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WO2025240841A1 - Thérapie antimicrobienne - Google Patents

Thérapie antimicrobienne

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Publication number
WO2025240841A1
WO2025240841A1 PCT/US2025/029732 US2025029732W WO2025240841A1 WO 2025240841 A1 WO2025240841 A1 WO 2025240841A1 US 2025029732 W US2025029732 W US 2025029732W WO 2025240841 A1 WO2025240841 A1 WO 2025240841A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
shc2
skin
composition
lantibiotic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/029732
Other languages
English (en)
Inventor
Richard L. Gallo
Teruaki NAKATSUJI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California Berkeley
University of California San Diego UCSD
Original Assignee
University of California Berkeley
University of California San Diego UCSD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of California Berkeley, University of California San Diego UCSD filed Critical University of California Berkeley
Publication of WO2025240841A1 publication Critical patent/WO2025240841A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • 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
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof

Definitions

  • the disclosure relates to methods and compositions for treating infection, and modulating skin and mucosal microflora to treat diseases or disorders that are related to or exacerbated by dysbiosis.
  • INCORPORATION BY REFERENCE OF SEQUENCE LISTING [0004] The instant application contains a Sequence Listing which has been submitted electronically in .xml format and is hereby incorporated by reference in its entirety for all purposes. Said .xml copy, created on May 16, 2025, is named 00015- 439WO1.xml and is 5,787 bytes in size.
  • BACKGROUND [0005] Small, cationic antimicrobial peptides (AMPs) are naturally occurring antibiotics of the innate immune system.
  • AMPs are widely distributed in animals and plants and are among the most ancient host defense factors. Their spectrum of activity includes Gram-positive and Gram-negative bacteria as well as fungi and certain infective agents. As resistance of pathogenic microbes to conventional antibiotics increases, researchers are exploring these endogenous antibiotics as a potential source or new therapies against variety of infectious diseases.
  • AD atopic dermatitis
  • SA Staphylococcus aureus
  • the increased susceptibility to SA has been associated with diminished innate immune defense including abnormal Attorney Docket No.00015-439WO1 barrier function and decreased induction of antimicrobial peptides (AMPs) such as cathelicidin and ⁇ -defensins.
  • AMPs antimicrobial peptides
  • Symptoms of atopic dermatitis also referred to as eczema or atopic eczema include: dry skin that forms a rash; scaly, swollen, and red skin; rash on the face, or inside the knees, elbows, or wrists; blisters that ooze; changes in skin color after repeated episodes; thickened, cracked, dry, scaly skin or skin that looks leathery in patches; and severe itchiness (pruritis), especially at night, along with raw, sensitive, swollen skin from scratching.
  • Atopic dermatitis signs and symptoms vary widely from person to person and may further include: red to brownish-gray patches, especially on the hands, feet, ankles, wrists, neck, upper chest, eyelids, inside the bend of the elbows and knees, and, in infants, the face, scalp, back of the head, ears, legs, feet, arms, hands and buttocks; small, raised bumps, which may leak fluid and crust over when scratched.
  • Atopic dermatitis most often begins before age 5 and may persist into adolescence and adulthood. For some people, it flares periodically and then clears up for a time, even for several years.
  • Dysbiosis comprises an imbalance in the cutaneous or mucosal flora, including the nasal, oral, ophthalmic, urogenital, intestinal flora, wherein species such as S. aureus become overrepresented and other species become underrepresented.
  • nonpathogenic bacteria may secrete inhibitors or simply occupy all available niches, thus either directly inhibiting or indirectly excluding pathogens that would otherwise be able to establish infectious states or foster the development of disease or disease-like states, such as atopic dermatitis.
  • the disclosure demonstrates a screen of the commensal Staphylococcus hominis strain (C2) from the skin of a healthy human donor.
  • C2 commensal Staphylococcus hominis strain
  • Staphylococcus hominis C2 kills a broad range of Staphylococcus aureus and Staphylococcus epidermidis Attorney Docket No.00015-439WO1 strains.
  • Staphylococcus hominis C2 strain or the lantibiotic produced by this bacterial strain can serve as an effective antimicrobial therapy for dermatological conditions associated with Staphylococcus aureus infection, such as Atopic Dermatitis, Netherton Syndrome, and wounds.
  • Staphylococcus hominis C2 produces both lantibiotic and autoinducing peptides.
  • the lantibiotic peptide exerts direct killing activity against a wide range of Staphylococcus aureus strains including MRSAs and the autoinducing peptide suppresses the production of various virulence factors from Staphylococcus aureus regulated under quorum sensing, such as phenol soluble modulins and proteases. These virulence factors are known to disrupt the integrity of the epidermal barrier and induce inflammation in the skin. Therefore, Staphylococcus hominis C2 can exert multi-therapeutic effects against diseases associated with Staphylococcus aureus infection.
  • Live Staphylococcus hominis C2 or purified lantibiotic can be utilized for treating Staphylococcus aureus infections. Long-term stability of bacterial viability for 5 days at room temperature has been achieved, 1 month at 4C and > 3 years at - 80C. Additionally, the pure lantibiotic peptide remains stable at room temperature. Therefore, either live bacteria or the lantibiotic peptide can be incorporated into topical medications for therapeutic use. [0012] The disclosure provides a lantibiotic peptide produced by the Staphylococcus hominis C2 strain.
  • This lantibiotic peptide exerts broad-spectrum antimicrobial activity, effectively eliminating a wide range of Staphylococcus aureus strains, including MRSA, as well as other Gram-positive skin pathogens such as Streptococcus pyogenes. Accordingly, the Staphylococcus hominis C2 strain itself or the lantibiotic peptide isolated from this bacterial strain can be utilized for the treatment of skin disorders associated with Staphylococcus aureus or Streptococcus pyogenes infection, such as atopic dermatitis or Netherton syndrome.
  • the disclosure provides compositions and methods for the treatment of disorders related to dysbiosis of the skin as well as Attorney Docket No.00015-439WO1 infection by S. aureus and methicillin-resistant Staphylococcus aureus (MRSA) and other gram-positive bacterial pathogens (e.g., Streptococcus pyogenes and Corynebacterium minitissimum).
  • MRSA methicillin-resistant Staphylococcus aureus
  • Other gram-positive bacterial pathogens e.g., Streptococcus pyogenes and Corynebacterium minitissimum.
  • the disclosure provides compositions comprising culture S. hominis strains (e.g., S. hominis C2) that produce a lantibiotic encoded by SEQ ID NO:1 and having antimicrobial activity against S. aureus.
  • the disclosure also provides compositions comprising extracts of S.
  • the lantibiotics, extracts comprising such lantibiotics and probiotic compositions comprising an S. hominis strain e.g., S. hominis C2
  • S. hominis C2 an S. hominis strain
  • MRSA microsomal spheral spheral spheral spheral spheral spheral spheral spheral spheral s s
  • S. hominis strain e.g., S. hominis C2
  • compositions and methods for treating these disorders by restoring the healthy cutaneous flora utilizing antimicrobial peptides derived from residents of the healthy cutaneous flora, or by directly administering probiotic compositions containing strains that are derived from a healthy cutaneous flora, or as the rare surviving florae cultured from the skin of diagnosed patients with a floral dysbiosis, and that are capable of either killing or inhibiting the growth of pathogenic species on the skin or species associated with a disease-like microbial imbalance.
  • the disclosure provides a thickened topical composition comprising one or more probiotic bacterial strains of the genus Staphylococcus, and more specifically comprising the strain Staphylococcus hominis C2.
  • the probiotic strains of the disclosure may be provided in live form, in freeze- dried form, or in a reconstitutable form.
  • the disclosure further provides a composition comprising strains of Staphylococcus hominis that produces a polypeptide of SEQ ID NO:2 as described herein Attorney Docket No.00015-439WO1 which may be formulated for topical administration to the skin, scalp, or mucosae.
  • the disclosure further provides a composition wherein the probiotic bacterial strains comprises Staphylococcus hominis C2 and optionally one or more of S. epidermidis strains such as Staphylococcus epidermidis strains MO34, MO38, A11, AMT1, AMT5-C5, and/or AMT5-G6 and/or Staphylococcus hominis strains A9, AMT2, AMT3, AMT4-C2, AMT4-G1, and/or AMT4-D12.
  • S. epidermidis strains such as Staphylococcus epidermidis strains MO34, MO38, A11, AMT1, AMT5-C5, and/or AMT5-G6 and/or Staphylococcus hominis strains A9, AMT2, AMT3, AMT4-C2, AMT4-G1, and/or AMT4-D12.
  • compositions of the disclosure may also comprise conditioned culture medium, or isolated antimicrobial compounds derived from the strains described herein, such as the peptides designated here as ShC2 lantibiotic (e.g., having a precursor peptide sequence that is at least 85%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to MTKITKDDLKKITENRIEARTHPTVVPVSAAVCGVATKLVPTSKCA SIVKPCNK (SEQ ID NO:2).
