THE SAME

Field of the Disclosure

[0001] The disclosure relates to formulations, methods, processes, and kits for a spore-based vaccine. In particular, the disclosure relates to formulations with improved stability and/or shelf-life and methods of making the same.

BACKGROUND OF THE DISCLOSURE

[0002] Vaccination is an effective way to provide prophylactic protection against infectious diseases, including, but not limited to, viral, bacterial, and/or parasitic diseases, such as influenza, AIDS, hepatitis virus infection, cholera, malaria and tuberculosis, and many other diseases. For example, Coccidioides species (C. immitis and C. posadasii) are the causative agents of coccidioidomycosis (Valley Fever), an important emerging disease in humans and animals endemic to the southwestern United States as well as pails 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.

[0003] 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 easily transferable to humans because of intolerable adverse effects and poor efficacy.

SUMMARY OF THE INVENTION

[0004] Aspects of the disclosure relate to a storage stable vaccine composition. In some embodiments, the composition comprises one or more live genetically modified Coccidioides fungal spore comprising an inactivated cyclic peptide synthase Cpsl gene product, a buffer, and about 2 to about 20% w/w of a stabilizing agent. In some embodiments, the stabilizing agent is at about 10% w/w to about 20% w/w. In some embodiments, the stabilizing agent is at about 10% w/w. Tn some embodiments, the stabilizing agent is DMSO. In some embodiments, the stabilizing agent is a mixture of BSA and DMSO. In some embodiments, the BSA is about 10% w/w and the DMSO is about 30% w/w. In some embodiments, the buffer is a phosphate buffer. In some embodiments, the composition retains at least about 50% of the one or more live genetically modified Coccidioides fungal spores for at least 90 days. In some embodiments, the composition retains at least about 80% of the one or more live genetically modified Coccidioides fungal spores for at least 90 days. In some embodiments, the composition maintains at least 50% of its original activity for at least 3 months. In some embodiments, the composition maintains at least 90% of its original activity for at least 3 months. In some embodiments, the composition is non-lyophilized. In some embodiments, the buffer is a pharmaceutically acceptable carrier. In some embodiments, the composition is maintained at a temperature of about -4 °C to about room temperature. In some embodiments, the composition is maintained at a relative humidity of at least 10%. In some embodiments, the genetically modified Coccidiodies fungal spore is avirulent. In some embodiments, the composition is formulated for use as a vaccine. In some embodiments, the live genetically modified Coccidioides fungal spore is obtained from a fungal cell selected from the group consisting of: Coccidioides posadasii, Coccidioides immitis, and Coccidioides silveira. In some embodiments, the composition is not subjected to lyophilization or foamdrying. In some embodiments, the composition is not subjected to freezing conditions. In some embodiments, a live, attenuated vaccine comprises the storage stable composition as described herein. In some embodiments, a non-virulent vaccine comprises the storage stable composition as described herein.

[0005] Additional aspects described herein relate to a kit. In some embodiments, a kit comprises the storage stable composition as described herein and instructions for providing the storage stable composition. In some embodiments, the kit further comprises at least one syringe.

[0006] Additional aspects described herein relate to a method for preparing a storage-stable composition. In some embodiments, the method comprises mixing one or more live genetically modified Coccidioides fungal spore comprising an inactivated cyclic peptide synthase Cpsl gene product, a buffer, and about 2 to about 20% w/w of a stabilizing agent, thereby producing a storage- stable composition. In some embodiments, the stabilizing agent is at about 10% w/w to about 20% w/w. Tn some embodiments, the stabilizing agent is at about 10% w/w. In some embodiments, the stabilizing agent is DMSO. In some embodiments, the stabilizing agent is a mixture of BSA and DMSO. In some embodiments, the BSA is about 10% w/w and the DMSO is about 30% w/w. In some embodiments, the buffer is a phosphate buffer. In some embodiments, the composition retains at least about 50% of the one or more live genetically modified Coccidioides fungal spores for at least 90 days. In some embodiments, the composition retains at least about 80% of the one or more live genetically modified Coccidioides fungal spores for at least 90 days. In some embodiments, the composition maintains at least 50% of its original activity for at least 3 months. In some embodiments, the composition maintains at least 90% of its original activity for at least 3 months. In some embodiments, the composition is non-lyophilized. In some embodiments, the buffer is a pharmaceutically acceptable carrier. In some embodiments, the composition is maintained at a temperature of about -4 °C to about room temperature. In some embodiments, the composition is maintained at a relative humidity of at least 10%. In some embodiments, the genetically modified Coccidiodies fungal spore is avirulent. In some embodiments, the composition is formulated for use as a vaccine. In some embodiments, the live genetically modified Coccidioides fungal spore is obtained from a fungal cell selected from the group consisting of: Coccidioides posadasii, Coccidioides immitis, and Coccidioides silveira.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, components. For the sake of brevity, reference numerals or features having a previously described function may or may not be described in connection with other drawings in which they appear.

