Nucleic acid vaccines against coccidioidomycosis

Abstract

A trivalent vaccine induces robust antibody and mucosal and systemic IFN-γ and Th17 T cell responses against Coccidioides and affords complete protection from fungal dissemination and disease. The combination of the three antigens of this trivalent vaccine can exert synergistic protection against lethality, disease and fungal dissemination. These robust responses can be further enhanced by use of one or more additional antigens described herein.

Description

NUCLEIC ACID VACCINES AGAINST COCCIDIOIDOMYCOSIS

[0001] This application claims benefit of United States provisional patent application number 63 / 571,835, filed March 29, 2024, the entire contents of which are incorporated by reference into this application. REFERENCE TO A SEQUENCE LISTING

[0002] The content of the XML file of the sequence listing named “UW085_seq”, which is 76 kb in size, created on March 27, 2025, and electronically submitted herewith the application, is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under Grant No.5U19AI166058- 02, awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. BACKGROUND

[0004] Coccidioidomycosis, also known as Valley Fever (VF) impacts residents in arid regions of the world including southwestern US, areas in South and Central America and in Mexico. Coccidioidomycosis is caused by Coccidioides posadasii (Cp) and Coccidioides immitis (Ci), which have very similar genomic sequences. Annual incidences are rising overall with estimated increases in recent years of more than 200% in some areas. In addition, epidemiological studies indicate that the geographical range of VF is expanding and up to 17-29% of community-acquired pneumonia in these areas can be attributed to Coccidioides infections. VF poses a significant and ongoing threat to human health, but to date, there is no safe and effective vaccine for VF (See Galgiani et al., 2022, J. Fungi 8:383). As such, there is an urgent unmet need to develop a vaccine that can provide protection from the disease. SUMMARY

[0005] The constructs, compositions, and methods described herein provide tools for addressing this need. The trivalent vaccine described herein induces robust antibody and mucosal and systemic IFN-γ and Th17 T cell responses against Coccidioides antigens and affords complete protection from fungal dissemination and disease in mice. The combination of the three antigens of this trivalent vaccine can exert synergistic protection against lethality, disease and fungal dissemination. Eight additional antigens are described that can further improve protection against Valley Fever.

[0006] In one embodiment, described herein is a nucleotide construct that expresses expression library immunization antigen 1 (ELI or ELI-Ag1), antigen 2 / proline-rich protein (PRA / Ag2), and / or peroxisomal matrix protein1 (PMP1) of Coccidioides posadasii. In some embodiments, the nucleotide construct expresses ELI, ARA / Ag2, and / or PMP1 of Coccidioides immitis. In some embodiments, the nucleotide construct is a single nucleotide construct expressing each of ELI, PRA / Ag2, and PMP1. In some embodiments, the nucleotide construct comprises multiple nucleotide constructs, each expressing one or more of these immunogens of Coccidioides posadasii and / or Coccidioides immitis.

[0007] In another embodiment, described herein is a nucleotide construct that expresses one or more of the 8 protective antigens listed in Table 4 (SEQ ID NOs: 11-26). The construct can be designed for use in combination with the construct that expresses ELI, ARA / Ag2, and / or PMP1, either as part of a single multi-antigen construct, or as an additional multi-antigen construct capable of co-administration. In some embodiments, the nucleotide construct comprises multiple nucleotide constructs, each expressing one or more of these 8 additional immunogens of Coccidioides posadasii and / or Coccidioides immitis.

[0008] In some embodiments, the nucleotide is deoxyribonucleic acid (DNA). In some embodiments, the DNA is manufactured using cells or in vitro, including, but not limited to, enzymatic methods, such as rolling circle amplification-DNA (RCA-DNA) or doggy bone DNA (dbDNA). The latter, dbDNA, (Touchlight, Inc) employs an in vitro enzymatic technology, similar to RCA technology, but uses distinct enzymes (phi29 DNA polymerase and a protelomerase) to generate covalently closed, linear DNA constructs. Also contemplated is “minicircle” DNA, such as synDNA(TM) (CytoGenix), or other selectable marker free, bacterial ori-free expression cassettes useful for vaccination. In some embodiments, the nucleotide is ribonucleic acid (RNA), including, but not limited to, messenger (mRNA), transfer RNA (tRNA), or self-amplifying RNA (saRNA or repRNA). The engineering of tRNA vaccines is described, for example, by Wang et al., 2023, Emerg. Microbes Infect. 12(1):2157339.

[0009] In some embodiments, the construct further expresses interleukin 12 (IL-12) and heat-labile enterotoxin of Escherichia coli (LT). In some embodiments, the IL-12 and LT are provided on a separate, or second construct from the first construct expressing ELI, PRA / Ag2, and PMP1, and / or one or more of the additional 8 antigens described herein. In some embodiments, the LT comprises both A and B subunits. In some embodiments, the LT comprises the A subunit without the B subunit and in others, it comprises a double mutant LT (dmLT; see www.ncbi.nlm.nih.gov / pmc / articles / PMC3647992).

[0010] Also described herein is a composition comprising the nucleotide construct of any of the embodiments described above. In some embodiments, the composition comprises one or more additional constructs. In some embodiments, the additional constructs comprise a second nucleotide construct, wherein the second nucleotide construct expresses interleukin 12 (IL-12) and heat-labile enterotoxin of Escherichia coli (LT). In some embodiments, the additional constructs comprise separate constructs, each encoding IL-12 or LT.

[0011] In some embodiments, the second construct expressing IL-12 and LT is used separately to enhance T cell and mucosal immunity, independent of the first construct. In some embodiments, the second construct expressing IL-12 and / or LT is administered as an adjuvant together with a different immunogenic composition. In some embodiments, the second construct expresses IL-12, and a third construct expresses LT.

[0012] In another embodiment, described herein is a method of eliciting an immune response to Coccidioides posadasii and Coccidioides immitis in a subject. Coccidioides posadasii and Coccidioides immitis infect humans and animals, and immunogens from one of these strains will also protect against the other (Campuzano A, et al. Vaccines (Basel). 2024 Jan 9;12(1):67. doi: 10.3390 / vaccines12010067. PMID: 38250880; PMCID: PMC10819930.) In some embodiments, the method is effective at eliciting an immune response to other fungal antigens sharing conserved regions. The method can also be used to prevent and / or treat fungal infections, such as Valley Fever.