  • ShC2 lantibiotic e.g., having a precursor peptide sequence that is at least 85%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to MTKITKDDLKKITENRIEARTHPTVVPVSAAVCGVATKLVPTSKCA SIVKPCNK (SEQ ID NO:2).
  • the disclosure contemplates the use of heterologously expressed or synthetic ShC2 lantibiotics, ShC2 lantibiotic derivatives, or ShC2 lantibiotic-like peptid
  • the disclosure also provides a composition comprising a ShC2 lantibiotic peptide, derivative or variant and a cathelicidin peptide, derivative or variant.
  • the disclosure further provides a composition of any of the foregoing embodiments, wherein the peptide comprises one or more D-amino acids, one or more non- naturally occurring amino acids, and/or one or more post- translational modifications.
  • the disclosure provides a composition of any of the foregoing embodiments, wherein the peptide is substantially purified from other peptides.
  • the disclosure provides a composition wherein the peptide is present in a crude extract.
  • the disclosure provides a composition of any of the foregoing embodiments in a formulation for topical administration.
  • the disclosure provides a composition of any of the foregoing embodiments, wherein the formulation comprises a lotion, ointment or spray or cream or oil suspension, but not limited to these formats.
  • the disclosure provides a composition of any of the foregoing embodiments, wherein the ShC2 lantibiotic peptide, derivative or variant comprises a sequence selected from SEQ ID NO:2 comprising a non-natural amino acid, SEQ ID NO:2 comprising a D-amino acid, or SEQ ID NO:2 comprising a fusion construct.
  • the disclosure also provides a method for inhibiting the spread and/or reducing the risk of infection with a microbe comprising contacting the microbe with an effective amount of a composition of the disclosure.
  • the contacting is in vivo.
  • the contacting in vivo is by topical administration.
  • the disclosure further provides a method of treating skin or mucosal infections, atopic dermatitis, psoriasis, acne, or other disorders related to skin dysbiosis by applying to the skin or mucosa an effective amount of the compositions disclosed herein to a subject in need thereof.
  • the disclosure provides a method of treating atopic dermatitis comprising contacting a subject having or suspected of having atopic dermatitis with an effective amount of a probiotic composition comprising one or more of the bacterial strains disclosed herein. [0022] The disclosure provides a method of treating atopic dermatitis comprising contacting a subject having or suspected of having atopic dermatitis with an effective amount of a ShC2 lantibiotic peptide, derivative or variant.
  • the disclosure provides a method of treating atopic dermatitis or dysbiosis of the skin by contacting the affected area with a composition comprising bacterial strains that secrete ShC2 lantibiotic peptide, such as Staphylococcus hominis strain C2.
  • a composition comprising bacterial strains that secrete ShC2 lantibiotic peptide, such as Staphylococcus hominis strain C2.
  • the disclosure provides methods and compositions as described above which further comprise a cathelicidin peptide.
  • the disclosure provides a composition comprising a thickened topical formulation of one or more probiotic bacterial strains and optionally, a prebiotic compound, a protectant, humectant, emollient, abrasive, salt, and/or surfactant; wherein the one or more probiotic bacterial strain comprises one or more bacterial strains of the genus Staphylococcus; and wherein the composition is formulated for the topical treatment of disorders of dysbiosis of the skin, scalp, or mucosae.
  • the one or more probiotic bacterial strain comprising Staphylococcus hominis.
  • one or more probiotic bacterial strains comprises Staphylococcus hominis strains C2.
  • the one or more probiotic bacterial strains produces a Attorney Docket No.00015-439WO1 peptide having a sequence of SEQ ID NO:2 or a sequence that is at least 85% identical thereto and has antimicrobial activity, wherein such peptide is optionally post-translationally modified.
  • the one or more probiotic bacterial strains is provided in a live form.
  • the one or more probiotic bacterial strains is provided in a lyophilized or freeze-dried or spray dried form.
  • the probiotic bacterium can be reconstituted into a live form.
  • the disclosure also provides a method of treating skin or mucosal infections, atopic dermatitis, psoriasis, mastitis, acne, or other disorders related to skin dysbiosis in humans or other mammals by applying to the skin or mucosa an effective amount of a composition as described herein and in the preceding paragraph(s).
  • the composition is applied topically.
  • the composition is formulated as a cream, ointment, unguent, spray, powder, oil, thickened formulation or poultice.
  • the disclosure also provides a composition comprising a ShC2 lantibiotic peptide, derivative or variant, and further comprising one or more thickeners, solvents, emulsifiers, or pharmaceutically acceptable carriers or excipients.
  • the composition further comprises a cathelicidin peptide, derivative or variant.
  • the ShC2 lantibiotic peptide, derivative or variant comprises one or more D-amino acids or non-naturally occurring amino acids.
  • the ShC2 lantibiotic peptide is produced in situ by Staphylococcus hominis strain C2.
  • the peptide is formulated for topical administration.
  • the formulation comprises a lotion, ointment cream, powder, unguent, oil, or spray.
  • the ShC2 lantibiotic peptide, derivative or variant comprises a sequence of SEQ ID NO:2 or an active fragment thereof having antimicrobial activity (e.g., a mature form).
  • the ShC2 lantibiotic peptide, derivative or variant is provided as an extract or lysate Staphylococcus hominis strain C2.
  • the disclosure also provides a method for treating skin or mucosal infection or atopic dermatitis in a subject comprising contacting the subject with an effective amount of a composition comprising a ShC2 lantibiotic peptide, derivative or variant.
  • the disclosure also provides a recombinant vector comprising a polynucleotide encoding a polypeptide that is at least 85%,90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2, or a biologically active fragment thereof having antimicrobial activity.
  • the vector comprises a polynucleotide that encodes a polypeptide of SEQ ID NO:2 or a sequence that is at least 85-99% identical thereto.
  • the vector comprises a polynucleotides that encodes a polypeptide of SEQ ID NO:2 from about amino acid 21 to about amino acid 54.
  • the vector comprises a polynucleotide that is at least 95% identical to SEQ ID NO:1 and encodes a polypeptide of SEQ ID NO:2.
  • the vector comprises a fragment of SEQ ID NO:1.
  • the vector is an expression vector.
  • the disclosure also provides a host cell engineered to express a recombinant vector of the disclosure.
  • the host cell is a non-pathogenic attenuated host cell.
  • the disclosure also provide a recombinant polypeptide produced by the host cell of the disclosure.
  • the recombinant polypeptide is purified from a host cell culture.
  • Attorney Docket No.00015-439WO1 [0031]
  • the disclosure also provides a composition comprising the host cell of disclosure.
  • the disclosure provides a composition consisting of Staphylococcus hominis C2 and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is a gel of glycerol, hydroxyethyl cellulose and phosphate- buffered saline.
  • the S is a gel of glycerol, hydroxyethyl cellulose and phosphate- buffered saline.
  • hominis C2 is present at about 5 x 10 CFU/gram.
  • the disclosure also provides a composition comprising a polypeptide having at least 85% identity to SEQ ID NO:2 or 3 and having antimicrobial activity; and an autoinducing peptide (AIP).
  • the AIP comprises a peptidic structure of Formula II: .
  • the upper panel illustrates the HPLC elution profile of peptides purified from culture supernatant of ShC2 strain.
  • the lower panel represent antimicrobial activity of indicated fraction against S. aureus. To test antimicrobial activity, each fraction was applied on tryptic soy broth agar inoculated with S. aureus. Dark zones (clear) indicate no growth of S. aureus. The fraction 31 exhibited a peak corresponding to anti-S. aureus activity.
  • Figure 2 shows MALDI-TOF Mass-spectrometry profile of Fraction 31 obtained from the HPLC purification of ShC2 culture supernatant.
  • Figure 3 shows a map of the large plasmid identified in ShC2 strain by high-resolution whole genome sequencing. This highlights the presence of lantibiotic-biosynthesis genes, including LanM and Lan C. Candidates for lantibiotic precursor genes are indicated as peg.2198, 2199 and 2200.
  • Figure 4 shows the secondary structure of lantibiotic purified from ShC2 strain (Formula I), predicted through combined analysis of HPLC purification, mass spectrometry and whole genome sequencing. Dha, 2,3-didehydroalanine; Dhb, (Z)-2,3- didehydrobutyrine.
  • FIG. 5 shows the secondary structure of an antoinducing peptide identified within the genome sequence of ShC2.
  • Figure 6 shows stability of ShC2 in the skin gel lotion formulation at various storage temperatures. Viability of ShC2 in the gel formulation was assessed by determining colony-forming units (CFU) on tryptic soy agar at the indicated time points following manufacture. Samples were stored at room temperature (RT), 4 ⁇ °C, ⁇ 20 ⁇ °C, and ⁇ 80 ⁇ °C.
