US007.914799B2

(12) United States Patent (10) Patent No.: US 7,914,799 B2 Jira et al. (45) Date of Patent: Mar. 29, 2011

(54) ANTI-FUNGAL COMPOSITION Ruedlet al. Clin. Diagn. Lab. Immunol. 1: 150-154, 1994.* Rutishauser et al. Adv. Ther. 15: 330-341, 1998.* (75) Inventors: Vic Jira, El Monte, CA (US); Vichai Cassone et al. Infect. Immun. 63: 2619-2624, 1995.* Jirathitikal, Chachoengsao (TH) Elahi et al. Vaccine 19: 2516-2521, 2001.* Nicoletti et al. Arzneimittelforschung 42: 1246-1250, 1992.* (73) Assignee: Immunitor USA, Inc., College Park, Browder et al. Int. J. Immunopharmacol. 6: 19-26, 1984.* MD (US) Jensen et al. J. Infect. Dis. 174: 133-140, 1996.* Williams et al. J. Reticuloendothel. Soc. 23: 479-490, 1978.* (*) Notice: Subject to any disclaimer, the term of this Suzuki et al. Int. J. Immunopharmacol. 11:761-769, 1989.* patent is extended or adjusted under 35 IMeister et al. J. Infect. Dis. 135: 235-242, 1977.* U.S.C. 154(b) by 433 days. Salvaggio et al. J. Immunol. 100: 1340-1352, 1968.* Kennedy et al. In: Bioactive Carbohydrates: In Chemistry, Biochem (21) Appl. No.: 10/228,280 istry and Biology. John Wiley & Sons, New York, p. 308, 1984.* Sakurai et al. Int. J. Pharmacol. 14: 821-830, 1992.* (22) Filed: Aug. 27, 2002 Numata et al. Org. Lett. 25. 4447-4450, 2004, abstract.* Koike K. Jpn. J. Antibiot. 29: 1098-1105, 1976, abstract.* (65) Prior Publication Data Marinova et al. Inter. J. Immunopharmacol. 22: 843-854, 2000.* US 2003/OO39667 A1 Feb. 27, 2003 Cohen et al. Am. J. Health-System Pharmacy, posted Sep. 14, 2007.* Hurtel et al. Infect. Immun. 31: 95-101, 1981.* Related U.S. Application Data Burford-Mason et al. J. Infect. 14: 147-157, 1987.* Levy et al. Vaccine 7:337-340, 1989.* (60) Provisional application No. 60/314,666, filed on Aug. Otero et al. J. Food Process. Preservat. 22:419-432, 1998.* 27, 2001. Koukalova et al. English translation of CS277558, pp. 1-10, 1993.* Koukalova et al. English translation of CS 277 558, pp. 1-9, 1993.* (51) Int. Cl. Hida et al. Biol. Pharm. Bull. 30: 1589-1592, 2007, abstract.* A6 IK39/00 (2006.01) Kondori et al. APMIS 116: 867-876, 2008, abstract.* A6 IK 45/00 (2006.01) Ishibashi et al. FEMS Immunology and Medical Microbiology 44: AOIN 63/00 (2006.01) 99-109, 2005, abstract.* (52) U.S. Cl...... 424/274.1; 424/184.1; 424/93.5; Edgington et al. Mol. Cell. Biol. 19: 1369-1380, 1999, abstract.* 424/278.1 Bougnoux et al. J. Clin. Microbiol. 31: 1644-1645, 1993.* Hazen et al. J. Clin. Microbiol. 37: 824-827, 1999, abstract.* (58) Field of Classification Search ...... 424/274.1, Tacket et al. Nat. Med. 4:607-609, 1998, abstract.* 424/1841, 93.5, 93.51; 530/823, 824 Tacket et al. Curr. Trop. Microbiol. Immunol. 332: 103-117, 2009, See application file for complete search history. abstract. Arakawa et al. Nature Biotechnol. 16: 292-297, 1998, abstract. (56) References Cited Stedman's Medical Dictionary, Williams & Wilkins, A Waverly Company, 26th Edition, pp. 29 and 853. U.S. PATENT DOCUMENTS Saccharomyces cerevisiae, Retrieved from Internet: URL: http://en. 4,229.434 A 10, 1980 Sarkisov et al...... 424,88 wikipedia.org/wiki/Saccharomyces cerevisiae, pp. 1-5. 4,368,191 A 1/1983 Sarkisov et al. ... 424/88 , Retrieved from URL Internet: http://en.wikipedia.org/wiki/ 5,003,750 A * 4/1991 Delgado ..... 52.741.11 Filamentous fungi, pp. 1-5. 5,277,904 A 1/1994 Pier ...... 424,88 Ceccati-Antonini, et al., “Filamentous growth in Saccharomyces 5,284,652 A 2, 1994 Pier 5,320,849 A * 6/1994 Hagiwara et al...... 424/442 cerevisiae.” Brazilian Journal of Microbiology, vol.35, No.3, (2004), 5.453,273 A 9, 1995 Werner et al...... 424,274.1 pp. 1-16. 5,641,761 A * 6/1997 Takahashi et al. ... 514/54 Pathogen, Retrieved from Internet URL: http://en.wikipedia.org/ 5,817,643 A * 10/1998 Jamas et al...... 514/54 wiki/Pathogen. 5,858,378 A * 1/1999 Bostwick ... 424,274.1 Wordsmyth, Retrieved from Internet URL: http:www.wordsmyth. 5.948,413 A 9, 1999 Mendoza . 424,274.1 net/live/home.php?script search&matchent pathogen&matchtype. 6,007,809 A * 12/1999 Chaykin ... 424/93.51 Definition of Pathogen, Retrieved from MedicineNet.com, Internet 6,020,324 A 2/2000 Jamas et al...... 514/54 URL: http://www.medterms.com/script/main/art. 6,132,733 A 10/2000 Werner et al...... 424,274.1 asp?articlekey=6383.

6,333,164 B1* 12/2001 Takesako et al...... 435/7.3 6,551,600 B2 * 4/2003 Hawkins et al. ... 424,278.1 * cited by examiner 6,939,864 B1* 9/2005 Johnson et al. ... 514/54 2002/01 19928 A1* 8/2002 McAnalley ...... 514/12 Primary Examiner — S. Devi FOREIGN PATENT DOCUMENTS (74) Attorney, Agent, or Firm — Dykema Gossett PLLC CS 277 558 * 2/1993 CS 277558 * 2/1993 (57) ABSTRACT EP O555618 A2 * 8, 1993 JP 6O126223. A * 7, 1985 A multivalent fungal vaccine comprising one or more heat JP 402218615 A * 8, 1990 inactivated fungal antigens, wherein at least one fungal anti WO WO 9735596 A1 * 10/1997 gen is effective in producing an immune response in a host WO WO99,38529 * 8/1999 when said vaccine is administered orally at a dose that is Sufficient for preventing or treating the fungal disease in said OTHER PUBLICATIONS host. Also described are methods for making and using an Markova et al. Int. J. Immunopharmac, 19: 205-214, 1997.* orally available anti-fungal vaccine. Manual of Clinical Microbiology, III Edition (Ed) Lenette et al. ASM, Washington D.C., 1980, pp. 562.563 & 577.* 1 Claim, No Drawings US 7,914,799 B2 1. 2 ANT-FUNGAL COMPOSITION enzyme systems (e.g., cytochrome P450) and inhibit fungal growth. Significant inhibition of mammalian P450 results in This application claims priority of provisional application important drug interactions. This group of agents includes Ser. No. 60/314,666 filed Aug. 27, 2001. ketoconazole, clotrimazole, miconazole, econazole, buto conazole, oxiconazole, Sulconazole, terconazole, FIELD OF THE INVENTION and itraconazole. These agents may be administered to treat systemic mycoses. Ketoconazole, an orally administered imi The invention relates to an oral composition useful for dazole, is used to treat nonmeningeal , histo treatment and prevention of fungal diseases. The invention plasmosis, and also relates to process of making an orally available antifun 10 in non-immunocompromised patients, and is also useful for gal vaccine and methods of use. oral and esophageal . Adverse effects include rare drug-induced hepatitis; ketoconazole is also contraindicated BACKGROUND OF THE INVENTION in pregnancy. Itraconazole appears to have fewer side effects than ketoconazole and is used for most of the same indica Pathogenic fungi occur worldwide and can cause diseases 15 tions. Fluconazole also has fewer side effects than ketocona in humans, animals and plants. Fungal infections in humans Zole and is used for oral and and range from Superficial, i.e., skin Surface to deeply invasive cryptococcal meningitis. Miconazole is a parenteral imida type or disseminated infection. Some of such infections espe Zole with efficacy in coccidioidomycosis and several other cially those that are disseminated are fatal. Thus fungal dis mycoses, but has side effects including hyperlipidemia and eases can be divided into the life-threatening systemic infec hyponatremia. tions, such as , systemic candidiasis, The third major group of antifungal agents includes ally , blastomycosis, coccidioidomycosis, paracoc lamnines-thiocarbamates, which are generally used to treat cidioidomycosis, and , and more common skin infections. This group includes tolnaftate and naftifine. ones which are non-life-threatening, like (ring Another antifungal agent is griseoflulvin, a fungistatic agent worm) infections, including tinea pedis (athlete's foot), tinea 25 which is administered orally for fungal infections of skin, cruris (jock itch), candidiasis, and actinomycosis. or nails that do not respond to topical treatment. The life-threatening fungal infections are a growing prob Limitations of current therapeutic options include: inad lem not only for immunosuppressed or immunocompromised equate spectrum of activity, lack of efficacy due to growing individuals but also in individuals with viral infections, such resistance, poor safety profile, multiple drug interactions, as cytomegalovirus (CMV), and influenza, for cancer patients 30 inadequate pharmacokinetic profile, and excessive cost. receiving chemotherapy or radiotherapy, for transplant Development of antifungal agents is a challenge because patients receiving antirejection agents, and for patients that there are very few potential drug targets unique to fungi. have received toxic chemicals, metals and radiation exposure. Experts in fungal field agree that new drugs needs to be Fungal opportunistic infections such as candidiasis, crypto developed. Three novel azoles that offer improved potency coccosis, and histoplasmosis, occur frequently in patients 35 and a wide spectrum of activity are in late-stage development: with AIDS. Among the opportunistic infections, fungal infec Voriconazole, posaconazole, and ravuconazole. Another new tions caused by Pneumocystis, Candida, Cryptococcus, or class of agents, the candins—are a novel generation of cell Histoplasma are very common and prevalence can be as high wall active semisynthetic 1.3 beta-glucan inhibitors—caspo as 85% among HIV-infected individuals. The incidence of fungin, micafungin, and anidulafungin. There is also systemic fungal infections increased 600% in teaching hos 40 so-called Nyotran, a novelliposomal formulation of nystatin. pitals and 220% in non-teaching hospitals during the 1980s. In addition, a new class of protein synthesis inhibitors, the The most common clinical isolate is (com Sordarins, are in preclinical development. Resistance to anti prising about 19% of all isolates). Nearly 40% of all deaths fungals has become more apparent in recent years and may from hospital-acquired infections were due to fungi. worsen with the increase in prophylatic therapy even with The treatment of fungal infections has lagged behind bac 45 new drugs being developed. A small number of experts terial chemotherapy. There are substantially fewer antifungal believe that in addition to new drug discovery strategy an drugs than antibacterial drugs. The majority of known anti alternative strategy for overcoming this and other problems is fungal agents fall into one of three main groups. The major development of a vaccine. group includes polyene derivatives, including amphotericin B A number of fungal vaccines have been proposed in the and the structurally related compounds nystatin and pimari 50 past: U.S. Pat. Nos. 4,229.434 and 4.368,191 issued to cin, which are only administered intravenously. These are Sarkisov et al., disclose a live vaccine for prophylaxis broad-spectrum antifungals that bind to ergosterol, a compo and treatment of trichopytosis caused by men nent of fungal cell membranes, and thereby disrupt the mem tagrophytes. U.S. Pat. Nos. 5.277,904 and 5,284,652 issued to branes, leading to cell death. Amphotericin B is usually effec Pier disclose a broad spectrum dermatophyte vaccine for the tive for systemic mycoses, but its administration is limited by 55 prophylaxis of dermatophyte infection in animals, such as toxic effects that include fever and kidney damage, and other guinea pigs, cats, rabbits, horses and lambs. This vaccine accompanying side effects such as anemia, low blood pres comprises a suspension of killed T. equinum, T. mentagro Sure, headache, nausea, vomiting and phlebitis. The unrelated phytes (var. granulare), M. Canis and M. gypseum in an effec antifungal agent flucytosine (5-fluorocytosine), an orally tive amount combined with an adjuvant. U.S. Pat. Nos. 5.453, absorbed drug, is frequently used as an adjunct to amphoteri 60 273 and 6,132,733 issued to Werner et al., disclose a cin B treatment for Some forms of candidiasis and cryptococ ringworm vaccine comprising an effective amount of a cal meningitis. Its adverse effects include bone marrow homogenized, formaldehyde-killed fungi, i.e., Microsporum depression with leukopenia and thrombocytopenia. canis culture in a carrier. U.S. Pat. No. 5,948,413 issued to The second major group of antifungal agents includes Mendoza discloses a method and vaccine for treatment of azole derivatives which impair synthesis of ergosterol via 65 pythiosis in humans and animals. Vaccine comprises a mix lanosterol demethylase and lead to accumulation of metabo ture of extracellular and intracellular proteins and enables lites that disrupt the function of fungal membrane-bound cures of chronic pythiosis. However, the number of patents US 7,914,799 B2 3 4 relating to a fungal vaccine is much smaller than those dis iosum, Pityrosporum ovale, Saccharomyces cerevisiae, Sac closing new antifungal drugs. Most of vaccine work was done charomyces boulardii, Saccharomyces pombe, Scedosporium by Scientists who were outside of mainstream of fungal drug apiosperum, , beigelii, developers since fungal experts' establishment was not really Penicillium marineffei, spp., Fonsecaea spp., concerned or convinced by the idea that vaccines would work. Wangiella