Immunigenic compositions against fungal infections and methods of making and using thereof

Abstract

Compositions and methods for generating an immune response to prevent or treat fungal infection in a subject in need thereof, are disclosed. Advantageously, the compositions and methods may be used prophylactically to immunize a subject against Coccidioides antigens or used therapeutically to treat or ameliorate the onset and severity of disease in a subject in need thereof. The compositions in one embodiment include markerless live attenuated Δcps1 Coccidioides species. In some preferred embodiments, the compositions additionally include an adjuvant and optionally, an excipient.

Description

[0001] IMMUNIGENIC COMPOSITIONS AGAINST FUNGAL INFECTIONS AND METHODS OF MAKING AND USING THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of and priority to U.S.S.N. 63 / 556,986 filed February 24, 2024, which is incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with Government Support under grant No. AI132140 awarded by National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD OF THE INVENTION

[0007] The disclosed invention is generally in the field of immunogenic compositions to elicit protective immunity against fungal infections.

[0008] BACKGROUND OF THE INVENTION

[0009] Coccidioides species (C. immitis and C. posadasii) are the causative agents of coccidioidomycosis (Valley Fever), an important emerging disease endemic to the southwestern US, as well as parts of Mexico and central and South America. Infection begins with inhalation of arthroconidia that initiate the parasitic phase in lungs and can result in a respiratory infection or if not controlled, a more serious disseminated disease.

[0010] Coccidioides spp. are dimorphic and produce a unique parasitic phase structure, the spherule, via a switch from polar to isotrophic growth with the spherule expanding from a barrel-shaped arthrocondium that is 3-6 pm by 2-4 pm in size to a sphere 80-100 microns in diameter. Internal septation and spore formation results in production of hundreds of endospores that if released can disseminate and reinitiate spherule formation at other places in the body. Most infections are mild and resolve without medical intervention although about 30% of infections cause flu-like symptoms that may take 1-4 months to resolve.

[0011] Fungal infections impose a significant economic burden on healthcare systems and societies due to costs associated with medical care, hospitalization, lost productivity, and reduced quality of life for affected individuals. It is estimated that the US economic burden of fungal diseases as SI 1.5 billion in 2019 (Benedict et al., Open Forum Infect Dis, 9(4) (2022)). Coccidioidomycosis is reported in up to 20,000 persons per year and has an economic impact close to $1.5 billion (Galgiani et al., J Fungi (Basel), 8(8): 838 (2022)). Development of the fungal vaccines will offer a valuable tool for preventing fungal infections, particularly in high- risk populations such as immunocompromised individuals and those living in endemic regions. CPS1 Coccidiodes species mutants are disclosed for example in U.S. patent no. 9,884,097, 10413602, 10758600, 11,260,092. Despite the need, no antifungal vaccines have been approved for use in humans to date.

[0012] Over the last 50 years, many approaches to vaccination against coccidioidomycosis have been tried, including whole killed cells, live mutant vaccines that have been modified in virulence, partially purified cellular extracts, and recombinant proteins that were identified by a myriad of both low and high technology methods. To date, killed whole cell vaccines provide the best protection in mice but are not transferable to humans because of intolerable adverse effects and poor efficacy. Recombinant proteins offer the safest approach but have modest efficacy in mice and have not been tried in a higher species.

[0013] There remains a need for compositions that can elicit an immune response in subjects such as human to pathogenic fungus such as Coccidioides species.

[0014] It is an object of the present invention to provide compositions that elicit an immune response in a subject such as human in need thereof, against Coccidioides species.

[0015] It is also an object the present invention to provide methods of creating genetic constructs for eliciting an immune response in a subject such as human in need thereof, against Coccidioides species.

[0016] BRIEF SUMMARY OF THE INVENTION

[0017] Methods for generating live attenuated Coccidioides species which do not include exogenous nucleic acid sequences, are disclosed, as well as compositions resulting therefrom, which are useful for generating an immune response against a fungal infection, in a subject in need thereof.

[0018] The methods include a two-step process to create a CPS1 gene deletion in Coccidioides ( / Icpsl Coccidiodes) which is avirulent and does not include exogenous nucleic acids (herein, markerless live attenuated Coccidioides). These attenuated forms of the fungi will not result in immunogen-induced pathology in an animal exposed to the immunogen. The disclosed methods for creating markerless gene deletion strains allow one to make strains with multiple mutations / gene deletion strains serially or in parallel.

[0019] The two-step process incorporates two vectors. The first vector includes a “suicide gene”, (herein after, “suicide vector”) for example, the Herpes simplex virus gene that encodes for thymidine kinase (HSV:tk) and a selectable marker for example, hygromycin phosphotransferase (hph) encoding gene, placed between flaking sequences of the gene to be replaced (gene A). The second vector includes flanking sequences for the gene to be replaced (i.e., gene A). Where CPS1 is the gene to be replaced, the first vector includes the Herpes simplex virus gene that encodes for thymidine kinase (HSV:tk) and a selectable marker for example, a hygromycin phosphotransferase (hph) encoding sequence, placed between flaking sequences of CPS1.

[0020] In the first step, the fungus is transfected with the suicide vector. The introduction of this construct into the fungus produces a gene replacement mutant strain of gene A (here, CPS1 ) and expresses the suicide gene as well as the selectable marker (herein , ACPSl / suicide / marker). Mutant strains containing the suicide gene and the gene encoding the selectable marker (ACPSl / suicide / marker) are selected by treatment with the drug corresponding to the marker. Thus, in some forms, mutant strains containing the suicide gene thymidine kinase (HSV:tk) and the gene encoding the selectable marker (hygromycin phosphotransferase) are selected by treatment with hygromycin.

[0021] In the second step, the selected ACPSl / suicide / marker mutants are transfected with a construct that contains the gene A flanking sequences fused to each other, and selected against the presence of the suicide gene to obtain mutants that now lack the suicide gene and selectable marker gene (herein, ACPS1 / suicide've / markerveor Acpsl ::nada). Where the suicide gene is HSV:tk , the cells are treated with F2dU. The F2dU drug is converted by the HSV-Tk protein to a toxic metabolite that is lethal to fungal cells expressing the HSV-Tk gene.

[0022] The compositions in one embodiment include a live attenuated Coccidioides mutant, Acpsl ::nada, which includes a deletion of CPS1 gene (Acpsl ) and which do not include exogenous nucleic acids. The live attenuated Acpsl Coccidioides mutants have in their genome, the S'CP l flank linked to the 3’ CPS1 flank. In some preferred embodiments, the compositions additionally include an adjuvant and optionally, an excipient.

[0023] Also provided are such compositions, wherein the composition is capable of inducing an immune response selected from the group consisting of: neutrophil invasion; granuloma formation; resistance to mycosis; and immunity to mycosis.

[0024] Also provided are such compositions, wherein the fungus is a Coccidioides spp.

[0025] Also provided are such compositions, wherein the composition is capable of inducing resistance to coccidioidomycosis (valley fever).

