WO2024020533A1 - Compositions immunogènes pour traitement ou prévention d'infections à actinomycètes - Google Patents

Compositions immunogènes pour traitement ou prévention d'infections à actinomycètes Download PDF

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Publication number
WO2024020533A1
WO2024020533A1 PCT/US2023/070682 US2023070682W WO2024020533A1 WO 2024020533 A1 WO2024020533 A1 WO 2024020533A1 US 2023070682 W US2023070682 W US 2023070682W WO 2024020533 A1 WO2024020533 A1 WO 2024020533A1
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seq
amino acid
lysin
acid sequence
sequence identity
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PCT/US2023/070682
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English (en)
Inventor
Jason Holder
Clinton DAWSON
Helen Bartlett
Cody GLICKMAN
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Endolytix Technology, Inc.
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Publication of WO2024020533A1 publication Critical patent/WO2024020533A1/fr

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    • 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
    • C12N1/06—Lysis of microorganisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • 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
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • 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
    • A61K39/05—Actinobacteria, e.g. Actinomyces, Streptomyces, Nocardia, Bifidobacterium, Gardnerella, Corynebacterium; Propionibacterium
    • 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
    • A61P31/06—Antibacterial agents for tuberculosis
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • 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
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/246—Isoamylase (3.2.1.68)
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium
    • C12R2001/16—Corynebacterium diphtheriae
    • C—CHEMISTRY; METALLURGY
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/32—Mycobacterium
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    • 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
    • C12R2001/365—Nocardia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01068—Isoamylase (3.2.1.68)

Definitions

  • Bacterial pathogens are a leading cause of infectious disease. Many bacteria are successfully detected by the human immune system and are rapidly cleared before onset of infection. However, many bacterial pathogens evade the host immune system by residing within a host cell. These intracellular bacteria have evolved diverse immune evasion techniques by residing and multiplying within host cells, such as immune cells (e.g., macrophages or dendritic cells), and the correct intracellular compartment (e.g., endosome, phagosome, lysosome, or cytosol) within the host cells.
  • Bacterial infections that propagate within a host cell often present a difficult treatment barrier due to lack of accessibility of the subcellular location of the infection. While certain antibacterial compositions may treat the infection (e.g., in vitro), delivering the treatment to the correct subcellular location in which the bacteria reside has proved to be a challenging endeavor.
  • Mycobacteria are actinomycetia (e.g., corynebacteriales or propionibacteriales) that are denoted by a thick cell wall that is rich in mycolic acids.
  • Mycobacteria contain an envelope that contains a cell membrane composed of a lipid bilayer and cell wall that includes a peptidoglycan layer and an arabinogalactan layer, and outer membrane that contains a hydrophobic mycolate layer.
  • Many mycobacteria also contain an outer capsule composed of polysaccharides, such as D-glucan, D- arabino-D-mannan, and D-mannan.
  • NTM non-tuberculosis mycobacteria
  • compositions and methods for treating and preventing intracellular bacterial infections are needed.
  • the invention described herein includes compositions and methods of treating or preventing a bacterial infection with an immunogenic composition.
  • the invention also features methods of producing an immunogenic composition.
  • the invention features a method of producing an immunogenic composition for treating or preventing an actinomycetia bacterial infection in a subject.
  • the method includes the step of (a) contacting a sample of actinomycetia cells with a composition that includes two or more (e.g., two, three, or four) of a lysin A, a lysin B, an isoamylase, and an a-amylase.
  • the composition lyses the cells of the sample to produce a plurality of biological particles or fragments thereof of the actinomycetia cells.
  • the method further includes the step of (b) collecting the plurality of biological particles or fragments thereof or a portion thereof of step (a) to produce the immunogenic composition.
  • the method further includes purifying the immunogenic composition that includes the plurality of biological particles or fragments thereof.
  • the method may include removing undesired biological particles or fragments thereof.
  • the method further includes administering the immunogenic composition to a subject to treat or prevent the actinomycetia bacterial infection.
  • the invention features a method of treating or preventing an actinomycetia bacterial infection in a subject.
  • the method includes administering to the subject an immunogenic composition that includes a plurality of biological particles or fragments thereof of actinomycetia cells.
  • the immunogenic composition may have been produced by the step of (a) contacting a sample of actinomycetia cells with a composition that includes two or more (e.g., two, three, or four) of a lysin A, a lysin B, an isoamylase, and an a-amylase.
  • the composition lyses the cells of the sample to produce a plurality of biological particles or fragments thereof of the actinomycetia cells.
  • the immunogenic may have also been produced by the step of (b) collecting the plurality of biological particles or fragments thereof or a portion thereof from to produce the immunogenic composition.
  • the method further includes administering the composition that includes two or more of (e.g., two, three, or all four of) a lysin A, a lysin B, an isoamylase, and an a-amylase.
  • the composition includes lysin A and lysin B.
  • the composition includes lysin A and isoamylase.
  • the composition includes lysin A and a-amylase.
  • the composition includes lysin B and isoamylase.
  • the composition includes lysin B and a-amylase.
  • the composition includes isoamylase and a-amylase. In some embodiments, the composition includes lysin A, lysin B, and isoamylase. In some embodiments, the composition includes lysin A, lysin B, and a-amylase. In some embodiments, the composition includes lysin A, isoamylase, and a-amylase. In some embodiments, the composition includes lysin B, isoamylase, and a-amylase. In some embodiments, the composition includes lysin A, lysin B, isoamylase, and a- amylase. Accordingly, such a composition may be used to treat and prevent an actinomycetia infection in a subject.
  • the composition includes one or more (e.g., two or more, three or more, or all four of) (a) a lysin A that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -182; (b) a lysin B that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241 ; (c) an isoamylase that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392; and (d) an a-amylase including an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%,
  • the composition includes one or more (e.g., two or more, three or more, or all four of) (a) a lysin A that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; (b) a lysin B that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184; (c) an isoamylase that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and (d) an a-amylase including an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%)
  • the composition includes lysin A and lysin B, and the lysin A includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -182; and the lysin B includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241.
  • the composition includes lysin A and lysin B, and the lysin A includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; and the lysin B includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184.
  • the lysin A includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2
  • the lysin B includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184.
  • the composition includes lysin A and isoamylase, and the lysin A includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -182; and the isoamylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392.
  • the composition includes lysin A and isoamylase, and the lysin A includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; and the isoamylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243.
  • the composition includes lysin A and a-amylase, and the lysin A includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -182; and the a-amylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.
  • the composition includes lysin A and a-amylase, and the lysin A includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; and the a-amylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-398.
  • the composition includes lysin B and isoamylase
  • the lysin B includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241
  • the isoamylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392.
  • the composition includes lysin B and isoamylase
  • the lysin B includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184
  • the isoamylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243.
