Targeting nanoparticles and their use in connection with fungal infections

Abstract

Provided herein are targeted nanoparticles for the diagnosis, treatment or prevention of fungal infections. Provided herein are targeting nanoparticles and their use in connection with fungal infections.

Description

[0001] This application is a divisional application of the invention application with the application date of January 8, 2020, the Chinese national application number 202080017115.5, and the invention name “Targeted nanoparticles and their uses related to fungal infections”. Technical Field

[0002] The present invention relates to targeted nanoparticles and their use in connection with fungal infections. Background Art

[0003] Hundreds of indigenous fungal species cause a wide variety of diseases, including aspergillosis, blastomycosis, candidiasis, coccidioidomycosis (valley fever), cryptococcosis, histoplasmosis, dermatophytoses, and Pneumocystis pneumonia (PCP), to name a few. Collectively, pathogenic fungi infect many different organs, but the skin and lungs are the most common sites. Some fungal diseases are merely disabling, while others are life-threatening. Despite advances in our understanding of the pathology of fungal infections, current methods for diagnosing and treating fungal infections remain inadequate. Summary of the Invention

[0004] Provided herein are targeted nanoparticles, such as liposomes, for diagnosing, treating, or preventing fungal infections. Some liposomes contain an antifungal agent and a targeting molecule that binds to a target antigen on a fungal cell. In the liposomes provided herein, the targeting molecule is incorporated into the outer surface of the liposome, while the antifungal agent is encapsulated within the liposome.

[0005] Further provided is a method of treating or preventing a fungal infection in a subject, comprising administering to a subject having or at risk of developing a fungal infection a multiplicity of liposomes, wherein each liposome in the multiplicity comprises an antifungal agent and a targeting molecule that binds to a target antigen on a fungal cell, wherein the targeting molecule is incorporated into the outer surface of the liposome and the antifungal agent is encapsulated within the liposome.

[0006] Also provided is a method for preparing a multiplicity of liposomes comprising an antifungal agent and a targeting molecule that binds to a target antigen on a fungal cell, wherein the targeting molecule is incorporated into the outer surface of each liposome and the antifungal agent is encapsulated in each liposome. The method comprises: (a) dissolving or suspending the antifungal agent in a solvent at about 60° C. for about 10 minutes to about 30 minutes; (b) encapsulating the antifungal agent into each liposome by mixing the multiplicity of liposomes in suspension form with the antifungal agent / solvent solution of step a) at about 60° C. for about 3 to about 5 hours, or at about 37° C. for about 24-120 hours; and (c) incorporating the targeting molecule into the outer surface of each liposome by contacting the liposomes comprising the encapsulated antifungal agent with the targeting molecule conjugated to the lipid at about 60° C. for about 45 minutes to about 90 minutes.

[0007] Also provided are liposomes comprising a targeting molecule that binds to a target fungal antigen, wherein the targeting molecule is incorporated into the outer surface of the liposome, and wherein the targeting molecule is linked to a molecule that generates a signal when the targeting molecule binds to the target fungal antigen. In some examples, the targeting molecule is linked to a C-terminal and / or N-terminal fragment of a fluorescent protein or a fragment of a fluorescent protein.

[0008] Further provided are methods for detecting a fungal infection in a subject or in a sample from a subject, comprising: a) contacting the subject or the subject's sample with a multiplicity of liposomes, wherein each liposome in the multiplicity comprises a targeting molecule that binds to a target fungal antigen, wherein the targeting molecule is incorporated into the outer surface of the liposome, and wherein the targeting molecule is linked to a molecule that produces a signal when the targeting molecule binds to the target fungal antigen; and b) detecting the signal, wherein the signal indicates the presence of a fungal infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] This application includes the following drawings. The drawings are intended to illustrate certain embodiments and / or features of the compositions and methods and are intended to supplement any one or more of the descriptions of the compositions and methods. The drawings do not limit the scope of the compositions and methods unless the written description clearly indicates otherwise.

[0010] Figure 1A The nucleic acid sequence encoding an exemplary codon-optimized soluble mouse Dectin-1 (sDectin-1) is shown (SEQ ID NO: 1). The 9-codon vector pET-45b+ sequence is boxed, and the start codon is underlined. The sites used for cloning into pET-45B+KpnI (GGTACC) (SEQ ID NO: 23) and PacI (TTAATTAA) (SEQ ID NO: 21) are underlined, respectively. The codon for the GlySer (G, S) flexible linker residue is shown in bold, and the codon for the reactive lys (K) residue (AAG) is shown in bold, with the lysine codon shown in italics. The mouse sDectin-1 sequence (CLEC7A, GenBank accession number AAS37670.1) is shown in plain text; the Ala codons GCT and the stop codons TAA and TTA are underlined, with the stop codon shown in bold. The alternative gene name is MmsDectin1lyshis. The nucleotide sequence is 604 base pairs in length, of which 597 base pairs encode a protein of 199 amino acids in length.The nucleic acid encoding the exemplary codon-optimized mouse sDectin-1 was cloned into pET-45B+.

[0011] Figure 1BThe amino acid sequence encoded by SEQ ID NO: 1 is shown (SEQ ID NO: 2). This is a polypeptide comprising the mouse sDectin-1 polypeptide. The N-terminal amino acid sequence from pET-45B+ and the (His)6(HHHHHH) (SEQ ID NO: 22) affinity tag are boxed. The GlySer (GS) flexible linker residue and the reactive lys (K) residue are shown in bold (with lysine shown in italics). The mouse sDectin-1 amino acid residues are shown in plain text (amino acids 23-199 of SEQ ID NO: 2), ending with the C-terminal Ala residue (A) shown in bold, the codon for which is used to place the stop codon and Pac1 site in frame. It should be understood that, optionally, the stop codon in any polypeptide sequence disclosed herein, if not part of the native polypeptide from which the polypeptide is derived, can be removed to produce a polypeptide that does not include one or more stop codons. The protein comprising the mouse sDectin-1 polypeptide is 199 amino acids long and has a molecular weight (MW) of 22,389.66 g / mol. The theoretical pI is 7.74. It should be understood that any protein described herein comprising an affinity tag (e.g., a (His)6 affinity tag) can be modified to remove the His tag. In some instances, any nucleotide sequence described herein can further comprise a protease cleavage site for post-translational and / or post-purification removal of the affinity tag.

[0012] Figure 1C The nucleotide sequence (SEQ ID NO: 3) encoding an exemplary codon-optimized soluble mouse Dectin-2 (sDectin-2) is shown. The 9-codon vector pET-45b+ sequence is boxed, and the start codon is underlined. The sites used for cloning into pET-45B+KpnI (GGTACC) (SEQ ID NO: 23) and PacI (TTAATTAA) (SEQ ID NO: 21) are underlined, respectively. The codons for the GlySer (G, S) flexible linker residues are shown in bold, and the codons for the reactive lys (K) residue (AAG) are shown in bold, with the lysine codon shown in italics. The codon-optimized sDectin-2 from the CLEC6A mouse Dectin 2 gene is shown in plain text, with the Ala codon (GCT) and stop codons TAA and TTA underlined, with the stop codon shown in bold. The alternative gene name is MmsDectin2lyshis. The nucleic acid sequence is 574 base pairs in length, of which 567 base pairs encode a protein of 190 amino acids in length.The nucleic acid encoding the exemplary codon-optimized mouse sDectin-2 was cloned into pET-45B+.

[0013] Figure 1D The amino acid sequence encoded by SEQ ID NO:3 is shown (SEQ ID NO:4). This polypeptide comprises the mouse sDectin-2 protein. The N-terminal amino acid and (His)6(HHHHHH) (SEQ ID NO:22) affinity tag from pET-45B+ are boxed. The GlySer (GS) flexible linker residue and reactive lys (K) residue are shown in bold, and lysine is shown in italics. The mouse sDectin-2 amino acid residues are shown in plain text (amino acids 23-189 of SEQ ID NO:4), ending with the C-terminal Ala residue (A) (bold), whose codon is used to place the stop codon and Pac1 site in frame. The resulting polypeptide comprising mouse sDectin-2 has 189 amino acids, a MW of 21,699.25 g / mol, and a theoretical pI of 6.33.

[0014] Figure 1E The nucleic acid sequence encoding an exemplary codon-optimized soluble mouse Dectin-3 (sDectin-3) is shown (SEQ ID NO: 5). The 9-codon vector pET-45b+ sequence is boxed, and the start codon is underlined. The sites used for cloning into pET-45b+KpnI (GGTACC) (SEQ ID NO: 23) and PacI (TTAATTAA) (SEQ ID NO: 21) are underlined, respectively. The codons for the GlySer (G, S) flexible linker residues are shown in bold. The reactive lysine (K) codon (AAG) is shown in bold, and lysine is shown in italics. The codon-optimized sDectin-3 from the CLEC4D mouse Dectin-3 gene (GenBank accession number NP_034949.3) is shown in plain text, with the Ala codon (GCT) and the stop codons TAA and TTA underlined. The stop codon is shown in bold. The alternative gene name is MmsDectin3lyshis. The nucleotide sequence is 604 base pairs long, of which 597 base pairs encode a protein of 199 amino acids in length. The nucleic acid encoding the exemplary codon-optimized mouse sDectin-3 was cloned into pET-45B+.

[0015] Figure 1FThe amino acid sequence encoded by SEQ ID NO:5 is shown (SEQ ID NO:6). This polypeptide comprises the mouse sDectin-3 protein. The N-terminal amino acid and (His)6(HHHHHH) (SEQ ID NO:22) affinity tag from pET-45B+ are boxed. The GlySer (GS) flexible linker residue and reactive lys (K) residue are shown in bold, and lysine is shown in italics. The mouse sDectin-3 amino acid residues are shown in plain text (amino acids 23-199 of SEQ ID NO:6), ending with the C-terminal Ala residue (A) shown in bold, whose codon is used to place the stop codon and Pac1 site in frame. The polypeptide is 199 amino acids in length, has a MW of 23,023.72 g / mol, and a theoretical pI of 6.52.

[0016] Figure 1G The nucleic acid sequence encoding an exemplary codon-optimized soluble human Dectin-1 (sDectin-1) is shown (SEQ ID NO: 7). The human sDectin-1 DNA sequence was expressed from the vector pET-45B+. The nine codons of the vector pET-45b+ and the His tag are boxed, and the start codon is underlined. The cloning sites BamHI (GGATCC) (SEQ ID NO: 24) and PacI (TTAATTAA) (SEQ ID NO: 21) are underlined, respectively. Codons for the enterokinase processing site are shown in lowercase, and codons for the GlySer (G, S) flexible linker residue and the reactive lys (K) residue (AAA and AAG) are shown in bold (with lysine codons shown in italics). The human sDectin-1 sequence (CLEC7A, GenBank Accession No. NM_197947) is shown in plain text and codons have been optimized for expression. The Ala codons GCT and the stop codons TAA and TTA are underlined, and the stop codon is in bold. An alternative name for this sequence is HssDectin1lyshis. The nucleotide sequence encoding human sDectin-1 is 649 base pairs long and encodes a polypeptide of 214 amino acids. The nucleic acid encoding the exemplary codon-optimized human sDectin-1 was cloned into pET-45B+.

[0017] Figure 1HThe amino acid sequence encoded by SEQ ID NO:7 is shown (SEQ ID NO:8). This is a polypeptide comprising the human sDectin-1 protein. The N-terminal amino acid and (His)6(HHHHHH) (SEQ ID NO:22) affinity tag from pET-45B+ are boxed. The enterokinase processing site is shown in lowercase. The GlySer (GS) flexible linker residue and reactive lys (K) residue are shown in bold, and lysine is shown in italics. The human sDectin-1 amino acid residues are shown in plain text (amino acids 35-214 of SEQ ID NO:8), ending with the C-terminal Ala residue (A) shown in bold, whose codon is used to place the stop codon and Pac1 site in frame. The polypeptide is 214 amino acids in length, has a MW of 23,703.20 g / mol, and a theoretical pI of 6.22.

[0018] Figure 1I The nucleic acid sequence encoding an exemplary codon-optimized soluble human Dectin-2 (sDectin-2) (SEQ ID NO: 9) is shown. The human sDectin-2 nucleotide sequence is expressed from the vector pET-45B+. The nucleotide sequence is approximately 580 base pairs in length, of which 616 bases encode a protein of 203 amino acids in length. The 9 codon vector pET-45b+ sequence (including the His tag) is boxed, and the start codon is underlined. The cloning sites BamHI (GGATCC) (SEQ ID NO: 24) and PacI (TTAATTAA) (SEQ ID NO: 21) are underlined, respectively. The codons for the enterokinase treatment site are shown in lowercase. The codons for the GlySer (G, S) flexible linker residue are shown in bold, and the reactive lys (K) residue (AAG) is shown in bold, with lysine shown in italics. The codon-optimized sDectin-2 from the CLEC6A human Dectin2 gene (cDNA GenBank accession number NM_001317999) is shown in plain text. The Ala codon (GCT) and the stop codons TAA and TTA are underlined, and the stop codon is shown in bold. The alternative gene name is HssDectin2lyshis. The nucleic acid encoding the exemplary codon-optimized human sDectin-2 was cloned into pET-45B+.

[0019] Figure 1JThe amino acid sequence encoded by SEQ ID NO:9 is shown (SEQ ID NO:10). This polypeptide comprises the human sDectin-2 protein. The N-terminal amino acid and (His)6(HHHHHH) (SEQ ID NO:22) affinity tag from pET-45B+ are boxed. The enterokinase processing site is shown in lowercase. The GlySer (GS) flexible linker residue and the reactive Lys (K) residue are shown in bold, and Lysine is shown in italics. The human sDectin-2 amino acid residues are shown in plain text (GenBank accession number NP_001007034.1) (amino acids 36-203 of SEQ ID NO:10), ending with the C-terminal Ala residue (A) shown in bold, whose codon is used to place the stop codon and Pac1 site in frame. The polypeptide is 203 amino acids in length, has a MW of 22,969 g / mol, and a theoretical pI of 5.91.

[0020] Figure 1K The nucleic acid sequence encoding an exemplary codon-optimized soluble human Dectin-3 (sDectin-3) is shown (SEQ ID NO: 11). The human sDectin-3 DNA sequence was expressed from the vector pET-45B+ in E. coli. The 9 codon vector pET-45b+ sequence and the hist tag are boxed, and the start codon is underlined. The sites used for cloning into pET-45b+BamHI (GGATCC) (SEQ ID NO: 24) and PacI (TTAATTAA) (SEQ ID NO: 21) are underlined, respectively. The codons for the enterokinase processing site are shown in lowercase. The codons for the GlySer (G, S) flexible linker residue are shown in bold, and the reactive lys (K) residue (AAG) is shown in bold, with lysine shown in italics. The codon-optimized sDectin-3 from the CLEC4D human Dectin-3 gene (GenBank accession number NM_080387) is shown in plain text. The Ala codon (GCT) and the stop codons TAA and TTA are underlined, and the stop codon is shown in bold. The alternative gene name is HssDectin3lyshis. The nucleotide sequence is 628 base pairs long and encodes a polypeptide of 207 amino acids in length. The nucleic acid encoding the exemplary codon-optimized human sDectin-3 was cloned into pET-45B+.

[0021] Figure 1LThe amino acid sequence encoded by SEQ ID NO:11 (SEQ ID NO:12) is shown. This polypeptide comprises the human Dectin-3 protein. The N-terminal amino acid and (His)6(HHHHHH) (SEQ ID NO:22) affinity tag from pET-45B+ are boxed. The enterokinase processing site is shown in lowercase. The GlySer (GS) flexible linker residue and the reactive lysine (K) residue are shown in bold, and lysine is shown in italics. The human sDectin-3 amino acid residues (GenBank accession number NP_525126) are shown in plain text (amino acids 35-207 of SEQ ID NO:12), ending with the C-terminal Ala residue (A) shown in bold, whose codon is used to place the stop codon and Pac1 site in frame. The protein is 207 amino acids long, has a MW of 23,662 g / mol, and a theoretical pI of 7.64.

[0022] Figure 1MThe nucleic acid sequence encoding an exemplary codon-optimized soluble mouse Dectin-1 (sDectin-1) fused to the N-terminal portion of Venus is shown (SEQ ID NO: 13). It should be understood that the soluble mouse Dectin-1 amino acid sequence that does not include the membrane domain can be interchanged with a soluble Dectin-1 amino acid sequence from another species, for example, the human Dectin-1 amino acid sequence set forth in SEQ ID NO: 8, or a fragment thereof. Dectin-2 and Dectin-3 amino acid sequences from mice or other species, or fragments thereof, can also be used in any of the constructs described herein to detect fungal infections. MmsDECTIN1VyN is a codon-optimized DNA sequence expressed in pET-45B+ that encodes half of the BiFC diagnostic. Sequence: 1,168 base pairs, of which 1,161 base pairs encode a protein of 385 amino acids in length. The 9-codon vector pET-45b+ sequence is boxed and the start codon is underlined. The cloning sites KpnI (GGTACC) (SEQ ID NO: 23) and PacI (TTAATTAA) (SEQ ID NO: 21) are underlined, respectively. The codon for the short GlySerGly flexible linker residue is shown in bold, followed by the nucleotide sequence (465 base pairs) encoding the VenusVyNT154M sequence (which is the N-terminal half of the mutant Venus protein modified from GenBank Accession No. AKA95335) in lowercase letters, followed by the long GlySer spacer shown in bold (with the reactive lysine shown in italics), followed by the 528-residue mouse sDectin-1 sequence (shown in plain text) (CLEC7A, GenBank Accession No. AAS37670.1), ending with the stop codons TAA and TTA (shown in bold).

[0023] Figure 1NShown is the amino acid sequence (SEQ ID NO:14) encoded by SEQ ID NO:13. This polypeptide comprises mouse MmsDectin1VyN protein. The N-terminal amino acid from pET-45B+ and the (His)6(HHHHHH) (SEQ ID NO 22) affinity tag are boxed. The first GlySer (GS) flexible linker is shown in bold, followed by the C-terminal portion of the Venus fluorescent protein VyN represented in lowercase letters, representing Venus residues 1-155 with T154M mutation (amino acids 15-169 of SEQ ID NO:14). This is followed by a long GlySer spacer sequence containing reactive lys (K) residues shown in italics for coupling to a lipid carrier, followed by mouse sDectin-1 amino acid residues (amino acids 211-387 of SEQ ID NO:14) shown in plain text. The peptide is 387 amino acids in length, has a MW of 42,181.62 g / mol, and a predicted pI of 6.55.

[0024] Figure 1O The nucleic acid sequence encoding an exemplary codon-optimized soluble mouse Dectin-1 (sDectin-1) fused to the C-terminal portion of Venus is shown (SEQ ID NO: 15). MmsDECTIN1VC is a codon-optimized DNA sequence that can be expressed in pET-45B+ and encodes half of the BiFC diagnostic. Length: 955, of which 948 encode a protein of 316 amino acids in length. The 9-codon vector pET-45b+ sequence is boxed, and the start codon is underlined. The cloning sites KpnI (GGTACC) (SEQ ID NO: 23) and PacI (TTAATTAA) (SEQ ID NO: 21) at the beginning and end are underlined, respectively. The codon for the GlySer (G, S) flexible linker residue is shown in bold. Then, the coding sequence for the C-terminal amino acid coding sequence of Venus (252 nucleotides) (aa residues 155 to 238, modified by GenBank accession number AKA95335 and containing the T154M mutation) is shown in lowercase. Then, the long GlySer flexible linker is shown in bold, with the reactive lysine for coupling to the lipid carrier shown in italics. This is followed by the mouse sDectin-1 sequence (CLEC7A, GenBank accession number NAAS37670.1) with a length of 528 residues (shown in plain text), ending in the stop codons TAA and TTA shown in bold.

[0025] Figure 1PShown is the amino acid sequence (SEQ ID NO:16) encoded by SEQ ID NO:15. This polypeptide comprises the mouse MmsDectin1VC protein. The N-terminal amino acid and (His)6(HHHHHH) (SEQ ID NO:22) affinity tag from pET-45B+ are boxed. The first GlySer (GS) flexible linker sequence is shown in bold. Then, the C-terminal half (Genbank accession number AKA95335) of the Venus fluorescent protein representing Venus residues 155 to 238 (amino acids 15-98 of SEQ ID NO:16) is shown in lowercase. Then, the long GlySer flexible spacer containing the reactive lys (K) residue (italics) is shown in bold. Mouse sDectin-1 amino acid residues are shown in plain text (amino acids 140-316), followed by the C-terminal Ala residue (A) for placing the stop codon in the frame. The peptide is 316 amino acids in length, has a MW of 34,081.21 g / mol, and a predicted pI of 6.8.

[0026] Figure 1Q : MmDEC2VyN, also the codon-optimized DNA sequence of MmDEC2VyN expressed in pET-45B (SEQ ID NO: 17). Length: 577 bp, the 9-codon vector pET-45b+ sequence is boxed, the start codon is underlined, the cloning sites KpnI (GGTACC) (SEQ ID NO: 23) and PacI (TTAATTAA) (SEQ ID NO: 21) are underlined, respectively, the codons for the Gly, Ser (G, S) flexible linker residues are shown in bold, the reactive Lys (K) residue AAG is shown in italics, the sDectin-2 sequence from the mouse Dectin2 gene CLEC6A codon-optimized for E. coli expression is shown in plain text, followed by a 15-residue Glyser-rich flexible spacer, followed by Venus VyN in lowercase letters. The coding sequence for T154M (i.e., the N-terminal half of the mutant Venus protein modified from AKA95335) is 465 nucleotides long, with one Ala (A) codon, GCT, and two stop codons, TAA and TTA, underlined; the stop codon is in bold. The alternative gene name is MmsDectin2lyshis, and its length is 1084 bp, minus 7 bp for the stop and following restriction sites, encoding a 359-residue protein. However, a shorter 1057 bp version, starting at the Kpn1 site, GGTACC (SEQ ID NO: 23), can be ordered from GenScript for subcloning into pET-45b+.

[0027] Figure 1R Figure 1: Final MmsDEC2VyN protein (SEQ ID NO: 18) being synthesized. The N-terminal amino acid and (His)6(HHHHHH) (SEQ ID NO: 22) affinity tag from pET-45B+ are boxed, the GlySer (GS) flexible linker residue and reactive Lys (K) residue are shown in bold (with Lysine in italics), and the 166 mouse sDectin-2 amino acid residues are shown in plain text (amino acids 23-188 of SEQ ID NO: 18), followed by a 15-residue GlySer-rich flexible spacer, Venus residues 1-155 with a T154M mutation (amino acids 204-359 of SEQ ID NO: 18), and ending with the C-terminal Ala residue (A) shown in bold, whose codon is used to place a stop codon and a PacI site in frame. 359 amino acids in total, MW 40,198 g / mol. PI 6.04. OD2801.940 / mg / mL.

[0028] Figure 1S : MmDEC2VC, also the codon-optimized DNA sequence of MmDEC2VC expressed in pET-45B (SEQ ID NO: 19). Length: 577 bp, the 9-codon vector pET-45b+ sequence is boxed, the start codon is underlined, and the cloning sites are KpnI (GGTACC) (SEQ ID NO: 23) and PacI (TTAATTAA) (SEQ ID NO: 19). NO: 21) are underlined, the codons for the Gly, Ser (G, S) flexible linker residues are bolded, and the reactive Lys (K) residue AAG is italicized. The sDectin-2 sequence, codon-optimized for E. coli expression from the mouse Dectin2 gene CLEC6A, is shown in plain text, followed by a 15-residue Glyser-rich flexible spacer, followed by the 252-nucleotide coding sequence for the C-terminal amino acids of Venus (aa residues 155 to 238, modified from AKA95335) in lowercase. One Ala (A) codon, GCT, and two stop codons, TAA and TTA, are underlined; the stop codon is shown in bold. Alternative gene name: MmsDectin2lyshis. Length: 871 bp, minus 7 bp for the stop codon and cleavage site, encoding a 288-residue protein. However, a shorter 844 bp version starting at the Kpn1 site GGTACC (SEQ ID NO: 23) was ordered from Genscript for subcloning into pET-45b+.

[0029] Figure 1TFigure 2: Final MmsDEC2VC2 protein (SEQ ID NO: 20) being synthesized. The N-terminal amino acid and (His)6(HHHHHH) (SEQ ID NO: 22) affinity tag from pET-45B+ are boxed, the GlySer (GS) flexible linker residue and reactive Lys (K) residue are shown in bold (with Lysine in italics), and the 166 mouse sDectin-2 amino acid residues are shown in plain text (amino acids 23-188 of SEQ ID NO: 20), followed by a flexible spacer rich in GlySer with 15 residues, Venus residues 155 to 238 (amino acids 204-288 of SEQ ID NO: 20), followed by two stop codons and a PacI site in the framework. 288 amino acids in total with a MW of 32,098 g / mol. PI 6.16, OD 280 2.02 OD / mg / mL.

[0030] Figure 2 Shown are SDS PAGE analyses of soluble Dectin-1 (sDectin-1) in cell extracts and after purification. The sDectin-1 protein was produced in E. coli BL21 strain grown overnight in Luria broth from the pET-45B plasmid without IPTG induction, dissolved in GuHCl buffer, purified by Ni-NTA resin and examined by SDS PAGE. Extraction into a buffer also containing the reducing agent β-mercaptoethanol and Triton-X100 detergent greatly improved recovery from insoluble inclusion bodies (center lane) relative to buffers without them (right lane). The protein was examined on a 12% acrylamide gel stained with Coomassie blue. Extraction of the cells with urea buffer produced very little protein.

[0031] Figure 3Schematic diagram of the DEC-AmB-LL model of liposomes loaded with sDectin-1, amphotericin B, and rhodamine. Amphotericin B (AmB, blue oval structure) is embedded in the lipid bilayer of liposomes with a diameter of 100 nm. sDectin-1 (DEC, green spherical structure) is coupled to the lipid carrier DSPE-PEG, and both DSPE-PEG-DEC and red fluorescent DHPE-rhodamine (red star) are also inserted into the liposome membrane. The molar ratio of sDectin-1, rhodamine, AmB, and liposome lipids is 1:2:13:100. Two sDectin-1 monomers (two DSEP-PEG-DEC molecules) must float together in the membrane to bind to cell wall β-glucan (the red sugar portion). The two liposome controls examined were BSA-AmB-LL containing an equal μg amount of 65 kDa BSA instead of 22 kDa sDectin-1 (i.e., 0.33 BSA:2:13:100 molar ratio) and AmBisome-like liposomes (AmB-LL) without any protein coating (0:2:13:100 molar ratio). Based on these molar ratios, the surface area of ​​liposomes with a diameter of 100 nm, and the published 6 nm 2 Lipid bilayer 5×10 6 Based on an estimate of the number of lipid molecules, it is estimated that there are approximately 3,000 rhodamine molecules in each liposome and approximately 1,500 sDectin-1 monomers in each DEC-AmB-LL.

[0032] Figures 4A-F show that sDectin-1-coated DEC-AmB-LL strongly binds to swollen conidia and germ tubes of germinating Aspergillus fumigatus (A. fumigatus), while AmB-LL does not. A. Rhodamine red fluorescent DEC-AmB-LL binds to swollen conidia (white arrows) and seedling germ tubes of A. fumigatus. B. Rhodamine red fluorescent AmBisome-like AmB-LL does not bind. Even with the red channel enhanced as shown in this image, no liposomes were detected. The smallest red dots represent individual 100 nm diameter liposomes observed based on fluorescence (orange arrows). Large clusters of liposomes form brighter red staining areas. C to F. C and D are stained with DEC-AmB-LL. E and F are stained with BSA-AmB-LL. A and B. Cells were grown at 37°C for 8 hours in VMM + 1% glucose. Labeling was performed in liposome dilution buffer LDB for 60 minutes. All three liposome preparations were diluted 1:100, resulting in a final concentration of liposome sDectin-1 and BSA protein of 1 μg / 100 μL. Seedlings were observed in the green channel for cytoplasmic EGFP expression only and in the red channel for liposomes. A and B were taken at 63X under oil immersion. C to F were taken at 20X on an inverted fluorescence microscope.

[0033] Figures 5A-F show that sDectin-1-coated DEC-AmB-LL binds to swollen conidia and hyphae of mature Aspergillus fumigatus cells, whereas non-targeted AmBisome-like AmB-LL does not. A. fumigatus conidia were germinated and grown in VMM + 1% glucose at 37°C for 16 hours and then stained with fluorescent liposomes. A to D show staining with a 1:100 dilution of rhodamine-fluorescent DEC-AmB-LL to provide 1 μg of sDectin-1 per 100 μL; E and F show staining with an equal amount of red-fluorescent AmB-LL for 60 minutes. A: DIC image alone. B: Combined DIC and red-fluorescent image. A and B show binding of rhodamine-fluorescent DEC-AmB-LL to swollen conidia (white arrows) and hyphae. C to F show examination of cytoplasmic green EGFP and red fluorescence from liposomes. The smallest red dots represent individual 100 nm liposomes (orange arrows) visible due to their intense fluorescence. C and D show that almost all conidia and most hyphae are stained by DEC-AmB-LL. E and F show that AmB-LL does not bind. A and B were taken at 63X under oil immersion and seven stacked images were merged. C to F were taken at 20X on an inverted fluorescence microscope.