[0013] In some embodiments, the method comprises administering to the subject a composition comprising one or more nucleic acid constructs as described herein. In some embodiments, the immune response comprises mucosal immunity, an antibody response, and / or a T cell response. In some embodiments, the T cell response comprises a systemic IFN-γ and / or Th17 T cell response. In some embodiments, the immune response protects the subject from lethality, fungal dissemination, and / or 15% weight loss within 30 days following a challenge with Coccidioides posadasii and / or Coccidioides immitis.

[0014] In some embodiments, the composition is administered by injection into the epidermis of the subject. In some embodiments, the composition is administered by intramuscular injection. Other routes of DNA or RNA vaccine delivery are known to those skilled in the art, including but not limited to, via mucosal, intradermal, intraperitoneal, intravenous administration. Examples of mucosal routes include, for example, intranasal, oral, inhaled, ocular, rectal or vaginal delivery. Those skilled in the art aware of various methods of DNA or RNA delivery, including but not limited to, gene gun, electroporation, topical, micro needles, patches, jet injector. In some embodiments, the DNA or RNA is formulated with lipid-nanoparticles or lipid nano-carriers.

[0015] The amount to be administered is effective to elicit an immune response, such as a mucosal, antibody, or T cell response. The amount that is effective can be determined by various means, including, for example, performing an assay to detect and / or measure the level of markers of an immune response. The level of such markers can be compared to a known reference level or to a pretreatment level in the same or similar subjects. In some embodiments, the amount to be administered is a prophylactically and / or therapeutically effective amount. In some embodiments, the amount to be administered is protective against challenge with the relevant immunogen(s). In some embodiments, the amount is sufficient to induce IFN-γ and / or Th17 T cell responses in a subject.

[0016] Also provided are kits and articles of manufacture for use with one or more methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG.1 illustrates the Valley Fever (VF) DNA and RNA vaccine immunogenicity study design. C57BL / 6J (n=10 / vaccination group) were vaccinated with 1μg or 10μg of LION formulated repRNA or were vaccinated with 1μg of gene gun delivered repRNA or DNA targeting ELI-A1, PRA / Ag2 or PMP1. Mice were primed on day 0 and were boosted 28 days post-prime. Mice were sacrificed 21 days post-boost (D49) and serum, spleens and lungs collected to analyze antibody responses by ELISA and T cell responses by ELISPOT.

[0018] FIG.2 is a bar graph showing the post-boost IgG levels in mg / ml for immunized mice. The concentration of IgG was determined in sera collected from N=5 Balb / c and N=5 C57Bl / 6 mice per group two weeks after the DNA booster immunization using an ELISA and recombinant proteins (for PMP1 and PRA-Ag1) or peptides (for ELI-A1) for capture antigen.

[0019] FIGS.3A-3B are bar graphs demonstrating that DNA and repRNA vaccines targeting VF ELI-A1, PRA / Ag2 and PMP1 elicit strong T cell responses in the spleen and lung. C57Bl / 6 mice were IM vaccinated with 1μg or 10μg of LION formulated repRNA or with 1μg of gene gun delivered DNA or repRNA vaccines expressing the Coccidioides antigens, ELI- A1, PRA / Ag2 or PMP1. Mice were primed at day 0 and boosted on day 28 and sacrificed 21 days post-boost to analyze T cell responses in the spleen and lung using a dual IFN-γ / IL-17 ELISPOT assay. Cells were stimulated with 1μg / ml of ELI-A1, PRA / Ag2 or PMP1 peptides as indicated and IFN-γ or IL-17 producing T cells quantified. N=8-10 mice / vaccination group. A two-way ANOVA was used. *p<0.05, ***p<0.001, ****p<0.0001.

[0020] FIGS.4A-4B are graphs demonstrating protective efficacy in a mouse model of C. posadasii infection. C57Bl / 6 mice (N=15 per group) were primed and boosted four weeks apart with either DNA vaccines delivered by gene gun into the skin, repRNA vaccinesformulated in LION and delivered IM, a live attenuated vaccine (TKO-LA) or with saline (PBS controls). Eight weeks after the booster dose, all mice were challenged with a high dose of 500 conidia of C. posadasii Silveira. Mice were weighed daily and those reaching 15% weight loss were euthanized. Shown are (4A) Survival and (4B) weight loss.

[0021] FIG.5 shows the fungal burden in lung, spleen and brain. Twenty-one days post- challenge, all mice were sacrificed, and lungs, spleen and brain were collected and analyzed for fungal CFU in each tissue. Shown are individual values for each mouse and medians. P value < 0.05*, < 0.01**, < 0.005***, < 0.001****.

[0022] FIG.6 is a survival plot showing the protective efficacy of bivalent DNA vaccines. Protective efficacy in mouse model of C. posadasii infection. C57Bl / 6 mice (N=15 per group) were primed and boosted four weeks apart with either DNA vaccines delivered by gene gun into the skin, repRNA vaccines formulated in LION and delivered IM, a live attenuated vaccine (TKO-LA) or with saline (PBS controls). Eight weeks after the booster dose, all mice were challenged with a high dose of 500 conidia of C. posadasii Silveira. Mice were weighed daily and those reaching 15% weight loss were euthanized.

[0023] FIG.7 is a schematic illustrating an overview of the rapid nucleic-acid based vaccine strategy to identify novel protective immunogens described in Example 2. Novel vaccine antigens are first selected based protection following DNA vaccination, 3 antigens co- delivered per group. Subsequent down-selection of individual antigens for further study was based on in vitro expression and / or immunogenicity (antibody response measured just before challenge). The individual antigens that meet these criteria (8 shown in Example 2) will then be tested separately as individual DNA vaccines for the ability to afford protection from Coccidioides challnge in mice. This process, using primarily protective efficacy as the primary criterion for selection, can rapidly identify new protective antigens for an effective vaccine. Adding one or more novel immunogens to the lead trivalent DNA vaccine (TriVFDNA) could further enhance its efficacy and immunogenicity by inducing broader immune responses targeting multiple proteins on the fungus (representative antigens shown in the schematic are coded by a SIL#. (SIL = P. Silvera, the strain of Coccidioides posadasii that the antigens were identified from.)

[0024] FIG.8 is a graph depicting the results of screening 24 candidate antigens based on protective efficacy in a mouse model of Coccidioides infection. N=10 female C57BL6 mice per group. Mice (6 weeks old) received a vaccine, plus a boost at 4 weeks, followed by a challenge with about 700 conidia, IN. Mice were challenged at 14 weeks of age. Endpoint was at 30 days. Percent survival is plotted as a function of time in days.DETAILED DESCRIPTION

[0025] The data presented herein show that the trivalent vaccine induced robust antibody and mucosal and systemic IFN-γ and Th17 T cell responses against each Coccidioides antigen and afforded complete protection from fungal dissemination and disease in mice. The discovery that antibodies contribute to the immune response contrasts with the current paradigm that antibodies do not play a role in protection from Valley Fever. The difference is likely due to the ability of the DNA vaccine described herein to drive a robust Th1 immune response, which influences the quality and type of antibody response induced. The data presented herein show that the combination of the three antigens of this trivalent vaccine can exert synergistic protection against lethality, disease and fungal dissemination. These robust responses can be further enhanced by use of one or more additional antigens described herein. Definitions

[0026] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.