  • the composition includes lysin B and a-amylase
  • the lysin B includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241
  • the a-amylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.
  • the composition includes lysin B and a-amylase
  • the lysin B includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184
  • the a-amylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-398.
  • the composition includes isoamylase and a-amylase, and the isoamylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392; and the a-amylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.
  • the composition includes isoamylase and a-amylase, and the isoamylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and the a-amylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-398.
  • the composition includes lysin A, lysin B, and isoamylase
  • the lysin A includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -182
  • the lysin B includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241
  • the isoamylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392.
  • the composition includes lysin A, lysin B, and isoamylase
  • the lysin A includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2
  • the lysin B includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184
  • the isoamylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243.
  • the composition includes lysin A, lysin B, and a-amylase
  • the lysin A includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -182
  • the lysin B includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241
  • the a-amylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.
  • the composition includes lysin A, lysin B, and a-amylase
  • the lysin A includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2
  • the lysin B includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184
  • the a-amylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-398.
  • the composition includes lysin A, isoamylase, and a-amylase
  • the lysin A includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -182
  • the isoamylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392
  • the a-amylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.
  • the composition includes lysin A, isoamylase, and a-amylase, and the lysin A includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2; the isoamylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243; and the a-amylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-398.
  • the composition includes lysin B, isoamylase, and a-amylase
  • the lysin B includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241
  • the isoamylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392
  • the a-amylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.
  • the composition includes lysin B, isoamylase, and a-amylase
  • the lysin B includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184
  • the isoamylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243
  • the a-amylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-398.
  • the composition includes lysin A, lysin B, isoamylase, and a-amylase
  • the lysin A includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -182
  • the lysin B includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 183-241
  • the isoamylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 242-392
  • the a-amylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-4
  • the composition includes lysin A, lysin B, isoamylase, and a-amylase
  • the lysin A includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2
  • the lysin B includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184
  • the isoamylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243
  • the a-amylase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs
  • the composition includes lysin A, lysin B, isoamylase, and a-amylase, and the lysin A includes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; the lysin B includes the amino acid sequence of SEQ ID NO: 183 or SEQ ID NO: 184; the isoamylase includes the amino acid sequence of SEQ ID NO: 242 or SEQ ID NO: 243; and the a-amylase includes the amino acid sequence of any one of SEQ ID NOs: 393-398.
  • the composition includes lysin A, lysin B, isoamylase, and a-amylase, and the lysin A includes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; the lysin B includes the amino acid sequence of SEQ ID NO: 183 or SEQ ID NO: 184; the isoamylase includes the amino acid sequence of SEQ ID NO: 242 or SEQ ID NO: 243; and the a-amylase includes the amino acid sequence of any one of SEQ ID NOs: 393-398.
  • the composition includes a lysin A of SEQ ID NO: 1 , a lysin B of SEQ ID NO: 183, an isoamylase of SEQ ID NO: 242, and an a-amylase of SEQ ID NO: 393.
  • the composition includes a lysin A of SEQ ID NO: 2, a lysin B of SEQ ID NO: 184, an isoamylase of SEQ ID NO: 243, and an a-amylase of SEQ ID NO: 394.
  • the composition includes a lysin A of SEQ ID NO: 1 , a lysin B of SEQ ID NO: 183, an isoamylase of SEQ ID NO: 242, and an a-amylase of SEQ ID NO: 395.
  • the composition includes a lysin A of SEQ ID NO: 2, a lysin B of SEQ ID NO: 184, an isoamylase of SEQ ID NO: 243, and an a-amylase of SEQ ID NO: 396.
  • the composition includes a lysin A of SEQ ID NO: 1 , a lysin B of SEQ ID NO: 183, an isoamylase of SEQ ID NO: 242, and an a-amylase of SEQ ID NO: 397.
  • the composition includes a lysin A of SEQ ID NO: 2, a lysin B of SEQ ID NO: 184, an isoamylase of SEQ ID NO: 243, and an a-amylase of SEQ ID NO: 398.
  • the bacterial infection is caused by or the cells are an actinomycetia bacterium.
  • the actinomycetia is a corynebacteriales or propionibacteriales.
  • the cornyebacteriales is a Mycobacterium species.
  • the Mycobacterium species is M. tuberculosis, M. leprae, M. lepromatosis, M. avium, M. kansasii, M. fortuitum, M. chelonae, M. marinum, M. intracellulare, M. abscessus, M. chimera, M. boletti, M. fortuitum, M. goodii, or M. masiliense.
  • the corynebacteriales is a Nocardia, Corynebacterium, or Rhodococcus species.
  • the propionibacteriales is a Cutibacterium species.
  • the compositions and methods described herein may be used to treat or prevent an infection caused by other actinomycetia (e.g., corynebacteriales or propionibacteriales) that have similar envelope components as mycobacteria.
  • the compositions and methods may be used for a Nocardia, Corynebacterium, or Rhodococcus species.
  • the Nocardia species may be, e.g., N. brasiliensis, N. cyriacigeorgica, N. farcinica, N. nova, N. asteroids, N. brasiliensis, and N. caviae.
  • the Corynebacterium species may be, e.g., C. glutamicum or C. diphtheriae.
  • the Rhodococcus species may be, e.g., R. fascians or R. equi.
  • compositions and methods may be used for a Cutibacterium species.
  • the Cutibacterium species may be, e.g., C. acnes.
  • the method further includes administering an antibiotic, e.g., to treat or prevent the infection.
  • the antibiotic may be administered with the composition or as its own treatment.
  • the antibiotic is a cephalosporin, a carbapenem, a penicillin, an aminoglycoside, a cephalosporin, a rifamycin, a macrolide, or a fluoroquinolone.
  • the antibiotic is thiacetazone, sq-109, bedaquiline, delamanid, pyrazinamide, or isoniazid.
  • the antibiotic is azithromycin, clarithromycin, ethambutol, rifampin, biapenem, or amikacin.
  • the antibiotic is a macrolide (e.g., azithromycin, clarithromycin, erythromycin).
  • the antibiotic is a macrolide (e.g., azithromycin, clarithromycin, erythromycin).
  • the antibiotic is an aminoglycoside (e.g., kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin (e.g., neomycin B, C, or E), streptomycin, or plazomicin).
  • aminoglycoside e.g., kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin (e.g., neomycin B, C, or E), streptomycin, or plazomicin).
  • composition and/or the antibiotic is administered intravenously, orally, or via inhalation (e.g., via aerosol).
  • the invention features an immunogenic composition produced a method as described herein.
  • the invention features an immunogenic composition produced by a method as described herein.
  • the method includes the step of (a) contacting a sample of actinomycetia cells with a composition that includes two or more (e.g., two, three, or four) of a lysin A, a lysin B, an isoamylase, and an a-amylase.
