EA046206B1 - BIOCONJUGATES OF POLYSACCHARIDE O-ANTIGENS OF E.COLI, METHODS OF THEIR PREPARATION AND METHODS OF THEIR APPLICATION - Google Patents

Description

Cross reference to related applications

This application claims benefit from U.S. Provisional Patent Application No. 62/819,746, filed March 18, 2019, the contents of which are incorporated herein by reference in their entirety.

Link to sequence listing submitted electronically

This application contains a sequence listing submitted electronically via EFSWeb as a sequence listing in ASCII format in a file called 004852_11560_Sequence-Listing, created on March 11, 2020, 198 kB in size. The sequence listing provided via EFS-Web is part of the specification and is incorporated herein by reference in its entirety.

Prior Art

Extraintestinal pathogenic strains of E. coH (ExPEC) are usually harmless inhabitants of the human gastrointestinal tract, along with commensal strains of E. coH. ExPEC isolates are difficult to distinguish from commensal isolates by serotype, despite the predominance of ExPEC in many clonal lineages, as determined by O-antigen, capsular and flagellar antigen (abbreviated O:K:H, e.g. O25:K1:H4) serotypes. Unlike commensal E. coH, ExPEC strains express a wide range of virulence factors, allowing them to colonize the gastrointestinal tract and also cause a wide range of extraintestinal infections associated with a significant burden on the health care system due to hospitalizations and deaths. Infants, the elderly, and immunocompromised patients are partially susceptible to ExPEC infections, including invasive ExPEC disease (IED).

ExPEC strains are the most common cause of urinary tract infections (UTIs) and are an important contributor to surgical wound infections and neonatal meningitis. These strains are also associated with abdominal and pelvic infections and hospital-acquired pneumonia, and sometimes accompany other extraintestinal infections such as osteomyelitis, cellulitis, and wound infections. All of these primary sites of infection can lead to ExPEC bacteremia. ExPEC is the most common cause of community-acquired bacteremia and the main causative agent of hospital-acquired bacteremia and is found in approximately 17-37% of clinically significant blood isolates. Patients with ExPEC-positive blood cultures usually present with septic syndrome, severe sepsis, or septic shock. There has been an increase in ExPEC resistance to first-line antibiotics, including cephalosporins, fluoroquinolones and trimethoprim/sulfamethoxazole. The emergence and rapid proliferation of ExPEC sequence type 131 (ST131) is considered a major driver of increased drug resistance, including multidrug resistance. Among clinical isolates of ExPEC, this clone is found in 12.5-30%, is mainly of the O25b:H4 serotype and demonstrates pronounced resistance to fluoroquinolones, which is often accompanied by resistance to trimethoprim/sulfamethoxazole, and has extended-spectrum beta-lactamases conferring resistance to cephalosporins . O-antigen comprises the immunodominant cell wall lipopolysaccharide (LPS) component of Gram-negative bacteria, including E. coH. Currently, more than 180 serologically unique E. coH O-antigens have been identified, with the vast majority of ExPEC isolates classified into fewer than 20 O-antigen serotypes. Full-length E. coH O-antigens typically consist of approximately 10–25 repeating sugar units attached to a highly conserved core LPS structure, with each component synthesized separately by enzymes encoded predominantly by the rfb and rfa gene clusters, respectively. After polymerization of the O-antigen, the polysaccharide O-antigen backbone can be modified, usually by the addition of acetyl or glucose residues. These modifications effectively increase serotype diversity by creating antigenically distinct serotypes that share a common polysaccharide backbone but differ in their side branches. Genes encoding O-antigen-modifying enzymes are usually found outside the rfb cluster on the chromosome, and in some cases these genes are found within lysogenic bacteriophages.

ExPEC isolates belonging to serogroup O4 have been commonly detected in modern monitoring studies of blood isolates in the US and EU. The structure of the O4 polysaccharide was determined as ->2) α-L-Rha (1->6) α-D-Glc (1->3) α-L-FucNAc (1->3) β-D-GlcNAc (1 -> from E.coH strain O4:K52 (Jann et al., Carbohydr. Res. (1993) v. 248, pp.241-250). For strains O4:K3, O4:K6 and O4:K12, a separate form of the O4 polysaccharide structure, in which the above structure is modified by the addition of α-D-Glc (1->3) linked to a rhamnose residue of the polysaccharide (Jann et al., 1993, supra), this form of the polysaccharide is mentioned below in this document as glycosylated O4. The enzymes responsible for modifying the O-antigen in E. coH O4 strains have not been identified. Efforts to develop a vaccine to prevent ExPEC infections have focused on polysaccharide O-antigen conjugates. A 12-valent O-antigen conjugate vaccine has been synthesized by isolation and purification of polysaccharide O-antigen and chemical conjugation with detoxified Pseudomonas aeruginosa exotoxin A and tested for safety and immunogenicity in clinical settings

- 1 046206 phase 1 study (Cross et al., J. Infect. Dis. (1994) v.170, pp.834-40). This vaccine candidate has never been licensed for clinical use. Recently, a bioconjugation system in E.coH was developed. in which the polysaccharide antigen and carrier protein are synthesized in vivo and subsequently conjugated in vivo through the activity of the oligosaccharyltransferase PglB, a Campylobacter jejuni enzyme expressed in E. coH (Wacker et al., Proc. Nat. Acad. Sci. (2006) v. 103, pp. 7088-93). This N-linked protein glycosylation system is capable of transferring various polysaccharides to a protein carrier, allowing for simple methods to purify the conjugate.

Bioconjugation has been successfully used to produce a conjugated polysaccharide for a quadrivalent E.coH O-antigen vaccine candidate (Poolman and Wacker, J. Infect. Dis. (2016) v.213(1), pp. 6-13). However, development of a successful ExPEC vaccine requires coverage of predominant serotypes, and the presence of additional O-antigen modifications in subsets of ExPEC isolates poses an additional challenge in covering isolates exhibiting unmodified and modified LPS.

Summary of the invention

Due to the increasing resistance of ExPEC isolates to antibiotics and the presence of additional O-antigen modifications among the predominant O-serotypes, there is a need for improved prevention and therapy of these infections.

The invention addresses this need by determining the genetic makeup of current clinical isolates, including identifying genes encoding Oantigen-modifying enzymes, thereby enabling the generation of recombinant host cells capable of synthesizing O-antigen bioconjugates, including bioconjugates containing selected O-antigen modifications.

In one aspect, provided herein is a bioconjugate of a glycosylated E.coH O4 polysaccharide antigen covalently linked to a carrier protein, wherein the glycosylated E.coH O4 polysaccharide antigen has the structure of the formula (O4-Glc+) as shown in Table. 1, where n is an integer from 1 to 100, preferably from 1 to 50, for example from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20.

In a specific embodiment, the glycosylated E.coH O4 polysaccharide antigen is covalently linked to an asparagine residue (Asn) at a glycosylation site in a carrier protein containing a glycosylation consensus sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2.

In some embodiments, the carrier protein is selected from the group consisting of detoxified P. aeruginosa exotoxin A (EPA), E. coH flagellin (FliC), CRM 197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified hemolysin A S. aureus, agglutinating factor A, agglutinating factor B, heat-labile enterotoxin E.coH, neutralized variants of heat-labile enterotoxin E.coH, subunit B of cholera toxin (CTB), cholera toxin, neutralized variants of cholera toxin, E.coli Sat protein, protein passenger domain Sat of E. coli, pneumolysin of Streptococcus pneumoniae, hemocyanin of Megathura crenulata (KLH), PcrV of P. aeruginosa, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from nontypeable Haemophilus influenzae. In a specific embodiment, the carrier protein is detoxified P. aeruginosa exotoxin A (EPA). In such embodiments, the EPA preferably contains from 1 to 20, preferably from 1 to 10, preferably from 2 to 4, consensus glycosylation sequences having SEQ ID NO: 1, the consensus sequences preferably having SEQ ID NO: 2.

In a specific embodiment, the carrier protein contains four consensus glycosylation sequences (EPA-4). In a preferred embodiment, the EPA carrier protein comprises SEQ ID NO: 3.

In another aspect, a composition or immunogenic composition is provided comprising a bioconjugate of a glycosylated E. coli O4 polysaccharide antigen covalently linked to a carrier protein as described herein. In some embodiments, the composition or immunogenic composition contains at least one additional polysaccharide antigen covalently linked to a carrier protein.

In some embodiments, the composition or immunogenic composition contains at least one additional polysaccharide antigen covalently linked to a carrier protein, wherein the at least one additional polysaccharide antigen is selected from the group consisting of E. coli O1A polysaccharide antigen, E. coli O2 polysaccharide antigen , E.coli polysaccharide antigen O6A, E.coli polysaccharide antigen O8, E.coli polysaccharide antigen O15, E.coli polysaccharide antigen O16, E.coli polysaccharide antigen O18, E.coli polysaccharide antigen 025b and E.coli polysaccharide antigen O75. In specific embodiments, the polysaccharide antigen O1A of E. coli contains the structure of formula (O1A), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. In certain embodiments, the polysaccharide antigen O2 E.coH contains the structure of the formula ( O2), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. In specific embodiments, the polysaccharide antigen O6AE.coH contains the structure of the formula (O6A ), as shown in table. 1, where n represents

- 2 046206 an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. In specific embodiments, the polysaccharide antigen 08 E.coH contains the structure of formula (O8), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. In specific embodiments, the E. coli O15 polysaccharide antigen contains the structure of the formula ( O15), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. In specific embodiments, the polysaccharide antigen O16 E.coH contains the structure of the formula ( O16), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. In specific embodiments, polysaccharide antigen 018a E.coH contains the structure of the formula (018 a), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. In specific embodiments, the polysaccharide antigen 025B E.coH contains the structure of the formula ( 025B), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. In specific embodiments, the polysaccharide antigen O75 E.coH contains the structure of the formula ( O75), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20.

In some embodiments, each additional E.coH O1 A, O2, O6A, O8, O15, O16, O18A, 025B, and/or O75 antigen is covalently linked to an Asn residue at a glycosylation site containing a glycosylation consensus sequence having SEQ ID NO: 1. preferably having SEQ ID NO: 2, in each carrier protein. In specific embodiments, each carrier protein is detoxified P. aeruginosa exotoxin A (EPA). Preferably, each EPA contains 110, preferably 2-4, preferably 4 glycosylation sites, each of which contains a consensus glycosylation sequence having SEQ ID NO: 2. In certain embodiments, each EPA contains SEQ ID NO: 3.

In specific embodiments, the composition or immunogenic composition comprises at least polysaccharide antigens O1 A, O2, glycosylated O4, O6A and O25B of E. coli, each of which is covalently linked to a carrier protein. In specific embodiments, the composition or immunogenic composition comprises at least polysaccharide antigens O1A, O2, glycosylated O4, O6A, O8, O15, O16, 025B and O75 of E. coli, each of which is covalently linked to a carrier protein. In specific embodiments, the composition or immunogenic composition contains at least the polysaccharide antigens O1A, O2, glycosylated O4, O6A, O8, O15, O16, O18A, 025B and O75 of E. coli, each of which is covalently linked to a carrier protein.

In a specific aspect, a composition or immunogenic composition is provided that contains:

(1) a bioconjugate of the glycosylated polysaccharide antigen O4 of E. coli covalently linked to a carrier protein rendered harmless by the exotoxin AR. aeruginosa (EPA-4) containing SEQ ID NO: 3, wherein the glycosylated polysaccharide antigen O4 of E. coli contains the structure of the formula (O4- Glc+);

(2) a bioconjugate of an E.coli O1A polysaccharide antigen covalently linked to an EPA-4 carrier protein, wherein the E.coli O1A polysaccharide antigen contains the structure of formula (O1A);

(3) a bioconjugate of an E.coli O2 polysaccharide antigen covalently linked to an EPA-4 carrier protein, wherein the E.coli O2 polysaccharide antigen contains the structure of formula (O2);

(4) a bioconjugate of an E.coli O6A polysaccharide antigen covalently linked to an EPA-4 carrier protein, wherein the E.coli O6A polysaccharide antigen contains the structure of formula (O6A);

(5) a bioconjugate of an E.coli O8 polysaccharide antigen covalently linked to an EPA-4 carrier protein, wherein the E.coli O8 polysaccharide antigen contains the structure of formula (O8);

(6) a bioconjugate of an E.coli O15 polysaccharide antigen covalently linked to an EPA-4 carrier protein, wherein the E.coli O15 polysaccharide antigen contains the structure of formula (O15);

(7) a bioconjugate of an E.coli O16 polysaccharide antigen covalently linked to an EPA-4 carrier protein, wherein the E.coli O16 polysaccharide antigen contains the structure of formula (O16);

(8) a bioconjugate of E.coli polysaccharide antigen 025B covalently linked to an EPA-4 carrier protein, wherein the E.coli polysaccharide antigen 025B contains the structure of formula (025B); and (9) a bioconjugate of an E.coli O75 polysaccharide antigen covalently linked to an EPA-4 carrier protein, wherein the E.coli O75 polysaccharide antigen contains the structure of the formula (O75), where the structure of each of the formulas (O4-Glc+), (O1A) , (O2), (O6A), (O8), (O15), (O16), (O18A), (025B) and (O75) is shown in table. 1, where n is an integer from 1 to 100, preferably from 1 to 50, for example from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20.

In some embodiments, such a composition or immunogenic composition further comprises:

(10) a bioconjugate of the polysaccharide antigen O18A E.coH, covalently linked to the carrier protein EPA-4, where the polysaccharide antigen O18A E.coH contains the structure of the formula (O18A), where the structure of the formula (O18A) is shown in table. 1, and n in this structure is an integer from 1 to 100, preferably from 1 to 50, for example from 3 to 50, for example from 5 to 40, for example from 7 to 25, on

- 3 046206 example from 10 to 20.

In some embodiments, the E. coli polysaccharide antigen 025B bioconjugate is present in the composition described herein at a concentration that is approximately 1.5 to 6 times, such as 2 to 4 times, higher than the concentration of any other bioconjugate in the composition.

In some embodiments, the composition described herein contains bioconjugates of the polysaccharide antigens O1A, O2, glycosylated O4, O6A, O8, O15, O16, 025b and O75 E.coH, wherein the bioconjugates are O1A:O2:glycosylated O4:O6A:O8:O15 :O16:O25B:O75 are present in a polysaccharide mass ratio of 1:1:1:1:1:1:1:2:1 or 2:1:1:2:1:1:1:4:1.

In some embodiments, the composition described herein contains bioconjugates of the polysaccharide antigens O1A, O2, glycosylated O4, O6A, O8, O15, O16, 018a, O25B and O75 E.coH, wherein the bioconjugates are O1A:O2:glycosylated O4:O6A:O8 :O15:O16:O18A:O25B:O75 are present in a polysaccharide mass ratio of 1:1:1:1:1:1:1:1:2:1 or 2:1:1:2:1:1:1:1 :4:1.

In some embodiments, the concentration of the E. coli polysaccharide antigen 025B bioconjugate in the composition described herein is 2 to 50 μg/ml, preferably 8 to 40 μg/ml, such as 16 to 32 μg/ml.

In another aspect, there is provided a method of inducing antibodies against a glycosylated E. coli O4 antigen in a subject, comprising administering to the subject a bioconjugate of a glycosylated E. coli O4 antigen described herein, or a composition or immunogenic composition described herein.

In a specific embodiment, the antibodies have opsonophagocytic activity.

In another aspect, a method of vaccinating a subject against extraintestinal pathogenic E. coli is provided, comprising administering to the subject a bioconjugate of E. coli glycosylated antigen 04 described herein, or a composition or immunogenic composition described herein. In some aspects, a bioconjugate of a glycosylated E. coli O4 polysaccharide antigen described herein, or a composition or immunogenic composition described herein, is provided for use in inducing antibodies against E. coli glycosylated O4 antigen. In some aspects, a bioconjugate of the glycosylated polysaccharide antigen 04 of E. coli described herein, or a composition or immunogenic composition described herein, is provided for use in vaccination against extraintestinal pathogenic E. coli (ExPEC). In some aspects, the use of a bioconjugate of a glycosylated polysaccharide E. coli antigen 04 described herein, or a composition or immunogenic composition described herein, is provided in the manufacture of a medicament for inducing antibodies against glycosylated E. coli antigen 04 in a subject. In some aspects, the use of a bioconjugate of a glycosylated polysaccharide E. coli O4 antigen described herein, or a composition or immunogenic composition described herein, is provided in the manufacture of a medicament for vaccinating a subject against extraintestinal pathogenic E. coli (ExPEC). In another aspect, provided herein is a recombinant host cell for producing a bioconjugate of a glycosylated E.coH O4 polysaccharide antigen covalently linked to a carrier protein, wherein the glycosylated E.coH O4 polysaccharide antigen contains a structure of the formula (O4-Glc+) as shown in Table . 1, where n is an integer from 1 to 100, preferably from 1 to 50, for example from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20, the host cell contains:

(1) the nucleotide sequence of the rfb gene cluster for the O4 polysaccharide antigen of E. coH;

(2) a nucleotide sequence encoding a glucosyltransferase, the sequence of which is at least 80% identical to SEQ ID NO: 4, wherein the glucosyltransferase is capable of modifying the O4 polysaccharide antigen of E.coH to produce a glycosylated polysaccharide antigen O4 of E.coH;

(3) nucleotide sequences encoding a translocase and a glycosyltransferase, the sequences of which are at least 80% identical to SEQ ID NO: 7 and 8, respectively, where the translocase is capable of translocating bactoprenol-bound glucose and the glycosyltransferase is capable of glucylating bactoprenol;

(4) a nucleotide sequence encoding a carrier protein; and (5) a nucleotide sequence encoding an oligosaccharyl transferase capable of covalently linking the E.coH O4 polysaccharide antigen to a carrier protein to produce a bioconjugate.

In a specific embodiment, the recombinant host cell contains a nucleotide sequence encoding a glucosyltransferase that is capable of modifying an E.coH O4 polysaccharide antigen to produce a glycosylated E.coH O4 polysaccharide antigen and has an amino acid sequence that is at least 95%, preferably at least 98% identical to the sequence of SEQ ID NO: 4. In some embodiments, the glucosyltransferase contains SEQ ID NO: 4.

In some embodiments, the recombinant host cell contains a nucleotide sequence encoding a translocase that is capable of translocating bactoprenol-bound glucose and

- 4 046206 has a sequence that is at least 90%, preferably at least 95%, preferably at least 98% identical to the sequence of SEQ ID NO: 7. In some embodiments, the translocase comprises SEQ ID NO: 7.

In some embodiments, the recombinant host cell contains a nucleotide sequence encoding a glycosyltransferase that is capable of glycosylating bactoprenol and has a sequence that is at least 90%, preferably at least 95%, preferably at least 98% identical to the sequence of SEQ ID NO : 8. In some embodiments, the glycosyltransferase comprises SEQ ID NO: 8. In some embodiments, the recombinant host cell contains a nucleotide sequence that encodes an oligosaccharyltransferase comprising the amino acid sequence of SEQ ID NO: 6. In preferred embodiments, the oligosaccharyltransferase comprises the amino acid sequence of SEQ ID NO: 6 having the N311V mutation, more preferably SEQ ID NO: 6 having both the Y77H and N311V mutations. In some embodiments, the recombinant host cell contains a nucleotide sequence that encodes a carrier protein containing at least one glycosylation site containing a consensus glycosylation sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2.

In some embodiments, the rfb gene cluster for E.coH O4 polysaccharide antigen contains a sequence that encodes enzymes that create E.coH O4 polysaccharide antigen (formula (O4-Glc-) in Table 1), and which is at least 80% , e.g., at least 90%, e.g., at least 95%, e.g., at least 98% identical to SEQ ID NO: 9. In some embodiments, the rfb gene cluster comprises SEQ ID NO: 9.

In some embodiments, the recombinant host cell contains a nucleotide sequence encoding a carrier protein, wherein the carrier protein is selected from the group consisting of detoxified P. aeruginosa exotoxin A (EPA), E. coH flagellin (FliC), CRM 197, maltose binding protein ( MBP), diphtheria toxoid, tetanus toxoid, neutralized S.aureus hemolysin A, agglutinating factor A, agglutinating factor B, heat-labile enterotoxin E.coH, neutralized variants of heat-labile enterotoxin E.coH, subunit B of cholera toxin (CTS), cholera toxin, neutralized cholera toxin variants, E. coli Satis protein, E. coli Satis protein passenger domain, Streptococcus pneumoniae pneumolysin, Megathura crenulata hemocyanin (KLH), P. aeruginosa PcrV, Neisseria meningitidis outer membrane protein (OMPC), and D protein from nontypeable Haemophilus influenzae .

In a specific embodiment, the recombinant host cell encodes an inactivated Pseudomonas aeruginosa exotoxin A (EPA) carrier protein, preferably EPA, containing 1-10, preferably 2-4, preferably 4 glycosylation sites, each of which contains a consensus glycosylation sequence having SEQ ID NO : 2. In a preferred embodiment, the EPA comprises the amino acid sequence of SEQ ID NO: 3.

Preferably, the recombinant host cell is E. coH, for example, E. coH strain K-12, such as strain W3110.

In another aspect, a method is provided for producing a bioconjugate of a glycosylated E.coH O4 polysaccharide antigen covalently linked to a carrier protein, wherein the glycosylated E.coH O4 polysaccharide antigen contains a structure of the formula (O4-Glc+) as shown in Table 1. 1, where n is an integer from 1 to 100, preferably from 1 to 50, for example from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20, the method includes culturing a recombinant host cell according to the invention under conditions for the production of a bioconjugate. In some embodiments, the method further includes isolating the bioconjugate from the recombinant host cell. In another aspect, a bioconjugate prepared by the described method is provided. In another aspect, a composition is provided containing a bioconjugate obtained by the described method.

In another aspect, a method is provided for producing a recombinant host cell to produce a bioconjugate of a glycosylated E.coH O4 polysaccharide antigen covalently linked to a carrier protein, wherein the glycosylated E.coH O4 polysaccharide antigen contains a structure of the formula (O4-Glc+) as shown in Table. 1, where n is an integer from 1 to 100, preferably from 1 to 50, for example from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20, the method includes administering one or more one recombinant nucleic acid molecule per cell for producing a recombinant host cell, wherein the recombinant host cell contains:

(1) the nucleotide sequence of the rfb gene cluster for the O4 polysaccharide antigen of E. coH;

(2) a nucleotide sequence encoding a glucosyltransferase, the sequence of which is at least 80% identical to SEQ ID NO: 4, wherein the glucosyltransferase is capable of modifying the O4 polysaccharide antigen of E.coH to produce a glycosylated polysaccharide antigen O4 of E.coH;

(3) nucleotide sequences encoding a translocase and a glycosyltransferase, the sequences of which are at least 80% identical to SEQ ID NO: 7 and 8, respectively, where trans

- 5 046206 locase is capable of translocating bactoprenol-bound glucose, and glycosyltransferase is capable of glucylating bactoprenol;

(4) a nucleotide sequence encoding a carrier protein; and (5) a nucleotide sequence encoding an oligosaccharyltransferase capable of covalently linking the glycosylated E.coH O4 polysaccharide antigen to a carrier protein to produce a bioconjugate.

In a specific embodiment, the glucosyltransferase that is capable of modifying the E.coH O4 polysaccharide antigen to produce a glycosylated E.coH O4 polysaccharide antigen has an amino acid sequence that is at least 90%, at least 95%, preferably at least 98% identical to the sequence of SEQ ID NO: 4. In some embodiments, the glucosyltransferase contains SEQ ID NO: 4.

In some embodiments, the oligosaccharyltransferase contains the amino acid sequence of SEQ ID NO: 6, preferably SEQ ID NO: 6, containing the amino acid mutation N311V. In some embodiments, the oligosaccharyltransferase contains SEQ ID NO: 6 having amino acid mutations Y77H and N311V.

In some embodiments, the rfb gene cluster for E.coH O4 polysaccharide antigen contains a sequence that encodes enzymes that create E.coH O4 polysaccharide antigen (formula (O4-Glc-) in Table 1), and which is at least 80% , e.g., at least 90%, e.g., at least 95%, e.g., at least 98% identical to SEQ ID NO: 9. In some embodiments, the rfb gene cluster comprises SEQ ID NO: 9.

In some embodiments, the translocase is capable of translocating bactoprenol-linked glucose and has a sequence identity of at least 90%, preferably at least 95%, preferably at least 98% to SEQ ID NO: 7. In some embodiments, the translocase comprises SEQ ID NO: 7. In some embodiments, the glycosyltransferase is capable of glycosylating bactoprenol and has at least 90%, preferably at least 95%, preferably at least 98% sequence identity to SEQ ID NO: 8. In some embodiments, the glycosyltransferase comprises SEQ ID NO: 8. In some embodiments, the carrier protein comprises at least one glycosylation site comprising a consensus glycosylation sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2. In some embodiments, the carrier protein is selected from the group consisting of from neutralized exotoxin A of P.aeruginosa (EPA), flagellin E.coH (FliC), CRM 197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, neutralized hemolysin A of S.aureus, agglutinating factor A, agglutinating factor B, thermolabile enterotoxin E.coH, neutralized variants of the thermolabile enterotoxin E.coH, subunit B of cholera toxin (CTS), cholera toxin, neutralized variants of cholera toxin, E.coli Sat protein, E.coli Sat protein passenger domain, Streptococcus pneumoniae pneumolysin, Megathura hemocyanin crenulata (KLH), PcrV of P. aeruginosa, Neisseria meningitidis outer membrane protein (OMPC), and protein D from nontypeable Haemophilus influenzae. In a specific embodiment, the carrier protein is detoxified Pseudomonas aeruginosa exotoxin A (EPA), preferably EPA, containing 1-10, preferably 2-4, preferably 4 glycosylation sites, each containing a consensus glycosylation sequence having SEQ ID NO: 2. In specific embodiments, the carrier protein is EPA with four consensus glycosylation sequences (EPA-4), preferably the carrier protein comprises SEQ ID NO: 3.

In some embodiments, the recombinant host cell is an E. coH cell, for example, from E. coH strain K12, such as strain W3110. In another aspect, a method is proposed for producing a bioconjugate of a polysaccharide antigen _Ox covalently linked to a carrier protein, the method comprising the steps of:

(1) obtaining a recombinant host cell containing:

A. the nucleotide sequence of the rfb gene cluster for the polysaccharide antigen _Ox ;

b. a nucleotide sequence encoding a carrier protein comprising at least one glycosylation site comprising a consensus glycosylation sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2, and

V. nucleotide sequence encoding oligosaccharyltransferase PglB _y ; and (2) culturing the recombinant host cell under conditions for producing the bioconjugate, wherein:

when the _Ox antigen is a polysaccharide antigen O1A, PglBy contains amino acid mutations N311V, K482R, D483H and A669V;

when the Ox antigen is a glycosylated polysaccharide antigen O4, PglBy contains the amino acid mutation N311V or the amino acid mutations Y77H and N311V, and the recombinant host cell further contains a sequence encoding GtrS, the sequence of which is at least 80% identical to SEQ ID NO: 4, and a nucleotide sequence encoding GtrA and GtrB, the sequence of which is at least 80% identical to SEQ ID NO: 7 and

- 6 046206

8, respectively;

when the O _x antigen is an O6A polysaccharide antigen, PglB _y contains the amino acid mutations N311V, K482R, D483H and A669V;

when the O _x antigen is an O8 polysaccharide antigen, PglBy does not contain amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669;

when the O _x antigen is an O15 polysaccharide antigen, PglBy contains the amino acid mutations N311V, K482R, D483H and A669V;

when the O _x antigen is an O16 polysaccharide antigen, PglB _y contains the amino acid mutations Y77H, S80R, Q287P, K289R and N311V;

when the Ox antigen is an O18A polysaccharide antigen, PglBy does not contain amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669; and when the _Ox antigen is an O75 polysaccharide antigen, PglBy contains the amino acid mutation N311V, wherein in each case the amino acid mutations are wild type PglBy having the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the carrier protein is detoxified P. aeruginosa exotoxin A (EPA). Preferably, the EPA carrier protein contains 1-10, preferably 2-4, preferably 4 glycosylation sites, each of which contains a consensus glycosylation sequence having SEQ ID NO: 2. In a particular embodiment, the EPA carrier protein contains SEQ ID NO: 3. In another In this aspect, a bioconjugate is provided, obtained by a method for producing a bioconjugate of a polysaccharide antigen O _x covalently linked to a carrier protein, as described herein. In another aspect, a composition is provided containing such a bioconjugate. In such embodiments, the composition contains at least 2, preferably at least 3, more preferably at least 5, even more preferably at least 7 such bioconjugates.

Brief description of graphic materials

The following brief description, as well as a detailed description of the invention, will be more understandable when read in conjunction with graphic materials. It should be understood that the invention is not limited to the specific embodiments shown in the drawings.

In graphic materials:

in fig. 1A-B show ELISA-measured IgG titers against unmodified (GLC-) or glucose-modified (GLC+) O4 LPS in serum from two rabbits immunized with a Glc-modified O4 polysaccharide bioconjugate as described in Example 4; titers were determined by ELISA in four replicates;

in fig. 2 shows IgG titers in ELISA with whole cells and pooled serum of rabbits immunized with Glc-modified O4 bioconjugate against E. coH O4 isolates with characterized gtrS status, as described in example 4; the following isolates were gtrS negative: A2625, stGVXN4988, OC24784, OC24787, and OC24788; the following isolates were gtrS positive: Y1382, E551, OS24334, stGVXN4983, stGVXN4994, and OS24794; strain OS9487 (ATCC 35383; serotype O75) was also included to serve as a negative control; In fig. 3A-B show Western blots of LPS extracted from gtrS-positive and O4-negative isolates, where pooled serum from rabbits immunized with the modified O4 polysaccharide was used for detection;

in fig. 4A and 4B show humoral responses induced by glycosylated O4 (O4-Glc+)-EPA bioconjugates; in fig. 4A shows serum antibody levels measured by ELISA on days 0, 14 and 42 post-immunization; individual titers (log10 EC50) and GMT ± 95% CI are shown; the gray dotted line indicates the threshold above which sample dilution curves are fit by a 4-parameter logistic regression model; in fig. 4B shows the results of an opsonophagocytic assay (OPK) to determine the functionality of antibodies in serum samples obtained on day 42 after immunization with a glycosylated O4 (O4-Glc+)-EPA bioconjugate (4.0 μg); Wilcoxon rank sum test and Bonferroni correction; *P<0.05, ***P<0.0001;

in fig. 5 shows the booster effect of glycosylated O4 bioconjugate (O4 Glc+) in Sprague Dawley rats immunized with 3 different doses as described in Example 4; Serum antibody levels were measured by ELISA at days 0, 14, and 42 postimmunization; individual titers (log 10 EC50) are shown for each animal; lines between data points connect IgG titers for each animal over time; the gray dotted line indicates the threshold above which sample dilution curves are fit by a 4-parameter logistic regression model; statistical analysis was performed using the Wilcoxon signed rank test and Bonferroni correction for multiple comparisons (day 14 vs. day 0, P = 0.012 for 4.0 μg/dose; day 42 vs. day 0, P = 0.006 for all doses; day 42 vs. day 14, P = 0.006 for all doses);

in fig. 6 shows antibody functionality induced by O4-Glc+-EPA bioconjugate; Sprague Dawley rats were immunized 3 times intramuscularly with composition buffer or O4 (Glc+)-EPA bioconjugate at a dose of 4.00 μg/dose; the functionality of antibodies is determined

- 7 046206 was determined by studying opsonophagocytic activity (OPKA) using O4(Glc+) and O4(Glc-) E. coli strains; individual opsonic titres (O1) and GMT ± 95% CI are shown;

in fig. 7A-B show blot-like electropherograms obtained from screening PglB by capillary electrophoresis using monoclonal antibodies to detect O4-Glc+ bioconjugate in the periplasmic fraction, allowing visualization of O4-Glc+ bioconjugate production for each strain tested. A monoglycosylated product of approximately 180 kDa, a diglycosylated product of approximately 320 kDa, and a triglycosylated product of approximately 450 kDa. A) First round of screening for wild type PglB in lane 3, N311V-PglB in lanes 2 and 4, blank control strain in lane 1, and other PglB variants in lanes 5 and 6. B) Second round of screening for N311V PglB in lane 3, N311V+ Y77H PglB in lane 9, blank control strain in lanes 1 and 2, other PglB variants in remaining lanes;

in fig. 8A-K show the humoral response induced by the ExPEC10V vaccine in New Zealand White rabbits. Animals received 3 intramuscular injections of ExPEC10V or saline, administered 2 weeks apart. ExPEC10V vaccine was administered at 3 different concentrations (Group 1: high dose, Group 2: medium dose and Group 3: low dose, Table 11) and the control group received only saline. solution (group 4, 0.9% (w/v) sodium chloride solution). Antibody levels were measured by ELISA on day 0 (pre-vaccination) and days 14, 27 and 42 (post-vaccination). Individual titers (EC50 values) and geometric mean titers (GMT) ± 95% CI are shown. Wilcoxon rank sum test with Bonferroni correction for multiple comparisons. ExPEC10V-vaccinated animals (groups 1, 2 and 3) were compared with controls that received saline. solution (group 4). *p<0.05, **p<0.01; ***p<0.001; ****p<0.0001. LOD: limit of detection;

in fig. 9A-K show the humoral response induced by ExPEC10V. New Zealand White rabbits received 3 intramuscular immunizations with ExPEC10V (total 105.6 μg polysaccharide) or 0.9% w/v sodium chloride solution (control). IgG titers were determined by ELISA on day 1 (pre-immunization, n=20/group), day 31 (post-immunization, n=20/group) and day 50 (post-immunization, n=10/group). The graphs show individual titers and geometric mean ± 95% confidence interval for each group. Differences in IgG titers between the ExPEC10V-treated and control groups were analyzed using a Tobit model with a likelihood ratio test. P values <0.05 were considered significant. *P<0.05, ****P<0.0001;

in fig. 10A-B show the general design of the Phase 1/2a clinical trial of the ExPEC10V vaccine in humans. In fig. 10A shows the overall study design for Cohort 1, and FIG. 10B shows the overall study design for Cohort 2. Details are described in Example 11.

Detailed Description of the Invention

All publications, articles and patents that are cited or described in the Background Art section or elsewhere in the specification are incorporated by reference in their entirety. The discussion of documents, acts, materials, devices, products, and the like that are included in this description serves to provide context for the invention. Such discussion is not an assumption that any or all of the stated subject matter is part of the prior art with respect to any of the inventions presented or claimed. Unless otherwise specified, all technical and scientific terms used herein have their usual meaning known to those skilled in the art to which this invention relates. Otherwise, certain terms mentioned have the meaning set forth in the specification.

It should be noted that in the specification and claims, all terms in the singular include those entities in the plural unless the context clearly indicates the contrary. Unless otherwise specified, the term at least preceding a series of elements should be understood to refer to each element in the series.

Those skilled in the art will recognize, or be able to discover through routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be within the scope of this invention.

Throughout this specification and in the following claims, unless the context otherwise requires, the word contain and variations such as contains and containing are intended to include the specified integer or stage or group of integers or stages, but not to exclude any other integer or stages or groups of integers or stages. When used herein, the term containing may be replaced by the term comprising or including, and sometimes when used herein by the term having. Consisting of, as used herein, excludes any element, step or ingredient not specified in the claim element. The term essentially consisting of does not exclude materials or steps that do not significantly affect the essential and novel characteristics of the claim. Any of the above terms containing, comprising, including and having, whenever used herein in the context of an aspect or embodiment of the invention, may be replaced by the term consisting of or essentially with

- 8 046206 standing out for changing the opening volume.

In the context of the present description, the union and/or between a plurality of listed elements should be understood as covering both individual and combined options. For example, if two elements are connected and/or, the first option refers to the applicability of the first element without the second. The second option refers to the applicability of the second element without the first. The third option concerns the applicability of the first and second elements together. Either of these options is intended to fall within the meaning of, and therefore satisfy, the requirement of the term and/or as used herein. The simultaneous use of more than one of the options is intended to fall within this meaning and therefore satisfy the requirement of the term and/or.

The identification of a structural modification of the O antigen, namely the glucose branch, in E. coH serotype O4 (Jann et al., 1993) poses a challenge for the discovery and development of a glycoconjugate vaccine targeting bacterial isolates in this serotype. The proportion of modern clinical O4 isolates expressing unmodified (without glucose side branches) and modified (with glucose side branches) forms of O4 O antigen is unknown. Gaining information about this characteristic is critical for selecting the appropriate antigenic structure. Additionally, the extent to which vaccine-induced antibodies against one form of O4 polysaccharide will cross-react with another form has not been determined. Purification of lipid A-free Oantigen and subsequent chemical conjugation to a carrier protein is a lengthy and labor-intensive process. In addition, the processes of purification, lipid A detoxification, and chemical conjugation can lead to loss of epitopes, antigen heterogeneity, and decreased immunogenicity of the conjugated polysaccharide. Synthesis of glycoconjugates by bioconjugate can overcome these limitations of classical purification and chemical conjugation, but in vivo synthesis of O4 glucose branch Oantigen requires the activity of a polysaccharide branching enzyme that resides outside the rfb gene cluster. To date, the O-antigen-modifying enzyme responsible for the glucose branch in O4 E. coH strains has not been identified. Cloning the rfb O4 gene cluster into a bioconjugate strain of E. coH expressing PglB will not be sufficient to synthesize an O4 glycoconjugate with a glucose branch, but rather will result in the formation of only O4 bioconjugates without glucose branches (their glycan structure is shown in formula (O4) in Table 1 ). As used herein, the terms glycosylated O4, glucose branch O4, O4 Glc+ and Glc+ O4 O-antigen refer to the glucose side-branch O-antigen O4, and its structure is shown in the formula (O4-Glc+) in Table 1. 1.

Disclosed herein is a gene encoding an O-antigen-modifying enzyme responsible for the glucose branch of the O4 polysaccharide antigen of E. coH. Also described are host cells, for example, genetically modified host cells containing nucleic acid encoding enzymes capable of producing bioconjugates containing a glycosylated O4 polysaccharide antigen covalently linked to a carrier protein in vivo. Such host cells can be used to create bioconjugates containing a glycosylated O4 antigen linked to a carrier protein, which can be used, for example, in therapeutic and/or prophylactic compositions (eg, vaccines). Further provided herein are compositions containing bioconjugates of glycosylated polysaccharide O4 antigen, alone or in combination with other E. coH antigens (e.g., O1, O2, O6, O8, O15, O16, O18, O25, and/or O75 polysaccharide antigens and their subserotypes). The compositions can be used in methods of prevention and/or therapy, for example, vaccinating hosts against E. coH infection, and they can find use in the production of antibodies that can be used, for example, in methods of therapy, such as immunizing subjects.

As used herein, the terms O antigen, polysaccharide O antigen, saccharide O antigen, and OPS refer to the O antigen of gram-negative bacteria. Typically, the O antigen is a polymer of immunogenic repeating polysaccharide units. In a specific embodiment, the terms O-antigen, polysaccharide O-antigen and OPS refer to the O-antigen of Escherichia coli. Different serotypes of E.coH express different O-antigens. In E.coH, gene products involved in O-antigen biogenesis are encoded by the rfb gene cluster. As used herein, the terms rfb cluster and rfb gene cluster refer to a gene cluster encoding an enzyme apparatus capable of synthesizing the O-antigen backbone structure. The term rfb cluster can be applied to any O-antigen biosynthetic cluster and preferably refers to a cluster of genes from the genus Escherichia, in particular E. coH.

As used herein, the term O1A refers to the O1A antigen of E.coH (subserotype E.coH serotype O1). The term O2 refers to the O2 antigen of E.coH (subserotype E.coH serotype O2). The term 06a refers to the 06A antigen of E.coH (subserotype E.coH serotype O6). The term O8 refers to the O8 antigen of E.coH (subserotype E.coH serotype O8). The term O15 refers to the O15 antigen of E.coH (subserotype E.coH serotype O15). The term 016 refers to the O16 antigen of E.coH (subserotype E.coH serotype O16). The term O18A refers to the O18A antigen of E.coH (subserotype E.coH serotype O18). The term 025B refers to the 025B antigen of E.coH (subserotype E.coH serotype O25). The term O75 refers to the O75 antigen of E. coli (a subserotype of E. coli serotype O75).

The structures of the polysaccharide O-antigens of E. coli mentioned in this application are shown in table below. 1. One repeat unit for each polysaccharide O-antigen of E. coH is shown.

Table 1. Structures of polysaccharide O-antigens of E.coH

Polysaccharide- new O- antigens E. coli Repeating unit structure ¹ Non-glycosylated polysaccharide antigen 04 (O4-G1C-) [—2)-a-L-Rhap-(1 ->6)-a-D-Glcp-(1 ->3)-a-L-FucpNAc-( 1 ->3)-p-D-GlcpNAc-( 1 ->]„ Glycosylated polysaccharide antigen 04 (O4-G1C+) aD-GIcp 1 3 [->2)-aL-Rhap-(1 ->6)-aD-Glcp-(1 -^3)-aL-FucpNAc-(1 -^3>pD-GlcpNAc-(1^ ] _n Polysaccharide antigen O1A(O1A) [—3)-a-L-Rhap-(1 —3>a-L-Rhap-(1 ->3> P-L-Rhap-(1 ->4)^-D-GlcpNAc-(1 -»]„ 2 t 1 β-D-ManpNAc Polysaccharide antigen 02 (02) [->3)-aL-Rhap-(1 ->2)-aL-Rhap-(1 -3)-pL-Rhap-(1 ->4)-pD-GlcpNAc-(1 -*] _n 2 t 1 aD-Fucp3NAc Polysaccharide antigen O6A (Ob) [-*4)-aD-GalpNAc-(1 -»3)-pD-Manp-(1 -»4)-fJ-D-Manp-(1 ->3)-aD-GlcpNAc-(1 -*] _n 2 t 1 β-D-Glcp Polysaccharide antigen 08 (08) aD-Manp3Me-(1 ->[3)-β-0-Μ3ηρ-(1 -*2)-aD-Manp-(1 ->2)-aD-Manp-(1 -*] _n Polysaccharide antigen 015 (015) [^2)-pD-Galp-(1 ^3)-oc-i_-FucpNAc-(1 ->3)-pD-GlcpNAc-(1 ->] _n Polysaccharide antigen 016(016) [->2)^-D-Galf-(1 ->6)-aD-Glcp-(1 ->3)-aL-Rhap-(1 -*3)-aD-GlcpNAc-(1 -] _n 2 f Ac

- 10 046206

Polysaccharide antigen O18A (O18A) [-»2)-a-l_-Rhap-(1 ^6)^ao-Glcp-(1 -4)-aD-Galp-(1 -*3)-aD-GlcpNAc-(1 -*] _n 3 t 1 β-D-GlcpNAc Polysaccharide antigen 025B (025B) β-D-Glcp 1 I 6 [-*4)-aD-Glcp-(1 -»3)-aL-Rhap-(1 -»3)^-D-GlcpNAc-(1 ->] _n 3 2 tt 1 Ac aL-Rhap Polysaccharide antigen 075 (075) β-D-Manp 1 Φ 4 [^•3)-aD-Galp-(1 -4-4)-aL-Rhap-(1 -4-3)-3-D-GlcpNAc-(1 -e] _p

Each n is independently an integer from 1 to 100, such as 1-50, 1-40, 1-30, 1-20 and 1-10, 3-50, 3-40, such as at least 5, such as 5-40, for example 7-30, for example 7 to 25, for example 10 to 20, but in some cases it can be 1-2.

All monosaccharides described herein have a general meaning known in the art. Monosaccharides can have the D or L configuration. If D or L is not specified, the sugar is considered to have the D configuration. Monosaccharides are generally referred to by abbreviations that are widely known and used in the art. For example, Glc refers to glucose, D-Glc refers to D-glucose, and L-Glc refers to L-glucose. Other common abbreviations for monosaccharides include: Rha, rhamnose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; Fuc, fucose; Man, mannose; Man3Me, 3-O-methyl-mannose; Gal, galactose; FucNAc, N-acetylfucosamine and Rib, ribose. The suffix f' refers to furanose and the suffix p refers to pyranose.

The terms RU, repeat unit and repeat unit used in relation to Oantigen refer to the biological repeat unit (BRU) of the O antigen as it is synthesized in vivo by cellular mechanisms (eg, glycosyltransferases). The amount of RU O-antigen may vary depending on the serotype, and in embodiments of the invention typically ranges from about 1 to 100 RU, preferably from about 1 to 50 RU, for example, 1-50 RU, 1-40 RU, 1-30 RU, 1-20 RU and 1-10 RU, and more preferably at least 3 RU, at least 4 RU, at least 5 RU, for example 3-50 RU, preferably 5-40 RU, for example 7-25 RU, for example 10-20 RU. However, in some cases the number of RU O-antigen may be 1-2. The structure of each O-antigen that is specifically described herein is shown to contain one RU with a variable n indicating the number of RUs. In each polysaccharide O-antigen in the bioconjugate of the invention, n is independently equal to an integer from 1 to 100, such as 1-50, 1-40, 1-30, 1-20, 1-10, preferably at least 3 or more preferably at least 5, for example 3-50, preferably 5-40 (for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40), but in some cases there may be 1- 2. In some embodiments, n is independently an integer from 7 to 25, such as about 10-20. Values may vary between individual polysaccharide O-antigens in the composition and are presented here as average values, i.e. if the bioconjugate is described herein as having an n that is independently equal to an integer of 5-40, most of the polysaccharide O-antigens contained in the composition have 5-40 repeat units, but some polysaccharide O-antigens may also be contained having less than 5 repeat units or more than 40 repeating units. The term glycoconjugate refers to a conjugate of a sugar or saccharide antigen (eg, oligo- and polysaccharide) to a protein associated with another chemical entity, including, but not limited to, proteins, peptides, lipids, etc. Glycoconjugates can be prepared chemically, for example by chemically (synthetically) coupling a protein and a sugar or saccharide antigen. The term glycoconjugate also includes bioconjugates.

The term bioconjugate refers to a conjugate between a protein (e.g., a carrier protein) and a sugar or saccharide antigen (e.g., oligo- and polysaccharide) produced in a host cell, preferably a bacterial host cell, e.g., an E. coH host cell, wherein the apparatus of the cell -the host binds the antigen to the protein (for example, binds to N). Preferably, the term bioconjugate refers to a conjugate between a protein (eg, a carrier protein) and an O-antigen,

- 11 046206 respectfully O-antigen of E. coli (for example, O1A, O2, glycosylated O4, O6A, O8, O15, O16, O18A, 025B, O75, etc.) obtained in the host cell, where the cell apparatus is the host binds the antigen to the protein (for example, binds to N). Since bioconjugates are produced in host cells by the host cell machinery, the antigen and protein are covalently linked within the bioconjugate through a glycosidic bond. Bioconjugates can be produced in recombinant host cells that have been genetically modified to express the cellular machinery required for O-antigen synthesis and/or O-antigen binding to the target protein. The bioconjugates that are described herein offer advantages over chemically derived glycoconjugates in which the glycans are isolated from the bacterial cell wall and subsequently chemically linked to a carrier protein, for example, bioconjugates require fewer chemicals to produce and are more uniform in terms of view of the resulting final product and contain less or no free (i.e. not associated with the carrier protein) glycan. Thus, in typical embodiments, bioconjugates are preferred over chemically produced glycoconjugates. The term approximately, when used in conjunction with a number, refers to any number within ±1, ±5 or ±10% of the stated number.

The term percentage (%) sequence identity or % identity describes the number of identical amino acid matches of two or more aligned amino acid sequences compared to the number of amino acid residues comprising the total length of the amino acid sequences. In other words, using alignment, it is possible to establish the percentage of amino acid residues that are the same between two or more sequences (e.g., 90, 95, 97, or 98% identity) when the sequences are compared and aligned to a maximum match determined using a sequence comparison algorithm known in the art. technical field, or by manual alignment and visual inspection. The sequences that are compared to determine sequence identity may thus differ in amino acid substitution(s), addition(s) or deletion(s). Programs suitable for aligning protein sequences are known to those skilled in the art. The percentage identity of protein sequences can be determined, for example, using programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, for example, using the NCBI BLAST algorithm (Altschul SF, et al (1997), Nucleic Acids Res. 25:3389-3402).

For example, for amino acid sequences, sequence identity and/or similarity can be determined using standard techniques known in the art, including, without limitation, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, Algorithm for Aligning Identical Regions by Needleman and Wunsch, 1970, J. Mol. Biol. 48:443), a method for searching for similarities by Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. U.S.A. 85:2444, using a computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al, 1984, Nucl. Acid Res. 12:387-395, preferably using default settings, or by testing. In some embodiments, the percent identity is calculated using FastDB based on the following parameters: mismatch penalty 1; penalty for opening a gap 1; penalty for gap length 0.33; and concatenation penalty 30, Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

Another example of a useful algorithm is the BLAST algorithm, described by: Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al, 1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873–5787. A particularly useful BLAST program is the WUBLAST-2 program, which was obtained from Altschul et al, 1996, Methods in Enzymology 266: 460-480. WUBLAST-2 uses several search parameters, most of which have default values. An additional useful algorithm is gapped BLAST, published by Altschul et al, 1993, Nucl. Acids Res. 25:3389–3402. The term invasive disease caused by extraintestinal pathogenic Escherichia coli (ExPEC) (IED) is defined here as an acute illness with systemic bacterial infection that is microbiologically confirmed either by the isolation and identification of E. coli from the blood or other normally sterile body sites, or by the isolation and identification of is. coli from the urine of a patient with signs and symptoms of invasive disease (systemic inflammatory response syndrome (SIRS), sepsis, or septic shock) and no other identifiable source of infection.

Bioconjugates of glycosylated polysaccharide antigen O4 E.coH.

In one aspect, a bioconjugate of a glycosylated E. coli O4 polysaccharide antigen is provided, covalently linked to a carrier protein. As used herein, the term O4 refers to the O4 antigen of E. coli (E. coli serotype O4). It is known that E.coH serotype O4 has a structural modification of the Oantigen. In particular, some O4 serotypes express a modified O antigen that has a branched glucose unit. The terms glycosylated O4 antigen, glycosylated O4 polysaccharide antigen, O4-Glc+ polysaccharide antigen, and O4 antigen as used herein

- 12 046206

Glc+ refers to an O4 antigen (eg, E. coli O4 antigen) having a glucose branch in which D-glucose is linked to L-rhamnose in the L-Rha^D-Glc^L-FucNAc^D-GlcNAc repeat unit. In a specific embodiment, the glycosylated polysaccharide antigen O4 E.coH contains the structure of the formula (O4-Glc+), as shown in table. 1, where n is an integer from 1 to 100. In preferred embodiments, n is an integer from 3 to 50, such as 5 to 40, such as 7 to 25, such as 10 to 20.

E.coH O4 strains, regardless of glucose branch status, carry an essentially identical rfb gene cluster encoding genes responsible for the production of the O4 polysaccharide antigen. However, in vivo synthesis of the modified glucose branch O4 antigen requires the activity of a polysaccharide branching enzyme located outside the rfb gene cluster. To the best of the inventors' knowledge, the enzyme responsible for the branching of the polysaccharide responsible for the modification of the O4 antigen with glucose is not known to date. The inventors discovered the sequence of the enzyme that provides branching of the polysaccharide responsible for the modification of the O4 antigen by glucose. The discovery of this enzyme makes it possible to obtain bioconjugates of a modified polysaccharide antigen O4, which has a glucose branch. A glucose-modified form of the O4 polysaccharide antigen is present in predominant serotypes and thus can be used to provide an improved immune response, for example, for prophylactic or therapeutic use.

In particular, the present document provides the sequence of the gtrS gene encoding a glucosyltransferase enzyme specific for E. coli serotype O4 that glycosylates the O4 antigen. As a rule, the gtrA, gtrB and gtrS genes encode enzymes responsible for the glycosylation of Oantigen. While the gtrA and gtrB genes are highly homologous and interchangeable across serotypes, the gtrS gene encodes a serotype-specific O-antigen glucosyltransferase. The gtrS gene of serotype O4 E. coli encodes the GtrS enzyme, which modifies the O4 antigen by introducing a glucose branch. Characterization of modern clinical isolates of E. coH serotype O4 revealed the presence of gtrS in 78% of isolates tested, indicating that the polysaccharide E. coH O4 antigen, modified by the addition of a glucose residue, predominates in modern infectious isolates.

In one embodiment, a gtrS gene nucleic acid from E. coli serotype O4 is provided, encoding a GtrS glucosyltransferase comprising the amino acid sequence of SEQ ID NO: 4. In another embodiment, the gtrS nucleic acid encodes a GtrS protein from E. coli serotype O4, which is approximately 80, 85 , 90, 95, 96, 97, 98, 99 or 100% identical to the amino acid sequence of SEQ ID NO: 4, preferably 98, 99 or 100% identical to the amino acid sequence of SEQ ID NO: 4. A GtrS protein that is at least 80% identical to the amino acid sequence of SEQ ID NO: 4. % identical to the amino acid sequence of SEQ ID NO: 4, is capable of specifically glycosylating the polysaccharide O4 antigen E.coH to obtain a glycosylated O4 antigen having the structure of the formula (O4-Glc+), as shown in table. 1. One skilled in the art will be able to generate mutated forms of the GtrS protein SEQ ID NO: 4 that are at least 80% identical to the sequence of SEQ ID NO: 4, and test such sequences for glycosylation activity of the E. coH O4 antigen, taking into account the provisions herein inventions. Recombinant host cells containing a nucleic acid sequence encoding the gtrS glucosyltransferase gene of E. coli serotype O4, and the use of recombinant host cells to produce glucose-modified O4 polysaccharide antigens and bioconjugates thereof are described in more detail below.

Sequences for the proteins encoded by gtrA and gtrB, which function as bactoprenol-bound glucose translocase (GtrA, pulls bactoprenol-bound glucose across the inner membrane into the periplasm) and bactoprenol glucosyltransferase (GtrB, binds glucose to bactoprenol), respectively, may contain amino acid sequences that are at least about 80% identical to SEQ ID NO: 7 and 8, respectively. In some embodiments, nucleic acid sequences encoding the GtrA and GtrB proteins that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 and 8, respectively, and having bactoprenol-linked glucose translocase activity and bactoprenol glucosyltransferase, respectively, are also present in host cells of the invention that further contain the O4-specific rfb locus, the O4-specific GtrS coding sequence described above, the oligosaccharyltransferase described above, and the sequence encoding a carrier protein having one or more than one glycosylation consensus sequence described herein to produce glycosylated E. coli O4 bioconjugates (containing the glycan structure of the formula (O4-Glc+) of Table 1).

The E. coli glycosylated polysaccharide antigen O4 bioconjugates provided herein are covalently linked to a carrier protein, preferably through a glycosidic linkage. Any suitable carrier protein known to those skilled in the art may be used, given the specification herein.

Suitable carriers include detoxified P. aeruginosa exotoxin A (EPA), flagellin E. coH (FHC), CRM 197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid,

- 13 046206 neutralized hemolysin A of S.aureus, agglutinating factor A, agglutinating factor B, heat-labile enterotoxin E.coH, neutralized variants of the heat-labile enterotoxin E.coH, subunit B of cholera toxin (CTB), cholera toxin, neutralized variants of cholera toxin, Sat protein E. coli, E. coli Sat protein passenger domain, Streptococcus pneumoniae pneumolysin, Megathura crenulata hemocyanin (KLH), P. aeruginosa PcrV, Neisseria meningitidis outer membrane protein (OMPC), and protein D from non-typeable Haemophilus influenzae, but are not limited to the above. Bioconjugation to various carrier proteins containing the desired glycosylation consensus sequence has been described, demonstrating that a wide range of proteins can be glycosylated using this technology (see e.g. WO 06/119987, WO 2015/124769, WO 2015/158403, WO 2015/ 82571, WO 2017/216286 and WO 2017/67964, together demonstrating a wide range of protein carriers that have been successfully used in bioconjugation).

In some embodiments, the carrier protein is modified such that the protein is less toxic and/or more susceptible to glycosylation. In a specific embodiment, the carrier proteins used in this invention are modified such that the number of glycosylation sites in the carrier proteins is maximized such that the proteins can be administered at lower concentrations, for example, in an immunogenic composition, especially in the form of bioconjugates thereof.

Thus, in some embodiments, the carrier proteins described herein are modified to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more more glycosylation sites than typically associated with the carrier protein (e.g., compared to the number of glycosylation sites associated with the carrier protein in its native/natural state, i.e., wild type). Introduction of glycosylation sites into a carrier protein can be accomplished by inserting a consensus glycosylation sequence anywhere in the protein's primary structure, for example by adding new amino acids to the protein's primary structure so that a glycosylation site is added in whole or in part, or by mutating existing amino acids in the protein to creation of a glycosylation site. One of ordinary skill in the art will appreciate that the amino acid sequence of a protein can be easily modified using approaches known in the art, for example, recombinant approaches that involve modification of the nucleic acid sequence encoding the protein. In specific embodiments, glycosylation consensus sequences are introduced into specific regions of the carrier protein, for example, into surface structures of the protein, at the N- or C-termini of the protein, and/or into loops that are stabilized by disulfide bridges at the base of the protein. In some embodiments, the glycosylation consensus sequence can be extended by adding lysine residues for more efficient glycosylation.

Illustrative examples of glycosylation consensus sequences that can be inserted or created in a carrier protein include Asn-X-Ser(Thr), where X can be any amino acid other than Pro (SEQ ID NO: 1); and Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any amino acid other than Pro (SEQ ID NO: 2).

In some embodiments, the glycosylated E.coH O4 polysaccharide antigen is covalently linked to an asparagine (Asn) residue at a glycosylation site (e.g., linked to N), wherein the Asn residue is present within the glycosylation site containing the glycosylation consensus sequence having SEQ ID NO: 1. more preferably having SEQ ID NO: 2. Typically, the carrier protein contains 1-10 glycosylation sites, preferably 2 to 4 glycosylation sites, most preferably 4 glycosylation sites, each containing a consensus glycosylation sequence having the amino acid sequence of SEQ ID NO : 1 and most preferably the amino acid sequence of SEQ ID NO: 2.

In specific embodiments, the carrier protein is detoxified P. aeruginosa exotoxin A (EPA). Various detoxified EPA variants are described in the literature and can be used as carrier proteins. For example, neutralization can be achieved by mutation and deletion of residues important for catalytic activity, such as L552V and ΔΕ553 according to Lukac et al., 1988, Infect Immun, 56: 3095-3098, and Ho et al., 2006, Hum Vaccin, 2:89-98. As used herein, EPA refers to detoxified exotoxin A from P. aeruginosa. In those embodiments where the carrier protein is EPA, the glycosylated E.coH O4 polysaccharide antigen may be covalently linked to an Asn residue at a glycosylation site containing the glycosylation consensus sequence having SEQ ID NO: 1, and is preferably covalently linked to an Asn residue at a glycosylation site containing a consensus glycosylation sequence having SEQ ID NO: 2. Preferably, the EPA carrier protein contains 1-10 glycosylation sites, preferably 2 to 4 glycosylation sites, most preferably 4 glycosylation sites, each containing a consensus glycosylation sequence, having the amino acid sequence of SEQ ID NO: 1 and, most preferably, the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the EPA carrier protein contains four glycosylation sites, each of which contains a consensus glycosylation sequence, e.g., a glycosylation site

- 14 046206 containing a glycosylation consensus sequence having SEQ ID NO: 2. As used herein, the terms EPA-4 carrier protein and EPA-4 refer to a carrier protein that is a detoxified exotoxin A from P. aeruginosa, containing four glycosylation sites, each of which contains consensus glycosylation sequences having SEQ ID NO: 2. A preferred example of a carrier protein is an EPA carrier protein that contains the amino acid sequence of SEQ ID NO: 3.

Compositions.

In another aspect, a composition is provided comprising a bioconjugate of a glycosylated E. coli O4 polysaccharide antigen covalently linked to a carrier protein. The compositions provided herein may include any bioconjugate of the glycosylated E.coH O4 polysaccharide antigen covalently linked to a carrier protein (eg, EPA) described herein.

In some embodiments, the composition is an immunogenic composition. As used herein, the term immunogenic composition refers to a composition that can elicit an immune response in the host or subject to whom the composition is administered. The immunogenic compositions may further comprise a pharmaceutically acceptable carrier. In some embodiments, the composition is a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier. The term pharmaceutically acceptable carrier as used herein refers to a diluent, adjuvant, excipient or carrier medium with which the composition is administered and which is non-toxic and should not interfere with the effectiveness of the active ingredient. For example, saline solutions and aqueous solutions of dextrose and glycerol can be used as liquid carriers, in particular for injection solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, ethanol and the like. Other examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences, edited by E.W. Martin.

In one embodiment, the composition of the invention contains the bioconjugates of the invention in Tris-buffered saline (TBS) pH 7.4 (eg, containing Tris, NaCl and KCl, eg 25 mM, 137 mM and 2.7 mM, respectively). In other embodiments, the compositions of the invention contain the bioconjugates of the invention in a KH ₂ PO ₄ /Na ₂ HPO ₄ buffer at a concentration of approximately 10 mM and a pH of approximately 7.0, containing approximately 5% (w/v) sorbitol, approximately 10 mM methionine and about 0.02% (w/v) polysorbate 80. In other embodiments, the compositions of the invention contain the bioconjugates of the invention in a KH ₂ PO ₄ /Na ₂ HPO ₄ buffer at a concentration of about 10 mM and a pH of about 7.0, containing approximately 8% (w/v) sucrose, approximately 1 mM EDTA and approximately 0.02% (w/v) polysorbate 80 (see, e.g., WO 2018/077853 for suitable buffers for O-antigen E bioconjugates .coli covalently bound to the EPA carrier protein).

In some embodiments, the disclosed compositions are monovalent formulations and contain a single E.coH polysaccharide O-antigen, for example, in an isolated form or as part of a glycoconjugate or bioconjugate, such as a glycosylated E.coH O4 polysaccharide antigen. Also provided are compositions (eg, pharmaceutical and/or immunogenic compositions) that are multivalent compositions, eg, divalent, trivalent, tetravalent, etc. compositions. For example, the multivalent composition contains more than one antigen, such as E. coH O-antigen, a glycoconjugate or a bioconjugate thereof. In specific embodiments, the proposed multivalent composition contains a bioconjugate of the glycosylated E.coH O4 polysaccharide antigen and at least one additional antigen.

In one embodiment, the composition (eg, a pharmaceutical composition and/or an immunogenic composition) is a monovalent composition comprising a bioconjugate of a glycosylated E. coli O4 polysaccharide antigen covalently linked to a carrier protein as described herein. In one embodiment, the composition (eg, a pharmaceutical composition and/or an immunogenic composition) is a multivalent composition comprising a glycosylated E. coli O4 polysaccharide antigen covalently linked to a carrier protein as described herein, and at least one additional antigen.

In some embodiments, the additional antigen is a saccharide or polysaccharide antigen, more preferably an E.coH polysaccharide O-antigen, such as the E.coH O-antigens of one or more of the serotypes O1, O2, O6, O8, O15, O16, O18, O25 and O75 and their subserotypes. In some embodiments, each of the additional E.coH polysaccharide O-antigens is a glycoconjugate, which means that the E.coH polysaccharide O-antigen is covalently linked to another chemical entity, e.g., a protein, peptide, lipid, etc., most preferably , a carrier protein, for example, by a chemical or enzymatic method. In preferred embodiments, each of the additional E.coH polysaccharide O-antigens is a bioconjugate in which the polysaccharide O-antigen is covalently linked, for example, to a carrier protein via gly

- 15 046206 cosid bond enzymatically through the host cell apparatus. In some embodiments, the compositions provided herein may contain 1-20 additional glycoconjugates, more preferably E. coH polysaccharide O-antigen bioconjugates such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional glycoconjugates or preferably bioconjugates of E. coH polysaccharide O-antigens. Other antigens, such as peptide, protein or lipid antigens, etc., may be included in the compositions provided herein.

