp Gln Asp Asp Ile Trp 85 90 95 Leu Glu Asp Lys Leu Glu Val Thr Leu Leu Glu Ala Gln Lys His Asp 100 105 110 Leu Ser Lys Pro Leu Leu Val Tyr Thr Asp Leu Lys Val Val Asn Gln 115 120 125 Gln Leu Glu Ile Thr His Ala Ser Met Ile Lys Thr Gln Ser Ala His 130 135 140 Ala Asn Thr Thr Leu Leu Gln Glu Leu Thr Glu Asn Thr Val Thr Gly 145 150 155 160 Gly Thr Met Met Ile Asn Gln Ala Leu Ala Lys Glu Trp Asn Thr Cys 165 170 175 Glu Gly Leu Leu Met His Asp Trp Tyr Leu Ala Leu Val Ala Ala Ala 180 185 190 Arg Gly Lys Leu Val Tyr Leu Asp Ile Pro Thr Glu Leu Tyr Arg Gln 195 200 205 His Asp Ala Asn Val Leu Gly Ala Arg Thr Trp Ser Lys Arg Met Lys 210 215 220 His Trp Leu Arg Pro His Gln Leu Ile Arg Lys Tyr Trp Trp Leu Ile 225 230 235 240 Thr Ser Ser Gln Gln Gln Ala Gln Leu Leu Leu Asp Leu Pro Leu Gln 245 250 255 Pro Lys Asp Arg Asp Met Val Glu Ala Tyr Val Ser Leu Leu Thr Met 260 265 270 Ser Leu Thr Lys Arg Leu Ala Thr Leu Lys Thr Tyr Gly Phe Arg Lys 275 280 285 Asn Arg Ala Phe His Thr Leu Val Phe Trp Ser Leu Val Ile Thr Leu 290 295 300 Phe Gly Tyr Arg Arg Lys 305 310 <210> 7 <211> 311 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 7 Met Lys Val Asn Ile Leu Met Ser Thr Tyr Asn Gly Gln Glu Phe Ile 1 5 10 15 Ala Gln Gln Ile Gln Ser Ile Gln Lys Gln Thr Phe Glu Asn Trp Asn 20 25 30 Leu Leu Ile Arg Asp Asp Gly Ser Ser Asp Gly Thr Pro Lys Ile Ile 35 40 45 Ala Asp Phe Ala Lys Ser Asp Ala Arg Ile Arg Phe Ile Asn Ala Asp 50 55 60 Lys Arg Glu Asn Phe Gly Val Ile Lys Asn Phe Tyr Thr Leu Leu Lys 65 70 75 80 Tyr Glu Lys Ala Asp Tyr Tyr Phe Phe Ser Asp Gln Asp Asp Val Trp 85 90 95 Leu Pro Gln Lys Leu Glu Leu Thr Leu Ala Ser Val Glu Lys Glu Asn 100 105 110 Asn Gln Ile Pro Leu Met Val Tyr Thr Asp Leu Thr Val Val Asp Arg 115 120 125 Asp Leu Gln Val Leu His Asp Ser Met Ile Lys Thr Gln Ser His His 130 135 140 Ala Asn Thr Ser Leu Leu Glu Glu Leu Thr Glu Asn Thr Val Thr Gly 145 150 155 160 Gly Thr Met Met Val Asn His Cys Leu Ala Lys Gln Trp Lys Gln Cys 165 170 175 Tyr Asp Asp Leu Ile Met His Asp Trp Tyr Leu Ala Leu Leu Ala Ala 180 185 190 Ser Leu Gly Lys Leu Ile Tyr Leu Asp Glu Thr Thr Glu Leu Tyr Arg 195 200 205 Gln His Glu Ser Asn Val Leu Gly Ala Arg Thr Trp Ser Lys Arg Leu 210 215 220 Lys Asn Trp Leu Arg Pro His Arg Leu Val Lys Lys Tyr Trp Trp Leu 225 230 235 240 Val Thr Ser Ser Gln Gln Gln Ala Ser His Leu Leu Glu Leu Asp Leu 245 250 255 Pro Ala Ala Asn Lys Ala Ile Ile Arg Ala Tyr Val Thr Leu Leu Asp 260 265 270 Gln Ser Phe Leu Asn Arg Ile Lys Trp Leu Lys Gln Tyr Gly Phe Ala 275 280 285 Lys Asn Arg Ala Phe His Thr Phe Val Phe Lys Thr Leu Ile Ile Thr 290 295 300 Lys Phe Gly Tyr Arg Arg Lys 305 310 <210> 8 <211> 317 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 8 Met Lys Ile Asn Ile Leu Met Ser Thr Tyr Asn Gly Glu Lys Phe Leu 1 5 10 15 Ala Glu Gln Ile Glu Ser Ile Gln Lys Gln Thr Val Thr Asp Trp Thr 20 25 30 Leu Leu Ile Arg Asp Asp Gly Ser Ser Asp Arg Thr Pro Glu Ile Ile 35 40 45 Gln Asp Phe Val Ala Lys Asp Ser Arg Ile His Phe Ile Asn Ala Asp 50 55 60 His Arg Ile Asn Phe Gly Val Ile Lys Asn Phe Phe Thr Leu Leu Lys 65 70 75 80 Tyr Glu Glu Ala Asp Tyr Tyr Phe Phe Ser Asp Gln Asp Asp Val Trp 85 90 95 Leu Pro His Lys Ile Glu Thr Ser Leu Asn Lys Ala Lys Glu Leu Glu 100 105 110 Lys Asn Arg Pro Phe Leu Ile Tyr Thr Asp Leu Thr Ile Val Asn Gln 115 120 125 Ser Leu Glu Thr Ile His Glu Ser Met Ile Ser Phe Gln Ser Asp His 130 135 140 Ala Asn Thr Thr Leu Leu Glu Glu Leu Thr Glu Asn Thr Val Thr Gly 145 150 155 160 Gly Thr Ala Leu Ile Asn His Ala Leu Ala Glu Leu Trp Thr Asp Asp 165 170 175 Lys Asp Leu Leu Met His Asp Trp Phe Leu Ala Leu Leu Ala Ser Ala 180 185 190 Met Gly Asn Leu Val Tyr Ile Asn Glu Ala Thr Glu Leu Tyr Arg Gln 195 200 205 His Asp Arg Asn Val Leu Gly Ala Arg Thr Trp Ser Lys Arg Leu Lys 210 215 220 Thr Trp Ser Lys Pro His Leu Met Leu Asn Lys Tyr Trp Trp Leu Ile 225 230 235 240 Gln Ser Ser Gln Gln Gln Ala Gln Lys Leu Leu Asp Leu Pro Leu Ser 245 250 255 Ser Asp Lys Arg Lys Leu Val Glu His Tyr Val Thr Leu Leu Glu Lys 260 265 270 Pro Leu Met Thr Arg Leu Arg Asp Leu Lys Lys Tyr Gly Tyr Lys Lys 275 280 285 Asn Arg Ala Phe His Thr Phe Val Phe Arg Met Leu Ile Ile Thr Lys 290 295 300 Ile Gly Tyr Arg Arg Thr Val Lys Asn Gly Ile Ile Gln 305 310 315 <210> 9 <211> 581 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 9 Met Asn Arg Val Leu Leu Tyr Val His Phe Asn Lys Tyr Asn Lys Val 1 5 10 15 Ser Lys His Ile Tyr Tyr Gln Leu Glu Lys Leu Arg Pro Leu Phe Thr 20 25 30 Thr Val Val Phe Ile Ser Asn Ser Lys Val Glu Gln Lys Glu Leu Glu 35 40 45 Asn Leu Gln Lys Gln Arg Leu Ile Asp Ser Phe Ile Gln Arg Glu Asn 50 55 60 Lys Gly Phe Asp Phe Ala Ala Trp His Asp Gly Met Met Lys Ile Gly 65 70 75 80 Phe Asp Asp Leu Thr Leu Cys Asp Ser Leu Thr Ile Met Asn Asp Thr 85 90 95 Cys Phe Gly Pro Leu Trp Gly Met Ala Pro Tyr Phe Glu Lys Phe Asp 100 105 110 Asn Asn Gln Ser Val Asp Phe Trp Gly Leu Thr Asn Asn Arg Lys Thr 115 120 125 Ser Ser Phe Lys Glu His Ile Gln Ser Tyr Phe Ile Thr Phe Lys Gln 130 135 140 His Val Ile Gln Ser Asp Ala Phe Leu Asn Phe Trp Lys Thr Ile Lys 145 150 155 160 Glu Tyr Asp Asp Val Gln Glu Val Ile Gln Lys Tyr Glu Thr Gln Val 165 170 175 Thr Thr Thr Leu Leu Glu Ala Gly Phe Asn Tyr Gln Thr Val Phe Asp 180 185 190 Thr Arg Glu Ala Asp Ser Ser Phe Met Leu His Pro Asp Phe Ser Tyr 195 200 205 Tyr Asn Pro Thr Ala Ile Leu Gln His Arg Val Pro Phe Ile Lys Val 210 215 220 Lys Ala Ile Asp Ala Asn Gln His Ile Thr Pro Tyr Leu Leu Asn Met 225 230 235 240 Ile Glu Glu Glu Thr Thr Tyr Pro Val Asp Leu Ile Ile Ser His Met 245 250 255 Ser Gln Val Gly Leu Pro Asp Ala Lys Tyr Leu Leu Ala Arg Lys Tyr 260 265 270 Leu Pro Phe Glu Ser Leu Val Thr Gln Asn Val Pro Arg Ile Ala Val 275 280 285 His Leu His Val Phe Tyr Val Asp Leu Leu Asn Glu Phe Leu Glu Gly 290 295 300 Phe Ala Ser Trp Glu Phe Gln Tyr Asp Leu Tyr Ile Thr Thr Asp Thr 305 310 315 320 Gln Glu Lys Lys Glu Ala Ile Glu Lys Leu Leu Val Gln Ser Asn Arg 325 330 335 His Ala His Leu Tyr Val Thr Gly Asn Val Gly Arg Asp Val Leu Pro 340 345 350 Methionine, Leucine, Leucine, Leucine, Lysine, Aspartic acid, Lysine, Leucine, Arginine, Aspartic acid, Tyrosine, Aspartic acid, Tyrosine, Isoleucine, Glycine, Histidine 355 360 365 Phenylalanine, Histidine, Threonine, Lysine, Lysine, Serine, Lysine, Glutamic acid, Alanine, Aspartic acid, Phenylalanine, Tryptophan, Alanine, Glycine, Glutamic acid, Serine 370 375 380 Tryptophan, Arginine, Lysine, Glutamic acid, Leucine, Isoleucine, Asparagine, Methionine, Leucine, Isoleucine, Lysine, Proline, Alanine, Asparagine, Glutamic acid, Isoleucine 385 390 395 400 Valine, Arginine, Serine, Phenylalanine, Glutamic acid, Asparagine, Asparagine, Aspartic acid, Isoleucine, Glycine, Isoleucine, Valine, Isoleucine, Alanine, Aspartic acid, Isoleucine 405 410 415 Proline, Serine, Phenylalanine, Phenylalanine, Arginine, Phenylalanine, Asparagine, Lysine, Isoleucine, Valine, Aspartic acid, Alanine, Tryptophan, Asparagine, Glutamic acid, Histidine 420 425 430 Leucine, Isoleucine, Alanine, Proline, Glutamic acid, Methionine, Methionine, Arginine, Leucine, Tryptophan, Lysine, Glutamic acid, Methionine, Glycine, Leucine, Lysine 435 440 445 Lysine, Glutamic acid, Isoleucine, Aspartic acid, Phenylalanine, Glutamine, Serine, Methionine, Aspartic acid, Threonine, Phenylalanine, Valine, Methionine, Serine, Tyrosine, Glycine 450 455 460 Threonine, Phenylalanine, Valine, Tryptophan, Phenylalanine, Lysine, Phenylalanine, Aspartic acid, Alanine, Leucine, Lysine, Proline, Leucine, Phenylalanine, Aspartic acid, Leucine 465 470 475 480 Aspartic acid, Leucine, Threonine, Valine, Aspartic acid, Aspartic acid, Isoleucine, Proline, Lysine, Glutamic acid, Proline, Leucine, Proline, Glutamine, Asparagine, Serine 485 490 495 Isoleucine, Leucine, Histidine, Alanine, Isoleucine, Glutamic acid, Arginine, Leucine, Leucine, Valine, Tyrosine, Isoleucine, Alanine, Tryptophan, Aspartic acid, Arginine 500 505 510 Phe Tyr Asp Phe Arg Ile Val Lys Asn Pro Tyr Asn Leu Ser Pro Phe 515 520 525 Ile Asp Asn Lys Leu Leu Asn Leu Arg Glu Ser Gly Gly Ala Arg Thr 530 535 540 Tyr Val Asn Phe Asp His Met Gly Gly Ile Lys Gly Ala Leu Lys Tyr 545 550 555 560 Ile Ile Ile Gly Pro Ala Arg Ala Met Lys Tyr Ile Val Lys Arg Val 565 570 575 Leu Lys Ser Lys Arg 580 <210> 10 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 10 Met Asn Arg Val Leu Leu Tyr Val His Phe Asn Lys Tyr Asn Lys Val 1 5 10 15 Ser Lys His Ile Tyr Tyr Gln Leu Glu Lys Leu Arg Pro Leu Phe Thr 20 25 30 Thr Val Val Phe Ile Ser Asn Ser Lys Val Glu Gln Lys Glu Leu Glu 35 40 45 Asn Leu Gln Lys Gln Arg Leu Ile Asp Ser Phe Ile Gln Arg Glu Asn 50 55 60 Lys Gly Phe Asp Phe Ala Ala Trp His Asp Gly Met Met Lys Ile Gly 65 70 75 80 Phe Asp Asp Leu Thr Leu Cys Asp Ser Leu Thr Ile Met Asn Asp Thr 85 90 95 Cys Phe Gly Pro Leu Trp Gly Met Ala Pro Tyr Phe Glu Lys Phe Asp 100 105 110 Asn Asn Gln Ser Val Asp Phe Trp Gly Leu Thr Asn Asn Arg Lys Thr 115 120 125 Ser Ser Phe Lys Glu His Ile Gln Ser Tyr Phe Ile Thr Phe Lys Gln 130 135 140 His Val Ile Gln Ser Asp Ala Phe Leu Asn Phe Trp Lys Thr Ile Lys 145 150 155 160 Glu Tyr Asp Asp Val Gln Glu Val Ile Gln Lys Tyr Glu Thr Gln Val 165 170 175 Thr Thr Thr Leu Leu Glu Ala Gly Phe Asn Tyr Gln Thr Val Phe Asp 180 185 190 Thr Arg Glu Ala Asp Ser Ser Phe Met Leu His Pro Asp Phe Ser Tyr 195 200 205 Tyr Asn Pro Thr Ala Ile Leu Gln His Arg Val Pro Phe Ile Lys Val 210 215 220 Lys Ala Ile Asp Ala Asn Gln His Ile Thr Pro Tyr Leu Leu Asn Met 225 230 235 240 Ile Glu Glu Glu Thr Thr Tyr Pro Val Asp Leu Ile Ile Ser His Met 245 250 255 Ser Gln Val Gly Leu Pro Asp Ala Lys Tyr Leu Leu Ala Arg Lys Tyr 260 265 270 Leu Pro Phe Glu Ser Leu Val Thr Gln Asn Val Pro Arg Ile Ala Val 275 280 285 His Leu His Val Phe Tyr Val Asp Leu Leu Asn Glu Phe Leu Glu Gly 290 295 300 Phe Ala Ser Trp Glu Phe Gln Tyr Asp Leu Tyr Ile Thr Thr Asp Thr 305 310 315 320 Gln Glu Lys Arg Lys Gln Leu Lys Asn Tyr 325 330 <210> 11 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 11 Met Gly Val Ser Val Arg Pro Leu Tyr Tyr Asn Arg Tyr Ser Arg Lys 1 5 10 15 Lys Glu Ala Ile Glu Lys Leu Leu Val Gln Ser Asn Arg His Ala His 20 25 30 Leu Tyr Val Thr Gly Asn Val Gly Arg Asp Val Leu Pro Met Leu Leu 35 40 45 Leu Lys Asp Lys Leu Arg Asp Tyr Asp Tyr Ile Gly His Phe His Thr 50 55 60 Lys Lys Ser Lys Glu Ala Asp Phe Trp Ala Gly Glu Ser Trp Arg Lys 65 70 75 80 Glu Leu Ile Asn Met Leu Ile Lys Pro Ala Asn Glu Ile Val Arg Ser 85 90 95 Phe Glu Asn Asn Asp Ile Gly Ile Val Ile Ala Asp Ile Pro Ser Phe 100 105 110 Phe Arg Phe Asn Lys Ile Val Asp Ala Trp Asn Glu His Leu Ile Ala 115 120 125 Pro Glu Met Met Arg Leu Trp Lys Glu Met Gly Leu Lys Lys Glu Ile 130 135 140 Asp Phe Gln Ser Met Asp Thr Phe Val Met Ser Tyr Gly Thr Phe Val 145 150 155 160 Trp Phe Lys Phe Asp Ala Leu Lys Pro Leu Phe Asp Leu Asp Leu Thr 165 170 175 Val Asp Asp Ile Pro Lys Glu Pro Leu Pro Gln Asn Ser Ile Leu His 180 185 190 Ala Ile Glu Arg Leu Leu Val Tyr Ile Ala Trp Asp Arg Phe Tyr Asp 195 200 205 Phe Arg Ile Val Lys Asn Pro Tyr Asn Leu Ser Pro Phe Ile Asp Asn 210 215 220 Lys Leu Leu Asn Leu Arg Glu Ser Gly Gly Ala Arg Thr Tyr Val Asn 225 230 235 240 Phe Asp His Met Gly Gly Ile Lys Gly Ala Leu Lys Tyr Ile Ile Ile 245 250 255 Gly Pro Ala Arg Ala Met Lys Tyr Ile Val Lys Arg Val Leu Lys Ser 260 265 270 Lys Arg <210> 12 <211> 408 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 12 Met Ile Gly Lys Ile Ile Arg Ser Tyr Gln Asp Glu Gly Gly Arg Ala 1 5 10 15 Thr Leu Arg Lys Ile Arg Gln Arg Leu Gln Gly Gly Gly His Pro Gln 20 25 30 Ser Ala Gly Lys Ile Asp Leu Asn Arg Ile Pro Ile Met Pro Gln Leu 35 40 45 Glu Asp Ile Ala Gln Ala Asp Tyr Ile Asn His Pro Tyr Gln Arg Pro 50 55 60 Ala Lys Leu Asp Lys Lys Gln Leu Asn Ile Ala Trp Val Ser Pro Pro 65 70 75 80 Val Gly Lys Gly Gly Gly Gly His Thr Thr Ile Ser Arg Phe Val Lys 85 90 95 Tyr Leu Gln Ser Gln Gly His His Ile Thr Phe Tyr Ile Tyr His Asn 100 105 110 Asn Thr Ile Glu Gln Ser Ala Lys Glu Ala Gln Glu Ile Phe Ser Lys 115 120 125 Ala Tyr Gly Ile Glu Val Ala Val Asp Asp Leu Lys Asn Phe Ser Asn 130 135 140 Gln Asp Leu Val Phe Ala Thr Ser Trp Glu Thr Ala Tyr Ala Val Phe 145 150 155 160 Asn Leu Lys Ser Glu Asn Leu His Lys Phe Tyr Phe Val Gln Asp Phe 165 170 175 Glu Pro Ile Phe Tyr Gly Val Gly Ser Arg Tyr Lys Leu Ala Glu Ala 180 185 190 Thr Tyr Lys Phe Gly Phe Tyr Gly Ile Thr Ala Gly Lys Trp Leu Thr 195 200 205 His Lys Leu Lys Asp Tyr His Met Asp Ala Asp Tyr Phe Asn Phe Gly 210 215 220 Ala Asp Thr Asp Ile Tyr Lys Pro Lys Ala Pro Leu Gln Lys Lys Lys 225 230 235 240 Lys Ile Ala Phe Tyr Ala Arg Ala His Thr Glu Arg Arg Gly Phe Glu 245 250 255 Leu Gly Val Met Ala Leu Lys Ile Phe Lys Asp Lys His Pro Glu Tyr 260 265 270 Asp Ile Glu Phe Phe Gly Gln Asp Met Ser His Tyr Asp Ile Pro Phe 275 280 285 Asp Phe Ile Asp Arg Gly Ile Leu Asn Lys Glu Glu Leu Ala Ala Ile 290 295 300 Tyr His Glu Ser Val Ala Cys Leu Val Leu Ser Leu Thr Asn Val Ser 305 310 315 320 Leu Leu Pro Leu Glu Leu Leu Val Ala Gly Cys Ile Pro Val Met Asn 325 330 335 Ser Gly Asp Asn Asn Thr Met Val Leu Gly Glu Asn Asp Asp Ile Ala 340 345 350 Tyr Ala Glu Ala Tyr Pro Val Ala Leu Ala Glu Glu Leu Cys Lys Ala 355 360 365 Val Glu Arg Ser Asp Ile Asp Thr Tyr Ala Asn Glu Met Ser Gln Lys 370 375 380 Tyr Asp Gly Val Ser Trp Glu Asn Ser Tyr Arg Lys Val Glu Glu Ile 385 390 