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WO2025140679A1 - Souche bactérienne vivante de staphylococcus sp. - Google Patents

Souche bactérienne vivante de staphylococcus sp. Download PDF

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Publication number
WO2025140679A1
WO2025140679A1 PCT/CN2024/143562 CN2024143562W WO2025140679A1 WO 2025140679 A1 WO2025140679 A1 WO 2025140679A1 CN 2024143562 W CN2024143562 W CN 2024143562W WO 2025140679 A1 WO2025140679 A1 WO 2025140679A1
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Prior art keywords
live bacteria
bacteria strain
staphylococcus
strain
genes
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Inventor
Mengya ZHANG
Qiubin LIN
Ming-Chun Lee
Mingrui WANG
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Shanghai Yuguan Biotech Co Ltd
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Shanghai Yuguan Biotech Co Ltd
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Publication of WO2025140679A1 publication Critical patent/WO2025140679A1/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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/44—Staphylococcus
    • C12R2001/445—Staphylococcus aureus

Definitions

  • S. aureus has evolved many mechanisms to circumvent killing by these potent innate immune cells.
  • the production of secreted virulence factors during pathogenesis is primarily controlled by the combined influence of two-component systems (TCSs) that sense the host environment and respond accordingly.
  • TCSs two-component systems
  • the Sae TCS has been shown to be essential for evasion of human neutrophil killing.
  • the system is composed of a histidine kinase sensor, SaeS, a response regulator, SaeR, and two accessory proteins, SaeP and SaeQ.
  • the sensor protein SaeS detects environmental cues, such as changes in pH or oxygen levels, and then phosphorylates the response regulator, SaeR.
  • Embodiment 2 The live bacteria strain of Embodiment 1, wherein the expression of one or more genes of the sae two component system in the live bacteria strain is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%or more, as compared to corresponding control strain.
  • Embodiment 3 The live bacteria strain of Embodiment 1 or 2, wherein the expression of one or more genes selected from SaeS, SaeR, SaeP and SaeQ in the live bacteria strain is reduced, preferably, the expression of all of SaeS, SaeR, SaeP and SaeQ in the live bacteria strain is reduced, more preferably, none of SaeS, SaeR, SaeP and SaeQ expresses in the live bacteria strain.
  • Embodiment 4 The live bacteria strain of Embodiment 1, wherein the live bacteria strain contains one or more mutations in one or more genes selected from SaeS, SaeR, SaeP and SaeQ, preferably, all of SaeS, SaeR, SaeP and SaeQ in the live bacteria strain are mutated.
  • Embodiment 5 The live bacteria strain of any one of Embodiments 1-4, wherein the mutation in the one or more genes of the sae two component system results in reduced or no expression of the one or more genes of the sae two component system, or results in expression of one or more proteins of the sae two component system with reduced or no activity.
  • Embodiment 6 The live bacteria strain of any one of Embodiments 1-5, wherein the mutation comprises a deletion of the one or more genes of the sae two component system, preferably, all genes of the sae two component system in the live bacteria strain are completely or partially deleted, more preferably, all genes of the sae two component system in the live bacteria strain are completely deleted.
  • Embodiment 7 The live bacteria strain of Embodiment 6, wherein one or more genes selected from SaeS, SaeR, SaeP and SaeQ in the live bacteria strain is deleted, preferably, all of SaeS, SaeR, SaeP and SaeQ in the live bacteria strain are completely or partially deleted, more preferably, all of SaeS, SaeR, SaeP and SaeQ in the live bacteria strain are completely deleted.
  • Embodiment 8 The live bacteria strain of any one of Embodiment 1-7, wherein the live bacteria strain also has reduced adenosine synthase A (adsA) activity, preferably, lacks adenosine synthase A (adsA) activity, for example, as compared to a corresponding control strain.
  • adsA adenosine synthase A
  • Embodiment 9 The live bacteria strain of Embodiment 8, wherein the live bacteria strain comprises a mutation in the adsA gene.
  • Embodiment 10 The live bacteria strain of Embodiment 9, wherein the mutation results in reduced or no expression of the adsA gene, or results in expression of adsA protein with reduced or no activity.
  • Embodiment 11 The live bacteria strain of Embodiment 9 or 10, wherein the mutation comprises a deletion of the adsA gene in the live bacteria strain, for example, the adsA gene is partially or completely deleted.
