OA20646A - Bioconjugates of E. coli O-antigen polysaccharides, methods of production thereof, and methods of use thereof. - Google Patents

Bioconjugates of E. coli O-antigen polysaccharides, methods of production thereof, and methods of use thereof. Download PDF

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OA20646A
OA20646A OA1202100435 OA20646A OA 20646 A OA20646 A OA 20646A OA 1202100435 OA1202100435 OA 1202100435 OA 20646 A OA20646 A OA 20646A
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coli
antigen
antigen polysaccharide
carrier protein
bioconjugate
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OA1202100435
Inventor
Jeroen Geurtsen
Jan Theunis Poolman
Kellen Cristhina Fae
Pieter Jan Burghout
Eveline Marleen WEERDENBURG
Patricia Ibarra Yon
Darren Robert Abbanat
Stefan Jochen KEMMLER
Michael Thomas KOWARIK
Manuela Mally
Veronica GAMBILLARA FONCK
Martin Edward Braun
Maria Paula Carranza Sandmeier
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Janssen Pharmaceuticals, Inc.
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Publication of OA20646A publication Critical patent/OA20646A/en

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Abstract

A bioconjugate of an E. coli glucosylated O4 antigen polysaccharide covalently linked to a carrier protein and compositions thereof are provided. Also provided are recombinant host cells for producing the bioconjugate, and methods of producing the bioconjugate using the recombinant host cells. The recombinant host cells contain a nucleic acid encoding a glucosyl transferase capable of modifying the E. coli O4 antigen with glucose branching to produce the glucosylated O4 antigen polysaccharide. Bioconjugates of an E. coli glucosylated O4 antigen polysaccharide described herein can be used alone or in combination with one or more additional E. coli O-antigen polysaccharides to induce antibodies against an E. coli glucosylated antigen, and to vaccinate a subject against extraintestinal pathogenic E. coli (ExPEC).

Description

TITLE OF THE INVENTION
Bioconjugates of E. coli O-Antigen Polysaccharides, Methods of Production Thereof, and Methods of Use Thereof
CROSS REFERENCE TO RELATED APPLICATION
[001] This application claims priority to U.S. Provisional Application No. 62/819,746 filed on March 18,2019, the disclosure of which is incorporated herein by reference in its entirety.
.i
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[002] This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file naine “004852_l 1560_Sequence-Listing”, création date of March 11, 2020, and having a size of 198 KB. The sequence listing submitted via EFS-Web is paît of the spécification and is herein incorporated by reference in its entirety.
· v BACKGROUNDOF THE INVENTION '
[003] Extraintestinal pathogenic Escherichia coli (ExPEC) strains are normally harmless ' inhabitants of the human gastrointestinal tract, alongside commensal E. coli strains. ExPEC isolâtes cannot readily be distinguished from commensal isolâtes by serotype, although many clonal lineages are dominated by ExPEC, as defined by O-antigen, capsule and flagellai’ antigen serotypes (abbreviated as O:K:H, for example O25:K1 :H4). In contrast to commensal E. coli, ExPEC strains express a broad array of virulence factors enabling them to colonize the gastrointestinal tract, as well as to cause a wide range of extraintestinal infections, which are associated with a significant healthcare cost burden due to hospitalization and death.
Neonates, the elderly, and immunocompromised patients are particularly susceptible to ExPEC infection, including. invasive ExPEC disease (IED).
[004] ExPEC strains arc the most common cause of urinary tract infections (UTI) and important contributors to surgical site infections and néonatal meningitis. The strains are also associated with abdominal and pelvic infections and nosocomial pneumonia, and are occasionally involved in other extraintestinal infections, such as osteomyelitis, cellulitis, and '
. ' i . | wound infections. Ail these primary sites of infection can resuit in ExPEC bacteremia. ExPEC is the most common cause of community-onset bacteremia and a major causative pathogen in nosocomial bacteremia and is found in about 17% to 37% of clinically signifîcant blood isolâtes. Patients with an ExPEC-positive blood culture typically suffer sepsis syndrome, severe sepsis, or septic shock. Increasing résistance of ExPEC against first-line antibiotics including the cephalosporins, fluoroquinolones, and trimethoprim/sulfamethoxazole has been observed. The emergence and rapid global dissémination of ExPEC sequence type 131 (STI 31) is considered a main driver of increased drug résistance, including multi-drug résistance. This clone is found in 12.5% to 30% of ail ExPEC clinical isolâtes, exhibits mostly serotype O25b:H4, and shows high levels of résistance to fluoroquinolones, which is often accompanied by trimethoprim/sulfamethoxazole résistance and extended-spectrum beta-lactamases conferring résistance to cephalosporins.
[005] The O-antigen comprises the immunodominant component of the cell wall lipopolysaccharide (LPS) in Gram-negative bacteria, including E. coli. There are currently >180 serologically unique E. coli O-antigens identified, with the vast majority of ExPEC isolâtes classified within less than 20 O-antigen serotypes. Full-length E. coliQantigens are typically comprised of about 10 to 25 repeating sugar units attached to the highly conserved LPS core structure, with each component synthesized separately by enzymes encoded predominantly in the rfb and rfa gene clusters, respectively. Following polymerization of the O-antigen, the O-antigen polysaccharide backbone may be modified, typically through 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 side branches. Genes encoding O-antigen modifying enzymes typically résidé outside of the rfb cluster on the chromosome, and in some cases, these genes are found within lysogénie bactériophages.
[006] ExPEC isolâtes belonging to the 04 serogroup hâve been commonly identified in contemporary surveillance studies of U.S. and EU blood isolâtes. The structure of the 04 polysaccharide was determined as —>2) α-L-Rha (1 ->6) α-D-Glc (1 ->3) a-L-FucNAc (l->3) β-D-GlcNAc (l-> from an E. coli O4:K52 strain (Jann et al., Carbohydr. Res. (1993) v. 248, pp.241-250). A distinct form of the 04 polysaccharide structure was determined for O4:K3, O4:K6 and O4:K12 strains, in which the structure above was modified by the addition of an α-D-Glc (l->3) linked to the rhamnose residue of the polysaccharide (Jann et al., 1993, supra), this form of the polysaccharide referred to herein below as ‘glucosylated 04’. The enzymes responsible for the O-antigen modification within E. coli 04 strains were not identified.
[007] Efforts toward the development of a vaccine to prevent ExPEC infections hâve focused on O-antigen polysaccharide conjugates. A 12-valent O-antigen conjugate vaccine was synthesized through extraction and purification of O-antigen polysaccharide and Chemical conjugation to detoxified Pseudomonas aeruginosa exotoxin A and tested for safety and immunogenicity in a Phase 1 clinical study (Cross et al., J. Infect. Dis. (1994) v.170, pp.834-40). This candidate vaccine was never licensed for clinical use. A bioconjugation System in E. coli has been developed recently, in which the polysaccharide antigen and the carrier protein are both synthesized in vivo and subsequently conjugated in vivo through the activities of the oligosaccharyl transferase PglB, a Campylobacter jejuni enzyme, expressed in E. coli (Wacker et al., Proc. Nat. Acad Sci. (2006) v. 103, pp. 7088-93). This N-linked protein glycosylation System is capable of the transfer of diverse polysaccharides to a carrier protein, allowing for straightforward methods to purify the conjugate.
[008] Bioconjugation has been used successfully to produce conjugate polysaccharide for an E. coli four-valent O-antigen candidate vaccine (Poolman and Wacker, J. Infect. Dis.
(2016) v.213(l), pp. 6-13). However, the development of a successful ExPEC vaccine requires coverage of prédominant serotypes, and the presence of further O-antigen modifications in subsets of ExPEC isolâtes présents a further challenge in covering isolâtes displaying unmodified and modified LPS.
BRIEF SUMMARY OF THE INVENTION
[009] In view of increasing antibiotic résistance among ExPEC isolâtes and the presence of further O-antigen modifications among prédominant O-serotypes, there is a need for improved prophylactic and therapeutic treatments for these infections. The invention satisfîes this need by defining the genetic composition of contemporary clinical isolâtes, including îdentifying the genes encoding O-antigen modifying enzymes, thus allowing for the engineering of recombinant host cells capable of synthesizing bioconjugates of the Oantigens including bioconjugates comprising selected O-antigen modifications.
(0010] In one aspect, provided herein is a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein, wherein the E. coli glucosylated 04 antigen polysaccharide comprises the structure of Formula (O4-Glc+) as shown in Table 1, wherein n is an integer of 1 to 100, preferably 1 to 50, e.g. 3 to 50, e.g. 5 to 40, e.g. 7 to 25, e.g. 10 to 20.
[0011] In a particular embodiment, the E. coli glucosylated 04 antigen polysaccharide is covalently linked to an Asn residue in a glycosylation site comprising a glycosylation consensus sequence having SEQID NO: 1, preferably having SEQ ID NO: 2, in the carrier protein.
[0012] In some embodiments, the carrier protein is selected from the group consisting of detoxified Exotoxin A of P. aeruginosa (EPA), E. coli flagellin (FliC), CRM197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of N. aureus, dumping factor A, dumping factor B, E. coli heat labile enterotoxin, detoxified variants of K coli heat labile enterotoxin, Choiera toxin B subunit (CTB), choiera toxin, detoxified variants of choiera toxin, E. coli Sat protein, the passenger domain of E. coli Sat protein, Streptococcus pneumoniae Pneumolysin, Keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.
[0013] In a particular embodiment, the carrier protein is a detoxified exotoxin A of Pseudomonas aeruginosa (EPA). In such embodiments, the EPA preferably comprises 1 to 20, preferably 1 to 10, preferably 2 to 4, glycosylation consensus sequences having SEQ ID NO: 1, the consensus sequences preferably having SEQ ID NO: 2.
[0014] In a particular embodiment, the carrier protein comprises four glycosylation consensus sequences (EPA-4). In a preferred embodiment, the carrier protein comprises SEQ IDNO:3.
[0015] In another aspect, provided herein is a composition or immunogenic composition comprising a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein as described herein.
[0016] In some embodiments, a composition or immunogenic composition comprises at least one additional antigen polysaccharide covalently linked to a carrier protein.
[0017] In some embodiments, a composition or immunogenic composition comprises at least one additional antigen polysaccharide covalently linked to a carrier protein, wherein the at least one additional antigen polysaccharide is selected from the group consisting of E. coli 01A antigen polysaccharide, E. coli 02 antigen polysaccharide, E. coli 06Α antigen polysaccharide, E. coli 08 antigen polysaccharide, E. coli 015 antigen polysaccharide, E. coli 016 antigen polysaccharide, E. coli 018Α antigen polysaccharide, E, coli O25B antigen polysaccharide, and E. coli 075 antigen polysaccharide. In spécifie embodiments, the E. coli 01A antigen polysaccharide comprises the structure of Formula (01 A) as shown in Table 1, wherein n is an integer of 1 to 100, preferably 3 to 50, for example 5 to 40, e.g. 7 to 25, e.g. 10 to 20. In spécifie embodiments, the E. coli 02 antigen polysaccharide comprises the structure of Formula (02) as shown in Table 1, wherein n is an integer of 1 to 100, preferably 3 to 50, for example 5 to 40, e.g. 7 to 25, e.g. 10 to 20. In spécifie embodiments, the E. coli 06A antigen polysaccharide comprises the structure of Formula (06A) as shown in Table 1, wherein n is an integer of 1 to 100, preferably 3 to 50, for example 5 to 40, e.g. 7 to 25, e.g. 10 to 20. In spécifie embodiments, the E. coli 08 antigen polysaccharide comprises the structure of Formula (08) as shown in Table 1, wherein n is an integer of 1 to 100, preferably 3 to 50, for example 5 to 40, e.g. 7 to 25, e.g. 10 to 20. In spécifie embodiments, the E. coli 015 antigen polysaccharide comprises the structure of Formula (015) as shown in Table 1, wherein n is an integer of 1 to 100, preferably 3 to 50, for example 5 to 40, e.g. 7 to 25, e.g. 10 to 20. In spécifie embodiments, the E. coli 016 antigen polysaccharide comprises the structure of Formula (016) as shown in Table 1, wherein n is an integer of 1 to 100, preferably 3 to 50, for example 5 to 40, e.g. 7 to 25, e.g. 10 to 20. In spécifie embodiments, the E. coli O18A antigen polysaccharide comprises the structure of Formula (O18A) as shown in Table 1, wherein n is an integer of 1 to 100, preferably 3 to 50, for example 5 to 40, e.g. 7 to 25, e.g. 10 to 20. In spécifie embodiments, the E. coli O25B antigen polysaccharide comprises the structure of Formula (O25B) as shown in Table 1, wherein n is an integer of 1 to 100, preferably 3 to 50, for example 5 to 40, e.g. 7 to 25, e.g. 10 to 20. In spécifie embodiments, the E, coli 075 antigen polysaccharide comprises the structure of Formula (075) as shown in Table 1, wherein n is an integer of 1 to 100, preferably 3 to 50, for example 5 to 40, e.g. 7 to 25, e.g. 10 to 20.
[0018] In certain embodiments, each of the additional E. coli O1 A, 02, O6A, 08, 015, \
016, O18A, O25B, and/or 075 antigen polysaccharides is covalently bound to an Asn résidé in a glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2, in each of the carrier protein. In particular embodiments, i J 5 | i ? ! i i each of the carrier protein is a detoxifïed exotoxin A of Pseudomonas aeruginosa (EPA). Preferably, each EPA comprises 1-10, preferably 2-4, preferably 4 glycosylation sites each comprising a glycosylation consensus sequence having SEQ ID NO: 2. In particular embodiments, each EPA comprises SEQ ID NO: 3.
[0019] In particular embodiments, the composition or immunogenic composition comprises at least the E.coli 01 A, 02, glucosylated 04, 06A and O25B antigen polysaccharides each covalently linked to a carrier protein. In particular embodiments, the composition or immunogenic composition comprises at least the E. coli 01 A, 02, glucosylated 04, O6A, 08, 015, 016, O25B, and 075 antigen polysaccharides each covalently linked to a carrier protein. In particular embodiments, the composition or immunogenic composition comprises at least the E. coli Ol A, 02, glucosylated 04, 06A, 08, 015, 016, 018A, O25B, and 075 antigen polysaccharides each covalently linked to a carrier protein.
[0020] In a particular aspect, provided is a composition or immunogenic composition that comprises:
(i) a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a detoxifïed Exotoxin A of P. aeruginosa (EPA-4) carrier protein comprising SEQ ID NO: 3, wherein the E. coli glucosylated 04 antigen polysaccharide comprises the structure of Formula (04-Glc+);
(ii) a bioconjugate of an E. coli 01A antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 01A antigen polysaccharide comprises the structure of Formula (01 A);
(iii) a bioconjugate of an E. coli 02 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 02 antigen polysaccharide comprises the structure of Formula (02);
(iv) a bioconjugate of an E. coli 06A antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 06A antigen polysaccharide comprises the structure of Formula (06A);
(v) a bioconjugate of an E. coli 08 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 08 antigen polysaccharide comprises the structure of Formula (08);
(vi) a bioconjugate of an E. coli 015 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 015 antigen polysaccharide comprises the structure of Formula (015);
(vii) a bioconjugate of an E. coli 016 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 016 antigen polysaccharide comprises the structure of Formula (016);
(viii) a bioconjugate of an E. coli O25B antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O25B antigen polysaccharide comprises the structure of Formula (O25B); and (ix) a bioconjugate of an E. coli 075 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 075 antigen polysaccharide comprises the structure of Formula (075), wherein the structure of each of Formulas (O4-Glc+), (OlA), (02), (06A), (08), (015), (016), (018A), (O25B), and (075) is shown in Table 1, and each n is independently an integer of 1 to 100, preferably 1 to 50, e.g. 3 to 50, e.g. 5 to 40, e.g. 7 to 25, e.g. 10 to 20. [0021] In certain embodiments, such composition or immunogenic composition further comprises:
(x) a bioconjugate of an E. coli 018A antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 018A antigen polysaccharide comprises the structure of Formula (018 A), wherein the structure of Formula (018 A) is shown in Table 1, and n in this structure is an integer of 1 to 100, preferably 1 to 50, e.g. 3 to 50, e.g. 5 to 40, e.g. 7 to 25, e.g. 10 to 20.
[0022] In certain embodiments, a bioconjugate of an E. coli 025B antigen polysaccharide is présent in a composition described herein at a concentration that is about 1.5-6 times, e.g. about 2 to 4 times, higher than a concentration of any other bioconjugate in the composition. [0023] In certain embodiments, a composition described herein comprises bioconjugates of E. coli OlA, 02, glucosylated 04, O6A, 08, 015, 016, O25B, and 075 antigen polysaccharides, wherein the bioconjugates of OlA:O2:glucosylated O4:O6A:O8:O15:O16:O25B:O75 are présent in a ratio by weight of polysaccharide of 1:1:1:1:1:1:1:2:1,or 2:1:1:2:1:1:1:4:1.
[0024] In certain embodiments, a composition described herein comprises bioconjugates of E. coli OlA, 02, glucosylated 04, O6A, 08, 015, 016, O18A, O25B, and 075 antigen polysaccharides, wherein the bioconjugates of 01 A:O2:glucosylated O4:O6A:O8:O15:O16:O18A:O25B:O75 are présent in a ratio by weight of polysaccharide of 1:1:1:1:1:1:1:1:2:1, or 2:1:1:2:1:1:1:1:4:1.
[0025] In certain embodiments, a concentration of a bioconjugate of an E. coli O25B antigen polysaccharide in a composition described herein is 2 to 50 pg/mL, preferably 8 to 40 pg/mL, e.g. 16-32 pg/mL.
[0026] In another aspect, provided herein is a method of inducing antibodies against an E. coli glucosylated 04 antigen in a subject, comprising administering to the subject a bioconjugate of an E. coli glucosylated 04 antigen as described herein, or a composition or immunogenic composition as described herein.
[0027] In a particular embodiment, the antibodies hâve opsonophagocytic activity.
[0028] In another aspect, provided herein is a method of vaccinating a subject against extraintestinal pathogenic E. coli (ExPEC), comprising administering to the subject a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide as described herein, or a composition or immunogenic composition as described herein. In certain aspects, provided herein is a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide as described herein, or a composition or immunogenic composition as described herein, for use in inducing antibodies against an E. coli glucosylated 04 antigen. In certain aspects, provided herein is a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide as described herein, or a composition or immunogenic composition as described herein, for use in vaccination against extra-intestinal pathogenic E. coli (ExPEC). In certain aspects, provided herein is the use of a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide as described herein, or a composition or immunogenic composition as described herein, for the manufacture of a médicament for inducing antibodies against an E. coli glucosylated 04 antigen in a subject. In certain aspects, provided herein is the use of a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide as described herein, or a composition or immunogenic composition as described herein, for the manufacture of a médicament for vaccinating a subject against extraintestinal pathogenic E. coli (ExPEC).
[0029] In another aspect, provided herein is a recombinant host cell for producing a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein, wherein the glucosylated 04 antigen polysaccharide comprises the structure of Formula (O4-Glc+) as shown in Table 1, wherein n is integer of 1 to 100, preferably 1 to 50, e.g. 3 to 50, e.g. 5 to 40, e.g. 7 to 25, e.g. 10 to 20, the host cell comprising:
(i) a nucléotide sequence of an rfb gene cluster for the E. coli 04 antigen polysaccharide;
(ii) a nucléotide sequence encoding a glucosyl transferase having at least 80% sequence identity to SEQ ID NO: 4, wherein the glucosyl transferase is capable of modifying the E. coli 04 antigen polysaccharide to produce the E. coli glucosylated 04 antigen polysaccharide;
(iii) nucléotide sequences encoding a translocase and a glycosyltransferase having at least 80% sequence identity to SEQ ID NOs: 7 and 8 respectively, wherein the translocase is capable of translocating bactoprenol-linked glucose and the glycosyltransferase is capable of glucosylating bactoprenol;
(iv) a nucléotide sequence encoding the carrier protein; and (iv) a nucléotide sequence encoding an oligosaccharyl transferase capable of covalently linking the E. coli glucosylated 04 antigen polysaccharide to the carrier protein to produce the bioconjugate.
[0030] In a particular embodiment, a recombinant host cell comprises a nucléotide sequence encoding a glucosyl transferase that is capable of modifying the E. coli 04 antigen polysaccharide to produce the E. coli glucosylated 04 antigen polysaccharide and having an amino acid sequence that has at least 90%, preferably at least 95%, preferably at least 98% sequence identity to SEQ ID NO: 4. In certain embodiments, the glucosyl transferase comprises SEQ ID NO: 4.
[0031] In certain embodiments, the recombinant host cell comprises a nucléotide sequence encoding a translocase that is capable of translocating bactoprenol-linked glucose and having at least 90%, preferably at least 95%, preferably at least 98% sequence identity to SEQ ID NO: 7. In certain embodiments, the translocase comprises SEQ ID NO: 7.
[0032] In certain embodiments, the recombinant host cell comprises a nucléotide sequence encoding a glycosyltransferase that is capable of glucosylating bactoprenol and having at least 90%, preferably at least 95%, preferably at least 98% sequence identity to SEQ ID NO: 8. In certain embodiments, the glycosyltransferase comprises SEQ ID NO: 8. In certain embodiments, the recombinant host cell comprises a nucléotide sequence that encodes an oligosaccharyl transferase comprising the amino acid sequence of SEQ ID NO: 6. In preferred embodiments, the oligosaccharyl transferase comprises the amino acid sequence of SEQ ID NO: 6 having mutation N311V, more preferably SEQ ID NO: 6 having both mutations Y77H and N311V.
[0033] In certain embodiments, the recombinant host cell comprises a nucléotide sequence encoding a carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2. [0034] In certain embodiments, the rfh gene cluster for the E. coli 04 antigen I polysaccharide comprises a sequence that encodes the enzymes that create the E. coli 04 antigen polysaccharide (Formula (O4-Glc-) in Table 1) and 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 certain embodiments the rfb gene cluster comprises SEQ ID NO: 9.
[0035] In certain embodiments, the recombinant host cell comprises a nucléotide sequence encoding a carrier protein, wherein the carrier protein is selected from the group consisting of detoxified Exotoxin A of P. aeruginosa (EPA), E. coli flagellin (FliC), CRM197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of Si aureus, dumping factor A, dumping factor B, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Choiera toxin B subunit (CTB), choiera toxin, detoxified variants of choiera toxin, E. coli Sat protein, the passenger domain of E. coli Sat protein, Streptococcuspneumoniae Pneumolysin, Keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.
[0036] In a particular embodiment, a recombinant host cell encodes a detoxified exotoxin i
A of Pseudomonas aeruginosa (EPA) carrier protein, preferably EPA comprising 1-10, preferably 2-4, preferably 4 glycosylation sites each comprising a glycosylation consensus sequence having SEQ ID NO: 2. In a preferred embodiment, EPA comprises the amino acid sequence of SEQ ID NO: 3.
[0037] Preferably, a recombinant host cell is E. coli, e.g. an E. coli K-12 strain, such as strain W3110.
[0038] In another aspect, provided is a method of producing a bioconjugate of an E. coli \ glucosylated 04 antigen polysaccharide covalently linked to a carrier protein, wherein the E.
coli glucosylated 04 antigen polysaccharide comprises the structure of Formula (O4-Glc+) as i
shown in Table 1, wherein n is an integer of 1 to 100, preferably 1 to 50, e.g. 3 to 50, e.g. 5 to 40, e.g. 7 to 25, e.g. 10 to 20, the method comprising culturing the recombinant host cell of the invention under conditions for production of the bioconjugate. In some embodiments, the method further comprises isolating the bioconjugate from the recombinant host cell.
[0039] In another aspect, provided is a bioconjugate produced by a method as described herein. In another aspect, provided is a composition comprising a bioconjugate produced by a method as described herein.
[0040] In another aspect, provided is a method for making a recombinant host cell for producing a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein, wherein the glucosylated 04 antigen polysaccharide comprises the structure of Formula (O4-Glc+) as shown in Table 1, wherein n is an integer of 1 to 100, preferably 1 to 50, e.g. 3 to 50, e.g. 5 to 40, e.g. 7 to 25, e.g. 10 to 20, the method comprising introducing one or more recombinant nucleic acid molécules into a cell to produce the recombinant host cell, wherein the recombinant host cell comprises:
(i) a nucléotide sequence of an rfb gene cluster for the E. coli 04 antigen polysaccharide;
(ii) a nucléotide sequence encoding a glucosyl transferase having at least 80% sequence identity to SEQ ID NO: 4, wherein the glucosyl transferase is capable of modifying the E. coli 04 antigen polysaccharide to produce the E. coli glucosylated 04 antigen polysaccharide;
(iii) nucléotide sequences encoding a translocase and a glycosyltransferase having at least 80% sequence identity to SEQ ID NOs: 7 and 8 respectively, wherein the translocase is capable of translocating bactoprenol-linked glucose and the glycosyltransferase is capable of glucosylating bactoprenol;
(iv) a nucléotide sequence encoding the carrier protein; and (v) a nucléotide sequence encoding an oligosaccharyl transferase capable of covalently linking the E. coli glucosylated 04 antigen polysaccharide to the carrier protein to produce the bioconjugate.
[0041] In a particular embodiment thereof the glucosyl transferase that is capable of modifying the E. coli 04 antigen polysaccharide to produce the E, coli glucosylated 04 antigen polysaccharide has an amino acid sequence that has at least 90%, preferably at least 95%, preferably at least 98% sequence identity to SEQID NO: 4. In certain embodiments, the glucosyl transferase comprises SEQ ID NO: 4.
[0042] In certain embodiments, the oligosaccharyl transferase comprises the amino acid sequence of SEQ ID NO: 6, preferably of SEQ ID NO: 6 comprising the amino acid mutation N31IV. In certain embodiments, the oligosacccharyl transferase comprises SEQ ID NO: 6 having the amino acid mutations Y77H and N311V.
[0043] In certain embodiments, the rfi gene cluster for the E. coli 04 antigen polysaccharide comprises a sequence that encodes the enzymes that create the E. coli 04 antigen polysaccharide (Formula (O4-Glc-) in Table 1) and 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 certain embodiments the rfb gene cluster comprises SEQ ID NO: 9.
[0044] In certain embodiments, the translocase is capable of translocating bactoprenollinked glucose and has at least 90%, preferably at least 95%, preferably at least 98% sequence identity to SEQ ID NO: 7. In certain embodiments, the translocase comprises SEQ ID NO: 7.
[0045] In certain embodiments, the glycosyltransferase is capable of glucosylating bactoprenol and has at least 90%, preferably at least 95%, preferably at least 98% sequence identity to SEQ ID NO: 8. In certain embodiments, the glycosyltransferase comprises SEQ IDNO: 8.
[0046] In certain embodiments, 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. In certain embodiments, the carrier protein is selected from the group consisting of detoxified Exotoxin A of P. aeruginosa (EPA), E. coli flagellin (FliC), CRM 197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of S. aureus, dumping factor A, dumping factor B, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Choiera toxin B subunit (CTB), choiera toxin, detoxified variants of choiera toxin, E. coli Sat protein, the passenger domain of E. coli Sat protein, Streptococcus pneumoniae Pneumolysin, Keyhole limpet hemocyanin (KLH), P, aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.
[0047] In a particular embodiment, the carrier protein is a detoxified exotoxin A of Pseudomonas aeruginosa (EPA), preferably wherein the EPA comprises 1-10, preferably 24, preferably 4, glycosylation sites each comprisîng a glycosylation consensus sequence having SEQ ID NO: 2, In particular embodiments, the carrier protein is EPA with four glycosylation consensus sequences (EPA-4), preferably wherein the carrier protein comprises SEQIDNO: 3.
[0048] In some embodiments, the recombinant host cell is an E. coli cell, e.g. from an E. coli K12 strain, such as from a W3110 strain.
[0049] In another aspect, provided is a method of preparing a bioconjugate of an Ox antigen polysaccharide covalently linked to a carrier protein, the method comprising:
(i) providing a recombinant host cell comprising:
a. a nucléotide sequence of an rfb gene cluster for the Ox-antigen polysaccharide;
b. a nucléotide sequence encoding the carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2; and
c. a nucléotide sequence encoding an oligosaccharyl transferase PglBy; and (ii) culturing the recombinant host cell under conditions for production of the bioconjugate, wherein:
when the Ox- antigen is 01A antigen polysaccharide, the PglBy comprises the amino acid mutations of N31IV, K482R, D483H, and A669V;
when the Ox-antigen is glucosylated 04 antigen polysaccharide, the PgiBy comprises the amino acid mutation N31IV or the amino acid mutations Y77H and N311V, and the recombinant host cell further comprises a sequence encoding a GtrS having at least 80% identity to SEQID NO: 4, and nucléotide sequences encoding a GtrA and a GtrB having at least 80% sequence identity to SEQ ID NOs: 7 and 8 respectively;
when the Οχ-antigen is O6A antigen polysaccharide, the PglBy comprises the amino acid mutations of N31IV, K482R, D483H, and A669V;
when the Ox-antigen is 08 antigen polysaccharide, the PglBy comprises no amino acid mutations at positions ΊΊ, 80, 287,289,311,482,483 and 669;
when the Ox-antigen is 015 antigen polysaccharide, the PglBy comprises the amino acid mutations of N31IV, K482R, D483H, and A669V;
when the Ox-antigen is 016 antigen polysaccharide, the PglBy comprises the amino acid mutations of Y77H, S80R, Q287P, K289R, and N31IV;
when the Οχ-antigen is 018A antigen polysaccharide, the PglBy comprises no amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669; and when the Οχ-antigen is 075 antigen polysaccharide, the PglBy comprises the amino acid mutation of N31IV, wherein in each case the amino acid mutations are relative to the wild-type PglBy having the amino acid sequence of SEQ ID NO: 6.
[0050] In certain embodiments, the carrier protein is detoxified exotoxin A of I
Pseudomonas aeruginosa (EPA). Preferably, the EPA carrier protein comprises 1-10, preferably 2-4, preferably 4, glycosylation sites each comprising a glycosylation consensus sequences having SEQ ID NO: 2. In a particular embodiment, the EPA carrier protein comprises SEQ ID NO: 3.
[0051] In another aspect, provided is a bioconjugate produced by a method of preparing a I bioconjugate of an Ox antigen polysaccharide covalently linked to a carrier protein as described herein. In another aspect, provided is a composition comprising such a bioconjugate. In such embodiments, a composition comprises at least 2, preferably at least 3, more preferably at least 5, still more preferably at least 7 of such bioconjugates.
BRIEF DESCRIPTION OF THE FIGURES j
' . I |0052] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the précisé embodiments shown in the drawings.
[0053] In the drawings:
[0054] FIGS. 1A-B show ELISA IgG titers against unmodified (GLC-) or glucosemodified (GLC+) 04 LPS in sera from two rabbits immunized with Glc-modified 04 polysaccharide bioconjugate as described in Example 4; ELISA titers were determined in quadruplicate;
[0055] FIG. 2 shows IgG titers in whole cell ELI S As with pooled sera from rabbits immunized with a Glc-modified 04 bioconjugate against E. coli 04 isolâtes with characterized gtrS status as described in Example 4; the following isolâtes were gtrSnegative: A2625, stGVXN4988, OC24784, OC24787 and OC24788; the following isolâtes were g/rS-positive: Y1382, E551, OC24334, stGVXN4983, stGVXN4994 and OC24794; the négative control strain OC9487 (ATCC 35383; serotype 075) was also included;
[0056] FIGS. 3A-B show Western blots of LPS extracted from gfrS-positive and -négative 04 isolâtes probed with pooled sera from rabbits immunized with modified 04 polysaccharide;
[0057] FIGS. 4A and 4B show antibody responses induced by glucosylated 04 (O4-Glc+)EPA bioconjugates; FIG. 4A shows sérum antibody levels measured by ELISA at day 0,14 and 42 post-immunization; individual titers (loglO EC50 titer) and GMT ± 95% CI are shown; the grey dotted line indicates the threshold above which the dilution curves of the samples hâve a 4PL fitting; FIG. 4B shows the results of the opsonophagocytic (OPK) assay to détermine the functionality of the antibodies in sérum samples obtained at day 42 postimmunization with glucosylated 04 (O4-Glc+)-EPA bioconjugate (4.0 pg); Wilcoxon rank sum test and Bonferroni correction; *P<0.05, ***P<0.0001;
[0058] FIG. 5 shows the boost effect of glucosylated 04 (04 Glc+)-EPA bioconjugate in Sprague Dawley rats immunized at 3 different doses as described in Example 4; sérum antibody levels were measured by ELISA at day 0,14 and 42 post-immunization; individual titers (loglO EC50 titer) are shown for each animal; the fines between the data points connect IgG titers for each animal in time; the grey dotted line indicates the threshold above which the dilution curves of the samples hâve a 4PL fitting; statistical analysis was performed with
Wilcoxon signed-rank test and Bonferroni correction for multiple comparisons (day 14 vs day 0, P = 0.012 for 4.0 pg/dose; day 42 vs day 0, P = 0.006 for ail doses; day 42 vs day 14, P = 0.006 for ail doses);
[0059] FIG. 6 shows the functionality of antibodies induced by O4-Glc+-EPA bioconjugate; Sprague Dawiey rats were immunized intramuscularly 3 times with formulation buffer or O4(Glc+)-EPA bioconjugate at 4.00 pg/dosc; functionality of the antibodies was determined by opsonophagocytic killing assay (OPKA) using O4(Glc+) and O4(Glc-) E. coli strains; individual opsonic titers (01) and GMT ± 95% CI are shown;
[0060] FIGS. 7A-B show capillary electrophoresis readout of PglB screen visualizing 04Glc+ bioconjugate production for each tested strain in a blot-like image, using monoclonal antibodies to detect O4-Glc+ bioconjugate in the periplasmic fraction. Mono-glycosylated product approximately 180 kDa, di-glycosylated product approximately 320 kDa and triglycosylated product approximately 450 kDa. A) First screening round. Wt PglB in lane 3, N311 V-PglB in lanes 2 and 4, empty control strain in lane 1 and other PglB variants in lanes 5 and 6. B) Second screening round. N311V PglB in lane 3, N311V+Y77H PglB in lane 9, empty control strain in lanes 1 and 2, other PglB variants in remaining lanes.
[0061] FIGS. 8A-K show antibody responses induced by ExPEClOV vaccine in New Zealand White rabbits. Animais received 3 intramuscular immunizations with ExPEClOV or saline administered 2 weeks apart. ExPEClOV vaccine was administered at 3 different concentrations (group 1 : hîgh dose, group 2: medium dose and group 3: low dose, Table 11) and a control group received only saline (group 4, 0.9% (w/v) sodium chloride solution).
Antibody levels were measured by ELISA at day 0 (pré-vaccination) and days 14, 27 and 42 (post-vaccination). Individual titers (EC50 titer) and géométrie mean titers (GMT) ± 95% CI are shown. Wilcoxon Rank Sum test with Bonferroni correction for multiple comparisons. Comparisons ExPEClOV vaccinated animais (group 1, 2 and 3) versus saline control (group 4). *p < 0.05, **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001. LOD: limit of détection.
[0062] FIGS. 9A-K show antibody responses induced by ExPEClOV. New Zealand White rabbits received 3 intramuscular immunizations with ExPEClOV (105.6 pg total polysaccharide) or 0.9% w/v sodium chloride solution (control). IgG titers were determined by ELISA at day 1 (pre-immunization, n = 20/group), day 31 (post-immunization, n = 20/group) and day 50 (post-immunization, n = 10/group). Plots show individual titers and |
! i géométrie mean ± 95% confidence interval for each group. Différences in IgG titers between the ExPEClOV and control group were analyzed using a Tobit model with a likelihood ratio test. P-values < 0.05 were considered significant. *P < 0.05, ****P < 0.0001.
[0063] FIGS. 10A-B show the overall study design for a phase l/2a clinical trial with ExPEClOV vaccine in humans. FIG. 10A shows the overall study design for Cohort 1, and FIG. 10B shows the overall study design for Cohort 2. See Example 11 for details.
DETAILED DESCRIPTION OF THE INVENTION
[0064] Various publications, articles and patents are cited or described in the background and throughout the spécification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the présent spécification is for the purpose of providing context for the invention. Such discussion is not an admission that any or ail of these matters form part of the prier art with respect to any inventions disclosed or claimed.
[0065] Unless defined otherwise, ail technical and scientifîc ternis used herein hâve the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain ternis used herein hâve the meanings as set forth in the spécification.
[0066] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictâtes otherwise.
[0067] Unless otherwise indicated, the terni “at least” preceding a sériés of éléments is to be understood to refer to every élément in the sériés.
[0068] Those skilled in the art will recognize or be able to ascertain using no more than routine expérimentation, many équivalents to the spécifie embodiments of the invention described herein. Such équivalents are intended to be encompassed by the invention.
[0069] Throughout this spécification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the terni “containing” or “including” or sometimes when used herein with the term “having”.
[0070] When used herein “consisting of ’ excludes any element, step, or ingrédient not specified in the claim element. When used herein, “consisting essentially of ’ does not exclude materials or step s that do not materially affect the basic and novel characteristics of the claim. Any of the aforementioned ternis of “comprising,” “containing,” “including,” and “having,” whenever used herein in the context of an aspect or embodiment of the invention can be replaced with the term “consisting of ’ or “consisting essentially of ’ to vary scopes of the disclosure.
[0071] As used herein, the conjunctive term “and/or” between multiple recited éléments is understood as encompassing both individual and combined options. For instance, where two éléments are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second éléments together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”
[0072] The identification of an O-antigen structural modification, namely glucose branching, within the E. coli 04 serotype (Jann et al., 1993) présents a challenge to the discovery and development of a glycoconjugate vaccine targeting bacterial isolâtes within this serotype. The proportion of clinical contemporary 04 isolâtes expressing the unmodified (not having a glucose side-branch) and modified (having a glucose side-branch) forms of the 04 O-antigen is unknown. Obtaining information on this characteristic is critical for selecting the relevant antigenic structure. In addition, the extent to which vaccine induced antibodies elicited to one form of the 04 polysaccharide will cross-react with the other form has not been determined. Purification of O-antigen free from lipid A and subséquent Chemical conjugation to a carrier protein is a lengthy and laborious process. Additionally, the purification, lipid A détoxification and Chemical conjugation processes can resuit in loss of epitopes, antigen heterogeneity and reduced immunogenicity of the conjugated polysaccharide. Synthesis of glycoconjugates by bioconjugation can overcome these limitations of classical purification and Chemical conjugation, but the in vivo synthesis of glucose-branched 04 O-antigen requîtes the activity of a polysaccharide branching enzyme, which lies outside of the rft> gene cluster. To date, the O-antigen modifying enzyme responsible for glucose-branching in 04 E. coli strains has not been identified. Cloning the 04 rfb gene cluster into the bioconjugation E. coli strain expressing PglB will not be sufficient to synthesize the glucose-branched 04 glycoconjugate, but rather would only produce non-glucose-branched 04 bioconjugates (the structure of the glycan thereof is shown in Formula (04) in Table 1). As used herein, the ternis “glucosylated 04”, “glucose-branched 04”, “04 Glc+” and “Glc+ 04” O-antigen refer to 04 O-antigen with a glucose side-branch, and the structure thereof is shown in formula (O4-Glc+) in Table 1.
[0073] Disclosed herein is the gene encoding the O-antigen modifying enzyme responsible for glucose branching of the E. coli 04 antigen polysaccharide. Also disclosed herein are host cells, e.g., recombinantly engineered host cells comprising nucleic acid encoding enzymes capable of producing bioconjugates comprising the glucosylated 04 antigen polysaccharide covalently bound to a carrier protein in vivo. Such host cells can be used to generate bioconjugates comprising the glucosylated 04 antigen linked to a carrier protein, which can be used in, e.g., the formulation of therapeutic and/or prophylactic compositions (e.g., vaccines). Further provided herein are compositions comprising bioconjugates of the glucosylated 04 antigen polysaccharide, alone or in combination with other E. coli antigens (e.g., 01, 02,06, 08, 015, 016, 018, 025, and/or 075 antigen polysaccharides and subserotypes thereof). The compositions can be used in prophylactic and/or therapeutic methods, e.g., vaccination of hosts against infection with E. coli, and are useful in the génération of antibodies, which can be used, e.g., in therapeutic methods such as for immunization of subjects.
[0074] As used here, the terms “O-antigen,” “O-antigen polysaccharide,” “O-antigen saccharide,” and “OPS” refer to the O-antigen of Gram-negative bacteria. Typically, an Oantigen is a polymer of immunogenic repeating polysaccharide units. In a particular embodiment, the terms “O-antigen,” “O-antigen polysaccharide,” and “OPS” refer to the Oantigen of Escherichia coli. Different serotypes of E. coli express different O-antigens. In E. coli, the gene products involved in O-antigen biogenesis are encoded by the rfb gene cluster. {0075] As used herein, “rfb cluster” and “rfb gene cluster” refer to a gene cluster that encodes enzymatic machinery capable of synthesizing an O-antigen backbone structure. The term rfb cluster can apply to any O-antigen biosynthetic cluster, and preferably refers to a gene cluster from the genus Escherichia, particularly E. coli.
[0076] As used herein, the term “01 A” refers to the 01A antigen of E. coli (a subserotype of E. coli serotype Ol). The term “02” refers to the 02 antigen of E. coli (E. coli serotype 02). The term “06A” refers to the 06A antigen of E. coli (a subserotype of E. coli serotype 06). The term “08” refers to the 08 antigen of E. coli (E. coli serotype 08). The term “015” refers to the 015 antigen of E. coli (E. coli serotype 015). The term “016” refers to the 016 antigen of E. coli (E. coli serotype 016). The term “018A” refers to the 018A antigen of E. coli (a subserotype of E. coli serotype 018). The term “O25B” refers to the O25B antigen from E. coli (a subserotype of E. coli serotype 025). The term “075” refers to the 075 antigen of E. coli (E. coli serotype 075).
[0077] The structures of E. coli O-antigen polysaccharides referred to throughout this application are shown below in Table 1. A single repeating unit for each E. coli O-antigen polysaccharide is shown.
Table 1: Structures of E. coli O-antigen Polysaccharides
E. coli Oantigen Polysaccharide Structure of Repeating Unit1
Nonglucosylated 04 antigen polysaccharide (04-Glc-) [-»2)-a-L-Rhap-(1 -»6)-a-D-Glcp-(1 -*3)-a-L-FucpNAc-(1 -»3)-p-D-GlcpNAc-(1
Glucosylated 04 antigen polysaccharide (O4-G1C+) a-D-Gfcp 1 ! 3 [^2)-a-L-Rhap-(1 ^6)-a-D-Glcp-(1 -»3)-a-L-FucpNAc-(1 -»3)-p-D-GlcpNAc-(1 -»]„
01A antigen polysaccharide (01A) [-»3)-a-L-Rhap-(1 -*3)-a-L-Rhap-(1 -^3)-p-L-Rhap-(1 -»4)^-D-GlcpNAc-(1 2 î 1 β-D-ManpNAc
02 antigen polysaccharide (02) [-»3)-a-L-Rhap-(1 -*2)-a-L-Rhap-(1 ->3)-p-L-Rhap-(1 -»4)-p-D-GlcpNAc-(1 ->]n 2 î 1 a-D-Fucp3NAc
06A antigen polysaccharide (06) [-»4)-a-D-GalpNAc-(1 -^3)-p-D-Manp-(1 -»4)-p-D-Manp-(1-»3)-a-D-GlcpNAc-(1 -*]n 2 î 1 p-D-GIcp
08 antigen polysaccharide (08) a-D-Manp3Me-(1 ->[3)-p-D-Manp-(1 -*2)-a-D-Manp-(1-*2)-a-D-Manp-(1 -*]n
015 antigen polysaccharide (015) [-»2)-p-D-Galp-(1 ->3)-a-L-FucpNAc-(1 ->3)-p-D-GlcpNAc-(1 -*]n
016 antigen polysaccharide (016) [-»2)-p-D-Galf-(1 -*6)-a-D-Glcp*(1 ^3)-a-L-Rhap-(1 ~*3)-a-D-GlcpNAc-(1 2 î Ac
O18A antigen polysaccharide (018 A) [^2)-a-L-Rhap-(1 -»6)-a-D-Glcp-(1 ->4)-a-D-Galp-(1 ->3)-a-D-GlcpNAc-(1 Tn 3 î 1 β-D-GlcpNAc
O25B antigen polysaccharide (O25B) β-D-Glcp 1 l 6 [-*4)-a-D-Gicp-(1 -»3)-a-L-Rhap-{1 -»3)^-D-GlcpNAc-(1 ->]n 3 2 t t 1 Ac a-L-Rhap
075 antigen polysaccharide (075) β-D-Manp 1 Φ 4 [->3)-a-D-Galp-(1 ->4)-a-L-Rhap-(1 ->3)-p-D-GlcpNAc-(1->]n
1 Each n is independently an integer of 1 to 100, such as 1-50,1-40, 1-30,1-20, and 1-10, 3-50, 3-40,
e.g. at least 5, such as 5-40, e.g. 7-30, e.g. 7 to 25, e.g. 10 to 20, but in some instances can be 1-2.
[0078] Ail monosaccharides described herein hâve their common meaning known in the art. Monosaccharides can hâve the D or L configuration. If D or L is not specified, the sugar is understood to hâve the D configuration. Monosaccharides are typically referred to by abbreviations commonly 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, Nacetylgalactosamine; Fuc, fiicose; Man, mannose; Man3Me, 3-O-methyl-mannose; Gai, galactose; FucNAc, N-acetylfucosamine; and Rib, ribose. The suffix “f’ refers to furanose and the suffix “p” refers to pyranose.
[0079] The terrns “RU,” “repeat unit,” and ”repeating unit” as used with respect to an Oantigen refer to the biological repeat unit (BRU) of an O-antigen as it is synthesized in vivo by cellular machinery (e.g., glycosyltransferases). The number of RUs of an O-antigen may vary per serotype, and in embodiments of the invention typically varies from about 1-100 RUs, preferably about 1 to 50 RUs, such as 1-50 RUs, 1-40 RUs, 1-30 RUs, 1-20 RUs, and 1-10 RUs, and more preferably at least 3 RUs, at least 4 RUs, at least 5 RUs, such as 3-50 RUs, preferably 5-40 RUs, e.g. 7-25 RUs, e.g. 10-20 RUs. However, in some instances, the number of RUs of an O-antigen can be 1-2. The structure of each O-antigen that is specifically described herein is shown containing one RU with the variable “n” designating the number of RUs. In each Oantigen polysaccharide in a bîoconjugate of the invention, n is independently an integer of 1-100, such as 1-50, 1-40, 1-30, 1-20, 1-10, preferably at least 3, more preferably at least 5, such as 350, preferably 5-40 (e.g. 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 instances can be 1-2. In some embodiments n is indepently an integer of about 7-25, e.g. about 10-20. The values may vary between individual O-antigen polysaccharides in a composition, and are provided here as average values, i.e. if a bîoconjugate is described herein as having an n that is independently an integer of 5-40, the composition contains a majority of O-antigen polysaccharides with 5-40 repeat units, but may also contain some O-antigen polysaccharides that hâve less than 5 repeat units or more than 40 repeat units.
[00801 The term “glycoconjugate” refers to a sugar or saccharide antigen (e.g., oligo- and polysaccharide)-protein conjugate linked to another Chemical species, including but not limited to proteins, peptides, lipids, etc. Glycoconjugates can be prepared chemically, e.g., by Chemical (synthetic) linkage of the protein and sugar or saccharide antigen. The term glycoconjugate also includes bioconjugates.
[0081] 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) prepared in a host cell background, preferably a bacterial host cell, e.g. an E.coli host cell, wherein host cell machinery links the antigen to the protein (e.g., N-links). Preferably, the term “bioconjugate” refers to a conjugate between a protein (e.g., carrier protein) and an O-antigen, preferably an E. coli Oantigen (e.g., O1A, 02, glucosylated 04,06A, 08, 015,016, O18A, O25B, 075, etc.) prepared in a host cell background, wherein host cell machinery links the antigen to the protein (e.g., Nlinks). Because bioconjugates are prepared in host cells by host cell machinery, the antigen and protein are covalently linked via a glycosidic linkage or bond in a bioconjugate. Bioconjugates can be prepared in recombinant host cells engineered to express the cellular machinery needed to synthesize the O-antigen and/or link the O-antigen to the target protein. Bioconjugates, as described herein, hâve advantageous properties over chemically prepared glycoconjugates where the glycans are purified from bacterial cell walls and subsequently chemically coupled to a carrier protein, e.g., bioconjugates require fewer Chemicals in manufacture and are more consistent in terms of the final product generated, and contain less or no free (i.e. unbound to carrier protein) glycan. Thus, in typical embodiments, bioconjugates are preferred over chemically produced glycoconjugates.
[0082] The term “about,” when used in conjunction with a number, refers to any number within ±1, ±5 or ±10% of the referenced number.
[0083] The term “percent (%) sequence identity” or “% identity” describes the number of matches (“hits”) of identical amino acids of two or more aligned amino acid sequences as compared to the number of amino acid residues making up the overall length of the amino acid sequences. In other terms, using an alignaient, for two or more sequences the percentage of amino acid residues that are the same (e.g. 90%, 95%, 97% or 98% identity) may be determined, when the sequences are compared and aligned for maximum correspondence as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visuaily inspected. The sequences which are compared to déterminé sequence identity may thus differ by substitution(s), addition(s) or deletion(s) of amino acids, Suitable programs for aligning protein sequences are known to the skilled person. The percentage sequence identity of protein sequences can, for example, be determined with programs such as CLUSTALW, Clustal Oméga, FASTA or BLAST, e,g using the NCBIBLAST algorithm (Altschul SF, et al (1997), Nucleic Acids Res. 25:3389-3402).
[0084] For example, for amino acid sequences, sequence identity and/or similarity can be determined by using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. U.S.A. 85:2444, computerized implémentations 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 the default settings, or by inspection. In certain embodiments, percent identity is calculated by FastDB based upon the following parameters: mismatch penalty of 1 ; gap penalty of 1 ; gap size penalty of 0.33; and joining penalty of 30, Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.
[0085] Another example of a useful algorithm is the BLAST algorithm, described in: 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 WU-BLAST-2 program which was obtained from Altschul et al, 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to the default values.
[0086] An additional useful algorithm is gapped BLAST as reported by Altschul et al, 1993, Nucl. Acids Res. 25:3389-3402.
[0087] The terni “Invasive Extraintestinal pathogenic Escherichia coli (ExPEC) disease (IED)” is defined herein as an acute illness consistent with systemic bacterial infection, which is microbiologically confirmed either by the isolation and identification of E. coli from blood or other normally stérile body sites, or by the isolation and identification of E. coli from urine in a patient with presence of signs and symptoms of invasive disease (systemic inflammatory response syndrome (SIRS), sepsis or septic shock) and no other identifiable source of infection.
[0088] Bioconî usâtes of E. coli glucosylated 04 Antigen Polysaccharides
[0089] In one aspect, provided herein is a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein. As used herein, the term “04” refers to the 04 antigen from E. coli (E. coli serotype 04). O-antigen structural modification is known to exist within the E. coli 04 serotype. In particular, some 04 serotypes express a modified Oantigen having a branched glucose unit. As used herein, “glucosylated 04 antigen,” “glucosylated 04 antigen polysaccharide, “O4-Glc+ antigen polysaccharide,” and “O4-Glc+ antigen” refer to an 04 antigen (e.g., E. coli 04 antigen) having a glucose branch, in which Dglucose is linked to L-rhamnosc in the repeating unit L-Rha—>D-Glc—»L-FucNAc—>D-GlcNAc. In a particular embodiment, an E. coli glucosylated 04 antigen polysaccharide comprises the structure of formula (04-Glc+), as shown in Table 1, wherein n is an integer of 1 to 100. In preferred embodiments, n is an integer of 3 to 50, e.g. 5 to 40, e.g. 7 to 25, e.g. 10 to 20.
[0090] E. coli 04 strains, independent of glucose branching status, carry a substantially identical rfb gene cluster encoding the genes responsible for production of the 04 antigen polysaccharide. However, in vivo synthesis of the modified 04 antigen having glucose branching requires the activity of a polysaccharide branching enzyme, which lies outside of the rfb gene cluster. To the best of the knowledge of the inventors, the identity of the polysaccharide branching enzyme responsible for glucose modification of the 04 antigen has remained unknown to date. Here, the inventors discovered the sequence of the polysaccharide branching enzyme responsible for glucose modification ofthe 04 antigen. Identification of this enzyme enables production of bioconjugates of the modified 04 antigen polysaccharide having glucose branching. The glucose modified form of the 04 antigen polysaccharide is présent in prédominant serotypes and can thus be used to provide an improved immune response, e.g for prophylactic or therapeutic use.
[0091] In particular, provided herein is the sequence of a gtrS gene encoding a glucosyltransferase enzyme spécifie for E. coli serotype 04 that glucosylates the 04 antigen. In general, the gtrA, gtrB, and gtrS genes encodes the enzymes responsible for O-antigen glucosylation. While the gtrA and gtrB genes in different serotypes are highly homologous and interchangeable, the gtrS gene encodes a serotype spécifie O-antigen glucosyl transferase. The gtrS gene of E. coli serotype 04 encodes the GtrS enzyme that modifies the 04 antigen by introducing glucose branching. Characterization of contemporary clinical E. coli isolâtes of the 04 serotype revealed the presence of gtrS in 78% of tested isolâtes, indicating that E. coli 04 antigen polysaccharide modified with the addition of a glucose residue is prédominant in current infecting isolâtes,
[0092] In one embodiment, provided herein is a nucleic acid of a gtrS gene from E. coli serotype 04 encoding a GtrS glucosyltransferase comprising the amino acid sequence of SEQ ID NO: 4. In another embodiment, a gtrS nucleic acid encodes a GtrS protein from E. coli serotype 04 that is about 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 is capable of specifically glucosylatîng the E. coli 04 antigen polysaccharide to obtain a glucosylated 04 antigen having the structure of Formula (O4-Glc+) as shown in Table 1. One of ordinary skill in the art will be able to make mutated forms of the GtrS protein of SEQ ID NO: 4 having at least 80% sequence identity to SEQ ID NO: 4, and test such sequences for glucosylation activity of the E. coli 04 antigen in view of the présent disclosure. Recombinant host cells comprising nucleic acid sequence encoding the glucosyl transferase gtrS gene of E. coli serotype 04, and use of the recombinant host cells in production of the glucose modified 04 antigen polysaccharides and bioconjugates thereof are described in greater detail below.
[0093] Sequences for gtrA and gtrB encoded proteins, which function as bactoprenol-linked glucose translocase (GtrA, flips the bactoprenol-linked glucose over the inner membrane to the periplasm) and bactoprenol glucosyl transferase (GtrB, links glucose to bactoprenol), respectively, may comprise amino acid sequences that are at least about 80% identical to SEQ ID NOs: 7 and 8, respectively. In certain embodiments, nucleic acid sequences encoding GtrA and GtrB proteins that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 7 and 8, respectively, and having bactoprenol-linked glucose translocase and bactoprenol glucosyl transferase activity, respectively, are also présent in the host cells ofthe invention, that further comprise an 04-specific rfh locus, the 04-specific GtrS encoding sequence described above, an oligosaccharyl transferase as described herein, and a sequence encoding a carrier protein having one or more glycosylation consensus sequences as described herein, to produce bioconjugates of E.coli glucosylated 04 serotype (comprising glycan structure of Formula (04-Glc+) in Table 1).
[0094] Bioconjugates of an E. coli glucosylated 04 antigen polysaccharide provided herein are covalently linked to a carrier protein, preferably by a glycosidic linkage. Any carrier protein known to those skilled in the art in view of the présent disclosure can be used. Suitable carrier proteins include, but are not limited to, detoxified Exotoxin A of P. aeruginosa (EPA), E. coli flagellin (FliC), CRM197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of 5. aureus, dumping factor A, dumping factor B, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Choiera toxin B subunit (CTB), choiera toxin, detoxified variants of choiera toxin, E. coli Sat protein, the passenger domain of E. coli Sat protein, Streptococcus pneumoniae Pneumolysin, Keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein oÎNeisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae. Bioconjugation with various different carrier proteins containing the required consensus glycosylation sequence has been described, showing 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 showing a wide variety of carrier proteins that were successfully used in bioconjugation).
[0095] In certain embodiments a carrier protein is modified, e.g., modified in such a way that the protein is less toxic and/or more susceptible to glycosylation. In a spécifie embodiment, the carrier proteins used herein are modified such that the number of glycosylation sites in the carrier proteins is maximized in a manner that allows for lower concentrations ofthe protein to be administered, e.g., in an immunogenic composition, particularly in its bioconjugate form.
[0096] Thus, in certain embodiments, the carrier proteins described herein are modified to include 1, 2, 3,4, 5, 6, 7, 8, 9,10 or more glycosylation sites than would normally be associated with the carrier protein (e.g., relative to the number of glycosylation sites associated with the carrier protein in its native/natural, i.e., “wild-type” State). Introduction of glycosylation sites into a carrier protein can be accomplished by insertion of a glycosylation consensus sequence anywhere in the primary structure of the protein by, e.g., adding new amino acids to the primary structure of the protein such that a glycosylation site is added in full or in part, or by mutating existing amino acids in the protein in order to generate a glycosylation site. One of ordinary skill in the art will recognize that the amino acid sequence of a protein can be readily modified using approaches known in the art, e.g., recombinant approaches that include modification of the nucleic acid sequence encoding the protein, In spécifie embodiments, glycosylation consensus sequences are introduced into spécifie régions ofthe carrier protein, e.g., surface structures of the protein, at the N or C termini of the protein, and/or in loops that are stabilized by disulfide bridges at the base of the protein. In some embodiments, a glycosylation consensus sequence can be extended by addition of lysine residues for more efficient glycosylation.
[0097] Exemplary examples of glycosylation consensus sequences that can be inserted into or generated in a carrier protein include Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 1); and Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any amino acid except Pro (SEQ ID NO: 2).
[0098] In some embodiments, the E. coli glucosylated 04 antigen polysaccharide is covalently linked to an asparagine (Asn) residue in the carrier protein (e.g., N-linked), wherein the Asn residue is présent in a glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, more preferably having SEQ ID NO: 2. Typically, a carrier protein comprises ΙΙΟ glycosylation sites, preferably 2 to 4 glycosylation sites, most preferably 4 glycosylation sites, such as 1-10, preferably 2-4, and more preferably 4 glycosylation sites each comprising a glycosylation consensus sequences having the amino acid sequence of SEQ ID NO: 1, and more preferably the amino acid sequence of SEQ ID NO: 2.
[0099] In particular embodiments, a carrier protein is a detoxifïed Exotoxin A of P. aeruginosa. For EPA, various detoxifïed protein variants hâve been described in literature and could be used as carrier proteins. For example, détoxification can be achieved by mutating and deleting the catalytically essential residues 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 a detoxifïed Exotoxin A of P. aeruginosa. In those embodiments, wherein the carrier rice, flour, chalk, silica gel, sodium stéarate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, éthanol and the like. Other examples of suitable pharmaceutically acceptable carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin.
[00104] In one embodiment, a composition of the invention comprises the bioconjugates of the invention in a Tris-buffered saline (TBS) pH 7.4 (e.g. containing Tris, NaCl and KO, e.g. at 25 mM, 137 mM and 2.7 mM, respectively). In other embodiments, the compositions ofthe invention comprise bioconjugates ofthe invention in about 10 mM KH2PO4/Na2HPO4 buffer at pH of about 7.0, about 5% (w/v) sorbitol, about 10 mM méthionine, and about 0.02% (w/v) polysorbate 80. In other embodiments, the compositions ofthe invention comprise bioconjugates of the invention in about 10 mM KH2PO4/Na2FTPO4 buffer at pH of about 7.0, about 8% (w/v) sucrose, about 1 mM EDTA, and about 0.02% (w/v) polysorbate 80 (see e.g. WO 2018/077853 for suitable buffers for bioconjugates of E. coli O-antigens covalently bound to EPA carrier protein).
[00105] In some embodiments, the compositions described herein are monovalent formulations, and contain one E. coli O-antigen polysaccharide, e.g., in isolated form or as part of a glycoconjugate or bioconjugate, such as the E. coli glucosylated 04 antigen polysaccharide. Also provided herein are compositions (e.g., pharmaceutical and/or immunogenic compositions) that are multivalent compositions, e.g., bivalent, trivalent, tetravalent, etc. compositions. For example, a multivalent composition comprises more than one antigen, such as an E. coli Oantigen, glycoconjugate, or bioconjugate thereof. In particular embodiments, multivalent compositions provided herein comprise a bioconjugate of an£. coli glucosylated 04 antigen polysaccharide, and at least one additional antigen.
[00106] In one embodiment, a composition (e.g., pharmaceutical and/or immunogenic composition) is a monovalent composition comprising a biconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein as described herein.
[00107] In another embodiment, a composition (e.g., pharmaceutical and/or immunogenic composition) is a multivalent composition comprising an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein as described herein, and at least one additional antigen.
[00108] In some embodiments, the additional antigen is antigen saccharide or polysaccharide, more preferably an E. coli O-antigen polysaccharide, such as E. coli O-antigens of one or more of the 01, 02, 06, 08, 015, 016, 018, 025, and 075 serotypes and subserotypes thereof. In some embodiments, each of the additional E. coli O-antigen polysaccharides is a glycoconjugate, meaning that the E. coli O-antigen polysaccharide is covalently linked to another Chemical species, e.g., protein, peptide, lipid, etc., most preferably a carrier protein, such as by Chemical or enzymatic methods. In preferred embodiments, each of the additional E. coli O-antigen polysaccharides is a bioconjugate in which the O-antigen polysaccharide is covalently linked to, e.g. a carrier protein, via a glycosidic bond enzymatically by host cell machinery. Compositions provided herein in certain embodiments can comprise 1-20 additional glycoconjugates, more preferably bioconjugates of E. coli O-antigen polysaccharides, 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. coli O-antigen polysaccharides. Other antigens can be included in the compositions provided herein, such as peptide, protein, or lipid antigens, etc.
[00109] In some embodiments, a composition (e.g., pharmaceutical and/or immunogenic composition) comprises a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide, and at least one additional antigen polysaccharide selected from the group consisting of E, coli 01A antigen polysaccharide, E. coli 02 antigen polysaccharide, E. coli O6A antigen polysaccharide, E. coli 08 antigen polysaccharide, E. coli 015 antigen polysaccharide, E. coli 016 antigen polysaccharide, E. coli 018A antigen polysaccharide, E. coli O25B antigen polysaccharide, and E. coli 075 antigen polysaccharide. Preferably, each of the additional O-antigen polysaccharides is covalently linked to a carrier protein, and is more preferably a bioconjugate.
[00110] In one embodiment, an 01A antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in a composition provided herein (e.g., in combination with a glucosylated 04 antigen polysaccharide or bioconjugate thereof). In a spécifie embodiment, the 01A antigen polysaccharide comprises the structure of formula (01 A) as shown in Table 1, wherein n is an integer of 1-100, preferably 3-50, e.g. 5-40, e.g. 7 to 25, e.g. 10 to 20. Preferably, the 01A antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein, e.g., EPA.
[00111] In one embodiment, an 02 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in a composition provided herein (e.g., in combination with a glucosylated 04 antigen polysaccharide or bioconjugate thereof). In a spécifie embodiment, the 02 antigen polysaccharide comprises the structure of formula (02) as shown in Table 1, wherein n is an integer of 1-100, preferably 3-50, e.g. 5-40, e.g. 7 to 25, e.g. 10 to 20. Preferably, the 02 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein, e.g., EPA.
[00112] In one embodiment, an O6A antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in a composition provided herein (e.g., in combination with a glucosylated 04 antigen polysaccharide or bioconjugate thereof). In a spécifie embodiment, the O6A antigen polysaccharide comprises the structure of formula (06A) as shown in Table 1, wherein n is an integer of 1-100, preferably 3-50, e.g. 5-40, e.g. 7 to 25, e.g. 10 to 20. Preferably, the O6A antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein, e.g., EPA.