In some embodiments, the composition (e.g., pharmaceutical and/or immunogenic composition) contains a bioconjugate of a glycosylated E. coli O4 polysaccharide antigen and at least one additional polysaccharide antigen selected from the group consisting of E. coli O1A polysaccharide antigen, E. coli O2 polysaccharide antigen. coli, polysaccharide antigen 06a E.coli, polysaccharide antigen O8 E.coli, polysaccharide antigen O15 E.coli, polysaccharide antigen O16 E.coli, polysaccharide antigen O18 E.coli, polysaccharide antigen 025B E.coli and polysaccharide antigen O75 E.coli . Preferably, each of the additional polysaccharide O-antigens is covalently linked to a carrier protein and more preferably is a bioconjugate. In one embodiment, the O1A polysaccharide antigen (eg, in isolated form or as part of a glycoconjugate or bioconjugate) is used in a composition of the invention (eg, in combination with a glycosylated O4 polysaccharide antigen or a bioconjugate thereof). In a specific embodiment, the polysaccharide antigen O1A contains the structure of formula (O1A), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. Preferably, the O1A polysaccharide antigen is part of a bioconjugate and is covalently linked to a protein carrier, such as EPA.

In one embodiment, an O2 polysaccharide antigen (eg, in isolated form or as part of a glycoconjugate or bioconjugate) is used in a composition of the invention (eg, in combination with a glycosylated O4 polysaccharide antigen or a bioconjugate thereof). In a specific embodiment, the polysaccharide antigen O2 contains the structure of formula (O2), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. Preferably, the O2 polysaccharide antigen is part of a bioconjugate and is covalently linked to a protein carrier, such as EPA.

In one embodiment, the O6A polysaccharide antigen (eg, in isolated form or as part of a glycoconjugate or bioconjugate) is used in a composition of the invention (eg, in combination with a glycosylated O4 polysaccharide antigen or a bioconjugate thereof). In a specific embodiment, the O6A polysaccharide antigen contains the structure of the formula (O6A), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. Preferably, the O6A polysaccharide antigen is part of a bioconjugate and is covalently linked to a protein carrier, such as EPA.

In one embodiment, an O8 polysaccharide antigen (eg, in isolated form or as part of a glycoconjugate or bioconjugate) is used in a composition of the invention (eg, in combination with a glycosylated O4 polysaccharide antigen or a bioconjugate thereof). In a specific embodiment, the O8 polysaccharide antigen contains the structure of formula (O8), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. Preferably, the O8 polysaccharide antigen is part of a bioconjugate and is covalently linked to a protein carrier, such as EPA.

In one embodiment, the O15 polysaccharide antigen (eg, in isolated form or as part of a glycoconjugate or bioconjugate) is used in a composition of the invention (eg, in combination with a glycosylated O4 polysaccharide antigen or a bioconjugate thereof). In a specific embodiment, the O15 polysaccharide antigen contains the structure of formula (015), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. Preferably, the O15 polysaccharide antigen is part of a bioconjugate and is covalently linked to a protein carrier, such as EPA.

In one embodiment, the O16 polysaccharide antigen (eg, in isolated form or as part of a glycoconjugate or bioconjugate) is used in a composition of the invention (eg, in combination with a glycosylated O4 polysaccharide antigen or a bioconjugate thereof). In a specific embodiment, the O16 polysaccharide antigen contains the structure of formula (O16), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. Preferably, the O16 polysaccharide antigen is part of a bioconjugate and is covalently linked to a protein carrier, such as EPA.

In one embodiment, the O18A polysaccharide antigen (eg, in isolated form or as part of a glycoconjugate or bioconjugate) is used in a composition of the invention (eg, in combination with a glycosylated O4 polysaccharide antigen or a bioconjugate thereof). In a specific embodiment, the O18A polysaccharide antigen contains the structure of the formula (O18A), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. Preferably the O18A polysaccharide antigen is part of a bio

- 16 046206 conjugate and is covalently linked to a carrier protein, such as EPA.

In one embodiment, the 025b polysaccharide antigen (eg, in isolated form or as part of a glycoconjugate or bioconjugate) is used in a composition of the invention (eg, in combination with a glycosylated O4 polysaccharide antigen or a bioconjugate thereof). In a specific embodiment, polysaccharide antigen 025B contains the structure of formula (025B), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. Preferably, the polysaccharide antigen 025B is part of a bioconjugate and is covalently linked to a protein carrier, such as EPA.

In one embodiment, the O75 polysaccharide antigen (eg, in isolated form or as part of a glycoconjugate or bioconjugate) is used in a composition of the invention (eg, in combination with a glycosylated O4 polysaccharide antigen or a bioconjugate thereof). In a specific embodiment, the O75 polysaccharide antigen contains the structure of formula (O75), as shown in table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. Preferably, the O75 polysaccharide antigen is part of a bioconjugate and is covalently linked to a protein carrier, such as EPA.

In another embodiment, the composition (e.g., pharmaceutical and/or immunogenic composition) contains at least the polysaccharide antigens O1A, O2, glycosylated O4, O6A and 025B of E. coli, preferably bioconjugates of the polysaccharide antigens O1A, O2, glycosylated O4, O6A and 025B, covalently associated with a carrier protein (eg, a pentavalent composition). In a preferred embodiment, the composition (e.g., pharmaceutical and/or immunogenic composition) contains at least polysaccharide antigens O1A, O2, glycosylated O4, O6A, O8, O15, O16, 025B and O75 E. coli, preferably bioconjugates of polysaccharide antigens O1A, O2, glycosylated O4, O6A, O8, O15, O16, 025B and O75 covalently linked to a carrier protein (for example, a 9-valent composition).

In another preferred embodiment, the composition (e.g., pharmaceutical and/or immunogenic composition) contains at least E. coli polysaccharide antigens O1A, O2, glycosylated O4, O6A, O8, O15, O16, O18A, O25B and O75, preferably bioconjugates of O1A polysaccharide antigens , O2, glycosylated O4, O6A, O8, O15, O16, O18A, 025B and O75 covalently linked to a carrier protein (eg, 10-valent composition).

Also contemplated herein are compositions that optionally further contain additional O-antigens (eg, in isolated form or as part of a glycoconjugate or bioconjugate) of E. coH from other serotypes.

In some embodiments, each additional polysaccharide antigen O1A, O2, O6A, O8, O15, O16, O18A, 025B and/or O75 E.coH is covalently linked to a carrier protein. The polysaccharide O-antigen may be linked to a carrier protein by chemical or other synthetic means, or the polysaccharide O-antigen may be part of a bioconjugate and is preferably part of a bioconjugate. Any suitable carrier protein known to those skilled in the art may be used, given the specification herein. Suitable carriers include inactivated P. aeruginosa exotoxin A (EPA), E. coH flagellin (FliC), CRM 197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, inactivated S. aureus hemolysin A, agglutinating factor A, agglutinating factor B , thermolabile enterotoxin E.coH, neutralized variants of the thermolabile enterotoxin E.coH, subunit B of cholera toxin (CTS), cholera toxin, neutralized variants of cholera toxin, E.coli Sat protein, E.coli Sat protein passenger domain, Streptococcus pneumoniae pneumolysin, hemocyanin Megathura crenulata (KLH), PcrV of P. aeruginosa, Neisseria meningitidis outer membrane protein (OMPC), and protein D from nontypeable Haemophilus influenzae, but are not limited to. Preferably, the carrier protein is EPA.

In some embodiments, each of the additional E.coH polysaccharide antigens O1A, O2, O6A, O8, O15, O16, O18A, O25B, and/or O75, especially when part of a bioconjugate, is covalently linked to an asparagine (Asn) residue in the carrier protein wherein the Asn residue is present in a glycosylation site containing the consensus glycosylation sequence Asn-X-Ser (Thr), where X can be any amino acid except Pro (SEQ ID NO: 1), preferably where the Asn residue is present in the glycosylation site containing the consensus glycosylation sequence Asp (Glu)-X-Asn-Z-Ser (Thr), where X and Z are independently selected from any amino acid except Pro (SEQ ID NO: 2). The carrier protein may contain 1-10 glycosylation sites, preferably 2 to 4 glycosylation sites, most preferably 4 glycosylation sites, each containing a consensus glycosylation sequence. In a specific embodiment, the carrier protein is an EPA-4 carrier protein, for example an EPA-4 carrier protein comprising the amino acid sequence of SEQ ID NO: 3.

In a specific embodiment, a composition is provided (e.g., a pharmaceutical and/or immunogenic composition) containing: (1) a bioconjugate of a glycosylated E. coli O4 polysaccharide antigen covalently linked to a carrier protein detoxified by P. aeruginosa exotoxin A containing SEQ ID NO: 3 ( EPA-4 carrier protein), where the glycosylated polysaccharide antigen O4

- 17 046206

E.coli contains the structure of Formula (O4-Glc+); (2) a bioconjugate of the glycosylated polysaccharide antigen O1A of E.coH covalently linked to the carrier protein EPA-4, wherein the polysaccharide antigen O1a of E.coH contains the structure of Formula (O1A); (3) a bioconjugate of a glycosylated E.coH O2 polysaccharide antigen covalently linked to an EPA-4 carrier protein, wherein the E.coH O2 polysaccharide antigen contains the structure of Formula (O2); (4) a bioconjugate of the glycosylated polysaccharide antigen O6A E.coH covalently linked to the carrier protein EPA-4, where the polysaccharide antigen O6A E.coH contains the structure of Formula (O6A); (5) a bioconjugate of a glycosylated E.coH O8 polysaccharide antigen covalently linked to an EPA-4 carrier protein, wherein the E.coH O8 polysaccharide antigen contains the structure of Formula (O8); (6) a bioconjugate of a glycosylated E.coH O15 polysaccharide antigen covalently linked to an EPA-4 carrier protein, wherein the E.coH O15 polysaccharide antigen contains the structure of Formula (O15); (7) a bioconjugate of a glycosylated E.coH O16 polysaccharide antigen covalently linked to an EPA-4 carrier protein, wherein the E.coH O16 polysaccharide antigen contains the structure of Formula (O16); (8) a bioconjugate of the glycosylated polysaccharide antigen 025B E.coH covalently linked to the carrier protein EPA-4, where the polysaccharide antigen 025B E.coH contains the structure of Formula (025B) and (9) a bioconjugate of the glycosylated polysaccharide antigen O75 E.coH covalently linked with the carrier protein EPA-4, where the polysaccharide antigen O75 E.coH contains the structure of Formula (O75), where each of the Formulas is given in table. 1, and independently for each of the formulas n represents an integer from 1 to 100, for example from 1 to 50, preferably from 3 to 50, for example from 5 to 40. In a specific embodiment, said composition (for example, pharmaceutical and/or immunogenic composition) additionally contains: (10) a bioconjugate of the polysaccharide antigen O18A E.coH, covalently linked to the carrier protein EPA-4, where the polysaccharide antigen O18A E.coH contains the structure of the formula (O18A) given in table. 1, where n in this structure is an integer from 1 to 100, for example from 1 to 50, preferably from 3 to 50, for example from 5 to 40.

In some embodiments, the present composition comprises a bioconjugate of a glycosylated E. coli polysaccharide antigen O4 and at least a bioconjugate of an E. coli 025B polysaccharide antigen, wherein the bioconjugate of an E. coli 025B antigen is present in the composition at a concentration that is approximately 1.5 to 6 times, e.g. , 2-4 times higher, for example 1.5, 2, 3, 4, 5 or 6 times higher, than the concentration of any other bioconjugate present in the composition.

In specific embodiments, the composition contains bioconjugates of polysaccharide antigens O1A, 02, glycosylated 04, O6A, 08, 015, 016, 025B and O75 E.coH, and bioconjugates O1A:O2: glycosylated O4:O6A:O8:O15:O16:O25B:O75 are present in the ratio (by mass of O-antigen polysaccharides) 1:1:1:1:1:1:1:2:1 or 2:1:1:2:1:1:1:4:1.

In specific embodiments, the composition contains bioconjugates of polysaccharide antigens O1A, 02, glycosylated 04, O6A, 08, 015, 016, O18A, 025B and O75 E.coH, and bioconjugates O1A:O2: glycosylated O4:O6A:O8:O15:O16:O18A :O25B:O75 are present in the ratio (by mass of O-antigen polysaccharides) 1:1:1:1:1:1:1:1:2:1 or 2:1:1:2:1:1:1:1 :4:1. In some embodiments, the proposed composition contains a bioconjugate of glycosylated E. coli polysaccharide antigen 04 and at least a bioconjugate of E. coli O25B polysaccharide antigen, wherein the bioconjugate of E. coli O25B antigen is present in the composition at a concentration of 2 to 50 μg/ml, preferably 8 to 40 μg/ml, more preferably 16 to 32 μg/ml, such as 16, 18, 20, 22, 24, 26, 28, 30 or 32 μg/ml. In such embodiments, the concentration of the E. coli O25B polysaccharide antigen bioconjugate is preferably about 1.5-6 times, e.g., 2-4 times higher, e.g., 1.5, 2, 3, 4, 5, or 6 times higher than the concentration of any other bioconjugate present in the composition.

In some embodiments, the compositions described herein (eg, pharmaceutical and/or immunogenic compositions) contain or are administered in combination with an adjuvant. The adjuvant for administration in combination with the composition described herein may be administered before (e.g., 72, 48, 24, 12, 6, 2, 1 hour, 10 minutes), concurrently, or after (e.g., 72, 48, 24, 12, 6, 2, 1 hour, 10 minutes) introduction of the specified composition. As used herein, the term adjuvant refers to a compound that, when administered in combination or as part of a composition described herein, enhances and/or enhances the immune response to the E. coH polysaccharide O-antigen in the bioconjugate, however, when only the adjuvant compound is administered , an immune response to the polysaccharide O-antigen E.coH is not produced in the bioconjugate. In some embodiments, the adjuvant enhances the immune response to the E.coH polysaccharide O-antigen in its bioconjugate and does not cause an allergy or other adverse reaction. The enhancement of the immune response by adjuvants can be mediated by several mechanisms, including, for example, lymphocyte recruitment, stimulation of B and/or T cells, or stimulation of macrophages. Examples of suitable adjuvants include aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, aluminum sulfate and alumina, including nanoparticles containing alum or nanoaluminium formulations), calcium phosphate, monophosphoryl lipid A (MPL) or 3-de-O- acylated monophosphoryl lipid A (3D-MPL) (see eg GB 2220211, EP 0971739, EP 1194166, US 6491919), AS01, AS02, AS03 and AS04 (all GlaxoSmithKline; see eg EP 1126876, US 7357936 for AS04, EP 0671948, EP 0761231, US 5750110

- 18 046206 for AS02), MF59 (Novartis), imidazopyridine compounds (see WO 2007/109812), imidazoquinoxaline compounds (see WO 2007/109813), delta inulin, STING-activating synthetic cyclic dinucleotides (for example US20150056224), combinations of lecithin and carbomer homopolymers (eg US 6676958) and saponins such as QuilA and QS21 (see eg Zhu D and W Tuo, 2016, Nat Prod Chem Res 3: e113 (doi:10.4172/2329-6836.1000e113) , Matrix M, Iscoms, Iscomatrix, etc., possibly in combination in QS7 (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); US Patent No. 5,057,540), but are not limited to the above. In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants are oil-in-water emulsions (such as squalene or peanut oil), possibly in combination with immunostimulants, such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)). Another adjuvant is CpG (Bioworld Today, Nov. 15, 1998). Other examples of adjuvants are liposomes containing immunostimulants such as MPL and QS21, such as AS01E and AS01B (eg US 2011/0206758). Other examples of adjuvants are CpG (Bioworld Today, November 15, 1998) and imidazoquinolines (such as imiquimod and R848). See, for example, Reed G, et al., 2013, Nature Med, 19: 1597-1608. In some embodiments, the adjuvant contains a toll-like receptor 4 (TLR4) agonist. TLR4 agonists are well known in the art, see, for example, Ireton GC and SG Reed, 2013, Expert Rev Vaccines 12: 793-807. In some embodiments, the adjuvant contains a TLR4 agonist containing lipid A or an analog or derivative thereof, such as MPL, 3D-MPL, RC529 (e.g., EP1385541), PET-Lipid A, GLA (glycopyranosyl lipid adjuvant, synthetic disaccharide glycolipid; e.g., US 20100310602, US 8722064), SLA (e.g. Carter D et al, 2016, Clin Transl Immunology 5: el08 (doi: 10.1038/cti.2016.63), which describes a structure-function approach to optimize TLR4 ligands for human vaccines), PHAD (phosphorylated hexaacyl -disaccharide), 3D-PHAD (which has the same structure as GLA), 3D-(6acyl)-PHAD (3D(6A)-PHAD) (PHAD, 3D-PHAD and 3D(6A)PHAD are synthetic variants of the lipid And, see, for example, avantilipids.com/divisions/adjuvants, where the structures of these molecules are also given), E6020 (CAS number 287180-63-6), ONO4007, OM-174 and the like. In some embodiments, the compositions described herein do not contain an adjuvant and are not administered in combination with an adjuvant.

In some embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to the subject. For example, the compositions (e.g., pharmaceutical and/or immunogenic) described herein can be formulated for subcutaneous, parenteral, oral, sublingual, buccal, intradermal, transdermal, colorectal, intraperitoneal, rectal, intravenous, intranasal, intratracheal, intramuscular , local, transdermal or intradermal administration. In a specific embodiment, the proposed composition (eg, pharmaceutical and/or immunogenic) is formulated for intramuscular injection.

Methods of application.

The bioconjugates and compositions provided herein can be used to induce antibodies against glycosylated E.coH O4 antigen in a subject and to vaccinate a subject against E.coH, particularly extraintestinal pathogenic E.coH (ExPEC). As used herein, subject means any animal, preferably a mammal, to which the bioconjugate and composition provided herein will be or has been administered. The term mammal as used herein includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates such as monkeys or apes, humans, etc. In some embodiments, the subject is a human. A person can be of any age. In some embodiments, the subject is a human between about two months and about 18 years of age, such as 1 to 18 years of age. In some embodiments, the subject is a person at least 18 years of age. In some embodiments, the subject is a person between 15 and 50 years of age, such as 18 to 40 years, such as 20 to 45 years. In some embodiments, the subject is a male human. In some embodiments, the subject is a female human. In some embodiments, the subject is immunocompromised. In some embodiments, the subject is a person at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old. In some embodiments, the subject is a person no older than 100 years of age, no more than 95 years of age, no more than 90 years of age, no more than 85 years of age, no more than 80 years of age, or no more than 75 years of age. In some embodiments, the subject is a person at least 60 years of age and no older than 85 years of age. In some embodiments, the subject is a human in stable health. In some embodiments, the subject is a person in stable health, at least 60 years of age and not more than 85 years of age. In some embodiments, the subject is a person who has a history of urinary tract infection (UTI; i.e., a bacterial infection in the urethra, bladder, ureters, and/or kidneys), i.e., has had at least one episode of UTI in his or her her life. In some embodiments, the subject is a person who has had a history of UTI within the last twenty, fifteen,

- 19 046206 twelve, ten, nine, eight, seven, six, five, four, three, two years or one year. In some embodiments, the subject is a person who has had a history of UTI within the past two years. In some embodiments, the subject is a person with a history of recurrent UTIs, i.e. had at least two UTIs within six months or at least three UTIs within a year. In some embodiments, the subject is a person who has had a history of UTI within the past two years. In some embodiments, the subject is a person in stable health, 60 years of age or older. In some embodiments, the subject is a person 60 years of age or older with a history of UTI within the past two years. In some embodiments, the subject is a person 60 years of age or less than 75 years of age with a history of UTI within the past two years. In some embodiments, the subject is a person 75 years of age or older with a history of UTI within the past two years. In some embodiments, the subject is a patient scheduled for elective urogenital and/or abdominal procedures or surgeries, for example, transrectal ultrasound-guided prostate biopsy (TRUS-PNB).

In one aspect, there is provided a method of inducing antibodies against a glycosylated E.coH O4 antigen in a subject, comprising administering to the subject any of the bioconjugates of a glycosylated E.coH O4 antigen covalently linked to a carrier protein described herein, or a composition comprising a bioconjugate of a glycosylated O4 antigen. E.coH covalently bound to a protein, alone or in combination with other E.coH polysaccharide O-antigens or their glycoconjugates or bioconjugates.

In some embodiments, antibodies induced, elicited, or detected against the glycosylated E. coH O4 antigen have opsonophagocytic activity. In specific embodiments, the induced, evoked or detected antibodies are cross-reactive antibodies capable of mediating opsonophagocytic killing of both glycosylated and non-glycosylated O4 strains of E. coli.

In some embodiments, antibodies raised, elicited, or detected against the glycosylated E.coH O4 antigen specifically recognize unmodified and glucose-modified O4 polysaccharide antigen. In some embodiments, antibodies induced, elicited, or detected against the glycosylated E.coH O4 antigen specifically recognize E.coH serotype O4. In some embodiments, antibodies raised, elicited, or detected against the glycosylated E.coH O4 antigen preferentially bind to the glycosylated O4 antigen over the non-glycosylated O4 antigen. Antibodies induced by the bioconjugates and compositions described herein may include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen-binding site that specifically binds to an E. coli polysaccharide O antigen, such as a glycosylated O4 polysaccharide antigen. Antibodies induced, evoked or detected using the bioconjugates or compositions provided herein can be used to monitor the effectiveness of therapy and/or disease progression. Any immunoassay system known in the art may be used for this purpose, including competitive and non-competitive assay systems using methods such as radioimmunoassays, ELISA (enzyme-linked immunosorbent assay), electrochemiluminescence (ECL) immunoassays, sandwich immunoassays, precipitation reactions, reactions gel diffusion precipitation tests, immunodiffusion tests, immunoradiometric tests, fluorescence immunoassays, protein A immunoassays and immunoelectrophoretic tests, but not limited to these. Some of these assays, such as ECL-based immunoassays, can be performed in a multiplex format, and multiplex assay formats are generally preferred.

Antibodies induced, evoked or detected using a bioconjugate of the glycosylated polysaccharide antigen E. coH O4 can be used to detect E. coH O4 strains, in particular strains with glycosylated O4, for example, among a variety of E. coH strains and/or to diagnose infection , caused by a strain of E.coH with O4 or glycosylated O4.

In another aspect, provided herein is a method of vaccinating a subject against E.coH (e.g., extraintestinal pathogenic E.coH, ExPEC), comprising administering to the subject any of the bioconjugates of the glycosylated E.coH O4 antigen covalently linked to a carrier protein described herein , or a composition containing a bioconjugate of a glycosylated E.coH O4 antigen covalently linked to a carrier protein, alone or in combination with other E.coH O-antigens or their glycoconjugates or bioconjugates. One skilled in the art will understand that the subject will be vaccinated against strains of E. coli whose O-antigens or glycoconjugates or bioconjugates thereof are present in the administered composition. For example, administration of a composition containing polysaccharide antigens O1A, O2, glycosylated O4, 06a and 025b can be used to vaccinate a subject against E. coH serotypes O1A, O2, O4, O6A and 025B. In some embodiments, vaccination is intended to prevent invasive ExPEC disease (IED), such as urosepsis, bacteremia, sepsis, etc. In some embodiments, vaccination is intended to prevent or reduce

- 20 046206 occurrence or severity of urinary tract infections. In some embodiments, the IED may be purchased in a hospital setting, for example, from patients undergoing urogenital and/or abdominal procedures or surgeries. In some embodiments, the IED may be associated with medical care, such as in patients receiving medical care for another medical condition, such as through central lines, etc., such as in a hospital, ambulatory surgery center, end-stage renal disease unit, facility long-term care, etc. In some embodiments, the IED may be of community-acquired origin, such as in a patient who has not recently been exposed to health risks.

In another aspect, there is provided a method of inducing an immune response against E. coli (eg, ExPEC) in a subject, comprising administering to the subject any of the E. coli glycosylated O4 antigen bioconjugates. covalently linked to a carrier protein described herein, or a composition comprising a bioconjugate of a glycosylated E. coli O4 antigen covalently linked to a carrier protein, alone or in combination with other E. coli O-antigens or glycoconjugates or bioconjugates thereof. In one embodiment, at the time of administration, the subject has an E. coli infection (eg, ExPEC). In a preferred embodiment, the subject is free of E. coli infection (eg, ExPEC) at the time of administration.

In some embodiments, the described compositions and bioconjugates can be administered to a subject to induce an immune response that includes the production of antibodies, preferably antibodies having opsonophagocytic activity. Such antibodies can be isolated by methods known to one skilled in the art (eg, immunoaffinity chromatography, centrifugation, precipitation, etc.).

The ability of the bioconjugates and compositions described herein to elicit an immune response in a subject can be assessed using any approach known to those skilled in the art or described herein. In some embodiments, the ability of a bioconjugate to elicit an immune response in a subject can be assessed by immunizing a subject (e.g., a mouse, rat, rabbit, or monkey) or a number of subjects with the bioconjugate described herein, as well as immunizing an additional subject (e.g., a mouse, rat, rabbit, or monkey) or a series of control subjects (PBS). The subject or series of subjects can subsequently be infected with ExPEC and the ability of the ExPEC to cause disease (eg, UTI, bacteremia, or other disease) in the subject or series of subjects can be determined. Those skilled in the art will appreciate that if a subject or series of subjects immunized with a control suffers from disease following infection with ExPEC, the subject or series of subjects immunized with the bioconjugate(s) or composition thereof described herein suffers less or no disease , the bioconjugate is capable of inducing an immune response in a subject. The ability of the bioconjugate(s) or composition thereof described herein to generate antiserum that cross-reacts with the ExPEC Oantigen can be tested, for example, using an immunoassay such as ELISA (see, for example, Van den Dobbelsteen et al, 2016, Vaccine 34: 4152-4160) or ECL (electrochemiluminescence) immunoassay. For example, the ability of the bioconjugates described herein to elicit an immune response in a subject can be assessed using a serum bactericidal test (SBA) or an opsonophagocytic test (OPK or OPKA), which is an established and accepted method used to obtain vaccine approval. based on glycoconjugates. Such tests are well known in the art and, briefly, involve the steps of generating and releasing antibodies against a target of interest (e.g., E. coli O-polysaccharide antigen, e.g., E. coli glycosylated O4 polysaccharide antigen) by administering to a subject (e.g. mouse, rat, rabbit or monkey) of a compound that causes the production of such antibodies. Subsequently, the bactericidal ability of the antibodies can be assessed, for example, by culturing the bacteria in question (for example, E. coli of the corresponding serotype), in the presence of said antibodies and complement and, depending on the test, neutrophils, and assessing the ability of the antibodies to mediate death and/or neutralization of bacteria, for example, using standard microbiological approaches. An example of OPK analysis for bioconjugate E. coli vaccines is given, for example, by Abbanat et al, 2017, Clin. Vaccine Immunol. 24:e00123-17. The OPK assay can be performed in a monoplex or multiplex format, of which the multiplex format is generally preferred (eg, testing multiple serotypes simultaneously). The multiplex OPK assay is sometimes referred to as MOPA in this document.

In some embodiments, the methods described include administering an effective amount of E. coli glycosylated O4 antigen bioconjugates covalently linked to a carrier protein described herein, or a composition comprising a bioconjugate of E. coli glycosylated O4 antigen covalently linked to a carrier protein, alone or in combination with other E. coli O-antigens or their glycoconjugates or bioconjugates. In one embodiment, the effective amount is an amount that vaccinates a subject against E. coli (eg, ExPEC). In another embodiment, the effective amount is an amount that elicits an immune response against E. coli (e.g., ExPEC) in a subject, such as an immune response including production of

- 21 046206 ku antibodies, preferably antibodies with opsonophagocytic activity.

In specific embodiments where the composition of the present invention comprises a bioconjugate of a glycosylated E. coli polysaccharide antigen O4 and at least a bioconjugate of an E. coli 025b polysaccharide antigen, the effective amount of the E. coli 025b polysaccharide antigen is approximately 1.5 to 6 times, for example, approximately 2 times -4 times higher, for example 1.5, 2, 3, 4, 5 or 6 times higher, than the concentration of any other bioconjugate present in the composition. In such embodiments, the effective amount of E.coH 025B polysaccharide antigen is, for example, about 5 to 18 μg per administration, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 mcg per administration.

In some embodiments, the bioconjugate or composition of the invention is administered to a subject once. In some embodiments, the bioconjugate or composition of the invention is administered to a subject more than once, for example, in a prime-boost regimen. In some embodiments, the time between two administrations is at least two weeks, at least one month, at least two months, at least three months, at least six months, at least one year, at least two years, at least five years, at least ten years or at least fifteen years. In humans, the desired immune response can generally be induced by a single administration of a bioconjugate or composition of the invention. In some embodiments, repeated administration is provided, for example, after ten years.

Host cells.

Provided herein are host cells, eg prokaryotic host cells, capable of producing E.coH O-antigens and bioconjugates containing such E.coH O-antigens. The proposed host cells are preferably modified (eg, through genetic engineering) to contain one or more nucleic acids encoding the host cell machinery (eg, glycosyltransferases) used to produce polysaccharide E. coH Oantigens and/or bioconjugates thereof.

To produce the E.coH polysaccharide O-antigens described herein (e.g., E.coH glycosylated polysaccharide antigen O4) and bioconjugates containing the E.coH polysaccharide O-antigens described herein (e.g., E.coH glycosylated polysaccharide antigen bioconjugate .coH) any host cells known to those skilled in the art may be used, including archaebacteria, prokaryotic host cells, and eukaryotic host cells. In a preferred embodiment, the host cell is a prokaryotic host cell. Examples of prokaryotic host cells for use in the production of the E.coH polysaccharide O-antigens described herein and bioconjugates containing the E.coH polysaccharide O-antigens described herein include Escherichia spp., Shigella spp., Klebsiella spp., Xhantomonas spp., Salmonella spp., but not limited to, Yersinia spp., Lactococcus spp., Lactobacillus spp., Pseudomonas spp., Corynebacterium spp., Streptomyces spp., Streptococcus spp., Staphylococcus spp., Bacillus spp., and Clostridium spp. In a specific embodiment, the host cell used to produce the E.coli polysaccharide O-antigens described herein and bioconjugates containing the E.coli polysaccharide O-antigens described herein is a prokaryotic host cell and is preferably E.coH. In some embodiments, the host cells used to produce the E. coH polysaccharide O-antigens and bioconjugates described herein are engineered to contain heterologous nucleic acids, for example, heterologous nucleic acids containing rfb gene clusters of the desired serotype O- antigen, heterologous nucleic acids that encode one or more carrier proteins and/or glycosyltransferases. In a specific embodiment, heterologous rfb genes and/or heterologous nucleic acids that encode proteins involved in glycosylation pathways (eg, prokaryotic and/or eukaryotic glycosylation pathways) can be introduced into the host cells described herein. Such nucleic acids may encode proteins including, but not limited to, oligosaccharyltransferases and/or glycosyltransferases. This document describes the sequences of various genes and gene clusters encoding glycosyltransferases that may find use in the production of recombinant host cells that can, for example, be used to produce E. coH polysaccharide O-antigens and their bioconjugates. Those skilled in the art will appreciate that due to the degeneracy of the genetic code, a protein having a particular amino acid sequence can be encoded by many different nucleic acids. Thus, those skilled in the art will understand that the nucleic acid provided herein can be modified so that its sequence differs from the sequence presented herein without affecting the amino acid sequence of the protein encoded by the nucleic acid. Provided herein are host cells (e.g., recombinant host cells) for producing E.coH glycosylated polysaccharide antigen O4 bioconjugate, O1A polysaccharide antigen, O2 polysaccharide antigen, O6A polysaccharide antigen, O8 polysaccharide antigen, O15 polysaccharide antigen, O16 polysaccharide antigen, polysaccharide antigen O18A, polysaccharide antigen 025B or polysaccharide antigen O75. Proposed host cells contain

- 22 046206 harvest nucleic acids encoding enzymes (for example, glycosyltransferases) capable of producing the polysaccharide O-antigen E. coH. The host cells provided herein may naturally express nucleic acids that are capable of producing the O-antigen of interest, or host cells may be made to express such nucleic acids. In some embodiments, the nucleic acids are heterologous to host cells and are introduced into host cells using genetic approaches known in the art. For example, nucleic acids can be introduced into a host cell by genetic manipulation (for example, a gene cluster is expressed on a plasmid or plasmids or integrated into the host cell genome (see, for example, published international patent applications WO 2014/037585, WO 2014/057109, WO 2015/052344) In one embodiment, a host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of a glycosylated E. coli O4 polysaccharide antigen covalently linked to a carrier protein is provided. -precursor, a nucleic acid sequence encoding the gtrS gene, which, as far as the authors know, was identified for the first time as encoding an enzyme that provides polysaccharide branching, capable of transferring glucose to the O4 antigen E.coH (i.e., a glucosyltransferase specific to the O4 polysaccharide antigen E.coH), and, in particular, to L-Rha through an a-1,3-glycosidic bond. An example of the amino acid sequence of such a branching enzyme is provided in SEQ ID NO: 4. Other examples contain amino acid sequences that are at least 80% identical thereto. Illustrative examples of nucleic acids encoding gtrS genes specific for the E. coH O4 polysaccharide antigen include SEQ ID NO: 5, or degenerate nucleic acid sequences thereof, which encode SEQ ID NO: 4, or nucleic acid sequences which encode functional O4-specific GtrS enzymes that are at least 80% identical to, but not limited to, SEQ ID NO: 4.

In a specific embodiment, a host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of a glycosylated E. coli O4 polysaccharide antigen covalently linked to a carrier protein contains a nucleotide sequence encoding a glucosyltransferase that is at least 80% identical to SEQ ID NO : 4, for example, approximately 80, 85, 90, 91, 92, 93, 94, 95, 95, 97, 98, 99 or 100% identical to the sequence of SEQ ID NO: 4. Taking into account the redundancy of the genetic code, one skilled in The field can generate nucleic acid variants encoding the amino acid sequences of glucosyltransferases, for example, using codon optimized sequences, if desired.

In some embodiments, a host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of a glycosylated E. coli O4 polysaccharide antigen covalently linked to a carrier protein that contains a nucleotide sequence encoding a glucosyltransferase (GtrS) that is at least 80 identical % SEQ ID NO: 4, further contains a nucleotide sequence encoding bactoprenol-linked glucose translocase (GtrA) identical to at least 80% of SEQ ID NO: 7, and a nucleotide sequence encoding bactoprenol glucosyltransferase (GtrB) identical at least 80% of the sequence of SEQ ID NO: 8. In some embodiments, said nucleic acid sequences encode GtrA and GtrB proteins that are at least about 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 7 and 8, respectively, and have bactoprenol-linked glucose translocase (SEQ ID NO: 7) and bactoprenol glucosyltransferase (SEQ ID NO: 8) activity, respectively. Taking into account the redundancy of the genetic code, one of ordinary skill in the art can generate variants of the nucleic acid encoding the amino acid sequences of the bactoprenol-linked glucose translocases and bactoprenol glucosyltransferases, for example, using codon optimized sequences, if desired.

The proposed host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of a glycosylated E. coli O4 polysaccharide antigen covalently bound to a carrier protein further comprises the nucleotide sequence of the rfb gene cluster for the E. coli O4 polysaccharide antigen. An example of an rfb gene cluster that may have utility for the production of the E.coH O4 polysaccharide antigen is provided herein as SEQ ID NO: 9. Another example is the AY568960 locus, which can be found in GenBank. Degenerate nucleic acid sequences encoding the same enzymes encoded by the sequence, or sequences encoding enzymes that are at least 80% identical, preferably at least 85, 90, 91, 92, 93, 94, may also be used. , 95, 96, 97, 98, 99%.

In a specific embodiment provided herein is a host cell (e.g., a recombinant host cell, preferably a recombinant prokaryotic host cell, preferably a recombinant E.coH host cell) that produces a glycosylated O4 polysaccharide antigen, wherein the host cell contains gtrS, the rfb gene cluster of the polysaccharide antigen O4 E.coH and a nucleic acid encoding a carrier protein. Such host cells may be

- 23 046206 engineered using recombinant approaches to contain one or more plasmids containing the gtrS gene, the rfb gene cluster and/or a nucleic acid encoding a carrier protein, or to contain some or all of the corresponding genes, such as gtrS, the rfb cluster and/or a nucleic acid encoding a carrier protein integrated into the genome of a host cell. In some embodiments, genes or gene clusters are integrated into the genome of a host cell using homologous recombination. The advantage of gene integration into the host cell genome is stability in the absence of antibiotic selection.

In another specific embodiment, provided herein is a host cell (e.g., a recombinant host cell, preferably a recombinant prokaryotic host cell) that produces a glycosylated O4 polysaccharide antigen, wherein the host cell contains GtrS (glucosyltransferase) as well as enzymes encoded by rfb gene cluster. In some embodiments, some or all of the above enzymes are heterologous to the host cell.

In other specific embodiments, provided herein is a host cell (e.g., a recombinant host cell, preferably a recombinant prokaryotic host cell) that produces a glycosylated E. coH O4 polysaccharide antigen, wherein the host cell further comprises a nucleotide sequence encoding an oligosaccharyltransferase and /or a nucleotide sequence encoding a carrier protein. In one specific embodiment, the oligosaccharyltransferase is heterologous to the host cell. In another specific embodiment, the carrier protein is heterologous to the host cell. Preferably, the host cell contains a heterologous nucleotide sequence encoding a glucosyltransferase that is at least 80% sequence identical to SEQ ID NO: 4. In preferred embodiments, the O4 cluster rfb genes are heterologous to the host cell. Preferably, a sequence encoding an enzyme that is capable of introducing a branched glucose side chain into the O4 antigen, i.e. the gtrS gene (encoding a glucosyltransferase with at least 80% sequence identity to SEQ ID NO: 4) is heterologous to the host cell. A nucleic acid is heterologous to a host cell if the same sequence is not naturally present in the host cell. A heterologous nucleic acid may, for example, be introduced into a parent cell by genetic engineering, for example, by transformation (eg, chemical transformation or electroporation) and/or recombination. In some embodiments, a heterologous nucleic acid, such as a desired rfb locus, a gtrS coding sequence, a carrier protein coding sequence, and/or a glycosyltransferase coding sequence is integrated into the genome of a host cell, preferably a bacterial host cell, preferably an E. coH host cell. . In preferred embodiments, the endogenous rfb locus and, if applicable, the gtrS coding sequence have been inactivated, preferably by deletion from the genome of the recombinant host cell relative to its predecessor, and preferably they are replaced by the desired heterologous rfb locus, and, if applicable, the desired gtrS coding sequence , respectively. In some embodiments, the host cell is E. coH K-12 (by way of non-limiting example, strain K-12 is E. coH strain W3110), or E. coH strain B (by way of non-limiting example, strain B is E. coH strain). coH BL21), or any other well-defined strain of E. coH, such as laboratory strains or industrial strains, as opposed to primary wild-type isolates. In preferred embodiments, the host cell is derived from E. coli that does not express O4 antigen or glycosylated O4 antigen by introducing into such E. coli an O4 rfb locus and a gtrS gene encoding a glucosyltransferase that is at least 80% identical to the sequence of SEQ ID NO : 4. The advantage of using well-characterized strains such as E. coH K-12 or E. coH B as host cell precursors is the ability to use a similar production process for various O-antigen bioconjugates since the characteristics of the producing strain are well defined . Although bioconjugates of different O-antigens will behave differently and expression processes may be optimized for each production strain, at least the basic process for producing O-antigen bioconjugates using such well-defined precursor strains will be more predictable. than when unknown strains, such as wild-type isolates, are used as precursors to obtain host strains. Thus, the experience of obtaining previously described E. coH O-antigen bioconjugates, such as bioconjugates O1A, O2, 06a and 025b, as described, for example, in WO 2015/124769 and WO 2017/035181, can be used as a basis for the development production of other bioconjugates of the E.coH O-antigen. Unlike gtrS, the gtrA and gtrB genes are not serotype-specific, and in some embodiments they are homologous to the host cell (e.g., E. coli K12 strain W3110 includes gtrA and gtrB genes that are capable of functioning together with the serotype-specific O4 recombinantly introduced gtrS gene encoding a glucosyltransferase SEQ ID NO: 4 or a glucosyltransferase that is at least 80% identical to it, replacing the endogenous gtrS gene). In other embodiments, one or both of the gtrA and gtrB genes (encoding GtrA and GtrB proteins that are at least about 80% identical to SEQ ID

- 24 046206

NO: 7 and 8, respectively, and have bactoprenol-linked glucose translocase and bactoprenol glucosyltransferase activities, respectively, are also recombinantly introduced into the host cell, for example, in the case where the host cell does not have endogenous gtrA and/or gtrB genes.

Also provided herein are host cells (eg, recombinant host cells) capable of producing a bioconjugate of an E. coli O1A, O2, O6A, O8, O15, O16, O18A, 025b, or O75 polysaccharide antigen covalently linked to a carrier protein. Such host cells (eg, recombinant host cells) contain the nucleotide sequence of the rfb gene cluster specific for the polysaccharide O-antigen. rfb gene clusters can be isolated from wild-type E. coli strains and combined with nucleic acids encoding an oligosaccharyltransferase (eg, PglB) and a carrier protein (eg, EPA) in a single host cell to produce a recombinant host cell that produces the interest O-antigen E.coH or its bioconjugate. For example, such host cells can be engineered using recombinant approaches to contain one or more plasmids containing the rfb gene cluster, an oligosaccharyltransferase (eg, PglB) and a carrier protein (eg, EPA), using bioconjugation technology such as described in WO 2014/037585, w0 2009/104074 and WO 2009/089396. Preferably, host cells contain rfb gene clusters integrated into their genome. In some embodiments, the nucleic acids encoding the oligosaccharyltransferase, the carrier protein, and, where applicable, the gtrS gene are also integrated into the genome of the host cell. In some embodiments, heterologous or homologous gtrA and gtrB genes are also integrated into the genome of the host cell. The preparation of bioconjugates for antigens O1A, O2, O6A and 025B is described in detail in WO 2015/124769 and WO 2017/035181. Examples of gene clusters for each E.coH O-antigen (rfb loci) are described by Iguchi A, et al, DNA Research, 2014, 1-7 (doi: 10.1093/dnares/dsu043) and DebRoy C, et al, PLoS One. 2016, 11(1):e0147434 (doi: 10.1371/journal.pone.0147434; corrections in: Plos One. 2016, 11(4):e0154551, doi:

10.1371/journal.pone.0154551). Nucleic acid sequences for rfb clusters and amino acid sequences for the proteins encoded within them can also be found in public databases such as GenBank. Examples of rfb cluster sequences that can be used in producer strains for bioconjugates with polysaccharide antigens of the serotypes disclosed herein are also provided in SEQ ID NOs: 9 and 11-19. Thus, for each of the desired bioconjugates mentioned above, the corresponding rfb cluster can be introduced into a host cell to obtain host cells containing the specific rfb cluster for the desired O-antigen, as well as containing a nucleic acid encoding an oligosaccharyltransferase and a carrier protein . For the reasons stated above, it is preferable that the host cells are recombinant host cells and preferably come from strains with relatively well-known characteristics, such as laboratory or production strains of E. coH, for example E. coH K12 or E. coH BL21, etc. . Preferably, the rfb clusters are heterologous to the host cell, eg introduced into a progenitor cell of the host cell and preferably integrated into its genome. Preferably, the original rfb gene cluster, if present in the progenitor cell, is replaced in the host cell by the rfb gene cluster for the O-antigen of interest to allow production of a bioconjugate of the O-antigen of interest. Preferably, the oligosaccharyltransferase is heterologous to the host cell, and in some embodiments, the nucleic acid encoding such oligosaccharyltransferase is integrated into the genome of the host cell.

Any of the host cells provided herein (e.g., recombinant host cells, preferably recombinant prokaryotic host cells) contain nucleic acids encoding additional enzymes active in N-glycosylation of proteins, for example, the proposed host cell may further comprise a nucleic acid encoding an oligosaccharyltransferase, or nucleic acids encoding other glycosyltransferases.

Host cells provided herein contain a nucleic acid encoding an oligosaccharyltransferase. Oligosaccharyltransferases transfer lipid-bound oligosaccharides to aspartic residues of the resulting polypeptide chains, which contain a consensus Nglycosylation motif. The nucleic acid encoding the oligosaccharyltransferase may be native to the host cell or may be introduced into the host cell using genetic approaches. In preferred embodiments, the oligosaccharyltransferase is heterologous to the host cell. E.coH does not naturally contain an oligosaccharyltransferase, and therefore, if E.coH is used as a host cell for the production of bioconjugates, the host cell contains a heterologous oligosaccharyltransferase, for example, introduced through genetic engineering. The oligosaccharyltransferase may come from any source known in the art given the present disclosure. In some embodiments, as an alternative to an oligosaccharyltransferase with N-glycosyltransferase activity, such as an O-glycosyltransferase, for example, PglL can be used as a non-limiting example in combination with its own, different consensus glycosylation sequence in a carrier protein, as, for example, described in WO 2016/82597. Thus, according to the invention, other glycosyltransferases, such as O-glycosyltransferases, can also be used as oligosaccharyltransferases.

- 25 046206

In some preferred embodiments, the oligosaccharyltransferase is an oligosaccharyltransferase from Campylobacter. For example, in one embodiment, the oligosaccharyltransferase is an oligosaccharyltransferase from Campylobacter jejuni (i.e., pglB; see, e.g., Wacker et al., 2002, Science 298:1790-1793; see also, e.g., NCBI Gene ID: 3231775 , Uniprot accession number 086154). In another embodiment, the oligosaccharyltransferase is an oligosaccharyltransferase from Campylobacter lari (see, for example, NCBI Gene ID: 7410986).

In specific embodiments, the oligosaccharyltransferase is the PglB oligosaccharyltransferase from Campylobacter jejuni, including the naturally occurring protein (wild type) or any variant thereof, for example, as described in published international patent applications WO 2016/107818 and WO 2016/107819. PglB can transfer lipid-linked oligosaccharides to asparagine residues in the consensus sequences SEQ ID NO: 1 and SEQ ID NO: 2. In certain embodiments, the PglB oligosaccharyltransferase contains SEQ ID NO: 6 or a variant thereof. In some embodiments, one or more endogenous consensus glycosylation sequences in wild-type PglB have been mutated to avoid autoglycosylation of PglB, for example SEQ ID NO: 6 containing the N534Q mutation. Examples of PglB oligosaccharyltransferase variants suitable for use in recombinant host cells provided herein include PglB oligosaccharyltransferase of SEQ ID NO: 6 containing at least one mutation selected from the group consisting of N311V, K482R, D483H, A669V, Y77H, S80R, Q287P and K289R. In one specific embodiment, the PglB oligosaccharyltransferase variant has SEQ ID NO: 6 containing the N311V mutation. In another specific embodiment, the PglB oligosaccharyltransferase variant has SEQ ID NO: 6 containing mutations Y77H and N311V. In another specific embodiment, the PglB oligosaccharyltransferase variant has SEQ GO NO: 6 containing mutations N311V, K482R, D483H and A669V. In another specific embodiment, the PglB oligosaccharyltransferase variant has SEQ GO NO: 6 containing mutations Y77H, S80R, Q287P, K289R and N311V. It has been discovered and described herein that certain variants of the PglB oligosaccharyltransferase unexpectedly provide improved yield in the production of E. coH Oantigen bioconjugates of certain serotypes. The improved or optimal PglB variant for a given E.coH Oantigen cannot be predicted. Thus, some aspects of the invention also provide methods for producing bioconjugates of specific E. coH O-antigens using specific PglB variants as the oligosaccharyltransferase. Additional PglB variants that are at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to SEQ ID NO: 6 and still have oligosaccharyltransferase activity, preferably having in combination one or more specific amino acids at specified positions set forth herein (e.g., 77Y, 80S, 287Q, 289K, 311N, 482K, 483D, 669A; or 311V; or 311V, 482R, 483H, 669V; or 77H, 80R, 287P , 289R, 311V; or 77H, 311V; etc.) can also be used for the production of bioconjugates.

In a specific embodiment, a host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of a glycosylated E. coli O4 polysaccharide antigen covalently linked to a carrier protein further comprises a nucleotide sequence encoding a Campylobacter jejuni PglB oligosaccharyltransferase having the amino acid sequence of SEQ ID NO : 6, or preferably SEQ ID NO: 6 containing the N311V mutation, or more preferably SEQ ID NO: 6 containing the Y77H and N311V mutations. In other specific embodiments, a host cell (e.g., a recombinant host cell) capable of producing an E. coli O1A, 06a, or O15 polysaccharide antigen bioconjugate covalently linked to a carrier protein further comprises a nucleotide sequence encoding a Campylobacter jejuni PglB oligosaccharyltransferase having the amino acid sequence of SEQ ID NO: 6, or preferably SEQ ID NO: 6, containing mutations N311V, K482R, D483H and A669V.

In a specific embodiment, a host cell (e.g., a recombinant host cell) capable of producing an E. coli O16 polysaccharide antigen bioconjugate covalently linked to a carrier protein further comprises a nucleotide sequence encoding a Campylobacter jejuni PglB oligosaccharyltransferase having the amino acid sequence of SEQ ID NO: 6, or preferably SEQ ID NO: 6 containing mutations Y77H, S80R, Q287P, K289R and N311V.

In a specific embodiment, a host cell (e.g., a recombinant host cell) capable of producing an E. coli O75 polysaccharide antigen bioconjugate covalently linked to a carrier protein further comprises a nucleotide sequence encoding a Campylobacter jejuni PglB oligosaccharyltransferase having the amino acid sequence of SEQ ID NO: 6, or preferably SEQ ID NO: 6 containing the N311V mutation.

In a specific embodiment, a host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of an E. coli O8, O18A, 025B, or O2 polysaccharide antigen covalently linked to a carrier protein further comprises a nucleotide sequence encoding a Campylobacter jejuni PglB oligosaccharyltransferase having the amino acid sequence of SEQ ID NO: 6, preferably wherein SEQ ID NO: 6 does not contain amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669.

In some embodiments, any of the host cells provided herein comprise

- 26 046206 a nucleic acid encoding a carrier protein, for example a protein, to which polysaccharide O-antigen(s) produced by the glycosylation machinery of the host cell can be attached to form a bioconjugate . The host cell may contain a nucleic acid encoding any carrier protein known to those skilled in the art in light of the present invention, including detoxified P. aeruginosa exotoxin A (EPA), E. coH flagellin (FHC), CRM 197, maltose binding protein (MBP) , diphtheria toxoid, tetanus toxoid, neutralized hemolysin A S.aureus, agglutinating factor A, agglutinating factor B, heat-labile enterotoxin E.coH, neutralized variants of the thermolabile enterotoxin E.coH, subunit B of cholera toxin (CTS), cholera toxin, neutralized variants of cholera toxin, E. coli Sat protein, E. coli Sat protein passenger domain, Streptococcus pneumoniae pneumolysin, Megathura crenulata hemocyanin (KLH), P. aeruginosa PcrV, Neisseria meningitidis outer membrane protein (OMPC), and D protein from nontypeable Haemophilus influenzae, but not limited to the above.

In preferred embodiments, the host cell further comprises a nucleic acid encoding detoxified P. aeruginosa exotoxin A (EPA). Preferably, the EPA carrier protein contains 1-10 glycosylation sites, preferably 2 to 4 glycosylation sites, most preferably 4 glycosylation sites, each containing a consensus glycosylation sequence having the amino acid sequence of SEQ ID NO: 1 and most preferably having the amino acid sequence SEQ ID NO: 2. In a specific embodiment, the host cell further comprises a nucleic acid encoding an EPA-4 carrier protein comprising SEQ ID NO: 3. In some embodiments, carrier proteins used in the creation of bioconjugates by host cells described in herein, contain a tag, that is, a sequence of amino acids that allows the isolation and/or identification of the protein carrier. For example, adding a tag to a carrier protein may be useful for purifying the protein and therefore purifying conjugate vaccines containing the tagged carrier protein. Examples of tags that may be used herein include, but are not limited to, histidine (HIS) tags (eg, hexahistidine tags or 6XHis-Tag), FLAG-TAGs, and HA (hemagglutinin) tags. In some embodiments, the tags used herein are removable, for example, removed by chemical agents or enzymatic methods when they are no longer needed, for example, after protein purification. In other embodiments, the carrier protein does not contain a tag.

In some embodiments, a carrier protein described herein comprises a signal sequence that directs the carrier protein to the periplasmic space of a host cell expressing the carrier protein. In a specific embodiment, the signal sequence is derived from E. coH DsbA, E. coH outer membrane porin A (OmpA), E. coH maltose binding protein (MalE), Erwinia carotovorans pectate lyase (PelB), Flgl, NikA or endoxylanase (XynA) of Bacillus, sp ., heat labile enterotoxin LTIIb of E. coli, endoxylanase XynA of Bacillus, or flagellin (Flgl) of E. coli. In one embodiment, the signal sequence comprises SEQ ID NO: 10. The signal sequence may be cleaved after translocation of the protein into the periplasm and thus may more not present in the final carrier protein of the bioconjugate.

In some embodiments, additional modifications (eg, using recombinant methods) described herein can be introduced into the host cells. For example, in the background genotype (genome) of a host cell, there may be a deletion or modification of host cell nucleic acids (e.g., genes) that encode proteins that form part of a possible competing or interfering glycosylation pathway (e.g., compete with or interfere with one or more heterologous genes involved in glycosylation that are recombinantly introduced into the host cell) in such a way as to inactivate them/render them dysfunctional (i.e., host cell nucleic acids that are deleted/modified do not encode a functional protein). In some embodiments, when nucleic acids are deleted from the genome of host cells provided herein, they are replaced with a desired sequence, for example, a sequence that is useful for the production of a polysaccharide O-antigen or a bioconjugate thereof. Examples of genes or gene clusters that can be deleted in host cells (and, in some cases, replaced with other desired nucleic acid sequences) include host cell genes or gene clusters involved in glycolipid biosynthesis, such as waaL (see eg Feldman et al. , undecaprenol-P biosynthesis genes (e.g., uppS, uppP), und-P recycling genes, metabolic enzymes involved in the biosynthesis of nucleotide-activated sugars, the common Enterobacteriaceae (EC) antigen cluster, and prophage O-antigen modification clusters such as the gtrABS cluster or its area. In a specific embodiment, host cells described herein are modified such that they do not produce any polysaccharide O antigen other than the desired polysaccharide O antigen, for example, glycosylated polysaccharide O4 antigen.

In a specific embodiment, the waaL gene is removed from the genome of the host cell (e.g., recombinant

- 27 046206 host cell) proposed herein, or functionally inactivated. The terms waaL and waaL gene refer to the O-antigen ligase gene, encoding a membrane-bound enzyme with an active site located in the periplasm. The encoded enzyme transfers undecaprenyl phosphate (UPP)-linked O-antigen to the lipid A core, forming lipopolysaccharide. Deletion or disruption of the endogenous waaL gene (eg, in AwaaL strains) impairs O-antigen transfer to lipid A and may instead enhance O-antigen transfer to another biomolecule, such as a carrier protein.

In another specific embodiment, the original genome of the prokaryotic host cell provided herein has a deletion or functional inactivation of one or more of the waaL gene, the gtrA gene, the gtrB gene, the gtrS gene, and the rfb gene cluster.

In one embodiment, the host cell used is an E.coH that produces a bioconjugate of a glycosylated O4 polysaccharide antigen, wherein the genome of the host cell has a deletion or functional inactivation of the waaL gene and insertion of a gtrS gene specific for the E.coH O4 polysaccharide antigen. In some embodiments, for strains producing glycosylated O-antigen O4 bioconjugates, a gtrS gene encoding a glucosyltransferase with at least 80% sequence identity to SEQ ID NO: 4 is inserted in place of the gtrS gene of the parent strain to replace the gtrS gene in that parent strain with the gene that is responsible for the glycosylation of the O4 antigen. An example of such a parent strain is E. coli K-12 strain W3110. The gtrA and gtrB genes may be homologous to the parental strain, or, alternatively, one or both of these genes may be heterologous to the parental strain. As a rule, unlike the gtrS gene, these gtrA and gtrB genes are nonspecific for the O-antigen structure. This document also provides methods for generating recombinant host cells. Recombinant host cells produced by the methods described herein can be used to produce bioconjugates of E. coH O-antigens. The methods include introducing one or more recombinant nucleic acid molecules into a cell to produce a recombinant host cell. As a rule, recombinant nucleic acid molecules are heterologous. Any suitable method known in the art may be used to introduce recombinant nucleic acid molecules into a host cell. Recombinant nucleic acids can be introduced into the host cells described herein using any methods known to those skilled in the art, for example, electroporation, chemical transformation, heat shock, natural transformation, phage transduction and conjugation. In specific embodiments, recombinant nucleic acids are introduced into host cells described herein using a plasmid. For example, heterologous nucleic acids can be expressed in host cells using a plasmid (eg, an expression vector). In another specific embodiment, heterologous nucleic acids are introduced into host cells described herein using a genome insertion method, such as those described in published international patent applications WO 2014/037585, WO 2014/057109 or WO 2015/052344.

In one embodiment, a method of producing a recombinant host cell for producing a bioconjugate of a glycosylated E.coH O4 polysaccharide antigen covalently linked to a carrier protein comprises introducing into a cell, preferably an E.coH cell, one or more recombinant nucleic acid molecules to produce a recombinant host cell . In such embodiments, the recombinant nucleic acid molecules introduced into the cell include (1) the nucleotide sequence of the rfb gene cluster for the E. coH O4 polysaccharide antigen; (2) a nucleotide sequence encoding a glucosyltransferase, the sequence of which is at least 80% identical to SEQ ID NO: 4, wherein the glucosyltransferase is capable of modifying the O4 polysaccharide antigen of E.coH to form a glycosylated polysaccharide antigen O4 of E.coH; (3) a nucleotide sequence encoding a carrier protein; and (4) a nucleotide sequence encoding an oligosaccharyl transferase capable of covalently linking the glycosylated E.coH O4 polysaccharide antigen to a carrier protein to produce a bioconjugate. In preferred embodiments, a nucleotide sequence encoding a glucosyltransferase that is at least 80% sequence identical to SEQ ID NO: 4 replaces the endogenous gtrS gene. Deletion of endogenous gtrS has the advantage that it does not interfere with the formation of the polysaccharide structure of the glycosylated O4 antigen. In some embodiments, the nucleotide sequence of the rfb gene cluster for the E. coH O4 polysaccharide antigen replaces the endogenous rfb gene cluster of the parent strain that is used to generate the recombinant host cell. If the cell does not already encode the gtrA and/or gtrB genes, nucleotide sequences encoding a translocase (gtrA) and a glucosyltransferase (gtrB) that are at least 80% sequence identical to SEQ ID NO: 7 and 8, respectively, can be introduced into the cell. If the cell already encodes the gtrA and gtrB genes (as, for example, in the case of E.coH K-12 strain W3110), there is no need to introduce or change these genes.

In a specific embodiment, the glucosyltransferase (gtrS, specific for adding a glucose branch to the O4 antigen) has SEQ ID NO: 4.

In a specific embodiment, the oligosaccharyltransferase is PglB from ^jejuni. In one such embodiment, the oligosaccharyltransferase comprises the amino acid sequence of SEQ ID

- 28 046206

NO: 6. In another such embodiment, the oligosaccharyltransferase contains the amino acid sequence of SEQ ID NO: 6 containing the N311V mutation. In another such embodiment, the oligosaccharyltransferase contains the amino acid sequence of SEQ ID NO: 6 containing the Y77H and N311V mutations.

In another specific embodiment, the carrier protein contains at least one glycosylation site containing a consensus glycosylation sequence having SEQ ID NO: 1, preferably SEQ ID NO: 2. In another specific embodiment, the carrier protein is EPA, preferably EPA-4, such as EPA-4 containing SEQ ID NO: 3.

E. coH strains, which are commonly used in molecular biology as a tool and as a model organism, can, for example, be used as parent cells for host cells in some embodiments of the invention. Non-exhaustive examples include E. coH K12 strains (eg, such as W1485, W2637, W3110, MG1655, DH1, DH5a, DH10, etc.), B strains (eg, BL21, REL606, etc.), C strains or W strains. In one particular embodiment, the host strain is derived from the parent strain W3110. This strain can, for example, be obtained from the E. coH culture museum at Yale University. For more information on E.coH, see for example Ecoliwiki.net.

Methods for obtaining conjugates and bioconjugates.

Also provided are methods for preparing glycoconjugates of E. coH polysaccharide O-antigens described herein. Glycoconjugates, including bioconjugates, can be produced in vitro or in vivo, for example, using recombinant host cells described herein. In some embodiments, glycoconjugates can be prepared by chemical synthesis, i.e. obtained outside host cells (in vitro). For example, the E.coH polysaccharide O-antigen can be conjugated to carrier proteins using methods known to those skilled in the art, including methods involving activation of reactive groups of the polysaccharide/oligosaccharide as well as the carrier protein. See, for example, Pawlowski et al., 2000, Vaccine 18:1873-1885; and Robbins, et al., 2009, Proc Natl Acad Sci USA 106:7974-7978), the contents of which are incorporated by reference in their entirety. Such approaches include extracting antigenic polysaccharides/oligosaccharides from host cells, purifying the polysaccharides/oligosaccharides, chemically activating the polysaccharides/oligosaccharides, and conjugating the polysaccharides/oligosaccharides to a carrier protein. In some embodiments, the host cells described herein can be used to produce bioconjugates containing E. coH polysaccharide O-antigen covalently linked to a carrier protein. Methods for preparing such bioconjugates are known in the art. See, for example, WO 2003/074687 and WO 2006/119987. Such methods include culturing any recombinant host cell described herein under conditions for producing a bioconjugate. Bioconjugates can be isolated, separated and/or purified from recombinant host cells using any method known in the art in light of the present disclosure. For example, bioconjugates can be purified by any protein purification method known in the art, for example, by chromatography (e.g., ion exchange, anion exchange, affinity and size separation column chromatography), centrifugation, solubility differences, or any other standard protein purification method . See, for example, the methods described in WO 2009/104074. In addition, to facilitate purification, bioconjugates can be fused to heterologous polypeptide sequences. The actual conditions used to purify a particular bioconjugate will depend in part on factors such as net charge, hydrophobicity and/or hydrophilicity of the bioconjugate, and will be apparent to those skilled in the art. The preparation of bioconjugates for O1A, O2, 06a and 025b, as well as vaccine compositions containing them, for example, has been described in WO 2015/124769 and WO 2017/035181. Also provided are bioconjugates prepared by the methods described herein, that is, using the recombinant host cells described herein.

In some embodiments, a method of producing an E. coH polysaccharide O-antigen bioconjugate covalently linked to a carrier protein comprises: (1) producing a recombinant host cell containing (a) the nucleotide sequence of the rfb gene cluster for the polysaccharide O-antigen; (b) a nucleotide sequence encoding a carrier protein, preferably an EPA, containing at least one glycosylation site, containing a consensus glycosylation sequence having SEQ ID NO: 1, preferably SEQ ID NO: 2, and more preferably containing four glycosylation sites each of which contains a consensus glycosylation sequence having SEQ ID NO: 2; and (c) a nucleotide sequence encoding an oligosaccharyltransferase, for example, PglB oligosaccharyltransferase or a variant thereof. In some embodiments, E.coH polysaccharide O-antigens produced using the recombinant host cells described herein are covalently linked to a carrier protein at a specific polysaccharide to protein weight ratio (w/w). This mass ratio of the amount of polysaccharide O-antigen covalently bound to a carrier protein is called the glycan/protein ratio, or the polysaccharide/protein ratio, or the PS/protein ratio. In some embodiments, the polysaccharide O-antigen is covalently linked to a carrier protein at a polysaccharide ratio

- 29 046206 charide and protein (w/w) from about 1:20 to 20:1, preferably from 1:10 to 10:1, more preferably from 1:3 to 3:1. In some non-limiting embodiments of the bioconjugates described herein, the glycan/protein ratio is from about 0.1 to 0.5, for example, 0.1, 0.15, 0.2, 0.25, 0.3, 0. 35, 0.4, 0.45 or 0.5. In such embodiments, the weight ratio of polysaccharide Oantigen:protein is from about 1:10 to 1:2, for example, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3 or 1:2, depending on the specific O-antigen serotype. In some embodiments, the glycan/protein ratio is from about 0.15 to about 0.45. In general, a higher glycan/protein ratio of O-antigen polysaccharide to carrier protein is preferred because in some cases a high amount of carrier protein may lead to immunological interference. In addition, a higher glycan/protein ratio will help obtain sufficient amounts of polysaccharide O-antigen dosed in bioconjugate form while maintaining a relatively low amount of carrier protein, which is especially useful for multivalent compositions in which the composition must cover multiple serotypes, e.g. containing bioconjugates of at least 4 different O-antigens, at least 5 different O-antigens, at least 6 different O-antigens, at least 7 different O-antigens, at least 8 different Oantigens, at least 9 different O-antigens, at least 10 different O-antigens, etc.

