WO2019043245A1 - Cellules microbiennes exprimant des sérotypes streptococciques - Google Patents

Cellules microbiennes exprimant des sérotypes streptococciques Download PDF

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WO2019043245A1
WO2019043245A1 PCT/EP2018/073714 EP2018073714W WO2019043245A1 WO 2019043245 A1 WO2019043245 A1 WO 2019043245A1 EP 2018073714 W EP2018073714 W EP 2018073714W WO 2019043245 A1 WO2019043245 A1 WO 2019043245A1
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microbial cell
microbial
nucleotide sequence
seq
nucleic acid
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Emily KAY
Brendan Wren
Jon CUCCUI
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London School of Hygiene and Tropical Medicine
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London School of Hygiene and Tropical Medicine
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Priority claimed from GBGB1714128.4A external-priority patent/GB201714128D0/en
Priority claimed from GBGB1807669.5A external-priority patent/GB201807669D0/en
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Publication of WO2019043245A1 publication Critical patent/WO2019043245A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines

Definitions

  • the disclosure relates to a glycoconjugate vaccine comprising one or more polysaccharides covalently linked to one or more carrier polypeptides wherein said glyconjugate is manufactured by a modified microbial cell; vaccines and immunogenic compositions comprising said glycoconjugate(s); uses and methods of treatment of microbial infections using said vaccines and immunogenic compositions; bioreactors for the manufacture of said glycoconjugates and methods to prepare said glycoconjugate(s) from said modified microbial cells.
  • the disclosure provides an efficient means to manufacture vaccines effective in protecting human subjects from microbial infection, particularly in relation to the manufacture of pneumococcal glycoconjugate vaccines, for example, vaccines protective against Streptococcus pneumoniae.
  • a “carrier” is an immunogenic molecule which, when bound to a second molecule that augments immune responses to the latter.
  • Some antigens are not intrinsically immunogenic, for example glycans, yet may be capable of generating antibody responses when associated with a foreign protein molecule such as keyhole-limpet haemocyanin or tetanus toxoid.
  • Such antigens contain B-cell epitopes but no T-cell epitopes.
  • the protein moiety of such a conjugate (the "carrier” protein) provides T-cell epitopes which stimulate helper T-cells that in turn stimulate antigen-specific B-cells to differentiate into plasma cells and produce antibody against the antigen.
  • Protein D from Haemophilus influenzae is Protein D from Haemophilus influenzae, CRM197 a non-toxic recombinant variant of the diphtheria toxin of Corynebacterium diphtheria, or the tetanus toxoid of Clostridium tetani.
  • Streptococcus pneumoniae, or pneumococcus is an important human pathogen causing a variety of invasive illnesses such as pneumonia, meningitis and bacteraemia. Serious pneumococcal infections are most common in infants, toddlers, the elderly, and subjects with deficient immune systems such as HIV patients or other underlying conditions.
  • S. pneumoniae is the causative agent for pneumonia related deaths in developing countries.
  • S. pneumoniae is the causative agent for pneumonia related deaths in developing countries.
  • Prevnar® comprises seven serotypes: 4, 6B, 9V, 14, 18C, 19F and 23F, and is suitable for immunisation of infants less than 2 years old and is crosslinked to a diphtheria toxin carrier protein.
  • An alternative thirteen component vaccine also linked to diphtheria toxin comprises serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F and is suitable for human subjects over 65 years old.
  • a further ten serotype pneumococcal vaccine comprises the following serotypes; 1 , 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. These vaccines are effective in protection against pneumococcal infection and are manufactured by chemical conjugation which is expensive.
  • vaccine and immunogenic compositions comprising carrier polypeptides conjugated with bacterial polysaccharides wherein said bacterial polysaccharides (serotypes) are conjugated to a carrier polypeptide in a microbial based cell expression system adapted to express said carrier polypeptide, one bacterial polysaccharide and an oligosaccharyltransferase.
  • bacterial cells that are genetically modified to facilitate the production of said polysaccharide conjugate vaccines that include modifications to the microbial genome which result in the synthesis of full length native or near native bacterial capsular polysaccharides that are immunogenic.
  • Further pneumococcal vaccines comprising serotype 8 are disclosed.
  • a microbial cell wherein said cell comprises:
  • nucleic acid molecule comprising a nucleotide sequence encoding one or more polypeptides involved in the synthesis of one or more heterologous bacterial capsular polysaccharides not expressed by said microbial cell; ii) a nucleic acid molecule comprising a nucleotide sequence encoding at least one heterologous polysaccharide chain length regulator;
  • nucleic acid molecule comprising a nucleotide sequence encoding one or more carrier polypeptides that comprise one or more glycosylation motifs;
  • nucleic acid molecule comprising a nucleotide sequence encoding an oligosaccharyltransferase.
  • the genome of said microbial cell is modified in one or more endogenous genes encoding a polysaccharide chain length regulator characterised in that said endogenous bacterial capsular polysaccharide chain length regulator is modified or inactivated and substantially lacks expression and/or activity.
  • Means to mutate and/or inactivate microbial genes are known in the art and include the use of mutagens, homologous recombination and transposon integration to disrupt gene function. More recently CRISPR/Cas 9 has enabled the targeted modification of genomic DNA to introduce permanent genetic modifications with high precision.
