Treatment and detection of infections and diseases associated with various fungal pathogens

Abstract

Immunogenic compositions comprising non-native pan-fungal Kex peptides and methods of using the compositions for the treatment or prevention of infection and / or disease associated with fungal pathogens (e.g., Pneumocystis, Aspergillus, Candida, or Cryptococcus) in a subject are described. Also provided are compositions and kits for detecting or quantifying the presence of antibodies to the non-native pan-fungal Kex peptides in a subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This international PCT application claims priority to and benefit of U.S. Provisional Patent Application No. 62 / 796,120, filed January 24, 2019, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] background Pathogenic fungal organisms are ubiquitous in the environment and can cause global health threats. Pathogenic fungi are usually not harmful to individuals with healthy and normally functioning immune systems, even after exposure, for example, by inhalation. However, individuals with weakened or impaired immune systems or individuals with pre-existing medical conditions, such as pulmonary disease or viral infections (e.g., HIV / AIDS infection), are at higher risk of experiencing serious health problems and adverse reactions after exposure to and infection by fungal pathogens.

[0003] Due to the serious impact of infection by fungal organisms on individuals with poor medical health and weakened immune systems, there is a continuing and urgent need for methods and compositions for treating or preventing infections and associated diseases caused by these pathogens.Especially in less affluent areas of the world, there is a need for cost-effective and efficient methods of treating and protecting against fungal pathogens.Effective methods and approaches for treating or preventing infections by fungal pathogens, for which there is currently no or insufficient adequate treatment, are particularly desirable in order to mitigate fungal pathogens that constantly threaten at-risk individuals worldwide. Summary of the Invention

[0004] overview Provided and described herein are methods for treating or preventing infections (e.g., opportunistic infections) by fungal pathogens, as well as methods for treating or preventing pulmonary disease and pulmonary dysfunction associated with infections by fungal pathogens.

[0005] In one aspect, Provided are immunogenic peptides, or polynucleotides encoding immunogenic peptides, having at least about 95% or more amino acid sequence identity to TIFF0007636331000001.tif32150, or a fragment thereof.

[0006] In another aspect, There is provided an immunogenic peptide, or a polynucleotide encoding the immunogenic peptide, comprising or consisting of TIFF0007636331000002.tif32150, or a fragment thereof.

[0007] In some embodiments, the immunogenic composition comprises an effective amount of the immunogenic peptide or the polynucleotide encoding the immunogenic peptide and a pharma- ceutically acceptable excipient.In some embodiments, the immunogenic composition further comprises an adjuvant.In one embodiment, the adjuvant is α-galactosylceramide (αGC).In one embodiment, the adjuvant is alum.

[0008] In another aspect, there is provided a method of eliciting an immune response in a subject, the method comprising administering to the subject a peptide, polynucleotide, or immunogenic composition as described above.

[0009] In another aspect, provided herein is a method for treating or protecting a subject against a fungal infection, comprising administering to the subject an immunogenic peptide, polynucleotide, or composition as described above.

[0010] Also provided herein is a method of treating or protecting a subject against a fungal infection, comprising administering to a subject an immunogenic peptide, polynucleotide, or composition of any of the preceding claims, an isolated antiserum comprising an antibody or antigen-binding fragment thereof that specifically binds to an immunogenic peptide of any of the preceding claims, or an isolated or purified antibody or antigen-binding fragment thereof that specifically binds to an immunogenic peptide of any of the preceding claims, in an amount effective to treat or protect the subject against a fungal infection. In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof.

[0011] In some embodiments of the methods disclosed herein, the fungal infection is associated with the genus Aspergillus, the genus Candida, and / or the genus Cryptococcus. In some embodiments, the method treats a pulmonary disease, pulmonary dysfunction, or a symptom thereof. In some embodiments, the pulmonary disease or pulmonary dysfunction is Pneumocystis pneumonia. In some embodiments, the pulmonary disease or pulmonary dysfunction is aspergillosis.

[0012] In another aspect, a method is provided for treating or protecting a subject against a fungal infection, comprising administering to the subject an immunogenic composition comprising SEQ ID NO:2 or a fragment thereof, or a polynucleotide encoding SEQ ID NO:2 or a fragment thereof, in an amount effective to induce an immune response against one or more of the fungal pathogens of the genera Pneumocystis, Aspergillus, Candida, or Cryptococcus.

[0013] In another aspect, a method of treating or protecting a subject against a fungal infection is provided, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof that specifically binds to SEQ ID NO: 2. In some embodiments, the antibody or antigen-binding fragment thereof is present in or isolated from antisera derived from a donor subject. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a Kex peptide of Pneumocystis, Aspergillus, Candida, or Cryptococcus.

[0014] In another aspect, a method is provided for eliciting an immune response in a subject, comprising administering to the subject a wild-type Kex peptide or a polynucleotide encoding same, and alpha-galactosylceramide (α-GC).

[0015] In yet another aspect, a method is provided for treating or protecting a subject against a fungal infection, comprising administering to the subject a wild-type Kex peptide or a polynucleotide encoding the same, and alpha-galactosylceramide (α-GC). In some embodiments of the method, the fungal infection is associated with Aspergillus, Candida, and / or Cryptococcus. In some embodiments of the method, or of the method for inducing an immune response in a subject, comprising administering to the subject a wild-type Kex peptide or a polynucleotide encoding the same, and α-GC, the method treats a pulmonary disease, pulmonary dysfunction, or a symptom thereof.

[0016] In another aspect, a method for treating or protecting a subject against a fungal infection is provided, comprising administering to a subject an immunogenic composition comprising a wild-type Kex peptide or a fragment thereof, or a polynucleotide encoding the same, and α-galactosylceramide (αGC) in an amount effective to induce an immune response against one or more fungal pathogens of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus.

[0017] In yet another aspect, a method is provided for treating or protecting a subject against infection by a fungal pathogen of the genus Aspergillus or Pneumocystis, comprising administering to the subject an immunogenic composition comprising an effective amount of an immune response to the fungal pathogen of the genus Pneumocystis, the immunogenic composition comprising a Kex peptide (pan-fungal peptide 2) of SEQ ID NO: 2 or a fragment thereof, or a polynucleotide encoding the pan-fungal peptide 2 peptide or a fragment thereof. In one embodiment of the method, the subject is also administered an adjuvant. In one embodiment, the adjuvant is alpha-galactosylceramide (α-GC). In another embodiment, the adjuvant is alum. In an embodiment of the method, the induced immune response treats or protects the subject against a pulmonary disease or pulmonary dysfunction and / or symptoms thereof, or against an Aspergillus-related disease and / or symptoms thereof. In one embodiment of the method, the subject has an immunodeficiency disease or acquired immune deficiency disease syndrome, such as HIV, AIDS, or SIV. In particular embodiments, the pulmonary disease is Pneumocystis pneumonia (PCP) and / or symptoms thereof and the Aspergillus-associated disease is aspergillosis or invasive pulmonary aspergillosis (IPA) and / or symptoms thereof.

[0018] In some embodiments of any of the methods disclosed herein, the subject is a mammal, and in some embodiments, the subject is a human.In some embodiments, the subject is immunocompromised or immunosuppressed.In some embodiments, the immunocompromised or immunosuppressed subject is treated for cancer, immunodeficiency disease, congenital disease, or autoimmune disease.In some embodiments, the immunocompromised or immunosuppressed subject is a pre-transplant or post-transplant subject.In some embodiments, the immunocompromised or immunosuppressed subject is treated for HIV, rheumatoid arthritis, or psoriasis.

[0019] Other features and advantages of the described embodiments will become apparent from the detailed description, and from the claims.

[0020] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.The following references provide those skilled in the art with the general definitions of many terms used in the embodiments described herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified.

[0021] By "Kex1" or "kexin" or "KEX" protein is meant a polypeptide or peptide fragment thereof having at least about 85% or more amino acid identity to the amino acid sequence provided in GenBank Accession No. EU918304.1, NCBI Accession No. XM_746441.1, GenBank Accession No. AF022372.1, or NCBI Accession No. XM_572303.1, and having immunogenic activity. In one embodiment, the Kex peptide is an antigenically stable active site peptide sequence. (Kutty, G. and Kovacs, JA, 2003, Infect. Immun., 71(1):571-574; Lee. LH et al., 2000, Gene, 242(1-2):141-150; and Russian, DA et al., 1999, Proc. Assoc. Am. Physicians, 111(4):347-356). In one embodiment, the Kex peptide is a fragment of a naturally occurring kexin protein or a non-naturally occurring pan-fungal peptide or fragment thereof.

[0022] "Pan-fungal peptide 1" refers to a KEX peptide that includes a sequence that has 95, 96, 97, 98, 99, or 100% identity to the amino acid sequence of pan-fungal peptide 1 and has immunogenic activity. In some embodiments, pan-fungal peptide 1 includes one, two, or three additional amino acids at the carboxy and / or amino termini of the peptide, which do not alter the ability of the peptide to stimulate an immune response in a subject. The sequence of pan-fungal peptide 1 is as follows: TIFF0007636331000003.tif18137

[0023] "Pan-fungal peptide 2" refers to a KEX peptide that contains a sequence that has 95, 96, 97, 98, or 99% identity to pan-fungal peptide 2 and has immunogenic activity. In some embodiments, pan-fungal peptide 2 contains one, two, or three additional amino acids at the carboxy and / or amino termini that do not alter the immunogenicity of the peptide. The sequence of pan-fungal peptide 2 is as follows: TIFF0007636331000004.tif18138

[0024] An exemplary Pneumocystis Kex1 polypeptide fragment isolated from a Pneumocystis colonized non-human primate (cynomolgus macaque) and having GenBank Accession No. EU918304.1 is provided below: TIFF0007636331000005.tif8149

[0025] An exemplary polynucleotide sequence encoding a Kex1 polypeptide fragment provided in GenBank Accession No. EU918304.1 is provided below: TIFF0007636331000006.tif24138

[0026] An exemplary Kex (KexB endoprotease) polypeptide sequence from Aspergillus fumigatus (Af293), having NCBI accession number XM_746441.1, is provided below: TIFF0007636331000007.tif75130

[0027] An exemplary Kex (Kex2 proteinase) polypeptide sequence from Candida albicans, having GenBank Accession No. AF022372.1, is provided below: TIFF0007636331000008.tif81128

[0028] An exemplary Kex polypeptide sequence for Cryptococcus neoformans (JEC21), having NCBI accession number XM_572303.1, is provided below: TIFF0007636331000009.tif81128

[0029] By "agent" is meant a peptide, a nucleic acid molecule, or a small chemical compound.

[0030] By "ameliorate" is meant to lessen, inhibit, attenuate, decrease, arrest, or stabilize the onset or progression of a disease.

[0031] By "alteration" is meant a change (increase or decrease) in the expression level or activity of a gene or polypeptide as detected by standard methods known in the art (e.g., those described herein). As used herein, alteration includes a 10% change in expression levels, preferably a 25% change in expression levels, more preferably a 40% change, and most preferably a 50% or greater change.

[0032] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. Methods of preparing antibodies are well known to those skilled in the science of immunology. Antibodies can be intact immunoglobulins from natural sources or from recombinant sources, or can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Tetramers may be naturally occurring or reconstituted from single chain antibodies or antibody fragments. Antibodies also include dimers that may be naturally occurring or constructed from single chain antibodies or antibody fragments. Antibodies of the described embodiments include, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab') 2Antibodies can exist in a variety of forms, including fragments, as well as single chain antibodies (scFv), humanized antibodies, and human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0033] The term "antibody fragment" refers to a portion of an intact antibody, and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, VFv antibodies that exhibit sufficient affinity for a target. L or V H Examples of antibodies that can be used include, but are not limited to, single domain antibodies, such as camelid antibodies (Riechmann, 1999, Journal of Immunological Methods 231:25-38), which are composed of either a single domain or a single antibody, as well as multispecific antibodies formed from antibody fragments. Antibody fragments also include human or humanized antibodies, or portions of human or humanized antibodies.

[0034] Antibodies can be produced by any of the methods known in the art that utilize a polypeptide (e.g., a kexin polypeptide) or an immunogenic peptide fragment thereof as an immunogen. One method of obtaining antibodies is to immunize a suitable host animal with the immunogen and follow standard procedures for polyclonal or monoclonal antibody production. The immunogen facilitates the display of the immunogenic fragment on the cell surface. Immunization of a suitable host can be carried out in several ways. A nucleic acid sequence encoding the polypeptide described herein, or an immunogenic fragment thereof, can be provided to the host in a delivery vehicle that is taken up by the host's immune cells. The cells then express a receptor on the cell surface, generating an immunogenic response in the host. Alternatively, a nucleic acid sequence encoding the polypeptide or an immunogenic fragment thereof can be expressed in a cell in vitro, followed by isolation of the polypeptide and administration of the polypeptide to a suitable host where antibodies are produced.

[0035] Alternatively, antibodies to the polypeptide may be derived from an antibody phage display library, if desired. Bacteriophage are capable of infecting and propagating within bacteria, which, when combined with human antibody genes, can be engineered to display human antibody proteins. Phage display is the process by which phages are made to "display" human antibody proteins on their surface. Genes from a human antibody gene library are inserted into a population of phages. Each phage carries genes for a different antibody and therefore displays a different antibody on its surface.

[0036] The produced antibodies can then be purified from the host by any method known in the art. Antibody purification methods can include salt precipitation (e.g., with ammonium sulfate), ion exchange chromatography (e.g., on a cation or anion exchange column, preferably performed at neutral pH and eluted with a step gradient of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as Protein A, Protein G, hydroxyapatite, and anti-immunoglobulin.

[0037] Antibodies can be conveniently produced from hybridoma cells engineered to express the antibody. Methods for making hybridomas are well known in the art. Hybridoma cells can be cultured in a suitable medium, and spent medium can be used as a source of antibody. Polynucleotides encoding the antibody of interest can then be obtained from the hybridoma producing the antibody, and the antibody can then be produced synthetically or recombinantly from these DNA sequences. To produce large amounts of antibody, it is generally more convenient to obtain ascites fluid. Methods for producing ascites fluid generally include injecting hybridoma cells into immunologically naive histocompatible or immunotolerant mammals, particularly mice. The mammal can be primed for ascites fluid production by prior administration of a suitable composition (e.g., pristane).

[0038] By "anti-kexin antibody," "anti-Kex antibody," or "anti-Kex1 antibody" is meant an antibody or antigen-binding fragment thereof that selectively binds to a kexin polypeptide or a peptide fragment thereof, including, for example, the Kex1 peptide fragments of fungal pathogens such as Candida albicans, Pneumocystis hominis, Pneumocystis jirovecii (also known as carinii), Aspergillus fumigatus, and Cryptococcus neoformans, as described herein. In various embodiments, an anti-kexin antibody or anti-Kex1 antibody specifically binds to a binding site of a kexin protein or peptide. In certain embodiments, the anti-kexin or anti-Kex1 antibody specifically binds to a binding site of one or more kexin proteins or peptides of fungal pathogens, such as fungal pathogens of the genera Candida, Pneumocystis, Aspergillus, and / or Cryptococcus.

[0039] "Antisera" refers to serum containing one or more antibodies against a particular antigen. Antisera containing antibodies can be obtained from the blood or serum of an animal (mammal), such as a human, that has been immunized or inoculated with an immunogen (or antigenic material), typically by injection into the bloodstream or tissue, or by infection. In one embodiment, the animal (mammal), such as a human, can be immunized or inoculated with the blood or serum of an organism or individual whose immune system has been stimulated to produce an immune response (e.g., antibody production) by natural contact or infection with an antigenic material or immunogen. In this case, the antisera contains a population of anti-Kex peptide antibodies, e.g., polyclonal or monoclonal antibodies, generated or produced by a donor subject that has been immunized, inoculated, or exposed to a Kex peptide immunogen, or a polynucleotide encoding the Kex peptide immunogen, from a fungal pathogen, e.g., Pneumocystis jiroveci. Such antisera, isolated (and / or purified) from a donor subject, are used to immunize (i.e., administer to) another (unrelated) subject to provide immunity (acquired immunity) against infections or diseases caused by or associated with not only Pneumocystis pathogens as the original source of immunogen, but also other fungal pathogens that have Kex peptides similarly targeted and recognized by the antibodies in the antisera. In embodiments, the fungal pathogens include Pneumocystis spp. and one or more of Candida spp. or Candida albicans, Aspergillus spp. or Aspergillus fumigatus, or Cryptococcus spp. or Cryptococcus neoformans. In this manner, a subject receiving the antisera, i.e., the antibodies in the antisera, is treated or protected against infections and / or diseases caused by more than one fungal pathogen. Such antisera-derived immune protection against multiple fungal pathogens constitutes the adaptive or passive immunity acquired by the recipient subject conferred from the isolated antisera of the donor subject. As will be understood by those skilled in the art, serum is the amber-colored, protein-rich liquid component of blood that separates from the clot when blood clots.A serum component containing one or more antibodies (cross-protective antibodies) is called an "antiserum". In one embodiment, the antiserum is an isolated antiserum, e.g., isolated from a donor subject. In one embodiment, the isolated antiserum may be processed by methods used by those skilled in the art, e.g., by dilution, concentration (e.g., by filtration or centrifugation or both), chromatography, purification to remove ions or extraneous proteins, etc., prior to its use as a therapeutic or protective therapy as described herein. In one embodiment, the isolated antiserum may be further purified after isolation. In one embodiment, the isolated antiserum is not further processed or purified. In one embodiment, the antibody or antigen-binding fragment thereof contained in the isolated antiserum may be further isolated by methods practiced by those skilled in the art, e.g., but not limited to, affinity chromatography, size exclusion chromatography, immunoprecipitation, dialysis, HPLC chromatography, etc.

[0040] "Biological sample" refers to any fluid, cell, or tissue obtained from a subject. In some embodiments, the biological sample is blood, serum, plasma, cerebrospinal fluid, bronchoalveolar lavage fluid, sputum, tears, saliva, urine, semen, feces, etc.

[0041] In this disclosure, "comprises," "comprising," "containing," "having," and the like can have the meaning ascribed to them under U.S. Patent Law and can mean "includes," "including," and the like; "consisting essentially of" or "consisting essentially of" likewise has the meaning under U.S. Patent Law, where the term is open-ended and permits the presence of more than what is recited so long as the basic or novel characteristics of what is recited are not altered by the presence of more than what is recited, but rather it excludes aspects of the prior art.

[0042] "Detecting" refers to identifying the presence, absence, or amount of the analyte being detected or to be detected.

[0043] "Disease" refers to any condition, dysfunction, or disorder that damages or interferes with the normal function of cells, tissues, or organs.In various embodiments, the disease is a lung disease or brain disease, such as meningitis.Non-limiting examples of lung diseases include chronic obstructive pulmonary disease (COPD), which is a progressive lung disease including emphysema, chronic bronchitis, refractory (irreversible) asthma, pneumonia (e.g., Pneumocystis pneumonia), and some forms of bronchiectasis.

