Methods of inhibiting porcine cytomegalovirus

Abstract

Disclosed are methods of inhibiting infection of a cell by porcine cytomegalovirus (PCMV), methods of inhibiting reactivation of PCMV in a porcine cell infected with PCMV, methods of reducing or preventing viral activation in a xenotransplantation donor tissue or organ, methods of reducing the risk of rejection of a xenotransplant tissue or organ in a subject in need thereof or preventing rejection of a xenotransplant tissue or organ in a subject in need thereof, and method of treating PCMV infection in a subject in need thereof.

Description

Atty. Dkt. No. 650053.01243 METHODS OF INHIBITING PORCINE CYTOMEGALOVIRUS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of priority of United States Provisional Patent Application No. 63 / 728,798, filed December 6, 2024, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (“650053_01243.xml” which is 4,369 bytes in size and was created on December 3, 2025) are herein incorporated by reference in its entirety.BACKGROUND

[0003] Recent advances have been made in xenotransplantation, with genetically modified pigs emerging as optimal xenotransplant donors. However, infection with porcine CMV (PCMV) remains a major challenge, as PCMV reduces the survival time of pig xenotransplants in nonhuman primates, in association with high viral loads; cells producing PCMV proteins and possibly infectious virus are found in many organs of baboon recipients. Also, during the first pig heart xenotransplantation into a human patient at the University of Maryland, PCMV was transmitted to the patient. PCMV is closely related to the human herpesviruses (HHV)-6A, -6B and -7. It is only distantly related to the human cytomegalovirus (HCMV). Therefore, improved therapies for treating PCMV are needed in the art, especially therapies that are tolerated by human recipients of xenotranspl anted pig tissues or organs.SUMMARY

[0004] In an aspect of the current disclosure, methods of inhibiting infection of a cell by porcine cytomegalovirus (PCMV) are provided. In some embodiments, the methods comprise contacting the cell with one or more compound selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, or pritelivir. In some embodiments, the one or more compound is selected from brincidofovir, cidofovir. maribavir. artesunate, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, MLS8091, digitoxin, or MLS8544. In some embodiments, contacting comprises contacting the cell with the compound at a concentration of about 1 nM to about 100 nM. In some embodiments, the cell comprises a porcine cell. In some embodiments, the cell comprises a porcine heart cell, a porcine liver cell, a porcine vascular cell, a porcine kidney cell, a porcineAtty. Dkt. No. 650053.01243 pancreas cell, a porcine lung cell. In some embodiments, the porcine heart cell comprises a cardiomyocyte. In some embodiments, the porcine liver cell comprises a hepatocyte, a Kupffer cell, a hepatic stellate cell, or a cholangiocyte. In some embodiments, the porcine vascular cell comprises a vascular endothelial cell. In some embodiments, the porcine kidney cell comprises a kidney epithelial cell, a podocyte, a mesangial cell, or a kidney endothelial cell. In some embodiments, the porcine pancreas cell comprises an acinar cell, an alpha cell, a beta cell, or a pancreatic polypeptide (PP) cell. In some embodiments, the porcine lung cell comprises a lung epithelial cell, a lung fibroblast, an alveolar macrophage, an alveolar type 2 (ATII) cell, an airway basal cell, a bronchioalveolar stem cell (BASC), a mast cell, an alveolar epithelial cell, or a bronchiolar epithelial cell. In some embodiments, the one or more compound comprises two or more compounds selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, or pritelivir. In some embodiments, the one or more compounds may comprise MLS8969, NFU1827, MLS8554, MLS8091, or NCGC2955. In some embodiments, the one or more compounds may comprise oxazolidinone amide derivatives.

