IMMUNOGENIC COMPOSITION

专利摘要:
The present invention relates to a conjugate comprising an antigen (eg a saccharide antigen) covalently linked to a Pseudomonas aeruginosa PcrV carrier protein comprising an amino acid sequence having at least 80% identity to the sequence of SEQ ID NOs. : 1 to 4, wherein the antigen is bound (directly or via a linker) to an amino acid residue of the P. aeruginosa PcrV carrier protein. The invention also relates to Pseudomonas aeruginosa PcrV proteins which contain consensus sequences comprising glycosylation sites.
公开号:BE1024361B1
申请号:E2016/5783
申请日:2016-10-19
公开日:2018-02-05
发明作者:Christiane Marie-Paule Simone Jeanne Feron；Stefan Jochen Kemmler；Michael Thomas Kowarik；Julien Laurent Quebatte
申请人:Glaxosmithkline Biologicals Sa；
IPC主号:

专利说明:

(73) Holder (s):
GLAXOSMITHKLINE BIOLOGICALS SA
1330, RIXENSART
Belgium (72) Inventor (s):
FERON Christiane Marie-Paule Simone Jeanne
1330 RIXENSART
Belgium
KEMMLER Stefan Jochen 8952 SCHLIEREN Switzerland
KOWARIK Michael Thomas 8952 SCHLIEREN Switzerland
QUEBATTE Julien Laurent 8952 SCHLIEREN Switzerland (54) IMMUNOGENIC COMPOSITION (57) The present invention relates to a conjugate comprising an antigen (for example a saccharide antigen) covalently linked to a PcrV carrier protein of Pseudomonas aeruginosa comprising an amino acid sequence having at least 80% identity with the sequence of SEQ ID NO: 1 to 4, in which the antigen is linked (directly or via a linker) to an amino acid residue of the PcrV carrier protein of P aeruginosa. The invention also relates to PcrV proteins from Pseudomonas aeruginosa which contain consensus sequences comprising glycosylation sites.
BELGIAN INVENTION PATENT
FPS Economy, SMEs, Middle Classes & Energy
Publication number: 1024361 Deposit number: BE2016 / 5783
Intellectual Property Office International Classification: A61K 39/104 A61K 39/385 C07K 14 / 21A61P 31/04 Date of issue: 05/02/2018
The Minister of the Economy,
Having regard to the Paris Convention of March 20, 1883 for the Protection of Industrial Property;
Considering the law of March 28, 1984 on patents for invention, article 22, for patent applications introduced before September 22, 2014;
Considering Title 1 “Patents for invention” of Book XI of the Code of Economic Law, article XI.24, for patent applications introduced from September 22, 2014;
Having regard to the Royal Decree of 2 December 1986 relating to the request, the issue and the maintenance in force of invention patents, article 28;
Considering the patent application received by the Intellectual Property Office on 19/10/2016.
Whereas for patent applications falling within the scope of Title 1, Book XI of the Code of Economic Law (hereinafter CDE), in accordance with article XI. 19, §4, paragraph 2, of the CDE, if the patent application has been the subject of a search report mentioning a lack of unity of invention within the meaning of §ler of article XI.19 cited above and in the event that the applicant does not limit or file a divisional application in accordance with the results of the search report, the granted patent will be limited to the claims for which the search report has been drawn up.
Stopped :
First article. - It is issued to
GLAXOSMITHKLINE BIOLOGICALS SA, Rue de l'Institut 89, 1330 RIXENSART Belgium;
represented by
PRONOVEM - Office Van Malderen, Avenue Josse Goffin 158, 1082, BRUXELLES;
a Belgian invention patent with a duration of 20 years, subject to payment of the annual fees referred to in article XI.48, §1 of the Code of Economic Law, for: IMMUNOGENIC COMPOSITION.
INVENTOR (S):
FERON Christiane Marie-Paule Simone Jeanne, GlaxoSmithKline Biologicals SA Rue de l'Institut 89, 1330, RIXENSART;
KEMMLER Stefan Jochen, LimmaTech Biologies AG, Grabenstrasse 3, 8952, SCHLIEREN;
KOWARIK Michael Thomas, LimmaTech Biologies AG, Grabenstrasse 3,8952, SCHLIEREN;
QUEBATTE Julien Laurent, LimmaTech Biologies AG, Grabenstrasse 3, 8952, SCHLIEREN;
PRIORITY (S):
10/21/2015 GB 1518668.7;
DIVISION:
divided from the basic application: filing date of the basic application:
Article 2. - This patent is granted without prior examination of the patentability of the invention, without guarantee of the merit of the invention or of the accuracy of the description thereof and at the risk and peril of the applicant (s) ( s).
Brussels, 02/05/2018, By special delegation:
BE2016 / 5783
IMMUNOGENIC COMPOSITION
Technical area
The present invention relates to the field of immunogenic compositions and conjugate-based vaccines, their preparation and the use of such compositions in medicine. More particularly, it relates to the use of a PcrV as a new carrier protein derived from Pseudomonas aeruginosa. A PcrV can be used as a carrier protein for other antigens, in particular saccharide antigens or other antigens having no epitope associated with T lymphocytes. The PcrV carrier protein can act both as a carrier protein and as an antigen. -even.
Context
The conjugation of T cell independent antigens to carrier proteins has long been a well-established method of involving T cells in an immune response against an antigen normally independent of T cells. In this way, an immune response can be enhanced by allowing the establishment of an immune memory and a better stimulated response. Those skilled in the art know that conjugate-based vaccines, which have been developed by conjugating bacterial capsular saccharides with carrier proteins, have been successful; the carrier protein has the known effect of changing the T cell independent polysaccharide antigen to a T cell dependent antigen which can elicit a response associated with immune memory. In this case, the document WO 02/58737
BE2016 / 5783 describes a vaccine comprising purified capsular polysaccharides from serogroups A, C, W135 and Y of N. meningitidis conjugated with a carrier protein
Several carrier proteins are known in the art, tetanus toxoid, diphtheria toxoid, CRM197 and protein D of Haemophilus influenzae being used as carrier protein in commercial vaccines. Vaccines have also used diphtheria toxin, and mutant forms like CRM197, as a T cell dependent carrier for saccharides, effective and safe. CRM197 is currently used in vaccines conjugating CRM197 with a Haemophilus influenzae type b oligosaccharide (HibTitre®; Lederle Praxis Biologicals, Rochester, N. Y.).
The disease caused by infection with Pseudomonas strains (eg P. aeruginosa) poses a considerable threat worldwide. Although the development of vaccines against this infection is in progress, there is still a severe shortage of effective vaccines against Pseudomonas infection, which can be produced safely in large quantities.
The present invention relates to a novel carrier protein. The Pseudomonas aeruginosa PcrV protein is not usually used as a carrier protein. The present invention relates to conjugates in which the PcrV protein serves both as a carrier protein for a saccharide antigen, and itself acts as an antigen, so that a neutralizing immune response develops against PcrR, and a opsonic response develops against an LPS.
BE2016 / 5783
Consequently, according to one aspect of the present invention, it relates to a conjugate comprising an antigen covalently linked to a PcrV carrier protein of Pseudomonas aeruginosa comprising an amino acid sequence having at least 80% identity with the sequence SEQ ID NOs: 1 to 4, the antigen being linked (directly or via a linker) to an amino acid residue of the carrier protein PcrV of P. aeruginosa.
According to a second aspect of the invention, it relates to a PcrV protein having an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 1 to 4, said acid sequence amino acids comprising a consensus sequence D / EX-NXS / T, in which X represents any amino acid except proline.
According to a further aspect of the invention, it relates to an immunogenic composition comprising the conjugate or the PcrV proteins according to the invention and a pharmaceutically acceptable excipient.
According to a further aspect of the invention, this relates to a method for preparing an immunogenic composition according to the invention, comprising the step of mixing the conjugate or a PcrV protein according to the invention with a pharmaceutically acceptable excipient .
According to a further aspect of the invention, it relates to a conjugate or a PcrV protein according to the invention intended to be used in the treatment of an infection, and methods of treatment using
BE2016 / 5783 the conjugate or the PcrV protein according to the invention are also an additional aspect of the invention.
According to a further aspect of the invention, the latter relates to a polynucleotide coding for a PcrV protein of P. aeruginosa according to the invention and a polynucleotide coding for a PcrV protein, the nucleotide sequence of which codes for a polypeptide having a sequence d amino acids having at least 80% identity with any of SEQ ID NOs: 1 to 4.
According to a further aspect of the invention, it relates to a polynucleotide vector according to the invention.
According to a further aspect of the invention, it relates to a host cell comprising:
i) a nucleic acid encoding a glycosyltransferase;
ii) an acid comprising for the nucleic acid coding for oligosaccharyl transferase; and iii) a nucleic acid coding for a PcrV protein of P. aeruginosa according to the invention.
According to a further aspect of the invention, it relates to a method for producing a bioconjugate comprising a PcrV protein of P. aeruginosa linked to a saccharide, said method comprising the culture of the host cell according to the invention under conditions suitable for protein production.
According to a further aspect of the invention, it relates to a bioconjugate produced by the method according to the invention, said bioconjugate comprising a saccharide linked to a PcrV protein of P. aeruginosa.
BE2016 / 5783
Description of the figures
Figure 1. Western blot of periplasm extracts from modified host cells producing bioconjugates. The strains indicated are described in the Examples. "Int" means an integrated component. “*” Indicates an integration by an approach of induction by a transposon.
Figure 2. Description of the repeat unit of the Pseudomonas aeruginosa 06 O-antigen. * Indicates positions whose chemical composition may vary depending on the identity of the sub-serotype. This variability is introduced by the activity of amidases which convert the acid functions of the GalNacA residues to C6 into amides, to give GalNacAN (or GalNFmA to GalNFmAN; in certain sub-serotypes, an acetyl group substitutes the GalNAcAN * residue for C3) . The genes leading to polymerization of the repeat unit (wzy), acetylation, formylation and amidation of one of the GalNX residues are unknown. L-Rha, L-Rhamnose; D-GalNAcAN, 6-amido-2-Nacetyl-D-galactosaminuronic acid; D-GalNFmAN, 2-Nformyl-D-galactosaminuronic acid; D-QuiNAc, N-acetyl-Dquinosamine.
Figure 3. Functional test of Pseudomonas aeruginosa 06 formyltransferase.
3A: Detection of the unique repeat unit of 06 formylated linked to the lipid A nucleus by Western blotting. An E. coli W3110 Awec strain was transformed using a cosmid encoding the rfbO6 aggregate (incomplete) and an expression plasmid encoding the 06-formyltransferase (fmt06; SEQ ID NO: 2) . Of
BE2016 / 5783 cell extracts were collected after a night of induction during growth at 37 ° C. in LB medium, digested with proteinase K, separated by SDSPAGE, and electrically transferred to nitrocellulose membranes. An induced signal was observed, in the presence of fmt06, following immunodetection carried out with an antiserum specific for 06, but not in the control containing the empty vector. This result clearly shows that formylation is a relevant antigen for P. aeruginosa cells, as well as a prerequisite for detection by this antiserum.
3B: Confirmation of the formylation on a single repeat unit of 06 released from undecaprenylpyrophosphate. An E. coli W3110 Awec AwaaL strain was transformed with the same plasmids as above and grown in shake flasks, in order to produce only repeat units of O-antigen 06 (in these strains, wzy polymerase is absent). The glycolipids were then analyzed. Briefly, the repeat units were extracted from the dried cells, in the form of glycolipids, affinity purified on C18 cartridges, hydrolysed (in order to remove the undecaprenylpyrophosphate from the O-antigen 06 repeat units), labeled by reductive amination with 2-aminobenzamide, and analyzed by HPLC in normal phase. The co-expression of fmt06 produced an additional signal after 61 'of elution, which contained oligosaccharides corresponding to the repeating unit 06 formylated and labeled, whereas in the absence of the gene, the main signal appeared
BE2016 / 5783 after 58 ', and contained the repeat unit 06 Nacetylated and labeled.
Figure 4. Functional test of the candidate wzy polymerase associated with P. aeruginosa 06. E. coli W3110 Awec cells containing a cosmid encoding the rfb aggregate (incomplete, deprived of the fmtO6 and wzy genes) were transformed using plasmids encoding fmtO6 and the wzy candidate PAK_01823 (SEQ ID NO: 3 ) or using the corresponding empty vectors. The cell extracts were treated with proteinase K and the LPS were analyzed by immunodetection following an SDS-PAGE and an electrical transfer on nitrocellulose membranes.
Figure 5. Cloning of the expression aggregate of the artificial O-antigen of Pseudomonas aeruginosa 06. First, the rfb aggregate of the stGVXN4017 strain of P. aeruginosa 06 (“PAK” strain of Pseudomonas aeruginosa 06 ) was cloned into a cosmid vector by PCR cloning using standard techniques. Bioinformatics-based homology searches identified formyltransferase (FT) and O-antigen polymerase (wzy), which were then inserted one after the other downstream of the aggregate. rfb. The resulting gene aggregates make it possible to carry out the biosynthesis of the complete repeat unit of O-antigen 06 of P. aeruginosa (rfbO6 +, no polymer) and the biosynthesis of the polysaccharide (rfbO6 ++, in which wzy is included). , in derivatives of E. coli W3110.
FIG. 6 describes a Western transfer of extracts of the periplasm of modified host cells.
BE2016 / 5783 producing bioconjugates. The strains indicated are described in the Examples.
6A: results obtained for strain E. coli "S17 3 4 3" modified to contain an integrated pglB and one integrated rfb aggregate from of 06 of P. aeruginosa. 6B: results obtained for strain E. coli "S17 2 0 9" modified to contain a pglB carried by a plasmid and an integrated rfb aggregate from 06 from P. aeruginosa • 6C: results obtained for strain E. coli
"St2182" modified to contain a pglB carried by a plasmid and an rfb aggregate carried by a plasmid originating from P. aeruginosa 06.
Figure 7. Purified EPA-O6 glycoconjugate. EPA-O6 was purified from extracts of the periplasm of host cells modified by metal chelate affinity chromatography, anion exchange chromatography and size exclusion chromatography (SEC). The final SEC eluate was characterized by SDSPAGE followed by Coomassie blue staining or Western blotting using the indicated antibodies.
Figure 8. Plasmid retention (PR) for plasmid systems 1 and 3 in the presence or absence of selection pressure by an antibiotic. RA is expressed as the% of cells containing the corresponding plasmid. Figures A and B show the PR of the plasmid EPA (Kanamycin, black) in host cells modified to contain an rfb aggregate and a pglB integrated in the presence (A) or in the absence (B) of kanamycin. Figures C and D show the PR of the plasmid
BE2016 / 5783
EPA (Kanamycin, black), plasmid pglB (Spectinomycin, white) and plasmid of the rfb aggregate (Tetracycline, dotted) in modified host cells in the presence (C) and in the absence (D) of the three antibiotics. The percentage of cells in which the three plasmids are stored is shown in gray. Inoc = inoculum; U = uninduced cells; 14 = cells after 6 hours of induction; 16 = cells after induction overnight.
Figure 9. Biological activity of the vaccine-induced anti06 antiserum.
9A: Median titers obtained by ELISA on sera from mice collected from the different vaccination groups, following the third injection. No ads = without adjuvant, O / W: indicates the adjuvant used, an adjuvant in oil-in-water emulsion. O / W alone designates a control group which does not contain glycoconjugate.
9B: The median titers of elimination by opsonophagocytosis (inducing a 50% reduction in cfu compared to the control) are indicated. The fold and pill sets represent pooled sera collected after the second or third injection.
Figure 10. Rate of inhibition of hemolysis by PcrV of sera collected on day 14 for pill (day 42).
Figure 11. Titers of anti-06 IgG obtained by ELISA in individual sera, 14 days after II (day 42).
Figure 12. Titers of anti-06 opsonophagocytosis in individual sera, 14 days after III (day 24).
Figure 13. Titers of anti-PcrV IgG obtained by ELISA in rat sera 14 days after II (day 28) and 14 days after III (day 42).
BE2016 / 5783
Figure 14. Titers of anti-06 IgG obtained by ELISA in sera of rats collected 14 days after III (day 42).
detailed description
The present invention relates to a conjugate comprising an antigen covalently linked to a PcrV carrier protein of Pseudomonas aeruginosa comprising an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97 %, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 1 to 4, the antigen being linked (directly or via a linker) to an amino acid residue of the protein PcrV carrier of P. aeruginosa.
In one embodiment, the amino acid residue to which the antigen is bound is not an asparagine residue, and in this case the conjugate is typically produced by chemical conjugation, many methods of which are well known in the art. For example, the amino acid is chosen from the group consisting of: Ala, Arg, Asp, Cys, Gly, Glu, Gin, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr , and Val. The amino acid is optionally: an amino acid comprising an amino terminal group, a lysine an arginine, a glutamic acid, an aspartic acid, a cysteine, a tyrosine, a histidine, an arginine, or a tryptophan.
In one embodiment, the antigen is covalently linked to a PcrV carrier protein from Pseudomonas aeruginosa by means of a chemical bond obtainable using a chemical conjugation process.
BE2016 / 5783
In one embodiment, the chemical conjugation method is chosen from the group consisting of carbodiimide chemistry, reductive amination, cyanylation chemistry (CDAP chemistry for example), maleimide chemistry, hydrazide chemistry, ester chemistry, and Nhydroxysuccinimide chemistry. Optionally, the antigen is covalently linked to an amino acid aspartic acid, glutamic acid, lysine cysteine, tyrosine, histidine, arginine, or tryptophan on the carrier protein PcrV of P. aeruginosa.
In one embodiment, the antigen is linked directly to the PcrV carrier protein of P. aeruginosa.
In one embodiment, the antigen is linked to the PcrV carrier protein of P. aeruginosa via a linker. Optionally, the linker is chosen from the group consisting of linkers containing 4 to 12 carbon atoms, bifunctional linkers, linkers containing 1 or 2 reactive amino groups at their ends, B-propionamide, nitrophenylethylamine, halides haloacyl, 6-aminocaproic acid and DHA.
In general, the following types of chemical groups present on a carrier protein can be used to effect coupling / conjugation:
A) Carboxyl (for example those of an aspartic acid or a glutamic acid). In one embodiment, this group is directly linked to amino groups present on the saccharides or on amino groups of a linker, thanks to the chemistry of carbodiimides, for example with EDAC.
BE2016 / 5783
B) Amino group (for example that of a lysine). In one embodiment, this group is directly linked to carboxyl groups present on the saccharides or on carboxyl groups of a linker, thanks to the chemistry of the carbodiimides, for example with EDAC. In another embodiment, this group is directly linked to hydroxyl groups present on the saccharides, activated using CDAP or CNBr or on these same groups belonging to a linker; saccharides or linkers having an aldehyde group; to saccharides or linkers having a succinimide ester group.
C) Sulfhydryl (for example that of a cysteine). In one embodiment, this group is linked to a saccharide or to a bromo- or chloroacetylated linker thanks to the chemistry of maleimides. In one embodiment, this group is activated / modified by a bis-diazobenzidine.
D) Group hydroxyl (for example the one of a tyrosine). In one embodiment, this group is activated / modified with a bis-diazobenzidine. E) Group imidazolyle (e.g. the one of a histidine). In one embodiment, this group is activated / modified with a bis-diazobenzidine. F) Group guanidyle (for example the one of a arginine). G) Group indolyle (for example the one of a tryptophan).
In general, the groups present on a following saccharide can be used for coupling: OH, COOH or NH2. The aldehyde groups can be generated by various treatments known in the art, such as
BE2016 / 5783 by: periodate, acid hydrolysis, hydrogen peroxide, etc.
Direct coupling approaches:
Saccharide-OH + CNBr or CDAP ----> cyanate ester + Nth-Prot ----> conjugate
Saccharide aldehyde + NPh-Prot ----> Base
Schiff + NaCNBPh ----> conjugate
Saccharide-COOH + NPh-Prot + EDAC ----> conjugate
Saccharide-NPh + COOH-Prot + EDAC ----> conjugate
Indirect coupling approaches via a spacer (linker):
Saccharide-OH + CNBr or CDAP ----> cyanate ester + NH2 ---- NH2 ----> saccharide ---- NH2 + COOH-Prot + EDAC
----> conjugate
Saccharide-OH + CNBr or CDAP ----> cyanate ester + NH2 ---- SH ----> saccharide ---- SH + SH-Prot (native protein of which a cysteine is exposed, or obtained after modification amino groups of the protein by SPDP for example) ----> saccharide S-S-Prot
Saccharide-OH + CNBr or CDAP ----> cyanate ester + NH2 ---- SH ----> saccharide ---- SH + maleimide-Prot (modification of amino groups) ----> conjugate
Saccharide-COOH + EDAC + NH2 ---- NH2 ---> saccharide --- NH2 + EDAC + COOH-Prot ----> conjugate
Saccharide-COOH + EDAC + NH2 ---- SH ---> saccharide --- SH + SH-Prot (native protein of which a cysteine is exposed, or obtained after modification of amino groups
BE2016 / 5783 protein by SPDP for example) ----> saccharide S-S-Prot
Saccharide-COOH + EDAC + NH2 ---- SH ----> saccharide --- SH + maleimide-Prot (modification of amino groups)
----> conjugate
Saccharide aldehyde + NH2 ---- NH2 ---->
saccharide --- NH2 + EDAC + COOH-Prot ----> conjugate
Note: Any suitable carbodiimide can be used in place of the EDAC above.
In one embodiment, the conjugate according to the invention contains an amino acid residue to which the antigen is linked, the amino acid residue being an asparagine residue.
In one embodiment, the asparagine residue is not part of the consensus sequence D / EXN-XS / T introduced in the amino acid sequence which has at least 50%, 60%, 70%, 80%, 85% , 90%,%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 1 to 4, X representing any amino acid except proline.
However, in one embodiment
additional, the asparagine residue made part of the sequence consensus D / E-X-N-X-S / T introduced in the sequence acids amines which presents at less 50 0Gold 60%, 70 %, 80%, 85%, 90%, 92%, 95%, 96%, 97 0θ r 98%, 99 % or 100 % identity with sequence of SEQ ID NO : 1 to 4, X representing any acid
amino except proline, the asparagine residue being located at a position corresponding to amino acids 23 to 166, or amino acids 281 to 317, or to amino acid
BE2016 / 5783
317 of SEQ ID NO: 3. For example, 24 to 100, amino acids 24 to 50, 310 to 317.
amino acids amino acids
In one embodiment, the asparagine residue is part of the consensus sequence D / EXNXS / T introduced into the amino acid sequence which has at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 1 to 4, X representing any amino acid except proline, the asparagine residue being located between amino acids 1 and 143, or amino acids 258 and 294, or at amino acid 294 of SEQ ID NO: 4. For example, at amino acids 1 to 100, or acids amino acids 1 to 50, or amino acids 1 to 25, or amino acids 290 to 294 of SEQ ID NO: 4.
In one embodiment, the asparagine residue is part of the consensus sequence D / EXNXS / T, X representing any amino acid except proline, the asparagine residue not being introduced by a mutation in the sequence of SEQ ID NO: 5, or a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% d identity with SEQ ID NO: 5.
In one embodiment, a peptide comprising the consensus sequence D / EXNXS / T is introduced into the amino acid sequence by the elimination of a sequence of the peptide PcrV and its replacement by the peptide comprising a consensus sequence D / EXNXS / T. In one embodiment, the sequence of the PcrV peptide
BE2016 / 5783 eliminated contains from 1 to 7 amino acids or 7, 6, 5, 4, 3, 2 or an amino acid.
In one embodiment, the peptide comprising the consensus sequence D / EXNXS / T is introduced into the amino acid sequence at a position located between amino acid residues 23 and 166 of SEQ ID NO: 3 or between the acid residues amino acids 1 and 143 of SEQ ID NO: 4 or at a position located between amino acid residues 23 and 100 or 23 and 50 of SEQ ID NO: 3, or between amino acid residues 1 and 100, 1 and 50 or 1 and 24 of SEQ ID NO: 4.
In one embodiment, at least 1, 2, 3, 4 or 5 consensus sequences D / E-X-N-X-S / T are introduced into the sequence corresponding to any one of the
SEQ ID NO: 1 to 4 or in a sequence presenting at less 50%, 60% , 70%, 80%, 85%, 90% , 92%, 95 oGold 96%, 97%, 98%, 99% or 100% identity with one of
these.
In one embodiment, the PcrV carrier protein has a sequence comprising at least one of SEQ ID NO: 6 to 62, for example SEQ ID NO: 6 to 12 and 33.