  • Figure 7A-C shows ShC2 strain with anti-Streptococcus and anti-S. aureus activity decreases bacterial growth and protects against inflammation in mouse AD model. ShC2 (10 CFU) was co- applied with S.
  • references to “a probe” includes a plurality of such cells and reference to “the cell” includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.
  • the use of “or” means “and/or” unless stated otherwise.
  • “comprise,” “comprises,” “comprising” Attorney Docket No.00015-439WO1 “include,” “includes,” and “including” are interchangeable and not intended to be limiting.
  • Atopic dermatitis is a common, chronic skin disorder characterized by dysfunction of the epidermal barrier and relapsing skin inflammation. The severity of this disease is associated with dysbiosis of the skin microbiome and the high susceptibility of these patients to colonization and infections by Staphylococcus aureus.
  • Childhood AD is more prevalent than adult AD and is characterized by a more complex microbial dysbiosis, including elevated colonization by both S. aureus and various Streptococcus species.
  • AMPs antimicrobial peptides
  • aureus further exacerbates symptoms of atopic dermatitis and leads to immune dysfunction such as TH2 lymphocyte skewing, reduced AMPs, exacerbated allergic reactions and disruption of the skin barrier.
  • immune dysfunction such as TH2 lymphocyte skewing, reduced AMPs, exacerbated allergic reactions and disruption of the skin barrier.
  • Prior studies of patients with atopic dermatitis have shown that the bacterial flora present on these patients is different than the bacteria found on the skin of non-atopic subjects.
  • the microbiome of patients with atopic dermatitis is less diverse and typically has a higher abundance of Staphylococcal species. Without intending to be bound by any particular theory, it has been hypothesized that dysbiosis of the skin microbiome could contribute to the pathophysiology of this disease.
  • the diverse community of microorganisms that normally comprise the microbiome have been suggested to contribute to cutaneous homeostasis.
  • Staphylococcus epidermidis can control inflammation after injury, influence T-cell development and induce expression of AMPs.
  • the microbiome may produce its own AMPs that could synergize with AMPs produced by host cells. Therefore, in addition to the deleterious effects of colonization by S. aureus, dysbiosis of the microbiome in atopic dermatitis could contribute to disease by loss of their beneficial functions.
  • Existing antibiotic therapies non-specifically kill bacteria, which impacts the homeostasis of the resident microflora.
  • Imbalanced microflora contribute to the pathogenesis of skin inflammatory diseases, such as atopic dermatitis, rosacea, acne vulgaris etc.
  • This disclosure provides compositions and formulations for disinfecting surfaces or treating infections but does not pose the safety risks of non-specific antibiotics. Further the disclosure provides for probiotic approaches wherein subjects may be provided with live S. hominis alone or in combination with other commensal bacterial strains which may Attorney Docket No.00015-439WO1 produce the necessary antimicrobial compounds in situ while simultaneously restoring the characteristics of a healthy cutaneous flora.
  • Preclinical data from a mouse model of AD colonized with both S. aureus and Streptococcus support the therapeutic potential of topically applied ShC2.
  • Staphylococcus hominis (S. hominis) is a major constituent of the microflora of healthy human skin. Recent studies indicate that S. epidermidis protect human skin by preventing pathogenic infections by producing phenol-soluble modulins (PSMs) and small molecule antibiotic, named “Firmocidin”, which function as additional antimicrobial compounds on normal human skin (see, e.g., U.S. Pat. Publ. No. 2013/0331384A1, the disclosure of which is incorporated herein by reference). The present disclosure provides the use of live S.
  • PSMs phenol-soluble modulins
  • Firmocidin small molecule antibiotic
  • AMPs such as LL-37 and hBDs-2 and -3 have lower levels of expression in inflamed skin of atopic patients than inflamed skin of normal subjects, but these AMP expressions are low in non- inflamed skin. Therefore, the increased capacity of the non- inflamed normal skin to kill bacteria is not likely due to the expression of these host AMPs.
  • the disclosure describes novel antimicrobial peptides (AMPs) from culture supernatant of a clinical isolate of S. hominis (S. hominis C2; “ShC2”).
  • S. hominis C2 S. hominis C2
  • ShC2 novel antimicrobial peptides
  • ShC2 lantibiotics exert antimicrobial and Attorney Docket No.00015-439WO1 bactericidal activity against Staphylococcus aureus (S. aureus), but do not inhibit the growth of commensal bacteria on the skin such as S. epidermidis. Therefore, the disclosure provides antibiotics with potent but selective activity against pathogens, and high safety profile as they are found normally in the human skin microbiome, as well as probiotic approaches to treating these conditions.
  • ShC2 can eliminate a broader range of S. aureus strains including MRSA and exhibits more potent antimicrobial activity compared to ShA9 (Table 1).
  • the disclosure provides the following: (I) lantibiotic precursor coding sequence: atgactaaaataactaaagatgatttgaaaagattacagaaaatcgtattgaagcacgt acacatccaaccgttgttcctgtaagtgctgctgtatgcggagttgctactaaattagta ccaacatcgaaatgtgcttcaattgtaaaccatgtaataaataaaa (SEQ ID NO:1)
  • Precursor peptide MTKITKDDLKKITENRIEARTHPTVVPVSAAVCGVATKLVPTSKCASIVKPCNK (SEQ ID NO:2)
  • III a
  • ShC2 also produces an autoinducing peptide (AIP) that potently antagonizes quorum sensing activity of S. aureus.
  • AIP autoinducing peptide
  • the ShC2-derived lantibiotic demonstrates potent antimicrobial activity against a broad array of S. aureus clinical isolates from AD patients (see Table 1), as well as several Streptococcus species, including S. pyogenes, S. salivarius, and S. oralis (see Table 2). These streptococcal species are frequently isolated from the skin of children with AD, highlighting the relevance of targeting them in pediatric populations.
  • Staphylococcus hominis C2 (ShC2), isolated from healthy human skin, represents a promising new candidate for bacteriotherapy in atopic dermatitis (AD).
  • ShC2 synthesizes an autoinducing peptide (AIP) that antagonizes the S. aureus Agr quorum sensing system, further suppressing its virulence factor expression.
  • AIP autoinducing peptide
  • an embodiment comprising a topical gel formulation containing live ShC2 showed acceptable shelf life stability under 4C and retained antimicrobial activity.
  • ShC2 application significantly suppressed growth of both S. aureus and S. pyogenes and inflammation induced by the synergistic action between both pathogens.
  • antimicrobial means that the peptide destroys, or inhibits or prevents the growth or proliferation of, a microbe (e.g., a bacterium, fungus, and/or virus).
  • antiviral means that a peptide destroys, or inhibits or prevents the growth or proliferation of a virus or a virus-infected cell.
  • anti- tumor means that a peptide prevents, inhibits the growth of, or destroys, a tumor cell(s).
  • antifungal means that a peptide prevents, destroys, or inhibits the growth of a fungus.
  • probiotic refers to the process of providing live or attenuated microbial cultures, or lysates, lyophiles or extracts of such cultures, in order to supplement or replace elements of a healthy cutaneous or mucosal flora.
  • An attenuated vector for delivery to the skin can be include a virus or bacteria that has been genetically modified to (a) make the vector non-pathogenic, (b) have reduced pathogenicity, (c) be replication defective, or (d) to be non-antigenic. Other attenuation are known in the art. The attenuation is typically performed by knocking out a gene or disrupting a gene coding sequence or expression control element such that the (a)-(c) or (d) is accomplished.
  • the "ShC2 lantibiotics" are composed of two distinct domains: an N-terminal "prosequence” domain and the C-terminal domain of the mature ShC2 lantibiotic.
  • the mature ShC2 lantibiotic comprises a sequence of SEQ ID NO:2 from about amino acid 21 to Attorney Docket No.00015-439WO1 about amino acid 54 (e.g., beginning at about amino acid 19, 20, 21, 22, or 23 of SEQ ID NO:2 and extending to about amino acid 50, 51, 52, 53, or 54 of SEQ ID NO:2).
  • the pre-pro form of ShC2 lantibiotic is about 54 amino acids in length and is post-translationally process to provide the mature form. Based upon the expression system and organism, the mature form may be processed slightly differently depending upon the proteases present. Moreover, it will also be readily apparent that the pre-pro form of ShC2 lantibiotic can be used in the methods, compositions and kits of the disclosure, wherein prior to or after administration the pre- pro form can be processed in vitro or in vivo.
  • polypeptide comprising SEQ ID NO:2 is typically cleaved following amino acid number 20 of SEQ ID NO:2, however, one of skill in the art will recognize that depending upon the enzyme used, the expression system used and/or the conditions under which proteolytic cleavage of the polypeptide takes place, the cleavage site may vary from 1 to 3 amino acid in either direction of amino acid number 21 of SEQ ID NO:2.