  • Embodiment 12 The live bacteria strain of any one of Embodiments 1-11,
  • live bacteria strain also has reduced capsule production, preferably, lacks capsule production, and/or
  • live bacteria strain contains one or more mutations in one or more capsular polysaccharide synthesis related genes
  • Embodiment 13 The live bacteria strain of Embodiment 12, wherein the capsule production in the live bacteria strain is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%or more, as compared to a corresponding control strain, preferably, the live bacteria strain does not produce capsules.
  • Embodiment 14 The live bacteria strain of Embodiment 12 or 13, wherein the production of capsular polysaccharides in the live bacteria strain is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%or more, as compared to a corresponding control strain, preferably, the live bacteria strain does not produce capsular polysaccharides.
  • Embodiment 17 The live bacteria strain of any one of Embodiments 12-16, wherein all capsular polysaccharide synthesis related genes in the live bacteria strain are mutated.
  • Embodiment 19 The live bacteria strain of any one of Embodiments 12-18, wherein the mutation comprises a deletion of the one or more capsular polysaccharide synthesis related genes, preferably, all capsular polysaccharide synthesis related genes in the live bacteria strain are partially or completely deleted, more preferably, all capsular polysaccharide synthesis related genes in the live bacteria strain are completely deleted.
  • Embodiment 20 The live bacteria strain of any one of Embodiments 12-19, wherein the one or more capsular polysaccharide synthesis related genes are selected from the group consisting of: capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO, and capP.
  • Embodiment 21 The live bacteria strain of Embodiment 20, wherein all the polysaccharide synthesizing genes of capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO, and capP are deleted, preferably, completely deleted.
  • Embodiment 22 The live bacteria strain of any one of Embodiments 1-21, wherein the mutation is achieved by homologous recombination or by targeted mutagenesis, such as via CRISPR, TALEN or ZFN technologies.
  • Embodiment 23 The live bacteria strain of any one of Embodiments 1-22, wherein the live bacteria strain has a reduced virulence, for example, the virulence of the live bacteria strain is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%or more, as compared to a corresponding control strain.
  • Embodiment 24 The live bacteria strain of any one of Embodiments 1-23, wherein the live bacteria strain has an increased immunogenicity, for example, the immunogenicity of the live bacteria strain is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%or more, as compared to a corresponding control strain.
  • Embodiment 25 The live bacteria strain of any one of Embodiments 1-24, wherein the species from Staphylococcus sp. is selected from the group consisting of Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus carnosus, Staphylococcus epidermidis, Staphylococcus intermedius, Staphylococcus lentus, Staphylococcus lugdunensis, Staphylococcus pettenkoferi, Staphylococcus simulans, Staphylococcus vitulinus, Staphylococcus xylosus, and Staphylococcus subspecies anaerobius,
  • the species from Staphylococcus sp. is Staphylococcus aureus.
  • Embodiment 26 The live bacteria strain of any one of Embodiments 1-25, wherein the live bacteria strain is derived from a parent strain which is a clinical isolate.
  • Embodiment 27 The live bacteria strain of any one of Embodiments 1-25, wherein the live bacteria strain is derived from a parent strain which already has low virulence.
  • Embodiment 28 The live bacteria strain of any one of Embodiments 1-27, wherein the live bacteria strain is derived from Staphylococcus aureus serotype 5 or serotype 8, more preferably, the live bacteria strain is derived from Staphylococcus aureus serotype 5.
  • Embodiment 29 The live bacteria strain of Embodiment 28, wherein the live bacteria train is derived from Staphylococcus aureus Newman strain, FPR3735, JE2 or ATCC29213.
  • Embodiment 30 The live bacteria strain of any one of Embodiments 1-29, which is for use as a live expression vector for expression of a desired protein.
  • Embodiment 31 The live bacteria strain of any one of Embodiments 1-30, wherein the live bacteria strain further comprises a coding sequence of a desired protein, and thereby being able to express the desired protein.
  • Embodiment 34 The live bacteria strain of any one of Embodiments 30-33, wherein the desired protein is expressed and displayed on the surface of the cell of the live bacteria strain; or is expressed and secreted out of the cell of the live bacteria strain.
  • Embodiment 35 The live bacteria strain of any one of Embodiments 30-34, wherein the desired protein is an antigen.
  • Embodiment 36 The live bacteria strain of any one of Embodiments 30-35, wherein the desired protein is selected from EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA, mLukB, or any combination thereof.
  • Embodiment 47 The use, the composition, or the method of Embodiment 46, wherein the bacterial infection is Staphylococcus aureus infection.