The glycan/protein ratio of the conjugate of the invention can be determined by determining the amount of protein and the amount of glycan. The amount of protein can be determined by measuring the UV absorbance at 280 nm (A280). The number of glycans can be determined based on ion chromatography with pulsed amperometric detection (IC-PAD) of the sugar in the repeat unit (e.g., Man for O8 from Table 1 and GlcNAc for other glycans from Table 1), followed by structural information about the repeat unit can be used to calculate the total number of glycans (e.g., an O1A repeating unit has a molar mass of 845 Da, and one mole of such a repeating unit contains one mole of GlcNAc, allowing the total number of glycans to be calculated when the amount of GlcNAc has been determined by IC-PAD).

In some embodiments, the E. coH polysaccharide antigen 025b bioconjugate covalently linked to a carrier protein produced using a recombinant host cell in accordance with the cells and methods described herein has a certain degree of acetylation at the 2-position of the L-Rh sugar. The degree of O-acetylation of the 025B polysaccharide antigen in the bioconjugate is preferably at least 30%, preferably at least 50%, for example at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%.

Likewise, the degree of O-acetylation of the E.coH O16 polysaccharide antigen in the bioconjugate is preferably at least 30%, preferably at least 50%, for example at least 50, 55, 60, 65, 70, 75, 80, 85 , 90, 95 or 100%.

In specific embodiments, a method for producing a polysaccharide O-antigen bioconjugate involves producing a recombinant host cell containing a nucleic acid sequence encoding a particular oligosaccharyltransferase enzyme, particularly PglB oligosaccharyltransferase or a variant thereof, depending on the polysaccharide O-antigen bioconjugate that is desired to be produced. A particular variant of the oligosaccharyltransferase enzyme may influence the yield of the bioconjugate produced by the host cell. Generally, a higher yield is preferred since the yield will influence the cost of obtaining a particular bioconjugate, which is especially important for multivalent compositions containing several different bioconjugates.

In one particular embodiment, when the O antigen is an O1A, O6A, or O15 polysaccharide antigen, the PglB oligosaccharyltransferase contains amino acid mutations N311V, K482R, D483H, and A669V, wherein the amino acid mutations are wild-type PglB having the amino acid sequence of SEQ ID NO: 6.

In another specific embodiment, when the O antigen is an O4 polysaccharide antigen, the PglB oligosaccharyltransferase contains amino acid mutation N311V or amino acid mutations Y77H and N311V, wherein the amino acid mutations are wild-type PglB having the amino acid sequence of SEQ ID NO: 6.

In another specific embodiment, when the O antigen is an O16 polysaccharide antigen, the PglB oligosaccharyltransferase contains amino acid mutations Y77H, S80R, Q287P, K289R and N311V, where the amino acid mutations are wild type PglB having the amino acid sequence of SEQ ID NO: 6.

In another specific embodiment, when the O antigen is an O75 polysaccharide antigen, the PglB oligosaccharyltransferase contains the amino acid mutation N311V, where the amino acid mutations are wild type PglB having the amino acid sequence of SEQ ID NO: 6.

In another specific embodiment, when the O antigen is an O8, O18A, 025B, or O2 polysaccharide antigen, the PglB oligosaccharyltransferase contains the amino acid sequence of SEQ ID NO: 6, wherein SEQ ID NO: 6 does not contain amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669. In some embodiments, the PglB oligosaccharyltransferase contains the amino acid sequence of SEQ ID NO: 6.

- 30 046206

In some embodiments, polysaccharide O-antigen bioconjugates produced by recombinant host cells encoding oligosaccharyltransferase enzymes from the Oantigen/oligosaccharyltransferase PglB pairs identified above preferably have one or more of the preferred characteristics described herein, such as glycan/protein ratio and/or percentage multiglycosylated carrier protein.

Incarnations

Embodiment 1 is a bioconjugate of a glycosylated E.coli O4 polysaccharide antigen covalently linked to a carrier protein, wherein the glycosylated E.coli O4 polysaccharide antigen contains the structure of the formula (O4-GlC+)a-D-GIcp

I h [->2)-aL-Rhap-(1 ->6)-aD-Glcp-( 1 ->3)-aL-FucpNAc-(1 ->3)-pD-GlcpNAc-( 1 ->] _n where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20.

Embodiment 1 is the bioconjugate of Embodiment 1, wherein the glycosylated polysaccharide E.coH O4 antigen is covalently linked to an Asn residue at a glycosylation site in a carrier protein containing a glycosylation consensus sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2.

Embodiment 3 is the bioconjugate of embodiment 1 or embodiment 2, wherein the carrier protein is selected from the group consisting of detoxified P. aeruginosa exotoxin A (EPA), E. coH flagellin (FliC), CRM 197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, neutralized hemolysin A S.aureus, agglutinating factor A, agglutinating factor B, heat-labile enterotoxin E.coH, neutralized variants of the thermolabile enterotoxin E.coH, subunit B of cholera toxin (CTB), cholera toxin, neutralized variants of cholera toxin, Sat protein E. coli, E. coli Sat protein passenger domain, Streptococcus pneumoniae pneumolysin, Megathura crenulata hemocyanin (KLH), P. aeruginosa PcrV, Neisseria meningitidis outer membrane protein (OMPC), and protein D from nontypeable Haemophilus influenzae.

Embodiment 4 is the bioconjugate of embodiment 3, wherein the carrier protein is detoxified P. aeruginosa exotoxin A (EPA), preferably containing 1 to 20, preferably 1 to 10, preferably 2 to 4, consensus glycosylation sequences having SEQ ID NO: 1, consensus sequences preferably have SEQ ID NO: 2.

Embodiment 5 is a bioconjugate of embodiment 4, wherein the carrier protein contains four consensus glycosylation sequences (EPA-4), preferably where the carrier protein contains SEQ ID NO: 3.

Embodiment 6 is a composition containing the bioconjugate of any one of embodiments 1-5.

Embodiment 7 is an immunogenic composition containing a bioconjugate according to any of embodiments 1-5.

Embodiment 8 is the composition of embodiment 6 or the immunogenic composition of embodiment 7 comprising at least one additional polysaccharide antigen covalently linked to a carrier protein. Embodiment 9 is the composition or immunogenic composition of embodiment 8, wherein the at least one additional polysaccharide antigen is selected from the group consisting of E.coli O1A polysaccharide antigen, E.coli O2 polysaccharide antigen, E.coli O6a polysaccharide antigen, polysaccharide antigen O8 E.coli, polysaccharide antigen O15 E.coli, polysaccharide antigen O16 E.coli, polysaccharide antigen O18A E.coli, polysaccharide antigen 025B E.coli and polysaccharide antigen O75 E.coli. Embodiment 10 is the composition or immunogenic composition of embodiment 9, wherein (1) the E. coli O1A polysaccharide antigen contains the structure of formula (O1A)

[—3)-aL-Rhap-(1 —3)-aL-Rhap-(1 ->3)-₽-L-Rhap-(1 -*4)-pD-GlcpNAc-(1 ->] _n 2 t 1 β-D-ManpNAc (2) polysaccharide antigen O2 E.coH contains the structure of the formula (O2) [-*3)-aL-Rhap-(1 ^2)-aL-Rhap-(1 ->3)-pL -Rhap-(1 -»4)-pD-GlcpNAc-(1 -] _n 2 t 1 aD-Fucp3NAc (3) polysaccharide antigen O6AB.coH contains the structure of the formula (O6A)

- 31 046206

[->4)-aD-GalpNAc-(1 ->3)-β-0-Μ3ηρ-(1 -4)-pD-Manp-(1 ->3)-aD-GlcpNAc-(1 -*] _n 2 t 1 β-D-Glcp (4) polysaccharide antigen O8 E. coli contains the structure of the formula (O8) aD-Manp3Me-(1 -*[3)-pD-Manp-(1 ->2)-aD-Manp- (1 ->2)-aD-Manp-(1 -»] _n (5) polysaccharide antigen O15 of E. coli contains the structure of the formula (O15) [^2)-pD-Galp-(1^3)-aL-FucpNAc -(1^3)-pD-GlcpNAc-(1^] _n (6) polysaccharide antigen O16 E.coH contains the structure of the formula (O16)

[“►2)-pD-Galf-(1-*6)4x-D-Glcp-(1-*3)4x-L-Rhap-(1-*3)-aD-GlcpNAc-(1-*] _n t Ac (7) polysaccharide antigen 018a of E. coli contains the structure of the formula (O18A) [^2)-aL-Rhap-(1->6)-aD-Glcp-(1-4)-aD-Galp-(1 ^3)-aD-GlcpNAc-(1^] _n t 1 β-D-GlcpNAc (8) polysaccharide antigen 025B E.coH contains the structure of the formula (025B) β-D-Glcp !

[-*4)-aD-Glcp-(1 -»3)-aL-Rhap-(1 -^3)^-D-GlcpNAc-(1 -^] _n 3 t 1 aL-Rhap ,.

;

(9) polysaccharide antigen O75 E.coH contains the structure of the formula (O75) β-D-Manp g 4

[^3)-a-D-Galp-(1 ->4)-a-L-Rhap-(1 ->3)-3-D-GlcpNAc-(1 where each n independently represents an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20.

Embodiment 11 is the composition or immunogenic composition of embodiment 10, wherein each of the additional polysaccharide antigens O1A, O2, O6A, O8, O15, O16, O18A, O25B and/or O75 E.coH is covalently linked to an Asn residue at the glycosylation site in each a carrier protein containing a consensus glycosylation sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2.

Embodiment 12 is the composition or immunogenic composition of embodiment 11, wherein each carrier protein is a detoxified Pseudomonas aeruginosa exotoxin A (EPA).

Embodiment 13 is the composition or immunogenic composition of embodiment 12, wherein each EPA contains 1-10, preferably 2-4 glycosylation sites, each of which contains a consensus glycosylation sequence having SEQ ID NO: 2.

Embodiment 14 is the composition or immunogenic composition of embodiment 12, wherein each EPA contains four glycosylation sites, each of which contains a glycosylation consensus sequence having SEQ ID NO: 2.

Embodiment 15 is the composition or immunogenic composition of embodiment 12, wherein each EPA contains SEQ ID NO: 3.

Embodiment 16 is a composition or immunogenic composition according to any of embodiments 9-15, which contains at least polysaccharide antigens O1A, O2, glycosylated O4, O6A and O25B of E. coli, each of which is covalently linked to a carrier protein.

Embodiment 17 is a composition or immunogenic composition according to any of embodiments 9-15, which contains at least polysaccharide antigens O1A, O2, glycosylated O4, O6A, O8, O15, O16, O25B and O75 of E. coli, each of which is covalently linked with a carrier protein.

Embodiment 18 is a composition or immunogenic composition according to any of embodiments 9-15, which contains at least polysaccharide antigens O1A, O2, glycosylated O4, O6A, O8, O15, O16, O18A, O25B and O75 E. coli, each of which covalently bound to a carrier protein.

Embodiment 19 is a composition comprising:

- 32 046206

1. bioconjugate of the glycosylated polysaccharide antigen O4 of E.coli, covalently linked to the neutralized exotoxin A of P.aeruginosa (carrier protein EPA-4), containing SEQ ID NO: 3, where the glycosylated polysaccharide antigen O4 of E.coH contains the structure of the formula (O4- Glc+);

2. a bioconjugate of the polysaccharide antigen O1A of E. coli covalently linked to the carrier protein EPA-4, where the polysaccharide antigen O1A of E. coli contains the structure of the formula (O1A);

3. a bioconjugate of the polysaccharide antigen O2 of E. coli covalently linked to the carrier protein EPA-4, where the polysaccharide antigen O2 of E. coli contains the structure of the formula (O2);

4. bioconjugate of the polysaccharide antigen 06a of E. coli, covalently linked to the carrier protein EPA-4, where the polysaccharide antigen O6A of E. coli contains the structure of the formula (O6A);

5. a bioconjugate of the E.coli O8 polysaccharide antigen covalently linked to the EPA-4 carrier protein, wherein the E.coli O8 polysaccharide antigen contains the structure of formula (O8);

6. bioconjugate of the polysaccharide antigen O15 of E. coli covalently linked to the carrier protein EPA-4, where the polysaccharide antigen O15 of E. coli contains the structure of the formula (O15);

7. bioconjugate of the polysaccharide antigen O16 of E. coli covalently linked to the carrier protein EPA-4, where the polysaccharide antigen O16 of E. coli contains the structure of the formula (O16);

8. bioconjugate of the polysaccharide antigen 025B of E. coli, covalently linked to the carrier protein EPA-4, where the polysaccharide antigen 025B of E. coli contains the structure of the formula (025B); And

9. bioconjugate of the polysaccharide antigen O75 of E. coli covalently linked to the carrier protein EPA-4, where the polysaccharide antigen O75 of E. coli contains the structure of the formula (O75);

where each of the structures of the formulas (O4-Glc+), (01A), (O2), (06A), (O8), (O15), (O16), (025B) and (O75) is shown in table. 1, and each n is an integer from 1 to 100, preferably from 3 to 50, such as 5 to 40, such as 7 to 25, such as 10 to 20.

Embodiment 20 is a composition according to embodiment 19, further comprising:

(10) a bioconjugate of the polysaccharide antigen O18A E.coH, covalently linked to the carrier protein EPA-4, where the polysaccharide antigen O18A E.coH contains the structure of the formula (O18A) shown in table. 1, and n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20.

Embodiment 21 is a method of inducing antibodies against E. coli glycosylated O4 antigen in a subject, comprising administering to the subject a bioconjugate of any of embodiments 1-5 or a composition or immunogenic composition of any of embodiments 6-20.

Embodiment 22 is the method of embodiment 21 wherein the antibodies have opsonophagocytic activity.

Embodiment 23 is a method of vaccinating a subject against E. coli, particularly extraintestinal pathogenic E. coli (ExPEC), comprising administering to the subject a bioconjugate of any of embodiments 1-5 or a composition or immunogenic composition of any of embodiments 6-20.

Embodiment 24 is a bioconjugate of any of embodiments 1-5 or a composition or immunogenic composition of any of embodiments 6-20 for use in inducing antibodies against the glycosylated E.coH O4 antigen.

Embodiment 25 is a bioconjugate of any of embodiments 1-5 or a composition or immunogenic composition of any of embodiments 6-20 for use in vaccination against extraintestinal pathogenic E. coli (ExPEC).

Embodiment 26 is a recombinant host cell for producing a bioconjugate of a glycosylated O4 polysaccharide antigen E.coH covalently linked to a carrier protein, wherein the glycosylated O4 polysaccharide antigen contains the structure of the formula (O4-Glc+) shown in Table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, the host cell contains:

(1) the nucleotide sequence of the rfb gene cluster for the E. coH O4 polysaccharide antigen (2) the nucleotide sequence encoding a glucosyltransferase, the sequence of which is at least 80% identical to SEQ ID NO: 4, where the glucosyltransferase is capable of modifying the E. coH O4 polysaccharide antigen with obtaining glycosylated polysaccharide antigen O4 E.coH;

(3) nucleotide sequences encoding a translocase and a glycosyltransferase, the sequences of which are at least 80% identical to SEQ ID NO: 7 and 8, respectively, where the translocase is capable of translocating bactoprenol-bound glucose and the glycosyltransferase is capable of glycosylating bactoprenol;

(4) a nucleotide sequence encoding a carrier protein; and (5) a nucleotide sequence encoding an oligosaccharyltransferase capable of covalently linking the glycosylated E.coH O4 polysaccharide antigen to a carrier protein to produce a bioconjugate.

Embodiment 27 is the recombinant host cell of embodiment 26, wherein: the glucosyltransferase has the amino acid sequence of SEQ ID NO: 4; oligosaccharyltransferase contains the amino acid sequence of SEQ ID NO: 6, preferably SEQ ID NO: 6, containing

- 33 046206 amino acid mutation N311V, more preferably SEQ ID NO:6 containing amino acid mutations Y77H and N311V; and the carrier protein comprises at least one glycosylation site comprising a consensus glycosylation sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2.

Embodiment 28 is the recombinant host cell of embodiment 26 or embodiment 27, wherein the carrier protein is selected from the group consisting of detoxified P. aeruginosa exotoxin A (EPA), E.coH flagellin (FliC), CRM 197, maltose binding protein (MBP) ), diphtheria toxoid, tetanus toxoid, neutralized hemolysin A S.aureus, agglutinating factor A, agglutinating factor B, heat-labile enterotoxin E.coH, neutralized variants of heat-labile enterotoxin E.coH, subunit B of cholera toxin (CTS), cholera toxin, neutralized variants cholera toxin, E. coli Sat protein, E. coli Sat protein passenger domain, Streptococcus pneumoniae pneumolysin, Megathura crenulata hemocyanin (KLH), P. aeruginosa PcrV, Neisseria meningitidis outer membrane protein (OMPC), and protein D from nontypeable Haemophilus influenzae.

Embodiment 29 is a recombinant host cell according to any of embodiments 2628, wherein the carrier protein is detoxified Pseudomonas aeruginosa exotoxin A (EPA).

Embodiment 30 is the recombinant host cell of embodiment 29, wherein the EPA contains 1-10, preferably 2-4 glycosylation sites containing a consensus glycosylation sequence having SEQ ID NO: 2.

Embodiment 31 is the recombinant host cell of embodiment 30, wherein the carrier protein is EPA with four consensus glycosylation sequences (EPA-4), preferably where the carrier protein comprises SEQ ID NO: 3.

Embodiment 32 is a recombinant host cell according to any one of embodiments 2631, which is an E. coH cell, for example, E. coH strain K-12, such as strain W3110.

Embodiment 33 is a method of producing a bioconjugate of a glycosylated E.coH O4 polysaccharide antigen covalently linked to a carrier protein, wherein the glycosylated E.coH O4 polysaccharide antigen contains the structure of the formula (O4-Glc+) shown in Table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, the method includes culturing a recombinant host cell according to any one of claims. 26-32 under conditions for producing the bioconjugate.

Embodiment 34 is the method of embodiment 33, further comprising isolating a bioconjugate from a recombinant host cell.

Embodiment 35 is a bioconjugate prepared by the method of embodiment 33 or 34.

Embodiment 36 is a composition containing the bioconjugate of embodiment 35.

Embodiment 37 is a method of producing a recombinant host cell to produce a bioconjugate of a glycosylated polysaccharide antigen O4 E.coH covalently linked to a carrier protein, wherein the glycosylated polysaccharide antigen O4 contains the structure of the formula (O4-Glc+) shown in Table. 1, where n is an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, the method includes introducing one or more recombinant nucleic acid molecules into a cell to obtain a recombinant host cell, where the recombinant cell -the owner contains:

(1) the nucleotide sequence of the rfb gene cluster for the O4 polysaccharide antigen of E. coH;

(2) a nucleotide sequence encoding a glucosyltransferase, the sequence of which is at least 80% identical to SEQ ID NO: 4, wherein the glucosyltransferase is capable of modifying the E.coH O4 polysaccharide antigen to produce a glycosylated E.coH O4 polysaccharide antigen;

(3) nucleotide sequences encoding a translocase and a glycosyltransferase, the sequences of which are at least 80% identical to SEQ ID NO: 7 and 8, respectively, where the translocase is capable of translocating bactoprenol-bound glucose and the glycosyltransferase is capable of glycosylating bactoprenol;

(4) a nucleotide sequence encoding a carrier protein; and (5) a nucleotide sequence encoding an oligosaccharyltransferase capable of covalently linking the glycosylated E.coH O4 polysaccharide antigen to a carrier protein to produce a bioconjugate.

Embodiment 38 is the method of embodiment 37, wherein the glucosyltransferase has SEQ ID NO: 4; the oligosaccharyltransferase contains the amino acid sequence of SEQ ID NO: 6, preferably SEQ ID NO: 6, containing the amino acid mutation N311V, and the carrier protein contains at least one glycosylation site containing the consensus glycosylation sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2.

Embodiment 39 is the method of embodiment 37 or embodiment 38, wherein the carrier protein is selected from the group consisting of detoxified P. aeruginosa exotoxin A (EPA), E. coH flagellin (FliC), CRM 197, maltose binding protein (MBP), diphtheria toxoid, tetanus

- 34 046206 toxoid, neutralized hemolysin A of S.aureus, agglutinating factor A, agglutinating factor B, heat-labile enterotoxin E.coH, neutralized variants of the thermolabile enterotoxin E.coH, subunit B of cholera toxin (CTS), cholera toxin, neutralized variants of cholera toxin, E. coli Sat protein, E. coli Sat protein passenger domain, Streptococcus pneumoniae pneumolysin, Megathura crenulata hemocyanin (KLH), P. aeruginosa PcrV, Neisseria meningitidis outer membrane protein (OMPC), and D protein from nontypeable Haemophilus influenzae.

Embodiment 40 is the method of any one of embodiments 37-39, wherein the carrier protein is detoxified Pseudomonas aeruginosa exotoxin A (EPA).

Embodiment 41 is the method of embodiment 40, wherein the EPA contains 1-10, preferably 2-4 glycosylation sites, each of which contains a glycosylation consensus sequence having SEQ ID NO: 2.

Embodiment 42 is the method of embodiment 41, wherein the carrier protein is an EPA with four consensus glycosylation sequences (EPA-4), preferably where the carrier protein comprises SEQ ID NO:

Embodiment 43 is the method of any one of embodiments 37-42, where the cell is an E.coH cell, for example, E.coH strain K12, such as strain W3110.

Embodiment 44 is the composition of embodiment 19 or embodiment 20, wherein the polysaccharide antigen 025b bioconjugate is present in the composition at a concentration that is about 1.5 to 6 times, such as about 2 to 4 times, higher than the concentration of either of the other bioconjugates.

Embodiment 45 is a composition according to embodiment 44, wherein the O1A:O2:glycosylated O4:O6A:O8:O15:O16:O25B:O75 bioconjugates are present in a polysaccharide weight ratio of 1:1:1:1:1:1:1:2: 1 or 2:1:1:2:1:1:1:4:1.

Embodiment 46 is a composition according to embodiment 44, wherein the O1A:O2:glycosylated O4:O6A:O8:O15:O16:O18A:O25B:O75 bioconjugates are present in a polysaccharide weight ratio of 1:1:1:1:1:1:1: 1:2:1 or 2:1:1:2:1:1:1:1:4:1.

Embodiment 47 is a composition according to any of embodiments 44-46, wherein the concentration of the 025B polysaccharide antigen bioconjugate is 2 to 50 μg/ml, preferably 8 to 40 μg/ml, for example 16 to 32 μg/ml.

Description of embodiments of the invention

The following examples are intended to further illustrate the invention. It should be understood that the following examples do not limit the scope of the invention and that the scope of the invention is defined by the following claims.

Example 1: Epidemiological data on E. coli infections.

Global monitoring studies were conducted to determine the distribution of E.coH O-serotypes causing bacteremia. Between 2011 and 2017, more than 3,200 E. coli isolates were collected from the blood of hospitalized patients 60 years of age or older in North America, Europe, Asia-Pacific, and South America. For each strain, the O-antigen serotype was analyzed using classical agglutination methods, and the O-antigen was genotyped by sequence. See table. 2. Isolates from human blood samples were analyzed to determine the pathogens within them and their patterns of antibiotic resistance. E.coH isolates were obtained from samples after analysis. The identification of E. coH was confirmed using MALDI-TOF MS. Further analysis of E. coH isolates was performed using an antiserum agglutination assay to determine their O-antigen serotype (DebRoy et al. (2011) Animal health research reviews/Conference of Research Workers in Animal Diseases 12, 169-185). Isolates not typeable by agglutination were further analyzed by whole-genome sequencing followed by O-antigen genotyping based on O-antigen serotype-specific wzy and wzx gene sequences.

Table 2. Distribution of the most common O-serotypes of E. coN associated with bacteremia from a collection of 3217 isolates from blood samples collected worldwide between 2011 and 2017, based on O-serotyping by agglutination and O-genotyping of isolates. not typed by agglutination. Subjects were hospitalized in the following countries: USA, Canada, Argentina, Brazil, UK, Germany, Spain, Italy, Netherlands, France, Japan, Thailand,

South Korea and Australia

O-Serotype Distribution η (%) 025 737 (22.9%) 02 268 (8.3%) About 261 (8.1%) 01 255 (7.9%)

- 35 046206

075 145 (4.5%) 015 110(3.4%) 08 104 (3.2%) 016 103 (3.2%) 04 96 (3.0%) 018 91 (2.8%)

Stratification by geographic location of bacteremia-associated E. coli globally showed a predominance of 10 major O serotypes regardless of location, suggesting that these are the predominant O serotypes associated with bacteremia-associated E. coli worldwide.

Globally, the prevalence of the 10 major O-serotypes among bacteremia-associated multidrug-resistant E.coH isolates (n=345), that is, those strains that are resistant to at least three classes of clinically relevant antimicrobials, is 75.4%. According to the totality of epidemiological analysis data, the predominant 10 Oserotypes can cover 60-80% of bacterial infections associated with E.coH, taking into account the coverage of some non-typeable strains.

A multivalent vaccine covering a significant proportion of the E. coH serotypes causing bacteremia would be very useful. Thus, O-serotypes from table. 2 may be good candidates for a multivalent O-antigen vaccine. Such a vaccine can be successfully obtained using bioconjugation technology.

One of the 10 main serotypes (Table 2) is O4. Thus, it would be useful to obtain a bioconjugate vaccine that includes the polysaccharide O-antigen of E. coli serotype O4 associated with a carrier protein.

Example 2. Characterization of modern clinical isolates of O4 by genes encoding enzymes that modify O-antigen.

Two variants of the E.coH polysaccharide antigen O4 have been described (see, for example, Jann B, et al., 1993, Carbohydr. Res. 248: 241-250), one of which has a non-branched structure (the structure is shown in Table 1 as ( O4-Glc-)), and the other variant is replaced by an additional glucose side branch (the structure is shown in Table 1 as (O4-Glc+)). The proportion of these two variants occurring among contemporary clinical isolates was unknown. Although both variants react with O4 antiserum, it was also not known whether immunological differences existed between these variants. Moreover, the enzyme responsible for the addition of the glucose side branch to form the polysaccharide antigen (O4-Glc+) has not yet been identified, and its putative coding sequence is likely located outside the rfb O4 gene cluster.

A set of 32 agglutination-confirmed clinical isolates of E. coH O4, initially isolated in the period 2011-2012. from subjects in the United States and the European Union, were analyzed by whole-genome sequencing. The extracted rfb gene cluster sequences of the 32 sequenced O4 isolates were aligned with those of the reference strain and compared at the nucleotide level. With the exception of some naturally occurring single nucleotide polymorphisms, all characterized isolates had an rfb cluster that was identical to the O4 reference strain, indicating that E. coH O4 strains, regardless of their Glc branching status, carry an identical rfb gene cluster. Thus, the creation of the E. coli O4-Glc+ polysaccharide antigen likely requires a gene of unknown sequence that encodes an E. coH O4-specific branching enzyme and which must be located somewhere outside the E. coH O4 rfb gene cluster. The sequence of this unknown gene must be identified and used if one wishes to produce bioconjugates to O4-Glc+ E. coli polysaccharide antigens in a strain that would otherwise produce only bioconjugates to O4-Glc- E. coli polysaccharide antigens. Whole-genome sequencing data were then analyzed for the presence of genes outside the rfb gene cluster that may encode O-antigen-modifying enzymes. Homologues of gtrAB in Shigella flexneri were first identified in E. coH O4. An open reading frame downstream of gtrAB in E.coH has been putatively identified as the E.coH O4-specific gtrS gene, which may encode the putative E.coH O4-specific branching enzyme GtrS responsible for adding a glucose branch to the O4 E antigen .dream.

The amino acid sequence of the GtrS enzyme specific for E.coH O4 is given as SEQ ID NO: 4. An example of a nucleotide sequence encoding this protein is given as SEQ ID NO: 5.

Of the characterized E. coH O4 isolates, approximately 80% were found to carry the gtrS gene identified here (26 of 32). The abundance of the E. coH O4-specific gtrS sequence was also determined by PCR using sequence-specific

- 36 046206 primers, in an independent set of 20 agglutination-confirmed clinical isolates of E. coli O4 isolated in the period 2014-2016. from entities in the United States and the European Union. This analysis showed that 17 of 20 isolates carried the gtrS O4 sequence, corresponding to a prevalence of 85%.

Example 3. Cloning of gtrS O4 in E. coli W3110, preparation and confirmation of the structure of Glc-modified O4 bioconjugates.

To test whether bioconjugates containing polysaccharide O4 antigen modified with branching glucose could be produced, E. coli strains with a putative branching enzyme could be generated to produce a bioconjugate of O4 antigen and EPA. To do this, the endogenous gtrS O16 gene was replaced with the putative gtrS O4 gene (SEQ ID NO: 5, see example 2), and the rfb O16 cluster in E. coli strain W3110 ΔwzzE-wecG ΔwaaL ΔwbbI-J-K was replaced with the rfb O4 cluster by homologous recombination. Alternatively, in some strains the rfb O4 cluster was encoded by a plasmid.

These strains were then introduced with plasmids encoding the carrier protein neutralized Pseudomonas aeruginosa exotoxin A (EPA) (a variant having 2 or 4 consensus glycosylation sites, designated EPA-2 and EPA-4, respectively), and the oligosaccharyltransferase PglB. Glc-modified 04-Epa bioconjugates were prepared by culturing E. coli producer strains in bioreactors and inducing the expression of PglB and EPA with IPTG and arabinose, respectively. 04-EPA bioconjugates were isolated from periplasmic biomass extract.

To confirm the detailed polysaccharide composition and binding of the 04-EPA bioconjugates, several NMR experiments were performed on bioconjugates having the EPA-4 carrier protein (data not shown). Signal assignments were consistent with those published in the literature (Jansson, P.E., et al., 1984, Carbohydr. Res. 134(2): 283-291; Jann B, et al., 1993, Carbohydr. Res. 248: 241-250) . The 1D spectrum recorded at 313K showed an intense HOD signal and low-intensity sharp signals from the pentasaccharide repeat units (RU) of O4 with five anomeric, two NAc and two H6 signals (Rha and FucNAc).

The assignment of proton signals in the one-dimensional experiment was confirmed using two-dimensional proton-proton correlation and proton-carbon correlation NMR spectroscopy. First, two-dimensional TOCSY (total correlation spectroscopy) experiments (120 ms) demonstrated the expected cross-peaks at H1 and H6 (for Rha and FucNAc) of the O4 pentasaccharide RU and small peaks from the terminal RU and EPA. In the methyl region in the TOCSY spectrum, cross peaks were observed at H6 with H1 for α-Rha and at H6 with H5 for α-FucNAc for RU O4. Other peaks observed belonged to the amino acids EPA and terminal Rha (tRha). Second, the carbon NMR spectrum contained well-dispersed and identifiable single peaks for RU O4. Carbons were identified indirectly through attached protons using a HSQC (heteronuclear single quantum correlation spectroscopy) experiment. The HSQC-DEPT experiment produced inverted peaks for CH2 groups. In the HSQC experiment, cross-peaks were observed for the pentasaccharide RU O4 [5 anomeric, ring, two N-acetyl and two methyl (Rha and FucNAc)] groups, as well as EPA amino acids in characteristic regions. It was possible to find each of the proton/carbon pairs for O4 based on the proton signal assignments and literature data.

Thus, structural characterization experiments confirmed that using the putative E. coli O4 gtrS gene identified in Example 2, O4 bioconjugates with Glc branches (containing the polysaccharide antigenic structures shown in the formula (O4-Glc+) in Table . 1).

Example 4. Immunogenicity of O4 bioconjugate with Glc branch in rabbits.

Glc-modified O4 bioconjugates (ie, having glycans with the structure of the formula (O4Glc+) as shown in Table 1) were used to immunize rabbits using the speedy-rabbit protocol (Eurogentec). Sera from immunized rabbits were analyzed by ELISA for anti-O4 IgG titers against purified O4 lipopolysaccharide (LPS) with (Glc+; i.e., containing a glycosylated O4 polysaccharide) or without the Glc branch (Glc-; i.e., containing a non-glycosylated O4 polysaccharide). As a result of immunization with the bioconjugate, high IgG titers were observed in both rabbits (Fig. 1). In both cases, antibody titers against Glc+ LPS induced by the O4 bioconjugate were higher than those against Glc-LPS.

Sera were also pooled and used in whole cell ELISA studies and test sets of E. coli O4 isolates with characterized gtrS status. Five gtrS-negative (no Glc branch) and six gtrS-positive (with Glc branch) E. coli O4 isolates and a strain serving as a negative control were tested. Pooled sera from rabbits immunized with the Glc-modified O4 bioconjugate contained high titers of IgG that specifically recognized the O4 isolates tested (Fig. 2). According to the LPS ELISA, all O4 isolates tested were recognized by the antisera. gtrS-positive isolates generally exhibited more intense binding than gtrS-negative isolates (Fig. 2). In particular, the following

- 37 046206 isolates were gtrS-positive: Y1382, E551, OS24334, stGVXN4983, stGVXN4994 and OS24794, and the following isolates were gtrS-negative: A2625, stGVXN4988, OC24784, OS24787 and OS24788. The antisera did not bind to the unrelated O serotype E. coli strain OC9487 (ATCC 35383), which served as a negative control.

Profiles of LPS isolated from the study set of gtrS-positive and -negative isolates on silver-stained polyacrylamide gels did not reveal detectable differences between isolates expressing unmodified and modified forms of the O4 antigen, confirming that the observed differences are not explained by quantitative differences in LPS expression levels (data not shown).

Western blots of extracted LPS using pooled antiserum were used to assess O-antigen recognition of O4 by immunoglobulins G produced in response to immunization with a Glc-modified O4 bioconjugate. Binding of both modified and unmodified O4 LPS by immunoglobulin Gs from rabbits immunized with modified O4 was observed and included specific recognition of LPS bands across a wide range of sizes, including high molecular weight LPS bands (Fig. 3).

Unless specifically stated otherwise, in further experiments described below, when reference is made to O4 bioconjugate or producer strains or EcoO4, it refers to the bioconjugate or O4 producing strain with Glc branches (having the glycan structure (O4-Glc+) in Table 1), (thus Thus, in these experiments, the terms O4 and O4-Glc+ are used interchangeably for bioconjugates or producing strains).

Example 5. Immunogenicity of O4 bioconjugate with Glc branch in rats.

Sprague Dawley rats were immunized 3 times intramuscularly with formulation buffer or (O4-Glc+)-EPA bioconjugate (i.e., a bioconjugate of glycosylated polysaccharide O4 antigen covalently linked to an EPA carrier protein; the carrier protein was EPA- 2, as described in Example 3 above) at 3 different doses (0.04, 0.40 or 4.0 μg). Serum antibody levels were measured by ELISA on days 0, 14, and 42 postimmunization. Immunization with 0.04 μg, 0.40 μg, and 4.00 μg of (O4-Glc+)-EPA bioconjugate caused a significant increase in IgG antibody levels at day 42 post-immunization compared to formulation buffer (Figure 4A). Antibodies induced by the (O4-Glc+) conjugate were functional, that is, capable of mediating the killing of the (O4-Glc+) E. coli strain (Fig. 4B).

Antibody levels induced by 0.04, 0.40, and 4.0 μg of (O4-Glc+)-EPA bioconjugate were significantly increased on day 42 compared to baseline (day 42 vs. day 0, P = 0.006 for all doses) and on day 14 after immunization (day 42 vs. day 14, P = 0.006 for all doses) (Fig. 5). In the 4.0 μg bioconjugate group, titers also increased significantly on day 14 compared to day 0, indicating that a single dose of 4.0 μg (O4-Glc+)-EPA bioconjugate caused a significant increase IgG titers (day 14 vs. day 0, P = 0.012). The significant increase in IgG titers observed between days 14 and 42 for all three bioconjugate concentrations tested indicated that the third dose of (O4-Glc+)-EPA bioconjugate was able to enhance humoral responses (Figure 5).

The functionality of antibodies induced by the O4-Glc+-EPA conjugate in rats immunized intramuscularly 3 times with the composition preparation buffer or bioconjugate at a dose of 4.00 μg was determined by opsonophagocytic activity (OPKA) using E. coli strains O4(Glu+) and O4( Glu-). Antibodies induced by the (O4-Glc+)-EPA bioconjugate were functional, i.e. capable of mediating the destruction of the E. coli strain (O4-Glc+) (Fig. 4B, Fig. 6). It is noteworthy that antibodies induced by the (O4-Glc+)-EPA bioconjugate were able to mediate the destruction of both (O4-Glc+) and (O4-Glc-), that is, E. coli strains having glycans with the structure of the formula (O4- Glc-) in table. 1, i.e. O4 without Glc branch) (Fig. 6).

In conclusion, antibodies induced by the O4-Glc+-EPA bioconjugate are cross-reactive and are able to mediate the killing of E. coli O4 strains with and without the glucose branch.

Example 6. Strains producing E. coli O-antigen bioconjugates and the resulting bioconjugate products.

In addition to the (O4-Glc+)-EPA bioconjugates prepared as described above, nine (9) other bioconjugates were prepared. In particular, the additionally obtained bioconjugats included Bioconjugat E.Coli O1A-EPA, Bioconjugat 02-Ryu, Bioconjugat O6A-Ra-Era, Bioconjugat 08-Rajugat 015-Rye, Bioconjugat 016-Rye, Bioconjugat O18A-ORA, O25V-ORA and BIOK bioconjugate O75EPA. The chemical structures of the glycans of these conjugates can be seen in the corresponding formulas in Table. 1. The composition containing 10 bioconjugates is referred to herein as ExPEC10V. The composition containing the bioconjugates O1A-EPA, 02-EPA, O6A-EPA and O25B-EPA is referred to herein as ExPEC4V (and was previously described, for example, in WO 2015/124769 and WO 2017/035181).

Parental strain of Escherichia coli W3110.

The non-pathogenic E. coli strain K12 W3110 was used as the parent strain to generate all ten producer strains. E. coli strain K12 W3110 was obtained from the E. coli culture museum

- 38 046206 (Yale University, New Haven (CT), USA, product number CGSC#4474). The corresponding genotype was described previously (E.coH W3110, F-, lambda-, IN(rrnD-rrnE)1, rph-1), and its genomic sequence was published previously (Hayashi K, et al., 2006, Mol. Syst Biol 2006.0007 (doi:10.1038/msb4100049) E.coH strain W3110 was genetically modified to produce each of the O-antigen-E.coI bioconjugates (Table 3).

Strains producing bioconjugates.

Compositions ExPEC4V and ExPEC10V contain bioconjugates 02-EPA and O25B-EPA from the same producing strains. The ExPEC4V composition contains an O1A-EPA bioconjugate from the production strains stGVXN4411 or stLMTB10217, while the ExPEC10V composition contains an O1A-EPA bioconjugate from the production strain stLMTB10217. The ExPEC4V composition contains the O6A-EPA bioconjugate from the production strain stGVXN4112, while the ExPEC10V composition contains the O6A-EPA bioconjugate from the production strain stLMTB10923. In addition, the ExPEC10V composition contains bioconjugates O4-EPA (i.e. (O4-Glc+)-EPA), O8-EPA, O15-EPA, O16-EPA, O18AEPA and 075-EPA from production strains that are not used for ExPEC4V . Different producing strains may vary the plasmids for expression of the EPA carrier protein and/or PglB oligosaccharyltransferase, as indicated below. An overview of several producing strains is given in Table. 3 below.

Table 3. Summary of data on genetic engineering of E.coH producing strains for O-antigen bioconjugates for vaccine compositions ExPEC4V and ExPEC10V

Serotype Strain name Gene mutations Plasmids rfb gene cluster waaL gtrABS PglB EPA O1A (ExPEC4V) stGVXN4411 Arfb::01А rfb upecGVXN_032 AwaaL - pGVXN970 pGVXN1076 O1A (ExPEC4V; ExPEC 10 V) stLMTB 10217 Arfb::01Arfb upecGVXN_032 AwaaL - pGVXN122 1 pGVXN1076 02 stGVXN4906 Arfb::02 rfb upecGVXN_116 AwaaL - pGVXN971 pGVXN1076 04 BVEC-L- 00684 Ar/b::04 rfb CCUG11450 AwaaL \gtrS::gtrS 04 pGVXN121 7 pGVXN1076 O6A (ExPEC4V) stGVXN4112 Arfb::O6A rfb CCUG11309 AwaaL - pGVXN114 pGVXN659 O6A (ExPEC 10V) stLMTB 10923 Arfb::06A rfb CCUG11309 AwaaL - pGVXN122 1 pGVXN1076 08 stLMTB 11734 Ar/&::08 r/& E2420 AwaaL AgtrABS pGVXN970 pGVXN1076 015 stLMTB 1173 8 Ar/&::015 rfb OC24891 AwaaL AgtrABS pGVXN122 1 pGVXN1076 016 stLMTB 11739 Ar/&::016 rfb OC24208 AwaaL AgtrABS pGVXN238 1 pGVXN1076 O18A BVEC-L- 00559 Ar/&::O18A rfb OC24255 AwaaL AgtrABS pGVXN970 pGVXN1076 O25B stGVXN4459 Ar#::O25B rfb upecGVXN_138 AwaaL AgtrABS pGVXN970 pGVXN1076 075 stLMTB 11737 Arfb:: 075 rfb CCUG31 AwaaL AgtrABS pGVXN121 7 pGVXN1076

O-antigen biosynthesis gene cluster (rfb).

In all O-antigen-producing E.coH strains, the natural genomic O16::IS5 (rfb) E.coli W3110 biosynthesis gene cluster was replaced with selected Oantigen-specific biosynthesis gene clusters from E.coH strains of the selected serotype, encoding serotype-specific Oantigen structures ( see Table 1 for these O-antigen structures). After genome-wide analysis of E. coH blood isolates, ten rfb donor clusters were selected or confirmed. Replacement of the rfb O16::IS5 gene cluster W3110,

- 39 046206 which is defective in the biosynthesis of the O-antigen, was achieved through a single act of homologous recombination. In the case of the rfb gene clusters O16 and 018a, the donor DNA was recombined through the flanking genes gnd and rmlCA, while the rfb gene cluster for other strains was recombined through the flanking genes gnd and galF. The sequences of the rfb clusters in the producer strains are presented in SEQ ID NOs: 9 and 11-19.

O-antigen ligase gene (waaL).

All O-antigen-producing E. coli strains carry an artificially introduced deletion of the genomic O-antigen ligase of E. coli W3110, encoded by the waaL gene. AwaaL strains disrupt the transfer of Oantigen to lipid A and instead transfer the O antigen to a carrier protein to increase product yield.

O-antigen glycosylation genes fgtrABS).

In the genomes of the producer strains E. coli O8, O15, O16, O18A, 025B and O75, the gtrABS genes of E. coli W3110, which are responsible for the glycosylation of the O-antigen O16, were deleted. While the gtrA and gtrB genes are highly homologous and interchangeable across serotypes, the gtrS gene encodes a serotype-specific O-antigen glycosyltransferase. In E. coli W3110, GtrS can transfer a glucose (Glc) residue to a GlcNAc sugar in the a-L-Rha-(1^3)-D-GlcNAc motif of E. coli O16 O-antigen. In the E. coli producer strains O1A, O2 and O6A, there is no deletion or substitution of the gtrABS gene. These O antigens lack the a-L-Rha-(1^3)-D-GlcNAc motif, which is a natural substrate for E. coli O16 gtrS. In the E. coli O4 production strain, the gtrS gene W3110 was replaced by the E. coli O4 gtrS gene to ensure proper glycosylation of the E. coli O4 O antigen.

Oligosaccharyltransferase pglB.

All E. coli O-antigen-producing strains expressed a variant of the C. jejuni glycosyltransferase PglB, which can transfer the O-antigen to a consensus amino acid sequence on a carrier protein by N-glycosylation. PglB recognizes a wide range of substrates, but due to low yield, several production strains have been developed expressing a variant of PglB having altered substrate specificity, resulting in improved yield (see, for example, WO 2016/107818, WO 2016/107819). The pglB gene was placed on a plasmid downstream of an isopropyl-β-O-thiogalactopyranoside (IPTG)-inducible promoter. In table 4 below lists the PglB variants encoded by the plasmids that were used to obtain E. coli O-antigen producing strains for the bioconjugates for the ExPEC4V and ExPEC10V compositions described above. Other plasmids with variations in the vector backbone, antibiotic resistance marker, and/or alternative PglB variants have also been successfully tested for bioconjugate production.

Table 4. PglB and EPA plasmids used in E. coli O-antigen producing strains

Name plasmids Gene Description ¹ pGVXN114 PglB Codon usage of C. jejuni., SpR pGVXN970 pglB Codon usage optimized for E. coli\SpR pGVXN971 _F //A ⁵³⁴ ° Codon usage optimized for E. coli\ natural glycosylation site PglB inactivated; SpR pGVXN1217 pglB™™ Codon usage optimized for E. coli\ PglB with substrate optimization; SpR pGVXN1221 /?g/B ^N311v,K482R ' ^D483H ' ^A669V Codon usage optimized for E. coli\ PglB with substrate optimization; SpR pGVXN2381 /?g/B ^Y77H ' ^S80R ' ^Q287P ' ^K289R ' ^N311v Codon usage optimized for E. coli\ PglB with substrate optimization; SpR pGVXN659 EPA-4 EPA with four sites bioconjugation; AmpR pGVXN1076 EPA-4 EPA with four sites bioconjugation; KanR

- 40 046206

SpR - spectinomycin resistance; AmpR - ampicillin resistance; KanR - kanamycin resistance.

Carrier protein (EPA).

All strains producing E. coli O-antigen expressed genetically neutralized P. aeruginosa ADP-ribosyltransferase (EPA) toxoid as a carrier protein for Oantigen. EPA toxin differs from wild-type EPA toxin by two residues: Leu552 is replaced by Val and Glu553 is deleted (in the catalytic domain). Deletions of Glu553 have been reported to significantly reduce toxicity. In addition to the toxicity-reversing mutation, four N-glycosylation site consensus motifs (EPA-4) were introduced. The gene was placed on a plasmid after the inducible L-arabinose (Ara) promoter (Table 4). Table 4 is limited to plasmids used in the bioconjugate producer strains used in the ExPEC4V and ExPECIOV compositions described above. Plasmids with variations in the vector backbone, antibiotic resistance marker, and/or EPA variants, for example, differing in the number of N-glycosylation site consensus motifs (eg, having two such motifs, EPA-2), have also been successfully tested for the production of bioconjugates.

Example 7. Optimization of oligosaccharyltransferase to create bioconjugates with glycosylated O4 antigen (O4-Glc+).

Bioconjugate production can be optimized by modifying the Cjejuni oligosaccharyltransferase PglB, which can result in more efficient or more pronounced N-glycosylation of the O-antigen of interest with the EPA carrier protein. In the E. coli strain, an optimization strategy was used to produce a bioconjugate with the glycosylated polysaccharide O-antigen O4 (O4-Glc+), which made it possible to obtain an optimized (O4-Glc+)-specific variant of PglB, improving the yield of the bioconjugate.

In this approach, an O4-Glc+ polysaccharide O-antigen-producing strain containing an EPA-expressing plasmid was transformed with a number of different PglB-expressing plasmids, each containing different amino acid substitutions in the PglB protein that altered substrate specificity. The level of bioconjugate production and the profile of each strain were assessed at the level of shake flasks in experiments with osmotic shock; immunological analysis of periplasmic extracts was carried out by capillary electrophoresis using O4-O1c+ specific monoclonal antibodies for detection.

One of the PglB variants tested, containing the amino acid substitution N311V, was found to significantly improve the yield of glycosylated O4 bioconjugates (Fig. 7A).

In further development, when the N311V PglB variant was further modified, the Y77H amino acid substitution further increased the O4-O1c+ specific yield and increased the level of di- and triglycosylated product compared to the N311V PglB variant, when other modifications were neutral or had a negative effect on yield product (Fig. 7B). Plasmid pLMTB4008 (SpR) encodes an E. coli codon frequency optimized substrate optimized (O4-Glc+) variant of PglB containing the Y77H and N311V mutations.

It was found that the PglB variant with optimized substrate specificity for the polysaccharide O-antigen O4-Glc+, containing amino acid substitutions N311V and Y77H compared to the wild-type C. jejuni glycosyltransferase PglB (wt), doubles the yield of the bioconjugate compared to the optimized PglB-N311V variant in the first cycle.

Similarly, using screening, we also determined the most optimal PglB variants in terms of productivity for the production of the bioconjugate of the E.coH O-antigen of other nine serotypes in the ExPECIOV composition. It was found that PglB with amino acid mutations N311V, K482R, D483H and A669V gives the highest yield of bioconjugates containing the polysaccharide antigen O1A, 06a or O15.

Wild-type PglB (i.e., without amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483, and 669) was found to give the highest yield of bioconjugates containing polysaccharide antigen O2, O8, O18A, or 025B. It was found that PglB with amino acid mutations Y77H, S80R, Q287P, K289R and N311V gives the highest yield of bioconjugates containing polysaccharide antigen 016.

It was found that PglB with the amino acid mutation N311V gives the highest yield of bioconjugates containing the O75 polysaccharide antigen. These results indicate that the optimal PglB variant varies for different O-antigens and that it is not possible to predict the optimal PglB variant to produce a bioconjugate with a given polysaccharide O-antigen.

Example 8. Bioconjugates of O-antigens from 10 serotypes of E. coli and their qualitative characteristics.

O-glycan residues of target O-antigens are structurally diverse and have different repeat units. The specificity and affinity of the glycosyltransferase PglB is related to the glycan structure. Thus, creating a bioconjugate with desired quality characteristics, such as purity, glycan/protein ratio, etc., is complex and challenging. Correct

- 41 046206 The combination of PglB and the EPA carrier protein determines the yield and may influence the efficiency of glycosylation. By optimizing PglB and carrier proteins, bioconjugates with the desired quality characteristics were obtained. It may also be important to maintain a lower threshold of total carrier protein, especially when one or more O-antigen bioconjugates are combined together and administered as part of a single formulation or vaccine, since very large amounts of carrier protein may result in immunological interference. To avoid this phenomenon, conjugates with a higher glycan/protein ratio are preferred. Consequently, bioconjugates with at least comparable (to the previously described ExPEC4V vaccine that was the subject of clinical trials) degree of glycosylation were developed for the ExPEC10V vaccine. Each bioconjugate was prepared by culturing the appropriate host cells (Example 6, Table 3) in bioreactors (10 and/or 200 L volume) and expressing the bioconjugates according to previously described methods. Each drug substance was produced batchwise by bacterial fed-batch fermentation to produce biomass containing expressed polysaccharide bioconjugates of the corresponding serotype. Cells were cultured and induced with IPTG (isopropylthiogalactoside) and arabinose. Bioconjugates were isolated from the periplasm of cells in bioreactor cultures by osmotic shock followed by chromatographic purification. This process was carried out for each of the 10 bioconjugates.

The thus obtained bioconjugates of E.coH O-antigen, which are drug substances (DS) for ExPEC10V and ExPEC4V, showed comparable key quality indicators: (1) the purity associated with the technological process (measured using RP-HPLC) was higher 95%, (2) polysaccharide/protein ratio varied from 0.1 to 0.5, mainly from 0.15 to 0.45, (3) bacterial endotoxin (European Pharmacopoeia 2.2.3) was less than 0.5 EU/ mcg of polysaccharide. The average length of individual polysaccharide chains was typically approximately 10-20 repeat units (measured using high resolution SDS-PAGE).

The structures of the repeating polysaccharide units were confirmed (by NMR and tandem mass spectrometry (MS/MS) of intact or trypsin-digested conjugates) to be as shown in the Formulas for the corresponding serotypes in Table. 1, for all ten bioconjugates that are drugs for the ExPEC10V composition described above.

Among the ten serotypes bioconjugated for the ExPEC10V formulation, serotype O18 had the lowest bioconjugate yield.

The ExPEC10V drug product (DP) contains a mixture of the ten monovalent drugs described above.

Example 9: Toxicology of ExPEC10V Vaccine.

A pilot study of the toxicity and local tolerability of a single dose of ExPEC10V (non-GLP compliant) was conducted in female NZW (New Zealand White) rabbits. One group (n=2) received an intramuscular (IM) injection (on day 0) of control (saline), and the second group (n=4) received an intramuscular injection of ExPEC10V at 105.6 μg total polysaccharide^)/dose (9.6: 9.6: 9.6: 9.6: 9.6: 9.6: 9.6: 9.6: 19.2: 9.6 mcg PS per dose, for O-serotypes O1A , O2, O4, O6A, O8, O15, O16, O18A, 025b and O75, respectively), the injected volume was 0.6 ml (176 μg PS/ml). Necropsy was performed on the 2nd day. No mortality was observed. In addition, there were no vaccine-related events recorded for clinical observation (including injection site events according to the Draize scale), body weight, food intake, or body temperature. On histological examination, no vaccine-related pathological changes were observed at the injection site or draining (ileal) lymph node. One of the four exposed animals (day 2) showed a slight increase in germinal center formation in the spleen, which was considered a normal immunological response to the vaccine administered. Overall, administration of a single IM dose of ExPEC10V to female rabbits was well tolerated.

Example 10. Immunogenicity of the mixed composition of ExPEC10V in rabbits.

The ExPEC4V vaccine (containing bioconjugates of E. coH serotypes O1A, O2, O6A and 025B) has previously been shown to be immunogenic for these four serotypes in rats, rabbits and humans (see, for example, WO 2015/124769; WO 2017/035181; Huttner et al, 2017, Lancet Infect Dis, http://dx.doi.org/10.1016/S1473-3099(17)30108-1; RW Frenck Jr., et al., abstract 5587, ASM Microbe 2018). Examples 4 and 5 above demonstrated the immunogenicity of the new bioconjugates of the invention having the glycosylated O4 E.coH serotype. The immunogenicity of bioconjugates of E. coH serotypes O8, O15, O16, O18A, and O75 (all of which in this experiment contained EPA-2 as a carrier protein) when administered separately (monovalently) to rats confirmed that each of these bioconjugates was also immunogenic, because ELISA data showed that each of these bioconjugates could produce large amounts of antibodies specific to the E. coH O-antigen (not shown). Also, the immunogenicity of a 10-valent vaccine, which contained a mixture of 10 bioconjugates described above, was studied. New Zealand White (NZW) rabbits (females 12-16 weeks old) received 3 i.v.

- 42 046206 remuscular immunizations with ExPEC10V or saline, administered at 2-week intervals (Table 5; administration on days 0, 14, and 27). The 10 polysaccharides that are part of the ExPEC10V vaccine used in these experiments were conjugated to the EPA carrier protein containing 4 glycosylation sites (EPA-4). The vaccine was formulated in 3 different doses: Group 1 (high dose): 8 μg/dose O1A, O2, O6A, O4, O8, O15, O16, O18 and O75 and 16 μg/dose 025B; Group 2 (medium dose): 4 mcg/dose O2, O4, O8, O15, O16, O18 and O75, 8 mcg/dose O1A and 06a, and 16 mcg/dose 025b; Group 3 (low dose): 0.4 mcg/dose O2, O4, O8, O15, O16, O18 and O75, 0.8 mcg/dose O1A and O6A, and 1.6 mcg/dose 025B. Animals from the control group (Group 4) received only saline solution (0.9% (w/v) sodium chloride solution) (Table 5). Antibody levels were assessed on day 0 (pre-immunization) and days 14, 27 and 42 (post-immunization). Serum antibody levels induced by each of the bioconjugates included in the vaccine and the EPA carrier protein were measured by ELISA (total IgG) using type-specific LPS as immobilizer. Antibody titers were presented as EC50 values, which correspond to the half-maximal effective concentration, calculated from titration curves constructed from 12 dilutions in duplicate using a four-parameter logistic nonlinear regression model. Functional activity was determined by OPK.

Table 5. Description of experimental groups

Experimental groups Administration (µg/PS) O1A:O2:O6A:O25B:O4:O8:O15:O16:O18A:O75 Sample size Group 1 (high dose) 8:8:8:16:8:8:8:8:8:8 7 Group 2 (medium dose) 8:4:8:16:4:4:4:4:4:4 7 Group 3 0.8:0.4:0.8:1.6:0.4:0.4:0.4:0.4:0.4:0.4 7 (low dose) Group 4 (control) 0.9% (w/v) sodium chloride solution 7

The results are shown in Fig. 8 and summarized in table. 6.

Table 6. Summary indicators of the production of antibodies specific to the E.coH O-antigen in NZW rabbits in response to ExPEC10V

Dark gray boxes indicate serotype-specific humoral responses for which p values were statistically significant. Light gray boxes indicate serotype-specific humoral responses for which p values were not statistically significant (ns). Wilcoxon rank sum test with Bonferroni correction for multiple comparisons. Comparison of animals vaccinated with ExPEC10V (groups 1, 2 and 3) with a control group receiving saline (group 4). *p < 0.05, **p < 0.01. * P values were statistically significant after excluding one animal outlier from the control group (sensitivity analysis).

High dose ExPEC10V (group 1) induced significantly higher levels of IgG antibodies at all time points tested (days 14, 27, and 42 postimmunization) compared to saline control for O1A, O2, O4, O6A, O16, O18A and 025B (Fig. 8, Table 6). Significantly higher antibody titers induced by O8 and O75 conjugates compared to saline control were observed on days 27 and 42 after immunization (Fig. 8, Table 6).

ExPEC10V at a medium dose (group 2) and a low dose (group 3) induced significantly more

- 43 046206 high levels of IgG antibodies at all time points studied (14, 27 and 42 days after immunization) compared with saline, which served as a control, for O1A, O2, O4, 06a, O16 and 025b (Fig. 8, table. 6). Significantly higher antibody titers induced by O8, O18A, and O75 conjugates compared with saline control were observed on days 27 and 42 postimmunization, suggesting that booster dosing enhanced the response to these O serotypes in rabbits (Fig. 8, table 6).

For the O15 conjugates, sensitivity analysis with one control animal outlier showed that all three doses of ExPEC10V vaccine produced a significant increase in antibody response compared to saline control on days 14, 27, and 42 postimmunization (Fig. 8, table 6).

Antibodies induced by EPA carrier protein were significantly higher than EPA antibody titers in the saline group (control) for the three ExPECIOV doses tested (high, medium, and low) at all time points studied (days 14, 27, and 42). ) (Fig. 8).

Dose-to-dose comparisons (not shown) demonstrated that at day 14 post-vaccination, the high dose of ExPEC10V induced significantly higher antibody responses compared to the low dose for the majority of conjugates tested (O1A, O2, O4, O6A, O15, O16, O18A and O25B ). The medium dose of ExPEC10V also induced significantly higher humoral responses compared to the low dose for O1A, O2, O4, O18A, 025B and O75. For the O8 conjugate, all three ExPEC10V formulations induced similar antibody levels at day 14 postvaccination.

Low dose ExPEC10V caused a significant increase in antibody response at day 42 post-vaccination (after primary and two booster doses) compared with high and medium doses of ExPEC10V for conjugates O1A, O2, O4, O16, 025B and O75.

These results are consistent with other experience with conjugate vaccines, where, for example, there was no clear relationship between dose and magnitude of antibody response to primary vaccination in infants vaccinated with pneumococcal conjugate vaccine (Poolman JT, et al. Expert Rev Vaccines. 2013, 12(12 ): 1379-94).

For conjugates O6A, O8 and O15, no significant differences were observed between the three ExPEC10V doses tested at day 42 post-vaccination. For the O18A conjugate, the high dose of ExPEC10V induced a significantly greater antibody response compared with the medium dose at 42 days post-vaccination.

For carrier protein (EPA), high and medium doses of ExPEC10V induced significantly greater antibody responses compared with low doses at day 14 postvaccination. The high dose vaccine also induced a significantly greater antibody response compared with the low dose at 42 days post-vaccination.

In conclusion, three formulations of ExPEC10V (high, medium and low dosage) administered by intramuscular injection on days 0, 14, 27 are immunogenic in rabbits.

So far, functional antibodies capable of eradicating E. coli strains induced by this vaccine in rabbits have been shown for serotypes O1A, O2, O4, O6A, O15, O16 and 025B.

In an additional experiment, a GMP batch of ExPEC10V vaccine was prepared (see Example 8 above for production) and administered to NZW rabbits as part of a toxicological study (Table 7). In this study, NZW rabbits (male and female) received 3 intramuscular injections (0.6 ml) of ExPEC10V vaccine (days 1, 15 and 29), and a control group received 0.9% (w/v) sodium chloride solution ( saline). Each vaccine dose contained 9.6 μg of polysaccharide (PS) for serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A and O75 and 19.2 μg of PS for serotypes 025B, corresponding to 105.6 μg of total PS ( 176 µg total PS/ml) and 382.8 µg total EPA (638 µg EPA/ml). IgG titers against O-antigens and carrier protein (EPA) were determined in samples collected during the pre-exposure period (day 1) and on days 31 and 50 after immunization.

A significant increase in the humoral response against all O-antigens and the EPA carrier protein was observed at 31 and 50 days after vaccination in the ExPEC10V-treated group compared with the saline-only control group (Fig. 9, Table 8). For serotype O1A, a significantly greater antibody response was also observed on day 1 (baseline) when vaccinated animals were compared with control animals. These results suggest that some animals are pre-exposed to E. coli or have antibodies that cross-react with O1A-LPS.

- 44 046206

Table 7. Experimental groups and dose of ExPEC10V used in NZW rabbits

Groups Impact Dose Days of introduction Main (31 days) (males/females) Recovery (day 50) (males/females) 1 control 0 1, 15.29 10 10 2 ExPEClOV 105.6 µg PS* 1, 15.29 10 10

*Each dose (0.6 ml administered volume) contains 9.6: 9.6: 9.6: 9.6: 9.6: 9.6: 9.6: 9.6: 19.2: 9. 6 µg polysaccharide (PS) for serotypes Θ1Ά, O2, O4, O6A, O8, O15, O16, O18A, 025b, O75, respectively (176 µg total PS/ml). Each dose contains 382.8 mcg EPA protein (638 mcg EPA/ml).

Table 8. Immunogenicity of ExPEC10V in NZW rabbits in a toxicology study.

BoMciici vis Humoral responses to 31 laziness after vak'pnnapnn

ExPEClOV O1A laziness **** laziness ****

О6А 025В **** **** **** **** **** ****

08 015 **** **** **** **** **** ****

016 018A 075 **** **** **** **** **** ****

Humoral responses induced by ExPEClOV. Light gray rectangles indicate serotypes for which a significant increase in humoral responses was observed in the vaccinated group compared to the control. Tobit model with likelihood ratio test ****p<0.0001.

Example 11: Phase 1/2a Study of ExPEC10V Vaccine in Humans.

There are currently no vaccines to prevent IED. The serotypes that make up the ExPEC10V vaccine (O1A, O2, O4, O6A, O8, Θ15, Θ16, O18A, 025B and O75) were selected to address the problem of invasive disease caused by the majority of clinically significant ExPEC strains, which also represent the majority of ExPEC isolates causing resistance. to antimicrobial IEDs, including ST131. The selected serotypes are representative of the ten predominant ExPEC O serotypes that cause circulating blood infections in the elderly population and are responsible for approximately 70% of ExPEC circulating blood infections.

