Sequence Listing

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 10, 2023, is named 51486-010WO2_Sequence_Listing_7_10_23.xml and is 660,329 bytes in size.

Background

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.

One group of challenging intracellular bacterial infections is caused by mycobacteria. 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. This complex cell envelope contributes to the hardiness of the mycobacteria and is particularly difficult to penetrate and destroy. Pathogenic mycobacteria are often partitioned into two groups: M. tuberculosis and non-tuberculosis mycobacteria (NTM). In contrast to tuberculosis, person-to-person transmission of NTM is rare. Nonetheless, the number of NTM infections is a growing health concern, particularly in people with lung disease.

Improved compositions and methods for treating and preventing intracellular bacterial infections, such as those caused by mycobacteria, are needed.

Summary of the Invention

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.

In one aspect, 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.

In some embodiments, the method further includes purifying the immunogenic composition that includes the plurality of biological particles or fragments thereof. For example, the method may include removing undesired biological particles or fragments thereof.

In some embodiments, the method further includes administering the immunogenic composition to a subject to treat or prevent the actinomycetia bacterial infection.

In another aspect, 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.

In some embodiments of any of the above aspects, 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. In some embodiments, the composition includes lysin A and lysin B. In some embodiments, the composition includes lysin A and isoamylase. In some embodiments, the composition includes lysin A and a-amylase. In some embodiments, the composition includes lysin B and isoamylase. In some embodiments, the composition includes lysin B and a-amylase. In some embodiments, 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.

In some embodiments of any of the above aspects, 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%, or 100%) sequence identity to any one of SEQ ID NOs: 393-445.

In some embodiments, 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%) sequence identity to any one of SEQ ID NOs: 393-398.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the composition includes lysin B and isoamylase, 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 ; 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. In some embodiments, the composition includes lysin B and isoamylase, 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; 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.

In some embodiments, the composition includes lysin B and a-amylase, 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 ; 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.

In some embodiments, the composition includes lysin B and a-amylase, 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; 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the composition includes lysin A, lysin B, 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; 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 ; 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.

In some embodiments, the composition includes lysin A, lysin B, 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; 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; 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.

In some embodiments, the composition includes lysin A, lysin B, 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; 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 ; 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.

In some embodiments, the composition includes lysin A, lysin B, 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 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; 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.

In some embodiments, 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 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; 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.

In some embodiments, 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.

In some embodiments, the composition includes lysin B, isoamylase, and a-amylase, 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 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.

In some embodiments, the composition includes lysin B, isoamylase, and a-amylase, 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 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.

In some embodiments, the composition includes lysin A, lysin B, 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 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; 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.

In some embodiments, the composition includes lysin A, lysin B, 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 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; 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. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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.

In some embodiments the bacterial infection is caused by or the cells are an actinomycetia bacterium. In some embodiments, the actinomycetia is a corynebacteriales or propionibacteriales. In some embodiments, the cornyebacteriales is a Mycobacterium species.

In some embodiments, 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. In some embodiments, the corynebacteriales is a Nocardia, Corynebacterium, or Rhodococcus species.

In some embodiments, the propionibacteriales is a Cutibacterium species.

For example, in some embodiments, 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. For example, the compositions and methods may be used for a Nocardia, Corynebacterium, or Rhodococcus species. For example, 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.

In some embodiments, the compositions and methods may be used for a Cutibacterium species. The Cutibacterium species may be, e.g., C. acnes.

In some embodiments of any of the above aspects, 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. In some embodiments, the antibiotic is a cephalosporin, a carbapenem, a penicillin, an aminoglycoside, a cephalosporin, a rifamycin, a macrolide, or a fluoroquinolone. In some embodiments, the antibiotic is thiacetazone, sq-109, bedaquiline, delamanid, pyrazinamide, or isoniazid. In some embodiments, the antibiotic is azithromycin, clarithromycin, ethambutol, rifampin, biapenem, or amikacin. In some embodiments, the antibiotic is a macrolide (e.g., azithromycin, clarithromycin, erythromycin). In some embodiments, the antibiotic is a macrolide (e.g., azithromycin, clarithromycin, erythromycin). In some embodiments, 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).

In some embodiments, the composition and/or the antibiotic is administered intravenously, orally, or via inhalation (e.g., via aerosol).

In another aspect, the invention features an immunogenic composition produced a method as described herein.

In another aspect, 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. In some embodiments, the composition comprises lysin A and lysin B. In some embodiments, the composition comprises lysin A and isoamylase. In some embodiments, the composition comprises lysin A and a-amylase. In some embodiments, the composition comprises lysin B and isoamylase. In some embodiments, 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. 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.

Definitions

As used herein, the term “about” refers to +/- 10% of a recited value.

The term “antibacterial lytic protein,” as used herein, 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.