[0008] FIG. 1 is a block diagram of an embodiment of a method for preparing a storage-stable composition.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0009] The compositions, kits, and methods disclosed herein each have several aspects, no single one of which is solely responsible for their desirable attributes. Without limiting the scope of the claims, some prominent features will now he discussed briefly. Numerous other embodiments arc also contemplated, including embodiments that have fewer, additional, and/or different components, steps, features, objects, benefits, and any other desirous properties. The components, aspects, and steps may also be arranged and ordered differently. After considering this discussion, and particularly after reading the section entitled “Detailed Description”, one will understand how the features of the compositions, kits, and methods disclosed herein provide desirous properties over other known compositions, kits, and methods.

[0010] It was recently discovered that a live genetically modified Coccidioides spore with an inactivated cyclic peptide synthase Cpsl gene (herein referred to as Cpsl) is useful as a potential immune response-provoking agent for protection against coccidioidomycosis. However, these live genetically modified Coccidioides spores were not found to be very shelf-stable or resistant to extreme conditions as the native strains. Thus, the production and stability of the genetically modified Coccidioides spores for the use in vaccines was limited to a narrow window from the time they were produced to the time they would need to be shipped and administered.

[0011] Embodiments of the invention relate to formulations which improve the stability and shelf-life of the genetically modified Coccidioides spores. Accordingly, aspects of the disclosure described herein relate to vaccine formulations including a live genetically modified Coccidioides spore with improved stability, shelf-life longevity, and methods for preparing the same. In some embodiments, the formulations include varying amounts of glycerol, albumin (bovine serum albumin; BSA), and DMSO which were found to stabilize the genetically modified Coccidioides spores for at least 90 days with less than 90% degradation of the spores in the sample. In some embodiments, the formulation is found to stabilize the genetically modified Coccidioides spores for at least 45 weeks. In other embodiments, the formulation may stabilize the genetically modified Coccidioides spores for at least 10, 20, 30, 40, 50, 52 weeks or more.

Definitions

[0012] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, and up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5- fold, and within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0013] The term “buffer” or “buffer system” refers to a pharmaceutically acceptable aqueous buffer system comprising pyrogen-free water system with the ability to resist a change in pH upon addition of an inorganic compound, organic compound, acid, alkali, or dilution with a solvent or diluent. Buffer characteristics are defined in detail in, for example, Remington's Pharmaceutical Sciences, 18th Ed. Gennaro Ed., Mack Publishing Co., Easton Pa. (1990) pp. 241-243.

[0014] Coccidioides cyclic peptide synthase Cpsl, herein referred to as Cpsl, is a protein encoded by the CPS 1 gene and CPS 1 mRNA, and may encode either wild type or a mutant. A wild type or mutant CPS1 gene product will encode for a Cpsl protein.

[0015] 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 CPS 1 can be effected by inactivating the CPS 1 gene. Inactivation which renders the gene or protein dysfunctional includes such methods as deletions, mutations, substitutions, interruptions or insertions in the nucleic acid gene sequence.

[0016] General techniques of genetic recombination, including vector construction, transformation, selection of transformants, host cell expression, etc., are further described in Maniatis et al, 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley Interscience, N.Y.; Innis et al. (eds), 1995, PCR Strategies, Academic Press, Inc., San Diego, Calif.; and Erlich (ed), 1992, PCR Technology, Oxford University Press, New York. Agrobacterium transformation and replacement of Coccidioides genes was as described in Abuodeh et al. 2000, Genetic Transformation of Coccidioides immitis facilitated by Agrohacterium tumefaciens. Journal of Infectious Diseases, 181:2106-2110 and Kellner ct al., 2005, Coccidioides posadasii contains a single 1,3-beta-glucan synthase gene that appears to be essential for growth, Eukaryotic Cell, 4:111-120.

[0017] As used herein, a “therapeutically effective amount” means a quantity of a specified pharmaceutical or therapeutic compound or composition sufficient to achieve a desired effect in a subject, or in a cell, being treated with the compound or composition. The effective amount of the agent will be dependent on several factors, including, but not limited to, the subject or cells being treated, and the manner of administration of the therapeutic composition.

[0018] As is conventional in the art, the term “attenuated” refers to a cell, culture, or strain of fungus (e.g. Coccidioides spp.) exhibiting a detectable reduction in infectivity or virulence in vitro and/or in vivo as compared to that of the parent strain of the fungus from which the attenuated cell, culture, or strain is derived. 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.

