Corynebacterium mastitidis-derived mycolates for treating barriers

Abstract

The invention provides a composition comprising, as an active principal, a mycolate, or salt, conjugate, or hydrate thereof, and a pharmaceutically acceptable carrier. Also provided are methods and uses involving treatment and / or prophylaxis of an infection and involving stimulating an immune response in a subject through administering the inventive composition.

Description

CORYNEBACTERIUM MASTITIDIS-DERIVED MYCOLATES FOR TREATING BARRIERSSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with Government support under project number EY000184, National Institutes of Health, National Eye Institute. The Government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0002] Infectious keratitis is a global corneal disease that constitutes the fifth leading cause of blindness. Infectious keratitis occurs when corneal barrier function is compromised. Several studies have revealed a protective effect attributed to commensal bacteria in maintaining immune homeostasis of the ocular surface. Specifically, the topical association of an ocular commensal Corynebacterium mastitidis (C. mast) has been demonstrated to protect mice against keratitis induced by Pseudomonas ceruginosa, the most common cause of contact lens-associated keratitis. C. mast protects against corneal fungal and bacterial infections by eliciting interleukin- 17 (IL-17) responses from local yd T cells. This promotes bacterial clearance and permits corneal epithelial healing.

[0003] This commensal relationship between C. mast prokaryotes and mammals (such as mice and humans) presents an opportunity for probiotics as therapy and prophylaxis against infection, particularly involving barrier tissue (e.g., cornea, nasal epithelium, oral epithelium, rectal epithelium, skin, vaginal epithelium, and the like) and other structures, such as hair, nails, teeth, etc., which can harbor pathogenic bacteria. However, the barrier tissues and other structures of some human and animal hosts may not be suitable for colonization. For example, individual eye chemistry and / or competition with other commensals or infectious organisms may impede commensal ocular colonization by C. mast, posing a barrier to C. mast therapy and / or prophylaxis using a probiotic approach. Also, bacterial and fungal infection is commonly treated with antibiotics and corticosteroids; human and veterinary patients also may be treated with systemic pharmaceutical agents (e.g., antibiotics) able to penetrate the tissues of the eye and other barrier tissues. Such chemical therapies also may impede colonization by C. mast, therebycountervailing potential probiotic C. mast therapy and prophylaxis as against such infectious agents. Moreover, extended antibiotic use poses a risk of numerous undesirable side effects and can lead to the evolution of antibiotic-resistant strains of infectious agents, such as pathogenic bacteria and fungi.

[0004] Accordingly, the potential unavailability of C. mast commensal colonization presents a technical problem and a clinical need for a substitute for and / or augmentation of C. mast probiotic therapy and prophylaxis. The present invention provides a solution to this problem and addresses this clinical need.BRIEF SUMMARY OF THE INVENTION

[0005] The invention provides a composition comprising, as an active principal, a mycolate, or salt, conjugate, or hydrate thereof, and a carrier. Also provided are methods and uses involving treatment and / or prophylaxis of infection of barrier tissues and other structures and involving stimulating an immune response in a subject through administering the inventive composition.

[0006] The inventive composition and methods can combat infections and reduce or obviate extended use of antibiotics, which, as noted above, has undesirable side effects. Accordingly, the present invention addresses the foregoing technical problem and clinical need.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0007] Figure 1 presents the design and results of a study demonstrating that lipid components derived from C. mast induce local IL-17A responses from VyV85 T cells. Panel A depicts the of the experimental design. C57BL / 6J were inoculated with either crude lipid components of C. mast (25 pg / per eye) or corn oil daily for 7 days. Panels B and C present data concerning the percentage of IL-17A+ and Ki67+ in Vy4 cells (panel B) of eye-draining cervical LNs (DLNs) with PMA and ionomycin. Representative flow cytometric analysis and neutrophils (panel C) of the conjunctiva from mice depicted in panel A.

[0008] Figure 2 is an image of thin layer chromatography (TLC) results of Fractions 1-5 from the C. mast lipidome, as separated using a silica-based extraction.

[0009] Figure 3 graphically presents the results of LCMS analysis of chromatography of Fractions 1 and 3-5 from the C. mast lipidome, as separated using a silica-based extraction. Thetop trace represents Fraction 1, the second highest trace represents Fraction 2, the third highest trace represents Fraction 4, and the bottom trace represents Fraction 5.

[0010] Figure 4 graphically displays the results of flow cytometry data concerning intracellular IL-17A detected from y8 T cells that had been co-cultured with CD1 lc+ dendritic cells (DCs) in the presence of the indicated C. mast lipid Fractions for three days.

[0011] Figure 5 is a bar graph displaying the amount (pg / ml) of IL-17A detected from y8 T cells that had been co-cultured with CD1 lc+ dendritic cells (DCs) in the presence of the indicated C. mast lipid Fractions for three days.

[0012] Figures 6-9 present information concerning an experiment investigating whether C. mast-derived mycolates elicit IL-17A production by y8 T cells in vitro. Figure 6 depicts the protocol for a second extraction of pooled C. mast Fractions 3 and 4 and data showing the enrichment of mycolates in the resulting Fraction 3.

[0013] Figure 7 graphically presents the results of an experiment in which y8 T cells were Co-cultured with CD1 lc+ DCs in the presence of indicated lipid Fractions of C. mast for 3 days. Representative flow cytometry data showed intracellular IL-17A detected in y8 T cells (left panel); supernatants were collected and tested for IL-17A production (right panel).

[0014] Figure 8 presents the results of an experiment in which sorted y8 T cells cultured with WT DCs and mycolates in the presence of indicated blocking antibodies for 3 days. Representative flow cytometry data (left column) showed Intracellular IL-17A detected in y8 T cells in each condition; supernatants were collected and tested for IL-17A production (right column).

