Immunogenic antifungal polypeptides, compositions and methods thereof

Abstract

Immunogenic compositions comprising non-naturally occurring generic fungal kexin peptides are described and characterized and the use of such compositions in the treatment or prevention of various diseases or severe diseases and / or symptoms thereof in a subject, in particular in the treatment or prevention of various diseases or severe diseases and / or symptoms thereof, in particular in the treatment or prevention of diseases associated with fungal pathogens (e.g., fungal pathogens). The present invention relates to a method for treating diseases or severe diseases and / or symptoms thereof associated with infection by genus Pneumocystis, Aspergillus, Candida, or Cryptococcus, e.g., Aspergillus, Candida, or Cryptococcus. The immunogenic peptides or immunogenic fragments thereof may be recombinantly produced, recombined, and / or isolated.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the priority and benefit of U.S. Provisional Application No. 63 / 409,491, filed on September 23, 2022, the content of which is incorporated herein by reference in its entirety.

[0003] Statement of Government Support

[0004] This invention was made with government support under Grant No. R01 AI148365 awarded by the National Institutes of Health. The government has certain rights in the invention. Background Art

[0005] Pathogenic fungal organisms are ubiquitous in the environment and can pose a global health threat. Pathogenic fungi typically do not harm individuals with a healthy and functioning immune system, even after exposure (e.g., by inhalation). However, individuals with a weakened or impaired immune system or those with pre - existing medical conditions (e.g., those with lung diseases or viral infections such as HIV / AIDS infections) are at a higher risk of developing severe health problems and adverse reactions after exposure to and infection by fungal pathogens.

[0006] Due to the severe consequences of fungal organism infections for 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 related diseases caused by these pathogens. There is a need for cost - effective and efficient methods of treating and preventing fungal pathogens, especially in the less affluent regions of the world. Given the continuing health threat posed by fungal pathogens to at - risk individuals globally, there is a particular need for immunogens with improved characteristics for use in methods of treating or preventing fungal - pathogen - related diseases for which there are currently no suitable therapies or the therapies are inadequate. Summary of the Invention

[0007] Provided and described herein are immunogens, compositions, and methods for treating or preventing diseases associated with or caused by fungal pathogen infections (e.g., opportunistic infections), as well as for treating or preventing lung diseases and poor lung function and other diseases and / or their symptoms associated with fungal pathogen infections.

[0008] In one aspect, there is provided an immunogenic peptide or a polynucleotide encoding the immunogenic peptide, the immunogenic peptide comprising an amino acid sequence (in the direction from the amino - terminus (NH2) to the carboxy - terminus (COOH)):

[0009] 1DDDGKTVDGPSPLVLRAFINGVNNGRNGLGSIYVFASGNGGIYEDNSNFD

[0010] 51GYANSVFTITIGGIDKHGKRPKYSEASSSQLAVTYAGGSA(SEQ ID NO:1), (referred to herein as "pan-fungal peptide 1b" or "PF-KEX1b" peptide). In the examples, functional fragments of the PF-KEX1b immunogenic peptide are provided.

[0011] In one aspect, provided is an immunogenic peptide or a polynucleotide encoding the immunogenic peptide, the immunogenic peptide comprising an amino acid sequence (in the direction from the amino terminus (NH2) to the carboxyl terminus (COOH)):

[0012] 1PDDGKTMEGPDILVLRAFINGVQNGRDGKGSIYVFASGNGGGFEDNSNFD

[0013] 51GYTNSIYSITVGAIDRKGLHPSYSEASSAQLVVTYSSGSG(SEQ ID NO:2), (referred to herein as "pan-fungal peptide 2b" or "PF-KEX2b" peptide). In the examples, functional fragments of the PF-KEX2b immunogenic peptide are provided.

[0014] In one aspect, provided is an immunogenic peptide or a polynucleotide encoding the immunogenic peptide, the immunogenic peptide consisting of the following amino acid sequence:

[0015] 1DDDGKTVDGPSPLVLRAFINGVNNGRNGLGSIYVFASGNGGIYEDNSNFD

[0016] 51GYANSVFTITIGGIDKHGKRPKYSEASSSQLAVTYAGGSA(SEQ ID NO:1) (pan-fungal peptide 1b or PF-KEX1b).

[0017] In one aspect, provided is an immunogenic peptide or a polynucleotide encoding the immunogenic peptide, the immunogenic peptide consisting of the following amino acid sequence:

[0018] 1PDDGKTMEGPDILVLRAFINGVQNGRDGKGSIYVFASGNGGGFEDNSNFD

[0019] 51GYTNSIYSITVGAIDRKGLHPSYSEASSAQLVVTYSSGSG(SEQ ID NO:2) (pan-fungal peptide 2b or PF-KEX2b).

[0020] In one aspect, there is provided an immunogenic peptide or a polynucleotide encoding an immunogenic peptide, said immunogenic peptide having at least 98% sequence identity with the following amino acid sequence:

[0021] DDDGKTVDGPSPLVLRAFINGVNNGRNGLGSIYVFASGNGGIYEDNSNFD

[0022] GYANSVFTITIGGIDKHGKRPKYSEASSSQLAVTYAGGSA (SEQ ID NO:1) (PF-KEX1b), wherein said peptide contains serine (S) amino acid residues at least at positions 47 and 77 of said amino acid sequence.

[0023] In one aspect, there is provided an immunogenic peptide or a polynucleotide encoding an immunogenic peptide, said immunogenic peptide having at least 98% sequence identity with the following amino acid sequence:

[0024] PDDGKTMEGPDILVLRAFINGVQNGRDGKGSIYVFASGNGGGFEDNSNFD

[0025] GYTNSIYSITVGAIDRKGLHPSYSEASSAQLVVTYSSGSG (SEQ ID NO:2), (PF-KEX2b), wherein said peptide contains serine (S) amino acid residues at least at positions 47 and 77 of said amino acid sequence.

[0026] In an embodiment of any of the above-described aspects, there is provided an immunogenic peptide of an immunogenic polypeptide or an immunogenic fragment of an immunogenic peptide. In an embodiment of any of the above-described aspects and their embodiments, the immunogenic PF-KEX1b and PF-KEX2b peptides, or immunogenic portions thereof, may provide benefits related to manufacturing and formulation methods. As a non-limiting example, the peptides are stable and are not expected to crosslink and / or aggregate. In an embodiment of any of the above-described aspects and / or their embodiments, the immunogenic polypeptide, the immunogenic peptide of the immunogenic polypeptide, or the immunogenic fragment of the immunogenic polypeptide or peptide is recombinant and / or recombinantly produced. In an embodiment of any of the above-described aspects and / or their embodiments, the immunogenic polypeptide, the immunogenic peptide of the immunogenic polypeptide, or the immunogenic fragment of the immunogenic polypeptide or peptide is isolated. The terms "polypeptide" and "peptide" may also be used interchangeably herein.

[0027] In some embodiments, the polypeptide or its functional peptide, or the peptide or its functional fragment has at least 85%, at least 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% amino acid sequence identity with the sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0028] In another aspect, there is provided an immunogenic composition comprising an effective amount of an immunogenic peptide or a polynucleotide encoding the immunogenic peptide described above, and a pharmaceutically acceptable carrier, vehicle or excipient. In some embodiments, the immunogenic composition further comprises an adjuvant. In an embodiment, the adjuvant is α-galactosylceramide (αGC), alum or

[0029] In another aspect, there is provided a method of eliciting an immune response in a subject, wherein the method involves administering to the subject an immunogenic peptide, a coding polynucleotide, or an immunogenic composition of any one of the aspects and / or their embodiments described above.

[0030] In another aspect, there is provided a method of treating or protecting a subject from a disease or its symptoms associated with or caused by a fungal infection, wherein the method involves administering to the subject an immunogenic peptide, a coding polynucleotide, or an immunogenic composition of any one of the aspects and / or their embodiments described above in an amount effective to treat or protect the subject from the fungal disease or its symptoms. In an embodiment, the peptide is recombinant, recombinantly produced and / or isolated.

[0031] In another aspect, there is provided a method of treating or protecting a subject from a disease or its symptoms associated with or caused by a fungal infection, wherein the method comprises administering to the subject an isolated antiserum in an amount effective to treat or protect the subject from the fungal disease or its symptoms, the isolated antiserum comprising an antibody or an antigen-binding fragment thereof that specifically binds to an immunogenic peptide of any one of the aspects and / or their embodiments described above, or an isolated or purified antibody or an antigen-binding fragment thereof. In an embodiment, the peptide is recombinant, recombinantly produced and / or isolated. In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody or an antigen-binding fragment thereof.

[0032] In some embodiments of any of the above-described methods and / or their embodiments, the disease or its symptoms are associated with or caused by fungal pathogens of the genus Aspergillus, Candida, Pneumocystis, and / or Cryptococcus. In an embodiment, the disease or its symptoms are associated with or caused by the Aspergillus fumigatus fungal pathogen. In some embodiments, the method treats a lung disease, a lung dysfunction, or its symptoms. In some embodiments, the lung disease or lung dysfunction is Pneumocystis pneumonia (PCP), aspergillosis, or invasive pulmonary aspergillosis (IPA).

[0033] In another aspect, there is provided a method of treating or protecting a subject from a disease or a serious disease or its symptoms associated with or caused by a fungal infection, wherein the method involves administering to the subject an immunogenic composition 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 immunogenic composition comprising an immunogenic peptide of SEQ ID NO:1 or an immunogenic peptide of SEQ ID NO:2, or an immunogenic fragment thereof, or a polynucleotide encoding the peptide of SEQ ID NO:1 or SEQ ID NO:2 or a functional fragment thereof. In an embodiment, the fungal pathogen is of the genus Aspergillus or Aspergillus fumigatus. In an embodiment, the peptide of SEQ ID NO:1 or 2 is recombinant, recombinantly produced, and / or isolated.

[0034] In another aspect, there is provided a method of treating or protecting a subject from a fungal infection, wherein the method comprises administering to the subject an effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to the PF-KEX2b peptide of SEQ ID NO:2 or the PF-KEX1b peptide of SEQ ID NO:1. In an embodiment, the PF-KEX2b peptide of SEQ ID NO:2 or the PF-KEX1b peptide of SEQ ID NO:1 is recombinant, recombinantly produced, and / or isolated. In an embodiment, the antibody or an antigen-binding fragment thereof is present in or isolated from an antiserum derived from a donor subject. In some embodiments, the antibody or an antigen-binding fragment thereof specifically binds to the Kex peptide of one or more fungal pathogens of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus.

[0035] In an embodiment of the method of any of the above-described aspects and / or its embodiments, the subject is a mammal or a human (human patient). In some embodiments, the subject is an immunocompromised or immunosuppressed subject. In some embodiments, the subject is a pre-transplant or post-transplant subject. In some embodiments, the immunocompromised or immunosuppressed subject is being treated for an immunodeficiency disease (e.g., HIV, AIDS, HIV / AIDS), an autoimmune disease, rheumatoid arthritis, or psoriasis. In some embodiments, the disease or its symptoms are associated with or caused by an infection with fungal species of Aspergillus, Pneumocystis, Candida, and / or Cryptococcus. In an embodiment, the disease or its symptoms are associated with or caused by an infection with fungal species of Aspergillus. In other embodiments of the above-described method and / or its embodiments, the subject has, is at risk of, or is predisposed to a fungal infection with fungal species of Aspergillus, Pneumocystis, Candida, and / or Cryptococcus, and a disease or its symptoms associated with or caused by said infection. In an embodiment, the method treats or prevents a lung disease, lung dysfunction, aspergillosis, invasive pulmonary aspergillosis (IPA), or its symptoms.

[0036] In another aspect, there is provided a method of treating or protecting a subject from a disease or a severe disease or its symptoms, wherein the method involves administering to the subject an immunogenic peptide or polynucleotide or a pharmaceutical composition thereof of any of the above-described aspects and / or its embodiments in an amount effective to treat or protect the subject from the disease or the severe disease or its symptoms. In an embodiment, the peptide is recombinant, recombinantly produced, and / or isolated.

[0037] In another aspect, there is provided a method of treating or protecting a subject from a disease or a severe disease or its symptoms, wherein the method involves administering to the subject a separated antiserum containing an antibody or an antigen-binding fragment thereof that specifically binds to an immunogenic peptide of any of the above-described aspects and / or its embodiments in an amount effective to treat or protect the subject from the disease or the severe disease or its symptoms.

[0038] In another aspect, there is provided a method of treating or protecting a subject from a disease or a severe disease or its symptoms, wherein the method involves administering to the subject a separated or purified antibody or antigen-binding fragment that specifically binds to an immunogenic peptide of any of the above-described aspects and / or its embodiments in an amount effective to treat or protect the subject from the disease or the severe disease or its symptoms. In an embodiment, the antibody is a monoclonal antibody, a polyclonal antibody, or an antigen-binding fragment thereof.

[0039] In embodiments of the methods described above, the disease or severe disease or its symptoms are selected from lung diseases, asthma, severe asthma, refractory asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, pneumonia, Pneumocystis pneumonia, bronchiectasis, aspergillosis, invasive pulmonary aspergillosis (IPA), vaginitis, urinary tract infection (UTI), organ transplantation, tissue transplantation, immunodeficiency diseases, HIV, AIDS, HIV / AIDS, congenital diseases, autoimmune diseases, rheumatoid arthritis, psoriasis, inflammation-related diseases, diabetes, type 1 diabetes or type 2 diabetes.

[0040] In another aspect, there is provided a method of treating or protecting a subject from a disease or severe disease or its symptoms associated with or caused by an Aspergillus fungal pathogen, wherein the method involves administering to the subject an amount of the PF-KEX2b peptide of SEQ ID NO:2, or a polynucleotide encoding PF-KEX2b, or an immunogenic composition comprising the PF-KEX2b peptide or a polynucleotide encoding PF-KEX2b, effective to induce an immune response and treat or prevent a disease, severe disease or its symptoms associated with infection by the Aspergillus fungal pathogen. In embodiments, the PF-KEX2b peptide of SEQ ID NO:2 is recombinant, recombinantly produced and / or isolated.

[0041] In another aspect, there is provided a method of treating or protecting a subject from a disease or severe disease or its symptoms associated with or caused by an Aspergillus fungal pathogen, wherein the method involves administering to the subject an amount of the PF-KEX1b peptide of SEQ ID NO:1, or a polynucleotide encoding PF-KEX1b, or an immunogenic composition comprising the PF-KEX1b peptide or a polynucleotide encoding PF-KEX1b, effective to induce an immune response and treat or prevent a disease, severe disease or its symptoms associated with infection by the Aspergillus fungal pathogen. In embodiments, the PF-KEX1b peptide of SEQ ID NO:1 is recombinant, recombinantly produced and / or isolated.

[0042] In embodiments of the methods of the aspects described above, an adjuvant is administered to the subject. In embodiments, the adjuvant is alum or In an embodiment of the method, an induced immune response treats or protects a subject from a lung disease or lung dysfunction and / or its symptoms, and / or an Aspergillus-related disease and / or its symptoms. In an embodiment, the Aspergillus-related disease is aspergillosis or invasive pulmonary aspergillosis (IPA) and / or its symptoms. In an embodiment of the method, the subject is a mammal or a human (human patient). In an embodiment, the subject is an immunocompromised or immunosuppressed subject. In an embodiment, the immunocompromised or immunosuppressed subject is a pre-transplant subject or a post-transplant subject. In an embodiment of the method, the immunocompromised or immunosuppressed subject is being treated for cancer, an immunodeficiency disorder, a congenital disorder, or an autoimmune disease. In an embodiment of the method, the immunocompromised or immunosuppressed subject is being treated for an immunodeficiency disorder (e.g., HIV, AIDS, HIV / AIDS), an autoimmune disease, rheumatoid arthritis, or psoriasis.

[0043] In another aspect, a vaccine is provided that comprises an effective amount of a PF-KEX2b peptide as shown in SEQ ID NO:2 or a polynucleotide encoding PF-KEX2b. In an embodiment, the PF-KEX2b peptide as shown in SEQ ID NO:2 is recombinant, recombinantly produced, and / or isolated.

[0044] In another aspect, a vaccine is provided that comprises an effective amount of a PF-KEX1b peptide as shown in SEQ ID NO:1 or a polynucleotide encoding PF-KEX1b. In an embodiment, the PF-KEX1b peptide as shown in SEQ ID NO:1 is recombinant, recombinantly produced, and / or isolated.

[0045] Other features and advantages of the described embodiments will be apparent from the detailed description and the claims.

[0046] Definitions

[0047] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide a general definition of many of the terms used in the embodiments described herein to those skilled in the art: 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 and Marham, The Harper Collins Dictionary of Biology (1991). Unless otherwise indicated, the following terms as used herein have the meanings given to them below.

[0048] The "Kex1" or "Kexin" or "KEX" protein means a polypeptide or peptide (e.g., a fragment of a KEX polypeptide) having at least about 85% or greater amino acid identity with the amino acid sequences provided by GenBank accession number EU918304.1, NCBI accession number XM_746441.1, GenBank accession number AF022372.1, or NCBI accession number XM_572303.1 and having immunogenic activity. In an embodiment, the Kex peptide is an antigen-stable active site peptide sequence. (Kutty, G. and Kovacs, J.A., 2003, Infect. Immun., 71(1):571-574; Lee, L.H. et al., 2000, Gene, 242(1-2):141-150; and Russian, D.A. et al., 1999, Proc. Assoc. Am. Physicians, 111(4):347-356). In an embodiment, the Kex peptide is a fragment of a naturally occurring Kexin protein or a non-naturally occurring pan-fungal peptide or a fragment thereof.

[0049] As used herein, "pandemic fungal peptide 1b" (PF-KEX1b) refers to a KEX peptide that comprises a sequence having at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% identity to the amino acid sequence of pandemic fungal peptide 1b (PF-KEX1b) (SEQ ID NO:1) and having immunogenic activity. In some embodiments, pandemic fungal peptide 1b (PF-KEX1b) comprises 1, 2 or 3 additional amino acids at the carboxyl and / or amino terminus of the peptide, and the additional amino acids do not alter the ability of the peptide to stimulate an immune response in a subject. In an embodiment, pandemic fungal peptide 1b (PF-KEX1b) herein contains the amino acid serine at positions 47 and 77 of the sequence, which provides favorable properties, for example, for manufacturing purposes and shelf life. In an embodiment, the PF-KEX1b peptide can facilitate manufacturing and formulation methods. As a non-limiting example, the peptide is stable and not expected to cross-link and / or aggregate. The sequence of pandemic fungal peptide 1b (PF-KEX1b) described herein is as follows:

[0050] 1DDDGKTVDGPSPLVLRAFINGVNNGRNGLGSIYVFASGNGGIYEDNSNFD

[0051] 51GYANSVFTITIGGIDKHGKRPKYSEASSSQLAVTYAGGSA (pandemic fungal peptide 1b, (PF-KEX1b), (SEQ ID NO:1)). In an embodiment, immunogenic activity or functional fragments of the sequence are encompassed. In some cases, the terms PF-KEX1b polypeptide and PF-KEX1b peptide may be used interchangeably herein.

[0052] As used herein, "pan-fungal peptide 2b" (PF-KEX2b) refers to a KEX peptide that comprises a sequence having at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% identity to pan-fungal peptide 2b (PF-KEX2b) (SEQ ID NO: 2) and having immunogenic activity. In some embodiments, pan-fungal peptide 2 (PF-KEX2b) comprises 1, 2 or 3 additional amino acids at the carboxyl and / or amino terminus, and the additional amino acids do not alter the immunogenicity of the peptide. In an embodiment, pan-fungal peptide 2 (PF-KEX2b) herein includes the amino acid serine at positions 47 and 77 of the sequence, which provides favorable properties, for example, for manufacturing and formulation purposes. As a non-limiting example, the PF-KEX2b peptide is stable and is not expected to cross-link and / or aggregate. The sequence of pan-fungal peptide 2 (PF-KEX2b) described herein is as follows:

[0053] 1PDDGKTMEGPDILVLRAFINGVQNGRDGKGSIYVFASGNGGGFEDNSNFD

[0054] 51GYTNSIYSITVGAIDRKGLHPSYSEASSAQLVVTYSSGSG (pan-fungal peptide 2, (PF-KEX2b), (SEQ ID NO: 2)). In an embodiment, immunogenic activity or functional fragments of the sequence are encompassed. In some cases, the terms PF-KEX2b polypeptide and PF-KEX2b peptide may be used interchangeably herein.

[0055] The following provides an exemplary Kex1 polypeptide fragment (peptide) of Pneumocystis isolated from a Pneumocystis-colonized non-human primate (cynomolgus monkey) and having GenBank accession number EU918304.1:

[0056] 1DDDGKTVDGPSPLVLRAFIN GVNNGRNGLG SIYVFASGNG GIYDDNCNFD GYANSVFTIT

[0057] 61IGGIDKHGKR FAYSEACSSQ LAVTYAGGSA

[0058] The following provides an exemplary polynucleotide sequence encoding the Kex1 polypeptide fragment (peptide) and having GenBank accession number EU918304.1:

[0059]

[0060]

[0061] The following provides an exemplary Kex (KexB endoprotease) polypeptide sequence of Aspergillus fumigatus (Af293) with the NCBI accession number XM_746441.1:

[0062] MRFLGSIALVLSSISVASANVRSRSYDTHEFFALHLDDSASPSHVAQLLGARHEGQIGE

[0063] LANHHTFSIPRERSSDLDALLERARAARKIRRRARDDATSQEQHNDALGGILWSQKLAP

[0064] KKRLVKRVPPPERLARTFATGKEDPVAAQSQKRIASTLGITDPIFNGQWHLFNTVQLGH

[0065] DLNVTGVWMEGITGKGVTTAVVDDGLDMYSNDLKPNYFPEGSYDFNDHTPEPRPRLSDD

[0066] KHGTRCAGEIAAARNDVCGVGVAYDSRVAGVRILSKAIDDADEATAINFAYQENDIFSC

[0067] SWGPPDDGATMEGPGILIKRAFVNGVQNGRGGKGSIFVFAAGNGASFEDNCNFDGYTNS

[0068] IYSITVGAIDREGNHPSYSESCSAQLVVAYSSGSGDAIHTTDVGTDKCYSFHGGTSAAG

[0069] PLAAGTVALALSARPELTWRDAQYLMVETAVPIHEDDGSWQVTKAGRKFSHDWGYGKVD

[0070] AYALVQKAKTWELVKPQAWFHSPWLRVQHKVPQGDQGLASSYEVTEQMMKNANIARLEH

[0071] VTVTMNVNHTRRGDLSVELRSPEGIVSHLSTTRKSDNEKAGYVDWTFMTVAHWGESGVG

[0072] RWTVIVKDTNVNEFTGEFIDWRLNLWGEAIDGANQKPHPFPDEHDDDHSIEDAIVATTS

[0073] VETGPTKTGVPGSTDDTINRPVNAKPVETQTPSPAETTATKLAPPAETRPAATATSSPT

[0074] PPAASDSFLPSFMPTFGASKRTQIWIYAAIGSIIVFCIGLGIYFQVQRRKRILNNPRDD

[0075] YDFEMIEDENALHGGNGRSGRTQRRGGELYNAFAGESDEEEPLFSDEDDEPYRDRAPSE

[0076] DRLRDTSSDDRSLRHGDH

[0077] The following provides an exemplary Kex (Kex2 protease) polypeptide sequence of Candida albicans with GenBank accession number AF022372.1:

[0078] MLPIKLLIFILGYLLSPTLQQYQQIPPRDYENKNYFLVELNTTNSQKPLIDFISHYRG

[0079] HYNFEHQLSSLDNHYVFSIDKSHPHNSFLGNHNSNEYNLMKRQLGHEQDYDELISHVE

[0080] SIHLLPMKKLSKRIPVPIEMEDVVFDNRDDTGSDNHEATDEAHQKLIEIAKKLDIHDP

[0081] EFTTQWHLINLKYPGHDVNVTGLWLEDILGQGIVTALVDDGVDAESDDIKQNFNSEGS

[0082] WDFNNKGKSPLPRLFDDYHGTRCAGEIAAVKNDVCGIGVAWKSQVSGIRILSGPITSS

[0083] DEAEAMVYGLDTNDIYSCSWGPTDNGKVLSEPDVIVKKAMIKGIQEGRDKKGAIYVFA

[0084] SGNGGRFGDSCNFDGYTNSIYSITVGAIDYKGLHPQYSEACSAVMVVTYSSGSGEHIH

[0085] TTDIKKKCSATHGGTSAAAPLASGIYSLILSANPNLTWRDVQYISVLSATPINEEDGN

[0086] YQTTALNRKYSHKYGYGKTDAYKMVHFAKTWVNVKPQAWYYSDIIEVNQTITTTPEQK

[0087] APSKRDSPQKIIHSSVNVSEKDLKIMNVERVEHITVKVNIDSTYRGRVGMRIISPTGV

[0088] ISDLATFRVNDASTRGFQNWTFMSVAHWGETGIGEWKVEVFVDDSKGDQVEINFKDWQ

[0089] FRIFGESIDGDKAEVYDITKDYAAIRRELLEKEKQNSKSTTTTSSTTTATTTSGGEGD

[0090] QKTTTSAENKESTTKVDNSASITTSQTASLTSSNEQHQPTESNSDSDSDTDDENKQEG

[0091] EEDNDNDNDNGNKKANSDNTGFYLMSIAVVGFIAVLLVMKFHKTPGSGRRRRRRDGYE

[0092] FDIIPGEDYSDSDDDEDDSDTRRADDDSFDLGHRNDQRVVSASQQQRQYDRQQDEARD

[0093] RLFDDFNAESLPDYENDMFKIGDEEEEEEEEEEGQQSAKAPSNSEGNSGTSTKK

[0094] The following provides an exemplary Kex polypeptide sequence of Cryptococcus neoformans (JEC21) with NCBI accession number XM_572303.1:

[0095] MRTLLSLWGILLALIVPPSLALQRPQPRSYDTHAYYALELDPSISPAAALQLSKSLGV

[0096] ELVERIGELDGHWLVRTEGWTPEHASITKRSVSHDPILKRWEALPSSLGKKSLTPLSL

[0097] KQRAKRHKSYSPRSRHSRDDRTELLYAQNELHLADPMLDQQWHLINTQMKDIELNVTG

[0098] LWGRGITGEGVHVVIIDDGLDVESKDLKDNFFAEGSYDFNDHTELPIPRLKDDQHGTR

[0099] CAGEIAAVPNDVCGVGVAYDSKIAGVRILSAPISDADEAAALNYAYQLNDIYSCSWGP

[0100] PDDGRSMEAPDGLILKAMVNGVQKGRDGKGSVFVFAAGNGGGSDDQCNFDGYTNSIFS

[0101] VTVGAVDRKGLHPYYSEMCAAMMVVAPSSGSGDHIHTTDVGKDKCSHSHGGTSAAAPL

[0102] AVGVFALALSVRPDLTWRDIQHLAVRHAVFFNPDDPAWELTAAGRHFSYKYGYGKLDA

[0103] GLFVEAAEKWQLVKPQTWYDSPSVYLPTTSPADVTRRQDEAADGPTSSDEETSNPPPV

[0104] VEPSGSFITEDGVISTYEVTQSMLFDANFERLEHVTVRVWIDHQRRGDVEVELTSPNG

[0105] VVSVLCRQRRFDNADSGFPGWKFMSLKHWDENPVGTWTIKVKDQVNPDKTGRFVAWSL

[0106] QLWGESVDPALAKLWAPAEEGQPDEEQTGSNPSTTVSQKPKPTALLPGDHGEASGEAT

[0107] QPGLGSATAHPQPTSTTGDAGNVAEPTGPTDADADEGFFSGISNLASSSTWLAGAGAI

[0108] IILSGAAIGAFFFIRARRQKRNLFGLSNNGQGARGAYEPVDDVQMSLLERGRRKFGKS

[0109] KSESQGTKDLYDAFGDGPSDEEEEDLDERTALRYHDGFLEDDEPNEVGPKTEYKDEPE

[0110] SEPETFKDGEETVGTKDKGKGKGPSEGESGSGSSSSWQDAADEEARV

[0111] "Agent" means a peptide, nucleic acid molecule or small compound.

[0112] "Improve" means to reduce, inhibit, attenuate, alleviate, prevent or stabilize the development or progression of a disease.

[0113] "Alter" means a change (increase or decrease) in the expression level or activity of a gene or polypeptide as detected by methods known by standard techniques (such as those described herein). As used herein, an alter includes a 10% change in the expression level, preferably a 25% change in the expression level, more preferably a 40% change, and most preferably a 50% or greater change.

[0114] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Methods for preparing antibodies are well known to those of ordinary skill in the immunology art. An antibody can be a full-length immunoglobulin derived from a natural source or a recombinant source and can be an immunoreactive portion of a full-length immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. The tetramers can be naturally occurring or reconstructed from single-chain antibodies or antibody fragments. Antibodies also include dimers that can be naturally occurring or constructed from single-chain antibodies or antibody fragments. The antibodies of the described embodiments can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies (Mabs), Fv, Fab, and F(ab')2, 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, New York; 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).

[0115] The term "antibody fragment" refers to a portion of a full-length antibody. An antibody fragment can refer to the antigen-determining variable regions of a full-length antibody (e.g., the heavy and light chain variable regions). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, single-domain antibodies, such as camelid antibodies (Riechmann, 1999, Journal of Immunological Methods, 231:25-38) (composed of V L or V H domains and exhibiting sufficient affinity for a target), and multispecific antibodies formed from antibody fragments. Antibody fragments can be derived from chimeric antibodies, human antibodies, humanized antibodies, or portions of human antibodies or humanized antibodies.

[0116] Antibodies can be prepared using a polypeptide (e.g., Kexin polypeptide) or an immunogenic peptide fragment thereof as an immunogen by any method known in the art. One way to obtain antibodies is to immunize a suitable host animal with the immunogen and follow standard procedures for polyclonal or monoclonal antibody production. The immunogen aids in the presentation of the immunogenic fragment on the cell surface. The suitable host can be immunized in a variety of ways. The nucleic acid sequence encoding the polypeptide or its immunogenic fragment as described herein can be provided to the host in a delivery vehicle that is taken up by the host's immune cells. The cells will then express the receptor on the cell surface, thereby generating an immunogenic response in the host. Alternatively, the nucleic acid sequence encoding the polypeptide or its immunogenic fragment can be expressed in vitro cells, and then the polypeptide is isolated and administered to a suitable host for antibody production.

[0117] Alternatively, if desired, antibodies against the polypeptide can be derived from an antibody phage display library. Phage are able to infect bacteria and replicate within the bacteria, and when combined with human antibody genes, the phage can be engineered to display human antibody proteins. Phage display is the process by which phage "display" human antibody proteins on their surface. Genes from a human antibody gene library are inserted into a population of phage. Each phage carries the gene for a different antibody and thus displays a different antibody on its surface.

[0118] Antibodies prepared by any method known in the art can then be purified from the host. Antibody purification methods can include salt precipitation (e.g., with ammonium sulfate), ion exchange chromatography (e.g., on cation or anion exchange columns, preferably run at neutral pH and eluted with a step gradient increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and affinity resin chromatography using, for example, Protein A, Protein G, hydroxyapatite, and anti-immunoglobulin.

[0119] Antibodies can be conveniently produced by hybridoma cells engineered to express the antibody. Methods for preparing hybridomas are well known in the art. Hybridoma cells can be cultured in a suitable medium, and the spent medium can be used as a source of the antibody. The polynucleotide encoding the antibody of interest can then be obtained from the hybridoma producing the antibody, and then the antibody can be produced synthetically or recombinantly from these DNA sequences. To produce large amounts of antibody, it is usually more convenient to obtain ascitic fluid. Methods for producing ascites typically involve injecting the hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal (especially a mouse). The mammal can be prepared for ascites production by prior administration of a suitable composition (e.g., pristane).

[0120] "Anti-Kexin antibody", "anti-Kex antibody", or "anti-Kex1 antibody" means an antibody or an antigen-binding fragment thereof that selectively binds to a Kexin polypeptide or peptide as described herein (including, for example, Kex1 peptide fragments of fungal pathogens such as Candida albicans, Pneumocystis hominis, Pneumocystis jirovecii (formerly known as Pneumocystis carinii), Aspergillus fumigatus, and Cryptococcus neoformans) or PF-KEX1b or PF-KEX2b as described herein. In various embodiments, the anti-Kexin antibody or anti-Kex1 antibody specifically binds to the binding site of the Kexin protein or peptide. In particular embodiments, the anti-Kexin antibody or anti-Kex1 antibody specifically binds to the binding site of the Kexin protein or peptide of a fungal pathogen (e.g., one or more of fungal pathogens of the genus Candida, Pneumocystis, Aspergillus, and / or Cryptococcus). In an embodiment, the antibody binds to PF-KEX1b. In an embodiment, the antibody binds to PF-KEX2b.

[0121] "Antiserum" refers to serum containing one or more antibodies against a specific antigen. Antiserum containing antibodies can be obtained from the blood or serum of animals (mammals), including humans, that have been immunized or inoculated with an immunogen (or antigenic material) by injection (typically into the blood or tissue) or by infection. In embodiments, animals (mammals), including humans, 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 infection with or natural contact with an antigenic material or immunogen. In such cases, the antiserum contains anti-Kex peptide antibodies, such as polyclonal antibodies, or a population of monoclonal antibodies, produced or generated by an immunized, inoculated, or exposed donor subject against a Kex peptide immunogen or a polynucleotide encoding a Kex peptide immunogen (derived from a fungal pathogen such as Pneumocystis (e.g., Pneumocystis jirovecii), e.g., PF-KEX1b or PF-KEX2b peptide immunogen). Such antiserum, isolated (and / or purified) from the donor subject, is used to immunize (i.e., administer to) another (unrelated) subject to provide immunity (acquired immunity) against an infection or disease caused by or associated with not only the Pneumocystis pathogen that was the original source of the immunogen but also other fungal pathogens having a Kex peptide (which is also targeted and recognized by the antibodies in the antiserum). In embodiments, the fungal pathogens include one or more species of Pneumocystis, as well as one or more species of Candida or Candida albicans, Aspergillus or Aspergillus fumigatus, or Cryptococcus or Cryptococcus neoformans. In this way, the subject receiving the antiserum (i.e., the antibodies in the antiserum) is treated or protected from infection and / or disease caused by more than one fungal pathogen. Such antiserum-derived immunoprotection against multiple fungal pathogens constitutes acquired or passive immunity obtained by the recipient subject and conferred by the isolated antiserum of the donor subject. Those skilled in the art will understand that serum is the amber, protein-rich liquid blood component that separates from the clot when blood coagulates. The serum component containing one or more antibodies (cross-protective antibodies) is called "antiserum". In embodiments, the antiserum is isolated antiserum, e.g., isolated from a donor subject. In embodiments, before being used as a therapeutic or protective therapeutic agent as described herein, the isolated antiserum can be processed by methods used by those skilled in the art, such as dilution, concentration (e.g., via filtration or centrifugation or both), chromatography, purification to remove ions or foreign proteins, etc. In embodiments, the isolated antiserum can be further purified after isolation. In embodiments, the isolated antiserum is not further processed or purified.In embodiments, the antibodies or antigen-binding fragments thereof contained in the isolated antiserum can be further isolated by methods practiced by those skilled in the art, such as but not limited to further isolation by affinity chromatography, size exclusion chromatography, immunoprecipitation, dialysis, HPLC chromatography, etc.

[0122] "Biological sample" means any liquid, 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. In embodiments, tissue preparations are encompassed, where the tissue is homogenized or otherwise prepared to yield a suspension containing cells.

[0123] In this disclosure, "comprises", "comprising", "containing", "having", etc. can have the meanings given to them by United States patent law and can mean "includes", etc.; "consisting essentially of" likewise has the meaning given by United States patent law, and the term is open-ended, allowing the presence of more than what is recited, so long as the basic or novel characteristics of what is recited are not changed by the presence of more than what is recited, excluding prior art embodiments.

[0124] "Detect" refers to identifying the presence, absence, or amount of an analyte to be detected.

[0125] "Disease" means any illness, dysfunction, or disorder that impairs or interferes with the normal function of cells, tissues, or organs. In various embodiments, non-limiting examples of diseases or their symptoms associated with fungal pathogen infections include pulmonary (lung) diseases, brain diseases such as meningitis. Non-limiting examples of pulmonary diseases include chronic obstructive pulmonary disease (COPD), a progressive lung disease that includes emphysema, chronic bronchitis, refractory (irreversible) asthma, pneumonia (e.g., Pneumocystis pneumonia), and some forms of bronchiectasis. Non-limiting examples of diseases associated with Aspergillus species infections include aspergillosis or invasive pulmonary aspergillosis (IPA) and / or their symptoms. Non-limiting examples of diseases associated with Candida species (e.g., Candida albicans, Candida auris, Candida glabrata, Candida tropicalis, Candida parapsilosis, or Candida krusei) infections can include, but are not limited to, vaginitis and urinary tract infections (UTIs). Non-limiting examples of diseases, conditions, pathologies, or their symptoms associated with immunosuppressed or immunocompromised subjects (patients) include organ or tissue transplantation or post-transplantation cancer, immunodeficiency diseases (e.g., HIV, AIDS, HIV / AIDS), congenital diseases, or autoimmune diseases, rheumatoid arthritis or psoriasis, etc. In an embodiment, the term disease includes immunocompromised subjects or patients, or immunosuppressed subjects or patients. In an embodiment, due to immunocompromise or immunosuppression, a subject may be at high risk of, or be susceptible to, fungal infections and related diseases. In an embodiment, the PF-KEX1b and PF-KEX2b peptide immunogens and / or antibodies or antisera generated against these peptide immunogens may have prophylactic and / or therapeutic use in a subject suffering from or at risk of suffering from inflammation-related diseases, diabetes (type 1 or type 2 diabetes), asthma, severe asthma, COPD, and immune or autoimmune disorders (e.g., HIV, AIDS, HIV / AIDS), including their symptoms.

[0126] "Effective amount" means the amount required to improve the symptoms of a disease relative to an untreated patient. The effective amount of one or more active compounds for practicing the methods described herein for the therapeutic treatment of a disease varies depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the treating physician or veterinarian will determine the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. The immunologically effective amount of an isolated antiserum as 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, the effective amount of an isolated antiserum 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 dosages for passive immunotherapy (i.e., administration of antibody-containing antiserum) range from about 0.3 mg to about 100 mg / kg total body weight. After passive immunotherapy, the therapeutic efficacy is typically evaluated due to the different responses of individual patients to the therapy. Dosage adjustments can be made as needed. The treatment regimen can be determined by methods known and practiced by those skilled in the art. In one embodiment, the amount is sufficient to induce an immune response.

[0127] "Fragment" means a portion of a polypeptide or nucleic acid molecule. This portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. Fragments can 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. In embodiments, the fragment or portion retains activity or function, such as but not limited to antigen-binding function or immunogenicity.

[0128] "Genetic vaccine" means an immunogenic composition comprising a polynucleotide encoding an antigen.

[0129] "Immune response" means any activity of the immune system 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. In some embodiments, the immune response encompasses a B cell response, a T cell response, or both a B cell and T cell response.

[0130] The term "immunocompromised" refers to a subject whose immune system and / or related immune responses to pathogens, pathogenic antigens, diseases, etc. are weakened or impaired. A subject may be immunocompromised due to taking immunosuppressive drugs or having a disease or pathology that affects the subject's immune system (such as certain congenital diseases). The term "immunosuppression" refers to the partial or complete suppression of a subject's immune system and related immune responses to pathogens, pathogenic antigens, diseases, etc., for example, by reducing the activity or efficiency of the immune system. Immunosuppression of a subject's immune system or immune response may occur naturally due to the subject's disease or disorder, or may be induced in the subject by administering immunosuppressive agents, drugs (such as anticancer drugs, compounds, etc.). In some cases, a subject who is immunosuppressed or undergoing immunosuppression, or whose immune system is weakened due to a disease or condition (such as chemotherapy or an immunodeficiency disease) is referred to as immunocompromised.

[0131] An "immunogenic composition" means a composition comprising an antigen or immunogen or a polynucleotide encoding said antigen or immunogen, wherein the composition elicits an immune response in an immunized subject.

[0132] The terms "isolated", "purified" or "biologically pure" refer to materials that are, to varying degrees, free of the components that are normally associated with them in their natural state or environment. "Isolated" indicates the degree of separation from the initial source or environment. "Purified" indicates a degree of separation higher than isolated. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide as described herein is purified if it is substantially free of cellular material, viral material or culture medium when produced by recombinant DNA techniques, or is substantially free of chemical precursors or other chemicals when produced by chemical synthesis. Analytical chemical techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography, are typically used to determine purity and homogeneity. The term "purified" may indicate that a nucleic acid or protein produces substantially a single band on an electrophoretic gel. For proteins that are subject to modification (e.g., phosphorylation or glycosylation), different modifications can result in different separated proteins, which can be purified separately.

[0133] "Isolated polynucleotide" means a nucleic acid (e.g., DNA) that does not contain genes flanking the gene in the naturally occurring genome of the organism from which the nucleic acid molecule is derived. Thus, the term includes, for example, recombinant DNA incorporated into a vector; incorporated into an autonomously replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryote or eukaryote; or existing as an independent molecule separate from other sequences (e.g., cDNA or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion). In addition, the term includes RNA molecules transcribed from DNA molecules and recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0134] "Isolated polypeptide" or "isolated peptide" means a polypeptide or peptide that has been separated from its natural accompanying components. Typically, a polypeptide is isolated when it is at least 60% by weight free of the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight the polypeptide or peptide as described herein. The isolated polypeptides or peptides as described herein can be obtained, for example, by extraction from natural sources, by expression of recombinant nucleic acids encoding such polypeptides, or by chemical synthesis of the protein. Purity can be measured by any suitable method (e.g., column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis).

[0135] As used herein, "obtaining an agent" includes synthesizing, purchasing, isolating, purifying, or otherwise obtaining the agent.

[0136] "Opportunistic infection" means an infection caused by a pathogen (e.g., a fungal pathogen, bacterium, virus, protozoan, or parasite) that takes advantage of an opportunity to infect a subject (host) that is normally not accessible to the infection, such as a host with a weakened immune system, an immunocompromised host, an immunosuppressed host, a host with an altered microbiota or microbiome, or a host whose protective epithelial barrier has been breached or broken. In an embodiment, the opportunistic infection is caused by one or more fungal pathogens as described herein.

[0137] "Reduce" or "diminish" means a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0138] "Reference" means a standard or control condition. A "reference sequence" is a defined sequence that is used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a designated sequence; for example, a segment of a full-length cDNA or gene sequence, or the full-length 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, and 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, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabove or below or in between.

[0139] "Specifically binds" means that a compound or antibody or an antigen-binding fragment thereof recognizes and binds to a polypeptide or peptide, but essentially does not recognize and bind to other molecules in a sample (e.g., a biological sample that naturally includes a polypeptide or peptide as described herein). Cross-reactive binding includes specific binding to the original polypeptide or peptide antigen / immunogen (e.g., by an antibody or an antigen-binding fragment thereof) and binding to polypeptides or peptides other than the original antigen / immunogen.

[0140] Nucleic acid molecules that can be used to generate a recombinant immunogen or vaccine include any nucleic acid molecule encoding a polypeptide or a peptide fragment thereof (e.g., a Kex wild-type or pan-fungal polypeptide or a Kex wild-type or pan-fungal peptide as described herein). Such nucleic acid molecules need not be 100% identical to the endogenous nucleic acid sequence, but will typically exhibit substantial identity to the endogenous sequence. Polynucleotides having "substantial identity" to the endogenous sequence are typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. A nucleic acid molecule can include any nucleic acid molecule encoding a polypeptide or a peptide fragment thereof. Polynucleotides having "substantial identity" to the endogenous sequence are typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" means pairing under various stringent conditions to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene as described herein) or portions thereof. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

[0141] "Substantially identical" means that a polypeptide or nucleic acid molecule exhibits at least 50% identity relative to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such a sequence has at least 60%, more preferably 80% or 85%, and even more preferably 90%, 95%, or even 99% identity at the amino acid level or nucleic acid relative to the sequence used for comparison.

[0142] Sequence identity is typically measured using sequence analysis software (e.g., the sequence analysis software package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705, the 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 method of determining the degree of identity, the BLAST program can be used, where the probability score between e -3 and e -100 indicates closely related sequences.

[0143] "Subject" means 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 mammal. In an embodiment, the subject is a human. In an embodiment, the subject is a human patient who is undergoing treatment for an infection or disease caused by one or more pathogenic fungi (e.g., Pneumocystis, Aspergillus, Candida, or Cryptococcus). In an embodiment, the subject is a human patient at risk of an infection (e.g., an opportunistic infection) or disease caused by one or more pathogenic fungi (e.g., Pneumocystis, Aspergillus, Candida, or Cryptococcus). In an embodiment, the subject is a mammalian (e.g., human; non-human primate) donor subject from whom an antiserum containing an anti-fungal Kex peptide antibody has been obtained or isolated. In an embodiment, the subject is a mammalian (e.g., human; non-human primate) recipient subject who receives the isolated antiserum and obtains protective immunity (and treatment) against a variety of fungal pathogens.

[0144] As used herein, "vector" refers to a nucleic acid (polynucleotide) molecule into which foreign nucleic acid can be inserted without disrupting the ability of the vector to replicate in a host cell and / or integrate into the host cell. A vector can include nucleic acid sequences that permit its replication in a host cell, such as an origin of replication. An insertable vector is capable of inserting itself into host nucleic acid. A vector can also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to permit transcription and translation of one or more inserted genes in 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 to be inserted into the vector to maintain the correct reading frame.

[0145] "Vaccine" means a preparation of immunogenic material (such as a protein or nucleic acid; particle) that is capable of stimulating (eliciting) an immune response and is administered to a subject to treat a disease, disorder or pathology, or to prevent a disease, disorder or pathology such as an infectious disease (such as an infectious disease caused by a fungal infection). The immunogenic material can include, for example, attenuated or inactivated microorganisms (such as attenuated viruses), or antigenic proteins, peptides or DNA from such microorganisms. A vaccine may elicit a prophylactic (preventative) immune response in a subject; a vaccine may also elicit a therapeutic immune response in a subject. As described above, the method of administering a vaccine varies depending on the vaccine and can include, for example, routes or means such as inoculation (intravenous or subcutaneous injection), ingestion, inhalation or other forms of administration. Inoculation can be carried out by a variety of routes, including parenterally, such as intravenously, subcutaneously or intramuscularly. A vaccine can also be administered with an adjuvant to enhance the immune response.

[0146] The ranges provided herein are understood to be shorthand for all values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers or sub-range 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.

[0147] As used herein, the term "treat (treat, treating, treatment, etc.)" refers to reducing, alleviating, mitigating or improving a disease, disorder and / or its associated symptoms. It will be understood that, although not excluded, treating a disease, disorder and / or its associated symptoms does not necessarily require elimination of the disease, disorder, condition or its associated symptoms.

[0148] As used herein, a therapeutic agent that "prevents" a disorder or condition refers to a compound or material that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to an untreated control sample. In an 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 containing an anti-Kex peptide antibody or an antigen-binding fragment thereof as described herein.

[0149] Unless explicitly stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless explicitly stated or obvious from context, as used herein, the terms "a, an" and "the" are understood to be singular or plural.

[0150] Unless explicitly stated or obvious from context, as used herein, the term "about" is understood to be within the normal tolerances in the art, e.g., 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 clear from the context, all numerical values provided herein are modified by the term about.

[0151] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0152] Exemplary embodiments as described herein are provided below in the form of various and non-limiting aspects related to the described embodiments in the accompanying drawings and schematics.

[0153] Figure 1 The figure shows the following: The PF.KEX2b immunogen elicited an antibody response, and antibody production (as measured by antibody titers in the plasma of animals (mice) that received the PF-KEX2b immunogen). The results present the mean reciprocal endpoint titer (RET) of plasma anti-PF.KEX2b specific immunoglobulin G (IgG antibody) after three immunizations of mice with 10 μg (represented by an "x" on the graph) or 20 μg (represented by a solid black circle on the graph) of the PF.KEX2b immunogen and alum ( InvivoGen, San Diego, California) adjuvant, as determined by enzyme-linked immunosorbent assay (ELISA). The arrows on the graph indicate the time points of animal immunization. "Wpv": weeks post-vaccination.