[0034] Figure 6A-FFigure 3. sDectin-1 coated liposomes (DEC-AmB-LL) show strong binding to Candida albicans and Cryptococcus neoformans cells. A, C, and E are bright field images of Candida albicans strain Sc5314 and Cryptococcus neoformans strain H99 labeled with DEC-AmB-LL diluted 1:100 in LDB. B, D, and F are a combination of bright field and red fluorescence images showing strong binding of rhodamine-labeled DEC-AmB-LL to these cells. Ordinary, uncoated AmB-LL showed no detectable binding to these cells. A and B were taken at 63X under oil immersion, and C to F were taken at 20X on an inverted fluorescence microscope.

[0035] Figure 7: sDectin-1-coated DEC-AmB-LL binds to Aspergillus fumigatus at a frequency several orders of magnitude higher than control AmB-LL or BSA-AmB-LL, and binding is inhibited by soluble β-glucan. A sample of 4,500 A. fumigatus conidia was germinated and grown in VMM + 1% glucose at 37°C for 36 hours, fixed in formalin or examined for viable cells, and incubated for 1 hour with liposomes diluted 1:100 in liposome dilution buffer. Unbound liposomes were washed away. As shown in Figures 4 and 5, multiple fields of red fluorescence images were captured at 20X magnification. Each field of view contained approximately 25 enlarged conidia and an extensive hyphal network. A, B, C. Labeling of formalin-fixed cells. D, E, F. Labeling of viable cells. G, H, I. Inhibitory effect of 1 mg / mL laminarin (soluble β-glucan) on DEC-AmB-LL labeling of fixed cells compared to 1 mg / mL sucrose as a control. A, D, and G. The number of red fluorescent liposomes and liposome clusters was counted, averaged for each field, and plotted on a log10 scale. The numerical mean is indicated above each bar and on the vertical axis. The standard error is shown. Example fields of liposomes used to construct the bar graphs are shown in B, C, E, F, H, and I.

[0036] Fig. 8 A-G is presented at the AmB concentration close to the ED50 of AmB, and DEC-AmB-LL kills or inhibits the efficiency of Aspergillus fumigatus (A.fumigatus) growth much higher than AmB-LL.A sample of 4,500 Aspergillus fumigatus (A.fumigatus) conidia is allowed to germinate and grow in Vogel minimal medium (VMM+1% glucose) in 96-well microtiter plates, and simultaneously treated with liposome dilution buffer or a liposome preparation of AmB (AD 3uM AmB, E.0.09uM AmB, F.0.18uM AmB) delivered to the growth medium at a specified concentration.Use Cell Titer Blue (CTB) reagent (A and C) (it measures total cell esterase activity) or by hypha length (B and D) or by germination percentage (E and F) to estimate viability and growth.Subtract the background fluorescence of the wells containing CTB but lacking cells and liposomes in the culture medium.Indicate standard error. The insets in B and D show examples of hyphal length measurements for samples treated with AmB-LL and DEC-AmB-LL. In B and D, one hyphal length unit is equal to 5 microns. A and B, and C and D compare the results of two complete biological replicates prepared and stored in different buffers. In A and B, liposomes were prepared in RN#5 buffer (0.1M NaH2PO4, 10mM triethanolamine pH 7.2, 1M L-arginine, 100mM NaCl, 5mM EDTA, 5mM BME (β-mercaptoethanol)). In C to F, liposomes were prepared in RN#5 buffer (0.1M NaH2PO4, 10mM triethanolamine pH 7.2, 1M L-arginine, 100mM NaCl, 5mM EDTA, 5mM BME). Cells were grown for 56 hours (C, D) or 36 hours (A, B, E, F). G. Dose-response curve based on the percentage of conidial germination. Conidia were plated in microtiter plate wells along with liposomes delivering different concentrations of AmB in VMM + glucose.After 8 hours at 37°C, the percentage of conidia that had germinated was quantified.

[0037] Figure 9A-B Based on cytoplasmic GFP fluorescence, the ED of AmB was close to and below 50DEC-AmB-LL inhibits the growth of Aspergillus fumigatus cells more effectively than AmBisome-like AmB-LL at AmB concentrations of 100 μM. A sample of 4,500 conidia of Aspergillus fumigatus was germinated and grown in Vogel minimal medium (VMM) + 1% glucose in a 96-well microtiter plate, and the sample was treated with a liposome dilution buffer control or a dilution of an AmB-loaded liposome preparation delivering 2 μM AmB (A.) and 0.67 μM AmB (B.), and incubated at 37°C for 36 hours. Cell integrity was determined based on the amount of cytoplasmic green fluorescence of the EGFP reporter gene in the A1163 strain of Aspergillus fumigatus. The fluorescence background of wells containing culture medium but lacking cells and liposomes was subtracted. The liposomes were stored in RN#5 buffer before use and diluted with liposome dilution buffer LDB.

[0038] Figure 10 sDectin-1-coated DEC-AmB-LL showed less toxicity to HEK293 cells than uncoated AmBisome-like AmB-LL. Human embryonic kidney HEK293 cells were treated with liposomes for 2 hours, after which the liposomes were washed away. The cells were then incubated for an additional 16 hours and assayed. The liposomes delivered a total of 30 or 15 μM AmB to the culture medium. Cell viability and survival were assessed using the CellTiter Blue esterase assay.

[0039] Figure 11 A schematic model of an exemplary immobilized Dectin-coated liposome for detecting fungal β-glucan and mannan polysaccharides in a subject or sample from the subject is shown. DEC-BiFC-L is a liposome coated with 500 sDectin monomers (blue) fused to the N-terminal half of the green fluorescent protein VenusVyN155 (e.g., DEC-VN-) and 500 sDectin monomers fused to the C-terminal half of Venus VC155 (DEC-VC-). It will rapidly recognize and bind to fungal β-glucan or mannan to form sDectin dimers, resulting in the formation of assembled Venus, which will produce a strong green bimolecular fluorescence complementation (BiFC) signal. These liposomes will only produce a green fluorescent signal when in contact with fungal cells or released β-glucan or mannan. When these liposomes are attached to an insoluble matrix by, for example, biotin-streptavidin binding, they can be used to detect low concentrations of fungal polysaccharides in large volumes of serum using standard fluorescence instrumentation.

[0040] Figure 12Schematic diagram of the DEC-HRP-L (Dectin-coated liposome) model for detecting fungal β-glucans and mannans. sDectin (DEC, green spherical structures) and horseradish peroxidase (HRP, hexagonal structures) are conjugated to the lipid carriers DSPE-PEG or DSPE and incorporated into the liposome membrane. The molar ratio of sDectin, HRP, PEG, and liposome lipids is approximately 1:1:13:100. Two sDectin monomers (two DSEP-PEG-DEC molecules) must float together in the membrane to bind to cell wall β-glucans or mannans (the red sugar moiety). After these liposomes are bound to fixed fungal samples, excess unbound liposomes are washed away. Then, substrates for HRP enzymatic activity (4-chloro-1-naphthol and peroxide) are added. After substrate addition, the purple precipitate produced by HRP is measured to determine the total amount of bound DEC-HRP-L and the total amount of fungal polysaccharides.

[0041] Figure 13A The modified mouse sDectin-2 DNA, MmsDectin2lyshis DNA sequence (SEQ ID NO: 3), is shown. The codon-optimized DNA sequence of MmsDectin2lyshis was cloned into pET-45B. NCBI Bankit #MN104679. Length: 577 bp. The 9-codon vector pET-45b+ sequence is boxed, the start codon is underlined, and the cloning sites KpnI (GGTACC) (SEQ ID NO:23) and PacI (TTAATTAA) (SEQ ID NO:21) are underlined, respectively. Codons for the Gly, Ser (G, S) flexible linker residues are bold, and the reactive Lys (K) residue AAG is bold and italicized. The sDectin-2 sequence from the mouse Dectin 2 gene CLEC6A, codon-optimized for E. coli expression, is shown in plain text and ends with an Ala (A) codon, GCT, and two stop codons, TAA and TTA, in a PacI site, shown in bold. Length of the coding sequence and two stop codons: 574 bp, encoding a protein of 189 residues. Alternative gene name: MmsDectin2lyshis.

[0042] Figure 13BsDectin-2 (DEC2) protein (SEQ ID NO: 4) synthesized in E. coli is shown. The N-terminal amino acid peptide sequence and (His)6(HHHHHH) (SEQ ID NO: 22) affinity tag from pET-45B+ are boxed, the GlySer (GS) flexible linker residue and the reactive lys (K) residue are shown in bold (with lysine shown in italics), and the 166 mouse sDectin-2 amino acid residues are shown in plain text, ending with the C-terminal Ala residue (A) shown in bold. A total of 189 amino acids with a MW of 21,763.29 g / mol and a theoretical pI of 6.33. The sDectin-2 sequence represents aa amino acid residues 44 to 209 from the native mouse Dectin-2 sequence. Alternative protein name: MmsDectin2lyshis protein.

[0043] Figure 14 Figure 2 shows SDS PAGE analysis of purified sDectin-2. Crude extracts of E. coli BL21 cells expressing and not expressing sDectin-2 and purified sDectin-2 protein were examined by SDS PAGE on a 12% gel stained with Coomassie blue. Molecular weight markers and the approximate molecular weight of the modified sDectin-2 of 22 kDa are indicated on the left.

[0044] Figure 15 A model of sDectin-2 coated liposomes loaded with rhodamine and amphotericin B is shown. Amphotericin B (AmB, blue oval structure) is embedded in the lipid bilayer of 100 nm diameter liposomes. sDectin-2 (DEC2, green spherical structure) is coupled to the lipid carrier DSPE-PEG, and DEC2-PEG-DSPE and red fluorescent DHPE-rhodamine (red star) are inserted into the liposome membrane via their lipid moieties DSPE and DHPE. The molar ratio of sDectin-2, rhodamine, AmB and liposome lipids is 1:2:11:100. Two sDectin-2 monomers (two DEC2-PEG-DSPE molecules) must float together in the membrane to bind to the fungal mannan (red sugar part). Based on these molar ratios, the surface area of ​​100 nm diameter liposomes and the published 6 nm 2 Lipid bilayer 5×10 6 Based on an estimate of lipid molecules, we calculated that in each DEC2-AmB-LL, there were approximately 1,500 sDectin-1 monomers, 3,000 rhodamine molecules, and approximately 16,500 AmB molecules per liposome.

[0045] Figure 16A-F shows sDectin-2-coated liposomes DEC2-AmB-LL bound to the extracellular matrix associated with a variety of morphological Candida albicans cells. A: Yeast cells. Cells are highlighted by differential interference contrast microscopy (green, DIC) and rhodamine-fluorescently labeled DEC2-AmB-LL (red). B&C: Pseudohyphae (Ps-Hyp) and hyphae (Hyp). Cells are highlighted by the cells' endogenous GFP fluorescence. DEC2-AmB-LL is associated with the extracellular matrix (Ex) in large clusters. D, E, F: Mature hyphae. D: Brightfield microscopy shows the extracellular matrix surrounding hyphae of the stained cells in E. E: Combined brightfield image and red fluorescence of the liposomes. F: Additional images parallel to the image in E re-emphasize the typical staining of the matrix. All cells were stained with DEC2-AmB-LL (e.g., 0.5 μg sDectin-2 / 100 μL) diluted 1:200 in LDB2 buffer for 1 hour. DEC2-AmB-LL stained (Ex+) or unstained or weakly stained (Ex-) extracellular matrix is ​​indicated. Arrows indicate individual liposomes. Photos were taken at 63X magnification under oil immersion. Similar images were obtained from several independent fungal cell labeling studies.

[0046] Figure 17A-G shows DEC2-AmB-LL bound to the extracellular matrix associated with Cryptococcus neoformans (C. neoformans) and Aspergillus fumigatus (A. fumigatus) cells. Rhodamine red fluorescent DEC2-AmB-LL binds to the extracellular matrix (Ex) in large clusters and rarely binds to the cell wall of these two species. A, B, C, and D: Cryptococcus neoformans (C. neoformans). Yeast cells were co-stained with capsular glucuronoxylomannan (GXM) and secondary goat anti-mouse antibody Alexa488 (green) using DEC2-AmB-LL and mouse monoclonal antibody 18B7. All cells were stained with DEC2-AmB-LL (e.g., 0.5 μg sDectin-2 / 100 μL) diluted 1:200 in LDB2 for 1 hour. A: Bright field image of cells. B: Green fluorescence image of cells stained with GXM-specific antibody. C: Merged fluorescence image of B and D. D: Red fluorescent DEC2-AmB-LL. E, F and G: Aspergillus fumigatus. E and F: Bright field and combined fluorescence images of seedlings grown for 10 hours and stained with DEC2-AmB-LL. G: Mature hyphae grown for 24 hours. Both species were stained with DEC2-AmB-LL (e.g., 0.5 μg sDectin-2 / 100 μL) diluted 1:200 into LDB2 for 1 hour. Extracellular matrix stained with DEC2-AmB-LL (Ex+) or unstained or weakly stained (Ex-) or extracellular matrix not stained with 18B7 or DEC2-AmB-LL (Ex- / -) is indicated. Photos were taken at 63X or 20X (Plate 2G) magnification under oil immersion (Panels AF). Similar images were obtained from three independent fungal cell labeling studies.

[0047] Figure 18A-DDEC2-Rhod (rhodamine-labeled DEC2) and DEC2-AmB-LL are shown to bind to the exopolysaccharide matrix surrounding A. fumigatus hyphal cells in a similar pattern. A. fumigatus conidia were germinated at low density on microscope chamber slides in VMM + 1% glucose + 0.5% BSA and grown for 24 hours at 37°C. The cells were fixed and then stained for one hour with either rhodamine-labeled DEC2 protein, DEC2-Rhod, or DEC2-coated liposomes. A & B: DEC2-Rhod. C & D: DEC2-AmB-LL. Cells were imaged at 20X, and differential interference contrast images (DIC, panels A and C) and combined DIC and red fluorescence images (panels B and D) were captured. Because cells were highly dispersed and we wanted to show several example cells on one plate, these images are composites made from images of cells taken from separate imaging fields and placed next to each other (see dashed outlines of cells moved into a common field). Images are representative of 90% of the fungal cell colonies examined.

[0048] Figures 19A-I show that sDectin-2-coated DEC2-AmB-LL binds to Candida albicans, Cryptococcus neoformans, and A. fumigatus cells at an efficiency one to two orders of magnitude higher than the control AmB-LL. Dense fields of fixed fungal cells were incubated for 1 hour with a 1:200 dilution of liposomes in liposome dilution buffer LDB2 (e.g., 0.5 μg sDectin-2 / 100 μL). Unbound liposomes were washed away after one hour. Multiple fields of red fluorescence images were captured at 20X, and the area of ​​fluorescence was estimated in Image J. Examples of captured images are shown to the right of the bar graph. A, B, and C: C. albicans, D, E, and F: C. Cryptococcus neoformans. G, H, and I: A. fumigatus. In A, D, and G, standard errors from the mean are given, and fold differences and p values ​​are indicated to distinguish binding of DEC2-AmB-LL from binding of AmB-LL.

[0049] Figure 20A-H shows the specificity, stability, and rate of DEC2-AmB-LL binding. A, B, and C: Binding specificity. DEC-AmB-LL labeling of C. albicans was inhibited by soluble yeast mannan, but not by sucrose or laminarin. Each polysaccharide was added at 10 mg / mL during a 1-hour staining procedure. D, E, and F: Binding stability. Figure 20A-CFigure 2 Data from the 100 μg sDectin-2 assay. Plates of Candida albicans cells stained with DEC2-AmB-LL and control liposomes were left in PBS, stored in the dark for 2 months, re-photographed, and the area of ​​liposome binding re-quantified. G&H: Binding rate. Mature cultures of C. albicans consisting of a few pseudohyphae and hyphae grown in VMM + 20% FBS on the surface of a 24-well microtiter plate were fixed, blocked, and treated with DEC2-AmB-LL for the indicated times. In all three experiments, DEC2-AmB-LL was diluted 1:200 w / v with LDB2 (0.5 μg in 100 μL sDectin-2) and washed 4 times with LDB2. For each time point, multiple red fluorescent images were taken at 20X magnification on an inverted fluorescence microscope, and the average area of ​​red fluorescent liposome staining was estimated. The standard error of the mean is shown in A, D, G, and H. In A and D, the numerical mean and number of fields examined are indicated above each bar and on the vertical axis. In A and D, the fold difference and p-value of the performance of DEC-AmB-LL relative to mannan inhibition (A) or relative to AmB-LL (D) are indicated. These results are representative of two biological replicates.

[0050] Figure 21A-D shows the inhibition and killing of Candida albicans (C.), Cryptococcus neoformans (C. neoformans), and Aspergillus fumigatus (A. fumigatus) by sDectin-2-coated amphotericin B-loaded liposomes. A: C. albicans with DEC2-AmB-LL. Cells at the pseudohyphae and early hyphal stages were grown in 96-well polystyrene microtiter plates in RPMI medium + 0.5% BSA. Cells were treated with liposomes delivering 1.0, 0.5, 0.25, and 0.12 μM AmB to the indicated medium for 30 minutes, washed twice with medium, and grown for 16 hours before their metabolic activity was determined using CellTiter-Blue (CTB) reagent. B: C. neoformans with DEC2-AmB-LL. C. neoformans cells were grown in liquid YPD medium + 0.5% BSA with vigorous shaking for 2 hours and treated with liposomes delivering 0.4, 0.2, or 0.1 μM AmB into the medium as indicated for 4 hours or overnight. Cells were diluted, plated on YPD medium, and colony forming units (CFU) were counted from multiple plates. C: A. fumigatus with DEC2-AmB-LL. Conidia were germinated in VMM + glucose + 0.5% BSA in 96-well polystyrene microtiter plates for 9 hours, treated with liposomes delivering 0.5 and 0.25 μM AmB into the medium as indicated for 2 hours, washed twice with medium, grown overnight, and their metabolic activity was determined using CTB reagent in RPMI + 0.5% BSA without phenol red indicator. Control wells were overgrown with hyphae extending from the medium and therefore had low metabolic activity, and even though there were more cells in these wells, the signal produced was lower. D: A. fumigatus with DEC1-AmB-LL. The assay conditions were similar to those in C, except that the liposomes were first diluted into LDB1 buffer (PBS + 0.5% BSA + 1 mM BME) and then diluted into growth medium. For the CTB assays in A, C, and D, the fluorescence background from the medium incubated with the CTB reagent was subtracted. The standard error of all values ​​is shown, and the fold difference and p-value are estimated to compare the performance of AmB-LL with DEC2-AmB-LL. Two or more biological replicates gave similar results.

[0051] Figure 22A-B Two immunosuppressive models of pulmonary aspergillosis and the timeline used to examine the efficacy of Dectin-2-targeted antifungal-loaded liposomes are shown. A: Steroid model. B: Leukopenia model.

[0052] Figure 23 Dectin-targeted antifungal drug-loaded liposomes are shown. Antifungal drug-loaded liposomes coated with carbohydrate recognition domains such as Dectin-1 or Dectin-2 specifically bind to invasive fungal cells and biomembranes and actively deliver antifungal drugs to fungal cells. Targeted liposomes bind to fungal cells (right) more efficiently than non-targeted drug-loaded liposomes (e.g., ) or detergent-solubilized antifungal drugs (left) by several orders of magnitude. Therefore, targeted liposomes have a lower relative affinity for animal cells than non-targeted liposomes and deliver much higher doses of antifungal drugs to fungal cells at lower total drug concentrations. By reducing the effective dose required to kill fungal cells, they are less toxic to animal cells.

[0053] Figure 24A-B Conidia of A. fumigatus strain CEA10 germinate rapidly to establish infection centers in the lungs of CD1 mice. On day 0 (Figure 21), CD1 Swiss mice were given 2×10 6 On day 2, 48 hours after infection, mice were euthanized and manually prepared thin sections (e.g., approximately 0.5 mm thick) of individual lobes of the infected lung were fixed in formalin and stained for fungal chitin with Calcein White for one hour. The cells were then stained with DAPI filter set Ex on a Leica DM6000 compound microscope. A360 / Em A470 Epifluorescence images. A: Most sections show a large infection center with hundreds of stained hyphae. Photographed at 5X magnification. B: The infection center consists of clusters of extended hyphae. No ungerminated conidia were observed. Photographed at 20X magnification.

[0054] Figure 25A-D shows that DEC2-AmB-LL is significantly more effective than AmB-LL in reducing lung fungal burden in a steroid mouse model of immunosuppression-mediated aspergillosis. Figure 21A ) using 2×10 6 fumigatus conidia CEA10 (day 0) and the next day (day 1) were treated with DEC2-AmB-LL or or treated with the buffer used to dilute the liposomes (buffer control) (see Figure 21ATimeline in ). Three mice in each treatment group that survived to day 4 were euthanized, and the fungal burden in each lung was determined by two independent methods. AC: Colony forming units (CFU). A: Bar graph comparing the mean number of CFU per lung for the three treatment groups. B and C are example photographic images of fields of cells examined for the AmB-LL and DEC2-AmB-LL treatment groups, respectively, taken at 20X magnification. CFU was estimated by plating homogenized lung tissue on rich growth medium, incubating the plates overnight, and counting fungal cell microcolonies. Standard errors are represented by lines and whiskers. D: Relative quantity of rDNA. The relative quantity (RQ) of the Aspergillus fumigatus (A. fumigatus) rDNA intergenic spacer (IGS) was determined by performing qPCR on parallel samples of lung homogenate from the same three groups of three lungs as those determined in Figure 24A. Data were normalized to the level of A. fumigatus rDNA in the one buffer control mouse with the highest level (ie, RQ was set to 1.0 for this mouse).

[0055] Figure 26A-B DEC2-AmB-LL was significantly more effective than AmB-LL in reducing lung fungal burden in a leukopenic mouse model of immunosuppression-mediated aspergillosis. Figure 21B ) using 5×10 5 fumigatus conidia CEA10 (day 0) and the next day the mice were infected with DEC2-AmB-LL or or treated with the buffer used to dilute the liposomes (control) (see Figure 21B Timeline in ). Three mice per treatment group were euthanized, and the fungal burden per lung was determined by two independent methods. A: Colony forming units (CFU) of A. fumigatus. Bar graph comparing the mean number of CFU per lung for the three treatment groups. Standard errors are represented by lines and whiskers. B: Relative quantity of fungal cell rDNA. The relative quantity (RQ) of the A. fumigatus rDNA intergenic spacer (IGS) was determined by qPCR on replicate samples of lung homogenate from the same three groups of three lungs as determined in A. The data were normalized to the level of A. fumigatus rDNA in one control mouse with the highest level (i.e., the mouse with an RQ of 1.0).

[0056] Figure 26A-BDEC2-AmB-LL was shown to be two orders of magnitude more effective than AmB-LL in reducing lung fungal burden in a leukopenic mouse model of immunosuppression-mediated aspergillosis. Figure 21B ) using 5×10 5 fumigatus conidia CEA10 (day 0) and the next day (day 1) were treated with DEC2-AmB-LL or or treated with the buffer used to dilute the liposomes (buffer control) (see Figure 21B Timeline in ). Three mice per treatment group were euthanized, and the fungal burden per lung was determined by two independent methods. A: Colony forming units (CFU) of A. fumigatus. Bar graph comparing the mean number of CFU per lung for the three treatment groups. Standard errors are represented by lines and whiskers. B: Relative quantity of fungal cell rDNA. The relative quantity (RQ) of the A. fumigatus rDNA intergenic spacer (IGS) was determined by qPCR on replicate samples of lung homogenate from the same three groups of three lungs as determined in A. The data were normalized to the level of A. fumigatus rDNA in one control mouse with the highest level (i.e., the mouse with an RQ of 1.0).

[0057] Figure 27A -D shows the DNA coding and protein sequences of Dectin-2 Venus fusions used for BiFC detection of fungal mannans. A and C: DNA sequences of MmDEC2VyN and MmDEC2VC, respectively. B and D: Protein sequences of DEC2-VyN and DEC2-VC, respectively.

[0058] A: Codon-optimized DNA sequence of MmDEC2VyN expressed in pET-45B (SEQ ID NO: 17). Length: 577 bp. The 9-codon vector pET-45b+ sequence is boxed (with the start codon underlined). The cloning sites KpnI (GGTACC) (SEQ ID NO: 23) and PacI (TTAATTAA) (SEQ ID NO: 21) are underlined, respectively. Codons for the Gly, Ser (G, S) flexible linker residues are shown in bold and the reactive Lys (K) residue AAG is shown in italics. The sDectin-2 sequence from the mouse Dectin 2 gene CLEC6A, codon-optimized for E. coli expression, is shown in plain text, followed by a 15-residue Gly-rich residue. The flexible spacer region of ser is followed by the 465-nucleotide coding sequence of VenusVyNT154M (the N-terminal half of the mutant Venus protein modified from AKA95335) shown in lowercase letters. One Ala (A) codon, GCT, and two stop codons, TAA and TTA, are underlined (with the stop codon shown in bold). The alternative gene name: MmsDectin2lyshis, length: 1084 bp, minus 7 bp for the stop and following restriction sites (i.e., 1077 bp), encoding a 359-residue protein. However, the shorter 1057 bp version ordered from GenScript begins at the Kpn1 site, GGTACC (SEQ ID NO: 23), for subcloning into pET-45b+.

[0059] B: Final DEC2-VyN protein (SEQ ID NO: 18) being synthesized. The N-terminal amino acid and (His)6(HHHHHH) (SEQ ID NO: 22) affinity tag from pET-45B+ are boxed, the GlySer (GS) flexible linker residue and reactive Lys (K) residue are shown in bold (with Lysine in italics), and the 166 mouse sDecetin-1 amino acid residues are shown in plain text, followed by a 15-residue GlySer-rich flexible spacer, followed by Venus residues 1-155 with a T154M mutation, ending with the C-terminal Ala residue (A) shown in bold, whose codon is used to place a stop codon and PacI site in frame. A total of 359 amino acids, MW 40,198 g / mol. pI 6.04. OD2801.940 / mg / mL.

[0060] C: MmDEC2VC, also codon-optimized DNA sequence of MmDEC2VC expressed in pET-45B (SEQ ID NO: 19). Length: 577 bp, the 9-codon vector pET-45b+ sequence is boxed (with the start codon underlined), the cloning sites KpnI (GGTACC) (SEQ ID NO: 23) and PacI (TTAATTAA) (SEQ ID NO: 21) are underlined, respectively, the codons for the Gly, Ser (G, S) flexible linker residues are shown in bold and the reactive Lys (K) residue AAG is shown in italics, the sDectin-2 sequence from the mouse Dectin 2 gene CLEC6A, codon-optimized for E. coli expression, is shown in plain text, followed by a 15-residue Gly-rich residue. The flexible spacer region of ser is followed by the 252-nucleotide coding sequence for the C-terminal amino acid sequence of Venus (aa residues 155 to 238, modified from AKA95335) in lowercase letters. One Ala (A) codon, GCT, and two stop codons, TAA and TTA, are underlined (the stop codon is shown in bold). Alternative gene name: MmsDectin2lyshis, length: 871 bp, minus 7 bp for the stop codon and restriction site, encoding a 288-residue protein. However, a shorter 844 bp version starting at the Kpn1 site, GGTACC (SEQ ID NO: 23), can be ordered from Genscript for subcloning into pET-45b+.

[0061] D: Final DEC2-VC protein being synthesized (SEQ ID NO: 20). The C-terminal amino acid and (His)6(HHHHHH) (SEQ ID NO: 22) affinity tag from pET-45B+ are boxed, the GlySer (GS) flexible linker residue and reactive Lys (K) residue are shown in bold (with Lys in italics), and the 166 mouse sDecetin-2 amino acid residues are shown in plain text, followed by a 15-residue GlySer-rich flexible spacer, Venus residues 155-238, and ending with the added C-terminal Ala residue (A) shown in bold. The codon for this Ala residue is used to place a stop codon and PacI site in frame. A total of 288 amino acids, MW 32,098 g / mol. PI 6.16, OD280 2.02 OD / mg / mL.