In one embodiment, the PcrV carrier protein has a sequence comprising at least 1, 2, 3, 4 or 5 of SEQ ID NOs: 6 to 12 and 33. optionally at least 3 of SEQ ID NOs: 6 to 12 and 33 .
In one embodiment, the PcrV carrier protein has a sequence comprising SEQ ID NO: 6 and / or SEQ ID NO: 9 and / or SEQ ID NO: 11 and / or SEQ ID NO: 33.
BE2016 / 5783
In one embodiment, the antigen is a saccharide of the bacterial capsular saccharide type, a lipopolysaccharide or a bacterial lipooligosaccharide.
The saccharides can be chosen from a group comprising: the capsular saccharide of N. meningitidis serogroup A (MenA), the capsular saccharide of N. meningitidis serogroup C (MenC), the capsular saccharide of N. meningitidis serogroup Y (MenY) ), the capsular saccharide of serogroup W of N. meningitidis (MenW), the capsular saccharide of H. influenzae type b (Hib), the capsular saccharide of group I of group B of Streptococcus, the capsular saccharide of group II B of Streptococcus, the capsular saccharide of group III of group B of Streptococcus, the capsular saccharide of group IV of group B of Streptococcus, the capsular saccharide of group V of group B of Streptococcus, the capsular saccharide of
Staphylococcus type 5 aureus, the saccharide capsular Staphylococcus aureus of type 8, the saccharide vi of Salmonella typhi, a LPS (such as L3 and / or L2) of N . meningitidis, an LPS of Mr. catarrhalis,
an LPS of H. influenzae, O-antigens of Shigella, O-antigens of P. aeruginosa, O-antigens of E. coli and among all the saccharides of pneumococcus such as those corresponding to the serotype: 1, 2, 3, 4, 5,
6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F or 33F.
In one embodiment, the antigen is a lipopolysaccharide from P. aeruginosa. Optionally, the antigen is an O-antigen originating from P. aeruginosa, possibly 01, 02, 03, 04, 05, 06,
BE2016 / 5783
07, 08, 09, OIO, Oll, 012, 013, 014, 015, 016, 017, 018, 019 or 020, for example 06 or 011. In one embodiment, this relates to a bioconjugate comprising a carrier protein PcrV linked to a Pseudomonas aeruginosa O-antigen, said Pseudomonas aeruginosa O-antigen being one of the serotypes described by Knirel et al., 2006, Journal of Endotoxin Research 12 (6): 324336, said publication being incorporated here in reference in its entirety. In specific embodiments, the P. aeruginosa O-antigen is 06 or 011.
In a further aspect of the invention, this relates to a PcrV protein of which an amino acid sequence has at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of SEQ ID NO: 1 to 4, said amino acid sequence comprising a consensus sequence D / EXNXS / T, X representing any what amino acid except proline.
In one embodiment, the consensus sequence D / EXNXS / T in which X represents any amino acid except proline, is at a position located between amino acids 23 and 166, or amino acids 281 and 317, or at amino acid 317 of SEQ ID NO: 3.
In one embodiment, the consensus sequence D / EXNXS / T in which X represents any amino acid except proline, is located between amino acids 1 and 143, or amino acids 258 and 294, or at amino acid 294 of SEQ ID NO: 4.
BE2016 / 5783
In one embodiment, a peptide comprising the consensus sequence D / EXNXS / T is introduced into the amino acid sequence by the elimination of a sequence of the peptide PcrV and its replacement by a peptide comprising the consensus sequence D / EXNXS / T. In one embodiment, the sequence of the PcrV peptide contains from 1 to 7 amino acids, optionally 7, 6, 5, 4, 3, 2 or an amino acid.
In one embodiment, the peptide comprising the consensus sequence D / EXNXS / T is introduced into the amino acid sequence at a position located between amino acid residues 23 and 166 of SEQ ID NO: 3 or between the acid residues amino 1 and 143 of SEQ ID NO: 4.
In one embodiment, the peptide comprising the consensus sequence D / EXNXS / T is introduced into the amino acid sequence at a position located between amino acid residues 23 and 100 or 23 and 48 of SEQ ID NO: 3, or between amino acid residues 1 and 75 or 1 and 24 of SEQ ID NO: 4.
In one embodiment, at least 2, 3 or 4 consensus sequences D / EXNXS / T, or exactly 1, 2, 3, 4, 5 or 6 consensus sequences D / EXNXS / T are introduced into the sequence corresponding to the any of SEQ ID NOs: 1 to 4 or in a sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identity with one of these.
In one embodiment, the PcrV protein has an amino acid sequence comprising at least one of SEQ ID NOs: 6 to 62, optionally at
BE2016 / 5783 minus one of SEQ ID NO: 6 to 12 and 33, possibly at least 3 of SEQ ID NO: 6 to 12 and 33.
In one embodiment, the PcrV protein according to the invention has an amino acid sequence comprising SEQ ID NO: 6 and / or SEQ ID NO: 9 and / or SEQ ID NO: 11 and / or SEQ ID NO: 33.
In one embodiment, the PcrV carrier proteins used to produce the bioconjugates of the present invention comprise a "marker", that is to say an amino acid sequence making it possible to isolate and / or identify the protein carrier. For example, the addition of a marker to a carrier protein of the present invention may prove useful during the purification of this protein, and therefore during the purification of conjugate-based vaccines comprising the labeled carrier protein. Examples of markers that can be used according to the invention include, but are not limited to, histidine (His-tag) markers (e.g., hexahistidine marker, or 6xHis-tag marker), FLAG marker, and HA markers . In certain embodiments, the markers used according to the invention are labile, and can for example be removed by chemical agents or by enzymatic means, when they are no longer useful, for example once the protein has been purified .
In certain embodiments, the carrier proteins of the present invention include a signal sequence which directs the carrier protein to the periplasm of the host cell which expresses the carrier protein. In a specific embodiment, the
BE2016 / 5783 signal sequence comes from the DsbA of E. coli, porin A of the outer membrane (OmpA) of E. coli, maltose binding protein (MalE) from E. coli, pectin lyase (PelB) from Erwinia carotovorans, Flgl, NikA, or endoxylanase (XynA) from Bacillus species, the heat-labile enterotoxin LTIIb from E. coli. coli, Bacillus XynA endoxylanase, or E. coli flagellin (Flgl). coli. In one embodiment, the PcrV protein according to the invention comprises a leader sequence capable of directing the PcrV protein towards the periplasm of the bacterium. Optionally, the leader sequence is an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO : 63. In one embodiment, an alanine residue is added between the leader sequence and the start of the sequence of the mature protein. The advantage of such an alanine residue is that it induces a more efficient cleavage of the leader sequence.
In one embodiment, the PcrV protein according to the invention has an amino acid sequence comprising a peptide marker which can be used for the purification of the PcrV protein. Optionally, the peptide marker is located at the C-terminus of the amino acid sequence. Optionally, the peptide marker includes six histidine residues.
In a further aspect of the invention, it relates to a process for the preparation of an immunogenic composition according to the invention, comprising the step of mixing the conjugate or the PcrV protein with a pharmaceutically acceptable excipient.
BE2016 / 5783
The PcrV proteins and the conjugates according to the invention are particularly suitable as components of immunogenic compositions and vaccines. A method according to the invention may therefore comprise the step of formulating the PcrV protein or the conjugate in the form of an immunogenic composition or a vaccine. In a further aspect of the invention, this relates to a composition immunogen comprising the conjugate according to the invention or the PcrV protein according to the invention and a pharmaceutically acceptable excipient. The immunogenic composition according to the invention can also comprise additional antigens. Here are examples of such additional antigens: a conjugate of an O-antigen and a carrier protein, a conjugate of a bacterial capsular polysaccharide and a carrier protein, a conjugate of an LOS, of a carrier protein and a protein. Among the suitable conjugates one can find 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of 01, 02, 03, 04, 05, 06, 07, 08, 09, OlO, 011, 012 , 013, 014, 015, 016, 017, 018, 019 or 020 of P. aeruginosa Among the suitable proteins there are also proteins of P. aeruginosa, such as for example the exoprotein A of P. aeruginosa or one of its variants, such as those described in the document WO 13/36 574, the OmpI, OmpF or PopB of P. aeruginosa (YpoB, YopD, FliC) or the hybrid proteins OprF-OmpI (see documents US 5,955,090 or US 6,300,102).
Immunogenic compositions and vaccines according to the invention typically include "pharmaceutically acceptable excipients" which include any excipient which does not itself induce production
BE2016 / 5783 of antibodies harmful to the individual to whom the composition is administered. The compositions also typically contain a diluent, for example water, saline, glycerol, etc. In addition, they can contain auxiliary substances, such as wetting or emulsifying agents, pH buffers, polyols and the like.
The compositions comprising the PcrV conjugates or proteins of the present invention can include any component the use of which is suitable for pharmaceutical administration. In specific embodiments, the compositions of the present invention are monovalent formulations. In other embodiments, the compositions of the present invention are multivalent formulations, for example bivalent, trivalent and tetravalent formulations. For example, a multivalent formulation comprises more than one antigen, for example more than one conjugate.
In certain embodiments, the compositions of the present invention additionally comprise a preservative, for example thimerosal, a mercurial derivative. In a specific embodiment, the pharmaceutical compositions of the present invention comprise from 0.001% to 0.01% thimerosal. In other embodiments, the pharmaceutical compositions of the present invention do not include a preservative.
In certain embodiments, the compositions of the present invention (eg immunogenic compositions) include, or are
BE2016 / 5783 administered in combination with an adjuvant. When administered in combination with the composition, the adjuvant of the present invention can be administered before, at the same time, or after administration of said composition. In certain embodiments, the term "adjuvant" designates a compound which, when administered in conjunction with the composition of the present invention, or when it is one of its constituents, increases, improves and / or stimulates the immune response towards a bioconjugate, but which, when administered alone, does not generate any immune response towards the bioconjugate. In some embodiments, the adjuvant elicits an immune response against the conjugate or the PcrV protein, and does not cause allergies or other adverse reactions. Adjuvants can improve the immune response through several mechanisms, including, for example, lymphocyte recruitment, stimulation of B and / or T lymphocytes, and stimulation of macrophages.
Specific examples of adjuvants include, but are not limited to, aluminum salts (alums) (such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate), monophosphoryl lipid A (MPL) 3-de-O-acylated (see English patent GB2220211), MF59 (Novartis), AS03 (GlaxoSmithKline), AS04 (GlaxoSmithKline), polysorbate 80 (Tween 80; ICL Americas, Inc. ), imidazopyridine compounds (see international patent application No. PCT / US 2007/064 857, published under the international publication number WO 2007/109 812), imidazoquinoxaline compounds (see international patent application
BE2016 / 5783
PCT / US No. 2007/064 858, published under the international publication number WO 2007/109 813) and saponins, such as QS2 (see Kensil et al., In Vaccine Design: The Subunit and Adjuvant Approach (eds
Plenum & Newman,
U.S. Pat. No. 5,057,540), realization, adjuvant
Powell
1995); de Freund's modes
Press, NY,
In some is the adjuvant (complete or incomplete). Among the adjuvants, there are also oil-in-water emulsions (including squalene or peanut oil), possibly combined with other stimulants of the immune system, such as monophosphoryl-lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)). Another possible adjuvant is CpG (Bioworld Today, Nov. 15, 1998).
In certain embodiments, the formulation of the compositions of the present invention is adapted to the desired route of administration for a patient. For example, the formulation of the compositions of the present invention can be adapted for subcutaneous, parenteral, oral, intradermal, transdermal, colorectal, intraperitoneal or rectal administration. In a specific embodiment, the formulation of the pharmaceutical compositions can be adapted for administration by the intravenous, oral, intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular, topical, intradermal, transdermal or pulmonary route.
In certain embodiments, the compositions of the present invention additionally comprise one or more buffers, for example a phosphate buffer and a sucrose-phosphate-glutamate buffer.
BE2016 / 5783
In other embodiments, the compositions of the present invention do not include buffers.
In certain embodiments, the compositions of the present invention additionally comprise one or more salts, for example sodium chloride, calcium chloride, sodium phosphate, monosodium glutamate and aluminum salts (for example 1 aluminum hydroxide, aluminum phosphate, alum (aluminum and potassium sulfate), or a mixture of these aluminum salts). In other embodiments, the compositions of the present invention do not include salts.
The compositions of the present invention may be contained in a container, package or dispenser, together with instructions for their administration.
The compositions of the present invention can be stored before use: they can for example be stored frozen (for example at around -20 ° C or at around -70 ° C); stored under refrigerated conditions (for example at around 4 ° C); or stored at room temperature.
The immunogenic compositions used as vaccines comprise an immunologically effective amount of the PcrV protein or of the conjugate according to the invention, as well as any other necessary component. The term "immunologically effective amount" means that administration of this amount to an individual, either as a single dose or as part of a series, is effective for treatment or prevention. This quantity is variable, and depends on health and
BE2016 / 5783 the physical condition of the individual to be treated, his age, the degree of protection desired, the formulation of the vaccine and other relevant factors. It is expected that this amount will fall within a relatively wide range which can be determined during routine testing.
The vaccines of the present invention preferably include adjuvants. Among the suitable adjuvants, there is an aluminum salt such as an aluminum hydroxide (alum) or aluminum phosphate gel, but also a calcium, magnesium, iron or zinc salt, or also an insoluble suspension of acylated tyrosine, or acylated sugars, cationic or anionic derivatives of polysaccharides, or polyphosphazenes.
It is preferable that the choice of adjuvants is based on an inducer promoting a Thl or Th2 type response. High levels of Th1-type cytokines tend to favor, for a given antigen, the induction of cell-based immune responses, while high levels of Th2-type cytokines tend to favor, for the antigen, induction of humoral immune responses.
It is important to remember that the distinction is not absolute between Thl and Th2 type immune responses. In reality, the immune response developed by an individual can be described as mainly of Thl type or mainly of Th2 type. However, it is often more practical to consider families of cytokines based on those described in murine CD4 + ve T cell clones by Mosmann and Coffman (Mosmann, T.R. and Coffman,
BE2016 / 5783
R.L. (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annual Review of Immunology, 7, pl45-173). Traditionally, Thl-type responses have been associated with the production by T lymphocytes of INF-γ and IL-2 cytokines. Other cytokines, which are not produced by T lymphocytes, are often directly associated with the induction of Thl-type immune responses, such as, for example, IL-12. In contrast, Th2-type responses are associated with the secretion of IL-4, IL-5, IL-6, or IL-10. The adjuvant systems favoring a predominance of the Th1 response include: monophosphoryl-lipid A or one of its derivatives, in particular monophosphoryllipid A 3-de-O-acylated (3D-MPL) (for its preparation, see the document GB 2220211 A); and a combination of a monophosphoryl-lipid A, preferably monophosphoryl-lipid A 3-de-O-acylated, and an aluminum salt (e.g. aluminum phosphate or aluminum hydroxide) or an oil in water emulsion. In such associations, the antigen and 3D-MPL are included in the same particle structures, which allows more efficient presentation of antigenic and immunostimulatory signals. Studies have shown that 3D-MPL is capable of further improving the immunogenic character of an antigen adsorbed on an alum [Thoelen et al. Vaccine (1998) 16: 708-14; EP 689 454-B1].
An improved system contains a monophosphoryllipid A associated with a saponin derivative, in particular QS21 associated with 3D-MPL, as described in the
BE2016 / 5783 document WO 94/00 153, or a less reactogenic composition in which QS21 is attenuated with cholesterol, as described in document WO 96/33 739. Document WO 95/17 210 describes an adjuvant formulation particularly potent comprising QS21, 3D-MPL and tocopherol in an oil-in-water emulsion, which is a preferred formulation. Preferably, the vaccine additionally contains a saponin, more preferably QS21. The formulation may also include an oil-in-water emulsion and tocopherol (WO 95/17210). The present invention also relates to a method for producing a vaccine formulation comprising mixing a protein of the present invention with a pharmaceutically acceptable excipient, such as for example 3D-MPL. Other preferred inducers of the Th1 response are unmethylated CpGs containing oligonucleotides (WO 96/02 555), which are suitable for use according to the present invention.
The compositions according to the invention may contain an oil in water emulsion. Indeed, these seem to be useful as components of adjuvants (EP 399 843; WO 95/17 210). It is possible to use oil in water emulsions such as those described in document WO 95/17 210 (which describes oil in water emulsions comprising from 2 to 10% of squalene, from 2 with 10% of alpha-tocopherol and 0.3 to 3% of tween 80, and their use alone or in combination with QS21 and / or 3D-MPL), the document WO 99/12 565 (which describes oil emulsion compositions in
BE2016 / 5783 water comprising a metabolizable oil, a saponin and a sterol, and MPL) or the document WO 99/11 241. Other oil in water emulsions, described also suitable document.
The vaccine preparations according to the present invention can be used to protect or in or as WO 09/127 676 those and in WO 09/127 677, are treating a mammal subject to infection, administering said vaccine by mucosal systemic route . These administrations may include injection by intramuscular, intraperitoneal, intradermal or subcutaneous route; or mucosally, via the oral / food, respiratory or urogenital tract. For the treatment of pneumonia or otitis, intranasal administration of vaccine is preferred (since the nasopharyngeal transport of pneumococci can then be effectively limited, which reduces infection at an early stage). Although the vaccine according to the invention can be administered in a single dose, its components can also be administered jointly at the same time or at different times (for example, the pneumococcal polysaccharides could be administered separately, at the same time or 1 to 2 weeks after the administration of a bacterial protein of the composition, to optimally coordinate the immune responses with respect to each other). In case of co-administration, the possible Thl adjuvant may be present in all or part of the different administrations, however, it is preferable that it is present in association with a bacterial protein.
BE2016 / 5783 vaccine composition. In addition to a single route of administration, it is possible to use 2 different routes of administration. For example, polysaccharides can be administered IM (or ID) and bacterial proteins can be administered IN (or ID). In addition, the vaccines according to the invention can be administered IM for sensitization doses and IN route for booster doses.
The amount of conjugated antigen in each dose of vaccine is chosen to be sufficient to induce a protective immune response without inducing undesirable side effects typical of vaccines. This amount will be different depending on the specific immunogen used and how it is presented. Generally, each dose should contain 0.1 to 100 µg of polysaccharide, preferably 0.1 to 50 µg for polysaccharide conjugates, preferably 0.1 to 10 µg, more preferably 1 to 10 pg, the interval from 1 to 5 pg being all the more preferred.
The content of protein antigens in the vaccine will typically be in the range of 1 to 100 pg, preferably 5 to 50 pg, most preferably in the range of 5 to 25 pg. Following an initial vaccination, patients may receive one or more booster immunizations at appropriate time intervals.
The preparation of vaccines is generally described in the document Vaccine Design (The subunit and adjuvant approach (eds Powell M.F. & Newman M.J.) (1995) Plenum Press New York). Encapsulation at
BE2016 / 5783 the interior of liposomes is described by Fullerton, US patent publication 4,235,877.
The vaccines according to the present invention can be stored in solution or freeze-dried. Preferably, the solution is lyophilized in the presence of a sugar such as sucrose, trehalose or lactose. It is all the more preferable that they are lyophilized and reconstituted immediately before use.
In one embodiment, the PcrV conjugate or protein according to the invention is intended to be used in the treatment of an infection, in particular in the treatment of an infection by P. aeruginosa, for example for a human patient who would need it.
In a further aspect of the invention, it relates to a polynucleotide encoding the PcrV protein according to the invention. For example, a polynucleotide coding for a PcrV protein, whose nucleotide sequence codes for a polypeptide whose
sequence amino acids present in less 50 oθ r 60%, 70%, 80 %, 85%, 90%, 92%, 95%, 96%, 97 oθ r 98%, 99% or 100% identity with the sequence of 1 'a any of SEQ ID NOs: 1 to 4. In a aspect
additional to the invention, it relates to a vector comprising such a polynucleotide.
In a further aspect of the invention,
this concerns a host cell including: i) an acid nucleic coding for a glycosyltransferase; ii) an acid nucleic coding for a
oligosaccharyl transferase; and
BE2016 / 5783 iii) a nucleic acid coding for a PcrV protein of P. aeruginosa according to the invention.
A prokaryotic host cell thus modified comprises nucleic acids encoding enzymes capable of producing a bioconjugate comprising an antigen, for example a saccharide antigen attached to the PcrV protein. Such host cells can naturally express nucleic acids specific for the production of a saccharide antigen, or the host cells can be designed to express such nucleic acids. In other words, in certain embodiments, said nucleic acids are heterologous with respect to the host cells. In certain embodiments, at least one of said nucleic acids specific for the production of a saccharide antigen is heterologous with respect to the host cell and integrated into the genome of the host cell. In certain embodiments, the host cells according to the present invention comprise nucleic acids coding for additional enzymes, the activity of which is the N-glycosylation of proteins, the host cells according to the invention further comprising, for example, a nucleic acid. coding for an oligosaccharyl transferase and / or for at least one nucleic acid coding for other glycosyltransferases. In certain embodiments, the host cells according to the present invention comprise a nucleic acid encoding a carrier protein, for example a protein to which oligosaccharides and / or polysaccharides can be linked to form a bioconjugate. In a specific embodiment, the host cell is E. coli.
BE2016 / 5783
The nucleic acid sequences comprising the aggregates of rfb genes which can be inserted into the host cells of the present invention are known in the art. In a specific embodiment, the rfb gene aggregate inserted into a host cell of the present invention is an rfb gene aggregate from E. coli, for example an rfb gene aggregate from E. coli. coli from any O / O-antigen known in the art, e.g. 01, 02, 03, 04, 05, 06, 07, 08, 09, OlO, 011, 012, 013, 014, 015 , 016, 017, 018, 019, 020, 021, 022, 023, 024, 025, 026, 027, 028, 029, 030, 032, 033, 034, 035, 036, 037, 038, 039, 040, 041 , 042, 043, 044, 045, 046, 048, 049, 050, 051, 052, 053, 054, 055, 056, 057, 058, 059, 060, 061, 062, 063, 064, 065, 066, 068 , 069, 070, 071, 073, 074, 075, 076, 077, 078, 079, 080, 081, 082, 083, 084, 085, 086, 087, 088, 089, 090, 091, 092, 093, 095 , 096, 097, 098, 099, OlOO, O101, 0102, 0103, 0104, 0105, 0106, 0107, 0108, 0109, OllO, YES, 0112, 0113, 0114, 0115, 0116, 0117, 0118, 0119, 0120 , 0121, 0123, 0124, 0125, 0126, 0127, 0128, 0129, 0130, 0131, 0132, 0133, 0134, 0135, 0136, 0137, 0138, 0139, 0140, 0141, 0142, 0143, 0144, 0145, 0146 , 0147, 0148, 0149, 0150, 0151, 0152, 0153, 0154, 0155, 0156, 0157, 0158, 0159, 0160, 0161, 0162, 0163, 0164, 0165, 0166, 0167, 0168, 0169, 0170, 0171 , 0172, 0173, 0174, 0175, 017 6, 0177, 0178, 0179, 0180, 0181, 0182, 0183, 0184, 0185, 0186, or 0187, and the associated sub-serotypes. In another specific embodiment, the rfb gene aggregate inserted into a host cell of the present invention is an rfb gene aggregate from a strain of Pseudomonas (e.g. a strain of