[0154] Figure 2A and Figure 2B presents a study design figure and a figure showing the following: survival curves related to a fungal challenge study of invasive pulmonary aspergillosis in an immunosuppressed mouse model. Figure 2A presents the design of a fungal challenge study in animals. Figure 2B shows the survival curves of animals immunized with the PF.KEX2b peptide immunogen (represented by the "x" markers on the figure) compared to sham-immunized control animals. The PF.KEX2b-immunized animals were significantly protected against the development of aspergillosis after immunosuppression (induced using tacrolimus and cortisol) compared to sham-immunized controls (*p = 0.0323, Mantel-Cox test). The results indicate that immunosuppressed animals immunized with the PF.KEX2b peptide immunogen in combination with an adjuvant had a significantly reduced mortality associated with Aspergillus fungal infection (p = 0.0323).

[0155] Figure 3A and Figure 3B shows Coomassie staining gels and Western blot analyses of the PF-KEX2a peptide (i.e., pan-fungal peptide 2; see, e.g., U.S. Publication No. 2022 / 0184190, the content of which is incorporated herein by reference) and the PF-KEX2b peptide described herein. Figure 3A : PF-KEX2a protein quality analysis before use in animal immunization / vaccination and after extended storage at -80°C. After dialyzing the PF-KEX2a peptide overnight in PBS, the dialyzed protein was aliquoted and frozen for later in vivo use. The quality of the protein was evaluated by Coomassie staining after gel electrophoresis and PF-KEX2a Western blot analysis immediately after dialysis (left sub-panel), after storage at -80°C for 47 days (middle sub-panel), and after storage for 90 days (right sub-panel). Asterisks (*) indicate the post-dialysis pool, which was aliquoted and stored at -80°C for subsequent immunization / vaccination use at 47 and 90 days. The PF-KEX2a peptide ran on the gel at a similar molecular weight in the presence or absence of β-mercaptoethanol (left sub-panel) and did not degrade over time. When measured by the Bradford assay, the concentration of each aliquot was 1.23 mg / ml, 1.20 mg / ml, and 1.28 mg / ml after dialysis, after storage at -80°C for 47 days, and after storage for 90 days, respectively. It is expected that the PF-KEX1b and PF-KEX2b peptides described herein will exhibit similar protein quality analysis results. Figure 3B: Animal immunization / vaccination and quality analysis of PF-KEX2b protein prior to storage at -80°C. After dialyzing the PF-KEX2b peptide overnight in PBS, the dialyzed protein was aliquoted and frozen for later in vivo use. After dialysis and storage at -80°C, the protein quality was evaluated by Coomassie staining (left sub-panel) and PF-KEX2b immunoblot analysis (right sub-panel) after gel electrophoresis. Detailed Description

[0156] Compositions and methods for treating or preventing infections by one or more fungal pathogens, or diseases associated with infections by more than one fungal pathogen, are described herein, where the pathogens have different etiologies. By way of specific example, fungal pathogens that cause disease include Aspergillus (a common mold that causes aspergillosis, allergic reactions, lung infections, and other health problems); Pneumocystis (colonizes lung tissue and causes severe pneumonia after infection); Candida (typically present in the gut and mucous membranes and can cause thrush, infections, and invasive candidiasis, especially in people with poor health or a weak immune system) when systemically infected; and Cryptococcus (can infect the lungs and cause pneumonia-like diseases in the lungs and can also infect the brain and cause meningitis in the brain). In embodiments, the compositions and methods can be used to treat or prevent diseases associated with infection by fungal pathogens of the genus Aspergillus, such as aspergillosis.

[0157] The described embodiments are based, at least in part, on the discovery that mammalian subjects immunized with non-naturally occurring polypeptides or peptides (referred to herein as pan-fungal (PF) peptides) having sequence similarity to the Kexin (Kex or Kex1) proteins described herein, such as pan-fungal peptide 1b (referred to herein as "PF-KEX1b") (SEQ ID NO:1) and pan-fungal peptide 2 (referred to herein as "PF-KEX2b") (SEQ ID NO:2), produce antibodies that specifically bind Kex peptides of different fungal pathogens, including Pneumocystis, Candida, Aspergillus, and Cryptococcus. Thus, PF peptides as described herein can be used to treat or prevent diseases caused by or associated with infection by any one or more of the fungal pathogens Pneumocystis, Candida, Aspergillus, and Cryptococcus. In embodiments, the PF-KEX1b and PF-KEX2b peptides described herein can be used to treat or prevent diseases caused by or associated with infection by fungal pathogens of the genus Aspergillus, such as aspergillosis. The PF-KEX1b and PF-KEX2b peptides (stable peptides) can also provide beneficial properties, such as reduced or absent cross-linking and / or aggregation, which are useful for manufacturing or formulating peptides (e.g., for commercial production and use).

[0158] An antibody-containing antiserum produced in response to the administration of a non-naturally occurring pan-fungal Kex peptide PF-KEX1b and / or PF-KEX2b, or a polynucleotide encoding PF-KEX1b or PF-KEX2b, can also be used for the treatment of diseases caused by infection with one or more fungal pathogens and can provide immunity against one or more fungal pathogens to a subject or to another unrelated subject (i.e., a recipient subject) that receives the antiserum via a suitable mode and route of administration. One of ordinary skill in the art will understand that, as used herein, the subject from which the antiserum is obtained or isolated is the "donor subject," and the subject to which the isolated antiserum 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 for or protection from a disease caused by infection with one or more fungal pathogens of the genus Pneumocystis, Candida, Aspergillus, and / or Cryptococcus. In embodiments, the fungal pathogen is Aspergillus.

[0159] The generation of such immunologically cross-reactive antisera (and antibodies therein) in a subject (e.g., a donor subject) immunized or exposed to PF-KEX1b or PF-KEX2b or a polynucleotide encoding a pan-fungal Kex peptide that reacts with Kex peptides of a variety of fungal pathogens, such as fungal pathogens of the genus Candida, Aspergillus, and Cryptococcus, is surprising and unexpected, particularly in view of the low amount of amino acid sequence identity (a variability rate of about 48%-70%) between the Kex peptides of fungal pathogens of the genus Pneumocystis, Candida, Aspergillus, and Cryptococcus, and in view of the total amount of amino acid sequence variability between the Kex1 peptide of Pneumocystis and the Kex peptides of fungal organisms of the genus Candida, Aspergillus, and Cryptococcus (ranging from a variability rate of about 70% to 96%).

[0160] Also included herein are immunogenic compositions comprising PF-KEX1b or PF-KEX2b, or a polynucleotide encoding a pan-fungal peptide, which, when administered to a subject and an antiserum containing an antibody or an antigen-binding fragment thereof is produced, elicit a potent immune response in the subject, wherein the antibody or its antigen-binding fragment binds (reacts) not only to the immunizing PF-KEX1b or PF-KEX2b peptide immunogen but also to a similar but non-identical kexin antigen produced by other fungal pathogens. In embodiments, the PF-KEX2b peptide or a composition thereof (used as an immunogen administered to a subject) produces an immune response in the subject that treats a disease (e.g., aspergillosis) and / or reduces the mortality associated with infection with a fungal pathogen of the genus Aspergillus (e.g., Aspergillus fumigatus). See, e.g., Figure 2A and Figure 2B . In embodiments, the subject is an immunosuppressed or immunocompromised subject.

[0161] One benefit of the methods described is the treatment or prevention of diseases, their symptoms and adverse reactions associated with infection by one or more different fungal pathogens using only one therapeutic agent, said therapeutic agent being a composition comprising a non-naturally occurring PF-KEX1b or PF-KEX2b peptide or a polynucleotide encoding a pan-fungal Kex peptide, or an antiserum (isolated antiserum) raised in response to a non-naturally occurring PF-KEX1b or PF-KEX2b peptide or a polynucleotide encoding a pan-fungal Kex peptide, treating multiple fungal organisms and / or providing cross-protection against multiple fungal organisms, and treating or preventing diseases and their symptoms associated with infection (and colonization) by different fungal pathogens (e.g., one or more, or at least two or more fungal pathogens), such as lung diseases and poor lung function. In embodiments, the different fungal pathogens include Pneumocystis, Aspergillus, Candida, and Cryptococcus, particularly one or more, or at least two or more thereof. In one embodiment, a composition is provided that comprises a non-naturally occurring PF-KEX1b or PF-KEX2b peptide or a polynucleotide encoding a pan-fungal Kex peptide that elicits the production of antibodies that specifically react with Kex peptides from one or more of Pneumocystis (Pneumocystis jirovecii), Aspergillus (Aspergillus fumigatus), Candida (Candida albicans), or Cryptococcus (Cryptococcus neoformans). Another embodiment provides an antibody-containing antiserum produced in a subject immunized with a non-naturally occurring PF-KEX1b or PF-KEX2b peptide, or a polynucleotide encoding a pan-fungal Kex peptide, said antibodies also specifically reacting with Kex peptides from one or more of Pneumocystis (Pneumocystis jirovecii), Aspergillus (Aspergillus fumigatus), Candida (Candida albicans), or Cryptococcus (Cryptococcus neoformans). Thus, the antiserum is cross-protective (e.g., cross-reacts with Kex peptides of multiple fungal types) and, when provided to another (e.g., unrelated) subject in need, provides treatment and / or protection against diseases as described herein, including those associated with infection by one or more fungal organisms. In embodiments, the antiserum is an isolated antiserum. In embodiments, the isolated antiserum is administered in a pharmaceutically acceptable composition.

[0162] The methods and compositions described herein that comprise PF-KEX1b or PF-KEX2b peptides provide economic, medical, and practical benefits in the treatment and prevention of fungal diseases associated with infection and colonization by different types of fungal pathogens, such as lung diseases or various brain infections. In embodiments, the methods and compositions can comprise PF-KEX1b and PF-KEX2b peptide immunogens.

[0163] Treatment methods

[0164] The methods and compositions provided herein can be used to treat or prevent diseases associated with or caused by the following: fungal pathogens of the genera Pneumocystis, Aspergillus, Candida, and Cryptococcus, and in particular one or more of Pneumocystis jirovecii or Pneumocystis carinii, Aspergillus fumigatus, Candida albicans, or Cryptococcus neoformans. Against severe or serious diseases and their symptoms caused by infection with at least one and in particular more than one of these fungal organisms, the methods and compositions provided herein can provide immune protection to a subject. The methods and compositions provided herein can immunize a recipient subject to prevent infection by at least one and in particular more than one of these fungal organisms and / or the diseases caused thereby. Generally, an immunogenic composition comprising a non-naturally occurring PF-KEX1b or PF-KEX2b peptide, a polynucleotide encoding a pan-fungal Kex peptide, or a purified antiserum containing one or more antibodies generated against a non-naturally occurring PF-KEX1b or PF-KEX2b peptide can be administered therapeutically and / or prophylactically to provide immunity against other pathogenic fungal organisms expressing Kex proteins or peptide antigens. The methods include administering to an individual, alone or in a physiologically acceptable carrier, excipient, or diluent, an immunologically effective amount of an immunogenic composition as described herein comprising PF-KEX1b or PF-KEX2b, a polynucleotide encoding a pan-fungal Kex peptide, a purified antiserum, or immune serum or immune plasma. In an embodiment, the immunogenic composition comprising PF-KEX1b or PF-KEX2b, a polynucleotide encoding a pan-fungal Kex peptide, or a purified antiserum is in a pharmaceutically acceptable composition.

[0165] Methods for treating or preventing diseases, or the severity of diseases, disorders or symptoms affected by, or caused by, infection with one or more fungal pathogens (e.g., of the genera Pneumocystis, Aspergillus, Candida, or Cryptococcus, and more particularly one or more of the fungi Pneumocystis jirovecii or Pneumocystis carinii, Aspergillus fumigatus, Candida albicans, or Cryptococcus neoformans) are provided and described herein, the methods comprising administering to a recipient subject in need thereof (e.g., a mammal, such as a human patient) a therapeutically effective amount of an immunogenic composition as described herein, the immunogenic composition comprising a non-naturally occurring PF-KEX1b or PF-KEX2b peptide, a polynucleotide encoding a pan-fungal Kex peptide, or a purified antiserum raised in response to immunization with a non-naturally occurring PF-KEX1b or PF-KEX2b peptide. In an embodiment, the antiserum contains antibodies that specifically target a Kexin protein or Kex peptide to neutralize the activity of Kex protease. In an embodiment, the purified antiserum containing antibodies against a PF-KEX1b or PF-KEX2b peptide allows the recipient subject to achieve and passively acquire protective immunity and / or treatment against a plurality of fungal pathogens.

[0166] In an embodiment, the method as described herein involves treating a subject suffering from or susceptible to an infection with a fungus of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus, or a disease or its symptoms associated with or caused by one or more of these fungal pathogens (e.g., lung disease, COPD, aspergillosis, etc.). The method comprises the step of administering to the subject (e.g., a mammalian or human patient) PF-KEX1b or PF-KEX2b peptide, a polynucleotide encoding PF-KEX1b or PF-KEX2b peptide, an immunogenic composition comprising PF-KEX1b or PF-KEX2b peptide or the encoding polynucleotide, or an isolated antiserum raised against PF-KEX1b or PF-KEX2b peptide, in an amount sufficient to treat the infection, disease, disorder, or its symptoms, under conditions for treating an infection, disease, disorder, or its symptoms caused by one or more different types of fungal organisms. In an embodiment, the isolated antiserum is in a pharmaceutically acceptable composition.

[0167] Methods for treating or preventing a disease, a severe or serious disease, and / or its symptoms caused by or associated with an infection by one or more types of fungal pathogens are also provided and described, wherein the methods comprise administering a therapeutically effective amount of an immunogenic composition comprising a non-naturally occurring PF-KEX1b or PF-KEX2b peptide as described herein, a polynucleotide encoding a pan-fungal Kex peptide, or an isolated antiserum (e.g., comprising one or more antibodies or antigen-binding fragments thereof) generated in response to an immunogen of a non-naturally occurring PF-KEX1b peptide or PF-KEX2b peptide administered to a subject (e.g., a mammal, such as a human). In various embodiments, the method prevents a disease, a severe disease, and / or death associated with an infection by one or more fungal pathogens (selected from the group consisting of Pneumocystis, Aspergillus, Candida, or Cryptococcus) in a subject susceptible to fungal infection, disease, severe disease, or its symptoms (e.g., COPD, lung / pulmonary disease, poor lung function, aspergillosis, etc.). In an embodiment, the method comprises the step of administering to a recipient mammal, in an amount sufficient to prevent or treat the disease, disorder, or its symptoms (and severe forms thereof), and under conditions for treating the disease, disorder, or its symptoms, a therapeutically effective amount of an immunogenic composition comprising a non-naturally occurring PF-KEX1b or PF-KEX2b peptide, a polynucleotide encoding a pan-fungal Kex peptide, or an isolated antiserum generated in response to immunization of a subject with a non-naturally occurring PF-KEX1b or PF-KEX2b peptide. In an embodiment, the method comprises the step of administering to a recipient mammal, in an amount sufficient to treat or prevent the infection, disease, disorder, or its symptoms, and under conditions for treating or preventing the infection, disease, disorder, or its symptoms, a therapeutic, prophylactic, or preventive amount of an immunogenic composition comprising a non-naturally occurring PF-KEX1b or PF-KEX2b peptide, a polynucleotide encoding a pan-fungal Kex peptide, or an antiserum generated in response to immunization of a subject with a non-naturally occurring PF-KEX1b or PF-KEX2b peptide. In an embodiment, the isolated antiserum is in a pharmaceutically acceptable composition. In an embodiment, the recipient mammal is a human patient in need of treatment.

[0168] The treatment will be suitably administered to subjects, particularly humans, who are suffering from, have, are susceptible to, or at risk of having: an infection by one or more fungal organisms (particularly organisms of the genus Pneumocystis, Aspergillus, Candida or Cryptococcus, and particularly one or more of Pneumocystis jirovecii or Pneumocystis carinii, Aspergillus fumigatus, Candida albicans or Cryptococcus neoformans), or a disease, pathogenic condition or symptom thereof associated with an infection by one or more of said fungal pathogens. Those “at risk” subjects can be determined by any objective or subjective judgment by diagnostic tests or the opinion of the subject or healthcare provider (e.g., genetic tests, enzyme tests or assays, or protein markers (e.g., anti-Kex antibody levels, such as anti-Kex antibody levels in serum), family history, etc.). The methods herein also include administering to a recipient subject (including a subject identified as in need of such treatment or at risk of infection) an effective amount of an antifungal pathogen immunogenic composition as described herein, which comprises a non-naturally occurring PF-KEX1b or PF-KEX2b peptide, a polynucleotide encoding a pan-fungal Kex peptide, or an antiserum produced in response to immunization of a subject with a non-naturally occurring PF-KEX1b or PF-KEX2b peptide and isolated from said subject. Identification of subjects in need of such treatment can involve the judgment of the recipient subject or healthcare or medical professional and can be subjective (e.g., opinion) or objective (e.g., measurable by test or diagnostic methods). In an embodiment, the isolated antiserum is provided in a pharmaceutically acceptable composition.

[0169] In some aspects, methods of treating or preventing fungal pathogen-related diseases or conditions (e.g., pulmonary infections, pulmonary diseases or disorders, pneumonia, COPD, aspergillosis, etc.) in a subject are characterized, wherein the methods involve administering to a subject in need thereof an effective amount of an immunogenic composition comprising a non-naturally occurring PF-KEX1b or PF-KEX2b peptide, a polynucleotide encoding a pan-fungal Kex peptide, or an isolated antiserum (produced in an individual in response to administration of a non-naturally occurring PF-KEX1b or PF-KEX2b peptide and obtained from an individual that has mounted an antibody immune response against the PF-KEX1b or PF-KEX2b peptide), such that the subject is therapeutically and / or prophylactically treated against the disease or its symptoms associated with the fungal pathogen.

[0170] In another aspect, a method is provided for treating or preventing a disease and / or its symptoms associated with a fungal infection in a patient who is receiving or has received an immunosuppressive drug or medication, either within or outside a hospital environment, and who is susceptible or may become susceptible (or at risk of) an infection by a pathogenic fungus (e.g., one or more of the fungal pathogens of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus) due to drug-induced immunosuppression of the immune system. For example, such a patient may be preparing for transplantation (pre-transplant patient) or may have undergone transplantation (post-transplant patient) and is being administered one or more immunosuppressive drugs or medications (anti-rejection medications) and / or otherwise pharmacologically treated to reduce the likelihood of rejection of the transplanted organ or tissue, thereby rendering the patient more vulnerable, susceptible, or at risk of an infection and / or disease caused by one or more of the fungal pathogens of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus. Patients with other types of diseases and conditions (e.g., but not limited to cancer, immunodeficiency diseases (HIV, AIDS, HIV / AIDS), autoimmune diseases, rheumatoid arthritis, or psoriasis, etc.) may also be administered medications with immunosuppressive effects to treat or manage their conditions and, as a result, are at risk of or have a risk of one or more fungal pathogen infections. Such patients may thus be immunocompromised. 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), mycophenolate (CELLCEPT, MYFORTIC); biologic agents and monoclonal antibodies or monoclonal antibody-based antibodies or antigen-binding fragments thereof such as abatacept (ORENCIA); adalimumab (HUMIRA); 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 an embodiment, a patient is about to receive or has received a transplant of an organ selected from the group consisting of a kidney, liver, heart, bone marrow, pancreas, lung, gallbladder, bladder, and the like.

[0171] An immunogenic composition comprising a non-naturally occurring PF-KEX1b or PF-KEX2b peptide, a polynucleotide encoding a pan-fungal Kex peptide, or an antibody (or antiserum containing such an antibody) against a non-naturally occurring PF-KEX1b or PF-KEX2b peptide can be administered in an effective amount to a patient receiving a transplant rejection drug or other immunosuppressive drug, the effective amount increasing, augmenting, or enhancing the immune response of the immunosuppressed patient to a disease or its symptoms associated with or caused by an infection with one, one or more, two or more, three or more, or all four of the fungal pathogens Pneumocystis, Aspergillus, Candida, or Cryptococcus. In an embodiment, the presence (and antibody titer) of antibodies against one or more of the fungal pathogens in a patient receiving an immunosuppressive drug during treatment with the immunosuppressive drug can be evaluated and monitored by employing the methods and kits described herein.

[0172] Optionally, an immunogenic composition comprising a non-naturally occurring PF-KEX1b or PF-KEX2b peptide, a polynucleotide encoding a pan-fungal Kex peptide, or a separated antiserum produced in response to a PF-KEX1b or PF-KEX2b peptide used as an immunogen, as described herein, can be administered in combination with one or more of any other treatment or therapy (e.g., an antifungal therapy). For example, an immunogenic composition comprising a non-naturally occurring PF-KEX1b or PF-KEX2b peptide, a polynucleotide encoding a pan-fungal Kex peptide, or a separated antiserum or immunop plasma produced in response to a non-naturally occurring PF-KEX1b or PF-KEX2b peptide immunogen (wherein the separated antiserum or immunop plasma contains anti-PF-KEX1b or anti-PF-KEX2b peptide antibodies) can be administered to a subject in combination with other antibodies or antibody mixtures having antifungal activity (including, for example, immunop plasma), or in combination with one or more drugs to provide protective immunity in the recipient against one or more of the fungal organisms of the genus Pneumocystis, Aspergillus, Candida, and / or Cryptococcus. As non-limiting examples, one or more drugs having antifungal activity include trimethoprim-sulfamethoxazole, azithromycin-sulfamethoxazole, clarithromycin-sulfamethoxazole, atovaquone, sulfadoxine-pyrimethamine, erythromycin-sulfisoxazole, PS-15, and dapsone-trimethoprim, as well as intravenous pentamidine and clindamycin-primaquine. In an embodiment of any of the foregoing, an immunogenic composition comprising a PF-KEX1b or PF-KEX2b peptide, a polynucleotide encoding a pan-fungal Kex peptide, or a separated antiserum produced in response to a PF-KEX1b or PF-KEX2b peptide immunogen is provided in a pharmaceutically acceptable composition.

[0173] In an embodiment of any of the foregoing aspects, a PF-KEX1b or PF-KEX2b peptide immunogen, an immunogenic composition comprising a non-naturally occurring PF-KEX1b or PF-KEX2b peptide, a polynucleotide encoding a pan-fungal Kex peptide, or an antibody-containing antiserum produced in response to immunization with a PF-KEX1b or PF-KEX2b peptide immunogen and separated from a donor subject can be administered to a recipient subject to allow the recipient subject to obtain immunoprotection against infection or disease caused by one or more of the fungal pathogens, including memory immunoprotection.

[0174] Methods for administering both single and combination therapies (e.g., simultaneously or in other forms) are known to those of skill in the art and are described, for example, in Remington's Pharmaceutical Sciences, 12th Edition, edited by E.W. Martin, Mack Publishing Co. In an embodiment, the antiserum provides a therapeutic antibody-containing composition that treats an infection or disease caused by one or more fungal pathogens as described herein. In another embodiment, the antiserum provides a prophylactic antibody-containing composition that prevents and protects against a disease, a serious disease, and / or its symptoms caused by one or more fungal pathogens as described herein. In an embodiment, the isolated antiserum is in a pharmaceutically acceptable composition.

[0175] Additional methods

[0176] Currently, there is a lack of methods and reagents to determine whether patients asymptomatic for infection with one or more, two or more, three or more, or each of the fungal pathogens of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus are susceptible, vulnerable, or at risk for infection with one or more of these pathogens. It is currently difficult to isolate these fungal organisms because their amounts may be very low or they do not grow under the culture conditions available for assessing their presence in a subject. Thus, it is difficult for healthcare providers and patients to know whether a patient is actually infected with one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus, or whether the patient is likely to become infected (e.g., after a medical procedure, surgery, or transplant).

[0177] It is also difficult to identify, demonstrate, or stratify patients who are susceptible or likely to be susceptible to infection with one or more fungal pathogens, and to monitor changes in a patient's susceptibility over time (e.g., during recovery from surgery or during immunosuppressive therapy, such as after an organ transplant, or during other chemotherapy treatments) or to monitor the reduction or elimination of infection over time in patients being treated for a fungal infection or a related disease. The methods described herein provide a viable solution to such medical needs. In addition, methods are provided that allow a patient to be treated with an appropriate or more targeted fungal therapy by stratifying the patient based on whether the patient has antibodies specific for a particular fungal pathogen to prevent a disease or infection, or by stratifying the patient based on whether the patient has antifungal Kexin peptide antibodies specific for a given type of pathogenic fungus.