spp., Sporothrix spp., Basidiobolus spp., Conid Indeed only two commercial antifungal vaccines are cur iobolus spp., Rhizopus spp. Mucor spp., Absidia spp., Mortier rently available. The pioneering work in the former Soviet ella spp., Cunninghamella spp., Saksenaea spp. Union has resulted in ringworm vaccine for which is The concept of present vaccine is directly applicable to sold as Ringvac bovis LTF-130 (Alpharma, Oslo, Norway). fungi causing disease in plants. These parasites include fungi Another veterinarian vaccine similar to Soviet live vaccine is 10 from generalike Alternaria spp. Curvularia spp., Helminthos Czech live vaccine Bioveta (Bioveta, Ivanovice na Hane, porium spp., spp., spp., Penicillium spp. Czech Republic). Monolinia spp., Rhizoctonia spp., Paecilomyces spp., Pithony While thus there are commercial fungal vaccines and ces spp., and Cladosporium spp. Due to immune cross-reac experimental prototype vaccines which have potential thera tion between some of plant/food fungi with animal fungi peutic and prophylactic properties, without exception, they 15 these organisms are equally effective as immunogenic prepa are all delivered by an injection. No oral fungal vaccines with ration for creating vaccine against human or veterinarian proven clinical benefit exist at the present time. This is mainly fungal infections. due to the deep-rooted conviction that vaccine antigens will The method for preparing the vaccine according to the degrade in the stomach and thus the activity of the vaccine present invention for prophylaxis and treatment of fungal will be annihilated effectively rendering such a vaccine use infection involves growing the fungi of interest on a nutrient less. This notion applies not only to oral fungal vaccine but all medium containing sources of carbon, nitrogen, and other other vaccines in general. biologically active ingredients to an optimal level, processing Currently scientists are trying to obviate this problem by fungi such that obtained components thereof are not degrad developing special encapsulation means to prevent a vaccine able upon oral absorption. In accordance with the present degradation. However, oral vaccine development is proving 25 invention the immunogenic character of a specific fungus lengthy and complex and, at the present time, there is no species, the Suspension of fungus cells is lyzed, dried, and completely satisfactory antifungal vaccine that is orally avail formulated into an oral pill preferably without any conven able. tional immune adjuvant. The incidence of fungal infections caused by the opportu While preferred method of administering is oral other nistic fungal infections has increased significantly in recent 30 means of delivery, e.g., parenteral, are equally contemplated. years. The use of antifungal drugs still causes major problems Vaccine can be administered alone or together with other due to widespread drug resistance and the toxicity related vaccines. Vaccine can be advantageously administered along effects of present chemotherapy. The availability of non-toxic with established and experimental antifungal drugs. Vaccine and effective oral vaccine against pathogenic fungi would be can be also administered with immune adjuvant. Non-limit advantageous. Present inventors have Surprisingly discovered 35 ing examples Quil ATM, Freund's Incomplete Adjuvant, anhy an effective broad-spectrum antifungal vaccine which is drous lipids, cholera toxin produced by various strains of readily available orally. Vibrio cholerae, the heat-labile enterotoxin produced by Some enterotoxigenic strains of Escherichia coli, and alumi SUMMARY OF THE INVENTION num hydroxide among many others. Other strategies are not 40 excluded to enhance the mucosal immunization, including The preferred embodiment of this invention comprises an the use of attenuated mutants of bacteria (e.g., Salmonella oral antifungal Vaccine prepared according to processes of the spp.) as carriers of heterologous antigens, encapsulation of invention. Accordingly another embodiment provides a pro antigens into microspheres, gelatin capsules, different formu cess of making oral antifungal vaccine that is effective against lations of liposomes, adsorption onto nanoparticles, use of various fungal pathogens including but not limited to Der 45 lipophilic immune stimulating complexes, and addition of matophytres like Epidermophyton floccusum, Microsporum bacterial products with known adjuvant properties. audouini, , Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum DETAILED DESCRIPTION OF THE INVENTION nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton 50 Fungi are eukaryotic cells that may reproduce sexually or megnini, Trichophyton mentagrophytes, Trichophyton asexually and may be biphasic, with one form in nature and a quinckeanum, , Trichophyton Schoen– different form in the infected host. Fungal diseases are leini, , Trichophyton verrucosum, T. referred to as mycoses. Some mycoses are endemic, i.e. infec verrucosum var. album, var. discoides, var. ochraceum, Tri tion is acquired in the geographic area that is the natural chophyton violaceum, and/or Trichophyton faviforme, 55 habitat of that fungus. These endemic mycoses are usually Other contemplated fungal pathogens comprise Aspergil self-limited and minimally symptomatic. Some mycoses are lus filmigatus, Aspergillus flavus, Aspergillus niger, Aspergil chiefly opportunistic, occurring in immunocompromised lus nidulans, Aspergillus terreus, Aspergillus sydowi, patients such as organ transplant patients, cancer patients Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces undergoing chemotherapy, burn patients, AIDS patients, or capitatus, Candida albicans, Candida enolase, Candida 60 patients with diabetic ketoacidosis. Fungal infections are tropicalis, , , Candida becoming a major health concern for a number of reasons, parapsilosis, Candida Stellatoidea, Candida parakawsei, including the limited number of antifungal agents available, , Candida pseudotropicalis, Candida the increasing incidence of species resistant to older antifun guilliermondi, Cladosporium carrionii, immi gal agents, and the growing population of immunocompro tis, Blastomyces dermatidis, , 65 mised patients at risk for opportunistic fungal infections. Geotrichum clavatum, , Paracoc Neutropenic patients (due to, e.g., chemotherapy, immuno cidioides brasiliensis, Pneumocystis carinii, Pythium insid Suppressive therapy, infection, including AIDS, or an other US 7,914,799 B2 5 6 wise dysfunctional immune system) are predisposed to the commonly fatal, so aggressive diagnosis and treatment is development of invasive fungal infections, most commonly required. The vaccine of the invention provides effective including Candida species and Aspergillus species, and, on therapy against aspergillosis. occasion, Fusarium, TrichospOron and Dreschlera. Cryptoc is a chronic fungal infection of the skin, cocus infection is also common in patients on immunosup hair or nails by , which include members of the pressive agents. Histoplasmosis, caused by Histoplasma, is species Trichophyton, Microsporum and Epidermophyton. the most common endemic respiratory in the United Infection of the foot (tinea pedis), Scalp () are States; over 40 million people have been infected. The disease most common, although widespread infection on non-hair is noncontagious and ordinarily self-limited, but chronic pull bearing skin () also occurs. Clinical manifesta monary infection and disseminated infection may occur. Pul 10 tions vary and may present on the skin as fissuring or lesions monary infection rarely requires treatment, but disseminated with Scaling, vesicles or pustules (and alopecia on the scalp), infection may be treated with amphotericin B. Coccidioido or on the nails as discolored or chalky, crumbling nails. Both mycosis, caused by Coccidioides, is a noncontagious respi topical and systemic therapies may be used to treat dermato ratory mycosis prevalent in the southwest United States. It phyte infection, including topically administered imidazoles also is usually self-limited but may lead to chronic pulmonary 15 and triazoles (such as itraconazole, miconazole, ketoconZaole infection or disseminated infection. Blastomycosis, caused and econZaole), haloprogin, undecylic acid, ciclopiroX ola by Blastomyces is a noncontagious, Subacute or chronic mine, tolnaftate and . Often these chemotherapies endemic mycosis most commonly seen in the Southeast fail to provide relief but patients who took the present vaccine United States. Most pulmonary infections are probably self are Surprisingly responding at higher rate than with conven limited. Paracoccidioidomycosis, caused by Paracoccidio tional therapy. ides, is a noncontagious respiratory mycosis that is the most Fusarium species can cause localized or hematogenously common systemic mycosis in South America. It may be acute disseminated infection (fusariosis), most frequently in and self-limited or may produce progressive pulmonary dis patients who have a hemopoietic malignancy and neutrope ease or extrapulmonary dissemination. Disseminated disease nia. Abrupt onset of fever, sometimes with myalgia, is fol is generally fatal in the absence of therapy. The present inven 25 lowed in the majority of cases by distinctive skin lesions tion addresses the problem of the prior art and provides an resembling ecthyrna gangrenosum. Infection can be treated effective vaccine and methods of administration. with amphotericin B but recovery depends ultimately on alle Cryptococcosis is a noncontagious, often opportunistic viation of neutropenia. Mortality typically exceeds 90%. The mycosis. It is characterized by respiratory involvement or vaccine of the invention does not cause any adverse reactions hematogenous dissemination, often with meningitis. A major 30 let alone neutropenia. etiologic agent is C. neoformans. Most pulmonary infections is an acute Suppurative opportunistic are probably overlooked, but cryptococcal meningitis, which mycosis that produces rhinocerebral, pulmonary or dissemi accounts for 90% of reported disease, is dramatic and seldom nated disease in immunocompromised patients, and local or overlooked. Cryptococcosis is a particular problem in immu disseminated disease in patients with burns or open wounds. nocompromised patients; cryptococcal meningitis occurs in 7 35 Infection is caused by fungi in the class Zygomycetes, and to 10% of AIDS patients. The principal symptom of menin include Basidiobolus, Conidiobolus, Rhizopus, Mucor, gitis is headache; associated findings include mental changes, Absidia, Mortierella, Cunninghamella, and Saksenaea. Rhi ocular symptoms, hearing deficits, nausea, vomiting, and sei nocerebral mucormycosis accounts for about half of all cases Zures. Without treatment, 80% of patients die within two of mucormycosis. It is one of the most rapidly fatal fungal years. In meningitis, cryptococci can be observed in India ink 40 diseases, with death occurring within 2-10 days in untreated preparations of cerebrospinal fluid sediment, and can be cul patients. Early clinical signs include nasal stuffiness, bloody tured from the cerebrospinal fluid. Treatment is generally nasal discharge, facial Swelling and facial pain. The infection with fluconazole or the combination of amphotericin B and then spreads to the eyes, cranial nerves and brain. Pulmonary flucytosine, although amphotericin B does not cross the blood mucormycosis is nearly as common as rhinocerebral disease brain barrier. The vaccine of the invention is effective against 45 and manifests with the same necrotizing and infarction as fungal infection in the brain. aspergillosis. Fungi are virtually never seen or cultured from Aspergillosis is a term that encompasses a variety of dis blood, sputum or cerebrospinal fluid. Disseminated mucor ease processes caused by Aspergillus species. Aspergillus mycosis may follow pulmonary or burn wound infection. species are ubiquitous; their spores are constantly being Treatment is with amphotericin B. In this clinical application inhaled. Of the more than 300 species known, only a few are 50 the vaccine of the invention is more effective than amphot ordinarily pathogenic for man: A. fumigatus, A. flavus, A. ericin B. niger, A. nidulans, A. terreus, A. Sydowi, A. flavatus, and A. Candidiasis is a general term for a variety of local and glaucus. Aspergillosis is increasing in prevalence and is par systemic processes caused by colonization or infection of the ticularly a problem among patients with chronic respiratory host by species of the Candida. Candidiasis occurs disease or immunocompromised patients. Among immuno 55 worldwide; superficial infections of the skin, mouth and other compromised patients, aspergillosis is second only to can mucus membranes are universal. Invasive systemic disease didiasis as the most common opportunistic mycosis and has become a problem due to the use of high doses of antibi accounts for about 15% of the systemic mycoses in this otics that destroy normal bacterial flora, immunosuppressive