  • Embodiment 49 The use, composition or the method of Embodiment 48, wherein the invasive disease is bloodstream infection, endocarditis, Morel’s disease or sepsis.
  • Embodiment 50 The use, composition or the method of any one of Embodiments 47-49, wherein the Staphylococcus aureus infection is methicillin-resistant S. aureus (MRSA) infection or methicillin-sensitive S. aureus (MSSA) infection, preferably, the infection is a recurring S. aureus infection.
  • MRSA methicillin-resistant S. aureus
  • MSSA methicillin-sensitive S. aureus
  • Embodiment 51 The use, composition or the method of any one of Embodiments 45-50, wherein the subject is a mammal such as human, mouse, rat, monkey, dog, pig, sheep, goat, cow, horse, donkey, cattle, cat; a poultry such as chicken, duck, goose.
  • a mammal such as human, mouse, rat, monkey, dog, pig, sheep, goat, cow, horse, donkey, cattle, cat
  • a poultry such as chicken, duck, goose.
  • Figure. 1 Deletion regions of target KO genes (adsA, CPs, saeRS, saePQRS) .
  • FIG. 3 IgG antibody titer of enrolled animals.
  • FIG. 1 Bacterial load of 10-antigen strain.
  • Sequence identity has recognized meaning in the art, and the percentage of sequence identity between two nucleotide sequences or amino acid sequences can be calculated using the disclosed techniques. Sequence identity can be measured along the entire length of the specified nucleotide sequence or amino acid sequence or along a region of the sequence. In some embodiments, the sequence identity is measured along the entire length of the specified nucleotide sequence or amino acid sequence. Many methods for measuring the sequence identity are available in the art and can be applied in the present invention.
  • the invention provides a live bacteria strain of a species from Staphylococcus sp., wherein
  • the live bacteria strain has reduced activity of the sae two component system, preferably, lacks the activity of the sae two component system;
  • capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO, and capP are deleted in the live strain of the invention, preferably, completely deleted.
  • the virulence of the live bacteria strain of the invention may be reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%or more, as compared to a corresponding control strain.
  • the immunogenicity of the live bacteria strain of the invention may be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%or more, as compared to a corresponding control strain.
  • the live bacteria strain of the invention may be derived from a parent strain which is a wildtype strain of the same species.
  • a wildtype strain may be a strain that has not been genetically engineered.
  • a wildtype strain may be a strain whose Sae two component system genes, adsA gene and or capsular polysaccharide synthesis related genes have not been genetically engineered.
  • a wildtype strain may be a clinical isolate.
  • the live bacteria strain of the invention may be derived from a parent strain which already has low virulence.
  • the parent strain is an attenuated strain.
  • the live bacteria strain of the invention may contain other modification (s) that may result in attenuation.
  • the live bacteria strain of the invention may be derived from a species from Staphylococcus sp..
  • the live bacteria strain of the invention may be derived from Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus carnosus, Staphylococcus epidermidis, Staphylococcus intermedius, Staphylococcus lentus, Staphylococcus lugdunensis, Staphylococcus pettenkoferi, Staphylococcus simulans, Staphylococcus vitulinus, Staphylococcus subspecies anaerobius, or Staphylococcus xylosus.
  • the live bacteria strain of the invention may be derived from any isolate strain of the specific bacterium species.
  • the live bacteria strain of the invention may be derived from a bacteria strain of various serotypes.
  • the serotype of a bacteria strain is determined by the capsular polysaccharides produced by the bacteria strain.
  • the live bacteria strain of the invention is derived from Staphylococcus aureus, and thus also refers to a live S. aureus strain of the invention.
  • the live S. aureus strain of the invention is derived from S. aureus of various serotypes, including but not limited to serotype 5, serotype 8 and the like.
  • the live S. aureus strain of the invention is derived from serotype 5 S. aureus.
  • the live S. aureus strain of the invention is derived from serotype 8 S. aureus.
  • the live S. aureus strain of the invention is derived from S. aureus Newman, FPR3735, JE2 or ATCC29213 strain. In some preferred embodiments, the live S. aureus strain of the invention is derived from S. aureus Newman strain.
  • the Staphylococcus aureus Newman strain is a serotype 5 strain commercially available from the National Collection of Type Cultures (NCTC) in the United Kingdom under the accession number NCTC 8178.