[00113] In one embodiment, an 08 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in a composition provided herein (e.g., in combination with a glucosylated 04 antigen polysaccharide or bioconjugate thereof). In a spécifie embodiment, the 08 antigen polysaccharide comprises the structure of formula (08) as shown in Table 1, wherein n is an integer of 1-100, preferably 3-50, e.g. 5-40, e.g. 7 to 25, e.g. 10 to 20. Preferably, the 08 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein, e.g., EPA.
[00114] In one embodiment, an 015 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in a composition provided herein (e.g., in combination with a glucosylated 04 antigen polysaccharide or bioconjugate thereof). In a spécifie embodiment, the 015 antigen polysaccharide comprises the structure of formula (015) as shown in Table 1, wherein n is an integer of 1-100, preferably 3-50, e.g. 5-40, e.g. 7 to 25, e.g. 10 to 20. Preferably, the 015 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein, e.g., EPA.
[00115] In one embodiment, an 016 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in a composition provided herein (e.g., in combination with a glucosylated 04 antigen polysaccharide or bîoconjugate thereof). In a spécifie embodiment, the 016 antigen polysaccharide comprises the structure of formula (016) as shown in Table 1, wherein n is an integer of 1-100, preferably 3-50, e.g. 5-40, e.g. 7 to 25, e.g. 10 to 20. Preferably, the 016 antigen polysaccharide is part of a bîoconjugate and is covalently linked to a carrier protein, e.g., EPA.
[00116] In one embodiment, an 018A antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bîoconjugate) is used in a composition provided herein (e.g., in combination with a glucosylated 04 antigen polysaccharide or bîoconjugate thereof). In a spécifie embodiment, the 018A antigen polysaccharide comprises the structure of formula (O18A) as shown in Table 1, wherein n is an integer of 1-100, preferably 3-50, e.g. 5-40, e.g. 7 to 25, e.g. 10 to 20. Preferably, the 018A antigen polysaccharide is part of a bîoconjugate and is covalently linked to a carrier protein, e.g., EPA.
[00117] In one embodiment, an O25B antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bîoconjugate) is used in a composition provided herein (e.g., in combination with a glucosylated 04 antigen polysaccharide or bîoconjugate thereof). In a spécifie embodiment, the O25B antigen polysaccharide comprises the structure of formula (O25B) as shown in Table 1, wherein n is an integer of 1-100, preferably 3-50, e.g. 5-40, e.g. 7 to 25, e.g. 10 to 20. Preferably, the O25B antigen polysaccharide is part of a bîoconjugate and is covalently linked to a carrier protein, e.g., EPA.
[00118] In one embodiment, an 075 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bîoconjugate) îs used in a composition provided herein (e.g., in combination with a glucosylated 04 antigen polysaccharide or bîoconjugate thereof). In a spécifie embodiment, the 075 antigen polysaccharide comprises the structure of formula (075) as shown in Table 1, wherein n is an integer of 1-100, preferably 3-50, e.g. 5-40, e.g. 7 to 25, e.g. 10 to 20. Preferably, the 075 antigen polysaccharide is part of a bîoconjugate and is covalently linked to a carrier protein, e.g., EPA.
[00119] In another embodiment, a composition (e.g., a pharmaceutical and/or immunogenic composition) comprises at least the E. coli 01 A, 02, glucosylated 04, O6A and O25B antigen polysaccharides, preferably bioconjugates ofthe 01 A, 02, glucosylated 04, O6A and O25B antigen polysaccharides covalently linked to a carrier protein, e.g., EPA (i.e., apentavalent composition).
[00120] In a preferred embodiment, a composition (e.g., a pharmaceutical and/or immunogenic composition) comprises at least the E. coli O1A, 02, glucosylated 04, O6A, 08, 015, 016, O25B and 075 antigen polysaccharides, preferably bioconjugates of the 01A, 02, glucosylated 04, 06A, 08, 015, 016, 025B and 075 antigen polysaccharides covalently linked to a carrier protein, e.g., EPA (i.e., a 9-valent composition).
[00121] In another preferred embodiment, a composition (e.g., a pharmaceutical and/or immunogenic composition) comprises at least the E. coli 01 A, 02, glucosylated 04, 06A, 08, 015, 016, 018A, 025B and 075 antigen polysaccharides, preferably bioconjugates of the OlA, 02, glucosylated 04, 06A, 08, 015, 016, 018A, O25B and 075 antigen polysaccharides covalently linked to a carrier protein, e.g., EPA (i.e., a 10-valent composition).
[00122] Also contemplated herein are compositions which optionally further comprise additional O-antigens (e.g., in isolated form, or as part of a glycoconjugate or bioconjugate) from other E. coli serotypes.
[00123] In some embodiments, each of the additional E. coli OlA, 02, 06A, 08, 015, 016, 018A, O25B, and/or 075 antigen polysaccharides is covalently linked to a carrier protein. The O-antigen polysaccharide can be linked to a carrier protein by chemical or other synthetic methods, or the O-antigen polysaccharide can be part of a bioconjugate, and is preferably part of a bioconjugate. Any carrier protein known to those skilled in the art in view of the présent disclosure can be used. Suitable carrier proteins include, but are not limited to, detoxified Exotoxin A of P. aeruginosa (EPA), E. coli flagellin (FliC), CRM197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of S. aureus, dumping factor A, dumping factor B, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Choiera toxin B subunit (CTB), choiera toxin, detoxified variants of choiera toxin, E. coli Sat protein, the passenger domain of E. coli Sat protein, Streptococcus pneumoniae Pneumolysin, Keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (0MPC), and protein D from non-typeable Haemophilus influenzae. Preferably, the carrier protein is EPA.
[00124] In some embodiments, each ofthe additional E. coli 01 A, 02, O6A, 08, 015, 016, 018A, O25B, and/or 075 antigen polysaccharides, particularly when part of a bioconjugate, is covalently linked to an asparagine (Asn) residue in the carrier protein, wherein the Asn residue is présent in a glycosylation site comprising a glycosylation consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 1), preferably wherein the Asn residue is présent in a glycosylation site comprising a glycosylation consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any amino acid except Pro (SEQ ID NO: 2), The carrier protein can comprise 1-10 glycosylation sites, preferably 2 to 4 glycosylation sites, most preferably 4 glycosylation sites, each comprising a glycosylation consensus sequence. In a particular embodiment, the carrier protein is EPA-4 carrier protein, for instance EPA-4 carrier protein comprising the amino acid sequence of SEQ ID NO: 3.
[00125] In a particular embodiment, provided herein is a composition (e.g., pharmaceutical and/or immunogenic composition) comprising: (i) a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a detoxified Exotoxin A of P. aeruginosa carrier protein comprising SEQ ID NO: 3 (EPA-4 carrier protein), wherein the E. coli glucosylated 04 antigen polysaccharide comprises the structure of Formula (O4-Glc+); (ii) a bioconjugate of an E. coli 01A antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 01A antigen polysaccharide comprises the structure of Formula (01A); (iii) a bioconjugate of an E. coli 02 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 02 antigen polysaccharide comprises the structure of Formula (02); (iv) a bioconjugate of an E. coli 06A antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 06A antigen polysaccharide comprises the structure of Formula (06A); (v) a bioconjugate of an E. coli 08 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 08 antigen polysaccharide comprises the structure of Formula (08); (vi) a bioconjugate of an E. coli 015 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 015 antigen polysaccharide comprises the structure of Formula (015); (vii) a bioconjugate of an E. coli 016 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 016 antigen polysaccharide comprises the structure of Formula (016); (viii) a bioconjugate of an E. coli 025B antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O25B antigen polysaccharide comprises the structure of Formula (O25B); and (ix) a bioconjugate of an E. coli 075 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 075 antigen polysaccharide comprises the structure of Formula (075), wherein each of the Formulas is provided in Table 1, and for each of the Formulas independently n is an integer of 1 to 100, e.g. 1 to 50, preferably 3 to 50, e.g. 5 to 40.
[00126] In a particular embodiment, said composition (e.g. pharmaceutical and/or immunogenic composition) further comprises; (x) a bioconjugate of an E. coli O18A antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 018A antigen polysaccharide comprises the structure of Formula (O18A) as shown in Table 1, wherein n for this structure is is an integer of 1 to 100, e.g. 1 to 50, preferably 3 to 50, e.g. 5 to 40.
[00127] In some embodiments, a composition provided herein comprises a biconjugate of an E. coli glucosylated 04 antigen polysaccharide, and at least a bioconjugate of an E. coli O25B antigen polysaccharide, wherein the bioconjugate of the E. coli O25B antigen polysaccharide is présent in the composition at a concentration that is about 1.5 to 6 times, e.g. about 2 to 4 times higher, such as 1.5,2, 3, 4, 5 or 6 times higher than the concentration of any of the other bioconjugates présent in the composition.
[00128] In particular embodiments, a composition comprises bioconjugates of E. coli 01 A, 02, glucosylated 04, 06A, 08, 015, 016, 025B, and 075 antigen polysaccharides, wherein the bioconjugates of OlA:O2:glucosylated O4:O6A:O8:O15:O16:O25B:O75 are présent in a ratio (by weight of O-antigen polysaccharide) of 1:1:1:1:1:1:1:2:1, or 2:1:1:2:1:1:1:4:1.
[00129] In particular embodiments, a composition comprises bioconjugates of E. coli 01 A, 02, glucosylated 04, O6A, 08, 015, 016, O18A, O25B, and 075 antigen polysaccharides, wherein the bioconjugates of OlA:O2:glucosylated 04:06A:08:015:016:018A:025B:075 are présent in aratio (by weight of O-antigen polysaccharide) of 1:1:1:1:1:1:1:1:2:1, or 2:1:1:2:1:1:1:1:4:1.
[00130] In some embodiments, a composition provided herein comprises a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide, and at least a bioconjugate of an E. coli O25B antigen polysaccharide, wherein the bioconjugate ofthe E. coli O25B antigen polysaccharide is présent in the composition at a concentration of 2 to 50 pg/mL, preferably 8 to 40 pg/mL, more preferably 16-32 pg/mL, such as 16, 18, 20, 22, 24, 26, 28, 30, or 32 pg/mL. In such embodiments, the concentration of the bioconjugate of the E. coli O25B antigen polysaccharide is preferably about 1.5 to 6 tîmes, e.g., about 2 to 4 times higher, such as 1.5, 2, 3, 4, 5, or 6 times higher than the concentration of any of the other bioconjugates présent in the composition. [00131] In certain embodiments, the compositions described herein (e.g., pharmaceutical and/or immunogenic compositions) comprise, or are administered in combination with, an adjuvant. The adjuvant for administration in combination with a composition described herein may be administered before (e.g. within 72 hours, 48 hours, 24 hours, 12 hours, 6 hours, 2 hours, 1 hour, 10 minutes), concomitantly with, or after (e.g. within 72 hours, 48 hours, 24 hours, 12 hours, 6 hours, 2 hours, 1 hour, 10 minutes) administration of said composition. As used herein, the term “adjuvant” refers to a compound that when administered in conjunction with or as part of a composition described herein augmente, enhances and/or boosts the immune response to an E. coli O-antigen polysaccharide in a bioconjugate, but when the adjuvant compound is administered alone does not generate an immune response to the E. coli O-antigen polysaccharide in the bioconjugate. In some embodiments, the adjuvant enhances an immune response to an E. coli O-antigen polysaccharide in a bioconjugate thereof and does not produce an allergy or other adverse reaction. Adjuvants can enhance an immune response by several mechanisms including, e.g., lymphocyte recruitment, stimulation of B and/or T cells, and stimulation of macrophages.
[00132] Examples of suitable adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, aluminum sulfate and aluminum oxide, including nanoparticles comprising alum or nanoalum formulations), calcium phosphate, monophosphoryl lipid A (MPL) or 3-de-O-acylated monophosphoryl lipid A (3D-MPL) (see e.g., United Kingdom Patent GB2220211, EP0971739, EPI 194166, US6491919), AS01, AS02, AS03 and AS04 (ail GlaxoSmithKline; see e.g. EPI 126876, US7357936 for AS04, EP0671948, EP0761231, US5750110 for AS02), MF59 (Novartis), imidazopyridine compounds (see WO2007/109812), imidazoquinoxaline compounds (see WO2007/109813), delta-inulin, STINGactivating synthetic cyclic-di-nucleotides (e.g. US20150056224), combinations of lecithin and carbomer homopolymers (e.g. US6676958), and saponins, such as QuilA and QS21 (see e.g. Zhu D and W Tuo, 2016, Nat Prod Chem Res 3: el 13 (doi: 10.4172/2329-6836.1000e! 13), Matrix M,
Iscoms, Iscomatrix, etc, optionally in combination with QS7 (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plénum Press, NY, 1995); U.S. Pat. No. 5,057,540). In some embodiments, the adjuvant is Freund’s adjuvant (complété or incomplète). Other adjuvants are oil in water émulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, 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). Further examples of adjuvants are liposomes containing immune stimulants such as MPL and QS21 such as in AS01E and AS01B (e.g. US 2011/0206758). Other examples of adjuvants are CpG (Bioworld Today, Nov. 15, 1998) and imidazoquinolines (such as imîquimod and R848). See, e.g., Reed G, et al., 20\3, Nature Med, 19:1597-1608. In certain embodiments, the adjuvant contains a toll-like receptor 4 (TLR4) agonist. TLR4 agonists are well known in the art, see e.g. Ireton GC and SG Reed, 2013, Expert Rev Vaccines 12: 793-807. In certain embodiments, the adjuvant comprises a TLR4 agonist comprising lipid A, or an analog or dérivative thereof, such as MPL, 3D-MPL, RC529 (e.g. EP1385541), PET-lipid A, GLA (glycopyranosyl lipid adjuvant, a synthetic disaccharide glycolipid; e.g. US20100310602, US8722064), SLA (e.g. Carter D et al, 2016, Clin Transi 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 (the structure of which is the same as that of GLA), 3D-(6-acyl)-PHAD (3D(6A)-PHAD) (PHAD, 3D-PHAD, and 3D(6A)PHAD are synthetic lipid A variants, see e.g.
avantilipids.com/divisions/adjuvants, which also provide structures of these molécules), E6020 (CAS Number 287180-63-6), ONO4007, OM-174, and the like.
[00133] In certain embodiments, the compositions described herein do not comprise, and are not administered in combination with, an adjuvant.
[00134] In certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions (e.g., pharmaceutical and/or immunogenic) described herein can be formulated for subcutaneous, parentéral, oral, sublingual, buccal, intradermal, transdermal, colorectal, intraperitoneal, rectal administration, intravenous, intranasal, intratracheal, intramuscular, topical, transdermal, or intradermal administration. In a spécifie embodiment, a composition provided herein (e.g., pharmaceutical and/or immunogenic) is formulated for intramuscular injection.
[00135] Methods of Use
[00136] Bioconjugates and compositions provided herein can be used to induce antibodies against an E. coli glucosylated 04 antigen in a subject, and to vaccinate a subject against E. coli, in particular extra-intestinal pathogenic E. coli (ExPEC). As used herein, “subject” means any animal, preferably a mammal, to whom will be or has been administered a bioconjugate or composition provided herein. The terni “mammal” as used herein, encompasses 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 (NHPs) such as monkeys or apes, humans, etc. In certain embodiments, a subject is a human. A human subject may be of any âge. In certain embodiments, a subject is a human of about two months to about 18 years old, e.g. of 1 year to 18 years old. In certain embodiments, a subject is a human of at least 18 years old. In certain embodiments, a subject is a human of 15 to 50 years old, e.g. 18 to 45 years old, e.g. 20 to 40 years old. In certain embodiments, a subject is a human male. In certain embodiments, a subject is a human female. In certain embodiments, a subject is immunocompromised. In certain embodiments, a subject is a human of at least 50 years, at least 55 years, at least 60 years, at least 65 years old. In certain embodiments, a subject is a human that is not older than 100 years, not older than 95 years, not older than 90 years, not older than 85 years, not older than 80 years, or not older than 75 years. In certain embodiments, a subject is a human of at least 60 years old, and not older than 85 years old. In certain embodiments, a subject is a human in stable health. In certain embodiments, a subject is a human adult of at least 60 and not more than 85 years old in stable health. In certain embodiments, a subject is a human that has a history of a urinary tract infection (UTI, i.e. a bacterial infection in the urethra, bladder, ureters, and/or kidneys), i.e. having had at least one UTI épisode in his or her life. In certain embodiments, a subject is a human that has a history of UTI in the past twenty, fifteen, twelve, ten, nine, eight, seven, six, five, four, three, two or one years. In certain embodiments, a subject is a human that has a history of UTI in the past two years. In certain embodiments, a subject is a human subject that has a history of récurrent UTI, i.e. having had at least two UTIs in six months or at least three UTIs in one year. In certain embodiments, a subject is a human subject that has a history of récurrent UTI î l j in the past two years. In certain embodiments, a subject is a human of 60 years or older in stable health. In certain embodiments, a subject is a human of 60 years or older that has a history of UTI in the past two years. In certain embodiments, a subject is a human of at least 60 years and less than 75 years old that has a history of UTI in the past two years. In certain embodiments, a subject is a human subject of 75 years or older that has a history of UTI in the past two years. In certain embodiments, a subject is a patient scheduled for undergoing elective urogénital and/or abdominal procedures or surgeries, e.g. transrectal ultrasound-guided prostate needle biopsy (TRUS-PNB).
[00137] In one aspect, provided herein is a method of inducing antibodies against an E. coli glucosylated 04 antigen in a subject, comprising administering to the subject any of the bioconjugates of an E. coli glucosylated 04 antigen covalently linked to a carrier protein described herein, or a composition comprising a bioconjugate of an E. coli glucosylated 04 antigen covalently linked to a protein, alone or further in combination with other E. coli Oantigen polysaccharides or glycoconjugates or bioconjugates thereof.
[00138] In certain embodiments, the antibodies induced, elicited or identified against an E. coli glucosylated 04 antigen hâve opsonophagocytic activity. In particular embodiments, the antibodies induced, elicited or identified are cross-reactive antibodies capable of mediating opsonophagocytic killing of both E. coli glucosylated and non-glucosylated 04 strains.
[00139] In certain embodiments, the antibodies induced, elicited or identified identified against an E. coli glucosylated 04 antigen specifically recognize unmodified and glucose modified 04 antigen polysaccharide. In certain embodiments, the antibodies induced, elicited or identified against an E. coli glucosylated 04 antigen specifically recognize E. coli of the 04 serotype. In certain embodiments, the antibodies induced by a bioconjugate of an E. coli glucosylated 04 antigen bind preferentially to glucosylated 04 antigen as compared to non-glucosylated 04 antigen.
[00140] Antibodies induced by the bioconjugates and compositions described herein can | include immunoglobulin molécules and immunologically active portions of immunoglobulin molécules, i.e., molécules that contain an antigen binding site that specifically binds to an E. coli O-antigen polysaccharide, e.g., glucosylated 04 antigen polysaccharide.
ί i I i i
[00141] Antibodies induced, elicited or identified using the bioconjugates or compositions provided herein can be used to monitor the effîcacy of a therapy and/or disease progression. Any immunoassay System known in the art can be used for this purpose including, but not limited to, compétitive and noncompetitive assay Systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assays), electrochemiluminescence (ECL)-based immunoassays, “sandwich” immunoassays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays and immunoelectrophoresis assays. Several of these assays, e.g. ECLbased immunoassays, can be done in multiplex format, and typically multiplex assay formats are preferred.
[00142] Antibodies induced, elicited or identified using a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide can be used to detect E. coli 04 strains, particularly glucosylated 04 strains, for example, from a plurality of£. coli strains and/or to diagnose an infection by an E. coli 04 or glucosylated 04 strain.
[00143] In another aspect, provided herein is a method of vaccinating a subject against E. coli (e.g. extra-intestinal pathogenic E. coli, ExPEC), comprising administering to the subject any of the bioconjugates of an E. coli glucosylated 04 antigen covalently linked to a carrier protein described herein, or a composition comprising a bioconjugate of an E. coli glucosylated 04 antigen covalent linked to a carrier protein, alone or further in combination with other E. coli Oantigens or glycoconjugates or bioconjugates thereof. One skilled in the art will understand that the subject will be vaccinated against E. coli strains whose O antigens or glycoconjugates or bioconjugates thereof are présent in the composition administered. For example, administration of a composition comprising 01 A, 02, glucosylated 04, O6A, and O25B antigen polysaccharides can be used to a vaccinate a subject against E. coli serotypes 01 A, 02, 04, 06A, and O25B.
[00144] In certain embodiments, vaccination is for preventing an invasive ExPEC disease (IED), e.g., urosepsis, bacteremia, sepsis, etc. In certain embodiments, vaccination is to prevent or reduce the occurrence or severity of urinary tract infections. In certain embodiments, an IED can be hospital-acquired, e.g. in patients undergoing urogénital and/or abdominal procedures or surgeries. In certain embodiments, an IED can be healthcare-associated, e.g. in patients receiving health care for another condition, for instance via central lines, cathéters, etc, e.g. in a hospital, ambulantory surgical center, end-stage rénal disease facility, long-term care facility, etc. In certain embodiments, the IED can be community-acquired, e.g. in a patient that was not recently exposed to healthcare risks.
[00145] In another aspect, provided herein is a method of inducing an immune response against E. coli (e.g., ExPEC) in a subject, comprising administering to the subject any of the bioconjugates of an E. coli glucosylated 04 antigen covalently linked to a carrier protein described herein, or a composition comprising a bioconjugate of an E. coli glucosylated 04 antigen covalently linked to a carrier protein, alone or further in combination with other E. coli O-antigens or glycoconjugates or bioconjugates thereof. In one embodiment, the subject has an E. coli (e.g., ExPEC) infection at the time of administration. In a preferred embodiment, the subject does not hâve an E. coli (e.g., ExPEC) infection at the time of administration.
[00146] In certain embodiments, the compositions and bioconjugates described herein can be administered to a subject to induce an immune response that includes the production of antibodies, preferably antibodies having opsonophagocytic activity. Such antîbodîes can be isolated using techniques known to one of skill in the art (e.g., immunoaffinity chromatography, centrifugation, précipitation, etc.).
[00147] The ability of the bioconjugates and compositions described herein to generate an immune response in a subject can be assessed using any approach known to those of skill in the art or described herein. In some embodiments, the ability of a bioconjugate to generate an immune response in a subject can be assessed by immunizing a subject (e.g., amouse, rat, rabbit, or monkey) or set of subjects with a bioconjugate described herein and immunizing an additional subject (e.g., a mouse, rat, rabbit, or monkey) or set of subjects with a control (PBS). The subjects or set of subjects can subsequently be challenged with ExPEC and the ability of the ExPEC to cause disease (e.g., UTI, bacteremia, or other disease) in the subjects or set of subjects can be determined. Those skilled in the art will recpgnize that if the subject or set of subjects immunized with the control suffer(s) from disease subséquent to challenge with the ExPEC but the subject or set of subjects immunized with a bioconjugate(s) or composition thereof described herein suffer less from or do not suffer from disease, then the bioconjugate is able to generate an immune response in a subject. The ability of a bioconjugate(s) or composition thereof described herein to induce antiserum that cross-reacts with an 0 antigen from ExPEC can be tested by, e.g., an immunoassay, such as an ELISA (see e.g., Van den Dobbelsteen et al, 2016, Vaccine 34: 4152-4160), or an ECL-based immunoassay.
[00148] For example, the ability of the bioconjugates described herein to generate an immune response in a subject can be assessed using a sérum bactericidal assay (SBA) or opsonophagocytic killing assay (OPK assay, or OPKA), which represents an established and accepted method that has been used to obtain approval of glycoconjugate-based vaccines. Such assays are well-known in the art and, briefly, comprise the steps of generating and isolating antibodies against a target of interest (e.g., an O antigen polysaccharide, e.g., E. coli glucosylated 04 antigen polysaccharide) by administering to a subject (e.g., a mouse, rat, rabbit, or monkey) a compound that elicits such antibodies. Subsequently, the bactericidal capacity ofthe antibodies can be assessed by, e.g., culturing the bacteria in question (e.g., E. coli ofthe relevant serotype) in the presence of the antibodies and complément and - depending on the assay - neutrophilie cells and assaying the ability ofthe antibodies to médiate killing and/or neutralization ofthe bacteria, e.g., using standard microbiological approaches. For an example of OPK assay for E.coli bioconjugate vaccines, see e.g. Abbanat et al, 2017, Clin. Vaccine Immunol. 24: e0012317. An OPK assay can be performed in monoplex or multiplex format, of which multiplex format (e.g. testing multiple serotypes at the same time) is typically preferred. A multiplex OPK assay is sometimes referred to herein as “ΜΟΡΑ”.
[00149] In some embodiments, the methods described herein comprise administering an effective amount of bioconjugates of an E. coli glucosylated 04 antigen covalently linked to a carrier protein described herein, or a composition comprising a bioconjugate of an E. coli glucosylated 04 antigen covalently linked to a carrier protein, alone or further in combination with other E. coli O-antigens or glycoconjugates or bioconjugates thereof. In one embodiment, an “effective amount” is an amount that vaccinâtes a subject against E. coli (e.g., ExPEC). In another embodiment, an “effective amount” is an amount that induces an immune response against E. coli (e.g., ExPEC) in a subject, such as an immune response including the production of antibodies, preferably antibodies having opsonophagocytic activity.
[00150] In particular embodiments, wherein a composition provided herein comprises a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide and at least a bioconjugate of an E. coli O25B antigen polysaccharide, an effective amount of the E. coli O25B antigen polysaccharide is about 1.5 to 6 times, e.g. about 2 to 4 times higher, such as 1.5, 2, 3, 4, 5 or 6 times higher than the concentration of any of the other bioconjugates présent in the composition. In such embodiments, an effective amount of the E. coli O25B antigen polysaccharide is for instance about 5 to 18 pg per administration, such as 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18 pg per administration.
[00151] In certain embodiments, a bioconjugate or composition according to the invention is administered to a subject once. In certain embodiments, a bioconjugate or composition according to the invention is administered to a subject more than once, e.g. in a prime-boost regimen. In certain 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 y ears, or at least fifteen years. In humans, a desired immune response can typically be generated by a single administration of a bioconjugate or composition according to the invention. In certain embodiments, a repeat administration after for instance ten years is provided.
[00152] Host Cells
[00153] Provided herein are host cells, e.g., prokaryotic host cells, capable of producing E, coli O antigens and bioconjugates comprising such E. coli O antigens. The host cells provided herein preferably are modified to comprise (e.g., through genetic engineering) one or more of the nucleic acids encoding host cell machinery (e.g., glycosyltransferases) used to produce E. coli Oantigen polysaccharides and/or bioconjugates thereof.
[00154] Any host cells known to those of skill in the art can be used to produce the E. coli O antigen polysaccharides described herein (e.g., E. coli glucosylated 04 antigen polysaccharide) and bioconjugates comprising the E. coli O antigen polysaccharides described herein (e.g., a bioconjugate of E. coli glucosylated 04 antigen polysaccharide) including archaea, prokaryotic host cells, and eukaryotic host cells. In a preferred embodiment, a host cell is a prokaryotic host cell. Exemplary prokaryotic host cells for use in production of the E. coli O antigen polysaccharides described herein and bioconjugates comprising the E. coli O antigen polysaccharides described herein include, but are not limited to, Escherichia species, Shigella species, Klebsiella species, Xhantomonas species, Salmonella species, Yersinia species,
Lactococcus species, Lactobacillus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Staphylococcus species, Bacillus species, and Clostridium species.
[00155] In a spécifie embodiment, the host cell used to produce the E. coli O antigen polysaccharides described herein and bioconjugates comprising the E. coli O antigen polysaccharides described herein is a prokaryotic host cell, and is preferably E. coli.
[00156] In certain embodiments, the host cells used to produce the E. coli O antigen polysaccharides and bioconjugates described herein are engineered to comprise heterologous nucleic acids, e.g., heterologous nucleic acids comprising rfb gene clusters of a desired O antigen serotype, heterologous nucleic acids that encode one or more carrier proteins and/or glycosyltransferases. In a spécifie embodiment, heterologous rfb genes, and/or heterologous nucleic acids that encode proteins involved in glycosylation pathways (e.g., prokaryotic and/or eukaryotic glycosylation pathways) can be introduced into the host cells described herein. Such nucleic acids can encode proteins including, but not limited to, oligosaccharyl transferases and/or glycosyltransferases.
[00157] Sequences of various genes and gene clusters encoding glycosyltransferases useful in making recombinant host cells that can, e.g., be used to préparé E. coli O antigen polysaccharides and bioconjugates thereof are described herein. Those skilled in the art will appreciate that due to the degeneracy of the genetic code, a protein having a spécifie amino acid sequence can be encoded by multiple different nucleic acids. Thus, those skilled in the art will understand that a nucleic acid provided herein can be altered in such a way that its sequence differs from a sequence provided herein, without affecting the amino acid sequence of the protein encoded by the nucleic acid.
[00158] Provided herein are host cells (e.g., recombinant host cells) for producing a bîoconjugate of an E. coli glucosylated 04 antigen polysaccharide, 01A antigen polysaccharide, 02 antigen polysaccharide, O6A antigen polysaccharide, 08 antigen polysaccharide, 015 antigen polysaccharide, 016 antigen polysaccharide, O18A antigen polysaccharide, O25B antigen polysaccharide, or 075 antigen polysaccharide. The host cells provided herein comprise nucleic acids encoding enzymes (e.g., glycosyltransferases) capable of producing the E. coli O antigen polysaccharide. The host cells provided herein can naturally express nucleic acids capable of producing an O antigen of interest, or the host cells can be made to express such nucleic acids. In certain embodiments the nucleic acids are heterologous to the host cells and introduced into the host cells using genetic approaches known in the art. For example, the nucleic acids can be introduced into the host cell by genetic manipulation (e.g., the gene cluster is expressed on a plasmid or plasmids or integrated into the host cell genome (see, e.g., International Patent Application Publications WO 2014/037585, WO 2014/057109, WO 2015/052344).
[00159] In one embodiment, provided herein is a host cell (e.g., recombinant host cell) capable of producing a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein. Such a host cell comprises, preferably by engineering a precursor cell, a nucleic acid sequence encoding a gtrS gene, which, to the best of the knowledge of the inventors, was identified herein for the first time as encoding a polysaccharide branching enzyme capable of transferring glucose to the E. coli 04 antigen (i.e., a glucosyltransferase spécifie to the E. coli 04 antigen polysaccharide), and particularly to L-Rha via an a-l,3-glycosidic linkage. An example of an amino acid sequence of such branching enzyme is provided in SEQ ID NO: 4. Other examples comprise amino acid sequences that are at least 80% identical thereto.
Exemplary examples of nucleic acid sequence encoding gtrS genes spécifie to the E. coli 04 antigen polysaccharide include, but are not limited to, SEQ ID NO: 5, or degenerate nucleic acid sequences thereto that encode SEQ ID NO: 4, or nucleic acid sequences that encode functional 04-specific GtrS enzymes that hâve at least 80% identity to SEQ ID NO: 4.
[00160] In a spécifie embodiment, a host cell (e.g., recombinant host cell) capable of producing a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein, comprises a nucléotide sequence encoding a glucosyl transferase having at least 80% sequence identity to SEQ ID NO: 4, such as about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4. In view of the redundancy in the genetic code, one of ordinary skill in the art can make variants of nucleic encoding the amino acid sequences of glucosyl transferases, e.g., using codon optimized sequences, if desired.
[00161] In certain embodiments, a host cell (e.g., recombinant host cell) capable of producing a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein, comprising a nucléotide sequence encoding a glucosyl transferase (GtrS) having at least 80% sequence identity to SEQ ID NO: 4, further comprises a nucléotide sequence encoding a bactoprenol-linked glucose translocase (GtrA) having at least 80% sequence identity to SEQ ID NO: 7, and a nucléotide sequence encoding a bactoprenol glucosyl transferase (GtrB) having at least 80% sequence identity to SEQ ID NO: 8. In certain 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 NOs: 7 and 8, respectively, and hâve bactoprenollinked glucose translocase (SEQ ID NO: 7) and bactoprenol glucosyl transferase (SEQ ID NO: 8) activity, respectively. In view of the redundancy in the genetic code, one of ordinary skill in the art can make variants of nucleic encoding the amino acid sequences of bactoprenol-linked glucose translocases and of bactoprenol glucosyl transferases, e.g., using codon optimized sequences, if desired.
[00162] A host cell (e.g., recombinant host cell) capable of producing a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein provided herein further comprises a nucléotide sequence of an rfb gene cluster for the E. coli 04 antigen polysaccharide. An example of an rfb gene cluster useful for production of the E. coli 04 antigen polysaccharide is provided herein as SEQ ID NO: 9. Another example can be found in GenBank, locus AY568960. Degenerate nucleic acid sequences encoding the sarne enzymes as encoded by this sequence, or sequences that encode enzymes that are at least 80% identical, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, can also be used.
[00163] In a spécifie embodiment, provided herein is a host cell (e.g., a recombinant host cell, preferably a recombinant prokaryotic host cell, preferably a recombinant E. coli host cell) that produces glucosylated 04 antigen polysaccharide, wherein the host cell comprises gtrS, an rfb gene cluster for the E.coli 04 antigen polysaccharide, and nucleic acid encoding a carrier protein. Such host cells can be engineered using recombinant approaches to comprise one or more plasmids comprising the gtrS gene, the rfb gene cluster, and/or nucleic acid encoding a carrier protein, or to comprise some or ail of the relevant genes such as gtrS, the rfb cluster and/or the nucleic acid encoding the carrier protein integrated into the host cell genome. In i certain embodiments, the genes or gene clusters hâve been integrated into the genome of the host I cell using homologous recombination. An advantage of intégration of genes into the genome of the host cell is stability in the absence of antibiotic sélection.
[00164] In another spécifie embodiment, provided herein is a host cell (e.g., a recombinant host cell, preferably a recombinant prokaryotic host cell) that produces glucosylated 04 antigen polysaccharide, wherein the host cell comprises GtrS (glucosyltransferase), as well as the enzymes encoded by the 04 rjb cluster. In certain embodiments, some or ail ofthe aforementioned enzymes are heterologous to the host cell.
[00165] In other spécifie embodiments, provided herein is a host cell (e.g. a recombinant host cell, preferably a recombinant prokaryotic host cell) that produces E. coli glucosylated 04 antigen polysaccharide, preferably a bioconjugate of £ coli glucosylated 04 antigen polysaccharide, wherein the host cell further comprises a nucléotide sequence encoding an oligosaccharyl transferase and/or a nucléotide sequence encoding a carrier protein. In one spécifie embodiment, the oligosaccharyl transferase is heterologous to the host cell. In another spécifie embodiment, the carrier protein is heterologous to the host cell. Preferably, the host cell comprises a heterologous nucléotide sequence encoding a glucosyl transferase having at least 80% sequence identity to SEQ ID NO: 4. In preferred embodiments, the rfb genes ofthe 04 cluster are heterologous to the host cell. Preferably the sequence encoding the enzyme that is capable of introducing the branched glucose side chain to the 04 antigen, i.e. the gtrS gene (encoding a glucosyl transferase having at least 80% sequence identity to SEQ ID NO:4) is heterologous to the host cell. A nucleic acid is heterologous to the host cell if the same sequence is notnaturally présent in said host cell. Heterologous nucleic acid can for instance be introduced...... in a parent cell by genetic engineering, e.g by transformation (e.g. Chemical transformation or electroporation) and/or recombination. In certain embodiments, heterologous nucleic acid such as a desired rjb locus, gtrS coding sequence, carrier protein encoding sequence, and/or glycosyltransferase encoding sequence are integrated into the genome of the host cell, preferably a bacterial host cell, preferably an E. coli host cell. In preferred embodiments, the endogenous rjb locus and if applicable gtrS coding sequence hâve been inactivated, preferably deleted from the genome ofthe recombinant host cell as compared to apredecessor thereof, and preferably these are replaced by the desired heterologous rjb locus, and if applicable desired gtrS coding sequence, respectively. In certain embodiments the host cell is a K-12 of E. coli (as a non50 I i ί !
limiting example, E. coli strain W3110 is a K-12 strain), or aB strain of E. coli (as a nonlimiting example, E. coli strain BL21 is a B strain), or any other well-defined strain of E. coli, e.g. laboratory strains or production strains, in contrast to primary wild-type isolâtes. In preferred embodiments, the host cell is derived from E. coli that does not express 04 antigen or glucosylated 04 antigen, by introduction into such E. coli of the 04 rjb locus and the gtrS gene encoding a glucosyl transferase having at least 80% sequence identity to SEQ ID NO:4.
Advantages of using well-characterized strains, such as E. coli K-12 or E. coli B, as precursors for host cells is the possibility to use a similar production process for different O-antigen bioconjugates, since the characteristics of the production strain are well-defined. Even though bioconjugates of different O-antigens will behave differently and expression processes can be optimized per production strain, at least the basic process for production of O-antigen bioconjugates will be more predictable using such well-defined precursor strains than when unknown strains such as wild-type isolâtes are used as precursors for production of host strains. This way, expérience with production of earlier described E. coli O-antigen bioconjugates such as O1A, 02, 06A and O25B bioconjugates as described in for instance WO 2015/124769 and WO 2017/035181 can be used as basis to design production of other E. coli O-antigen bioconjugates. Unlike gtrS, the gtrA and gtrB genes are not serotype-specific, and in certain embodiments these are homologous to the host cell (e.g. E.coliK12 strain W3110 includes gtrA and gtrB genes that are capable of functioning together with the O4-serotype spécifie recombinantly introduced gtrS gene encoding a glucosyl transferase of SEQ ID NO: 4 or a glucosyl transferase that is at least 80% identical thereto, replacing the endogenous gtrS gene). In other embodiments, one or both of gtrA and gtrB genes (encoding GtrA and GtrB proteins that are at least about 80% identical to SEQ ID NOs: 7 and 8, respectively, and having bactoprenollinked glucose translocase and bactoprenol glucosyl transferase activity respectively, are also recombinantly introduced in the host cell, for instance in case the host cell does not hâve endogenous gtrA and/or gtrB genes.
[00166] Also provided herein are host cells (e.g., recombinant host cells) capable of producing a bioconjugate of an E. coli O1A, 02, O6A, 08, 015, 016, O18A, O25B, or 075 antigen polysaccharide covalently linked to a carrier protein. Such host cells (e.g., recombinant host cells) comprise nucléotide sequence of an rfb gene cluster spécifie to the O-antigen polysaccharide. The gene clusters can be isolated from wild-type E. coli strains, and combined with nucleic acids encoding an oligosaccharyl transferase (e.g., PglB) and carrier protein (e.g., EPA) within one host cell to obtain a recombinant host cell that produces the E. coli O-antigen of interest or bioconjugate thereof. For example, such host cells can be engineered using recombinant approaches to comprise one or more plasmids comprising the rfb gene cluster, oligosaccharyl transferase (e.g., PglB) and carrier protein (e.g., EPA) using bioconjugation technology such as that described in WO 2014/037585, WO 2009/104074, and WO 2009/089396. Preferably the host cells comprise the rfb gene clusters integrated into their genome. The nucleic acids encoding oligosaccharyl transferase, carrier protein, and where applicable gtrS gene, are in certain embodiments also integrated into the genome ofthe host cell. Heterologous or homologous gtrA and gtrB genes are in certain embodiments also integrated into the genome of the host cell.
[00167] Préparation of bioconjugates for OlA, 02,06A and O25B antigens has been described in detail in WO 2015/124769 and WO 2017/035181. Examplary gene clusters for each E. coli O antigen (rfb loci) hâve been described in Iguchi A, et al, DNA Research, 2014, 1-7 (doi: 10.1093/dnares/dsu043), and in DebRoy C, et al, PLoS One. 2016, 1 l(l):e0147434 (doi: 10.1371/joumal.pone.0147434; correction in: Plos One. 2016, 1 l(4):e0154551, doi: 10.1371/journal.pone.0154551). Nucleic acid sequences for the rfb clusters and amino acid sequences for proteins encoded therein can also be found in public databases, such as GenBank. Exemplary sequences for rfb clusters that can be used in production 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 ofthe desired bioconjugates mentioned above, the respective rfb cluster can be introduced into a host cell, to obtain host cells with the spécifie rfb cluster for the desired O-antigen, as well as containing nucleic acid encoding oligosaccharyltransferase and carrier protein. For reasons indicated above, preferably the host cells are recombinant host cells, and preferably are derived from strains with relatively well-known characteristics, such as E. coli laboratory or production strains, e.g. E. coli K12 or E. coli BL21, etc. Preferably, the rfb clusters are heterologous to the host cell, e.g. introduced into a precursor cell ofthe host cell, and i preferably integrated into the genome thereof. Preferably an original rfb gene cluster, if such was | présent in a precursor cell, has been replaced by the rfb gene cluster for the O-antigen of interest I
I !
in the host cell, to enable production of bioconjugate of the O-antigen of interest. Preferably the oligosaccharyltransferase is heterologous to the host cell, and in certain embodiments nucleic acid encoding such oligosaccharyltransferase is integrated into the genome of the host cell.
[00168] Any of the host cells provided herein (e.g., recombinant host cells, preferably recombinant prokaryotic host cells) comprise nucleic acids encoding additional enzymes active in the Λ-glycosylation of proteins, e.g., the host cell provided herein can further comprise a nucleic acid encoding an oligosaccharyl transferase or nucleic acids encoding other glycosyltransferases.
[00169] The host cells provided herein comprise a nucleic acid that encodes an oligosaccharyl transferase. Oligosaccharyl transferases transfer lipid-linked oligosaccharides to asparagine residues of nascent polypeptide chains that comprise an Af-glycosylation consensus motif. The nucleic acid that encodes an oligosaccharyl transferase can be native to the host cell, or can be introduced into the host cell using genetic approaches. In preferred embodiments, the oligosaccharyl transferase is heterologous to the host cell. E. coli does not naturally comprise an oligosaccharyl transferase, and hence if E.coli is used as a host cell for production of bioconjugates, a heterologous oligosaccharyl transferase is comprised in such host cell, e.g. upon introduction by genetic engineering. The oligosaccharyl transferase can be from any source known in the art in view of the présent disclosure.
[00170] In certain embodiments, an alternative to an oligosaccharyl transferase with Nglycosyltransferase activity, such as an O-glycosyltransferase, e.g. as a non-limiting example PglL, can be used, in conjunction with its own, different, glycosylation consensus sequence in the carrier protein, as for instance described in WO 2016/82597. Other glycosyltransferases, such as O-glycosyltransferases, can thus also be used as an oligosaccharyltransferase according to the invention.
[00171] In certain preferred embodiments, the oligosaccharyl transferase is an oligosaccharyl transferase from Campylobacter. For example, in one embodiment, the oligosaccharyl transferase is an oligosaccharyl transferase from Campylobacter jejuni (î.e.,pglB; see, e.g., Wacker et al., 2002, Science 298:1790-1793; see also, e.g., NCBI Gene ID: 3231775, UnîProt Accession No. 086154). In another embodiment, the oligosaccharyl transferase is an oligosaccharyl transferase from Campylobacter lari (see, e.g., NCBI Gene ID: 7410986).
[00172] In spécifie embodiments, the oligosaccharyl transferase is PglB oligosaccharyl transferase from Campylobacter jejuni, including the natural (wild-type) protein or any variant thereof, such as those described in International Patent Application Publications 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 particular embodiments, the PglB oligosaccharyl transferase comprises SEQ ID NO: 6, or a variant thereof. In certain embodiments one or more endogenous glycosylation consensus sequences in a wildtype PglB hâve been mutated to avoid PglB autoglycosylation, e.g. SEQ ID NO: 6 comprising the mutation N534Q. Examples of variant PglB oligosaccharyl transferases suitable for use in the recombinant host cells provided herein include the PglB oligosaccharyl transferase of SEQ ID NO: 6 comprising at least one mutation selected from the group consisting of N31IV, K482R, D483H, A669V, Y77H, S80R, Q287P, and K289R. In one particular embodiment, a variant PglB oligosaccharyl transferase has SEQ ID NO: 6 comprising the mutation N31IV. In another particular embodiment, a variant PglB oligosaccharyl transferase has SEQ ID NO: 6 comprising the mutations Y77H and N311V. In another particular embodiment, a variant PglB oligosaccharyl transferase has SEQ ID NO: 6 comprising the mutations N31IV, K482R, D483H, and A669V. In another particular embodiment, a variant PglB oligosaccharyl transferase has SEQ ID NO: 6 comprising the mutations Y77H, S80R, Q287P, K289R, and N31IV. It was found and described herein that certain PglB oligosaccharyl transferase variants give surprisingly improved yields in production of E. coli O-antigen bioconjugates of spécifie serotypes. The improved or optimal PglB variant for a given E. coli O-antigen was not predictable. The invention in certain aspects therefore also provides methods for producing bioconjugates of spécifie E. coli O-antigens, using spécifie PglB variants as the oligosaccharyl transferase. Further variants of PglB that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 6 and still hâve oligosaccharyl transferase activity, preferably having one or more of the spécifie amino acids on the indicated positions disclosed in combination herein (e.g. 77Y, 80S, 287Q, 289K, 3 UN, 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 production of bioconjugates.
[00173] In a spécifie embodiment, a host cell (e.g., recombinant host cell) capable of producing a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein further comprises a nucléotide sequence encoding PglB oligosaccharyl transferase from Campylobacter jejuni having the amino acid sequence of SEQ ID NO: 6, or preferably SEQ ID NO: 6 comprising the mutation N31IV, or more preferably SEQ ID NO: 6 comprising the mutations Y77H and N311V.
[00174] In other spécifie embodiments, a host cell (e.g., recombinant host cell) capable of producing a bioconjugate of an E. coli 01 A, O6A, or 015 antigen polysaccharide covalently linked to a carrier protein further comprises a nucléotide sequence encoding PglB oligosaccharyl transferase from Campylobacter jejuni having the amino acid sequence of SEQ ID NO: 6, or preferably SEQ ID NO: 6 comprising the mutations N311V, K482R, D483H, and A669V.
[00175] In a spécifie embodiment, a host cell (e.g., recombinant host cell) capable of producing a bioconjugate of an E. coli 016 antigen polysaccharide covalently linked to a carrier protein further comprises a nucléotide sequence encoding PglB oligosaccharyl transferase from Campylobacter jejuni having the amino acid sequence of SEQ ID NO: 6, or preferably SEQ ID NO: 6 comprising the mutations Y77H, S80R, Q287P, K289R, and N31IV.
[00176] In a spécifie embodiment, a host cell (e.g., recombinant host cell) capable of producing a bioconjugate of an E. coli 075 antigen polysaccharide covalently linked to a carrier protein further comprises a nucléotide sequence encoding PglB oligosaccharyl transferase from Campylobacter jejuni having the amino acid sequence of SEQ ID NO: 6, or preferably SEQ ID NO: 6 comprising the mutation N311V.
[00177] In a spécifie embodiment, a host cell (e.g., recombinant host cell) capable of producing a bioconjugate of an E. coli 08, O18A, O25B, or 02 antigen polysaccharide covalently linked to a carrier protein further comprises a nucléotide sequence encoding PglB oligosaccharyl transferase from Campylobacter jejuni having the amino acid sequence of SEQ ID NO: 6, preferably wherein SEQ ID NO: 6 comprises no amino acid mutations at positions 77, 80, 287, 289, 311,482,483, and 669.
[Θ0178] In some embodiments, any of the host cells provided herein comprise a nucleic acid encoding a carrier protein, e.g., a protein to which the O-antigen polysaccharide(s) produced by the host cell glycosylation machinery can be attached to form a bioconjugate. The host cell can comprise a nucleic acid encoding any carrier protein known to those skîlled in the art in view of the présent disclosure including, but not limited to, detoxified Exotoxin A of P. aeruginosa (EPA), E. coli flagellin (FliC), CRM197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of 5. aureus, dumping factor A, dumping factor B, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Choiera toxin B subunit (CTB), choiera toxin, detoxified variants of choiera toxin, E. coli Sat protein, the passenger domain of E. coli Sat protein, Streptococcus pneumoniae Pneumolysin, Keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.
[00179] In preferred embodiments, a host cell further comprises a nucleic acid encoding detoxified Exotoxin A ofP. aeruginosa (EPA). Preferably, the EPA carrier protein comprises 110 glycosylation sites, preferably 2 to 4 glycosylation sites, most preferably 4 glycosylation sites, such as 1-10, preferably 2-4, and more preferably 4 glycosylation sites each comprising a glycosylation consensus sequence having the amino acid sequence of SEQ ID NO: 1, and more preferably having the amino acid sequence of SEQ ID NO: 2. In a spécifie embodiment, a host cell further comprises a nucleic acid encoding EPA-4 carrier protein comprising SEQ ID NO: 3. [00180] In certain embodiments, the carrier proteins used in the génération of the bioconjugates by the host cells described herein comprise a “tag,” i.e., a sequence of amino acids that allows for the isolation and/or identification of the carrier protein. For example, adding a tag to a carrier protein can be useful in the purification of that protein and, hence, the purification of conjugate vaccines comprising the tagged carrier protein. Exemplary tag s that can be used herein include, without limitation, histidine (HIS) tags (e.g., hexa-histidine-tag, or 6XHis-Tag), FLAG-TAG, and HA tags. In certain embodiments, the tags used herein are removable, e.g., removal by Chemical agents or by enzymatic means, once they are no longer needed, e.g., after the protein has been purified. In other embodiments, the carrier protein does not comprise a tag. [00181] In certain embodiments, the carrier proteins described herein comprise a signal sequence that targets the carrier protein to the periplasmic space of the host cell that expresses the carrier protein. In a spécifie embodiment, the signal sequence is from E. coli DsbA, E. coli outer membrane porin A (OmpA), E. coli maltose binding protein (MalE), Erwinia carotovorans pectate lyase (PelB), Flgl, NikA, or Bacillus sp. endoxylanase (XynA), heat labile E. coli enterotoxin LTIIb, Bacillus endoxylanase XynA, or E. coli flagellin (Flgl). In one embodiment, the signal sequence comprises SEQ ID NO: 10. A signal sequence may be cleaved off after translocation of the protein to the periplasm and may thus no longer be présent in the final carrier protein of a bioconjugate.
[00182] In certain embodiments, additional modifications can be introduced (e.g., using recombinant techniques) into the host cells described herein. For example, host cell nucleic acids (e.g., genes) that encode proteins that form part of a possibly competing or interfering glycosylation pathway (e.g., compete or interfère with one or more heterologous genes involved in glycosylation that are recombinantly introduced into the host cell) can be deleted or modified in the host cell background (genome) in a manner that makes them inactive/dysfiinctional (i.e., the host cell nucleic acids that are deleted/modified do not encode a fiinctional protein). In certain embodiments, when nucleic acids are deleted from the genome of the host cells provided herein, they are replaced by a désirable sequence, e.g., a sequence that is useful for production of an O antigen polysaccharide or bioconjugate thereof.
[00183] Exemplary genes or gene clusters that can be deleted in host cells (and, in some cases, replaced with other desired nucleic acid sequences) include genes or gene clusters of host cells involved in glycolipid biosynthesis, such as waaL (see, e.g., Feldman et al., 2005, PNAS USA 102:3016-3021), the lipid A core biosynthesis cluster (uw), galactose cluster (gai), arabinose cluster (ara), colonie acid cluster (w), capsular polysaccharide cluster, undecaprenol-p biosynthesis genes (e.g. uppS, uppP), und-P recycling genes, metabolic enzymes involved in nucléotide activated sugar biosynthesis, enterobacterial common antigen cluster (eca), and prophage O antigen modification clusters like the gtrABS cluster or régions thereof. In a spécifie embodiment, the host cells described herein are modified such that they do not produce any O antigen polysaccharide other than a desired O antigen polysaccharide, e.g., glucosylated 04 antigen polysaccharide.
[00184] In a spécifie embodiment, the waaL gene is deleted or functionally înactivated from the genome of a host cell (e.g., recombinant host cell) provided herein. 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 undecaprenylphosphate (UPP)-bound O antigen to the lipid A core, forming lipopolysaccharide. Délétion or disruption ofthe endogenous waaL gene (e.g., NwaaL strains) disrupts transfer ofthe O-antigen to lipid A, and can instead enhance transfer ofthe O-antigen to another biomolecule, such as a carrier protein.
[00185] In another spécifie embodiment, one or more of the waaL gene, gtrA gene, gtrB gene, gtrS gene, and the rfi gene cluster is deleted or functionally inactivated from the original genome of a prokaryotic host cell provided herein.
[00186] In one embodiment, a host cell used herein is E. coli that produces a bioconjugate of glucosylated 04 antigen polysaccharide, wherein the waaL gene is deleted or functionally inactivated from the genome ofthe host cell, and agir S gene spécifie to E. coli 04 antigen polysaccharide is inserted. In certain embodiments for production strains for bioconjugates of the glucosylated 04 O-antigen, a gtrS gene encoding a glucosyl transferase having at least 80% sequence identity to SEQID NO:4 is inserted in the place of a gtrS gene of the parent strain, so as to replace the gtrS gene in that parent strain with the one that is responsible for glucosylation of the 04 antigen. An example of such a parent strain is E. coli K-12 strain W3110. The gtrA and gtrB genes can be homologous to the parent strain, or altematively one or both of these genes can be heterologous to the parent strain. Typically, and unlike the gtrS gene, these gtrA and gtrB genes are not spécifie for the O-antigen structure.
[00187] Also provided herein are methods of making recombinant host cells. Recombinant host cells produced by the methods described herein can be used to produce bioconjugates of E. coli O antigens. The methods comprise introducing one or more recombinant nucleic acid molécules into a cell to produce the recombinant host cell. Typically, the recombinant nucleic acid molécules are heterologous. Any method known in the art in view of the présent disclosure can be used to introduce recombinant nucleic acid molécules into a host cell. Recombinant nucleic acids can be introduced into the host cells described herein using any methods known to those of ordinary skill in the art, e.g., electroporation, Chemical transformation, by heat shock, natural transformation, phage transduction, and conjugation. In spécifie embodiments, recombinant nucleic acids are introduced into the host cells described herein using a plasmid. For example, the heterologous nucleic acids can be expressed in the host cells by a plasmid (e.g., an expression vector). In another spécifie embodiment, heterologous nucleic acids are introduced into the host cells described herein using the method of insertion into the genome as for instance described in International Patent Application Publication WO 2014/037585, WO 2014/057109, or WO 2015/052344.
[00188] In one embodiment, a method of making a recombinant host cell for producing a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein comprises introducing one or more recombinant nucleic acid molécules into a cell, preferably an E. coli cell, to produce the recombinant host cell. In such embodiments, the recombinant nucleic acid molécules introduced into the cell include (i) a nucléotide sequence of an rfb gene cluster for the E. coli 04 antigen polysaccharide; (ii) a nucléotide sequence encoding a glucosyl transferase having at least 80% sequence identity to SEQ ID NO: 4, wherein the glucosyl transferase is capable of modily ing the E. coli 04 antigen polysaccharide to produce the E. coli glucosylated 04 antigen polysaccharide; (iii) a nucléotide sequence encoding a carrier protein; and (iv) a nucléotide sequence encoding an oligosaccharyl transferase capable of covalently linking the E. coli glucosylated 04 antigen polysaccharide to the carrier protein to produce the bioconjugate. In preferred embodiments, the nucléotide sequence encoding a glucosyl transferase having at least 80% sequence identity to SEQ ID NO: 4 replaces the endogenous gtrS gene. Deleting the endogenous gtrS has the advantage that it will not interfère with génération of the glucosylated 04 antigen polysaccharide structure. In certain embodiments, the nucléotide sequence of the rfb gene cluster for the E. coli 04 antigen polysaccharide replaces the endogenous rfb gene cluster of the parent strain that is used to make the recombinant host cell. If the cell does not yet encode gtrA and/or gtrB genes, nucléotide sequences encoding a translocase (gtrA) and a glycosyltransferase (gtrB), having at least 80% identity to SEQ ID NOs: 7 and 8, respectively, can be introduced into the cell. If the cell already encodes gtrA and gtrB genes (such as for instance the case in E. coli K-12 strain W3110), there is no need to introduce or change these genes.
[00189] In a spécifie embodiment, the glucosyl transferase (gtrS spécifie for adding glucose branch to 04 antigen) has SEQ ID NO: 4.
[00190] In a spécifie embodiment, the oligosaccharyl transferase is PglB from C. jejuni. In one such embodiment, the oligosaccharyl transferase comprises the amino acid sequence of SEQ ID NO: 6. In another such embodiment, the oligosaccharyl transferase comprises the amino acid sequence of SEQ ID NO: 6 comprising the mutation N31IV. In another such embodiment, the oligosaccharyl transferase comprises the amino acid sequence of SEQ ID NO: 6 comprising the mutations Y77H and N311V.
[00191] In another spécifie embodiment, the carrier protein comprises at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably SEQ ID NO: 2. In another spécifie embodiment, the carrier protein is EPA, preferably EPA-4, such as EPA-4 comprising SEQ ID NO: 3.
[00192] E. coli strains that are used routinely in molecular biology as both a tool and a model organism can for instance be used as parents for host cells in certain embodiments according to the invention. Non-limiting examples include E. coli K12 strains (for example, such as W1485, W2637, W3110, MG1655, DH1, DH5a, DH10, etc.), B strains (e.g. BL-21, REL606, etc.), C strains, or W strains. In one particular embodiment, the host strain is derived from parent strain W3110. This strain can for instance be obtained from the E. coli Genetic Stock Center at Yale. For more information on E. coli, see e.g, Ecoliwiki.net.
[00193] Methods of Producing Conjugates and Bioconjugates
[00194] Also provided are methods of producing glycoconjugates of the E. coli O antigen polysaccharides described herein. Glycoconjugates, including bioconjugates, can be prepared in vitro or in vivo, e.g., using the recombinant host cells described herein for production.
[00195] In some embodiments, glycoconjugates can be prepared by Chemical synthesis, i.e., prepared outside of host cells (in vitro'). For example, an E. coli O antigen polysaccharide can be conjugated to carrier proteins using methods known to those of ordinary skill in the art, including by means of using activation reactive groups in the polysaccharide/oligosaccharide as well as the carrier protein. See, e.g., Pawlowski et al., 2000, Vaccine 18:1873-1885; and Robbins, et al., 2009, Proc Natl AcadSci USA 106:7974-7978), the disclosures of which are herein incorporated by reference. Such approaches comprise extraction of antigenic polysaccharides/ oligosaccharides from host cells, purifying the polysaccharides/oligosaccharides, chemically activating the polysaccharides/oligosaccharides, and conjugating the polysaccharides/ oligosaccharides to a carrier protein.
[00196] In some embodiments, the host cells described herein can be used to produce bioconjugates comprising an E. coli O antigen polysaccharide covalently linked to a carrier protein. Methods of producing such bioconjugates using host cells are known in the art. See,
e.g., WO 2003/074687 and WO 2006/119987. Such methods comprise culturing any of the recombinant host cells described herein under conditions for production of the bioconjugate. Bioconjugates can be isolated, separated, and/or purified from recombinant host cells using any method known in the art in view of the présent disclosure. For example, bioconjugates can be purified by any method known in the art for purification of a protein, for instance, by chromatography (e.g., ion exchange, anionic exchange, affmity, and sizîng column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. See, e.g., methods described in WO 2009/104074. Further, the bioconjugates can be fused to heterologous polypeptide sequences to facilitais purification. The actual conditions used to purify a paiticular bioconjugate will dépend, 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. Préparation of bioconjugates for 01 A, 02, O6A, and O25B, as well as vaccine compositions comprising these, hâve for instance been described in WO 2015/124769 and in WO 2017/035181.
[00197] Also provided are bioconjugates produced by the methods described herein, i.e., using the recombinant host cells described herein.
[00198] In some embodiments, a method of preparing a bioconjugate of an E. coli O-antigen polysaccharide covalently linked to a carrier protein comprises: (i) providing a recombinant host cell comprising (a) nucléotide sequence of an rfb gene cluster for the O-antigen polysaccharide; (b) a nucléotide sequence encoding a carrier protein, preferably EPA, comprising at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably SEQ ID NO: 2, and more preferably comprising four glycosylation sites each comprising a glycosylation consensus sequence having SEQ ID NO: 2; and (c) nucléotide sequence encoding an oligosaccharyl transferase, for instance PglB oligosaccharyl transferase or variant thereof.
[00199] In certain embodiments, E. coli O-antigen polysaccharides produced using the recombinant host cells described herein are covalently bound to the carrier protein at a particular polysaccharide to protein ratio by weight (w/w). This ratio of amount of O-antigen polysaccharide by weight covalently bound to the carrier protein by weight is referred to as the “glycan/protein ratio” or “polysaccharide/protein ratio” or “PS/protein ratio”. In some embodiments, the O-antigen polysaccharide is covalently bound to the carrier protein at a polysaccharide to protein (w/w) ratio of about 1: 20 to 20:1, preferably 1:10 to 10:1, more preferably 1:3 to 3:1. In certain non-limiting embodiments for bioconjugates described herein, glycan/protein ratio is about 0.1 to 0.5, such as 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 the O-antigen polysaccharide: protein is about 1:10 to 1:2, such as 1:10: 1:9:1:8, 1:7, 1:6, 1:5, 1:4,1:3, or 1:2, depending on the particular O-antigen serotype. In certain 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 a high amount of carrier protein can lead to immunological interférence in some instances. Also, a higher glycan/protein ratio would help getting sufficient O-antigen polysaccharide dosed in the form of bîoconjugate, while keeping the amount of carrier protein relatively low, which is especially bénéficiai for multivalent compositions where multiple serotypes are to be covered by the composition, e.g. compositions comprising bioconjugates from at least 4 different O-antigens, at least 5 different O-antigens, at least 6 different Oantigens, at least 7 different O-antigens, at least 8 different O-antigens, at least 9 different Oantigens, at least 10 different O-antigens, etc.
[00200] A glycan/protein ratio of a conjugate according to the invention can be determined by determining the protein amount and the glycan amount. Protein amount can be determined by measurement of UV absorbance at 280 nm (A280). Glycan amount can be determined based on ion chromatography with pulsed amperometric détection (IC-PAD) of a sugar in the repeat unit (e.g. of Man for 08 in Table 1, and of GlcNAc for the other glycans in Table 1), after which the structural information of the repeat unit can be used to calculate the total glycan amount (e.g. the repeat unit of 01A has a molar mass of 845 Da and one mole of such a repeat unit contains one mole of GlcNAc, enabling calculation of the total glycan amount when the amount of GlcNAc has been determined by IC-PAD).
[00201] In some embodiments, a bîoconjugate of an E. coli O25B antigen polysaccharide covalently linked to a carrier protein produced using a recombinant host cell according to the cells and methods described herein has a certain degree of acétylation at position 2 of the L-Rh sugar. The degree of O-acetylation of O25B antigen polysaccharide in a bîoconjugate is preferably at least 30%, preferably at least 50%, such as at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
[00202] Similarly, the degree of O-acetylation of an E. coli 016 antigen polysaccharide in a bioconjugate is preferably at least 30%, preferably at least 50%, such as at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
[00203] In spécifie embodiments, a method of preparing a bioconjugate of an O-antigen polysaccharide comprises providing a recombinant host cell comprising nucleic acid sequence encoding a particular oligosaccharyl transferase enzyme, particularly a PglB oligosaccharyl transferase or variant thereof, depending on the O-antigen polysaccharide bioconjugate to be produced. The particular oligosaccharyl transferase enzyme variant may impact the yield of bioconjugate produced by the host cell. Typically, a higher yield is preferred, since the yield will impact the costs for producing a spécifie bioconjugate, which is especially important for multivalent compositions comprising several different bioconjugates.
[00204] In one particular embodiment, when the O- antigen is O1A, O6A, or 015 antigen polysaccharide, the PglB oligosaccharyl transferase comprises the amino acid mutations of N31IV, K482R, D483H, and A669V, wherein the amino acid mutations are relative to the wildtype PglB having the amino acid sequence of SEQ ID NO: 6.
[00205] In another particular embodiment, when the O-antigen is glucosylated 04 antigen polysaccharide, the PglB oligosaccharyl transferase comprises the amino acid mutation N31IV, or the amino acid mutations of Y77H and N311V, wherein the amino acid mutations are relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6.
[00206] In another particular, embodiment, when the O-antigen is 016 antigen polysaccharide, the PglB oligosaccharyl transferase comprises the amino acid mutations of Y77H, S80R, Q287P, K289R, and N311V, wherein the amino acid mutations are relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6.