[0019] The term “avirulent”, as used herein, 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. “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 CPS 1 gene described herein.

[0020] 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 CPS1 gene.

[0021] As used herein, “Vaccine” 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.

[0022] As used herein, “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.

[0023] As used herein, “thermally- stable” relates to an enhanced persistence of an active substance or pharmaceutical product as a function of time under the influence of a variety of environmental factors, primarily temperature, and is also affected by other conditions such as humidity and light in comparison with a control preparation that is not thermally stable. In making this determination, product-related factors also influence the stability, e.g., the chemical and physical properties of the active substance and the pharmaceutical excipients, the dosage form and its composition, the manufacturing process, the nature of the container-closure system, and the properties of the packaging materials. Also, the stability of excipients that may contain or form reactive degradation products are considered.

[0024] As used herein, “stability” means the prevention of a reaction, reduction or degradation of components, such as genetically modified spores in a composition or formulation. A composition may be “stable” if the components, such as genetically modified spores, are viable for a reasonable period of time.

[0025] As used herein, “maintaining,” “maintain,” and “maintenance” when referring to compositions or active agents means keeping, sustaining, or retaining the bioactivity of at least one genetically modified spore.

[0026] 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.

[0027] 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 non-human 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.

Immunogenic Composition [0028] The current disclosure provides for compositions comprising a fungus having a dysfunctional Cpsl gene product. The fungus having a dysfunctional Cpsl gene product and methods for making the same are described in U.S. Pat. Nos. 9,884,097 and 10,758,600, which are hereby incorporated by reference. In some embodiments, a fungus having a dysfunctional Cpsl gene product is included in an immunogenic composition. In some embodiments, the immunogenic composition may be formulated as an ingredient in a pharmaceutical composition, and this formulation can aid in administration of the composition. In some embodiments, the immunogenic composition may routinely contain pharmaceutically acceptable concentrations of salts, buffering agents, preservatives and various compatible carriers or diluents. For all forms of delivery, the vectors may be formulated in a physiological salt solution. In one embodiment, the immunogenic composition is a spore-based vaccine.

[0029] In some embodiments, the immunogenic composition includes one or more fungi having a dysfunctional Cpsl gene product. The live attenuated fungi described herein are capable of triggering an immune response that protects a mammal against fungal infection or colonization after one or more administrations as a live vaccine. A “protective immune response” refers to any immunological response, either antibody or cell-mediated immunity, or both, occurring in the subject that either prevents or detectably reduces subsequent infection, or eliminates or detectably reduces the severity, or detectably slows the rate of progression, of one or more clinical symptoms or conditions associated with fungal infection.

[0030] In some embodiments, the immunogenic composition includes a live genetically modified Coccidioid.es fungal spore including an inactivated cyclic peptide synthase Cpsl gene product. In some embodiments, the fungus is avirulent. In some embodiments, the dysfunctional Cpsl gene product is a result of a deletion of at least a portion of the Cpsl gene. In some embodiments, the dysfunctional Cpsl gene product is a result of a deletion in a region of the Cpsl gene selected from the group consisting of at least about the entire Cpsl gene. In some embodiments, the dysfunctional Cpsl gene product is a result of at least about the entire DMAP region of the Cpsl gene. In some embodiments, the dysfunctional Cpsl gene product is at least about an entire AMP binding domain region of the Cpsl gene.

[0031] In some embodiments, the dysfunctional Cpsl gene product is a regulatory element of the Cpsl gene. In some embodiments, the dysfunctional Cpsl gene product is a regulatory element of the Cpsl gene with at least 95% the coding sequence of the Cpsl gene. Tn some embodiments, the dysfunctional Cpsl gene product is a regulatory element of the Cpsl gene with at least 90% the coding sequence of the Cpsl gene. In some embodiments, the dysfunctional Cpsl gene product is a regulatory element of the Cpsl gene with at least 70% the coding sequence of the Cpsl gene. In some embodiments, the dysfunctional Cpsl gene product is a regulatory element of the Cpsl gene. at least 60% the coding sequence of the Cpsl gene. In some embodiments, the dysfunctional Cpsl gene product is a regulatory element of the Cpsl gene. at least 50% the coding sequence of the Cpsl gene. In some embodiments, the dysfunctional Cpsl gene product is a regulatory element of the Cpsl gene with at least 40% the coding sequence of the Cpsl gene. In some embodiments, the dysfunctional Cpsl gene product is a regulatory element of the Cpsl gene with at least 30% the coding sequence of the Cpsl gene. In some embodiments, the dysfunctional Cpsl gene product is a regulatory element of the Cpsl gene with at least 20% the coding sequence of the Cpsl gene. In some embodiments, the dysfunctional Cpsl gene product is a regulatory element of the Cpsl gene with at least 10% the coding sequence of the Cpsl gene.