[0026] Also provided are such compositions, wherein the composition is capable of inducing immunity to coccidioidomycosis (valley fever).

[0027] Also provided are such compositions, wherein the fungal cell is selected from the group consisting of: Coccidioides immitis or Coccidioides posadasii.

[0028] Also provided are such compositions, which are formulated as a vaccine. Also provided are such compositions, wherein the composition comprises further avirulence protection means.

[0029] Also provided are such compositions, which is a mammalian immunogen.

[0030] Also provided are such compositions, which is a human immunogen.

[0031] The present invention also provides methods of eliciting an immune response in a mammal comprising administering to a mammal a pharmaceutically-effective dose of a composition herein. The compositions and methods may be used prophylactically to immunize a subject against Coccidioides antigens, or used therapeutically to treat or ameliorate the onset and severity of disease in a subject in need thereof.

[0032] Methods for eliciting an immune response against a fungal species are disclosed. The methods include administering the markerless cpsl Coccidioides species to the subject. In one embodiment, the administration is not followed by administration of an antifungal agent that is capable of preventing / blocking fungal growth. In some forms, markerless / Icp l Coccidioides species are administered in the form of spores.

[0033] Also provided are such methods wherein the composition is administered by injection. Also provided are such methods wherein the composition is administered intranasally. Also provided are such methods wherein the pharmaceutical composition is formulated for subcutaneous, intramuscular, and / or intraperitoneal administration.

[0034] Also provided are such methods wherein the pharmaceutical composition is formulated for intranasal administration.

[0035] Also provided are such methods wherein the fungal virulence is attenuated or eliminated in any mammal susceptible to the fungus.

[0036] Also provided are such methods wherein the mammalian subject is selected from the group consisting of: laboratory animal; companion animal; draft animal; meat animal; zoo animal; and human.

[0037] Also provided are such methods wherein the subject is a mammal selected from the group consisting of: cat; dog; horse; bovine; camelids; and human.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 is a schematic showing a two-step transformation process to generate markerless CPS1 deletion mutant. The Herpes simplex virus gene that encodes for thymidine kinase (HSV- tk) was used as a toxic gene and 5-fluoro-2’ -deoxyuridine (F2dU) for counter-selection. First a cpsi strain with CPS1 replaced by an Hph / HSV-tk cassette was created i / lcyxs / : :hphRZtk+) using selection for hygromycin resistance. This strain was then transformed with a construct that contains the fused 5’ and 3’ flanking sequences of the C. posadasii CPS1 gene using selection on F2dU. This compound is lethal for strains that express the HSV-tk gene. Strains that grew on F2dU were screened for deletion of the Hph / HS V -tk sequences via homologous recombination with the fused CPS1 flanking sequences. The resulting strain has a CPS1 deletion containing the fused flanking sequences and no foreign sequences.

[0040] FIGs. 2A-2B are a pair of plots showing protection from fungal burden after vaccination with CPS1 deletion mutants. CFU (Colony forming units) are significantly lower in lung (FIG. 2A) and spleen (FIG. 2B) of mice vaccinated with CPS1 deletion mutants (cpsl 19 and cpsl 1714) compared with the unvaccinated mice control group.

[0041] FIG. 3. Life cycle of Coccidioides. Coccidioides spp. alternate between saprobic (my celia) (left) and parasitic (spherules) (right). The saprobic cycle is found in the environment and produces infectious arthroconidia. They may become airborne and be inhaled by the host or may return to the environment to continue the saprobic life cycle.

[0042] DETAILED DESCRIPTION OF THE INVENTION

[0043] The disclosed method and compositions can be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0044] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0045] I. DEFINITIONS

[0046] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if an aptamer is disclosed and discussed and a number of modifications that can be made to a number of molecules or compositions including the aptamer are discussed, each and every combination and permutation of the aptamer and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials. These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0047] Coccidioides acyl Co A ligase-like protein (Cpsl), is a protein encoded by a CPS 1 gene and / or CPS1 mRNA. The CPS1 gene and / or CPS1 mRNA encodes wild type version of the protein, dcpsl strain refers to Coccidiodes species in which the CPS1 gene has been deleted from its genome.

[0048] As used herein, “attenuated” refers to refers to procedures that weaken an agent of disease (a pathogen). An attenuated pathogen is a weakened, less vigorous pathogen. A vaccine against a pathogenic fungal disease such as disease caused by Coccidioides can be made from an attenuated, less virulent strain of the fungus, resulting in a fungus capable of stimulating an immune response and creating immunity but not causing illness or less severe illness. Reduction in virulence encompasses any detectable decrease in any attribute of virulence, including infectivity in vitro and / or in vivo, or any decrease in the severity or rate of progression of any clinical symptom or condition associated with infection.

[0049] The term “avirulent”, as used herein in reference to a microbe, does not mean that a microbe of that genus or species cannot ever function as a pathogen, but that the particular microbe being used is avirulent with respect to the particular animal being treated. The microbe may belong to a genus or even a species that is normally pathogenic but must belong to a strain that is avirulent. The microbe may also be modified genetically or through avirulence protection means to make the microbe avirulent. Examples of avirulent means include, but are not limited to, genetic engineering to knock out genes required for virulence, amino acid biosynthesis knockout, truncation of the viral genome, aging, killing, formulation, resistance to reversion to wild type, and fusion.

[0050] “Pathogenic,” as used herein, means capable of causing disease or impairing normal physiological functioning. An “avirulent strain” is incapable of inducing the full set of symptoms of the disease that is normally associated with its virulent pathogenic counterpart. The term “microbes,” as used herein, includes bacteria, protozoa, and fungi. Derivatives of avirulent Coccidioides spp. are also contemplated to be within the scope of this disclosure. By “derivative” it is meant sexually or asexually derived progeny and mutants of the avirulent strains including single or multiple base substitutions, deletions, insertions or inversions which retain the inability to produce functional Cpsl protein. For example, the Coccidioides posadasii Silveira strain that has a deletion of the CPS1 gene described herein.

[0051] The term “dysfunctional,” “non- functional,” “inactivated,” or “inactivation” when referring to a gene or a protein means that the known normal function or activity of the gene or protein has been eliminated or highly diminished. For example, inactivation of CPS1 gene renders the gene or protein dysfunctional. This can be accomplished using methods such as deletions, mutations, substitutions, interruptions or insertions in the sequence of the gene or the protein.

[0052] As used herein, the term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease state being treated or to otherwise provide a desired pharmacologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the age of the subject.

