WO2025240443A1 - Candida for use in prevention or mitigation of cutaneous viral infections - Google Patents

Abstract

The present invention relates to yeast compositions and methods to prevent or mitigate cutaneous viral infections.

Description

PREVENTION OR MITIGATION OF CUTANEOUS VIRAL INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing date of U.S. Provisional Patent No. 63/647,218, filed on May 14, 2024, which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under AI152011 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to yeast compositions and methods for preventing, treating, or mitigating cutaneous viral infections.

BACKGROUND

In higher vertebrates, skin is the largest body organ and serves as an important environmental interface, providing a protective envelope that is crucial for homeostasis. Many infectious disorders, such as viral infection, have skin manifestations. Conversely, patients with skin disorders, such as atopic dermatitis, often have enhanced susceptibility to viral infections of the skin. Cutaneous or skin viral infections are a group of conditions caused by various viruses that result in skin manifestations, such as rashes, blisters, or lesions. Such infections can be localized or systemic and may be contagious, depending on the causative virus. For example, global incidence of human papillomavirus (HPV) infection varies depending on geographic region, but it is estimated that up to 45% of men and 44% of women are infected with genital HPV [1], HPV is categorized into two groups with regard to cancer causing potential: low-risk and high-risk [2], Low-risk HPV subtypes commonly associate with anogenital and cutaneous warts [1], While there are no HPV-specific treatments for cutaneous warts, anecdotal studies and a recent clinical trial (NCT05383625) have suggested that the HPV vaccine can be used for resolution of cutaneous warts [3], Despite this, the standard of care for warts as well as other skin viral infections is typically based on destruction of infected tissue through topical medication, cauterization, and/or freezing, demonstrating a critical need for additional research [4], Similarly, herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) are also highly prevalent and the rates of HSV-1/2 infection in some locations can reach 70 to 80% (WHO, who.int/news-room/fact-sheets/detail/herpes-simplex-virus). There is no treatment other than broad spectrum antivirals. Similarly, there are no specific treatments for shingles (also known as herpes zoster), a disease caused by the varicella zoster virus (VZV) and characterized by a painful skin rash with blisters in a localized area, other than broad spectrum antivirals. There is a need for compositions and methods to prevent or mitigate cutaneous viral infections.

SUMMARY

This disclosure addresses the need mentioned above in a number of aspects.

In one aspect, the disclosure provides a method for treating or limiting a skin infection with a virus in a subject in need thereof. The method comprises administering to the subject an effective amount of a pharmaceutical composition that comprises at least one extract of Candida albicans, Candida parapsilosis, or Candida tropicalis.

The virus can be any other virus in which the infection results in skin manifestations. In some embodiments, the virus is selected from the group consisting of herpes simplex virus (HSV) -1 or -2, monkeypox virus, SARS-CoV-2, measles virus, rubella virus, varicella zoster virus, parvovirus, herpesvirus 6 and 7 virus, orf virus, variola minor/major (more commonly known as smallpox), enterovirus, human papillomavirus (HPV), molloscum contagiosum virus and vaccinia virus (VV). In some embodiments, the VV comprises a component from a microbial organism selected from the group consisting of influenza virus, varicella zoster virus, human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), polio virus, variola virus, rabies virus, rotavirus, human papillomavirus, Ebola virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, lyssavirus, measles virus, mumps virus, and Rubella virus. In some embodiments, the pharmaceutical composition is administered to the subject before, after, or concurrent with the VV.

In some embodiments, the extract comprises a filtered extract of a Candida albicans, such as that deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126019 or PTA-126020. In some embodiments, the extract comprises filtered extracts of both ATCC PTA-126019 and ATCC PTA-126020, such as Candin®. In some embodiments, the extract comprises a filtered extract of one or more Candida albicans, Candida parapsilosis, and Candida tropicalis, such as one or more of ATCC PTA-126019, ATCC PTA- 126020, ATCC9968, and ATCC22019. In some embodiments, when an extract of Candida albicans is administered, the virus is not human papillomavirus or molloscum contagiosum virus.

In some embodiments, the pharmaceutical composition is administered to the subject via injection or topical application. In one embodiment, the subject is a mammal. In one embodiment, the subject is a human. The details of one or more embodiments of the invention are set forth in the description below. Other features, objectives, and advantages of the invention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are diagrams showing that Candin® treatment decreases viral spread after infection of keratinocytes with VV or HSV-1 and that keratinocytes treated with Candin® are less susceptible to the two different viruses. FIG. 1A shows keratinocytes infected with VV and then stained with crystal violet to visualize viral plaque formation and cytopathic effect. FIG. IB depicts the dose responsive reduction observed in cytopathic effect. FIG. 1C shows keratinocytes infected with a fluorescent reporter HSV-1 and then visualized with fluorescent microscopy to enumerate viral plaque formation and cytopathic effect. FIG. ID shows graphs of a dose responsive reduction observed in cytopathic effect.

FIGS. 2A-2C are diagrams showing different impacts on cytopathic effect of VV on keratinocytes by three different yeasts: C. albicans, S. cerevisiae, and AT. sympodialis. FIG. 2 A shows keratinocytes infected with W and stained with crystal violet to visualize viral plaque formation and cytopathic effect. FIG. 2B is a graph of dose responsive reduction observed in cytopathic effects by the yeasts. FIG. 2C is a graph of dose responsive reductions observed in number of plaques per well by the yeast.

FIG. 3 is a diagram showing that neither Candin® nor live yeast C. albicans impact viability of keratinocytes. Keratinocytes were differentiated for 24 hours in the presence of Candin® or live replicating yeast. Viability was assessed via an LDH assay, n = 2-5 experiments. Data are shown as mean ± SEM. CFU, colony forming units.

FIG. 4 illustrates that Candida exposure diminishes keratinocyte (KC) viral susceptibility with varying efficiency. Each experiment is encoded by a unique symbol. Columns represent mean and error bars are standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0 001, **** p < 0.0001.

FIG. 5 shows that treatment with in-house generated Candida extracts diminishes KC viral susceptibility (CP: C. parapsilosis CA: C. albicans,' CT: C. tropicalis). Columns represent mean and error bars are standard error of the mean. # p < 0.05, ## p < 0.01, ### p < 0.001.

DETAILED DESCRIPTION OF THE INVENTION

This application relates to compositions and methods to mitigate cutaneous viral infections. Certain aspects of this application are based, at least in part, on unexpected findings of a previously unknown consequence of the interaction between a common member of the cutaneous my cobiome (Candida albicans, Candida parapsilosis, or Candida tropicalis) and the human skin epithelium.

It is hypothesized that Candida species stimulate pattern recognition receptors (PRRs) present on or in keratinocytes, thereby activating pathways that additionally promote an antiviral response. Keratinocytes express a host of PRRs such as nucleotide-binding oligomerization (NOD)-like receptors, retinoic acid-inducible gene-I (RIG-I)-like receptors, toll-like receptors (TLRs), and C-type Lectin Receptors (CLRs) [11], Given that yeast are likely too large to be internalized by keratinocytes and that keratinocytes have limited phagocytic capability, it is most likely that the PRRs responsible for findings disclosed herein are cell surface TLRs and/or CLRs [11], Notably, keratinocytes express receptors previously demonstrated to be important for yeast recognition including TLR2 and dectin- 1 (a CLR) [11], These receptors are thought to respond to Candida lipopeptides or P-glucans, and activation of these receptors leads to production of reactive oxygen species and antimicrobial peptides, which could limit viral infection [11], Ongoing studies are focused on using CRISPR/Cas9 gene editing to knockout these receptors and examine whether diminished viral susceptibility resulting from Candida exposure is abrogated.

Taken together, the results disclosed herein highlight the importance of the interaction between skin resident yeast species and host epidermal cells. It is possible that individuals with altered yeast species present on their skin may be predisposed to cutaneous viral infections (e.g., warts caused by HPV). Research is limited regarding changes in fungal composition on healthy versus diseased skin, though some studies have implicated Malassezia species as contributing to diseases such as atopic dermatitis [12], Additionally, the inventors have demonstrated that keratinocytes treated with Candin® are more resistant to viral infections. These findings argue for the importance of the downstream signaling events that lead to the antiviral state observed.

Accordingly, the presently disclosed compositions (including topical formulations) and methods can be used to treat viral skin diseases, which may include, for example, those caused by poxviruses, herpes simplex (e.g., cold sores), fifth disease, roseola, zoonotic diseases, chickenpox, , measles, melioidosis, and shingles. One skilled in the art will appreciate that these and related embodiments of the present disclosure may be used to limit or treat skin conditions, disorders, complications, diseases, infections, or otherwise, in addition to those listed herein. Extracts and Pharmaceutical Compositions

In one aspect, this application discloses extracts and/or secreted antigens from yeast (such as Candida albicans, Candida parapsilosis, or Candida tropicalis) and related pharmaceutical compositions comprising the extracts and/or secreted antigens. Any strains or isolates of these Candida species can be used. Examples include a random contaminant from the environment, strains known in the art, or those available from a depository (e.g., ATCC). Such extracts and/or secreted antigens can be prepared according to the methods described in US11116808B2, US11963991B2, and W02020033329A1, all of which are incorporated by reference in their entireties.

A pharmaceutical composition of the present disclosure comprising at least one filtered extract of Candida albicans, Candida parapsilosis, or Candida tropicalis and/or secreted antigens can be prepared by a series of steps comprising growing one or more strains of Candida albicans, Candida parapsilosis, or Candida tropicalis separately, pooling cultures of one or more strains of Candida albicans, Candida parapsilosis, or Candida tropicalis and dialyzing the mixture, heating the mixture, lyophilizing the heated dialyzed material, producing a dry powder, extracting the dry powder, filtering the extract, and producing a master lot filtered solution. In an aspect, a pharmaceutical composition of the present disclosure can be produced by the protocol as outlined in Example 1 of US 11116808B2, the content of which is incorporated by reference in its entirety.

In an aspect, a pharmaceutical composition of the present disclosure may be prepared using one or more strains of Candida albicans (or Candida parapsilosis, or Candida tropicalis), such as using two strains, three strains, four strains, five strains, six strains, seven strains, eight strains, nine strains, or ten strains. In one aspect, a pharmaceutical composition of the present disclosure is prepared using two strains of Candida albicans, Candida parapsilosis, or Candida tropicalis. In an aspect, a pharmaceutical composition of the present disclosure is prepared using strains of Candida albicans provided in Hasenclever HF and Mitchell WO, “Antigenic Studies c Candida, ” Journal of Bacteriology 1961; 82:578-581. In an aspect, a pharmaceutical composition of the present disclosure is prepared using two strains of Candida albicans, where a representative sample of a first strain has been deposited with the American Type Culture Collection (ATCC; located at 10801 University Boulevard, Manassas, Va. 20110) under ATCC Accession No. PTA-126019, and a representative sample of a second strain has been deposited with the ATCC under ATCC Accession No. PTA-126020. In an aspect, a representative sample of a first strain has been deposited with the ATCC under ATCC Accession No. ATCC-10231. In one embodiment, a pharmaceutical composition of the present disclosure is prepared using strains of Candida tropicalis deposited under ATCC Accession No. ATCC9968. In one embodiment, a pharmaceutical composition of the present disclosure is prepared using strains of Candida parapsilosis deposited under ATCC Accession No. ATCC 22019.