  • the genetic code is well understood by one of skill in the art and it is routine in generating polynucleotides encoding a desired polypeptides sequence; the disclosure also provides polynucleotides encoding the polypeptides of the disclosure.
  • the disclosure provides SEQ ID NO:1: atgactaaaataactaaagatgatttgaaaagattacagaaaatcgtattgaagcacgt acacatccaaccgttgttcctgtaagtgctgctgtatgcggagttgctactaaattagta ccaacatcgaaatgtgcttcaattgtaaaccatgtaataaataataaaa, which encode the polypeptides of SEQ ID NO:2.
  • the term "ShC2 lantibiotic peptide” refers to the mature form of ShC2 lantibiotic comprising a chain of amino acids that is about 32 to about 34 amino acids in length and comprises a sequence that is at least 85-100% identical to the sequence as set forth in SEQ ID NO:3: THPTVVPVSAAVCGVATKLVPTSKCASIVKPCNK (SEQ ID NO:3) wherein Serine and Threonine residue (bold/ital) are dehydrated by Lantibiotic producing enzymes and cross linked with Cysteine (Bolded) with mono-sulfide bond, forming lanthionine, or post-translationally modified versions thereof (Formula I): Attorney Docket No.00015-439WO1 [0071]
  • the method provides ShC2 lantibiotic derivatives comprising (a) peptides that are at least 85-100% identical to a ShC2 lantibiotic peptide of SEQ ID NO:2 and having antimicrobial activity
  • the method provides ShC2 lantibiotic derivatives comprising lanthionine or methyllanthionine residues, or ShC2 lantibiotic derivatives modified such that they contain lanthionine or methyllanthionine residues. It is not necessary that the analog, derivative, variation, or variant have activity identical to the activity of the ShC2 lantibiotic peptide from which the analog, derivative, conservative variation, or variant is derived so long as it has some antimicrobial activity. In another embodiment, the disclosure provides a ShC2 lantibiotic polypeptide comprising at least one conservative amino acid difference compared to polypeptide of SEQ ID NO:2 or 3.
  • compositions comprising a pharmaceutically acceptable excipient and comprising a substantially pure ShC2 lantibiotic peptide or derivative.
  • compositions comprising a probiotic formulation which includes one or more ShC2 lantibiotic producing bacterial strains.
  • purified refers to a peptide that is substantially free of other proteins, lipids, and polynucleotides (e.g., cellular components with which an in vivo Attorney Docket No.00015-439WO1 produced peptide would naturally be associated).
  • a peptide of the disclosure is at least 70%, 80%, or most commonly 90% or more pure by weight.
  • a “variant” is an antimicrobial peptide (e.g., a ShC2 lantibiotic peptide of the disclosure) that is an altered form of an ShC2 lantibiotic.
  • the term “variant” includes an antimicrobial peptide produced by the method disclosed herein in which at least one amino acid (e.g., from about 1 to 10 amino acids; 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) of a reference peptide is substituted with another amino acid (typically conservative substitutions).
  • reference peptide means any of the antimicrobial peptides of the disclosure (e.g., a polypeptide consisting of SEQ ID NO:2 or 3 or a mature form thereof), from which a variant, derivative, analog, or conservative variation is derived. Included within the term “derivative” is a hybrid peptide that includes at least a portion of an ShC2 lantibiotic peptide. Derivatives can be produced by adding one or a few (e.g., 1 to 5) amino acids to an antimicrobial peptide without completely inhibiting the antimicrobial activity of the peptide. In addition, C-terminal derivatives, e.g., C-terminal methyl esters, can be produced and are encompassed by the disclosure.
  • the disclosure also includes peptides that are conservative variations of those peptides as exemplified herein.
  • conservative variation denotes a polypeptide in which at least one amino acid is replaced by another, biologically, chemically, or structurally similar residue.
  • Examples of conservative variations include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, and the like.
  • Neutral hydrophilic amino acids that can be substituted for one another include asparagine, glutamine, serine and threonine.
  • Structurally conservative variations include the substitution of alanine for serine (and vice versa), isoleucine for threonine (and vice versa), arginine for lysine (and vice versa), Attorney Docket No.00015-439WO1 and the replacement of any of tyrosine, phenylalanine, tryptophan, and histidine for any other member of that group.
  • the term "conservative variation” also encompasses a peptide having a substituted amino acid in place of an unsubstituted parent amino acid; typically, antibodies raised to the substituted polypeptide also specifically bind the unsubstituted polypeptide.
  • a “ShC2-lantibiotic” refers to a compound comprising a post-translationally modified peptide produced by S. hominis C2, which optionally contains one or more lanthionine or methyllanthionine moieties, and shows antimicrobial activity against one or more non-S. hominis species.
  • a “ShC2-antimicrobial” refers to a compound comprising a non-lantibiotic compound produced or secreted by S. hominis C2, which may optionally comprise a non-lantibiotic peptide, and which shows antimicrobial activity against one or more non-S.
  • a ShC2 lantibiotic peptide variant can be a peptide mimetic, which is a non-amino acid chemical structure that mimics the structure of, for example, a ShC2 lantibiotic peptide of SEQ ID NO:2 or 3 (or a mature form thereof) yet retains antimicrobial/antibacterial activity.
  • a mimetic generally is characterized as exhibiting similar physical characteristics such as size, charge or hydrophobicity in the same spatial arrangement found in the ShC2 lantibiotic peptide counterpart.
  • a specific example of a peptide mimetic is a compound in which the amide bond between one or more of the amino acids is replaced, for example, by a carbon--carbon bond or other bond well known in the art (see, for example, Sawyer, Peptide Based Drug Design, ACS, Washington (1995)).
  • the amino acids of a ShC2 lantibiotic peptide, variant or peptidomimetic of the disclosure can be selected from the twenty naturally occurring amino acids, including, unless stated otherwise, L-amino acids and D-amino acids. The use of D-amino acids are particularly useful for increasing the life of a protein or peptide. Polypeptides incorporating D-amino acids are resistant to proteolytic digestion.
  • amino acid also refers to Attorney Docket No.00015-439WO1 compounds such as chemically modified amino acids including amino acid analogs, naturally occurring amino acids that are not usually incorporated into proteins such as norleucine, and chemically synthesized compounds having properties known in the art to be characteristic of an amino acid, provided that the compound can be substituted within a peptide such that it retains its biological activity.
  • glutamine can be an amino acid analog of asparagine, provided that it can be substituted within an active fragment of a ShC2 lantibiotic peptide, variant and the like such that it retains its antimicrobial/antibacterial activity.
  • amino acids and amino acids analogs are listed in Gross and Meienhofer, The Peptides: Analysis, Synthesis, Biology, Academic Press, Inc., New York (1983).
  • An amino acid also can be an amino acid mimetic, which is a structure that exhibits substantially the same spatial arrangement of functional groups as an amino acid but does not necessarily have both the "-amino" and "-carboxyl” groups characteristic of an amino acid.
  • Polypeptides and peptides of the disclosure can be synthesized by commonly used methods such as those that include t- BOC or FMOC protection of alpha-amino groups.
  • Polypeptide and peptides of the disclosure can also be synthesized by the well-known solid phase peptide synthesis methods such as those described by Merrifield, J. Am. Chem. Soc., 85:2149, 1962) and Stewart and Young, Solid Phase Peptides Synthesis, Freeman, San Francisco, 1969, pp. 2762). If desired, the peptides can be quantitated by the solid phase Edman degradation. Such synthesized peptides/polypeptides can be exposed to a culture of S.
  • the disclosure also includes isolated polynucleotides (e.g., DNA, cDNA, or RNA) encoding the polypeptide and peptides of Attorney Docket No.00015-439WO1 the disclosure. Included are polynucleotides that encode analogs, mutants, conservative variations, and variants of the polypeptides and peptides described herein.
  • isolated refers to a polynucleotide that is substantially free of proteins, lipids, and other polynucleotides with which an in vivo-produced polynucleotides naturally associated.
  • the polynucleotide is at least 70%, 80%, or 90% or more isolated from other matter, and conventional methods for synthesizing polynucleotides in vitro can be used in lieu of in vivo methods.
  • polynucleotide refers to a polymer of deoxyribonucleotides or ribonucleotides, in the form of a separate fragment or as a component of a larger genetic construct (e.g., by operably linking a promoter to a polynucleotide encoding a peptide of the disclosure).
  • Numerous genetic constructs e.g., plasmids and other expression vectors
  • prokaryotic or eukaryotic e.g., yeast, insect, or mammalian
  • DNA (see, e.g., SEQ ID NO:1) encoding the ShC2 lantibiotic peptide, derivatives of variants thereof of the disclosure can be inserted into an "expression vector.”