[00207] In another particular embodiment, when the O-antigen is 075 antigen polysaccharide, the PglB oligosaccharyl transferase comprises the amino acid mutation of N31IV, wherein the amino acid mutations are relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6.
[00208] In another particular embodiment, when the O-antigen is 08, 018A, O25B, or 02 antigen polysaccharide, the PglB oligosaccharyl transferase comprises the amino acid sequence of SEQ ID NO: 6, wherein SEQ ID NO: 6 comprises no amino acid mutations at positions 77, 80, 287,289, 311,482, 483, and 669. In certain embodiments thereof, the PglB oligosaccharyl transferase comprises the amino acid sequence of SEQ ID NO: 6.
[00209] In some embodiments, bioconjugates of O-antigen polysaccharides produced by recombinant host cells encoding the oligosaccharyl transferase enzymes per the O-antigen/PglB oligosaccharyl transferase pairings indicated above preferably hâve one or more ofthe preferred attributes described herein, e.g., glycan/protein ratio and/or percent of multi-glycosylated carrier protein.
[00210] EMBODIMENTS
[00211] Embodiment 1 is a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein, wherein the E. coli glucosylated 04 antigen polysaccharide comprises the structure of Formula (O4-Glc+):
a-D-GIcp
[-»2)-a-L-Rhap-(1 ->6)-a-D-Glcp-(1 -»3)-a-L-FucpNAc-(1-*3)-p-D-GlcpNAc-(1 ->]n wherein n is an integer of 1 to 100, preferably 3 to 50, for example 5 to 40, for examp le 7 to 25, for example 10 to 20.
[00212] Embodiment 2 is the bioconjugate of embodiment 1, wherein the E. coli glucosylated 04 antigen polysaccharide is covalently linked to an Asn residue in a glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2, în the carrier protein.
[00213] Embodiment 3 is the bioconjugate of embodiment 1 or embodiment 2, wherein the carrier protein is selected from the group consisting of detoxified Exotoxin A of P. aeruginosa (EPA), E. coli flagellin (FliC), CRM 197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of S. aureus, dumping factor A, dumping factor B, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Choiera toxin B subunit (CTB), choiera toxin, detoxified variants of choiera toxin, E. coli Sat protein, the passenger domain of E. coli Sat protein, Streptococcuspneumoniae Pneumolysin, Keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.
[00214] Embodiment 4 is the bioconjugate of embodiment 3, wherein the carrier protein is detoxified exotoxin A of Pseudomonas aeruginosa (EPA), preferably comprising 1 to 20, preferably 1 to 10, preferably 2 to 4, glycosylation consensus sequences having SEQ ID NO: 1, the consensus sequences preferably having SEQ ID NO: 2.
[00215] Embodiment 5 is the bioconjugate of embodiment 4, wherein the carrier protein comprises four glycosylation consensus sequences (EPA-4), preferably wherein the carrier protein comprises SEQ ID NO: 3.
[00216] Embodiment 6 is a composition comprising the bioconjugate of any one of embodiments 1-5.
[00217] Embodiment 7 is an immunogenic composition comprising the bioconjugate of any one of embodiments 1-5.
[00218] Embodiment 8 is the composition of embodiment 6 or immunogenic composition of embodiment 7, comprising at least one additional antigen polysaccharide covalently linked to a carrier protein.
[00219] Embodiment 9 is the composition or immunogenic composition of embodiment 8, wherein the at least one additional antigen polysaccharide is selected from the group consisting of E. coli OlA antigen polysaccharide, E. coli 02 antigen polysaccharide, E. coli O6A antigen polysaccharide, E. coli 08 antigen polysaccharide, E. coli 015 antigen polysaccharide, E. coli 016 antigen polysaccharide, E. coli O18A antigen polysaccharide, E. coli 025B antigen polysaccharide, and E. coli 075 antigen polysaccharide.
[00220] Embodiment 10 is the composition or immunogenic composition of embodiment 9, wherein (i) the E. coli OlA antigen polysaccharide comprises the structure of Formula (OlA):
I ί
[->3)-a-L-Rhap~(1 -*3)-a-L-Rhap-(1 -»3)-p-L-Rhap-(1 ->4)-p-D-GlcpNAc-(1 ->]n 2 î
β-D-ManpNAc (il) the E. coli 02 antigen polysaccharide comprises the structure of Formula (02):
[->3)-a-L-Rhap-(1 ->2)-ot-L-Rhap-(1 —>3)-f-L-Rhap-(1 -»4)-β-θ-ΘΙορΝΑο(1 -*]n 2 î
a-D-Fucp3NAc (iii) the E. coli 06A antigen polysaccharide comprises the structure of Formula (06A):
[-*4)-a-D-GalpNAc-(1 -»3)-fl-D-Manp-(1 -Μ)-β-0-Μ3ηρ-(1 “>3)-a-D-GlcpNAc-(1 -»]n î
β-D-Glcp .
?
(iv) the E. coli 08 antigen polysaccharide comprises the structure of Formula (08):
ct-D-Manp3Me-(1 -^[3)-p-D-Manp-(1 -»2)-a-D-Manp-( 1 -*2)-a-D-Manp-(1 Tn (v) the E. coli 015 antigen polysaccharide comprises the structure of Formula (015):
[^2)-p-D-Galp-(1->3)-a-L-FucpNAc-(1->3)-p-D-GlcpNAc-(1->]n !» (vi) the E. coli 016 antigen polysaccharide comprises the structure of Formula (016):
[-»2)^-D-Galf-(1 ->6)-a-D-Glcp-(1 -^3)-a-L-Rhap-(1 ^3)-a-D-GlcpNAc-(1 -»]n î
Ac .
(vii) the E. coli O ISA antigen polysaccharide comprises the structure of Formula (018A):
[->2)-a-L-Rhap-(1 ~»6)-a-D-Glcp-(1 ->4)-a-D-Galp-(1 -*3)-a-D-GlcpNAc-(1 -»]n î
β-D-GlcpNAc .
?
(viii) the E. coli O25B antigen polysaccharide comprises the structure of Formula (O25B):
β-D-Glcp i
|>4)-a-D-Glcp-(1 ^3)-cc-L-Rhap-(1 -*3)-p-D-GlcpNAc-(1 -^]rt tt
1Ac a-Î-Rhap.
(ix) the E. coli 075 antigen polysaccharide comprises the structure of Formula (075):
β-D-Manp
[->3)-a-D-Galp-(1 -»4)“a-L-Rhap-(1 -4-3)-|j-D-GlcpNAc-(1 ->]n J wherein each n is independently an integer of 1 to 100, preferably of 3 to 50, e.g. 5 to 40, e.g. 7 to 25, e.g. 10 to 20.
[00221] Embodiment 11 is the composition or immunogenic composition of embodiment 10, wherein each of the additional E. coli O1A, 02, O6A, 08, 015, 016, O18A, O25B, and/or 075 antigen polysaccharides is covalently bound to an Asn residue in a glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2, in each of the carrier protein.
[00222] Embodiment 12 is the composition or immunogenic composition of embodiment 11, wherein each of the carrier protein is a detoxified exotoxin A of Pseudomonas aeruginosa (EPA).
{00223] Embodiment 13 is the composition or immunogenic composition of embodiment 12, wherein each EPA comprises 1-10, preferably 2-4, glycosylation sites each comprising a glycosylation consensus sequence having SEQID NO: 2.
[00224] Embodiment 14 is the composition or immunogenic composition of embodiment 12, wherein each EPA comprises four glycosylation sites each comprising a glycosylation consensus sequence having SEQ ID NO; 2.
[00225] Embodiment 15 is the composition or immunogenic composition of embodiment 12, wherein each EPA comprises SEQ ID NO: 3.
[00226] Embodiment 16 is the composition or immunogenic composition of any one of embodiments 9-15, comprising at least the E. coli OlA, 02, glucosylated 04, O6A, and O25B antigen polysaccharides each covalently linked to a carrier protein.
[00227] Embodiment 17 is the composition or immunogenic composition of any one of embodiments 9-15, comprising at least the E. coli OlA, 02, glucosylated 04, O6A, 08, 015, 016, 025B and 075 antigen polysaccharides each covalently linked to a carrier protein.
[00228] Embodiment 18 is the composition or immunogenic composition of any one of embodiments 9-15, comprising at least the E, coli OlA, 02, glucosylated 04, O6A, 08, 015, 016, 018A, O25B, and 075 antigen polysaccharides each covalently linked to a carrier protein.
[00229] Embodiment 19 is a composition comprising:
(i) a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a detoxified Exotoxin A of P. aeruginosa (EPA-4 carrier protein) comprising SEQ ID NO: 3, wherein the E. coli glucosylated 04 antigen polysaccharide comprises the structure of Formula (O4-Glc+);
(ii) a bioconjugate of an E. coli OlA antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli OlA antigen polysaccharide comprises the structure of Formula (OlA);
(iii) a bioconjugate of an E. coli 02 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 02 antigen polysaccharide comprises the structure of Formula (02);
(iv) a bioconjugate of an E. coli 06A antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O6A antigen polysaccharide comprises the structure of Formula (06 A);
(v) a bioconjugate of an E. coli 08 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 08 antigen polysaccharide comprises the structure of Formula (08);
(vi) a bioconjugate of an E. coli 015 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 015 antigen polysaccharide comprises the structure of Formula (015);
(vii) a bioconjugate of an E. coli 016 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 016 antigen polysaccharide comprises the structure of Formula (016);
(viii) a bioconjugate of an E. coli O25B antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O25B antigen polysaccharide comprises the structure of Formula (O25B); and (ix) a bioconjugate of an E. coli 075 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 075 antigen polysaccharide comprises the structure of Formula (075), wherein each ofthe structures of Formulas (04-Glc+), (01A), (02), (06A), (08), (015), (016), (O25B), and (075) is shown in Table 1, and each n is independently an integer of 1 to 100, preferably 3 to 50, e.g. 5 to 40, e.g. 7 to 25, e.g. 10 to 20.
[00230] Embodiment 20 is the composition of embodiment 19, further comprising:
(x) a bioconjugate of an E. coli O18A antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O18A antigen polysaccharide comprises the structure of Formula (O18A) shown in Table 1, and n is an integer of 1 to 100, preferably 3 to 50, e.g. 5 to 40, e.g. 7 to 25, e.g. 10 to 20.
[00231] Embodiment 21 is a method of inducing antibodies against an E. coli glucosylated 04 antigen in a subject, comprising administering to the subject the bioconjugate of any one of embodiments 1-5, or the composition or immunogenic composition of any one of embodiments 6-20.
[00232] Embodiment 22 is the method of embodiment 21, wherein the antibodies hâve opsonophagocytic activity.
[00233] Embodiment 23 is a method of vaccinating a subject against E. coli, in particular extra-intestinal pathogenic E. coli (ExPEC), comprising administering to the subject the bioconjugate of any one of embodiments 1-5, or the composition or immunogenic composition of any one of embodiments 6-20.
[00234] Embodiment 24 is the bioconjugate of any one of embodiments 1 -5, or the composition or immunogenic composition of any one of embodiments 6-20 for use in inducing antibodies against an E. coli glucosylated 04 antigen.
[00235] Embodiment 25 is the bioconjugate of any one of embodiments 1 -5, or the composition or immunogenic composition of any one of embodiments 6-20 for use in vaccination against extra-intestinal pathogenic E. coli (ExPEC).
[00236] Embodiment 26 is a recombinant host cell for producing a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein, wherein the glucosylated 04 antigen polysaccharide comprises the structure of Formula (O4-Glc+) as shown in Table 1, wherein n is an integer of 1 to 100, preferably of 3 to 50, e.g. of 5 to 40, the host cell comprising:
(i) a nucléotide sequence of an rfb gene cluster for the E. coli 04 antigen polysaccharide;
(ii) a nucléotide sequence encoding a glucosyl transferase having at least 80% sequence identity to SEQ ID NO: 4, wherein the glucosyl transferase is capable of modifying the E. coli 04 antigen polysaccharide to produce the E. coli glucosylated 04 antigen polysaccharide;
(iii) nucléotide sequences encoding a translocase and a glycosyltransferase having at least 80% sequence identity to SEQ ID NOs: 7 and 8 respectively, wherein the translocase is capable of translocating bactoprenol-linked glucose and the glycosyltransferase is capable of glucosylating bactoprenol;
(iv) a nucléotide sequence encoding the carrier protein; and (v) a nucléotide sequence encoding an oligosaccharyl transferase capable of covalently linking the E. coli glucosylated 04 antigen polysaccharide to the carrier protein to produce the bioconjugate.
[00237] Embodiment 27 is the recombinant host cell of embodiment 26, wherein:
the glucosyl transferase has the amino acid sequence of SEQ ID NO: 4;
the oligosaccharyl transferase comprises the amino acid sequence of SEQ ID NO: 6, preferably SEQ ID NO: 6 comprising the amino acid mutation N31IV, more preferably SEQ ID NO:6 comprising the amino acid mutations Y77H and N31IV; 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.
[00238] 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 Exotoxin A of P. aeruginosa (EPA), E. coli flagellin (FliC), CRM197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of S. aureus, dumping factor A, dumping factor B, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Choiera toxin B subunit (CTB), choiera toxin, detoxified variants of choiera toxin, E. coli Sat protein, the passenger domain of E. coli Sat protein, Streptococcuspneumoniae Pneumolysin, Keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein oÎNeisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.
]00239] Embodiment 29 is the recombinant host cell of any one of embodiments 26-28, wherein the carrier protein is a detoxified exotoxin A of Pseudomonas aeruginosa (EPA). [00240] Embodiment 30 is the recombinant host cell of embodiment 29, wherein the EPA comprises 1-10, preferably 2-4, glycosylation sites each comprising a glycosylation consensus sequence having SEQ ID NO: 2.
[00241] Embodiment 31 is the recombinant host cell of embodiment 30, wherein the carrier protein is EPA with four glycosylation consensus sequences (EPA-4), preferably wherein the carrier protein comprises SEQ ID NO: 3.
[00242] Embodiment 32 is the recombinant host cell of any one of embodiments 26-31, which is an E. coli cell, e.g. an E. coli K-12 strain, such as strain W3110.
[00243] Embodiment 33 is a method of producing a bîoconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein, wherein the E. coli glucosylated 04 antigen polysaccharide comprises the structure of Formula (O4-Glc+) as shown in Table 1, wherein n is an integer of 1 to 100, preferably of 3 to 50, e.g. of 5 to 40, the method comprising culturing the recombinant host cell of any one of embodiments 26-32 under conditions for production of the bîoconjugate.
[00244] Embodiment 34 is the method of embodiment 33, further comprising isolating the bîoconjugate from the recombinant host cell.
[00245] Embodiment 35 is a bîoconjugate produced by the method of embodiment 33 or 34. [00246] Embodiment 36 is a composition comprising the bîoconjugate of embodiment 35. [00247] Embodiment 37 is a method for making a recombinant host cell for producing a bîoconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein, wherein the glucosylated 04 antigen polysaccharide comprises the structure of Formula (O4-Glc+) as shown in Table 1, wherein n is an integer of 1 to 100, preferably of 3 to 50, e.g. of 5 to 40, the method comprising introducing one or more recombinant nucleic acid molécules into a cell to produce the recombinant host cell, wherein the recombinant host cell comprises: (ï) a nucléotide sequence of an rfb gene cluster for the E. coli 04 antigen polysaccharide;
(ii) a nucléotide sequence encoding a glucosyl transferase having at least 80% sequence identity to SEQ ID NO: 4, wherein the glucosyl transferase is capable of modifying the E. coli 04 antigen polysaccharide to produce the E. coli glucosylated 04 antigen polysaccharide;
(iiî) nucléotide sequences encoding a translocase and a glycosyltransferase having at least 80% sequence identity to SEQ ID NOs: 7 and 8 respectively, wherein the translocase is capable of translocating bactoprenol-linked glucose and the glycosyltransferase is capable of glucosylating bactoprenol;
(iv) a nucléotide sequence encoding the carrier protein; and (v) a nucléotide sequence encoding an oligosaccharyl transferase capable of covalently linking the E. coli glucosylated 04 antigen polysaccharide to the carrier protein to produce the bîoconjugate.
[00248] Embodiment 38 is the method of embodiment 37, wherein:
the glucosyl transferase has SEQ ID NO: 4;
the oligosaccharyl transferase comprises the amino acid sequence of SEQ ID NO: 6, preferably SEQ ID NO: 6 comprising the amino acid mutation N31IV; 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.
[00249] Embodiment 39 is the method of embodiment 37 or embodiment 38, wherein the carrier protein is selected from the group consisting of detoxified Exotoxin A of P. aeruginosa (EPA), E. coli flagellin (FliC), CRM197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of S. aureus, dumping factor A, dumping factor B, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxîn, Choiera toxin B subunit (CTB), choiera toxin, detoxified variants of choiera toxin, E. coli Sat protein, the passenger domain of E. coli Sat protein, Streptococcus pneumoniae Pneumolysin, Keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.
[00250] Embodiment 40 is the method of any one of embodiments 37-39, wherein the carrier protein is a detoxified exotoxin A of Pseudomonas aeruginosa (EPA).
[00251] Embodiment 41 is the method of embodiment 40, wherein the EPA comprises 1-10, preferably 2-4, glycosylation sites each comprising a glycosylation consensus sequence having SEQ ID NO: 2.
[00252] Embodiment 42 is the method of embodiment 41, wherein the carrier protein is EPA with four glycosylation consensus sequences (EPA-4), preferably wherein the carrier protein comprises SEQ IDNO: 3.
[00253] Embodiment 43 is the method of any one of embodiments 37-42, wherein the cell is an E. coli cell, e.g. from an E. coli K12 strain, such as from a W3110 strain.
[00254] Embodiment 44 is a composition according to embodiment 19 or embodiment 20, wherein the bioconjugate of the O25B antigen polysaccharide is présent in the composition at a concentration that is about 1.5-6 times, e.g. about 2 to 4 times, higher than the concentration of any of the other bioconjugates.
]00255] Embodiment 45 is a composition according to embodiment 44, wherein the bioconjugates of OlA:O2;glucosylated O4:O6A:O8:O15:O16:O25B:O75 are présent in a ratio by weight of polysaccharide of 1:1:1:1:1:1:1:2:1, or 2:1:1:2:1:1:1:4:1.
[00256] Embodiment 46 is a composition according to embodiment 44, wherein the bioconjugates ofOlA:O2:glucosylated O4:O6A:O8:O15:O16:O18A:O25B:O75 are présent in a ratio by weight of polysaccharide of 1:1:1:1:1:1:1:1:2:1, or 2:1:1:2:1:1:1:1:4:1.
[00257] Embodiment 47 is a composition according to any one of embodiments 44 to 46, wherein a concentration of the bioconjugate of the O25B antigen polysaccharide is 2 to 50 pg/mL, preferably 8 to 40 pg/mL, e.g. 16-32 pg/mL.
EXAMPLES
[00258] The following examples of the invention are to further illustrate the nature of the invention. It should be understood that the following examples do not limit the invention and the scope of the invention is to be determined by the appended claims.
Example 1: Epidemiological data of E.coli infections
[00259] To détermine the O-serotype distribution of bacteremia-causing E. coli, global surveillance studies were performed. Between 2011 and 2017, more than 3200 E. coli bloodstream isolâtes were collected from patients >60 years of âge hospitalized in countries within North America, Europe, the Asia-Pacific région, and South America. Each strain was analyzed for O antigen serotype using classical agglutination techniques and sequence-based Ogenotyping. See Table 2.
[00260] Isolated human blood samples were analyzed to détermine the identity of pathogens therein and their antibiotic résistance patterns. E. coli isolâtes were obtained from the samples following the analysis. E. coli identity was verified by MALDI-TOF MS. Further analysis on the E. coli isolâtes was performed using an antisera-based agglutination assay to détermine their Oantigen serotype (DebRoy et al. (2011) Animal health research reviews / Conférence of Research Workers in Animal Diseases 12,169-185). Isolâtes un-typeable by the agglutination method, were further analyzed by whole-genome sequencîng followed by O-genotyping based on Oserotype spécifie wzy and wzx gene sequences.
[00261] Table 2: distribution of the most common bacteremia-associated E. coli O-serotypes from a collection of 3217 blood isolâtes collected globally between 2011 and 2017, based on Oserotyping by agglutination plus O-genotyping of isolâtes un-typeable by agglutination. Subjects were hospitalized in the following countries: USA, Canada, Argentina, Brazil, UK, Germany,
Spain, Italy, The Netherlands, France, Japan, Thailand, South Korea and Australia.
__O-serotype Prevalence n (%)
025 _______________737 (22.9%)________________
02 _______________268(8.3%)_______________
______ 06 261 (8.1%)
______ 01 ________________255 (7.9%)_________________
_________ 075 _________________145 (4.5%)_________________
______ 015 _______________110(3.4%)_______________
08 __________104 (3.2%)_________________
______ 016 103 (3.2%)
04 _________________96 (3.0%)__________________
_________________018_______________ ______________ 91 (2.8%) .
[00262] Stratification of on geographical location in the global set of bacteremia-associated E. coli showed a prevalence ofthe top 10 O-serotypes independent of location, suggesting these to be the prédominant O-serotypes globally associated with bacteremia-causing E. coli.
[00263] In the global set of bacteremia-associated multi-drug résistant E. coli isolâtes (n=345), i.e. those strains that are résistant to at least three classes of clinically relevant antimicrobîal drugs, the prevalence of the top 10 O-serotypes is 75.4%.
[00264] Ail information from epidemiology analysis taken together, the 10 prédominant Oserotypes could cover an estimated 60-80% of E. co/i-associated bacteremia infections, assumîng coverage of subportions of the un-typeable strains.
[00265] A multivalent vaccine covering a signifîcant proportion of bacteremia-causing E. coli serotypes would be very useful. The O-serotypes of Table 2 would thus be good candidates for an O-antigen based multivalent vaccine. Such a vaccine could beneficially be prepared using bioconjugation technology.
[00266] One of the serotypes in the top-10 (Table 2) is 04. It would thus be bénéficiai to préparé a bioconjugate vaccine that includes O-antigen polysaccharide of E. coli serotype 04 coupled to a carrier protein.
Example 2: Characterization of Contemporary 04 Clinical Isolâtes for Genes Encoding Oantigen Modifying Enzymes
[00267] Two variants of E. coli 04 antigen polysaccharide hâve been described (see, e.g. Jann B, et al., 1993, Carbohydr. Res. 248: 241-250), one having an unbranched structure (structure shown as (04-Glc-) in Table 1) and another variant substituted with an additional glucose sidebranch (structure shown as (O4-Glc+) in Table 1). The proportion in which these two variants are found in contemporary clinical isolâtes was not known. Although both variants react with 04 antisera, it was also not known whether immunological différences between these variants exist. Moreover, an enzyme responsible for attaching the glucose side-branch to generate the (04Glc+) antigen polysaccharide was hitherto not identified, and a putative coding sequence thereof is likely residing outside the 04 rfb gene cluster.
[00268] A set of 32 agglutination-confirmed E. coli 04 clinical isolâtes originally isolated during the period of 2011-2012 from subjects in the United States and the European Union were subjected to whole genome sequence analysis. Extracted rjb gene cluster sequences from the 32 sequenced 04 isolâtes were aligned with those ofthe reference strain and compared atthe nucléotide level. Except for some naturally occurring single nucléotide polymorphisms, the characterized isolâtes ail displayed an rjb cluster that was identical to the 04 reference strain, indicating that E. coli 04 strains, independent of their Glc-branching status, carry an identical rjb gene cluster. Thus, to generate the E. coli O4-Glc+ antigen polysaccharide, a gene with unknown sequence that encodes an E. coli O4-specific branching enzyme and that must résidé somewhere outside ofthe E. coli 04 rfb gene cluster is likely needed. The sequence ofthis unknown gene needs to be identified and employed if one wants to produce bioconjugates with the E. coli 04Glc+ antigen polysaccharides in a strain that would otherwise only produce bioconjugates with E. coli O4-GIc- antigen polysaccharides.
[00269] The whole-genome sequence data were then analyzed for the presence of genes outside ofthe ηβ gene cluster that may encode O-antigen modifying enzymes. Homologs of gtrAB in Shigellaflexneri were first identified in E. coli 04. An open reading frame downstream oigirAB in E. coli was then putatively identified as the E. coli 04-specific gene gtrS, that could encode the putative E. coli 04 spécifie branching enzyme GtrS responsible for adding a glucose branch to the E. coli 04 antigen.
[00270] The amino acid sequence of the E. coli 04 spécifie GtrS enzyme is provided as SEQ ID NO. 4. An exemplary nucleic acid sequence encoding this protein is provided as SEQ ID NO 5. *
[00271] Of the characterized E. coli 04 isolâtes, approximately 80% were found to carry the here identifîed gtrS gene (26 out of 32). Prevalence of the E. coli 04-specific gtrS sequence was also determined by PCR using sequence spécifie primers in an independent set of 20 agglutination-confirmed E. coli 04 clinical isolâtes isolated during the period of2014-2016 from subjects in the United States and the European Union. This analysis demonstrated that 17 out of 20 isolâtes carried the 04 gtrS sequence, which corresponds to a prevalence of 85%.
Exam£le 3: Cloning of 04 gtrS into E. coli W3U0, Production and structural confirmation of GIc-Modifîed 04 Bioconjugates
[00272] To test whether bioconjugates comprising 04-antigen polysaccharide modified with a branching glucose could be prepared, E. coli O4-antigen EPA bioconjugate production strains with the putative branching enzyme were constructed. For this, the endogenous O16-gtrS gene was substituted by the putative O4-gtrS gene (SEQ ID NO: 5, see Example 2) and the 016 rfi cluster was replaced with the 04 rfb cluster in E. coli strain W3110 SwzzE-wecG MvaaL EwbblJ-K by homologous recombination. Altematively, in some strains, the 04 rfb cluster was encoded on a plasmid.
[00273] Subsequently, plasmids encoding a detoxified exotoxin A of Pseudomonas aeruginosa (EPA) carrier protein (a variant either having 2 or 4 consensus glycosylation sites, referred to as ΈΡΑ-2’ and ΈΡΑ-4’, respectively), and oligosaccharyl transferase PglB were introduced into the strains. O4-EPA bioconjugates modified with Glc were produced by growing the E. coli production strains in bioreactor cultures, and induction of PglB and EPA expression by IPTG and arabinose, respectively. The O4-EPA bioconjugates were extracted from the biomass periplasmic extract.
[00274] To confïrm the detailed polysaccharide composition and linkage of the O4-EPA bioconjugates, multiple NMR experiments were performed on the bioconjugates having EPA-4 carrier protein (data not shown). The assignments obtained agreed with literature published (Jansson, P.E, et al, 1984, Carbohydr. Res. 134(2); 283-291; Jann B, et al, 1993, Carbohydr.
Res. 248: 241-250). The ID spectrum recorded at 313K showed a large HOD signal and small sharp signais from the 04 pentasaccharide RU with five anomeric, two NAc and two H6 signais (Rha and FucNAc).
[00275] The ID proton assignments were confïrmed by use of 2D proton-proton and protoncarbon corrélation NMR experiments. First, 2D TOCSY (120 ms) experiments demonstrated the expected cross peaks from H1 and H6 (for Rha and FucNAc) for the 04 pentasaccharide RU and small peaks from the terminal RU and EPA. In the methyl région, TOCSY showed cross peaks from H6 to H1 for α-Rha and H6 to H5 for α-FucNAc for the 04 RU. Other peaks observed were from EPA amino acids and terminal Rha (tRha). Second, a carbon NMR spectrum contained well-dispersed and diagnostic single peaks for the 04 RU. The carbons were profded indîrectly through the attached protons by use of the HSQC experiment. The HSQC-DEPT experiment gave inverted peaks for CH2 groups. The HSQC gave cross peaks for the 04 pentasaccharide RU [5 anomeric, ring, two N-acetyl and two methyl (Rha & FucNAc)] groups as well as EPA amino acids in characteristic régions. Each of theproton/carbon pairs for the 04 could be assigned based on the proton assignments and literature.
[00276] The structural characterization experiments thus confïrmed that Glc-branched 04 bioconjugates (comprising polysaccharide antigen structures as indicated by Formula (04-Glc+) in Table 1) could be produced, using the putative E. coli Μ-gtrS gene identified in Example 2.
Example 4: Immunogenicity of a Glc-Branched 04 Bioconjugate in Rabbits
[00277] Glc-modified 04 bioconjugates (i.e. having glycans with the structure of Formula (O4-Glc+) as shown in Table 1) were used for rabbit immunization by applying a speedy-rabbit protocol (Eurogentec). Sera from immunized rabbits were anaiyzed by ELISA for anti-04 IgG titers against purified 04 lipopolysaccharide (LPS) with (Glc+; i.e. containing glucosylated 04 polysaccharide) or without Glc-branching (Glc-; i.e. containing non-glucosylated 04 polysaccharide). Immunization with the bioconjugate resulted in high IgG titers in both rabbits (FIG. 1). In both cases, antibody titers induced by the 04 bioconjugate were higher against Glc+ LPS as compared to Glc- LPS.
[00278] Sera were also pooled and used in whole cell ELISA studies with test sets of E. coli 04 isolâtes with characterized gtrS status. Five gfnS’-ncgative (no Glc-branching) and six gtrS positive (Glc-branching) E. coli 04 isolâtes and a négative control strain were tested. Pooled sera from rabbits immunized with a Glc-modified 04 bioconjugate contained high titers of IgG specifîcally recognizing the tested 04 isolâtes (FIG. 2). In concordance with the LPS ELISA, ail tested 04 isolâtes were recognized by the immune sera. The gtoS-positivc isolâtes displayed an overall higher binding than the g/rYnegative isolâtes (FIG. 2). In particular, the following isolâtes were g/nYpositive: Y1382, E551, OC24334, stGVXN4983, stGVXN4994 and 0C24794, and the following isolâtes were g/rS-negative: A2625, stGVXN4988, OC24784, 0C24787, and OC24788. Immune sera did not bind the négative control strain of a non-related O-serotype, E. coli OC9487 (ATCC 35383).
[00279] The profiles of LPS extracted from the test set of ^-positive and -négative isolâtes in silver-stained polyacrylatnide gels did not reveal marked différences between isolâtes expressing unmodifîed and modified forms of the 04 antigen confirming that the observed différences are not explained by quantitative différences in LPS expression levels (data not shown).
[00280] Western blots of extracted LPS using pooled immune sera were performed to assess récognition of 04 O-antigen by IgGs elicited in response to immunization with a Glc-modified 04 bioconjugate. Binding of both modified and unmodifîed 04 LPS by IgGs from modified 04 immunized rabbits was observed and included spécifie récognition of LPS bands spanning a wide range of sizes, including high molecular weight LPS bands (FIG. 3).
[00281] In the further experiments below, when reference is made to Ό4’ bioconjugate or production strains or ‘EcoO4’, the bioconjugate or production strain of Glc-branched 04 (having glycan structure (O4-Glc+) in Table 1) is meant, unless specifîcally indicated otherwise (the terms ‘04’ and O4-Glc+’ are thus used interchangeably for bioconjugates or production strains in those experiments).
Example 5: Immunogenicity of a Glc-Branched 04 Bioconjugate in Rats
[00282] Sprague Dawley rats were immunized intramuscularly 3 times with formulation buffer or (O4-GIc+)-EPA bioconjugate (i.e. bioconjugate of glucosylated 04 antigen polysaccharide covalently coupled to EPA carrier protein; carrier protein was EPA-2 as described in Example 3 above) at 3 different doses (0.04 gg, 0.40 gg or 4.0 gg). Sérum antibody levels were measured by ELISA at day 0, 14 and 42 post-immunization.
[00283] Immunization with 0.04 gg, 0.40 gg and 4.00 gg of (O4-Glc+)-EPA bioconjugate induced significant increase in the levels of IgG antibodies at day 42 post-immunization when compared to formulation buffer (FIG. 4A). The antibodies induced by (O4-Glc+)-conjugate were functional, i.e., capable of mediating killing of (O4-Glc+) E. coli strain (FIG. 4B).
[00284] Antibody levels induced by 0.04 gg, 0.40 gg and 4.0 gg of (O4-Glc+)-EPA bioconjugate were significantly increased at day 42 as compared to those detected at baseline (day 42 vs day 0, P = 0.006 for ail doses) and at day 14 post-immunization (day 42 vs day 14, P 0.006 for ali doses) (FIG. 5). In the group that received 4.0 gg of bioconjugate, titers were also significantly increased at day 14 compared to day 0, indicating that a single dose of 4.0 gg of (O4-Glc+)-EPA bioconjugate induces significant increase in IgG titers (day 14 vs day 0, P=0.012). The significant increase in IgG titers observed between day 14 and 42, for ali three concentrations of bioconjugate tested showed that a third dose of (O4-Glc+)-EPA bioconjugate is able to boost antibody responses (FIG. 5).
[00285] Functionality of antibodies induced by O4-Glc+-EPA conjugate in the rats immunized intramuscularly 3 times with formulation buffer or the bioconjugate at 4.00 gg/dose was determined by opsonophagocytic killing assay (OPKA) using O4(Glu+) and O4(Glu-) E. coli strains. The antibodies induced by (O4-Glc+)-EPA bioconjugate were functional, i.e., capable of mediating killing of an (O4-GIc+) E. coli strain (FIG. 4B, FIG. 6). Notably, antibodies induced by (O4-Glc+)-EPA bioconjugate were capable of mediating killing of both (O4-Glc+) and (04Glc-, i.e. having glycans with structure of Formula (04-Glc-) in Table 1, i.e. 04 polysaccharide without Glc-branching) E, coli strains (FIG. 6).
[00286] In conclusion, antibodies induced by O4-Glc+-EPA bioconjugate are cross-reactive and capable of mediating killing of E. coli 04 strains with and without glucose branching.
Example 6: Production Strains for E. coli O-antigen Bioconjugates and resulting Bioconjugate products
[00287] In addition to (O4-Glc+)-EPA bioconjugates prepared as described above, nine (9) other bioconjugates were produced. In particular, the additionally produced bioconjugates included E. coli O1A-EPA bioconjugate, 02-EPA bioconjugate, 06A-EPA bioconjugate, 08EPA bioconjugate, 015-EPA bioconjugate, 016-EPA bioconjugate, 018A-EPA bioconjugate, O25B-EPA bioconjugate, and 075-EPA bioconjugate. The Chemical structures of the glycans of these conjugales can be seen in the respective Formulas in Table 1. A composition comprising the 10 bioconjugates is referred to herein as ‘ExPEClOV’. A composition comprising the O1AEPA, 02-EPA, O6A-EPA and O25B-EPA bioconjugates is referred to as ‘ExPEC4V’ (and was previously described in for instance WO 2015/124769 and WO 2017/035181).
[00288] Escherichia coli W3110 Parental Strain
[00289] The non-pathogenic E. coli K12 strain W3110 was used as the parental strain for the construction of ail ten production strains. The E. coli K12 strain W3110 was obtained from the Coli Genetic Stock Center (Yale University, New Haven (CT), USA, product number CGSC#4474). Its relevant génotype was previously described (E. coli W3110, F-, lambda-, IN(rmD-rmE)l, rph-1) and its genomic sequence was previously published (Hayashi K, et al., 2006, Mol. Syst. Biol. 2006.0007 (doi:10.1038/msb4100049). The E. coli W3110 strain was genetically modified to enable production of each of the E. coli O-antigen bioconjugates (Table 3)·
[00290] Bioconjugate production strains
[00291] The “ExPEC4V” and “ExPEClOV” compositions both comprise the 02-EPA and O25B-EPA bioconjugates from the sanie production strains. The “ExPEC4V” composition comprises the O1A-EPA bioconjugate from the stGVXN4411 or stLMTB 10217 production strains, while the “ExPEClOV” composition comprises the O1A-EPA bioconjugate from the stLMTB 10217 production strain. The “ExPEC4V” composition comprises the O6A-EPA bioconjugate from the stGVXN4112 production strain, while the “ExPEClOV” composition comprises the O6A-EPA bioconjugate from the stLMTB10923 production strain. Furthermore, the “ExPEClOV” composition comprises the O4-EPA (i.e. (O4-Glc+)-EPA), O8-EPA, 015-EPA, 016-EPA, O18A-EPA, and 075-EPA bioconjugates from production strains that are not used for ExPEC4V . Different production strains could vary in the plasmids for expression of the EPA carrier protein and/or the oligosaccharyl transferase PglB, as indicated below. An overview of several production strains is given in Table 3 below.
Table3: OverView of genetic engineering of E. coli production strains for O-antigen bioconjugates for ExPEC4V and ExPEClOV vaccine compositions
Serotype Strain name Genomic mutations Plasmids
r/bgene duster waaL gtrABS pglB epa
O1A (ExPEC4V) stGVXN4411 brfb;:01Arfb upecGVXN_032 AwoaL - PGVXN970 PGVXN1076
O1A (ExPEC4V; ExPEClOV) StLMTB10217 ArftirOlA rfb upecGVXN_032 AwaaL - PGVXN1221 PGVXN1076
02 stGVXN4906 àrfb;;02 rfb upecGVXN_116 AwaaL PGVXN971 PGVXN1076
04 BVEC-L-00684 Ar/ü::04 rfb CCUG11450 AwaaL &gtrS::gtrS 04 PGVXN1217 PGVXN1076
06A (ExPEC4V) stGVXN4112 Ar/b::06A rfb CCUG11309 AwaaL - PGVXN114 PGVXN659
06A (ExPEClOV) stLMTB10923 Az/b06A rfb CCUG11309 AwaaL - PGVXN1221 PGVXN1076
08 stLMTB11734 &rfb08 rfb E2420 AwaaL AgtrABS PGVXN970 PGVXN1076
015 stLMTB11738 Arfi):;015 rfb 0C24891 Awooi, AgtrABS PGVXN1221 PGVXN1076
016 stLMTB11739 &rfb;:01B rfb OC24208 AwaaL ^gtrABS PGVXN2381 PGVXN1076
018A BVEC-L-00559 Ar/8O18A rfb OC24255 àwaaL AgtrABS PGVXN970 PGVXN1076
O25B stGVXN4459 Ai/b::O25B rfb upecGVXN_138 AwaaL AgtrABS PGVXN970 PGVXN1076
075 stLMTB11737 Ar/&::075 rfb CCUG31 &waaL AgtrABS __ PGVXN1217 pGVXN1076
[00292] O-antigen Biosynthesis (rfb} Gene Cluster
[00293] In ail E. coli O-antigen production strains, the naturally occurring E. coli W3110 genomic O16::IS5 -antigen biosynthesis (rfb) gene cluster was replaced by the selected Oantigen-specific biosynthesis clusters from E. coli strains ofthe selected serotype, encoding for the serotype-specific O-antigen structures (see Table 1 for these O-antigen structures). The ten donor rfb clusters were selected or confirmed after whole-genome analysis of E. coli blood isolâtes. Replacement ofthe W3110 O16::IS5 rfi gene cluster, which is defective in O-antigen biosynthesis, has been achieved in a single homologous recombination event. In case ofthe 016 and O18A rfb gene clusters, the donor DNA recombined via the flanking gVand rmlCA genes, while the rfb gene cluster for the other strains recombined via the flanking gnd and galF genes. Sequences of the rfb clusters in the production strains are provided in SEQ ID NOs: 9 and 11-19.
[00294] O-antigen ligase (waaL} gene
[00295] Ail E. coli O-antigen production strains carry an artificially introduced délétion ofthe E. coli W3110 genomic O-antigen ligase encoded by the waaL gene. In the SwaaL strains the transfer ofthe O-antigen to lipid A is disrupted, which instead directs transfer ofthe O-antigen to the carrier protein to increase product yield.
[00296] O-antigen glucosylation (gtrABS) genes
[00297] In the E. coli 08, 015, 016, O18A, O25B, and 075 production strains the E. coli W3110 genomic gtrABS genes, which are responsible for 016 O-antigen glucosylation, hâve been deleted. While the gtrA and gtrB genes in different serotypes are highly homologous and interchangeable, the gtrS gene encodes a serotype-specific O-antigen glycosyl transferase. In E. coli W3110 GtrS can transfer a glucose (Glc) residue to the GlcNAc sugar in the a-L-Rha(1^3)-d-G1cNAc motif ofthe E. coli 016 O-antigen. In the E. coli O1A, 02 and O6A production strains no délétion or replacement of the gtrABS gene has occurred. These O-antigens miss the a-L-Rha-(l-G)-D-GlcNAc motif that is the natural substrate for E. coli 016 gtrS. In the E. coli 04 production strain, the W3110 gtrS gene has been replaced with the E. coli 04 gtrS gene to accommodate proper glucosylation ofthe E. coli 04 O-antigen.
[00298] Oligosaccharyl transferase PglB
[00299] Ail E. coli O-antigen production strains expressed a variant of the C. jejuni glycosyl transferase PglB, which can transfer the O-antigen onto an amino acid consensus sequence on a carrier protein by V-glycosylation. PglB has broad substrate récognition, but due to lowproduct yields several production strains were prepared expressing a PglB variant having modified substrate specifîcities, which resulted in improved product yield (see e.g. WO 2016/107818, WO 2016/107819). The^g/R gene was placed behind an Isopropyl β-D-l-thiogaIactopyranoside (IPTG) inducible promoter on a plasmid. Table 4 below lists the PglB variants encoded by the plasmids used for production of the E. coli O-antigen production strains for the bioconjugates for the ExPEC4V and ExPEClOV compositions described above. Further plasmids with variation in vector backbone, antibiotic résistance marker, and/or alternative PglB variants hâve also been tested successfiilly for bîoconjugate production.
[00300] Table 4: PglB and EPA plasmids used in E. coli O-antigen Production Strains
Plasmid name Gene Description1
pGVXN114 PglB C. jejuni codon usage; SpR
pGVXN970 PglB E. coli codon usage optimized; SpR
pGVXN971 pglB^3^ E. coli codon usage optimized; The natural glycosylation site of PglB was inactivated; SpR
pGVXN1217 pglB^^^’ E. coli codon usage optimized; Substrate optimized PglB; SpR
pGVXN1221 PglB™1 V,K482R,D483H,A669V E. coli codon usage optimized; Substrate optimized PglB; SpR
pGVXN2381 ^g/£Y77H,S80R,Q287P,K289R,N311V E. coli codon usage optimized; Substrate optimized PglB; SpR
pGVXN659 JEPA-4 EPA with four bioconjugation sites; AmpR
pGVXN1076 EPA-4 EPA with four bioconjugation sites; KanR
SpR, spectinomycin résistant; AmpR, ampicill in résistant; KanR, kanamycin résistant
[00301] Carrier protein (EPA)
[00302] Ail E. coli O-antigen production strains expressed a genetically detoxified P. aeruginosa ADP-ribosyltransferase toxoid (EPA) as a carrier protein for the O-antigen. The EPA toxoid differs from wild-type EPA toxin in two residues: Leu552 was changed to Val and Glu553 (in the catalytîc domain) was deleted. Glu553 délétions were reported to significantly reduce toxicity. In addition to the détoxifîcation mutation, four (EPA-4) consensus Nglycosylation site motifs were introduced. The epa gene was placed behind a L-Arabinose (Ara) inducible promoter on a plasmid (Table 4). Table 4 is limited to the plasmids used in production strains for bioconjugates used in the “ExPEC4V” and “ExPEClOV” compositions described above, Plasmids with variation in vector backbone, antibiotic résistance marker, and/or EPA variants, e.g. varying in the number of consensus TV-glycosylation site motifs (e.g. having two such motifs, EPA-2), hâve also been tested successfully for bioconjugate production.
Example 7: Optimizing the oligosaccharyltransferase for génération of bioconjugates with glucosylated 04 (O4-Glc+) antigen
[00303] Yield optimization for bioconjugate production can be achieved by modification of the C.jejuni oligosaccharyl transferase PglB, which can lead to a more efficient or higher degree of .V-glycosylation of the O-antigen of interest to the EPA carrier protein. In an E. coli strain for production of bioconjugate with glucosylated 04 (04-Glc+) O-antigen polysaccharide, such optimization strategy was applied and resulted in an (O4-Glc+)-specific optimized PglB variant improving bioconjugate product yield.
[00304] In this approach, an O4-Glc+ O-antigen polysaccharide producing strain containing an EPA-expression plasmid was transformed with a variety of different PglB expression plasmids, each of which contained different amino acid substitutions in the PglB protein, altering substrate specificity. Bioconjugate production level and profile of each strain was assessed at shake-flask level in osmotic shock experiments, and readout was performed by capillary electrophoresis immunoassays on the periplasmic extract using O4-Glc+ -spécifie monoclonal antibodies.
[00305] One of the tested PglB variants containing an N31IV amino acid substitution was found to improve product yield of glucosylated 04 bioconjugates significantly (FIG. 7A). [00306] In a further improvement where the N31IV PglB-variant was further modified, an Y77H amino acid substitution further enhanced O4-Glc+-specific product yield and showed an increased degree of di-and tri-glycosylated product compared to the N31IV PglB-variant, where other modifications were found to be neutral or had a négative effect on product yield (FIG. 7B).
Plasmid pLMTB4008 (SpR) encodes E. coli codon usage optimized, (O4-Glc+)-substrate optimized, PglB variant with mutations Y77H and N31IV.
[00307] The PglB variant with optimized substrate specificity for O4-Glc+ O-antigen polysaccharide, containing N311V and Y77H amino acid substitutions relative to wild-type (wt) C.jejuni glycosyl transferase PglB, was found to double bioconjugate yield compared to the first round optimized PglB-N31 IV variant.
[00308] Similarly using screens, the most optimal yielding PglB variants were also determined for E. coli O-antigen bioconjugate production of the of the other nine serotypes in the ExPEC 10V composition.
[00309] For bioconjugates having the 01 A, 06A, or 015 antigen polysaccharide, PglB with amino acid mutations N311V, K482R, D483H, and A669V was found to give the highest yields. [00310] For bioconjugates having the 02, 08, 018A, or O25B antigen polysaccharide, wildtype PglB (i.e. not having amino acid mutations at positions 77, 80, 287, 289, 311, 482,483 and 669) was found to give the highest yields.
[00311] For bioconjugates having the 016 antigen polysaccharide, PglB with amino acid mutations Y77H, S80R, Q287P, K289R, and N311V was found to give the highest yields. [00312] For bioconjugates having the 075 antigen polysaccharide, PglB with amino acid mutation N31IV was found to give the highest yields.
[00313] It can be seen from these results that the optimal PglB variant is different for different O-antigens, and that the optimal PglB variant for producing a bioconjugate with a given Oantigen polysaccharide is unpredictable.
Example 8: Bioconjugates of O-antigens from 10 E. coli serotypes and their quality attributes
[00314] O-glycan residues ofthe target O-antigens are structurally diverse and hâve variable repeating units. The specificity and affinity ofthe glycosyl transferase PglB is linked to the glycan structure. Thus, making a bioconjugate that has the desired quality attributes, e.g., purity, glycan/protein ratio, etc., is a challenging, non-straightforward, task. The right combination of PglB and EPA carrier protein détermines the yield and may influence glycosylation efficiency. By optimizing the PglB and carrier proteins, bioconjugates having the desired quality attributes were produced. It may be also important to maintain a lower threshold value of total carrier protein, particularly when one or more O-antigen bioconjugates are combined together and administered in a single composition or vaccine, because very high amounts of carrier protein may lead to immunological interférence. In order to avoid such a phenomenon, conjugates having a higher glycan/protein ratio are preferred. Hence, for ExPEC 10V vaccine, bioconjugates with at least comparable (to the previously described ExPEC4V vaccine that has been subject to clinical trials) glycosylation ratio were developed.
[00315] The bioconjugates were each produced by culturing the respective host cells (Example 6, Table 3) in bioreactors (10L and/or 200L volumes) and expression ofthe bioconjugates, following methods previously described. Each drug substance was manufactured batch-wise by bacterial fed-batch fermentation to generate biomass containing the expressed bioconjugates of the corresponding polysaccharide serotype. Cells were cultured and induced with IPTG and arabinose. The bioconjugates were isolated from the periplasm of the cells in the bioreactor cultures by osmotic shock followed by chromatographie purification. This process was performed for each of the 10 bioconjugates.
[00316] The E. coli O-antigen bioconjugates thus prepared that are drug substances (DS s) for ExPEC 10V and ExPEC4V showed comparable critical quality attributes: (1) process-related purity (measured by RP-HPLC) was higher than 95%, (2) polysaccharide/protein ratio ranged between about 0.1-0.5, mostly between 0.15 and 0.45, (3) bacterial endotoxin (Ph. Eur. 2.2.3) was less than 0.5 EU/pg polysaccharide. The average length ofthe individual polysaccharide chains was typically between about 10-20 repeating units (measured using high resolution SDSPAGE).
[00317] The structures of the polysaccharide repeat units were confirmed (by NMR and MS/MS of the conjugates, intact or trypsin-digested) to be the ones shown in the Formulas for the corresponding serotypes in Table 1, for ail ten bioconjugates that are DSs for the ExPEC 10V composition described above.
[00318] The 018 serotype had the lowest yields of bioconjugate production amongst the ten serotypes of which bioconjugates were made for the ExPEC 10V composition.
[00319] ExPEC 10V drug product (DP) comprises a mixture of the ten monovalent DSs described above.
Example 9: Toxicology of ExPEClOV vaccine
[00320] A single-dose pilot toxicity and local tolérance study (non-GLP) with ExPEClOV was conducted in female NZW rabbits. One group (n=2) received an intramuscular (IM) injection (on Day 0) of the control (saline), and a second group (n=4) received an IM injection of ExPEClOV at 105.6 pg total polysaccharide (PS)/dose (9.6: 9.6: 9.6: 9.6: 9.6: 9.6: 9.6: 9.6: 19.2: 9.6 pg PS per dose, for respectively O-serotypes 01 A, 02, 04, O6A, 08, 015, 016, O18A, O25B and 075) using a dosing volume of 0.6 mL (176 pg PS/mL). Necropsy was performed on Day 2. [00321] There were no mortalities observed. In addition, there were no vaccine-related effects noted for clinical observations (including injection site effects using Draize scoring), body weight, food consomption, and body température. Histopathologically, there were no vaccinerelated changes observed at the administration site or draining (iliac) lymph node. A minimal increase in germinal center formation in the spleen was observed in one out of four treated animais (Day 2), and was considered a normal, immunological response to the injected vaccine. Overall, the administration of a single IM dose of ExPEClOV to female rabbits was welltolerated.
Example 10: Immunogenicity of ExPEClOV blended formulation in rabbits
[00322] An ExPEC4V vaccine (comprising bioconjugates of £ .coli 01 A, 02, O6A, and O25B serotypes) has previously been shown to be immunogenic for these four serotypes in rats, rabbits, and humans (see e.g. 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). The novel bioconjugates of the invention having the E. coli glucosylated 04 serotype were shown to be immunogenic in Examples 4 and 5 above. Immunogenicity of the bioconjugates of E. coli serotypes 08, 015, 016, O18A, and 075 (ail having EPA-2 as carrier protein in this experiment) when separately administered (monovalent) to rats confirmed that also each of these bioconjugates was immunogenic, since ELISA data indicated that each of these bioconjugates could elicit high levels of E. coli O-antigen spécifie antibodies (not shown).
[00323] Immunogenicity of the 10-valent vaccine that contained a mixture of the 10 bioconjugates as described above was also tested. New Zealand White (NZW) rabbits (female, 12-16 weeks old) received 3 intramuscular immunizations with ExPEClOV or saline administered 2 weeks apart (Table 5; administration at days 0, 14, and 27). The 10 polysaccharides that are part of the ExPEClOV vaccine used in these experiments were conjugated to the carrier protein EPA containing 4 sites of glycosylation (EPA-4). The vaccine was formulated in 3 different doses: Group 1 (‘high dose’): 8 ug/dose of O1A, 02, O6A, 04, 08, 015, 016, 018 and 075 and 16 ug/dose of O25B; Group 2 (‘medium dose’): 4 ug/dose of 02, 04, 08, 015, 016, 018 and 075, 8 ug/dose of 01A and 06A and 16 ug/dose of O25B; Group 3 (‘low dose’): 0.4 ug/dose of 02, 04, 08, 015, 016, 018 and 075, 0.8 ug/dose of 01A and O6A and 1.6 ug/dose of O25B. Animais from the control group (Group 4) received only saline (0.9% (w/v) sodium chloride solution) (Table 5).
[00324] Antibody responses were evaluated at day 0 (pre-immunization) and days 14, 27 and 42 post-immunization. Sérum antibody levels induced by each of the bioconjugates included in the vaccine and the carrier protein EPA were measured by ELISA (total IgG), using type-specifîc LPS as coating material. The antibody titers were reported as EC50 values that correspond to the half maximal effective concentration based on duplicates of 12-step titration curves plotted in a 4-parameter logistic nonlinear régression model. Functional activity was determined by OPK.
Table 5. Description of experimental groups.
Experimental groups Dosing (pg/PS) O1A:O2:O6A:O25B:O4:O8:O15:O16:O18A:O75 Sam pie 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 (low dose) 0.8:0.4:0.8:1.6:0.4:0.4:0.4:0.4:0.4:0.4 7
Group 4 (control) 0.9% (w/v) sodium chloride solution 7
[00325]
Results are shown in FIG. 8 and summarized in Table 6.
[00326] Table 6. Summary of E. coli O-antigcn spécifie antibody responses induced by ExPEClOV inNZW rabbits.
ExPEClOV __________________________Antibody responses d ay 14 post-vaccination 1
dose OlA 02 06A O25B 04 08 015* 016 018A 1 075
High * · ·:** · · : .. ** . . ** - ·.· ** . . : ** *
Mid ** : • ··** · : ** · .. HuH :
Low ··· ·. *.-·. . * ··: . - *. . · * î Hk : .¾¾..
ExPEClOV dose . _ . . -_____________________Antibody responses day 27 post-vaccination 1
OlA 02 06A O25B 04 08 015* 016 018A 075
High ** ** ·. ** . ; ' ** ' · . * : . ** ** **
Mid . ** . ** : c ** · . ·· . .... ·- ** · . - *
Low ' ** ·. · ·. * * . c· ** ·· : ** ';· ·' · ** . ' ' ’· ?
ExPEClOV dose ; OlA i 02 i 06A intibody responses day 42 post-vaccinatio O25B 04 i___08 ί 015* n 016 018A 075
H'Sh ** *+ ·· . ** ' ! ** . ·. ' ** .. ·'** ' ' ; ** **
Mid . ** ** .. ** ' ** ; ** • **
Low ** - - ** ' % : · ** .. .· . · * * ' ** ; ' ' ' ** · ; **
Dark gray squares show serotype-specific antibody responses in which p values were statistically significant. Light gray squares show serotype-specific antibody responses in which p values were not statistically significant (ns). Wilcoxon Rank Sum test with Bonferroni correction for multiple comparisons. Comparisons ExPEClOV vaccinated animais (Group 1,2 and 3) versus saline control (Group 4). *p<0.05, **p<0.01.# P values were statistically significant after excluding an outlier animal from the control group (sensitivity analysis).
[00327] The high dose of ExPEClOV (Group 1) induced signiflcantly higher IgG antibody levels at ail time-points investigated (Days 14, 27 and 42 post-immunization) when compared to saline control for OlA, 02, 04, O6A, 016, 018A and O25B (FIG. 8, Table 6). Signiflcantly higher antibody titers induced by 08 and 075 conjugates when compared to saline control were observed at Days 27 and 42 post-immunization (FIG. 8, Table 6).
[00328] The medium dose of ExPEClOV (Group 2) and the low dose (Group 3) induced significantly higher antibody levels at ail time-points investigated (Days 14, 27 and 42 postimmunization) when compared to saline control for OlA, 02, 04, 06A, 016 and O25B (FIG. 8, Table 6). Signiflcantly higher antibody titers induced by 08, O18A and 075 conjugates when compared to saline control were observed at Days 27 and 42 post-immunization suggesting that the boost dose in rabbits increases the response to these O-serotypes (FIG. 8, Table 6).
[00329] For 015 conjugates, sensitivity analysis omitting an outlier animal from the control group showed that ail three doses of ExPEClOV vaccine induced a significant increase in antibody responses when compared to saline control at Days 14,27 and 42 post-immunization (FIG. 8, Table 6).
[00330] Antibodies induced by the carrier protein EPA were signifïcantly higher than EPA antibody titers in the saline-treated (control) group for the three doses of ExPEClOV tested (high, medium and low) at ail time points investigated (Days 14, 27 and 42) (FIG. 8).
[00331] Between dose comparisons (not shown) showed that at Day 14 post-vaccination, the high dose of ExPEClOV induced signifïcantly higher antibody responses when compared to the low dose for most of the conjugates tested (O1A, 02, 04, O6A, 015, 016, O18A and O25B). The medium dose of ExPEClOV also induced signifïcantly higher antibody responses compared to the low dose for 01 A, 02, 04, 018A, O25B and 075. For 08 conjugate, ail three formulations of ExPEClOV induced similar levels of antibodies at Day 14 post-vaccinai ion.
[00332] The low dose of ExPEClOV induced a significant increase in antibody responses at Day 42 post vaccination (after a prime and two boost doses) when compared to the high and medium doses of ExPEClOV for 01A, 02, 04, 016, O25B and 075 conjugates. These findings are in line with other expériences with conjugate vaccines, where for instance no clear relationship between dose and the magnitude of the antibody response to primary vaccination was observed in infants vaccinated with pneumococcal conjugate vaccine (Poolman JT, et al. Expert Rev Vaccines. 2013, 12(12):1379-94).
[00333] There were no significant différences between the three doses of ExPEClOV tested at Day 42 post-vaccination for 06A, 08 and 015 conjugates. For the O18A conjugate, the high dose of ExPEClOV induced a signifïcantly higher antibody response when compared to the medium dose at Day 42 post-vaccination.
[00334] For the carrier protein (EPA), the high and medium dose of ExPEC 1OV induced signifïcantly higher antibody responses when compared to the low dose at day 14 postvaccination. The high dose of the vaccine also induced signifïcantly higher antibody responses when compared to the low dose at day 42 post-vaccination.
[00335] In conclusion, the three formulations of ExPEClOV (high, medium and low), administered via intramuscular injection on Days 0,14, 27 are immunogenic in rabbits.
[00336] So far, functional antibodies capable of killing E.coli strains induced by this vaccine in rabbits were shown for serotypes 01 A, 02, 04, O6A, 015,016 and O25B.
[00337] In a further experiment, a GMP batch of the ExPEC 1OV vaccine (see Example 8 above for production) was prepared and injected into NZW rabbits as part of a toxicology study (Table 7). In this study, NZW rabbits (males and females) received 3 intramuscular injections (0.6 mL) of the ExPEClOV vaccine (day 1, 15 and 29) and a control group received 0.9% (w/v) sodium chloride solution (saline). Each dose of the vaccine contained 9.6 pg polysaccharide (PS) for serotypes O1A, 02, 04, O6A, 08, 015, 016, O18A and 075 and 19.2 pg PS for serotypes O25B, corresponding to 105.6 pg total PS (176 pg total PS/mL) and 382.8 pg of total EPA (638 pg EPA/mL). IgG titers against O-antigens and carrier protein (EPA) were determined from samples collected during the pre-treatment period (day 1) and days 31 and 50 post-immunization. [00338] A significant increase in antibody responses against ail O-antigens and the carrier protein EPA were observed at day 31 and 50 post-vaccination in the group that received ExPEClOV when compared to the control group that received only saline (Fig. 9, Table 8). For 01A serotype, a significantly higher antibody response was also observed at day 1 (baseline) when vaccinated animais were compared with the Controls. These results suggest that some animais were pre-exposed to E. coli or hâve antibodies that cross-react with OlA-LPS.
[00339] Table 7. Experimental groups and ExPEC 1 OV dose used in NZW rabbits.
Groups Treatment Dose Dosing days Main (day 31) (males/females) Recovery (day 50) (males/females)
1 control 0 1, 15, 29 10 10
2 ExPEClOV 105.6 pg PS* 1,15,29 10 10
*Each dose (0.6 mL dosing volume) contains 9.6:9.6:9.6:9.6:9.6:9.6:9.6:9.6:19.2:9.6 pg polysaccharide (PS) for serotypes O1A, 02, 04, O6A, 08, 015, 016, O18A, O25B, 075, respectively (176 pg total PS/mL). Each dose contains 382.8 pg EPA protein (638 pg EPA/mL).
Table 8. Immunogencity of ExPEClOV in NZW rabbits as part to a toxicology study.
Treatment Antibody responses day 31 post-vacci nation _____
ExPEClOV O1A 02 O6A O25B 04 08 015 016 O18A 075
Day 31 -(1 J ] F** [ # j **** **** **** ’
Day 50 *·**·*··': W* 1 L A <-J 1***'*\ 1 **** __________________________________________________________________________________________________________________________________________1 ______________________________________________________________________________________________________________________________________________________i
Antibody responses induced by ExPEClOV. Light gray squares show serotypes in which a significant increase in antibody responses was observed in the vaccine group compared to control. Tobit model with a likelihood ratio test. ****P< 0.0001.
Example 11: Phase l/2a trial with the ExPEClOV vaccine in hnmans
[00340] At présent, there is no vaccine available to prevent IED, The serotypes comprising the ExPEClOV vaccine (O1A, 02, 04, O6A, 08, 015, 016, O18A, O25B and 075) were selected to address invasive disease caused by the majority of clinically relevant ExPEC strains that also representthe majority of ExPEC isolâtes causing antimicrobial résistant IED, including ST 131. The selected serotypes are représentative for the ten prévalent ExPEC O-serotypes causing bloodstream infections in the older population and responsible for approximately 70% of bloodstream infections caused by ExPEC.
[00341] Since the mechanism of action of conjugate vaccines in the prévention of invasive disease is not expected to be affected by antibiotic résistance mechanisms, it is believed that ExPEClOV vaccine provides protection against IED caused by drug-resistant- and drugsusceptible O1A, 02, 04, O6A, 08, 015, 016, O18A, O25B and 075 serotypes.
[00342] There is preceding clinical expérience with ExPEC4V, an earlier vaccine candidate which comprised a subset of four of the E. coli O-antigen conjugales (01 A, 02, O6A and O25B) also found in ExPEC 1OV. Based on the results from four clinical studies (two completed phase 1 studies, one completed phase 2 study and an ongoing phase 2 study), ExPEC4V was well-tolerated by the study participants and no vaccine-related safety signais were observed at doses up to 16 pg polysaccharide (PS) per serotype (01 A, 02, O6A and O25B). Most adverse events (AEs) were Grade 1 and 2, very few Grade 3 AEs were reported. Late-onset solicited local AEs (AEs which start after Day 5 post-vaccination) were observed mainly with the higher doses of ExPEC4V. In each study, the ExPEC4V vaccine was shown to be immunogenic, demonstrating a dose-dependent vaccine immune response, and O-antigen spécifie Immunoglobulin G (IgG) titer increases, as measured by enzyme-linked immunosorbent assay (ELISA). Functional activity of the antibodies was demonstrated with an ExPEC4V-optimized opsonophagocytic killing assay (OPKA). Co-analysis of ELISA and OPKA test results showed corrélation between the assay responses (Pearson corrélation coefficients >0.61 and >0.48 for Day 30 and Day 360, respectively in a Phase 2 clinical trial [study 4V-BAC2001]), substantiating the use of ELISA as a primary measure of ExPEC4V antibody titers and to predict functional antibody activity. Analysis ofthe immunogenicity data has demonstrated the durability of the immune response through three years after vaccination with ExPEC4V. It has now also been observed that sera from humans vaccinated with ExPEC4V and that had high titers of serotype-specific opsonophagocytic antibodies, when passively transfered into mice that were subsequently intraperitoneally challenged with E. coli strains of O25B or 02 serotype, were able to médiate protection in vivo (not shown). Hence, ExPEC4V-specîfic opsonophagocytic human antibodies médiate bacterial killing in vivo, which is in line with other conjugate vaccines in which the proposed mechanîsm of protection is by induction of opsonophagocytic antibodies that médiate bacterial killing.
