Coccidioides antigens and methods of their use

Abstract

The present invention concerns methods and compositions for treating or preventing fungal infection, particularly infection by a Coccidioides species. The invention provides methods and compositions for stimulating an immune response against the fungus. In certain embodiments, the methods and compositions involve a recombinant vaccine.

Description

COCCIDIOIDES ANTIGENS AND METHODS OF THEIR USERELATED APPLICATIONS

[0001] This Application is an International Application claiming priority to US Provisional Application 63 / 537,194 filed September 7, 2023 which is incorporated herein by reference in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under 1U19AI166761-01 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTIONI. FIELD OF THE INVENTION

[0003] The present invention relates generally to the fields of immunology, microbiology, and pathology. More particularly, it concerns methods and compositions involving recombinant antigens and vaccines for invoking an immune response against a Coccidioides fungus.II. BACKGROUND

[0004] Coccidioides posadasii and C. immitis are human fungal pathogens which cause a mild to severe respiratory disease (coccidioidomycosis, or San Joaquin Valley fever) in both immunocompetent and immunocompromised individuals. This fungal infection poses a great risk to over 30 million people (approximately 10% of the US population) who live in the endemic regions, most notably the southwestern United States (McCotter et al., Medical mycology, 2019, 57:S30-S40). It is estimated about 350,000 people in the US become infected with Coccidioides every year (Chiller, Overview of endemic mycosis. Vaccine Strategies for Endemic Fungal Pathogens, Rockville, MD, NIAID 2019). Approximately 60% of individuals infected with Coccidioides are asymptomatic judging by skin test reactivity (Smith, American journal of public health, 1940, 30:600-11; Smith et al., JAMA, 1956, 160:546-52). The remaining 40% of individuals exposed to this microbe develop symptomatic disease, which can manifest as an acute or progressive pulmonary syndrome resembling other community- acquired pneumonia with formation of pulmonary nodules and cavities, extrapulmonary non-meningeal mycosis, or meningitis (Pappagianis, Clinical presentation of infectious entities. In: Einstein H, editor, Coccidioidomycosis, Washington, D.C.: National Foundation for Infectious Disease, 1996, p. 9-11; Galgiani et al., Clin Infect Dis 2005, 41 : 1217-23).

[0005] The overall mortality is greater than 50% in multifocal dissemination cases. The disseminated form of this mycosis is difficult to treat and typically requires long courses of antifungal drug administration. For preventive intervention, there is currently no approved vaccines against coccidioidomycosis. Coccidioidal vaccines have been developed using candidates including live attenuated strains, formalin-killed spherules (FKS) and subunit vaccines (Hung et al. Infection and immunity, 86(1 l):e00070-18, 2018; Pappagianis, The American review of respiratory disease, 1993,148:656-60; Xue et al., Infection and immunity, 2009, 77:3196-208). Despite the apparent ability of a live, attenuated vaccine to elicit and maintain long-term T cell memory to Coccidioides infection, it poses significant concerns for individuals with underlying conditions of compromised cell-mediated immune systems (Spring et al., Vaccine, 2013, 31 :4975-83). Meanwhile, FKS vaccine did not demonstrate protective efficacy in a human Phase III trial (Pappagianis, The American review of respiratory disease, 1993,148:656-60). Generation of a recombinant protein subunit vaccine is a safer strategy in the design of a clinically acceptable reagent that is pure and chemically defined (Cole et al., PLoS pathogens, 2013, 9:el003768; Cole et al., Current fungal infection reports, 2012, 6:235- 44). Several Coccidioides antigens have been identified and evaluated for subunit vaccine efficacy using a murine model of pulmonary coccidioidomycosis (Li et al., Infection and immunity, 2001, 69:2878-87; Orsbom et al., Infection and immunity, 2006, 74: 1865-72; Tarcha et al., Infection and immunity, 2006, 74:5802-13; Tarcha et al., Infection and immunity, 2006, 74:516-27).

[0006] Recent success in mRNA-based vaccines in protecting human COVID-19 has provided a new vaccine platform for coccidioidal vaccine development. Development of a mRNA vaccine against Coccidioides infection is an attractive vaccination strategy in large part due to the speed and low cost of manufacturing. Efficient delivery of mRNA vaccines to the cytoplasm is essential to ensure antigen production and immune stimulation. Various antigen or mRNA delivery systems have been shown to stimulate both Thl and Th 17 responses that are essential for vaccine immunity against fungal infections.

[0007] There is an urgent unmet need to develop an effective vaccine against Coccidioides infection, which continues to be a major cause of morbidity within endemic areas. Currently, there is no Coccidioides vaccine approved for general use.SUMMARY OF THE INVENTION

[0008] In addressing the problems associated with Coccidioides infection the inventors have developed effective vaccines against Coccidioides infection. Provided are examples of novel polypeptide antigen formulations and mRNA vaccine formulations that include Coccidioides antigens (e.g. Cpal, Cpa2, Cpa3, Cpa4, Cpa5) and / or rnRNAs encoding Coccid oides antigens (e g. Cpal, Cpa2, Cpa3, Cpa4, Cpa5). These vaccines can be used to prevent, for example, coccidioidomycosis in humans and domestic animals (e.g., dogs).

[0009] Disclosed herein are engineered Coccidioides polypeptide antigens (rCpas) and mRNA encoding Coccidioides polypeptide antigens (rCpas). The Coccidioides antigen(s) can include all or a portion of two or more polypeptides or peptides selected from all or a segment of Ag2 / Pra, Cs-Ag, Ure, Gell, Pmpl, Pepl, Amnl, Plb, peroxisomal matrix protein, vacuolar protease A, vacuolar protease A, urease, glucan 1,3 -beta-glucosidase, beta-glucosidase, glycosyl transferase, disulfide-isomerase, enolase, glycolipid-anchored surface protein 5, CPC735_052600, CPAG_08847, ecm33, CPC735_035760, CPC735_033430, and / or trehalose synthase (Thl). In certain aspects the rCpas include Coccidioides polypeptide antigen 1 (rCpal), Coccidioides polypeptide antigen 2 (rCpa2), Coccidioides polypeptide antigen 3 (rCpa3), Coccidioides polypeptide antigen 4 (rCpa4), and / or Coccidioides polypeptide antigen 5 (rCpa5).

[0010] The rCpas can include 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, or more of the following peptides or fragments thereof in any order: Ag2 / Pra (e.g, SEQ ID NO: 12); Cs-Ag (e.g, SEQ ID NO: 13); Pmpl (e.g, SEQ ID NO: 14); Pepl -Pl (SEQ ID NO: 7), Pepl-P2 (SEQ ID NO: 8), Amnl-P9 (SEQ ID NO: 9), Amnl -P10 (SEQ ID NOTO), and / or Plb (SEQ ID NO: 11) of a Coccidioides species, e.g, Coccidioides posadasii, a DCpep (SEQ ID NO: 15); Ag2 / Pra (e.g., amino acids 19 to 106 of SEQ ID NO: 12); Peroxisomal matrix protein (e.g., amino acid 5 to 35 of SEQ ID NO: 16); Pmpl (e.g., amino acids 9 to 33 of SEQ ID NO: 14); vacuolar protease A (amino acid 7 to 22 of SEQ ID NO: 17); vacuolar protease A (amino acid 43 to 64 of SEQ ID NO: 17); vacuolar protease A (amino acid 368 to 383 of SEQ ID NO: 17); Amnl-P9 (SEQ ID NO: 9); Amnl -P10 (amino acids 5 to 20 of SEQ ID NOTO); Plb (SEQ ID NO: 11); urease (amino acids 744 to 778 of SEQ ID NO: 18);glucan 1,3 -beta-glucosidase (amino acids 57 to 71 of SEQ ID NO: 19); beta-glucosidase (amino acids 189 to 212 of SEQ ID NO:20); glycosyl transferase (amino acids 496 to 511 of SEQ ID NO:21); glycosyl transferase (amino acids 362 to 383 of SEQ ID NO:21); glycosyl transferase (amino acids 413 to 432 of SEQ ID NO:21); disulfide-isomerase (amino acids 273 to 291 of SEQ ID NO:22); disulfide-isomerase (amino acids 186 to 204 of SEQ ID NO:22); enolase (amino acids 161 to 183 SEQ ID NO:23); glycolipid-anchored surface protein 5 (amino acids 193 to 208 of SEQ ID NO:24); CPC735_052600 (amino acids 210 to 231 of SEQ ID NO:25); CPC735_052600 (amino acids 229 to 247 of SEQ ID NO:25); CPAG_08847 (amino acids 239 to 260 of SEQ ID NO:26); CPAG 08847 (amino acids 313 to 331 of SEQ ID NO:26); ecm33 (amino acids 223 to 244 of SEQ ID NO:27); ecm33 (amino acids 112 to 140 of SEQ ID NO:27); CPC735_035760 (amino acids 109 to 136 of SEQ ID NO:28); CPC735_033430 (amino acids 34 to 56 of SEQ ID NO:29); Pepl-POB (SEQ ID NO:40); Ure-PAB (SEQ ID NO:41); and / or Thl (SEQ ID NO:42), - including variants of the aforementioned polypeptides / peptides. The rCpa can further include an amino terminal affinity tag, e.g, hexahistidine tag. In certain aspects two or more, or each polypeptide or peptide is separated by a spacer, e.g., GPGPG or an AAY spacer. In certain aspects a peptide or fragment can be present more than once, adjacent to or non-adjacent to another copy of the peptide or fragment. Certain embodiments are directed to an mRNA vaccine encoding a rCpa as described herein.

[0011] In certain embodiments rCpas can include all or a segment of Ag2 / Pra (e.g., SEQ ID NO: 12); Pmpl (e.g, SEQ ID NO: 14); Amnl-P9 (SEQ ID NO:9), Amnl-PIO (SEQ ID NO: 10), and Plb (SEQ ID NO: 11). The rCpa can further include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, additional polypeptide(s) or peptide(s) selected from Cs-Ag (e.g, SEQ ID NO: 13); Pep 1 -Pl (SEQ ID NO: 7); Pepl-P2 (SEQ ID NO: 8); DCpep (SEQ ID NO: 15); Peroxisomal matrix protein (e.g, amino acid 5 to 35 of SEQ ID NO: 16); vacuolar protease A (amino acid 7 to 22 of SEQ ID NO: 17); vacuolar protease A (amino acid 43 to 64 of SEQ ID NO: 17); vacuolar protease A (amino acid 368 to 383 of SEQ ID NO: 17); Amnl-P9 (SEQ ID NO: 9); Amnl-PIO (amino acids 5 to 20 of SEQ ID NO: 10); Plb (SEQ ID NO: 11); urease (amino acids 744 to 778 of SEQ ID NO: 18); glucan 1,3-beta-glucosidase (amino acids 57 to 71 of SEQ ID NO: 19); beta-glucosidase (amino acids 189 to 212 of SEQ ID NO:20); glycosyl transferase (amino acids 496 to 511 of SEQ ID NO:21); glycosyl transferase (amino acids 362 to 383 of SEQ ID NO:21); glycosyl transferase (amino acids 413 to 432 of SEQ ID NO:21); disulfide-isomerase (amino acids 273 to 291 of SEQ ID NO:22); disulfide-isomerase (amino acids 186 to 204 of SEQ ID NO:22); enolase (amino acids 161 to 183 SEQ ID NO:23);glycolipid-anchored surface protein 5 (amino acids 193 to 208 of SEQ ID NO:24); CPC735_052600 (amino acids 210 to 231 of SEQ ID NO:25); CPC735_052600 (amino acids 229 to 247 of SEQ ID NO:25); CPAG_08847 (amino acids 239 to 260 of SEQ ID NO:26); CPAG 08847 (amino acids 313 to 331 of SEQ ID NO:26); ecm33 (amino acids 223 to 244 of SEQ ID NO:27); ecm33 (amino acids 112 to 140 of SEQ ID NO:27); CPC735_035760 (amino acids 109 to 136 of SEQ ID NO:28); and / or CPC735 033430 (amino acids 34 to 56 of SEQ ID NO:29) - including variants of the aforementioned polypeptides / peptides. The rCpa can further include an amino terminal affinity tag, e.g., hexahistidine tag. In certain aspects two or more, or each polypeptide or peptide is separated by a spacer, e.g., GPGPG or an AAY spacer. In certain aspects a polypeptide or peptide can be present more than once, adjacent to or nonadj acent to another copy of the polypeptide or peptide. Certain embodiments are directed to an mRNA vaccine encoding an rCpas as described herein.

[0012] In further aspects, the rCpa described herein is a multimer, e.g., a fusion of two or more polypeptides or peptide segments. In certain aspects of the invention, a rCpa comprises multimers or concatemers of 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 or more polypeptides or segments thereof. Concatemers are linear polypeptides having one or more repeating peptide units. The proteins or peptide fragments can be consecutive or separated by a spacer or other peptide sequences, e.g., one or more additional fungal peptides. In a further aspect, polypeptides or peptides contained in the multimer or concatemer can include, but are not limited to Ag2 / Pra, Cs-Ag, Ure, Gell, Pmpl, Pepl, Amnl, Plb, peroxisomal matrix protein, vacuolar protease A, vacuolar protease A, urease, glucan 1,3 -beta-glucosidase, beta-glucosidase, glycosyl transferase, disulfideisomerase, enolase, glycolipid-anchored surface protein 5, CPC735_052600, CPAG_08847, ecm33, CPC735_035760, CPC735_033430, trehalose synthase, or immunogenic fragments thereof.

[0013] The rCpal antigen includes the following peptides or peptide fragments: Ag2 / Pra (e g., SEQ ID NO: 12), Cs-Ag (e g., SEQ ID NO: 13), Pmpl (e.g, SEQ ID NO: 14), and 5 promiscuous immunodominant T cell epitopes derived from Pepl-Pl (SEQ ID NO:7), Pepl- P2 (SEQ ID NO: 8), Amnl-P9 (SEQ ID NO: 9), Amnl -P10 (SEQ ID NO: 10), Plb (SEQ ID NO: 11) of a Coccidioides species, e.g, Coccidioides posadasii, a DCpep (SEQ ID NO: 15); and / or optionally an amino terminal affinity tag, e.g., hexahistidine tag. In certain aspects each element is separated by a spacer, e.g., GPGPG spacer. In certain embodiments the rCpal hasthe amino acid sequence of SEQ ID NO:2. In certain aspects the rCpal antigen is encoded by an mRNA vaccine having a nucleotide sequence of SEQ ID NO: 30, or a nucleotide sequence including SEQ ID NO:30, or a nucleotide sequence that is at least 80, 85, 90, 95, or 99 percent similar or identical to SEQ ID NO:30.