Brief Description of the Drawings

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

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. The 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 are bright field images as in FIGS. 1 A and 1 B except performed at pH 5.3.

FIGS. 3A-3F are graphs from a flow cytometry experiment. Gates were established by the size and features of particles as seen in forward and side scattered light, respectively, via flow cytometry. Gates were detected that could separate mycobacterial populations of single cells, cellular debris, and large aggregates (aggrs) at t=1 , 2, and 12 hours after treatment with ABIa in vitro. 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 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.

Detailed Description

The invention described herein includes methods of treating or preventing a bacterial infection with an immunogenic composition and methods of producing the same. 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. By utilizing enzymatic degradation of peptidoglycan, mycolic acid, 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. In general, 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. Such a combination of lytic proteins is particularly advantageous in killing and rupturing a mycobacterial cell. To come up with the protein components, we first rationally attacked three layers of the mycobacterial envelope, the capsule, the junction between the mycolic acids and the AGL layer, and the peptidoglycan layer. 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.

Methods of Producing an Immunogenic Composition

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.

In some embodiments, the method further includes purifying the immunogenic composition that includes the plurality of biological particles or fragments thereof. For example, 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.

Methods of Treatment and Prevention

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.

In some embodiments, 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. For example, the subject may have been administered an immunogenic composition. Following the initial administration, the subject may continue to be administered a composition containing lytic enzymes, e.g., to treat the bacterial infection of prevent subsequent infection.

For example, 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.

In some embodiments, 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. For example, the subject may have been administered an immunogenic composition. Following the initial administration, the subject may continue to be administered the immunogenic composition, e.g., to treat the bacterial infection of prevent subsequent infection.

For example, 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.

In some embodiments, 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. In some embodiments, treating the infection includes administering an antibiotic. In some embodiments, treating the infection includes administering the composition containing the lytic enzymes and the antibiotic. In some embodiments, 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. In some embodiments, 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. For example, the methods may decrease a level of infection (e.g., number of bacteria or size of infection), as compared to a reference. For example, 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%. In some embodiments, 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. If the infection increases, a higher dose of treatment (e.g., a plurality of actinomycetia particles or fragments thereof, a composition containing lytic enzymes, or antibiotic) can be administered to the subject. Alternatively, if the infection decreases, a lower dose of treatment can be administered to the subject. If 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.

Biological Particles and Fragments

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.

Specific products known to be released from actinmycetia after degradation can be used in the compositions and methods described herein. Examples 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.

Antibacterial lytic proteins

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.

Table 1. Lytic protein sequences Protein SEQ ID NO: Sequence

The 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.

The 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.

The 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.

The 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.

In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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.

Additional sequences were identified that may be useful in the compositions and methods described herein. In some embodiments, 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.

Table 2. Lysin A Sequences

Additional sequences were identified that may be useful in the compositions and methods described herein. In some embodiments, 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.

Table 3. Lysin B Sequences

Additional sequences were identified that may be useful in the compositions and methods described herein. In some embodiments, 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.

Table 4. Isoamylase Sequences

Additional sequences were identified that may be useful in the compositions and methods described herein. In some embodiments, 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

One of skill in the art would appreciate that the lytic proteins described herein may be recombinantly produced. Accordingly, 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. One of skill in the art would also appreciate that 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.

In some embodiments, 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.,

I I mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, or 20 mg/mL). In some embodiments, the composition includes a concentration of lysin A, lysin B, isoamylase, and/or a-amylase of from 1 mg/mL to 10 mg/mL.

Bacterial Infections

The 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). In some embodiments, 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. In particular embodiments, the mycobacterium is an NTM. In some embodiments, the NTM is M. abscessus, M. intracellulare, M. avium, M. chimera, M. boletti, M. fortuitum, M. goodii, and M. masiliense.

In some embodiments, 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. For example, the compositions and methods may be used to for a Nocardia, Corynebacterium, or Rhodococcus species. For example, 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. The composition and methods may be used for a propionibacteriales, such as a Cutibacterium species. The Cutibacterium species may be, e.g., C. acnes.

Pharmaceutical Compositions

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.

The 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. In one embodiment, the composition is administered via aerosol. Parenteral administration may be by continuous infusion over a selected period of time. In some preferred embodiments, 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. For example, 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.

Certain 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 (e.g., aerosol formulations) described herein 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. Alternatively, 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. Where 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. 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. Alternatively, an inhalable liquid dosage form described herein may be administered using a metered-dose inhaler. Metered-dose inhalers are known in the art and typically include a canister, actuator, and a metering valve.

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. For oral therapeutic administration, 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. In all cases the form must be sterile and must be fluid to the extent that may be easily administered via syringe. 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.

The 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, e.g., a composition including biological particles or fragments thereof or a lytic protein, described herein, 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. In some embodiments, the dosage of a composition, e.g., a composition including a lytic protein, is a prophylactically or a therapeutically effective amount. Furthermore, it is understood that 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.