[00343] ExPEClOV includes a total of ten serotypes and increases coverage from about 50% (ExPEC4V) to approximately 70% of bloodstream infections caused by ExPEC in adults aged 60 years and older. Based on the clinical expérience with ExPEC4V, and on the pre-clinical data for ExPEClOV as discussed in the examples above, it is expected that administration of ExPEClOV will induce immune responses to E. coli serotypes OlA, 02, 04, O6A, 08, 015, 016, O18A, O25B and 075 also in humans.
[00344] A randomized, observer-blind, first-in-human phase l/2a study to evaluate the safety, I reactogenicity, and immunogenicity of three different doses of the ExPEClOV vaccine is conducted in humans aged 60 to 85 years in stable health (study 10V-BAC1001). The study design includes 2 cohorts: A total of 1,004 participants are enrolled in the study with 404 participants (100 participants/ExPEC 1OV dose) aged >60 to <85 years in stable health in Cohort 1 and an additional of 600 participants aged >60 years in stable health with a history of UTI in the past 5 years in Cohort 2.
[00345] ExPEClOV is a 10-valent vaccine candidate in development for the prévention of ί invasive extraintestinal pathogenîc Escherichia coli (ExPEC) disease (IED) in adults 60 years of âge and older. ExPEClOV consists ofthe O-antigen polysaccharides (PSs) of the ExPEC serotypes OlA, 02, 04, O6A, 08, 015, 016, O18A, O25B and 075 separately bioconjugated to the carrier protein, a genetically detoxified form of exotoxin A (EPA) derived from Pseudomonas aeruginosa, and its production has been described above. The 04 PS is the glucosylated form, having the structure of Formula (04-Glc+) in Table 1. i ί 95 |
[00346] OBJECTIVES AND ENDPOINTS
[00347] COHORT 1 - Phase l/2a observer-blind period with open-label long-term follow-up period (N=404):
Objectives Endpoints
Primary
• To evaluate the safety and reactogenicity of different doses of ExPEClOV in participants >60 to <85 years of âge • Solicited local and systemic adverse events (AEs) collected for 14 days post-vaccination (from Day 1 to Day 15) • Unsolicited AEs collected from the administration of the study vaccine until 29 days post-vaccination (from Day 1 to Day 30) • Serious adverse events (SAEs) collected from the administration of the study vaccine until Day 181
• To evaluate the dose-dependent immunogenicity of ExPEClOV on Day 15 in participants >60 to <85 years of âge • Antibody titers for ExPEClOV, as determined by multiplex electrochemiluminescent (ECL)-based immunoassay and multiplex opsonophagocytic assay (ΜΟΡΑ) on Day 15
Secondary
• To evaluate the corrélation between multiplex ECL-based immunoassay (total antibody) and ΜΟΡΑ (functional antibody) sérum titers on Day 15 • Antibody titers for ExPEClOV, as determined by multiplex ECL-based immunoassay and ΜΟΡΑ on Day 15
• To evaluate the dose-dependent immunogenicity of ExPEClOV on Days 30 and 181 in participants >60 to <85 years of âge • Antibody titers for ExPEClOV, as determined by multiplex ECL-based immunoassay and ΜΟΡΑ on Days 30 and 181
Objectives End points
• To evaluate, in the long-term follow-up (LTFU) period, the safety of the ExPEC 10V dose selected for further clinical development based on the Day 30 primary analysis in participants >60 to <85 years of âge • SAEs related to the study vaccine or study procedures collected from Day 182 until the end of the study
• To evaluate, in the LTFU period, the immunogenicity of the ExPEC 10V dose selected for further clinical development based on the Day 30 primary analysis • Antibody titers for ExPEC 10 V, as determined by multiplex ECL-based immunoassay and ΜΟΡΑ at Year 1 (Day 366), Year 2 (Day 731) and Year 3 (Day 1096)
COHORT 2 - Double-blind period with double-blind long-term folio w-up period (N=600):
Objectives Endpoints
Primary
• To evaluate the safety and reactogenicity of the selected dose of ExPEC 10V in participants >60 years of âge with a histoiy of UTI in the past 5 years • Solicited local and systemic AEs collected for 14 days post-vaccination (from Day 1 to Day 15) • Unsolicited AEs collected from the administration of the study vaccine until 29 days post-vaccination (from Day 1 to Day 30) • SAEs collected from the administration of the study vaccine until Day 181
• To evaluate the immunogenicity of the selected dose of ExPEClOV on Day 30 in participants >60 years of âge with a history of UTI in the past 5 years • Antibody titers for ExPEClOV, as determined by multiplex ECL-based immunoassay and ΜΟΡΑ on Day 30
Secondary
• To evaluate the corrélation between multiplex ECL-based immunoassay (total antibody) and ΜΟΡΑ (functional antibody) sérum titers on Day 30 in participants >60 years of âge with a history of UTI in the past 5 years • Antibody titers for ExPEClOV, as determined by multiplex ECL-based immunoassay and ΜΟΡΑ on Day 30
• To evaluate the immunogenicity of the selected dose of ExPEClOV on Days 15 and 181 in participants >60 years of âge with a history of UTI in the past 5 years • Antibody titers for ExPEClOV, as determined by multiplex ECL-based immunoassay and ΜΟΡΑ on Days 15 and 181
Objectives Endpoints
• To evaluate, in the LTFU period, the safety of the selected dose of ExPEClOV in participants >60 years of âge with a history of UTI in the past 5 years • SAEs related to the study vaccine or study procedures collected from Day 182 until the end of the study
• To evaluate, in the LTFU period, the immunogenicity of the selected dose of ExPEClOV in participants >60 years of âge with a history of UTI in the past 5 years • Antibody titers for ExPEClOV, as determined by multiplex ECL-based immunoassay and ΜΟΡΑ at Year 1 (Day 366), Year 2 (Day 731), and Year 3 (Day 1096)
Exnloratory
• To evaluate the effect of ExPEClOV on the intestinal (stool) microbiome by metagenomic analyses • Metagenomics of stool samples from a selected subset of participants to evaluate the effect of ExPEClOV on: - Prevalence of pathogens (eg, Clostridium difficile) in the intestinal flora - Prevalence of ExPEClOV serotypes in the intestinal flora
[00348] OVERALL DESIGN
[00349] This is a randomized, multicenter, interventional study including two cohorts.
[00350] For Cohort 1, the study has an observer-blind, active-controlled design, and a total of 404 adult participants aged >60 to <85 years in stable health with or without a history of UTI are included. The study design for Cohort 1 is comprised of three periods: a maximum of28-day screening period, an observer-blinded 181-day follow-up period with vaccination on Day 1 and an open-label LTFU period which lasts from Day 182 until 3 years (Day 1096) post-vaccination (FIG. 10A). Only participants from the ExPEClOV selected dose group (approximately 100 participants) and participants from the Prevnar 13 group progress to the LTFU period. The end of Cohort 1 is the last participant’s Year 3 visit (Day 1096).
[00351] For Cohort 2, the study has a double-blind, placebo-controlled design, and a total of 600 adult participants aged >60 years in stable health with a history of UTI in the past 5 years is included. Enrollment commences after completion ofthe Phase l/2a primary analysis and ExPEClOV dose sélection from Cohort 1. The study design for Cohort 2 is comprised of three periods: a maximum 28-day screening period, a double-blind 181-day follow-up period with vaccination on Day 1, and a double-blind LTFU period which lasts from Day 182 until 3 years (Day 1096) post-vaccination (FIG. 10B). Ail participants in Cohort 2 progress to the LTFU period. The end of study is the last participant’s Year 3 visit (Day 1096) in Cohort 2.
[00352] Cohort 1 : Phase 1
[00353] In Phase 1 of Cohort 1, a total of 84 participants are enrolled in a staggered approach following stepwise dose-escalating procedures with safety évaluations in place before progressing from one step to the next. An internai Data Review Committee (DRC) is commissioned for this study to review the physical examination data (baseline as well as targeted), baseline démographie data and the 14-day post-vaccination safety data (including solicited local and systemic AEs, unsolicited AEs, SAEs, clinical laboratory data and vital signs) of these 84 Phase 1 participants. In this phase of the study, participants were enrolled and randomized in six steps:
Step 1: Four sentinel participants were enrolled and randomized; two participants in the ExPEClOV low dose group (Table 11), and one participant each in the ExPEC4V and Prevnar 13 groups.
Step 2: Twenty-four participants were enrolled and randomized; 18 participants in the ExPEClOV low dose group (Table 11), and three participants each in the ExPEC4V and Prevnar 13 groups.
Step 3: Four sentinel participants were enrolled and randomized; two participants in the ExPEClOV medium dose group (Table 11), and one participant each in the ExPEC4V and Prevnar 13 groups.
Step 4: Twenty-four participants were enrolled and randomized; 18 participants in the ExPEClOV medium dose group (Table 11), and three participants each in the ExPEC4V and Prevnar 13 groups.
Step 5: Four sentinel participants were enrolled and randomized; two participants in the ExPEClOV high dose group (Table 11), and one participant each in the ExPEC4V and Prevnar 13 groups.
Step 6: Twenty-four participants were enrolled and randomized; 18 participants in the ExPEC 10V high dose group (Table 11), and three participants each in the ExPEC4V and Prevnar 13 groups.
[00354] AH participants received a single intramuscular (IM) injection of either ExPEC 10V (1 of 3 doses), ExPEC4V or Prevnar 13 on Day 1 per the assigned study vaccination groups. The four sentinel participants at each of Steps 1, 3 and 5 were contacted by téléphoné 24 hours postvaccination to collect safety information. The blinded 24-hour post-vaccination safety data in each group of four sentinel participants were reviewed by the principal investigator (PI), study responsible physician (SRP) and sponsor medical lead (SML). Randomization of additional participants for the next step was halted until this Day 2 sentinel safety évaluation was completed.
[00355] In the absence of any clinically significant findings, an additional 24 participants (for Steps 2, 4, and 6) were enrolled and randomized to one of three study vaccination groups (Table 11) to receive a single IM injection of either ExPEC 10V (1 of 3 doses), ExPEC4V or Prevnar 13 on Day 1.
[00356] After vaccination of an additional 24 participants at each dose level (low dose in Step 2, medium dose in Step 4, and high dose in Step 6), 14-day post-vaccination safety data of ail 28 (4+24) participants at each dose level was reviewed by the DRC before progressing to the next dose level or Phase 2a.
[00357] Cohort 1 : Phase 2a
[00358] Based on acceptable safety and reactogenicity (in the absence of any safety concems or any events meeting a spécifie study pausing rule) as determined by DRC after the review of 14-day post-vaccination safety data for the initial 84 participants, the remaining 320 participants from Cohort 1 were randomized and dosed in Phase 2a of the study. These additional 320 participants were enrolled and randomized in parallel in a ratio of 2:2:2:1 ;1 to one of the five study vaccination groups to receive a single IM injection of either ExPEC 10V (1 of 3 doses), ExPEC4V or Prevnar 13 on Day 1 (Table 11).
[00359] In addition to performing the 14-day safety review for the initial 84 participants, the DRC also évaluâtes safety data of Cohort 1 over the course of the study and review any events that meet a spécifie study vaccination pausing rule or any other safety issue that may arise.
100
[00360] For Cohort 1, the primary analysis occurs when ail participants hâve completed the Day 30 visit (Visit 4) or hâve discontinued earlier. The final analysis occurs when ail participants hâve completed the Day 181 visit or hâve discontinued earlier. For participants progressing to the open-label long-term follow-up (LTFU) period (ExPEClOV selected dose group and Prevnar 13 group) , yearly follow-up analyses include safety and immunogenicity data (multiplex ECLbased immunoassay and ΜΟΡΑ) collected up to the time of the visit at Year 1 (Day 366), Year 2 (Day 731) and Year 3 (Day 1096) after vaccination.
[00361] Cohort 2
[00362] In Cohort 2, the safety, reactogenicity, and immunogenicity of the selected dose of ExPEClOV (based on the primary analysis results of Cohort 1) is evaluated in participants aged >60 years in stable health with a history of UTI in the past 5 years. For Cohort 2, the study has a double-blind, placebo-controlled design, and a total of 600 participants are enrolled and randomized in parallel in a 2:1 ratio (400 participants in the ExPEClOV group and 200 in the placebo group).
[00363] AH participants receive a single IM injection of either the selected dose of ExPEC 10V or placebo on Day 1 per the assigned study vaccination groups (Table 12).
[00364] For Cohort 2, the primary analysis includes safety and immunogenicity data and occurs when ail participants hâve completed the Day 30 visit (Visit 4) or hâve discontinued earlier. The final analysis occurs when ail participants hâve completed the Day 181 visit or hâve discontinued earlier. For ail participants, yearly follow-up analyses include safety and immunogenicity data (multiplex ECL-based immunoassay and ΜΟΡΑ) collected up to the time of the visit at Year 1 (Day 366), Year 2 (Day 731), and Year 3 (Day 1096) after vaccination.
[00365] A stool sample analysis is performed in a selected subset of participants to evaluate the effect of ExPEClOV on the prevalence of pathogens (eg, Clostridium difficile) and ExPEClOV serotypes in the intestinal flora using metagenomics.
[00366] NUMBER OF PARTICIPANTS
[00367] A total of 1004 participants is enrolled in the study; 404 participants in Cohort 1 and 600 participants in Cohort 2.
[00368] INTERVENTION GROUPS
[00369] Description of Interventions i !
101
[00370] ExPEClOV: E. coli bioconjugate vaccine in phosphate buffered solution containing O-antigen PS ofExPEC serotypes OlA, 02, 04, 06A, 08, 015, 016, 018A, O25B and 075 separately bioconjugated to the EPA carrier protein. Single 0.5 mL IM (deltoid) injection of one of the three doses of ExPEClOV on Day 1.
[00371] ExPEC4V: E. coli bioconjugate vaccine in saline buffer solution containing O-antigen PS ofExPEC serotypes OlA, 02, O6A, O25B (4:4:4:8 pg PS/ExPEC serotypes) separately bioconjugated to the EPA carrier protein. Single 0.5 mL IM (deltoid) injection of ExPEC4V on Day 1.
[00372] Prevnar 13: Stérile suspension of saccharides of the capsular antigens of Streptococcuspneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14,18C, 19A, 19F, and 23F, individually linked to non-toxic Diphtheria CRM197 protein. Single 0.5 mL IM (deltoid) injection on Day 1, supplied in a single-dose prefilled syringe.
[00373] Placebo: normal saline. Single 0.5 mL IM (deltoid) injection of placebo on Day 1.
[00374] The ExPEC study intervention material s are described in Table 9.
[00375] Table 9. BAC1001MV ExPEC Study Vaccines.
Study Arm OlA (pg) 02 (pg) 04 (pg) O6A (pg) 08 (pg) 015 (pg) 016 (pg) O18A (pg) O25B (pg) 075 (pg) EPA (pg) PS (Total) (pg)
Low dose ExPEClOV 4 4 4 4 4 4 4 4 8 4 160 44
Medium dose 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=a genetically detoxified form of exotoxin A derived from Pseudomonas aeruginosa', PS=polysaccharide ExPEC4V consists of the O-antigen polysaccharides (PSs) of the ExPEC serotypes OlA, 02, O6A, and O25B separately bioconjugated to the EPA carrier protein.
ExPEClOV consists of the O-antigen polysaccharides (PSs) ofthe ExPEC serotypes OlA, 02, 04, O6A, 08, 015, 016,018A, O25B and 075 separately bioconjugated to the EPA carrier protein.
Dose is based on PS only. The EPA (pg) are measured values.
[00376] ExPEClOV is composed of 10 monovalent drug substances (DSs). For this clinical study, 2 different concentrations (medium and high) of drug product (DP) are produced (Table
102
0). A third (low) concentration is obtained in the clinic by diluting the high concentration 1:1 with dilution buffer, which is the same as the formulation buffer. Each DP is formulated in Sodium/Potassium phosphate buffer at pH 7.0 (0.02% [w/w] Polysorbate 80, 5% [w/w] sorbitol, 10 mM méthionine).
[00377] Table 10: Composition of ExPEClOV vaccine for phase l/2a clinical study
Ingrédient Amount (jig/mL)a
Active11 Low Concentration b Medium Concentration High Concentration
O-anügen polysaccharide
EcoOlA 8 16 16
EcoO2 8 8 16
EcoO4 8 8 16
EcoO6A 8 16 16
EcoO8 8 8 16
EcoO15 8 8 16
EcoO16 8 8 16
EcoO18A 8 8 16
EcoO25B 16 32 32
EcoO75 8 8 16
Carrier protein
EPA 320 441 640
Excipients
KH2PO4 6.19 mM
Na2HPO4 3.81 mM
Sorbitol 5% (w/w)
Méthionine lOmM
Polysorbate 80 0.02% (w/w)
EPA^genetically detoxified P. aeruginosa exotoxin A used as carrier protein a The active ingrédient is a biologically synthesized conjugate composed of the PS antigen and a carrier protein (EPA); the dose is calculated on the PS moiety only.
b The “low concentration” is obtained in the clinic by diluting the “high concentration” 1:1 with dilution buffer [00378] SAFETY EVALUATIONS
[00379] Key safety assessments include solicited local and systemic AEs, unsolicited AEs, SAEs, physical examinations, vital sign measurements, and clinical laboratory tests.
[00380] IMMUNOGENICITY EVALUATIONS
[00381] Key immunogenicity assessments of collected sera include the assessment of ExPEClOV and ExPEC4V serotype-specific total IgG antibody levels elicited by the vaccine as measured by a multiplex ECL-based immunoassay, and ExPEClOV and ExPEC4V serotypespecific functional antibodies as measured by an opsonophagocytic killing assay (OPKA) in multiplex format (ΜΟΡΑ). Immunogenicity assessments of pneumococcal antibody titers elicited by Prevnar 13 are not performed.
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[00382] The levels of sérum antibodies înduced by ExPEC 10V are measured by a multiplex electrochemiluminescent (ECL)-based immunoassay. This assay combines high binding carbon électrodes in a multi-spot 96-well format microplate that is coated with different E. coli O-LPS antigens or the carrier protein EPA. The levels of antigen-specific antibodies présent in sérum samples are detected using a secondary antibody (anti-human IgG) labeled with SULFO-TAG. The SULFO-TAG emits light in the presence of electrical stimulation at an intensity that increases proportionally to the amount of bound IgG antibodies. This assay was qualified according to International Conférence on Harmonisation (ICH) recommendations.
[00383] The levels of functional antibodies înduced by ExPEC 10V are measured by a multiplex opsonophagocytic assay (ΜΟΡΑ). Briefly, heat-inactivated sérum samples are serially diluted and incubated with different E. coli strains that are specifically resitant to different types of antibiotics. After that, human complément and phagocytic cells (HL60) are added to the reaction and, after a second incubation period, an aliquot of the reaction mix is transferred to different PVDF hydrophilic membrane fîlter plates containing media supplemented with spécifie antibiotic that selectively allow growth of a strain that is résistant to that particular antibiotic. After ovemight grown, the colony forming units (CFUs) are counted to détermine the number of surviving bacteria. This assay was qualified according to ICH recommendations.
[00384] For ExPEC 10V serotype antibodies as measured by multiplex ECL-based immunoassay and ΜΟΡΑ, and EPA as measured by multiplex ECL-based immunoassay only, the following measures of immunogenicity are evaluated and tabulated by the study vaccination groups, for ail immunogenicity time points:
- proportion of participants with a >2-fold and >4-fold increase in sérum antibody titers from Day 1 (pre-vaccination)
- géométrie mean titer (GMT)
- GMR: fold change from baseline, calculated from the post-baseline/baseline value.
For the LTFU period, descriptive summaries of immunogenicity are provided for each serotype. [00385] Dose sélection for later phases considers the totality of the evidence available at the time of the primary analysis of Cohort 1 (Day 30 results).
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[00386] Table 11: Cohort 1: Vaccination Schedule
Phase 1 Phase 2a Tota 1
Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7
Study Vaccin atiou Group Vaccination on Day 1 Sentinel participants (Low dose) Additionai participan ts (Low dose) Sentinel participants (Medium dose) Additionai participants (Medium dose) Sentinel participai! ts (High dose) Additionai participants (High dose) Additionai Phase 2a Participants
G1 Low dose ExPEC 10 V* 2 18 80 100
G2 Mediu m dose ExPEC 10V* 2 18 80 100
G3 High dose ExPEC 10V* 2 18 80 100
G4 ExPEC 4y** 1 3 1 3 1 3 40 52
G5 Prevnar iq*** 1 3 1 3 1 3 40 52
Total 4 24 4 24 4 24 320 404
* ExPEC 10V consists of the O-antigen polysaccharides (PSs) of the ExPEC serotypes 01 A, 02,
04, 06A, 08, 015, 016, 018A, O25B and 075 separately bioconjugated to the carrier protein, a genetically detoxified form of exotoxin A (EPA) derived from Pseudomonas aeruginosa.
** ExPEC4V consists ofthe O-antigen polysaccharides (PSs) ofthe ExPEC serotypes 01 A, 02, 06A, and O25B separately bioconjugated to the carrier protein, a genetically detoxified form of exotoxin A (EPA) derived from Pseudomonas aeruginosa.
*** Prevnar 13, Pneumococcal 13-valent conjugate vaccine (Diphtheria CRM 197 protein) is a stérile suspension of saccharides of the capsular antigens of Streptococcus pneumoniae serotypes 1, 3,4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, individually linked to non-toxic Diphtheria CRM 197 protein.
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[00387] Table 12: Cohort 2: Vaccination Schedule
Study Vaccination Group Vaccination on Day 1 Total
G6 ExPEClOV3 400
G7 Placebo 200
Total 600
a ExPEClOV consista ofthe O-antigen polysaccharides (PSs) ofthe ExPEC serotypes 01 A, 02, 04, O6A, 08, 015, 016, 018A, O25B, and 075 separately bîoconjugated to the carrier protein, a genetically detoxified form of exotoxin A (EPA) derived from Pseudomonas aeruginosa. [00388] The randomization ratio for the participants enrolled in Cohort 2 of the study is 2:1 (ExPEC 10 V:Placebo). The ExPEClOV dose used in Cohort 2 is based on the primary analysis (Day 30) results of Cohort 1.
[00389] STATUS
[00390] Enrollment and vaccination of Cohort 1 of the study described above was completed. The study is ongoing in a blinded manner. Based on ongoing review ofthe safety data, no major safety issues were identified, and the ExPEClOV vaccine has an acceptable safety profile.
[00391] The analysis of the immunogenicity of the Cohort 1 clinical samples is ongoing in a blinded fashion. The ECL data were 100% Acceptance Quality Limits (AQL) checked and uploaded for data management. Analysis ofthe ΜΟΡΑ samples is ongoing. Data unblinding and statistical analysis is performed by using a clinical research organization (CRO).
[00392] The Cohort 2 vaccinations are started once the ExPEC 1OV dose for that Cohort has been identified based on the fînalized primary analysis of the Day 30 results from Cohort 1.
[00393] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope ofthe présent invention as defined by the présent description.
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SEQUENCES
SEQ ID NO: 1 (Glycosylation consensus sequence)
Asn-X-Ser(Thr), wherein X can be any amino acid except Pro
SEP ID NO: 2 (Optimized glycosylation consensus sequence)
Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any amino acid except Pro
SEQ ID NO: 3^EPA carrier protein comprising 4 glycosylation consensus sequences (EPA-4))
G SGGGDQNATG SGGGKLAEEA FDLWNECAKA CVLDLKDGVR SSRMSVDPAI ADTNGQGVLH YSMVLEGGND ALKLAIDNAL SITSDGLTIR LBGGVEPNKP VRYSYTRQAR GSWSLNWLVP IGHEKPSHIK VFIHELNAGN QLSHMSPIYT IEKGDELLAK LARDATFFVR AHESNEMQPT LAISHAGVSV VMAQAQPRRE KRWSEWASGK VLCLLDPLDG VYNYLAQQRC NLDDTWEGKI YRVLAGNPAK HDLDIKDNHN STPTVISHRL HFPEGGSLAA LTAHQACHLP LEAFTRHRQP RGWEQLEQCG YPVQRLVALY LAARLSWNQV DQVIRNALAS PGSGGDLGEA IREQPEQARL ALTLAAAESE RFVRQGTGND EAGAASADW SLTCPVAKDQ NRTKGECAGP ADSGDALLER NYPTGAEFLG DGGDVSFSTR GTQNWTVERL LOAHRÛLEBR GYVFVGYHGT FLEAAQSIVF GGVRARSQDL DAIWRGFYIA GDPALAYGYA QEQEPDARGR IRNGALLRVY VPRWSLPGFY RTGLTLAAPE AAGEVERLIG HPLPLRLDAI TGPEEEGGRV TILGWPLAER TWIPSAIPT DPRNVGGDLD PSSIPDKEQA ISALPDYASQ PGKPPREDLK LGSGGGDQHA T.
SEQ ID NO: 4 (04 GtrS amino acid sequence)
MNNLIMNNWCKL SI FI I AF ILLWLRRPDILTNAQFWAEDS VFWY KD AYENGFLS SLTTPRNGYFQTVSTFI VGLTALLNPDYAPFVSNFFGIMIRSVIIWFLFTERFNFLTLTTRIFLSIYFLCMPGLDEVHANITNAHWYL 3ΣΥν3ΜΙΕΙΑΕΝΡβ3Κ3ΚΡΡΗΌΙΡΡΙΒη3σΒ8σΡΡΙΙΡΙΒΑΑ80ΡΚΡΙΝΝ0ΚηΗΙ3νΡ3ΡΙΝΡΥΒΚ0ΡΥΑΑ Μΐν0ΑΡΙ2σΤ8ΙΙΒΤΡΝΘΤΕ33ΑΡΕσΡ3ΡΌνΐ33ΙΙ83ΝΙΡΕΡΤΡνΡΚΌΙΑΚΑαΝϋΝΕΐ2Ε3ΥΡΕ3ν3ΙΕ30 ΑΑΡνΡνκαΤΜΕΜΚνΡΑΤΕΡΕΕΙΙΙΡδΜΆΚΡβΣΤηβΑΡΟΕΡΤΕΙΝαΟΟβΕΎΡνΝΙΗΙΑΙΡΒΙΙΕΟνΥΕΒΕΟνΡ ΟΚνΑΤΙ,ΡβΚΙΥΕΤΙΒΕΡνΜΟΟΕΝΡνίΤΡΣΡΝΜΝνίΕΕΟΑΤΕΙΝΝΑΚΤαηνίΒΙΟνΣΡΡσΒΤΕΕΒΚΚΚ
SEQ ID NO: 5 (Example 04 gtrS nucleic acid sequence)
ATGAATAATTTAATTATGAATAACTGGTGTAAATTATCTATATTTATTATTGCATTTATTTTGCTATGGCT TAGAAGGCCGGATATACTCACAAACGCACAATTTTGGGCAGAAGATTCCGTTTTCTGGTATAAGGACGCCT ATGAGAACGGATTCTTAAGTTCACTAACAACGCCTAGGAATGGGTATTTCCAGACTGTTTCTACATTTATA GTTGGTCTGACTGCTTTATTAAATCCAGATTATGCACCTTTTGTTTCTAATTTTTTTGGCATAATGATTCG CTCAGTAATTATATGGTTTTTATTTACAGAAAGATTCAACTTCCTCACATTGACTACTAGGATTTTCTTAT CTATTTATTTTCTATGCATGCCTGGATTGGATGAAGTTCATGCAAATATAACAAATGCACATTGGTATTTG TCATTATATGTATCAATGATCCTGATAGCTCGCAATCCAAGTTCAAAATCATGGAGGTTTCATGATATATT CTTTATCTTGCTATCCGGGCTCAGTGGCCCATTTATAATTTTCATTTTAGCAGCTTCATGCTTTAAATTTA TAAATAATTGTAAAGATCATATTAGTGTAAGATCTTTCATAAATTTC TACTTGCGT C AGC C ATACGCATTA ATGATTGTTTGCGCTTTAATTCAAGGAACTTCTATAATTCTAACTTTCAATGGCACACGTTCCTCAGCACC GCTAGGATTCAGTTTTGATGTGATTTCGTCTATTATATCATCGAATATTTTTTTATTTACATTTGTCCCAT
107
GGGATATTGCAAAGGCTGGGTGGGATAATTTACTGTTATCTTATTTTTTGTCTGTTTCGATTTTGTCGTGT GCGGCCTTTGTTTTTGTTAAAGGTACGTGGCGAATGAAAGTATTTGCAACTTTACCATTGCTAATTATAAT ATTTTCAATGG C AAAAC CACAATTGACAGACTCGGCACCTCAATTGC CAACACTTATTAATGGGCAAGGTT CAAGATACTTCGTAAATATACATATTGCGATATTCTCTTTGCTATGTGTTTACTTACTTGAGTGCGTCAGG GGGAAAGTGGCAACTTTATTTTCCAAAATATACTTAACAATTTTGCTATTCGTGATGGGATGTTTGAATTT TGTTATC AC C C C ACTC C CAAACATGAACTGGAGGGAAGGTGCTACTTTGATTAATAATGCAAAAACTGGTG ATGTCATTTCGATTCAAGTGCTACCACCTGGCCTAACACTTGAACTAAGGAAAAAATAA
SEQ ID NO: 6 (Example PglB sequence (‘wild-type’))
ML·KKEYL·KNPYL·VL·FAMIIL·AYVFSVFCRFYWVWWASEFNEYFFNNQLMIISNDGYAFAEGARDMIAGFHQ ΡΝΌΕ3ΥΥ633Ι13ΑΕΤΥΐ7ΕΥΚΙΤΡΕ3ΡΕ3ΙΙΕΥΜ3ΤΡΕ33ΕννΐΡΤΙΕΕΑΝΕΎΚΕΡ1ΜΟΡνΑΑΒΒΑ3ΙΑΝ3Υ ΥΝΕΤΜβΟΥΥΟΤΟΜΕνίνΕΡΜΕΙΒΕΕΜνΕΜΙΒΚΚΌΕΕβΕΙΑΕΡΕΕΙΘΙΥΕνίΜΥΡββΥΤΕΝνΑΕίαΕΕηΐΥΤΒ IFHRKEKIFYIAVIL·SSLTL·SNIAWFYQSAIIVILFALFALEQKRLNFMIIGIL·GSATLIFLIL·SGGVDPI LYQLKFYIFRSDESANLTQGFMYFNVNQTIQEVENVDLSEFMRRISGSEIVFLFSLFGFVWLLRKHKSMIM ALPILVL·GFLΆLKGGLRFTIYSVPVMAL·GFGFLLSΞFKAIMVKKYSQLTSNVCIVFATILTLAPVFIHIYN YKAPTVFSQNEASLLNQLKNIANREDYWTWWDYGYPVRYYSDVKTLVDGGKHLGKDNF F PS FAL S KDE QA AANMARLSVEYTEKSFYAPQNDILKTDILQAMMKDYNQSNVDL.FLASLSKPDFKIDTPKTRDIYLYMPARM 3ΕΙΡ3ΤνΑ3Ε8ΡΙΝηθΤθνΕϋΚΡΡΤΡ3ΤΑΥΡΕΒνΚΝΟΕΙΥΕ3ΝσννΒ3ηϋΡΕ3ΡΚΙ6ϋΝνν3νΝ3ΐνΕΙΝ0 IKQGE YKIT PIDDKAQ FYIFYLKDSAI PYAQFILMDKTMFNSAYVQMFFLGNYDKNL FDLVINSRDAKVFK LKI
SEQ ID NO: 7 (example gtrA amino acid sequence; E. coli W3110 yfdG, GenBank: BAA16209.1)
MLKLFAKYTSIGVLNTLIHWWFGVCIYVAHTNQAIjAISIFAGFWAVSFSFFANAKFTFKASTTTMRYMLYV GFMGTLS ATVGWAADR.C AL PPMITLVTFS AI S LVCGFVYSKFIVFRDAK
SEQ ID NO: 8 (example gtrB amino acid sequence -Æ coli W3110 vfdHL GenBank: BAA16210.1)
MKISLWPVFNEEEAIPIFYKTVREFEELKSYEVEIVFINDGSKDATESIINALAVSDPLWPLSFTRNFG KEPALFAGLDHATGDAIIPIDVDLQDPIEVIPHLIEKWQAGADMVLAKRSDRSTDGRLKRKTAEWFYKLHN ΚΙδΝΡΚΙΕΕΝνσϋΡΕΣΜβΕΟννΕΝΙΚΕΜΡΕΕΝΕΕΜΚΟΙΣβΝνααΚΤϋΐνΕΥνΡΑΕΕΙΑΟΠΤΚΡΝαΜΚΕνίΝΕ ΑΕΕαΐΤ3Ε8ΤΕΡΕΕΐνίΤΥΐσΣννΑ3νΑΡΙΥ6ΑΝΜΙΕΟΤΙΙΕΟΝΑνΕαΥΡ3ΕΕν8ΙΕΕΒαθΙ0ΜΐσΐσνΕσΕ YIGRTYIETKKRPKYIIKRVKK
SEP ID NO: 9 (example 04 rfb locus nucléotide sequence — 04-EPA production strain BVEC-L00684f)
ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGAT AC C CAAAGAGATGCTAC CAATCGTCGACAAGCC AATG ATTC AGTACATTGTTGACGAGATTGTGGCTGC AG GGATCAAAGAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTAT GAGTTAGAATCACTC CTTGAGCAGCGCGTGAAGCGTCAACTGCTGGCGGAAGTACAGTC CATC TGTCCGC C GGGCGTGACCATTATGAACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGAC CTGCCATTGGTGACAACCCATTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCG CTACGTTACAACCTTGCTGCCATGATTGCACGTTTCAACGAAACGGGCCGCAGCCAGGTGCTGGCAAAACG TATGCCGGGTGACCTCTCTGAATACTCCGTCATCCAGACTAAAGAGCCGCTGGACCGTGAGGGTAAAGTCA GCCGCATTGTTGAATTTATCGAAAAACCGGATCAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGT CGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTGGAACGTACTCAGCCTGGTGCATGGGGACGTATTCA GCTGACTGATGCTATTGCCGAGCTGGCGAAAAAACAATCCGTTGATGCAATGCTGATGACCGGCGACAGTT ACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAGTATGGCCTACGCAACCTGAAAGAAGGG GCGAAGTTCCGTAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAATCTGACCGGATGTAACGGTTGAT AAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAATCTTCCTGCCGTTGTTTTA TATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAGTTTTCCAGGATTTTCCTTGTTT CCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTAACGCTCGTCAC ATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCATTAATACCTCTATT
108
AATCAAACTGAGAGCCGCTTATTTCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGTGAAAATACTT GTTACTGGTGGCGCAGGATTTATTGGTTCAGCTGTAGTTCGTCACATTATAAATAATACGCAGGATAGTGT TGTTAATGTCGATAAATTAACGTACGCCGGAAACCGGGAATCACTTGCTGATGTTTCTGATTCTGAACGCT ATGTTTTTGAACATGCGGATATTTGCGATGCACCTGCAATGGCACGGATTTTTGCTCAGCATCAGCCGGAT GCAGTGATGCACCTGGCTGCTGAAAGCCATGTTGACCGTTCAATTACAGGCCCTGCGGCATTTATTGAAAC CAATATTGTTGGTACTTATGTCCTTTTGGAAGCCGCTCGCAATTACTGGTCTGCTCTTGATAGCGACAAGA aaaatagcttccgttttcatcatatttctactgacgaagtatatggtgatttgcctcatcctgacgaggta AATAATACAGAAGAATTACCCTTATTTACTGAGACAACAGCTTACGCGCCAAGCAGCCCTTATTCCGCATC CAAAGCAT C CAGCG ATC ATTT AGTCCG CG CGTGGAAAC GTACCTATGGTTT ACCGACCATTGTGACTAATT GCTCTAACAATTATGGT C CTTAT CATTTC C CGGAAAAATTGATTCCATTGGTTATTCTCAATGCTC TGGAA GGTAAAGCATTACCTATTTATGGTAAAGGGGATCAAATTCGCGACTGGCTGTATGTTGÀAGATCATGCGCG TGCGTTATATACCGTCGTAACCGAAGGTAAAGCGGGTGAAACTTATAACATTGGTGGGCACAACGAAAAGA AAAACATAGATGTAGTGCTCACTATTTGTGATTTGCTGGATGAGATTGTACCGAAAGAGAAATCTTATCGT GAGCAAATCACTTATGTTGCCGATCGTCCGGGACACGATCGCCGTTATGCGATTGATGCTGAGAATATTGG TCGCGAATTGGGATGGAAACCACAGGAAACGTTTGAGAGCGGGATTCGGAAGACAGTGGAATGGTATCTGT CCAATACAAAATGGGTTGATAATGTGAAAAGTGGTGCCTATCAATCGTGGATTGAAGAGAACTATGAGGGC CGCCAGTAATGAATATCCTCCTTTTTGGCAAAACAGGGCAGGTAGGTTGGGAACTACAGCGTGCTCTGGCA CCTCTGGGTAACTTGATTGCTCTTGATGTTCATTCCACTGATTATTGTGGCGATTTCAGTAACCCCGAAGG TGTGGCTGAAAC CGTCAAAAAAATTCGCCCAGATGTTATTGTTAATGCTGCTGCTCATAC CGC GGTAG ATA AGGCTGAGTCAGAACCAGAATTTGCACAATTACTCAATGCGACCAGCGTTGAAGCAATTGCAAAAGCGGCT AATGAAGTTGGGGCTTGGGTAATTCATTACTCAACTGACTACGTCTTCCCTGGAAATGGCGACATGCCATG GCTCGAGACTGATGTAACCGCTCCGCTCAATGTTTATGGCAAAACCAAATTGGCTGGAGAAAGAGCATTAC AAGAACATTGCGCAAAGCATCTTATTTTCCGTACCAGCTGGGTATATGCAGGTAAAGGAAATAACTTTGCC AAAACAATGTTACGTCTGGCAAAAGAGCGCGAAGAACTGGCTGTGATAAACGATCAGTTTGGCGCACCAAC AGGTGCTGAATTGCTGGCTGATTGCACCGCTCATGCCATTCGCGTGGCATTAAAAAAACCAGAAGTTGCTG GCTTGTACCATCTGGTAGCAAATGGCACAACAACCTGGCACGATTACGCCGCGCTAGTATTCGAAGAAGCC CGTAAAGCAGGGATTGACCTTGCACTTAACAAACTCAACGCCGTACCAACAACGGCTTATCCTACTCCAGC CCGCCGTCCTCATAATTCTCGCCTCAATACCGAAAAGTTTCAGCAGAACTTTGCGCTTGTCTTGCCTGACT GGCAGGTGGGCGTGAAACGTATGCTCAACGAATTATTTACGACTACGGCAATTTAACAAATTTTTGCATCT CGCTCATGATGCCAGAGCGGGATGAATTAAAAGGAATGGTGAAATGAAAACGCGTAAAGGTATTATTCTGG CTGGTGGTTCCGGCACTCGTCTTTATCCTGTGACGATGGCAGTGAGTAAACAACTGCTGCCGATTTATGAT AAGCCGATGATTTATTATCCGCTTTCAACGCTTATGTTAGCGGGTATTCGCGATATTCTTATTATCAGTAC GCCACAGGATACACCGCGTTTCCAACAATTGTTGGGGGACGGGAGTCAGTGGGGGCTTAATCTACAGTATA AAGTACAACCGAGTCCGGATGGCCTGGCGCAAGCGTTTATTATTGGTGAAGACTTTATTGGTGGTGATGAT TGTGCACTCGTACTTGGCGATAATATCTTCTATGGACACGACTTGCCGAAATTAATGGAAGCTGCTGTTAA CAAAGAAATCGGTGCAACGGTATTTGCTTATCACGTCAATGATCCTGAACGTTATGGTGTCGTGGAGTTTG ATAATAACGGTACTGCAATTAGCCTGGAAGAAAAACCGCTGGAAC C AAAAAGTAACT ATGCGGTTACTGGG CTTTATTTCTATGACAATGATGTTGTAGAAATGGCGAAAAACCTTAAGCCTTCTGCCCGTGGCGAACTGGA AATTACCGATATTAACCGTATTTATATGGAGCAGGGACGTTTGTCTGTCGCTATGATGGGGCGTGGTTATG CCTGGTTGGATACTGGTACACATCAAAGTCTTATTGAAGCAAGTAACTTCATTGCCACCATTGAAGAGCGT CAGGGATTAAAGGTATCTTGCCCGGAAGAGATTGCTTACCGTAAAGGGTTTATTGATGCTGAGCAGGTGAA AGTATTAGCCGAACCGCTGAAGAAAAATGATTATGGTCAGTATCTGCTAAAAATGATTAAAGGTTATTAAT AAAATGAACGTAATTAAAACTGAAATTCCTGATGTGCTGATTTTTGAACCAAAAGTTTTTGGTGATGAACG TGGCTTCTTTTTTGAGAGTTTTAACCAGAAAGTATTTGAAGAAGCTGTAGGACGGAAGGTTGAATTTGTTC AGGATAACCATTCTAAGTCTAAAATAAATGTATTGCGTGGGATGCATTATCAAACACAAAATACTCAAGGA .
AAACTGGTTCGGGTAATTTCTGGTTCAGTATATGATGTTGCCGTAGATTTAAGAGAAAAATCAAAGACATT TGGCAAATGGGTGGGTGTAGAATTATCTGGGAATAATAAAAGACAATTGTGGATC C C CGAAGGTTTTGC CC ATGGTTTTTATGTGTTGGAGGAGAATAC CGAATTTGTTTATAAATGTACCGATACTTATAACC CTGCT CAT
GAACACACATTGCTATGGAATGATCCAACTATCAATATAAGTTGGCCAATCATACAAAACTGCAAGCCAAT |
TATTT CTGAAAAAGATG CT AATGGAC ATCTTTTTTCAC ATAAAAC CTATTTCTGAAATGC AATATTATGAG TTTAATTAGAAACAGTTTCTATAATATTGCTGGTTTTGCTGTGCCGACATTAGTTGCAGTCCCTGCTTTGG GGATTCTTGCCAGGCTGCTTGGACCGGAGAATTTTGGACTTTTCACACTAGCATTCGCTTTGATAGGATAT GCAAGTATTTTCGACGCCGGGATTAGTCGAGCTGTAATCAGAGAAATCGCTCTTTATCGAGAAAGTGAAAA AGAGCAAATACAAATTATTTCGACAGCAAGTGTAATCGTACTATTCTTAGGGGTGGTTGCAGCTTTGTTAC TTTATTTTAGTAGTAATAAAGTTGTTGAGTTATTGAATGTTAGTTCCGTTTATATTGAAACAGCAGTGCGT GCATTCTCTGTTATTTCATTTATAATACCTGTGTATCTGATTAACCAGATTTGGCTTGGTTATCTGGAAGG
109 gctagaaaaatttgcaaatataaatgttcagagaatgatttctagcacaagcttggctatattaccagtga TATTTTGTTATTACAATCCCTCGTTGCTTTATGCTATGTATGGGTTGGTGGTTGGGCGTGTGATTTCATTT TTGATTAGCGCAATAATTTGTCGAGATATTATTCTTAAAAGTAAACTTTACTTTAATGTGGCAACTTGCAA TCGTCTTAT CTCTTTTGGTGGATGGATAACAGTTAGTAATATC ATAAGC C CAAT C ATGGCATATTTCGACC GCTTTATCATCTCTCATATTATGGGGGCTTCGAGAATTGCATTTTATACAGCGCCCTCAGAGGGTGTATCA AGGTTAATTAATATCCCATATGCTTTGGCAAGAGCTCTATTTCCTAAATTGGCATATAGCAATAATGATGA TGAACGAAAAAAATTACAACTACAGAGCTACGCAATTATAAGCATTGTATGTCTACCCATAGTTGTTATTG GTGTCATTTTTGCCTCATTCATAATGACAACATGGATGGGACCTGATTATGCCTTAGAAGCAGCAACTATC ATGAAAATACTTCTTGCTGGTTTTTTCTTTAACTCTTTAGCGCAAATACCTTATGCATACTTGCAATCTAT CGGAAAGTCAAAAATTACCGCATTTGTGCATCTCATAGAACTTGCGCCATACTTATTATTATTGTATTACT TCACAATGCATTTCGGCATAATTGGCACGGCAATCGCTTGGTCACTTAGAACATTTTGTGATTTTGTTATA CTACTTTCGATATCGAGAAGAAAATGATTGCGGTTGATATTGCGCTTGCAACCTACAATGGTGCTAATTTT ATTCGGCAACAGATTGAATCTATCCAGAAACAAACTTATAGAAATTGGCGTCTTATAATAAGTGATGATAA CTCGAGTGATGATACTGTTGATATTATTAAGGATATGATGTCTAACGACAGTCGTATCTATTTGGTAGGAA ATAAAAGACAAGGAGGGGTTATTCAGAACTTTAATTATGCTCTTTCACAAACTACATCTGAAATTGTGTTA CTATGTGACCAGGATGACATTTGGCCGGAGGAGCGTCTGGAAATTCTTATAGATAAATTTAAGGCCTTGCA GCGTAATGATTTTGTTCCGGCAATGATGTTTACTGATTTGAAATTAGTAGACGAAAATAATTGTTTGATTG CAGAAAGTTTTTATCGAACGAATÀATATTAATCCACAAGATAATCTGAAAAATAATAATCTTCTCTGGCGT TCAACGGTATATGGCTGTACTTGCATCATGAATAAGAAACTTGTTGATATTGCATTGCCTATACCTACATA TGCACATATGCATGATCAATGGTTGGCATTATTAGCGAAGCAATATGGTAACATTTTTTATTTCGACTATG CGTCTGTTCGTTATAGGCAACATTCTACAAATGTTGTTGGTGGTAGAAATAAAACGCCATTTCAAAAATTT AATTCCATACAAAAAAACCTAAAAAGGATTAATTTGCTAGTGGATAGAACTGTTGCTTTAATTAAATCAAA TAACGATTTCTATCCAGGGAATAAAATGGAAAATAAAATTGATTACTTAAAATTTGGAGTGAATGAAGTAT TACCTTATCTTTTTAAAGGAAACAAGAAAGTTTTTTCACTTTGTGTATTAATTAGTTTGGCATTACAAAAA TGATATATTTATTATTTTTTTTTGCACTGTTTATGATCTGTACGTTTTTAACACACAGGCGACAGGCATTA TATGTTGTATCTGCGTTAGTATTTCTTTTTTTGGCTTTAACCTATCCATCAGGAGGGGACTGGATAGGTTA TTTTCTCCATTATGACTGCATGGTTAATGAGCAGTGTAATAATGGTTTTATAATGTTTGAACCTGGATATG AATTAATTGTTTCCTTATTTGGATATTTGGGATTTCAGACAATTATTATTTTTATAGCCGCTGTAAATGTA ATTCTAATATTAAATTTTGCAAAGCATTTTGAAAACGGAAGTTTTGTTATTGTTGCGATAATGTGCATGTT CCTTTGGAGTGTTTATGTTGAGGCGATTAGACAGGCTCTGGCCTTATCTATAGTTATATTTGGGATTCATT CTCTTTTTTTGGGTAGAAAAAGGAAATTTATAACATTAGTATTATTTGCGTCAACTTTCCATATAACTGCT TTGATTTGTTTTCTTCTAATGACTCCTCTATTTTCAAAGAAATTAAGCAAGATAATAAGTTATAGCCTATT AATTTTCAGTAGCTTCTTTTTCGCTTTTTCTGAAACCATATTAAGTGCACTCCTTGCAATTTTGCCAGAAG GATCCATTGCCAGTGAAAAATTAAGTTTTTACTTAGCAACCGAGCAATACAGGCCACAGTTATCTATTGGG AGTGGCACTATTCTTGACATTATACTTATTTTTCTGATATGTGTAAGTTTTAAACGAATAAAGAAATATAT GCTCGCTAATTATAATGCTGCAAATGAGATATTGCTTATTGGTTGCTGTCTTTATATTTCTTTCGGTATTT TTATCGGGAAAATGATGCCAGTTATGACTCGCATTGGTTGGTATGGTTTTCCATTTGTTATAGTACTTCTT TATATTAACTTGGGTTATTCAGAATATTTTAAGAGGTATATAAATAAAAGAGGGTGTGGGTATAGCAAATT ATTAATTGCTTTTTATTTTTTGCTACAAATTTTGCGACCATTAACATATGATTATAGCTATTATAATATAA TGCACCAGGATACTTTGCTGAATAGGTTTGATGCATTAGATGATGCATCATTAAGACAATCAGCGAAGAGA AAATGTTTCGATTTGGGAAAGATAGGATATGGTTTCTTATGTAGTATATAATATCCTGCATTCATTCGGAT AATTTCCTATGGAAGTGTCCTTTGCTCTGTCTGTCCTCATTTGTTGAAATTTTATGTTAATAAGAAGCTTT AGATAACCACTTAGGAACTGTATGTTTGATCTGTCCAAAAATTATATTATTGTAAGTGCGACGGCGCTGGC TTCCGGAGGTGCATTAACTATATTAAAGCAATTTATAAAACATGCATCACAAAATTCAAATGACTATATTA TGTTTGTATCTGCGGGATTGGAGTTGCCGGTCTGTGATAACATCATTTACATAGAAAACACACCAAAAGGA TGGTTGAAAAGAATATATTGGGATTGGTTCGGTTGTCGGTLAGTTTATCTCGGTiACATAAGATTAACGTTAA GAAAGTAATTTCTCTACAAAATTCCAGTTTGAATGTTCCTTACGAACAGATTATTTACTTGCACCAGCCAA TTC CTTTTAGTAAAGTTGATTCTTTTTTAAAAAATATC ACAT C CGATAACGTAAAGCTTTTTTTATATAAA AAGTTTTATTCCTATTTTATATTTAAATATGTGAATGCCAATACAACCATCGTAGTGCAAACGAATTGGAT GAAAAAAGGAGTGCTGGAGCAATGTGATAAAATTAGTACCGAAAGGGTCCTTGTTATAAAACCTGATATCA AAGCATTTAATAATACTAATTTTGATGTAGATATGGATGTATCTGCATiAAACACTCTTATATCCAGCGACA CCACTTACCTATAAAAATCATTTGGTCATTCTGAAGGCGTTGGTTATTTTAAAGAAAAAGTATTTTATAGA TGAT CTGAAATT CC AAGTGACTTTTGAAZkAGAATAGGTACAAAAATTTTGATAAGTTTGTGC AATTAAAT A ACTTAAGCAAAAACGTTGATTATCTCGGCGTTCTTTCATACTCGAACTTGCAAAAAAAATATATGGCGGCA TCTTTAATCGTTTTTCCTAGCTATATCGAATCATATGGGTTACCACTCATCGAAGCTGCTAGTTTAGGAAA AAAAATCATTAGTAGTGATCTTCCTTATGCCCGGGATGTTTTAAAGGATTATAGCGGCGTAGATTTTGTTkA