[0044] By "effective amount" is meant the amount required to improve the symptoms of a disease compared to an untreated patient. The effective amount of active compound used to practice the methods described herein for therapeutic treatment of a disease will vary depending on the method of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosing regimen. Such an amount is referred to as an "effective" amount. An immunologically effective amount of the isolated antisera described herein is the amount required to treat a fungal infection or disease associated with one or more of the fungal pathogens described herein. By way of example, an effective amount of the isolated antisera can be determined by measuring the amount or titer of antibodies against the desired immunogen present in the serum by methods known and practiced in the art. Typical dosage ranges for passive immunotherapy (i.e., administration of antisera containing antibodies) include from about 0.3 mg to about 100 mg / kg total body weight. Passive immunotherapy is usually followed by a cure, as individual patients respond differently to the therapy. Dosage adjustments can be altered as needed. The treatment regimen can be determined by methods known and practiced by those of skill in the art. In one embodiment, the amount is sufficient to induce an immune response.

[0045] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. The portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0046] By "genetic vaccine" is meant an immunogenic composition that includes a polynucleotide encoding an antigen.

[0047] "Immune response" refers to any activity of the immune system that is directed against an antigen. In some embodiments, the immune response is an innate or adaptive immune response that protects a subject from infection by a pathogen (e.g., a fungal pathogen) or treats a pathogen infection. In some embodiments, the immune response involves the production of antibodies against an antigen.

[0048] The term "immunocompromised" refers to a subject having a weakened or impaired immune system and / or immune response associated with a pathogen, pathogenic antigen, disease, etc. A subject may be immunocompromised as a result of taking immunosuppressive drugs or by suffering from a disease or pathological condition that affects the subject's immune system, such as a certain congenital disease. The term "immunosuppressed" refers to a subject whose immune system and associated immune response to a pathogen, pathogenic antigen, disease, etc. is partially or completely suppressed, for example, by a reduction in the activity or efficiency of the immune system. Immunosuppression of a subject's immune system or immune response may occur naturally due to a disease or disorder in the subject, or may be induced in the subject by administration of an immunosuppressant, drug, e.g., an anti-cancer drug, compound, etc. In some cases, a subject who is or has been immunosuppressed, or who has a weakened immune system due to a disease or condition (e.g., chemotherapy or an immunodeficiency disease), is said to be immunocompromised.

[0049] By "immunogenic composition" is meant a composition comprising an antigen or immunogen, or a polynucleotide encoding an antigen or immunogen, which elicits an immune response in an immunized subject.

[0050] The terms "isolated," "purified," or "biologically pure" refer to a material that is more or less free from components that are normally associated with it as found in its natural state or environment. "Isolated" refers to some degree of separation from the original source or surroundings. "Purified" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free from other materials such that any impurities do not substantially affect the biological properties of the protein or cause other deleterious consequences. That is, a nucleic acid or peptide described herein is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In the case of proteins that can undergo modifications, such as phosphorylation or glycosylation, different modifications can give rise to different isolated proteins that can be purified separately.

[0051] "Isolated polynucleotide" refers to a nucleic acid (e.g., DNA) that does not contain the genes adjacent to the gene in the naturally occurring genome of the organism from which the nucleic acid molecule is derived.Thus, this term includes, for example, recombinant DNA that is incorporated into a vector; recombinant DNA that is incorporated into an autonomously replicating plasmid or virus; or recombinant DNA that is incorporated into the genomic DNA of a prokaryotic or eukaryotic organism; or that exists as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion).In addition, this term includes RNA molecules that are transcribed from DNA molecules, as well as recombinant DNA that is part of a hybrid gene that codes for additional polypeptide sequences.

[0052] "Isolated polypeptide" or "isolated peptide" refers to a polypeptide or peptide that is separated from naturally associated components. Typically, a polypeptide is isolated when it is at least 60% by weight free from the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight, of the polypeptide or peptide described herein. An isolated polypeptide or peptide described herein may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, such as column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0053] As used herein, "obtaining" in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.

[0054] "Opportunistic infection" refers to an infection caused by a pathogen, such as a fungal pathogen, a bacterium, a virus, a protozoan, or a parasite, that takes advantage of an opportunity to infect a normally unavailable subject (host), such as a host with a weakened immune system, an immunocompromised host, an immunosuppressed host, a host with an altered microbiota or bacterial flora, or a host with a damaged or compromised protective integumentary barrier. In one embodiment, the opportunistic infection is caused by one or more fungal pathogens described herein.

[0055] By "reduce" or "decrease" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0056] "Reference" refers to a standard or control state. "Reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a particular sequence; for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides or about 300 nucleotides or any integer therebetween.

[0057] By "specifically binds" is meant a compound or antibody or antigen-binding fragment thereof that recognizes and binds to a polypeptide or peptide but does not substantially recognize and bind to other molecules in a sample, such as a biological sample that naturally contains the polypeptides or peptides described herein. Cross-reactive binding includes not only specific binding (e.g., by an antibody or antigen-binding fragment thereof) to the original polypeptide or peptide antigen / immunogen, but also binding to polypeptides or peptides other than the original antigen / immunogen.

[0058] Nucleic acid molecules useful for generating recombinant immunogens or vaccines include any nucleic acid molecule that encodes a polypeptide or peptide fragment thereof, such as the Kex wild-type or pan-fungal polypeptide or Kex wild-type or pan-fungal peptide described herein. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantial identity to the endogenous sequence. A polynucleotide that has "substantial identity" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. The nucleic acid molecule may include any nucleic acid molecule that encodes a polypeptide or peptide fragment thereof. A polynucleotide that has "substantial identity" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to a pair that forms a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof under various stringency conditions. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507.)

[0059] By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, more preferably 90%, 95% or even 99% identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0060] Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach for determining the degree of identity, e -3 and e -100 The BLAST program may be used, in which a probability score between indicates closely related sequences.

[0061] "Subject" refers to a mammal, including but not limited to a human or non-human mammal, such as a non-human primate, or a murine, bovine, equine, canine, ovine, or feline. In one embodiment, the subject is a human. In one embodiment, the subject is a human patient undergoing treatment for an infection or disease caused by one or more pathogenic fungi, such as Pneumocystis, Aspergillus, Candida, or Cryptococcus. In one embodiment, the subject is a human patient at risk for an infection (e.g., an opportunistic infection) or disease caused by one or more pathogenic fungi, such as Pneumocystis, Aspergillus, Candida, or Cryptococcus. In one embodiment, the subject is a mammalian (e.g., human; non-human primate) donor subject from which antisera containing antifungal Kex peptide antibodies are obtained or isolated. In one embodiment, the subject is a mammalian (eg, human; non-human primate) recipient subject who receives the isolated antisera and acquires protective immunity (and treatment) against multiple fungal pathogens.

[0062] As used herein, "vector" refers to a nucleic acid (polynucleotide) molecule into which a foreign nucleic acid can be inserted without interfering with the vector's ability to replicate and / or integrate into a host cell. A vector can contain a nucleic acid sequence that allows replication in a host cell, such as an origin of replication. An insertion vector can insert itself into a host nucleic acid. A vector can also contain one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of a gene inserted into a host cell. Those skilled in the art will recognize that depending on the expression vector used, it may be necessary to add additional nucleotides to the 5' end of the foreign nucleic acid inserted into the vector to maintain the proper reading frame.

[0063] "Vaccine" refers to a preparation of immunogenic material (e.g., protein or nucleic acid; vaccine) that can stimulate (elicit) an immune response and is administered to a subject to treat a disease, condition, or pathology, such as an infectious disease (e.g., caused by dengue virus infection), or to prevent a disease, condition, or pathology. The immunogenic material may include, for example, an attenuated or killed microorganism (such as an attenuated virus), or an antigenic protein, peptide, or DNA derived from such a microorganism. Vaccines may elicit a preventative immune response in a subject; they may also elicit a therapeutic immune response in a subject. As noted above, the method of vaccine administration varies from vaccine to vaccine and may include routes or means, such as inoculation (intravenous or subcutaneous injection), ingestion, inhalation, or other forms of administration. Inoculation can be delivered by any number of routes, including parenterally, such as intravenously, subcutaneously, or intramuscularly. Vaccines may be administered with adjuvants to enhance the immune response.

[0064] It is understood that the ranges provided herein are shorthand for all values ​​within the range.For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting 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, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0065] As used herein, the terms "treat," "treatment," and the like refer to reducing, alleviating, attenuating, or improving a disease, disorder, and / or its associated symptoms. Of course, but not excluded, treating a disease, disorder, and / or its associated symptoms does not require that the disease, disorder, condition, or its associated symptoms be eliminated.

[0066] As used herein, a therapeutic agent that "prevents" a disorder or condition refers to a compound or agent that reduces the occurrence of a disorder or condition in a statistical sample in a treated sample compared to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of a disorder or condition compared to an untreated control sample.In one embodiment, the prophylactic therapeutic agent is an antibody or an antigen-binding fragment thereof.In a particular embodiment, the prophylactic therapeutic agent is an isolated antiserum that contains an anti-Kex peptide antibody or an antigen-binding fragment thereof as described herein.

[0067] As used herein, unless otherwise stated or clear from context, the term "or" is understood to be inclusive. As used herein, unless otherwise stated or clear from context, the terms "a," "an," and "the" are understood to be singular or plural.

[0068] As used herein, unless otherwise specified or clear from the context, the term "about" is understood to be within the normal tolerance in the art, for example, within 2 standard deviations of the mean.About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0069] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein. [The present invention 1001] TIFF0007636331000010.tif31150 or a fragment thereof, having at least about 95% or more amino acid sequence identity thereto, or a polynucleotide encoding an immunogenic peptide. [The present invention 1002] TIFF0007636331000011.tif32150 or a fragment thereof, comprising or consisting of an immunogenic peptide, or a polynucleotide encoding an immunogenic peptide. [The present invention 1003] An immunogenic composition comprising an effective amount of a peptide of the present invention 1001 or 1002 or a polynucleotide encoding the peptide, and a pharma- ceutically acceptable carrier or excipient. [The present invention 1004] The immunogenic composition of the present invention 1003, further comprising an adjuvant. [The present invention 1005] The immunogenic composition of the present invention 1004, wherein said adjuvant is α-galactosylceramide (αGC). [The present invention 1006] A method for inducing an immune response in a subject, the method comprising administering to the subject any of the peptides, polynucleotides, or immunogenic compositions of the present inventions 1001 to 1005. [The present invention 1007] A method for treating or protecting a subject against a fungal infection, comprising administering to the subject any of the immunogenic peptides, polynucleotides, or compositions of the present invention 1001 to 1005. [The present invention 1008] A method for treating or protecting a subject against a fungal infection, the method comprising the step of administering to the subject an immunogenic peptide, polynucleotide, or composition of any of claims 1001-1005 of the present invention; an isolated antiserum comprising an antibody or antigen-binding fragment thereof that specifically binds to an immunogenic peptide of any of claims 1001-1005 of the present invention; or an isolated or purified antibody or antigen-binding fragment thereof that specifically binds to an immunogenic peptide of any of claims 1001-1005 of the present invention, in an amount effective to treat or protect the subject against the fungal infection. [The present invention 1009] The method of claim 1008, wherein said antibody is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof. [The present invention 1010] The method of any of claims 1007 to 1009, wherein said fungal infection is associated with an Aspergillus, Candida, Cryptococcus, or Pneumocystis infection. [The present invention 1011] The method according to any one of claims 1007 to 1009, for treating a pulmonary disease, pulmonary dysfunction, or a symptom thereof. [The present invention 1012] The method of claim 1011, wherein said lung disease or pulmonary dysfunction is Pneumocystis pneumonia (PCP). [The present invention 1013] The method of claim 1011, wherein said lung disease or pulmonary dysfunction is aspergillosis or invasive pulmonary aspergillosis (IPA). [The present invention 1014] A method for treating or protecting a subject against a fungal infection, comprising administering to the subject an immunogenic composition comprising SEQ ID NO:2 or a fragment thereof, or a polynucleotide encoding SEQ ID NO:2 or a fragment thereof, in an amount effective to induce an immune response against one or more fungal pathogens of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus. [The present invention 1015] A method for treating or protecting a subject against a fungal infection, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof that specifically binds to SEQ ID NO:2. [The present invention 1016] The method of claim 10, wherein said antibody or fragment thereof is present in or isolated from antisera derived from a donor subject. [The present invention 1017] The method of any one of claims 1015 to 1016, wherein the antibody or antigen-binding fragment thereof specifically binds to a Kex peptide of Pneumocystis, Aspergillus, Candida, or Cryptococcus. [The present invention 1018] The method according to any one of claims 1006 to 1017, wherein the subject is a mammal. [The present invention 1019] The method of claim 1018, wherein the subject is a human. [The present invention 1020] The method according to any one of claims 1006 to 1019, wherein the subject is in an immunodeficient state or an immunosuppressed state. [The present invention 1021] The method of claim 1020, wherein said immunocompromised or immunosuppressed subject is a pre-transplant subject or a post-transplant subject. [The present invention 1022] The method of claim 1020, wherein the immunocompromised or immunosuppressed subject is being treated for rheumatoid arthritis or psoriasis. [The present invention 1023] A method for inducing an immune response in a subject, comprising administering to the subject a wild-type Kex peptide or a polynucleotide encoding the wild-type Kex peptide and alpha-galactosylceramide (α-GC). [The present invention 1024] A method for treating or protecting a subject against a fungal infection, comprising administering to the subject a wild-type Kex peptide or a polynucleotide encoding the wild-type Kex peptide and alpha-galactosylceramide (α-GC). [The present invention 1025] The method of claim 1024, wherein said fungal infection is associated with or caused by an infection with Aspergillus, Candida, and / or Cryptococcus species. [The present invention 1026] The method of any one of claims 1023 to 1025, for treating a lung disease, pulmonary dysfunction, invasive pulmonary aspergillosis (IPA), or a symptom thereof. [The present invention 1027] The wild-type Kex peptide TIFF0007636331000012.tif11148 Any of the methods of 1023 to 1026 of the present invention, comprising: [The present invention 1028] A method for treating or protecting a subject against a fungal infection, comprising administering to the subject an immunogenic composition comprising a wild-type Kex peptide or a fragment thereof, or a polynucleotide encoding the wild-type Kex peptide, and alpha-galactosylceramide (α-GC), in an amount effective to induce an immune response against one or more fungal pathogens of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus. [The present invention 1029] A method for treating or protecting a subject against infection by a fungal pathogen of the genus Aspergillus or Pneumocystis, comprising administering to the subject an immunogenic composition comprising a Kex peptide of SEQ ID NO: 2 (pan-fungal peptide 2) or a fragment thereof, or a polynucleotide encoding the pan-fungal peptide 2 peptide or a fragment thereof, in an amount effective to induce an immune response against fungal pathogens of the genus Pneumocystis and Aspergillus. [The present invention 1030] The method of claim 1029, further comprising administering to said subject an adjuvant. [The present invention 1031] The method of claim 1029 or 1030, wherein the adjuvant is alpha-galactosylceramide (α-GC) or alum. [The present invention 1032] The method of any of claims 1029 to 1031, wherein the induced immune response treats or protects the subject against a pulmonary disease or pulmonary dysfunction and / or symptoms thereof, or against an Aspergillus-associated disease and / or symptoms thereof. [The present invention 1033] The method of claim 1032, wherein said lung disease or pulmonary dysfunction is Pneumocystis pneumonia (PCP) and / or symptoms thereof and said Aspergillus associated disease is invasive pulmonary aspergillosis (IPA) and / or symptoms thereof. [The present invention 1034] The method according to any one of claims 1023 to 1033, wherein the subject is a mammal. [The present invention 1035] The method of claim 1034, wherein the subject is a human. [The present invention 1036] The method according to any one of claims 1023 to 1035, wherein the subject is in an immunodeficient state or an immunosuppressed state. [The present invention 1037] The method of claim 1036, wherein said immunocompromised or immunosuppressed subject is a pre-transplant subject or a post-transplant subject. [The present invention 1038] The method of claim 1036, wherein the immunocompromised or immunosuppressed subject is being treated for cancer, an immunodeficiency disease, a congenital disease, or an autoimmune disease. [The present invention 1039] The method of claim 1036, wherein said immunocompromised or immunosuppressed subject is being treated for HIV, rheumatoid arthritis, or psoriasis. [The present invention 1040] The wild-type Kex peptide TIFF0007636331000013.tif11148 Any of the methods of 1023 to 1028 or 1034 to 1039 of the present invention, comprising: [The present invention 1041] A vaccine comprising an effective amount of panfungal peptide 1 or 2. [Brief description of the drawings]

[0070] Exemplary embodiments described herein are provided below as figures and illustrations relating to the described embodiments in their various non-limiting aspects.