[0005] In an aspect of the current disclosure, methods of inhibiting reactivation of porcine cytomegalovirus (PCMV) in a porcine cell infected with PCMV are provided. In some embodiments, the methods comprise contacting the cell with one or more compounds selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, MLS8091, digitoxin, or MLS 8544. In some embodiments, the one or more compound is selected from brincidofovir, cidofovir, maribavir, artesunate, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, MLS8091, digitoxin, or MLS8544. In some embodiments, contacting comprises contacting the cell with the compound at a concentration of about 1 nM to about 100 nM. In some embodiments, the cell comprises a porcine cell. In some embodiments, the cell comprises a porcine heart cell, a porcine liver cell, a porcine vascular cell, a porcine kidney cell, a porcine pancreas cell, a porcine lung cell. In some embodiments, the porcine heart cell comprises a cardiomyocyte. In some embodiments, the porcine liver cell comprises a hepatocyte, a Kupffer cell, a hepatic stellate cell, or a cholangiocyte. In some embodiments, the porcine vascular cell comprises a vascular endothelial cell. In some embodiments, the porcine kidney cell comprises a kidney epithelial cell, a podocyte, a mesangial cell, or a kidney endothelial cell. In some embodiments, the porcine pancreas cell comprises an acinar cell, an alpha cell, a beta cell, or a pancreatic polypeptide (PP) cell. In some embodiments, the porcine lung cell comprises a lungAtty. Dkt. No. 650053.01243 epithelial cell, a lung fibroblast, an alveolar macrophage, an alveolar type 2 (ATII) cell, an airway basal cell, a bronchioalveolar stem cell (BASC). a mast cell, an alveolar epithelial cell, or a bronchiolar epithelial cell. In some embodiments, the one or more compound comprises two or more compounds selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, or pritelivir. In some embodiments, the one or more compounds may comprise MLS8969, NFU1827, MLS8554, MLS8091, or NCGC2955. In some embodiments, the one or more compounds may comprise oxazolidinone amide derivatives.

[0006] In an aspect of the current disclosure, methods of reducing or preventing viral activation in a xenotransplantation donor tissue or organ are provided. In some embodiments, the methods compnse contacting the xenotransplantation donor tissue or organ with one or more compound selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, digitoxin, MLS8091, or MLS 8544. In some embodiments, the one or more compound is selected from brincidofovir, cidofovir. maribavir. artesunate, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, MLS8091, digitoxin, or MLS8544. In some embodiments, the therapeutically effective amount results in the donor tissue or organ being contacted with the compound at a concentration of about 1 nM to about 100 nM. In some embodiments, the donor tissue or organ comprises porcine heart tissue, porcine liver tissue, porcine vascular tissue, porcine kidney tissue, porcine pancreas tissue, or porcine lung tissue. In some embodiments, the donor tissue or organ comprises a porcine heart, a porcine liver, a porcine blood vessel, a porcine kidney, a porcine pancreas, or a porcine lung. In some embodiments, the tissue or organ is located in a living recipient and the tissue or organ is connected to the recipient’s circulatory system. In some embodiments, the recipient is a primate. In some embodiments, the recipient is a baboon. In some embodiments, the recipient is a human subject. In some embodiments, the one or more compound comprises two or more compounds selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, or pritelivir. In some embodiments, the one or more compounds may comprise MLS8969, NFU1827, MLS8554, MLS8091, or NCGC2955. In some embodiments, the one or more compounds may comprise oxazolidinone amide derivatives.

[0007] In an aspect of the current disclosure, methods of reducing the risk of rejection of a xenotransplant tissue or organ in a subject in need thereof or preventing rejection of a xenotransplant tissue or organ in a subject in need thereof are provided. In some embodiments.Atty. Dkt. No. 650053.01243 the method comprising administering a therapeutically effective amount of one or more compound selected from ganciclovir, maribavir, foscamet. brincidofovir, cidofovir. emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, digitoxin, MLS8091, or MLS8544. In some embodiments, the one or more compound is selected from brincidofovir, cidofovir, maribavir, artesunate, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, digitoxin, MLS8091, or MLS8544. In some embodiments, the therapeutically effective amount results in the donor tissue or organ being contacted with the compound at a concentration of about 1 nM to about 100 nM. In some embodiments, the donor tissue or organ comprises porcine heart tissue, porcine liver tissue, porcine vascular tissue, porcine kidney tissue, porcine pancreas tissue, or porcine lung tissue. In some embodiments, the donor tissue or organ comprises a porcine heart, a porcine liver, a porcine blood vessel, a porcine kidney, a porcine pancreas, or a porcine lung. In some embodiments, the recipient is a primate. In some embodiments, the recipient is a baboon. In some embodiments, the recipient is ahuman subject. In some embodiments, the one or more compound comprises two or more compounds selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, or pritelivir. In some embodiments, the one or more compounds may comprise MLS8969, NFU1827, MLS8554, MLS8091, or NCGC2955. In some embodiments, the one or more compounds may comprise oxazolidinone amide derivatives.