[0015] Figure 9 presents the results of an experiment in which sorted y8 T cells cultured with TLR2-deficient DCs and mycolates for 3 days. Representative flow cytometry data (left column) showed intracellular IL-17A detected in y8 T cells in each condition; supernatants were collected and tested for IL-17A production (right column).

[0016] Figure 10 presents the design and results of in vivo experiments investigating the effects of ocular surface application of C. mast-derived mycolates on y8 T cells from the eyedraining LNs and conjunctiva. Panel (A) depicts the experimental design. Panel (B) presents representative flow cytometry data (left) and bar graphs (right) showed Intracellular IL-17A and Ki-67 detected in y8 T cells from eye-draining lymph nodes (DLN) from mice described in (A).Panel (C) presents representative flow cytometry the percentage of yd T cells data (left) and a bar graph showed the percentage and total number of yd T cells in conjunctiva yd T cells described in (A). Panel (D) is an illustration of a mouse model of keratitis. Panel (E) depicts the experimental design to evaluate the efficacy of mycolates in the keratitis model. (F) presents the clinical scores and bacterial burden from mice described in (E).

[0017] Figure 11 graphically presents the results of an experiment (Example 5 herein), which demonstrate that mycolates from C. mast but not from Mycobacterium tuberculosis (M. tb) stimulate IL-17A response in yd T cells.

[0018] Figure 12 graphically presents the results of an experiment (Example 6 herein), which demonstrate that trehalose monocorynomycolate (TMCM), but not trehalose dicorynomycolate. (TDCM) or unconjugated corynomycolate, is a potent IL-17A stimulant. (A) Schematic of synthetic corMyc(al8: l / Myl8: l) (corMyc(36:2)), D-(+)-trehalose 6- monoMyc(al8: l / Myl8: l) (TMCM(36:2)) and D-(+)-trehalose 6,6'-diMyc(al8:l / Myl8:l) (TDCM(72:4)). (B) Synthetic corMyc(36:2), TMCM(36:2), or TDCM(72:4) were dissolved in ethanol (EtOH). 1 pg / ml of each synthetic lipid was added in cocultures with sorted ydT cells and CD1 lc+ DCs for 3 days. The frequency of Ki67+ IL-17A producing Vy4+ T cells and the level of IL-17A in the culture supernatant are shown. Each dot represents one experiment. Bars represent mean ± SE. *p<0.05, **p<0.01, ***p <0.001. Statistical significance was determined by the Mann- Whitney test (B).

[0019] Figure 13 graphically presents the results of an experiment (Example 7 herein), which demonstrate that TNF-a and IL-6 affect TMCM-induced IL- 17 responses when present together, but not individually. (A) Sorted ydT cells alone or with CD1 lc+ DCs in the presence of TMCM(36:2) for 3 days. Representative flow cytometry plot showing the percentage of Ki67+IL-17A+ on gated ydT cells, and levels of IL-17A in the culture supernatant are shown in dot plot. Each dot represents one experiment. (B) Blockade antibodies targeting anti-TNF-a or anti-IL-6R were added in cocultures with sorted ydT cells with CD1 lc+ DCs in the presence of TMCM(36:2) for 2 days. Representative flow cytometry plot showing the percentage of Ki67+IL-17A+ on gated ydT cells. Representative data of at least 2 independent experiments. Bars represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined by the Mann-Whitney test (A).

[0020] Figure 14 graphically presents the results of an experiment (Example 7 herein), which demonstrate that TLR2 and Mincle expressed by dendritic cells (“DCs”) sense TMCM and regulate y8 T cell IL-17A production. (A-C) DCs were stimulated with either vehicle control ethanol or 1 pg / ml synthetic TMCM(36:2) for 2 days. (A) WT DCs, (B) TLR2 deficient DCs, or (C) WT DCs pretreated with IgG or anti-Mincle antibody, were stimulated with TMCM(36:2) for 2 days. Cytokine levels in the culture supernatants were measured by a Legendplex mouse inflammation panel for 13 analytes. Bar plots list five cytokines that are induced by TMCM. Each dot represents one experiment. (D) Sorted y8 T cells were cocultured with WT or TLR2 deficient CD1 lc+ DCs in the presence of 1 pg / ml synthetic TMCM(36:2) for 72 hours. Representative FACS plot showing the percentage of Ki67+ IL-17A+ y6 T cells and the scatter plot showing the level of IL-17A in the culture supernatant. Each dot represents one experiment. (E-G) FACS plots showing the percentage of Ki67+ IL-17A+ y8 T cells and the scatter plot showing levels of 1L-17A production in the supernatants from y8 T cells co-cultured with WT DCs (E) with or without anti-Mincle for 48 hours; or (F) with or without anti -IL- 1R for 48 hours; or (G) cultures with or without anti-IL-6R + anti-TNF-oi for 48 hours. Each dot represents one experiment. Bars are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined by the Mann-Whitney test (A-G).

[0021] Figure 15 graphically presents the results of an experiment (Example 8 herein), which demonstrate that y8 TCR signaling does not affect IL-7R expression. (A) Schematic representation of CD3^ IT AM motif in Ert2-cre(-) and Ert2-cre(+) CD3(^ 6Y / 6Y(6F(induced)) mice before and after tamoxifen induced modification. The tyrosines (Y) in the wild-type ITAM motif were replaced by phenylalanines (F) in mutant CD3^, impairing TCR signaling. (B) The efficiency of mutant CD3 induction shown as Myc tag expression after tamoxifen treatment. Lymphocytes from WT (left trace) and mutated CD3c mice (right trace) stained with anti-Myc. (C) The expression of IL-7Ra on CD44+CD27- y8 T cells from WT and mutated CD3(^ mice 8 days after tamoxifen treatment. Each dot represents an individual mouse. The results were representative of at least 2 independent experiments. Bars represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined by the Mann-Whitney test (C).