[0071] [Figure 1] FIG. 1 presents a multiple sequence alignment comparing the amino acid sequences of two pan-fungal Kex peptides (i.e., pan-fungal peptide 1 and pan-fungal peptide 2) derived from Pneumocystis (human and macaque) with the amino acid sequences of the human Pneumocystis Kex1 peptide, the macaque Pneumocystis Kex1 peptide, the Aspergillus Kex peptide, the Cryptococcus Kex peptide, and the Candida Kex peptide. [Diagram 2] Figure 2 presents a multiple sequence alignment comparing the amino acid sequences of pan-fungal peptide 1 and pan-fungal peptide 2 with the amino acid sequence of the human Pneumocystis Kex1 peptide. Pan-fungal peptide 1 shows 97% amino acid sequence identity to the human Pneumocystis Kex1 peptide, and pan-fungal peptide 2 shows 69% amino acid sequence identity to the human Pneumocystis Kex1 peptide. As described herein (e.g., Example 1), pan-fungal peptide 1 and pan-fungal peptide 2 comprise the amino acid sequences shown in Figures 1 and 2, respectively, and shown above. [Figure 3A]Figures 3A-3F illustrate prophylactic vaccination of panfungal peptide 2 in SIV-infected rhesus macaques. Figure 3A is a timeline illustrating the administration and exposure schedule used to determine the immunogenicity of panfungal 2 peptide in macaques. [Figure 3B] Figures 3B-3E graphically show reciprocal endpoint titers (RET) obtained by ELISA analysis of serum samples collected from macaques immunized with panfungal peptide 2 and alum adjuvant prior to SIV infection and exposure with Pneumocystis fungal pathogens to determine serum immunoglobulin (IgG) titers against targets Cryptococcus Kex, Pneumocystis Kex1, and Aspergillus Kex. In these figures, the arrows indicate the time the test subject was immunized with panfungal peptide 2 and the time the test subject received a booster immunization. The dotted vertical lines indicate the time the test subject was infected with simian immunodeficiency virus (SIV). Figure 3B: RET measured by ELISA analysis of panfungal peptide 2. Figure 3C: RET measured by ELISA analysis against Cryptococcus Kex. Figure 3D: RET measured by ELISA analysis against Pneumocystis Kex1. FIG. 3E is a graph of RET measured by ELISA assay against Aspergillus Kex. [Figure 3C] See legend to Figure 3B. [Figure 3D] See legend to Figure 3B. [Figure 3E] See legend to Figure 3B. [Figure 3F] FIG. 3F is a graph showing the rate of Pneumocystis pneumonia (PcP) observed in animals vaccinated with the pan-fungal 2 peptide (n=1) or sham vaccine (n=7) up to 40 weeks post-SIV infection. [Figure 4A]Figures 4A and 4B show an outline of prophylactic vaccination of SIV-infected rhesus macaques with Pneumocystis Kex1 peptide and alum adjuvant, followed by therapeutic vaccination of the animals with Pneumocystis Kex1 peptide and alpha-galactosylceramide (α-GC) as an adjuvant. Figure 4A is a timeline illustrating the administration and exposure schedule used to study the efficacy of Pneumocystis Kex1 prophylactic vaccination with alum as an adjuvant, and therapeutic vaccination with αGC as an adjuvant. Figure 4B is a graph of RET measured by ELISA analysis of serum immunoglobulin G (IgG) titers against target Pneumocystis Kex1. [Figure 4B] See legend to Figure 4A. [Figure 4C] FIG. 4C is a graph showing the rate of Pneumocystis pneumonia (PcP) observed in animals vaccinated with Pneumocystis Kex1 and alum adjuvant and boosted after SIV infection with Pneumocystis Kex1 (n=6) or sham vaccine (n=7) with αGC adjuvant. [Diagram 5] Figures 5A and 5B show an outline of therapeutic vaccination of Pneumocystis Kex1 with αGC adjuvant in SIV-infected rhesus macaques. Figure 5A is a timeline illustrating the administration and exposure schedule used to study the immunogenicity of therapeutic vaccination of Pneumocystis Kex1 with αGC. Figure 5B is a graph of serum immunoglobulin G (IgG) titers RET against target Pneumocystis Kex1 measured by ELISA analysis in the late chronic phase of SIV infection. [Figure 6]Figures 6A and 6B illustrate panfungal peptide 2-specific humoral responses following panfungal peptide 2 immunization in mice. Figure 6A shows the immunization (vaccination) study design. CF-1 mice (7 per group) were immunized with panfungal peptide 2 + adjuvant (TiterMax) or PBS + TiterMax (sham vaccination). Blood was collected for serological analysis at 0, 14, and 28 days post-immunization. Figure 6B is a graph showing the mean plasma reciprocal endpoint titers (RET) of panfungal peptide 2-specific immunoglobulin G (IgG) titers determined by enzyme-linked immunosorbent assay (ELISA) from a single vaccination with panfungal peptide 2 + TiterMax1 or PBS + TiterMax. Panfungal peptide 2-vaccinated animals achieved a mean peak RET of 5 x 105 compared to those of sham-vaccinated animals (**p=0.002). [Figure 7] Figure 7 is a schematic illustrating the design of a study in which mice were vaccinated with panfungal peptide 2 and then challenged with Aspergillus fumigatus pathogen. Following the panfungal peptide 2 peptide and sham vaccination regimen described in Figure 6A, all mice were immunosuppressed by administration of 2.5 mg cortisone acetate per day for 6 days prior to intranasal challenge with 5 x 106 Aspergillus sp. (Af293) conidial spores. All mice were then observed twice daily for signs of invasive pulmonary aspergillosis (IPA). The association between panfungal peptide 2 vaccination and IPA-related deaths is presented in Table 2 of Example 9 below. [Figure 8] Figure 8 is a graph showing pulmonary fungal burden following A. fumigatus challenge, as described above in Figure 7. Quantification of fungal burden in the lungs was performed using Grocott's methenamine silver (GMS) staining. A. fumigatus fungal burden was significantly reduced in pan-fungal peptide 2 vaccinated animals (*p=0.026). [Figure 9]Figures 9A and 9B show panfungal peptide 2-specific humoral responses following panfungal peptide 2 immunization in non-human primates. Figure 9A is a schematic showing the vaccination study design. In this study, 15 rhesus macaques (n=7 panfungal peptide 2 vaccinated, n=8 sham vaccinated) were immunized and boosted with panfungal peptide 2 + adjuvant (alum) or PBS + alum. Blood was collected for serological analysis at 1, 2, 4, 6, and 8 weeks after each vaccination. Eight weeks after the second vaccination, all animals were infected with SIV and continuously exposed to Pneumocystis by co-housing. All animals were monitored monthly for Pneumocystis infection. Figure 9B is a graph showing the mean plasma reciprocal endpoint titer (RET) of panfungal peptide 2-specific immunoglobulin G (IgG) titers determined by enzyme-linked immunosorbent assay (ELISA). The timing of the first and second sets of immunizations is indicated by arrows. Pan-fungal peptide 2 immunized (vaccinated) macaques achieved a mean peak RET of 4x105 two weeks after the second immunization (post vaccination 2 (wpv2)) compared to sham vaccinated animals (*p=0.019). [Figure 10] FIG. 10 is a graph showing chronic Pneumocystis colonization in pan-fungal peptide 2 vaccinated and sham vaccinated cohorts of rhesus macaques. The graph shows the proportion of SIV-infected animals (pan-fungal peptide 2 vaccinated (n=7) or sham vaccinated (n=8)) that were chronically colonized with Pneumocystis (nested PCR positive for ≥2 consecutive months) over the course of SIV infection (28 weeks post-SIV inoculation) compared by Kaplan-Meier (*p=0.05). At 28 weeks post-infection, chronic Pneumocystis colonization was significantly less in pan-fungal peptide 2 immunized SIV-infected animals compared to sham immunized animals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] DETAILED DESCRIPTION OF EMBODIMENTS The method for treating or preventing the disease associated with infection by one or more fungal pathogens, or infection by two or more fungal pathogens, where the pathogen is of different etiology, is addressed herein.Specific examples of fungal pathogens that cause disease include Pneumocystis, which colonizes lung tissue and causes severe pneumonia after infection; Aspergillus, which is a common mold that causes aspergillosis, allergic reactions, lung infections and other health problems; Candida, which is usually present in the intestinal tract and mucous membranes, and can cause thrush, infections and invasive candidiasis when systemically infected, especially in those with poor health or weak immune systems; and Cryptococcus, which can infect the lungs and cause pneumonia-like illness, and infect the brain and cause meningitis.

[0073] The described embodiments are based, at least in part, on the discovery that mammalian subjects immunized with non-natural peptides having sequence similarity to the kexin (Kex or Kex1) protein (referred to herein as pan-fungal peptides) generated antibodies that specifically bind to Kex peptides of several different fungal pathogens, including Pneumocystis, Candida, Aspergillus, and Cryptococcus. Thus, the pan-fungal peptides described herein are useful for treating or preventing fungal infections by any one or more of the genera Pneumocystis, Candida, Aspergillus, and Cryptococcus.

[0074] Antisera containing antibodies generated in response to administration of a non-natural pan-fungal Kex peptide or a polynucleotide encoding the pan-fungal Kex peptide can also serve as a treatment for infection and / or disease caused by more than one fungal pathogen, and can provide immunity against multiple fungal pathogens in another or unrelated subject (i.e., a recipient subject) to which the antisera is administered via an appropriate method and route of administration. As used herein, it will be understood by those skilled in the art that the subject from which the antisera is obtained or isolated is the "donor subject" and the subject to which the isolated antisera is administered or provided is the "recipient subject." In embodiments, the subject is a mammal, particularly a human or non-human primate. The recipient subject can be a patient or individual in need of treatment or protection against an infection or disease caused by one or more of the Pneumocystis, Candida, Aspergillus and / or Cryptococcus fungal pathogens.

[0075] The production of such immunologically cross-reactive antisera (and antibodies therein) produced in subjects (e.g., donor subjects) immunized with or exposed to pan-fungal Kex peptides, or polynucleotides encoding pan-fungal Kex peptides, such as the non-native consensus Kex1 peptides described herein, reactive with Kex peptides of multiple fungal pathogens, such as Candida, Aspergillus, and Cryptococcus fungal pathogens, was surprising and unexpected, especially considering the low amount of amino acid sequence identity between the Kex peptides of Pneumocystis, Candida, Aspergillus, and Cryptococcus fungal pathogens (about 48%-70% variation), as well as the overall amount of amino acid sequence variation between the Kex1 peptides of Pneumocystis and the Kex peptides of Candida, Aspergillus, and Cryptococcus fungal organisms (ranging from about 70% to 96% variability).

[0076] Also encompassed herein is an immunogenic composition comprising a pan-fungal peptide or a polynucleotide encoding the pan-fungal peptide, which induces a strong immune response in a subject following administration of the composition and induces the production in the subject of antisera containing one or more antibodies or antigen-binding fragments thereof that react not only with the immunizing antigen but also with similar but non-identical antigens found in other distinct fungal pathogens.

[0077] One advantage of the described methods is the provision of treatment or prevention of infection by and disease associated with several different fungal pathogens using only one therapeutic agent, i.e., a composition comprising a non-natural pan-fungal Kex peptide or a polynucleotide encoding the pan-fungal Kex peptide, or an antiserum (isolated antiserum) generated in response to administration of the non-natural pan-fungal Kex peptide or a polynucleotide encoding the pan-fungal Kex peptide, which cross-protects against multiple fungal organisms and treats or prevents disease and its symptoms (e.g., lung disease and reduced lung function) associated with infection (and colonization) by different fungal pathogens (e.g., at least two or more fungal pathogens). In one embodiment, the different fungal pathogens include the genera Pneumocystis, Aspergillus, Candida, and Cryptococcus, particularly at least two or more of them. In one embodiment, a composition is provided comprising a non-natural pan-fungal Kex peptide or a polynucleotide encoding the pan-fungal Kex peptide that elicits the production of antibodies that specifically react with a Kex peptide from one or more of the genera Pneumocystis (Pneumocystis jiroveci), Aspergillus (Aspergillus fumigatus), Candida (Candida albicans), or Cryptococcus (Cryptococcus neoformans). In another embodiment, there is provided an antiserum produced in a subject immunized with a non-natural pan-fungal Kex peptide or a polynucleotide encoding the pan-fungal Kex peptide, the antiserum also containing antibodies specifically reactive with Kex peptides from one or more of the genera Pneumocystis (Pneumocystis jiroveci), Aspergillus (Aspergillus fumigatus), Candida (Candida albicans), or Cryptococcus (Cryptococcus neoformans). Thus, the antiserum is cross-protective (e.g., cross-reactive with Kex peptides of multiple fungal types) and, when provided to another (e.g., unrelated) subject in need thereof, provides treatment and / or protection against infection and / or disease associated with multiple fungal organisms. In one embodiment, the antiserum is an isolated antiserum.In one embodiment, the isolated antisera is administered in a pharma- ceutically acceptable composition.

[0078] The methods and compositions described herein provide economic, medical and practical benefits in the treatment and prevention of fungal infections and diseases, such as types of pulmonary diseases, or brain infections, associated with infection and colonization by different types of fungal pathogens.

[0079] Treatment method The methods and compositions provided herein can be used to treat or prevent infections and / or associated diseases caused by the fungal pathogens Pneumocystis, Aspergillus, Candida, and Cryptococcus, particularly Pneumocystis hominis or jiroveci, Aspergillus fumigatus, Candida albicans, or Cryptococcus neoformans. The methods and compositions provided herein can provide immune protection in a subject against infections and diseases caused by at least one, particularly two or more of these fungal organisms. The methods and compositions provided herein can immunize a recipient subject against infections by at least one, particularly two or more of these fungal organisms. In general, immunogenic compositions comprising a non-natural pan-fungal Kex peptide or a polynucleotide encoding the pan-fungal Kex peptide, or isolated antisera containing one or more antibodies generated against the non-natural pan-fungal Kex peptide, can be administered therapeutically and / or prophylactically to provide immunity against other pathogenic fungal organisms expressing Kex proteins or peptide antigens. The method includes administering to an individual an immunologically effective amount of an immunogenic composition comprising a pan-fungal Kex peptide or a polynucleotide encoding the pan-fungal Kex peptide, an isolated antiserum, or an immune serum or plasma, as described herein, alone or in a physiologically acceptable carrier, excipient, or diluent. In one embodiment, the immunogenic composition comprising a pan-fungal Kex peptide or a polynucleotide encoding the pan-fungal Kex peptide, or isolated antiserum, is in a pharma- ceutically acceptable composition.

[0080] Provided and described herein are methods of treating or preventing infection with one or more fungal pathogens (e.g., Pneumocystis, Aspergillus, Candida, or Cryptococcus, more specifically, one or more of Pneumocystis hominis or jiroveci, Aspergillus fumigatus, Candida albicans, or Cryptococcus neoformans fungi), and / or diseases, disorders, or symptoms thereof, comprising administering a therapeutically effective amount of an immunogenic composition comprising a non-natural pan-fungal Kex peptide described herein, or a polynucleotide encoding the pan-fungal Kex peptide, or isolated antisera generated in response to the non-natural pan-fungal Kex peptide, to a recipient subject (e.g., a mammal such as a human patient) in need thereof. In one embodiment, the antisera contain antibodies that specifically target the kexin protein or Kex peptide thereof and neutralize the activity of the Kex proteinase. In one embodiment, the isolated antisera enable a recipient subject to achieve and passively acquire protective immunity against multiple fungal pathogens.

[0081] In one embodiment, the method described herein involves treating a subject suffering from or susceptible to an infection with Pneumocystis, Aspergillus, Candida, or Cryptococcus, or a disease or symptom thereof caused by one or more of these fungal pathogens (e.g., pulmonary disease or COPD). The method includes administering to a subject (e.g., a mammal or human patient) an effective amount of a pan-fungal Kex peptide or an immunogenic composition comprising a polynucleotide encoding the pan-fungal Kex peptide, or isolated antisera generated in response to the non-natural pan-fungal Kex peptide, sufficient to treat an infection, disease, disorder, or symptom thereof caused by one or more different types of fungal organisms under conditions such that the infection, disease, disorder, or symptom thereof is treated. In one embodiment, the isolated antisera is in a pharma- ceutically acceptable composition.

[0082] Also provided and described are methods of treating or preventing infection with two or more types of fungal pathogens, and / or diseases or disorders or symptoms thereof, comprising administering to a subject (e.g., a mammal such as a human) a therapeutically effective amount of an immunogenic composition comprising a non-natural pan-fungal Kex peptide or a polynucleotide encoding the pan-fungal Kex peptide, or isolated antisera (e.g., comprising one or more antibodies or antigen-binding fragments thereof) generated in response to the non-natural pan-fungal Kex peptide. In various embodiments, the methods prevent infection with two or more fungal pathogens selected from the genera Pneumocystis, Aspergillus, Candida, or Cryptococcus in a subject susceptible to an infection, disease, or symptom thereof (e.g., COPD, lung disease, reduced lung function, or symptom thereof). In one embodiment, the method comprises administering to a recipient mammal a therapeutic amount of a non-natural pan-fungal Kex peptide or an immunogenic composition comprising a polynucleotide encoding the pan-fungal Kex peptide, or isolated antisera generated in response to the non-natural pan-fungal Kex peptide, sufficient to treat the infection, disease, disorder, or symptom thereof, under conditions such that the infection, disease, disorder, or symptom thereof is treated. In one embodiment, the method comprises administering to a recipient mammal a prophylactic or preventive amount of a non-natural pan-fungal Kex peptide or an immunogenic composition comprising a polynucleotide encoding the pan-fungal Kex peptide, or isolated antisera generated in response to the non-natural pan-fungal Kex peptide, sufficient to prevent the infection, disease, disorder, or symptom thereof, under conditions such that the infection, disease, disorder, or symptom thereof is prevented. In one embodiment, the isolated antisera is in a pharma- ceutically acceptable composition. In one embodiment, the recipient mammal is a human patient in need of treatment.

[0083] The treatment is suitably administered to a subject (particularly a human) suffering from, having, susceptible to, or at risk for infection with, or a disease, condition, or symptom thereof, by two or more fungal organisms, particularly two or more of the genera Pneumocystis, Aspergillus, Candida, or Cryptococcus, particularly Pneumocystis hominis or jiroveci, Aspergillus fumigatus, Candida albicans, or Cryptococcus neoformans. The determination of a subject being "at risk" may be made by any objective or subjective determination, such as by a diagnostic test or opinion of the subject or a health care provider (e.g., a genetic test, an enzyme test or assay, or a protein marker (e.g., the level of anti-Kex antibodies in serum), family history, etc.). The methods herein also include administering an effective amount of an anti-fungal pathogen immunogenic composition comprising a non-natural pan-fungal Kex peptide or a polynucleotide encoding the pan-fungal Kex peptide described herein, or an antiserum generated in response to the natural pan-fungal Kex peptide and isolated from a donor subject, to a recipient subject, including subjects identified as in need of such treatment or at risk of infection. Identifying a subject in need of such treatment may involve the judgment of the recipient subject or a medical worker or professional, and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method). In one embodiment, the isolated antiserum is provided in a pharmaceutically acceptable composition.

[0084] In some aspects, methods are provided for treating or preventing a fungal infection or a disease or condition associated with a fungal pathogen (e.g., a pulmonary infection, a pulmonary disease or disorder, pneumonia, COPD, etc.) in a subject, comprising administering to a subject in need thereof an effective amount of a non-natural pan-fungal Kex peptide or an immunogenic composition comprising a polynucleotide encoding a pan-fungal Kex peptide, as described herein, or isolated antisera obtained from an individual that has raised an antibody immune response to the non-natural pan-fungal kex peptide, generated in response to the non-natural pan-fungal Kex peptide, such that the subject is therapeutically and / or prophylactically treated against an infection or disease associated with a different fungal pathogen.

