Modified Exotoxin A Protein

Abstract

This invention provides modified proteins, immunogenic compositions, and vaccines containing modified proteins, their manufacture, and the use of such compositions in pharmaceuticals. [Solution] A modified EPA (Exotoxin A of Pseudomonas aeruginosa) protein containing a specific amino acid sequence is provided, which is modified in that it contains one (or more) consensus sequences selected from three specific amino acid sequences, and can be used as a carrier protein for other antigens, particularly monosaccharide antigens, or other antigens lacking a T cell epitope.

Description

Technical Field

[0001] The present invention relates to the fields of modified proteins, immunogenic compositions, and vaccines containing modified proteins, their production, and the use of such compositions in medicine. More particularly, it relates to modified EPA (Exotoxin A of Pseudomonas aeruginosa) proteins. The modified EPA can be used as a carrier protein for other antigens, particularly monosaccharide antigens, or other antigens lacking T cell epitopes.

Background Art

[0002] Protein glycosylation is a common post-translational modification in bacteria, where glycans are covalently bonded to, for example, surface proteins, flagella, or fimbriae. Glycoproteins play a role in adhesion, stabilization of proteins against proteolysis, and evasion of host immune responses. Two protein glycosylation mechanisms are distinguished by the mode in which glycans are transferred to proteins: one mechanism involves the direct transfer of carbohydrates from nucleotide-activated sugars to acceptor proteins (e.g., used in protein O glycosylation in the Golgi apparatus of eukaryotic cells and flagellin O glycosylation in some bacteria). The second mechanism involves the transfer of polysaccharides to protein acceptors by oligosaccharide transferases (OTases) following pre-assembly of polysaccharides onto lipid carriers (by glycotransferases) (Faridmoayer et al., J. Bacteriology, pp. 8088-8098, 2007). This second mechanism is used, for example, in N-glycosylation in the endoplasmic reticulum of eukaryotic cells, the well-characterized N-linked glycosylation system of Campylobacter jejuni, and the more recently characterized O-linked glycosylation systems of Neisseria meningitidis, Neisseria gonococcus, and Pseudomonas aeruginosa. For O-linked glycosylation (O-glycosylation), glycans typically bind to serine or threonine residues on protein acceptors. For N-linked glycosylation (N-glycosylation), glycans typically bind to asparagine residues on protein acceptors. In Escherichia coli (E. coli), it is possible to reconstitute N-glycosylation of C. jejuni protein by simultaneously expressing the pgl gene locus and acceptor glycoprotein by recombination (Wacker et al. (2002), Science, Vol. 298, pp. 1790-1793).

[0003] International Publication No. 2006 / 119987 (Aebi et al.) describes efficient means and methods for protein synthesis, including in vivo N-glycosylation in prokaryotes. It further describes the introduction of N-glycans into recombinant proteins to modify their immunogenicity, stability, biological activity, prophylactic activity, and / or therapeutic activity, as well as the supply of host cells presenting the recombinant N-glycosylated proteins of the present invention on their surface. In addition, it describes recombinant N-glycosylated proteins comprising one or more of the following optimized amino acid sequences: D / EXNZS / T, where X and Z may be any native amino acids other than Pro. By introducing such optimized amino acid sequences into a protein, the protein is N-glycosylated at the introduced site by oligosaccharide transferase.

[0004] Conjugate vaccines (vaccines containing carrier proteins that covalently bind to immunogenic antigens) have been a successful approach to vaccination against various bacterial infections. The conjugation of T-independent antigens, such as monosaccharides, to carrier proteins has long been established as a means to enable T cells to participate in the immune response to generally T-independent antigens. Thus, the immune response can be enhanced by enabling the development of immunological memory and the boostability of the response. To increase the efficiency of conjugate vaccine production, in vivo methods (for producing "bioconjugate vaccines") have been developed. These in vivo methods incorporate the N-glycosylation and O-glycosylation systems described above. For example, International Publication No. 2009 / 104074 describes a Shigella bioconjugate vaccine containing a protein carrier with Pseudomonas aeruginosa exotoxin (EPA) modified to contain at least one consensus sequence D / EXNZS / T, and International Publication No. 2017 / 035181 describes an Escherichia coli O antigen that covalently binds to the detoxified exotoxin A (EPA) of the Pseudomonas aeruginosa carrier protein. However, certain antigens have been found to be less immunogenic than others when conjugated to the EPA carrier protein.

[0005] Exotoxin A (also known as "EPA" or "ETA") of Pseudomonas aeruginosa is a secreted bacterial toxin that is a member of the ADP-ribosyltransferase toxin family. The natural protein is a single polypeptide chain of 613 amino acids (67 kDa). The protein consists of three major domains: domains Ia and b (receptor-binding domains), domain II (transmembrane domain), and domain III (catalytic NAD-ribosyltransferase domain) (Allured et al., Proc. Natl. Acad. Sci. USA, Vol. 83, pp. 1320-1324, March 1986). The last four residues of domain II (400-404), together with domain III (405-613), form the catalytic subunit of the toxin that has ADP-ribosyltransferase activity (Siegall et al., J. Biol. Chem., Vol. 264, pp. 14256-14261, 1989). A mutant form of Pseudomonas aeruginosa exotoxin A (ETA) lacking the important active site residue glutamate-553 was shown to be expressed in ETA-negative strains of Pseudomonas aeruginosa and exported from cells as efficiently as wild-type ETA. The mutant protein purified from culture medium lacked ADP-ribosyltransferase activity (Kileen et al., Biochimica et Biophysica Acta, Vol. 1138 (1992), pp. 162-166). [Overview of the project] [Problems that the invention aims to solve]

[0006] Further EPA carrier proteins suitable for in vivo conjugation are needed. Further EPA carrier proteins are needed to enable selection of EPA carrier proteins for various (mono)saccharide antigens. Additionally, further EPA carrier proteins containing at least one consensus sequence D / EXNZS / T and exhibiting promising or improved properties, such as increased glycosylation efficiency, are also needed. [Means for solving the problem]

[0007] The present invention provides a modified EPA protein containing at least one consensus sequence (e.g., D / EXNZS / T) for glycosylation, which is used for conjugation to an antigen (e.g., bacterial polysaccharide). In the modified EPA protein of the present invention, the glycosylation consensus sequence is introduced into a specific region of the EPA carrier protein. The inventors have found that the position of the consensus sequence within the EPA amino acid sequence can increase glycosylation efficiency and / or optimize the manipulation of the N glycosylation site. The modified EPA protein of the present invention exhibits higher site occupancy, a higher sugar-to-protein ratio, and / or higher yield. Furthermore, differences in protein glycosylation can affect the bioactivity, antigenicity, stability, and / or half-life of the protein. In addition, increased glycosylation can assist in the purification of the protein by chromatography. In certain embodiments, the modified EPA protein described herein is modified so that the number of glycosylation sites within the carrier protein is optimized. This makes it possible to administer EPA protein at lower concentrations in its conjugate form, for example, through an immunogenic composition or vaccine. Furthermore, the present invention provides additional EPA carrier proteins by selecting EPA carrier proteins with different numbers of consensus sequence sites for various monosaccharide antigens. The number of glycosites may be selected to optimize the sugar:protein ratio. For example, for shorter monosaccharide antigens (glycans), a larger number of glycosites may be used (using the modified EPA carrier proteins of the present invention described herein) to increase the sugar:protein ratio.

[0008] Accordingly, provided in one embodiment of the present invention is a modified EPA (Exotoxin A of Pseudomonas aeruginosa) protein having an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, wherein the amino acid sequence is modified in that it includes one (or more) consensus sequences selected from :D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the one (or more) consensus sequences are each one or more amino acids between amino acid residues 198-218 at the same position in the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1 (e.g., one or more amino acids between amino acid residues 203-213). (ii) one or more amino acids independently selected from (ii) amino acid residue Y208, (iii) one or more amino acids between amino acid residues 264-284 (e.g., one or more amino acids between amino acid residues 269-279, e.g., amino acid residue R274), (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), and (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519), are added next to or substitute for one or more amino acids.

[0009] Also provided are the amino acid sequence of SEQ ID NO: 1, or modified EPA (Exotoxin of Pseudomonas aeruginosa) having an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. A) A protein, which is modified in that its amino acid sequence contains one (or more) consensus sequences selected from:D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the one (or more) consensus sequences are at the same position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to:SEQ ID NO: 1 (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213; or one or more amino acids between 205-211; e.g., amino acid residue D218; e.g., amino acid residue Y208), or (ii) one or more amino acids between amino acid residues 264-284 (iii) one or more amino acids independently selected from amino acid residues 269-279 (e.g., one or more amino acids between amino acid residues 271-277, e.g., amino acid residue R279, e.g., amino acid residue R274), (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, or one or more amino acids between amino acid residues 315-321, e.g., amino acid residue G323, e.g., amino acid residue S318), and (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524, or one or more amino acids between amino acid residues 516-522, e.g., amino acid residue G525, e.g., amino acid residue A519), are added next to or substitute for one or more amino acids.

[0010] According to a further aspect of the present invention, what is provided is a conjugate (e.g., a bioconjugate) comprising the modified EPA protein of the present invention linked to an antigen (e.g., a monosaccharide antigen, optionally a bacterial polysaccharide antigen).

[0011] According to a further aspect of the present invention, a polynucleotide encoding the modified EPA protein of the present invention is provided.

[0012] According to a further aspect of the present invention, a vector comprising a polynucleotide encoding the modified EPA protein of the present invention is provided.

[0013] According to a further aspect of the present invention, the following is provided: i) Depending on the circumstances, one or more nucleotide sequences containing polysaccharide synthesis genes incorporated into the host cell genome for generating bacterial polysaccharide antigens (e.g., O antigens derived from Gram-negative bacteria, depending on the circumstances, from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, or capsular polysaccharides derived from Gram-positive bacteria, depending on the circumstances, from Streptococcus pneumoniae or Staphylococcus aureus) or yeast polysaccharide antigens or mammalian polysaccharide antigens; ii) Nucleotide sequences that encode heterologous oligosaccharide transferases, sometimes within plasmids; iii) A nucleotide sequence that optionally encodes the modified EPA protein of the present invention within a plasmid. It is a host cell that contains [the specified element].

[0014] A further aspect of the present invention provides a process (method) for producing a bioconjugate comprising (or comprising) a modified EPA protein linked to a polysaccharide, the process comprising (i) culturing the host cells of the present invention under conditions suitable for glycoprotein production, and (ii) isolating the bioconjugate produced by the host cells, optionally from a peripheral extract derived from the host cells.

[0015] In a further aspect of the present invention, an immunogenic composition comprising the conjugate of the present invention (e.g., a bioconjugate) and optionally pharmaceutically acceptable excipients and / or carriers is provided.

[0016] A further aspect of the present invention provides a vaccine comprising the immunogenic composition of the present invention and optionally an adjuvant.

[0017] A further aspect of the present invention provides a method for inducing an immune response in a subject (e.g., a human), comprising administering a therapeutically or prophylactically effective amount of the conjugate (e.g., a bioconjugate) of the present invention, the immunogenic composition of the present invention, or the vaccine of the present invention to a subject (e.g., a human) in need of induction.

[0018] In a further aspect of the present invention, the provided items are a conjugate (e.g., a bioconjugate), an immunogenic composition, or a vaccine of the present invention, used to induce an immune response in a subject (e.g., a human).

[0019] In a further aspect of the present invention, the present invention provides a conjugate (e.g., a bioconjugate), an immunogenic composition, or a vaccine, which are used in the production of a drug that induces an immune response in a subject (e.g., a human). This invention also relates to the following: [Item 1] A modified EPA (Exotoxin A of Pseudomonas aeruginosa) protein having an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence is modified in that it includes one (or more) consensus sequences selected from: D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the one (or more) consensus sequences are, respectively: (i) one or more amino acids between amino acid residues 198-218 of SEQ ID NO: 1 (e.g., one or more amino acids between amino acid residues 203-213, e.g., amino acid residue Y208), (ii) one or more amino acids between amino acid residues 264-284 (e.g., one or more amino acids between amino acid residues 269-279, e.g., ami Modified EPA protein in which (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), and (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519) are independently selected from, or are added next to or substitute for one or more amino acids at the same position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. [Item 2] The amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and is modified in that the amino acid sequence includes two (or more) consensus sequences selected from:D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the two (or more) consensus sequences are, respectively: (i) one or more amino acids between amino acid residues 198-218 of SEQ ID NO: 1 (e.g., one or more amino acids between amino acid residues 203-213, e.g., amino acid residue Y208), (ii) one or more amino acids between amino acid residues 264-284 (e.g., one or more amino acids between amino acid residues 269-279, e.g., amino acid residue R274). , (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230-250 (e.g., one or more amino acids between amino acid residues 235-245; e.g., amino acid residue K240), or a modified EPA (Exotoxin A of Pseudomonas aeruginosa) protein as described in item 1, which is independently selected from (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230-250 (e.g., one or more amino acids between amino acid residues 235-245; e.g., amino acid residue K240), or which is added next to or substitutes one or more amino acids in the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. [Item 3] The amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and is modified in that the amino acid sequence includes three (or more) consensus sequences selected from:D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the three (or more) consensus sequences are, respectively: (i) one or more amino acids between amino acid residues 198-218 of SEQ ID NO: 1 (e.g., one or more amino acids between amino acid residues 203-213, e.g., amino acid residue Y208), (ii) one or more amino acids between amino acid residues 264-284 (e.g., one or more amino acids between amino acid residues 269-279, e.g., amino acid residue R274). , (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230-250 (e.g., one or more amino acids between amino acid residues 235-245; e.g., amino acid residue K240), or a modified EPA (Exotoxin A of Pseudomonas aeruginosa) protein as described in item 1, which is independently selected from (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230-250 (e.g., one or more amino acids between amino acid residues 235-245; e.g., amino acid residue K240), or which is added next to or substitutes one or more amino acids in the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. [Item 4] A modified EPA protein according to any one of items 1 to 3, wherein a consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) independently substitutes one or more amino acids in the amino acid sequence of SEQ ID NO: 1, or in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (for example, each consensus sequence substitutes a single amino acid residue, e.g., a single amino acid residue selected from Y208, R274, S318, and A519). [Item 5] A modified EPA protein according to any one of items 1 to 4, wherein a further consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and JD / EXNZS / TU (SEQ ID NO: 5) (where X and Z are independently any amino acid other than proline, and J and U are independently 1 to 5 naturally occurring amino acid residues) is added to the N-terminus of SEQ ID NO: 1, or adjacent to or substituting one or more amino acids at the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. [Item 6] A modified EPA protein according to any one of items 1 to 5, wherein a further consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and JD / EXNZS / TU (SEQ ID NO: 5) (where X and Z are independently any amino acid other than proline, and J and U are independently 1 to 5 naturally occurring amino acid residues) is added to the C-terminus of SEQ ID NO: 1, or adjacent to or substituting one or more amino acids at the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. [Item 7] At least one consensus sequence selected from D / E-X-N-Z-S / T (SEQ ID NO: 2) and K-D / E-X-N-Z-S / T-K (SEQ ID NO: 3) (where X and Z are each independently any amino acid other than proline) is (i) one or more amino acids between amino acid residues 198 and 218 of SEQ ID NO: 1 (for example, one or more amino acids between amino acid residues 203 and 213, for example amino acid residue Y208), (ii) one or more amino acids between amino acid residues 308 and 328 of SEQ ID NO: 1 (for example, one or more amino acids between amino acid residues 313 and 323, for example amino acid residue S318), or (iii) one or more amino acids between amino acid residues 509 and 529 of SEQ ID NO: 1 (for example, one or more amino acids between amino acid residues 514 and 524; for example amino acid residue A519), or is added adjacent to or substitutes for an amino acid at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. The modified EPA protein according to any one of items 1 to 6. [Item 8] The modified EPA protein according to any one of items 1 to 7, wherein the amino acid sequence comprises a substitution of leucine 552 (or at the position equivalent to L552 of SEQ ID NO: 1) with valine (L552V) and a deletion of glutamine 553 (or at the position equivalent to E553 of SEQ ID NO: 1) (ΔE553). [Item 9] A conjugate (for example, a bioconjugate) comprising the modified EPA protein according to any one of items 1 to 8, covalently linked to an antigen (for example, a sugar antigen, optionally a bacterial polysaccharide antigen). [Item 10] The conjugate (for example, a bioconjugate) according to item 9, wherein the antigen is a sugar, optionally a bacterial polysaccharide (for example, derived from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or Staphylococcus aureus). [Item 11] i) one or more nucleotide sequences, optionally containing polysaccharide synthesis genes integrated into the host cell genome, for generating bacterial polysaccharide antigens (e.g., O antigens derived from Gram-negative bacteria, optionally from Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Gram-positive bacteria, optionally from Streptococcus pneumoniae or Staphylococcus aureus) or yeast polysaccharide antigens or mammalian polysaccharide antigens; ii) Nucleotide sequences that encode heterologous oligosaccharide transferases, sometimes within plasmids; iii) A nucleotide sequence that may be encoded within the plasmid, which may contain the modified EPA protein described in any one of items 1 to 8. Host cells containing these cells. [Item 12] A method for producing a bioconjugate containing a modified EPA protein linked to a polysaccharide, comprising (i) culturing host cells as described in item 11 under conditions suitable for glycoprotein production, and (ii) isolating the bioconjugate, optionally from a peripheral extract derived from the host cells. [Item 13] An immunogenic composition comprising a conjugate (e.g., a bioconjugate) as described in item 9 or 10, and optionally pharmaceutically acceptable excipients and / or carriers. [Item 14] A vaccine comprising the immunogenic composition described in item 13, and optionally an adjuvant. [Item 15] A method for inducing an immune response in a subject (e.g., a human), comprising administering a therapeutically or prophylactically effective amount of a conjugate (e.g., a bioconjugate) described in item 9 or 10, an immunogenic composition described in item 13, or a vaccine described in item 14 to a subject (e.g., a human) in need thereof. [Brief explanation of the drawing]

[0020] [Figure 1]This figure shows the 3D structure of the EPA, represented by an illustration, and the selected positions Y208, R274, S318, and A519 for insertion of the glycosite, represented by a sphere. [Figure 2-1] This figure shows the sequence alignment of EPA mutants containing a single glycosite at positions Y208, R274, S318, and A519, compared to EPA without a glycosite. EPA_detox (SEQ ID NO: 1), EPA_mut_Y208 (SEQ ID NO: 34), EPA_mutR274 (SEQ ID NO: 35), EPA_mut_S318 (SEQ ID NO: 36), and EPA_mut_A519 (SEQ ID NO: 37). [Figure 2-2] This is a continuation of Figure 2-1. [Figure 2-3] This is a continuation of Figure 2-2. [Figure 2-4] This is a continuation of Figure 2-3. [Figure 3] This figure shows the SDS-PAGE analysis of IMAC (fixed metal affinity chromatography) enriched peripheral extracts of Escherichia coli strains expressing an EPA variant in which KpO antigen (Klebsiella pneumoniae O antigen) polysaccharide is generated and PglB and glycosite KDQNATK (SEQ ID NO: 4) are introduced at the following positions: Y208 (lane 1), K240 (lane 2), R274 (lane 3), S318 (lane 4), A376 (lane 5), A519 (lane 6), and K240 and A376 (lane 7). The non-glycosylated EPA carrier is labeled with bands corresponding to KpO antigen-EPA bioconjugates, which occupy one and two glycosites. [Figure 4]This figure shows the SDS-PAGE analysis of IMAC-enriched peripheral extracts of E. coli strains expressing EPA mutants that generate KpO antigen polysaccharide and introduce PglB and glycosite KDQNATK (SEQ ID NO: 4) at the following positions: K240 and A376 (lane 1), Y208 and R274 (lane 2), Y208 and S318 (lane 3), Y208 and A519 (lane 4), R274 and S318 (lane 5), R274 and A519 (lane 6), S318 and A519 (lane 7), and Y208, R274, and A519 (lane 8). The non-glycosylated EPA carrier is labeled with bands corresponding to KpO antigen-EPA bioconjugates occupying one, two, and three glycosites. [Figure 5-1]It generates KpO antigen polysaccharide and PglB, and 1 to 7 glycosites at the following positions: Y208 (lane 1), K240 (lane 2), R274 (lane 3), S318 (lane 4), A376 (lane 5), A519 (lane 6), K240 and A376 (lane 7), Y208 and R274 (lane 8), Y208 and S318 (lane 9), Y208 and A519 (lane 8) N10), R274 and S318 (lane 11), R274 and A519 (lane 12), S318 and A519 (lane 13), Y208, R274, and A519 (lane 14), N-terminal glycotag, and K240 and A376, and C-terminal glycotag (lane 15), N-terminal glycotag, and Y208, R274, and A519 (lane 16), N-terminal glycotag, and Y208, R274, and A519, as well as the C-terminal glycotag (lane 17), N-terminal glycotag, and Y208, S318, and A519, as well as the C-terminal glycotag (lane 18), N-terminal glycotag, and R274, S318, and A519, as well as the C-terminal glycotag (lane 19), N-terminal glycotag, and Y208, R274, S318, and A519, and This figure shows immunoblot analysis of peripheral extracts of Escherichia coli strains expressing EPA mutants introduced into the C-terminal glycotag (lane 20), N-terminal glycotag, and Y208, K240, R274, S318, and A519 (lane 21), as well as the N-terminal glycotag, and Y208, K240, R274, S318, and A519, as well as the C-terminal glycotag (lane 22). The upper panel shows immunoblots probed with anti-KpO antigen antiserum, while the lower panel shows immunoblots probed with anti-EPA antibody. The non-glycosylated EPA carrier is labeled with a band corresponding to the KpO antigen-EPA bioconjugate, which occupies 1 to 7 glycosites. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 5-3] This is a continuation of Figure 5-2. [Figure 6]It generates KpO antigen polysaccharide and PglB and glycosite KDQNATK (SEQ ID NO: 4) at the following positions: N-terminal glycotag, and Y208, R274, and A519, and C-terminal glycotag (lane 1), N-terminal glycotag, and Y208, S318, and A519, and C-terminal glycotag (lane 2), N-terminal glycotag, and R274, S318, and A519, and C-terminal glycotag (lane 3), N-terminal glycotag, and Y208, R274, S3 This figure shows the SDS-PAGE analysis of peripheral extracts of Escherichia coli strains expressing EPA variants introduced into 18, A519, and the C-terminal glycotag (lane 4), N-terminal glycotag, and Y208, K240, R274, S318, and A519 (lane 5), N-terminal glycotag, and Y208, K240, R274, S318, and A519, and the C-terminal glycotag (lane 6), N-terminal glycotag, and Y208, R274, and A519 (lane 7). Bands corresponding to the KpO antigen-EPA bioconjugate are labeled with arrows. The first row of the table shows the number of glycosites. [Figure 7] This figure shows the SDS-PAGE analysis of IMAC-enriched peripheral extracts of Escherichia coli strains that produce Sf2a Flexner's Shigella 2a (left) or Sp11A Streptococcus pneumoniae 11A (right) polysaccharides and express EPA mutants in which glycosites are introduced at PglB and K240, a second glycosite at A376 (lane 1), or three glycosites at positions Y208, R274, and A519 (lane 2). [Figure 8] This figure shows the SDS-PAGE analysis of IMAC-enriched peripheral extracts of Escherichia coli strains that produce two different Klebsiella pneumoniae O antigen polysaccharides (left and right) and express EPA mutants that have PglB and glycosites at positions Y208, R274, and A519 (lane 1), or an N-terminal glycotag and glycosites at positions Y208, R274, and A519 (lane 2). [Figure 9]This figure shows immunoblot analysis of peripheral extracts from E. coli strains expressing EPA mutants that generate Sf2a, Sp33F, PaO6, or PaO11 antigen polysaccharides (lower panel), and have PglB and one glycosite KDQNATK (SEQ ID NO: 4) introduced at the following positions: Y208 (lane 1), D218 (lane 2), R274 (lane 3), R279 (lane 4), S318 (lane 5), G323 (lane 6), A376 (lane 7), A519 (lane 8), and G525 (lane 9). The EPA bioconjugate and the band corresponding to the non-glycosylated EPA carrier protein are labeled with arrows. [Modes for carrying out the invention]

[0021] definition Carrier proteins are proteins that can covalently bind to antigens (e.g., monosaccharide antigens, bacterial polysaccharide antigens) to create conjugates (e.g., bioconjugates). Carrier proteins activate T cell-mediated immunity against the conjugated antigen.

[0022] EPA: Exotoxin A of Pseudomonas aeruginosa (also known as "Exotoxin of Pseudomonas aeruginosa," "EPA," or "ETA")

[0023] All amino acids other than proline (pro, P): This refers to amino acids selected from the group consisting of alanine (ala, A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0024] Naturally occurring amino acid residues: Amino acids that are naturally incorporated into polypeptides. In particular, the 20 amino acids encoded by the universal genetic code: alanine (ala, A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0025] Glycosyltransferases (GTFs, Gtf): Enzymes that establish glycosidic bonds. Glycotransferases are enzymes that catalyze the formation of glycosidic bonds, thereby creating glycosides. For example, they catalyze the transfer of a monosaccharide moiety from an activated nucleotide sugar (also known as a "glycosyl donor") to a nucleophilic glycosyl acceptor molecule. This nucleophilic molecule can be oxygen-, carbon-, nitrogen-, or sulfur-based.

[0026] O antigen (also known as O-specific polysaccharide or O side chain): A component of surface lipopolysaccharide (LPS) in Gram-negative bacteria. Examples include O antigens derived from Pseudomonas aeruginosa and Klebsiella pneumoniae.

[0027] Lipopolysaccharide (LPS): A large molecule composed of lipids and polysaccharides linked together by covalent bonds.

[0028] Capsular polysaccharides (CPs): Polysaccharides found on the cell walls of bacteria. Examples include capsular polysaccharides derived from Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, and Staphylococcus aureus.

[0029] wzy: A polysaccharide polymerase gene that encodes an enzyme that catalyzes polysaccharide polymerization. The encoded enzyme moves oligosaccharide units to the non-reducing end to form glycosidic bonds.

[0030] waaL: An O antigen ligase gene that encodes a membrane-binding enzyme. The encoded enzyme transfers undecaprenyl diphosphate (UPP)-bound O antigen to lipid A core oligosaccharides, forming lipopolysaccharides.

[0031] As used herein, the term "conjugate" refers to a carrier protein that covalently binds to an antigen.

[0032] As used herein, the term "bioconjugate" refers to a conjugate between a protein (e.g., a carrier protein) and an antigen (e.g., a monosaccharide antigen, e.g., a bacterial polysaccharide antigen) prepared in a host cell background, where the host cell mechanism binds the antigen to the protein (e.g., N-linked glycosylation).

[0033] As used herein, the term "modified protein" means a protein that has been altered (by one or more means) compared to the wild type (for example, "modified EPA protein" excludes the wild-type EPA protein).