  • said microbial cell is a bacterial cell, for example an Escherichia coli cell.
  • said bacterial cell is a lactic acid bacteria of the genus Lactococcus or Lactobacillus.
  • said microbial cell is a fungal, algal or yeast cell.
  • said nucleic acid encoding polypeptides involved in the synthesis of heterologous bacterial capsular polysaccharide is isolated from a Gram-negative or Gram-positive bacterial species.
  • said Gram negative bacterial species is selected from the genus: Nesseria spp, Klebsiella spp, Haemophilus spp, Pseudomonas spp and Salmonella spp.
  • said Gram positive bacterial species is selected from the genus: Bacillus spp, Lactococcus spp, Lactobacillus spp, Streptococcus spp and Staphylococcus spp.
  • Streptococcus species is Streptococcus pneumoniae.
  • said nucleic acid molecule encoding one or more polypeptides involved in the synthesis of one or more heterologous bacterial capsular polysaccharides synthesizes a Streptococcus pneumoniae polysaccharide serotype selected from one of the 96 serotypes expressed by Streptococcus pneumoniae.
  • nucleic acid molecule encoding one or more polypeptides involved in the synthesis of a heterologous bacterial capsular polysaccharides synthesizes a Streptococcus pneumoniae polysaccharide serotype wherein said polysaccharide comprises an acetylated reducing end sugar.
  • acetylated reducing end sugar comprises a 2-acetamido group.
  • nucleic acid molecule encoding one or more polypeptides involved in the synthesis of a heterologous bacterial capsular polysaccharides synthesize a Streptococcus pneumoniae polysaccharide serotype wherein said polysaccharide comprises glucose as reducing end sugar.
  • nucleic acid molecule encoding one or more polypeptides involved in the synthesis of a heterologous bacterial capsular polysaccharides synthesize a Streptococcus pneumoniae polysaccharide serotype wherein said polysaccharide comprises galactose or a galactose derivative as reducing end sugar.
  • said polysaccharide comprising an acetylated reducing end sugar is selected from the group of serotypes consisting of: 1 , 4, 5, 12F,12A, 12B, 25F, 25A, 38, 44, 45 or 46.
  • said polysaccharide comprising glucose as reducing end sugar is serotype 8.
  • said polysaccharide comprises galactose or a galactose derivative as the reducing end sugar, either structurally confirmed or suggested by the presence of wcjG or wcjH: 10F, 10A, 10B, 10C, 29, 31 , 33C, 35F, 39, 43, 47F, 47A.
  • said serotypes are selected from a group consisting of: 1 , 4, 5, 12F, 12A, 12B, 25F, 25A, 38, 44, 45, 46 or 8.
  • the operons encoding genes involved in the synthesis of capsular polysaccharides are known in the art, for example see Table 1 and Table 5 and accession numbers therein.
  • said nucleic acid molecule encoding one or more polypeptides involved in the synthesis of one or more heterologous bacterial capsular polysaccharides synthesizes a native Streptococcus pneumoniae serotype 4 polysaccharide.
  • nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 1.
  • said microbial cell expresses one or more AATGal synthesis genes encoded by SEQ ID NO: 69 and SEQ ID NO: 70
  • said nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NO: 57.
  • said nucleic acid molecule encoding one or more polypeptides involved in the synthesis of one or more heterologous bacterial capsular polysaccharides synthesizes a Streptococcus pneumoniae polysaccharide serotype selected from the group consisting of: 1 or 4 or 5.
  • said nucleic acid molecule encoding one or more polypeptides involved in the synthesis of a heterologous bacterial capsular polysaccharides synthesizes a Streptococcus pneumoniae polysaccharide serotype 8 or 1 or 4 or 5.
  • said microbial cell expresses the serotype capsular polysaccharides 1 , 4 and 5.
  • said microbial cell expresses the serotype capsular polysaccharide 8, or serotype 1 , 4, 5 and 8.
  • said microbial cell expresses a serotype capsular polysaccharide selected from the group consisting of: 4, 6B, 9V, 14, 18C, 19F and 23F.
  • said microbial cell expresses a serotype capsular polysaccharide selected from the group consisting of: 1 , 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.
  • said microbial cell expresses serotype 1 .
  • said microbial cell expresses serotype 1 encoded by the nucleotide sequence set forth in SEQ ID NO:58.
  • said microbial cell expresses serotype 3.
  • Methods for making immunogenic conjugates comprising Streptococcus pneumoniae serotype 3 are known in the art and disclosed in patent application US2010316666 which is hereby incorporated by reference in its entirety.
  • said microbial cell expresses serotype 4.
  • said microbial cell expresses serotype 4 encoded by the nucleotide sequence set forth in SEQ ID NO: 1 .
  • said microbial cell expresses serotype 5
  • said microbial cell expresses serotype 5 encoded by the nucleotide sequence set forth in SEQ ID NO: 59.
  • said microbial cell expresses serotype 6A.
  • said microbial cell expresses serotype 6A encoded by the nucleotide sequence set forth in SEQ ID NO: 74.
  • said microbial cell expresses serotype 6B.
  • said microbial cell expresses serotype 6A encoded by the nucleotide sequence set forth in SEQ ID NO: 75.
  • said microbial cell expresses serotype 7F.
  • said microbial cell expresses serotype 7F encoded by the nucleotide sequence set forth in SEQ ID NO: 76.