[0062] Figures 28A and B show the protein design and model of liposomes coated with Dectin-2 fused to a complementary fragment of the Venus protein for the detection of fungal mannans containing polysaccharides. A. Diagnostic liposome model employed. Liposomes coated with sDectin-2 monomers (blue) fused to the N-terminal fragment of the green fluorescent protein Venus VyN155 (e.g., DEC2-VyN) and liposomes coated with sDectin-2 monomers fused to the C-terminal fragment of Venus VC (DEC2-VC) recognize and bind to fungal mannans to form sDectin-2 dimers, resulting in the production of assembled Venus proteins and a strong green bimolecular fluorescence complementation (BiFC) signal. These fungal diagnostic liposomes only generate a green fluorescent signal when exposed to mannans in the fungal cell wall, the exopolysaccharide matrix, biofilms, or released soluble polysaccharides containing mannans. Both DEC2-VyN and DEC2-VC were conjugated to the lipid carrier DSPE-PEG and incorporated into the liposome membrane. DEC2-conjugated monomers were incorporated into liposomes such that the total DEC2 protein and liposome lipids were at a molar ratio of 1:100, or with 1 mole percent of DEC2. Based on this molar ratio and the surface area of ​​100 nm diameter liposomes and published data, the total DEC2 protein and liposome lipids were 1:100, respectively. 6 nm 2 Lipid bilayer 5×10 6 Estimated number of lipid molecules 78 , we calculated that there are approximately 1,500 DEC2 monomers (i.e., 750 DEC2-VyN and 750 DEC2-VC fusion proteins) in each DEC2-BiFC reagent liposome. See Table 2. These liposomes can be easily attached to an insoluble matrix by, for example, biotin-streptavidin binding (right side of the figure). In this configuration, the DEC2-BiFC reagent should detect very low concentrations of fungal mannan released into serum using standard fluorescence instrumentation. B. Design of Dectin-2 Venus fusion proteins. Two separate Dectin-2 fusion proteins were expressed in Escherichia coli. One was fused to the N-terminus of the mutant Venus protein VyN and the other was fused to the C-terminal fragment of the Venus protein VC. These two well-characterized complementary fragments are known to assemble and produce a BiFC signal (i.e., DEC2 dimer in this article) when they come into proximity due to the assembly of interacting carrier proteins. Flexible (gly ser) of different lengths n The Gly spacer separates the functional domains to allow a degree of independence of movement and function.

[0063] Figure 29DEC2-BiFC reagent generates a mannan-specific Venus green fluorescence signal. Each polysaccharide was added to a single column of a 96-well microtiter plate at 1 mg / mL during a 2-hour incubation with DEC2-BiFC reagent liposomes at 23°C. The average background signal from incubations with glucans was subtracted from all other samples. Fold differences are indicated between the expected target yeast α-mannan and fungal β-glucans and sucrose. Standard errors are indicated by lines and whiskers.

[0064] Figure 30A-J DEC2-BiFC reagent liposomes are shown to produce an A. fumigatus cell-dependent green fluorescence signal. 4,500 A. fumigatus conidia (CEA10 strain) were germinated and grown for 72 hours at 37°C in VMM + 1% glucose on lysine-coated 24-well microtiter plates, fixed in formalin, and washed into LDB2 buffer. AH: Cells were incubated with liposomal DEC2-BiFC reagent diluted 1:100 in liposomal dilution buffer LDB2. The final DEC2 protein concentration was approximately 1 ug / 100 uL. Fungal cell colonies were photographed in pairs at 20X magnification. Paired bright field images and merged fluorescence images are shown. I and J images showing control cells incubated with dilution buffer and the red channel are simplified to show negligible fluorescence background from the green channel. A, C, E, G, and I on the left are bright-field images of Aspergillus fumigatus cell colonies. B, D, F, H, and J on the right are bright-field images (red) merged with the Venus green fluorescent protein image from the adjacent bright-field image on the left. The white arrow in 30B indicates rare hyphae that do not produce a signal. DETAILED DESCRIPTION

[0065] The following description lists various aspects and embodiments of the compositions and methods of the present invention. Specific embodiments are not intended to limit the scope of the compositions and methods. Instead, the embodiments merely provide non-limiting examples that are at least included within the scope of the disclosed compositions and methods.

[0066] Globally, more than 300 million people suffer from fungal infections. Some fungal diseases are acute and serious. Other fungal infections are recurrent, and some are chronic. Each year, there are approximately 200,000, 400,000 and 1,000,000 cases of aspergillosis, candidiasis and cryptococcosis, respectively, with an alarmingly high mortality rate. Skin fungal infections and dermatophytes are the most common fungal infections. It is estimated that 4% to 10% of the world's population (e.g., more than 300 million people) suffer from fungal infections of the feet (athlete's foot infection, Tinapedia), especially toenail infections (onychomycosis), which may be disabling.

[0067] Aspergillosis is caused by Aspergillus fumigatus and related Aspergillus species. Patients at greatest risk for life-threatening aspergillosis have weakened immune systems, such as those caused by stem cell transplantation or organ transplantation, or have various lung diseases, including tuberculosis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, or asthma. In immunocompromised patients, aspergillosis is the second most common fungal infection after candidiasis. The additional costs associated with treating invasive aspergillosis are estimated to be $40,000 per child and $10,000 per adult. Patients with aspergillosis are treated with antifungal drugs, such as amphotericin B, itraconazole, voriconazole, fluconazole, and the like. However, even with antifungal therapy, the one-year survival rate for immunocompromised patients with aspergillosis is only 25% to 60%. In addition, all known antifungal agents for the treatment of aspergillosis are highly toxic to human cells (Allen et al., Antifungal agents for the treatment of systemic fungal infections in children. Pediatrics & Child Health 15: 603-608 (2010)). Targeted liposomes are provided herein that can improve antifungal drug delivery and enhance the therapeutic efficacy of antifungal agents against a wide range of fungal pathogens and / or reduce the toxicity of antifungal agents when administered to a subject.

[0068] Nanoparticles

[0069] Provided herein are nanoparticles for drug delivery to fungal cells. As used in the full text, nanoparticles can be, but are not limited to, lipid nanoparticles, for example, liposomes or non-liposomal lipid nanoparticles (for example, lipid nanoparticles (LNP) with a non-aqueous core), dendrimers, polymer micelles, nanocapsules or nanospheres, to name a few. For example, provided herein are nanoparticles, for example liposomes, which comprise antifungal agents and targeting molecules for the target antigen in conjunction with the fungal cells, wherein the targeting molecules are incorporated into the outer surface of the nanoparticles, and the antifungal agents are encapsulated in the nanoparticles. The nanoparticles of the drug-loaded materials of various compositions can be targeted to the fungal cells for targeting molecules for making nanoparticles (for example liposomes) targeted to fungi as described herein. Other examples include, but are not limited to, iron oxide nanoparticles, polysaccharide gel nanoparticles and silicon dioxide nanoparticles.

[0070] As used herein, the term liposome refers to an aqueous or aqueous buffer compartment surrounded by at least one lipid bilayer. Liposome can carry an aqueous solution, compound, drug or other substance in a compartment (i.e., an inner cavity or space) surrounded by at least one lipid bilayer. The size of the liposome, i.e., diameter, can vary. For example, the liposome can have an approximately 1000 nanometer (nm) or smaller size. For example, the liposome particle can have an approximately 50nm to approximately 1000nm, approximately 50nm to approximately 900nm, approximately 50nm to approximately 800nm, approximately 50nm to approximately 700nm, approximately 50nm to approximately 600nm, approximately 50nm to approximately 500nm, approximately 50nm to approximately 400nm, approximately 50nm to approximately 300nm, approximately 50nm to approximately 200nm or approximately 50nm to approximately 100nm. Liposomes include liposomes comprising a compartment for encapsulating a kind of agent (e.g., antifungal), liposomes comprising a targeting molecule that is attached to or incorporated into the liposome exterior, and liposomes comprising the antifungal of encapsulation. An encapsulated antifungal agent is an antifungal agent that is completely or partially located in the interior space of the liposome. For example, in any liposome described herein, at least about 75%, 80%, 85%, 90%, 95% or 99% of the antifungal agent is incorporated into the interior space of the liposome or into the lipid bilayer of the liposome.

[0071] Any of the nanoparticles described herein, such as liposomes, can contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 molar or higher percentage of the antifungal agent relative to the lipid. In other words, the nanoparticle can comprise a molar ratio of 1:100, 2:100, 3:100, 4:100, 5:100, 6:100, 7:100, 8:100, 9:100, 10:100, 11:100, 12:100, 13:100, 14:100, 15:100, 16:100, 17:100, 18:100, 19:100, 20:100 or higher of the antifungal agent to the liposome lipid. As used herein, a molar ratio is the ratio between the amounts (moles) of two components, such as the ratio between the moles of targeting molecule and the moles of lipid (moles of targeting molecule:moles of lipid) or the ratio between the moles of antifungal agent and the moles of lipid (moles of antifungal agent:moles of lipid). Similarly, the nanoparticles can comprise a molar ratio of targeting protein to liposomal lipid of 0.002:100, 0.05:100, 0.1:100, 0.5:100, 1:100, 2:100, 3:100, 4:100, 5:100, 10:100, 15:100, 20:100, 25:100 or more.

[0072] Also provided are multimers of two or more of any of the liposomes described herein. For example, a multimer of liposomes can contain from about 2 to about 1×10 14 (100 trillion) liposomes. For example, a multisome can have at least 100, 250, 500, 750, 1000, 5000, 10,000, 25,000, 50,000, 100,000, 500,000, 1 million or more liposomes. Liposomes can be prepared by any suitable method known to those skilled in the art or later discovered. In general, liposomes can be prepared by thin film hydration techniques followed by several freeze-thaw cycles. Liposomal suspensions can also be prepared according to methods known to those skilled in the art. Akbarzadeh et al. (“Liposome: classification, preparation and applications,” Nanoscale Res. Lett. 8(1):102(2013)) describe exemplary methods for preparing liposomes, which is incorporated herein by reference in its entirety.

[0073] In general, a variety of lipid compositions can be used to make liposomes. These lipids include neutral lipids that exist in an uncharged form or a neutral zwitterionic form at physiological pH. Such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. Synthetic derivatives of any lipid described herein can also be used to make lipid nanoparticles. Lipid nanoparticles can also include sterols, such as cholesterol. Lipid nanoparticles can also include cationic lipids that have a net positive charge at approximately physiological pH. Such cationic lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl-N,N,N-triethylammonium chloride (DOTMA); N,N-distearoyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 1,2-dioleyloxy-3-trimethylaminopropane chloride salt (DOTAP·Cl); 3-β-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Cho l”), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-trifluoroacetate (“DOSPA”), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA) and N-(1,2-dimyristoyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). Anionic lipids are also suitable for use in the lipid nanoparticles described herein. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-lauroylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups attached to neutral lipids.

[0074] In some examples, the liposomes include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine, distearoylphosphatidylcholine (DSPC), dilinoleoylphosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol (DSPE-PEG), sphingomyelin, cholesterol, or any combination thereof. In some examples, PEG can be PEG-molecular weight (MW500) to PEG-MW20000. In addition to the components of the liposomes described herein, any of the lipids described herein can be conjugated to a targeting molecule or fragment thereof that binds to an antigen on a fungal cell. In some examples, PEGylated versions of any of the lipids described herein can be conjugated to targeting molecules or fragments thereof that bind to antigens on fungal cells (eg, fungal cell wall antigens or fungal cell exopolysaccharide matrix antigens).

[0075] As used in the full text, targeting molecules have binding affinity to the antigen on the fungal cell, optionally with a molecule of specific binding affinity, and can include but are not limited to antibodies, polypeptides, peptides, aptamers or small molecules. As used in the full text, the antigen on the fungal cell or the target fungal cell antigen can be any antigen associated with the fungal cell at any stage of the fungal cell cycle. For example, but not limited to, the antigen can be an antigen associated with the fungal cell (for example, an antigen embedded in the fungal cell wall, an antigen attached to the fungal cell wall or a fungal cell surface antigen). The antigen associated with the fungal cell can also be directly or indirectly combined with the fungal cell, for example, directly or indirectly combined with the fungal cell wall. Antigen can also be an antigen of the fungal extracellular polysaccharide matrix (for example, biofilm) produced by the fungal cell or associated with the fungal cell. In some instances, the extracellular polysaccharide matrix is ​​attached to or is bound to the fungal cell or fungal cell group. It should be understood that the extracellular polysaccharide matrix associated with the fungal cell can but not necessarily be produced by the fungal cell or fungal cell group associated with it. As used in the full text, the antigen can be but not limited to protein, lipid or carbohydrate.

[0076] As used throughout, "polypeptide," "protein," and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the term encompasses amino acid chains of any length in which the amino acid residues are linked by covalent peptide bonds, including full-length proteins.

[0077] As used throughout the text, the term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0078] In each case of listing a specific nucleic acid or polypeptide sequence, embodiments are also provided comprising a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 99%) identity to the sequence recited. Sequence identity and similarity are defined as the percentage of amino acid residues (i.e., identical residues) in the candidate sequence that are identical to the starting amino acid residue after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percentage of sequence identity. For example, provided herein are polypeptides and nucleic acid sequences having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 99%) identity to SEQ ID NOs: 1-20. Polypeptides and nucleic acid sequences that do not include the histidine tags and / or linker sequences listed in SEQ ID NOs: 1-20 are also provided. Also provided herein are polypeptides and nucleic acid sequences that are at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 99%) identical to the polypeptides and nucleic acid sequences excluding the histidine tags and / or linker sequences listed in SEQ ID NOs: 1-20. Also provided are nucleic acids encoding the polypeptides described herein. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), the method of similarity of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0079] Any polypeptide disclosed herein may comprise one or more conservative amino acid substitutions. As non-limiting examples, the following list summarizes possible substitutions that may generally be made without significantly altering the biological activity of the corresponding variant:

[0080] 1) Alanine (A), serine (S), threonine (T), valine (V), glycine (G) and proline (P);

[0081] 2) Aspartic acid (D), glutamic acid (E);

[0082] 3) Asparagine (N), glutamine (Q);

[0083] 4) Arginine (R), Lysine (K), Histidine (H);

[0084] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V) and

[0085] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0086] See also Creighton, Proteins, WH Freeman and Co. (1984).

[0087] When making such changes / substitutions, the hydropathic index of amino acids may also be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on proteins is generally understood in the art (Kyte and Doolittle; (1982) J Mol Biol. 157(1):105-32). It is generally recognized that the relative hydropathicity of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).

[0088] Any polypeptide provided herein, such as soluble Dectin-1, Dectin-2 or Dectin-3 monomers, can comprise N-terminal or C-terminal deletions or truncations. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids can be deleted from the N-terminal or C-terminal of any polypeptide provided herein, but still retain at least one function, such as dimerization with another soluble Dectin monomer. As shown in the accompanying drawings, soluble Dectin monomer polypeptide sequences are shown in plain text. As described above, these sequences that do not include histidine tags or linker sequences can be truncated by deleting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids from the N-terminal and / or C-terminal of the polypeptide.

[0089] Also provided are compositions comprising any of the polypeptides described herein. For example, provided herein are compositions comprising a polypeptide comprising a soluble Dectin-1, Dectin-2, or Dectin-3 monomeric polypeptide or a fragment thereof. Optionally, the polypeptide can comprise or consist of amino acids 23-199 of SEQ ID NO:2, amino acids 23-189 of SEQ ID NO:4, amino acids 23-100 of SEQ ID NO:6, amino acids 35-214 of SEQ ID NO:8, amino acids 36-203 of SEQ ID NO:10, or amino acids 35-207 of SEQ ID NO:12. Also provided are fragments of a polypeptide comprising or consisting of amino acids 23-199 of SEQ ID NO: 2, amino acids 23-189 of SEQ ID NO: 4, amino acids 23-100 of SEQ ID NO: 6, amino acids 35-214 of SEQ ID NO: 8, amino acids 36-203 of SEQ ID NO: 10, or amino acids 35-207 of SEQ ID NO: 12. By way of example, and not limitation, also provided are fragments of a polypeptide comprising amino acids 23-199 of SEQ ID NO: 2, amino acids 23-189 of SEQ ID NO: 4, amino acids 23-100 of SEQ ID NO: 6, amino acids 35-214 of SEQ ID NO: 8, amino acids 36-203 of SEQ ID NO: 10, or amino acids 35-207 of SEQ ID NO: 12 that contain a deletion of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids at the N-terminus or C-terminus. Optionally, the polypeptide is connected or conjugated to a fluorescent moiety, such as, but not limited to, rhodamine. As described in the Examples section, the composition can include a buffer, such as, for example, a refolding buffer containing about 0.5M to about 1.5M L-arginine. For example, but not limited to, the composition can include a pH value of 7.2 containing the following buffer: between about 0.05 and 0.15M NaH2PO4, between about 10mM and 20mM triethanolamine, between about 0.5M and 1.5M L-arginine, between about 50 and 200mM NaCl, between about 2.5mM to 7.5mM EDTA, and between about 0.25 to 7.5mM BME. Optionally, the composition can include a pH value of 7.2 containing the following buffer: about 0.1M NaH2PO4, about 10mM triethanolamine, about 1ML-arginine, about 100mM NaCl, about 5mMEDTA, and 5mM BME. Also provided are kits comprising any of the compositions. Optionally, the kit comprises a denaturing buffer or a reducing buffer, for example, a reducing buffer comprising beta-mercaptoethanol.Also provided are kits comprising any of the liposomes described herein.

[0090] As used throughout, the term "antibody" includes, but is not limited to, nanometer bodies, complete immunoglobulins (i.e., complete antibodies) of any class (including polyclonal and monoclonal antibodies) and antibody fragments that retain the ability to bind their specific antigens. Also useful are conjugates of antibody fragments and antigen-binding proteins (single-chain antibodies), such as, for example, described in U.S. Patent No. 4,704,692, the contents of which are incorporated herein by reference in their entirety.

[0091] As used throughout, an aptamer is an oligonucleotide (single-stranded DNA or single-stranded RNA) or peptide molecule that selectively binds to a target antigen. See, for example, Lakhin et al., "Aptamers: Problems, Solutions and Prospects," Acta Naturae 5(4):34-43 (2013); and Reverdatto et al., "Peptide aptamers: development and applications," Curr. Top Med. Chem. 15(12):1082-101 (2015), which are incorporated herein by reference in their entirety.

[0092] As used herein, the terms "specifically bind" or "selectively bind" refer to binding that is measurably distinct from nonspecific or nonselective interactions. Specific binding can be measured, for example, by determining the binding of a molecule to a target antigen compared to the binding of a control molecule. Specific binding can be determined by competition with a control molecule similar to the target antigen, such as an excess of unlabeled target antigen. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target antigen.

[0093] Optionally, from two targeting molecules to about 10,000 targeting molecules can be incorporated into the liposomes provided herein. For example, about 5 to about 100, about 5 to about 200, about 5 to about 300, about 5 to about 400, about 5 to about 500, about 5 to about 600, about 5 to about 700, about 5 to about 800, about 5 to about 900, about 5 to about 1000, about 5 to about 1100, about 5 to about 1200, about 5 to about 1300, about 5 to about 1400, about 5 to about 1500, about 5 to about 1600, about 5 to about 1700, about 5 to about 1800, about 5 to about 1900, about 5 to about 2000, about 5 to about 2250, about 5 to about 2500, About 5 to about 3000 targeting molecules, about 5 to about 3500, about 5 to about 4000 targeting molecules, about 5 to about 4500 targeting molecules, about 5 to about 5000 targeting molecules, about 5 to about 5500 targeting molecules, about 5 to about 6000 targeting molecules, about 5 to about 6500 targeting molecules, about 5 to about 7000 targeting molecules, about 5 to about 7500 targeting molecules, about 5 to about 8000 targeting molecules, about 5 to about 8500 targeting molecules, about 5 to about 9000 targeting molecules, about 5 to about 9500 targeting molecules, or about 5 to about 10,000 targeting molecules are incorporated into one or more liposomes described herein. In some examples, about 2 molecules to about 3,000 targeting molecules are incorporated into nanoparticles having a diameter of 100 nm. One skilled in the art would not know how to calculate the number of targeting molecules that can be incorporated into a nanoparticle, for example between about 2 and 10,000 targeting molecules or more, depending on the size of the nanoparticle.

[0094] As used in the full text, the targeting molecule is incorporated into the outer surface of the liposome, meaning that the targeting molecule is incorporated into the outer lipid bilayer of the liposome or is attached to the liposome. Incorporation can occur by inserting or embedding the targeting molecule into the lipid bilayer. Attachment to the liposome can, for example, occur by affinity with the molecule incorporated into the outer lipid bilayer of the liposome. For example, the liposome can be coated with biotin (for example, DSPE-PEG-biotin inserted into the lipid bilayer) and the targeting molecule connected with streptavidin. Alternatively, the targeting molecule can be conjugated to the outer surface of the liposome. The targeting molecule can be conjugated to the liposome by many methods known in the art (for example, Arruebo et al., " Antibody-Conjugated Nanoparticles for Biomedical Applications, " Journal of Nanomaterials vol. 2009, paper ID 439389 (2009)). Liposomes can also be conjugated to targeting molecules through streptavidin / biotin bonds, thiol / maleimide chemistry, azide / alkyne chemistry, tetrazine / cyclooctyne chemistry and other click chemistries. These chemical handles are prepared during or after the phosphoramidite synthesis process. As used herein, the term "click chemistry" refers to a biocompatible reaction primarily intended to bind a selected substrate to a specific biomolecule. Click chemistry reactions are not interfered with by water, produce minimal and non-toxic by-products, and are characterized by a high thermodynamic driving force that can quickly and irreversibly drive it to obtain a high yield of a single reaction product with high reaction specificity.

[0095] The targeting molecule may bind to an antigen on one or more types of fungal cells or fungal cell populations, including but not limited to cells from animal fungal pathogens (e.g., human fungal pathogens) and plant pathogens. Examples of human fungal pathogens include but are not limited to Alternaria sp. alternata), Aspergillus species (such as A. fumigatus), Blastomyces species (such as B. dermatitidis), Candida species (such as A. fumigatus, C. glabrata, C. krusei, C. auris), Coccidioides species (such as C. immitis and C. posadasii), Cryptococcus species (such as C. gattii and C. neoformans), Histoplasma species (such as H. capsulatum); Pneumocystis species (such as P. jirovecii), Sporothrix species (such as S. schenckii), Talaromyces marneffei marneffei) (formerly Penicillium marneffei) and Trichophyton rubrum.

[0096] Examples of plant fungal pathogens include, but are not limited to, Aecidium glycines, Aecidium mori, Alternaria japonica, Alternaria padwickii, Alternaria triticina, Alternaria yaliinficiens, Amylostereumareolatum, Apiognomonia erythrostoma, Arkoola nigra, Balansia oryzae-sativae, Botryosphaeria berengeriana, and Echinococcus spp. Calonectria pseudonaviculata- Boxwood dieback pathogens - Ceratocystis fagacearum, Ceratocystis fagacearum, Chalarafraxinea, Chrysomyxa abietis, Chrysomyx ahimalensis, Chrysomyxa rhododendri, Ciborinia allii, Claviceps fusiformis, Claviceps gigantea, Claviceps sorghi, Claviceps sorghicola, Cronartium flaccidum, Crumenulopsis sororia, Diaporthe vexans), Didymella fabae-Ascochyta fabae, Endophyllum kaernbachii, Exobasidium vexans, Gerwasia imperialis, Gerwasia mayorii, Gerwasia rosae, Gerwasia rubi, Gerwasia variabilis, Goplanadioscoreae, Gymnosporangium miyabei, Gymnosporangium yamadae, Hamaspora acutissima, Hamaspora australis, Hamaspora hashiokai), Hamaspora longissimi, Hamaspora rubi-sieboldii, Hamaspora sinica, Harpophoramaydis, Hemileia vastatrix, Kuehneola japonica, Kuehneola loeseneriana, Lachnellula willkommii, Leptographium wingfieldii, Mainsia rubi, Melampsoracapraearum), Melampsora larici-epitea, Melampsora larici-pentandrae, Melampsora larici-pentandrae, Monilia polystroma, Monilinia fructigena, Ochropsora ariae, Olivetectonae, Ophiostoma longicollum, Peronospora digitalis, Peronospora radii, Phakopsora ampelopsidis, Phakopsora euvitis, Phakopsora meibomiae, Phakopsora truncatula, pachyrhizi), Phomatracheiphila, Phragmidium acuminatum, Phragmidium arcticum, Phragmidium arisanense, Phragmidium assamense, Phragmidium barclayi, Phragmidium bulbosum, Phragmidium butleri, Phragmidium formosanum, Phragmidium griseum, Phragmidium hiratsukanum, Phragmidium kamtschatkae, Phragmidium nambuanum, Phragmidium pauciloculare, Phragmidium rosae-moschatae, Phragmidium rosae-multiflorae, Phragmidiumrosae-rugosae), Phragmidium rubi-thunbergii, Phragmidium yamadanum, Phyllacoramaydis, Pileolaria pistaciae, Pileolaria terebinthi, Plasmopara obducens, Pseudocercospora angolensis, Puccinia agrophila, Puccinia buxi, Puccinia erythropus, Puccinia gladioli, Puccinia glyceriae), Puccinia hemerocallidis, Puccinia horiana, Puccinia akuehnii, Puccinia mccleanii, Puccinia melanocephala, Puccinia miscanthi, Puccinia pittieriana, Puccinia psidii, Puccinia substriata, Puccinia veronicae-longifoliae, Pucciniastrum coryli, Setomelanomma holmii, Sphaceloma poinsettiae, Sporisporium pulverulentum, Sporisorium sacchari, Thecaphorasolani, Thekopsoraareolate, Urocystisagropyri, Uromycesgladioli, Uromyces nyikensis, Uromycestransversalis, and Uromycladiumtepperianum. It should be understood that in addition to detecting, treating, or preventing fungal infections in subjects, any of the methods provided herein can also be used to detect, treat, or prevent fungal infections in plants. For example, but not by way of limitation, fungal infections can be detected, treated, or prevented on the surface or on ground samples or extracts of leaves, stems, roots, petals, sepals, stamens, carpels, and seeds of a plant.

[0097] In some instances, the targeting molecule is a C-type lectin receptor or a fragment thereof. In other instances, the targeting molecule is a lectin binding protein or a fragment thereof. Fragments of the targeting molecules described herein are polypeptides that bind to a target antigen on a fungal cell with the same or different binding affinity as the protein or polypeptide from which the fragment is derived. Examples of C-type lectin receptors that can be used as targeting molecules include Dectin-1 (CLEC7A, mouse GenBank accession number: AAS37670 and human GenBank accession number: NP_922938), Dectin-2 (CLEC6A mouse GenBank accession number: NP_064385 and human GenBank accession number: Q6EIG7), Dectin-3 (CLEC4D mouse GenBank accession number: NP_034949 and human GenBank accession number: NP_034949), and fragments of Dectin-1, Dectin-2, and Dectin-3. Fragments of Dectin-1, Dectin-2, and Dectin-3 that contain amino acid sequences that bind to β-glucans and / or mannans on fungal cells can also be used as targeting molecules. Exemplary targeting molecules include, but are not limited to, SEQ ID NO:2, which contains the amino acid sequence of a mouse Dectin-1 β-glucan binding fragment. SEQ ID NO:2 contains an N-terminal His tag that can be removed. Any nucleic acid construct used to prepare soluble Dectin-1 (e.g., SEQ ID NOs:7-14) can further contain a selective protease cleavage site to remove the N-terminal His tag sequence after expression of the Dectin-1 polypeptide. These include, but are not limited to, peptide protease processing sites recognized by tobacco etch virus (TEV) protease, enteropeptidase, thrombin, factor Xa, and rhinovirus 3C protease. Another exemplary targeting molecule is a soluble Dectin-3 polypeptide comprising SEQ ID NO:6. SEQ ID NO:6 contains amino acids 44 to 219 of mouse Dectin-3. Additional exemplary targeting molecules include soluble human Dectin-1, Dectin-2, and Dectin-3 polypeptides comprising SEQ ID NOs: 8, 10, and 12, respectively. SEQ ID NOs: 8, 10, and 12 comprise amino acids 69 to 248 of human Dectin-1, amino acids 42 to 209 of human Dectin-2, and amino acids 44 to 215 of human Dectin-3, respectively.Also provided are fragments of amino acids 69 to 248 of human Dectin-1, amino acids 42 to 209 of human Dectin-2, and amino acids 44 to 215 of human Dectin-3, for example, fragments of polypeptides comprising or consisting of amino acids 69 to 248 of human Dectin-1, amino acids 42 to 209 of human Dectin-2, and amino acids 44 to 215 of human Dectin-3 that contain a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids at the C-terminus and / or N-terminus, which can be used in any of the described liposomes, polypeptides, or compositions.