395 400 Ile Arg Arg Glu Val Ile Asn Asp 405 <210> 13 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 13 Met Thr Asp Lys Ile Lys Ala Thr Val Phe Ile Pro Val Tyr Asn Gly 1 5 10 15 Glu Asn Asp His Leu Glu Glu Thr Leu Thr Ala Leu Tyr Thr Gln Lys 20 25 30 Thr Asp Phe Ser Trp Asn Val Met Ile Thr Asp Ser Glu Ser Lys Asp 35 40 45 Arg Ser Val Ala Ile Ile Glu Thr Phe Ala Glu Arg Tyr Gly Asn Leu 50 55 60 Gln Leu Ile Lys Leu Lys Lys Ser Asp Tyr Ser His Gly Ala Thr Arg 65 70 75 80 Gln Met Ala Ala Glu Leu Ser Ser Ala Glu Tyr Met Val Tyr Leu Ser 85 90 95 Gln Asp Ala Val Pro Ala Asn Glu His Trp Leu Ala Glu Met Leu Lys 100 105 110 Pro Phe Thr Ile His His Asp Ile Val Ala Val Leu Gly Lys Gln Lys 115 120 125 Pro Arg Ile Gly Cys Phe Pro Ala Met Lys Tyr Asp Ile Asn Ala Val 130 135 140 Phe Asn Glu Gln Gly Val Ala Gly Ala Ile Thr Leu Trp Thr Arg Gln 145 150 155 160 Glu Glu Ser Leu Lys Gly Lys Tyr Thr Lys Glu Ser Phe Tyr Ser Asp 165 170 175 Val Cys Ser Ala Ala Pro Arg Asp Phe Leu Val Asn Glu Ile Gly Tyr 180 185 190 Arg Ser Val Pro Tyr Ser Glu Asp Tyr Glu Tyr Gly Lys Asp Ile Leu 195 200 205 Asp Ala Gly Tyr Met Lys Ala Tyr Asn Ser Asp Ala Ile Val Glu His 210 215 220 Ser Asn Asp Val Leu Leu Ser Glu Tyr Lys Gln Arg Ile Phe Asp Glu 225 230 235 240 Thr Tyr Asn Val Arg Arg Asn Ser Gly Val Thr Thr Pro Ile Ser Val 245 250 255 Ser Thr Val Leu Ile Gln Phe Leu Lys Ser Ser Val Lys Asp Ala Met 260 265 270 Lys Ile Val Ser Asp Gln Asp Tyr Ser Trp Lys Arg Lys Leu Tyr Trp 275 280 285 Leu Ala Val Asn Pro Leu Phe His Phe Glu Lys Trp Arg Gly Met Arg 290 295 300 Leu Ala Asn Ser Val Asp Met Thr Lys Asp Asn Ser Lys His Ser Leu 305 310 315 320 Glu Asn Ser Lys Ser Lys Gly 325 <210> 14 <211> 585 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 14 Met Lys Arg Leu Leu Leu Tyr Val His Phe Asn Lys Tyr Asn Arg Leu 1 5 10 15 Ser Pro His Val Leu Tyr Gln Leu Lys Lys Met Arg Pro Leu Phe Ser 20 25 30 Asn Leu Ile Phe Ile Ser Asn Ser Ser Leu Asn Asp Ser Asp Arg Gln 35 40 45 Glu Leu Leu Ser Ser Gly Leu Val Asn Glu Val Ile Gln Arg Gln Asn 50 55 60 Ile Gly Phe Asp Phe Ala Ala Trp Arg Asp Gly Met Ala Thr Val Gly 65 70 75 80 Phe Glu Ser Leu Ser Glu Tyr Asp Asn Val Thr Ile Met Asn Asp Thr 85 90 95 Cys Phe Gly Pro Leu Trp Asp Met Lys Pro Tyr Phe Leu Thr Tyr Glu 100 105 110 Asp Asp Glu Glu Val Asp Phe Trp Gly Leu Thr Asn Asn Arg Gln Thr 115 120 125 Lys Glu Phe Asp Glu His Ile Gln Ser Tyr Phe Ile Ser Phe Lys Lys 130 135 140 Thr Val Leu Ser Asn Glu Thr Phe Leu His Phe Trp Arg Thr Val Gln 145 150 155 160 Asp Phe Thr Asp Val Gln Asp Val Ile Lys Asn Tyr Glu Thr Gln Val 165 170 175 Thr Thr Gly Leu Leu Lys Glu Gly Phe Arg Tyr Lys Cys Ile Phe Asn 180 185 190 Thr Val Thr Ala Asp Ala Ser Gly Met Leu His Ala Asp Phe Ser Tyr 195 200 205 Tyr Asn Pro Thr Ala Ile Leu Lys His Gln Val Pro Phe Ile Lys Val 210 215 220 Lys Thr Ile Asp Ala Asn Gln Ser Ile Ala Pro Tyr Leu Leu Gln Val 225 230 235 240 Ile Lys Asn Gln Thr Asp Tyr Pro Val Asp Leu Ile Val Ser His Met 245 250 255 Ser Asp Ile His Tyr Pro Asp Ala Pro Tyr Leu Leu Ser Gln Lys Tyr 260 265 270 Leu Glu Lys Gln Glu Glu Ser Asp Leu Lys Val Ser Glu His Ser Ile 275 280 285 Ala Val His Leu His Val Phe Tyr Val Asp Leu Leu Glu Glu Phe Leu 290 295 300 His Ala Phe Thr Ser Phe Lys Phe Pro Phe Asp Leu Tyr Ile Thr Thr 305 310 315 320 Asp Lys Ser Glu Lys Glu Ser Glu Ile Lys Ala Ile Leu Asp Ser Phe 325 330 335 Arg Val Ser Ala Lys Ile Val Val Thr Gly Asn Ile Gly Arg Asp Val 340 345 350 Leu Pro Met Leu Lys Leu Lys Asp Glu Leu Ser Gln Tyr Asp Tyr Ile 355 360 365 Gly His Phe His Thr Lys Lys Ser Lys Glu Ala Asp Phe Trp Ala Gly 370 375 380 Glu Ser Trp Arg Asn Glu Leu Ile Asp Met Leu Ile Lys Pro Ala Asn 385 390 395 400 Thr Ile Ile Asn Gln Phe Glu Asp Pro Ala Ile Gly Ile Ile Ile Ala 405 410 415 Asp Ile Pro Ser Phe Phe Arg Phe Asn Lys Ile Val Thr Pro Leu Asn 420 425 430 Glu His Leu Ile Ala Pro Glu Met Asn Lys Leu Trp Glu Lys Met Asn 435 440 445 Leu Ser Lys Thr Ile Asp Phe Glu Gln Phe Asp Thr Phe Val Met Ser 450 455 460 Tyr Gly Thr Phe Val Trp Phe Lys Tyr Asp Ala Leu Lys Pro Leu Phe 465 470 475 480 Asp Leu Asn Leu Lys Asp Gly Asp Val Pro Lys Glu Pro Leu Pro Gln 485 490 495 Asn Ser Ile Leu His Ala Val Glu Arg Leu Leu Ile Tyr Ile Ala Trp 500 505 510 Asp Ser His Phe Asp Phe Arg Ile Ala Lys Asn Asn Val Glu Leu Thr 515 520 525 Pro Phe Leu Asp Asn Lys Leu Leu Asn Asp Lys Ser Asn Ser Leu Pro 530 535 540 Asn Thr Tyr Val Asp Phe Thr Tyr Met Gly Gly Ile Lys Gly Ala Leu 545 550 555 560 Lys Tyr Ile Phe Ile Gly Pro Ala Arg Ala Ile Lys Tyr Ile Tyr Ile 565 570 575 Arg Thr Lys Glu Lys Ile Phe Asn Gly 580 585 <210> 15 <211> 583 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 15 Met Lys Arg Leu Leu Leu Tyr Val His Phe Asn Lys Tyr Asn Arg Val 1 5 10 15 Ser Ser His Val Val Tyr Gln Leu Thr Gln Met Arg Ser Leu Phe Ser 20 25 30 Lys Val Ile Phe Ile Ser Asn Ser Gln Val Ala Asp Ala Asp Val Lys 35 40 45 Met Leu Arg Glu Lys His Leu Ile Asp Asp Phe Ile Gln Arg Gln Asn 50 55 60 Ser Gly Phe Asp Phe Ala Ala Trp Arg Asp Gly Met Val Phe Val Gly 65 70 75 80 Phe Asp Glu Leu Val Thr Tyr Asp Ser Val Thr Thr Met Asn Asp Thr 85 90 95 Cys Phe Gly Pro Leu Trp Glu Met Tyr Ser Ile Tyr Gln Glu Phe Glu 100 105 110 Thr Lys Thr Thr Val Asp Phe Trp Gly Leu Thr Asn Asn Arg Ala Thr 115 120 125 Lys Ser Phe Arg Glu His Ile Gln Ser Tyr Phe Ile Ser Phe Lys Ala 130 135 140 Ser Val Leu Arg Ser Thr Ala Phe Arg Asp Phe Trp Glu Asn Ile Lys 145 150 155 160 Glu Tyr Gln Asp Val Gln Lys Val Ile Asp Gln Tyr Glu Thr Lys Val 165 170 175 Thr Thr Thr Leu Leu Asp Ala Gly Phe Gln Tyr Asp Val Val Phe Asp 180 185 190 Thr Thr Lys Glu Asp Ala Ser His Met Leu His Ala Asp Phe Ser Tyr 195 200 205 Tyr Asn Pro Thr Ala Ile Leu Asn His Arg Val Pro Phe Ile Lys Val 210 215 220 Lys Ala Ile Asp Asn Asn Gln His Ile Thr Pro Tyr Leu Leu Asn Asp 225 230 235 240 Ile Gln Lys Asn Ser Thr Tyr Pro Ile Asp Leu Ile Val Ser His Met 245 250 255 Ser Glu Ile Asn Tyr Pro Asp Phe Ser Tyr Leu Leu Gly His Lys Tyr 260 265 270 Val Lys Lys Arg Glu Arg Val Asp Leu Lys Asn Gln Lys Val Ala Val 275 280 285 His Leu His Val Phe Tyr Val Asp Leu Leu Glu Glu Phe Leu Thr Ala 290 295 300 Phe Lys Gln Phe His Phe Ser Tyr Asp Leu Phe Ile Thr Thr Asp Ser 305 310 315 320 Asp Asp Lys Lys Ala Glu Ile Glu Glu Ile Leu Ser Ala Asn Gly Gln 325 330 335 Glu Ala Gln Val Phe Val Thr Gly Asn Ile Gly Arg Asp Val Leu Pro 340 345 350 Met Leu Lys Leu Lys Asn Tyr Leu Ser Ala Tyr Asp Phe Val Gly His 355 360 365 Phe His Thr Lys Lys Ser Lys Glu Ala Asp Phe Trp Ala Gly Gln Ser 370 375 380 Trp Arg Glu Glu Leu Ile Asp Met Leu Val Lys Pro Ala Asp Asn Ile 385 390 395 400 Leu Ala Gln Leu Gln Gln Asn Pro Lys Ile Gly Leu Val Ile Ala Asp 405 410 415 Met Pro Thr Phe Phe Arg Tyr Asn Lys Ile Val Asp Ala Trp Asn Glu 420 425 430 His Leu Ile Ala Pro Glu Met Asn Thr Leu Trp Gln Lys Met Gly Met 435 440 445 Thr Lys Lys Ile Asp Phe Asn Ala Phe His Thr Phe Val Met Ser Tyr 450 455 460 Gly Thr Phe Val Trp Phe Lys Tyr Asp Ala Leu Lys Pro Leu Phe Asp 465 470 475 480 Leu Asn Leu Thr Asp Asp Asp Val Pro Glu Glu Pro Leu Pro Gln Asn 485 490 495 Ser Ile Leu His Ala Ile Glu Arg Leu Leu Ile Tyr Ile Ala Trp Asn 500 505 510 Glu His Tyr Asp Phe Arg Ile Ser Lys Asn Pro Val Asp Leu Thr Pro 515 520 525 Phe Ile Asp Asn Lys Leu Leu Asn Glu Arg Gly Asn Ser Ala Pro Asn 530 535 540 Thr Phe Val Asp Phe Asn Tyr Met Gly Gly Ile Lys Gly Ala Phe Lys 545 550 555 560 Tyr Ile Phe Ile Gly Pro Ala Arg Ala Val Lys Tyr Ile Leu Lys Arg 565 570 575 Ser Leu Gln Lys Ile Lys Ser 580 <210> 16 <211> 304 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 16 Met Leu Glu Asn Thr Lys Ile Leu Arg Lys Val Phe Tyr Leu Trp Gln 1 5 10 15 Lys Gly Glu Leu Met Ile Leu Ile Thr Gly Ser Asn Gly Gln Leu Gly 20 25 30 Thr Glu Leu Arg Tyr Leu Leu Asp Glu Arg Gly Val Asp Tyr Val Ala 35 40 45 Val Asp Val Ala Glu Met Asp Ile Thr Asn Glu Asp Lys Val Glu Ala 50 55 60 Val Phe Ala Gln Val Lys Pro Thr Leu Val Tyr His Cys Ala Ala Tyr 65 70 75 80 Thr Ala Val Asp Ala Ala Glu Asp Glu Gly Lys Ala Leu Asn Glu Ala 85 90 95 Ile Asn Val Thr Gly Ser Glu Asn Ile Ala Lys Ala Cys Gly Lys Tyr 100 105 110 Gly Ala Thr Leu Val Tyr Ile Ser Thr Asp Tyr Val Phe Asp Gly Asn 115 120 125 Lys Pro Val Gly Gln Glu Trp Val Glu Thr Asp His Pro Asp Pro Lys 130 135 140 Thr Glu Tyr Gly Arg Thr Lys Arg Leu Gly Glu Leu Ala Val Glu Arg 145 150 155 160 Tyr Ala Glu His Phe Tyr Ile Ile Arg Thr Ala Trp Val Phe Gly Asn 165 170 175 Tyr Gly Lys Asn Phe Val Phe Thr Met Glu Gln Leu Ala Glu Asn His 180 185 190 Ser Arg Leu Thr Val Val Asn Asp Gln His Gly Arg Pro Thr Trp Thr 195 200 205 Arg Thr Leu Ala Glu Phe Met Cys Tyr Leu Thr Glu Asn Gln Lys Ala 210 215 220 Phe Gly Tyr Tyr His Leu Ser Asn Asp Ala Lys Glu Asp Thr Thr Trp 225 230 235 240 Tyr Asp Phe Ala Lys Glu Ile Leu Lys Asp Lys Ala Val Glu Val Val 245 250 255 Pro Val Asp Ser Ser Ala Phe Pro Ala Lys Ala Lys Arg Pro Leu Asn 260 265 270 Ser Thr Met Asn Leu Asp Lys Ala Lys Ala Thr Gly Phe Val Ile Pro 275 280 285 Thr Trp Gln Glu Ala Leu Lys Ala Phe Tyr Gln Gln Gly Leu Lys Lys 290 295 300 <210> 17 <211> 824 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 17 Met Ile Lys Asp Thr Phe Leu Lys Thr Asn Trp Leu Asn Ile Ser His 1 5 10 15 His Ile Ile Leu Leu Val Phe Gly Phe Tyr Phe Ser Phe Tyr Ser Leu 20 25 30 Ala Lys Glu Leu Val Ser Ser Thr Ala Gln Pro Val Asn Tyr Tyr Ala 35 40 45 His Leu Leu Asn Val Ser Phe Val Gly Tyr Ile Ile Ser Leu Ile Gly 50 55 60 Leu Ser Tyr Tyr Leu Ser Arg Gln Val Ser Arg Gln Leu Phe Leu Lys 65 70 75 80 Thr Ser Phe Ile Val Ile Ser Tyr Leu Ile Val Ser Tyr Trp Val Gln 85 90 95 Ile Thr Gln His Leu Asn Asp Lys Arg Phe Asp Ile Trp Ser Leu Thr 100 105 110 Lys Asn Gln Phe Tyr Gln Phe Gln Ala Leu Pro Ser Leu Leu Ile Ile 115 120 125 Leu Val Met Ala Thr Leu Ile Lys Ile Leu Val Ala Tyr Phe Ala Ile 130 135 140 Glu Lys Asp Arg Phe Gly Leu Leu Gly Tyr Gln Gly Asn Thr Phe Ser 145 150 155 160 Val Ala Leu Ile Leu Ala Val Val Pro Ile Asn Asp Ile His Leu Leu 165 170 175 Lys Leu Ile Ser Ser Arg Phe Ser Glu Leu Val Thr Ala Gly Asn Ser 180 185 190 Gln Ile Ala Leu Leu Lys Ile Ser Gly Leu Leu Ile Val Leu Leu Val 195 200 205 Ile Phe Ala Thr Ile Ile Tyr Val Val Leu Asn Ala Leu Lys His Leu 210 215 220 Lys Ser Asn Lys Pro Ser Phe Ser Val Ala Ala Thr Thr Ser Leu Phe 225 230 235 240 Leu Ala Leu Val Phe Asn Tyr Thr Phe Gln Tyr Gly Val Lys Gly Asp 245 250 255 Glu Ala Leu Leu Gly Tyr Tyr Val Phe Pro Gly Ala Thr Leu Phe Gln 260 265 270 Ile Val Ala Ile Thr Leu Val Ala Leu Leu Ala Tyr Val Ile Thr Asn 275 280 285 Arg Tyr Trp Pro Thr Thr Phe Phe Leu Leu Ile Leu Gly Thr Ile Ile 290 295 300 Ser Val Val Asn Asp Leu Lys Glu Ser Met Arg Ser Glu Pro Leu Leu 305 310 315 320 Val Thr Asp Phe Val Trp Leu Gln Glu Leu Gly Leu Val Thr Ser Phe 325 330 335 Val Lys Lys Ser Val Ile Val Glu Met Val Val Gly Leu Ala Ile Cys 340 345 350 Ile Val Val Ala Trp Tyr Leu His Gly Arg Val Leu Ala Gly Lys Leu 355 360 365 Phe Met Ser Pro Val Lys Arg Ala Ser Ala Val Leu Gly Leu Phe Ile 370 375 380 Val Ser Cys Ser Met Leu Ile Pro Phe Ser Tyr Glu Lys Glu Gly Lys 385 390 395 400 Ile Leu Ser Gly Leu Pro Ile Ile Ser Ala Leu Asn Asn Asp Asn Asp 405 410 415 Ile Asn Trp Leu Gly Phe Ser Thr Asn Ala Arg Tyr Lys Ser Leu Ala 420 425 430 Tyr Val Trp Thr Arg Gln Val Thr Lys Lys Ile Met Glu Lys Pro Thr 435 440 445 Asn Tyr Ser Gln Glu Thr Ile Ala Ser Ile Ala Gln Lys Tyr Gln Lys 450 455 460 Leu Ala Glu Asp Ile Asn Lys Asp Arg Lys Asn Asn Ile Ala Asp Gln 465 470 475 480 Thr Val Ile Tyr Leu Leu Ser Glu Ser Leu Ser Asp Pro Asp Arg Val 485 490 495 Ser Asn Val Thr Val Ser His Asp Val Leu Pro Asn Ile Lys Ala Ile 500 505 510 Lys Asn Ser Thr Thr Ala Gly Leu Met Gln Ser Asp Ser Tyr Gly Gly 515 520 525 Gly Thr Ala Asn Met Glu Phe Gln Thr Leu Thr Ser Leu Pro Phe Tyr 530 535 540 Asn Phe Ser Ser Ser Val Ser Val Leu Tyr Ser Glu Val Phe Pro Lys 545 550 555 560 Met Ala Lys Pro His Thr Ile Ser Glu Phe Tyr Gln Gly Lys Asn Arg 565 570 575 Ile Ala Met His Pro Ala Ser Ala Asn Asn Phe Asn Arg Lys Thr Val 580 585 590 Tyr Ser Asn Leu Gly Phe Ser Lys Phe Leu Ala Leu Ser Gly Ser Lys 595 600 605 Asp Lys Phe Lys Asn Ile Glu Asn Val Gly Leu Leu Thr Ser Asp Lys 610 615 620 Thr Val Tyr Asn Asn Ile Leu Ser Leu Ile Asn Pro Ser Glu Ser Gln 625 630 635 640 Phe Phe Ser Val Ile Thr Met Gln Asn His Ile Pro Trp Ser Ser Asp 645 650 655 Tyr Pro Glu Glu Ile Val Ala Glu Gly Lys Asn Phe Thr Glu Glu Glu 660 665 670 Asn His Asn Leu Thr Ser Tyr Ala Arg Leu Leu Ser Phe Thr Asp Lys 675 680 685 Glu Thr Arg Ala Phe Leu Glu Lys Leu Thr Gln Ile Asn Lys Pro Ile 690 695 700 Thr Val Val Phe Tyr Gly Asp His Leu Pro Gly Leu Tyr Pro Asp Ser 705 710 715 720 Ala Phe Asn Lys His Ile Glu Asn Lys Tyr Leu Thr Asp Tyr Phe Ile 725 730 735 Trp Ser Asn Gly Thr Asn Glu Lys Lys Asn His Pro Leu Ile Asn Ser 740 745 750 Ser Asp Phe Thr Ala Ala Leu Phe Glu His Thr Asp Ser Lys Val Ser 755 760 765 Pro Tyr Tyr Ala Leu Leu Thr Glu Val Leu Asn Lys Ala Ser Val Asp 770 775 780 Lys Ser Pro Asp Ser Pro Glu Val Lys Ala Ile Gln Asn Asp Leu Lys 785 790 795 800 Asn Ile Gln Tyr Asp Val Thr Ile Gly Lys Gly Tyr Leu Leu Lys His 805 810 815 Lys Thr Phe Phe Lys Ile Ser Arg 820 <210> 18 <211> 284 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 18 Met Ile Leu Ile Thr Gly Ala Asn Gly Gln Leu Gly Ser Glu Leu Arg 1 5 10 15 His Leu Leu Asp Glu Arg Thr Gln Glu Tyr Val Ala Val Asp Val