In an aspect, the present disclosure provides for a pharmaceutical composition comprising at least 80% mannose, such as from 80% mannose to 85% mannose, from 85% mannose to 90% or mannose from 80% to 90% mannose. In an aspect, the present disclosure provides for a resulting pharmaceutical composition comprising at least 8% glucose, such as from 8% glucose to 10% glucose, from 10% glucose to 12% glucose, or from 8% glucose to 12% glucose. In an aspect, the present disclosure provides for a resulting pharmaceutical composition comprising at least 1% galactose, such as from 1% galactose to 2.5% galactose, from 2.5% galactose to 5% galactose, or from 1% to 5% galactose.

In an aspect, the present disclosure provides for a pharmaceutical composition that is prepared by diluting a filtrate, such as a master lot filtered solution obtained according to Example 1 ofUS11116808B2, For instance, 1.7 mL of this master lot filtered solution is diluted with 998.3 mL of a diluent, which consists of 5.0 g/L NaCl, 2.5 g/L NaHCCh, 4.5 mL/L phenol, 1.2 mL/L 20% solution human serum albumin, 0.8 mL/L polysorbate 80.

In an aspect, the present disclosure provides for a pharmaceutical composition comprising antigens having a molecular weight of about 167 kilodaltons, such as from 157 kilodaltons to 177 kilodaltons. In an aspect, a molecular weight is measured by a method using a superpose 12 column calibrated with dextran standards having molecular weights between 1 kilodalton and 512 kilodalton.

The therapeutic agents can be present in a stock composition optionally containing other components, including, for example, a storage solution, such as a suitable buffer, e.g., a physiological buffer. The stock composition can be diluted as needed. In a preferred embodiment, the composition is a pharmaceutical composition and the other component is a pharmaceutically acceptable carrier, such as are described in Remington's Pharmaceutical Sciences (1980) 16th editions, Osol, ed., 1980.

A pharmaceutically acceptable carrier suitable for use in the invention is non-toxic to cells, tissues, or subjects at the dosages employed, and can include a buffer (such as a phosphate buffer, citrate buffer, and buffers made from other organic acids), an antioxidant (e.g., ascorbic acid), a low-molecular weight (less than about 10 residues) peptide, a polypeptide (such as serum albumin, gelatin, and an immunoglobulin), a hydrophilic polymer (such as polyvinylpyrrolidone), an amino acid (such as glycine, glutamine, asparagine, arginine, and/or lysine), a monosaccharide, a disaccharide, and/or other carbohydrates (including glucose, mannose, and dextrins), a chelating agent (e.g., ethylenediaminetetratacetic acid [EDTA]), a sugar alcohol (such as mannitol and sorbitol), a salt-forming counterion (e.g., sodium), and/or an anionic surfactant (such as TWEEN, PLURONICS, and PEG). In one embodiment, the pharmaceutically acceptable carrier is an aqueous pH-buffered solution.

In some embodiments of the disclosure, aqueous suspensions of a therapeutic agent contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkyl oxide (e.g. ethylene oxide, propylene oxide) with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.

In some embodiments of the disclosure, the pharmaceutical composition of a therapeutic agent is in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.

Topical Formulations

Pharmaceutical compositions suitable for topical administration are useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the particle described herein include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active particle suspended or dissolved in a carrier with suitable emulsifying agents. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyl dodecanol, benzyl alcohol and water.

As noted above, some embodiments described herein relate to topical formulations of the described compositions, which formulations comprise the extract in a pharmaceutically acceptable carrier, excipient or diluent and in a therapeutic amount, as disclosed herein, when administered topically to an animal, preferably a mammal, and most preferably a human.

Topical administration of the agents described herein, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of topical administration of agents for serving similar utilities. Topical application or administration of a composition means, in preferred embodiments, directly contacting the composition (e.g., a topical formulation) with skin of the subject undergoing treatment, which may be at one or more localized or widely distributed skin sites and which may generally refer to contacting the topical formulation with intact stratum comeum or epidermis but need not be so limited; for instance, certain embodiments contemplate as a topical application the administration of a topical formulation described herein to injured, abraded or damaged skin, or skin of a subject, such that contact of the topical formulation may take place not only with stratum corneum or epidermis but also with skin granular cell, spinous cell, and/or basal cell layers, and/or with dermal or underlying tissues, for example, as may accompany certain types of wound repair or wound healing or other skin tissue remodeling.

The topical formulations (e.g., cosmeceutical and pharmaceutical compositions) of the disclosure may be prepared by combining the agents described herein with an appropriate pharmaceutically acceptable carrier, diluent or excipient for use in a topical formulation preparation, and may be formulated into preparations in solid, semi-solid, gel, cream, colloid, suspension or liquid or other topically applied forms, such as powders, granules, ointments, solutions, washes, gels, pastes, plasters, paints, bioadhesives, microsphere suspensions, and aerosol sprays. Pharmaceutical compositions of the disclosure are formulated so as to allow the active ingredients contained therein to be bioavailable upon topical administration of the composition to skin of a subject, such as a mammal, including a human, and in certain preferred embodiments a human patient having a skin condition that results from infection. The topical formulations described herein deliver a therapeutically effective amount of the agent described herein to skin fibroblasts and keratinocytes. Preferred formulations therefore exhibit ready permeability into the skin, as can be determined according to any of a number of established methodologies known to the art for testing the skin permeability of a drug composition (see, e.g., Wagner et al., 2002 J. Invest. Dermatol. 118:540, and references cited therein; Bronaugh et al., 1985 ./. Pharm. Sci. 74:64; Bosman et al., 1998.7 Pharm. Biomed. Anal. 17:493-499; Bosman et al., 1996 J. Pharm Biomed Anal. 1996 14: 1015-23; Bonferoni et al., 1999 Pharm Dev TechnoL 4:45-53; Frantz, Instrumentation and methodology for in vitro skin diffusion cells in methodology for skin absorption. In: Methods for Skin Absorption (Kemppainen & Reifenrath, Eds), CRC Press, Florida, 1990, pp. 35-59; Tojo, Design and calibration of in vitro permeation apparatus. In: Transdermal Controlled Systemic Medications (Chien Y W, Ed), Marcel Dekker, New York, 1987, 127-158; Barry, Methods for studying percutaneous absorption. In: Dermatological Formulations: Percutaneous absorption, Marcel Dekker, New York, 1983, 234-295).

Compositions that will be administered to the skin of a subject or patient may in certain embodiments take the form of one or more dosage units, where for example, a liquid-filled capsule or ampule may contain a single dosage unit, and a container of a topical formulation as described herein in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain a therapeutically effective amount of an agent of the disclosure, or a pharmaceutically acceptable salt thereof, for treatment of a skin condition that results from infection in skin of a subject, in accordance with the present teachings.

As noted above, the present topical formulations may take any of a wide variety of forms, and include, for example, creams, lotions, solutions, sprays, gels, ointments, pastes or the like, and/or may be prepared so as to contain liposomes, micelles, and/or microspheres. See, e.g., U.S. Pat. No. 7,205,003. For instance, creams, as is well known in the arts of pharmaceutical and cosmeceutical formulation, are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are water-washable, and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also called the “internal” phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant.

Lotions, which are preferred for delivery of cosmetic agents, are preparations to be applied to the skin surface without friction, and are typically liquid or semi-liquid preparations in which solid particles, including the active agent, are present in a water or alcohol base. Lotions are usually suspensions of solids, and preferably comprise a liquid oily emulsion of the oil-in- water type. Lotions are preferred formulations herein for treating large body areas, because of the ease of applying a more fluid composition. It is generally preferred that the insoluble matter in a lotion be finely divided. Lotions will typically contain suspending agents to produce better dispersions as well as compounds useful for localizing and holding the active agent in contact with the skin, e.g., methylcellulose, sodium carboxymethyl-cellulose, or the like.

Solutions are homogeneous mixtures prepared by dissolving one or more chemical substances (solutes) in a liquid such that the molecules of the dissolved substance are dispersed among those of the solvent. The solution may contain other pharmaceutically acceptable and/or cosmeceutically acceptable chemicals to buffer, stabilize or preserve the solute. Common examples of solvents used in preparing solutions are ethanol, water, propylene glycol or any other pharmaceutically acceptable and/or cosmeceutically acceptable vehicles.

Gels are semisolid, suspension-type systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol, and, optionally, an oil. Preferred “organic macromolecules,” z.e., gelling agents, may be chemically crosslinked polymers such as crosslinked acrylic acid polymers, for instance, the “carbomer” family of polymers, e.g., carboxypolyalkylenes, that may be obtained commercially under the Carbopol® trademark. Also preferred in certain embodiments may be hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers and polyvinylalcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methyl cellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing or stirring, or combinations thereof.

Ointments are semisolid preparations that are typically based on petrolatum or other petroleum derivatives. The specific ointment base to be used, as will be appreciated by those skilled in the art, is one that will provide for a number of desirable characteristics, e.g., emolliency or the like. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating, and nonsensitizing. As explained in Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), at pages 1399 1404, ointment bases may be grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin, and hydrophilic petrolatum. Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin, and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight (see, e.g., Remington, Id.).

Pastes are semisolid dosage forms in which the active agent is suspended in a suitable base. Depending on the nature of the base, pastes are divided between fatty pastes or those made from single-phase aqueous gels. The base in a fatty paste is generally petrolatum or hydrophilic petrolatum or the like. The pastes made from single-phase aqueous gels generally incorporate carboxymethylcellulose or the like as a base.

Formulations may also be prepared with liposomes, micelles, and microspheres. Liposomes are microscopic vesicles having one (unilamellar) or a plurality (multilamellar) of lipid walls comprising a lipid bilayer, and, in the present context, may encapsulate and/or have adsorbed to their lipid membranous surfaces one or more components of the topical formulations described herein or certain carriers or excipients. Liposomal preparations herein include cationic (positively charged), anionic (negatively charged), and neutral preparations. Cationic liposomes are readily available. For example, N[l-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the tradename Lipofectin® (GIBCO BRL, Grand Island, N.Y.). Similarly, anionic and neutral liposomes are readily available as well, e.g., from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with DOTMA in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

Micelles are known in the art as comprised of surfactant molecules arranged so that their polar headgroups form an outer spherical shell, while the hydrophobic, hydrocarbon chains are oriented towards the center of the sphere, forming a core. Micelles form in an aqueous solution containing surfactant at a high enough concentration so that micelles naturally result. Surfactants useful for forming micelles include, but are not limited to, potassium laurate, sodium octane sulfonate, sodium decane sulfonate, sodium dodecane sulfonate, sodium lauryl sulfate, docusate sodium, decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, tetradecyltrimethyl-ammonium chloride, dodecylammonium chloride, polyoxyl-8 dodecyl ether, polyoxyl-12 dodecyl ether, nonoxynol 10, and nonoxynol 30.