[0085] In another aspect, a method is provided for treating or preventing fungal infection and / or disease in a patient who is receiving or has received an immunosuppressant drug or medication and who is susceptible or may be susceptible (or at risk) to infection with a pathogenic fungus, such as one or more of the fungal pathogens Pneumocystis, Aspergillus, Candida, or Cryptococcus, either in or outside of a hospital environment, as a result of drug-induced immune system suppression. By way of example, such a patient may be preparing to receive a transplant (pre-transplant patient) or may have received a transplant (post-transplant patient) and has been administered one or more immunosuppressant drugs or medications (anti-rejection drugs) and / or otherwise been treated with drugs to reduce the likelihood of rejection of the transplanted organ or tissue, such that the patient is susceptible, predisposed, or at high risk to infection and / or disease caused by one or more of the fungal pathogens Pneumocystis, Aspergillus, Candida, or Cryptococcus. Patients with other types of diseases and conditions, such as rheumatoid arthritis or psoriasis, may also be administered drug therapies with immunosuppressive effects to treat or manage their condition and may therefore suffer from or be at risk of infection with one or more fungal pathogens.Non-limiting classes of immunosuppressive drugs and medications include, for example, corticosteroids, such as prednisone (e.g., DELATSONE, ORASONE); budesonide (ENTOCORT EC) or prednisolone (MLLIPRED) calcineurin inhibitors, such as cyclosporine (NEORAL, SANDIMMUNE, SANGCYA); or tacrolimus (ASTAGRAF XL, ENVARSUS XR, PROGRAF); mTOR inhibitors, such as sirolimus (RAPAMUNE), everolimus (AFINITOR, ZORTRESS); inosine monophosphate dehydrogenase (IMDH) inhibitors, such as azathioprine (AZASAN, IMURAN), leflunomide (ARAVA), mycophenolic acid (CELLCEPT, MYFORTIC); biologics and monoclonal or monoclonal antibody-based antibodies or antigen-binding fragments thereof, such as abatacept (ORENCIA); adalimumab (HUMIRA); These include anakinra (KINERET); certolizumab (CIMZIA); etanercept (ENBREL); golimumab (SIMPONI); infliximab (REMICADE); ixekizumab (TALTZ); natalizumab (TYSABRI); rituximab (RITIXAN); secukinumab (COSENTYX); tocilizumab (ACTEMRA); ustekinumab (STELARA); and vedolizumab (ENTYVIO). In one embodiment, the patient is about to receive or has received an organ transplant selected from kidney, liver, heart, bone marrow, pancreas, lung, gallbladder, bladder, etc.

[0086] An immunogenic composition comprising a non-natural pan-fungal Kex peptide or a polynucleotide encoding the pan-fungal Kex peptide, or an antibody against the non-natural pan-fungal Kex peptide (or an antiserum containing such an antibody) can be administered to a patient undergoing transplant rejection or other immunosuppressive drug therapy in an amount effective to enhance an immune response against infection with one or more, two or more, three or more, or all four of these fungal pathogens in an immunosuppressed patient. In one embodiment, a patient receiving immunosuppressive drugs can be evaluated and monitored for the presence (and antibody titer) of antibodies against one or more of the fungal pathogens during treatment with the immunosuppressive drug by utilizing the methods and kits described herein.

[0087] Optionally, the immunogenic compositions comprising the non-natural pan-fungal Kex peptides or polynucleotides encoding the pan-fungal Kex peptides described herein, or isolated antisera generated in response to the non-natural pan-fungal Kex peptides, may be administered in combination with one or more of any other treatments or therapies (e.g., anti-fungal therapies). For example, the immunogenic compositions comprising the non-natural pan-fungal Kex peptides or polynucleotides encoding the pan-fungal Kex peptides described herein, or isolated antisera or immune plasma generated in response to the non-natural pan-fungal Kex peptides containing anti-Kex non-natural pan-fungal peptide antibodies or antigen-binding fragments thereof, may be administered in combination with other antibodies or antibody cocktails having anti-fungal activity (e.g., including immune plasma), or in combination with one or more drugs, e.g., one or more drugs having anti-fungal activity (e.g., Trimethoprim-sulfamethoxazole, azithromycin-sulfamethoxazole, clarithromycin-sulfamethoxazole, atovaquone, sulfadoxine-pyrimethamine, erythromycin-sulfisoxazole, PS-15, and dapsone-trimethoprim, as well as intravenous pentamidine and clindamycin-primaquine) may be administered in combination to provide protective immunity in the recipient against Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms. In any of the foregoing embodiments, the non-natural pan-fungal Kex peptide or an immunogenic composition comprising a polynucleotide encoding the pan-fungal Kex peptide, or isolated antisera generated in response to the non-natural pan-fungal Kex peptide, is provided in a pharma- ceutically acceptable composition.

[0088] In one embodiment of any of the foregoing aspects, an immunogenic composition comprising a non-natural pan-fungal Kex peptide or a polynucleotide encoding the pan-fungal Kex peptide, or antisera generated in response to a non-natural pan-fungal Kex peptide isolated from a donor subject immunized, inoculated or exposed to a Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogen (e.g., against a Kex peptide expressed by such a fungal pathogen), enables a recipient subject to acquire immune protection (including memory immune protection) against infection or disease caused by two or more of these fungal pathogens.

[0089] Methods for administering both monotherapy and combination therapy (e.g., simultaneously or otherwise) are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences, 12th Edition, E.W. Martin, Mack Publishing Co. In one embodiment, the antisera provide therapeutic antibody-containing compositions that treat infections or diseases caused by one or more fungal pathogens as described herein. In another embodiment, the antisera provide prophylactic antibody-containing compositions that prevent and protect against infections or diseases caused by one or more fungal pathogens as described herein. In one embodiment, the isolated antisera is in a pharma- ceutically acceptable composition.

[0090] Additional methods Currently, there is a lack of methods and reagents for determining whether a patient who is asymptomatic for infection with one or more, two or more, three or more, or each of the fungal pathogens Pneumocystis, Aspergillus, Candida, or Cryptococcus is susceptible, prone, or at risk of infection with one or more of these pathogens.Currently, it is difficult to plate out these fungal organisms because they may exist in very small amounts or do not grow under the culture conditions available to evaluate their presence in subjects.As a result, it is difficult for doctors and patients to know whether a patient is actually infected with one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus, or whether they are likely to be infected (e.g., after medical procedures, surgery, or transplants).

[0091] It is difficult to identify, qualify, or stratify patients who are or may be susceptible to infection by one or more fungal pathogens, and to monitor patients for changes in susceptibility over time, for example, during recovery from surgery or during immunosuppressive therapy, for example, after organ transplantation, or during other chemotherapy treatment, or for reduction or elimination of infection in patients undergoing treatment for fungal infection or related disease over time.The methods described herein provide a viable solution to such medical needs.Furthermore, methods are provided that stratify patients based on whether they have or do not have antibodies specific to a particular fungal pathogen, thereby allowing patients to be treated with appropriate or more directed fungal therapy by preventing infection, or based on whether they have or do not have antifungal Kex peptide antibodies specific to a given type of pathogenic fungus.

[0092] In one embodiment, a method is provided for detecting antibodies against a non-natural Kex protein or peptide in a sample obtained from a subject, the method comprising: (a) contacting a biological sample obtained from the subject with a non-natural Kex peptide; and (b) detecting binding of the non-natural Kex peptide to antibodies in the sample that specifically bind to the Kex peptide, wherein detection of said binding indicates the presence of antibodies against the Kex peptide of a fungal organism in the sample from the subject. In one embodiment, the Kex peptide is attached to a solid support or substrate. In one embodiment, binding is detected by performing an immunoassay, such as an enzyme-linked immunosorbent assay.

[0093] In another embodiment, a method of monitoring or detecting antibodies to a fungal organism associated with an infection in a subject undergoing a transplant or a subject who is to undergo a transplant procedure, for example to determine whether the subject is or will be protected from infection by one or more fungal pathogens selected from the genera Pneumocystis, Aspergillus, Candida, or Cryptococcus, the method comprising: (a) measuring a level of antibodies that bind to a non-naturally occurring pan-fungal Kex protein or peptide in a sample obtained from the subject at an initial time point, before the subject undergoes the transplant procedure; (b) measuring a level of antibodies that bind to a non-naturally occurring pan-fungal Kex protein or peptide in a sample obtained from the subject at one or more time points after the subject undergoes the transplant procedure; and (c) detecting that the subject sample contains a level of antibodies that specifically bind to the non-naturally occurring pan-fungal Kex peptide compared to a predetermined or threshold level or compared to a control level, wherein a high level of antibodies that bind to the non-naturally occurring pan-fungal Kex peptide in the subject's sample indicates that the subject has mounted an immune response to the fungal organism. In one embodiment, antibodies detected in a subject's sample that bind to the non-natural pan-fungal Kex peptide may be useful in protecting the subject from infection with Pneumocystis, Aspergillus, Candida, or Cryptococcus by the methods and compositions described herein.

[0094] Repeating the practice of the above method over time (at different time intervals or for different periods of time) allows for monitoring of the subject's antibody levels against the non-natural pan-fungal Kex peptide (i.e., the level of antibodies that bind to the non-natural Kex peptide), which can inform a physician or clinician as to whether continued, new, or different treatment of the subject with an appropriate anti-fungal drug or therapy is required or justified, or whether no or less anti-fungal treatment is justified based on the antibody titers measured in the subject's sample.

[0095] In other embodiments, methods are provided for detecting the presence of antibodies that bind to a non-natural pan-fungal Kex peptide in a biological sample from a subject, e.g., blood, serum, plasma, lymph, bronchoalveolar lavage fluid, wherein the level of antibodies to the non-natural pan-fungal Kex peptide in the biological sample is simultaneously determined. For example, in one embodiment, the method includes the steps of (a) contacting a biological sample obtained from the subject with a non-natural pan-fungal Kex peptide that selectively binds to a plurality of antibodies in the subject's sample for a period of time sufficient to allow for the formation of bound Kex peptide-antibody complexes; (b) detecting binding of the Kex peptide to the plurality of antibodies in the subject's sample, thereby determining the level of antibodies to the Kex peptide in the sample; and (c) comparing the level of the plurality of antibodies in the sample to a predetermined threshold, where a level of antibodies that bind to at least one of the plurality of Kex peptides above or below the predetermined threshold indicates, for example, that the subject has an antibody titer to a Kex peptide derived from one or more of the fungal organisms and has mounted an immune response. Thus, subjects having an antibody response measured against the Kex peptide are protected from infection and disease associated with one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms.

[0096] In another embodiment, a method is provided for assessing antibodies that bind to one or more Kex peptides of the fungal organisms Pneumocystis, Aspergillus, Candida, or Cryptococcus in a subject, the method comprising: (a) contacting a biological sample obtained from the subject with a composition comprising one or more (multiple) Kex peptides from Pneumocystis, Aspergillus, Candida, or Cryptococcus for a period of time sufficient to allow the formation of antibody-Kex peptide complexes; (b) detecting binding of the multiple Kex peptides to antibodies in the sample, thereby detecting the level or titer of anti-Kex peptide antibodies in the sample; and (c) comparing the level or titer of anti-Kex peptide antibodies in the biological sample with a predetermined threshold or control value, wherein at least one level of anti-Kex peptide antibodies above or below the predetermined threshold indicates whether the subject has or does not have an immune response (antibody response) appropriate to prevent infection with one or more fungal organisms, respectively.

[0097] In another embodiment, a composition is provided that includes a solid substrate and a number of Kex peptides from one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus immobilized on the substrate. In one embodiment, Kex peptides from each of Pneumocystis, Aspergillus, Candida, or Cryptococcus are immobilized on the substrate at different, indexable locations. In another embodiment, a mixture of Kex peptides from two or more, three or more, or all four of Pneumocystis, Aspergillus, Candida, or Cryptococcus are immobilized on the substrate at different, indexable locations. Anti-Kex peptide antibody binding from a sample obtained from a subject previously vaccinated with a non-natural pan-fungal Kex peptide can be measured or detected by measuring or detecting a complex of anti-Kex peptide antibody bound to the Kex peptide localized on the substrate. In one embodiment, the compositions are included in kits for performing immunoassays for detecting and / or measuring antibody-peptide complexes, as well as for determining or measuring the level or amount of antibody present in a subject's sample.

[0098] antibody As described herein, antisera containing antibodies that specifically bind to a non-naturally occurring kexin peptide and cross-react with kexin peptides of one or more different fungal organisms, such as one or more of the genera Pneumocystis, Aspergillus, Candida, or Cryptococcus, to provide immune protection against infections and diseases caused by two or more of these fungal pathogens are useful in therapeutic methods. For example, isolated antisera containing antibodies that target and / or inhibit or neutralize the activity of a non-naturally occurring pan-fungal kexin peptide and cross-react with two or more of these fungal pathogens are particularly useful in the methods described herein. In certain embodiments, methods of using isolated antisera (or immune plasma) comprising antibodies or antigen-binding fragments thereof that specifically bind to a non-native pan-fungal Kex peptide and cross-react with kexin polypeptides of Pneumocystis, Aspergillus, Candida, or Cryptococcus are provided for the treatment or prevention of infections by these fungal pathogens and / or diseases and conditions associated therewith, such as pulmonary diseases and various types of disorders, pneumonia, COPD, invasive pulmonary aspergillosis (IPA), and the like.

[0099] Methods for preparing antibodies are well known to those skilled in the scientific field of immunology. As used herein, the term "antibody" refers not only to complete antibody molecules, but also to fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are routinely utilized both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to complete immunoglobulin molecules, but also to the well-known active fragments F(ab') 2 F(ab') , which lacks the Fc fragment of a complete antibody. 2Fragments and Fab fragments are cleared from the circulation more rapidly and may have less non-specific tissue binding than complete antibodies (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Antibodies may include complete natural antibodies, bispecific antibodies; chimeric antibodies; Fab fragments, Fab' fragments, single chain V region fragments (scFv), fusion polypeptides, and non-conventional antibodies.

[0100] Non-conventional antibodies include, but are not limited to, nanobodies, linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062, (1995)), single domain antibodies, single chain antibodies, and antibodies with multiple valencies (e.g., diabodies, tribodies, tetrabodies, and pentabodies). Nanobodies are the smallest fragments of naturally occurring heavy chain antibodies that have evolved to be fully functional in the absence of light chains. Nanobodies are only half the size of single chain Fv fragments, yet have the affinity and specificity of conventional antibodies. As a result of this unique structure combined with extreme stability and a high degree of homology with human antibody frameworks, nanobodies can bind to therapeutic targets that are inaccessible to conventional antibodies. Multivalent recombinant antibody fragments offer high binding avidity and unique targeting specificity to cancer cells. These multimeric scFvs (e.g., diabodies, tetrabodies) represent an improvement over parental antibodies, as small molecules of about 60-100 kDa in size provide faster clearance from the blood and rapid tissue uptake. See, for example, Power et al. (Generation of recombinant multimeric antibody fragments for tumor diagnosis and therapy, Methods Mol Biol, 207, 335-50, 2003); and Wu et al., Anti-carcinoembryonic antigen (CEA) diabody for rapid tumor targeting and imaging, Tumor Targeting, 4, 47-58, (1999)).

[0101] Various techniques for making and using non-conventional antibodies have been described. Bispecific antibodies generated using leucine zippers are described by Kostelny et al. (J. Immunol. 148(5):1547-1553, (1992)). Diabody technology is described by Hollinger et al. (Proc. Natl. Acad. Sci. USA 90:6444-6448, (1993)). Another strategy for making bispecific antibody fragments using single-chain Fv (sFv) dimers is described by Gruber et al. (J. Immunol. 152:5368, (1994)). Trispecific antibodies are described by Tutt et al. (J. Immunol. 147:60, (1991)). Single chain Fv polypeptide antibodies can be prepared by conjugating V polypeptides either directly or via a peptide-encoding linker as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, (1988)). H Coding sequence and V L Covalently linked VH::VL heterodimers that can be expressed from a nucleic acid containing the coding sequence. See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754.

[0102] In various embodiments, the antiserum (isolated antiserum) contains antibodies or antigen-binding fragments thereof that specifically bind to the non-natural Kex peptide, either monoclonal or polyclonal. Also included are methods of obtaining or isolating antibodies from immune serum (antiserum) or immune plasma and producing hybrid or chimeric antibodies therefrom. In such hybrid or chimeric antibodies, one pair of heavy and light chains is obtained from a first antibody and the other pair of heavy and light chains is obtained from a different second antibody. Such hybrid or chimeric antibodies may be formed using humanized heavy and light chains. Methods for isolating antibodies and producing hybrid or chimeric antibodies are known and practiced by those skilled in the art.

[0103] Generally, a complete antibody is said to contain an "Fc" region and a "Fab" region. The Fc region is involved in complement activation and is not involved in antigen binding. "F(ab') 2 Antibodies from which the Fc region has been enzymatically cleaved or which have been produced without the Fc region, designated "Fab" fragments, retain both of the antigen-binding sites of the intact antibody. Similarly, antibodies from which the Fc region has been enzymatically cleaved or which have been produced without the Fc region, designated "Fab" fragments, retain one of the antigen-binding sites of the intact antibody. Fab fragments consist of a covalently linked antibody light chain and a portion of the antibody heavy chain, designated "Fd." The Fd fragment is the primary determinant of antibody specificity (one Fd fragment can associate with up to 10 different light chains without altering antibody specificity). Isolated Fd fragments retain the ability to specifically bind to an immunogenic epitope.

[0104] Antibodies (and immune sera or plasma containing antibodies) can be produced or generated by any of the methods known in the art that utilize a soluble polypeptide or an immunogenic fragment thereof (e.g., a Kex pan-fungal peptide) as an immunogen. One way to obtain antibodies is to immunize a suitable host animal or subject with an immunogen, i.e., a polynucleotide encoding the pan-fungal Kex peptide, and follow standard procedures for polyclonal or monoclonal antibody production. Briefly, the immunogen will facilitate the presentation of the immunogen (or an immunogenic fragment of the immunogen) on the cell surface. Immunization of a suitable host can be carried out in several ways. By way of example, a nucleic acid sequence encoding an immunogenic pan-fungal kexin peptide can be provided to the host in a delivery vehicle (or molecular expression construct) that is taken up by the host's immune cells. The cells will then process and appropriately express the pan-fungal Kex peptide in a manner that results in an immunogenic response in the host. In an embodiment, a non-natural pan-fungal Kex peptide can be expressed by a delivery vehicle or expression construct. In other exemplary embodiments, a nucleic acid sequence encoding a non-natural pan-fungal Kex peptide may be expressed intracellularly in vitro and the expressed recombinant pan-fungal Kex peptide product may be isolated and used as an immunogen in a suitable immunized host to produce anti-Kex peptide antibodies and generate anti-Kex antisera.

[0105] Alternatively, antibodies against non-natural pan-fungal Kex peptides may be derived from an antibody phage display library, if desired. Bacteriophage are capable of infecting and propagating within bacteria, which, when combined with human immunoglobulin (antibody) genes, can be engineered to display human antibody proteins. Phage display is the process by which phages are made to "display" human antibody proteins on their surface. Genes from a human antibody gene library are inserted into a population of phages. Each phage carries genes for a different antibody and therefore displays a different antibody on its surface.

[0106] Antibodies produced by any method known in the art can then be purified from the immunized host. Antibody purification methods include, but are not limited to, salt precipitation (e.g., with ammonium sulfate), ion exchange chromatography (e.g., on a cation or anion exchange column, preferably performed at neutral pH and eluted with a step gradient of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as Protein A, Protein G, hydroxyapatite, or anti-immunoglobulin.