[0053] The term “immunogen,” “immunogens,” “antigen,” or “antigens” means a material that can induce an immune response and is therefore antigenic. By “immune response” means any reaction by the immune system. These reactions include the alteration in the activity of an organism's immune system in response to an antigen and may involve, for example, antibody production, induction of cell-mediated immunity, complement activation or development of immunological tolerance. Immune response to antigens is well studied and widely reported. A survey of immunology is given in Barrett, James, T., Textbook of Immunology: Fourth Edition, C. V. Mosby Co., St. Louis, Mo. (1983). More specifically, the present disclosure provides a live, attenuated fungus (e.g. Coccidioides spp.) that can be used as an immunogenic composition or a vaccine. It will be appreciated that the attenuated fungus contains a dysfunctional or deletion of CPS 1 gene. “Vaccine,” as used herein, means an agent used to stimulate the immune system of a living organism so that protection against future harm is provided “Immunization” refers to the process of inducing a continuing high level of antibody and / or cellular immune response in which T-lymphocytes can either kill a pathogen and / or activate other cells (e.g., phagocytes) to do so in an organism, which is directed against a pathogen or antigen to which the organism has been previously exposed.

[0054] The term “adjuvant” is intended to mean a composition with the ability to enhance an immune response to an antigen generally by being delivered with the antigen at or near the site of the antigen. Ability to increase an immune response is manifested by an increase in immune mediated protection. Enhancement of humoral immunity can be determined by, for example, an increase in the titer of antibody raised to the antigen. Enhancement of cellular immunity can be measured by, for example, a positive skin test, cytotoxic T-cell assay, ELISPOT assay for IFN- gamma or IL-2. Adjuvants are well known in the art. Exemplary adjuvants include, for example, Freud's complete adjuvant, Freud's incomplete adjuvant, aluminum adjuvants, MF59 and QS21. Adjuvants can also be genetically encoded such as mRNA adjuvants.

[0055] As used herein, “inhibit,” “inhibiting,” or “inhibition” includes any measurable or reproducible reduction in the infectivity of a fungus in the subject. “Reduction in infectivity” means the ability of the subject to prevent or limit the spread of the fungus in tissues or organs exposed to or infected by the fungus. Furthermore, “amelioration,” “protection,” “prevention,” and “treatment” mean any measurable or reproducible reduction, prevention, or removal of any of the symptoms associated with fungal infectivity, and particularly, the prevention, or amelioration of infection and resultant pathology itself.

[0056] As used herein, “subject” means a patient or individual having symptoms of, or at risk for, fungal infection, coccidioidomycosis, or other malignancy. A subject may be human or nonhuman and may include, for example, laboratory animal, companion animal; draft animal, meat animal, zoo animal, and human. The subjects may include either adults or juveniles (e.g., children). Moreover, subject may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. II. COMPOSITIONS

[0057] A. Live Attenuated Fungal Species

[0058] Live attenuated fungal mutants which are avirulent are disclosed. They include a CPS1 deletion. The live attenuated fungal mutants species are are genetically modified to delete the CPS1 gene (Pepsi)' , retain the 5’ flank of the CPS1 linked to the 3’flank of CPS1 and additionally, do not include exogenous nucleic acids. The live attenuated cpsl fungal mutants have in their genome, the 5'CPSl flank linked to the 3'CPSl flank i.e., Acpsl / suicide~'e / marker'veor Acpsl ::nada. The disclosed live attenuated fungal mutants (i.e., Acpsl ::nada) can be further subjected to the disclosed two-step process, to provide mutants with a further deletion of additional gene (s) of interest which include the 5 ’ flank of the additional gene(s) of interest linked to the 3 ’flank of the gene(s) of interest without having to have different selectable markers for each mutation. Additional mutations, (for example, deletions) that could be made to further attenuate the Acpsl ::nada strain could be genes in amino acid biosynthetic pathways such as a-IPM synthase (LeuC), and a-IPM isomerase (Leu A), that have been implicated in virulence in other fungi, or genes involved in cell stress responses such as HAC1 and IRE1 which regulate the unfolded protein response, or heat shock protein genes such as Hsp90, Hsp70, and Hsp20-40 that are important for proper protein folding and induced during the parasitic phase.

[0059] The fungus is selected from the group consisting of: Coccidioides immitis or Coccidioides posadasii.

[0060] In a preferred embodiment, the disclosed compositions include a live attenuated Acpsl ::nada Coccidiodes species.

[0061] Coccidioides immitis and Coccidioides posadasii reside in the soil in certain parts of the southwestern United States, most notably in California and Arizona. It is also prevalent in northern Mexico, and parts of Central and South America. It is dormant during long dry spells, then develops as a mold with long filaments when the rains come and that matures to break off into spores. The spores, known as arthroconidia, are swept into the air by disruption of the soil, such as during construction, farming, windstorms or an earthquake.

[0062] In soil, Coccidioides spp. exists in filament form. It forms hyphae in horizontal and vertical direction. With time, cells within hyphae degenerate to form alternating barrel shaped cells, approximately 3-5 microns in size, called arthroconidia. Arthroconidia are lightweight and carried by air currents. They can easily be inhaled without a person knowing. On arriving in alveoli, they enlarge in size and internal septations are developed, forming a structure termed a spherule. Internal spores, termed endospores develop within the spherule as it matures. Rupture of the spherules release these endospores, which in turn repeat the cycle and spread the infection locally and can disseminate to any organ via the blood and lymph systems. Nodules can form in lungs surrounding these spherules. When these rupture, they release their contents into bronchus, forming thin-walled cavities (FIG. 3, reproduced from Donovan, et al., Clin Microbiol Rev 33:e00112-19, 2020). These cavities can result in symptoms like characteristic chest pain, meoptysis and persistent cough. B. Adjuvants

[0063] The disclosed Acpsl::nada strains can be administered in conjunction with other immunoregulatory agents, including adjuvants. Useful adjuvants but are not limited to one or more set forth below:

[0064] Glycolipids, Mineral Containing Adjuvant Compositions include mineral salts, such as aluminum salts and calcium salts. Exemplary mineral salts include hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates), sulfates, and the like or mixtures of different mineral compounds (e.g., a mixture of a phosphate and a hydroxide adjuvant, optionally with an excess of the phosphate), with the compounds taking any suitable form (e.g., gel, crystalline, amorphous, and the like), and with adsorption to the salt(s) being preferred. The mineral containing compositions can also be formulated as a particle of metal salt (WO / 0023105). Aluminum salts can be included in compositions of the invention such that the dose of A13+ is between 0.2 and 1.0 mg per dose.