[0027] As used herein, a “significant difference” means a difference that can be detected in a manner that is considered reliable by one skilled in the art, such as a statistically significant difference, or a difference that is of sufficient magnitude that, under the circumstances, can be detected with a reasonable level of reliability. In one example, an increase or decrease of 10% relative to a reference sample is a significant difference. In other examples, an increase or decrease of 20%, 30%, 40%, or 50% relative to the reference sample is considered a significant difference. In yet another example, an increase of two-fold relative to a reference sample is considered significant.

[0028] “Nucleotide sequence” refers to a heteropolymer of deoxyribonucleotides, ribonucleotides, or peptide-nucleic acid sequences that may be assembled from smaller fragments, isolated from larger fragments, or chemically synthesized de novo or partially synthesized by combining shorter oligonucleotide linkers, or from a series of oligonucleotides, to provide a sequence which is capable of expressing the encoded protein.

[0029] The term "primer," as used herein, means an oligonucleotide designed to flank a region of DNA to be amplified. In a primer pair, one primer is complementary to nucleotides present on the sense strand at one end of a polynucleotide fragment to be amplified and another primer is complementary to nucleotides present on the antisense strand at the other end of the polynucleotide fragment to be amplified. A primer can have at least about 11 nucleotides, and preferably, at least about 16 nucleotides and no more than about 35nucleotides. Typically, a primer has at least about 80% sequence identity, preferably at least about 90% sequence identity with a target polynucleotide to which the primer hybridizes.

[0030] As used herein, the term “probe” refers to an oligonucleotide, naturally or synthetically produced, via recombinant methods or by PCR amplification, that hybridizes to at least part of another oligonucleotide of interest. A probe can be single-stranded or double- stranded.

[0031] As used herein, the term “active fragment” refers to a substantial portion of an oligonucleotide that is capable of performing the same function of specifically hybridizing to a target polynucleotide.

[0032] As used herein, "hybridizes," "hybridizing," and "hybridization" means that the oligonucleotide forms a noncovalent interaction with the target DNA molecule under standard conditions. Standard hybridizing conditions are those conditions that allow an oligonucleotide probe or primer to hybridize to a target DNA molecule. Such conditions are readily determined for an oligonucleotide probe or primer and the target DNA molecule using techniques well known to those skilled in the art. The nucleotide sequence of a target polynucleotide is generally a sequence complementary to the oligonucleotide primer or probe. The hybridizing oligonucleotide may contain nonhybridizing nucleotides that do not interfere with forming the noncovalent interaction. The nonhybridizing nucleotides of an oligonucleotide primer or probe may be located at an end of the hybridizing oligonucleotide or within the hybridizing oligonucleotide. Thus, an oligonucleotide probe or primer does not have to be complementary to all the nucleotides of the target sequence as long as there is hybridization under standard hybridization conditions.

[0033] The term "complement" and "complementary" as used herein, refers to the ability of two nucleic acid molecules to base pair with each other. For example, in DNA, adenine (A) is complementary to thymine (T). In RNA, adenine (A) is complementary to uracil (U). In some embodiments, complementarity refers to an antisense compound that is capable of base pairing with its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity. Typically, two DNA molecules are complementary if they hybridize under the standard conditions referred to above. Typically, two DNA molecules are complementary if they have at least about 80% sequence identity, preferably at least about 90% sequence identity.

[0034] As used herein, "pharmaceutically acceptable carrier" or “excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil / water emulsion, and various types of wetting agents.

[0035] Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990).

[0036] As used herein, the term "preventing" refers to the prophylactic treatment of a patient in need thereof. The prophylactic treatment can be accomplished by providing an appropriate dose of a therapeutic agent or vaccine to a subject at risk of suffering from an ailment, thereby substantially averting onset of the ailment. It will be understood by those skilled in the art that it is not always possible to distinguish between "preventing" and "suppressing" since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, as used herein the term "prophylaxis" is intended as an element of "treatment" to encompass both "preventing" and "suppressing" as defined herein. The term "protection," as used herein, is meant to include "prophylaxis."

[0037] The term "effective amount" refers to that amount of a therapeutic agent or vaccine that is sufficient to effect treatment when administered to a subject in need of such treatment. The effective amount will vary depending upon the specific activity of the therapeutic agent being used, the severity of the patient's disease state, and the age, physical condition, existence of other disease states, and nutritional status of the patient. Additionally, other medication the patient may be receiving will affect the determination of the effective amount of the therapeutic agent to administer. As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects. In a typical embodiment, the subject is a human. In some embodiments, the subject is a llama, alpaca, dog, cat, horse, or monkey.

[0038] As used herein, “a” or “an” means at least one, unless clearly indicated otherwise.

[0039] Constructs and Compositions

[0040] Provided are nucleic acid constructs, as well as compositions comprising same, that can be used to elicit an immune response and provide protection against Valley Fever. In one embodiment, described herein is a nucleotide construct that expresses expression library immunization antigen 1 (ELI or ELI-Ag1), antigen 2 / proline-rich protein (PRA / Ag2), and / or peroxisomal matrix protein1 (PMP1) of Coccidioides posadasii. In some embodiments, the nucleotide construct expresses ELI, ARA / Ag2, and / or PMP1 of Coccidioides immitis. In some embodiments, the nucleotide construct is a single nucleotide construct expressing each of ELI, PRA / Ag2, and PMP1. In some embodiments, the nucleotide construct comprises multiple nucleotide constructs, each expressing one or more of these immunogens of Coccidioides posadasii and / or Coccidioides immitis. In some embodiments, the antigen has the amino acid sequence shown in SEQ ID NO: 4-6. In some embodiments, the antigen has a sequence with at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more sequence homology with a polypeptide selected from the group consisting of SEQ ID NOs: 4-6.