[0008] In an aspect of the current disclosure, methods of treating porcine cytomegalovirus (PCMV) infection in a subject in need thereof are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of one or more compound selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, digitoxin, MLS8091, or MLS8544 to the subject. In some embodiments, the subject is a pig. In some embodiments, the subject is a primate. In some embodiments, the subject is a baboon. In some embodiments, the subject is ahuman subject. In some embodiments, the subject comprises porcine tissue. In some embodiments, the subject comprises a porcine xenograft. In some embodiments, treating comprises reducing one or more sign or symptom of PCMV infection in the subject. In some embodiments, the one or more compounds may comprise MLS8969, NFU1827, MLS8554, MLS8091, or NCGC2955. In some embodiments, the one or more compounds may comprise oxazolidinone amide derivatives.Atty. Dkt. No. 650053.01243 BRIEF DESCRIPTION OF THE FIGURES

[0009] FIGS. 1A and IB. FIG. 1A shows the region of DNA polymerase used to construct gBlocks. FIG. IB shows a standard curve of the PCMV DNA FOL gBlock amplification.

[0010] FIG. 2 shows a metagenomic sequence analysis of human, porcine, and baboon CMV genomes. Anvi'o pangenome plot of human (pink), porcine (purple), and baboon (teal) CMV genome comparison. Bins labeled across the bottom indicate gene clusters belonging to their respective genome alone (human = pink; porcine = bright blue; baboon = dark blue) or shared across multiple genomes (genes present in all three genomes = red; genes present in human and baboon only = orange: no genes were unique to human and porcine or porcine and baboon genomes). Bar graphs on the right indicate total genome lengths and total number of genes per 1000 bp. Homogeneity indexes (Comb. = combined, Func. = functional, Geo. = geometric, Ind. = index) are shown. Only two HCMV genes (UL53 and UL92) share high sequence homology7with PCMV and BCMV.

[0011] FIG. 3 shows ECso and plaque assays results for ganciclovir and cyclopropavir inhibition of PCMV.

[0012] FIG. 4 shows ECso and plaque assays results for cidofovir and brincidovir inhibition of PCMV.

[0013] FIG. 5 shows EC50 and plaque assays results for artesunate (AS) and artemisinin dimer 838 (838) inhibition of PCMV.

[0014] FIG. 6 shows EC50 and plaque assays results for emetine and digitoxin inhibition of PCMV.

[0015] FIG. 7 shows EC50 and plaque assays results for MLS854 and cyclopropavir and plaque assays results for MLS8091 (8091) inhibition of PCMV.

[0016] FIG. 8 shows plaque assays results for the combination of ganciclovir and artemisinin (GCV+AS) and cyclopropavir, artemisinin (cyclopropavir+AS), cidofovir and artemisinin (cidofovir+AS), and brincidofovir and artemisinin (brincidofovir+AS).DETAILED DESCRIPTION

[0017] Disclosed herein are methods of inhibiting infection of a cell by porcine cytomegalovirus (PCMV), methods of inhibiting reactivation of PCMV in a porcine cell infected with PCMV, methods of reducing or preventing viral activation in a xenotransplantation donor tissue or organ, and methods of reducing the risk of rejection of a xenotransplant tissue or organ in a subject in need thereof or preventing rejection of aAtty. Dkt. No. 650053.01243 xenotransplant tissue or organ in a subject in need thereof. Also disclosed are compositions, systems, kits, and platforms for performing one or more of the methods disclosed herein.Methods of inhibiting infection of a cell by porcine cytomegalovirus

[0018] In an aspect of the current disclosure, methods of inhibiting infection of a cell by porcine cytomegalovirus (PCMV) are provided. In some embodiments, the methods comprise contacting the cell with one or more compounds selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, digitoxin. MLS8091, or MLS8544. The one or more compounds may also be derivatives of ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, digitoxin, MLS8091, or MLS8544. The one or more compounds may also be pharmaceutically acceptable salts of ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, digitoxin, MLS8091, or MLS8544. In embodiments, the methods comprise contacting the cell with two or more of the compounds above, three or more of the compounds, four or more of the compounds, five or more of the compounds, six or more of the compounds, seven or more of the compounds, eight or more of the compounds, nine or more of the compounds, ten or more of the compounds, or eleven of the compounds.