  • the composition comprises lysin A and lysin B.
  • the composition comprises lysin A and isoamylase.
  • the composition comprises lysin A and a-amylase.
  • the composition comprises lysin B and isoamylase.
  • the composition comprises lysin B and a-amylase. In some embodiments, the composition comprises isoamylase and a-amylase. In some embodiments, the composition comprises lysin A, lysin B, and isoamylase. In some embodiments, the composition comprises lysin A, lysin B, and a-amylase. In some embodiments, the composition comprises lysin A, isoamylase, and a-amylase. In some embodiments, the composition comprises lysin B, isoamylase, and a-amylase. In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and a-amylase. In some embodiments, the composition comprises lysin A, lysin B, isoamylase, and a-amylase.
  • the composition lyses the cells of the sample to produce a plurality of biological particles or fragments thereof of the actinomycetia cells.
  • the method further includes the step of (b) collecting the plurality of biological particles or fragments thereof or a portion thereof of step (a) to produce the immunogenic composition.
  • antibacterial lytic protein refers to a protein that has bactericidal and/or bacteriolytic activity against bacteria.
  • Non-limiting examples of antibacterial lytic proteins include holins, lysins (e.g., lysin A and/or lysin B), amylases (e.g., isoamylase or a-amylase), capsule depolymerases (e.g., hydrolase, metallohydrolase, epoxide hydrolase, peptidoglycan hydrolase, polysaccharase, polysaccharide lyase, endosialidase, hyaluronan lyase, or alginate lyase), beta lactamases, and lysozyme.
  • holins e.g., lysin A and/or lysin B
  • amylases e.g., isoamylase or a-amylase
  • FIGS. 1 A and 1 B are bright field images of mycobacterial bacilli rod shaped cells that are shredded by ABIa (i.e., composition containing lysin A, lysin B, isoamylase, and a-amylase) into subcellular fragments that contain stainable lipids giving rise to light grey staining bodies that are separated from the genomic DNA.
  • ABIa i.e., composition containing lysin A, lysin B, isoamylase, and a-amylase
  • FIGS. 1 A and 1 B are bright field images of mycobacterial bacilli rod shaped cells that are shredded by ABIa (i.e., composition containing lysin A, lysin B, isoamylase, and a-amylase) into subcellular fragments that contain stainable lipids giving rise to light grey staining bodies that are separated from the genomic DNA.
  • FIG. 1 A shows untreated mycobacterial cells without fragments. Lipid
  • FIGS. 2A and 2B are bright field images as in FIGS. 1 A and 1 B except performed at pH 5.3.
  • FIGS. 3A and 3B show data in the absence (FIG. 3A) and presence (FIG. 3B) of ABIa at 1 hour.
  • FIGS. 3C and 3D show data in the absence (FIG. 3D) and presence (FIG. 3D) of ABIa at 2 hours.
  • FIGS. 3E and 3F show data in the absence (FIG. 3E) and presence (FIG. 3F) ABIa at 12 hours.
  • FIGS. 4A-4F are graphs from a flow cytometry experiment showing single cells. Cells and the resulting debris were stained with SYTOX Green Nucleic Acid Stain and lipophilic FM4-64FX stain. Graphs of particle size/ feature were used to establish gates for fluorescent analysis plots of treated and untreated cell populations. The x and y axes represent increasing FM 4-64FX and SYTOX fluorescence intensity, respectively, in logarithmic scale and range incrementally from 10 3 -to-10 6 .
  • FIGS. 4A and 4B show data in the absence (FIG. 4A) and presence (FIG. 4B) of ABIa at 1 hour.
  • FIGS. 4C and 4D show data in the absence (FIG.
  • FIGS. 4C and 4D show data in the absence (FIG. 4E) and presence (FIG. 4F) of ABIa at 12 hours.
  • FIGS. 5A-5F are graphs from a flow cytometry experiment showing smaller cellular debris. Cells and the resulting debris were stained with SYTOX Green Nucleic Acid Stain and lipophilic FM4- 64FX stain. Graphs of particle size/ feature were used to establish gates for fluorescent analysis plots of treated and untreated cell populations. The x and y axes represent increasing FM 4-64FX and SYTOX fluorescence intensity, respectively, in logarithmic scale and range incrementally from 10 -3 -to- 10 6 .
  • FIGS. 5A and 5B show data in the absence (FIG. 5A) and presence (FIG. 5B) of ABIa at 1 hour.
  • FIGS. 5C and 5D show data in the absence (FIG. 5C) and presence (FIG. 5D) of ABIa at 2 hours.
  • FIGS. 5E and 5F show data in the absence (FIG. 5E) and presence (FIG. 5F) of ABIa at 12 hours.
  • FIGS. 6A-6F are graphs from a flow cytometry experiment showing aggregated cell debris. Cells and the resulting debris were stained with SYTOX Green Nucleic Acid Stain and lipophilic FM4- 64FX stain. Graphs of particle size/ feature were used to establish gates for fluorescent analysis plots of treated and untreated cell populations. The x and y axes represent increasing FM 4-64FX and SYTOX fluorescence intensity, respectively, in logarithmic scale and range incrementally from 10 -3 -to- 10 6 .
  • FIGS. 6A and 6B show data in the absence (FIG. 6A) and presence (FIG. 6B) of ABIa at 1 hour.
  • FIGS. 6C and 6D show data in the absence (FIG. 6C) and presence (FIG. 6D) of ABIa at 2 hours.
  • FIGS. 6E and 6F show data in the absence (FIG. 6E) and presence (FIG. 6F) of ABIa at 12 hours.
  • Mycobacteria are actinomycetia (e.g., corynebacteriales or propionibacteriales) that are denoted by a thick cell wall that is rich in mycolic acids.
  • Mycobacteria contain, from outside to inside, a capsule, mycolic acid layer, arabinogalactan (AGL) layer, peptidoglycan (PG), plasma membrane, and cytoplasm.
  • This complex cell envelope contributes to the hardiness of the mycobacteria and is particularly difficult to identify, penetrate and destroy, which is needed for the effective treatment of mycobacterial infections.
  • This invention features the generation a highly immunogenic cell derived particles that contain the complex antigenic substrates built by actinomycetia that can be neutralized by antibodies and drive cell death of infected cells by allowing an effective acquired immunity response. This could control disseminated cells as well as those in intracellularly infected with actinomycetia bacteria.
  • the remarkable feature with this approach is the antigens have been built by mycobacteria through very complicated biosynthesis performed stepwise in multiple compartments within actinmycetia cells: cytosol, plasma membrane, periplasm, outer membrane, and capsule.
  • the methods described herein will generate smaller antigenic pieces that are released from the cell and of a suitable size to bind to B cell receptors that go on to make antibodies to these complex antigens.