Microspheres, similarly, may be incorporated into the presently described topical formulations. Like liposomes and micelles, microspheres essentially encapsulate one or more components of the present formulations. They are generally, but not necessarily, formed from lipids, preferably charged lipids such as phospholipids. Preparation of lipidic microspheres is well known in the art.

Various additives, as known to those skilled in the art, may also be included in the topical formulations. For example, solvents, including relatively small amounts of alcohol, may be used to solubilize certain formulation components. It may be desirable, for certain topical formulations or in cases of particularly severe skin conditions to include in the topical formulation an added skin permeation enhancer in the formulation. Examples of suitable enhancers include, but are not limited to, ethers such as diethylene glycol monoethyl ether (available commercially as Transcutol®) and diethylene glycol monomethyl ether; surfactants such as sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride, Poloxamer® (231, 182, 184), Tween® (20, 40, 60, 80), and lecithin (U.S. Pat. No. 4,783,450); alcohols such as ethanol, propanol, octanol, benzyl alcohol, and the like; polyethylene glycol and esters thereof such as polyethylene glycol monolaurate (PEGML; see, e.g., U.S. Pat. No. 4,568,343); amides and other nitrogenous compounds such as urea, dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone, l-methyl-2- pyrrolidone, ethanolamine, diethanolamine, and triethanolamine; terpenes; alkanones; and organic acids, particularly citric acid and succinic acid. Azone® and sulfoxides such as DMSO and CioMSO may also be used, but are less preferred.

Most preferred skin permeation enhancers are those lipophilic co-enhancers typically referred to as “plasticizing” enhancers, z.e., enhancers that have a molecular weight in the range of about 150 to 1000 daltons, an aqueous solubility of less than about 1 wt %, preferably less than about 0.5 wt %, and most preferably less than about 0.2 wt %. The Hildebrand solubility parameter of plasticizing enhancers is in the range of about 2.5 to about 10, preferably in the range of about 5 to about 10. Preferred lipophilic enhancers are fatty esters, fatty alcohols, and fatty ethers. Examples of specific and most preferred fatty acid esters include methyl laurate, ethyl oleate, propylene glycol monolaurate, propylene glycerol dilaurate, glycerol monolaurate, glycerol monooleate, isopropyl n-decanoate, and octyldodecyl myristate. Fatty alcohols include, for example, stearyl alcohol and oleyl alcohol, while fatty ethers include compounds wherein a diol or triol, preferably a C2-C4 alkane diol or triol, are substituted with one or two fatty ether substituents. Additional skin permeation enhancers will be known to those of ordinary skill in the art of topical drug delivery, and/or are described in the relevant literature. See, c.g, Percutaneous Penetration Enhancers, eds. Smith et al. (CRC Press, 1995).

Various other additives may be included in the topical formulations according to certain embodiments of the present disclosure, in addition to those identified above. These include, but are not limited to, antioxidants, astringents, perfumes, preservatives, emollients, pigments, dyes, humectants, propellants, and sunscreen agents, as well as other classes of materials whose presence may be cosmetically, medicinally or otherwise desirable. Typical examples of optional additives for inclusion in the formulations of the disclosure are as follows: preservatives such as sorbate; solvents such as isopropanol and propylene glycol; astringents such as menthol and ethanol; emollients such as polyalkylene methyl glucosides; humectants such as glycerine; emulsifiers such as glycerol stearate, PEG- 100 stearate, polyglyceryl-3 hydroxylauryl ether, and polysorbate 60; sorbitol and other polyhydroxy alcohols such as polyethylene glycol; sunscreen agents such as octyl methoxyl cinnamate (available commercially as Parsol MCX) and butyl methoxy benzoylmethane (available under the tradename Parsol 1789); antioxidants such as ascorbic acid (vitamin C), a-tocopherol (Vitamin E), P-tocopherol, y-tocopherol, 5-tocopherol, 8-tocopherol, y-tocopherol, y-tocopherol, r|-tocopherol, and retinol (vitamin A); essential oils, ceramides, essential fatty acids, mineral oils, vegetable oils (e.g., soy bean oil, palm oil, liquid fraction of shea butter, sunflower oil), animal oils (e.g., perhydrosqualene), synthetic oils, silicone oils or waxes (e.g., cyclomethicone and dimethicone), fluorinated oils (generally perfluoropoly ethers), fatty alcohols (e.g., cetyl alcohol), and waxes (c.g, beeswax, carnauba wax, and paraffin wax); skin-feel modifiers; and thickeners and structurants such as swelling clays and crosslinked carboxypolyalkylenes that may be obtained commercially under the Carbopol® trademark.

Other additives include beneficial agents such as those materials that condition the skin (particularly, the upper layers of the skin in the stratum comeum) and keep it soft by retarding the decrease of its water content and/or protect the skin. Such conditioners and moisturizing agents include, by way of example, pyrrolidine carboxylic acid and amino acids; organic antimicrobial agents such as 2,4,4'-trichloro-2-hydroxy diphenyl ether (triclosan) and benzoic acid; anti-inflammatory agents such as acetylsalicylic acid and glycyrrhetinic acid; anti- seborrhoeic agents such as retinoic acid; vasodilators such as nicotinic acid; inhibitors of melanogenesis such as kojic acid; and mixtures thereof. Other advantageously included cosmeceutically active agents may be present, for example, a-hydroxyacids, a-ketoacids, polymeric hydroxyacids, moisturizers, collagen, marine extracts, and antioxidants such as ascorbic acid (vitamin C), a-tocopherol (Vitamin E) or other tocopherols such as those described above, and retinol (vitamin A), and/or cosmetically acceptable salts, esters, amides, or other derivatives thereof. Additional cosmetic agents include those that are capable of improving oxygen supply in skin tissue, as described, for example, in WO 94/00098 and WO 94/00109. Sunscreens may also be included.

Other embodiments may include a variety of non-carcinogenic, non-irritating healing materials that facilitate treatment with the formulations of the disclosure. Such healing materials may include nutrients, minerals, vitamins, electrolytes, enzymes, herbs, plant extracts, glandular or animal extracts, or safe therapeutic agents that may be added to the formulation to facilitate the healing of dermal disorders. The amounts of these various additives are those conventionally used in the cosmetics field, and range, for example, from about 0.01% to about 20% of the total weight of the topical formulation.

The formulations of the disclosure may also include conventional additives such as opacifiers, fragrance, colorant, gelling agents, thickening agents, stabilizers, surfactants, and the like. Other agents may also be added, such as antimicrobial agents, to prevent spoilage upon storage, z.e., to inhibit growth of microbes such as viruses, bacteria, yeasts, or/and molds. Suitable antimicrobial agents are typically selected from methyl and propyl esters of p- hydroxybenzoic acid (e.g., methyl and propyl paraben), sodium benzoate, sorbic acid, imidurea, and combinations thereof. The formulations may also contain irritation-mitigating additives to minimize or eliminate the possibility of skin irritation or skin damage resulting from the chemical entity to be administered, or other components of the composition. Suitable irritationmitigating additives include, for example: a-tocopherol; monoamine oxidase inhibitors, particularly phenyl alcohols such as 2-phenyl-l-ethanol; glycerin; salicylates; ascorbates; ionophores such as monensin; amphiphilic amines; animonium chloride; N-acetylcysteine; capsaicin; and chioroquine. The irritation-mitigating additive, if present, may be incorporated into the topical formulation at a concentration effective to mitigate irritation or skin damage, typically representing not more than about 20 wt %, more typically not more than about 5 wt %, of the formulation. The topical formulations may also contain, in addition to extract(s) described herein, a therapeutically effective amount of one or more additional pharmacologically active agents suitable for topical administration. Such agents may include an asymmetrical lamellar aggregate consisting of phospholipids and oxygen-loaded fluorocarbon or a fluorocarbon compound mixture, which are capable of improving oxygen supply in skin tissue, as described, for example, in International Patent Publication Nos. WO 94/00098 and WO 94/00109.

A pharmacological acceptable carrier may also be incorporated in the topical formulation of certain present embodiments and may be any carrier conventionally used in the art. Examples include water, lower alcohols, higher alcohols, polyhydric alcohols, monosaccharides, disaccharides, polysaccharides, hydrocarbon oils, fats and oils, waxes, fatty acids, silicone oils, nonionic surfactants, ionic surfactants, silicone surfactants, and water-based mixtures and emulsion-based mixtures of such carriers.

Embodiments of the present disclosure may be used cosmetically, pharmaceutically, or both at the same time. Cosmetic and pharmaceutical applications may include such products as aerosols, baby products, bath oils, bubble baths, cleansers, color cosmetic products, conditioners, concealers, creams, deodorants, disinfectants, drops, eye and facial makeup, fingernail polish, foundation, gels, lip balm, lip gloss, lipstick, masks, milks, moisturizing creams, night cream, ointments, oils, perfumes, patches (including transdermal patches), powders, shampoos, shaving gels or lotions, skin benefit creams and lotions, soaps, sponges, sprays, toners, tonics, wipes, and the like. One skilled in the art will appreciate that embodiments of the present disclosure are not limited to the examples provided herein.

Topical formulation embodiments of the present disclosure may be applied regularly to whatever skin area requires treatment with the frequency and in the amount necessary to achieve the desired results. The frequency of treatment depends on the nature of the skin condition (e.g., a skin condition that results from viral infection in skin), the degree of damage or deterioration of the skin, the responsiveness of the user's skin, the strength of the active ingredients (e.g., the extract described herein and optionally one or more additional pharmaceutically or cosmeceutically active ingredients) in the particular embodiment, the effectiveness of the vehicle used to deliver the active ingredients into the appropriate layer of the skin, the ease with which the formula is removed by physical contact with clothing or its removal by sweat or other intrinsic or extrinsic fluids, and the convenience to the user's lifestyle.

Typical concentrations of biochemically active substances such as the novel treatment composition described herein can range, for example, from about 0.001-30% by weight based on the total weight of the composition, to about 0.01-5.0%, and more preferably to about 0.1- 2.0%. As one representative example, compositions of the present disclosure may be applied to the skin at a rate equal to from about 0.1 mg/cm² of skin to about 100 mg/cm² of skin. Representative examples of topical formulations include, but are not limited to, aerosols, alcohols, anhydrous bases (such as lipsticks and powders), aqueous solutions, creams, emulsions (including either water-in-oil or oil-in-water emulsions), fats, foams, gels, hydro-alcoholic solutions, liposomes, lotions, microemulsions, ointments, oils, organic solvents, polyols, polymers, powders, salts, silicone derivatives, and waxes. Topical formulations may include, for example, chelating agents, conditioning agents, emollients, excipients, humectants, protective agents, thickening agents, or UV absorbing agents. One skilled in the art will appreciate that formulations other than those listed may be used in embodiments of the present disclosure.