[0065] Oil-Emulsion Adjuvants suitable for use as adjuvants in the invention can include squalene-water emulsions, such as MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer). See, e.g., WO90 / 14837 and, Podda, Vaccine 19: 2673-2680, 2001. Additional adjuvants for use in the compositions are submicron oil-in-water emulsions. Examples of submicron oil-in-water emulsions for use herein include squalene / water emulsions optionally containing varying amounts of MTP-PE, such as a submicron oil-in-water emulsion containing 4-5% w / v squalene, 0.25-1.0% w / v Tween 80 (polyoxyelthylenesorbitan monooleate), and / or 0.25-1.0% Span 85 (sorbitan trioleate), and, optionally, N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(T-2'-dipalmitoyl-s- -n- glycero-3-huydroxyphosphophoryloxy)-ethylamine (MTP-PE), for example, the submicron oil- in-water emulsion known as "MF59" (International Publication No. WO90 / 14837; U.S. Pat. Nos. 6,299,884 and 6,451,325, incorporated herein by reference in their entirety. MF59 can contain 4-5% w / v Squalene (e.g., 4.3%), 0.25-0.5% w / v Tween 80, and 0.5% w / v Span 85 and optionally contains various amounts of MTP-PE, formulated into submicron particles using a microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, Mass.). For example, MTP-PE can be present in an amount of about 0-500 pg / dose, or 0-250 pg / dose, or 0-100 pg / dose. Submicron oil-in-water emulsions, methods of making the same and immunostimulating agents, such as muramyl peptides, for use in the compositions, are described in detail in International Publication No. WO90 / 14837 and U.S. Pat. Nos. 6,299,884 and 6,451,325.

[0066] Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) can also be used as adjuvants in the invention. Saponin Adjuvant Formulations can also be used as adjuvants in the invention. Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root). Saponin adjuvant formulations can include purified formulations, such as QS21, as well as lipid formulations, such as Immunostimulating Complexs (ISCOMs; see below). Saponin compositions have been purified using High Performance Thin Layer Chromatography (HPLC) and Reversed Phase High Performance Liquid Chromatography (RP-HPLC). Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. A method of production of QS21 is disclosed in U.S. Pat. No. 5,057,540. Saponin formulations can also comprise a sterol, such as cholesterol (see WO96 / 33739). Combinations of saponins and cholesterols can be used to form unique particles called ISCOMs. ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. For example, an ISCOM can include one or more of Quil A, QHA and QHC. ISCOMs are described in EP0109942, WO96 / 11711, and WO96 / 33739. Optionally, the ISCOMS can be devoid of additional detergent. See WO00 / 07621. A description of the development of saponin based adjuvants can be found at Barr, et al., Advanced Drug Delivery Reviews 32: 247-27, 1998 and Sjolander, et al., Advanced Drug Delivery Reviews 32: 321-338, 1998.

[0067] Virosomes and Virus Like Particles (VLPs) can also be used as adjuvants. These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome. The viral proteins can be recombinantly produced or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and- Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, QB-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein pl).

[0068] Bacterial or Microbial Derivatives useful as adjuvants include: (i) Non-Toxic Derivatives of Enterobacterial Lipopolysaccharide (LPS); (ii) lipid derivatives, (iii) immunostimulatory oligonucleotides and ADP-Ribosylating Toxins and Detoxified Derivatives Thereof, (iv) ADP- Ribosylating Toxins and Detoxified Derivatives Thereof. Examples of Non-Toxic Derivatives of LPS Monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3 dMPL). 3 dMPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. An example of a "small particle" form of 3 De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454. Such "small particles" of 3 dMPL are small enough to be sterile filtered through a 0.22 micron membrane (see EP 0 689 454). Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g., RC-529 (Johnson et al., Bioorg Med Chem Lett, 9: 2273-2278, 1999). Examples of lipid A derivatives can include derivatives of lipid A from Escherichia coli such as OM-174. OM-174 is described for example in Meraldi et al., Vaccine 21: 2485-2491, 2003; and Pajak, et al., Vaccine 21: 836-842, 2003. Examples of immunostimulatory oligonucleotides nucleotide sequences containing a CpG motif (a sequence containing an unmethylated cytosine followed by guanosine and linked by a phosphate bond). Bacterial double stranded RNA or oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.

[0069] The CpG’s can include nucleotide modifications / analogs such as phosphorothioate modifications and can be double- stranded or single-stranded. Optionally, the guanosine can be replaced with an analog such as 2'-deoxy-7-deazaguanosine. See Kandimalla, et al., Nucleic Acids Research 31: 2393-2400, 2003; WO02 / 26757 and WO99 / 62923 for examples of analog substitutions. The adjuvant effect of CpG oligonucleotides is further discussed in Krieg, Nature Medicine (2003) 9(7): 831-835; McCluskie, et al., FEMS Immunology and Medical Microbiology (2002) 32: 179-185; W098 / 40100; U.S. Pat. No. 6,207,646; U.S. Pat. No. 6,239,116 and U.S. Pat. No. 6,429,199. The CpG sequence can be directed to Toll-like receptor (TLR9), such as the motif GTCGTT or TTCGTT. See Kandimalla, et al., Biochemical Society Transactions (2003) 31 (part 3): 654-658. The CpG sequence can be specific for inducing a Thl immune response, such as a CpG-A ODN, or it can be more specific for inducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in Blackwell, et al, J. Immunol. 170: 4061-4068, 2003; Krieg, TRENDS in Immunology 23: 64-65, 2002, and WO01 / 95935. In some aspects, the CpG oligonucleotide can be constructed so that the 5' end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences can be attached at their 3' ends to form "immunomers". See, for example, Kandimalla, et al., BBRC 306: 948-95, 2003; Kandimalla, et al., Biochemiccd Society Transactions 31: 664-658, 2003; Bhagat et al., BBRC 300: 853-861, 2003, and WO03 / 035836. Bacterial ADP-ribosylating toxins and detoxified derivatives thereof can be used as adjuvants in the invention. For example, the toxin can be derived from E. coli (i.e., E. coli heat labile enterotoxin (LT)), cholera (CT), or pertussis (PTX). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in WO95 / 17211 and as parenteral adjuvants in WO98 / 42375. In some aspects, the adjuvant can be a detoxified LT mutant such as LT-K63, LT-R72, and LTR192G. The use of ADP-ribosylating toxins and detoxified derivatives thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in the following references, each of which is specifically incorporated by reference herein in their entirety: Beignon, et al., Infection and Immunity 70: 3012-3019, 2002; Pizza, et al., Vaccine 19: 2534-2541, 2001; Pizza, et al., Int. J. Med. Microbiol 290: 455-461, 2003; Scharton- Kersten et al., Infection and Immunity 68: 5306-5313, 2000; Ryan et al., Infection and Immunity 67: 6270-6280, 2003; Partidos et al., Immunol. Lett. 67: 09-216, 1999; Peppoloni et al., Vaccines 2: 285-293, 2003; and Pine et al., J. Control Release 85: 263-270, 2002.

[0070] Bioadhesives and mucoadhesives can also be used as adjuvants in the invention. Suitable bioadhesives can include esterified hyaluronic acid microspheres (Singh et al., J. Cont. Rele. 70:267-276, 2001) or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof can also be used as adjuvants in the invention disclosed for example in WO99 / 27960.

[0071] Adjuvant Microparticles: Microparticles can also be used as adjuvants. Microparticles (i.e., a particle of about 100 nm to about 150 pm in diameter, or 200 nm to about 30 pm in diameter, or about 500 nm to about 10 pm in diameter) formed from materials that are biodegradable and / or non-toxic (e.g., a poly(alpha-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, and the like), with poly(lactide-co- glycolide) are envisioned, optionally treated to have a negatively-charged surface (e.g., with SDS) or a positively-charged surface (e.g., with a cationic detergent, such as CTAB).