110
TTTACAATAATGAAGATGGCTGGGCTAAGGCGTTGTTTAATGTTTTAAATGGCAATTCGAAGCTCAATTTT AGGCCTTATGAAAAAGATAGTCGTTCATCTTGGCCACAGTTCTTCTCTATTTTGAAATAAGGTGTATTATG TTTAATGGTAAAATATTGTTAATTACTGGTGGTACGGGGTCTTTCGGTAATGCTGTTCTAAGACGTTTTCT TGACACTGAT AT CAAAGAAATACGTATTTTTTC C CGGGATGAAAAAAAACAAGATGACATGAGGAAAAAAT ATAATAATCCGAAACTTAAGTTCTATATAGGTGATGTTCGCGACTATTCGAGTATCCTCAATGCTTCTCGA GGTGTTGATTTTATTTATCATGCTGCAGCTCTGAAGCAAGTACCTTCCTGCGAATTCCACCCAATGGAAGC TGTAAAAACGAATGTTTTAGGTACGGAAAACGTACTGGAAGCGGCAATAGCTAATGGAGTTAGGCGAATTG TATGTTTGAGTACAGATAAAGCTGTATATCCTATCAATGCAATGGGTATTTCCAAAGCGATGATGGAAAAA GTAATGGTAGCAAAATCGCGCAATGTTGACTGCTCTAAAACGGTTATTTGCGGTACACGTTATGGCAATGT AATGGCATCTCGTGGTTCAGTTATCCCATTATTTGTCGATCTGATTAAATCAGGTAGACCAATGACGATAA CAGACCCTAATATGACTCGTTTCATGATGACTCTCGAAGACGCTGTTGATTTGGTTCTTTACGCATTTGAA CATGGCAATAATGGTGATATTTTTGTCCAAAAGGCACCTGCGGCTACCATCGAAACGTTGGCTATTGCACT CAAAGAATTACTTAATGTAAACCAACACCCTGTAAATATAATCGGCACCCGACACGGGGAAAAACTGTACG AAGCGTTATTGAGCCGAGAGGAAATGATTGCAGCGGAGGATATGGGTGATTATTATCGTGTTCCACCAGAT CTCCGCGATTTGAACTATGGAAAATATGTGGAACATGGTGACCGTCGTATCTCGGAAGTGGAAGATTATAA CTCTCATAATACTGATAGGTTAGATGTTGAGGGAATGAAAAAATTACTGCTAAAACTTCCTTTTATCCGGG CACTTCGGTCTGGTGAAGATTATGAGTTGGATTCATAATATGAAAATTTTAGTTACTGGCGCTGCAGGGTT TATCGGTCGAAATTTGGT ATT C CGGCTTAAGG AAGCTGGATATAACGAACT C ATTACGATAGATC GTAACT CTTCTTTGGCGGATTTAGAGCAGGGACTTAAGCAGGCAGATTTTATTTTTCACCTTGCTGGGGTAAATCGT C C CGTGAAGGAGTGTGAATTTGAAGAGGGAAATAGTAATCTAACTCAACAGATTGTTGAT ATCCTGAAAAA AAACAATAAAAATACTCCTATCATGCTGAGTTCTTCCATCCAGGCTGAATGTGATAACGCTTATGGAAAGA GTAAAGCAGCTGCGGAAAAAATCATTCAGCAGTATGGGGAAACGACAAACGCTAAATATTATATTTATCGC TTGCCGAATGTATTCGGTAAGTGGTGTCGAC C AAATTATAACTCCTTTATAGCAACTTTCTGC CAT CG CAT TGCAAATGATGAAGCTATTACAATTAATGATCCTTCAGCAGTTGTAAATCTGGTGTATATAGATGACTTTT GTTCTGACATATTAAAGCTATTAGAAGGAGCGAACGAAACTGGTTACAGGACATTTGGTCCAATTTATTCT GTTACTGTTGGTGAAGTGGCACAATTAATTTACCGGTTTAAAGAAAGTCGCCAAACATTAATCACCGAAGA TGTAGGTAATGGATTTACACGTGCATTGTACTCAACATGGTTAAGTTACCTGTCTCCTGAACAGTTTGCGT ATACGGTTCCTTCTTATAGTGATGACAGAGGGGTATTCTGTGAAGTATTGAAAACGAAAAACGCGGGCCAG TTTTCGTTCTTTACTGCGCATCCAGGAATTACTCGGGGTGGTCATTATCATCATTCCAAAAATGAGAAATT TATTGTCATCCGAGGAAGTGCTTGTTTCAAATTTGAAAATATTGTCACGAGTGAACGATATGAACTTAATG TTTCCTCTGATGATTTTAAAATTGTTGAAACAGTTCCGGGATGGACGCATAACATTACTAATAATGGCTCG GATGAGCTAGTTGTTATGCTTTGGGCAAATGAAATATTTAATCGTTCTGAACCAGATACTATAGCGAGAGT TTTATCGTGAAAAAATTGAAAGTCATGTCGGTTGTTGGGACTCGTCCAGAAATTATTCGACTCTCGCGTGT CCTTGCÀAAATTAGATGAATATTGTGACCACCTTATTGTTCATACCGGGCAAAACTACGATTATGAACTGA ATGAAGTTTTTTTCAAAGATTTGGGTGTTCGCAAACCTGATTATTTTCTTAATGCCGCAGGTAAAAATGCA GCAGAGACTATTGGACAAGTTATCATTAAAGTTGATGAGGTCCTTGAACAGGAAAAACCAGAAGCCATGTT AGTACTTGGCGATACTAACTCCTGTATTTCAGCAATACCAGCAAAGCGTCGAAGAATTCCGATCTTCCATA TGGAGGCTGGGAATCGTTGTTTTGACCAACGCGTACCGGAAGAAACTAACAGAAAAATAGTTGATCATACC GCTGATATCAATATGACATATAGTGATATCGCGCGTGAATATCTTCTGGCTGAAGGTGTACCAGCCGATAG AATTATTAAAACCGGTAGCCCAATGTTTGAAGTACTCACTCATTATATGCCGCAGATTGATGGTTCCGATG TACTTTCTCGCCTGAATTTAACACCTGGGAATTTCTTTGTGGTÀAGTGCCCACAGAGAAGAAAATGTTGAT ACCCCTAAACAACTTGTGAAACTGGCGAATATACTTAATACCGTGGCTGAAAAATATGATGTCCCGGTAGT TGTTTCTACTCATCCTCGCACTCGTAACCGCATCAACGAAAACGGTATTCAATTCCATAAAAATATCTTGC TTCTTAAGCCATTAGGATTTCACGATTACAACCATCTGCAAAAAAATGCACGTGCTGTTTTATCGGATAGT GGGACTATTACAGAAGAGTCCTCCATTATGAACTTCCCTGCACTCAATATACGAGAAGCGCACGAACGCCC GGAAGGCTTCGAAGAAGGGGCAGTAATGATGGTCGGTCTTGAATCTGATCGCGTTTTACAGGCATTAGAAA TT ATTGCAACAC AGC CTCGTGGAGAAGTACGCTTACTTCGT C AGGTTAGTGACTAT AGC ATGC CAAATGTT T C AGATAAAGTTCTGCGT ATT AT C CATTC AT ATACTGACT ACGTTAAACGGGTTGTCTGGAAGCAATAC TA ATGAAACTTGCATTAATCATTGATGATTATTTGCCCCATAGCACACGCGTTGGGGCTAAAATGTTTCATGA GTTAGGCCTTGAATTACTGAGCAGAGGCCATGATGTTkACTGTAATTACGCCTGACATCTCATTACAAGCAA TTTATTCTATTAGTATGATTGATGGTATAAAGGTTTGGCGTTTCAAAAGTGGACCTTTAAAGGATGTAGGT AAGGCTAAACGTGCCATAAATGAAACTCTTTTATCTTTTCGCGCATGGCGCGCATTTAAGCACCTCATTCA ACATGATACATTTGATGGTATCGTTTATTATTCCCCCTCTATTTTTTGGGGCGACTTGGTTAAAAAAATAA AACAACGATGCCAGTGCCCAAGCTATCTGATCCTAAGGGATATGTTTCCACAGTGGGTCATTGATGCAGGT ATGTTGAAAGCCGGTTCACCAATTGAAAAATATTTTAGGTATTTTGAAAAAAAGTCATATCAGCAGGCTGG CCGGATAGGGGTAATGTCTGATAAGAATCTTGAGATATTTCGCCAGACCAATA^GGTTATCCGTGTGAAG
111
TTTTACGTAATTGGGCC TCAATGACTCCTGTGTCTGCCAGCGATGATTAT CATTCACTTCGTCAAAAATAC GATCTAAAAGATAAAGTCATTTTTTTCTATGGCGGTAATATTGGGCATGCTCAGGATATGGCAAACTTAAT GCGCCTTGCGCGTAATATGATGCGTTATCATGATGCTCATTTCCTGTTTATAGGGCAGGGTGATGAAGTTG AGCTGATAAAATCTCTTGCTGCAGAATGGAATTTAACTAATTTCACTCATCTACCTTCAGTGAACCAGGAA GAGTTTAAATTAATTTTATCTGAAGTTGATGTCGGCCTGTTCTCCCTTTCATCTCGCCATTCTTCACATAA TTTCC C CGGAAAATTACTAGGGTATATGGTTCAAT CAATCC CGATCCTTGGGAGTGTGAATGGCGG C AATG ATTTAATGGATGTAATTAATAAGCACAGAGCCGGTTTCATTCATGTTAATGGTGAAGATGATAAACTGTTT GAATCTGCACAATTGCTTCTTAGTGATTCAGTTTTAAGAAAACAGCTAGGTCAGAACGCTAATGTGTTGTT AAAGTCTCAATTTTCGGTTGAATCGGCGGCACATACTATCGAAGTCCGACTGGAGGCTGGAGAATGCGTTT AGTTGATGACAATATTCTGGATGAACTTTTTCGCACTGCAGCAAATTCTGAACGTTTGCGCGCTCATTATT TATTGCACGCATCTCATCAGGAGAAGGTTCAACGTTTACTTATTGCATTTGTACGCGACAGCTATGTTGAA C C C CATTGGCATG AGTT ACCG C ATC AGTGGGAAATGTTTGTCGTC ATGCAAGGGCAATTAGAAGTTTGTTT GTATGAGCAAAATGGTGAGATCCAAAAACAGTTTGTTGTTGGAGACGGTACGGGAATAAGCGTCGTGGAAT TTTCCCCAGGAGATATACATAGTGTCAAATGCCTGTCACCAAAAGCCCTTATGTTGGAGATAAAGGAGGGG CCATTTGACCCACTCAAAGCTAAGGCTTTTTCTAAGTGGTTATAGGGCGATACACCACCGTTTATTCTTCT ATCTTATTCTATACATGCTGGGTTACCATCTTAGCTTCTTCAAGCCGCGCAACCCCGCGGTGACCACCCCT GACAGGAGTAGCTAGCATTTGACCACCCCTGACAGGATTAGCTAGCATATGAGCTCGAGGATATCTACTGT GGGTACCCGGGATCCGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGG AATAGGAACTAAGGAGGATATTCATAT
SEO ID NO: 10 (example signal sequence for EPA carrier protein)
MKKIWLALAG LVLAFSASA
SEQ ID NO: 11 (example O1A rfb locus nucléotide sequence - O1A-EPA production strain stG VXN4411 and stLMTB 10217)
ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGAT ACCCAAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAG GGATCAAAGAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTAT GAGTTAGAATCACTCCTTGAGCAGCGCGTGAAGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCC GGGCGTGACCATTATGAACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGAC CTGCCATTGGTGACAACCCATTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCG CTACGTTACAACCTTGCTGCCATGATTGCACGTTTCAACGAAACGGGCCGCAGCCAGGTGCTGGCAAAACG TATGC CGGGTGACCT CTCTGAATACTCCGTCATCCAGACTAAAGAGC CGCTGGACCGTGAGGGTAAAGT CA GCCGCATTGTTGAATTTATCGAAAAACCGGATCAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGT CGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTGGAACGTACTCAGCCTGGTGCATGGGGACGTATTCA GCTGACTG ATGCTATTG C CG AGCTGGCGAAAAAACAAT C CGTTGATGCAATGCTGATGACCGGCGACAGTT ACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAGTATGGCCTACGCAACCTGAAAGAAGGG GCGAAGTT C CGTAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAATCTGAC CGGATGTAACGGTTGAT AAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAATCTTCCTGCCGTTGTTTTA TATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAGTTTTCCAGGATTTTCCTTGTTT CCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTAACGCTCGTCAC ATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCATTAATACCTCTATT AATCAAACTGAGAGCCGCTTATTTCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGCTAGCGTGAAG ATACTTGTTACTAGGGGCGCAGGATTTATTGGTTCTGCTGTAGTTCGTCACATTATAAATAATACGCAGGA TAGTGTTGTTAATGTCGATAAATTAACGTACGCCGGAAACCTGGAATCACTTGCTGATGTTTCTGACTCTG AACGCTATGTTTTTGAACATGCGGATATTTGCGATGCTGCTGCAATGGCGCGGATTTTTGCTCAGCATCAG CCGGATGCAGTGATGCACCTGGCTGCTGAAAGCCATGTGGATCGTTCAATTACAGGCCCTGCGGCATTTAT TGAAACCAATATTGTTGGTACTTATGTCCTTTTGGAAGCGGCTCGCAATTACTGGTCTGCTCTTGATGGCG ACAAGAAAAATAGCTTC CGTTTTCATCATATTTCTACTGACGAAGTCTATGGTGATTTGCCT CAT CCTGAC GAAGTAAATAATAAAGAACAATTACCCCTCTTTACTGAGACGACAGCTTACGCGCCTAGTAGTCCTTATTC CGCATCAAAAGCATCCAGCGATCATTTAGTCCGCGCGTGGAAACGTACCTATGGTTTACCGACTATTGTGA CTAACTGTTCGAATAACTACGGTCCTTATCACTTTCCGGAAAAATTGATTCCACTAGTAATTCTTAATGCT CTGGAAGGTAAGGCATTACCTATTTATGGCAAAGGGGATCAAATTCGTGACTGGCTGTATGTTGAAGATCA TGCGCGTGCGTTATATACCGTAGTTACTGAAGGTCAAGCGGGTGAAACCTATAACATTGGCGGACACAACG
112
AAAAGAAAAACATCGATGTTGTGCTGACTATTTGTGATTTGTTGGACGAGATAGTCCCGAAAGAGAAATCT TATCGTGAGCAAATTACTTATGTTGCTGATCGCCCAGGGCATGATCGCCGTTATGCGATTGATGCTGAGAA GATTGGTCGCGAATTGGGATGGAAACCACAGGAAACGTTTGAGAGTGGGATTCGTAAAACGGTGGAATGGT ATTTGGCTAATGCAAAATGGGTTGATAATGTGAAAAGTGGTGCCTATCAATCGTGGATTGAACAGAACTAT GAGGGCCGCCAGTAATGAATATCCTCCTTTTTGGCAAAACAGGGCAGGTAGGTTGGGAACTACAGCGTGCT CTGGCACCTCTGGGTAATTTGATTGCTCTTGATGTTCACTCCACTGATTACTGTGGTGATTTTAGTAACCC TGAAGGTGTGGCTGAAACAGTCAAAAGAATTCGACCTGATGTTATTGTTAATGCTGCGGCTCACACCGCAG TAGATAAGGCTGAGTCAGAACCCGAATTTGCACAATTACTCAATGCGACTAGCGTTGAATCAATTGCAAAA GCGGCAAATGAAGTTGGGGCTTGGGTAATTCATTACTCAACTGACTACGTATTCCCTGGAAATGGCGACAC GCCATGGCTGGAGATGGATGCAACCGCACCGCTAAATGTTTACGGTGAAACCAAGTTAGCTGGAGAAAAAG CATTACÀAGAGCATTGTGCGAAGCACCTAATTTTCCGTACCAGCTGGGTCTATGCAGGTAAAGGAAATAAT TT CGCCAAAACGATGTTG CGT CTGGC AAAAGAGCGTGAAGAACTAGC CGTTATTAATGAT C AGTTTGGTGC GCCAACAGGTGCTGAACTGCTGGCTGATTGTACGGCACATGCCATTCGTGTCGCACTGAATAAACCGGATG TCGCAGGCTTGTACCATTTGGTAGCCAGTGGTACCACAACCTGGTACGATTATGCTGCGCTGGTTTTTGAA GAGGCGCGCAATGCAGGCATTCCTCTTGCACTCAACAAGCTCAACGCAGTACCAACAACTGCCTATCCTAC ACCAGCTCGTCGTCCACATAACTCTCGCCTTAATACAGAAAAATTTCAGCAGAATTTTGCGCTTGTATTGC CTGACTGGCAGGTTGGTGTGAAACGCATGCTCAACGAATTATTTACGACTACAGCAATTTAATAGTTTTTG CATCTTGTTCGTGATGGTGGAGCAAGATGAATTAAAAGGAATGATGAAATGAAAACGCGTAAAGGTATTAT TTTAGCGGGTGGTTCTGGTACTCGTCTTTATCCTGTGACTATGGTCGTCAGTAAACAGCTATTACCTATAT ATGATAAACCGATGATCTATTATCCGCTTTCTACACTGATGTTAGCGGGTATTCGCGATATTCTGATTATT AGTACGCCACAGGATACTCCTCGTTTTCAACAACTGCTGGGTGACGGTAGCCAGTGGGGCCTGAATCTTCA GTACAAAGTGCAACCGAGTCCGGATGGTCTTGCGCAGGCATTTATTATCGGTGAAGAGTTTATTGGTGGTG ATGATTGTGCTTTGGTACTTGGTGATAATATCTTCTACGGTCACGACCTGCCTAAGTTAATGGATGCCGCT GTTAACAAAGAAAGTGGTGCAACGGTATTTGCCTATCACGTTAATGATCCTGAACGCTATGGTGTCGTTGA , GTTTGATAAAAACGGTACGGCGATCAGCCTGGAAGAAAAACCGCTACAACCAAAAAGTAATTATGCGGTAA CCGGGCTTTATTTTTATGATAACGACGTTGTCGAAATGGCGAAAAATCTTAAGCCTTCTGCCCGCGGTGAA CTGGAAATTACCGATATTAACCGTATCTATATGGAACAAGGGCGTTTATCTGTTGCCATGATGGGGCGTGG TTATGCGTGGTTAGACACGGGGACACATCAGAGCCTGATTGAGGCAAGCAACTTTATTGCAACAATTGAAG AGCGTCAGGGGCTGAAAGTTTCCTGCCCGGAAGAAATTGCTTACCGTAAAGGGTTTGTTGATGCTGAGCAG GTGAAAGTATTAGCTGAACCTCTGAAAAAAAATGCTTATGGTCAGTATCTGCTGAAAATGATTAAAGGTTA TTAATAAAATGAACGTAATTAAAACAGAAATTCCTGATGTACTGATTTTTGAACCGAAAGTTTTTGGTGAT GAGCGTGGTTTCTTTTTTGAGAGCTTTAACCAGAAGGTTTTTGAGGAAGCTGTAGGCCGCAAAGTTGAATT TGTTCAGGATAACCATTCGAAGTCTAGTAAAGGTGTTTTACGCGGGCTGCATTATCAGTTGGAACCTTATG CACAAGGAAAATTGGTGCGTTGCGTTGTCGGTGAAGTTTTTGACGTAGCTGTTGATATTCGTAAATCGTCA TCGACTTTTGGCAAATGGGTTGGGGTGAATTTATCTGCTGAGAATAAGCGGCAATTGTGGATTCCTGAGGG ATTTGCACATGGTTTTTTAGTGCTGAGTGAGACGGCGGAGTTTTTGTATAAGACGACAAATTATTATCATC CT C AGAGTGATAGAGGAATAAAATGGGATGAT C CAAGCATCAATATTT C ATGGCC AGTCGATTCACAAGTG CTGCTATCAGCTAAAGATAATAAGCATCCTCCATTAACAAAGATTGAAATGTATAGTTAAGATCACGATAA ATCTTGGAAGGGTTGCAAAATTGAATAAAATAGTGAGCAAAAGTGAAATAAGGAACGTAATCCACAATGCT GGCTATATGATGATTACTCAGATAGCTTTATATGTTGCACCATTATTTATACTGAGTTATCTGTTAAAAAC ACTGGGGGTTGCACAGTTTGGTAATTATGCCTTAATACTATCAATCGTTGCATATTTACAGATTATAACGG ATTATGGTTTTTCTTTTAGTGCAAGTCGTGCGATCTCACAGAATAGAGAGGACAAAGAATATATATCAAAA ATTTATCTGTCAACTATGACTATCAAGTTGGCGATATGCGCTTTCTTATTCTTATTGCTCATGCTATTTTT AAATCTTTTGCCTGTGCAAGCTGAATTAAAACAAGGAATATTATATGGATATCTTCTTGTAATAGGAAATA CTTTC C AACC AC AATGGTTTTTC CAAGGT ATCGAAAAATTAAAAATCAT AGC C CTTTCTAATGTTATATC A AGATGCGCCGCGTGTTTACTTGTATTTATCTATGTGAGGAATAGCGAGGATTTACAAAAAGCACTTTTAGT ACAGTCACTTCCATTAGTAATTTCTGCGATTGGATTAAATATATTTATATTGAAATATATCAATATTATTT TTCCGGAAAAAAAATTATTTAAGGTAATTTTAAAAGAAGGTAAGGATTTTTTTCTTGCATCACTTTATTCT GTTATTCTCAATAATAGTGGCATTTTTCTATTAGGGATTTTTACTAATCCTGTTATTGTTGGTGTATATGC CGC CGCTGAAAAGAT AGT CAAGGC CGTATTGT CGCTATTTACACC ACTGACGCAAGC TATAT ATCC TT ATA ATTGTCGTAAGTTTT CACTAT CCGTATTTGACGGCATTGAGGCAGCAAAAAAAACTGGTATACCAATT ATA ATTTTAGCATTTATAGCTGCTGTTATCGTTGCAATTACCTTACCTGTTGCÀATCGACTATCTTAATTTTCC AAAAGAAACAATTTTTGTAGGTCAAATATTAAGTGCATGGATCTTTTTTGGTGTTCTTAATAATGTATTCG GCATTCAGATATTGAGTGCATCAGGAAGAAGTAAAATATATAGTAGGATGGTATTCGTATCAGCGCTTATA ACATTACTTTTGATTACTCTATTATTGCAGTTTTGTAACGCCACTGGAGTGGCATGTGCAATATTATTGGG TGAAATGTTCTTATCAATATTGTTACTTAAGCGATATAAAAAAATAATTTAAGGAATAGTTATGAAGAAGT
113
TATTATTAGTGTTCGGTACTAGGCCTGAAGCAATAAAGATGGCCTCTATCATTGAATTATTAAAAAAAGAT TGTAGATTCGAATATAAAATATGTGTGACAGGCCAACATAAAGAGATGCTTGATCAAGTTATGCAAGTATT TGATGTTAAACCTGATTATAATTTACGGATTATGCAGCCTGGGCAAACATTAGTATCTATAGCAACAAATA TACTCTCACGGTTAAGTGAAGTTTTAATTATAGAAAAGCCAGATATTATACTTGTGCATGGGGATACAACG ACTACCCTTGCTGCTACTTTAGCTGGGTATTACCACCAAATAAAAGTTTGTCATGTGGAAGCAGGATTAAG AACAGGGGATATTTACTCTCCTTGGCCTGAAGAGGGCAATCGTAAAGTTACAGGGGCATTAGCATGTATTC ATTTCGCCC C AACAGAGAGATCAAAAGATAATCTCCTGAGGGAGGGGGTCAAAGTAAATAATATATTTGTA ACGGGTAATACCGTCATCGACTCTTTATTTATTGCAAAAGATATCATAGATAATGACCCTAATATAAAGAA CGCTTTACATAATAAATTTAATTTTCTTGATAAAAGCCGACGAGTAGTACTTATAACAGGTCATCGAAGAG AAAATTTCGGGAAAGGTTTTGAAGATATATGCTTTGCAATAAAGGAATTAGCTTTCATTTATCCTAATGTA GATTTTATTTATCCGGTGCATCTTAATCCCAATGTAATGGAACCAGTACATCGTATATTAGATAATATATG TAATATTTACCTTATTGAGCCCTTGGATTATTTGCCTTTTGTTTATTTAATGAATGAGTCATATTTAATAT TGACTGATTCAGGGGGGATACAAGAAGAAGCGCCTTCGTTAGGTAAACCGGTTTTGGTTATGCGTGATACT ACTGAACGCCCTGAGGCGGTTGAGGCTGGTACTGTTGTATTAGTGGGGACTTCTAAGATAAAAATAGTAAA TAAAGTAACGGAGCTATTAAACAATGCTGATATCTACAATGCTATGTCTCTGTTACATAATCCATATGGCG ATGGAACAGCTGCTCAAAAAATTCTTAATGTGCTCGCCCAAGAGCTAATTTAATTTAAGCTAAAAATATGT TATTAATTATTGCTGATTATCCAAACGAAATGAATATGCGCGAGGGAGCTATGCAACGAATAGATGCGATA GACTCTCTCATTCGAGATCGCAAGCGAGTGTATTTGAATATTTCATTCAAAAAGCATCTAGTTCGCTCAAA TAGTTCCTTTAATAATGTTATAGTTGAAAATCTAAATGCAATTATTCACAGAAACATCATAAAACAGTACA TGCAAAAATCAACAACTATATATGTTCATTCTGTTTATAATTTATTAAAGGTTATAACGCTCATTGATCTA AAAAAAACAATTCTTGATATACATGGTGTTGTACCGGAAGAACTTTTGGCAGATAATAAAAAATTACTTAG TAAAGTATATAACATGGTGGAAAAAAAAGGTGTCCTTGGATGCAAAAAATTAATACACGTCAGTACAGAAA TGCAAAAACACTATGAAGCAAAATATGGAGTAAACTTGGCTGAAAGGTCAATAGTGCTCCCGATTTTTGAA T ATAAAAAT ATAAC C CAATCGCAAAACAAATGGACAGAAAATAAAAT ACGAAGTAT CTATCTTGGAGGATT ACAAACATGGCAAAATATTGATAAAATGATTCAAGTTTGTGATGACACAGTGATAAACAATGAAGCAGGTA AGTATGAATTCAACTTTTTCATCCCACAGAGTAACTTGGAAGGGTTTATAGATAAATATTCGTTAAAATTA CATAATATCAATGCTAATGCATCTACGCTATCACGTGATGAAGTAATTCCCTTTCTAAAAGAATGTCATAT TGGTTTTGTATTGCGCGATGATATAATAGTAAACAGAGTTGCGTGCCCTACAAAATTGGTTGAATATTTAG AGTGTGGTGTCGTTCCAGTTGTGCTCTCCCCACTTATAGGTGATTTTTATTCGATGGGATATCAATACATT ACTACAGAGGAAATGGCTAACAGAAGTATAAGTTTGTTGGATCTTGAAAAAATGGCTGCACATAATTTACA AATTTTGACTTCTTATCAGAAGAGAACCTACAAGGCACAGAAAGAACTTATTGCTCAACTGTGCTGAATTT TTTACATATATAAAATTATGTAAGCATATCGCGGGTCAGGTAATTGTATGCGTATCAAATATAAAGATAAC GGTTATATATTATGTTTTCTATTATGTTTCATTTTGAGCTACTTAGTTTTACTCAAATCTGACTACTTTCC TGCTGATTTTCTGCCATATACAGAAATATACGATGGGACATACGGAGAAATCAATAATATTGAGCCTGCCT TTTTATATTTAACACGGTTGTTTCATTATTTAAATTTCCCCTATATATTTTTTGCAATGTTAGTTTGTGCC TTATGTTTAAGTTGGAAAATAAAATATGCAAGAAAAATAATTAAAGATAGTTATATATATTTGTTCTTGTA TGTATATGTATCATTTTATGTGTTTTTGCATGAAATGACTCAATTGCGCATAGCAATTGCAGTCACTATGT GCTATGTGTCGGTTTATTATTACTTTTATAAAAATTGTATTAAACATGCACTGCCATGGATGGTGTTGGCT ATTTTGTTTCATTACAGCGCCTTGCTTTTATTTATGTCATTATTTATATACAGTTATAGGAGGTTATTAAT AGTAATTATAGGGTTTGTAATATGTATGAGCTTTTTAAACGTGTATGCAGATACAATTGCACTATATTTGC CAAATGAAAAZÏATAGTAAATTATTTATATAGTATTTCATCATCATTAGACAATAGAAATGATTTGGCAATA TTCAACCTGAATAATATAATATTTTTATCAATATTTATTTTGATCTTTTATCTTAGCCGATATATAAAATT AAATGATAATGAGGCGAAGTTTATTAAGTATGTGCAATGTTCAGGAATATTAGCCTTTTGTATTTTCTTTC TGGCTAGTGGAGTCCCGGTCATTGCTTATCGAACTGCAGAGTTGCTGCGAATATTTTATCCGATGGCTTTA GTATTAATCCTTTCGCATATAAAAAATAATAATATGCGTTATTTTATTGCAGTCATTATAGTTATCCTTTC AGGCTTAATGTTGTTTATAACACTAAGGGCTGTATCAATAGTTGGTCAAGGATTATAAAATGAATGTTGCT ATTTTGTTGTCTACGTATAATGGCGAAZÏAATATTTAGAGGAACAACTGGATTCATTGCTGCTTCAAAGTTA TCAGGATTTTGTAGTGTATATCCGTGATGACGGATCATCTGATAGAACTGTAAATATAATAAACCAATACG TAATGAAAGATAACAGATTTATTAACGTGGGTAATTCAGAAAATCTTGGTTGTGCTGCTTCGTTTATTAAT TTATTAAGAAATGCTTCAGCCGATATTTATATGTTTTGTGACCAAGATGATTATTGGCTTCCGAATAAATT ACAGCGTGCTGTGGATTATTTTTCGGCTATTGATCCTTTACAACCTACCTTGTATCATTGCGATCTAAGCG TTGTTGATGAAAAACTTAATATTATACAAAATT CATTTTTGCAGCATCAGAAAATGT CAGCGTATGATT CA ATGAGAAAAAATAATCTTTTCATACAAAATTTTGTTGTTGGTTGTTCATGTGCTGTTAATGCTTCACTTGC GGAATTTGTTCTTTCGCGAATTGGAGAGCAGCATGTAAAAATGATAGCTATGCATGACTGGTGGTTAGCCG TGACTGCAAAACTTTTTGGT CGAATC CATTTTGATAATACTCAAACGATT CTTTATCGACAACATCAGGGC AATGTATTAGGTGCAAAATCATCAGGTATGATGCGTTTTATTCGATTAGGATTAAATGGGCAAGGGATTTC
114
GCGAGTAGTATCTTTTAGAAAAAAAGTTTGTGCGCAAAATAAGCTTCTTTTAGATGTCTATGATAAAGATT TAAATCTTGAGCAAAAAAAATCTATCAGGCTTGTAATTGAGGGCCTTAAAGAGAACTCTTCAATTGCTGAC CTTTTAAAATGTTTCTATCATGGTAGCTATATGCAAGGTTTTAAACGTAATCTTGCCTTAATATATTCAGT TCTTTACACAAAAAAAAGAAGATAGTGTATCCTTATGAAAAAAATTGCTATTATCGGTACTGTTGGCATAC CAGCATCATATGGCGGATTTGAAACATTAGTTGAAAATTTAACAAGATACAATTCCTCGGGAGTTGAATAT AATGTTTTTTGTTC ATCGTTT CACTACAAAT CC CACCAAAAAAAACATAATGGGGCCCGTTT AATTTAT AT TCCGCTTAAAGCCAATGGATGGCAGAGCATTGCGTATGACATAATTTCGTTAGCATATTCTATTTTTTTGA AGCCTGATGTGATTCTGATTTTAGGGGTTTCTGGTTGTTCATTTTTGCCTTTCTTCAAACTCTTAACACGC GCTAAGTTTATTACTAATATTGATGGCCTGGAATGGCGAAGAGATAAATGGAATTCAAAAGTGAAACGTTT CTTAAAATTTTCAGAAAAAATCGCAGTTCAATATTCGGATGTCGTTATTACGGATAATGAGGCAATTTCTG AGTACGTTTTTAACGAGTATAATAAAGATAGCCGAGTTATTGCCTATGGAGGGGATCATGCATGGTTAAAT ACTGAGGATGTATTTACAACAAGAAATTATAAAAGCGATTACTACCTTTCTGTATGTCGTATCGAACCCGA AAACAATGTAGAATTAATTTTAAAAACATTTTCAAAGCTAAAATATAAAATAAAATTTATTGGAAATTGGA ATGGCAGCGAGTTTGGAAAGAAACTTAGGCTGCATTATTCTAACTATCCAAATATTGAAATGATTGATCCG ATTTATGATCTTCAACAATTATTTCACTTACGAAATAATTGCATAGGATATATACATGGTCATTCGGCTGG AGGAACAAACCCTTCTTTAGTCGAGGCAATGCATTTTAGTAAACCTATATTTGCATATGATTGTAAGTTTA ATAGGTACACTACTGAAAATGAAGCATGTTATTTTTCTAATGAATCTGACCTCGCAGAGAAAATCATAATG CATTGTGAGCTATCATTAGGTGTCTCTGGCACGAAAATGAAAGAAATTGCTAACCAGAAATACACTTGGAG ACGAATAGCAGAAATGTATGAGGATTGCTATTAACTCTGTTAAACTTCAAATCTTTTACAATATATGGCAT GACTATAAGCGCATTAATTGTTTTTCAAGCCGCTCTCGCGGTGACCACCCCCTGACAGGGGATCCGTGTAG GCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTAAGGAGGATAT TCATATGGATAAAGCCGTAAGCATATAAGCATGGATAAGCTATTTATACTTTAATAAGTACTTTGTATACT TATTTGCGAACATT CC AGGC C GCGAGCATTCAGCGCGGTGATCACACCTGACAGGAGTATGTAATGTC CAA GCAACAGATCGGCGTAGTCGGTATGGCAGTGATGGGACGCAACCTTGCGCTCAACATCGAAAGCCGTGGTT AT ACC GTCTCTATTTTCAACCGTTC C CGTGAGAAGACGGAAGAAGTGATTGCCGAAAATCCAGGCAAGAAA CTGGTTCCTTACTATACGGTGAAAGAGTTTGTCGAATCTCTGGAAACGCCTCGTCGCATCCTGTTAATGGT GAAAGCAGGTGC AGGCACGGATGCTGCTATTGATTCCCTCAAAC CATATCTCGATAAAGGAG ACAT CAT CA TTGATGGTGGTAACACCTTCTTCCAGGACACTATTCGTCGTAATCGTGAGCTTTCAGCAGAGGGCTTTAAC TTCATCGGTACCGGTGTTTCTGGCGGTGAAGAGGGGGCGCTGAAAGGTCCTTCTATTATGCCTGGTGGCCA gaaagaagcctatgaattggtagcaccgatcctgaccaaaatcgccgccgtagctgaagacggtgaaccat GCGTTACCTATATTGGTGCCGATGGCGCAGGTCACTATGTGAAGATGGTTCACAACGGTATTGAATACGGC GATATGCAGCTGATTGCTGAAGCCTATTCTCTGCTTAAAGGTGGCCTGAACCTCACCAACGAAGAACTGGC GC AGAC CTTTACCGAGTGGAATAACGGTGAACTGAGCAGTTAC CTG ATCGACATCACCAAAG AT AT CTT CA CCAAAAAAGATGAAGACGGTAACTACCTGGTTGATGTGATCCTGGATGAAGCGGCTAACAAAGGTACCGGT AAATGGACCAGCCAGAGCGCGCTGGATCTCGGCGAACCGCTGTCGCTGATTACCGAGTCTGTGTTTGCACG TTATATCTCTTCTCTGAAAGATCAGCGTGTTGCCGCATCTAAAGTTCTCTCTGGTCCGCAAGCACAGCCAG CAGGCGACAAGGCTGAGTTCATCGAAAAAGTTCGTCGTGCGCTGTATCTGGGCAAÀATCGTTTCTTACGCC CAGGGCTTCTCTCAGCTGCGTGCTGCGTCTGAAGAGTACÀACTGGGATCTGAACTACGGCGAAATCGCGAA GATTTTCCGTGCTGGCTGCATCATCCGTGCGCAGTTCCTGCAGAAAATCACCGATGCTTATGCCGAAAATC CACAGATCGCTAACCTGTTGCTGGCTCCGTACTTCAAGCAAATTGCCGATGACTACCAGCAGGCGCTGCGT GATGTCGTTGCTTATGCAGTACAGAACGGTATTCCGGTTCCGACCTTCTCCGCAGCGGTTGCCTATTACGA CAGCTACCGTGCTGCTGTTCTGCCTGCGAACCTGATCCAGGCACAGCGTGACTATTTTGGTGCGCATACTT ATAAGCGTATCGATAAAGAAGGTGTGTTCCATACCGAATGGCTGGATTAA
SEQ ID NO: 12 (example 02 rfb locus nucléotide sequence - 02-EPA production strain stGVXN4906')
ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGAT ACCCAAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAG GGATCAAAGAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTAT GAGTTAGAATCACTCCTTGAGCAGCGCGTGAAGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCC GGGCGTGACCATTATGAACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGAC CTGCCATTGGTGACAACCCATTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCG CTACGTTACAACCTTGCTGCCATGATTGCACGTTTCAACGAAACGGGCCGCAGCCAGGTGCTGGCAAAACG TATGCCGGGTGACCTCTCTGAATACTCCGTCATCCAGACTAAAGAGCCGCTGGACCGTGAGGGTAAAGTCA GCCGCATTGTTGAATTTATCGAAAAACCGGATCAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGT CGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTGGAACGTACTCAGCCTGGTGCATGGGGACGTATTCA
115
GCTGACTGATGCTATTGCCGAGCTGGCGAAAAAACAATCCGTTGATGCAATGCTGATGACCGGCGACAGTT ACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAGTATGGCCTACGCAACCTGAAAGAAGGG GCGAAGTTCCGTAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAATCTGACCGGATGTAACGGTTGAT AAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAATCTTCCTGCCGTTGTTTTA TATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAGTTTTCCAGGATTTTCCTTGTTT CCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTAACGCTCGTCAC ATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCATTAATACCTCTATT AATCAAACTGAGAGCCGCTTATTTCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGTGAAAATACTT GTTACTGGTGGCGCAGGATTTATTGGTTCAGCTGTAGTTCGTCACATTATAAATAATACGCAGGATAGTGT TGTTAATGTCGATAAATTAACGTACGCCGGAAACCGGGAATCACTTGCTGATGTTTCTGATTCTGAACGCT ATGTTTTTGAACATGCGGATATTTGCGATGCACCTGCAATGGCACGGATTTTTGCTCAGCATCAGCCGGAT GCAGTGATGCACCTGGCTGCTGAAAGCCATGTTGACCGTTCAATTACAGGCCCTGCGGCATTTATTGAAAC CAATATTGTTGGTACTTATGTCCTTTTGGAAGCCGCTCGCAATTACTGGTCTGCTCTTGATAGCGACAAGA AAAATAGCTTCCGTTTTCATCATATTTCTACTGACGAAGTCTATGGTGATTTGCCTCATCCAGATGAAGTA AATAATACAGAAGAATTACCCTTATTTACTGAGACGACAGCTTACGCGCCAAGCAGCCCTTATTCCGCATC CAAAGCAT CCAGCGATCATTTAGT C CG CG C ATGGAAACGTACGTATGGTTT ACCGACC ATTGTGACTAATT GCTCGAAC AACTATGGT CCGT AT C ACTTC C CGGAAAAGCTTATT CC ATTGGTT ATTCTTAATGCACTGGAA GGTAAGGCATTACCTATTTATGGCAAAGGGGATCAAATTCGCGACTGGTTGTATGTAGAGGATCATGCTCG TGCGTTATATACCGTCGTAACCGAAGGTAAAGCGGGTGAAACTTATAACATTGGCGGACACAACGAAAAGA AAAACATCGATGTTGTGCTGACTATTTGTGATTTGTTGGATGAGATTGTACCGAAAGAGAAATCTTATCGT GAGCAAATTACTTATGTTGCTGATCGCCCAGGGCATGATCGCCGTTATGCAATTGATGCCGATAAAATTAG CCGCGAATTGGGCTGGAAACCACAGGAAACGTTTGAGAGCGGGATTCGCAAAACGGTGGAATGGTATCTGG CTAATACAAATTGGGTTGAGAATGTGAAAAGCGGTGCTTATCAGTCATGGATCGAACAAAACTATGAGGGC CGTCAGTAATGAATATCCTGCTTTTCGGCAAAACAGGGCAGGTGGGTTGGGAACTGCAGCGTGCTCTGGCG CCGCTGGGTAATCTGATCGCTCTTGATGTTCACTCCACTAATTATTGTGGAGATTTCAGCAACCCCGAAGG TGTGGCAGAAACCGTCAAAAAAATTCGTCCTGACGTTATTGTTAATGCTGCTGCTCACACTGCAGTAGATA AAGCAGAATCAGAACCGGATTTCGCACAATTACTTAACGCGACAAGCGTCGAAGCGATTGCAAAAGCTGCT AATGAAGTCGGGGCCTGGGTTATACACTACTCTACTGATTATGTTTTCCCAGGCAGTGGTGACGCGCCATG GCTGGAAACGGATGCAACAGCACCGCTAAATGTTTACGGTGAAACAAAATTAGCTGGGGAAAAGGCATTAC AAGAACATTGCGCAAAGCATCTTATTTTCCGTACCAGCTGGGTATACGCTGGTAAAGGAAATAACTTTGCT AAAACGATGTTGCGTTTGGCAAAAGAACGCGAAGAACTGGCTGTGATAAACGATCAGTTTGGCGCACCAAC AGGTGCTGAATTGCTGGCTGATTGCACCGCTCATGCCATTCGCGTGGCATTAAAAAAACCAGAAGTCGCTG GCTTGTACCATCTGGTAGCAAGTGGCACAACAACCTGGCACGATTATGCTGCGCTGGTTTTTGAAGAGGCG CGCAAAGCAGGGATTAATCTTGCACTTAACAAACTTAACGCCGTGCCAACAACGGCCTATCCCACACCAGC CCGTCGACCCCATAACTCTCGCCTCAATACAGAAAAGTTTCAGCAGAACTTTGCGCTTGTCTTGCCTGACT GGCAGGTGGGCGTGAAACGTATGCTCAACGAATTATTTACGACTACGGCAATTTAACAAATTTTTGCATCT CG CTCATGATGC C AGAGCGGG ATGAATTAAAAGGAATGGTGAAATGAAAACGC GTAAAGGTATTATTCTGG CTGGTGGTTCCGGCACTCGTCTTTATCCTGTGACGATGGCAGTGAGTAAACAATTGCTGCCGATTTATGAT AAGCCGATGATTTATTATCCGCTTTCAACGCTTATGTTAGCGGGTATTCGCGATATTCTTATTATTAGTAC GC C AC AGGATACAC CGCGTTT C CAAC AATTATTGGGGGACGGGAGCC AGTGGGGTCTTAATCTAC AGT AT A AAGTACAACCGAGTCCGGATGGCCTGGCGCAAGCGTTTATTATTGGCGAAGACTTTATTGGTGGTGATGAT TGTGCACTCGTACTTGGCGATAATATCTTCTATGGACACGACTTGCCGAAATTGATGGAAGCTGCTGTTAA CAAAGAAAGCGGTGCAACGGTATTTGCTTATCACGTTAATGATCCTGAACGCTATGGTGTCGTGGAGTTTG ATAATAACGGTACGGCZkATTAGCCTGGAAGAAAAACCGCTGGAGCCAAAAAGCAACTATGCGGTTACTGGG CTTTATTTCTATGACAATGACGTTGTGGAAATGGCTAAAAACCTTAAGCCTTCTGCCCGTGGCGAACTGGA AATTACCGATATTAACCGTATTTATATGGAACAAGGACGTTTGTCTGTAGCCATGATGGGGCGTGGCTATG CATGGTTGGATACAGGGACGCATCAAAGCCTTATTGAAGCAAGTAACTTCATTGCAACAATTGAAGAGCGT CAGGGATTAAAGGTATCTTGCCCGGAAGAGATTGCTTACCGTAAAGGGTTTATTGATGCCGAGCAGGTGAA AGT ATTAG C CGAAC CGCTT AT CAAGAATCAATATGGTC AAT ATTTGCTGAAAATGATCAGCGAATAGT AT A TGGGAACTCAATGATGGATATTAAATTAATCTCTTTGCAAAAACATGGGGATGAGCGCGGTGCATTAATTG CTCTTGAAGAGCAACGAAATATACCTTTCGAAGTCAAAAGAATATATTACATACTTGAGACTCTTAATGGA GTAAGACGCGGATTTCATGCGCACAAGGTTACTCGTCAGTTAGCTATTGTAGTCAAGGGAGCTTGTAAATT TCATCTGGATAATGGTAAAGAAACAAAGCAGGTGGAACTTAATGATCCAACAATTGCGTTGCTGATAGAAC CCTATATATGGCATGAZkATGTATGATTTTAGTGATGATTGTGTGCTGCTTGTAATTGCGGATGATTTCTAT AAAGAGTCTGATTATATCCGCAATTATGATGATTTTATTAGAAGAGTAAATTCAATTGAGAATTCATAAGC TAAGTGACGTCCAGACAACATCAATTGGTGATGGAACAACTATCTGGCAGTTTGTTGTGATACTAAAAGGT
116
GCTGTAATTGGTAATAATTGCAACATCTGTGCAAATACCTTAATTGAAAATAACGTTGTAATTGGTAACAA TGTCACAGTCAAAAGCGGTGTGTATATTTGGGATGGCGTTAAAATAGAGGATAATGTTTTTATTGGTCCTT GTGTAGCATTTACAAATGATAAGTATCCTCGCTCTAAAGTCTATCCTGATGAATTTTTGCAAACAATAATA CGCAAAGGAGCATCAATAGGTGCTAACGCAACCATCCTGCCAGGAATTGAAATTGGTGAAAAAGCAATCGT TGGTGCGGGGAGTGTTGTAACCAAAAATGTACCGCCATGCGCAATAGTAGTAGGTAATCCAGCTCGATTTA TTAAATGGGTAGAGGATAATGAATAAAATTGATTTTTTAGATCTTTTTGCAATTAACCAGCGACAGCACAA AGAATTAGTCTCTGCGTTTAGTAGGGTGCTAGATTCTGGTTGGTATATCATGGGCGAAGAACTTGAGCAGT TCGAGAAAGAGTTCGCAGAATACTGTGGAGTTAAGTATTGCATTGGTGTAGCAAATGGCCTTGATGCGTTG ATACTAGTATTGAGGGCATGGAAAGAACTTGGCTATCTTGAAGACGGTGACGAGGTATTAGTACCGGCAAA TACATATATTGCTTCTATTCTTGCTATAACAGAGAACAAACTTGTTCCTGTTCTTGTTGAACCAGATATAG AAACTTATAATATTAATCCTGCTTTAATTGAAAATTACATTACGGAAAAAACTAAAGCAATATTACCGGTT CACTTATATGGTCTATTGTGCAATATGCCAGAAATTAGTGCAATCGCCAGAAAATATAATCTGTTGATTCT TGAAGATTGTGCACAAGCACATGGTGCAATACGTGATGGTCGCAAAGCTGGAGCTTGGGGGGATGCTGCAG GATTTAGTTTTTATCCAGGAAAAAACCTTGGAGCTTTGGGGGATGCGGGAGCTGTTACTACAAATAATGCA GAATTATCCTCAACTATAAAAGCTTTGCGAAATTATGGGTCACATAAGAAATATGAAAATATTTATCAGGG ATTGAATAGTCGATTGGATGAACTGCAAGCAGCCTTATTGCGTGTAAAAATCCATACATTACCGGAAGATA CTGCGATTCGGCAAAGGATTGCTGAAAAATATATTCGTGAAATAAAAAACCCTGCGATTACGTTACCAGTG TACGAAGGCCAAGGTGCGCATGTTTGGCATTTATTTGTAGTAAGAATCGCTAATCGTGAAAAATTCCAGTC ATACTTATTAGAGAAGGGTATCAAAACCTTAATTCACTATCCATTACCACCCCATAAGCAGCAAGCATATC AAAATATGTCTAGCCTTAGCCTTCCAATTACTGAGCAAATTCATGATGAAGTCATTTCTTTACCTATAAGT CCGGTAATGAGTGAAGATGATGTCAATTATGTAATCAAAATGGTCAATGATTACAAGTAATGAAAAAATTT CTTCAGGTAACTATATTATCCGCTATCTATACATTCATTAAAATGATTGCGGGTTTTATCATCGGTAAGGT AGTAGCAATTTATACAGGGCCATCAGGGGTAGCAATGCTTGGCCAAGTGCAAAGTTTAATCACAATAGTTG CAGGTACTACCTCTGCACCTGTAAGCACAGGCCTTGTTCGATATACTGCGGAAAATTGGCAAGAAGGACAA GAAGCATGCGCGCCATGGTGGCGCGCATGCTTAAGGGTTACTCTGTTTTTATTCTTGCTTATTATTCCCGT TGTTATTATATTGTCGAAAAATATTAGTGAGTTACTTTTTAGCGATGGACAATACACATGGTTAATCATTT TCGCATGTTGTATATTGCCATTCTCCATTATAAATACATTGATCGCTTCAGTTTTAAATGGTCAACAATTT TATAAGCAATATATATTGGTTGGGATGTTTTCTGTATTCATTTCTACTATGTTTATGATTTTGTTGATTGT AGCTTATAATCTTAAAGGTGCATTGATTGCCACAGCTATAAATAGTGCTATTGCTGGTCTTGTATTGGTTT TATTTTGTCTCAATAAATCTTGGTTTAGATTTAAATATTGGTGGGGTAAAACGGATAAAGACAAAATTATA AAAATTATTCATTATACTCTGATGGCTCTGGTTTCTGTTATCTCCATGCCTACAGCATTGATGTGTATTAG AAAAATATTGATTGCTAAAACTGGTTGGGAGGATGCAGGGCAATGGCAGGCCGTATGGAAGATATCTGAGG TTTATCTTGGTGTTGTGACAATTGCTTTGTCAACATATTTCTTACCAAGATTGACAATTATAAAAACAAGT TTCCTTATAATkAAAAGAAGTAAATAGTACTATATTATACATAATATCTATTACTTCATTCATGGCGTTGAG TATCTATTTATTCCGCGATTTGGTAATAACAGTTTTATTTACTGAACAGTTTCGCTCAGCTCGTGAATTAT TTTTATTACAACTTATAGGGGATGTAATAA7ÏAATTGCTGGGTTTCTTTATGCATACCCTCTTCAAAGTCAG GGGCATACTAAACTATTCATCAGTTCAGAAGTGATTTTTTCTATGCTCTTTATCATTACCACCTATATTTT TGTTGTAAATTATGGAGTACATGGTGCTAACATAAGTTATGTCATTACATATAGTTTATATTTTGTGTTTG CATTTGTGTTTACTAATTTTATTAATGTTAGAAGAAATAATTAAAAACAGAGGTTGAATTTTGAAAATAAT TATACCTGTCTTAGGATTTGGCAGGGCTGGTGGTGAAAGAGTTCTTTCTAAGCTGGCAACTGAATTGATGA ATTATGGACATGATGTAAGTTTTGTTGTTCCAGATAATAGAACTAATCCATATTATGCTACCACAGCAAAA ATTGTCACGAGTAAATCTAGTCAAAACCGTGTAATkAATATTGAGAATCATTAAAAATTACTATAATCTGTG GCGTAAATGCATAGAGTTAAATCCTGATGCTGTAGTTGCTAGTTTTCATTTGACTGCCTATCTTGTCGCAT TATTACCAATCACCCGTCGTAAGAAATATTATTATATTCAGGCGTATGAAGTTAATTTTTTTGATAATATA ATATGGAAATTAATAGCGGGTTTAACATATTATTTACCGCTTAAAAAAATACTAAATAGTCCTAATTTGCT TCCTCATAAACATGATGATTTTATAGGAGTAGTTCCTGCAGGAGTAGATTTAAACGTTTTCTATCCGAAAC CATCAAATAGGTTATTAAATGGTCACACATCAATAGGGATTATTGGTAGAAAAGAGAAGCACAAAGGAACT AGCGAAATTATTTCAGTATTGTGTTCACTGGAAAATAAAGCTGGAATTATAATCAATATTGCGATCTATCT TGAAGAAGTTGATAAGCAGCGTTTAATCGCTGCCGGGTTTCAGGTTAATTTTTTTCCGATTACTTCTGATT TAGAATTGGCATCCTTTTATCGAAGCAATGACATCATGATTGCTGTTGGGTTAATTGAAGATGGCGCTTTC CATTATCCTTGTGCTGAATCAATGGCTTGTGGTTGTCTTGTTATTTCAAATTATGCGCCACTTACTGAAAC TAACAGTGTACTTAAATTAGTCAAGTTTGATGCTTGCAAACTTGGTGAAGCAATTAATCTTTGTCTCAATC TTGACCTAGAAGAAAAAAGCAAAGAAATCCAATCTAATATTTCTGTGTTGAATAAATATGACTGGAAAATT GTTGGTGAAACTTTCAATAGTTTATTGTTAGATGCAAATAAATAGTATACGTTGATGGGGAAAATATGAAT ATTGTTAAAACTGATATTCCAGATCTGATCGTTCTTGZ\ACCAAAAGTGTTTAGTGATGAACGCGGCTTTTT TATGGAGAGTTATAATCAGATTGAATTTGAGAAGGCAATAGGAAGGCACGTAAATTTTGTTCAGGATAATC
117
ATTCAAAATCTAGTAAAGGCGTACTACGTGGGTTGCATTATCAATTAGCACCGTATGCACAGGCTAAATTA GTTCGATGTGTTGTAGGTCAGGTATTTGATGTTGCTGTTGATCTTAGAAAAAATTCACCAACGTTCAAAAA ATGGTTTGGAATAACCCTTTCCGCAGAAAATAAACGACAATTATGGATACCCGAAGGATTTGCTCATGGTT TCTTGGTGACCAGTGATGAAGCTGAGTTCATTTATAAGACAACTAACTACTATGCTCCTGGTCATCAGCAA GCAATTATTTACAATGATCCTATTTTAAACATCGATTGGCCTTTCTGCAGTAGTGCTCTGTCATTATCACA AAAAGATCAAGAAGCAAAATTATTTTCAGAATTATTGGACAGTGAACTGTTCTAATAAAGTGTGCCACCTT ATCCGTCTGAAGGATAGGTGGTTGCTTATATTTTTTTGAGTATGTTTGTATAATGACAGAAAATAGTCCGA AATATAAACACGATAAAAGCTTAATAAGTTTTATCTACTTATTTTTTATATTTACACTTATTGTAGGCTTT ATTATCGCAAATACCCAGTTTTTGGGGCGAAGTAGAGACTATGATAATTATATACAGATCTTTTCTGGTAA AGAAGGGGAGGGGGTTCTTGAATTATTTTATCGCGGATTGATGTTAATAACGACCAGCTATGAAACTATCA TTTTTATAATTTTAACATGTTCTTTTTTTATAAAGGCAAGGTTTCTCGCTAACTATTCGCGTAATTTTTCA GGCTTGACCTTATTCTTTATTTATTATGCAAGCGTTGCACTTTGGGTTTTAGATTATACTCAATTCAGAAA TGGTCTATGTATTTCCATTTTAATGTTTTCCGTATACTATTTATTTATAAATAAACCGACTTATTTTTATT TCTCGGTATTATGTGCAATTGCAACTCATTGGTCTGCTTTGCCTTTTTTGCTTTTATATCCTTTTGTCTAT TCAACAAAAATAAGACGCCTTGGTTATTTTTGTTTCAGTATTCTTGTTTTGATTGCGATCTCAGGAGAAGG AAAAGAGATCATATCTTTTATAAGAAATTTTGGAGTGGGACAAAAAATAGGAAATGAAGCTGGTGTAAATT TAATAAATTCATTATCCCTTACCGCTATTTCCTGGTTTATTATTAGTTACATATCAAGCATTGGAAATGAA AGGAGAAATTTAAGGCTTTTCTTTTGTTATGGTGTCATGCAATACGTGACTTTTAGCCTTTTCTCTCTACC TGTTATGGCTTTCCGTATTTTGGAAATGTATTTTTTCCTTATGCTAACCATTGGGGTGTTTATTAAGCAAA AAAAGAATTATTATTTTATTTTTTGCAAAGTGTTAATTTTATTGTATCTAACATACTATTATCATATGGTC TTTGGAGTGATTAATGTGTAAGGCTAAGGTGTTGGCTATAATTGTTACTTACAACCCGGAAATTATTCGAT TGACGGAATGTATTAACTCTTTAGCCCCACAAGTTGAGAGAATAATTCTTGTAGATAATGGCTCAAATAAT AGTGATTTGATAAAAAATATCAGTATTAATAACCTTGAAATTATTTTACTTTCGGAAAACAAAGGCATTGC ATTTGCTCAGAACCATGGTGTTAAGAAGGGCCTGGAAGCAAAAGAGTTTGACTATTTATTTTTCTCAGATC AGGATACTTGCTTTCCTAGCGATGTTATTGAAAAACTTAAGAGTACATTTACGAAAAATAATAAAAAAGGT AAAAATGTTGCTTGTGCTTCTCCTTTTTTTAAAGACCATCGTTCAAATTATATGCATCCGTCAGTCAGCCT AAATATTTTTACGAGTACAAAAGTTATATGTAGTGAAGTAGACGATGATCTTTATCCCTCGCATGTTATTG CTTCTGGGATGTTAATGTCTCGTGAAGCATGGCGCGTCGTCGGACCATTTTGTGAAAAACTCTTTATAGAC TGGGTTGATACAGAATGGTGTTGGCGTGCATTAGCTAATAATATGATTATTGTTCAGACACCATCAGTCAT CATTTCTCATGAACTTGGGTATGGGCAGAAAATTTTTGCTGGTCGATCTGTTACAATACATAATTCTTTCA GAAATTTTTATAAAATACGCAATGCAATATACTTAATGCTGCATTCAAATTATAGCTTCAAGTATCGTTAT CATGCTTTTTTTCATGCGACAAAGAATGTTGTATTTGAAATTTTATATTCGAAAGAAAAATTAAATTCACT GAAGGTTTGTTTTAAAGCTGTACGTGATGGTATGTTCAATAATTTTTAATACGAAAATAGTTAGGCTCAAG GTGTTTAAATGGAAGAAAATAATATGAAGACGGTCGCTGTAGTTGGCACAGTGGGTGTTCCTGCTTGTTAT GGTGGGTTCGAATCACTTGTTCAGAATCTAATTGATTATCAATCTGATGGTATACAATATCAGATATTTTG CTCTTCAAAAAAATATGATAAAAAATTTAAAAATTATAAAAATGCAGAATTAATCTATTTGCCGATAAATG CCAATGGCGTCTCTAGCATAATTTATGATATTATGTGTTTAATTATTTGTTTATTCAAAAGGCCAGATGTT GTTTTAATATTGGGGGTGTCTGGTTGTTTATTTCTACCAATTTATAAACTATTTTCAAAATCAAAGATTAT TGTCAATATTGATGGGCTTGAATGGCGTAGAAATAAATGGGGAACGTTTGCTZiAGAAATTTCTTAAAATAT CTGAGGCGATATCTATTAG?\ATAGCTGATATTATCATTTCAGATAATCAAGCAATAGCTGATTATGTGGAA AATAAGTACAAGAAAAAAAGTGTAGTTATAGCTTATGGCGGAGATCATGCCACTAATCTTAGTACACCGAT AGACAATGATCAAAAAAAAGAAGGTTATTATTTGGGGCTTTGTAGGATAGAGCCTGAG2\ATAATATAGAAA TGATTCTGAATGCCTTCATTAATACAGATAAAAAAATTAAATTTATGGGTAATTGGGATAACAGCGAGTAT GGACGCCAGCTAAAAAAATATTATTCAAACTATCCAAATATCACCCTACTAGAACCTAACTATAATATTGA AGAGCTTTATAAACTAAGAAAAAATTGTCTTGCATACATTCATGGACACTCGGCTGGTGGAACAAACCCTT CTTTAGTTGAAGCGATGCATTTTAATATTCCTATTTTTGCTTTCGATTGTGACTTTAATCGTTACACAACT AACAATTTAGCTCATTACTTTAATGATTCTGAACAACTTAGCTTATTAGCAGAAAGTTTGTCTTTTGGAAA TCTTAAATGTCGAGTATTAGATTTAAAAAATTATGCTGAAGATATGTATAACTGGAGGCATATAGCTGCTA TGTATGAATCTATTTATTAAACGCATTAACAATAATATAATTGACCTTATATAGCAGGGAAAGATCACGTA ACGCTGCGGCGCGCCGATCCCCATATGAATATCCTCCTTAGTTCCTATTCCGAAGTTCCTATTCTTTCTAG AGAATAGGAACTTCGGAATAGGAACTAAGGAGGATATTCATATGGATAAAGCCGTAAGCATATAAGCATGG ATAAGCTATTTATACTTTAATAAGTACTTTGTATACTTATTTGCGAACATTCCAGGCCGCGAGCATTCAGC GCGGTGATCACACCTGACAGGAGTATGTAATGTCCAAGCAACAGATCGGCGTAGTCGGTATGGCAGTGATG GGACGCAACCTTGCGCTCAACATCGAAAGCCGTGGTTATACCGTCTCTATTTTCAACCGTTCCCGTGAGAA GACGGAAGAAGTGATTG CCGAAAATC C AGGCAAGAAACTGGTTCCTTACTATACGGTGAAAGAGTTTGT CG AATCTCTGGAAACGCCTCGTCGCATCCTGTTAATGGTGAAAGCAGGTGCAGGCACGGATGCTGCTATTGAT
118
TCCCTCAAACCATATCTCGATAAAGGAGACATCATCATTGATGGTGGTAACACCTTCTTCCAGGACACTAT TCGTCGTAATCGTGAGCTTTCAGCAGAGGGCTTTAACTTCATCGGTACCGGTGTTTCTGGCGGTGAAGAGG GGGCGCTGAAAGGTCCTTCTATTATGCCTGGTGGCCAGAAAGAAGCCTATGAATTGGTAGCACCGATCCTG ACCAAAATCGCCGCCGTAGCTGAAGACGGTGAACCATGCGTTACCTATATTGGTGCCGATGGCGCAGGTCA CTATGTGAAGATGGTTCACAACGGTATTGAATACGGCGATATGCAGCTGATTGCTGAAGCCTATTCTCTGC TTAAAGGTGGCCTGAACCTCACCAACGAAGAACTGGCGCAGACCTTTACCGAGTGGAATAACGGTGAACTG AGCAGTTACCTGATCGACATCACCAAAGATATCTTCACCAAAAAAGATGAAGACGGTAACTACCTGGTTGA TGTGATCCTGGATGAAGCGGCTAACAAAGGTACCGGTAAATGGACCAGCCAGAGCGCGCTGGATCTCGGCG AACCGCTGTCGCTGATTACCGAGTCTGTGTTTGCACGTTATATCTCTTCTCTGAAAGATCAGCGTGTTGCC GCATCTAAAGTTCTCTCTGGTCCGCAAGCACAGCCAGCAGGCGACAAGGCTGAGTTCATCGAAAAAGTTCG TCGTGCGCTGTATCTGGGCAAAATCGTTTCTTACGCCCAGGGCTTCTCTCAGCTGCGTGCTGCGTCTGAAG AGTACAACTGGGATCTGAACTACGGCGAAATCGCGAAGATTTTCCGTGCTGGCTGCATCATCCGTGCGCAG TTCCTGCAGAAAATCACCGATGCTTATGCCGAAAATCCACAGATCGCTAACCTGTTGCTGGCTCCGTACTT CAAGCAAATTGCCGATGACTACCAGCAGGCGCTGCGTGATGTCGTTGCTTATGCAGTACAGAACGGTATTC CGGTTCCGACCTTCTCCGCAGCGGTTGCCTATTACGACAGCTACCGTGCTGCTGTTCTGCCTGCGAACCTG ATCCAGGCACAGCGTGACTATTTTGGTGCGCATACTTATAAGCGTATCGATAAAGAAGGTGTGTTCCATAC CGAATGGCTGGATTAA
SEQ ID NO: 13 iexample O6A rfb locus nucléotide sequence - O6A-EPA production strain stGVXN4112 and stLMTB10923)
ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGAT ACCCAAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAG GGATCAAAGAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTAT GAGTTAGAATCACTCCTTGAGCAGCGCGTGAAGCGTCAACTGCTGGCGGAAGTACAGTCCATTTGCCCGCC GGGCGTGACAATTATGAACGTGCGTCAGGGCGAACCTTTAGGTTTGGGCCACTCCATTTTATGTGCACGAC CTGCCATTGGTGACAATCCATTTGTCGTGGTGCTGCCAGACGTTGTGATCGACGACGCCAGCGCCGACCCG CTGCGCTACAACCTTGCTGCCATGATTGCGCGCTTCAACGAAACGGGCCGCAGCCAGGTGCTGGCAAAACG TATGCCGGGTGACCTCTCTGAATACTCTGTCATCCAGACCAAAGAGCCGCTGGACCGCGAAGGTAAAGTCA GCCGCATTGTTGAATTCATCGAAAAACCGGATCAGCCGCAGACGCTGGACTCAGACATCATGGCCGTTGGT CGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTTGAACGCACTCAGCCTGGTGCATGGGGGCGTATTCA GCTGACTGATGCCATTGCCGAACTGGCGAAAAAACAGTCCGTTGATGCCATGCTGATGACCGGCGACAGCT ACGACTGCGGTAAAAAAATGGGTTATATGCAAGCGTTCGTGAAGTATGGACTACGCAACCTCAAAGAAGGG GCGAAGTTCCGTAAAGGGATTGAGAAGCTGTTAAGCGAATAATGAAAATCTGACCGGATGTAACGGTTGAT AAGAAAATTATAACGGCAGTGAAGATTAGCGGCGAAAGTAATTTGTTGCGAATTTTCCTGCCGTTGTTTTA TATAAACAATCAGAATAACAACGACTTAGCAATAGGATTTTCGTCAAAGTTTTCCAGGATTTTCCTTGTTT CCAGAGCGGATTGGTAAGACAATTAGCATTTGAATTTTACGGGTTTAGCGCGAGTGGGTAACGCTCGTCAC ATCGTAGACATGCATGCAGTGCTCTGGTAGCTGTAÀAGCCAGGGGCGGTAGCGTGCTGAAATTATAAAGTC ATTCTTATAGAACATCGCATTTCAATAATATAATTACACCTAAATGAATAGGATACAACGTGTGCACAATT ATTTAAGGCTTAAAGATAAAATAAAAAACGTATTTTTAGGGTTGTATATATTGCAGTTATTTAATTATATC GCGCCATTGGTAATTATCCCTATCCTGATAAAATATATTGGGTTGGGGGAATATGGGGAATTGGTCTATAT TACATCTATTTATCAAATAGTGGCTTTGATTATTGATTTTGGCTTTACTTACACAGGACCTGTGGTTGCTG CGAGACATAGATGTGAGAC C CAAAATTTAC AGC GCTATTACTCAATAGTTGTTCTTTTAAAAT CATTGCTT TTTATAATTGCATTAACATGTGTATTTTTATTGTGCAGATTAAATATAGTCCACTTGTCATTTTTTGGGTT TTTGTCAATTTTTCTATGCACTATTGGTAATATATTATCGCCCAATTGGTTTTTGCAGGGGATTGGTGATT TTAAAAAACTTTCATACTCACAAGTAATAGTGAGAATAACATTGTTTATCATACTTCTTGTTTATGTCTGT AGTGGCGGAGATAATGTTTTTATCCTAAGTTTTTTGCAAAATGCAACATTACTCATATGCTGTATATACTT ATGGC CAAAT ATTC ATATT AGC C ATGTTGTTC ATCTTAAAC CTAATGAATGCATTGTGGZkATTTAAGAAGG CAGGAAATGTTTTTATTGGCGTAATAGGTACGATTGGTTAC3\ATGGTCTAATTCCTGTGTTAATTGGAAAC CTTTGCGGTAATACGAGTCTTGGTGTTTTTTCAATCGTTCAAAAAATGACAACAGCATGTCAAAGTCTAAT TAATCCAATATCACAGTATATGTTATCTCAAGTTTCAGAAATTAAACCTCAAGATAAACTGTTTTATTATA GAATTAAAAAAAGTTTTTTTGTGCATTTAACAATTAGCAT7)ATTGCATGTTTATGTTATATGGGGTTAGGG CAATATGTGGCGACTTTTATAGGTAAAGTTGACGTTTCATTTGTTATTATTTTATTTGCGTCAATAATTAC CATTTTTTCATCTTTAAATAATGTCCTTGGTATACAGTTTCTTATACCGACAGATAATGTAAAAATACTAC GAAGTATAAATGTTATGGCGGGAATTATTGTTGTTAGTTTGTCCTGGCTGTTAATATCACGCTTTGACATT CTGGGGGGGGTTTTATTAAACCTAATTGGTGAGTTTCTTGTATTCAGTATGCTAGCTTTTATTGCCCATCG