[0107] In certain aspects, antibodies can be conveniently produced from hybridoma cells engineered to express the antibody. Methods for making hybridomas are well known in the art. Hybridoma cells can be cultured in a suitable medium, and spent medium can be used as a source of antibodies. Polynucleotides encoding the antibody of interest can then be obtained from the hybridoma producing the antibody, and the antibody can then be produced synthetically or recombinantly from these nucleic acid sequences. To produce large amounts of antibodies, it is generally more convenient to obtain ascites fluid. Methods for producing ascites fluid generally include injecting hybridoma cells into immunologically naive, histocompatible or immunotolerant mammals, particularly mice. The mammals can be primed for ascites fluid production by prior administration of a suitable composition (e.g., pristane). Ascites fluid, which contains antibodies, usually in high concentration, can be obtained from the peritoneal fluid of animals carrying the injected hybridoma cells.

[0108] Monoclonal antibodies (Mabs) can also be "humanized" by methods known in the art. A "humanized" antibody is an antibody in which at least part of the sequence has been altered from its earlier form to make it more similar to human immunoglobulins derived from a human source. Techniques for humanizing antibodies are particularly useful when the antibody is generated in a non-human animal (e.g., mouse, rat). Non-limiting examples of methods for humanizing murine antibodies are provided in U.S. Pat. Nos. 4,816,567, 5,530,101, 5,225,539, 5,585,089, 5,693,762, and 5,859,205.

[0109] In the foregoing embodiments, one or more antibodies or antigen-binding fragments thereof generated against a non-natural Kex peptide can be used alone or in combination in a pharmaceutical composition to provide immune protection against disease or infection caused by one or more of these fungal pathogens in a subject in need thereof. Such antibodies may be isolated or purified from the antisera described herein, or may be produced, for example, by recombinant molecular biology techniques, and purified and formulated for pharmaceutical use in a subject in need thereof. Such preparations of antibodies may have similar immune protective properties as those provided by the isolated antisera comprising the anti-fungal Kex peptide antibodies described herein.

[0110] vaccine A vaccine is a biological preparation that provides active adaptive immunity (e.g., protective immunity) in a subject against a particular disease. Vaccines usually contain an agent that resembles the disease-causing pathogen, e.g., a microorganism, fungus, etc., and are often made with a weakened or killed form of the pathogen, or a toxin or surface protein or peptide of the pathogen. After administration of the vaccine to a subject, the pathogen is expressed and recognized as foreign (or "non-self") to the subject, stimulating the subject's immune system to mount an immune response (B cell (antibody) and / or T cell (cellular) immune response) to destroy the pathogen. Furthermore, cells of the immune system (e.g., B cells) exposed to the vaccinating agent retain a memory of the pathogen, so that the pathogen is recognized and destroyed by the memory cells upon later or subsequent encounters. Vaccines can be prophylactic (e.g., to prevent or ameliorate the effects of future infection by a pathogen) or therapeutic (e.g., to treat a disease or infection caused by or associated with a pathogen or disease-causing pathogen at the time or after a subject has been infected with or encountered by the pathogen).

[0111] While many vaccines are prepared from attenuated forms of pathogens, or from inactivated disease-causing organisms, or suitable portions of such pathogens or organisms, such as toxins, proteins / peptides, or harmful enzymes, the antigens to which the immune system responds often comprise a relatively small number of amino acids, such as peptides (e.g., non-natural pan-fungal Kex peptides). Protein or peptide portions of a pathogen may comprise the vaccine. A peptide vaccine is any peptide that serves to immunize an organism (elicit an immune response or a protective immune response, such as an antibody (B cell) response and / or an immune cell (T cell) response in the immunized organism) against a pathogen. In embodiments, the peptide antigen may be a non-natural pan-fungal Kex peptide. In one embodiment, a vaccine containing a non-natural Kex peptide antigen may be used to provide immune protection against each of the other fungal organisms after administration to a recipient subject in need.

[0112] In the case of non-attenuated vaccines, peptide sequences that elicit a protective immune response are identified, and synthetic (or recombinantly produced) forms of the peptides are utilized as vaccine agents. Because they are synthetic and not naturally occurring, peptide vaccines pose little or no risk of mutation or reversion, and little or no risk of contamination with pathogenic or toxic substances. Furthermore, chemical manipulation or modification of the peptide structure can result in increased stability and reduced undesirable or adverse effects that may be associated with natural proteins or peptide sequences.

[0113] Synthetic or recombinantly produced peptide antigens can be easily prepared in large quantities as a component of vaccines. Such materials may also expose portions of protein antigens that are not recognized by the immune system during natural infection, possibly as a result of protein masking or post-translational modification. Sequencing new strains and serotypes of microbial, fungal pathogens and other pathogenic organisms allows for rapid modification of peptide antigens to produce strain-specific immune responses, particularly against antigenic epitopes that are recognized and targeted by the antibodies and cells of the host's or recipient's immune system. In some cases, precise epitope or antigenic sites on peptide antigens may be synthetically generated using modeling of three-dimensional epitope or antigenic sites of pathogens.

[0114] In one aspect, a vaccine (or immunogenic composition) is provided that includes a synthetically (recombinantly) produced peptide, i.e., a pan-fungal kexin peptide, that is not identical among several different types of fungal pathogens (e.g., fungal pathogens of the genera Pneumocystis, Aspergillus, Candida, and Cryptococcus), but is capable of being immunologically targeted and is useful for treating or preventing infections or diseases caused by or associated with multiple fungal pathogens after administration (immunization) to a subject. In one embodiment, a peptide vaccine or immunogenic composition from a non-natural pan-fungal Kex peptide, when used to immunize an individual, elicits an immune response in the form of production of antisera (or immune plasma) containing cross-reactive antibodies that protect against all of the etiologically distinct fungal pathogens Pneumocystis, Aspergillus, Candida and Cryptococcus, particularly Pneumocystis hominis, Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. Thus, antisera or immune plasma generated by a vaccine or immunogenic composition comprising a non-natural pan-fungal Kex peptide may be used as the sole therapeutic or protective agent required to treat or prevent infections or diseases caused by or associated with two or more different fungal pathogens, i.e., Pneumocystis, Aspergillus, Candida and / or Cryptococcus fungal pathogens, particularly infections or diseases caused by or associated with Pneumocystis hominis, Aspergillus fumigatus, Candida albicans and / or Cryptococcus neoformans. In one embodiment, antisera generated by such a peptide vaccine is isolated. In one embodiment, the isolated antisera is used in a pharmaceutical composition.

[0115] In some aspects, a genetic vaccine is provided. A genetic vaccine is any vaccine that includes a polynucleotide sequence that encodes an immunogen that, once expressed, serves to immunize an organism. Administration of the genetic vaccine induces an immune response or a protective immune response, such as an antibody (B cell) response and / or an immune cell (T cell) response in the immunized organism against a pathogen after the immunogen is expressed intracellularly. In some embodiments, the genetic vaccine provides a polynucleotide sequence that encodes a Kex peptide, such as a pan-fungal peptide or a wild-type peptide. In some embodiments, the peptide is a non-naturally occurring peptide. In some embodiments, the genetic vaccine provides a polynucleotide sequence that encodes two or more Kex peptides. In some embodiments, the polynucleotide encoding the Kex peptide is in a vector that has elements, such as a promoter and enhancer, to facilitate expression of the encoded Kex peptide. The nucleic acid in the genetic vaccine may also be integrated into the genome of the subject, where expression of the immunogen may be driven by an endogenous promoter or a promoter encoded by the inserted nucleic acid. The polynucleotide encoding the immunogen in the genetic vaccine may be a DNA polynucleotide or an RNA polynucleotide. A polynucleotide may contain nucleotide analogues that can inhibit degradation of the polynucleotide.

[0116] Pharmaceutical Compositions Also featured herein is a method for treating or preventing infection and disease associated with one or more fungal pathogens in a subject.The method comprises administering to a subject in need thereof an immunologically effective amount of an immunogenic composition comprising a non-natural pan-fungal Kex peptide or a polynucleotide encoding said pan-fungal Kex peptide, or an isolated antiserum generated in response to said non-natural pan-fungal Kex peptide, which cross-treats and / or protects said subject from infection and / or disease associated with one or more different fungal pathogens selected from the genera Pneumocystis, Aspergillus, Candida or Cryptococcus.In one embodiment, the isolated antiserum is used in a pharmaceutical composition.

[0117] Typically, the carrier or excipient for the immunogenic compositions or vaccines described herein is a pharma- ceutically acceptable carrier or excipient, such as sterile water, saline solution, buffered saline solution, aqueous dextrose, aqueous glycerol, ethanol, or a combination thereof. Preparation of such solutions to ensure sterility, pH, isotonicity, and stability is performed according to protocols established in the art. Usually, the carrier or excipient is selected to minimize allergies and other undesirable effects, and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, etc. Such methods also include administering an adjuvant, such as an oil-in-water emulsion, saponin, cholesterol, phospholipids, CpG, polysaccharides, variants thereof, and combinations thereof, with the compositions described herein. Optionally, the formulation for prophylactic administration may also contain one or more adjuvants to enhance the immune response to the antigen or immunogen, such as a pan-fungal Kex peptide antigen or immunogen. Suitable adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, α-galactosylceramide (α-GC), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, Bacillus Calmette-Guerin (BCG), Corynebacterium parvum, and the synthetic adjuvants QS-21 and MF59. In one embodiment, the isolated antisera are used in a pharmaceutical composition.

[0118] Administration of a non-naturally occurring pan-fungal Kex peptide or an immunogenic composition comprising a polynucleotide encoding the pan-fungal Kex peptide, or an antiserum, such as an isolated antiserum, monoclonal antibody, or polyclonal antibody against a non-naturally occurring pan-fungal kexin protein or peptide, as a therapeutic agent for treating or preventing infection with a fungal pathogen described herein, or a disease or condition associated with a fungal pathogen (e.g., pulmonary infection or disease, pulmonary dysfunction, COPD, pneumonia, etc.), can be by any suitable means that results in a concentration of the therapeutic agent effective to ameliorate, reduce, eliminate, attenuate, or stabilize the disease or disease symptoms in a subject, optionally in combination with other components. The therapeutic agent may be administered systemically, and may be formulated, for example, in a pharma- ceutically acceptable composition or buffer, such as saline. Preferred routes of administration include, for example, subcutaneous, intravenous, intraperitoneal, intramuscular, intrathecal, or intradermal injections that provide a continuous, sustained level of the therapeutic agent in the subject. The amount of therapeutic agent administered varies depending on the method of administration, the age and weight of the subject, and the clinical symptoms of fungal infection or related disease.Usually, the amount will be within the range of that used for other drugs used in the treatment of pulmonary disease or dysfunction, but in some cases, a smaller amount may be appropriate because the scope of protection and treatment provided by the therapeutic agent is increased.The composition is administered at a dosage that improves, reduces, reduces, weakens, alleviates, or eliminates the effects of fungal pathogen infection or disease (e.g., pulmonary infection and disease and its symptoms) as determined by methods known to those skilled in the art.In one embodiment, the isolated antiserum is administered or provided to the recipient subject at or near the site of infection or colonization by pathogenic fungal organisms.

[0119] In some embodiments, the therapeutic or prophylactic treatment agent may be included in any suitable amount in any suitable carrier material, generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal) administration route. Pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. AR Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York).

[0120] Pharmaceutical compositions may be formulated to release the active agent substantially immediately upon administration or after any predetermined time or period following administration, in some cases. The latter type of composition is typically known as a controlled release formulation, and includes: (i) formulations that provide a substantially constant concentration of a therapeutic agent or drug in the body for an extended period of time; (ii) formulations that provide a substantially constant concentration of a therapeutic agent or drug in the body for an extended period of time after a predetermined lag time; (iii) formulations that sustain action for a predetermined period of time by maintaining a relatively constant effective level in the body while simultaneously minimizing undesirable side effects associated with fluctuations in the plasma level of the active agent (sawtooth kinetic pattern); (iv) formulations that localize action, for example by spatially placing the controlled release composition adjacent to or in contact with an organ such as the heart; (v) formulations that allow convenient dosing, for example, a dose administered once a week or once every two weeks; and (vi) formulations that target a disease using carriers or chemical derivatives to deliver the therapeutic agent or drug to a specific cell type. For some applications, controlled release formulations eliminate the need for frequent dosing during the day to maintain therapeutic agent levels in the plasma, serum, or blood. In one embodiment, the isolated antisera may be formulated with one or more additional components for administration to a subject.

[0121] In order to obtain controlled release, in which the release rate exceeds the metabolic rate of the therapeutic agent or drug in question, any of several strategies can be pursued.In one example, controlled release is obtained by appropriate selection of various formulation parameters and components, including, for example, various types of controlled release compositions and coatings.Thus, therapeutic agent or drug can be formulated with suitable excipients into a pharmaceutical composition that releases therapeutic agent or drug in a controlled manner when administered.Examples include single or multiple unit tablet or capsule composition, oily solution, suspension, emulsion, microcapsule, microsphere, molecular complex, nanoparticle, patch, and liposome.

[0122] The pharmaceutical composition can be administered parenterally by injection, infusion, or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, etc.), in dosage forms, formulations, or via suitable delivery devices or implants containing conventional non-toxic pharmacologic carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

[0123] Compositions for parenteral use may be provided in unit dosage form (e.g., in single-dose ampoules) or in vials containing several doses and to which suitable preservatives may be added (see below). The compositions may be in the form of a solution, suspension, emulsion, injection device, or delivery device for implantation, or may be provided as a dry powder to be reconstituted with water or another suitable medium before use. Apart from the active agent that reduces or improves a disease or dysfunction, such as a pulmonary disease or dysfunction, the composition may contain suitable parenterally acceptable carriers and / or excipients. In some cases, the active therapeutic agent may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, etc. for controlled release. In addition, the composition may contain suspending agents, solubilizing agents, stabilizing agents, pH adjusting agents, tonicity adjusting agents, and / or dispersing agents.

[0124] In some embodiments, a pharmaceutical composition comprising an active ingredient (e.g., an immunogenic composition comprising a non-natural pan-fungal Kex peptide or a polynucleotide encoding the pan-fungal Kex peptide described herein, or an isolated anti-fungal antiserum generated in response to the non-natural pan-fungal Kex peptide) is formulated for intravenous delivery, e.g., intravenous injection, or intrathecal delivery. In one embodiment, the antiserum is an isolated antiserum. To prepare such a composition, a suitable therapeutic agent is dissolved or suspended in a parenterally acceptable liquid vehicle, excipient, or solvent. Among the acceptable vehicles and solvents that may be utilized are, for example, water, water adjusted to a suitable pH by the addition of an appropriate amount of hydrochloric acid, sodium hydroxide, or a suitable buffer; 1,3-butanediol; Ringer's solution; and isotonic sodium chloride solution and dextrose solution. Aqueous formulations may also contain one or more preservatives (e.g., methyl, ethyl, or n-propyl p-hydroxybenzoate). If one of the agents is only sparingly or only slightly soluble in water, a solubility enhancer or solubilizer can be added or the solvent may include 10-60% w / w propylene glycol or the like.

[0125] Kits and compositions for detecting and / or quantifying antibodies reactive with fungal-derived Kex peptides - Patents.com In another embodiment, kits and compositions are provided that conveniently allow the detection and / or quantification of the presence of antibodies against one or more Kex proteins or peptides of one or more fungal pathogens of Pneumocystis, Aspergillus, Candida, or Cryptococcus in a sample (e.g., blood or serum) of a subject, or the level of one or more antibodies that may be present.In one embodiment, the subject is a human patient.In one embodiment, the patient has undergone or is about to undergo a transplant, such as an organ or tissue transplant, and may therefore be at high risk of infection with one or more fungal pathogens.In one embodiment, the transplant patient, or the patient who will undergo a transplant, is immunosuppressed and / or otherwise treated with drugs to reduce the possibility of rejection of the transplanted organ or tissue, which makes the patient susceptible or susceptible to infection and / or disease caused by one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens. In one embodiment, the patient has received or is to receive a transplant of an organ selected from kidney, liver, heart, bone marrow, pancreas, lung, and the like.

[0126] Such kits described herein fulfill a long-standing need in the art to detect or qualify whether any patient, particularly a transplant patient, has a sufficient level (titer) of anti-fungal pathogen antibodies to ensure that the patient will not be infected with one or more fungal pathogens described herein during or after a medical procedure or treatment (e.g., surgery or transplantation), for example, while hospitalized or performed on either an inpatient or outpatient basis. Currently, due to a lack of suitable reagents and assays, it is difficult to assess whether a patient who is about to undergo a medical procedure or surgery, particularly an immunosuppressed patient who is about to undergo a transplantation procedure, or a patient who is about to begin other immunosuppressive therapy, will contract a fungal infection, for example, an infection and / or disease caused by one or more of the fungal pathogens of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus, after or during the immunosuppressive therapy and treatment. The use of a kit that can test a patient sample to determine whether the patient has an antibody titer (e.g., high or low antibody titer to one or more of the fungal pathogens) to one or more of these fungal pathogens will greatly enhance the patient's post-operative or post-transplant recovery and the success of direct treatment. For example, if after testing a patient sample (e.g., a blood or serum sample from a transplant patient) with a kit described herein, the patient is determined to have low, negligible, or no antibody (antiserum) titer to one or more of the fungal pathogens, particularly to one or more Kex peptides of the fungal pathogens, it can be inferred that the patient is not naturally protected from potential or actual infection by one or more of the genera Pneumocystis, Aspergillus, Candida, or Cryptococcus.

[0127] The kit described herein will allow the tester and the patient to determine and know whether the patient's sample (serum sample) contains antibodies to one or more, two or more, three or more, or four Kex proteins / peptides of Pneumocystis, Aspergillus, Candida, or Cryptococcus organisms. If the results obtained from the use of the kit indicate that the patient has no specific antifungal Kex peptide antibodies targeting the specific antifungal Kex peptide or a low titer of the antibodies (e.g., no specific antifungal Kex peptide antibodies in serum), the patient will be identified as potentially susceptible or susceptible to infection with the specific fungal pathogen, and can then be administered the appropriate antifungal treatment against the specific fungal pathogen to which the patient has no specific antibodies or has negligible or reduced titers. In one embodiment, the patient is administered a prophylactic antifungal treatment or therapy. In one embodiment, treatment involves administering to the patient an appropriate drug or drug therapy best designed to treat an infection or disease associated with an infection by a particular fungal pathogen or by two or more fungal pathogens, i.e., Pneumocystis, Aspergillus, Candida, or Cryptococcus. In one embodiment, treatment involves administering to the patient a composition described herein that includes a non-natural pan-fungal Kex peptide or a polynucleotide encoding the pan-fungal Kex peptide to produce a cross-reactive (cross-protective) antibody immune response in the patient, thereby reducing or eliminating one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus organisms associated with the infection. Antibodies generated against a non-natural pan-fungal Kex peptide of one of the fungal organisms can recognize the Kex peptides of other fungal organisms described herein, thereby conferring protection (cross-protection) against two or more of the fungal organisms in the patient.