Because the mode of action of conjugate vaccines is not expected to be influenced by antibiotic resistance mechanisms in the prevention of invasive disease, ExPEC10V is considered to provide protection against IEDs caused by drug-resistant and drug-susceptible serotypes O1A, O2, O4, O6A, O8 , Θ15, Θ16, O18A, 025B and O75. There is prior clinical experience with ExPEC4V, an earlier vaccine candidate that contained a set of four E. coli O-antigen conjugates (01A, O2, 06A and 025B) also present in ExPEC10V. Based on the results of four clinical studies (two completed phase 1 studies, one completed phase 2 study, and an ongoing phase 2 study), ExPEC4V was well tolerated by study participants, and at doses up to 16 μg of polysaccharide (PS) per serotype (01A, O2, 06A, and 025B) there were no safety warning signs associated with the vaccine. Most adverse events (AEs) were grade 1 and 2, with very few grade 3 AEs reported. On-demand delayed local AEs (AEs that begin after day 5 post-vaccination) were observed primarily at higher doses of ExPEC4V. In each study, the ExPEC4V vaccine was shown to be immunogenic, demonstrating a dose-dependent immune response to the vaccine and an increase in O-antigen-specific immunoglobulin G (IgG) titers as measured by enzyme-linked immunosorbent assay (ELISA) results. An opsonophagocytic activity assay (OPKA) optimized for ExPEC4V demonstrated the functional activity of the antibodies. Combined analysis of ELISA and OPKA results showed a correlation between the results of these tests (Pearson correlation coefficients >0.61 and >0.48 for day 30 and day 360, respectively, in a phase 2 clinical trial [4V-BAC2001 study]), justifying the use of ELISA as a primary measurement of antibody titers to ExPEC4V and to predict functional antibody activity. Analysis of immunogenicity data demonstrated the persistence of the immune response over three years after ExPEC4V vaccination. It has now also been shown that sera from ExPEC4V-vaccinated individuals that had high titers of serotype-specific opsonophagocytic antibodies, when passively transferred to mice that were subsequently intraperitoneally infected with E. coli serotype 025B or O2 strains, were able to provide protection in vivo (not shown) . Therefore, ExPEC4V-specific opsonophagocytic human antibodies mediate bacterial killing in vivo, which is consistent with data from other conjugate vaccines in which the proposed mechanism of protection is the induction of opsonophagocytic antibodies that mediate bacterial killing.

ExPEC10V includes a total of ten serotypes and increases coverage from approximately 50% (ExPEC4V) to approximately 70% of circulating blood infections caused by ExPEC in adults aged 60 years and older. Based on clinical experience with ExPEC4V and preclinical data for Ex

- 45 046206

PEC10V discussed in the Examples above, administration of ExPEClOV is also expected to induce immune responses to E. coH serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, 025b and O75 in humans. A randomized, blinded, first-in-human Phase 1/2a trial to evaluate the safety, reactogenicity, and immunogenicity of three different doses of ExPECIOV vaccine is being conducted in healthy people aged 60 to 85 years (Study 10V-BAC1001). The study design includes 2 cohorts: a total of 1004 participants were included in the study, 404 participants (100 participants/dose of ExPEC10V) aged 60 (inclusive) to 85 (inclusive) years in stable health in cohort 1 and an additional 600 participants aged 60 years and older in stable health with a history of UTI in the last 5 years in cohort 2.

ExPEC10V is a 10-valent vaccine candidate in development for the prevention of invasive disease caused by extraintestinal pathogenic Escherichia coli (ExPEC) (IED) in adults aged 60 years and older. ExPEC10V consists of polysaccharide (PS) O-antigens of ExPEC serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, 025B and O75, individually bioconjugated to a carrier protein, a genetically neutralized form of exotoxin A (EPA), obtained from Pseudomonas aeruginosa, and its preparation was described above. PS O4 is a glycosylated form having the structure of the formula (O4-Glc+) in Table. 1.

Tasks and endpoints.

Cohort 1 - Phase 1/2a, investigator-blinded, open-label, long-term follow-up period (N=404)

Tasks Endpoints Basic • Assess the safety and reactogenicity of different doses of ExPECIOV in participants aged >60 and <85 years • Local and systemic adverse events (AEs) received upon request, recorded within 14 days after vaccination (from day 1 to day 15) • Spontaneously reported AEs, registered from the moment of introduction study vaccine until 29 days after vaccination (from 1 day to 30 days) • Serious adverse events (AEs) registered from the moment administration of the study vaccine up to 181 days • Assess dose-dependent immunogenicity of ExPECIOV at day 15 in participants aged >60 and <85 years. • Antibody titers to ExPECIOV, measured in multiplex immunoassay based electrochemiluminescence (ECL) and multiplex analysis opsonophagocytic activity (MORA) on day 15 Minor • Assessing the correlation between serum titers obtained in a multiplex immunoassay based on ECL (total antibodies) and MOPA (functional antibodies) on day 15 • Antibody titers to ExPECIOV, measured in multiplex immunoassay based on ECL and MOPA on day 15 • Assess dose-dependent immunogenicity of ExPECIOV at 30 and 181 days in participants aged >60 and <85 years • Antibody titers to ExPECIOV, measured in multiplex immunoassay based on ECL and MOPA at 30 and 181 days

- 46 046206

• Long-term evaluation • Serious AEs associated with observation (OS) of the safety of the dose of ExPEClOV selected for further clinical development based on the primary analysis at 30 days in participants aged >60 and <85 years study vaccine or study procedures, from day 182 until the end of the study • Assessment during the DN period of the immunogenicity of the dose of ExPEClOV selected for further clinical development based on the primary analysis on day 30 • Antibody titers to ExPEClOV, measured in multiplex immunoassay based on ECL and MORA after 1 year (366 days), 2 years (731 days) and 3 years (1096 days)

Cohort 2 - double-blind period with double-blind long-term follow-up (N=600)

Tasks Endpoints Basic • Assess the safety and reactogenicity of a selected dose of ExPEClOV in participants aged >60 years with a history of UTI in the past 5 years • Local and systemic AEs received upon request, recorded within 14 days after vaccination (from 1 day to 15 days) • Spontaneously reported AEs, registered from the moment of administration of the study vaccine until the 29th day after vaccination (from 1 day to 30 days) • Serious AEs recorded from the date of administration of the study vaccine to 181 days • Assess the immunogenicity of a selected dose of ExPEClOV at day 30 in participants aged >60 years with a history of UTI in the past 5 years • Antibody titers to ExPEClOV, measured in multiplex immunoassay based on ECL and MOPA on day 30 Minor

- 47 046206

• Estimation of correlation between serum titers obtained in a multiplex immunoassay based on ECL (total antibodies) and MOPA (functional antibodies) at day 30 in participants aged >60 years with a history of UTI in the past 5 years • Antibody titers measured in wasp immunoassay on day 30 to ExPEClOV, multiplex :novs ECL and MORA • Evaluation of the immunogenicity of a selected dose of ExPEClOV on days 15 and 181 in participants aged >60 years with a history of UTI in the past 5 years • Antibody titers to ExPEClOV, measured in multiplex immunoassay based on ECL and MOPA on days 15 and 181

Tasks Endpoints • A DN assessment of the safety of a selected dose of ExPEClOV in participants aged >60 years with a history of UTI in the last 5 years • Serious AEs associated with the study vaccine or provided by the study procedures, from day 182 until the end of the study • DN assessment of the immunogenicity of a selected dose of ExPEClOV in participants aged >60 years with a history of UTI in the last 5 years • Antibody titers to ExPEClOV, measured in multiplex immunoassay based on ECL and MORA after 1 year (366 days), 2 years (731 days) and 3 years (1096 days) Search engines • Assessment of the effect of ExPEClOV on intestinal microbial (stool) based on the results of metagenomic analysis • Metagenomic analysis of stool samples from a selected subset of participants to assess the impact of ExPEClOV on: - Pathogen prevalence (eg Clostridium difficile') in intestinal flora - Seroprevalence ExPEClOV in the intestinal flora

General design of the study

This is a randomized, multicentre, interventional study including two cohorts.

For Cohort 1, the study is using the active drug as a control, with investigator blinding, and includes a total of 404 adult participants aged 60 years and older and up to and including 85 years of age in stable health with or without a history of UTI. The study design for Cohort 1 includes three periods: a maximum 28-day screening period, a 181-day follow-up period with investigator blinding with vaccination on day 1, and an open-label period that lasts from day 182 to 3 years ( 1096 days) after vaccination (Fig. 10A). Only participants in the selected dose group of ExPEC10V (approximately 100 participants) and participants in the Prevnar 13 group enter the DN period. The end of the study for Cohort 1 is the participant's final visit after 3 years (day 1096). For Cohort 2, the study is conducted using placebo as a control, with investigator and participant blinding, and includes a total of 600 adult participants aged 60 years or older in stable health with a history of UTI in the past 5 years. Enrollment begins after completion of the primary phase 1/2a analysis and dose selection of ExPEC10V in Cohort 1. The study design for Cohort 2 includes three periods: a maximum 28-day screening period, a double-blind 181-day follow-up period with vaccination on day 1, and double-blind DN period, which lasts from day 182 to 3 years (day 1096) after vaccination (Fig. 10B). All participants in Cohort 2 enter the DV period. The end of the study is the last visit of the Cohort 2 participant after 3 years (day 1096).

Cohort 1. Phase 1.

In Phase 1, a total of 84 participants from Cohort 1 were treated with a staged approach with stepwise dose escalation, assessing safety before moving from one phase to the next. An internal data review committee (DRC) was commissioned to conduct this study to review the data.

- 48 046206 physical examination data (baseline and target), baseline demographic data and safety data on day 14 after vaccination (including local and systemic AEs obtained upon request, spontaneously reported AEs, serious AEs, clinical laboratory data and vital signs ) of these 84 Phase 1 participants. At this stage of the study, participants were recruited and randomized in six stages:

Stage 1. The first four participants were selected and randomized; two participants in the low dose ExPEC10V group (Table 11) and one participant each in the ExPEC4V and Prevnar 13 groups.

Stage 2: 24 participants were recruited and randomized; There were 18 participants in the low dose ExPEC10V group (Table 11) and three participants each in the ExPEC4V and Prevnar 13 groups.

Stage 3. The first four participants were selected and randomized; two participants in the medium dose group ExPEC10V (Table 11) and one participant each in the ExPEC4V and Prevnar 13 groups.

Stage 4: 24 participants were recruited and randomized; There were 18 participants in the medium dose ExPEC10V group (Table 11) and three participants each in the ExPEC4V and Prevnar 13 groups.

Stage 5. The first four participants were selected and randomized; two participants in the high dose ExPEC 10V group (Table 11) and one participant each in the ExPEC4V and Prevnar 13 groups.

Stage 6: 24 participants were recruited and randomized; There were 18 participants in the high dose ExPEC10V group (Table 11) and three participants each in the ExPEC4V and Prevnar 13 groups.

On Day 1, all participants received a single intramuscular (IM) injection of either ExPEC10V (1 of 3 doses), ExPEC4V, or Prevnar 13 according to the vaccination group in which they were assigned to the study. The first four participants in each of phases 1, 3, and 5 were contacted by telephone 24 hours after vaccination to collect safety information. Masked safety data at 24 hours post-vaccination for the first 4 participants in each arm were reviewed by the principal investigator (PI), physician in charge (SRP), and sponsor's medical lead (SML). Randomization of additional participants into the next phase was suspended until safety assessments were completed on day 2 in the first 4 participants.

In the absence of any clinically significant abnormalities, an additional 24 participants were enrolled (for phases 2, 4, and 6) and randomized to one of three study vaccine groups (Table 11) to receive one IM injection or ExPEC10V (1 of 3 doses). , ExPEC4V or Prevnar 13 in 1 day.

After vaccinating an additional 24 participants at each dose level (low dose in Stage 2, medium dose in Stage 4, and high dose in Stage 6), the DRC analyzed safety data at day 14 post-vaccination for all 28 (4+24) participants for each dose levels before moving to the next dose level or Phase 2a.

Cohort 1. Phase 2a.

Based on acceptable safety and reactogenicity (in the absence of any safety concerns or any events meeting specific study stop rules) established by the DRC after reviewing safety data at day 14 post-vaccination for the first 84 participants, the remaining 320 participants were randomized from Cohort 1 was enrolled and administered as part of a phase 2a study. These additional 320 participants were enrolled in the study and randomized in parallel in a 2:2:2:1:1 ratio to one of five study vaccine groups to receive on day 1 a single intramuscular injection of either ExPEC10V (1 of 3 doses), ExPEC4V or Prevnar 13 (Table 11). In addition to analyzing day 14 safety in the first 84 participants, the DRC is also assessing safety data in Cohort 1 of the study and considering any events that meet the specific study vaccination hold rule or any other safety concerns that may arise.

For Cohort 1, the primary analysis is performed when all participants have completed the day 30 visit (visit 4) or have left the study early. The final analysis occurs when all participants have completed the day 181 visit or have discontinued study participation earlier. For participants entering the open-label long-term follow-up (LOF) period (ExPEC10V dose-selected group and Prevnar 13 group), annual follow-up analyzes include safety and immunogenicity data (ECL-based multiplex immunoassay and MOPA) at the 1-year visit (366 day), 2 years (731 days) and 3 years (1096 days) after vaccination.

Cohort 2.

In Cohort 2, the safety, reactogenicity and immunogenicity of the selected dose of ExPEC10V (based on the results of the primary analysis of Cohort 1) were assessed in participants aged 60 years or older with stable health status and a history of UTI in the last 5 years. For Cohort 2, the study is using placebo in as a control, with investigator and participant blinding, and included a total of 600 participants randomized in parallel in a 2:1 ratio (400 participants in the ExPEC10V group and 200 in the placebo group).

On Day 1, all participants received a single IM injection of either the selected dose of ExPECIOV or placebo according to the vaccination group in which they were assigned to the study (Table 12).

For Cohort 2, the primary analysis includes safety and immunogenicity data and conducts

- 49 046206 when all participants completed the visit on day 30 (visit 4) or discontinued participation in the study earlier. The final analysis occurs when all participants have completed the day 181 visit or have discontinued study participation earlier. For participants, annual follow-up analyzes include safety and immunogenicity data (ECL and MOPA multiplex immunoassay) at visits 1 year (366 days), 2 years (731 days), and 3 years (1096 days) after vaccination.

A study of stool samples is conducted in a selected subset of participants to assess the impact of ExPEC10V on the prevalence of pathogens (eg, Clostridium difficile) and ExPEC10V serotypes in the intestinal flora using metagenomic analysis.

Number of participants.

A total of 1004 participants took part in the study; 404 participants in cohort 1 and 600 participants in cohort 2.

Impact groups.

Description of the impact.

ExPEC10V. Bioconjugate vaccine E.coH in a phosphate buffer solution containing PS O-antigen of serotypes ExPEC O1A, O2, O4, O6A, O8, O15, O16, 018a, 025b and O75, bioconjugated separately with the EPA carrier protein.

Single IM (deltoid) injection of 0.5 ml of one of three doses of ExPEC10V on day 1.

ExPEC4V. Bioconjugate vaccine E.coH in a physiological buffer solution containing PS O-antigen of serotypes ExPEC O1A, O2, O6A, 025B (4:4:4:8 μg PS/ExPEC serotypes), bioconjugated separately with the EPA carrier protein.

Single intramuscular (deltoid) injection of 0.5 ml ExPEC4V on day 1.

Prevnar 13. Sterile suspension of saccharides of capsular antigens of Streptococcuspneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, separately associated with the non-toxic diphtheria protein CRM197. A single 0.5 ml IM (deltoid) injection supplied in a pre-filled syringe containing a single dose.

Placebo: saline solution. Single intramuscular (deltoid) injection of 0.5 ml placebo on day 1.

The materials of the ExPEC study are described in Table. 9.

Table 9. Study vaccines BAC1001MV (ExPEC4V)

Impact group Viya O1A (mcg) 02 (mcg) 04 (mcg) O6A (mcg) 08 (mcg) 015 (mcg) 016 (mcg) O18A (mcg) О25В (mcg) 075 (mcg) EPA (mcg) PS (sum -rno) (mcg) Low dose ExPEClOV 4 4 4 4 4 4 4 4 8 4 160 44 Average dose of ExPEClOV 8 4 4 8 4 4 4 4 16 4 221 60 High dose ExPEClOV 8 8 8 8 8 8 8 8 16 8 320 88 ExPEC4V 4 4 - 4 - - - - 8 - 72 20

EPA is a genetically neutralized form of exotoxin A obtained from Pseudomonas aeruginosa; PSpolysaccharide;

ExPEC4V consists of polysaccharide (PS) O-antigens of the ExPEC serotypes O1A, O2, O6A and 025B, individually bioconjugated to the EPA carrier protein;

ExPEC10V consists of polysaccharide (PS) O-antigens of the ExPEC serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, 025B and O75, individually bioconjugated to the EPA carrier protein. The dose is determined only by PS. EPA (µg) - measured values;

ExPEC10V consists of 10 monovalent drug substances (DS). For this clinical study, 2 different concentrations (medium and high) of drug product (DP) are produced (Table 10). The third (low) concentration is prepared in the clinic by diluting the high concentration 1:1 with a dilution buffer that is the same as the formulation buffer. Each DP is prepared in sodium potassium phosphate buffer pH 7.0 (0.02% [wt/wt] Polysorbate 80, 5% [wt/wt] sorbitol, 10 mM methionine).

- 50 046206

Table 10. ExPEClOV Vaccine Composition for Phase 1/2a Clinical Study

Ingredient Amount (µg/ml) ³ Active ³ Low ^{concentration} Medium concentration High concentration Polysaccharide ABOUT- antigen EcoOlA 8 16 16 EcoO2 8 8 16 EcoO4 8 8 16 EsoOBA 8 16 16 EsoO8 8 8 16 EsoO15 8 8 16 EsoO16 8 8 16 Eso018A 8 8 16 EsoO25V 16 32 32 EsoO75 8 8 16 Carrier protein EPA 320 441 640 Excipients KN ₂ PO ₄ Na ₂ HPO ₄ Sorbitol Methionine Polysorbate 80 6.19 mM 3.81 mM 5% (w/w) 10 mM 0.02% (w/w)

EPA - genetically neutralized exotoxin A of P. aeruginosa, used as a protein carrier;

and the active ingredient is a biologically synthesized conjugate consisting of a PS antigen and a carrier protein (EPA); the dose is calculated only according to the PS group;

^b low concentration is prepared in the clinic by diluting the high concentration 1:1 with dilution buffer.

Security assessment

Key safety assessments include on-demand local and systemic AEs, spontaneously reported AEs, serious AEs, physical examination, vital signs, and clinical laboratory tests.

Immunogenicity assessment

Key assessments of the immunogenicity of collected sera include assessment of the levels of total IgG antibodies specific for vaccine serotypes ExPEC10V and ExPEC4V, as measured by an ECL-based multiplex immunoassay, and functional antibodies specific for ExPEC10V and ExPEC4V serotypes, as measured by an opsonophagocytic activity assay. (OPKA) in multiplex format (MORA). The immunogenicity of pneumococcal antibody titers induced by Prevnar 13 has not been assessed.

Levels of serum antibodies induced by ExPEC10V are measured using a multiplex electrochemiluminescence (ECL) immunoassay. This assay is performed in 96-well microplates, where each well contains high-binding carbon electrodes, on the surface of which various E. coli O-LPS antigens or EPA protein carrier are immobilized in the form of separate spots. Levels of antigen-specific antibodies present in serum samples are determined using a secondary antibody (anti-human IgG) labeled with SULFO-TAG. When electrically stimulated, SULFO-TAG emits light, the intensity of which increases in proportion to the amount of bound IgG antibodies. This analysis meets the requirements specified in the recommendations of the International Conference on Harmonization (ICH).

Levels of functional antibodies induced by ExPEC10V are measured using a multiplex opsonophagocytic activity assay (MOPA). Briefly, a series of dilutions of heat-inactivated serum samples are prepared and incubated with different strains of E. coH that are specifically resistant to different types of antibiotics. After this, human complement and phagocytic cells (HL60) are added to the reaction and after a second period of incubation, an aliquot of the reaction mixture is transferred to various filter plates with a hydrophilic PVDF membrane containing

- 51 046206 a growing medium to which a certain antibiotic has been added, which selectively promotes the growth of a strain resistant to this antibiotic. After growing overnight, colony-forming units (CFUs) are counted to determine the number of surviving bacteria. This assay meets the requirements specified in the ICH guidelines.

For antibodies to ExPEC10V serotypes measured by the ECL- and MOPA-based multiplex immunoassay, and to EPA measured only by the ECL-based multiplex immunoassay, the following immunogenicity scores were assessed and tabulated by vaccination group of interest for all time points, at which determine immunogenicity:

proportion of participants with >2-fold and >4-fold increase in serum antibody titers from day 1 (before vaccination);

geometric mean titer (GMT);

GMR: fold change from baseline, calculated from the ratio of post-baseline values to baseline values.

For the DN period, a summary descriptive analysis of immunogenicity is presented for each serotype.

Dose selection for later phases takes into account the totality of data available at the time of the initial analysis of Cohort 1 (Day 30 results).

Table 11. Cohort 1: vaccination schedule

Phase 1 Phase 2a Sum mar but Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 Stage 6 Stage 7 Issl Let's go May corpse pa vaccine tions Vac cp nation in 1st day First participants (low dose) Additional participant and (low dose) First participants (medium dose) Additional participants (medium dose) First participant and (high dose) Additional participants (high dose) Additional Phase 2a Participants G1 Low dose ExPEC 10V* 2 18 80 100 G2 Average dose ExPEC 10V* 2 18 80 100 G3 High 1st dose ExPEC 10V* 2 18 80 100 G4 ExPEC 4V** 1 3 1 3 1 3 40 52 G5 Prevna g 13*** 1 3 1 3 1 3 40 52 Total 4 24 4 24 4 24 320 404

* ExPEC10V consists of polysaccharide (PS) O-antigens of ExPEC serotypes O1A, O2, O4, 06a, O8, O15, O16, O18A, 025B and O75, individually bioconjugated to a carrier protein, a genetically neutralized form of exotoxin A (EPA) , obtained from Pseudomonas aeruginosa.

** ExPEC4V consists of polysaccharide (PS) O-antigens of the ExPEC serotypes O1A, O2, O6A and 025B, individually bioconjugated to a carrier protein, a genetically neutralized form of exotoxin A (EPA) derived from Pseudomonas aeruginosa.

*** Prevnar 13, pneumococcal 13-valent conjugate vaccine (diphtheria protein CRM197) is a sterile suspension of saccharides of capsular antigens of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, individually associated with the non-toxic diphtheria protein CRM197.

- 52 046206

Table 12. Cohort 2: vaccination schedule

Researched vaccination group Vaccination in the 1st day Total G6 ExPEC10V ^a 400 G7 Placebo 200 Total 600

* ExPEC10V consists of polysaccharide (PS) O-antigens of ExPEC serotypes O1A, O2, O4, 06 a, O8, O15, O16, O18A, 025B and O75, bioconjugated separately to a carrier protein, a genetically neutralized form of exotoxin A (EPA ), obtained from Pseudomonas aeruginosa.

Participants included in Study Cohort 2 were randomized in a 2:1 ratio (ExPEC10V:placebo). The dose of ExPEC10V used in Cohort 2 is based on the results of the primary analysis (day 30) of Cohort 1.

Status.

Recruitment and vaccination of Cohort 1 of the study described above have been completed.

The study is conducted with data masking. Based on the current analysis of safety data, no serious safety concerns were identified and ExPEC10V vaccine has an acceptable safety profile.

Immunogenicity analysis of cohort 1 clinical samples is performed with data masking. The ECL data was 100% Acceptable Quality Level (AQL) tested and loaded for data processing. Analysis of MORA samples is ongoing. Data unmasking and statistical analysis are performed with the assistance of a clinical research organization (CRO). Vaccination of Cohort 2 begins after determination of the ExPEC10V dose for that Cohort based on the final primary analysis of the 30-day results for Cohort 1.

Those skilled in the art will appreciate that the embodiments described above may be modified without changing the spirit of the invention. It is therefore understood that this invention is not limited to the specific embodiments disclosed, but also covers modifications that fall within the spirit of the present invention as set forth herein and are within the scope of the present invention.

- 53 046206

Sequences

SEQ ID NO: 1 (Glycosylation Consensus Sequence)

Asn-X-Ser(Thr), where X can be any amino acid except Pro

SEQ ID NO: 2 (optimized glycosylation consensus sequence) Asp(Glu)-X-Asn-Z-Ser(Thr), where X and Z are independently selected from any amino acid except Pro

SEQ ID NO: 3 (EPA carrier protein containing 4 consensus glycosylation sequences (EPA-4))

G SGGGDQNATG SGGGKLAEEA FDLWNECAKA CVLDLKDGVR SSRMSVDPAI ADTNGQGVLH YSMVLEGGND ALKLAIDNAL SITSDGLTIR LEGGVEPNKP VRYSYTRQAR GSWSLNWLVP IGHEKPSNIK VFIHELNAGN QLSHMSPIYT IEMGDELLAK LARDATFFVR AHESNEMQPT LAISHAG VSV VMAQAQPRRE KRWSEWASGK VLCLLDPLDG VYNYLAQQRC NLDDTWEGKI YRVLAGNPAK HDLDIKDNNN STPTVISHRL HFPEGGSLAA LTAHQACHLP LEAFTRHRQP RGWEQLEQCG YPVQRLVALY LAARLSWNQV DQVIRNALAS PGSGGDLGEA IREQPEQARL ALTLAAAESE RFVRQG TGND EAGAASADW SLTCPVAKDQ NRTKGECAGP ADSGDALLER NYPTGAEFLG DGGDVSFSTR GTQNWTVERL LQAHRQLEER GYVFVGYHGT FLEAAQSIVF GGVRARSQDL DAIWRGFYIA GDPALAYGYA QDQEPDARGR IRNGALLRVY VPRWSLPGFY RTGLTLAAPE AAGEVERLIG HPLPLRLDAI TGPEEEGGRV TILGWPLAER TWIPSAIPT DPRNVGGDLD PSSIPDKEQA ISALPDYASQ PGKPPREDLK LGSGGGDQNA T

SEQ ID NO: 4 (amino acid sequence of GtrS 04)

MNNLIMNNWCKLSIFIIAFILLWLRRPDILTNAQFWAEDSVFWYKDAYENGFLSSLTTPRNGYFQTVSTFIVGLT ALLNPDYAPFVSNFFGIMIRSVIIWFLFTERFNFLTLTTRIFLSIYFLCMPGLDEVHANITNAHWYLSLYVSMIL IARNPSSKSWRFHDIFFILLSGLSGPFIIFILAASCFKFINNCKDHISVRSFINFYLRQPYAL MIVCALIQGTSI ILTFNGTRSSAPLGFSFDVISSIISSNIFLFTFVPWDIAKAGWDNLLLSYFLSVSILSCAAFVFVKGTWRMKVFA TLPLLIIIFSMAKPQLTDSAPQLPTLINGQGSRYFVNIHIAIFSLLCVYLLECVRGKVATLFSKIYLTILLFVMG CLNFVITPLPNMNWREGATLINNAKTGDVISIQVLPPGLTLELRKK

SEQ ID NO: 5 (Example nucleotide sequence of gtrS 04)

ATGAATAATTTAATTATGAATAACTGGTGTAAATTATCTATATTTATTATTGCATTTATTTTGCTATGGCTTAGA AGGCCGGATATACTCACAAACGCACAATTTTGGGCAGAAGATTCCGTTTTCTGGTATAAGGACGCCTATGAGAAC GGATTCTTAAGTTCACTAACAACGCCTAGGAATGGGTATTTCCAGACTGTTTCTACATTTATAGTTGGTCTGACT GCTTTATTAAATCCAGATTATGCAC CTTTTGTTTCTAATTTTTTTGGCATAATGATTCGCTCAGTAATTATATGG TTTTTATTTACAGAAAGATTCAACTTCCTCACATTGACTACTAGGATTTTCTTATCTATTTATTTTCTATGCATG CCTGGATTGGATGAAGTTCATGCAAATATAACAAATGCACATTGGTATTTGTCATTATATGTATCAATGATCCTG ATAGCTCGCAATCCAAGTTCAAAATCATGGAGGTTTCATGATATATTCTTTATCT TGCTATCCGGGCTCAGTGGC CCATTTATAATTTTCATTTTAGCAGCTTCATGCTTTAAATTTATAAATAATTGTAAAGATCATATTAGTGTAAGA TCTTTCATAAATTTCTACTTGCGTCAGCCATACGCATTAATGATTGTTTGCGCTTTAATTCAAGGAACTTCTATA ATTCTAACTTTCAATGGCACACGTTCCTCAGCACCGCTAGGATTCAGTTTTGATGTGATTTCGTCTATTATATCA TCGAATATT TTTTTATTTACATTTGTCCCATGGGATATTGCAAAGGCTGGGTGGGATAATTTACTGTTATCTTAT TTTTTGTCTGTTTCGATTTTGTCGTGTGCGGCCTTTGTTTTTGTTAAAGGTACGTGGCGAATGAAAGTATTTGCA ACTTTACCATTGCTAATTATAATATTTTCAATGGCAAAACCACAATTGACAGACTCGGCACCTCAATTGCCAACA

- 54 046206

CTTATTAATGGGCAAGGTTCAAGATACTTCGTAAATATACATATTGCGATATTCTCTTTGCTATGTGTTTACTTA CTTGAGTGCGTCAGGGGGAAAGTGGCAACTTTATTTTCCAAAATATACTTAACAATTTTGCTATTCGTGATGGGA TGTTTGAATTTTGTTATCACCCCACTCCCAAACATGAACTGGAGGGAAGGTGCTACTTTGATTAATAATGCAAAA ACTGGTGATGTCATTTCGATT CAAGTGCTACCACCTGGCCTAACACTTGAACTAAGGAAAAAATAA

SEQ ID NO: 6 (Example PglB (“wild type”) sequence)

MLKKEYLKNPYLVLFAMIILAYVFSVFCRFYWVWWASEFNEYFFNNQLMIISNDGYAFAEGARDMIAGFHQPNDL SYYGSSLSALTYWLYKITPFSFESIILYMSTFLSSLWIPTILLANEYKRPLMGFVAALLASIANSYYNRTMSGY YDTDMLVIVLPMFILFFMVRMILKKDFFSLIALPLFIGIYLWWYPSSYTLNVALIGLFLI YTLIFHRKEKIFYIA VILSSLTLSNIAWFYQSAIIVILFALFALEQKRLNFMIIGILGSATLIFLILSGGVDPILYQLKFYIFRSDESAN LTQGFMYFNVNQTIQEVENVDLSEFMRRISGSEIVFLFSLFGFVWLLRKHKSMIMALPILVLGFLALKGGLRFTI YSVPVMALGFGFLLSEFKAIMVKKYSQLTSNVCIVFATILT LAPVFIHIYNYKAPTVFSQNEASLLNQLKNIANR EDYWTWWDYGYPVRYYSDVKTLVDGGKHLGKDNFFPSFALSKDEQAAANMARLSVEYTEKSFYAPQNDILKTDI LQAMMKDYNQSNVDLFLASLSKPDFKIDTPKTRDIYLYMPARMSLIFSTVASFSFINLDTGVLDKPFTFSTAYPL DVKNGEIYLSNGWLS DDFRSFKIGDNWSVNSIVEINSIKQGEYKITPIDDKAQFYIFYLKDSAIPYAQFILMD KTMFNSAYVQMFFLGNYDKNLFDLVINSRDAKVFKLKI

SEQ ID NO: 7 (example amino acid sequence of gtrA; E. coli W3110 yfdG, GenBank: VLL16209.1)

MLKLFAKYTSIGVLNTLIHWWFGVCIYVAHTNQALANFAGFWAVSFSFFANAKFTFKASTTTMRYMLYVGFMG T L SATVGWAAD RCAL P PMIT LVT FSAIS LVCGFVYS КFIVFRDAK

SEQ ID NO: 8 (example amino acid sequence of gtrB-E. coli W3110 yfdH, GenBank: BAA16210.1)

MKISLWPVFNEEEAIPIFYKTVREFEELKSYEVEIVFINDGSKDATESIINALAVSDPLWPLSFTRNFGKEPA LFAGLDHATGDAIIPIDVDLQDPIEVIPHLIEKWQAGADMVLAKRSDRSTDGRLKRKTAEWFYKLHNKISNPKIE ENVGDFRLMSRDWENIKLMPERNLFMKGILSWVGGKTDIVEYVRAERIAGDTKF NGWKLWNLALEGITSFSTFP LRIWTYIGLWASVAFIYGAWMILDTIIFGNAVRGYPSLLVSILFLGGIQMIGIGVLGEYIGRTYIETKKRPKYI IKRVKK

SEQ ID NO: 9 (example of nucleotide sequence of the rfb 04 locus - producer strain 04-EPA BVEC-L-00684f)

ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGATACCC AAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAGGGATCAAA GAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTATGAGTTAGAATCA CTCCTTGAGCAGCGCGTGA AGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCCGGGCGTGACCATTATG AACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGACCTGCCATTGGTGACAACCCA TTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCGCTACGTTACAACCTTGCTGCCATG ATTGCACGTTTCAACGAAACGGGCCGCAGCCAGG TGCTGGCAAAACGTATGCCGGGTGACCTCTCTGAATACTCC GTCATCCAGACTAAAGAGCCGCTGGACCGTGAGGGTAAAGTCAGCCGCATTGTTGAATTTATCGAAAAACCGGAT CAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGTCGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTG GAACGTACTCAGCCTGGTGCATGGGGACGTATTCAGCTGACTGATGCTATTGCCGAGCTGGCGAAA AAACAATCC

- 55 046206

GTTGATGCAATGCTGATGACCGGCGACAGTTACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAG TATGGCCTACGCAACCTGAAAGAAGGGGCGAAGTTCCGTAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAA TCTGACCGGATGTAACGGTTGATAAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAA TCTTCCTGCCGTTGTTTT ATATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAGTTTTCCA GGATTTTCCTTGTTTCCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTA ACGCTCGTCACATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGGCGGTAGCGTGCATTAATAC CTCTATTAATCAAACTGAGAGCCGCTTATTTCACAG CATGCTCTGAAGTAATATGGAATAAATTAAGTGAAAATA CTTGTTACTGGTGGCGCAGGATTTATTGGTTCAGCTGTAGTTCGTCACATTATAAATAATACGCAGGATAGTGTT GTTAATGTCGATAAATTAACGTACGCCGGAAACCGGGAATCACTTGCTGATGTTTCTGATTCTGAACGCTATGTT TTTGAACATGCGGATATTTGCGATGCACCTGCAATGGCACGGATTTTTGCTCAGCATCAGCC GGATGCAGTGATG CACCTGGCTGCTGAAAGCCATGTTGACCGTTCAATTACAGGCCCTGCGGCATTTATTGAAACCAATATTGTTGGT ACTTATGTCCTTTTGGAAGCCGCTCGCAATTACTGGTCTGCTCTTGATAGCGACAAGAAAAATAGCTTCCGTTTT CATCATATTTCTACTGACGAAGTATATGGTGATTTGCCTCATCCTGACGAGGTAAATAATACAGAAGAATTACCC TTATTTACTGA GACAACAGCTTACGCGCCAAGCAGCCCTTATTCCGCATCCAAAGCATCCAGCGATCATTTAGTC CGCGCGTGGAAACGTACCTATGGTTTACCGACCATTGTGACTAATTGCTCTAACAATTATGGTCCTTATCATTTC CCGGAAAAATTGATTCCATTGGTTATTCTCAATGCTCTGGAAGGTAAAGCATTACCTATTTATGGTAAAGGGGAT CAAATTCGCGACTGGCTGTATGTTGAAGATCATGC GCGTGCGTTATATACCGTCGTAACCGAAGGTAAAGCGGGT GAAACTTATAACATTGGTGGGCACAACGAAAAGAAAAACATAGATGTAGTGCTCACTATTTGTGATTTGCTGGAT GAGATTGTACCGAAAGAGAAATCTTATCGTGAGCAAATCACTTATGTTGCCGATCGTCCGGGACACGATCGCCGT TATGCGATTGATGCTGAGAATATTGGTCGCGAATTGGGATGGAAACCACAGGAAA CGTTTGAGAGCGGGATTCGG AAGACAGTGGAATGGTATCTGTCCAATACAAAATGGGTTGATAATGTGAAAAGTGGTGCCTATCAATCGTGGATT GAAGAGAACTATGAGGGCCGCCAGTAATGAATATCCTCCTTTTTGGCAAAACAGGGCAGGTAGGTTGGGAACTAC AGCGTGCTCTGGCACCTCTGGGTAACTTGATTGCTCTTGATGTTCATTCCACTGATTATTGTGGCGATTTCAGTA ACCCCGA AGGTGTGGCTGAAACCGTCAAAAAAATTCGCCCAGATGTTATTGTTAATGCTGCTGCTCATACCGCGG TAGATAAGGCTGAGTCAGAACCAGAATTTGCACAATTACTCAATGCGACCAGCGTTGAAGCAATTGCAAAAGCGG CTAATGAAGTTGGGGCTTGGGTAATTCATTACTCAACTGACTACGTCTTCCCTGGAAATGGCGACATGCCATGGC TCGAGACTGATGTAACCGCTCCGCTCAATGTTTA TGGCAAAACCAAATTGGCTGGAGAAAGAGCATTACAAGAAC ATTGCGCAAAGCATCTTATTTTCCGTACCAGCTGGGTATATGCAGGTAAAGGAAATAACTTTGCCAAAACAATGT TACGTCTGGCAAAAGAGCGCGAAGAACTGGCTGTGATAAACGATCAGTTTGGCGCACCACAGGTGCTGAATTGC TGGCTGATTGCACCGCTCATGCCATTCGCGTGGCATTAAAAAAACCAGAAGTTGCT GGCTTGTACCATCTGGTAG CAAATGGCACAACAACCTGGCACGATTACGCCGCGCTAGTATTCGAAGAAGCCCGTAAAGCAGGGATTGACCTTG CACTTAACAAACTCAACGCCGTACCAACAACGGCTTATCCTACTCCAGCCCGCCGTCCTCATAATTCTCGCCTCA ATACCGAAAAGTTTCAGCAGAACTTTGCGCTTGTCTTGCCTGACTGGCAGGTGGGCGTGAAACGTATGCTCAACG AATTA TTTACGACTACGGCAATTTAACAAATTTTTGCATCTCGCTCATGATGCCAGAGCGGGATGAATTAAAAGG AATGGTGAAATGAAAACGCGTAAAGGTATTATTCTGGCTGGTGGTTCCGGCACTCGTCTTTATCCTGTGACGATG GCAGTGAGTAAACAACTGCTGCCGATTTATGATAAGCCGATGATTTATTATCCGCTTTCAACGCTTATGTTAGCG GGTATTCGCGATATTCTTATTATCAGTA CGCCACAGGATACACCGCGTTTCCAACAATTGTTGGGGGACGGGAGT CAGTGGGGGCTTAATCTACAGTATAAAAGTACAACCGAGTCCGGATGGCCTGGCGCAAGCGTTTATTATTGGTGAA GACTTTATTGGTGGTGATGATTGTGCACTCGTACTTGGCGATAATATCTTCTATGGACACGACTTGCCGAAATTA ATGGAAGCTGCTGTTAACAAAGAAATCGGTGCAACGGTATTTG CTTATCACGTCAATGATCCTGAACGTTATGGT

- 56 046206

GTCGTGGAGTTTGATAATAACGGTACTGCAATTAGCCTGGAAGAAAAACCGCTGGAACCAAAAAGTAACTATGCG GTTACTGGGCTTTATTTCTATGACAATGATGTTGTAGAAATGGCGAAAAACCTTAAGCCTTCTGCCCGTGGCGAA CTGGAAATTACCGATATTAACCGTATTTATATGGAGCAGGGACGTTTGTCTGTCGCTATGATGGGGCGTGGTTAT GCCTGGTTGGATACTGGTA CACATCAAAGTCTTTATTGAAGCAAGTAACTTCATTGCCACCATTGAAGAGCGTCAG GGATTAAAGGTATCTTGCCCGGAAGAGATTGCTTACCGTAAAGGGTTTATTGATGCTGAGCAGGTGAAAGTATTA GCCGAACCGCTGAAGAAAAATGATTATGGTCAGTATCTGCTAAAAATGATTAAAGGTTATTAATAAAATGAACGT AATTAAAACTGAAATTCCTGATGTGCTGATTTTTGAACCAAA AGTTTTTGGTGATGAACGTGGCTTCTTTTTTGA GAGTTTTAACCAGAAAGTATTTGAAGAAGCTGTAGGACGGAAGGTTGAATTTGTTCAGGATAACCATTCTAAGTC TAAAATAAATGTATTGCGTGGGATGCATTATCAAACACAAAATACTCAAGGAAAACTGGTTCGGGTAATTTCTGG TTCAGTATATGATGTTGCCGTAGATTTAAGAGAAAAAATCAAAGACATTTGGCAAATGGGTGGGTGTA GAATTATC TGGGAATAATAAAAGACAATTGTGGATCCCCGAAGGTTTTGCCCATGGTTTTTATGTGTTGGAGGAGAATACCGA ATTTGTTTATAAAATGTACCGATACTTATAACCCTGCTCATGAACACACATTGCTATGGAATGATCCAACTATCAA TATAAGTTGGCCAATCATACAAAACTGCAAGCCAATTATTTCTGAAAAAGATGCTAATGGACATCTTTTTTCACA TAAAACCTATTTCTGAAATGCA ATATTATGAGTTTAATTAGAAACAGTTTCTATAATATTGCTGGTTTTGCTGTG CCGACATTAGTTGCAGTCCCTGCTTTGGGGATTCTTGCCAGGCTGCTTGGACCGGAGAATTTTGGACTTTTCACA CTAGCATTCGCTTTGATAGGATATGCAAGTATTTTTCGACGCCGGGATTAGTCGAGCTGTAATCAGAGAAATCGCT CTTTATCGAGAAAGTGAAAAAAGAGCAAATACAAATTATTTCGAC AGCAAGTGTAATCGTACTATTCTTAGGGGTG GTTGCAGCTTTGTTACTTTATTTTAGTAGTAATAAAGTTGTTGAGTTATTGAATGTTAGTTCCGTTTATATTGAA ACAGCAGTGCGTGCATTCTCTGTTATTTCATTTATAATACCTGTGTATCTGATTAACCAGATTTGGCTTGGTTAT CTGGAAGGGCTAGAAAAATTTGCAAATATAAAATGTTCAGAGAATGATTTCTAGCACAAGCTTCT ATATTACCA GTGATATTTTGTTATTACAATCCCTCGTTGCTTTATGCTATGTATGGGTTGGTGGTTGGGCGTGTGATTTCATTT TTGATTAGCGCAATAATTTGTCGAGATATTATTCTTAAAAGTAAACTTTACTTTAATGTGGCAACTTGCAATCGT CTTATCTCTTTTGGTGGATGGATAACAGTTAGTAATATCATAAGCCCAATCATGGCATATTTCGACCGCTTTATC ATCTCTCATATTATG GGGGCTTCGAGAATTGCATTTTATACAGCGCCCTCAGAGGGTGTATCAAGGTTAATTAAT ATCCCATATGCTTTGGCAAGAGCTCTATTTCCTAAATTGGCATATAGCAATAATGATGATGAACGAAAAAAATTA CAACTACAGAGCTACGCAATTATAAGCATTGTATGTCTACCCATAGTTGTTATTGGTGTCATTTTTGCCTCATTC ATAATGACAACATGGATGGGACCTGATTATGCCTTAGAAGCAGCAACTAT CATGAAAATACTTCTTGCTGGTTTT TTCTTTAACTCTTTAGCGCAAATACCTTATGCATACTTGCAATCTATCGGAAAGTCAAAAATTACCGCATTTGTG CATCTCATAGAACTTGCGCCATACTTATTATTATTGTATTACTTCACAATGCATTTCGGCATAATTGGCACGGCA ATCGCTTGGTCACTTAGAACATTTTGTGATTTTGTTATACTACTTTCGATATCGAGAAGAAAATGATTGCGGTTG ATATTGCGC TTGCAACCTACAATGGTGCTAATTTTATTCGGCAACAGATTGAATCTATCCAGAAACAAACTTATA GAAATTGGCGTCTTATAATAAGTGATGATAACTCGAGTGATGATACTGTTGATATTATTAAGGATATGATGTCTA ACGACAGTCGTATCTATTTGGTAGGAAATAAAAGACAAGGAGGGGTTATTCAGAACTTTAATTATGCTCTTTCAC AAACTACATCTGAAATTGTGTTACTATGTGAC CAGGATGACATTTGGCCGGAGGAGCGTCTGGAAAATTCTTATAG ATAAATTTAAGGCCTTGCAGCGTAATGATTTTGTTCCGGCAATGATGTTTACTGATTTGAAATTAGTAGACGAAA ATAAT T GT T T GAT T G CAGAAAGT T T T TAT C GAAC GAATAATAT TAAT C CACAAGATAAT C T GAAAAATAATAAT C TTCTCTGGCGTTCAACGGTATATGGCTGTACTTGCATCATGAATAAGAAACTTGTTGATATTGCATTGCCTATAC CTACATATGCACATATGCATGATCAATGGTTGGCATTATTAGCGAAGCAATATGGTAACATTTTTTATTTCGACT ATGCGTCTGTTCGTTATAGGCAACATTCTACAAATGTTGTTGGTGGTAGAAATAAAACGCCATTTCAAAATTTA ATTCC ATACAAAAAAACCTAAAAAGGATTAATTTGCTAGTGGATAGAACTGTTGCTTTAATTAAAATCAAATAACG

- 57 046206

ATTTCTATCCAGGGAATAAAATGGAAAATAAAATTGATTACTTAAAATTTGGAGTGAATGAAGTATTACCTTATC T T T T Τ AAAGGAAACAAGAAAGT Τ Τ Τ Τ Τ CACT Τ Τ GT GT AT Τ ΑΑΤ AGT Τ Τ GG CAT Τ ACAAAAAT GAT AT AT Τ TAT Τ AT TTTTTTTTGCACTGTTTATGATCTGTACGTTTTTAACACACAGGCGACAGGCATTATATGTTGTATCTGCGTT AGTATTTCTTTTTGGCTTTAACCTATCCATCAGGAGGGGACTGGATAGGTTATTTTCTCCATTATGACTGCAT GGTTAATGAGCAGTGTAATAATGGTTTTATAATGTTTGAACCTGGATATGAATTAATTGTTTCCTTATTTGGATA TTTGGGATTTCAGACAATTATT ATTTTTATAGCCGCTGTAAATGTAATTCTAATATTAAATTTTGCAAAGCATTT TGAAAACGGAAGTTTTGTTATTGTTGCGATAATGTGCATGTTCCTTTGGAGTGTTTATGTTGAGGCGATTAGACA GGCTCTGGCCTTATCTATAGTTATATTTGGGATTCATTCTCTTTTTTTGGGTAGAAAAAGGAAATTTATAACATT AGTATTATTTGCGTCAACTTTCCATATAACTGCTTTGATTTGTT TTCTTCTAATGACTCCCTTATTTCAAAGAA ATTAAGCAAGATAATAAGTTATAGCCTATTAATTTTCAGTAGCTTCTTTTTCGCTTTTTCTGAAACCATATTAAG TGCACTCCTTGCAATTTTGCCAGAAGGATCCATTGCCAGTGAAAAATTAAGTTTTTATAGCAACCGAGCAATA CAGGCCACAGTTATCTATTGGGAGTGGCACTATTCTTGACATTATACTTATTTTTCTGATATGTGTAAGTTTTAA ACGAATAAAGAAATATATGCTCGCTAATTATAATGCTGCAAATGAGATATTGCTTATTGGTTGCTGTCTTTATAT TTCTTTCGGTATTTTTATCGGGAAAATGATGCCAGTTATGACTCGCATTGGTTGGTATGGTTTTTCCATTTGTTAT AGTACTTCTTTATATTAACTTGGGTTATTCAGAATATTTTAAGAGGTATATAAATAAAAGAGGGTGTGGGTATAG CAAATTATTAATTGCTTTTT ATTTTTTGCTACAAATTTTGCGACCATTAACATATGATTATAGCTATTATAATAT AATGCACCAGGATACTTTGCTGAATAGGTTTGATGCATTAGATGATGCATCATTAAGACAATCAGCGAAGAGAAA ATGTTTCGATTTGGGAAAGATAGGATATGGTTTCTTATGTAGTATATAATATCCTGCATTCATTCGGATAATTTC CTATGGAAGTGTCCTTTGCTCTGTCTGTCCTCATTTGTTGAAATTTT ATGTTAATAAGAAGCTTTAGATAACCAC TTAGGAACTGTATGTTTGATCTGTCCAAAAATTATATTATTGTAAGTGCGACGGCGCTGGCTTCCGGAGGTGCAT TAACTATATTAAAGCAATTTATAAAACATGCATCACAAAATTCAAATGACTATATTATGTTTGTATCTGCGGGAT TGGAGTTGCCGGTCTGTGATAACATCATTTACATAGAAAACACACCAAAAGGATGGTTGAAAAGAATATATTGGG ATTGGTTCGGTTGTCGGAAGTTTATCTCGGAACATAAGATTAACGTTAAGAAAGTAATTTCTCTACAAAATTCCA GTTTGAATGTTCCTTACGAACAGATTATTTACTTGCACCAGCCAATTCCTTTTAGTAAAGTTGATTCTTTTTTAA AAAATAT CACAT C GATA AC GTAAAG C Τ Τ Τ Τ Τ Τ TATATAAAAAGT TTTATTCCTATTTTATATT ΤAAATAT GT GA ATGCCAATACAACCATCGTAGTGCAAACGAATTGGATGAAAAAAGGAGTGCTGGAGCAATGTGATAAAATTAGTA CCGAAAGGGTCCTTGTTATAAAACCTGATATCAAAGCATTTAATAATACTAATTTTGATGTAGATATGGATGTAT CTGCAAAAAC ACTCTTATATCCAGCGACACCACTTACCTATAAAAATCATTTGGTCATTCTGAAGGCGTTGGTTA Τ Τ Τ TAAAGAAAAAGTAT Τ Τ TATAGAT GAT S T GAAAT T S CAAGT GAC Τ Τ Τ T GAAAAGAATAG GTACAAAAAT Τ Τ T G ATAAGTTTGTGCAATTAAATAACTTAAGCAAAAACGTTGATTATCTCTC CGTTCTTTCATACTCGAACTTGCAAA AAAAATATATGGCGGCATCTTTAATCGTTTTTCCTAGCTATATCGAATCATATGGGTTACCACTCATCGAAGCTG CTAGTTTAGGAAAAAAAATCATTAGTAGTGATCTTCCTTATGCCCGGGATGTTTTAAAGGATTATAGCGGCGTAG ATTTTGTAATTTACAATAATGAAGATGGCTGGGCTAAGGCGTTGTTTAATGTTTTAAATGGCAATTCGAAGCTCA ATTTTAGGCCTTATGAAAAAGATAGTCGTTCATCTTGGCCACAGTTCTTCTCTATTTTGAAATAAGGTGTATTAT GTTTAATGGTAAAATA TTGTTAATTACTGGTGGTACGGGGTCTTTCGGTAATGCTGTTCTAAGACGTTTTCTTGA CACT GAT AT C AAAGAAAT AC GTATTTTTTCCCGG GAT GAAAAAAAC AAGAT GAC AT GAG GAAAAAAT AT ΑΑΤ AA TCCGAAACTTAAGTTCTATATAGGTGATGTTCGCGACTATTCGAGTATCCTCAATGCTTCTCGAGGTGTTGATTT TATTTATCATGCT GCAGCTCTGAAGCAAGTACCTTTCCTGCGAATTCCACCCAATGGAAGCTGTAAAAACGAATGT TTTAGGTACGGAAAACGTACTGGAAGCGGCAATAGCTAATGGAGTTAGGCGAATTGTATGTTTGAGTACAGATAA AGCTGTATATCCTATCAATGCAATGGGTATTTCCAAAGCGATGATGGAAAAAGTAATGGTAGCAAAATCGCGCAA

- 58 046206

TGTTGACTGCTCTAAAACGGTTATTTGCGGTACACGTTATGGCAATGTAATGGCATCTCGTGGTTCAGTTATCCC AT TAT T T GT C GAT CT GAT TAAAT CAGGTAGAC CAAT GAC GATAACAGAC C C TAATAT GACT C GT TT CAT GAT GAC TCTCGAAGACGCTGTTGATTTGGTTCTTTACGCATTTGAACATGGCAATAATGGTGATATTTTTGTCCAAAA GGC ACCTGCGGCTACCATCGAAACGTTGGCTATTGCACTCAAAGAATTACTTAATGTAAACCAACACCCTGTAAATAT AATCGGCACCCGACACGGGGAAAAACTGTACGAAGCGTTATTGAGCCGAGAGGAAATGATTGCAGCGGAGGATAT GGGTGATTATTATCGTGTTCCACCAGATCTCCGCGATTTGAACTATGGAAAATATGTGGAACATGGTGACCGTCG TAT CT CGGAAGT GGAAGATTATAACT CT CATAATACT GATAGGTTAGAT GT T GAGGGAAT GAAAAAATTACT GCT AAAACTTCCTTTTATCCGGGCACTTCGGTCTGGTGAAGATTATGAGTTGGATTCATAATATGAAAATTTTAGTTA CTGGCGCTGCAGGGTTTATCGGTCGAAATTTGGTATTCCGGCTTAAGGAAGCTGGATATAACGAACTCATTACGA TAGATCGTAACTCTTCTTTGGCGGATTTAGAGC AGGGACTTAAGCAGGCAGATTTTATTTTTCACCTTGCTGGGG TAAATCGTCCCGTGAAGGAGTGTGAATTTGAAGAGGGAAATAGTAATCTAACTCAACAGATTGTTGATATCCTGA AAAAAAACAATAAAAATACTCCTATCATGCTGAGTTCTTCCATCCAGGCTGAATGTGATAACGCTTATGGAAAGA GTAAAGCAGCTGCGGAAAAAATCATTCAGCAGTATGGGGAAACGACAAACGCTAAATATTATATTTA TCGCTTGC CGAATGTATTCGGTAAGTGGTGTCGACCAAATTATAACTCCTTTATAGCAACTTTCTGCCATCGCATTGCAAATG ATGAAGCTATTACAATTAATGATCCTTCAGCAGTTGTAAATCTGTGTATATAGATGACTTTTGTTCTGACATAT TAAAGCTATTAGAAGGAGCGAACGAAACTGGTTACAGGACATTTGGTCCAATTTATTCTGTTACTGTTGGTGAAG TGGCACAATTAATTTA CCGGTTTTAAAGAAAGTCGCCAAACATTAATCACCGAAGATGTAGGTAATGGATTTACAC GTGCATTGTACTCAACATGGTTAAGTTACCTGTCTCCTGAACAGTTTGCGTATACGGTTCCTTCTTATAGTGATG ACAGAGGGGTATTCTGTGAAGTATTGAAAACGAAAAACGCGGGCCAGTTTTCGTTCTTTACTGCGCATCCAGGAA TTACTCGGGGTGGTCATTATCATCATTCCAAAAATGAGAAATTTATTGTCATCCGAGGAAGTGCTTGTTTCAAAT T GAAAATAT T GT CAC GAGT GAAC GATAT GAACT TAAT GT T T C CT CT GAT GAT T T TAAAAT T GT T GAAACAGT T C CGGGATGGACGCATAACATTACTAATAATGGCTCGGATGAGCTAGTTGTTATGCTTTGGGCAAATGAAATATTTA ATCGTTCTGAACCAGATACTATAGCGAGAGTTTTATCGTGAAAAAATTGAAAGTCATGTCGGTTGTTGGGACTCG TCCAGAAATT ATTCGACTCTCGCGTGTCCTTGCAAAATTAGATGAATATTGTGACCACCTTATTGTTCATACCGG GCAAAACTACGATTATGAACTGAATGAAGTTTTTTTCAAAGATTTGGGTGTTCGCAAACCTGATTATTTTCTTAA TGCCGCAGGTAAAAATGCAGCAGAGACTATTGGACAAGTTATCATTAAAGTTGATGAGGTCCTTGAACAGGAAAA ACCAGAAGCCATGTTAGTACTTGGCGATACTAACTCC TGTATTTCAGCAATACCAGCAAAGCGTCGAAGAATTCC GATCTTCCATATGGAGGCTGGGAATCGTTGTTTTGACCAACGCGTACCGGAAGAAACTAACAGAAAAATAGTTGA TCATACCGCTGATTCAATATGACATATAGTGATATCGCGCGTGAATATCTTCTGGCTGAAGGTGTACCAGCCGA TAGAATTATTAAAACCGGTAGCCCAATGTTTGAAGTACTCACTCATTATATGCCGCAGAT TGATGGTTCCGATGT ACTTTCTCGCCTGAATTTAACACCTGGGAATTTCTTTGTGGTAAGTGCCCACAGAAGAAAATGTTGATACCCC TAAACAACTTGTGAAACTGGCGAATATACTTAATACCGTGGCTGAAAAATATGATGTCCCGGTAGTTGTTTCTAC TCATCCTCGCACTCGTAACCGCATCAACGAAAACGGTATTCAATTCCATAAAAATATCTTGCTTCTTAAGCCATT AGGATTTCA CGATTACAACCATCTGCAAAAAAATGCACGTGCTGTTTTTATCGGATAGTGGGACTATTACAGAAGA GTCCTCCATTATGAACTTCCCTGCACTCAATATACGAGAAGCGCACGAACGCCCGGAAGGCTTCGAAGAAGGGGC AGTAATGATGGTCGGTCTTGAATCTGATCGCGTTTTACAGGCATTAGAAATTATTGCAACACAGCCTCGTGGAGA AGTACGCTTACTTCGTCAGGTTAGTG ACTATAGCATGCCAAATGTTTCAGATAAAGTTCTGCGTATTATCCATTC ATATACTGACTACGTTAAACGGGTTGTCTGGAAGCAATACTAATGAAACTTGCATTAATCATTGATGATTATTTG CCCCATAGCACACGCGTTGGGGCTAAAATGTTTCATGAGTTAGGCCTTGAATTACTGAGCAGAGGCCATGATGTA ACTGTAATTACGCCTGACATCTCATTACAAGCAATTTATTCTATTAGTATGATTGAT GGTATAAAAGGTTTGGCGT

- 59 046206

TTCAAAAGTGGACCTTTTAAAGGATGTAGGTAAGGCTAAACGTGCCATAAATGAAACTCTTTTATCTTTTCGCGCA TGGCGCGCATTTAAGCACCTCATTCAACATGATACATTTGATGGTATCGTTTATTATTCCCCCTCTATTTTTTGG GGCGACTTGGTTAAAAAAATAAAACAACGATGCCAGTGCCCAAGCTATCTGATCCTAAGGGATATGTTTCCACAG TGGGTCATTGATGCAGGTATGTT GAAAGCCGGTTCACCAATTGAAAAATATTTTAGGTATTTTGAAAAAAAGTCA TATCAGCAGGCTGGCCGGATAGGGGTAATGTCTGATAAGAATCTTGAGATATTTCGCCAGACCAATAAAGGTTAT CCGTGTGAAGTTTTACGTAATTGGGCCTCAATGACTCCTGTGTCTGCCAGCGATGATTATCATTCACTTCGTCAA AAATACGATCTAAAAGATAAAGTCATTTTTTCTAGGCGGTAATA TTGGGCATGCTCAGGATATGGCAAACTTA ATGCGCCTTGCGCGTAATATGATGCGTTATCATGATGCTCATTTCCTGTTTATAGGGCAGGGTGATGAAGTTGAG CTGATAAAATCTCTTGCTGCAGAATGGAATTTAACTAATTTCACTCATCTACCTTCAGTGAACCAGGAAGAGTTT AAATTAATTTTATCTGAAGTTGATGTCGGCCTGTTCTCCCTTTCATCTCGCCATTCTTCACATAATTTCCCCGGA AAATTACTAGGGTATATGGTTCAATCAATCCCGATCCTTGGGAGTGTGAATGGCGGCAATGATTTAATGGATGTA ATTAATAAGCACAGAGCCGGTTTCATTCATGTTAATGGTGAAGATGATAAACTGTTTTGAATCTGCACAATTGCTT CTTAGTGATTCAGTTTTAAGAAAACAGCTAGGTCAGAACGCTAATGTGTTGTTAAAGTCTCAATTTTCGGTTGAA TCGGCGGCACATACTATC GAAGTCCGACTGGAGGCTGGAGAATGCGTTTAGTTGATGACAATATTCTGGATGAAC TTTTTCGCACTGCAGCAAATTCTGAACGTTTGCGCGCTCATTATTTATTGCACGCATCTCATCAGGAGAAGGTTC AACGTTTACTTATTGCATTTGTACGCGACAGCTATGTTGAACCCCATTGGCATGAGTTACCGCATCAGTGGGAAA TGTTTGTCGT CAT GCAAGGGCAAT TAGAAG T T T GT T T GTAT GAGCAAAAT G GT GAGAT C CAAAAACAGT T T GT T G TTGGAGACGGTACGGGAATAAGCGTCGTGGAATTTTCCCCAGGAGATATACATAGTGTCAAATGCCTGTCACCAA AAGCCCTTATGTTGGAGATAAAGGAGGGGCCATTTGACCCACTCAAAGCTAAGGCTTTTTCTAAGTGGTTATAGG GCGATACACCACCGTTTATTCTTCTATCTTATTCTATACATGC TGGGTTACCATCTTAGCTTCTTCAAGCCGCGC AACCCCGCGGTGACCACCCCTGACAGGAGTAGCTAGCATTTGACCACCCCTGACAGGATTAGCTAGCATATGAGC TCGAGGATTCTACTGTGGGTACCCGGGATCCGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGA ATAGGAACTTCGGAATAGGAACTAAGGAGGATATTCATAT

SEQ ID NO: 10 (example signal sequence of ERL carrier protein) MKKIWLALAG LVLAFSASA

SEQ ID NO: 11 (example of nucleotide sequence of the O1L rfb locus - O1L-ERL producer strain stGVXN4411 and stLMTB10217)

ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGATACCC AAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAGGGATCAAA GAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTATGAGTTAGAATCA CTCCTTGAGCAGCGCGTGA AGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCCGGGCGTGACCATTATG AACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGACCTGCCATTGGTGACAACCCA TTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCGCTACGTTACAACCTTGCTGCCATG ATTGCACGTTTCAACGAAACGGGCCGCAGCCAGG TGCTGGCAAAACGTATGCCGGGTGACCTCTCTGAATACTCC GTCATCCAGACTAAAGAGCCGCTGGACCGTGAGGGTAAAGTCAGCCGCATTGTTGAATTTATCGAAAAACCGGAT CAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGTCGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTG GAACGTACTCAGCCTGGTGCATGGGGACGTATTCAGCTGACTGATGCTATTGCCGAGCTGGCGAAA AAACAATCC GTTGATGCAATGCTGATGACCGGCGACAGTTACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAG TATGGCCTACGCAACCTGAAAGAAGGGGCGAAGTTCCGTAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAA

- 60 046206

TCTGACCGGATGTAACGGTTGATAAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAA

TCTTCCTGCCGTTGTTTTATATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAGTTTTCCA

GGATTTTCCTTGTTTCCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTA

ACGCTCGTCACATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGGCGGTAGCGTGCATTAATAC

CTCTATTAATCAAACTGAGAGCCGCTTATTTCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGCTAGCGTG

AAGATACTTGTTACTAGGGGCGCAGGATTTATTGGTTCTGCTGTAGTTCGTCACATTATAAATAATACGCAGGAT

AGTGTTGTTAATGTCGATAAATTAACGTACGCCGGAAACCTGGAATCACTTGCTGATGTTTCTGACTCTGAACGC

TATGTTTTTGAACATGCGGATATTTGCGATGCTGCTGCAATGGCGCGGATTTTTGCTCAGCATCAGCCGGATGCA

GTGATGCACCTGGCTGCTGAAAGCCATGTGGATCGTTCAATTACAGGCCCTGCGGCATTTATTGAAACCAATATT

GTGGTACTTATGTCCTTTTGGAAGCGGCTCGCAATTACTGGTCTGCTCTTGATGGCGACAAGAAAAATAGCTTC

CGTTTTCATCATATTTCTACTGACGAAGTCTATGGTGATTTGCCTCATCCTGACGAAGTAAATAATAAAGAACAA

TTACCCCTCTTTACTGAGACGACAGCTTACGCGCCTAGTAGTCCTTATTCCGCATCAAAAGCATCCAGCGATCAT

TTAGTCCGCGCGTGGAAACGTACCTATGGTTTACCGACTATTGTGACTAACTGTTCGAATAACTACGGTCCTTAT

CACTTTCCGGAAAAATTGATTCCACTAGTAATTCTTAATGCTCTGGAAGGTAAGGCATTACCTATTTATGGCAAA

GGGGATCAAATTCGTGACTGGCTGTATGTTGAAGATCATGCGCGTGCGTTATATACCGTAGTTACTGAAGGTCAA

GCGGGTGAAACCTATAACATTGGCGGACACAACGAAAAGAAAAAACATCGATGTTGTGCTGACTATTTGTGATTTG

TTGGACGAGATAGTCCCGAAAGAGAAATCTTATCGTGAGCAAATTACTTATGTTGCTGATCGCCCAGGGCATGAT

CGCCGTTATGCGATTGATGCTGAGAAGATTGGTCGCGAATTGGGATGGAAACCACAGGAAACGTTTGAGAGTGGG

ATTCGTAAAACGGTGGAATGGTATTTGGCTAATGCAAAATGGGTTGATAATGTGAAAAGTGGTGCCTATCAATCG

TGGATTGAACAGAACTATGAGGGCCGCCAGTAATGAATATCCTCCTTTTTGGCAAAACAGGGCAGGTAGGTTGGG

AACTACAGCGTGCTCTGGCACCTCTGGGTAATTTGATTGCTCTTGATGTTCACTCCACTGATTACTGTGGTGATT

TTAGTAACCCTGAAGGTGTGGCTGAAACAGTCAAAAGAATTCGACCTGATGTTATTGTTAATGCTGCGGCTCACA

CCGCAGTAGATAAGGCTGAGTCAGAACCCGAATTTGCACAATTACTCAATGCGACTAGCGTTGAATCAATTGCAA

AAGCGGCAAATGAAGTTGGGGCTTGGGTAATTCATTACTCAACTGACTACGTATTCCCTGGAAATGGCGACACGC

CATGGCTGGAGATGGATGCAACCGCACCGCTAAATGTTTACGGTGAAACCAAGTTAGCTGGAGAAAAAGCATTAC

AAGAGCATTGTGCGAAGCACCTAATTTTCCGTACCAGCTGGGTCTATGCAGGTAAAGGAAATAATTTCGCCAAAA

CGATGTTGCGTCTGGCAAAAGAGCGTGAAGAACTAGCCGTTATTAATGATCAGTTTGGTGCGCCAACAGGTGCTG

AACTGCTGGCTGATTGTACGGCACATGCCATTCGTGTCGCACTGAATAAACCGGATGTCGCAGGCTTGTACCATT

TGGTAGCCAGTGGTACCACAACCTGGTACGATTATGCTGCGCTGGTTTTTGAAGAGGCGCGCAATGCAGGCATTC

CTCTTGCACTCAACAAGCTCAACGCAGTACCAACAACTGCCTATCCTACACCAGCTCGTCGTCCACATAACTCTC

GCCTTAATACAGAAAAATTTCAGCAGAATTTTGCGCTTGTATTGCCTGACTGGCAGGTTGGTGTGAAACGCATGC

TCAACGAATTATTTACGACTACAGCAATTTAATAGTTTTTGCATCTTGTTCGTGATGGTGGAGCAAGATGAATTA

AAAGGAATGATGAAATGAAAACGCGTAAAGGTATTATTTTAGCGGGTGGTTCTGGTACTCGTCTTTATCCTGTGA

CTATGGTCGTCAGTAAACAGCTATTACCTATATATGATAAACCGATGATCTATTATCCGCTTTCTACACTGATGT

TAGCGGGTATTCGCGATATTCTGATTATTAGTACGCCACAGGATACTCCTCGTTTTCAACAACTGCTGGGTGACG

GTAGCCAGTGGGGCCTGAATCTTCAGTACAAAGTGCAACCGAGTCCGGATGGTCTTGCGCAGGCATTTATTATCG

GTGAAGAGTTTATTGGTGGTGATGATTGTGCTTTGGTACTTGGTGATAATATCTTCTACGGTCACGACCTGCCTA

AGTTAATGGATGCCGCTGTTAACAAAGAAAGTGGTGCAACGGTATTTGCCTATCACGTTAATGATCCTGAACGCT

ATGGTGTCGTTGAGTTTGATAAAAACGGTACGGCGATCAGCCTGGAAGAAAAACCGCTACAACCAAAAAGTAATT

ATGCGGTAACCGGGCTTTATTTTTATGATAACGACGTTGTCGAAATGGCGAAAAATCTTAAGCCTTCTGCCCGCG

- 61 046206

GTGAACTGGAAATTACCGATATTAACCGTATCTATATGGAACAAGGGCGTTTATCTGTTGCCATGATGGGGCGTG GTTATGCGTGGTTAGACACGGGGACACATCAGAGCCTGATTGAGGCAAGCAACTTTATTGCAACAATTGAAGAGC GTCAGGGGCTGAAAGTTTCCTGCCCGGAAGAAATTGCTTACCGTAAAGGGTTTGTTGATGCTGAGCAGGTGAAAG TATTAGCTGAACCTCTGA AAAAAAATGCTTATGGTCAGTATCTGCTGAAAATGATTAAAGGTTATTAATAAAATG AACGTAATTAAAACAGAAATTCCTGATGTACTGATTTTTGAACCGAAAGTTTTTGGTGATGAGCGTGGTTTCTTT TTTGAGAGCTTTAACCAGAAGGTTTTTGAGGAAGCTGTAGGCCGCAAAGTTGAATTTGTTCAGGATAACCATTCG AAGTCTAGTAAAGGTGTTTTACGCGGGCTGCATTATC AGTTGGAACCTTATGCACAAGGAAAATTGGTGCGTTGC GTTGTCGGTGAAGTTTTTGACGTAGCTGTTGATATTCGTAAATCGTCATCGACTTTTGGCAAATGGGTTGGGGTG AATTTATCTGCTGAGAATAAGCGGCAATTGTGGATTCCTGAGGGATTTGCACATGGTTTTTTAGTGCTGAGTGAG ACGGCGGAGTTTTTGTATAAGACGACAAATTTATTATCATCCTCAGAGTGATAGAGGA ATAAAATGGGATGATCCA AGCATCAATATTTCATGGCCAGTCGATTCACAAGTGCTGCTATCAGCTAAAGATAATAAGCATCCTCCATTAACA AAGATTGAAATGTATAGTTAAGATCACGATAAATCTTGGAAGGGTTGCAAAATTGAATAAAATAGTGAGCAAAAG TGAAATAAGGAACGTAATCCACAATGCTGGCTATATGATGATTACTCAGATAGCTTTATATGTTGCACCATTATT TATACTGAGTT ATCTGTTAAAAACACTGGGGGTTGCACAGTTTGGTAATTATGCCTTAATACTATCAATCGTTGC ATATTTACAGATTATAACGGATTATGGTTTTTCTTTTAGTGCAAGTCGTGCGATCTCACAGAATAGAGAGGACAA AGAATATATATCAAAATTTATCTGTCAACTATGACTATCAAGTTGGCGATATGCGCTTTCTTATTCTTATTGCT CATGCTATTTTTAAATCTTTTGCCTGTGCAAGCT GAATTAAAACAAGGAATATTATATGGATATCTTCTTGTAAT AGGAAATACTTTCCAACCACAATGGTTTTTCCAAGGTATCGAAAAATTAAAAATCATAGCCCTTTCTAATGTTAT ATCAAGATGCGCCGCGTGTTTACTTGTATTTATCTATGTGAGGAATAGCGAGGATTTACAAAAGCACTTTTAGT ACAGTCACTTCCATTAGTAATTTCTGCGATTGGATTAAATATATTTATTGAAATATATCAATA TTATTTTTCC GGAAAAAAAATTATTTAAGGTAATTTTAAAAGAAGGTAAGGATTTTTTTCTTGCATCACTTTATTCTGTTATTCT CAATAATAGTGGCATTTTTCTATTAGGGATTTTTACTAATCCTGTTATTGTTGGTGTATATGCCGCCGCTGAAAA GATAGTCAAGGCCGTATTGTCGCTATTTACACCACTGACGCAAGCTATATATCCTTATAATTGTCGTAAGTTTTTC ACTATCCGTATT TGACGGCATTGAGGCAGCAAAAAAAACTGGTATACCAATTATAATTTTAGCATTTATAGCTGC TGTTATCGTTGCAATTACCTTACCTGTTGCAATCGACTATCTTAATTTTCCAAAAGAAACAATTTTTGTAGGTCA AATATTAAGTGCATGGATCTTTTTTGGTGTTCTTAATAATGTATTCGGCATTCAGATATTGAGTGCATCAGGAAG AAGTAAAATATATAGTAGGATGGTATTCGTATCAGCG CTTATAACATTACTTTTGATTACTCTATTATTGCAGTT TTGTAACGCCACTGGAGTGGCATGTGCAATATTATTGGGTGAAATGTTCTTATCAATATTGTTACTTAAGCGATA TAAAAAAATAATTTAAGGAATAGTTATGAAGAAGTTATTATTAGTGTTCGGTACTAGGCCTGAAGCAATAAAGAT GGCCTCTATCATTGAATTATTAAAAAAAGATTGTAGATTCGAATATAAAATATGTGTGACAGG CCAACATAAAGA GATGCTTGATCAAGTTATGCAAGTATTTGATGTTAAACCTGATTATAATTTACGGATTATGCAGCCTGGGCAAAC ATTAGTATCTATAGCAACAAATATACTCTCACGGTTAAGTGAAGTTTTAATTATAGAAAAGCCAGATATTATACT TGTGCATGGGGATACAACGACTACCCTTGCTGCTACTTTAGCTGGGTATTACCACCAAATAAAAGTTTGTCATGT GGAAGCAGG ATTAAGAACAGGGGATATTACTCTCCTTGGCCTGAAGAGGGCAATCGTAAAGTTACAGGGGCATT AGCATGTATTCATTTCGCCCCAACAGAGAGATCAAAAGATAATCTCCTGAGGGAGGGGGTCAAAGTAAATAATAT ATTTGTAACGGGTAATACCGTCATCGACTCTTTATTTATTGCAAAAGATATCATAGATAATGACCCTAATATAAAA GAACGCTTTACATAATAAATTTAATTTTCTTGATA AAAGCCGACGAGTAGTACTTATAACAGGTCATCGAAGAGA AAATTTCGGGAAAGGTTTTGAAGATATATGCTTTGCAATAAAGGAATTAGCTTTCATTTATCCTAATGTAGATTT TAT T TAT C C GGT GCAT CT TAAT C C CAAT GTAAT GGAAC CAGTACAT C GTATAT TAGATAATATAT GTAATAT T TA CCTTATTGAGCCCTTGGATTATTTGCCTTTTGTTTATTTAATGAATGAGTCATATTTAATATTGACTGATTCAGG

- 62 046206

GGGGATACAAGAAGAAGCGCCTTCGTTAGGTAAACCGGTTTTGGTTATGCGTGATACTACTGAACGCCCTGAGGC GGTTGAGGCTGGTACTGTTGTATTAGTGGGGACTTCTAAGATAAAAATAGTAAATAAAGTAACGGAGCTATTAAA CAATGCTGATATCTACAATGCTATGTCTCTGTTACATAATCCATATGGCGATGGAACAGCTGCTCAAAAAATTCT TAATGTGCTCGCCCAAGAGCTAA TTTAATTTAAGCTAAAAATATGTTATTAATTATTGCTGATTATCCAAACGAA ATGAATATGCGCGAGGGAGCTATGCAACGAATAGATGCGATAGACTCTCTCATTCGAGATCGCAAGCGAGTGTAT TTGAATATTTCATTCAAAAAGCATCTAGTTCGCTCAAATAGTTCCTTTAATAATGTTATAGTTGAAAATCTAAAT GCAAT TAT T CACAGAAACAT CATAAAACAGTACAT GCAAAAAT CAACA ACTATATAT GT T CAT T CT GT T TATAAT TTATTAAAGGTTATAACGCTCATTGATCTAAAAAAAACAATTCTTGATATACATGGTGTTGTACCGGAAGAACTT TTGGCAGATAATAAAAAATTACTTAGTAAAGTATATAACATGGTGGAAAAAAAGGTGTCCTTGGATGCAAAAAA TTAATACACGT CAGTACAGAAAT GCAAAAACACTAT GAAGCAAAATAT GGAGTAAACTT GGCT GAAA GGT CAATA GTGCTCCCGATTTTTGAATATAAAAATATAACCCAATCGCAAAACAAATGGACAGAAAATAAAATACGAAGTATC TATCTTGGAGGATTACAAACATGGCAAAATATTGATAAAATGATTCAAGTTTGTGATGACACAGTGATAAACAAT GAAGCAGGTAAGTATGAATTCAACTTTTTCATCCCACAGAGTAACTTGGAAGGGTTTATAGATAAATATTCGTTA AAATTACATAATATCAATGCTAA TGCATCTACGCTATCACGTGATGAAGTAATTCCCTTTCTAAAAGAATGTCAT ATTGGTTTTGTATTGCGCGATGATATAATAGTAAACAGAGTTGCGTGCCCTACAAAATTGGTTGAATATTTAGAG TGTGGTGTCGTTCCAGTTGTGCTCTCCCCACTTATAGGTGATTTTTATTCGATGGGATATCAATACATTACTACA GAGGAAATGGCTAACAGAAGTATAAGTTTGTTGGATCTTG AAAAAATGGCTGCACATAATTTACAAATTTTGACT TCTTATCAGAAGAGAACCTACAAGGCACAGAAAGAACTTATTGCTCAACTGTGCTGAATTTTTTACATATAAAA ATTATGTAAGCATATCGCGGGTCAGGTAATTGTATGCGTATCAAATATAAAGATAACGGTTATATATTATGTTTT CTATTATGTTTCATTTTGAGCTACTTAGTTTTACTCAAATCTGACTACTTTCCTGCTGATTTTCTGCCATATA CA GAAATATACGATGGGACATACGGAGAAATCAATAATATTGAGCCTGCCTTTTTATATTTAACACGGTTGTTTCAT TATTTAAATTTCCCCTATATATTTTTTGCAATGTTAGTTTGTGCCTTATGTTTAAGTTGGAAAATAAAATATGCA AGAAAAATAAT TAAAGATAGT TATATATAT TTGTTCTT GTAT GTATAG GTAT CAT T T TAT GT GT TT T T GCAT GAA ATGACTCAATTGCGCATAGCAATTGCAGTCACTATGTGCTATGTGTCGGTTTATTATTACTTTTATAAAAATTGT ATTAAACATGCACTGCCATGGATGGTGTTGGCTATTTTGTTTCATCAGCGCCTTGCTTTTATTTATGTCATTA TTTATATACAGTTATAGGAGGTTATTAATAGTAATTATAGGGTTTGTAATATGTATGAGCTTTTTAAA CGTGTAT G CAGATACAAT T G CAC TATAT T T G WITH CAAAT GAAAAAATAGTAAAT TATTTATATAGTATTT CAT CAT CAT TAGAC AATAGAAATGATTTGGCAATATTCAACCTGAATAATATAATATTTTTATCAATATTTATTTTGATCTTTATCTT AGCCGATATAAAATTAAATGATAATGAGGCGAAGTTTATTAAGTATGTGCAATGTTCAGGAATATTAGCCTTT TGTATTTTCTTTCTGGCTAGTGGAGTCCCGGTCATTGCTTATCGAACTGCAGAGTTGCTGCGAATATTTTATCCG ATGGCTTTAGTATTAATCCTTTCGCATATAAAAAATAATAATATGCGTTATTTTATTGCAGTCATTATAGTTATC CTTTCAGGCTTAATGTTGTTTATAACACTAAGGGCTGTATCAATAGTTGGTCAAGGATTATAAAATGAATGTTGC TATTTTGTTGTCTACGTATAATG GCGAAAAATATTTAGAGGAACAACTGGATTCATTGCTGCTTCAAAGTTATCA GGAT T T T GTAGT GTATAT C C GT GAT GAC GGAT CAT CT GATAGAACT GTAAAATATAATAAAC CAATAC GTAAT GAA AGATAACAGATTTATTAACGTGGGTAATTCAGAAAATCTTGGTTGTGCTGCTTCGTTTATTAATTTATTAAGAAA TGCTTCAGCCGATATTTATATGTTTTGTGACCAAGATGATTATTGGCTTCCGAATAAATTACAGCGTGCTGTGGA TTATTTTTCGGCTATTGATCCTTTACAACCTACCTTGTATCATTGCGATCTAAGCGTTGTTGATGAAAAACTTAA TAT TATACAAAAT T CAT T T T T G CAG CAT CAGAAAAT GT CAG C GTAT GAT T CAAT GAGAAAAAATAAT C T T T T CAT ACAAAATTTTGTTGTTGGTTGTTCATGTGCTGTTAATGCTTCACTTGCGGAATTTGTTCTTTCGCGAATTGGAGA GCAGCATGTAAAAATGATAGCTATGCATGACTGGTGGTTAGCCGTGACTGCAAAACTTTTTGGTCGAATCCATTT

- 63 046206

TGATAATACTCAAACGATTCTTTATCGACAACATCAGGGCAATGTATTAGGTGCAAAATCATCAGGTATGATGCG TTTTATTCGATTAGGATTAAATGGGCAAGGGATTTCGCGAGTAGTATCTTTTAGAAAAAAAGTTTGTGCGCAAAA TAAGCTTCTTTTAGATGTCTATGATAAAGATTTAAATCTTGAGCAAAAATCTATCAGGCTTGTAATTGAGGG CCTTAAAGAGAACTCTTCAATTGCTG ACCTTTTAAAATGTTTCTATCATGGTAGCTATATGCAAGGTTTTAAACG TAATCTTGCCTTAATATATTCAGTTCTTTACACAAAAAAAAGAAGATAGTGTATCCTTATGAAAAAAATTGCTAT TATCGGTACTGTTGGCATACCAGCATATGGCGGATTTGAAACATTAGTTGAAAATTTAACAAGATACAATTC CTCGGGAGTTGAATATAATGTTTTTTGTTCATCGTTTCACTACAAATCCCACCAAAA AAAACATAATGGGGCCCG TTTAATTTATATTCCGCTTAAAGCCAATGGATGGCAGAGCATTGCGTATGACATAATTTCGTTAGCATATTCTAT _{TTTTTTGAAGCCTGATGTGATTCTGATTTTAGGGGTTTCTGGTTGTTCATTTTTGCCTTTCTTCAAACTCTTAAC} ACGCGCTAAGTTTATTACTAATATTGATGGCCTGGAATGGCGAAGAGATAAATGGAATTCAAAAGTGAAACGTTT CTTAAAATTTTCAGAAAAAATCGCAGTTCAATATTCGGATGTCGTTATTACGGATAATGAGGCAATTTCTGAGTA CGTTTTTAACGAGTATAATAAAGATAGCCGAGTTATTGCCTATGGAGGGGATCATGCATGGTTAAATACTGAGGA TGTATTTACAACAAGAAATTATAAAA GCGATTACTACCTTTCTGTATGTCGTATCGAACCCGAAAACAATGTAGA ATTAATTTTAAAAACATTTTCAAAGCTAAAATATAAAATAAAATTTATTGGAAATTGGAATGGCAGCGAGTTTGG AAAGAAACTTAGGCTGCATTATTCTAACTATCCAAATATTGAAATGATTGATCCGATTTATGATCTTCAACAATT ATTTCACTTACGAAATAATTGCATAGGATATATACATGGTCATTCGGCTGGAGGAAC AAACCCTTCTTTAGTCGA GGCAATGCATTTTAGTAAACCTATTTGCATATGATTGTAAGTTTAATAGGTACACTACTGAAAATGAAGCATG TTATTTTTCTAATGAATCTGACCTCGCAGAGAAAATCATAATGCATTGTGAGCTATCATTAGGTGTCTCTGGCAC GAAAATGAAAGAAATTGCTAACCAGAAATACACTTGGAGACGAATAGCAGAAATGTATGAGGATTGCTATTAACT CTGTTAAACTTC AAATCTTTTACAATATATGGCATGACTATAAGCGCATTAATTGTTTTTCAAGCCGCTCTCGCG GTGACCACCCCCTGACAGGGGATCCGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAA CTTCGGAATAGGAACTAAGGAGGATATTCATATGGATAAAGCCGTAAGCATATAAGCATGGATAAGCTATTTATA CTTTAATAAGTACTTTGTATACTTATTTGCGAACATTCCAGGCCGC GAGCATTCAGCGCGTGATCACACCTGAC AGGAGTATGTAATGTCCAAGCAACAGATCGGCGTAGTCGGTATGGCAGTGATGGGACGCAACCTTGCGCTCAACA TCGAAAGCCGTGGTTATACCGTCTCTATTTTCAACCGTTCCCGTGAGAAGACGGAAGAAGTGATTGCCGAAAATC CAGGCAAGAAACTGGTTCCTTACTATACGGTGAAAGAGTTTGTCGAATCTCTGGAAACGCCTCGTC GCATCCTGT TAATGGTGAAAGCAGGTGCAGGCACGGATGCTGCTATTGATTCCCTCAAACCATATCTCGATAAAGGAGACATCA TCATTGATGGTGGTAACACCTTCTTCCAGGACACTATTCGTCGTAATCGTGAGCTTTCAGCAGAGGGCTTTAACT TCATCGGTACCGGTGTTTCTGGCGGTGAAGAGGGGGCGCTGAAAGGTCCTTCTATTATGCCTGGTGGCCAGAAAG AAGCCTATG AATTGGTAGCACCGATCCTGACCAAAATCGCCGCCGTAGCTGAAGACGGTGAACCATGCGTTACCT ATATTGGTGCCGATGGCGCAGGTCACTATGTGAAGATGGTTCACAACGGTATTGAATACGGCGATATGCAGCTGA TTGCTGAAGCCTATTCTCTGCTTAAAGGTGGCCTGAACCTCACCAACGAAGAACTGGCGCAGACCTTTACCGAGT GGAATAACGGTGAACTGAGCAGTTACCT GATCGACATCACCAAAGATATCTTCACCAAAAAAGATGAAGACGGTA ACTACCTGGTTGATGTGATCCTGGATGAAGCGGCTAACAAAGGTACCGGTAAATGGACCAGCCAGAGCGCGCTGG ATCTCGGCGAACCGCTGTCGCTGATTACCGAGTCTGTGTTTGCACGTTATATCTCTTCTTGAAAGATCAGCGTG TTGCCGCATCTAAAGTTCTCTCTGGTCCGCAAGCACAGCCAGCAGGCGACAAG GCTGAGTTCATCGAAAAAGTTC GTCGTGCGCTGTATCTGGGCAAAATCGTTTCTTACGCCCAGGGCTTCTCAGCTGCGTGCTGCGTCTGAAGAGT ACAACTGGGATCTGAACTACGGCGAAATCGCGAAGATTTTCCGTGCTGGCTGCATCATCCGTGCGCAGTTCCTGC AGAAAATCACCGATGCTTATGCCGAAAATCCACAGATCGCTAACCTGTTGCTGGCTCCGTACTTCAAGCAAATT G CCGATGACTACCAGCAGGCGCTGCGTGATGTCGTTGCTTATGCAGTACAGAACGGTATTCCGGTTCCGACCTTCT

- 64 046206

CCGCAGCGGTTGCCTATTACGACAGCTACCGTGCTGCTGTTCTGCCTGCGAACCTGATCCAGGCACAGCGTGACT ATTTTGGTGCGCATACTTATAAGCGTATCGATAAAGAAGGTGTGTTCCATACCGAATGGCTGGATTAA

SEQ ID NO: 12 (example of nucleotide sequence of the rfb 02 locus - producer strain 02-EPA stGVXN4906)

ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGATACCC AAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAGGGATCAAA GAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTATGAGTTAGAATCA CTCCTTGAGCAGCGCGTGA AGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCCGGGCGTGACCATTATG AACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGACCTGCCATTGGTGACAACCCA TTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCGCTACGTTACAACCTTGCTGCCATG ATTGCACGTTTCAACGAAACGGGCCGCAGCCAGG TGCTGGCAAAACGTATGCCGGGTGACCTCTCTGAATACTCC GTCATCCAGACTAAAGAGCCGCTGGACCGTGAGGGTAAAGTCAGCCGCATTGTTGAATTTATCGAAAAACCGGAT CAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGTCGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTG GAACGTACTCAGCCTGGTGCATGGGGACGTATTCAGCTGACTGATGCTATTGCCGAGCTGGCGAAA AAACAATCC GTTGATGCAATGCTGATGACCGGCGACAGTTACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAG TATGGCCTACGCAACCTGAAAGAAGGGGCGAAGTTCCGTAAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAA TCTGACCGGATGTAACGGTTGATAAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAA TCTTCCTGCCGTT GTTTTATATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAGTTTTCCA GGATTTTCCTTGTTTCCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTA ACGCTCGTCACATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCATTAATAC CTCTATTAATCAAACTGAGAGCCGCTTATT TCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGTGAAAATA CTTGTTACTGGTGGCGCAGGATTTATTGGTTCAGCTGTAGTTCGTCACATTATAAATAATACGCAGGATAGTGTT GTTAATGTCGATAAATTAACGTACGCCGGAAACCGGGAATCACTTGCTGATGTTTCTGATCTGAACGCTATGTT TTTGAACATGCGGATATTTGCGATGCACCTGCAATGGCACGGATTTTTGCTCAGCAT CAGCCGGATGCAGTGATG CACCTGGCTGCTGAAAGCCATGTTGACCGTTCAATTACAGGCCCTGCGGCATTTATTGAAACCAATATTGTTGGT ACTTATGTCCTTTTGGAAGCCGCTCGCAATTACTGGTCTGCTCTTGATAGCGACAAGAAAAATAGCTTCCGTTTT CATCATATTTCTACTGACGAAGTCTATGGTGATTTGCCTCATCCAGATGAAGTAAATAATACAGAAGAATTACCC TTAT TTACTGAGACGACAGCTTACGCGCCAAGCAGCCCTTATTCCGCATCCAAAGCATCCAGCGATCATTTAGTC CGCGCATGGAAACGTACGTATGGTTTACCGACCATTGTGACTAATTGCTCGAACAACTATGGTCCGTATCACTTC CCGGAAAAGCTTATTCCATTGGTTATTCTTAATGCACTGGAAGGTAAGGCATTACCTATTTATGGCAAAGGGGAT CAAATTCGCGACTGGTTGTATGTAGAGGATCATGC TCGTGCGTTATATACCGTCGTAACCGAAGGTAAAGCGGGT GAAACTTATAACATTGGCGGACACAACGAAAAGAAAAACATCGATGTTGTGCTGACTATTTGTGATTTGTTGGAT GAGATTGTACCGAAAGAGAAATCTTATCGTGAGCAAATTACTTATGTTGCTGATCGCCCAGGGCATGATCGCCGT TATGCAATTGATGCCGATAAAATTAGCCGCGAATTGGGCTGGAAACCACAGGAAA CGTTTGAGAGCGGGATTCGC AAAACGGTGGAATGGTATCTGGCTAATACAAATTGGGTTGAGAATGTGAAAAGCGGTGCTTATCAGTCATGGATC GAACAAAACTATGAGGGCCGTCAGTAATGAATATCCTGCTTTTCGGCAAAACAGGGCAGGTGGGTTGGGAACTGC AGCGTGCTCTGGCGCCGCTGGGTAATCTGATCGCTCTTGATGTTCACTCCACTAATTATTGTGGAGATTTCAGCA ACCCC GAAGGTGTGGCAGAAACCGTCAAAAAAATTCGTCCTGACGTTATTGTTAATGCTGCTGCTCACACTGCAG TAGATAAAGCAGAATCAGAACCGGATTTCGCACAATTACTTAACGCGACAAGCGTCGAAGCGATTGCAAAAGCTG CTAATGAAGTCGGGGCCTGGGTTATACACTACTCTACTGATTATGTTTTCCCAGGCAGTGGTGACGCGCCATGGC

- 65 046206

TGGAAACGGATGCAACAGCACCGCTAAATGTTTACGGTGAAACAAAATTAGCTGGGGAAAAGGCATTACAAGAAC ATTGCGCAAAGCATCTTATTTTCCGTACCAGCTGGGTATACGCTGGTAAAGGAAATAACTTTGCTAAAACGATGT TGCGTTTGGCAAAAGAACGCGAAGAACTGGCTGTGATAAACGATCAGTTTGGCGCACCAACAGGTGCTGAATTGC TGGCTGATTGCACCGCTCAT GCCATTCGCGTGGCATTAAAAAAACCCAGAAGTCGCTGGCTTGTACCATCTGGTAG CAAGTGGCACAACAACCTGGCACGATTATGCTGCGCTGGTTTTTTGAAGAGGCGCGCAAAGCAGGGATTAATCTTG CACTTAACAAACTTAACGCCGTGCCAACAACGGCCTATCCCACACCAGCCCGTCGACCCCATAACTCTCGCCTCA ATACAGAAAAGTTTCAGCAGAACTTTGCGCTTGTCTTGCCTGACT GGCAGGTGGGCGTGAAACGTATGCTCAACG AATTATTTACGACTACGGCAATTTAACAAATTTTTGCATCTCGCTCATGATGCCAGAGCGGGATGAATTAAAAGG AATGGTGAAATGAAAACGCGTAAAGGTATTATTCTGGCTGGTGGTTCCGGCACTCGTCTTTATCCTGTGACGATG GCAGTGAGTAAACAATTGCTGCCGATTTATGATAAGCCGATGATTTATTATCCGCTTTCAACGCTTA TGTTAGCG GGTATTCGCGATATTCTTATTATTAGTACGCCACAGGATACACCGCGTTTCCAACAATTATTGGGGGACGGGAGC CAGTGGGGTCTTAATCTACAGTATAAAGTACAACCGAGTCCGGATGGCCTGGCGCAAGCGTTTATTATTGGCGAA GACTTTATTGGTGGTGATGATTGTGCACTCGTACTTGGCGATAATATCTTCTATGGACACGACTTGCCGAAATTG ATGGA AGCTGCTGTTAACAAAGAAAGCGGTGCAACGGTATTTGCTTATCACGTTAATGATCCTGAACGCTATGGT GTCGTGGAGTTTGATAATAACGGTACGGCAATTAGCCTGGAAGAAAACCGCTGGAGCCAAAAAGCAACTATGCG GTTACTGGGCTTTATTTCTATGACAATGACGTTGTGGAAATGGCTAAAAACCTTAAGCCTTCTGCCCGTGGCGAA CTGGAAATTACCGATATTAACCGTATT TATATGGAACAAGGACGTTTGTCTGTAGCCATGATGGGGCGTGGCTAT GCATGGTTGGATACAGGGACGCATCAAAGCCTTATTGAAGCAAGTAACTTCATTGCAACAATTGAAGAGCGTCAG GGATTAAAGGTATCTTGCCCGGAAGAGATTGCTTACCGTAAAGGGTTTATTGATGCCGAGCAGGTGAAAGTATTA GCCGAACCGCTTATCAAGAATCAATATGGTCAATATTTGCTGAAAATGAT C AAAGCAGGTGGAACTTAATGATCCAACAATTGCGTTGCTGATAGAACCCTATATATGGCATGAAATGTATGATT TTAGTGATGATTGTGTGCTGCTTGTAATTGCGGATGATTTCTATAAAGAGTCTGATTATATCCGCAATTATGATG ATTTTATTAGAAGAGTAAATTCAATTGAGAATTCATAAGCTAAGTGACGTCCAGACAACATCAATTGGTGATGGA ACAACTATCTGGCAGTT TGTTGTGATACTAAAAGGTGCTGTAATTGGTAATAATTGCAACATCTGTGCAAATACC TTAATTGAAAATAACGTTGTAATTGGTAACAATGTCACAGTCAAAAGCGGTGTGTATATTTGGGATGGCGTTAAA ATAGAGGATAATGTTTTTATTGGTCCTTGTGTAGCATTTACAAATGATAAGTATCCTCGCTCTAAAGTCTATCCT GATGAATTTTTGCAAACAATAATACGGAAAGGAGCATCAATA TGCTAACGCAACCATCCTGCCAGGAATTGAA ATTGGTGAAAAAGCAATCGTTGGTGCGGGGAGTGTTGTAACCAAAAATGTACCGCCATGCGCAATAGTAGTAGGT AATCCAGCTCGATTTATTAAATGGGTAGAGGATAATGAATAAAATTGATTTTTTAGATCTTTTTGCAATTAACCA GCGACAGCACAAAGAATTAGTCTCTGCGTTTAGTAGGGTGCTAGATTCTGGTTGGTATATC ATGGGCGAAGAACT TGAGCAGTTCGAGAAAGAGTTCGCAGAATACTGTGGAGTTAAGTATTGCATTGGTGTAGCAAATGGCCTTGATGC GTTGATACTAGTATTGAGGGCATGGAAAGAACTTGGCTATCTTGAAGACGGTGACGAGGTATTAGTACCGGCAAA TACATATATTGCTTCTATTCTTGCTATAACAGAGAACAAACTTGTTCCTGTTCTTGTTGAACCAGATATAGAAAC TTATAATATT AATCCTGCTTTAATTGAAAATTACATTACGGAAAAAACTAAAGCAATATTACCGGTTCACTTATA TGGTCTATTGTGCAATATGCCAGAAATTAGTGCAATCGCCAGAAAATATAATCTGTTGATTCTTGAAGATTGTGC ACAAGCACATGGTGCAATACGTGATGGTCGCAAAGCTGGAGCTTGGGGGGATGCTGCAGGATTTAGTTTTTATCC AGGAAAAAACCTTGGAGCTTTGGGGGATGCGGGAGCTG TTACTACAAATAATGCAGAATTATCCTCAACTATAAA

- 66 046206

AGCTTTGCGAAATTATGGGTCACATAAGAAATATGAAAATATTTATCAGGGATTGAATAGTCGATTGGATGAACT GCAAGCAGCCTTATTGCGTGTAAAAATCCATACATTACCGGAAGATACTGCGATTCGGCAAAGGATTGCTGAAAA ATATATTCGTGAAATAAAAAACCCTGCGATTACGTTACCAGTGTACGAAGGCCAAGGTGCGCATGTTTGGCATTT ATTTGTAGTAAGAATCGCTAATCGTGAAA AATTCCAGTCATACTTATTAGAGAAGGGTATCAAAACCTTAATTCA CTATCCATTACCACCCCATAAGCAGCAAGCATATCAAAATATGTCTAGCCTTAGCCTTCCAATTACTGAGCAAAT T CAT GAT GAAGT CAT T T CT T TAC CTATAAGT C C GGTAAT GAGT GAAGAT GAT GT CAAT TAT GTAAT CAAAAT GGT CAATGATTACAAGTAATGAAAAAATTTC TTCAGGTAACTATATTATCCGCTATCTATACATTCATTAAAATGATT GCGGGTTTTATCATCGGTAAGGTAGTAGCAATTTATACAGGGCCATCAGGGGTAGCAATGCTTGGCCAAGTGCAA AGTTTAATCACAATAGTTGCAGGTACTACCTCTGCACCTGTAAGCACAGGCCTTGTTCGATATACTGCGGAAAAT TGGCAAGAAGGACAAGAAGCATGCGCGCCATGGTGGCGCGCATGCTTAAG GGTTACTCTGTTTTTATTCTTGCTT ATTATTCCCGTTGTTATTATATTGTCGAAAAATATTAGTGAGTTACTTTTTAGCGATGGACAATACACATGGTTA ATCATTTTCGCATGTTGTATATTGCCATTCTCCATTATAAATACATTGATCGCTTCAGTTTTAAATGGTCAACAA TTTTATAAGCAATATATATTGGTTGGGATGTTTTCTGTATTCATTTCTACTATGTTTATGATTTTGTTGATTGTA GCTTATAATCTTAAAGGTGCATTGATTGCCACAGCTATAAATAGTGCTATTGCTGGTCTTGTATTGGTTTTATTT TGTCTCAATAAATCTTGGTTTAGATTTAAATATTGGTGGGGTAAAACGGATAAAGACAAAATTATAAAAATTATT CATTATACTCTGATGGCTCTGGTTTCTGTTATCTCCATGCCTACAGCATTGATGTGTATTAGAAAAATATTGATT GCTAAAACTGGTTGGGAGGATGCAGGG CAATGGCAGGCCGTATGGAAGATATCTGAGGTTTATCTTGGTGTTGTG AC AAT TGCTTTGT CAACATAT T T C T TAC CAAGAT T GACAAT TATAAAAACAAGT T T C C T TATAAAAAAAGAAGTA AATAGTACTATATTATACATAATATCTATTACTTCATTCATGGCGTTGAGTATCTATTTATTCCGCGATTTGGTA ATAACAGTTTTATTTACTGAACAGTTTCGCTCAGCTCGTGAATTATTTTTATTACAACTTATAGGGGATGTAATA AAAATTGCTGGGTTTCTTTATGCATACCCTCTTCAAAGTCAGGGGCATACTAAACTATTCATCAGTTCAGAAGTG AT TTTTTCTATGCTCTTTATCATTACCACCTATATTTTTGTTGTAAAATTATGGAGTACATGGTGCTAACATAAGT TAT GT CAT T ACAT AT AGT T TAT AT TTTGTGTT T GCAT T T GT GT T TACT AAT T T TAT T AAT GT T AGAAGAAAT AAT TAAAAACAGAGGTTGAATTTTGAAAATAATTATACCTGTCTTAGGATTTGGCAGGGCTGGTGGTGAAAGAGTTCT TTCTA AGCTGGCAACTGAATTGATGAATTATGGACATGATGTAAGTTTTGTTGTTCCAGATAATAGAACTAATCC ATATTATGCTACCACAGCAAAAATTGTCACGAGTAAATCTAGTCAAAACCGTGTAAAAATATTGAGAATCATTAA AAATTACTATAATCTGTGGCGTAAATGCATAGAGTTAAATCCTGATGCTGTAGTTGCTAGTTTTCATTTGACTGC CTATCTTGTCGCATTATTACCAATCACCCG TCGTAAGAAATATTATTATATTCAGGCGTATGAAGTTAATTTTTT TGATAATATAATATGGAAATTAATAGCGGGTTTAACATATTATTTACCGCTTAAAAAAATACTAAATAGTCCTAA TTTGCTTCCTCATAAACATGATGATTTTATAGGAGTAGTTCCTGCAGGAGTAGATTTAAACGTTTTCTATCCGAA ACCATCAAATAGGTTATTAAATGGTCACACATCAATAGGGATTATTGGTAGAAAAGA GAAGCACAAAGGAACTAG CGAAATTATTTCAGTATTGTGTTCACTGGAAAATAAAGCTGGAATTATAATCAATATTGCGATCTATCTTGAAGA AGTTGATAAGCAGCGTTTAATCGCTGCCGGGTTTCAGGTTAATTTTTTTCCGATTACTTCTGATTTAGAATTGGC ATCCTTTTATCGAAGCAATGACATCATGATTGCTGTTGGGTTAATTGAAGATGGCGCTTTCCATTATCCTTGTGC TGAATCAATGGCTTGTGGTTGTCTTGTTATTTTCAAATTATGCGCCACTTACTGAAACTAACAGTGTACTTAAATT AGTCAAGTTTGATGCTTGCAAACTTGGTGAAGCAATTAATCTTTGTCTCAATCTTGACCTAGAAGAAAAAAGCAA AGAAATCCAATCTAATATTTCTGTGTTGAATAAATATGACTGGAAAATTGTTGGTGAAACTTTCAATAGTTTATT GTTAGAATGCAAATAAATAGTATACGTTGATGGGGAAAATATGAATATTGTTAAAACTGATATTCCAGATCTGATC GTTCTTGAACCAAAAGTGTTTAGTGATGAACGCGGCTTTTTTATGGAGAGTTATAATCAGATTGAATTTGAGAAG GCAATAGGAAGGCACGTAAATTTTGTTCAGGATAATCATTCAAAATCTAGTAAAGGCGTACTACGTGGGTTGCAT

- 67 046206

TATCAATTAGCACCGTATGCACAGGCTAAATTAGTTCGATGTGTTGTAGGTCAGGTATTTGATGTTGCTGTTGAT CTTAGAAAAAATTCACCAACGTTCAAAAAATGGTTTGGAATAACCCTTTCCGCAGAAAATAAACGACAATTATGG ATACCCGAAGGATTTGCTCATGGTTTCTTGGTGACCAGTGATGAAGCTGAGTTCATTTATAAGACAACTAACTAC TATGCTCCTGGTCATCAGCAA GCAATTATTTACAATGATCCTATTTTAAACATCGATTGGCCTTTCTGCAGTAGT GCTCTGTCATTATCACAAAAAGATCAAGAAGCAAAATTATTTTCAGAATTATTGGACAGTGAACTGTTCTAATAA AGTGTGCCACCTTATCCGTCTGAAGGATAGGTGGTTGCTTATATTTTTTTGAGTATGTTTGTATAATGACAGAAA ATAGTCCGAAATATAAACACGATAAAAGCTTAATAAGTTTTATCT ACTTATTTTTTATTTTACACTTATTGTAG GCTTTATTATCGCAAATACCCAGTTTTTGGGGCGAAGTAGAGACTATGATAATTATATACAGATCTTTTCTGGTA AAGAAGGGGAGGGGGTTCTTGAATTATTTTATCGCGGATTGATGTTAATAACGACCAGCTATGAAACTATCATTT TTATAATTTTAACATGTTCTTTTTTTATAAAGGCAAGGTTTCTCGCTAACTATTCGCGTAATTTTT CAGGCTTGA CCTTATTCTTTATTTATTATGCAAGCGTTGCACTTTGGGTTTTAGATTATACTCAATTCAGAAATGGTCTATGTA TTTCCATTTTAATGTTTTCCGTATACTATTTATTTATAAAATAAACCGACTTATTTTTATTTCTCGGTATTATGTG CAATTGCAACTCATTGGTCTGCTTTGCCTTTTTTGCTTTTATATCCTTTTGTCTATTCAACAAAAATAAGACGCC TTGGTTATTT TTGTTTCAGTATTCTTGTTTTGATTGCGATCTCAGGAGAAGGAAAAGAGATCATATCTTTTATAA GAAATTTTGGAGTGGGACAAAAAATAGGAAATGAAGCTGGTGTAAATTTAATAAATTCATTATCCCTTACCGCTA TTTCCTGGTTTATTATTAGTTACATATCAAGCATTGGAAATGAAAGGAGAAATTTAAGGCTTTTCTTTTGTTATG GTGTCATGCAATACGTGACTTTTAGCCTTTTCTCTCTA CCTGTTATGGCTTTCCGTATTTTGGAAATGTATTTTTT TCCTTATGCTAACCATTGGGGTGTTTATTAAGCAAAAAAAGAATTATTATTTTATTTTTTGCAAAGTGTTAATTT TATTGTATCTAACATACTATTATCATATGGTCTTTGGAGTGATTAATGTGTAAGGCTAAGGTGTTGGCTATAATT GTTACTTACAACCCGGAAATTATTCGATTGACGGAATGTATTAACTCTTTAGCCCCCA CAAGTTGAGAGAATAATT CTTGTAGATAATGGCTCAAATAATAGTGATTTGATAAAAAATATCAGTATTAATAACCTTGAAATTTATTTTACTT TCGGAAAACAAAGGCATTGCATTTGCTCAGAACCATGGTGTTAAGAAGGGCCTGGAAGCAAAAGAGTTTGACTAT TTATTTTTCTCAGATCAGGATACTTGCTTTCCTAGCGATGTTATTGAAAAACTTAAGAGTACATTTACGAAAAAT AATAAAAAAGGTA AAAATGTTGCTTGTGCTTCCTTTTTTTAAAGACCATCGTTCAAATTATATGCATCCGTCA GTCAGCCTAAATATTTTTACGAGTACAAAAGTTATATGTAGTGAAGTAGACGATGATCTTTATCCCTCGCATGTT ATTGCTTCTGGGATGTTAATGTCTCGTGAAGCATGGCGCGTCGTCGGACCATTTTGTGAAAAACTCTTTATAGAC TGGGTTGATACAGAATGGTGTTGGCGTGCATTAGC TAATAATATGATTATTGTTCAGACACCATCAGTCATCATT TCTCATGAACTTGGGTATGGGCAGAAAATTTTTGCTGGTCGATCTGTTACAATACATAATTCTTTCAGAAATTTT TATAAAATACGCAATGCAATATACTTAATGCTGCATTCAAATTATAGCTTCAAGTATCGTTATCATGCTTTTTTT CAT GC GACAAAGAAT GT T GTAT T T GAAAT T T TATAT T C GAAAGAAAAAT TAAAT T CACT GAAGGT T T GT T T TAAA GCTGTACGTGATGGTATGTTCAATAATTTTTAATACGAAAATAGTTAGGCTCAAGGTGTTTAAATGGAAGAAAAT AATATGAAGACGGTCGCTGTAGTTGGCACAGTGGGTGTTCCTGCTTGTTATGGTGGGTTCGAATCACTTGTTCAG AATCTAATTGATTATCAATCTGATGGTATACAATATCAGATATTTTGCTCTTCAAAAAAATATGATAAAAAAATTT AAAAATTATAAAAATGCAGAATTAATCTATTTGCCGATAAATGCCAATGGCGTCTCTAGCATAATTTATGATATT ATGTGTTTAATTATTTGTTTATT CAAAAGGCCAGATGTTGTTTTAATATTGGGGGTGTCTGGTTGTTTATTTCTA CCAATTTATAAACTATTTTCAAAATCAAAGATTATTGTCAATATTGATGGGCTTGAATGGCGTAGAAATAAATGG GGAACGTTTTGCTAAGAAATTTCTTAAAATATCTGAGGCGATATCTATTAGAATAGCTGATATTATCATTTCAGAT AAT CAAGCAATAGCT GATTAT GT GGAAAATAAGTACAAGAAA AAAAGT GTAGTTATAGCTTAT GGCGGAGAT CAT GCCACTAATCTTAGTACACCGATAGACAATGATCAAAAAAAAGAAGGTTATTATTTGGGGCTTTGTAGGATAGAG CCTGAGAATAATATAGAAATGATTCTGAATGCCTTCATTAATACAGATAAAAAAATTAAATTTATGGGTAATTGG

- 68 046206

GATAACAGCGAGTATGGACGCCAGCTAAAAAAATATTATTCAAACTATCCAAATATCACCCTACTAGAACCTAAC TATAATATTGAAGAGCTTTATAAACTAAGAAAAAATTGTCTTGCATACATTCATGGACACTCGGCTGGTGGAACA AACCCTTCTTTAGTTGAAGCGATGCATTTTAATATTCCTATTTTTGCTTTCGATTGTGACTTTAATCGTTACACA ACTAACAATTTAGCTCATTACTTTAATGAT TCTGAACAACTTAGCTTATTAGCAGAAAGTTTGTCTTTTGGAAAT CTTAAATGTCGAGTATTAGATTTAAAAAATTATGCTGAAGATATGTATAACTGGAGGCATATAGCTGCTATGTAT GAATCTATTTATTAAACGCATTAACAATAATATAATTGACCTTATATAGCAGGGAAAGATCACGTAACGCTGCGG CGCGCCGATCCCCATATGAATATCCTCCTTAGTTCCTATTCCGAAGTTCCTATTCTT TCTAGAGAATAGGAACTT CGGAATAGGAACTAAGGAGGATATTCATATGGATAAAGCCGTAAGCATATAAGCATGGATAAGCTATTTATACTT TAATAAGTACTTTGTATACTTATTTGCGAACATTCCAGGCCGCGAGCATTCAGCGCGGTGATCACACCTGACAGG AGTATGTAATGTCCAAGCAACAGATCGGCGTAGTCGGTATGGCAGTGATGGGACGCAACCTTGCGCTCAACATCG AAA GCCGTGGTTATACCGTCTCTATTTTCAACCGTTCCCGTGAGAAGACGGAAGAAGTGATTGCCGAAAATCCAG GCAAGAAACTGGTTCCTTACTATACGGTGAAAGAGTTTGTCGAATCTCTGGAAACGCCTCGTCGCATCCTGTTAA TGGTGAAAGCAGGTGCAGGCACGGATGCTGCTATTGATTCCCTCAAACCATATCTCGATAAAGGAGACATCATCA TTGATGGTGGTAACACCTTCTTCCAG GACACTATTCGTCGTAATCGTGAGCTTTCAGCAGAGGGCTTTAACTTCA TCGGTACCGGTGTTTCTGGCGGTGAAGAGGGGGCGCTGAAAGGTCCTTCTATTATGCCTGGTGGCCAGAAAGAAG CCTATGAATTGGTAGCACCGATCCTGACCAAAATCGCCGCCGTAGCTGAAGACGGTGAACCATGCGTTACCTATA TTGGTGCCGATGGCGCCAGGTCACTATGTGAAGATGGTTCACA ACGGTATTGAATACGGCGATATGCAGCTGATTG CTGAAGCCTATTCTCTGCTTAAAGGTGGCCTGAACCTCACCAACGAAGAACTGGCGCAGACCTTTACCGAGTGGA ATAACGGTGAACTGAGCAGTTACCTGATCGACATCACCAAAGATATCTTCACCAAAAAAGATGAAGACGGTAACT ACCTGGTTGATGTGATCCTGGATGAAGCGGCTAACAAAGGTACCGGTAAATGGACCAGCCAGAGCGCGCT GGATC TCGGCGAACCGCTGTCGCTGATTACCGAGTCTGTGTTTGCACGTTATATCTCTTCTCTGAAAGATCAGCGTGTTG CCGCATCTAAAGTTCTCTCTGGTCCGCAAGCACAGCCAGCAGGCGACAAGGCTGAGTTCATCGAAAAAGTTCGTC GTGCGCTGTATCTGGGCAAAATCGTTTCTTACGCCCAGGGCTTCTCTCAGCTGCGTGCTGCGTCTGAAGAGTACA ACTGGGATCTGAACT ACGGCGAAATCGCGAAGATTTTCCGTGCTGGCTGCATCATCCGTGCGCAGTTCCTGCAGA AAATCACCGATGCTTATGCCGAAAATCCACAGATCGCTAACCTGTTGCTGGCTCCGTACTTCAAGCAAATTGCCG ATGACTACCAGCAGGCGCTGCGTGATGTCGTTGCTTATGCAGTACAGAACGGTATTCCGGTTCCGACCTTCTCCG CAGCGGTTGCCTATTACGACAGCTACCGTGCTGCTGTT CTGCCTGCGAACCTGATCCAGGCACAGCGTGACTATT TTGGTGCGCATACTTATAAGCGTATCGATAAAGAAGGTGTGTTCCATACCGAATGGCTGGATTAA

SEQ ID NO: 13 (example of nucleotide sequence of the rfb locus O6L - producer strain ObL-ERL stGVXN4112 and stLMTB 10923)

ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGATACCC AAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAGGGATCAAA GAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTATGAGTTAGAATCA CTCCTTGAGCAGCGCGTGA AGCGTCAACTGCTGGCGGAAGTACAGTCCATTTGCCCGCCGGGCGTGACAATTATG AACGTGCGTCAGGGCGAACCTTTAGGTTTGGGCCACTCCATTTTATGTGCACGACCTGCCATTGGTGACAATCCA TTTGTCGTGGTGCTGCCAGACGTTGTGATCGACGACGCCAGCGCCGACCCGCTGCGCTACAACCTTGCTGCCATG ATTGCGCGCTTCAACGAAACGGGCCGCAGCCAGGTG CTGGCAAAACGTATGCCGGGTGACCTCTCTGAATACTCT GTCATCCAGACCAAAGAGCCGCTGGACCGCGAAGGTAAAGTCAGCCGCATTGTTGAATTCATCGAAAAACCGGAT CAGCCGCAGACGCTGGACTCAGACATCATGGCCGTTGGTCGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTT GAACGCACTCAGCCTGGTGCATGGGGGCGTATTCAGCTGACTGATGCCATTGCCGAACTGGCGAAAAAA CAGTCC

- 69 046206

GTTGATGCCATGCTGATGACCGGCGACAGCTACGACTGCGGTAAAAAAATGGGTTATATGCAAGCGTTCGTGAAG TATGGACTACGCAACCTCAAAGAAGGGGCGAAGTTCCGTAAAGGGATTGAGAAGCTGTTAAGCGAATAATGAAAA TCTGACCGGATGTAACGGTTGATAAGAAAATTATAACGGCAGTGAAGATTAGCGGCGAAAGTAATTTGTTGCGAA TTTTCCTGCCGTTGTTTTAT ATAAACAATCAGAATAACAACGACTTAGCAATAGGATTTTCGTCAAAGTTTTCCA GGATTTTCCTTGTTTCCAGAGCGGATTGGTAAGACAATTAGCATTTGAATTTTACGGGTTTAGCGCGAGTGGGTA ACGCTCGTCACATCGTAGACATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCTGAAATTA TAAAGTCATTCTTATAGAACATCGCATTTCAATAATATAATTA CACCTAAATGAATAGGATACAACGTGTGCACA ATTATTTAAGGCTTAAAGATAAAATAAAAAACGTATTTTTAGGGTTGTATATATTGCAGTTATTTAATTATATCG CGCCATTGGTAATTATCCCTATCCTGATAAAATATATTGGGTTGGGGGAATATGGGGAATTGGTCTATATTACAT CTATTTATCAAATAGTGGCTTTGATTATTGATTTTGGCTTTACTTACACAGGACCTGTGGTTGCTG CGAGACATA GATGTGAGACCCAAAATTTACAGCGCTATTACTCAATAGTTGTTCTTTTAAAATCATTGCTTTTTATAATTGCAT TAACATGTGTATTTTTATTGTGCAGATTAAATATAGTCCACTTGTCATTTTTTGGGTTTTTGTCAATTTTTCTAT GCACTATTGGTAATATATTATCGCCCAATTGGTTTTTGCAGGGGATTGGTGATTTTAAAAACTTTCATACTCAC AAGTAATAGTGA GAATAACATTGTTTATCATACTTCTTGTTTATGTCTGTAGTGGCGGAGATAATGTTTTTATCC TAAGTTTTTTGCAAAATGCAACATTACTCATATGCTGTATATACTTATGGCCAAATATTCATATTAGCCATGTTG TTCATCTTAAACCTAATGAATGCATTGTGGAATTTAAGAAGGCAGGAAATGTTTTTATTGGCGTAATAGGTACGA TTGGTTACAATGGTCTAATTCCTGTGTTAATT GGAAACCTTTTGCGGTAATACGAGTCTTGGTGTTTTTTCAATCG T T CAAAAAAT GACAACAGCAT GT CAAAGT CTAAT TAAT WITH CAATAT CACAGTATAT GT TAT CT CAAGT T T CAGAAA TTAAACCCTCAAGATAAACTGTTTTATTATAGAATTAAAAAAAGTTTTTTTGTGCATTTAACAATTAGCATAATTG CATGTTTATGTTATATGGGGTTAGGGCAATATGTGGCGACTTT TATAGGTAAAGTTGACGTTTCATTTGTTATTA TTTTATTTGCGTCAATAATTACCATTTTTTCATCTTTAAATAATGTCCTTGGTATACAGTTTTСTTATACCGACAG ATAATGTAAAAATACTACGAAGTATAAATGTTATGGCGGGAATTATTGTTGTTAGTTTGTCCTGGCTGTTAATAT CACGCTTTGACATTCTGGGGGGGGTTTTATTTAAACCTAATTGGTGAGTTTCTTGTA TTCAGTATGCTAGCTTTTA TTGCCCATCGAAAGTGGGGAGCGAGAGTATAATGAAAGTGAAGGCGGTTCCTGCTATTACATTCTATTTAAGTTT AATGCTGACAATTTTAGTGTTACTGTTTGGTAATGAACCAAATAAAATCACAATATATCCTTGTTATAGCAACGAT AACAGTTTTTTATATCGCATATATCACTAATAAAATAACTTCTCCGGCCAGCCTTCTCGTTATATCATCTTTTGT GTT TTTAGGTTGTCGCCCTTTATTATCTTTGTTTGCAAACTATGATTATAGGATTGCCGATTGGTTTATTGAAGG ATATATGGATGACGATGTGATTTTGGCTAACTATGCTATAACACTAATGTATTATGGTTATACATTGGGACTAAT TCTATGCAAAAATACTGAAAAATTTTATCCGCATGGTCCTTATCCTGAAAAACAATTGCTAAAAATAAAGTTTCT TTTGACTTTATTTTTTCTGGGTTCGATAGGTATG GTTGTAAAAGGGATATTCTTTTTTAACTTTATAGAATCTAA TAGTTATGTTGATATTTATCAATCAAATATAACAACGCCAATAGGTTATGATTTTCTATCTTATTTATTTTATTG TTCTTTTTTCCTTATATGTGCGTTTCATATACAGTTCAGAACAAAATAAAAAATTTCTTTTTATTGCGATATGCAT TGCTGCATTTAGCACCTTGAAGGGTAGTCGTAGTGAAGCTATAACGTTTCTTTTTAAC GGTTACATGTATATATTT TAATGAAGTAAAGACAAGAAACTTACGTCTGCTGATTACAATGATTTTTGTTTTTAGCGTCATTTTTGTGATTAG TGAATTTATCTCAATGTGGCGCACTGGAGGGAGTTTTTTTCAATTAATGCAGGGTAATAATCCTGTTATAAACTT TGTATACGGCATGGGAGTATCATATCTTTCCATTTATCAATCAGTAAAACTACAACTATTGTCAGGGGGATATAA TGTTACCTATCTATTCAGCCAGTTAATAATAACTTGCTCGTCAATATTTAATGTCAAATTGAGCTTGCCGGAAAT AAGCTATAGCCATTTGGCCTCATACACAGCAAACCCAGAACTATATAATCTTGGGTTCGGACTTGGGGGGAGTTA TTTAGCAGAATCGTTTTTAGCAT TTGGTCTGATTGGATGTTTCATTATACCCTTTTTACTTTTACTTAATTTAAA T GTAT T GGAAAAATATACAAAAAACAAAC CAAT TATATAT T T T GT T TAT TATAGT GT GT T GC CACCTATAT TAT T

- 70 046206

CACACCAAGAGAGACTTTGTTCTATTTCTTCCCCTATCTTGTCAAAAGTATATTTGTTGCTTTTTTAGTTACATT ATACAT C CAGTATAAAAAGGAT T GAC CAAAAT GT CAGAAAAAAAT GT CAGCATAATAAT C C CAAGT TATAACAGG GCTCATATTCTTAAGGAGGTCATACCAAGTTATTTTCAGGATGAGACTTTAGAGGTTATAGTTATCAATGATGGA TCAACAGATAATACAA ATAGTGTATTAGCTGAACTGAAGGAAAAATATTCTCAGTTAGTTATTTTAGAAAATGAA ACGAATAAAAAACAGATGTATTCTAAAAACCGAGGGATTGAAATAGCCAAAGGGAATATATTTTTTTTGGTGAT GATGACTCTTACCTCTTACCCGGTGTTATATCTCGGTTATTGGCTACAAAATATGAGACAGGCGCTGATGTAATC GGCGCAAGAATACTTTATATGAATAATAACGAGAAAACAATTGAAGAT TGCATAAATCGACATAAAAAAGAGGGG CGTTTTGT TAGT GAT CTAAATAGAT T GGAT T T TAGT TATACAT GT GAT T T G GAC CAT C C GAT T GAAT GT T T T TAT GCACAGCCTTTTGTTCTAGCTGAAAGGGAACTAATATCGAAATATCGATTTGATATATCTTATACGGGAAACTGC TATCGTGAGGAAACTGATTTCATGCTATCTCTATTTATTAAAAATAAAAAAA ATTTATATATGATTCAAAGGCTTTG TTAATAAATTTACCTCCAAGAAAAGCGACGGGAGGGGCAAGAACAGCTAATCGATTAAAATATCATTACGAAAGT TGCATAAATAATTATAGATTTTTAAAAAAATATAATGATAATTTGAATCTTCTTTCAGGACAAAAGCATGCTATA TTTTACCGACAGTGTCAATTCGTTCTGCTAAAAATGAAGTCGTTTATCGGGAAGTTTTTAAAATGATTATATATA TC GCCGCGTATAATGGTTCAGGAGGGCAAGGTGGGGTGGAAAGGGTTGTTGCCCAACAATGTAACATTCTTAAAA ATTTGGGGGTTAAAGTCATTATACTTGATAAAACATACTTCAAAATTTCTAACAAAATTCGTAACAAAAAAATAC AAGTAGCACTTTATCCAATATTAGTTTCTCTTTATTTAACCTTACAAAAATTACGTGGCGTGACGTTTTAAAGTTA TTGCACATGGCTATTGTTCTCCTTTTTATAGGAATGACATCTTAATAGCTCATGGCAATATGAAATGTTATTTTC AAACAGTCATGAATAAAAAACCTAATCGGTTGTCTGGCAGTGGTCTTTTATCTTTCTATGAGCGTTGGGCTGGAG CATTTTCAAAAAATATCTGGGCTGTTTCAAATAAGGTTAAAAGTGAATGGAATGAGCTTTACAATATTAATTCAC ATAAAAT CAAAGT T GT T C GAAAT T T TATAAAT S T T G CACAAT T T GAT TACAC T GAT GT TAAT GAAG CAGAATAT G TGACATTTGTCGGGCGATTGGAAAAAGGAAAAGGAATAGATGATCTGTATTACATATGTAAAAATCTGCCAGATA CTTCCTTCCATTTAGTTTCAAGTATTCCCGCCCCACAAAATTTTGCTTCGCTAAATAATGTTCTGACCAGCATTG CTGTCCCCTATGCGAAAATGCCAGAAA TATTTAAGAAATCCAGAGTACTTATTTTACCGTCCTATTATGAAGGAT ATGAGCTGGTTACTATTGAAGCGCTATGCTGTGGTTGCCCTGTGATAGGCTATAATGTTGGTGCAATTAGAGAGT TGTATGCAGAAAGTTTTCCTGGCGTATTTATTGCCAATAATAAAGAAGATTTAGCACAAGTAGCCTACAAATTAA TTAGTCTTGATAATGAAAAATATTATCATTTGAGACAAACTATTTATAG CAAGCGTGAGCTTTTTTCTGAAGAGA GATATGCGGAAATTTTAACGGCGGCATTTAATGAAAAAAAATAAGAAACTCTGTCTCATTTCAATTAACTCATAT AATGAACTTACCGGAGGAGGAGTATATTTACGTACGCTTGTTAGTTTTCTACAAAAACAGAATGTTAATTTAACA CTTATTGATAAAAAATCCTCAGGTAAACTATTCGAAGACAATACTTTTCAACATATATCATTTTATTAAAGGTAAA CGTC AGGATATAATATCCAGGCTTTTTTTTATACCATCATTTTATGTCCCTTATATTTTCTCAATAATTAAAATT TTACGGAAGCAAGATATTCTTGCTTTTCACAACTCTCGGCTTGGATTGTTATGTCTGCTTTTTTAGAATACTCATG C C C CACAAAAAGAT CATAT TGTTTACG GATAAC T T C GAATAT GAC T TAATAAGACAAAAAGATAAAAACATAAC T ACTTTTATTGAAAAATTAATTG TTTATCTCAATGAATTTATCGGGCTTAAGAATTCAGATTTAGTTAGCTATATT ACCCGGCAAGATAAAAATGCAATGGATAAATTTTATGGGATTAAAAAAAGCAGAAATTTAATTCTCCCTGTGATA TTTAGTAGAGAAAAACCAACTGATGTATTGTCAGCTCACTTTATTAATGAGTATAATCGATTGAATAATGATAAT AGGAAAAAAGTAGTATTTACTGCATCTTTTGATTTTTTTCCAAATATAGATGCTGCCAACTATGTTTTAAATGCA GCAAAGTCTAATAATGATTATTGCTATATTTTGGCAGGTAGGAAAAGTACTACTTTGAATCTTCCTGATTTGGAT AATTTATTTTTTTTCGATAATC TATCTAATAGTGAAATGTCATATTTATTATCTGCTTGTGATGTTTTTTATTCT CCTATAGTTTTAGGAAGTGGAATGAAAACAAAAATTGCAGAAGCACTATCATATGGATTATATATTTATGCGACA GAGCATTCCTTAATCGGCTATGATGAAATTATACACAATAAGGAGTGTGTTAAAAAAATCTCACATTTGGATGAG