Adjuvants

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. In some embodiments, an adjuvant and a plurality of biological particles or fragments thereof are coadministered in separate compositions. In some embodiments, an adjuvant is mixed or formulated with a plurality of biological particles or fragments thereof in a single composition and administered to a subject. In some embodiments, an adjuvant and plurality of biological particles or fragments thereof are co-administered in separate compositions. In some embodiments, 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).

Small molecules. 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. Preferably, the ISCOM includes one or more of QuilA, QHA & QHC. Optionally, the ISCOMS may be devoid of additional detergent.

Adjuvants suitable for use in the disclosure include non-toxic derivatives of enterobacterial lipopolysaccharide (LPS). 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.

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).

DNA based adjuvants. 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. In some embodiments, an adjuvant is an inorganic nanoparticle, such as gold nanorods or silica-based nanoparticles (e.g., mesoporous silica nanoparticles (MSN)). In some embodiments, 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).

Combination Therapies

The pharmaceutical compositions described herein may be administered as part of a combination therapy. 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. In some embodiments, the delivery of the two or more agents is simultaneous or concurrent and the agents may be coformulated. In some embodiments, 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.

In any of the combination embodiments described herein, 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.

The pharmaceutical compositions described herein may further include an additional antibacterial agent that is administered in conjunction with the immunogenic composition or antibacterial lytic proteins.

The 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.

Antibiotics

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, ceftibuten, cefdinir, cefpirome, cefepime, chlorhexidine, BAL5788, BAL9141 , imipenem, ertapenem, meropenem, astreonam, clavulanate, sulbactam, tazobactam, streptomycin, neomycin, kanamycin, paromycin, gentamicin, tobramycin, amikacin, netilmicin, spectinomycin, sisomicin, dibekalin, isepamicin, tetracycline, chlortetracycline, demeclocycline, minocycline, oxytetracycline, methacycline, doxycycline, erythromycin, azithromycin, clarithromycin, telithromycin, ABT-773, lincomycin, clindamycin, vancomycin, oritavancin, dalbavancin, teicoplanin, quinupristin and dalfopristin, sulphanilamide, para-aminobenzoic acid, sulfadiazine, sulfisoxazole, sulfamethoxazole, sulfathalidine, linezolid, nalidixic acid, oxolinic acid, norfloxacin, perfloxacin, enoxacin, ofloxacin, ciprofloxacin, temafloxacin, lomefloxacin, fleroxacin, grepafloxacin, sparfloxacin, trovafloxacin, clinafloxacin, gatifloxacin, moxifloxacin, gemifloxacin, sitafloxacin, metronidazole, daptomycin, garenoxacin, ramoplanin, faropenem, polymyxin, tigecycline, AZD2563, trimethoprim, ethambutol, rifamycin, and rifampin. In some embodiments, multiple antibiotics are administered in combination with the compositions described herein. In some embodiments, the antibiotic is a cephalosporin, carbapenem (e.g., biapenem), penicillin, a macrolide, an aminoglycoside, or a fluoroquinolone. In some embodiments, the antibiotic is selected from the group consisting of thiacetazone, sq-109, bedaquiline, delamanid, pyrazinamide, and isoniazid.

In some embodiments, the antibiotic is a macrolide (e.g., azithromycin, clarithromycin, erythromycin). In some embodiments, 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).

Advantageously, in some embodiments, 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. In some preferred embodiments, the additional antibiotic is amikacin. The amikacin may be liposomal amikacin that is formulated, e.g., for inhalation.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.

Example 1. Fragmentation of Mycobacterium abscessus bacilli

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. The increase in FM 4-64FX expression together with the loss of the bacilli phenotype in the treatment (+ ABIa) suggests ABIa is capable of shredding M. abscessus.

FIGS. 3A-3F are graphs from a flow cytometry experiment. Gates were established by the size and features of particles as seen in forward and side scattered light, respectively, via flow cytometry. Gates were detected that could separate mycobacterial populations of single cells, cellular debris, and large aggregates (aggrs) at t=1 , 2, and 12 hours after treatment with ABIa in vitro. 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 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. Overall, these results indicate that actinomycetia can be shredded using the ABIa cocktail, and the biological particles or fragments thereof can be subsequently used to produce an immunogenic composition that can be used to treat or prevent bacterial infection in a subject.

Example 2. Production of an immunogenic composition

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) may be administered the immunogenic composition, e.g., via intravenous injection, e.g., with an adjuvant. The subject may then produce an immunogenic response to the mycobacterial particles and fragments without risking infection, thereby preventing future onset of infection.

Other Embodiments

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.

While the invention has been described in connection with specific embodiments thereof, it will be understood that invention is capable of further modifications and that this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.