[0014] The rCpa2 antigen includes the following peptides or peptide fragments: Ag2 / Pra (e.g., SEQ ID NO:38), Cs-Ag (e.g., SEQ ID NO:39), Pmpl (e.g., SEQ ID NO: 14), and 5 promiscuous immunodominant T cell epitopes derived from Pepl-Pl (SEQ ID NO:7), Pepl- P2 (SEQ ID NO: 8), Amnl-P9 (SEQ ID NO: 9), Amnl-PIO (SEQ ID NO: 10), and Plb (SEQ ID NO: 11) of a Coccidioides species, e.g, Coccidioides immitis; a DCpep (SEQ ID NO: 15); and / or an amino terminal affinity tag, e.g., hexahistidine tag. In certain aspects each element is separated by a spacer, e.g., GPGPG spacer. In certain embodiments the rCpa2 has the amino acid sequence of SEQ ID NO:34. In certain aspects the rCpa2 antigen is encoded by an mRNA vaccine having a nucleotide sequence of SEQ ID NO:33.

[0015] The rCpa3 antigen includes the following peptides or peptide fragments: Ag2 / Pra (e.g., amino acids 19 to 106 of SEQ ID NO: 12); Cs-Ag (e.g., SEQ ID NO: 13); Pmpl (e.g., SEQ ID NO: 14); 5 promiscuous, immunodominant T cell epitopes derived from Pepl-Pl (SEQ ID NO: 7), Pepl-P2 (SEQ ID NO: 8), Amnl-P9 (SEQ ID NO: 9), Amnl-PIO (SEQ ID NO: 10), Plb (SEQ ID NO: 11); a DCpep (SEQ ID NO: 15); and an amino terminal affinity tag, e.g., hexahistidine tag. In certain aspects one or more elements are separated by a spacer, e.g., GPGPG spacer. In certain embodiments the rCpa3 has the amino acid sequence of SEQ ID NO:4. In certain aspects the rCpa3 antigen is encoded by an mRNA vaccine having a nucleotide sequence of SEQ ID NO: 31, or a nucleotide sequence including SEQ ID NO: 31, or a nucleotide sequence that is at least 80, 85, 90, 95, or 99 percent similar or identical to SEQ ID NO:31.

[0016] The rCpa4 antigen includes an immunogenic fragment of Ag2 / Pra (e.g., amino acids 19 to 106 of SEQ ID NO: 12); Peroxisomal matrix protein (e.g., amino acid 5 to 35 of SEQ ID NO: 16); Pmpl (e.g., amino acids 9 to 33 of SEQ ID NO: 14); vacuolar protease A (amino acid 7 to 22 of SEQ ID NO: 17); vacuolar protease A (amino acid 43 to 64 of SEQ ID NO: 17); vacuolar protease A (amino acid 368 to 383 of SEQ ID NO: 17); Amnl-P9 (SEQ ID NO: 9); Amnl-PIO (amino acids 5 to 20 of SEQ ID NO: 10); Plb (SEQ ID NO: 11); urease (amino acids 744 to 778 of SEQ ID NO: 18); glucan 1,3-beta-glucosidase (amino acids 57 to 71 of SEQ ID NO: 19); beta-glucosidase (amino acids 189 to 212 of SEQ ID NO:20); glycosyl transferase (amino acids 496 to 511 of SEQ ID NO:21); glycosyl transferase (amino acids 362to 383 of SEQ ID NO:21); glycosyl transferase (amino acids 413 to 432 of SEQ ID NO:21); disulfide-isomerase (amino acids 273 to 291 of SEQ ID NO:22); disulfide-isomerase (amino acids 186 to 204 of SEQ ID NO:22); enolase (amino acids 161 to 183 SEQ ID NO:23); glycolipid-anchored surface protein 5 (amino acids 193 to 208 of SEQ ID NO:24); CPC735_052600 (amino acids 210 to 231 of SEQ ID NO:25); CPC735_052600 (amino acids 229 to 247 of SEQ ID NO:25); CPAG_08847 (amino acids 239 to 260 of SEQ ID NO:26); CPAG 08847 (amino acids 313 to 331 of SEQ ID NO:26); ecm33 (amino acids 223 to 244 of SEQ ID NO:27); ecm33 (amino acids 112 to 140 of SEQ ID NO:27); CPC735_035760 (amino acids 109 to 136 of SEQ ID NO:28); and / or CPC735 033430 (amino acids 34 to 56 of SEQ ID NO:29). In certain aspects each element is separated by a spacer, e.g, GPGPG or AAY spacer. In certain embodiments the rCpa4 has the amino acid sequence of SEQ ID NO:6. In certain aspects the rCpa4 antigen is encoded by an mRNA vaccine having a nucleotide sequence of SEQ ID NO:32, or a nucleotide sequence including SEQ ID NO:32, or a nucleotide sequence that is at least 80, 85, 90, 95, or 99 percent similar or identical to SEQ ID NO:32.

[0017] The rCpa5 antigen includes the following peptides or peptide fragments: Ag2 / Pra (e.g., amino acids 19 to 106 of SEQ ID NO: 12), Pepl-POB (SEQ ID NO:40), Ure-PAB (SEQ ID N0:41), Thl (SEQ ID NO:42), Cs-Ag (e.g., SEQ ID NO: 13), Pmpl (e.g, SEQ ID NO: 14), and 5 promiscuous immunodominant T cell epitopes derived from Pepl-Pl (SEQ ID NO:7), Pepl-P2 (SEQ ID NO: 8), Amnl-P9 (SEQ ID NO: 9), Amnl-PIO (SEQ ID NO: 10), and Plb (SEQ ID NO: 11) of a Coccidioides species, e.g, Coccidioides posadasii; optionally a DCpep (SEQ ID NO: 15); and / or an optional amino terminal affinity tag, e.g., hexahistidine tag. In certain aspects each element is separated by a spacer, e.g, GPGPG spacer. In certain embodiments the rCpa5 has the amino acid sequence of SEQ ID NO:36. In certain aspects the rCpa2 antigen is encoded by an mRNA vaccine having a nucleotide sequence of SEQ ID NO 35.

[0018] In some aspects, the rCpa or variants thereof is at least 80% identical or similar to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. In further aspects, the recombinant antigen or variant thereof disclosed herein is at least 85, 90, 95, 98, 99, to 100% identical or similar to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

[0019] The antigen(s) or mRNA(s) encoding the antigen(s) can be incorporated into an adjuvant / delivery system. The adjuvant / delivery system can be a microparticle deliverysystem. In certain aspects the adjuvant / delivery system augments Thl and / or Thl7 immunity to improve protective efficacy of the antigen(s) or mRNA encoded antigen(s) against Coccidioides infection. In certain aspects a vaccine composition further includes an adjuvant, e.g., a TLR ligand / agonist. In certain aspects the adjuvant is an oligonucleotide adjuvant comprising a plurality of CpG motifs (ODN).

[0020] Certain embodiments are directed to an immunogenic composition comprising a rCpa or mRNA vaccine as described herein. The immunogenic composition can include one or more Coccidioides antigen(s) or mRNA(s). The immunogenic composition may also include an adjuvant and / or be encapsulated in a microparticle.

[0021] Embodiments include the use of a composition comprising one or more rCpa and / or one or more mRNA encoding a rCpa to treat or prevent a Coccidioides infection in a subject. Embodiments include the use of a rCpa or an mRNA encoding the same in methods and compositions for the amelioration of fungal and / or Coccidioides infection. Certain embodiments provide methods and compositions that can be used to limit Coccidioides growth and / or persistence in a subject. In some cases, the methods include stimulating an immune response by administering to a subject an effective amount of an immunogenic composition described herein. A protective or therapeutic immune response against a Coccidioides fungus can be generated by administering to the subject an effective amount of a composition including (i) a rCpa protein disclosed herein or a variant thereof or (ii) a mRNA comprising a sequence encoding a rCpa disclosed herein or variant thereof or both (i) and (ii). In certain aspects the subject is a human or animal (e.g., livestock or companion animal). In a further aspect the composition is formulated in a pharmaceutically acceptable formulation. In certain aspects other anti-fungal compounds, compositions, or treatments can be administered in combination with the rCpa(s) and / or mRNA(s). The rCpa or mRNA encoding a rCpa can be formulated in a pharmaceutically acceptable composition. In certain aspects the Coccidioides for which a subject is being treated may be Coccidioides posadasii or Coccidioides immitis. A composition may be administered more than one time to a subject, and may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more times. The administration of the composition includes but is not limited to oral, parenteral, subcutaneous, intramuscular, intravenous, or various combinations thereof, including injection, inhalation, instillation, or aspiration.

[0022] Compositions discussed herein are typically administered to human subjects, but administration to other animals capable of eliciting an immune response to a Coccidioides fungi is contemplated, particularly cattle, horses, goats, sheep and other domestic animals.

[0023] An immune response refers to a humoral response, a cellular response, or both a humoral and cellular response in an organism. An immune response can be measured by assays that include, but are not limited to, assays measuring the presence or amount of antibodies that specifically recognize a protein or cell surface protein, assays measuring T-cell activation or proliferation, and / or assays that measure modulation in terms of activity or expression of one or more cytokines. In certain embodiments the immune response is a protective immune response. In still further aspects, the methods and compositions of the invention can be used to prevent, ameliorate, reduce, or treat infection of tissues or glands, e.g., lungs, and other infections. Other methods include but are not limited to prophylactically reducing fungal burden in a subject not exhibiting signs of infection, particularly those subjects suspected of or at risk of being colonized by a target fungus, e.g., patients that are or will be at risk or susceptible to infection during a hospital stay, treatment, and / or recovery.

[0024] The subject can have (e.g., are diagnosed with a Coccidioides infection), can be suspected of having, or can be at risk of developing a Coccidioides infection. Compositions of the present invention include immunogenic compositions wherein the antigen(s) or epitope(s) are contained in an amount effective to achieve the intended purpose. More specifically, an effective amount means an amount of active ingredients necessary to stimulate or elicit an immune response, or provide resistance to, amelioration of, or mitigation of infection. In more specific aspects, an effective amount prevents, alleviates or ameliorates symptoms of disease or infection, or prolongs the survival of the subject being treated. Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any preparation used in the methods of the invention, an effective amount or dose can be estimated initially from in vitro studies, cell culture, and / or animal model assays. For example, a dose can be formulated in animal models to achieve a desired immune response or circulating antibody concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0025] The term “providing” is used according to its ordinary meaning to indicate “to supply or furnish for use.” In some embodiments, the protein is provided directly by administering the protein, while in other embodiments, the protein is effectively provided byadministering a nucleic acid that encodes the protein. In certain aspects the invention contemplates compositions comprising various combinations of nucleic acid, antigens, peptides, and / or epitopes.

[0026] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” It is also contemplated that anything listed using the term “or” may also be specifically excluded.

[0027] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0028] Following long-standing patent law, the words “a” and “an,” when used in conjunction with the word “comprising” in the claims or specification, denotes one or more, unless specifically noted.

[0029] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.DESCRIPTION OF THE DRAWINGS

[0030] So that the matter in which the above-recited features, advantages and objects of the invention as well as others which will become clear are attained and can be understood in detail, more particular descriptions and certain embodiments of the invention briefly summarized above are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate certain embodiments of the invention and therefore are not to be considered limiting in their scope.

[0031] FIG 1A-1D. Illustration of an example of a typical coccidioidal mRNA vaccine structure. (A) A basic mRNA vaccine consists of a 5 ’cap, a 5’UTR, and open reading frame (encoding an optional His-tag and Coccidioides antigens), a 3’UTR and a poly(A) tail. (B) A dendritic cell peptide coding sequence is added to the downstream of Coccidioides antigens forenhancing antigen presentation. (C) A signal sequence plus two alanine (AA) are added at the N-terminus to facilitate the secretion of the His-tagged Ci / Cp antigens. (D) A vaccine construct contains both signal peptide and dendritic cell peptide coding sequences for Ci / Cp antigen secretion and dendritic cell targeting.

[0032] FIG. 2. Illustration of examples of Ci / Cp antigen constructs, rCpal-5. Coccidioidal antigenic proteins and peptides are linked in tandem with GPGPG or AAY spacer. The N- terminal His(6x) Tag is designed for easy protein purification. Also indicated are GenBank accession numbers for rCpal and rCpa2 proteins.

[0033] FIG. 3 A-3D. Microparticles efficiently deliver mCherry mRNA that is expressed in vivo. Mice were injected with a dose of 5 pg of mCherry mRNA packaged in polysaccharide based microparticles (MPs) by the subcutaneous route and single cells were prepared from the injection sites (IS), spleen (SPL), and bone marrow (BM) at 36 hr post injection for determining in vivo expression of mCherry in phagocytes by imaging flow cytometry analysis. (A) MP- mCherrymRNA construct, (B) experimental design. (C) Representative macrophage cells (CD45+F4 / 80+) that take up MPs (stained with calcofluor white, CFW) and express mCherry. (D) Measurement of mCherry expression (mean fluorescence intensity, MFI) in phagocytic immune cells.

[0034] FIG. 4A-4C. Microparticles deliver Cpal mRNA that is expressed in vivo. Mice were injected with an escalated dose of (0-10 pg) of Cpal mRNA packaged in polysaccharide based microparticles (MPs) by the subcutaneous route and single cells were prepared from the injection sites (IS), spleen (SPL), and bone marrow (BM) at 36 hr post injection for determining in vivo expression of His-tagged Cpal protein in immune cells by flow cytometry analysis. (A) MP-Cpal mRNA construct. (B) Representative macrophages (CD45+F4 / 80+) that take up MPs (stained with calcofluor white, CFW) and express His-tag Cpal. (C) Measurement of Cpal protein (mean fluorescence intensity, MFI) in antigen presenting cells (B cells, dendritic cells (DC), and macrophages (Mac)).