[0178] In an embodiment, a method for detecting an antibody in a sample obtained from a subject is provided, wherein the antibody binds to or reacts against a non-naturally occurring PF-KEX1b or PF-KEX2b peptide, and wherein the method comprises: (a) contacting a biological sample obtained from the subject with a non-naturally occurring PF-KEX1b or PF-KEX2b peptide; and (b) detecting an antibody in the sample that specifically binds to the non-naturally occurring PF-KEX1b or PF-KEX2b peptide, wherein detection of binding indicates the presence of an antibody against the Kex peptide of a fungal organism in the sample of the subject. In an embodiment, the PF-KEX1b and / or PF-KEX2b peptide is attached to a solid support or substrate. In an embodiment, binding is detected by performing an immunoassay (e.g., an enzyme-linked immunosorbent assay (ELISA) or a chip assay).

[0179] In another embodiment, a method for monitoring or detecting an antibody against a fungal organism associated with an infection in a subject who has undergone or is about to undergo transplantation to determine, for example, whether the subject is protected or will be protected from infection by one or more fungal pathogens selected from the group consisting of Pneumocystis, Aspergillus, Candida, or Cryptococcus, wherein the method comprises: (a) measuring the level of an antibody that binds to a non-naturally occurring PF-KEX1b or PF-KEX2b peptide in a sample obtained from the subject prior to transplantation surgery at a first time point; (b) measuring the level of an antibody that binds to the non-naturally occurring PF-KEX1b or PF-KEX2b peptide in a sample obtained from the subject at one or more time points after the subject has undergone transplantation surgery; and (c) detecting that the sample of the subject contains a level of an antibody that specifically binds to the non-naturally occurring PF-KEX1b or PF-KEX2b peptide relative to a predetermined or threshold level or relative to a control level, wherein a high level of an antibody that binds to the non-naturally occurring PF-KEX1b or PF-KEX2b peptide in the sample of the subject indicates that the subject has mounted an immune response against the fungal organism (e.g., the kexin peptide of the fungal organism). In an embodiment, an antibody that binds to a non-naturally occurring pan-fungal PF-KEX1b or PF-KEX2b peptide detected in a sample of a subject according to the methods described herein can be used to protect the subject from infection by Pneumocystis, Aspergillus, Candida, or Cryptococcus. In an embodiment of the foregoing method, one of PF-KEX1b peptide and / or PF-KEX2b peptide may be included.

[0180] Repeating the practice of the above method over time (at different time intervals or different time periods) allows monitoring of the levels of antibodies in a subject that bind to non-naturally occurring PF-KEX1b or PF-KEX2b peptides, and based on the measured titers of anti-PF-KEX1b or anti-PF-KEX2b peptide antibodies in a subject's sample, a healthcare provider or clinician can be informed whether it is necessary or reasonable to initiate continued, new, or different treatment of the subject with an appropriate antifungal drug or therapy, or whether it is reasonable to discontinue antifungal treatment or administer less antifungal treatment.

[0181] Other embodiments also provide methods for detecting the presence of antibodies in a biological sample of a subject (e.g., blood, serum, plasma, lymph fluid, bronchoalveolar lavage fluid) that bind to non-naturally occurring PF-KEX1b or PF-KEX2b peptides, wherein the level of antibodies in the biological sample against the non-naturally occurring PF-KEX1b or PF-KEX2b peptides is determined simultaneously. For example, in one embodiment, the method comprises: (a) contacting a biological sample obtained from a subject with a non-naturally occurring PF-KEX1b or PF-KEX2b peptide that selectively binds a plurality of antibodies in the subject's sample for a sufficient period of time to form bound PF-KEX1b or PF-KEX2b peptide-antibody complexes; (b) detecting the binding of the PF-KEX1b or PF-KEX2b peptide to the plurality of antibodies in the subject's sample, thereby determining the level of antibodies against the kexin peptide in the sample; and (c) comparing the level of the plurality of antibodies in the sample to a predetermined threshold, wherein a level of antibodies that bind to at least one of PF-KEX1b or PF-KEX2b peptides that is higher or lower than the predetermined threshold indicates, for example, that the subject has an antibody titer and has mounted an immune response against the Kex peptide of one or more of the fungal organisms. Thus, a subject having a measured antibody response to the Kex peptide is protected from diseases, severe diseases, or symptoms associated with or caused by infection with one or more of the fungal organisms of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus.

[0182] In another embodiment, there is provided a method for evaluating in a subject an antibody that binds to a Kex peptide of one or more of the fungal organisms Pneumocystis, Aspergillus, Candida, or Cryptococcus, the method comprising: (a) contacting a biological sample obtained from the subject with a composition comprising a PF-KEX1b peptide and / or a PF-KEX2b peptide for a sufficient period of time to form an antibody-Kex peptide complex; (b) detecting the binding of the Kex peptide to the antibody in the sample, thereby detecting the level or titer of the anti-Kex peptide antibody in the sample; and (c) comparing the level or titer of the anti-Kex peptide antibody in the biological sample with a predetermined threshold or control value, wherein a level of at least one of the anti-Kex peptide antibodies being higher or lower than the predetermined threshold indicates that the subject correspondingly has or does not have a sufficient immune response (antibody response) to prevent infection by one or more fungal organisms.

[0183] antibody

[0184] As described herein, an antiserum comprising an antibody that specifically binds to a non-naturally occurring PF-KEX1b or PF-KEX2b peptide and cross-reacts with a Kexin peptide of one or more different fungal organisms (e.g., one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus) provides therapeutic treatment and / or immunoprotection against diseases and their symptoms caused by or associated with infection by one or more of these fungal pathogens, and is thus useful in a method of treatment. In a particular embodiment, there is provided a method of using an isolated antiserum (or immunoplate) comprising an antibody or an antigen-binding fragment thereof (specifically binding to a non-naturally occurring PF-KEX1b or PF-KEX2b peptide and cross-reacting with a Kexin peptide of one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus) for treating or preventing diseases and / or their symptoms caused by or associated with infection by these fungal pathogens, such as various types of pulmonary diseases and disorders, pneumonia, COPD, aspergillosis, invasive pulmonary aspergillosis (IPA), etc.

[0185] Methods for preparing antibodies are well known to those of ordinary skill in the art of immunology. As used herein, the term "antibody" refers not only to intact antibody molecules, but also to antibody molecule fragments that retain the ability to bind to an immunogen (immunogenic antigen). Such fragments are also well known in the art and are often employed both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules, but also to the well-known active fragments F(ab')2 and Fab. The F(ab')2 and Fab fragments, which lack the Fc fragment of the intact antibody, are cleared more rapidly from the circulation and may have less non-specific tissue binding than intact antibodies (see, e.g., Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Antibodies can include all natural antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single-chain V-region fragments (scFv), fusion polypeptides, and unconventional antibodies.

[0186] Unconventional 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 multivalency (e.g., diabodies, triabodies, tetra-bodies, and pentabodies). Nanobodies are the smallest fragments of naturally occurring heavy chain antibodies that have evolved full functionality in the absence of a light chain. Nanobodies possess the affinity and specificity of conventional antibodies, although they are only half the size of a single-chain Fv fragment. The consequence of this unique structure combined with their extreme stability and high homology to the human antibody framework is that nanobodies can bind therapeutic targets inaccessible to conventional antibodies. Recombinant antibody fragments with multivalency provide high binding avidity and unique targeting specificity for cancer cells. These multimeric scFvs (e.g., diabodies, tetra-bodies) are improved compared to the parental antibodies because small molecules of approximately 60-100 kDa size provide faster blood clearance 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)).

[0187] A variety of techniques for making and using unconventional antibodies have been described. Bispecific antibodies generated using leucine zippers were described by Kostelny et al. (J. Immunol. 148(5):1547-1553, (1992)). The diabody technology was 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 was described by Gruber et al. (J. Immunol. 152:5368, (1994)). Trispecific antibodies were described by Tutt et al. (J. Immunol. 147:60, (1991)). Single-chain Fv polypeptide antibodies include covalently linked VH::VL heterodimers that can be expressed from nucleic acids that include V H - and V L -encoding sequences that are directly joined or joined by a peptide-encoding linker as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, (1988)). See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754.

[0188] In various embodiments, an antiserum (isolated antiserum) contains an antibody or an antigen-binding fragment thereof that is monoclonal or polyclonal and specifically binds a non-naturally occurring PF-KEX1b or PF-KEX2b peptide. Also covered are methods for obtaining or isolating antibodies from immune sera (antisera) or immune plasma and generating hybrid or chimeric antibodies therefrom. In such hybrid or chimeric antibodies, one pair of heavy and light chains is obtained from a first antibody, while the other pair of heavy and light chains is obtained from a different second antibody. Such hybrid or chimeric antibodies can also be formed using humanized antibody heavy and light chains. Methods for isolating antibodies and generating hybrid or chimeric antibodies are known and practiced by those skilled in the art.

[0189] Generally speaking, intact antibodies are considered to contain "Fc" and "Fab" regions. The Fc region is involved in complement activation and not in antigen binding. Antibodies that have been enzymatically cleaved in the Fc region or that have been produced without an Fc region are called "F(ab')2" fragments, which retain the two antigen-binding sites of the intact antibody. Similarly, antibodies that have been enzymatically cleaved in the Fc region or that have been produced without an Fc region are called "Fab'" fragments, which retain one of the antigen-binding sites of the intact antibody. The Fab fragment consists of a covalently bound antibody light chain and a portion of the antibody heavy chain (designated "Fd"). The Fd fragment is the major determinant of antibody specificity (a single Fd fragment can associate with up to ten different light chains without altering antibody specificity). Isolated Fd fragments retain the ability to specifically bind immunogenic epitopes.

[0190] Antibodies (and immune sera or plasma containing antibodies) can be produced or generated using a soluble polypeptide, peptide, or an immunogenic fragment thereof (e.g., the PF-KEX1b or PF-KEX2b peptide) as an immunogen by any method known in the art. One way to obtain an antibody is to immunize a suitable host animal or subject with the PF-KEX1b or PF-KEX2b peptide immunogen or a polynucleotide encoding a pan-fungal Kex peptide, and follow standard procedures for polyclonal or monoclonal antibody production. Briefly, the administered immunogen will promote the presentation of the immunogen (or an immunogenic fragment of the immunogen) on the cell surface. The suitable host can be immunized in a variety of ways. For example, a nucleic acid sequence encoding the immunogenic PF-KEX1b or PF-KEX2b 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 PF-KEX1b or PF-KEX2b peptide in a manner that generates an immunogenic response in the host. In an embodiment, the non-naturally occurring PF-KEX1b or PF-KEX2b peptide can be expressed via a delivery vehicle or expression construct. In other exemplary embodiments, the nucleic acid sequence encoding the non-naturally occurring PF-KEX1b or PF-KEX2b peptide can be expressed in cells in vitro, and the expressed recombinant PF-KEX1b or PF-KEX2b peptide product can be isolated and used as an immunogen to generate anti-Kex peptide antibodies and anti-Kex peptide antisera in a suitable immunized host.

[0191] Alternatively, if desired, antibodies against non-naturally occurring PF-KEX1b or PF-KEX2b peptides can be derived from antibody phage display libraries. Phage are able to infect bacteria and multiply within bacteria, and can be engineered to display human antibody proteins when combined with human immunoglobulin (antibody) genes. Phage display is the process by which phage "display" human antibody proteins on their surface. Genes from a human antibody gene library are inserted into a population of phage. Each phage carries the gene for a different antibody and thus displays a different antibody on its surface.

[0192] Antibody purification methods include, but are not limited to, salt precipitation (e.g., with ammonium sulfate), ion exchange chromatography (e.g., on cation or anion exchange columns, preferably run at neutral pH and eluted with a step gradient increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and affinity resin chromatography using, for example, Protein A, Protein G, hydroxyapatite, or anti-immunoglobulin.

[0193] In certain aspects, antibodies can be conveniently produced by hybridoma cells engineered to express the antibody. Methods for preparing hybridomas are well known in the art. Hybridoma cells can be cultured in a suitable medium, and the spent medium can be used as a source of the antibody. The polynucleotide 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 antibody, it is usually more convenient to obtain ascitic fluid. Methods for producing ascitic fluid generally include injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal (especially a mouse). The mammal can be prepared for ascites production by prior administration of a suitable composition (e.g., pristane). Ascitic fluid containing the antibody (typically at high concentration) can be obtained from the peritoneal fluid of the animal containing the injected hybridoma cells.

[0194] Monoclonal antibodies (Mabs) can also be "humanized" by methods known in the art. A "humanized" antibody is an antibody in which at least a portion of the sequence has been altered from its original form to make it more closely resemble immunoglobulins derived from human sources. Techniques for humanizing antibodies are particularly useful when the antibody is produced in a non-human animal (e.g., mouse, rat). Non-limiting examples of methods for humanizing murine antibodies are provided, for example, in U.S. Patent Nos. 4,816,567, 5,530,101, 5,225,539, 5,585,089, 5,693,762, and 5,859,205.

[0195] In the foregoing embodiments, one or more antibodies or antigen-binding fragments thereof generated against the non-naturally occurring PF-KEX1b or PF-KEX2b peptides can be used alone or in combination in a pharmaceutical composition to provide immunotherapy and / or protection against a disease or its symptoms caused by one or more of the fungal pathogens in a subject in need thereof. Such antibodies can be isolated or purified from antiserum as described herein, or they can be generated, purified, and formulated for pharmaceutical use in a subject in need thereof by, for example, recombinant molecular biology techniques. Such formulations of antibodies that bind to the PF-KEX1b or PF-KEX2b peptides can have immunoprotective properties similar to those provided by the isolated antiserum containing antifungal Kex peptide antibodies.

[0196] Vaccine

[0197] A vaccine is a biological agent that can provide active, acquired immunity (e.g., protective immunity) against a specific disease in a subject. Vaccines typically contain substances similar to the disease-causing agents (e.g., microorganisms, fungi, etc.) that cause the disease and are generally made from a weakened or inactivated form of such substances, or from toxins or surface proteins, peptides, or polynucleotides encoding them. After administration of a vaccine to a subject, the substance is expressed and recognized by the subject as a foreign substance (or "non-self substance") and stimulates the subject's immune system to mount an immune response (a B cell (antibody) and / or T cell (cellular) immune response) and eliminate the substance. In addition, cells of the immune system (e.g., B cells) that are exposed to the vaccine retain a memory of the vaccine, such that upon later or subsequent encounter, the antibodies produced by the memory cells recognize and eliminate the vaccine. Vaccines can be prophylactic (e.g., preventing or ameliorating the effects of future infections by a pathogen) or therapeutic (e.g., treating a disease or infection caused by or associated with a pathogen or disease-causing agent when or after a subject has been infected or encountered the pathogen).

[0198] While many vaccines are prepared from attenuated forms of pathogens, or inactivated disease-causing organisms, or suitable portions of such pathogens or organisms such as toxins, proteins / peptides (e.g., non-naturally occurring PF-KEX1b or PF-KEX2b peptides) or virulent enzymes, the antigens to which the immune system responds typically consist of a relatively small number of amino acids, such as peptides that retain immunogenicity and antigenicity. The protein or peptide portion of a pathogen can constitute a vaccine. A peptide vaccine is any peptide used to immunize an organism against a pathogenic agent, protein, peptide, toxin, or pathogen (eliciting 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). In embodiments, the peptide antigen can be a non-naturally occurring PF-KEX1b or PF-KEX2b peptide. In embodiments, after administration to a recipient subject in need, a vaccine comprising a non-naturally occurring PF-KEX1b or PF-KEX2b peptide antigen can be used to provide immune protection against fungal organisms of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus.

[0199] For non-attenuated vaccines, it is necessary to identify the peptide sequences that elicit a protective immune response and to use peptides in synthetic (or recombinantly produced) form as the vaccine substance. Since peptide vaccines are non-naturally occurring and synthetic, there is little or no risk of mutation or reversion, and there is also little or no risk of contamination by pathogenic or toxic substances. In addition, chemical manipulation or modification of the peptide structure can improve stability and reduce adverse side effects or adverse reactions associated with natural protein or peptide sequences.

[0200] Synthetic or recombinantly produced peptide antigens can be easily prepared in large quantities as components of a vaccine. Such substances may also expose protein antigen portions that are not recognized by the immune system during natural infection, which may be due to protein masking or post-translational modification. Sequencing of new strains and serotypes of microorganisms, fungal pathogens, and other pathogenic organisms allows for rapid modification of peptide antigens to generate strain-specific immune responses, particularly against antigenic epitopes recognized and targeted by antibodies and cells of the host or recipient immune system. In some cases, three-dimensional epitope or antigen site modeling of a pathogen can be used to synthesize one or more correct epitopes or antigen sites on the peptide antigen.

[0201] In one aspect, there is provided a vaccine (or immunogenic composition) comprising a synthetic (recombinant) - produced peptide, namely the PF - KEX1b or PF - KEX2b peptide, which is distinct among several different types of fungal pathogens (e.g., Pneumocystis, Aspergillus, Candida, and Cryptococcus fungal pathogens), but is immunologically targetable and can be used to treat or prevent diseases caused by or associated with one or more than one fungal pathogen upon administration to a (immunized) subject. In an embodiment, when used to immunize an individual, a peptide vaccine or immunogenic composition comprising a non - naturally occurring PF - KEX1b or PF - KEX2b peptide elicits an immune response in the form of production of an antiserum (or immune plasma) containing antibodies that cross - react with and provide cross - protection against the kex peptides of the etiologically distinct fungal pathogens Pneumocystis, Aspergillus, Candida, and Cryptococcus, and in particular, Pneumocystis jirovecii, Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans. Thus, the antiserum or immune plasma produced by a vaccine or immunogenic composition comprising a non - naturally occurring PF - KEX1b or PF - KEX2b peptide can be used as the sole therapeutic or protective agent required to treat or prevent diseases or their symptoms caused by or associated with more than one kexin - producing fungal pathogen (i.e., Pneumocystis, Aspergillus, Candida, and / or Cryptococcus fungal pathogens), and in particular, infections or diseases caused by or associated with Pneumocystis jirovecii, Aspergillus fumigatus, Candida albicans, and / or Cryptococcus neoformans. In an embodiment, the antiserum produced by such a peptide vaccine is isolated. In an embodiment, the isolated antiserum is used in a pharmaceutical composition.

[0202] In some aspects, gene vaccines are provided. A gene vaccine is any vaccine that contains a polynucleotide sequence encoding an immunogen, where the immunogen, once expressed, serves to immunize an organism. Administration of the gene vaccine elicits an immune response or a protective immune response against a pathogen or a product produced by the pathogen, such as an antibody (B cell) response and / or an immune cell (T cell) response, in the immunized organism after the immunogen is expressed in cells. In some embodiments, the gene vaccine provides a polynucleotide sequence encoding a Kex peptide (e.g., the PF-KEX1b or PF-KEX2b peptide described herein). In some embodiments, the gene vaccine provides a polynucleotide sequence encoding both the PF-KEX1b peptide and the PF-KEX2b peptide. In some embodiments, the polynucleotide encoding the PF-KEX1b or PF-KEX2b peptide is present in a vector having elements such as a promoter and an enhancer to facilitate the expression of the encoded PF-KEX1b or PF-KEX2b peptide. In some cases, the nucleic acid in the gene vaccine can integrate into the genome of the subject, where the expression of the immunogen can be driven by an endogenous promoter or a promoter encoded by the inserted nucleic acid. In other cases, the nucleic acid in the gene vaccine does not integrate into the genome of the subject. The polynucleotide encoding the immunogen in the gene vaccine can be a DNA polynucleotide or an RNA polynucleotide. The polynucleotide can include nucleotide analogs, which can inhibit the degradation of the polynucleotide.

[0203] Pharmaceutical composition

[0204] Also characterized herein are methods of treating or preventing a disease and / or its symptoms associated with or caused by infection of a subject with one or more fungal pathogens. In one aspect, the method includes administering to a subject in need thereof an immunologically effective amount of the PF-KEX1b or PF-KEX2b peptide, an immunogenic composition comprising the PF-KEX1b or PF-KEX2b peptide, a polynucleotide encoding the PF-KEX1b or PF-KEX2b peptide, or a purified antiserum generated in response to immunization with a non-naturally occurring PF-KEX1b or PF-KEX2b peptide, which treats and / or protects the subject from a disease and / or its symptoms associated with infection with at least one of different fungal pathogens selected from the group consisting of Pneumocystis, Aspergillus, Candida, or Cryptococcus. In an embodiment, the purified antiserum is used in a pharmaceutical composition.

[0205] Typically, the carriers or excipients for the immunogenic compositions or vaccines as described herein are pharmaceutically acceptable carriers or excipients, such as sterile water, saline solution, buffered saline solution, aqueous glucose solution, aqueous glycerol solution, ethanol, or combinations thereof. Such solutions are prepared according to established protocols in the art to ensure sterility, pH, isotonicity, and stability. Generally, the carrier or excipient is selected to minimize hypersensitivity and other adverse effects and to be suitable for a particular route of administration, such as subcutaneous, intramuscular, intravenous, intranasal, etc. Such methods also include administering an adjuvant (such as an oil-in-water emulsion, saponin, cholesterol, phospholipid, CpG, polysaccharide, variants thereof, and combinations thereof) together with the composition as described herein. Optionally, the formulation for prophylactic administration also contains one or more adjuvants for enhancing the immune response against the antigen or immunogen (such as the PF-KEX1b or PF-KEX2b peptide antigen or immunogen). Suitable adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, alpha-galactosylceramide (α-GC), alum, mineral gels (such as aluminum hydroxide), surfactants (such as lysolecithin), pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, Bacillus Calmette-Guérin (BCG), Corynebacterium parvum, and the synthetic adjuvants QS-21 and MF59. In the examples, isolated antiserum is used in the pharmaceutical composition.

[0206] An immunogenic composition (comprising a PF-KEX1b or PF-KEX2b peptide, a polynucleotide encoding a pan-fungal Kex peptide, or an antiserum such as a purified antiserum, a monoclonal or polyclonal antibody against a PF-KEX1b or PF-KEX2b peptide) can be administered as a therapeutic agent for treating or preventing a disease, a severe or serious disease or its symptoms (e.g., a pulmonary infection or disease, poor lung function, COPD, pneumonia, aspergillosis, invasive pulmonary aspergillosis (IPA), etc.) caused by or associated with an infection by a fungal pathogen as described herein, thereby generating a certain concentration of the therapeutic agent, which, if desired, in combination with other components, can effectively improve, reduce, eliminate, alleviate, mitigate or stabilize the disease or the disease symptoms of a subject. The therapeutic agent can be administered systemically, e.g., formulated in a pharmaceutically acceptable composition or buffer (such as saline). Preferred routes of administration include, for example, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrathecal injection or intradermal injection, e.g., to provide a continuous, sustained therapeutic level in the subject. The amount of the therapeutic agent to be administered varies depending on the mode of administration, the age and weight of the subject, and varies due to the disease and / or clinical symptoms associated with the fungal infection. Generally, the amount will be within the range of the amounts of other agents used to treat pulmonary diseases or dysfunctions, although in some cases, the protective and therapeutic scope provided by the therapeutic agent is increased, so a lower amount may be appropriate. The composition is administered in a dose to improve, reduce, alleviate, mitigate, relieve or eliminate the effects of a fungal pathogen infection or disease (e.g., a pulmonary infection and disease and its symptoms), as determined by methods known to those skilled in the art. In an embodiment, a purified antiserum is administered or provided to a recipient subject at or near the site of infection or colonization by one or more pathogenic fungal organisms.

[0207] In an embodiment, a therapeutic or prophylactic therapeutic agent can be included in any suitable carrier substance in any appropriate amount and is typically present in an amount of 0.1%-95%, 0.5%-95%, or 1%-95% (by weight) of the total weight of the composition. The composition can be provided in a dosage form suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal) routes of administration. The pharmaceutical composition can be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th Edition), edited by A.R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, edited by J. Swarbrick and J.C. Boylan, 1988-1999, Marcel Dekker, New York).

[0208] In some cases, the pharmaceutical composition can be formulated such that the active drug is released substantially immediately after administration or at any predetermined time or time period after administration. The latter type of composition is generally referred to as a controlled-release formulation, which includes (i) a formulation that maintains a substantially constant concentration of the therapeutic agent or drug in the body over an extended period of time; (ii) a formulation that maintains a substantially constant concentration of the therapeutic agent or drug in the body over an extended period of time after a predetermined lag time; (iii) a formulation that functions continuously over a predetermined period of time by maintaining a relatively constant effective level in the body while minimizing adverse side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) a formulation that localizes the action by, for example, the spatial arrangement of the controlled-release composition adjacent to or in contact with an organ (e.g., the heart); (v) a formulation that allows convenient dosing such that, for example, a dose is administered once a week or once every two weeks; and (vi) a formulation that targets a disease by delivering the therapeutic agent or drug to a specific cell type or tissue using a carrier or chemical derivative. For some applications, the controlled-release formulation can maintain a therapeutic level in plasma, serum, or blood without the need for frequent dosing during the day. In an embodiment, the isolated antiserum can be formulated with one or more additional components for administration to a subject.