group. Opportunistic pulmonary aspergillosis is character agents, and agents toxic to bone marrow, e.g., during cancer ized by widespread bronchial erosion and ulceration, fol 60 therapy. Neutropenia is a major risk factor for Candida dis lowed by invasion of the pulmonary vessels, with thrombosis, semination. Candidiasis is also seen among immunocompro embolization and infarction. Clinically, infection manifests mised individuals such as AIDS patients, organ transplant as a necrotizing patchy bronchopneumonia, sometimes with patients, patients receiving parenteral nutrition, and cancer hemorrhagic pulmonary infarction. In about 40% of cases, patients undergoing radiation treatment and chemotherapy. It there is hematogenous spread to other sites. Aspergillosis is 65 is the most common opportunistic mycosis in the world. The also a rare but devastating complication of burn wounds; most common etiologic agent is Candida albicans. Other amputation is often required for cure. Invasive aspergillosis is infectious species include C. tropicalis, C. parapsilosis, C. US 7,914,799 B2 7 8 Stellatoidea, C. krusei, C. parakawsei, C. lusitaniae, C. include C. tropicalis, C. parapsilosis, C. Stellatoidea, C. pseudotropicalis, C. guillermondi and C. glabrata. Candida kusei, C. parakwisei, C. lusitaniae, C. pseudotropicalis, C. albicans is normally found in the mouth, throat, gastrointes guilliermondi and C. glabrata. Candida albicans is normally tinal tract and vagina of humans. Non-albicans species fre found in the mouth, throat, gastrointestinal tract and vagina of quently colonize skin. Candida species occur in two forms humans. Non-albicans species frequently colonize skin. Can that are not temperature- or host-dependent. The usual colo dida species occur in two forms that are not temperature- or nizing forms are that may assume a pseudomycelial host-dependent. The usual colonizing forms are yeasts that configuration, especially during tissue invasion. Pseudo may assume a pseudomycelial configuration, especially dur myceliae result from the sequential budding of yeasts into ing tissue invasion. Pseudomyceliae result from the sequen branching chains of elongated organisms. Candida albicans 10 contains cell wall mannoproteins that appear to be respon tial budding of yeasts into branching chains of elongated sible for attachment of the yeast cells to specific host tissues. organisms. Candida albicans contains cell wall mannopro C. albicans also binds avidly to extracellular matrix (ECM) teins that appear to be responsible for attachment of the yeast proteins such as fibronectin, laminin, and types I and IV cells to specific host tissues. C. albicans also binds avidly to collagen, all of which contain heparin-binding domains. 15 extracellular matrix (ECM) proteins such as fibronectin, Clinically, candidiasis manifests as Superficial mucocutane laminin, and types I and IV collagen, all of which contain ous infections, chronic mucocutaneous candidiasis, or sys heparin-binding domains. Clinically, candidiasis manifests temic infection. Superficial mucocutaneous infections can as Superficial mucocutaneous infections, chronic mucocuta occur in any area of skin or mucus membrane. Thrush, com neous candidiasis, or systemic infection. Superficial muco monly seen in AIDS patients, is characterized by a patchy or cutaneous infections can occur in any area of skin or mucus continuous, creamy pseudomembrane that covers the tongue, membrane. Thrush, commonly seen in AIDS patients, is char mouth, or other oropharyngeal Surfaces and may be accom acterized by a patchy or continuous, creamy pseudomem panied by ulceration and necrosis. Intestinal candidiasis is brane that covers the tongue, mouth, or other oropharyngeal commonly asymptomatic, but is a major source of hematog Surfaces and may be accompanied by ulceration and necrosis. enous invasion in immunocompromised individuals. Inter 25 Intestinal candidiasis is commonly asymptomatic, but is a trigo involves the axillae, groins, inframammary folds, and major source of hematogenous invasion in immunocompro other warm, moist areas, and may manifest as red, oozing or mised individuals. Intertrigo involves the axillae, groins, dry, scaly lesions. Infections may occur in other areas, includ inframammary folds, and other warm, moist areas, and may ing perianal and genital areas. , infection of the manifest as red, oozing or dry, Scaly lesions. Infections may nails, often follows chronic exposure of the hands or feet to 30 occur in other areas, including perianal and genital areas. moisture. Some patients with limited T-cell immunodefi Paronychia, infection of the nails, often follows chronic expo ciency develop chronic mucocutaneous candidiasis. These sure of the hands or feet to moisture. Some patients with patients Suffer from persistent Superficial Candida infection limited T-cell immunodeficiency develop chronic mucocuta of the skin, Scalp, nails and mucus membranes. Most cases of neous candidiasis. These patients suffer from persistent systemic candidiasis are caused by Candida albicans and C. 35 Superficial Candida infection of the skin, Scalp, nails and tropicalis, and increasingly, C. glabrata. Clinical manifesta mucus membranes. Most cases of systemic candidiasis are tions of Candida infection appear mainly in the eyes, kidneys caused by Candida albicans and C. tropicalis, and increas and skin. In the eyes, there may be single or multiple raised, ingly, C. glabrata. Clinical manifestations of Candida infec white, fluffy chorioretinal lesions. These lesions are a poten tion appear mainly in the eyes, kidneys and skin. In the eyes, tial cause of blindness. Involvement of the kidneys includes 40 there may be single or multiple raised, white, fluffy chori diffuse abscesses, capillary necrosis and obstruction of the oretinal lesions. These lesions are a potential cause of blind ureters. Infection may result in progressive renal insuffi ness. Involvement of the kidneys includes diffuse abscesses, ciency. Systemic Candida infection can also manifest as capillary necrosis and obstruction of the ureters. Infection maculonodular skin lesions Surrounded by a reddened area; may result in progressive renal insufficiency. Systemic Can these lesions have an appearance similar to acne but are a 45 dida infection can also manifest as maculonodular skin major clue to a potentially lethal disease. Other manifesta lesions Surrounded by a reddened area; these lesions have an tions of systemic candidiasis may include osteomyelitis, appearance similar to acne but area major clue to a potentially arthritis, meningitis, and abscesses in the brain, heart, liver, lethal disease. Other manifestations of systemic candidiasis spleen and thyroid. Involvement of the lungs is also common, may include osteomyelitis, arthritis, meningitis, and but pulmonary lesions are usually too small to be seen on 50 abscesses in the brain, heart, liver, spleen and thyroid. chest X-ray. Finally, Candida endocarditis can occur in Involvement of the lungs is also common, but pulmonary patients receiving prolonged intravenous therapy or cardiac lesions are usually too small to be seen on chest X-ray. valve implants, or in intravenous drug abusers. Fungallesions Finally, Candida endocarditis can occur in patients receiving appear on the valves, and can embolize and occlude large prolonged intravenous therapy or cardiac valve implants, or blood vessels. The mortality rate from systemic candidiasis is 55 in intravenous drug abusers. Fungal lesions appear on the about 50%. Systemic candidiasis may be treated with flu valves, and can embolize and occlude large blood vessels. conazole, a fungistatic agent, or amphotericin B, a fungicidal The mortality rate from systemic candidiasis is about 50%. agent although systemic use of the latter is limited by its Systemic candidiasis may be treated with fluconazole, a toxicity. Infection of the cornea and conjunctiva, including fungistatic agent, or amphotericin B, a fungicidal agent keratoconjunctivitis, can result from infection by amoeba, 60 although systemic use of the latter is limited by its toxicity. viruses, fungi and bacteria. Debilitated patients can develop Infection of the cornea and conjunctiva, including keratocon keratitis from fungi such as Candida or Fusarium which is junctivitis, can result from infection by amoeba, Viruses, often associated with corneal ulceration and can lead to scar fungi and bacteria. Debilitated patients can develop keratitis ring with severe visual loss. Over 80% of patients with Can from fungi such as Candida or Fusarium which is often asso dida respond to the therapy with instant composition. com 65 ciated with corneal ulceration and can lead to Scarring with mon opportunistic mycosis in the world. The most common severe visual loss. Over 80% of patients with Candida etiologic agent is Candida albicans. Other infectious species respond to the therapy with instant composition. US 7,914,799 B2 9 10 Many common ailments are actually dermatophyte infec efits the most. The dose finding study is a routine clinical tions. Tinea pedis (athlete's foot or ringworm of the feet) is exercise well established in the clinical art. associated with Epidermophyton floccuSum, various species The compounds may also be used to treat infections caused of Trichophyton and, rarely, species of Microsporum and by protozoa Such as Toxoplasma, Cryptosporidium, Leish other fungi. Tinea unguium (ringworm of the nails) is caused mania, Tripanosoma, Giardia and Trichomonas. For example, by Trichophyton rubrum. (“Jock itch' of ring they can be used in agriculture or related fields for the control worm of the groin) results from infection with Epidermophy of phytopathogenic fungi such as Alternaia Solani, Blumeria ton floccusum and species of Trichophyton. Tinea corporis graminis, Botrytis cinerea, Cercospora beticola, Cladospo (ringworm of the body) is caused by various species of Tri rium herbarum, Corticium rolfsii, Guignardia bidwellii, Hel 10 minthosporium tritici repentis, Leptosphaeria nodorum, chophyton and Microsporum, involves the Smooth and hair Magnaporthe grisea f. sp. oryzae, Micronectriella nivalis, less skin and results in either simple Scaling or deep granu Monilinia fructigena, Mycosphaerella fijiensis, Mycosphaer lomas. Tinea imbricata (scaly ringworm) is a disease of the ella musicola, Mycosphaerella ligulicola, Mycosphaerella tropics and is apparently caused by a single fungus, Tricho pinodes, Phomopsis viticola, Plasmopara viticola, phyton concentricum. (barber's itch or ring 15 Podosphaera leucotricha, Pseudopeziza tracheiphila, Phy worm of the beard) is caused by various species of Tricho tophthora infestans, Puccinia recondita, Pyrenophora teres, phyton and Microsporum. Tinea capitis (ringworm of the Rhizoctonia Solani, Venturia inaequalis, Uncinula necator Scalp and hair) is most common in children but may affect and Scierotinia Scierotiorum, in particular for the residual adults. The causative organisms, various species of Tricho control of Blumeria graminis, Plasmopara viticola, Puccinia phyton and Microsporum, may be acquired by contact with recondita and Pyrenophora teres, and for the curative control infected animals or children. Microsporum audouini is most of Puccinia recondita. commonly involved but Microsporum canis and For example the compounds of the invention may be used Microsporum gypsum (gypseum) produce deeper, more in combination with one or more other antifungal agents. Such severe lesions. Trichophyton tonsurans is also known to pro as a polyenic derivative e.g. (Amphotericin B. Nystatin, a duce widespread infections in the scalp. The significance of 25 lipid formulation of Amphotericin B), 5-fluctyosine, anazole skin pH in the development of ringworm is widely known. derivative e.g. (Fluconazole, Intraconazole, Ketoconazole, The Susceptibility of humans to ringworm is much greater Miconazole, Clotrimazole, ZD-08070, UK-109496, SCH before puberty than afterwards when the skin pH falls from 56592), 5-Fluorocytosine, a Pneumocandin or Echinocandin about 6.5 to about 4.0. This change is largely due to excretion derivative such as Cilofungin, LY-303366, L733560, of fatty acids in the sebum and these fatty acids are often 30 L-743872 or other cell wall active compound such as Nikko highly fungistatic. For this reason, various kinds of topically mycin Z. Sordarin, Steroids, and/or one or more immuno applied agents have been used to kill the infecting fungus and modulating agents such as an interferon e.g. IFN-), interleu relieve the condition. Many treatments for ringworm are kine e.g. (IL-1, IL-2, IL-3 and IL-8) and colony stimulating based upon alteration of skin pH by topically applying Vari factors, (G)-CSF, (M)-CSF and (GM)-CSF and defensins. ous agents (e.g., propionic acid, undecylenic acid). Other 35 Particularly advantageous compounds for use with com ringworm therapies have relied upon other topically applied pounds of the invention include Intraconazole, Flucytosine, commercially available products such as Conofite