[0035] FIG. 5. MP-CpalmRNA vaccination induces Thl memory responses. Four groups of C57BL / 6 or HLA-DR4tgmice were primed and boosted twice with 0-10 pg of MP-CpalmRNA vaccine at 14-day intervals. Splenocytes were prepared at day 14 post-final vaccination and stimulated with 200 nM rCpal or media as a negative control in an IFN-y ELISpot assay.Shown are representative IFN-y ELISpot blots and calculated spot forming units per million splenocytes for each vaccine group.

[0036] FIG. 6A-6D. MP-CpalmRNA (MP-CPAl-Dcpep) vaccination protects mice challenged with a lethal dose of Coccidioides posadasii arthroconidia. C57BL / 6 mice (n=10 per group) were immunized and boosted twice with the 5 pg dosage of MP-encapsulated CPAl-DCpep mRNA or empty MPs. Two non-vaccinated groups were also included. Mice in groups 1-3 were challenged by oropharyngeal route with 50 Cp C735 spores 3 weeks following the final boost (A). Immunization with the MP-CpalmRNA vaccine resulted in a significant reduction in weight loss compared to unvaccinated mice (B). Additionally, there was a significant reduction in fungal burden in both the lungs (C) and spleens (D) of mice that received the MP-CpalmRNA vaccine. * p<0.05, ** p<0.01, **** P<0.0001. Kruskal -Wallis test.

[0037] FIG. 7A-7B. MP-rCpa5 vaccination protects HLA-DR4 transgenic mice challenged with a lethal dose of Coccidioides posadasii C735 arthroconidia. (A) HLA-DR4 Tg mice (n=10 mice per group) were immunized and boosted at a two-week interval with 10 pg of microparticle-encapsulated rCpa5 protein (MP-rCpa5). A non-vaccinated group was included as a control. Four weeks following the final boost, mice in both groups were challenged by the oropharyngeal route with 54 C735 spores. (B) Immunization with the MP-rCpa5 vaccine resulted in a significant reduction in fungal burden in both the lungs and spleens of mice that received the MP-rCpa5 vaccine. Significance was determined by Mann-Whitney t tests (# p<0.0001, *** p<0.001).DETAILED DESCRIPTION

[0038] Disclosed herein are recombinant Coccidioides polypeptide antigens (rCpas) and mRNA vaccines encoding the rCpas. In certain embodiments the antigen is a rCpal, rCpa2, rCpa3, rCpa4, and / or rCpa5 antigen. The recombinant antigens or mRNA encoding the same can be incorporated into a composition comprising an adjuvant or a delivery system.I. COCCIDIOIDES POLYPEPTIDE ANTIGENS (CPAS)

[0039] Certain aspects of the invention include methods and compositions concerning proteinaceous compositions including Cpas (e.g., peptide vaccines) or nucleic acids encoding one or more Cpas (e.g., mRNA vaccines). Various Cpas or mRNA encoding the same can be used in the context of the present invention.

[0040] Disclosed herein are engineered Coccidioides polypeptide antigens (rCpas) and mRNA encoding Coccidioides polypeptide antigens (rCpas). The antigen(s) included in a rCpas can include all or a portion of two or more polypeptides or peptides selected from all or a segment of Ag2 / Pra, Cs-Ag, Ure, Gell, Pmpl, Pepl, Amnl, Plb, peroxisomal matrix protein, vacuolar protease A, vacuolar protease A, urease, glucan 1,3 -beta-glucosidase, betaglucosidase, glycosyl transferase, disulfide-isomerase, enolase, glycolipid-anchored surface protein 5, CPC735_052600, CPAG_08847, ecm33, CPC735_035760, CPC735_033430 and / or trehalose synthase. In certain aspects the rCpas include Coccidioides polypeptide antigen 1 (rCpal), Coccidioides polypeptide antigen 2 (rCpa2), Coccidioides polypeptide antigen 3 (rCpa3), Coccidioides polypeptide antigen 4 (rCpa4), and Coccidioides polypeptide antigen 5 (rCpa5).

[0041] rCpas can include 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, to 27 or more of the following peptides or fragments thereof in any order: Ag2 / Pra (e.g., SEQ ID NO: 12); Cs-Ag (e.g., SEQ ID NO: 13); Pmpl (e.g., SEQ ID NO: 14); Pepl -Pl (SEQ ID NO: 7), Pepl-P2 (SEQ ID NO: 8), Amnl-P9 (SEQ ID NO: 9), Amnl -P10 (SEQ ID NO: 10), and / or Plb (SEQ ID NO: 11) of a Coccidioides species, e.g, Coccidioides posadasii, aDCpep (SEQ ID NO: 15); Ag2 / Pra (e.g., amino acids 19 to 106 of SEQ ID NO: 12); Peroxisomal matrix protein (e.g., amino acid 5 to 35 of SEQ ID NO: 16); Pmpl (e.g., amino acids 9 to 33 of SEQ ID NO: 14); vacuolar protease A (amino acid 7 to 22 of SEQ ID NO: 17); vacuolar protease A (amino acid 43 to 64 of SEQ ID NO: 17); vacuolar protease A (amino acid 368 to 383 of SEQ ID NO: 17); Amnl-P9 (SEQ ID NO: 9); Amnl -P10 (amino acids 5 to 20 of SEQ ID NO: 10); Plb (SEQ ID NO: 11); urease (amino acids 744 to 778 of SEQ ID NO: 18); glucan 1,3 -beta-glucosidase (amino acids 57 to 71 of SEQ ID NO: 19); beta-glucosidase (amino acids 189 to 212 of SEQ ID NO:20); glycosyl transferase (amino acids 496 to 511 of SEQ ID NO:21); glycosyl transferase (amino acids 362 to 383 of SEQ ID NO:21); glycosyl transferase (amino acids 413 to 432 of SEQ ID NO:21); disulfide-isomerase (amino acids 273 to 291 of SEQ ID NO:22); disulfide-isomerase (amino acids 186 to 204 of SEQ ID NO:22); enolase (amino acids 161 to 183 SEQ ID NO:23); glycolipid-anchored surface protein 5 (amino acids 193 to 208 of SEQ ID NO:24); CPC735_052600 (amino acids 210 to 231 of SEQ ID NO:25); CPC735_052600 (amino acids 229 to 247 of SEQ ID NO:25); CPAG_08847 (amino acids 239 to 260 of SEQ ID NO:26); CPAG 08847 (amino acids 313 to 331 of SEQ ID NO:26); ecm33 (amino acids 223 to 244 of SEQ ID NO:27); ecm33 (amino acids 112 to 140 of SEQ ID NO:27); CPC735 035760 (amino acids 109 to 136 of SEQ ID NO:28); Pepl-POB (SEQ ID NO:40),Ure-PAB (SEQ ID N0:41); Thl (SEQ ID NO:42); and / or CPC735 033430 (amino acids 34 to 56 of SEQ ID NO:29) - including variants of the aforementioned polypeptides / peptides. The rCpa can have an amino terminal or carboxy terminal tag, e.g., hexahistidine tag, or variants thereof. In certain aspects each element is separated by an amino acid spacer, e.g., GPGPG or AAY spacer. In certain aspects an element can be present more than once, adjacent to or nonadj acent to another copy of the element.

[0042] The rCpal antigen includes the following peptides or peptide fragments: Ag2 / Pra (e g., SEQ ID NO: 12), Cs-Ag (e g., SEQ ID NO: 13), Pmpl (e.g, SEQ ID NO: 14), and 5 promiscuous immunodominant T cell epitopes derived from Pepl-Pl (SEQ ID NO:7), Pepl- P2 (SEQ ID NO: 8), Amnl-P9 (SEQ ID NO: 9), Amnl-PIO (SEQ ID NO: 10), and Plb (SEQ ID NO: 11) of a Coccidioides species, e.g, Coccidioides posadasii; a DCpep (SEQ ID NO: 15); and / or an amino terminal affinity tag, e.g., hexahistidine tag. In certain aspects each element is separated by a spacer, e.g., GPGPG spacer. In certain embodiments the rCpal has the amino acid sequence of SEQ ID NO:2. In certain embodiments the rCpal has the amino acid sequence of SEQ ID NO:2. In certain aspects the rCpal antigen is encoded by an mRNA vaccine having a nucleotide sequence of SEQ ID NO:30.

[0043] The rCpa2 antigen includes the following peptides or peptide fragments: Ag2 / Pra (e g., SEQ ID NO:38), Cs-Ag (e.g, SEQ ID NO:39), Pmpl (e.g, SEQ ID NO: 14), and 5 promiscuous immunodominant T cell epitopes derived from Pepl-Pl (SEQ ID NO:7), Pepl- P2 (SEQ ID NO: 8), Amnl-P9 (SEQ ID NO: 9), Amnl-PIO (SEQ ID NO: 10), and Plb (SEQ ID NO: 11) of a Coccidioides species, e.g, Coccidioides immitis; a DCpep (SEQ ID NO: 15); and / or an amino terminal affinity tag, e.g., hexahistidine tag. In certain aspects each element is separated by a spacer, e.g., GPGPG spacer. In certain embodiments the rCpa2 has the amino acid sequence of SEQ ID NO:34. In certain aspects the rCpa2 antigen is encoded by an mRNA vaccine having a nucleotide sequence of SEQ ID NO:33.

[0044] The rCpa3 antigen includes the following peptides or peptide fragments: Ag2 / Pra (e.g., amino acids 19 to 106 of SEQ ID NO: 12); Cs-Ag (e.g., SEQ ID NO: 13); Pmpl (e.g., SEQ ID NO: 14); 5 promiscuous, immunodominant T cell epitopes derived from Pepl-Pl (SEQ ID NO: 7), Pepl-P2 (SEQ ID NO: 8), Amnl-P9 (SEQ ID NO: 9), Amnl-PIO (SEQ ID NO: 10), Plb (SEQ ID NO: 11); a DCpep (SEQ ID NO: 15); and an amino terminal affinity tag, e.g., hexahistidine tag. In certain aspects one or more elements are separated by a spacer, e.g., GPGPG spacer. In certain embodiments the rCpa3 has the amino acid sequence of SEQ IDN0:4. In certain aspects the rCpa3 antigen is encoded by an mRNA vaccine having a nucleotide sequence of SEQ ID NO: 31.

[0045] The rCpa4 antigen includes an immunogenic fragment of Ag2 / Pra (e.g., amino acids 19 to 106 of SEQ ID NO: 12); Peroxisomal matrix protein (e.g., amino acid 5 to 35 of SEQ ID NO: 16); Pmpl (e.g., amino acids 9 to 33 of SEQ ID NO: 14); vacuolar protease A (amino acid 7 to 22 of SEQ ID NO: 17); vacuolar protease A (amino acid 43 to 64 of SEQ ID NO: 17); vacuolar protease A (amino acid 368 to 383 of SEQ ID NO: 17); Amnl-P9 (SEQ ID NO: 9); Amnl-PIO (amino acids 5 to 20 of SEQ ID NO: 10); Plb (SEQ ID NO: 11); urease (amino acids 744 to 778 of SEQ ID NO: 18); glucan 1,3-beta-glucosidase (amino acids 57 to 71 of SEQ ID NO: 19); beta-glucosidase (amino acids 189 to 212 of SEQ ID NO:20); glycosyl transferase (amino acids 496 to 511 of SEQ ID NO:21); glycosyl transferase (amino acids 362 to 383 of SEQ ID NO:21); glycosyl transferase (amino acids 413 to 432 of SEQ ID NO:21); disulfide-isomerase (amino acids 273 to 291 of SEQ ID NO:22); disulfide-isomerase (amino acids 186 to 204 of SEQ ID NO:22); enolase (amino acids 161 to 183 SEQ ID NO:23); glycolipid-anchored surface protein 5 (amino acids 193 to 208 of SEQ ID NO:24); CPC735_052600 (amino acids 210 to 231 of SEQ ID NO:25); CPC735_052600 (amino acids 229 to 247 of SEQ ID NO:25); CPAG_08847 (amino acids 239 to 260 of SEQ ID NO:26); CPAG 08847 (amino acids 313 to 331 of SEQ ID NO:26); ecm33 (amino acids 223 to 244 of SEQ ID NO:27); ecm33 (amino acids 112 to 140 of SEQ ID NO:27); CPC735_035760 (amino acids 109 to 136 of SEQ ID NO:28); and / or CPC735 033430 (amino acids 34 to 56 of SEQ ID NO:29). In certain aspects each element is separated by a spacer, e.g., GPGPG or AAY spacer. In certain embodiments the rCpa4 has the amino acid sequence of SEQ ID NO:6. In certain aspects the rCpa4 antigen is encoded by an mRNA vaccine having a nucleotide sequence of SEQ ID NO:32.

[0046] The rCpa5 antigen includes the following peptides or peptide fragments: Ag2 / Pra (e.g., amino acids 19 to 106 of SEQ ID NO: 12), Pepl-POB (SEQ ID NO:40), Ure-PAB (SEQ ID NO:41), Thl (SEQ ID NO:42), Cs-Ag (e.g, SEQ ID NO: 13), Pmpl (e.g, SEQ ID NO: 14), and 5 promiscuous immunodominant T cell epitopes derived from Pepl-Pl (SEQ ID NO:7), Pepl-P2 (SEQ ID NO: 8), Amnl-P9 (SEQ ID NO: 9), Amnl-PIO (SEQ ID NO: 10), and Plb (SEQ ID NO: 11) of a Coccidioides species, e.g, Coccidioides posadasii; optionally a DCpep (SEQ ID NO: 15); and / or an optional amino terminal affinity tag, e.g., hexahistidine tag. In certain aspects each element is separated by a spacer, e.g, GPGPG spacer. In certainembodiments the rCpa5 has the amino acid sequence of SEQ ID NO:36. In certain aspects the rCpa2 antigen is encoded by an mRNA vaccine having a nucleotide sequence of SEQ ID NO 35.

[0047] As used herein, a “protein” or “polypeptide” refers to a molecule comprising at least ten amino acid residues. A peptide is typically shorter, less than 10 amino acid residues in length. In certain embodiments a modified or variant protein or polypeptide can be employed. A “modified protein” or “modified polypeptide” or “modified peptide” or a “variant” refers to a protein or polypeptide or peptide whose chemical structure, particularly its amino acid sequence, is altered with respect to a reference protein or polypeptide. It is specifically contemplated that a modified / variant protein or polypeptide retains immunogenicity.