  • expression vector refers to a genetic construct such as a plasmid, virus or other vehicle known in the art that can be engineered to contain a polynucleotide encoding a polypeptide of the disclosure.
  • Such expression vectors are typically plasmids that contain a promoter sequence that facilitates transcription of the inserted genetic sequence in a host cell.
  • the expression vector typically contains an origin of replication, and a promoter, as well as genes that allow phenotypic selection of the transformed cells (e.g., an antibiotic resistance gene).
  • Various promoters, including inducible and constitutive promoters, can be utilized in the disclosure.
  • the expression vector contains a Attorney Docket No.00015-439WO1 replicon site and control sequences that are derived from a species compatible with the host cell.
  • competent cells that are capable of DNA uptake can be prepared using the CaCl, MgCl or RbCl methods known in the art.
  • physical means such as electroporation or microinjection can be used. Electroporation allows transfer of a polynucleotide into a cell by high voltage electric impulse.
  • polynucleotides can be introduced into host cells by protoplast fusion, using methods well known in the art. Suitable methods for transforming eukaryotic cells, such as electroporation and lipofection, also are known.
  • Host cells encompassed by of the disclosure are any cells in which the polynucleotides of the disclosure can be used to express the ShC2 lantibiotic peptide, derivatives or variants of the disclosure.
  • the term also includes any progeny of a host cell.
  • Host cells which are useful, include bacterial cells, fungal cells (e.g., yeast cells), plant cells and animal cells.
  • host cells can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology (1986)).
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS or Bowes melanoma
  • plant cells and the like.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • a host cell can comprise a bacterial cell present in a normal bacterial flora of the skin that has been engineered to express or over express a ShC2 lantibiotic peptide or other antimicrobial peptide of the disclosure. These engineered Attorney Docket No.00015-439WO1 bacterial cells can then be used as a probiotic such that they are applied to skin.
  • Host cells can be eukaryotic host cells (e.g., mammalian cells). In one embodiment, the host cells are mammalian production cells adapted to grow in cell culture.
  • CHO Chinese hamster ovary (CHO) cells are widely used for the production of several complex recombinant proteins, e.g. cytokines, clotting factors, and antibodies (Brasel et al., Blood 88:20042012 (1996); Kaufman et al., J. Biol Chem 263: 63526362 (1988); McKinnon et al., J Mol Endocrinol 6:231239 (1991); Wood et al., J.
  • DHFR dihydrofolate reductase
  • the dihydrofolate reductase (DHFR)-deficient mutant cell lines are the CHO host cell lines commonly used because the efficient DHFR selectable and amplifiable gene expression system allows high level recombinant protein expression in these cells (Kaufman, Meth Enzymol 185:527566 (1990)).
  • these cells are easy to manipulate as adherent or suspension cultures and exhibit relatively good genetic stability.
  • CHO cells and recombinant proteins expressed in them have been extensively characterized and have been approved for use in clinical manufacturing by regulatory agencies.
  • Polynucleotides encoding the polypeptide and peptides of the disclosure can be isolated from a cell (e.g., a bacterial cell such as S. hominis C2 or a recombinant cultured cell), or they can be produced in vitro.
  • a DNA sequence encoding a ShC2 lantibiotic peptide of interest can be obtained by: 1) isolation of a double- stranded DNA sequence from genomic DNA; 2) chemical manufacture of a polynucleotide such that it encodes the ShC2 lantibiotic peptide of interest; or 3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a donor cell (i.e., to produce cDNA).
  • Carrier peptides can facilitate isolation of fusion proteins that include the peptides of the disclosure.
  • Purification tags can be operably linked to a ShC2 lantibiotic peptide of the disclosure.
  • GST glutathione-S- transferase
  • purification can be accomplished in a single step using an IgG-sepharose affinity column.
  • the pOprF-peptide which is the N-terminal half of the P. aeruginosa outer membrane protein F, can readily be purified because it is the prominent protein species in outer membrane preparations.
  • the fusion peptides can be purified using reagents that are specifically reactive with (e.g., specifically bind) the ShC2 lantibiotic peptide of the fusion peptide.
  • reagents that are specifically reactive with (e.g., specifically bind) the ShC2 lantibiotic peptide of the fusion peptide For example, monoclonal or polyclonal antibodies that specifically bind the ShC2 lantibiotic peptide can be used in conventional purification methods. Techniques for producing such antibodies are well known in the art.
  • a fusion construct comprising a polypeptide linked to a ShC2 lantibiotic peptide of the disclosure can be linked at either the amino or carboxy terminus of the peptide.
  • the polypeptide that is linked to the ShC2 lantibiotic peptide is sufficiently anionic such that the ShC2 lantibiotic peptide has a net charge that is neutral or negative.
  • the anionic polypeptide can correspond in sequence to a naturally occurring protein or can be entirely artificial in design.
  • the polypeptide linked to a ShC2 lantibiotic peptide may help stabilize the ShC2 lantibiotic peptide and protect it from proteases, although the carrier polypeptide need not be shown to serve such a purpose.
  • the carrier polypeptide may Attorney Docket No.00015-439WO1 facilitate transport of the fusion peptide.
  • carrier polypeptides examples include anionic pre-pro peptides and anionic outer membrane peptides.
  • carrier polypeptides include glutathione- S-transferase (GST), protein A of Staphylococcus aureus, two synthetic IgG-binding domains (ZZ) of protein A, outer membrane protein F of Pseudomonas aeruginosa, protein transduction domains and the like.
  • GST glutathione- S-transferase
  • ZZ two synthetic IgG-binding domains
  • the disclosure is not limited to the use of these polypeptides; others suitable carrier polypeptides are known to those skilled in the art.
  • a linker moiety comprising a protease cleavage site may be operably linked to a ShC2 lantibiotic peptide or variant of the disclosure.
  • the linker may be operable between to domains of a fusion protein (e.g., a fusion protein comprising a ShC2 lantibiotic peptide and a carrier polypeptide).
  • a fusion protein e.g., a fusion protein comprising a ShC2 lantibiotic peptide and a carrier polypeptide.
  • the linker moiety can include the recognition sequence within flexible spacer amino acid sequences, commonly used in the art.
  • the spacer DNA sequence can encode a protein recognition site for cleavage of the carrier polypeptide from the ShC2 lantibiotic peptide.
  • spacer DNA sequences include, but are not limited to, protease cleavage sequences, such as that for Factor Xa protease, the methionine, tryptophan and glutamic acid codon sequences, and the pre-pro defensin sequence.
  • Factor Xa is used for proteolytic cleavage at the Factor Xa protease cleavage sequence, while chemical cleavage by cyanogen bromide treatment releases the peptide at methionine or related residues.
  • the fused product can be cleaved by insertion of a codon for tryptophan (cleavable by o-iodosobenzoic acid) or glutamic acid (cleavable by Staphylococcus protease). Insertion of such spacer DNA sequences is not a requirement for the production of functional ShC2 lantibiotic peptides, such sequences can enhance the stability of the fusion peptide.
  • the pre-pro defensin sequence is negatively charged, so accordingly, it is envisioned within the disclosure that other DNA sequences encoding negatively charged peptides also can be used as spacer DNA sequences to stabilize the fusion peptide.
  • the disclosure also provides a method for inhibiting the growth of a bacterium (e.g., S. aureus) by contacting the bacterium with an inhibiting effective amount of a peptide of the disclosure.
  • a bacterium e.g., S. aureus
  • the term "contacting" refers to exposing the bacterium to the peptide so that the peptide can inhibit, kill, or lyse bacteria.
  • the disclosure also provides a method for inhibiting skin disease or disorder and/or bacterial infection comprising placing on or within a subject a probiotic formulation comprising bacteria which secrete a peptide or antimicrobial molecule of the disclosure such that the growth of the pathogen or undesirable microbe is inhibited or prevented.
  • Contacting of an organism with a ShC2 lantibiotic peptide of the disclosure can occur in vitro, for example, by adding the peptide to a bacterial culture to test for susceptibility of the bacteria to the peptide, or contacting a bacterially contaminated surface with the peptide (e.g., on a subject or an inanimate object).
  • contacting can occur in vivo, for example by administering the peptide to a subject afflicted with a bacterial infection or susceptible to infection.
  • contacting can occur by exposing the bacterium to a probiotic formulation comprising bacterial strains that produce the ShC2 lantibiotic peptide (e.g., S.
  • Inhibiting or “inhibiting effective amount” refers to the amount of peptide that is sufficient to cause, for example, a bacteriostatic or bactericidal effect.
  • Bacteria that can be affected by the peptides of the disclosure include both gram- negative and gram-positive bacteria.
  • bacteria that can be affected include Staphylococcus aureus, Streptococcus pyogenes (group A), Streptococcus sp. (viridans group), Streptococcus agalactiae (group B), S.