[0209] Any of a variety of strategies can be followed to obtain controlled release, where the release rate exceeds the metabolic rate of the therapeutic agent or drug under discussion. In one instance, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, for example, various types of controlled release compositions and coatings. Thus, a therapeutic agent or drug can be formulated with a suitable excipient into a pharmaceutical composition that releases the therapeutic agent or drug in a controlled manner upon administration. Examples include single or multi-unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, plasters, and liposomes.

[0210] The pharmaceutical composition can be administered parenterally by injection, infusion, or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, etc.) in a dosage form, formulation, or via a suitable delivery device or implant (containing conventional, non-toxic pharmaceutically acceptable 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.

[0211] Compositions for parenteral use can be provided in unit dosage form (e.g., single-dose ampoules), or in vial form containing several doses, and suitable preservatives can be added thereto (see below). The composition can be in the form of a solution, suspension, emulsion, infusion device, or delivery device for implantation, or can be presented as a dry powder to be reconstituted with water or another suitable vehicle prior to use. In addition to the active drug that reduces or ameliorates a disease or dysfunction (e.g., a pulmonary disease or pulmonary dysfunction), the composition can include a suitable parenterally acceptable carrier and / or excipient. In some cases, one or more active therapeutic agents can be incorporated into microspheres, microcapsules, nanoparticulates, liposomes, etc. to achieve controlled release. Additionally, the composition can include suspending agents, solubilizing agents, stabilizers, pH regulators, tonicity regulators, and / or dispersing agents.

[0212] In some embodiments, pharmaceutical compositions comprising an active therapeutic agent (e.g., an immunogenic composition comprising a non-naturally occurring PF-KEX1b peptide or PF-KEX2b peptide, a polynucleotide encoding a pan-fungal Kex peptide, or a purified antifungal antiserum responsive to immunization with a non-naturally occurring PF-KEX1b peptide or PF-KEX2b peptide as described herein) are formulated for intravenous delivery (e.g., intravenous injection) or intrathecal delivery. In an embodiment, the antiserum is a purified antiserum. To prepare such a composition, one or more suitable therapeutic agents are dissolved or suspended in a parenterally acceptable liquid vehicle, excipient, or solvent. Acceptable vehicles and solvents that may be employed are, for example, water; water adjusted to a suitable pH by the addition of a suitable amount of hydrochloric acid, sodium hydroxide, or a suitable buffer; 1,3-butanediol; Ringer's solution; and isotonic sodium chloride and dextrose solutions. Aqueous formulations may also contain one or more preservatives (e.g., methyl, ethyl, or n-propyl p-hydroxybenzoate). In the case where one of the agents is only slightly or sparingly soluble in water, a solubilizing or wetting agent may be added, or the solvent may comprise, for example, 10%-60% w / w propylene glycol, and the like.

[0213] Kits and compositions for detecting and / or quantifying antibodies that react with a PF-KEX1b peptide or a PF-KEX2b peptide

[0214] In another embodiment, kits and compositions are provided that advantageously allow the detection and / or quantification of the presence of antibodies against a Kex protein or peptide of one or more fungal pathogens of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus, or the level of one or more such antibodies that may be present, in a sample (e.g., blood or serum) of a subject. In an embodiment, the subject is a human patient. In an embodiment, the patient has had a transplant, e.g., an organ or tissue transplant, or is about to have a transplant, and is thus at a higher risk of being infected by one or more fungal pathogens. In an embodiment, the transplant patient or the patient about to have a transplant is immunosuppressed and / or otherwise medicated to reduce the likelihood of rejection of the transplanted organ or tissue, thereby making the patient more vulnerable or susceptible to infection and / or disease caused by one or more fungal pathogens of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus. In an embodiment, the patient has received or is about to receive a transplant of an organ selected from the group consisting of kidney, liver, heart, bone marrow, pancreas, lung, and the like.

[0215] A kit such as described herein meets a long-felt need in the art to detect or demonstrate whether any patient, particularly a transplant patient, has a sufficient level (titer) of antibodies against a fungal pathogen to ensure that the patient will not die from the disease and / or be treated and / or protected after being infected with one or more fungal pathogens as described herein, e.g., during hospitalization, or during or after a medical procedure or treatment (such as surgery or transplantation) performed in a hospital or outpatient setting. Currently, due to the lack of appropriate reagents and assays, it is difficult to assess whether a patient about to undergo a medical procedure or surgery, particularly an immunosuppressed patient about to undergo transplantation, or a patient about to start other immunosuppressive therapies, will develop a fungal infection, such as an infection and / or disease caused by one or more fungal pathogens of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus, during or after the immunosuppressive therapy and treatment. Using a kit that tests a patient's sample to determine whether the patient has an antibody titer against one or more of these fungal pathogens (e.g., a high or low antibody titer against kexin peptides of one or more of the fungal pathogens) will greatly increase the success rate of the patient's recovery and targeted treatment after surgery or transplantation. For example, if after testing a patient's sample (e.g., a blood, plasma, or serum sample from a transplant patient) using a kit as described herein, it is determined that the patient has a low, negligible, or zero antibody (antiserum) titer against one or more of the fungal pathogens, particularly the Kex peptides of one or more of the fungal pathogens, it can be presumed that the patient does not have natural or sufficient protection against a possible or actual infection by one or more of the fungal pathogens of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus.

[0216] The kits as described herein will allow testers and patients to determine and understand whether a patient's sample (serum or plasma sample) contains one or more, two or more, three or more, or four of the Kex proteins / peptides against organisms of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus. If the results obtained using the kit indicate that the patient does not have specific antifungal Kex peptide antibodies against a specific antifungal Kex peptide, or that such antibody titers are low (e.g., no specific antifungal Kex peptide antibodies in serum or plasma), the patient is identified as potentially being vulnerable or predisposed to disease or severe disease following infection with a specific fungal pathogen, and then appropriate antifungal treatment can be administered against the specific fungal pathogen for which the patient has no specific antibodies or negligible specific antibodies or reduced antibody titers. In an embodiment, prophylactic antifungal treatment or therapy is administered to the patient. In an embodiment, the treatment comprises administering to the patient an appropriate drug or pharmaceutical that is optimally designed to treat an infection with a specific fungal pathogen or two or more fungal pathogens (i.e., Pneumocystis, Aspergillus, Candida, or Cryptococcus) or a disease associated with such infection. In an embodiment, the treatment comprises administering to the patient a composition as described herein comprising a non-naturally occurring PF-KEX1b peptide or PF-KEX2b peptide or a polynucleotide encoding a pan-fungal Kex peptide to generate a cross-reactive (cross-protective) antibody immune response in the patient, thereby reducing or eliminating a disease, severe disease, and / or its symptoms caused by or associated with an infection with one or more of the organisms of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus. Antibodies generated against the non-naturally occurring PF-KEX1b peptide or PF-KEX2b peptide as described herein can recognize Kex peptides of other fungal organisms as described herein, thereby providing treatment and / or protection (cross-protection) against more than one of the fungal organisms in the patient.

[0217] In an embodiment, a kit is provided for detecting an antibody against a fungal Kex protein or peptide against organisms of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus in a biological sample of a patient or demonstrating the level of said antibody, wherein the kit comprises a substrate attached with a non-naturally occurring PF-KEX1b peptide and / or PF-KEX2b peptide for measuring the level of antibody in the sample. The substrate is contacted with a biological sample obtained from a patient, and a labeled detection molecule is used to detect and measure the level of antibody that binds to the PF-KEX1b peptide and / or PF-KEX2b peptide on the substrate. In an embodiment, detecting an anti-fungal Kex peptide antibody in a sample or measuring the level of such an antibody present in a patient's sample is compared with a positive and / or negative control. In an embodiment, detecting an anti-fungal Kex peptide antibody in a sample or measuring the level of such an antibody present in a patient's sample is compared with a cut-off value. In an embodiment, the substrate is attached with a non-naturally occurring PF-KEX1b peptide and / or PF-KEX2b peptide and Kex peptides from each of the genus Pneumocystis, Aspergillus, Candida, and Cryptococcus. In an embodiment, the substrate is attached with a non-naturally occurring PF-KEX1b peptide and / or PF-KEX2b peptide and a Kex peptide from one of the genus Pneumocystis, Aspergillus, Candida, and Cryptococcus. In an embodiment, the substrate is attached with a PF-KEX1b peptide and / or PF-KEX2b peptide and Kex peptides from the genus Pneumocystis and Aspergillus. In an embodiment, the substrate is attached with a PF-KEX1b peptide and / or PF-KEX2b peptide and Kex peptides from the genus Pneumocystis and Candida. In an embodiment, the substrate is attached with a PF-KEX1b peptide and / or PF-KEX2b peptide and Kex peptides from the genus Pneumocystis and Cryptococcus. In an embodiment, the substrate is attached with a PF-KEX1b peptide and / or PF-KEX2b peptide and Kex peptides from the genus Candida and Aspergillus. In an embodiment, the substrate is attached with a PF-KEX1b peptide and / or PF-KEX2b peptide and Kex peptides from the genus Cryptococcus and Aspergillus. In an embodiment, the substrate is attached with a PF-KEX1b peptide and / or PF-KEX2b peptide and Kex peptides from the genus Candida and Cryptococcus. In an embodiment, the substrate is attached with a PF-KEX1b peptide and / or PF-KEX2b peptide and Kex peptides from the genus Pneumocystis, Aspergillus, and Candida. In an embodiment, the substrate is attached with a PF-KEX1b peptide and / or PF-KEX2b peptide and Kex peptides from the genus Pneumocystis, Aspergillus, and Candida. In an embodiment, the substrate is attached with a PF-KEX1b peptide and / or PF-KEX2b peptide and Kex peptides from the genus Pneumocystis, Aspergillus, and Cryptococcus.In an embodiment, the substrate is attached with PF-KEX1b peptide and / or PF-KEX2b peptide and Kex peptides derived from Pneumocystis, Cryptococcus, and Candida. In an embodiment, the substrate is attached with PF-KEX1b peptide and / or PF-KEX2b peptide and Kex peptides derived from Aspergillus, Cryptococcus, and Candida. In an embodiment, the PF-KEX1b peptide and / or PF-KEX2b peptide are recombinantly produced. In an embodiment, the detection of antibodies binding to the fungus-derived Kex peptides in a sample is performed using an immunoassay (e.g., ELISA). In an embodiment, ELISA detects the complex formed between the antifungal Kex peptide antibody present in the sample and the Kex peptide that binds to the antibody. In an embodiment, the detection of antibodies binding to the Kex peptides derived from fungi in a sample is performed using an immunosorbent assay, by immunoprecipitation, by immunoblotting, or a combination thereof.

[0218] A kit is also provided, which comprises reagents that allow the assessment, measurement, evaluation, or detection of antibodies against PF-KEX1b peptide and / or PF-KEX2b peptide. Such antibodies may be contained in a biological sample obtained from a subject who is tested, evaluated, or assessed using the kit. In particular, the biological sample may be a blood, serum, plasma, urine, cerebrospinal fluid, sputum, bronchoalveolar lavage fluid, tear, saliva, fecal, or semen sample, or a tissue or cell sample obtained from the subject. In particular, the reagents of the kit comprise PF-KEX1b peptide and / or PF-KEX2b peptide.

[0219] In particular embodiments, the kit is provided as an enzyme-linked immunosorbent assay (ELISA) kit comprising a PF-KEX1b peptide and / or a PF-KEX2b peptide. In other embodiments, the provided kit allows for the detection of cross-reactive antibodies, where the antibodies are generated by immunization with a PF-KEX1b peptide and / or a PF-KEX2b peptide. In such embodiments, the kit is provided as an ELISA kit comprising a Kex peptide of Pneumocystis, Aspergillus, Candida, or Cryptococcus attached to a solid support or substrate. Thus, the peptide attached to the substrate acts as a "capture" reagent that binds to antibodies present in a sample obtained from a subject being tested. For example, the ELISA kit can comprise a solid support such as a chip, a microtiter plate containing a plurality of wells (e.g., a 96-well plate), beads, or a resin to which the peptide capture reagent is attached. In one embodiment, the kit comprises Kex peptides from each of Pneumocystis, Aspergillus, Candida, or Cryptococcus as described herein, with the peptides independently attached to different regions or components of the solid substrate or support, e.g., the Kex peptides of each fungal organism are attached to separate and distinct wells of a microtiter plate or independently attached to beads to produce a population of beads to which the Kex peptides of each of Pneumocystis, Aspergillus, Candida, or Cryptococcus are attached. In another embodiment, the kit comprises a combination or mixture of Kex peptides from Pneumocystis, Aspergillus, Candida, or Cryptococcus attached to a region or component of the solid substrate or support, e.g., all of the Kex peptides of Pneumocystis, Aspergillus, Candida, or Cryptococcus are attached to a single well of a microtiter plate or a single bead. In a further embodiment, the kit comprises a combination of one, two or more, three or more, or four of the Kex peptides from Pneumocystis, Aspergillus, Candida, or Cryptococcus attached to a given region (e.g., a single well of a microtiter plate) of the solid substrate or support.

[0220] In an embodiment, in an ELISA platform, the wells of a microtiter plate may be coated with PF-KEX1b peptide and / or PF-KEX2b peptide, Aspergillus Kex peptide, Candida Kex peptide, or Cryptococcus Kex peptide. In an ELISA platform, the wells of a microtiter plate may be coated with Pneumocystis Kex peptide and Aspergillus Kex peptide. In an ELISA platform, the wells of a microtiter plate may be coated with Pneumocystis Kex peptide and Candida Kex peptide. In an ELISA platform, the wells of a microtiter plate may be coated with Pneumocystis Kex peptide and Cryptococcus Kex peptide. In an ELISA platform, the wells of a microtiter plate may be coated with Aspergillus Kex peptide and Candida Kex peptide. In an ELISA platform, the wells of a microtiter plate may be coated with Aspergillus Kex peptide and Cryptococcus Kex peptide. In an ELISA platform, the wells of a microtiter plate may be coated with Candida Kex peptide and Cryptococcus Kex peptide. In an ELISA platform, the individual wells of a microtiter plate may be coated with Pneumocystis Kex peptide, Aspergillus Kex peptide, and Candida Kex peptide. In an ELISA platform, the individual wells of a microtiter plate may be coated with Pneumocystis Kex peptide, Aspergillus Kex peptide, and Cryptococcus Kex peptide. In an ELISA platform, the individual wells of a microtiter plate may be coated with Pneumocystis Kex peptide, Candida Kex peptide, and Cryptococcus Kex peptide. In an ELISA platform, the individual wells of a microtiter plate may be coated with Aspergillus Kex peptide, Candida Kex peptide, and Cryptococcus Kex peptide.

[0221] The kit may further comprise means for detecting the peptide or any antibody that binds thereto, such as a detectable antibody, a secondary antibody-signal complex, such as a goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (HRP) or 3,3′,5,5′-tetramethylbenzidine (TMB) as an HRP substrate.

[0222] In another embodiment, the kit can be provided as an immunochromatographic strip that includes a membrane on which one, two, three, or four fungal Kex peptides are immobilized at different positions or, alternatively, are immobilized together at one position on the membrane, and a means for detecting antibody binding in a test sample, such as a detectably labeled peptide or a secondary antibody conjugated to gold particles, where the membrane can be a nitrocellulose (NC)-based membrane, a PVDF membrane, or other suitable types of membranes used in the art. The kit can include a plastic plate or substrate on which the sample is applied, and an immobilized detection agent, such as a detectably labeled Kex peptide, for example, a peptide conjugated to gold particles that is immobilized at intervals and on a substrate, such as a glass fiber filter or nitrocellulose membrane, or a labeled detection agent that can detect complexes of antibodies that bind to the Kex peptide in one or more bands on the substrate. In such a platform, a sample, such as blood or serum, is maintained above the detection reagent immobilized on the substrate by continuous capillary flow such that sample antibodies that bind to the labeled Kex peptide or sample antibodies that are complexed with the Kex peptide reagent can be detected. Generally, ELISA assays and immunosorbent assays, including ELISA membrane-based immunosorbent assays, and variations of these assays are known and practiced by those skilled in the art.

[0223] 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 made of, for example, 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. US2010 / 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.

[0224] In an embodiment, multiplex assays (e.g., multiplex ELISA) can be used to simultaneously detect different specific antibodies in a test sample. In an embodiment, such methods employ an array in which multiple binders (e.g., capture peptides) for multiple antibodies are immobilized on a substrate (e.g., a membrane), with each capture agent positioned at a specific, predetermined location on the substrate. Methods of performing assays using such arrays include, for example, those described in U.S. Patent Application Publication Nos. US 2010 / 0093557 A1 and US 2010 / 0190656 A1, the disclosures of which are hereby incorporated by reference in their entirety. If flow cytometry, chemiluminescence, or electrochemiluminescence techniques are employed, the multiplex arrays can be used in several different formats. Illustratively, flow cytometry multiplex arrays, also known as bead-based multiplex arrays, include the Flow Cytometry Bead Array (CBA) system from BD Biosciences (Bedford, MA) and the Multi-Analyte Profiling technology from Luminex Corp. (Austin, TX) (both of which employ sets of beads distinguishable by flow cytometry) and other techniques known and used in the art.

[0225] In another embodiment, the multiplex ELISA from Quansys Biosciences (Logan, UT) involves coating multiple specific capture reagents (one reagent per spot) at multiple spots within the same well of a 96-well microtiter plate. Multiple antibodies bound at the corresponding spots on the plate are then detected using chemiluminescence techniques.

[0226] In certain embodiments, a patient can be diagnosed by adding a biological sample of the patient (e.g., blood, plasma, or serum) to a kit or a component thereof and detecting a sample antibody that specifically binds to a Kex peptide reagent. For example, the method includes: (i) collecting a blood, plasma, or serum sample from a subject; (ii) adding the subject's sample to a component (e.g., a storage tube or a substrate) in the kit; and (iii) detecting the peptide reagent that has bound to the sample antibody. In this method, the subject's sample (e.g., blood, plasma, or serum) is contacted with one or more Kex peptide reagents (e.g., PF-KEX1b peptide and / or PF-KEX2b peptide). 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, the blood is not collected from the patient (i.e., the blood has already been collected), and the presence of antibodies in the blood that bind / react with the PF-KEX1b peptide and / or PF-KEX2b peptide is determined. In some embodiments, using a kit, sample antibodies generated against one or more, two or more, three or more, or four of the kexin peptides or proteins of organisms of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus bind or react with the PF-KEX1b peptide or PF-KEX2b peptide. Additionally, in other embodiments, the sample can comprise a tissue sample or a clinical sample, which can be processed (e.g., homogenized and / or suspended) in a culture medium or a buffer before the assay. In an embodiment, any one or more antibodies found to be present in a test sample from a subject can be isolated, or isolated and purified, and further characterized.

[0227] The kit can also comprise a washing solution or instructions for preparing a washing solution, wherein the combination of the capture reagent and the washing solution allows for the capture of anti-Kex antibodies on a solid support for subsequent detection, e.g., by a secondary antibody, a labeled reagent peptide, or mass spectrometry. In a further embodiment, the kit can comprise instructions regarding suitable operating parameters in the form of a label or a separate insert (package insert). For example, the instructions can 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, how to interpret the results, etc. In yet another embodiment, the kit can comprise one or more containers containing suitable positive and negative controls or control samples to be used as one or more standards for detection, calibration, or normalization.

[0228] On the other hand, there is provided a kit for treating or preventing an infection or disease caused by or associated with two or more of fungal pathogens of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus. In some embodiments, the kit includes an effective amount of a therapeutic or prophylactic antiserum that contains anti-Kex peptide antibodies or antigen-binding fragments thereof that bind / react with PF-KEX1b peptide and / or PF-KEX2b peptide. In some embodiments, these antibodies cross-react in unit dosage form with one or more of the kexin peptides or polypeptides of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus. In embodiments, the antiserum is a purified antiserum. In other embodiments, the kit includes a therapeutic or prophylactic composition that contains an effective amount of an antifungal immunoprotectant such as the antiserum in unit dosage form. In some embodiments, the kit contains a device for dispensing the composition (e.g., a nebulizer, metered-dose inhaler) or a sterile container containing the pharmaceutical composition; such a container can be in the form of a box, ampoule, bottle, vial, tube, pouch, sachet, blister pack, or other suitable container forms known in the art. Such a container can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing a drug.

[0229] If desired, there is provided a pharmaceutical composition together with instructions for administering the pharmaceutical composition to a subject suffering from or at risk of infection or developing a disease and / or its symptoms caused by an infection with one or more of the fungal organisms of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus, the pharmaceutical composition containing a purified antiserum. The instructions will generally include information regarding the use of the composition for treating or preventing a disease and / or its symptoms caused by an infection with one or more of the fungal organisms of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus. In other embodiments, the instructions include at least one of the following: a description of the therapeutic / prophylactic agent; a dosage regimen and administration for treating or preventing a disease or its symptoms caused by one or more of the fungal organisms of the genus Pneumocystis, Aspergillus, Candida, or Cryptococcus; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions can be printed directly on the container (if present), or affixed to the container as a label, or provided as a separate sheet, booklet, card, or folder, placed inside or together with the container.

[0230] Unless otherwise indicated, the practice of the presently described embodiments employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the capabilities of those of ordinary skill in the art. Such techniques have been explained fully in the literature, such as "Molecular Cloning: A Laboratory Manual", 2nd ed. (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 thus, may be considered for use in the preparation and practice of the aspects and embodiments described herein. Techniques that are particularly useful for specific embodiments will be discussed in the following sections.

[0231] Examples

[0232] The following examples are given to provide a complete disclosure and description to those of ordinary skill in the art of how to make and use the products, compositions, assays, screens, and treatment methods as described herein, and are not intended to limit the scope of the described aspects and embodiments.

[0233] Example 1: Pan-fungal Immunogenic Peptides

[0234] Described herein is a pan-fungal Kexin peptide, used as an immunogen to generate a potent immune response against more than one fungal pathogen. When administered to a subject, the peptide is immunogenic (eliciting an anti-peptide antibody response). The peptide comprises the following:

[0235] Panfungal peptide 1b (PF-KEX1b) (which has the following amino acid sequence: DDDGKTVDGPSPLVLRAFINGVNNGRNGLGSIYVFASGNGGIYEDNSNFDGYANSVFTITIGGIDKHGKRPKYSEASSSQLAVTYAGGSA (SEQ ID NO: 1)) or an immunogenic fragment thereof.

[0236] Panfungal peptide 2b (PF-KEX2b) (which has the following amino acid sequence: PDDGKTMEGPDILVLRAFINGVQNGRDGKGSIYVFASGNGGGFEDNSNFDGYTNSIYSITVGAIDRKGLHPSYSEASSAQLVVTYSSGSG (SEQ ID NO: 2)) or an immunogenic fragment thereof.

[0237] In embodiments, PF-KEX1b and PF-KEX2b peptides containing serine residues at positions 47 and 77 can provide beneficial properties for the manufacture and formulation of peptides (e.g., for commercial production and use), such as lack or reduction of stability, cross-linking, and / or aggregation.

[0238] The PF-KEX1b and PF-KEX2b peptide sequences are related but not identical to the Kex1 panfungal consensus sequence, which was identified by performing a multiple sequence alignment of the KEX1 peptide sequences from Pneumocystis (accession number EU918304.1) (isolated from rhesus macaque), Aspergillus fumigatus Kexin (accession number XM746441), Candida albicans Kexin (accession number AF022372), and Cryptococcus neoformans Kexin (accession number XP572303.1). The alignment was performed using Clustal Omega (http: / / www.ebi.ac.uk / Tools / msa / clustalo / ) to analyze sequence identity and similarity, as described in WO 2020 / 154510 (published on July 30, 2020, the entire content of which is incorporated herein by reference). The 90-mer panfungal consensus KEX1 peptides (panfungal peptide 1 and panfungal peptide 2) in WO 2020 / 154510 have approximately 97% and 69% amino acid sequence identity, respectively, with the amino acid sequence of Pneumocystis KEX1 isolated from humans. The PF-KEX1b peptide (SEQ ID NO: 1) and the PF-KEX2b peptide (SEQ ID NO: 2) contain serine residues at positions 47 and 77 of the amino acid sequence.