[0048] A scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score. BLASTP “Positives” shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. A polypeptide comprising an amino acid sequence that when optimally aligned with a sequence will generate a percent similarity of at least 80 percent using the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1, wherein the polypeptide functions as an immunogenic polypeptide. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. AppL Math. 2:482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC / Gene program by Intelligenetics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA. The BLAST family of programs, which can be used for database similarity searches, includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein databasesequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley- Interscience, New York (1995).

[0049] Polypeptides / peptides may be altered by truncation, rendering them shorter than the corresponding reference or parent, but they may also be altered by fusing or conjugating a heterologous polypeptide / peptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc. .

[0050] Proteins, polypeptides, or peptides may be made by known techniques, including (i) expression of proteins, polypeptides, or peptides through standard molecular biological techniques, (ii) isolation of proteinaceous compounds from natural sources, or (iii) chemical synthesis.

[0051] Amino acid sequence variants of rCpas disclosed herein can be used to produce similar polypeptides. Variants can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the invention may affect 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous or contiguous amino acids of reference or parent polypeptide. A variant can comprise an amino acid sequence that is at least 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical or similar to a sequence provided or referenced herein. A variant can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids, in particular aspects conservative substitution(s). A polypeptide of a Coccidioides species, such as Ag2 / Pra, Cs-Ag, Ure, Gell, Pmpl, Pepl, Amnl, Plb, Peroxisomal matrix protein, vacuolar protease A, urease, glucan 1,3-beta-glucosidase, beta-glucosidase, glycosyl transferase, disulfide-isomerase, enolase, glycolipid-anchored surface protein 5, CPC735_052600, CPAG_08847, ecm33, CPC735_035760, CPC735_033430 and / or trehalose synthase are contemplated for use in compositions and methods described herein.

[0052] Deletion variants typically lack one or more residues of the native or wild-type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleicacid sequence to generate a truncated protein. Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of one or more residues. Terminal additions, called fusion proteins, may also be generated. These fusion proteins include multimers or concatemers of one or more peptide or polypeptide described or referenced herein. A multimer or concatemer can comprise the same polypeptide repeated or a plurality of polypeptides.

[0053] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative such that a function or activity of the polypeptide is affected. Nonconservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.

[0054] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids. Codons include the following amino acids and related codons: Alanine / Ala / A GCA GCC GCG GCU; Cysteine / Cys / C UGC UGU; Aspartic acid / Asp / D GAC GAU; Glutamic acid / Glu / E GAA GAG; Phenylalanine / Phe / F UUC UUU; Glycine / Gly / G GGA GGC GGG GGU; Histidine / His / H CAC CAU; Isoleucine / Ile / I AUA AUC AUU; Lysine / Lys / K AAA AAG; Leucine / Leu / L UUA UUG CUA CUC CUG CUU; Methionine / Met / M AUG; Asparagine / Asn / N AAC AAU; Proline / Pro / P CCA CCC CCG CCU; Glutamine / Gln / Q CAA CAG; Arginine / Arg / R AGA AGG CGA CGC CGG CGU; Serine / Ser / S AGC AGU UCA UCC UCG UCU; Threonine / Thr / T ACA ACC ACG ACU; Valine / Val / V GUA GUC GUG GUU; Tryptophan / Trp / W UGG; Tyrosine / Tyr / Y UAC UAU.

[0055] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still comprises or be essentially as set forth in one of the sequences disclosed herein while maintaining immunogenicity. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various noncoding sequences flanking either of the 5' or 3' portions of the coding region.

[0056] The polypeptides or rCpas described herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more variant amino acids within at least, or at most 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, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids, or any range derivable therein, of the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 to SEQ ID NO:29, or SEQ ID NO:38 to SEQ ID NO:42 including immunogenic fragments thereof.

[0057] An immunogenic fragment as described herein may include 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, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids, or any range derivable therein, of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 to SEQ ID NO:29, or SEQ ID NO:38 to SEQ ID NO:42.

[0058] In certain embodiments, a composition may include a polynucleotide that is or is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to a nucleic acid sequence encoding a rCpa or antigen disclosed herein. In certain aspects, the nucleic acid sequence will have all or part of the nucleic acid sequence provided herein.

[0059] Polypeptides, peptides, antigens, or immunogens can be conjugated or linked covalently or noncovalently to other moieties such as adjuvants, proteins, peptides, supports, fluorescence moieties, or labels. The term “conjugate” or “immunoconjugate” is broadly used to define the operative association of one moiety with another agent and is not intended to refer solely to any type of operative association and is particularly not limited to chemical “conjugation.” Compositions of the invention may further comprise an adjuvant or a pharmaceutically acceptable excipient. An adjuvant may be covalently or non-covalently coupled to a polypeptide or peptide of the invention. In certain aspects, the adjuvant is chemically conjugated to a protein, polypeptide, or peptide.

[0060] It is contemplated that in compositions of the invention there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein). Of this, about, at least about, or at most 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100% may be one or more recombinant antigen disclosed herein or its variant and may be used in combination with other peptides or polypeptides, such as other fungal peptides and / or antigens.

[0061] The present invention contemplates the administration of recombinant antigen or mRNA encoding an antigen disclosed herein and variants thereof to affect a preventative therapy or therapeutic effect against the development of a disease or condition associated with infection by a Coccidioides pathogen(s).

[0062] In certain aspects, combinations of Coccidioides antigens or mRNAs encoding antigens are used in the production of an immunogenic composition that is effective at treating or preventing Coccidioides infection. Different molecules on the surface of the fungus are involved in different steps of the infection cycle. Combinations of certain antigens can elicit an immune response which protects against multiple stages of Coccidioides infection. The effectiveness of the immune response can be measured either in animal model assays and / or using an opsonophagocytic assay.

[0063] Another embodiment of the present invention uses autologous B lymphocyte cell lines, which are transfected with a viral vector that expresses an immunogen product, and more specifically, a protein having immunogenic activity. Other examples of mammalian host cell lines include, but are not limited to Vero and HeLa cells, other B- and T- cell lines, such as CEM, 721.221, H9, Jurkat, Raji, as well as cell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cells. In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or that modifies and processes the gene product in the manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.

[0064] Immunogenic fragments are immunologically reactive with antibodies generated against the Coccidioides proteins or with antibodies generated by infection of a mammalian host with Coccidioides. Immunogenic fragments also include fragments that when administered at an effective dose, (either alone or as a hapten bound to a carrier), elicit a protective or therapeutic immune response against Coccidioides infection, in certain aspects it is protective against Coccidioides posadasii and / or Coccidioides immitis infection. Such an immunogenic fragment may include, for example, the protein lacking an N-terminal leader sequence, and / or a transmembrane domain and / or a C-terminal anchor domain. In a preferred aspect the immunogenic fragment according to the invention comprises substantially all of theextracellular domain of a protein which has at least 80%, 85%, 90%, 95%, or 97 to 99% identity or similarity, including all values and ranges there between, to a sequence selected segment of a polypeptide described or referenced herein.II. NUCLEIC ACIDS

[0065] Certain embodiments concern recombinant polynucleotides encoding the proteins, polypeptides, peptides described herein, e.g., rCpal, rCpa3, rCpa4, and variants thereof. The nucleic acid sequences for all or segments of Ag2 / Pra, Cs-Ag, Ure, Gell, Pmpl, Pepl, Amnl, Plb, Peroxisomal matrix protein, vacuolar protease A, urease, glucan 1,3 -beta-glucosidase, beta-glucosidase, glycosyl transferase, di sulfide-isom erase, enolase, glycolipid-anchored surface protein 5, CPC735_052600, CPAG_08847, ecm33, CPC735_035760,CPC735 033430, trehalose synthase, and other proteins are included, all of which are incorporated by reference, and can be used to prepare peptides or polypeptides. In further embodiments, a polynucleotide molecule comprising a nucleic acid sequence encoding a recombinant antigen disclosed herein is contemplated. In further aspects, an expression vector comprises the nucleic acid sequence operably linked to an expression control sequence. In still further aspects, a host cell comprising the expression vector is also contemplated.

[0066] As used in this application, the term “polynucleotide” refers to a nucleic acid molecule that is recombinant. Included within the term “polynucleotide” are oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.

[0067] In particular embodiments the invention concerns isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a recombinant antigen disclosed herein or variants thereof. The term “recombinant” may be used in conjunction with a polynucleotide or polypeptide and generally refers to a polypeptide or polynucleotide produced and / or manipulated in vitro or that is a replication product of such a molecule. In various embodiments the nucleic acids of the invention may be used in genetic vaccines.

[0068] The nucleic acid segments used in the present invention can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.

[0069] In certain embodiments, the invention concerns isolated nucleic acid segments and recombinant vectors that include within their sequence a contiguous nucleic acid sequence encoding one or more of the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more of SEQ ID NO:7 to SEQ ID NO:29 SEQ, SEQ ID NO:38 to SEQ ID NO:42 or fragments thereof.

[0070] In certain embodiments, the present invention provides polynucleotide variants having identity or similarity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity or similarity, including all values and ranges there between, compared to a polynucleotide sequence of this invention (e.g., SEQ ID NO:30, 31, or 32) using the methods described herein (e.g., BLAST analysis using standard parameters). The invention also contemplates the use of polynucleotides which are complementary to all the polynucleotides described herein.A. mRNA Vaccines

[0071] mRNA vaccines can be categorized based on their structure and function: linear (non-replicating) mRNA vaccines, circular mRNA vaccines, and / or self-amplifying mRNA (saRNA) vaccines. Linear mRNA vaccines are typically a single strand of mRNA that encodes for a specific antigen. The mRNA is modified to enhance stability, translation efficiency, and reduce immune stimulation. Once administered, typically encapsulated in microparticles (MPs) for protection and delivery, the linear mRNA enters the host cell's cytoplasm. Here, it is directly translated by ribosomes into the target protein. This protein then triggers an immune response, leading to the production of antibodies and T-cell activation.

[0072] Self-Amplifying mRNA (saRNA) Vaccines include not only the gene for the antigen but also the genes for RNA replication machinery (replicase genes) derived from alphaviruses or other RNA viruses. This makes the RNA molecule longer than conventional mRNA. Upon entering the cell, the saRNA is translated into both the antigen and the replicase proteins. The replicase proteins then replicate the RNA, leading to an increase in the number of mRNA molecules within the cell. This results in more antigen production from a single RNA molecule, amplifying the immune response.

[0073] In certain aspects, provided herein are circular RNA polynucleotides comprising a 3' post splicing group I intron fragment, optionally a first spacer, an Internal Ribosome Entry Site (IRES), an expression sequence, optionally a second spacer, and a 5' post splicing group I intron fragment. In some embodiments, these regions are in that order.

[0074] In certain embodiments, transcription of a vector comprising a 5' homology region, a 3' group I intron fragment, optionally a first spacer, an Internal Ribosome Entry Site (IRES), an expression sequence, optionally a second spacer, a 5' group I intron fragment, and a 3' homology region results in the formation of a precursor linear RNA polynucleotide capable of circularizing. In some embodiments, this precursor linear RNA polynucleotide circularizes when incubated in the presence of guanosine nucleotide or nucleoside (e.g., GTP) and divalent cation (e.g., Mg2+).

[0075] In one embodiment, a 5' spacer sequence is a polyA sequence. In another embodiment, the 5' spacer sequence is a poly AC sequence. In one embodiment, a spacer comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% poly AC content. In one embodiment, a spacer comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polypyrimidine (C / T or C / U) content.

[0076] In certain embodiments, the vectors, precursor RNA and circular RNA provided herein comprise an internal ribosome entry site (IRES). Inclusion of an IRES permits the translation of one or more open reading frames from a circular RNA (e.g., open reading frames that form the expression sequence). The IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. In some embodiments, the polynucleotides herein comprise an expression sequence or exon segment, e.g., encoding a Cpa described herein. In certain embodiments, the vectors provided herein comprise a 3' UTR. In some embodiments. In some embodiments, the vectors provided herein comprise a 5' UTR. Insome embodiments, the vector provided herein comprises a polyA region. In some embodiments the polyA region is at least 30 nucleotides long or at least 60 nucleotides long.

[0077] In certain embodiments, the polynucleotides provided herein are circular RNA polynucleotides or are useful for making circular RNA polynucleotides.

[0078] In some embodiments, provided herein is a vector. In certain embodiments, the vector comprises, in the following order, (a) a 5' homology region, (b) a 3' group I intron fragment, (c) optionally, a first spacer sequence, (d) an IRES, (e) an expression sequence or exon segment, (f) optionally, a second spacer sequence, (g) a 5' group I intron fragment, and (h) a 3' homology region. In some embodiments, the vector comprises a transcriptional promoter upstream of the 5' homology region.

[0079] In certain embodiments, the circular RNA provided herein is injected into an animal (e.g., a human), such that a polypeptide encoded by the circular RNA molecule (e.g., a Cpa) is expressed inside the animal.

[0080] According to certain embodiments, an mRNA vaccine encodes at least one Cpa polypeptide of interest (antigen). They may have any combination of the features described herein. In one embodiment, the polynucleotides of the present invention may encode at least one peptide or polypeptide of interest, e.g., a rCpa, rCpal, rCpa2, rCpa3, rCpa4, rCpa5, a variant thereof, or combinations of antigens selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 to SEQ ID NO:29, SEQ ID NO:38 to SEQ ID NO:42 or fragments thereof. As used herein, such a region may be referred to as a “coding region” or “region encoding” or “open reading frame (ORF)”. In one embodiment, the polynucleotides of the present invention is or functions as a messenger RNA (mRNA). As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide which encodes at least one peptide or polypeptide of interest and which is capable of being translated to produce the encoded peptide polypeptide of interest in vitro, in vivo, in situ or ex vivo. According to certain embodiments, the coding RNA is an mRNA, a self-replicating RNA, a circular RNA, a viral RNA, or a replicon RNA.

[0081] In embodiments, the coding RNA is a circular RNA. As used herein, “circular RNA” or “circRNAs” are circular polynucleotide constructs that encode at least one antigenic peptide or protein as defined herein. Accordingly, in preferred embodiments, a circRNAcomprises at least one antigenic peptide or protein as defined herein, or an immunogenic fragment or an immunogenic variant thereof.