[0019] The one or more compounds may comprise MLS8969, NFU1827, MLS8554, MLS8091, or NCGC2955. See, e.g., Kapoor et al., “Validation and characterization of five distinct novel inhibitors of human cytomegalovirus” J Med Chem. 2020 Apr 23;63(8):3896-3907. which is incorporated by reference herein.

[0020] As used herein, “artesunate” refers to a compound with the structure:

[0021] As used herein, “artemisinin” refers to a compound with the structure:Atty. Dkt. No. 650053.01243

[0022] As used herein, “artemisinin dimer 838” refers to a compound with the structure:

[0023] As used herein, “emetine” refers to a compound with the structure:

[0024] As used herein, “MLS8969” refers to a compound with the systematic name N-{4-[(5-chloro-2-methoxybenzoyl)amino]phenyl}-2-furamide or the structure:Atty. Dkt. No. 650053.01243

[0025] As used herein, “NFU1827” refers to a compound with the structure:

[0026] As used herein. “MLS8091?’ refers to a compound with the structure:

[0027] As used herein, " MLS8554” refers to a compound with the structure:

[0028] As used herein, “NCGC2955” refers to a compound with the structure:Atty. Dkt. No. 650053.01243

[0029] As used herein, “hPMPA” refers to a compound with the structure:OH

[0030] The one or more compounds may comprise oxazolidinone amide derivatives. See, e.g., Plotkin et al., ‘'Discovery of Broad-Spectrum Herpes Antiviral Oxazolidinone Amide Derivatives and Their Structure-Activity Relationships” ACS Med Chem Lett. 2024 Jul 9;15(8): 1232-1241, which is incorporated by reference herein.

[0031] The one or more compounds may comprise brincidofovir, cidofovir, maribavir, artesunate, artemisinin dimer 838, emetine, cyclopropavir, digitoxin, MLS8091, or MLS8544.

[0032] The one or more compounds may comprise one, two, three, four, five, or more of the compounds, in any combination.

[0033] In some embodiments, the one or more compounds are present in a composition for administration to a subject, organ, or tissue. In some embodiments, the composition comprises one or more pharmaceutically acceptable carriers, vehicles, diluents, etc.

[0034] As used herein, “inhibiting infection” refers to reducing the likelihood that a particular cell will be infected by a virus, e.g., PCMV, as compared to a control cell. The level to which a compound inhibits infection may be determined by methods known in the art. e.g., by quantitative polymerase chain reaction (qPCR), e.g., as shown in Example 1, plaque assay.Atty. Dkt. No. 650053.01243 enzy me linked immunosorbent assay (ELISA), or other suitable methods. In one example, a first population of cells may be contacted with a compound, e.g., ganciclovir, maribavir. foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, digitoxin, MLS8091, or MLS8544 and the level of infection of a virus, e.g., PCMV, is measured. A second population of the same ty pe of cells is contacted with a control compound, e.g., a vehicle or diluent, and the level of infection of the virus, e.g., PCMV, is measured. Inhibition of infection occurs where the level of infection is reduced in the first population of cells as compared to the second population of cells.

[0035] Contacting refers to contacting a cell or sample directly or indirectly in vitro, ex vivo, or in vivo (i.e., within a subject as defined herein). Contacting a sample may include addition of a compound to a sample, or administration to a subject. Contacting encompasses administration to a solution, cell, tissue, mammal, subject, patient, or human. Further, contacting a cell includes adding an agent to a cell culture. In embodiments, contacting comprises contacting the cell with the compound at a concentration of about 1 nM to about 100 nM, or any value or subrange therein. In embodiments, the concentration of the compound is less than 1 nM. In embodiments, the concentration is more than 100 nM. The concentration of the compound may be a range within about 1 nM to about 100 nM, including but not limited to, about 10 nM to about 90 nM, about 20 nM to about 80 nM, about 30 nM to about 70 nM, about 40 nM to about 60 nM.