Ala 20 25 30 Glu Met Asp Ile Thr Asn Ala Glu Met Val Asp Lys Val Phe Glu Glu 35 40 45 Val Lys Pro Ser Leu Val Tyr His Cys Ala Ala Tyr Thr Ala Val Asp 50 55 60 Ala Ala Glu Asp Glu Gly Lys Glu Leu Asp Phe Ala Ile Asn Val Thr 65 70 75 80 Gly Thr Glu Asn Val Ala Lys Ala Ala Ala Lys His Asp Ala Thr Leu 85 90 95 Val Tyr Ile Ser Thr Asp Tyr Val Phe Asp Gly Glu Lys Pro Val Gly 100 105 110 Gln Glu Trp Glu Val Asp Asp Leu Pro Asp Pro Lys Thr Glu Tyr Gly 115 120 125 Arg Thr Lys Arg Met Gly Glu Glu Leu Val Glu Lys Tyr Ala Ser Lys 130 135 140 Phe Tyr Thr Ile Arg Thr Ala Trp Val Phe Gly Asn Tyr Gly Lys Asn 145 150 155 160 Phe Val Phe Thr Met Gln Asn Leu Ala Lys Thr His Lys Thr Leu Thr 165 170 175 Val Val Asn Asp Gln His Gly Arg Pro Thr Trp Thr Arg Thr Leu Ala 180 185 190 Glu Phe Met Thr Tyr Leu Ala Glu Asn Gln Lys Asp Phe Gly Tyr Tyr 195 200 205 His Leu Ser Asn Asp Ala Lys Glu Asp Thr Thr Trp Tyr Asp Phe Ala 210 215 220 Val Glu Ile Leu Lys Asp Thr Asp Val Glu Val Lys Pro Val Asp Ser 225 230 235 240 Ser Gln Phe Pro Ala Lys Ala Lys Arg Pro Leu Asn Ser Thr Met Ser 245 250 255 Leu Glu Lys Ala Lys Ala Thr Gly Phe Val Ile Pro Thr Trp Gln Asp 260 265 270 Ala Leu Lys Glu Phe Tyr Lys Gln Glu Val Lys Lys 275 280 <210> 19 <211> 284 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 19 Met Ile Leu Ile Thr Gly Ser Asn Gly Gln Leu Gly Thr Glu Leu Arg 1 5 10 15 Tyr Leu Leu Asp Glu Arg His Val Asp Tyr Val Ala Val Asp Val Ala 20 25 30 Glu Met Asp Ile Thr Asp Ala Asp Lys Val Glu Ala Val Phe Ala Gln 35 40 45 Val Lys Pro Thr Leu Val Tyr His Cys Ala Ala Tyr Thr Ala Val Asp 50 55 60 Ala Ala Glu Asp Glu Gly Lys Ala Leu Asn Glu Ala Ile Asn Val Thr 65 70 75 80 Gly Ser Glu Asn Ile Ala Lys Ala Cys Gly Lys Tyr Gly Ala Thr Leu 85 90 95 Val Tyr Ile Ser Thr Asp Tyr Val Phe Asp Gly Asn Lys Pro Val Gly 100 105 110 Gln Glu Trp Leu Glu Thr Asp Val Pro Asp Pro Gln Thr Glu Tyr Gly 115 120 125 Arg Thr Lys Arg Leu Gly Glu Leu Ala Val Glu Gln Tyr Ala Glu His 130 135 140 Phe Tyr Ile Ile Arg Thr Ala Trp Val Phe Gly Asn Tyr Gly Lys Asn 145 150 155 160 Phe Val Phe Thr Met Gln Gln Leu Ala Glu Lys His Pro Arg Leu Thr 165 170 175 Val Val Asn Asp Gln His Gly Arg Pro Thr Trp Thr Arg Thr Leu Ala 180 185 190 Glu Phe Met Cys Tyr Leu Ala Glu Asn Gln Lys Ala Phe Gly Tyr Tyr 195 200 205 His Leu Ser Asn Asp Ala Lys Glu Asp Thr Thr Trp Tyr Asp Phe Ala 210 215 220 Lys Glu Ile Leu Lys Asp Lys Ala Val Glu Val Val Pro Val Asp Ser 225 230 235 240 Ser Ala Phe Pro Ala Lys Ala Lys Arg Pro Leu Asn Ser Thr Met Asn 245 250 255 Leu Asp Lys Ala Lys Ala Thr Gly Phe Val Ile Pro Thr Trp Gln Glu 260 265 270 Ala Leu Lys Glu Phe Tyr Gln Gln Asp Arg His Gln 275 280 <210> 20 <211> 284 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 20 Met Ile Leu Ile Thr Gly Ser Asn Gly Gln Leu Gly Thr Glu Leu Arg 1 5 10 15 Tyr Leu Leu Asp Glu Arg His Val Asp Tyr Val Ala Val Asp Val Ala 20 25 30 Glu Met Asp Ile Thr Asp Ala Asp Lys Val Glu Ala Val Phe Ala Gln 35 40 45 Val Lys Pro Thr Leu Val Tyr His Cys Ala Ala Tyr Thr Ala Val Asp 50 55 60 Ala Ala Glu Asp Glu Gly Lys Ala Leu Asn Glu Ala Ile Asn Val Thr 65 70 75 80 Gly Ser Glu Asn Ile Ala Lys Ala Cys Gly Lys Tyr Gly Ala Thr Leu 85 90 95 Val Tyr Ile Ser Thr Asp Tyr Val Phe Asp Gly Asn Lys Pro Val Gly 100 105 110 Gln Glu Trp Leu Glu Thr Asp Val Pro Asp Pro Gln Thr Glu Tyr Gly 115 120 125 Arg Thr Lys Arg Leu Gly Glu Leu Ala Val Glu Gln Tyr Ala Glu His 130 135 140 Phe Tyr Ile Ile Arg Thr Ala Trp Val Phe Gly Asn Tyr Gly Lys Asn 145 150 155 160 Phe Val Phe Thr Met Gln Gln Leu Ala Glu Lys His Pro Arg Leu Thr 165 170 175 Val Val Asn Asp Gln His Gly Arg Pro Thr Trp Thr Arg Thr Leu Ala 180 185 190 Glu Phe Met Cys Tyr Leu Ala Glu Asn Gln Lys Ala Phe Gly Tyr Tyr 195 200 205 His Leu Ser Asn Asp Ala Lys Glu Asp Thr Thr Trp Tyr Asp Phe Ala 210 215 220 Lys Glu Ile Leu Lys Asp Lys Ala Ile Glu Val Val Pro Val Asp Ser 225 230 235 240 Ser Ala Phe Pro Ala Lys Ala Lys Arg Pro Leu Asn Ser Thr Met Asn 245 250 255 Leu Asp Lys Ala Lys Ala Thr Gly Phe Val Ile Pro Thr Trp Gln Glu 260 265 270 Ala Leu Lys Glu Phe Tyr Gln Gln Asp Arg His Gln 275 280 <210> 21 <211> 284 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 21 Met Ile Leu Ile Thr Gly Ser Asn Gly Gln Leu Gly Thr Glu Leu Arg 1 5 10 15 His Leu Leu Asn Glu Arg Asn Glu Asp Tyr Val Ala Val Asp Val Ala 20 25 30 Glu Met Asp Ile Thr Lys Ala Glu Lys Val Asp Glu Val Phe Leu Gln 35 40 45 Val Lys Pro Ser Leu Val Tyr His Cys Ala Ala Tyr Thr Ala Val Asp 50 55 60 Ala Ala Glu Asp Glu Gly Lys Glu Leu Asp Tyr Ala Ile Asn Val Thr 65 70 75 80 Gly Thr Glu Asn Ile Ala Lys Ala Cys Glu Lys Tyr Asn Ala Thr Leu 85 90 95 Val Tyr Ile Ser Thr Asp Tyr Val Phe Asp Gly Glu Lys Pro Val Gly 100 105 110 Gln Glu Trp Glu Val Asp Asp Lys Pro Asp Pro Lys Thr Glu Tyr Gly 115 120 125 Arg Thr Lys Arg Leu Gly Glu Glu Ala Val Glu Lys Tyr Val Lys Asn 130 135 140 Phe Tyr Ile Ile Arg Thr Ala Trp Val Phe Gly Asn Tyr Gly Lys Asn 145 150 155 160 Phe Val Phe Thr Met Gln His Leu Ala Lys Ser His Asn Ser Leu Thr 165 170 175 Val Val Asn Asp Gln His Gly Arg Pro Thr Trp Thr Arg Thr Leu Ala 180 185 190 Glu Phe Met Thr Tyr Leu Ala Glu Asn Gln Lys Glu Tyr Gly Tyr Tyr 195 200 205 His Leu Ser Asn Asp Ala Thr Glu Asp Thr Thr Trp Tyr Asp Phe Ala 210 215 220 Leu Glu Ile Leu Lys Asp Thr Asp Val Val Val Lys Pro Val Asp Ser 225 230 235 240 Ser Gln Phe Pro Ala Lys Ala Lys Arg Pro Leu Asn Ser Thr Met Ser 245 250 255 Leu Thr Lys Ala Lys Ala Thr Gly Phe Val Ile Pro Thr Trp Gln Glu 260 265 270 Ala Leu Gln Glu Phe Tyr Lys Gln Asp Val Lys Lys 275 280 <210> 22 <211> 284 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 22 Met Ile Leu Ile Thr Gly Ser Asn Gly Gln Leu Gly Thr Glu Leu Arg 1 5 10 15 Tyr Leu Leu Asp Glu Arg Asn Val Glu Tyr Val Ala Val Asp Val Ala 20 25 30 Glu Met Asp Ile Thr Asn Pro Asp Met Val Asp Glu Val Phe Ala Gln 35 40 45 Val Lys Pro Thr Leu Val Tyr His Cys Ala Ala Tyr Thr Ala Val Asp 50 55 60 Ala Ala Glu Asp Glu Gly Lys Ala Leu Asn Gln Ala Ile Asn Val Asp 65 70 75 80 Gly Thr Val Asn Ile Ala Lys Ala Cys Gln Lys Tyr Asn Ala Thr Leu 85 90 95 Val Tyr Ile Ser Thr Asp Tyr Val Phe Asp Gly Thr Lys Thr Val Gly 100 105 110 Gln Glu Trp Leu Glu Thr Asp Ile Pro Asp Pro Lys Thr Glu Tyr Gly 115 120 125 Arg Thr Lys Arg Leu Gly Glu Glu Ala Val Glu Lys Tyr Val Asp Gln 130 135 140 Phe Tyr Ile Ile Arg Thr Ala Trp Val Phe Gly His Tyr Gly Lys Asn 145 150 155 160 Phe Val Phe Thr Met Gln Asn Leu Ala Lys Thr His Pro Lys Leu Thr 165 170 175 Val Val Asn Asp Gln Tyr Gly Arg Pro Thr Trp Thr Arg Thr Leu Ala 180 185 190 Glu Phe Met Cys His Leu Thr Glu Asn Gln Lys Asp Tyr Gly Tyr Tyr 195 200 205 His Leu Ser Asn Asp Ser Lys Glu Asp Thr Ser Trp Tyr Asp Phe Ala 210 215 220 Lys Glu Ile Leu Lys Asp Thr Asp Val Glu Val Val Pro Val Asp Ser 225 230 235 240 Ser Ala Phe Pro Ala Lys Ala Lys Arg Pro Leu Asn Ser Thr Met Asn 245 250 255 Leu Asp Lys Ala Lys Ala Thr Gly Phe Val Ile Pro Thr Trp Gln Glu 260 265 270 Ala Leu Asn Glu Phe Tyr Lys Gln Glu Val Lys Lys 275 280 <210> 23 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 23 Met Asn Phe Leu Thr Lys Lys Asn Arg Ile Leu Leu Arg Glu Met Val 1 5 10 15 Lys Thr Asp Phe Lys Leu Arg Tyr Gln Gly Ser Ala Ile Gly Tyr Leu 20 25 30 Trp Ser Ile Leu Lys Pro Leu Met Met Phe Thr Ile Met Tyr Leu Val 35 40 45 Phe Ile Arg Phe Leu Arg Leu Gly Gly Asn Ile Pro His Phe Pro Val 50 55 60 Ala Leu Leu Leu Ala Asn Val Ile Trp Ser Phe Phe Ser Glu Ala Thr 65 70 75 80 Ser Met Gly Met Val Ser Ile Val Ser Arg Gly Asp Leu Leu Arg Lys 85 90 95 Leu Asn Phe Ser Lys His Ile Ile Val Phe Ser Ala Ile Leu Gly Ala 100 105 110 Leu Ile Asn Phe Leu Ile Asn Leu Val Val Val Leu Ile Phe Ala Leu 115 120 125 Ile Asn Gly Val Thr Ile Ser Asn Tyr Ala Tyr Phe Ser Phe Phe Leu 130 135 140 Phe Ile Glu Leu Val Val Phe Val Val Gly Ile Ala Leu Leu Leu Ser 145 150 155 160 Thr Val Phe Val Tyr Tyr Arg Asp Leu Ala Gln Val Trp Glu Val Leu 165 170 175 Leu Gln Ala Gly Met Tyr Ala Thr Pro Ile Ile Tyr Pro Ile Thr Phe 180 185 190 Val Leu Glu Gly His Pro Leu Ala Ala Lys Ile Leu Met Leu Asn Pro 195 200 205 Ile Ala Gln Met Ile Gln Asp Phe Arg Tyr Leu Leu Ile Asp Arg Ala 210 215 220 Asn Val Thr Ile Trp Gln Met Ser Thr Asn Trp Phe Tyr Ile Ala Ile 225 230 235 240 Pro Tyr Leu Ile Pro Phe Ile Leu Leu Phe Ile Gly Ile Thr Val Phe 245 250 255 Lys Lys Asn Ala Thr Lys Phe Ala Glu Ile Ile 260 265 <210> 24 <211> 401 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 24 Met Thr Asn Asn Lys Ile Ala Val Lys Val Glu His Val Ser Lys Ser 1 5 10 15 Phe Lys Leu Pro Thr Glu Ala Thr Lys Ser Phe Arg Thr Thr Leu Val 20 25 30 Asn Arg Phe Arg Gly Ile Lys Gly Phe Thr Glu Gln Gln Val Leu Lys 35 40 45 Asp Ile Asn Phe Glu Val His Lys Gly Asp Phe Phe Gly Ile Val Gly 50 55 60 Arg Asn Gly Ser Gly Lys Ser Thr Leu Leu Lys Ile Ile Ser Gln Ile 65 70 75 80 Tyr Val Pro Glu Lys Gly Gln Val Thr Val Asp Gly Lys Met Val Ser 85 90 95 Phe Ile Glu Leu Gly Val Gly Phe Asn Pro Glu Leu Thr Gly Arg Glu 100 105 110 Asn Val Tyr Met Asn Gly Ala Met Leu Gly Phe Thr Lys Glu Glu Ile 115 120 125 Asn Ala Met Tyr Asp Asp Ile Val Asp Phe Ala Glu Leu His Asp Phe 130 135 140 Met Asn Gln Lys Leu Lys Asn Tyr Ser Ser Gly Met Gln Val Arg Leu 145 150 155 160 Ala Phe Ser Val Ala Ile Lys Ala Gln Gly Asp Val Leu Ile Leu Asp 165 170 175 Glu Val Leu Ala Val Gly Asp Glu Ala Phe Gln Arg Lys Cys Asn Asp 180 185 190 Tyr Phe Met Glu Arg Lys Asp Ser Gly Lys Thr Thr Ile Leu Val Thr 195 200 205 His Asp Met Gly Ala Val Lys Lys Tyr Cys Asn Arg Ala Val Leu Ile 210 215 220 Glu Asp Gly Leu Val Lys Ala Tyr Gly Glu Pro Phe Asp Val Ala Asn 225 230 235 240 Gln Tyr Ser Val Asp Asn Thr Glu Thr Lys Glu Glu Leu Gln Asp Ser 245 250 255 Glu Lys Val Ala Ile Ser Asp Ile Val Gln Gln Leu Arg Val Asn Leu 260 265 270 Thr Ser Lys Gln Arg Ile Thr Pro Lys Glu Ile Ile Ser Phe Glu Val 275 280 285 Ser Tyr Glu Val Leu Arg Asp Glu Pro Thr Tyr Ile Ala Phe Ser Leu 290 295 300 Thr Asp Met Asp Arg Asn Ile Trp Val Tyr Asn Asp Asn Ser Arg Asp 305 310 315 320 Gln Leu Val Glu Gly Ile Gly Lys Lys Thr Ile Ser Tyr Gln Cys His 325 330 335 Leu Ser His Leu Asn Asp Ile Lys Leu Lys Leu Glu Val Thr Val Arg 340 345 350 Asp Lys Asp Gly Gln Met Leu Leu Phe Ser Thr Ala Glu Gln Ser Pro 355 360 365 Lys Ile Ile Ile Gln Arg Asp Asp Ile Thr Ser Asp Asp Phe Ser Ala 370 375 380 Leu Asp Ser Ala Ser Gly Leu Tyr Gln Arg Asn Gly Gln Trp Thr Phe 385 390 395 400 Ser <210> 25 <211> 335 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 25 Met His Lys Val Ser Ile Ile Cys Thr Asn Tyr Asn Lys Ala Pro Trp 1 5 10 15 Leucine, Glycine, Glutamic acid, Alanine, Leucine, Aspartic acid, Serine, Phenylalanine, Leucine, Asparagine, Glutamine, Lysine, Threonine, Asparagine, Phenylalanine, Glutamic acid 20 25 30 Valine, Aspartic acid, Isoleucine, Isoleucine, Valine, Isoleucine, Aspartic acid, Aspartic acid, Alanine, Serine, Threonine, Aspartic acid, Glutamic acid, Serine, Lysine, Threonine 35 40 45 Isoleucine, Leucine, Glutamic acid, Aspartic acid, Tyrosine, Glutamine, Threonine, Arginine, Phenylalanine, Proline, Glutamic acid, Lysine, Isoleucine, Threonine, Leucine, Leucine 50 55 60 Phenylalanine, Asparagine, Aspartic acid, Histidine, Asparagine, Leucine, Glycine, Isoleucine, Threonine, Lysine, Threonine, Tryptophan, Isoleucine, Lysine, Alanine, Cysteine 65 70 75 80 Leucine, Tyrosine, Alanine, Lysine, Glycine, Lysine, Tyrosine, Isoleucine, Alanine, Arginine, Cysteine, Aspartic acid, Glycine, Aspartic acid, Aspartic acid, Tyrosine 85 90 95 Tryptophan, Threonine, Aspartic acid, Aspartic acid, Leucine, Lysine, Leucine, Glutamine, Lysine, Glutamine, Valine, Aspartic acid, Alanine, Leucine, Glutamic acid, Alanine 100 105 110 Serine, Lysine, Tyrosine, Serine, Lysine, Tryptophan, Serine, Asparagine, Threonine, Aspartic acid, Phenylalanine, Aspartic acid, Phenylalanine, Valine, Aspartic acid, Asparagine 115 120 125 Lysine, Glycine, Lysine, Valine, Leucine, Histidine, Serine, Asparagine, Valine, Phenylalanine, Glutamic acid, Threonine, Glycine, Tyrosine, Isoleucine, Proline 130 135 140 Phenylalanine, Threonine, Aspartic acid, Threonine, Tyrosine, Glutamic acid, Lysine, Valine, Leucine, Alanine, Leucine, Lysine, Glycine, Methionine, Threonine, Methionine 145 150 155 160 Alanine, Serine, Threonine, Tryptophan, Valine, Valine, Aspartic acid, Alanine, Glutamic acid, Leucine, Methionine, Arginine, Phenylalanine, Valine, Asparagine, Glutamine 165 170 175 Lys Ile Asn Ile Glu Thr Pro Asp Asp Thr Phe Asp Met Gln Leu Glu 180 185 190 Leu Phe Gln Leu Thr Ser Leu Thr Tyr Ile Asn Asp Ser Thr Thr Val 195 200 205 Tyr Arg Met Thr Ser Asn Ser Asp Ser Arg Pro Ala Asp Lys Lys Arg 210 215 220 Met Ile His Arg Ile Lys Gln Leu Leu Gln Thr Gln Val Phe Tyr Leu 225 230 235 240 Ala Lys Tyr Pro Gln Ala Asn Ile Pro Gln Ile Ala Asn Leu Leu Met 245 250 255 Glu Gln Asp Gly Lys Asn Glu Leu Arg Ile His Glu Leu Ser Cys Leu 260 265 270 Ile Asn Asp Leu Arg Gln Glu Leu Asn Glu Lys Thr Glu Gln Gln Lys 275 280 285 Glu Arg Glu Phe Glu Ile Lys Glu Ile Ile Glu Asn Gln Ser Arg Gln 290 295 300 Ile Cys Glu Leu Thr His Gln Tyr Asn Cys Val Ile Asn Ser Arg Arg 305 310 315 320 Trp Lys Tyr Met Ser Lys Leu Ile Asp Phe Ile Arg Arg Lys Lys 325 330 335 <210> 26 <211> 268 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 26 Met Asn Phe Leu Thr Lys Lys Asn Arg Ile Leu Leu Arg Glu Met Val 1 5 10 15 Lys Thr Asp