[0041] In another embodiment, described herein is a nucleotide construct that expresses one or more of the 8 antigens listed in Table 4 that were down-selected based on protection in mice (SEQ ID NOs: 11-26). In some embodiments, the antigen has the amino acid sequence shown in SEQ ID NO: 4-6. In some embodiments, the antigen has a sequence with at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more sequence homology with a polypeptide selected from the group consisting of SEQ ID NOs: 11-26. The construct can be designed for use in combination with the construct that expresses ELI, ARA / Ag2, and / or PMP1, either as part of a single multi-antigen construct, or as an additional multi-antigen construct capable of co-administration. In some embodiments, the nucleotide construct comprises multiple nucleotide constructs, each expressing one or more of these 8 additional immunogens of Coccidioides posadasii and / or Coccidioides immitis.

[0042] The nucleic acid constructs described herein as expressing one or more antigens comprise nucleic acid sequences engineered to express the amino acid sequence of the one or more antigens. In some embodiments, the construct comprises the sequence capable of antigen expression. In some embodiments, the construct further comprises additional sequence. Additional sequence can include, for example, linker or other sequence for optimized expression of one or a plurality of antigens. In some embodiments, the additional sequence can include sequence that enhances immunogenicity and / or provides additional antigens beyond those described herein. In some embodiments, the construct consists of the antigen expression sequence.

[0043] In some embodiments, the nucleotide is deoxyribonucleic acid (DNA). In some embodiments, the DNA is manufactured using cells or in vitro, including, but not limited to,enzymatic methods, such as rolling circle amplification-DNA (RCA-DNA) or doggy bone DNA (dbDNA). The latter, dbDNA, (Touchlight, Inc) employs an in vitro enzymatic technology, similar to RCA technology, but uses distinct enzymes (phi29 DNA polymerase and a protelomerase) to generate covalently closed, linear DNA constructs. Also contemplated is “minicircle” DNA, such as synDNA(TM) (CytoGenix), or other selectable marker free, bacterial ori-free expression cassettes useful for vaccination. In some embodiments, the nucleotide is ribonucleic acid (RNA), including, but not limited to, messenger (mRNA), transfer RNA (tRNA), or self-amplifying RNA (saRNA or repRNA). The engineering of tRNA vaccines is described, for example, by Wang et al., 2023, Emerg. Microbes Infect. 12(1):2157339.

[0044] In some embodiments, the construct further expresses interleukin 12 (IL-12) and heat-labile enterotoxin of Escherichia coli (LT). In some embodiments, the IL-12 and LT are provided on a separate, or second construct from the first construct expressing ELI, PRA / Ag2, and PMP1, and / or one or more of the additional 8 antigens described herein. In some embodiments, the LT comprises both A and B subunits. In some embodiments, the LT comprises the A subunit without the B subunit and in others, it comprises a double mutant LT (dmLT; see www.ncbi.nlm.nih.gov / pmc / articles / PMC3647992).

[0045] Also described herein is a composition comprising the nucleotide construct of any of the embodiments described above. In some embodiments, the composition comprises one or more additional constructs. In some embodiments, the additional constructs comprise a second nucleotide construct, wherein the second nucleotide construct expresses interleukin 12 (IL-12) and heat-labile enterotoxin of Escherichia coli (LT). In some embodiments, the additional constructs comprise separate constructs, each encoding IL-12 or LT. Exemplary sequences for LT are provided in SEQ ID NOs: 7 and 8. Exemplary murine sequences for IL-12 are provided in SEQ ID NOs: 9 and 10. Exemplary human IL-12 sequences are provided in SEQ ID NOs: 63 and 64.

[0046] In some embodiments, the second construct expressing IL-12 and LT is used separately to enhance T cell and mucosal immunity, independent of the first construct. In some embodiments, the second construct expressing IL-12 and / or LT is administered as an adjuvant together with a different immunogenic composition. In some embodiments, the second construct expresses IL-12, and a third construct expresses LT.

[0047] Methods

[0048] Provided herein is a method of eliciting an immune response to Coccidioides posadasii and Coccidioides immitis in a subject. Coccidioides posadasii and Coccidioides immitis infect humans and other animals, and immunogens from one of these strains willalso protect against the other (Campuzano A, et al. Vaccines (Basel).2024 Jan 9;12(1):67. doi: 10.3390 / vaccines 12010067. PMID: 38250880; PMCID: PMC10819930.) In some embodiments, the method is effective at eliciting an immune response to other fungal antigens sharing conserved regions. The method can also be used to prevent and / or treat fungal infections, such as Valley Fever.

[0049] In some embodiments, the method comprises administering to the subject a composition comprising one or more nucleic acid constructs as described herein. In some embodiments, the immune response comprises mucosal immunity, an antibody response, and / or a T cell response. In some embodiments, the T cell response comprises a systemic IFN-γ and / or Th17 T cell response. In some embodiments, the immune response protects the subject from lethality, fungal dissemination, and / or 15% weight loss within 30 days following a challenge with Coccidioides posadasii and / or Coccidioides immitis.

[0050] In some embodiments, the composition is administered by injection into the epidermis of the subject. In some embodiments, the composition is administered by intramuscular injection. Other routes of DNA or RNA vaccine delivery are known to those skilled in the art, including but not limited to, via mucosal, intradermal, intraperitoneal, intravenous administration. Examples of mucosal routes include, for example, intranasal, oral, inhaled, ocular, rectal or vaginal delivery. Those skilled in the art are aware of various methods of DNA or RNA delivery, including but not limited to, gene gun, electroporation, topical, micro needles, patches, jet injector. In some embodiments, the DNA or RNA is formulated with lipid-nanoparticles or lipid nano-carriers.

[0051] The amount to be administered is effective to elicit an immune response, such as a mucosal, antibody, or T cell response. The amount that is effective can be determined by various means, including, for example, performing an assay to detect and / or measure the level of markers of an immune response. The level of such markers can be compared to a known reference level or to a pretreatment level in the same or similar subjects. In some embodiments, the amount to be administered is a prophylactically and / or therapeutically effective amount. In some embodiments, the amount to be administered is protective against challenge with the relevant immunogen(s). In some embodiments, the amount is sufficient to induce IFN-γ and / or Th17 T cell responses in a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a veterinary subject. In some embodiments, the subject is a llama, alpaca, dog, cat, horse, or monkey. Dogs, llamas, alpacas, cats, horses and monkeys are particularly susceptible. Zoo animals and wildlife (e.g., coyotes, rodents, etc.) can also benefit from preventative protection. The subject may be at elevatedrisk of contracting Valley Fever. In some embodiments, the subject has already been exposed to Valley Fever.