[0072] Examples of liposome formulations suitable for use as adjuvants are described in U.S. Pat. No. 6,090,406, U.S. Pat. No. 5,916,588, and EP 0 626 169.

[0073] Additional adjuvants include polyoxyethylene ethers and polyoxyethylene esters. WO99 / 52549. Such formulations can further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol (WO 01 / 21207) as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol (WO 01 / 21152). In some aspects, polyoxyethylene ethers can include: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8- steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35 -lauryl ether, or polyoxyethylene-23-lauryl ether.

[0074] PCPP formulations for use as adjuvants are described, for example, in Andrianov et al., Biomaterials 19: 109-115, 1998.1998. Examples of muramyl peptides suitable for use as adjuvants in the invention can include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-l-alanyl-d-isoglutamine (nor-MDP), and N-acetylmuramyl-l-alanyl-d- isoglutaminyl-l-alanine-2-(r-2'-dipalmitoyl-s- -n-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE). Examples of imidazoquinolone compounds suitable for use as adjuvants in the invention can include Imiquimod and its homologues, described further in Stanley, Clin Exp Dermatol 27: 571-577, 2002 and Jones, Curr Opin Investig Drugs 4: 214-218, 2003. Human immunomodulators suitable for use as adjuvants in the invention can include cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, and the like), interferons (e.g., interferon-gamma), macrophage colony stimulating factor, and tumor necrosis factor.

[0075] Adjuvant Combinations: adjuvants are used in some preferred embodiments as combinations. For example, adjuvant compositions can include: a saponin and an oil-in-water emulsion (WO99 / 11241); a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g., 3 dMPL) (see W094 / 00153); a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g., 3 dMPL)+a cholesterol; a saponin (e.g., QS21)+3 dMPL+IL-12 (optionally+a sterol) (WO98 / 57659); combinations of 3 dMPL with, for example, QS21 and / or oil-in-water emulsions (See European patent applications 0835318, 0735898 and 0761231); SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic- block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion. Ribi adjuvant system (RAS), (Ribi Immunochem) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox); and one or more mineral salts (such as an aluminum salt)+a non-toxic derivative of LPS (such as 3 dPML).

[0076] Aluminum salts and MF59 are examples of adjuvants for use with injectable influenza vaccines. Bacterial toxins and bioadhesives are examples of adjuvants for use with mucosally- delivered vaccines, such as nasal vaccines. All adjuvants noted above, and others as generally known in the art to one of ordinary skill, can be formulated for intranasal administration using techniques well known in the art.

[0077] C. Formulations and Carriers

[0078] The composition of the invention can be formulated in pharmaceutical compositions. These compositions can comprise, in addition to one or more of the strains, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials well known to those skilled in the art. Such materials should typically be non-toxic and should not typically interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal routes.

[0079] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. A tablet can include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline solution, dextrose, or other saccharide solution or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol can be included. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition (e.g., immunogenic or vaccine formulation) is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. The formulation should be selected according to the mode of administration.

[0080] For intravenous, cutaneous, or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity, and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants, and / or other additives can be included, as required.

[0081] The compositions may be formulated for intranasal administration with a pharmaceutically acceptable carrier such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) suitable mixtures thereof, or vegetable oils. If necessary, the action of contaminating microorganisms may be prevented by various antibacterial agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. It will often be preferable to include in the formulation isotonic agents, for example, glucose or sodium chloride. Such formulation may be administered intranasally as an aerosol or atomized spray, or as liquid drops.

[0082] Mucosal compositions may be, for example, liquid dosage forms, such as pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Excipients suitable for such vaccine preparations include, for example, inert diluents commonly used in the art, such as, water, saline, dextrose, glycerol, lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol. Excipients also can comprise various wetting, emulsifying, suspending, flavoring (e.g., sweetening), and / or perfuming agents.

[0083] Administration is preferably in a “therapeutically effective amount” or “prophylactically effective amount” (as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of disease being treated. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in the latest edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa. (“Remington's”).

[0084] III. METHODS OF MAKING

[0085] Several methods are known in the art to achieve gene deletion (Pratt and Aramayo, Fungal Genet Biol, 37(1 ) :56-71 (2002); Dohn, et al., Fungal Genet Biol. 1 1 1 : 1-6 (2018)). Once cells have undergone successful gene deletion via homologous recombination, they are selected using selectable markers that can get integrated into the organism’s genome through this process. Cells are grown in a selective medium containing the antibiotic or selective agent to which the marker gene confers resistance. Only cells that contain the integrated selectable marker gene through homologous recombination will survive. However, this technique leaves a selectable marker as a footprint in the genome. Therefore, the presence of foreign selective marker genes in surviving cells can pose challenges, particularly when these cells are intended for therapeutic use or regulatory approval in humans. Consequently, developing a method to generate deletion strains without marker genes is crucial.

[0086] Further, although techniques described in Dohn, et al., Fungal Genet Biol. 111:1-6 (2018) can be used to produce markerless strain however, it requires a pyrG mutant strain to start the selection which is not always feasible.; the pyrG selection has not worked in Coccidioides.

[0087] Construction of the marker less cpsl strain

[0088] Deletion of the Coccidioides CPS1 gene resulted in a mutant strain that shows efficacy and safety as a live attenuated vaccine to prevent coccidioidomycosis that is being developed for commercial production as a canine vaccine. The original mutant (Narra et al., Infection and Imunology, 84(10) (2016)) was made by replacing the six kilobase CPS1 gene with the hygromycin phosphotransferase drug resistance marker (Hph), allowing selection for the transformed line. For development of a human vaccine candidate, it would be desirable to have a CPS1 deletion strain that does not contain an exogenous gene such as a drug resistance marker. To create such a strain, a system using a two-step process to eliminate a gene of interest by incorporating a “suicide gene” vector system was designed. The two-step process incorporates two vectors. The first vector includes a “suicide gene”, for example, HSV:tk and a selectable marker for example, hph, placed between flaking sequences of the gene to be replaced, for example the CPS1 gene (herein after, “suicide vector”) (See FIG. V, Acpsl::hph, HSV: tk construct). The second vector includes flanking sequences for the gene to be replaced, for example, CPS1 gene, fused to each other (See FIG. 1; Acpsl ::nada construct).

[0089] In the first step, the fungus is transfected with the suicide vector. The introduction of this construct into the fungus produces a gene replacement mutant strain of CPS1 gene that lacks the CPS1 gene coding sequences, and expresses the suicide gene (See FIG. 1) i.e., a ACPSl / suicide. Mutant strains contain the suicide and the gene encoding the selectable marker (herein A CPS 1 / suicide / marker) are selected by treatment with the drug corresponding to the marker. For example, where the marker is hygromycin phosphotransferase (hph), mutants are selected by treatment with hygromycin. Where the marker is bleomycin resistance protein (hie), mutants are selected by treatment with bleomycin.