  • the invention provides a live bacteria strain of a species from Staphylococcus sp., wherein
  • the live bacteria strain of the invention may comprise a coding sequence of a desired protein, and thereby be able to express the desired protein.
  • the coding sequence of the desired protein is introduced into the live bacteria strain of the invention, for example, through a nucleic acid expression construct. In some embodiments, the introduced coding sequence of the desired protein is integrated into the genome of the live bacteria strain of the invention.
  • the desired protein can be expressed and displayed on the surface of the cell of the live bacteria strain of the invention. In some embodiments, the desired protein can be expressed and secreted out of the cell of the live bacteria strain of the invention.
  • the desired protein includes but is not limited to an antibody, an antigen, and the like.
  • Exemplary antigen proteins include but are not limited to EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA, mLukB, or the like, or any combinations thereof.
  • the EsxA may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%or even 100%sequence identity with SEQ ID NO: 44.
  • the EsxB may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%or even 100%sequence identity with SEQ ID NO: 46.
  • the mLukS-PV may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%or even 100%sequence identity with SEQ ID NO: 48.
  • the mLukF-PV may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%or even 100%sequence identity with SEQ ID NO: 50.
  • the live bacteria strain of the invention expresses (e.g., overexpresses) one or more antigens selected from EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA, and mLukB.
  • the live bacteria strain of the invention expresses (e.g., overexpresses) the antigen mHla.
  • the live bacteria strain of the invention expresses (e.g., overexpresses) the antigens EsxA, and EsxB.
  • the live bacteria strain of the invention expresses (e.g., overexpresses) the antigens mHla, and mLukS-PV. In some embodiments, the live bacteria strain of the invention expresses (e.g., overexpresses) the antigens mHla, EsxA, EsxB and mLukS-PV. In some embodiments, the live bacteria strain of the invention expresses (e.g., overexpresses) the antigens EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA, and mLukB.
  • the live bacteria strain of the invention is for use in preventing and/or treating a bacterial infection in a subject.
  • the invention provides a method for preventing and/or treating a bacterial infection in a subject, which comprises administering an effective amount of the live bacteria strain of the invention or the composition of the invention to the subject.
  • preventing and/or treating a bacterial infection also encompasses preventing and/or treating diseases or clinical signs or symptoms caused by the bacterial infection.
  • the composition may further comprise an adjuvant.
  • adjuvant refers to additional components in a vaccine to enhance the immune response, or ancillary molecules added to the vaccine or generated by the body after the respective induction by such additional components, like but not restricted to interferons, interleukins or growth factors.
  • adjuvants can include aluminum hydroxide and aluminum phosphate, saponins, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion.
  • composition may further comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, salt solutions (such as Ringer's solution) , alcohol, oil, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone and coloring agents.
  • pharmaceutically acceptable carriers include water, NaCl, physiological saline, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, salt solutions (such as Ringer's solution) , alcohol, oil, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone and coloring agents.
  • the composition is formulated in a form for intramuscular administration, intraperitoneal administration, subcutaneous administration, oral administration or intranasal administration. In one embodiment, the composition is not for intravenous administration. In some embodiments, the composition is in a lyophilized form, which can be reconstituted before use.
  • an effective amount refers to an amount of a substance, compound, material, or composition containing a compound (such as the live bacteria strain of the invention or the composition of the invention) which is at least sufficient to produce a preventive or therapeutic effect after administration to a subject. Therefore, it is an amount necessary to prevent, cure, improve, retard or partially retard the symptoms of a disease or disorder, such as bacterial infection.
  • the actual dosage of the live strain or composition of the present invention to be administered to a subject can be determined according to the following physical and physiological factors: weight, sex, severity of symptoms, type of diseases to be treated, previous or current therapeutic intervention, unknown etiological disease of the patient, administration time, administration route and the like.
  • the amount of the live strains in the composition and the appropriate dose for an individual subject will be determined by the medical personnel responsible for administration.
  • the bacterial infection is caused by a bacterial species from which the live bacteria strain of the invention is derived.
  • the bacterial infection is caused by a bacteria strain of a serotype different from serotype of the live bacteria strain of the invention.
  • the bacterial infection is caused by a species from Staphylococcus sp..
  • the bacterial infection is an infection caused by Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus carnosus, Staphylococcus epidermidis, Staphylococcus intermedius, Staphylococcus lentus, Staphylococcus lugdunensis, Staphylococcus pettenkoferi, Staphylococcus simulans, Staphylococcus vitulinus, Staphylococcus subspecies anaerobius or Staphylococcus xylosus, or any combinations thereof.