[0036] The cell may be contacted with about 0.1 nM to about 1000 nM or about 0.1 nM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, about 19 nM, about 20 nM, about 21 nM, about 22 nM, about 23 nM, about 24 nM, about 25 nM, about 26 nM, about 27 nM, about 28 nM, about 29 nM, about 30 nM, about 31 nM. about 32 nM, about 33 nM. about 34 nM, about 35 nM. about 36 nM, about 37 nM, about 38 nM, about 39 nM, about 40 nM, about 41 nM, about 42 nM, about 43 nM, about 44 nM, about 45 nM, about 46 nM, about 47 nM, about 48 nM, about 49 nM, about 50 nM, about 51 nM, about 52 nM, about 53 nM, about 54 nM, about 55 nM, about 56 nM. about 57 nM, about 58 nM, about 59 nM, about 60 nM, about 61 nM. about 62 nM, about 63 nM, about 64 nM, about 65 nM, about 66 nM, about 67 nM, about 68 nM, about 69 nM, about 70 nM, about 71 nM, about 72 nM, about 73 nM, about 74 nM, about 75 nM, about 76 nM, about 77 nM, about 78 nM, about 79 nM, about 80 nM, about 81 nM, about 82 nM, about 83 nM, about 84 nM, about 85 nM, about 86 nM, about 87 nM, about 88 nM, about 89 nM, about 90 nM, about 91 nM, about 92 nM, about 93 nM, about 94 nM, about 95 nM,Atty. Dkt. No. 650053.01243 about 96 nM, about 97 nM, about 98 nM, about 99 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM. about 600 nM, about 700 nM, about 800 nM, about 900 nM about 1000 nM, or more of the one or more compounds.

[0037] The cell may be a vertebrate cell, a mammalian cell, a porcine cell, or a primate cell. The primate cell may be a human cell. In embodiments, the cell comprises a heart cell, a liver cell, a vascular cell, a kidney cell, a pancreas cell, or a lung cell. In exemplary embodiments, the cell comprises a porcine cell. In embodiments, the cell comprises a porcine heart cell, a porcine liver cell, a porcine vascular cell, a porcine kidney cell, a porcine pancreas cell, or a porcine lung cell. In embodiments, the porcine heart cell comprises a cardiomyocyte. In embodiments, the porcine liver comprises a hepatocyte, a Kupffer cell, a hepatic stellate cell, or a cholangiocyte. In embodiments, the porcine vascular cell compnses a vascular endothelial cell. In embodiments, the porcine kidney cell comprises a kidney epithelial cell, a podocyte, a mesangial cell, or a kidney endothelial cell. In embodiments, the porcine pancreas cell comprises an acinar cell, an alpha cell, a beta cell, or a pancreatic polypeptide (PP) cell. In embodiments, the porcine lung cell comprises a lung epithelial cell, a lung fibroblast, an alveolar macrophage, an alveolar type 2 (ATII) cell, an airway basal cell, a bronchioalveolar stem cell (BASC), a mast cell, an alveolar epithelial cell, or a bronchiolar epithelial cell.

[0038] As indicated above, pharmaceutically acceptable salts of the compounds are contemplated and also may be utilized in the disclosed methods, compositions, systems, kits, and platforms. The term "pharmaceutically acceptable salt” as used herein, refers to salts of the compounds, which are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds as disclosed herein with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts. It will be appreciated by the skilled reader that most or all of the compounds as disclosed herein are capable of forming salts and that the salt forms of pharmaceuticals are commonly used, often because they are more readily crystallized and purified than are the free acids or bases.

[0039] Acids commonly employed to form acid addition salts may include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of suitable pharmaceutically acceptable salts may include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate.Atty. Dkt. No. 650053.01243 dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleat-, butyne-.1,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate. phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, α-hydroxybutyrate. glycolate. tartrate, methanesulfonate, propanesulfonate, naphthalene- 1 -sulfonate, naphthal ene-2-sulfonate, mandelate, and the like.

[0040] Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Bases useful in preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.

[0041] The counterion forming a part of any salt of a compound disclosed herein may not be critical to the activity of the compound, so long as the salt as a whole is pharmacologically acceptable and as long as the counter-ion does not contribute undesired qualities to the salt as a whole. Undesired qualities may include undesirable solubility or toxicity.