[0082] The term “replicon RNA” will be recognized and understood by the person of ordinary skill in the art and is intended to be an optimized self-replicating RNA. Such constructs may include replicase elements derived from, for example and not limited to alphaviruses (e.g. SFV, SIN, VEE, or RRV) and the substitution of the structural virus proteins with the nucleic acid of interest, e.g., at least one antigenic peptide or protein as defined herein. Alternatively, the replicase may be provided on an independent coding RNA construct. Downstream of the replicase may be a sub-genomic promoter that controls replication of the replicon RNA.

[0083] In one embodiment, the polynucleotides of the present invention may be structurally modified or chemically modified. In certain aspects, the polynucleotides have a uniform chemical modification of all or any of the same nucleoside type or a population of modifications, or a measured percent of a chemical modification of all any of the same nucleoside type but with random incorporation, such as where all uridines are replaced by a uridine analog, e.g., pseudouridine. In another embodiment, the polynucleotides may have a uniform chemical modification of two, three, or four of the same nucleoside type throughout the entire polynucleotide (such as all uridines and all cytosines, etc. are modified in the same way).

[0084] Polynucleotide Architecture. Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5'UTR, a 3'UTR, a 5' cap, and a poly- A tail. The polynucleotides described herein may function as mRNA and include one or more of these elements.

[0085] In order to further enhance protein production, polynucleotides of the present invention can be designed to be conjugated to other polynucleotides, dyes, intercalating agents (e.g., acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG- 40K), MPEG, [MPEG], polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport / absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins, e.g., glycoproteins, or peptides, e.g., molecules having aspecific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a lung cell, endothelial cell, or bone cell, hormones and hormone receptors, non- peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or a drug. In a preferred embodiment, the polynucleotides of the present invention which encode a peptide are conjugated to one or more cell markers. Conjugation may result in increased stability and / or half-life and may be particularly useful in targeting the polynucleotides to specific sites in the cell, tissue or organism.

[0086] Cell-Penetrating Polypeptides. The polynucleotides disclosed herein may also encode or be conjugated to one or more cell-penetrating polypeptides. As used herein, “cellpenetrating polypeptide” or CPP refers to a polypeptide which may facilitate the cellular uptake of molecules. The cell-penetrating peptide may also include a signal sequence. As used herein, a “signal sequence” refers to a sequence of amino acid residues bound at the amino terminus of a nascent protein during protein translation.

[0087] In one embodiment, the polynucleotides may also encode a fusion protein / peptide. The fusion protein / peptide may be created by operably linking a heterologous protein or peptide to a therapeutic protein / peptide. As used herein, “operably linked” refers to the therapeutic protein and the heterologous protein or peptide being connected in such a way to permit the expression of the complex when introduced into the cell. Preferably, the therapeutic protein may be covalently linked to the heterologous protein or peptide in the formation of the fusion protein.

[0088] Polynucleotides Having Untranslated Regions (UTRs). The polynucleotides of the present invention (e.g., rCpa encoding polynucleotides) may comprise one or more regions or parts which act or function as an untranslated region. Where polynucleotides are designed to encode at least one polypeptide of interest, the polynucleotides may comprise one or more of these untranslated regions or UTRs. By definition, untranslated regions (UTRs) of a gene or nucleic acid are transcribed but not translated. In mRNA, the 5'UTR starts at the transcription start site and continues to the start codon of the coding region but does not include the start codon; whereas the 3'UTR starts immediately following the stop codon and continues until the transcriptional termination signal. The regulatory features of UTR can be incorporated into the polynucleotides of the present invention to among other things, enhance the stability of the molecule.

[0089] Natural 5'UTRs bear features which play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. 5'UTR also has been known to form secondary structures which are involved in elongation factor binding. By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of the polynucleotides of the invention.

[0090] Other non-UTR sequences may also be used as regions or subregions within the polynucleotides. For example, introns or portions of intron sequences may be incorporated into regions of the polynucleotides of the invention. Incorporation of intronic sequences may increase protein production as well as polynucleotide levels.

[0091] Combinations of features may be included in flanking regions and may be contained within other features. For example, the ORF may be flanked by a 5' UTR which may contain a strong Kozak translational initiation signal and / or a 3' UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail. 5'UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes.

[0092] A UTR from various gene(s) may be incorporated into the regions of the polynucleotide. Furthermore, multiple UTRs of any known gene may be utilized. It is also within the scope of the present invention to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as the transcript from which they were selected or may be altered in orientation or location. Hence a 5' or 3' UTR may be inverted, shortened, lengthened, made with one or more other 5' UTRs or 3' UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3' or 5' UTR may be altered relative to a wild type or native UTR by the change in orientation or location or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3' or 5') comprise a variant UTR.

[0093] In one embodiment, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature, or property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue, or at some time during development. The UTRs from any of these genes may be swapped forany other UTR of the same or different family of proteins to create a new polynucleotide. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.

[0094] 3' UTR and the A U Rich Elements. Natural or wild type 3 ' UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes: Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C- Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class.

[0095] Regions Having a 5' Cap. The 5' cap structure of a natural mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5' proximal introns removal during mRNA splicing. Endogenous mRNA molecules may be 5 '-end capped generating a 5 '-ppp-5 '-triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the mRNA molecule. These 5 '-guanylate caps may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or ante-terminal transcribed nucleotides of the 5' end of the mRNA may optionally also be 2'-O-methylated. 5'- decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.

[0096] In some embodiments, polynucleotides may be designed to incorporate a cap moiety. Modifications to the polynucleotides of the present invention may generate a non- hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5 '-ppp-5' phosphodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass.) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkagein the 5 '-ppp-5' cap. Additional modified guanosine nucleotides may be used such as a-methyl- phosphonate and seleno-phosphate nucleotides.

[0097] Additional modifications include, but are not limited to, 2'-O-methylation of the ribose sugars of 5 '-terminal and / or 5'-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2'-hydroxyl group of the sugar ring. Multiple distinct 5 '-cap structures can be used to generate the 5 '-cap of a nucleic acid molecule, such as a polynucleotide which functions as an mRNA molecule.

[0098] Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5 '-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or linked to the polynucleotides of the invention.

[0099] For example, the Anti -Reverse Cap Analog (ARC A) cap contains two guanines linked by a 5 '-5 '-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5 '-triphosphate-5 '-guanosine (m7G- 3'mppp-G; which may equivalently be designated 3' O-Me-m7G(5')ppp(5')G). The 3'-0 atom of the other, unmodified, guanine becomes linked to the 5 '-terminal nucleotide of the capped polynucleotide. The N7- and 3 '-O-methlyated guanine provides the terminal moiety of the capped polynucleotide. Another example of a cap is mCAP, which is similar to ARCA but has a 2'-O-methyl group on guanosine (i.e., N7,2'-O-dimethyl-guanosine-5 '-triphosphate-5 '- guanosine, m7Gm-ppp-G).

[0100] Viral Sequences. Viral sequences such as, but not limited to, the translation enhancer sequence of the barley yellow dwarf virus (BYDV-PAV), the Jaagsiekte sheep retrovirus (JSRV) and / or the Enzootic nasal tumor virus (See e.g., International Pub. No. WO2012129648; herein incorporated by reference in its entirety) can be engineered and inserted in the polynucleotides of the invention and can stimulate the translation of the construct in vitro and in vivo. Transfection experiments can be conducted in relevant cell lines and protein production can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day 7 post-transfection.

[0101] IRES Sequences. Polynucleotides (e.g., rCpa encoding polynucleotides) which may contain an internal ribosome entry site (IRES). First identified as a feature Picorna virus RNA, IRES plays an important role in initiating protein synthesis in absence of the 5' cap structure.An IRES may act as the sole ribosome binding site or may serve as one of multiple ribosome binding sites of an mRNA. Polynucleotides containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes (“multicistronic nucleic acid molecules”). When polynucleotides are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the invention include without limitation, those from coxsackievirus B3 (CVB3), picomaviruses (e.g., FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).

[0102] Pofy-A Tails. During RNA processing, a long chain of adenine nucleotides (poly-A tail) may be added to a polynucleotide such as an mRNA molecule in order to increase stability. Immediately after transcription, the 3 ' end of the transcript may be cleaved to free a 3 ' hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that can be between, for example, approximately 80 to approximately 250 residues long, including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 residues long. Terminal groups on the poly A tail may be incorporated for stabilization into polynucleotides of the invention. Polynucleotides of the present invention may include des-3' hydroxyl tails. They may also include structural moieties or 2'-Omethyl modifications as taught by Junjie Li, et al. (Current Biology, Vol. 15, 1501-1507, Aug. 23, 2005, the contents of which are incorporated herein by reference in its entirety).

[0103] The polynucleotides may be designed to encode transcripts with alternative polyA tail structures including histone mRNA. These mRNAs are distinguished by their lack of a 3' poly(A) tail, the function of which is instead assumed by a stable stem-loop structure and its cognate stem-loop binding protein (SLBP); the latter carries out the same functions as those of PABP on polyadenylated mRNAs

[0104] Unique poly-A tail lengths provide certain advantages to the polynucleotides of the present invention. Generally, the length of a poly-A tail, when present, is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400,1.500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the polynucleotide or region thereof includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to1.500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1.000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

[0105] In one embodiment, the poly-A tail is designed relative to the length of the overall polynucleotide or the length of a particular region of the polynucleotide. This design may be based on the length of a coding region, the length of a particular feature or region or based on the length of the ultimate product expressed from the polynucleotides.

[0106] In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or feature thereof. The poly-A tail may also be designed as a fraction of the polynucleotides to which it belongs. In this context, the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, a construct region or the total length of the construct minus the poly-A tail. Further, engineered binding sites and conjugation of polynucleotides for Poly-A binding protein may enhance expression.

[0107] Start Codon Region. In some respects the polynucleotides may have regions that are analogous to or function like a start codon region. In one embodiment, the translation of a polynucleotide may initiate on a codon which is not the start codon AUG. Translation of the polynucleotide may initiate on an alternative start codon such as, but not limited to, ACG, AGG, AAG, CTG / CUG, GTG / GUG, ATA / AU A, ATT / AUU, TTG / UUG. As a non-limiting example, the translation of a polynucleotide begins on the alternative start codon ACG. As another non-limiting example, polynucleotide translation begins on the alternative start codon CTG or CUG. Yet another non-limiting example, the translation of a polynucleotide begins on the alternative start codon GTG or GUG. Nucleotides flanking a codon that initiates translation such as, but not limited to, a start codon or an alternative start codon, are known to affect thetranslation efficiency, the length and / or the structure of the polynucleotide. (See e.g., Matsuda and Mauro, PLoS ONE, 2010 5: 11; the contents of which are herein incorporated by reference in its entirety). Masking any of the nucleotides flanking a codon that initiates translation may be used to alter the position of translation initiation, translation efficiency, length and / or structure of a polynucleotide.

[0108] Stop Codon Region. In one aspect, the polynucleotides may include at least one or two stop codons before the 3' untranslated region (UTR). The stop codon may be selected from TGA, TAA and TAG. In one aspect, the polynucleotides include the stop codon TGA and one additional stop codon. In a further embodiment the addition stop codon may be TAA. In another embodiment, the polynucleotides of the present invention include three stop codons.

[0109] Signal Sequences. The polynucleotides described herein may also encode additional features which facilitate trafficking of the polypeptides to therapeutically relevant sites. One such feature which aids in protein trafficking is the signal sequence. As used herein, a “signal sequence” or “signal peptide” is a polynucleotide or polypeptide, respectively, which is from about 9 to 200 nucleotides (3-60 amino acids) in length which is incorporated at the 5' (or N- terminus) of the coding region or polypeptide encoded, respectively. Addition of these sequences result in trafficking of the encoded polypeptide to the endoplasmic reticulum through one or more secretory pathways. Some signal peptides are cleaved from the protein by signal peptidase after the proteins are transported.

[0110] Protein Cleavage Signals and Sites. In certain aspects, polypeptides of the invention (e.g., peptide polypeptides) may include various protein cleavage signals and / or sites. In one embodiment, the polypeptides of the present invention may include at least one protein cleavage signal containing at least one protein cleavage site. The protein cleavage site may be located at the N-terminus, the C-terminus, at any space between the N- and the C-termini such as, but not limited to, half-way between the N- and C-termini, between the N-terminus and the half-way point, between the half-way point and the C-terminus, and combinations thereof. In one embodiment, the polynucleotides of the present invention may be engineered such that the polynucleotide contains at least one encoded protein cleavage signal. The encoded protein cleavage signal may include, but is not limited to, signalase cleavage signal, a proprotein convertase (or prohormone convertase), thrombin and / or Factor Xa protein cleavage signal.

[0111] Codon Optimization. The polynucleotides of the invention, their regions or parts or subregions may be codon optimized. Codon optimization methods are known in the art and may be useful in efforts to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post translation modification sites in encoded protein (e.g. glycosylation sites); add, remove, or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; to adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and / or proprietary methods. In one embodiment, the ORF sequence is optimized using optimization algorithms.

[0112] In Vitro Transcription-Enzymatic Synthesis. cDNA or polynucleotides (e.g., expression vectors) encoding the polynucleotides described herein may be transcribed using an in vitro transcription (IVT) system. The system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs. The polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as, but not limited to, polymerases able to incorporate polynucleotides (e.g., modified nucleic acids).

[0113] Solid-Phase Chemical Synthesis. Polynucleotides described herein may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of polynucleotides or nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Impurities and excess reagents are washed away and no purification is required after each step. The automation of the process is amenable on a computer-controlled solid-phase synthesizer. Solid-phase synthesis allows rapid production of polynucleotides or nucleic acids in a relatively large scale that leads to the commercial availability of some polynucleotides or nucleic acids. Furthermore, it is useful in site-specific introduction of chemical modifications in the polynucleotide ornucleic acid sequences. It is an indispensable tool in designing modified derivatives of natural nucleic acids.

[0114] Liquid Phase Chemical Synthesis. The synthesis of polynucleotides of the present invention by the sequential addition of monomer building blocks may be carried out in a liquid phase. A covalent bond is formed between the monomers or between a terminal functional group of the growing chain and an incoming monomer. Functional groups not involved in the reaction must be temporarily protected. After the addition of each monomer building block, the reaction mixture is purified before adding the next monomer building block. The functional group at one terminal of the chain is deprotected to be able to react with the next monomer building blocks.

[0115] Combination of Synthetic Methods. The synthetic methods discussed above each has its own advantages and limitations. Attempts have been conducted to combine these methods to overcome the limitations. Such combinations of methods are within the scope of the present invention.