and Cap Fluconazole, Amphotericin B, Natamycin, primaricin, whey, tan. Orally-administered agents (e.g., and Keto and other well known in the art. conazole) are also available. The compositions of the present invention can include Unfortunately, however, post-infection treatment cannot 40 antiinflammatory steroids. Such steroids are exemplified, but completely prevent in many instances. Once therapy is dis not limited to, Betamethasone dipropionate, Clobetasol pro continued, reinfection usually occurs. It would therefore be pionate. Diflorasone diacetate, Halbetosal, 2 Amicinonide, desirable to provide a vaccine against fungus to prevent infec Desoximetasone, Diflorasonediacetate, Fluocinonide, Halci tion before these adverse effects are suffered. One of the nonide, MometaSone, Triamcinolone acetonide, 3 Amicino objects of the present invention is to provide a prophylactic 45 nide, Betamethasone benzoate, Diflorasone diacetate, Fluti vaccine which contains fungal antigen or immunogen and casone, Fluocinonide, Halocinonide, Fluocinolone, effectively prevents the fungal infection. Preferably this vac Flurandrenolide, Halcinonide, Hydrocortisone, Clocortal cine is oral or delivered transmucosally. one, Predincarbate, 6 Aclometasone, Desonide, and/or Meth The term “immunogen' or “antigen' as used hereinafter ylprednisolone. comprises any entity capable of producing a protective anti 50 Mycoplasmas of veterinary or medical interest include body or cell-mediated immunological response against a Mycoplasma bovis, M. gallisepticum, M. agalactiae, M. pathogenic organism in an animal. The antigen or immuno hyopneumoniae, M. pneumoniae, M. Synoviae, M. arthritidis, gen can be whole pathogen or or part of the pathogen includ M. capricolium, M. dispar; M. hominis, M. mycodies subs ing any cell component, a protein, glycoprotein, glycolipid, capri, M. Orale, M. Oripneumoniae, M. pulmonis, M. Cynos, polysaccharide or lipopolysaccharide which belongs or is 55 M. hyorhinis, M. mycoides, M. Salvarium and M. fermentans. associated with the pathogen, or it may be a polypeptide or A vaccine in accordance with the invention is formulated in other entity which mimics all or part of Such a pathogen or conjunction with or without at least one pharmaceutically protein, glycoprotein, glycolipid, polysaccharide or acceptable carrier. There are numerous and diverse types of lipopolysaccharide thereof. An example of fungal polysac acceptable carriers which are readily appreciated by those charide is schizophyllan. 60 skilled in the art depending on the route of vaccine adminis In general the immunological preparation used according tration. While preferred route is oral other routes of vaccine to the present invention comprise by weight 0.0000001 to administration that are appropriate in the practice of the 20%, and more often 0.000001 to 5% of an immunogen. No invention include rectal, vaginal, intraintestinal, transdermal, specific adjuvant is required as in all other vaccines of the intranasal, parenteral, i.e., intramuscular, intraperitoneal, prior art. The precise effective dose is determined by titration 65 intradermal, intravenous, and implant. Optimized efficacy or serial dilution of the immunogen to determine the most can be achieved in certain instances by combining two dif effective concentration from which the treated patient ben ferent routes of administration in a course of therapy. For US 7,914,799 B2 11 12 example, intraintestinal administration followed by oral tab erably, the nasal dosage form should be isotonic with nasal lets or liquid formulation or intravenous administration fol secretions. Although vaccine can be formulated in water, lowed by intraperitoneal administration. more preferably it will beformulated in a solution buffered to For oral administration, vaccine can be formulated with a a pH of between about 3.0 and 8.0, and most preferably pH pharmaceutically acceptable solid or liquid carrier. Solid 5.0–5.4 using e.g., a buffer system Such as an acetate buffer, a form preparations include powders, tablets, pills, capsules, phosphate buffer, a citrate buffer, and a succinate buffer. cachets, and dispersible granules. A solid carrier can be one or Formulations Suitable for parenteral administration conve more Substances which may also act as diluents, flavoring niently comprise sterile aqueous preparations of vaccine agents, solubilizers, lubricants, Suspending agents, binders which are preferably isotonic with the blood of the recipient. like calcium and magnesium, preservatives, tablet disinte 10 Suitable carrier solutions include phosphate buffered saline, grating agents, or an encapsulating material. saline, water, lactated ringers or dextrose. Such formulations In powders, the carrier is a finely divided solid which is in may be conveniently prepared by admixing vaccine with a mixture with the finely divided active component. In tablets, water to produce a solution or Suspension which is filled into the active component is mixed with the carrier having the a sterile container and sealed against bacterial contamination. necessary binding properties in Suitable proportions and com 15 Preferably, sterile materials are used under aseptic manufac pacted in the shape and size desired. turing conditions to avoid the need for terminal sterilization. The powders, capsules and tablets preferably contain from Such formulations may optionally contain one or more 0.00001 to about seventy percent of the active compound. additional ingredients among which may be mentioned pre Suitable carriers are magnesium carbonate, or chloride, mag servatives (when the formulations are presented in multi-dose nesium Stearate, talc, Sugar, lactose, pectin, dextrin, starch containers), buffers to provide a suitable pH value for the gelatin, tragacanth, methylcellulose, sodium carboxymethyl formulation, and Sodium chloride, or glycerin, to render a cellulose, microcrystalline cellulose, a low melting wax, formulation isotonic with the blood. cocoa butter, and the like. For parenteral administration, vaccine can be used in free The term “preparation” as used herein is intended to or salt form (for example, salts of alkali and alkaline earth include the formulation of the active compound with encap 25 metals such as Sodium, magnesium and calcium, salts if min Sulating material as a carrier providing a capsule in which the eral acids such as HCl and Sulfuric acid, or salts of organic active component, with or without other carriers, is Sur acids, such as acetic acid. Amine addition salts can also be rounded by a carrier, which is thus in association with it. used in the practice of the invention, for example a phosphate Similarly, cachets and lozenges are included. Tablets, pow amine addition salt. Examples of typical carriers are sterilized ders, capsules, pills cachets, and lozenges can be used as Solid 30 water, saline, and phosphate buffered saline. Optional addi dosage forms suitable for oral administration. tives include isotonic agents, stabilizers, pH controlling Another type of solid carrier useful in the practice of the agents, agents necessary for the proper infusion of solutions, invention is a foodstuff. Solid foodstuffs suitable for admix and water soluble nutrients. ture with a therapeutic dosage or unit dosage of vaccine are, Transdermal administration can be accomplished using for example, cereals, chewing gum, crackers, candies, meats, 35 preparations in the form of ointments, emulsions, lotions, vegetable and fruit preparations for babies, and cookies. For Solutions, creams or transdermal patches. Suitable pharma Veterinary applications, vaccine can be admixed directly into ceutical carriers for transdermal administration include, for a grain ration or incorporated into a salt block. Likewise, example, polyethylene glycol, propylene glycol, isopropanol, vaccine can be formulated with a liquid foodstuff, for ethanol, oleic acid, N-methylpyrrolidone, Sesame oil, olive example, milk, infant formula, juices, liquid vitamin Supple 40 oil, wood alcohol ointments, Vaseline, and paraffin, or mix ments, soft drinks, e.g., CocaCola, and oral rehydration solu tures thereof. When vaccine is formulated in a transdermal tions. patch, the therapeutic dose can be incorporated either directly Liquid form preparations include Solutions, Suspensions, in an adhesive layer that fixes a drug impermeable backing to emulsions, for example, of water aqueous solution, or other the skin of the treated-Subject, or can be incorporated in a liquids, half-liquid bases, or optionally in pharmaceutically 45 matrix layer and released therefrom in controlled fashion. acceptable solvents (e.g., DMSO-propylene glycol solu Suitable adhesive layer carriers for vaccine include, for tions). example, polyisobutenes, polyisobutylenes, polyacrylates, Also included are solid form preparations which are polyurethanes, polysiloxanes, polystyrene copolymers, intended to be converted, shortly before use, to liquid for EVA-copolymer, and polyether amide block copolymers. preparation for oral or rectal administration. Such liquid 50 Suitable drug-releasing matrices include, for example, forms include solutions, Suspensions, and emulsions. These natural or synthetic rubbers, polymeric materials. Such as preparations may contain, in addition to the active compo EVA copolymers, thickened mineral oil, and petroleum jelly. nent, colorants, flavors, stabilizers, buffers, artificial and Optional constituents for the transdermal administration of natural Sweeteners, dispersants, thickeners, Solubilizing vaccine include drug permeable rate-controlling membranes agents, thermal converting agents capable of converting Solid 55 and penetration enhancers which are well known to those into liquid at body temperature, and the like. skilled in the transdermal formulation art. For intranasal administration of vaccine, the choice of suit Suppository administration is particularly well suited for able carriers is highly dependent upon the exact nature of the patients with disorders in digestive organs and for infant nasal dosage form desired, e.g., Solutions, Suspensions, oint patients, and affords constant release over an extended period ments, or gels. Nasal dosage forms can generally contain 60 of time. Typical base carriers for Suppositories include, for large amounts of water in addition to the active ingredient. example, natural, synthetic or partially synthetic fats, waxes Minor amounts of other ingredients such as pH adjusters, and derivative thereof from animal, vegetable, or mineral emulsifiers or dispersing agents, preservatives (e.g., antimi origin. Specific examples include olive oil, corn oil, castor oil, crobials), Surfactants (e.g., non-ionics such as polysorbates) hydrogenated oils, petrolatum, Solid paraffin, ligind paraffin, jelling agents, buffering and other stabilizing agents (e.g., 65 carnuba wax, bees wax, lanolin partially or totally synthetic antioxidants and metal chelating agents) and solubilizing esters of glycerol fatty acid, mono, di, or triglycerides of agents (e.g., solubility enhancers) may also be present. Pref saturated or unsaturated fatty acids, and others well known in US 7,914,799 B2 13 14 the art. Other additives suitable for incorporation into a sup present invention can also be administered following incor pository of the invention include preservative, stabilizers, poration into liposomes or other micro-carriers, or after con Surfactants, pigments, pH modifiers and purified water. jugation to polysaccharides, proteins or polymers or in com Vaccine can also be made as an implant so that need for bination with Quil-ATM. Other adjuvants suitable for use in frequent administration is not required anymore. A preferred the present invention include conjugates comprising the implant matrix is made of a biocompatible, biodegradable, immunogen together with an integral membrane protein of bioabsorbable and/or bioerodible polymeric material that will prokaryotic origin, like TraT. become gradually disintegrated by the animals system When needed the fungal vaccine of the invention can be through enzymatic, chemical and/or cellular hydrolytic advantageously combined with other types of vaccines. Such action, and release the immunogen for Sustained delivery into 10 strategy will facilitate spontaneous vaccination programs surrounding tissue fluids over an about 0-2 year period. The against several pathogens at once. Contemplated vaccines implant may be formulated, for example, from cholesterol, include but are not limited to: Diphtheria vaccine, Pertussis cellulosic polymers, polylactide, polycaprolactone, polygly vaccine, Tetanus Vaccine, H. influenzae, Branhamella collide or other like polymers or copolymers thereof. The catarrhalis, Moraxella catarrhalis, S. pneumoniae, all sero implant may include immunostimulants such as aluminum 15 types, E. coli, endotoxin or J5 antigen (LPS, Lipid A and hydroxide, muramyldipeptide, lipophilic amines, saponins, Gentabiose), E. coli, O polysaccarides, Klebsiella, polysac Freund's incomplete adjuvant (FIA), polymeric adjuvants, charides, S. aureus, S. epidermidis, serotype polysaccharide, among other adjuvants. The implant may also contain an I, II and III (and common protective antigens), N. meningidi immunomodulator Such as a cytokine, complex carbohy tis, serotype specific or protein antigens, Polio vaccine, drate, and the like to enhance or modulate the immune Mumps, measles, rubella vaccine, Respiratory Syncytial response, and other additives as desired. Such as preserva Virus, Rabies, Hepatitis A, B, C, and others, Human immu tives, buffering agents, and the like. Other slow release forms nodeficiency virus I and II (GP120, GP41, GP160, p24, and are well known in the art, e.g., dermal patches, pumps, slow others like peptide, DNA, chimera), Herpes simplex types 1 release tablets, Suppositories, etc., they can be equally Suit and 2, CMV, EBV, Varicella/Zoster, Malaria, Tuberculosis, able to replace the implant. 