[0052] Kits

[0053] Kits and articles of manufacture are also provided herein for use with one or more methods described herein. The kits can contain one or more of the constructs, compositions and / or one or more of the nucleic acid molecules described herein, such as the nucleic acid molecules encoding antigens identified as SEQ ID NOs: 4-26, or encoding polypeptides having a sequence at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more sequence homology with a polypeptide selected from the group consisting of SEQ ID NOs: 4-26. The kits can also contain nucleic acids that encode one or more of the polypeptides described herein. The kits can further contain adjuvants, reagents, and buffers necessary for the makeup and delivery of the vaccines.

[0054] The kits can also include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements, such as the immunogens and adjuvants, to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic.

[0055] The articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

[0056] A kit typically includes labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included. EXAMPLES

[0057] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.

[0058] Example 1: Trivalent DNA vaccine for Coccidioidomycosis delivered by gene gun induces robust antibody and T cell responses and affords complete protection from fungal dissemination following a high dose challenge with C. posadasii

[0059] IFN-γ and Th17 T cell responses are associated with control of Coccidioides infections indicating an effective vaccine will likely need to induce these responses. In addition, localization of these responses to the lung mucosa may provide better protection atthe site of exposure. Nucleic acid vaccines, including both DNA and RNA vaccines, induce robust CD4+ and CD8+ T cell responses including IFN-γ and Th-17 responses. Previous work showed that gene gun delivery of DNA vaccines formulated with potent genetic adjuvants into the skin can substantially increase these responses in the spleen and lung mucosa. This Example describes the immunogenicity and protective efficacy of gene gun delivered adjuvanted DNA vaccines and self-amplifying RNA vaccines (repRNA) vaccines encoding three known Coccidioides immunogens including the expression library immunization antigen 1 (ELI), the antigen 2 / proline-rich protein (PRA / Ag2), and peroxisomal matrix protein1 (PMP1).

[0060] DNA vaccines expressing these immunogens were administered separately or as a trivalent combination. The DNA vaccine immunogens were co-delivered with two genetic adjuvants, IL-12 and LT (A and B subunits) as a strategy to increase T cell responses and mucosal immune responses against these immunogens. The gene gun delivered adjuvanted DNA vaccines induced robust antibody responses as well as systemic and mucosal IFN-γ and Th17 responses in the spleen and lung against each immunogen. Following challenge, the PRA / Ag2 and trivalent DNA vaccines afforded 90 and 100% protection, respectively, from weight loss and mortality. In contrast, mice immunized with the separate PMP1 and ELI DNA vaccines and each repRNA vaccine exhibited significant weight loss and mortality that was not significantly different from the unvaccinated controls. Analysis of fungal burden showed the PRA / Ag2 and trivalent DNA vaccines both reduced fungal burden by an average of 1 log and a substantial 2.5 logs, respectively. Notably, CFU were undetectable in the spleen and brain in the trivalent DNA vaccine group whereas CFU were low but detectable in the PRA / Ag2 DNA vaccine group, a result that indicates synergistic effect in protection when the 3 immunogens are combined.

[0061] A second challenge study of bivalent combinations of the 3 immunogens showed that a bivalent DNA vaccine expressing only the PMP1+ ELI immunogens without PRA / Ag2 afforded 100% protection from a less virulent challenge providing strong evidence that co- delivery of the 3 immunogens provides a synergistic effect that likely contributes to the robust protective efficacy observed when delivered as a trivalent DNA vaccine. These studies also provide strong evidence supporting implementation of this trivalent DNA vaccine for protection from Coccidioides infections including C. Posadasii and C. Immitis.

[0062] Coccidioidomycosis, also known as Valley Fever (VF) impacts residents in arid regions of the world including southwestern US, areas in South and Central America and in Mexico. Annual incidences are rising overall with estimated increases in recent years of more than 200% in some areas. In addition, epidemiological studies indicate that the geographical range of VF is expanding and up to 17-29% of community-acquired pneumoniain these areas can be attributed to Coccidioides spp. infections. VF poses a significant and ongoing threat to human health, but to date, there is no safe and effective vaccine licensed for VF. As such, there is an urgent unmet need to develop a vaccine that can provide protection from the disease. Although to date, there are no vaccines for fungal diseases, evidence suggests an effective vaccine for VF should be possible. Individuals who recover from VF will generally have lifelong immunity against re-exposure. Furthermore, early studies employing live attenuated vaccines have demonstrated significant protection in mouse and nonhuman primate models of infection although due to the risk of severe reactogenicity and genetic reversion, a live attenuated vaccine for human use may not be a viable approach.

[0063] Nucleic acid vaccines, including both DNA and RNA vaccines, result in the intracellular expression of antigens, mimicking a live infection including induction of robust antibody and T cell responses, but without the risks associated with a live infection. Recent advances with both DNA and mRNA vaccine technologies have moved them to the forefront as one of the most effective vaccine strategies to induce protective immunity in humans, as evident by the current, highly efficacious licensed COVID-19 mRNA vaccines. IFN-γ and Th17 T cell responses are associated with control of Coccidioides infections, indicating an effective vaccine will likely need to induce these responses. In addition, localization of these responses to the lung mucosa may provide better protection at the site of exposure. Nucleic acid vaccines, including both DNA and RNA vaccines, induce robust CD4+ and CD8+ T cell responses including IFN-γ and Th-17 responses. It has been shown that gene gun delivery of DNA vaccines formulated with potent genetic adjuvants into the skin can substantially increase these responses in the spleen and lung mucosa. In this Example, the immunogenicity and protective efficacy of gene gun delivered adjuvanted DNA vaccines and self-amplifying RNA vaccines (repRNA) vaccines encoding known Coccidioides immunogens is demonstrated.

[0064] Monovalent DNA vaccines expressing Coccidioides genes induce robust antibody and T cell responses in mice

[0065] Self-amplifying replicon RNA (repRNA) and DNA vaccines expressing three Coccidioides immunogens including the expression library immunization antigen 1 (ELI), the antigen 2 / proline-rich protein (PRA / Ag2), and peroxisomal matrix protein1 (PMP1) were administered separately or as a trivalent combination. Each repRNA and DNA vaccine was delivered at 1mg doses directly into epidermal cells in the skin. To increase mucosal and Th1 T cell responses, the DNA vaccines were co-formulated with two plasmids expressing genetic adjuvants, the heat-labile enterotoxin from E. coli (LT) and IL-12. Mice were primed and boosted 4 weeks apart (Figure 1).

[0066] Three weeks after the boost, 5 mice per group were euthanized to analyze immune responses. The serum was analyzed for IgG antibodies against each immunogen by ELISA, and splenocytes and lung lymphocytes were stimulated with peptide pools representing each vaccine immunogen and then analyzed for IL-17 and IFN-γ T cell responses using a dual IFN-γ / IL-17 ELISPOT.