[0128] In one embodiment, a kit is provided for detecting or qualifying the level of antibodies against a non-natural pan-fungal Kex protein or peptide that cross-reacts with one, two, three, or four of Pneumocystis, Aspergillus, Candida, or Cryptococcus organisms in a patient sample, comprising a substrate to which a biological sample obtained from a patient is applied, to which non-natural pan-fungal Kex peptides from one or more, two or more, three or more, or each of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms are attached for measuring the level of antibodies in the sample that bind or immunologically react with the peptides; and a labeled detection molecule for detecting and measuring the level of antibodies that bind to the Kex peptides on the substrate. In one embodiment, the detection of anti-fungal Kex peptide antibodies in the sample or the measurement of the level of such antibodies present in the patient sample is compared to a positive and / or negative control. In one embodiment, the detection of anti-fungal Kex peptide antibodies in a sample or the measurement of the level of such antibodies present in a patient sample is compared to a cut-off value.In one embodiment, a substrate is attached with a non-natural pan-fungal Kex peptide and a Kex peptide from each of the genera Pneumocystis, Aspergillus, Candida, and Cryptococcus.In one embodiment, a substrate is attached with a non-natural pan-fungal Kex peptide and a Kex peptide from one of the genera Pneumocystis, Aspergillus, Candida, and Cryptococcus.In one embodiment, a substrate is attached with a non-natural pan-fungal Kex peptide and a Kex peptide from Pneumocystis and Aspergillus.In one embodiment, a substrate is attached with a non-natural pan-fungal Kex peptide and a Kex peptide from Pneumocystis and Candida. In one embodiment, a substrate has attached thereto a non-natural pan-fungal Kex peptide and Kex peptides derived from Pneumocystis and Cryptococcus.In one embodiment, a substrate has attached thereto a non-natural pan-fungal Kex peptide and a Kex peptide from Candida and Aspergillus. In one embodiment, a substrate has attached thereto a non-natural pan-fungal Kex peptide and a Kex peptide from Cryptococcus and Aspergillus. In one embodiment, a substrate has attached thereto a non-natural pan-fungal Kex peptide and a Kex peptide from Candida and Cryptococcus. In one embodiment, a substrate has attached thereto a non-natural pan-fungal Kex peptide and a Kex peptide from Pneumocystis, Aspergillus and Candida. In one embodiment, a substrate has attached thereto a non-natural pan-fungal Kex peptide and a Kex peptide from Pneumocystis, Aspergillus and Candida. In one embodiment, a substrate has attached thereto a non-natural pan-fungal Kex peptide and a Kex peptide from Pneumocystis, Aspergillus and Candida. In one embodiment, a substrate has attached thereto a non-natural pan-fungal Kex peptide and a Kex peptide from Pneumocystis, Aspergillus and Candida. In one embodiment, the substrate is attached with a non-natural pan-fungal Kex peptide and a Kex peptide from Pneumocystis, Cryptococcus and Candida. In one embodiment, the substrate is attached with a non-natural pan-fungal Kex peptide and a Kex peptide from Aspergillus, Cryptococcus and Candida. In one embodiment, the pan-fungal Kex peptide is recombinantly produced. In one embodiment, the detection of antibodies in the sample that bind to the fungal-derived Kex peptide is performed using an immunoassay, such as an ELISA. In one embodiment, the ELISA detects complexes between Kex peptides bound to anti-fungal Kex peptide antibodies present in the sample. In one embodiment, the detection of antibodies in the sample that bind to the fungal-derived Kex peptide is performed using an immunosorbent assay, by immunoprecipitation, by immunoblotting, or a combination thereof.

[0129] Also provided are kits that include reagents that allow for the evaluation, measurement, assessment or detection of antibodies against non-natural Kex peptides. Such antibodies may be contained in a biological sample obtained from a subject being tested, evaluated or assessed using the kit. In particular, the biological sample may be a blood, serum, plasma, urine, cerebrospinal fluid, sputum, bronchial lavage, tears, saliva or semen sample, or a tissue or cell sample obtained from the subject. In particular, the reagents of the kit include the non-natural Kex peptide.

[0130] In certain embodiments, the kit is provided as an enzyme-linked immunosorbent assay (ELISA) kit comprising a non-natural pan-fungal Kex peptide. In other embodiments, the kit provided allows for the detection of cross-reactive antibodies produced by immunization with a non-natural pan-fungal Kex peptide. In such embodiments, the kit is provided as an ELISA kit comprising a Pneumocystis, Aspergillus, Candida, or Cryptococcus Kex peptide attached to a solid support or substrate. Thus, the peptide attached to the substrate serves as a "capture" reagent that binds to antibodies present in a sample obtained from a subject being tested. By way of example, the ELISA kit may comprise a solid support, such as a chip, a microtiter plate containing many wells (e.g., a 96-well plate), beads, or a resin to which a peptide capture reagent is attached. In one embodiment, the kit comprises a Kex peptide from each of the genera Pneumocystis, Aspergillus, Candida, or Cryptococcus described herein independently attached to separate regions or components of a solid substrate or support, e.g., the Kex peptide of each fungal organism is attached to separate and distinct wells of a microtiter plate, or independently attached to beads, resulting in a population of beads having attached thereto a Kex peptide from each of the genera Pneumocystis, Aspergillus, Candida, or Cryptococcus. In another embodiment, the kit comprises a combination or mixture of Kex peptides from Pneumocystis, Aspergillus, Candida, or Cryptococcus attached to regions or components of a solid substrate or support, e.g., all of the Kex peptides from Pneumocystis, Aspergillus, Candida, or Cryptococcus are attached to a single well of a microtiter plate or to a single bead. In further embodiments, the kit comprises a combination of one, two or more, three or more, or four of Kex peptides from Pneumocystis, Aspergillus, Candida, or Cryptococcus attached to a given area of ​​a solid substrate or support, such as a single well of a microtiter plate.

[0131] In embodiments, in an ELISA platform, the wells of a microtiter plate may have a non-natural pan-fungal Kex peptide, an Aspergillus Kex peptide, a Candida Kex peptide, or a Cryptococcus Kex peptide attached thereto. In an ELISA platform, the wells of a microtiter plate may have a Pneumocystis Kex peptide and an Aspergillus Kex peptide attached thereto. In an ELISA platform, the wells of a microtiter plate may have a Pneumocystis Kex peptide and a Candida Kex peptide attached thereto. In an ELISA platform, the wells of a microtiter plate may have a Pneumocystis Kex peptide and a Cryptococcus Kex peptide attached thereto. In an ELISA platform, the wells of a microtiter plate may have an Aspergillus Kex peptide and a Candida Kex peptide attached thereto. In an ELISA platform, the wells of a microtiter plate may have an Aspergillus Kex peptide and a Candida Kex peptide attached thereto. In an ELISA platform, the wells of a microtiter plate may have an Aspergillus Kex peptide and a Cryptococcus Kex peptide attached thereto. In an ELISA platform, the wells of a microtiter plate can have a Candida Kex peptide and a Cryptococcus Kex peptide attached thereto. In an ELISA platform, individual wells of a microtiter plate can have a Pneumocystis Kex peptide, an Aspergillus Kex peptide and a Candida Kex peptide attached thereto. In an ELISA platform, individual wells of a microtiter plate can have a Pneumocystis Kex peptide, an Aspergillus Kex peptide and a Cryptococcus Kex peptide attached thereto. In an ELISA platform, individual wells of a microtiter plate can have a Pneumocystis Kex peptide, a Candida Kex peptide and a Cryptococcus Kex peptide attached thereto. In an ELISA platform, individual wells of a microtiter plate can have a Pneumocystis Kex peptide, a Candida Kex peptide and a Cryptococcus Kex peptide attached thereto.

[0132] The kit may further include a means for detecting the peptide or any antibody bound thereto, e.g., a detectable antibody, such as a goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (HRP), a secondary antibody-signal conjugate, or tetramethylbenzidine (TMB) as a substrate for HRP.

[0133] In another embodiment, the kit may be provided with an immunochromatographic strip comprising a membrane on which one, two, three or four Kex peptides are immobilized at separate locations on the membrane or in combination at one location on the membrane, and a means for detecting the binding of antibodies in a test sample, such as detectably labeled peptides or secondary antibodies bound to gold particles, where the membrane may be a nitrocellulose (NC) based membrane, a PVDF membrane, or other suitable type of membrane used in the art. The kit may include a sample to which is applied and an immobilized detection agent, such as a detectably labeled Kex peptide, for example, a plastic plate or substrate on which the gold particle-bound peptide is immobilized at time intervals on a substrate, such as a glass fiber filter or nitrocellulose membrane, or a labeled detection agent capable of detecting a complex of the antibody bound to the Kex peptide as one or more bands on the substrate. In such a platform, a continuous capillary flow of the sample, such as blood or serum, is maintained over the detection reagent immobilized on the substrate, such that the sample antibody bound to the labeled Kex peptide or the sample antibody complexed with the Kex peptide reagent can be detected. In general, immunosorbent assays, including ELISA assays and ELISA membrane-based immunosorbent assays, as well as variations of these assays, are known and practiced by those of skill in the art.

[0134] Solid or solid-phase substrates or carriers that can be effectively used in such assays are well known to those skilled in the art, and include, for example, 96-well microtiter plates, glass, paper, and microporous membranes, such as those made of nitrocellulose, nylon, polyvinylidene fluoride, polyester, cellulose acetate, mixed cellulose esters, and polycarbonate.Suitable microporous membranes include, for example, those described in U.S. Patent Application Publication No. 2010 / 0093557 A1.Methods for automating immunoassays are well known in the art, and include, for example, those described in U.S. Patent Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691.

[0135] In one embodiment, multiplex assays, such as multiplex ELISA, can be used to simultaneously detect different specific antibodies in test samples.In an embodiment, such methods utilize an array in which multiple binding agents (e.g., capture antibodies) specific for multiple antibodies are immobilized on a substrate, such as a membrane, with each capture agent being located at a specific, predefined position on the substrate.Methods for carrying out such array-based assays include, for example, those described in U.S. Patent Application Publication Nos. US 2010 / 0093557A1 and US 2010 / 0190656A1, the disclosures of which are specifically incorporated herein by reference.When flow cytometry, chemiluminescence, or electrochemiluminescence technology is utilized, multiplex arrays can be used in several different formats. By way of illustration, flow cytometry multiplexed arrays, also known as bead-based multiplexed arrays, include the Cytometric Bead Array (CBA) system from BD Biosciences (Bedford, MA) and Multi-Analyte Profiling (xMAP®) technology from Luminex Corp. (Austin, TX), both of which utilize bead sets that are distinguishable by flow cytometry.

[0136] In another embodiment, the multiplex ELISA from Quansys Biosciences (Logan, UT) involves coating multiple specific capture reagents (one reagent per spot) to multiple spots in the same well on a 96-well microtiter plate, then detecting multiple antibodies bound to corresponding spots on the plate using chemiluminescence technology.

[0137] In certain embodiments, a patient can be diagnosed by adding a biological sample (e.g., blood or serum) from the patient to a kit, or a component thereof, and detecting the relevant sample antibody that specifically binds to the Kex peptide reagent. By way of example, the method includes the steps of (i) collecting a blood or serum sample from the subject; (ii) adding the subject's sample to a component in the kit, such as a holding tube or substrate; and (iii) detecting the peptide reagent to which the sample antibody has bound. In this method, the subject's sample, such as blood or serum, is contacted with the Kex peptide reagent. If anti-Kex peptide antibodies are present in the sample, the antibodies will bind to the Kex peptide reagent, or a subset thereof. In other kit and diagnostic embodiments, blood is not collected from the patient (i.e., has already been collected) and is assayed for the presence of antibodies to non-natural Kex peptides. In some embodiments, antibodies against a non-native pan-fungal Kex peptide cross-react with one or more, two or more, three or more, or four of Pneumocystis, Aspergillus, Candida, or Cryptococcus organisms using the kit. Additionally, in other embodiments, the sample may include a tissue sample or a clinical sample, which may be treated, e.g., homogenized and / or suspended in a medium or buffer prior to the assay. In embodiments, any antibodies found to be present in a test sample from a subject may be isolated, or isolated and purified, and further characterized.

[0138] The kit can also include a wash solution or instructions for making a wash solution, where the combination of capture reagent and wash solution allows for capture of anti-Kex antibodies on a solid support for subsequent detection, for example, by a secondary antibody, a labeled peptide reagent, or mass spectrometry. In further embodiments, the kit can include instructions for appropriate operating parameters in the form of a label or a separate insert. For example, the instructions may inform the consumer or user how to collect the sample, how to wash the anti-Kex peptide antibody and Kex peptide reagent complex after binding has occurred, how to interpret the results, etc. In yet another embodiment, the kit can include one or more containers with appropriate positive and negative controls or control samples to be used as standards for detection, calibration, or normalization.

[0139] In another aspect, a kit is provided for the treatment or prevention of an infection or disease caused by or associated with two or more of the fungal pathogens Pneumocystis, Aspergillus, Candida, or Cryptococcus. In some embodiments, the kit comprises an effective amount of a therapeutic or prophylactic antiserum containing an anti-Kex peptide antibody or antigen-binding fragment thereof that binds / reacts with a non-natural Kex peptide. In some embodiments, the antibodies cross-react with one or more of the genera Pneumocystis, Aspergillus, Candida, or Cryptococcus in a unit dosage form. In one embodiment, the antiserum is an isolated antiserum. In other embodiments, the kit comprises a therapeutic or prophylactic composition containing an effective amount of an antifungal immune protective agent, such as an antiserum, in a unit dosage form. In some embodiments, the kit includes a device for dispensing the composition (e.g., a nebulizer, a metered dose inhaler) or a sterile container containing the pharmaceutical composition; such a container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. Such a container can be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding medicaments.

[0140] Optionally, the pharmaceutical composition is provided with instructions for administering the pharmaceutical composition containing the isolated antiserum to a subject having or at risk of developing a fungal infection, particularly infections and diseases caused by Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms and symptoms thereof. The instructions will generally include information about the use of the composition for the treatment or prevention of infections caused by one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms. In other embodiments, the instructions include at least one of the following: description of the therapeutic / prophylactic agent; dosing schedule and administration for the treatment or prevention of infections caused by one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms or symptoms thereof; usage instructions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical trials; and / or references. The instructions may be printed directly on the container (if present), or as a label affixed to the container, or as a separate sheet, pamphlet, card or holder supplied in or with the container.

[0141] The practice of the embodiments described herein will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the control of those of ordinary skill in the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of polynucleotides and polypeptides, such as those described herein, and may be considered, for example, in the creation and practice of the aspects and embodiments described herein. Particularly useful techniques for particular embodiments are discussed in the following sections. EXAMPLES

[0142] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assays, screening and treatment methods described herein, and are not intended to limit the scope of the described aspects and embodiments.

[0143] Example 1: Identification of a pan-fungal consensus sequence for Kex1 To identify a pan-fungal consensus sequence for Kex1, multiple sequence alignments of KEX1 peptide sequences from Pneumocystis spp. (accession no. EU918304.1) (isolated from a macaque), Aspergillus fumigatus kexin (accession no. XM746441), Candida albicans kexin (accession no. AF022372), and Cryptococcus neoformans kexin (accession no. XP572303.1) were performed using Clustal Omega (http: / / www.ebi.ac.uk / Tools / msa / clustalo / ) and analyzed for sequence identity and similarity (Figure 1). The resulting 90-mer pan-fungal consensus KEX1 peptides (pan-fungal peptide 1 and pan-fungal peptide 2), shown in Figure 2, have approximately 97% and 69% amino acid sequence identity, respectively, to the amino acid sequence of Pneumocystis KEX1 isolated from humans. The corresponding DNA sequences of the pan-fungal 2 consensus Kex1 peptides were cloned into an Escherichia coli (E. coli) expression vector, and the recombinant proteins were produced, isolated, and purified.

[0144] Example 2: Production of recombinant kexin protein The amino acid sequences of Aspergillus Kex, Cryptococcus Kex, Candida Kex, and panfungal Kex peptides 1 and 2 shown in Figure 1 were reverse translated with Escherichia coli K-12 codon bias (www.ebi.ac.uk / Tools / st / emboss_backtranseq / ) and inserted into the expression vector pET-28b(+) (GenScript) using NcoI and BamHI restriction sites. Pneumocystis Kex1 was cloned using the Genbank: EU918304.1 sequence. Each insert contained an additional GC 5' to the Kex sequence and a CG after the Kex sequence to keep the Kex sequence insert in frame. Plasmids were transformed into E. coli BL21 (DE3) cells and transformed clones were selected by plating on LB agar supplemented with 40 μg / mL kanamycin. All recombinant kexin proteins were expressed and purified as described in Example 3 below.

[0145] Example 3: Purification procedure of recombinant kexin protein This example describes protocols for purifying histidine-tagged, recombinantly produced (pET28b vector) (Millipore-Sigma, US) Pneumocystis Kex1, Aspergillus Kex, Cryptococcus Kex, Candida Kex, panfungal Kex peptides 1 and 2. Aspergillus Kex and Cryptococcus Kex proteins have different elution conditions due to the insoluble nature of these recombinant proteins.

[0146] Materials and Equipment A. LB (Lysogeny Broth) growth medium with kanamycin (40 μg / mL), typically pH 7.5 in a volume of 1 L. Mix 10 g NaCl, 5 g yeast extract, and 10 g tryptone peptone; distilled / deionized H2O 2 Bring the volume to 1 L with O. B. 1 M IPTG solution. C. Extraction Buffer: (sterile filtered), 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, 6 M guanidine-HCl, pH 7.4. D. Wash Buffer: (sterile filtered) 50 mM Sodium Phosphate, 300 mM NaCl, 10 mM Imidazole pH 7.4. E. 1 M imidazole solution in 50 mM sodium phosphate, 300 mM NaCl, pH 7.4, 0.2 μm sterile filter (sterile filtration). F. HisPur™ Cobalt Resin (ThermoFisher #89966) G. Disposable 5 mL polypropylene column (Thermo P#29922). H. His-tag protease inhibitor cocktail (PIC) (Sigma P#8849). I. Bradford Dye (Bio Rad P#500-0006). J. Bovine serum albumin standard (ThermoFisher #23209). K. Coomassie blue staining solution containing 0.2% Coomassie blue, 7.5% acetic acid and 50% ethanol. L. 50% methanol, 10% acetic acid and 40% dHO 2 Coomassie blue destaining solution containing O. M. Acrylamide Bis 30% (Sigma P#1001356385) N. N,N,N',N'-Tetramethylethylenediamine (TEMED) (Sigma P#1001434505). O. Sodium dodecyl sulfate (SDS) (10% stock solution). P. Ammonium persulfate (APS) (10% stock solution). Q. 1.5 Tris buffer pH 8.8 (187 g Tris base in 1 L dHO 2 0 and adjust the pH to 8.8). R. 0.5 Tris buffer pH 6.5 (0.5 g Tris base 6 in 1 L dHO 2 0 and adjust the pH to 6.5). S. Spectra™ Multicolor Ladder--wide range stain (ThermoP#22634). T. SDS-PAGE sample buffer (4x). U. Hoefer gel casting system (model SE250).