[0032] In some embodiments, the dysfunctional Cpsl gene product is a result of deletion of the entire Cpsl gene. In certain embodiments, the dysfunctional Cpsl gene product is a result of a deletion of at least about the entire Cpsl gene, at least about the entire DMAP region of the Cpsl gene, at least about an entire AMP binding domain region of the Cpsl gene, a regulatory element of the Cpsl gene, at least the coding sequence of the Cpsl gene, at least about 90% of the Cpsl gene, at least about 80% of the Cpsl gene, at least about 70% of the Cpsl gene, at least about 60% of the Cpsl gene, at least about 50% of the Cpsl gene, at least about 40% of the Cpsl gene, at least about 30% of the Cpsl gene, at least about 20% of the Cpsl gene, and at least about 10% of the Cpsl gene. In another embodiment, the dysfunctional Cpsl gene product is a result of deletion of the entire Cpsl gene.

[0033] In some embodiments, fungus is genetically engineered to have a dysfunctional Cpsl gene product. In some embodiments, the fungal cell is selected from the group including Coccidioides immilis; Coccidioides posadasii; Aspergillus fumigalus; Aspergillus flavus; Histoplasma capsulatum; Blastomyces dermatitidis; Cryptococcus neoformans; Cryptococcus laurentii and Cryptococcus albidus; Cryptococcus gattii; Candida albicans; Candida glabrata; Saccharomyces boulardii; Candida tropicalis; Candida krusei; and Candida parapsilosis. In some embodiments, fungus is Magnaporthe oryzae. In some embodiments, the fungus is one or more Coccidioides spp. In some embodiments, the fungus is selected from Coccidioides posadasii and Coccidioides immitis.

[0034] In some embodiments, the immunogenic composition is capable of inducing resistance to coccidioidomycosis (valley fever). In some embodiments, the immunogenic composition is capable of inducing immunity to coccidioidomycosis (valley fever). In some embodiments, the immunogenic composition is capable of inducing an immune response. In some embodiments, the immunogenic composition is capable of inducing an immune response as a result of a physical reaction selected from the group consisting of: early disruption of spherules; secretion of a metabolite; and secretion of a small molecule. In some embodiments the immunogenic 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. In some embodiments, the immunogenic composition includes further avirulence protection means. In some embodiments, the further avirulence protection means is selected from the group of amino acid biosynthesis knockout; truncation; aging; modification; killing; formulation; resistance to reversion to wild type; and fusion. In some embodiments, the composition is a mammalian immunogen. In some embodiments, the composition is a human immunogen. In some embodiments, the fungal virulence is attenuated or eliminated in any mammal susceptible to the fungus.

Formulations

[0035] An aspect of the disclosure provided herein is a storage stable vaccine formulation. In some embodiments, the storage stable vaccine formulation includes an immunogenic composition described herein. In some embodiments, the storage stable vaccine formulation is a spore-based vaccine formulation with improved stability. In some embodiments, the spore-based vaccine formulation includes one or more immunogenic compositions as described herein. In some embodiments, the spore-based vaccine formulation includes one or more genetically modified spores as described herein. In some embodiments, the spore-based vaccine further includes a buffer. In some embodiments, the spore-based vaccine formulation further includes a stabilizing agent.

[0036] In some embodiments, the stabilizing agent includes DMSO. In some embodiments, the stabilizing agent includes BSA. In some embodiments, the stabilizing agent includes a mixture of DMSO and BS A. Tn some embodiments, the stabilizing agent includes one or more dextrans. Other stabilizing agents known in the art, c.g., for stabilizing other vaccines, can also be included in the compositions described herein, for example, amino acids, such as sodium glutamate, arginine, lysine, and cysteine; monosaccharides, such as glucose, galactose, fructose, and mannose; disaccharides, such as sucrose, maltose, and lactose; sugar alcohols such as sorbitol and mannitol; polysaccharides, such as oligosaccharide, starch, cellulose, and derivatives thereof; human serum albumin and bovine serum albumin; gelatin, and gelatin derivatives, such as hydrolyzed gelatin; and ascorbic acid as an antioxidant. These materials are described in publications, e.g., “Toketsu-Kanso To Hogo Busshitsu (Lyophilization And Protective Materials)” written by Nei, p. 1-176, published by Tokyo Daigaku Shuppan Kai (Publishing Association of the University of Tokyo), Japan in 1972; and “Shinku Gijutsu Koza (8): Sinku Kanso (Lecture on Vacuum Technology (8): Vacuum Drying)” written by Ota et al., p. 176-182, published by Nikkan Kogyo Shimbun Co., Ltd., Japan in 1964.