[0034] As used herein, the term “immunogenic fragment” means a portion of an antigen smaller than the whole that is specific to the fragment and capable of inducing a humoral and / or cellular immune response in an animal host, such as a human. Protein fragments can be produced using techniques known in the art, for example, by recombination, proteolytic digestion, or chemosynthesis. Internal or terminal fragments of a polypeptide can be produced by removing one or more nucleotides from one end (for terminal fragments) or both ends (for internal fragments) of the nucleic acid encoding the polypeptide. Typically, the fragments contain at least 10, 20, 30, 40, or 50 consecutive amino acids of the full-length sequence. Fragments can be readily modified by adding or removing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 amino acids from either the N-terminus or the C-terminus, or both. Modified EPA protein fragments still include the modifications described to be made into an EPA protein.

[0035] As used herein, the term “conservative amino acid substitution” encompasses substitutions of native amino acid residues with non-native residues that have little to no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position, and that do not reduce immunogenicity. For example, this may be substitutions within the following group: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Conservative amino acid modifications to the sequence of a polypeptide (and corresponding modifications to the coding nucleotide) may produce polypeptides having similar functional and chemical properties to those of the parent polypeptide.

[0036] As used herein, the term "deletion" refers to the removal of one or more amino acid residues from a protein sequence. Typically, approximately 1 to 6 residues (e.g., 1 to 4 residues) are deleted at any single site within the protein molecule.

[0037] As used herein, the terms “insertion” or “addition” (including other tenses, e.g., “inserted”) mean the addition of one or more non-native amino acid residues within a protein sequence, or, as the context may require, the addition of one or more non-native nucleotides within a polynucleotide sequence. Typically, about 1 to 10 residues (e.g., 1 to 7 residues, 1 to 6 residues, or 1 to 4 residues) are inserted into any one site within a protein molecule.

[0038] As used herein, the term "adjacent to" refers to the addition of one or more non-native amino acid residues in a protein sequence at a position adjacent to a reference amino acid or amino acid region. For example, "adjacent to one or more amino acids between amino acid residues 198–218" means an addition at a position adjacent to any one of amino acid residues 198–218 (including adjacent to amino acid residues 198 or 218).

[0039] As used herein, the term "glycocyte" refers to an amino acid sequence recognized by bacterial oligosaccharide transferases, such as PglB of C. jejuni.

[0040] A "consensus sequence" is a sequence having a specific structure and / or function. As used herein, the term "consensus sequence" refers to a sequence containing a glycosite. The consensus sequence may be selected from: the 5-amino acid consensus sequence D / EXNZS / T (SEQ ID NO: 2), the 7-amino acid consensus sequence KD / EXNZS / TK (SEQ ID NO: 3), or an extended consensus sequence (e.g., JD / EXNZS / TU (SEQ ID NO: 5)).

[0041] As used herein, the term "introduced into" is used to refer to the location and manner in which a consensus sequence is inserted into an amino acid sequence. A glycosite introduced at the N-terminal or C-terminal position of a protein may be added adjacent to the N-terminal or C-terminal amino acid sequence, but a consensus sequence (or glycosite) introduced at a specific amino acid residue in a protein, for example, Y208, may substitute for an amino acid.

[0042] Unless otherwise specified, a numerical range (e.g., "25-30") includes the endpoint (i.e., values ​​25 and 30). For example, "between amino acids 198-218 of SEQ ID NO: 1" refers to the position within the amino acid sequence of SEQ ID NO: 1 between amino acids 198 and 218, including both amino acids 198 and 218.

[0043] The term “identical” or “identity” percentage refers to a nucleotide or amino acid sequence that is identical or has a specified percentage of identical nucleotide or amino acid residues (e.g., 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity across a specified region) when compared and aligned for the greatest match, for example, using a sequence comparison algorithm or by manual alignment and visual inspection. Identity between polypeptides can be calculated using various algorithms. Generally, when calculating identity percentages, the two sequences being compared are aligned to give the greatest correlation between the sequences. This may include inserting “gaps” into one or both sequences to enhance the degree of alignment. For example, the Needleman-Wunsch algorithm for global alignment (Needleman and Wunsch 1970, J. Mol. Biol. Vol. 48: pp. 443-453) or the Smith-Waterman algorithm for local alignment (Smith and Waterman 1981, J. Mol. Biol. Vol. 147: pp. 195-197) may be used, for example, with default parameters (Smith-Waterman uses the BLOSUM 62 scoring matrix with a gap opening penalty of 10 and a gap extension penalty of 1). A preferred algorithm is described by Dufresne et al. (Nature Biotechnology, 2002, Vol. 20, pp. 1269-1271) and is used in the software GenePAST (Genome Quest Life Sciences, Inc., Boston, MA). The GenePAST "identity percentage" algorithm finds the best fit between the query sequence and the subject sequence and expresses the alignment as an accurate percentage. GenePAST does not perform alignment scoring adjustments that take into account the biological relevance between the query sequence and the subject sequence.The identity between the two sequences is calculated over the entire length of both sequences and expressed as a percentage of the reference sequence (e.g., sequence number 1 of the present invention).

[0044] As used herein, the term “recombinant” means artificial or synthetic. In certain embodiments, “recombinant protein” refers to a protein produced using a recombinant nucleotide sequence (a nucleotide sequence introduced into a host cell). In certain embodiments, the nucleotide sequence encoding the “recombinant protein” is heterologous to that of the host cell.

[0045] As used herein, the terms “isolated” or “purified” mean a protein, conjugate (e.g., bioconjugate), polynucleotide, or vector in a form not found in nature. Examples include proteins, conjugates (e.g., bioconjugate), polynucleotides, or vectors isolated from host cells or organisms (including crude extracts) or extracted from their natural environment. In certain embodiments, an isolated or purified protein is a protein that is essentially free from all other polypeptides to which it is naturally bound (or naturally in contact).

[0046] As used herein, the term "subject" refers to animals, in particular mammals, such as primates (e.g., humans).

[0047] As used herein, the term “effective amount” refers to an amount of treatment that has a preventive and / or therapeutic effect in the context of administering treatment (e.g., the immunogenic composition or vaccine of the present invention) to a subject. In a particular embodiment, “effective dose” means a therapeutic dose sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or resolving the severity of a bacterial infection or associated symptoms; (ii) shortening the duration of a bacterial infection or associated symptoms; (iii) preventing the progression of a bacterial infection or associated symptoms; (iv) causing regression of a bacterial infection or associated symptoms; (v) preventing the development or onset of a bacterial infection or associated symptoms; (vi) preventing the recurrence of a bacterial infection or associated symptoms; (vii) reducing organ failure associated with a bacterial infection; (viii) reducing hospitalizations in subjects with a bacterial infection; (ix) shortening the length of hospitalizations in subjects with a bacterial infection; (x) increasing the survival of subjects with a bacterial infection; (xi) eliminating a bacterial infection in a subject; (xii) inhibiting or reducing bacterial replication in a subject; and / or (xiii) enhancing or improving the preventive or therapeutic effect of another treatment.

[0048] The term "comprises" is open-ended and means "includes." Therefore, unless the context requires a different interpretation, the words "comprises" or "has," and their variations (including "comprise" and "comprising," or "have" and "having," respectively) will be understood to mean the inclusion of the explicitly stated compound, molecule, composition, or process, but not to exclude any other compound, molecule, composition, or process. The terms "comprises" and "has," when used as transitional clauses herein, are open-ended, while the term "consists of," when used as a transitional clause herein, is closed (i.e., limited to those listed, and nothing more). In certain embodiments, and for readability, the word "is" may be used instead of "consists of" or "consists of." The abbreviation "e.g." (eg) originates from the Latin "exempli gratia" (for example) and is used in this specification to indicate non-restrictive examples. Therefore, the abbreviation "e.g." (eg) is synonymous with the term "for example."

[0049] EPA protein Exotoxin A (also known as "EPA" or "ETA") of Pseudomonas aeruginosa is a secreted bacterial toxin that is a member of the ADP-ribosyltransferase toxin family. The EPA protein useful in this invention can be produced by methods known in the art, taking into consideration this disclosure. See, for example, Ihssen et al. (2010) Microbial Cell Factories Vol. 9:p. 61, International Publication No. 2006 / 119987, International Publication No. 2009 / 104074, and International Publication No. 2015 / 124769A1. Exotoxin A from Pseudomonas aeruginosa lineage PA103 was cloned and sequenced by Gray et al. (1984) Proc. Nati. Acad. Sci. USA Vol. 81, pp. 2645-2649. Comparison of the derived NH2-terminal amino acid sequence with that determined by sequence analysis of the secreted protein showed that EPA is produced as a 638-amino acid precursor, from which 25 highly hydrophobic leader peptides are removed during the secretion process (see Figure 1 in Gray et al. (1984)). The sequence within the EPA structural gene appears to be well conserved across strains (Vasil et al. (1986), Infection and Immunity, May 1986, pp. 538-548).

[0050] Since EPA is a toxin, it needs to be detoxified before it can be administered in vivo (i.e., made non-toxic to mammals, such as humans, when provided in a protective dosage). The modified EPA protein of the present invention may be genetically detoxified (i.e., by mutation). The genetically detoxified sequence may have unwanted activity, such as ADP-ribosyltransferase activity, removed, while reducing toxicity and retaining the ability to induce anti-EPA protective and / or neutralizing antibodies after administration to humans. The genetically detoxified sequence may retain its immunogenic epitope. The modified EPA protein may be genetically detoxified by one or more point mutations. For example, detoxification can be achieved by catalytically mutating and deleting essential residues, as described by Lukac et al. (1988), Infect Immun, Vol. 56: pp. 3095-3098, and Ho et al. (2006), Hum Vaccin, Vol. 2: pp. 89-98, for example, by substitution of leucine 552 with valine (L552V) and deletion of glutamic acid 553 (ΔE553). Detoxification can be achieved by catalytically mutating / deleting the essential residue L552VΔE553 using quick-change mutagenesis (Stratagene) and phosphorylated oligonucleotides 5'-GAAGGCGGGCGCGTGACCATTCTCGGC (SEQ ID NO: 38) and 5'-GCCGAGAATGGTCACGCGCCCGCCTTC (SEQ ID NO: 39), resulting in the construct pGVXN70. Detoxification can be measured by determining the inhibition of ADP-ribosyltransferase and cytotoxic activity according to the methodology described in Lukac et al. (1988), Infect Immun, Vol. 56: pp. 3095-3098, and the references cited herein, namely Douglas et al. (1987), J. Bacteriol, Vol. 169: pp. 4962-4966 and Douglas et al. (1987).Detoxified EPA has lower ADP-ribosyltransferase activity and cytotoxic activity than wild-type EPA, and is appropriately the same as or lower than that of modified EPA described by Lukac et al. (1988), i.e., ΔE553 EPA (EPA with glutamic acid 533 deletion). Therefore, the modified EPA protein of the present invention may be the amino acid sequence of SEQ ID NO: 1 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1). The modified EPA protein may include a substitution of leucine 552 with valine (L552V) and a deletion of glutamine 553 (ΔE553) with respect to the amino acid sequence of SEQ ID NO: 1 (or the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1). The modified EPA protein of the present invention may be the amino acid sequence of SEQ ID NO: 1 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1), including the substitution of leucine 552 with valine (L552V). The modified EPA protein of the present invention may be the amino acid sequence of SEQ ID NO: 1 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1), including the deletion of glutamine 553 (ΔE553). Preferably, the modified EPA protein of the present invention is modified by the substitution of leucine 552 with valine (L552V) and the deletion of glutamine 553 (ΔE553). Preferably, the modified EPA protein of the present invention has the amino acid sequence of SEQ ID NO: 1 (or the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1), which includes a substitution of leucine 552 with valine (L552V) and a deletion of glutamine 553 (ΔE553).

[0051] In one embodiment of the present invention, the EPA protein has the amino acid sequence of SEQ ID NO: 1: Sequence ID 1 EPA sequence AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFV RAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWN QVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARS QDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGLDPSSIPDKEQAISALPDYASQPGKPPREDLK Sequence ID 1: EPA sequence (numbered amino acids 1-612) 10 20 30 40 50 60 AEEAFDLWNE CAKACVLDLK DGVRSSRMSV DPAIADTNGQ GVLHYSMVLE GGNDALKLAI 70 80 90 100 110 120 DNALSITSDG LTIRLEGGVE PNKPVRYSYT RQARGSWSLN WLVPIGHEKP SNIKVFIHEL 130 140 150 160 170 180 NAGNQLSHMS PIYTIEMGDE LLAKLARDAT FFVRAHESNE MQPTLAISHA GVSVVMAQAQ 190 200 210 220 230 240 PRREKRWSEW ASGKVLCLLD PLDGVYNYLA QQRCNLDDTW EGKIYRVLAG NPAKHDLDIK 250 260 270 280 290 300 PTVISHRLHF PEGGSLAALT AHQACHLPLE AFTRHRQPRG WEQLEQCGYP VQRLVALYLA 310 320 330 340 350 360 ARLSWNQVDQ VIRNALASPG SGGDLGEAIR EQPEQARLAL TLAAAESERF VRQGTGNDEA 370 380 390 400 410 420 GAASADVVSL TCPVAAGECA GPADSGDALL ERNYPTGAEF LGDGGDVSFS TRGTQNWTVE 430 440 450 460 470 480 RLLQAHRQLE ERGYVFVGYH GTFLEAAQSI VFGGVRARSQ DLDAIWRGFY IAGDPALAYG 490 500 510 520 530 540 YAQDQEPDAR GRIRNGALLR VYVPRWSLPG FYRTGLTLAA PEAAGEVERL IGHPLPLRLD 550 560 570 580 590 600 AITGPEEEGG RVTILGWPLA ERTVVIPSAI PTDPRNVGGD LDPSSIPDKE QAISALPDYA 610 SQPGKPPRED PAGE

[0052] The term "modified EPA protein" refers to an EPA amino acid sequence (for example, the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequence to SEQ ID NO: 1), and the EPA amino acid sequence is modified by the addition, substitution, or deletion of one or more amino acids (for example, D / EXNZS / T (SEQ ID NO: 2), KD / EXNZS / TK (SEQ ID NO: 3), and / or the extended consensus sequence (for example, JD / EXNZS / TK)). The modified EPA protein may be the EPA amino acid sequence of SEQ ID NO: 1, which is modified by the addition of a consensus sequence selected from U (SEQ ID NO: 5); and / or by the substitution of one or more amino acids with a consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3). For example, the modified EPA protein may be the EPA amino acid sequence of SEQ ID NO: 1, which is modified by the addition of a consensus sequence selected from U (SEQ ID NO: 5); and / or by the substitution of one or more amino acids with a consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2), KD / EXNZS / TK (SEQ ID NO: 3), and / or an extended consensus sequence (e.g., JD / EXNZS / TU (SEQ ID NO: 3)). 5)) is modified in that it includes one (or more) consensus sequences selected from the above. As used herein, X and Z in the consensus sequences of the present invention are independently any amino acid other than proline; preferably, X is Q (glutamine) and Z is A (alanine). The modified EPA protein may also further include modifications (additions, substitutions, deletions). Preferably, the modified EPA protein of the present invention includes SEQ ID NO: 1 (or SEQ ID NO: 1 and at least 80%, 85%, 90%, 92%, 95%, The modified EPA protein of the present invention includes a substitution of leucine 552 with valine (L552V) and a deletion of glutamine 553 (ΔE553) with respect to the amino acid sequence at the same position within the 96%, 97%, 98%, or 99% identical amino acid sequence. In one embodiment, the modified EPA protein of the present invention is an EPA protein that does not exist in nature (i.e., is not natural). The modified EPA protein of the present invention may have an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.The modified EPA protein of the present invention may have an amino acid sequence that is at least 80% identical to SEQ ID NO: 1. The modified EPA protein of the present invention may have an amino acid sequence that is at least 85% identical to SEQ ID NO: 1. The modified EPA protein of the present invention may have an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. The modified EPA protein of the present invention may have an amino acid sequence that is at least 91% identical to SEQ ID NO: 1. The modified EPA protein of the present invention may have an amino acid sequence that is at least 92% identical to SEQ ID NO: 1. The modified EPA protein of the present invention may have an amino acid sequence that is at least 93% identical to SEQ ID NO: 1. The modified EPA protein of the present invention may have an amino acid sequence that is at least 94% identical to SEQ ID NO: 1. The modified EPA protein of the present invention may have an amino acid sequence that is at least 95% identical to SEQ ID NO: 1. The modified EPA protein of the present invention may have an amino acid sequence that is at least 96% identical to SEQ ID NO: 1. The modified EPA protein of the present invention may have an amino acid sequence that is at least 97% identical to SEQ ID NO: 1. The modified EPA protein of the present invention may have an amino acid sequence that is at least 98% identical to SEQ ID NO: 1. The modified EPA protein of the present invention may have an amino acid sequence that is at least 99% identical to SEQ ID NO: 1.

[0053] The present invention provides a modified EPA (Exotoxin A of Pseudomonas aeruginosa) protein having the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, wherein the amino acid sequence is modified in that it includes one (or more) consensus sequences selected from :D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), each of which is either adjacent to or substitutes one or more amino acids selected from specific amino acid residues within the EPA protein (consensus sequence region). These consensus sequence sites are independently selected from (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213, e.g., amino acid residue Y208), (ii) one or more amino acids between amino acid residues 264-284 (e.g., one or more amino acids between amino acid residues 269-279, e.g., amino acid residue R274), (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), and (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519) at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.Therefore, the present invention provides a modified EPA (Exotoxin A of Pseudomonas aeruginosa) protein having the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, which is modified in that the amino acid sequence includes one (or more) consensus sequences selected from :D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the one (or more) consensus sequences each include one or more amino acids between amino acid residues 198-218 at the same position in the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1 (e.g., one or more amino acids between amino acid residues 203-213). (ii) one or more amino acids independently selected from (ii) amino acid residue Y208, (iii) one or more amino acids between amino acid residues 264-284 (e.g., one or more amino acids between amino acid residues 269-279, e.g., amino acid residue R274), (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), and (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519), are added next to or substitute for one or more amino acids. In this specification, for example, the numbering of amino acid residues specified in (i) to (iv) above refers to amino acid positions within Sequence ID No. 1 (or, if the amino acid sequence is aligned with the amino acid sequence of Sequence ID No. 1 to maximize sequence identity between the two sequences, then this sequence is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to Sequence ID No. 1 in the positions corresponding to those of Sequence ID No. 1).

[0054] Furthermore, the present invention relates to a modified EPA (Exotoxin of Pseudomonas aeruginosa) having an amino acid sequence identical to that of SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of SEQ ID NO: 1. A) Provides a protein which is modified in that its amino acid sequence includes one (or more) consensus sequences selected from:D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the one (or more) consensus sequences are at the same position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to:SEQ ID NO: 1 (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213; or one or more amino acids between 205-211; e.g., amino acid residue D218; e.g., amino acid residue Y208), or (ii) one or more amino acids between amino acid residues 264-284 (iii) one or more amino acids independently selected from amino acid residues 269-279 (e.g., one or more amino acids between amino acid residues 271-277, e.g., amino acid residue R279, e.g., amino acid residue R274), (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, or one or more amino acids between amino acid residues 315-321, e.g., amino acid residue G323, e.g., amino acid residue S318), and (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524, or one or more amino acids between amino acid residues 516-522, e.g., amino acid residue G525, e.g., amino acid residue A519), are added next to or substitute for one or more amino acids.In this specification, for example, the numbering of amino acid residues specified in (i) to (iv) above refers to amino acid positions within Sequence ID No. 1 (or, if the amino acid sequence is aligned with the amino acid sequence of Sequence ID No. 1 to maximize sequence identity between the two sequences, then this sequence is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to Sequence ID No. 1 in the positions corresponding to those of Sequence ID No. 1).

[0055] Furthermore, the present invention provides a modified EPA (Exotoxin A of Pseudomonas aeruginosa) protein having the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, wherein the amino acid sequence is modified in that it includes two (or more) consensus sequences selected from :D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), and each of the two (or more) consensus sequences is either added next to or substitutes one or more amino acids selected from specific amino acid residues within the EPA protein (consensus sequence region). These consensus sequence sites are (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213, e.g., amino acid residue Y208) at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or (ii) one or more amino acids between amino acid residues 264-284 (e.g., one or more amino acids between amino acid residues 269-279, e.g., amino acid residue R27) 4) (iii) one or more amino acids between amino acid residues 308 to 328 (e.g., one or more amino acids between amino acid residues 313 to 323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509 to 529 (e.g., one or more amino acids between amino acid residues 514 to 524; e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230 to 250 (e.g., one or more amino acids between amino acid residues 235 to 245; e.g., amino acid residue K240) are independently selected.Therefore, the present invention relates to a modified EPA (Exotoxin of Pseudomonas aeruginosa) having an amino acid sequence identical to that of Sequence ID No. 1 by at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%. A) is provided, which is modified in that the amino acid sequence includes two (or more) consensus sequences selected from:D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the two (or more) consensus sequences are at equal positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to:SEQ ID NO: 1 (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213, e.g., amino acid residue Y208), or (ii) one or more amino acids between amino acid residues 264-284 It is either adjacent to or substitutes one or more amino acids independently selected from the following amino acids (e.g., one or more amino acids between amino acid residues 269-279, e.g., amino acid residue R274), (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230-250 (e.g., one or more amino acids between amino acid residues 235-245; e.g., amino acid residue K240).Furthermore, the present invention provides a modified EPA (Exotoxin A of Pseudomonas aeruginosa) having the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, which is modified in that the amino acid sequence includes two (or more) consensus sequences selected from :D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), each of which two (or more) consensus sequences is at the same position in the amino acid sequence of SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (e.g., one or more amino acids between amino acid residues 203-213, e.g., amino acid residue Y208), ( ii) It is attached next to or substitutes for one or more amino acids independently selected from (ii) one or more amino acids between amino acid residues 264-284 (e.g., one or more amino acids between amino acid residues 269-279, e.g., amino acid residue R274), (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), and (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519).

[0056] Furthermore, two (or more) consensus sequences are: at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences at the same positions: (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213; or one or more amino acids between 205-211; e.g., amino acid residue D218; e.g., amino acid residue Y208), (ii) one or more amino acids between amino acid residues 264-284 (e.g., one or more amino acids between amino acid residues 269-279; or one or more amino acids between amino acid residues 271-277; e.g., amino acid residue R279; e.g., amino acid residue R274), (i ii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323; or one or more amino acids between amino acid residues 315-321, e.g., amino acid residue G323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; or one or more amino acids between amino acid residues 516-522; e.g., amino acid residue G525, e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230-250 (e.g., one or more amino acids between amino acid residues 235-245; e.g., amino acid residue K240) can be independently selected.

[0057] Furthermore, the present invention provides a modified EPA (Exotoxin A of Pseudomonas aeruginosa) protein having the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, wherein the amino acid sequence is modified in that it includes three (or more) consensus sequences selected from :D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), each of which is either adjacent to or substitutes one or more amino acids selected from specific amino acid residues within the EPA protein (consensus sequence region). These consensus sequence sites are (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213, e.g., amino acid residue Y208) at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or (ii) one or more amino acids between amino acid residues 264-284 (e.g., one or more amino acids between amino acid residues 269-279, e.g., amino acid residue R27) 4) (iii) one or more amino acids between amino acid residues 308 to 328 (e.g., one or more amino acids between amino acid residues 313 to 323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509 to 529 (e.g., one or more amino acids between amino acid residues 514 to 524; e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230 to 250 (e.g., one or more amino acids between amino acid residues 235 to 245; e.g., amino acid residue K240) are independently selected.Therefore, the present invention relates to a modified EPA (Exotoxin of Pseudomonas aeruginosa) having an amino acid sequence identical to that of Sequence ID No. 1 by at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%. A) is provided, which is modified in that the amino acid sequence includes three (or more) consensus sequences selected from:D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the three (or more) consensus sequences are each (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213, e.g., amino acid residue Y208) at the same position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or (ii) one or more amino acids between amino acid residues 264-284 It is either adjacent to or substitutes one or more amino acids independently selected from the following amino acids (e.g., one or more amino acids between amino acid residues 269-279, e.g., amino acid residue R274), (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230-250 (e.g., one or more amino acids between amino acid residues 235-245; e.g., amino acid residue K240).

[0058] Furthermore, three (or more) consensus sequences are: at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences at the same positions: (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213; or one or more amino acids between 205-211; e.g., amino acid residue D218; e.g., amino acid residue Y208), (ii) one or more amino acids between amino acid residues 264-284 (e.g., one or more amino acids between amino acid residues 269-279; or one or more amino acids between amino acid residues 271-277; e.g., amino acid residue R279; e.g., amino acid residue R274), (i ii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323; or one or more amino acids between amino acid residues 315-321, e.g., amino acid residue G323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; or one or more amino acids between amino acid residues 516-522; e.g., amino acid residue G525, e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230-250 (e.g., one or more amino acids between amino acid residues 235-245; e.g., amino acid residue K240) can be independently selected.

[0059] Furthermore, the present invention provides a modified EPA (Exotoxin A of Pseudomonas aeruginosa) protein having the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, wherein the amino acid sequence is modified in that it includes four (or more) consensus sequences selected from :D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), each of which is either adjacent to or substitutes one or more amino acids selected from specific amino acid residues within the EPA protein (consensus sequence region). These consensus sequence sites are (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213, e.g., amino acid residue Y208) at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or (ii) one or more amino acids between amino acid residues 264-284 (e.g., one or more amino acids between amino acid residues 269-279, e.g., amino acid residue R27) 4) (iii) one or more amino acids between amino acid residues 308 to 328 (e.g., one or more amino acids between amino acid residues 313 to 323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509 to 529 (e.g., one or more amino acids between amino acid residues 514 to 524; e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230 to 250 (e.g., one or more amino acids between amino acid residues 235 to 245; e.g., amino acid residue K240) are independently selected.Therefore, the present invention relates to a modified EPA (Exotoxin of Pseudomonas aeruginosa) having an amino acid sequence identical to that of Sequence ID No. 1 by at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%. A) is provided, which is modified in that the amino acid sequence includes four (or more) consensus sequences selected from:D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the four (or more) consensus sequences are each (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213, e.g., amino acid residue Y208) at the same position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to:SEQ ID NO: 1, or (ii) one or more amino acids between amino acid residues 264-284 It is either adjacent to or substitutes one or more amino acids independently selected from the following amino acids (e.g., one or more amino acids between amino acid residues 269-279, e.g., amino acid residue R274), (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230-250 (e.g., one or more amino acids between amino acid residues 235-245; e.g., amino acid residue K240).

[0060] Furthermore, four (or more) consensus sequences are: at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences at the same positions within the same amino acid sequence: (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213; or one or more amino acids between 205-211; e.g., amino acid residue D218; e.g., amino acid residue Y208), (ii) one or more amino acids between amino acid residues 264-284 (e.g., one or more amino acids between amino acid residues 269-279; or one or more amino acids between amino acid residues 271-277; e.g., amino acid residue R279; e.g., amino acid residue R274), (i ii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323; or one or more amino acids between amino acid residues 315-321, e.g., amino acid residue G323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; or one or more amino acids between amino acid residues 516-522; e.g., amino acid residue G525, e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230-250 (e.g., one or more amino acids between amino acid residues 235-245; e.g., amino acid residue K240) can be independently selected.

[0061] The sequence of EPA sequence number 1 (with numbering) has amino acids Y208, K240, R274, S318, and A519 underlined. TIFF2026097994000001.tif98160

[0062] In one embodiment, the modified EPA protein of the present invention may be derived from an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, which is an immunogenic fragment and / or mutant of SEQ ID NO: 1.