  • Gram-negative cocci such as, for example, Neisseria gonorrhoeae, Neisseria meningitidis, and Branhamella catarrhalis
  • Gram-positive bacilli such as Bacillus anthracis, Bacillus subtilis, P.acne Corynebacterium diphtheriae and Corynebacterium species which are diptheroids (aerobic and anerobic), Listeria monocytogenes, Attorney Docket No.00015-439WO1 Clostridium tetani, Clostridium difficile, Escherichia coli, Enterobacter species, Proteus mirablis and other sp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella, Shigella, Serratia sp., and Campylo
  • Infection with one or more of these bacteria can result in diseases such as bacteremia, pneumonia, meningitis, osteomyelitis, endocarditis, sinusitis, arthritis, urinary tract infections, tetanus, gangrene, colitis, acute gastroenteritis, impetigo, acne, acne posacue, wound infections, born infections, fascitis, bronchitis, and a variety of abscesses, nosocomial infections, and opportunistic infections.
  • Fungal organisms may also be affected by the ShC2 lantibiotic peptides of the disclosure and include dermatophytes (e.g., Microsporum canis and other Microsporum sp.; and Trichophyton sp. such as T.
  • yeasts e.g., Candida albicans, C. Tropicalis, or other Candida species
  • Saccharomyces cerevisiae Saccharomyces cerevisiae
  • Torulopsis glabrata Saccharomyces cerevisiae
  • Epidermophyton floccosum Malassezia furfur (Pityropsporon orbiculare, or P.
  • the method for inhibiting the growth of bacteria or microbe can also include contacting the bacterium or microbial entity with the peptide in combination with one or more antibiotics or antifungal agents.
  • a peptide(s) of the disclosure can be administered to any host, including a human or non-human animal, in an amount effective to inhibit growth of a bacterium, virus, or fungus.
  • the peptides are useful as antimicrobial agents, antiviral agents, and/or antifungal agents.
  • the bacterial strains that produce the peptides are useful as probiotic agents.
  • Any of a variety of art-known methods can be used to administer the peptide to a subject.
  • the peptide of the disclosure can be administered parenterally by injection or by gradual infusion over time.
  • the peptide can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, topically or transdermally.
  • a ShC2 lantibiotic peptide of the disclosure may be formulated for Attorney Docket No.00015-439WO1 topical administration (e.g., as a lotion, cream, spray, gel, oil suspension, or ointment).
  • topical administration e.g., as a lotion, cream, spray, gel, oil suspension, or ointment.
  • formulations in the market place include topical lotions, creams, soaps, wipes, powders, devices like gauze pads to cover wounds, and the like. It may be formulated into liposomes to reduce toxicity or increase bioavailability or stability.
  • Preparations for parenteral administration of a peptide of the disclosure include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate.
  • aqueous carriers examples include water, saline, and buffered media, alcoholic/aqueous solutions, and emulsions or suspensions.
  • parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives such as, other antimicrobial, anti-oxidants, chelating agents, inert gases and the like also can be included.
  • the disclosure provides a method for inhibiting a topical bacterial or fungal-associated disorder by contacting or administering a therapeutically effective amount of a peptide or skin-probiotic of the disclosure to a subject who has, or is at risk of having, such a disorder.
  • inhibiting means preventing or ameliorating a sign or symptoms of a disorder (e.g., a rash, sore, and the like).
  • signs that can be ameliorated include an increase in a subject's blood level of TNF, fever, hypotension, neutropenia, leukopenia, thrombocytopenia, disseminated intravascular coagulation, adult respiratory distress syndrome, shock, and organ failure.
  • subjects who can be treated in the disclosure include human or animal subjects at risk Attorney Docket No.00015-439WO1 for, or those suffering from, a toxemia, such as endotoxemia resulting from a gram-negative bacterial infection, venom poisoning, or hepatic failure.
  • a toxemia such as endotoxemia resulting from a gram-negative bacterial infection, venom poisoning, or hepatic failure.
  • Other examples include subjects having a dermatitis as well as those having skin infections such as mastitis and especially bovine mastitis, or injuries subject to infection with gram-positive or gram-negative bacteria or a fungus.
  • candidate patients include those suffering from infection by E. coli, Hemophilus influenza B, Neisseria meningitides, staphylococci, or pneumococci.
  • terapéuticaally effective amount as used herein for treatment of a subject afflicted with a disease or disorder means an amount of ShC2 lantibiotic peptide sufficient to ameliorate a sign or symptom of the disease or disorder.
  • a therapeutically effective amount can be measured as the amount sufficient to decrease a subject's symptoms of dermatitis or rash by measuring the frequency of severity of skin sores.
  • the subject is treated with an amount of ShC2 lantibiotic peptide sufficient to reduce a symptom of a disease or disorder by at least 50%, 90% or 100%.
  • the optimal dosage of the peptide will depend upon the disorder and factors such as the weight of the patient, the type of bacterial or fungal infection, the weight, sex, and degree of symptoms. Nonetheless, suitable dosages can readily be determined by one skilled in the art.
  • a suitable dosage is 0.5 to 40 mg peptide/kg body weight, e.g., 1 to 8 mg peptide/kg body weight.
  • a suitable therapy regime can combine administration of a peptide(s) or probiotic composition of the disclosure with one or more additional therapeutic agents (e.g., an inhibitor of TNF, an antibiotic, other probiotic compositions, other lantibiotics, autoinducing peptides, and the like).
  • additional therapeutic agents e.g., an inhibitor of TNF, an antibiotic, other probiotic compositions, other lantibiotics, autoinducing peptides, and the like.
  • the Attorney Docket No.00015-439WO1 peptide(s), other therapeutic agents, and/or antibiotic(s) can be administered, simultaneously, but may also be administered sequentially.
  • Suitable antibiotics include aminoglycosides (e.g., gentamicin), beta-lactams (e.g., penicillins and cephalosporins), quinolones (e.g., ciprofloxacin), and novobiocin.
  • the antibiotic is administered in a bactericidal amount.
  • the peptide provides for a method of increasing antibiotic activity.
  • the ShC2 lantibiotic peptide and antibiotic are administered within 48 hours of each other (e.g., 2 to 8 hours, or may be administered simultaneously).
  • a "bactericidal amount” is an amount sufficient to achieve a bacteria-killing blood concentration in the subject receiving the treatment.
  • an "antibiotic,” as used herein, is a chemical substance that, in dilute solutions, inhibits the growth of, or kills microorganisms. Also encompassed by this term are synthetic antibiotics (e.g., analogs) known in the art.
  • the peptides of the disclosure can be used, for example, as preservatives or sterilants of materials susceptible to microbial or viral contamination.
  • the peptides can be used as preservatives in processed foods (e.g., to inhibit organisms such as Salmonella, Yersinia, Listeria and Shigella).
  • the peptides can be used in combination with antibacterial food additives, such as lysozymes.
  • the peptides and/or probiotics of the disclosure also can be used as a topical agent, for example, to inhibit Pseudomonas or Streptococcus or kill odor-producing microbes (e.g., Micrococci).
  • the optimal amount of a ShC2 lantibiotic peptide of the disclosure for any given application can be readily determined by one of skill in the art.
  • the ShC2 lantibiotics and/or probiotics of the disclosure are also useful in promoting wound repair and tissue regeneration.
  • Matrix metalloproteinases (MMPS) are inflammatory enzymes that degrade proteins in various tissues.
  • proteases e.g., MMPs
  • MMPs proteases
  • compositions provided herein can be used concurrently with other antibacterial agents including sulfa drugs such as sulfamethizole, sulfisoxazole, sulfamonomethoxine, sulfamethizole, salazosulfapyridine, silver sulfadiazine and the like; quinoline antibacterial agents such as nalidixic acid, pipemidic acid trihydrate, enoxacin, norfloxacin, ofloxacin, tosufloxacin tosilate, ciprofloxacin hydrochloride, lomefloxacin hydrochloride, sparfloxacin, fleroxacin and the like; antiphthisics such as isoniazid, ethambutol (ethambutol hydrochloride), p-aminosalicylic acid (calcium p-aminosalicylate), pyrazinamide, ethionamide, protionamide, rifam
  • AMPs antimicrobial peptides
  • Cathelicidins are found in several mammalian species. Production of cathelicidins is induced in response to epithelial wounding or infectious challenge, or suppressed by the Attorney Docket No.00015-439WO1 virulence mechanisms of certain bacterial pathogens, e.g., Shigella dysenteriae. Cathelicidin expression is also differentially effected in certain chronic inflammatory disorders.
  • a formulation, composition and method comprise both a ShC2 lantibiotic and cathelicidin.
  • a topical formulation e.g., a lotion, ointment or aerosol spray
  • a cathelicidin and ShC2 lantibiotic peptide or derivatives thereof.