[0067] The gene gun delivered adjuvanted DNA vaccines induced robust antibody responses (Figure 2) as well as systemic and mucosal IFN-γ and Th17 responses in the spleen (Figure 3A) and lung (Figure 3B) against each immunogen. The PMP1 and the PRA / Ag2 antigens induced the strongest antibody responses. Notably, Th-17 responses were higher in the lung than in the spleen (Figures 3A & B). In contrast, the repRNA vaccines expressing the same immunogens induced low to undetectable T cell responses (Figure 3) and antibody responses. The gene gun delivered repRNA vaccines also induced T cell responses to each antigen, but the responses were considerably lower than in mice immunized with the DNA vaccines, and IgG responses were low to undetectable (Figure 3). The PMP1 antigen induced the highest IFN-γ systemic T cell responses (Figure 3A) and the PMP1 and PRA / Ag2 antigens both induced strong and comparable mucosal T cell responses (Figure 3B). Overall, the PMP1 antigen was the most immunogenic inducing robust antibody and systemic T cell responses as well as significant mucosal T cell responses in the lung. The PRA / Ag2 antigen also induced robust antibody responses. Systemic T cell responses induced by this antigen were notably lower than for PMP1 but both PRA / Ag2 and PMP1 induced comparable mucosal IFN-g and Th-17 T cell responses in the lung.

[0068] Analysis of repRNA expression of the three Coccidioides proteins was low. It’s possible that small antigens expressed by this platform may be outcompeted by the large expression cassette required for the repRNA platform. Additional work can determine the cause for the relatively low immunogenicity of the repRNA vaccines compared to the DNA vaccines and to optimize repRNA vaccine expression of these immunogens before concluding which nucleic acid vaccine platform (DNA or repRNA) is more immunogenic for Valley Fever immunogens.

[0069] The PRA / Ag2 and trivalent DNA vaccine afford significant protection from lethality and weight loss following a high dose challenge with C. Posadasii in mice.

[0070] Next the ability of the vaccines to protect from Coccidioides infection was evaluated in mice. Four groups of N=15 C57Bl / 6 mice / group were primed and boosted with DNA or repRNA vaccines expressing each antigen separately or in a trivalent DNA vaccine combination using LION (repRNA) or the gene gun (DNA or repRNA). For all DNA vaccines,a dual IL-12 / LT genetic adjuvant that increases mucosal and T cell responses was co- administered at a 10:1 vaccine-to-adjuvant ratio. For comparison, an additional group was primed and boosted with a live attenuated vaccine (TKO) previously shown to afford protection from C. posadasii (Hung CY, et al. Infect Immun.2014 Feb;82(2):903-13. doi: 10.1128 / IAI.01148-13. Epub 2013 Dec 9. PMID: 24478103; PMCID: PMC3911407; Narra HP, et al. Infect Immun.2016 Sep 19;84(10):3007-16. doi: 10.1128 / IAI.00633-16. PMID: 27481239; PMCID: PMC5038059.) and naïve controls received saline inoculations. Eight weeks after the booster dose, all vaccinated mice and the naïve control mice were intranasally challenged with a high dose (500 arthroconidia) of C. posadasii Silveira. The remaining N=10 mice per group were monitored for weight loss for 21 days to analyze protection from disease and to measure fungal dissemination into other tissues. Mice reaching 15% weight loss were euthanized. At day 21, all remaining mice were sacrificed the lungs, spleen and brain were collected to analyze for fungal burden.

[0071] Following the challenge, the PRA / Ag2 and trivalent DNA vaccines exhibited 90 and 100% protection from lethality (Figure 4A) and weight loss (Figure 4B), respectively. In contrast, mice immunized with the separate PMP1 and ELI DNA vaccines and the TKO live attenuated vaccine exhibited significant mortality (Figure 4A) and weight loss (Figure 4B) that was not significantly different from the unvaccinated controls. The nearly equal protection from the PRA / Ag2 immunogen alone suggests this immunogen likely contributed to most of the protection observed in the trivalent DNA vaccine.

[0072] The trivalent DNA vaccine but not the PRA / Ag2 DNA vaccine affords complete protection from fungal dissemination.

[0073] Analysis of fungal burden in the primary site of infection (i.e. lungs) in the DNA vaccine groups vs the TKO live attenuated vaccine and naïve controls showed the PRA / Ag2 and trivalent DNA vaccines reduced fungal burden by an average of 1 log and a substantial 2.5 logs, respectively (Figure 5). Notably, CFU were undetectable in the spleen and brain in the trivalent DNA vaccine group whereas CFU in spleen and brain were low but still detectable in the PRA / Ag2 DNA vaccine group. CFU in the mice immunized with the PMP1 and ELI-A1 This result shows the trivalent vaccine, but not the PRA / Ag2 monovalent vaccine, completely prevented dissemination of arthroconidia to other tissues since with the PRA / Ag2 vaccine, there was low but detectable CFU in the spleen and brain (Figure 5). Together, these data suggested an additive effect in protection when the immunogens were combined in the trivalent DNA vaccine that provided an advantage in protection from fungal dissemination when compared to each monovalent DNA vaccine. Together, these results indicated a synergistic effect in protection when the 3 immunogens are combined.

[0074] To determine if this synergistic effect could be achieved with a combination of only two immunogens, a 2nd challenge study was performed to investigate all possible bivalent combinations of the three immunogens. Three groups of N=15 mice were immunized with a prime and boost (4 weeks apart) of each bivalent DNA vaccine composition consisting of PRA / Ag2 + PMP1, PRA / Ag2 + ELI-A1 and PMP1 + ELI-A1. All bivalent DNA vaccines were co-delivered with the IL-12 / LT genetic adjuvant. Two additional groups of N=15 mice were immunized with a prime and boost of the TKO live attenuated vaccine and a delta-CPS1 live attenuated vaccine that was recently shown to afford significant protection in a canine challenge model (pubmed.ncbi.nlm.nih.gov / 34696935 / ). The live attenuated vaccines were administered as a prime and boost at the same timepoints as the DNA vaccine.

[0075] As shown in Figure 6, the challenge in this study resulted in significantly milder disease when compared to the first challenge study since lethality in the controls was less than 100%, suggesting the actual challenge dose administered may have been lower than in the first challenge study (Figure 4). Nevertheless, the results in Figure 6 show that all three bivalent combinations of the DNA vaccine afforded 100% protection from lethality and protection was significantly better than the controls and the two live attenuated vaccines. Notably, the bivalent combination consisting of only the PMP1+ ELI immunogens without the potent PRA / Ag2 antigen still afforded 100% protection (Figure 6) providing strong evidence that all 3 of these immunogens likely contribute to the robust protective efficacy observed with the trivalent DNA vaccine.