[0063] In one embodiment, the modified EPA protein of the present invention may be derived from an immunogenic fragment of SEQ ID NO: 1 comprising at least about 15, at least about 20, at least about 40, or at least about 60 consecutive amino acid residues of SEQ ID NO: 1, the polypeptide being able to induce antibodies that bind to SEQ ID NO: 1.

[0064] It is well known that natural EPA consists of three distinct structural domains (Allured et al., Proc. Natl. Acad. Sci. USA, Vol. 83, pp. 1320-1324, March 1986): Domain I is an antiparallel β structure. It contains residues 1-252 and 365-404. It has 17 β chains. The first 13 chains form the structural core of the long β barrel. After chain 13 of Domain I, the peptide chains traverse one side of the barrel and lead to the second domain. Domain II (residues 253-364) consists of six consecutive a-helices, along with a disulfide linking helix A and helix B. Helices B and E are approximately 30 Å long; helices C and D are approximately 15 Å long. Domain III consists of the carboxyl terminus (one-third) of the molecule, residues 405-613. The most prominent structural feature of Domain III is its elongated clft. This domain has a less regular secondary structure than Domains I and II. The immunogenic fragments of the EPA protein of the present invention may be produced by the removal and / or modification of one or more of these domains. In one embodiment, the immunogenic fragment of SEQ ID NO: 1 may contain amino acid residues of domain I of SEQ ID NO: 1 (residues 1-252 and 365-404). In another embodiment, the immunogenic fragment of SEQ ID NO: 1 may contain amino acid residues of domain II of SEQ ID NO: 1 (residues 253-364). In another embodiment, the immunogenic fragment of SEQ ID NO: 1 may contain at least amino acid residues of domain III of SEQ ID NO: 1 (residues 405-612). In another embodiment, the immunogenic fragment of SEQ ID NO: 1 may contain amino acid residues of domain I of SEQ ID NO: 1 (residues 1-252 and 365-404) and amino acid residues of domain II of SEQ ID NO: 1 (residues 253-364). In another embodiment, the immunogenic fragment of SEQ ID NO: 1 may contain at least amino acid residues of domain II of SEQ ID NO: 1 (residues 253-364) and amino acid residues of domain III of SEQ ID NO: 1 (residues 405-612).

[0065] In EPA, there are eight cysteine ​​forms that form disulfides in a sequential order: Cys-11 forms a disulfide with Cys-15, Cys-197 forms a disulfide with Cys-214, Cys-265 forms a disulfide with Cys-287, and Cys-372 forms a disulfide with Cys-379. Appropriately, the immunogenic fragment of SEQ ID NO: 1 contains the eight cysteines of SEQ ID NO: 11, 15, 197, 214, 265, 287, 372, and 379 (i.e., these residues are unmodified). Therefore, appropriately, the modified EPA protein of the present invention contains the eight cysteines of SEQ ID NO: 1: Cys-11, Cys-15, Cys-197, Cys-214, Cys-265, Cys-287, Cys-372, and Cys-379, or equivalent cysteines in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0066] In one embodiment, the modified EPA protein of the present invention may be derived from an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, which is a variant of SEQ ID NO: 1 and differs from SEQ ID NO: 1 only by the deletion and / or addition and / or substitution of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids). The amino acid substitution may be conserved or non-conserved. In one embodiment, the amino acid substitution is conserved. Substitutions, deletions, additions, or any combination thereof may be combined within a single variant, as long as the variant is an immunogenic polypeptide. In one embodiment, the modified EPA protein of the present invention may be derived from a variant of SEQ ID NO: 1 in which amino acids 1-10, 5-10, 1-5, 1-3, 1-2, or one amino acid of SEQ ID NO: 1 are substituted or deleted.

[0067] Appropriately, the immunogenic fragment and / or variant of Sequence ID No. 1 contains a B cell or T cell epitope. Such epitopes may be predicted using, for example, 2D structural prediction using the PSIPRED program (David Jones, Brunel Bioinformatics Group, Dept. Biological Sciences, Brunel University, Uxbridge UB8 3PH, UK) and a combination of antigenicity indices calculated based on the method described by Jameson and Wolf (CABIOS 4: pp. 181-186

[1988] ).

[0068] In the modified EPA protein of the present invention, one or more consensus sequences are added next to one or more amino acids of SEQ ID NO: 1, or adjacent to an EPA amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or substitute one or more amino acids. In one embodiment of the present invention, one or more amino acids (e.g., 1 to 7 amino acids, e.g., 1 amino acid) of an EPA amino acid sequence (e.g., the amino acid sequence of SEQ ID NO: 1, or an EPA amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1) are substituted by a five-amino acid D / EXNZS / T (SEQ ID NO: 2) or a seven-amino acid KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence. For example, a single amino acid in an EPA amino acid sequence (e.g., SEQ ID NO: 1) may be substituted (i.e., replaced) by a D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence. In addition to the above, two, three, four, five, six, or seven amino acids in the EPA amino acid sequence (e.g., SEQ ID NO: 1, or an EPA amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1) may be substituted (i.e., replaced) with the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence. Preferably, a single amino acid in the EPA amino acid sequence (e.g., SEQ ID NO: 1) is substituted (i.e., replaced) with the KDQNATK (SEQ ID NO: 4) consensus sequence at an internal consensus sequence site (i.e., a consensus sequence site within the EPA amino acid sequence, rather than being added next to an N-terminal or C-terminal amino acid).Furthermore, the classic five-amino acid glycosylation consensus sequence (D / EXNZS / T (SEQ ID NO: 2)) may be extended on either side of the consensus sequence by 1 to 5 other amino acid residues for more effective glycosylation. For example, the extended consensus sequence may be JD / EXNZS / TU (SEQ ID NO: 5). As used herein, J and U are independently 1 to 5 naturally occurring amino acid residues, preferably J and U are independently 1 to 5 amino acid residues independently selected from glycine and / or serine, for example, B may be GSGGG and U may be GSGG. For example, the extended consensus sequence may be GSGGGD / EXNZS / TGSGG (SEQ ID NO: 25). Preferably, an extended consensus sequence, such as JD / EXNZS / TU (SEQ ID NO: 5) or GSGGGD / EXNZS / TGSGG (SEQ ID NO: 25), is used, where the consensus sequence is added next to the N-terminal or C-terminal amino acid of the EPA protein.

[0069] A combination of consensus sequences selected from the 5-amino acid consensus sequence D / EXNZS / T (SEQ ID NO: 1), the 7-amino acid consensus sequence KD / EXNZS / TK (SEQ ID NO: 3), and the extended consensus sequence (e.g., JD / EXNZS / TU (SEQ ID NO: 5)) may be used. For example, one, two, three, four, or five amino acids in the amino acid sequence of the carrier protein may each be independently substituted (i.e., replaced) by the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) consensus sequence (where X and Z are independently any amino acid other than proline (preferably X is Q (glutamine) and Z is A (alanine)) (e.g., KDQNATK (SEQ ID NO: 4)), and one or two consensus sequences JD / EXNZS / TU (SEQ ID NO: 5) (where J and U are independently one to five naturally occurring amino acid residues (preferably J and U are independently one to five amino acid residues independently selected from glycine and / or serine, e.g., GSGGGD / EXNZS / TGSGG (SEQ ID NO: 25))) may be added adjacent to the N-terminal or C-terminal amino acid of the carrier protein. Therefore, the carrier protein may contain one, two, three, four, or five consensus sequences selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline (preferably, X is Q (glutamine) and Z is A (alanine)). The carrier protein may further contain one or two elongated consensus sequences JD / EXNZS / TU (SEQ ID NO: 5), where J and U are independently one to five naturally occurring amino acid residues (preferably, J and U are independently one to five amino acid residues independently selected from glycine and / or serine, e.g., GSGGGD / EXNZS / TGSGG (SEQ ID NO: 25)).The modified EPA protein can be glycosylated by introducing one or more consensus sequences selected from the 5-amino acid consensus sequence D / EXNZS / T (SEQ ID NO: 2), the 7-amino acid consensus sequence KD / EXNZS / TK (SEQ ID NO: 3), and / or an extended consensus sequence (e.g., JD / EXNZS / TU (SEQ ID NO: 5)) according to the present invention. Thus, the present invention also provides the modified EPA protein of the present invention, which is glycosylated.

[0070] Location of the consensus array As previously described, the inventors have found that the position of a consensus sequence in a specific region / amino acid within the EPA amino acid sequence enables increased glycosylation efficiency and / or optimization of the manipulation of the N glycosylation site.

[0071] In the modified EPA protein of the present invention, the consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) may be added next to one or more amino acid residues between amino acids 198 and 218 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or it may substitute for one or more amino acid residues. More specifically, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may be added next to or substitute for one or more amino acid residues between amino acids 203-213 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. For example, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may substitute amino acid residue Y208 or D218 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. More specifically, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may be added next to or substitute for one or more amino acid residues between amino acids 205-211 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.Preferably, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may substitute amino acid residue Y208 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0072] In the modified EPA protein of the present invention, the consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) may be added next to one or more amino acid residues between amino acids 264 and 284 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or it may substitute for one or more amino acid residues. More specifically, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may be added next to or substitute for one or more amino acid residues between amino acids 269 and 279 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. For example, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may substitute amino acid residues R274 or R279 at the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. More specifically, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may be added next to or substitute for one or more amino acid residues between amino acids 271-277 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.Preferably, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may substitute amino acid residue R274 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0073] In the modified EPA protein of the present invention, the consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) may be added next to one or more amino acid residues between amino acids 308 and 328 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or it may substitute for one or more amino acid residues. More specifically, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may be added next to or substitute for one or more amino acid residues between amino acids 313-323 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. For example, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may substitute amino acid residue S318 or G323 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. More specifically, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may be added next to or substitute for one or more amino acid residues between amino acids 313-323 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.Preferably, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may substitute amino acid residue S318 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0074] In the modified EPA protein of the present invention, the consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) may be added next to one or more amino acid residues between amino acids 509 and 529 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or it may substitute for one or more amino acid residues. More specifically, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may be added next to or substitute for one or more amino acid residues between amino acids 514-524 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. For example, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may substitute amino acid residue A519 or G525 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. More specifically, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may be added next to or substitute for one or more amino acid residues between amino acids 516-522 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.Preferably, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may substitute amino acid residue A519 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0075] In the modified EPA protein of the present invention, the consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) may be added next to one or more amino acid residues between amino acids 230 and 250 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or it may substitute for one or more amino acid residues. More specifically, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may be added next to or substitute for one or more amino acid residues between amino acids 235 and 245 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. More specifically, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may be added next to or substitute for one or more amino acid residues between amino acids 237-243 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. Preferably, the D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) consensus sequence may substitute for amino acid residue K240 at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0076] In the modified EPA protein of the present invention, the consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2), KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)), or the elongated consensus sequence (e.g., JD / EXNZS / TU (SEQ ID NO: 5)) may be added next to one or more amino acid residues in the N-terminal 10 amino acids at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or may substitute for one or more amino acid residues. More specifically, a consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2), KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)), or an extended consensus sequence (e.g., JD / EXNZS / TU (SEQ ID NO: 5)) may be added next to one or more amino acid residues in the N-terminal 5 amino acids at the same position within the amino acid sequence of SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or may substitute for one or more amino acid residues. Preferably, a consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2), KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)), or an extended consensus sequence (e.g., JD / EXNZS / TU (SEQ ID NO: 5)) may be added next to the N-terminal amino acid at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0077] In the modified EPA protein of the present invention, the consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2), KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)), or the elongated consensus sequence (e.g., JD / EXNZS / TU (SEQ ID NO: 5)) may be added next to one or more amino acid residues within the C-terminal 10 amino acids at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or may substitute for one or more amino acid residues. More specifically, a consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2), KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)), or an extended consensus sequence (e.g., JD / EXNZS / TU (SEQ ID NO: 5)) may be added next to one or more amino acid residues within the same position in the C-terminal 5 amino acids within the amino acid sequence of SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequence to SEQ ID NO: 1, or may substitute for one or more amino acid residues. Preferably, a consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2), KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)), or an extended consensus sequence (e.g., JD / EXNZS / TU (SEQ ID NO: 5)) may be added next to the C-terminal amino acid at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0078] In the modified EPA protein of the present invention, the consensus sequences selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) may each independently substitute one or more amino acids in the amino acid sequence of SEQ ID NO: 1, or in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (for example, each consensus sequence substitutes a single amino acid residue, e.g., a single amino acid residue selected from Y208, D218, R274, R279, S318, G323, A519, and G525; for example, each consensus sequence substitutes a single amino acid residue, e.g., a single amino acid residue selected from Y208, R274, S318, and A519). In the modified EPA protein of the present invention, two or more consensus sequences selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) may each independently substitute one or more amino acids in the amino acid sequence of SEQ ID NO: 1, or in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (for example, each consensus sequence substitutes a single amino acid residue, e.g., a single amino acid residue selected from Y208, D218, R274, R279, S318, G323, A519, G525, and K240; for example, each consensus sequence substitutes a single amino acid residue, e.g., a single amino acid residue selected from Y208, R274, S318, A519, and K240).In the modified EPA protein of the present invention, three or more consensus sequences selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) may each independently substitute one or more amino acids in the amino acid sequence of SEQ ID NO: 1, or in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (for example, each consensus sequence substitutes a single amino acid residue, e.g., a single amino acid residue selected from Y208, D218, R274, R279, S318, G323, A519, G525, and K240; for example, each consensus sequence substitutes a single amino acid residue, e.g., a single amino acid residue selected from Y208, R274, S318, A519, and K240). In the modified EPA protein of the present invention, four or more consensus sequences selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) may each independently substitute one or more amino acids in the amino acid sequence of SEQ ID NO: 1, or in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (for example, each consensus sequence may substitute a single amino acid residue, e.g., a single amino acid residue selected from Y208, D218, R274, R279, S318, G323, A519, G525, and K240; for example, each consensus sequence may substitute a single amino acid residue, e.g., a single amino acid residue selected from Y208, R274, S318, A519, and K240).In the modified EPA protein of the present invention, five or more consensus sequences selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) may each independently substitute one or more amino acids in the amino acid sequence of SEQ ID NO: 1, or in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (for example, each consensus sequence substitutes a single amino acid residue, e.g., a single amino acid residue selected from Y208, D218, R274, R279, S318, G323, A519, G525, and K240; for example, each consensus sequence substitutes a single amino acid residue, e.g., a single amino acid residue selected from Y208, R274, S318, A519, and K240).

[0079] In the modified EPA protein of the present invention, the consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2), KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)), or the elongated consensus sequence JD / EXNZS / TU (SEQ ID NO: 5) (e.g., GSGGGD / EXNZS / TGSGG (SEQ ID NO: 25)) may be added next to one or more N-terminal amino acid residues at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or may substitute one or more amino acid residues. Therefore, the modified EPA protein of the present invention may include a further consensus sequence selected from:D / EXNZS / T (SEQ ID NO: 2) and JD / EXNZS / TU (SEQ ID NO: 5) (where X and Z are independently any amino acid other than proline, and J and U are independently 1 to 5 naturally occurring amino acid residues), which is added next to or substitutes one or more N-terminal amino acid residues at the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. For example, the modified EPA protein of the present invention may include a further consensus sequence selected from:D / EXNZS / T (SEQ ID NO: 2) and JD / EXNZS / TU (SEQ ID NO: 5) (where X and Z are independently any amino acid other than proline, and J and U are independently 1 to 5 naturally occurring amino acid residues), which is added next to the N-terminal amino acid residue at the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0080] In the modified EPA protein of the present invention, the consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2), KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)), or the elongated consensus sequence JD / EXNZS / TU (SEQ ID NO: 5) (e.g., GSGGGD / EXNZS / TGSGG (SEQ ID NO: 25)) may be added next to one or more C-terminal amino acid residues at the same position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or may substitute one or more amino acid residues. Therefore, the modified EPA protein of the present invention may include a further consensus sequence selected from:D / EXNZS / T (SEQ ID NO: 2) and JD / EXNZS / TU (SEQ ID NO: 5) (where X and Z are independently any amino acid other than proline, and J and U are independently 1 to 5 naturally occurring amino acid residues), which is added next to or substitutes one or more amino acids at the same position in the C-terminal amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 1 at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. For example, the modified EPA protein of the present invention may include a further consensus sequence selected from:D / EXNZS / T (SEQ ID NO: 2) and JD / EXNZS / TU (SEQ ID NO: 5) (where X and Z are independently any amino acid other than proline, and J and U are independently 1 to 5 naturally occurring amino acid residues), which is added next to the C-terminal amino acid at the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0081] In the modified EPA protein of the present invention, three or more consensus sequences selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) may each independently substitute one or more amino acids (for example, each consensus sequence may substitute a single amino acid residue, e.g., a single amino acid residue selected from Y208, R274, S318, A519, and K240), and the consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) may be added adjacent to one or more amino acid residues at the N-terminus. The consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) may be added next to one or more C-terminal amino acid residues at the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or may be added next to one or more C-terminal amino acid residues (e.g., added next to a C-terminal amino acid).

[0082] In the modified EPA protein of the present invention, four or more consensus sequences selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) may each independently substitute one or more amino acids (for example, each consensus sequence substitutes a single amino acid residue, e.g., a single amino acid residue selected from Y208, R274, S318, A519, and K240), and the consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) may be added adjacent to one or more amino acid residues at the N-terminus. The consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) may be adjacent to one or more C-terminal amino acid residues at the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or may be adjacent to one or more C-terminal amino acid residues (e.g., adjacent to a C-terminal amino acid).

[0083] In the modified EPA protein of the present invention, five or more consensus sequences selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) may have one or more amino acid substitutions (for example, each consensus sequence may have one amino acid residue substituted, e.g., one amino acid residue selected from Y208, R274, S318, A519, and K240), and the consensus sequences selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) may be added adjacent to one or more amino acid residues at the N-terminus. The consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) may be added next to one or more amino acid residues at the same position within the same amino acid sequence as SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or may be added next to one or more amino acid residues at the same position within the same amino acid sequence as SEQ ID NO: 1, or may be added next to one or more amino acid residues (e.g., next to a C-terminal amino acid).

[0084] The modified EPA protein of the present invention may contain at least one consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) (where X and Z are independently any amino acid other than proline), which is added next to or substitutes for (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213; e.g., amino acid residue Y208), (ii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323; e.g., amino acid residue S318), or (iii) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519) at the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. For example, the modified EPA protein of the present invention may contain a consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), which is added next to or substitutes for one or more amino acids between amino acid residues 198-218 at the same position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (e.g., one or more amino acids between amino acid residues 203-213, e.g., amino acid residue Y208). For example, the modified EPA protein of the present invention may contain a consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), which is added next to or substitutes for one or more amino acids between amino acid residues 308-328 at the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318).For example, the modified EPA protein of the present invention may contain a consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), which is added next to or substitutes for one or more amino acids between amino acid residues 509-529 at the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519).

[0085] The modified EPA protein of the present invention may contain two consensus sequences. The modified EPA protein may have the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, which is modified in that the amino acid sequence contains two consensus sequences, for example, the two consensus sequences are added next to or substitute for two amino acid residues of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. Therefore, the modified EPA protein of the present invention may contain two consensus sequences which optionally substitute amino acid residues selected from (i) Y208 and R274, (ii) Y208 and S318, (iii) Y208 and A519, (iv) R274 and S318, (v) R274 and A519, or (vi) S318 and A519 of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. The modified EPA protein of the present invention may contain two consensus sequences which substitute amino acid residues Y208 and R274 of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. For example, the modified EPA protein of the present invention may contain (or be derived from) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6. The modified EPA protein of the present invention may contain two consensus sequences in which amino acid residues Y208 and S318 of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, are substituted. For example, the modified EPA protein of the present invention may contain (or be derived from) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28.The modified EPA protein of the present invention may contain two consensus sequences in which amino acid residues Y208 and A519 of an amino acid sequence identical to or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO: 1 are substituted. For example, the modified EPA protein of the present invention may contain (or be derived from) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29. The modified EPA protein of the present invention may contain two consensus sequences in which amino acid residues R274 and S318 of an amino acid sequence identical to or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 are substituted. For example, the modified EPA protein of the present invention may contain (or be derived from) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30. The modified EPA protein of the present invention may contain two consensus sequences in which amino acid residues R274 and A519 of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, are substituted. For example, the modified EPA protein of the present invention may contain (or be derived from) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31. The modified EPA protein of the present invention may contain two consensus sequences in which amino acid residues S318 and A519 of an amino acid sequence identical to or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 are substituted. For example, the modified EPA protein of the present invention may contain (or be derived from) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[0086] The modified EPA protein of the present invention may contain three consensus sequences. The modified EPA protein may have an amino acid sequence identical to SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, which is modified in that the amino acid sequence contains three consensus sequences, for example, the three consensus sequences are added next to or substitute for three independently selected amino acid residues of the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1. Therefore, the modified EPA protein of the present invention may contain three consensus sequences, which may optionally substitute amino acid residues Y208, R274, and A519 of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. For example, the modified EPA protein of the present invention may contain (or be derived from) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[0087] The modified EPA protein of the present invention may contain four consensus sequences. The modified EPA protein may have an amino acid sequence identical to SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, which is modified in that the amino acid sequence contains four consensus sequences, for example, the four consensus sequences are added next to or substitute for four independently selected amino acid residues of the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. Therefore, the modified EPA protein of the present invention may contain four consensus sequences which optionally substitute amino acid residues Y208, R274, and A519 of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and are added adjacent to the N-terminal amino acid. For example, the modified EPA protein of the present invention may contain (or be derived from) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.

[0088] The modified EPA protein of the present invention may contain five consensus sequences. The modified EPA protein may have an amino acid sequence identical to SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, which is modified in that the amino acid sequence contains five consensus sequences, for example, the five consensus sequences are added next to or substitute for five independently selected amino acid residues of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. Therefore, the modified EPA protein of the present invention may contain five consensus sequences, which may be selected from substitutions of amino acid residue Y208, substitutions of amino acid residue R274, substitutions of amino acid residue S318, substitutions of amino acid residue A519, additions at the N-terminus (i.e., added to the N-terminus), and additions at the C-terminus (i.e., added to the C-terminus), which are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1. For example, the modified EPA protein may contain (or be derived from) (i) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to (i) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to (ii) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to (iii) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to (iii) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to (iii) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to (i) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to (iiii) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to (ii) an amino acid sequence that is at least 95%, 96%, 97%, 98%,

[0089] The modified EPA protein of the present invention may contain six consensus sequences. The modified EPA protein may have an amino acid sequence identical to SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, which is modified in that the amino acid sequence contains six consensus sequences, for example, the six consensus sequences are added next to or substitute for six independently selected amino acid residues of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. Therefore, the modified EPA protein of the present invention may contain six consensus sequences, which may be selected from substitutions of amino acid residue Y208, K240, R274, S318, A519, N-terminal additions (i.e., added to the N-terminus), and C-terminal additions (i.e., added to the C-terminus) of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1.

[0090] The modified EPA protein of the present invention may contain seven consensus sequences. The modified EPA protein may have an amino acid sequence identical to SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, which is modified in that the amino acid sequence contains seven consensus sequences, for example, the seven consensus sequences are added next to or substitute for seven independently selected amino acid residues of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. Therefore, the modified EPA protein of the present invention may contain seven consensus sequences, which may optionally substitute amino acid residues Y208, K240, R274, S318, and A519 of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and are added at the N-terminus (i.e., at the N-terminus) and at the C-terminus (i.e., at the C-terminus). For example, the modified EPA protein of the present invention may contain (or be derived from) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14.

[0091] A reference to "amino acids...between" (for example, between amino acids 198 and 218) will be understood by those skilled in the art to refer to the amino acid numbers counted sequentially from the N-terminus of the amino acid sequence, for example, "between amino acids 198 and 218 of SEQ ID NO: 1" will refer to the position within the amino acid sequence between amino acids 198 and 218 (including both amino acids 198 and 218) of SEQ ID NO: 1. Therefore, in the embodiment in which "a consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) (e.g., KDQNATK (SEQ ID NO: 4)) is added next to or substitutes for one or more amino acids between amino acid residues 198-218", the consensus sequence may be added next to or substitutes for any one (or more) amino acid numbers 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218 in SEQ ID NO: 1. Those skilled in the art will understand that if the EPA amino acid sequence is a variant and / or fragment of the amino acid sequence of SEQ ID NO: 1, for example, an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, then the reference to "aminotherium..." will refer to a position equal to the defined position if this sequence is aligned with the amino acid sequence of SEQ ID NO: 1 to maximize sequence identity between the two sequences (the sequence alignment tool is not limited to Clustal Omega (www.ebi.ac.ac.uk), MUSCLE (www.ebi.ac.uk), or T-coffee (www.tcoffee.org)). In one embodiment, the sequence alignment tool used is Clustal Omega (www.ebi.ac.ac.uk).

[0092] The amino acid numbers referred to herein correspond to the amino acids in SEQ ID NO: 1, and as previously described, those skilled in the art can determine by alignment the equivalent amino acid positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. Addition of amino acids to or deletion of amino acids from mutants and / or fragments of SEQ ID NO: 1 may result in different actual amino acid positions in the consensus sequence within the mutated sequence. However, by aligning the mutated sequence with the reference sequence, amino acids can be identified at positions equivalent to the corresponding amino acids in the reference sequence, thereby establishing suitable positions for addition or substitution of the consensus sequence.

[0093] The modified EPA protein of the present invention may be an isolated modified EPA protein. The modified EPA protein of the present invention may be a recombinant modified EPA protein. The modified EPA protein of the present invention may be an isolated recombinant modified EPA protein.

[0094] Composer array The modified EPA protein of the present invention comprises a D / EXNZS / T (SEQ ID NO: 2), KD / EXNZS / TK (SEQ ID NO: 3), or JD / EXNZS / TU (SEQ ID NO: 5) consensus sequence, where X and Z are independently any amino acid other than proline, and J and U are independently 1 to 5 naturally occurring amino acid residues. The classic five-amino acid glycosylation consensus sequence (D / EXNZS / T (SEQ ID NO: 2)) may be extended by 1 to 5 other amino acid residues to either side of the consensus sequence JD / EXNZS / TU (SEQ ID NO: 5) for more effective glycosylation (e.g., GSGGGD / EXNZS / TGSGG (SEQ ID NO: 25)). The classic five-amino acid glycosylation consensus sequence (D / EXNZS / T (SEQ ID NO: 2)) may be extended by lysine residues for more effective glycosylation (e.g., KD / EXNZS / TK (SEQ ID NO: 3)). Therefore, the consensus sequence within the modified EPA protein of the present invention may include (or be derived from) the D / EXNZS / T (Sequence ID 2) consensus sequence.