BE2016 / 5783
P. aeruginosa), of a strain of Salmonella (for example a strain of S. enterica), of a strain of Yersinia, of a strain of Klebsiella pneumoniae, of a strain of Francisella (for example F. tularensis) , a strain of Acinetobacter baumannii, a strain of Burkholderia, or a strain of Shigella.
Nucleic acid sequences comprising the aggregates of capsular polysaccharide genes which can be inserted into the host cells of the present invention are known in the art. In a specific embodiment, the capsular polysaccharide gene aggregate inserted into a host cell of the present invention is a capsular polysaccharide gene aggregate from an E. coli strain. coli, a strain of Streptococcus (e.g. S. pneumoniae, S. pyrogenes, S. agalacticae), a strain of Staphylococcus (e.g. S. aureus), or a strain of Burkholderia (e.g. B. mallei, B. pseudomallei, B. thailandensis). Methods for making such host cells capable of producing conjugates are described in WO 06/119 987, WO 09/104 074, WO 11/62 615, WO 11/138 361, WO 14/57 109 , WO 14/72 405.
In a specific embodiment, the present invention relates to a modified prokaryotic host cell comprising nucleic acids encoding enzymes capable of producing a bioconjugate comprising a saccharide antigen, said host cell comprising an rfb aggregate originating from Pseudomonas or a glycosyltransferase derived from an rfb aggregate from Pseudomonas. In a specific embodiment,
BE2016 / 5783 said rfb aggregate from Pseudomonas, or a glycosyltranferase derived from an rfb aggregate from Pseudomonas, is integrated into the genome of said host cell. In another specific embodiment, said rfb aggregate from Pseudomonas, or a glycosyltranferase derived from an rfb aggregate from Pseudomonas, is an rfb aggregate from Pseudomonas aeruginosa.
In another specific embodiment, said host cell comprises a nucleic acid coding for an oligosaccharyl transferase (for example pglB from Camphylobacter jejuni). In another specific embodiment, said nucleic acid coding for an oligosaccharyl transferase (for example Camglylobacter jejuni pglB) is integrated into the genome of the host cell. In a specific embodiment, said host cell comprises a nucleic acid encoding a carrier protein. In another specific embodiment, the host cell is E. coli.
In another specific embodiment, the present invention relates to a modified prokaryotic host cell comprising (i) an rfb aggregate from Pseudomonas, said rfb aggregate being integrated into the genome of said host cell; (ii) a nucleic acid coding for an oligosaccharyl transferase (for example pglB from Camphylobacter jejuni), said nucleic acid coding for an oligosaccharyl transferase being integrated into the genome of the host cell; and (iii) a carrier protein, said carrier protein being either carried by a plasmid or integrated into the genome of said host cell. In another embodiment
BE2016 / 5783 specific, said rfb aggregate from Pseudomonas is an rfb aggregate from Pseudomonas aeruginosa. In another specific embodiment, the host cell is E. coli.
In another specific embodiment, the present invention relates to a modified prokaryotic host cell comprising (i) a glycosyltranferase derived from an rfb aggregate originating from Pseudomonas, said glycosyltransferase being integrated into the genome of said host cell; (ii) a nucleic acid coding for an oligosaccharyl transferase (for example Camphylobacter jejuni pglB}, said nucleic acid coding for an oligosaccharyl transferase being integrated into the genome of the host cell; and (iii) a carrier protein, said carrier protein being either carried by a plasmid or integrated into the genome of said host cell In another specific embodiment, said glycosyltranferase derived from an rfb aggregate originating from Pseudomonas is an rfb aggregate originating from Pseudomonas aeruginosa. a specific embodiment, the host cell is E. coli.
In a specific embodiment, the rfb aggregate from Pseudomonas, or a glycosyltranferase derived from an rfb aggregate from Pseudomonas, is an rfb aggregate or glycosyltransferase from Pseudomonas aeruginosa. In another specific embodiment, said rfb aggregate originating from Pseudomonas, or a glycosyltranferase derived from an rfb aggregate originating from Pseudomonas, is an rfb aggregate or a glycosyltransferase originating from serotypes 01, 02, 03, 04, 05, 06, 07, 08, 09, 010, 011, 012, 013, 014,
BE2016 / 5783
015, 016, 017, 018, 019, or 020 of Pseudomonas aeruginosa In another specific embodiment, said rfb aggregate from Pseudomonas aeruginosa is the rfb aggregate of any of the serotypes described by Knirel et al., 2006 , Journal of Endotoxin Research 12 (6): 324-336, said publication being incorporated herein by reference in its entirety. In a specific embodiment, said rfb aggregate originating from Pseudomonas, or a glycosyltranferase derived from an rfb aggregate originating from Pseudomonas, is an rfb aggregate or glycosyltransferase originating from the serotype 06 PAK strain of Pseudomonas aeruginosa. In a specific embodiment, said rfb aggregate originating from Pseudomonas, or a glycosyltranferase derived from an rfb aggregate originating from Pseudomonas, is an rfb aggregate or a glycosyltransferase originating from serotype 011 of Pseudomonas aeruginosa, for example of the strain PA103 of Pseudomonas aeruginosa (see for example Genbank Accession No. KF364633.1). In a specific embodiment, the genes encoding a formyltransferase enzyme (GenBank: EOT23134.1; NCBI protein ID: PAK_01412; SEQ ID NO: 2) and a wzy polymerase (GenBank: EOT19368.1; NCBI protein ID: PAK_01823; SEQ ID NO: 3) are introduced (by means of a plasmid or by integration for example) in addition to said rfb aggregate originating from the strain of serotype 06 PAK of Pseudomonas aeruginosa in order to introduce a functional extension there.
In a specific embodiment, a modified prokaryotic host cell according to the present invention comprises a nucleic acid encoding a
BE2016 / 5783 formyltransferase. In another specific embodiment, said formyltransferase is formyltransferase presented in SEQ ID NO: 65, or a homolog thereof. In another specific embodiment, said formyltransferase is incorporated (inserted for example into the genome or expressed via a plasmid) in said host cell as part of an rfb aggregate of Pseudomonas, said rfb aggregate of Pseudomonas having been modified to include formyltransferase. In another specific embodiment, said Pseudomonas rfb aggregate is an rfb aggregate of Pseudomonas aeruginosa serotype 06.
In another specific embodiment, a modified prokaryotic host cell according to the present invention comprises a nucleic acid encoding a wzy polymerase. In another specific embodiment, said wzy polymerase is the wzy polymerase presented in SEQ ID NO: 66, or a homolog thereof. In another specific embodiment, said wzy polymerase is incorporated (inserted for example into the genome or expressed via a plasmid) in said host cell as part of an rfb aggregate of Pseudomonas, said rfb aggregate of Pseudomonas having been modified to include wzy polymerase. In another specific embodiment, said Pseudomonas rfb aggregate is an rfb aggregate of Pseudomonas aeruginosa serotype 06.
In another specific embodiment, a modified prokaryotic host cell according to the present invention comprises (i) a nucleic acid encoding
BE2016 / 5783 a formyltransferase and (ii) a nucleic acid encoding a wzy polymerase. In a specific embodiment, said formyltransferase is the formyltransferase presented in SEQ ID NO: 65, or a homolog thereof having at least 85%, 90% or 95% identity with SEQ ID NO: 65. In another specific embodiment, said wzy polymerase is the wzy polymerase presented in SEQ ID NO: 66, or a homolog thereof having at least 85%, 90% or 95% identity with SEQ ID NO : 66. In a specific embodiment, said formyltransferase and said wzy polymerase are incorporated (inserted for example into the genome or expressed via a plasmid) in said host cell as part of an rfb aggregate of Pseudomonas , said Pseudomonas rfb aggregate having been modified to include formyltransf erase and wzy polymerase. In another specific embodiment, said Pseudomonas rfb aggregate is an rfb aggregate of Pseudomonas aeruginosa serotype 06.
The nucleic acids coding for formyltransferases and the nucleic acids coding for wzy polymerases which are used to generate the rfb aggregates of modified Pseudomonas, such as for example the rfb aggregates of serotype 06 of Pseudomonas aeruginosa, can be inserted into the rfb aggregates to multiple positions and in multiple orientations.
In a specific embodiment, the gene coding for said formyltransferase and / or the gene coding for said wzy polymerase is / are inserted downstream of the genes of the pseudomonas rfb aggregate, by
BE2016 / 5783 example the rfb aggregate of serotype 06 of Pseudomonas aeruginosa. In a specific embodiment, the gene coding for said formyltransferase and / or the gene coding for said wzy polymerase is / are inserted downstream of the wbpM gene of the rfb aggregate of Pseudomonas aeruginosa serotype 06.
In a specific embodiment, the gene coding for said formyltransferase and / or the gene coding for said wzy polymerase is / are inserted upstream of the genes of the rfb aggregate of Pseudomonas, for example the rfb aggregate of the serotype 06 of Pseudomonas aeruginosa. In a specific embodiment, the gene coding for said formyltransferase and / or the gene coding for said wzy polymerase is / are inserted downstream of the wzz gene of the rfb aggregate of serotype 06 of Pseudomonas aeruginosa.
In a specific embodiment, the gene coding for said formyltransferase and / or the gene coding for said wzy polymerase is / are inserted in a clockwise direction with respect to the genes of the rfb aggregate of Pseudomonas , for example the rfb aggregate of serotype 06 of Pseudomonas aeruginosa.
In a specific embodiment, the gene coding for said formyltransferase and / or the gene coding for said wzy polymerase is / are inserted anticlockwise with respect to the genes of the rfb aggregate of Pseudomonas, for example the rfb aggregate of Pseudomonas aeruginosa serotype 06.
In a specific embodiment, the present invention relates to a modified prokaryotic host cell.
BE2016 / 5783 comprising nucleic acids encoding enzymes capable of producing a bioconjugate comprising a 06 antigen of Pseudomonas. In a specific embodiment, said host cell comprises the rfb aggregate of Pseudomonas aeruginosa serotype 06, a nucleic acid coding for a wzy polymerase, and a formyltransferase. In a specific embodiment, the wzy polymerase is the wzy polymerase from P. aeruginosa (SEQ ID NO: 66), or a homologue thereof (the wzy polymerase from the PAK or LESB58 strain of Pseudomonas aeruginosa for example). In another specific embodiment, the formyltransferase is formyltransferase from P. aeruginosa 06 (SEQ ID NO: 65), or a homolog thereof (formyltransferase from the PAK or LESB58 strain of Pseudomonas aeruginosa for example). In certain embodiments, at least one nucleic acid encoding the rfb aggregate, the wzy polymerase, and / or the formyltransferase is integrated into the genome of the host cell, for example using a method of the present invention. In a specific embodiment, each of the nucleic acids coding for the rfb aggregate, the wzy polymerase and the formyltransferase, is integrated into the genome of the host cell, for example using a method of the present invention. In certain embodiments, the host cell further comprises a nucleic acid coding for an oligosaccharyl transferase (for example Camglylobacter jejuni pglB), said nucleic acid coding for an oligosaccharyl transferase being either carried by a plasmid or integrated into the cell genome
BE2016 / 5783 host; and a nucleic acid coding for a carrier protein, said nucleic acid coding for said carrier protein being either carried by a plasmid or integrated into the genome of the host cell. In a specific embodiment, said nucleic acid encoding said oligosaccharyl transferase is integrated into the genome of the host cell.
Genetic background
Examples of host cells which can be used to generate the modified host cells according to the present invention include, but are not limited to, Escherichia species, Shigella species, Klebsiella species, Xhantomonas species, species of Salmonella, Yersinia species, Lactococcus species, Lactobacillus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Staphylococcus species, Bacillus species, and Clostridium species. In a specific embodiment, the host cell according to the invention is E. coli.
In certain embodiments, the genetic context of the host cell is for example modified by a deletion of at least one gene. Examples of genes whose deletion can be carried out in host cells (and in some cases, which can be replaced by other desired nucleic acid sequences) include the genes of host cells involved in the biosynthesis of glycolipids, such as waaL (see for example Feldman et al., 2005, PNAS USA 102: 3016-3021),
BE2016 / 5783 the O-antigen aggregate (rfb or wb), aggregates of antigens common in enterobacteria (wec), the aggregate allowing the synthesis of the lipid A nucleus (waa), and the aggregates allowing modification of the prophage O-antigen, such as the gtrABS aggregate. In a specific embodiment, the host cells according to the present invention are modified so that they produce no other antigen than those desired, among the O-antigens of Pseudomonas for example. In a specific embodiment, the functional deletion or deactivation is carried out of at least one gene from the waaL gene, the gtrA gene, the gtrB gene, the gtrS gene, or from at least one of the aggregate genes wec, or at least one of the genes of the rfb gene aggregate present in the genome of a prokaryotic host cell according to the invention. In one embodiment, a host cell used according to the invention is E. coli, in which the deletion or functional inactivation of the waaL gene, of the gtrA gene, of the gtrB gene, of the gtrS gene present in the genome is carried out. the host cell. In another embodiment, a host cell used according to the invention is E. coli, in which the deletion or functional inactivation of the waaL gene and of the gtrS gene present in the genome of the host cell is carried out. In another embodiment, a host cell used according to the invention is E. coli, in which the deletion or functional inactivation of the waaL gene and of genes of the wec aggregate present in the genome of the host cell is carried out .
Carrier proteins
BE2016 / 5783
Any carrier protein suitable for use in the production of conjugate vaccines (for example bioconjugates which can be used in vaccines) can be used according to the invention: nucleic acids coding for the carrier protein can for example be introduced into a host according to the invention, in order to produce a bioconjugate comprising a carrier protein linked to a Pseudomonas antigen. Examples of carrier proteins include, but are not limited to, detoxified exotoxin A from P. aeruginosa (EPA; see, for example, Ihssen, et al., (2010) Microbial cell factories 9, 61), CRM197, the protein maltose binding agent (MBP), diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of S. aureus, agglutination factor A, agglutination factor B, FimH of E. coli, the FimC of E. coli, the heat-labile enterotoxin from E. coli, detoxified variants of E. coli heat-labile enterotoxin. coli, cholera toxin B (CTB), cholera toxin, detoxified variants of cholera toxin, the Sat protein of E. coli, the cloned DNA domain of the Sat protein of E. coli, Streptococcus pneumoniae pneumolysin and its detoxified variants, C. jejuni AcrA, Pseudomonas PcrV protein, and C. jejuni natural glycoproteins. The PcrV protein is used in many embodiments of the invention.
In specific embodiments, the carrier proteins expressed by the modified host cells according to the invention are expressed from a nucleic acid which has been integrated into the genome of the modified host cell. This means that an acid
BE2016 / 5783 nucleic acid encoding a carrier protein has been integrated into the genome of the host cell. In certain embodiments, the carrier proteins expressed by the modified host cells according to the invention are expressed from a plasmid which has been introduced into the modified host cell.
In certain embodiments, the carrier proteins for producing the bioconjugates of the present invention are modified, for example modified so that the protein is less toxic and / or less subject to glycosylations. In a specific embodiment, the carrier proteins used to produce the bioconjugates according to the invention are modified so as to maximize the number of glycosylation sites on the carrier proteins in order to allow the administration of lower concentrations of the protein, for example in an immunogenic composition, in its form of bioconjugate.
In certain embodiments, the carrier proteins according to the invention are modified to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional glycosylation sites compared to those normally associated with the carrier protein (relative to the number of glycosylation sites associated with the carrier protein in its native / natural state for example, or its “wild type” state). In specific embodiments, the introduction of the glycosylation sites is carried out by means of the insertion of consensus glycosylation sequences (such as, for example, Asn-X-Ser (Thr), X representing any amino acid except Pro; or Asp (Glu) -X-Asn-Z-Ser (Thr),
BE2016 / 5783
X and Z being independently chosen from all natural amino acids, except Pro (see document WO 2006/119987)) at any location in the primary structure of the protein. The introduction of such glycosylation sites can be carried out by adding for example new amino acids to the primary structure of the protein (which amounts to adding all or part of the glycosylation sites), or by mutating the amino acids existing in the protein, in order to generate the glycosylation sites (in other words, the amino acids are not added to the protein, but amino acids chosen from those of the protein undergo a mutation, in order to form glycosylation sites). Those skilled in the art will recognize that the amino acid sequence of a protein can be easily modified by approaches known in the art, such as, for example, recombinant approaches which include modifying the nucleic acid sequence encoding protein. In specific embodiments, the consensus glycosylation sequences are introduced into specific regions of the carrier protein, such as, for example, the surface structures of the protein, at the C-terminal or N-terminal ends of the protein, and / or in the loops which are stabilized by disulfide bridges at the base of proteins. In certain embodiments, the conventional consensus glycosylation sequence comprising 5 amino acids can be extended using lysine residues, to make glycosylation more efficient, the inserted consensus sequence then being able to code for 5, 6 or 7 amino acids.
BE2016 / 5783 which can be inserted or which replace the amino acids of the acceptor protein.
In some embodiments, the carrier proteins used to produce the bioconjugates of the present invention include a "tag", that is, an amino acid sequence for isolating and / or identifying the carrier protein. For example, the addition of a marker to a carrier protein of the present invention may prove useful in the purification of this protein, and therefore in the purification of conjugate vaccines comprising the labeled carrier protein. . Examples of markers that can be used according to the invention include, but are not limited to, histidine (His-tag) markers (e.g., hexahistidine marker, or 6xHis-tag marker), FLAG marker, and HA markers . In certain embodiments, the markers used according to the invention are labile, and can for example be removed by chemical agents or by enzymatic means, when they are no longer useful, for example once the protein has been purified .
In certain embodiments, the carrier proteins of the present invention include a signal sequence which directs the carrier protein to the periplasm of the host cell which expresses the carrier protein. In a specific embodiment, the signal sequence comes from the DsbA of E. coli, outer membrane porin A (OmpA) from E. coli, maltose binding protein (MalE) from E. coli, pectin lyase (PelB) from Erwinia carotovorans, from Flgl , from
BE2016 / 5783
NikA, or endoxylanase (XynA) from Bacillus species, heat-labile enterotoxin LTIIb from E. coli, endoxylanase XynA from Bacillus, or flagellin (Flgl) from E. coli.
Glycosylation machinery
Oligosaccharyl-transferases
The oligosaccharyl transferases transfer lipid-linked oligosaccharides onto the asparagine residues of nascent polypeptide chains which comprise a consensus motif of N-glycoxylation, such as for example Asn-X-Ser (Thr), X representing any amino acid except Pro; or Asp (Glu) -X-Asn-ZSer (Thr), X and Z being chosen independently from all natural amino acids, except Pro (see document WO 2006/119 987). See, for example, documents WO 2003/074 687 and WO 2006/119 987, said publications being incorporated herein by reference in their entirety.
In certain embodiments, the host cells according to the present invention comprise a nucleic acid encoding an oligosaccharyl transferase. The nucleic acid encoding an oligosaccharyltransferase may be native to the host cell, or may be introduced into the host cell by approaches using genetics, as described above. The oligosaccharyl transferase can be from any source known in the art. In a specific embodiment, the oligosaccharyl transferase is an oligosaccharyl transferase from Campylobacter. In another embodiment
BE2016 / 5783 specific, oligosaccharyl-transferase is an oligosaccharyl-transferase from Campylobacter jejuni, (i.e. pglB, see e.g. Wacker et al., 2002, Science 298: 1790-1793; see also NCBI Gene ID: 3,231,775, UniProt Accession No. 086154). In another specific embodiment, the oligosaccharyl transferase is an oligosaccharyltransferase from Campylobacter lari, (see for example NCBI Gene ID: 7,410,986).
In a specific embodiment, the modified host cells according to the present invention comprise a nucleic acid sequence coding for an oligosaccharyl transferase, said nucleic acid sequence coding for an oligosaccharyl transferase being integrated into the genome of the host cell. .
Auxiliary enzymes
In certain embodiments, nucleic acids encoding at least one helper enzyme are introduced into the host cells modified according to the invention. Such nucleic acids encoding at least one helper enzyme can either be carried by a plasmid or integrated into the genome of the host cell. Examples of helper enzymes include, but are not limited to, epimerases, branching enzymes, modification, amidation, chain length regulation, acetylation, formylation, polymerization.
The nucleic acid sequences encoding epimerases which can be inserted into the host cells of the present invention are known in the art. In
BE2016 / 5783 certain embodiments, the epimerase inserted into the host cell according to the invention is an epimerase described in international patent publication No. WO 2011/062 615, said publication being incorporated herein by reference in its entirety. In a specific embodiment, the epimerase is the epimerase for which the gene Z3206 of strain 0157 of E. coli. See for example document WO 2011/062 615 and Rush et al., 2009, The Journal of Biological Chemistry 285: 1671-1680, which is incorporated herein by reference in its entirety. In a specific embodiment, the modified host cells according to the present invention comprise a nucleic acid sequence coding for an epimerase, said nucleic acid sequence coding for an epimerase being integrated into the genome of the host cell.
In some embodiments, a nucleic acid sequence encoding a formyltransferase is inserted into, or expressed by, a host cell according to the invention. Formyltransferases are enzymes that catalyze the transfer of formyl groups to an acceptor molecule. In a specific embodiment, a nucleic acid sequence coding for the formyltransferase of Pseudomonas aeruginosa 06 (fmtO6) (SEQ ID NO: 65), or a homologue thereof, is inserted into a host cell according to the invention or expressed by it. In another specific embodiment, a nucleic acid sequence encoding a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or homology with
BE2016 / 5783 SEQ ID NO: 65, is inserted into the host cells according to the invention or expressed by them.
Certain formyltransferases involved in the biosynthesis of polysaccharides are known, and can be inserted into the host cells according to the invention or expressed by them. For example, vioF is an enzyme from serotype 030 of P. alcalifaciens, which is 48% identical with formyltransferase from Francisella tularensis (Nagaraja et al. 2005). It converts dTDP-D-Qui4N into dTDP-D-Qui4NFo, and is involved in the biosynthesis of O-antigen (Liu et al. 2012, Glycobiology 22 (9): 1236-1244). Another formyltransferase involved in the biosynthesis of polysaccharides is ArnA (from, for example, E. coli), a bifunctional enzyme in which the N-terminal domain converts UDP-Ara4N to UDP-AraNFo, while the C domain -terminal is involved in the oxidative decarboxylation of UDP-glucuronic acid. The two enzymatic activities are necessary for the L-Ara4N modification of lipid A and the resistance to polymyxin (Breazeale et al., 2005, The Journal of Biological Chemistry 280 (14): 14154-14167). Another formyltransferase involved in the biosynthesis of polysaccharides is wekD, an enzyme originating from serotype 0119 of E. coli, involved in the biosynthesis of TDP-DRhaNAc3NFo (Anderson et al., 1992, Carbohydr Res 237: 249-62).