[0098] Dectin-1 is a transmembrane receptor expressed in T cells and is encoded by the CLEC7A (C-type lectin domain containing 7A, beta-glucan receptor, GR) gene in mice and humans. Dectin-1 binds to various beta-glucans in the fungal cell wall and is the main receptor that signals the presence of exposed cell wall components on the surface of pathogenic and non-pathogenic fungi across the membrane to stimulate the innate immune response. Although human and mouse Dectin-1 are plasma membrane proteins of 244 and 247 amino acids in length, respectively, there are mRNA splice variants that produce shorter human isoforms. Dectin-1 floats on the membrane as a monomer but binds to beta-glucans as a dimer, as Figure 3 The design of the Dectin-1 targeted liposomes shown in is mimicked. The extracellular C-terminal domain, which is 176 amino acids (20 kDa) in length, can be manipulated alone as soluble sDectin-1 and contains a β-glucan binding domain. β1→3 glucans are structurally distinct classes of polysaccharides, and therefore, sDectin-1 binds to various β-glucans in different ways with Kd affinity constants ranging from 2.6 mM to 2.2 pM. Dectin-1 and fragments thereof having pan-fungal binding activity, for example, β-glucan binding fragments, are exemplary targeting molecules that can be used to kill one or more types of fungal cells, such as, but not limited to, Aspergillus, Candida, and / or Cryptococcus cells.

[0099] Other mammalian proteins with mannan and mannose binding domains that can be used for liposome targeting of fungal polysaccharides are mannose receptor C type 1 (human MRC1, NG_047011), mannose binding protein 2 (MBL2, NG_033955) and C-type lectin domain family 4 member L (CD209, CLEC4L or DC-SIGN, human NG_012167) or fragments thereof. Examples of non-mammalian proteins with fungal carbohydrate binding domains that can be used as targeting molecules include, but are not limited to, bacterial and insect glucan binding proteins CBM11 (e.g., TYP77495 from Paenibacillus methanolicus) and CBM39 (e.g., EZA53410 from Ooceraea biori), bacterial mannan binding proteins CBM27 (e.g., PWV98652 from Paenibacillus cellulosilyticus), CBM35 (e.g., PYE65467 from Paenibacillus sp. OV191), CBM46 (e.g., GBF77546 from Paenibacillus sp. 598K), and MVL (e.g., AZB35797 from Chryseobacterium bernardetii), or fragments thereof.

[0100] Examples of mammalian proteins with chitin-binding domains that can be used as targeting molecules include, but are not limited to, chitinase-1 (CHIT1, human NG_012867), chitinase-3-like-1 (HCGP39, CHI3L1, human NG_013056), chitinase-3-like-2 (CHI3L2, human NM_001025197), chitinase acidic (CHIA, human gene card number GC01P111291), and chitobiase (CTBS, human gene card GC01M084549), or fragments thereof. Examples of non-mammalian proteins with chitin-binding domains that can be used as targeting molecules include, but are not limited to, bacterial CBM5 (e.g., TDX99194 from Lysinibacillus xylanilyticus) and plant and / or fungal CBM18 (e.g., AYU56549 from Verticillium alfalfae) and CBM19 (e.g., XP_018290979 from Phycomyces blakesleeanus NRR1555).

[0101] Antifungal agents that can be incorporated into or encapsulated in the targeted liposomes described herein include, but are not limited to, polyene or azole antifungal agents. Examples of polyene antifungal agents include, but are not limited to, amphotericin B (AmB), candidatin, filipin, hamycin, natamycin, nystatin, hitachimycin, and rimocidin. Examples of azole antifungal agents include, but are not limited to, imidazoles (e.g., bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sukonazole, and tioconazole), triazoles (e.g., albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole, voriconazole), thiazoles (e.g., abafungin), and echinocandins (e.g., caspofungin, micafungin, and anidulafungin).

[0102] Amphotericin B (AmB) is the most commonly used agent for many types of fungal infections, including aspergillosis. Side effects of amphotericin B include neurotoxicity and / or nephrotoxicity and / or hepatotoxicity, and often lead to patient death. AmB is hydrophobic and is embedded in the lipid bilayer of liposomes, such as Figure 3 As shown. Commercial non-targeted spherical AmB-loaded liposomes AmB-LL are commonly referred to as AmBisomes. Compared to the second most commonly used AmB product, deoxycholate detergent-solubilized AmB, AmB-LL penetrates into various organs more effectively, penetrates cell walls and shows reduced toxicity at slightly higher, more effective AmB doses. However, AmB-LL still produces AmB human toxicity, such as nephrotoxicity in 50% of patients. When infected mice are treated with AmB-LL, large fungal cell populations are often retained. This large residual population may be the reason why human patients treated with detergent-solubilized AmB and AmB-LL have a high relapse rate and subsequent mortality rate after treatment. The targeted liposomes provided herein are designed to effectively target fungal cells and / or reduce the toxicity of antifungal agents such as AmB.

[0103] In some examples, the concentration of the antifungal drug is reduced compared to the concentration of the antifungal drug incorporated into or encapsulated in liposomes that do not contain a targeting molecule or fragment thereof incorporated into their outer surface, wherein the targeting molecule binds to an antigen on a fungal cell. By forming protein-coated liposomes (i.e., coating the liposomes with a polypeptide, such as a C-lectin type receptor or fragment thereof), lower concentrations of antifungal agents can be used to treat or prevent fungal infections, thereby reducing the toxicity associated with administering antifungal drugs to a subject. Lower toxicity allows for extended use of targeted antifungal agents over longer periods of time, which will reduce fungal burden beyond the poor reduction currently achieved. Lower toxicity may allow for the prophylactic use of the targeted liposomes described herein, for example, as a nasal spray, to prevent lung infections before they are established.

[0104] In some liposomes, the concentration of the antifungal drug is reduced or decreased by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or any percentage in between. In some examples, the reduction in toxicity is a reduction in renal cell toxicity and / or hepatocellular toxicity in vitro and / or in vivo. In another example, the reduction in AmB concentration in the liposomes is from about 11 molar percent relative to the liposome lipids to about 1 to 10 molar percent relative to the liposome lipids. In some targeted liposomes, the concentration of the antifungal agent relative to the liposome lipid percentage can range from about 1 to about 20 molar percent of the antifungal agent. For example, the concentration of the antifungal agent relative to the liposome lipid percentage can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 molar percent of the antifungal agent.

[0105] In some instances, the targeted liposomes have reduced affinity and / or less toxicity to animal cells (e.g., human cells) compared to liposomes that do not contain a targeting molecule that binds to an antigen on a fungal cell, e.g., a targeting molecule incorporated into the outer surface of the liposome. In some instances, the affinity of the targeted liposomes for fungal cells in the lungs of a subject is higher than the affinity of the targeted liposomes for lung, kidney, or liver cells of the subject. Thus, the liposomes provided herein can be used to deliver an antifungal agent to a subject while minimizing the effect of the antifungal agent on non-fungal cells (e.g., human lung, kidney, or liver cells), thereby reducing the toxicity of the antifungal agent. Any liposome comprising an antifungal agent as described herein can be used to reduce or decrease fungal infection in vitro, ex vivo, or in vivo.

[0106] Method for preparing targeted liposomes

[0107] Provided herein is a method for preparing a multiplicity of liposomes comprising an antifungal agent and a targeting molecule that binds to a target antigen on a fungal cell, wherein the targeting molecule is incorporated into the outer surface of each liposome and the antifungal agent is encapsulated within each liposome, the method comprising the steps of: (a) dissolving the antifungal agent in a solvent at about 60° C. for about 10 minutes to about 30 minutes; (b) encapsulating the antifungal agent in each liposome by mixing the multiplicity of liposomes in suspension with the antifungal agent / solvent solution of step (a) at about 60° C. for about 3 to about 5 hours, or at about 37° C. for about 24-120 hours; and (c) incorporating the targeting molecule into the outer surface of each liposome by contacting the liposomes comprising the encapsulated antifungal agent with the targeting molecule conjugated to the lipid at 60° C. for about 45 minutes to about 90 minutes.

[0108] In the methods for preparing liposomes provided herein, the antifungal agent can be dissolved in any suitable solvent. Depending on the antifungal agent and its properties, one skilled in the art will know how to select an effective solvent for dissolving the antifungal agent. In some instances, the antifungal agent, such as amphotericin B, is dissolved in aqueous DMSO or formamide. Other hydrophobic solvents or amphiphobic solvents may also be used. Optionally, the antifungal agent may be dissolved for about 10 to about 120 minutes. For example, the antifungal agent can be dissolved in about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120 minutes.

[0109] Optionally, the antifungal agent can be dissolved at a temperature of about 55° C. to about 65° C., for example, at about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65° C. Optionally, the antifungal agent can be encapsulated into each liposome by mixing the multimerization of liposomes in suspension with the antifungal agent / solvent solution (dissolved antifungal agent) at a temperature of about 55° C. to about 65° C., for example, at about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65° C., for about 3 to about 5 hours. In another example, the antifungal agent is encapsulated into each liposome by mixing a multiplicity of liposomes in suspension with an antifungal agent / solvent solution (dissolved antifungal agent) at about 35° C. to about 45° C., e.g., at 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45° C. for about 24-120 hours. In another example, the antifungal agent is encapsulated into each liposome by mixing a multiplicity of liposomes in suspension with an antifungal agent / solvent solution (dissolved antifungal agent) at about 35° C. to about 45° C., e.g., at 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45° C. for about 72-100 hours.

[0110] Optionally, in any of the methods for preparing liposomes described herein, a C-type lectin receptor or fragment thereof selected from the group consisting of Dectin-1, Dectin-2, and Dectin 3, or binding fragments thereof, is maintained in a refolding buffer comprising arginine and denatured prior to incorporation into the liposomes. Optionally, any of the methods for preparing liposomes described herein may further comprise storing the liposomes comprising the antifungal agent and the targeting molecule in a refolding buffer comprising arginine. Optionally, the refolding buffer may comprise from about 0.5 to about 1.5 M arginine. Optionally, the refolding buffer may comprise about 0.1 M NaH2PO4, about 10 mM triethanolamine, about 1 M L-arginine, about 100 mM NaCl, about 5 mM EDTA, and 5 mM BME, pH 7.2.

[0111] In some methods, the targeting molecule is conjugated to a lipid. The lipid conjugated to the targeting molecule can be a PEGylated or non-PEGylated lipid. Examples of lipids that can be conjugated to targeting molecules and examples of antifungal agents that can be incorporated into targeted liposomes are described above. In some examples, the targeting molecule incorporated into the outer surface of each liposome is a C-type lectin receptor, e.g., Dectin-1, Dectin-2, Dectin-3, or a fragment thereof, a chitin binding protein, an exopolysaccharide binding protein, or a fragment thereof, or an antibody.

[0112] Multimers of targeted liposomes prepared by the described methods can produce multimers containing any number of liposomes, for example, from about two to about 100,000,000 liposomes. It should be understood that during the preparation of targeted liposomes, there may be some liposomes that do not encapsulate the antifungal agent or that do not have the targeting molecule incorporated into their outer surface. Thus, provided herein are multimers of targeted liposomes, wherein at least 70%, 80%, 90%, 95%, 99%, 99.5% or 99.9% of the liposomes contain an encapsulated antifungal agent and a targeting molecule that binds to an antigen on a fungal cell, wherein the targeting molecule is incorporated into the outer surface of the liposomes.

[0113] Fungal infection testing

[0114] Also provided herein are liposomes comprising a targeting molecule and a signal generating molecule in conjunction with a target fungal cell antigen, wherein the targeting molecule is incorporated into the outer surface of the liposome, and the signal generating molecule produces a detectable signal when the targeting molecule is in conjunction with the target fungal cell antigen. In some instances, the signal generating molecule is connected or attached to the targeting molecule. In other instances, the signal generating molecule is incorporated into the outer surface of the liposome or attached to the outer surface of the liposome. These liposomes can be used for in vivo, in vitro or in vitro detection of fungi or fungal infections. Fungal cells or one or more fungal cells, i.e., fungal cell colonies, can be detected. Detectable signals can be detected directly or indirectly. For example, the signal generating molecule can be a fluorescent dye, a label or a probe (e.g., rhodamine, fluorescein, green fluorescent protein, acridine orange, etc.) that is directly detected.

[0115] In some instances, the targeting molecule is linked to a molecule that can be directly detected in vivo using imaging techniques (including but not limited to magnetic resonance imaging, radiography, positron emission tomography (PET), computed tomography (CT) scanning, to name a few). Examples of molecules that can be used to detect fungal infections using in vivo imaging include but are not limited to metalloproteins, ferritin, transferrin, aquaporins, and chemical exchange saturation transfer (CEST) reporters, to name a few. See, for example, Silindir et al., “Liposomes and the irapplications in molecular imaging,” J. Drug Target 20(5):401-415 (2012); and Mukherjee et al., “Biomolecular MRI Reporters: evolution of new mechanisms,” Prog. Nucl. Magn Reson. Spectrosc. 102-103:32-42 (2017)). In another example, the targeting molecule is linked to a primary antibody or a fragment thereof (e.g., an Fc fragment of an antibody) that can be indirectly detected using a secondary antibody. In some instances, the target fungal antigen is located on a fungal cell. In other examples, target fungal antigens such as fungal cell wall components are released from fungal cells into a biological sample.

[0116] In some embodiments, the liposome itself generates a signal upon binding to the fungal cell antigen. This signal can be generated in one or more steps after liposome binding. Examples of single-step signaling systems include Figure 11and 28A, wherein the liposomes may or may not be optionally attached to a solid support. In this embodiment, a subject or biological sample is contacted with liposomes comprising a fusion protein comprising a soluble Dectin (targeting molecule) linked to a C-terminal fragment of a fluorescent protein and a liposome comprising a soluble Dectin (targeting molecule) linked to an N-terminal fragment of a fluorescent protein. Examples of fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP, such as Venus), green fluorescent protein (GFP), and red fluorescent protein (RFP), as well as derivatives of these proteins, such as mutant derivatives. See, for example, Chudakov et al., "Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues," Physiological Reviews 90(3): 1103-1163 (2010); and Specht et al., "A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging," Annual Review of Physiology 79: 93-117 (2017)).

[0117] In some examples, targeting molecules such as soluble Dectin-1, Dectin-2, Dectin-3, or fragments thereof, upon binding to β-glucans or mannans on fungal cells, form dimers on the surface of liposomes. When a liposome having a first targeting molecule linked to an N-terminal fragment of a fluorescent protein and a second targeting molecule linked to a C-terminal fragment of a fluorescent protein on its surface is contacted with a subject with a fungal infection (e.g., the subject's eye, ear, throat, vagina, nasal passages, skin, or nails, to name a few) or a sample from the subject (e.g., urine, blood, serum, tears, sputum, lung lavage fluid, tissue scrapings, or homogenates), the soluble Dectin monomers incorporated into the liposome surface will form dimers upon binding to β-glucans or mannans present on or derived from fungal cells. The first and second targeting molecules can be the same or different.

[0118] Dimer formation affects the interaction between the C-terminal fragment of the fluorescent protein and the N-terminal fragment of the fluorescent protein, i.e., complementation (bimolecular fluorescence complementation (BiFC)), so that the signal generated by this interaction can be detected by fluorescence, for example, by a handheld fluorescent lamp, a fluorescence microscope, a fluorescence microtiter plate reader, a fluorescence flow cytometer, or other fluorescence detection instrument. Schematic model of Dectin-coated liposomes that produce a fluorescent signal when bound to fungal glucan or mannan or the fungus itself is shown in Figure 11 and Figure 28A. This construct enables a single-step assay in which the binding of liposomes to fungal polysaccharides and subsequent signal generation occur in a single clinical step, without the need for wash steps or the addition of additional reagents. See, for example, Kilpatrick et al., "AG Protein-Coupled Receptor Dimer Imaging Assay Reveals Selectively Modified Pharmacology of Neuropeptide Y Y1 / Y5 Receptor Heterodimers," Mol. Pharm. 87:718-732 (2015).

[0119] use Figure 3 Rhodamine fluorescent liposomes and Figure 12 The horseradish peroxidase-linked liposomes in the present invention exemplify exemplary constructs for implementing two-step and three-step diagnostic assays. For both diagnostic assays, excess non-bound diagnostic liposomes are washed away in the second step. The fluorescence of the remaining bound rhodamine-labeled liposomes ( Figure 3 ) can be directly measured as shown in Figures 4, 5 and 6. HRP-coated liposomes ( Figure 12 ) requires the addition of 4-chloro-1-naphthol and peroxide, substrates for HRP enzyme activity, and a third step of incubating them for about 10 to about 30 minutes. The product of the enzyme is a purple precipitate that can be measured spectrophotometrically or under a microscope. This signal is a measure of the amount of bound liposomes and, therefore, the amount of fungal polysaccharide. Other signal-generating enzymes can be used instead of HRP, including but not limited to luciferase (for luminescence), β-glucuronidase and β-galactosidase (for color generation) and protamine (for MRI signals). Multi-step liposomal detection systems for fungal cells and fungal cell components can be constructed using any fungal cell antigen targeting molecule (e.g., antibodies or fragments thereof, aptamers, small molecules, etc.), and Dectin dimerization is not required during the binding process.

[0120] Also provided herein are liposomes comprising targeting molecules that bind to target antigens on fungal cells, wherein the targeting molecules are incorporated into the outer surface of the liposomes, and wherein the targeting molecules are connected or fused to the C-terminal or N-terminal fragment of a fluorescent protein. Therefore, the liposomes comprise a fusion protein that comprises a targeting molecule that binds to a fungal cell antigen and the C-terminal or N-terminal fragment of a fluorescent protein. Multimers of these liposomes are also provided. In some instances, the multimers comprise a first subset of liposomes containing a targeting molecule that is connected to the N-terminal fragment of a fluorescent protein and a second subset of liposomes containing a targeting molecule that is connected to the C-terminal fragment of a fluorescent protein.

[0121] In some examples, the targeting molecules (e.g., soluble Dectin-1, Dectin-2, or fragments thereof) form dimers upon binding to β-glucans or mannans on fungal cells. When liposomes having a first targeting molecule linked to an N-terminal fragment of a fluorescent protein and a second targeting molecule linked to a C-terminal fragment of a fluorescent protein on their surface are contacted with a subject infected with a fungus (e.g., the subject's eye, ear, throat, vagina, nasal passages, skin, or nails, to name a few) or a sample from the subject (e.g., urine, blood, serum, tears, sputum, lung lavage fluid, tissue scrapings, or homogenates), the soluble Dectin-1, Dectin-2, or Dectin-3 monomers incorporated into the liposome surface will form dimers upon binding to β-glucans or mannans on any fungal cells present in the subject or in the subject's sample, as well as any soluble β-glucans or mannans released from these fungi. Dimer formation will affect the interaction, i.e., complementation, between the C-terminal fragment of the fluorescent protein and the N-terminal fragment of the fluorescent protein (e.g., bimolecular fluorescence complementation (BiFC)), so that the signal generated by this interaction can be detected by fluorescence, for example, by using a fluorescence microscope, a fluorescence microtiter plate reader, a fluorescence flow cytometer, or other fluorescence detection instrument. See, for example, Kilpatrick et al., “AG Protein-Coupled Receptor Dimer Imaging Assay Reveals Selectively Modified Pharmacology of Neuropeptide Y Y1 / Y5 Receptor Heterodimers,” Mol. Pharm. 87: 718-732 (2015)). A schematic model of Dectin-coated liposomes that produce a fluorescent signal when bound to fungal glucans or mannans or the fungus itself is shown in Figure 11 and Figure 28A. This construct enables a single-step assay in which the binding of liposomes to the fungal polysaccharide and subsequent signal generation all occur in a single clinical step, without the need for wash steps or the addition of additional reagents. See, for example, Kilpatrick et al., "AG Protein-Coupled Receptor Dimer Imaging Assay Reveals Selectively Modified Pharmacology of Neuropeptide Y Y1 / Y5 Receptor Heterodimers," Mol. Pharm. 87:718-732 (2015).

[0122] Further provided herein are fusion polypeptides comprising a targeting molecule that binds to a target fungal cell antigen and the N-terminal or C-terminal portion of a fluorescent polypeptide. These fusion polypeptides can be used to detect fungal infections. In some instances, the targeting molecule is a C-type lectin receptor, an antibody, a fungal cell wall binding protein, a chitin binding protein, or a fragment thereof. In some instances, the targeting molecule is Dectin-1, Dectin-2, Dectin-3, or a fragment thereof. In some instances, the fluorescent protein is yellow fluorescent protein or a derivative thereof.

[0123] Provided herein are methods for detecting a fungal infection in a subject or in a sample from the subject, comprising: a) contacting the subject or the subject's sample with a multiplicity of liposomes, wherein each liposome in the multiplicity comprises a targeting molecule that binds to a target fungal cell antigen and a signal-generating molecule, wherein the targeting molecule is incorporated into the outer surface of the liposome, and the signal-generating molecule generates a detectable signal when the targeting molecule binds to the target fungal cell antigen; and b) detecting the signal, wherein the signal indicates the presence of a fungal infection. This method can be used to detect the presence of a fungal infection in vivo, in vitro, or in vitro. In some instances, the fungal cell antigen is a fungal cell antigen on a cell. In other instances, the fungal cell antigen is a soluble fungal cell antigen, such as β-glucan or mannan in a biological sample. In some instances, the targeting molecule is linked to the signal-generating molecule. In other instances, the signal-generating molecule is incorporated into or attached to the outer surface of the liposome. In some instances, the targeting molecule is linked to a signal-generating enzyme, such as HRP, luciferase, β-glucuronidase, and β-galactosidase. In other examples, the targeting molecule is linked to a fluorescent protein, such as rhodamine, GFP, YFP, RFP, etc. A fragment of a fluorescent protein, for example, the N-terminus or C-terminus of any fluorescent protein, can be linked to the targeting molecule. In other examples, the targeting molecule is linked to an antibody or a fragment thereof.

[0124] Provided herein are methods for detecting a fungal infection in a subject or a sample of a subject, comprising: (a) contacting the subject or the sample of the subject with a multiplicity of liposomes, wherein each liposome in the multiplicity comprises a targeting molecule linked to an N-terminal fragment of a fluorescent protein and a targeting molecule linked to a C-terminal fragment of the fluorescent protein, wherein the targeting molecule binds to a target antigen on a fungal cell, and (b) detecting a fluorescent signal generated by the interaction between the N-terminal and C-terminal fragments of the fluorescent protein, wherein the signal indicates the presence of a fungal infection. In some methods, the fluorescent signal is detected using ultraviolet light or a fluorescence microscope or an instrument for quantitative fluorescence detection of bimolecular fluorescence complementation (i.e., BiFC analysis).

[0125] In some methods for detecting fungal infection, each liposome in the multisome comprises: a) at least about 500 targeting molecules linked to an N-terminal fragment of a fluorescent protein; and b) at least about 500 targeting molecules linked to a C-terminal fragment of a fluorescent protein. For example, each liposome can comprise at least about 250, 500, 100, 1500, 2000, or 2500 targeting molecules linked to an N-terminal fragment of a fluorescent protein and at least about 250, 500, 100, 1500, 2000, or 2500 targeting molecules linked to a C-terminal fragment of a fluorescent protein.

[0126] Further provided are methods for detecting a fungal infection in a subject or a sample from the subject, comprising: a) contacting the subject or the sample from the subject with a first fusion polypeptide multimer comprising a targeting molecule that binds a target fungal cell antigen and the N-terminal portion of a fluorescent polypeptide, and a second fusion polypeptide multimer comprising a targeting molecule that binds a target fungal cell antigen and the C-terminal portion of a fluorescent polypeptide; and b) detecting a fluorescent signal resulting from the interaction between the N-terminal and C-terminal fragments of the fluorescent protein, wherein the signal indicates the presence of a fungal infection.

[0127] In some instances, the multimers of the liposomes are immobilized on a solid support. Non-limiting examples of solid support materials include glass, modified or functionalized glass, plastics (including copolymers of acrylates, polystyrene and styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane or Teflon J, nylon, nitrocellulose, polysaccharides, resins), silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glass and plastics. The size and shape of the solid support can vary. The solid support can be planar, the solid support can be a hole, or alternatively, the solid support can be a bead or a slide. In some instances, the solid support is a hole in a porous plate. In other instances, the solid support can be a magnetic bead, a resin based on agarose or agarose beads. In other instances, the solid support comprises a non-agarose chromatographic medium, a monolith or nanoparticles. For example, the chromatographic medium can be, for example, methacrylate, cellulose or glass. In other instances, the nanoparticles are gold nanoparticles or magnetic nanoparticles.

[0128] As used throughout, subject means individual. Subject can be an adult subject or a pediatric subject. Pediatric subjects include subjects whose age ranges from birth to eighteen years old. Therefore, pediatric subjects less than about 10 years old, 5 years old, 2 years old, 1 year old, 6 months, 3 months, 1 month, 1 week or 1 day are also included in the subject. Preferably, the subject is a mammal, such as a primate, and more preferably a person. Non-human primates are also subjects. The term "subject" includes domesticated animals (such as cats, dogs, etc.), livestock (such as cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (such as ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.). Therefore, veterinary use and medical preparations are contemplated herein.

[0129] As used herein, a biological sample is a sample derived from a subject and includes, but is not limited to, any cell, tissue, or biological fluid. The sample can be, but is not limited to, blood, plasma, serum, sputum, urine, saliva, bronchoalveolar lavage fluid, biopsy (e.g., tissue or cells isolated from organ tissue, e.g., tissue or cells isolated from lung, liver, kidney, skin, etc.), vaginal secretions, nasal secretions, skin, gastric secretions, or bone marrow specimens.

[0130] The subject may also be a plant or a seed from a plant. The biological sample may also be from a plant, but is not limited to any cell, tissue, or plant exudate. The sample may be, but is not limited to, the surface of a leaf, stem, root, petal, sepal, stamen, carpel, or seed, or a crushed sample or extract thereof.

[0131] Methods of treating or preventing fungal infections

[0132] Also provided are methods for treating or preventing a fungal infection in a subject. The methods comprise administering to a subject suffering from or at risk of suffering from a fungal infection an effective amount of any of the liposome multimers described herein, wherein each liposome in the multimer comprises an antifungal agent and a targeting molecule that binds to a target antigen on a fungal cell, wherein the targeting molecule is incorporated into the outer surface of the liposome and the antifungal agent is encapsulated within the liposome. Any of the liposomes or multimers of liposomes provided herein can be in the form of a pharmaceutical composition.

[0133] The method can be used to treat or prevent fungal infections in any animal (e.g., humans). Examples of human fungal infections include, but are not limited to, Alternaria alternata, Aspergillus species (such as A. fumigatus), Blastomyces species (such as B. dermatitidis), Candida species (such as A. fumigatus, C. glabrata, C. krusei, C. auris), Coccidioides species (such as C. immitis and C. posadasii), Cryptococcus species (such as Cryptococcus gattii), and C. gattii and C. neoformans), Histoplasma species such as H. capsulatum; Pneumocystis species such as P. jirovecii, Sporothrix species such as S. schenckii, Talaromyces marneffei (formerly Penicillium marneffei), and Trichophyton rubrum.

[0134] Throughout the text, treat, treat, and treat (treatment) refer to methods that reduce or delay one or more effects or symptoms of a fungal infection. A subject can be diagnosed with a fungal infection. Treatment can also refer to a method that reduces underlying pathology rather than just symptoms. The effect of administering to a subject can have, but is not limited to, the following effects: reducing one or more symptoms of the disease, reducing the severity of the disease, completely eliminating the disease, or delaying the onset or worsening of one or more symptoms. For example, if one or more symptoms of the subject's disease are reduced by about 10% compared to the subject before treatment or compared to a control subject or control value, the disclosed method is considered to be treatment. Thus, the reduction can be a reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any number therebetween.

[0135] As used herein, prevent, prevent, or prevent (prevention) means to exclude, delay, prevent, avoid, pre-empt, stop or hinder the onset, incidence, severity or recurrence of a disease or condition. For example, if the onset, incidence, severity or recurrence of a fungal infection in a subject susceptible to fungal infection or susceptible to recurrence of a fungal infection is reduced or delayed compared to an untreated control subject susceptible to fungal infection or susceptible to recurrence of a fungal infection, the disclosed method is considered to be preventive. Thus, the reduction or delay in the onset, incidence, severity or recurrence of a fungal infection can be a reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any amount therebetween.

[0136] In some methods, the subject is immunocompromised. For example, the subject can be a subject who has received a stem cell, organ, tissue, or bone marrow transplant, a subject who has cancer, a subject who is receiving cancer therapy (e.g., chemotherapy, immunotherapy, or radiation therapy), a subject who is taking corticosteroids, a subject who is infected with HIV or has acquired immunodeficiency syndrome, a subject who has hepatitis, a subject who has a B cell deficiency, or a subject who has a T cell deficiency, to name a few.