[0076] These results show that a novel trivalent DNA vaccine delivered by gene gun induced robust antibody and mucosal and systemic IFN-γ and Th-17 T cell responses against each Coccidioides antigens and afforded complete protection from fungal dissemination and disease in mice. Although the PRA-Ag1 immunogen alone afforded comparable protection from lethality and weight loss as the trivalent DNA vaccine, only the trivalent vaccine achieved complete protection from fungal dissemination and the greatest reduction in fungal burden in the lungs, a result that indicates all 3 immunogens likely contribute synergistically to the overall enhanced protection in this group that even exceeded protective efficacy of two live attenuated vaccines. Notably, all 3 bivalent combinations also afforded significant protection from lethality including the PMP1 + ELI-A1 combination that lacks the dominant PRA-Ag1. These results provide further evidence that all three antigens contributed to protection. These studies provide the first evidence that the combination of these three antigens can exert synergistic protection against lethality, disease and fungal dissemination. Previous studies of live attenuated vaccines for Valley Fever have shown cross-protection against C. immitis, another strain that causes Valley Fever in humans and animals, indicating that vaccines that afford protection from C. posadasii can cross-protectagainst C. immitis. These studies demonstrate feasibility of developing a DNA vaccine for Valley Fever.

[0077] Example 2: Identification of novel vaccine immunogens that protect from Coccidioides when expressed as nucleic acid vaccines

[0078] This Example describes a rapid nucleic acid-based vaccine strategy to identify novel protective immunogens. The Example makes use of an inclusive, unbiased approach to immunogen discovery using transcriptomic analyses of Coccidioides-infected mouse lungs, identifying 26 small, secreted proteins (SSPs) that play a role in pathogenesis of VF and could be effective immunogens to include in a vaccine. Additionally, this Example implements an approach to sequence T cell receptors and characterizes T cell clones and associated epitopes present in specimens isolated from VF patients as candidate T cell immunogens. The approach involves cross-referencing the T cell repertoire within a subset of 24 of the 26 SSPs. To determine if any of these 24 proteins could work as a protective vaccine antigen, a rapid nucleic acid vaccine-based screening strategy was developed to identify candidates that could contribute to protection. An overview of this approach is described below and shown in Figure 7.

[0079] First, DNA vaccines expressing each of the 24 antigens were constructed. Groups of mice were then immunized with a co-delivery of 3 antigens per group (8 groups total). Following a booster dose, the mice were then challenged with Coccidioides. Five of the 8 groups of mice exhibited some degree of protection (delayed or reduced mortality) relative to the unvaccinated controls.

[0080] The individual antigens within each of these groups were then further analyzed for their ability to (a) express the encoded antigen in vitro, (b) induce an antibody response, (c) include sequences matching Valley Fever T cell epitopes in human and / or nonhuman primates naturally infected with Valley Fever. From this analysis, 8 candidate antigens were identified for further study.

[0081] As a final step, one can then determine which of the individual antigens contribute to protection by immunizing 8 groups of mice with one of each of the 8 candidate antigens from step 5. From this final step, antigens that confer some degree of protection can be identified. These new antigens can then be employed in combination with the trivalent DNA vaccine encoding PRA-Ag2, PMP1 and ELI-1 to determine if protection can be further improved by inducing immune responses against more antigens.

[0082] Construction of Valley Fever DNA Vaccine Candidates

[0083] Native sequences of proteins expressed by Coccidiodes posadasii (CP) were retrieved from NCBI databases.

[0084] The methionine initiation codon of the native sequences was removed (initiation codon is native to vaccine plasmid UW7563 just upstream of Nhe1 site). To enable expression confirmation of CP proteins in a western blot a hexa-his tag sequence was added to the carboxy-terminal amino acid of the native protein, followed by two stop codons.

[0085] The modified protein sequences were codon-optimized with human biased codons to create coding sequences.

[0086] To facilitate cloning into the UW DNA vaccine plasmid (UW7563), a Nhe1 restriction enzyme sequence encoding the amino acids alanine-serine was added 5’, and a BglII site added 3’ to the coding sequences. The Nhe1-BglII flanked coding sequences were synthesized by a vendor and supplied in a plasmid.

[0087] UW7563 and plasmids with coding sequences were restricted with both Nhe1 and BglII, and appropriate DNA bands retrieved after agarose gel purification. UW7563 backbone and coding sequence inserts were ligated, transformed into e.coli, and colonies screened for recombinants.

[0088] Analysis of in vitro expression

[0089] Vaccine candidates were transfected into B16 cells. After 24 hours cells were lysed, and lysates western blotted using an anti-hexa His antibody as probe. To date, expression from 75% of vaccine candidate proteins has been confirmed.

[0090] The coding sequences of candidates that show protective efficacy will be resynthesized to remove the N-terminal Nhe1 site and C-terminal hexa-his tag and inserted in UW7563 for use in further experiments.

[0091] In vivo analysis of protective efficacy and immunogenicity

[0092] Each group of 6-week old C57B6 mice (N=10 per group) were primed at week 0 and boosted at week 4 with a co-delivery of 3 DNA vaccine plasmids each expressing a novel immunogen (.67 ug per DNA vaccine x 3 = 2.0 ug total DNA) + genetic adjuvant (IL-12 + LT, total 0.2 ug). A total of 8 groups of mice were each immunized with 3 DNA vaccines each. Two weeks after the booster dose, serum was collected and analyzed for antibody responses by ELISA using tagged recombinant protein representing each protein produced in E. coli. All mice, and an additional group of naïve controls were challenged 4 weeks after the booster dose with a high dose of Coccidioides posadasii (Silveira strain, challenge dose, approximately 700 conidia). Mice were evaluated daily for morbidity and mortality and thosereaching 20% weight loss were sacrificed. Protection (delayed and / or reduced mortality) was evaluated relative to the unvaccinated controls.

[0093] Analysis of cross-reactivity to human and NHP T cell epitopes

[0094] To confirm reactivity of vaccine antigens blood was collected from PTM with a history of VF (n=4) or from active human VF patients (n=2). Peripheral blood mononuclear cells were isolated and T cells were expanded non-specifically using anti-CD3 antibody and purified IL-2 and IL-15. After expansion for 7-10 days the cells were stimulated with overlapping antigen peptide pools using the Multiplexed Identification of T cell Receptor Antigen (MIRA) specificity scheme (Klinger M, et al. PLoS One.2015 Oct 28;10(10):e0141561). Activated cells were sorted based on cell type (Lymphocyte, Live, CD3+, CD4+) and activation (CD137+) and RNA was extracted from the sorted activated T cells. T cell receptor (TCR) sequences were amplified and sequenced. TCRs found in three of seven stimulating addresses according to the MIRA scheme, indicating antigen responding clonally expanded T cell clones, were identified and the associated stimulating antigen were identified.