[0095] In the modified EPA protein of the present invention, the consensus sequence may be selected from D / EXNZS / T (SEQ ID NO: 2), KD / EXNZS / TK (SEQ ID NO: 3), or JD / EXNZS / TU (SEQ ID NO: 5), where X is Q (glutamine) and Z is A (alanine). In the modified EPA protein of the present invention, the consensus sequence may be selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X is Q (glutamine) and Z is A (alanine). In one embodiment, the consensus sequence is D / EXNZS / T (SEQ ID NO: 2) (where X is Q (glutamine) and Z is A (alanine)), for example, DQNAT (SEQ ID NO: 25), also known as "DQNAT". In one embodiment, the consensus sequence is KD / EXNZS / TK (SEQ ID NO: 3) (where X is Q (glutamine) and Z is A (alanine)), for example, KDQNATK (SEQ ID NO: 4), also known as "KDQNATK". In the modified EPA protein of the present invention, the consensus sequence may be selected from D / EXNZS / T (SEQ ID NO: 2), KD / EXNZS / TK (SEQ ID NO: 3), or JD / EXNZS / TU (SEQ ID NO: 5), where X is Q (glutamine) and Z is A (alanine), and J and U are 1 to 5 amino acid residues independently selected from glycine and / or serine.

[0096] In one embodiment, the modified EPA protein of the present invention contains at least two D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) consensus sequences. In one embodiment, the modified EPA protein of the present invention contains at least three D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) consensus sequences. In one embodiment, the modified EPA protein of the present invention contains at least four D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) consensus sequences. In one embodiment, the modified EPA protein of the present invention contains at least five D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) consensus sequences. In one embodiment, the modified EPA protein of the present invention contains at least six D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) consensus sequences. In one embodiment, the modified EPA protein of the present invention contains at least seven D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) consensus sequences. In one embodiment, the modified EPA protein of the present invention contains three to seven D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) consensus sequences. In one embodiment, the modified EPA protein of the present invention contains four to seven D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) consensus sequences. In one embodiment, the modified EPA protein of the present invention contains five to seven D / EXNZS / T (SEQ ID NO: 2) or KD / EXNZS / TK (SEQ ID NO: 3) consensus sequences.

[0097] The introduction of such glycosylation sites can be achieved, for example, by adding a new amino acid to the primary structure of a protein (i.e., by completely or partially adding a glycosylation site), or by mutating an existing amino acid in the protein to generate a glycosylation site (i.e., by mutating a selected amino acid in the protein rather than adding an amino acid to the protein to form a glycosylation site). In one embodiment, the consensus sequence is introduced by recombination into the EPA amino acid sequence of SEQ ID NO: 1, or into an EPA amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0098] The modified EPA protein of the present invention may further include a "peptide tag" or "tag," i.e., a sequence of amino acids that enables the isolation and / or identification of the modified EPA protein. For example, attaching a tag to the modified EPA protein of the present invention may be useful for the purification of the protein and therefore for the purification of a conjugate (e.g., bioconjugate) vaccine containing the tagged modified EPA protein. Exemplary tags that can be used herein include, but are not limited to, a histidine (HIS) tag (e.g., a hexahistidine tag or a 6XHis- tag), a FLAG-TAG, and an HA tag. In one embodiment, the tag is a hexahistidine tag. The tags used herein can be removed when they are no longer needed, for example, after the protein has been purified, for example, by chemical agents or by enzymatic means. Thus, the modified EPA protein of the present invention may further include a peptide tag. Optionally, the peptide tag is positioned at the C-terminus of the amino acid sequence. Optionally, the peptide tag contains six histidine residues at the C-terminus of the amino acid sequence. In one embodiment, the modified EPA protein of the present invention comprises (or comprises) an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to any one of the sequences of SEQ ID NOs. 6 to 14, and a peptide tag (e.g., six histidine residues at the C-terminus of the amino acid sequence).

[0099] In one embodiment, the modified EPA protein of the present invention includes a signal sequence that can direct the EPA protein towards the periplasm of a host cell (e.g., bacteria). The signal sequence, such as the periplasmic signal sequence, is typically removed by a signal peptidase during the transposition of the protein, for example, into the periplasm (i.e., the mature protein is a protein from which at least the signal sequence has been removed). The signal sequences include E. coli flagellin (FlgI) [MIKFLSALILLLVTTAAQA (SEQ ID NO: 15)], E. coli outer membrane porin A (OmpA) [MKKTAIAIAVALAGFATVAQA (SEQ ID NO: 16)], E. coli maltose-binding protein (MalE) [MKIKTGARILALSALTTMMFSASALA (SEQ ID NO: 17)], soft rot fungus (Erwinia carotovora) pectin lyase (PelB) [MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 18)], heat-labile E. coli enterotoxin LTIIb [MSFKKIIKAFVIMAALVSVQAHA (SEQ ID NO: 19)], and Bacillus subtilis. The signal sequence may be derived from subtilis endoxylanase XynA [MFKFKKKFLVGLTAAFMSISMFSATASA (SEQ ID NO: 20)], Escherichia coli DsbA [MKKIWLALAGLVLAFSASA (SEQ ID NO: 21)], TolB [MKQALRVAFGFLILWASVLHA (SEQ ID NO: 22)], or SipA [MKMNKKVLLTSTMAASLLSVASVQAS (SEQ ID NO: 23)]. In a particular embodiment, the signal sequence is derived from Escherichia coli DsbA [MKKIWLALAGLVLAFSASA (SEQ ID NO: 21)].

[0100] Therefore, the present invention provides a modified EPA protein whose amino acid sequence further includes a signal sequence that can direct the EPA protein towards the periplasm of a host cell (e.g., bacteria), and optionally the signal sequence is DsbA (SEQ ID NO: 21). The signal peptide of the E. coli-derived protein DsbA can be genetically fused to the N-terminus of a mature EPA sequence. For example, a plasmid derived from pEC415 [Schulz, H., Hennecke, H., and Thony-Meyer, L., Science, vol. 281, pp. 1197-1200, 1998] containing an RNase sequence following the DsbA signal peptide code can be digested to remove the RNase insertion while maintaining the DsbA signal (NdeI~EcoRI). Next, EPA is amplified using PCR (forward oligo 5'-AAGCTAGCGCCGCCGAGGAAGCCTTCGACC (SEQ ID NO: 32) and reverse oligo 5'-AAGAA TTCTCAGTGGTGGTGGTGGTGGTGCTTCAGGTCCTCGCGCGGCGG (SEQ ID NO: 33)), digested with Nhel / EcoRI, and ligated to replace the previously removed RNase sequence. The resulting construct (pGVXN69) encodes a protein product containing a DsbA signal peptide, a mature EPA sequence, and a hexa-histag.

[0101] A further aspect of the present invention is a polynucleotide encoding the modified EPA protein of the present invention. For example, a polynucleotide encoding the modified EPA protein has a nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 6 to 14. For example, a nucleotide sequence according to SEQ ID NO: 40, or a nucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 40. For example, a nucleotide sequence according to SEQ ID NO: 41, or a nucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41. For example, a nucleotide sequence according to SEQ ID NO: 42, or a nucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 42. For example, a nucleotide sequence following SEQ ID NO: 43, or a nucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 43. The nucleotide sequence includes a nucleotide encoding an amino acid corresponding to one (or more) consensus sequences selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3). For example, it encodes a modified EPA protein having a consensus sequence substituted at positions Y208, R274, S318, and / or A519 in the amino acid sequence of SEQ ID NO: 1, or at one or more equivalent positions in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0102] A vector containing such polynucleotides is a further embodiment of the present invention.

[0103] Conjugate Furthermore, the present invention provides a conjugate (e.g., a bioconjugate) comprising (or comprising) the modified EPA protein of the present invention linked to an antigen (e.g., a monosaccharide antigen, or optionally a bacterial polysaccharide antigen). The antigen may be a bacterial polysaccharide antigen, a yeast polysaccharide antigen, or a mammalian polysaccharide antigen.

[0104] In one embodiment, the conjugate comprises a conjugate (e.g., a bioconjugate) containing (or comprising) the modified EPA protein of the present invention covalently linked to an antigen (e.g., a monosaccharide antigen, optionally a bacterial polysaccharide antigen), the antigen being linked (directly or via a linker). In one embodiment, the antigen is directly linked to the modified EPA protein of the present invention. In one embodiment, the antigen is directly linked to an amino acid residue of the modified EPA protein.

[0105] In one embodiment, the modified EPA protein is covalently linked to the antigen via a chemical bond available by a chemical conjugation method (i.e., the conjugate is produced by chemical conjugation). The chemical conjugation method may be selected from the group consisting of carbodiimide chemistry, reduction animation, cyanylation chemistry (e.g., CDAP chemistry), maleimide chemistry, hydrazide chemistry, ester chemistry, and N-hydroxysuccinimide chemistry. The conjugate can be prepared by direct reduction amination, as described in U.S. Patent Application Publication No. 2007 / 10184072 (Hausdorff), U.S. Patent No. 4,365,170 (Jennings), and U.S. Patent No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188, EP-208375, and EP-0-477508. The conjugation method may also rely on the activation of monosaccharides by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP), such as by forming cyanate esters. Such conjugates are described in Uniformed Services University's International Publication No. 93 / 15760, as well as International Publication Nos. 95 / 08348 and 96 / 29094. See also Chu C. et al., Infect. Immunity, 1983, pp. 245-256.

[0106] Generally, the following types of chemical groups on modified EPA proteins can be used for coupling / conjugation: A) Carboxyl group (e.g., via aspartic acid or glutamic acid). In one embodiment, this group is linked directly to an amino group on a monosaccharide, or to an amino group on a linker having carbodiimide chemistry, such as EDAC. B) Amino group (e.g., via lysine). In one embodiment, this group is linked directly to a carboxyl group on a monosaccharide or to a carboxyl group on a linker having carbodiimide chemistry, such as EDAC. In another embodiment, this group is linked directly to a hydroxyl group activated by CDAP or CNBr on a monosaccharide or to such a group on a linker; to a monosaccharide or linker having an aldehyde group; or to a monosaccharide or linker having a succinimide ester group. C) Sulfhydryl (e.g., via cysteine). In one embodiment, this group is linked to a bromo- or chloroacetylated monosaccharide or linker by maleimide chemistry. In one embodiment, this group is activated / modified by bisdiazobenzidine. D) A hydroxyl group (e.g., via tyrosine). In one embodiment, this group is activated / modified by bisdiazobenzidine. E) Imidazolyl group (e.g., via histidine). In one embodiment, this group is activated / modified by bisdiazobenzidine. F) Guanidyl group (e.g., via arginine). G) Indolyl group (e.g., via tryptophan).

[0107] Generally, the following groups on monosaccharides can be used for coupling: OH, COOH, or NH2. Aldehyde groups may be generated after various treatments, such as periodate, acid hydrolysis, hydrogen peroxide, and others.

[0108] Conjugates can be purified by any method known in the art of protein purification, such as chromatography (e.g., ion exchange, anion exchange, affinity, and sizing column chromatography), centrifugation, solubility difference, or any other standard technique for protein purification. See, for example, Saraswat et al., 2013, Biomed. Res. Int. ID0312709 (pp. 1-18); also see the methods described in International Publication No. 2009 / 104074. The actual conditions used to purify a particular conjugate will depend on the synthetic strategy (e.g., synthetic versus recombinant) and the bioconjugate's characteristics, such as net charge, hydrophobicity, and / or hydrophilicity.

[0109] In one embodiment, the amino acid residue on the modified EPA protein to which the antigen is linked is selected from the group consisting of Ala, Arg, Asp, Cys, Gly, Glu, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. Optionally, the amino acid is an amino acid containing a terminal amine group of lysine, arginine, glutamic acid, aspartic acid, cysteine, tyrosine, histidine, or tryptophan. In one embodiment, the amino acid residue on the modified EPA protein to which the antigen is linked is not an asparagine residue, in which case the conjugate is typically generated by chemical conjugation. In addition, the antigen may be linked to an amino acid (e.g., asparagine) on a modified EPA protein selected from asparagine, aspartic acid, glutamic acid, lysine, cysteine, tyrosine, histidine, arginine, or tryptophan. In the case of asparagine, the conjugate may be a bioconjugate (e.g., enzymatic conjugation using oligosaccharide transferase, e.g., PglB). In one embodiment, the amino acid residue on the modified EPA protein to which the antigen is linked is an asparagine residue. Preferably, the amino acid residue on the modified EPA protein to which the antigen is linked is an asparagine residue within a consensus sequence, such as D / EXNZS / T (SEQ ID NO: 2), KD / EXNZS / TK (SEQ ID NO: 3), or JD / EXNZS / TU (SEQ ID NO: 5) consensus sequence.

[0110] The conjugate of the present invention may be a conjugate of recombinant modified EPA protein (e.g., a chemical conjugate or a bioconjugate). The conjugate of the present invention may be a conjugate of isolated recombinant modified EPA protein and recombinant antigen, for example, a recombinant monosaccharide (i.e., a bioconjugate).

[0111] antigen The antigen may be a monosaccharide antigen, such as a bacterial polysaccharide, such as an O antigen or capsular polysaccharide, a yeast polysaccharide, or a mammalian polysaccharide. The polysaccharide contains two or more monosaccharides, typically more than 10 monosaccharides. In one embodiment, the antigen in the conjugate (e.g., bioconjugate) of the present invention is a bacterial polysaccharide selected from the genera Shigella, Pseudomonas, Klebsiella, Streptococcus, or Staphylococcus (e.g., Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or Staphylococcus aureus). In one embodiment, the antigen is a bacterial polysaccharide antigen (for example, an O antigen derived from Gram-negative bacteria, possibly from Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Gram-positive bacteria, possibly from Streptococcus pneumoniae or Staphylococcus aureus). In one embodiment, the antigen is an O antigen derived from Gram-negative bacteria. In one embodiment, the antigen in the conjugate of the present invention (e.g., bioconjugate) is a bacterial polysaccharide selected from Shigella species, Klebsiella species, or Streptococcus species (e.g., Shigella flexner, Shigella sonnei, Klebsiella pneumoniae, or Streptococcus pneumoniae). In one embodiment, the antigen in the conjugate of the present invention (e.g., bioconjugate) is a bacterial polysaccharide selected from Shigella flexner, Klebsiella pneumoniae, and Streptococcus pneumoniae. In one embodiment, the antigen is a bacterial polysaccharide derived from Klebsiella pneumoniae. Therefore, the present invention provides a conjugate (e.g., a bioconjugate) comprising the modified EPA protein of the present invention linked to an antigen, wherein the antigen is a monosaccharide, and optionally a bacterial polysaccharide (e.g., derived from Shigella shiga, Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or Staphylococcus aureus). In one embodiment, the antigen is an O antigen. In another embodiment, the antigen is a capsular polysaccharide.

[0112] In certain embodiments, the antigen is, for example, an O antigen derived from Gram-negative bacteria. In certain embodiments, the antigen is an O antigen derived from a Salmonella, Shigella, Pseudomonas, or Klebsiella species. In certain embodiments, the antigen is an O antigen derived from a Shigella, Pseudomonas, or Klebsiella species (e.g., Shigella shiga, Shigella flexner, Shigella sonne, Pseudomonas aeruginosa, or Klebsiella pneumoniae). In one embodiment, the antigen is an O antigen derived from Shigella shiga, Shigella flexner, or Shigella sonne. For example, the antigen may be the O antigen derived from Shigella 1, Shigella sonnei, Shigella flexneri 6, and Shigella flexneri 2a and 3a O (Dmitriev, BA et al. Somatic Antigens of Shigella Eur J. Biochem, 1979, Vol. 98: p. 8; Liu et al. Structure and genetics of Shigella O antigens FEMS Microbiology Review, 2008, Vol. 32: p. 27). In one embodiment, the antigen is the O antigen derived from Pseudomonas aeruginosa. For example, the antigen may be the O antigen derived from Pseudomonas aeruginosa serotypes 1 to 20 (Raymond et al., J Bacteriol. 2002, Vol. 184 (No. 13): pp. 3614-3622). In one embodiment, the antigen is the O antigen derived from Klebsiella pneumoniae.

[0113] In certain embodiments, the antigen is a capsular polysaccharide derived from meningococcal serogroup A (MenA), meningococcal serogroup C (MenC), meningococcal serogroup Y (MenY), meningococcal serogroup W (MenW), Haemophilus influenzae type b (Hib), group B streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus. In certain embodiments, the antigen is a capsular polysaccharide derived from a Streptococcus or Staphylococcus species (e.g., Streptococcus pneumoniae or Staphylococcus aureus). In one embodiment, the antigen is a capsular polysaccharide derived from Staphylococcus aureus. For example, the antigen may be a capsular polysaccharide derived from Staphylococcus aureus types 5 and 8. In one embodiment, the antigen is a capsular polysaccharide derived from Streptococcus pneumoniae.

[0114] host cell Furthermore, the present invention: i) one or more nucleotide sequences, optionally containing polysaccharide synthesis genes integrated into the host cell genome, for generating bacterial polysaccharide antigens (e.g., O antigens derived from Gram-negative bacteria, optionally from Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Gram-positive bacteria, optionally from Streptococcus pneumoniae or Staphylococcus aureus) or yeast polysaccharide antigens or mammalian polysaccharide antigens; ii) Nucleotide sequences that encode heterologous oligosaccharide transferases, sometimes within plasmids; iii) A nucleotide sequence that optionally encodes the modified EPA protein of the present invention within a plasmid. Provides host cells containing the following:

[0115] Disclosures of methods for producing host cells capable of generating bioconjugates can be found in International Publication No. 06 / 119987, International Publication No. 09 / 104074, International Publication No. 11 / 62615, International Publication No. 11 / 138361, International Publication No. 14 / 57109, International Publication No. 14 / 72405, and International Publication No. 16 / 20499.

[0116] Examples of host cells that can be used to generate the bioconjugate of the present invention include archaea, prokaryotic host cells, and eukaryotic host cells. In certain embodiments, the host cell is a non-human host cell. Examples of prokaryotic host cells used to generate the bioconjugate of the present invention include species of the genera Escherichia, Shigella, Klebsiella, Xanthomonas, Salmonella, Yersinia, Lactococcus, Lactobacillus, Pseudomonas, Corynebacterium, Streptomyces, Streptococcus, Staphylococcus, Bacillus, and Clostridium. Preferably, the host cell is Escherichia coli (e.g., E. coli K12 W3110).

[0117] The host cell may be modified to delete or alter genes in the host cytogenetic background (genome) that compete with or interfere with the synthesis of the polysaccharide of interest (e.g., compete with or interfere with one or more heterologous polysaccharide synthesis genes introduced into the host cell by recombination). These genes may be deleted or altered in the host cell background (genome) to be inactive / dysfunctional (i.e., the deleted / altered host cell nucleotide sequence does not encode any functional protein and does not encode any protein). In one embodiment, if a nucleotide sequence is deleted from the genome of the host cell of the present invention, it is replaced with a desired sequence, for example, a sequence useful for glycoprotein synthesis. Exemplary genes that can be deleted (and, in some cases, replaced by other desired nucleotide sequences) within host cells include host cell genes involved in glycolipid biosynthesis, e.g., waaL (see, e.g., Feldman et al. 2005, PNAS USA vol. 102: pp. 3016-3021), O antigen clusters (rfb or wb), enterobacteria common antigen clusters (wec), lipid A core biosynthesis clusters (waa), galactose clusters (gal), arabinose clusters (ara), colonic acid clusters (wc), capsular polysaccharide clusters, undecaprenol-pyrophosphate biosynthesis genes (e.g., uppS (undecaprenyl pyrophosphate synthase), uppP (undecaprenyl diphosphatase)), Und-P recycling genes, metabolic enzymes involved in nucleotide-activated sugar biosynthesis, enterobacteria common antigen clusters, and prophage O antigen modification clusters such as the gtrABS cluster. In one embodiment, one or more of the following genes are deleted or functionally inactivated from the genome of the prokaryotic host cell of the present invention: the waaL gene, the gtrA gene, the gtrB gene, the gtrS gene, a gene derived from the wec cluster, a gene derived from the colonic acid cluster (wc), or a gene derived from the rfb gene cluster.In another embodiment, one or more of the waaL gene, gtrA gene, gtrB gene, gtrS gene, genes derived from the wec cluster, or genes derived from the rfb gene cluster are deleted or functionally inactivated from the genome of the prokaryotic host cell of the present invention. In a particular embodiment, the host cell of the present invention is Escherichia coli, and the natural Enterobacteriaceae Common Antigen Cluster (ECA, wec), cholanaic acid cluster (wca), and O16 antigen cluster are deleted, except for wecA. In addition, the natural lipopolysaccharide O antigen ligase waaL may be deleted from the host cell of the present invention. In addition, the natural gtrA gene, gtrB gene, and gtrS gene may be deleted from the host cell of the present invention.

[0118] The host cells of the present invention are engineered to contain heterologous nucleotide sequences. The host cells of the present invention are engineered to contain nucleotide sequences that optionally encode the modified EPA protein of the present invention within a plasmid. Furthermore, the host cells of the present invention contain one or more nucleotide sequences including polysaccharide synthesis genes. Therefore, the host cells of the present invention can generate bioconjugates containing antigens, such as monosaccharide antigens (e.g., bacterial, yeast, or mammalian polysaccharide antigens) that bind to the modified EPA protein of the present invention. One or more heterologous nucleotide sequences may encode polysaccharide synthesis proteins that produce bacterial polysaccharide antigens, yeast polysaccharide antigens, or mammalian polysaccharide antigens. Thus, the present invention also: i) One or more heteronucleotide sequences, which may include polysaccharide synthesis genes incorporated into the host cell genome, for generating bacterial polysaccharide antigens (e.g., O antigens derived from Gram-negative bacteria, possibly Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, or capsular polysaccharides derived from Gram-positive bacteria, possibly Streptococcus pneumoniae or Staphylococcus aureus) or yeast polysaccharide antigens or mammalian polysaccharide antigens; ii) Nucleotide sequences that encode heterologous oligosaccharide transferases, sometimes within plasmids; iii) A nucleotide sequence that optionally encodes the modified EPA protein of the present invention within a plasmid. Provides host cells containing the following:

[0119] The host cell of the present invention may contain one or more nucleotide sequences sufficient to produce monosaccharide antigens (e.g., bacterial polysaccharide antigens), particularly monosaccharide antigens (e.g., bacterial polysaccharide antigens) that are heterologous to the host cell. For example, if the host cell is E. coli, the host cell may contain one or more nucleotide sequences containing polysaccharide synthesis genes sufficient to produce bacterial polysaccharide antigens of bacteria that are not E. coli polysaccharide antigens. The bacterial polysaccharide antigen may be an O antigen or a capsular polysaccharide antigen. Thus, the present invention also: i) One or more nucleotide sequences, possibly containing polysaccharide synthesis genes integrated into the host cell genome, for generating bacterial polysaccharide antigens (e.g., O antigens derived from Gram-negative bacteria, possibly from Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, or capsular polysaccharides derived from Gram-positive bacteria, possibly from Streptococcus pneumoniae or Staphylococcus aureus); ii) Nucleotide sequences encoding heterologous oligosaccharide transferases, possibly within a plasmid; iii) A nucleotide sequence that optionally encodes the modified EPA protein of the present invention within a plasmid. Provides host cells containing the following:

[0120] Polysaccharide synthesis genes encode proteins (polysaccharide synthesis proteins) involved in the synthesis of polysaccharides. In one embodiment, the host cell may contain one or more nucleotide sequences including a polysaccharide synthesis gene for producing an O antigen derived from a Gram-negative bacterium selected from Shigella shiga, Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, and Klebsiella pneumoniae, or a polysaccharide synthesis gene for producing a capsular polysaccharide derived from a Gram-positive bacterium selected from Streptococcus pneumoniae and Staphylococcus aureus. In another embodiment, the host cell may contain one or more nucleotide sequences including an O antigen derived from a Gram-negative bacterium selected from Shigella flexner and Klebsiella pneumoniae, or a polysaccharide synthesis gene for producing a capsular polysaccharide derived from a Gram-positive bacterium selected from Streptococcus pneumoniae and Staphylococcus aureus.

[0121] Host cells for the production of bacterial polysaccharide antigens The host cell of the present invention may contain one or more nucleotide sequences including a polysaccharide synthesis gene for generating the O antigen. In certain embodiments, the host cell contains one or more nucleotide sequences including a polysaccharide synthesis gene for generating the O antigen derived from a Salmonella, Shigella, Pseudomonas, or Klebsiella species. In certain embodiments, the host cell contains one or more nucleotide sequences including a polysaccharide synthesis gene for generating the O antigen derived from a Shigella, Pseudomonas, or Klebsiella species (e.g., Shigella shiga, Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, or Klebsiella pneumoniae). In certain embodiments, the host cell contains one or more nucleotide sequences including a polysaccharide synthesis gene for generating the O antigen derived from a Shigella or Klebsiella species (e.g., Shigella shiga, Shigella flexner, Shigella sonnei, or Klebsiella pneumoniae). In one embodiment, the host cell contains one or more nucleotide sequences containing polysaccharide synthesis genes for generating O antigens derived from Shigella shiga, Shigella flexner, or Shigella sonnei. For example, the host cell may contain one or more nucleotide sequences containing polysaccharide synthesis genes for generating O antigens derived from Shigella shiga type 1, Shigella sonnei, and Shigella flexneri type 6, as well as Shigella flexneri 2a and 3a0 (Dmitriev, BA et al. Somatic Antigens of Shigella Eur J. Biochem, 1979, Vol. 98: p. 8; Liu et al. Structure and genetics of Shigella O antigens FEMS Microbiology Review, 2008, Vol. 32: p. 27). In one embodiment, the host cell contains one or more nucleotide sequences containing polysaccharide synthesis genes for generating O antigens derived from Pseudomonas aeruginosa, for example, Pseudomonas aeruginosa serotypes 1 to 20. In one embodiment, the host cell contains one or more nucleotide sequences including a polysaccharide synthesis gene for producing the O antigen derived from Klebsiella pneumoniae.

[0122] The host cell of the present invention may contain one or more nucleotide sequences including a polysaccharide synthesis gene for producing capsular polysaccharides. In certain embodiments, the host cell contains one or more nucleotide sequences including a polysaccharide synthesis gene for producing capsular polysaccharides derived from meningococcal serogroup A (MenA), meningococcal serogroup C (MenC), meningococcal serogroup Y (MenY), meningococcal serogroup W (MenW), Haemophilus influenzae type b (Hib), group B streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus. In certain embodiments, the host cell contains one or more nucleotide sequences including a polysaccharide synthesis gene for producing capsular polysaccharides derived from a Streptococcus or Staphylococcus species (e.g., Streptococcus pneumoniae or Staphylococcus aureus). In one embodiment, the host cell contains one or more nucleotide sequences including a polysaccharide synthesis gene for producing capsular polysaccharides derived from Staphylococcus aureus, for example, Staphylococcus aureus types 5 and 8. In one embodiment, the host cell contains one or more nucleotide sequences including a polysaccharide synthesis gene for producing capsular polysaccharides derived from Streptococcus pneumoniae.