[0137] In some methods, the subject has one or more conditions that affect the subject's lung function, such as pulmonary fibrosis, pneumonia, asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, tuberculosis, emphysema, or sarcoidosis.

[0138] The methods provided herein optionally include selecting a subject suffering from a fungal infection or at risk of a fungal infection. Those skilled in the art know how to diagnose a subject suffering from a fungal infection. For example, a medical examination can be performed. One or more of the following tests can also be used: microscopic examination of clinical samples, histopathological tests, culture tests, and serological tests. Molecular diagnosis and antigen detection of clinical samples can also be used (see, for example, Kozel and Wickes "Fungal Diagnostics", Cold Spring Harb. Perspect. Med. 4(4): a019299 (2014)).

[0139] The methods provided herein optionally further comprise administering to the subject an effective amount of a second therapeutic agent or therapy. The second therapeutic agent or therapy can be administered to the subject before, simultaneously with, or after administration of the liposome multimers. In some methods, the second therapeutic therapy is surgery. In some methods, the second therapeutic agent is a second antifungal agent. The antifungal agent can be any of the above-mentioned polyene antifungals, azole antifungals, imidazoles, triazoles, or echinocandins.

[0140] Pharmaceutical composition

[0141] The term "effective amount" as used throughout the text is defined as any amount required to produce a desired physiological response, such as to treat or prevent a fungal infection. The dosage range used is large enough to produce those dosage ranges of the desired effect that one or more symptoms of a disease or condition are affected (e.g., reduced or delayed). The dosage should not be large enough to cause significant adverse side effects, such as unwanted cross-reactions, unwanted cell death, etc. Typically, the dosage will vary with the type of inhibitor, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, excretion rate, drug combination, and the severity of the specific condition, and can be determined by those skilled in the art. If any contraindications occur, the dosage can be adjusted by a personal physician. The dosage can vary and can be administered in a single dose or in multiple doses administered daily or at extended intervals.

[0142] Any liposome described herein can be provided in the form of a composition, such as a pharmaceutical composition. The composition may comprise one or more liposomes disclosed herein. Optionally, the composition comprising one or more liposomes is located in a kit. Pharmaceutical compositions include, for example, pharmaceutical compositions comprising a therapeutically effective amount of any liposome described herein and a pharmaceutical carrier. The term "carrier" refers to a compound, composition, substance, or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, effectiveness, selectivity, or any other characteristic of the compound or composition to achieve its intended use or purpose. For example, the carrier can be selected to minimize any degradation of the active ingredient and minimize any adverse side effects in the subject. Such pharmaceutically acceptable carriers include sterile biocompatible pharmaceutical carriers, including but not limited to saline, buffered saline, artificial cerebral spinal fluid, dextrose, and water.

[0143] Pharmaceutical compositions comprising any of the liposomes described herein can be prepared according to standard techniques and further comprise a pharmaceutically acceptable carrier. Typically, physiological saline will be used as a pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water or saline, 0.4% saline, 0.3% glycine, dextrose, and the like, including glycoproteins such as albumin, lipoproteins, and globulins for enhancing stability. These compositions are typically sterile. The pharmaceutical compositions may also comprise a pharmaceutically acceptable excipient. Such excipients include any agent that does not itself induce an immune response harmful to the individual receiving the composition and that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugars, and ethanol. Pharmaceutically acceptable salts may be included, for example, inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like may be present in such vehicles. The preparation of pharmaceutically acceptable carriers, excipients, and formulations containing these substances is described in, for example, Remington: The Science and Practice of Pharmacy, 22nd ed., Loyd V. Allen et al., eds., Pharmaceutical Press (2012).

[0144] The aqueous solution can be packaged for use or can be filtered and lyophilized under aseptic conditions, and the lyophilized product can be mixed with a sterile aqueous solution before use. The composition can contain pharmaceutically acceptable auxiliary substances required for close physiological conditions, such as pH adjustment and buffer, tension regulator, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride and calcium chloride. In addition, the liposomal suspension can include a lipid protective agent, which protects lipid from free radical and lipid peroxidation damage when stored. Lipophilic free radical quenchers such as alpha-tocopherol and water-soluble iron-specific chelating agents, such as ferrioxamine, are suitable.

[0145] In pharmaceutical preparation, the concentration of liposome can change significantly, i.e. from being less than about 0.05 % by weight, generally or at least about 2-5 % by weight to as many as 10 % by weight to 30 % by weight change, and will be selected mainly according to the specific mode of administration selected by fluid volume, viscosity.Perhaps liposome can be dried or lyophilized, and in use, is resuspended to desired concentration in water or buffer. The amount of activating agent in the amount of liposome or the liposome used depends on the specific marker used, the morbid state diagnosed and the judgment of the clinician, but usually between about 0.01 and about 150mg every kilogram of body weight, preferably between about 0.1 and about 20mg / kg body weight, between about 0.1 to about 10mg / kg body weight or between about 0.1 to about 5mg / kg body weight, it can be pressed single dose or with the form of each dosage (such as every day 1 to 4 times) and use. Administration can be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or more. One skilled in the art can adjust the dosage as described below based on the specific characteristics of the agent and the subject receiving it.

[0146] The compositions disclosed herein are administered in a variety of ways, depending on whether local or systemic treatment is needed and the area to be treated. The compositions are administered by any of the following routes of administration: oral, intranasal, by inhalation, by nebulizer, parenteral, intravenous, intraperitoneal, intracranial, intraspinal, intrathecal, intraventricular, intramuscular, subcutaneous, intracavitary or transdermal. The pharmaceutical compositions can also be delivered locally to the area in need of treatment, for example, by topical application or local injection. The pharmaceutical compositions can also be delivered by pump or at the surgical site. The effective dose of any of the methods of administration described herein can be extrapolated from a dose-response curve derived from an in vitro or animal model test system.

[0147] Disclosed are materials, compositions, and components that can be used in, used in conjunction with, or used to prepare the disclosed methods and compositions or the products of the disclosed methods and compositions. These and other materials are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed, while specific reference to each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed, and a number of modifications that can be made to a number of molecules included in the method are discussed, each combination and permutation of the method, as well as possible modifications, is specifically contemplated unless expressly indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of the present disclosure, including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are multiple additional steps that can be performed, it should be understood that each of these additional steps can be performed as any specific method step or combination of method steps of the disclosed method, and each such combination or subset of combinations is specifically contemplated and considered disclosed.

[0148] The publications cited herein, and the materials for which they are cited, are expressly incorporated by reference in their entirety.

[0149] Example

[0150] The following examples are provided for illustrative purposes only and are not intended to be limiting. Those skilled in the art will readily recognize a variety of noncritical parameters that can be changed or modified to produce essentially the same or similar results.

[0151] Example 1

[0152] Dectin-1

[0153] Fungal growth

[0154] Aspergillus fumigatus strain A1163 was cloned using the plasmid pBV126 carrying the green fluorescent protein EGFP described by Kang et al. (“A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum. Fungal Genet Biol 42:483-492 (2005)) was transformed under the control of the ribosomal protein 27 promoter of Magnaporthe grisea and used in some experiments to monitor fungal cells to generate strain AEK012. Aspergillus fumigatus spores were grown on plates in Vogel's minimal medium (VMM, 1% glucose, 1.5% agar) for 7 days, and conidia were harvested in PBS + 0.1% Tween. For fluorescent liposome localization and growth inhibition and killing assays, 20,000 and 4,500 AEK012 conidia were plated on 24-well and 96-well poly-L-lysine-coated plates in VMM, 1% glucose, 0.5% BSA, respectively, and incubated at 37°C for periods ranging from 8 hours to 4 days (2001, Chapter 7. Using Microscopy to Explore the Duplication Cycle, p 119-125. In Talbot NJ (ed.), Molecular Biology, 2001, pp. 119-125). and cellular biology of filamentous fungi: practical approach Oxford University Press, University of Exeter, Exeter, UK; and Sasaki et al. (Heterochromatin controls gammaH2A localization in Neurosporacrassa. Eukaryot Cell 13: 990-1000 (2014)). Candida albicans Sc5314 and Cryptococcus neoformans H99 were pre-grown overnight in potato dextrose broth. The cells were then washed three times with sterile water and resuspended in Roswell Park Memorial Institute (RPMI) medium and grown on poly-L-lysine-coated plates at 37°C for 10 hours. All fungal cell growth was performed in a BSL2 laboratory.For fluorescence imaging of liposome-stained fungi, all three fungi were washed three times with PBS, fixed in 4% formaldehyde in PBS for 15 to 60 minutes, washed once and stored in PBS at 4°C.

[0155] Production of soluble Dectin-1

[0156] The sequence of an exemplary codon-optimized E. coli expression construct with Ms-sDectin-1 cloned into pET-45B (GenScript) is shown in Figure 1. This construct encodes a slightly modified 198aa long sDectin-1 protein containing a vector-specified N-terminal (His)6 affinity tag, a flexible spacer, two lysine residues, another flexible spacer, followed by a C-terminal 176aa murine sDectin-1 domain. Starting from a 1 L bacterial culture (BL21 strain) grown overnight in Luria broth without IPTG induction, approximately 45 mg / L of 22 kDa sDectin-1 ( Figure 2 sDectin-1 was extracted from cell pellets in 6 M guanidine hydrochloride (GuHCl, Fisher BioReagents BP178), 0.1 M Na2HPO4 / NaH2PO4, 10 mM triethanolamine, 100 mM NaCl, 5 mM BME, 0.1% Triton-X100 at pH = 8.0. In this buffer, sDectin-1 was bound to nickel affinity resin (QiaGen, #30210), washed in this buffer adjusted to pH 6.3, and eluted in this buffer adjusted to pH 4.5. The pH of the eluted protein was immediately neutralized to pH 7.2 with 1 M pH 10.0 M triethanolamine for long-term storage. 40 mg of protein with a purity greater than 95% was recovered ( Figure 2sDectin-1 samples at 6 μg / μL in the same GuHCl buffer supplemented with fresh 5 mM BME were further adjusted to pH 8.3 with triethanolamine and reacted with a 4-molar excess of the lipid carrier reagent DSPE-PEG-3400-NHS (Nanosoftpolymers, 1544-3400) at 23°C for 1 hour to prepare DSPE-PEG-DEC. Gel exclusion chromatography was performed on Bio-Gel P-6 acrylamide resin (Bio-Rad #150-0740) in refolding and storage buffer RN#5 (0.1 M NaH2PO4, 10 mM triethanolamine, pH 7.2, 1 M L-arginine, 100 mM NaCl, 5 mM EDTA, 5 mM BME) to remove unincorporated DSPE-PEG and GuHCl. The composition of RN#5 was determined empirically by testing a number of mildly denaturing and crowding buffers, most of which caused the modified Dectin-1 protein to fall out of solution after a few days. When stored in RN#5, the protein remains in solution indefinitely, but if freshly reduced with BME, it can readily renature to the active carbohydrate-bound form when diluted from RN#5 into normal biological buffers. DSPE-PEG-BSA was prepared from bovine serum albumin (BSA) (Sigma, A-8022) using the same protocol.

[0157] Remote loading of amphotericin B, sDectin-1, BSA, and rhodamine into liposomes

[0158] Sterile PEGylated liposomes were obtained from FormuMax Sci. Inc. (DSPC:CHOL:mPEG2000-DSPE, 50:45:5 mole%, 100 nm in diameter, liposome suspension containing 60 μmol / mL lipid, ~4×10 12liposomes / mL, #F10203A). Commercial AmBisomes (amphotericin b liposomes for injection, Gilead, Avanti) contain approximately 11 molar percent of AmB. 11 molar percent of amphotericin B (AmB, Sigma A4888) relative to liposome lipids was remotely loaded into small batches of liposomes to prepare AmBisome-like AmB-LL used throughout the study. For example, AmB (1.8 mg, 1.95 umol) was dissolved in 13 uL DMSO by heating at 60 ° C for 10 to 20 minutes and occasionally mixing to prepare an oily, transparent brown AmB solution. 250 uL of sterile liposome suspension (15 micromoles liposome lipids) was added to the AmB oil and mixed at 37 ° C on a rotating platform for 96 hours, at which time most of the AmB was embedded in the liposomes. As quantified by analysis at A406, unincorporated AmB (0.3 μmol) remained in the oil phase, while 1.65 μmol was incorporated into the liposomes, resulting in the liposomes containing 11 mole percent of AmB relative to the moles of lipid. In a separate preparation, gel exclusion chromatography was performed on BioGel A-0.5M agarose resin (BioRad 151-0140) and the excluded fractions at A406 were examined, confirming that 11 mole percent of AmB was retained in the liposomes. By starting with an oil phase containing a higher or lower amount of AmB, larger or smaller amounts of AmB can be loaded into the liposomes. These liposomes, containing approximately 11 mole percent of AmB, are referred to throughout as AmB-LL.

[0159] DEC-AmB-LL and BSA-AmB-LL were prepared by incubating at 60°C for 60 minutes to allow DSPE-PEG-sDectin-1 and DSPE-PEG-BSA conjugates in RN#5 buffer to be incorporated into the phospholipid bilayer membrane of AmB-LL via their DSPE moieties at 1.0 and 0.33 molar percent of protein relative to the molar number of liposome lipids. During this same 60°C incubation period, a red fluorescent tag, rhodamine lissamine B-DHPE triethanolamine salt (Invitrogen, #L1392), was also incorporated at 2 molar percent relative to the liposome lipids (Yao et al., pHLIP(R)-mediated delivery of PEGylated liposomes to cancer cells. J Control Release 167:228-237 (2013); He et al., Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system. J Nanobiotechnology 10:26 (2012); and Garrett et al., Liposomes fuse with sperm cells and induce activation by delivery of impermeant agents. Biochim Biophys Acta 1417:77-88 (1999)). Gel exclusion chromatography on BioGel A-0.5M resin confirmed that at these molar ratios, the insertion of rhodamine-DHPE and DSPE-PEG-protein into liposomes was essentially quantitative. DEC-AmB-LL stored at 4°C maintains binding specificity for approximately 2 months. Storage under free conditions can extend the shelf life.

[0160] Microscopy of liposome binding

[0161] Formalin-fixed or living fungi or animal cells are incubated with DEC-AmB-LL, BSA-AmB-LL and AmB-LL liposomes in liposome dilution buffer LDB (PBS pH 7.2, 0.5% BSA, 5mM BME), and unbound liposomes are washed away after incubation for 15 minutes to 2 hours in the same LDB (see accompanying drawings). Images of rhodamine red fluorescent liposomes, green EGFP Aspergillus fumigatus (A.fumigatus) and differential interference contrast (DIC) illumination cells are taken on a Leica DM6000B automated microscope at 63X under oil immersion (Fig. 4A, 4B, 5A, 5B, 6A, 6B). Cells are removed from the plate and spread on a microscope slide. Seven Z stack images are recorded at intervals of one micron and merged in Adobe Photoshop CC2018. Brightfield and / or red and / or green fluorescence images of cells directly on microtiter plates were captured at 10X, 20X, or 40X on an Olympus IX70 inverted microscope and an Olympus PENE-PL7 digital camera, and the brightfield and / or color layers were merged in Photoshop (Figures 4C-4F, 5C-5F, 6C-6F).

[0162] Cell growth and viability assays

[0163] Liposome stock solution is stored in 800uM AmB, which is first diluted 2 to 20 times in liposome dilution buffer (LDB) or growth medium, and then further diluted 10 times or 20 times in growth medium containing cells to use at a specified concentration. The total dilution factor is generally 250 times to 4,000 times. According to the manufacturer's instructions (Promega, document #G8080), CellTiter-Blue (CTB) cell viability assay is performed as follows: 100 or 200uL of fungal or animal cells in growth medium are treated with 20uL substrate and incubated at 37°C for 4 hours, followed by the addition of 50uL 3% SDS to stop the reaction. The red fluorescence of the esterase CTB product (Ex485 / Em590) is measured in a Biotek Synergy HT microtiter plate reader. For each data point, the data from six wells are averaged and the standard error is calculated (Figure 8A, 8C). Data for germination (Figures 8E-8F) and hyphal length (Figures 8B, 8D) were manually collected from multiple images taken at 10X and / or 20X. After fixation in 4% formaldehyde and PBS, the viability of EGFP-expressing A. fumigatus A1163 cells was measured by green fluorescence on an Ex495 / Em520 microtiter plate reader (Figure 9).

[0164] result

[0165] Preparation of sDectin-1-coated liposomes loaded with amphotericin B

[0166] The control AmB-loaded liposomes AmB-LL were prepared by remote loading of 11 molar percent of AmB relative to the molar number of liposome lipids in PEGylated liposomes, which are similar to commercial AmBisomes (Gilead AmBisomes) in structure and AmB concentration. Figure 2 ) and bovine serum albumin (BSA) were modified with the PEGylated lipid carrier DSPE-PEG. 1 mol% DSPE-PEG-DEC was incorporated into AmB-LL to prepare sDectin-1 coated DEC-AmB-LL ( Figure 3 ), and 0.33 mole percent of DSPE-PEG-BSA was incorporated into AmB-LL to prepare BSA-Amb-LL. This molar ratio of sDectin-1 (MW 22 kDa) and BSA (MW 65 kDa) resulted in both sets of liposomes being coated with equivalent microgram amounts of protein. Because these protein-coated liposomes were made from the same AmB-LL, all three liposome preparations contained 11 mole percent of AmB relative to the moles of lipid. Two mole percent of DHPE-rhodamine was loaded into all three liposome types to prepare red fluorescent AmB-LL, BSA-AmB-LL, and DEC-AmB-LL.

[0167] sDectin-1-coated liposomes DEC-AmB-LL strongly bind to fungal cells

[0168] In an assay conducted on Aspergillus fumigatus seedlings, rhodamine red fluorescent DEC-AmB-LL strongly bound to enlarged conidia and germ tubes, as shown in Figure 4. sDectin-1-targeted liposomes often bound in large numbers or aggregated to specific areas. Although 100 nm liposomes are too small to be resolved by light microscopy, individual liposomes can be seen as small, red fluorescent dots of somewhat uniform size (orange arrows, Figure 4A). Because each liposome contains more than a thousand rhodamine molecules ( Figure 3), so each of them fluoresces strongly enough to be visualized as a single liposome. Essentially all seedlings bound DEC-AmB-LL (Figures 4C and 4D). No binding to ungerminated conidia was detected (not shown). AmBisome-like AmB-LL (Figure 4B) and bovine serum albumin-coated liposomes, BSA-AmB-LL (Figures 4E and 4F) did not detectably bind to conidia or germ tubes. Maximum labeling of DEC-AmB-LL was reached within 15 to 30 minutes, and the strong red fluorescence signal of cell-bound DEC-AmB-LL was maintained for several weeks when the plates were stored in PBS at 4°C in the dark.

[0169] DEC-AmB-LL also bound to enlarged conidia and hyphae from more mature cells, as shown in Figure 5. Similarly, sDectin-1-targeted liposomes generally bound in clumps, but some small red dots of fairly uniform size were visible (orange arrows, Figure 5A), which appeared to be single fluorescent liposomes. Unlike the labeling of germ tubes, DEC-AmB-LL bound only to a subset of mature hyphae (Figures 5C and 5D), as shown in earlier reports on the binding of various sDectin-1 preparations. AmB-LL did not detectably bind to mature hyphae (Figures 5E and 5F) or BSA-AmB-LL (not shown). Finally, DEC-AmB-LL also labeled Candida albicans hyphae and Cryptococcus neoformans H99 cells (Figure 6). In short, Dectin-coated amphotericin B-loaded liposomes bound efficiently to fungal cells, whereas uncoated AmBisome-like liposomes and BSA-coated liposomes did not.

[0170] Binding of DEC-AmB-LL and control LL to fixed and live fungal cells was quantified by counting the number of single fluorescent liposomes and fluorescent liposome clumps bound to dense areas of A. fumigatus hyphae after washing away unbound liposomes. Figures 7A-F show that DEC-AmB-LL bound to fixed and live hyphae more than 100-fold more frequently than control liposomes, BSA-AmB-LL, or AmB-LL. Figure 7G-I It was shown that laminarin inhibited the binding of sDectin-1-coated DEC-AmB-LL by more than 50-fold by the addition of soluble β-glucan, but sucrose did not inhibit the binding, demonstrating that the binding to fungal cells is β-glucan-specific.

[0171] Killing and growth inhibition of fungi by DEC-AmB-LL

[0172] Using liposomes that delivered AmB at concentrations approaching the estimated ED of 2 to 3 uM AmB for various A. fumigatus strains 50 And estimated MIC of 0.5 to 1uM) after treating Aspergillus fumigatus (A.fumigatus), various fungal cell growth and viability assays were performed. In most of these experiments, 4,500 conidia were germinated and incubated in 96-well microtiter plates together with drug-loaded liposomes for 36 to 56 hours. Figure 8 shows that targeted DEC-AmB-LL (Dectin-1 coated liposomes loaded with AmB) kills Aspergillus fumigatus (A.fumigatus) cells or inhibits the growth of Aspergillus fumigatus cells more effectively than BSA-AmB-LL or uncoated AmB-LL. The assays performed using CellTiterBlue (CTB) reagent showed that cells treated with DEC-AmB-LL delivering 3uM AmB could kill Aspergillus fumigatus (A.fumigatus), and the killing effect was one order of magnitude higher than AmBisome-like AmB-LL or BSA-coated liposomes BSA-AmB-LL carrying the same amount of drug (Fig. 8A). The total cytoplasmic esterase activity of CTB reagent is substituted as cell integrity and viability. As the second method that liposome activity is scored, hyphae length is checked. Hyphae length determination has given surprising similar results, shows that the DEC-AmB-LL that sends 3uM AmB is much more effective than AmB-LL or BSA-AmB-LL aspect suppressing hyphae growth (Fig. 8 B). In the complete biological replication experiment that uses different AmB remote loading methods, independent s-Dectin-1 and BSA and rhodamine loading and different liposome dilution buffers to carry out, similar but slightly less significant result (Fig. 8 C and 8D) is obtained when sending 3uM AmB. CTB reagent and hyphae length determination show that DEC-AmB-LL is killing Aspergillus fumigatus (A.fumigatus) or suppressing the effectiveness aspect AmB-LL height almost an order of magnitude.

[0173] An additional assay using liposomal AmB activity measured the percentage of conidia that germinated in the presence of various liposomal preparations (Figures 8E and 8F). DEC-AmB-LL, delivering as little as 0.09 uM and 0.187 uM AmB, was several times more effective than AmB-LL or BSA-AmB-LL in inhibiting conidial germination of A. fumigatus.

[0174] The fourth assay using liposome activity examined the endogenous green fluorescence signal ( FIG. 9 ) produced by the EGFP-expressing Aspergillus fumigatus (A. fumigatus) AEK012 strain. When measuring green fluorescence levels, the DEC-AmB-LL delivered at 2 uM or 0.67 uM AmB was more effective than AmB-LL or BSA-AmB-LL in inhibiting the growth of fungal cells. In short, various assays showed that targeting DEC-AmB-LL was more effective in killing Aspergillus fumigatus (A. fumigatus) cells or inhibiting the growth or germination of Aspergillus fumigatus cells than uncoated AmB-LL or BSA-coated BSA-AmB-LL.

[0175] The dose-response curve for determining the percentage of conidia germination is shown in Figure 8G The results illustrate the wide differences in performance among the three liposome types. DEC-AmB-LL outperformed the other two liposome types, with activity proportional to concentration, and they outperformed AmB-LL over a wide range of concentrations. BSA-AmB-LL consistently performed the worst, likely because they block the random channel separating the liposome membrane from the fungal plasma membrane that allows AmB-LL to bind. The timing of these assays influences the precise shape of these curves, with assays performed earlier better resolving differences at the lowest concentrations, while longer incubation times resolve differences at the highest AmB concentrations by allowing for more highly inhibited cell growth. This makes it difficult to determine the optimal differences between liposome preparations across the entire concentration range where DEC-AmB-LL outperforms the control liposomes. Overall, however, the relationship between the three liposome classes remains consistent.

[0176] Decreased animal cell binding and toxicity of DEC-AmB-LL

[0177] The Cell Titer Blue assay of human embryonic kidney HEK293 cell viability showed that AmB-LL and AmB in deoxycholate micellar suspension were more toxic to HEK293 cells than DEC-AmB-LL or BSA-AmB-LL. Figure 10 Coating liposomes with protein presumably slows their uptake by the plasma membrane of animal cells. Cells were treated for two hours with various preparations delivering 15 or 30 micromolar AmB, the excess AmB-containing material was washed off, and the cells were grown overnight at 37°C before being assayed.

[0178] Diagnostic methods

[0179] Liposomes coated with 500 targeting molecules (e.g., sDectin-1 monomers fused to the N-terminal half of Venus green fluorescent protein via a flexible linker (e.g., DEC1-VN)) and also coated with 500 targeting molecules (e.g., sDectin-1 monomers fused to the C-terminal half of Venus via a flexible linker (DEC1-VC)) will rapidly recognize and bind to low concentrations of fungal β-glucan to form sDectin-1 dimers and assembled Venus, resulting in a strong green bimolecular fluorescence complementation (BiFC) signal. The DNA and protein sequences of these exemplary BiFC reporter gene constructs are shown in SEQ ID NOs: 13, 14, 15, 16 ( Figure 1M 、 1N , 10 and 1P). For additional sequences, see also SEQ ID NOs: 17, 18, 19, 20 for Dectin-2 BiFC fusions ( Figure 1Q 、 1R , 1S, 1T and Figure 27D In any of the constructs provided herein, N-terminal or C-terminal fragments of the targeting sequence can be used. For example, N-terminal or C-terminal fragments of Dectin-1, Dectin-2, or Dectin-3. In some examples, the presence of hundreds or thousands of sDectin-1 monomers per liposome ensures rapid and sensitive detection. This assay relies on the fact that Dectins bind tightly and irreversibly to glucans or mannans on cells or in solution only as dimers.

[0180] Optionally, when these fungus-targeting liposomes are attached to an insoluble matrix, they can be used to detect even low concentrations of fungal cell surface molecules, such as β-glucan, in large volumes, control buffers, and serum in vitro using standard fluorescence instrumentation. When exposed to large volumes of sample material, the insoluble matrix allows the fluorescent signal to be concentrated in a small volume for more sensitive detection. In addition to serum, immobilized liposomes can be used to measure fungal and soluble fungal cell wall material in urine, lung lavage fluid, or solubilized tissue extracts.

[0181] In some instances, targeted liposomes can be used to detect fungal mannans. This can be achieved, for example, by coating liposomes with approximately 500 sDectin-2 monomers fused to the N-terminal half of a fluorescent protein (e.g., VenusDEC2-VN) and coating the same liposomes with approximately 500 sDectin-2 or Dectin-3 monomers fused to the C-terminal half of a complementary fluorescent protein (Venus, DEC2-VC, DEC3-VC). Dectin-2 homodimers strongly bind to fungal wall mannans, but monomers do not significantly bind. Heterodimers between Dectin-2 and Dectin-3 can bind to mannans more strongly than their individual homodimers.

[0182] The system would enable assays that: (1) can be completed within 60 minutes of combining serum with the liposome matrix; (2) can be performed in a single step; (3) can detect very low concentrations of polysaccharides with high sensitivity; (4) are inexpensive; (5) require minimal operator training and expertise; and / or (6) can detect virtually all fungal pathogens. These liposomes should enable one-step assays for invasive fungi in blood, serum, urine, lung exudates, lungs, eyes, throat, vagina, skin, and fingernails and toenails. Figure 11 A possible model for free-floating or immobilized Dectin-coated liposomes for one-step detection of fungal β-glucan and mannan polysaccharides is shown. One-step detection means that the binding of the liposomes to the target cell wall component and the generation of the signal are biochemically correlated, i.e., no additional processing steps or reagents are required for detection.

[0183] Biochemical production of mouse or human sDectin-1 is complicated because the protein readily aggregates in aqueous buffers and becomes insoluble and inactive. As shown here, the solubility problem of sDectin was overcome by combining several approaches, including the use of a very short charged peptide tag, the inclusion of 6M GuHCl during protein extraction, purification, and chemical modification, by performing refolding, liposome loading, and storage in a buffer containing the protein solubilizer 1M arginine, and the inclusion of a thiol reducing agent.