Chelating agents may be optionally included in topical formulations, and may be selected from any agent that is suitable for use in a cosmetic composition, and may include any natural or synthetic chemical which has the ability to bind divalent cationic metals such as Ca²⁺, Mn²⁺, or Mg²⁺. Examples of chelating agents include, but are not limited to EDTA, disodium EDTA, EGTA, citric acid, and dicarboxylic acids.

Conditioning agents may also be optionally included in topical formulations. Examples of skin conditioning agents include, but are not limited to, acetyl cysteine, N-acetyl dihydrosphingosine, acrylates/behenyl acrylate/dimethicone acrylate copolymer, adenosine, adenosine cyclic phosphate, adensosine phosphate, adenosine triphosphate, alanine, albumen, algae extract, allantoin and deriviatives, aloe barbadensis extracts, aluminum PCA, amyloglucosidase, arbutin, arginine, azulene, bromelain, buttermilk powder, butylene glycol, caffeine, calcium gluconate, capsaicin, carbocysteine, carnosine, beta-carotene, casein, catalase, cephalins, ceramides, chamomilla recutita (malricaria) flower extract, cholecalciferol, cholesteryl esters, coco-betaine, coenzyme A, corn starch modified, crystallins, cycloethoxymethicone, cysteine DNA, cytochrome C, darutoside, dextran sulfate, dimethicone copolyols, dimethylsilanol hyaluronate, DNA, elastin, elastin amino acids, epidermal growth factor, ergocalciferol, ergosterol, ethylhexyl PCA, fibronectin, folic acid, gelatin, gliadin, betaglucan, glucose, glycine, glycogen, glycolipids, glycoproteins, glycosaminoglycans, glycosphingolipids, horseradish peroxidase, hydrogenated proteins, hydrolyzed proteins, jojoba oil, keratin, keratin amino acids, and kinetin, lactoferrin, lanosterol, lauryl PCA, lecithin, linoleic acid, linolenic acid, lipase, lysine, lysozyme, malt extract, maltodextrin, melanin, methionine, mineral salts, niacin, niacinamide, oat amino acids, oryzanol, palmitoyl hydrolyzed proteins, pancreatin, papain, PEG, pepsin, phospholipids, phytosterols, placental enzymes, placental lipids, pyridoxal 5-phosphate, quercetin, resorcinol acetate, riboflavin, RNA, saccharomyces lysate extract, silk amino acids, sphingolipids, stearamidopropyl betaine, stearyl palmitate, tocopherol, tocopheryl acetate, tocopheryl linoleate, ubiquinone, vitis vinifera (grape) seed oil, wheat amino acids, xanthan gum, and zinc gluconate. Skin conditioning agents other than those listed above may be combined with a disclosed composition or preparation provided thereby, as can be readily appreciated by one skilled in the art.

Topical formulations may also optionally include one or more emollients, examples of which include, but are not limited to, acetylated lanolin, acetylated lanolin alcohol, acrylates/Cio- 30 alkyl acrylate crosspolymer, acrylates copolymer, alanine, algae extract, aloe barbadensis extract or gel, althea officinalis extract, aluminum starch octenyl succinate, aluminum stearate, apricot (primus armeniaca) kernel oil, arginine, arginine aspartate, arnica montana extract, ascorbic acid, ascorbyl palmitate, aspartic acid, avocado (persea gratissima) oil, barium sulfate, barrier sphingolipids, butyl alcohol, beeswax, behenyl alcohol, betasitosterol, BHT, birch (betula alba) bark extract, borage (borago officinalis) extract, 2-bromo- 2-nitropropane-l,3-diol, butcherbroom (ruscus aculeatus) extract, butylene glycol, calendula officinalis extract, calendula officinalis oil, candelilla (euphorbia cerifera) wax, canola oil, caprylic/capric triglyceride, cardamon (elettaria cardamomum) oil, carnauba (copernicia cerifera) wax, carrageenan (chondrus crispus). carrot (daucus carota sativa) oil, castor (ricinus communis) oil, ceramides, ceresin, ceteareth-5, ceteareth-12, ceteareth-20, cetearyl octanoate, ceteth-20, ceteth-24, cetyl acetate, cetyl octanoate, cetyl palmitate, chamomile (anthemis nobilis) oil, cholesterol, cholesterol esters, cholesteryl hydroxystearate, citric acid, clary (salvia sclarea) oil, cocoa (theobroma cacao) butter, coco-caprylate/caprate, coconut (cocos nucifera) oil, collagen, collagen amino acids, corn (zea mays) oil, fatty acids, decyl oleate, dextrin, diazolidinyl urea, dimethicone copolyol, dimethiconol, dioctyl adipate, dioctyl succinate, dipentaerythrityl hexacaprylate/hexacaprate, DMDM hydantoin, DNA, erythritol, ethoxydiglycol, ethyl linoleate, eucalyptus globulus oil, evening primrose (oenothera biennis) oil, fatty acids, fructose, gelatin, geranium maculatum oil, glucosamine, glucose glutamate, glutamic acid, glycereth-26, glycerin, glycerol, glyceryl distearate, glyceryl hydroxystearate, glyceryl laurate, glyceryl linoleate, glyceryl myristate, glyceryl oleate, glyceryl stearate, glyceryl stearate SE, glycine, glycol stearate, glycol stearate SE, glycosaminoglycans, grape (vitis vinifera) seed oil, hazel (corylus americana) nut oil, hazel (corylus avellana) nut oil, hexylene glycol, honey, hyaluronic acid, hybrid safflower (carthamus tinctorius) oil, hydrogenated castor oil, hydrogenated coco-glycerides, hydrogenated coconut oil, hydrogenated lanolin, hydrogenated lecithin, hydrogenated palm glyceride, hydrogenated palm kernel oil, hydrogenated soybean oil, hydrogenated tallow glyceride, hydrogenated vegetable oil, hydrolyzed collagen, hydrolyzed elastin, hydrolyzed glycosaminoglycans, hydrolyzed keratin, hydrolyzed soy protein, hydroxylated lanolin, hydroxyproline, imidazolidinyl urea, iodopropynyl butylcarbamate, isocetyl stearate, isocetyl stearoyl stearate, isodecyl oleate, isopropyl isostearate, isopropyl lanolate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, isostearamide DEA, isostearic acid, isostearyl lactate, isostearyl neopentanoate, jasmine (jasminum officinale) oil, jojoba (buxus chinensis) oil, kelp, kukui (aleurites moluccana) nut oil, lactamide MEA, laneth-16, laneth-10 acetate, lanolin, lanolin acid, lanolin alcohol, lanolin oil, lanolin wax, lavender (lavandula angustifolia) oil, lecithin, lemon (citrus medica limonum) oil, linoleic acid, linolenic acid, macadamia ternifolia nut oil, magnesium stearate, magnesium sulfate, maltitol, matricaria (chamomilla recutita) oil, methyl glucose sesquistearate, methylsilanol PCA, microcrystalline wax, mineral oil, mink oil, mortierella oil, myristyl lactate, myristyl myristate, myristyl propionate, neopentyl glycol dicaprylate/dicaprate, octyl dodecanol, octyldodecyl myristate, octyldodecyl stearoyl stearate, octyl hydroxystearate, octyl palmitate, octyl salicylate, octyl stearate, oleic acid, olive (olea europaea) oil, orange (citrus aurantium dulcis) oil, palm (elaeis guineensis) oil, palmitic acid, pantethine, panthenol, panthenyl ethyl ether, paraffin, PCA, peach (prunus persica) kernel oil, peanut (arachis hypogaea) oil, PEG-8 C12 18 ester, PEG-15 cocamine, PEG-150 distearate, PEG-60 glyceryl isostearate, PEG-5 glyceryl stearate, PEG-30 glyceryl stearate, PEG-7 hydrogenated castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-20 methyl glucose sesquistearate, PEG-40 sorbitan peroleate, PEG-5 soy sterol, PEG- 10 soy sterol, PEG-2 stearate, PEG-8 stearate, PEG-20 stearate, PEG-32 stearate, PEG-40 stearate, PEG-50 stearate, PEG-100 stearate, PEG- 150 stearate, pentadecalactone, peppermint (mentha piperita) oil, petrolatum, phospholipids, polyamino sugar condensate, polyglyceryl-3 diisostearate, polyquaternium-24, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, polysorbate 85, potassium myristate, potassium palmitate, potassium sorbate, potassium stearate, propylene glycol, propylene glycol dicaprylate/dicaprate, propylene glycol dioctanoate, propylene glycol dipelargonate, propylene glycol laurate, propylene glycol stearate, propylene glycol stearate SE, PVP, pyridoxine dipalmitate, quaternium-15, quaternium-18 hectorite, quaternium-22, retinol, retinyl palmitate, rice (oryza sativa) bran oil, RNA, rosemary (rosmarinus officinalis) oil, rose oil, safflower (carthamus tinctorius) oil, sage (salvia officinalis) oil, salicylic acid, sandalwood (santalum album) oil, serine, serum protein, sesame (sesamum indicum) oil, shea butter (butyrospermum parkd' ), silk powder, sodium chondroitin sulfate, sodium DNA, sodium hyaluronate, sodium lactate, sodium palmitate, sodium PCA, sodium polyglutamate, sodium stearate, soluble collagen, sorbic acid, sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan sesquioleate, sorbitan stearate, sorbitol, soybean (glycine soja) oil, sphingolipids, squalane, squalene, stearamide MEA-stearate, stearic acid, stearoxy dimethicone, stearoxytrimethylsilane, stearyl alcohol, stearyl glycyrrhetinate, stearyl heptanoate, stearyl stearate, sunflower (helianlhus annuus) seed oil, sweet a/mond (primus amygdalus dulcis) oil, synthetic beeswax, tocopherol, tocopheryl acetate, tocopheryl linoleate, tribehenin, tridecyl neopentanoate, tridecyl stearate, triethanolamine, tristearin, urea, vegetable oil, water, waxes, wheat (triticum vulgar e) germ oil, and ylang ylang (cananga odorata) oil.