[0123] Host cells containing heterologous nucleotide sequences for generating bacterial polysaccharide antigens The host cells of the present invention may naturally express one or more nucleotide sequences containing polysaccharide synthesis genes for the production of monosaccharide antigens (e.g., bacterial polysaccharide antigens), or the host cells may be engineered to express one or more such nucleotide sequences. For example, the host cells of the present invention may utilize cytosolic, endogenous, or heterologous sugar transferases (cytosolic sugar transferases) for the sequential assembly of oligosaccharides. Heterologous nucleotide sequences (e.g., nucleotide sequences encoding carrier proteins and / or other proteins, e.g., nucleotide sequences encoding proteins involved in glycosylation) can be introduced into the host cells of the present invention using methods such as electroporation, chemical transformation by heat shock, spontaneous transformation, phage transduction, and conjugation. In certain embodiments, heterologous nucleotide sequences are introduced into the host cells of the present invention using plasmids, for example, heterologous nucleotide sequences are expressed in the host cells by plasmids (e.g., expression vectors). In another particular embodiment, heterologous nucleotide sequences are introduced into the host cells of the present invention using insertion methods described in International Publication No. 14 / 037585. In certain embodiments, the host cell of the present invention comprises one or more nucleotide sequences containing polysaccharide synthesis genes heterologous to the host cell. In certain embodiments, one or more of the nucleotide sequences containing polysaccharide synthesis genes heterologous to the host cell are incorporated into the genome of the host cell. The heterologous nucleotide sequences may encode, but are not limited to, sugar transferases, oligosaccharide transferases, epimerases, flippases, and / or polymerases. In certain embodiments, the host cell of the present invention comprises one or more heterologous nucleotide sequences encoding a sugar transferase. The sugar transferase may be derived, for example, from species of the genera Escherichia, Shigella, Klebsiella, Salmonella, Pseudomonas, Streptococcus, or Staphylococcus.

[0124] The host cell of the present invention may contain one or more heterologous nucleotide sequences containing polysaccharide synthesis genes for generating the O antigen. In certain embodiments, the host cell contains one or more nucleotide sequences derived from a Salmonella, Shigella, Pseudomonas, or Klebsiella species that encode a polysaccharide synthesis protein for generating the O antigen. In certain embodiments, the host cell contains one or more nucleotide sequences derived from a Shigella, Pseudomonas, or Klebsiella species (e.g., Shigella shiga, Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, or Klebsiella pneumoniae) that encode a polysaccharide synthesis protein for generating the O antigen. In certain embodiments, the host cell contains one or more nucleotide sequences derived from a Shigella or Klebsiella species (e.g., Shigella shiga, Shigella flexner, Shigella sonnei, or Klebsiella pneumoniae) that encode a polysaccharide synthesis protein for generating the O antigen. In one embodiment, the host cell contains one or more nucleotide sequences derived from Shigella shiga, Shigella flexner, or Shigella sonnei that encode a polysaccharide synthesis protein for producing the O antigen. For example, the host cell may contain one or more nucleotide sequences derived from Shigella shiga type 1, Shigella sonnei, and Shigella flexneri type 6, as well as Shigella flexneri 2a and 3a, that encode a polysaccharide synthesis protein for producing the O antigen. In one embodiment, the host cell contains one or more nucleotide sequences derived from Pseudomonas aeruginosa, for example, serotypes 1 to 20, that encode a polysaccharide synthesis protein for producing the O antigen. In one embodiment, the host cell contains one or more nucleotide sequences derived from Klebsiella pneumoniae that encode a polysaccharide synthesis protein for producing the O antigen. The nucleotide sequences encoding the O antigen may be rfb clusters. As used herein, rfb clusters refer to gene clusters that encode an enzymatic mechanism capable of synthesizing the O antigen. The host cells may contain rfb gene clusters derived from Shigella shiga, Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

[0125] The host cell of the present invention may contain one or more heterologous nucleotide sequences containing polysaccharide synthesis genes for producing capsular monosaccharides. In certain embodiments, the host cell contains one or more nucleotide sequences derived from Neisseria meningitidis serogroup A (MenA), Neisseria meningitidis serogroup C (MenC), Neisseria meningitidis serogroup Y (MenY), Neisseria meningitidis serogroup W (MenW), Haemophilus influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus that encode polysaccharide synthesis proteins for producing capsular monosaccharides. In certain embodiments, the host cell contains one or more nucleotide sequences derived from a Streptococcus or Staphylococcus species (e.g., Streptococcus pneumoniae or Staphylococcus aureus) that encode polysaccharide synthesis proteins for producing capsular polysaccharides. In one embodiment, the host cell contains one or more nucleotide sequences derived from Staphylococcus aureus, for example, Staphylococcus aureus types 5 and 8, that encode polysaccharide synthesis proteins for producing capsular polysaccharides. In one embodiment, the host cell contains one or more nucleotide sequences containing polysaccharide synthesis genes for producing capsular polysaccharides from Streptococcus pneumoniae. The nucleotide sequences may be a capsular polysaccharide gene cluster. The host cell may contain capsular polysaccharide gene clusters derived from streptococcal lineages (e.g., Streptococcus pneumoniae, Group A hemolytic streptococcus (S. pyogenes), Group B hemolytic streptococcus (S. agalacticae)) or staphylococcal lineages (e.g., Staphylococcus aureus). For Streptococcus pneumoniae, the capsular polysaccharide gene cluster maps between dexB and aliA in the Streptococcus pneumoniae chromosome (Llull et al., 1999, J. Exp. Med. 190, pp. 241-251). Typically, the capsular polysaccharide gene cluster contains four relatively conserved genes: (wzg), (wzh), (wzd), and (wze) at its 5' end (Jiang et al., 2001, Infect. Immun. Vol. 69, pp. 1244-1255). In addition, the capsular polysaccharide gene cluster of Streptococcus pneumoniae contains wzx (polysaccharide flippase gene) and wzy (polysaccharide polymerase gene).The CP gene clusters for all 90 serotypes of Streptococcus pneumoniae have been sequenced by the Sanger Institute (http: / / www.sanger.ac.uk / Projects / S_pneumoniae / CPS / ), and the wzx and wzy genes for 89 serotypes have been annotated and analyzed (Kong et al., 2005, J. Med. Microbiol. Vol. 54, pp. 351-356). Capsular biosynthesis genes of Streptococcus pneumoniae are further described by Bentley et al. (PLoS Genet. March 2006; Vol. 2 (No. 3): p. e31), and their sequences are available in GenBank. Therefore, in one embodiment, the host cells of the present invention may further include nucleotide sequences encoding polymerase (e.g., wzy), flippase (e.g., wzx), and optionally nucleotide sequences encoding chain length regulators (e.g., wzz).

[0126] In certain embodiments, the host cell may also contain heterologous nucleotide sequences located outside the RFB cluster or capsular polysaccharide cluster. For example, nucleotide sequences encoding sugar transferases and acetyltransferases, found outside the RFB cluster or capsular polysaccharide cluster and modifying recombinant polysaccharides, may be introduced into the host cell.

[0127] Oligosaccharide transferase N-linked protein glycosylation (the addition of a carbohydrate molecule to an asparagine residue within the polypeptide chain of a target protein) is the most common type of post-translational modification occurring in the endoplasmic reticulum of eukaryotes. The process is accomplished by the enzyme oligosaccharide transferase complex (OST), which is responsible for the transfer of pre-assembled oligosaccharides from the lipid carrier (dolichol phosphate) to the asparagine residue of developing proteins within the conserved sequence Asn-X-Ser / Thr (where X is any amino acid other than proline) in the endoplasmic reticulum.

[0128] Furthermore, it has been shown that the foodborne pathogen Campylobacter jejuni, a bacterium, can N-glycosylate proteins due to the fact that it possesses its own glycosylation mechanism (Wacker et al., Science, 2002; Vol. 298 (No. 5599): pp. 1790-1793). The mechanism responsible for this reaction is encoded by a cluster called "pgl" (for protein glycosylation). The C. jejuni glycosylation mechanism can be translocated to E. coli and enable glycosylation of recombinant proteins expressed by E. coli cells. Previous studies have demonstrated methods for generating E. coli strains capable of performing N-glycosylation (e.g., Wacker et al., Science. 2002; Vol. 298 (No. 5599): pp. 1790-1793; Nita-Lazar et al., Glycobiology. 2005; Vol. 15 (No. 4): pp. 361-367; Feldman et al., Proc Natl Acad Sci US A. 2005; Vol. 102 (No. 8): pp. 3016-3021; ​​Kowarik et al., EMBO J. 2006; Vol. 25 (No. 9): pp. 1957-1966; Wacker et al., Proc Natl Acad Sci US A. 2006; Vol. 103 (No. 18): pp. 7088-7093; (See International Publication Nos. 2003 / 074687, 2006 / 119987, 2009 / 104074, 2011 / 06261, and 2011 / 138361).

[0129] The host cells of the present invention optionally contain a nucleotide sequence encoding a heterologous oligosaccharide transferase within the plasmid. In certain embodiments, the oligosaccharide transferase is an oligosaccharide transferase derived from the genus Campylobacter. In other specific embodiments, the oligosaccharide transferase is optionally pglB derived from Campylobacter jejuni (i.e., pglB; see, e.g., Wacker et al. 2002, Science vol. 298: pp. 1790-1793; also see, e.g., NCBI Gene ID: 3231775, UniProt Accession No. O86154) Sequence ID No. 24: MLKKEYLKNPYLVLFAMIILAYVFSVFCRFYWVWWASEFNEYFFNNQLMIISNDGYAFAEGARDMIAGFHQPNDLSYYGSSLSALTYWLYKITPFSFESIILYMSTFLSSLVVIPTILLANEYKRPLMGFVAALLASIANSYYNRTMSGYYDTDMLVIVLPMFILFFMVRMILKKDFF SLIALPLFIGIYLWWYPSSYTLNVALIGLFLIYTLIFHRKEKIFYIAVILSSLTLSNIAWFYQSAIIVILFALFALEQKRLNFMIIGILGSATLIFLILSGGVDPILYQLKFYIFRSDESANLTQGFMYFNVNQTIQEVENVDLSEFMRRISGSEIVFLFSLFGFVWLLRKHKSMIMA LPILVLGFLALKGGLRFTIYSVPVMALGFGFLLSEFKAIMVKKYSQLTSNVCIVFATILTLAPVFIHIYNYKAPTVFSQNEASLLNQLKNIANREDYVVTWWDYGYPVRYYSDVKTLVDGGKHLGKDNFFPSFALSKDEQAAANMARLSVEYTEKSFYAPQNDILKTDILQAMMKDYN QSNVDLFLASLSKPDFKIDTPKTRDIYLYMPARMSLIFSTVASFSFINLDTGVLDKPFTFSTAYPLDVKNGEIYLSNGVVLSDDFRSFKIGDNVVSVNSIVEINSIKQGEYKITPIDDKAQFYIFYLKDSAIPYAQFILMDKTMFNSAYVQMFFLGNYDKNLFDLVINSRDAKVFKLKI

[0130] Therefore, the host cells of the present invention may optionally contain within the plasmid a nucleotide sequence encoding pglB, optionally a nucleotide sequence encoding pglB derived from Campylobacter jejuni, optionally a nucleotide sequence encoding pglB derived from Campylobacter jejuni having a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 24.

[0131] polymerase Furthermore, the host cell of the present invention may contain a nucleotide sequence encoding a polymerase (e.g., wzy). In one embodiment, the polymerase (e.g., wzy) is introduced into the host cell of the present invention (i.e., the polymerase is heterogeneous to the host cell). In one embodiment, the polymerase is a bacterial polymerase. In one embodiment, the polymerase is a capsular polysaccharide polymerase (e.g., wzy) or an O antigen polymerase (e.g., wzy). In one embodiment, the polymerase is an O antigen polysaccharide polymerase (e.g., wzy) derived from, for example, a Shigella, Pseudomonas, or Escherichia species (e.g., Shigella flexneri, Shigella sonnei, Pseudomonas, or Escherichia coli). In one embodiment, the polymerase is a capsular polysaccharide polymerase (e.g., wzy) derived from, for example, meningococcal serogroup A (MenA), meningococcal serogroup C (MenC), meningococcal serogroup Y (MenY), meningococcal serogroup W (MenW), Haemophilus influenzae type b (Hib), group B streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus. In one embodiment, the polymerase is a capsular polysaccharide polymerase (e.g., wzy) from Streptococcus pneumoniae. The wzy polymerase may be incorporated into the host cell as part of an rfb cluster or a capsular polysaccharide cluster (e.g., inserted into the genome or expressed by a plasmid). Thus, the host cell of the present invention may further contain a nucleotide sequence encoding a heterologous wzy polymerase.

[0132] Flippa Furthermore, the host cell of the present invention may include a nucleotide sequence encoding a flippase (e.g., wzx), for example, a heterologous flippase. The flippase translocates wild-type repeat units and / or their corresponding engineered (hybrid) repeat units from the cytoplasm to the periplasm of the host cell (e.g., Escherichia coli). In one embodiment, the flippase is a bacterial flippase, for example, a flippase of the polysaccharide biosynthesis pathway of interest. In a particular embodiment, the host cell of the present invention includes a nucleotide sequence encoding a flippase (e.g., the wzx gene) of a polysaccharide biosynthesis pathway in a Streptococcus, Shigella, Escherichia, Pseudomonas, or Staphylococcus species (e.g., Streptococcus pneumoniae, Shigella flexner, Shigella sonnei, Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus). In one embodiment, the flippase is a capsular polysaccharide flippase (e.g., wzx) of Streptococcus pneumoniae. Other flippases that can be introduced into host cells according to the present invention include, for example, those derived from Campylobacter jejuni (e.g., pglK).

[0133] Accessory enzymes In one embodiment, a nucleotide sequence encoding one or more accessory enzymes is introduced into the host cell of the present invention. Thus, the host cell of the present invention may further contain one or more of these accessory enzymes. Such nucleotide sequences encoding one or more accessory enzymes may be held in a plasmid or incorporated into the genome of the host cell of the present invention. Exemplary accessory enzymes include, but are not limited to, epimerases (e.g., International Publication No. 2011 / 062615), branching enzymes, modification enzymes (e.g., adding choline, glycerol phosphate, or Pilbert), amidases, chain length regulators, acetylases, formylases, and polymerases. Thus, the host cell of the present invention may also contain a nucleotide sequence encoding a chain length regulator (e.g., wzz), such as a heterologous chain length regulator. In one embodiment, the chain length regulator is a capsular polysaccharide chain length regulator of Streptococcus pneumoniae (e.g., wzz).

[0134] Bioconjugate The present invention provides a bioconjugate comprising a modified EPA protein of the present invention linked to an antigen (e.g., a bacterial polysaccharide antigen, a yeast polysaccharide antigen, or a mammalian polysaccharide antigen). In certain embodiments, the antigen is an O antigen or a capsular polysaccharide. In one embodiment, the antigen is an O antigen derived from Gram-negative bacteria. In one embodiment, the present invention provides a bioconjugate comprising a modified EPA protein of the present invention linked to an antigen, wherein the antigen is a monosaccharide, optionally a bacterial polysaccharide (e.g., derived from Shigella shiga, Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or Staphylococcus aureus). In one embodiment, the present invention provides a bioconjugate comprising a modified EPA protein of the present invention linked to an antigen, wherein the antigen is a bacterial polysaccharide (e.g., derived from Shigella shiga, Shigella flexner, Shigella sonnei, Klebsiella pneumoniae, or Streptococcus pneumoniae). In another embodiment, the present invention provides a bioconjugate comprising a modified EPA protein of the present invention linked to an antigen, the antigen being a bacterial polysaccharide derived from Shigella flexneri, Klebsiella pneumoniae, or Streptococcus pneumoniae. The antigen is linked to an amino acid (e.g., asparagine) on a modified EPA protein selected from asparagine, aspartic acid, glutamic acid, lysine, cysteine, tyrosine, histidine, arginine, or tryptophan. The bioconjugates described herein have advantages over chemical conjugates of antigen-carrier proteins in that they require fewer chemicals in production and are more immutable with respect to the final product produced.

[0135] A further aspect of the present invention is a process for producing a bioconjugate comprising (or comprising) a modified EPA protein linked to a monosaccharide, the process comprising (i) culturing the host cells of the present invention under conditions suitable for glycoprotein production, and (ii) isolating the bioconjugate produced by the host cells, optionally by isolating the bioconjugate from a peripheral extract derived from the host cells.

[0136] For example, bioconjugates can be produced using a shaking flask process, for example, in an LB shaking flask. In an embodiment of the present invention, a fed-add process for the production of recombinant glycosylated proteins in bacteria can be used to produce the bioconjugates of the present invention. The objective is to maintain simplicity and reproducibility in the process while increasing glycosylation efficiency and recombinant protein yield per cell. The bioconjugates of the present invention can be produced on a commercial scale by developing an optimized production method using a typical E. coli production process. Various types of feed strategies, such as batch, chemostat, and fed-add, can be used.

[0137] The bioconjugates of the present invention can be purified, for example, by chromatography (e.g., ion exchange, anion exchange, affinity, and sizing column chromatography), centrifugation, solubility difference, or by any other standard technique for protein purification. See, for example, Saraswat et al. 2013, Biomed. Res. Int. ID#312709 (pp. 1-18); also see the method described in International Publication No. 2009 / 104074. Furthermore, the bioconjugates may be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0138] Analysis method Using various methods, the structural composition and sugar chain length of the bioconjugate of the present invention can be analyzed, and the use of glycosylation sites can be determined.

[0139] Glycans can be analyzed using hydrazine degradation. First, polysaccharides are released from the protein carrier by incubation with hydrazine according to the manufacturer's instructions (Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK). The nucleophile hydrazine attacks the glycosidic bond between the polysaccharide and the carrier protein, enabling the release of the bound glycan. The N-acetyl group is lost during this procedure and must be reconstituted by re-N-acetylation. The free glycan is purified on a carbon column and then labeled at the reducing end with the fluorophore 2-aminobenzamide. See Bigge JC, Patel TP, Bruce JA, Goulding PN, Charles SM, Parekh RB: Nonselective and efficient fluorescent labeling of glycans using 2-aminobenzamide and anthranilic acid. Anal Biochem 1995, vol. 230 (no. 2): pp. 229-238. Labeled polysaccharides are separated by a GlycoSep-N column (GL Sciences) according to the HPLC protocol of Royle et al. (Royle L, Mattu TS, Hart E, Langridge JI, Merry AH, Murphy N, Harvey DJ, Dwek RA, Rudd PM: An analytical and structural database provides a strategy for sequencing O-glycans from microgram quantities of glycoproteins. See Anal Biochem 2002, Vol. 304 (No. 1): pp. 70-90). The resulting fluorescence chromatogram shows the polysaccharide length and number of repeating units. Structural information can be collected by collecting individual peaks and then performing MS / MS analysis. This allows for confirmation of the monosaccharide composition and sequence of the repeating units, and in addition, identification of the homogeneity of the polysaccharide composition.In addition, the size of the complete bioconjugate can be measured using high-mass MS and size exclusion HPLC.

[0140] Yield may be measured as the amount of carbohydrates derived from 1 liter of bacterial culture grown in a bioreactor under controlled and optimized conditions. After purification of the bioconjugate, the carbohydrate yield can be measured directly by an anthron assay or ELISA using carbohydrate-specific antiserum. Indirect measurement is possible by calculating the theoretical carbohydrate content per gram of protein using protein content (measured by BCA, Lowry, or Bardford assay), as well as glycan length and structure. In addition, yield can also be measured by drying the glycoprotein preparation from a volatile buffer and weighing it using a balance.

[0141] For example, various methods such as SDS-PAGE or capillary gel electrophoresis can be used to analyze the conjugates of the present invention. Polymer length is defined by the number of linearly assembled repeating units. This means that a typical ladder-like pattern is the result of different numbers of repeating units constituting the glycan. Thus, two adjacent bands in SDS-PAGE (or other techniques for size separation) differ by only a single repeating unit. This discrete difference is utilized when analyzing glycoproteins for glycan size: non-glycosylated carrier proteins and bioconjugates with different polymer chain lengths separate according to their electrophoretic mobility. The first detectable number of repeating units (n1) and the average number of repeating units (n) present on the bioconjugate are determined. average These parameters are measured. Using these parameters, for example, batch consistency or polysaccharide stability can be demonstrated.

[0142] The use of glycosylation sites may be quantified, for example, by glycopeptide LC-MS / MS: the conjugate is digested by a protease, the peptides are separated by an appropriate chromatographic method (C18, hydrophilic interaction HPLC HILIC, GlycoSepN column, SE HPLC, AE HPLC), and different peptides are identified using MS / MS. This method may or may not be used in conjunction with prior glycan shortening by chemical (Smith degradation) or enzymatic methods. Quantification of glycopeptide peaks using UV detection at 215–280 nm allows for a relative determination of glycosylation site use. In another embodiment, site use may be quantified by size exclusion HPLC: higher glycosylation site use is reflected by earlier elution times from the SE HPLC column. In yet another embodiment, the use of the site may be quantified by quantitative concentration measurement of the purified bioconjugate stained with Coomassie Brilliant Blue after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0143] Immunogenic compositions and vaccines The conjugates (e.g., bioconjugates) of the present invention are particularly suitable for incorporation into immunogenic compositions and vaccines.

[0144] The present invention provides an immunogenic composition comprising the conjugate (e.g., bioconjugate) of the present invention, and optionally pharmaceutically acceptable excipients and / or carriers.

[0145] The immunogenic composition comprises an immunologically effective amount of the modified EPA protein or conjugate (e.g., bioconjugate) of the present invention, and any other components. “Immunologically effective amount” means that the amount administered to an individual, either as a single dose or as part of a series, is effective for treatment or prevention. The amount varies depending on the health and physical condition of the individual being treated, age, the desired level of protection, the vaccine formulation, and other relevant factors.

[0146] Pharmaceutically acceptable excipients and carriers are listed, for example, in Remington's Pharmaceutical Sciences (EW Martin, Mack Publishing Co., Easton, PA, 5th edition (1975)). Pharmaceutically acceptable excipients include buffers, e.g., phosphate buffer (e.g., sodium phosphate). Pharmaceutically acceptable excipients include salts, e.g., sodium chloride. Pharmaceutically acceptable excipients include solubilizers / stabilizers, e.g., polysorbate (e.g., TWEEN 80). Pharmaceutically acceptable excipients include preservatives, e.g., 2-phenoxyethanol or thiomersal. Pharmaceutically acceptable excipients include carriers, e.g., water or physiological saline.

[0147] Also provided is a method for producing an immunogenic composition of the present invention, comprising the step of mixing the modified EPA protein or conjugate (e.g., bioconjugate) of the present invention with a pharmaceutically acceptable excipient and / or carrier.

[0148] Furthermore, the present invention provides immunogenic compositions (e.g., vaccine compositions) that optionally include an adjuvant.

[0149] The term "adjuvant" refers to a compound that, when administered in conjunction with or as part of the immunogenic composition of the vaccine of the present invention, increases, enhances, and / or boosts the immune response to the modified EPA protein conjugate / bioconjugate, but, when administered alone, does not produce an immune response to the modified EPA protein conjugate / bioconjugate. Adjuvants can enhance the immune response through several mechanisms, including, for example, lymphocyte replacement, B and / or T cell stimulation, and macrophage stimulation. Specific examples of adjuvants include, but are not limited to, aluminum salts (alum) (e.g., aluminum hydroxide, aluminum phosphate, and aluminum sulfate), three de-O-acylated monophosphoryllipids A (MPL) (see UK Patent No. 2220211), MF59 (Novartis), AS01 (GlaxoSmithKline), and saponins, such as QS21 (Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (Powell & Newman, eds., Plenum Press, NY, 1995); see U.S. Patent No. 5,057,540). In some embodiments, the adjuvant is a Freund's adjuvant (complete or incomplete). Other adjuvants may include immunostimulants, such as oil-in-water emulsions (e.g., squalene or peanut oil) combined with monophosphoryl lipid A (see Stoute et al. N. Engl. J. Med. 336, pp. 86-91 (1997)).

[0150] Also provided is a method for producing an immunogenic composition of the present invention, comprising the step of mixing the modified EPA protein or conjugate (e.g., bioconjugate) of the present invention with a pharmaceutically acceptable excipient and / or carrier, and an adjuvant. Vaccine preparations are typically described in Vaccine Design ("The subunit and adjuvant approach" (edited by Powell MF and Newman MJ) (1995), Plenum Press, New York).

[0151] The immunogenic composition of the present invention may be contained in a container, pack, or dispenser, along with instructions for administration.

[0152] The immunogenic composition or vaccine of the present invention can be stored before use, for example, by freezing (e.g., about -20°C or about -70°C); storing under refrigerated conditions (e.g., about 4°C); or storing at room temperature. The immunogenic composition or vaccine of the present invention may be stored in solution or lyophilized. In one embodiment, the solution is lyophilized in the presence of a sugar, such as sucrose, trehalose, or lactose. In another embodiment, the vaccine of the present invention is lyophilized and immediately reconstituted before use.

[0153] Dosage and dosage The immunogenic composition or vaccine of the present invention may be used to protect or treat a subject (e.g., a mammal) by administering the immunogenic composition or vaccine via a systemic route or a mucosal route. These administrations may include injection via intramuscular (IM), intraperitoneal, intradermal (ID), or subcutaneous (SC) routes; or via mucosal administration through the mouth / food, respiration, or genitourinary tract.

[0154] In one embodiment, the immunogenic composition or vaccine of the present invention is administered by an intramuscular delivery route. Intramuscular administration may be performed in the thigh or upper arm. The injection is typically performed via a needle (e.g., a subcutaneous injection needle), but needleless injections may also be used. A typical intramuscular dose is 0.5 ml.

[0155] In another embodiment, the immunogenic composition or vaccine of the present invention is administered by intradermal administration. Human skin includes an outer layer of "keratin" cuticles called the stratum corneum, which covers the epidermis. Beneath the epidermis is a layer called the dermis, which in turn covers the subcutaneous tissue. The conventional technique of intradermal injection, the "Mantou procedure," involves cleaning the skin, stretching it with one hand, and inserting a small-gauge needle (26-31 gauge) with the bevel facing upward at an angle of 10-15°. As soon as the bevel of the needle is inserted, slight pressure is applied to lift it under the skin while lowering the shank of the needle and advancing it further. Subsequently, the liquid is injected very slowly to form a bleb or bump on the skin surface, and then the needle is slowly withdrawn.

[0156] In another embodiment, the immunogenic composition or vaccine of the present invention is administered by intranasal administration. Typically, the immunogenic composition or vaccine is administered topically to the nasopharynx region without, for example, inhalation into the lungs. It is desirable to use an intranasal delivery device that delivers the immunogenic composition or vaccine formulation to the nasopharynx region without entering the lungs, or substantially entering them. A suitable device for intranasal administration of vaccines according to the present invention is a spray device. A suitable commercially available nasal spray device is ACCUSPRAY® (Becton Dickinson).

[0157] The amount of conjugate (e.g., bioconjugate) in each immunogenic composition or vaccine dose is selected as the amount that induces an immunoprotective response without serious adverse side effects in a typical vaccine. Such an amount varies depending on which specific immunogen is used and how it is demonstrated. The conjugate (e.g., bioconjugate) content is typically in the range of 1 to 100 μg, and preferably 5 to 50 μg.

[0158] Preventive and therapeutic uses Furthermore, the present invention provides an immunogenic composition or vaccine of the present invention for use in pharmaceuticals.