[0116] As used herein, a "conservative substitution" in a polypeptide or peptide is the replacement of an amino acid with another amino acid that has the same net electronic charge and approximately the same size and shape. Amino acids with aliphatic or substituted aliphatic amino acid side chains have approximately the same size when the total number of carbon and heteroatoms in their side chains differs by no more than about four. They have approximately the same shape when the number of branches in their side chains differs by no more than one. Amino acids with phenyl or substituted phenyl groups in their side chains are considered to have about the same size and shape. Listed below are five groups of amino acids. Replacing an amino acid in a polypeptide or peptide with another amino acid from the same group results in a conservative substitution:

[0117] Group I: glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, and non-naturally occurring amino acids with C1-C4 aliphatic or C1-C4 hydroxyl substituted aliphatic side chains (straight chained or monobranched).

[0118] Group II: glutamic acid, aspartic acid and non-naturally occurring amino acids with carboxylic acid substituted C1-C4 aliphatic side chains (unbranched or one branch point).

[0119] Group III: lysine, ornithine, arginine and non-naturally occurring amino acids with amine or guanidino substituted C1-C4 aliphatic side chains (unbranched or one branch point).

[0120] Group IV: glutamine, asparagine and non-naturally occurring amino acids with amide substituted Cl -C4 aliphatic side chains (unbranched or one branch point).

[0121] Group V: phenylalanine, phenylglycine, tyrosine and tryptophan.

[0122] As used herein, a "highly conservative substitution" in a polypeptide is the replacement of an amino acid with another amino acid that has the same functional group in the side chain and nearly the same size and shape. Amino acids with aliphatic or substituted aliphatic amino acid side chains have nearly the same size when the total number of carbon and heteroatoms in their side chains differs by no more than two. They have nearly the same shape when they have the same number of branches in their side chains. Examples of highly conservative substitutions include valine for leucine, threonine for serine, aspartic acid for glutamic acid and phenylglycine for phenylalanine. Examples of substitutions which are not highly conservative include alanine for valine, alanine for serine and aspartic acid for serine.B. Vectors

[0123] Polypeptides of the invention may be encoded by a nucleic acid molecule comprised in a vector. The term “vector” is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell where it can be replicated and expressed. A nucleic acid sequence can be “heterologous,” which means that it is in a context foreign to the cell in which the vector is being introduced or to the nucleic acid in which is incorporated, which includes a sequence homologous to a sequence in the cell or nucleic acid but in a position within the host cell or nucleic acid where it is ordinarily not found. Vectors include DNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques. In addition to encoding a recombinant antigen disclosed herein or variant thereof, the vector can encode other polypeptide sequences such as a one or more other fungal peptide, a tag, or an immunogenicity enhancing peptide. Useful vectors encoding such fusion proteins include pIN vectors, vectors encoding a stretch of histidines, and pGEX vectors, for use in generating glutathione S- transferase (GST) soluble fusion proteins for later purification and separation or cleavage.

[0124] The term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described herein.III. IMMUNE RESPONSE AND ASSAYS

[0125] As discussed above, the invention concerns evoking or inducing an immune response in a subject against a recombinant antigen or variants thereof. In one embodiment, the immune response can protect against or treat a subject having, suspected of having, or at risk of developing an infection or related disease, particularly those related to Coccidioides. One use of the immunogenic compositions of the invention is to prevent infections by inoculating a subject prior to entering an environment having an increased risk of infection.

[0126] In other embodiments the antigens described herein can used in a diagnostic method to detect the presence of specific antibodies in a biological sample, which indicates a previous or current infection or exposure to a particular pathogen, such as Coccidioides. Antigen-based tests detect the presence of an antigen from the pathogen. Serology tests can use antigens to detect antibodies in the blood, serum, or other biological samples. The antigen is often immobilized on a surface (like in ELISA), and if antibodies specific to that antigen are present in the sample, they will bind to it. This binding can be visualized or measured through various methods. In an ELISA (Enzyme-Linked Immunosorbent Assay) assay an antigen (e.g., Cpa described herein) is coated onto a plate. A sample is added, and if it contains antibodies against the antigen, they will bind. Detection systems then indicate this binding, often through color change. Agglutination test (direct or indirect) use the antigen to detect antibodies in the sample where visible clumping indicates a positive result. Indirect or passive agglutination might involve antigens attached to particles like latex beads, enhancing visibility of the reaction.

[0127] Antigens in diagnostics serve as the target for detecting either the pathogen itself or the immune response against it, offering a balance between speed, cost, and sometimes sensitivity, depending on the method used. These methods are crucial for rapid diagnosis,especially in outbreaks or pandemics, providing quick insights into infection status or immune response.A. Immunoassays

[0128] The present invention includes the implementation of serological assays to evaluate whether and to what extent an immune response is induced or evoked by compositions of the invention. There are many types of immunoassays that can be implemented. Immunoassays encompassed by the present invention include, but are not limited to, those described in U.S. Patent 4,367,110 (double monoclonal antibody sandwich assay) and U.S. Patent 4,452,901 (western blot). Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo.

[0129] Immunoassays generally are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. In one example, antibodies or antigens are immobilized on a selected surface, such as a well in a polystyrene microtiter plate, dipstick, or column support. Then, a test composition suspected of containing the desired antigen or antibody, such as a clinical sample, is added to the wells. After binding and washing to remove nonspecifically bound immune complexes, the bound antigen or antibody may be detected. Detection is generally achieved by the addition of another antibody, specific for the desired antigen or antibody, that is linked to a detectable label. This type of ELISA is known as a “sandwich ELISA.” Detection also may be achieved by the addition of a second antibody specific for the desired antigen, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

[0130] Competition ELISAs are also possible implementations in which test samples compete for binding with known amounts of labeled antigens or antibodies. The amount of reactive species in the unknown sample is determined by mixing the sample with the known labeled species before or during incubation with coated wells. The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal. Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove nonspecifically bound species, and detecting the bound immune complexes.

[0131] Antigen or antibodies may also be linked to a solid support, such as in the form of plate, beads, dipstick, membrane, or column matrix, and the sample to be analyzed is applied to the immobilized antigen or antibody. In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period. The wells of the plate will then be washed to remove incompletely-adsorbed material. Any remaining available surfaces of the wells are then “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein, and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.B. Diagnosis of Fungal Infection

[0132] In addition to the use of proteins, polypeptides, and / or peptides, as well as antibodies binding these polypeptides, proteins, and / or peptides, to treat or prevent infection as described above, the present invention contemplates the use of these polypeptides, proteins, peptides, and / or antibodies in a variety of ways, including the detection of the presence of Coccidioides to diagnose an infection, whether in a patient or on medical equipment which may also become infected. In accordance with the invention, a preferred method of detecting the presence of infections involves the steps of obtaining a sample suspected of being infected by one or more Coccidioides species or strains, such as a sample taken from an individual, for example, from one's lung, blood, saliva, tissues, bone, muscle, cartilage, or skin. Following isolation of the sample, diagnostic assays utilizing the polypeptides, proteins, peptides, and / or antibodies of the present invention may be carried out to detect the presence of Coccidioides, and such assay techniques for determining such presence in a sample are well known to those skilled in the art and include methods such as radioimmunoassay, western blot analysis, and ELISA assays. In general, in accordance with the invention, a method of diagnosing an infection is contemplated wherein a sample suspected of being infected with Coccidioides has added to it the polypeptide, protein, peptide, antibody, or monoclonal antibody in accordance with the present invention, and Coccidioides are indicated by antibody binding to the polypeptides, proteins, and / or peptides, or polypeptides, proteins, and / or peptides binding to the antibodies in the sample.

[0133] Methods include those for diagnosing an infection caused by Coccidioides spp. in a subject. The methods can include obtaining a biological sample from the subject or theenvironment. The sample can include, but is not limited to swab (or other environmental sampling technique), blood, serum, respiratory secretions, and tissue samples. The sample can be contacted with an antigen specific to Coccidioides spp. The antigen can be, but is not necessarily immobilized on a solid support. The presence of antibodies specific to Coccidioides spp. antigen in the sample can be detected by binding the antigen. Detection can be performed using an enzyme-linked immunosorbent assay (ELISA) or an immunodiffusion assay or other technique known in the art. An ELISA can use a secondary antibody conjugated with an enzyme, followed by a substrate that produces a detectable signal upon enzymatic cleavage. Immunodiffusion can be used by formation of a visible precipitation band indicating the presence of specific antibodies. The presence of a detectable signal or precipitation band indicates the presence of Coccidioides spp. specific antibodies, thereby diagnosing an infection by Coccidioides spp. The amount of specific antibodies can be quantified by serial dilution of the sample and determining the highest dilution at which the signal is still detectable, thereby providing a titer indicative of the severity of the infection.

[0134] An antigen can be included in a diagnostic kit for detecting Coccidioides spp. A diagnostic kit can include an antigen, as described herein, specific to Coccidioides spp. immobilized on a solid support. The kit can include detection reagents, including secondary antibody conjugated with a label, such as an enzyme for ELISA or a medium for immunodiffusion. Control samples for positive and negative reactions can also be include in a diagnostic kit as well as instructions for use of the kit. The kit can include a solid support for the antigen such as microtiter plates, membranes, beads, dipsticks, and / or gel matrices.

[0135] Accordingly, antibodies in accordance with the invention may be used for the prevention of infection from Coccidioides fungus (i.e., passive immunization), for the treatment of an ongoing infection, or for use as research tools. The term "antibodies" as used herein includes monoclonal, polyclonal, chimeric, single chain, bispecific, simianized, and humanized or primatized antibodies as well as Fab fragments, such as those fragments which maintain the binding specificity of the antibodies, including the products of an Fab immunoglobulin expression library. Accordingly, the invention contemplates the use of single chains such as the variable heavy and light chains of the antibodies. Generation of any of these types of antibodies or antibody fragments is well known to those skilled in the art.

[0136] Any of the polypeptides, proteins, peptides, and / or antibodies described above may be labeled directly with a detectable label for identification and quantification of Coccidioidesfungus. Labels for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances, including colored particles such as colloidal gold or latex beads. Suitable immunoassays include enzyme-linked immunosorbent assays (ELISA).C. Protective Immunity

[0137] In some embodiments of the invention, proteinaceous compositions confer protective immunity to a subject. Protective immunity refers to a body’s ability to mount a specific immune response that protects the subject from developing a particular disease or condition that involves the agent against which there is an immune response. An immunogenically effective amount confers protective immunity to the subject.

[0138] As used herein in the specification and in the claims section that follows, the term polypeptide or peptide refer to a stretch of amino acids covalently linked there amongst via peptide bonds. Different polypeptides have different functionalities according to the present invention. While according to one aspect, a polypeptide is derived from an immunogen designed to induce an active immune response in a recipient, according to another aspect of the invention, a polypeptide is derived from an antibody which results following the elicitation of an active immune response in, for example, an animal, and which can serve to induce a passive immune response in the recipient. In both cases, however, the polypeptide is encoded by a polynucleotide according to any possible codon usage.

[0139] As used herein the phrase “immune response” or its equivalent “immunological response” refers to the development of a humoral (antibody mediated), cellular (mediated by antigen-specific T cells or their secretion products) or both humoral and cellular response directed against a protein, peptide, carbohydrate, or polypeptide of the invention in a recipient patient. Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody, antibody containing material, or primed T-cells. A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules, to activate antigen-specific CD4 (+) T helper cells and / or CD8 (+) cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils or other components of innate immunity. As used herein “active immunity” refers to any immunity conferred upon a subject by administration of an antigen.

[0140] As used herein “passive immunity” refers to any immunity conferred upon a subject without administration of an antigen to the subject. “Passive immunity” therefore includes, but is not limited to, administration of activated immune effectors including cellular mediators or protein mediators (e.g., monoclonal and / or polyclonal antibodies) of an immune response. A monoclonal or polyclonal antibody composition may be used in passive immunization for the prevention or treatment of infection by organisms that carry the antigen recognized by the antibody. An antibody composition may include antibodies that bind to a variety of antigens that may in turn be associated with various organisms. The antibody component can be a polyclonal antiserum. In certain aspects the antibody or antibodies are affinity purified from an animal or second subject that has been challenged with an antigen(s). Alternatively, an antibody mixture may be used, which is a mixture of monoclonal and / or polyclonal antibodies to antigens present in the same, related, or different microbes or organisms, such as fungus, including but not limited to Coccidioides.

[0141] Passive immunity may be imparted to a patient or subject by administering to the patient immunoglobulins (Ig) and / or other immune factors obtained from a donor or other nonpatient source having a known immunoreactivity. In other aspects, an antigenic composition of the present invention can be administered to a subject who then acts as a source or donor for globulin, produced in response to challenge with the antigenic composition ("hyperimmune globulin"), that contains antibodies directed against Coccidioides or other organisms. A subject thus treated would donate plasma from which hyperimmune globulin would then be obtained, via conventional plasma-fractionation methodology, and administered to another subject in order to impart resistance against or to treat Coccidioides infection. Hyperimmune globulins according to the invention are particularly useful for immune-compromised individuals, for individuals undergoing invasive procedures or where time does not permit the individual to produce their own antibodies in response to vaccination. See U.S. Patents 6,936,258, 6,770,278, 6,756,361, 5,548,066, 5,512,282, 4,338,298, and 4,748,018, each of which is incorporated herein by reference in its entirety, for exemplary methods and compositions related to passive immunity.

[0142] For purposes of this specification and the accompanying claims the terms "epitope” and "antigenic determinant” are used interchangeably to refer to a site on an antigen to which B and / or T cells respond or recognize. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopesformed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols (1996). Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen. T-cells recognize continuous epitopes of about nine amino acids for CD8 cells or about 13-15 amino acids for CD4 cells. T cells that recognize the epitope can be identified by in vitro assays that measure antigen-dependent proliferation, as determined by3H-thymidine incorporation by primed T cells in response to an epitope (Burke et al., 1994), by antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., 1996) or by cytokine secretion.

[0143] The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4 (+) T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating IgG and T-cells from an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject.

[0144] As used herein and in the claims, the terms "antibody” or "immunoglobulin” are used interchangeably and refer to any of several classes of structurally related proteins that function as part of the immune response of an animal or recipient, which proteins include IgG, IgD, IgE, IgA, IgM and related proteins.