  • said microbial cell expresses serotype 9V.
  • said microbial cell expresses serotype 9V encoded by the nucleotide sequence set forth in SEQ ID NO:77.
  • said microbial cell expresses serotype 14.
  • said microbial cell expresses serotype 14 encoded by the nucleotide sequence set forth in SEQ ID NO: 78.
  • said microbial cell expresses serotype 18C.
  • said microbial cell expresses serotype 18C encoded by the nucleotide sequence set forth in SEQ ID NO: 79.
  • said microbial cell expresses serotype 19A.
  • said microbial cell expresses serotype 19A encoded by the nucleotide sequence set forth in SEQ ID NO: 80.
  • said microbial cell expresses serotype 19F.
  • said microbial cell expresses serotype 19F encoded by the nucleotide sequence set forth in SEQ ID NO: 81 .
  • said microbial cell expresses the seroptype 23F.
  • said microbial cell expresses serotype 23F encoded by the nucleotide sequence set forth in SEQ ID NO: 82.
  • said microbial cell expresses one or more ribitol genes encoded by the nucleotide sequence set forth in SEQ ID NO: 73.
  • nucleic acid molecules comprising nucleotide sequences encoding the serotypes described herein can vary due to e.g. naturally occurring sequence polymorphisms and can comprise a nucleotide sequence which is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical over the full-length nucleotide sequence of the sequences set forth in SEQ ID NOs 1 , 57-68 and 73-82.
  • nucleic acid molecule encoding one or more polypeptides involved in the synthesis of one or more heterologous bacterial capsular polysaccharides is provided on an episomal plasmid.
  • nucleic acid molecule encoding one or more polypeptides involved in the synthesis of one or more heterologous bacterial capsular polysaccharides is stably integrated into the genome of said microbial cell.
  • nucleic acid encoding a heterologous polysaccharide chain length regulator is encoded by the wzd gene and wze gene.
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • SEQ ID NO: 15 that has at least 70% nucleotide sequence identity over the full nucleotide sequence and encodes a polypeptide with polysaccharide chain length regulator activity.
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • SEQ ID NO: 14 that has at least 70% nucleotide sequence identity over the full nucleotide sequence and encodes a polypeptide with polysaccharide chain length regulator activity.
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • nucleic acid encoding a heterologous polysaccharide chain length regulator is selected from the group consisting of: i) a nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 5 and SEQ ID NO: 17;
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • SEQ ID NO: 17 that has at least 70% nucleotide sequence identity over the full nucleotide sequence and encodes a polypeptide with polysaccharide chain length regulator activity.
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • SEQ ID NO: 18 that has at least 70% nucleotide sequence identity over the full nucleotide sequence and encodes a polypeptide with polysaccharide chain length regulator activity.
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • SEQ ID NO: 19 that has at least 70% nucleotide sequence identity over the full nucleotide sequence and encodes a polypeptide with polysaccharide chain length regulator activity.
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • SEQ ID NO: 20 that has at least 70% nucleotide sequence identity over the full nucleotide sequence and encodes a polypeptide with polysaccharide chain length regulator activity.
  • said nucleic acid encoding a heterologous polysaccharide chain length regulator is selected from the group consisting of: i) a nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 9 and SEQ ID NO: 21 ;
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • SEQ ID NO: 21 that has at least 70% nucleotide sequence identity over the full nucleotide sequence and encodes a polypeptide with polysaccharide chain length regulator activity.
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • SEQ ID NO: 23 that has at least 70% nucleotide sequence identity over the full nucleotide sequence and encodes a polypeptide with polysaccharide chain length regulator activity.
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • nucleic acid encoding a heterologous polysaccharide chain length regulator is selected from the group consisting of: i) a nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO: 13 and SEQ ID NO: 25;
  • nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NO:
  • said oligosaccharyltransferase is a Campylobacter oligosaccharyltransferase.
  • Campylobacter oligosaccharyltransferase is a Campylobacter y ' e/i/n/ ' oligosaccharyltransferase.
  • said Campylobacter oligosaccharyltransferase is a Campylobacter sputorum oligosaccharyltransferase.
  • said oligosaccharyltransferase is encoded by a nucleic acid molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 26, or a nucleotide sequence that has at least 50% nucleotide sequence identity over the full length of the nucleotide sequence set forth in SEQ ID NO: 26.
  • said oligosaccharyltransferase is encoded by a nucleic acid molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 27 or 28, or a nucleotide sequence that has at least 50% nucleotide sequence identity over the full length of the nucleotide sequence set forth in SEQ ID NO: 27 or SEQ ID NO: 28.
  • said oligosaccharyltransferase is represented by the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence that is at least 50% identical over the full length of the amino acid sequence set forth in SEQ ID NO: 29.
  • said oligosaccharyltransferase is represented by the amino acid sequence set forth in SEQ ID NO: 30, or an amino acid sequence that is at least 50% identical over the full length of the amino acid sequence set forth in SEQ ID NO: 30.
  • said oligosaccharyltransferase has at least 55% identity, more preferably at least 60% identity, even more preferably at least 65% identity, still more preferably at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% identity, and most preferably at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the full length amino or nucleotide acid sequences as set forth in SEQ ID NOs 26 to 30.