[0239] The coding DNA sequence of the PF-KEX2b peptide was cloned into an Escherichia coli expression vector, and the recombinant protein was produced, isolated, and purified for the studies described below.

[0240] Example 2: Evaluation of the immunogenicity of PF-KEX2b peptide

[0241] To evaluate the immunogenicity of the PF-KEX2b peptide (SEQ ID NO:2) as described herein, BALB / c mice (50% male) were immunized intramuscularly (IM) with 10 μg (the line with "x" on the graph) or 20 μg (the solid line on the graph) of the PF-KEX2b peptide (mixed with adjuvant at a 1:1 ratio). Twenty-one (21) days after the first immunization, the mice were boost-immunized with the same dose of the PF-KEX2b peptide plus The same dose was administered for the third immunization 21 days after the second immunization. Figure 1 The immunization time points and the antibody titers at different time points are shown. The research results indicate that immunizing the animals with 10 μg or 20 μg of the PF-KEX2b peptide and adjuvant resulted in strong antibody titers after each vaccination (immunization).

[0242] Example 3: Evaluation of the immunogenicity and protective efficacy of PF-KEX2b peptide and immunization in a murine model of invasive aspergillosis associated with Aspergillus fumigatus (AF) infection

[0243] A murine model of invasive pulmonary aspergillosis (IPA) was used to evaluate whether mice immunized with the PF-KEX2b peptide and adjuvant as described herein developed a humoral immune response and immune antiserum containing anti-PF-KEX2b antibodies, and whether these antibodies protected the immunized animals from subsequent infection with Aspergillus pathogens.

[0244] Vaccine construction and purification

[0245] The polynucleotide encoding the PF-KEX2b (SEQ ID NO:2) peptide was cloned into the pET28b(+) expression vector (Novagen) of Escherichia coli BL21(DE3)pLysS (ThermoFisher Scientific) and purified by affinity chromatography.

[0246] Immunization and immunosuppression

[0247] Mice were immunized with 10 or 20 μg of the PF-KEX2b immunogen (mixed with adjuvant at a 1:1 ratio according to the adjuvant guidelines, e.g., ) Preparation together) Mice were immunized via intramuscular (IM) injection. In some embodiments, for example using TiterMax adjuvant, mice were immunized subcutaneously at the base of the tail. Control mice were sham-immunized with PBS and the same adjuvant. Two weeks after immunization, all mice received an immunosuppressive regimen using subcutaneous injection (SC) of tacrolimus and cortisol. This regimen was administered for six days, during which trimethoprim was added to the drinking water to control secondary infections.

[0248] Aspergillus fumigatus challenge and monitoring

[0249] Aspergillus fumigatus Af293 conidia were maintained in solid 1% glucose minimal medium for 72 hours, harvested in 0.01% Tween-20, counted using a hemocytometer, and then diluted in PBS. After six days of immunosuppression as described above, mice were inoculated intranasally with 5 x 10 6 conidia in 40 μl PBS.

[0250] After challenge, changes in mouse weight, body temperature, and appearance were monitored twice daily. In addition to showing ruffled fur and dyspnea, if the weight loss exceeded 20% of the baseline weight or the body temperature dropped below 29°C, the animal was humanely euthanized. Six days after challenge, all remaining animals were humanely euthanized and their lungs were collected for analysis.

[0251] Fungal burden

[0252] After euthanasia, the right lungs of all animals were stored in 10% neutral buffered formalin. The fixed lung tissues were embedded in paraffin, sectioned, and stained with Gomori-modified methenamine silver staining. Images of five different regions were taken and the fungal burden was quantified according to the guidelines provided by Stolz et al., 2018, J. Vis. Exp. [Journal of Visualized Experiments], (133) e57155:1-8.

[0253] Humoral immune response to PF-KEX2b peptide

[0254] This representative experiment used the PF-KEX2b peptide as an immunogen to evaluate the protective efficacy of the peptide and adjuvant immunization in a murine model of invasive aspergillosis. Therefore, BALB / c mice (female) were immunized intranasally three times (at 21-day intervals) with 10 μg of the PF-KEX2b peptide (mixed with the adjuvant at a ratio of 1:1). Control mice were sham-immunized with PBS and Two (2) weeks after the third immunization, the mice were immunosuppressed with tacrolimus (1 mg / kg / day, i.p.) and cortisol (125 mg / kg, every 3 days) and challenged with 5 x 10 6Intranasal challenges were performed with Aspergillus fumigatus conidia. Figure 2A The challenge study design is shown. The study results showed that mice immunized with PF-KEX2b peptide and immunized (vaccinated) (represented by the line with “x” on the graph in Figure 2B ) had a significantly reduced mortality rate compared to sham-immunized control mice (represented by the solid line on the graph in Figure 2B ) (p = 0.0323). As observed in Figure 2B , immunization of animals with PF-KEX2b peptide and significantly reduced the mortality rate associated with Aspergillus infection and invasive aspergillosis in an immunosuppressed mouse model.

[0255] Statistical analysis

[0256] All statistical analyses were performed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). The reduction in IPA-related deaths was evaluated using Fisher’s exact test. The relative risk of IPA-mortality was determined using the Koopman asymptotic score. The differences in fungal burden by GMS staining were analyzed by Mann-Whitney U tests.

[0257] Example 4: Production of recombinant Kexin proteins

[0258] The pan-fungal Kex peptides PF-KEX1b (PF-KEX1b) and PF-KEX2b (PF-KEX2b) were reverse-translated according to Escherichia coli K-12 codon preferences (www.ebi.ac.uk / Tools / st / emboss_backtranseq / ) and inserted into the expression vector pET-28b(+) using NcoI and BamHI restriction sites (GenScript). Each insert contained an additional GC at the 5' of the Kex sequence, followed by CG, to keep the Kex sequence insert in-frame. The plasmids were transformed into Escherichia coli BL21(DE3) cells and plated on LB agar supplemented with 40 μg / mL kanamycin to select the transformed clones. The recombinant PF-KEX1b and PF-KEX2b proteins were expressed and purified as described in Example 5 below.

[0259] Example 5: Procedure for purifying (His-tagged) recombinant Kexin peptides

[0260] This example describes a protocol for purifying recombinantly produced PF-KEX1b and PF-KEX2b peptides (pET28b vector) (Millipore-Sigma, USA) tagged with histidine (His).

[0261] Materials and Equipment

[0262] A. LB (Lysogeny Broth) growth medium containing kanamycin (40 μg / mL), typically in a 1 L volume, pH adjusted to 7.5. Mix 10 g NaCl, 5 g yeast extract, and 10 g tryptone; bring the volume to 1 L with distilled / deionized H2O.

[0263] B. 1 M IPTG solution.

[0264] C. Extraction buffer: (sterile filtered), 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, 6 M guanidine-HCl, pH 7.4.

[0265] D. Wash buffer: (sterile filtered), 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 7.4.

[0266] E. 1 M imidazole solution in 50 mM sodium phosphate, 300 mM NaCl, pH 7.4, and a 0.2 μm sterile filter (sterile filtered).

[0267] F. HISPUR TM Cobalt resin (Thermo Fisher Scientific, catalog number 89966)

[0268] G. Disposable 5 mL polypropylene column (Thermo part number (P#) 29922).

[0269] H. His-tagged protease inhibitor cocktail (PIC) (Sigma catalog number 8849).

[0270] I. Bradford Dye (Bio Rad catalog number 500-0006).

[0271] J. Bovine serum albumin standard (Thermo Fisher Scientific, catalog number 23209).

[0272] K. Coomassie blue staining solution containing 0.2% Coomassie blue, 7.5% acetic acid, and 50% ethanol.

[0273] L. Coomassie blue de-staining solution containing 50% methanol, 10% acetic acid, and 40% dH2O.

[0274] M. Bisacrylamide 30% (Sigma, part number 1001356385)

[0275] N.N,N,N’,N’-Tetramethylethylenediamine (TEMED) (Sigma, part number 1001434505).

[0276] O. Sodium dodecyl sulfate (SDS) (10% stock solution).

[0277] P. Ammonium persulfate (APS) (10% stock solution).

[0278] Q. 1.5 Tris buffer pH 8.8 (Add 187 g Tris base to 1 L dH2O and adjust the pH to 8.8).

[0279] R. 0.5 Tris buffer pH 6.5 (Add 60.5 g Tris base to 1 L dH2O and adjust the pH to 6.5).

[0280] S. Spectra TM Multicolor Standards - Broad Spectrum Stain (Thermo, part number 22634).

[0281] T. SDS-PAGE Sample Buffer (4X).

[0282] U. Hoefer Gel Casting System (Model SE250).

[0283] The procedures used are as follows:

[0284] Culture in E. coli and induce protein expression: Streak the Kexin construct on pET28b(+) onto an LB KAN agar plate and incubate overnight (O / N) at 37°C or incubate on the bench top at room temperature until sufficient bacterial growth / colonization is obtained. (The plates can be stored at 4°C for approximately 1 month). Inoculate a single colony into 10 mL of liquid LB KAN 40 (10 μL of 40 mg / mL KAN per 10 mL LB) (allowing for a liquid to gas ratio of approximately 1:5) and grow overnight at 37°C with shaking. After overnight incubation, dilute the culture 1:50 into liquid LB KAN 40 (dilute 4 mL of the overnight culture into 196 mL of fresh medium) and place on a 37°C shaker. The culture is grown to an OD 6000.5, then add 1 mM IPTG to induce expression; place on a shaker at 37 °C for 4 - 5 hours. Divide the total volume of the culture into five 50 mL Oakridge tubes (about 40 mL of culture per tube). For scale-up: for larger volumes, 250 mL Oakridge tubes can be used. Harvest the cells by centrifuging at 6,000 x g for 25 minutes at 4 °C (SS-34 or SLA-1500 rotors can be used). Pour off the supernatant and freeze the cell pellet at -80 °C until use. The storage time of the E. coli pellet should not exceed two weeks before protein extraction.

[0285] Protein purification using Talon metal affinity resin: Thaw the pellet on ice and resuspend the cell pellet in 10 mL of extraction buffer + 200 μL of PIC. Incubate at 4 °C for 2 hours on a nutator. Centrifuge the suspension at 10,000 x g for 20 minutes at 4 °C (using an SS-34 rotor). Collect the supernatant and place on ice until the Talon resin is prepared. Prepare a polypropylene elution column by suspending the column in an upright position; add a few drops of wash buffer to the porous plate and then use the reverse end of a Pasteur pipette to evenly press the plate to the bottom of the column.

[0286] Prepare Talon resin: Gently swirl to resuspend the Talon resin, 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. Add 10 mL of deionized water to wash the resin and spin again at 500 x g for 5 minutes. Carefully remove the supernatant and discard. Equilibrate the resin in 10 mL of extraction buffer and spin at 500 x g for 5 minutes. Carefully remove the supernatant and discard. Perform batch binding by mixing the clarified lysate and the equilibrated resin together and incubating at 4 °C for 1 hour. Add the lysate and resin suspension to the prepared polypropylene elution column. Discard the flow-through. Wash the resin with 15 column volumes of extraction buffer, followed by 15 column volumes of wash buffer. Elution of panfungal peptide 2b. Elute with 1.5 mL fractions with increasing imidazole concentration in wash buffer and collect the elution fractions.

[0287] Elution gradient: Add 1.5 mL of 75 mM imidazole in wash buffer and collect the fraction. Add 1.5 mL of 100 mM imidazole in wash buffer and collect the fraction. Add 1.5 mL of 125 mM imidazole in wash buffer and collect the fraction. Add 1.5 mL of 150 mM imidazole in wash buffer and collect the fraction. Add 1.5 mL (x2) of 175 mM imidazole in wash buffer and collect the fraction. Add 1.5 mL (x3) of 200 mM imidazole in wash buffer and collect the fraction.

[0288] Elution of Pneumocystis Kex1 and Panzymopeptide 2.

[0289] Elution of Aspergillus Kex and Cryptococcus Kex (insoluble proteins): Resuspend the resin in 1.5 mL of 1% SDS and transfer to two 1.5 mL tubes. Boil the suspension in a 95 °C heating block. Centrifuge the suspension at 500 x g for 5 minutes. Collect the supernatant fraction containing the denatured protein. Repeat the above four steps three times to recover additional protein. Add 20 μL of PIC to each fraction of interest and store at 4 °C. (The imidazole solution should be stored on ice before use.)

[0290] Quantification of proteins in elution fractions (Bradford assay - low concentration standard curve): Remove BSA - 100 μg / mL from the refrigerator (4 °C) and thaw on ice. Set up the cuvettes for the standard curve and add the specified amounts of thawed BSA / dH2O as shown in the table below (Table 1).

[0291] Table 1

[0292]

[0293] Add an additional cuvette for each fraction and dilute the sample 1:50 (20 μL sample + 180 μL dH2O). Prepare 1:4 Bradford dye in dH2O (10 mL dye + 30 mL dH2O) and add 800 μL (final volume 1 mL) to each cuvette. Mix the cuvettes individually by inverting and incubate at room temperature for 15 minutes. 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, then add 0.25 μg / mL BSA standard to B1 and B2. Continue adding increasing concentrations of BSA standards to the plate in the same order. Once all BSA standards have been added to the plate, load the samples in duplicate into the wells immediately below until there are no remaining rows, then proceed with the top row of the next two rows. After all samples have been loaded onto the plate, read at 595 nm - "low concentration standard curve." Record the linear regression (R 2 ) and BSA standard curve values (data obtained from assays with R 2 < 0.95 should not be used). The raw value of the sample represents a 1:50 dilution, so it should be multiplied by 50 to convert back to μg / mL. Once the protein concentration has been determined, the fractions for coating plates (e.g., ELISA / ELISPOT), injection, etc. must be run on a 15% 2 - mm SDS - PAGE gel to evaluate purity.

[0294] Identifying proteins via Coomassie blue staining of SDS-PAGE gels: For each gel, wash 1 glass cover plate, 1 white aluminum back plate, 2 black plate spacers, and 1 white 10-well stacking comb with dH2O. Rinse with 70% ethanol solution. Confirm that all solidified gel residues left after the last use have been removed before pouring. After all materials are air-dried, remove the back plate and lay it flat on the workbench. Place one spacer on each side of the plate, and then clamp it with a transparent glass cover plate. Confirm that the notches of the spacers are correctly aligned with the edges of the two plates. Loosen all the screws on the casting block and slide the sandwich plate with the spacers into the gel maker. Confirm that all plates and spacers are flat and aligned. Before carefully tightening the screws, let the sandwich plate protrude about 3 mm from the bottom of the casting block to avoid cracking the plate. Place the casting block in the bracket and insert the black plastic plug into the bracket. Turn the plug to press the casting block into the black rubber pad of the bracket. Confirm that the bottom of the plate is well sealed by the rubber of the bracket to avoid leakage. Prepare the separating / running gel according to the following recipe for 15%-SDS PAGE gel, and add the solution to the cavity between the aluminum back plate and the glass cover plate. Leave about 1.5 - 2 cm of space at the top of the sandwich plate for the stacking gel. Add about 1 mL of dH2O to the casting block. Once APS / TEMED is added to the solution, the gel will start to polymerize.

[0295] Separating gel 15％ Bisacrylamide 30% 5 mL <![CDATA[Water (dH2O)]]> 2.34 mL 1.5 Tris buffer pH 8.8 2.5 mL SDS (10%) 100 μL APS (10%) 50 μL TEMED 10 μL

[0296] Once the gel has hardened (about 35 minutes), remove the water layer and prepare the stacking gel solution according to the following recipe. Add the solution quickly.

[0297]

[0298]

[0299] Immediately place the white 10-well stacking comb into the stacking gel and allow it to polymerize fully (about 10 minutes). Prepare the samples to run on the gel: (5 μg of protein per well). If the fraction concentration < 165 μg / mL, use 22.5 μL of sample + 7.5 μL of 4X sample buffer. If the fraction concentration > 165 μg / mL => sample volume = 5 μg / (concentration (μg / mL) / 1000) and 4X sample buffer volume = (1 / 3) * sample volume. Heat inactivate all samples in a water bath at 56 °C for 10 minutes. Remove the 15% SDS-PAGE gel from the casting block and fix it to the running device with 2 red clips. Fill the cavity and the bottom tray of the running device with 1x SDS-PAGE running buffer. The stacking comb can now be removed. Add 10 μL of broad-spectrum stain (part number 26634) SPECTRA TMThe multicolor standard is added to the first well of the gel, and then 30 μL of the prepared sample is added to the subsequent wells. Once all the samples are loaded, attach the electrodes to the appropriate terminals and turn on the power (red to red, black to black). Allow the gel to run at approximately 80 - 120 volts for 1.5 - 2.5 hours until the dye band runs out from the bottom of the gel. At this point, turn off the machine and disconnect the electrodes (Note: Lower voltage and shorter time intervals will improve the quality of the resulting gel). Drain the running buffer from the running device. Remove the red clips, spacers, and gently separate the glass cover plate from the gel casting frame. Use the straight hard plastic edge of the gel scraper to cut off the stacking gel and throw it into the trash. Cut the separating gel as needed for further assays (i.e., Western blotting, etc.) (Since stained standards were used, there is no need to notch a corner of the gel to determine orientation). For the separating gel to be stained, wash it 3 times with dH2O for 15 minutes each. If necessary, add approximately 25 mL of Coomassie blue stain to the gel for 2+ hours or overnight. Decolorize using Coomassie blue decolorizer until the optimal band color / gel transparency is obtained. Take a photo and save it as a JPG / TIF file.

[0300] Example 6: PF-KEX2a and PF-KEX2b Peptide Enzyme-Linked Immunosorbent Assay (ELISA)

[0301] This example describes a protocol for performing an ELISA immunoassay using the pan-fungal peptide 2a (PF-KEX2a) or pan-fungal peptide 2b (PF-KEX2b) peptide antigens used as immunogens in the examples described above. The ELISA is performed to detect (and quantify) the presence of anti-fungal PF-KEX2a or PF-KEX2b antibodies in samples such as blood, plasma, serum, bronchoalveolar lavage fluid, or biological fluid samples. The antibodies to be detected (and quantified) react with and / or bind to, for example, the PF-KEX2b peptide immunogen or the PF-KEX2a peptide immunogen used as the immunogen. As a representative example, antibodies are generated against the PF-KEX2b peptide used as the immunogen, as described in Examples 1 and 2 above and as shown in Figure 1 , Figure 2A and Figure 2B shown.

[0302] Materials and equipment:

[0303] PF-KEX2b protein, which can be purified as described in the examples herein. 1x PBS; Immulon high-binding (4HBX) flat-bottom microtiter plates (Thermo Fisher Scientific, catalog number 3855); blocking buffer: 5% non-fat milk in 1x PBS; wash buffer: 1x phosphate-buffered saline (PBS) + 0.05% Tween-20; secondary antibody: horseradish peroxidase conjugated to goat anti-human immunoglobulin (IgG at 1:10,000; Sigma-Aldrich); normal human plasma (Atlanta Biologicals, Inc., Lawrenceville, Georgia). Negative / normal control plasma, with undetectable absorbance (i.e., equal to or less than that of the dilution buffer alone) at a dilution of 1:100 in the KEX-ELISA (enzyme-linked immunosorbent assay) at OD 450 is used as a negative control; substrate: 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase substrate (e.g., SureBlue TMB substrate, 1-component; KPL, Inc.); stop solution: 1M H2SO4; microplate adhesive sealing film (plate sealer) (e.g., SealPlate sterile film from Excel Scientific, catalog number 100-SEAL-PLT); 96-well plate reader (any system capable of reading OD at a wavelength of 450 nm).

[0304] The procedure for performing ELISA is as follows:

[0305] Coat / block the ELISA plate with PF-KEX2b protein: Prepare PF-KEX2b protein at 5 μg / mL in 1x PBS. Add 50 μL of the diluted PF-KEX2b to each well of an Immulon 4HBX flat-bottom ELISA plate. Cover the plate tightly with Parafilm or a plate sealer and incubate overnight at 4°C. After overnight incubation, remove the buffer by flicking into a sink or bucket, then tap the plate on a blotting pad or paper towel to remove additional buffer. Wash the plate 2 times with wash buffer (PBS 0.05% Tween-20) (approximately 200 μL of wash buffer per well per wash, flicking and tapping the plate between washes). Add 100 μL of blocking buffer (5% milk / PBS) to each well and incubate at 37°C for 1 hour. Empty the plate and wash 2 times with wash buffer. At this step, the plate can be sealed and frozen at -20°C until ready to use.

[0306] Processing plasma or other infectious fluids (e.g., bronchoalveolar lavage (BAL) fluid supernatant, etc.) – First use: Remove plasma aliquots from the -80°C freezer.

[0307] Option 1: Heat-inactivate the entire aliquot at 56°C for 30 minutes.

[0308] Option 2: If heat-inactivation of the plasma sample will have an adverse effect on other potential uses, the sample should be thawed at 4°C or on ice. Remove an aliquot (approx. 100 μL), transfer to a new tube, and heat-inactivate (30 minutes, 56°C). Return the remaining sample to the -80°C freezer and label it as thawed 1x.

[0309] Before use, centrifuge the sample at >10,000 x g for 1 - 2 minutes to pellet aggregates. To prevent contamination during storage, add approximately 0.01% to 0.02% NaN3. Store sample aliquots at 4°C for up to 6 months. For subsequent assays, no further heat-inactivation is required; however, the sample should be briefly centrifuged before each use.

[0310] ELISA for endpoint titer determination (plasma): Dilute plasma 1:100 in blocking buffer. Add 50 μL of the diluted plasma and perform 2-fold (or 4-fold if needed) serial dilutions directly in the plate (final volume in each well should be 50 μL) to generate the endpoint titer. Perform the assay in duplicate; set up enough plates for all isotypes of interest. For example, if there are 10 samples and endpoint titers are to be generated for IgG and IgM anti-PF-KEX2b antibodies, 4 plates need to be set up (duplicate plates for both IgG and IgM). Include negative / normal controls on each plate. Cover the plate with a plate sealer and incubate overnight at 4°C. Empty the plate (flick and tap), and wash 4 times with wash buffer. Add 50 μL of the secondary antibody (diluted in blocking buffer) (see appropriate dilution under Materials and Equipment above) to each well. Incubate at 37°C for 1 hour. Empty the plate and wash 6 times with wash buffer. Add 100 μL of TMB to each well, protect from light, and incubate at 37°C for 30 minutes. Add 25 - 50 μL of the stop solution (1 M H2SO4) to each well. Read the OD of the plate at 450 nm (on any standard plate reader) within 20 minutes of adding the stop solution.

[0311] Most healthy adult individuals (including humans and non-human primates) have circulating antibodies against Pneumocystis; therefore, when selecting control samples for calculating endpoint titers, plasma samples must be screened from healthy donors to identify and obtain appropriate controls. In appropriate normal / negative control plasma, the PF-KEX2b OD at a 1:100 dilution 450It should not exceed 0.1; however, the lower the OD of the normal / negative control plasma, the better the control. To control inter-plate variability, after selecting appropriate normal / negative controls, the same normal / negative controls should be used on all plates.