25 Candida albicans, other candida, Pneumocystis carinii, The pharmaceutical preparation is preferably in unit dos Mycoplasma Influenzae virus A and B, Adenovirus, Group A age form. In Such form, the preparation is Subdivided into unit streptococcus, Group B streptococcus, serotypes, Ia, Ib, II doses containing appropriate quantities of the active compo and III, Pseudomonas servinosa (serotype specific), Rhinovi nent. The unit dosage form can be a packaged preparation, the rus, Parainfluenae, types 1, 2 and 3, Coronaviruses, Salmo package containing discrete quantities of preparation, such as 30 nella, Shigella, Rotavirus, Enteroviruses, and Chlamydia tra packeted tablets, capsules, lotions, ointments and powders in chomatis, Smallpox, yellow fever, distemper, cholera, fowl vials or ampoules. Also, the unit dosage form can be a cap pox, scarlet fever, diphtheria, otitis, whooping couch, foot Sule, tablet, cachet, or lozenge itself, or it can be the appro and mouth disease, poliomyelitis and combination thereof. priate number of any of these in packaged form. Instant vaccine can also be combined with anti-parasite It is also within the scope of the invention to administer 35 vaccines against for example nematodes like Ancylotoma vaccine in a time-release formulation Such as a bolus for caninum, Strongylus vulgaris, Tricho strongylus colubrifor veterinary therapies. A wide variety of methods are now avail mis, Haemonchus contortus, Ostertagia Ostertagi, Ascaris able in the art for preparing time-release or long-acting com suum, Toxascaris leonina, Uncinaria Stenocephala, Trichuris positions. Any of these time-release or long-acting formula vulpis, Dirofiaria immitis, Toxocara spp., Necator america tions are Suitable in the practice of the present invention as 40 nus, Ancylostoma duodenale, Ascaris lumbricoides, Trichu long as it does not adversely affect the effectiveness of vac ris trichiura, Enterobius vermicularus, Strongyloides ster cine in the treatment or prevention of fungal infection. Advan coralis or Wuchereria bancrofti. tages of time-release formulations include a lower concentra Without limiting to above parasites instant vaccine can be tion of peak serum absorption which Substantially reduces also used in combination with vaccines against Plasmodium any possible adverse side effects and/or toxicity of the active 45 falciparum, trypanosomiasis, leishmaniasis, amoebiasis, tri administered. In addition, a reduced frequency of administra chomoniasis, primary amoebic meningitis, chronic acan tion results, which Substantially improves patient compli thamoebic encephalitis and keratitis, babesiosis or combina ance. A frequency of administration of every 12 to 24 hours tion thereof. would be preferred although not necessary. In addition, a The fungal vaccine preparation of the invention can be more constant concentration of vaccine would result, and 50 combined with a vaccine which contains an antigen from a consequently, a more consistent relief or prophylaxis of dis microbe selected from the group consisting of Streptococcus ease symptoms. pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoea, Present vaccine will contain pharmaceutically and/or vet Neisseria meningitidis, Corynebacterium diphtheriae, erinarally acceptable carrier, diluent, excipient and/or adju Clostridium botulinum, Clostridium perfiringens, vant. These can comprise Marcol 52, Montanide 888, 55 Clostridium tetani, Haemophilus influenzae, Klebsiella squalane or squalene, Adjuvant 65 (containing peanut oil, pneumoniae, Klebsiella Ozaenae, Klebsiella rhinosclerona mannide monooleate and aluminium monoStearate), tis, Staphylococcus aureus, Vibrio cholerae, Escherichia coli, lipopolysaccharide, mineral gels such as aluminium hydrox Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, ide, aluminium phosphate, calcium phosphate and alum, Sur Campylobacter jejuni, Aeromonas hydrophila, Bacillus factants like hexadecylamine, octadecylamine, lysolecithin, 60 cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia dimethyldioctadecylammonium bromide, N,N-dioctadecyl pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, N, N'-bis(2-hydroxyethyl)propanediamine, methoxyhexa Shigella flexneri, Shigella sonnei, Salmonella typhimurium, decylglycerol and pluronic polyols, polyanions like pyran, Treponema pallidum, Treponema pertenue, Treponema car dextran Sulfate, polyacrylic acid and carbopol, peptides and ateneum, Borrelia vincentii, Leptospira icterohemorrhagiae, amino acids like muramyl dipeptide, glucan, dimethylgly 65 Mycobacterium tuberculosis, Toxoplasma gondii, Pneu cine, tuftsin and trehalose dimycolate. The antigens, precur mocystis carinii, Francisella tularensis, Brucella abortus, sors, expression products and/or synthetic polypeptides of the Brucella suis, Brucella melitensis, Mycoplasma spp., Rick US 7,914,799 B2 15 16 ettsia prowazeki, Rickettsia tsutsugamushi, Chlamydia spp., group by an ether bond. Examples of such solvents are meth Helicobacter pylori, , Aspergillus fumi oxy ethanol and ethoxyethanol. In addition solubilisers such gatus, Candida albicans, Blastomyces dermatitidis, Crypto as Surfactants can be added to the solvent. Examples of Suit coccus neoformans, Histoplasma capsulatum, Entamoeba able solubilizers are sodium lauryl sulphate, dioctyl sulpho histolytica, Trichomonas tenas, Trichomonas hominis, Tri Succinate, calcium chloride or Surfactants of the non-ionic chomonas vaginalis, Trypanosoma gambiense, Trypanosoma type, for instance those which are sold under the trade marks rhodesiense, Trypanosoma Cruzi, Leishmania donovani, Tween, Span, Brij and Myr. Adhesion or complex formation Leishmania tropica, Leishmania braziliensis, Pneumocystis between the fungus and the Suitable adjuvant or carrier, can be pneumonia, Enterobius vermicularis, Trichuris trichiura, achieved by adding the protein to the solution or vice versa. Ascaris lumbricoides, Trichinella spiralis, Strongyloides 10 The protein can already be present in an aqueous environment Stercoralis, Schistosoma japonicum, Schistosoma mansoni, Such as water, milk, or blood. The complex of fungus and the Schistosoma haematobium, variola virus, vaccinia virus, one or more proteins can be used directly in the desired cowpox virus, varicella-Zoster virus, Herpes Simplex virus 1, application or can be processed further. For example the Herpes Simplex virus 2, influenza viruses, parainfluenza complex can be isolated and/or purified using well known virus, mumps virus, measles virus, respiratory syncytial 15 methods. Examples of drying methods to separate the com virus, rubella virus, Hepatitis A virus, Hepatitis B virus, plex from a liquid are filtration, centrifugation, evaporation of Hepatitis C virus, Hepatitis Evirus, and Non-A/Non-B Hepa the liquid, spray drying, freeze drying and fluid bed drying. titis virus. The purified complex of fungus and one or more proteins is The invention also contemplates a method of producing a advantageous for the treatment of fungal infections. The com diagnostic and/or research reagent to detect agents that are plex can be added to edible Substances e.g., cheeses or sau characteristic of diseases caused by fungi by immunizing a Sages, agricultural products e.g., grain and Vegetables. A host with a vaccine described above so that the host produces composition of the invention may also be in the form of an antibodies or cells-mediated immune response against the emulsion (e.g. for coating) of the oil-in-water or water-in-oil agents. The antibodies and/or T cells may be then isolated type. Examples of Suitable preparations for treating agricul using standard procedures. Thus, as used herein, diagnostic 25 tural products such as fruits are films of polymeric materials reagent refers to a composition of antibodies (polyclonal or Such as for example polyolefins, e.g., polyethylene and monoclonal) that may be used to detect agents that are char polypropylene. The amount of fungus in a liquid composition acteristic of diseases. As used herein, research reagent refers for immersion or spraying treatment may be from 0.00001% to a composition of antibodies (polyclonal or monoclonal) to 2% w/v or higher as desired. Preferably the amount is from that may be used in the laboratory. Cell-mediated reaction can 30 0.0001% to 1% w/v. In principle the immersion liquid may be also be used make diagnostic reagents (e.g., Elispot-type of any kind. When an aqueous system is used, the addition of reagent) and to follow the patient’s response to immunization a surfactant may be of advantage, in particular for treating objects with a hydrophobic surface. Useful surfactants are for EXAMPLE1 example anionic tensides such as Sodium lauryl Sulphate or 35 polyethylene alkyl ethers such as Cetomacrogol(R) 1000 or One method to produce the antifungal preparation is by polyoxyethylene ethers e.g. Tween 60, 61 and 65. dissolving or hydrolyzing the Suitable fungi by increasing or reducing the pH using methods known in the art. Preferably EXAMPLE 2 the pH is above 10 or below 4. Examples of suitable aqueous An effective treatment for arresting the growth of undes acids are aqueous solutions of HCl, H2SO4, citric acid and 40 ired microbes in the preparation comprises pasteurization. lactic acid. Examples of Suitable alkaline Solutions are solu Pasteurization generally comprises partial sterilization at a tions of hydroxides such as NaOH, KOH and NH4OH. The temperature and for a period of time that destroys objection dissolved fungus can be mixed with the suitable carrier or able organisms, without major chemical alteration of the adjuvant compound, like a protein or amino acid. Preferably product. To arrest the activity of the bacteria in the composi a food grade proteins is used. Examples of Suitable proteins 45 tion of the invention, pasteurization can comprise heating the are milk proteins, e.g. whey proteins, caseins and caseinates composition to a temperature of at least about 110. degree. F., or other compounds, such as L-glutamine. Also mixtures of more preferably at least about 120. degree. F., and maintain different proteins and mixtures of proteins with other com ing that temperature for at least about 30 minutes. According pounds such as fats e.g. vegetable and animal fats or oils such to the so-called “holding method' or “batch pasteurization', as milk fat, butter fat, soya bean oil and sunflower oil can be 50 the preparation can beheated to 145. degree. F. and held at this used. Also compositions or products which contain mixtures temperature for 30 minutes with constant stirring with an of these compounds can be used. Examples of such mixtures agitator at 60 rpm. Alternatively, a high-temperature, short are cow milk or soya bean milk. Another example is blood time process, can be equally employed. This requires a tem from a normal donor or from fungus-infected patient. After perature of 162. degree. F. for as little as approximately 16 mixing the dissolved fungus with the Suitable compound, the 55 seconds. Even shorter duration processes (278. degree. F. for pH is adjusted to neutral, e.g., between 5 and 9, preferably 4-6 seconds, or 285. degree. F. for 2 seconds) can be between 6 and 8. When the suitable carrier compound was not employed, albeit at added expense. Any combination of time present, the fungus preparation can crystallize. The presence and temperature can be utilized bu those of skill in the art so of the suitable adjuvant can prevent the formation of visible long as it achieves adequate pasteurization and arrests the crystals under microscope. 