[0022] Figure 16 graphically presents the results of an experiment (Example 8 herein), which demonstrate that y8 TCR signaling supports IL-1R1 expression and is required for IL-17Aproduction in response to TMCM. (A) Experimental design for inducing expression of WT or mutant CD3 in Ert2-cre(-) CD3 6Y / 6Y(6F(induced)) (WT CD3Q and Ert2-cre(+) CD3 6Y / 6Y(6F (induced)) (Mutant CD3Q mice. (B) Percent of CD44+CD27- (left) and IL-17A+ (right) cells among y5 T cells in LNs of WT and mutated CD3i^ mice 8 days after tamoxifen induction. Each dot represents an individual mouse. (C) Overlaid histograms and scatter plots showing the expression of IL-1R1 on CD44+CD27- y8 T cells from WT (right trace) and mutated (middle trace) CD3^ mice 8 days after tamoxifen treatment. Each dot represents an individual mouse. Fluorescence minus one (FMO) (left trace) was used as controls. (D-E) y8 T lymphocytes purified from WT and mutated CD3^ mice on day 8 after tamoxifen treatment were stimulated with IL- 1 [3 and analyzed by phospho-flow (D). The mean fluorescence intensity (MFI) of phospho-Erk (left) and phospho-P38 (right) in Rorgt+ y8 T cells (y8 T cells with IL- 17A producing phenotype) at indicated time points after IL- 10 stimulation is displayed. (E) Representative data from three independent experiments. (F) y3 T cells were sorted from WT or mutated CD3(^ mice 8 days after tamoxifen treatment and co-cultured with DCs in the presence of TMCM(36:2) for 48 hours. A representative FACS plot illustrates the proportion of Ki67+ IL- 17A+ y8 T cells in WT or mutant CD3(^ y8 T cells. The scatter plot displays the IL-17A levels in culture supernatants quantified by ELISA. Data are pooled from two independent experiments. The results were representative of at least 2 independent experiments. Bars represent mean ± SEM with *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined by the Mann-Whitney test (B-C, F) and two-way ANOVA (E).DETAILED DESCRIPTION OF THE INVENTION

[0023] In an embodiment, the invention provides a composition comprising, as an active principal, a mycolate, or salt, conjugate, or hydrate thereof, and a carrier.

[0024] Mycolates, or mycolic acids, are fatty acids comprising acyl tails. For use in the inventive composition, the mycolate, or salt, conjugate, or hydrate thereof, desirably comprises an acyl tail comprising between 14 and 22 carbons (e. , 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 carbons in the acyl tail). Such mycolates were identified as among those in the active Fractions (Fractions 3 and 4) derived from C. mast, as reported below in the Examples.Accordingly, the mycolate, or salt, conjugate, or hydrate thereof, for inclusion in the inventive composition can be derived from or purified from C. mast. Mycolates having acyl chains longerthan about 22 carbons do not appear to provide protection from at least ocular infection as do those within the range of 14-22 carbons. Mycolates, or salts, conjugates, or hydrates thereof, comprising acyl tails shorter than 14 carbons can be included within the inventive composition; however, such were not identified as among those in the active Fractions (Fractions 3 and 4) derived from C. mast, as reported below in the Examples.

[0025] While, as noted, the mycolate, or salt, conjugate, or hydrate thereof, for inclusion in the inventive composition can be derived from or purified from C. mast, such also can be chemically synthesized. Methods of synthesizing lipids and conjugating such to sugar moieties are known, and such can be employed to manufacture the mycolate, or salt, conjugate, or hydrate thereof, for inclusion in the inventive composition.

[0026] As noted, the mycolate for inclusion in the inventive composition can be in the form of derivatives, such as salts, conjugates, hydrates, etc. Preferably, such derivatives are pharmaceutically and / or ophthalmically compatible in that they enhance the ease of formulation and do not pose a risk of toxicity in pharmaceutical and / or ophthalmic formulations. In one embodiment involving a mycolate derivative, the mycolate can be conjugated to a sugar moiety (e.g., a monohexose, dihexose, et.), such as trehalose, galactose, glucose, sucrose, lactose, or other synthetic or naturally occurring mono-, di-, and trisaccharides, such as where the sugar moieties are in a cyclic form. While not wishing to be bound by hypothesis, it may be that conjugation of the mycolate with a sugar moiety that does not naturally conjugate to the mycolic acid may enhance the activity further or change the nature of the activity in a therapeutically and / or prophylactically advantageous manner.

[0027] Of course, while the mycolate for inclusion in the inventive composition can be derivatized (e.g., conjugated with a sugar moiety as discussed above), it also can be present as an underivatized carboxyl. It will be observed that, while data provided in the Examples herein suggest that sugar-derivation of mycolates (e.g., trehalose-conjugated mycolates) have superior antibacterial properties as opposed to underivatized forms, the latter are within the scope of the present invention. Such can be in a composition, for example, which can be packaged and then derivatized with the desired sugar prior to use.

[0028] Exemplary, non-limiting, mycolates for use in the present invention can have structures such as the following. These examples are termed corynomycolate (“corMyc”) andderivatized compounds thereof, referring to a subfamily of mycolates present in naturally high abundance in C. mast and related Corynebacterium species:A: corMyc(al8: l / Myl8: l), i.e., corMyc(36:2)B: TDCM(al8: 1 / 18: 1, al8: 1 / 18: 1), i.e., TDCM(72:4)or C: TMCM(al8:l / 18: l), i.e., TMCM(36:2)

[0029] While use of monomycolates (such as structures A and C) appear to be more effective than dimycolates when treating tissues, particularly ophthalmic tissues, dimycolates (e.g., structure B) or trimycolates (as well as monomycolates) can be effectively used to coat or treat surfaces, such as barrier tissues (e.g., cornea, nasal epithelium, oral epithelium, rectal epithelium, skin, vaginal epithelium, and the like) and other structures, such as hair, nails, teeth, etc., which can harbor pathogenic bacteria. Indeed, the mycolates as described herein can be employed to treat inanimate surfaces as well, such as bandages, catheters, needles, other wound dressing, and the like, to assist in preventing the growth of pathogenic fungi or bacteria, or the formation of biofilms comprising such.