[0042] Pharmaceutically acceptable esters and amides of the compounds can also be employed in the compositions and methods disclosed herein. Examples of suitable esters include alkyl, aryl, and aralkyl esters, such as methyl esters, ethyl esters, propyl esters, dodecyl esters, benzyl esters, and the like. Examples of suitable amides include unsubstituted amides, monosubstituted amides, and disubstituted amides, such as methyl amide, dimethyl amide, methyl ethyl amide, and the like.

[0043] In addition, the methods disclosed herein may be practiced using solvate forms of the compounds or salts, esters, and / or amides, thereof. Solvate forms may include ethanol solvates, hydrates, and the like.Methods of inhibiting reactivation of porcine cytomegalovirus

[0044] In an aspect of the current disclosure, methods of inhibiting reactivation of porcine cytomegalovirus (PCMV) in a porcine cell infected with PCMV are provided. In some embodiments, the methods comprise contacting the cell with one or more compounds selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate,Atty. Dkt. No. 650053.01243 artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, digitoxin, MLS8091, or MLS8544.

[0045] The cell may be contacted with an amount of the one or more compound sufficient to inhibit reactivation of PCMV in the cell, e.g., about 0.1 nM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM. about 12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM. about 17 nM, about 18 nM, about 19 nM, about 20 nM, about 21 nM, about 22 nM, about 23 nM, about 24 nM, about 25 nM, about 26 nM, about 27 nM, about 28 nM, about 29 nM, about 30 nM, about 31 nM, about 32 nM, about 33 nM, about 34 nM, about 35 nM, about 36 nM, about 37 nM, about 38 nM, about 39 nM, about 40 nM, about 41 nM, about 42 nM, about 43 nM, about 44 nM, about 45 nM, about 46 nM, about 47 nM, about 48 nM, about 49 nM, about 50 nM, about 51 nM, about 52 nM, about 53 nM, about 54 nM, about 55 nM, about 56 nM, about 57 nM, about 58 nM, about 59 nM, about 60 nM, about 61 nM, about 62 nM, about 63 nM, about 64 nM, about 65 nM, about 66 nM, about 67 nM, about 68 nM, about 69 nM, about 70 nM, about 71 nM. about 72 nM, about 73 nM, about 74 nM, about 75 nM, about 76 nM. about 77 nM, about 78 nM, about 79 nM, about 80 nM, about 81 nM, about 82 nM, about 83 nM, about 84 nM, about 85 nM, about 86 nM, about 87 nM, about 88 nM, about 89 nM, about 90 nM, about 91 nM, about 92 nM, about 93 nM, about 94 nM, about 95 nM, about 96 nM, about 97 nM, about 98 nM, about 99 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM about 1000 nM, or more.

[0046] A cell may be latently infected with PCMV and the virus may “reactivate” during times of stress. Latently infected cells may not actively produce any viral proteins, or low or undetectable levels of viral proteins. Reactivation includes a change in the viral proteins being produced by the infected cells, which may be measured by, e.g., qPCR and antibody-based methods, as described above.Methods of reducing or preventing viral activation in a xenotransplantation donor tissue or organ

[0047] In an aspect of the current disclosure, methods of reducing or preventing viral activation in a xenotransplantation donor tissue or organ are provided. In some embodiments, the methods comprise contacting the xenotransplantation donor tissue or organ with one or more compound selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate,Atty. Dkt. No. 650053.01243 artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, digitoxin, MLS8091, or MLS8544.

[0048] In some embodiments, the donor tissue or organ comprises porcine tissue. The porcine tissue may comprise porcine heart tissue, porcine liver tissue, porcine vascular tissue, porcine kidney tissue, porcine pancreas tissue, or porcine lung tissue. In some embodiments, the donor tissue or organ comprises a porcine heart, a porcine liver, a porcine blood vessel, a porcine kidney, a porcine pancreas, or a porcine lung.

[0049] In embodiments, the donor tissue or organ is located in a living recipient (e.g., a primate, such as a baboon or a human), and may be connected to the recipient’s circulatory system such that the donor organ is perfused by the recipient's circulatory system.

[0050] The tissue or organ may be contacted with the one or more compounds, similarly to the contacting of cells.