  • the complexity includes the glycoproteins, glycolipids, unusual lipids, glycopolymers, and free proteins. In some cases, it may be advantageous to further fractionate the antigenic particles that have the best antigenic effects and lowest negative effects on the patient.
  • the present invention features the use of a cocktail of lytic enzymes that was rationally designed to degrade the mycobacterial envelope.
  • the methods described herein include using a cocktail of antibacterial lytic proteins that degrade the mycobacterial envelope to lyse the bacteria.
  • the composition may be used, e.g., in vitro, to contact a sample of actinomycetia cells. Upon lysis, the ruptured bacterial cell produces a plurality of biological particles or fragments thereof, such as lipids, carbohydrates, polypeptides, and nucleic acids. These biological particles or fragments thereof may be used to create an immunogenic composition that can be administered to a subject to treat or prevent a bacterial infection caused by the same or related species of bacteria.
  • the cocktail includes two or more of lysin A, lysin B, isoamylase, and a-amylase.
  • a combination of lytic proteins is particularly advantageous in killing and rupturing a mycobacterial cell.
  • the components of envelopes at the basic structure levels are observed for many actinomycetia, such as corynebacteriales (e.g., mycobacteria) and propionibacteriales, such as cutibacteria. Therefore, these cocktails can be used to produce biological particles or fragments thereof by rupturing a variety of mycobacteria and related actinomycetia (e.g., corynebacteriales or propionibacteriales) with similar envelope structures.
  • the methods described herein can be used for producing an immunogenic composition that can be used to produce an immune response in a subject.
  • the immunogenic composition ca be used to treat or prevent a bacterial infection, e.g., an actinomycetia infection.
  • the methods described herein include the step of (a) contacting a sample of actinomycetia cells with a composition that includes two or more (e.g., two, three, or four) of a lysin A, a lysin B, an isoamylase, and an a- amylase.
  • the composition lyses the cells of the sample to produce a plurality of biological particles or fragments thereof of the actinomycetia cells.
  • the method may further include the step of (b) collecting the plurality of biological particles or fragments thereof or a portion thereof of step (a) to produce the immunogenic composition.
  • the method further includes purifying the immunogenic composition that includes the plurality of biological particles or fragments thereof.
  • the method may include removing undesired biological particles or fragments thereof, e.g., that do not produce an immunogenic response from the composition. Such methods may be used to enrich the immunogenic composition to elicit a robust immune response in a subject.
  • the methods described herein include treating or preventing an actinomycetia bacterial infection in a subject.
  • the method includes administering to the subject an immunogenic composition that includes a plurality of biological particles or fragments thereof of actinomycetia cells.
  • the immunogenic composition may have been produced by the step of (a) contacting a sample of actinomycetia cells with a composition that includes two or more (e.g., two, three, or four) of a lysin A, a lysin B, an isoamylase, and an a-amylase.
  • the composition lyses the cells of the sample to produce a plurality of biological particles or fragments thereof of the actinomycetia cells.
  • the immunogenic may have also been produced by the step of (b) collecting the plurality of biological particles or fragments thereof or a portion thereof from to produce the immunogenic composition.
  • the methods described herein further include administering the composition, e.g., that includes the two or more (e.g., two, three, or four) of a lysin A, a lysin B, an isoamylase, and an a-amylase, to the subject, e.g., to treat or prevent the actinomycetia bacterial infection.
  • the subject may have been administered an immunogenic composition.
  • the subject may continue to be administered a composition containing lytic enzymes, e.g., to treat the bacterial infection of prevent subsequent infection.
  • the composition can be repeatedly given one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) times and the course of infection can be monitored. If the infection exhibits a reduced amount (e.g., reduced by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or substantially all) of the infection, e.g., as compared to a previous sample, this may indicate the infection is being properly treated.
  • a reduced amount e.g., reduced by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or substantially all
  • the methods described herein includes administering the immunogenic composition, multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) times to treat or prevent the actinomycetia bacterial infection.
  • the subject may have been administered an immunogenic composition.
  • the subject may continue to be administered the immunogenic composition, e.g., to treat the bacterial infection of prevent subsequent infection.
  • the composition can be repeatedly given one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) times and the course of infection can be monitored. If the infection exhibits a reduced amount (e.g., reduced by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or substantially all) of the infection, e.g., as compared to a previous sample, this may indicate the infection is being properly treated.
  • a reduced amount e.g., reduced by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or substantially all
  • treating the infection includes administering the composition containing the lytic enzymes, e.g., containing two or more of (e.g., two, three, or all four of) a lysin A, a lysin B, an isoamylase, and an a-amylase.
  • treating the infection includes administering an antibiotic.
  • treating the infection includes administering the composition containing the lytic enzymes and the antibiotic.
  • treating the infection includes administering the immunogenic composition containing a plurality of biological particles or fragments thereof of the actinmycetia cells and the composition containing the lytic enzymes.
  • treating the infection includes administering an immunogenic composition containing the plurality of biological particles or fragments thereof of the actinmycetia cells and an antibiotic. In some embodiments, treating the infection includes administering an immunogenic composition containing a plurality of biological particles or fragments thereof of the actinmycetia cells, a composition containing the lytic enzymes, and an antibiotic.
  • the methods of treatment described herein may be used to reduce a level of infection.
  • the methods may decrease a level of infection (e.g., number of bacteria or size of infection), as compared to a reference.
  • the infection may decrease by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • the methods described herein may be used to monitor or guide a course of treatment. For example, if a subject has a bacterial infection, a sample from the subject can be repeatedly probed to identify a level of infection over time.
  • a higher dose of treatment e.g., a plurality of actinomycetia particles or fragments thereof, a composition containing lytic enzymes, or antibiotic
  • a lower dose of treatment can be administered to the subject.
  • the level of infection stays the same (e.g., within a level of +/- 20%, e.g., +/- 10%)
  • the same dose of treatment can be administered to the subject. If the infection reaches a baseline level or is no longer present, the treatment may be discontinued.
  • the components of the compositions described herein can lyse a plurality of bacterial cells to produce a plurality of biological particles or fragments thereof.
  • the biological particles or fragments thereof may include, for example, lipids, nucleic acids (e.g., DNA or RNA), polypeptides, carbohydrates, and the like. Such biological particles or fragments thereof may be identified by any suitable method known to the skilled artisan.
  • the presence of the biological particles or fragments thereof, e.g., associated with the bacterial infection may be identified, for example, by evaluating the concentration or relative abundance of the biological particles or fragments thereof in a sample. These biological particles or fragments thereof can be assayed for immunogenic activity and purified for subsequent use.
  • compositions and methods described herein include a portion of the peptidoglycan cell wall, glucose or glucose polymers, gluco-oligomers, crosslinked glucose with a 1 -4 linkages and a 1 -6 linkages, araginogalactan oligomers, proteins, or unique mycobacterial lipids.