[0159] The present invention provides a method for inducing an immune response in a subject (e.g., a human), comprising administering a therapeutically or prophylactically effective amount of the conjugate (e.g., a bioconjugate), the immunogenic composition, or the vaccine of the present invention to a subject (e.g., a human) that requires induction. The present invention also provides the conjugate (e.g., a bioconjugate), the immunogenic composition, or the vaccine of the present invention used to induce an immune response in a subject (e.g., a human). The present invention also provides the conjugate (e.g., a bioconjugate), the immunogenic composition, or the vaccine of the present invention used to manufacture a drug for inducing an immune response in a subject (e.g., a human).

[0160] Also provided herein are methods for inducing an immune response to bacteria in a subject, comprising administering to the subject a conjugate (e.g., a bioconjugate), an immunogenic composition, or a vaccine of the present invention. The conjugate (e.g., a bioconjugate), the immunogenic composition, or the vaccine of the present invention can be used to induce an immune response to bacteria, such as Shigella species, Pseudomonas aeruginosa, Klebsiella pneumoniae, Neisseria meningitidis, Haemophilus influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus. In one embodiment, the conjugate (e.g., bioconjugate), the immunogenic composition, or the vaccine of the present invention can be used to induce an immune response against bacteria, such as species of Streptococcus, Shigella, Pseudomonas, Klebsiella, or Staphylococcus (e.g., Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or Staphylococcus aureus). In one embodiment, the subject has a bacterial infection at the time of administration. In another embodiment, the subject does not have a bacterial infection at the time of administration.

[0161] Furthermore, provided herein are methods for inducing the production of opsonophagocytic antibodies against bacteria in a subject, comprising administering to the subject a conjugate (e.g., a bioconjugate), an immunogenic composition, or a vaccine of the present invention. The conjugate (e.g., a bioconjugate), an immunogenic composition, or a vaccine of the present invention can be used to induce the production of opsonophagocytic antibodies against bacteria, such as Shigella species, Pseudomonas aeruginosa, Klebsiella pneumoniae, Neisseria meningitidis, Haemophilus influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus in a subject. In one embodiment, the conjugate (e.g., bioconjugate), the immunogenic composition, or the vaccine of the present invention can be used to induce the production of opsonizing antibodies against bacteria, such as Streptococcus species, Shigella species, Pseudomonas species, Klebsiella species, or Staphylococcus species (e.g., Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or Staphylococcus aureus) in a subject.

[0162] The present invention also provides a method for treating and / or preventing a yeast infection or bacterial infection in a subject, comprising administering the subject a conjugate (e.g., a bioconjugate) of the present invention. The conjugate (e.g., a bioconjugate) may be in the form of an immunogenic composition or a vaccine. Thus, the present invention provides a method for treating and / or preventing a yeast infection or bacterial infection in a subject (e.g., a human), comprising administering a therapeutically or prophylactically effective amount of the conjugate (e.g., a bioconjugate), the immunogenic composition, or the vaccine of the present invention to a subject (e.g., a human) in need of treatment and / or prevention. The present invention also provides a conjugate (e.g., a bioconjugate), the immunogenic composition, or the vaccine of the present invention used for treating and / or preventing a yeast infection or bacterial infection in a subject (e.g., a human). The present invention also provides a conjugate (e.g., a bioconjugate), the immunogenic composition, or the vaccine of the present invention used for producing a drug for treating and / or preventing a yeast infection or bacterial infection in a subject (e.g., a human). In certain embodiments, the immunogenic compositions or vaccines of the present invention are used to prevent a target bacterial infection. Bacterial infections that can be treated and / or prevented using the conjugates (e.g., bioconjugates) of the present invention include those caused by Neisseria meningitidis, Haemophilus influenzae type b (Hib), Streptococcus species, Shigella species, Pseudomonas species, Klebsiella species, or Staphylococcus species (e.g., Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or Staphylococcus aureus).

[0163] Embodiments of the present invention are further described in the following numbered paragraphs: 1. Modified EPA (Exotoxin of Pseudomonas aeruginosa) having an amino acid sequence identical to that of SEQ ID NO: 1 by at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO: 1. A) A protein which is modified in that its amino acid sequence includes one (or more) consensus sequences selected from:D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the one (or more) consensus sequences are at the same position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to:SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to: (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213; or one or more amino acids between 205-211; e.g., amino acid residue D218; e.g., amino acid residue Y208), or (ii) one or more amino acids between amino acid residues 264-284 (e.g., A A modified EPA protein in which one or more amino acids are added next to or substituted for one or more amino acids independently selected from (iii) one or more amino acids between amino acid residues 269-279; or one or more amino acids between amino acid residues 271-277 (e.g., amino acid residue R279, e.g., amino acid residue R274), (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323; or one or more amino acids between amino acid residues 315-321, e.g., amino acid residue G323, e.g., amino acid residue S318), and (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; or one or more amino acids between amino acid residues 516-522; e.g., amino acid residue G525, e.g., amino acid residue A519). 2. The amino acid sequence of SEQ ID NO: 1, or a modified EPA (Exotoxin of Pseudomonas aeruginosa) of paragraph 1 having an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 1. A) which is modified in that the amino acid sequence includes two (or more) consensus sequences selected from:D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the two (or more) consensus sequences are at the same position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to:SEQ ID NO: 1 (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213, e.g., amino acid residue Y208), or (ii) one or more amino acids between amino acid residues 264-284 Modified EPAs that are attached to or substitute for one or more amino acids independently selected from (iii) amino acids (e.g., one or more amino acids between amino acid residues 269-279, e.g., amino acid residue R274), (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230-250 (e.g., one or more amino acids between amino acid residues 235-245; e.g., amino acid residue K240). 3. The amino acid sequence of SEQ ID NO: 1, or a modified EPA (Exotoxin of Pseudomonas aeruginosa) of paragraph 1 having an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 1. A) A protein which is modified in that its amino acid sequence includes three (or more) consensus sequences selected from:D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the three (or more) consensus sequences are at the same position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to:SEQ ID NO: 1 (i) one or more amino acids between amino acid residues 198-218 (e.g., one or more amino acids between amino acid residues 203-213, e.g., amino acid residue Y208), or (ii) one or more amino acids between amino acid residues 264-284 A modified EPA protein in which one or more amino acids are added next to or substituted for one or more amino acids independently selected from (iii) amino acids (e.g., one or more amino acids between amino acid residues 269-279, e.g., amino acid residue R274), (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230-250 (e.g., one or more amino acids between amino acid residues 235-245; e.g., amino acid residue K240). 4. A consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) independently substitutes one or more amino acids in the amino acid sequence of SEQ ID NO: 1, or in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (for example, each consensus sequence substitutes a single amino acid residue, e.g., a single amino acid residue selected from Y208, R274, S318, and A519), a modified EPA protein according to any of paragraphs 1-3. 5. A further consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and JD / EXNZS / TU (SEQ ID NO: 5) (where X and Z are independently any amino acid other than proline, and J and U are independently 1 to 5 naturally occurring amino acid residues) is added next to or substitutes one or more amino acids at the same position in the N-terminal amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 1, or is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, for any modified EPA protein of any of paragraphs 1 to 4. 6. A further consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and JD / EXNZS / TU (SEQ ID NO: 5) (where X and Z are independently any amino acid other than proline, and J and U are independently 1 to 5 naturally occurring amino acid residues) is added next to or substitutes one or more amino acids at the same position in the C-terminal amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 1, or is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, in any of the modified EPA proteins of paragraphs 1 to 5. 7. At least one consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) (where X and Z are independently any amino acid other than proline) is one or more amino acids between amino acid residues 198-218 at the same position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (e.g., one between amino acid residues 203-213) (ii) one or more amino acids (e.g., amino acid residue Y208), (ii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), or (iii) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519), which are added next to or substituted for any of the modified EPA proteins described in paragraphs 1-6. 8. A modified EPA protein according to any of paragraphs 1-7, wherein the modified EPA protein contains two consensus sequences, which optionally substitute amino acid residues selected from (i) Y208 and R274, (ii) Y208 and S318, (iii) Y208 and A519, (iv) R274 and S318, (v) R274 and A519, or (vi) S318 and A519, of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. 9. The modified EPA protein contains two consensus sequences in which amino acid residues Y208 and R274 of the amino acid sequence identical to or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and optionally SEQ ID NO: 6: AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAH ESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNKDQNATKLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTKDQNATKHRQPRGWEQLEQCGYPVQRLVALYLA ARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVR ARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK A modified EPA protein according to paragraph 8, comprising (or comprising) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to that of the modified EPA protein according to paragraph 8. 10. The modified EPA protein contains three consensus sequences, which optionally substitute amino acid residues Y208, R274, and A519 of the amino acid sequence identical to or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO: 1, as any of the modified EPA proteins in paragraphs 1 to 7. 11. The modified EPA protein contains three consensus sequences in which amino acid residues Y208, R274, and A519 of the amino acid sequence identical to or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and optionally SEQ ID NO: 7: AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHE SNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNKDQNATKLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTKDQNATKHRQPRGWEQLEQCGYPVQRLVALYLAARL SWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQ DLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLKDQNATKAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK A modified EPA protein according to paragraph 10, comprising (or comprising) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to that of the modified EPA protein according to paragraph 10. 12. The modified EPA protein contains four consensus sequences which optionally substitute amino acid residues Y208, R274, A519 of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and is added adjacent to this N-terminal amino acid, the modified EPA protein of any of paragraphs 1 to 7. 13. The modified EPA protein has the amino acid residues Y208, R274, A519 of the amino acid sequence of SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and contains four consensus sequences added adjacent to this N-terminal amino acid, and optionally SEQ ID NO: 8: GSGGGDQNATGSGGGKLAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAK LARDATFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNKDQNATKLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTKDQNATKHRQPRGWEQLEQCGYPVQRL VALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVR ARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLKDQNATKAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK A modified EPA protein according to paragraph 12, which may contain (or consist of) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to that of the modified EPA protein according to paragraph 12. 14. The modified EPA protein contains five consensus sequences, which may be selected from substitutions of amino acid residue Y208, amino acid residue R274, amino acid residue S318, amino acid residue A519, N-terminal addition, and C-terminal addition of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or any of the modified EPA proteins in paragraphs 1 to 7. 15. The modified EPA protein contains five consensus sequences selected from substitutions of amino acid residue Y208, amino acid residue R274, amino acid residue S318, amino acid residue A519, N-terminal addition, and C-terminal addition of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or SEQ ID NO: 1, which may include SEQ ID NOs: 9, 10, or 11: Sequence ID 9 TIFF2026097994000002.tif60169 Sequence ID 10 TIFF2026097994000003.tif60169 Sequence ID 11 A modified EPA protein according to paragraph 14, comprising (or comprising) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF2026097994000004.tif60169. 16. The modified EPA protein contains six consensus sequences, which may be selected from substitutions of amino acid residue Y208, amino acid residue K240, amino acid residue R274, amino acid residue S318, amino acid residue A519, N-terminal addition, and C-terminal addition of any of the modified EPA proteins in paragraphs 1 to 7, which may be at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, or any of the following: substitution of amino acid residue Y208, substitution of amino acid residue K240, substitution of amino acid residue R274, substitution of amino acid residue S318, substitution of amino acid residue A519, N-terminal addition, and C-terminal addition. 17. The modified EPA protein contains six consensus sequences selected from substitutions of amino acid residue Y208, K240, R274, S318, A519, N-terminal addition, and C-terminal addition of an amino acid sequence identical to or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, which may include SEQ ID NO: 12 or 13: Sequence ID 12 TIFF2026097994000005.tif66167 Sequence ID 13 A modified EPA protein according to paragraph 16, comprising (or consisting of) an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF2026097994000006.tif48167TIFF2026097994000007.tif15166. 18. A modified EPA protein comprising seven consensus sequences, which optionally substitute amino acid residues Y208, K240, R274, S318, A519 of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and is added at the N-terminus and at the C-terminus, as any of the modified EPA proteins in paragraphs 1 to 7. 19. The modified EPA protein contains seven consensus sequences, which are added at the N-terminus and C-terminus, in which amino acid residues Y208, K240, R274, S318, A519 of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or SEQ ID NO: 1, are substituted. Sequence ID 14 A modified EPA protein according to paragraph 8, having (or being) at least 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to TIFF2026097994000008.tif66166. 20.X is Q (glutamine) and Z is A (alanine), one of the modified EPA proteins from paragraphs 1-19. 21. A modified EPA protein of any of paragraphs 1-20, wherein the amino acid sequence includes a substitution of leucine 552 (or at the position equivalent to L552 in SEQ ID NO: 1) with valine (L552V) and a deletion of glutamine 553 (or at the position equivalent to E553 in SEQ ID NO: 1) (ΔE553). 22. A modified EPA protein according to any of paragraphs 1 to 21, wherein the amino acid sequence further comprises a peptide tag, which optionally comprises six histidine residues, and which optionally is positioned at the C-terminus of the amino acid sequence. 23. The amino acid sequence further includes a signal sequence that can direct the EPA protein toward the periplasm of a host cell (e.g., bacteria), wherein the signal sequence is DsbA (SEQ ID NO: 21), a modified EPA protein according to any of paragraphs 1-22. 24. A conjugate (e.g., a bioconjugate) containing any modified EPA protein from paragraphs 1-23, linked to an antigen (e.g., a monosaccharide antigen, or possibly a bacterial polysaccharide antigen). 25. The modified EPA protein is conjugated according to paragraph 24, either covalently directly to the antigen via chemical bonds available using chemical conjugation methods, or via a linker. 26. The conjugate (e.g., bioconjugate) of paragraph 24, wherein the antigen is covalently linked to an amino acid on a modified EPA protein selected from asparagine, aspartic acid, glutamic acid, lysine, cysteine, tyrosine, histidine, arginine, or tryptophan (e.g., asparagine). 27. The antigen is a conjugate (e.g., bioconjugate) of any of the following paragraphs 24-26: monosaccharide, possibly bacterial polysaccharide (e.g., derived from Shigella shiga, Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or Staphylococcus aureus), possibly Gram-negative bacterial O antigen. 28. A polynucleotide encoding any of the modified EPA proteins described in paragraphs 1-23. 29. A vector containing the polynucleotides from paragraph 28. 30.i) Depending on the circumstances, one or more nucleotide sequences containing a polysaccharide synthesis gene, depending on the circumstances, incorporated into the host cell genome, for generating a bacterial polysaccharide antigen (e.g., O antigen from Gram-negative bacteria, depending on the circumstances, Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, or capsular polysaccharide from Gram-positive bacteria, depending on the circumstances, Streptococcus pneumoniae or Staphylococcus aureus) or a yeast polysaccharide antigen or a mammalian polysaccharide antigen; ii) Nucleotide sequences that encode heterologous oligosaccharide transferases, sometimes within plasmids; iii) A modified EPA protein according to any of paragraphs 1-23, optionally encoding a nucleotide sequence within the plasmid. Host cells containing these cells. 31. A host cell according to paragraph 30, further comprising nucleotide sequences encoding polymerases (e.g., wzy), flippases (e.g., wzx), and optionally nucleotide sequences encoding chain length regulators (e.g., wzz). 32. Oligosaccharide transferase is, in some cases, pglB derived from Campylobacter jejuni, in host cells according to paragraph 30 or paragraph 31. 33. The host cell is Escherichia coli (e.g., E. coli K12 W3110), according to paragraph 30 or paragraph 31. 34. A method for producing a bioconjugate containing a modified EPA protein linked to a polysaccharide, comprising (i) culturing one of the host cells described in paragraphs 30 to 33 under conditions suitable for glycoprotein production, and (ii) isolating the bioconjugate, optionally from a peripheral extract derived from the host cell. 35. An immunogenic composition comprising any of the conjugates (e.g., bioconjugates) described in paragraphs 24-27, and optionally pharmaceutically acceptable excipients and / or carriers. 36. A vaccine comprising the immunogenic composition of paragraph 35, and optionally an adjuvant. 37. A method for inducing an immune response in a subject (e.g., a human), comprising administering to a subject (e.g., a human) in need of induction a therapeutically or prophylactically effective amount of any of the conjugates (e.g., bioconjugates) of paragraphs 24-27, an immunogenic composition of paragraph 35, or a vaccine of paragraph 36. 38. A conjugate (e.g., a bioconjugate) from any of paragraphs 24-27, an immunogenic composition from paragraph 35, or a vaccine from paragraph 36, used to induce an immune response in a subject (e.g., a human). 39. A conjugate (e.g., a bioconjugate) from any of paragraphs 24-27, an immunogenic composition from paragraph 35, or a vaccine from paragraph 36, used in the manufacture of a drug for inducing an immune response in a subject (e.g., a human). 40. A method for treating and / or preventing a yeast infection or bacterial infection in a subject (e.g., a human), comprising administering to a subject (e.g., a human) in need of treatment and / or prevention a therapeutically or prophylactically effective amount of any of the conjugates (e.g., bioconjugates) of paragraphs 24-27, an immunogenic composition of paragraph 35, or a vaccine of paragraph 36. 41. A conjugate (e.g., a bioconjugate) from any of paragraphs 24-27, an immunogenic composition from paragraph 35, or a vaccine from paragraph 36, used to treat and / or prevent a yeast infection or bacterial infection in a subject (e.g., a human). 42. A conjugate (e.g., a bioconjugate) from any of paragraphs 24-27, an immunogenic composition from paragraph 35, or a vaccine from paragraph 36, used in the manufacture of a drug for treating and / or preventing a yeast infection or bacterial infection in a subject (e.g., a human).

[0164] The following examples are provided to allow for a better understanding of the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0165] [Examples] Materials and methods EPA engineering for glycosylation with antigenic glycans The crystalline structure of EPA was analyzed to predict suitable sites for glycosite insertion. Sixty-seven solvent-accessible amino acid residues were selected for site-directed mutagenesis. Genetically detoxified EPA containing the mutation L552VΔE553 was used as a template for mutagenesis (Lukac et al. (1988), Infect Immun, Vol. 56: pp. 3095-3098, and Ho et al. (2006), Hum Vaccin, Vol. 2: pp. 89-98). This gene, along with the N-terminal DsbA signal sequence and the C-terminal His6 tag, was cloned into a pEC415-derived plasmid (Schulz, H., Hennecke, H., Thony-Meyer, L., Prototype of a heme chaperone essential for cytochrome c maturation. Science, Vol. 281, pp. 1197-1200, 1998). Each selected amino acid residue was substituted with the glycosylation secone KDQNATK (SEQ ID NO: 4) to yield a total of 67 EPA mutants (each containing a single glycosylation site (glycosylate)). Additional rounds of mutagenesis were performed on available and selected single-site EPA mutants to generate EPA mutants containing two, three, and four glycosites. Gene synthesis was used to generate EPA mutants containing more than four glycosites. From the 67 mutants tested, four sites: Y208, R274, S318, and A519 were selected for further combinations shown in this study. These mutations were found to be advantageous because (i) they did not reduce protein expression levels (indicating that the overall protein structure was not affected, i.e., not destabilized by the insertion of glycosites at these sites), and (ii) the selected sites were found to achieve effective glycosylation, i.e., high site occupancy. Additionally, we investigated the suitability of additional locations near Y208, R274, S318, and A519 for glycosite insertion (D218, R279, G232, and G525).

[0166] Glycosylation test using manipulated EPA containing one or more glycosites Sixty-seven EPA mutants containing a single inserted glycosite were tested for in vivo glycosylation efficiency using various antigenic glycans. For such glycosylation tests, E. coli strain W3110ΔwaaL was transformed with three plasmids: a pEC415 plasmid containing an EPA mutant, a plasmid expressing PglB, and a plasmid expressing an enzyme for the biosynthesis of the polysaccharide of interest (described below). Non-pathogenic E. coli K12 strain W3110 was obtained from the Coli Genetic Stock Center (Yale University, New Haven (CT), USA, product number CGSC#4474). In some cases, glycosylation tests were performed using E. coli strains in which a cluster of genes for polysaccharide biosynthesis was incorporated into the E. coli genome, enabling transformation using only two plasmids expressing EPA mutant and PglB (see International Publication No. 2014 / 057109 and International Publication No. 2015 / 052344 for further details on the incorporation).

[0167] The selection criteria for EPA variants with a single glycosite included total expression level and the level of complex carbohydrates produced. The latter indicates the suitability of the glycosite location for modification by PglB.

[0168] The dataset presented in this study uses a derivative of strain W3110, which includes deletions in the lipopolysaccharide O antigen ligase gene waaL, deletions or substitutions of the O16 O antigen cluster rfb, and substitutions of genomic clusters by clusters responsible for the biosynthesis of desired recombinant glycans of Klebsiella pneumoniae O antigen (KpO antigen), Shigella flexner 2a (Sf2a), or Streptococcus pneumoniae 11A (Sp11A) and 33F (Sp33F) capsular polysaccharides, as well as Pseudomonas aeruginosa O antigens (PaO6 and PaO11).

[0169] Figures 6 and 8 (P018-0183) show that the Escherichia coli strains used for recombinant generation of the Klebsiella pneumoniae O antigen in glycans also contain potentially interfering elements: the wzzE-wecG gene derived from the Enterobacteria Common Antigen (ECA) wec cluster and the cholanic acid wca cluster, and genomic deletions of the gtrABS and wzzB genes involved in O16 biosynthesis.

[0170] E. coli strains producing the KpO antigens shown in Figures 1-5 or the KpO antigen of the glycan shown in Figure 8 (P018_0167) were transformed with the pEC415 plasmid containing the EPA mutant and a plasmid expressing PglB. To prepare the preculture, colony streaks from the transformed plates were inoculated into 5 ml of Terrific Broth (TB) medium containing 10 mM MgCl2 and appropriate antibiotics, and grown o / n (overnight) at 37°C. Using the preculture, the cells were inoculated into 50 ml of supplemental TB medium in a shaking flask and started the process. 600 =0.1 was given. The culture medium was heated at 37°C while shaking at 200 rpm. 600 The cells were grown until the gene count reached 0.8-1, and then induced by the addition of 0.001% arabinose (EPA) and 0.1 mM IPTG (PglB). Expression and glycosylation of the EPA mutant were continued over and over at 37°C.

[0171] The E. coli strains that produce the KpO antigen shown in Figures 6 and 8 (P018-0183) were transformed with plasmids expressing both the EPA mutant and PglB. These cultures were then induced by the addition of 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) alone, and the expression of the EPA mutant and glycosylation were continued over and over at 37°C.

[0172] Preparation of periplasm extract OD 600The amount of cells derived from the o / n culture medium corresponding to =60 (measured using a spectrophotometer) was collected by centrifugation. The cell pellet was resuspended in 1.5 ml of lysis buffer (30 mM Tris-HCl pH 8.5, 1 mM EDTA (ethylenediaminetetraacetic acid), 20% sucrose), and lysozyme was added to a final concentration of 1 mg / ml. The suspension was incubated at 4°C for 25 minutes with gentle shaking, and then centrifuged at 16,000 rcf for 10 minutes. After centrifugation, the supernatant corresponding to the peripheral extract (PPE) was transferred to a fresh tube.

[0173] Enrichment of peripheral substance extracts by fixed-metal affinity chromatography (IMAC) To enrich the peripheral extract with EPA mutants and enable more direct readout by SDS-PAGE, His-tagged EPA mutants were purified using a one-step purification method with Ni-NTA (nickel nitrilotriacetate) agarose. 1 ml of PPE was mixed with 250 μl of 5× binding buffer (150 mM Tris HCl pH 8.0, 50 mM imidazole, 2.5 M NaCl, 20 mM MgCl2), then 200 μl of pre-equilibriumized Ni-NTA slurry was added, and the mixture was incubated for 30 minutes with gentle shaking. The resin was then washed with 1× binding buffer (30 mM Tris pH 8.0, 10 mM imidazole, 500 mM NaCl), and the binding protein was eluted with elution buffer (30 mM Tris pH 8.0, 500 mM imidazole, 200 mM NaCl). IMAC-enriched PPE was analyzed by SDS-PAGE (Laemmli, UK (1970). "Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4". Nature. Vol. 227 (No. 5259): pp. 680-685. Bibcode: 1970Natur.227..680L. doi: 10.1038 / 227680a0. ISSN 0028-0836. PMID 5432063). Non-glycosylated carriers and EPA complex carbohydrates glycosylated at one or more positions were detected on the gel by Coomassie staining (Fazekas de St. Groth, S.; Webster, RG; Datyner, A. (1963). "Two new staining procedures for quantitative estimation of proteins on electrophoretic strips". Biochimica et Biophysica Acta. Vol. 71: pp. 377-391. doi: 10.1016 / 0006-3002(63)91092-8. PMID 18421828).

[0174] Western blot analysis of peripheral substance extracts Furthermore, peripheral extracts were analyzed by immunoblotting against EPA (Sigma-Aldrich, Cat. number P2318) and polysaccharides bound to EPA. Antiserum against KpO antigens of Klebsiella pneumoniae was used for detection.

[0175] The results of these experiments are shown in Figures 3 to 6 and are described in the following examples.

[0176] [Example 1] (Figure 3) SDS-PAGE analysis was performed on IMAC-enriched peripheral extracts of E. coli strains expressing EPA mutants that produced KpO antigen polysaccharide and introduced PglB and the glycosite KDQNATK (SEQ ID NO: 4) at the following positions in SEQ ID NO: Y208 (lane 1), K240 (lane 2), R274 (lane 3), S318 (lane 4), A376 (lane 5), A519 (lane 6), and K240 and A376 (lane 7). The bands shown in Figure 3 correspond to KpO antigen-EPA bioconjugates with a non-glycosylated EPA carrier and one and two glycosites occupied.

[0177] Conclusion: EPA glycosylation by KpO antigens at each of the new sites was found to be equally good or better compared to sites K240 and A376. In particular, Y208, S318, and A519 appeared to be superior to sites K240 and A376. Y208 showed higher total expression, and all three showed higher conjugate levels than sites K240 and A376.

[0178] [Example 2] (Figure 4) SDS-PAGE analysis was performed on IMAC-enriched peripheral extracts of E. coli strains expressing EPA mutants that generate KpO antigen polysaccharides and have PglB and glycosite KDQNATK (SEQ ID NO: 4) introduced at the following positions: K240 and A376 (lane 1, SEQ ID NO: 26), Y208 and R274 (lane 2, SEQ ID NO: 6), Y208 and S318 (lane 3, SEQ ID NO: 28), Y208 and A519 (lane 4, SEQ ID NO: 29), R274 and S318 (lane 5, SEQ ID NO: 30), R274 and A519 (lane 6, SEQ ID NO: 31), S318 and A519 (lane 7, SEQ ID NO: 7), and Y208, R274, and A519 (lane 8). The bands shown in Figure 4 correspond to KpO antigen-EPA bioconjugates with a non-glycosylated EPA carrier and one, two, and three glycosites.

[0179] Conclusion: Glycosylation of EPA-2S variants containing glycosites at novel locations (i.e., variants of EPA with two additional glycosylation sites) by KpO antigen was confirmed to be equally good or better than that of EPA-2S with glycosites at positions K240 and A376. From the gel shown in Figure 4, Y208+A519 and S318+A519 had higher total protein levels, and all 2S combinations had a high ratio of 2x glycosylated EPA to single glycosylated EPA. In addition, EPA-3S (i.e., variants of EPA with three additional glycosylation sites) with glycosites at positions Y208, R274, and A519 was sufficiently glycosylated by KpO antigen.