[0051] The donor or the recipient may be administered the one or more compounds. The donor or the recipient may be administered the one or more compounds at about, e.g., about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg. about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg. about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg. about 4.1 mg / kg. about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg, about 5.0 mg / kg, about 5.1 mg / kg, about 5.2 mg / kg, about 5.3 mg / kg, about 5.4 mg / kg, about 5.5 mg / kg, about 5.6 mg / kg, about 5.7 mg / kg, about 5.8 mg / kg, about 5.9 mg / kg, about 6.0 mg / kg, about 6.1 mg / kg, about 6.2 mg / kg, about 6.3 mg / kg, about 6.4 mg / kg. about 6.5 mg / kg, about 6.6 mg / kg, about 6.7 mg / kg, about 6.8 mg / kg, about 6.9 mg / kg, about 7.0 mg / kg, about 7.1 mg / kg, about 7.2 mg / kg, about 7.3 mg / kg, about 7.4 mg / kg, about 7.5 mg / kg, about 7.6 mg / kg, about 7.7 mg / kg, about 7.8 mg / kg, about 7.9 mg / kg, about 8.0 mg / kg, about 8.1 mg / kg, about 8.2 mg / kg, about 8.3 mg / kg, about 8.4 mg / kg, about 8.5 mg / kg, about 8.6 mg / kg, about 8.7 mg / kg, about 8.8 mg / kg, about 8.9 mg / kg, about 9.0 mg / kg, about 9.1 mg / kg, about 9.2 mg / kg, aboutAtty. Dkt. No. 650053.01243 9.3 mg / kg, about 9.4 mg / kg, about 9.5 mg / kg, about 9.6 mg / kg. about 9.7 mg / kg, about 9.8 mg / kg, about 9.9 mg / kg, or about 10.0 mg / kg, or more, as determined by a physician or veterinarian.Methods of reducing the risk of rejection of a xenotransplant tissue or organ or preventing rejection of a xenotransplant tissue or organ

[0052] In an aspect of the current disclosure, methods of reducing the risk of rejection of a xenotransplant tissue or organ in a subject in need thereof or preventing rejection of a xenotransplant tissue or organ in a subject in need thereof are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of one or more compound selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, digitoxin, MLS8091, or MLS8544 to the transplant subject, before, during, and / or after transplant. Administration may be prophylactic (e.g.. before transplant, and / or before any signs or symptoms of PCMV are noted in the transplant subject after transplant).

[0053] In some embodiments, the donor tissue or organ comprises porcine heart tissue, porcine liver tissue, porcine vascular tissue, porcine kidney tissue, porcine pancreas tissue, or porcine lung tissue. In some embodiments, the donor tissue or organ comprises a porcine heart, a porcine liver, a porcine blood vessel, a porcine kidney, a porcine pancreas, or a porcine lung. The donor tissue or organ may be located in a living recipient, e.g., a primate, such as a baboon or a human, and may be connected to the recipient’s circulatory system such that the donor organ is perfused by the recipient's circulatory system.

[0054] As used herein, “therapeutically effective amount” or “effective amount” refers to the amount of the compound necessary to improve one sign or symptom of a subject’s condition. In embodiments, the therapeutically effective amount results in the donor tissue or organ being contacted with the compound at a concentration of about 1 nM to about 100 nM. In embodiments, the concentration of the compound is less than 1 nM. In embodiments, the concentration is more than 100 nM. The concentration of the compound may be a range within about 1 nM to about 100 nM, including but not limited to, about 10 nM to about 90 nM, about 20 nM to about 80 nM, about 30 nM to about 70 nM, about 40 nM to about 60 nM.