In addition, the domains linked to formyltransferase activity have been characterized. The domain called FMT_core is present in the majority of formyltransferases.
BE2016 / 5783
Mention may be made, for example, of methionyl-tRNAformyltränsterase, formyltransferase, phosphoribosylglycinamide1, UDP-glucuronic acid decarboxy1ase / UDP-4-amino-4-deoxy-L-arabinoseformyltransferase, vioF from Providencia alcalifaciens 030, and EL coli. The formyltransferases mentioned above use FTHF (N-10-formyltetrahydrofolate) as the formyl donor. Formate-producing enzymes using FTHF (10-formyltetrahydrofolate) as a substrate also contain this domain. In addition, AICARFT is present in phosphoribosylaminoimidazolecarboxamideformyltransferase / IMP-cyclohydrolase and FDH_GDH is present in phosphoribosylglycinamideformyltransferase 2.
In some embodiments, a nucleic acid sequence encoding an O-antigen polymerase (wzy gene) is inserted into or expressed by host cells according to the invention. O-antigen polymerases are multiple overlapping transmembrane proteins. They use repeat units of O-antigen linked to undecaprenylpyrophosphate as substrates to generate a linear polymer composed of repeat units. O-antigen (wzy) polymerases are present in Gram-negative bacteria that synthesize O-antigen polymers through a mechanism involving wzy.
In a specific embodiment, a nucleic acid sequence encoding the wzy polymerase
BE2016 / 5783 of Pseudomonas aeruginosa (SEQ ID NO: 66), or a homologue thereof (the wzy polymerase of the PAK strain or LESB58 of Pseudomonas aeruginosa for example), is inserted into the host cells according to the invention or expressed by these. Among the bacteria known to contain wzy polymerases, there are for example Escherichia coli, Pseudomonas aeruginosa, Shigella flexneri and Salmonella typhimurium. In another specific embodiment, a nucleic acid sequence encoding a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or homology with SEQ ID NO: 66, is inserted into the host cells according to the invention or expressed by them.
Number of copies of a gene
In certain embodiments, the number of copies of one or more gene (s) integrated into a modified host cell according to the invention is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 In a specific embodiment, the number of copies of one or more gene (s) integrated into a modified host cell according to the invention is 1 or 2.
Benefits
The host cells modified according to the present invention are particular in terms of commercial importance to perform because they on a large scale allow, as well as by and of interest, the bioconjugate fermentation, including of saccharides, for example the antigens of Pseudomonas which can be used as therapeutic compounds (e.g. in
BE2016 / 5783 immunogenic compositions, vaccines), the risks of which are reduced due to the greater stability of the DNA inserted into the chromosome and consequently of the expression of the DNA of interest during fermentation. The host cells modified according to the invention have advantages over host cells which rely on the expression, via a plasmid, of the nucleic acids necessary for the production of the bioconjugates of the invention, since, among other things, selection by antibiotic at During fermentation is no longer necessary once the heterologous DNA is inserted into the genome of the host cell. This means that when the inserted DNA is inserted into the chromosome, it does not need to be selected because it spreads during the replication of the host genome. In addition, a known drawback of systems carried by a plasmid is the risk, with each generation (that is to say with each replication cycle of the host cell), of losing the plasmid. This loss of the plasmid is due to the sometimes inappropriate distribution of the plasmids in the daughter cells during the cell separation step during cell division. On a larger scale, bacterial cell cultures require more duplication to achieve high cell densities than for small scale fermentations. Thus, the increase in cell stability and in the expression of the inserted DNA leads to better yields of products, which is clearly advantageous. In addition, cell stability is a criterion of acceptability of the process when it is rated by
BE2016 / 5783 regulatory authorities, whereas, for various reasons, antibiotic selection is generally not desired at the time of fermentation, for example, the antibiotics residual in the final therapeutic compounds present the risk of causing allergic reactions, and antibiotics can promote antibiotic resistance (for example by gene transfer or by the selection of resistant pathogens).
The present invention relates to modified host cells which can be used to make bioconjugates, comprising saccharide antigens which can be used as therapeutic compounds (e.g. in immunogenic compositions, vaccines), in which certain genetic elements necessary to cause the production of Bioconjugates are stably integrated without the genome of the host cell.
Therefore, the host cell may contain a reduced number of plasmids, a single plasmid or no plasmid. In certain embodiments, the presence of a single plasmid can allow greater flexibility of the productive strain and make it possible to modify the nature of the conjugation (means the saccharide or the carrier protein contained), which easily increases the flexibility of the productive strain.
In general, reducing the use of plasmids leads to a productive strain more suitable for use in the production of medicinal products. A disadvantage linked to the presence of genetic material
BE2016 / 5783 essential on plasmids is that it requires selection pressure to maintain the episomal elements in the host cell. Selection pressure necessitates the use of antibiotics, which is undesirable for the production of medicinal products, due to the risk of allergic reaction against antibiotics for example, and higher manufacturing costs. In addition, the selection pressure is often not complete, which leads to inhomogeneous bacterial cultures in which certain clones have lost the plasmid, and therefore do not produce the bioconjugate. The host cells according to the present invention are therefore capable of producing a safer product, which can be obtained in high yields.
Bioconj ugués
The modified host cells according to the present invention can be used to produce bioconjugates comprising a saccharide antigen, for example a Pseudomonas antigen linked to a carrier protein. The methods for producing bioconjugates using host cells are known in the art, see for example the documents WO 2003/074 687 and WO 2006/119 987. The bioconjugates according to the present invention have advantageous properties compared to antigen-carrier protein chemical conjugates, in that less chemicals are required for their manufacture and in that the production of the final product is more homogeneous.
BE2016 / 5783
In a specific embodiment, the present invention relates to a bioconjugate comprising a carrier protein linked to a Pseudomonas antigen. In a specific embodiment, said Pseudomonas antigen is a Pseudomonas aeruginosa O-antigen. In a specific embodiment, the present invention relates to a bioconjugate comprising an O-antigen of P. aeruginosa and a carrier protein, said carrier protein being EPA, PcrV (also known under the name of LcrV, EspA, SseB) , PopB (YopB, YopD, FliC), or OprF, Oprl. The carrier proteins used in the exemplary embodiments use 1ΈΡΑ and PcrV.
In a specific embodiment, the present invention relates to a bioconjugate comprising a carrier protein linked to an O-antigen of Pseudomonas aeruginosa, said O-antigen of Pseudomonas aeruginosa being an O-antigen originating from serotypes 01, 02, 03, 04, 05, 06, 07, 08, 09, OlO, 011, 012, 013, 014, 015,
016, 017, 018, 019, or 020 of Pseudomonas aeruginosa.
In a specific embodiment, the present invention relates to a bioconjugate comprising a carrier protein linked to an O-antigen of Pseudomonas aeruginosa, said O-antigen of Pseudomonas aeruginosa being one of the serotypes described by Knirel et al., 2006, Journal of Endotoxin Research 12 (6): 324-336, said publication being incorporated herein by reference in its entirety.
In a specific embodiment, the present invention relates to a bioconjugate comprising a carrier protein linked to a Pseudomonas O-antigen
BE2016 / 5783 aeruginosa, said O-antigen of Pseudomonas aeruginosa being an O-antigen originating from serotype 06 of Pseudomonas aeruginosa.
In a specific embodiment, the present invention relates to a bioconjugate comprising a carrier protein linked to an O-antigen of Pseudomonas aeruginosa, said O-antigen of Pseudomonas aeruginosa being an O-antigen originating from serotype 011 of Pseudomonas aeruginosa. In a specific embodiment, said O-antigen originating from Pseudomonas aeruginosa serotype 011 originating from Pseudomonas aeruginosa strain PA103 (see for example Genbank Accession No. KF364633.1).
The bioconjugates according to the invention can be purified by any method known in the art intended for the purification of a protein, by chromatography for example (such as for example ion exchange chromatography, ion exchange anions, affinity and steric exclusion), or by centrifugation, differential solubility, or by any other standard protein purification technique. See for example Saraswat et al., 2013, Biomed. Res. Int. ID # 312 709 (p. 1-18); see also the methods described in document WO 2009/104 074. In addition, the bioconjugates can be fused to sequences of heterologous polypeptides according to the invention, or known elsewhere in the art, in order to facilitate their purification. The conditions actually used to purify a particular bioconjugate will depend in part on the synthesis strategy, and on factors such as the net charge, the hydrophobic character, and / or the
BE2016 / 5783 hydrophilic nature of the bioconjugate, and will be obvious to those skilled in the art.
Analytical methods
Various methods can be used to analyze the structural compositions and the lengths of the saccharide chains of the bioconjugates of the present invention.
In one embodiment, the glycans can be analyzed by hydrazinolysis. First, the polysaccharides are released from their carrier proteins upon incubation with hydrazine, according to the manufacturer's instructions (Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK). The nucleophilic hydrazine attacks the glycosidic bond between the polysaccharide and the carrier protein, and allows the release of the glycans which are linked to it. The N-acetyl groups are lost during this treatment, and must be replaced by a new N-acetylation. The free glycans are purified on carbon columns, and then labeled at their reducing end using the 2-amino-benzamide fluorophore. See Bigge JC, Patel TP, Bruce JA, Goulding PN, Charles SM, Parekh RB: Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Anal Biochem 1995, 230 (2): 229-238. The labeled polysaccharides are separated on a GlycoSep-N column (GL Sciences) according to the HPLC protocol published by Royle et al. See Royle L, Mattu TS, Hart E, Langridge JI, Merry AH, Murphy N, Harvey DJ, Dwek RA, Rudd PM: An analytical and structural database provides
BE2016 / 5783 a strategy for sequencing O-glycans from microgram quantities of glycoproteins. Anal Biochem 2002, 304 (1): 70-90. The fluorescence chromatogram obtained indicates the length of the polysaccharides and the number of repeat units. Structural information can be gathered by collecting each of the peaks individually and then performing an SM / SM analysis. The monosaccharide composition and the sequence of the repeat unit can thus be confirmed, and it is also possible to identify a homogeneity of the composition of the polysaccharides.
In another embodiment, the glycans and the bioconjugates are analyzed by SDS-PAGE or capillary gel electrophoresis. The length of the polymers constituting the glycans of the O-antigen is defined by the number of repeating units assembled in a linear fashion. This means that the typical ladder pattern is the consequence of the different numbers of repeating units making up the glycan. Thus, two bands next to each other, obtained by SDS-PAGE or by other separation techniques according to size, differ by only one repetition unit. It is these distinct differences which are exploited during the analysis of the size of glycans in glycoproteins: The non-glycosylated carrier protein and the bioconjugate carrying different lengths of polymer chain separate according to their respective electrophoretic mobilities. The first number of detectable repeat units (ni) and the average number of repeat units (nm) present are measured.
BE2016 / 5783 on a bioconjugate. These parameters can be used to demonstrate the homogeneity or stability of the polysaccharides from one batch to another.
In another embodiment, the size of the complete bioconjugates is measured by high mass MS and steric exclusion HPLC.
In another embodiment, an anthrone sulfuric acid assay can be used to measure the polysaccharide yields. See Leyva A, Quintana A, Sanchez M, Rodriguez EN, Cremata J, Sanchez JC: Rapid and sensitive anthrone-sulfuric acid assay in microplate format to quantify carbohydrate in biopharmaceutical products: method development and validation. Biologicals: journal of the International Association of Biological Standardization 2008, 36 (2): 134-141. In another embodiment, an α-methylpentose assay can be used to measure the yields of polysaccharides. See for example Dische et al., J Biol Chem. 1948 Sep; 175 (2): 595-603.
Modification of the use of the glycosylation site
In order to show that the use of a site of a particular protein is modified in a multiple plasmid system contrary to what is observed in an inserted system, the use of the glycosylation site must be quantified. This can be done by the methods below.
CL-SM / SM of glycopeptides: the bioconjugates are digested by protease (s), and the peptides are separated by an appropriate chromatography method
BE2016 / 5783 (C18, HILIC hydrophilic interaction HPLC, GlycoSepN columns, HPLC-ES, HPLC-EA), and the different peptides are identified by SM / SM. This method can be used with or without prior shortening of the saccharide chain by chemical (Smith degradation) or enzymatic methods. The quantification of the glycopeptide peaks by UV detection at 215 and 280 nm allows the determination of the relative use of the glycosylation sites.
HPLC of steric exclusion: the greater use of glycosylation sites is reflected by a shorter elution time on a column of HPLC-ES.
Homogeneity
The homogeneity of the bioconjugates (in other words, the homogeneity of the saccharide residues which are linked to it) can be analyzed using methods which measure the length of the glycans and the hydrodynamic radius.
Other Clinical Applications / Potential Practices
The integrated strains can lead to a better yield of bioconjugates, the drawbacks linked to antibiotic selection being reduced compared to systems with three plasmids. In addition, less proteolytic degradation is observed, the cells undergoing less metabolic load.
The integrated strains produce bioconjugates whose distributions of polysaccharides are shorter and less spread out. Thus, bioconjugates are easier to characterize and are better defined. In addition, insertion can reduce the extent of stress
BE2016 / 5783 periplasmic undergone by the cells, which can result in a lesser proteolysis of the product during the fermentation process, the disadvantages linked to the selection by antibiotic being reduced compared to the systems with three plasmids.
Protein glycosylation systems require the presence of three recombinants in the productive host: DNA expressing a carrier protein, DNA expressing an oligosaccharyl transferase, and DNA expressing a polysaccharide. In the prior art, these three elements of bacterial production systems are contained in plasmids. Thus, manufacturing risks becoming unstable due to the loss of plasmids, in particular because the antibiotics used to maintain the presence of the plasmids must not be present during the fermentation of products labeled BPF. The inserted strains containing one plasmid less, they prove more stable after many generations. This means that the fermentations can be carried out on a larger scale, and with longer incubation times (higher number of generations). In addition, the absence of the use of antibiotics for selection makes the product safer, due to the absence of residual antibiotics which can cause allergic reactions in sensitive patients. See COMMITTEE WE, BIOLOGICAL O,
STANDARDIZATION: WHO Technical Report Sériés 941. In: Fifty-sixth Report. Edited by Organization WH. Geneva: World Health Organization; 2007.
BE2016 / 5783
The inserted strains are genetically more stable, the insertion into the chromosomes being fixed, which leads to greater reproducibility of the desired protein products during the production process, for example during the culture of host cells comprising an inserted heterologous DNA.
Analytical methods for estimating benefit
Yield. The yield is measured as the quantity of bacterial carbohydrates derived from a liter of productive bacterial culture medium grown in a bioreactor under controlled and optimized conditions Following the purification of the bioconjugate, the carbohydrate yields can be measured directly, either by an anthrone assay, or by an ELISA assay using carbohydrate-specific antisera. Indirect measurements are possible using the amount of protein (measured by the well known BCA, Lowry or Bradford assays) and the length and structure of the glycans in order to calculate a theoretical amount of carbohydrate per gram of protein. In addition, the yield can also be measured by drying the glycoprotein preparation from a volatile buffer, and measuring the mass using a balance.
Homogeneity. The term homogeneity designates the variability in the length of the glycans and possibly the number of glycosylation sites. The methods listed above can be used for this purpose. The HPLC-ES makes it possible to measure the hydrodynamic radius. A higher number of
BE2016 / 5783 glycosylation on the support results in large variations in the hydrodynamic radius compared to a support having fewer glycosylation sites. However, homogeneity can be greater when single chains of glycans are analyzed, their length being better controlled. The length of the glycans is measured by hydrazinolysis, SDS-PAGE and ECG. In addition, the term homogeneity can also mean that certain patterns of use of the glycosylation sites can vary within a more or less wide range. These factors can be measured by CL-SM / SM of glycopeptides.
Stability and reproducibility of the strain. The stability of the strain is measured, during bacterial fermentation in the absence of selective pressure, by direct and indirect methods which confirm the presence or absence of recombinant DNA in cells in productive culture. The influence of the culture volume can be simulated by longer culture times, corresponding to longer generation times. The more generations there are in fermentation, the more likely it is that a recombinant element will be lost. The loss of a recombinant element is considered to be instability. Indirect methods are based on the association of cassettes with recombinant DNA, such as for example antibiotic resistance cassettes in a plasmid. The cells in productive culture are deposited on plates coated with selective media, such as for example LB plates supplemented with antibiotics or other chemical substances.
BE2016 / 5783 related to a selection system, and the resistant colonies are considered to be positive for the recombinant DNA respectively associated with the selection chemicals. In the case of a multiple plasmid system, colonies resistant to multiple antibiotics are counted, and the proportion of cells containing all three resistors is considered to be a stable population. It is also possible to use instead a quantitative PCR to measure the quantity of recombinant DNA corresponding to the three elements in the presence or in the absence of selection, and at different times of fermentation. Thus, the relative and absolute amounts of recombinant DNA are measured and compared. The reproducibility of the production process is measured by the complete analysis of uniform batches using the methods set out in the present invention.
In one embodiment, this relates to a host cell in which the nucleic acid which codes for a glycosyltransferase is derived from an rfb aggregate of Pseudomonas, said nucleic acid sequence possibly being integrated in a stable manner into the genome of the host cell. The rfb aggregate possibly comes from Pseudomonas aeruginosa, possibly from serotype 06 or 011.
In one embodiment, the host cell comprises an oligosaccharyl transferase derived from Campylobacter, the oligosaccharyl transferase being for example PglB from C. jejuni.
BE2016 / 5783
In one embodiment, the host cell comprises the nucleic acid encoding a PcrV protein from P. aeruginosa in a plasmid of the host cell.
In one embodiment, the host cell further comprises a formyltransferase enzyme, the nucleic acid encoding a protein having about, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or homology with SEQ ID NO: 65, or said nucleic acid coding for SEQ ID NO: 65.
In one embodiment, the host cell according to the invention further comprises a nucleic acid coding for a wzy polymerase, the nucleic acid coding for a protein having approximately, or at least 80%, 85%, 90%, 95 %, 96%, 97%, 98% or 99% of identity or homology with SEQ ID NO: 66, or said nucleic acid coding for SEQ ID NO: 66.
In one embodiment, the host cell comprises a nucleic acid encoding a formyltransferase enzyme and / or the nucleic acid encoding a wzy polymerase which are stably integrated into the genome of the host cell. Optionally, a gene encoding a formyltransferase enzyme and / or a gene encoding a wzy polymerase is present in a host cell plasmid.
In one embodiment, the host cell is E. coli.
In a further aspect of the invention, this relates to a method for producing a bioconjugate comprising a PcrV protein of P. aeruginosa linked to a saccharide, said method comprising the culture of the host cell according to
BE2016 / 5783
The invention under conditions suitable for the production of proteins.
In a further aspect of the invention, it relates to a bioconjugate produced by the method according to the invention, said bioconjugate comprising a saccharide linked to a PcrV protein of P. aeruginosa.
The terms "comprising", "includes" and "include" used herein may be replaced by the terms "consisting of", "consists of" and "consist of", respectively, in all cases.
The expression "the asparagine residue being located at a position located between the amino acids ... of SEQ ID NO: ..." is understood as the introduction of the asparagine residue into an amino acid sequence at a position which would correspond to the defined position, if the reference sequence and the mutated sequence were aligned so as to maximize the sequence identity between the two sequences. Addition or deletion of amino acids from a mutated sequence could imply a difference in the effective position of the asparagine residue among the amino acids of the mutated sequence, however, by aligning the mutated sequence with the reference sequence, the appropriate insertion position for the amino acid asparagine can be established.
The expression "the peptide comprising the consensus sequence D / E-X-N-X-S / T is located at a position between the amino acids ... of SEQ ID NO: ..." is understood as the introduction of the
BE2016 / 5783 consensus sequence in an amino acid sequence at a position that would correspond to the defined position, if the reference sequence and the mutated sequence were aligned to maximize the sequence identity between the two sequences. Addition or deletion of amino acids from a mutated sequence could imply a difference in the effective position of the consensus sequence among the amino acids of the mutated sequence, however, by aligning the mutated sequence mutation with the reference sequence, the appropriate insertion position for the consensus sequence can be established.
The P. aeruginosa O-antigens (01 to 020) are defined according to the classification of serotypes corresponding to the IATS nomenclature.
All the references or patent publications cited in the present description are incorporated herein by reference.
The examples presented below are intended to make the present invention more understandable. These examples are offered for illustrative purposes only, and should in no way be construed as limiting the scope of the present invention.
Protein and nucleic acid sequences
SEQ ID NO: 1 - Sequence of the wild-type PcrV protein
MEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPL
SEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSL
HGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQ
BE2016 / 5783
GIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSP
KQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYN
SAVEALNRFIQKYDSVLRDILSAI
SEQ ID NO: 2 - PcrV
AKDQNATKVRNLNAARELFKDQNATKDELLAASKDQNATKAPASAEQEEL LALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQ PGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAEL KVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSN LDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLN DKVNEKTTLLNDTSSRYNSAVEALNRFIQ KYDSVLRDILSAI