  • said oligosaccharyltransferase is encoded by the PgIL gene, for example encoded by the PgIL gene of Actinobacteria and encoded by the nucleotide sequence set forth in SEQ ID NO: 72.
  • said microbial cell is adapted to express a glycosyltransferase encoded by a nucleotide sequence as set forth in SEQ ID NO: 71 (NGT).
  • said oligosaccharyltransferase has at least 55% identity, more preferably at least 60% identity, even more preferably at least 65% identity, still more preferably at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% identity, and most preferably at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the full length nucleic acid sequences as set forth in SEQ ID NOs 71 or 72.
  • said nucleic acid molecule encoding said oligosaccharyltransferase or glycosyltransferase is stably integrated into the genome of said microbial or bacterial cell.
  • said one or more carrier polypeptide(s) includes the amino acid motif: Asn-X-Ser or Asn-X-Thr where X is any amino acid except proline.
  • said one or more carrier polypeptide(s) includes the amino acid motif: D/E-X-N-X-S/T, wherein X is any amino acid except proline.
  • said one or more carrier polypeptide(s) includes the amino acid motif D/E-X-N-X-S/T, wherein X is any amino acid except proline and is selected from the group consisting of: DVNVT, EVNAT, DQNAT, DNNNT, DNNNS, DQNRT, ENNFT, DSNST, DQNIS, DQNVS, DNNVS, DYNVS, DFNVS, DFNAS, DFNSS, DVNAT, DFNVT or DVNAS.
  • X is any amino acid except proline and is selected from the group consisting of: DVNVT, EVNAT, DQNAT, DNNNT, DNNNS, DQNRT, ENNFT, DSNST, DQNIS, DQNVS, DNNVS, DYNVS, DFNVS, DFNAS, DFNSS, DVNAT, DFNVT or DVNAS.
  • nucleic acid encoding said carrier polypeptide(s) is stably integrated into the microbial genome.
  • said endogenous bacterial serotype polysaccharide chain length regulator is modified and encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: i) a nucleic acid molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 31 ;
  • nucleic acid molecule comprising a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);
  • nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the nucleotide sequence in i) and ii) above wherein said nucleic acid molecule encodes a bacterial serotype polysaccharide chain length regulator and is at least 75% identical over the full-length nucleotide sequence set forth in SEQ ID NO: 31 , wherein said nucleic acid molecule is mutated or deleted for all or part of the nucleotide sequence encoding said endogenous bacterial serotype polysaccharide chain length regulator and substantially lacks expression and/or encodes a polypeptide that substantially lacks enzyme activity.
  • Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other.
  • the stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001 ); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993).
  • the T m is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand.
  • the following is an exemplary set of hybridization conditions and is not limiting: Very High Stringency (allows sequences that share at least 90% identity to hybridize) Hybridization: 5x SSC at 65°C for 16 hours
  • polypeptide comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 32;
  • a polypeptide comprising or consisting of an amino acid sequence that is at least 75% identical over the full length of the amino acid sequence set forth in SEQ ID NO: 32 and is a bacterial serotype polysaccharide chain length regulator and
  • the bacterial serotype polysaccharide chain length regulator has at least 75% 80%, 85% or 90%, or most preferably at least 95%, 96%, 97%, 98% or 99% identity with the full-length nucleotide or amino acid sequences as set forth in SEQ ID NO 31 or 32.
  • a vaccine or immunogenic composition comprising a glycoconjugate vaccine obtained or obtainable from the microbial cell according to the invention.
  • said composition further includes an adjuvant
  • a vaccine or immunogenic composition according to the invention for use in the treatment of microbial infection or a condition resulting from a microbial infection in a subject.
  • said subject is human. In an alternative embodiment of the invention said subject is a non-human animal.
  • said microbial infection is a streptococcal infection or a condition resulting from a streptococcal infection.
  • the streptococcal infection or condition is caused by a streptococcal species selected from the group consisting of: Streptococcus pyogenes; S.agalactiae, S.dysgalactiae, S. bovis, S. sanguinis, S.suis, S.mitis, S.mutans and S. pneumoniae.
  • the streptococcal infection or condition is caused by S. pneumoniae.
  • the subject infected with a streptococcal infection or suffering from a condition associated with a streptococcal infection is an elderly human subject.
  • the subject infected with a streptococcal infection or suffering from a condition associated with a streptococcal infection is an infant human subject.
  • a microbial culture comprising a cell according to the invention.
  • a cell culture vessel comprising a microbial culture according to the invention.
  • said cell culture vessel is a fermenter.
  • a method for the manufacture of a glycoconjugate according to the invention comprising the steps: i) providing a microbial cell culture according to the invention;
  • glycoconjugate obtained or obtainable by the method according to the invention.
  • a cell culture vessel comprising a bacterial cell culture according to the invention.
  • said cell culture vessel is a fermenter.
  • a microbial cell wherein said cell comprises a modified bacterial serotype polysaccharide chain length regulator selected from the group consisting of: i) a nucleic acid molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 31 ;
  • nucleic acid molecule comprising a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);
  • nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the nucleotide sequence in i) and ii) above wherein said nucleic acid molecule encodes a bacterial serotype polysaccharide chain length regulator and is at least 75% identical to the nucleotide sequence set forth in SEQ ID NO: 31 ,
  • nucleic acid molecule is mutated or deleted for all or part of the nucleotide sequence encoding said bacterial serotype polysaccharide chain length regulator and substantially lacks expression and/or encodes a polypeptide that substantially lacks enzyme activity.