[0090] In the second step, the selected A CPS 1 / suicide / marker mutants are transfected with a construct that contains the CPS1 gene flanking sequences fused to each other, and selected against the presence of the suicide gene to obtain mutants that now lack the suicide gene and selectable marker gene (herein, ACPSl / suicide-ve / marker-ve or Acpsl::nada). For example, where the suicide gene is the Herpes simplex virus thymidine kinase gene (HSV-Tk), gene, the desired mutants are selected by treatment with drug 5-fhioro-2’ -deoxyuridine (F2dU). For strains to survive, the suicide gene and selectable marker at the CPS1 gene locus need to be removed and replaced by the construct of the CPS1 gene flanking sequences that was introduced in step 2.

[0091] Using this two-step process, a CPS1 gene replacement mutant that contains no foreign sequences (i.e., foreign to the fungus being transformed) at the CPS1 gene locus was generated in the Examples below; instead, it contains the normal (endogenous) gene A flanking sequences fused to each other with the entire gene A coding sequences deleted. This system uses the ability to counter- select against a suicide gene such as the Herpes simplex virus thymidine kinase gene (HSV-Tk) using the drug 5-fluoro-2’ -deoxyuridine (F2dU). The F2dU drug is converted by the HSV-Tk protein to a toxic metabolite that is lethal to fungal cells expressing the HSV-Tk gene.

[0092] As shown in the Examples, in the first step, a Coccidioides posadasii strain Silveira (AM1801) mutant was created, where the CPS1 gene was deleted by replacement with the Hph gene and the HSV-Tk gene, via selection for resistance to hygromycin. This mutant strain was then used as a transformation recipient introducing a construct AM1714 that contained the 5’ and 3’ flanking sequences of the C. posadasii CPS1 gene with selection on F2dU at 400 micromolar (Figure 1). This compound is lethal for untransformed AM1801 strains.

[0093] Strains that grew on the drug were screened and demonstrated to be CPS1 deletion mutants where the Hph-HSV-Tk sequences were deleted by homologous recombination between the AM1714 construct and the flanking sequences at the AM1801 strain locus, resulting in a CPS1 locus that only contained the fused flanking sequences of the CPS1 gene, and no foreign sequences, as described above. Thus, these strains are markerless mutants. This twostep process is key for two reasons: 1) one is able to create markerless mutant strains, which would allow one to make strains with multiple mutations (for example, the cpsl deletion mutant (Acpsl::nada) can now be used as a recipient for deletion of a second gene using the same process as used to create the cpsl deletion strain); 2) the markerless cpsl deletion mutant provides an improved live attenuated cpsl vaccine for humans.

[0094] Another suicide gene system uses a Tet-On inducible promoter to regulate two lethal bacterial endonuclease genes, the E. coli mazF gene, and the Serratia marcescens nucA gene. This system works by having these suicide genes transformed in like HSV-Tk, and when the second construct is transformed in, inducing the endonucleases by growing the transformants in the presence of tetracycline, which induces the nuclease and kills cells in which the genes are still present.

[0095] Methods of culturing fungi and generating spores the form, are known in the art (Mead, et al., Curr Protoc Microbiol. 2020 Sep;58(l):el l3).

[0096] IV. METHODS OF USING

[0097] Coccidioidomycosis is a fungal infection (Valley Fever) known to be caused by the two endemic fungal species, Coccidioides immitis and Coccidioides posadasii. The disease can range from an asymptomatic infection that renders humans immune for life to a fatal respiratory or disseminated infection. Approximately 40% of 150,000 people infected annually become sick, and approximately 5% develop severe and life-threatening illness that may leave them disabled, under continuous treatment, or deceased. Among patients that develop disseminated disease, most are treated for months to years, and many who discontinue treatment suffer relapse at some point because the current drugs available suppress but do not eradicate the fungus from the body. Treatment with antifungal medication may cost between $5000-$20,000 per year, not including the costs of ancillary care such as hospitalization, rehabilitation, frequent medical care, and disability. Coccidioidomycosis is an endemic fungal infection that is reported in up to 20,000 persons per year and has an economic impact close to $1.5 billion (Galgiani et al., J Fungi (Basel), 8(8): 838 (2022)).

[0098] Coccidioidomycosis is commonly known as cocci or “Valley Fever”, as well as “California Fever”, “desert rheumatism”, and “San Joaquin Valley Fever”, is endemic in certain parts of Arizona, California, Nevada, New Mexico, Texas, Utah and northern Mexico.

[0099] Infection is caused by inhalation of the particles. The disease is not transmitted from person to person. The infection ordinarily resolves leaving the patient with a specific immunity to re-infection. However, in some cases the infection may manifest itself repeatedly or permanently over the life of the host. Coccidioides immitis or Coccidioides posadasii is a dimorphic saprophytic organism that grows as a mycelium in the soil and produces a spherule form in the host organism.

[0100] Methods for eliciting an immune response against Coccidioides species that cause Coccidioidomycosis are disclosed. Besides humans, dogs, and cats, the fungus can be shown to infect most mammals, even if they do not get sick from it very often. Species in which Valley Fever has been found include livestock such as cattle and horses; llamas; marine mammals, including sea otter; zoo animals such as monkeys and apes, kangaroos, tigers, etc.; and wildlife endemic to the geographic area such as cougar, skunk, and javelina.

[0101] The methods include administering the avirulent Coccidioides posadasii strain, Acpsl , to the subject. In one embodiment, the administration is not followed by administration of an antifungal agent that is capable of preventing / blocking fungal growth. The subject is preferably a mammal, such as a human subject.

[0102] The compositions in some forms include markerless CPS1 Coccidioides posadasii or markerless CPS1 Coccidioides immitis.

[0103] The compositions in some forms include any avirulent forms of Coccidioides posadasii or avirulent Coccidioides immitis.

[0104] In some forms, the subject is administered a composition containing an effective amount of markerless Acpsl spores of Coccidioides posadasii. In some forms the subject is administered a composition containing an effective amount of markerless Acpsl spores of Coccidioides immitis. In some forms the Acpsl strains are chemically attenuated Coccidioides. In some forms the Acpsl strains are genetically attenuated Coccidioides. In some forms the Acpsl strains of Coccidioides have been irradiated to provide attenuation.

[0105] In certain embodiments, a pharmaceutical composition comprising one or more species of live-attenuated Coccidioides is co-administered with an adjuvant such as a glycolipid adjuvant. Useful glycolipid analogs are disclosed for example in U.S. Patent No. 9,642,909. In some embodiments, the co-administration is by the same or a different route of administration. For example, a pharmaceutical composition comprising one or more species of cpsl strains of Coccidioides administered by an intravenous, intramuscular, intradermal, or subcutaneous route can be co- administered with a glycolipid adjuvant administered by an intravenous, intramuscular, intradermal, or subcutaneous route.