[0030] Within the composition, the mycolate, or salt, conjugate, or hydrate thereof, can be present in an amount suitable for delivering a concentration to the aqueous humor and / or tears orto barrier tissues or other structures equal to or greater than the 50% or greater (such as 90%) minimum inhibitory concentration (MICso or MIC90) level of the mycolate, or salt, conjugate, or hydrate thereof, relative to bacteria and / or fungi commonly associated with ophthalmic infections or infections of barrier tissues or other structures. While a suitable concentration for formulating the mycolate, or salt, conjugate, or hydrate thereof, can thus be calculated and determined using ordinary skill, such concentration can be in the range of from about 10 pg / ml to about 100 pg / ml or between about 0.1 % and 1.0% by weight of the inventive composition, although these ranges are non-limiting. In the examples presented below, for instance, in vivo application employed a formulation of C. mctsl-Acvw' cA mycolates with a concentration of 5 mg / ml.

[0031] The carrier for inclusion in embodiments of the inventive composition can be “pharmaceutically acceptable,” i.e., can be such as is typically employed for application of pharmaceutical, cosmetic, or other substances to the surfaces of barrier tissues or other structures. For example, when formulated for direct administration to the eye, the carrier should be ophthalmically acceptable. In this sense, the carrier is compatible with the chemical microenvironment of tissues of the eye and / or lacrimal secretions (tears). Thus, inventive composition, in its ophthalmic embodiments, desirably has a pH and osmotic values compatible with tears, eye tissues and the aqueous humor. Thus, for example, the pH of the inventive composition can be between about 6.0 to about 8.0. Osmotic values of the inventive composition can be between about 200 to about 400 mOsm / L. These pH and osmotic values are exemplary, and the actual pH and mOsm / L values of any embodiment of the inventive composition can vary somewhat from these ranges.

[0032] Moreover, the properties of the composition, and the carrier within it, can vary depending on the tissue or other structure to which the inventive composition is to be applied. Indeed, for application to barrier tissues or other structures, the inventive composition can include the mycolate (including salt, conjugate, or hydrate thereof) admixed with compositions typically applied to such barrier tissues or other structures. Thus, the composition can be in the form of eye drops, mouthwash, skin cream or lotion, suppositories, toothpaste, etc., in which the mycolate (including salt, conjugate, or hydrate thereof) is admixed. Such compositions areknown and many commercial formulations of such compositions are available to consumers and healthcare professionals.

[0033] Additionally, the inventive composition can comprise one or more additional active principal agents, such as an antibiotic and / or anti-infective drug substance (e.g., an antibacterial, antifungal, or antiviral agent, or combination thereof), which can be any such agent approved or under investigation for treatment or prophylaxis of eye infections or infections of other barrier tissues or structures. Examples of such other antibiotic and / or anti-infective active principals include, but are not limited to, fluoroquinolones (e.g., besifloxacin, ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, ofloxacin, and the like), aminoglycosides (e.g., azithromycin, erythromycin, gentamicin, natamycin, neomycin, tobramycin, and the like) and other commonly employed anti-infectives (e.g., bacitracin, chloramphenicol, ganciclovir, gramicidin, lotilaner, oxytetracycline, polymyxin b, povidone iodine, trifluridine, trimethoprim, vidarabine, etc.). Any such additional active principal, or combination thereof, can be present within the inventive composition in an amount (percent by weight) or concentration typically employed in compositions applied to barrier tissues or other structures. Indeed, the inventive composition can take the form of an existing (e.g., an FDA-approved or investigational) formulation of any such additional active principal to which the mycolate, or salt, conjugate, or hydrate thereof, is added. Any such formulation can be adapted to comprise mycolate, or salt, conjugate, or hydrate thereof, provided it is screened for compatibility with the mycolate, or salt, conjugate, or hydrate thereof, active principal.

[0034] The inventive composition can include excipients to aid in formulation, promote stability, enhance solubility, and increase bioavailability and thus activity. For example, in an embodiment, the carrier can comprise a lipid component or be a lipid carrier to optimize the mycolate structure as a micelle or vesicle. Also, in an embodiment, it is desirable to formulate the inventive as part of a liposome, which will integrate the inventive into the liposome structure stabilizing its solubility and enabling control over its delivery properties. Any suitable liposome compatible with the mycolate, or salt, conjugate, or hydrate thereof, can be employed in the inventive composition, many of which are commercially available. However, it can be advantageous to employ liposomes comprising the mixture of lipids present in C. mast membranes, including, for example, phosphatidylglycerol, cardiolipin, phosphatidylinositol, andfree fatty acids, which are known to naturally support the my colic acid. Thus, a liposome for inclusion in the inventive composition can comprise, consist of, or consist essentially of any combination of phosphatidylglycerol, cardiolipin, phosphatidylinositol, and free fatty acids. Any suitable loading of mycolic acids in the liposome of choice that maximizes mycolic acid bioavailability and activity can be included.

[0035] In addition to the mycolate, or salt, conjugate, or hydrate thereof, and optional additional active principal agent and / or lipid or liposomal components, the inventive composition can comprise other components, such as excipients commonly employed in pharmaceutical or cosmetic compositions. For example, the inventive composition can comprise one or more steroidal or non-steroidal anti-inflammatory agent. One or more preservatives, such as those commonly included in commercial formulations, also can be included. Indeed, when packaged in multi-dose form, as is typical of commercial medications, inclusion of one or more preservatives is preferred to maintain the sterility of the formulation once opened by a consumer. Often, such preservatives are included in ophthalmic and other formulations in an amount of from 0.001% to 1.0% by weight, which can be suitable for the inventive composition as well, although this range is by no means limiting in the context of the inventive composition.