[0312] ELISA for endpoint titer determination (BAL supernatant): Dilute the BAL supernatant 1:100 in saline. Determine the urea concentration of the BAL supernatant and the corresponding plasma samples using the QuantiChrom urea assay (BioAssay Systems, catalog number DIUR-500). Dilute the plasma 1:10 in distilled water and use the undiluted BAL supernatant according to the instructions on the kit insert. Place the plasma samples in the wells of a 96-well plate and add 5 μL of standard (1:10 dilution), blank (distilled water), and sample (1:10 dilution) in duplicate. Place the BAL supernatant in the wells of a 96-well plate and add 50 μL of standard (diluted to 5 mg / dL), blank (distilled water), and sample (undiluted). Add 200 μL of working reagent (included in the kit) and tap to mix. Incubate the plasma plate at room temperature (RT) for 20 minutes and read at OD 520 and incubate the BAL supernatant plate at room temperature for 50 minutes and read at OD 430Read as follows. Calculate the urea concentration ([urea]) in plasma and BAL supernatant: [urea] = (OD sample - OD blank) / (OD standard - OD blank) * [standard]. The standard concentration for plasma will be 50 mg / dL and for BAL supernatant will be 5 mg / dL. Calculate the 1:100 dilution of the BAL supernatant as follows: Find the 1:100 dilution factor of bal to plasma (dilution factor = 100 / (plasma [urea] / bal [urea])). Calculate the dilution volume for a total 500 ul sample (sample volume = 500 μL / dilution factor; saline volume = 500 μL - sample volume). Add the sample volume and saline volume to prepare a 1:100 diluted BAL supernatant sample. Add 50 μL of the diluted BAL supernatant and make serial 2-fold (or 4-fold if required) dilutions directly in the plate with saline (final volume in each well should be 50 μL) to generate endpoint titers. Perform the assay in duplicate; set up enough plates for all isotypes of interest, e.g., if there are 10 samples and endpoint titers are to be generated for IgG and IgM anti-PF-KEX2b antibodies, 4 plates need to be set up (duplicate plates for both IgG and IgM). Include negative / normal controls in each plate as described above. Cover the plates with a plate sealer and incubate overnight at 4°C. Empty the plates (flick and tap), and wash 4 times with wash buffer. Add 50 μL of the secondary antibody (diluted in blocking buffer, see appropriate dilution under Materials and Equipment) to each well. Incubate at 37°C for 1 hour. Empty the plates and wash 6 times with wash buffer. Add 100 μL of TMB to each well, protect from light, and incubate at 37°C for 30 minutes. Add 25 - 50 μL of the stop solution (1 M H2SO4) to each well. Read the OD of the plates at 450 nm (on any standard plate reader) within 20 minutes of adding the stop solution.

[0313] Determine the endpoint titer: Plot the OD readings for each sample (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. The endpoint titer is defined as the dilution at which the test sample gives the same OD reading as the negative control (i.e., where the lines intersect). Usually, the reciprocal endpoint titer is reported; thus, if the dilution is 1:1600, the endpoint titer is reported as 1600. Calculate the endpoint titer for each duplicate plate to confirm that the results between plates are consistent. The acceptable error is within one dilution. If the reciprocal endpoint titers (RET) from duplicate plates are within one dilution, take the average titer (e.g., when making doubling dilutions, the RET for a sample from plate 1 is 1600 and from plate 2 is 3200, then the average titer is 2400). If the endpoint titers on duplicate plates differ by more than one dilution, repeat the ELISA on another plate and take the average of the two closest values.

[0314] Example 7: Procedure for Purifying (Non-His-tagged) Recombinant Kexin 2a and 2b Peptides

[0315] This example describes the purification of PF.KEX2a (or PF.KEX2b) peptide expressed from the pET-28b+ vector in BL21(DE3)pLys Escherichia coli cells. If PF.KEX2a (pET-28B(+)) is expressed in BLR(DE3)pLys cells, then for each step, half the volume of cell culture is used in the same buffer volume. For anion exchange chromatography, the elution gradient is 0 - 2M NaCl instead of 0 - 1M NaCl.

[0316] Materials and equipment:

[0317] Animal-free Luria-Bertani (LB) agar (containing kanamycin 40 μg / ml + chloramphenicol 34 μg / ml); BBL Select APS LB Broth Base 500 g. BD, catalog number 292438 (Dissolve 10 g of powder + 7.5 g of Bacto Agar (BD, catalog number 214010) in 500 mL of purified water. Mix well. Autoclave at 121 °C for 15 minutes. Cool until lukewarm). Chloramphenicol 1000x stock solution [34 μg / ml]. (Sigma, catalog number C1919-5G, dissolve the powder in 100% ethanol, filter through a 0.2 μm sterile filter, store at -20 °C). Kanamycin 1000x stock solution [40 mg / ml]. (Dissolve the powder in water, filter through a 0.2 μm sterile filter, store at -20 °C). Add 500 μl of chloramphenicol 1000x stock solution [34 mg / ml] and 500 μl of kanamycin 1000x stock solution [40 mg / ml] to 500 mL of warm LB agar. Final concentrations: chloramphenicol [34 μg / ml], kanamycin [40 μg / ml]. Pour approximately 25 plates every 6 months as needed.

[0318] Animal-free Luria-Bertani (LB) broth (containing 40 μg / ml kanamycin + 34 μg / ml chloramphenicol); BBL Select APS LB Broth Base, 500 g. BD, catalog number 292438. (Dissolve 40 g of the powder in 2 L of purified water. Mix well. Autoclave at 121 °C for 15 minutes). Add 1 mL of chloramphenicol 1000x stock solution [34 mg / ml] and 1 mL of kanamycin 1000x stock solution [40 mg / ml] to 1 L of LB. Final concentration: chloramphenicol [34 μg / ml], kanamycin [40 μg / ml]. Prepare 4 L of LB broth + kanamycin / chloramphenicol for each purification. Kimble 20 x 150 mm borosilicate glass culture tubes (Duran Wheaton Kimble, catalog number 7500-20150) - for overnight culture. 4 X 2000 mL Erlenmeyer flasks for subculture. 1 M IPTG solution (0.22 μm sterile filter, 200 μl aliquots). Oakridge tubes (4 x 500 ml, Beckman, catalog number 361691; 4 x 50 ml, Beckman, catalog number 357003). Centrifuges: (Beckman Coulter Avanti J-E high-speed centrifuge (JLA-10.500 fixed-angle rotor (Max 10,000 RPM; 18,600 g). (JA-25.50 fixed-angle rotor (max 25,000 RPM; 75,600 g). Labnet Prism-R refrigerated microcentrifuge. Prepare 500 ml of 20 mM Tris HCl, pH 8.0 (10 mL of 1 M Tris HCl, pH 8.0). Fill to 500 ml with dH2O. 0.22 μm sterile filter, 250 mL, PES membrane (Genesee Scientific, catalog number 25-227); store at room temperature. CellLytic B cell lysis reagent (Sigma, catalog number B7435) - 500 ML, store at room temperature. Benzonase (5000x) (Sigma, catalog number E1014-25KU), store at -20 °C. Protease inhibitor cocktail (PIC) (Sigma, part number 8849).

[0319] A. Millex-GP syringe filter device, 0.22 μm, polyethersulfone, 33 mm, gamma sterilized. Millipore Express PES membrane with fast flow and low binding. Millipore Corporation, part number SLGPR33RS (replacement for Millipore part number SLGP033RS).

[0320] B. BD 60 mL syringe. LUER-LOK TM Needle. (BD Corporation, part number 309653).

[0321] C. Anion exchange chromatography buffer A (running buffer): Prepare 500 ml of 6 M urea, 20 mM Tris HCl, pH 8.0 (reagents: 1 M Tris-HCl, pH 8.0; (Quality Biological, catalog number 351-007-101); urea: MW = 60.06 g / mol).

[0322] Procedure: Weigh out 180.18 g of urea; add 10 mL of 1 M Tris-HCl, pH 8.0; dissolve the mixture in approximately 400 ml of dH2O and stir with a stir bar; after stirring, sonicate in a 30 °C water bath for approximately 10 minutes; fill to 500 ml with dH2O; sterile filter through a 0.22 μm 500 mL, PES membrane (Jena Scientific, part number 25-225); prepare fresh on the day of ion exchange chromatography. Store at 4 °C to prevent urea decomposition and protein carbamylation.

[0323] D. Anion exchange chromatography buffer B (elution buffer): Prepare 250 ml of 6 M urea, 1 M NaCl, 20 mM Tris HCl, pH 8.0. (Reagents: 1 M Tris-HCl, pH 8.0, Quality Biological, catalog number 351-007-101; NaCl: MW = 58.44 g / mol; urea: MW = 60.06 g / mol).

[0324] ProcedureWeigh out 90.09 g of urea; weigh out 14.61 g of NaCl; add 5 mL of 1 M Tris-HCl, pH 8.0; dissolve the mixture in approximately 200 ml of dH2O and stir with a stir bar; after stirring, sonicate in a 30 °C water bath for approximately 10 minutes; fill to 250 ml with dH2O; sterile filter through a 0.22 μm 250 mL, PES membrane (Genesee Scientific, catalog number 25-225); prepare fresh on the day of performing ion exchange chromatography. Store at 4 °C to prevent urea decomposition and protein carbamylation.

[0325] FPLC solutions for column storage. Sterile filter all solutions. 20% ethanol, 0.2 μm sterile filter. For AKTA cleaning and column storage buffer and dH2O.

[0326] FPLC solutions for cleaning HiTrap Capto Q columns. Sterile filter all solutions. 2 M NaCl and 1 M NaOH.

[0327] Anion exchange column: HiTrap Capto Q 5x 5 mL GE Healthcare Life Sciences / Cytiva (catalog number 11001303); store the column at 4 °C in 20% ethanol.

[0328] Size exclusion chromatography column: Superdex 75 Increase 10 / 30 GL; (GE Healthcare Life Sciences / Cytiva Life Sciences, catalog number 29148721); store the column at 4 °C in 20% ethanol.

[0329] Size exclusion chromatography buffer: Prepare 500 mL of 6 M urea, 250 mM NaCl, 20 mM Tris-HCl, pH 8.0. Reagents: 1 M Tris-HCl, pH 8.0 (Quality Biologicals, catalog number 351-007-101), NaCl (MW = 58.44 g / mol); urea (MW = 60.06 g / mol)

[0330] ProcedureWeigh out 180.18 g of urea; weigh out 7.305 g of NaCl; add 10 mL of 1 M Tris-HCl, pH 8.0. Dissolve the mixture in approximately 400 ml of dH2O and stir with a stir bar. After stirring, sonicate at 30 °C for approximately 10 minutes. Fill to 500 ml with dH2O. Sterile filter through a 0.22 μm 500 mL, PES membrane (Genesee Scientific, catalog number 25-225). Prepare fresh on the day of ion exchange chromatography. Store at 4 °C to prevent urea decomposition and protein carbamylation. Fast Protein Liquid Chromatography (FPLC) system: Trent Lab FPLC or CVI Protein Core. Superloop 50 mL (GE Biosciences / Cytiva, catalog number 18111382). Ultra-4 centrifugal filtration device, Ultracel-3 regenerated cellulose membrane, 4 mL sample volume. (SigmaMillipore, catalog number UFC800324). Millex-GP syringe filter device, 0.22 μm, polyethersulfone (PES) membrane, 33 mm, gamma sterilized. SigmaMillipore, catalog number SLGP033RS. Pierce Detergent Compatible Bradford Assay Kit (Thermo Fisher Scientific, catalog number 23246). Western blotting reagents. Target: PF-KEX2a.

[0331] 1x SDS PAGE Running Buffer: 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3; 6x SDS Loading Dye: 12% SDS, 47% glycerol, 60 mM Tris HCl pH 6.8, 0.06% bromophenol blue, 5% β-mercaptoethanol. Prepare 10 mL: 1.2 g SDS, 6 mg bromophenol blue, 4.7 mL glycerol, 1.2 mL 0.5 M Tris HCl pH 6.8, 2.5 mL H2O. Heat at 37 °C to dissolve SDS in glycerol, Tris buffer and water. Prepare 950 μL aliquots of the loading buffer and store at -20 °C. Add 50 μL of 14.3 M pure β-mercaptoethanol (βME) before use. The final 2βME concentration should be 5% before use. Membrane: iBlot Transfer Stacks Nitrocellulose (0.2 μM), Thermo Fisher Scientific, cat. no. IB401001. 1° Antibody: High-titer serum from a monkey (cat. no. 5617) vaccinated with PFKEX-Histidine vaccine at 1 wpv2 on 4 / 3 / 19; RET = 1,152,000; store at -4 °C. 2° Antibody: Goat anti-monkey IgG (H+L)-HRP [1 mg / mL], (Thermo Fisher Scientific, cat. no. PA1-84631). Blocking Buffer: 5% non-fat dry milk in PBS-T (0.05% Tween-20) or TBS-T (0.05% Tween-20). Prepare at least 1 hour before blocking. Wash Buffer: PBS or TBS. Standard laboratory protocol. SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, cat. no. 34577). PRECISION PLUS PROTEIN TM DualXtra Prestained Protein Standards (cat. no. 1610377). Ponceau S Staining Buffer (0.2% Ponceau S, 5% glacial acetic acid). Coomassie Staining: Coomassie Brilliant Blue R-250, (Fisher Scientific, cat. no. BP101-25). Destaining Buffer (per liter: 10% acetic acid (100 mL), 40% methanol (400 mL), 50% dH2O (500 mL)). Mix 1.25 g of Coomassie Brilliant Blue R-250 with 500 mL of destaining buffer. Stir overnight to dissolve.)

[0332] When purifying the protein from BL21(DE3)pLys cells, prepare 2 x 4L cell cultures before purification; prepare a protein purification worksheet for each 4L cell culture. For each anion exchange run, purify the protein from 4L cell culture. For size exclusion chromatography, pool the best fractions from 2 x 4L anion exchange chromatography runs. Up to 20 mg of protein can be injected through the size exclusion chromatography column; thus, in order to purify the protein from the anion exchange chromatography enriched fractions obtained from 2 x 4L cell cultures, 2 - 3 size exclusion chromatography runs are required.

[0333] Culture and induce the protein in 2 x 4L = 8L total Escherichia coli. Since it is difficult to culture all 8L in one day, the cultures are divided into 2 x 4L cell culture batches.

[0334] Culture 4 L cell culture : Streak the untagged PFKEX on pET28b in BL21(DE3)pLyS onto an LB agar + kanamycin / chloramphenicol plate and incubate overnight at 37 °C and select single colonies. Inoculate 8 single colonies into 8 x 10 mL LB broth + kanamycin / chloramphenicol (liquid to gas ratio is approximately 1:5) in Kimble 20 x 150 mm borosilicate glass culture tubes (Duran Wheaton Kimble, catalog number 7500 - 20150), overnight at 37 °C, shaking at 225 rpm. Culture 4L liquid cultures: After overnight incubation, dilute the cultures 1:50 into liquid LB broth + kanamycin / chloramphenicol. Add 10 mL of the culture to 500 ml. Repeat for 8 x 500 ml cultures. Place each 500 ml volume into a 2L Erlenmeyer flask for culture. Shake at 37 °C for approximately 2 hours or until OD 600 = 0.5. Induce protein expression with 0.5 mM IPTG by adding 250 ul of 1M IPTG to each approximately 500 mL culture. Shake at 37 °C for 4 hours. Harvest the cells in 500 mL Oakridge tubes and centrifuge at 6,000 x g for 20 minutes at 4 °C in a Beckman Avanti J-E centrifuge (JLA-10.500 fixed angle rotor). Discard the supernatant. Resuspend each pellet in approximately 50 mL of 20 mM Tris HCl (pH 8.0) to wash away the residual medium. Pool all the pellets into a single Oakridge tube containing the cell paste from 4L cell culture. Centrifuge at 6,000 x g for 20 minutes at 4 °C. Discard the supernatant. Remove as much of the residual liquid as possible to prevent dilution of CelLytic B when the cells lyse. Store the cell pellet at -80 °C until use. Repeat the Escherichia coli culture and induction for another 4L cell culture.

[0335] Isolate inclusion bodies from 4 L cell culture:Thaw the precipitated cells on ice. Resuspend the cell pellet from 4 L of cell culture in 40 mL of CelLytic B cell lysis reagent + 8 μL of benzonase nuclease + 400 μl of protease inhibitor cocktail (PIC) (Sigma P8849). Vortex the cell suspension for 1 - 2 minutes until completely resuspended and shake the cells at room temperature for 10 min. Centrifuge at 15,000 g for 10 min at 4 °C in a 50 mL Oakridge tube. Discard the cell lysate supernatant. Perform a second cell lysis on the 4 L cell culture using a total of 20 mL of CelLytic B lysis reagent. Pipette forcefully and vortex the cell pellet to facilitate cell lysis and breakdown of the precipitated material. Centrifuge at 15,000 g for 10 min at 4 °C. Prepare 60 mL of inclusion body wash buffer by diluting 6 mL of CelLytic B + 54 mL of dH2O. Wash the pellet with 20 mL of the prepared inclusion body wash buffer. Pipette forcefully and vortex the suspended material to facilitate cell lysis and inclusion body separation. Centrifuge at 15,000 g for 10 min at 4 °C. Repeat 2 times. Wash the pellet with 20 mL of 20 mM Tris HCl (pH 8.0). Pipette forcefully and vortex the suspended material to facilitate cell lysis and inclusion body separation. Centrifuge at 15,000 g for 10 min at 4 °C. Discard the supernatant. Store the purified inclusion bodies at -80 °C until anion exchange chromatography is performed. Repeat the procedure for the second 4 L cell culture pellet.

[0336] Dissolve and extract His-tag-free PF-KEX peptide from inclusion bodies: For each anion exchange chromatography run, use 4 L of cell culture. Thaw the purified inclusion bodies on ice. Resuspend the pellet in 50 mL of "Buffer A" (running buffer): 6 M urea, 20 mM Tris HCl, pH 8.0 plus 200 ul of protease inhibitor cocktail in a 50 mL Oakridge tube. Pipette forcefully and vortex the suspended material to break down the inclusion bodies and facilitate protein extraction. If necessary, sonicate in a 30 °C water bath for 10 min to dissolve the pellet. Shake the suspension at room temperature for 2 hours. Place the suspension (in a 50 mL conical tube) on ice and store overnight at 4 °C to precipitate unwanted non-Kexin proteins (e.g., to prevent the Capto Q column from reaching the pressure limit. Non-Kexin proteins will precipitate on the column at 4 °C). The next morning, centrifuge at 15,000 g for 15 min at 4 °C. Harvest the supernatant and record the supernatant volume. Save 100 μl of the suspension before filtration (labeled "before filtration") to determine losses due to filtration. Save some of the pellet to analyze extraction efficiency (labeled "urea pellet"). Load the sample / supernatant into a BD 60 mL syringe. LUER-LOK TMNeedle. BD, catalog number 309653, and connect the syringe to a Millex-GP syringe filter unit, 0.22 μm, Millipore, catalog number SLGPR33RS. Filter the sample with the syringe to remove particulates and remove gas from the sample before anion exchange chromatography. Save 100 μl of the suspension (labeled "filtered / AIEX starting material") before anion exchange to estimate the amount of protein before anion exchange chromatography. Store the filtered sample on ice before anion exchange chromatography.

[0337] Anion exchange chromatography (AIEX): Using FPLC equipped with 50 mL superloop: Perform one anion exchange run per 4 L of cell culture.

[0338] Protocol: Turn on the Unicorn software program. Connect the HiTrap Capto Q column. Do not introduce air bubbles into the column. Wash the 20% ethanol storage solution from the column with 5 mL of dH2O. Exchange the storage buffer to AIEX buffer (Buffer A: 6 M urea, 20 mM Tris-HCl, pH 8.0; Buffer B: 6 M urea, 1 M NaCl, 20 mM Tris-HCl, pH 8.0). Equilibrate the column with 5 CV / 5 mL of Buffer A →, 5 CV / 5 mL of Buffer B →, 5 CV / 5 mL of Buffer A. Inject the sample, avoiding air entry. Enter the approximate injection volume to the computer. Record the injection volume to estimate the purification efficiency. Run program conditions: Bind approximately the sample volume to the column. Collect approximately the sample volume of the flow-through. Save 100 μl of the "flow-through" for analysis. Wash with 100 mL / 20 CV of Buffer A. Elute with a 0-100% gradient of Buffer B at 25 mL / 5 CV (fraction size = 1.0 mL; flow rate: 0.8 mL / min; pressure limit: 0.5 MPa). Wash the column with 75 ml / 15 CV of Buffer B. Wash the column with 25 ml / 5 CV of Buffer A.

[0339] Column reuse (repeat after each run): Wash with at least 2 CV of 2 M NaCl. Wash with at least 4 CV of 1 M NaOH. Wash with at least 2 CV of 2 M NaCl. Rinse with at least 5 CV of dH2O. If repeating the AIEX run, equilibrate the column with 25 ml / 5 CV of Buffer A. Wash and store the column in 20% ethanol at 4 °C according to the final stripping / in-place cleaning protocol. After sample run, collect the "flow-through", "wash", and elution fractions. Print the chromatogram after the run. Store the sample at 4 °C for up to 1 week while performing Bradford assay and SDS-PAGE analysis. For long-term storage, store at -80 °C. Figure 3A and Figure 3BCoomassie-stained gels and immunoblot analyses were provided, which showed purified PF-KEX2a peptide ( Figure 3A ) and PF-KEX2b peptide ( Figure 3B )(after dialysis). Figure 3A The stability of the purified PF-KEX2a peptide over time (e.g., stored at -80 °C for 47 days and 90 days) was demonstrated; similar results were expected for the PF-KEX2b peptide.

[0340] Quality control analysis after anion exchange chromatography / ion exchange chromatography (IEX) Typically, up to 30 samples from each anion exchange chromatography run were analyzed by SDS-PAGE (western blotting and Coomassie staining). As many anion exchange chromatography fractions as possible with the highest protein concentration were analyzed, adapted to each gel, to obtain the highest protein recovery level. Control samples were included in the analysis as they were necessary for calculating the purification efficiency. All protein fractions were measured by the Bradford assay using a kit compatible with 6M urea buffer and following the attached protocol. For example, the Pierce detergent-compatible Bradford assay kit protocol (Thermo Fisher Scientific catalog number 23246) and the BioRad protein assay (catalog number 500-0006); the linear range of this microtiter assay is 0.05 - 0.5 mg / ml.

[0341] The elution fractions and standards were measured in duplicate. Samples collected as follows were included for QC analysis: Urea precipitation: For checking extraction and solubilization efficiency. A small volume was boiled for 5 min at 1:1 in approximately 100 μl of 1% SDS. Centrifuge at 16,000 x g for 5 min. Collect the supernatant for SDS-PAGE analysis. Sample before filtration: Confirm the amount of protein collected in the supernatant after extraction. Use the sample volume before filtration to calculate the yield. Sample after filtration / starting material for AIEX: Determine the amount of protein loaded onto the column. Use the sample volume recorded above to calculate the amount of protein loaded onto the column. Flow-through: For checking anion exchange column binding efficiency. Wash. Determine if any precipitate drops out of the solution (i.e., if peaks appear during stripping or otherwise). Anion exchange fraction: Read the samples at 595 nm. Infer the fraction concentration from the standard curve and calculate the protein yield by anion exchange chromatography.

[0342] Dilute the sample with water and add 6x Laemmli Buffer. Coomassie staining (10 μg / well or up to 30 μl). Western blotting (5 μg / well or up to 30 μl). Add 6 μl of 6x Laemmli Buffer (5% 2-mercaptoethanol) to 30 μl of diluted sample. Centrifuge at full speed for 5 minutes to pellet debris. To avoid potential protein modification issues, samples containing urea were not boiled. If necessary, store the prepared samples at -80 °C until the next day.

[0343] Gel electrophoresis : Prepare a 15% separating / 4% stacking polyacrylamide gel according to the manufacturer's protocol. (Handcasting Polyacrylamide Gels; Bio-Rad Laboratories, Inc., Bulletin No. 6201). Use a 15-well comb. Assemble the running apparatus and fill the inner chamber and bottom tray with 1x SDS-PAGE running buffer. Remove the comb and rinse the wells. Do not reuse the inner chamber buffer. Load 10 μL of broad-spectrum staining (Part No. 26634) SPECTRA TM Multicolor standards (diluted in 1x Laemmli Buffer). If all samples / standards are the same volume and all wells are full, the gel will run straighter. Load the samples. Run the gel at a constant voltage (approx. 80 - 120 volts) for 1.5 - 2.5 hours until the dye front runs off the bottom of the gel. Once the run is complete, remove the stacking gel with a spatula. Wash the gel with dH2O before Coomassie staining or Western blotting (see Western blotting protocol).

[0344] Membrane transfer: Assemble the Invitrogen IBLOT TM Gel transfer system. Open the device lid and place the anode stack with the red plastic bottom directly on the blotting surface, aligned with the right gel barrier. Place the pre-run 15% SDS Page gel on the transfer membrane of the anode stack. Wet the IBLOT with dH2O TMPlace the filter paper on top of the gel and make sure to remove air bubbles using the blotting roller. Remove the seal of the cathode stack and place the gel face down on the filter paper. Use the roller to remove all air bubbles. Connect the removable sponge to the metal contact in the upper right corner of the lid. Close the lid, secure the latch, and turn on the power switch (located on the back of the machine). Select Program #3 (P4) at approximately 20 volts and adjust the default time from 5 minutes. Press the Start / Stop button and wait for the light to change from red to green. Once the gel running time is over, the machine will beep and the “Start / Stop” button should be pressed again until the light changes from green to red. At this point, turn off the power switch and disconnect the power cord. Open the lid and make sure the stained standards have been properly transferred to the nitrocellulose membrane. Trim the membrane to the desired size with a knife and transfer it to a light-resistant tray using forceps.