60 Alternatively the complex of fungus and the Suitable com undesired microbial growth. pound is made by dissolving the fungus in a suitable solvent EXAMPLE 3 system. Suitable solvent systems are lower alcohols such as methanol, ethanol, propanol, ethylene glycol, propylene gly High-pressure commercially available CO2 gas is an ideal col and glycerol or mixtures thereof. Further, in a suitable 65 Solvent. It has the density of a liquid, but is also highly alcoholic solvent, one or more hydroxyl groups may be linked penetrable and diffusible since it has the properties of a gas at by an ester bond to a fatty acid or linked to another alcohol the same time. US 7,914,799 B2 17 18 EXAMPLE 4 agents, colorants, and ionizing radiation. Equally suitable are enzymatic treatments; including endogenous and/or exog Liquid nitrogen can be used to freeze and to cryogenically enous enzyme reactions. grind fungus preparation. For freezing, isolated fungus prepa The cryoground powder then can be further extracted with ration is immersed into liquid nitrogen for a few (1-15) sec liquid or Super-critical CO2 as a solvent. In one example the onds, with equilibration at 0. degree. F., to form Small crys solvent to feed ratio of 300 (1 lb./min.) is used. The extractor tals. This method is often more cost efficient than the use of a is maintained at 37. degree. C. at 3500 psig, first separator at mechanical freezer. The resulting cryogenic gas can be 40. degree. C. at about 1500 psig, and second separator at 29. recycled in the assembly line as fungus is brought to the degree.-30. degree. C. at about 1000 psig, where the bulk of freezing equilibrium (e.g. 0. degree. F.). Depending on the 10 extract is collected. The solvent from the second separator is needs temperature can vary from -5. degree. F. to -40. fed into a knock-out vessel at about 1500 psig and then degree. F. for less than one minute for grinding in liquid recycled. The residue in the extractor is in the range of 8-17% nitrogen. Fungus treated according to the invention can be of the feed. CO2 extracts about 83-92% of the feed which is brought to a temperature as low as -320. degree. F. According much higher than for example chloroform/methanol extrac to the present invention, fungus is Submerged in liquid nitro 15 tion. gen for a sufficient time, generally several minutes. Depend While CO2 extraction is easy and simple process one of ing on the strain of fungus, a contact time of 15-30 minutes skill in the art will know that other extraction means can be can be used in order to insure maximal processing. Liquid used to substitute this particular extraction method. Super nitrogen is also viricidal, bactericidal, and fungicidal, par critical fluid extraction may be accomplished with many dif ticularly at longer and lower cryogenic temperatures. ferent gases and Substances including but not limited Helium; The freezing the pieces of the desired fungal tissue and Neon: Argon; Krypton; Xenon; Nitrogen; Hydrogen: Oxy lowering its temperature to a critical brittles state will convert gen; Ozone: Fluorine: Ammonia; Boron Trifluoride; Carbon the unbreakable, Viscous, and sticky material like fungus into Monoxide; Hydrogen Chloride; Hydrogen Sulfide: Nitric an extremely brittle and fragile substance. Brittleness tem Oxide: Nitrogen Dioxide: Nitrous Oxide: Silane; Silane perature depends primarily on the species and composition of 25 Chlorotrifluoro; Silicon Tetra Fluoride; Sulfur Dioxide: Sul fungal tissue (water, lipids, proteins, carbohydrates, miner fur Hexafluoride; Water; Methane; Ethane: Propane; n-Bu als) and therefore its thermal properties. The rate of crystal tane; iso-Butane; Ethene (Ethylene); Propene (Propylene); lization (i.e., nucleation and crystal growth) of water will n-Butene; Ethyne (acetylene); Monofluoro Methane; Trif affect the size of crystals. Slow rate of crystallization results luoro Methane; Tetrafluoro Methane; Monochlorodifluoro; in formation of large extracellular water crystals which can 30 Methane Monochlorotrifluoro; Methane Dichlorodifluoro; cause some rupture of cell membranes, but such effects and Methane Monobromotrifluoro; Methane Monofluoro Ethane; even repeated freeze-thaw process have negligible effects in Hexafluoro Ethane; Chloropenatfluoro Ethane: Perfluoro comparison to cell rupture by cryogrinding. It is preferred, butane; and 1,1-difluro Ethylene among many others. however, to keep the physical structure of the tissue cells relatively intact up to the cryogrinding step. This means that, 35 EXAMPLE 5 all other factors being equal, the fastest possible freezing rate should be employed. In a typical run appropriately diluted fungus culture solu Once frozen brittle pieces of tissue are obtained, they are tion is poured into the batch reactor and the lid is secured. transferred to any size reduction equipment such as Waring Fungal preparation can be hydrolyzed and reduced as blender and homogenized (ground) for the desired length of 40 described in Examples above. Carbon dioxide is fed into the time, i.e., a few minutes at 22,000 RPM. At larger scales, roll reactor until pressures of about 2760 kPa, 4140 kPa or 5520 mills with both attrition and impact grinding can be used with kPa are reached. The contents of the reactor is then rapidly the same efficiency. It is important that the tissue is kept at or heated to the desired temperature, thereby increasing the below its brittle temperature throughout the grinding process. pressure further. The reactor content is then mixed for 30 min. Grinding below the brittleness temperature is required in 45 Pressure is released and the reactor content is cooled to 10. order to produce the necessary Small particle size. For this degree. C. The resulting slurry is either centrifuged using a purpose one can add, if needed, Sufficient quantities of liquid Model RC-5B Sorval Refrigerated Superspeed Centrifuge at nitrogen to the sizereduction equipment. Upon completion of approximately 5000 g for 1 hr or filtered over paper or the grinding process, an extremely fine, free-flowing and ceramic filter. The precipitate is then washed repeatedly and non-sticky cryoground tissue (“fungus powder) is produced. 50 allowed to dry. Precipitates are usually obtained at tempera The resultant powder is then can be sieved at or below its tures greater than 50. degree. C. and at pressures greater than brittleness temperature to obtain uniform particles. For this 2760 kPa. The yield of precipitate increases as the time of purpose, one of skill in the art can use Stacked Stainless Steel reaction increases. pH values during reaction are measured standard sieves. Thus, cryogenic treatment of tissue followed with a highpressure probe designed to withstand pressures up by cryogrinding of the frozen tissue, provides one with mate 55 to 6.9 Mpa. pH values decrease dramatically with increasing rials with very small particle size and enormous Surface area pressure up to 2760 kPa. The original pH is restored after the in a form Suitable for obtaining improved quantities and depressurization step in the process. The greatest recovery is qualities of desired extracts. While it is clear the above pro obtained using conventional salting methods known in the art, cess is highly advantageous the desired material can also be but these methods have the disadvantage of contaminating the treated with one or any combination of more than one of the 60 protein fractions with salt. following methods of treating the samples including crush ing, comminuting, high and low pressure pressing, flaking, EXAMPLE 6 Sonication, freeze-thaw treatment, emulsification, homogeni Zation, filtration, high speed mixing, centrifugation, mechani Filamentous fungi are Suitable for use in the present inven cal separation and thermal treatments including microwave 65 tion for the production of antifungal preparation. Preferred treatment. Without limiting the sample can be treated with filamentous fungi include those of the class known as inorganic and organic acids, bases, solvents, Surface active Oomycetes. Members include Pythium ultimum (ATCC US 7,914,799 B2 19 20 11123), Pythium debaryanum (ATCC 9998), Pythium peripheral nervous system tissues and organs (cranial nerves, sp(ATCC 11270) and Pythium irregulare (ATCC 10951) spinal nerves, etc.); myocardial and vascular tissues and Pythium irregulare (ATCC 10951). Preferred fungi are those organs (heart, arteries, and veins); circulatory tissues and that cause pathogenesis in humans, e.g., Aspergillus fumiga organs (whole blood or components thereof, e.g., erythro tus. Any suitable fermenter can be used in the practice of the cytes, lymphocytes leukocytes, platelets, plasma); lymphatic present invention. Because filamentous fungi exhibit a strong system tissues and organs (lymph nodes, spleen, thymus); affinity for adhering to Surfaces and for Surface colonization, respiratory system tissues and organs (upper respiratory tract, a fermenter designed to take advantage of such properties can lungs); digestive system tissues and organs (including mouth, be used. The particular type of fermenter used in the practice teeth, tongue, salivary glands, pharynx, esophagus, perito of the present invention is not critical as long as the fermenter 10 neum, Stomach, Small and large intestine, liver, gallbladder, is capable of providing the conditions for filamentous fungi pancreas); skeletal tissue and organs (axial and appendicular growth. Non-limiting examples of Suitable fermenters skeleton, bone marrow); muscles (Smooth and skeletal); include Submerged culture fermenters, rotating fermentors, endothelial and epithelial tissue; membranes, cartilage tis as well as the so-called film reactors. Typically, mycelia of a Sues (tendons, ligaments, joints); sensory organs (eyes, ear, fungus are grown from an inoculum grown in Yeast Mainte 15 nose, tongue); endocrine or other glandular tissue (thyroid nance (YM) medium comprised of 3 g/l yeast extract (YE), 3 gland, parathyroid gland, pituitary gland, adrenal gland); uri g/1 malt extract (ME), and 5 g/l peptone. The inoculum is nary tissue and organs (kidneys, urinary bladder, urethra); macerated for 30 sec at low speed in a Waring-type blender. reproductive organs and tissues (testes, ovaries, placenta, Two centiliters of the macerated inoculum are added to 100 etc.); and adipose tissues (fat) Such as is contained in Subcu ml of growth medium in a 250 ml Erlenmeyer flask. The taneous and internal organs, as well as biological exudates, growth medium is 10 g/l spray-dried Sweet whey permeate Such as feces, sputum, urine, Sweat, mucus, semen, milk, and powder (SWP), 3 g/1YE, and 3 g/l KHPO, at a pH of about So forth. In each case processing conditions are chosen which 6.0. The culture is incubated at 24. degree. C. under orbital optimizes the physical and rheological characteristics of the shaking at 135 rpm and is harvested at 3-4 days by filtering desired powder. over filter paper and washed. 25 EXAMPLE 9 EXAMPLE 7 Standard Sabouraud's Dextrose Broth and Sabouraud’s In this example, yeast of the species Candida is cultivated Dextrose Agar plates are commercially available Suppliers at a temperature of about 25-40. degree. C in a fermentor 30 like from Difco and Gibco. SDB is a broth that contains having a useful volume of approximately 500 liters on a neopeptone and bacto-dextrose in a proportion of 1:4 while conventional carbohydrate medium to produce about 10 SD agar contains neopeptone, bacto-dextrose and agar in grams per liter per hour of yeast cells. The total hourly output proportions of 2:8:3. These fungal culture media may contain of this fermentor is accordingly 5 kilograms of yeast cells. adequate Small amounts of cyclohexamide and chloram Fermentor is a closed tank or kettle that is provided with a 35 phenicol to prevent concomitant bacterial growth. stirrer driven by an electric motor and includes a mechanical A fungal lesion from a human having the desired fungus foam breaker in the form of baffle Vanes to provide for the infection is wiped with 70% alcohol solution and allowed to separation of gas from the liquid in the foam that may form air dry. The surface of the lesion is then scraped with a scalpel during fermentation. The fermentor is also provided with to remove some of the infected skin tissue. The scrapings are cooling elements or heat exchanger through which a coolant 40 then placed in SDB and cultured. After significant growth is is passed. The fermentor is also provided with a pipe for observed, a sample from the culture is plated on SD agar Supplying the culture medium or Substrate or other nutrients plates to check the purity of the sample fungal population. to the fermentor and an outlet pipe is provided for withdraw Pure cultures are then used as an inoculate to propagate larger ing the cells of the yeast or other microorganisms that have batches of fungi. Alternatively the original inoculate is first been grown or cultivated therein. Compressed air is Supplied 45 subcultured in a separate 10 ml vial containing SDB and then to the fermenting Substrate through a pipe from a compressor incubated at room temperature for 4 days with constant shak or blower. The exhaust air or gases are discharged from the ing. Once fungus is grown to maximum the contents of vial is fermentor through a pipe that is connected to the mechanical then added to a larger growth chamber containing SDB. The foam breaker. The yeast that is grown in the fermentor is chambers are then grown at room temperature until growth is separated from the fermentation brew therein by means of a 50 saturated (i.e., no increase from previous