[0037] In some embodiments, the stabilizing agent is in the amount from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40% w/w, or ranges including and/or spanning the aforementioned values.

[0038] In some embodiments, the buffer is a buffer system. In some embodiments, the buffer is one or more buffer systems. For example, a buffer system may be selected from the group consisting of acetate, succinate, citrate, prolamine, histidine, borate, carbonate and phosphate buffer systems. In some embodiments, the buffer is a phosphate buffer. In some embodiments, the buffer is a buffer salt. For example, the buffer salt may be selected from one or more of the group consisting of sodium succinate, potassium succinate, sodium phosphate and potassium phosphate.

[0039] In some embodiments, the buffer is in the amount from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40% w/w, or ranges including and/or spanning the aforementioned values.

[0040] In some embodiments, the storage stable vaccine formulation further includes a vaccine adjuvant. The adjuvant (s) may be a substance that has a direct (e.g., cytokine or Bacille Calmette-Guerin (BCG)) or indirect effect (liposomes) on cells of the subject's immune system. Examples of often suitable adjuvants include oils (e.g., mineral oils), metallic salts (e.g., aluminum hydroxide or aluminum phosphate), bacterial components (e.g., bacterial liposaccharides, Freund's adjuvants, and/or MDP), plant components (e.g., Quil A), cytokines and/or one or more substances that have a carrier effect (e.g., bentonite, latex particles, liposomes, and/or Quil A). Adjuvants also include, for example, CARBIGEN adjuvant and carbopol. It should be recognized that this disclosure encompasses both compositions that include an adjuvant(s), as well as compositions that do not include any adjuvant.

[0041] In some embodiments, the storage stable vaccine formulation optionally further includes one or more additional therapeutic agents. In certain embodiments, the additional therapeutic agent or agents are selected from the group consisting of growth factors, anti-inflammatory agents, vasopressor agents, collagenase inhibitors, topical steroids, matrix metalloproteinase inhibitors, ascorbates, angiotensin II, angiotensin III, calreticulin, tetracyclines, fibronectin, collagen, thrombospondin, transforming growth factors (TGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factors (IGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), neu differentiation factor (NDF), hepatocyte growth factor (HGF), and hyaluronic acid.

[0042] In some embodiments, the storage stable vaccine formulation optionally further includes a pharmaceutically acceptable carrier. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as glucose, and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. Tn some embodiments, the preservatives include an antibiotic.

[0043] In some embodiments, the storage stable vaccine formulation is administered at a dose 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 of the composition, at least about 400,000 of the composition, at least about 500,000 spores of the composition, or ranges including and/or spanning the aforementioned values.

[0044] In some embodiments, the storage stable vaccine formulation is a storagestable vaccine formulation. In some embodiments, the storage stable vaccine formulation is a shelf-stable vaccine formulation. In some embodiments, the storage stable vaccine formulation is a thermally- stable vaccine formulation. In some embodiments, the thermally- stable vaccine composition includes an immunogenic composition as described herein, a buffer, and a stabilizing agent.

[0045] In some embodiments, the storage stable vaccine formulation with improved stability maintains the original bioactivity. In some embodiments, the spore-based vaccine formulation with improved stability maintains at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70% at least 65%, at least 60%, at least 55%, at least 50%, or ranges including and/or spanning the aforementioned values, of the original bioactivity. In some embodiments, the spore -based vaccine formulation retains at least about 30% of its original bioactivity e.g., at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% of the original bioactivity or higher activity at about 4 °C, at about 25 °C, at about 37 °C, at about 45 °C, or greater, for at least up to 6 months. In some embodiments, the spore-based vaccine formulation retains at least about 8% of the original bioactivity at temperatures of about 37° C, or greater, for at least 6 months. In some embodiments, the spore-based vaccine formulation retains at least about 10% of the original bioactivity at temperatures from about -80 °C to about 45 °C for at least 6 months. In some embodiments, the spore-based vaccine formulation retains some of the original bioactivity at temperatures from about 4°C to about room temperature for at least 10 months. In some embodiments, the spore-based vaccine formulation retains at least some of the original bioactivity at temperatures from about 4°C to about room temperature for at least 45 weeks.