Phe Lys Leu Arg Tyr Gln Gly Ser Phe Ile Gly His Leu 20 25 30 Trp Ser Ile Leu Lys Pro Met Leu Leu Phe Thr Ile Met Tyr Leu Val 35 40 45 Phe Val Arg Phe Leu Lys Phe Asp Asp Gly Thr Pro His Tyr Ala Val 50 55 60 Ser Leu Leu Leu Gly Met Val Thr Trp Asn Phe Phe Thr Glu Ala Thr 65 70 75 80 Asn Met Gly Met Leu Ser Ile Val Ser Arg Gly Asp Leu Leu Arg Lys 85 90 95 Ile Asn Phe Pro Lys Glu Ile Ile Val Ile Ser Ser Val Val Gly Ala 100 105 110 Thr Ile Asn Tyr Phe Ile Asn Ile Leu Val Val Phe Ala Phe Ala Leu 115 120 125 Ile Asn Gly Val Gln Pro Ser Phe Gly Val Phe Ile Leu Ile Pro Leu 130 135 140 Phe Leu Glu Leu Phe Leu Phe Ala Thr Gly Val Ala Phe Ile Leu Ala 145 150 155 160 Thr Leu Phe Val Lys Tyr Arg Asp Met Gly Pro Ile Trp Glu Val Met 165 170 175 Leu Gln Ala Gly Met Tyr Gly Thr Pro Ile Ile Tyr Ser Ile Thr Tyr 180 185 190 Ile Ile Gln Arg Gly His Leu Gly Ile Ala Lys Val Met Met Met Asn 195 200 205 Pro Leu Ala Gln Ile Ile Gln Glu Leu Arg His Phe Ile Val Tyr Ser 210 215 220 Gly Ala Thr Ile Asn Trp Asp Ile Phe Glu Asn Lys Phe Phe Thr Leu 225 230 235 240 Ile Pro Ile Ile Leu Ser Leu Ser Ala Phe Val Ile Gly Tyr Val Ile 245 250 255 Phe Lys Arg Asn Ala Lys Lys Phe Ala Glu Ile Leu 260 265 <210> 27 <211> 388 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 27 Met Ser Glu Lys Lys Val Val Leu Ser Val Asp Ser Val Ser Lys Ser 1 5 10 15 Phe Lys Leu Pro Thr Glu Ala Ser Asn Ser Leu Arg Thr Ser Leu Val 20 25 30 Asn Tyr Phe Lys Gly Ile Lys Gly Tyr Thr Glu Gln His Val Leu Asp 35 40 45 Asp Ile Ser Phe Gln Val Glu Glu Gly Asp Phe Phe Gly Ile Val Gly 50 55 60 Arg Asn Gly Ser Gly Lys Ser Thr Leu Leu Lys Ile Ile Ser Lys Ile 65 70 75 80 Tyr Glu Pro Glu Lys Gly Thr Val Thr Val Asp Gly Lys Leu Val Pro 85 90 95 Phe Ile Glu Leu Gly Val Gly Phe Asn Pro Glu Leu Thr Gly Arg Glu 100 105 110 Asn Val Phe Met Asn Gly Ala Leu Leu Gly Phe Ser Arg Asp Glu Val 115 120 125 Ala Ala Met Tyr Asp Asp Ile Val Ser Phe Ala Glu Leu His Asp Phe 130 135 140 Met Asp Gln Lys Leu Lys Asn Tyr Ser Ser Gly Met Gln Val Arg Leu 145 150 155 160 Ala Phe Ser Ile Ala Ile Lys Ala Lys Gly Asp Ile Leu Ile Leu Asp 165 170 175 Glu Val Leu Ala Val Gly Asp Glu Ala Phe Gln Arg Lys Cys Phe Asp 180 185 190 Tyr Phe Ala Gln Leu Lys Arg Glu His Lys Thr Val Ile Leu Val Thr 195 200 205 His Ser Met Glu Gln Val Gln Arg Phe Cys Asn Lys Ala Met Leu Ile 210 215 220 Asp Lys Gly His His Met Glu Val Gly Thr Pro Leu Glu Ile Ser Gln 225 230 235 240 Ile Tyr Lys Gln Leu Asn Gly Leu Asn Val Ala Lys Glu Ser Ala Lys 245 250 255 Glu Thr Glu Asn Asn Gly Ile Ser Leu Ser Ser Gln Phe Ile Asn His 260 265 270 Lys Asp Asp Thr Leu Thr Phe Thr Phe Asp Val His Phe Glu Gln Thr 275 280 285 Ile Glu Asp Pro Val Leu Thr Phe Thr Ile His Lys Asp Thr Gly Glu 290 295 300 Leu Leu Tyr Arg Trp Val Ser Asp Glu Glu Val Glu Gly Ser Ile Met 305 310 315 320 Ile Lys Asn His Lys Val Ser Ile Asp Phe Ala Ile Gln Asn Ile Phe 325 330 335 Pro Asn Gly Lys Phe Thr Thr Glu Phe Gly Val Lys Ser Arg Asp Arg 340 345 350 Ser Lys Glu Tyr Ala Met Phe Ser Gly Ile Cys Asn Phe Glu Leu Ile 355 360 365 Asn Arg Gly Lys Ser Gly Asn Asn Ile Tyr Trp Lys Pro Glu Thr Thr 370 375 380 Val Lys Leu Ser 385 <210> 28 <211> 427 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 28 Met Arg Met Tyr Gln Gly Lys Arg Phe Leu Leu Thr His Ile Trp Leu 1 5 10 15 Arg Gly Phe Ser Gly Ala Glu Ile Asn Ile Leu Glu Leu Ala Thr Tyr 20 25 30 Leu Lys Glu Ala Gly Ala Gln Val Glu Val Phe Thr Phe Leu Ala Lys 35 40 45 Ser Pro Met Leu Asp Glu Phe Gln Lys Asn Gly Ile Pro Val Ile Asp 50 55 60 Asp Ser Asp Tyr Pro Phe Asp Val Ser Gln Tyr Asp Val Val Cys Ser 65 70 75 80 Ala Gln Asn Ile Ile Pro Pro Ala Met Ile Glu Ala Leu Gly Lys Ser 85 90 95 Gln Glu Lys Leu Pro Lys Phe Ile Phe Phe His Met Ala Ala Leu Pro 100 105 110 Glu His Val Leu Glu Gln Pro Tyr Ile Tyr Gln Leu Glu Lys Lys Ile 115 120 125 Ser Ser Ala Thr Leu Ala Ile Ser Glu Glu Ile Val Asn Lys Asn Leu 130 135 140 Lys Arg Phe Phe Lys Asp Ile Pro Asn Leu His Tyr Tyr Pro Asn Pro 145 150 155 160 Ala Pro Glu Ser Tyr Ala Ala Met Glu His Leu Lys Lys Gln Ser Pro 165 170 175 Glu Arg Ile Leu Val Ile Ser Asn His Pro Pro Gln Glu Val Ile Asp 180 185 190 Met Glu Pro Leu Leu Ala Lys Lys Gly Ile His Val Asp Tyr Phe Gly 195 200 205 Val Trp Ser Asp His Tyr Glu Leu Val Thr Pro Glu Leu Leu Ala Ser 210 215 220 Tyr Asp Cys Val Val Gly Ile Gly Lys Asn Ala Gln Tyr Cys Leu Val 225 230 235 240 Met Gly Lys Pro Ile Tyr Ile Tyr Asp His Phe Lys Gly Pro Gly Tyr 245 250 255 Leu Thr Glu Thr Asn Phe Glu Ala Ala Ala Leu Asn Asn Phe Ser Gly 260 265 270 Arg Gly Phe Glu Glu Gln Glu Lys Thr Ala Glu Glu Leu Val Asp Asp 275 280 285 Leu Leu Glu His Tyr Gln Ser Ala Gln Ala Phe Gln His Asn His Leu 290 295 300 Tyr Asp Tyr Arg Ser Arg Tyr Thr Ile Ser Thr Ile Val Asp His Ile 305 310 315 320 Tyr Lys Ser Ile Asn Ile Ile Pro Lys Ala Ile Ala Pro Leu Glu Gln 325 330 335 Val Asp Val Glu Tyr Ile Lys Ala Ile Thr Leu Phe Ile Arg Thr Arg 340 345 350 Leu Val Arg Leu Glu Asn Asp Val Ala Asn Leu Trp Glu Ala Val His 355 360 365 Arg Tyr Glu Gln Leu Asp Arg Lys Ala Thr Ala Lys Arg Glu Ala Leu 370 375 380 Glu Gln Leu Leu Thr Ala Lys Thr Thr Glu Leu Asn Leu Ile Lys Thr 385 390 395 400 Ser Arg Met Phe Lys Leu Tyr Gln Leu Leu Trp Arg Ile Lys Gly Phe 405 410 415 Phe Phe Arg Lys Glu His Leu Lys Arg Ala Lys 420 425 <210> 29 <211> 269 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 29 Met Asp Phe Phe Ser Arg Lys Asn Arg Ile Leu Leu Lys Glu Leu Ile 1 5 10 15 Lys Thr Asp Phe Lys Leu Arg Tyr Gln Gly Ser Ala Ile Gly Tyr Leu 20 25 30 Trp Ser Ile Leu Lys Pro Leu Met Leu Phe Ala Ile Met Tyr Ile Val 35 40 45 Phe Val Arg Phe Leu Pro Leu Gly Gly Asp Val Pro His Trp Pro Val 50 55 60 Ala Leu Leu Leu Gly Asn Val Ile Trp Thr Phe Phe Gln Glu Thr Thr 65 70 75 80 Met Met Gly Met Val Ser Val Val Thr Arg Gly Asp Leu Leu Arg Lys 85 90 95 Leu Asn Phe Ser Lys Gln Thr Ile Val Phe Ser Ala Val Ser Gly Ala 100 105 110 Ala Ile Asn Phe Gly Ile Asn Val Ile Val Val Leu Ile Phe Ala Leu 115 120 125 Leu Asn Gly Val Thr Phe Thr Phe Arg Trp Asn Leu Phe Leu Leu Ile 130 135 140 Pro Leu Phe Leu Glu Leu Leu Leu Phe Ser Thr Gly Ile Ala Phe Ile 145 150 155 160 Leu Ser Thr Leu Tyr Val Arg Tyr Arg Asp Ile Gly Pro Val Trp Glu 165 170 175 Val Ile Leu Gln Gly Gly Phe Tyr Gly Thr Pro Ile Ile Tyr Ser Leu 180 185 190 Thr Tyr Ile Ala Thr Arg Ser Val Val Gly Ala Lys Leu Leu Leu Leu 195 200 205 Ser Pro Ile Ala Gln Ile Ile Gln Asp Met Arg His Ile Leu Ile Asp 210 215 220 Pro Ala Asn Val Thr Ile Trp Gln Met Ile Asn His Lys Ser Ile Ala 225 230 235 240 Val Ile Pro Tyr Leu Val Pro Ile Phe Val Phe Ile Ile Gly Phe Leu 245 250 255 Val Phe Asn Tyr Asn Ala Lys Lys Phe Ala Glu Ile Ile 260 265 <210> 30 <211> 405 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 30 Met Thr Lys Asn Asn Ile Ala Val Lys Val Asp His Val Ser Lys Tyr 1 5 10 15 Phe Lys Leu Pro Val Glu Ser Thr Gln Ser Leu Arg Thr Ala Leu Val 20 25 30 Asn Arg Phe Lys Gly Ile Lys Gly Tyr Lys Lys Gln His Val Leu Arg 35 40 45 Asp Ile Asp Phe Glu Val Glu Lys Gly Asp Phe Phe Gly Ile Val Gly 50 55 60 Arg Asn Gly Ser Gly Lys Ser Thr Leu Leu Lys Ile Ile Ser Gln Ile 65 70 75 80 Tyr Val Pro Glu Gln Gly Lys Val Thr Val Asp Gly Lys Leu Val Ser 85 90 95 Phe Ile Glu Leu Gly Val Gly Phe Asn Pro Glu Leu Thr Gly Arg Glu 100 105 110 Asn Val Tyr Met Asn Gly Ala Met Leu Gly Phe Thr Thr Glu Glu Val 115 120 125 Asp Thr Met Tyr Gln Asp Ile Val Asp Phe Ala Glu Leu Gln Asp Phe 130 135 140 Met Asn Gln Lys Leu Lys Asn Tyr Ser Ser Gly Met Gln Val Arg Leu 145 150 155 160 Ala Phe Ser Val Ala Ile Lys Ala Gln Gly Asp Val Leu Ile Leu Asp 165 170 175 Glu Val Leu Ala Val Gly Asp Glu Ala Phe Gln Arg Lys Cys Asn Asp 180 185 190 Tyr Phe Leu Glu Arg Lys Asn Ser Gly Lys Thr Thr Ile Leu Val Thr 195 200 205 His Asp Met Ala Ala Val Lys Lys Tyr Cys Asn Lys Ala Val Leu Ile 210 215 220 Asp Asp Gly Leu Ile Lys Ala Ile Gly Glu Pro Phe Asp Val Ala Asn 225 230 235 240 Gln Tyr Ser Leu Asp Asn Thr Asp Gln Ile Val Glu Asp Lys Gln Glu 245 250 255 Glu Glu Ala Ala Val Gln Glu Glu Glu Gln Ile Val Val Asp Asn Leu 260 265 270 Glu Val Lys Leu Leu Ser Ala Asn Arg Met Thr Pro Arg Asp Ser Ile 275 280 285 Arg Phe Glu Ile Ser Tyr Asn Val Leu Ala Asp Val Gly Thr Tyr Ile 290 295 300 Ala Leu Ser Leu Thr Asp Val Asp Arg Asn Ile Trp Ile Tyr Asn Asp 305 310 315 320 Asn Ser Leu Asp Tyr Leu Ser Ser Gly Ser Gly Lys Lys Arg Val Phe 325 330 335 Tyr Glu Cys His Leu Lys Ser Leu Asn Asp Ile Lys Leu Lys Leu Glu 340 345 350 Val Thr Val Arg Asp Lys Gln Gly Gln Met Leu Ala Phe Ser Ser Ala 355 360 365 Thr Asn Thr Pro Ile Ile Ser Ile Asn Arg Asp Asp Leu Glu Gly Asp 370 375 380 Asp Lys Ser Ala Met Asp Ser Ala Ser Gly Leu Ile Gln Arg Asn Gly 385 390 395 400 Gln Trp Gln Phe Ser 405 <210> 31 <211> 465 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 31 Met Val Lys Val Ser Ile Ile Cys Thr Asn Tyr Asn Lys Gly Ser Trp 1 5 10 15 Ile Gly Glu Ala Ile Asp Ser Phe Leu Lys Gln Glu Thr Ser Phe Pro 20 25 30 Tyr Glu Ile Ile Ile Val Asp Asp Ala Ser Thr Asp His Ser Val His 35 40 45 Ile Ile Lys Thr Tyr Gln Lys Gln Tyr Pro Asp Leu Ile Arg Ala Phe 50 55 60 Phe Asn Gln Glu Asn Gln Gly Ile Thr Lys Thr Trp Ser Asp Ile Cys 65 70 75 80 Lys Lys Ala Arg Gly Gln Tyr Ile Ala Arg Cys Asp Gly Asp Asp Tyr 85 90 95 Trp Ile Asp Pro Phe Lys Leu Gln Lys Gln Ile Asp Leu Leu Glu Thr 100 105 110 Ser Pro Glu Ser Lys Trp Ser Asn Thr Asp Phe Asp Met Val Asp Ser 115 120 125 Lys Gly Asn Ile Ile His Lys Asp Val Leu Lys Asn Asn Ile Ile Pro 130 135 140 Phe Met Asp Ser Tyr Glu Lys Met Leu Ala Leu Lys Gly Met Thr Met 145 150 155 160 Ala Ser Thr Trp Leu Val Glu Thr Lys Leu Met Leu Glu Ile Asn Asp 165 170 175 Arg Ile Asn Lys Asp Ala Val Asp Asp Thr Phe Asn Ile Gln Leu Glu 180 185 190 Leu Phe Lys Lys Thr Lys Leu Ala Phe Leu Arg Asp Ser Thr Thr Val 195 200 205 Tyr Arg Met Asp Ala Glu Ser Asp Ser Arg Ser Lys Asp Ser Glu Lys 210 215 220 Leu Ala Gln Arg Phe Asp Arg Leu Leu Glu Thr Gln Leu Glu Tyr Ile 225 230 235 240 Glu Lys Tyr Pro Asp Ser Asp Tyr Lys Lys Val Leu Glu Tyr Leu Leu 245 250 255 Pro Lys His Asn Asp Phe Glu Lys Val Leu Ala Gln Asp Gly Lys Asn 260 265 270 Val Trp Asp Asn Gln Gln Ile Thr Ile Tyr Leu Ala Lys Gly Asp Asp 275 280 285 Gln Glu Phe Ser Glu Glu Asn Cys Phe Gln Phe Pro Leu Gln His Ser 290 295 300 Gly Asn Ile Gln Leu Thr Phe Pro Glu Asn Ile Arg Lys Ile Arg Ile 305 310 315 320 Asp Leu Ser Glu Ile Pro Ser Tyr Tyr Arg Gln Val Ser Leu Val Asn 325 330 335 Thr Thr Val Asn Thr Glu Leu Leu Pro Thr Trp Thr Asn Ala Lys Val 340 345 350 Phe Gly Tyr Ser Tyr Tyr Phe Ile Ala Pro Asp Pro Gln Met Ile Tyr 355 360 365 Asp Leu Thr Ala Gln Glu Gly Gln Asp Phe Lys Leu Thr Tyr Glu Trp 370 375 380 Phe Asn Val Asp Gln Pro Ser Gln Pro Asp Phe Leu Ala Asn His Leu 385 390 395 400 Val Lys Glu Leu Asp Gln Lys Lys Val Glu Leu Lys Met Leu Ser Pro 405 410 415 Tyr Lys Tyr Gln Tyr Gln Lys Ala Val Ala Glu Arg Asp Leu Tyr Leu 420 425 430 Glu Gln Leu Asn Glu Met Val Val Arg Tyr Asn Ser Val Thr His Ser 435 440 445 Arg Arg Trp Thr Ile Pro Thr Lys Ile Ile Asn Leu Phe Arg Arg Lys 450 455 460 Lys 465 <210> 32 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 32 Met Glu Leu Phe Ser Lys Lys Asn Arg Ile Leu Leu Lys Glu Leu Val 1 5 10 15 Lys Thr Asp Phe Lys Leu Arg Tyr Gln Gly Ser Ala Ile Gly Tyr Leu 20 25 30 Trp Ser Ile Leu Lys Pro Leu Leu Met Phe Thr Ile Met Tyr Leu Val 35 40 45 Phe Ile Arg Phe Leu Arg Leu Gly Gly Ser Val Pro His Phe Pro Val 50 55 60 Ala Leu Leu Leu Ala Asn Val Ile Trp Ser Phe Phe Ser Glu Ala Thr 65 70 75 80 Gly Met Gly Met Val Ser Ile Val Thr Arg Gly Asp Leu Leu Arg Lys 85 90 95 Leu Asn Phe Ser Lys His Thr Ile Val Phe Ser Ala Val Leu Gly Ala 100 105 110 Leu Ile Asn Phe Ser Ile Asn Leu Val Val Val Leu Ile Phe Ala Leu 115 120 125 Ile Asn Gly Val Thr Ile Ser Pro Phe Ala Tyr Met Ala Ile Pro Leu 130 135 140 Phe Ile Glu Leu Leu Ile Leu Ala Val Gly Val Ala Leu Leu Leu Ser 145 150 155 160 Thr Leu Phe Val Tyr Tyr Arg Asp Leu Ala Gln Val Trp Glu Val Leu 165 170 175 Met Gln Ala Ala Met Tyr Ala Thr Pro Ile Ile Tyr Pro Ile Thr Phe 180 185 190 Val Ser Asp Lys Asn Pro Leu Ala Ala Lys Ile Leu Met Leu Asn Pro 195 200 205 Leu Ala Gln Met Ile Gln Asp Leu Arg Phe Leu Leu Ile Asp Arg Ala 210 215 220 Asn Ala Thr Ile Trp Gln Met Ser Asn His Trp Tyr Tyr Val Met Ile 225 230 235 240 Pro Tyr Leu Ile Pro Phe Leu Val Leu Ala Leu Gly Ile Leu Val Phe 245 250 255 Asn Lys Asn Ala Lys Lys Phe Ala Glu Ile Ile 260 265 <210> 33 <211> 403 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 33 Met Ser Thr Arg Asp Ile Ala Val Lys Val Glu His Val Ser Lys Ser 1 5 10 15 Phe Lys Leu Pro Thr Glu Ala Thr Lys Ser Phe Arg Thr Thr Leu Val 20 25 30 Asn Arg Phe Arg Gly Ile Lys Gly Tyr Thr Glu Gln Lys Val Leu Lys 35 40 45 Asp Ile Asn Phe Glu Val Lys Lys Gly Asp Phe Phe Gly Ile Val Gly 50 55 60 Arg Asn Gly Ser Gly Lys Ser Thr Leu Leu Lys Ile Ile Ser Gln Ile 65 70 75 80 Tyr Val Pro Glu Lys Gly Thr Val Thr Val Glu Gly Lys Met Val Ser 85 90 95 Phe Ile Glu Leu Gly Val Gly Phe Asn Pro Glu Leu Thr Gly Arg Glu 100 105 110 Asn Val Tyr Met Asn Gly Ala Met Leu Gly Phe Thr Gln Glu Glu Val 115 120 125 Asp Ala Met Tyr Glu Asp Ile Val Asp Phe Ala Glu Leu His Asp Phe 130 135 140 Met Asn Gln Lys Leu Lys Asn Tyr Ser Ser Gly Met Gln Val Arg Leu 145 150 155 160 Ala Phe Ser Val Ala Ile Lys Ala Gln Gly Asp Val Leu Ile Leu Asp 165 170 175 Glu Val Leu Ala Val Gly Asp Glu Ala Phe Gln