[0036] When a consistency more viscous than water is desired (for example, to increase ocular absorption of the active principal(s) or increase the retention time in the eye or on the surface of the cornea or for application to barrier tissues or other structures), the inventive composition can comprise one or more viscosity enhancing agents. Any suitable viscosity enhancing agents typically employed in pharmaceutical or cosmetic formulations can be included in the inventive composition, if desired. Such viscosity enhancing agents include, for example, cellulose derivatives (such as carboxymethyl cellulose, hydroxy propyl cellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose, methyl cellulose, and the like), and substances such as polyvinyl alcohol and / or polyvinyl pyrrolidone, although these listed agents are provided merely as examples and by no means intended to limit the scope of the invention. Viscosity enhancing agents commonly employed in ophthalmic formulations can be present in a range of between about 0.01% to 2% by weight, which can be suitable for the inventive composition as well, although this range is by no means limiting in the context of the inventive composition. Indeed, for embodiments in which the inventive composition is in the form of acream, salve, lotion, paste (such as toothpaste), etc., to be applied to skin or other structures, viscosity enhancing agents can be present in higher amounts to achieve the desired consistency.

[0037] In certain embodiments, inventive composition can be formulated as an ophthalmic composition and, in such embodiments, is typically presented in a dosage form suitable for topical delivery to the eye. Thus, the composition can be formulated as a sterile solution, suspension, gel, cream, or other type of composition. For example, in certain embodiments, the inventive composition can take the form of a liquid, which conveniently can be applied to the eye as drops. In certain other embodiments, the inventive composition can be formulated as an irrigating solution, such as can be applied to the eye, nose, skin, and other epithelial tissues, for example, during surgical procedures. In yet other embodiments, when used ophthalmically, the inventive composition can take the form of formulations suitable for cleaning and handling of contact lenses.

[0038] The inventive composition can be made using standard methods of pharmaceutics involved in the formulation of pharmaceutical or cosmetic formulations, such as dental, dermal, ocular, nasal, otic, oral, etc., irrigating solutions, and formulations suitable for cleaning and handling of contact lenses. Thus, for example, the mycolate, or salt, conjugate, or hydrate thereof, and optional additional active principal agent and / or lipid or liposomal components, as well as any other desired excipients, can be admixed with water or other suitable carrier to form a suitable solution, suspension, gel, cream, paste (e.g., toothpaste), or other type of composition. This, then, can be sterilized and packaged as appropriate for the desired method of application, such as in multi-dose containers.

[0039] Using the inventive composition, in an aspect, the invention provides methods involving delivering the composition to a subject, such as to an eye of such subject or to other barrier tissues or other structures, uses of the composition involving such delivery, and formulation of the composition for such uses. In the performance of such methods, and in connection with such uses, the inventive composition is administered to a portion of a barrier tissue or other structure (e.g., cornea (e.g., one or both eyes), nasal epithelium, oral epithelium, rectal epithelium, skin, vaginal epithelium, and the like) and other structures, such as hair, nails, teeth, etc., of a subject in an amount and according to a dosing regimen sufficient to achieve the purpose of administration.

[0040] In the performance of the inventive method, and in connection with the inventive use of the composition, the “subject” to be treated is typically human (i.e., a patient), but the subject can be a non-human animal instead and is typically mammalian (such as a pet animal, livestock, or animal typically employed in laboratory research, such as cats, cows, dogs, horses, mice, pigs, rabbits, and rats, among others).

[0041] Typically, the performance of such methods involves administering the inventive composition topically to the barrier tissue or other structure, but it can involve perfusion or injection into wet tissues, such as the eye. In certain embodiments, the inventive method can involve a single dose, such as when the composition is used as an irrigating solution, such as for example, during surgical procedures. In other embodiments, the composition can be applied, either by a physician, laboratory researcher, or veterinarian, or indeed by the subject, for example as a solution, suspension, cream, lotion, paste, or eyedrops, applied one or several (e.g., from one to five or more) times daily. When drops are applied, such can be metered or dispensed via gravity, and will typically comprise between about 100 pl to about 500 pl of solution, depending on the physical parameters of the dropper or packaging and viscosity of the inventive composition. Other types of the inventive composition (e.g., creams, lotions, pastes, etc.) can be applied to skin, other epithelial tissue, teeth, etc., or inanimate surfaces as well, such as bandages, catheters, needles, other wound dressing, and the like, using standard methods.

[0042] In one embodiment, the method and use can be employed for treatment or prophylaxis of an infection impacting a barrier tissue or other structure (e.g., cornea (e.g., one or both eyes), nasal epithelium, oral epithelium, rectal epithelium, skin, vaginal epithelium, and the like) or other structures, such as hair, nails, teeth, etc. The infection can be due to any organism (e.g., a bacterium or fungus), such as those commonly causing opportunistic or pathogenic infections of barrier tissues or other structures. Some of the many opportunistic or pathogenic organisms that can be combatted through the application of the inventive method and use include, but are not limited to, Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter Iwoffii, Aspergillus sp., Candida sp. Chlamydia pneumoniae, Chlamydia trachomatis, Citrobacter freundii, Citrobacter koseri, Clostridium perfringens, Corynebacterium sp., Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Fusarium sp., Fusobacterium sp., Haemophilus influenzae, Haemophilus parainfluenzae, Klebsiella oxytoca,Klebsiella pneumonia, Legionella pneumophila, Listeria monocytogenes. Micrococcus luteus, Moraxella catarrhalis, Morganella morganii, Mycobacterium avium, Mycobacterium marinum, Mycoplasma pneumonia, Neisseria gonorrhoea, Prevotella sp. , Propionibacterium acnes, Proteus mirabilis, Proteus vulgaris, Pseudomonas aruginosa, Pseudomonas stutzeri, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hamolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, Staphylococcus warneri, Streptococcus agalactia, Streptococcus mitis, Streptococcus pneumonia, Streptococcus pyogenes, Streptococcus viridans, among others.