- 71 046206

GAATTTCCTAAAGATTTCAAGATGAAAAGTATCAATAAACAGCTAATAATGTCTTATCAGCAAAAATATTATTCA CATTATCGGTTTAATGGCCATGAACTTGATATAATAAATTTTGACGATTAGTTAGTGGAGATATAATATGAACAT ATTAGTAACTGGTGGTGCTGGATATATCGGATCTCATACGGCTATTGAATTACTGAATGCAGGTCATGAGATTAT CGTTCTGGACAATTTCAGTAATGCT TCATACAAGTGTATCGAAAAAATAAAAGAAATTACTCGACGTGATTTTAT AACAATTACTGGAGATGCTGGGTGTAGGAAGACACTCTCCGCTATTTTCGAGAAACACGCCATAGATATAGTTAT TCATTTTGCTGGCTTTAAATCTGTTTCAGAGTCTAAAAGTGAACCCTTAAAGTATTACCAGAATAATGTTGGAGT GACCATTACTTTATTACAGGTAATGGAAGAGTACAGAATTAAAAAATTTATC TTTAGTTCATCTGCGACAGTCTA TGGTGAACCAGAGATAATTCCAATTCCAGAAACAGCTAAAATTGGAGGAACTACGAATCCATATGGCACATCGAA GTATTTTGTTGAAAAAATTCTAGAGGATGTTAGTTCCACGGGAAAACTGGATATAATTTGCTTGAGATATTTTAA TCCTGTCGGTGCTCATTCTAGTGGTAAAATAGGTGAGGCTCCATCTGGTATCCCTAATAATCTTGTTCCTTATTT ATTGGATG TTGCGAGTGGTAAAACGTGATAAATTATTTATTTATGGCAATGATTACCCTACTAATGATGGAACAGG TGTAAGGGATTTTATTCATGTTGTTGACTTAGCGAAAGGTCATTTGGCTGCAATGAATTATTTAAGTATCAATTC GGGATATAATATCTTTAATCTTGGTACAGGAAAAGGTTATTCGGTACTTGAATTAATCACTACATTTGAAAAATT AACAAACATTAAGGTCAATAAATCTTTT ATAGAGAGAAGGGCAGGGGATGTTGCGTCTTGTTGGGCTGATGCAGA TAAAGCTAATTCTTTATTGGACTGGCAAGCCGAACAAACTCTAGAACAGATGTTATTGGACTCGTGGCGTTGGAA AAAAAATTAT СCAGACGGATT CT GAATATAAAAGGTTTCAGTTTTAT GAAT СAAT CAGAGCAGAGAAAAAAAATA CTGGTTCTTACACCTCGCTTTCCCTACCGGTCATTGGAGGGGTCATG ATAGATTAAGAGTCTATATGTTATGTAAAGAA CTTTCCAAAAAATATGATCTTATTCTTCTGAGCTTATGTGATCAACCACTAGAACTTGAAATAAATATAAAATGAC TCGGTCTTCAAAGAAATTCATCGTGTCTATCTACCAAAATAATAAATCATATTATAATGTATTAAAAGCTTTGGTT ACGCAAAAACCGTTGCAAATTGCTTATTATCAATCGGACACATTTAAGAATAAATACAATAAATTAATTAAACA A TGCGATGCAGTATTTTGTCATCTGATAAGAGTTGCTGATTATGTTAAGGATACAGACAAGTTCAAAATTCTTGAT ATGACAGATGCAATATCTTTGAATTACAGTCGCGTTAAAAAATTAGCAAGTAAAAAAAGTTTGCGTGCAATTATT TATTCTCTGGAACAAAAAAGATTAGAATCATATGAACGTTCTGTGGCGAATCTTTTTGATTTGACCACTTTTATT TCATCCGTAGACCGTGACTATCTCT ACCCTAATCTGGGCAGTAATATCCATATAGTCAATAATGGGGTTGATACA TCAGCCTTGAGATATATAAAAAGAGAAATAAAAATCGATAAGCCTGTGGAACTTATATTTATCGGAAATATGTAT TCTTTACAAAATATGGATGCTGCAAAACATTTTGCTAAGAATATTTTACCTTGCTTGTATGATGAGTTTAATATT ATTTTTAAAGTGATTGGTAAGATCTCAGAAACTAATAAAAATATATTAAATTCATTTAAAAATA CAATTGCTTTA GGTACTGTTGATGATATCAATTCTTCCGCTTCTACAGGGCATATAGGTATATGTCCTGTTCGTCTTGGAGCAGGC GTACAAAATAAAATTCTTGAATACATGGCTTTAGGTTTACCATGTATTACATCTAGCATTGGTTATGAAGGTATT AATGCAAAATCAGGTAGCGAAATTTTTGTTGCAGATACAGTAGAGCAATATAAAAACGTACTAAGAGAAATAATT TACGATTATA ATCGTTATACTGAAGTGGCTGAAAATGCCCGTAGTTTTGTAGAAAATAATTTTTCTTGGGAATCA AAAGT TGC CΑΑΤ Τ TAAT GAATACAT TAGAT GAGAAAT TATAT GAACΑΑΤΑΑΤAAAAT TAT TACAC C G CCTGTTGGGCGTAACACTGCCCCTGCAATTGCATTAGCGGCGTTGCTGATGTCTAAGTCTGATAAAAGTGCAGA TGATCTTATGCTCGTACTGGCTGCAGATCACGTTATACACGATGAAGAAAAATTTTGTAACGCTGTTAGATCGGC AATTCCATACGCTGCTGATGGGAAATTGGTAACATTTGGTATAATTCCAGACAAAGCAGAAACTGGTTATGGTTA TATACATCGAGGACAATATATTAATCAG GAAGATTCGGATGCATTTATAGTGTCATCATTTGTTGAAAAGCCAAA TCATGAGACAGCCACTAAATATCTTGCTTCCGGTGAGTATTATTGGAATAGCGGTATGTTTTTGTTTAGTGCAAA

- 72 046206

TCGTTATATAGAGGAACTTAAACAATTTCGGCCTGATATTTTATCCGCTTGTGAAAAAGCAATTGCTTCAGCGAA CTTTGACCTTGATTTTGTGCGTTTAGATGAAAGTTCTTTCTCTAAGTGCCCTGAAGAATCAATTGATTACGCTGT AATGGAAAAAACAAAAGACGCAATTGTTATTCCAATGGATGCTGGCTGGAGTGATGTCGGTTCATGGTCTTCTCT TTGGGAAATTAATGATAAAG ACTCAGACGGCAACGTAATAGTTGGGGATATTTTCTCTCATGAAACAAAGAATTC TTTCATATATGCCGAATCGGGAATTGTTGCTACAGTTGGAGTGGAAAATTTAGTTGTTGTCCAAACAAAGGATGC T GT T CT T GT CT CAGAGAGAAATAAAGT T CAGGAT GTAAAGAAAATAGTAGAACAAAT TAAAAAT T CAGGT C GTAG CGAGCATTATGTTCATCGCGAAGTATATCG TCCTTGGGGTAAATATGATTCCATTGACACAGGGGAGCGTTATCA GGTCAAACGTATAACAGTAAATCCTGGTGAAGGACTTTCTTTACAAATGCACCATCATAGGGCAGAACATTGGAT CATAGTTTCTGGAACTGCAAGGGTGACTATAGGTTCTGAAACTAAGATTCTTAGCGAAAATGAATCTGTTTACAT ACCTCTTGGTGTAATACACTGCTTGGAAAATCCAGGGAAAATTCCTCTTGATTTAATTGAAG TTCGTTCTGGATC T TAT T TAGAAGAAGAC GAT GT TAT C C GT T T T CAGGAC C GATAT GGT C GTAG CTAAAT T T T T GATAAT GTAAC GTT AGTAGAAGAGCGCTAATATTTTTAGTTAATCTGTAATAAGTATTATTTGTTTAAGGTATATCATGTCGAGTTTAC CCTGCTTTAAAGCCTATGATATTCGCGGGAAATTAGGCGAAGAACTGAATGAAGATA TTGCCTGGCGCATTGGTC GCGCTTATGGCGAATTTCTCAAACCGAAAACCATTGTGTTAGGCGGTGACGTCCGACTCACCAGCGAAACCTTAA AACTGGCGCTGGCGAAGGGGTTACAGGATGCGGGCGTCGATGTGCTGGATATTGGCATGTCCGGCACCGAAGAGA TCTATTTCGCCACGTTCCATCTCGGCGTGGATGGCGGCATCGAAGTTACCGCCAGCCATAACCCGATGGATTACA ACGGCATGAAACTGGTGCGCGAAGGGGCTCGCCCGATCAGCGGTGATACCGGACTGCGCGACATCCAGCGTCTGG CAGAAGCCAACGACTTTCCTCCCG TTGATGAAACCAAACCGGTCGCTATCAGCAAATCAATCTGCGTGACGCTT ACGTTGATCACCTGTTCGGTTATATCAACGTCAAAAACCTCACGCCGCTCAAGCTGGTGATTAACTCCGGGAACG GCGCGGCGGGTCCGGTGGTGGACGCCATTGAAGCCCGCTTTAAAGCCCTCGGCGCACCCGTGGAATTAATCAAAG TGCACAACACGCCGGACGGCAATTTCCCCAACGGTATTCCTA ACCCGCTACTGCCGGAATGTCGCGACGACACCC GCAATGCGGTCATCAAACACGGCGCGGATATGGGCATTGCCTTTGATGGCGATTTTGACCGCTGTTTCCTGTTTG ACGAAAAAGGGCAGTTTATTGAGGGCTACTACATTGTCGGCCTGCTGGCAGAAGCGTTCCTCGAAAAAAATCCCG GCGCGAAGATCATCCACGATCCACGTCTCTCCTGGAACACCGTTGATGTGGTGACTGCCGCAG GCGGCACCCCGG TAATGTCGAAAACCGGACACGCCTTTATTAAAGAACGTATGCGCAAGGAAGACGCTATCTACGGTGGCGAAATGA GCGCCCACCATTACTTCCGTGATTTCGCTTACTGCGACAGCGGCATGATCCCGTGGCTGCTGGTCGCCGAACTGG TGTGCCTGAAAGGAAAACGCTGGGCGAACTGGTGCGCGACCGGATGGCAGCGTTTCCGGCAAGCGGTGAGATCA ACAGCAAACT GGCACACCCCGTTGAGGCGATTAACCGCGTGGAACAGCACTTTAGCCGCGAGGCGCTGGCGGTGG ATCGCACCGATGGCATCAGCATGACCTTTGCCGACTGGCGCTTTAACCTGCGCTCCTCTAACACCGAACCGGTGG TGCGGTTGAATGTGGAATCGCGCGGCGATGTACCGCTGATGGAAGAAAAGACAAAACTTATCCTTGAGTTACTGA ACAAGTAATTCAGTAATTTCATATAAATGGGTT TTAAAAAACGGAAAAGATGAGATATCCGGTGGGTATATCCA AGGTAATGCTATTCAGTATCTCTATGAGTGAGTTAACATCTATACCACATTTAAGCCGCACACTTCGGGATCCCC ATATGAATATCCTCCTTAGTTCCTATTCCGAAGTTCCTATTCTTTCTAGAGAATAGGAACTTCGGAATAGGAACT AAGGAGGATATTCATATGGATAAAGCCGTAAGCATATAAGCATGGATAAGCTATTTATACTTTAATA AGTACTTT GTATACTTATTTGCGAACATTCCAGGCCGCGAGCATTCAGCGCGGTGATCACACCTGACAGGAGTATGTAATGTC CAAGCAACAGATCGGCGTAGTCGGTATGGCAGTGATGGGACGCAACCTTGCGCTCAACATCGAAAGCCGTGGTTA TACCGTCTCTATTTTCAACCGTTCCCGTGAGAAGACGGAAGAAGTGATTGCCGAAAATCCAGGCAAGAAACTGGT TCCTTACTATACG GTGAAAGAGTTTGTCGAATCTCTGGAAACGCCTCGTCGCATCCTGTTAATGGTGAAAGCAGG TGCAGGCACGGATGCTGCTATTGATTCCCTCAAACCATATCTCGATAAAGGAGACATCATCATTGATGGTGGTAA CACCTTCTTCCAGGACACTATTCGTCGTAATCGTGAGCTTTCAGCAGAGGGCTTTAACTTCATCGGTACCGGTGT

- 73 046206

TTCTGGCGGTGAAGAGGGGGCGCTGAAAGGTCCTTCTATTATGCCTGGTGGCCAGAAAGAAGCCTATGAATTGGT AGCACCGATCCTGACCAAAATCGCCGCCGTAGCTGAAGACGGTGAACCATGCGTTACCTATATTGGTGCCGATGG CGCAGGTCACTATGTGAAGATGGTTCACAACGGTATTGAATACGGCGATATGCAGCTGATTGCTGAAGCCTATTC TCTGCTTAAAGGTGGC CTGAACCTCACCAACGAAGAACTGGCGCAGACCTTTACCGAGTGGAATAACGGTGAACT GAGCAGTTACCTGATCGACATCACCAAAGATATCTTCACCAAAAAAGATGAAGACGGTAACTACCTGGTTGATGT GATCCTGGATGAAGCGGCTAACAAAGGTACCGGTAAATGGACCAGCCAGAGCGCGCTGGATCTCGGCGAACCGCT GTCGCTGATTACCGAGTCTGTGTTTGCACGTTATATCTCTTCT CTGAAAGATCAGCGTGTTGCCGCATCTAAAGT TCTCTCTGGTCCGCAAGCACAGCCAGCAGGCGACAAGGCTGAGTTCATCGAAAAAGTTCGTCGTGCGCTGTATCT GGGCAAAATCGTTTCTTACGCCCAGGGCTTCTCTCAGCTGCGTGCTGCGTCTGAAGAGTACAACTGGGATCTGAA CTACGGCGAAATCGCGAAGATTTTCCGTGCTGGCTGCATCATCCGTGCGCAGTTCCTGCA GAAAATCACCGATGC TTATGCCGAAAATCCACAGATCGCTAACCTGTTGCTGGCTCCGTACTTCAAGCAAATTGCCGATGACTACCAGCA GGCGCTGCGTGATGTCGTTGCTTATGCAGTACAGAACGGTATTCCGGTTCCGACCTTCTCCGCAGCGGTTGCCTA TTACGACAGCTACCGTGCTGCTGTTCTGCCTGCGAACCTGATCCAGGCACAGCGTGACTATTTTGGTGCGCATAC TT ATAAGCGTATCGATAAAGAAGGTGTGTTCCATACCGAATGGCTGGATTAA

SEQ ID NO: 14 (example of nucleotide sequence of the rfb 08 locus - producer strain 08-EPA stLMTB11734)

ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGATACCC AAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAGGGATCAAA GAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTATGAGTTAGAATCA CTCCTTGAGCAGCGCGTGA AGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCCGGGCGTGACCATTATG AACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGACCTGCCATTGGTGACAACCCA TTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCGCTACGTTACAACCTTGCTGCCATG ATTGCACGTTTCAACGAAACGGGCCGCAGCCAGG TGCTGGCAAAACGTATGCCGGGTGACCTCTCTGAATACTCC GTCATCCAGACTAAAGAGCCGCTGGACCGTGAGGGTAAAGTCAGCCGCATTGTTGAATTTATCGAAAAACCGGAT CAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGTCGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTG GAACGTACTCAGCCTGGTGCATGGGGACGTATTCAGCTGACTGATGCTATTGCCGAGCTGGCGAAA AAACAATCC GTTGATGCAATGCTGATGACCGGCGACAGTTACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAG TATGGCCTACGCAACCTGAAAGAAGGGGCGAAGTTCCGTAAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAA TCTGACCGGATGTAACGGTTGATAAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAA TCTTCCTGCCGTT GTTTTATATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAGTTTTCCA GGATTTTCCTTGTTTCCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTA ACGCTCGTCACATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCATTAATAC CTCTATTAATCAAACTGAGAGCCGCTTATT TCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGCTAGCGAT CGCTTAAGATCTAGGATTTCATTATGTTACTTCCTGTAATTATGGCTGGTGGTACCGGCAGTCGTCTCTGGCCGA TGTCACGCGAGCTTTATCCGAAACAGTTCCTCCGCCTGTTCGGGCAGAACTCCATGCTGCAGGAAACCATCACCC GACTCTCGGGCCTTGAAATCCATGAACCGATGGTCATCTGTAACGAAGAG CACCGCTTCCTGGTGGCTGAACAGC TACGCCAGCTCAATAAGCTGTCGAATAATATTATTCTTGAGCCGGTCGGGCGCAACACCGCCCCGGCCATCGCCC TGGCAGCCCTTCAGGCCACCCGCGACGGCGACGACCCGCTGATGCTGGTTCTCGCCGCTGACCATATCATCAATA ACCAGTCGGCCTTCCACGACGCCATCCGGGTCGCCGAGCAGTATGCTGATGAAGGTCATCTGGTCACCTTC TA TCGTGCCGAATGCCCCGGAAACTGGCTACGGTTACATTCAGCGCGGCGTGGCGCTCACCGATAGTGCCCATTCCG

- 74 046206

CGTACCAGGTGGCCCGCTTTGTGGAGAAGCCGGATCGCGAGCGCGCCGAGGCTTACCTCGCCTCCGGGGAGTACT ACTGGAACAGCGGCATGTTTATGTTCCGCGCCAAGAAATACCCTCATCGAGCTGGCCAAATACCGTCCGGATATCC TGGAAGCCTGCCAGGCTGCGGTGAATGCCGCCGATAATGGCAGCGATTTCATCAATATCCCGCATGATATTTTCT GCGAGTGCCCGGATGAGTC CGTGGACTATGCCGTTATGGAGAAAACCGCCGATGCGGTGGTGGTCGGTCTCGATG CTGACTGGAGCGACGTCGGCTCCTGGTCCGCACTATGGGAGGTCAGCCCGAAAGACGAGCAGGGCAATGTCCTCA GCGGTGACGCGTGGGTACACAACAGCGAAAACTGCTACATCAACAGCGACGAGAAGCTAGTGGCGGCCATTGGCG TAGAGAATCTGGTGATTGTCAGCACTAAGGACGCCGTG CTGGTGATGAATCGCGAGCGTTCCCAGGACGTGAAGA AGGCGGTCGAGTTCCTCAAGCAGAACCAGCGCAGCGAGTACAAGCGCCACCGTGAGATTTACCGCCCCTGGGGCC GTTGCGACGTAGTGGTCCAGACCCCGCGCTTCAACGTCAACCGCATCACGGTGAAACCAGGCGGTGCCTTCTCGA TGCAGATGCACCACCATCGCGCCGAGCATTGGGTTATTCTCGCCGGCACC GGTCAGGTGACTGTCAACGGTAAGC AGTTCCTGTTGTCCGAGAACCAGTCCACCTTTATTCCGATTGGCGCCGAGCACTGCCTGGAAAACCCTGGCTGTA TTCCGCTGGAAGTGCTGGAGATCCAGTCGGGGGCGTACCTTGGCGAGGACGACATTATTCGTATTAAAGACCAGT ATGGTCGTTGCTAATTATTTTCGGGACAAGACGCAGAATGACACAGTTAACTTGTTTTAAAGCTTAT GACATCCG TGGTGAACTGGGTGAGGAACTGAACGAGGACATCGCCTACCGTATCGGTCGCGCCTACGGCGAATTTCTGAAACC CGGGAAGATAGTGGTGGGGGGCGATGTGCGCCTCACAAGCGAGTCGCTGAAGCTGGCGCTGGCCCGCGGGTTAAT GGACGCCGGTACCGACGTGCTGGACATCGGCCTGAGCGGTACCGAAGAGATTTACTTTGCCACCTCCACCTTGG GGTAGAT GGTGGCATCGAGGTGACCGCGAGCCACAATCCTATGAACTACAACGGCATGAAGCTGGTGCGCGAGAA TGCGAAGCCCATCAGCGGCGACACCGGCCTGCGGGATATCCAGCGCCTGGCGGAGGAAAACCAGTTCCCGGCCGTCGCGGGACCCTGAGCAAGATATCGGTACTGAAGGAGTATGTTGACCATCTGATGAGCTACGT GGACTTCTCGAACTTCACCCGTCC ACTGAAGTTGGTGGTGAACTCCGGAAACGGGGCTGCGGGGCACGTGATTGA TGAGGTGGAGAAACGCTTCGCGGCGGCTGGGGTGCCGGTAACCTTTATCAAGGTGCATCACCAGCCGGATGGCCA TTTCCCTAACGGTATCCCGAATCCGCTGCTGCCGGAGTGCCGCCAGGATACCGCCGACGCGGTGCGCGAGCATCA GGCCGACATGGGGATTGCCTTTGACGGCGACTTCGATCGCTGCT TCCTGTTCGATGACGAAGCTTCGTTTATCGA GGGGTATTACATTGTCGGCCTGCTGGCTGAGGCGTTCCTGCAGAAGCAGCCGGGAGCGAAAATCATTCACGACCC GCGCTTGACGTGGAACACGGTAGACATCGTGACCCGCAACGGCGGCCAGCCGGTGATGTCGAAGACGGGGCATGC GTTCATCAAGGAGCGGATGCGTCAGGAAGACGCTATCTACGGCGGGGAGATGAGTGCGCACCAT TACTTCCGCGA TTTCGCCTACTGCGATAGCGGGATGATCCCGTGGCTGCTGGTGGCGGAGCTGCTGTGTCTGAAGAACAGCTCGCT GAAATCGCTGGTGGCGGACCGCCAGAAGGCGTTCCCTGCGTCGGGAGAGATCAACCGCAAGCTAAGTAATGCTGC TGAGGCGATCGCCCGCATCCGGGCGCAGTATGAGCCGGCGGCTGCACACATCGACACAACGGACGGGATCAGTAT TGAATACC CTGAATGGCGCTTTAACCTGCGCACGTCTAACACCGAGCCGGTGGTGCGTCTGAACGTTGAGTCCAG AGCTGATGTGGCGCTTATGAATGAAAAAACGACCGAGCTGTTACACCTGTTAAGCGGGGAATAAGGTGAGAGATT TACTAACGACGATTTATCGTTATCGGGGATTTATCTGGAGCAGTGTTAAACGTGATTTTCAGGCACGCTATCAAA CTAGTATGCTGGGCGCACTATCT CGTTTTACAACCGCTCTCTATGATTCTGGTCTATACCCTGGTTTTTTCCG AGGTGATGAAGGCAAGAATGCCCGATAATACCGGGTCGTTTTGCCTATAGTATTTATCTCTGTTCCGGGGTACTGA CCTGGGGATTATTTACTGAGATGCTGGATAAAGGTCAGAGCGTATTTATTAACAATGCTAATCTGATCAAGAAAC TCAGTTTTCCGAAAATCTGTCTGCCGATCATCGTGACGTTATCG GCGGTGCTAAATTTCGCGATTATTTTCAGTC TGTTTCTAATTTTTATCATTGTCACCGGTAACTTCCCCGGCTGGCTCTTTCTCTCGGTGATACCGGTCCTGCTTT TGCAGATCCTGTTTGCCGGTGGGCTGGGGATGATCCTTGGTGTCATGAACGTCTTTTTCAGGGATGTGGGGCAAC TGGTTGGCGTTGCGCTGCAATTCTGGTTTTGGTTCACACCCATTGTTTATGTACTGAATTCAT TACCTGCATGGG CAAAAAATCTGATGATGTATAACCCGATGACTCGGATCATGCAATCTTATCAGTCCATCTTCGCCTATCATCTGG

- 75 046206

CCCCCAACTGGTATTCGCTATGGCCAGTATTGGCTCTCGCCATTATTTTCTGCGTCATCGGTTTCAGGATGTTCC GCAAGCATGCGGCGGATATGGTGGATGAATTATAATGAGTTATATCAGAGTAAATAATGTCGGTAAGGCGTATCG CCAGTATCACTCAAAGACCGGGAGACTGATCGAATGGTTATCCCCTCTGAATACCAAACGCCATAATTTGAAATG GATCCTCCGCGATATTAATTTC GAAGTCGCTCCGGGCGAGGCTGTCGGTATTATCGGTATCAACGGTGCAGGCAA GAGTACCCTGCTTAAACTCATAACCGGGACGTCCAGGCCGACGACTGGAGAAATTGAAATCTCCGGACGTGTCGC TGCATTACTCGAATTGGGGATGGGGTTTCATTCGATTTTCACTGGTCGGCAGAATGTTTATATGTCTGGGCAACT GTTGGGGTTATCGTCAGAGAAAATAACTGAACTGATGCCGCAAATTGAAG AGTTTGCTGAGATTGGGGACTATAT CGATCAACCTGTGCGCGTCTACTCCAGTGGGATGCAAGTTCGATTAGCTTTTAGTGTAGCGACGGCTATCCGTCC TGATGTGCTAATTATCGATGAGGCATTATCTGTTGGGGATGCATATTTCCAGCATAAAAGCTTTGAGCGTATTCG AAAATTTCGTCAGGAAGGGACCACGCTGTTGCTGGTATCCCATGATAAACAAGCGATCCAAAGCATTTGCGACC G GGCCATTTTATTGAATAAAGGCCAAATTGAAATGGAAGGTGAACCTGAAGCAGTGATGGATTATTACAATGCTCT TCTGGCCGATAAACAAAATCAGTCCATTAAACAAGTTGAGCATAATGGTAAAACGCAAACTGTTTCAGGCACTGG TGAGGTGACTATCTCTGAGGTTCATCTTCTCGATGAACAGGGCAATGTGACTGAATTTGTTTCGGTAGGGCATCG TGTCAGCTTGCAGGTCAACGTTGAGG TCAAGGACGATATTCCTGAGCTTGTTGTCGGATATATGATTAAGGATCG ACTTGGGCAGCCGATTTTCGGGACCAATACGTACCATCTCAATCAGACACTCACCTCCCTGAAAAAAGGAGAAAA GCGTTCGTTCTTATTTTCTTTCGATGCGAGATTGGGGGTTGGCTCCTATTCTGTCGCTGTCGCGTTGCATACTTC CAGTACGCACCTCGGCAAAAACTATGAATGGCGCGATCTGGCCGTGGTA TTCAACGTCGTTAACACGGAACAACA AGAGTTTGTCGGCGTGTCCTGGTTGCCGCCTGAACTGGAGATTTCTTAATGGGTTCGTCGTTTTATCGTTCATTT GAAGAACGACACAGAGGTTCGGTTGAAGAAATCAAGCGCCGCTTGAGTTTTTATTTACCTTTTCTTGCAGGTCTG AAGGACATTTATCCTGATGGCGTGATTGCGGATATTGGTTGCGGACGTGGCGAATGGTTGGAGATCC TGACTGAA AATGGCATTGCGAACATCGGCGTCGATCTCGATGATGGCATGCTGGCGCGCGCCAGGGAGGCCGGACTGAATGTG CAGAAAATGGATTGTCTGCAGTTTTTTGCAAAGTCAGGCGGATCAGAGCCTGATAGCGTTGACCGGTTTTTCATATT GCTGAGCATTTGCCGTTTGAGGTCCTGCAGCAACTCGCCATGCATACCCTACGGGTGCTGAAACCAGGTGGTTTG CTGATCCTCG AAACGCCGAACCCGGAGAATGTAAGCGTCGGCACCTGTTCATTTTATATGGATCCAACGCATAAT CATCCTCTGCCACCGCCACTGCTTGAGTTTTTACCTATTCATTATGGTTTTACCCGAGCAATTACCGTTCGTCTG CAGGAAAAAGAGGTTCTTCAATCTCCGGATGCAGCCGTTAATTTGGTCGATGTACTCAAAGGGGTGAGCCCCGAC TACAGCATCATTGCTCAAGAAAGCAGCGCCAACAGATA TTCTTGAACGCTTTGACACCCTGTTTACCCAGCAGTAC GGTCTGACGCTGGATGCTCTGAGCAACCGTTACGATGCGATTTTGCGCCAACAGTTTTCGTCCGTTGTCTCACGG CTGGAGACGTTGAACCAAACCTATATGCAACAGATAAGCCAAATGTCAGAGACTATTCAGACGTTGCAAGGTGAG GTTGACGATCTGAGTCATGTCATCGATCAGAACCATCAGCTTCATCAGCAAATGGC ATTTACATAACAGTCGT TCATGGCGTATTACTCAACCACTACGCTGGTTGTCTTTGCAACGTCAATTATTACGTCAGGAAGGGGCTAAAGTG CGAGCCCGTAGGGCTGGGAAAAAAATATTGCGCAAAGGGATGGCGCTCTCGCTGGTCTTTTTCCATCGTTACCCT AAGTCTAAGGTTTATCTGTTTAAGGTTCTGAGAAAAACTGGCTGCTATACATTGCTACAACGTTTGTTCCAACGC GTA AT GCT GGT GCAAT CT GACACGAT GAT GATGCAGT CCAGAAGATAT GAT GT GGGTACT GAAGAAAT GACAAGT CGCGCGATGAGTATTTATAACGAATTAAAAAAAATAAAAATACGGAGAAATAACGATGCGTATTGTCATAGATTTAC AAGGCGCACAGACGGAAAGCCGCTTTCGTGGCATCGTCGTTATAGTATCGCAATCGCCAGAGGCATAATCAGAA ATAACAG CCGGCATGAGATTTTCATCGCGCTATCCGCCATGCTGGATGAGTCGATTGCAAATATTAAGGCGCAAT TTGCCGATCTCCTGCCGGCAGAAAATATAGTCGTATGGCATGCCGTAGGCCCTGTTCGTGCGATGGACCAAGGTA ATGAATGGCGTCGGGAGAGCGCAGAACTGATTCGGGAAGCGTTTCTTGAATCATTGTGTCCAGATGTCGTTTTCA TTACGAGTTTGTTTGAAGGTCATGTCGACGATGCGGCTACATCGGTACACAAATTTAGTCGTCAGTATAAAGTAG

- 76 046206

CCGTACTGCACCACGATCTTATCCCCCTCGTGCAGGCGGAAACCTATCTGCAGGACGATGTATACAAACCCTACT ATTTACAGAAAGTTGAGTGGTTAAAAAACGCTGACCTTTTGTTGACTAACTCTGCTTATACCGCACAGGAAGCGA TCGAGCATCTGCATTTACAGGGCGATCATGTGCAGAATATTGCAGCCGCAGTCGATTCTCAGTTTTGTATGGCGG AGGTGGCAGCGAGCGAAAA AGAGACCGTCCTTGGCCATTACGGTATTCAGCGCGAGTTCATGTTGTATGCGCCCG GAGGATTTGACTCAAGGAAAAACTTTAAACGGTTGATTGAGGCCTATGCCGGGCTCAGTGATGCCTTACGTCGCA GTCATCAACTGGTCATCGTCAGTAAGCTTTCCATCGGTGATCGTCAGTATCTGGAATCCCTTGCGTCAGGTAATG GTTTACAGCAGGGCGAACTGGTACTCACTGGTTATGTGCCG GAAGATGAGCTGATCCAGCTCTATCGCCTATGTA AGCTGTTCATCTTTGCTTCACTACATGAAGGTTTTGGTTGCCGGTTCTGGAAGCAATGTCGTGCGGTGCGCCGG TGATTGGCTCAAATGTCACCAGTATTCCTGAAGTCATCGGTAATCCTGAGGCATTATTCGACCCGTATTCTGTCT CTTCCATGAGGGATAAGATCGCGCAATGTTTGACTGATGATACCTTCCTCGCGC GTCTGAAAGAAATGGCGCAGC AGCAAGCGCGTAATTTCTCTTGGGATAAAGCTGCGGTGACTGCTCTGGAAGCTTTCGAAAAGATCGCGGTAGAAG ACACCGGTACTGCGCAGGTTTTGCCTGAAGCTTTGATTCAGAAGATCCTTGCTATCTCACAAGGGCAGCCAGATG ACCGCGATCTGCGCTTGTGCGCAACGGCCATTGATTACAATCTGAAAACGGCAGAACTTTATCAAATCGACGATA A ATCGCTGAACTGGCGTGTGGAAGGCCCATTCGATAGCTCATATAGTCTGGCGTTGGTCAACCGCGAATTTGCCC GGGCACTCTCAGCCGATGGTGTAGAGGTTTTATTGCATTCCACTGAAGGACCAGGTGATTTTGCCCCAGATGCCT CGTTTATGGCACAGTCGGAAAATAGTGATCTTCTGGCATTTTATAATCAATGTCAGACCCGCAAGAGTAACGAAA AGATAGATATTATTAGCAGAAA TATCTATCCACCGGGTTACCAAAATGGATGCCAAAGTAAAATTCCTTCATT GTTATGCTTGGGAAGAAACGGGCTTTCCGCAACCGTGGATCAATGAATTTAATCGGGAACTTGACGGAGTGCTGT GTACTTCGGAACATGTTCGTAAAATACTGATTGATAACGGACTGAATGTGCCCGCATTTGTTGTTGGCAATGGCT GTGACCATTGGCTCAATATCCCAGCCGAGACGACAAAAGATGTGGA TCACGGAACATTCCGTTTCCTGCACGTCT CTTCTTGTTTCCCACGCAAAGGGATACAGGCAATGCTTCAGGCTTGGGGGAAGGCGTTCACTCGTCGTGACAATG TTATCTTAATCATTAAGACTTTTAACAATCCGCACAATGAAATTGACGCATGGCTGGCTCAGGCCCAGGCTCAAT TCATAGACTATCCCAAAGTTGAAGTGATCAAAGAGGATATGTCAGCCACCGAGCTTAAAGGGCTTTATGAAA GCT GTGATGTTTTGGTTGCTCCAGGTTGCGCTGAAGGCTTTGGTTTACCTATTGCTGAAGCAATGCTGAGTGGGCTAC CGGCTATCGTCACCAATTGGAGCGGGCAACTTGATTTTGTTAATTCACAAAATTCATGGCTGGTTGACTATCAGT TCACTCGGGTAAAAACGCACTTTGGTCTGTTTTCCTCAGCCTGGGCCAGTGTGGATATTGACAACTTAACAGATG CATTAAAAGCGGCAGCCTCA ACCGATAAATCAGTGCTGCGTGACATGGCCAATGCTGGTCGCGAGCTTCTTCTGC AGCAGTTTACCTGAAAGCGGTGGCTGATCGTTCTTGCCAGGCGGTCAAGACTCTGCGTGCGCATATTGATATTG CACAGCATCGGGCGCGCATTGGCTGGGTGACGACCTGGAACACGAAATGTGGGATCGCAACCTATTCCCAGCATC TGGTGGAAAGCGCACCTCATGGCGCGGATGTTGTTTTTGCTCC CCAGGTCAGCGCTGGCGATCTTGTGTGTGCAG ACGAAGAGTTTGTACTTCGCAACTGGATTGTAGGTAAAGAGAGCAACTATCTGGAAAACCTCCAGCCACACATTG ATGCTCTGAGACTCGATGTCATTGTGATCCAATTCAACTATGGATTCTTTAATCATCGAGAACTGTCGGCGTTTA TTCGTCGCCAGCATGACGCCGGTCGTTCAGTTGTTATGACGATGCACTCAACTGTGGATCCGCT GGAAAAAGAGC CGAGCTGGAATTTCCGTCTTGCTGAAATGAAAGAGGCGCTGGCACTTTGCGACCGGTTGTTGGTGCATTCGATTG CCGATATGAACCGCCTTAAAGATTTAGGCTTAACTGCGAATGTTGCTTTATTCCCGCACGGTGTTATCAACTACT CCGCAGCGAGCGTCACACGTCAACAGCAGTCTTTACCGCTAATTGCGAGCTATGGCTTCTGCTTACCGCATAAGG GCCTGATGGAACT AGTAGAATCCGTCCATAGACTCAAGCAAGCCGGTAAACCGGTTCGTTTACGACTGGTGAACG CAGAGTATCCTGTTGGGGAGTCACGCGATCTGGTGGCAGAGCTTAAAGCTGCTGCTCAGCGGTTAGGTGTTACCG ATCTGATTGAGATGCATAATGATTTCCTACCTGATGCGGAGAGTCTGCGGTTGCTTTCAGAAGCCGATCTTCTGA TTTTTGCTTATCAGAATACTGGGGAGTCTGCTAG CGGGGCGGTACGTTATGGTATGGCGACTCAAAAACCTGTTG

- 77 046206

CGGTAACGCCCCTGGCGATATTTGATGATTTGGACGATGCCGTCTTTTAAATTTGATGGATGCAGCGTCGATGATA TCAGTCAGGGGATTGACCGGATCCTGAATTCCATCCGTGAACAGAACTCTTGGGCAACCAGGACTCAACAACGTG CCGATGCATGGCGGGAACAACATGATTATCAAGCTGTTTCACGCCGTCTGGTTAATATGTGTCAAGGCTTAGCTA AAGCTAAATATTTTAAATAAAAATA TCTCTCTTGTATTTTTTGCCTTTGAATACAAGAGGGGTTAGATAATGTGT CATTTATTATGAAAATTATTTTTGCTACTGAGCCAATTAAATACCCATTAACGGGCATCGGTCGGTATTCCCTGG AGCTGGTTAAGCGGCTGGCGGTCGCCCGCGAAATTGAAGAATTAAAGCTATTTCACGGTGCGTCGTTTATAGAAC AGATCCCTTTGGTGGAGAATAAAAGCGATACCAAAGCCAAGCAATCAT GGTCGTCTGTCGGCGTTTCTACGCCGAC AGACGCTGTTGATTGAGGCTTATCGCTTGCTGCATCCGCGGCGCCAGGCGTGGGCATTGCGCGACTATAAGGATT ATATCTACCATGGCCCCAATTTTTATCTGCCGCATAAACTGGAACGCGCCGTGACCACGTTTCATGACATATCCA TTTTTACCTGCCCGGAATATCATCCAAAAGATCGGGTTCGCTATATGGAGAAGTCCCTGCATGAGAGTCTGGA TT CGGCAAAGCTGATCCTGACCGTTTCTGATTTCTCGCGCAGTGAAATTATCCGCTTGTTCAACTATCCGGCGGAGC GGATCGTAACCACCAAGCTAGCCTGCAGCAGTGACTATATCCCACGCAGCCCGGCAGAGTGTCTGCCGGTACTGC AGAAATATCAGCTGGCGTGGCAGGCCTACGCGCTATATATCGGCACTATGGAGCCACGTAAAAATATCCGAGGCC TGCTGCATGCCTATCAGCT GCTACCGATGGAGATCCGCATGCGCTATCCGCTAATCCTTAGCGGCTATCGCGGCT GGGAAGACGATGTGCTGTGGCAGTTAGTCGAGCGCGGTACTCGGGAAGGCTGGATCCGTTACCTCGGATATGTTC CGGATGAAGACCTGCCGTATCTGTACGCAGCGGCCAGAGTCTTTGTTTATCCCTCCTTCTACGAGGGATTCGGTT TACCTATTCTTGAAGCGATGGGTCTTGCGGTGTGCC TAGTATGCTCCAATGTCACCTCTTTGCCTGAGGTTGTTG GCGATGCCGGCCTCGTTGCCGATCCTAATGATATAGACGCGATTAGCGCGCAAATTTTGCAGAGCCTGCAAGATG ATAGCTGGCGGGAAATCGCCACCGCGCGCGGTCTTGCTCAGGCGAAACAGTTTTCGTGGGAGAACTGTGCGACAC AGACCATTAACGCCTATAAATTACTCTAAGGGTGTCAGTTGAGAGTTCTACACG TCTATAAGACTTACTATCCCG ATACCTACGGCGGTATTGAGCAGGTCATTTATCAGCTAAGTCAGGGCTGCGCCCGCCGGGGAATCGCAGCCGATG TTTTCACTTTTAGCCCGGACAAAGATACAGGTCCTGTCGCTTACGAAGATCATCGGGTCATTTATAATAAACAGC TTTTTGAAATTGCCTCCACGCCGTTTTCGCTGAAAGCGTTAAAGCGTTTTAAAGCTGATTAAAGATGACTACGATA TCAT CAACTACCATTTTCCGTTTCCCTTTATGGATATGCTGCATCTTTCGGCGCGGCCTGACGCCAGGACTGTGG TGACCTATCACTCTGATATAGTGAAACAAAAACGGTTAATGAAGCTGTACCAGCCGCTGCAGGAGCGATTTCTCA GCGGCGTAGATTGCATCGTTGCCTCGTCGCCCAATTACGTGGCTTCCAGCCAGACCCTGAAAAAATATCTGGATA AAACGGTGGTGATCCCGTTTGGTCTG GAGCAGCAGGACGTGCAGCACGATCCGCAGAGGGTCGCGCACTGGCGGG AAACTGTCGGCGATAAGTTCTTTCTTCGTCGGCACTTTCCGCTACTACAAAGGGCTGCATATTCTGATGGATG CCGCTGAGCGTAGCCGACTGCCAGTGGTGGTTGTAGGGGGCGGGCCGCTGGAATCGGAAGTGCGGCGTGAAGCGC AGCAGCGCGGGCTGAGCAATGTGATGTTTACCGGCATGCTCAA CGACGAAGATAAGTACATTCTCTTCCAGCTCT GCCGGGGCGTGGTATTCCCCTCGCATCTGCGCTCTGAGGCGTTTGGCATTACGTTATTGGAAGGCGCACGCTTTG CAAGGCCGCTGATCTCTTGCGAGATCGGTACAGGTACCCTTTCATTAACCAGGACAAAGTGAGTGGTTGCGTGA TTCCGCCGAATGATAGCCAGGCGCTGGTGGAGGCGATGAATGAGCTCTGGAATAACGAGGAAACC TCCAACCGCT ATGGCGAAAACTCGCGTCGTCGTTTTGAAGAGATGTTTACTGCCGACCATATGATTGACGCCTATGTCAATCTCT ACACTACATTGCTGGAAAGCAAATCCTGAGCGGCCGCGAGCTCGTCGACTCGAGGATCCGTGTAGGCTGGAGCTG CTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTAAGGAGGATATTCATATGGATAAAG CCGTAAGCATATAAG CATGGATAAGCTATTTATACTTTAATAAGTACTTTGTATACTTATTTGCGAACATTCCAG GCCGCGAGCATTCAGCGCGGTGATCACACCTGACAGGAGTATGTAATGTCCAAGCAACAGATCGGCGTAGTCGGT ATGGCAGTGATGGGACGCAACCTTGCGCTCAACATCGAAAGCCGTGGTTATACCGTCTCTATTTTCAACCGTTCC CGTGAGAAGACGGAAGAAGTGATTGCCGAAAA TCCAGGCAAGAAACTGGTTCCTTACTATACGGTGAAAGAGTTT

- 78 046206

GTCGAATCTCTGGAAACGCCTCGTCGCATCCTGTTAATGGTGAAAGCAGGTGCAGGCACGGATGCTGCTATTGAT TCCCTCAAACCATATCTCGATAAAGGAGACATCATTGATGGTGGTAACACCTTCTTCCAGGACACTATTCGT CGTAATCGTGAGCTTTCAGCAGAGGGCTTTAACTTCATCGGTACCGGTGTTTCTGGCGGTGAAGAGGGGGCGCTG AAAGGTCCTTCTATT ATGCCTGGTGGCCAGAAAGAAGCCTATGAATTGGTAGCACCGATCCTGACCAAAATCGCC GCCGTAGCTGAAGACGGTGAACCATGCGTTACCTATATTGGTGCCGATGGCGCAGGTCACTATGTGAAGATGGTT CACAACGGTATTGAATACGGCGATATGCAGCTGATTGCTGAAGCCTATTCTCTGCTTAAAGGTGGCCTGAACCTC ACCAACGAAGAACTGGCGCAGACCTTTACCGAGTGGA ATAACGGTGAACTGAGCAGTTACCTGATCGACATCACC AAAGATATCTTCACCAAAAAAGATGAAGACGGTAACTACCTGGTTGATGTGATCCTGGATGAAGCGGCTAACAAA GGTACCGGTAAATGGACCAGCCAGAGCGCGCTGGATCTCGGCGAACCGCTGTCGCTGATTACCGAGTCTGTGTTT GCACGTTATATCTCTTCTCTGAAAGATCAGCGTGTTGCCGCATCTAAAGTTCTCTCTGG CGCAAGCACAGCCA GCAGGCGACAAGGCTGAGTTCATCGAAAAAGTTCGTCGTGCGCTGTATCTGGGCAAAATCGTTTCTTACGCCCAG GGCTTCTCTCAGCTGCGTGCTGCGTCTGAAGAGTACAACTGGGATCTGAACTACGGCGAAATCGCGAAGATTTTC CGTGCTGGCTGCATCATCCGTGCGCAGTTCCTGCAGAAAATCACCGATGCTTATGCCGAAAATCCACAGATCGCT AACCT GTTGCTGGCTCCGTACTTCAAGCAAATTGCCGATGACTACCAGCAGGCGCTGCGTGATGTCGTTGCTTAT GCAGTACAGAACGGTATTCCGGTTCCGACCTTCTCCGCAGCGGTTGCCTATTACGACAGCTACCGTGCTGCTGTT CTGCCTGCGAACCTGATCCAGGCACAGCGTGACTATTTTGGTGCGCATACTTATAAGCGTATTGATAAAGAAGGT GTGTTCCATACCGAATGGCTGGAT TAA

SEQ ID NO: 15 (example of nucleotide sequence of the rfb 015 locus - producer strain 015-EPA stLMTB11738)

ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGATACCC AAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAGGGATCAAA GAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTATGAGTTAGAATCA CTCCTTGAGCAGCGCGTGA AGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCCGGGCGTGACCATTATG AACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGACCTGCCATTGGTGACAACCCA TTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCGCTACGTTACAACCTTGCTGCCATG ATTGCACGTTTCAACGAAACGGGCCGCAGCCAGG TGCTGGCAAAACGTATGCCGGGTGACCTCTCTGAATACTCC GTCATCCAGACTAAAGAGCCGCTGGACCGTGAGGGTAAAGTCAGCCGCATTGTTGAATTTATCGAAAAACCGGAT CAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGTCGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTG GAACGTACTCAGCCTGGTGCATGGGGACGTATTCAGCTGACTGATGCTATTGCCGAGCTGGCGAAA AAACAATCC GTTGATGCAATGCTGATGACCGGCGACAGTTACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAG TATGGCCTACGCAACCTGAAAGAAGGGGCGAAGTTCCGTAAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAA TCTGACCGGATGTAACGGTTGATAAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAA TCTTCCTGCCGTT GTTTTATATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAGTTTTCCA GGATTTTCCTTGTTTCCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTA ACGCTCGTCACATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCATTAATAC CTCTATTAATCAAACTGAGAGCCGCTTATT TCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGCTAGCATG AGCAAAACTAAACTAAATGTTCTTTACCTTGCAATAAGTCAGGGTGCCAATTACCTACTGCCATTATTAATTTTT CCTTATCTTGTTAGAGTCATTGGTGTATCGAATTTTGGTGATCTGAGTTTTTCATTGATAACTATACAAGTGTTG TTAATGGTTGTTGAATATGGTTTTGGATATAGTGGGACAAGAGAAATAGCACTAAATAAC GATAAAAATACCAT TCTGAATTTTTTTGCGGTGTGGTGCTTGCTCGTTTTATATTAATGCTAATTGCAGCTATAATACTCATAATACTC

- 79 046206

TGTTTTTTTTATGTTTTTAACGACGTTAAGTCTTTGTTATGTGTTGGTTTTCTGTCCGTAATTGCAGGTGTTTTC AATCCAAATTGGTTTTTGCAAGGTAAGGAAATGATGAGTGTGATGGCTGTGCTGTCACTATTTTCACGAGGCATA GCAGTCGTTGCAGTTTATCTAATTATAAAACCCGCAACGCCGATGTACATCAGTGCCTTATTATTGAGCATGCCA TATATTTTG TATTCATTCTGTGGCGTTGCCTACTTACTTATTATCAAGGAGATTTTTTTATGTAGGCCACCGATA AAGAAAATTCAAGTAATTTTAAAAATGGATTTCATTTTTTTTGTTCAACACTTGCGACTAGTGCATACACAATG TTGACCCCTCTTGTATTGGGTGGCGTATCTGGAAAGTTTGATGTAGGCATCTTTAACTCAGCTAACATGATCAAA CAAGGTTTGGCTGGACTTGCATCACCATTAGTCCA AGCTTTTTATCCAAGAATTAACATTTTGCAAAGAGAGAAT CCATATATTGCAAACTTAAAATCTAGAATGATTCTTAAATACTTGCTTGTTTTTTACATGGCTTTAGCAATACCA TTTTTACTTTTTGCCAACCAATTATCATTATTAATAATTCGGCATGAAAGGTGAAGTAATTGCAGGTGCAATGCAA TTAATGACATTGCTTCCTATATTCATAGGTTTTTAATACAGTTGTCGGGTTACTTGTATTAG TACCTAATGGGATG CAAAAACAGTATTTCAAATCTATTTTCCTAGGAACTATTACTTGTTTAAGCATAGTTTATCCAGCATGTAAATAT TATGGAGCAACGGGTGCGATTGTGAGTCTTATTGTAGCTGAAATTTTCGTTGGCATGGGAATGCTTAAACAATTC ATTAAAGTAAATAAAACCGTATGTAGGCCTCATAAATTATGAATATCTCGGTAATAATATCTGTTTGGAAACGCC CAGTTCAATTAGAAT TGATTCTCTCTGAGCTCGATTCTCAGGCTAAAGACAATAGTCTACACCTAGAAGTAATTG T T T C GATAGT CATAGT GGTAAAGAAAT T GAT GAT GTAGT T GCT GATAATAT T CATAAAAAGAAAATAT TAATA TTATCCATCAACATACTAAAAATATCTCCGCTAAGCGCAATTTCGGAGCATCCCTAGCCCATGGGGATTATT TAATATTTCTTGATGATGATTG TATACCCGCAAGTGGATATATCATCGTTGCTGAACTATTTAAAAAAAATGA ATAGTAAAAGCGTTTTATGTGGGGAAGTTAGATTCGAAAATGAACTCATTGAGACCAGCAATTACTATCGCTACA GGAACTCTTTACACCCTAAGTTTAGTGATAGTCCTGATATCTCTATGAATGCCTGGACTTTTGTCGCAATGAATT GTGTTCTTGATAGAAAGGCATTTTCATCAGGTATAGTTTCATATAATG T TAGTAAAATAT G WITH CAAT CAATAT T T T T CAATAAAT T TAT T T T TAAAAAAATAATACAAT T T T TAAAAAAAACAG ATGCAAATAAAAAAACTCTATTTCCCAATTCTTTACAGATATGTGTTGATTTCGGCATATACATGGTATTGGAG AGCGTGGCACCTCAAAAACAGATGATTTGCTTAAGAACTGGTATATATAGATGATGCTATCTTCATTTATTAAGA CATTTGTATGGAAGGTAAAAAACAATGAAGTATAATGCATTGATGGCTTTTTTATTATTTTTTGTTGTTTTTTTT AGATTG TCGCTGATAATACCTTTCTTATATTTGGCATTTATTCCTGCATTTTTTGGTATTATGTATTTAGTGCGT AATTTTATGATTACTATGGGCAATGGATTGGTATCTATAGATCGTAAAAATTTGTTGCTGTTATCTATATTCATA ATTATTTTTTTATTTTGTTTGGTTTTTCGATTGTTTCAAAAAAGCCATTCTTTTTCAAAGTTATTTTACCGTTAGA TTATTTATGTTGTTTTTATTTTCAT TTGTTCCTGCGTATTATTTAGTAAATAGATTCATAAAGGGTGACTTGAAA TTAATGGAGCGAATATTAGTGTATTCTCTTGGGTTCAAATAGTTATTTTTTTTGGTATGTATATAAGTCCAGAG TTAAAAAGATTGTTATATACTTTCTTTGGTATGTCTGACTCTGTTAATCTTTGGGAACAAAATGCTAAAGTAAGA GGATTTGGGTTGTCGGGTGAAATAAATTTCATGACACCATTTTTGA TGATCTATATGTCATTTTTATTGATGAAA AGGCGTTATGCTTTAATTACTTTAATTTGTCTGACTCAAATCGTAAATTCTAACATGGCTGTGATTGCAGCCATT ATTGGTATCGGTTGCTCTAGACTTAATATTAATATAAAAATTGCAACAGTATTGATTTTGGGAGTTTTAGTTTAT AGCTTAGGAGCGGTGTTCTTTCCTCGATTTTATGATGAGTTCGTTTCTGGAGATGGCACAAGAACTCTGG ATATC T TAT TACAGCAACAT GTGTTTGTT GTAGGTAAT T TAGAT T T T T TTAATATTATATTT GGATTACAGCAAAACATA TCTTCATCAATCCCCGATATTAAACAAAGTTCGGATATGGGCTGGGTTATACTGTTTAATTACGGTGGGTTAACA TTTATTACACTCTTTTATTTTTAATCTTTACTATTTCTATTGCGACATTTGGAATGACATATCAAGCAATTATA

- 80 046206

TGGATGTTAATTGGGATAATTTTCAATACCAAAGGTTTAGTTTTAGGATCTAACGGCTATTTCTTTCTATCTTTT ATATATAT GT T T T T GAATAGAGTAACACT TAGT GGACAGAGT T CAAT TACTAATAAGT TAGGT CAAGTAAGTAAA TAGCTTCCAGAGTATATTTGTCAATGATTTGAGGTTCGGTTATTATGTTTTCATCTAAAACACTGTTAATTACTG GTGG TACTGGCTCTTTCGGGAATGCTGTATTAAATAGATTTCTTGATACAGATATTGCAGAAATCCGTATATTTA GT CGT GAT GAAAAAAAACAAGAT GATAT GCGGAAAAAATACAATAAT CAAAAAT TAAAGTT CTATATT GGT GAT G TCAGAGATTACCGTAGTATTTTGAATGCGACTCCGCGTGTTGATTTTATATATCATGCAGCGGCACTTAAGCAAG TTCCATCATGTGAATTTCAT CCTATGGAAGCCGTTAAAACTAATATCCTTGGTACGGAAAATGTTCTTGAAGCAG CTATAGCGAATGAAGTGAAGAGGGTTGTATGCCTAAGTACTGATAAAGCTGTATACCCGATTAACGCAATGGGTA TTTCAAAAGCTATGATGGAAAAGGTCATGGTCGCGAAATCCCGTAATGTTGATCGCAATAAAACAGTAATATGTG GTACCCGTTATGGGAATGTTATGGCATCTCGCGGTTCAG TTATTCCATTATTTGTTGATCTTATTAGAGCGGGCA AGCCACTCACAATAACTGATCCTAATATGACCCGCTTTATGATGACTCTTGAGGATGCGGTAGATTTAGTTCTTT ATGCGTTTGAACATGGTAATAATGGTGATATCTTTGTGCAAAAAGCACCTGCAGCAACTATTGACACATTAGCTA TTGCTTTAAAGGAATTACTAAATGTTCCTGACCATCCGGTAAATGTCATTGGAACGCGTCATGGCGAG AAATTAT ATGAAGCTCTACTTAGTCGTGAGGAAATGATCGCTGCTATAGATATGGGCGATTATTACCGTGTCCCGCCAGATC TTCGTGACCTTAATTATGGCAAATATGTTGAGCAAGGTGATAGCCGAATATCTGAAATAGAAGATTATAACTCTC ATAATACTCAACGGTTAGATGTTGAAGGCATGAAAGAGCTCTTGCTAAAATTAGCCTTTATTCGAGCAATTCGTG CTGGTGAAAAATATAAT CTGGATTCATGATATGAAAATATTAGTTACTGGTGCAAATGGTTTTATTGGTCGTAAT TTATGTTTGAGGCTTGAGGAACTTGGTTATAAAGATCTTATTAGAATTGATCGAGAATCAACGAAGCAAGATCTT GACAAGGCTTACAGGATGCCGATTTTATTTATCACTTAGCTGGTATCAATAGACCTAAGACTGATGATGAGTTTT ATTTCTGGAAACAGTGATTTAACAAAGCATATAGTTGA GTATCTCCTTTCTATTGGTAAGAATACACCAATTATG CTAAGTTCTTCGATACAAGCTGAACTTAATAATGCTTATGGGGTTAGCAAAGCTGTAGCTGAAAGCTATGTCGAA AAATATGCTGCTGCTAGTGGTTCTTCGTATTATATTTTCAGATATCCAAACGTTTTTGGTAAAATGGTGTAAGCCA AACTATAATTCTTTTATAGCAACTTTTTGCTACAATATTTCCAATGATATTGAGATTACT ATCAATGATGCAGCA GCGCCAGTCAATCTGGTCTATATTGATGATGTTTGTACTGATGCTATAGCTCTTCTCTGGGACGGTTGAAAGT GGATATAAAAGTTGTTGCACCAATTTATTCAACAACAGTTGGTGAAGTTGCAGAATTAATTTATAGCTTCAAAAAT AGCCGTTCCACCCTGATCACAGAGGCTGTCGGGGCGGGATTTACCCGTGCATTGTATTCTACATGGCTGAGTT AT TTACCAGCAGAGAAGTTTGCGTACAAGGTACCTTTTTATGGGGATGCCCGCGGAGTCTTTTGTGAGATGTTGAAA ACGCCTTCAGCGGGGCAGTTTTCATTTTTTACTGCTCACCCTGGTATTACGCGTGGCGGACATTACCATCACAGT AAAAATGAGAAGTTTTTGGTCATTCGAGGTCAGGCATGCTTTAAATTTGAACATGTGATTACCGGTGAGCGATAT A GAATACTGCGAGCATATTATTGTCCATACTGGTCAAAATTATGATTACGAATTAAATGAAG TGTTCTTCAATGACTTGGGTGTTCGAAAACCTGATTATTTTTTAAATGCAGCGGGTAAAAATGCGGCGGAAACCA TTGGTCAGGTTATTATTAAGGTAGATGAAGTATTAGAAATCGAAAAACCTGAAGCAATACTGGTATTGGGCGATA CGAATTCATGTATTTCTGCCATTCCGCCAAACGCCGT AAAGTGCCTATATTTCATATGGAAGCAGGTAACCGTT GTTTCGATCAACGCGTGCCTGAAGAAACCAACAGACGTATTGTTGACCATACGGCTGATATCAATATGACCTACA GTGATATTGCTCGTGAATATCTCTTGGCTGAAGGTATCCCAGCTGATCGGATCATAAAAACTGGTAGCCCTATGT TTGAGGTTCTTTCATATTATATGCCCCAAATTGATGGTTCAGATGTGCTATCGCGTTTGA ATCTACAGTCTGGTG AGTTTTTTGTAGTAAGTGCGCATCGTGAAGAGAATGTTGATTCTCCAAAACAGCTCGTAAAGCTTGCGAACATTC

- 81 046206

TAAATACTGTTGCTGAAAAATATAATCTTCCAGTTATTGTCTCCACACACCCAAGGACACGTAACCGAATCCGTG AGCAAGGAATTGAATTTCATTCAAATATAAATCTACTGAAACCATTGGGTTTCCATGATTATAACCACTTGCAGA AGAACTCACGAGCTGTGCTTTCAGATAGCGGTACTATCACTGAAGAGTCATCCATCATGAATTTCCCAGCGGTAA ACATCCGGGAAGCGCATGAGCGTC CGGAAGGCTTTGAGGAAGCATCCGTCATGATGGTGGGGTTAGAGTGTGAAC GCGTATTACAAGCGCTGGATATTCTGGCAACACAACCGCGAGGTGAAGTCCGTCTTTTACGTCAGGTTAGTGATT ACAGCATGCCAAATGTGTCGGATAAAGTTGTCAGAATTGTTCACTCTTACACAGATTATGTTAAGAGAGTCGTCT GGAAAGAATATTGATGAAACTTGCTTTAATCATAGATG ATTACCTGCCCAACAGTACTCGTGTTGGTGCAAAAAT GTTTCATGAACTTGCTCAAGAATTTATCCAGCGTGGGCACGATGTTACGGTAATTACTCCTGGTACGGGCATGCA AGAAGAGATTTCTTTTGATACCTTTCAGGGGGTAAAAACATGGCGTTTTAAAAGCGGGCCGCTCAAGGATGTAAG TAAAATTCAGCGAGCGGTCAATGAAACGCTTTTGTCCTATCGGGCGTGGAAAGCCATCAAAAAATG GGTAAAAAA AGAGACCTTTGAGGGGGTGATTTATTATTCACCTTCCATATTCTGGGGGCCTTTAGTTAAAAAAATTAAAGCTCG TTGCCAATGTCCTGCTTATCTTATTTTAAGAGATATGTTTCCACAATGGGTAATTGATGCAGGAATGCTTAATGC TGGTTCCCCAATAGAACGCTACTTTCGTCTTTTTGAAAAATATCTTATCGTCAGGCAAATCGTATTGGACTTAT GTCTGATAAGAATCTT A ATTAATTAATTCATTAGCATCTGAGTGGGC ATTGACGAATTTTCACCTATTTGCCCTCGGTTAACCAAGATGAATTTAAGTTCATTTTGTCGGAAATGGATATCGG CTTGTTTTCTCTTTCCGCTAGACACTCTTCCCATAATTTTCCTGGTAAGTTATTAGGCTATATGGTTCAGTCGCT ACCTATTTTAGGTAGCGTAAATGCCGGAAATGATTTGCTCGACATTGTCAATCAAAATAATGCGGGATT AATCCA TGTCAATGGTGAGGACGATAAATTATGTCAATCTGCGCTATTAATGTTGCATGATATTGATGTGCGCCGGCAACT TGGTTCGGGGGCGAATATATTGTTGAAAGAACAATTCTCCGTTGAGTCTGCGGCACAGACGATAGAAATGAGGTT GGAGGCATGCAATGCGATTAATTGATAATGACCAACTCGACGAATTATATGATCAAGCCGGGCAATCGGAACGTT TACGTTCCCA CCTTATGATGCACGGCTCGCATCAAGAAAAGGTACAGCGTTTACTTATTGCATTAGTAAAGGGCA GCTATGTTGAACCGCATTATCACGAACTTCCTCATCAGTGGGAAATGTTCATTGTTATGGAGGGGCAACTTCAGG TTTGTTTGTATGGTAGAAATGGTGAGGTTATAAAGCAATTTATAGCAGGAGATAATACTGGAATGAGCATTGTGG AGTTTTCTCCGGGCGATATACACAGTG TCGAATGCCTATCTCCGCGTGCTCTTATGGTGGAAGTTAAGGAGGGGC CATTTGACCCTTCTTTTGCAAAATCGTTCGTGTGAGCGGCCGCGAGCTCGTCGACTCGAGGATCCGTGTAGGCTG GAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTAAGGAGGATATTCATATGG ATAAAGCCGTAAGCATATAAGCATGGATAAGCTATTTATACTTTAATAAGTACT TTGTATACTTATTTGCGAACA TTCCAGGCCGCGAGCATTCAGCGCGGTGATCACACCTGACAGGAGTATGTAATGTCCAAGCAACAGATCGGCGTA GTCGGTATGGCAGTGATGGGACGCAACCTTGCGCTCAACATCGAAAGCCGTGGTTATACCGTCTCTATTTTCAAC CGTTCCCGTGAGAAGACGGAAGAAGTGATTGCCGAAAATCCAGGCAAGAAACTGGTTCCTTACTATACGGTG AAA GAGTTTGTCGAATCTCTGGAAACGCCTCGTCGCATCCTGTTAATGGTGAAAGCAGGTGCAGGCACGGATGCTGCT ATTGATTCCCTCAAACCATATCTCGATAAAGGAGACATCATCATTGATGGTGGTAACACCTTCTTCCAGGACACT ATTCGTCGTAATCGTGAGCTTTCAGCAGAGGGCTTTAACTTCATCGGTACCGGTGTTTCTGGCGGTGAAGAGGGG GCGCTGAAAGGTCCTTCTA TTATGCCTGGTGGCCAGAAAGAAGCCTATGAATTGGTAGCACCGATCCTGACCAAA ATCGCCGCCGTAGCTGAAGACGGTGAACCATGCGTTACCTATATTGGTGCCGATGGCGCAGGTCACTATGTGAAG ATGGTTCACAACGGTATTGAATACGGCGATATGCAGCTGATTGCTGAAGCCTATTCTCTGCTTAAAGGTGGCCTG AACCTCACCAACGAAGAACTGGCGCAGACCTTTACCGAGTG GAATAACGGTGAACTGAGCAGTTACCTGATCGAC