Arg Lys Cys Asn Asp 180 185 190 Tyr Phe Met Glu Arg Lys Glu Ser Gly Lys Thr Thr Ile Leu Val Thr 195 200 205 His Asp Met Ala Ala Val Lys Lys Tyr Cys Asn Arg Ala Val Leu Ile 210 215 220 Glu Asp Gly Leu Val Lys Ala Leu Gly Asp Pro Asp Asp Val Ala Asn 225 230 235 240 Gln Tyr Ser Phe Asp Asn Ala Ile Ala Ser Glu Thr Val Glu Lys Lys 245 250 255 Glu Asp Gly Lys Ser Thr Glu Lys Lys Glu Ser Gln Leu Ile Ser Asp 260 265 270 Phe Ser Ala Gln Leu Leu Thr Lys Pro Gln Ile Ser Pro Asp Glu Asp 275 280 285 Ile Thr Ile Ser Phe Ser Tyr Asn Val Leu Lys Asn Met Glu Thr His 290 295 300 Val Ala Leu Ser Phe Ile Asp Ile Asp Thr Asn Leu Gly Leu Tyr Asn 305 310 315 320 Asp Asn Ser Met Ser Leu Lys Thr Asn Gly Gln Gly Gln Lys Thr Val 325 330 335 Thr Met Thr Cys Gln Met Ser Tyr Leu Asn His Ala Lys Leu Lys Leu 340 345 350 Ala Ala Thr Val Arg Asp Lys Asp Lys His Pro Leu Ala Phe Leu Pro 355 360 365 Val Asn Glu Ile Pro Val Ile Leu Ile Asp Arg Lys Val Asp Ala Ser 370 375 380 Asn Glu Ser Glu Trp Asp Ala Asn Thr Gly Ile Leu Arg Arg Ser Ser 385 390 395 400 Gln Trp Thr <210> 34 <211> 590 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 34 Met Lys Lys Ile Leu Phe Val Ser Pro Thr Gly Thr Leu Asp Asn Gly 1 5 10 15 Ala Glu Ile Ser Ile Thr Asn Leu Met Val Leu Leu Thr Gln Glu Gly 20 25 30 Tyr Asp Ile Ile Asn Val Ile Pro Lys Ile Lys His Ser Thr His Asp 35 40 45 Ala Tyr Leu His Lys Met Arg Glu Asn Gln Ile Lys Val Tyr Glu Leu 50 55 60 Asp Tyr Thr Asn Trp Trp Trp Glu Ser Ala Pro Gly Asp Lys Ile Gly 65 70 75 80 His Leu Glu Asp Arg Ser Ala Tyr Tyr Gln Lys Tyr Ile Tyr Glu Ile 85 90 95 Arg Lys Ile Ile Ala Glu Glu Ala Val Asp Leu Val Ile Thr Ser Thr 100 105 110 Ala Asn Leu Phe Gln Gly Ala Leu Ala Ala Ala Cys Glu Arg Ile Pro 115 120 125 His Tyr Trp Ile Ile His Glu Phe Pro Leu Asp Glu Phe Ala Tyr Tyr 130 135 140 Lys Glu Leu Ile Pro Phe Ile Glu Glu Tyr Ser Asp Lys Ile Phe Thr 145 150 155 160 Val Glu Gly Lys Leu Thr Glu Phe Leu Arg Pro Leu Leu Lys Glu Ser 165 170 175 Gln Lys Leu Phe Pro Phe Val Pro Phe Val Asn Ile Lys Lys Asn Asn 180 185 190 Asn Leu Lys Thr Gly Glu Glu Thr Arg Leu Ile Ser Ile Ser Arg Ile 195 200 205 Asn Glu Asn Lys Asn Gln Leu Glu Leu Leu Lys Ala Tyr Gln Ser Met 210 215 220 Ala Glu Pro Lys Pro Glu Leu Leu Phe Val Gly Asp Trp Asp Asp Ser 225 230 235 240 Tyr Lys Glu Lys Cys Asp Asp Phe Ile Gln Ser His Gln Leu Lys Thr 245 250 255 Val Arg Phe Leu Gly His Gln Ser Asn Pro Trp Asn Leu Met Thr Asp 260 265 270 Lys Asp Ile Leu Val Leu Asn Ser Lys Met Glu Thr Phe Gly Leu Val 275 280 285 Phe Val Glu Ala Leu Ile Gln Gly Ile Pro Val Leu Ala Ser Asn Asn 290 295 300 Tyr Gly Tyr Ser Ser Val Val Asp Tyr Phe Gly Cys Gly Lys Leu Tyr 305 310 315 320 His Leu Gly Asp Glu Lys Glu Leu Val Ala Leu Leu Asn Glu Phe Val 325 330 335 Thr Asn Phe Ser Glu Glu Lys Lys Lys Ser Leu Thr Gln Ser Phe Met 340 345 350 Val Glu Glu Lys Tyr Thr Ile Glu Lys Ser Tyr Cys Ala Leu Leu Asp 355 360 365 Ala Ile Ser Asn Glu Asn Ser Val Lys Ser Asp Arg Pro Ile Trp Leu 370 375 380 Ser Gln Phe Leu Gly Ala Tyr Asn Pro Leu Ser Thr Phe Ser Pro Ala 385 390 395 400 Gly Lys Glu Ser Ile Ser Ile Tyr Tyr Arg Asp Glu Asn Gly Asn Trp 405 410 415 Ser Glu Asn Gln Lys Leu Val Phe Ser Leu Phe Asn Arg Asp Ser Phe 420 425 430 Thr Phe Ser Val Pro Lys Gly Met Thr Arg Ile Arg Leu Asp Met Ser 435 440 445 Glu Arg Pro Ser Tyr Tyr Asp Lys Ile Thr Leu Val Asp Ser Asp Thr 450 455 460 Met Thr Gln Leu Leu Pro Thr Asn Val Ser Gly Phe Glu Glu Asn Asn 465 470 475 480 Ser Phe Tyr Phe Asn His Ser Asp Pro Gln Met Glu Phe Asn Val Ser 485 490 495 Phe Ser Lys Asn Asn Val Phe Gln Leu Ser Tyr Gln Leu Ala Asn Leu 500 505 510 Glu Asn Ile Phe Gln Asp Ser Phe Leu Pro Asn Gln Leu Val Gln Lys 515 520 525 Leu Leu Ser Phe Lys Glu Lys Gln Ser Asp Leu Glu Met Leu Lys Ile 530 535 540 Glu Asn His Gln Leu Gln Glu Lys Asn Lys Leu Lys Gln Glu Gln Leu 545 550 555 560 Glu Glu Met Val Val Arg Tyr Asn Ser Val Ile His Ser Arg Arg Trp 565 570 575 Ser Ile Pro Thr Lys Met Ile Asn Phe Leu Arg Arg Lys Lys 580 585 590 <210> 35 <211> 846 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 35 Met Lys Gln Leu Lys Lys Ile Trp Asp Met Leu Gly Lys Gln Lys Leu 1 5 10 15 Leu Ile Phe Ile Phe Ile Phe Ala Leu Asn Val Thr Leu Arg Asn Tyr 20 25 30 Asp Leu Leu Ile Gly Arg Arg Ala Asn Ser Ser Leu Ser Phe Lys Val 35 40 45 Ile Ser Lys Asn Phe Asp Ile Met Ile Glu His Trp Glu Ala Leu Pro 50 55 60 Ser His Phe Lys Ile Ile Gly Gly Val Cys Leu Val Ile Tyr Val Leu 65 70 75 80 Ser Ile Leu Gly Leu Ser Phe Tyr Leu Ser Lys Asn Leu Lys Lys Thr 85 90 95 Phe Phe Ile Glu Leu Leu Leu Gly Tyr Gly Leu Tyr Ile Val Ile Ser 100 105 110 Tyr Phe Leu Ala Val Thr Arg Glu Leu Asn Asn Glu Ser Phe Lys Ile 115 120 125 Trp Asp Leu Ala Lys Asn His Phe Phe Gln Pro Tyr Phe Leu Pro Thr 130 135 140 Leu Val Leu Ile Ile Val Cys Thr Leu Ala Leu Asn Tyr Leu Ile Arg 145 150 155 160 Val Lys Met Lys Arg Ser His Leu Ser Arg Lys Met Thr Leu Leu Leu 165 170 175 Glu Asn Phe Ser Glu Thr Glu Phe Leu Leu Thr Gly Leu Ile Val Ser 180 185 190 Phe Ile Leu Ser Asp Thr Leu Tyr Val Lys Leu Leu Gln Glu Ser Leu 195 200 205 Arg Ala Tyr Tyr His Lys Pro Leu Ala Tyr Glu Ser Leu Leu Phe Leu 210 215 220 Tyr Thr Leu Leu Thr Leu Ile Leu Phe Ser Val Ile Val Glu Ala Cys 225 230 235 240 Phe Asn Ala Tyr Arg Ser Ile Lys Leu Asn Arg Pro Asn Leu Ser Leu 245 250 255 Ala Phe Val Ser Ser Leu Leu Phe Ala Thr Ile Phe Asn Tyr Ala Phe 260 265 270 Gln Tyr Gly Leu Lys Asn Asp Ala Asp Leu Leu Gly Lys Tyr Ile Val 275 280 285 Pro Gly Ala Thr Ala Tyr Gln Ile Leu Val Leu Thr Ala Ala Gly Phe 290 295 300 Phe Leu Tyr Leu Ile Ile Asn Arg Tyr Leu Leu Val Thr Phe Leu Ile 305 310 315 320 Val Ile Leu Gly Ser Ile Ile Thr Val Val Asn Val Leu Lys Val Gly 325 330 335 Met Arg Asn Glu Pro Leu Leu Val Thr Asp Phe Ala Trp Val Thr Asn 340 345 350 Ile Arg Leu Leu Ala Arg Ser Val Asn Ala Asn Ile Ile Phe Ser Thr 355 360 365 Leu Leu Ile Leu Ala Ala Leu Ile Leu Leu Tyr Leu Phe Leu Arg Lys 370 375 380 Arg Leu Leu Gln Gly Lys Ile Thr Glu Asn His Arg Leu Lys Val Gly 385 390 395 400 Leu Ile Ser Ser Ile Cys Leu Leu Gly Phe Ser Ile Phe Ile Ile Phe 405 410 415 Arg Asn Glu Lys Gly Ser Lys Ile Val Asn Gly Ile Pro Val Ile Ser 420 425 430 Gln Val Asn Asn Trp Val Asp Ile Gly Tyr Gln Gly Phe Tyr Ser Asn 435 440 445 Ala Ser Tyr Lys Ser Leu Met Tyr Val Trp Thr Lys Gln Val Thr Lys 450 455 460 Ser Ile Met Asp Lys Pro Ser Asp Tyr Ser Lys Glu Arg Ile Leu Lys 465 470 475 480 Leu Ala Lys Lys Tyr Asn Asn Val Ala Asn Lys Ile Asn Lys Val Arg 485 490 495 Thr Glu Asn Ile Ser Asn Gln Thr Val Ile Tyr Ile Leu Ser Glu Ser 500 505 510 Phe Ser Asp Pro Asp Arg Val Lys Gly Val Asn Leu Ser Arg Asp Val 515 520 525 Ile Pro Asn Ile Lys Gln Ile Lys Glu Lys Thr Thr Ser Gly Leu Met 530 535 540 His Ser Asp Gly Tyr Gly Gly Gly Thr Ala Asn Met Glu Phe Gln Ser 545 550 555 560 Leu Thr Gly Leu Pro Tyr Tyr Asn Phe Asn Ser Ser Val Ser Thr Leu 565 570 575 Tyr Thr Glu Val Val Pro Asp Met Ser Val Phe Pro Ser Ile Ser Asn 580 585 590 Gln Phe Lys Ser Lys Asn Arg Val Val Ile His Pro Ser Ser Ala Ser 595 600 605 Asn Tyr Ser Arg Lys Tyr Val Tyr Asp Lys Leu Lys Phe Pro Thr Phe 610 615 620 Val Ala Ser Ser Gly Thr Ser Asp Lys Ile Thr His Ser Glu Lys Val 625 630 635 640 Gly Leu Asn Val Ser Asp Lys Thr Thr Tyr Gln Asn Ile Leu Asp Lys 645 650 655 Ile Asn Pro Ser Gln Ser Gln Phe Phe Ser Val Met Thr Met Gln Asn 660 665 670 His Val Pro Trp Ala Ser Asp Glu Pro Ser Asp Val Val Ala Thr Gly 675 680 685 Lys Gly Tyr Thr Lys Asp Glu Asn Gly Ser Leu Ser Ser Tyr Ala Arg 690 695 700 Leu Leu Thr Tyr Thr Asp Lys Glu Thr Lys Asp Phe Leu Ala Gln Leu 705 710 715 720 Ser Gln Leu Lys His Lys Val Thr Val Val Phe Tyr Gly Asp His Leu 725 730 735 Pro Gly Leu Tyr Pro Glu Ser Ala Phe Lys Lys Asp Pro Asp Ser Gln 740 745 750 Tyr Gln Thr Asp Tyr Phe Ile Trp Ser Asn Tyr Asn Thr Lys Thr Leu 755 760 765 Asn His Ser Tyr Val Asn Ser Ser Asp Phe Thr Ala Glu Leu Leu Glu 770 775 780 His Thr Asn Ser Lys Val Ser Pro Tyr Tyr Ala Leu Leu Thr Glu Val 785 790 795 800 Leu Asp Asn Thr Thr Val Gly His Gly Lys Leu Thr Lys Glu Gln Lys 805 810 815 Glu Ile Ala Asn Asp Leu Lys Leu Ile Gln Tyr Asp Ile Thr Val Gly 820 825 830 Lys Gly Tyr Ile Arg Asn Tyr Lys Gly Phe Phe Asp Ile Arg 835 840 845 <210> 36 <211> 390 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 36 Met Lys Gln Ser Val Tyr Ile Ile Gly Ser Lys Gly Ile Pro Ala Lys 1 5 10 15 Tyr Gly Gly Phe Glu Thr Phe Val Glu Lys Leu Thr Glu Tyr Gln Lys 20 25 30 Asp Gly Asn Ile Gln Tyr Tyr Val Ala Cys Met Arg Glu Asn Ser Ala 35 40 45 Lys Ser Gly Phe Thr Ala Asp Thr Phe Glu Tyr Asn Gly Ala Ile Cys 50 55 60 Tyr Asn Ile Asp Val Pro Asn Ile Gly Pro Ala Arg Ala Ile Ala Tyr 65 70 75 80 Asp Ile Ala Ala Val Asn Lys Ala Ile Glu Leu Ser Lys Gly Asn Lys 85 90 95 Asp Glu Ala Pro Ile Phe Tyr Ile Leu Ala Cys Arg Ile Gly Pro Phe 100 105 110 Ile Ser Gly Leu Lys Lys Lys Ile Arg Ser Ile Gly Gly Arg Leu Leu 115 120 125 Val Asn Pro Asp Gly His Glu Trp Leu Arg Ala Lys Trp Ser Leu Pro 130 135 140 Val Arg Lys Tyr Trp Lys Phe Ser Glu Gln Leu Met Val Lys His Ala 145 150 155 160 Asp Leu Leu Val Cys Asp Ser Lys Asn Ile Glu Lys Tyr Ile Arg Glu 165 170 175 Asp Tyr Lys Gln Tyr Gln Pro Lys Thr Thr Tyr Ile Ala Tyr Gly Thr 180 185 190 Asp Thr Thr Pro Ser Ser Leu Lys Ser Glu Asp Ala Lys Val Arg Asn 195 200 205 Trp Tyr Arg Glu Lys Gly Val Ser Glu Asn Gly Tyr Tyr Leu Val Val 210 215 220 Gly Arg Phe Val Pro Glu Asn Asn Tyr Glu Thr Met Ile Arg Glu Phe 225 230 235 240 Ile Lys Ser Lys Ser Asn Lys Asp Phe Val Leu Ile Thr Asn Val Glu 245 250 255 Gln Asn Lys Phe Tyr Asp Gln Leu Leu Lys Glu Thr Gly Phe Asp Lys 260 265 270 Asp Leu Arg Val Lys Phe Val Gly Thr Val Tyr Asp Gln Glu Leu Leu 275 280 285 Lys Tyr Ile Arg Glu Asn Ala Phe Ala Tyr Phe His Gly His Glu Val 290 295 300 Gly Gly Thr Asn Pro Ser Leu Leu Glu Ala Leu Ala Ser Thr Lys Leu 305 310 315 320 Asn Leu Leu Leu Asp Val Gly Phe Asn Arg Glu Val Gly Glu Asp Gly 325 330 335 Ala Ile Tyr Trp Lys Lys Asp Glu Leu Ala His Val Ile Glu Glu Val 340 345 350 Glu Arg Phe Asp Glu Gly Asp Ile Thr Glu Leu Asp Glu Lys Ser Ser 355 360 365 Gln Arg Ile Ala Asp Ala Phe Thr Trp Glu Lys Ile Val Ser Asp Tyr 370 375 380 Glu Glu Val Phe Thr Val 385 390 <210> 37 <211> 282 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 37 Met Asn Lys Tyr Cys Ile Leu Val Leu Phe Asn Pro Asp Ile Ser Val 1 5 10 15 Phe Ile Asp Asn Val Lys Lys Ile Leu Ser Leu Asp Val Ser Leu Phe 20 25 30 Val Tyr Asp Asn Ser Ala Asn Lys His Ala Phe Leu Ala Leu Ser Ser 35 40 45 Gln Glu Gln Thr Lys Ile Asn Tyr Phe Ser Ile Cys Glu Asn Ile Gly 50 55 60 Leu Ser Lys Ala Tyr Asn Glu Thr Leu Arg His Ile Leu Glu Phe Asn 65 70 75 80 Lys Asn Val Lys Asn Lys Ser Ile Asn Asp Ser Val Leu Phe Leu Asp 85 90 95 Gln Asp Ser Glu Val Asp Leu Asn Ser Ile Asn Ile Leu Phe Glu Thr 100 105 110 Ile Ser Ala Ala Glu Ser Asn Val Met Ile Val Ala Gly Asn Pro Ile 115 120 125 Arg Arg Asp Gly Leu Pro Tyr Ile Asp Tyr Pro His Thr Val Asn Asn 130 135 140 Val Lys Phe Val Ile Ser Ser Tyr Ala Val Tyr Arg Leu Asp Ala Phe 145 150 155 160 Arg Asn Ile Gly Leu Phe Gln Glu Asp Phe Phe Ile Asp His Ile Asp 165 170 175 Ser Asp Phe Cys Ser Arg Leu Ile Lys Ser Asn Tyr Gln Ile Leu Leu 180 185 190 Arg Lys Asp Ala Phe Phe Tyr Gln Pro Ile Gly Ile Lys Pro Phe Asn 195 200 205 Leu Cys Gly Arg Tyr Leu Phe Pro Ile Pro Ser Gln His Arg Thr Tyr 210 215 220 Phe Gln Ile Arg Asn Ala Phe Leu Ser Tyr Arg Arg Asn Gly Val Thr 225 230 235 240 Phe Asn Phe Leu Phe Arg Glu Ile Val Asn Arg Leu Ile Met Ser Ile 245 250 255 Phe Ser Gly Leu Asn Glu Lys Asp Leu Leu Lys Arg Leu His Leu Tyr 260 265 270 Leu Lys Gly Ile Lys Asp Gly Leu Lys Met 275 280 <210> 38 <211> 264 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 38 Met Val Tyr Ile Ile Ile Val Ser His Gly His Glu Asp Tyr Ile Lys 1 5 10 15 Lys Leu Leu Glu Asn Leu Asn Ala Asp Asp Glu His Tyr Lys Ile Ile 20 25 30 Val Arg Asp Asn Lys Asp Ser Leu Leu Leu Lys Gln Ile Cys Gln His 35 40 45 Tyr Ala Gly Leu Asp Tyr Ile Ser Gly Gly Val Tyr Gly Phe Gly His 50 55 60 Asn Asn Asn Ile Ala Val Ala Tyr Val Lys Glu Lys Tyr Arg Pro Ala 65 70 75 80 Asp Asp Asp Tyr Ile Leu Phe Leu Asn Pro Asp Ile Ile Met Lys His 85 90 95 Asp Asp Leu Leu Thr Tyr Ile Lys Tyr Val Glu Ser Lys Arg Tyr Ala 100 105 110 Phe Ser Thr Leu Cys Leu Phe Arg Asp Glu Ala Lys Ser Leu His Asp 115 120 125 Tyr Ser Val Arg Lys Phe Pro Val Leu Ser Asp Phe Ile Val Ser Phe 130 135 140 Met Leu Gly Ile Asn Lys Thr Lys Ile Pro Lys Glu Ser Ile Tyr Ser 145 150 155 160 Asp Thr Val Val Asp Trp Cys Ala Gly Ser Phe Met Leu Val Arg Phe 165 170 175 Ser Asp Phe Val Arg Val Asn Gly Phe Asp Gln Gly Tyr Phe Met Tyr 180 185 190 Cys Glu Asp Ile Asp Leu Cys Leu Arg Leu Ser Leu Ala Gly Val Arg 195 200 205 Leu His Tyr Val Pro Ala Phe His Ala Ile His Tyr Ala His His Asp 210 215 220 Asn Arg Ser Phe Phe Ser Lys Ala Phe Arg Trp His Leu Lys Ser Thr 225 230 235 240 Phe Arg Tyr Leu Ala Arg Lys Arg Ile Leu Ser Asn Arg Asn Phe Asp 245 250 255 Arg Ile Ser Ser Val Phe His Pro 260 <210> 39 <211> 301 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 39 Met Val Ala Val Thr Tyr Ser Pro Gly Pro His Leu Glu Arg Phe Leu 1 5 10 15 Ala Ser Leu Ser Leu Ala Thr Glu Arg Pro Val Ser Val Leu Leu Ala 20 25 30 Asp Asn Gly Ser Thr Asp Gly Thr Pro Gln Ala Ala Val Gln Arg Tyr 35 40 45 Pro Asn Val Arg Leu Leu Pro Thr Gly Ala Asn Leu Gly Tyr Gly Thr 50 55 60 Ala Val Asn Arg Thr Ile Ala Gln