[0043] In another embodiment, the method and use can be employed for stimulating an immune response in a subject. For example, the inventive composition comprising the mycolate, or salt, conjugate, or hydrate thereof can be employed to induce the production of IL-17A in tears or barrier tissue to which the composition is applied. It will be observed that one advantage of the inventive composition, method, and use thereof is in supplying or supplementing the beneficial effect of mycolates, such as those derived from C. mast, to subjects to combat eye and other barrier tissue infections while reducing or obviating extended use of antibiotics and their attendant undesirable side effects. Additionally or alternatively, the inventive composition, method, and use thereof can provide the beneficial therapeutic and prophylactic effect of mycolates, such as those derived from C. mast, to subjects whose particular eye chemistry, microbiota, or use of other agents (e.g., antibiotics) impede the commensal ocular colonization by C. mast and for whom probiotic therapy may not be a viable option.

[0044] Additionally, while the inventive composition, methods, and uses can be used as monotherapy, the inventive compositions also can be used in conjunction with standard antibiotic treatment of subjects. For example, the inventive composition can be administered to a subject having received, or receiving, a course of antibacterial or antifungal therapy. Thus, the inventive composition can be administered alone, prior to, concurrently with, or following such course or antibacterial or antifungal therapy. In embodiments, particularly those in which the inventive composition is administered following antibacterial or antifungal therapy, the mycolate within the composition can help immune cells (such as invariant T-cell populations, and others) maintain tolerance while retaining protective capabilities in the treated tissue to protect against opportunistic, pathogenic and / or antibiotic resistant organisms from populating or outgrowing inthe tissue. Such application of the inventive method and composition advantageously helps the treated subject recover from the pathogenic infection and avoid secondary opportunistic infections.EXAMPLES

[0045] The following experimental Examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. In brief, the experiments discussed in the Examples demonstrate the identification of active components of C. mast responsible for the protective function of this commensal, the activation of y8 T cells in response to the identified active components, and the provision of protection against keratitis attributed to the identified active components.

[0046] This was achieved by systematically fractionating the lipidome of C. mast into five components, each of which was then screened for IL-17A induction in vitro. One fraction (“fraction 3”) exhibiting the highest IL- 17 stimulatory effect was found to contain mycolates as its active component. Topical instillation of these mycolates onto the mouse ocular surface resulted in IL-17 production in eye-draining lymph nodes, increased numbers of IL-17A- producing y8 T cells in the conjunctiva and exhibited protection of the treated mice from infectious keratitis. Additional in vivo experiments reveal the specificity of mycolates derived from C. mast as opposed to derived from Mycobacterium tuberculosis .EXAMPLE 1

[0047] This example demonstrates that lipid components of C. mast induce local IL-17A responses from Vy4V85 T cells at the ocular surface.

[0048] C57BL / 6J mice were inoculated with either crude lipid components of C. mast or corn oil daily for 7 days and sacrificed on day 8 (Figure 1, panel A). Thereafter, eye-draining cervical LNs (DLNs) were assayed to determine the number and percentage of IL-17A+ and Ki67+ in Vy4 cells therein. As revealed in Figure 1, panels B and C, the percentage of both IL- 17A+ and Ki67+ cells obtained from the mice treated with crude lipid components of C. mast greatly, and significantly, exceeded those obtained from the mice treated with corn oil.

[0049] Vy4+CD44+CD27- cells sorted from lipid-associated mice also were subjected to single-cell TCR sequencing. Animo acid sequences and counts of clones that are exclusively in lipid-associated mice are shown in Table 1.Table 1EXAMPLE 2

[0050] This example demonstrates the identification of immunologically active mycolates derived from C. mast.

[0051] Liquid chromatography was employed to separate components from crude lipid extracted from C. mast via MeOH. Five components were extracted (Figures 2 and 3) and analyzed. SPE1 analysis by thin layer chromatography (TLC) (Figure 2) and liquid chromatography mass spectrophotometry (LCMS) (Figure 3) revealed the following:• Fraction 1 recovered the least polar corynomycolic acids (Figure 2, highest band), the most glycosylated lipids in the mixture.• Fraction 2 did not contain much recoverable material.• Fraction 3 recovered a second family of lipids that are likely corynomycolic acids (CorMycs) that migrate just below the top band in Figure 2.• Fraction 4 recovered a mixture of those same CorMycs as Fraction 3 as well as cardiolipin (CL).• Fraction 5 recovered other phospholipids (PG, CL, PI, and PIM).

[0052] The ability of each component to activate yb T cells was examined by co-culturing CD1 lc+ dendritic cells and sorted yb T cells for three days in the presence of each respective Fraction or control. Flow cytometry data demonstrated intracellular IL-17 highest in Fraction 3 (Figure 4). Supernatants also were collected and tested for IL-17A production, revealing as well that Fraction 3 resulted in a significantly higher production of 1L-17A than any other fraction or control cohort (Figure 5). Fractions 1 & 2 and their corresponding mock extracts did not induce IL-17A production by yb T cells. Fraction 3, containing fatty acids and CorMycs, induced thehighest level of IL-17A production from y8 T cells, followed by Fraction 4, containing CorMycs and cardiolipin. The phospholipid-containing Fraction 5 had only a minimal IL-17A stimulatory effect.EXAMPLE 3

[0053] This example demonstrates that C. mast-derived mycolates elicit IL-17A production by y8 T cells in vitro.

[0054] Pooled C. mast Fraction 3 and Fraction 4 (described in Example 2) were loaded on a Cl 8 cartridge for a second extraction (SPE2). As shown in Figure 5, the mycolates were found to be enriched in SPE2 Fraction 3.