[0180] [Example 3] (Figure 5) It generates KpO antigen polysaccharide and PglB, as well as 1 to 7 glycosites at the following positions: Y208 (lane 1), K240 (lane 2), R274 (lane 3), S318 (lane 4), A376 (lane 5), A519 (lane 6), K240 and A376 (lane 7), Y208 and R274 (lane 8, SEQ ID NO: 6), Y208 and S318 (lane 9), Y208 and A519 (lane 10), R27 4 and S318 (lane 11), R274 and A519 (lane 12), S318 and A519 (lane 13), Y208, R274, and A519 (lane 14, sequence number 7), N-terminal glycotag, and K240 and A376, and C-terminal glycotag (lane 15), N-terminal glycotag, and Y208, R274, and A519 (lane 16, sequence number 8), N-terminal glycotag, and Y208, R27 4, and A519, and C-terminal glycotag (lane 17; SEQ ID NO: 9), N-terminal glycotag, and Y208, S318, and A519, and C-terminal glycotag (lane 18, SEQ ID NO: 10), N-terminal glycotag, and R274, S318, and A519, and C-terminal glycotag (lane 19, SEQ ID NO: 11), N-terminal glycotag, and Y208, R274, S318, and A519, and C-terminal Immunoblotting was performed on peripheral extracts of E. coli strains expressing EPA variants introduced into the glycotag (lane 20, SEQ ID NO: 12), N-terminal glycotag, and Y208, K240, R274, S318, and A519 (lane 21, SEQ ID NO: 13), N-terminal glycotag, and Y208, K240, R274, S318, and A519, as well as the C-terminal glycotag (lane 22, SEQ ID NO: 14). In Figure 5, the upper panel shows immunoblots probed with anti-KpO antigen antiserum, while the lower panel shows immunoblots probed with anti-EPA antibody. The bands correspond to the non-glycosylated EPA carrier and the KpO antigen-EPA bioconjugate occupying 1 to 7 glycosites.

[0181] Conclusion: This demonstrates that when using a variant of EPA containing a combination of seven consensus sequences, glycosylation at up to seven glycosites is possible by combining glycosite locations.

[0182] [Example 4] (Figure 6) The KpO antigen polysaccharide (of a different serotype than those shown in Figures 1-5 and Examples 1-3) is produced, and PglB and the glycosite KDQNATK (SEQ ID NO: 4) are located at the following positions: N-terminal glycotag, and Y208, R274, and A519, and C-terminal glycotag (lane 1, SEQ ID NO: 9), N-terminal glycotag, and Y208, S318, and A519, and C-terminal glycotag (lane 2, SEQ ID NO: 10), N-terminal glycotag, and R274, S318, and A519, and C-terminal glycotag (lane 3, SEQ ID NO: 11), N-terminal glycotag, and Y2 SDS-PAGE analysis was performed on peripheral extracts of E. coli strains expressing EPA mutants introduced into 08, R274, S318, and A519, as well as the C-terminal glycotag (lane 4, SEQ ID NO: 12), the N-terminal glycotag, and Y208, K240, R274, S318, and A519 (lane 5, SEQ ID NO: 13), the N-terminal glycotag, and Y208, K240, R274, S318, and A519, as well as the C-terminal glycotag (lane 6, SEQ ID NO: 14), the N-terminal glycotag, and Y208, R274, and A519 (lane 7, SEQ ID NO: 8). In Figure 6, the bands corresponding to the KpO antigen-EPA bioconjugate are labeled with arrows.

[0183] Conclusion: Using a different Klebsiella pneumoniae serotype O antigen, the glycosylation efficiency of EPA with more than three glycosites can be demonstrated.

[0184] [Example 5] (Figure 7) SDS-PAGE analysis was performed on IMAC-enriched peripheral extracts of Escherichia coli strains expressing EPA mutants that produce Sf2a (see Figure 7 - left) or Sp11A (see Figure 7 - right) polysaccharides and have glycosites at PglB and K240, a second glycosite at A376 (lane 1, SEQ ID NO: 26), or three glycosites at positions Y208, R274, and A519 (lane 2, SEQ ID NO: 7).

[0185] result

[0186] [Table 1]

[0187] Conclusion: EPA3S (Y208, R274, and A519) yield higher site occupancy, a higher sugar-to-protein ratio, and higher sugar yield compared to EPA2S (K240 and A376). The data show increased diglycosylation and triglycosylation with EPA3S compared to EPA2S.

[0188] [Example 6] (Figure 8) SDS-PAGE analysis was performed on IMAC-enriched peripheral extracts of Escherichia coli strains that produced two different Klebsiella pneumoniae O antigen polysaccharides (left and right) and expressed EPA mutants having PglB and glycosites at positions Y208, R274, and A519 (lane 1, SEQ ID NO: 7), or an N-terminal glycotag and glycosites at positions Y208, R274, and A519 (lane 2, SEQ ID NO: 8).

[0189] Conclusion: Addition of the N-terminal glycosite to the 3-site EPA (Y208, R274, and A519) increased the modal molecular weight of the conjugate. This indicates a high site occupancy rate for this additional site.

[0190] [Example 7] (Figure 9) Immunoblotting was performed on peripheral extracts of E. coli strains expressing EPA mutants that generate Sf2a, Sp33F, PaO6, or PaO11 antigen polysaccharides and have one glycosite introduced at PglB and the following positions: Y208 (lane 1), D218 (lane 2), R274 (lane 3), R279 (lane 4), S318 (lane 5), G323 (lane 6), A376 (lane 7), A519 (lane 8), and G525 (lane 9). In Figure 9, the Sf2a panel shows immunoblots probed with anti-EPA antibodies, while the other three panels show immunoblots probed with anti-His antibodies. The bands correspond to the O antigen-EPA bioconjugate with a non-glycosylated EPA carrier and one glycosite occupying it.

[0191] Conclusion: This comparative data demonstrates that, in addition to successful glycosylation at positions Y208, R274, S318, and A519, glycosites can be introduced at additional positions within the sequence ranges 198–218 (e.g., position D218), 264–284 (e.g., position R279), 308–328 (e.g., position G323), and 509–529 (e.g., position G525). While glycosylation at these additional positions is effective in most cases, it is not superior to the primarily selected positions within the specified range. When comparing glycosylation at positions D218, R279, G323, and G525 with position A376, it can be concluded that there is a dependency on the type of antigen polysaccharide. Overall, the additional positions D218, R279, G323, and G525 are not always superior to A376 and often appear to show equivalent glycosylation levels. These additional sites for glycosylation offer further possibilities for combination with other glycosylation sites, which can lead to a higher total sugar-to-protein ratio, i.e., a higher glycan yield, and ultimately reduce the amount of bioconjugate needed for immunization. Furthermore, the more suitable sites for glycosylation there are, the greater the flexibility in bioconjugate design, allowing for the selection of the best combination for each antigen polysaccharide.

[0192] Sequence List Sequence ID 1: EPA sequence derived from Pseudomonas aeruginosa AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFV RAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWN QVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARS QDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGLDPSSIPDKEQAISALPDYASQPGKPPREDLK Sequence ID 2: Consensus Sequence (Artificial Sequence) D / EXNZS / T Sequence ID 3: Consensus Sequence (Artificial Sequence) KD / EXNZS / TK Sequence ID 4: Consensus Sequence (Artificial Sequence) KDQNATK Sequence ID 5: Consensus Sequence (Artificial Sequence) JD / EXNZS / TU Sequence ID 6: Modified EPA sequence (artificial sequence) with the consensus sequence inserted at Y208+R274. AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAH ESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNKDQNATKLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTKDQNATKHRQPRGWEQLEQCGYPVQRLVALYLA ARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVR ARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK Sequence ID 7: Modified EPA sequence (artificial sequence) with the consensus sequence inserted at Y208+R274+A519. AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHE SNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNKDQNATKLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTKDQNATKHRQPRGWEQLEQCGYPVQRLVALYLAARL SWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQ DLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLKDQNATKAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK Sequence ID 8: Modified EPA sequence (artificial sequence) with the consensus sequence inserted at the N-terminus + Y208 + R274 + A519. GSGGGDQNATGSGGGKLAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAK LARDATFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNKDQNATKLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTKDQNATKHRQPRGWEQLEQCGYPVQRL VALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVR ARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLKDQNATKAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK Sequence ID 9: Modified EPA sequence (artificial sequence) with the consensus sequence inserted at the N-terminus + Y208 + R274 + A519 + C-terminus. GSGGGDQNATGSGGGAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDA TFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNKDQNATKLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTKDQNATKHRQPRGWEQLEQCGYPVQRLVALYLAAR LSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIW RGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLKDQNATKAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLGSGGGDQNATGSGG Sequence ID 10: A modified EPA sequence (artificial sequence) in which the consensus sequence is inserted at the N-terminus + Y208 + S318 + A519 + C-terminus. GSGGGDQNATGSGGGAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDA TFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNKDQNATKLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQV DQVIRNALAKDQNATKSPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIW RGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLKDQNATKAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLGSGGGDQNATGSGG Sequence ID 11: A modified EPA sequence (artificial sequence) in which the consensus sequence is inserted at the N-terminus + R274 + S318 + A519 + C-terminus. GSGGGDQNATGSGGGAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDA TFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTKDQNATKHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQV DQVIRNALAKDQNATKSPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIW RGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLKDQNATKAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLGSGGGDQNATGSGG Sequence ID 12: A modified EPA sequence (artificial sequence) in which the consensus sequence is inserted at the N-terminus + Y208 + R274 + S318 + A519 + C-terminus. GSGGGDQNATGSGGGAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATF FVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNKDQNATKLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTKDQNATKHRQPRGWEQLEQCGYPVQRLVALYLAARLSW NQVDQVIRNALAKDQNATKSPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDA IWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLKDQNATKAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLGSGGGDQNATGSGG Sequence ID 13: Modified EPA sequence (artificial sequence) with the consensus sequence inserted at the N-terminus + Y208 + K240 + R274 + S318 + A519. GSGGGDQNATGSGGGAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDA TFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNKDQNATKLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKDNNNSTPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTKDQNATKHRQPRGWEQLEQCGYPVQRLVA LYLAARLSWNQVDQVIRNALAKDQNATKSPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFG GVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLKDQNATKAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGLDPSSIPDKEQAISALPDYASQPGKPPREDLK Sequence ID 14: A modified EPA sequence (artificial sequence) in which the consensus sequence is inserted at the N-terminus + Y208 + K240 + R274 + S318 + A519 + C-terminus. GSGGGDQNATGSGGGAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFF VRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNKDQNATKLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKDNNNSTPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTKDQNATKHRQPRGWEQLEQCGYPVQRLVALYLAAR LSWNQVDQVIRNALAKDQNATKSPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLD AIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLKDQNATKAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLGSGGGDQNATGSGG Sequence ID No. 15: Flagellin (FlgI) signal sequence of Escherichia coli. MIKFLSALILLLVTTAAQA Sequence ID No. 16: E. coli outer membrane porin A (OmpA) signal sequence MKKTAIAIAVALAGFATVAQA Sequence ID No. 17: E. coli maltose-binding protein (MalE) signal sequence MKIKTGARILALSALTTMMFSASALA Sequence ID 18: Pectinate lyase (PelB) signal sequence of *Cordyceps militaris* (soft rot). MKYLLPTAAAGLLLLAAQPAMA Sequence ID 19: Signal sequence of enterotoxin LTIIb in thermolabile Escherichia coli. MSFKKIIKAFVIMAALVSVQAHA Sequence ID No. 20: Bacillus subtilis endoxylanase XynA signal sequence MFKFKKKFLVGLTAAFMSISMFSATASA Sequence ID No. 21: E. coli DsbA signal sequence MKKIWLALAGLVLAFSASA Sequence ID No. 22: E. coli TolB signal sequence MKQALRVAFGFLILWASVLHA Sequence ID 23: SipA signal sequence of Streptococcus agalactiae MKMNKKVLLTSTMAASLLSVASVQAS Sequence ID No. 24: pglB derived from Campylobacter jejuni MLKKEYLKNPYLVLFAMIILAYVFSVFCRFYWVWWASEFNEYFFNNQLMIISNDGYAFAEGARDMIAGFHQPNDLSYYGSSLSALTYWLYKITPFSFESIILYMSTFLSSLVVIPTILLANEYKRPLMGFVAALLASIANSYYNRTMSGYYDTDMLVIVLPMFILFFMVRMILKKDFF SLIALPLFIGIYLWWYPSSYTLNVALIGLFLIYTLIFHRKEKIFYIAVILSSLTLSNIAWFYQSAIIVILFALFALEQKRLNFMIIGILGSATLIFLILSGGVDPILYQLKFYIFRSDESANLTQGFMYFNVNQTIQEVENVDLSEFMRRISGSEIVFLFSLFGFVWLLRKHKSMIMA LPILVLGFLALKGGLRFTIYSVPVMALGFGFLLSEFKAIMVKKYSQLTSNVCIVFATILTLAPVFIHIYNYKAPTVFSQNEASLLNQLKNIANREDYVVTWWDYGYPVRYYSDVKTLVDGGKHLGKDNFFPSFALSKDEQAAANMARLSVEYTEKSFYAPQNDILKTDILQAMMKDYN QSNVDLFLASLSKPDFKIDTPKTRDIYLYMPARMSLIFSTVASFSFINLDTGVLDKPFTFSTAYPLDVKNGEIYLSNGVVLSDDFRSFKIGDNVVSVNSIVEINSIKQGEYKITPIDDKAQFYIFYLKDSAIPYAQFILMDKTMFNSAYVQMFFLGNYDKNLFDLVINSRDAKVFKLKI Sequence ID 25: Consensus Sequence (Artificial) GSGGGD / EXNZS / TGSGG Sequence ID 26: A modified EPA sequence (artificial sequence) in which the consensus sequence is inserted at K240+A376. AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAH ESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKDNNNSTPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWN QVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAKDQNRTKGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVR ARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK Sequence ID 27: A modified EPA sequence (artificial sequence) in which the consensus sequence is inserted at the N-terminus + K240 + A376 + C-terminus. GSGGGDQNATGSGGGKLAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLA RDATFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKDNNNSTPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTRHRQPRGWEQLEQCGYPVQRLVALYLAARL SWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAKDQNRTKGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQ DLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLKLGSGGGDQNAT Sequence ID 28: Modified EPA sequence (artificial sequence) with the consensus sequence inserted at Y208+S318. TIFF2026097994000010.tif60166 Sequence ID 29: Modified EPA sequence (artificial sequence) with the consensus sequence inserted at Y208+A519. TIFF2026097994000011.tif60166 Sequence ID 30: Modified EPA sequence (artificial sequence) with the consensus sequence inserted at R274+S318. TIFF2026097994000012.tif60166 Sequence ID 31: A modified EPA sequence (artificial sequence) in which the consensus sequence is inserted into R274+A519. TIFF2026097994000013.tif60166 Sequence ID 32 Forward primer (artificial sequence) AAGCTAGCGCCGCCGAGGAAGCCTTCGACC Sequence ID 33: Reverse primer (artificial sequence) AAGAATTCTCAGTGGTGGTGGTGGTGGTGCTTCAGGTCCTCGCGCGGCGG Sequence ID 34 EPA_mut_Y208 AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAI DNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHEL NAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQAQ PRREKRWSEWASGKVLCLLDPLDGVYNKDQNATKLAQQRCNLDDTWEGKIYRVLAGNPAK HDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCG YPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTL AAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLG DGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDL DAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRPSLPGFYRTGLTLA APEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRN VGGDLPSSIPDKEQAISALPDYASQPGKPPREDLK sequence number 36 EPA_mut_S318 AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAI DNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHEL NAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQAQ PRREKRWSEWASGKVCLLDPLDGVYNYLAQQRCNLDDTWEGKIIRVLAGNPAK HDLDIKPTVISHRLLHFPEGGSLAALTAHQACHLPLEAFTRHRQPRGWEQLEQCG YPVQRLVALYLAARLSWNQVDQVIRNALAKDQNATKPGSGGDLGEAIREQPEQARLALTL AAAESERFVRQGTGNDEAGASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLG DGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDL DAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLA APEAAGEVERLIGHPLPLLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRN VGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK sequence no. 37 EPA_mut_A519 AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAI DNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHEL NAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQAQ PRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAK HDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLEAFTRHRQPRGWEQLEQCG YPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTL AAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLG DGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDL DAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRWSLPGFYRTGLTLKDQN ATKAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPSAIPTDPRN VGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK SEQ ID NO: 38 Primer GAAGGCGGGCGCGTGACCATTCTCGGC SEQ ID NO: 39 Primer GCCGAGAATGGTCACGCGCCCGCCTTC SEQ ID NO: 40 Nucleotide sequence EPA with mutation Y208>KDQNATK KDQNATK Sequence ID No. 41: Nucleotide sequence EPA containing mutation R274>KDQNATK KDQNATK Sequence ID No. 42: Nucleotide sequence EPA containing mutation S318>KDQNATK KDQNATK Sequence ID No. 43: Nucleotide sequence EPA containing mutation A519>KDQNATK KDQNATK