Leu Gly Glu Met Ala Gly Asp Ala 65 70 75 80 Gly Glu Pro Trp Gly Asp Asp Trp Val Ile Val Ala Asn Pro Asp Val 85 90 95 Gln Trp Gly Pro Gly Ser Ile Asp Ala Leu Leu Asp Ala Ala Ser Arg 100 105 110 Trp Pro Arg Ala Gly Ala Leu Gly Pro Leu Ile Arg Asp Pro Asp Gly 115 120 125 Ser Val Tyr Pro Ser Ala Arg Gln Met Pro Ser Leu Ile Arg Gly Gly 130 135 140 Met His Ala Val Leu Gly Pro Phe Trp Pro Arg Asn Pro Trp Thr Thr 145 150 155 160 Ala Tyr Arg Gln Glu Arg Leu Glu Pro Ser Glu Arg Pro Val Gly Trp 165 170 175 Leu Ser Gly Ser Cys Leu Leu Val Arg Arg Ser Ala Phe Gly Gln Val 180 185 190 Gly Gly Phe Asp Glu Arg Tyr Phe Met Tyr Met Glu Asp Val Asp Leu 195 200 205 Gly Asp Arg Leu Gly Lys Ala Gly Trp Leu Ser Val Tyr Val Pro Ser 210 215 220 Ala Glu Val Leu His His Lys Ala His Ser Thr Gly Arg Asp Pro Ala 225 230 235 240 Ser His Leu Ala Ala His His Lys Ser Thr Tyr Ile Phe Leu Ala Asp 245 250 255 Arg His Ser Gly Trp Trp Arg Ala Pro Leu Arg Trp Thr Leu Arg Gly 260 265 270 Ser Leu Ala Leu Arg Ser His Leu Met Val Arg Ser Ser Leu Arg Arg 275 280 285 Ser Arg Arg Arg Lys Leu Lys Leu Val Glu Gly Arg His 290 295 300 <210> 40 <211> 296 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 40 Met Asn Ser Asn Ile Tyr Ala Val Ile Val Thr Tyr Asn Pro Glu Leu 1 5 10 15 Lys Asn Leu Asn Ala Leu Ile Thr Glu Leu Lys Glu Gln Asn Cys Tyr 20 25 30 Val Val Val Val Asp Asn Arg Thr Asn Phe Thr Leu Lys Asp Lys Leu 35 40 45 Ala Asp Ile Glu Lys Val His Leu Ile Cys Leu Gly Arg Asn Glu Gly 50 55 60 Ile Ala Lys Ala Gln Asn Ile Gly Ile Arg Tyr Ser Leu Glu Lys Gly 65 70 75 80 Ala Glu Lys Ile Ile Phe Phe Asp Gln Asp Ser Arg Ile Arg Asn Glu 85 90 95 Phe Ile Lys Lys Leu Ser Cys Tyr Met Asp Asn Glu Asn Ala Lys Ile 100 105 110 Ala Gly Pro Val Phe Ile Asp Arg Asp Lys Ser His Tyr Tyr Pro Ile 115 120 125 Cys Asn Ile Lys Lys Asn Gly Leu Arg Glu Lys Ile His Val Thr Glu 130 135 140 Gly Gln Thr Pro Phe Lys Ser Ser Val Thr Ile Ser Ser Gly Thr Met 145 150 155 160 Val Ser Lys Glu Val Phe Glu Ile Val Gly Met Met Asp Glu Glu Leu 165 170 175 Phe Ile Asp Tyr Val Asp Thr Glu Trp Cys Leu Arg Cys Leu Asn Tyr 180 185 190 Gly Ile Leu Val His Ile Ile Pro Asp Ile Glu Met Val His Ala Ile 195 200 205 Gly Asp Lys Ser Val Lys Ile Cys Gly Ile Asn Ile Pro Ile His Ser 210 215 220 Pro Val Arg Arg Tyr Tyr Arg Val Arg Asn Ala Phe Leu Leu Leu Arg 225 230 235 240 Lys Asn His Val Pro Leu Leu Leu Ser Ile Arg Glu Val Val Phe Ser 245 250 255 Leu Ile His Thr Thr Leu Ile Ile Ala Thr Gln Lys Asn Lys Ile Glu 260 265 270 Tyr Met Lys Lys His Ile Leu Ala Thr Leu Asp Gly Ile Arg Gly Ile 275 280 285 Thr Gly Gly Gly Arg Tyr Asn Ala 290 295 <210> 41 <211> 289 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 41 Met Asp Ile Ser Ile Ile Ile Val Asn Tyr Asn Thr Pro Lys Leu Thr 1 5 10 15 Val Glu Ala Ile Glu Ser Ile Leu Lys Ser Lys Thr Lys Tyr Ser Tyr 20 25 30 Glu Ile Ile Val Val Asp Asn His Ser Ser Asp Asp Ser Val Arg Ile 35 40 45 Leu Lys Gly Lys Phe Pro Asn Ile Val Val Ile Glu Asn Lys Gln Asn 50 55 60 Val Gly Phe Ser Lys Ala Asn Asn Gln Ala Ile Lys Leu Ser Lys Gly 65 70 75 80 Arg Tyr Ile Leu Leu Leu Asn Ser Asp Thr Ile Val Lys Glu Asp Thr 85 90 95 Ile Glu Lys Met Ile Glu Phe Met Asp Lys Ser Lys Lys Val Gly Ala 100 105 110 Ser Gly Cys Glu Val Val Leu Pro Asn Gly Glu Leu Asp Arg Ala Cys 115 120 125 His Arg Gly Phe Pro Thr Pro Glu Ala Ser Phe Tyr Tyr Leu Val Gly 130 135 140 Leu Ala Arg Leu Phe Pro Arg Ser Arg Arg Phe Asn Gln Tyr His Leu 145 150 155 160 Gly Tyr Met Asn Leu Asn Glu Pro His Pro Ile Asp Cys Leu Val Gly 165 170 175 Ala Phe Met Met Val Arg Arg Glu Val Ile Glu Gln Val Gly Leu Leu 180 185 190 Asp Glu Glu Phe Phe Met Tyr Gly Glu Asp Ile Asp Trp Cys Tyr Arg 195 200 205 Ile Lys Gln Ala Gly Trp Glu Ile Tyr Tyr Cys Pro Phe Thr Ser Ile 210 215 220 Ile His Tyr Lys Gly Ala Ser Ser Lys Lys Lys Pro Phe Lys Ile Val 225 230 235 240 Tyr Glu Phe His Arg Ala Met Phe Leu Phe His Arg Lys His Tyr Ala 245 250 255 Arg Lys Tyr Pro Phe Ile Val Asn Cys Leu Val Tyr Thr Gly Ile Ala 260 265 270 Ala Lys Phe Ile Leu Ser Ala Ile Ile Asn Thr Phe Arg Lys Ile Gly 275 280 285 Gly <210> 42 <211> 377 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 42 Met Lys Ile Ser Ile Ile Gly Asn Thr Ala Asn Ala Met Ile Leu Phe 1 5 10 15 Arg Leu Asp Leu Ile Lys Thr Leu Thr Lys Lys Gly Ile Ser Val Tyr 20 25 30 Ala Phe Ala Thr Asp Tyr Asn Asp Ser Ser Lys Glu Ile Ile Lys Lys 35 40 45 Ala Gly Ala Ile Pro Val Asp Tyr Asn Leu Ser Arg Ser Gly Ile Asn 50 55 60 Leu Ala Gly Asp Leu Trp Asn Thr Tyr Leu Leu Ser Lys Lys Leu Lys 65 70 75 80 Lys Ile Lys Pro Asp Ala Ile Leu Ser Phe Phe Ser Lys Pro Ser Ile 85 90 95 Phe Gly Ser Leu Ala Gly Ile Phe Ser Gly Val Lys Asn Asn Thr Ala 100 105 110 Met Leu Glu Gly Leu Gly Phe Leu Phe Thr Glu Gln Pro His Gly Thr 115 120 125 Pro Leu Lys Thr Lys Leu Leu Lys Asn Ile Gln Val Leu Leu Tyr Lys 130 135 140 Ile Ile Phe Pro His Ile Asn Ser Leu Ile Leu Leu Asn Lys Asp Asp 145 150 155 160 Tyr His Asp Leu Ile Asp Lys Tyr Lys Ile Lys Leu Lys Ser Cys His 165 170 175 Ile Leu Gly Gly Ile Gly Leu Asp Met Asn Asn Tyr Cys Lys Ser Thr 180 185 190 Pro Pro Thr Asn Glu Ile Ser Phe Ile Phe Ile Ala Arg Leu Leu Ala 195 200 205 Glu Lys Gly Val Asn Glu Phe Val Leu Ala Ala Lys Lys Ile Lys Lys 210 215 220 Thr His Pro Asn Val Glu Phe Ile Ile Leu Gly Ala Ile Asp Lys Glu 225 230 235 240 Asn Pro Gly Gly Leu Ser Glu Ser Asp Val Asp Thr Leu Ile Lys Ser 245 250 255 Gly Val Ile Ser Tyr Pro Gly Phe Val Ser Asn Val Ala Asp Trp Ile 260 265 270 Glu Lys Ser Ser Val Phe Val Leu Pro Ser Tyr Tyr Arg Glu Gly Val 275 280 285 Pro Arg Ser Thr Gln Glu Ala Met Ala Met Gly Arg Pro Ile Leu Thr 290 295 300 Thr Asn Leu Pro Gly Cys Lys Glu Thr Ile Ile Asp Gly Val Asn Gly 305 310 315 320 Tyr Val Val Lys Lys Trp Ser His Glu Asp Leu Ala Glu Lys Met Leu 325 330 335 Lys Leu Ile Asn Asn Pro Glu Lys Ile Ile Ser Met Gly Glu Glu Ser 340 345 350 Tyr Lys Leu Ala Arg Glu Arg Phe Asp Ala Asn Val Asn Asn Val Lys 355 360 365 Leu Leu Lys Ile Leu Gly Ile Pro Asp 370 375 <210> 43 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 43 Met Val Lys Val Ile Arg Gly Arg Glu Arg Phe Leu Thr Lys Leu Tyr 1 5 10 15 Ala Phe Val Asp Phe Ala Met Met Gln Gly Ala Phe Phe Leu Ala Trp 20 25 30 Val Leu Lys Phe Lys Val Phe His Asn Gly Val Gly Gly His Leu Pro 35 40 45 Leu Glu Asp Tyr Leu Phe Trp Ser Phe Val Tyr Gly Ala Ile Ala Ile 50 55 60 Val Ile Gly Tyr Leu Val Glu Leu Tyr Ala Pro Lys Arg Lys Glu Lys 65 70 75 80 Phe Ser Asn Glu Leu Ala Lys Val Leu Gln Val His Thr Leu Ser Met 85 90 95 Phe Val Leu Leu Ser Val Leu Phe Thr Phe Lys Thr Val Asp Val Ser 100 105 110 Arg Ser Phe Leu Leu Leu Tyr Phe Ala Trp Asn Leu Ile Leu Val Ser 115 120 125 Ile Tyr Arg Tyr Ile Val Lys Gln Ser Leu Arg Thr Leu Arg Lys Lys 130 135 140 Gly Tyr Asn Lys Gln Phe Val Leu Ile Ile Gly Ala Gly Ser Ile Gly 145 150 155 160 Arg Lys Tyr Phe Glu Asn Leu Gln Met His Pro Glu Phe Gly Leu Glu 165 170 175 Val Val Gly Phe Leu Asp Asp Phe Arg Thr Lys His Ala Pro Glu Phe 180 185 190 Ala His Tyr Lys Pro Ile Ile Gly Gln Thr Ala Asp Leu Glu His Val 195 200 205 Leu Ser His Gln Leu Ile Asp Glu Val Ile Val Ala Leu Pro Leu Gln 210 215 220 Ala Tyr Pro Lys Tyr Arg Glu Ile Ile Ala Val Cys Glu Lys Met Gly 225 230 235 240 Val Arg Val Ser Ile Ile Pro Asp Phe Tyr Asp Ile Leu Pro Ala Ala 245 250 255 Pro His Phe Glu Ile Phe Gly Asp Leu Pro Ile Ile Asn Val Arg Asp 260 265 270 Val Pro Leu Asp Glu Leu Arg Asn Arg Val Leu Lys Arg Ser Phe Asp 275 280 285 Ile Val Phe Ser Leu Val Ala Ile Ile Val Thr Ser Pro Ile Met Leu 290 295 300 Leu Ile Ala Ile Gly Ile Lys Leu Thr Ser Pro Gly Pro Ile Ile Phe 305 310 315 320 Lys Gln Glu Arg Val Gly Leu Asn Arg Arg Thr Phe Tyr Met Tyr Lys 325 330 335 Phe Arg Ser Met Lys Pro Met Pro Gln Ser Val Ser Asp Thr Gln Trp 340 345 350 Thr Val Glu Ser Asp Pro Arg Arg Thr Lys Phe Gly Ala Phe Leu Arg 355 360 365 Lys Thr Ser Leu Asp Glu Leu Pro Gln Phe Phe Asn Val Leu Lys Gly 370 375 380 Asp Met Ser Ile Val Gly Pro Arg Pro Glu Arg Pro Phe Phe Val Glu 385 390 395 400 Lys Phe Lys Lys Glu Ile Pro Lys Tyr Met Ile Lys His His Val Arg 405 410 415 Pro Gly Ile Thr Gly Trp Ala Gln Val Cys Gly Leu Arg Gly Asp Thr 420 425 430 Ser Ile Gln Glu Arg Ile Glu His Asp Leu Phe Tyr Ile Glu Asn Trp 435 440 445 Ser Leu Trp Leu Asp Ile Lys Ile Ile Leu Leu Thr Ile Thr Asn Gly 450 455 460 Leu Val Asn Lys Asn Ala Tyr 465 470 <210> 44 <211> 324 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 44 Met Glu Met Pro Leu Val Ser Ile Val Val Ala Thr Tyr Phe Pro Arg 1 5 10 15 Thr Asp Phe Phe Glu Lys Gln Leu Gln Ser Leu Asn Asn Gln Thr Tyr 20 25 30 Glu Asn Ile Glu Ile Ile Ile Cys Asp Asp Ser Ala Asn Asp Ala Glu 35 40 45 Tyr Glu Lys Val Lys Lys Met Val Glu Asn Ile Ile Ser Arg Phe Pro 50 55 60 Cys Lys Val Ile Arg Asn Glu Lys Asn Val Gly Ser Asn Lys Thr Phe 65 70 75 80 Glu Arg Leu Thr Gln Glu Ala Asn Gly Asp Tyr Ile Cys Tyr Cys Asp 85 90 95 Gln Asp Asp Ile Trp Leu Ser Glu Lys Val Glu Arg Leu Val Asn His 100 105 110 Ile Thr Lys His His Cys Thr Leu Val Tyr Ser Asp Leu Ser Leu Ile 115 120 125 Asp Glu Asn Asp Arg Ile Ile His Lys Ser Phe Lys Arg Ser Asn Phe 130 135 140 Arg Leu Lys His Val His Gly Asp Asn Thr Phe Ala His Leu Ile Asn 145 150 155 160 Arg Asn Ser Val Thr Gly Cys Ala Met Met Ile Arg Ala Asp Val Ala 165 170 175 Lys Ser Ala Ile Pro Phe Pro Asp Tyr Asp Glu Phe Val His Asp His 180 185 190 Trp Leu Ala Ile His Ala Ala Val Lys Gly Ser Leu Gly Tyr Ile Lys 195 200 205 Glu Pro Leu Val Trp Tyr Arg Ile His Leu Gly Asn Gln Ile Gly Asn 210 215 220 Gln Arg Leu Val Asn Ile Thr Asn Ile Asn Asp Tyr Ile Arg His Arg 225 230 235 240 Ile Glu Lys Gln Gly Asn Lys Tyr Arg Leu Thr Leu Glu Arg Leu Ser 245 250 255 Leu Thr Leu Gln Gln Lys Gln Leu Val Tyr Phe Gln Ile His Leu Thr 260 265 270 Glu Ala Arg Lys Lys Phe Ser Gln Lys Pro Cys Leu Gly Asn Phe Phe 275 280 285 Lys Ile Val Pro Leu Ile Lys Tyr Asp Ile Ile Leu Phe Leu Phe Glu 290 295 300 Leu Met Ile Phe Thr Val Pro Phe Thr Cys Ser Ile Trp Ile Phe Lys 305 310 315 320 Lys Leu Lys Tyr <210> 45 <211> 1127 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 45 Met Glu Arg Cys Arg Met Asn Lys Lys Ile Pro Phe Asp Gln Tyr Gln 1 5 10 15 Arg Tyr Lys Asn Ala Ala Glu Ile Ile Asn Leu Ile Arg Glu Glu Asn 20 25 30 Gln Ser Phe Thr Ile Leu Glu Val Gly Ala Asn Glu His Arg Asn Leu 35 40 45 Glu His Phe Leu Pro Lys Asp Gln Val Thr Tyr Leu Asp Ile Glu Val 50 55 60 Pro Glu His Leu Lys His Met Thr Asn Tyr Ile Glu Ala Asp Ala Thr 65 70 75 80 Asn Met Pro Leu Asp Asp Asn Ala Phe Asp Phe Val Ile Ala Leu Asp 85 90 95 Val Phe Glu His Ile Pro Pro Asp Lys Arg Asn Gln Phe Leu Phe Glu 100 105 110 Ile Asn Arg Val Ala Lys Glu Gly Phe Leu Ile Ala Ala Pro Phe Asn 115 120 125 Thr Glu Gly Val Glu Glu Thr Glu Ile Arg Val Asn Glu Tyr Tyr Lys 130 135 140 Ala Leu Tyr Gly Glu Gly Phe Arg Trp Leu Glu Glu His Arg Gln Tyr 145 150 155 160 Thr Leu Pro Asn Leu Glu Glu Thr Glu Asp Ile Leu Arg Lys Glu Asn 165 170 175 Ile Glu Tyr Val Lys Phe Glu His Gly Ser Leu Leu Phe Trp Glu Lys 180 185 190 Leu Met Arg Leu His Phe Leu Val Ala Asp Arg Asn Val Leu His Asp 195 200 205 Tyr Arg Phe Met Ile Asp Asp Phe Tyr Asn Lys Asn Ile Tyr Glu Val 210 215 220 Asp Tyr Ile Gly Pro Cys Tyr Arg Asn Phe Ile Val Val Cys Arg Asp 225 230 235 240 Lys Ala Lys Arg Glu Phe Ile Gln Ser Ile Tyr Glu Lys Arg Lys Gln 245 250 255 Asn Ser Tyr Leu Lys Asn Ser Thr Ile Ser Lys Leu Asn Glu Leu Glu 260 265 270 Asn Ser Ile Tyr Ser Leu Lys Ile Ile Asp Lys Glu Asn Gln Ile Tyr 275 280 285 Lys Lys Ser Leu Glu Ile Thr Glu Gln Leu Leu Glu Asp Leu Lys Leu 290 295 300 Lys Glu Gln Gln Ile Ile Glu Lys Ile Gln Thr Ile Lys Lys Lys Thr 305 310 315 320 Glu Met Ile Glu Leu Gln Asn Gln Lys Ile Gln Glu Leu Lys Ile Glu 325 330 335 Cys Glu Asn Lys Ser Ile Glu Asn Asn Asn Leu Tyr Ser Gln Leu Leu 340 345 350 Glu Lys Glu Asn Tyr Ile Lys Gln Leu Gln Asn Gln Ala Glu Ser Met 355 360 365 Arg Ile Lys Asn Arg Leu Lys Lys Ile Leu Asn Phe Ser Phe Ile Lys 370 375 380 Tyr Val Arg Lys Ile Ile Asn Ile Ile Phe Arg Arg Lys Phe Lys Phe 385 390 395 400 Lys Leu Gln Pro Val His His Leu Glu Trp Ser Asn Gly Lys Trp Leu 405 410 415 Val Leu Gly Arg Asp Pro His Phe Ile Leu Lys Gly Gly Ser Tyr Pro 420 425 430 Ser Ser Trp Thr Ile Ile Gln Trp Arg Ala Ser Ala Asn Ser Ser Ala 435 440 445 Leu Leu Arg Leu Tyr Tyr Asp Thr Gly Gly Gly Phe Ser Glu Asn Gln 450 455 460 Ser Phe Asn Leu Gly Lys Ile Gly Asn Asp Ile Asn Arg Asp Tyr Glu 465 470 475 480 Cys Val Ile Cys Leu Pro Glu Asn Ile His Leu Leu Arg Leu Asp Ile 485 490 495 Glu Gly Glu Ile Ser Glu Phe Glu Leu Glu Asn Leu Thr Phe Thr Ser 500 505 510 Ile Ser Arg Leu Glu Val Phe Tyr Lys Ser Phe Ile Asn His Cys Arg 515 520 525 Lys Arg Asn Ile Lys Asn Tyr Lys Glu Leu Tyr Ser Leu Ile Lys Lys 530 535 540 Leu Phe Ile Leu Val Arg Arg Glu Gly Leu Lys Ser Ile Trp Tyr Arg 545 550 555 560 Ala Lys Gln Lys Leu Ser Met Glu Leu Leu Ser Glu Asp Pro Tyr Glu 565 570 575 Val Phe Leu Asn Val Ser Ser Lys Val Asp Lys Glu Ile Val Leu Ser 580 585 590 Glu Ile Lys Lys Leu Lys Tyr Lys Pro Lys Phe Ser Val Ile Leu Pro 595 600 605 Val Tyr Asn Val Glu Glu Lys Trp Leu Arg Lys Cys Ile Asp Ser Val 610 615 620 Leu Asn Gln Trp Tyr Pro Tyr Trp Glu Leu Cys Ile Val Asp Asp Asn 625 630 635 640 Ser Ser Lys Asp Tyr Ile Lys Pro Val Leu Glu Glu Tyr Ser Asn Arg 645 650 655 Asp Ser Arg Ile Lys Thr Val Phe Arg Ser Asn Asn Gly His Ile Ser 660 665 670 Glu Ala Ser Asn Thr Ala Leu