[0055] Sorted y8 T cells and CD1 lc+ DCs were co-cultured in the presence of SPE2 C. mast Fractions for three days and assayed as described in Example 2. Representative flow cytometry data are presented in Figure 6, left panel, and data demonstrating respective levels of IL-17A production are presented in Figure 6, right panel.

[0056] Sorted y5 T cells and wild type DCs were co-cultured in the presence of mycolates anti-IL-lR or anti-CDld antibodies. Representative data from flow cytometry and the production of IL-17A from pooled supernatants are presented in Figure 7 (left and right columns, respectively). Sorted y8 T cells and either wild type or TLR-deficient DCs were co- cultured in the presence of mycolates. Representative data from flow cytometry and the production of IL-17A from pooled supernatants are presented in Figures 8 and 9, respectively. The data demonstrate that mycolate-induced IL-17A production depends on IL1R signaling, but not TLR2 and TLR2 signaling.EXAMPLE 4

[0057] This example demonstrates that C. mast-derived mycolates elicit local y8 T responses and a trend to clear more Pseudomonas in treated mice.

[0058] The ocular surface of C57BL / 6J mice was treated by instillation of the Fraction 3 mycolates or vehicle control for 7 consecutive days. The responses of y8 T cells from the eye-draining lymph nodes (DLNs) and conjunctiva were compared. The results are presented in Figure 10.

[0059] Collectively, the in vivo data demonstrate that mycolates from Fraction 3 were more effective in inducing Ki-67 and IL-17A in yd T cells from both conjunctiva and DLNs in mice compared to the control group treated with the vehicle and exhibited a trend to clear more Pseudomonas in treated mice.EXAMPLE 5

[0060] This example demonstrates that the protective and immunostimulatory effects of mycolates derived from C. mast are specific.

[0061] 25 pg mycolates (5 pl of 5 mg / ml mycolates on ocular surface) either fromMycobacterium tuberculosis M. th. Sigma, M4537) or purified from C. mast were instilled in mouse conjunctiva daily for five days. Eye-draining lymph nodes were collected to assess the IL-17A cytokine production from and proliferation of (assessed via the marker, Ki67+) of Vy4+ cells. The results (Figure 11) demonstrate that mycolates from C. mast but not from A / , tb stimulate IL-17A response in y8 T cells.EXAMPLE 6

[0062] This example demonstrates that trehalose monocorynomycolate (TMCM) induces IL- 17A production in y8 T cells.

[0063] To identify the specific corynomycolates with IL-17A stimulatory capacity, a representative naked (unconjugated) corynomycolate (corMyc (al8: l / myl8:l)), a trehalose monocorynomycolate (TMCM (al8: l / myl8: 1)) and a trehalose dicorynomycolate (TDCM (al 8 : 1 / my 18 : 1 , al 8 : 1 / my 18: 1)) were synthesized (AVANTI POLAR LIPIDS) based on the most abundant corynomycolate species detected in the crude lipid extract from C. mast (Figure 12A). The IL-17A stimulatory capacity of these compounds was again evaluated in DC and 78 T cell cocultures. While TMCM(al8: l / myl8:l) (a.k.a., TMCM(36:2)) potently stimulated IL-17A production, neither corMyc (al 8: 1 / my 18: 1) (a.k.a., corMyc(36:2)) nor TDCM(al8: 1 / my 18: 1, al8: l / myl8: l) (a.k.a., TDCM(72:4)), showed significant IL-17A inducing effects (Figure 12B). Taken together, these results identify TMCM as the primary C. w?c7.s / -derived stimulant that activated Vy4+78 T cells.EXAMPLE 7

[0064] This example demonstrates that recognition of TMCM(36:2) by TLR2 and Mincle contributes to induction of IL-17A from yb T cells.

[0065] After identifying the specific stimulant, the synthetic TMCM was used to dissect the downstream signaling events leading to IL- 17 production. DCs are essential for the induction of IL-17A from yb T cells after stimulation with TMCM(36:2) (Figure 13A). Thus, how DCs may sense and respond to TMCM(36:2) was investigated. First, the cytokine secretion profile of DCs stimulated with TMCM(36:2) was investigated, which showed that TMCM(36:2) induces the production of IL-la, TNF-a, MCP-1, IL-ip and IL-6 by DCs (Figure 14A) while IL-23, IFN-P, IL-10, IL-27, !L-12p70, GM-CSF were below the limit of detection (data not shown).

[0066] Since TLR2 and Mincle can recognize glycolipids from Corynebacteria, whether TLR2 and / or Mincle on DCs was required for TMCM(36:2) induction of IL-17. TLR2 deficient DCs exhibited a marked reduction (p<0.05) in the production of IL-la, TNF- a, IL-lb, and IL-6 (Figure 14B) was investigated. Anti -Mincle pretreated DCs displayed reduced IL-l and TNF- a in response to TMCM (36:2) stimulation, compared to IgG-pretreated DCs (Figure 14C). In line with this, yb T cells cocultured with TMCM(36:2)-pulsed TLR2-deficient or Mincle-blocked DCs produced less IL-17A (Figure 14D-E), suggesting that recognition of TMCM(36:2) by TLR2 and / or Mincle on DCs influences IL-17A production by yb T cells though cytokines IL-1, IL-6 and / or TNF-a. Blockade of IL-1R signaling almost completely abolished IL-17A production in response to synthetic TMCM(36:2) (Figure 14F).

[0067] To investigate the roles of IL-6 and TNF-a in IL-17A production, cocultures were treated with individual blocking antibodies or a combination of both. Blocking IL-6 or TNF-a individually did not reduce IL- 17 production; however, simultaneous blockade of IL-6 and TNF-a led to a partial decrease in IL-17 production (Figures 13B and 14G). These findings demonstrate that TMCM(36:2) activates the innate immune system at least in part through TLR2 and / or Mincle receptors, triggering an IL- 1 -dependent pathway similar to wild-type C. mast stimulation of IL- 17 production from yb T cells.EXAMPLE 8

[0068] This example demonstrates that TCR signaling is critical for IL-1R1 upregulation to support IL-17A response of y8 T cells responding to TMCM.