  • the bacterial serotype polysaccharide chain length regulator has at least 75% 80%, 85% or 90%, and most preferably at least, 91 %, 93%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the full-length nucleotide or amino acid sequences as set forth in SEQ ID NO 31 or 32.
  • kits comprising one or more microbial cells according to the invention wherein each microbial cell is engineered to express at least one serotype.
  • said kit comprises a microbial cell genetically engineered to express a serotype selected from the group consisting of: 4, 6B, 9V, 14, 18C, 19F or 23F.
  • said kit comprises a microbial cell genetically engineered to express a serotype selected from the group consisting of: 1 , 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F.
  • said kit comprises a microbial cell genetically engineered to express a serotype selected from the group consisting of: 1 , 4, 5, 6B, 7F, 9V, 14, 18C, 19F or 23F.
  • said kit comprises a microbial cell genetically engineered to express a serotype selected from the group consisting of: 4, 6B, 9V, 14, 18C, 19F or 23F.
  • said kit comprises microbial cells that express each of said serotypes.
  • the kit provides therefore a microbial cell or a plurality of microbial cells which express individually one serotype.
  • said microbial cells express serotypes that can provide a multivalent vaccine composition.
  • Microbial Cell Culture Bacterial cultures used in the process according to the invention are grown or cultured in the manner with which the skilled worker is familiar, depending on the host organism.
  • bacteria are grown in a liquid medium comprising a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0°C and 100°C, preferably between 10°C and 60°C, while gassing in oxygen.
  • the pH of the liquid medium can either be kept constant, that is to say regulated during the culturing period, or not.
  • the cultures can be grown batchwise, semi-batchwise or continuously. Nutrients can be provided at the beginning of the fermentation or fed in semi- continuously or continuously.
  • the products produced can be isolated from the bacteria as described above by processes known to the skilled worker, for example by extraction, distillation, crystallization, if appropriate precipitation with salt, and/or chromatography.
  • the pH value is advantageously kept between pH 4 and 12, preferably between pH 6 and 9, especially preferably between pH 7 and 8.
  • the culture medium to be used must suitably meet the requirements of the bacterial strains in question. Descriptions of culture media for various bacteria can be found in the textbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981 ).
  • these media which can be employed in accordance with the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements.
  • Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Examples of carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
  • Sugars can also be added to the media via complex compounds such as molasses or other by-products from sugar refining. The addition of mixtures of a variety of carbon sources may also be advantageous.
  • oils and fats such as, for example, soya oil, sunflower oil, peanut oil and/or coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as, for example, glycerol, methanol and/or ethanol, and/or organic acids such as, for example, acetic acid and/or lactic acid.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds.
  • nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as cornsteep liquor, soya meal, soya protein, yeast extract, meat extract and others.
  • the nitrogen sources can be used individually or as a mixture.
  • Inorganic salt compounds which may be present in the media comprise the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
  • Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur- containing fine chemicals, in particular of methionine.
  • Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts may be used as sources of phosphorus.
  • Chelating agents may be added to the medium in order to keep the metal ions in solution.
  • Particularly suitable chelating agents comprise dihydroxyphenols such as catechol or protocatechuate and organic acids such as citric acid.
  • the fermentation media used according to the invention for culturing bacteria usually also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine. Growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium.
  • composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the optimization of media can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach” (Editors P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.
  • All media components are sterilized, either by heat (20 min at 1 .5 bar and 121 °C) or by filter sterilization.
  • the components may be sterilized either together or, if required, separately. All media components may be present at the start of the cultivation or added continuously or batchwise, as desired.
  • the culture temperature is normally between 15°C and 45°C, preferably at from 25°C to 40°C, and may be kept constant or may be altered during the experiment.
  • the pH of the medium should be in the range from 5 to 8.5, preferably around 7.0.
  • the pH for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid.
  • Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters.
  • suitable substances having a selective effect for example antibiotics.
  • Aerobic conditions are maintained by introducing oxygen or oxygen-containing gas mixtures such as, for example, ambient air into the culture.
  • the temperature of the culture is normally 20°C to 45°C and preferably 25°C to 40°C.
  • the culture is continued until formation of the desired product is at a maximum. This aim is normally achieved within 10 to 160 hours.
  • the fermentation broth can then be processed further.
  • the biomass may, according to requirement, be removed completely or partially from the fermentation broth by separation methods such as, for example, centrifugation, filtration, decanting or a combination of these methods or be left completely in said broth. It is advantageous to process the biomass after its separation.
  • the fermentation broth can also be thickened or concentrated without separating the cells, using known methods such as, for example, with the aid of a rotary evaporator, thin-film evaporator, falling-film evaporator, by reverse osmosis or by nanofiltration.
  • this concentrated fermentation broth can be processed to obtain the fatty acids present therein.
  • Capsular Polysaccharides Bacterial capsules are formed primarily from long-chain polysaccharides with repeat-unit structures. A given bacterial species can produce a range of capsular polysaccharides with different structures and this help distinguish isolates by serotyping, as is the case with Streptococcal antigens. Capsules are virulence factors for many pathogenic bacteria. Capsular polysaccharides are often linked to the cell surface of the bacterium via covalent attachments to either phospholipid or lipid-A molecules although some capsular polysaccharides may be associated with the cell in the absence of a membrane anchor capsular polysaccharides can be either homo- or heteropolymers composed of repeating monosaccharides joined by glycosidic linkages.