[0046] In some embodiments, the storage stable vaccine formulation with improved stability may be maintained for any period of time e.g., hours, days, weeks, months or years. In some embodiments, the spore-based vaccine formulation described herein can be maintained at a temperature above 0 °C. for at least about 3 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours, at least about 24 hours or longer. In some embodiments, the spore-based vaccine formulation described herein can be maintained for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days or longer. In some embodiments, the sporebased vaccine formulation described herein can be maintained for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks or longer. In some embodiments, the spore-based vaccine formulation described herein can be maintained for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months or longer.

[0047] In some embodiments, the storage stable vaccine formulation as described herein may be maintained at any temperature. In some embodiments, the spore-based vaccine formulation as described herein may be maintained at a manufacturer’s recommended temperature. In some embodiments, the spore-based vaccine formulation can be maintained in liquid nitrogen or in dry ice. In some embodiments, the storage stable vaccine formulation can be maintained, for example, between about -80 °C and about -20 °C, inclusive, or between about -20 °C and about 0 °C, inclusive. In some embodiments, the spore-based vaccine formulation can be maintained at a temperature above 0 °C. In those embodiments, the sporebased vaccine formulation can be maintained at a temperature from about 0 °C. to about an ambient temperature. As used herein, the term “ambient temperature” is used to describe a surrounding temperature at which the storage stable vaccine formulation described herein are maintained and it includes temperatures between 0 °C. and 60 °C, between 0 °C and 50 °C, or between 0 °C and 40 °C. In some embodiments, the ambient temperature is the fridge temperature (e.g., between 0 °C and 15 °C, inclusive). In some embodiments, the ambient temperature is about the body temperature of a subject (e.g., between 36 °C and 38 °C, inclusive, for a human subject, or a higher or lower body temperature range for other animals). In some embodiments, the ambient temperature is the room temperature, e.g., between 20 °C and 35 °C, and it can vary with geographical conditions. For example, the room temperature in warm-climate regions, e.g., Africa, can be generally warmer than that in cool-climate regions, e.g., the United States or United Kingdom. In some embodiments, the storage stable vaccine formulation can be maintained at a temperature of at least about 37 °C or greater than 37 °C. In some embodiments, the storage stable vaccine formulation can be maintained at a temperature of at least about 40 °C or greater than 40 °C. In some embodiments, the storage stable vaccine formulation can be maintained at a temperature of at least about 45 °C or greater than 45 °C.

[0048] In some embodiments, the storage stable vaccine formulation described herein can be maintained under exposure to light, e.g., light of different wavelengths and/or from different sources. In some embodiments, the spore-based vaccine formulation described herein can be maintained under exposure to UV or infra-red irradiation. In some embodiments, the compositions described herein can be maintained under visible lights.

[0049] In some embodiments, the storage stable vaccine formulation described herein when stored or transported can be subjected to at least one state-changing cycle. The term “state-changing cycle” as used herein refers to a change of a material state, including, but not limited to, from a solid state to a fluid state, or from a fluid state to a solid state. A fluid state can include, but is not limited to, liquids, gases, slurries, flowable paste, plasmas, and any combinations thereof. A solid state refers to a state that is not flowable, and it can also encompass semi-solids, e.g., a gel. The storage stable vaccine formulation described herein can be maintained at a certain state for any period of time, e.g., seconds, minutes, hours, weeks, months, or years, before changing to another state. A state-changing cycle can be resulted from at least one change in an environmental condition described herein, e.g., a temperature change, a change in ambient air pressure, light condition, humidity, or any combinations thereof. [0050] Tn one embodiment, the state-changing cycle refers to a freeze-thaw cycle. In such embodiments, the storage stable vaccine formulation described herein when stored or transported can be subjected to at least one freeze-thaw cycle, at least two freeze-thaw cycles, at least three freeze-thaw cycles, at least four freeze-thaw cycles, at least five freeze-thaw cycles, at least six freeze -thaw cycles, at least seven freeze-thaw cycles, at least eight freezethaw cycles, at least nine freeze-thaw cycles, at least ten free-thaw cycles or more. The term “freeze-thaw cycles” is used herein to describe a series of alternating freezing and thawing, and also encompasses a series of alternating frozen (solid) and fluid state. For example, one freeze-thaw cycle involves a change of state between a frozen (solid) state and a fluid state. The time interval between freezing and thawing, or frozen and fluid state, can be any period of time, e.g., hours, days, weeks or months. For example, once a storage stable vaccine formulation has been frozen or is in a frozen state, it can be continually stored in the frozen state at sub-zero temperatures, e.g., between about -20 °C and -80 °C, until it needs to be thawed for use again. Freezing of a composition can be performed rapidly, e.g., in liquid nitrogen, or gradually, e.g., in a freezing temperature, e.g., between about -20 °C and -80 °C . Thawing of a frozen composition can be performed at any temperature above 0 °C rapidly, e.g., at room temperature, or gradually, e.g., on ice. Typically, an active agent in non-silk fibroin matrix can lose its bioactivity over one or more freeze-thaw cycles. As described herein, distributing an active agent in a silk fiborin matrix can increase the stability of the active agent and thus retain its bioactivity during one or more freeze-thaw cycles.