SEQ ID NO: 3
MSFKKIIKAFVIMAALVSVQAHAAEVRNLNAARELFLDELLAASAAPASA EQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVL QARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKA LTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEY ALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDR SRPLNDKVNEKTTLLNDTSSRYNSAVEALNR FIQKYDSVLRDILSAI
SEQ ID NO: 4 - mature
AEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPL
SEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSL
HGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQ
GIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSP
KQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYN
SAVEALNRFIQKYDSVLRDILSAI
SEQ ID NO: 5 - Sequence of the epitope
BE2016 / 5783
VYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYA
LLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRS
RPLNDKVNE
SEQ ID NO: 6 AKDQNATKVRNLNAARELF
SEQ ID NO: 7 VRNKDQNATKNAARELF
SEQ ID NO: 8 VRNLNAAKDQNATKELF
SEQ ID NO: 9 ELFKDQNATKDELLAAS
SEQ ID NO: 10 DELKDQNATKAAS
SEQ ID NO: 11 DELLAASKDQNATKAP
SEQ ID NO: 12 APKDQNATKSAEQEEL
SEQ ID NO: 13 ALLRSEKDQNATKI
SEQ ID NO: 14 ALLRSERIKDQNATKLAH
BE2016 / 5783
SEQ ID NO: 15 LAHKDQNATKGQPL
SEQ ID NO: 16 GQPLKDQNATKEAQVLKA
SEQ ID NO: 17 EAKDQNATKVLKALA
SEQ ID NO: 18 VLKALAKDQNATKLLAA
SEQ ID NO: 19 VLKALAWKDQNATKLAA
SEQ ID NO: 20 LAAKDQNATKPSA
SEQ ID NO: 21 PSAKDQNATKPGQG
SEQ ID NO: 22 PSAPPKDQNATKQG
SEQ ID NO: 23 QGKDQNATKEVLR
SEQ ID NO: 24 QGLEKDQNATKLR
SEQ ID NO: 25
BE2016 / 5783
LRKDQNATKVLQAR
SEQ ID NO: 26
VLGARKDQNATKQ
SEQ ID NO: 27
VLGARRQKDQNATKGAQW
SEQ ID NO: 28
VLQARRQPGKDQNATKQW
SEQ ID NO: 29
QWKDQNATKLREFLVSAYF
SEQ ID NO: 30
LREFLVSAYFSLKDQNATKG
SEQ ID NO: 31
GKDQNATKLDEDVIGVYKD
SEQ ID NO: 32
KDVLQTKDQNATKDGKRKAL
SEQ ID NO: 33
KYDSVLRDILSAKDQNATK
SEQ ID NO: 34
MSFKKIIKAFVIMAALVSVQAHA
SEQ ID NO: 35
AXD / EXNXS / TXVRNLNAARELF
BE2016 / 5783
SEQ ID NO: 36
VRNX D / EXNX S / TXNAARE L F
SEQ ID NO: 37
VRNLNAAXD / EXNXS / TXELF
SEQ ID NO: 38
ELFXD / EXNXS / TXDELLAAS
SEQ ID NO: 39
DELXD / EXNXS / TXAAS
SEQ ID NO: 40
DELLAASXD / EXNXS / TXAP
SEQ ID NO: 41
APXD / EXNXS / TXSAEQEEL
SEQ ID NO: 42
ALLRSEXD / EXNXS / TXI
SEQ ID NO: 43
ALLRSERIXD / EXNXS / TXLAH
SEQ ID NO: 44
LAHXD / EXNXS / TXGQPL
SEQ ID NO: 45
GQPLXD / EXNXS / TXEAQVLKA
BE2016 / 5783
SEQ ID NO: 46 EAXD / EXNXS / TXVLKALA
SEQ ID NO: 47 VLKALAXD / EXNXS / TXLLAA
SEQ ID NO: 48 VLKALAWXD / EXNXS / TXLAA
SEQ ID NO: 49 LAAXD / EXNXS / TXPSA
SEQ ID NO: 50 PSAXD / EXNXS / TXPGQG
SEQ ID NO: 51 PSAPPXD / EXNXS / TXQG
SEQ ID NO: 52 QGXD / EXNXS / TXEVLR
SEQ ID NO: 53 QGLEXD / EXNXS / TXLR
SEQ ID NO: 54 LRXD / EXNXS / TXVLQAR
SEQ ID NO: 55 VLGARXD / EXNXS / TXQ
SEQ ID NO: 56
BE2016 / 5783
VLGARRQXD / EXNXS / TXGAQW
SEQ ID NO: 57
VLQARRQPGXD / EXNXS / TXQW
SEQ ID NO: 58
QWXD / EXNXS / TXLREFLVSAYF
SEQ ID NO: 59
LREFLVSAYFSLXD / EXNXS / TXG
SEQ ID NO: 60
GXD / EXNXS / TXLDEDVIGVYKD
SEQ ID NO: 61
KDVLQTXD / EXNXS / TXDGKRKAL
SEQ ID NO: 62
KYDSVLRDILSAKDQNATK
SEQ ID NO: 63
MSFKKIIKAFVIMAALVSVQAHA
SEQ ID NO: 64
D / E-X-N-X-S / T
SEQ ID NO: 65 - formyltransferase Met Ser Trp Gin Leu Phe Ser Glu Lily Cys Arg Phe Leu Gly To the Val Glue lie Ser Gin His Phe Trp Gly Phe lie Val Leu Glue To the Ser Phe Gly Met Lily lie Lily To the To the Leu lie Val Asp Asp Leu Ser Leu Ser Glu Trp Gin Lily Arg To the lie
BE2016 / 5783
Glue Asp Ser Ser Glue Tyr Leu Asp Isle Gin Leu Val Leu Ser Cys Arg Asn Ser To the Thr Lily Lily Ser Val Isle Lily His Cys Gly Tyr Tyr Phe Leu Asn Isle Leu Ser Leu Lily Asn Asp Met Thr Arg Arg Val Gin Leu Asp Ser Arg Gly Ser Glue Val Isle His Phe Asp Ser Asp Tyr Glue Gly To the Trp Gin Arg Isle Pro Glue Asp Val Cys To the Arg Isle Leu Asp Lily Gly Isle Lily Leu Val Isle Lily Phe Gly Met Ser Leu Leu Arg Isle Asp Gly Gly Leu Gin Arg Leu Asp Isle Leu Ser Tyr His His Gly Asp Pro Glue Tyr Tyr Arg Gly Arg Pro To the Gly Phe Tyr Glue Isle Tyr Glue Asn To the Asp Ser Val Gly Isle Isle Val Gin Lily Leu Ser Asn Lily Leu Asp To the Gly Glue Val Leu Val Arg Gly Tyr Ser Lily Val His His His Ser Tyr Lily Lily Thr Ser Arg Asn Phe Tyr Leu Asn Ser Val Val Leu Leu Arg Lily To the Leu Val Asn Tyr Ser Arg Gly Glue Gin Val Val Leu Glue Lily Leu Gly Lily Asn Tyr Arg Leu Pro Ser Asn Phe Thr Val Phe Lily Phe Phe Cys Lily Thr Isle Phe Arg Gly Leu To the Arg Leu Ser Tyr Gly To the Phe Phe Glue Lily Lily Trp Asn Val Val To the Leu Pro Tyr Asn Asp Isle Pro Ser Leu Gin Glue Leu Ser Val Ser To the Gly Lily Isle Pro Lily Val Glue Lily Gly Tyr Thr Phe Tyr To the Asp Pro Phe Phe Ser To the Asp Gly Lily Leu Isle Arg Leu Glue To the Leu Asn To the Ser Asn Gly Leu Gly Glue Isle Isle Glue Leu Lily To the Gin Ser Leu Asp Phe Ser Arg Val Isle Leu Lily Gly Asn His Phe Ser Tyr Pro Tyr Ser Phe Glue To the Ser Gly Val Glue Tyr Leu Isle Pro Glue Val To the Ser His Ser To the Pro Cys Leu Leu Pro Pro Pro Phe To the Leu Glue Ser Lily Lily Leu Phe Gin Gly Met Glue Gly Glue Arg Isle Leu Asp Gly Thr Leu Phe Glue His Gly Gly Arg Tyr Tyr Leu Phe Cys Gly Gin To the Val Ser Gly Ser Asp Asn Leu Tyr Leu Tyr Val Gly Glue Ser Leu Glue Gly Pro Tyr Thr Ser His Pro Cys Asn Pro Val Val Met Asn Pro Gly Ser To the Arg Met Gly Gly Arg Isle Phe Lily Glue Gly Gly Lily Leu Tyr Arg Phe Gly Gin Asn Asn Ser Tyr Gly Tyr
BE2016 / 5783
Gly Ser Ser Leu To the Val Asn Glue lie Glue Val Leu Asp Pro Glue His Tyr Ser Glue Lily Arg Val To the Asn Leu To the Phe Gin Asp To the Arg Gly Pro His Thr lie Asp lie His Gly Gin Thr Met lie Leu Asp Phe Tyr Gin Asp Arg Phe Ser Leu Leu To the Gly Tyr Arg Arg Leu Val To the Arg Leu Leu Ser Arg Gly SEQ ID NO: 66 - polymerase : wzy Met . Tyr  To the Met Leu Thr Gly To the Thr Leu Leu lie Phe To the Val To the To the Arg Leu Leu To the Arg Ser To the lie His Pro Ser Val To the Met Pro lie Thr Trp Gly Leu Gly Leu lie Gly Val Ser Leu To the Ser Leu lie Gly Phe Tyr Arg Val Glue Ser Asp To the Leu Leu lie Phe Leu Phe Gly Val Met Ser Phe Ser Leu Ser To the Gly Cys Phe Ser Phe Leu Tyr Asn Gly Tyr Phe Arg To the Pro Ser Ser Asn Phe Leu Phe Asp Ser Glue Leu Arg Thr Arg To the Leu Val lie Phe Phe Cys Leu To the His lie Val Phe Leu Thr Val lie Tyr Arg Asp Leu Ser Ser lie To the Pro Thr Leu Arg Glue To the To the Tyr Met To the Arg To the Gin Ser Val Ser Gly Glue Pro Val Leu Ser Ser Leu Ser Met Asn Tyr Leu Gin Leu Gly Gin Thr Val lie Pro Leu Val Val Leu Leu Tyr Leu Arg Gly Lily Cys Gly Val Leu Gly Phe Leu To the lie Ser Val Pro Trp Met Gly Val lie Leu Leu To the Ser Gly Arg To the Ser Leu Met Gin Met Leu Val Gly Leu Phe Phe lie Tyr lie Leu Val Lily Gly Ser Pro Ser Leu Lily Ser Leu Leu Val lie Gly Leu To the Met Phe Leu Val lie To the Val Gly To the Val To the Thr Ser Lily lie Gin Phe His Glue Gly Asp Gly lie Ser Thr Leu Phe lie Glue Leu Tyr Arg His Val To the Gly Tyr To the Leu Gin Gly Pro Val Leu Phe Asp Arg Tyr Tyr Gin Gly Ser lie His Leu Glue Pro Tyr Trp Ser Pro Leu Asn Gly Phe Cys Ser lie Leu To the Thr Val Gly Leu Cys Gin Lily Pro Pro Leu His Leu Asp Phe Tyr Glue Tyr To the Pro Gly Glue Leu Gly Asn Val Tyr Ser Met Phe Phe Ser Met Tyr Pro His Tyr Gly To the Leu
BE2016 / 5783
Gly Val Isle Gly Val Met To the Leu Tyr Gly Met Leu Cys Ser Tyr To the Tyr Cys Lily To the Lily Lily Gly Ser Leu Tyr Phe Thr Val Leu Ser Ser Tyr Leu Phe Ser To the Isle Val Phe Ser Leu Phe Ser Asp Gin Isle Ser Thr Ser Trp Trp Phe Tyr Val Lily Met Thr Isle Isle Leu Gly Isle Leu Cys Phe Val Phe Arg Arg Asp Arg Met Phe Val Isle Arg Leu Pro Gin To the Gly
SEQ ID NO: 67 - nucleotide sequence of PcrV ATGGAAGTCAGAAACCTTAATGCCGCTCGCGAGCTGTTCCTGGACGAGCT
CCTGGCCGCGTCGGCGGCGCCTGCCAGTGCCGAGCAGGAGGAACTGCTGGCCCTG
TTGCGCAGCGAGCGGATCGTGCTGGCCCACGCCGGCCAGCCGCTGAGCGAGGCGC
AAGTGCTCAAGGCGCTCGCCTGGTTGCTCGCGGCCAATCCGTCCGCGCCTCCGGG
GCAGGGCCTCGAGGTACTCCGCGAAGTCCTGCAGGCACGTCGGCAGCCCGGTGCG
CAGTGGGATCTGCGCGAGTTCCTGGTGTCGGCCTATTTCAGCCTGCACGGGCGTC
TCGACGAGGATGTCATCGGTGTCTACAAGGATGTCCTGCAGACCCAGGACGGCAA
GCGCAAGGCGCTGCTCGACGAGCTCAAGGCGCTGACCGCGGAGTTGAAGGTCTAC
AGCGTGATCCAGTCGCAGATCAACGCCGCGCTGTCGGCCAAGCAGGGCATCAGGA
TCGACGCTGGCGGTATCGATCTGGTCGACCCCACGCTATATGGCTATGCCGTCGG
CGATCCCAGGTGGAAGGACAGCCCCGAGTATGCGCTGCTGAGCAATCTGGATACC
TTCAGCGGCAAGCTGTCGATCAAGGATTTTCTCAGCGGCTCGCCGAAGCAGAGCG
GGGAACTCAAGGGCCTCAGCGATGAGTACCCCTTCGAGAAGGACAACAACCCGGT
CGGCAATTTCGCCACCACGGTGAGCGACCGCTCGCGTCCGCTGAACGACAAGGTC
AACGAGAAGACCACCCTGCTCAACGACACCAGCTCCCGCTACAACTCGGCGGTCG
AGGCGCTCAACCGCTTCATTCAGAAATACGACAGCGTCCTGCGCGACATTCTCAG
CGCGATCTAG
SEQ ID NO: 68 X-S / T-X-N-X-D / E
Examples
BE2016 / 5783
Example 1: The bacterial strains in which an oligosaccharyl transferase and an rfb aggregate are inserted are stable and produce bioconjugates
This example demonstrates that it is possible to successfully produce bioconjugates using a bacterial host strain which has been genetically modified by the insertion of (i) a nucleic acid coding for an oligosaccharyl transferase and (ii) a nucleic acid encoding an rfb aggregate.
Host cells of E. modified coli were produced by inserting the following directly into the genome of the host cells: (i) a nucleic acid coding for C. jejuni oligosaccharyl transferase (PglB) and (ii) a nucleic acid coding for the aggregate rfb beyond strain PA103 of Pseudomonas aeruginosa. Said rfb aggregate contains the genes necessary for the synthesis of O-antigen of the serogroup 011 of Pseudomonas aeruginosa. The insertions were carried out using a new insertion method described in document PCT / EP 2013/071 328 (see section 5.2 above) or the pUT-mini system (Biomedal Lifescience). The insertion method described in document PCT / EP 2013/071 328 is regiospecific and uses homologous recombination, while the pUT-mini system is based on a random approach by means of a transposon, the result of which is random insertion of a nucleic acid sequence of interest into the genome of a host cell. The cells of E. coli were further modified by the introduction of a plasmid expressing, as a carrier protein, the exoprotein A (EPA) of detoxified Pseudomonas in the host cells. So,
BE2016 / 5783 host cells of E. modified coli described in the present example express (i) C. jejuni oligosaccharyltransferase (PglB), following the integration of a nucleic acid coding for said oligosaccharyl transferase into the genome of the host cell; (ii) the genes of an rfb aggregate of Pseudomonas aeruginosa which produce the O-antigen, following the integration of a nucleic acid coding for said rfb aggregate originating from the strain PA103 of Pseudomonas aeruginosa in the genome of the host cell; and (iii) the EPA carrier protein, following the transformation of the host cell using a plasmid comprising a nucleic acid encoding the carrier protein.
Host cells of E. additional coli were produced in order to compare the capacity of the modified host cells comprising double integrations (integration of an oligosaccharyl transferase and integration of an rfb aggregate) to produce bioconjugates (EPA-O11) with the production of bioconjugates by host cells containing (i) a single integration only, either oligosaccharyltransferase or rfb aggregate, the remaining components (carrier protein and oligosaccharyl transferase or rfb aggregate) expressed through a host cell plasmid ; or (ii) no integrated component, all the components (carrier protein, oligosaccharyltransferase and rfb aggregate) being expressed by means of a plasmid.
Three different basic strains of E. coli were used for the analysis: (i) St4167 (W3110 OwaaL, OrfbOl6:: rfbP.a.011), which includes a deletion of the gene
BE2016 / 5783 waaL of E. coli, a deletion of the rfb 016 aggregate from E. coli, and an insertion of the rfb 011 aggregate of P. aeruginosa (PCT / EP 2013/071 328); (ii) Stll28 (W3110 OwaaL), which includes a deletion of the waaL gene from E. coli: and (irr) Stl935 (W3110 OwaaL, OwzzE-wecG,
OwbbIJK), which includes a deletion of the indicated genes. To allow the insertion of the rfb 011 aggregate of P. aeruginosa into the st4167 strain, the rfb 011 aggregate was cloned into a plasmid pDOC then the method used was that described in document PCT / EP 2013/071 328 Strains St4167 represent the strains with double integrations.
The specific plasmids used to introduce EPA into host cell strains are called "pl077" and "pl50". The latter is described by Ihssen, et al., (2010) Microbial cell factories 9, 61, and the plasmids are the same, with the exception that for pl077, the amp cassette of pl50 is replaced by a Kan cassette.
The following variants of St4167 were produced: (i) St4167 where pglB is inserted in place of the yahL gene of the host cell (using the method of document PCT / EP 2013/071 328) and the EPA is expressed by plasmid P1077; (ii) St4167 where pglB is inserted in place of the host cell ompT gene (using the pUT-mini system) and the EPA is expressed by the plasmid P150; (iii) Stl467 where the pglB is expressed by the plasmid pl769 (pglB in pDOC) and the EPA is expressed by the plasmid P1077; (iv) Stl467 where the pglB is expressed by the plasmid p939 (expression plasmid based on pEXT21, for the pglB provided with an HA marker, after
BE2016 / 5783 optimization of the codons) and 1ΈΡΑ is expressed by the plasmid P1077; and (v) Stl467 where the pglB is expressed by the plasmid pl762 (pglB in pDOC) and 1ΈΡΑ is expressed by the plasmid P1077.
The following variants of Stll28 were produced: (i) Stll28 where pglB is expressed by the plasmid p939, the rfb aggregate of 011 of P. aeruginosa is expressed by the plasmid pl64 (plasmid pLAFR designed to contain the rfb aggregate of 011 of P. aeruginosa), and the EPA is expressed by the plasmid P1077; and (ii) St1128 where the pglB is inserted in place of the yahL gene of the host cell (using the method of document PCT / EP 2013/071 328), the rfb aggregate of 011 of P. aeruginosa is expressed by plasmid pl64, and the EPA is expressed by plasmid P1077.
The following variants of Stl935 were produced: (i) Stl935 where pglB is inserted in place of the ompT gene of the host cell (using the method of document PCT / EP 2013/071 328), the aggregate rfb of P. aeruginosa 011 is expressed by plasmid pl64, and EPA is expressed by plasmid P1077; (ii) Stl935 where the
pglB is inserted to the gene place yahL of the cell host (using of the method of document PCT / EP 2013/071 328), The aggregate rfb of 011 of
P. aeruginosa is expressed by plasmid pl64, and EPA is expressed by plasmid P1077; and St1935 where pglB is expressed by plasmid p939, the rfb aggregate of 011 from P. aeruginosa is expressed by plasmid pl64, and the EPA is expressed by plasmid P1077.
As shown in Figure 1, all strains expressing an oligosaccharyl transferase, a protein
BE2016 / 5783 carrier, and an rfb aggregate produce bioconjugates. This can be observed on the blots described between the markers at 100 and 130 kDa, corresponding to EPA011. It is important to note that this observation includes strains comprising the double integration of an oligosaccharyl transferase and an rfb aggregate. See, in particular, the results presented for St4167. Thus, this example demonstrates not only that stable host cells can be produced following a double insertion of genes / gene aggregates into the genome of host cells, but also that these genes retain their function. In particular, the inserted oligosaccharyltransferase and the inserted rfb aggregate retained their function, resulting in the production of bioconjugates.
Example 2: Identification of a uncomfortable of formyltransferase who contributes to the synthesis of 1 oligo / polysaccharide of the O- antigen native of
P. aeruginosa 06
This example describes the identification of the formyltransferase from Pseudomonas aeruginosa 06.
The data concerning the proteome of the Pseudomonas aeruginosa 06 strain "LESB58", the genome of which is known, were studied in search of homologies with domains corresponding to a query on domain prototypes "Formyltransferase" and "Similar to C-terminal domain of an FMT ”using the algorithm provided on the site www.supfam.org/SUPERFAMILY/. Research has identified
BE2016 / 5783 protein sequences having domains which can be related.
In order to determine which one or the other of the 9 identified candidates was specific for 06 (and therefore for a repeat unit of formylated O-antigen), it was decided to attest to their absence in the proteome d another Pseudomonas aeruginosa serotype (05, strain PAO1) by searching by BLAST (NCBI website) The structure of the O-antigen of Pseudomonas aeruginosa 05 is not related to that of Pseudomonas aeruginosa 06. in particular, there is no formyl group in the structure of 05. 8 of the 9 candidates had homologs in serotype 05 of Pseudomonas aeruginosa, thereby indicating that these proteins were not specific for the strain of Pseudomonas aeruginosa
06 LESB58. The candidate remaining (locus tag = PLES 12061, GenBank: CAW25933 .1; SEQ ID NO: 2) did not have no counterpart obvious in the serotype 05 from Pseudomonas aeruginosa , and a by therefore summer classroom as
of
Aeruginosa pseudomonas formyltransferase (SEQ ID NO specific for LESB58 / aeruginosa serotype.
In order to confirm the specificity of 06, it was decided to verify the presence of formyltransferase of serotype 06 of Pseudomonas aeruginosa (SEQ ID NO: 2) discovered in other strains of serotype 06 of Pseudomonas aeruginosa. Proteins equivalent to Pseudomonas serotype 06 2) have been identified in four other Pseudomonas aeruginosa serotype 06 strains, including the locator marker PAK 01412
BE2016 / 5783 in the strain “PAK” and the locus marker PAM18_1171 in the strain M18.
Formyltransferases with weak sequence identity with Pseudomonas aeruginosa serotype 06 formyltransferase (SEQ ID NO: 2) have also been identified in
Methylob acterium WP 020093860)
IDENTITY ACCESSION, (33% identity, _ in Thiothrix nivea (30
ACCESSION WP_002707142), Anaerophaga thermohalophila (28% identity, ACCESSION WP_010422313), Halorubrum californiense (27% identity, ACCESSION WP_008445073), Azorhizobium caulinodans (25% identity, ACCESSION WP_012170036) and Burkholderia glathei ( identity, ACCESSION KDR39707). Taken together, these homology analyzes indicate that related genes code for specific activity of 06 linked to formylation.
The gene of SEQ ID NO: 2 was cloned in order to test the activity of the formyltransferase of the serotype 06 of Pseudomonas aeruginosa (SEQ ID NO: 2) on the structure of repeat units of unformylated 06. The rare TTG primer codon has been replaced by ATG. FIG. 5 proposes a schematic representation of the cloning of the formyltransferase of the serotype 06 of Pseudomonas aeruginosa (SEQ ID NO: 2) in the rfb aggregate of Pseudomonas aeruginosa 06, and the relative organization of the genes.
Once identified, the function of the formyltransferase from Pseudomonas aeruginosa 06 was analyzed. The functionality of Pseudomonas aeruginosa serotype 06 formyltransferase (SEQ ID NO: 2) was tested by co-expression of
BE2016 / 5783 functional the rfb aggregate of Pseudomonas aeruginosa 06 in strains of E. coli not having an ECA aggregate To highlight (wec;
On formylation, unique repeat units of the antigen were analyzed linked to the lipid A nucleus (in a waaL positive strain). The repeat unit of the formylated Oantigen of 06 was identified by immunodetection using a specific antibody to 06 (Figure 3A), indicating that the formyl group is a relevant epitope of the structure of O- Pseudomonas aeruginosa 06 antigen.
In order to highlight the formylation at the molecular level, the repeat units of 06 were analyzed by MALDI-SM / SM. The purified repeat units labeled with 2-AB showed that the coexpression of formyltransferase of Pseudomonas aeruginosa serotype 06 (SEQ ID NO: 2) with the genes of the rfb aggregate of Pseudomonas aeruginosa 06 led to a main peak of which the fluorescence signal was shifted by 2 to 3 minutes (from 58 to 61 ', Fig. 3B).
Analysis by MALDI-SM / SM of the substance contained in the peak at 58 'is the result of a fragmentation in Y series ions in accordance with the repetition unit of 06 non-formylated, N-acetylated, marked at 2-AB. The protonated precursor ion of m / z = 905 is fragmented according to the series of ions 905-> 759-> 543-> 326, corresponding to the loss of mass units 146 (deoxyhexose), 216 (N-acetylhexosaminuronic acid amidated), 217 (N-acetylhexosaminuronic acid). The substance collected after 61 ′ obtained from cells expressing the pseudomonas serotype 06 formyltransferase gene
BE2016 / 5783 aeruginosa contains a majority precursor ion of mass 891, which fragments according to the series 891-> 745-> 529-> 326, corresponding to the loss of mass units 146 (as above), 216 (as above) and 203 (N-formylhexosaminuronic acid amide). These data demonstrate that the formylation depends on the expression of the formyltransferase of the serotype 06 of Pseudomonas aeruginosa, and that the respective gene codes for the formyltransferase. The gene coding for formyltransferase of Pseudomonas aeruginosa serotype 06 was therefore named fmtO6. The replacement of an acetyl group of N-acetylhexosaminuronic acid amidated by a formyl group suggests a two-step mechanism during which the
group acetyl East first eliminated before I add out group formyl . This model implies that an amine group free present in position C2 plays on role of a intermediate before the binding of group formyl to
monosaccharide by the formyltransferase domain. The deacetylated and unformylated O-antigen may therefore be an important and immunologically relevant polysaccharide form, present in sub-stoichiometric amounts, of serotype 06 of P. aeruginosa.
Example 3: Identification and analysis of the wzy gene allowing the polymerization of the P. aeruginosa 06 O-antigen.
This example describes the identification of the wzy polymerase from Pseudomonas aeruginosa 06.
O-antigen polysaccharides are constituents of the outer surface of many
BE2016 / 5783 Gram-negative bacteria. The enzymatic machinery responsible for the biosynthesis of O-antigen is often coded by a single gene aggregate called rfb aggregate. Pseudomonas aeruginosa serotype 06 strains express a polymerized O-antigen (Figure 2) However, in the corresponding aggregate coding for O-antigen, no gene coding for an O-antigen polymerase (wzy) is present. This means that, in order to recombinantly express the O-antigen 06 of P. aeruginosa in E. coli, it is necessary to identify the wzy gene. O-antigen (wzy) polymerases are integrally internal membrane proteins which catalyze the polymerization of O-antigen repeat units in the periplasmic space before their block binding to the lipid nucleus oligosaccharide A, thus forming an LPS. Wzy polymerases are extremely specific for their oligomeric repeat unit, and there is little homology among the wzy genes.
The structure of the O-antigen of Pseudomonas aeruginosa 019 has structural similarities with that of Pseudomonas aeruginosa 06. It was therefore assumed that the wzy proteins recognizing the two structures could also have similar properties, for example concerning their structure, sequence or number of transmembrane domains. The sequence of the wzy 019 protein from Pseudomonas aeruginosa 019 (ACCESSION AAM27560) is known and was used in a first analysis request by BLAST, using the proteome of the 06 PAK strain of Pseudomonas aeruginosa as a base for the search for homology.
BE2016 / 5783
In order to assess whether one of the candidates identified was specific for Pseudomonas aeruginosa 06, it was decided to attest to their presence in the proteome of another serotype of Pseudomonas aeruginosa (05, strain PAO1). The structure of the O-antigen of 05 is not related to that of 06 and 019. The first 100 results were analyzed individually by BLAST to determine if they were present in the proteome of Pseudomonas aeruginosa 05. 97 of 100 candidates from the PAK proteome had homologs in serotype 05 of Pseudomonas aeruginosa, indicating that these proteins were generally present among the strains of Pseudomonas aeruginosa, and probably not linked to the biosynthesis of O-antigen 06. Three of the 100 Candidates had no obvious counterpart in the Pseudomonas aeruginosa serotype 05 proteome, and were therefore considered specific to Pseudomonas aeruginosa 06.
In order to test whether one of the three candidate proteins identified was the wzy of Pseudomonas aeruginosa 06, the three proteins were chosen as requests for analysis by BLAST. One of the three candidates, PAK_01823 (06wzy PAK_01823; SEQ ID NO: 3), presented identity with the amino acid sequences of other polymerases of known oligosaccharide repeating units, such as for example 25% of identity with the polymerases of oligosaccharide repeating units of Streptococcus sanguinis (ACCESSION WP_004192559) and 22% of identity with the polymerases of oligosaccharide repeating units of Escherichia coli 0139 (ACCESSION AAZ85718). PAK_01823 (06wzy
BE2016 / 5783
PAK_01823; SEQ ID NO: 3) has therefore been identified as a wzy of Pseudomonas aeruginosa 06.
To confirm that SEQ ID NO: 3 corresponds to the protein encoded by the wzy of Pseudomonas aeruginosa 06, the localization in the cell of the protein was predicted by a bioinformatic method using PSORTb (www.psort.org/psortb/) . Predictions indicated that the protein was located in the cytoplasmic membrane and comprised 11 transmembrane domains, a common feature among O-antigen polymerases.
Proteins equivalent to PAK_01823 (06wzy PAK_01823; SEQ ID NO: 3) have been identified in other strains of P. aeruginosa 06-positive, including strain LESB58 (of which the wzy protein from Pseudomonas aeruginosa 06 only a difference in the sequence of aa compared to the PAK strain and to a strain tested internally).
The functional analysis of the Pseudomonas aeruginosa 06 wzy was then carried out. The rfb aggregate of Pseudomonas aeruginosa 06, the fmt06 gene (in other words the gene coding for SEQ ID NO: 2, see example 2 above), and the gene coding for the wzy of Pseudomonas aeruginosa 06 (in other words the gene coding for SEQ ID NO: 3) were co-expressed in these E coli Awec cells, and the lipopolysaccharide formed was analyzed by immunoblotting (FIG. 4). A ladder-like signal was detected by the anti-06 antiserum only in the sample from cells containing all three transgenes, indicating that PAK_01823 (06wzy PAK_01823; SEQ ID NO: 3) is