  • Cathelicidin proteins are composed of two distinct domains: an N-terminal “cathelin-like” or “prosequence” domain and the C-terminal domain of the mature AMP. The C-terminal domains of cathelicidins were among the earliest mammalian AMPs to show potent, rapid, and broad-spectrum killing activity.
  • cathelin-like derives from the similarity of the N-terminal sequence with that of cathelin, a 12 kDa protein isolated from porcine neutrophils that shares similarity with the cystatin superfamily of cysteine protease inhibitors.
  • Cathelicidins are expressed in neutrophils and myeloid bone marrow cells and most epithelial sources, and were the first AMPs discovered in mammalian skin due to their presence in wound fluid. In the neutrophil, cathelicidins are synthesized as full length precursor and targeted to the secondary granules where they are stored.
  • LL- 37 The C-terminal 37 amino acids of human cathelicidin (LL- 37) has been characterized. LL-37 was originally referred to as FALL39, named for the first 4 N-terminal amino acids of this domain Attorney Docket No.00015-439WO1 and the total number of residues (i.e., 39).
  • LL-37 is a peptide predicted to contain an amphipathic alpha helix and lacks cysteine, making it different from all other previously isolated human peptide antibiotics of the defensin family, each of which contain 3 disulfide bridges.
  • Full length human cathelicidin (sometimes referred to as full length LL-37) comprises the cathelin-like precursor protein and the C-terminal LL-37 peptide.
  • the cathelicidin polypeptide comprising SEQ ID NO:4 has a number of distinct domains. For example, a signal domain comprising a sequence as set forth from about 1 to about 29-31 of SEQ ID NO:4 is present.
  • the signal domain is typically cleaved following amino acid number 30 of SEQ ID NO:4, however, one of skill in the art will recognize that depending upon the enzyme used, the expression system used and/or the conditions under which proteolytic cleavage of the polypeptide takes place, the cleavage site may vary from 1 to 3 amino acid in either direction of amino acid number 30 of SEQ ID NO:4.
  • Another domain comprises the N- terminal domain, referred to as the cathelin-like domain.
  • the cathelin-like domain comprises from about amino acid 29 (e.g., 29- 31) to about amino acid 128 (e.g., 128-131) of SEQ ID NO:4.
  • Yet another domain of SEQ ID NO:4 comprises the C-terminal domain referred to as LL-37.
  • the LL-37 domain comprises from about amino acid 128 (e.g., 128-134) to amino acid 170 of SEQ ID NO:4.
  • LL-37 comprises the amino acid sequence set forth in SEQ ID NO:4.
  • Vitamin D3 (or its analogs) with ShC2 lantibiotic can be administered systemically to treat systemic infections, in particular pneumonia, sepsis and TB.
  • compositions and methods of the disclosure may also comprise treating disorders of skin dysbiosis by administration of Attorney Docket No.00015-439WO1 an antimicrobial compound or an organism secreting an antimicrobial compound, or administration of a probiotic composition comprising organisms that support skin health.
  • the composition includes a second active agent (e.g., an antibiotic, vitamin D3, cathelicidin etc.).
  • the compositions described herein comprise a probiotic organism.
  • the probiotic organism is a bacterium.
  • the bacterium comprises a component of the normal skin flora. In further embodiments, the bacterium comprises a strain of Staphylococcus hominis. In other embodiments, the probiotic organism comprises a mixture of strains. In some embodiments, the mixture of strains comprises multiple strains of S. hominis. In other embodiments, the mixture of strains comprises one or more strains of S. hominis and one or more strains of S. epidermidis. In some embodiments, the composition comprises one or more strains in addition to S. hominis and/or S. epidermidis.
  • the additional strain or strains comprise one or more strains from the genus Staphylococcus, Lactobacillus or Lactococcus.
  • specific formulations may comprise Staphylococcus hominis or Staphylococcus epidermidis, in particular, Staphylococcus hominis strain A9, Staphylococcus hominis strain C2, Staphylococcus hominis strain AMT2, Staphylococcus hominis strain AMT3, Staphylococcus hominis strain AMT4-C2, Staphylococcus hominis strain AMT4-G1, Staphylococcus hominis strain AMT4-D12, Staphylococcus epidermidis strain AMT1, Staphylococcus epidermidis strain SE-A11, Staphylococcus epidermidis strain AMT5-C5, and/or Staphylococcus epidermidis strain AMT5-G6.
  • Such formulations typically comprise sufficient quantities of bacterial cells as to provide a final density of 10 10 CFU/cm when applied to the skin of a subject.
  • Such formulations may comprise concentrations of from about 10 to about 10 CFU/g, or alternatively, from 10 to about 10 CFU/g, or alternatively, from about 10 to about 10 CFU/g.
  • Such formulations may comprise multiple strains of S. hominis and/or S.
  • epidermidis may further comprise Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Attorney Docket No.00015-439WO1 and/or other such species or strains as are known in the art to form a part of the normal healthy cutaneous or mucosal flora.
  • S. hominis strains as described above comprise 100% of the bacterial cells in a formulation.
  • S. hominis strains as described above comprise 100% of the bacterial cells in a formulation.
  • hominis C2 comprises 90-100%, 85-95%, 70-80%, 75- 85%, 60-70%, 65-75%, 50-60%, 55-65%, 40-50%, 45-55%, 30-40%, 35- 45%, 20-30%, 25-35%, 10-20%, 15-20%, 1-10%, 5-15%, or less than 1% of the bacterial cells in a given formulation, wherein the remainder of the colony forming units are provided by S. epidermidis, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, and/or other such strains as are known in the art to form a part of the normal healthy cutaneous or mucosal flora.
  • the formulations comprise about 60% S. hominis C2 and about 40% S. epidermidis of the strains listed above. In some embodiments, the formulations comprise about 50% S. hominis C2 and about 50% S. epidermidis of the strains listed above. In some embodiments, the formulations comprise about 40% S. hominis C2 and about 60% S. epidermidis of the strains listed above. In some embodiments, the formulations comprise about 70% S. hominis C2 and about 30% S. epidermidis. In some embodiments, the formulations comprise about 30% S. hominis C2 above and about 70% S. epidermidis of the strains listed above. In some embodiments, the formulations comprise about 80% S. hominis C2 above and about 20% S.
  • the formulations comprise about 20% S. hominis C2 and about 80% S. epidermidis of the strains listed above. In some embodiments, the formulations comprise about 90% S. hominis C2 and about 10% S. epidermidis of the strains listed above. In some embodiments, the formulations comprise greater than about 90% S. hominis C2 above and less than about 10% S. epidermidis of the strains listed above. In some embodiments, the formulations comprise less than about 10% S. hominis C2 and greater than about 90% S. epidermidis of the strains listed above.
  • an autologous transplant refers to the transplantation of bacterial strains from one site to another on the same subject or to the same site, regardless of whether the strains are cultured prior to administration or not.
  • the bacterial strains obtained from the subject are expanded in culture and then transplanted back to the subject.
  • an allogeneic transplant refers to the transplantation of bacterial strains from one subject to another subject, or to the administration to a subject of a composition comprising bacterial strains that were not collected from upon or within their own body.
  • Such collection can be carried out by swabbing, scraping, wiping, or cutting and removing tissue on which resides one of the bacterial strains as described herein; optionally growing and isolating single colonies from agar plates or otherwise using methods known in the art; optionally growing expanded cultures of the isolated bacteria or crude swabs, wipes, scrapes, tissue, or other isolate in liquid or solid culture according to methods known in the art; optionally harvesting bacteria from said expanded culture by centrifugation, filtration, gravity settling, scraping, or by other means known in the art; formulating the bacteria or the crude isolate with a thickener, carrier, or excipient; and contacting the subject in an area determined to be in need of the transplant, with said formulation.
  • a prebiotic compound comprises a polysaccharide, hydrolysate, salt, herbal extract, or any other compound sufficient to foster the growth of an associated probiotic strain when used in combination with that strain, such as yeast hydrolysate in concentrations of less than about 40% (w/w), microcrystalline cellulose in concentrations of less than about 10% (w/w), and/or sucrose in concentrations of less than about 10% (w/w).
  • prebiotics that may be adapted for use with cutaneous bacteria include inulin, glucooligosaccharides, isomaltooligosaccharides, lactosucrose, polydextrose, soybean oligosaccharides, and xylooligosaccharides, and those disclosed in Gibson, G.R. and Roberfroid, M,(Eds.) Handbook of Prebiotics, CRC press (2008); Roberfroid, M., J. Nutr. 137(3):830S-837 (2007) and Slavin, J. Nutrients 5(4):1417-1435 (2013), each of which is incorporated herein by reference in its entirety.
  • the method comprises contacting a subject with a probiotic and/or prebiotic composition as described Attorney Docket No.00015-439WO1 herein.
  • a contact comprises an autologous transplant.