  • the cellular contents can be fractionated for enhanced purification, e.g., to separate the genomic DNA, RNA, and proteins from the envelope fragments. This may facilitate analysis of either subcellular fraction in order to identify the biological particles and fragments thereof that produce a robust immunogenic response.
  • the invention features methods of using one or more of (e.g., one, two, three, or four) of lysin A, lysin B, isoamylase, and a-amylase to produce a plurality of biological particles or fragments thereof for treating or preventing an actinomycetia bacterial infection.
  • Exemplary proteins suitable for use in the compositions described herein are shown in Table 1 below.
  • compositions described herein may include a lysin A that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.
  • the lysin A may include or consist of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • compositions described herein may include a lysin B that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 183 or SEQ ID NO: 184.
  • the lysin B may include or consist of the amino acid sequence of SEQ ID NO: 183 or SEQ ID NO: 184.
  • the compositions described herein may include an isoamylase that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 242 or SEQ ID NO: 243.
  • the isoamylase may include or consist of the amino acid sequence of SEQ ID NO: 242 or SEQ ID NO: 243.
  • compositions described herein may include an a-amylase that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 393-398.
  • the a-amylase may include or consist of the amino acid sequence of any one of SEQ ID NOs: 393-398.
  • compositions described herein may include an a-amylase that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 393 or SEQ ID NO: 394.
  • the a-amylase may include or consist of the amino acid sequence of SEQ ID NO: 393 or SEQ ID NO: 394.
  • the composition includes a lysin A of SEQ ID NO: 1 , a lysin B of SEQ ID NO: 183, an isoamylase of SEQ ID NO: 242, and an a-amylase of SEQ ID NO: 393.
  • the composition includes a lysin A of SEQ ID NO: 2, a lysin B of SEQ ID NO: 184, an isoamylase of SEQ ID NO: 243, and an a-amylase of SEQ ID NO: 394.
  • the composition includes a lysin A of SEQ ID NO: 1 , a lysin B of SEQ ID NO: 183, an isoamylase of SEQ ID NO: 242, and an a-amylase of SEQ ID NO: 395.
  • the composition includes a lysin A of SEQ ID NO: 2, a lysin B of SEQ ID NO: 184, an isoamylase of SEQ ID NO: 243, and an a-amylase of SEQ ID NO: 396.
  • the composition includes a lysin A of SEQ ID NO: 1 , a lysin B of SEQ ID NO: 183, an isoamylase of SEQ ID NO: 242, and an a-amylase of SEQ ID NO: 397.
  • the composition includes a lysin A of SEQ ID NO: 2, a lysin B of SEQ ID NO: 184, an isoamylase of SEQ ID NO: 243, and an a-amylase of SEQ ID NO: 398.
  • the composition includes a lysin A that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to of any one of SEQ ID NOs: 1 -182 as shown in Table 2 below.
  • the composition includes a lysin B that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to of any one of SEQ ID NOs: 183-241 as shown in Table 3 below.
  • the composition includes an isoamylase that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to of any one of SEQ ID NOs: 242-392 as shown in Table 4 below.
  • the composition includes an a-amylase that includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to of any one of SEQ ID NOs: 393-445 as shown in Table 5 below. Table 5. a-amylase Sequences
  • the lytic proteins described herein may be recombinantly produced.
  • the protein may contain a suitable purification tag, such as a His tag containing, e.g., three, four, five, six, seven, eight, nine, ten, or more histidine residues present at the N-terminus or C-terminus of the protein.
  • the protein may also contain a removable signal sequence present at the N-terminus or C-terminus of the protein.
  • the lytic proteins described herein may include biologically active fragments thereof, e.g., fragments of a lytic protein as described herein that may be truncated, e.g., by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more amino acids but still substantially retain their biological activity.
  • the composition includes a concentration of proteins (e.g., lysin A, lysin B, isoamylase, and/or a-amylase) of from 0.1 mg/mL to 20 mg/mL (e.g., e.g., from 0.1 mg/mL to
  • I mg/mL e.g., 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, or 1 mg/mL, e.g., from 1 mg/mL to 10 mg/mL, e.g., 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, e.g., from 10 mg/mL to 20 mg/mL, e.g.,
  • the composition includes a concentration of lysin A, lysin B, isoamylase, and/or a-amylase of from 1 mg/mL to 10 mg/mL.
  • compositions and methods described herein may be used to treat or prevent an infection caused by a mycobacterium, such as an intracellular mycobacterium that resides in a professional antigen presenting cell (e.g., macrophage or dendritic cell).
  • a mycobacterium such as an intracellular mycobacterium that resides in a professional antigen presenting cell (e.g., macrophage or dendritic cell).
  • the mycobacterial species is M. tuberculosis, M. leprae, M. lepromatosis, M. avium, M. kansasii, M. fortuitum, M. chelonae, M. marinum, M. intracellulare, M. abscessus, M. chimera, M. boletti, M. fortuitum, M. goodii, or M. masiliense.
  • the mycobacterium is an NTM.
  • the NTM is M. abscessus, M. intracellulare, M. avium, M. chimera, M. boletti, M. fortuitum, M. goodii, and M. masiliense.
  • compositions and methods described herein may be used to treat or prevent an infection caused by other actinomycetia (e.g., corynebacteriales or propionibacteriales) that have similar envelope components as mycobacteria.
  • actinomycetia e.g., corynebacteriales or propionibacteriales
  • the compositions and methods may be used to for a Nocardia, Corynebacterium, or Rhodococcus species.
  • the Nocardia species may be, e.g., N. brasiliensis, N. cyriacigeorgica, N. farcinica, N. nova, N. asteroids, N. brasiliensis, and N. caviae.
  • the Corynebacterium species may be, e.g., C.
  • the Rhodococcus species may be, e.g., R. fascians or R. equi.
  • the composition and methods may be used for a propionibacteriales, such as a Cutibacterium species.
  • the Cutibacterium species may be, e.g., C. acnes.
  • the biological particles and fragments thereof and lytic enzymes described herein are preferably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.
  • compositions described herein may be administered to a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art.
  • the composition may be administered, for example, by oral, parenteral, intrathecal, intracerebroventricular, intraparenchymal, buccal, sublingual, nasal, rectal, patch, pump, or transdermal administration and the pharmaceutical compositions formulated accordingly.
  • Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, intracerebroventricular, intraparenchymal, rectal, and topical modes of administration.
  • the composition is administered via aerosol.
  • Parenteral administration may be by continuous infusion over a selected period of time.
  • the compositions described herein are administered via inhalation.
  • Administration of more than one antibacterial agent may be by the same route or by different routes and may occur sequentially or substantially simultaneously.
  • a first antibacterial agent of the combination may be administered by intravenous injection while a second therapeutic agent of the combination may be administered orally.
  • compositions described herein may be administered, e.g., by inhalation.
  • Inhalation may be oral inhalation or nasal inhalation.