BE2016 / 5783 actually the P. aeruginosa 06 polymerase. The gene coding for PAK_01823 was therefore named O6wzy
In order to generate a single gene aggregate containing all of the genetic elements necessary to allow E. coli to express by recombination the P. aeruginosa 06 O-antigen, the fmtO6 and 06wzy genes (in other words, the genes encoding respectively for SEQ ID NO: 2 and 3) were cloned downstream of the rfb aggregate of P. aeruginosa 06. FIG. 5 provides a schematic representation of the cloning of the polymerase 06wzy of the O-antigen of Pseudomonas aeruginosa 06 optimizing the use of codons in the rfb aggregate of Pseudomonas aeruginosa 06 cloned in association with the formyltransferase of 06, and the relative organization of genes. In addition, it was determined that the fmt06 and 06wzy genes (in other words, the genes coding for SEQ ID NOs: 2 and 3 respectively) could be inserted at multiple positions in the rfb aggregate of P. aeruginosa 06. in particular, the fmt06 gene could be inserted clockwise with respect to the rfb aggregate downstream of the rfb aggregate, or upstream of the rfb aggregate under the control of a separate promoter. In addition, the fmt06 gene could be inserted counterclockwise relative to the rfb aggregate upstream of the rfb aggregate, or downstream of the rfb aggregate. The 06wzy gene could be inserted clockwise relative to the rfb aggregate upstream or downstream of the rfb aggregate or upstream of the rfb aggregate under the control of a separate promoter. All the constructions described ci94
BE2016 / 5783 above were active considering the biosynthesis of the P. aeruginosa 06 O-antigen (data not shown).
Example 4 The bacterial strains into which an oligosaccharyl transferase and a complete rfbO6 aggregate are inserted are stable and produce bioconjugates
Example 1 demonstrates that the bioconjugates can be successfully produced by a host bacterial strain which has been genetically modified by the insertion of (i) a nucleic acid coding for an oligosaccharyltransferase and (ii) a nucleic acid coding for an rfb aggregate . In this example, experiments similar to those described in Example 1 were carried out, using the Pseudomonas PcrV protein as carrier protein.
In its natural state, the primary amino acid sequence of PcrV (see for example UniProt 030527) does not contain a consensus N-glycosylation sequence ("glycosite"). Using the methods described in document WO 2006/119 987, recombinant variants of PcrV were designed comprising one, two, three, four or five glycosites. In particular, manipulation of the nucleic acid sequence coding for PcrV has led to PcrV variants expressing one, two, three, four or five of the optimized N-glycosylation consensus sequences Asp (Glu) -X-AsnZ -Ser (Thr), X and Z being chosen independently from all the natural amino acids, except Pro.
Host cells of E. modified coli were produced by inserting the following directly
BE2016 / 5783 in the genome of the host cells: (i) a nucleic acid coding for the C. jejuni oligosaccharyl transferase (PglB) and (ii) a nucleic acid coding for the rfb aggregate of the serotype O6_PAK strain of Pseudomonas aeruginosa. Said rfb aggregate contains the genes necessary for the synthesis of O-antigen of Pseudomonas aeruginosa serogroup 06. The insertions were carried out using a new insertion method described in document PCT / EP 2013/071 328 (see section 5.2 above) or the pUT-mini system (Biomedal Lifescience). Host cells of E. coli were further modified by the introduction of a PcrV expressing plasmid comprising from one to five glycosites, as described above. Thus, the host cells of E. modified coli described in the present example express (i) C. jejuni oligosaccharyl transferase (PglB), following the integration of a nucleic acid coding for said oligosaccharyl transferase into the genome of the host cell; (ii) the genes of an rfb aggregate of Pseudomonas aeruginosa which produce the antigen 06, following the integration of a nucleic acid coding for said rfb aggregate originating from the PAK strain of Pseudomonas aeruginosa in the genome of the host cell ; and (iii) the modified PcrV carrier protein, following the transformation of the host cell using a plasmid comprising a modified nucleic acid coding for the carrier protein (the nucleic acid having been modified to code for a to five glycosites, as described above).
Host cells of E. additional modified coli were produced to be able to compare
BE2016 / 5783 the capacity of the modified host cells comprising double integrations (integration of an oligosaccharyltransferase and integration of an rfb aggregate) to produce bioconjugates (PcrV-06) with the production of bioconjugates by host cells containing (i) a single integration only, either of oligosaccharyltransferase or of rfb aggregate, the remaining constituents (carrier protein and oligosaccharyl transferase or rfb aggregate) being expressed through a plasmid of the host cell; or (ii) no integrated component, all the components (carrier protein, oligosaccharyltransferase and rfb aggregate) being expressed by means of a plasmid.
Three different strains of E. coli were used and compared during the analysis: (i) St7343, which includes both pglB and the complete 06 rfb aggregate inserted into the genome of the host cell (i.e. a double integration strain ), and a plasmid encoding a PcrV protein (with one, two, three, four or five glycosites); (ii) St7209, which comprises a pglB expressed by a plasmid, the rfb aggregate of 06 inserted into the genome of the host cell, and a plasmid coding for a PcrV protein (with one, two, three, four or five glycosites ); and (iii) St2182, which comprises a pglB expressed by a plasmid, an rfb aggregate of 06 expressed by a plasmid, and a plasmid coding for a PcrV protein (with one, two, three, four or five glycosites). Figure 6 shows the characteristics of each strain (6A: St7343; 6B: St7209; 6C: St2182).
BE2016 / 5783
As shown in Figure 6, all strains expressing an oligosaccharyl transferase, a carrier protein, and an rfb aggregate produce bioconjugates. This can be seen on the blotches described between
markers Between 40 and 70 kDa (to the around marker at 55 kDa), corresponding at PcrV-06. It is important of to note that like he East shown in The example 1, this observation includes strains
comprising the double integration of an oligosaccharyltransferase and an rfb aggregate. See in particular the results presented in Figure 6A. Thus, like example 1, this example not only demonstrates that stable host cells can be produced following a double insertion of genes / gene aggregates into the genome of host cells, but also that these genes retain their function. In particular, the inserted oligosaccharyl transferase and the inserted rfb aggregate retained their function, resulting in the production of bioconjugates.
Example 5 Production and Purification of EPA-O6 Bioconjugates
This example describes the production of bioconjugates comprising the Pseudomonas aeruginosa 06 antigen.
E. coli W3110 AwaaL Awec Arfb was transformed using plasmids comprising the rfb aggregate of Pseudomonas aeruginosa 06, the oligosaccharyl transferase pglB from C. jejuni, the gene encoding the detoxified EPA carrier protein, and the wbpVLM transferase genes associated with QuiNAc biosynthesis (from a strain of Pseudomonas aeruginosa 06).
BE2016 / 5783
The results of the plasmid retention analysis are presented in FIG. 8. Host cells containing all the four plasmids were inoculated into a medium (LB broth) supplemented with Tetracycline, Spectinomycin, Kanamycin and Ampicillin. The preculture was grown overnight at 37 ° C. The next day, the preculture was inoculated by dilution to an OD 600 of 0.1 in a medium (TB) supplemented with MgCl 2, Tetracycline, Spectinomycin, Kanamycin and Ampicillin. The cells were grown at 37 ° C. until reaching an OD 600 of 0.8 to 1.0, then the expression of pglb, epa and wbpVLM were induced by the addition of 1 mM IPTG and 0.1% arabinosis. The cells were harvested by centrifugation after overnight induction.
The EPA-O6 bioconjugates were purified from extracts of the periplasm of host cells modified by metal chelate affinity chromatography (IMAC), anion exchange chromatography (Source Q) and steric exclusion chromatography (SEC ). The eluted fractions containing glycoconjugates were combined and then introduced into the next chromatography step. The final SEC eluates were characterized by SDS-PAGE followed by staining with Coomassie blue or Western blotting using the antibodies indicated in FIG. 7.
The EPA-O6 bioconjugate was characterized using a series of analytical methods. The endotoxin level was measured by LAL assay (13 EU / ml). The
BE2016 / 5783 purity was determined by SDS-PAGE and capillary gel electrophoresis (ECG, 86% purity). The amount of protein was measured using a BCA assay (1.75 mg / ml). The amount of polysaccharide was measured using an anthrone assay (Dubois et al., 1956; 311.6 pg / ml). The average size of the polymer 06 was determined using a high resolution SDS-PAGE of the "degree of glycosylation" (DOG) (average of 7.9 repetition units per polymer). The electrical isoforms of the bioconjugate were determined by electrofocusing (EF). Finally, the identity of the bioconjugate was confirmed by immunoblotting using antibodies directed against the protein (EPA) or the polysaccharide (06).
Example 6: Immunization Studies
This example demonstrates that the O6-EPA bioconjugate of
P. aeruginosa is immunogenic.
6 week old female BALB / c OlaHsd mice (in groups of 20) were immunized intramuscularly on days 0, 14 and 28 with 0.2 pg or pg of O6-EPA conjugate (see Example 5) in a formulation containing or not an adjuvant (with an adjuvant in oil-in-water emulsion). A control group of 10 mice was vaccinated with the adjuvant (O / W) alone. Anti-06 ELISA assays were performed in the individual sera collected on day 42 (14 after
III) and the opsonic titers were determined on pooled post-II and post-III sera. The results are shown in Figure 9 and described in detail below.
100
BE2016 / 5783
Figure 9A shows the anti-06 ELISA response. The purified LPS-O6 from 06 (PaO6a, 6c) was deposited, in physiological solution with phosphate buffer (PBS) at 8 pg / ml, on high affinity micro-titration plates (Nunc Maxisorp), at 4 ° C for a night. The plates were blocked with 1% PBS-BSA for 30 min with stirring at RT. The polyvalent antisera of mice were pre-diluted to 1/100 or 1/10, then new dilutions by two were made in the microplates, before incubation with shaking at room temperature for 30 minutes. After washing, the bound murine antibody was assayed using diluted goat anti-mouse IgG AffiniPure (H + L) conjugated to a peroxidase, supplied by Jackson ImmunoLaboratories Inc. (ref: 115-035-003) 1/5000 with PBS-0.05% tween-0.2% BSA. The detection antibodies were incubated with shaking for 30 minutes at room temperature. The color was developed using 4 mg of OPD + 5 μL of H2O2 for 10 ml of 0.1 M citrate buffer at pH 4.5 for 15 minutes in the absence of light at room temperature. The reaction was stopped with 50 μΐ of HCl, and the optical density (OD) was determined at 490 nm compared to 620 nm.
The level of anti-06 antibodies present in the serum is expressed by median titers. A TMG of the individual sera was calculated for the 20 samples from each of the treated groups (10 for the control group).
An immune response was observed in mice after injection of the bioconjugate formulated with the adjuvant. No difference was observed between the doses. Similar observations have been made
101
BE2016 / 5783 concerning the percentage of seroconversion. Very weak or undetectable responses were observed with the formulation containing no adjuvant.
FIG. 9B shows the opsonic titers in HL60 cells originating from mice immunized with the O6-EPA bioconjugate formulated with or without adjuvant.
The opsonophagocytosis (OPA) assay was carried out in round-bottom microplates with 15 μΐ of phagocytic HL-60 cells (suspension adjusted to 5.10 ⁶ cells / mL), 15 μL of P. aeruginosa bacteria (cultured in TSA medium in Petri dishes), 15 μΐ of dilutions of the test serum, and 15 μL of porcine supplement. The combined inactivated test sera were diluted for the first time (for a final dilution of 1/16 or 1/50) in 1% HBSS-BSA and added to a strain of P. aeruginosa 06 (strain of ID: HNCMB 170009, obtained from the Hungarian National Collection of Medical Bacteria) diluted to arrive at a count of 200 to 250 CFU / well at the end of the test.
The HL-60 cells (suspension adjusted to 5.10 ⁶ cells / mL) and the porcine complement (12.5% in the final medium) were added to each well. Each control sample corresponded to a control containing inactivated complement.
The reaction mixture was incubated at 37 ° C for 90 minutes with shaking. After a 1/200 dilution, 50 µL of the volume obtained was transferred to a flat-bottom microplate. 50 µL of MH agar was added, followed by PBS-0.9% agar. An automatic colony count was performed after overnight incubation at 30 ° C.
102
BE2016 / 5783
The opsonophagocytic activity is expressed as the inverse of the dilution of serum giving at least 50% elimination.
The data demonstrate the functionality of the antibodies induced on after injection of the formulations of the group containing an adjuvant.
In conclusion, this example demonstrates that the O6-EPA bioconjugate of P. aeruginosa is both immunogenic and functional (in other words, induces the presence of antibodies which eliminate P. aeruginosa 06 in vivo).
Example 7
This example demonstrates that the 06-PcrV bioconjugates of P. aeruginosa are immunogenic.
Immunization
Groups of 20 6-week-old BALB / c mice and groups of 20 6-week-old female OFA-SD rats were immunized IM on days 0, 14 and 28 with 0.2 µg of 06-PcrV bioconjugate in a formulation without adjuvant or containing an adjuvant based on an oil in water emulsion.
The immune response of IgG was determined by an ELISA assay (with an anti-06 and anti-PcrV ELISA). The functionality of the antibodies was evaluated, for 06, by an opsonophagocytic assay, and for PcrV, by a hemolytic assay. The immune response was evaluated in the individual sera collected on day 42 (postIII) and on combined sera post-II and post-III.
Conjugates tested
103
BE2016 / 5783
Three 06-PcrV conjugates of P. aeruginosa were tested. In each case, three glycosylation sites were introduced into the PcrV and the short, medium and long 06 chains were added to the glycosylation sites.
long 06-PcrV way 06-PcrV short 06-PcrV Conjugate GO GO GO H- Vi 3 3 I— ¹ Ο C + • e Ω o O Fr Fr ω ω vs ω H- I— ¹C + <T ω ω C + Η- H- C + o ω 3 ω Fr ω I— ¹3 Ω Ό im o o O O ο 3 VS I— ¹ 3 ω e r> 3 r + 3 VS fl> ω e vs ω e o Fr vs o o o ω ο 3 3 ο cn 1— ¹co r> 3 GO Gn Ω 1— ¹ 3 Ό ω U ¹ e 3 - ' ο C + C + e ω- <r Η- C + 3 Η- ω ο ω 3 O o O ω Ο 3 3 Ο o 1— ¹ r> 3 Gn <1 σ> Ω O GO GO 3 ω I— ¹3 - ' C + ω e £ <r Ω C + e Η- ω ο ω 3 COI— ¹ω GO <τ Ό Ό <1 co Ό Ο o ° o ° o ° e C + AT AT ω 3 Gn Gn Fr GO ο Gn C + ο X Η- 3 ω I— ¹GO GO Ό o <1 <1 VSo e <1 GO ω o ° C + o ° o ° ω-
BE2016 / 5783
BE2016 / 5783
Anti-06 ELISA (on rat sera)
The purified 06-LPS was deposited, in physiological solution with phosphate buffer (PBS) at 8 μg / ml, on high affinity micro-titration plates (Nunc Maxisorp), at 4 ° C. overnight. The plates were blocked with 1% PBS-BSA for 30 minutes with shaking at RT. The rat antisera were pre-diluted to 1/100 or 1/10, then new dilutions by two were made in the microplates, before incubation with shaking at RT for 30 minutes. After washing, the bound rat antibodies were assayed using IgG anti-goat AffiniPure rat (H + L) conjugated to a peroxidase, supplied by Jackson ImmunoLaboratories Inc. (ref: 112-035-003) diluted 1/2 500 with PBS 0.05% tween. The detection antibodies were incubated with shaking for 30 minutes at room temperature. The color was developed using 4 mg of OPD + 5 μL of H2O2 for 10 ml of 0.1 M citrate buffer at pH 4.5 for 15 minutes in the absence of light at room temperature. The reaction was stopped with 50 μΐ of HCl, and the optical density (OD) was determined at 490 nm compared to 620 nm. The level of anti06 antibodies present in the serum is expressed by median titers. A TMG was calculated for the 20 individual samples from each of the treated groups.
Anti-PcrV ELISA (on rat sera)
The purified histidine-labeled PcrV was deposited, in physiological solution with phosphate buffer (PBS) at 1 μg / ml, on high affinity micro-titration plates (Nunc Maxisorp), at 4 ° C. overnight. The
106
BE2016 / 5783 plates were blocked with 1% PBS-BSA for 30 minutes with stirring at RT. The rat antisera were pre-diluted 1/400, then further dilutions by two were made in the microplates, before incubation with shaking at RT for 30 minutes. After washing, the bound rat antibodies were assayed using IgG anti-goat AffiniPure rat (H + L) conjugated to a peroxidase, supplied by Jackson ImmunoLaboratories Inc. (ref: 112-035-003) diluted 1/5000 with PBS 0.05% tween. The detection antibodies were incubated with shaking for 30 minutes at room temperature. The color was developed using 4 mg of OPD + 5 μL of EJCh for 10 ml of 0.1 M citrate buffer at pH 4.5 for 15 minutes in the absence of light at room temperature. The reaction was stopped with 50 µL HCl, and the optical density (OD) was determined at 490 nm compared to 620 nm.
The level of anti-PcrV antibodies present in the serum is expressed by median titers. A TMG was calculated for the 20 samples from each of the treated groups.
Anti-06 ELISA (on mouse sera)
The purified O6-LPS was deposited, in physiological solution with phosphate buffer (PBS) at 8 pg / mL, on high affinity micro-titration plates (Nunc Maxisorp), at 4 ° C overnight. The plates were blocked with 1% PBS-BSA for 30 minutes with shaking at RT. The polyvalent antisera of mice were pre-diluted to 1/100 or 1/10, then new dilutions by two were carried out in micro107
BE2016 / 5783 plates, before incubation with shaking at RT for 30 minutes. After washing, the antibodies of bound mice were assayed using IgG anti-mouse goat AffiniPure (H + L) conjugated to a peroxidase, supplied by Jackson ImmunoLaboratories Inc. (ref: 115-035-003) diluted 1/2 500 with PBS-0.05% tween. The detection antibodies were incubated with shaking for 30 minutes at room temperature. The color was developed using 4 mg of OPD + 5 μL of EJCg for 10 ml of 0.1 M citrate buffer at pH 4.5 for 15 minutes in the absence of light at room temperature. The reaction was stopped with 50 µL HCl, and the optical density (OD) was determined at 490 nm compared to 620 nm. The level of anti-06 antibodies present in the serum is expressed by median titers. A TMG was calculated for the 20 individual samples from each of the treated groups.
Anti-PcrV ELISA (on mouse sera)
The purified histidine-labeled PcrV was deposited, in physiological solution with phosphate buffer (PBS) at 1 μg / ml, on high affinity micro-titration plates (Nunc Maxisorp), at 4 ° C. overnight. The plates were blocked with 1% PBS-BSA for 30 minutes with shaking at RT. The mouse antisera were pre-diluted 1/400, then new dilutions by two were made in the microplates, before incubation with shaking at RT for 30 minutes. After washing, the bound mouse antibodies were assayed using AffiniPure goat anti-mouse IgG (H + L) conjugated to a peroxidase, supplied by Jackson.
108
BE2016 / 5783
ImmunoLaboratories Inc. (ref: 115-035-003) diluted 1/5000 with PBS-0.05% tween. The detection antibodies were incubated with shaking for 30 minutes at room temperature. The color was developed using 4 mg of OPD + 5 μL of H2O2 for 10 ml of 0.1 M citrate buffer at pH 4.5 for 15 minutes in the absence of light at room temperature. The reaction was stopped with 50 µL HCl, and the optical density (OD) was determined at 490 nm compared to 620 nm.
The level of anti-PcrV antibodies present in the serum is expressed by median titers. A TMG was calculated for the 20 samples from each of the treated groups.
Response to an anti-06 opsonophagocytosis (OPA) assay
The opsonophagocytosis (OPA) assay was carried out in round bottom microplates with 15 μL of phagocytic HL-60 cells (suspension adjusted to 5.10 ⁶ cells / ml), 15 μL of P. aeruginosa bacteria (cultured in TSA medium in Petri dishes), 15 μL of dilutions of the test serum and 15 μL of porcine complement.
The inactivated test sera were diluted for the first time (1/4) in 1% HBSS-BSA and added to a strain of P. aeruginosa (GVXN 5871 diluted to arrive at a count of 200 to 250 CFU / well at the end of test).
The HL-60 cells (suspension adjusted to 5.10 ⁶ cells / mL) and the porcine complement (12.5% in the final medium) were added to each well. Every
109
BE2016 / 5783 tested sample corresponded to a control containing inactivated complement.
The reaction mixture was incubated at 37 ° C for 90 minutes with shaking. After a 1/200 dilution, 50 µL of the volume obtained was transferred to a flat-bottom microplate. 50 µL of MH agar was added, followed by PBS-0.9% agar (and an additional 50 µL for safety reasons). An automatic colony count was performed after overnight incubation at 30 ° C.
The opsonophagocytic activity was expressed as the inverse of the dilution of serum giving at least 50% elimination.
Anti-PcrV hemolysis inhibition response
ATCC 29260 (PCRV +) was cultured overnight at 37 ° C. with 5% CO 2, in TSA Agar medium in Petri dishes and harvested with 5 ml of MinS liquid medium. A few pL were inoculated into a Wyame vial and grown for 4 hours.
Two-fold dilutions of the test sera were made with 80 µL of phosphate buffered saline (DPBS) in 96-well U-bottom microplates.
pL of ATCC 29260 diluted 3 times were added (dilution allowing the lysis of 100% of the rabbit erythrocytes to be carried out). 80 μL of purified and diluted rabbit erythrocytes were then added to each well. The dilution of rabbit erythrocytes was determined for each assay in order to obtain an identical and standard inhibition of hemolysis. The plates were
110
BE2016 / 5783 centrifuged at 1000 rpm, at 4 ° C for 10 min and incubated at 37 ° C for 2 hours.
The plates were then centrifuged at
1,000 rpm for 10 minutes at 4 ° C. 150 µL (supernatant) of each well was then transferred to a flat bottom microplate and the optical density was determined at 405 nm with a microtiter plate reader.
The hemolysis inhibition activity was expressed by the median titers (50% inhibition) of the pooled sera.
Anti-06 IgG ELISA sets of individual post III and post II sera from 4 rats were evaluated by ELISA on all of the groups in the experiment. The results are shown in Figure 14.
A stimulating effect is observed for the sera collected post II to post III. The bioconjugate 06 without adjuvant has a low immunogenicity compared to all the bioconjugates 06 formulated in an oil-in-water emulsion containing an adjuvant, which are more immunogenic.
Anti-PcrV immune response sets of individual post and post III sera
II of 4 rats were evaluated by ELISA on all of the PcrV groups of the experiment (G5 to G9).
The geometric means of the median titers obtained for both the post II and post III rat sera are presented in FIG. 13.
111
BE2016 / 5783
A stimulating effect was observed between the collected sera post II and post III. A very weak anti-PcrV immune response or no anti-PcrV immune response was observed with the 06-PcrV bioconjugate without adjuvant. In contrast, a good immune response was observed against the PcrV support protein for the three 06 bioconjugates formulated in an oil-in-water adjuvant.
The highest antibody response was observed with the group 5 bioconjugate 06-PcrV comprising 20% of sugars / proteins (the highest protein concentration).
PcrV hemolysis inhibition assay:
The results of the hemolysis inhibition assays by PcrV are presented in FIG. 10. In mice immunized with the 06-PcrV conjugates, good rates of inhibition of hemolysis by PcrV were obtained for the of the conjugates tested. PcrV made it possible to generate functional antibodies with good inhibition rates of hemolysis by PcrV for all of the 06-PcrV bioconjugates tested on mice Although shorter chains of saccharides with a saccharide / protein ratio by 20% revealed a trend towards better rates than other conjugates, the improvement was not found to be statistically significant.
In both mice and rats, the presence of an oil in water emulsion improved the immune response against PcrV.
112
BE2016 / 5783
Results of opsonophagocytosis
The results of the opsonophagocytosis assays are shown in Figure 12. A good opsonophagocytosis response was obtained in samples for which the conjugate formulation contained an adjuvant in an oil-in-water emulsion. This has not been tested in samples not containing adjuvants. Subsequent studies have revealed very weak opsonic responses in sera from mice immunized with conjugates containing no adjuvants.
The scope of the present invention should in no case be limited by the specific embodiments described in the present description. Indeed, various modifications of the subject provided in the present description, in addition to those described, will become obvious to those skilled in the art after reading the previous description and the related figures. Such modifications are intended to fall within the scope of the appended claims.
Various publications, various patents and patent applications are cited in the present description, the descriptions of which are incorporated herein by reference in their entirety.
113
BE2016 / 5783