day measured by centrifuge or other Suitable apparatus (filter) for separating eye). The chambers are shaken vigorously once a day or solids from liquids. The fermentation brew is withdrawn from shaken constantly in a rotating shaker at 150 rpm. When the fermentor through a pipe that discharges into a separator, maximum growth is reached, a sample from each fermentoris from which the concentrated solid fungus in the brew is plated onto SD plates to check the purity of the cultures. The separated from the liquid portion. Other pathogenic or gen 55 duration of culture varies with individual fungus species. erally considered non-pathogenic yeast (for example, com Maximum growth for Microsporum canis, Microsporum gyp mon yeast Saccharomyces cerevisiae causes in sum and Alternaria sp., is approximately 4 days, 7 days and 4 weak and immunosuppressed patients) can be mass-culti days, respectively. T. equinum when grown in agar plates vated and used for preparation of the vaccine of the invention. grows as a flat, buff to brown, granular colony with a reddish 60 brown reverse pigmentation. Microconidia are produced in EXAMPLE 8 great abundance, but macroconidia are rare. T. mentagro phytes (var. granulare) grows as a flat, granular, buff colored In addition to fungus preparation obtained from a fungal colony with tannish, brown reverse pigmentation. It produces culture, one can prepare fungus preparation from any tissue, microconidia in great abundance but macroconidia are infre organs, and cells which host fungal infection. For example, 65 quent. The organism grows equally well on casein basal agar one can select fungus-infected central nervous system tissues ("CBA") and CBA supplemented with nicotinic acid. M. and organs (brain, spinal chord, spinal fluid, appendages); canis produces abundant macroconidia with prominent Sur US 7,914,799 B2 21 22 face projections (vesicles) but relatively sparse microconidia. recombinant fungal antigenic Substance is provided as fol The organism grows as a flat to moderately folded, silky to lows. Transformed E. coli cells from a culture expressing cottony white Surface colony with a yellow reverse pigment. optimally high levels of recombinant fungal antigens are The culture is identified by its pigmentation and typical mor streaking onto an L-Broth plate containing 100 microg/ml phology of its macroconidia. M. gypseum grows as a flat 5 ampicillin and the plate is incubated overnight at 37° C. A granular to Velvety buff to orange colored colony that single colony is inoculated into 10 ml of L-Broth, 100 micro becomes white and cottony with age. The organism produces gram/ml amplicillin and grown overnight at 37° C. Andali abundant rough walled (vesiculate) macroconidia and numer quotis used to Verify plasmid structure by restriction mapping ous microconidia. The obtained fungal cell mass can be with Sal1 and Pstl. A second aliquot is used to induce expres washed, e.g., with sterile distilled water and then disrupted by 10 sion of fungal antigens and the rest of the culture is made 15% sonication until 100% of the hyphae are fragmented. Other glycerol by adding 4 volume of 75% sterile glycerol. Glyc disruption methods are equally suitable using for example a erol cell stocks are aliquoted in 1 ml and quickly frozen in French press. The fragmented mixture is then centrifuged at liquid nitrogen or dry-ice ethanol bath. These master seed 5,000xg for 20 minutes and pellet is processed as described in stocks are stored at -70° C. When needed the master seed other examples. 15 stock is scraped with a sterile applicator which is used to streak an L-Broth plate containing 100 microg/ml amphicil EXAMPLE 10 lin. Single colonies from this plate are used to inoculate 20-50 ml of L-Broth/amp, which is incubated at 37° C. overnight. Alternatively instead of growing fungi of interest in a clas An aliquot of the overnight culture is used to inoculate larger sical mycotic culture the genes that encode relevant immu- 20 volumes (1-6 liters) of L-Broth/amp. Cells are incubated at nogenic proteins or immunogens of fungi of interest can be 37° C. overnight and reach an OD650 of approximately 5 cloned according to standard molecular biology procedures prior to use as inoculum for the fermenter run. Fermenters well known in the art. The genes can be used to express the (capacity about 16 liters) containing 101 of L-Broth and 1 ml proteins in an expression vector for example in E. coli and of antifoam are inoculated with 100-500 ml from the inocu combined to provide the starting material for a recombinant 25 lum culture. Cells are grown at 37° C. to an or) of about 1. vaccine. With advent of recombinant DNA technology it Expression of fungal antigens is induced by addition of 100 became possible to employ prokaryotic and eukaryotic hosts ml of an IPTG solution (100 mM) to yield a 1 mM final Such as E. coli, yeast, fungi, insect, and mammalian cells in concentration fermenter. Cells are grown for 3 additional culture to produce useful antigens and fragments thereof. For hours and Subsequently harvested using continuous flow cen example aspartyl proteinase (Sap2) family of proteins or the 30 trifugation. At this step cells can be frozen and kept at -20°C. 65 kDa mannoprotein (MP65) of Candida albicans can be until further proceedings. Alternatively, 250 liter fermentors useful antigens to be obtained by recombinant means. The are inoculated with 1-5 liter from the inoculum culture. gene encoding for the immunodominant antigen gp43 from Growth, induction, and harvest are as indicated before. Fro the Paracoccidioides brasiliensis is Zen E. coli cells are thawed and suspended in 2.5 volumes of another convenient target. Pneumocystis carinii p55 antigen 35 lysis buffer (0.1M sodium phosphate (NaPi), pH 7.5, 1 mM or the major surface glycoprotein (MSG or gpA) are yet EDTA, 0.1 M NaCl). Cells are broken in a non-continuous another attractive antigens. Antigenic Protein 1, referred to as system using a 300 ml glass unit of a Dyno-mill at 3000 rpm “PMAP1 is an ideal antigen candidate for vaccine against and 140 ml of acid-washed glass beads for 15 min. The pathogenic fungus Penicillium marineffei. The major Surface jacketed chamber is kept cool by a -20° C. ethylene glycol antigen (P30) of the Toxoplasma gondii can be expressed in 40 solution. Broken cells are centrifuged at 27,000xg for 25 an insect cell culture system infected with recombinant bacu minutes to remove debris and glass beads. The Supernatant is lovirus. Vector DNA can comprise plasmid, phage or viral recovered and kept at 4°C. The cell extract is made 30% DNA and be easily selected from the group consisting of (NH4)2SO4 by slowly adding the ammonium sulfate at 4°. lambdagt11, pluR290, PUR 291, pluR282, p OK270, puC8, The extract is stirred for 10 min after the final concentration is pUC9, pZipNeo, an SV40 based vector, lambda gt10, an 45 achieved, followed by centrifugation at 27,000xg for 20 min. EMBL vector, pBR327, pBR329, pBR329 containing a par The pellet is resuspended in 1M NaCl, 1 mM EDTA, 1% locus, baculovirus and vaccinia virus among many others. Triton X-100TM, and 5% SDS, and then boiled for 5 min. The Representative promoters for driving gene expression Suit fraction obtained by selective precipitation is submitted to gel able for use within the present invention include both eukary filtration using a G50 Sephadex column equilibrated in otic (e.g., pol I, II or III) and prokaryotic promoters, and 50 0.03M NaPi, pH 6.8. Chromatograpy is developed in the inducible or non-inducible (i.e., constitutive) promoters, such same solution. Fractions are collected and absorbance at 280 as, for example, Murine Leukemia virus promoters (e.g., inn is determined. Antigen-containing fractions are pooled MoMLV), metallothionein promoters, the glucocorticoid and characterized by protein gel electrophoresis, Western promoter, Drosophila actin SC distal promoter, SV 40 pro analysis, and ELISA and are further processed into the com moter, heat shock protein 65 promoter, heat shock protein 70 55 position of the invention. It is also equally possible to use promoter, immunoglobulin promoters, Mouse polyomavirus these recombinantantigens without these steps of purification promoter (“Py’), nous sarcoma virus (“RSV), BK virus and since polyvalent vaccine containing E. coli antigens will be JC virus promoters, MMTV promoter, alphavirus junction equally desirable against enterotoxigenic strains of E. coli. region, CMV promoter, Adenovirus VAIRNA, rPna pro Alternatively a process for purifying one or more antigenic or moter, tRNA methionine promoter, CaMV 35S promoter, 60 immunogenic Substances from a source liquid can comprise nopaline synthetase promoter, and the lac promoter among steps of contacting the source liquid with a chromatography many others. Based on these one can easily imagine a gene resin; incubating the soure liquid with the chromatography expression unit comprising a DNA coding sequence for a resin for a sufficient contact time to allow the resin to bind a fungal heterologous protein and a regulatory element for desired fraction of one or more antigenic Substances, recircu transcription of said DNA sequence and translation with in a 65 lating the chromatography resin in a cross-flow filter, concen host cell wherein said regulatory element comprises a host trating the chromatography resin and separating contami specific promoter. A typical example of mass-producing nants from the chromatography-resin-bound antigens by US 7,914,799 B2 23 24 concentration and/or diafiltration; eluting the antigenic Sub cines and immunomodulators, antioxidants, amino acids, stance from the chromatography resin; and separating the Sources of amino acids and amino acid analogs, antibiotics, immunogenic Substance from the chromatography resin by Vitamins, and minerals. diafiltration; recovering the desired immunogen(s); and optionally concentrating the antigenic or immunogenic fun 5 EXAMPLE 1.4 gus-specified Substance. The effect of immunization with vaccine of the invention EXAMPLE 11 can be tested in vitro. Various methods are available to test immune response to vaccine. One of examples to evaluate Yet another recombinant approach is to grow recombinant 10 cell-mediated response is disclosed as follows: Peripheral attenuated fungi and use them for the preparation of the blood mononuclear cells (PBMCs) of vaccinated and present vaccine. For example pulmonary infection with the untreated guinea pigs or of human vaccines and normal non Blastomyces dermatitidis often progresses vaccinated Subjects are isolated from the blood samples using and requires treatment to prevent fatality. A recombinant Ficoll-HypaqueTM (Pharmacia, Piscataway, N.J.) gradient strain of the fungus lacking the WI-1 adhesin gene and 15 and centrifuged at 400xg for 30 min. PBMCs that are col accordingly the pathogenicity can be created and propagated lected at the interface are harvested and washed twice with in the culture. By mutating a pathogenetic locus in a fungus RPMI 1640 medium. When needed, the PBMCs are separated one can create an attenuated vaccine strain. into plastic-adherent and non-adherent cells by first incubat EXAMPLE 12 ing 5/10 mln cells in 10 ml of RPMI supplemented with 25 mM HEPES 0.05 mM.beta-mercaptoethanol, and 10% FCS In this example vaccine is prepared against mycoplasma (v/v). After 2 hours of attachment time, the non-adherent cells infection. Mycoplasma hyopneumoniae is first propagated in are removed and the adherent cells are washed three times a modification of Friis medium. Each Suspension for vaccine with growth medium. PBMCS, non-adherent cells, or adher incorporation is prepared by inoculating 500 ml of broth with 25 ent cells are seeded at 2x10 cells/100 microL growth 4 ml of the stock culture at pH 7.4 and then incubated at 37. medium in 96-well plates and are challenged with isolated degree. C. for 3 days or until the pH drops to 6.8. By this time fungal antigen of Candida albicans for 6 days in a 37°C., 5% the organism reaches the log phase of growth. The cultures CO2 incubator. For antibody blocking experiments, fungal are inactivated using ultraviolet irradiation, concentrated antigen is pre-incubated with equal Volume of human antise using tangential flow ultrafiltration, and washed with phos 30 rum known to have antibodies to the antigen for 1 hour before phate buffered saline (PBS). The concentration of antigen being added to the cells. On the 6' day, these cells are pulsed was adjusted such that it contains 10 mcg of total protein per with 1 microCi/well of radioactive thymidine for 6 hours to 1 gram dose of vaccine. assess the degree of cell proliferation. Radiactive isotope incorporated into cellular DNA is collected on glass fibers EXAMPLE 13 35 with a cell harvester and the degree of proliferation is deter mined by Scintillation counting of radioactivity. Data col The vaccine of the invention does not require the express lected are expressed as meantSD of the quadruplicates. addition of immune adjuvants, which are obligatory with