[0106] In some forms the subject is administered one or more doses or a pharmaceutical composition containing no more than 150,000 spores; no more than 50,000 spores or no more than 25,000 spores. In some forms no more than 3 doses are administered, no more than two doses are administered or no more than one dose is administered.

[0107] In some forms, the dose of composition administered is selected from the group consisting of: at least about 500 spores of the composition; at least about 1,000 spores of the composition; at least about 10,000 spores of the composition; at least about 20,000 spores of the composition; at least about 30,000 spores of the composition; at least about 40,000 spores of the composition; at least about 50,000 spores of the composition; at least about 60,000 spores of the composition; at least about 70,000 spores of the composition; at least about 80,000 spores of the composition; at least about 90,000 spores of the composition; at least about 100,000 spores of the composition; at least about 150,000 spores of the composition; at least about 200,000 spores of the composition; at least about 300,000 spores; at least about 500,000 spores.

[0108] Also provided are such methods which further comprise administering at least a second subsequent dose of the composition to the mammal.

[0109] Also provided are such methods wherein the at least second subsequent dose is administered at a time interval selected from the group consisting of: approximately one week after the first dose; approximately two weeks after the first dose; approximately three weeks after the first dose; approximately four weeks after the first dose; approximately five weeks after the first dose; approximately six weeks after the first dose; approximately seven weeks after the first dose; and approximately eight weeks after the first dose.

[0110] The invention can be further understood by way of the following non-limiting examples.

[0111] Examples

[0112] Methods

[0113] Mice: C57BL / 6 8 wk. old F mice - 44 mice

[0114] Fungal strains: strain 19 / hyg; markerless Acpsl; Silveira

[0115] IN challenge / Vx with cpsl: Grp 1,2 IN - 6 / 20; SC Vx Grp 3-5 - #1 - 6 / 21, #2 - 7 / 5

[0116] Infect (Silveira): Grp 3-5 - 7 / 31

[0117] Sacrifice: Grp 1,2 - 7 / 18; Grp 3-5 - 8 / 14 Group 1: strain 19 - IN 10 mice

[0118] Group 2 markerless - IN 10 mice

[0119] Group 3: strain 19 SC x2 8 mice

[0120] Group 4: markerless SC x2 8 mice

[0121] Group 5 : saline SC x 2 8 mice

[0122] Inoculate group 1 and 2 mice once IN with 10K spores of cpsl and monitor for 4 weeks. Sacrifice on day 28 and culture entire lungs. This will compare clearance / persistence. 8 of these will be cultured and histopathology performed on 2. Mice should be negative by day 28.

[0123] Vaccinate groups 3 and 4 twice 2 weeks apart with 10K spores of Icpsl and challenge with Silveira 4 weeks later to verify ability to protect. Mice will be sacrificed for fungal burden at 14 days.

[0124] Groups 1 and 2: IN inoculation - Mice were anesthetized with ketamine 80 mg / kg and xylazine 10 mg / kg IP. Anesthesia was good but one mouse was briefly placed in the anesthesia chamber with isoflurane. Infection dose target was incorrect and mice received higher than planned doses because LFS wrote 10,000 spores for inoculation rather than the usual 1000 spores used to test avirulence of cpsl knockouts. Post-infection plate counts showed Strain 19 mice received 8,667 spores and Strain 1714 mice received 5,933 spores.

[0125] Mice did not become ill during the 28 day study period. At dissection, 6 / 10 of the strain 19 mice had no gross evidence of cpsl. The other four mice had visible spots on the lung tissue that were not solid in nature. Three of the 1714 mice had 1-2 granulomas each and the remainder had normal lungs. There was no evidence of dissemination in any mice. Spleens were plated whole and lungs were collected for quantitative fungal burden in #1-8. #9 and 10 had lungs and spleens fixed for histopathology.

[0126] Groups 3-5: SC vaccination - Mice were vaccinated twice in the right (prime) and left (boost) groin SC 2 weeks apart. Post- vaccination counts were lower than the planned 10K target dose (str 19: P7,133 B 8,000; 1714: P 6,533 B 8,467). The group 5 mice received saline SC on the same schedule. About one week after booster vaccination, small reactions at the prime site of both groups of mice began to develop in 30-40% of the mice. None were very large or required treatment and there did not seem to be any difference between the groups. At necropsy, only a few mice had significant reactions. One had a large primary site and a small booster site, neither of which was draining or appeared painful. Another in the other group had a moderate prime site, and one had evidence of prior drainage and scarring. (I failed to record the animal numbers, I think, but I have some photos). A novel observation in these mice, most of which were vaccinated by a single person holding and injecting the mice, is that there were body wall granulomas and two mice (strain 19 #6, strain 1714 #5) had a string of 2-4 small white nodules in the area of the mesentery between the pancreas and the spleen and two very white 1-3 mm lesions each within the liver near the diaphragm. Both of these tissues grew on GYE plates, and the growth from the strain 19 group subsequently grew on hyg plates. There were no other grossly visible abdominal abnormalities in either mouse. I think some of the mice may have been accidentally peritoneally vaccinated by struggling during the vaccination process and having the needle slip into the abdomen. This was not related to evidence of protection, as both of these mice had low lung fungal burdens and no splenic dissemination. Obviously, the vaccine strains did not disseminate widely or expand significantly.

[0127] Three mice (1 strain 19, 2 strain 1714) had progressive disease of lungs with weight loss (range 3.4%- 19%). While the fungal burdens did not approach the quantities of the unvaccinated mice by more than 1 log, these would be classified as poor vaccine responses.

[0128] Silveira IN challenge - Mice were challenged with a target dose of 100 spores of strain Silveira IN under anesthesia. Anesthesia was routine with 80 mg ketamine and 10 mg xylazine and all mice were adequately anesthetized. Some recovery times were extended and mice required heat support with disposable chemical handwarmers. AU mice were sternal or ambulatory within 40 minutes. Post infection plate counts were that mice received 47 spores, approximately half of the target dose.

[0129] Results:

[0130] 1) A virulence of Strain 1714

[0131] Mice were given 6-8 fold higher doses of the vaccine strains into the lungs than normally tested to determine avirulence. Lungs and spleens from mice given strain 19 were all culture negative, while 3 / 8 mice given strain 1714 had some growth in lungs but none in spleens. One mouse (1714 #3) exhibited mild expansion from the initial inoculation (lung fungal burden 12,300 cfu), and the other two were lower than starting inoculum (910 cfu, 2500 cfu). The conclusion from this is that strain 1714, the markerless Acpsl, is avirulent in mice. Strain 19 continues to robustly demonstrate a lack of pathogenicity along with a general lack of persistence when given as a single lung inoculation.

[0132] Histopathology supported the observations and culture results. From the strain 19 group: #9 - 1 very small residual granuloma, possibly 1 endospore observed. #10 - Normal lungs, no lesions.