Glu Ile Ala Thr Gly Asp Phe Ile Ala 675 680 685 Leu Leu Asp His Asp Asp Glu Leu Ala Pro Glu Ala Leu Tyr Glu Asn 690 695 700 Ala Val Leu Leu Asn Glu His Pro Asp Ala Asp Met Ile Tyr Ser Asp 705 710 715 720 Glu Asp Lys Ile Thr Lys Asp Gly Lys Arg His Ser Pro Leu Phe Lys 725 730 735 Pro Asp Trp Ser Pro Asp Thr Leu Arg Ser Gln Met Tyr Ile Gly His 740 745 750 Leu Thr Val Tyr Arg Thr Asn Leu Val Arg Gln Leu Gly Gly Phe Arg 755 760 765 Lys Gly Phe Glu Gly Ser Gln Asp Tyr Asp Leu Ala Leu Arg Val Ala 770 775 780 Glu Lys Thr Asn Asn Ile Tyr His Ile Pro Lys Ile Leu Tyr Ser Trp 785 790 795 800 Arg Glu Ile Glu Thr Ser Thr Ala Val Asn Pro Ser Ser Lys Pro Tyr 805 810 815 Ala His Glu Ala Gly Leu Lys Ala Leu Asn Glu His Leu Glu Arg Val 820 825 830 Phe Gly Lys Gly Lys Ala Trp Ala Glu Glu Thr Glu Tyr Leu Phe Val 835 840 845 Tyr Asp Val Arg Tyr Ala Ile Pro Glu Asp Tyr Pro Leu Val Ser Ile 850 855 860 Ile Ile Pro Thr Lys Asp Asn Ile Glu Leu Leu Ser Ser Cys Ile Gln 865 870 875 880 Ser Ile Leu Asp Lys Thr Thr Tyr Pro Asn Tyr Glu Ile Leu Ile Met 885 890 895 Asn Asn Asn Ser Val Met Glu Glu Thr Tyr Ser Trp Phe Asp Lys Gln 900 905 910 Lys Glu Asn Ser Lys Ile Arg Ile Ile Asp Ala Met Tyr Glu Phe Asn 915 920 925 Trp Ser Lys Leu Asn Asn His Gly Ile Arg Glu Ala Asn Gly Glu Val 930 935 940 Phe Val Phe Leu Asn Asn Asp Thr Ile Val Ile Ser Glu Asp Trp Leu 945 950 955 960 Gln Arg Leu Val Glu Lys Ala Leu Arg Glu Asp Val Gly Thr Val Gly 965 970 975 Gly Leu Leu Leu Tyr Glu Asp Asn Thr Ile Gln His Ala Gly Val Val 980 985 990 Ile Gly Met Gly Gly Trp Ala Asp His Val Tyr Lys Gly Met His Pro 995 1000 1005 Val His Asn Thr Ser Pro Phe Ile Ser Pro Val Ile Asn Arg Asn 1010 1015 1020 Val Ser Ala Ser Thr Gly Ala Cys Leu Ala Ile Ala Lys Lys Val 1025 1030 1035 Ile Glu Lys Ile Gly Gly Phe Asn Glu Glu Phe Ile Ile Cys Gly 1040 1045 1050 Ser Asp Val Glu Ile Ser Leu Arg Ala Leu Lys Met Gly Tyr Val 1055 1060 1065 Asn Ile Tyr Asp Pro Tyr Val Arg Leu Tyr His Leu Glu Ser Lys 1070 1075 1080 Thr Arg Asp Ser Phe Ile Pro Glu Arg Asp Phe Glu Leu Ser Ala 1085 1090 1095 Lys Tyr Tyr Ser Pro Tyr Arg Glu Ile Gly Asp Pro Tyr Tyr Asn 1100 1105 1110 Gln Asn Leu Ser Tyr Asn His Leu Ile Pro Thr Ile Arg Ser 1115 1120 1125 <210> 46 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 46 Met Ala Arg Ser Gly Gly Val Val Ile Lys Lys Lys Val Ala Ala Ile 1 5 10 15 Ile Ile Thr Tyr Asn Pro Asp Leu Thr Ile Leu Arg Glu Ser Tyr Thr 20 25 30 Ser Leu Tyr Lys Gln Val Asp Lys Ile Ile Leu Ile Asp Asn Asn Ser 35 40 45 Thr Asn Tyr Gln Glu Leu Lys Lys Leu Phe Glu Lys Lys Glu Lys Ile 50 55 60 Lys Ile Val Pro Leu Ser Asp Asn Ile Gly Leu Ala Ala Ala Gln Asn 65 70 75 80 Leu Gly Leu Asn Leu Ala Ile Lys Asn Asn Tyr Thr Tyr Ala Ile Leu 85 90 95 Phe Asp Gln Asp Ser Val Leu Gln Asp Asn Gly Ile Asn Ser Phe Phe 100 105 110 Phe Glu Phe Glu Lys Leu Val Ser Glu Glu Lys Leu Asn Ile Val Ala 115 120 125 Ile Gly Pro Ser Phe Phe Asp Glu Lys Thr Gly Arg Arg Phe Arg Pro 130 135 140 Thr Lys Phe Ile Gly Pro Phe Leu Tyr Pro Phe Arg Lys Ile Thr Thr 145 150 155 160 Lys Asn Pro Leu Thr Glu Val Asp Phe Leu Ile Ala Ser Gly Cys Phe 165 170 175 Ile Lys Leu Glu Cys Ile Lys Ser Ala Gly Met Met Thr Glu Ser Leu 180 185 190 Phe Ile Asp Tyr Ile Asp Val Glu Trp Ser Tyr Arg Met Arg Ser Tyr 195 200 205 Gly Tyr Lys Leu Tyr Ile His Asn Asp Ile His Met Ser His Leu Val 210 215 220 Gly Glu Ser Arg Val Asn Leu Gly Leu Lys Thr Ile Ser Leu His Gly 225 230 235 240 Pro Leu Arg Arg Tyr Tyr Leu Phe Arg Asn Tyr Ile Ser Ile Leu Lys 245 250 255 Val Arg Tyr Ile Pro Leu Gly Tyr Lys Ile Arg Glu Gly Phe Phe Asn 260 265 270 Ile Gly Arg Phe Leu Val Ser Met Ile Ile Thr Lys Asn Arg Lys Thr 275 280 285 Leu Ile Leu Tyr Thr Ile Lys Ala Ile Lys Asp Gly Ile Asn Asn Glu 290 295 300 Met Gly Lys Tyr Lys Gly 305 310 <210> 47 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 47 tacctcgagg gcaaagccgt ttttccatag gctccgccc 39 <210> 48 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 48 tacggatccg ttatttcctc ccgttaaata atagataac 39 <210> 49 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 49 agactcgaga tgcaggatgt ttttatcatt ggtagc 36 <210> 50 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 50 agactcgaga tgttcattta aaaataaagc ctcgtac 37 <210> 51 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 51 tctgaattca tgcaggatgt ttttatcatt ggtagc 36 <210> 52 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 52 acactgcagt taatgttcat ttaaaaataa agcctcgtac 40 <210> 53 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 53 cactctaacc cagctggatt gataaaaaag cg 32 <210> 54 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 54 caatccagct gggttagagt ggaaacggtc t 31 <210> 55 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 55 cgtaattatt tgcaggaaca aagcgtccta aaatg 35 <210> 56 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 56 cgctttgttc ctgcaaataa ttacgaaacc gc 32 <210> 57 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 57 caatgccaat attagctgaa atgaccaaat c 31 <210> 58 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 58 ggtcatttca gctaatattg gcattgaccg c 31 <210> 59 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 59 gtctgcgttc cagcagcaat aaaacatgtt ttag 34 <210> 60 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 60 gttttattgc tgctggaacg cagacacaac cttc 34 <210> 61 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 61 ctctaacccg tttggattga taaaaaagcg tccacctcg 39 <210> 62 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 62 cgctttttta tcaatccaaa cgggttagag tggaaacggt c 41 <210> 63 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 63 ggtttcgtaa ttattttgag gaacaaagcg 30 <210> 64 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 64 gttcctcaaa ataattacga aaccgc 26 <210> 65 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 65 tgccaatatt atttgaaatg accaaatcag cc 32 <210> 66 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 66 gatttggtca tttcaaataa tattggcatt gaccgctacc 40 <210> 67 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 67 ggttgtgtct gcgttccgaa agcaataaaa catgttttag acc 43 <210> 68 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 68 gttttattgc tttcggaacg cagacacaac cttcacg 37 <210> 69 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 69 tttagaccgc gtccactcta acccgtctgg 30 <210> 70 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 70 agagtggacg cggtctaaat ggtcaagacc 30 <210> 71 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 71 ttcggatcca actattagcc tacattcgag aacagg 36 <210> 72 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 72 acactgcagt taatgttcat ttaaaaataa agcctcgtac 40 <210> 73 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 73 ctttaagaag gagactcgag atgggacgct tttttatcaa tccagac 47 <210> 74 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 74 gtctggattg ataaaaaagc gtcccatctc gagtctcctt cttaaag 47 <210> 75 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 75 ctttaagaag gagactcgag atggggttag agtggaaacg gtc 43 <210> 76 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 76 gaccgtttcc actctaaccc catctcgagt ctccttctta aag 43 <210> 77 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 77 ggatccatga tggcaattac ctatgccctg tc 32 <210> 78 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 78 acactgcagt taatgttcat ttaaaaataa agcctcgtac 40 <210> 79 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 79 ggatccatgg aagagttgat tagtcatcaa tcatct 36 <210> 80 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 80 acactgcagt taatgttcat ttaaaaataa agcctcgtac 40 <210> 81 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 81 ggtaccatgc gtcatatatt catcatagga agtcgcg 37 <210> 82 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 82 atattctaga attataggta ccccttatta aagttaaaca aaattatttc 50 <210> 83 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 83 gctatccgtg agttcatgac ttcg 24 <210> 84 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 84 ctgcagttaa ctttcatgta agaacaagtc ctcgtac 37 <210> 85 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 85 cgaagtcatg aactcacgga tagc 24 <210> 86 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 86 ggaggaattc accttgcgtc atatattcat cataggaagt cgcg 44 <210> 87 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 87 tctgaattca tgaaacagtc agtttatatc attggttcaa 40 <210> 88 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 88 ggttgtgtct gcgttccata agcaataaag gtcgtcttgg gctgatactg 50 <210> 89 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 89 ccagattcag aaccctattt tttatgtgtt ggcgtgtcga gtaggcccat ttattgcgcc 60 atttgtgaag cagattcaca atcg 84 <210> 90 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 90 cgattgtgaa tctgcttcac aaatggcgca ataaatgggc ctactcgaca cgccaacaca 60 taaaaaatag ggttctgaat ctgg 84 <210> 91 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 91 caatccagac gggcacgagt ggaaactgtc taaatggtca agac 44 <210> 92 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 92 gtcttgacca tttagacagt ttccactcgt gcccgtctgg attg 44 <210> 93 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 93 tgccaatatt atttgaaatg accaaatcag cc 32 <210> 94 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 94 gatttggtca tttcaaataa tattggcatt gaccgctacc 40 <210> 95 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 95 ggttgtgtct gcgttccgaa agcaataaaa catgttttag acc 43 <210> 96 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 96 gttttattgc tttcggaacg cagacacaac cttcacg 37 <210> 97 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 97 atctgaattc atgcaggatg ttttcatcat tggtagc 37 <210> 98 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 98 acactgcagt taatgttcat ctaaaaataa agcctcatac 40 <210> 99 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 99 tctgaattca tgcaagatgt tttcattata gg 32 <210> 100 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 100 acactgcagt taactttcgt tcaagaacaa gtcctc 36 <210> 101 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 101 atgaattcat gcaggatgtt ttcatcattg gtagcaga 38 <210> 102 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 102 atctgcagtt aatgttcatc taaaaataaa gcctcatact ccccaacaat 50 <210> 103 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 103 tctgaattca tgaaacagtc agtttatatc attggttcaa 40 <210> 104 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 104 atatctgcag gcatcataca gtaaacactt cctcataatc tgac 44 <210> 105 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 105 ccagattcag aaccctattt tttatgtgtt ggcgtgtcga gtaggcgctt ttattgcgcc 60 atttgtgaag cagattcaca atcg 84 <210> 106 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 106 cgattgtgaa tctgcttcac aaatggcgca ataaaagcgc ctactcgaca cgccaacaca 60 taaaaaatag ggttctgaat ctgg 84 <210> 107 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 107 aagttctgtt tcagggcccg aacattaata ttttactatc cacctac 47 <210> 108 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 108 atggtctaga aagctttact ttctcctgta accaaataag gtaac 45 <210> 109 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 109 aagttctgtt tcagggcccg aaggttaata tcttaatggc cacctac 47 <210> 110 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 110 atggtctaga aagctttatc tcttattgta ataatttgtt gcaatcaacc 50 <210> 111 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 111 aagttctgtt tcagggcccg aaagttaata ttttaatgtc cacctac 47 <210> 112 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 112 atggtctaga aagctttatt ttctcctata accaaattta g 41 <210> 113 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 113 aagttctgtt tcagggcccg agtaacaagc aaattg 36 <210> 114 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 114 atggtctaga aagctttaaa taaacattaa ctcaccg 37 <210> 115 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 115 cttaaatctc ttatccattg tacccgcccc caaaac 36 <210> 116 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 116 gttttggggg cgggtacaat ggataagaga tttaag 36 <210> 117 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 117 cgaagtatct taaatctacc atccattgtc ctc 33 <210> 118 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 118 gaggacaatg gatggtagat ttaagatact tcg 33 <210> 119 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 119 gaccttcacg aagtatacca aatctcttat cc 32 <210> 120 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 120 ggataagaga tttggtatac ttcgtgaagg tc 32 <210> 121 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 121 tagatttagg accttcacca agtatcttaa atctc 35 <210> 122 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 122 gagatttaag atacttggtg aaggtcctaa atc 33 <210> 123 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 123 gcagatgtct attttttcag tgcccaagat gatatatggt tagac 45 <210> 124 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 124 gtctaaccat atatcatctt gggcactgaa aaaatagaca tctgc 45 <210> 125 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 125 cttgatattc caacagaatt attccgtcag cacgatgc 38 <210> 126 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 126 gcatcgtgct gacggaataa ttctgttgga atatcaag 38 <210> 127 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 127 caacagaatt ataccgtcag gccgatgcta acgtgttggg 40 <210> 128 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 128 cccaacacgt tagcatcggc ctgacggtat aattctgttg 40