  • the genetic loci necessary for the production of bacterial capsules are known and primarily clustered at a single chromosomal locus, which allows for the coordinate regulation of a large number of genes that may be involved in both the biosynthesis and export of capsular polysaccharides. In most bacterial species, the capsule gene clusters demonstrate conserved sequence and genetic organization.
  • the Escherichia coli K1 antigen a homopolymer of a2,8-linked N- acetylneuraminic acid (NeuNAc)
  • NeuNAc N- acetylneuraminic acid
  • bacterial species may demonstrate great structural diversity in synthesizing capsules
  • chemically identical capsular polysaccharides may also be synthesized by different bacterial species.
  • the Neisseria meningitidis group B capsular polysaccharide is identical to the K1 polymer of E. coli, and the E. coli K18, K22, and K100 antigens have the same constituents and structure as the Haemophilus influenza serotype b capsule.
  • capsular polysaccharide serotypes between bacterial species.
  • S. pneumonia is encapsulated presenting specific carbohydrates on its cell wall.
  • the different polysaccharides determine the bacterium's serotype.
  • these polysaccharide capsule antigens do not elicit protective levels of antibodies or induce immunological memory especially in children under two years of age and in individuals who are immune suppressed.
  • WO201 1/151760 which is incorporated by reference in its entirety, discloses a vaccine protective against serotype 1 , 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. Also see WOOO/56360 and WOOO/62801 each of which is incorporated by reference it their entirety.
  • Prevnar® comprises seven serotypes: 4, 6B, 9V, 14, 18C, 19F and 23F, and is suitable for immunisation of infants less than 2 years old and is crosslinked to a diphtheria toxin carrier protein.
  • An alternative thirteen component vaccine also linked to diphtheria toxin comprises serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F and is suitable for human subjects over 65 years old.
  • a further ten serotype pneumococcal vaccine comprises the following serotypes; 1 , 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. These vaccines are effective in protection against pneumococcal infection and are manufactured by chemical conjugation which is expensive.
  • Adjuvants and Vaccines are effective in protection against pneumococcal infection and are manufactured by chemical conjugation which is expensive.
  • adjuvants immunomodulators
  • adjuvants have been used for decades to improve the immune response to vaccine antigens.
  • the incorporation of adjuvants into vaccine formulations is aimed at enhancing, accelerating and prolonging the specific immune response to vaccine antigens.
  • Advantages of adjuvants include the enhancement of the immunogenicity of weaker antigens, the reduction of the antigen amount needed for a successful immunisation, the reduction of the frequency of booster immunisations.
  • adjuvants can also be employed to optimise a desired immune response, e.g. with respect to immunoglobulin classes and induction of cytotoxic or helper T lymphocyte responses.
  • certain adjuvants can be used to promote antibody responses at mucosal surfaces.
  • Adjuvants can be classified according to their source, mechanism of action and physical or chemical properties.
  • the most commonly described adjuvant classes are gel-type, microbial, oil-emulsion and emulsifier-based, particulate, synthetic and cytokines. More than one adjuvant may be present in the final vaccine product according to the invention.
  • the origin and nature of the adjuvants currently being used or developed is highly diverse.
  • MDP is derived from bacterial cell walls; saponins are of plant origin, squalene is derived from shark liver and recombinant endogenous immunomodulators are derived from recombinant bacterial, yeast or mammalian cells.
  • adjuvants licensed for veterinary vaccines such as mineral oil emulsions that are too reactive for human use.
  • complete Freund's adjuvant is one of the most powerful adjuvants known.
  • the vaccine compositions of the invention can be administered by any conventional route, including injection.
  • the administration may be, for example, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or intradermally.
  • the vaccine compositions of the invention are administered in effective amounts.
  • An "effective amount" is that amount of a vaccine composition that alone or together with further doses, produces the desired response. In the case of treating a particular bacterial disease the desired response is providing protection when challenged by an infective agent.
  • Figure 1 Immunoblot of pB4 expressed in various £ coli backgrounds. Lysed, whole cell samples were separated by SDS-PAGE on a 4-12 % Bis-Tris gel and detected using anti- serotype 4 and anti-Prevnar primary antisera, and fluorescent secondary antibody. Lane 1 : £. coli W31 10 carrying recombinant pB-4 plasmid; Lane 2: E. coli W31 10 carrying empty vector pBBR1 MCS3; Lane 3: £ coli CLM37 carrying recombinant pB-4 plasmid and expressing GalE; Lane 4: £ coli W31 l OACId carrying recombinant pB-4 plasmid; Lane 5: £.
  • Fig 2 Immunoblot of pB5 expressed in CLM37. Lysed, whole cell samples were separated by SDS-PAGE on a 10 % Bis-Tris gel and detected using anti-serotype 5 and fluorescent secondary antibody. M: Molecular weight marker PageRuler Plus. Lane 1 : £ co// W31 10 carrying recombinant pB-5 plasmid.