[0184] Previous reports have shown that mouse sDectin-1 efficiently binds to inflated conidia and germ tubes of Aspergillus fumigatus, but binds inefficiently (if at all) to mature hyphae and is completely ineffective against ungerminated conidia (Steele et al., The beta-glucan receptor dectin-1 recognizes specific morphologies of Aspergillus fumigatus. PLoS Pathog 1:e42 (2005)). The lack of hyphal binding may be due to greatly reduced levels of β-glucan on the surface of older, resting cells, or it may be that β-glucan in the mature cell wall is less accessible. Here, sDectin-1-coated fluorescent DEC-AmB-LL efficiently bound to a subset of inflated conidia, germ tubes, and hyphae, indicating that the modified sDectin-1 described herein, presented on the liposome surface, retains its normal affinity for β-glucan. These binding data demonstrate for the first time that chemically modified forms of sDectin-1 (e.g., DSPE-PEG-Dectin-1) can maintain their fungal cell binding specificity. Furthermore, fluorescent DEC-AmB-LL binds rapidly and remains stably bound to cells for weeks. Purified sDectin-1 has been reported to bind to round yeast cells of Candida albicans and to hyphae in the region between the parent cell and the mature bud, but not to hyphae. Although binding to A. fumigatus hyphae was less efficient than that of DEC-AmB-LL to C. albicans, we demonstrated reasonable binding of DEC-AmB-LL at multiple sites along C. albicans hyphae (Figure 6). The greater avidity of liposomes coated with more than one thousand sDectin-1 molecules, likely similar to the avidity of pentameric IgM antibodies, ensures rapid binding and very slow release of bound liposomes. The presence of thousands of rhodamine molecules on each liposome also increases the chances of detecting a clear fluorescent signal. In numerous experiments using different binding buffers containing BSA blocking agents and various incubation periods, no significant affinity of uncoated AmB-LL or BSA-AmB-LL for fungal cells was detected. Although BSA-AmB-LL bound weakly to inflated conidia of Aspergillus fumigatus (A. fumigatus) in preliminary experiments without incorporating BSA blocking agents into the incubation,

[0185] However, Aspergillus, Candida, and Cryptococcus species belong to three evolutionarily distinct fungal groups—the Hemiascomycetes, Euascomycetes, and Hymenococcus—that are separated from their common ancestor by hundreds of millions of years. DEC-AmB-LL specifically bound to all three fungal groups, indicating that sDectin-1-targeted liposomes can access β-glucans found in the outer cell walls of many pathogenic fungi. However, compared with Aspergillus, Dectin-1 bound relatively weakly to Candida, while Dectin-2 bound more strongly. This suggests that robust panfungal detection of fungal pathogens may require assays that detect both glucans and mannans.

[0186] As shown herein, in various biological experimental replicates using different cell-based assays, DEC-AmB-LL was more effective in killing or inhibiting A. fumigatus cells than AmBisome-like AmB-LL, which delivered the same level of AmB. In all our experiments, DEC-AmB-LL was more effective at killing or inhibiting A. fumigatus cells at various AmB concentrations tested (which were close to or below the estimated ED of 3 uM). 50 ) showed several times to more than an order of magnitude higher fungicidal activity than the control liposomes. DEC-AmB-LL was detected to be significantly more active than AmB-LL, even at AmB concentrations as low as 0.094 uM AmB (well below the MIC of AmB). These studies indicate that DEC-AmB-LL significantly reduced the ED of AmB to kill A. fumigatus and inhibit the growth of A. fumigatus. 50 and MIC.

[0187] The lipid membrane of uncoated liposomes is passively bound to the lipid membrane of animal cells (which are different from fungal cells and are not protected by cell walls). However, the protein coating of liposomes and the coating carried out with mouse or human serum albumin, especially reduced the clearance rate of liposomes from animal models, thus increasing the half-life of liposomes. It is speculated that the protein coating slows down the direct interaction between the liposome membrane and the plasma membrane, thereby reducing the passive delivery of the drug. Liposomes are protein coated with sDectin-1 to reduce the direct interaction between the liposome membrane and the plasma membrane of animal cells, thereby reducing their interaction and fusion with animal cells. The results described here (which show that DEC-AmB-LL reduces the toxicity of human kidney cells relative to AmB-LL) are consistent with this viewpoint.

[0188] In summary, when optionally conjugated to a PEGylated lipid carrier and incorporated into liposomes as a monomer, sDectin-1 is able to form functional complexes and effectively bind to β-glucans in the cell walls of a wide variety of fungal species. Multiple growth inhibition and viability assays performed on DEC-AmB-LL delivering AmB concentrations ranging from 0.094 to 3 uM demonstrated that sDectin-1-targeted liposomes reduced the ED50 of liposomal AmB to well below the ED50 reported for non-targeted AmBisomes, as modeled by our AmB-LL. Furthermore, DEC-AmB-LL had lower affinity for and was less toxic to animal cells than AmBisome-like liposomes. Taken together, these results suggest the rationale for using sDectin-1-coated liposomes as pan-fungal carriers for targeted antifungal therapy.

[0189] Example 2

[0190] Dectin-2

[0191] Cell culture

[0192] Candida albicans CAI4, A. fumigatus A1163, and wild-type Cryptococcus neoformans H99-α expressing GFP under the control of the ADH1 promoter were grown in liquid form in Vogel's minimal medium (VMM) + 1% glucose (85) + 0.5% BSA or RPMI 1640 medium (ThermoFisher SKU-11835-030) + 0.5% BSA or YPD (1% yeast extract, 2% peptone, 2% dextrose) with shaking in 24 or 96-well polystyrene microtiter plates or on glass microscope chamber slides and incubated at 37°C for 3 to 36 hours. For A. fumigatus, plates were pre-coated with poly-L-lysine, and for all three species, glass microscope slides were pre-coated. All fungal cell growth was performed in a BSL2 laboratory. Before treating the cells with fluorescent liposomes for microscopic analysis of binding, fungal cells were washed three times with PBS, fixed in 4% formaldehyde in PBS for 60 min, washed three times, and stored in PBS at 4°C.

[0193] Human colorectal adenocarcinoma cell line HT-29 (ATCC HTB-38) and human embryonic kidney cell line HEK-293 (ATCC CRL-1573) were grown in 96-well microtiter plates in RPMI medium without red indicator dye plus 10% fetal bovine serum in an incubator at 37°C in an atmosphere supplemented with 5% CO 2 . The viability and metabolic activity of the cell lines after overnight antifungal treatment were determined using CTB reagent diluted 1:10 in culture medium and incubated at 37°C for 60 to 90 minutes, with 8 wells per treatment.

[0194] Production and chemical modification of sDectin-2

[0195] The carboxyl terminus of Dectin-2 contains its mannan recognition domain, sDectin-2. Figure 13 shows the sequence of a codon-optimized E. coli expression construct with MmsDectin-2 lyshis synthesized by GenScript and cloned into pET-45B. The 577 base pair DNA sequence encodes a slightly modified 189a.a. sDectin-2 protein containing a vector-specified N-terminal (His)6 affinity tag, an additional flexible GlySer spacer, a sequence LysGlyLys containing a glycine residue for cross-linking, another flexible spacer, followed by a C-terminal 166a.a. long murine sDectin-2 domain. The modified sDectin-2 protein, DEC2, was expressed in E. coli and purified as described above for mouse sDectin-1, followed by an additional gel exclusion chromatography step on Sephacryl S-100HR (GE Healthcare, #17061210). Proteins were visualized on SDS-PAGE gels stained with Coomassie blue. Figure 14A 5 μg / uL sample of sDectin-2 in the same GuHCl buffer (to which 5 mM 2-mercaptoethanol was freshly added) was adjusted to pH 8.3 using 1 M pH 10 triethanolamine and reacted with a 4 molar excess of the reactive succinimidyl ester NHS moiety of DSPE-PEG-3400-NHS (1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-conjugated polyethylene glycol (PEG), from Nanosoftpolymers, 1544-3400) at 23°C for 1 hour to prepare DEC2-PEG-DSPE (Supplementary Figure S1). Unincorporated DSPE-PEG and GuHCl were removed by size exclusion chromatography on Bio-Gel P-6 acrylamide resin (Bio-Rad #150-0740) in renaturation and storage buffer RN#5 (0.1 M NaH2PO4, 10 mM triethanolamine, pH 8.0, 1 M L-arginine, 100 mM NaCl, 5 mM EDTA, 5 mM 2-mercaptoethanol) (51). BSA-PEG-DSPE was prepared using BSA (bovine serum albumin, Sigma, A-8022) according to the same protocol, but using a buffer without GuHCl during DSPE-PEG labeling and L-arginine during Bio-Gel P6 chromatography. Rhodamine-labeled sDectin-2 (DEC2-Rhod) was prepared using the same procedure as DEC2-PEG-DSPE in the same GuHCl buffer, but labeling was performed on sDectin-2 using a 4-molar excess of rhodamine-NHS reagent (Thermo Fisher #46406). Hydrolyzed, unbound rhodamine reagent and unwanted salts were removed from DEC2-Rhod by size exclusion chromatography on Bio-Gel P2 resin in RN#5 buffer. Like DEC2-PEG-DSPE, RN#5 maintains DEC2-Rhod in a state where it readily renatures to the active carbohydrate-bound form upon dilution into milder biological buffers.

[0196] Remote loading of AmB, sDectin-2, BSA and rhodamine into liposomes

[0197] Starting from sterile PEGylated liposomes from FormuMax Sci. Inc. (DSPC:CHOL:mPEG2000-DSPE, FormuMax #F10203A), a small batch of liposomes with 11 mole percent AmB relative to 100% liposomal lipid (AmB, Sigma A4888) was prepared to prepare AmB-LL as described above (see Table 1).

[0198] Table 1

[0199]

[0200] In parallel with the above protocol, DEC2-PEG-DSPE- and BSA-PEG-DSPE conjugates in RN#5 buffer and PBS were integrated into the phospholipid bilayer membrane of AmB-LL via their lipid DSPE portion by incubation at 60°C for 30 minutes to prepare DEC2-AmB-LL and BSA-AmB-LL. During these same 60°C incubations, 2 mol% of the red fluorescent label rhodamine lissamine B-DHPE (Invitrogen, #L1392) was also incorporated into sDectin-2-coated and BSA-coated liposomes and AmB-LL. Duplicate samples of DEC2-AmB-LL were subjected to gel exclusion chromatography on Bio-Gel A 0.5M resin (Bio-Rad, #151-0140). The fluorescent liposomes were effectively excluded from the resin. Since sDectin-2 was not detected in the low molecular weight fraction contained in the gel (2.2 OD A 280 1 mg / mL) or rhodamine, we conclude that both can be efficiently loaded into liposomes. Fresh 2 mM BME was added to DEC2-AmB-LL before each use in binding or killing assays. DEC2-AmB-LL stored at 4°C in RN#5 appears to retain full fungal cell binding specificity and killing activity for at least 12 months.

[0201] Microscopic examination of liposomes and DEC2-Rhod bound to fungal cells

[0202] Formalin-fixed fungal cells were incubated with liposomes at 23°C in liposome dilution buffer (LDB2) (20 mM HEPES, 10 mM triethanolamine, 150 mM NaCl, 10 mM CaCl2, 1 mM β-mercaptoethanol (BME), 5% BSA, pH 8.0), with fresh BME added. The liposome stock solution was diluted 1:200 and then incubated with the cells to give a sDectin-2 protein concentration of 0.5 μg / 100 μL. After incubation for 15 minutes, 1 hour, or longer, unbound liposomes were washed away with four changes of LDB2. Merged images of rhodamine red fluorescent liposomes, green fluorescent cells, and differential interference contrast (DIC) illuminated cells were taken from cells grown on microscope slides on a Leica DM6000B automated microscope under oil immersion at 63X. The DEC2-Rhod stock solution was also diluted 1:200 and incubated with cells, resulting in a sDectin-2 protein concentration of 0.5 μg / 100 μL. Brightfield, DIC, and red (Ex560 / Em645) and green (Ex500 / Em535) fluorescence images of cells in microtiter plates were acquired at 20X magnification using an Olympus IX70 inverted microscope and an Olympus PENE-PL7 digital camera. The brightfield and / or fluorescence color layers were merged in Photoshop. Liposome-bound areas at 20X magnification were quantified by importing 8-bit grayscale copies of unmodified red fluorescence TIF images into Image J (imagej.nih.gov / ij). Image > Adjust > Threshold > Apply was used to capture only the red fluorescent areas illuminated by liposomes. The area data for each image was then transferred to the file using Analyze > Measure. Six to ten images were typically analyzed, and the average of the majority of area estimates was calculated. However, due to the large variability in staining intensity between C. neoformans cells in different photographic fields, 90 images were analyzed for each treatment. Brightfield, DIC, and fluorescence images of cells grown in microscope chamber slides were also taken at 20× or at 63× under oil immersion on a Leica DM6000B automated microscope.

[0203] Glucuronide lignan (GXM)-specific monoclonal antibody 18B7 was obtained from Sigma-Aldrich (MABF2069) and used at a dilution of 1:200 (0.5 μg / 100 μL). The cells were developed with goat anti-mouse secondary antibody Alexa 488 (Life Technologies, A11001) also diluted 1:200 and photographed using a GFP filter (Ex500 / Em535).

[0204] Growth inhibition and viability assays after liposome treatment

[0205] Liposome stocks were stored at 615 to 800 μM AmB and were typically diluted 30 to 600-fold into liposome dilution buffer LDB2 and then diluted 1:11 into growth medium to achieve the indicated final bactericidal concentrations ranging from 2 μM to 0.1 μM. Control cells received an equal amount of LDB2. CellTiter-Blue (CTB) cell viability and metabolic activity assays for Candida albicans and A. fumigatus were performed as described recently for A. fumigatus: incubated with CTB reagent for 3 to 4 hours and 96-well plates analyzed in a Bio-Tek Synergy HT fluorescence microtiter plate reader. The fluorescence background of the control wells was subtracted from the experimental wells. For each data point, the data from eight wells were averaged. These assays have a large amount of background and are less sensitive if the cells are assayed in VMM. When performing parallel cell viability assays on Cryptococcus neoformans using this protocol or other CTB protocols published for this species, we were unable to detect any fluorescent signal from the CTB reagent. As an alternative measure of cell viability for C. neoformans and Candida albicans, the assay was performed by growing 1 mL of cells in YPD, adding drug-loaded liposomes for the specified growth time, diluting the cells, plating on YPD, and counting colony-forming units (CFU). The fraction of dead C. neoformans cells in cells grown in YPD after treatment with bactericide-loaded liposomes was determined by adding 50 μg / mL propidium iodide to the culture medium and incubating at 37°C for 60 min. The culture medium was removed and replaced with PBS for fluorescence microscopy using the red fluorescent protein channel (Ex560 / Em595), and the percentage of dead stained cells was scored relative to the total number of stained and unstained cells. In experiments using DEC1-AmB-LL, all liposomes were diluted with LDB1 (PBS + 5% BSA + 1 mM BME) (51) rather than LDB2.

[0206] Dectin-2 is encoded by the human and mouse C-type LECtin receptor gene, CLEC6A. Dectin-2 binds to α-mannans, as well as N-linked and O-linked mannans, found in mannoproteins (42-46). Dectin-2 is expressed on the plasma membrane of some lymphocytes, with its mannan-binding domain (sDectin-2) located externally and its signaling domain in the cytoplasm. Dectin-2 functions as an innate immune receptor, signaling active fungal infection to the host.

[0207] As described in Example 1, Dectin-1-coated liposomes loaded with amphotericin B (AmB) target β-glucans in the inner cell wall and effectively bind to various cell types of A. fumigatus and Cryptococcus neoformans yeast cells. Dectin-1-coated liposomes loaded with AmB (DEC1-AmB-LL) can effectively inhibit and kill A. fumigatus cells. However, Dectin-1 liposomes bind poorly to Candida albicans, presumably due to the presence of a thick mannan and mannoprotein outer layer that masks their β-glucans. Here, AmB-loaded liposomes were coated with the mannan binding domain of mouse Dectin-2, sDectin-2. Compared with non-targeted drug-loaded liposomes, sDectin-2-coated AmB-loaded liposomes effectively bound to Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, and significantly reduced cell growth and viability.

[0208] result

[0209] Preparation of bactericide-loaded sDectin-2-coated fluorescent liposomes

[0210] Figure 15 The model of the bactericide-loaded sDectin-2 coated liposome constructed herein is shown in FIG. The liposome construction method and liposome composition are closely similar to those of the sDectin-1 coated liposomes described above. 11 molar percent of amphotericin B (AmB) relative to the molar number of liposome lipids was remotely loaded into the membrane of the PEGylated liposome to prepare the AmB-loaded liposome AmB-LL. For reference, the widely used commercial non-targeted liposome product loaded with AmB The murine sDectin-2 sequence was designed to contain a small lysine tag at its amino terminus ( FIG. 13 ). It was expressed in E. coli ( Figure 14 ), and the purified sDectin-2 protein was conjugated to NHS-PEG-DSPE via this lysine tag to make DEC2-PEG-DSPE. DEC2-PEG-DSPE was then incorporated into AmB-LL via its DSPE lipid portion at 1 molar percent of protein molecules relative to the molar number of liposome lipids (1,500 sDectin-2 molecules per liposome) to prepare DEC2-AmB-LL. Similarly, and as a control for protein-coated liposomes, 0.33 molar percent of bovine serum albumin was incorporated into AmB-LL via a lipid carrier to prepare BSA-AmB-LL. This resulted in equivalent μg amounts of 22kDa sDectin-2 and 66kDa BSA proteins on the surface of these two groups of liposomes. Compared to commercial products The very similar uncoated AmB-LL was also used as a liposome control. 2 mol % DHPE-rhodamine was also incorporated into the liposome membranes of all three liposome preparations. Thus, all three sets of liposomes contained the same 11 mol % AmB and 2 mol % rhodamine. The composition of DEC2-AmB-LL was compared to that of BSA-AmB-LL, AmB-LL, and compared with the composition of AmB / micelles.

[0211] sDectin-2-coated liposomes DEC2-AmB-LL bind to a variety of fungal species better than control lipids Stronger body

[0212] sDectin-2 coated red fluorescent DEC2-AmB-LL binds strongly to Candida albicans yeast cells, pseudohyphae and hyphae (Figure 16). The vast majority of sDectin-2 coated liposomes bind in large clusters to the extracellular polysaccharide matrix associated with these cells. In addition, DEC2-AmB-LL binds to a large subset of the extracellular matrix (Ex+) surrounding these cells, while some areas of the matrix clearly do not bind to sDectin-2 coated liposomes (Ex-) (Figure 16E). Although 100 nm liposomes are too small to be resolved by light microscopy, the estimated 3,000 rhodamine molecules ( Figure 15) can visualize the fluorescent signal from individual liposomes. It was rare to see individual DEC2-AmB-LL (white arrows, Figure 16A) or liposome clusters directly bound to the cell wall of Candida albicans. In contrast, individual sDectin-1-coated liposomes frequently bound to the cell wall of A. fumigatus cells.

[0213] DEC2-AmB-LL is strongly combined with Cryptococcus neoformans yeast cells (Figure 17). Monoclonal antibody 18B7 is specific for glucuronoxylomannan (GXM) found in the capsule and exopolysaccharide of Cryptococcus neoformans (C.neoformans). Antibody 18B7 dyed most (Figure 17 B) but not all of the cell capsules and most but not all of the exopolysaccharides visible in the bright field image (Ex+, Figure 17A). DEC2-AmB-LL and the GXM areas of most 18B7 dyeing in the exopolysaccharide matrix are strongly co-stained, but the 187B-labeled GXM of the capsule is not dyed (Figure 17C, 17D). In addition, there are some extracellular matrix areas that are not dyed with 18B7 or DEC2-AmB-LL (Ex- / -, Figure 17A).

[0214] DEC2-AmB-LL was also bound in large clusters to the exopolysaccharide matrix produced by germinating conidia and hyphae of A. fumigatus (Figures 17E, 17F, 17G). Similarly, little, if any, binding was associated with the cell wall itself. In addition, there appeared to be areas where the exopolysaccharide matrix was not stained or was poorly stained (e.g., Figures 17E and 17F).

[0215] Because DEC2-AmB-LL binds poorly or not at all to mannans within the tightly cross-linked polysaccharides of the cell walls of all three fungal species examined, we believe that the 100-nanometer diameter size of our liposomes limits their entry or that sDectin-2 is somehow confined and unable to achieve full activity while present within the liposome membrane. The rotational diameter of DEC2-AmB-LL in solution is even larger than estimated for their physical size, given that they are coated with sDectin-2 protein and associated water molecules. sDectin-2 itself has an atomic weight of 22 kDa, so the rotational diameter in solution can be estimated to be approximately 4 nm. Rhodamine-conjugated sDectin-2, DEC2-Rhod, was prepared. The atomic weights of rhodamine (0.48 kDa) and one or two rhodamine conjugate molecules had little effect on this size estimate for DEC2-Rhod. Red fluorescent DEC2-Rhod strongly bound to the majority of the exopolysaccharide matrix surrounding A. fumigatus hyphal cells ( FIG. 18 , panels A and B). The binding pattern and intensity were indistinguishable from those of DEC2-AmB-LL ( FIG. 18 , panels C and D).

[0216] Compared with the BSA-AmB-LL of uncoated AmB-LL and BSA-coated, the efficiency of DEC2-AmB-LL being combined with fungal cells is quantified.The area of ​​fluorescent liposome signal is a plurality of red fluorescence photographic images taken from the liposome staining culture that almost converges with fungal cells and measures (Figure 19).The efficiency that DEC2-AmB-LL is combined with Candida albicans (C.albicans) pseudohyphae and hyphae, new cryptococcus (C.neoformans) yeast cell and Aspergillus fumigatus (A.fumigatus) hyphae is higher than AmB-LL or BSA-AmB-LL by 50 to 150 times (Figure 19 A, 19D, 19G).For carrying out these measurements, the example of quantitative fluorescent liposome photographic image is listed adjacent to each bar graph (Figure 19 B, 19C, 19E, 19F, 19H, 19I).

[0217] The specificity, stability, and rate of DEC2-AmB-LL binding were characterized using the same quantitative assay for the area of ​​fluorescent liposomes bound to pseudohyphae and hyphae of C. albicans ( FIG. 20 ). During the binding assay, DEC2-AmB-LL labeling was inhibited by 75% by inclusion of solubilized yeast mannan, but not by the same concentration of the soluble β-glucan laminarin or the glucose-fructose containing disaccharide sucrose ( FIG. 20A , 20B , 20C ). This result confirms that the binding of sDectin-2-targeted liposomes to the extracellular matrix is ​​mannan-specific, consistent with the published carbohydrate specificity of sDectin-2. The stability of DEC2-AmB-LL binding to C. albicans cells was examined by taking the DEC2-AmB-LL stained preparations examined in FIG. 19A and storing them in phosphate-buffered saline at 4°C in the dark. After 2 months, the cells were re-photographed and liposome staining was quantified. The fluorescence intensity of DEC2-AmB-LL binding to cells is still very strong, estimated to be 50 times stronger than the non-specific binding of AmB-LL (Figures 20D, 20E, 20F). This result shows that DEC2-AmB-LL itself is relatively stable, and their binding to cells is also relatively stable. The DEC2-AmB-LL binding rate is estimated by exposing dense areas of Candida albicans (C.albicans) pseudohyphae and hyphal cells to liposomes for 10 seconds to 90 minutes before washing out unbound liposomes (Figures 20G, 20H). The area marked by DEC2-AmB-LL increases exponentially rapidly in the first 15 minutes (Figure 20G), then slows down, but does not seem to be complete after 90 minutes (Figure 20H).

[0218] In summary, Dectin-2-coated AmB liposomes, DEC2-AmB-LL, specifically, stably, and rapidly bound to fungal mannans present in the extracellular matrix of Candida albicans grown in vitro, whereas almost no nonspecific binding was observed with control liposomes. Only trace amounts of DEC2-AmB-LL bound to the cell wall. DEC2-AmB-LL also efficiently bound to portions of the extracellular matrix surrounding Cryptococcus neoformans and Aspergillus fumigatus cells.

[0219] Growth inhibition and killing by DEC2-AmB-LL

[0220] Various fungal cell growth and viability assays were performed after treating actively growing cultures of Candida albicans (C. albicans), Cryptococcus neoformans (C. neoformans), and Aspergillus fumigatus (A. fumigatus) with sDectin-2-coated liposomes and control liposomes that delivered AmB at concentrations close to the minimum inhibitory concentration (MIC) of the fungicide (Figure 21). Depending on the assay conditions and delivery method of AmB, the estimated MIC of AmB for these species ranged from 0.06 to 1.3 μM.

[0221] 4,000 C. albicans yeast cells were inoculated into each well of a 96-well microtiter plate and grown for 6 hours to the pseudohyphae and early hyphae stages before being treated with drug-loaded liposomes. After a 30-minute incubation, the liposomes were washed away and the cells were grown for an additional 16 hours. Figure 21A Targeted DEC2-AmB-LL, which delivers 1 μM AmB to 0.125 μM AmB, was shown to kill or inhibit C. albicans cells 90 to 3 times more efficiently than uncoated AmB-LL or BSA-AmB-LL, which deliver the same concentration of AmB. The difference is significant given that cells were only exposed to the liposomal drug for 30 minutes in these experiments. These data were obtained using CellTiter-Blue reagent to assess cytoplasmic reductase activity as a surrogate for cell integrity and viability. Dead cells or metabolically inactivated cells do not reduce the resazurin substrate to a fluorescent resorufin product. Continuous treatment of C. albicans cells with liposomes for a full 16-hour period or with higher drug concentrations resulted in excessive cell death in all three drug-loaded liposome samples, making it impossible to clearly answer the differences between the different liposome preparations. DEC2-AmB-LL preparations retained their full antifungal activity for six months as long as they were freshly reduced. The number of viable cells after liposome treatment was also measured. C. albicans yeast cells grown in liquid culture in rich medium were incubated with liposomes delivering 2 μM AmB. The liposomes were washed away after 30 minutes. After an additional 6 hours of growth, the cultures were diluted and colony-forming units (CFU) were determined on agar plates using rich medium. Based on CFU, DEC2-AmB-LL was three times more effective than AmB-LL or BSA-AmB-LL in inhibiting or killing C. albicans yeast cells in liquid. Figure 21A ).

[0222] Liquid-grown C. neoformans yeast cells were treated with liposomes delivering 0.4 μM AmB for 4 h and with liposomes delivering 0.4, 0.2, and 0.1 μM AmB overnight ( Figure 21B ). At the end of each treatment, cells were diluted and colony forming units (CFU) were determined on agar plates with rich medium. DEC2-AmB-LL was 2.5 to 11 times more effective at killing C. neoformans than AmB-LL or BSA-AmB-LL, with the best treatment being 0.2 μM AmB overnight. As an alternative assay, Cryptococcus neoformans yeast cells grown on microtiter plates were treated with liposomes delivering 1 μM AmB for 5 hours. Cell death of the cells was immediately determined by incubating the cells with propidium iodide. Propidium iodide enters dead cells, but not live cells, and emits red fluorescence when embedded in short regions of double-stranded DNA or double-stranded RNA. The propidium iodide assay showed that under these treatment conditions, DEC2-AmB-LL was 5 times more effective than uncoated AmB-LL in killing C. neoformans cells ( Figure 21B ).

[0223] Use delivery close to and below the target Estimated MIC 0.5 μ M AmB concentration of liposomes treated Aspergillus fumigatus (A.fumigatus). When the germ tube first began to converge from 95% conidia, conidia germinated and grew to a very early seedling stage. Then, cells were treated with liposomes or liposome dilution buffer containing AmB for 2 hours, and unbound liposomes were washed away with growth medium. The cells were grown for 19 more hours, and the viability and metabolic activity of the cells were determined with CellTiterBlue reagent. The DEC2-AmB-LL that delivered 0.5 μ M and 0.25 μ M AmB killed Aspergillus fumigatus (A.fumigatus) or inhibited the growth of Aspergillus fumigatus 20 times and 36 times higher than AmB-LL (Figure 21 C), respectively. It should be noted that the Aspergillus fumigatus (A.fumigatus) cells treated with dilution buffer control overgrew during this assay and produced thick hyphae mats in microtiter wells. Therefore, the metabolic activity and CellTiterBlue signal from these control cells were low.

[0224] Dectin-1-loaded liposomes targeting AmB also effectively bind to A. fumigatus inflated conidia, seedlings, and hyphal cells, and inhibit and kill these cells, but they bind to β-glucan instead of α-mannan. In this previous study, cells were continuously incubated with liposomes throughout the assay without washing. To more directly compare the drug targeting efficiency of the two Dectins, DEC1-AmB-LL was examined using the same assay design used for DEC2-AmB-LL in this article, and the liposomes were washed away after 2 hours, but the liposomes were initially diluted into LDB1 and the cells were grown for 16 hours. DEC2-AmB-LL, which delivers 0.5 μM and 0.25 μM AmB, was 28-fold and 5-fold more efficient than AmB-LL in killing A. fumigatus or inhibiting A. fumigatus growth, respectively (Figure 21D). Using this assay condition, the results of Dectin-1 and Dectin-2 targeting to AmB-loaded liposomes were very similar.