In some embodiments a topical formulation may contain a suitable excipient, which typically should have a high affinity for the skin, be well tolerated, stable, and yield a consistency that allows for easy utilization. Suitable topical excipients and vehicles can be routinely selected for a particular use by those skilled in the art, and especially with reference to one of many standard texts in the art, such as Remington's Pharmaceutical Sciences, Vol. 18, Mack Publishing Co., Easton, Pa. (1990), in particular Chapter 87 (which is herein incorporated by reference in its entirety). Optionally one or more humectants are also included in the topical formulation. Examples of humectants include, but are not limited to, amino acids, chondroitin sulfate, di glycerin, erythritol, fructose, glucose, glycerin, glycerol, glycol, 1,2,6-hexanetriol, honey, hyaluronic acid, hydrogenated honey, hydrogenated starch hydrolysate, inositol, lactitol, maltitol, maltose, mannitol, natural moisturization factor, PEG- 15 butanediol, polyglyceryl sorbitol, salts of pyrollidone carboxylic acid, potassium PCA, propylene glycol, sodium glucuronate, sodium PCA, sorbitol, sucrose, trehalose, urea, and xylitol.

Certain embodiments contemplate topical formulations containing one or more additional skin protective agent. Examples of skin protective agents may include, but are not limited to, algae extract, allantoin, aluminum hydroxide, aluminum sulfate, betaine, camellia sinensis leaf extract, cerebrosides, dimethicone, glucuronolactone, glycerin, kaolin, lanolin, malt extract, mineral oil, petrolatum, potassium gluconate, and talc. One skilled in the art will readily appreciate that skin protectants other than those listed above may also be combined with a disclosed composition of the present disclosure or preparation provided thereby.

Surfactants may also desirably be included in certain topical formulations contemplated herein, and can be selected from any natural or synthetic surfactants suitable for use in cosmetic compositions, such as cationic, anionic, zwitterionic, or non-ionic surfactants, or mixtures thereof. (See Rosen, M., “Surfactants and Interfacial Phenomena,” Second Edition, John Wiley & Sons, New York, 1988, Chapter 1, pages 4 31). Examples of cationic surfactants may include, but are not limited to, DMDAO or other amine oxides, long-chain primary amines, diamines and polyamines and their salts, quaternary ammonium salts, polyoxyethylenated long-chain amines, and quaternized polyoxyethylenated long-chain amines. Examples of anionic surfactants may include, but are not limited to, SDS; salts of carboxylic acids (e.g., soaps); salts of sulfonic acids, salts of sulfuric acid, phosphoric and polyphosphoric acid esters; alkylphosphates; monoalkyl phosphate (MAP); and salts of perfluorocarboxylic acids. Examples of zwitterionic surfactants may include, but are not limited to, cocoamidopropyl hydroxysultaine (CAPHS) and others which are pH-sensitive and require special care in designing the appropriate pH of the formula (z.e., alkylaminopropionic acids, imidazoline carboxylates, and betaines) or those which are not pH-sensitive (e.g., sulfobetaines, sultaines). Examples of non-ionic surfactants may include, but are not limited to, alkylphenol ethoxylates, alcohol ethoxylates, polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated mercaptans, long-chain carboxylic acid esters, alkonolamides, tertiary acetylenic glycols, polyoxyethylenated silicones, N-alkylpyrrolidones, and alkylpolyglycosidases. Any combination of surfactants is acceptable. Certain embodiments may include at least one anionic and one cationic surfactant, or at least one cationic and one zwitterionic surfactant which are compatible, z.e., do not form complexes which precipitate appreciably when mixed.

Examples of thickening agents that may also be present in certain topical formulations include, but are not limited to, acrylamides copolymer, agarose, amylopectin, bentonite, calcium alginate, calcium carboxymethyl cellulose, carbomer, carboxymethyl chitin, cellulose gum, dextrin, gelatin, hydrogenated tallow, hydroxytheylcellulose, hydroxypropylcellulose, hydroxpropyl starch, magnesium alginate, methylcellulose, microcrystalline cellulose, pectin, various PEG'S, polyacrylic acid, polymethacrylic acid, polyvinyl alcohol, various PPG's, sodium acrylates copolymer, sodium carrageenan, xanthan gum, and yeast beta-glucan. Thickening agents other than those listed above may also be used in embodiments of this disclosure.

According to certain embodiments contemplated herein, a topical formulation for use in treating a skin condition may comprise one or more sunscreening or UV absorbing agents. Where ultraviolet light- (UVA and UVB) absorbing properties are desired, such agents may include, for example, benzophenone, benzophenone- 1, benzophenone-2, benzophenone-3, benzophenone-4, benzophenone-5, benzophenone-6, benzophenone-7, benzophenone-8, benzophenone-9, benzophenone- 10, benzophenone- 11, benzophenone- 12, benzyl salicylate, butyl PABA, cinnamate esters, cinoxate, DEA-methoxycinnamate, diisopropyl methyl cinnamate, ethyl dihydroxypropyl PABA, ethyl diisopropylcinnamate, ethyl methoxycinnamate, ethyl PABA, ethyl urocanate, glyceryl octanoate dimethoxycinnamate, glyceryl PABA, glycol salicylate, homosalate, isoamyl p-methoxycinnamate, oxides of titanium, zinc, zirconium, silicon, manganese, and cerium, PABA, PABA esters, Parsol 1789, and isopropylbenzyl salicylate, and mixtures thereof. One skilled in the art will appreciate that sunscreening and UV absorbing or protective agents other than those listed may be used in the present disclosure.

Topical formulations disclosed herein are typically effective at pH values between about 2.5 and about 10.0. Preferably, the pH of the composition is at or about the following pH ranges: about pH 5.5 to about pH 8.5, about pH 5 to about pH 10, about pH 5 to about pH 9, about pH 5 to about pH 8, about pH 3 to about pH 10, about pH 3 to about pH 9, about pH 3 to about pH 8, and about pH 3 to about pH 8.5. Most preferably, the pH is about pH 7 to about pH 8. One of ordinary skill in the art may add appropriate pH adjusting ingredients to the compositions of the present disclosure to adjust the pH to an acceptable range.

The compositions of this disclosure can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art. Controlled release drug delivery systems include osmotic pump systems and dissolutional systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled release systems are given in U.S. Pat. Nos. 3,845,770 and 4,326,525 and in P. J. Kuzma et al, Regional Anesthesia 22 (6): 543-551 (1997), all of which are incorporated herein by reference.

Some embodiments include sustained-release pharmaceutical compositions. An exemplary sustained-release composition has a semipermeable matrix of a solid hydrophobic polymer to which a therapeutic agent is attached or in which the therapeutic agent is encapsulated. Examples of suitable polymers include a polyester, a hydrogel, a polylactide, a copolymer of L-glutamic acid and T-ethyl-L-glutamase, non-degradable ethylene-vinylacetate, a degradable lactic acid-glycolic acid copolymer, and poly-D-(-)-3 -hydroxybutyric acid. Such matrices are typically in the form of shaped articles, such as films, or microcapsules. In another embodiment, a sustained-release composition includes a liposomally entrapped inhibitor. Liposomes are small vesicles composed of various types of lipids, phospholipids, and/or surfactants. These components are typically arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing a therapeutic agent can be prepared by known methods, such as, for example, those described in Epstein, et al. (1985) PNAS USA 82:3688-92, and Hwang, et al., (1980) PNAS USA, 77:4030-34.

The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The pharmaceutical composition can be prepared to provide easily measurable amounts for administration. Pharmaceutical compositions of the disclosure can be stored in any standard form, including, e.g., an aqueous solution or a lyophilized cake. Such compositions are typically sterile when administered to subjects. Sterilization of an aqueous solution is readily accomplished by filtration through a sterile filtration membrane. If the composition is stored in lyophilized form, the composition can be filtered before or after lyophilization and reconstitution.

Therapeutic Uses

Pharmaceutical compositions according to the disclosure can be generally administered systemically. The most suitable route will depend on the nature and severity of the condition being treated. Depending on the disorder to be treated, the pharmaceutical compositions described herein may be administered orally, parenterally (e.g., via intravenous, subcutaneous, intracutaneous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection), topically, mucosally (e.g., rectally or vaginally), nasally, buccally, ophthalmically, via inhalation spray (e.g, delivered via nebulization, propellant or a dry powder device) or via an implanted reservoir. Those skilled in the art are also familiar with determining administration methods (e.g, sprays, creams, open application, occlusive dressing, soaks, washes, etc.), dosage forms, suitable pharmaceutical excipients and other matters relevant to the delivery of the compounds to a subject in need thereof.

In certain embodiments, the disclosure provides methods of treating or preventing skin infection with a virus comprising administering an effective amount of a pharmaceutical composition comprising an active pharmaceutical agent disclosed herein to a subject in need thereof.

In one aspect, the disclosure provides a method for treating or preventing a skin infection with a virus in a subject in need thereof. The method comprises administering to the subject an effective amount of a pharmaceutical composition that comprises an extract of Candida albicans. The virus is not human papillomavirus (HPV) or molloscum contagiosum virus.

In some embodiments, the virus is selected from the group consisting of herpes simplex virus (HSV) -1 or -2, monkeypox virus, SARS-CoV-2, measles virus, rubella virus, varicella zoster virus, parvovirus, herpesvirus 6 and 7 virus, orf virus, variola minor/major, enterovirus, smallpox, and vaccinia virus (VV).

In some embodiments, the VV comprises a component from a microbial organism selected from the group consisting of influenza virus, varicella zoster virus, human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), polio virus, variola virus, rabies virus, rotavirus, human papillomavirus, Ebola virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, lyssavirus, measles virus, mumps virus, and Rubella virus.

In some embodiments, the pharmaceutical composition is administered to the subject before, after, or concurrent with the VV.

In some embodiments, the extract comprises a filtered extract of a Candida albicans deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126019 or PTA-126020.

In some embodiments, the extract comprises filtered extracts of both ATCC PTA- 126019 and ATCC PTA-126020.

In some embodiments, the extract comprises filtered extracts of one or more Candida albicans, Candida parapsilosis, and Candida tropicalis, such as one or more ATCC PTA- 126019, ATCC PTA-126020, ATCC9968, and ATCC22019.

In some embodiments, the pharmaceutical composition comprises or is Candin®.

In some embodiments, the pharmaceutical composition is administered to the subject via injection or topical application.

In one embodiment, the subject is a mammal.

In one embodiment, the subject is a human.

In some embodiments, a cream, lotion, gel, ointment, paste or the like may be spread on the affected surface and gently rubbed in. A solution may be applied in the same way, but more typically will be applied with a dropper, swab, or the like, and carefully applied to the affected areas. The application regimen will depend on a number of factors that may readily be determined, such as the severity of the condition and its responsiveness to initial treatment, but will normally involve one or more applications per day on an ongoing basis. One of ordinary skill may readily determine the optimum amount of the formulation to be administered, administration methodologies and repetition rates. In general, it is contemplated that the formulations of the disclosure will be applied in the range of once or twice weekly up to once, twice or thrice daily.

For topical administration the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical or cosmeceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. Topical formulations may contain a concentration of the compound of the disclosure from about 0.1 to about 10% w/v (weight per unit volume). A topical formulation may be provided in the form of a cream, lotion, solution, spray, gel, ointment, paste or the like, and/or may contain liposomes, micelles, microspheres and/or other microparticle or nanoparticle delivery elements.