权利要求:
Claims (58)
[1]
1. Conjugate comprising an antigen covalently linked to a PcrV carrier protein from Pseudomonas aeruginosa comprising an amino acid sequence having at least 70% or 80% identity with the sequence of SEQ ID NO: 1 to 4, in which the antigen is linked (directly or via a linker) to an asparagine amino acid residue of the PcrV carrier protein of P. aeruginosa in which the asparagine residue is part of the consensus sequence D / E-XN-XS / T introduced into the amino acid sequence having at least 70% or 80% identity with the sequence of SEQ ID NO: 1 to 4, in which X represents any amino acid except proline, the asparagine residue being located at a position corresponding to amino acids between amino acids 24 to 166 or between amino acids 281 to 317, or at amino acid 317 of SEQ ID NO: 3 or at a position corresponding to amino acids between am acids ines 1 to 143 or between amino acids 258 to 294, or at amino acid 2 94 of the
SEQ ID NO: 4.
[2]
2. Conjugate according to claim 1, in which the asparagine residue is part of the consensus sequence D / EXNXS / T, in which X represents any amino acid except proline, the asparagine residue not being introduced by a mutation in the sequence of SEQ ID NO: 5, or a sequence having at least 80% identity with the sequence of SEQ ID NO: 5.
114
BE2016 / 5783
[3]
3. Conjugate according to any one of claims 1 or 2, in which a peptide comprising the consensus sequence D / EXNXS / T is introduced into the amino acid sequence by the elimination of a sequence of the PcrV peptide and its replacement by the peptide comprising the consensus sequence D / EXNXS / T.
[4]
4. Conjugate according to claim 3, in which the sequence of the PcrV peptide contains from 1 to 7 amino acids.
[5]
5. Conjugate according to claim 3, in which the sequence of the peptide PcrV contains an amino acid.
[6]
6. Conjugate according to any one of claims 1 to 5, in which the peptide comprising the consensus sequence D / EXNXS / T is introduced into the amino acid sequence at a position located between amino acid residues 24 and 143 of the SEQ ID NO: 3 or between amino acid residues 1 and 120 of SEQ ID NO: 4.
[7]
7. Conjugate according to claim 6, in which the peptide comprising the consensus sequence D / E-X-N-XS / T is introduced into the amino acid sequence at a position located between amino acid residues 24 and 48
from SEQ ID NO: 3 or between acid residues amines 1 and 24 of the SEQ ID NO: 4. 8. Conjugate according to any one of claims 1 at 7, in which at least 2, 3 or 4
consensus sequences D / E-X-N-X-S / T are introduced into the sequence corresponding to any one of SEQ ID NOs: 1 to 4 or a sequence having at least 70% or 80% identity with them.
115
BE2016 / 5783
[8]
9. Conjugate according to any one of claims 1 to 8, in which the carrier protein PcrV has a sequence comprising at least one of
SEQ ID NO: 6 to 62.
[9]
10. Conjugate according to claim 9, in which the PcrV carrier protein present comprising at least one of SEQ ID NO a sequence
6 to 12 and 33.
[10]
11. Conjugate according to claim 10, in which the carrier protein PcrV has a sequence comprising at least 3 of SEQ ID NOs: 6 to 12 and 33.
[11]
12. Conjugate according to claim 10 or 11, in which the carrier protein PcrV has a sequence comprising SEQ ID NO: 6 and / or SEQ ID NO: 9 and / or SEQ ID NO: 11 and / or SEQ ID NO: 33.
[12]
13. Conjugate according to any one of claims 1 to 12, in which the antigen is a saccharide.
[13]
14. The conjugate of claim 13, wherein the antigen is a bacterial capsular saccharide.
[14]
15. Conjugate according to claim 13, in which the antigen is a lipopolysaccharide or a bacterial lipooligosaccharide.
[15]
16. The conjugate according to claim 15, wherein the antigen is a lipopolysaccharide originating from P. aeruginosa.
[16]
17. Conjugate according to claim 16, in which the antigen is an O-antigen originating from P. aeruginosa, optionally 01, 02, 03, 04, 05, 06, 07, 08, 09, OlO, 011, 012, 013 , 014, 015, 016, 017, 018, 019 or 020, for example 06 or 011.
116
BE2016 / 5783
[17]
18. PcrV protein comprising an amino acid sequence having at least 70% or 80% identity with the sequence of SEQ ID NO: 1 to 4, said amino acid sequence comprising a consensus sequence D / EX-NXS / T, in which X represents any amino acid except proline.
[18]
19. The PcrV protein according to claim 18, in which the consensus sequence D / EXNXS / T, in which X represents any amino acid except proline, is located at a position between amino acids 23 and 166 or between amino acids 281 and 317 or at amino acid 317 of SEQ ID NO: 3.
[19]
20. The PcrV protein according to claim 18, in which the consensus sequence D / EXNXS / T, in which X represents any amino acid except proline, is located between amino acids 1 and 143 or between amino acids 258 and 294 or at amino acid 294 of SEQ ID NO: 4.
[20]
21. PcrV protein according to any one of claims 18 to 20, in which a peptide comprising the consensus sequence D / EXNXS / T is introduced into the amino acid sequence by the elimination of a sequence of the PcrV peptide and its replacement with a peptide comprising the consensus sequence D / EXNXS / T.
[21]
22. PcrV protein according to claim 21, in which the sequence of the PcrV peptide contains from 1 to 7 amino acids.
117
BE2016 / 5783
[22]
23. The PcrV protein according to claim 21, wherein the sequence of the PcrV peptide contains an amino acid.
[23]
24. PcrV protein according to any one of claims 18 to 23, in which the peptide comprising the consensus sequence D / EXNXS / T is introduced into the amino acid sequence at a position located between amino acid residues 24 and 143 of SEQ ID NO: 3 or between amino acid residues 1 and 120 of SEQ ID NO: 4.
[24]
25. The PcrV protein according to claim 24, in which the peptide comprising the consensus sequence D / EXNXS / T is introduced into the amino acid sequence at a position located between amino acid residues 24 and 48 of SEQ ID NO: 3 or between amino acid residues 1 and 24 of SEQ ID NO: 4.
[25]
26. PcrV protein according to any one of claims 18 to 25, in which at least 2, 3 or 4 consensus sequences D / EXNXS / T are introduced into the sequence corresponding to any one of SEQ ID NO: 1 to 4 or in a sequence having at least 80% identity with them.
[26]
27. PcrV protein according to any one of claims 18 to 26, which has an amino acid sequence which comprises at least one sequence from SEQ ID NO: 6 to 62.
[27]
28. The PcrV protein according to claim 27, which has an amino acid sequence which comprises at least one sequence from SEQ ID NOs: 6 to 12 and 33.
118
BE2016 / 5783
[28]
29. The PcrV protein according to claim 28, which has an amino acid sequence which comprises at least 3 sequences from SEQ ID NOs: 6 to 12 and 33.
[29]
30. PcrV protein according to claim 28 or 29,
who at a sequence amino acids which understands the SEQ ID NO: 6 and or the SEQ ID NO: 9 and or the SEQ ID NO: 11 and or the SEQ ID NO: 33.
[30]
31. The PcrV protein according to any one of claims 18 to 30, in which the amino acid sequence comprises a peptide marker which can be used for the purification of the PcrV protein.
[31]
32. The PcrV protein according to claim 31, wherein the peptide marker is located at the C-terminus of the amino acid sequence.
[32]
33. The PcrV protein according to any one of claims 18 to 32, in which the peptide marker comprises six histidine residues.
[33]
34. The PcrV protein according to any one of claims 18 to 33, wherein the amino acid sequence comprises a leader sequence which is capable of directing the PcrV protein to the periplasm of a bacterium.
[34]
35. The PcrV protein according to claim 34, in which the leader sequence has an amino acid sequence having at least 80% identity with SEQ ID NO: 63.
[35]
36. Immunogenic composition comprising the conjugate according to any one of claims 1 to 17, or the PcrV proteins according to any one of claims 18 to 35, and a pharmaceutically acceptable excipient.
119
BE2016 / 5783 according to the antigens
[36]
37. The immunogenic composition according to claim 36 further comprising additional antigens.
[37]
38. The immunogenic composition of claim 37 wherein additional are selected from the group consisting of a conjugate of an O-antigen and a carrier protein, a conjugate of a bacterial capsular polysaccharide and a carrier protein, a conjugate of an LOS, a carrier protein and a protein.
[38]
39. A method of preparing the immunogenic composition according to any one of claims 36 to 38 comprising the step of mixing the conjugate or the PcrV protein with a pharmaceutically acceptable excipient.
[39]
40. Conjugate or protein PcrV according to any one of claims 1 to 35 intended to be used in the treatment of an infection.
[40]
41. Conjugate or protein PcrV according to any one of claims 1 to 35 intended for use in the treatment of an infection by P. aeruginosa.
[41]
42. A PcrV conjugate for use according to claim 40 or claim 41, said treatment being that for a human subject in need thereof.
[42]
43. Conjugate, PcrV protein or immunogenic composition according to any one of the preceding claims 1 to 42, used in the treatment or prevention of a Pseudomonas infection.
120
BE2016 / 5783
[43]
44. Conjugate, PcrV protein or immunogenic composition according to any one of the preceding claims 1 to 42, used in treatment or prevention
Pseudomonas by
P.
aerugrnosa, from infection
[44]
45. Polynucleotide coding for the PcrV protein according to any one of claims 18 to 35,
[45]
46. Polynucleotide coding for a PcrV protein, the nucleotide sequence of which codes for a polypeptide having an amino acid sequence having at least 70% or 80% identity with any of SEQ ID NO: 1 to 4.
[46]
47. A vector comprising the polynucleotide according to claim 43 or claim 46.
[47]
48. Host cell comprising: nucleic
i) An acid glycosyltransferase;
ii) An acid coding for a nucleic acid oligosaccharyl transferase; and iii) A nucleic acid encoding a PcrV protein of P. aeruginosa according to any one of claims 18 to 35.
[48]
49. The host cell according to claim 48, in which the nucleic acid which codes for a glycosyltransferase is derived from an rfb aggregate of Pseudomonas, in which said nucleic acid sequence is optionally stably integrated into the genome of the host cell. .
[49]
50. Host cell according to claim 49, in which said Pseudomonas is Pseudomonas aeruginosa, optionally of serotype 06 or 011.
121
BE2016 / 5783
[50]
51. Host cell according to any one of claims 48 to 50, in which the oligosaccharyltransferase is derived from Campylobacter.
[51]
52. Host cell according to claim 51, in which the oligosaccharyl transferase is PglB from C. jejuni.
[52]
53. Host cell according to any one of claims 48 to 50, in which the nucleic acid coding for a PcrV protein of P. aeruginosa is present in a plasmid of the host cell.
[53]
54. A host cell according to any one of claims 48 to 53, further comprising a formyltransferase enzyme, wherein said nucleic acid encodes a protein having about or at least or 99
80%, 85%, 90%, 95%, 96%, 97%, identity or homology with SEQ ID NO: 65, or in which said nucleic acid codes for the
SEQ ID NO: 65.
[54]
55. Host cell according to any one of claims 48 to 54, further comprising a nucleic acid coding for a wzy polymerase, in which said nucleic acid codes for a protein having approximately or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of identity or homology with SEQ ID NO: 66, or in which said nucleic acid codes for SEQ ID NO: 66.
[55]
56. Host cell according to any one of claims 54 and 55, in which the nucleic acid coding for a formyltransferase enzyme and / or the nucleic acid coding for a wzy polymerase have been stably integrated into the genome of the cell. host.
122
BE2016 / 5783
[56]
57. Host cell according to any one of claims 54 and 55, in which a gene coding for a formyltransferase enzyme and / or a gene coding for a wzy polymerase is present in a plasmid.
5 the host cell.
[57]
58. Host cell according to any one of claims 48 to 57, said host cell being E. coli.
[58]
59. A method of producing a bioconjugate comprising a PcrV protein of P. aeruginosa linked to a saccharide, said method comprising culturing the host cell according to any one of claims 48 to 58, under conditions suitable for production protein.
60. A bioconjugate produced by the method of claim 59, said bioconjugate comprising a saccharide linked to a PcrV protein of P. aeruginosa.
123
BE2016 / 5783
124
BE2016 / 5783 y
<
Z '5 σ
Û <t
E
u.
z
TO ü
I
Q
Z <
y <
Z "
O <~ i _ôh
E
TO
C ££ n
125
BE2016 / 5783
Of
126
BE2016 / 5783 fmtO6
06wzy
Φ
TJ
Φ "-Φ
E o
O_
LD
O
Q
O c
CD
C5 u
<u
C5 ci:
LO
Gold
Ö .SP
127
BE2016 / 5783
IT) üb
E
128
BE2016 / 5783
Line 1 4 . .. .1 .... ΓΖΖ 5, I ϋ ιύ Sample " i 2 ₅ i - γτ ^ , -, Strain St 343 Ρ <ί06 genomics jKjIB genomics P.ciV ϊΊίί3ζΐ E * 153S rWS *. ..plSOÇi. . P-W95 i »2öW ρ301 © P202Ü (j iycosh 1 i mutatîonot ί 2 + - 1 l + i-k. î «4 + î I + UÏ444-