119
AAAGTGGGGAGCGAGAGTATAATGAAAGTGAAGGCGGTTCCTGCTATTACATTCTATTTAAGTTTAATGCT GACAATTTTAGTGTTACTGTTTGGTAATGAACCAAATAAATCACAATATATCCTTGTTATAGCAACGATAA CAGTTTTTTATATCGCATATATCACTAATAAAATAACTTCTCCGGCCAGCCTTCTCGTTATATCATCTTTT GTGTTTTTAGGTTGTCGCCCTTTATTATCTTTGTTTGCAAACTATGATTATAGGATTGCCGATTGGTTTAT TGAAGGATATATGGATGACGATGTGATTTTGGCTAACTATGCTATAACACTAATGTATTATGGTTATACAT TGGGACTAATTCTATGCAAAAATACTGAAAAATTTTATCCGCATGGTCCTTATCCTGAAAAACAATTGCTA AAAATAAAGTTTCTTTTGACTTTATTTTTTCTGGGTTCGATAGGTATGGTTGTAAAAGGGATATTCTTTTT TAACTTTATAGAATCTAATAGTTATGTTGATATTTATCAATCAAATATAACAACGCCAATAGGTTATGATT TTCTATCTTATTTATTTTATTGTTCTTTTTTCCTTATATGTGCGTTTCATATACAGTTCAGAACAAATAAA AAATTTCTTTTTATTGCGATATGCATTGCTGCATTTAGCACCTTGAAGGGTAGTCGTAGTGAAGCTATAAC GTTTCTTTTAACGGTTACATGTATATATTTTAATGAAGTAAAGACAAGAAACTTACGTCTGCTGATTACAA TGATTTTTGTTTTTAGCGTCATTTTTGTGATTAGTGAATTTATCTCAATGTGGCGCACTGGAGGGAGTTTT TTTCAATTAATGCAGGGTAATAATCCTGTTATAAACTTTGTATACGGCATGGGAGTATCATATCTTTCCAT TTATCAATCÀGTAAAACTACAACTATTGTCAGGGGGATATAATGTTACCTATCTATTCAGCCAGTTAATAA TAACTTGCT CGT CAATATTTAATGTCAAATTGAGCTTGC CGGAAATAAGCTATAGC CATTTGGCC T CATAC ACAGCAAACCCAGAACTATATAATCTTGGGTTCGGACTTGGGGGGAGTTATTTAGCAGAATCGTTTTTAGC ATTTGGTCTGATTGGATGTTTCATTATACCCTTTTTACTTTTACTTAATTTAAATGTATTGGAAAAATATA CAAAAAACAAACCAATTATATATTTTGTTTATTATAGTGTGTTGCCACCTATATTATTCACACCAAGAGAG ACTTTGTTCTATTTCTTCCCCTATCTTGTCAAAAGTATATTTGTTGCTTTTTTAGTTACATTATACATCCA GTATAAAAAGGATTGAC CAAAATGTCAGAAAAAAATGTCAGCATAATAAT CC CAAGTTATAACAGGGCTCA TATTCTTAAGGAGGTCATACCAAGTTATTTTCAGGATGAGACTTTAGAGGTTATAGTTATCAATGATGGAT CAACAGATAATACAAATAGTGTATTAGCTGAACTGAAGGAAAAATATTCTCAGTTAGTTATTTTAGAAAAT GAAACGAATAAAAAACAGATGTATTCTAAAAACCGAGGGATTGAAATAGCCAAAGGGAAATATATTTTTTT TGGTGATGATGACTCTTACCTCTTACCCGGTGTTATATCTCGGTTATTGGCTACAAAATATGAGACAGGCG CTGATGTAATCGGCGCAAGAATACTTTATATGAATAATAACGAGAAAACAATTGAAGATTGCATAAATCGA CATAAAAAAGAGGGGCGTTTTGTTAGTGATCTAAATAGATTGGATTTTAGTTATACATGTGATTTGGACCA TCCGATTGAATGTTTTTATGCACAGCCTTTTGTTCTAGCTGAAAGGGAACTAATATCGAAATATCGATTTG ATATATCTTATACGGGAAACTGCTATCGTGAGGAAACTGATTTCATGCTATCTCTATTTATTAAAAATAAA AAATTTATATATGATTCAAAGGCTTTGTTAATAAATTTACCTCCAAGAAAAGCGACGGGAGGGGCAAGAAC AG CTAATC GATTAAAAT ATC ATT ACGAAAGTTGC ATAAATAATTAT AGATTTTTAAAAAAATATAATGATA ATTTGAATCTTCTTTCAGGACAAAAGCATGCTATATTTTACCGACAGTGTCAATTCGTTCTGCTAAAAATG AAGTCGTTTATCGGGAAGTTTTTAAAATGATTATATATATCGCCGCGTATAATGGTTCAGGAGGGCAAGGT GGGGTGGAAAGGGTTGTTGCCCAACAATGTAACATTCTTAAAAATTTGGGGGTTAAAGTCATTATACTTGA TAAAACATACTTCAAAATTTCTAACAAAATTCGTAACAAAAAAATACAAGTAGCACTTTATCCAATATTAG TTTCTCTTTATTTAACCTTACAAAAATTACGTGGCGTGACGTTTAAAGTTATTGCACATGGCTATTGTTCT CCTTTTTATAGGAATGACATCTTAATAGCTCATGGCAATATGAAATGTTATTTTCAAACAGTCATGAATAA AAAACCTAATCGGTTGTCTGGCAGTGGTCTTTTATCTTTCTATGAGCGTTGGGCTGGAGCATTTTCAAAAA ATATCTGGGCTGTTTCAAATAAGGTTAAAAGTGAATGGAATGAGCTTTACAATATTAATTCACATAAAATC AAAGTTGTTCGAAATTTTATAAATCTTGCACAATTTGATTACACTGATGTTAATGAAGCAGAATATGTGAC ATTTGTCGGGCGATTGGAAAAAGGAAZiAGGAATAGATGATCTGTATTACATATGTAAAAATCTGCCAGATA CTTCCTTCCATTTAGTTTCAAGTATTCCCGCCCCACAAAATTTTGCTTCGCTJkAATAATGTTCTGACCAGC ATTGCTGTCCCCTATGCGAAAATGCCAGAAATATTTAAGAAATCCAGAGTACTTATTTTACCGTCCTATTA TGAAGGATATGAGCTGGTTACTATTGAAGCGCTATGCTGTGGTTGCCCTGTGATAGGCTATAATGTTGGTG CAATTAGAGAGTTGTATGCAGAAAGTTTTCCTGGCGTATTTATTGCCAATAATAAAGAAGATTTAGCAC2ÏA GTAGCCTACAAATTAATTAGTCTTGATAATGAAAAATATTATCATTTGAGACAAACTATTTATAGCAAGCG TGAGCTTTTTTCTGAAGAGAGATATGCGGAAATTTTAACGGCGGCATTTAATGAAAAAAAATAAGAAACTC TGTCTCATTTCAATTAACTCATATAATGAACTTACCGGAGGAGGAGTATATTTACGTACGCTTGTTAGTTT TCTACAAAAACAGAATGTTAATTTAACACTTATTGATAAAAAATCCTCAGGTAAACTATTCGAAGACAATA CTTTTCAACATATATCATTTATTAAAGGTAAACGTCAGGATATAATATCCAGGCTTTTTTTTATACCATCA TTTTATGTCCCTTATATTTTCTCAATAATTAAAATTTTACGGAAGCAAGATATTCTTGCTTTTCACAACTC TCGGCTTGGATTGTTATGTCTGCTTTTTAGAATACTCATGCCCCACAAAAAGATCATATTGTTTACGGATA ACTTCGAATATGACTTAATAAGACAAAAAGATAAAAACATAACTACTTTTATTGAAAAATTAATTGTTTAT CTCAATGAATTTATCGGGCTTAAGAATTCAGATTTAGTTAGCTATATTACCCGGCZtAGATAAAAATGCAAT GGATAAATTTTATGGGATTAAAAAAAGCAGAAATTTAATTCTCCCTGTGATATTTAGTAGAGAAAAACCAA CTGATGTATTGTCAGCTCACTTTATTAATGAGTATAATCGATTGAATAATGATAATAGGAAAAAAGTAGTA TTTACTGCATCTTTTGATTTTTTTCCAAATATAGATGCTGCCAACTATGTTTTAAATGCAGCATtAGTCTAA
120
TAATGATTATTGCTATATTTTGGCAGGTAGGAAAAGTACTACTTTGAATCTTCCTGATTTGGATAATTTAT TTTTTTTCGATAATCTATCTAATAGTGAAATGTCATATTTATTATCTGCTTGTGATGTTTTTTATTCTCCT ATAGTTTTAGGAAGTGGAATGAAAACAAAAATTGCAGAAGCACTATCATATGGATTATATATTTATGCGAC AGAGCATTCCTTAATCGGCTATGATGAAATTATACACAATAAGGAGTGTGTTAAAAAAATCTCACATTTGG ATGAGGAATTTC CTAAAGATTTCAAGATGAAAAGTATCAATAAACAGCT AATAATGT CTTATCAGCAAAAA TATTATTCACATTATCGGTTTAATGGCCATGAACTTGATATAATAAATTTTGACGATTAGTTAGTGGAGAT ATAATATGAACATATTAGTAACTGGTGGTGCTGGATATATCGGATCTCATACGGCTATTGAATTACTGAAT GCAGGTCATGAGATTATCGTTCTGGACAATTTCAGTAATGCTTCATACAAGTGTATCGAAAAAATAAAAGA AATTACTCGACGTGATTTTATAACAATTACTGGAGATGCTGGGTGTAGGAAGACACTCTCCGCTATTTTCG AGAAACACGCCATAGATATAGTTATTCATTTTGCTGGCTTTAAATCTGTTTCAGAGTCTAAAAGTGAACCC TTAAAGTATTACCAGAATAATGTTGGAGTGACCATTACTTTATTACAGGTAATGGAAGAGTACAGAATTAA AAAATTTATCTTTAGTTCATCTGCGACAGTCTATGGTGAACCAGAGATAATTCCAATTCCAGAAACAGCTA AAATTGGAGGAACTACGAATCCATATGGCACATCGAAGTATTTTGTTGAAAAAATTCTAGAGGATGTTAGT TCCACGGGAAAACTGGATATAATTTGCTTGAGATATTTTAATCCTGTCGGTGCTCATTCTAGTGGTAAAAT AGGTGAGGCTCCATCTGGTATCCCTAATAATCTTGTTCCTTATTTATTGGATGTTGCGAGTGGTAAACGTG ATAAATTATTTATTTATGGCAATGATTACCCTACTAATGATGGAACAGGTGTAAGGGATTTTATTCATGTT GTTGACTTAGCGAAAGGTCATTTGGCTGCAATGAATTATTTAAGTATCAATTCGGGATATAATATCTTTAA TCTTGGTACAGGAAAAGGTTATTCGGTACTTGAATTAATCACTACATTTGAAAAATTAACAAACATTAAGG TCAATAAATCTTTTATAGAGAGAAGGGCAGGGGATGTTGCGTCTTGTTGGGCTGATGCAGATAAAGCTAAT TCTTTATTGGACTGGCAAGCCGAACAAACTCTAGAACAGATGTTATTGGACTCGTGGCGTTGGAAAAAAAA TTATCCAGACGGATTCTGAATATAAAAGGTTTCAGTTTTATGAATCAATCAGAGCAGAGAAAAAAAATACT GGTTCTTACACCTCGCTTTCCCTACCCTGTCATTGGAGGGGATAGATTAAGAGTCTATATGTTATGTAAAG AACTTTCCAAAAAATATGATCTTATTCTTCTGAGCTTATGTGATCAACCACTAGAACTTGAAATAAATATA AATGACTCGGTCTTCAAAGAAATTCATCGTGTCTATCTACCAAAATATAAATCATATTATAATGTATTAAA AGCTTTGGTTACGCAAAAACCGTTGCAAATTGCTTATTATCAATCGGACACATTTAAGAATAAATACAATA AATTAATTAAACAATGCGATGCAGTATTTTGTCATCTGATAAGAGTTGCTGATTATGTTAAGGATACAGAC AAGTTCAAAATTCTTGATATGACAGATGCAATATCTTTGAATTACAGTCGCGTTAAAAAATTAGCAAGTAA AAAAAGTTTGCGTGCAATTATTTATTCTCTGGAACAAAAAAGATTAGAATCATATGAACGTTCTGTGGCGA ATCTTTTTGATTTGACCACTTTTATTTCATCCGTAGACCGTGACTATCTCTACCCTAATCTGGGCAGTAAT ATCCATATAGTCAATAATGGGGTTGATACATCAGCCTTGAGATATATAAAAAGAGAAATAAAAATCGATAA GCCTGTGGAACTTATATTTATCGGAAATATGTATTCTTTACAAAATATGGATGCTGCAAAACATTTTGCTA AGAATATTTTACCTTGCTTGTATGATGAGTTTAATATTATTTTTAAAGTGATTGGTAAGATCTCAG71AACT AATAAAAATATATTAAATTCATTTAAAAATACAATTGCTTTAGGTACTGTTGATGATATCAATTCTTCCGC TTCTACAGGGCATATAGGTATATGTCCTGTTCGTCTTGGAGCAGGCGTACAAAATAAAATTCTTGAATACA TGGCTTTAGGTTTACCATGTATTACATCTAGCATTGGTTATGAAGGTATTAATGCAAAATCAGGTAGCGAA ATTTTTGTTGCAGATACAGTAGAGCAATATAAAAACGTACTAAGAGAAATAATTTACGATTATAATCGTTA TACTGAAGTGGCTGAAAATGCCCGTAGTTTTGTAGAAAATAATTTTTCTTGGGAATCAAAAGTTGCCAATT TAATGAATACATTAGATGAGAAATTATATGAACAATAATAAAATTATTACACCTATCATTATGGCTGGTGG TTCAGGCAGTCGGTTGTGGCCACTATCAAGAATTCTCTATCCGAAACAATTTCTTAGCCTAATCGGTAGTC ATACCATGCTTCAAACAACGGCTAATCGTCTGGATGGTTTGGATTGTACCAACCCTTATGTCATTTGTAAT GAACAATACCGCTTTATAGTTGCTGAACAGCTTAGAAAAAT CGATAGATTGACTTCAAAGAATATCAT CCT TGAGCCTGTTGGGCGTAACACTGCCCCTGCAATTGCATTAGCGGCGTTGCTGATGTCTAAGTCTGATAAAA GTGCAGATGATCTTATGCTCGTACTGGCTGCAGATCACGTTATACACGATGAAGAAAAATTTTGTAACGCT GTTAGATCGGCAATTCCATACGCTGCTGATGGGAAATTGGTAACATTTGGTATAATTCCAGACAAAGCAGA AACTGGTTATGGTTATATACATCGAGGACAATATATTAATCAGGAAGATTCGGATGCATTTATAGTGTCAT CATTTGTTGAAAAGCCAAATCATGAGACAGCCACTAAATATCTTGCTTCCGGTGAGTATTATTGGAATAGC GGTATGTTTTTGTTTAGTGCAAATCGTTATATAGAGGAACTTAAACAATTTCGGCCTGATATTTTATCCGC TTGTGAAAAAGCAATTGCTTCAGCGAACTTTGACCTTGATTTTGTGCGTTTAGATGAAAGTTCTTTCTCTA AGTGCCCTGAAGAATCAATTGATTACGCTGTAATGGAAAAAACAAAAGACGCAATTGTTATTCCAATGGAT GCTGGCTGGAGTGATGTCGGTTCATGGTCTTCTCTTTGGGAAATTAATGATAAAGACTCAGACGGCAACGT AATAGTTGGGGATATTTTCTCTCATGAAACAAAGAATTCTTTCATATATGCCG7LATCGGGAATTGTTGCTA CAGTTGGAGTGGAAAATTTAGTTGTTGTCCAAACAAAGGATGCTGTTCTTGTCTCAGAGAGAAATAAAGTT CAGGATGTAAAGAAAATAGTAGAACAAATTAAAAATTCAGGTCGTAGCGAGCATTATGTTCATCGCGAAGT ATATCGTCCTTGGGGTAAAT ATGATT CC ATTGACACAGGGGAGCGTTAT CAGGT CAAACGTATAAC AGTAA ATCCTGGTGTiAGGACTTTCTTTACAAATGCACCATCATAGGGCAGAACATTGGATCATAGTTTCTGGAACT GCAAGGGTGACTATAGGTTCTGAAACTAAGATTCTTAGCGAAAATGAATCTGTTTACATACCTCTTGGTGT
121
AATACACTGCTTGGAAAATCCAGGGAAAATTCCTCTTGATTTAATTGAAGTTCGTTCTGGATCTTATTTAG AAGAAGACGATGTTATCCGTTTTCAGGACCGATATGGTCGTAGCTAAATTTTTGATAATGTAACGTTAGTA GAAGAGCGCTAATATTTTTAGTTAATCTGTAATAAGTATTATTTGTTTAAGGTATATCATGTCGAGTTTAC CCTGCTTTAAAGCCTATGATATTCGCGGGAAATTAGGCGAAGAACTGAATGAAGATATTGCCTGGCGCATT GGTCGCGCTTATGGCGAATTTCTCAAACCGAAAACCATTGTGTTAGGCGGTGACGTCCGACTCACCAGCGA AACCTTAAAACTGGCGCTGGCGAAGGGGTTACAGGATGCGGGCGTCGATGTGCTGGATATTGGCATGTCCG GCACCGAAGAGATCTATTTCGCCACGTTCCATCTCGGCGTGGATGGCGGCATCGAAGTTACCGCCAGCCAT AÀCCCGATGGATTACAACGGCATGAAACTGGTGCGCGAAGGGGCTCGCCCGATCAGCGGTGATACCGGACT GCGCGACATCCAGCGTCTGGCAGAAGCCAACGACTTTCCTCCCGTTGATGAAACCAAACGCGGTCGCTATC AGCAAATCAATCTGCGTGACGCTTACGTTGATCACCTGTTCGGTTATATCAACGTCAAAAACCTCACGCCG CTCAAGCTGGTGATTAACTCCGGGAACGGCGCGGCGGGTCCGGTGGTGGACGCCATTGAAGCCCGCTTTAA AGCCCTCGGCGCACCCGTGGAATTAATCAAAGTGCACAACACGCCGGACGGCAATTTCCCCAACGGTATTC CTAACCCGCTACTGCCGGAATGTCGCGACGACACCCGCAATGCGGTCATCAAACACGGCGCGGATATGGGC ATTGCCTTTGATGGCGATTTTGACCGCTGTTTCCTGTTTGACGAAAAAGGGCAGTTTATTGAGGGCTACTA CATTGTCGGCCTGCTGGCAGAAGCGTTCCTCGAAAAAAATCCCGGCGCGAAGATCATCCACGATCCACGTC TCTCCTGGAACACCGTTGATGTGGTGACTGCCGCAGGCGGCACCCCGGTAATGTCGAAAACCGGACACGCC TTTATTAAAGAACGTATGCGCAAGGAAGACGCTATCTACGGTGGCGAAATGAGCGCCCACCATTACTTCCG TGATTTCGCTTACTGCGACAGCGGCATGATCCCGTGGCTGCTGGTCGCCGAACTGGTGTGCCTGAAAGGAA AAACGCTGGGCGAACTGGTGCGCGACCGGATGGCAGCGTTTCCGGCAAGCGGTGAGATCAACAGCAAACTG GCACACCCCGTTGAGGCGATTAACCGCGTGGAACAGCACTTTAGCCGCGAGGCGCTGGCGGTGGATCGCAC CGATGGCATCAGCATGACCTTTGCCGACTGGCGCTTTAACCTGCGCTCCTCTAACACCGAACCGGTGGTGC GGTTGAATGTGGAATCGCGCGGCGATGTACCGCTGATGGAAGAAAAGACAAAACTTATCCTTGAGTTACTG AACAAGTAATTCAGTAATTTCATATAAATGGGTTTTAAAAAACGGAAAAGATGAGATATCCGGTGTGGTAT AT C CAAGGTAATGCT ATT CAGTATCTCT ATGAGTGAGTTAAC ATCTATAC CAC ATTTAAGCCGCACACTTC GGGATCCCCATATGAATATCCTCCTTAGTTCCTATTCCGAAGTTCCTATTCTTTCTAGAGAATAGGAACTT CGGAATAGGAACTAAGGAGGATATTCATATGGATAAAGCCGTAAGCATATAAGCATGGATAAGCTATTTAT ACTTTAATAAGTACTTTGTATACTTATTTGCGAACATTCCAGGCCGCGAGCATTCAGCGCGGTGATCACAC CTGACAGGAGTATGTAATGTCCAAGCAACAGATCGGCGTAGTCGGTATGGCAGTGATGGGACGCAACCTTG CGCTCAACATCGAAAGCCGTGGTTATACCGTCTCTATTTTCAACCGTTCCCGTGAGAAGACGGAAGAAGTG ATTGCCGAAAATCCAGGCAAGAAACTGGTTCCTTACTATACGGTGAAAGAGTTTGTCGAATCTCTGGAAAC GCCTCGTCGCATCCTGTTAATGGTGAAAGCAGGTGCAGGCACGGATGCTGCTATTGATTCCCTCAAACCAT ATCTCGATAAAGGAGACATCATCATTGATGGTGGTAACACCTTCTTCCAGGACACTATTCGTCGTAATCGT GAGCTTTCAGCAGAGGGCTTTAACTTCATCGGTACCGGTGTTTCTGGCGGTGAAGAGGGGGCGCTGAAAGG TCCTTCTATTATGCCTGGTGGCCAGAAAGAAGCCTATGAATTGGTAGCACCGATCCTGACCAAAATCGCCG CCGTAGCTGAAGACGGTGAACCATGCGTTACCTATATTGGTGCCGATGGCGCAGGTCACTATGTGAAGATG GTTCACAACGGTATTGAATACGGCGATATGCAGCTGATTGCTGAAGCCTATTCTCTGCTTAAAGGTGGCCT GAACCTCACCAACGAAGAACTGGCGCAGACCTTTACCGAGTGGAATAACGGTGAACTGAGCAGTTACCTGA TCGACATCACCAAAGATATCTTCACCAAAAAAGATGAAGACGGTAACTACCTGGTTGATGTGATCCTGGAT GAAGCGGCTAACAAAGGTACCGGTAAATGGACCAGCCAGAGCGCGCTGGATCTCGGCGAACCGCTGTCGCT GATTACCGAGTCTGTGTTTGCACGTTATATCTCTTCTCTGAAAGATCAGCGTGTTGCCGCATCTAAAGTTC TCTCTGGTCCGCAAGCACAGCCAGCAGGCGACAAGGCTGAGTTCATCGAAAAAGTTCGTCGTGCGCTGTAT CTGGGCAAAATCGTTTCTTACGCCCAGGGCTTCTCTCAGCTGCGTGCTGCGTCTGAAGAGTACAACTGGGA TCTGAACTACGGCGAAATCGCGAAGATTTTCCGTGCTGGCTGCATCATCCGTGCGCAGTTCCTGCAGAAAA TCACCGATGCTTATGCCGAAAATCCACAGATCGCTAACCTGTTGCTGGCTCCGTACTTCAAGCAAATTGCC GATGACTACCAGCAGGCGCTGCGTGATGTCGTTGCTTATGCAGTACAGAACGGTATTCCGGTTCCGACCTT CTCCGCAGCGGTTGCCTATTACGACAGCTACCGTGCTGCTGTTCTGCCTGCGAACCTGATCCAGGCACAGC GTGACTATTTTGGTGCGCATACTTATAAGCGTATCGATAAAGAAGGTGTGTTCCATACCGAATGGCTGGAT TAA
SEP ID NO: 14 (example 08 rfb locus nucléotide sequence - 08-EPA production strain stLMTB11734)
ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGAT ACCCAAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAG GGATCAAAGAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTAT GAGTTAGAATCACTCCTTGAGCAGCGCGTGAAGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCC
122
GGGCGTGACCATTATGAACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGAC CTGCCATTGGTGACAACCCATTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCG CTACGTTACAACCTTGCTGCCATGATTGCACGTTTCAACGAAACGGGCCGCAGCCAGGTGCTGGCAAAACG TATGCCGGGTGACCTCTCTGAATACTCCGTCATCCAGACTAAAGAGCCGCTGGACCGTGAGGGTAAAGTCA GCCGCATTGTTGAATTTATCGAAAAACCGGATCAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGT CGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTGGAACGTACTCAGCCTGGTGCATGGGGACGTATTCA GCTGACTGATGCTATTGCCGAGCTGGCGAAAAAACAATCCGTTGATGCAATGCTGATGACCGGCGACAGTT ACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAGTATGGCCTACGCAACCTGAAAGAAGGG GCGAAGTTCCGTAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAATCTGACCGGATGTAACGGTTGAT AAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAATCTTCCTGCCGTTGTTTTA TATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAGTTTTCCAGGATTTTCCTTGTTT CCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTAACGCTCGTCAC ATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCATTAATACCTCTATT AATCAAACTGAGAGCCGCTTATTTCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGCTAGCGATCGC TTAAGATCTAGGATTTCATTATGTTACTTCCTGTAATTATGGCTGGTGGTACCGGCAGTCGTCTCTGGCCG ATGTCACGCGAGCTTTATCCGAAACAGTTCCTCCGCCTGTTCGGGCAGAACTCCATGCTGCAGGAAACCAT CACCCGACTCTCGGGCCTTGAAATCCATGAACCGATGGTCATCTGTAACGAAGAGCACCGCTTCCTGGTGG CTGAACAGCTACGCCAGCTCAATAAGCTGTCGAATAATATTATTCTTGAGCCGGTCGGGCGCAACACCGCC CCGGCCATCGCCCTGGCAGCCCTTCAGGCCACCCGCGACGGCGACGACCCGCTGATGCTGGTTCTCGCCGC TGACCATATCATCAATAACCAGTCGGCCTTCCACGACGCCATCCGGGTCGCCGAGCAGTATGCTGATGAAG GTCATCTGGTCACCTTCGGTATCGTGCCGAATGCCCCGGAAACTGGCTACGGTTACATTCAGCGCGGCGTG GCGCTCACCGATAGTGCCCATTCCGCGTACCAGGTGGCCCGCTTTGTGGAGAAGCCGGATCGCGAGCGCGC CGAGGCTTACCTCGCCTCCGGGGAGTACTACTGGAACAGCGGCATGTTTATGTTCCGCGCCAAGAAATACC TCATCGAGCTGGCCAAATACCGTCCGGATATCCTGGAAGCCTGCCAGGCTGCGGTGAATGCCGCCGATAAT GGCAGCGATTTCATCAATATCCCGCATGATATTTTCTGCGAGTGCCCGGATGAGTCCGTGGACTATGCCGT TATGGAGAAAACCGCCGATGCGGTGGTGGTCGGTCTCGATGCTGACTGGAGCGACGTCGGCTCCTGGTCCG CACTATGGGAGGTCAGCCCGAAAGACGAGCAGGGCAATGTCCTCAGCGGTGACGCGTGGGTACACAACAGC GAAAACTGCTACATCAACAGCGACGAGAAGCTAGTGGCGGCCATTGGCGTAGAGAATCTGGTGATTGTCAG CACTAAGGACGCCGTGCTGGTGATGAATCGCGAGCGTTCCCAGGACGTGAAGAAGGCGGTCGAGTTCCTCA AGC AGAAC CAGCGCAGCGAGT ACAAGCGCCAC CGTGAGATTTACCGCC CCTGGGGC CGTTGCGACGTAGTG GTCCAGACCCCGCGCTTCAACGTCAACCGCATCACGGTGAAACCAGGCGGTGCCTTCTCGATGCAGATGCA CCACCATCGCGCCGAGCATTGGGTTATTCTCGCCGGCACCGGTCAGGTGACTGTCAACGGTAAGCAGTTCC TGTTGTCCGAGAACCAGTCCACCTTTATTCCGATTGGCGCCGAGCACTGCCTGGAAAACCCTGGCTGTATT CCGCTGGAAGTGCTGGAGATCCAGTCGGGGGCGTACCTTGGCGAGGACGACATTATTCGTATTAAAGACCA GTATGGTCGTTGCTAATTATTTTCGGGACAAGACGCAGAATGACACAGTTAACTTGTTTTAAAGCTTATGA CATCCGTGGTGAACTGGGTGAGGAACTGAACGAGGACATCGCCTACCGTATCGGTCGCGCCTACGGCGAAT TTCTGAAACCCGGGAAGATAGTGGTGGGGGGCGATGTGCGCCTCACAAGCGAGTCGCTGAAGCTGGCGCTG GCCCGCGGGTTAATGGACGCCGGTACCGACGTGCTGGACATCGGCCTGAGCGGTACCGAAGAGATTTACTT TGCCACCTTCCACCTTGGGGTAGATGGTGGCATCGAGGTGACCGCGAGCCACAATCCTATGAACTACAACG GCATGAAGCTGGTGCGCGAGAATGCGAAGCCCATCAGCGGCGACACCGGCCTGCGGGATATCCAGCGCCTG GCGGAGGAAAAC C AGTT C C CGC CAGTGGAC CCGGCGCGTCG CGGGAC CCTGAGCAAGAT ATCGGTACTGAA GGAGTATGTTGACCATCTGATGAGCTACGTGGACTTCTCGAACTTCACCCGTCCACTGAAGTTGGTGGTGA ACTCCGGAAACGGGGCTGCGGGGCACGTGATTGATGAGGTGGAGAAACGCTTCGCGGCGGCTGGGGTGCCG GTAACCTTTATCAAGGTGCATCACCAGCCGGATGGCCATTTCCCTAACGGTATCCCGAATCCGCTGCTGCC GGAGTGCCGCCAGGATACCGCCGACGCGGTGCGCGAGCATCAGGCCGACATGGGGATTGCCTTTGACGGCG ACTTCGATCGCTGCTTCCTGTTCGATGACGAAGCTTCGTTTATCGAGGGGTATTACATTGTCGGCCTGCTG GCTGAGGCGTTCCTGCAGAAGCAGCCGGGAGCGAAAATCATTCACGACCCGCGCTTGACGTGGAACACGGT AGACATCGTGACCCGCAACGGCGGCCAGCCGGTGATGTCGAAGACGGGGCATGCGTTCATCAAGGAGCGGA TGCGTCAGGAAGACGCTATCTACGGCGGGGAGATGAGTGCGCACCATTACTTCCGCGATTTCGCCTACTGC GATAGCGGGATGATCCCGTGGCTGCTGGTGGCGGAGCTGCTGTGTCTGAAGAACAGCTCGCTGAAATCGCT GGTGGCGGACCG CC AGAAGGCGTT C C CTGCGTCGGGAGAGAT CAACCGCAAGCTAAGTAATGCTGC TGAGG CGATCGCCCGCATCCGGGCGCAGTATGAGCCGGCGGCTGCACACATCGACACAACGGACGGGATCAGTATT GAATACCCTGAATGGCGCTTTAACCTGCGCACGTCTAACACCGAGCCGGTGGTGCGTCTGAACGTTGAGTC CAGAGCTGATGTGGCGCTTATGAATGAAAAAACGACCGAGCTGTTACACCTGTTAAGCGGGGZkATZkAGGTG AGAGATTTACTAACGACGATTTATCGTTATCGGGGATTTATCTGGAGCAGTGTTAAACGTGATTTTCAGGC ACGCTATCAAACTAGTATGCTGGGCGCACTATGGCTCGTTTTACAACCGCTCTCTATGATTCTGGTCTATA
123
CCCTGGTTTTTTCCGAGGTGATGAAGGCAAGAATGCCCGATAATACCGGGTCGTTTGCCTATAGTATTTAT CTCTGTTCCGGGGTACTGACCTGGGGATTATTTACTGAGATGCTGGATAAAGGTCAGAGCGTATTTATTAA CAATGCTAATCTGATCAAGAAACTCAGTTTTCCGAAAATCTGTCTGCCGATCATCGTGACGTTATCGGCGG TGCTAAATTTCGCGATTATTTTCAGTCTGTTTCTAATTTTTATCATTGTCACCGGTAACTTCCCCGGCTGG CTCTTTCTCTCGGTGATACCGGTCCTGCTTTTGCAGATCCTGTTTGCCGGTGGGCTGGGGATGATCCTTGG TGTCATGAACGTCTTTTTCAGGGATGTGGGGCAACTGGTTGGCGTTGCGCTGCAATTCTGGTTTTGGTTCA CACCCATTGTTTATGTACTGAATTCATTACCTGCATGGGCAAAAAATCTGATGATGTATAACCCGATGACT CGGATCATGCAATCTTATCAGTCCATCTTCGCCTATCATCTGGCCCCCAACTGGTATTCGCTATGGCCAGT ATTGGCTCTCGCCATTATTTTCTGCGTCATCGGTTTCAGGATGTTCCGCAAGCATGCGGCGGATATGGTGG atgaattataatgagttatatcagagtaaataatgtcggtaaggcgtatcgccagtatcactcaaagaccg GGAGACTGATCGAATGGTTATCC C CTCTGAATACCAAACGC CATAATTTGAAATGGATCCTC CGCGATATT AATTTCGAAGTCGCTCCGGGCGAGGCTGTCGGTATTATCGGTATCAACGGTGCAGGCAAGAGTACCCTGCT TAAACTCATAAC CGGGACGTCC AGGC CGACGACTGGAGAAATTGAAATCTC CGGACGTGT CGCTGCATTAC TCGAATTGGGGATGGGGTTTCATTCTGATTTCACTGGTCGGCAGAATGTTTATATGTCTGGGCAACTGTTG GGGTTATCGTCAGAGAAAATAACTGAACTGATGCCGCAAATTGAAGAGTTTGCTGAGATTGGGGACTATAT CGATCAACCTGTGCGCGTCTACTCCAGTGGGATGCAÀGTTCGATTAGCTTTTAGTGTAGCGACGGCTATCC GTCCTGATGTGCTAATTATCGATGAGGCATTATCTGTTGGGGATGCATATTTCCAGCATAAAAGCTTTGAG CGTATTCGAAAATTTCGTCAGGAAGGGACCACGCTGTTGCTGGTATCCCATGATAAACAAGCGATCCAAAG CATTTGCGACCGGGCCATTTTATTGAATAAAGGCCAAATTGAAATGGAAGGTGAACCTGAAGCAGTGATGG ATTATTACAATGCTCTTCTGGCCGATAAACAAAATCAGTCCATTAAACAAGTTGAGCATAATGGTAAAACG CAAACTGTTTC AGGCACTGGTGAGGTGACTAT CTCTGAGGTT CATCTT CTCGATGAACAGGGCAATGTGAC TGAATTTGTTTCGGTAGGGCATCGTGTCAGCTTGCAGGTCAACGTTGAGGTCAAGGACGATATTCCTGAGC TTGTTGTCGGATATATGATTAAGGATCGACTTGGGCAGCCGATTTTCGGGACCAATACGTACCATCTCAAT CAGACACTCACCTCCCTGAAAAAAGGAGAAAAGCGTTCGTTCTTATTTTCTTTCGATGCGAGATTGGGGGT TGGCTCCTATTCTGTCGCTGTCGCGTTGCATACTTCCAGTACGCACCTCGGCAAAAACTATGAATGGCGCG ATCTGGCCGTGGTATTCAACGTCGTTAACACGGAACAACAAGAGTTTGTCGGCGTGTCCTGGTTGCCGCCT GAACTGGAGATTTCTTAATGGGTTCGTCGTTTTATCGTTCATTTGAAGAACGACACAGAGGTTCGGTTGAA GAAATCAAGCGCCGCTTGAGTTTTTATTTACCTTTTCTTGCAGGTCTGAAGGACATTTATCCTGATGGCGT GATTGCGGATATTGGTTGCGGACGTGGCGAATGGTTGGAGATCCTGACTGAAAATGGCATTGCGAACATCG GCGTCGATCTCGATGATGGCATGCTGGCGCGCGCCAGGGAGGCCGGACTGAATGTGCAGAAAATGGATTGT CTGCAGTTTTTGCAAAGTCAGGCGGATCAGAGCCTGATAGCGTTGACCGGTTTTCATATTGCTGAGCATTT GCCGTTTGAGGTCCTGCAGCAACTCGCCATGCATACCCTACGGGTGCTGAAACCAGGTGGTTTGCTGATCC TCGAAACGCCGAACCCGGAGAATGTÀAGCGTCGGCACCTGTTCATTTTATATGGATCCAACGCATAATCAT CCTCTGCCACCGCCACTGCTTGAGTTTTTACCTATTCATTATGGTTTTACCCGAGCAATTACCGTTCGTCT GCAGGAAAAAGAGGTTCTTCAATCTCCGGATGCAGCCGTTAATTTGGTCGATGTACTCAAAGGGGTGAGCC C CGACTACAGC AT C ATTGCTC AGAAAGC AGCGCCAAC AGATATTCTTGAACGCTTTGACAC C CTGTTTAC C CAGCAGTACGGTCTGACGCTGGATGCTCTGAGCAACCGTTACGATGCGATTTTGCGCCAACAGTTTTCGTC CGTTGTCTCACGGCTGGAGACGTTGAACCAAACCTATATGCAACAGATAAGCCAAATGTCAGAGACTATTC AGACGTTGCAAGGTGAGGTTGACGATCTGAGT C ATGTC ATCGATCAGAAC C ATCAGCTT C ATC AGC AAATG GCGGATTTACATAACAGTCGTTCATGGCGTATTACTCAACCACTACGCTGGTTGTCTTTGCAACGTCAATT ATTACGTCAGGAAGGGGCTAAAGTGCGAGCCCGTAGGGCTGGGAAAAAAATATTGCGCAAAGGGATGGCGC TCTCGCTGGTCTTTTTCCATCGTTACCCTAAGTCTAAGGTTTATCTGTTTAAGGTTCTGAGAAAAACTGGC TGCTATACATTGCTACAACGTTTGTTCCAACGCGTAATGCTGGTGCAATCTGACACGATGATGATGCAGTC CAGAAGATATGATGTGGGTACTGAAGAAATGACAAGTCGCGCGATGAGTATTTATAACGAATTAAAAAATA AAAATACGGAGAAATAACGATGCGTATTGTCATAGATTTACAAGGCGCACAGACGGAAAGCCGCTTTCGTG GCATCGGTCGTTATAGTATCGCAATCGCCAGAGGCATAATCAGAAATAACAGCCGGCATGAGATTTTCATC GCGCTATCCGCCATGCTGGATGAGTCGATTGCAAATATTAAGGCGCAATTTGCCGATCTCCTGCCGGCAGA AAATATAGTCGTATGGCATGCCGTAGGCCCTGTTCGTGCGATGGACCAAGGTAATGAATGGCGTCGGGAGA GCGCAGAACTGATTCGGGAAGCGTTTCTTGAATCATTGTGTCCAGATGTCGTTTTCATTACGAGTTTGTTT GAAGGTCATGTCGACGATGCGGCTACATCGGTACACAAATTTAGTCGTCAGTATAAAGTAGCCGTACTGCA CCACGATCTTATCC C C CTCGTGCAGGCGGAAACCTATCTGCAGGACGATGTATACAAACC CTACTATTTAC AGAAAGTTGAGTGGTTAAAAAACGCTGACCTTTTGTTGACTAACTCTGCTTATACCGCACAGGAAGCGATC GAGCATCTGCATTTACAGGGCGATCATGTGCAGAATATTGCAGCCGCAGTCGATTCTCAGTTTTGTATGGC GGAGGTGGCAGCGAGCGAAAAAGAGACCGTCCTTGGCCATTACGGTATTCAGCGCGAGTTCATGTTGTATG CGCCCGGAGGATTTGACTCAAGGAAAAACTTTAAACGGTTGATTGAGGCCTATGCCGGGCTCAGTGATGCC TTACGTCGCAGTCATCAACTGGTCATCGTCAGTAAGCTTTCCATCGGTGATCGTCAGTATCTGGAATCCCT
124
TGCGTCAGGTAATGGTTTACAGCAGGGCGAACTGGTACTCACTGGTTATGTGCCGGAAGATGAGCTGATCC AGCTCTATCGCCTATGTAAGCTGTTCATCTTTGCTTCACTACATGAAGGTTTTGGGTTGCCGGTTCTGGAA GCAATGTCGTGCGGTGCGCCGGTGATTGGCTCAAATGTCACCAGTATTCCTGAAGTCATCGGTAATCCTGA GGCATTATTCGACCCGTATTCTGTCTCTTCCATGAGGGATAAGATCGCGCAATGTTTGACTGATGATACCT TCCTCGCGCGTCTGAAAGAAATGGCGCAGCAGCAAGCGCGTAATTTCTCTTGGGATAAAGCTGCGGTGACT GCTCTGGAAGCTTTCGAAAAGATCGCGGTAGAAGACACCGGTACTGCGCAGGTTTTGCCTGAAGCTTTGAT TCAGAAGATCCTTGCTATCTCACAAGGGCAGCCAGATGACCGCGATCTGCGCTTGTGCGCAACGGCCATTG ATTACAATCTGAAAACGGCAGAACTTTATCAAATCGACGATAAATCGCTGAACTGGCGTGTGGAAGGCCCA TTCGATAGCTCATATAGTCTGGCGTTGGTCAACCGCGAATTTGCCCGGGCACTCTCAGCCGATGGTGTAGA GGTTTTATTGCATTCCACTGAAGGACCAGGTGATTTTGCCCCAGATGCCTCGTTTATGGCACAGTCGGAAA ATAGTGATCTTCTGGCATTTTATAATCAATGTCAGACCCGCAAGAGTAACGAAAAGATAGATATTATTAGC AGAAATAT CT ATCC ACCG CGGGTT ACCAAAATGGATGC C AAAGTAAAATT CCTTC ATTGTTATGCTTGGGA AGAAACGGGCTTTCCGCAACCGTGGATCAATGAATTTAATCGGGAACTTGACGGAGTGCTGTGTACTTCGG AACATGTTCGTAAAATACTGATTGATAACGGACTGAATGTGCCCGCATTTGTTGTTGGCAATGGCTGTGAC CATTGGCTCAATATCCCAGCCGAGACGACAAAAGATGTGGATCACGGAACATTCCGTTTCCTGCACGTCTC TTCTTGTTTCCCACGCAAAGGGATACAGGCAATGCTTCAGGCTTGGGGGAAGGCGTTCACTCGTCGTGACA ATGTTATCTTAATCATTAAGACTTTTAACAATCCGCACAATGAAATTGACGCATGGCTGGCTCAGGCCCAG GCTCAATTCATAGACTATCCCAAAGTTGAAGTGATCAAAGAGGATATGTCAGCCACCGAGCTTAAAGGGCT TTATGAAAGCTGTGATGTTTTGGTTGCTCCAGGTTGCGCTGAAGGCTTTGGTTTACCTATTGCTGAAGCAA TGCTGAGTGGGCTACCGGCTATCGTCACCAATTGGAGCGGGCAACTTGATTTTGTTAATTCACAAAATTCA TGGCTGGTTGACTATCAGTTCACTCGGGTAAAAACGCACTTTGGTCTGTTTTCCTCAGCCTGGGCCAGTGT GGATATTGACAACTTAACAGATGCATTAAAAGCGGCAGCCTCAACCGATAAATCAGTGCTGCGTGACATGG CCAATGCTGGTCGCGAGCTTCTTCTGCAGCAGTTTACCTGGAAAGCGGTGGCTGATCGTTCTTGCCAGGCG GTCAAGACTCTGCGTGCGCATATTGATATTGCACAGCATCGGGCGCGCATTGGCTGGGTGACGACCTGGAA CACGAAATGTGGGATCGCAACCTATTCCCAGCATCTGGTGGAAAGCGCACCTCATGGCGCGGATGTTGTTT TTGCTCCCCAGGTCAGCGCTGGCGATCTTGTGTGTGCAGACGAAGAGTTTGTACTTCGCAACTGGATTGTA GGTAAAGAGAGCAACTATCTGGAAAAC CT CCAGCCACACATTGATGCTCTGAGACTCGATGTCATTGTGAT CCAATTCAACTATGGATTCTTTAATCATCGAGAACTGTCGGCGTTTATTCGTCGCCAGCATGACGCCGGTC GTTCAGTTGTTATGACGATGCACTCAACTGTGGATCCGCTGGAAAAAGAGCCGAGCTGGAATTTCCGTCTT GCTGAAATGAAAGAGGCGCTGGCACTTTGCGACCGGTTGTTGGTGCATTCGATTGCCGATATGAACCGCCT TAAAGATTTAGGCTTAACTGCGAATGTTGCTTTATTCCCGCACGGTGTTATCAACTACTCCGCAGCGAGCG TCACACGTCAACAGCAGTCTTTACCGCTAATTGCGAGCTATGGCTTCTGCTTACCGCATAAGGGCCTGATG GAACTAGTAGAATCCGTCCATAGACTCAAGCAAGCCGGTAAACCGGTTCGTTTACGACTGGTGAACGCAGA GTATCCTGTTGGGGAGTCACGCGATCTGGTGGCAGAGCTTAAAGCTGCTGCTCAGCGGTTAGGTGTTACCG ATCTGATTGAGATGCATAATGATTTCCTACCTGATGCGGAGAGTCTGCGGTTGCTTTCAGAAGCCGATCTT CTGATTTTTGCTTATCAGAATACTGGGGAGTCTGCTAGCGGGGCGGTACGTTATGGTATGGCGACTCAAAA ACCTGTTGCGGTAACGCCCCTGGCGATATTTGATGATTTGGACGATGCCGTCTTTAAATTTGATGGATGCA GCGTCGATGATATCAGTCAGGGGATTGACCGGATCCTGAATTCCATCCGTGAACAGAACTCTTGGGCAACC AGGACTCAACAACGTGCCGATGCATGGCGGGAACAACATGATTATCAAGCTGTTTCACGCCGTCTGGTTAA TATGTGTCAAGGCTTAGCTAAAGCTAAATATTTTAAATAAAAATATCTCTCTTGTATTTTTTGCCTTTGAA TACAAGAGGGGTTAGATAATGTGTCATTTATTATGAAAATTATTTTTGCTACTGAGCCAATTAAATACCCA TTAACGGGCATCGGTCGGTATTCCCTGGAGCTGGTTAAGCGGCTGGCGGTCGCCCGCGAAATTGAAGAATT AAAGCTATTTCACGGTGCGTCGTTTATAGAACAGATCCCTTTGGTGGAGAATAAAAGCGATACCAAAGCCA GCAATCATGGTCGTCTGTCGGCGTTTCTACGCCGACAGACGCTGTTGATTGAGGCTTATCGCTTGCTGCAT CCGCGGCGCCAGGCGTGGGCATTGCGCGACTATAAGGATTATATCTACCATGGCCCCAATTTTTATCTGCC GCATAAACTGGAACGCGCCGTGACCACGTTTCATGACATATCCATTTTTACCTGCCCGGAATATCATCCAA AAGATCGGGTTCGCTATATGGAGAAGTCCCTGCATGAGAGTCTGGATTCGGCAAAGCTGATCCTGACCGTT TCTGATTTCTCGCGCAGTGAAATTATCCGCTTGTTCAACTATCCGGCGGAGCGGATCGTAACCACCAAGCT AGCCTGCAGCAGTGACTATATCCCACGCAGCCCGGCAGAGTGTCTGCCGGTACTGCAGAAATATCAGCTGG CGTGGCAGGCCTACGCGCTATATATCGGCACTATGGAGCCACGTAAAAATATCCGAGGCCTGCTGCATGCC TATCAGCTGCTACCGATGGAGATCCGCATGCGCTATCCGCTAATCCTTAGCGGCTATCGCGGCTGGGAAGA CGATGTGCTGTGGCAGTTAGTCGAGCGCGGTACTCGGGAAGGCTGGATCCGTTACCTCGGATATGTTCCGG ATGAAGACCTGCCGTATCTGTACGCAGCGGCCAGAGTCTTTGTTTATCCCTCCTTCTACGAGGGATTCGGT TTACCTATTCTTGAAGCGATGTCTTGCGGTGTGCCGGTAGTATGCTCCAATGTCACCTCTTTGCCTGAGGT TGTTGGCGATGCCGGCCTCGTTGCCGATCCTAATGATATAGACGCGATTAGCGCGCAAATTTTGCAGAGCC TGCAAGATGATAGCTGGCGGGAAATCGCCACCGCGCGCGGTCTTGCTCAGGCGAAACAGTTTTCGTGGGAG
125
AACTGTGCGACACAGACCATTAACGCCTATAAATTACT CTAAGGGTGT CAGTTGAG AGTT CTACACGTCTA TAAGACTTACTATCCCGATACCTACGGCGGTATTGAGCAGGTCATTTATCAGCTAAGTCAGGGCTGCGCCC GCCGGGGAATCGCAGCCGATGTTTTCACTTTTAGCCCGGACAAAGATACAGGTCCTGTCGCTTACGAAGAT CAT CGGGT C ATTTATAATAAAC AGCTTTTTGAAATTGC CTC C ACGCCGTTTTCGCTGAAAGCGTTAAAGCG TTTTAAGCTGATTAAAGATGACTACGATATCATCAACTACCATTTTCCGTTTCCCTTTATGGATATGCTGC ATCTTTCGGCGCGGCCTGACGCCAGGACTGTGGTGACCTATCACTCTGATATAGTGAAACAAAAACGGTTA ATGAAGCTGTACCAGCCGCTGCAGGAGCGATTTCTCAGCGGCGTAGATTGCATCGTTGCCTCGTCGCCCAA TTACGTGGCTTCCAG CCAGAC CCTGAAAAAATATCTGGATAAAACGGTGGTGATCC CGTTTGGTCTGGAGC AGCAGGACGTGCAGCACGATCCGCAGAGGGTCGCGCACTGGCGGGAAACTGTCGGCGATAAGTTCTTTCTC TTCGTCGGCACTTTCCGCTACTACAAAGGGCTGCATATTCTGATGGATGCCGCTGAGCGTAGCCGACTGCC AGTGGTGGTTGTAGGGGGCGGGCCGCTGGAATCGGAAGTGCGGCGTGAAGCGCAGCAGCGCGGGCTGAGCA ATGTGATGTTTAC CGGCATGCT C AACGACGAAG ATAAGTAC ATTCT CTTC C AGCTC TGCCGGGGCGTGGTA TTCCCCTCGCATCTGCGCTCTGAGGCGTTTGGCATTACGTTATTGGAAGGCGCACGCTTTGCAAGGCCGCT GATCTCTTGCGAGATCGGTACAGGTACCTCTTTCATTAACCAGGACAAAGTGAGTGGTTGCGTGATTCCGC CGAATGATAGCCAGGCGCTGGTGGAGGCGATGAATGAGCTCTGGAATAACGAGGAAACCTCCAACCGCTAT GGCGAAAACTCGCGTCGTCGTTTTGAAGAGATGTTTACTGCCGACCATATGATTGACGCCTATGTCAATCT CTACACTACATTGCTGGAAAGCAAATCCTGAGCGGCCGCGAGCTCGTCGACTCGAGGATCCGTGTAGGCTG GAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTAAGGAGGATATTCAT ATGGATAAAGCCGTAAGCATATAAGCATGGATAAGCTATTTATACTTTAATAAGTACTTTGTATACTTATT TGCGAACATTCCAGGCCGCGAGCATTCAGCGCGGTGATCACACCTGACAGGAGTATGTAATGTCCAAGCAA CAGATCGGCGTAGTCGGTATGGCAGTGATGGGACGCAACCTTGCGCTCAACATCGAAAGCCGTGGTTATAC CGTCTCTATTTTCAACCGTTCCCGTGAGAAGACGGAAGAAGTGATTGCCGAAAATCCAGGCAAGAAACTGG TTCCTTACTATACGGTGAAAGAGTTTGTCGAATCTCTGGAAACGCCTCGTCGCATCCTGTTAATGGTGAAA GCAGGTGCAGGCACGGATGCTGCTATTGATTCCCTCAAACCATATCTCGATAAAGGAGACATCATCATTGA TGGTGGTAACACCTTCTTCCAGGACACTATTCGTCGTAATCGTGAGCTTTCAGCAGAGGGCTTTAACTTCA TCGGTACCGGTGTTTCTGGCGGTGAAGAGGGGGCGCTGAAAGGTCCTTCTATTATGCCTGGTGGCCAGAAA GAAGCCTATGAATTGGTAGCACCGATCCTGACCAAAATCGCCGCCGTAGCTGAAGACGGTGAACCATGCGT TACCTATATTGGTGCCGATGGCGCAGGTCACTATGTGAAGATGGTTCACAACGGTATTGAATACGGCGATA TGCAGCTGATTGCTGAAGCCTATTCTCTGCTTAAAGGTGGCCTGAACCTCACCAACGAAGAACTGGCGCAG ACCTTTACCGAGTGGAATAACGGTGAACTGAGCAGTTACCTGATCGACATCACCAAAGATATCTTCACCAA AAAAGATGAAGACGGTAACTACCTGGTTGATGTGATCCTGGATGAAGCGGCTAACAAAGGTACCGGTAAAT GGACCAGCCAGAGCGCGCTGGATCTCGGCGAACCGCTGTCGCTGATTACCGAGTCTGTGTTTGCACGTTAT ATCTCTTCTCTGAAAGATCAGCGTGTTGCCGCATCTAAAGTTCTCTCTGGTCCGCAAGCACAGCCAGCAGG CGACAAGGCTGAGTTCATCGAAAAAGTTCGTCGTGCGCTGTATCTGGGCAAAATCGTTTCTTACGCCCAGG GCTTCTCTCAGCTGCGTGCTGCGTCTGAAGAGTACAACTGGGATCTGAACTACGGCGAAATCGCGAAGATT TTCCGTGCTGGCTGCATCATCCGTGCGCAGTTCCTGCAGAAAATCACCGATGCTTATGCCGAAAATCCACA GATCGCTAACCTGTTGCTGGCTCCGTACTTCAAGCAAATTGCCGATGACTACCAGCAGGCGCTGCGTGATG TCGTTGCTTATGCAGTACAGAACGGTATTCCGGTTCCGACCTTCTCCGCAGCGGTTGCCTATTACGACAGC TACCGTGCTGCTGTTCTGCCTGCGAACCTGATCCAGGCACAGCGTGACTATTTTGGTGCGCATACTTATAA GCGTATTGATAAAGAAGGTGTGTTCCATACCGAATGGCTGGATTAA
SEP ID NO: 15 (example 015 rfb locus nucléotide sequence 015-EPA production strain stLMTB11738)
ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGAT ACCCAAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAG GGATCAAAGAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTAT GAGTTAGAATCACTCCTTGAGCAGCGCGTGAAGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCC GGGCGTGACCATTATGAACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGAC CTGCCATTGGTGACAACCCATTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCG CTACGTTACAACCTTGCTGCCATGATTGCACGTTTCAACGAAACGGGCCGCAGCCAGGTGCTGGCAAAACG TATGC CGGGTGAC CT CTCTGAATACT C CGT CATC C AGACTAAAGAGC CGCTGGAC CGTGAGGGTAAAGTCA GCCGCATTGTTGAATTTATCGAAAAACCGGATCAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGT
126
CGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTGGAACGTACTCAGCCTGGTGCATGGGGACGTATTCA GCTGACTGATGCTATTGCCGAGCTGGCGAAAAAACAATCCGTTGATGCAATGCTGATGACCGGCGACAGTT ACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAGTATGGCCTACGCAACCTGAAAGAAGGG GCGAAGTTCCGTAAAGGTATTGAGAAGCTGTTAAGCGAATAATGÀAAATCTGACCGGATGTAACGGTTGAT AÀGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAATCTTCCTGCCGTTGTTTTA TATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAGTTTTCCAGGATTTTCCTTGTTT CCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTAACGCTCGTCAC ATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCATTAATACCTCTATT AATCAAACTGAGAGCCGCTTATTTCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGCTAGCATGAGC AAAACTAAACTAAATGTT CTTTAC CTTGCAATAAGTCAGGGTGC C AATTACCTACTGCC ATT ATT AATTTT TCCTTATCTTGTTAGAGTCATTGGTGTATCGAATTTTGGTGATCTGAGTTTTTCATTGATAACTATACAAG TGTTGTTAATGGTTGTTGAATATGGTTTTGGATATAGTGGGACAAGAGAAATAGCACTAAATAACGATAAA AAATACCATTCTGAATTTTTTTGCGGTGTGGTGCTTGCTCGTTTTATATTAATGCTAATTGCAGCTATAAT ACTCATAATACTCTGTTTTTTTTATGTTTTTAACGACGTTAAGTCTTTGTTATGTGTTGGTTTTCTGTCCG TAATTGCAGGTGTTTTCAATCCAAATTGGTTTTTGCAAGGTAAGGAAATGATGAGTGTGATGGCTGTGCTG TCACTATTTTCACGAGGCATAGCAGTCGTTGCAGTTTATCTAATTATAAAACCCGCAACGCCGATGTACAT CAGTGCCTTATTATTGAGCATGCCATATATTTTGTATTCATTCTGTGGCGTTGCCTACTTACTTATTATCA AGGAGATTTTTTTATGTAGGCCACCGATAAAGAAAATTCAAGTAATTTTAAAAAATGGATTTCATTTTTTT TGTTCAACACTTGCGACTAGTGCATACACAATGTTGACCCCTCTTGTATTGGGTGGCGTATCTGGAAAGTT TGATGTAGGCATCTTTAACTCAGCTAACATGATCAAACAAGGTTTGGCTGGACTTGCATCACCATTAGTCC AAGCTTTTTATCCAAGAATTAACATTTTGCAAAGAGAGAATCCATATATTGCAAACTTAAAATCTAGAATG ATTCTTAAATACTTGCTTGTTTTTTACATGGCTTTAGCAATACCATTTTTACTTTTTGCCAACCAATTATC ATTATTAATATTCGGCATGAAAGGTGAAGTAATTGCAGGTGCAATGCAATTAATGACATTGCTTCCTATAT TCATAGGTTTTAATACAGTTGTCGGGTTACTTGTATTAGTACCTAATGGGATGCAAAAACAGTATTTCAAA TCTATTTTCCTAGGAACTATTACTTGTTTAAGCATAGTTTATCCAGCATGTAAATATTATGGAGCAACGGG TGCGATTGTGAGTCTTATTGTAGCTGAAATTTTCGTTGGCATGGGAATGCTTAAACAATTCATTAAAGTAA ATAAAACCGTATGTAGGCCTCATAAATTATGAATATCTCGGTAATAATATCTGTTTGGAAACGCCCAGTTC AATTAGAATTGATTCTCT CTGAGCTCGATT CTC AGGCTAAAGACAATAGT CTACACCTAGAAGTAATTGTT TC CGATAGTC ATAGTGGTAAAGAAATTGATGATGTAGTTGCTGATAATATT CATAAAAAGAAAAAT ATTAA TATTATCCATCAACATACTAAAAATATACTCT C CGCTAAGCGCAATTTCGGAGCAT C C CTAGCCCATGGGG ATTATTTAATATTTCTTGATGATGATTGTATACCCGCAAGTGGATATATATCATCGTTGCTGAACTATTTA AAAAAAATGAATAGTAAAAGCGTTTTATGTGGGGAAGTTAGATTCGAAAATGAACTCATTGAGACCAGCAA TTACTATCGCTACAGGAACTCTTTACACCCTAAGTTTAGTGATAGTCCTGATATCTCTATGAATGCCTGGA CTTTTGTCGCAATGAATTGTGTTCTTGATAGAAAGGCATTTTCATCAGGTATAGTTTCATATAATGAAAAT TTTATTGGTTATGGTTGTGAAGATCATGAGTTTGGGTGGCAACTTGAAAAAAATGACTTCAAAATTATTTT TGCTGATTTTAAAATATTACATCACGAATACAGTGGCGATATAGAAGGATATACAAAAAAAATTCGTGCTA CAGCACGTGATGGTATGAATGTATTAAGCAAAGTAAGGCCTGAAATGTTTTCTACTAATAAAAAATTATTC CTAGTTGAGAAAATATTTAGTAAACACAAAACGTTTAGTTiAAATATGCCAATCAATATTTTTCAATAAATT TATTTTTAAAAAAATAATACAATTTTTAAAAAAAACAGATGCAAATAAAAAACTCTATTTCCCAATTCTTT ACAGATATGTGTTGATTTCGGCATATATACATGGTATTGGAGAGCGTGGCACCTCAAAAACAGATGATTTG CTTAAGAACTGGTATATATAGATGATGCTATCTTCATTTATTAAGACATTTGTATGGAAGGTAA2\AAACAA TGAAGTATAATGCATTGATGGCTTTTTTATTATTTTTTGTTGTTTTTTTTAGATTGTCGCTGATAATACCT TTCTTATATTTGGCATTTATTCCTGCATTTTTTGGTATTATGTATTTAGTGCGTAATTTTATGATTACTAT GGGCAATGGATTGGTATCTATAGATCGTAAAAATTTGTTGCTGTTATCTATATTCATAATTATTTTTTTAT TTTGTTTGGTTTTCGATTTGTTTCAAAAAAGCCATTCTTTTCAAAGTTATTTTACCGTTAGATTATTTATG TTGTTTTTATTTTCATTTGTTCCTGCGTATTATTTAGTAAATAGATTCATAAAGGGTGACTTGAAATTAAT GGAGCGAATATTAGTGTATTCTCTCTGGGTTCAAATAGTTATTTTTTTTGGTATGTATATAAGTCCAGAGT TAAAAAGATTGTTATATACTTTCTTTGGTATGTCTGACTCTGTTAATCTTTGGGAACAAAATGCTAAAGTA AGAGGATTTGGGTTGTCGGGTGAAATAAATTTCATGACACCATTTTTGATGATCTATATGTCATTTTTTAT GATGAAAAGGCGTTATGCTTTAATTACTTTAATTTGTCTGACTCAAATCGTAAATTCTAACATGGCTGTGA TTGCAGCCATTATTGGTATCGGTTGCTCTAGACTTAATATT7ÏATATAAAAATTGCAACAGTATTGATTTTG GGAGTTTTAGTTTATAGCTTAGGAGCGGTGTTCTTTCCTCGATTTTATGATGAGTTCGTTTCTGGAGATGG CACAAGAACTCTGGATATCTTATTACAGCAACATGTGTTTGTTGTAGGTAATTTAGATTTTTTTAATATTA TATTTGGATTACAGCAAAACATATCTTCATCAATCCCCGATATTAAACAAAGTTCGGATATGGGCTGGGTT ATACTGTTTAATTACGGTGGGTTAACATTTATTACACTCTTTTTATTTTTAATCTTTACTATTTCTATTGC GACATTTGGAATGACATATCAAGCAATTATATGGATGTTAATTGGGATAATTTTCAATACCAAAGGTTTAG
127
TTTTAGGATCTAACGGCTATTTCTTTCTATCTTTTATATATATGTTTTTGAATAGAGTAACACTTAGTGGA CAGAGTTCAATTACTAATAAGTTAGGTCAAGTAAGTAAATAGCTTCCAGAGTATATTTGTCAATGATTTGA GGTTCGGTTATTATGTTTTCATCTAAAACACTGTTAATTACTGGTGGTACTGGCTCTTTCGGGAATGCTGT ATTAAATAGATTTCTTGATACAGATATTGCAGAAATCCGTATATTTAGTCGTGATGAAAAAAAACAAGATG ATATGCGGAAAAAATACAATAATCAAAAATTAAAGTTCTATATTGGTGATGTCAGAGATTACCGTAGTATT TTGAATGCGACTCGCGGTGTTGATTTTATATATCATGCAGCGGCACTTAAGCAAGTTCCATCATGTGAATT TCATCCTATGGAAGCCGTTAAAACTAATATCCTTGGTACGGAAAATGTTCTTGAAGCAGCTATAGCGAATG AAGTGAAGAGGGTTGTATGCCTAAGTACTGATAAAGCTGTATACCCGATTAACGCAATGGGTATTTCAAAA GCTATGATGGAAAAGGT C ATGGT CGCGAAATCCCGTAATGTTGATCG CAAT AAAACAGTAATATGTGGT AC CCGTTATGGGAATGTTATGGCATCTCGCGGTTCAGTTATTCCATTATTTGTTGATCTTATTAGAGCGGGCA AGCCACTCACAATAACTGATCCTAATATGACCCGCTTTATGATGACTCTTGAGGATGCGGTAGATTTAGTT CTTTATGCGTTTGAACATGGTAATAATGGTGATATCTTTGTGCAAAAAGCACCTGCAGCAACTATTGACAC ATTAGCTATTGCTTTAAAGGAATTACTAAATGTTCCTGACCATCCGGTAAATGTCATTGGAACGCGTCATG GCGAGAAATTATATGAAGCTCTACTTAGTCGTGAGGAAATGATCGCTGCTATAGATATGGGCGATTATTAC CGTGTCCCGCCAGATCTTCGTGACCTTAATTATGGCAAATATGTTGAGCAAGGTGATAGCCGAATATCTGA AATAGAAGATTATAACTCTCATAATACTCAACGGTTAGATGTTGAAGGCATGAAAGAGCTCTTGCTAAAAT TAGCCTTTATTCGAGCAATTCGTGCTGGTGAAAAATATAATCTGGATTCATGATATGAAAATATTAGTTAC TGGTGCAAATGGTTTTATTGGTCGTAATTTATGTTTGAGGCTTGAGGAACTTGGTTATAAAGATCTTATTA GAATTGATCGAGAATCAACGAAGCAAGATCTTGAACAAGGCTTACAGGATGCCGATTTTATTTATCACTTA GCTGGTATCAATAGACCTAAGACTGATGATGAGTTTATTTCTGGAAACAGTGATTTAACAAAGCATATAGT TGAGTATCTCCTTTCTATTGGTAAGAATACACCAATTATGCTAAGTTCTTCGATACAAGCTGAACTTAATA ATGCTTATGGGGTTAGCAAAGCTGTAGCTGAAAGCTATGTCGAAAAATATGCTGCTGCTAGTGGTTCTTCG TATTATATTTTCAGATATCCAAACGTTTTTGGTAAATGGTGTAAGCCAAACTATAATTCTTTTATAGCAAC TTTTTGCTACAATATTTCCAATGATATTGAGATTACTATCAATGATGCAGCAGCGCCAGTCAATCTGGTCT ATATTGATGATGTTTGTACTGATGCTATAGCTCTTCTCTCTGGGACGGTTGAAAGTGGATATAAAGTTGTT GCACCAATTTATTCAACAACAGTTGGTGAAGTTGCAGAATTAATTTATAGCTTCAAAAATAGCCGTTCCAC CCTGATCACAGAGGCTGTCGGGGCGGGATTTACCCGTGCATTGTATTCTACATGGCTGAGTTATTTACCAG CAGAGAAGTTTGCGTACAAGGTACCTTTTTATGGGGATGCCCGCGGAGTCTTTTGTGAGATGTTGAAAACG CCTTCAGCGGGGCAGTTTTCATTTTTTACTGCTCACCCTGGTATTACGCGTGGCGGACATTACCATCACAG TAAAAATGAGAAGTTTTTGGTCATTCGAGGTCAGGCATGCTTTAAATTTGAACATGTGATTACCGGTGAGC GATATGAACTGAAAGTTTCATCGGGTGAGTTTAAGATTGTTGAAACAGTTCCTGGTTGGACACATGACATT ACAAATATTGGAACTGATGAATTAATAGTCATGCTCTGGGCAAATGAAATTTTCAACCGTGATGAGCCCGA TACTATTGCGAGACCTCTATAATGAAAAAATTAAAAGTTATGTCTGTTGTTGGAACCCGTCCTGAGATTAT C C GTTTGTCGAGGGTT CTTG CTAAGTTTGATGAATACTGCGAGCATATTATTGTC CATACTGGTCAAAATT ATGATTACGAATTAAATGAAGTGTTCTTCAATGACTTGGGTGTTCGAAAACCTGATTATTTTTTAAATGCA GCGGGTAAAAATGCGGCGGAAACCATTGGTCAGGTTATTATTAAGGTAGATGAAGTATTAGAAATCGAAAA ACCTGAAGCAATACTGGTATTGGGCGATACGAATTCATGTATTTCTGCCATTCCGGCCAAACGCCGTTiAAG TGCCTATATTTCATATGGAAGCAGGTAACCGTTGTTTCGATCAACGCGTGCCTGAAGAAACCAACAGACGT ATTGTTGACCATACGGCTGATATCAATATGACCTACAGTGATATTGCTCGTGAATATCTCTTGGCTGAAGG TATCCCAGCTGATCGGATCATAAAAACTGGTAGCCCTATGTTTGAGGTTCTTTCATATTATATGCCCCAAA TTGATGGTTCAGATGTGCTATCGCGTTTGAATCTACAGTCTGGTGAGTTTTTTGTAGTAAGTGCGCATCGT GAAGAGAATGTTGATTCTCCAAAACAGCTCGTAAAGCTTGCGAACATTCTAAATACTGTTGCTGAAAAATA TAATCTTCCAGTTATTGTCTCCACACACCCAAGGACACGTAACCGAATCCGTGAGCAAGGAATTGAATTTC ATTCAAATATAAATCTACTGAAAC CATTGGGTTTCCATGATTATAACCACTTGCAGAAGAACTCAC GAG CT GTGCTTTCAGATAGCGGTACTATCACTGAAGAGTCATCCATCATGAATTTCCCAGCGGTAAACATCCGGGA AGCGCATGAGCGTCCGGAAGGCTTTGAGGAAGCATCCGTCATGATGGTGGGGTTAGAGTGTGAACGCGTAT TACAAGCGCTGGATATTCTGGCAACACAACCGCGAGGTGAAGTCCGTCTTTTACGTCAGGTTAGTGATTAC AGCATGCCAAATGTGTCGGATAAAGTTGTCAGAATTGTTCACTCTTACACAGATTATGTTAAGAGAGTCGT CTGGAAAGAATATTGATGAAACTTGCTTTAATCATAGATGATTACCTGCCCAACAGTACTCGTGTTGGTGC AAAAATGTTTCATGAACTTGCTCAAGAATTTATCCAGCGTGGGCACGATGTTACGGTAATTACTCCTGGTA CGGGCATGCAAGAAGAGATTTCTTTTGATACCTTTCAGGGGGTAAAAACATGGCGTTTTAAAAGCGGGCCG CTCAAGGATGTAAGTAAAATTCAGCGAGCGGTCAATGAAACGCTTTTGTCCTATCGGGCGTGGAAAGCCAT CAAAAAATGGGTAAAAAAAGAGACCTTTGAGGGGGTGATTTATTATTCACCTTCCATATTCTGGGGGCCTT TAGTTAÀAAAAATTAAAGCTCGTTGCCAATGTCCTGCTTATCTTATTTTAAGAGATATGTTTCCACAATGG GTAATTGATGCAGGAATGCTTAATGCTGGTTCCCCAATAGAACGCTACTTTCGTCTTTTTGAAAAAATATC TTATCGTCAGGCAAATCGTATTGGACTTATGTCTGATAAGTkATCTTGATGTTTTTCGGAAAGATAATAAAG