[0147] The procedure used is as follows: A. Cultivation in E. coli and induction of protein expression: a. Streak the kexin on pET28b(+) construct onto an LB KAN agar plate and incubate at 37°C overnight (O / N) or on the bench at RT until sufficient bacterial growth / colony formation is obtained. (Plates can be stored at 4°C for approximately 1 month) b. Inoculate a single colony into 10 mL liquid LB KAN 40 (10 μL of 40 mg / mL KAN per 10 mL LB) (approximately 1:5 liquid to air ratio) and grow overnight at 37° C. with shaking. c. After overnight incubation, the culture is diluted 1:50 into liquid LB KAN 40 (4 mL of overnight culture into 196 mL of fresh medium) and placed on a shaker at 37°C. d. OD of the culture 600 Grow to 0.5 and then induce expression by adding 1 mM IPTG; leave at 37°C on a shaker for 4-5 hours. e. Divide the total culture volume into five 50 mL Oakridge tubes (approximately 40 mL of culture per tube). For scale-up: 250 mL Oakridge tubes for larger volumes can be used. f. Harvest the cells by centrifugation at 6,000 x g and 4°C for 25 minutes (SS-34 or SLA-1500 rotors can be used). g. Discard the supernatant and freeze the cell pellet at -80°C until use. Do not store the E. coli pellet for longer than 2 weeks prior to protein extraction.

[0148] B. Protein purification using Talon metal affinity resin: a. Thaw the pellet on ice and resuspend the cell pellet in 10 mL extraction buffer + 200 μL PIC. b. Incubate on a nutator at 4°C for 2 hours minutes. c. Centrifuge the suspension at 10,000 x g and 4°C for 20 minutes (using an SS-34 rotor). d. Collect the supernatant and keep on ice until the Talon resin is prepared. e. Prepare a polypropylene elution column by hanging the column in an upright position; add a few drops of wash buffer to the porous disc and then use the reverse end of a Pasteur pipette to press the disc evenly down to the bottom of the column. f. Prepare Talon Resin: Resuspend the Talon resin by gently shaking and add 3.5 mL of resin to a 15 mL conical tube and spin at 500 x g for 5 minutes. Carefully remove the ethanol layer without disturbing the resin. Wash the resin by adding 10 mL of deionized water and spin again at 500 x g for 5 minutes. Carefully remove and discard the supernatant. Equilibrate the resin in 10 mL of extraction buffer and spin at 500 x g for 5 minutes. Carefully remove and discard the supernatant. g. The clarified lysate and equilibrated resin are mixed together and batch bound by rocking at 4° C. for 1 hour. h. Add the lysate and resin suspension to the prepared polypropylene elution column. Discard the flow-through. i. Wash the resin with 15 column volumes of Extraction Buffer followed by 15 column volumes of Wash Buffer. j. Elution of Pneumocystis Kex1, Candida Kex, and panfungal peptide 2. Elute with increasing concentrations of imidazole in the wash buffer in 1.5 mL fractions and collect the eluted fractions. Elution gradient a. Add 1.5 mL of 75 mM imidazole in wash buffer and collect fractions. b. Add 1.5 mL of 100 mM imidazole in wash buffer and collect fractions. c. Add 1.5 mL of 125 mM imidazole in wash buffer and collect fractions. d. Add 1.5 mL of 150 mM imidazole in wash buffer and collect fractions. e. Add 1.5 mL (x2) of 175 mM imidazole in wash buffer and collect fractions. f. Add 1.5 mL (x3) of 200 mM imidazole in wash buffer and collect fractions. j. Pneumocystis Kex1, panfungal peptide 2 elution. k. Elution of Aspergillus sp. Kex and Cryptococcus sp. Kex (insoluble proteins) a. Resuspend the resin in 1.5 mL of 1% SDS and transfer into two 1.5 mL tubes. b. Boil the suspension in a heating block at 95°C. c. Centrifuge the suspension at 500 x g for 5 minutes. d. Collect the supernatant fraction containing the denatured protein. e. Steps a-d are repeated three times to recover additional protein. l. Add 20 μL of PIC to each fraction of interest and store at 4° C. (Imidazole solution should be stored on ice prior to use.)

[0149] C. Quantification of protein in elution fractions (Bradford Assay - Low Concentration Standard Curve): a. Remove BSA-100 μg / mL from the freezer (4° C.) and thaw on ice. b. Set up the standard curve cuvettes and add the thawed BSA / dH 2 Add the indicated amounts of both O and HCl (Table 1). (Table 1) TIFF0007636331000014.tif38128c. Add a cuvette to each fraction and dilute the sample 1:50 (20 µL sample + dHO 2 O 180μL). d.dH2 Prepare one-quarter Bradford dye in dHO (10 mL dye + dHO). 2 HO 30 mL) and add 800 µL to each cuvette (final volume 1 mL). e. Mix the cuvettes individually by inversion and incubate at RT for 15 min. f. After incubation, add 200 μL of 0 μg / mL BSA standard in duplicate to wells A1 and A2 of a 96-well flat-bottom plate, followed by 0.25 μg / mL BSA standard to wells B1 and B2. Continue adding increasing concentrations of BSA standards to the plate in the same order. g. Once all the BSA standards have been added to the plate, place duplicate samples in the wells directly below, then proceed to the top rows of the next two columns until the remaining rows are exhausted. h. After all samples are plated, read at 595 nm - "Low-conc. Std. Curve." i. Linear regression (R 2 ) and record the BSA standard curve values ​​(R 2 (Data from assays with a p < 0.95 should not be used.) Raw sample values ​​represent a 1:50 dilution and should be multiplied by 50 to reconvert to μg / mL. Once protein concentration has been determined, fractions intended for plate coating (e.g., ELISA / ELISPOT), injection, etc. should be run on a 15% 2 mm SDS-PAGE gel to assess purity.

[0150] D. Protein Identification by SDS-PAGE Gel Coomassie Blue Staining: a. For each gel, mix 1x glass cover plate, 1x white aluminum backing plate, 2x black plate spacers, and 1x white 10-lane stacking comb in dHO. 2 Wash with O. Rinse with 70% ethanol solution. Before casting, ensure that all solidified gel residue from previous use is removed. b. Once all materials have air dried, take the backing plate, lay it flat on the tabletop and place a spacer on either side of the plate before sandwiching it with a clear glass cover plate, making sure the notches on the spacer are properly aligned with the edges of both plates. c. Loosen all screws on the casting block and slide the sandwich plate with spacers onto the casters. Ensure all plates and spacers are even and aligned. Allow approximately 3 mm of the sandwich plate to protrude from the bottom of the casting block before carefully tightening the screws to avoid cracking the plate. d. Place the casting block in the holder and set the black plastic plug into the holder. Turn the plug to press the casting block into the black rubber mat of the holder. Make sure the bottom of the plate is tightly sealed with the rubber of the holder to prevent leakage. e. Prepare the separating / running gel according to the recipe below for a 15%-SDS PAGE gel and add the solution to the cavity between the aluminum backing plate and the glass cover plate. Leave approximately 1.5-2 cm of space above the sandwich plate for the stacking gel. Add dHO to the casting block. 2 Add approximately 1 mL of O. Once the APS / TEMED is added to the solution, the gel will begin to polymerize. Once the gel has set (approximately 35 minutes), remove the water layer and prepare the stacking gel solution from the recipe below. Add the solution quickly. TIFF0007636331000016.tif31128g. Immediately place the white 10-lane stacking comb into the stacking gel and allow to polymerize completely (approximately 10 min). h. Prepare samples to run on the gel: (5 µg protein per well) i. If the fraction concentration is less than 165 μg / mL, use 22.5 μL sample + 7.5 μL 4× sample buffer; ii. If fraction concentration is > 165μg / mL → Sample volume = 5μg / (Concentration(μg / mL) / 1000), 4x Sample buffer volume = 1 / 3 of sample volume. i. Heat inactivate all samples at 56°C in a water bath for 10 minutes. j. Remove the 15% SDS-PAGE gel from the casting block and attach it to the electrophoresis apparatus with 2x red clips. Fill the electrophoresis apparatus cavity and bottom tray with 1x SDS-PAGE running buffer. At this point, the stacking comb can be removed. Add 10 μL of Broad Range Stain (P# 26634) SPECTRA™ Multicolor Ladder to the first well of the gel, followed by 30 μL of prepared sample to subsequent wells. k. Once all samples are in, attach the electrodes to their appropriate terminals and turn the power on (red to red, black to black). Run the gel at 80-120 volts for approximately 1.5-2.5 hours until the dye bands leave the bottom of the gel. At that point, turn off the instrument and remove the electrodes (Note: lower voltages and shorter time intervals will result in better quality gels). l. Drain the running buffer from the electrophoresis apparatus. Remove the red clips, spacers and gently remove the glass cover plate from the gel casting frame. Using the hard plastic straight edge of a gel scraper, cut the stacking gel and place in the trash. Split the gel as needed for further assays, i.e. Western Blot, etc. (Since a stain ladder is being used, it is not necessary to score the corners of the gel to establish orientation). m. For resolving gels that are going to be stained, rinse with dHO for 15 min. 2 Wash 3 times with 500 mL of O. Add approximately 25 mL of Coomassie Blue stain to the gel for 2+ hours or overnight as needed. Destain with Coomassie Blue destain until optimal band color / gel clarity is achieved. Take a photo and save as a JPG / TIF file.

[0151] Example 4: Generation of immune sera Immunization of rhesus macaques with recombinant panfungal 2 peptides (Study design, Fig. 3A) An adult rhesus macaque (monkey subject no. 2116) was immunized intramuscularly with 100 μg of panfungal peptide 2. The monkey was boosted 8 weeks after dosing and infected with SIV 8 weeks after the boost. With reference to Figures 3B-3E, ELISA analysis of serum samples collected from monkey subject no. 2116 was used to determine the reciprocal endpoint titers (RET) for the targets panfungal peptide 2, Cryptococcus Kex, Pneumocystis Kex1, and Aspergillus Kex. As used herein, RET is the reciprocal of the highest dilution of an analyte (e.g., antibody or antiserum) that gives a reading above a cutoff value. For RET analysis, serum obtained from the test animal is assayed to determine the presence of immunoglobulins (IgG) that target, bind to, and / or have activity against the target protein. Peak titers for each target were 10 5 The antibody titers exceeded 100% in the control group, indicating the presence of primary antibodies that recognize kexin peptides from multiple fungal species. As illustrated in Figure 3F, more than half of the sham-vaccinated monkeys developed Pneumocystis pneumonia, whereas none of the pan-fungal-vaccinated monkeys developed pneumonia.

[0152] Example 5: Kex peptide enzyme-linked immunosorbent assay (ELISA) This example describes a protocol for performing an ELISA immunoassay utilizing Pneumocystis Kex1 protein, panfungal peptide 2, or Kex peptides from one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus. The ELISA is performed to detect the presence (and quantitate) of anti-fungal Kex peptide antibodies in a sample, such as blood, plasma, serum, bronchoalveolar lavage fluid, or a biological fluid sample. The anti-Kex peptide antibodies detected (and quantified) may target, react with, and / or bind to panfungal peptide 2, or one or more Kex peptides from Pneumocystis, Aspergillus, Candida, or Cryptococcus Kex peptides.

[0153] Materials and Equipment A. KEX1 protein, which can be purified as described in Example 2. B. 1x PBS C. Immulon high binding (4HBX) flat bottom microtiter plate (Thermo #3855) D. Blocking buffer: 5% skim milk in 1x PBS E. Wash Buffer: 1x Phosphate Buffered Saline (PBS) + 0.05% Tween-20 F. Secondary antibody: goat anti-human immunoglobulin-conjugated horseradish peroxidase (1:10,000 for IgG; Sigma-Aldrich). G. Normal human plasma (Atlanta Biologicals, Inc., Lawrenceville, GA). OD 2.000 in KEX-ELISA at a dilution of 1:100. 450 Negative / normal control plasma with undetectable absorbance at (i.e., below dilution buffer only) is used as a negative control. H. Substrate: 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase substrate (e.g., SureBlue TMB Substrate, 1-component; KPL, Inc.) I. Stop solution: 1 MH 2 SO4 J. Adhesive sealing film for microplates (such as SealPlate nonsterile film from Excel Scientific, catalog number 100-SEAL-PLT) K. 96-well plate reader (any system capable of reading OD at 450 nm wavelength).

[0154] The procedure for performing the ELISA is as follows: A. Coating / blocking ELISA plates with Kex1 protein: a. Prepare mkKex protein at 5 μg / mL in 1× PBS. Add 50 μL of diluted KEX1 per well of an Immulon 4HBX flat-bottom ELISA plate. Cover plate tightly with parafilm or plate sealer and incubate at 4° C. overnight. b. After overnight incubation, remove the buffer by flicking into a sink or bucket and tap the plate onto an absorbent pad or paper towel to remove excess. Wash the plate twice with wash buffer (PBS 0.05% Tween-20) (approximately 200 μL wash buffer per well per wash, flicking and tapping the plate between washes). c. Add 100 μL of blocking buffer (5% milk / PBS) to each well and incubate at 37° C. for 1 hour. d. Empty the plates and wash twice with Wash Buffer. Plates can be sealed and frozen at -20°C at this stage until ready for use.

[0155] B. Working with plasma or other infectious fluids (e.g., bronchoalveolar lavage (BAL) fluid supernatants) - First time use. a. Remove the plasma aliquot from the -80°C freezer. b. Option 1: Heat inactivate the entire aliquot at 56°C for 30 minutes. Option 2: If heat inactivation of the plasma sample would adversely affect other potential applications, thaw the sample at 4°C or on ice. Remove an aliquot (approximately 100 μL), transfer to a new tube, and heat inactivate (30 min, 56°C). Place the remaining sample back into the -80°C freezer, noting that it has been thawed once. c. Centrifuge samples at >10,000 x g for 1-2 minutes to pellet aggregates prior to use. d. Approximately 0.01-0.02% NaN to prevent contamination during storage. 3 Add 0.5% CO. Store sample aliquots at 4 °C for up to 6 months. No further heat inactivation is required for subsequent assays; however, samples should be briefly centrifuged before each use.

[0156] C. ELISA for endpoint titer determination (plasma): a. Dilute plasma 1:100 in blocking buffer. Add 50 μL of diluted plasma and perform serial 2-fold (or 4-fold if necessary) dilutions directly in the plate for generation of endpoint titers (final volume in each well should be 50 μL). Perform assays in duplicate: Set up enough plates for all isotypes of interest, for example, if there are 10 samples and one is generating endpoint titers for both IgG and IgM-KEX1 antibodies, this will require setting up 4 plates (duplicate plates for both IgG and IgM). Include negative / normal controls on each plate. Cover plates with plate sealers and incubate at 4° C. overnight. b. Empty the plate (flicking and tapping) and wash 4 times with wash buffer. c. Add 50 μL of secondary antibody (diluted in block) to each well (see appropriate dilution in Materials and Equipment section above). Incubate at 37° C. for 1 hour. d. Empty the plate and wash six times with wash buffer. e. Add 100 μL of TMB to each well and incubate at 37° C. for 30 minutes, protected from light. f. Add stop solution (1M H 2 SO 4 )Add 25-50μL. g. Read the OD of the plate (in any standard plate reader) at 450 nm within 20 minutes of adding the Stop Solution.

[0157] The majority of healthy adults (both humans and non-human primates) have circulating antibodies against Pneumocystis; therefore, when selecting control samples to be used in calculating endpoint titers, it is necessary to screen plasma samples from healthy donors to determine and obtain appropriate controls. Plasma from appropriate normal / negative controls should have a KEX1 OD at a dilution of 1:100. 450 should be below 0.1; however, the lower the OD of the normal / negative control plasma, the better the control. To control for plate-to-plate variability, after selection of the appropriate normal / negative control, the same normal / negative control should be used on all plates.

[0158] D. ELISA for endpoint titer determination (BAL supernatants). a. Dilute BAL supernatant 1:100 in normal saline. i. Determine urea concentrations of BAL supernatants and corresponding plasma samples using the QuantiChrom Urea Assay (BioAssay Systems Cat. No. DIUR-500). 1. Dilute plasma 1:10 in distilled water according to kit insert instructions and use BAL supernatant undiluted. 2. Plasma samples are plated into wells of a 96-well plate and 5 μL of standards (1:10 dilution), blank (distilled water) and samples (1:10 dilution) are added in duplicate. 3. Plate the BAL supernatant into wells of a 96-well plate and add 50 μL of standards (diluted to 5 mg / dL), blanks (distilled water) and samples (undiluted). 4. Add 200 μL of Working Reagent (included in the kit) and tap gently to mix. 5. Incubate the plasma plate at room temperature (RT) for 20 minutes and measure the OD in a spectrophotometer. 520 Read in. 6. Incubate the BAL supernatant plate at RT for 50 min and measure the OD 430 Read in. 7. Calculate the urea concentration ([Urea]) in plasma and BAL supernatant as follows: [Urea] = (OD Sample - OD Blank) / (OD Standard - OD Blank) x [Standard]. The standard concentration in plasma will be 50 mg / dL and in BAL supernatant will be 5 mg / dL. 8. Calculate a 1:100 dilution of BAL supernatant as follows: a. Find the 1:100 dilution ratio of bal to plasma i. Dilution factor = 100 / (plasma[urea] / bal[urea]) b. Calculate the volume of the dilution for a total sample of 500 μl i. Sample volume = 500μL / dilution factor ii. Volume of saline = 500μL-volume of sample 9. Add sample volume and saline volume to generate a 1:100 diluted BAL supernatant sample. b. Add 50 μL of diluted BAL supernatant and perform serial 2-fold (or 4-fold if necessary) dilutions directly in the plate with normal saline for generation of endpoint titers (final volume in each well should be 50 μL). Perform assays in duplicate: Set up enough plates for all isotypes of interest, for example, if there are 10 samples and endpoint titers are generated for both IgG and IgM-KEX1 antibodies, this will require setting up 4 plates (duplicate plates for both IgG and IgM). Include negative / normal controls in each plate as above. Cover plates with plate sealers and incubate at 4° C. overnight. c. Empty the plate (flicking and tapping) and wash 4 times with wash buffer. d. Add 50 μL of secondary antibody (diluted in block, see Materials and Equipment section for appropriate dilution) to each well. Incubate at 37° C. for 1 hour. e. Empty the plate and wash six times with wash buffer. f. Add 100 μL of TMB to each well and incubate at 37° C. for 30 minutes, protected from light. g. Add stop solution (1M H 2 SO 4 ) Add 25~50μL. h. Read the OD of the plate (in any standard plate reader) at 450 nm within 20 minutes of adding the Stop Solution.