- 82 046206

ATCACCAAAGATATCTTCACCAAAAAAGATGAAGACGGTAACTACCTGGTTGATGTGATCCTGGATGAAGCGGCT AACAAAGGTACCGGTAAATGGACCAGCCAGAGCGCGCTGGATCTCGGCGAACCGCTGTCGCTGATTACCGAGTCT GTGTTTGCACGTTATATCTCTTCTCTGAAAGATCAGCGTGTTGCCGCATCTAAAGTTCTCTCTGGTCCGCAAGCA CAGCCAGCAGGCGACAAGGCTGAG TTCATCGAAAAAGTTCGTCGTGCGCTGTATCTGGGCAAAATCGTTTCTTAC GCCCAGGGCTTCTCTCAGCTGCGTGCTGCGTCTGAAGAGTACAACTGGGATCTGAACTACGGCGAAATCGCGAAG ATTTTCCGTGCTGGCTGCATCATCCGTGCGCAGTTCCTGCAGAAAATCACCGATGCTTATGCCGAAAATCCACAG ATCGCTAACCTGTTGCTGGCTCCGTACTTCAAGCAAATTGCCGATGACT ACCAGCAGGCGCTGCGTGATGTCGTT GCTTATGCAGTACAGAACGGTATTCCGGTTCCGACCTTCTCCGCAGCGGTTGCCTATTACGACAGCTACCGTGCT GCTGTTCTGCCTGCGAACCTGATCCAGGCACAGCGTGACTATTTTGGTGCGCATACTTATAAGCGTATTGATAAA GAAGGTGTGTTCCATACCGAATGGCTGGATTAA

SEQ ID NO: 16 (example of nucleotide sequence of the rfb 016 locus - producer strain 016-EPA stLMTB11739)

ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGATACCC AAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAGGGATCAAA GAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTATGAGTTAGAATCA CTCCTTGAGCAGCGCGTGA AGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCCGGGCGTGACCATTATG AACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGACCTGCCATTGGTGACAACCCA TTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCGCTACGTTACAACCTTGCTGCCATG ATTGCACGTTTCAACGAAACGGGCCGCAGCCAGG TGCTGGCAAAACGTATGCCGGGTGACCTCTCTGAATACTCC GTCATCCAGACTAAAGAGCCGCTGGACCGTGAGGGTAAAGTCAGCCGCATTGTTGAATTTATCGAAAAACCGGAT CAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGTCGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTG GAACGTACTCAGCCTGGTGCATGGGGACGTATTCAGCTGACTGATGCTATTGCCGAGCTGGCGAAA AAACAATCC GTTGATGCAATGCTGATGACCGGCGACAGTTACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAG TATGGCCTACGCAACCTGAAAGAAGGGGCGAAGTTCCGTAAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAA TCTGACCGGATGTAACGGTTGATAAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAA TCTTCCTGCCGTT GTTTTATATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAGTTTTCCA GGATTTTCCTTGTTTCCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTA ACGCTCGTCACATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCATTAATAC CTCTATTAATCAAACTGAGAGCCGCTTATT TCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGTGAAAATA CTTGTTACTGGTGGCGCAGGATTTATTGGTTCAGCTGTAGTTCGTCACATTATAAATAATACGCAGGATAGTGTT GTTAATGTCGATAAATTAACGTACGCCGGAAACCGGGAATCACTTGCTGATGTTTCTGATCTGAACGCTATGTT TTTGAACATGCGGATATTTGCGATGCACCTGCAATGGCACGGATTTTTGCTCAGCAT CAGCCGGATGCAGTGATG CACCTGGCTGCTGAAAGCCATGTTGACCGTTCAATTACAGGCCCTGCGGCATTTATTGAAACCAATATTGTTGGT ACTTATGTCCTTTTGGAAGCCGCTCGCAATTACTGGTCTGCTCTTGATAGCGACAAGAAAAATAGCTTCCGTTTT CATCATATTTCTACTGACGAAGTCTATGGTGATTTGCCTCATCCAGATGAAGTAAATAATACAGAAGAATTACCC TTAT TTACTGAGACGACAGCTTACGCGCCAAGCAGCCCTTATTCCGCATCCAAAGCATCCAGCGATCATTTAGTC CGCGCGTGGAAACGTACATATGGTTTACCGACAATTGTGACTAATTGCTCGAACAACTATGGTCCTTATCATTTC CCGGAAAAGCTTATTCCACTGGTTATTCTTAATGCACTGGAAGGTAAGGCATTACCTATTTATGGCAAAGGAGAT CAGATCCGCGACTGGTTGTATGTTGAAGATCATGC GCGTGCGTTATATACCGTCGTAACCGAAGGTAAAGCGGGT GAAACTTATAACATTGGTGGGCACAACGAAAAGAAAAACATCGATGTAGTGCTCACTATTTGTGATTTGCTGGAT

- 83 046206

GAGATTGTACCGAAAGAGAAATCTTATCGTGAGCAAATCACTTATGTTGCTGATCGTCCGGGACACGATCGCCGC TATGCTATTGATGCTGAGAAGATTGGTCGCGCATTGGGATGGAAACCACAGGAAACGTTTGAGAGCGGGATTCGT AAAACGGTGGAATGGTACCTGTCCAATACAAAATGGGTTGATAATGTGAAAAGTGGTGCCTATCAATCGTGGATT GAACAGAACTATGAGGGC CGCCAGTAATGAATATCCTCCTTTTTGGCAAAACAGGGCAGGTAGGTTGGGAACTAC AGCGTGCTCTGGCACCTTTGGGTAATTTGATTGCTTTTGATGTTCACTCTACTGATTATTGCGGTGATTTTAGTA ATCCTGAAGGTGTAGCTGAAACCGTAAGAAGCATTCGGCCGGATATTATTGTCAATGCAGCCGCTCACACCGCAG TAGACAAAGCAGAATCAGAACCGGAGTTTGCACAATTAATTAAC GCAACAAGTGTCGAAGCGATTGCGAAAGCAG CAAATGAAGTTGGAGCCTGGGTTATCCATTACTCGACTGATTACGTCTTCCCTGGAAATGGCGATATGCCATGGC TGGAGACGGATGCAACCGCACCACTAAATGTTTACGGTGAAACCAAGTTAGCCGGAAAAAGCGTTACAGGAAT ATTGCGCGAAGCATCTTATTTTCCGGACCAGCTGGGTCTATGCAGGAAAAGGAAATAACTTCGCCAAAAC GATGT TACGTCTGGCAAAAGAGCGTGAAGAATTAGCGGTTATTAACGATCAGTTTGGTGCGCCAACAGGTGCTGAACTGC TGGCTGATTGTACAGCACATGCCATTCGTGTCGCACTGAATAAACCGATGTCGCAGGCTTGTACCATTTGGTAG CCAGTGGTACCACAACCTGGTACGATTATGCTGCGCTGGTTTTTTGAAGAGGCGCGCAAAGCAGGCATTCCCCTTG CACTCAACAAG CTCAACGCAGTACCAACAACAGCCTATCCTACACCAGCTCGTCGTCCACATAACTCTCGCCTTA ATACAGAAAAATTTCAGCAGAACTTTGCGCTTGTCTTGCCTGACTGGCAGGTTGGCGTGAAACGAATGCTCAATG AATTATTTACGACTACAGCAATTTAATAGTTTTTGCATCTTGTTCGTGATGGTGGAGCAAGATGAATTAAAAGGA ATGATGAAATGAAAATGCGTAAAAGGTATTATTTTAGC GGGTGGTTCTGGTACACGTCTTTATCCTGTGACTATGG CTGTCAGTAAACAGCTATTACCTATTTATGATAAACCGATGATCTATTACCCGCTCTCTACACTGATGTTGGCGG GTATTCGCGATATTTTGATTATCAGTACACCTCAGGATACTCCTCGTTTTCAACAATTGCTGGGTGACGGTAGCC AGTGGGGCCTGAATCTTCAGTACAAAGTGCAACCTAGCCCAGATGGCCTCGCG CAGGCATTTATCATCGGTGAAG AGTTTATTGGTGGTGATGATTGTGCTTTGGTTCTTGGTGATAATATCTTTTACGGTCACGATCTGCCGAAGCTAA TGGAGGCCGCTGTTAACAAAGAAAGTGGTGCAACGGTATTTGCCTATCACGTTAATGATCCAGAACGCTATGGTG TCGTTGAGTTTGATAAAAACGGTACGGCAATCAGTCTGGAAGAAAAAACCGTTAGAACCAAAGAGTAATTA CGCCG TTACAGGTCTGTACTTTTATGATAACGACGTGGTTCAGATGGCGAAAAACTTGAAGCCGTCTGCACGTGGTGAGT TAGAAATTACAGATATTAACCGTATTTATCTTGAGCAGGGACGTCTGTCTGTCGCGATGATGGGGCGTGGCTACG CGTGGCTGGACACGGGGACTCATCAGAGTCTGATAGAAGCAAGTAATTTTATTGCGACAATTGAAGAGCGCCAGG GATTGAAGGTTTCCT GTCCTGAAGAGATTGCATTTCGTAAAGGTTTTATTGATGTTGAGCAAGTAAGAAAATTAG CTGTACCACTAATAAAGAATAATTATGGGCAGTATCTTTATAAAATGACGAAGGATTCAAATTAATGAATGTGAT TAGAACTGAAATTGAAGATGTGCTAATTCTGGAGCCAAGAGTATTTGGTGATGATAGAGGTTTCTTTTATGAGAG CTTTAATCAATCAGCATTTGAACATATTCTAGG CTATCCGGTCAGCTTTGTTCAAGACAATCACTCACGTTCATC AAAAAATGTACTCAGAGGCCTTCACTTTCAACGCGGCGAGTACGCACAAGATAAACTTGTACGCTGCACTCATGG AGCAGTTTTTGATGTTGCTGTTGATATTCGACCCAATTCGGTATCCTTTGGTAAATGGGTTGGTGTTCTGCTTTC AGCTGATAATAAGCAGCAGTTGTGGATACCAAAAGGGTTTGCTCATGGCTTTTTTTTTCT GTCTGATATCGCTGA ATTTCAATATAAAAACTACAAACTATTATCATCCTGAAAGCGATTGTGGAATATGTTGGAATGATGAACGCATTGC AATTGATTGGCCCCAAACATCAGGGTTAATCCTTTCGCCAAAAGATGAAAGGCTCTTTACGTTAGATGAGCTTAT CAGATTAAAATTAATTGCATGAATACGAATAAATTATCTTTAAGAAGAAACGTTATATATCTGGCTGTCGTTCAA GGTAGCAATTA TCTTTTACCATTGCTTACATTTCCATATCTTGTAAGAACACTTGGTCCTGAAAATTTCGGTATA TTCGGTTTTTGGCCAAGCGACTATGCTATATATGATAATGTTTGTTGAATATGGTTTCAATCTCACAGCAACTCAG AGTATTGCCAAAGCAGCAGATAGTAAAGATAAAGTAACGTCTATTTTTTGGGCGGTGATATTTTCAAAAATAGTT CTTATCGTCATTACATTGATTTTCTTAACGTCGA TGACCTTGCTTGTTCCCTGAATATAACAAGCATGCCGTAATT

- 84 046206

ATATGGTCGTTTGTTCCTGCATTAGTCGGGAATTTAATCTACCCTATCTGGCTGTTTCAGGGAAAAGAAAAAATG AAATGGCTGACTTTAAGTAGTATTTTATCCCGCTTGGCTATTATCCCTCTAACATTTATTTTTGTGAACACAAAG TCAGATATAGCAATTGCCGGTTTTATTCAGTCAAGTGCAAATCTGGTTGCTGGAATTATTGCACTAGCTATCGTT GTTCATGAAGGTTGGATTGGTAAAGTTACGCTATCATTACATAATGTGCGTCGATCTTTAGCAGACGGTTTTCAT _{GTTTTTATTTCCACATCTGCTATTAGTTTATATTCTACGGGAATAGTTATTATCCTGGGATTTATATCTGGACCA} ACGTCCGTAGGGAATTTTAATGCGGCCAATACTATAAGAAACGCGCTTCAAGGGCTATTAAATCCTATCACCCAA GCAATATACCCAAGAATATCAAGTACGCTTGTTCTTAATCGTGTGAAGGGTGTGATTTTAATTAAAAAATCATTG ACCTGCTTGAGTTTGATTGGTGGTGCTTTTTCATTAATTCTGCTCTTGGGTGCATCTATACTAGTAAAAATAAGT ATAGGGCCGGGATATGATAATGCAGTGATTGTGCTAATGATTATATCGCCTCTGCCTTTTCTTATTTCATTAAGT AATGTCTATGGCATTCAAGTTATGCTGACCCATAATTATAAGAAAGAATTCAGTAAGATTTTAATCGCTGCGGGT TTGTTGAGTTTGTTGTTGATTTTTCCGCTAACAACTCTTTTTAAAGAGATTGGTGCAGCAATAACATTGCTTGCA ACAGAGTGCTTAGTTACGTCACTCATGCTGATGTTCGTAAGAAATAATAAATTACTGGTTTGCTGAGGATTTTAT GTACGATTATATCATTGTTGGTTCTGGTTTGTTTGGTGCCGTTTGTGCGAATGAGTTAAAAAAGCTAAACAAAAA AGTTTTAGTGATTGAGAAAAGAAATCATATCGGTGGAAATGCGTACACAGAGGACTGTGAGGGTATCCAGATTCA TAAATAT G GT G СACATAT TTTT CATAC CAAT GATAAATATATAT GG GAT TAG GT TAAT GAT T TAGTAGAAT T TAA TCGTTTTACTAATTCTCCACTGGCGATTTATAAAGACAAATTATTCAACCTTCCTTTTAATATGAATACTTTCCA CCAAATGTGGGGAGTTAAAGATCCTCAAGAAGCTCAAAATATCATTAATGCTCAGAAAAAAAAGTATGGTGACAA GGTACCTGAAAATTTGGAGGAGCAGGCGATTTCATTAGTTGGGGAGGACTTATACCAAGCATTGATAAAGGGTTA TACGGAGAAGCAGTGGGGAAGAAGTGCAAAAGAATTGCCTGCATTTATTATTAAGCGAATCCCAGTGAGATTTAC GTTTGATAACAATTATTTTTCCGATCGCTATCAAGGTATTCCGGTGGGAGGCTACACTAAGCTTATTGAAAAAAT GCTTGAAGGTGTGGACGTAAAATTAGGCATTGATTTTTTGAAAGACAAAGATTCTCTAGCGAGTAAAGCCCATAG AATCATCTACACTGGACCCATTGATCAGTACTTCGACTATAGGTTTGGAGCGTTAGAATATCGCTCTTTAAAATT TGAGACGGAACGCCATGAATTTCCAAACTTCCAAGGGAATGCAGTAATAAATTTCACTGATGCTAATGTACCATA ТАС CAGAATAAT T GAG CATAAACAT TTTGACTATGTT GAGACAAAG CATAC G GT T GT TACAAAAGAATAT C CAT T AGAGTGGAAAGTTGGCGACGAACCCTACTATCCAGTTAATGATAATAAAAACATGGAGCTTTTTAAGAAATATAG AGAGTTAGCTAGCAGAGAAGACAAGGTTATATTTGGCGGGCGTTTGGCCGAGTATAAATATTATGATATGCATCA AGTGATATCTGCCGCTCTTTATCAAGTGAAAAATATAATGAGTACGGATTAATGATCTATCTTGTA ATTAGTGTC TTTCTCATTACAGCATTTATCTGTTTATATCTTAAGAAGGATATATTTTATCCAGCCGTATGCGTTAATATCATC TTCGCACTGGTCTTATTGGGATATGAAATAACGTCAGATATATATGCTTTTCAGTTAAATGACGCTACGTTGATT TTTCTACTTTGCAATGTTTTGACATTTACCCTGTCATGTTTATTGACGGAAAGTGTATTAGATCTAAATATCAGA AAAGTCAATAAT GCTATTTATAGCATACCATCGAAGAAAGTGCATAATGTAGGCTTGTTAGTTATTTCTTTTTCG ATGATATATATATGCATGAGGTTAAGTAACTACCAGTTCGGGACTAGCTTACTTAGCTATATGAATTTGATAAGA GATGCTGATGTTGAAGACACATCAAGAAATTTCTCAGCATACATGCAGCCAATCATTCTAACTACTTTTGCTTTA TTTATTTGGTCTAAAAATTTACTAATACAAAGGTAAGTA AAACATTTACTTTACTTGTTTTTATTGTATTCATC TTTGCAATTATACTGAATACTGGTAAGCAAATTGTCTTTATGGTTATCATCTCTTATGCATTCATCGTAGGTGTT AATAGAGTAAAACATTATGTTTATCTTATTACAGCTGTAGGTGTTCTATTCTCCTTGTATATGCTCTTTTTACGT GGACTGCCTGGGGGGATGGCATATTATCTATCCATGTATTTGGTCAGCCCTATAATCGCGTTTCAGGA GTTTTAT TTTCAGCAAGTATCTAACTCTGCCAGTTCTCATGTCTTTTGGTTTTTTGAAAGGCTGATGGGGCTATTAACAGGT GGAGTCTCTATGTCGTTGCATAAAGAATTTGTGTGGGTGGGTTTTGCCAACAAATGTTTATACTGCTTTTTCGGAT TATGTTTATATTTCCGCGGAGCTAAGCTATTTGATGATGGTTATTCATGGCTGTATTTCAGGTGTTTTATGGAGA

- 85 046206

TTGTCTCGAAATTACATATCTGTGAAAATATTTTATTCATATTTTATTTATACCTTTTCTTTCATTTTTTATCAT GAAAGCTTCATGACTAATATTAGCAGTTGGATACAAATAACTCTTTGTATCATAGTATTCTCTCAATTTCTTAAG GCCCAGAAAATAAAGTGAAAATGTATTTTTTGAATGATTTAAATTTCTCTAGACGCGATGCTGGATTTAAAGCAA GAAAAGATGCACTGGACATTGCTTCAGA TTATGAAAACATTTCTGTTGTTAACATTCCTCTATGGGGTGGAGTAG TCCAGAGAATTATTAGTTCTGTTAAGCTTAGTACATTTCTCTGCGGTCTTGAAAATAAAGATGTTTTAATTTTCA ATTCCCGATGGCCAAACCATTTTGGCATATATTGTCATTCTTTCACCCGCCTTCTAAAATTTAGAATAGTACCTC TGATTCATGATATTGATGAATTAAGAGGAGGAGGGGGTAGTGATTCTGTG CGGCTTGCTACCTGTGATATGGTCA TAAGT CACAAT WITH CACAAAT GACAAAGTAC WITH T TAGTAAATATAT GT CT CAGGATAAAAT CAAAGACATAAAAATAT TTGATTACCTCGTCTCATCTGATGTGGAGCATCGAGATGTTACGGATAAGCAACGAGGGGTCATATATGCTGGCA ACCTTTCTAGGCATAAATGTTCTTTCATATATACTGAAGGATGCGATTTTACTCTCTTTGGTGTCA ACTATGAAA ATAAAGATAATCCTAAATATCTTGGAAGTTTTGATGCTCAATCTCCGGAAAAGATTAACCTCCCAGGCATGCAAT TTGGACTCATTTGGGATGGAGATTCTGTCGAAACCTGTAGTGGTGCCTTTGGCGACTATTTAAAGTTTAATAACC CTCATAAGACATCTCTTTATCTTTCAATGGAACTTCCAGTATTTATATGGGATAAAGCCGCCCTTGCGGATTTCA T T GTAGATAATAGA ATAG GATAT G CAGT G G GAT СAAT CAAAGAAAT G CAAGAGAT TGTTGACTC CAT GACAATAG AAACTTATAAGCAAATTAGTGAGAATACAAAAATTATTTCTCAGAAAATTCGAACAGGAAGTTACTTCAGGGATG TTCTTGAAGAGGTGATCGATGATCTTAAAACTCGCTAAACGATATGGTCTCTGTGGTTTTATTCGGCTTGTTAGA GATGTCTTATTGACTCGTGTA TTTTACCGGAACTGTAGAATTATTCGATTTCCCTGCTATATTCGCAATGATGGT AGCATTAATTTTGGTGAAAATTTCACAAGTGGAGTCGGTCTCAGGCTGGATGCATTTGGACGTGGCGTGATTTTT TTTTCCGATAATGTGCAAGTTAACGACTATGTTCATATCGCCTCAATTGAGAGCGTTACGATAGGTCGGGATACG CTTATTGCAAGTAAAGTATTTATTACCGATCATAATCAC GGTTCCTTTAAGCACTCTGATCCAATGAGTTCGCCA AATATACCTCCAGACATGCGCACGTTGGAATCTTCAGCTGTTGTAATTGGCCAGAGGGTTTGGTTGGGTGAGAAT GTGACGGTTTTGCCTGGAACAATTATTGGTAATGGAGTCGTAGTCGGCGCCAATTCTGTTGTTAGAGGTTCTATT CCCGAAAATACTGTCATTGCGGGAGTACCAGCAAAAATCATAAAGAAATACAAT CATGAGACCAAATTATGGGAA AAAGCATAGTCGTTGTTTCTGCGGTCAATTTTACCACTGGCGGTCCATTTACCATTTTGAAAAAATTTTTGGCAG CAACTAATAATAAAGAAAATGTCAGTTTTATCGCATTAGTCCATTCTGCTAAAGAGTTAAAAGAAAGTTATCCAT GGGTTAAATTCATTGAGTTTCCTGAGGTTAAAGGGTCGTGGCTAAAACGTTTGCACTTTGAATATGTAGTTTGTA AAAAACTTTCAAAAGAGCTGAATGCTACGCATTGGATTTGTCTGCATGATATTACGGCCAATGTCGTCACTAAAA AAAGATATGTGTATTGTCATAACCCTGCCCCTTTTTATAAAGGAATTTTATTCCGTGAAATTCTTATGGAGCCTA GCTTTTTCTTATTTAAAATGCTATACGGGCTGATATATAAAATAAACATTAAAAAAAATACTGCAGTGTTTGTTC AACAAT T C T G GAT GAAAGAAAAAT T TAT CAAGAAATAT T C TATAAATAACAT CAT TGTCAGTCGGC CAGAAAT TA AATTATCTGATAAAAGCCAACTTACTGATGATGATTCTCAATTTAAGAATAACCCTTCTGAGTTGACAATATTTT ACCCTGCTGTTCCACGAGTATTTAAAAATTACGAGCTTATTATTAGTGCAGCAAGGAAATTGAAAGAACAATCCA ATATTAAATTTCTGCTTACTATCAGTGGTACAGAAAATGCG TATGCAAAATATATTATCAGTCTTGCAGAAGGAC TGGATAATGTTCATTTCCTCGGGTACTTGGATAAAGAAAAAATCGATCATTGTTATAATATTTCAGATATAGTTT GTTTTCCCTCTAGGTTAGAAACATGGGGATTGCCGTTGTCTGAGGCTAAAGAGCGAGGTAAGTGGGTATTAGCAT CAGATTTCCCATTTACTAGAGAAACTCTTGGTAGTTATGAAAAGAAAGCTTTTTTTGATTCTAATAACG ATGACA TGTTAGTTAAACTTATTATTGACTTCAAAAAAGGTAACCTCAAAAAAGATATCTCTGATGCAAATTTCATTTATC GTAATGAAAATGTATTAGTTGGGTTTGATGAACTAGTTAATTTTATTACTGAAGAACATTGAAATGGTATATATA ATAATCGTTTCCCACGGACATGAAGACTACATCAAAAAATTACTCGAAAATCTTAATGCTGACGATGAGCACTAC AAGATTATCGTACGCGACAACAAA GACTCTCTATTATTGAAACAAATATGCCAGCATTATGCAGGCCTGGACTAT

- 86 046206

ATTAGTGGAGGTGTATACGGCTTTGGTCATAATAATAATATTGCGGTGGCGTATGTAAAGGAAAAATATAGACCC G CAGAT GAT GAT TAGAT TTTGTTTT GAAT CCC GATAT CAT CAT GAAG CAT GAT GAT T T G S T GACATATAT TAAA TATGTCGAAAGTAAGCGTTATGCTTTTAGTACATTATGCCTGTTCCGAGATGAAGCGAAATCTTTACATGATT AT TCCGTAAGAAAATTTCCTGTGCTTTCTGATTTTATTGTGTCATTTATGTTAGGGATTAATAAAACAAAAATTCCT AAAGAAAGTATCTATTCTGATACGGTTGTTGATTGGTGCGCAGGATCATTTATGCTGGTACGTTTTTCAGATTTT GTGCGTGTAAATGGCTTCGATCAAGGTTACTTTATGTACTGTGAAGATATTGACCTGTGCTTGAGGCTTAGCCTG GCTGGTGTCAGACTTC ATTATGTTCCCCGCTTTTCATGCGATACATTATGCTCATCATGACAATCGAAGTTTTTTTT TCAAAAGCCTTCAGATGGCACTTAAAAAGTACTTTTAGATATTTAGCCAGAAAACGTATTTTTATCAAATCGCAAC TTTGATCGAATTTCATCAGTTTTTCACCCGTAAGAGCTCGGTACCCGGGCCTAGGGTGTAGGCTGGAGCTGCTTC GAAGTTCCTATACTTTCTAGAATAGGAACTTCG GAATAGGAACTAAGGAGGATATTCATATCCGTCGACGGCG GCCGCCCTGCAGGCATGCAAGCTTGATCCATATGGATCGCTAGCTTAATTAAATAAAGCCGTAAGCATATAAGCA TGGATAAGCTATTTATACTTTAATAAGTACTTTGTATACTTATTTGCGAACATTCCAGGCCGCGAGCATTCAGCG CGGTGATCACACCTGACAGGAGTATGTAATGTCCAAGCAACAGATCGCGTAGTCGGTA TGGCAGTGATGGGACG CAACCTTGCGCTCAACATCGAAAGCCGTGGTTATACCGTCTATTTTCAACCGTTCCCGTGAGAAGACGGAAGA AGTGATTGCCGAAAATCCAGGCAAGAAACTGGTTCCTTACTATACGGTGAAAGAGTTTGTCGAATCTCTGGAAAC GCCTCGTCGCATCCTGTTAATGGTGAAAGCAGGTGCAGGCACGGATGCTGCTATTGATTCCCTCAAACCATATCT CGAT AAAGGAGACATCATCATTGATGGTGGTAACACCTTCTTCCAGGACACTATTCGTCGTAATCGTGAGCTTTC AGCAGAGGGCTTTAACTTCATCGGTACGGGTGTTTCTGGCGGTGAAGAGGGGGCGCTGAAAGGTCCTTCTATTAT GCCTGGTGGCCAGAAAGAAGCCTATGAATTGGTAGCACCGATCCTGACCAAAATCGCCGCCGTAGCTGAAGACGG TGAACCATGCGTTACCTATATTGG TGCCGATGGCGCAGGTCACTATGTGAAGATGGTTCACAACGGTATTGAATA CGGCGATATGCAGCTGATTGCTGAAGCCTATTCTCTGCTTAAAGGTGGCCTGAACCTCACCAACGAAACTGGC GCAGACCTTTACCGAGTGGAATAACGGTGAACTGAGCAGTTACCTGATCGACATCACCAAAGATATCTTCACCAA AAAAGATGAAGACGGTAACTACCTGGTTGATGTGATCCTGGATGAAGCG GCTAACAAAGGTACGGGTAAATGGAC CAGCCAGAGCGCGCTGGATCTCGGCGAACCGCTGTCGCTGATTACCGAGTCTGTGTTTGCACGTTATATCTCTTC TCTGAAAGATCAGCGTGTTGCCGCATCTAAAGTTCTCTCTGGTCCGCAAGCACAGCCAGCAGGCGACAAGGCTGA GTTCATCGAAAAAGTTCGTCGTGCGCTGTATCTGGGCAAAATCGTTTCTTACGCCCAGGGCTTCTCTCAG CTGCG TGCTGCGTCTGAAGAGTACAACTGGGATCTGAACTACGGCGAAATCGCGAAGATTTTCCGTGCTGGCTGCATCAT CCGTGCGCAGTTCCTGCAAAAAATCACCGATGCTTATGCCGAAAATCCACAGATCGCTAACCTGTTGCTGGCTCC GTACTTCAAGCAAATTGCCGATGACTACCAGCAGGCGCTGCGTGATGTCGTTGCTTATGCAGTACAGAACGGTAT TCCGGTTCCGACCTTCT CCGCAGCGGTTGCCTATTACGACAGCTACCGTGCTGCTGTTCTGCCTGCGAACCTGAT CCAGGCACAGCGTGACTATTTTGGTGCGCATACTTATAAGCGTATTGATAAAGAAGGTGTGTTCCATACCGAATG GCTGGATTAA

SEQ ID NO: 17 (example of nucleotide sequence of the rfb locus O18L - producer strain O18L-ERL BVEC-L-00559)

ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGATACCC AAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAGGGATCAAA GAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTATGAGTTAGAATCA CTCCTTGAGCAGCGCGTGA AGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCCGGGCGTGACCATTATG AACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGACCTGCCATTGGTGACAACCCA TTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCGCTACGTTACAACCTTGCTGCCATG

- 87 046206

ATTGCACGTTTCAACGAAACGGGCCGCAGCCAGGTGCTGGCAAAACGTATGCCGGGTGACCTCTCTGAATACTCC GTCATCCAGACTAAAGAGCCGCTGGACCGTGAGGGTAAAGTCAGCCGCATTGTTGAATTTATCGAAAAACCGGAT CAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGTCGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTG GAACGTACTCAGCCTGGTGCATGGGGACG TATTCAGCTGACTGATGCTATTGCCGAGCTGGCGAAAAAACAATCC GTTGATGCAATGCTGATGACCGGCGACAGTTACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAG TATGGCCTACGCAACCTGAAAGAAGGGGCGAAGTTCCGTAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAA TCTGACCGGATGTAACGGTTGATAAGAAAATTATAACGGCAGTGAAAATTCGCAG CAAAAGTAATTTGTTGCGAA TCTTCCTGCCGTTGTTTTATATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAGTTTTCCA GGATTTTCCTTGTTTCCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTA ACGCTCGTCACATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGCGGTAGCGTGCAT TAATAC CTCTATTAATCAAACTGAGAGCCGCTTATTTCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGTGAAAATA CTTGTTACTGGTGGCGCAGGATTTATTGGTTCAGCTGTAGTTCGTCACATTATAAATAATACGCAGGATAGTGTT GTTAATGTCGATAAATTAACGTACGCCGGAAACCGGGAATCACTTGCTGATGTTTCTGATCTGAACGCTATGTT TTTGAACATGCGGATATTTG CGATGCACCTGCAATGGCACGGATTTTTGCTCAGCATCAGCCGGATGCAGTGATG CACCTGGCTGCTGAAAGCCATGTTGACCGTTCAATTACAGGCCCTGCGGCATTTATTGAAACCAATATTGTTGGT ACTTATGTCCTTTTGGAAGCCGCTCGCAATTACTGGTCTGCTCTTGATAGCGACAAGAAAAATAGCTTCCGTTTT CATCATATTTCTACTGACGAAGTCTATGGTGATTTGCCTCATCC AGATGAAGTAAATAATACAGAAGAATTACCC TTATTTACTGAGACGACAGCTTACGCGCCAAGCAGCCCTTATTCCGCATCCAAAGCATCCAGCGATCATTTAGTC CGCGCGTGGAAACGTACATATGGTTTACCGACAATTGTGACTAATTGCTCGAACAACTATGGTCCTTATCATTTC CCGGAAAAGCTTATTCCACTGGTTATTCTTAATGCACTGGAAGGTAAGGCATTACCTATTTATGGCAAAAG AT CAGATCCGCGACTGGTTGTATGTTGAAGATCATGCGCGTGCGTTATATACCGTCGTAACCGAAGGTAAAGCGGGT GAAACTTATAACATTGGTGGGCACAACGAAAAGAAAAACATCGATGTAGTGCTCACTATTTGTGATTTGCTGGAT GAGATTGTACCGAAAGAGAAATCTTATCGTGAGCAAATCACTTATGTTGCTGATCGTCCGGGACACGATCGCCGC TATGCTATTGATGCT GAGAAGATTGGTCGCGCATTGGGATGGAAACCACAGGAAACGTTTGAGAGCGGGATTCGT AAAACGGTGGAATGGTACCTGTCCAATACAAAATGGGTTGATAATGTGAAAAGTGGTGCCTATCAATCGTGGATT GAACAGAACTATGAGGGCCGCCAGTAATGAATATCCTCCTTTTTGGCAAAACAGGGCAGGTAGGTTGGGAACTAC AGCGTGCTCTGGCACCTTTGGGTAATTTGATTGCTTTT GATGTTCACTCTACTGATTATTGCGGTGATTTTAGTA ATCCTGAAGGTGTAGCTGAAACCGTAAGAAGCATTCGGCCGGATATTATTGTCAATGCAGCCGCTCACACCGCAG TAGACAAAGCAGAATCAGAACCGGAGTTTGCACAATTAATTAACGCAACAAGTGTCGAAGCGATTGCGAAAGCAG CAAATGAAGTTGGAGCCTGGGTTATCCATTACTCGACTGATTACGTCTTCCCTGGAAATGGCG ATATGCCATGGC TGGAGACGGATGCAACCGCACCACTAAATGTTTACGGTGAAACCAAGTTAGCCGGAGAAAAAGCGTTACAGGAAT ATTGCGCGAAGCATCTTATTTTCCGGACCAGCTGGGTCTATGCAGGAAAAGGAAATAACTTCGCCAAAACGATGT TACGTCTGGCAAAAGAGCGTGAAGAATTAGCGGTTATTAACGATCAGTTTGGTGCGCCAACAGGTGCTGAACTGC TGGCTGATTG TACAGCACATGCCATTCGTGTCGCACTGAATAAACCGGATGTCGCAGGCTTGTACCATTTGGTAG CCAGTGGTACCACAACCTGGTACGATTATGCTGCGCTGGTTTTTTGAAGAGGCGCGCAAAGCAGGCATTCCCCTTG CACTCAACAAGCTCAACGCAGTACCAACAACAGCCTATCCTACACCAGCTCGTCGTCCACATAACTCTCGCCTTA ATACAGAAAAATTTCAGCAGAACTTTGCG CTTGTCTTGCCTGACTGGCAGGTTGGCGTGAAACGAATGCTCAATG AATTATTTACGACTACAGCAATTTAATAGTTTTTGCATCTTGTTCGTGATGGTGGAGCAAGATGAATTAAAAGGA ATGATGAAATGAAAATGCGTAAAGGTATTATTTTAGCGGGTGGTTCTGGTACACGTCTTTATCCTGTGACTATGG CTGTCAGTAAACAGCTATTACCTATTTATGATAAACCGATGATCTATTACCCG CTCTCTACACTGATGTTGGCGG

- 88 046206

GTATTCGCGATATTTTGATTATCAGTACACCTCAGGATACTCCTCGTTTTCAACAATTGCTGGGTGACGGTAGCC AGTGGGGCCTGAATCTTCAGTACAAAGTGCAACCTAGCCCAGATGGCCTCGCGCAGGCATTTATCATCGGTGAAG AGTTTATTGGTGGTGATGATTGTGCTTTGGTTCTTGGTGATAATATCTTTTACGGTCACGATCTGCCGAAGCTAA TGGAGGCCGCTGTTAACAAAGAAAGTGGTGCAACGGTATTTGCCTATCACGTTAATGATCCAGAACGCTATGGTG TCGTTGAGTTTGATAAAAACGGTACGGCAATCAGTCTGGAAGAAAAACCGTTAGAACCAAAGAGTAATTACGCCG TTACAGGTCTGTACTTTTATGATAACGACGTGGTTCAGATGGCGAAAAACTTGAAGCCGTCTGCACGTGGTGAGT TAGAAATTACAGATATTAACCGTATTTATCTTGAGCAGGGACGTCTGTCTGTCGCGATGATGGGGCGTGGCTACG CGTGGCTGGACACGGGGACTCATCAGAGTCTGATAGAAGCAAGTAATTTTATTGCGACAATTGAAGAGCGCCAGG GATTGAAGGTTTCCTGTCCTGAAGAGATTGCATTTCGTAAAGGTTTTATTGATGTTGAGCAAGTAAGAAAATTAG CTGTACCACTAATAAAGAATAATTATGGGCAGTATCTTTATAAAATGACGAAGGATTCAAATTAATGAATGTGAT TAGAACTGAAATTGAAGATGTGCTAATTCTGGAGCCAAGAGTATTTGGTGATGATAGAGGTTTCTTTTATGAGAG CTTTAATCAATCAGCATTTGAACATATTCTAGGCTATCCGGTCAGCTTTGTTCAAGACAATCACTCACGTTCATC AAAAAATGTACTCAGAGGCCTTCACTTTCAACGCGGCGAGTACGCACAAGATAAACTTGTACGCTGCACTCATGG AGCAGTTTTTGATGTTGCTGTTGATATTCGACCCAATTCGGTATCCTTTGGTAAATGGGTTGGTGTTCTGCTTTC AGCTGATAATAAGCAGCAGTTGTGGATACCAAAAGGGTTTGCTCATGGCTTTTTGGTTCTGTCTGATATCGCTGA ATTTCAATATAAAACTACAAACTATTATCATCCTGAAAGCGATTGTGGAATATGTTGGAATGATGAACGCATTGC AATTGATTGGCCCCAAACATCAGGGTTAATCCTTTCGCCAAAAGATGAAAGGCTCTTTACGTTAGATGAGCTTAT CAGATTAAAATTAATTGCATGAGGCCGGCCTTAAGGAGGACTAGTCCCGGCGCGCCATGAGTTTAATCAAAAACA _{GTTTTTGGAACCTTTGCGGGTATGTACTTCCAGCTATTGTGACACTACCAGCTTTGGGTATTATGGGGCGAAAAT} TAGGCCCAGAATTATTTGGTGTATTCACTTTGGCATTAGCTGTTGTGGGTTATGCAAGCATTTTTGATGCAGGCC TTACTCGCGCAGTGATACGAGAAGTCGCAATTGAAAAAGATAATGAAGAAAATAAGTTGAAAATTATTTCTTCAG CGACAGTTGTAATTATTTATTTGAGTTTGGCCGCCTCACTCTTATTATTTTTTTTTAGTGGTCATATCGCATTGC TACTGAACATTAGTGAGACTTTTTTTCATAATGTAAGTGTCTCGCTTAAAATTCTCGCAGCATCCATACCATTAT TTTT GAT ТАС T CAAATAT GGT T GT CAAT TT TAGAAGGT GAAGAAAGAT TTG GT T TACT TAATAT CTACAAAT CAA TTACGGGAGTGATATTAGCAATCTCACCGGCATTATTTATACTTATTAAACCCTCTTTGATGTATGCGATAATAG GCTTAGTTCTAGCAAGGTTTTTATGTTTTATTTTGGCTTTTATAATTTGTCACGATAAAGTGCTTAAAGCTAAAC TAACAAT C GATATAC CAACAAT TAAAAGAT T GT T TAT GTTCGGTGGT T GGAT TACAGTAAGTAATAT CAT CAGC C CTGTGCTATCATATTTTGATAGGTTTATTGTTTCAAATCAACTTGGGGCTGCTAATGTTGCTTTTTATACTGCAC CATCAGAAATTATTTCTCGGCTTAGTATAATTCCAGGTGCGTTTTCAAGAGCCTTATTTCCAAGATTAGCTAATG CAAAT AAT T C CG C T GAAAGAT AT AAAAC GAAAAGAT T AAT TAG AAT TT WITH AC TTTT AAT AAT CAT CACCCCTATTT TTTGTATTGGCGTGTTATTTTCAGAGAAGATAATGGTTTTATGGATGGGGGCATCATTTTTTGGTGAGCCTGGTT TGGTATTATCAATATTACTGATTGGCTTTATTTTTAATGGATTGGCACAAGTACCATTTGCCAGTATTCAATCCCC GAGGTCATGCTAAGATAACTGCATTTGTTCATCTCTTAGAGTT GTTTCCTTATTTATTACTTTTATTTTACCTCA TAAAAGCACATGGGGTTGTTGGCGCGGGTATTGCGTGGTCAGTGAGGATGATAGTAGATTATATAGCATTAAGTC TTTTGGACGGTAAGTATATTAATAAATAAAATTCAAAATGCAAGTTAATAACTCATGGCTTTATTTGGGTAGGTG ACAATTTATAATGATATATATTAACTTTAACTCTTCTTCTAGTTATAGCCATAATGTTTTCTCTTCTC T GGAT CAT T GT CAT C C CAAT TT CAT TATATAC CAT TTTC GAAATATATAT GGT T GGTAT

- 89 046206

GGGGGGAGCAGATGGATTCTTTCTCAATTTACGTCTTGCAAATACATTGGAGGGCTATACGGGTAAAAAATTTAT CTTAATGCCTGCTGTATATCCTCTAATGATGGCTATGTTCGCAATTGTTTGTCTAACAAAAACTTCCAAATTAAA TAAATACTCCATTTATTTCTGGATGTTTTTGTATTGTATTGGCACAATGGGAAAATTTTCAATATTAACGCCAAT AT T GACATAT Τ TA AT TAT T TAT GACT T CAAACATAGAT TAAAAGTAAAAAAACAATAAAGT Τ TACAT T GT T GAT AATTATATTAGCTTTAACTTTGCATTTTACACGTATGGCTGAGAATGACCACTCAACATTTTTATCTATTTTAGG GCTCTATATTTATTCACAATAATTGCTTTAGGCCAGTTGAATGAAGTAAATAGTAGTCATTTTGGTGAGTATAC GTTTAGATTCATATATGCTATA ACTAATAAAATTGGCCTTATTAAAGAATTGCCAGTAAATACTATTCTTGACTA TTCATACGTTCCTGTACCAACAAATGTATATACTGCACTTCAACCATTTTACCAGGATTTTGGTTATACTGGCAT CATATTTGGAGCAGTATTATACGACTAATATATGTGAGTTTATACACGGCGGTGTTCGTGGAAATAATACACA GGCATTACTGATTTACGCATTGTTTTCAGTTAGCAGTGCAACGGCTTTCT TCGCTGAAACGCTAGTAACGAATTT AGCTGGAAATGTGATGTTAGTATTATGTACCATCTTACTATGGCGATTTACAGTAATATGCAAACCAGTACAGTA ACCATTCTAATGGCCACCTACAATGGCGAGGCCTTCATCAAAAATCAGATTTTGTCACTACAACAACAAACATTT TCTAACTGGCGGTTATTTATTCAGGATGATGGGTCTACAGACAATACTATATCTATAATAAAAAACTTCCAAAAA TC TGACTCCAGAATTCGGCTAGTTGATGATAATTTGAAAGGTCAAGGTGCAGGAAAAAATTTTTTATCGCTGATA AAGTACAG C GAGACAGAT TATACAAT T TAT T GT GAC CAAGAT GATAT Τ T G G Τ TAGAAAACAAAATAT Τ T GAAT TA GTAAAGTATGCAAATGAAATTAAATTGAATGTATCAGATGCGCCTTCGCTAGTTTATGCTGATGGCTATGCTTAT ATGGATGGTGAGGGTACAATCGATTTTTCTGGGTATCTAACAATCATGCTGATCAATTAAAGGATTTTCTTTTT TTTAATGGTGGATACCAAGGATGTTCTATTATGTTCAATCGTGCAATGACCAAATTTCTTCTGAATTATCGAGGA TTTGTATATCTACATGA CGATATCACAACATTAGCTGCATACGCTCTTGGTAAAGTTTATTTTCTCCCGAAATAC CTTATGTTATATAGACAGCACACGAATGCGGTAACTGGTATCAAAACATTCCGCAATGGATTGACTTCTAAATTT AAATCACCAGTAAACTATCTTTTATCACGAAAACATTATCAGGTAAAAAAATCTTTTTTTGAATGTAACAGCTCT AT C Τ TAT CAGAGAC GAATAAAAAAGT Τ C TCTTATATTACAAACTGATAAAGATTTTGAGTGGATAATAATTGATGATGGTAGTATAGATGCAACAGCGGTAC TTGTAGAAGATTTTAGAAAAAAATGTGATTTTGACTTGATTTATTGCTATCAGGAAAATAATGGTAAGCCCATGG CTTTAAACGCTGGTGTTAAAGCTTGTAGAGGCGATTATATCTTTATTGTTGACAGTGATGATGCACTAACTCCCG ATGCCATAAAATTAATTAAAGAATCAATA CATGATTGCTTATCTGAGAAGGAAAGTTTCAGCGGAGTCGGTTTTA GAAAAGCATATAAAAGGGGGGATTATTGGTAATGATTTAAATAATTCTTCAGAACATATACTATTTAAATG CGACTGAGATTAGCAATTTAATAAATGGTGATGTTGCATATTGTTTTAAAAAAGAAAGTTTGGTAAAAAATCCAT T C C C C GTATAGAAGAT GAAAAAT Τ T GT T C CAGAAT TATATAT Τ T G GAATAAAATAAC T GACAAGG C GAAGAT T C GATTTAACATAAGCAAAGTTATATATCTTTGTGAGTATCTTGATGATGGTCTTTCTAAAAATTTCCATAACCAGC Τ ΤAAAAAATAC C CAAAG G G GT Τ TAAGAT Τ TAT TACAAAGAT CAAAGAAAAC GAGAGAAAAC Τ TATATAAAAAAAA C AAAGATGCTAATTAGATATTTGCAATGTTGTTATTATGAGAAAATAAAATGAAAATACTATTTGTCATTACAGG TTTAGGCCTTGGAGGTGCTGAGAAGCAGGTTTGTCTTTTAGCTGATAAATTAAGTTTAAGCGGGCACCATGTAAA GATTATTTCACTTGGACATATGTCTAATAATAAAGTCTTTCCTAGCGAAAATAATGTTAATGTCATTAATGTAAA TATGTCAAAAAACATTTCTGGAGTTATAAAA GGTTGTGTCAGAATTAGAGATGTTATAGCTAATTTCAAACCAGA CATTGTACACAGTCATATGTTTCATGCAAACATTATCACTAGATTGTCTGTAATTGGAATCAAAAACAGACCTGG TATTATATCAACTGCACATAATAAAAATGAAGGTGGGTATTTCAGAATGCTCACATATAGAATAACCGATTGTTT AAGTGATTGTTGTACAAATGTTAGCAAAGAAGCAGTGGATGAGTTTTTACGATAAAA GCCTTTAATCCCGCTAA

- 90 046206

AGCAATTACTATGTATAATGGGATAGATACCAATAAAATTTAAATTTGATTTATTGGCAAGGAGGGAAATTCGAGA CGGTATTAATATAAAAATGATGATATATTACTTGCTGCAGGTCGTTTAACGTTAGCTAAAGATTATCCTAA TTTATTGAATGCAATGACTCTGCTTCCTGAACACTTTAAACTTATTATTATTGGTGATGGTGAATTGCGTGACGA AATTAATATGCTTATAAAA AAATTGCAATTATCTAATAGGGTGTCCTTGTTGGGAGTTAAAAAAAATATTGCTCC CTATTTTTCTGCATGTGATATTTTTGTTCTCTCTTCTCGTTGGGAAGGATTTGGATTAGTCGTGGCAGAAGCTAT GTCATGTGAGCGAATTGTTGTTGGCACGGATTCAGGGGGAGTAAGAGAAGTTATTGGTGACGATGATTTTCTTGT ACCCATATCTGATTCAACACAACTTGCAAGCAAAATT GAAAAATTGTCTTTGAGCCAGATACGTGATCACATTGG TTTTCGGAATCGTGAGCGTATTTTAAAAAATTTCTCAATAGATACTATTATTATGCAGTGGCAAGAACTCTATGG AACTATAATTTGCTCAAAACATGAAAGGTAGATTTATATTTGGAACGTGTCTTTTGTTTGAATTTAATTCAATCT CAATTGAGATTTTTGTATTTCAAAATACCATCATAGCTAACGATGATTGGTATTTATTTTAAGA TGCTTTCTAT AAATATATTGACGTTTTTAATGCGCCGAAACGATTGGGCTGGGAACAGAGAAGTAAAACTGTTTTGAGAATGAAG AGT T T T T GAGAT GTT TAT GGATAT TAAAAAT T GAT C CAGT GAAT TAAT TAT T TATAATAAAT CAAGAT T TAAT GT TAATAAATGATAATCTTTTCTGACACTCATATTAATTATGAGTGGTACGTTTGGTAAAC TAAACTATTATATG ACAGCTAGAACAACTAAAGTTTTGCACTTACAATTACTCCCACTCTTAAGTGGCGTTCAAAGGGTAACATTAAAC GAAATTAGTGCGTTATATACTGATTATGATTATACACTAGTTTGCTCAAAAAAAGGTCCACTAACAAAAGCATTG CT GGAATAT GAT GT C GAT T GT CAT T GTAT C C C C GAACT TAC GAGAGAAAT TAC C GTAAAGAAT GAT T T TAAAGCA TTGTTCAAGCTTTATAAGTTCATAAAAAAAGAAAAATTTGACATTGTGCATACACATTCTTCAAAAACAGGTATT TTGGGGCGAGTTGCTGCCAAATTAGCACGTGTTGGAAAGGTGATCCACACTGTACATGGTTTTTCTTTTCCAGCC GCATCTAGTAAAAAAAGTTATTACCTTTATTTTTTCATGGAATGGATAGCAAAGTTCTTTAC GGATAAGTTAATC GTCTTGAATGTAGATGATGAATATATAGCAATAAACAAATTAAAATTCAAGCGGGATAAAGTTTTTTTAATTCCT AATGGAGTAGACACTGATAAGTTTTCTCCTTTAGAAAATAAAATTTATAGTAGCACCTTGAATCTAGTAATGGTT GGTAGATTATCCAAGCAAAAAGATCCTGAGACATTATTGCTTGCTGTTGAAAAACTGCTGAATGAAAATGTTAAT GTTAAGCTGACACT TGTAGGAGATGGTGAACTAAAAGAACAGTTAGAAAGCAGGTTCAAACGGCAAGATGGACGT ATAATTTTTCATGGATGGTCAGATAACATTGTTAATATTTTAAAAGTTAATGATCTTTTTATATTACCTTCTCTT TGGGAGGGTATGCCATTAGCAATTTTAGAAGCATTGAGCTGTGGACTTCCATGTATAGTCACTAATATTCCAGGT AATAATAGCTTAATAGAAGATGGCTATAATGGTTGTT TGTTTGAAATTAGAGATTGTCAGTTATTATCTCAAAAA ATCATGTCATATGTTGGTAAGCCAGAACTGATTGCACAGCAATCTACCAATGCACGATCATTTATTCTGAAAAAT TATGGATTAGTTAAAAGAAATAATAAGGTCAGACAGCTATATGATAATTAAGAGCTCGGTACCCGGGCCTAGGGT GTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACT AAGGAGGATAT TCATATCCGTCGACGGCGGCCGCCCTGCAGGCATGCAAGCTTGATCCATATGGATCGCTAGCTTAATTAAATAAA GCCGTAAGCATATAAGCATGGATAAGCTATTTATACTTTAATAAGTACTTTGTATACTTATTTGCGAACATTCCA GGCCGCGAGCATTCAGCGCGGTGATCACACCTGACAGGAGTATGTAATGTCCAAGCAACAGATCGGCGTAGTCGG TATGGCAGTG ATGGGACGCAACCTTGCGCTCAACATCGAAAGCCGTGGTTATACCGTCTCTATTTTCAACCGTTC CCGTGAGAAGACGGAAGAAGTGATTGCCGAAAATCCAGGCAAGAAACTGGTTCCTTACTATACGGTGAAAGAGTT TGTCGAATCTCTGGAAACGCCTCGTCGCATCCTGTTAATGGTGAAAGCAGGTGCAGGCACGGATGCTGCTATTGA TTCCCTCAAACCATATCTCGATAAAGGAGACAT CATCATTGATGGTGGTAACACCTTCTTCCAGGACACTATTCG TCGTAATCGTGAGCTTTCAGCAGAGGGCTTTAACTTCATCGGTACCGGTGTTTCTGGCGGTGAAGAGGGGGCGCT GAAAGGTCCTTCTATTATGCCTGGTGGCCAGAAAGAAGCCTATGAATTGGTAGCACCGATCCTGACCAAAATCGC CGCCGTAGCTGAAGACGGTGAACCATGCGTTACCTATATTGGTGCCGATGGCG CAGGTCACTATGTGAAGATGGT TCACAACGGTATTGAATACGGCGATATGCAGCTGATTGCTGAAGCCTATTCTCTGCTTAAAGGTGGCCTGAACCT

- 91 046206

CACCAACGAAGAACTGGCGCAGACCTTTACCGAGTGGAATAACGGTGAACTGAGCAGTTACCTGATCGACATCAC CAAAGATATCTTCACCAAAAAAGATGAAGACGGTAACTACCTGGTTGATGTGATCCTGGATGAAGCGGCTAACAA AGGTACGGGTAAATGGACCAGCCAGAGCGCGCTGGATCTCGGCGAACCGCTGTCGCTGATTACCGAGTCTGTGTT TGCACGTTATATCTCTTCTCT GAAAGATCAGCGTGTTGCCGCATCTAAAGTTCTCTCTGGTCCGCAAGCACAGCC AGCAGGCGACAAGGCTGAGTTCATCGAAAAAGTTCGTCGTGCGCTGTATCTGGGCAAAATCGTTTCTTACGCCCA GGGCTTCTCTCAGCTGCGTGCTGCGTCTGAAGAGTACAACTGGGATCTGAACTACGGCGAAATCGCGAAGATTTT CCGTGCTGGCTGCATCATCCGTGCGCAGTTCCTGCAAA AAATCACCGATGCTTATGCCGAAAATCCACAGATCGC TAACCTGTTGCTGGCTCCGTACTTCAAGCAAATTGCCGATGACTACCAGCAGGCGCTGCGTGATGTCGTTGCTTA TGCAGTACAGAACGGTATTCCGGTTCCGACCTTCTCCGCAGCGGTTGCCTATTACGACAGCTACCGTGCTGCTGT TCTGCCTGCGAACCTGATCCAGGCACAGCGTGACTATTTTGGTGCGCATACTTATAAG CGTATTGATAAAGAAGG TGTGTTCCATACCGAATGGCTGGATTAA

SEQ ID NO: 18 (example of nucleotide sequence of the rfb 025B locus - producer strain O25B-ERL stGVXN4459)

ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGATACCC AAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAGGGATCAAA GAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTATGAGTTAGAATCA CTCCTTGAGCAGCGCGTGA AGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCCGGGCGTGACCATTATG AACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGACCTGCCATTGGTGACAACCCA TTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCGCTACGTTACAACCTTGCTGCCATG ATTGCACGTTTCAACGAAACGGGCCGCAGCCAGG TGCTGGCAAAACGTATGCCGGGTGACCTCTCTGAATACTCC GTCATCCAGACTAAAGAGCCGCTGGACCGTGAGGGTAAAGTCAGCCGCATTGTTGAATTTATCGAAAAACCGGAT CAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGTCGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTG GAACGTACTCAGCCTGGTGCATGGGGACGTATTCAGCTGACTGATGCTATTGCCGAGCTGGCGAAA AAACAATCC GTTGATGCAATGCTGATGACCGGCGACAGTTACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAG TATGGCCTACGCAACCTGAAAGAAGGGGCGAAGTTCCGTAAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAA TCTGACCGGATGTAACGGTTGATAAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAA TCTTCCTGCCGTT GTTTTATATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAGTTTTCCA GGATTTTCCTTGTTTCCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTA ACGCTCGTCACATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCATTAATAC CTCTATTAATCAAACTGAGAGCCGCTTATT TCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGCTAGCAGT GAAGATACTTGTTACTGGTGGCGCAGGATTTATTGGTTCTGCTGTTGTTCGTCACATAATAAATAATACGCAAGA TAGTGTTGTTAATGTCGATAAATTAACATACGCCGGAAACCTGGAATCACTTGCAGATGTTTCTGATTCTGAACG CTATTTCTTTGAACATGCGGATATTTGTGATGCAGCTGCAATGGCACGGATTTTTGC TCAGCATCAGCCGGATGC AGTGATGCACCTGGCAGCTGAAAGCCATGTTGACCGTTCAATTACAGGCCCTGCGGCATTTATTGAAACCAATAT TGTGGGTACTTATGTCCTTTTAGAAGCGGCTCGGAATTATTGGTCTGGTCTGGATGATGAAAAGAAAAAAAACTT CCGTTTTCATCATATTTCTACTGATGAGGTGTATGGTGACTTACCCCATCCGGATGAAGTAAATAGCAATGAAAC GTTGCC GCTATTTACGGAAACGACAGCATACGCGCCAAGTAGTCCATATTCTGCTTCTAAAGCTTCCAGCGATCA TTTGGTTCGCGCATGGAAACGTACTTATGGTTTACCGACCATTGTGACTAATTGCTCGAACAACTATGGTCCTTA TCATTTCCCGGAAAAGCTTATTCCACTGGTTATTCTTAATTCACTGGAAGGTAAGGCATTACCTATTTATGGCAA AGGAGATCAGATCCGCGACTGGTTGTATGTAGA GGATCATGCTCGAGCGTTATATACCGTCGTAACCGAAGGTAA

- 92 046206

AGCGGGCGAAACTTATAACATTGGTGGACACAACGAAAAGAAAAACATCGACGTAGTGTTCACTATTTGTGATTT GTTGGATGAGATAGTCCCGAAAGAGAAATCTTACCGCGAGCAAATTACTTATGTTACCGATCGTCCGGGACACGA TCGCCGTTATGCGATTGATGCTGAGAAGATTGGTCGCGAATTGGGATGGAAACCACAGGAAACGTTTGAGAGTGG GATTCGTAAAACGGTG GAATGGTACCTGTCCAATACAAAATGGGTTGATAATGTGAAAAGTGGTGCCTATCAATC GTGGATTGAACAGAACTATGAGGGCCGCCAGTAATGAATATCCTCCTTTTTGGCAAAACAGGGCAGGTAGGTTGG GAACTACAGCGTGCTCTGGCACCTCTGGGTAATTTGATTGCTCTTGATGTTCACTCCACTGATTACTGTGGTGAT TTTAGTAATCCTGAAGGTGTAGCTGAAACCGTAAGAAGCATTC GGCCTGATATTATTGTCAACGCAGCCGCTCAC ACCGCAGTAGACAAAGCAGAATCAGAACCGAAGTTTGCACAATTACTGAACGCGACGAGTGTCGAAGCGATCGCG AAAGCAGCCAATGAAGTCGGCGCCTGGGTTATTCACTACTCTACTGACTACGTATTTCCGGGGACCGGTGAAATA CCATGGCAGGAGGAGGATGCAACCGCACCGCTAAATGTTTACGGTGAAACCAAGTTAGCGGGGAAAAAG CATTA CAAGAGCATTGTGCGAAGCACCTTATTTTCCGGACCAGCTGGGTCTATGCAGGTAAAGGAAATAACTTCGCCAAA ACAATGTTGCGTCTGGCAAAAGAGCGTGAAGAATTAGCCGTTATTAATGATCAGTTTGGTGCGCCAACTGGCGCA GAGTTACTGGCTGATTGTACGGCACATGCTATTCGTGTGGCACTGAATAAACCGGAAGTCGCAGGCTTGTACCAT CTGGTAGCTAGTGGTACC ACAACGTGGCACGATTATGCTGCGCTGGTTTTTTGAAGAGGCGCGCAAAGCAGGCATT CCCCTTGCACTCAACAAGCTCAACGCAGTACCAACAACAGCCTATCCTACACCAGCTCGTCGTCCACATAACTCT CGCCTTAATACAGAAAAATTTCAGCAGAACTTTGCGCTTGTCTTGCCTGACTGGCAGGTTGGCGTGAAACGAATG CTTAACGAATTATTTACGACTACAGCAATTTAATAGTTTT TGCATCTTGTTCGTAATGGTGGAGCAAGATGTATT AAAAGGAATGATGAAATGAAAACGCGTAAAGGTATTATTTTGGCGGGTGGTTCTGGTACTCGTCTTTATCCTGTG ACGATGGCCGTCAGTAAACAGCTGTTACCGATTTATGATAAACCGATGATCTATTACCCGCTCTCTACACTGATG TTAGCGGGTATTCGCGATATTCTGATTATCAGTACACCACAGGATACTCCTCGTTTTCAA CAACTGCTGGGTGAC GGGAGCCAGTGGGGCCTGAATCTTCAGTACAAAGTGCAACCGAGTCCGGATGGTCTTGCGCAGGCGTTTATTATC GGTGAAGAGTTTATTGGTGGTGATGATTGTGCTTTGGTACTTGGTGATAATATCTTCTACGGCCACGACCTGCCG AAGTTAATGGACGTAGCTGTTAACAAAGAAAGTGGTGCAACGGTATTTGCCTATCACGTTAATGATCCTGAA CGT TATGGTGTCGTGGAGTTTGATAATAACGGTACTGCAATTAGCCTGGAAGAAAAAACCGCTGGAACCAAAAAGTAAC TATGCGGTTACTGGGCTTTATTTCTATGACAATGACGTTGTGGAAATGGCGAAAAACCTTAAGCCTTCTGCCCGA GGTGAACTGGAAATTACCGATATTAACCGTATTTATATGGAACAAGGACGTTTGTCTGTCGCTATGATGGGGCGT GGCTATGCATGGCTGGATA CAGGGACGCATCAAAGTCTTATTGAAGCAAGCAACTTCATTGCCACCATTGAAGAG CGCCAGGGACTAAAGGTTTCCTGTCCGGAAGAAATTGCTTATCGTAAAGGGTTTATTGATGCTGAGCAGGTAAAA GTATTAGCCGAACCGTTGAAGAAAAATGCTTATGGTCAGTATCTGCTCAAAATGATTAAAGGTTATTAATAAGAT GAACGTAATTAAAACTGAAATTCCTGATGTGCTGATTTTTGAA CCAAAAGTTTTTGGGGATGAACGTGGCTTCTT TTTTGAGAGTTTTTAATCAGAGGATTTTTGAAGAAGCAGTAGGTCGTAAGGTTGAGTTTGTTCAGGATAACCATTC TAAGTCCAGTAAAGGTGTTTTACGTGGTCTTCATTATCAGTTAGAACCTTATGCTCAAGGAAAACTGGTGCGCTG TGTTGTTGGCGAGGTTTTTGATGTTGCGGTTGATATTCGTAAATCGTCACCTACAT TTGGGAAATGGGTTGGGGT GAATTTGTCTGCTGAGAATAAGCGTCAGTTGTGGATTCCTGAGGGATTTGCACATGGTTTTTTGGTGCTGAGTGA TTTAGCAGAAGTTTTATATAAAACGAATCAATATTATGCTCCATCACATGAAAAAAATATTATATGGAATGACCT CTTGCTTAATATTAAATGGCCGAGCACAGCACTGATCACTCTGTCTGATAAGGATGCAAATGGGGAAAGATTTGA ACTAAGTG AGTTTTGAAATGTCTCTCTTAAAACATAGTATATGGAATGTTGCGGGCTACTTTATACCAACATTAA TTGCAATTCCCGCCTTTGGATTAATTGCGAGGAAAATTGGTGTAGAACTATTTGGTTTGTATACGTTAGCAATGA TTTTTATAGGGTATGCAAGTATATTTGATGCTGGGTTAACAAGAGCTGTTGTGCGTGAAATAGCATTACTAAAA ACAGAGTGGACGATTGTAATACGATAATAGTAACTTC TATTATCGCTGTGATATTTTTAGGGTTTATCGGAGGCG