[0051] In some embodiments, the storage stable vaccine formulation described herein can be maintained at a relative humidity of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50% or higher. The term “relative humidity” as used herein is a measurement of the amount of water vapor in a mixture of air and water vapor. It is generally defined as the partial pressure of water vapor in the airwater mixture, given as a percentage of the saturated vapor pressure under those conditions.

[0052] In some embodiments, the storage stable vaccine formulation described herein can be lyophilized to decrease residual moisture during storage. In some embodiments, residual moisture is decreased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. [0053] Tn some embodiments, the storage stable vaccine formulation described herein can be maintained under or subjected to any air pressure. In some embodiments, the spore-based vaccine formulation described herein can be maintained under or subjected to about atmospheric pressure, or higher, e.g., about 1 atm, about 2 atms, about 3 atms, about 4 atms, about 5 atms, about 6 atms, about 7 atms, about 8 atms, about 9 atms or about 10 atms. In some embodiments, the spore-based vaccine formulation described herein can be maintained under or subjected to vacuum.

[0054] In some embodiments, the storage stable vaccine formulation may be maintained under two or more conditions specified herein.

Methods and Uses

[0055] In some aspects, methods for preparing a storage stable vaccine formulation are described herein. In some embodiments, the method for preparing a storage- stable composition includes (a) providing an immunogenic composition as described herein and (b) adding a stabilizing agent and buffer.

[0056] In some embodiments, as shown in FIG. 1, the method 100 includes mixing 102 one or more live genetically modified Coccidioides fungal spores comprising an inactivated cyclic peptide synthase Cps 1 gene product, a buffer, and about two to about 20% w/w of a stabilizing agent, thereby producing 104 a storage-stable composition. In some embodiments, the buffer includes one or more buffers according to any other embodiments disclosed herein. In some embodiments, the amount of the buffer includes an amount of one or more buffers according to any other embodiments disclosed herein. In some embodiments, the stabilizing agent includes one or more stabilizing agents according to any other embodiments disclosed herein. In some embodiments, the amount of the stabilizing agent includes an amount of the one or more stabilizing agents according to any other embodiments disclosed herein.

[0057] In general, the formulations and compositions described herein may be administered in an immunologically effective amount, which is an amount sufficient to induce a protective immune response in the subject against fungal infection (e.g. Coccidioides spp.).

[0058] It is contemplated that the formulations and compositions may be administered to a subject at a single time; or, alternatively, two or more times over days, weeks, months, or years. Tn some embodiments, the composition is administered at least two times. Tn some such embodiments, for example, the compositions arc administered twice, with the second dose (e.g., the booster) being administered 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. In the above embodiments, the first and subsequent dosages may vary, such as, for example, in amount and/or form. Often, however, the dosages are the same as to amount and form.

[0059] In certain embodiments, the formulations and compositions are administered to a subject that is immunogenically naive to the fungi, e.g., the subject has not been vaccinated for the fungus or exposed to the fungus. In accordance with this embodiment, the composition is administered before the subject recipient is infected with the fungus. In such embodiments, the vaccine preparation may, for example, be administered to prevent, reduce the risk of, or delay the onset of Coccidioides spp. infection or one or more (typically two or more) Coccidioides spp. symptoms.

[0060] In some embodiments, the formulations and compositions are administered to subjects in a population after a subject in the population has been infected with the fungus. In such embodiments, the compositions may, for example, ameliorate, suppress, or eradicate the fungus or one or more (typically two or more) fungal symptoms in the subjects of the population.

[0061] The formulations and compositions can be administered by conventional means, including, for example, mucosal administration, (such as intranasal, oral, intratracheal, and ocular), and parenteral administration (such as, without limitation, intraperitoneal, subcutaneous or intramuscular administration). The compositions may also be administered intradermally or transdermally (including, without limitation, via a skin patch or topical administration). Mucosal administration is often particularly advantageous for live attenuated vaccines.

[0062] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butancdiol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized prior to addition of spores, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. In order to prolong the effect of an active agent, it is often desirable to slow the absorption of the agent from subcutaneous or intramuscular injection. Delayed absorption of a parenterally administered active agent may be accomplished by dissolving or suspending the agent in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the agent in biodegradable polymers such as polylactidepolyglycolide. Depending upon the ratio of active agent to polymer and the nature of the particular polymer employed, the rate of active agent release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the agent in liposomes or microemulsions which are compatible with body tissues.