[0145] Under normal physiological conditions antibodies are found in plasma and other body fluids and in the membrane of certain cells and are produced by lymphocytes of the type denoted B cells or their functional equivalent. Antibodies of the IgG class are made up of four polypeptide chains linked together by disulfide bonds. The four chains of intact IgG molecules are two identical heavy chains referred to as H-chains and two identical light chains referred to as L-chains.

[0146] As used herein and in the claims, the phrase "an immunological portion of an antibody” includes a Fab fragment of an antibody, a Fv fragment of an antibody, a heavy chain of an antibody, a light chain of an antibody, a heterodimer consisting of a heavy chain and alight chain of an antibody, a variable fragment of a light chain of an antibody, a variable fragment of a heavy chain of an antibody, and a single chain variant of an antibody, which is also known as scFv. In addition, the term includes chimeric immunoglobulins which are the expression products of fused genes derived from different species, one of the species can be a human, in which case a chimeric immunoglobulin is said to be humanized. Typically, an immunological portion of an antibody competes with the intact antibody from which it was derived for specific binding to an antigen.

[0147] Optionally, an antibody or preferably an immunological portion of an antibody, can be chemically conjugated to, or expressed as, a fusion protein with other proteins. For purposes of this specification and the accompanying claims, all such fused proteins are included in the definition of antibodies or an immunological portion of an antibody.

[0148] As used herein the terms “immunogenic agent” or “immunogen” or “antigen” are used interchangeably to describe a molecule capable of inducing an immunological response against itself on administration to a recipient, either alone, in conjunction with an adjuvant, or presented on a display vehicle.D. Treatment Methods

[0149] A method of the present invention includes treatment for a disease or condition caused by a Coccidioides pathogen. An immunogenic polypeptide of the invention can be given to induce an immune response in a person infected with Coccidioides or suspected of having been exposed to Coccidioides. Methods may be employed with respect to individuals who have tested positive for exposure to Coccidioides or who are deemed to be at risk for infection based on possible exposure.

[0150] In particular, the invention encompasses a method of treatment for Coccidioides infection. The immunogenic compositions and vaccines of the invention are advantageous to use to inoculate health care workers.

[0151] In some embodiments, the treatment is administered in the presence of adjuvants or carriers or other Coccidioides antigens. Furthermore, in some examples, treatment comprises administration of other agents commonly used against fungal infection, such as one or more antifungal.

[0152] The use of peptides for vaccination can require, but not necessarily, conjugation of the peptide to an immunogenic carrier protein, such as hepatitis B surface antigen, keyhole limpet hemocyanin, or bovine serum albumin. Methods for performing this conjugation are well known in the art.IV. VACCINE AND OTHER PHARMACEUTICAL COMPOSITIONSA. Vaccines

[0153] The present invention includes methods for preventing or ameliorating Coccidioides infections. As such, the invention contemplates vaccines for use in both active and passive immunization embodiments. Immunogenic compositions, proposed to be suitable for use as a vaccine, may be prepared from immunogenic recombinant antigen disclosed herein, or a fragment thereof or a variant thereof. In certain aspects, antigenic material is extensively dialyzed to remove undesired small molecular weight molecules and / or lyophilized for more ready formulation into a desired vehicle.

[0154] Other options for a protein / peptide-based vaccine involve introducing nucleic acids encoding the antigen(s) as DNA vaccines. In this regard, reports described construction of recombinant vaccinia viruses expressing either 10 contiguous minimal CTL epitopes (Thomson, 1996) or a combination of B cell, cytotoxic T-lymphocyte (CTL), and T-helper (Th) epitopes from several microbes (An, 1997), and successful use of such constructs to immunize mice for priming protective immune responses. Thus, there is ample evidence in the literature for successful utilization of peptides, peptide-pulsed antigen presenting cells (APCs), and peptide-encoding constructs for efficient in vivo priming of protective immune responses. The use of nucleic acid sequences as vaccines is exemplified in U.S. Patents 5,958,895 and 5,620,896.

[0155] The preparation of vaccines that contain polypeptide or peptide sequence(s) as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all of which are incorporated herein by reference. Typically, such vaccines are prepared as injectables either as liquid solutions or suspensions: solid forms suitable for solution in or suspension in liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccines. In specific embodiments, vaccines are formulated with a combination of substances, as described in U.S. Patents 6,793,923 and 6,733,754, which are incorporated herein by reference.

[0156] Vaccines may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides: such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1% to about 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.

[0157] The polypeptides and polypeptide-encoding DNA constructs may be formulated into a vaccine as neutral or salt forms. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and those that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.

[0158] Typically, vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including the capacity of the individual’s immune system to synthesize antibodies and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms of active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.

[0159] The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application within a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection and the like. The dosage of the vaccine will depend on the route of administration and will vary according to the size and health of the subject.

[0160] In certain instances, it will be desirable to have multiple administrations of the vaccine, e.g., 2, 3, 4, 5, 6 or more administrations. The vaccinations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9 ,10, 11, 12 twelve-week intervals, including all ranges there between. Periodic boosters at intervals of 1-5 years will be desirable to maintain protective levels of the antibodies. The course of the immunization may be followed by assays for antibodies against the antigens, as described in U.S. Patents 3,791,932; 4,174,384 and 3,949,064.1. Carriers

[0161] A given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin, or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m- maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde, and bis-biazotized benzidine.2. Adjuvants

[0162] The immunogenicity of polypeptide or peptide compositions can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins, or synthetic compositions. A number of adjuvants can be used to enhance an antibody response against a recombination antigen disclosed herein. Adjuvants can (1) trap the antigen in the body to cause a slow release; (2) attract cells involved in the immune response to the site of administration; (3) induce proliferation or activation of immune system cells; or (4) improve the spread of the antigen throughout the subject’s body.

[0163] Adjuvants include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, mineral salts, polynucleotides, and natural substances. Specific adjuvants that may be used include Toll-like receptor (TLR) ligands (e.g., CpG oligonucleotides and the like), IL-1, IL-2, IL-4, IL-7, IL- 12, y-interferon, GMCSP, BCG, aluminum salts, such as aluminum hydroxide or other aluminum compound, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene / Tween 80 emulsion. MHC antigens may even be used. Others adjuvants or methods are exemplified in U.S. Patents 6,814,971, 5,084,269, 6,656,462, each of which is incorporated herein by reference).

[0164] Various methods of achieving adjuvant affect for the vaccine includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as about 0.05 to about 0.1% solution in phosphate buffered saline, admixture with synthetic polymers of sugars (CARBOPOL®) used as an about 0.25% solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between about 70° to about 101°C for a 30-second to 2-minute period, respectively. Aggregation by reactivating with pepsin-treated (Fab) antibodies to albumin; mixture with bacterial cells (e.g., C. parvum), endotoxins or lipopolysaccharide components of Gram-negative bacteria; emulsion in physiologically acceptable oil vehicles (e.g., mannide mono-oleate (Aracel A)); or emulsion with a 20% solution of a perfluorocarbon (FLUOSOL-DA®) used as a block substitute may also be employed to produce an adjuvant effect.

[0165] Examples of and often preferred adjuvants include complete Freund’s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund’s adjuvants, and aluminum hydroxide.

[0166] In some respects it is preferred that the adjuvant be selected to be a preferential inducer of either a Thl, a Th2, and / or Thl7 type of response. High levels of Thl- and Thl7- type cytokines tend to favor the induction of cell mediated immune responses to a given antigen, while high levels of Th2-type cytokines tend to favor the induction of humoral immune responses to the antigen.

[0167] The distinction of Thl, Th2, and Thl7-type immune response is not absolute. In reality an individual will support an immune response which is described as being predominantly Thl, predominantly Th2 or predominartly Th 17. However, it is often convenient to consider the families of cytokines in terms of that described in murine CD4+ T cell clones (O’Shea and Paul, Science 2010, 327:1098; Becattini et al., Science 2015 347:400).Traditionally, Th 1 -type responses are associated with the production of the INF-y and IL-2 cytokines by T-lymphocytes. Other cytokines often directly associated with the induction of Th 1 -type immune responses are not produced by T-cells, such as IL- 12. Th2-type responses are associated with the secretion of IL- 4, IL-5, and IL-10. Furthermore, Thl7-type responses are associated with the secretion of IL-17A, IL-17B, IL-17C, IL-17D, IL-17F, IL-22 and IL- 23. Cytokines IL-1 and IL-6 are produced by other immune cells to guide the development of Thl7-type response.

[0168] In addition to adjuvants, it may be desirable to co-administer biologic response modifiers (BRM) to enhance immune responses. BRMs have been shown to upregulate T cell immunity or downregulate suppresser cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg / d) (Smith / Kline, PA); or low-dose Cyclophosphamide (CYP; 300 mg / m2) (Johnson / Mead, NJ) and cytokines such as y-interferon, IL-1, IL-2, IL-6, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7.

[0169] As described herein, “particle” refers to any hollow and porous structure that can contain an antigen or a polynucleotide encoding an antigen. In some embodiments, the size of the particle is about 0.5 to about 5 pm.3. Microparticles (MPs)

[0170] In some embodiments, rCpa antigen and / or mRNA vaccines are formulated in a microparticle. A microparticle encapsulates the antigen or mRNA. In some embodiments, vaccines are formulated as a microparticle formed by one or more other polymer that forms a microparticle having an interior lumen. In some embodiments, mRNA vaccines are formulated in a polysaccharide-polycation complex. The formation of the polysaccharide microparticle may be accomplished by methods known in the art. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and / or polyarginine.

[0171] In some embodiments, a microparticle (e.g., a lipid microparticle) has a mean diameter of 10-500 nm, 20-400 nm, 30-300 nm, 40-200 nm. In some embodiments, a microparticle (e.g., a lipid microparticle) has a mean diameter of 50-150 nm, 50-200 nm, 80- 100 nm or 80-200 nm.B. Combination Therapy

[0172] The compositions and related methods of the present invention, particularly administration of the recombinant antigen disclosed herein or a variant thereof, may also be used in combination with the administration of traditional therapies. These include, but are not limited to, the administration of antifungals such as fluconazole, itraconazole, amphotericin B, voriconazole, and posaconazole.

[0173] In one aspect, it is contemplated that a polypeptide vaccine and / or therapy is used in conjunction with antifungal treatment. Alternatively, the therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agents and / or a proteins or polynucleotides are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and antigenic composition would still be able to exert an advantageously combined effect on the subject. In such instances, it is contemplated that one may administer both modalities within about 12-24 h of each other or within about 6-12 h of each other. In some situations, it may be desirable to extend the time period for administration significantly, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0174] Various combinations may be employed, for example antifungal therapy is “A” and the immunogenic molecule given as part of an immune therapy regime, such as an antigen, is “B”: A / B / A; B / A / B; B / B / A; A / A / B; A / B / B; B / A / A; A / B / B / B; B / A / B / B; B / B / B / A; B / B / A / B; A / A / B / B; A / B / A / B; A / B / B / A; B / B / A / A; B / A / B / A; B / A / A / B; A / A / A / B; B / A / A / A; A / B / A / A; A / A / B / A.

[0175] Administration of the immunogenic compositions of the present invention to a patient / subject will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the recombinant antigen, or other compositions described herein. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, such as hydration, may be applied in combination with the described therapy.C. General Pharmaceutical Compositions

[0176] In some embodiments, pharmaceutical compositions are administered to a subject. Different aspects of the present invention involve administering an effective amount of acomposition to a subject. In some embodiments of the present invention, the recombinant antigens disclosed herein may be administered to the patient to protect against infection by one or more Coccidioides pathogens. Alternatively, an expression vector encoding one or more such polypeptides or peptides may be given to a patient as a preventative treatment. Additionally, such compounds can be administered in combination with an antifungal. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0177] In addition to the compounds formulated for parenteral administration, such as those for intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; time release capsules; and any other form currently used, including creams, lotions, mouthwashes, inhalants and the like.

[0178] The active compounds of the present invention can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes. The preparation of an aqueous composition that contains an additional compound or compounds that increase the expression of MHC class I or MHC II molecules will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.

[0179] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0180] The proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic basessuch as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0181] The carrier also can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0182] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0183] Administration of the compositions according to the present invention will typically be via any common route. This includes, but is not limited to oral, nasal, or buccal administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, or intravenous injection. In certain embodiments, a vaccine composition may be inhaled (e.g., U.S. Patent 6,651,655, which is specifically incorporated by reference). Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier,” means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.

[0184] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in isotonic NaCl solution and either added to hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, Remington’s Pharmaceutical Sciences, 1990). Some variation in dosage will necessarily occur depending on the condition of the subject. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0185] An effective amount of therapeutic or prophylactic composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, z.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection desired.

[0186] Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.

[0187] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.D. In Vitro, Ex Vivo, or In Vivo Administration

[0188] As used herein, the term in vitro administration refers to manipulations performed on cells removed from or outside of a subject, including, but not limited to cells in culture. The term ex vivo administration as referred to herein is when cells that have been manipulated in vivo are subsequently assessed in vitro. The term in vivo administration includes all manipulations performed within a subject.

[0189] In certain aspects of the present invention, the compositions may be administered either in vitro, ex vivo, or in vivo. In certain in vitro embodiments, autologous T-lymphocyte cell lines are incubated with a microbial cell of the instant invention for 24 to 48 hours or with a recombinant protein(antigen) as described herein and / or a variant thereof and / or any other composition described herein for two hours. The transduced cells can then be used for in vitro analysis, or alternatively for ex vivo administration. U.S. Patents 4,690,915 and 5,199,942, both incorporated herein by reference, disclose methods for ex vivo manipulation of blood mononuclear cells and bone marrow cells for use in therapeutic applications.V. EXAMPLES

[0190] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.EXAMPLE 1CONSTRUCTION OF MRNA ENCODING COCCIDIOIDES ANTIGENS

[0191] The general structure of coccidioidal mRNA vaccines. The mRNA encoding Coccidioides antigens are synthesized using CleanCap® and CleanScript™ technologies by TriLink Biotechnologies (San Diego, CA). The coding sequences of coccidioidal (Ci / Cp) antigens with an N-terminus His-tag are flanked with the 5’ and 3’UTR of a human gene and cloned into Trilink selected plasmids (FIG. 1 A). The latter contains a T7 promoter for in vitrotranscription (IVT). The 5 ’cap and 3’ poly A tail (>80 bp) are added during IVT reaction. Nl- Methyl-Pseudouridine, a modified UTP, is incorporated into mRNA using a T7 RNA polymerase during IVT to reduce the immunogenicity of the resulting mRNA. Additionally, a dendritic cell targeting sequence (DCpep) that encodes a 12-mer peptide (FYPSYHSTPQRP (SEQ ID NO: 15)) can be included at the C-terminus of the mRNA vaccine construct (FIG. IB) to enhance antigen presenting and protective efficacy against coccidioidomycosis and other microbial infections. The mRNA vaccine construct can be further modified by inclusion of an N-terminus signal peptide (e.g. METDTLLLWVLLLWVPGSTG derived from mouse IgKVIII) plus two alanine (AA) encoding sequences to aid antigen secretion (Guler-Gane et al., PloS one, 2016) for MHC-II presentation by immune cells (FIG. 1C). Furthermore, the mRNA vaccine can contain both signal peptide / AA and DCpep coding sequences for Ci / Cp antigen secretion and dendritic cell targeting, respectively (FIG. ID).