The active agent for use in topical formulations are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific agent employed; the metabolic stability and length of action of the agent; the age, body weight, general health, sex, skin type and diet of the subject; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular skin condition; and the subject undergoing therapy. Generally, a therapeutically effective daily dose can be (for a 70 kg mammal) from about 0.0001 mg/kg (z.e., 0.007 mg) to about 10 g/kg (z.e., 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (z.e., 7 mg) to about 50 mg/kg (z.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (z.e., 70 mg) to about 25 mg/kg (z.e., 1.75 g).

The ranges of effective doses provided herein are not intended to be limiting and represent preferred dose ranges. However, the most preferred dosage will be tailored to the individual subject, as is understood and determined by one skilled in the relevant arts, (see, e.g., Berkow et al., eds., The Merck Manual, 16^th edition, Merck and Co., Rahway, N.J., 1992; Goodman et al., eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10^th edition, Pergamon Press, Inc., Elmsford, N.Y., (2001); Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, Ltd., Williams and Wilkins, Baltimore, Md. (1987); Ebadi, Pharmacology, Little, Brown and Co., Boston, (1985); Osolci al., eds., Remington's Pharmaceutical Sciences, 18^th edition, Mack Publishing Co., Easton, Pa. (1990); Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk, Conn. (1992)).

The total dose required for each treatment can be administered by multiple doses or in a single dose over the course of the day, if desired. Certain preferred embodiments contemplate a single application of the topical formulation per day. Generally, and in distinct embodiments, treatment may be initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. Kit and Articles of Manufacture

In another aspect, this disclosure provides a kit or an article of manufacture containing materials useful for the methods described above. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective (1) for improving delivery of a composition to a target site (2) for treating, preventing and/or diagnosing one or more of the conditions mentioned above. The container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used for treating the condition of choice.

Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a first therapeutic agent and/or (b) a second container with a composition contained therein, wherein the composition comprises a second, different therapeutic agent. The article of manufacture may comprise a third container with a composition contained therein, wherein the composition comprises a third therapeutic agent.

The article of manufacture in this embodiment of the disclosure may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a fourth container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

In some embodiments, the kit or article of manufacture further comprises instructional materials containing directions (z.e., protocols) for the practice of the methods described herein (e.g., instructions for using the kit for administering a composition). While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD-ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials. Definition

As used herein, a "subject" or "individual" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, sheep, goats, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a human or a nonhuman mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disorders. The terms, "individual," "patient" and "subject" can be used interchangeably herein. A subject can be male or female. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental, non-human animal or animal suitable as a disease model.

A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition or disorder in need of treatment or one or more complications related to such a condition or disorder, and optionally, have already undergone treatment for such a condition or disorder or the one or more complications related to the condition or disorder. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition or disorder or one or more complications related to the condition or disorder. For example, a subject can be one who exhibits one or more risk factors for the condition or disorder or one or more complications related to the condition or disorder or a subject who does not exhibit risk factors.

A "subject in need" of treatment for a particular condition or disorder can be a subject having that condition or disorder, diagnosed as having that condition or disorder, or at risk of developing that condition or disorder.

As used herein, the term "administering," refers to the placement of an agent as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the agents disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. The terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, ophthalmic administration, intraaural administration, intracerebral administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

As used herein, the terms “parenteral administration” and “administered parenterally” refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

As used herein, “unit dosage” formulations are those containing a dose or sub-dose of the administered ingredient adapted for a particular timed delivery. For example, exemplary “unit dosage” formulations are those containing a daily dose or unit or daily sub-dose or a weekly dose or unit or weekly sub-dose and the like.

As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a disorder. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is reduced. That is, "treatment" includes not just the improvement of symptoms or markers, but also a slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

A "therapeutically effective amount" is an amount sufficient to remedy a disease state or symptoms, particularly a state or symptoms associated with the disease state, or otherwise prevent, hinder, retard or reverse the progression of the disease state or any other undesirable symptom associated with the disease in any way whatsoever. As used herein, the term “therapeutically effective amount” of an agent of the present disclosure refers to an amount of the compound of the present disclosure that will elicit the biological or medical response of a subject, or ameliorate symptoms, slow or delay disease progression, or prevent a disease, etc. In one embodiment, the term refers to the amount that inhibits or reduces microbial (e.g., viral) colonization or infection. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. In one embodiment, the term refers to the individual dosage amounts or ranges of dosage amounts described in the present application.

The terms “prevent”, “preventing”, “prevention” and the like are used interchangeably herein to mean inhibit, hinder, retard, reduce or otherwise delay the development of and/or progression of a condition or disorder or a symptom thereof, in a subject. In the context of the present disclosure, the term “prevent” and variations thereof does not necessarily imply the complete prevention of the specified event. Rather, the prevention may be to an extent, and/or for a time, sufficient to produce the desired effect. Prevention may be inhibition, retardation, reduction or otherwise hindrance of the event, activity or function. Such preventative effects may be in magnitude and/or be temporal in nature.

A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of the disease state, or reducing the likelihood of the onset (or reoccurrence) of the disease state or associated symptoms. The full therapeutic or prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

As used herein, the term "pharmaceutical composition" refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. As used herein, the term “pharmaceutically acceptable carrier or excipient” refers to a carrier medium or an excipient which does not interfere with the effectiveness of the biological activity of the active ingredient(s) of the composition and which is not excessively toxic to the host at the concentrations at which it is administered. In the context of the present disclosure, a pharmaceutically acceptable carrier or excipient is preferably suitable for topical formulation. The term includes, but is not limited to, a solvent, a stabilizer, a solubilizer, a tonicity enhancing agent, a structure-forming agent, a suspending agent, a dispersing agent, a chelating agent, an emulsifying agent, an anti-foaming agent, an ointment base, an emollient, a skin protecting agent, a gel-forming agent, a thickening agent, a pH adjusting agent, a preservative, a penetration enhancer, a complexing agent, a lubricant, a demulcent, a viscosity enhancer, a bioadhesive polymer, or a combination thereof. The use of such agents for the formulation of pharmaceutically active substances is well known in the art (see, for example, "Remington 's Pharmaceutical Sciences", E. W. Martin, 18th Ed., 1990, Mack Publishing Co.: Easton, PA, which is incorporated herein by reference in its entirety).

The term "therapeutic agent" refers to any agent that is used to treat a disease. A therapeutic agent may be, for example, a polypeptide(s), an aptamer or a small molecule that can bind to a protein or a nucleic acid molecule that can bind to a nucleic acid molecule encoding a target (z.e., siRNA), etc.

As used herein, the term “a,” “an,” “the” and similar terms used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual 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 otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

As disclosed herein, a number of ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

The term “about” refers to within 10%, preferably within 5%, and more preferably within 1% of a given value or range. Alternatively, the term "about" refers to within an acceptable standard error of the mean, when considered by one of ordinary skill in the art.

EXAMPLES

Example 1

This example descibes material and methods used in Examples 2-8 below.

Cells and Culture Techniques

The immortalized human keratinocyte cell line N/TERT-2G was propagated as previously described in Brewer, M.G., el al., Conditions That Simulate the Environment of Atopic Dermatitis Enhance Susceptibility of Human Keratinocytes to Vaccinia Virus. Cells, 2022. 11(8). Briefly, N/TERT-2G cells were maintained in keratinocyte serum free medium (INVITROGEN/GIBCO, Grand Island, NY, USA) supplemented with bovine pituitary extract and epidermal growth factor (INVITROGEN/GIBCO, Grand Island, NY, USA), penicillin/streptomycin (INVITROGEN/GIBCO, Grand Island, NY, USA), and 0.3 mM CaCh (BOSTON BIOPRODUCTS, Ashland, MA, USA). Cells were grown to 30% confluency, trypsinized, and plated. Differentiation was initiated by exposing cells to high calcium- containing (1.8 mM) Dulbecco’s modified Eagle medium (DMEM — INVITROGEN/GIBCO, Grand Island, NY, USA). Every two days, cells received fresh medium. BSC40 cells were obtained from the ATCC and maintained in DMEM (CORNING, Manassas, VA, USA) supplemented with 10% fetal bovine serum (INVITROGEN/GIBCO, Grand Island, NY, USA).

Candida Culture Techniques

Clinical isolates of C. albicans were obtained from Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA. Candida strains were grown overnight at 37°C in yeast peptone dextrose medium (SIGMA-ALDRICH Inc., St. Louis, MO, USA). To calculate colony forming units (CFU), absorbance was read at 600 nm and compared to a standard curve. Input amounts for assays were validated through serial dilution and plating on CHROMagar Candida™ (CHROMagar, Paris, France).

Cell Viability Assay

Lactate dehydrogenase (LDH) assays were performed in the manner as previously described in Arnold, K.A., el al., JAK Signaling Is Critically Important in Cytokine-Induced Viral Susceptibility of Keratinocytes. Int J Mol Sci, 2023. 24(11). Briefly, N/TERT-2G cells were plated at a density of 75,000 cells per well in a 96-well plate. These cells were grown to confluency and then treated with Candin® (80 pg; Lots CA073 and CA074; NIELSON BIOSCIENCES, San Diego, CA, USA) or exposed to whole yeast (10² - 10⁴ CFU) for 24 hours. Supernatants were collected from each well and the LDH assay was performed (CYTOTOXICITY DETECTION KitPLUS LDH, MILLIPORE SIGMA Saint Louis, MO, USA). The LDH detection reaction was conducted at room temperature and terminated by the addition of Stop Solution after 10 minutes. Readings (absorbance at 490 nm and 620 nm) were taken using a SPECTRAMAX i3x Multi-Mode Plate Reader (MOLECULAR DEVICES, San Jose, CA, USA). The absorbance values from no cell control wells were subtracted from each condition, and all values were normalized to a lysed control sample (representing 100% death) generated through addition of a 1 : 10 dilution of the provided lysis buffer.

Viral Plaque Assay

The viruses used for infections included the Western Reserve (WR) strain of VV and a recombinant KOS strain of HSV-1 expressing the yellow fluorescent protein fluorophore (Etienne L. etal., Visualization of herpes simplex virus type 1 virions using fluorescent colors. J Virol Methods. 2017 Mar;241 :46-51. doi: 10.1016/j.jviromet.2016.12.012). Cells were seeded in a 24-well plate (150,000 cells/well) and then grown to confluence at which point they were switched to high calcium-containing medium (1.8 mM) to induce differentiation. At the time of differentiation, keratinocytes were exposed to yeast (10² - 10⁴ CFU) or treated with Candin® (40 - 80 pg). 24 hours later, cells were infected with a low multiplicity of infection ([MOI] 0.0001) of VV or HSV-1 for 24 or 48 hours, respectively. For VV infection, the medium was subsequently removed and replaced with crystal violet solution for 3 hours to stain for plaque visualization and quantitation. Monolayers were washed with water to remove crystal violet solution, and plates were allowed to dry completely prior to scanning and analysis on a BIOTEK LIONHEART FX Automated Microscope (AGILENT TECHNOLOGIES, Santa Clara, CA, USA). HSV-1 plaques were visualized and quantitated via fluorescent microscopy using the LIONHEART FX Automated Microscope.