  • such contact comprises an allogeneic transplant, wherein elements of the cutaneous or mucosal flora are transplanted to a first subject in need thereof from a second subject (the donor).
  • bacterial strains as disclosed above are identified and isolated from a second subject, amplified in an appropriate culture medium under such conditions as are known in the art to be conducive to bacterial growth, followed by harvest of the bacterial cells, mixing of the harvested cells at a predetermined concentration according to the disclosure with a predetermined formulation, and application of the mixture to the affected area of the first subject.
  • such composition comprises a standardized formulation, such as a formulation in which the concentrations of ingredients are fixed and are not varied from subject to subject.
  • the formulation is developed individually for each subject, based on criteria including but not limited to: the composition of the subject’s own cutaneous or mucosal flora; the subject’s disease state and treatment history; the nature and severity of the subject’s condition; the nature and severity of concurrent cutaneous or mucosal infections; the presence of other antimicrobial compounds including systemic antibiotics within the subject’s body; and other criteria such as are known to or would readily be apparent to those of skill in the art.
  • the composition comprises a cream, ointment, oil suspension or unguent wherein the probiotic bacteria as described above are incorporated within a moisturizer or emulsion such as those described below and in Nakatsuji, T. et al.
  • the composition comprises a patch or poultice wherein the bacteria are combined with a suitable excipient and are incorporated within a fabric, gel matrix, or polymer sheet.
  • suitable excipients and carriers for topical administration are known in the art and include thickeners, emulsifiers, fatty acids, polysaccharides, polyols, and polymers and copolymers, including, without limitation, alginate, Attorney Docket No.00015-439WO1 microcrystalline cellulose, polylactic acid, polylactic-co-glycolic acid, petrolatum, and numerous others known in the art.
  • the composition comprises a bacterial culture medium, a conditioned bacterial culture medium, and/or a bacterial culture. In some embodiments, the composition comprises a filtrate or supernatant of a bacterial culture medium. In some embodiments, the composition comprises a lyophilized culture medium. In some embodiments, the composition comprises a lyophilized conditioned culture medium produced from a filtrate or supernatant of a bacterial culture medium.
  • the method as described herein comprises supporting the health of the skin of a subject. In further embodiments, the method comprises providing a treatment for skin dysbiosis and disorders derived therefrom. In some embodiments the method comprises providing a treatment for bacterial infection of the skin.
  • the treatment comprises the steps of: identifying a subject with skin dysbiosis, bacterial infection, mastitis, burn or other wound, atopic dermatitis, psoriasis, or other chronic skin condition; and administering to the site of the condition in need of treatment the probiotic compositions as disclosed herein. Determination of the appropriate mode of administration of a given formulation (ointment, gel, patch, etc.) can be done by one of ordinary skill in the art of treating skin infections.
  • the probiotic compositions are re-applied at regularly timed intervals. In some embodiments, the probiotic compositions are reapplied every three days. In some embodiments, the probiotic compositions are reapplied every two days.
  • the probiotic compositions are reapplied every two days. In some embodiments, the probiotic compositions are reapplied daily. In some embodiments, the probiotic compositions are reapplied more than once per day. In some embodiments, the probiotic compositions are reapplied weekly. In some embodiments, the probiotic compositions are only applied a single time.
  • the method comprises providing a treatment for Staphylococcus aureus, including methicillin or oxacillin resistant S. aureus, infections. In some further Attorney Docket No.00015-439WO1 embodiments, the method comprises the steps of: diagnosing an S.
  • compositions comprising (i) S. hominis C2, (ii) an extract of an S. hominis C2 culture, (iii) a composition or preparation comprising an ShC2 lantibiotic, or (iv) any combination of (i)-(iii), wherein such compositions are capable of killing or inhibiting the growth of S. aureus, either by production of antimicrobial compounds, by competition for resources within the cutaneous or mucosal biota, or by other means. Determination of the appropriate mode of administration of a given formulation (ointment, gel, patch, etc.) can be done by one of ordinary skill in the art of treating skin infections.
  • the probiotic compositions are re-applied at regularly timed intervals (e.g., daily, every two days, every three days, weekly, etc.). It will be apparent to one of ordinary skill in the art that in other embodiments, similar or identical steps can be applied to provide a treatment for Pseudomonas aeruginosa infections or infections derived from bacteria of the genus Pseudomonas, Staphylococcus, Propionibacterium, Streptococcus, or Vibrio, or uncharacterized pathogens.
  • the method comprises providing a treatment for infections with unknown or uncharacterized pathogens. In some embodiments, the method comprises providing a treatment for polymicrobial infections.
  • the method comprises administering such treatment to a burn or wound. In some embodiments, the method comprises providing a treatment for a chronic skin condition. In some embodiments, the condition is atopic dermatitis, psoriasis, or other chronic skin condition.
  • the disclosure provides the following: (I) Precursor coding sequence: atgactaaaataactaaagatgatttgaaaagattacagaaaatcgtattgaagcacgt acacatccaaccgttgttcctgtaagtgctgctgtatgcggagttgctactaaattagta ccaacatcgaaatgtgcttcaattgtaaaccatgtaataaataaaaa (SEQ ID NO:1)
  • Precursor peptide MTKITKDDLKKITENRIEARTHPTVVPVSAAVCGVATKLVPTSKCASIVKPCNK (SEQ ID NO:2) Attorney Docket No.00015-439WO1
  • III a peptide cleaved by S8 serine protease THPTVVPVSAAVCGVATKLVPTSKCASIVKPCNK (SEQ ID NO:3) Ser
  • the disclosure also provides methods of isolating an S. hominis C2 strain that produces a lantibiotic of the disclosure.
  • the method comprises swabbing the skin of a subject to collect a sample.
  • the sample may be stored in 85% Tryptic Soy Broth (TSB) supplemented with 15% glycerol at –80°C.
  • TTB Tryptic Soy Broth
  • a swab sample is plated onto Mannitol Salt Agar containing egg yolk.
  • individual colonies of coagulase-negative Staphylococci (CoNS) are selected and cultured overnight in TSB at 37°C.
  • each culture plate can include control wells containing a non-antimicrobial S.
  • sterile conditioned media are prepared from each CoNS culture and mixed with 1 ⁇ 104 CFU of S. aureus (ATCC 35556).
  • Antimicrobial activity is defined as the Attorney Docket No.00015-439WO1 ability of the conditioned medium to inhibit S. aureus growth by at least 50% (I50) compared to the negative control after 22 hours of incubation. Cultures demonstrating inhibition can be further cultured or processed to determine if the culture expresses a lantibiotic of the invention (e.g., by PAGE analysis, sequencing, and the like).
  • Example 1 Screening for antimicrobial activity. Colonies of CoNS isolates from skin sites are randomly picked and transferred to a 96-well cluster tube containing TSB. Each plate also receives a non-antimicrobial strain of S. epidermidis (ATCC1457) as negative control, a known antimicrobial strain of Staphylococcus hominis A9 (see below) as positive control, and blank wells without bacteria. CoNS are cultured at 37C overnight with shaking. Growth is evaluated by OD .
  • ATCC1457 non-antimicrobial strain of S. epidermidis
  • Staphylococcus hominis A9 see below
  • ShC2 AIP The secondary structure of ShC2 AIP is identical to that of the S. hominis C5 strain.
  • a previous study has confirmed that the AIP from the S. hominis C5 strain potently antagonizes the quorum sensing of S. aureus (Williams et al., Science Translational Medicine, 2019, eaat8329). This suggests that ShC2 also possesses anti-quorum sensing activity against S. aureus.
  • the antimicrobial activity from each colony is evaluated by mixing with various S. aureus obtained from subjects with moderate to severe atopic dermatitis. [00125] Each S.
  • the ShC2 gel formulation comprises live ShC2 bacteria (5 ⁇ 10 CFU/gram) suspended in 15% glycerol, 2% hydroxyethyl cellulose, and 83% PBS.
  • Initial stability testing confirmed that the ShC2 gel maintains viability for at least 4 days at room temperature and for a minimum of 60 days when stored at or below 4 ⁇ °C (tested at 4 ⁇ °C, ⁇ 20 ⁇ °C, and ⁇ 80 ⁇ °C) (see Figure 6).

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Abstract

L'invention concerne des méthodes et des compositions comprenant un ou des peptide(s) lantibiotique(s) ShC2, des dérivés et des variantes. L'invention concerne également des méthodes et des compositions comprenant des compositions probiotiques utilisant des souches de S. hominis C2 qui produisent un lantibiotique ShC2, des peptides de type lantibiotique ShC2, ou d'autres inhibiteurs d'agents pathogènes de la peau. L'invention concerne également des méthodes de traitement pour des infections microbiennes cutanées et la dermatite atopique.
PCT/US2025/029732 2024-05-16 2025-05-16 Thérapie antimicrobienne Pending WO2025240841A1 (fr)

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