128
GCTATCCGTGCGAAGTTTTGCGTAATTGGGCATCCCTAACACCAACGATCATACCCAAGGATTATATACCA CTACGTAAGCGACTTGGCCTAGAGGATAAAACCATTTTCTTCTATGGTGGAAACATAGGTCATGCACAGGA CATGACAAACTTGATGCGACTTGTGAGAAACATGGCAGCATATCCTCAAGCTCATTTCCTATTTATTGGCC AGGGGGATGAAGTTGAATTAATTAATTCATTAGCATCTGAGTGGGCATTGACGAATTTCACCTATTTGCCC TCGGTTAACCAAGATGAATTTAAGTTCATTTTGTCGGAAATGGATATCGGCTTGTTTTCTCTTTCCGCTAG ACACTCTTCCCATAATTTTCCTGGTAAGTTATTAGGCTATATGGTTCAGTCGCTACCTATTTTAGGTAGCG TAAATGCCGGAAATGATTTGCTCGACATTGTCAATCAAAATAATGCGGGATTAATCCATGTCAATGGTGAG GACGATAAATTATGTCAATCTGCGCTATTAATGTTGCATGATATTGATGTGCGCCGGCAACTTGGTTCGGG GGCGAATATATTGTTGAAAGAACAATTCTCCGTTGAGTCTGCGGCACAGACGATAGAAATGAGGTTGGAGG CATGCAATGCGATTAATTGATAATGACCAACTCGACGAATTATATGATCAAGCCGGGCAATCGGAACGTTT ACGTTCCCACCTTATGATGCACGGCTCGCATCAAGAAAAGGTACAGCGTTTACTTATTGCATTAGTAAAGG GCAGCTATGTTGAACCGCATTATCACGAACTTCCTCATCAGTGGGAAATGTTCATTGTTATGGAGGGGCAA CTTCAGGTTTGTTTGTATGGTAGAAATGGTGAGGTTATAAAGCAATTTATAGCAGGAGATAATACTGGAAT GAGCATTGTGGAGTTTTCTCCGGGCGATATACACAGTGTCGAATGCCTATCTCCGCGTGCTCTTATGGTGG AAGTTAAGGAGGGGCCATTTGACCCTTCTTTTGCAAAATCGTTCGTGTGAGCGGCCGCGAGCTCGTCGACT CGAGGATCCGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGG AACTAAGGAGGATATTCATATGGATAAAGCCGTAAGCATATAAGCATGGATAAGCTATTTATACTTTAATA AGTACTTTGTATACTTATTTGCGAACATTCCAGGCCGCGAGCATTCAGCGCGGTGATCACACCTGACAGGA GTATGTAATGTCCAAGCAACAGATCGGCGTAGTCGGTATGGCAGTGATGGGACGCAACCTTGCGCTCAACA TCGAAAGCCGTGGTTATACCGTCTCT ATTTTCAACCGTTC C CGTGAGAAGACGGAAGAAGTGATTG C CGAA AATCCAGGCAAGAAACTGGTTCCTTACTATACGGTGAAAGAGTTTGTCGAATCTCTGGAAACGCCTCGTCG C ATC CTGTTAATGGTGAAAGCAGGTGCAGGCACGG ATGCTGCTATTG ATT CCCTCAAAC C AT ATCT CGATA AAGGAGACATCATCATTGATGGTGGTAACACCTTCTTCCAGGACACTATTCGTCGTAATCGTGAGCTTTCA GCAGAGGGCTTTAACTTCATCGGTACCGGTGTTTCTGGCGGTGAAGAGGGGGCGCTGAAAGGTCCTTCTAT TATGC CTGGTGGCCAGAAAGAAGCCTATGAATTGGTAGCAC CGATCCTGAC CAAAATCGC CGC CGTAGCTG AAGACGGTGAACCATGCGTTACCTATATTGGTGCCGATGGCGCAGGTCACTATGTGAAGATGGTTCACAAC GGTATTGAATACGGCGATATGCAGCTGATTGCTGAAGCCTATTCTCTGCTTAAAGGTGGCCTGAACCTCAC CAACGAAGAACTGGCGCAGACCTTTACCGAGTGGAATAACGGTGAACTGAGCAGTTACCTGATCGACATCA CCAAAGATATCTTCACCAAAAAAGATGAAGACGGTAACTACCTGGTTGATGTGATCCTGGATGAAGCGGCT AACAAAGGTACCGGTAAATGGACCAGCCAGAGCGCGCTGGATCTCGGCGAACCGCTGTCGCTGATTACCGA GTCTGTGTTTGCACGTTATATCTCTTCTCTGAAAGATCAGCGTGTTGCCGCATCTAAAGTTCTCTCTGGTC CGCAAGCACAGCCAGCAGGCGACAAGGCTGAGTTCATCGAAAAAGTTCGTCGTGCGCTGTATCTGGGCAAA ATCGTTTCTTACGCCCAGGGCTTCTCTCAGCTGCGTGCTGCGTCTGAAGAGTACAACTGGGATCTGAACTA CGGCGAAATCGCGAAGATTTTCCGTGCTGGCTGCATCATCCGTGCGCAGTTCCTGCAGAAAATCACCGATG CTTATGCCGAAAATCCACAGATCGCTAACCTGTTGCTGGCTCCGTACTTCAAGCAAATTGCCGATGACTAC CAGCAGGCGCTGCGTGATGTCGTTGCTTATGCAGTACAGAACGGTATTCCGGTTCCGACCTTCTCCGCAGC GGTTGCCTATTACGACAGCTACCGTGCTGCTGTTCTGCCTGCGAACCTGATCCAGGCACAGCGTGACTATT TTGGTGCGCATACTTATAAGCGTATTGATAAAGAAGGTGTGTTCCATACCGAATGGCTGGATTAA
SEQ ID NO: 16 (example 016 r/Z> locus nucléotide sequence - O16-EPA production strain stLMTB11739)
ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGAT ACCCAAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAG GGATCAAAGAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTAT GAGTTAGAATCACTCCTTGAGCAGCGCGTGAAGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCC GGGCGTGACCATTATGAACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGAC CTGCCATTGGTGACAACCCATTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCG CTACGTTACAACCTTGCTGCCATGATTGCACGTTTCAACGAAACGGGCCGCAGCCAGGTGCTGGCAAAACG TATGCCGGGTGACCTCTCTGAATACTCCGTCATCCAGACTAAAGAGCCGCTGGACCGTGAGGGTAAAGTCA GCCGCATTGTTGAATTTATCGAAAAACCGGATCAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGT CGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTGGAACGTACTCAGCCTGGTGCATGGGGACGTATTCA GCTGACTGATGCTATTGC CGAGCTGGCGAAAAAACAAT C CGTTGATGCAATGCTGATGACCGGCGAC AGTT
129
ACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAGTATGGCCTACGCAACCTGAAAGAAGGG GCGAAGTTCCGTAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAATCTGACCGGATGTAACGGTTGAT AAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAATCTTCCTGCCGTTGTTTTA TATAAAC C ATCAGAATAACAACGAGTT AGCAGT AGGGTTTTATTCAAAGTTTTC C AGGATTTTC CTTGTTT CCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTAACGCTCGTCAC ATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCATTAATACCTCTATT AATCAAACTGAGAGCCGCTTATTTCACAGCATGCTCTGÀAGTAATATGGAATAAATTAAGTGAAAATACTT GTTACTGGTGGCGCAGGATTTATTGGTTCAGCTGTAGTTCGTCACATTATAAATAATACGCAGGATAGTGT TGTTAATGTCGATAAATTAACGTACGCCGGAAACCGGGAATCACTTGCTGATGTTTCTGATTCTGAACGCT ATGTTTTTGAACATGCGGATATTTGCGATGCACCTGCAATGGCACGGATTTTTGCTCAGCATCAGCCGGAT GCAGTGATGCACCTGGCTGCTGAAAGCCATGTTGACCGTTCAATTACAGGCCCTGCGGCATTTATTGAAAC CAATATTGTTGGTACTTATGTCCTTTTGGAAGCCGCTCGCAATTACTGGTCTGCTCTTGATAGCGACAAGA AAAATAGCTTCCGTTTTCATCATATTTCTACTGACGAAGTCTATGGTGATTTGCCTCATCCAGATGAAGTA AATAATAC AGAAGAATTAC C C TTATTT ACTGAGACGAC AGCTTACGCGCCAAGCAGCCCTTATTC CGCATC CAAAGCATCCAGCGATCATTTAGTCCGCGCGTGGAAACGTACATATGGTTTACCGACAATTGTGACTAATT GCTCGAACAACTATGGTCCTTATCATTTCCCGGAAAAGCTTATTCCACTGGTTATTCTTAATGCACTGGAA GGTAAGGCATTACCTATTTATGGCAAAGGAGATCAGATCCGCGACTGGTTGTATGTTGAAGATCATGCGCG TGCGTTATATACCGTCGTAACCGAAGGTAAAGCGGGTGAAACTTATAACATTGGTGGGCACAACGAAAAGA AAAACATCGATGTAGTGCTCACTATTTGTGATTTGCTGGATGAGATTGTACCGAAAGAGAAATCTTATCGT GAGCAAATCACTTATGTTGCTGATCGTCCGGGACACGATCGCCGCTATGCTATTGATGCTGAGAAGATTGG TCGCGCATTGGGATGGAAACCACAGGAAACGTTTGAGAGCGGGATTCGTAAAACGGTGGAATGGTACCTGT CCAATACAAAATGGGTTGATAATGTGAAAAGTGGTGCCTATCAATCGTGGATTGAACAGAACTATGAGGGC CGC CAGTAATGAAT ATCCT C CTTTTTGGCAAAAC AGGGCAGGTAGGTTGGGAACTACAGCGTGCT CTGGC A CCTTTGGGTAATTTGATTGCTTTTGATGTTCACTCTACTGATTATTGCGGTGATTTTAGTAATCCTGAAGG TGTAGCTGAAACCGTAAGAAGCATTCGGCCGGATATTATTGTCAATGCAGCCGCTCACACCGCAGTAGACA AAGCAGAATCAGAACCGGAGTTTGCACAATTAATTAACGCAACAAGTGTCGAAGCGATTGCGAAAGCAGCA AATGAAGTTGGAGCCTGGGTTATCCATTACTCGACTGATTACGTCTTCCCTGGAAATGGCGATATGCCATG GCTGGAGACGGATGCAACCGCACCACTAAATGTTTACGGTGAAACCAAGTTAGCCGGAGAAAAAGCGTTAC AGGAATATTGCGCGAAGCATCTTATTTTCCGGACCAGCTGGGTCTATGCAGGAAAAGGAAATAACTTCGCC ÂAAACGATGTTACGTCTGGCAAAAGAGCGTGAAGAATTAGCGGTTATTAACGATCAGTTTGGTGCGCCAAC AGGTGCTGAACTGCTGGCTGATTGTACAGCACATGCCATTCGTGTCGCACTGAATAAACCGGATGTCGCAG GCTTGTACCATTTGGTAGCCAGTGGTACCACAACCTGGTACGATTATGCTGCGCTGGTTTTTGAAGAGGCG CGCAAAGCAGGCATTCCCCTTGCACTCAACAAGCTCAACGCAGTACCAACAACAGCCTATCCTACACCAGC TCGTCGTCCACATAACTCTCGCCTTAATACAGAAAAATTTCAGCAGAACTTTGCGCTTGTCTTGCCTGACT GGCAGGTTGGCGTGAAACGAATGCTCAATGAATTATTTACGACTACAGCAATTTAATAGTTTTTGCATCTT GTTCGTGATGGTGGAGCAAGATGAATTAAAAGGAATGATGAAATGAAAATGCGTAZkAGGTATTATTTTAGC GGGTGGTTCTGGTACACGTCTTTATCCTGTGACTATGGCTGTCAGTAAACAGCTATTACCTATTTATGATA AACCGATGATCTATTACCCGCTCTCTACACTGATGTTGGCGGGTATTCGCGATATTTTGATTATCAGTACA CCTCAGGATACTCCTCGTTTTCAACAATTGCTGGGTGACGGTAGCCAGTGGGGCCTGAATCTTCAGTACAA AGTGCAACCTAGCCCAGATGGCCTCGCGCAGGCATTTATCATCGGTGAAGAGTTTATTGGTGGTGATGATT GTGCTTTGGTTCTTGGTGATAATATCTTTTACGGTCACGATCTGCCGAAGCTAATGGAGGCCGCTGTTAAC AAAGAAAGTGGTGCAACGGTATTTGCCTATCACGTTAATGATCCAGAACGCTATGGTGTCGTTGAGTTTGA TAAAAACGGTACGGCAATCAGTCTGGAAGAAAAACCGTTAGAACCAAAGAGTAATTACGCCGTTACAGGTC TGTACTTTTATGATAACGACGTGGTTCAGATGGCGAAAAACTTGAAGCCGTCTGCACGTGGTGAGTTAGAA ATTACAGATATTAACCGTATTTATCTTGAGCAGGGACGTCTGTCTGTCGCGATGATGGGGCGTGGCTACGC GTGGCTGGACACGGGGACTCATCAGAGTCTGATAGAAGC7ÎAGTAATTTTATTGCGACAATTGAAGAGCGCC AGGGATTGAAGGTTTCCTGTC CTGAAG AGATTGC ATTT CGTAAAGGTTTTATTGATGTTGAG CAAGTAAGA AAATTAGCTGTACCACTAATAAAGAATAATTATGGGCAGTATCTTTATAAAATGACGAAGGATTCAAATTA ATGAATGTGATTAGAACTGAAATTGAAGATGTGCTAATTCTGGAGCCZiAGAGTATTTGGTGATGATAGAGG TTTCTTTTATGAGAGCTTTAATCAATCAGCATTTGAACATATTCTAGGCTATCCGGTCAGCTTTGTTCAAG ACAATC ACT C ACGTTCATCAAAAAATGTACTC AGAGGC CTTCACTTT CAACGCGGCGAGTACGCAC AAGAT AAACTTGTACGCTGCACTCATGGAGCAGTTTTTGATGTTGCTGTTGATATTCGACCCAATTCGGTATCCTT TGGTAAATGGGTTGGTGTTCTGCTTTCAGCTGATAATAAGCAGCAGTTGTGGATACCAAAAGGGTTTGCTC ATGGCTTTTTGGTTCTGTCTGATATCGCTGAATTTCJkATATAAAACTACAAACTATTATCATCCTGAAAGC GATTGTGGAAT ATGTTGGAATGATGAACGCATTGCAATTGATTGGCC CCAAACATCAGGGTTAATC CTTT C GCCAAAAGATGAAAGGCTCTTTACGTTAGATGAGCTTATCAGATTAAAATTAATTGCATGAATACGAATAA
130
ATTATCTTTAAGAAGAAACGTTATATATCTGGCTGTCGTTCAAGGTAGCAATTATCTTTTACCATTGCTTA CATTTCCATATCTTGTAAGAACACTTGGTCCTGAAAATTTCGGTATATTCGGTTTTTGCCAAGCGACTATG CTATATATGATAATGTTTGTTGAATATGGTTTCAATCTCACAGCAACTCAGAGTATTGCCAAAGCAGCAGA TAGTAAAGATAAAGTAACGTCTATTTTTTGGGCGGTGATATTTTCAAAAATAGTTCTTATCGTCATTACAT TGATTTTCTTAACGTCGATGACCTTGCTTGTTCCTGAATATAACAAGCATGCCGTAATTATATGGTCGTTT GTTCCTGCATTAGT CGGGAATTTAATCTAC C CTATCTGGCTGTTTC AGGGAAAAGAAAAAATGAAATGGCT GACTTTAAGTAGTATTTTATCCCGCTTGGCTATTATCCCTCTAACATTTATTTTTGTGAACACAAAGTCAG ATATAGCAATTGCCGGTTTTATTCAGTCAAGTGCAAATCTGGTTGCTGGAATTATTGCACTAGCTATCGTT GTTCATGAAGGTTGGATTGGTAAAGTTACGCTATCATTACATAATGTGCGTCGATCTTTAGCAGACGGTTT TCATGTTTTTATTTCCACATCTGCTATTAGTTTATATTCTACGGGAATAGTTATTATCCTGGGATTTATAT CTGGACCAACGTCCGTAGGGAATTTTAATGCGGCCAATACTATAAGAAACGCGCTTCAAGGGCTATTAAAT CCTATCAC C CAAGC AATAT AC C C AAGAAT ATC AAGTACGCTTGTTCTTAATCGTGTGAAGGGTGTGATTTT AATTAAAAAATCATTGACCTGCTTGAGTTTGATTGGTGGTGCTTTTTCATTAATTCTGCTCTTGGGTGCAT CTATACTAGTAAAAATAAGTATAGGGCCGGGATATGATAATGCAGTGATTGTGCTAATGATTATATCGCCT CTGCCTTTTCTTATTTCATTAAGTAATGTCTATGGCATTCAAGTTATGCTGACCCATAATTATAAGAAAGA ATTCAGTAAGATTTTAATCGCTGCGGGTTTGTTGAGTTTGTTGTTGATTTTTCCGCTAACAACTCTTTTTA AAGAGATTGGTGCAGCAATAACATTGCTTGCAACAGAGTGCTTAGTTACGTCACTCATGCTGATGTTCGTA AGAAATAATAAATTACTGGTTTGCTGAGGATTTTATGTACGATTATATCATTGTTGGTTCTGGTTTGTTTG GTGC CGTTTGTGCGAATGAGTTAAAAAAGCTAAACAAAAAAGTTTTAGTGATTGAGAAAAGAAAT C ATATC GGTGGAAATGCGTACACAGAGGACTGTGAGGGTATCCAGATTCATAAATATGGTGCACATATTTTTCATAC CAATGATAAATATATATGGGATTACGTTAATGATTTAGTAGAATTTAATCGTTTTACTAATTCTCCACTGG CGATTTATAAAGACAAATTATTCAACCTTCCTTTTAATATGAATACTTTCCACCAAATGTGGGGAGTTAAA GATCCTCAAGAAGCTCAAAATATCATTAATGCTCAGAAAAAAAAGTATGGTGACAAGGTACCTGAAAATTT GGAGGAGCAGGCGATTTCATTAGTTGGGGAGGACTTATACCAAGCATTGATAAAGGGTTATACGGAGAAGC AGTGGGGAAGAAGTGCAAAAGAATTGCCTGCATTTATTATTAAGCGAATCCCAGTGAGATTTACGTTTGAT AACAATTATTTTTCCGATCGCTATCAAGGTATTCCGGTGGGAGGCTACACTAAGCTTATTGAAAAAATGCT TGAAGGTGTGGACGTAAAATTAGGCATTGATTTTTTGAAAGACAAAGATTCTCTAGCGAGTAAAGCCCATA GAATCATCTACACTGGACCCATTGATCAGTACTTCGACTATAGGTTTGGAGCGTTAGAATATCGCTCTTTA AAATTTGAGACGGAACGC CATGAATTT CCAAACTT C CAAGGGAATGCAGTAATAAATTTCACTGATGCTAA TGTACCATATACCAGAATAATTGAGCATAAACATTTTGACTATGTTGAGACAAAGCATACGGTTGTTACAA AAGAATATCCATTAGAGTGGAAAGTTGGCGACGAACCCTACTATCCAGTTAATGATAATAAAAACATGGAG CTTTTTAAGAAATATAGAGAGTTAGCTAGCAGAGAAGACAAGGTTATATTTGGCGGGCGTTTGGCCGAGTA TAAATATTATGATATGCATCAAGTGATATCTGCCGCTCTTTATCAAGTGAAAAATATAATGAGTACGGATT AATGATCTATCTTGTAATTAGTGTCTTTCTCATTACAGCATTTATCTGTTTATATCTTAAGAAGGATATAT TTTATCCAGCCGTATGCGTTAATATCATCTTCGCACTGGTCTTATTGGGATATGAAATAACGTCAGATATA TATGCTTTTCAGTTAAATGACGCTACGTTGATTTTTCTACTTTGCAATGTTTTGACATTTACCCTGTCATG TTTATTGACGGAAAGTGT ATTAG ATCT AAATATC AGAAAAGT CAATAATGCTATTTATAGCATAC CAT CGA AGAAAGTGCATAATGTAGGCTTGTTAGTTATTTCTTTTTCGATGATATATATATGCATGAGGTTAAGTAAC TACCAGTTCGGGACTAGCTTACTTAGCTATATGAATTTGATAAGAGATGCTGATGTTGAAGACACATCAAG AAATTTCTCAGCATACATGCAGCCAATCATTCTAACTACTTTTGCTTTATTTATTTGGTCTAAAAAATTTA CTAATACAAAGGTAAGTAAAACATTTACTTTACTTGTTTTTATTGTATTCATCTTTGCAATTATACTGAAT ACTGGTAAGCAAATTGTCTTTATGGTTAT CAT CTCTTATGCATTCAT CGTAGGTGTTAATAGAGTAAAACA TTATGTTTATCTTATTACAGCTGTAGGTGTTCTATTCTCCTTGTATATGCTCTTTTTACGTGGACTGCCTG GGGGGATGGCATATTATCTATCCATGTATTTGGTCAGCCCTATAATCGCGTTTCAGGAGTTTTATTTTCAG CAAGTATCTAACTCTGCCAGTTCTCATGTCTTTTGGTTTTTTGAAAGGCTGATGGGGCTATTAACAGGTGG AGTCTCTATGTCGTTGCATAAAGAATTTGTGTGGGTGGGTTTGCCAACAAATGTTTATACTGCTTTTTCGG ATTATGTTTATATTTCCGCGGAGCTAAGCTATTTGATGATGGTTATTCATGGCTGTATTTCAGGTGTTTTA TGGAGATTGTCTCGAAATTACATATCTGTGAAAATATTTTATTCATATTTTATTTATACCTTTTCTTTCAT TTTTTATCATGAAAGCTTCATGACTAATATTAGCAGTTGGATACAAATAACTCTTTGTATCATAGTATTCT CTCAATTTCTTAAGGCCCAGAAAATAAAGTGAAAATGTATTTTTTGAATGATTTAAATTTCTCTAGACGCG ATGCTGGATTTAAAGCAAGAAAAGATGCACTGGACATTGCTTCAGATTATGAAAACATTTCTGTTGTTAAC ATTCCTCTATGGGGTGGAGTAGTCCAGAGAATTATTAGTTCTGTTAAGCTTAGTACATTTCTCTGCGGTCT TGJkAAATAAAGATGTTTTAATTTTCAATTTCCCGATGGCCAAACCATTTTGGCATATATTGTCATTCTTTC ACCGCCTTCTAAAATTTAGAATAGTACCTCTGATTCATGATATTGATGAATTAAGAGGAGGAGGGGGTAGT GATTCTGTGCGGCTTGCTACCTGTGATATGGTCATAAGTCACAATCCACAAATGACAAAGTACCTTAGTAA ATATATGTCTCAGGATAAAATCAAAGACATAAAAATATTTGATTACCTCGTCTCATCTGATGTGGAGCATC
131
GAGATGTTACGGATAAGCAACGAGGGGTCATATATGCTGGCAACCTTTCTAGGCATAAATGTTCTTTCATA TATACTGAAGGATGCGATTTTACTCTCTTTGGTGTCAACTATGAAAATAAAGATAATCCTAAATATCTTGG AAGTTTTG ATGCTCAATCTCCGGAAAAGATTAACCT CC C AGGCATGCAATTTGGACT C ATTTGGGATGGAG ATTCTGTCGAAACCTGTAGTGGTGCCTTTGGCGACTATTTAAAGTTTAATAACCCTCATAAGACATCTCTT TATCTTTCAATGGAACTTCCAGTATTTATATGGGATAAAGCCGCCCTTGCGGATTTCATTGTAGATAATAG AATAGGATATGCAGTGGGATCAATCAAAGAAATGCAAGAGATTGTTGACTCCATGACAATAGAAACTTATA AGCAAATTAGTGAGAATACAAAAATTATTTCTCAGAAAATTCGAACAGGAAGTTACTTCAGGGATGTTCTT GAAGAGGTGATCGATGATCTTAAAACTCGCTAAACGATATGGTCTCTGTGGTTTTATTCGGCTTGTTAGAG ATGTCTTATTGACTCGTGTATTTTACCGGAACTGTAGAATTATTCGATTTCCCTGCTATATTCGCAATGAT GGTAGCATTAATTTTGGTGAAAATTTCACAAGTGGAGTCGGTCTCAGGCTGGATGCATTTGGACGTGGCGT GATTTTTTTTTCCGATAATGTGCAAGTTAACGACTATGTTCATATCGCCTCAATTGAGAGCGTTACGATAG GTCGGGATACGCTTATTGCAAGTAAAGTATTTATTACCGATCATAATCACGGTTCCTTTAAGCACTCTGAT CCAATGAGTTCGCCAAATATACCTCCAGACATGCGCACGTTGGAATCTTCAGCTGTTGTAATTGGCCAGAG GGTTTGGTTGGGTGAGAATGTGACGGTTTTGCCTGGAACAATTATTGGTAATGGAGTCGTAGTCGGCGCCA ATTCTGTTGTTAGAGGTTCTATTCCCGAAAATACTGTCATTGCGGGAGTACCAGCAAAAATCATAAAGAAA TACAATCATGAGACCAAATTATGGGAAAAAGCATAGTCGTTGTTTCTGCGGTCAATTTTACCACTGGCGGT CCATTTACCATTTTGAAAAAATTTTTGGCAGCAACTAATAATAAAGAAAATGTCAGTTTTATCGCATTAGT CCATTCTGCTAAAGAGTTAAAAGAAAGTTATCCATGGGTTAAATTCATTGAGTTTCCTGAGGTTAAAGGGT CGTGGCTAAAACGTTTGCACTTTGAATATGTAGTTTGTAAAAAACTTTCAAAAGAGCTGAATGCTACGCAT TGGATTTGTCTGCATGATATTACGGCCAATGTCGTCACTAAAAAAAGATATGTGTATTGTCATAACCCTGC CCCTTTTTATAAAGGAATTTTATTCCGTGAAATTCTTATGGAGCCTAGCTTTTTCTTATTTAAAATGCTAT ACGGGCTGATATATAAAATAAACATTAAAAAAAATACTGCAGTGTTTGTTCAACAATTCTGGATGAAAGAA AAATTTATCAAGAAATATTCTATAAATAACATCATTGTCAGTCGGCCAGAAATTAAATTATCTGATAAAAG CCAACTTACTGATGATGATTCTCAATTTAAGAATAACCCTTCTGAGTTGACAATATTTTACCCTGCTGTTC CACGAGTATTTAAAAATTACGAGCTTATTATTAGTGCAGCAAGGAAATTGAAAGAACAATCCAATATTAAA TTTCTGCTTACTATCAGTGGTACAGAAAATGCGTATGCAAAATATATTATCAGTCTTGCAGAAGGACTGGA TAATGTTCATTTCCTCGGGTACTTGGATAAAGAAAAAATCGATCATTGTTATAATATTTCAGATATAGTTT GTTTTCCCTCTAGGTTAGAAACATGGGGATTGCCGTTGTCTGAGGCTAAAGAGCGAGGTAAGTGGGTATTA GCATCAGATTTCCCATTTACTAGAGAAACTCTTGGTAGTTATGAAAAGAAAGCTTTTTTTGATTCTAATAA CGATGACATGTTAGTTAAACTTATTATTGACTTCAAAAAAGGTAACCTCAAAAAAGATATCTCTGATGCAA ATTTCATTTATCGTAATGAAAATGTATTAGTTGGGTTTGATGAACTAGTTAATTTTATTACTGAAGAACAT TGAAATGGTATATATAATAATCGTTTCCCACGGACATGAAGACTACATCAAAAAATTACTCGAAAATCTTA ATGCTGACGATGAGCACTACAAGATTATCGTACGCGACAACAAAGACTCTCTATTATTGAAACAAATATGC CAGCATTATGCAGGCCTGGACTATATTAGTGGAGGTGTATACGGCTTTGGTCATAATAATAATATTGCGGT GGCGTATGTAAAGGAAAAATATAGACCCGCAGATGATGATTACATTTTGTTTTTGAATCCCGATATCATCA TGAAGCATGATGATTTGCTGACATATATTAAATATGTCGAAAGTAAGCGTTATGCTTTTAGTACATTATGC CTGTTCCGAGATGAAGCGAAATCTTTACATGATTATTCCGTAAGAAAATTTCCTGTGCTTTCTGATTTTAT TGTGTCATTTATGTTAGGGATTAATAAAACAAAAATTCCTAAAGAAAGTATCTATTCTGATACGGTTGTTG ATTGGTGCGCAGGATCATTTATGCTGGTACGTTTTTCAGATTTTGTGCGTGTAAATGGCTTCGATCAAGGT TACTTTATGTACTGTGAAGATATTGACCTGTGCTTGAGGCTTAGCCTGGCTGGTGTCAGACTTCATTATGT TCCCGCTTTTCATGCGATACATTATGCTCATCATGACAATCGAAGTTTTTTTTCAAAAGCCTTCAGATGGC ACTTAAAAAGTACTTTTAGATATTTAGCCAGAAAACGTATTTTATCAAATCGCAACTTTGATCGAATTTCA TCAGTTTTTCACCCGTAAGAGCTCGGTACCCGGGCCTAGGGTGTAGGCTGGAGCTGCTTCGAAGTTCCTAT ACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTAAGGAGGATATTCATATCCGTCGACGGCGGCCGCCC TGCAGGCATGCAAGCTTGATCCATATGGATCGCTAGCTTAATTAAATAAAGCCGTAAGCATATAAGCATGG ATAAGCTATTTATACTTTAATAAGTACTTTGTATACTTATTTGCGAACATTCCAGGCCGCGAGCATTCAGC GCGGTGATCACACCTGACAGGAGTATGTAATGTCCAAGCAACAGATCGGCGTAGTCGGTATGGCAGTGATG GGACGCAACCTTGCGCTCAACATCGAAAGCCGTGGTTATACCGTCTCTATTTTCAACCGTTCCCGTGAGAA GACGGAAGAAGTGATTGCCGAAAATCCAGGCAAGAAACTGGTTCCTTACTATACGGTGAAAGAGTTTGTCG AATCTCTGGAAACGCCTCGTCGCATCCTGTTAATGGTGAAAGCAGGTGCAGGCACGGATGCTGCTATTGAT TCCCTCAAACCATATCTCGATAAAGGAGACATCATCATTGATGGTGGTAACACCTTCTTCCAGGACACTAT TCGTCGTAATCGTGAGCTTTCAGCAGAGGGCTTTAACTTCATCGGTACGGGTGTTTCTGGCGGTGAAGAGG GGGCGCTGAAAGGTCCTTCTATTATGCCTGGTGGCCAGAAAGAAGCCTATGAATTGGTAGCACCGATCCTG ACCAAAATCGCCGCCGTAGCTGAAGACGGTGAACCATGCGTTACCTATATTGGTGCCGATGGCGCAGGTCA CTATGTGAAGATGGTTCACAACGGTATTGAATACGGCGATATGCAGCTGATTGCTGAAGCCTATTCTCTGC TTAAAGGTGGCCTGAACCTCACCAACGAAGAACTGGCGCAGACCTTTACCGAGTGGAATAACGGTGAACTG
132
AGCAGTTAC CTGAT CGACATC AC CAAAGATATCTTCACCAAAAAAGATGAAGACGGTAACT AC CTGGTTGA TGTGATCCTGGATGAAGCGGCTAACAAAGGTACGGGTAAATGGACCAGCCAGAGCGCGCTGGATCTCGGCG AACCGCTGTCGCTGATTACCGAGTCTGTGTTTGCACGTTATATCTCTTCTCTGAAAGATCAGCGTGTTGCC GCATCTAAAGTTCT CTCTGGT CCGCAAGCACAGCCAGCAGGCGACAAGGCTGAGTTC AT CGAAAAAGTTCG TCGTGCGCTGTATCTGGGCAAAATCGTTTCTTACGCCCAGGGCTTCTCTCAGCTGCGTGCTGCGTCTGAAG AGTACAACTGGGATCTGAACTACGGCGAAATCGCGAAGATTTTCCGTGCTGGCTGCATCATCCGTGCGCAG TTCCTGCAAAAAATCACCGATGCTTATGCCGAAAATCCACAGATCGCTAACCTGTTGCTGGCTCCGTACTT CAAGCAAATTGCCGATGACTACCAGCAGGCGCTGCGTGATGTCGTTGCTTATGCAGTACAGAACGGTATTC CGGTTCCGACCTTCTCCGCAGCGGTTGCCTATTACGACAGCTACCGTGCTGCTGTTCTGCCTGCGAACCTG ATCCAGGCACAGCGTGACTATTTTGGTGCGCATACTTATAAGCGTATTGATAAAGAAGGTGTGTTCCATAC CGAATGGCTGGATTAA
SEQ ID NO: 17 (example O18A rfb locus nucléotide sequence — O18A-EPA production strain BVEC1—00559)
ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGAT ACCCAAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAG GGATCAAAGAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTAT GAGTTAGAATCACTCCTTGAGCAGCGCGTGAAGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCC GGGCGTGACCATTATGAACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGAC CTGCCATTGGTGACAACCCATTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCG CTACGTTACAACCTTGCTGCCATGATTGCACGTTTCAACGAAACGGGCCGCAGCCAGGTGCTGGCAAAACG TATGCCGGGTGACCTCTCTGAATACTCCGTCATCCAGACTAAAGAGCCGCTGGACCGTGAGGGTAAAGTCA GCCGCATTGTTGAATTTATCGAAAAACCGGATCAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGT CGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTGGAACGTACTCAGCCTGGTGCATGGGGACGTATTCA GCTGACTGATGCTATTGCCGAGCTGGCGAAAAAACAATCCGTTGATGCAATGCTGATGACCGGCGACAGTT ACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAGTATGGCCTÀCGCAACCTGAAAGAAGGG GCGAAGTTCCGTAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAATCTGACCGGATGTAACGGTTGAT AAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAATCTTCCTGCCGTTGTTTTA TATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAGTTTTCCAGGATTTTCCTTGTTT CCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTAACGCTCGTCAC ATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCATTAATACCTCTATT AATCAAACTGAGAGCCGCTTATTTCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGTGAAAATACTT GTTACTGGTGGCGCAGGATTTATTGGTTCAGCTGTAGTTCGTCACATTATAAATAATACGCAGGATAGTGT TGTTAATGTCGATAAATTAACGTACGCCGGAAACCGGGAATCACTTGCTGATGTTTCTGATTCTGAACGCT ATGTTTTTGAACATGCGGATATTTGCGATGCACCTGCAATGGCACGGATTTTTGCTCAGCATCAGCCGGAT GCAGTGATGCACCTGGCTGCTGAAAGCCATGTTGACCGTTCAATTACAGGCCCTGCGGCATTTATTGAAAC CAATATTGTTGGTACTTATGTCCTTTTGGAAGCCGCTCGCAATTACTGGTCTGCTCTTGATAGCGACAAGA AAAATAGCTTCCGTTTTCATCATATTTCTACTGACGAAGTCTATGGTGATTTGCCTCATCCAGATGAAGTA AATAATACAGAAGAATTAC CCTTATTTACTGAGACGACAGCTTACGCGC CAAGCAGCCCTTATTC CGCATC CAAAGCATCCAGCGATCATTTAGTCCGCGCGTGGAAACGTACATATGGTTTACCGACAATTGTGACTAATT GCTCGAACAACTATGGTCCTTATCATTTCCCGGAAAAGCTTATTCCACTGGTTATTCTTAATGCACTGGAA GGTAAGGCATTACCTATTTATGGCAAAGGAGATCAGATCCGCGACTGGTTGTATGTTGAAGATCATGCGCG TGCGTTATATACCGTCGTAACCGAAGGTAAAGCGGGTGAAACTTATAACATTGGTGGGCACAACGAAAAGA AAAACATCGATGTAGTGCTCACTATTTGTGATTTGCTGGATGAGATTGTACCGAAAGAGAAATCTTATCGT GAGCAAATCACTTATGTTGCTGATCGTCCGGGACACGATCGCCGCTATGCTATTGATGCTGAGAAGATTGG TCGCGCATTGGGATGGAAACCACAGGAAACGTTTGAGAGCGGGATTCGTAAAACGGTGGAATGGTACCTGT CCAATACAAAATGGGTTGATAATGTGAAAAGTGGTGCCTATCAATCGTGGATTGAACAGAACTATGAGGGC CGCCAGTAATGAATATCCTCCTTTTTGGCAAAACAGGGCAGGTAGGTTGGGAACTACAGCGTGCTCTGGCA CCTTTGGGTAATTTGATTGCTTTTGATGTTCACTCTACTGATTATTGCGGTGATTTTAGTAATCCTGAAGG TGTAGCTGAAACCGTAAGAAGCATTCGGCCGGATATTATTGTCAATGCAGCCGCTCACACCGCAGTAGACA AAGCAGAATCAGAACCGGAGTTTGCACAATTAATTAACGCAACAAGTGTCGAAGCGATTGCGAAAGCAGCA AATGAAGTTGGAGCCTGGGTTATCCATTACTCGACTGATTACGTCTTCCCTGGAAATGGCGATATGCCATG GCTGGAGACGGATGCAACCGCACCACTAAATGTTTACGGTGAAACCAAGTTAGCCGGAGAAAAAGCGTTAC AGGAATATTGCGCGAAGCATCTTATTTTCCGGACCAGCTGGGTCTATGCAGGAAAAGGAAATAACTTCGCC AAAACGATGTTACGTCTGGCAAAAGAGCGTGAAGAATTAGCGGTTATTAACGATCAGTTTGGTGCGCCAAC
133
AGGTGCTGAACTGCTGGCTGATTGTACAGCACATGCCATTCGTGTCGCACTGAATAAACCGGATGTCGCAG GCTTGTACCATTTGGTAGCCAGTGGTACCACAACCTGGTACGATTATGCTGCGCTGGTTTTTGAAGAGGCG CGCAAAGCAGGCATTCCCCTTGCACTCAACAAGCTCAACGCAGTACCAACAACAGCCTATCCTACACCAGC TCGTCGTCCACATAACTCTCGCCTTAATACAGAAAAATTTCAGCAGAACTTTGCGCTTGTCTTGCCTGACT GGCAGGTTGGCGTGAAACGAATGCTCAATGAATTATTTACGACTACAGCAATTTAATAGTTTTTGCATCTT GTTCGTGATGGTGGAGCAAGATGAATTAAAAGGAATGATGAAATGAAAATGCGTAAAGGTATTATTTTAGC GGGTGGTTCTGGTACACGTCTTTATCCTGTGACTATGGCTGTCAGTAAACAGCTATTACCTATTTATGATA AACCGATGATCTATTACCCGCTCTCTACACTGATGTTGGCGGGTATTCGCGATATTTTGATTATCAGTACA CCTCAGGATACTCCTCGTTTTCAACAATTGCTGGGTGACGGTAGCCAGTGGGGCCTGAATCTTCAGTACAA AGTGCAACCTAGCCCAGATGGCCTCGCGCAGGCATTTATCATCGGTGAAGAGTTTATTGGTGGTGATGATT GTGCTTTGGTTCTTGGTGATAATATCTTTTACGGTCACGATCTGCCGAAGCTAATGGAGGCCGCTGTTAAC AAAGAAAGTGGTGCAACGGTATTTGCCTATCACGTTAATGATCCAGAACGCTATGGTGTCGTTGAGTTTGA TAAAAACGGTACGGCAATCAGTCTGGAAGAAAAACCGTTAGAACCAAAGAGTAATTACGCCGTTACAGGTC TGTACTTTTATGATAACGACGTGGTTCAGATGGCGAAAAACTTGAAGCCGTCTGCACGTGGTGAGTTAGAA ATTACAGATATTAACCGTATTTATCTTGAGCAGGGACGTCTGTCTGTCGCGATGATGGGGCGTGGCTACGC GTGGCTGGACACGGGGACTCATCAGAGTCTGATAGAAGCAAGTAATTTTATTGCGACAATTGAAGAGCGCC AGGGATTGAAGGTTTC CTGT C CTGAAGAGATTGCATTTCGTAAAGGTTTTATTGATGTTG AGCAAGTAAGA AAATTAGCTGTACCACTAATAAAGAATAATTATGGGCAGTATCTTTATAAAATGACGAAGGATTCAAATTA ATGAATGTGATTAGAACTGAAATTGAAGATGTGCTAATTCTGGAGCCAAGAGTATTTGGTGATGATAGAGG TTTCTTTTATGAGAGCTTTAATCAATCAGCATTTGAACATATTCTAGGCTATCCGGTCAGCTTTGTTCAAG ACAATCACTCACGTTCATCAAAAAATGTACTCAGAGGCCTTCACTTTCAACGCGGCGAGTACGCACAAGAT AAACTTGTACGCTGCACTCATGGAGCAGTTTTTGATGTTGCTGTTGATATTCGACCCAATTCGGTATCCTT TGGTAAATGGGTTGGTGTTCTGCTTTCAGCTGATAATAAGCAGCAGTTGTGGATACCAAAAGGGTTTGCTC ATGGCTTTTTGGTTCTGTCTGATATCGCTGAATTTCAATATAAAACTACAAACTATTATCATCCTGAAAGC GATTGTGGAATATGTTGGAATGATGAACGCATTGCAATTGATTGGCCCCAAACATCAGGGTTAATCCTTTC GCCAAAAGATGAAAGGCTCTTTACGTTAGATGAGCTTATCAGATTAAAATTAATTGCATGAGGCCGGCCTT AAGGAGGACTAGTCCCGGCGCGCCATGAGTTTAATCAAAAACAGTTTTTGGAACCTTTGCGGGTATGTACT TCCAGCTATTGTGACACTACCAGCTTTGGGTATTATGGGGCGAAAATTAGGCCCAGAATTATTTGGTGTAT TCACTTTGGCATTAGCTGTTGTGGGTTATGCAAGCATTTTTGATGCAGGCCTTACTCGCGCAGTGATACGA GAAGTCGCAATTGAAAAAGATAATGAAGAAAATAAGTTGAAAATTÀTTTCTTCAGCGACAGTTGTAATTAT TTATTTGAGTTTGGCCGCCTCACTCTTATTATTTTTTTTTAGTGGTCATATCGCATTGCTACTGAACATTA GTGAGACTTTTTTTCATAATGTAAGTGTCTCGCTTAAAATTCTCGCAGCATCCATACCATTATTTTTGATT ACTCAAATATGGTTGTCAATTTTAGAAGGTGAAGAAAGATTTGGTTTACTTAATATCTACAAATCAATTAC GGGAGTGATATTAGCAATCTCACCGGCATTATTTATACTTATTAAACCCTCTTTGATGTATGCGATAATAG GCTTAGTTCTAGCAAGGTTTTTATGTTTTATTTTGGCTTTTATAATTTGTCACGATAAAGTGCTTAAAGCT AAACTAACAATCGATATACCAACAATTAAAAGATTGTTTATGTTCGGTGGTTGGATTACAGTAAGTAATAT CATCAGCCCTGTGCTATCATATTTTGATAGGTTTATTGTTTCAAATCAACTTGGGGCTGCTAATGTTGCTT TTTATACTGCACCATCAGAAATTATTTCTCGGCTTAGTATAATTCCAGGTGCGTTTTCAAGAGCCTTATTT CCAAGATTAGCTAATGCAAATAATTCCGCTGAAAGATATAAAACGAAAAGATTAATTACAATTTCACTTTT AATAATCATCACCCCTATTTTTTGTATTGGCGTGTTATTTTCAGAGAAGATAATGGTTTTATGGATGGGGG CATCATTTTTTGGTGAGCCTGGTTTGGTATTATCAATATTACTGATTGGCTTTATTTTTAATGGATTGGCA CAAGTACC ATTTGC C AGTATTCAATC C CG AGGT CATGCTAAGATAACTGCATTTGTTC ATCT CTTAGAGTT GTTTCCTTATTTATTACTTTTATTTTACCTCATAAAAGCACATGGGGTTGTTGGCGCGGGTATTGCGTGGT CAGTGAGGATGATAGTAGATTATATAGCATTAAGTCTTTTGGACGGTAAGTATATTAATAAATAAAATTCA AAATGCAAGTTAATAACTCATGGCTTTATTTGGGTAGGTGACAATTTATAATGATATATATATTAACTTTA ACTCTTCTTCTAGTTATAGCCATAATGTTTTCTCTTCTCGGCACAAAAAGTAGGATCACATCTCCATTACC TTTGCATTTTTTACCATGGTTACTAACTTTAATTGTCGGGATAAGTAATTACGATCAATTTTACGAGTTTA ATGAAAGAAGCTTTTACTCTTTGTTGATTTGGTTTACAGTTATTTTTATATTTTATTTCATAGGGGAACTG GTTAATTATAAACGTGAAAATATAAATGTTTATTATGGTCTTTCACATATTAAATATGAATGTAAAAAATA TTGGATCATTGTCATCCCAATTTCATTATATACCATTTTCGAAATATATATGGTTGGTATGGGGGGAGCAG ATGGATTCTTTCTCAATTTACGTCTTGCAAATACATTGGAGGGCTATACGGGTAAAAAATTTATCTTAATG CCTGCTGTATATCCTCTAATGATGGCTATGTTCGCAATTGTTTGTCTAACAAAAACTTCCΑΑΆΤΤAAATAA ATACTCCATTTATTTCTGGATGTTTTTGTATTGTATTGGCACAATGGGAAAATTTTCAATATTAACGCCAA TATTGACATATTTAATTATTTATGACTTCAAACATAGATTAAAAGTAAAAATkAACAATAAAGTTTACATTG TTGATAATTATATTAGCTTTAACTTTGCATTTTACACGTATGGCTGAGAATGACCACTCAACATTTTTATC TATTTTAGGGCTCTATATTTATTCACCAATAATTGCTTTAGGCCAGTTGAATGAAGTAAATAGTAGTCATT
134
TTGGTGAGT AT ACGTTT AGATTC AT ATATGCTATAACTAATAAAATTGGCCTTATTAAAG AATTGC CAGTA AATACTATTCTTGACTATTCATACGTTCCTGTACCAACAAATGTATATACTGCACTTCAACCATTTTACCA GGATTTTGGTTATACTGGCATCATATTTGGAGCAGTATTATACGGACTAATATATGTGAGTTTATACACGG CCGGTGTTCGTGGAAATAATACACAGGCATTACTGATTTACGCATTGTTTTCAGTTAGCAGTGCAACGGCT TTCTTCGCTGAAACGCTAGTAACGAATTTAGCTGGAAATGTGATGTTAGTATTATGTACCATCTTACTATG GCGATTTACAGTAATATGCAAAC CAGT AC AGTAAC CATTCTAATGGC CAC CTACAATGGCGAGGCCTTC AT CAAAAATCAGATTTTGTCACTACAACAACAAACATTTTCTAACTGGCGGTTATTTATTCAGGATGATGGGT CTACAGACAATACTATATCTATAATAAAAAACTTCCAAAAATCTGACTCCAGAATTCGGCTAGTTGATGAT AATTTGAAAGGTCAAGGTGCAGGAAAAAATTTTTTATCGCTGATAAAGTACAGCGAGACAGATTATACAAT TTATTGTGACCAAGATGATATTTGGTTAGAAAACAAAATATTTGAATTAGTAAAGTATGCAAATGAAATTA AATTGAATGTATCAGATGCGCCTTCGCTAGTTTATGCTGATGGCTATGCTTATATGGATGGTGAGGGTACA ATCGATTTTTCTGGGATATCTAACAATCATGCTGATCAATTAAAGGATTTTCTTTTTTTTAATGGTGGATA CCAAGGATGTTCTATTATGTTCAATCGTGCAATGACCAAATTTCTTCTGAATTATCGAGGATTTGTATATC TACATGACGATATC ACAACATTAGCTGCATAC GCTCTTGGT AAAGTTT ATTTTCTCC CGAAATAC C TTATG TTATATAGACAGCACACGAATGCGGTAACTGGTATCAAAACATTCCGCAATGGATTGACTTCTAAATTTAA ATCACCAGTAAACTATCTTTTATCACGAAAACATTATCAGGTAAAAAAATCTTTTTTTGAATGTAACAGCT CTATCTTATCAGAGACGAATAAAAAAGTTTTTTTGGATTTTATTTCATTTTGTGAATCAAATAATAAATTT ACAGATTTTTTTAAGTTATGGCGAGGTGGGTTTAGATTAAATAACAGTAGAACTAAATTATTATTAAAATT CTTAATACGGAGAAAATTTAGCGAATGATTTCAATACTTACACCTACTTTTAATCGGCAACATACTTTATC AAGGCTATTCAATTCTCTTATATTACAAACTGATAAAGATTTTGAGTGGATAATAATTGATGATGGTAGTA TAGATGCAACAGCGGTACTTGTAGAAGATTTTAGAAAAAAATGTGATTTTGACTTGATTTATTGCTATCAG GAAAATAATGGTAAGCCCATGGCTTTAAACGCTGGTGTTAAAGCTTGTAGAGGCGATTATATCTTTATTGT TGAC AGTGATGATGCACTAACTC C CGATGCCATAAAATTAATTAAAGAATCAATACATGATTGCTT AT CTG AGAAGGAAAGTTTCAGCGGAGTCGGTTTTAGAAAAGCATATATAAAAGGGGGGATTATTGGTAATGATTTA AATAATTCTTCAGAACATATATACTATTTAAATGCGACTGAGATTAGCAATTTAATAAATGGTGATGTTGC ATATTGTTTTAAAAAAGAAAGTTTGGTAAAAAATCCATTCCCCCGTATAGAAGATGAAAAATTTGTTCCAG AATTATATATTTGGAATAAAATAACTGACAAGGCGAAGATTCGATTTAACATAAGCAAAGTTATATATCTT TGTGAGTATCTTGATGATGGTCTTTCTAAAAATTTCCATAACCAGCTTAAAAAATACCCAAAGGGGTTTAA GATTTATTACAAAGATCAAAGAAAACGAGAGAAAACTTATATAAAAAAAACAAAGATGCTAATTAGATATT TGCAATGTTGTTATTATGAGAAAATAAAATGAAAATACTATTTGTCATTACAGGTTTAGGCCTTGGAGGTG CTGAGAAGCAGGTTTGTCTTTTAGCTGATAAATTAAGTTTAAGCGGGCACCATGTAAAGATTATTTCACTT GGACATATGTCTAATAATAAAGTCTTTCCTAGCGAAAATAATGTTAATGTCATTAATGTAAATATGTCAAA AAACATTTCTGGAGTTATAAAAGGTTGTGTCAGAATTAGAGATGTTATAGCTAATTTCAAACCAGACATTG TACACAGTCATATGTTTCATGCAAACATTATCACTAGATTGTCTGTAATTGGAATCAAAAACAGACCTGGT ATTATATCAACTGCACATAATAAAAATGAAGGTGGGTATTTCAGAATGCTCACATATAGAATAACCGATTG TTTAAGTGATTGTTGTACAAATGTTAGCAAAGAAGCAGTGGATGAGTTTTTACGGATAAAAGCCTTTAATC CCGCTAAAGCAATTACTATGTATAATGGGATAGATACCAATAAATTTAAATTTGATTTATTGGCAAGGAGG GAAATTCGAGACGGTATTAATATAAAAAATGATGATATATTATTACTTGCTGCAGGTCGTTTAACGTTAGC TAAAGATTATCCTAATTTATTGAATGCAATGACTCTGCTTCCTGAACACTTTAAACTTATTATTATTGGTG ATGGTGAATTGCGTGACGAAATTAATATGCTTATAAAAAAATTGCAATTATCTAATAGGGTGTCCTTGTTG GGAGTTAAAAAAAATATTGCTCCCTATTTTTCTGCATGTGATATTTTTGTTCTCTCTTCTCGTTGGGAAGG ATTTGGATTAGTCGTGGCAGAAGCTATGTCATGTGAGCGAATTGTTGTTGGCACGGATTCAGGGGGAGTAA GAGAAGTTATTGGTGACGATGATTTTCTTGTACCCATATCTGATTCAACACAACTTGCAAGCAAAATTGAA AAATTGTCTTTGAGCCAGATACGTGATCACATTGGTTTTCGGAATCGTGAGCGTATTTTAAAAAATTTCTC AATAGATACTATTATTATGCAGTGGCAAGAACTCTATGGAACTATAATTTGCTCAAAACATGAAAGGTAGA TTTATATTTGGAACGTGTCTTTTGTTTGAATTTAATTCAATCTCAATTGAGATTTTTGTATTTCAAAAATA CCATCATAGCTAACGATGATTGGTATTTATTTTAAGATGCTTTCTATAAATATATTGACGTTTTTAATGCG CCGAAACGATTGGGCTGGGAACAGAGAAGTAAAACTGTTTTGAGAATGAAGAGTTTTTGAGATGTTTATGG ATATTAAAAATTGATCCAGTGAATTAATTATTTATAATAAATCAAGATTTAATGTTAATAAATGATAATCT TTTCTGACACTCATATTAATTATGAGTGGTACGTTTGGTAAACGGTAAACTATTATATGACAGCTAGAACA ACTAAAGTTTTGCACTTACAATTACTCCCACTCTTAAGTGGCGTTCAAAGGGTAACATTAAÀCGAAATTAG TGCGTTATATACTGATTATGATTATACACTAGTTTGCTCAAAAAAAGGTCCACTAACAAAAGCATTGCTGG AATATGATGTCGATTGTCATTGTATCCCCGAACTTACGAGAGAAATTACCGTAAAGAATGATTTTAAAGCA TTGTTCAAGCTTTATAAGTTCATAAAAAAAGAAAAATTTGACATTGTGCATACACATTCTTCAAAAACAGG TATTTTGGGGCGAGTTGCTGCCAAATTAGCACGTGTTGGAAAGGTGATCCACACTGTACATGGTTTTTCTT TTCCAGCCGCATCTAGTAAAAAAAGTTATTACCTTTATTTTTTCATGGAATGGATAGCAAAGTTCTTTACG
135
GATAAGTTAATCGTCTTGAATGTAGATGATGAATATATAGCAATAAACAAATTAAAATTCAAGCGGGATAA AGTTTTTTTAATTCCTAATGGAGTAGACACTGATAAGTTTTCTCCTTTAGAAAATAAAATTTATAGTAGCA CCTTGAATCTAGTAATGGTTGGTAGATTATCCAAGCAAAAAGATCCTGAGACATTATTGCTTGCTGTTGAA AAACTGCTGAATGAAAATGTTAATGTTAAGCTGACACTTGTAGGAGATGGTGAACTAAAAGAACAGTTAGA AAGCAGGTTCAAACGGCAAGATGGACGTATAATTTTTCATGGATGGTCAGATAACATTGTTAATATTTTAA AAGTTAATGATCTTTTTATATTACCTTCTCTTTGGGAGGGTATGCCATTAGCÀATTTTAGAAGCATTGAGC TGTGGACTTCCATGTATAGTCACTAATATTCCAGGTAATAATAGCTTAATAGAAGATGGCTATAATGGTTG TTTGTTTGAAATTAGAGATTGTCAGTTATTATCTCAAAAAATCATGTCATATGTTGGTAAGCCAGAACTGA TTGCACAGCAATCTACCAATGCACGATCATTTATTCTGAAAAATTATGGATTAGTTAAAAGAAATAATAAG GTCAGACAGCTATATGATAATTAAGAGCTCGGTACCCGGGCCTAGGGTGTAGGCTGGAGCTGCTTCGAAGT TCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTAAGGAGGATATTCATATCCGTCGACGGCGG CCGCCCTGCAGGCATGCAAGCTTGATCCATATGGATCGCTAGCTTAATTAAATAAAGCCGTAAGCATATAA GCATGGATAAGCTATTTATACTTTAATAAGTACTTTGTATACTTATTTGCGAACATTCCAGGCCGCGAGCA TTCAGCGCGGTGATCACACCTGACAGGAGTATGTAATGTCCAAGCAACAGATCGGCGTAGTCGGTATGGCA GTGATGGGACGCAACCTTGCGCTCAACATCGAAAGCCGTGGTTATACCGTCTCTATTTTCAACCGTTCCCG TGAGAAGACGGAAGAAGTGATTGCCGAAAATCCAGGCAAGAAACTGGTTCCTTACTATACGGTGAAAGAGT TTGTCGAATCTCTGGAAACGCCTCGTCGCATCCTGTTAATGGTGAAAGCAGGTGCAGGCACGGATGCTGCT ATTGATTCCCTCAAACCATATCTCGATAAAGGAGACATCATCATTGATGGTGGTAACACCTTCTTCCAGGA CACTATTCGTCGTAATCGTGAGCTTTCAGCAGAGGGCTTTAACTTCATCGGTACCGGTGTTTCTGGCGGTG AAGAGGGGGCGCTGAAAGGTCCTTCTATTATGCCTGGTGGCCAGAAAGAAGCCTATGAATTGGTAGCACCG ATCCTGACCAAAATCGCCGCCGTAGCTGAAGACGGTGAACCATGCGTTACCTATATTGGTGCCGATGGCGC AGGTCACTATGTGAAGATGGTTCACAACGGTATTGAATACGGCGATATGCAGCTGATTGCTGAAGCCTATT CTCTGCTTAAAGGTGGCCTGAACCTCACCAACGAAGAACTGGCGCAGACCTTTACCGAGTGGAATAACGGT GAACTGAGCAGTTACCTGATCGACATCACCAAAGATATCTTCACCAAAAAAGATGAAGACGGTAACTACCT GGTTGATGTGATCCTGGATGAAGCGGCTAACAAAGGTACGGGTAAATGGACCAGCCAGAGCGCGCTGGATC TCGGCGAACCGCTGTCGCTGATTACCGAGTCTGTGTTTGCACGTTATATCTCTTCTCTGAAAGATCAGCGT GTTGCCGCATCTAAAGTTCTCTCTGGTCCGCAAGCACAGCCAGCAGGCGACAAGGCTGAGTTCATCGAAAA AGTTCGTCGTGCGCTGTATCTGGGCAAAATCGTTTCTTACGCCCAGGGCTTCTCTCAGCTGCGTGCTGCGT CTGAAGAGTACAACTGGGATCTGAACTACGGCGAAATCGCGAAGATTTTCCGTGCTGGCTGCATCATCCGT GCGCAGTTCCTGCAAAAAATCACCGATGCTTATGCCGAAAATCCACAGATCGCTAACCTGTTGCTGGCTCC GTACTTCAAGCAAATTGCCGATGACTACCAGCAGGCGCTGCGTGATGTCGTTGCTTATGCAGTACAGAACG GTATTCCGGTTCCGACCTTCTCCGCAGCGGTTGCCTATTACGACAGCTACCGTGCTGCTGTTCTGCCTGCG AACCTGATCCAGGCACAGCGTGACTATTTTGGTGCGCATACTTATAAGCGTATTGATAAAGAAGGTGTGTT CCATACCGAATGGCTGGATTAA
SEQ ID NO: 18 (example O25B rfb locus nucléotide sequence - O25B-EPA production strain stGVXN4459)
ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGAT ACC CAAAGAGATGCT ACCAAT CGTCGACAAGC C AATGATTC AGTACATTGTTGACG AGATTGTGGCTGCAG GGATCAAAGAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTAT GAGTTAGAATCACTCCTTGAGCAGCGCGTGAAGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCC GGGCGTGACCATTATGAACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGAC CTGCCATTGGTGACAACCCATTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCG CTACGTTACAACCTTGCTGCCATGATTGCACGTTTCAACGAAACGGGCCGCAGCCAGGTGCTGGCAAAACG TATGCCGGGTGAC CTCTC TGAATACT CCGTCATC CAGACTAAAGAGC CGCTGGACCGTGAGGGTAAAGTCA GCCGCATTGTTGAATTTATCGAAAÀACCGGATCAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGT CGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTGGAACGTACTCAGCCTGGTGCATGGGGACGTATTCA GCTGACTGATGCTATTGCCGAGCTGGCGAAAAAACAATCCGTTGATGCAATGCTGATGACCGGCGACAGTT ACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAGTATGGCCTACGCAACCTGAAAGAAGGG GCGAAGTTCCGTAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAATCTGACCGGATGTAACGGTTGAT AAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAATCTTCCTGCCGTTGTTTTA TATAAACCATC AGAATAACAACG AGTTAGC AGT AGGGTTTT ATTCAAAGTTTTC C AGGATTTT CC TTGTTT CCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTAACGCTCGTCAC
136
ATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCATTAATACCTCTATT AATCAAACTGAGAGCCGCTTATTTCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGCTAGCAGTGAA GATACTTGTTACTGGTGGCGCAGGATTTATTGGTTCTGCTGTTGTTCGTCACATAATAAATAATACGCAAG ATAGTGTTGTTAATGTCG ATAAATTAAC ATACGC CGGAAAC CTGGAAT C ACTTGCAGATGTTTCTGATTCT GAACGCTATTTCTTTGAACATGCGGATATTTGTGATGCAGCTGCAATGGCACGGATTTTTGCTCAGCATCA GCCGGATGCAGTGATGCACCTGGCAGCTGAAAGCCATGTTGACCGTTCAATTACAGGCCCTGCGGCATTTA TTGAAACCAATATTGTGGGTACTTATGTCCTTTTAGAAGCGGCTCGGAATTATTGGTCTGGTCTGGATGAT GAAAAGAAAAAAAACTT C CGTTTTCAT CATATTTCTACTGATGAGGTGTATGGTGACTTAC CCCATCCGGA TGAAGTAAATAGCAATGAAACGTTGCCGCTATTTACGGAAACGACAGCATACGCGCCAAGTAGTCCATATT CTGCTTCTAAAGCTTCCAGCGATCATTTGGTTCGCGCATGGAAACGTACTTATGGTTTACCGACCATTGTG ACTAATTGCTCGAACAACTATGGTCCTTATCATTTCCCGGAAAAGCTTATTCCACTGGTTATTCTTAATTC ACTGGAAGGTAAGGCATTACCTATTTATGGCAAAGGAGATCAGATCCGCGACTGGTTGTATGTAGAGGATC ATGCTCGAGCGTTATATACCGTCGTAACCGAAGGTAAAGCGGGCGAAACTTATAACATTGGTGGACACAAC GAAAAGAAAAACATCGACGTAGTGTTCACTATTTGTGATTTGTTGGATGAGATAGTCCCGAAAGAGAAATC TTACCGCGAGCAAATTACTTATGTTACCGATCGTCCGGGACACGATCGCCGTTATGCGATTGATGCTGAGA AGATTGGTCGCGAATTGGGATGGAAACCACAGGAAACGTTTGAGAGTGGGATTCGTAAAACGGTGGAATGG TACCTGTCCAATACAAAATGGGTTGATAATGTGAAAAGTGGTGCCTATCAATCGTGGATTGAACAGAACTA TGAGGGCCGC CAGTAATGAATATCCT C CTTTTTGGCAAAACAGGGCAGGTAGGTTGGGAACTACAGCGTGC TCTGGCACCTCTGGGTAATTTGATTGCTCTTGATGTTCACTCCACTGATTACTGTGGTGATTTTAGTAATC CTGAAGGTGTAGCTGAAACCGTAAGAAGCATTCGGCCTGATATTATTGTCAACGCAGCCGCTCACACCGCA GTAGACAAAGCAGAATCAGAACCGAAGTTTGCACAATTACTGAACGCGACGAGTGTCGAAGCGATCGCGAA AGCAGCCAATGAAGTCGGCGCCTGGGTTATTCACTACTCTACTGACTACGTATTTCCGGGGACCGGTGAAA TACCATGGCAGGAGGAGGATGCAACCGCACCGCTAAATGTTTACGGTGAAACCAAGTTAGCGGGAGAAAAA GCATTACAAGAGCATTGTGCGAAGCACCTTATTTTCCGGACCAGCTGGGTCTATGCAGGTAAAGGAAATAA CTTCGCCAAAACAATGTTGCGTCTGGCAAAAGAGCGTGAAGAATTAGCCGTTATTAATGATCAGTTTGGTG CGCCAACTGGCGCAGAGTTACTGGCTGATTGTACGGCACATGCTATTCGTGTGGCACTGAATAAACCGGAA GTCGCAGGCTTGTACCATCTGGTAGCTAGTGGTACCACAACGTGGCACGATTATGCTGCGCTGGTTTTTGA AGAGGCGCGCAAAGCAGGCATTCCCCTTGCACTCAACAAGCTCAACGCAGTACCAACAACAGCCTATCCTA CACCAGCTCGTCGTCCACATAACTCTCGCCTTAATACAGAAAAATTTCAGCAGAACTTTGCGCTTGTCTTG CCTGACTGGCAGGTTGGCGTGAAACGAATGCTTAACGAATTATTTACGACTACAGCAATTTAATAGTTTTT GCATCTTGTTCGTAATGGTGGAGCAAGATGTATTAAAAGGAATGATGAAATGAAAACGCGTAAAGGTATTA TTTTGGCGGGTGGTTCTGGTACTCGTCTTTATCCTGTGACGATGGCCGTCAGTAAACAGCTGTTACCGATT TATGATAAACCGATGATCTATTACCCGCTCTCTACACTGATGTTAGCGGGTATTCGCGATATTCTGATTAT CAGTACACCACAGGATACTCCTCGTTTTCAACAACTGCTGGGTGACGGGAGCCAGTGGGGCCTGAATCTTC AGTACAAAGTGCAACCGAGTCCGGATGGTCTTGCGCAGGCGTTTATTATCGGTGAAGAGTTTATTGGTGGT GATGATTGTGCTTTGGTACTTGGTGATAATATCTTCTACGGCCACGACCTGCCGAAGTTAATGGACGTAGC TGTTAACAAAGAAAGTGGTGCAACGGTATTTGCCTATCACGTTAATGATCCTGAACGTTATGGTGTCGTGG AGTTTGATAATAACGGTACTGCAATTAGCCTGGAAGAAAAACCGCTGGAACCAAAAAGTAACTATGCGGTT ACTGGGCTTTATTTCTATGACAATGACGTTGTGGAAATGGCGAAAAACCTTAAGCCTTCTGCCCGAGGTGA ACTGGAAATTACCGATATTAACCGTATTTATATGGAACAAGGACGTTTGTCTGTCGCTATGATGGGGCGTG GCTATGCATGGCTGGATACAGGGACGCATCAAAGTCTTATTGAAGCAAGCAACTTCATTGCCACCATTGAA GAGCGCCAGGGACTAAAGGTTTCCTGTCCGGAAGAAATTGCTTATCGTAAAGGGTTTATTGATGCTGAGCA GGTAAAAGTATTAGCCGAACCGTTGAAGAAAAATGCTTATGGTCAGTATCTGCTCAAAATGATTAAAGGTT ATTAATAAGATGAACGTAATTAAAACTGAAATTCCTGATGTGCTGATTTTTGAACCAAAAGTTTTTGGGGA TGAACGTGGCTTCTTTTTTGAGAGTTTTAATCAGAGGATTTTTGAAGAAGCAGTAGGTCGTAAGGTTGAGT TTGTTCAGGATAACCATTCTAAGTCCAGTAAAGGTGTTTTACGTGGTCTTCATTATCAGTTAGAACCTTAT GCTCAAGGAAAACTGGTGCGCTGTGTTGTTGGCGAGGTTTTTGATGTTGCGGTTGATATTCGTAAATCGTC ACCTACATTTGGGAAATGGGTTGGGGTGAATTTGTCTGCTGAGAATAAGCGTCAGTTGTGGATTCCTGAGG GATTTGCACATGGTTTTTTGGTGCTGAGTGATTTAGCAGAAGTTTTATATAAAACGAATCAATATTATGCT CCATCACATGAAAAAAATATTATATGGAATGACCTCTTGCTTAATATTAAATGGCCGAGCACAGCACTGAT CACTCTGTCTGATAAGGATGCAAATGGGGAAAGATTTGAACTAAGTGAGTTTTGAAATGTCTCTCTTAAAA CATAGTATATGGAATGTTGCGGGCTACTTTATACCAACATTAATTGCAATTCCCGCCTTTGGATTAATTGC GAGGAAAATTGGTGTAGAACTATTTGGTTTGTATACGTTAGCAATGATTTTTATAGGGTATGCAAGTATAT TTGATGCTGGGTTAACAAGAGCTGTTGTGCGTGAAATAGCATTACTAAAAAACAGAGTGGACGATTGTAAT ACGATAATAGTAACTTCTATTATCGCTGTGATATTTTTAGGGTTTATCGGAGGCGGGGGAGTGTTTCTGCT TAAAGGCGATATTATTGAACTGTTAAATATCTCACCAATATATTACGCCGATTCGATAAAGTCTCTAGTAT