[0193] <110> GlaxoSmithKline Biologicals SA <120> MODIFIED EXOTOXIN A PROTEINS <130> PA26-101 <150> EP 20182138.6 <151> 2020-06-25 <150> EP 20182139.4 <151> 2020-06-25 <150> US 63 / 043,883 <151> 2020-06-25 <160> 43 <170> PatentIn version 3.5 <210> 1 <211> 612 <212> PRT <213> Pseudomonas aeruginosa <400> 1 Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val 1 5 10 15 Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro 20 25 30 Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val 35 40 45 Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu 50 55 60 Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu 65 70 75 80 Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser 85 90 95 Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn 100 105 110 Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His 115 120 125 Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys 130 135 140 Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu 145 150 155 160 Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met 165 170 175 Ala Gln Ala Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser 180 185 190 Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr 195 200 205 Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile 210 215 220 Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys 225 230 235 240 Pro Thr Val Ile Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu 245 250 255 Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Ala Phe 260 265 270 Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly 275 280 285 Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser 290 295 300 Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly 305 310 315 320 Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala 325 330 335 Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg 340 345 350 Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Ser Ala Asp Val Val 355 360 365 Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp 370 375 380 Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe 385 390 395 400 Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn 405 410 415 Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg 420 425 430 Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln 435 440 445 Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala 450 455 460 Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly 465 470 475 480 Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly 485 490 495 Ala Leu Leu Arg Val Tyr Val Pro Arg Trp Ser Leu Pro Gly Phe Tyr 500 505 510 Arg Thr Gly Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu 515 520 525 Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly 530 535 540 Pro Glu Glu Glu Gly Gly Arg Val Thr Ile Leu Gly Trp Pro Leu Ala 545 550 555 560 Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn 565 570 575 Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala 580 585 590 Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg 595 600 605 Glu Asp Leu Lys 610 <210> 2 <211> 5 <212> PRT <213> Artificial <220> <223> Consensus sequence <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa can be Asp or Glu <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa can be any naturally occurring amino acid except proline <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa can be any naturally occurring amino acid except proline <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa can be Ser or Thr <400> 2 Xaa Xaa Asn Xaa Xaa 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial <220> <223> Consensus sequence <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa can be Asp or Glu <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa can be any naturally occurring amino acid except proline <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa can be any naturally occurring amino acid except proline <220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa can be Ser or Thr <400> 3 Lys Xaa Xaa Asn Xaa Xaa Lys 1 5 <210> 4 <211> 7 <212> PRT <213> Artificial <220> <223> Consensus sequence <400> 4 Lys Asp Gln Asn Ala Thr Lys 1 5 <210> 5 <211> 7 <212> PRT <213> Artificial <220> <223> Consensus sequence <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa can be any naturally occurring amino acid <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa can be Asp or Glu <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa can be any naturally occurring amino acid except proline <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa can be any naturally occurring amino acid except proline <220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa can be Ser or Thr <220> <221> MISC_FEATURE <222> (7)..(7) <223> Xaa can be any naturally occurring amino acid <400> 5 Xaa Xaa Xaa Asn Xaa Xaa Xaa 1 5 <210> 6 <211> 624 <212> PRT <213> Artificial <220> <223> Modified EPA sequence with consensus sequences inserted at Y208+R274 <400> 6 Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val 1 5 10 15 Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro 20 25 30 Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val 35 40 45 Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu 50 55 60 Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu 65 70 75 80 Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser 85 90 95 Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn 100 105 110 Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His 115 120 125 Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys 130 135 140 Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu 145 150 155 160 Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met 165 170 175 Ala Gln Ala Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser 180 185 190 Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Lys 195 200 205 Asp Gln Asn Ala Thr Lys Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp 210 215 220 Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys 225 230 235 240 His Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe 245 250 255 Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His 260 265 270 Leu Pro Leu Glu Ala Phe Thr Lys Asp Gln Asn Ala Thr Lys His Arg 275 280 285 Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln 290 295 300 Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val 305 310 315 320 Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp 325 330 335 Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu 340 345 350 Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly 355 360 365 Asn Asp Glu Ala Gly Ala Ala Ser Ala Asp Val Val Ser Leu Thr Cys 370 375 380 Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala 385 390 395 400 Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly 405 410 415 Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu 420 425 430 Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe 435 440 445 Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe 450 455 460 Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly 465 470 475 480 Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp 485 490 495 Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg 500 505 510 Val Tyr Val Pro Arg Trp Ser Leu Pro Gly Phe Tyr Arg Thr Gly Leu 515 520 525 Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly 530 535 540 His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu 545 550 555 560 Gly Gly Arg Val Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val 565 570 575 Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp 580 585 590 Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu 595 600 605 Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys 610 615 620 <210> 7 <211> 630 <212> PRT <213> Artificial <220> <223> Modified EPA sequence with consensus sequences inserted at Y208+R274+A519 <400> 7 Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val 1 5 10 15 Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro 20 25 30 Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val 35 40 45 Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu 50 55 60 Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu 65 70 75 80 Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser 85 90 95 Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn 100 105 110 Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His 115 120 125 Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys 130 135 140 Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu 145 150 155 160 Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met 165 170 175 Ala Gln Ala Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser 180 185 190 Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Lys 195 200 205 Asp Gln Asn Ala Thr Lys Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp 210 215 220 Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys 225 230 235 240 His Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe 245 250 255 Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His 260 265 270 Leu Pro Leu Glu Ala Phe Thr Lys Asp Gln Asn Ala Thr Lys His Arg 275 280 285 Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln 290 295 300 Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val 305 310 315 320 Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp 325 330 335 Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu 340 345 350 Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly 355 360 365 Asn Asp Glu Ala Gly Ala Ala Ser Ala Asp Val Val Ser Leu Thr Cys 370 375 380 Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala 385 390 395 400 Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly 405 410 415 Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu 420 425 430 Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe 435 440 445 Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe 450 455 460 Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly 465 470 475 480 Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp 485 490 495 Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg 500 505 510 Val Tyr Val Pro Arg Trp Ser Leu Pro Gly Phe Tyr Arg Thr Gly Leu 515 520 525 Thr Leu Lys Asp Gln Asn Ala Thr Lys Ala Pro Glu Ala Ala Gly Glu 530 535 540 Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile 545 550 555 560 Thr Gly Pro Glu Glu Glu Gly Gly Arg Val Thr Ile Leu Gly Trp Pro 565 570 575 Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro 580 585 590 Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu 595 600 605 Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro 610 615 620 Pro Arg Glu Asp Leu Lys 625 630 <210> 8 <211> 647 <212> PRT <213> Artificial <220> <223> Modified EPA sequence with consensus sequences inserted at N-terminal+Y208+R274+A519 <400> 8 Gly Ser Gly Gly Gly Asp Gln Asn Ala Thr Gly Ser Gly Gly Gly Lys 1 5 10 15 Leu Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 20 25 30 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 35 40 45 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met 50 55 60 Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala 65 70 75 80 Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val 85 90 95 Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly 100 105 110 Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser 115 120 125 Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser 130 135 140 His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala 145 150 155 160 Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn 165 170 175 Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val 180 185 190 Met Ala Gln Ala Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala 195 200 205 Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn 210 215 220 Lys Asp Gln Asn Ala Thr Lys Leu Ala Gln Gln Arg Cys Asn Leu Asp 225 230 235 240 Asp Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala 245 250 255 Lys His Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His 260 265 270 Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys 275 280 285 His Leu Pro Leu Glu Ala Phe Thr Lys Asp Gln Asn Ala Thr Lys His 290 295 300 Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val 305 310 315 320 Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln 325 330 335 Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly 340 345 350 Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala 355 360 365 Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr 370 375 380 Gly Asn Asp Glu Ala Gly Ala Ala Ser Ala Asp Val Val Ser Leu Thr 385 390 395 400 Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp 405 410 415 Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp 420 425 430 Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val 435 440 445 Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val 450 455 460 Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val 465 470 475 480 Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg 485 490 495 Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln 500 505 510 Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu 515 520 525 Arg Val Tyr Val Pro Arg Trp Ser Leu Pro Gly Phe Tyr Arg Thr Gly 530 535 540 Leu Thr Leu Lys Asp Gln Asn Ala Thr Lys Ala Pro Glu Ala Ala Gly 545 550 555 560 Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala 565 570 575 Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Val Thr Ile Leu Gly Trp 580 585 590 Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp 595 600 605 Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys 610 615 620 Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys 625 630 635 640 Pro Pro Arg Glu Asp Leu Lys 645 <210> 9 <211> 658 <212> PRT <213> Artificial <220> <223> Modified EPA sequence with consensus sequences inserted at N-terminal+Y208+R274+A519+C-terminal <400> 9 Gly Ser Gly Gly Gly Asp Gln Asn Ala Thr Gly Ser Gly Gly Gly Ala 1 5 10 15 Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val Leu 20 25 30 Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro Ala 35 40 45 Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val Leu 50 55 60 Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu Ser 65 70 75 80 Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu Pro 85 90 95 Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser Trp 100 105 110 Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn Ile 115 120 125 Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His Met 130 135 140 Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys Leu 145 150 155 160 Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu Met 165 170 175 Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met Ala 180 185 190 Gln Ala Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly 195 200 205 Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Lys Asp 210 215 220 Gln Asn Ala Thr Lys Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr 225 230 235 240 Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His 245 250 255 Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro 260 265 270 Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu 275 280 285 Pro Leu Glu Ala Phe Thr Lys Asp Gln Asn Ala Thr Lys His Arg Gln 290 295 300 Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg 305 310 315 320 Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp 325 330 335 Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu 340 345 350 Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr 355 360 365 Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn 370 375 380 Asp Glu Ala Gly Ala Ala Ser Ala Asp Val Val Ser Leu Thr Cys Pro 385 390 395 400 Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu 405 410 415 Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly 420 425 430 Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg 435 440 445 Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val 450 455 460 Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly 465 470 475 480 Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe 485 490 495 Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln 500 505 510 Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val 515 520 525 Tyr Val Pro Arg Trp Ser Leu Pro Gly Phe Tyr Arg Thr Gly Leu Thr 530 535 540 Leu Lys Asp Gln Asn Ala Thr Lys Ala Pro Glu Ala Ala Gly Glu Val 545 550 555 560 Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr 565 570 575 Gly Pro Glu Glu Glu Gly Gly Arg Val Thr Ile Leu Gly Trp Pro Leu 580 585 590 Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg 595 600 605 Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln 610 615 620 Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro 625 630 635 640 Arg Glu Asp Leu Gly Ser Gly Gly Gly Asp Gln Asn Ala Thr Gly Ser 645 650 655 Gly Gly <210> 10 <211> 659 <212> PRT <213> Artificial <220> <223> Modified EPA sequence with consensus sequences inserted at N-terminal+Y208+S318+A519+C-terminal <400> 10 Gly Ser Gly Gly Gly Asp Gln Asn Ala Thr Gly Ser Gly Gly Gly Ala 1 5 10 15 Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val Leu 20 25 30 Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro Ala 35 40 45 Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val Leu 50 55 60 Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu Ser 65 70 75 80 Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu Pro 85 90 95 Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser Trp 100 105 110 Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn Ile 115 120 125 Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His Met 130 135 140 Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys Leu 145 150 155 160 Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu Met 165 170 175 Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met Ala 180 185 190 Gln Ala Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly 195 200 205 Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Lys Asp 210 215 220 Gln Asn Ala Thr Lys Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr 225 230 235 240 Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His 245 250 255 Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro 260 265 270 Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu 275 280 285 Pro Leu Glu Ala Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln 290 295 300 Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu 305 310 315 320 Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala 325 330 335 Leu Ala Lys Asp Gln Asn Ala Thr Lys Ser Pro Gly Ser Gly Gly Asp 340 345 350 Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu 355 360 365 Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly 370 375 380 Asn Asp Glu Ala Gly Ala Ala Ser Ala Asp Val Val Ser Leu Thr Cys 385 390 395 400 Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala 405 410 415 Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly 420 425 430 Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu 435 440 445 Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe 450 455 460 Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe 465 470 475 480 Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly 485 490 495 Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp 500 505 510 Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg 515 520 525 Val Tyr Val Pro Arg Trp Ser Leu Pro Gly Phe Tyr Arg Thr Gly Leu 530 535 540 Thr Leu Lys Asp Gln Asn Ala Thr Lys Ala Pro Glu Ala Ala Gly Glu 545 550 555 560 Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile 565 570 575 Thr Gly Pro Glu Glu Glu Gly Gly Arg Val Thr Ile Leu Gly Trp Pro 580 585 590 Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro 595 600 605 Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu 610 615 620 Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro 625 630 635 640 Pro Arg Glu Asp Leu Gly Ser Gly Gly Gly Asp Gln Asn Ala Thr Gly 645 650 655 Ser Gly Gly <210> 11 <211> 659 <212> PRT <213> Artificial <220> <223> Modified EPA sequence with consensus sequences inserted at N-terminal+R274+S318+A519+C-terminal <400> 11 Gly Ser Gly Gly Gly Asp Gln Asn Ala Thr Gly Ser Gly Gly Gly Ala 1 5 10 15 Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val Leu 20 25 30 Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro Ala 35 40 45 Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val Leu 50 55 60 Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu Ser 65 70 75 80 Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu Pro 85 90 95 Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser Trp 100 105 110 Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn Ile 115 120 125 Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His Met 130 135 140 Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys Leu 145 150 155 160 Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu Met 165 170 175 Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met Ala 180 185 190 Gln Ala Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly 195 200 205 Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr Leu 210 215 220 Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile Tyr 225 230 235 240 Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys Pro 245 250 255 Thr Val Ile Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu Ala 260 265 270 Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Ala Phe Thr 275 280 285 Lys Asp Gln Asn Ala Thr Lys His Arg Gln Pro Arg Gly Trp Glu Gln 290 295 300 Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu 305 310 315 320 Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala 325 330 335 Leu Ala Lys Asp Gln Asn Ala Thr Lys Ser Pro Gly Ser Gly Gly Asp 340 345 350 Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu 355 360 365 Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly 370 375 380 Asn Asp Glu Ala Gly Ala Ala Ser Ala Asp Val Val Ser Leu Thr Cys 385 390 395 400 Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala 405 410 415 Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly 420 425 430 Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu 435 440 445 Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe 450 455 460 Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe 465 470 475 480 Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly 485 490 495 Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp 500 505 510 Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg 515 520 525 Val Tyr Val Pro Arg Trp Ser Leu Pro Gly Phe Tyr Arg Thr Gly Leu 530 535 540 Thr Leu Lys Asp Gln Asn Ala Thr Lys Ala Pro Glu Ala Ala Gly Glu 545 550 555 560 Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile 565 570 575 Thr Gly Pro Glu Glu Glu Gly Gly Arg Val Thr Ile Leu Gly Trp Pro 580 585 590 Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro 595 600 605 Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu 610 615 620 Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro 625 630 635 640 Pro Arg Glu Asp Leu Gly Ser Gly Gly Gly Asp Gln Asn Ala Thr Gly 645 650 655 Ser Gly Gly <210> 12 <211> 665 <212> PRT <213> Artificial <220> <223> Modified EPA sequence with consensus sequences inserted at N-terminal+Y208+R274+S318+A519+C-terminal <400> 12 Gly Ser Gly Gly Gly Asp Gln Asn Ala Thr Gly Ser Gly Gly Gly Ala 1 5 10 15 Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val Leu 20 25 30 Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro Ala 35 40 45 Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val Leu 50 55 60 Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu Ser 65 70 75 80 Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu Pro 85 90 95 Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser Trp 100 105 110 Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn Ile 115 120 125 Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His Met 130 135 140 Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys Leu 145 150 155 160 Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu Met 165 170 175 Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met Ala 180 185 190 Gln Ala Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly 195 200 205 Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Lys Asp 210 215 220 Gln Asn Ala Thr Lys Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr 225 230 235 240 Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His 245 250 255 Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg Leu His Phe Pro 260 265 270 Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His Leu 275 280 285 Pro Leu Glu Ala Phe Thr Lys Asp Gln Asn Ala Thr Lys His Arg Gln 290 295 300 Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg 305 310 315 320 Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp 325 330 335 Gln Val Ile Arg Asn Ala Leu Ala Lys Asp Gln Asn Ala Thr Lys Ser 340 345 350 Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu 355 360 365 Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe 370 375 380 Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Ser Ala Asp 385 390 395 400 Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro 405 410 415 Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala 420 425 430 Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr 435 440 445 Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu 450 455 460 Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala 465 470 475 480 Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu 485 490 495 Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala 500 505 510 Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg 515 520 525 Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Trp Ser Leu Pro Gly 530 535 540 Phe Tyr Arg Thr Gly Leu Thr Leu Lys Asp Gln Asn Ala Thr Lys Ala 545 550 555 560 Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro 565 570 575 Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Val 580 585 590 Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser 595 600 605 Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser 610 615 620 Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala 625 630 635 640 Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Gly Ser Gly Gly Gly 645 650 655 Asp Gln Asn Ala Thr Gly Ser Gly Gly 660 665 <210> 13 <211> 658 <212> PRT <213> Artificial <220> <223> Modified EPA sequence with consensus sequences inserted at N-terminal+Y208+K240+R274+S318+A519 <400> 13 Gly Ser Gly Gly Gly Asp Gln Asn Ala Thr Gly Ser Gly Gly Gly Ala 1 5 10 15 Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val Leu 20 25 30 Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro Ala 35 40 45 Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val Leu 50 55 60 Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu Ser 65 70 75 80 Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu Pro 85 90 95 Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser Trp 100 105 110 Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn Ile 115 120 125 Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His Met 130 135 140 Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys Leu 145 150 155 160 Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu Met 165 170 175 Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met Ala 180 185 190 Gln Ala Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly 195 200 205 Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Lys Asp 210 215 220 Gln Asn Ala Thr Lys Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr 225 230 235 240 Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His 245 250 255 Asp Leu Asp Ile Lys Asp Asn Asn Asn Ser Thr Pro Thr Val Ile Ser 260 265 270 His Arg Leu His Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala 275 280 285 His Gln Ala Cys His Leu Pro Leu Glu Ala Phe Thr Lys Asp Gln Asn 290 295 300 Ala Thr Lys His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys 305 310 315 320 Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu 325 330 335 Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Lys Asp 340 345 350 Gln Asn Ala Thr Lys Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala 355 360 365 Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala 370 375 380 Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala 385 390 395 400 Gly Ala Ala Ser Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala 405 410 415 Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg 420 425 430 Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser 435 440 445 Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln 450 455 460 Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His 465 470 475 480 Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg 485 490 495 Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala 500 505 510 Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp 515 520 525 Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro 530 535 540 Arg Trp Ser Leu Pro Gly Phe Tyr Arg Thr Gly Leu Thr Leu Lys Asp 545 550 555 560 Gln Asn Ala Thr Lys Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu 565 570 575 Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu 580 585 590 Glu Glu Gly Gly Arg Val Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg 595 600 605 Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly 610 615 620 Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser 625 630 635 640 Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp 645 650 655 Leu Lys <210> 14 <211> 671 <212> PRT <213> Artificial <220> <223> Modified EPA sequence with consensus sequences inserted at N-terminal+Y208+K240+R274+S318+A519+C-terminal <400> 14 Gly Ser Gly Gly Gly Asp Gln Asn Ala Thr Gly Ser Gly Gly Gly Ala 1 5 10 15 Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val Leu 20 25 30 Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro Ala 35 40 45 Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val Leu 50 55 60 Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu Ser 65 70 75 80 Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu Pro 85 90 95 Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser Trp 100 105 110 Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn Ile 115 120 125 Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His Met 130 135 140 Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys Leu 145 150 155 160 Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu Met 165 170 175 Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met Ala 180 185 190 Gln Ala Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly 195 200 205 Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Lys Asp 210 215 220 Gln Asn Ala Thr Lys Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr 225 230 235 240 Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His 245 250 255 Asp Leu Asp Ile Lys Asp Asn Asn Asn Ser Thr Pro Thr Val Ile Ser 260 265 270 His Arg Leu His Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala 275 280 285 His Gln Ala Cys His Leu Pro Leu Glu Ala Phe Thr Lys Asp Gln Asn 290 295 300 Ala Thr Lys His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys 305 310 315 320 Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu 325 330 335 Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Lys Asp 340 345 350 Gln Asn Ala Thr Lys Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala 355 360 365 Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala 370 375 380 Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala 385 390 395 400 Gly Ala Ala Ser Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala 405 410 415 Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg 420 425 430 Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser 435 440 445 Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln 450 455 460 Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His 465 470 475 480 Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg 485 490 495 Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala 500 505 510 Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp 515 520 525 Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro 530 535 540 Arg Trp Ser Leu Pro Gly Phe Tyr Arg Thr Gly Leu Thr Leu Lys Asp 545 550 555 560 Gln Asn Ala Thr Lys Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu 565 570 575 Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu 580 585 590 Glu Glu Gly Gly Arg Val Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg 595 600 605 Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly 610 615 620 Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser 625 630 635 640 Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp 645 650 655 Leu Gly Ser Gly Gly Gly Asp Gln Asn Ala Thr Gly Ser Gly Gly 660 665 670 <210> 15 <211> 19 <212> PRT <213> E. coli <400> 15 Met Ile Lys Phe Leu Ser Ala Leu Ile Leu Leu Leu Val Thr Thr Ala 1 5 10 15 Ala Gln Ala <210> 16 <211> 21 <212> PRT <213> E. coli <400> 16 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala 20 <210> 17 <211> 26 <212> PRT <213> E. coli <400> 17 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala 20 25 <210> 18 <211> 22 <212> PRT <213> Erwinia carotovorans <400> 18 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala 20 <210> 19 <211> 23 <212> PRT <213> E. coli <400> 19 Met Ser Phe Lys Lys Ile Ile Lys Ala Phe Val Ile Met Ala Ala Leu 1 5 10 15 Val Ser Val Gln Ala His Ala 20 <210> 20 <211> 28 <212> PRT <213> Bacillus subtilis <400> 20 Met Phe Lys Phe Lys Lys Lys Phe Leu Val Gly Leu Thr Ala Ala Phe 1 5 10 15 Met Ser Ile Ser Met Phe Ser Ala Thr Ala Ser Ala 20 25 <210> 21 <211> 19 <212> PRT <213> E. coli <400> 21 Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser 1 5 10 15 Ala Ser Ala <210> 22 <211> 21 <212> PRT <213> E.coli <400> 22 Met Lys Gln Ala Leu Arg Val Ala Phe Gly Phe Leu Ile Leu Trp Ala 1 5 10 15 Ser Val Leu His Ala 20 <210> 23 <211> 26 <212> PRT <213> Streptococcus agalactiae <400> 23 Master Lys Master Asn Lys Lys Val Leu Leu Thr Ser Thr Master Ala Ala Ser 1 5 10 15 Leu Leu Ser Val Ala Ser Val Gln Ala Ser 20 25 <210> 24 <211> 713 <212> PRT <213> Campylobacter jejuni <400> 24 Put Leu Lys Lys Glu Tyr Leu Lys Asn Pro Tyr Leu Val Leu Phe Ala 1 5 10 15 Met Ile Ile Leu Ala Tyr Val Phe Ser Val Phe Cys Arg Phe Tyr Trp 20 25 30 Val Trp Trp Ala Ser Glu Phe Asn Glu Tyr Phe Phe Asn Asn Gln Leu 35 40 45 Met Ile Ile Ser Asn Asp Gly Tyr Ala Phe Ala Glu Gly Ala Arg Asp 50 55 60 Met Ile Ala Gly Phe His Gln Pro Asn Asp Leu Ser Tyr Tyr Gly Ser 65 70 75 80 Ser Leu Ser Ala Leu Thr Tyr Trp Leu Tyr Lys Ile Thr Pro Phe Ser 85 90 95 Phe Glu Ser Ile Ile Leu Tyr Met Ser Thr Phe Leu Ser Ser Leu Val 100 105 110 Val Ile Pro Thr Ile Leu Leu Ala Asn Glu Tyr Lys Arg Pro Leu Met 115 120 125 Gly Phe Val Ala Ala Leu Leu Ala Ser Ile Ala Asn Ser Tyr Tyr Asn 130 135 140 Arg Thr Met Ser Gly Tyr Tyr Asp Thr Asp Met Leu Val Ile Val Leu 145 150 155 160 Pro Met Phe Ile Leu Phe Phe Met Val Arg Met Ile Leu Lys Lys Asp 165 170 175 Phe Phe Ser Leu Ile Ala Leu Pro Leu Phe Ile Gly Ile Tyr Leu Trp 180 185 190 Trp Tyr Pro Ser Ser Tyr Thr Leu Asn Val Ala Leu Ile Gly Leu Phe 195 200 205 Leu Ile Tyr Thr Leu Ile Phe His Arg Lys Glu Lys Ile Phe Tyr Ile 210 215 220 Ala Val Ile Leu Ser Ser Leu Thr Leu Ser Asn Ile Ala Trp Phe Tyr 225 230 235 240 Gln Ser Ala Ile Ile Val Ile Leu Phe Ala Leu Phe Ala Leu Glu Gln 245 250 255 Lys Arg Leu Asn Phe Met Ile Ile Gly Ile Leu Gly Ser Ala Thr Leu 260 265 270 Ile Phe Leu Ile Leu Ser Gly Gly Val Asp Pro Ile Leu Tyr Gln Leu 275 280 285 Lys Phe Tyr Ile Phe Arg Ser Asp Glu Ser Ala Asn Leu Thr Gln Gly 290 295 300 Phe Met Tyr Phe Asn Val Asn Gln Thr Ile Gln Glu Val Glu Asn Val 305 310 315 320 Asp Leu Ser Glu Phe Met Arg Arg Ile Ser Gly Ser Glu Ile Val Phe 325 330 335 Leu Phe Ser Leu Phe Gly Phe Val Trp Leu Leu Arg Lys His Lys Ser 340 345 350 Met Ile Met Ala Leu Pro Ile Leu Val Leu Gly Phe Leu Ala Leu Lys 355 360 365 Gly Gly Leu Arg Phe Thr Ile Tyr Ser Val Pro Val Met Ala Leu Gly 370 375 380 Phe Gly Phe Leu Leu Ser Glu Phe Lys Ala Ile Met Val Lys Lys Tyr 385 390 395 400 Ser Gln Leu Thr Ser Asn Val Cys Ile Val Phe Ala Thr Ile Leu Thr 405 410 415 Leu Ala Pro Val Phe Ile His Ile Tyr Asn Tyr Lys Ala Pro Thr Val 420 425 430 Phe Ser Gln Asn Glu Ala Ser Leu Leu Asn Gln Leu Lys Asn Ile Ala 435 440 445 Asn Arg Glu Asp Tyr Val Val Thr Trp Trp Asp Tyr Gly Tyr Pro Val 450 455 460 Arg Tyr Tyr Ser Asp Val Lys Thr Leu Val Asp Gly Gly Lys His Leu 465 470 475 480 Gly Lys Asp Asn Phe Phe Pro Ser Phe Ala Leu Ser Lys Asp Glu Gln 485 490 495 Ala Ala Ala Asn Met Ala Arg Leu Ser Val Glu Tyr Thr Glu Lys Ser 500 505 510 Phe Tyr Ala Pro Gln Asn Asp Ile Leu Lys Thr Asp Ile Leu Gln Ala 515 520 525 Met Met Lys Asp Tyr Asn Gln Ser Asn Val Asp Leu Phe Leu Ala Ser 530 535 540 Leu Ser Lys Pro Asp Phe Lys Ile Asp Thr Pro Lys Thr Arg Asp Ile 545 550 555 560 Tyr Leu Tyr Met Pro Ala Arg Met Ser Leu Ile Phe Ser Thr Val Ala 565 570 575 Ser Phe Ser Phe Ile Asn Leu Asp Thr Gly Val Leu Asp Lys Pro Phe 580 585 590 Thr Phe Ser Thr Ala Tyr Pro Leu Asp Val Lys Asn Gly Glu Ile Tyr 595 600 605 Leu Ser Asn Gly Val Val Leu Ser Asp Asp Phe Arg Ser Phe Lys Ile 610 615 620 Gly Asp Asn Val Val Ser Val Asn Ser Ile Val Glu Ile Asn Ser Ile 625 630 635 640 Lys Gln Gly Glu Tyr Lys Ile Thr Pro Ile Asp Asp Lys Ala Gln Phe 645 650 655 Tyr Ile Phe Tyr Leu Lys Asp Ser Ala Ile Pro Tyr Ala Gln Phe Ile 660,665,670 Leu Met Asp Lys Thr Met Phe Asn Ser Ala Tyr Val Gln Met Phe Phe 675,680,685 Leu Gly Asn Tyr Asp Lys Asn Leu Phe Asp Leu Val Ile Ser Arg 690,695,700 Asp Ala Lys Val Phe Lys Leu Lys Ile 705,710 <210> 25 <211> 14 <212> PRT <213> Artificial <220> <223> Consensus sequence <220> <221> MISC_FEATURES <222> (6)…(6) <223> Whereas Asp or Glu <220> <221> MISC_FEATURES <222> (7)…(7) <223> Whatever can be any naturally occurring amino acid except proline <220> <221> MISC_FEATURE <222> (9)..(9) <223> Xaa can be any naturally occurring amino acid except proline <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa can be Ser or Thr <400> 25 Gly Ser Gly Gly Gly Xaa Xaa Asn Xaa Xaa Gly Ser Gly Gly 1 5 10 <210> 26 <211> 624 <212> PRT <213> Artificial <220> <223> Modified EPA sequence with consensus sequences inserted at K240+A376 <400> 26 Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val 1 5 10 15 Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro 20 25 30 Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val 35 40 45 Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu 50 55 60 Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu 65 70 75 80 Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser 85 90 95 Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn 100 105 110 Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His 115 120 125 Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys 130 135 140 Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu 145 150 155 160 Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met 165 170 175 Ala Gln Ala Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser 180 185 190 Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr 195 200 205 Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile 210 215 220 Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys 225 230 235 240 Asp Asn Asn Asn Ser Thr Pro Thr Val Ile Ser His Arg Leu His Phe 245 250 255 Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His 260 265 270 Leu Pro Leu Glu Ala Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu 275 280 285 Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr 290 295 300 Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn 305 310 315 320 Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg 325 330 335 Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu 340 345 350 Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala 355 360 365 Ala Ser Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Lys Asp Gln 370 375 380 Asn Arg Thr Lys Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala 385 390 395 400 Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly 405 410 415 Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu 420 425 430 Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe 435 440 445 Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe 450 455 460 Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly 465 470 475 480 Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp 485 490 495 Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg 500 505 510 Val Tyr Val Pro Arg Trp Ser Leu Pro Gly Phe Tyr Arg Thr Gly Leu 515 520 525 Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly 530 535 540 His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu 545 550 555 560 Gly Gly Arg Val Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val 565 570 575 Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp 580 585 590 Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu 595 600 605 Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys 610 615 620 <210> 27 <211> 652 <212> PRT <213> Artificial <220> <223> Modified EPA sequence with consensus sequences inserted at N-terminal+K240+A376+C-terminal <400> 27 Gly Ser Gly Gly Gly Asp Gln Asn Ala Thr Gly Ser Gly Gly Gly Lys 1 5 10 15 Leu Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys 20 25 30 Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp 35 40 45 Pro Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met 50 55 60 Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Al...

Claims

1. A modified EPA (Exotoxin A of Pseudomonas aeruginosa) protein having an amino acid sequence identical to SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, wherein the amino acid sequence is modified in that it includes one (or more) consensus sequences selected from: D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the one (or more) consensus sequences are, respectively: (i) one or more amino acids between amino acid residues 198–218 of SEQ ID NO: 1 (e.g., one or more amino acids between amino acid residues 203–213, e.g., amino acid residue Y208), (ii) one or more amino acids between amino acid residues 264–284 (e.g., one or more amino acids between amino acid residues 269–279, e.g., ami Modified EPA protein in which (iii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323, e.g., amino acid residue S318), and (iv) one or more amino acids between amino acid residues 509-529 (e.g., one or more amino acids between amino acid residues 514-524; e.g., amino acid residue A519) are independently selected from, or are added next to or substitute for one or more amino acids at the same position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

1.

2. The amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and is modified in that the amino acid sequence includes two (or more) consensus sequences selected from:D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the two (or more) consensus sequences are, respectively: (i) one or more amino acids between amino acid residues 198–218 of SEQ ID NO: 1 (e.g., one or more amino acids between amino acid residues 203–213, e.g., amino acid residue Y208), (ii) one or more amino acids between amino acid residues 264–284 (e.g., one or more amino acids between amino acid residues 269–279, e.g., amino acid residue R274), (iii) one or more amino acids between amino acid residues 308 to 328 (e.g., one or more amino acids between amino acid residues 313 to 323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509 to 529 (e.g., one or more amino acids between amino acid residues 514 to 524; e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230 to 250 (e.g., one or more amino acids between amino acid residues 235 to 245; e.g., amino acid residue K240), or added next to or substituting one or more amino acids at the same position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

1.

3. The amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and is modified in that the amino acid sequence includes three (or more) consensus sequences selected from:D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3), where X and Z are independently any amino acid other than proline, and the three (or more) consensus sequences are each: (i) one or more amino acids between amino acid residues 198–218 of SEQ ID NO: 1 (e.g., one or more amino acids between amino acid residues 203–213, e.g., amino acid residue Y208), (ii) one or more amino acids between amino acid residues 264–284 (e.g., one or more amino acids between amino acid residues 269–279, e.g., amino acid residue R274), (iii) one or more amino acids between amino acid residues 308 to 328 (e.g., one or more amino acids between amino acid residues 313 to 323, e.g., amino acid residue S318), (iv) one or more amino acids between amino acid residues 509 to 529 (e.g., one or more amino acids between amino acid residues 514 to 524; e.g., amino acid residue A519), and (v) one or more amino acids between amino acid residues 230 to 250 (e.g., one or more amino acids between amino acid residues 235 to 245; e.g., amino acid residue K240), or added next to or substituting one or more amino acids at the same position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

1.

4. A modified EPA protein according to any one of claims 1 to 3, wherein each consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) independently substitutes one or more amino acids in the amino acid sequence of SEQ ID NO: 1, or in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 (for example, each consensus sequence substitutes a single amino acid residue, e.g., a single amino acid residue selected from Y208, R274, S318, and A519).

5. A modified EPA protein according to any one of claims 1 to 4, wherein a further consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and JD / EXNZS / TU (SEQ ID NO: 5) (where X and Z are independently any amino acid other than proline, and J and U are independently 1 to 5 naturally occurring amino acid residues) is added to the N-terminus of SEQ ID NO: 1, or adjacent to or substituting one or more amino acids at the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

1.

6. A modified EPA protein according to any one of claims 1 to 5, wherein a further consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and JD / EXNZS / TU (SEQ ID NO: 5) (where X and Z are independently any amino acid other than proline, and J and U are independently 1 to 5 naturally occurring amino acid residues) is added to the C-terminus of SEQ ID NO: 1, or adjacent to or substituting one or more amino acids at the same position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

1.

7. At least one consensus sequence selected from D / EXNZS / T (SEQ ID NO: 2) and KD / EXNZS / TK (SEQ ID NO: 3) (where X and Z are independently any amino acid other than proline) is (i) one or more amino acids between amino acid residues 198-218 of SEQ ID NO: 1 (e.g., one or more amino acids between amino acid residues 203-213, e.g., amino acid residue Y208), or (ii) one or more amino acids between amino acid residues 308-328 (e.g., one or more amino acids between amino acid residues 313-323) The modified EPA protein according to any one of claims 1 to 6, wherein (iii) one or more amino acids between amino acid residues 509 to 529 (e.g., one or more amino acids between amino acid residues 514 to 524; e.g., amino acid residue A519), or is added next to or substituted for an equivalent position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

1.

8. A modified EPA protein according to any one of claims 1 to 7, wherein the amino acid sequence comprises a substitution of leucine 552 (or at a position equivalent to L552 in SEQ ID NO: 1) with valine (L552V), and a deletion of glutamine 553 (or at a position equivalent to E553 in SEQ ID NO: 1) (ΔE553).

9. A conjugate (e.g., a bioconjugate) comprising a modified EPA protein according to any one of claims 1 to 8, covalently linked to an antigen (e.g., a sugar antigen, or optionally a bacterial polysaccharide antigen).

10. The conjugate (e.g., bioconjugate) according to claim 9, wherein the antigen is a sugar, and optionally a bacterial polysaccharide (e.g., derived from Shigella shiga, Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or Staphylococcus aureus).

11. i) one or more nucleotide sequences, optionally containing polysaccharide synthesis genes integrated into the host cell genome, for generating bacterial polysaccharide antigens (e.g., O antigens derived from Gram-negative bacteria, optionally from Shigella flexner, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, or Gram-positive bacteria, optionally from Streptococcus pneumoniae or Staphylococcus aureus) or yeast polysaccharide antigens or mammalian polysaccharide antigens; ii) Nucleotide sequences that encode heterologous oligosaccharide transferases, sometimes within plasmids; iii) A nucleotide sequence optionally encoding the modified EPA protein according to any one of claims 1 to 8 within a plasmid. Host cells containing these cells.

12. A method for producing a bioconjugate containing a modified EPA protein linked to a polysaccharide, comprising (i) culturing the host cells described in claim 11 under conditions suitable for the production of glycoproteins, and (ii) isolating the bioconjugate, optionally isolating the bioconjugate from a peripheral extract derived from the host cells.

13. An immunogenic composition comprising the conjugate (e.g., bioconjugate) according to claim 9 or 10, and optionally pharmaceutically acceptable excipients and / or carriers.

14. A vaccine comprising the immunogenic composition according to claim 13, and optionally an adjuvant.

15. A method for inducing an immune response in a subject (e.g., a human), comprising administering to a subject (e.g., a human) in need of a therapeutically or prophylactically effective amount of the conjugate (e.g., a bioconjugate) described in claim 9 or 10, the immunogenic composition described in claim 13, or the vaccine described in claim 14.

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