- 93 046206

GGGGAGTGTTTCTGCTTAAAGGCGATATTATTGAACTGTTAAATATCTCACCAATATATTACGCCGATTCGATAA AGTCTCTAGTATTATTATCATCTCTGATACCTGTATTCTTAGTCACGCAAATACTATTAGCAGAGCTTGAGGGTC GGGAATATTTTGGGATTCTAAATATACAAAAAAGTGTAGGGAATTCTTTAATTGCAGGGTTACCTGCATTATTTG TTTTAATTAATCAAACG CTTTTTTCTGCAATTATTGGTGTAGCGATTGCAAGAGTTATATGCTTGTGGTTAAGCT ACATTATGAGCAGGGAAAGAATAACTATCGATATCTCATTTTTTTCAATAACTGTTTTAAAGCGGTTATTTAGAT ATGGCGGGTGGGTAACTATAAGTAACATAATATCTCTATTAGCGAGTATGGATAGATTTATTCTATCCCATA TCCAGGGAGCATCAAAAATTCATTCTATACAGTCCCTAATGAGCTGGTA ACTAGGCTTGGAATAGTTCCAGGCT CTCTTGGGAAAGCTGTTTTTCCAAAATTAAGTCATGCAAGGAATTTTACAGCGTCATATGCAGAGCAAAAAAAAG CTTATATATTAATGACTGTCATTGTAATGCCTTTGGTTTTATTTGTATATTATTACGCAAAGTTTATTTTAACAT TGTGGATGGGGGCTGAGTATGCAGGGATTTCGGTCGAAATATTACGGATTATGCTTATAGGGTATATTTT TAACT GTTATTCACAAATCTCTTTTGCCAACATACAGGCCTTTGGAAAAGCAAAATACACTGCATACATCCATATGATGG AATTTATTCCTTATTTGATAATGTTATATATAATTTCAAAGGAATATGGGGTTATTGGTGTTGCGTGGTTATGGA CAATTCGAGTAATAATTGATTTTTTGATGCTTTTATATATGAGTTATCGTTGTAATAATCTTATGAAAAAAGGGT AGCCTGATGATATATTGTGG TATTAAATTGGAATGGGGCTATAGATACCATTAATTGTGTTAAAAGTTTAATG GAT T TAAAT GT TAGC GAT TATAAAAT TAT CAT T GT T GATAACT GT T CTAT G GATAACT CATAT GATACTATAAAA GAAAATCTTAATTCATTATATATTGCTGATAAAAGTATCATTGAGGTGAAGTATGAGGATAGAAATAAATATAAAA ACCTTAGAAAACGATAAAATCATATTAATACAATCTCCGC AAAATAATGGGTACGCAAGTGGTAATAATATTGGC ATAGAGTTCGCTCTTAATCAGGAGAATATGAAATACGTCTGGGTTCTGAATAATGATACTGAAGTGGATAAAGAG GCTTTAACTCATTTAATTAGTAAATGTGATTCAGATAAAAGTATAGGGATTTGCGGTTCTCGTTTAGTCTATTTT GCCGACAGAGAGATGCAGCAAGGACTAGGTGGGGTGCATAACAAATGGTTATGCACTACAAAAAATT ATGAAATG GGAAGAT TAGT T T CCAAAAAATAT GAT GAT GAAGT CAT TAGTAAT GATATAGAT TATATAAT T GGC GCAT C GAT G TTTTTCTCTAGAGAATGTTTGGAAACAGTTGGATTGATGAATGAAGAATATTTTTTATACTATGAAGAGTTAGAT ATTTGCCTCAGAGCAAAAGCAAAGAACTTTAAATTAGGTATTTGCTCAGAAAGTTTGGTTTATCATAAAATAGGT GCAAGTACTGATGGGGGAAAGAGCATGATGGCTGATCTTTGCTCAATAAAAATAGGCTGGTCATTACAGAAAGG TTTTA TCCCCAATATTATTGGACGGTATGGTTGTCACTTTTTGTTGTAGCATTTAACCGTGCTAGAAGAGGTGAG T T T AAT AAGAT GAAAAGAT GT T T GAAT GTTATGTTTAACTT C AAAC GAAAC AAAG GT AG C AAAT G C CAT T AGAAT ATGCACTTAATCATGGTGTTAATAAATCTATAGTTTGATATGTTATTAAAGGGTATTTAATGAAAGTGGCTTTTT TATCTGCTTATGATCCACTATCTACATCCAGTTGGTCTGGCACACCTTATTATATGCTAAAGGCATTATCGAAGA GAAATAT T T C CAT T GAAATAT TAGGAC C GGTAAATAGCTATAT GATATACAT GT TAAAAGTATATAAAT TAATAT TAAGGTGTTTCGGAAAAGAATATGATTATAGTCATTCGAAGTTGCTTTCCAGGTATTACGGTAGAATATTCGGTA GGAAATTA AAAAAAATTGATGGTTTGGATTTTATTATCGCACCTGCAGGTTCCTCACAAATTGCTTTTTTAAAAA CAACCATACCAATAATATATCTATCGGATACAACATATGATCAATTAAAAAGCTATTATCCGAATTTAAATAAAA AAACAATTATAAAATGATGAGGATGCAAGTTTAATCGAACGCAAGGCTATTGAAAAAGCAACAGTAGTATCTTTCC CATCTAAATGGGCAATGGATTTTTGCAGGAATTATTACAG ATTAGATTTTGATAAATTAGTTGAAATACCATGGG GGGCTAATTTATTTGATGATATTCACTTTGCTAATAAAAATATAATTCAAAAGAATAGTTATACTTGTCTTTTCT TGGGAGTTGATTGGGAAAGAAAAGGTGGGAAAACAGCCTTGAAAGCAATTGAATATGTAAGGCAGTTATATGGGA TCGATGTTAGACTAAAAATTTGTGGATGTACTCCGAATCAAAAGATTTTACCTACTTGGGTTGAATTAATTGATA AAGTAGATAAAAATAACGTTGACGAATATCAGAAATTCATCGATGTGTTATCTAACGCTGATATACTTCTTTTAC CAACCATTGCTGAATGTTATGGAATGG TATTTTGTGAAGCTGCTGCTTTTGGATTGCCTGTTGTCGCTACAGATA CAGGTGGAGTCAGTTCTATAGTTATCAACGAAAGGACGGGGATATTAATTAAAGACCCGTTAGACTATAAGCACT

- 94 046206

TTGGAAATGCAATTCATAAAATAATTAGTTCCGTAGAGACTTATCAAAACTACTCCCAAAACGCAAGAATTAGAT ATAATAATATATTGCATTGGGACAATTGGGCTAAAAAGATAATTGAGATTATGTATGAGCATAAGAATAGAAGAA TСAAATAGCACAAAAAGAATTATATGTTTATTTATACTTTTTTTGTTTTCCCCTGATTTTTTGTTTTATACATTA GGGGTTGATAATTTTAGCATTTCAACG ATAATCTCAATTACATTGCTTTTTGTTTTTTTAAGAGCTAAAAATATT TGCAAAGATAATTTTCTAATAATAGTAGCGTTATTCATATTGTTGTGTTTTAACTGTTTGTTAAGTATGCTATTT AATATTGAACAGGCTTTAACATTTAAAGTTGTACTTTCAATATATAGCATCTTAATAATGGCATACGTCTCCTCT TGTTATGCACAGACGTTGTGGTTATGTTCTGAAGAAATACTTAAGAG ATCCGTCTTTTATTTGTTCGCATTTCTT TGCCTTATTGGCATTATAAGTATTCTTTTACAGAAGACTGAGATTATACATGATAAAAGTATGATTCTTTTTCCT GAACCATCAGCATTTGCATTGGTTTTTATACCTATCTTTTCATTTTGTTTATACTATACAAGAGGGGGGGGGCTA CTATTGCTCTATATATTATCTTTGGGTATTGCGTTAGGTATCCAGAATTTAACAATGTTGGTAGGCAT TGTGATT AGTGTTTTTGTGATGAAAAAAATAACTATAAGGCAAACTATTGTTATACTTTTGGGGGCATGGATTTTTTCCATG ATATTAAGTGATTTAGACATTTCTTACTATACATCCGGCTTGATTTTAAAAATACTACGAACCTATCAGTGCTT GTATATCTTTCAGGAATTGAAAGAGCTTTCTTGAATTTTATTACAAGTTATGGTCTTGGTATTGGTTTTCAACAA ATGGGAGTGAATGGGGAGATAG GAATATATCAACAAATTTTAGCTGAACTTGATGCCCCTATGTTAAATATATAC GATGGCTCATTTATTTCTTCTAAGTTAATATCTGAGTTTGGGGTTATTGGTGCATTAATGTGTATTTTTCTATTTT TTTTATTTTTCCCGATTTTATCTGCGTTTCAAAAAAAGTAAGAGATATTCACCGCAGTATATTTTAGCATATAGC TTCTACATGTGTTTCTTCATCCCTCTTTTTATACGTGGTGCTTTAT ATAAACCCCTATGTGTTTATGTTATTT TCATCAATATTTTTGTGCAAATATCACGCTAAAAATATCTTGATGAAATCTAATGTCCAGATAGCTATATAATAG TAGAT TATATAT CAT TAT CAC GTAAAT TAGATAT TAATAG CATATAT GATAAC TAG GACATAAATAAT GT G CAT TAAAAAAAACTTAAGTTAATTAAACGATATGGCCTTTATGGTGGTCTTAGGCTTCTTAAAGATATA TTCTTAAC AAAATTTTTATTTTGTT CAAAT GTTAGGATTAT TAGATTT CCAT GTTATAT TAGAAAAGAT GGAAGT GTTAGTTT TGGAAAAGGTTTTTACATCAGGTGTAGGATTACGAGTTGATGCATTTATGGATGCCGTAGTTTCCATTGGAGAAAA TGTTCAAATTAATGACTATGTTCACATCCGCGCTATTAATAATGTCATTATTGGTAGAGATACATTAATAGCAAG TAAAGTA T T TAT TAGT GAT CATAAT CAT GGTAT T T T T T CTAAAT C C GATAT C CATAGT T CAC CAACTAT TAT T C C TTCGTCTAGGCCCCTTGAATCTGCACCTGTGTATATTGGAGAGCGTGTGTGATTGGCGAAAATGTGACAATATT ACCAGGTGCGTGTATAGGTAATGGTGTAGTTATTGGCGCAAACAGTGTTGTTCGTGGTGAGATTCCTAATAATGT GATCATTG CTGGTGTTCCAGCTAAAATTGTTAAAAAATATAACTATGAGCGTATGCAATGGGAAAGAATATAGTT GTAATATCGGCTGTTAATTTTACAACCGGAGGCCCCTTTACCGTACTAAAAAATGTGCTTACAGCAACTAAAGAT AGACCGAATGTAAAATTTATTGCACTGGTTCATAGCTCTGCTGAACTAATGGAATTATTTCCGTGGGTTGAATTT ATAGAGTATCCAGAAGTCAAGTCTTCGTGGGTTAAAA GATTATATTTCGAATATATAACTTGCAATAGATTATCT AAGGTGATTAAGGCAACTCATTGGGTATGCTTACATGATATTACAGCAAATGTTAGTGTACCCTATAGATTTGTT TATTGCCACAATCCTGCACCGTTCTATAAATATTTAAGCTATCGAGATATTATAGGAGAACCTAAATTTTTATCTT TTTTATCTTTTTTATGGGCTTTTATACAATATCAATATAAAAAAAGAACACAGCAGTTTTTGTTCAGCAG CAGTGG CTAAAAAAAGAATTCGAAAAAAAATATAAGTTAAAGAATGTTGTTGTTAGTCGCCCTGAAGATATTTGCCCTTTT GAAAGTGATGGTTTGGTAAGAAAATAATAATAAAAAGGATGTGAGGATATTTTACCCAGCAGTGCCCCGTATATTT AAAAACTTTGAAGTTATCATACGTGCTGCACAAATATTACAAGATAAAAATATTCATTTTTTATCTTACTTTTGAT GGTACTGAAAATAAGTATGCA AAAAGAATATATAAAATTAGCTTCCGAACTGAAAAATGTACATTTCCTCGGTTAC CTTAATGCAACCGAGATGGTTAACTTTTATCAAGATTCAGATATTATTTGTTTCCCATCGAAACTAGAAACGTGG GGATTACCATTATCAGAAGCTAAAACATACAAAAAATGGATATTTGCGGCAGACTTACCTTATGCTCATGAAGTT TTATATAACTATTCAAAACTAGATATTTTTCCATTTGACGATGAGAAAATACTTG TTCGCTACATATTAGAGTAC

- 95 046206

ACAAGTAAAAATATGCATGAAGATATAAAAAATAGTAGGGTGAATTTTAATAATGATGCATTGACTGGTTTTGAA CAGTTTATTGAATATATCCTCAAGGGGAACTGACGTGGTTTATATTATAATCGTTTCACATGGCCATGATGACTA TATAGAAAATCTTTTATTAAAATTTAAAGTTGCCCTCTGGAAGATTTAAAATAATAGTTCGTGATAACAAAAGTTC AATGGTTTTAAAAAACATGCGAAAAAAATTGC GTAACCTATTTGCATGGAGGGCAATATGGATTTGGACATAA TAATAACATAGCAGTGTCATATATAATTAATAACTTCATGATTATGAATAATGATTATTTTCTCTTTCTTAACCC C GAT GTAT T CATAAC CAGT GAAAGT T T GAT TAAT TAT GTT GAT TATATAAT TAGTAAT GAT TATAAGT T TAGCAC ATTATGTCTTTATCGAGATTTTACTAAAAGCAAACATGATTATT CAATACGGAGTTTTTCCAACTTTATATGATTT TCTTTGTTCTTTTTTATTGGGGGTGAATAAAAGTAAAATTAAGAAGGAAAATATACTTTCTGATACTGTAGTTGA TTGGTGTGCTGGCTCATTTATGCTTATTCATGCTTTAAGTTTCTTAAATGTGAATGGTTTTGATCAAAAATATTT TATGTATTGTGAAGATATTGACCTTTGTATGCGTTTAAAATTAAGTGGAGTAGATCTTTACT ATACTCCCCATTT TGATGCTATTCATTATGCGCAGCATGAAAATAGAAGAATATTTACTAAAGCATTTCGATGGCATATAAGGAGTAT TACGCGCTACATATTACGGAAACCAATTCTTTCTTATAAAAACTATAGAAAAATTACATCCGAACTGGTAAAGTG ATTAAGGATCCGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAA CTAAGGAGGATATTCATAT GGATAAAGCCGTAAGCATATAAGCATGGATAAGCTATTTATACTTTAATAAGTACT TTGTATACTTATTTGCGAACATTCCAGGCCGCGAGCATTCAGCGCGGTGATCACACCTGACAGGAGTATGTAATG TCCAAGCAACAGATCGGCGTAGTCGGTATGGCAGTGATGGGACGCAACCTTGCGCTCAACATCGAAAGCCGTGGT TATACCGTCTCTATTTTCAACCGTTCCCGTGAGAAGACGGAA GAAGTGATTGCCGAAAATCCAGGCAAGAAACTG GTTCCTTACTATACGGTGAAAGAGTTTGTCGAATCTCTGGAAACGCCTCGTCGCATCCTGTTAATGGTGAAAGCA GGTGCAGGCACGGATGCTGCTATTGATTCCCTCAAACCATATCTCGATAAAGGAGACATCATCATTGATGGTGGT AACACCTTCTTCCAGGACACTATTCGTCGTAATCGTGAGCTTTCAGCAGAGGGCTTTAACTTC ATCGGTACCGGT GTTTCTGGCGGTGAAGAGGGGGCGCTGAAAGGTCCTTCTATTATGCCTGGTGGCCAGAAAGAAGCCTATGAATTG GTAGCACCGATCCTGACCAAAATCGCCGCCGTAGCTGAAGACGGTGAACCATGCGTTACCTATATTGGTGCCGAT GGCGCAGGTCACTATGTGAAGATGGTTCACAACGGTATTGAATACGGCGATATGCAGCTGATTGCTGAAGCCTAT TCTCT GCTTAAAGGTGGCCTGAACCTCACCAACGAAACTGGCGCAGACCTTTACCGAGTGGAATAACGGTGAA CTGAGCAGTTACCTGATCGACATCACCAAAGATATCTTCACCAAAAAAGATGAAGACGGTAACTACCTGGTTGAT GTGATCCTGGATGAAGCGGCTAACAAAGGTACCGGTAAATGGACCAGCCAGAGCGCGCTGGATCTCGGCGAACCG CTGTCGCTGATTACCGAGTCTGTGTTTGCACG TTATATCTCTTCTCTGAAAGATCAGCGTGTTGCCGCATCTAAA GTTCTCTCTGGTCCGCAAGCACAGCCAGCAGGCGACAAGGCTGAGTTCATCGAAAAAGTTCGTCGTGCGCTGTAT CTGGGCAAAATCGTTTCTTACGCCCAGGGCTTCTCTCAGCTGCGTGCTGCGTCTGAAGAGTACAACTGGGATCTG AACTACGGCGAAATCGCGAAGATTTTCCGTGCTGGCTGCATCATCCGTGCGCAG TTCCTGCAGAAAATCACCGAT GCTTATGCCGAAAATCCACAGATCGCTAACCTGTTGCTGGCTCCGTACTTCAAGCAAATTGCCGATGACTACCAG CAGGCGCTGCGTGATGTCGTTGCTTATGCAGTACAGAACGGTATTCCGGTTCCGACCTTCTCCGCAGCGGTTGCC TATTACGACAGCTACCGTGCTGCTGTTCTGCCTGCGAACCTGATCCAGGCACAGCGTGACTATTTTGGTGCGCAT ACTTATAAGCGTATTGATAAAGAAGGTGTGTTCCATACCGAATGGCTGGATTAA

SEQ ID NO: 19 (example of nucleotide sequence of the rfb 075 locus - producer strain 075-EPA stLMTBLl 737)

ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGATACCC AAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAGGGATCAAA GAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTATGAGTTAGAATCA CTCCTTGAGCAGCGCGTGA AGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCCGGGCGTGACCATTATG

- 96 046206

AACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGACCTGCCATTGGTGACAACCCA TTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCGCTACGTTACAACCTTGCTGCCATG ATTGCACGTTTCAACGAAACGGGCCGCAGCCAGGTGCTGGCAAAACGTATGCCGGGTGACCTCTCTGAATACTCC GTCATCCAGACTAAAGAGC CGCTGGACCGTGAGGGTAAAGTCAGCCGCATTGTTGAATTTATCGAAAAACCGGAT CAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGTCGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTG GAACGTACTCAGCCTGGTGCATGGGGACGTATTCAGCTGACTGATGCTATTGCCGAGCTGGCGAAAAAACAATCC GTTGATGCAATGCTGATGACCGGCGACAGTTACAAAAAA TGGGCTATATGCAGGCGTTTGTGAAG TATGGCCTACGCAACCTGAAAGAAGGGGCGAAGTTCCGTAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAA TCTGACCGGATGTAACGGTTGATAAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAA TCTTCCTGCCGTTGTTTTATATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAG TTTTCCA GGATTTTCCTTGTTTCCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTA ACGCTCGTCACATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCATTAATAC CTCTATTAATCAAACTGAGAGCCGCTTATTTCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGCTAGCAGT GAAGATACTTG TTACTGGTGGCGCAGGATTTATTGGTTCTGCTGTTGTTCGTCACATAATAAATAATACGCAAGA TAGTGTTGTTAATGTCGATAAATTAACATACGCCGGAAACCTGGAATCGCTCGCTGAAATTTCTGATTCTGAACG TTATTCATTTGAGCATGCAGATATCTGCGATGCCGAAGCGATGGCTCGTATTTTCGCACAGCACCAGCCAGACGC GGTGATGCACCTGGCAGCAGAGAGCCACGTTGA CCGCTCAATAACTGGCCCTGCGGCATTTATTGAAACCAATAT TGTGGGTACTTATGTTCTTTTAGAAGCGGCGCGCAATTATTGGTCTGGTCTGGATGATGAAAAGAAAAAAAACTT CCGCTTTCATCATATTTCTACTGATGAGGTGTATGGTGACTTACCCCATCCGGATGAAGTAAATAGCAATGAAAC GTTGCCGCTATTTACGGAAATGACAGCATACGCGCCAAGTAGTCCATATTCTGCTTCTAAA GCTTCCAGCGATCA TTTGGTTCGCGCATGGAAACGTACTTATGGTTTACCGACCATTGTGACTAATTGCTCGAACAACTATGGTCCTTA TCATTTCCCGGAAAAGCTTATTCCACTGGTTATTCTTAATGCACTGGAAGGTAAGGCATTACCTATTTATGGCAA AGGAGATCAGATCCGCGACTGGTTGTATGTAGAGGATCATGCTCGAGCGTTATATACCGTCGTAACCGGAAGTAA AGCGCG AAACTTATAACATTGGTGGACACAACGAAAAGAAAAACATCGACGTAGTGTTCACTATTTGTGATTT GTTGGATGAGATAGTCCCGAAAGAGAAATCTTATCGTGAGCAAATTACCTATGTTGCTGATCGCCCAGGGCATGA TCGCCGTTATGCAATTGATGCCGATAAAATTAGCCGCGAATTGGGCTGGAAACCACAGAAACGTTTGAGAGCGG GATTCGTAAAACTGTGGAATGGTATCTG TCCAATACAAAATGGGTTGATAATGTGAAAAGTGGTGCCTATCAATC GTGGATTGAACAGAACTATGGGGGCCGCCACTAATGAATATCCTCCTTTTTGGCAAAACAGGGCAGGTTGGTTGG GAACTACAGCGTGCTCTGGCACCTCTGGGTAATTTGATTGCTCTTGATGTTCACTCCACTGATTACTGTGGTGAT TTTAGTAACCCTGAAGGTGTGGCTGAAACCGTTAGAAGCATTCGGCCTGATATTAT TGTCAACGCAGCCGCTCAC ACCGCAGTAGACAAAGCAGAATCAGAACCGGAGTTTGCACAATTACTGAACGCGACGAGTGTCGAAGCGATCGCG AAAGCAGCCAATGAAGTCGGCGCTTGGGTTATTCACTACTCTACTGACTACGTATTTCCGGGGACCGGTGAAATA CCATGGCAGGAGGAGGATGCAACCGCACCGCTAAATGTTTACGGTGAAACCAAGTTAGCAGGAAAAAGCATTA CAAGAGCAT TGTGCGAAGCACCTTATTTTCCGGACCAGCTGGGTCTATGCAGGTAAAGGAAATAACTTCGCCAAA ACGATGTTGCGTCTGGCAAAAGAGCGTGAAGAATTAGCCGTTATTAATGATCAGTTTGGTGCGCCAACTGGCGCA GAGTTGCTGGCTGATTGTACGGCACATGCCATTCGTGTGGCACTGAATAAACCGGAAGTCGCAGGTTTGTACCAT CTGGTAGCCAGTGGTACCACAACCTGGCAC GATTATGCTGCGCTGGTTTTTTGAAGAGGCGCGCAAAGCAGGCATT CCCCTTGCACTCAACAAGCTCAACGCAGTACCAACAACAGTCTATCCTACACCAGCTCGTCGTCCACATAACTCT CGCCTTAATACAGAAAAATTTCAGCAGAACTTTGCGCTTGTCTTGCCTGACTGGCAGGTTGGTGTGAAACGCATG CTCAACGAATTATTTACGACTACAGCAATTTAATAGTTTTTGCATCTTGTTCG TGATGGTGGAACAAGATGAATT

- 97 046206

AAAAGGAATGATGGAATGAATACGCGTAAAGGTATTATTTTAGCGGGTGGTTCTGGTACACGTCTTTATCCTGTG

ACTATGGCTGTCAGTAAACAGCTGTTACCGATTTATGATAAACCGATGATCTATTACCCGCTCTCTACACTGATG

TTGGCGGGTATTCGCGATATTTTGATTATCAGCACGCCACAGGATACTCCTCGTTTTCAACAACTGCTGGGTGAT

GGGAGCCAGTGGGGGCTAAATCTTCACTACAAAGTGCAACCGAGTCCGGATGGTCTTGCGCAGGCATTTATCATC

GGTGAAGAGTTTATCGGTGGTGATGATTGTGCTTTGGTACTTGGTGATAATATCTTCTACGGTCACGACCTGCCT

AAGTTAATGGATGCCGCTGTTAACAAAGAAAGTGGTGCAACGGTATTTGCCTATCACGTTAATGATCCTGAACGC

TATGGTGTCGTTGAGTTTGATAAAAACGGTACTGCAATCAGCCTGGAAGAAAAACCGTTACAACCAAAAAGTAAT

TATGCGGTAACCGGGCTTTATTTCTATGATAACTACGTTGTGGAAATGGCGAAAAATCTTAAGCCTTCTGCCCGC

GGTGAACTGGAAATTACCGATATTAACCGTATCTATATGGAACAGGGGCATTTATCTGTTGCCATGATGGGACGT

GGATATGCCTGGCTGGACACGGGGACACATCAAAGTCTTATTGAAGCAAGCAACTTCATTGCCACCATTGAAGAG

CGCCAGGGCTTGAAAGTTTCCTGCCCGGAAGAAATTGCTTACCGTAAAGGGTTTATTGATGCTGAGCAGGTGAAA

GTATTAGCTAAACCGCTGAAAAAAAATGCTTATGGTCAGTATCTGCTAAAAATGATTAAAGGTTATTAATAAAAT

GAATGTTATTAAAACAGAAATTCCAGATGTACTGATTTTTTGAACCGAAAGTTTTTGGTGATGAGCGTGGTTTCTT

TATGGAAAGCTTTAATCAGAAAGTTTTTCGAAGAGGCTGTAGGGCGGAAGGTTGAATTTGTTCAGGATAATCATTC TAAATCGTGTAAAGGTGTACTTAGAGGTTTACACTTTCAGCTTCCTCCCTTTGAGCAGGCAAAATTAGTAAGGTG TATAGTTGGCGAGGTATTTGATGTTGCAGTAGACATTAGACCTAATTCTGAAACATTTGGTTCATGGGTTGGAGT AACTCTTTCGTCAGAAAATAAAA GGCAGCTATGGATTCCAGAAGGATTCGCCCATGGTTTTTTTAACTTTAAGTGA TATTGCAGAGTTTGTTTAATAAAACTAACAACTATTATTCTTTAAATCATGAAAGGGGAGTCATTTGGAACGATGA GGAAATTAACATTGCCTGGCCCTCTCAATCAGAGAAGATTCTGTCACAGAAAGATATTAATTTACCATCATTTAG

ATTTGTTCAAATGTTTAGCAAGTAGTGTTATCTTTACACTGCACATAGTCATCATTTTTTATGCTTTAAGTAAAT TATATTGCACATCTATAACACAAAGCGCAATAATATTTCGACCTGATGAAGGTTTGTGGTTATTTATCTTTCTAG GCGTTTTTTATGACTAAAATAGTTGTGGTTTCTACAGCTCCAATATTCCCGACAAATAATGGGTACAAAAGTTCT GTATTAGGAAGAATTGATGA GTTATTAAATGAGGATAATGAGGTCGTTTTGATTGAAATAAACCTTGAAAATGTT AC G GAAAAGAAAGAT GAAT TAATAC СAACAAGAT T TAATAATAT T CAAAGATAT GAAGTAAAAAAAATAT S TAGA TCATTTATTGCCGAGTTACAAATATTATTTGATATCAGAACTCGGTATGAACAATTATTTTCTTCTGCTGACATT AGAGATAACATAAAAAAGATAATTGATTTA GAAAACCTTCTATTATTATTGCTGAGTCTATATGGGCGTTGCAA GCATTGCCTATTGAAATTAGTGCGAGAATACACTGTGTTATTCATGATGTGGCAACTGATTTCTTTAAAGAAATG T T T GTAT CT CATAAT GAGGT T GTAC GAAAAAT TTTGTTTTTT TAAT GAT TAC CTAAAGT T GAAAAT TACT GAAGAA AATAT TAT СAAAC GT T T GAGAGT T GAG CAAT TTATCTTTCT GACAGAAGAGATAAAT GT T G GTATAAAACAAGA TACAATATTGATGAGGGTTGTTGTTCCTTAGCGAGCAATCATCTTTATGTAGAAAAGATTAAGAGAACTATCAAT TTCCAAACCCCTTTCCTGCTTATTCCCGGTAGCATTGAATTTTCACAAAATTTTTACGGCTTAAATTGGTTTATA AAAAATATATATCCTGGATTAAATAGGAAAATAAGAATAGTTGTAACAGGAAAGGCATCAGATAAAAAAATAAAG

ATGTTAAACTGTGGAGAGGAAATTACCTTTACGGGAGAGCTTGACTTTTCCACATATAATAAACTTAGCTCAACA

TGCTTGTGTGTTATTGCACCGATTACAACGGGCACTGGAATTAAAATAAAAATATTAGAAGCTGTACAAAAAGGT

ATTCCTGTACTTACAACAAAATTTGCTTCAAAAGGAATATGTTCCGATTTATGTTTTTATTGCGAGGAGGATACT

GACACAAACTTTGTCAATTTAATTAACAGTTTTCTTGAAACGACATTAAGAGTCCAAGAATGAATTTATTGCTTT

TTTCAGTCCTTGCGTTTGGTTTAATATTGGCTTTGGCCCATAATAATAAAAGTGGAGATATTAACGCATACTTAA

TGTTTTTTCTCGTGGTCCTAATGGTATTAATATCAGGGCTGCGTATGAATGATAGTGATTATATCGAATACAGGA

AAATGTATAATGAAGTGCCTATTTTATGTGACTTTAGTCTCGCATCTATAAGAGATATACATGGGGAGGTAGGCT

ATCTATTCTTATCATCAATCTTTAAAACTTTATGCTTGCCATTTCAATTATTTCTTTTTTTTATTGCTTTTTTAT

- 98 046206

CACTCCTGCTTACATATTTTTCATTCAGAAAAATAAGTTTAATACCGATACTATCGTTAGTTTTTTTATTTAAGCC ATGCTTTTATAGTTAGAGATTTGATTCAAATTAGGGCAGGATTAGCTGTTAGCATATCATTATATTCAATAATTA AATTTAAAGGAAATAAAAGTATAATTACAGGAGTTTTATTTGCTTCTTTGATTCATTCTGGGGCGCTTATTATTG CTCTTTGTTATСCTTTTTTCAAAAAAAAATA CATAACATTAAAAATGATGTTGTTTTTATTTTTAGTGTCAATTA TTTTTTCTTATTTGAATGGGCTTAATTTATCGATACAACTCTTATCTCAATATAGTTTGCTTCCAACTGCAATTT CGAATTATGTTGGTTGGGAAGAATATGATTATCGGGTGAGTATATTTACTAATCCGGTTTTTATTAAAGGTGTTT TTTTAATTGTCTTAATGCACAAATATGTACTTTCAGATATTAAAAATGAGA AAATTATAGTGCTTTATAACTTAT ATGTTTTAGGTGTATTAGCTATGGTTGCATTGAGTGGGATGGCTATTCTTTCAGGCCGTCTTTCATCCCTTTCTGA CACTAGGTGAAAGCATTTTAATTGTATATGCTCTGTTCTACAAAAGAAATACACCTCTGGCGTTTCTAATTTTTTT CTTTTTTAACAATTGTGCAATTAGGATATGATCTATTTATTTCTAATGTGCATCCTGAGCTTACTCTGATTATAT TT GGGT GAAT CTAAGT GAAAAATAATAAAATAGGCATACTTAT CT CTAAAATACAAAAT CTT GGACCT GT GAAT G TAGTACGAGGATTGATAAAAGAAAATAAAAAAAATATGCTTTACTGTTTTTTGTTTAACAAATAGCGTAGATAAAA ATATATATGATGAGTTATGCTGTTTAGGAGCCAAGGTTATATTAATACCAGATGGTACTTGGTTCAGCAAAATTT TATTTGTGAGA AGTTTTTTAAAGGAACATCCACATAATATCTTACATTCACATGGGATCACGGCCGATATGTTTT CTTACTTTCTGAATGGCGTGAAAATATCTACTATTCACAATAGACTAGATGAGGATTATATCCCATTATTTGGCG CGGTTAAAGGGAATGCTATATATTATCTTCATCGTTTTATATTACGAAGATTTAATCATATCGTTGCTTGCTCAG CAGCGGTCCAATCAAAACTGAAACAATCGAAAGTAAAAACTAAAATA ACCACCATCCAGAATGGGATTGATATAA CTAGGTTTAAGACACTTGAGTCTGATAAAAAAAAATTATTGAGGGAAAAACACGGATTTGATAGTGAAAAAAGAA TATTTATATATTGTGGCTCGTTATCATTAAGGAAAAATATTGCTTACCTCTTGGAACACTTAGCCATCGAAGAAA ATGATATATTTTTAATTCTAGGTGATGGTGAACTTTTTAGATATTGTAAGGATAAATATTCTAAAGATTTACGGT ATATA TTTATGGGGAAAGTTGAATGCCCTCTTGAATATTATCAATTATCAGATATTTTTGTTTCCGCTTCTTTAT CGGAAGGGCTCCCCTTGGCACTATTAGAAGCTGCCTCTACTGGGTGCTATTTATATGTTAGCGATATAGAGCCCC ATAGAGAAATTGCATCTCTATTAGGAGAGGAAAATATTTCTATGTTTAAAATTAAGGATGGATCATATAATTATT TGCAACCTAAAATAAAAAAAGCTGACTATAACGCTCTTT CTGACGATAAACTTTACAATATATCCGATAAAAAAA TGTCAAATCTTTATGACAAACTTTTTGTTTCTTTATTAGCAGAGGCACTAATATAATGATTTATGTTTCGGTA ATTTCTCATGGTCATTTCAAAACTCTTAAGGAATTAGGAGCAGTATCAAAATTAAATAATCACAGCAGAATTAAA GTTATCATCAAAGATAATTTAGGAGAGAGCGAGCTTTTGGATTTTTGTCAGGAAAACAAAATAACTTATTTA AGG TCTAAAGAGAAAAAAGGATTTGGAGAGAATAATAATGAAGTTTTTTTCCTTATCCTCCTTAATTACTAAGGAA GATTTTTTTGTGGTTATGAATCCTGATATATATTGAGTGCTCTGATCTATTAGATGTCGTAGATGAGTGTGGT TCAGCGAATGTTAATCTAGCAACGATAAATTTATACAGGGATTTTGATAAAAAAACATATGATAACTCAGTAAGG AAATTTTSSTCGGCA ATTGATTTTTTTATGTCATTTTTATTTAAGAAAAATGACTGTGTAGTAAATAAGAACAAA ATAACGAAACCAACATATGTTGATTGGGCTGCAGGTTCTTTTCTAATATTTAATGCCTTCTTTTATTCAAAACTC AACGGATTCAACGAAAAGTATTTTATGTATTGCGAAGATATTGATATATGTTGGCGAGCTAAAAAACACTTCAAT ACTTCAGTTTTATACTATCCATGCTAGGCAGCAATTCATTT CACAATTTAACAATCGTAGGATTTTTAGTAGA CATTTCATTTGGCATATAAAAAGTATTATCCTTTTTTTATTATATAAAAATGGTATGCTGCGTTCTAGTAAGTTG CTTTAATGCTAATATTCTTTTAAGAGGTGAGAATGATACCTGTTATTTTGGCTGGTGGTTCGGGAAGTCGCTTGT GGCCACTTTCACGAGAAAAGTTCCCCAAGCAGTTTTTTAAAGTTGACTGGCAGTTTGACAATGTT GCAGTCAACAT TGTCACGTCTTAATAATTTAAATGCTGATGATTCAATAGTTATATGCAACGAAGAGCATAGATTTATTGTTGCAG AACAATTAAGAGAGTTAGGCAAACTTTCAAATAACATTATTCTTGAACCCAAAGGTCGTAATACAGCCCCTGCTA TAACACTCGCAGCATTAGCAGCAAAAAGAAAATTCGCTGATGAAGATCCATTGATTCTTATTTTAGCTGCAGATC

- 99 046206

ACAACATCCAAGACGAACATGTTTTCTGTGAGGCAATTAATAAGGCGTCATCTTTAGCTAGTTATGGAAAACTAG TGACTTTTGGTATCGTTCCATTCAAACCTGAAACTGGGTATGGCTATATTCGTCGCGGTGATGAAGTGCCTGTAG ATGAGCAGCATGCGGTGGCCTTTGAAGTGGCGCAGTTTGTCGAAAAACGATCTGGAAACCGCGCAGGCCTATG TGGCAAGCGGCGAATATTACTGG AACAGCGGTATGTTCCTGTTCCGTGCCGGACGCTATCTCGAAGAACTGAAAA AGTATCGTCCGGATATTCTCGATGCCTGTGAAAAAGCGATGAGCGCCGTCGATCCGGATCTCGATTTTATTCGTG TGGATGAAGAGGCGTTTCTCGCTTGTCCGGAAGAGTCGGTGGATTACGCGGTCATGGAATGCACGGCAGATGCCG TTGTGGTGCCGATGGATGCGGGCTGGAGCGATGTC TTCCTGGTCTTCATTATGGGAGATCAGCGCCCACACCG CCGAGGGCAACGTTTGCCACGGCGATGTGATTAATCACAAAACTGAAAACAGCTATGTGTACGCCGAATCTGGCC TGGTCACCACCGTCGGGGTGAAAGATTTGGTGGTAGTGCAGACCAAAGATGCAGTGCTGATTGCCGACCGTAATG CGGTGCAGGATGTGAAGAAAGTGGTCGAGCAGATCAAAGCTGATGGTCGCCATGA GCATCGGGTGCATCGCGAAG TGTATCGTCCGTGGGGCAAATATGACTCTATCGACGCGGGCGACCGCTACCAGGTGAAACGCATCACCGTGAAAC CGGGCGAAGGTTTGTCGGTACAGATGCATTATCATCGCGCGGAACACTGGGTGGTTGTCGCGGGAACGGCAAAAG TCACTATCAACGGTGATATCAAACTGCTTGGTGAAAACGAGTCCATTTATATTCCGCTGGGGGGGATGCACTGCC T AAAACCCGGGGAAAATAGATTTAGAATTAATTGAAGTTCGCTCTGGTGCATATCTTGAAGAAGATGATGTTA T TAGAT GT TAT GAT C GCTAT GGAC GAAAGTAATATATAATAAT TAT T CAGAAT TAGAAAT GATAAT TATAAGT T TTCGTCTGGATAAACAATAGATAGTATGGGTTGGAAAATATGAGTTCTTTAACTTGTTTTAAAGCTTACGACATT CGCGGGAAATTAGGTG AAGAACTGAATGAAGATATCGCCTGGCGCATTGGTCGCCTATGGCGAATTTCTCAAA CCGAAAACCATTGTGTTAGGCGGTGATGTCCGTCTCACCAGCGAAACCTTAAAACTGGCGCTGGCAAAAGGTTTA CAGGATGCGGGCGTCGATGTGCTGGATATTGGCATGTCCGGCACCGAAGAGATTTATTTCGCCACGTTCCATCTC GGCGTGGATGGCGGCATTGAAGTTACCGCCAGCCATAATCCGAT GGATTACAACGGCATGAAGCTGGTGCGCGAA GGGGCTCGCCCGATCAGCGGTGATACCGGACTGCGCGACGTCCAGCGTCTGGCAGAAGCTAACGACTTTCCTCCC GTCGATGAAACCAAACGCGGTCGCTATCAGCAAATCAATCTGCGTGACGCTTACGTTGATCACCTGTTCGGTTAT ATCAATGTCAAAACCTTACGCCGCTCAAGCTGGTGATCAACTCCGGGAATGGCGCAGCGGGTCCG GTGGTGGAC GCTATCGAAGCCCGCTTTAAAGCCCTCGGCGCACCGGTGGAGTTAATCAAAGTGCATAACACGCCGGACGGCAAT TTCCCCAACGGTATTCCTAACCCGTTGCTGCCGGAATGTCGCGACGACACCCGCAATGCGGTCATCAAACACGGC GCGGATATGGGCATTGCCTTTGATGGCGATTTTGACCGCTGTTTCCTGTTTGACGAAAAAGGGCAGTTTATTGAG GGCT ACTACATTGTCGGCCTGCTGGCAGAAGCGTTCCTCGAAAAAAATCCCGGCGCGAAGATCATCCACGATCCA CGTCTCTCCTGGAACACCATTGATGTGGTGACGGCCGCGGGCGGCACGCCGGTGATGTCGAAAACAGGACACGCC TTTATTAAAGAACGTATGCGCAAGGAAGACGCCATCTACGGTGGCGAAATGAGCGCTCACCATTACTTCCGCGAT TTCGCTTACTGTGACAGCGGCATGA TCCCGTGGCTGCTGGTCGCCGAACTGGTGTGCCTGAAAGGAAAAACGCTG GGCGAACTGGTGCGCGACCGGATGGCGGCGTTTCCGGCAAGCGGTGAGATCAACAGAAAACTGGCGCACCCTGTT GAGGCGATTAACCGCGTGGAACAGCATTTTAGCCGTGAGGTGCTGGCGGTGGATCGCACCGATGGCATCAGCATG ACCTTTGCCGACTGGCGCTTTAACCTGCGCTCTTCCAACACCGAACC TGGTGCGCCTGAATGTGGAATCTCGC GGTGATGTTCAGGTTATGGTAATCCATACTCAAGAAATATTATCAATTTTGACGTCATAAAGAATAAGCCCTGAC AAGTTAGGGCTTAATTAATATATTTTTTTTGAATTGGGGATTTGTGGTAAGATTTTTTAATATGTTATTTAATG TGGTTGAATTAATGTTGACTGGAAAATAATAATGAGAACGAAAAAAGCATTACACAACTTTAAAGTTGATTTATT AATTACTTTTTTATTGGTTTTGCTAGGGTTTTATATTCGAACTGTTTTTGTTTCAAAATGGGAAGTGATATTAC TGGAGTGATGTTACTATTCACACAGTTGACAGCATATCTCAATTTGGCAGAATTAGGTATTGGAATTGCAGCTGC CAGCGTATTATAAAACCGCTCA GCGAGAATGAATACAATAAAATAACTTACATAATATCTTTGCTCTCAGTCAT ATACAAATATATATTTGTGTTTGTTTTGATTCTTGGCGTTGTTATAGGTATCTGTATTTATTACTTTATTGATTC

- 100 046206

TGTAAAGGTTGTAAAATGGCGTTTTTTTATATTGGGCTTTGTTCGTTTTTAATACATCGTTGACATATAGTTATGC TAAATACTCCACATTATTAACTGCTAATCAGCGGTACTCAGCAGTAAGAAAAATTCAAGGTGGCGGAAAAGTTAT AATAATTGTATTTCAGATATTAATTTTGTGCTTTACGCAAAGTTTCATACTTTATTTGTTAGTTGAGACTTTAGG TATTTTTTCTCAATATTTGAT TTTTAAAAAAATAATTGGGAACGGAAATCAATATCTCAGTAATGAGGTTTTACT TATTGAAAGCGATAAACTTTTGATAAAAAAAGAATTAAAAATAAGAATAAAAAAAATATGTTCTTCCATAAAATAGG TGCTGTGCTTGTCCTTAATACAGACTACCTGCTTGTATCAAAGTTTCTGACATTAAGTTATGTGACAATTTTTGG CAGCTATATGATGGTATTTCAGATAGTAACTGTTTTGATGTCAAGTTTTGTTA ATGCTATTACTGCAGGAATGGG TAATTACTTAATTAATAAAAGTAATTTAGAAATTAAGGAAATTACACGTCAATTTTATGTGATATTTATCGCCTT TGCAACATTCATATCACTAAATATGTTTTTTCTTGTTAATGATTTTATCGCAAAATGGATAGGTGTTAATTATAC ATTAAGTAACACCCTAGTTGCATTAATGATTGTTAACGTATTCATTAGTGTTGTCAGGGTACCTTCTGATATATT AAAAAA C G C AAGT G GAG AT T T T G GT GAT AT T TAT TAT C CAT TAT T AGAAG G T GT G C T GAAT AT TAG GAT AT C CAT CATTTTGGCTATCATTATTGGATTACCTGGCATTATTATAGGGACAATAGTATCTAACTTAATAGTAATAATGCT TGCGAAACCATTATATCTTTACTCTAAGTTATTTAATCTTAGAAATCCGACGAGGGTTTATTTTGAATTTATTTC TCGGCCTATGTTATATTCATTATGTGTGATTGGGGTGAGCTATTTATTGCGCGATGAAATATATTCATTTAAAGT AAGTACATGGTTGGATTTTATTAACAAGCTACTCTTAGTCTCTACTCCTAGCATATTGGTAATATGTGCTATTT CT CT AC G GAT AGT GACT T T AGAT TAT T T T T CAGAAAAAT TAT AT AT GT GAT TAT GAAGAAAT AAAAAT T T C GAAA ATGTATTAATCGAAATTATGCAACGAGCTTTATTTTTATAAATGATATGTGATCTTTTCGCGAATAGGAGTAAGG ATCCGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTAAGGA GGAT AT T CATAT GGATAAAGC C GT AAGCATATAAGCAT GGATAAGCTAT TT AT ACT T TAATAAGTACT T T GT ATA CTTATTTGCGAACATTCCAGGCCGCGAGCATTCAGCGCGGTGATCACACCTGACAGGAGTATGTAATGTCCAAGC AACAGATCGGCGTAGTCGGTATGGCAGTGATGGGACGCAACCTTGCGCTCAACATCGAAAGCCGTGGTTATACCG TCTCTATTTTCAACCGTTCC CGTGAGAAGACGGAAGAAGTGATTGCCGAAAATCCAGGCAAGAAACTGGTTCCTT ACTATACGGTGAAAGAGTTTGTCGAATCTCTGGAAACGCCTCGTCGCATCCTGTTAATGGTGAAAGCAGGTGCAG GCACGGATGCTGCTATTGATTCCCTCAAACCATATCTCGATAAAGGAGACATCATCATTGATGGTGGTAACACCT TCTTCCAGGACACTATTCGTCGTAATCGTGAGCTTTCAGCA GAGGGCTTTAACTTCATCGGTACCGGTGTTTCTG GCGGTGAAGAGGGGGCGCTGAAAGGTCCTTCTATTATGCCTGGTGGCCAGAAAGAAGCCTATGAATTGGTAGCAC CGATCCTGACCAAAATCGCCGCCGTAGCTGAAGACGGTGAACCATGCGTTACCTATATTGGTGCCGATGGCGCAG GTCACTATGTGAAGATGGTTCACAACGGTATTGAATACGGCGATATGCAGCTGATTGCTGAAG CCTATTCTCTGC TTAAAGGTGGCCTGAACCTCACCAACGAAGAACTGGCGCAGACCTTTACCGAGTGGAATAACGGTGAACTGAGCA GT TAC C T GAT C GACAT CAC CAAAGAT AT С T T С AC C AAAAAAGAT GAAGAC G GT AACT AC С T G GT T GAT GT GAT С C TGGATGAAGCGGCTAACAAAGGTACCGGTAAATGGACCAGCCAGAGCGCGCTGGATCTCGGCGAACCGCTGTCGC TGATTACCGAGTCTGTGTTTGCACGTTATATCTCTTCTCTGAAAGATCAGCGTGTTGCCGCATCTAAAGTTCTCT CTGGTCCGCAAGCACAGCCAGCAGGCGACAAGGCTGAGTTCATCGAAAAAGTTCGTCGTGCGCTGTATCTGGGCA AAATCGTTTCTTACGCCCAGGGCTTCTCTCAGCTGCGTGCTGCGTCTGAAGAGTACAACTGGGATCTGAACTACG GCGAAATCGCGAAGATTTTCCGTGCTGGCTGCATCATCCGTGCGCAGTTCCTGCAGAAAATCACCGATGCTTATG CCGAAAATCCACAGATCGCTAACCTGTTGCTGGCTCCGTACTTCAAGCAAATTGCCGATGACTACCAGCAGGCGC TGCGTGATGTCGTTGCTTATGCAGTACAGAACGGTATTCCGGTTCCGACCTTCTCCGCAGCGGTTGCCTATTACG ACAGCTACCGTGCTGCTGTTCTGCCTGCGAACCTGATCCAGGCACAGCGTGACTATTTTGGTGCGCATACTTATA AGCGTATTGATAAAGAAGGTGTGTTCCATACCGAATGGCTGGATTAA

Claims (19)

(1) the nucleotide sequence of the rfb gene cluster for the O4 polysaccharide antigen of E. coH; 1 β-D-GlcpNAc and n is an integer from 5 to 40. (1) a bioconjugate of the glycosylated polysaccharide antigen E.coH O4 covalently linked to detoxified P.aeruginosa exotoxin A (EPA-4 carrier protein) containing SEQ ID NO: 3, wherein the glycosylated polysaccharide antigen O4 E.coH contains the structure of the formula (O4 -Glc+) a-D-GIcp I (1) E. coli polysaccharide antigen O1A contains the structure of formula (01A) 1. A bioconjugate of a glycosylated polysaccharide antigen O4 of E. coli covalently linked to a carrier protein, wherein the glycosylated polysaccharide antigen O4 of E. coli contains the structure of the formula (O4-Glc+) a-D-GIcp J [^»2)-aL-Rhap-(1->6)-aD-Glcp-(1^»3)-aL-FucpNAc-(1-»3)-pD-GlcpNAc-(1->] _n where n is an integer from 1 to 100, the method includes culturing a recombinant prokaryotic host cell according to any one of claims 15 to 17 under conditions for producing a bioconjugate. (2) a nucleotide sequence encoding a glycosyltransferase, the sequence of which is at least 80% identical to SEQ ID NO: 4, where the glycosyltransferase is capable of modifying the E.coH O4 polysaccharide antigen to produce a glycosylated E.coH O4 polysaccharide antigen; [ >2)-aL-Rhap-(1 >6)-aD-Glcp-(1 >3)-<xL-FucpNAc-(1 >3)-|3-D-GlcpNAc-(1 >] _n where n is an integer from 1 to 100, where the host cell contains: [—»2)-aL-Rhap-(1 —»6)-«-D-Glcp-(1—*4)-aD-Galp-(1—»3)-aD-GlcpNAc-(1—*] _n t 2 t Ac (8) bioconjugate of E.coli polysaccharide antigen O25B covalently linked to an EPA-4 carrier protein containing SEQ ID NO: 3, where E.coli polysaccharide antigen 025b contains the structure of the formula (025b) β-D-Glcp I L >4)-dD-Glcp-(1 >3)-aL-Rhap-(1 >3H5-D-GlcpNAc-(1 >] _p tI 1Ac cc-L-Rhap; and (9) a bioconjugate of an E.coH O75 polysaccharide antigen covalently linked to an EPA-4 carrier protein containing SEQ ID NO: 3, wherein the E.coH O75 polysaccharide antigen contains the structure of the formula (O75) β-D-Manp F [^2)-p-D-Galf-(1 ->6)-a-D-Glcp-(1 ->3)-a-L-Rhap-(1 -3)-a-D-GlcpNAc-(1 ->]„ [^2)-pD-Galp-(1 ^3)-oc-L-FucpNAc-(1 ^3)-pD-GlcpNAc-(1 ^] _p (7) bioconjugate of the polysaccharide antigen O16 E.coH, covalently bound to an EPA-4 carrier protein containing SEQ ID NO: 3, wherein the polysaccharide antigen O16 E.coH contains the structure of the formula (O16) - 103 046206 2 ΐ 1 β-D-ManpNAc (3) a bioconjugate of the E.coH O2 polysaccharide antigen covalently linked to an EPA-4 carrier protein containing SEQ ID NO: 3, wherein the E.coH O2 polysaccharide antigen contains the structure of the formula (O2) [^2)-ot-L-Rhap-(1^6)-aD-Glcp-(1^3)-aL-FucpNAc-(1^3)-pD-GlcpNAc-(1^] _n (2) bioconjugate an E.coli O1A polysaccharide antigen covalently linked to an EPA-4 carrier protein containing SEQ ID NO: 3, wherein the E.coli O1A polysaccharide antigen contains the structure of the formula (O1 A) L >3)-aL-Rhap-(1 >3)-aL-Rhap-(1 >3)-pL-Rhap-(1 >4)-[3-D-GlcpNAc-(1 >] _n 2 ΐ Ac (7) polysaccharide antigen 018A E. coli contains the structure of the formula (018A) [->2)-a-l_-Rhap-(1 ->6)-«-D-Glcp-(1 ->4)- ct-D-Galp-(1 ->3)-aD-GlcpNAc-(1 ->] _n 3 t 1 β-D-GlcpNAc (8) polysaccharide antigen 025B E.coH contains the structure of the formula (025B) - 102 046206 β-D-Glcp I [^2)-pD-Galf-(1 —*6)-aD-Glcp-(1 —»3)-aL-Rhap-(1 —3)-aD-GlcpNAc-(1 ->] _l 2 t 1 a-D-Fucp3NAc (3) polysaccharide antigen 06A E.coH contains the structure of the formula (06A) 2 ΐ 1 β-D-ManpNAc (2) polysaccharide antigen 02 E.coH contains the structure of formula (02) 2. Bioconjugate according to claim 1, where n is an integer from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. [->2)-aL-Rhap-(1 ->6)-aD-Glcp-(1 -3)-«-L-FucpNAc-(1^3)-pD-GlcpNAc-(1 ->] _n where n represents an integer between 1 and 100. (3) nucleotide sequences encoding a translocase and a glycosyltransferase, the sequence of which is at least 80% identical to SEQ ID NO: 7 and 8, respectively, where the translocase is capable of translocating bactoprenol-bound glucose and the glycosyltransferase is capable of glycosylating bactoprenol; [->3)-aD-Galp-(1 ->4)-aL-Rhap-(1 ->3)-pD-GlcpNAc-(1 ->] _n where each n independently represents an integer from 5 to 40 . [->3)-aL-Rhap-(1 ->2)-aL-Rhap-(1 ^3)-pL-Rhap-(1 ->4)-pD-GlcpNAc-(1 -] _n 2 t 1 aD-Fucp3NAc (4) a bioconjugate of the polysaccharide antigen O6A of E.coH covalently linked to the carrier protein EPA-4 containing SEQ ID NO: 3, where the polysaccharide antigen O6A of E.coH contains the structure of the formula (O6A) [->3)-aD-Galp-(1 ->4)-aL-Rhap-(1 ->3)-3-D-GlcpNAc-(1 ->] _n where each n is independently an integer from 1 to 100, preferably from 3 to 50, for example from 5 to 40, for example from 7 to 25, for example from 10 to 20. [->3)-cc-L-Rhap-(1 ->2)-aL-Rhap-(1 ^3)-pL-Rhap-(1 -M)-pD-GlcpNAc-(1 ^] _p [ >3)-cr-L-Rhap-(1 >3)-aL-Rhap-(1 >3)-(%L-Rhap-(1 >4)-(5-D-GlcpNAc-(1 >] _n 3. The bioconjugate according to claim 1 or 2, wherein the glycosylated polysaccharide antigen O4 of E. coli is covalently linked to an Asn residue at the glycosylation site containing the glyco consensus sequence - 101 046206 sylation Asn-X-Ser(Thr), where X can be any amino acid other than Pro (SEQ ID NO: 1), preferably the consensus glycosylation sequence Asp(Glu)-X-Asn-ZSer(Thr), where X and Z are independently selected from any amino acid other than Pro (SEQ ID NO: 2) in the carrier protein. (4) a nucleotide sequence encoding a carrier protein; and (5) a nucleotide sequence encoding an oligosaccharyltransferase capable of covalently linking the glycosylated E.coH O4 polysaccharide antigen to a carrier protein to produce a bioconjugate. [-»4)-aD-GalpNAc-(1 -»3)-pD-Manp-(1 -^4)-pD-Manp-(1->3)-aD-GlcpNAc-(1 -»] _n I 1 β-D-Glcp (5) a bioconjugate of the polysaccharide antigen O8 E.coH covalently linked to the carrier protein EPA-4 containing SEQ ID NO: 3, where the polysaccharide antigen O8 E.coH contains the structure of the formula (O8) aD- MaprZM e-( 1 -· [3)-pD-Manp-( 1 ->2)-aDM anp-( 1 ->2)-aD-Manp-( 1 -*] _l (6) bioconjugate of polysaccharide antigen O15 E .coli covalently linked to an EPA-4 carrier protein containing SEQ ID NO: 3, wherein the polysaccharide antigen O15 E.coH contains the structure of formula (O15) [->4)-a-D-Glcp-(1 ->3)-a-L-Rhap-( 1 ->3)-0-D-GlcpNAc-(1 Tt 1Ac ct-L-Rhap; and (9) E. coli polysaccharide antigen O75 contains the structure of formula (O75) β-D-Manp F [-»4)-aD-GalpNAc-(1-»3)-pD-Manp-(1-»4)-pD-Manp-(1-»3)-aD-GlcpNAc-(1-*] _n I 1 β-ο-Glcp (4) polysaccharide antigen 08 E.coH contains the structure of the formula (08) aD-Manp3Me-(1 -^[3)-pD-Manp-(1 -»2)-ot-D-Manp -(1 -^2)-aD-Manp-(1 -^] _n (5) E. coli polysaccharide antigen 015 contains the structure of formula (015) [^2)-pD-Galp-(1 -^3)-a -l_-FucpNAc-(1 -H>3)-pD-GlcpNAc-(1 -Н>] _p (6) polysaccharide antigen 016 E.coH contains the structure of the formula (016) 4. Bioconjugate according to any one of claims 1 to 3, where the carrier protein is selected from the group consisting of detoxified P. aeruginosa exotoxin A (EPA), E. coH flagellin (FliC), CRM 197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, neutralized hemolysin A S.aureus, agglutinating factor A, agglutinating factor B, heat-labile enterotoxin E.coH, neutralized variants of the thermolabile enterotoxin E.coH, subunit B of cholera toxin (CTS), cholera toxin, neutralized variants of cholera toxin, E. coli Sat protein, E. coli Sat protein passenger domain, Streptococcus pneumoniae pneumolysin, Megathura crenulata hemocyanin (KLH), P. aeruginosa PcrV, Neisseria meningitidis outer membrane protein (OMPC), and protein D from nontypeable Haemophilus influenzae, preferably where the carrier protein is a detoxified Pseudomonas aeruginosa exotoxin A (EPA), preferably containing from 1 to 20, preferably from 1 to 10, preferably from 2 to 4 consensus glycosylation sequences having SEQ ID NO: 1, preferably the consensus sequences having SEQ ID NO: 2, more preferably, wherein the carrier protein comprises four consensus glycosylation sequences (EPA-4), more preferably wherein the carrier protein comprises SEQ ID NO: 3. 5. Pharmaceutical composition containing a bioconjugate according to any one of claims 1-4. 6. The pharmaceutical composition according to claim 5, containing at least one additional polysaccharide antigen covalently linked to a carrier protein. 7. The pharmaceutical composition according to claim 6, where at least one additional polysaccharide antigen is selected from the group consisting of polysaccharide antigen O1A E.coH, polysaccharide antigen O2 E.coH, polysaccharide antigen O6A E.coH, polysaccharide antigen O8 E.coH , polysaccharide antigen O15 E.coH, polysaccharide antigen O16 E.coH, polysaccharide antigen O18A E.coH, polysaccharide antigen 025B E.coH and polysaccharide antigen O75 E.coH, preferably where: 8. The pharmaceutical composition according to claim 7, wherein each carrier protein is an EPA containing SEQ ID NO: 3. 9. Pharmaceutical composition according to any one of claims 7 or 8, containing at least polysaccharide antigens O1A, O2, glycosylated polysaccharide antigens O4, 06a, O8, O15, O16, 018a, O25B and O75 E.coH, each of which is covalently linked with a carrier protein. (10) a bioconjugate of the polysaccharide antigen O18A of E.coH covalently linked to the carrier protein EPA-4 containing SEQ ID NO: 3, where the polysaccharide antigen O18A of E.coH contains the structure of the formula (O18A) 10. Pharmaceutical composition containing: 11. Pharmaceutical composition according to claim 10, additionally containing: 12. A method of inducing antibodies against the glycosylated O4 antigen E.coH in a subject, comprising administering to the subject a bioconjugate according to any one of claims 1-4 or a pharmaceutical composition according to any one of claims. 5-11. 13. A method of vaccinating a subject against E.coH, comprising administering to the subject a bioconjugate according to any one of claims 1-4 or a pharmaceutical composition according to any one of claims 5-11. 14. The vaccination method according to claim 13, where said E.coH is extraintestinal pathogenic E.coH (ExPEC). 15. Recombinant prokaryotic host cell to produce a bioconjugate of the glycosylated polysaccharide antigen O4 E.coH, covalently linked to a carrier protein, where the glycosylated polysaccharide antigen O4 contains the structure of the formula (O4-Glc+) a-D-GIcp I 16. The recombinant prokaryotic host cell according to claim 15, wherein the glycosyltransferase has SEQ ID NO: 4; - 104 046206 oligosaccharyltransferase contains the amino acid sequence SEQ ID NO: 6, preferably having SEQ ID NO: 6 containing the amino acid mutation N311V, more preferably SEQ ID NO: 6 containing the amino acid mutations Y77H and N311V; and the carrier protein comprises at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2, preferably wherein the carrier protein is detoxified Pseudomonas aeruginosa exotoxin A (EPA), more preferably wherein the EPA contains 1-10, preferably 2-4 glycosylation sites, each of which contains a consensus glycosylation sequence having SEQ ID NO: 2, more preferably where the carrier protein is an EPA with four consensus glycosylation sequences (EPA-4), more preferably wherein the protein carrier contains SEQ ID NO: 3. 17. The recombinant prokaryotic host cell according to any one of claims 15 or 16, which is an E. coH cell, for example E. coH strain K-12, such as strain W3110. 18. A method for producing a bioconjugate of the glycosylated polysaccharide antigen O4 E.coH, covalently linked to a carrier protein, where the glycosylated polysaccharide antigen O4 E.coH contains the structure of the formula (O4-Glc+) a-D-GIcp I 19. The composition according to any one of claims 10 or 11, wherein the bioconjugate of the polysaccharide antigen 025b is present in the composition at a concentration that is approximately 1.5-6 times, for example 2-4 times, higher than the concentration of any other bioconjugate, preferably where bioconjugates O1A:O2:glycosylated O4:O6A:O8:O15:O16:O18A:O25B:O75 are present in a polysaccharide mass ratio of 1:1:1:1:1:1:1:1:2:1 or 2:1 :1:2:1:1:1:1:4:1 and preferably wherein the concentration of the polysaccharide antigen 025B bioconjugate is from 2 to 50 μg/ml, preferably from 8 to 40 μg/ml, for example from 16 to 32 μg/ml .