  • An inhalable composition described herein may be provided as a liquid dosage form or dry powder dosage form.
  • a dry powder composition may be, e.g., administered by inhalation as is or after reconstitution in a vehicle (e.g., saline (e.g., isotonic saline), phosphate-buffered saline, or water).
  • Inhalable dry powder dosage forms may be prepared from liquid compositions described herein by drying (e.g., by freeze drying, spray drying, spray-freeze drying, or supercritical fluid technology).
  • Inhalable dry powder dosage forms described herein may include a carrier (e.g., lactose, sucrose, mannitol, and the like), cryoprotectant (e.g., trehalose, mannitol, and the like), and/or antiadherent (e.g., glycine, L-leucine, serine, and the like).
  • Inhalable dry powder dosage forms described herein may be administered using dry powder inhalers. Dry powder inhalers are known in the art and may or may not include a propellant. Non-limiting examples of dry powder inhalers can be found in Newman, Expert Opin. Biol. Then, 4:23-33, 2004, the disclosure of which is incorporated herein by reference in its entirety.
  • Inhalable liquid dosage forms may be prepared using techniques and methods useful in the preparation of liquid compositions containing lytic enzymes or biological particles or fragments thereof.
  • Inhalable liquid dosage forms typically include a suspension of the compositions described herein in a physiologically acceptable aqueous or nonaqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device.
  • the sealed container may be a unitary dispensing device, e.g., a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use.
  • the dosage form contains an aerosol dispenser, it will contain a propellant, which can be a compressed gas, e.g., compressed air or an organic propellant, e.g., hydrofluoroalkane.
  • a propellant which can be a compressed gas, e.g., compressed air or an organic propellant, e.g., hydrofluoroalkane.
  • the inhalable liquid dosage forms may be administered using a nebulizer.
  • the process of pneumatically converting a bulk liquid into small droplets is called atomization.
  • the operation of a pneumatic nebulizer requires a propellant as the driving force for liquid atomization.
  • Various types of nebulizers are described in Respiratory Care, 45:609-622, 2000, the disclosure of which is incorporated herein by reference in its entirety.
  • an inhalable liquid dosage form described herein may be administered using a metered-dose inhaler.
  • Metered-dose inhalers are known in the art and
  • a composition described herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard- or soft-shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • a composition described herein may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers.
  • a composition described herein may also be administered parenterally. Solutions of a composition described herein can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO, and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington’s Pharmaceutical Sciences (2012, 22nd ed.) and in The United States Pharmacopeia: The National Formulary (USP 41 NF 36), published in 2018.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, where the active ingredient is formulated with a carrier, such as sugar, acacia, tragacanth, gelatin, and glycerin.
  • Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base, such as cocoa butter.
  • composition described herein may be administered to an animal, e.g., a human, alone or in combination with pharmaceutically acceptable carriers, as noted herein, the proportion of which is determined by the solubility and chemical nature of the composition, chosen route of administration, and standard pharmaceutical practice.
  • the dosage of the compositions can vary depending on many factors, such as the pharmacodynamic properties of the antibacterial lytic proteins, the mode of administration, the age, health, and weight of the recipient, the nature and extent of the symptoms, the frequency of the treatment, and the type of concurrent treatment, if any, and the clearance rate of the composition in the animal to be treated.
  • the compositions described herein may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response.
  • the dosage of a composition e.g., a composition including a lytic protein
  • all dosages may be continuously given or divided into dosages given per a given time frame.
  • the composition can be administered, for example, every hour, day, week, month, or year. In some embodiments, the composition may be administered continuously or systemically.
  • the immunogenic compositions described herein may further include or be administered with an adjuvant.
  • An adjuvant enhances the immune responses (humoral and/or cellular) elicited in a subject who receives the adjuvant and/or an immunogenic composition including the adjuvant.
  • an adjuvant and a plurality of biological particles or fragments thereof are coadministered in separate compositions.
  • an adjuvant is mixed or formulated with a plurality of biological particles or fragments thereof in a single composition and administered to a subject.
  • an adjuvant and plurality of biological particles or fragments thereof are co-administered in separate compositions.
  • an adjuvant is mixed or formulated with a plurality of biological particles or fragments thereof in a single composition to obtain an immunogenic composition that is administered to a subject.
  • Adjuvants may be a TH1 adjuvant and/or a TH2 adjuvant. Further adjuvants contemplated by this disclosure include, but are not limited to, one or more of the following:
  • Mineral-containing compositions suitable for use as adjuvants in the disclosure include mineral salts, such as aluminum salts, and calcium salts.
  • the disclosure includes mineral salts such as hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates), sulphates, etc., or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g., gel, crystalline, amorphous, etc.).
  • Calcium salts include calcium phosphate (e.g., the "CAP").
  • Aluminum salts include hydroxides, phosphates, sulfates, and the like.
  • Oil-emulsion compositions suitable for use as adjuvants in the disclosure include squalene- water emulsions, such as MF59 (5% Squalene, 0.5% TWEEN 80 and 0.5% Span, formulated into submicron particles using a microfluidizer), AS03 (a-tocopherol, squalene and polysorbate 80 in an oil-in-water emulsion), Montanide formulations (e.g., Montanide ISA 51 , Montanide ISA 720), incomplete Freunds adjuvant (IFA) , complete Freund's adjuvant (CFA), and incomplete Freund's adjuvant (IFA).
  • squalene- water emulsions such as MF59 (5% Squalene, 0.5% TWEEN 80 and 0.5% Span, formulated into submicron particles using a microfluidizer
  • AS03 a-tocopherol, squalene and polysorbate 80 in an oil-in-water emulsion
  • Small molecules suitable for use as adjuvants in the disclosure include imiquimod or 847, resiquimod or R848, and gardiquimod.
  • Polymeric nanoparticles suitable for use as an adjuvant in the disclosure include polyphydroxy acids), polyhydroxy butyric acids, polylactones (including polycaprolactones), polydioxanones, polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosinederived polycarbonates, polyvinyl-pyrrolidinones or polyester-amides, and combinations thereof.
  • Saponin (i.e. , a glycoside, polycyclic aglycones attached to one or more sugar side chains) formulations suitable for use as an adjuvant in the disclosure include purified formulations, such as QS21 , as well as lipid formulations, such as ISCOMs and ISCOMs matrix.
  • QS21 is marketed as STIMULON (TM).
  • Saponin formulations may also include a sterol, such as cholesterol. Combinations of saponins and cholesterols can be used to form unique particles called immunostimulating complexes (ISCOMs).
  • ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs.
  • the ISCOM includes one or more of QuilA, QHA & QHC.
  • the ISCOMS may be devoid of additional detergent.
  • Adjuvants suitable for use in the disclosure include non-toxic derivatives of enterobacterial lipopolysaccharide (LPS).