[0063] In some embodiments, the method does not comprises a non-lyophilization step. In some embodiments, the method further comprises a lyophilization step. In some embodiments, the method does not comprises a foam-drying step. In some embodiments, the method further comprises a foam-drying step.

Kits

[0064] Packages and kits comprising at least one immunogenic composition or storage-stable composition or preparation are also described herein. The packages can be prepared in various types of containers, which can be selected from the group consisting of a vial, an ampule, a capsule, a tube, a delivery device, a bottle, and a packet. In some embodiments, the delivery device is a syringe. In some embodiments, the syringe can be needleless. [0065] An aspect of the disclosure is a vaccination kit. Tn some embodiments, the vaccination kit includes a unit dose of the vaccine formulation according to any of the above embodiments, a container, and instructions for use. In related embodiments, the instructions include storage at from about -4 °C to about 45 °C. and the like (calculation of 45 °C is that this temperature is the same as 113 °F).

EXAMPLES

[0066] To the extent not already indicated, it will be understood by those of ordinary skill in the art that any one of the various embodiments herein described and illustrated may be further modified to incorporate features shown in any of the other embodiments disclosed herein.

[0067] The following examples illustrate some embodiments and aspects of the invention. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be performed without altering the spirit or scope of the invention, and such modifications and variations are encompassed within the scope of the invention as defined in the claims which follow. The following examples do not in any way limit the invention.

Example 1 - Non-Lyophilized Formulations

[0068] Non-lyophilized formulation viability testing (vials stored at -20°C post formulation). The formulations include varying amounts of glycerol, albumin (BSA), and DMSO. The harvested spores and plates are described in Table 1.

Table 1

[0069] Four hundred microliters of harvest was spun down and resuspended in 2 ml for each formulation. The results are described in Table 2.

Table 2

Example 2

[0070] Stress testing of the active substance is used to identify the potential degradation products, and to establish degradation pathways and stability of the molecule. The nature of the stress testing depends on the individual active substance and the type of pharmaceutical product involved.

[0071] In general, an active substance is evaluated under storage conditions (with appropriate tolerances) that test thermal stability and, if applicable, sensitivity to moisture. The storage conditions and the lengths of time period for study are appropriate to consider storage, shipment, and subsequent use appropriate to the climatic zone or zones in which the active substance is likely intended to be stored. Long-term testing extends for a period about a month, or, about three months, about six months, about ten months, or about twelve months. Testing includes a number of product batches in conditions of packaging and temperatures that are representative of the product's intended use.

[0072] Data from an “accelerated” storage condition, if appropriate, are obtained to test the product at conditions beyond those intended, i.e., excessively high or low temperatures compared to potential actual ambient conditions. An accelerated storage condition includes tests of conditions that mimic handling issues such as prolonged exposure to excess moisture and variable volume delivery of a composition including the active substance. Calculations of the data obtained under accelerated conditions are then used to extrapolate the presumed stability of the product in normal environments and conditions, although these calculations are an estimate of stability of the product under normal conditions. Data obtained from accelerated storage conditions arc combined with other data including long-term testing described above to determine the stability of the product.

[0073] Genetically modified spores will be prepared following the teachings of U.S. Pat. Nos. 9,884,097 and 10,758,600. The genetically modified spores will be placed in 5%, 10%, 15%, and 20% DMSO at 25 °C (ambient temperature) for at least two months, at least four months, at least six months, at least eight months, at least ten months, or at least twelve months. CFU will be measured for each sample each week to determine viability of the spores.

Example 3

[0074] Formulations included DMSO (DMSO, HEPES, and NaCl (saline)). The formulation includes 2% DMSO, 10 mM HEPES at a pH of 7.0 and 0.15M saline. Genetically modified spores in ddH O were spun at 3,250 RPM for 30 minutes at 4 °C. After the supernatant was removed, spore pellets were resuspended, vortexed, and aliquoted at 1.1 mL. The fill potency of the formulation, the formulation including the spores, was about 1.5 x 10 ⁶ CFU/mL or lower. The estimated minimal infectious dose (MID) was at least 1 x 10 ⁴ . With storage of the formulation (including the spores) at room temperature and 4 °C, the storagestability of this formulation was found to be at least 45 weeks.

[0075] The scope of the present disclosure is not intended to be limited by the specific disclosures of preferred examples in this section or elsewhere in this specification, and may be defined by claims as presented in this section or elsewhere in this specification or as presented in the future. The language of the claims is to be interpreted broadly based on the language employed in the claims and not limited to the examples described in the present specification or during the prosecution of the application, which examples are to be construed as non-exclusive.