  • the bacterial infection is Staphylococcus aureus infection.
  • the Staphylococcus aureus infection is a skin infection, soft-tissue infection, or invasive disease.
  • the invasive disease is bloodstream infection, endocarditis, Morel’s disease, or sepsis.
  • the bacterial infection that can be prevented and/or treated by the invention may depend on the desired protein expressed by the live bacteria strain of the invention.
  • the present invention provides a method for attenuating a live bacteria strain and/or increasing the immunogenicity of a live bacteria strain, or a method for generating a live bacteria strain having lowered virulence and/or increased immunogenicity, said method comprises
  • AdsA adenosine synthase A
  • agarose gel of the PCR check of insertion genes are displayed in Figure 2. Briefly, upstream and downstream flanking regions of target insertion genes were PCR amplified from chromosomal DNA of S. aureus Newman ⁇ adsA ⁇ CP ⁇ saePQRS or Newman ⁇ adsA ⁇ CP strains with corresponding primers (Table 4) and the PCR products were separated by electrophoresis in a 1%agarose. The 5kb plus ladder was used as marker. Arrows indicate the expected bands for knock-in mutant and for the wild-type strain.
  • the recombinant plasmid was introduced into DH5a, followed by transformation into E. coli DC10B and subsequently into parental strains (Newman ⁇ adsA ⁇ CP ⁇ saePQRS/Newman ⁇ adsA ⁇ CP or engineered single/double/triple KI strains) .
  • the two-step selection of allelic replacement was performed as described previously[2] . Briefly, transformants were first cultured in BHI containing chloramphenicol (10 ⁇ g/ml) at 37°C for 2-3 days. The clones were screened by PCR to select for 1st recombination strains with specific primers (Table 3) .
  • mHla gene was successfully inserted into Newman ⁇ adsA ⁇ CP ⁇ saePQRS to generate Newman ⁇ adsA ⁇ CP ⁇ saePQRS+ mHla strain
  • EsxAB gene was successfully inserted into Newman ⁇ adsA ⁇ CP ⁇ saePQRS to generate Newman ⁇ adsA ⁇ CP ⁇ saePQRS+ EsxAB strain
  • antigen mLukS-PV gene was successfully inserted into Newman ⁇ adsA ⁇ CP ⁇ saePQRS + mHla strain to generate Newman ⁇ adsA ⁇ CP ⁇ saePQRS+ mHla + mLukS-PV strain
  • EsxAB-mLukS-PV gene was successfully inserted into Newman ⁇ adsA ⁇ CP ⁇ saePQRS + mHla strain to generate Newman ⁇ adsA ⁇ CP ⁇ saePQRS+mHla + Es
  • each BALB/c mouse was immunized intraperitoneally or intravenously with single dose fresh culture bacterial suspension (2x10 8 CFU or 2x10 7 CFU) .
  • the animals were monitored for survival daily for several days.
  • Challenge experiments were conducted on Day 49 intraperitoneally with Newman, (2x10 8 CFU) and survival rate was monitored for several days.
  • each BALB/c or C57BL/6 mouse was immunized subcutaneously with single dose fresh culture bacterial suspension (1x10 7 CFU or 5 x10 7 CFU) .
  • the animals were monitored for weight loss daily for 14 days and serum from BALB/c group were sampled on Day 21 and Day 28 for antibody titrations and examined by whole bacteria ELISA (Newman ⁇ spa ⁇ sbi) .
  • mice from BALB/c group were challenged on Day 42 subcutaneously with Newman (1x10 8 or 2x10 8 ) , HA-MRSA (4 x10 8 ) , FPR3735 (8x10 8 ) , Sau-HK3117 (2 x10 8 ) , MU3 strain (8 x10 8 ) and skin abscess and demonecrsis was monitored for several days.
  • a derived mutant strain deficient for the protein A and protein Sbi ( ⁇ spa ⁇ sbi) was used to avoid nonspecific binding with IgG or IgM antibodies.
  • 96-well ELISA plates were coated with Newman ⁇ spa ⁇ sbi, which were fixed to the bottom of the wells after overnight incubation at 4°C in 100mM carbonate-bicarbonate buffer, pH9.6. After coating, plates were washed five times with PBS to remove any unfixed bacteria. The residual sites were blocked with 200 ⁇ L per well of blocking solution (5%skim milk in PBS) for 2h at room temperature.