[0133] From 1714 group:

[0134] #9 - Normal lungs, no lesions

[0135] #10 - 1 small / medium granuloma with 4-5 spherules. Most spherules appear to have no contents. Granuloma has very well defined borders / marginal zone with abundant lymphoid aggregates.

[0136] 2) Vaccine protection from Strain 1714

[0137] Mice were vaccinated twice two weeks apart and challenged IN with Silveira 4 weeks later. Some animals in the control group began to look lethargic by day 9 and lost weight very fast, with the first animal appearing moribund on day 10 with 4 grams of weight loss (28%). Close monitoring daily led to all but three mice being sacrificed before day 14. One animal in this group was uninfected, but there were 9 mice and the uninfected mouse was removed from the analysis. All vaccinated mice looked clinically normal during the 14-day observation period, though two mice with higher lung fungal burdens had some weight loss. Results of lung and spleen fungal burden are shown in Figures 2A-2B.

[0138] Note that one of the mice in the cpsl 19 group (gray symbol with box) had growth from the spleen, but it was all cpsl that grew on hyg medium. PCR will be needed to determine if the growth from the 1714 strain mouse with a string of mesenteric granulomas and 2 liver granulomas was the knockout strain, as anticipated. There was no growth of any fungus from lung or spleen of that animal and it is anticipated that the nodules and the liver lesions are 1714 strain.

[0139] The three vaccinated mice with relatively high fungal burdens and dissemination, as well as weight loss and 3L lung scores (20-80% abnormal lung tissue) were considered vaccine failures or suboptimal vaccination, though their level of disease was less than the controls in terms of fungal burden, dissemination, and systemic clinical illness.

[0140] Discussion

[0141] The markerless CPS1 knockout strain 1714 is avirulent as predicted. The mild persistence in some of the mice shows the isolate picked might have minimally better growth competence in mice than vaccine strain 19, but the strain 19 has been selected for avirulence for some years and recloned to select avirulence. Generally, there is no difference in the avirulence of the markerless construct and it protects mice equally well.

[0142] Most mice show excellent protection and low fungal burdens in studies that have been performed till date. Source of mice is unchanged, along with infecting doses of spores, and the growth of challenge strain from Seed Bank only.

Claims

CLAIMSWe claim:

1. A method of making a live attenuated fungal mutant with a CPS1 gene deletion, comprising the following steps:(a) transfecting the fungus with a suicide vector, wherein the suicide vector comprises a suicide gene, a selectable marker and the 5’ and 3’ flanking sequence for the CPS1 gene;(b) selecting transformants to obtain ACPSl / suicide / marker mutants;(c) transfection the ACPSl / suicide / marker mutants with a vector comprising the 5’ and 3’ flanking sequence for the CPS1; and(d) selecting transformants against the suicide gene to obtain ACPSl / suicide -ve / marker- ve mutants.

2. The method of claim 1, wherein the suicide gene is the Herpes simplex virus thymidine kinase gene (HSV-Tk) or the E. coli mazF gene regulated by tetracycline inducible promoter, or Serralia marcescens nucA gene regulated by tetracycline inducible promoter.

3. The method of claim 1 or 2, wherein the selectable marker is the hygromycin phosphotransferase drug resistance gene.

4. The method of any one of claims 1-3, wherein selecting transformants to obtain ACPSl / suicide / marker mutants comprises treating the transfected fungus with a compound corresponding to the marker.

5. The method of any one of claims 1-4, wherein selecting transformants against the suicide gene to obtain A CPSl / suicide -ve / marker-ve mutants, comprises treating the ACPSl / suicide / marker mutants with an agent converted by the suicide gene into a compound toxic to the fungus and selecting for surviving fungus.

6. The method of claims 1-5, comprising selecting transformants to obtain ACPSl / suicide / marker mutants by contacting the transfected fungus with hygromycin if the marker gene is hygromycin phosphotransferase (hph) or bleomycin if the marker gene is bleomycin resistance protein (ble).

7. The method of anyone of claims 1-5, comprising selecting for A CPSl / suicide - ve / marker-ve mutants by treating ACPSl / suicide / marker mutants with 5-fluoro-2’ -deoxyuridine (F2dU) or selecting under the presence of tetracycline.

8. The method of claims 1-7, wherein the fungus is a Coccidioides sp.

9. The method of claims 1-7, wherein the fungus is selected from the group consisting of Coccidioides immitis or Coccidioides posadasii.

10. The method of claim 8, wherein the fungus is Coccidioides immitis or Coccidioides posadasii.

11. An immunogenic composition comprising: a live genetically modified Coccidioides fungal spore comprising a deletion of the cyclic peptide synthase Cpsl (CPS1 ) gene and the5’ CPS 1 flank linked to the 3’ CPS 1 flank regions; and a pharmaceutically acceptable carrier.

12. An immunogenic composition made according to the method of claims 1-10.

13. The immunogenic composition of claims 11 or 12, wherein the genetically modified Coccidioides fungal spore is avirulent.

14. The immunogenic composition of claim 1, wherein the spore is avirulent as a result of a deletion the CPS 1 gene.

15. The immunogenic composition of any one of claims 12-14, wherein the Coccidioides fungal spore is obtained from a fungal cell selected from the group consisting of: Coccidioides posadasii and Coccidioides immitis.

16. The immunogenic composition of any one of claims 12-15, wherein the genetically modified Coccidioides fungal spore is capable of inducing immunity to coccidioidomycosis.

17. The immunogenic composition of any one of claims 12-16, wherein the genetically modified Coccidioides fungal spore is capable of inducing an immune response.

18. The immunogenic composition of claim of any one of claims 12-19, wherein the immunogenic composition is formulated for use as a vaccine.

19. A method of preparing a pharmaceutical composition for passive immunization of a mammal in need of immunization comprising: a) mixing the immunogenic composition of claim 1 with a suitable excipient or carrier; and b) forming a pharmaceutical composition.

20. A method of eliciting an immune response in a mammal comprising: a) administering to a mammal a therapeutically effective amount of the immunogenic composition of any one of claims 12-18; and b) eliciting an immune response in the mammal.

21. The method of claim 20, wherein the immunogenic composition is formulated for subcutaneous, intramuscular, intranasal, oral and / or intraperitoneal administration.

22. A method of claim 20, wherein the mammal is selected from the group consisting of: laboratory animal; companion animal; draft animal; meat animal; zoo animal; and human.

23. The method of claim 21 or claim 22, further comprising administering at least a second subsequent dose of the immunogenic composition to the mammal.

24. The method of claim 23, wherein the at least second subsequent dose is administered at a time interval selected from the group consisting of: approximately one week after the first dose; approximately two weeks after the first dose; approximately three weeks after the first dose; approximately four weeks after the first dose; approximately five weeks after the first dose; approximately six weeks after the first dose; approximately seven weeks after the first dose; and approximately eight weeks after the first dose.

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