[0069] Even though TMCM-mediated IL-17 responses from 70 T cells appeared to rely on innate signaling in DCs, and the conserved 78 TCR repertoire in vivo, it remained uncertain whether TCR signaling was required for 78 T cell IL-17 production in response to TMCM. To address this, ‘knock-in CD3(^ switch’ mice (CD3(^ 6Y / 6Y(6F(induced))) were used, in which tyrosine (Y) in immunoreceptor tyrosine-based activation motif (IT AM) of CD3(^ proteins was mutated to phenylalanine (F) after tamoxifen induction (= mutant CD3 mice), impairing y8 TCR signaling (Figures 15A and 15B).

[0070] Eight days after tamoxifen induction, y8 T cells were isolated from peripheral LNs and analyzed for cell surface expression of CD44+ CD27 (marker for IL-17A producing y8 T cells) and IL-17 production after PMA / Ionomycin stimulation. By both criteria, y8 T cells with IL-17A- producing phenotype in CD3 mutant mice were unchanged (Figures 16A and 16B), indicating that y8 T cell development and effector function remained unaffected. Importantly, IL-1R1 expression by CD44+ CD27- y8 T cells was reduced in CD3 mutant mice (Figure 5C) and their IL-1R signaling after stimulation with IL-ip in vitro was decreased, as indicated by pErk and pP38 levels (Figures 16D and 16E). Of note, only IL-1R1 expression was affected, whereas IL- 7Ra expression remained normal (Figure 15C). In line with their decreased IL-1R signaling, mutant CD3 y8 T cells exhibited dramatically decreased proliferation and IL-17A production in response to synthetic TMCM-pulsed DCs (36:2) (Figure 16F). In view of the nonredundant role of 1L-1R signaling in y8 T cells for IL-17A production (Figure 16F), TCR signals may be essential for IL-17A production of Vy4 T cells in response to TMCM, at least in part by controlling their intrinsic IL-1R1 expression.

[0071] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0072] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are tobe construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms ( / .<?., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0073] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A composition comprising, as an active principal, a mycolate, or salt, conjugate, or hydrate thereof, and a pharmaceutically-acceptable carrier.

2. The composition according to claim 1, wherein the mycolate comprises an acyl tail comprising between 14 and 22 carbons.

3. The composition according to claim 1 or 2, wherein the mycolate is derived from Corynebacterium mastitidis (C. mast).

4. The composition according to any one of claims 1-3, wherein the mycolate is present as an underivatized carboxyl.

5. The composition according to claim 4, wherein the mycolate has the following structure:

6. The composition according to any one of claims 1-3, wherein the mycolate is conjugated to a sugar moiety.

7. The composition according to claim 6, wherein the conjugated mycolate has a structure selected from:

8. The composition according to claim 6, wherein the sugar moiety comprises trehalose.

9. The composition according to any one of claims 1-8, wherein the composition comprises a liposome.

10. The composition according to claim 9, wherein the liposome comprises one or more of phosphatidylglycerol, cardiolipin, phosphatidylinositol, and free fatty acids.

11. The composition according to any one of claims 1-10, which comprises at least one additional active principal.

12. The composition according to claim 11, wherein an additional active principal comprises an antibiotic drug substance.

13. The composition according to any one of claims 1-12, which is a cream, gel, paste, solution, or suspension.

14. The composition according to any one of claims 1-13, which is formulated for topical administration to the eye.

15. The composition according to claim 14, which is formulated for topical administration to the eye as drops.

16. The composition according to any one of claims 1-13, which is formulated for topical administration to barrier tissue.

17. The composition according to claim 16, wherein the barrier tissue comprises cornea, nasal epithelium, oral epithelium, rectal epithelium, skin, or vaginal epithelium.

18. The composition according to any one of claims 1-13, which is formulated for topical administration to hair, nails, or teeth.

19. A method for treatment or prophylaxis of an ocular infection, the method comprising administering the composition according to claim 14 or 15 to an eye of a subject suffering from or at risk of a pathogenic infection of the eye.

20. The composition according to claim 14 or 15 for use in treatment or prophylaxis of a pathogenic infection of the eye in a subject.

21. The method according to claim 19 or the composition-for-use according to claim 20, wherein the composition is administered topically to the eye.

22. A method for treatment or prophylaxis of barrier tissue infection, the method comprising administering the composition according to claim 16 or 17 to the barrier tissue of a subject suffering from or at risk of a pathogenic infection of the barrier tissue.

23. The composition according to claim 16 or 17 for use in treatment or prophylaxis of pathogenic barrier tissue infection in a subject.

24. The method according to claim 22 or the composition-for-use according to claim 23, wherein the barrier tissue comprises cornea, nasal epithelium, oral epithelium, rectal epithelium, skin, or vaginal epithelium.

25. The method or the composition-for-use according to any one of claims 22-24, wherein the composition is administered topically to the barrier tissue.

26. The method or the composition-for-use according to any one of claims 19-25, wherein the pathogenic infection comprises a pathogenic bacterium, fungus, or both.

27. The method or the composition-for-use according to claim 26, wherein the pathogenic infection comprises Pseudomonas ceruginosa.

28. A method for stimulating an immune response in a subject, the method comprising administering the composition according to any one of claims 1-18 to a subject in an amount sufficient to stimulate an immune response in the subject.

29. The composition according to any one of claims 1-18 for use in stimulating an immune response in a subject.

30. The method according to claim 28 or the composition-for-use according to claim 29, wherein the immune response comprises IL-17A induction.

31. The method or the composition-for-use according to any one of claims 19-30, wherein the composition is administered in a plurality of doses.

32. The method or the composition-for-use according to any one of claims 19-31, wherein the subject is human.

Images