Imaging and Quantification of Viral Plaques

Since viral plaques of VV and HSV are morphologically different, protocols were created relative to each virus to optimize data reduction and accurately quantify plaques to obtain similar results obtained by manual counts (through ImageJ). The BIOTEK LIONHEART FX and BIOTEK GEN5 IMAGE PRIME version 3.12 Microplate Reader and Imager Software were utilized for automated quantification of viral plaques on wells stained with crystal violet (VV) at the end of viral plaque assays.

Imaging and quantification ofW infection:

An imaging step for CORNING 24 well plates was used in the procedure section of the software, which utilized the 4X objective and the PL fluorescent TEXAS RED filter 586,647. This captures the autofluorescence associated with crystal violet. The illumination, integration time, and gain were left on the default settings (10, 204, 24, respectively). A montage of four- by-four images captured 93% of each monolayer. For the tile overlap on the image montage, the auto function for stitching was selected. Image stitching was performed for the TEXAS RED channel using the linear blend function, the option to crop stitched image to remove black rectangles on borders was selected, and the final images were downsized 20%. The next step included Image preprocessing where the background was dark, and the image smoothing strength utilized was 5 cycles. A step for cellular analysis to highlight and quantify plaques was then performed where the background was selected as dark, and the minimum and maximum object size were 100 pm and 4000 pm, respectively.

Imaging and quantification of HSV infection:

Images were captured using the 20X PL fluorescent phase objective set for GFP 469,525 and phase contrast. The illumination, integration time, and gain were left on the default settings (10, 16, 23.8, respectively). A montage of fifteen-by-fifteen images was taken to capture 56% of each monolayer. For the tile overlap on the image montage, the auto function for stitching was selected. Images were preprocessed with a dark background and background flattening selected. Images were stitched using linear blend, the option to fill gaps between montage tiles with local background color was selected, and final images were downsized 10%. The threshold was set to 12,000. Images were saved and exported to be analyzed in ImageJ for viral fluorescence. % monolayer containing viral fluorescence:

Once images were exported, ImageJ software was used to calculate the percentage of the monolayer within each image that was infected by virus (% Viral Fluorescence). Total image area was determined using Analyze Measure. The Threshold function was applied to the image so that fluorescent keratinocytes (plaques) were white: Image Adjust Threshold Apply. Finally, all areas considered to be infected were selected using the Edit Selection Create Selection, and the selection was inverted using the Make Inverse function. The infected area was measured (Analyze Measure), and the infected area was divided by the total area to obtain % Viral Fluorescence.

Statistical and Data Analysis

Statistical differences for all experiments were tested on experimental means via a oneway ANOVA (paired when appropriate) with the Geisser-Greenhouse correction or a parametric paired ratio t-test. To account for the fact that only 56% of HSV-1 infected monolayers were imaged and there were plaques present outside of the imaged field, these plaque numbers were adjusted to represent the total number of plaques per well. All statistical tests and graphs were performed with GRAPHPAD PRISM software v9.2.0 (GraphPad, San Diego, CA, USA).

Example 2

This example describes production of a composition comprising a sterile filtrate of a yeast strain such as Candida albicans, Candida parapsilosis, or Candida tropicalis and secreted antigens.

First, a pre-production culture is prepared from an oil-covered stock culture by aseptically removing one loopful of the stock culture of a yeast strain and placing it in Animal Free Trypticase Soy Broth (ATSB; TechNova, Dartmouth, Nova Scotia). This pre-production culture is incubated at 20-25° C. until a growth pellet is observed at the bottom of the tube, usually within 7 to 14 days. After evidence of fungal growth (e.g. milky yeast-like growth on the bottom of the tube) is observed, a portion of the growth is transferred to a plate of Animal Free Trypticase Soy Agar (ATSA; TechNova, Dartmouth, Nova Scotia) and is incubated at 20- 25° C. for 3 to 7 days. The growth from the ATSA plate is subcultured to a fresh ATSA plate and the plate is streaked to produce isolated colonies. The ATSA plate is incubated at 20-25° C. for 3 to 7 days.

From the ATSA plate, several isolated colonies with characteristic morphology are transferred to an Erlenmeyer flask containing Chemically Defined Candida Medium (CDCM; TechNova, Dartmouth, Nova Scotia). CDCM consists of 3.6 g/L KH2PO4, 1.2 g/L Na2HPO4, 8.0 g/L (NH₄)₂SO4, 0.2 g/L MgSO₄.7H₂O, 0.01 g/L ZnSO₄.7H₂O, 8.0 g/L sucrose, and 0.01 g/L biotin. The Erlenmeyer flask is incubated 3-7 days on a shaker set at approximately 60 rpm at a temperature of 20-25° C.

When the inoculum shows budding yeast pseudohyphae and if it is free of bacteria, each of the stock is transferred to separate production flasks containing CDCM at a dilution of approximately 1 : 100. For example, approximately 1.0 mL of each of the stock is transferred to separate production flasks containing 100 mL CDCM; or approximately 10 mL of each of the stock is transferred to separate production flasks containing 1000 mL CDCM. The production flasks are incubated at 20-25° C. for 7 days on a shaker, set at 60 rotations or oscillations per minute.

Upon completion of incubation, 1.0 mL of 1% phenol is added to each production flasks. The flasks are stored at 20-25° C. for 7 days for the fungus to be killed. The contents of all production flasks containing acceptable growth and no contamination are then pooled in a sterile glass bottle. The pooled material is cultured by adding 1.0 mL to each of three 40 mL tubes of Trypticase Soy Broth (TSB; TechNova, Dartmouth, Nova Scotia). Each TSB culture is further diluted 10-fold in TSB and is incubated at 20-25° C. for 7 days to ensure non-viability. During the culturing period, the poled production material is stored in a holding bottle at 20-25° C. for 7 days.

Dialyze the pooled culture in USP grade Water for Injection (WFI; TechNova, Dartmouth, Nova Scotia) at 1-8° C. using Spectrapor 6,000-8,000 MWCO dialysis tubing (size 40 mm). Dialyze with a 20-fold volume of WFI and repeat the procedure twice at 24-hour intervals. The dialyzed material is then heated uniformly for 60 minutes at 90-95° C. in a water bath.

200 mL of the heat treated dialyzed material is added to a 600 mL lyophilization flask and freeze dried in a Labconco lyophilizer. This procedure is repeated until the entire lot has been lyophilized. Lyophyilized material is then covered with petroleum ether at 20-25° C. for 6 to 8 hours with the supernatant being discarded via filtration. The resulting material is air dried in a fume hood. This is the dry powder denoted as the source material for further processing.

Dilute the source material with Coca's Glycerol Solution (0.25% NaCl, 0.125% NaHCCh, 53% glycerin; 47% WFI) having a pH in the range of 6 to 8.5 at a desired weight by volume extraction ratio. In an aspect, a volume extraction ratio is 1 :20 w/v, where 20 mL of Coca's Glycerol Solution is added to per gram of source material. This extract is mixed intermittently for approximately 71 hours on a magnetic stirrer at 1-8° C. until the source material appears homogenous throughout the mixture. The extract is then centrifuged at approximately 4000 RPMs for 20 minutes. After that, the extract is filtered by vacuum filtration with a Buchner funnel and Whatman No. 3 filter paper. Adjust the filtered extract to have a final concentration of 0.4% phenol and return it to 1-8° C. A sterile Sartorius 0.2 pm filter capsule (#5231307H) and filter the product under a class 100 laminar flow hood in a class 100,000 room.

In a sterile filtration room, use a sterile Sartorius “Sartobran” 0.45/0.2 pm [5235307H7OOA (0.05 m²) or 5231307H5OOB (0.03 m²)] to sterilize the product. Collect the filtered solution to an appropriately sized, sterile depyrogenated container, and store the filtered solution at 1-8° C. Finally, 1.7 mL of this master lot filtered solution is diluted with 998.3 mL of a diluent, which consists of 5.0 g/L NaCl, 2.5 g/L NaHCCh, 4.5 mL/L phenol, 1.2 mL/L 20% solution human serum albumin, 0.8 mL/L polysorbate 80.

Example 3

Clinical observations have demonstrated that patients treated with a non-replicating Candida albicans (C. albicans') extract called Candin® (Nielsen Bioscience, Inc.) exhibit clearance of both proximal (site of injection) and distal cutaneous warts. Of note, this is an off- label application of Candin® since it was initially developed for human intradermal administration to assess an individual’s cell-mediated immunity against C. albicans [5], To examine whether Candin® can function as a treatment for common cutaneous viral infections including molluscum contagiosum (caused by the molluscum contagiosum virus) and HPV, a retrospective chart review was undertaken [6], Treatment with Candin® for one year was found to lead to complete clearance of warts (HPV) in 87% of patients and complete resolution of molluscum contagiosum in 56% of patients. More contemporary, completed clinical trials (NCT02393417, NCT01757392) have also demonstrated that Candin® is effective for treatment of cutaneous viral infections.

The mechanism(s) responsible for Candin® to promote the clearance of cutaneous viral infections remain unknown. To better understand this, inventors utilized an in vitro infection model that quantifies changes in keratinocyte viral susceptibility [7], Human keratinocytes were concomitantly differentiated in high calcium containing medium and treated with Candin® for 24 hours. Since it has been demonstrated that keratinocytes are most susceptible to viral infections at early stages of differentiation this timepoint was chosen for these studies [8], Keratinocytes were infected with vaccinia virus (VV), a model for molluscum contagiosum virus because tissue culture practices currently do not exist, and herpes simplex virus-1 (HSV-1). It was confirmed that neither Candin® nor live yeast co-culture impacted keratinocyte viability as shown by the lactate dehydrogenase release assay (FIG. 3). Therefore, any decrease in viral susceptibility after Candin® exposure or yeast co-culture could not be explained by cell death.