Those Subjects that are vaccinated previously are expected to injectable vaccines of the prior art. However, if necessary, one have greater proliferation response than naive non-vaccinated of skill in the art can administer instant vaccine with an 40 individuals. Proliferation test can be easily replaced by cyto immune adjuvant. Typical adjuvants include but are not lim toxic lymphocyte (CTL) radioactive Chromium release a test ited Quil ATM which is a commercially available adjuvant using for example antigen loaded K562 cells as targets for manufactured by Sargent Chemical Co. (Clifton, N.J.) Quil CTL. Another simple technique for detecting the presence or ATM is a purified saponin extract from the bark of the Quillaja absence of previously exposed T-lymphocytes in a patients Saponaria Molina tree. It is currently used as a suspension 45 blood consists of incubating the whole blood with the antigen medium in a number of large animal vaccines throughout the for the Suspected fungus being diagnosed, which antigen is world, such as the vaccines utilized in the prevention of foot dispersed in a suitable pH-regulated medium, Such as a buff and mouth disease. Quil ATM is widely used, readily available ered saline Solution, for example. This incubation will cause and manufactured by many biologics producers (e.g., Sigma previously exposedlymphocytes to become activated. After a Chemical Co., St. Louis, Mo.). It is comprised primarily of 50 Suitable incubation period, a fluorescent dye, such as the light mineral oil. A number of anhydrous lipid based materi acetomethylester of the calcium-sensitive dye fura-2, or other als, such as “Lipovant” (Accurate Chemical and Scientific colorant having an affinity for activated lymphocytes or lym Co., Westbury, N.Y.) which consists of a peanut oil and leci phoblasts is added to the blood sample. The dye can obtain its thin mixture is suitable as well. Aluminum hydroxide gel sold attraction to the activated lymphocytes or resultant lympho by a number of companies including Accurate Chemical and 55 blasts through chemical means, by being combinable with the Scientific Co is another suitable immune adjuvant. Other excess calcium ions, or by other means, such as by being contemplated adjuvants and additives to the formulation tagged with a fluorescently-tagged antibody specific to the include various enterotoxins, tetanus toxoid, Saponin, tia transferrin, Leu-23, the surface receptor for T-cell growth benedeZole, tylorone, statolon, maleic anhydride-divinyl factor interleukin-2, or the like. The addition of the dye, ether, a pyran copolymer, amphotericin B, a liposome, silica, 60 colorant, or fluorescently tagged antibody will differentially calcium phosphate, glycerol betaine, protodyne, cyanidanol, highlight any responding lymphocytes or lymphoblasts in the imuthiol, picibanil, isoprinosine lentinan, azimexon, a leci blood sample. thin, levamisole, a retinol, a tocopherol, an antioxidant, an Other in vitro and in vivo assays are well know to those of aluminum salt, and aluminum oxide. The pharamaceutical skill in the art including Elispot test, neutralizing antibody formulation of instant vaccine can also contain bile salts, 65 measurement test, lactic acid release tests, MTT test, delayed Surfactants, enzymes, enzyme co-factors, hormones, prostag type hypersensitivity using the footpad Swelling response, landins, peptides, immunoglobulines, cytokines, other vac etc. US 7,914,799 B2 25 26 EXAMPLE 1.5 trobenzoic acid, 3,5-dibromo-2,6-dinitro benzoic acid, 2,4- dicholoro-3,5-dinitrobenzoic acid, methyl 3,5-dibromo-2,6- A 11 year-old male patient is presented with a history of dinitro benzoate, 1-aminocyclopropane carboxylic acid, progressive headache, mandibular soft tissue Swelling, and L-pyroglutamic acid (5-oxo-2-pyrrolidinecarboxylic acid, facial nerve palsy. A computerized tomography scan of the 5 albizziin (2-amino-3-ureido-propionic acid), L-phenylala head and neck shows abscesses in the bilateral retromolar nine ethyl ester, L-tryptophan ethyl ester, L-aZetidine-2-car fossa and in both ears. A non-sporulating fungus-like organ boxylic acid, 2-amino-4-(aminoxy)butanoic acid, 2-ami ism is isolated in pure culture after Surgical drainage of the nobutanoic acid, 2-aminohexanedioic acid, 2-amino-4- abscesses. The organism is identified as Pythium insidiosum. hydroxybutanoic acid, L-alpha.-amino-n-butyric acid, Despite treatment with amphotericin B, iodides, ketocona Zole, and Surgery, the infection did not disappear. A magnetic 2-amino-4-hydroxybutyric acid, 2-amino-5-hydroxypen resonance imaging (MRI) and magnetic resonance analysis tanoic acid, 2-amino-4-methyleneglutaric acid, 2-amino-4- (MRA) of the neck reveals regional lymph node enlargement, methylenepentanedioic acid, 0- (aminoiminomethyl)amino Stenosis and aneurysm in the external carotid artery. Surgical homoserine, 2,5-diamino-4-hydroxypentanoic acid, removal of the aneurysm is performed and Pythium insidio N-(carboxyacetyl)alanine, (2-amino-2-carboxyethyl) thio sum hyphae is histopathologically observed in the biopsied 15 Succinic acid, 2,3-diaminopropanoic acid, N.Sup.6-(ami tissue. A MRA performed reveals stenosis of the internal noiminomethyl) lysine, 2-amino-5-guanidino-4-hydroxy carotid artery indicating that Pythium insidiosum invaded the pentanoic acid, N.Sup.6-(aminoiminomethyl)-4- artery. The oral vaccine derived from culture Pythium insid hydroxylysine, N-(1H-indol-3-ylacetyl)aspartic acid, beta.- iosum is administered. Two pills of the vaccine are given per (2,4-dihydroxy-3-pyrimidinyl) alanine, 5-oxo-2- oS for 7 days. Remarkably, twenty-four hours post vaccina pyrrolidinecarboxylic acid, 2-piperidinecarboxylic acid, tion the patient's headache disappeared, his facial and left N-methylaminoacetic acid, 1.2.3,6-tetrahydro-2-pyridin tongue Swellings dramatically diminished, the enlarged cer ecarboxylic acid, alpha.-amino-beta.-uracil-1-ylpropanoic vical lymph node reduced in size, and the proximal left inter acid, N-carboxyacetyl-D-phenylalanine, (+)-threo-1-amino nal carotidartery Stenosis is significantly improved. One year 3-methylpentanoic acid, 4-hydroxymethyl-2-pyrrolidinecar 25 boxylic acid, 5-hydroxy-2-piperidinecarboxylic acid, 1-ami after the vaccination the patient remains free of disease symp nocyclopropane-1-carboxylic acid, 2-amino-3-ureido toms indicating that fungal infection is cleared due to admin propionic acid, istration of oral therapeutic vaccine of the invention. The following list of fungicides with which instant vaccine EXAMPLE 16 can be combined is intended to illustrate possible combina 30 tions but not to impose any restrictions. Examples of fungi Approximately 100 patients with HIV infection with oral cides which may be combined with compounds of the inven candidiasis are enrolled to the study. The oral vaccine of the tion: sulfur, dithiocarbamates and their derivatives, such as invention is administered to those who have oral thrush. At ferric dimethyldithiocarbamate, zinc dimethyidithiocarbam one and two weeks post-administration of the vaccine the oral ate, Zinc ethylenebisdithiocarbamate, manganese ethylenebi cavities of treated patients are examined again for signs of 35 Sdithiocarbamate, manganese Zinc ethylenediaminebis fungal infection. About 80% of patients have cleared or dithiocarbamate, tetramethylthiuram disulfides, ammonia reduced oral thrush. complex of zinc N,N'-ethylenebisdithiocarbamate, ammonia complex of zinc N,N'-propylenebisdithiocarbamate, zinc EXAMPLE 17 N,N'-propylenebisdithiocarbamate and N,N'-polypropyle 40 nebis(thiocarbamyl)disulfide: nitro derivative, such as dinitro 5 ml of the final vaccine was administered to cattle on (1-methylheptyl)-phenyl crotonate, 2-sec-butyl-4,6-dinitro several farms. Depending on the farm, 50-100% of the cattle phenyl 3,3-dimethylacrylate, 2-sec-butyl-4,6-dinitrophenyl treated were cured of pre-existing ringworm infection and isopropylcarbonate and diisopropyl 5-nitroisophthalate; het exhibited resistance to reinfection after treatment. Those erocyclic Substances, such as 2-heptadecylimidazol-2-yl infections not succumbing to treatment with the vaccine were 45 acetate, 2,4-dichloro-6-(o-chloroanilino)-s-triazine, O.O-di probably caused by infecting organisms not included in the ethyl phthalimdophosphonothioate, 5-amino-1-bis(dim vaccine (i.e., other than Microsporum canis or Microsporum ethylamino)-phosphinyl-3-phenyl-1,2,4-triazole, 2,3-dicy gypsum). 1 ml of the final vaccine also administered to cats. ano-1,4-dithioanthraquinone, 2-thio-1,3-dithio4,5-b] The cats treated exhibited resistance to ringworm infection up quinoxaline, methyl 1-(butylcarbamyl)2- to 18 months after administration of the vaccine. 50 benzimidazolecarbamate, 2-methoxycarbonylaminobenzimidazole, 2-(fur-2-yl)benz EXAMPLE18 imidazole, 2-(thiazol-4-yl)benzimidazole, N-(1,1,2,2-tetra chloroethylthio)tetrahydrophthalimide, N-trichlorometh Oral 850 mg pill containing the fungal antigens of the ylthiotetrahydrophthalimide, invention formulated by precipitation with magnesium chlo 55 N-trichloromethylthiophthalimide, N-dichlorofluorometh ride containing 6 molecules of water is administered to ylthio-N',N'-dimethyl-N-phenylsulfuric acid diamide, twenty police officers who usually wear boots that encourage 5-ethoxy-3-trichloromethyl-1,2,3-thiadiazole, 2-thiocy athlete's foot or pinea pedis growth. Half of officers receive anatomethylthiobenzothiazole, 1,4-dichloro-2,5-dimethoxy placebo pills and half receive the vaccine. In about one to two benzene, 4-(2-chlorophenylhydrazono)-3-methyl-5-isox weeks those who receive the vaccine have no signs or reduced 60 aZolone, 2-thiopyridine 1-oxide, 8-hydroxyquinoline and its signs of athlete's foot while in those on placebo the fungal copper salt, 2,3-dihydro-5-carboxanilido-6-methyl-1,4-ox growth is not affected. athiyne, 2,3-dihydro-5-carboxanilido-6-methyl-1,4-ox athiyne 4,4-dioxide, 2-methyl-5,6-dihydro-4H-pyran-3-car EXAMPLE19 boxanilide, 2-methylfuran-3-carboxanilide, 2.5- 65 dimethylfuran-3-carboxanilide, 2,4,5-trimethylfuran-3- The instant preparation is Suitably used along with other carboxanilide, 2,5-dimethyl-N-cyclohexylfuran-3- antifungals including but not limited to 3,5-dichloro-2,6-dini carboxamide, N-cyclohexyl-N-methoxy-2,5-diethylfuran-3- US 7,914,799 B2 27 28 carboxamide, 2-methylbenzanilide, 2-iodobenzanilide, vinyl-3-(3,5-dichlorophenyl)-2,4-dioxo-1,3-oxazolidine, N-formyl-N-morpholine-2.2.2-trichloroethylacetal, pipera 3-3,5-dichlorophenyl-5-methyl-5-methoxymethyl-1,3-ox Zine-1,4-diylbis-(1-(2.2.2-trichloroethyl)-formamide), 1-(3. aZolidine-2,4-dione, 3-(3,5-dichlorophenyl)-1-isopropylcar 4-dichloroanilino)-1-formylamino-2.2.2-trichloroethane, bamylhydantoin, N-(3,5-dichlorophenyl)-1,2-dimethylcy 2,6-dimethyl-N-tridecylmorpholine and its salts, 2,6-dim clopropane-1,2-dicarboximide, 2-cyano-N- ethyl-N-cyclododecylmorpholine and its salts, N3-(p-tert.- (ethylaminocarbonyl)-2-methoximino-acetamide, 1-2-(2, butylphenyl)-2-methylpropylcis-2,6-dimethylmorpholine, 4-dichlorophenyl)pentyl)-1H-1,2,4-triazole, 2,4-difluoro-a- N-3-(p-tert.-butylphenyl)-2-methylpropyl-piperidine, 1-2- (1H-1,2,4-triazol-1-ylmethyl)-benzhydryl alcohol, N-(3- (2,4-dichlorophenyl)-4-ethyl-1,3-dioxolan-2-yl-ethyl)-1H chloro-2,6-dinitro-4-trifluoromethylphenyl)-5- 1,2,4-triazole, 1-2(2,4-dichlorophenyl)-4-n-propyl-1,3-di 10 trifluoromethyl-3-chloro-2-aminopyridine, and 1-((bis-(4- oxolan-2-yl-ethyl)-1H-1,2,4-triazole, N-(n-propyl)-N-(2,4, fluorophenyl)-methylsilyl)-methyl)-1H-1,2,4-triazole. 6-trichlorophenoxyethyl)-N-imidazolylurea, 1-(4- While various embodiments of the present invention have chlorophenoxy)3.3-dimethyl-1-(1H-1,2,4-triazol-1-yl)- been described hereinabove, it is understood that they have butan-2-one, 1-(4-chlorophenoxy)-3.3-dimethyl-1-(1H-1.2. been presented by way of example only and not limitation. 4-triazol-1-yl)-butan-2-ol, alpha-(2-chlorophenyl)-alpha-(4- 15 Thus, the breadth and scope of the invention should not be chlorophenyl)-5-pyrimidinemethanol, 5-butyl-(2- limited by any of the above-described exemplary embodi dimethylamino-4-hydroxy-6-methylpyrimidine, bis-(p- ments, but should be defined only in accordance with the chlorophenyl)-3-pyridinemethanol, 1.2-bis-(3- following claims and their equivalents. ethoxycarbonyl-2-thioureido)-benzene, 1.2-bis-(3- What is claimed is: methoxycarbonyl-2-thioureido)benzene, and various 1. A heat-inactivated immunogen formulated as an oral pill fungicides, such as dodecylguanidine acetate, 3-3-(3,5-dim comprising a hydrolyzed, dry fungal immunogen, wherein ethyl-2-oxycyclohexyl)-2-hydroxyethyl-glutaramide, the fungal immunogen is a filamentous fungal antigen and hexachlorobenzene, DL-methyl-N-(2,6-dimethylphehyl)-N- wherein said immunogen upon administration retains the fur-2-yl alanate, methyl DL-N-(2,6-dimethylphenyl)-N-(2- ability to elicit an immune response to said immunogen in a methoxyacetyl)-alanate, N-(2,6-dimethylphenyl)-N-chloro 25 host. acetyl-DL-2-aminobutyrolactone, methyl DL-N-(2,6- dimethylphenyl)-N-(phenylacetyl)-alanate, 5-methyl-5-