[0192] Examples of Coccidioides spp. (Ci / Cp) antigens in mRNA vaccine constructs.

[0193] Herein, we describe 5 Coccidioides spp. (Ci / Cp) antigen constructs, rCpal-5 (FIG. 2). These antigens contain Coccidioides derived antigenic proteins and peptides linked in tandem with GPGPG or AAY spacer. These recombinant proteins also contain an N-terminal His(6x)-tag for easy protein purification by immobilized metal affinity chromatography. When the antigens are formulated as mRNA vaccines, the expression of His-tagged antigens in vaccinated host can be detected by anti-His antibody.

[0194] Coccidioides ppsadasii antigen 1 (Cpal): Recombinant Cpal (rCpal) is a well characterized protective multivalent antigen consisting of 3 Coccidioides posadasii-spQcidc, proteins and 5 T cell epitopes (FIG. 2; GenBank: AVH85517.1 (Hung et al. Infection and immunity, 86(1 l):e00070-18, 2018; Campuzano et al. J Immunol. 2020, 204:3296-306)). The mRNA encoding Cpal with an N-terminus His-tag (allowing for in vivo protein expression detection) and a C-terminus DCpep is codon-optimized for expression in mammalian cells. The DNA sequence for IVT and translated protein sequence of the CpalmRNA vaccine are SEQ ID NO: 1 and SEQ ID NO:2, respectively.

[0195] Coccidioides ppsadasii antigen 2 (Cpa2): Recombinant Cpa2 (rCpa2) is almost identical to Cpal, but is constructed using Coccidioides immitis-specific proteins and T cell epitopes (Fig. 2; GenBank: WAB54687.1). The mRNA encoding Cpa2 with an N-terminus His-tag (allowing for in vivo protein expression detection). The DNA sequence for IVT andtranslated protein sequence of the Cpa2mRNA vaccine are SEQ ID NO:33 and SEQ ID NO:34, respectively.

[0196] Coccidioides jjpsadasii antigen 3 (Cpa3): We have discovered that removing the signal peptide (first 18 amino acids) from the Ag2 / Pra in the rCpal improves protein expression and solubility. This rCpal derivative, His-tagged rCpa3 protein can be expressed and purified from asx E coli expression system by metal affinity chromatography. The rCpa3 protein can be further formulated with desirable adjuvants to protect coccidioidomycosis as a subunit protein vaccine. The Cpa3 antigen also can be developed as a coccidioidal mRNA vaccine. The DNA sequence for IVT and translated protein sequences of the Cpa3mRNA vaccine are provided in SEQ ID NO:3 and SEQ ID NO:4, respectively.

[0197] Coccidioides jjpsadasii antigen 4 (Cpa4): The recombinant Cpa4 (rCpa4) protein (SEQ ID NO:6) is a novel multivalent antigen vaccine consisting of 17 coccidioidal antigens (26 epitopes) linked in tandem with either a GPGPG or an AAY linker. The rCpa4 contains an N-terminus His-tag for easy protein purification by metal affinity chromatography. The rCpa4 can be formulated with desirable adjuvants to protect coccidioidomycosis as a subunit protein vaccine. The Cpa4 antigen also can be developed as a coccidioidal mRNA vaccine. The human codon optimized DNA sequence of the Cpa4mRNA for IVT is SEQ ID NO:5. The translated protein sequence of Cpa4mRNA is provided in SEQ ID NO:6.

[0198] Coccidioides jjpsadasii antigen 5 (Cpa5): The recombinant Cpa5 (rCpa5) protein (sequence displayed below) is a novel multivalent antigen vaccine derived from Cpa3 by insertion of 3 putative HLA-DR3 restricted coccidioidal peptides between Ag2 / Prai9-ioe and CS-Agi-i48 using a GPGPG linker. These 3 inserted peptides are Cp-Pepl-POB (GenBank accession number EER26423.1; aa 368-383), Cp-Ure-PIAB (GenBank EER29262.1; aa 738- 761) and Cp-Thl-POA (GenBank EER27481.1; aa 496-511). The rCpa5 contains a His-tag for easy protein purification by metal affinity chromatography. When constructing mRNA vaccine, the rCpa5 can be further modified by inclusion of an N-terminal signal peptide (METDTLLLWVLLLWVPGSTG (SEQ ID NO: 37)) derived from mouse IgKVIII followed by two alanine (AA) to facilitate vaccine antigen secretion for MHC-II process by antigen presenting cells. The Cpa5 antigen also can be developed as a coccidioidal mRNA vaccine. The human codon optimized DNA sequence of the Cpa5mRNA for IVT is shown below. The translated protein sequence of Cpa5mRNA is identical to rCpa5 exhibited above. The DNAsequence for IVT and translated protein sequences of the Cpa5mRNA vaccine are provided in SEQ ID NO:35 and SEQ ID NO:36, respectively.EXAMPLE 2MRNA VACCINE FORMULATIONS: ENCAPSULATING MRNAS

[0199] Microparticles have been generated for vaccine development against fungal infections.

[0200] To demonstrate delivery of mRNAs and subsequent expression of mRNA-encoding proteins mCherry mRNAs were encapsulated in a microparticle then injected into mice by the subcutaneous route. As shown in FIG. 3, 36 hours after MP 1 -mCherry mRNA and MP2- mCherrymRNA injection, immune cells at the injection site have already taken up these particles, migrated to spleen and bone marrow. More importantly, the packaged mRNAs were translated to express the fluorescent mCherry proteins in these antigen presenting cells.

[0201] Once mCherry mRNA delivery system was established, the Cpal mRNAs were synthesized and loaded into particles. The resulting encapsulated-CpalmRNA vaccine was delivered to mice by subcutaneous injection and the expression of Cpal protein in immune cells was analyzed by Flow cytometry using anti-His-tag antibody. As shown in FIG. 4, 36 hours after encapsulated-CpalmRNA injection, immune cells at the injection site have already taken up the vaccine, migrated to spleen and bone marrow. More importantly, the packaged mRNAs were translated to express the His-tagged Cpal protein in antigen presenting cells including B cells, dendritic cells, and macrophages.EXAMPLE 3VACCINATION OF MICE WITH ENCAPSULATED-CPAIMRNA MOUNTS ANTIGEN (CPA1) SPECIFIC TH1 MEMORY

[0202] It is essential for a vaccine to mount T cell memory for protection against coccidioidomycosis when the host encounter the fungus. As shown in FIG. 4 the expression of Cpal protein following encapsulated-CpalmRNA vaccination in mice. To further demonstrate the immunogenicity of encapsulated-CpalmRNA, conventional C57BL / 6 mice and humanized HLA-DR4 transgenic mice were vaccinated 3 times (14-day interval) with 0-10 pg of the vaccine. Spleen was collected from mice 14 days post final vaccination, single cell suspension was made and stimulated with recombinant Cpal protein for IFN-y ELISpot analysis. Asshown in FIG. 5, mice vaccinated with as low as 1 to 5 pg of the encapsulated-CpalmRNA were sufficient to mount a Thl-type memory response.EXAMPLE 4:VACCINATION WITH ENCAPSULATED-CPAIMRNA PROTECTS MICE AGAINST PULMONARY COCCIDIOIDOMYCOSIS

[0203] To evaluate protective efficacy of MP-CpalmRNA vaccine, we immunized C57BL6 mice (n=10 per group) with 5 pg dosage of MP-encapsulated Cpal-DCpep (Group 1) or empty MPs (Group 2) (FIG. 6A). The 2 groups of mice received 2 additional boosters with the same vaccine dose at a 2-week interval. Two non-vaccinated groups (Group 3 and 4) were also included. Mice in groups 1-3 were challenged by oropharyngeal route with 50 Coccidioides posadasii C735 spores 3 weeks following the final boost. Immunization with the MP-CpalmRNA vaccine resulted in a significant reduction in weight loss compared to unvaccinated mice (FIG. 6B). Additionally, at day 14 post challenge, there was a significant reduction in fungal burden in both the lungs (FIG. 6C) and spleens (FIG. 6D) of mice that received the MP-CpalmRNA vaccine. These data demonstrated that MP-CpalmRNA vaccine is effective in protection against pulmonary coccidioidomycosis.EXAMPLE 5VACCINATION WITH ENCAPSULATED-CPA5 PROTECTS MICE AGAINST PULMONARY COCCIDIOIDOMYCOSIS

[0204] To evaluate rCpa5 protein antigenicity, we encapsulated rCpa5 with microparticles (MP) and immunized HLA-DR4 transgenic mice with the MP-rCpa5 vaccine construct. Mice were vaccinated twice at a two-week interval, rested for 4 weeks and challenged with a lethal dose (54 spores) of C. posadasii C735 strain via the pulmonary route (FIG. 7A). Fungal burdens in the lungs and spleen were determined at day 12 post challenge. As shown in FIG. 7B, vaccination of mice with MP-rCpa5 generated protective immunity that significantly reduced fungal burden in both the lungs and spleens compared to PBS mock vaccination.

Claims

CLAIMS1. An immunogenic composition comprising a recombinant Coccidioides polypeptide antigen (rCpa) selected from one or more of a recombinant Coccidioides polypeptide antigen 2 (rCpa2) or a recombinant Coccidioides polypeptide antigen 3 (rCpa3) or a recombinant Coccidioides polypeptide antigen 4 (rCpa4) or a recombinant Coccidioides polypeptide antigen 5 (rCpa5).

2. The composition of claim 1, wherein the rCpa2 antigen has an amino acid sequence identical to SEQ ID NO:34, the rCpa3 antigen has an amino acid sequence identical to SEQ ID NO:4, the rCpa4 antigen has an amino acid sequence identical to SEQ ID NO:6, and the rCpa5 antigen has an amino acid sequence identical to SEQ ID NO:36.

3. The composition of claim 1, wherein the rCpa antigen is encapsulated in a microparticle.

4. The composition of claim 3, wherein the microparticle is a polysaccharide microparticle.

5. The composition of any one of claims 1 to 4, wherein the composition further comprises or is administered in conjunction with an adjuvant.

6. The composition of claim 5, wherein the adjuvant is a TLR ligand.

7. The composition of claim 6, wherein the TLR ligand is a CpG oligonucleotide.

8. A method of treating a subject having or at risk of Coccidioides infection comprising administering an effective amount of a composition from any of claims 1 to 7.

9. An engineered messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a recombinant Coccidioides polypeptide antigen 1 (rCpal), a recombinant Coccidioides polypeptide antigen 2 (rCpa2), a recombinant Coccidioides polypeptide antigen 3 (rCpa3), a recombinant Coccidioides polypeptide antigen 4 (rCpa4), and / or a recombinant Coccidioides polypeptide antigen 5 (rCpa5).

10. The engineered mRNA of claim 9, wherein the mRNA is linear, circular, or selfamplifying.

11. The engineered mRNA according to claim 9 or 10, further comprising a 5’ UTR.

12. The engineered mRNA according to any one of claims 9 to 11, further comprising a 3’ UTR.

13. The engineered mRNA according to any one of claims 9 to 12, further comprising a polyadenylation segment.

14. The engineered mRNA according to any one of claims 9 to 13, further comprising 5’ region comprising from 5’ to 3’ (i) a 5’ external homology segment, (ii) a 3’ intron and exon segment, (iii) a 5’ internal homology segment, and (iv) a poly adenosine / cytosine spacer.

15. The engineered mRNA according to any one of claims 9 to 14, further comprising 3’ region comprising from 5’ to 3’ (i) a poly adenosine / cytosine spacer, (ii) a 3’ internal homology segment, (iii) a 5’ intron and exon segment, and (iv) a 3’ external homology segment.

16. The engineered mRNA according to any one of claims 9 to 15, wherein the ORF has a nucleotide sequence identical to SEQ ID NO:33, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:35.

17. A DNA construct encoding the engineered mRNA according to any one of claims 9 to 16.

18. A method of treating a subject with a Coccidioides infection or a subject at risk of developing a Coccidioides infection comprising administering to the subject a formulation comprising an engineered mRNA according to any one of claim 9 to 16.

19. The method according to claim 18, wherein the formulation is administered to the subject by injection, inhalation, or instillation.

20. The method according to any one of claim 18 or 19, wherein the formulation is administered to the subject by intravenous (IV) injection.

21. The method according to any one of claim 18 or 19, wherein the formulation is administered to the subject by subcutaneous injection.

22. The method according to any one of claims 18 to 21, wherein the subject is diagnosed with a Coccidioides infection.

23. The method according to any one of claims 18 to 21, wherein the subject is diagnosed with a Coccidioides posadasii or Coccidioides immitis infection.

24. A method for diagnosing a Coccidioides spp. infection comprising: contacting a biological sample with an antigen specific to Coccidioides spp., - detecting antibodies specific to the Coccidioides spp. antigen in the sample; wherein presence of a detectable signal or precipitation band indicates the presence of Coccidioides spp. specific antibodies, thereby diagnosing an infection by Coccidioides spp.

25. A diagnostic kit for detecting Coccidioides spp. infection, comprising an antigen specific to Coccidioides spp. optionally immobilized on a solid support and detection reagents.

26. The kit of claim 25, wherein the solid support for the antigen is selected from the group consisting of microtiter plates, membranes, and gel matrices.

Images