  • Figure 3 Nucleotide and amino acid sequences of wze and wzd polypeptides
  • Figure 4 Immunoblot of pB8 expressed in various £ coli backgrounds. Lysed, whole cell samples were separated by SDS-PAGE on a 12 % Bis-Tris gel and detected using anti- serotype 8 primary antisera and fluorescent secondary antibody. Lane 1 : £.
  • Figure 5 sequence of capsule loci comprising reducing end sugars with glucose as reducing end sugar.
  • Table 1 Accession numbers for capsule loci with acetylated reducing end sugars.
  • Table 4 accession number for capsule loci comprising reducing end sugars with gl as reducing end sugar.
  • a construct was assembled consisting of flanks with homology to the ends of Cld and surrounding DNA, attached to a kanamycin resistance determinant.
  • the construct was assembled by PCR amplification of the kanamycin resistance determinant from pEXT22 using Q5 high-fidelity DNA polymerase and primers: KanPromF 5 ' -agcttgcagtgggcttacat- 3' (SEQ ID NO 83) and KanR S ' -gaaatctcgtgatggcaggt-S ' (SEQ ID NO 84).
  • KanPromF 5 ' -agcttgcagtgggcttacat- 3' SEQ ID NO 83
  • KanR S ' -gaaatctcgtgatggcaggt-S ' SEQ ID NO 84
  • PCR reactions were performed using the following cycling conditions: 98 °C for 30s, then 30 rounds of 98 °C 5s, 63 °C for 10s and 72 °C for 20s, followed by a final extension of 72 °C for 2 mins.
  • a soeing PCR was performed using 100ng of the purified PCR of the Kan cassette, cld left flank and cld right flank as templates and the primers cldLF and cldRR.
  • the cycling conditions were as follows: 98 °C for 30s, then 30 rounds of 98 °C 10s, 64 °C for 20s increasing by 0.2 °C every cycle and 72 °C for 1 min, followed by a final extension of 72 °C for 3 mins.
  • the correct size band was extracted from a gel and purified using a Qiagen gel extraction kit according to manufacturer's instructions.
  • Polynucleotide kinase was used to phosphorylate the ends of the PCR product to facilitate blunt end cloning into the Smal restriction enzyme site of an R6K ori containing plasmid (pBlueX).
  • the construct was verified by Sanger sequencing.
  • competent E co// ' W3110 cells were transformed with the CldAKan plasmid and recombinants selected for using kanamycin selection. Recombinant cells were screened to ensure the vector backbone was no longer present and that the wild type cld gene was interrupted with the kan cassette.
  • the S. pneumoniae genes wzD and wzE were amplified from a serotype 4 strain. wzdLBamHI 5 ' -TAAGGATCCggagaaatgatgaaagaacaa-3 " (SEQ ID NO 89) and wzeRSall 5'-TAAGTCGACtccccttccatactatattca-3 ' (SEQ ID NO 90). The genes were cloned into pEXT21 via the Bam HI and Sail restriction sites.
  • Electrocompetent E. coli W3110 or W3110ACId were prepared by three successive washes in an equal volume of 10% glycerol at 4 °C, followed by centrifugation at 4000 rpm 4°C for 10 mins. Plasmid DNA was introduced into the competent cells by electroporation at 2kV 200 ⁇ 25 pF in a 2mm gap cuvette. Cells were recovered at 37 °C in SOC broth for 1 hr before plating on LB agar containing appropriate antibiotics. CRISPR mutagenesis
  • SUBSTITUTE SHEET (RULE 26) were used as specified in the paper. gRNA design was carried out using ATUM crispr gRNA design tool (https://www.atum.bio/eCommerce/cas9/input)
  • E. coli cultures containing various plasmids were grown for 16 hrs with appropriate antibiotics before being diluted into fresh SSOB media to an OD 6 oo of 0.03.
  • the media was supplemented with 0.5 mM IPTG and 4 mM MnCI 2 and incubated at 28 °C for 24 hrs. All samples were ⁇ matched, washed with PBS, and then lysed using a Bioruptor ultrasonic processor (Diagenode, Belgium) set on a high pulse rate set for 30s on 30s off for 15 mins. Lysed samples were mixed with SDS-PAGE sample buffer and separated on 4-12% Bis-Tris gel in MOPS buffer (Invitrogen).
  • membranes were washed three times with PBS (0.1% Tween 20) and then incubated for 45 mins with a secondary goat anti-rabbit IgG IRDye800 and a goat anti-mouse IgG IRDye680 conjugate antibody at a dilution of 1 :10000.
  • Membranes were washed a further three times in PBS (0.1% Tween 20) and once with PBS before signal detection with the Odyssey LI-COR detection system (LI-COR Biosciences UK Ltd).
  • Group 1 capsule loci in E. coli have four conserved genes at the beginning of the locus, 3 of which control polymerization and export, independent of capsule structure (wza, wzb, wzc) (Whitfield 2006).
  • S. pneumoniae it is known that polymer length is controlled by a tyrosine kinase phosphoregulatory system.
  • S. pneumoniae the 4 initial genes in the capsule locus are highly conserved.
  • the 2 domains of WzC are contained in 2 separate proteins (CpsC and CpsD) (Yother 2011).