[0095] Results

[0096] Eight groups of mice (N=5 per group) were immunized with a co-delivery of 3 DNA vaccines, each representing one of the novel 24 antigens in Table.1. Figure 2 shows that 5 of the 8 groups of mice exhibited delayed and / or reduced mortality when compared to the controls, a result that indicates that at least one of the 3 antigens induced immune responses that protected against Coccidioides. Each of the 3 antigens included in the 5 test groups that showed protection for in vitro expression (measured by Western blot) and induced antibody responses (measured by ELISA of serum collected from each mouse prior to challenge) was further characterized.

[0097] Table 1: Nucleic acid vaccine target protection and the associated patient reactivity. Pigtail macaques (PTM) had a history of VF or VF serology in the past. Two human valley fever (VF) patients and four PTM were screened for T cell re-activation (CD137 expression) after stimulation with peptide pools from indicated targets. Reactivity indicates that target antigens can generate an immune response in the PTM VF model or Human VF patients. Future analysis will screen more patients and identify the reactive epitopes.

[0098] The results in Table 1 show 8 candidate antigens from the protected groups that meet the criteria of a) being included in one of the groups exhibiting some protection and b) being expressed in vitro and / or immunogenic. The rationale for further selecting antigens form the protective groups based on either in vitro expression or immunogenicity, and not both, is that some proteins that are immunogenic may not be detected by the in vitro expression assay method and alternatively, some proteins that express in vitro may not induce antibody responses (but may induce T cell responses that are not measured in the rapid screening approach).

[0099] These 8 antigens can be tested in a second mouse study for the ability to afford protection in mice when delivered as individual DNA vaccines. Antigens exhibiting significant protection from this final analysis (delayed or reduced mortality) will be further characterizedfor immunogenicity, including the ability to induce T cell responses. The most protective antigens from this final screening step can then be compared to and evaluated in combination with the trivalent DNA vaccine encoding PRA-Ag2, PMP1 and ELI-1.

[0100] The trivalent DNA vaccine currently affords 100% protection from challenge with 500 conidia. To determine if additional antigens enhance protection, future experiments can evaluate protection from challenge with 1000 conidia. As shown in Figure 1, when challenged with this very high dose, the trivalent DNA vaccine affords < 100% protection. It is expected that incorporating one or more novel protective fungal antigens into the trivalent DNA vaccine will broaden immunity and increase protection against this higher dose challenge.

[0101] Table 2: List of 24 Candidate Antigens

[0102] Table 3: Antigen Sequences

[0103] Table 4: Eight Selected Antigens

[0104] Example 3: Vaccine adjuvant considerations

[0105] In Example 1, mouse IL-12 sequences were used for mouse experiments. It is understood to those skilled in the art that the IL-12 sequence would be selected based on the species of the subject to be treated. Thus, for a human vaccine, one would use human IL-12. Likewise, for a canine vaccine, canine IL-12 would be employed. The LT used in the study described in Example 1 included both the A and B subunits and it’s the same one used in Arrington J, et al. J Virol 76: doi / full / 10.1128 / jvi.76.9.4536-4546.2002. The LT could be substituted for subunits of this adjuvant such as LTA1 (A subunit only) or (dmLT (double mutant LT) see for example: Valli E, et al. PLoS One.2020 Jan 13;15(1):e0227047. doi: 10.1371 / journal.pone.0227047. The LT could also be substituted for CT as shown in Arrington J, et al. J Virol 76: doi.org / 10.1128 / jvi.76.9.4536-4546.2002. This would include similar versions as the LT, such as CTA1 (A subunit only) and other mutants that retain its adjuvant activity.

[0106] Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains.

[0107] Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

What is claimed is:

1. A nucleotide construct that expresses expression library immunization antigen 1 (ELI), antigen 2 / proline-rich protein (PRA / Ag2), and / or peroxisomal matrix protein1 (PMP1) of Coccidioides posadasii and Coccidioides immitis.

2. The nucleotide construct of claim 1 that further expresses one or more antigens selected from SEQ ID NOs: 11-26.

3. The nucleotide construct of claim 1 or 2, wherein the nucleotide is DNA.

4. The nucleotide construct of claim 1 or 2, wherein the nucleotide is messenger (mRNA), transfer RNA (tRNA), or self-amplifying RNA (saRNA or repRNA).

5. The construct of claim 1, 2, 3, or 4, wherein the construct further expresses interleukin 12 (IL-12) and heat-labile enterotoxin of Escherichia coli (LT).

6. A composition comprising the nucleotide construct of any one of claims 1 to 5.

7. The composition of claim 6, further comprising one or more additional nucleotide constructs, and wherein the additional nucleotide constructs express interleukin 12 (IL-12) and heat-labile enterotoxin of Escherichia coli (LT).

8. A method of eliciting an immune response to Coccidioides posadasii and Coccidioides immitis in a subject, the method comprising administering to the subject a composition of claim 6.

9. The method of claim 8, wherein the immune response comprises mucosal immunity, an antibody response, and / or a T cell response.

10. The method of claim 9, wherein the T cell response comprises a systemic and / or mucosal IFN-γ and / or Th17 T cell response.

11. The method of claim 8, wherein the immune response protects the subject from lethality, fungal dissemination, and / or 15% weight loss within 30 days following a challenge with Coccidioides posadasii and / or Coccidioides immitis.

12. The method of claim 8, wherein the immune response results in a reduction in primary infection site fungal burden 13. The method of claim 8, wherein the composition is administered by injection into the epidermis of the subject.

14. The method of claim 8, wherein the composition is administered by intramuscular injection.

15. The method of claim 8, wherein the composition is administered by mucosal (i.e. intranasal, oral, inhaled, ocular, rectal or vaginal), intradermal, intraperitoneal, or intravenous delivery.

16. The method of claim 8, wherein the administering is by gene gun, electroporation, topical, micro needles, patches, or jet injector, including DNA or RNA formulated with lipid- nanoparticles or lipid nano-carriers.

17. The method of any of the preceding claims, wherein the subject is human.

18. The method of any of the preceding claims, wherein the subject is a llama, alpaca, dog, cat, horse, or monkey.

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