137
TATTATCATCTCTGATACCTGTATTCTTAGTCACGCAAATACTATTAGCAGAGCTTGAGGGTCGGGAATAT TTTGGGATTCTAAATATACAAAAAAGTGTAGGGAATTCTTTAATTGCAGGGTTACCTGCATTATTTGTTTT AATTAATCAAACGCTTTTTTCTGCAATTATTGGTGTAGCGATTGCAAGAGTTATATGCTTGTGGTTAAGCT ACATTATGAGCAGGGAAAGAATAACTATCGATATCTCATTTTTTTCAATAACTGTTTTAAAGCGGTTATTT AGATATGGCGGGTGGGTAACTATAAGTAACATAATATCTCCTATATTAGCGAGTATGGATAGATTTATTCT ATCCCATATCCAGGGAGCATCAAAAATATCATTCTATACAGTCCCTAATGAGCTGGTAACTAGGCTTGGAA TAGTTC C AGGCTCT CTTGGGAAAGCTGTTTTT C CAAAATTAAGTCATGCAAGGAATTTTACAGCGTCAT AT GCAGAGCAAAAAAAAGCTTATATATTAATGACTGTCATTGTAATGCCTTTGGTTTTATTTGTATATTATTA CGCAAAGTTTATTTTAACATTGTGGATGGGGGCTGAGTATGCAGGGATTTCGGTCGAAATATTACGGATTA TGCTTATAGGGTATATTTTTAACTGTTATTCACAAATCTCTTTTGCCAACATACAGGCCTTTGGAAAAGCA AAATACACTGCATACATCCATATGATGGAATTTATTCCTTATTTGATAATGTTATATATAATTTCAAAGGA ATATGGGGTTATTGGTGTTGCGTGGTTATGGACAATTCGAGTAATAATTGATTTTTTGATGCTTTTATATA TGAGTTATCGTTGTAATAATCTTATGAAAAAAGGGTAGCCTGATGATATATATTGTGGTATTAAATTGGAA TGGGGCTATAGATACCATTAATTGTGTTAAAAGTTTAATGGATTTAAATGTTAGCGATTATAAAATTATCA TTGTTGATAACTGTTCTATGGATAACTCATATGATACTATAAAAGAAAATCTTAATTCATTATATATTGCT GATAAAAGTATCATTGAGGTGAAGTATGAGGATAGAAATAAATATAAAACCTTAGAAAACGATAAAATCAT ATTAATACAATCTCCGCAAAATAATGGGTACGCAAGTGGTAATAATATTGGCATAGAGTTCGCTCTTAATC AGGAGAATATGAAATACGTCTGGGTTCTGAATAATGATACTGAAGTGGATAAAGAGGCTTTAACTCATTTA ATTAGTAAATGTGATTCAGATAAAAGTATAGGGATTTGCGGTTCTCGTTTAGTCTATTTTGCCGACAGAGA GATGCAGCAAGGACTAGGTGGGGTGCATAACAAATGGTTATGCACTACAAAAAATTATGAAATGGGAAGAT TAGTTTCCAAAAAATATGATGATGAAGTCATTAGTAATGATATAGATTATATAATTGGCGCATCGATGTTT TTCTCTAGAGAATGTTTGGAAACAGTTGGATTGATGAATGAAGAATATTTTTTATACTATGAAGAGTTAGA TATTTGCCTCAGAGCAAAAGCAAAGAACTTTAAATTAGGTATTTGCTCAGAAAGTTTGGTTTATCATAAAA TAGGTGCAAGTACTGATGGGGGAAAGAGCATGATGGCTGATCTTTGCTCAATAAAAAATAGGCTGGTCATT ACAGAAAGGTTTTATCCCCAATATTATTGGACGGTATGGTTGTCACTTTTTGTTGTAGCATTTAACCGTGC TAGAAGAGGTGAGTTTAATAAGATGAAAAGATGTTTGAATGTTATGTTTAACTTCAAACGAAACAAAGGTA GCAAATGCCATTAGAATATGCACTTAATCATGGTGTTAATAAATCTATAGTTTGATATGTTATTAAAGGGT ATTTAATGAAAGTGGCTTTTTTAT CTGCTTATG ATC CACTATCTAC ATC CAGTTGGTCTGGCAC AC CTTAT TATATGCTAAAGGCATTATCGAAGAGAAATATTTCCATTGAAATATTAGGACCGGTAAATAGCTATATGAT ATACATGTTAAAAGTATATAAATTAATATTAAGGTGTTTCGGAAAAGAATATGATTATAGTCATTCGAAGT TGCTTTCCAGGTATTACGGTAGAATATTCGGTAGGAAATTAAAAAAAATTGATGGTTTGGATTTTATTATC GCACCTGCAGGTTCCTCACAAATTGCTTTTTTAAAAACAACCATACCAATAATATATCTATCGGATACAAC ATATGATCAATTAAAAAGCTATTATCCGAATTTAAATAAAAAAACAATTATAAATGATGAGGATGCAAGTT TAATCGAACGCAAGGCTATTGAAAAAGCAACAGTAGTATCTTTCCCATCTAAATGGGCAATGGATTTTTGC AGGAATTATTACAGATTAGATTTTGATAAATTAGTTGAAATACCATGGGGGGCTAATTTATTTGATGATAT TCACTTTGCTAATAAAAATATAATTCAAAAGAATAGTTATACTTGTCTTTTCTTGGGAGTTGATTGGGAAA GAAAAGGTGGGAAAACAGCCTTGAAAGCAATTGAATATGTAAGGCAGTTATATGGGATCGATGTTAGACTA AAAATTTGTGGATGTACTCCGAATCAAAAGATTTTACCTACTTGGGTTGAATTAATTGATAAAGTAGATAA AAATAACGTTGACGAAT ATC AGAAATT CAT CGATGTGTTAT CTAACGCTGAT ATACTT CTTTTACCAACC A TTGCTGAATGTTATGGAATGGTATTTTGTGAAGCTGCTGCTTTTGGATTGCCTGTTGTCGCTACAGATACA GGTGGAGTCAGTTCTATAGTTATCAACGAAAGGACGGGGATATTAATTAAAGACCCGTTAGACTATAAGCA CTTTGGAAATGCAATTCATAAAATAATTAGTT C CGTAGAGACTTATCAAAACTACT C C CAAAACG CAAGAA TTAGATATAATAATATATTGCATTGGGACAATTGGGCTAAAAAGATAATTGAGATTATGTATGAGCATAAG AATAGAAGAATCAAATAG CACAAAAAGAATTATATGTTTATTTATACTTTTT CTTGTTTTCC CTGATTTTT TGTTTTATACATTAGGGGTTGATAATTTTAGCATTTCAACGATAATCTCAATTACATTGCTTTTTGTTTTT TTAAGAGCTAAAAATATTTGCAAAGATAATTTTCTAATAATAGTAGCGTTATTCATATTGTTGTGTTTTAA CTGTTTGTTAAGTATGCTATTTAATATTGAACAGGCTTTAACATTTAAAGTTGTACTTTCAATATATAGCA TCTTAATAATGGCATACGTCTCCTCTTGTTATGCACAGACGTTGTGGTTATGTTCTGAAGAAATACTTAAG AGATCCGTCTTTTATTTGTTCGCATTTCTTTGCCTTATTGGCATTATAAGTATTCTTTTACAGAAGACTGA GATTATACATGATAAAAGTATGATTCTTTTTCCTGAACCATCAGCATTTGCATTGGTTTTTATACCTATCT TTTCATTTTGTTTATACTATACAAGAGGGGGGGGGCTACTATTGCTCTATATATTATCTTTGGGTATTGCG TTAGGTATCCAGAATTTAACAATGTTGGTAGGCATTGTGATTAGTGTTTTTGTGATGAAAAAAATAACTAT AAGGCAAACTATTGTTATACTTTTGGGGGCATGGATTTTTTCCATGATATTAAGTGATTTAGACATTTCTT ACTATACATCGCGGCTTGATTTTAAAAATACTACGAACCTATCAGTGCTTGTATATCTTTCAGGAATTGAA AGAGCTTTCTTGAATTTTATTACAAGTTATGGTCTTGGTATTGGTTTTCAACAAATGGGAGTGAATGGGGA GATAGGAATATATCAACAAATTTTAGCTGAACTTGATGCCCCTATGTTAAATATATACGATGGCTCATTTA
138
TTTCTTCTAAGTTAATATCTGAGTTTGGGGTTATTGGTGCATTAATGTGTATTTTCTATTTTTTTTATTTT TCCCGATTTTATCTGCGTTTCAAAAAAAGTAAGAGATATTCACCGCAGTATATTTTAGCATATAGCTTCTA CATGTGTTTCTTCATCCCTCTTTTTATACGTGGTGCTGGTTATATAAACCCCTATGTGTTTATGTTATTTT CATCAATATTTTTGTGCAAATATCACGCTAAAAATATCTTGATGAAATCTAATGTCCAGATAGCTATATAA TAGTAGATTATATTATCATTATCACGTAAATTACATATTAATAGCATATATGATAACTAGGACATAAATAA TGTGCATTAAAAAAAAACTTAAGTTAATTAAACGATATGGCCTTTATGGTGGTCTTAGGCTTCTTAAAGAT ATATTCTTAACAAAATTTTTATTTTGTTCAAATGTTAGGATTATTAGATTTCCATGTTATATTAGAAAAGA TGGAAGTGTTAGTTTTGGAAAAGGTTTTACATCAGGTGTAGGATTACGAGTTGATGCATTTATGGATGCCG TAGTTTCCATTGGAGAAAATGTTCAAATTAATGACTATGTTCACATCGCGGCTATTAATAATGTCATTATT GGTAGAGATACATTAATAGCAAGTAAAGTATTTATTAGTGATCATAATCATGGTATTTTTTCTAAATCCGA TATCCATAGTTCACCAACTATTATTCCTTCGTCTAGGCCCCTTGAATCTGCACCTGTGTATATTGGAGAGC GTGTGTGGATTGGCGAAAATGTGACAATATTACCAGGTGCGTGTATAGGTAATGGTGTAGTTATTGGCGCA AACAGTGTTGTTCGTGGTGAGATTCCTAATAATGTGATCATTGCTGGTGTTCCAGCTAAAATTGTTAAAAA ATATAACTATGAGCGTATGCAATGGGAAAGAATATAGTTGTAATATCGGCTGTTAATTTTACAACCGGAGG CCCCTTTACCGTACTAAAAAATGTGCTTACAGCAACTAAAGATAGAGCCGAATGTAAATTTATTGCACTGG TTCATAGCTCTGCTGAACTAATGGAATTATTTCCGTGGGTTGAATTTATAGAGTATCCAGAAGTCAAGTCT TCGTGGGTTAAAAGATTATATTTCGAATATATAACTTGCAATAGATTATCTAAGGTGATTAAGGCAACTCA TTGGGTATGCTTACATGATATTACAGCAAATGTTAGTGTAC CCTATAGATTTGTTTATTGCCACAAT C CTG CACCGTTCTATAAATATTTAAGCTATCGAGATATTATAGGAGAACCTAAATTTTATCTTTTTTATCTTTTT TATGGGCTTTTATACAAT AT CAATATAAAAAAGAACACAGC AGTTTTTGTT CAGCAG CAGTGGCT AAAAAA AGAATTCGAAAAAAAATATAAGTTAAAGAATGTTGTTGTTAGTCGCCCTGAAGATATTTGCCCTTTTGAAA GTGATGGTTTGGTAAGAAATAATAATAAAAAGGATGTGAGGATATTTTAC C CAGCAGTG CCC CGTATATTT AAAAACTTTGAAGTTATCATACGTGCTGCACAAATATTACAAGATAAAAATATTCATTTTTATCTTACTTT TGATGGTACTGAAAATAAGTATGCAAAAAGAATATATAAATTAGCTTCCGAACTGAAAAATGTACATTTCC TCGGTTACCTTAATGCAACCGAGATGGTTAACTTTTATCAAGATTCAGATATTATTTGTTTCCCATCGAAA CTAGAAACGTGGGGATTACCATTATCAGAAGCTAAAACATACAAAAAATGGATATTTGCGGCAGACTTACC TTATGCTCATGAAGTTTTATATAACTATTCAAAAACTAGATATTTTCCATTTGACGATGAGAAAATACTTG TTCGCTACATATTAGAGTACACAAGTAAAAATATGCATGAAGATATAAAAAATAGTAGGGTGAATTTTAAT AATGATGCATTGACTGGTTTTGAACAGTTTATTGAATATATCCTCAAGGGGAACTGACGTGGTTTATATTA TAATCGTTTCACATGGCCATGATGACTATATAGAAAATCTTTTATTAAATTTAAAGTTGCCCTCTGGAAGA TTTAAAATAATAGTTCGTGATAACAAAAGTTCAATGGTTTTAAAAAAAACATGCGAAAAAAATTGCGTAAC CTATTTGCATGGAGGGCAATATGGATTTGGACATAATAATAACATAGCAGTGTCATATATAATTAATAACT TCATGATTATGAATAATGATTATTTTCTCTTTCTTAACCCCGATGTATTCATAACCAGTGAAAGTTTGATT AATTATGTTGATTATATAATTAGTAATGATTATAAGTTTAGCACATTATGTCTTTATCGAGATTTTACTAA AAGCAAACATGATTATTCAATACGGAGTTTTCCAACTTTATATGATTTTCTTTGTTCTTTTTTATTGGGGG TGAATAAAAGTAAAATTAAGAAGGAAAATATACTTTCTGATACTGTAGTTGATTGGTGTGCTGGCTCATTT ATGCTTATTCATGCTTTAAGTTTCTTAAATGTGAATGGTTTTGATCAAAAATATTTTATGTATTGTGAAGA TATTGACCTTTGTATGCGTTTAAAATTAAGTGGAGTAGATCTTTACTATACTCCCCATTTTGATGCTATTC ATTATGCGCAGCATGAAAATAGAAGAATATTTACTAAAGCATTTCGATGGCATATAAGGAGTATTACGCGC TACATATTACGGAAACCAATTCTTTCTTATAAAAACTATAGAAAAATTACATCCGAACTGGTAAAGTGATT AAGGATCCGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGA ACTAAGGAGGATATTCATATGGATAAAGCCGTAAGCATATAAGCATGGATAAGCTATTTATACTTTAATAA GTACTTTGTATACTTATTTGCGAACATTCCAGGCCGCGAGCATTCAGCGCGGTGATCACACCTGACAGGAG TATGTAATGTCCAAGCAACAGATCGGCGTAGTCGGTATGGCAGTGATGGGACGCAACCTTGCGCTCAACAT CGAAAGCCGTGGTTATACCGTCTCTATTTTCAACCGTTCCCGTGAGAAGACGGAAGAAGTGATTGCCGAAA ATCCAGGCAAGAAACTGGTTCCTTACTATACGGTGAAAGAGTTTGTCGAATCTCTGGAAACGCCTCGTCGC ATCCTGTTAATGGTGAAAGCAGGTGCAGGCACGGATGCTGCTATTGATTCCCTCAAACCATATCTCGATAA AGGAGACATCATCATTGATGGTGGTAACACCTTCTTCCAGGACACTATTCGTCGTAATCGTGAGCTTTCAG CAGAGGGCTTTAACTTCATCGGTACCGGTGTTTCTGGCGGTGAAGAGGGGGCGCTGAAAGGTCCTTCTATT ATGCCTGGTGGCCAGAAAGAAGCCTATGAATTGGTAGCACCGATCCTGACCAAAATCGCCGCCGTAGCTGA AGACGGTGAACCATGCGTTACCTATATTGGTGCCGATGGCGCAGGTCACTATGTGAAGATGGTTCACAACG GTATTGAATACGGCGATATGCAGCTGATTGCTGAAGCCTATTCTCTGCTTAAAGGTGGCCTGAACCTCACC AACGAAGAACTGGCGCAGAC CTTTAC CGAGTGGAATAACGGTGAACTGAGCAGTTAC CTGATCGACAT CAC CAAAGATATCTTCACCAAAAAAGATGAAGACGGTAACTACCTGGTTGATGTGATCCTGGATGAAGCGGCTA ACAAAGGTACCGGTAAATGGACCAGCCAGAGCGCGCTGGATCTCGGCGAACCGCTGTCGCTGATTACCGAG TCTGTGTTTGCACGTTATATCTCTTCTCTGAAAGATCAGCGTGTTGCCGCATCTAAAGTTCTCTCTGGTCC
139
GCAAGCACAGCCAGCAGGCGACAAGGCTGAGTTCATCGAAAAAGTTCGTCGTGCGCTGTATCTGGGCAAAA TCGTTTCTTACGCCCAGGGCTTCTCTCAGCTGCGTGCTGCGTCTGAAGAGTACAACTGGGATCTGAACTAC GGCGAAATCGCGAAGATTTTCCGTGCTGGCTGCATCATCCGTGCGCAGTTCCTGCAGAAAATCACCGATGC TTATGCCGAAAATCCACAGATCGCTAACCTGTTGCTGGCTCCGTACTTCAAGCAAATTGCCGATGACTACC AGCAGGCGCTGCGTGATGTCGTTGCTTATGCAGTACAGAACGGTATTCCGGTTCCGACCTTCTCCGCAGCG GTTGCCTATTACGACAGCTACCGTGCTGCTGTTCTGCCTGCGAACCTGATCCAGGCACAGCGTGACTATTT TGGTGCGCATACTTATAAGCGTATTGATAAAGAAGGTGTGTTCCATACCGAATGGCTGGATTAA
SEP ID NO: 19 (example 075 rfb locus nucléotide sequence - 075-EPA production strain
StLMTB11737)
ATGACGAATTTAAAAGCAGTTATTCCTGTAGCGGGTCTCGGGATGCATATGTTGCCTGCCACTAAGGCGAT ACCCAAAGAGATGCTACCAATCGTCGACAAGCCAATGATTCAGTACATTGTTGACGAGATTGTGGCTGCAG GGATCAAAGAAATCCTCCTGGTAACTCACGCGTCCAAGAACGCGGTCGAAAACCACTTCGACACCTCTTAT GAGTTAGAATCACTCCTTGAGCAGCGCGTGAAGCGTCAACTGCTGGCGGAAGTACAGTCCATCTGTCCGCC GGGCGTGACCATTATGAACGTGCGTCAGGGCGAACCTTTAGGTTTAGGCCACTCCATTTTGTGTGCGCGAC CTGCCATTGGTGACAACCCATTTGTCGTGGTACTGCCAGACGTTGTGATCGACGATGCCAGCGCCGACCCG CTACGTTACAACCTTGCTGCCATGATTGCACGTTTCAACGAAACGGGCCGCAGCCAGGTGCTGGCAAAACG TATGCCGGGTGACCTCTCTGAATACTCCGTCATCCAGACTAAAGAGCCGCTGGACCGTGAGGGTAAAGTCA GCCGCATTGTTGAATTTATCGAAAAACCGGATCAGCCGCAGACGCTGGACTCAGACATCATGGCCGTAGGT CGCTATGTGCTTTCTGCCGATATTTGGCCGGAACTGGAACGTACTCAGCCTGGTGCATGGGGACGTATTCA GCTGACTGATGCTATTGCCGAGCTGGCGAAAAAACAATCCGTTGATGCAATGCTGATGACCGGCGACAGTT ACGACTGCGGCAAAAAAATGGGCTATATGCAGGCGTTTGTGAAGTATGGCCTACGCAACCTGAAAGAAGGG GCGAAGTTCCGTAAAGGTATTGAGAAGCTGTTAAGCGAATAATGAAAATCTGACCGGATGTAACGGTTGAT AAGAAAATTATAACGGCAGTGAAAATTCGCAGCAAAAGTAATTTGTTGCGAATCTTCCTGCCGTTGTTTTA TATAAACCATCAGAATAACAACGAGTTAGCAGTAGGGTTTTATTCAAAGTTTTCCAGGATTTTCCTTGTTT CCAGAGCGGATTGGTAAGACAATTAGCGTTTGAATTTTTCGGGTTTAGCGCGAGTGGGTAACGCTCGTCAC ATCATAGGCATGCATGCAGTGCTCTGGTAGCTGTAAAGCCAGGGGCGGTAGCGTGCATTAATACCTCTATT AATCAAACTGAGAGCCGCTTATTTCACAGCATGCTCTGAAGTAATATGGAATAAATTAAGCTAGCAGTGAA GATACTTGTTACTGGTGGCGCAGGATTTATTGGTTCTGCTGTTGTTCGTCACATAATAAATAATACGCAAG ATAGTGTTGTTAATGTCGATAAATTAACATACGCCGGAAACCTGGAATCGCTCGCTGAAATTTCTGATTCT GAACGTTATTCATTTGAGCATGCAGATATCTGCGATGCCGAAGCGATGGCTCGTATTTTCGCACAGCACCA GCCAGACGCGGTGATGCACCTGGCAGCAGAGAGCCACGTTGACCGCTCAATAACTGGCCCTGCGGCATTTA TTGAAACCAATATTGTGGGTACTTATGTTCTTTTAGAAGCGGCGCGCAATTATTGGTCTGGTCTGGATGAT GAAAAGAAAAAAAACTTCCGCTTTCATCATATTTCTACTGATGAGGTGTATGGTGACTTACCCCATCCGGA TGAAGTAAATAGCAATGAAACGTTGCCGCTATTT ACGGAAATGACAGCATACGCGC CAAGTAGT C CATATT CTGCTTCTAAAGCTTCCAGCGATCATTTGGTTCGCGCATGGAAACGTACTTATGGTTTACCGACCATTGTG ACTAATTGCTCGAACAACTATGGTCCTTATCATTTCCCGGAAAAGCTTATTCCACTGGTTATTCTTAATGC ACTGGAAGGTAAGGCATTACCTATTTATGGCAAAGGAGATCAGATCCGCGACTGGTTGTATGTAGAGGATC ATGCTCGAGCGTTATATACCGTCGTAACCGAAGGTAAAGCGGGCGAAACTTATAACATTGGTGGACACAAC GAAAAGAAAAACATCGACGTAGTGTTCACTATTTGTGATTTGTTGGATGAGATAGTCCCGAAAGAGAAATC TTATCGTGAGCAAATTACCTATGTTGCTGATCGCCCAGGGCATGATCGCCGTTATGCAATTGATGCCGATA AAATTAGCCGCGAATTGGGCTGGAAACCACAGGAAACGTTTGAGAGCGGGATTCGTAAAACTGTGGAATGG TATCTGTCCAATACAAAATGGGTTGATAATGTGAAAAGTGGTGCCTATCAATCGTGGATTGAACAGAACTA TGGGGGCCGCC ACTAATGAATAT C CTC CTTTTTGGCAAAACAGGGC AGGTTGGTTGGGAACT ACAGCGTGC TCTGGCACCTCTGGGTAATTTGATTGCTCTTGATGTTCACTCCACTGATTACTGTGGTGATTTTAGTAACC CTGAAGGTGTGGCTGAAACCGTTAGAAGCATTCGGCCTGATATTATTGTCAACGCAGCCGCTCACACCGCA GTAGACAAAGCAGAATCAGAACCGGAGTTTGCACAATTACTGAACGCGACGAGTGTCGAAGCGATCGCGAA AGCAGCCAATGAAGTCGGCGCTTGGGTTATTCACTACTCTACTGACTACGTATTTCCGGGGACCGGTGAAA TACCATGGCAGGAGGAGGATGCAACCGCACCGCTAAATGTTTACGGTGAAACCAAGTTAGCAGGAGAAAAA GCATTACAAGAGCATTGTGCGAAGCACCTTATTTTCCGGACCAGCTGGGTCTATGCAGGTAAAGGAAATAA CTTCGCCAAAACGATGTTGCGTCTGGCAAAAGAGCGTGAAGAATTAGCCGTTATTAATGATCAGTTTGGTG CGCCAACTGGCGCAGAGTTGCTGGCTGATTGTACGGCACATGCCATTCGTGTGGCACTGAATAAACCGGAA
140
GTCGCAGGTTTGTACCATCTGGTAGCCAGTGGTACCACAACCTGGCACGATTATGCTGCGCTGGTTTTTGA AGAGGCGCGCAAAGCAGGCATTCCCCTTGCACTCAACAAGCTCAACGCAGTACCAACAACAGTCTATCCTA CACCAGCTCGTCGTCCACATAACTCTCGCCTTAATACAGAAAAATTTCAGCAGAACTTTGCGCTTGTCTTG CCTGACTGGCAGGTTGGTGTGAAACGCATGCTCAACGAATTATTTACGACTACAGCAATTTAATAGTTTTT GCATCTTGTTCGTGATGGTGGAACAAGATGAATTAAAAGGAATGATGGAATGAATACGCGTAAAGGTATTA TTTTAGCGGGTGGTTCTGGTACACGTCTTTATCCTGTGACTATGGCTGTCAGTAAACAGCTGTTACCGATT TATGATAAACCGATGATCTATTACCCGCTCTCTACACTGATGTTGGCGGGTATTCGCGATATTTTGATTAT CAGCACGCCACAGGATACTCCTCGTTTTCAACAACTGCTGGGTGATGGGAGCCAGTGGGGGCTAAATCTTC ACTACAAAGTGCAACCGAGTCCGGATGGTCTTGCGCAGGCATTTATCATCGGTGAAGAGTTTATCGGTGGT GATGATTGTGCTTTGGTACTTGGTGATAATATCTTCTACGGTCACGACCTGCCTAAGTTAATGGATGCCGC TGTTAACAAAGAAAGTGGTGCAACGGTATTTGCCTATCACGTTAATGATCCTGAACGCTATGGTGTCGTTG AGTTTGATAAAAACGGTACTGCAATCAGCCTGGAAGAAAAAC CGTTACAAC CAAAAAGTAATTATGCGGTA ACCGGGCTTTATTTCTATGATAACTACGTTGTGGAAATGGCGAAAAATCTTAAGCCTTCTGCCCGCGGTGA ACTGGAAATTACCGATATTAACCGTATCTATATGGAACAGGGGCATTTATCTGTTGCCATGATGGGACGTG GATATGCCTGGCTGGACACGGGGACACATCAAAGTCTTATTGAAGCAAGCAACTTCATTGCCACCATTGAA GAGCGCCAGGGCTTGAAAGTTTCCTGCCCGGAAGAAATTGCTTACCGTAAAGGGTTTATTGATGCTGAGCA GGTGAAAGTATTAGCTAAACCGCTGAAAAAAAATGCTTATGGTCAGTATCTGCTAAAAATGATTAAAGGTT ATTAATAAAATGAATGTTATTAAAACAGAAATTCCAGATGTACTGATTTTTGAACCGAAAGTTTTTGGTGA TGAGCGTGGTTTCTTTATGGAAAGCTTTAATCAGAAAGTTTTCGAAGAGGCTGTAGGGCGGAAGGTTGAAT TTGTTCAGGATAATCATTCTAAATCGTGTAAAGGTGTACTTAGAGGTTTACACTTTCAGCTTCCTCCCTTT GAGCAGGCAAAATTAGTAAGGTGTATAGTTGGCGAGGTATTTGATGTTGCAGTAGACATTAGACCTAATTC TGAAACATTTGGTTCATGGGTTGGAGTAACTCTTTCGTCAGAAAATAAAAGGCAGCTATGGATTCCAGAAG GATTCGCCCATGGTTTTTTAACTTTAAGTGATATTGCAGAGTTTGTTTATAAAACTAACAACTATTATTCT TTAAATCATGAAAGGGGAGTCATTTGGAACGATGAGGAAATTAACATTGCCTGGCCCTCTCAATCAGAGAA GATTCTGTCACAGAAAGATATTAATTTACCATCATTTAGATTTGTTCAAATGTTTAGCAAGTAGTGTTATC TTTACACTGCACATAGTCATCATTTTTTATGCTTTAAGTAAATTATATTGCACATCTATAACACAAAGCGC AATAATATTTCGACCTGATGAAGGTTTGTGGTTATTTATCTTTCTAGGCGTTTTTTATGACTAAAATAGTT GTGGTTTCTACAGCTCCAATATTCCCGACAAATAATGGGTACAAAAGTTCTGTATTAGGAAGAATTGATGA GTTATTAAATGAGGATAATGAGGTCGTTTTGATTGAAATAAACCTTGAAAATGTTACGGAAAAGAAAGATG AATTAATACCAACAAGATTTAATAATATTCAAAGATATGAAGTAAAAAAAATATCTAGATCATTTATTGCC GAGTTACAAATATTATTTGATATCAGAACTCGGTATGAACAATTATTTTCTTCTGCTGACATTAGAGATAA CATAAAAAAGATAATTGATTTAGAAAAACCTTCTATTATTATTGCTGAGTCTATATGGGCGTTGCAAGCAT TGCCTATTGAAATTAGTGCGAGAATACACTGTGTTATTCATGATGTGGCAACTGATTTCTTTAAAGAAATG TTTGTATCTCATAATGAGGTTGTACGAAAAATTTTGTTTTTTAATGATTACCTAAAGTTGAAAATTACTGA AGAAAATATTATCAAACGTTTGAGAGTTGAGCAATTTATCTTTCTGACAGAAGAAGATAAATGTTGGTATA AAACAAGATACAATATTGATGAGGGTTGTTGTTCCTTAGCGAGCAATCATCTTTATGTAGAAAAGATTAAG AGAACTATCAATTT CCAAAC C CCTTTC CTGCTTATTC C CGGTAGCATTGAATTTTCACAAAATTTTTACGG CTTAAATTGGTTTATAAAAAATATATATCCTGGATTAAATAGGAAAATAAGAATAGTTGTAACAGGAAAGG CATCAGATAAAAAAATAAAGATGTTAAACTGTGGAGAGGAAATTACCTTTACGGGAGAGCTTGACTTTTCC acatataataaacttagctcaacatgcttgtgtgttattgcaccgattacaacgggcactggaattaaaat aaaaatattagaagctgtacaaaaaggtattcctgtacttacaacaaaatttgcttcaaaaggaatatgtt ccgatttatgtttttattgcgaggaggatactgacacaaactttgtcaatttaattaacagttttcttgaa acgacattaagagtccaagaatgaatttattgcttttttcagtccttgcgtttggtttaatattggctttg gcccataataataaaagtggagatattaacgcatacttaatgttttttctcgtggtcctaatggtattaat atcagggctgcgtatgaatgatagtgattatatcgaatacaggaaaatgtataatgaagtgcctattttat gtgactttagtctcgcatctataagagatatacatggggaggtaggctatctattcttatcatcaatcttt aaaactttatgcttgccatttcaattatttcttttttttattgcttttttatcactcctgcttacatattt ttcattcagaaaaataagtttaataccgatactatcgttagttttttatttaagccatgcttttatagtta gagatttgattcaaattagggcaggattagctgttagcatatcattatattcaataattaaatttaaagga aataaaagtataattacaggagttttatttgcttctttgattcattctggggcgcttattattgctctttg ttatccttttttcaaaaaaaaatacataacattaaaaatgatgttgtttttatttttagtgtcaattattt tttcttatttgaatgggcttaatttatcgatacaactcttatctcaatatagtttgcttccaactgcaatt tcgaattatgttggttgggaagaatatgattatcgggtgagtatatttactaatccggtttttattaaagg tgtttttttaattgtcttaatgcacaaatatgtactttcagatattaaaaatgagaaaattatagtgcttt ataacttatatgttttaggtgtattagctatggttgcattgagtgggatggctattctttcaggccgtctt tcatcctttctgacactaggtgaaagcattttaattgtatatgctctgttctacaaaagaaatacacctct
141
GGCGTTTCTAATTTTTTCTTTTTTAACAATTGTGCAATTAGGATATGATCTATTTATTTCTAATGTGCATC CTGAGCTTACTCTGATTATATTTGGGTGAATCTAAGTGAAAAATAATAAAATAGGCATACTTATCTCTAAA ATACAAAATCTTGGACCTGTGAATGTAGTACGAGGATTGATAAAAGAAAATAAAAAATATGCTTTTACTGT TTTTTGTTTAACAAATAGCGTAGATAAAAATATATATGATGAGTTATGCTGTTTAGGAGCCAAGGTTATAT TAATACCAGATGGTACTTGGTTCAGCAAAATTTTATTTGTGAGÀAGTTTTTTAAAGGAACATCCACATAAT ATCTTACATTCACATGGGATCACGGCCGATATGTTTTCTTACTTTCTGAATGGCGTGAAAATATCTACTAT TCACAATAGACTAGATGAGGATTATATCCCATTATTTGGCGCGGTTAAAGGGAATGCTATATATTATCTTC ATCGTTTTATATTACGAAGATTTAATCATATCGTTGCTTGCTCAGCAGCGGTCCAATCAAAACTGAAACAA TCGAAAGTAAAAACTAAAATAACCACCATCCAGAATGGGATTGATATAACTAGGTTTAAGACACTTGAGTC TGATAAAAAAAAATTATTGAGGGAAAAACACGGATTTGATAGTGAAAAAAGAATATTTATATATTGTGGCT CGTTATCATTAAGGAAAAATATTGCTTACCTCTTGGAACACTTAGCCATCGAAGAAAATGATATATTTTTA ATTCTAGGTGATGGTGAACTTTTTAGATATTGTAAGGATAAATATTCTAAAGATTTACGGTATATATTTAT GGGGAAAGTTGAATGCCCTCTTGAATATTATCAATTATCAGATATTTTTGTTTCCGCTTCTTTATCGGAAG GGCTCCCCTTGGCACTATTAGAAGCTGCCTCTACTGGGTGCTATTTATATGTTAGCGATATAGAGCCCCAT AGAGAAATTGCATCTCTATTAGGAGAGGAAAATATTTCTATGTTTAAAATTAAGGATGGATCATATAATTA TTTGCAACCTAAftATAAAAAAAGCTGACTATAACGCTCTTTC TGACGATAAACTTTACAATAT ATC CGATA AAAAAATGTCAAATCTTTATGACAAACTTTTTGTTTCTTTATTAGAGCAGAGGCACTAATATAATGATTTA TGTTTCGGTAATTTCTCATGGTCATTTCAAAACTCTTAAGGAATTAGGAGCAGTATCAAAATTAAATAATC ACAGCAGAATTAAAGTTATCATCAAAGATAATTTAGGAGAGAGCGAGCTTTTGGATTTTTGTCAGGAAAAC AAAATAACTTATTTAAGGTCTAAAGAGAAAAAAGGATTTGGAGAGAATAATAATGAAGTTTTTTCCTCTAT ATCCTCCTTAATTACTAAGGAAGATTTTTTTGTGGTTATGAATCCTGATATATATATTGAGTGCTCTGATC TATTAGATGTCGTAGATGAGTGTGGTTCAGCGAATGTTAATCTAGCAACGATAAATTTATACAGGGATTTT GATAAAAAAACATATGATAACTCAGTAAGGAAATTTCCCTCGGCAATTGATTTTTTTATGTCATTTTTATT TAAGAAAAATGACTGTGTAGTAAATAAGAACAAAATAACGAAACCAACATATGTTGATTGGGCTGCAGGTT CTTTTCTAATATTTAATGCCTTCTTTTATTCAAAACTCAACGGATTCAACGAAAAGTATTTTATGTATTGC GAAGATATTGATATATGTTGGCGAGCTAAAAAACACTTCAATACTTCAGTTTTATACTATCCATGCTATGC AGCAATTCATTTGGCACAATTTAACAATCGTAGGATTTTTAGTAGACATTTCATTTGGCATATAAAAAGTA TTATCCTTTTTTTATTATATAAAAATGGTATGCTGCGTTCTAGTAAGTTGCTTTAATGCTAATATTCTTTT AAGAGGTGAGAATGATACCTGTTATTTTGGCTGGTGGTTCGGGAAGTCGCTTGTGGCCACTTTCACGAGAA AAGTTCCCCAAGCAGTTTTTAAAGTTGACTGGCAGTTTGACAATGTTGCAGTCAACATTGTCACGTCTTAA TAATTTAAATGCTGATGATTCAATAGTTATATGCAACGAAGAGCATAGATTTATTGTTGCAGAACAATTAA GAGAGTTAGGCAAACTTTCAAATAACATTATTCTTGAACCCAAAGGTCGTAATACAGCCCCTGCTATAACA CTCGCAGCATTAGCAGCAAAAAGAAAATTCGCTGATGAAGATCCATTGATTCTTATTTTAGCTGCAGATCA CAACATCCAAGACGAACATGTTTTCTGTGAGGCAATTAATAAGGCGTCATCTTTAGCTAGTTATGGAAAAC TAGTGACTTTTGGTATCGTTCCATTCAAACCTGAAACTGGGTATGGCTATATTCGTCGCGGTGATGAAGTG CCTGTAGATGAGCAGCATGCGGTGGCCTTTGAAGTGGCGCAGTTTGTCGAAAAACCGAATCTGGAAACCGC GCAGGCCTATGTGGCAAGCGGCGAATATTACTGGAACAGCGGTATGTTCCTGTTCCGTGCCGGACGCTATC TCGAAGAACTGAAAAAGTAT CGTCCGGATATTCTCGATGC CTGTGAAAAAGCGATGAGCGC CGTCGAT C CG GATCTCGATTTTATTCGTGTGGATGAAGAGGCGTTTCTCGCTTGTCCGGAAGAGTCGGTGGATTACGCGGT CATGGAATGCACGGCAGATGCCGTTGTGGTGCCGATGGATGCGGGCTGGAGCGATGTCGGTTCCTGGTCTT C ATTATGGGAGATC AGCGC C C ACAC CGCCGAGGGCAACGTTTGC CACGGCG ATGTGATTAATCACAAAACT GAAAACAGCTATGTGTACGCCGAATCTGGCCTGGTCACCACCGTCGGGGTGAAAGATTTGGTGGTAGTGCA GACCAAAGATGCAGTGCTGATTGCCGACCGTAATGCGGTGCAGGATGTGAAGAAAGTGGTCGAGCAGATCA AAGCTGATGGTCGCCATGAGCATCGGGTGCATCGCGAAGTGTATCGTCCGTGGGGCAAATATGACTCTATC GACGCGGGCGACCGCTACCAGGTGAAACGCATCACCGTGAAACCGGGCGAAGGTTTGTCGGTACAGATGCA TTATCATCGCGCGGAACACTGGGTGGTTGTCGCGGGAACGGCAAAAGTCACTATCAACGGTGATATCAAAC TGCTTGGTGAAAACGAGTCCATTTATATTCCGCTGGGGGCGATGCACTGCCTGGAAAACCCGGGGAAAATA GATTTAGAATTAATTGAAGTTCGCTCTGGTGCATATCTTGAAGAAGATGATGTTATTAGATGTTATGATCG CTATGGACGAAAGTAATATATAATAATTATTTCAGAATTAGAAATGATAATTATAAGTTTTCGTCTGGATA AACAATAGATAGTATGGGTTGGAAAATATGAGTTCTTTAACTTGTTTTAAAGCTTACGACATTCGCGGGAA ATTAGGTGAAGAACTGAATGAAGATATCGCCTGGCGCATTGGTCGCGCCTATGGCGAATTTCTCAAACCGA AAACCATTGTGTTAGGCGGTGATGTCCGTCTCACCAGCGAAACCTTAAAACTGGCGCTGGCAAAAGGTTTA CAGGATGCGGGCGTCGATGTGCTGGATATTGGCATGTCCGGCACCGAAGAGATTTATTTCGCCACGTTCCA TCTCGGCGTGGATGGCGGCATTGAAGTTACCGCCAGCCATAATCCGATGGATTACZkACGGCATGAAGCTGG TGCGCGAAGGGGCTCGCCCGATCAGCGGTGATACCGGACTGCGCGACGTCCAGCGTCTGGCAGAAGCTAAC GACTTTCCTCCCGTCGATGAAACCAAACGCGGTCGCTATCAGCAAATCAATCTGCGTGACGCTTACGTTGA
142
TCACCTGTTCGGTTATATCAATGTCAAAAACCTTACGCCGCTCAAGCTGGTGATCAACTCCGGGAATGGCG CAGCGGGTCCGGTGGTGGACGCTATCGAAGCCCGCTTTAAAGCCCTCGGCGCACCGGTGGAGTTAATCAAA GTGCATAACACGCCGGACGGCAATTTCCCCAACGGTATTCCTAACCCGTTGCTGCCGGAATGTCGCGACGA CACCCGCAATGCGGTCATCAAACACGGCGCGGATATGGGCATTGCCTTTGATGGCGATTTTGACCGCTGTT TCCTGTTTGACGAAAAAGGGCAGTTTATTGAGGGCTACTACATTGTCGGCCTGCTGGCAGAAGCGTTCCTC GAAAAAAATCCCGGCGCGAAGATCATCCACGATCCACGTCTCTCCTGGAACACCATTGATGTGGTGACGGC CGCGGGCGGCACGCCGGTGATGTCGAAAACAGGACACGCCTTTATTAAAGAACGTATGCGCAAGGAAGACG CCATCTACGGTGGCGAAATGAGCGCTCACCATTACTTCCGCGATTTCGCTTACTGTGACAGCGGCATGATC CCGTGGCTGCTGGTCGCCGAACTGGTGTGCCTGAAAGGAAAAACGCTGGGCGAACTGGTGCGCGACCGGAT GGCGGCGTTTCCGGCAAGCGGTGAGATCAACAGAAAACTGGCGCACCCTGTTGAGGCGATTAACCGCGTGG AACAGCATTTTAGCCGTGAGGTGCTGGCGGTGGATCGCACCGATGGCATCAGCATGACCTTTGCCGACTGG CGCTTTAACCTGCGCTCTTCCAACACCGAACCGGTGGTGCGCCTGAATGTGGAATCTCGCGGTGATGTTCA GGTTATGGTAATCCATACTCAAGAAATATTATCAATTTTGACGTCATAAAGAATAAGCCCTGACAAGTTAG GGCTTAATTAATATATATTTTTTTTGAATTGGGGATTTGTGGTAAGATTTTTAATATGTTATTTAATGTGG TTGAATTAATGTTGACTGGAAAATAATAATGAGAACGAAAAAAGCATTACACAACTTTAAAGTTGATTTAT TAATTACTTTTTTATTGGTTTTGCTAGGGTTTTATATTCGAACTGTTTTTGTTTCAAAAATGGGAAGTGAT ATTACTGGAGTGATGTTACTATTCACACAGTTGACAGCATATCTCAATTTGGCAGAATTAGGTATTGGAAT TGCAGCTGCCAGCGTATTATATAAACCGCTCAGCGAGAATGAATACAATAAAATAACTTACATAATATCTT TGCTCTCAGTCATATACAAATATATATTTGTGTTTGTTTTGATTCTTGGCGTTGTTATAGGTATCTGTATT TATTACTTTATTGATTCTGTAAAGGTTGTAAATGGCGTTTTTTTATATTGGGCTTTGTTCGTTTTTAATAC ATCGTTGACATATAGTTATGCTAAATACTCCACATTATTAACTGCTAATCAGCGGTACTCAGCAGTAAGAA AAATTCAAGGTGGCGGAAAAGTTATAATAATTGTATTTCAGATATTAATTTTGTGCTTTACGCAAAGTTTC ATACTTTATTTGTTAGTTGAGACTTTAGGTATTTTTTCTCAATATTTGATTTTTAAAAAAATAATTGGGAA CGGAAATCAATATCTCAGTAATGAGGTTTTACTTATTGAAAGCGATAAACTTTTGATAAAAAAAGAATTAA aaataagaataaaaaatatgttcttccataaaataggtgctgtgcttgtccttaatacagactacctgctt GTATCAAAGTTTCTGACATTAAGTTATGTGACAATTTTTGGCAGCTATATGATGGTATTTCAGATAGTAAC TGTTTTGATGTCAAGTTTTGTTAATGCTATTACTGCAGGAATGGGTAATTACTTAATTAATAAAAGTAATT TAGAAATTAAGGÀAATTACACGTCAATTTTATGTGATATTTATCGCCTTTGCAACATTCATATCACTAAAT ATGTTTTTTCTTGTTAATGATTTTATCGCAAAATGGATAGGTGTTAATTATACATTAAGTAACACCCTAGT TGCATTAATGATTGTTAACGTATTCATTAGTGTTGTCAGGGTACCTTCTGATATATTAAAAAACGCAAGTG GAC ATTTTGGTGAT ATTT ATTAT C C ATTATT AGTiAGGTGTGCTGAATATTACGATAT CC ATCATTTTGGCT ATCATTATTGGATTACCTGGCATTATTATAGGGACAATAGTATCTAACTTAATAGTAATAATGCTTGCGAA ACCATTATATCTTTACTCTAAGTTATTTAATCTTAGAAATCCGACGAGGGTTTATTTTGAATTTATTTCTC GGCCTATGTTATATTCATTATGTGTGATTGGGGTGAGCTATTTATTGCGCGATGAAATATATTCATTTAAA GTAAGTACATGGTTGGATTTTATTAACAAGCTACTCTTAGTCTCTACTCCTAGCATATTGGTAATATGTGC TATTTTCTCTACGGATAGTGACTTTAGATTATTTTTCAGAAAAATTATATATGTGATTATGAAGAAATAAA AATTTCGAAAATGTATTAATCGAAATTATGCAACGAGCTTTATTTTTATAAATGATATGTGATCTTTTCGC GAATAGGAGTAAGGATCCGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTT CGGAATAGGAACTAAGGAGGATATTCATATGGATAAAGCCGTAAGCATATAAGCATGGATAAGCTATTTAT ACTTTAATAAGTACTTTGTATACTTATTTGCGAACATTCCAGGCCGCGAGCATTCAGCGCGGTGATCACAC CTGACAGGAGTATGTAATGTCCAAGCAACAGATCGGCGTAGTCGGTATGGCAGTGATGGGACGCAACCTTG CGCTCAACATCGAAAGCCGTGGTTATACCGTCTCTATTTTCAACCGTTCCCGTGAGAAGACGGAAGAAGTG ATTGCCGAAAATCCAGGCAAGAAACTGGTTCCTTACTATACGGTGAAAGAGTTTGTCGAATCTCTGGAAAC GCCTCGTCGCATCCTGTTAATGGTGAAAGCAGGTGCAGGCACGGATGCTGCTATTGATTCCCTCAAACCAT ATCTCGATAAAGGAGACATCATCATTGATGGTGGTAACACCTTCTTCCAGGACACTATTCGTCGTAATCGT GAGCTTTCAGCAGAGGGCTTTAACTTCATCGGTACCGGTGTTTCTGGCGGTGAAGAGGGGGCGCTGAAAGG TCCTTCTATTATGCCTGGTGGCCAGAAAGAAGCCTATGAATTGGTAGCACCGATCCTGACCAAAATCGCCG CCGTAGCTGAAGACGGTGAACCATGCGTTACCTATATTGGTGCCGATGGCGCAGGTCACTATGTGAAGATG GTTCACAACGGTATTGAATACGGCGATATGCAGCTGATTGCTGAAGCCTATTCTCTGCTTAAAGGTGGCCT GAACCTCACCAACGAAGAACTGGCGCAGACCTTTACCGAGTGGAATAACGGTGAACTGAGCAGTTACCTGA TCGACATCACCAAAGATATCTTCACCAAAAAAGATGAAGACGGTAACTACCTGGTTGATGTGATŒTGGAT GAAGCGGCTAACAAAGGTACCGGTAAATGGACCAGCCAGAGCGCGCTGGATCTCGGCGAACCGCTGTCGCT GATTACCGAGTCTGTGTTTGCACGTTATATCTCTTCTCTGAAAGATCAGCGTGTTGCCGCATCTAAAGTTC TCTCTGGTCCGCAAGCACAGCCAGCAGGCGACAAGGCTGAGTTCATCGAAAAAGTTCGTCGTGCGCTGTAT CTGGGCAAAATCGTTTCTTACGCCCAGGGCTTCTCTCAGCTGCGTGCTGCGTCTGAAGAGTACAACTGGGA TCTGAACTACGGCGAAATCGCGAAGATTTTCCGTGCTGGCTGCATCATCCGTGCGCAGTTCCTGCAGAAAA
143
TCACCGATGCTTATGCCGAAAATCCACAGATCGCTAACCTGTTGCTGGCTCCGTACTTCAAGCAAATTGCC GATGACTACCAGCAGGCGCTGCGTGATGTCGTTGCTTATGCAGTACAGAACGGTATTCCGGTTCCGACCTT CTCCGCAGCGGTTGCCTATTACGACAGCTACCGTGCTGCTGTTCTGCCTGCGAACCTGATCCAGGCACAGC GTGACTATTTTGGTGCGCATACTTATAAGCGTATTGATAAAGAAGGTGTGTTCCATACCGAATGGCTGGAT TAA .
144

Claims (15)

1. A bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein, wherein the E. coli glucosylated 04 antigen polysaccharide comprises the structure of Formula (04-Glc+):
a-D-GIcp
[^2)-ct-L-Rhap-(1 -»6)-a-D-Glcp-(1 -»3)-a-L-FucpNAc-(1 ->3)-p-D-GlcpNAc-(1 ->]n wherein n is an integer of 1 to 100, preferably 3 to 50, for example 5 to 40, for example 7 to 25, for example 10 to 20.
2. The bioconjugate of claim 1, wherein the E. coli glucosylated 04 antigen polysaccharide is covalently linked to an Asn residue in a glycosylation site comprising a glycosylation consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 1), preferably a glycosylation consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any amino acid except Pro (SEQ ID NO: 2), in the carrier protein.
3. The bioconjugate of claim 1 or claim 2, wherein the carrier protein is selected from the group consisting of detoxified Exotoxin A of P. aeruginosa (EPA), E, coli flagellin (FliC), CRM197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemoiysin A of S. aureus, dumping factor A, dumping factor B, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Choiera toxin B subunit (CTB), choiera toxin, detoxified variants of choiera toxin, E. coli Sat protein, the passenger domain of E. coli Sat protein, Streptococcus pneumoniae Pneumolysin, Keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae, preferably wherein the carrier protein is detoxified exotoxin A of Pseudomonas aeruginosa (EPA), preferably comprising 1 to 20, preferably 1 to 10, preferably 2 to 4, glycosylation consensus sequences having SEQ ID NO: 1, the consensus sequences preferably having SEQ ID NO: 2, more preferably wherein the carrier protein comprises four glycosylation consensus sequences (EPA-4), more preferably wherein the carrier protein comprises SEQ ID NO: 3.
4. A composition comprising the bioconjugate of any one of daims 1-3.
5. The composition of claim 4, comprising at least one additional antigen polysaccharide covalently linked to a carrier protein.
145
6. The composition of claim 5, wherein the at least one additional antigen polysaccharide is selected from the group consisting of E. coli 01A antigen polysaccharide, E. coli 02 antigen polysaccharide, E. coli 06A antigen polysaccharide, E. coli 08 antigen polysaccharide, E. coli 015 antigen polysaccharide, E. coli 016 antigen polysaccharide, E. coli 018A antigen polysaccharide, E. coli O25B antigen polysaccharide, and E. coli 075 antigen polysaccharide, preferably wherein (i) the E. coli 01A antigen polysaccharide comprises the structure of Formula (01 A):
[->3)-a-L-Rhap-(1 ^3)-a-L-Rhap-(1 ->3)-p-L-Rhap-(1 ->4)-p-D-GlcpNAc-(1
2 t 1 β-D-ManpNAc .
(ii) the E. coli 02 antigen polysaccharide comprises the structure of Formula (02):
[-*3)-a-L-Rhap-(1 ^2)-a-L-Rhap-(1 ^3)^-L-Rhap-(1 -M)-p-D-GlcpNAc-(1 -*]n î
a-D-Fucp3NAc .
(iii) the E. coli 06A antigen polysaccharide comprises the structure of Formula (06A):
[->4)-a-D-GalpNAc-(1 ^3)-p-D-Manp-(1 —4)-p-D-Manp-(1 ^3)-a-D-GlcpNAc-(1 -»]n
2 î 1 β-D-Glcp .
(iv) the E. coli 08 antigen polysaccharide comprises the structure of Formula (08):
a-D-Manp3Me-(1 -43)-β-ϋ-Μ8ηρ-(1 ->2)-a-D-Manp-(1 ->2)-a-D-Manp-(1 -»]n ?
(v) the E. coli 015 antigen polysaccharide comprises the structure of Formula (015):
[^2)-p-D-Galp-(1 ->3)-a-L-FucpNAc-(1 ->3)-3-D-GlcpNAc-(1 ^]n , (vi) the E. coli 016 antigen polysaccharide comprises the structure of Formula (016):
146
[->2)^-D-GalA( 1 -»6)-a-D-Glcp-(1 ^3)-a-L-Rhap-(1 -»3)-a-D-GlcpNAc-(1^]n î Ac · ?
(vii) the E. coli O18A antigen polysaccharide comprises the structure of Formula (018A):
[->2)-a-L-Rhap-(1 -^6)-a-D-Glcp-(1 ^4)-a-D-Galp-(1 ^3)-a-D-GlcpNAc-(1 ^]n
3 f 1 β-D-GlcpNAc .
(viii) the E. coli O25B antigen polysaccharide comprises the structure of Formula (O25B):
β-D-Glcp !
[^4)-a-D-Glcp-(1 -3)-cr-L-Rhap-(1 ^3)-p-D-G!cpNAc-(1 ->]n îî
1Ac a-L-Rhap · and (ix) the E. coli 075 antigen polysaccharide comprises the structure of Formula (075):
β-D-Manp
1 ;
4 ,
[->3)-a-D-Galp-(1 ->4)-a-L-Rhap-(1 -^3)-p-D-GlcpNAc-(1 ?
wherein each n is independently an integer of 1 to 100, preferably of 3 to 50, e.g. 5 to 40, e.g. 7 to 25, e.g. 10 to 20.
7. The composition of claim 6, wherein each carrier protein is EPA comprising SEQ ID NO: 3.
8. The composition of any one of claims 6 or 7, comprising at least the E. coli 01 A, 02, glucosylated 04, O6A, 08, 015, 016, O18A, O25B, and 075 antigen polysaccharides each covalently linked to a carrier protein.
9. A composition comprising:
147 (i) a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a detoxified Exotoxin A of P. aeruginosa (EPA-4 carrier protein) comprising SEQ ID NO: 3, wherein the E, coli glucosylated 04 antigen polysaccharide comprises the structure of Formula (04-Glc+):
a-D-GIcp l
[->2)-a-L-Rhap-(1 -*6)-a-D-Glcp-( 1 ->3)-a-L-F ucpNAc-(1 ->3)-p-D-GlcpNAc-( 1 ^]n (ii) a bioconjugate of an E. coli 01A antigen polysaccharide covalently linked to an EPA4 carrier protein, wherein the E. coli 01A antigen polysaccharide comprises the structure of Formula (01 A):
[-*3)-a-L-Rhap-(1 -*3)-a-L-Rhap-(1 ^3)-p-L-Rhap-(1^4)-p-D-GlcpNAc-(1 -^]n t
1 β-D-ManpNAc .
(iii) a bioconjugate of an E. coli 02 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 02 antigen polysaccharide comprises the structure of Formula (02):
|>3)-a-L-Rhap-(1 ^2)-a-L-Rhap-(1 ^3)-p-L-Rhap-(1 ^4)-p-D-GlcpNAc-(1 ^]n t
a-D-Fucp3NAc .
(iv) a bioconjugate of an E. coli 06A antigen polysaccharide covalently linked to an EPA-
4 carrier protein, wherein the E. coli 06A antigen polysaccharide comprises the structure of Formula (O 6 A) :
[->4)-a-D-GalpNAc-(1 ->3)-β-0-Μθηρ-(1 -»4)-ft-D-Manp-(1~*3)-a-D-GlcpNAc-(1 -*]n î
β-D-Glcp .
148 (v) a bioconjugate of an E. coli 08 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli 08 antigen polysaccharide comprises the structure of Formula (08):
a-D-Manp3Me-(1 -»[3)-p-D-Manp-(1 ->2)-a-D-Manp-(1 -^2)-a-D-Manp-(1 -»]n (vi) a bioconjugate of an E. coli 015 antigen polysaccharide covalently linked to an EPA4 carrier protein, wherein the E. coli 015 antigen polysaccharide comprises the structure of F ormula (015) :
[^2)-p-D-Gaip-(1^3)-a-L-FucpNAc-(1^3)-p-D-GlcpNAc-(1^-]n.
(vii) abioconjugate of an E. coliO\6 antigen polysaccharide covalently linked to an EPA4 carrier protein, wherein the E. coli 016 antigen polysaccharide comprises the structure of Formula (016):
[^2)-p-D-Galf-(1^6)-a-D-Glcp-(1 ^3)-a-L-Rhap-(1 ^3)-a-D-GlcpNAc-(1
2 t Ac .
?
(viii) a bioconjugate of an E. coli O25B antigen polysaccharide covalently linked to an EPA4 carrier protein, wherein the E. coli O25B antigen polysaccharide comprises the structure of Formula (O25B):
β-D-Glcp
[^4)-a-D-Glcp-(1 ->3)-a-L-Rhap-(1 -3)-p-D-GlcpNAc-(1 tî
1Ac α-L-Rhap , ar|j (ix) a bioconjugate of an E. coli 075 antigen polysaccharide covalently linked to an EPA4 carrier protein, wherein the E. coli 075 antigen polysaccharide comprises the structure of Formula (075):
149 β-D-Manp
[-»3)-a-D-Galp-(1^4)-ot-L-Rhap-(1->3)^-D-GlcpNAc-(1->]n wherein each n is independently an integer of 1 to 100, preferably 3 to 50, e.g. 5 to 40, e.g. 7 to 25, e.g. 10 to 20, the composition preferably further comprising:
(x) a bioconjugate of an £ coli O18A antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O18A antigen polysaccharide comprises the structure of Formula (O18A):
[^2)-a-L-Rhap-(1 ->6)-ct-D-Glcp-(1 ^4)-a-D-Galp-(1 -^3)-a-D-GlcpNAc-(1
3 t 1 β-D-GlcpNAc , and n an integer of 1 to 100, preferably 3 to 50, e.g. 5 to 40, e.g. 7 to 25, e.g. 10 to 20.
10. (Currently Amended) Use of bioconjugate of any one of claims 1-3, or the composition of any one of claims 4-9, for the manufacture of a médicament for inducing antibodies against an E. coli glucosylated 04 antigen in a subject.
11. (Currently Amended) Use of the bioconjugate of any one of claims 1-3, or the composition of any one of claims 4-9, for the manufacture of a médicament for vaccinating a subject against E. coli, in particular extra-intestinal pathogenic E. coli (ExPEC).
12. A recombinant prokaryotic host cell for producing a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein, wherein the glucosylated 04 antigen polysaccharide comprises the structure of Formula (O4-Glc+):
a-D-GIcp l
[-*2)-a-L-Rhap-(1 -*6)-a-D-Glcp-(1 -»3)-a-L-FucpNAc-(1 ->3)-β-θ-ΘΙορΝΑο-(1 -*]n wherein n is an integer of 1 to 100, preferably of 3 to 50, e.g. of 5 to 40, the host cell comprising:
(i) a nucléotide sequence of an rfb gene cluster for the E. coli 04 antigen polysaccharide;
150 (ii) a nucléotide sequence encoding a glucosyl transferase having at least 80% sequence identity to SEQ ID NO: 4, wherein the glucosyl transferase is capable of modifying the E. coli 04 antigen polysaccharide to produce the E, coli glucosylated 04 antigen polysaccharide;
(iii) nucléotide sequences encoding a translocase and a glycosyltransferase having at least 80% sequence identity to SEQ ID NOs: 7 and 8 respectively, wherein the translocase is capable of translocating bactoprenol-linked glucose and the glycosyltransferase is capable of glucosylating bactoprenol;
(iv) a nucléotide sequence encoding the carrier protein; and (v) a nucléotide sequence encoding an oligosaccharyl transferase capable of covalently linking the E. coli glucosylated 04 antigen polysaccharide to the carrier protein to produce the bioconjugate.
13. The recombinant prokaryotic host cell of claim 12, wherein:
the glucosyl transferase has SEQ ID NO: 4;
the oligosaccharyl transferase comprises the amino acid sequence of SEQ ID NO: 6, preferably SEQ ID NO: 6 comprising the amino acid mutation N311V, more preferably SEQ ID N0:6 comprising the amino acid mutations Y77H and N31IV; 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 a detoxified exotoxin A of Pseudomonas aeruginosa (EPA), more preferably wherein the EPA comprises 1-10, preferably 2-4, glycosylation sites each comprising a glycosylation consensus sequence having SEQ ID NO: 2, more preferably wherein the carrier protein is EPA with four glycosylation consensus sequences (EPA-4), more preferably wherein the carrier protein comprises SEQ IDN0:3.
14. The recombinant prokaryotic host cell of any one of claims 12 or 13, which is an E. coli cell, e.g. an E. coli K-12 strain, such as strain W3110.
15. A method of producing a bioconjugate of an E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein, wherein the E. coli glucosylated 04 antigen polysaccharide comprises the structure of Formula (O4-Glc+):
151 α-D-Glcp 3 Ί
[^2)-ct-L-Rhap-(1 -^6)-a-D-Glcp-(1 ^3)-a-L-FucpNAc-(1 -^3)-p-D-GlcpNAc-(1 Y , wherein n is an integer of 1 to 100, preferably of 3 to 50, e.g. of 5 to 40, the method comprising cuituring the recombinant prokaryotic host cell of any one of daims 12-14 under conditions for production of the bioconjugate.
OA1202100435 2019-03-18 2020-03-18 Bioconjugates of E. coli O-antigen polysaccharides, methods of production thereof, and methods of use thereof. OA20646A (en)

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