Claims

1. A method for synthesizing rhamnose polysaccharide, wherein the rhamnose polysaccharide comprises a plurality of rhamnose moieties, the rhamnose moieties comprising a single straight chain of repeating units containing rhamnose, wherein each repeating unit consists of rhamnose bound to each other by α-1,3 and / or α-1,2 glycosidic bonds, or each repeating unit comprises rhamnose and one or more different monosaccharides, the method comprises: (i) using hexose-β-1,4-rhamnosyltransferase, hexose-α-1,2-rhamnosyltransferase and / or hexose-α-1,3-rhamnosyltransferase to transfer the rhamnose moiety to a hexose monosaccharide, disaccharide or trisaccharide to form a disaccharide, trisaccharide or tetrasaccharide comprising a rhamnose moiety at the non-reducing end of the disaccharide, trisaccharide or tetrasaccharide; wherein the hexose-β-1,4-rhamnosyltransferase is Glc-β-1,4-rhamnosyltransferase; wherein the hexose-α-1,2-rhamnosyltransferase is galactose-α-1,2-rhamnosyltransferase; wherein the hexose-α-1,3-rhamnosyltransferase is GlcNAc-α-1,3-rhamnosyltransferase, diNAcBac-α-1,3-rhamnosyltransferase, Glc-α-1,3-rhamnosyltransferase, or galactose-α-1,3-rhamnosyltransferase; (ii) generating a rhamnose polysaccharide by extension from the rhamnose moiety at the non-reducing end of the disaccharide, trisaccharide or tetrasaccharide using the bacterial enzymes Streptococcus pyogenes group A carbohydrate enzyme C (GacC) and / or Streptococcus pyogenes group A carbohydrate enzyme G (GacG), the bacterial enzymes Streptococcus pyogenes group A carbohydrate enzyme C (GacC) and / or Streptococcus pyogenes group A carbohydrate enzyme G (GacG) being heterologous to the bacterial species from which the hexose-β-1,4-rhamnosyltransferase, hexose-α-1,2-rhamnosyltransferase, hexose-α-1,3-rhamnosyltransferase used in step (i) are derived.

2. The method according to claim 1, wherein, the method is carried out in a bacterial species heterologous to the bacterial species from which the enzymes GacC and / or GacG are derived.

3. The method according to claim 1, wherein, the hexose-β-1,4-rhamnosyltransferase is not GlcNAc-β-1,4-rhamnosyltransferase.

4. The method according to claim 1, wherein, the Glc-β-1,4-rhamnosyltransferase comprises the WchF enzyme.

5. The method according to claim 4, wherein, the WchF enzyme comprises SEQ ID NO:

36.

6. The method according to claim 1, wherein, the galactose-α-1,2-rhamnosyltransferase comprises the WbbR enzyme.

7. The method according to claim 6, wherein, the WbbR enzyme comprises SEQ ID NO:

37.

8. The method according to claim 1, wherein, the GlcNAc-α-1,3-rhamnosyltransferase comprises the WbbL enzyme, and the galactose-α-1,3-rhamnosyltransferase comprises the WsaD enzyme.

9. The method according to claim 8, wherein, the WbbL enzyme comprises SEQ ID NO:

38.

10. The method according to claim 8, wherein, the WsaD enzyme comprises SEQ ID NO:

41.

11. The method according to claim 1, wherein, the enzymatic activity homologs of GacC and / or GacG are selected from the homologs of Group B, C, G streptococci, Streptococcus mutans, and Streptococcus uberis.

12. The method according to any one of the preceding claims, wherein, the method is carried out in Gram-negative bacteria.

13. The method according to claim 12, wherein, the method is carried out in Escherichia coli.

14. The method according to any one of claims 1 to 11, wherein, step ii) further comprises using one or more additional enzymes from the bacterial enzyme Gac cluster, or one or more enzymatic activity homologs or fragments thereof.

15. The method according to any one of claims 1 to 11, the method further comprises: (iii) conjugating the rhamnopolysaccharide with a receptor molecule to form a rhamnose glycoconjugate using an oligosaccharyltransferase capable of recognizing the reducing end of the rhamnopolysaccharide and hexose monosaccharides, wherein the receptor molecule comprises a peptide or protein.

16. The method according to claim 15, wherein, the oligosaccharyltransferase is heterologous to the bacteria in which the method is carried out.

17. The method according to claim 16, wherein, the oligosaccharyltransferase comprises PgIB, PgIL, PgIS or WsaB, or an enzymatic activity fragment thereof.

18. The method according to claim 15, wherein, the method further comprises purifying the rhamnose glycoconjugate.

19. A product obtainable using the method according to any one of claims 1 to 18.

20. A synthetic streptococcal polysaccharide having a non-reducing end of a single straight chain comprising repeating units containing rhamnose and a reducing end comprising hexose monosaccharides, disaccharides or trisaccharides, wherein each repeating unit consists of rhamnose bound to each other by α-1,3 and / or α-1,2 glycosidic bonds, or each repeating unit consists of rhamnose-galactose, wherein, the polysaccharide comprises an α-1,3 bond or an α-1,2 bond between the hexose monosaccharide, disaccharide or trisaccharide and the straight chain of the repeating unit; or the polysaccharide comprises a β-1,4 bond between the hexose monosaccharide, disaccharide or trisaccharide and the straight chain of the repeating unit, and the hexose monosaccharide, disaccharide or trisaccharide does not include N-acetylglucosamine.

21. The synthetic streptococcal rhamnopolysaccharide according to claim 20, wherein, the polysaccharide comprises an α-1,3 bond between the hexose monosaccharide, disaccharide or trisaccharide and the rhamnose partial straight chain, and the hexose includes N-acetylglucosamine, N,N'-diacetylbacillosamine, glucose or galactose.

22. The synthetic streptococcal rhamnopolysaccharide according to claim 20, wherein, the polysaccharide comprises an α-1,2 bond between the hexose monosaccharide, disaccharide or trisaccharide and the rhamnose partial straight chain, and the hexose includes galactose.

23. The synthetic Streptococcus rhamnose polysaccharide according to claim 20, wherein, the polysaccharide comprises a β-1,4 bond between the hexose monosaccharide, disaccharide or trisaccharide and the rhamnose moiety straight chain, and the hexose includes glucose.

24. The synthetic Streptococcus rhamnose polysaccharide according to claim 20, wherein, the polysaccharide comprises a polysaccharide or a fragment thereof selected from the group consisting of polysaccharides of groups A, B, C and G carbohydrates.

25. A Streptococcus rhamnose glycoconjugate comprising the Streptococcus rhamnose polysaccharide according to any one of claims 20 to 24 conjugated to a receptor.

26. The Streptococcus glycoconjugate according to claim 25, wherein, the polysaccharide is conjugated to the receptor at the reducing end of the polysaccharide.

27. The Streptococcus glycoconjugate according to claim 25, wherein, the receptor comprises a peptide or a protein.

28. An immunogenic composition or vaccine comprising the product according to claim 19, the synthetic Streptococcus rhamnose polysaccharide according to any one of claims 20 to 24 or the Streptococcus glycoconjugate according to any one of claims 25 to 27.

29. The immunogenic composition or vaccine according to claim 28, wherein, the immunogenic composition or vaccine further comprises a pharmaceutically acceptable and / or sterile excipient.

30. The immunogenic composition or vaccine according to claim 28, wherein, the immunogenic composition or vaccine further comprises an antigen, a polypeptide and / or an adjuvant.

31. Use of the product according to claim 19, the synthetic Streptococcus rhamnose polysaccharide according to any one of claims 20 to 24, or the Streptococcus glycoconjugate according to any one of claims 25 to 27 in the preparation of a medicament for the treatment or prevention of a disease, disorder or infection caused by a streptococcal etiology.

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