[0345] Ponceau staining : - To confirm protein transfer, wash the membrane with dH2O. Add approximately 1 mL of Ponceau S stain to the membrane and shake by hand for approximately 1 min. Remove the Ponceau S stain and rinse the membrane with dH2O until the double band of prohormone convertase 1 / 3 becomes visible (prohormone convertase 1 / 3, approximately 10 - 12 kDa). Take a picture of the membrane and save it as a TIFF. Continue to rinse the stain with dH2O until all stain is removed.

[0346] 0.025% Coomassie staining to measure fraction purity: Add approximately 25 mL of Coomassie blue stain. Cover the lid and microwave for 2 x 15 seconds. Cool by shaking at room temperature for 15 minutes. Discard the stain and wash with dH2O until the water runs clear. Add approximately 25 ml of decolorizer and a Kim Wipe to the gel. Shake overnight on a shaker at room temperature. The Kim Wipe will absorb the excess Coomassie stain, so there is no need to change the decolorizer overnight. Take a picture of the gel and save it as a TIFF file.

[0347] Western blot:Membrane: Nitrocellulose (0.2 μM), (Thermo Fisher Scientific, catalog number IB401001). 1° antibody: High-titer serum from a monkey (catalog number 5617) vaccinated with PFKEX-Histidine vaccine at 1 wpv2; RET = 1,152,000; stored at -4 °C. 2° antibody: Goat anti-monkey IgG (H+L)-HRP [1 mg / ml], (Thermo Fisher Scientific, catalog number PA1-84631). Blocking buffer: 5% non-fat milk in PBS-T (0.05% Tween-20) or TBS-T (0.05% Tween-20). Prepare at least 1 hour before blocking. Block at room temperature for 1 hour or overnight at 4 °C in 10 mL blocking buffer. Add primary antibody (1:5,000 - 10,000) in blocking buffer, no washing required (1 - 2 μl primary antibody + 10 mL blocking buffer). Incubate at room temperature for 1 - 2 hours or overnight at 4 °C. Wash 3 times with TBS-T (0.05% Tween-20) - 15 min, 10 min, and 5 min. Add secondary antibody (1:10,000) in blocking buffer and incubate at room temperature for 1 hour. (Add 1 secondary antibody + 10 mL blocking buffer). Wash 3 times in TBS-T. Develop using PicoWest. Take a picture of the membrane and save as a TIFF file.

[0348] After anion exchange chromatography: Pool the best fractions from two anion exchange chromatography runs for size exclusion chromatography. Do not perform size exclusion chromatography on samples with poor enrichment. The fractions should look relatively clean, otherwise the protein will precipitate onto the column and clog it. If protein precipitation occurs and an increase in column pressure is observed, the size exclusion chromatography column will need to be deeply cleaned with pepsin digestion. The size exclusion chromatography column can bind up to 20 mg per SEC run. The maximum loop size for size exclusion chromatography is 500 μl. The greater the sample concentration, the steeper the chromatographic peak. However, for a 500 μl sample per injection, the size exclusion chromatography column can only accommodate up to 20 mg of protein; therefore, the concentrated protein in 500 μl should be ≤ 20 mg. If the pooled sample contains > 20 mg, multiple size exclusion chromatography runs are required and adjusted proportionally accordingly. Concentrate the protein in discrete volumes at 500 μl intervals, corresponding to discrete numbers of runs. To perform an SEC run, if the amount of pooled protein is less than 20 mg, concentrate the protein to a volume of 500 μl. If the concentration of the pooled protein is between 20 - 40 mg, perform 2 SEC runs. Concentrate the protein to 1000 μl as above. If the concentration of the pooled protein is between 40 - 60 mg, perform 3 SEC runs. Concentrate the protein to 1500 μl.

[0349] Using Ultra-15 centrifugal filtration device (Sigma Millipore, catalog number UFC900324) for protein concentration: After anion-exchange chromatography, fractions with the highest protein purity and concentration are combined and the volume of the combined protein is recorded. Save 100 μl of this combined volume for downstream analysis and label it "AIEX combined" to calculate the approximate protein yield in the combined sample. Prepare an Ultra-15 centrifugal filter device (Sigma Millipore, catalog number UFC900324). Fill the column to its maximum volume with diH2O to equilibrate the filter for 5 min to remove residual storage glycerol on the column and prevent protein loss. Centrifuge at 3,000 g for 5 min at room temperature. Remove the water from the top and bottom of the spin column. Add the combined volume to the Ultra-15 centrifugal filter device (Sigma Millipore, catalog number UFC900324). Centrifuge at 3,000 g for 60 min at room temperature. Centrifuge until the desired estimated protein concentration and volume are reached.

[0350] Size exclusion chromatography (SEC): Connect a Superdex 75 Increase 10 / 300 GL column to the FPLC. Equip the FPLC with a 500 μl loop. Wash away the 20% ethanol storage solution with 1.5 CV / 36 ml dH2O. Equilibrate the column in 1.5 CV / 36 mL size-exclusion chromatography buffer (6 M urea, 250 mM NaCl, 20 mM Tris-HCl, pH 8.0). The SEC buffer must contain NaCl to maintain protein solubility during the SEC run and avoid protein precipitation. Flush the 500 μl loop with SEC buffer to confirm there is no leakage. Inject the sample, typically about 500 μl. Run the SEC program: injection: 500 μl (elution volume: 1.5 CV / 36 ml). Collect 1.0 ml fractions (flow rate: 0.8 mL / min; pressure limit: 5.0 MPa). Collect the fractions and the chromatogram. Typically, the fractions will elute in 15 ml units. After all runs are completed, wash the column with 1.5 CV / 36 ml H2O. Wash with 1.5 CV / 0.5 M NaOH. Wash with 2 CV / 48 mL H2O. Wash the column with 2 CV / 48 mL 20% ethanol. Store the column in 20% ethanol. If the column exceeds the pressure limit, perform a deep / rigorous cleaning: wash with 1.5 CV / 36 ml H2O. Fill the column with a 1 mg / ml pepsin solution in 0.1 M acetic acid containing 0.5 M NaCl and leave at room temperature overnight to digest and remove contaminants on the column. Wash with 2 CV / 48 ml H2O. Wash the column in 2 CV / 48 mL 20% ethanol. Store the column in 20% ethanol.

[0351] Quality control analysis after size exclusion chromatography (SEC):Typically, in each size exclusion chromatography run, up to about 15 samples are analyzed by SDS-PAGE (western blot and Coomassie staining) because one 1x15 well SDS-PAGE gel is used per analytical parameter and the gel can accommodate up to 14 samples (excluding molecular weight standards). The analysis includes as many size exclusion chromatography fractions as possible that have the highest protein concentration and are appropriate for each gel corresponding to the major peaks on the chromatogram. The "AIEX pooled samples" are analyzed to calculate the purification efficiency of the SEC purification step. The following parameters are calculated: the amount of protein in the AIEX pooled sample fractions; the total protein in the recovered fractions; the yield of purified protein recovered from the clarified fractions based on Coomassie and western blot analysis (recovery % = best SEC pooled fraction / AIEX pooled x 100); the yield of purified protein recovered from 8 L of cell culture, i.e., X mg protein / L cell culture.

[0352] To analyze protein purity and specificity, size exclusion chromatography (SEC) protein fractions are electrophoresed on gels and stained with Coomassie blue dye. Western blotting is also performed on the samples. For example, 5 μg of protein, such as the PF-KEX2a peptide, is loaded into each lane of the protein gel. In the experiment, it was found that prior to dialysis, the yield of purified protein in the pooled PF-KEX2a batches was 21 mg / 8 L of cell culture, or 2.63 mg / L of cell culture.

[0353] Urea removal (for in vivo use only): The following protocol pertains to dialysis of proteins for in vivo use. When performing buffer exchange by diafiltration, ensure that the PF.KEX2a protein does not precipitate out of solution (precipitate). Its solubility in 1x PBS is estimated to be between 1.5 - 2 mg / ml only after dialysis. If the dialyzed protein is for in vivo use, pool fractions ≥2 mg / ml. Pool at least 3 mL of purified protein to fill a single dialysis cassette. Dialysis must be performed at discrete intervals of 3 mL. If the cassette is not full or the protein concentration is below 1.5 mg / ml, the recovery % is below 50%. The typical recovery % is between 37% - 63.4%. Pool the remaining fractions with lower concentrations for in vitro assays (e.g., ELISA and ELISPOT assays). If the protein is used to coat plates for ELISA / ELISPOT, the protein remains soluble after dilution from urea to 1x PBS or sterile water. After size exclusion chromatography, fractions with the highest protein purity and concentration (≥1.5 mg / ml) are pooled. Calculate the approximate yield of protein in the pooled samples. Save 50 μl of the pooled volume for downstream analysis and label it "pre-concentration SEC pooled". Perform dialysis to remove urea. Dialysis promotes the highest solubility compared to diafiltration or TCA precipitation.

[0354] Dialysis (Pur-A-Lyzer Maxi3500 dialysis kit. Sigma, catalog number PURX35005-1KT ): Prepare 4 L of 1x PBS pH 7.4. Adjust the pH of the buffer for use in the donor. Fill the Pur-A-Lyzer with 2 - 3 mL of ultrapure water and incubate for at least 5 minutes. Empty the tube. Carefully check the tube for leaks, as water absorption by the membrane can cause the water level to drop. Load the sample into the Pur-A-Lyzer tube. Cover the tube with the provided cap (do not force). The sample volume should be in the range of 0.1 - 3 mL. If a smaller volume is used, load the sample close to the inner membrane. Set up the apparatus in a cold room at approximately 4 °C. Place the loaded Pur-A-Lyzer tube into the provided floating rack and then place the rack into a stirred beaker containing 2 L of 1x PBS pH 7.4. The floating rack can accommodate 1 - 7 Pur-A-Lyzer tubes. Adjust the stir bar speed. Dialyze for approximately 2 hours. Replace the dialysis buffer with 2 L of fresh 1x PBS pH 7.4. Dialyze overnight at 4 °C with stirring. In the morning, quickly remove the dialysis cassette from the dialysis solution. Carefully transfer the sample from the Pur-A-Lyzer to a clean tube. Analyze the dialyzed sample before and after analysis by Bradford assay, Coomassie staining, and Western blot analysis as described above. If necessary, perform endotoxin testing using the Pierce Chromogenic Endotoxin Quantitation Kit (Thermo Scientific, catalog number A39552). If pooled / concentrated protein is used for further use, such as immunization / vaccination, perform a Bradford assay to quantify the protein and repeat the Coomassie and Western blot assays to analyze potential protein degradation in the final product. Record the endotoxin value.

[0355] Other embodiments

[0356] From the foregoing description, it will be apparent that changes and modifications can be made to the aspects and embodiments described herein to adapt them to various uses and conditions. Such embodiments are also within the scope of the following claims.

[0357] The recitation of a list of elements in any definition of a variable herein includes defining the variable as any single element or combination (or sub - combination) of the listed elements. The recitation of embodiments herein includes embodiments for any single embodiment or embodiments in combination with any other disclosed embodiment or portions thereof.

[0358] All patents and publications mentioned in this specification are incorporated herein by reference in their entirety, and to the extent that each individual patent and publication is specifically and individually indicated to be incorporated by reference.

Claims

1. An immunogenic peptide or a polynucleotide encoding an immunogenic peptide or an immunogenic fragment thereof, wherein the immunogenic peptide comprises the amino acid sequence DDDGKTVDGPSPLVLRAFINGVNNGRNGLGSIYVFASGNGGIYEDNSNFD as shown in SEQ ID NO:1 GYANSVFTITIGGIDKHGKRPKYSEASSSQLAVTYAGGSA(PF-KEX1b).

2. An immunogenic peptide or a polynucleotide encoding an immunogenic peptide or an immunogenic fragment thereof, wherein the immunogenic peptide comprises the amino acid sequence PDDGKTMEGPDILVLRAFINGVQNGRDGKGSIYVFASGNGGGFEDNSNFD as shown in SEQ ID NO:2 GYTNSIYSITVGAIDRKGLHPSYSEASSAQLVVTYSSGSG(PF-KEX2b).

3. An immunogenic peptide or a polynucleotide encoding an immunogenic peptide or an immunogenic fragment thereof, wherein the immunogenic peptide consists of the amino acid sequence DDDGKTVDGPSPLVLRAFINGVNNGRNGLGSIYVFASGNGGIYEDNSNFD as shown in SEQ ID NO:1 GYANSVFTITIGGIDKHGKRPKYSEASSSQLAVTYAGGSA(PF-KEX1b).

4. An immunogenic peptide or a polynucleotide encoding an immunogenic peptide or an immunogenic fragment thereof, wherein the immunogenic peptide consists of the amino acid sequence PDDGKTMEGPDILVLRAFINGVQNGRDGKGSIYVFASGNGGGFEDNSNFD as shown in SEQ ID NO:2 GYTNSIYSITVGAIDRKGLHPSYSEASSAQLVVTYSSGSG(PF-KEX2b).

5. An immunogenic peptide or a polynucleotide encoding an immunogenic peptide or an immunogenic fragment thereof, wherein the immunogenic peptide has at least 98% sequence identity with the amino acid sequence DDDGKTVDGPSPLVLRAFINGVNNGRNGLGSIYVFASGNGGIYEDNSNFD as shown in SEQ ID NO:1 GYANSVFTITIGGIDKHGKRPKYSEASSSQLAVTYAGGSA(PF-KEX1b), and wherein the peptide contains serine (S) amino acid residues at least at positions 47 and 77 of the amino acid sequence.

6. An immunogenic peptide or a polynucleotide encoding an immunogenic peptide or an immunogenic fragment thereof, wherein the immunogenic peptide has at least 98% sequence identity with the amino acid sequence PDDGKTMEGPDILVLRAFINGVQNGRDGKGSIYVFASGNGGGFEDNSNFD as shown in SEQ ID NO:2 GYTNSIYSITVGAIDRKGLHPSYSEASSAQLVVTYSSGSG(PF-KEX2b) has at least 98% sequence identity, wherein the peptide contains serine (S) amino acid residues at least at positions 47 and 77 of the amino acid sequence.

7. The immunogenic peptide according to any one of claims 1-6, wherein the peptide is recombinant, recombinantly produced and / or isolated.

8. An immunogenic composition comprising an effective amount of the immunogenic peptide according to any one of claims 1-7 or a polynucleotide encoding the immunogenic peptide and a pharmaceutically acceptable carrier, vehicle or excipient.

9. The immunogenic composition according to claim 8, wherein the composition further comprises an adjuvant.

10. The immunogenic composition according to claim 9, wherein the adjuvant is selected from α-galactosylceramide (αGC), alum, or 11. A method of eliciting an immune response in a subject, the method comprising administering to the subject the immunogenic peptide according to any one of claims 1-10, a polynucleotide encoding the immunogenic peptide, or an immunogenic composition.

12. A method of treating or protecting a subject from a disease or its symptoms associated with or caused by a fungal infection, the method comprising administering to the subject an immunogenic peptide or polynucleotide according to any one of claims 1-7 in an amount effective to treat or protect the subject from the fungal disease or its symptoms.

13. A method of treating or protecting a subject from a disease or its symptoms associated with or caused by a fungal infection, the method comprising administering to the subject an immunogenic composition according to any one of claims 8-10 in an amount effective to treat or protect the subject from the fungal disease or its symptoms.

14. A method of treating or protecting a subject from a disease or its symptoms associated with or caused by a fungal infection, the method comprising administering to the subject a separated antiserum in an amount effective to treat or protect the subject from the fungal disease or its symptoms, the separated antiserum comprising an antibody or an antigen-binding fragment thereof that specifically binds to the immunogenic peptide according to any one of claims 1-7.

15. A method of treating or protecting a subject from a disease or its symptoms associated with or caused by a fungal infection, the method comprising administering to the subject a separated or purified antibody or antigen-binding fragment in an amount effective to treat or protect the subject from the fungal disease or its symptoms, the separated or purified antibody or antigen-binding fragment specifically binding to the immunogenic peptide according to any one of claims 1-7.

16. The method according to claim 14 or 15, wherein the antibody is a monoclonal antibody, a polyclonal antibody or an antigen-binding fragment thereof.

17. The method according to any one of claims 12-16, wherein the fungal infection is associated with or caused by a fungal pathogen of the genus Aspergillus, Candida, Cryptococcus or Pneumocystis.

18. The method according to any one of claims 12-17, wherein the method treats a pulmonary disease, a pulmonary dysfunction or its symptoms.

19. The method according to claim 18, wherein the lung disease or pulmonary dysfunction is Pneumocystis pneumonia (PCP), aspergillosis or invasive pulmonary aspergillosis (IPA).

20. A method for treating or protecting a subject from a disease or a severe disease or its symptoms, the method comprising: Administering to the subject an immunogenic peptide or polynucleotide, or a pharmaceutical composition thereof, as described in any one of claims 1-7, in an amount effective to treat or protect the subject from the disease or serious disease or symptoms thereof.

21. A method of treating or protecting a subject from a disease or serious disease or symptoms thereof, the method comprising administering to the subject, in an amount effective to treat or protect the subject from the disease or serious disease or symptoms thereof, a separated antiserum comprising an antibody or an antigen-binding fragment thereof that specifically binds to the immunogenic peptide as described in any one of claims 1-7.

22. A method of treating or protecting a subject from a disease or serious disease or symptoms thereof, the method comprising administering to the subject, in an amount effective to treat or protect the subject from the disease or serious disease or symptoms thereof, a separated or purified antibody or antigen-binding fragment that specifically binds to the immunogenic peptide as described in any one of claims 1-7.

23. The method according to claim 22, wherein the antibody is a monoclonal antibody, a polyclonal antibody or an antigen-binding fragment thereof.

24. The method according to any one of claims 20-23, wherein the disease or serious disease or symptoms thereof are selected from lung diseases, asthma, severe asthma, refractory asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, pneumonia, Pneumocystis pneumonia, bronchiectasis, aspergillosis, invasive pulmonary aspergillosis (IPA), vaginitis, urinary tract infection (UTI), organ transplantation, tissue transplantation, immunodeficiency diseases, HIV, AIDS, HIV / AIDS, congenital diseases, autoimmune diseases, rheumatoid arthritis, psoriasis, inflammation-related diseases, diabetes, type 1 diabetes or type 2 diabetes.

25. A method of treating or protecting a subject from a disease or serious disease or its symptoms associated with or caused by a fungal infection, the method comprising: Administering to the subject an immunogenic composition comprising the peptide of SEQ ID NO:2 or an immunogenic fragment thereof, or a polynucleotide encoding the peptide of 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.

26. A method of treating or protecting a subject from a disease or serious disease or its symptoms associated with or caused by a fungal infection, the method comprising: Administering to the subject an immunogenic composition comprising the peptide of SEQ ID NO:1 or an immunogenic fragment thereof, or a polynucleotide encoding the peptide of SEQ ID NO:1 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.

27. The method according to claim 25 or 26, wherein the peptide of SEQ ID NO:2 or an immunogenic fragment thereof is recombinant, recombinantly produced and / or isolated.

28. A method of treating or protecting a subject from a disease or serious disease or symptoms thereof associated with or caused by a fungal infection, the method comprising administering to the subject an effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to the PF-KEX2b peptide of SEQ ID NO:

2.

29. A method of treating or protecting a subject from a disease or serious disease or symptoms thereof associated with or caused by a fungal infection, the method comprising administering to the subject an effective amount of an antibody or an antigen-binding fragment thereof that specifically binds to the PF-KEX1b peptide of SEQ ID NO:

1.

30. The method according to claim 28 or 29, wherein the PF-KEX2b peptide of SEQ ID NO:2 or the PF-KEX1b peptide of SEQ ID NO:1 is recombinant, recombinantly produced, and / or isolated.

31. The method according to any one of claims 28-30, wherein the antibody or an antigen-binding fragment thereof is present in or isolated from antiserum derived from a donor subject.

32. The method according to any one of claims 28-31, wherein the antibody or an antigen-binding fragment thereof specifically binds to a Kex peptide of one or more of the genera Pneumocystis, Aspergillus, Candida, or Cryptococcus.

33. The method according to any one of claims 11-32, wherein the subject is a mammal.

34. The method according to claim 33, wherein the subject is a human or a human patient.

35. The method according to any one of claims 11-34, wherein the subject is an immunocompromised or immunosuppressed subject.

36. The method according to claim 35, wherein the immunocompromised or immunosuppressed subject is a pre-transplant subject or a post-transplant subject.

37. The method according to claim 35, wherein the immunocompromised or immunosuppressed subject is being treated for an immunodeficiency disease, an autoimmune disease, rheumatoid arthritis, or psoriasis.

38. The method according to any one of claims 11-34, wherein the disease or its symptoms are associated with or caused by an infection with a fungal species of the genus Aspergillus, Pneumocystis, Candida, and / or Cryptococcus.

39. The method according to claim 38, wherein the disease or its symptoms are associated with or caused by an infection with a fungal species of the genus Aspergillus.

40. The method according to any one of claims 11-37, wherein the subject has, is at risk of, or is predisposed to a fungal infection with a fungal species of the genus Aspergillus, Pneumocystis, Candida, and / or Cryptococcus, and a disease or its symptoms associated with or caused by the infection.

41. The method according to any one of claims 11-35 or 38-40, wherein the method treats or prevents a lung disease, a lung dysfunction, invasive pulmonary aspergillosis (IPA), or its symptoms.

42. A method of treating or protecting a subject from a disease or serious disease or its symptoms associated with or caused by an Aspergillus fungal pathogen, the method comprising: Administer to the subject a PF-KEX2b peptide of SEQ ID NO:2, or a polynucleotide encoding PF-KEX2b, or an immunogenic composition comprising a PF-KEX2b peptide or a polynucleotide encoding PF-KEX2b, in an amount effective to induce an immune response and treat or prevent a disease, severe disease, or symptoms thereof associated with infection by said Aspergillus fungal pathogen.

43. A method of treating or protecting a subject from a disease or serious disease or its symptoms associated with or caused by an Aspergillus fungal pathogen, the method comprising: Administer to the subject a PF-KEX1b peptide of SEQ ID NO:1, or a polynucleotide encoding PF-KEX1b, or an immunogenic composition comprising a PF-KEX1b peptide or a polynucleotide encoding PF-KEX1b, in an amount effective to induce an immune response and treat or prevent a disease, severe disease, or symptoms thereof associated with infection by said Aspergillus fungal pathogen.

44. The method according to claim 42 or 43, wherein the PF-KEX2b peptide of SEQ ID NO:2 or the PF-KEX1b peptide of SEQ ID NO:1 is recombinant, recombinantly produced, and / or isolated.

45. The method according to any one of claims 42-44, further wherein an adjuvant is administered to the subject.

46. The method according to claim 45, wherein the adjuvant is alum or 47. The method according to any one of claims 42-46, wherein the induced immune response treats or protects the subject from a lung disease or lung dysfunction and / or its symptoms, and / or an Aspergillus-related disease and / or its symptoms.

48. The method according to claim 47, wherein the Aspergillus-related disease is aspergillosis or invasive pulmonary aspergillosis (IPA) and / or its symptoms.

49. The method according to any one of claims 42-48, wherein the subject is a mammal.

50. The method according to claim 49, wherein the subject is a human or a human patient.

51. The method according to any one of claims 42-50, wherein the subject is an immunocompromised or immunosuppressed subject.

52. The method according to claim 51, wherein the immunocompromised or immunosuppressed subject is a pre-transplant subject or a post-transplant subject.

53. The method according to claim 51, wherein the immunocompromised or immunosuppressed subject is being treated for cancer, an immunodeficiency disease, a congenital disease, or an autoimmune disease.

54. The method according to claim 53, wherein the immunocompromised or immunosuppressed subject is being treated for HIV, AIDS, HIV / AIDS, rheumatoid arthritis, or psoriasis.

55. A vaccine comprising an effective amount of a PF-KEX2b peptide of SEQ ID NO:2 or a polynucleotide encoding PF-KEX2b.

56. A vaccine comprising an effective amount of a PF-KEX1b peptide of SEQ ID NO:1 or a polynucleotide encoding PF-KEX1b.

57. The vaccine according to claim 55 or 56, wherein the PF-KEX2b peptide of SEQ ID NO:2 or the PF-KEX1b peptide of SEQ ID NO:1 is recombinant, recombinantly produced, and / or isolated.

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