  • CpsC and CpsD CpsC and CpsD
  • CpsB The phosphatase CpsB is also involved in polymerization but the results of inactivation are variable depending on serotype. Removal of cpsB resulted in higher capsule production in D39 but no change to polymerization (Bender et ai 2003), and less capsule production in 19F (Morona et al 2000). CpsB can be compensated for by WzB from the colanic acid locus of E. coli, despite the two proteins having different structures and modes of action
  • chain length restriction via cld is likely to be a problem restricted to capsule types that have an acetylated reducing end sugar, which are also the serotypes that are substrates for CjPglB.
  • This list includes serotypes: 1 , 4, 5 » 12F, 12A, 12B, 25F, 25A, 38, 44, 45 and 46.
  • E. coli strains were grown in LB broth containing 0.1% glucose and induced after 2 hrs starter culture growth with 0.5 mM IPTG.
  • the expression of recombinant serotype 8 polysaccharide was compared in strains W3110 and W3110ACId (where the O-antigen chain length determinant has been interrupted).
  • the additional impact of expressing wzD and wzE was also evaluated.
  • Whole cell, OD-matched lysates, were run on an SDS-PAGE gel alongside positive controls of lysed S. pneumoniae 573/62 (serotype 8 strain from SSI, Denmark).
  • a Western blot was carried out using anti-SP8 antiserum.
  • Figure 4 shows that expressing wzDE improved polymerization, with or without the removal of the E. coli O- antigen chain length regulator (CId). This is in contrast to the results for serotype 4, where removal of CId was necessary to see improved polymerization with wzDE. This suggests that CId is not compensating for the native chain length regulators when the capsule is expressed in E. coli.
  • serotype 8 capsule does not have an acetylated reducing end sugar but instead has glucose at the reducing end.
  • Crystal structures of Wzb of Escherichia coli and CpsB of Streptococcus pneumoniae representatives of two families of tyrosine phosphatases that regulate capsule

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Abstract

L'invention concerne un vaccin glycoconjugué comprenant un ou plusieurs polysaccharides liés de manière covalente à un ou plusieurs polypeptides porteurs, ledit glycoconjugué étant fabriqué par une cellule microbienne modifiée; des vaccins et des compositions immunogènes comprenant ledit ou lesdits glycoconjugués; des utilisations et des méthodes de traitement d'infections microbiennes à l'aide desdits vaccins et compositions immunogènes; des bioréacteurs pour la fabrication desdits glycoconjugués et des procédés de préparation dudit ou desdits glycoconjugués à partir desdites cellules microbiennes modifiées.
PCT/EP2018/073714 2017-09-04 2018-09-04 Cellules microbiennes exprimant des sérotypes streptococciques Ceased WO2019043245A1 (fr)

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GBGB1714128.4A GB201714128D0 (en) 2017-09-04 2017-09-04 Glycoconjugate
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GBGB1807669.5A GB201807669D0 (en) 2018-05-11 2018-05-11 Glycoconjugate
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040202668A1 (en) * 2001-04-03 2004-10-14 Dominique Boutriau Vaccine composition
WO2007071707A2 (fr) * 2005-12-22 2007-06-28 Glaxosmithkline Biologicals Sa Vaccin
US20070231340A1 (en) * 2005-04-08 2007-10-04 Wyeth Multivalent pneumococcal polysaccharide-protein conjugate composition
WO2014072405A1 (fr) * 2012-11-07 2014-05-15 Glycovaxyn Ag Production d'un vaccin recombinant dans e. coli par conjugaison enzymatique
WO2015110942A2 (fr) * 2014-01-21 2015-07-30 Pfizer Inc. Polysaccharides capsulaires de streptococcus pneumoniae et leurs conjugués
WO2016020499A2 (fr) * 2014-08-08 2016-02-11 Glycovaxyn Ag Cellules hôtes modifiées utilisables dans la production de bioconjugués

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040202668A1 (en) * 2001-04-03 2004-10-14 Dominique Boutriau Vaccine composition
US20070231340A1 (en) * 2005-04-08 2007-10-04 Wyeth Multivalent pneumococcal polysaccharide-protein conjugate composition
WO2007071707A2 (fr) * 2005-12-22 2007-06-28 Glaxosmithkline Biologicals Sa Vaccin
WO2014072405A1 (fr) * 2012-11-07 2014-05-15 Glycovaxyn Ag Production d'un vaccin recombinant dans e. coli par conjugaison enzymatique
WO2015110942A2 (fr) * 2014-01-21 2015-07-30 Pfizer Inc. Polysaccharides capsulaires de streptococcus pneumoniae et leurs conjugués
WO2016020499A2 (fr) * 2014-08-08 2016-02-11 Glycovaxyn Ag Cellules hôtes modifiées utilisables dans la production de bioconjugués

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CALVIN C. DANIELS ET AL: "A Review of Pneumococcal Vaccines: Current Polysaccharide Vaccine Recommendations and Future Protein Antigens", THE JOURNAL OF PEDIATRIC PHARMACOLOGY AND THERAPEUTICS, vol. 21, no. 1, 1 January 2016 (2016-01-01), pages 27 - 35, XP055526395, ISSN: 1551-6776, DOI: 10.5863/1551-6776-21.1.27 *
EMILY J. KAY ET AL: "Recombinant expression of Streptococcus pneumoniae capsular polysaccharides in Escherichia coli", OPEN BIOLOGY, vol. 6, no. 4, 1 April 2016 (2016-04-01), pages 150243, XP055525931, DOI: 10.1098/rsob.150243 *

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