[0225] Toxicity of DEC2-AmB-LL to animal cells

[0226] Fast-growing human HEK293 and human HT-29 cell cultures were treated overnight with various liposome and deoxycholate micelle suspensions, each delivering 15 μM AmB. Based on the CellTiter-Blue assay of metabolic activity and viability, DEC2-AmB-LL was 10% to 20% more toxic than AmB-LL or BSA-AmB-LL and 2- to 5-fold less toxic than AmB deoxycholate (AmB / DOC) micelles ( Figure 14 When these cells were treated with lower AmB concentrations, for example, 3 μM AmB, only the AmB / DOC micelles showed measurable toxicity. When examined by fluorescence microscopy, the three liposome preparations did not appear to have any particular affinity for any of these cell lines.

[0227] In summary, the N-terminal domain of Dectin-2, sDectin-2, was coupled to a lipid carrier, and the conjugate was inserted into liposomes with the C-terminal carbohydrate recognition domain (CRD) of each monomer facing outward from the liposome membrane. Given that efficient binding to the extracellular matrix of three different human fungal pathogens was observed for these DEC2-AmB-LLs, sDectin-2 monomers must be conformationally free to form functional dimers required for efficient mannan binding, as C-type lectin receptors tend to bind weakly to some of their substrates. Published estimates of the effective concentration (EC50) for 50% binding of sDectin-2 to mannan-related polysaccharides range from approximately 20 mM (mannose), 2 mM (mannan-α-1-2-mannan), to 150 M (mannan). This is indeed weak binding compared to Dectin-2's closest paralog, Dectin-1, which binds to various β-glucans with an EC50 range of 2 mM to 2.2 picomolar. The higher avidity resulting from the approximately 1,500 sDectin-2 monomers per liposome likely accounts for the rapid, strong, and stable binding observed for DEC2-AmB-LL binding to fungal cells.

[0228] The data showing efficient binding of DEC2-AmB-LL to the extracellular matrix of all three species are consistent with the rich content of mannans in their exopolysaccharides. However, some regions of the matrix of Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus were not stained or stained poorly by sDectin-2-coated liposomes, suggesting that the distribution of mannans in the matrix is ​​heterogeneous or that mannans in these regions are masked from exposure to liposomal sDectin-2. The results indicate that the binding of DEC2-AmB-LL to the exopolysaccharide mannan is no more restricted than that of the much smaller DEC2-Rhod, indicating that size limitation may not be the primary constraint.

[0229] There is no convincing evidence that DEC2-AmB-LL binds to the cell walls of any of these fungal species above trace levels. Although cell wall mannan content varies greatly based on the growth medium and chemical analysis method, the estimated cell wall mannan polysaccharide content of Cryptococcus neoformans is 22%, that of Candida albicans is 40%, and that of A. fumigatus is 15 to 41%. Although rhodamine-labeled sDectin-2 DEC2-Rhod binds to the exopolysaccharide matrix of A. fumigatus with similar intensity and pattern as DEC2-AmB-LL, DEC2-Rhod does not stain the cell wall. Thus, once again, the size barrier of the much larger DEC2-AmB-LL does not appear to explain their lack of cell wall binding. Thus, these findings suggest that cell wall mannans may be chemically masked from sDectin-2-mediated liposome binding. This result is similar to the masking of Candida albicans cell wall β-glucans from sDectin-1 binding and from DEC1-AmB-LL binding.

[0230] Candida albicans can reversibly switch between a unicellular, oval yeast and a multicellular, oval, pseudohyphae and elongated hyphae morphology. All three stages are capable of producing extracellular matrix and adhering to host tissues. All three DEC2-AmB-LL matrices were bound, indicating that biocide-loaded Dectin-2-coated liposome therapy has the potential to reduce the virulence of Candida albicans.

[0231] DEC2-AmB-LL inhibited and killed Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus much more efficiently than plain uncoated AmB-LL or BSA-coated BSA-AmB-LL, which delivered the same concentration of AmB. A combination of a CTB reagent-based metabolic activity assay, a CFU-based cell growth assay, and propidium iodide staining of dead cells confirmed that cell inhibition and killing occurred. Incubation with DEC2-AmB-LL for as little as 30 minutes to several hours resulted in significant killing. When delivering AmB concentrations close to or below the reported MIC values ​​for AmB (at this concentration, DEC2-AmB-LL inhibited or killed fungal cells 3- to 90-fold more efficiently, while the equivalent uncoated AmB-LL had little or no effect on cell inhibition or viability. DEC2-AmB-LL bound to all three species 50- to 150-fold more efficiently than AmB-LL and, under certain experimental conditions, inhibited and killed them 11- to 94-fold more efficiently than AmB-LL.

[0232] A significant reduction in the MIC of AmB or other liposomally packaged therapeutic fungicides should lead to reduced fungicide doses and reduced dosing frequency, thereby reducing host toxicity. It is demonstrated herein that DEC2-AmB-LL is not particularly toxic to animal cells when delivered at 15 μM AmB (which is 15 to 150 times higher than the AmB concentration used here to kill fungal cells).

[0233] These studies were performed using the mouse sDectin-2 protein sequence to avoid concerns about sDectin-2 immunogenicity during future testing of sDectin-2-targeted antifungals in mouse models of candidiasis, aspergillosis, and cryptococcosis. The human sDectin-2 protein sequence is 72% identical to the mouse protein, lacking only two amino acids. Therefore, it may be possible to manipulate the human protein to target fungicide-loaded therapeutic liposomes for clinical studies.

[0234] In summary, new antifungal therapies are urgently needed. DEC2-AmB-LL effectively binds to the extracellular matrix produced by different cell stages of Candida albicans (C.albicans), Cryptococcus neoformans (C.neoformans) and Aspergillus fumigatus (A.fumigatus). DEC2-AmB-LL, which delivers AmB concentrations close to or below the MIC for growth inhibition and killing of all three species, demonstrates that sDectin-2 targeting of liposome-encapsulated drugs improves the antifungal effect by one or more orders of magnitude compared to non-targeted liposomal AmB. It is reasonable to propose that drug-loaded liposomes targeting fungal cells have great potential as a pan-antifungal therapy with broad application.

[0235] Example 3

[0236] Dectin-2-coated antifungal liposomes inhibit fungal burden in a mouse model of pulmonary aspergillosis

[0237] Fungal cell culture

[0238] A human clinical isolate of Aspergillus fumigatus CEA10 (CBS144.89, ATCC MYA1163) has previously been used in a mouse model of aspergillosis (see Desobeaux and Cray, "Rodent Models of Invasive Aspergillosis Due to Aspergillus fumigatus: Stilla Long Path toward Standardization," Front Microbiol. 8, 841 (2017)). Conidia were prepared by growing CEA10 at 37°C for 6 days on 1.5% agar plates containing Vogel's Minimal Medium (VMM) + 1% glucose + 100 ug / mL each of kanamycin and ampicillin. Conidia were harvested by gently shaking the plates with glass beads and phosphate-buffered saline + 0.05% Tween 20. Conidia were concentrated by filtering through a sterile 40 micron nylon mesh filter (Fisher Sci. #22363547, Hampton NH), sedimenting overnight at 1 x g, and the conidial cell density was determined in a hemocytometer. Germination efficiency was demonstrated to be close to 100%.

[0239] Antimetabolites and steroid products

[0240] A 35 mg / mL stock solution of cyclophosphamide (Cayman #13849) was prepared in pH 7.4 saline and delivered at 175 mg / kg mouse body weight. A cortisone acetate suspension (Cayman Chemical Co., #23798, Ann Arbor, MI) was prepared at 22.4 mg / mL in dihydrate and delivered at 112 mg / kg. A stock solution of triamcinolone (MilliporeSigma #T6376, Burlington, MA) was prepared at 40 mg / mL in DMSO and stored at 4°C. Immediately prior to intraperitoneal injection at 40 mg / kg, this stock solution was diluted 1:4 v / v in PBS to prepare an aqueous suspension.

[0241] Immunosuppression-mediated pulmonary aspergillosis mouse model

[0242] Outbred female CD1 (CD-1IGS) Swiss mice, 7 weeks old (25 g to 30 g each), were obtained from Charles River Labs. CD1 mice have been used for most experimental aspergillosis studies. 56 Steroid model (Figure 22A): Triamcinolone was injected intraperitoneally at 40 mg / kg of mouse body weight on day -1 (D-1) and day 3. 63Mice were immunosuppressed using a single IP injection of 175 mg / kg cyclophosphamide on day -3, followed by a single subcutaneous injection of 40 mg / kg triamcinolone on day -1. Since these mice were to be euthanized on day 4 to determine fungal burden, no subsequent immunosuppressant injections were given.

[0243] On day 1, immunosuppressed mice were infected by oropharyngeal aspiration of 50 μL conidia samples (Figure 22). The progression of symptoms began with grooming behavior and folding of the fur, followed by mild lethargy. Once mice exhibited severe lethargy and / or lost 25% of their body weight, they were declared clinically dead and euthanized by cervical dislocation after anesthesia. For fungal burden experiments, all animals were euthanized on day 4. All mouse protocols complied with the guidelines for the ethical treatment of nonhuman animals as outlined by the U.S. federal government and the University of Georgia Institutional Animal Care and Use Committee (IACUC).

[0244] Calcein RC (Blankophor BBH SV-2560, Bayer, Corp.) was used to stain fungal chitin in manually prepared mouse lung sections. A 25 mM stock solution was prepared by dissolving 5 mg in 218 μL of DMSO and storing at 4°C in the dark. Tissue was stained with a solution prepared by diluting the stock solution 1:1,000 in PBS + 5% DMSO to a final concentration of 25 μM Calcein.

[0245] Fungal load was estimated by colony-forming units (CFU) and real-time quantitative PCR (qPCR).

[0246] The lungs removed from animals that survived to day 4 were weighed and minced into hundreds of pieces with a diameter of approximately 1 mm. 3 Fragments of the lung tissue were then mixed so that a subset of the minced tissue could be used to accurately sample the entire lung. CFU: 25 mg of lung tissue was homogenized in 200 μL PBS and spread by shaking with sterile glass beads on YPD (yeast potato dextrose) agar plates containing 100 μg / mL each of kanamycin and ampicillin. After incubation for 16 hours at 37°C, micro-fungal colonies were counted and some colonies were photographed at 4X magnification on the EVOS imaging system (Figures 25B, 25C). The number of CFU reported in Figures 25A and 26A was corrected based on the area of ​​the entire plate relative to each microscope field and the weight of each lung. qPCR: Qiagen's D DNA was extracted from 25 mg replicates of each lung using the Qiagen Blood and Tissue Kit (#69504, Hilden, Germany). Tissue was mixed with 180 uL of buffer ATL and 20 uL of proteinase K according to the manufacturer's instructions. At this point, the protocol was modified to disrupt the fungal cell walls by adding glass beads and shaking the sample at medium speed using a bead mill (RETSCH MM300 Laboratory Mill) for 10 minutes at room temperature. The homogenate was a clear liquid, indicating that both lung cells and fungal cells were completely dissolved in the ATL buffer, but it contained some floating lipids. The material was filtered through a Qiagen Shredder spin column (Cat. No. 79654) to remove the floating lipids. At this point, we returned to the manufacturer's DNA preparation protocol, starting with the recommended incubation at 56°C for 10 minutes. Typically, 10 μg of DNA was obtained from 25 mg of lung tissue. As with the previously described approach 65 Similarly, quantitative real-time PCR (qPCR) was used to estimate the amount of Aspergillus fumigatus rRNA repeat sequences in 100 ng of lung DNA samples. Several new primer pairs were designed targeting the intergenic spacer (IGS) region in the rDNA gene of Aspergillus fumigatus. The optimal primer pair that produced the lowest cycle threshold (Ct) and a single dissociation peak had the following sequences (Af18SrRNA2S forward primer 5′-GGATCGGGCGGTGTTTCTATG A and Af18SrRNA2A reverse primer 5′-TTCTTTAAGTTTCAGCCTTGCGA CCAT). When uninfected lung tissue was examined, this primer pair did not produce a detectable product even after 45 PCR cycles. The relative quantity (RQ) of fungal rDNA IGS was determined using the dCt method (See Livak et al., "Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method," Methods 25, 402-408 (2001)) by normalizing all Ct values ​​to the lowest Ct value determined from the infected lung sample control.

[0247] result

[0248] As described herein, a new delivery system for antifungal drugs was developed using the C-type lectin receptors Dectin-1 and Dectin-2 to target drug-loaded liposomes to glucans and mannans, polysaccharides found in the cell walls and exopolysaccharide matrix of most fungal pathogens, respectively. Compared to the liposomal drugs, in some cases, Dectin-2 coated AmB liposomes reduced the effective dose of liposomal AmB in vitro by about 10 to 90 times to inhibit and kill Aspergillus fumigatus by 90%. In this paper, the in vivo efficacy of this novel therapeutic agent was studied using the steroid and leukopenia model of immunosuppression-mediated pulmonary aspergillosis in mice. These experiments were conducted based on the results reported in the literature. The MIC of AmB was delivered at a concentration of 0.2 mg AmB / kg mouse body weight. For both models, we demonstrated that Dectin-2-targeted AmB-loaded liposomes reduced lung fungal burden compared to non-targeted liposomes delivering the same low AmB concentration. By significantly reducing the effective dose of antifungal drugs, targeted pan-antifungal liposomes have the potential to create a new clinical paradigm for safer treatment of a wide range of fungal diseases, whether they are invasive or localized infections.

[0249] As described herein, the carbohydrate recognition domains of two C-type lectin receptors, Dectin-1 (DEC1) and Dectin-2 (DEC2), were used to target antifungal-loaded liposomes to fungal β-glucans and α-mannans, respectively. Dectin-targeted liposomes specifically bind to the cell walls and extracellular polysaccharide matrix of Aspergillus fumigatus (A. fumigatus), Candida albicans (C. albicans), and Cryptococcus neoformans (C. neoformans). Non-targeted liposomes do not bind specifically and can only passively deliver antifungal agents to fungal cells. As shown herein, DEC1-AmB-LL and DEC2-AmB-LL, DEC1- and DEC2-coated amphotericin B (AmB)-loaded liposomes, effectively bind to all stages of development of A. fumigatus (A. fumigatus), including uninflated and inflated spores, seedlings, and hyphae. They inhibit and / or kill A. fumigatus (A. fumigatus) with a higher efficiency than The results of the study investigated whether this fungal cell-targeting technology is effective in vivo in a mouse model of aspergillosis by targeting the fungal cell line AmB-LL, which is approximately 10- to 90-fold higher than the AmB-LL-like liposome and reduces the effective dose of the drug in vitro by an order of magnitude. Figure 23 The diagram in (right) illustrates that Dectin-coated liposomes loaded with antifungal drugs bind to fungal cells and their extracellular polysaccharide matrix, thereby specifically targeting the drug to the vicinity of the fungal cells. This also concentrates the antifungal agent away from the mammalian cell surface. In contrast, non-targeted drugs passively deliver the antifungal agent to all cells ( Figure 23 , left side).

[0250] Aspergillus fumigatus is the primary causative agent of aspergillosis, one of the four most life-threatening fungal diseases. Globally, there are an estimated 300,000 acute cases of aspergillosis. In 2017 alone, the cost of treating aspergillosis in the United States was between $58,000 and $105,000 per patient, and the annual medical costs of Aspergillus infections in the United States total $1.5 billion. Aspergillosis accounts for 17% of the costs of treating all fungal infections. A. fumigatus is a common soil organism, but is also found in homes and workplaces. Most people inhale thousands of spores every day without becoming infected. Patients at greatest risk for life-threatening invasive fungal infections, such as aspergillosis, typically have weakened immune systems and / or suffer from various lung diseases, which increase the chances of developing fungal infections. Among immunocompromised patients, aspergillosis is the second most common fungal infection after candidiasis. Furthermore, the number of immunocompromised individuals susceptible to various opportunistic fungal infections is increasing due to the rise in cancer, stem cell, and organ transplant patients using immunosuppressants.

[0251] Aspergillosis patients are treated with antifungal drugs such as amphotericin B (AmB), caspofungin and various azole drugs. Almost all antifungal agents are hydrophobic and their low water solubility poses a problem for drug delivery. Liposomal drug formulations with AmB embedded in a lipid bilayer membrane, such as or equivalents, such as AmB-LL, can more efficiently penetrate various organs, penetrate fungal cell walls, and show reduced nephrotoxicity and less infusion toxicity at higher AmB doses than detergent-solubilized AmB (e.g., AmB-DOC). Dectin and its equivalent, AmB-LL, are often used to kill Aspergillus fumigatus (A. fumigatus) residing in biofilms. Dectin-targeted technology can enhance antifungal activity in biofilms because Dectin binds to the extracellular polysaccharide matrix that helps form biofilms. However, all antifungal agents, whether packaged in liposomes or not, have serious limitations due to lack of sufficient fungicidal activity, the increase in drug-resistant fungal species, and host toxicity to cells and organs. For example, liposomal AMB can cause nephrotoxicity (see, e.g., Allen U. "Antifungal agents for the treatment of systemic fungal infections in children" Paediatrics & Child Health 15, 603-608 (2010); and Dupont B. "Overview of the lipid formulations of amphotericin B" JAntimicrob Chemother 49 Suppl 1, 31-36 (2002). Because of its side effects, clinicians often use the nickname "Amphoterrible" to describe amphotericin B. Even with drug treatment, the one-year mortality rate for patients with aspergillosis typically ranges from 10% to only 90%, depending on the patient's underlying condition. In approximately 10% of patients, invasive aspergillosis progresses to cerebral aspergillosis, a brain infection with a mortality rate as high as 99%.

[0252] One of the goals of using the Dectin-based technology described herein was to demonstrate increased efficacy of Dectin-2-coated liposomal drugs in inhibiting and killing A. fumigatus in vivo in an immunosuppression-mediated aspergillosis mouse model (Desoubeaux et al., "Animal Models of Aspergillosis" Comp Med 68, 109-123 (2018)). When working with concentrations of AmB that matched the minimum inhibitory concentration of AmB delivered by AmB-LL, we found that DEC2-AmB-LL significantly reduced lung fungal burden relative to AmB-LL delivering the same AmB concentration.

[0253] Different publications indicate that it is necessary to use 1 × 10 4 to 1×10 7fumigatus strain CEA10 was used to induce lethal aspergillosis infection in immunosuppressed CD1 outbred Swiss mice (see Desoubeaux et al.; and Herbst et al., "A new and clinically relevant murine model of solid-organ transplant aspergillosis" DisModelMech 6, 643-651 (2013)). When delivered to the lungs of mice, CEA10 germinated faster and showed greater virulence than the more commonly used A. fumigatus Af293 strain. In the in vivo model, as or The reported minimum inhibitory concentrations (MICs) of AmB delivered by AmB-LL range widely from 0.06 to 1.0 mg / kg mouse body weight and appear to vary greatly by A. fumigatus strain. By working at low AmB doses in AmB-LL, we sought to demonstrate that Dectin-coated liposomes targeted to fungal cells in vivo have the most improved performance relative to non-targeted liposomes in vivo. Treatment of A. fumigatus growth and viability in culture (previously demonstrated to be 1 or 2 orders of magnitude) improved the in vitro performance of Dectin-targeted liposomes when working at concentrations close to the in vitro MIC of AmB-LL. The experiments described herein show that The 0.2 mg AmB / kg delivered by AmB-LL was sufficient to measure a slight decrease in A. fumigatus levels in the lungs compared to infected control mice.

[0254] Healthy mice, like healthy humans, have a natural resistance to infection with certain species of Aspergillus. Therefore, mice must be immunosuppressed to develop acute aspergillosis. Two different immunosuppressive mouse models were used, the steroid model and the leukopenic model, which differ in the quality of the suppressed immune cell function and the severity of the immunosuppression. Figure 22 shows two protocols and timelines for examining the efficacy of Dectin-2-coated antifungal liposomes in a mouse model of pulmonary aspergillosis. Briefly, in the steroid model, CD1 mice were immunosuppressed with the synthetic steroid triamcinolone or cortisone (Figure 22A), while in the leukopenic model (Figure 22B), mice were immunosuppressed with the antimetabolite cyclophosphamide and steroids. In the steroid and leukopenic models of immunosuppression-mediated aspergillosis, immunosuppressed mice received 2×10 6and oropharyngeal inoculation of 100,000 conidia of Aspergillus fumigatus (A. fumigatus) strain CEA10. By day 2 (day 2 post infection), at least one large infection center of approximately 1 mm in diameter or greater was observed in almost every lobe of the mouse lungs examined. These infection centers consisted of clusters of short hyphae (Figure 24). On day 1 after fungal inoculation (Figure 22), the infected mice were randomly divided into three groups. One treatment group received Dectin-2-coated DEC2-AmB-LL that delivered 0.2 mg AmB / kg mouse body weight, and a second liposome-treated group received non-targeted AmB-LL that also delivered 0.2 mg AmB / kg; the control mock-treated group received only liposome dilution buffer. All conidia and liposomes were administered to mice via oropharyngeal delivery as previously described for lung disease models (see De Vooght et al., “Oropharyngeal aspiration: an alternative route for challenging in a mouse model of chemical-induced asthma,” Toxicology 259, 84-89 (2009)).

[0255] DEC2-AmB-LL treatment significantly reduced fungal burden compared with non-targeted AmB-LL.

[0256] The effect of antifungal liposome treatment on lung fungal burden was examined. First, a steroid model of immunosuppression-mediated aspergillosis was used (Figure 22A). CD1 Swiss mice were immunosuppressed with triamcinolone on days 1 and 3. On day 0, they were infected with 2×10 6 On day 1, mice were treated with DEC2-AmB-LL or with a mouse model that delivered 0.2 mg AmB / kg body weight. Mice were treated with DEC2-AmB-LL liposomes or with a control buffer for diluting liposomes. Between the 1st and 4th day, some animals in each treatment group died, but at least three animals survived to the 4th day in each treatment group. On the 4th day, three surviving animals were randomly selected from each group for euthanasia, and their lungs were collected and weighed. The fungal load in the lungs was checked by two methods (Figure 25). Homogenized lung tissue was plated on a rich growth medium, and after 16 hours, the average number of colony-forming units (CFU) in each lung was estimated, as shown in Figure 25 A. The number of live fungal cells that can form colonies shown by mice treated with DEC2-AmB-LL was 12.5 times lower than that of mice treated with AmB-LL, and 20.5 times lower than that of control mice treated with liposome dilution buffer. For the generation of image examples of fungal cell microcolony fields for these data, see Figure 25 B (mice treated with AmB-LL) and Figure 25 C (mice treated with DEC2-AmB-LL). To independently estimate the reduction in fungal burden caused by DEC2-AmB-LL, quantitative polymerase chain reaction (qPCR) was used to measure the relative number of A. fumigatus ribosomal rDNA gene copies in replicate samples of homogenized lung tissue from the same three mice in each of the three treatment groups. Figure 25D Based on the relative abundance estimates of rDNA, treatment with DEC2-AmB-LL resulted in a significantly lower fungal burden per lung than treatment with The levels in mice treated with AmB-LL were 22-fold lower and 40-fold lower than those in control mice. The concentration of AmB delivered in both fungal burden experiments was 0.2 mg / kg, which is within the reported range. The results were within the MIC range for Aspergillus fumigatus fungal burden in a mouse model of aspergillosis. An approximately 40% reduction in fungal burden was observed with AmB-LL compared to control animals, indicating that the concentration was at the high end of the MIC for non-targeted drugs.

[0257] Second, a leukopenic mouse model of aspergillosis mediated by immunosuppression was used. This model renders mice even more susceptible to aspergillosis and requires a lower dose of conidia to establish acute infection (Figure 22B). CD1 Swiss mice were immunosuppressed with triamcinolone on days -3 and -1. On day 0, they were infected with 5 × 10 5 On day 1, mice were treated with targeted DEC2-AmB-LL or non-targeted DEC2-AmB-LL to deliver 0.2 mg AmB / kg mouse body weight. AmB-LL liposomes were used to treat mice or mice were treated with a control buffer solution for diluting the liposomes. At 4 days (D4), except for one animal, all animals in the three treatment groups were still alive. An animal treated with a control buffer solution died on the morning of the 4th day, and the remaining control animals and one mouse treated with AMB-LL showed a reduction in grooming behavior. All remaining animals in the AmB-LL and DEC2-AmB-LL treatment groups looked relatively healthy. Three surviving animals from each treatment group were randomly selected, euthanized, and their lungs were collected and fungal load was estimated (Figure 26) by the two methods just described. The average number of colony forming units (CFU) per lung shown by the mice treated with DEC2-AmB-LL was 100 times lower than that of the mice treated with AmB-LL (Figure 26 A). Obviously, targeting antifungal liposomes to fungal cells significantly improved the efficacy of AmB packaged in the liposomes. The average number of CFU of the mice treated with AmB-LL was lower, but statistically indistinguishable from the animals treated with control buffer solution. The high fungal burden in the AmB-LL treated group was due to the fact that one mouse that had shown reduced grooming behavior had a very high fungal titer. In contrast, one mouse treated with DEC2-AmB-LL had no detectable fungal colonies, resulting in a very low mean CFU for this treatment group. As an independent estimate of fungal burden, the relative number of A. fumigatus rDNA gene copies was also examined on DNA prepared from parallel samples of homogenized lung tissue from the same mice ( FIG. 26B ). Treatment with DEC2-AmB-LL resulted in a significantly higher fungal burden per lung than treatment with DEC2-AmB-LL. Levels in mice treated with AmB-LL were 600-fold lower than in mice treated with AmB-LL. Clearly, targeting liposomal AmB to fungal cells using Dectin-2 significantly enhanced the drug's performance in reducing lung fungal burden.

[0258] As described herein, an alternative to antibody targeting of liposomes was developed by using the C-type lectin carbohydrate recognition domain of Dectin-2 in a pan-fungal delivery system to target liposome drugs to mannans in fungal cells and their exopolysaccharide matrix ( Figure 23). There is a concern that antibodies may not be as effective as Dectin-coated liposomes as a means of targeting liposomes to fungal pathogens for two main reasons. First, high affinity monoclonal antibodies may react only with a specific subset of glucans, mannans, xyloglucan cross-links and mannoproteins due to excessive specificity, thereby targeting only a small subset of fungal species or even a subset of fungal cells of the same species. The pan-antifungal activity of Dectin-targeted liposomes against a wide variety of invasive fungal pathogens should make them more worthy of extensive and expensive clinical development. Second, less cumbersome methods have been developed for producing the functional carbohydrate recognition domains of Dectin-1 and Dectin-2, DEC1 and DEC2, in Escherichia coli (E. coli) at a cost that is a few percent of the cost of producing the same molar concentration of monoclonal antibodies. Third, low reagent costs should encourage the clinical development of improved antifungal agents for the treatment of acute invasive fungal diseases and superficial fungal infections.

[0259] The data presented here demonstrate that, compared to non-targeted Like AmB-LL, DEC2-AmB-LL has significantly enhanced antifungal activity. Using two different immunosuppression-mediated mouse aspergillosis models, DEC2-AmB-LL reduced lung fungal burden relative to The equivalent AmB-LL reduced the fungal burden by one to two orders of magnitude. It is expected that treatment with DEC2-AMB-LL will provide improved mouse survival in the mouse model described herein. The significant reduction in fungal burden should be inferred to improve long-term patient survival.

[0260] Equally important, the significant effect was demonstrated using DEC2-AmB-LL, which delivered only 0.2 mg AmB / kg mouse body weight (a concentration close to the lower limit of the reported MIC of liposomal AmB against A. fumigatus in vivo in a mouse model of aspergillosis). In the studies described herein using both models, delivery of 0.2 mg AmB / kg of AmB-LL had modest or no effect on fungal burden. Studies of the efficacy of DEC2-AmB-LL in lung models of aspergillosis typically require concentrations of 5 to 20 mg AmB / kg to produce maximal antifungal effects on lung fungal burden and significantly improve mouse survival. Furthermore, multiple doses of AmB-LL are often required over several days post-infection to ensure significant reductions in fungal burden and mouse survival. However, here we demonstrate that low doses of AmB delivered by targeted DEC2-AmB-LL significantly reduce fungal burden, exceeding most studies in the literature to date. Even more striking reductions in fungal burden have been reported in mouse models. By significantly lowering the effective dose of AmB and reducing the number of doses required to control aspergillosis, Dectin-2-targeted liposomal AmB drugs, such as DEC2-AmB-LL, should be able to effectively control Aspergillus infections at concentrations that do not cause organ toxicity in humans.