  • LPS enterobacterial lipopolysaccharide
  • Such derivatives include monophosphoryl lipid A (MPLA), glucopyranosyl lipid A (GLA) and 3-O-deacylated MPL (3dMPL).
  • 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains.
  • Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g., RC- 529.
  • Liposomes suitable for use as an adjuvant in the disclosure include virosomes and CAF01 .
  • Adjuvants suitable for use in the disclosure include lipid nanoparticles (LNPs) and their components.
  • LNPs lipid nanoparticles
  • Lipopeptides i.e., compounds including one or more fatty acid residues and two or more amino acid residues
  • suitable for use as an adjuvant in the disclosure include Pam2 (Pam2CSK4) and Pam3 (Pam3CSK4).
  • Glycolipids suitable for use as an adjuvant in the disclosure include cord factor (trehalose dimycolate).
  • Peptides and peptidoglycans derived from (synthetic or purified) gram-negative or grampositive bacteria, such as MDP (N-acetyl-muramyl-L-alanyl-D-isoglutamine) are suitable for use as an adjuvant in the disclosure.
  • Carbohydrates (carbohydrate containing) or polysaccharides suitable for use as an adjuvant include dextran (e.g., branched microbial polysaccharide), dextran-sulfate, lentinan, zymosan, betaglucan, deltin, mannan, and chitin.
  • RNA based adjuvants suitable for use in the disclosure are poly IC, poly IC:LC, hairpin RNAs with or without a 5’triphosphate, viral sequences, polyU containing sequence, dsRNA natural or synthetic RNA sequences, and nucleic acid analogs (e.g., cyclic GMP-AMP or other cyclic dinucleotides e.g., cyclic di-GMP, immunostimulatory base analogs e.g., C8-substituted and N7,C8- disubstituted guanine ribonucleotides).
  • nucleic acid analogs e.g., cyclic GMP-AMP or other cyclic dinucleotides e.g., cyclic di-GMP, immunostimulatory base analogs e.g., C8-substituted and N7,C8- disubstituted guanine ribonucleotides.
  • DNA based adjuvants suitable for use in the disclosure include CpGs, dsDNA, and natural or synthetic immunostimulatory DNA sequences.
  • Proteins and peptides suitable for use as an adjuvant in the disclosure include flagelli n-fusion proteins, MBL (mannose-binding lectin), cytokines, and chemokines.
  • Viral particles suitable for use as an adjuvant include virosomes (phospholipid cell membrane bilayer).
  • An adjuvant for use in the disclosure may be bacterial derived, such as a flagellin, LPS, or a bacterial toxin (e.g., enterotoxins (protein), e.g., heat-labile toxin or cholera toxin).
  • An adjuvant for use in the disclosure may be a hybrid molecule such as CpG conjugated to imiquimod.
  • An adjuvant for use in the disclosure may be a fungal or oomycete microbe-associated molecular patterns (MAMPs), such as chitin or beta-glucan.
  • MAMPs microbe-associated molecular patterns
  • an adjuvant is an inorganic nanoparticle, such as gold nanorods or silica-based nanoparticles (e.g., mesoporous silica nanoparticles (MSN)).
  • an adjuvant is a multi-component adjuvant or adjuvant system, such as AS01 , AS03, AS04 (MLP5 + alum), CFA (complete Freund’s adjuvant: IFA + peptiglycan + trehalose dimycolate), CAF01 (two component system of cationic liposome vehicle (dimethyl dioctadecylammonium (DDA)) stabilized with a glycolipid immunomodulator (trehalose 6,6-dibehenate (TDB), which can be a synthetic variant of cord factor located in the mycobacterial cell wall).
  • DDA dimethyl dioctadecylammonium
  • TDB glycolipid immunomodulator
  • a combination therapy means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition.
  • the treatment regimen defines the doses and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap.
  • the delivery of the two or more agents is simultaneous or concurrent and the agents may be coformulated.
  • the two or more agents are not co-formulated and are administered in a sequential manner as part of a prescribed regimen.
  • Sequential or substantially simultaneous administration of each therapeutic agent can be by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues.
  • the therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination may be administered by intravenous injection or via aerosolization while a second therapeutic agent of the combination may be administered orally.
  • the first and second therapeutic agents may be administered simultaneously or sequentially, in either order.
  • the first therapeutic agent may be administered immediately, up to 15 minutes, up to 30 minutes, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to, 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, 14 hours, up to hours 16, up to 17 hours, up 18 hours, up to 19 hours up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours up to 24 hours or up to 1 -7, 1 -14, 1 -21 or 1 -30 days before or after the second therapeutic agent.
  • compositions described herein may further include an additional antibacterial agent that is administered in conjunction with the immunogenic composition or antibacterial lytic proteins.
  • compositions and methods described herein may further include treatment for an underlying lung condition, e.g., that may be exacerbated by a bacterial infection, e.g., NTM infection.
  • Suitable lung therapies include, without limitation, airway clearance, nebulizers, respirators, and inhalers, e.g., steroid inhalers.
  • the treatments described herein may include administration of an antibiotic.
  • the antibiotic may be administered alone or in combination with a composition containing lytic enzymes or immunogenic fragments.
  • Suitable antibiotics include, without limitation, penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, azlocillin, temocillin, cepalothin, cephapirin, cephradine, cephaloridine, cefazolin, cefamandole, cefuroxime, cephalexin, cefprozil, cefaclor, loracarbef, cefoxitin, cefmatozole, cefotaxime, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefixime, cefpodoxime, ceft
  • the antibiotic is a cephalosporin, carbapenem (e.g., biapenem), penicillin, a macrolide, an aminoglycoside, or a fluoroquinolone.
  • the antibiotic is selected from the group consisting of thiacetazone, sq-109, bedaquiline, delamanid, pyrazinamide, and isoniazid.
  • the antibiotic is a macrolide (e.g., azithromycin, clarithromycin, erythromycin).
  • the antibiotic is an aminoglycoside (e.g., kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin (e.g., neomycin B, C, or E), streptomycin, or plazomicin).
  • the synergy with the co-administered therapeutic agents may permit the antibiotic to be administered at a dose that would be subtherapeutic, if administered without the other therapeutic agents.
  • the antibiotic may be formulated with the composition containing the antibacterial lytic proteins or immunogenic fragments.
  • the antibiotic may be administered as a separate pharmaceutical composition.
  • the antibiotic may be administered at a different time than the pharmaceutical composition containing the lytic proteins or immunogenic fragments.
  • the additional antibiotic is amikacin.
  • the amikacin may be liposomal amikacin that is formulated, e.g., for inhalation.
  • FIGS. 1 A and 1 B are bright field images of mycobacterial bacilli rod shaped cells that are shredded by ABIa into subcellular fragments that contain stainable lipids giving rise to light grey staining bodies that are separated from the genomic DNA. Th experiment was performed at pH 6.2.