Example 4

Keratinocytes were concurrently differentiated and treated with Candin® (40, 60, or 80 pg) for 24 hours. These values are comparable to the mass of Candin® protein that is administered to patients (approximately 250 pg of protein in 0.1 mL of Candin®). Keratinocytes were infected with VV and 24 hours later monolayers were stained with crystal violet to visualize viral plaque formation and cytopathic effect (FIGS. 1A and 2A). Stained sections denoted regions of keratinocytes still adhered to the well, while cytopathic effect, or death of keratinocytes followed by detachment from the well, is represented by the absence of staining. These values were used to calculate the % monolayer cleared as a metric for cytopathic effect. A dose responsive reduction was observed in cytopathic effect, where keratinocytes treated with the highest dose of Candin® demonstrated the greatest reduction (52.3 ± 20.1%, p < 0.05; FIG. IB). No change in plaque number was observed at any dosage, but plaque size was significantly decreased (FIG. IB). To understand whether decreased viral susceptibility of keratinocytes treated with Candin® was driven by diminished viral dissemination, infected keratinocytes were harvested 24-hours post-infection to quantify production of infectious virions. Viral titer from keratinocytes treated with 80 pg Candin® was significantly decreased compared to media control VV infected keratinocytes (-5.73 ± 4.80-fold change, p < 0.01, Fig. IB). Together these results indicate that Candin® treatment decreases viral spread after infection of keratinocytes.

Example 5

Extending these findings to another family of cutaneous viruses, Candin® treated keratinocytes were infected with HSV-1. Similarly, there was a dose responsive reduction in infection, with the highest dose of Candin® treatment resulting in a 74.1 ± 10.5% reduction in HSV-1 induced cytopathic effect (p < 0.05; FIG. ID). In contrast to the results with VV infection, keratinocytes infected with HSV-1 had a 31.6 ± 8.4% reduction in plaque number when treated with the highest dose of Candin® (p < 0.01; FIG. ID). These results demonstrate that keratinocytes treated with Candin® are less susceptible to two different viruses and extend the potential benefit of this “therapy” from the Papillomaviridae family of viruses to both Poxviridae and Herpesviridae members. Example 6

Candin® is formulated from C. albicans extract (non-live material). To investigate whether the above observations were recapitulated with live replicating Candida species, with the idea that the whole organism could be used as an intervention, C. albicans and control yeasts (Saccharomyces cerevisiae and Malassezia sympodialis) were incorporated into the viral infection model. Therefore, to understand whether colonization by live yeast influence keratinocyte viral susceptibility, keratinocytes were exposed to 10², 10³, or 10⁴ colony forming units (CFU) 24 hours prior to VV infection. These CFU values were chosen to replicate what is thought to be clinically relevant levels on human skin [10],

Keratinocytes cultured with C. albicans (10⁴ CFU) and then infected with VV demonstrated a significant decrease in cytopathic effect (p < 0.05; FIG. 2B and plaque number following VV exposure (FIG. 2C). Keratinocytes exposed to Saccharomyces cerevisiae (10⁴ CFU) also demonstrated a significant decrease in cytopathic effect and plaque number (FIGS. 2B and 2C), but this effect was more modest when compared to C. albicans. Finally, no change in viral susceptibility was observed in keratinocytes co-cultured with Malassezia sympodialis.

These results are consistent with the results from Candin® treated keratinocytes and demonstrate that exposure to live Candida likewise diminishes viral susceptibility of keratinocytes, which promotes their usage as an antiviral intervention.

Example 7

In this study, it was determined that exposing KC to Candida spp results in diminished viral susceptibility. C. tropicalis reproducibly induces this effect at very low CFU (10¹ and 10²) as seen in a reduction in cytopathic effect, plaque number, and plaque size. C. parapsilosis exposure results in significantly diminished viral susceptibility at slightly higher CFU (10³ and 10⁴) (FIG. 4). C. albicans exposure only results in significantly diminished viral susceptibility at the highest CFU (10⁴), and C. glabrata exposure results in only very modest significantly reduced viral susceptibility. This data indicates that Candida spp are likely inducing a conserved mechanism for reducing viral susceptibility in KC, but differences at the species level influences this phenotype.

The following materials and methods were used in this study:

The immortalized KC cell line (N/TERT-2G) was plated on 24 well plates. Once confluent, KC were differentiated ± Candida (C.) parapsilosis, C. albicans, C. tropicalis, or C. glabrata at a range of colony forming units (CFU) spanning 10 to 10⁴ CFU. 24 hours post- differentiation, KC were infected with a low multiplicity of infection of vaccinia virus (VV). C. parapsilosis, C. albicans, and C. glabrata strains were identified by MALDI-TOF (Vitek MS) (Matrix- Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry). C. tropicalis identified by Deposit No. ATCC9968 was used. Multiple C. albicans were used including a primary isolate identified as mentioned above and the commonly used SC5314 strain. 30 hours post-infection, VV infection was stopped via crystal violet staining for 2 hours. The plate was then imaged on a BioTek Lionheart Automatic Imager. BioTek Gen 5.0 imaging software was used to analyze cytopathic effect (% of monolayer cleared), the number of plaques per well, and the plaque size of each plaque (pm). This data was then visualized using GraphPad Prism.

Example 8

Previously published work demonstrated that treating KC with Candin(R), a nonreplicating extract from C. albicans, results in diminished viral susceptibility. Given that the results utilizing replicating yeast (FIG. 4) demonstrated a greater efficacy with C. parapsilosis and reproducibly with C. tropicalis, it was investigated whether formulating extracts from either of these species would diminish KC viral susceptibility to a greater extent.

Briefly, immortalized KC cell line (N/TERT-2G) was plated on 24 well plates. Once confluent, KC were differentiated ± vehicle control (Coca’s Glycerol Solution, “Vehicle CTL”) or 0.5 pg/mL of Candida extract (CP: C . parapsilosis,' CA: C. albicans,' CT: C. tropicalis). To test for lot-to-lot variability, two Lots of Candida extract for C. parapsilosis were generated, where all preparations named “Lot 2” were generated at the same time. 24 hours postdifferentiation, KC were infected with a low multiplicity of infection of vaccinia virus (VV). 30 hours post-infection, VV infection was stopped via crystal violet staining for 2 hours. The plate was then imaged on a BioTek Lionheart Automatic Imager. BioTek Gen 5.0 imaging software was used to analyze cytopathic effect (% of monolayer cleared), the number of plaques per well, and the plaque size of each plaque (pm). This data was then visualized using GraphPad Prism. Each experiment is encoded by a different symbol, where matching symbols across different graphs are experimentally matched. The results are shown in FIG. 5.

As shown in FIG. 5, it was identified that treating KC with Candida extract results in diminished viral susceptibility. This effect is seen most robustly with preparations using C. parapsilosis, where there is a reduction in both cytopathic effect and plaque size, whereas the preparations using either of the other species only significantly reduced the average plaque size. These results indicate that there is a bioactive compound more prevalent in C. parapsilosis extracts that stimulates an antiviral response in KC.

References:

1. Kombe Kombe, A. J., B. Li, A. Zahid, H.M. Mengist, G.A. Bounda, Y. Zhou, and T. Jin, Epidemiology and Burden of Human Papillomavirus and Related Diseases, Molecular Pathogenesis, and Vaccine Evaluation. Front Public Health, 2020. 8: p. 552028.

2. Burd, E.M., Human papillomavirus and cervical cancer. Clin Microbiol Rev, 2003. 16(1): p.1-17.

3. Wyant, W.A., G.W. Burke, T. loannides, and A. J. Nichols, Systemic 9-valent human papillomavirus vaccine for recalcitrant common cutaneous warts in preparation for renal transplant. JAAD Case Rep, 2022. 22: p. 62-63.

4. Kore, V.B. and A. Anjankar, A Comprehensive Review of Treatment Approaches for Cutaneous and Genital Warts. Cureus, 2023. 15(10): p. e47685.

5. Ohri, L.K., J.M. Manley, A. Chatterjee, and N.E. Cornish, Pediatric case series evaluating a standardized Candida albicans skin test product. Ann Pharmacother, 2004. 38(6): p. 973-7.

6. Maronn, M., C. Salm, V. Lyon, and S. Galbraith, One-year experience with Candida antigen immunotherapy for warts and molluscum. Pediatr Dermatol, 2008. 25(2): p. 189-92.

7. Brewer, M.G., S.R. Monticelli, M.C. Moran, B.L. Miller, L.A. Beck, and B.M. Ward, Conditions That Simulate the Environment of Atopic Dermatitis Enhance Susceptibility of Human Keratinocytes to Vaccinia Virus. Cells, 2022. 11(8).

8. Moran, M.C., E. Chinchilli, H.M. Kenney, E.M. Pope, G. Scott, M.G. Brewer, and L.A. Beck, Stage of Keratinocyte Differentiation Is a Key Determinant of Viral Susceptibility in Human Skin. J Invest Dermatol, 2023.

9. Kuhbacher, A., A. Burger-Kenti scher, and S. Rupp, Interaction of Candida Species with the Skin. Microorganisms, 2017. 5(2).

10. Gao, Z., G.I. Perez-Perez, Y. Chen, and M. J. Blaser, Quantitation of major human cutaneous bacterial and fungal populations. J Clin Microbiol, 2010. 48(10): p. 3575-81.

11. Wang, J.-N. and M. Li, The Immune Function of Keratinocytes in Anti-Pathogen Infection in the Skin. International Journal of Dermatology and Venereology, 2020. 3(4): p. 231-238.

12. Jo, J.H., E. A. Kennedy, and H.H. Kong, Topographical and physiological differences of the skin mycobiome in health and disease. Virulence, 2017. 8(3): p. 324-333.

The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entireties.

Claims

CLAIMS What is claimed is:

1. A method for treating or preventing a skin infection with a virus in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition that comprises at least one extract of Candida albicans, Candida parapsilosis, or Candida tropicalis.

2. The method of claim 1, wherein the virus is selected from the group consisting of herpes simplex virus (HSV) -1 or -2, monkeypox virus, SARS-CoV-2, measles virus, rubella virus, varicella zoster virus, parvovirus, herpesvirus 6 and 7 virus, orf virus, variola minor/major, enterovirus, human papillomavirus (HPV), molloscum contagiosum virus and vaccinia virus (VV).

3. The method of claim 2, wherein the VV comprises a component from a microbial organism selected from the group consisting of influenza virus, varicella zoster virus, human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), polio virus, variola virus, rabies virus, rotavirus, human papillomavirus, Ebola virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, lyssavirus, measles virus, mumps virus, and Rubella virus.

4. The method of claim 3, wherein the pharmaceutical composition is administered to the subject before, after, or concurrent with the VV.

5. The method of any one of claims 1-4, wherein the extract comprises a filtered extract of a Candida albicans deposited with the American Type Culture Collection (ATCC) under ATCC Accession No. PTA-126019 or PTA-126020.

6. The method of claim 5, wherein the extract comprises filtered extracts of both ATCC PTA-126019 and ATCC PTA-126020.

7. The method of any one of claims 1-5, wherein the extract comprises a filtered extract of one or more of ATCC PTA-126019, ATCC PTA-126020, ATCC9968, and ATCC22019.

8. The method of any one of claims 1-7, wherein the pharmaceutical composition is administered to the subject via injection or topical application.

9. The method of any one of the preceding claims, wherein the subject is a mammal.

10. The method of claim 9, wherein the subject is a human.