[0261] Furthermore, the data reported herein on the use of Dectin-2 targeted liposomes that delivered only 0.2 mg AmB / kg and achieved an order of magnitude reduction in fungal burden suggest that targeting antifungal-loaded liposomes directly to fungal cells may be more effective than targeting adjacent lung cells and tissues. The close proximity of antifungal-loaded liposomes to fungal cells is likely to increase the local concentration of drug delivered to these cells. The in vitro data presented herein suggest that Dectin-2 primarily targets α-mannans in the exopolysaccharide matrix of A. fumigatus, rather than the mannan content of its cell wall. We have not yet confirmed that Dectin-2 primarily targets the exopolysaccharide matrix of A. fumigatus in vivo in a mouse model.

[0262] There are several different mouse models of aspergillosis that mimic the different types of immunosuppression suffered by the various patient populations that are most susceptible to aspergillosis. In this paper, a steroid and leukopenia model of immunosuppression was used to mediate fungal infection (Figure 22). In the steroid model, mice are pretreated with the synthetic glucocorticoid triamcinolone, which subverts the adaptive immune response by inhibiting the activity of some T cells and B cells, thereby increasing the proliferation of Aspergillus fumigatus hyphae into lung tissue. The steroid immunosuppressive mouse model effectively mimics the immunodeficient state of immunosuppressed patients who have received solid organ transplants and some patients who have received stem cell transplants, who are particularly susceptible to aspergillosis (see Desoubeaux et al.). However, a disadvantage of this model is the excessive recruitment of neutrophils to the affected tissues, especially the lungs, which leads to a vigorous inflammatory response. Because the immune system is only partially disabled, a relatively large fungal inoculum of at least 2×10 6 conidia to ensure that 100% of the mice develop acute aspergillosis. Most use between 500,000 and 10 6 Mice treated with smaller inocula ranging from 100 conidia to 2000 conidia survived without antifungal treatment, while at the other extreme, almost all mice treated with 5 × 10 6Treated mice died by day 3. In the steroid model, some vaccinated mice died as early as days 1 and 2, suggesting that the mice may have succumbed to inflammation and infection before the liposome treatment had time to take effect. Nevertheless, DEC2-AmB-LL, which targets fungal cells, reduced lung fungal burden compared to nontargeted AmB-LL, which delivers low doses of AmB.

[0263] In the next set of experiments, a leukopenic mouse model was employed (Figure 22B) in which mice were pretreated with cyclophosphamide and triamcinolone. Cyclophosphamide is a DNA synthesis inhibitor and antimetabolite that induces apoptosis and rapid reduction of various leukocyte populations, including B cells and memory T cells. This was added to the immunosuppressive effects of the steroid triamcinolone. This model abolishes most innate and immune responses and is much less restrictive to fungal cell proliferation than the steroid model. The severe leukopenia resembles the immunosuppressed state of hematopoietic stem cell transplant recipients. Because these mice develop severe leukopenia, a smaller fungal inoculum can be used, slowing the initial progression of the disease. It is hoped that slowing the initial progression of infection will allow time for treatment with AmB-LL or DEC2-AmB-LL to reduce the fungal burden and improve the survival of the mice in future experiments.

[0264] Both models produced statistically convincing evidence that liposomes targeted to fungal cells reduced the fungal burden in the lungs relative to non-targeted liposomes delivering low doses of AmB. This significant reduction in lung fungal burden at low doses of AmB, as shown in Figure 26, will undoubtedly translate into improved mouse survival and reduced animal toxicity.

[0265] As shown herein, DEC2-AmB-LL effectively binds to mannans in the extracellular polysaccharide matrix of in vitro grown Aspergillus fumigatus (A. fumigatus), Candida albicans (C. albicans), and Cryptococcus neoformans (C. neoformans) and improves the efficacy of killing all three fungal species. The above experiments show that Dectin-2 targeted liposome-mediated antifungal drug delivery significantly improves the efficacy of inhibiting and killing Aspergillus fumigatus (A. fumigatus) in two different immunosuppression-mediated aspergillosis mouse models. Considering the in vitro data showing effective activity against multiple fungal pathogens and the in vivo mouse data on A. fumigatus provided herein, Dectin-2 targeting of antifungal drugs should have pan-antifungal applications against many other invasive fungal diseases, including candidiasis and cryptococcosis, and possibly against dermatophytes (e.g., tinea pedis infections and nail onychomycosis caused by Trichophyton rubrum).

[0266] Example 4

[0267] Diagnostic methods

[0268] Cell culture

[0269] A. fumigatus strain CEA10 (ATCC MYA1163) was cultured in Vogel's minimal medium (VMM) + 1% glucose in 24-well polystyrene microtiter plates pre-coated with poly-L-lysine. 77 Or RPMI 1640 medium + 1% glucose (no red indicator) (ThermoFisher SKU-11835-030, Waltham, MA) at 37 ° C. for 12 to 16 hours. The cells were washed with PBS (150 mM NaCl, 10 mM phosphate, pH 7.4), fixed with 4% formaldehyde for 45 minutes and washed three times with PBS, and then the cells were incubated with DEC2-BiFC reagent liposomes.

[0270] Venus fusion protein

[0271] The sequences of the codon-optimized E. coli expression constructs DEC2-VyN and DEC2-VC are shown in Figure 27 along with the protein sequences and some predicted protein properties they produce. A flexible GlySer-rich spacer of 15 amino acids in length separates the DEC2 sequence from the Venus fragment. Spacers as short as 2 amino acids are commonly used in BiFC constructs, such as pET-BiFC. It should be understood that spacers of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acids or more can be used in these constructs. In this example, a relatively long spacer is used to prevent the complementary VyN and VC portions of the fusion protein from spontaneously associating when placed adjacent to each other on liposomes. Both gene sequences were synthesized by GenScript and subcloned into the pET-45B expression vector. 在于37 After IPTG induction for 6 hours at 4°C, the modified proteins were expressed in the BL21 strain of Escherichia coli. Both were extracted from cells, purified on a nickel affinity column, and stored in denaturing buffer #1 (pH = 8.0, 6M GuHCl (Fisher BioReagents BP178), 0.1M Na2HPO4 / NaH2PO4, 10mM triethanolamine, 100mM NaCl, 5mM 2-mercaptoethanol, 0.1% Triton-X100) as previously described for mouse sDectin-1.

[0272] DEC2-BiFC reagent construction

[0273] 5 μg / μL of both protein samples in this same GuHCl buffer (freshly supplemented with 5 mM 2-mercaptoethanol) were adjusted to pH 8.3 with 1 M pH 10 triethanolamine and reacted with a 4 molar excess of the reactive succinimidyl ester NHS portion of DSPE-PEG-3400-NHS (1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene glycol (PEG) (from Nanosoft polymers, 1544-3400) at 23°C for 1 hour. The PEG portion of the PEG-DSPE moiety makes these hydrophobic proteins slightly more soluble, while DSPE is a lipid that allows insertion into liposome membranes. The proteins were lysed by filtration using Bio-Gel P-6 acrylamide resin (Bio-Rad #150-0740) in renaturation and storage buffer RN #5 (0.1 M Size exclusion chromatography was performed in a 5% paraformaldehyde (NaH2PO4, 10 mM triethanolamine, pH 8.0, 1 mL of arginine, 100 mM NaCl, 5 mM EDTA, 5 mM 2-mercaptoethanol) solution to remove unincorporated DSPE-PEG and GuHCl. 69 Both modified proteins were stored in RN#5 at a concentration of approximately 5 μg / μL until used in activity assays or incorporated into liposomes. After removal of GuHCl by dialysis, protein purity levels were checked using SDS-PAGE.

[0274] Remote loading of DEC2-VyN and DEC2-VC into liposomes

[0275] Starting with sterile PEGylated liposomes (DSPC:CHOL:mPEG2000-DSPE, FormuMax #F10203A) from FormuMax Sci. Inc., a small batch of liposomes was prepared by adding 0.5 mole percent of each of DSPE-PEG-modified DEC2-VyN and DEC2-VC, still in RN#5 buffer, relative to 100% liposomal lipids used to prepare the DEC2-BiFC reagent (Table 2), and incubating in PBS at 60°C for 30 minutes. Table 2 shows the chemical composition of the DEC2-BiFC reagent liposomes discussed herein, expressed as moles of DEC2-VyN and DEC2-VC, where the total amount of liposomal lipids represents 100 mole percent, and compares this chemical composition with the recently described amphotericin B-loaded DEC2-AmB-LL. DEC2-BiFC reagent liposomes were diluted into liposome dilution buffer #2 (LDB2, 20 mM HEPES, 10 mM triethanolamine, 150 mM NaCl, 10 mM CaCl2, 1 mM β-mercaptoethanol (BME), 5% BSA pH 8.0) and then used for fluorescence measurement.

[0276] Table 2

[0277]

[0278] Binding of DEC2-BiFC reagent to soluble polysaccharides and cells

[0279] Cell-free assay. Soluble mannan from Saccharomyces cerevisiae (Sigma, Catalog #084K3789), laminarin from Laminaria digitate (Sigma-Aldrich, Catalog #L-9634), sucrose (Sigma), and polysaccharide stocks of dextran-T40 purified from Lactobacillus species and size-fractionated to 40,000 molecular weight (Pharmacosmos, Catalog #551000409007) were prepared at 10 mg / mL in PBS. These were then diluted 1:10 into LDB2 buffer containing DEC2-BiFC reagent liposomes and incubated at room temperature.

[0280] Cell-based assay. Formalin-fixed fungal cells were incubated with liposomes at 23°C in liposome dilution buffer LDB2 (20mM HEPES, 10mM triethanolamine, 150mM NaCl, 10mM CaCl2, 1mM β-mercaptoethanol (BME), 5% BSA, pH 8.0), with freshly added BME. DEC2-BiFC liposomes were diluted into LDB2 buffer and then incubated with cells to a w / v concentration of DEC2 protein components between 2μg / 100μL, 1.0μg / 100μL, or 0.5μg / 100μL, respectively. Incubations were performed at 23°C or 4°C. Images of fungal colonies stained with Venus green fluorescent DEC2-BiFC reagent liposomes were taken at 20X on an Olympus IX70 inverted microscope using a GFP filter, and parallel images were taken in bright field. Merged images were prepared in Adobe Photoshop by subtracting the green and blue channel data from the brightfield image and adding back the green channel data from the green fluorescence channel.

[0281] result

[0282] Dectin-2 drifts as a monomer in lymphocyte membranes but must form a dimer with its extracellular C-type lectin receptor domain to bind to α-mannans found in fungal cell walls, exopolysaccharide matrices, biomembranes, and polysaccharide fragments released into tissues and blood. Dimerization signals fungal infection to the immune system. We exploited the dimerization property of Dectin-2 to design a cell-free pan-fungal detection system based on bimolecular fluorescence complementation (BiFC). The carbohydrate recognition domain of Dectin-2 (DEC2) was fused to two complementary fragments of the green fluorescent protein VENUS, VyN and VC (DEC2-VyN). The DEC2-Venus fusion protein was modified with the lipid carrier DSPE-PEG and floated as a monomer in the liposome membrane to prepare the DEC2-BiFC reagent. The DEC2-BiFC reagent produced a fungal cell-specific green fluorescent signal upon binding to Aspergillus fumigatus (A. fumigatus). No cell-free specific fluorescent signal was generated. The reagent produces a mannan-specific signal upon binding to soluble polysaccharides. Future work will explore the signal produced upon binding to Candida albicans and Cryptococcus neoformans. DEC2-BiFC technology has the potential to serve as a simple, rapid, single-step diagnostic test for life-threatening invasive fungal infections.

[0283] Invasive fungal infections are often misdiagnosed as non-fungal related diseases. The delay of antifungal therapy significantly increases the risk of death in patients. The following are examples of aspergillosis, candidiasis and cryptococcosis being misdiagnosed as non-fungal diseases. Many symptoms of aspergillosis and tuberculosis (TB) are identical, resulting in initial misdiagnosis and the use of antibacterial drugs to treat TB rather than antifungal treatment. Similarly, invasive candidiasis is often misdiagnosed as bacterial infection, and these patients are treated with broad-spectrum antibiotics. In addition, some species of Candida species are promoted to overgrow with antibacterial drug treatment, and increase the chance that patients obtain acute cases of invasive candidiasis. Cryptococcal meningitis is often misdiagnosed as brain cancer, and therefore will not be treated with antifungal drugs immediately. This article provides a reliable, rapid, sensitive pan-fungal diagnostic method for differentiating fungal infections and distinguishing them from non-fungal diseases with similar symptoms.

[0284] Current methods for diagnosing fungal infections suffer from significant discriminability issues in terms of reliability, rapidity, and sensitivity. The most commonly used technique for diagnosing candidiasis is an in vitro culture method capable of detecting as few as 1 cell per milliliter. However, cell culture techniques and even PCR fail to detect candidiasis in 50% of patients with the disease because Candida albicans yeast cells can harbor in host tissues shortly after infection, thereby evading fungal cell-based serum detection. Because Aspergillus and Candida species release fungal-specific polysaccharides into serum, sputum, bronchoalveolar lavage fluid, and urine, assays for fungal polysaccharides, particularly immunoassays for mannans, galactomannans, and β-glucans, have been developed. However, current mannan and galactomannans assays (e.g., Platelia Aspergillus, Bio-Rad) and panfungal β-glucan assays (e.g., Fungitel, Beacon Diagnostics) for acute aspergillosis are primarily multistep immuno-ELISA sandwich assays. These commercial assays are not rigorously reliable and either fail to detect infection or produce false positives in a significant proportion of patients. Furthermore, these assays are not easily adaptable to point-of-care settings, further delaying diagnosis. Cryptococcal meningitis is diagnosed using antibody-based ELISAs and lateral flow assays for cryptococcal antigens, as well as PCR assays for fungal DNA, but these assays have similar limitations when applied to patient cerebrospinal fluid samples. Similarly, earlier detection would save the lives of many patients with invasive fungal diseases. Clearly, there is an urgent need for more reliable, simple, rapid, single-step, point-of-care fungal diagnostics to reduce the misdisposition of patients with invasive fungal diseases.

[0285] Dectin-2 (CLEC6A gene) is a C-type LECtin domain-containing transmembrane receptor expressed on the surface of certain lymphocyte types in mice and humans. Dectin-2 forms dimers or multimers when bound to fungal α-mannan and mannoproteins, thereby signaling fungal infection to the innate and adaptive immune systems. Although the affinity constants for the interaction of cell membrane receptors with high molecular weight polymorphic fungal polysaccharides are difficult to determine, the affinity estimates of sDectin-2-protein fusions for model mannans obtained from various publications indicate that it has only a modest affinity for mannan-containing polysaccharides. However, the wild-type and Clec4n - / - Comparison of responses in null mice showed that Dectin-2 signaling was maximal in response to a mannan polymer concentration of 1 ng / mL, implying an affinity constant, Kd, ​​in the subnanomolar range.

[0286] As shown herein, sDectin-2 (DEC2)-coated liposomes bind very rapidly, efficiently, and relatively irreversibly to the extracellular polysaccharide matrix of Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans. Thus, a new pan-fungal diagnostic method utilizing DEC2 has been developed. Candida, Cryptococcus, and Aspergillus species belong to three evolutionarily distinct fungal classes: Saccharomycetes (Ascomycota), Tremellomycetes (Basidiomycetes), and Eurotiomycetes (Ascomycota). It is estimated that they diverged from a common ancestor relatively early in the evolution of the fungal kingdom, between 80 and 1.3 billion years ago. Because DEC2-coated liposomes specifically bound to the extracellular matrix of all three genera, this suggests that the mannans found in the extracellular matrix of most pathogenic fungi are structurally conserved enough to be recognized by DEC2-based diagnostic methods.

[0287] Here, we exploit the property of DEC2 to form dimers upon binding to mannan-containing polysaccharides in order to pair Molecular fluorescence complementation ( DEC2 is fused to a complementary fragment of the fluorescent protein VENUS and combined in the same liposome. The resulting DEC2-BiFC reagent liposomes produce a clear fluorescent signal upon binding to Aspergillus fumigatus cells. This technology provides a simple, rapid, single-step, point-of-care assay for a wide variety of fungal pathogens, their exopolysaccharide matrices, biofilms, and released polysaccharides.

[0288] A model of a liposome-based single-step detection system using DEC2 is shown in Figure 28. It is based on the dimerization of DEC2 because it binds to fungal mannan and BiFC. Two separate sDectin-2 proteins were produced. The first protein was fused to the N-terminal portion of the green fluorescent protein Venus VyN (Venus residues 1-155, mutant T154M) to produce DEC2-VyN (Figures 28A, 28B). The advantage of the VyN Venus mutant fragment is that it has reduced spontaneous association with the C-terminal Venus fragment and a strong signal after association with the C-terminal fragment, while it has few advantages over the N-terminal fragments of other fluorescent proteins or unmodified VN fragments. The second protein produced was fused to the C-terminal portion of VenusVC (Venus residues 155-238) to produce DEC2-VC (Figures 28A, 28B). This VC fragment has previously cooperated with VyN in successful BiFC applications. Figure 27 shows the exact sequences of two coding DNAs and two BiFC fusion proteins, DEC2-VyN and DEC2-VC. Both coding sequences were expressed in Escherichia coli (E. coli), and the proteins were extracted into guanidine hydrochloride denaturing buffer and purified by affinity chromatography using the methods described herein. While still denatured, the complementary pairs of fusion proteins were each coupled to the amine-reactive, dual-lipid reagent DSPE-PEG-NHS via free lysine residues. The DSPE-PEG-modified proteins were then inserted into the membranes of identical 100-nanometer diameter liposomes via their coupled DSPE moieties in equal molar ratios to prepare DEC2-BiFC reagent liposomes (Figure 28A). The number of DEC2 fusion proteins inserted into each 100-nm liposome was limited to 1 molar percentage of DEC2, or 1,500 DEC2 molecules, relative to the number of liposome lipid molecules. In other words, approximately 750 DEC2-VyN and 750 DEC2-VC protein molecules were present in each liposome. The goal here was to use a protein concentration low enough to avoid spontaneous association of the two Venus fragments (such association would likely produce a high background fluorescence signal in the absence of any mannan binding), but a sufficiently high concentration of DEC2 to promote efficient binding, dimerization, and a strong signal when the reagent encounters fungal mannan. It has been proposed that DEC2-VyN and DEC2-VC monomers, floating together in the liposome membrane, form dimers or multimers almost exclusively when they bind to fungal cell mannan. It has also been proposed that DEC2 dimerization would promote the assembly of complementary portions of the Venus fluorescent protein -VyN and -VC to produce a BiFC signal.

[0289] A cell-free microtiter plate assay was performed to demonstrate that the DEC2-BiFC reagent liposomes produced a fluorescent signal specific for the polysaccharide α-mannan. DEC2-BiFC reagent liposomes delivered 1 μg / 100 μL of Venus

[0290] LDEC2 protein in the culture medium was incubated with soluble yeast mannan containing α-mannan, laminarin (soluble β-glucan), sucrose (a disaccharide of glucose and fructose), and dextran (α-glucan). The signal of the GFP channel was monitored in a fluorescence microtiter plate reader. Figure 29 The results show that statistically significantly higher levels of Venus green fluorescence were observed in wells containing mannan relative to wells containing other polysaccharides (p = <0.01). These results support the view that the signal is specific to mannan, as expected for Dectin-2. The maximum signal was observed after two hours and decreased with increasing incubation time.

[0291] A cell-based microscopy assay was performed to demonstrate that DEC2-BiFC reagent liposomes produce a fungal cell-specific fluorescent signal. Aspergillus fumigatus conidia were germinated on poly-L-lysine-coated microtiter plates and grown overnight to the early hyphal stage, forming small colonies with diameters of 300 to 500 microns. The cells were fixed in formalin and washed into liposome dilution buffer. The cells were incubated with DEC2-BiFC reagent that delivered 2 μg / 100 μL, 1 μg / 100 μL, and 0.5 μg / 100 μL of total DEC2 protein concentrations to the incubation medium.

[0292] In the GFP channel (Ex515 / Em528) of an inverted fluorescence microscope, the BiFC signal is easily seen. Figure 30 shows the fluorescent signal formed when the DEC2-BiFC reagent (1 μg DEC2 protein / 100 μL) is combined with an Aspergillus fumigatus (A. fumigatus) colony. The BiFC fluorescent signal is only observed when associated with fungal cells and is therefore fungal cell specific (Figure 30B, D, F, H). Each of more than one hundred fungal cell colonies was examined for the combined reagent. The signal is strongest at the center of each fungal colony. Please note that the center of each colony has the thickest cell layer and the oldest cells, and has the most time to produce adhesive exopolysaccharides rich in mannan to help them adhere to the lysine-coated plate surface. We have previously shown that DEC2 is particularly good at binding to exopolysaccharides produced by Aspergillus fumigatus (A. fumigatus), and exopolysaccharide deposition should enhance binding to the DEC2-BiFC reagent. There are long hyphae that are stained along most of their length. The maximum signal was observed after hatching for two to three days. There were some fungal hyphae that did not show fluorescent signal and may not be bound to the examples of reagents (white arrows, Figure 30 B). The fungal cell-specific signal of similar intensity was also detected using the liposomes delivering 2 μg DEC2 / 100 μL, and at 0.5 μg DEC2 / 100 μL, the signal was slightly weaker. No fluorescence was observed between cells (Figure 30 B, D, F, H) or in the control wells lacking fungal cells. The control cells hatched with buffer did not show green fluorescence signal (Figure 30 I and J).

[0293] Venus as the fluorescent protein of choice in BiFC constructs

[0294] Venus was chosen as the optimal fluorescent protein for constructing a BiFC pan-fungal diagnostic for two reasons. First, Venus was chosen because its two fragments (the C-terminal fragment VC and the mutant N-terminal fragment VyN) have the lowest reported spontaneous association rates among several of the best-studied yellow, green, and sky-blue fluorescent proteins when not forced together by a chaperone protein. Spontaneous association produces a fluorescent signal that is independent of binding of the fusion protein to the desired target and has been a major challenge in BiFC technology since its inception. A low spontaneous association rate is considered essential for developing diagnostic reagents that do not produce false positives (i.e., fluorescent signals in the absence of fungal cell mannans). Second, Venus is one of the brightest fluorescent proteins and is derived from a protein in the very bright yellow fluorescent protein (YFP) family. The quantum yield (the number of photons released per absorbed photon) is in the range of 0.5 to 0.6 (e.g., 50% to 60% of the theoretical 100% yield). 70 This is similar to the quantum yield of 0.7 for rhodamine, one of the most fluorescent low-molecular-weight fluorescent molecules commonly used in fluorescence cell chemistry.

[0295] BiFC reagent liposomes

[0296] The DEC2-BiFC reagent liposomes containing DEC2-VyN and DEC2-VC were designed for several reasons, rather than using the two soluble proteins, DEC2-VyN and DEC2-VC, in solution. First, DEC2 monomers are relatively insoluble, and by storing them in 6 M guanidine hydrochloride buffer, they can be kept soluble. Second, the addition of the DSPE-PEG moiety stabilizes and partially solubilizes the DEC2 monomers, allowing them to be stored for a period of time in a mildly denaturing buffer containing 1 M arginine. This modification is essential for the insertion of the modified proteins into the liposomes. However, this modified form of the two proteins has the potential for use as a diagnostic reagent without liposomes. Third, when using liposomes, the local concentration of DEC2 monomers can be controlled in a more easily manipulated, stable reagent. Again, the goal is to achieve a high DEC2 concentration, but not so high as to cause spontaneous association of the attached Venus fragments and a significant, measurable fluorescence background. For example, in these initial experiments, 1,500 DEC2 monomers (i.e., 750 DEC2-VyN plus 750 DEC2-VC monomers) were found to be effective. Even after storing the liposomes at 4°C for 2 months, no spontaneous signal formation was observed, understanding that the monomer concentration can be increased or decreased. Fourth, and perhaps most importantly, the avidity properties of the reagent liposomes should result in an exponential increase in fluorescence signal intensity. The potential for avidity is generated by having multiple mannan binding sites per liposome, such as observed for pentameric IgM antibodies. Once a DEC2-VyN and DEC2-VC pair binds to mannans in a mannan-rich fungal polysaccharide, the signal should be amplified as the binding spreads to adjacent DEC2 molecules on the liposome binding to adjacent mannan moieties in the mannan-rich polysaccharide. Once developed, the affinity should also stabilize the signal. Soluble BiFC diagnostic reagents do not offer these advantages. The data presented here were performed using DEC2-BiFC reagent liposomes. The results were clearly positive, and there was no doubt that the fluorescent signal produced by this reagent was specific to fungal cells.

[0297] In other examples, a shorter spacer can be used to force the VyN and VC segments together more efficiently to form a signal more quickly. The system described herein can be used to obtain a maximum signal within about 30 minutes, 40 minutes, or 60 minutes after combining the DEC2-BiFC reagent with the fungal sample.

[0298] The carbohydrate recognition domain of Dectin-2 binds tightly to α-mannan, accompanied by dimer formation on the surface of leukocytes in vivo. This property of the extracellular carbohydrate recognition domain DEC2 to form dimers has been successfully exploited to develop a fungal cell-specific diagnostic method that works in vitro. DEC2 was fused to two complementary fragments of the fluorescent protein VENUS to create DEC2-BiFC reagent liposomes. By incubating the DEC2-BiFC reagent liposomes with Aspergillus fumigatus (A. fumigatus) cells, they produced a clear fungal cell-specific green fluorescent signal. Therefore, this technology can be used as a simple, single-step, point-of-care fungal diagnostic method, which will reduce the mistreatment of patients with invasive fungal diseases and may be used as a diagnostic method for superficial fungal diseases.

[0299] Furthermore, there are dozens of C-type lectin-binding proteins that can form multimers, and because they bind a wide variety of target fungal polysaccharides, these polysaccharides can be used to expand this diagnostic technology (see, e.g., Hardison et al., “C-typelectin receptors orchestrate antifungal immunity,” Nat Immunol. 13, 817-822 (2012)).

Claims

1. A nanoparticle comprising an antifungal agent and a targeting molecule that binds to a target antigen on a fungal cell, wherein the targeting molecule is a soluble C-type lectin receptor incorporated into the outer surface of the nanoparticle, and wherein the antifungal agent is encapsulated in the nanoparticle.

2. The nanoparticle according to claim 1, wherein the soluble C-type lectin receptor is selected from the group consisting of soluble Dectin-1, soluble Dectin-2 and soluble Dectin-3.

3. The nanoparticle according to any one of claims 1-2, wherein the targeting molecule or fragment thereof is conjugated to a lipid or a pegylated lipid.

4. The nanoparticle according to any one of claims 1 to 3, wherein: a. the concentration of the antifungal drug is reduced compared to the concentration of the antifungal drug encapsulated in nanoparticles that do not comprise a targeting molecule incorporated into their outer surface; and / or b. The nanoparticles have reduced affinity and / or are less toxic to animal cells compared to nanoparticles that do not comprise a targeting molecule incorporated into their outer surface.

5. Multiplexes of the nanoparticles according to any one of claims 1 to 4.

6. A nanoparticle comprising a targeting molecule that binds to a target fungal cell antigen and a signal-generating molecule, wherein the targeting molecule is a C-type lectin receptor incorporated into the outer surface of the nanoparticle, and wherein the signal-generating molecule generates a signal when the targeting molecule binds to the target fungal cell antigen.

7. A pharmaceutical composition comprising the nanoparticle according to any one of claims 1 to 4 or the multiplex of the nanoparticle according to claim 5.

8. Use of the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating or preventing fungal infection in a subject suffering from or at risk of developing a fungal infection.

9. A method for preparing a multiplicity of nanoparticles comprising an antifungal agent and a targeting molecule that binds to a target antigen on a fungal cell, wherein the targeting molecule is a soluble C-type lectin receptor incorporated into the outer surface of each nanoparticle, and the antifungal agent is encapsulated in each nanoparticle, and wherein the nanoparticles are liposomes, the method comprising the steps of: a) dissolving the antifungal agent in a solvent at about 60° C. for about 10 minutes to about 30 minutes; b) encapsulating the antifungal agent into each liposome by mixing the multiplicity of liposomes in suspension with the antifungal agent / solvent solution of step a) at about 60° C. for about 3 to about 5 hours or at 37° C. for about 24-120 hours; and c) incorporating the targeting molecule into the outer surface of each liposome by contacting the liposomes comprising the encapsulated antifungal agent with the targeting molecule at 60°C for about 45 minutes to about 90 minutes.

10. A fusion polypeptide comprising a targeting molecule that binds to a target fungal cell antigen and the N-terminal or C-terminal portion of a fluorescent polypeptide, wherein the targeting molecule is a soluble C-type lectin receptor.

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