  • FIG. 1 A shows untreated mycobacterial cells without fragments. Lipids are shown in light grey, and DNA is shown in white. White staining DNA indicates dye access afforded by a compromised cell envelope. Intact cells do not readily uptake light grey dye.
  • FIG. 1 B shows ABIa treated cells and fragments. Lipids are shown in light grey, and DNA is shown in white. Subcellular fragments light up with the lipophilic light grey dye. The white DNA is spread out as compared to FIG. 1 A.
  • FIGS 2A and 2B show a similar experiment, with shredding observed at pH 5.3.
  • Dyes used in these images are as follows: SYTOX Green binds nucleic acid through a permeable membrane and FM 4-64FX binds to lipids on the outer leaflet of the plasmid membrane.
  • FM 4-64FX binds to lipids on the outer leaflet of the plasmid membrane.
  • FIGS. 3A and 3B show data in the absence (FIG. 3A) and presence (FIG. 3B) of ABIa at 1 hour.
  • FIGS. 3C and 3D show data in the absence (FIG. 3D) and presence (FIG. 3D) of ABIa at 2 hours.
  • FIGS. 3E and 3F show data in the absence (FIG. 3E) and presence (FIG. 3F) ABIa at 12 hours.
  • FIGS. 4A-4F are graphs from a flow cytometry experiment showing single cells. Cells and the resulting debris were stained with SYTOX Green Nucleic Acid Stain and lipophilic FM4-64FX stain. Graphs of particle size/ feature were used to establish gates for fluorescent analysis plots of treated and untreated cell populations. The x and y axes represent increasing FM 4-64FX and SYTOX fluorescence intensity, respectively, in logarithmic scale and range incrementally from 10 3 -to-10 6 .
  • FIGS. 4A and 4B show data in the absence (FIG. 4A) and presence (FIG. 4B) of ABIa at 1 hour.
  • FIGS. 4C and 4D show data in the absence (FIG. 4C) and presence (FIG. 4D) of ABIa at 2 hours.
  • FIGS. 4E and 4F show data in the absence (FIG. 4E) and presence (FIG. 4F) of ABIa at 12 hours.
  • FIGS. 5A-5F are graphs from a flow cytometry experiment showing smaller cellular debris.
  • Cells and the resulting debris were stained with SYTOX Green Nucleic Acid Stain and lipophilic FM4- 64FX stain.
  • Graphs of particle size/ feature were used to establish gates for fluorescent analysis plots of treated and untreated cell populations.
  • the x and y axes represent increasing FM 4-64FX and SYTOX fluorescence intensity, respectively, in logarithmic scale and range incrementally from 10 -3 -to- 10 6 .
  • FIGS. 5A and 5B show data in the absence (FIG. 5A) and presence (FIG. 5B) of ABIa at 1 hour.
  • FIGS. 5C and 5D show data in the absence (FIG. 5C) and presence (FIG. 5D) of ABIa at 2 hours.
  • FIGS. 5E and 5F show data in the absence (FIG. 5E) and presence (FIG. 5F) of ABIa
  • FIGS. 6A-6F are graphs from a flow cytometry experiment showing aggregated cell debris. Cells and the resulting debris were stained with SYTOX Green Nucleic Acid Stain and lipophilic FM4- 64FX stain. Graphs of particle size/ feature were used to establish gates for fluorescent analysis plots of treated and untreated cell populations. The x and y axes represent increasing FM 4-64FX and SYTOX fluorescence intensity, respectively, in logarithmic scale and range incrementally from 10 -3 -to- 10 6 .
  • FIGS. 6A and 6B show data in the absence (FIG. 6A) and presence (FIG. 6B) of ABIa at 1 hour.
  • FIGS. 6C and 6D show data in the absence (FIG.
  • FIGS. 6E and 6F show data in the absence (FIG. 6E) and presence (FIG. 6F) of ABIa at 12 hours.
  • a mycobacterial cell is obtained and cultured in vitro to propagate the culture.
  • the cell culture is treated with a composition that includes a lysin A, lysin B, an isoamylase, and an a-amylase.
  • the composition lyses the bacterial cells of the sample to produce a plurality of biological particles or fragments thereof of the mycobacterial cells.
  • the sample may then be purified to remove any undesired components and enrich for the desired biological particles or fragments thereof of the mycobacterial cells, thereby producing an immunogenic composition.
  • Example 3 Treating or preventing a bacterial infection with an immunogenic composition
  • the immunogenic composition produced as in Example 2 may be used to treat or prevent a mycobacterial infection in a human subject.
  • the composition containing a plurality of biological particles and fragments of the mycobacterial cell includes components associated with the mycobacterial cells, such as cell wall components (e.g., lipids and carbohydrates).
  • a subject at risk for developing a mycobacterial infection e.g., in the lung
  • the subject may then produce an immunogenic response to the mycobacterial particles and fragments without risking infection, thereby preventing future onset of infection.

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Abstract

La présente divulgation concerne des compositions et des méthodes pour le traitement d'infections à actinomycètes (par exemple, des infections corynébactériales), par exemple, provoquées par des cellules mycobactériennes, résidant à l'intérieur d'une cellule hôte (par exemple, une cellule de mammifère, par exemple, une cellule immunitaire, par exemple, un macrophage ou une cellule dendritique). Les procédés comprennent la mise en contact de cellules actinomycètes avec un mélange d'enzymes lytiques pour rompre les cellules bactériennes, libérant ainsi des particules biologiques et des fragments de celles-ci. Les produits de dégradation peuvent être utilisés en tant que composition immunogène pour traiter ou prévenir une infection future.
PCT/US2023/070682 2022-07-22 2023-07-21 Compositions immunogènes pour traitement ou prévention d'infections à actinomycètes WO2024020533A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150165013A1 (en) * 2012-06-29 2015-06-18 Lysando Ag Composition for use in mycobacteria vaccination
US20180271952A1 (en) * 2015-03-12 2018-09-27 Micreos Human Health B.V. Combination of Bactericidal Agent With a Lysosomotropic Alkalinising Agent for the Treatment of a Bacterial Infection
WO2021178969A1 (fr) * 2020-03-06 2021-09-10 Endolytix Technology, Inc. Compositions et méthodes pour le traitement d'infections bactériennes intracellulaires

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150165013A1 (en) * 2012-06-29 2015-06-18 Lysando Ag Composition for use in mycobacteria vaccination
US20180271952A1 (en) * 2015-03-12 2018-09-27 Micreos Human Health B.V. Combination of Bactericidal Agent With a Lysosomotropic Alkalinising Agent for the Treatment of a Bacterial Infection
WO2021178969A1 (fr) * 2020-03-06 2021-09-10 Endolytix Technology, Inc. Compositions et méthodes pour le traitement d'infections bactériennes intracellulaires

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