  • Healthy sheep within one year of age with clear background and without visible Staphylococcus aureus infection were first screened into the experimental group. A total of 294 animals were recruited for this experiment. Furthermore, screened serum-IgG response to S. aureus from total 294 healthy sheep, and enrolled 72 sheep with low IgG response (below 1.5) . All 72 screened animals were randomly grouped.
  • This experiment was divided into three groups, including two immunization groups (Sau0105 group and Sau0137 group) and one placebo group (physiological saline) , of which there were 30 in the Sau0105 group, 12 in the Sau0137 group and 30 in the Placebo group (shown in Table 5) .
  • the median and distribution of antibody levels of the final grouped animals were basically the same, indicating that the background of the three groups of animals was close to each other (as shown in Figure 3) .
  • mice immunized (i.p. ) with ⁇ saePQRS strains showed higher survival than mice injected with the same dosage (2x 10 8 CFU) of JE2 ⁇ adsA ⁇ saeRS ( Figure 4) . Similar results were observed in Newman strain. The mice were immunized (i.v.
  • ⁇ saePQRS strain Sau0126 showed significantly lower antibody titers both on Day 21 and Day 28 ( Figure 7) .
  • ⁇ saePQRS strains still retained their protection.
  • the mice immunized with Sau0021 and Sau0126 both reach 100%survival after challenge while control group with only 50%survival rate ( Figure 8) .

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Abstract

L'invention porte sur une souche bactérienne vivante de Staphylococcus sp. telle qu'une souche bactérienne vivante de Staphylococcus aureus, avec une activité réduite de saePQRS, une activité réduite d'adsA, et/ou une production réduite de capsules ; sur les utilisations de ladite souche bactérienne vivante ; sur un vaccin contre l'infection bactérienne contenant ladite souche bactérienne vivante ; et sur un procédé de prévention et/ou de traitement de l'infection bactérienne chez un sujet par l'administration de ladite souche bactérienne vivante.
PCT/CN2024/143562 2023-12-29 2024-12-29 Souche bactérienne vivante de staphylococcus sp. Pending WO2025140679A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
US20130064845A1 (en) * 2010-03-17 2013-03-14 Universite De Montreal Bacterial vaccine components from staphylococcus aureus and uses thereof
CN106177932A (zh) * 2016-07-03 2016-12-07 查文娟 一种耐甲氧西林金黄色葡萄球菌的疫苗
WO2021233420A1 (fr) * 2020-05-22 2021-11-25 Versitech Limited Souche vivante de staphylococcus aureus et ses utilisations

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130064845A1 (en) * 2010-03-17 2013-03-14 Universite De Montreal Bacterial vaccine components from staphylococcus aureus and uses thereof
CN106177932A (zh) * 2016-07-03 2016-12-07 查文娟 一种耐甲氧西林金黄色葡萄球菌的疫苗
WO2021233420A1 (fr) * 2020-05-22 2021-11-25 Versitech Limited Souche vivante de staphylococcus aureus et ses utilisations

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Title
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LIU QIAN, YEO WON€SIK, BAE TAEOK: "The SaeRS Two‐Component System of Staphylococcus aureus", GENES, MDPI AG, US, vol. 7, no. 10, US , pages 81, XP093331069, ISSN: 2073-4425, DOI: 10.3390/genes7100081 *
MOHAMED NAGLAA, TIMOFEYEVA YEKATERINA, JAMROZY DOROTA, ROJAS EDUARDO, HAO LI, SILMON DE MONERRI NATALIE C., HAWKINS JULIO, SINGH G: "Molecular epidemiology and expression of capsular polysaccharides in Staphylococcus aureus clinical isolates in the United States", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, US, vol. 14, no. 1, US , pages e0208356, XP093331071, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0208356 *
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ZHANG BAO-ZHONG, CAI JIANPIAO, YU BIN, XIONG LIFENG, LIN QIUBIN, YANG XIAO-YAN, XU CHEN, ZHENG SONGYUE, KAO RICHARD YI-TSUN, SZE K: "Immunotherapy Targeting Adenosine Synthase A Decreases Severity of Staphylococcus aureus Infection in Mouse Model", JOURNAL OF INFECTIOUS DISEASES, OXFORD UNIVERSITY PRESS, vol. 216, no. 2, 15 July 2017 (2017-07-15), pages 245 - 253, XP055874589, ISSN: 0022-1899, DOI: 10.1093/infdis/jix290 *

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