类似技术:

---

公开号 | 公开日 | 专利标题

---

BE1024361B1|2018-02-05|IMMUNOGENIC COMPOSITION

---

BE1022345A9|2016-11-16|USPA2 PROTEIN CONSTRUCTS AND USES THEREOF

---

BE1023838B1|2017-08-09|MODIFIED HOST CELLS FOR USE IN THE PRODUCTION OF BIOCONJUGATES

---

CA2883000A1|2014-03-13|Bioconjugates comprising modified antigens and uses thereof

---

BE1024767B1|2018-06-28|IMMUNOGENIC COMPOSITION

---

JP2020114232A|2020-07-30|Modified host cell and use of the same

---

BE1021938B1|2016-01-27|IMMUNOGENIC COMPOSITION FOR USE IN THERAPY

---

BE1025210B1|2018-12-12|IMMUNOGENIC CONJUGATES AND THEIR USE

---

JP6764872B2|2020-10-07|Acinetobacter O-oligosaccharide transferase and its use

---

BE1025443B1|2019-02-28|NOMV-ANTIGEN CONJUGATES AND THEIR USE

---

EP2429658B1|2016-04-20|Method for adjuvating the lipopolysaccharide | of gram-negative bacteria

---

FR2816844A1|2002-05-24|Composition containing enterobacterial outer membrane peptides, useful e.g. as vaccine carrier or adjuvant, derived from the protein&#39;s periplasmic domain

---

Wu et al.2005|Investigation of nontypeable Haemophilus influenzae outer membrane protein P6 as a new carrier for lipooligosaccharide conjugate vaccines

---

BE1024794B1|2018-07-10|IMMUNOGENIC COMPOSITIONS

---

JP6998866B2|2022-02-04|Pseudomonas aeruginosa PcrV-linked antigen vaccine

---

BE1023004B1|2016-10-31|PROCESSING PROCESS

---

TWI751513B|2022-01-01|BIOCONJUGATES OF E. coli O-ANTIGEN POLYSACCHARIDES, METHODS OF PRODUCTION THEREOF, AND METHODS OF USE THEREOF

---

EP2430151B1|2014-06-18|Meningococcal vaccine constituted of lipooligosaccharides from modified immunotype l6 neisseria meninigitidis

---

TW202102255A|2021-01-16|Bioconjugates of e. coli o-antigen polysaccharides, methods of production thereof, and methods of use thereof

---

WO2021259743A2|2021-12-30|Vaccine

---

BE1023004A1|2016-10-31|PROCESSING PROCESS

---

FR2828106A1|2003-02-07|Composition containing peptide from low molecular weight outer membrane protein, useful for preparing vaccines against infections or cancer

同族专利:

---

公开号 | 公开日

---

BE1024361A1|2018-01-30|

---

CN108778322A|2018-11-09|

---

US20190091319A1|2019-03-28|

---

JP2018535207A|2018-11-29|

---

WO2017067964A1|2017-04-27|

---

GB201518668D0|2015-12-02|

---

CA3002117A1|2017-04-27|

---

BR112018007960A2|2018-10-30|

---

MX2018004938A|2018-07-06|

---

EP3365004A1|2018-08-29|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US4235877A|1979-06-27|1980-11-25|Merck & Co., Inc.|Liposome particle containing viral or bacterial antigenic subunit|

---

US5057540A|1987-05-29|1991-10-15|Cambridge Biotech Corporation|Saponin adjuvant|

---

US4912094B1|1988-06-29|1994-02-15|Ribi Immunochem Research Inc.|Modified lipopolysaccharides and process of preparation|

---

WO1990014837A1|1989-05-25|1990-12-13|Chiron Corporation|Adjuvant formulation comprising a submicron oil droplet emulsion|

---

CA2138997C|1992-06-25|2003-06-03|Jean-Paul Prieels|Vaccine composition containing adjuvants|

---

EP0812593B8|1993-03-23|2010-11-10|SmithKline Beecham Biologicals S.A.|Vaccine compositions containing 3-0 deacylated monophosphoryl lipid a|

---

GB9326253D0|1993-12-23|1994-02-23|Smithkline Beecham Biolog|Vaccines|

---

DK0772619T4|1994-07-15|2011-02-21|Univ Iowa Res Found|Immunomodulatory oligonucleotides|

---

EP0717106B1|1994-12-16|2000-03-15|Chiron Behring Gmbh &amp; Co.|Immunogenic hybrid protein OprF-Oprl derived from Pseudomonas aeruginosa membrane proteins|

---

DE69637254T2|1995-04-25|2008-06-19|Glaxosmithkline Biologicals S.A.|Vaccines containing a saponin and a sterol|

---

EP1009382B1|1997-09-05|2003-06-18|GlaxoSmithKline Biologicals S.A.|Oil in water emulsions containing saponins|

---

GB9718901D0|1997-09-05|1997-11-12|Smithkline Beecham Biolog|Vaccine|

---

WO2002058737A2|2001-01-23|2002-08-01|Aventis Pasteur|Multivalent meningococcal polysaccharide-protein conjugate vaccine|

---

ES2353814T3|2005-05-11|2011-03-07|Eth Zuerich|RECOMBINANT N-GLYCOSILATED PROTEINS OF PROCEDURAL CELLS.|

---

AT539079T|2006-03-23|2012-01-15|Novartis Ag|IMIDAZOCHINOXALINE COMPOUNDS AS IMMUNOMODULATORS|

---

WO2007109812A2|2006-03-23|2007-09-27|Novartis Ag|Immunopotentiating compounds|

---

LT2257307T|2008-02-20|2018-08-10|Glaxosmithkline Biologicals S.A.|Bioconjugates made from recombinant n-glycosylated proteins from procaryotic cells|

---

ES2678694T3|2008-04-16|2018-08-16|Glaxosmithkline Biologicals Sa|Vaccine|

---

WO2009127677A1|2008-04-16|2009-10-22|Glaxosmithkline Biologicals S.A.|Vaccine|

---

CN101559222A|2009-05-31|2009-10-21|北京绿竹生物制药有限公司|Combined vaccine bonding bacterial polysaccharides and protein for human|

---

EP2501406B8|2009-11-19|2018-01-24|GlaxoSmithKline Biologicals S.A.|Biosynthetic system that produces immunogenic polysaccharides in prokaryotic cells|

---

PT2566507T|2010-05-06|2018-02-06|Glaxosmithkline Biologicals Sa|Capsular gram-positive bacteria bioconjugate vaccines|

---

EP2718320B1|2011-06-10|2018-01-10|MedImmune Limited|Anti-pseudomonas psl binding molecules and uses thereof|

---

US8932586B2|2011-09-06|2015-01-13|Intrexon Corporation|Modified forms of Pseudomonas exotoxin A|

---

CA2887133A1|2012-10-12|2014-04-17|Glycovaxyn Ag|Methods of host cell modification|

---

WO2014072405A1|2012-11-07|2014-05-15|Glycovaxyn Ag|Production of recombinant vaccine in e. coli by enzymatic conjugation|

---

EP3131577B1|2014-04-17|2020-04-22|GlaxoSmithKline Biologicals S.A.|Modified host cells and uses thereof|

---

ES2793023T3|2014-08-08|2020-11-12|Glaxosmithkline Biologicals Sa|Modified host cells and hybrid oligosaccharides for use in the production of bioconjugates|EP3894431A2|2018-12-12|2021-10-20|GlaxoSmithKline Biologicals SA|Modified carrier proteins for o-linked glycosylation|

---

PE20212265A1|2019-03-18|2021-11-30|Janssen Pharmaceuticals Inc|BIOCONJUGATES OF ANTIGENS-POLYSACCHARIDES OF E. COLI, PRODUCTION METHODS AND METHODS OF USE OF THE SAME|

---

KR20210134044A|2019-03-18|2021-11-08|얀센 파마슈티칼즈, 인코포레이티드|Method for producing bioconjugate of E. coli O-antigen polysaccharide, composition thereof and method of using same|

---

WO2021255684A1|2020-06-18|2021-12-23|Glaxosmithkline Biologicals Sa|Shigella-tetravalentbioconjugate|

法律状态:
2018-02-28| FG| Patent granted|Effective date: 20180205 |

---

2019-06-26| MM| Lapsed because of non-payment of the annual fee|Effective date: 20181031 |

优先权:

---

申请号 | 申请日 | 专利标题

---

GBGB1518668.7A|GB201518668D0|2015-10-21|2015-10-21|Immunogenic Comosition|

---

GB1518668.7|2015-10-21|

---