[0159] E. Determination of endpoint titers. Plot the OD readings from each sample (at all dilutions) as a line graph in Excel or a similar program. For normal / negative control samples, add 0.025 to each value before plotting as described below. b. The endpoint titer is defined by the dilution at which the test sample gives the same OD reading as that of the negative control (i.e., where the lines cross). Generally, the reciprocal endpoint titer is reported; thus, if the dilution is 1:1600, the endpoint titer is reported as 1600. c. Calculate the endpoint titer from each of the duplicate plates to ensure results are consistent between plates. The acceptable error is within 1 dilution. If the reciprocal endpoint titers (RET) from duplicate plates are within 1 dilution, average the titers (e.g., if 2-fold dilutions are made and the RET of a sample from plate 1 is 1600 and the RET from plate 2 is 3200, the average titer is 2400). If the endpoint titers of duplicate plates are not within 1 dilution of each other, repeat the ELISA on one additional plate and average the closest two values.

[0160] Example 6: Panfungal vaccination Adult rhesus macaques were immunized intramuscularly with 100 μg of the panfungal2 peptide and aluminum hydroxide (Imject Alum, ThermoScientific) mixed in a 1:1 ratio, and boosted 8 weeks later with 50 μg of the panfungal2 peptide and aluminum hydroxide. Eight weeks after the boost, the macaques were infected with simian immunodeficiency virus (SIV) and challenged with Pneumocystis (FIG. 3A). Seven additional macaques were vaccinated with a sham vaccine consisting of aluminum hydroxide but not containing any of the Kex peptides. See FIG. 3F, the percentage of monkeys diagnosed with Pneumocystis pneumonia steadily increased from week 10 to week 40 in sham-vaccinated monkeys. No monkeys vaccinated with the panfungal2 peptide developed pneumonia.

[0161] Example 7: Wild-type Pneumocystis Kex1 vaccination 100 μg of Pneumocystis sp. Kex1 mixed in a 1:1 ratio Adult rhesus macaques were immunized intramuscularly with TIFF0007636331000017.tif11137 and aluminum hydroxide (Imject Alum, Thermo Scientific), then boosted 8 weeks later with 50 μg wild-type Kex1 and aluminum hydroxide. Eight weeks after the boost, the macaques were infected with simian immunodeficiency virus (SIV) and challenged with Pneumocystis. Ten and 14 weeks after infection, the macaques were boosted with 100 μg wild-type Kex1 and 5 μg α-GC. Seven additional adult rhesus macaques were immunized intramuscularly with alum mixed at a 1:1 ratio, then boosted 8 weeks later with alum. Eight weeks after the boost, the macaques were infected with simian immunodeficiency virus (SIV), challenged with Pneumocystis (Figure 4A), and boosted with PBS at 10 and 14 weeks after infection. See FIG. 4B for ELISA analysis of serum samples collected from the Pneumocystis Kex1 vaccinated group and sham controls. After the second wild-type Kex1 + alum and wild-type Kex1 + α-GC boost, the peak titers in the wild-type Kex1 vaccinated group were 10 5With reference to FIG. 4C, only one of six wild-type Pneumocystis Kex1 vaccinated monkeys developed Pneumocystis pneumonia, which occurred at the end of 40 weeks of Pneumocystis challenge.

[0162] Example 8: Therapeutic vaccination in SIV immunocompromised non-human primates (NHPs) Adult rhesus macaques were infected with SIV and boosted 48, 55, and 61 weeks later with 50 μg Pneumocystis Kex1 and 5 μg α-galactosylceramide (αGC) as an adjuvant (Figure 5A). See Figure 5B for ELISA analysis of serum samples collected from Pneumocystis Kex1 vaccinated animals. After the second and third boosts at 57 and 63 weeks post-infection with α-GC, the peak reciprocal endpoint titers in the wild-type Kex1 vaccinated group were 10 4 It exceeded that.

[0163] Example 9: Assessment of immunogenicity and protective efficacy of PF, KEX1 in an Aspergillus fumigatus (AF)-infected mouse model As described below, an invasive pulmonary aspergillosis (IPA) mouse model was used to evaluate whether mice immunized with a panfungal Kex peptide (panfungal peptide 2) and adjuvant would mount a humoral immune response that protected immunized animals from subsequent infection with Aspergillus pathogens.

[0164] Panfungal peptide 2 Humoral immune response to The immunogenicity of the pan-fungal peptide 2 immunogen (referred to herein as the "vaccine candidate") was assessed by immunizing animals with recombinant pan-fungal peptide 2 and TiterMax adjuvant, or with PBS and TiterMax, according to the schedule shown in Figure 6A. Figure 6B shows that anti-pan-fungal peptide 2 antibody titers increased significantly following immunization of mice, peaking at 28 days post-immunization (average 5 x 10 antibody titers) compared to the sham-immunized cohort. 5 )( **p=0.002).

[0165] Panfungal peptide 2 Immunization protects against invasive pulmonary aspergillosis (IPA) in immunosuppressed CF-1 mice After vaccination, seven panfungal peptide 2-immunized mice and seven sham-immunized mice were immunosuppressed for 6 days and then immunized with 5 × 10 6 Mice were exposed intranasally to Aspergillus fumigatus (Af293) conidia (Figure 7). Mice were then observed twice daily for signs of aspergillosis: ruffled fur, respiratory distress, weight loss, and decreased body temperature. During the observation period, three of the sham-immunized cohort showed evidence of aspergillosis and met the criteria for euthanasia. Over the course of the experiment, none of the panfungal peptide 2-immunized mice showed severe symptoms or died of aspergillosis. The survival rate of panfungal peptide 2-immunized mice was significantly higher compared to that of sham-immunized mice ( * p=0.05). Furthermore, sham-immunized mice had a 1.7-fold relative risk of invasive pulmonary aspergillosis (IPA)-related death compared to the risk seen in panfungal peptide 2-immunized animals (Table 2).

[0166] Table 2. Association between panfungal peptide 2 vaccination and IPA-related deaths TIFF0007636331000018.tif101136

[0167] Table 2 presents the association and relative risk analysis comparing vaccination with observation of humane endpoint criteria. Panfungal peptide 2 vaccination showed a significant association with IPA-related death ( * p=0.05), whereas sham-vaccinated mice had a 1.75-fold relative risk of IPA-related death. These results were further supported by the analysis of fungal burden performed using GMS staining histological quantification. The A. fumigatus pulmonary fungal burden was significantly reduced in pan-fungal peptide 2-immunized mice compared to that of the sham-immunized cohort (Figure 8) ( * p=0.026).

[0168] The materials and methods used in this example are as follows. Study design Fourteen CF-1 mice (Charles River Laboratories) were randomly assigned to immune and sham cohorts. Blood was collected at baseline (day 0) and 14 and 28 days after immunization (Figure 6A). Plasma samples were stored at -80°C.

[0169] Vaccine construction and purification Pan-fungal peptide 2 (SEQ ID NO: 2), a recombinant protein consisting of 90 amino acids, was cloned into the pET28b(+) expression vector (Novagen) in E. coli BL21(DE3) pLysS (ThermoFisher Scientific) and purified by affinity chromatography.

[0170] Immunity and immunosuppression Seven mice were immunized subcutaneously at the base of the tail with 50 μg of panfungal peptide 2 prepared 1:1 in TiterMax adjuvant according to adjuvant guidelines. An additional seven mice were sham-immunized with PBS and TiterMax. 28 days after immunization, all mice were started on an immunosuppressive regimen with a subcutaneous injection of 2.5 mg cortisone acetate per mouse in PBS containing 0.5% methylcellulose and 0.01% Tween-80. This regimen was administered for 6 days (Figure 7), during which trimethoprim sulfamethoxazole was added to the drinking water to control secondary infections.

[0171] Aspergillus fumigatus Exposure and Monitoring A. fumigatus Af293 conidia were maintained on solid 1% glucose minimal medium for 72 h, harvested in 0.01% Tween-20, counted with a hemocytometer, and then diluted in PBS. 5 × 10 in 40 μl PBS were collected by intranasal inoculation after 6 days of cortisone acetate immunosuppression as described above. 6 Mice were inoculated with conidia.

[0172] After challenge, mice were monitored twice daily for changes in weight, temperature, and appearance. Animals were humanely killed if they exhibited ruffled fur and respiratory distress, as well as weight loss of more than 20% of baseline weight, or a temperature below 29°C. At 6 days post-challenge, all remaining animals were humanely killed and their lungs harvested for analysis.

[0173] fungal load After sacrifice, the right lungs of all animals were stored in 10% neutral buffered formalin. Fixed lung tissues were embedded in paraffin, sectioned, and stained with Gomori's modified methaneamine silver stain. Five different fields were taken and fungal burden was quantified according to the guidelines provided by Stolz et al., 2018, J. Vis. Exp., (133) e57155:1-8.

[0174] statistical analysis All statistical analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, CA). Reduction in IPA-associated mortality was assessed using Fisher's exact test. Relative risks of IPA mortality were also determined using the Koopman asymptotic score. Differences in fungal burden by GMS staining were analyzed by Mann-Whitney U test.

[0175] Example 10: Assessment of immunogenicity and protective efficacy of panfungal peptide 2 in a non-human primate model of Pneumocystis and HIV (SIV) co-infection Panfungal peptide 2 Humoral immune response to The immunogenicity of the pan-fungal peptide 2 immunogen (referred to herein as the "vaccine candidate") was assessed by immunization (vaccination) of non-human primates with recombinant pan-fungal peptide 2 and alum or with PBS and alum according to the schedule shown in Figure 9A. Anti-pan-fungal peptide 2 antibody titers increased significantly in immunized animals, peaking 2 weeks after the boost (mean = 4 x 10 5 ), but no significant changes in antibody titers were observed in the sham-immunized cohort ( Fig. 9B ).

[0176] Panfungal peptide 2 Immunity protects against Pneumocystis pneumonia All animals were infected with simian immunodeficiency virus (SIV) and monitored monthly for Pneumocystis spp., following the vaccination regimen diagrammed in FIG. 9A. Over the course of SIV infection, five macaques in the sham-immunized cohort tested positive for Pneumocystis spp. pneumonia (PCP) by PCR and bronchoalveolar lavage (BAL) smears by 32 weeks post-SIV infection. By this same time point, none of the macaques immunized with panfungal peptide 2 had developed symptoms or tested positive for PCP. This finding demonstrates a significant reduction in the number of PCP cases in panfungal peptide 2-immunized animals (p=0.035). Table 3 below presents data regarding the association of panfungal peptide 2 immunization (vaccination) with Pneumocystis spp. pneumonia (PCP) disease in the study animals, and the significant association of panfungal peptide 2 immunization (vaccination) with PCP (p=0.026). The association and relative risk between immunization of animals with panfungal peptide 2 and the diagnosis of PCP are shown in Table 3. Furthermore, the relative risk of sham-immunized (vaccinated) animals contracting PCP was determined to be 3.3 times the relative risk of panfungal peptide 2-immunized (vaccinated) animals (Table 3).

[0177] Table 3. Association between pan-fungal peptide 2 vaccination and Pneumocystis pneumonia. TIFF0007636331000019.tif83141

[0178] Panfungal peptide 2 Immunization reduces Pneumocystis colonization In addition to detection of Pneumocystis by first round PCR, BAL samples were evaluated for colonization by Pneumocystis by nested PCR. Figure 10 illustrates the time (in weeks) to chronic colonization (determined by 2 or more consecutive months of nested PCR positivity) in both the pan-fungal peptide 2-immunized and sham-immunized cohorts. Chronic colonization was found to be significantly reduced in the pan-fungal peptide 2-immunized cohort compared to the sham-immunized cohort up to week post infection (wpi) 28 ( * p=0.05).

[0179] The materials and methods used in this example are as follows.

[0180] animal Fifteen adult Chinese rhesus macaques ( Macaca mulatta ) were obtained from the National Primate Center or a supplier and housed in a BSL2+ primate facility in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

[0181] Vaccine construction and purification Pan-fungal peptide 2, a recombinant protein consisting of 90 amino acids, was cloned into the pET28b(+) expression vector (Novagen) in Escherichia coli BL21(DE3) pLysS (ThermoFisher, Scientific) and purified by affinity chromatography. The recombinant pan-fungal peptide 2 protein was used for immunoassay and enzyme-linked immunosorbent assay (ELISA).

[0182] Immunization and study design Seven macaques were immunized intramuscularly with 100 μg of panfungal peptide 2 and aluminum hydroxide (Imject Alum, Thermo Scientific) mixed in a 1:1 ratio. A control cohort of eight sham-immunized (vaccinated) macaques received PBS and alum. The animals were allowed to rest for 8 weeks, after which time the panfungal peptide 2-immunized animals were boosted with 50 μg of panfungal peptide 2 and alum, and the sham-immunized animals were sham-vaccinated as above. Eight weeks after the boost, NHP animals were immunized with SIV / Delta B670 (1:100 in PBS), tissue culture infectious dose 50% (TCID 50 =2.6×10 5 Mice were infected intravenously with Pneumocystis spp. Viral infection was monitored weekly for 4 weeks post-infection and monthly thereafter for up to 32 weeks. Bronchoalveolar lavage fluid (BAL) was collected monthly to monitor for the presence of Pneumocystis spp. infection.

[0183] Determination of Pneumocystis exposure and Pneumocystis infection Pneumocystis spp. (Pc) cannot be reliably cultured in vitro. Therefore, Pneumocystis exposure of panfungal peptide 2-immunized and sham-immunized macaques was performed via natural airborne infection by co-housing the animals as previously described by Kling et al. (2010, Infect. Immun., 78(10):4320-4330). Pneumocystis pneumonia (PCP) was defined as detection of Pneumocystis spp. in BAL fluid by first-round PCR and / or by microscopic-based detection of Pneumocystis spp. in BAL smears. Nested PCR was performed by amplifying 5 μL of the first-round product as previously described (Savoia, D. et al., 1997, Diagn Microbiol Infect Dis, 29(2):61-65). Chronic pulmonary Pneumocystis colonization was defined as detection of Pneumocystis DNA in nested rounds of PCR for ≥2 consecutive months. To control for DNA quality of BAL fluid samples, PCR to detect β-globin was also performed on BAL samples at each time point (method described by Croix, DA et al., 2002, AIDS Res Hum Retroviruses, 18(5):391-401).

[0184] statistical analysis All statistical analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, CA). Associations between panfungal peptide 2 immunization (vaccination) and PCP incidence in the vaccinated group compared with the sham-immunized group were determined using Fisher's exact test. Differences in PCP diagnosis between the panfungal peptide 2-immunized and sham-immunized cohorts were compared using the Kaplan-Meier test. Relative risks of PCP were calculated using the Koopman asymptotic score. Chronic Pc colonization was assessed by Mantel-Cox test.

[0185] Other Aspects From the foregoing description, it will be apparent that variations and modifications can be made to the aspects and embodiments described herein to accommodate various usages and conditions, such embodiments also falling within the scope of the following claims.

[0186] The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0187] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

1. An immunogenic peptide having 95% or greater amino acid sequence identity to PDDGKTMEGPDILVLRAFINGVQNGRDGKGSIYVFASGNGGGFEDNCNFDGYTNSIYSITVGAIDRKGLHPSYSEACSAQLVVTYSSGSG (SEQ ID NO: 2; pan-fungal peptide 2), or a polynucleotide encoding said immunogenic peptide.

2. An immunogenic peptide comprising or consisting of PDDGKTMEGPDILVLRAFINGVQNGRDGKGSIYVFASGNGGGFEDNCNFDGYTNSIYSITVGAIDRKGLHPSYSEACSAQLVVTYSSGSG (SEQ ID NO: 2; pan-fungal peptide 2), or a polynucleotide encoding said immunogenic peptide.

3. 10. An immunogenic composition comprising an effective amount of a peptide according to claim 1 or 2 or a polynucleotide encoding said peptide, and a pharma- ceutically acceptable carrier or excipient.

4. The immunogenic composition of claim 3, comprising an adjuvant comprising α-galactosylceramide (αGC).

5. 1. A pharmaceutical composition for inducing an immune response in a subject, comprising: A pharmaceutical composition comprising the immunogenic peptide, polynucleotide or immunogenic composition of any one of claims 1 to 4.

6. 1. A pharmaceutical composition for treating or protecting a subject against a fungal infection, or a disease, disorder or condition caused by or associated with a fungal infection, comprising: A pharmaceutical composition comprising an immunogenic peptide, polynucleotide or composition according to any one of claims 1 to 4.

7. 1. A pharmaceutical composition for treating or protecting a subject against a fungal infection, or a disease, disorder or condition caused by or associated with a fungal infection, comprising: The pharmaceutical composition comprises an immunogenic peptide, polynucleotide or composition according to any one of claims 1 to 4, The immunogenic peptide, polynucleotide, or composition comprises: Antisera or isolatable antibodies that specifically bind to an immunogenic peptide comprising the amino acid sequence set forth in SEQ ID NO: 2 and specifically bind to a kexin peptide of one or more fungal pathogens. The pharmaceutical composition induces the production of

8. 8. The pharmaceutical composition of claim 7, wherein the one or more fungal pathogens comprise Pneumocystis, Aspergillus, Cryptococcus, or Candida.

9. the fungal infection, disease, or disorder is caused by or associated with infection with Aspergillus, Candida, Cryptococcus, or Pneumocystis; 7. The pharmaceutical composition of claim 6.

10. 10. The pharmaceutical composition of any one of claims 7 to 9, for treating a pulmonary disease, pulmonary dysfunction, or a symptom thereof.

11. 11. The pharmaceutical composition of claim 10, wherein the lung disease or pulmonary dysfunction is Pneumocystis pneumonia (PCP), aspergillosis, or invasive pulmonary aspergillosis (IPA).

12. 1. A pharmaceutical composition for treating or protecting a subject against a fungal infection, or a disease or disorder caused by or associated with a fungal infection, comprising: An immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:2 or a polynucleotide encoding the amino acid sequence shown in SEQ ID NO:2 in an amount effective to induce an immune response against one or more of the fungal pathogens of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus. The pharmaceutical composition comprising:

13. 1. A pharmaceutical composition for treating or protecting a subject against a fungal infection, or a disease or disorder caused by or associated with a fungal infection, comprising: The pharmaceutical composition comprises an effective amount of an antibody that specifically binds to the amino acid sequence shown in SEQ ID NO:

2.

14. 14. The pharmaceutical composition of claim 13, wherein the antibody is present in or isolated from antisera derived from a donor subject.

15. The pharmaceutical composition of any one of claims 5 to 14, wherein the subject is a mammal or a human patient.

16. The pharmaceutical composition of any one of claims 5 to 15, wherein the subject is immunocompromised or immunosuppressed and / or a pre-transplant or post-transplant subject.

17. The pharmaceutical composition of any one of claims 5 to 16, wherein the subject is immunocompromised or immunosuppressed and / or is being treated for cancer, an immunodeficiency disease, a congenital disease, an autoimmune disease, HIV, rheumatoid arthritis or psoriasis.

18. A vaccine comprising an effective amount of the immunogenic peptide or polynucleotide of claim 1.

19. A vaccine comprising an effective amount of the immunogenic peptide or polynucleotide of claim 2.

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