[0055] The donor or the recipient may be administered the one or more compounds. The donor or the recipient may be administered the one or more compounds at about, e.g., about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06Atty. Dkt. No. 650053.01243 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg. about 0.8 mg / kg, about 0.9 mg / kg, about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg. about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3.0 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg. about 4.9 mg / kg, about 5.0 mg / kg, about 5.1 mg / kg, about 5.2 mg / kg, about 5.3 mg / kg, about 5.4 mg / kg, about 5.5 mg / kg, about 5.6 mg / kg, about 5.7 mg / kg, about 5.8 mg / kg, about 5.9 mg / kg, about 6.0 mg / kg, about 6.1 mg / kg, about 6.2 mg / kg, about 6.3 mg / kg, about 6.4 mg / kg, about 6.5 mg / kg, about 6.6 mg / kg, about 6.7 mg / kg, about 6.8 mg / kg, about 6.9 mg / kg, about 7.0 mg / kg, about 7.1 mg / kg, about 7.2 mg / kg, about 7.3 mg / kg. about 7.4 mg / kg, about 7.5 mg / kg, about 7.6 mg / kg, about 7.7 mg / kg, about 7.8 mg / kg, about 7.9 mg / kg, about 8.0 mg / kg, about 8.1 mg / kg, about 8.2 mg / kg, about 8.3 mg / kg, about 8.4 mg / kg, about 8.5 mg / kg, about 8.6 mg / kg, about 8.7 mg / kg, about 8.8 mg / kg, about 8.9 mg / kg, about 9.0 mg / kg, about 9.1 mg / kg, about 9.2 mg / kg, about 9.3 mg / kg, about 9.4 mg / kg, about 9.5 mg / kg, about 9.6 mg / kg. about 9.7 mg / kg, about 9.8 mg / kg, about 9.9 mg / kg, or about 10.0 mg / kg, or more, as determined by a physician or veterinarian.

[0056] The pharmaceutical compositions may be administered by any appropriate route. The one or more compounds may be administered by any suitable route, e.g., orally or parenterally, e.g., intravenously, subcutaneously, intramuscularly, intrathecally, intraperitoneally, etc. The particular dose and administration route can be determined by a physician.

[0057] The one or more compounds may be administered once per day, twice per day, three times per day, or four times per day for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days, or once per day, twice per day, three times per day, or four times per day for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks.Atty. Dkt. No. 650053.01243 about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51 weeks, about 52 weeks, or chronically, as determined by a physician.

[0058] A subject in need of reduction of the risk of rejection or prevention of rejection of a xenotransplanted tissue or organ includes any subject that has received a xenotransplant or will receive a xenotransplant, e g., a xenotransplant from a pig. The subject may be a primate, e.g., a baboon or a human.Methods of treating porcine cytomegalovirus infection in a subject in need thereof

[0059] In an aspect of the current disclosure, methods of treating porcine cytomegalovirus (PCMV) infection in a subject in need thereof are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of one or more compound selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, digitoxin, MLS8091, or MLS8544 to the subject.

[0060] The subject may comprise porcine tissue, e.g., the subject may comprise a porcine xenograft. The subject may be a primate subject comprising a porcine xenograft, e.g., a baboon or a human subject comprising a porcine xenograft. The one or more compounds may be administered as discussed above.

[0061] As used herein, ‘'treating PCMV infection” comprises reducing one or more sign or symptom of PCMV infection in the subject, e.g., pneumonia, rhinitis, a high mortality7rate in young piglets, porcine xenograft rejection, and / or reduced PCMV titer in the subject, tissue, or organ.Further Definitions

[0062] The disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.Atty. Dkt. No. 650053.01243

[0063] As used in this specification and the claims, the singular forms “a,” “an,"’ and “the’' include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.

[0064] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0065] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0066] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0067] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or B or “A and B.”Atty. Dkt. No. 650053.01243

[0068] All language such as "up to,’' “at least,'’ “greater than,’" “less than,"’ and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0069] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0070] Embodiments

[0071] Embodiment 1. A method of inhibiting infection of a cell by porcine cytomegalovirus (PCMV), the method comprising contacting the cell with one or more compound selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, or pritelivir.

[0072] Embodiment 2. A method of inhibiting reactivation of porcine cytomegalovirus (PCMV) in a porcine cell infected with PCMV, the method comprising contacting the cell with one or more compound selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, or pritelivir.

[0073] Embodiment 3. The method of embodiment 1 or 2, wherein the one or more compound is selected from brincidofovir, cidofovir, maribavir. artesunate, artemisinin dimer 838, or emetine.

[0074] Embodiment 4. The method of any one of the preceding embodiments, wherein contacting comprises contacting the cell with the compound at a concentration of about 1 nM to about 100 nM.

[0075] Embodiment 5. The method of any one of the preceding embodiments, wherein the cell comprises a porcine cell.

[0076] Embodiment 6. The method of any one of the preceding embodiments, wherein the cell comprises a porcine heart cell, a porcine liver cell, a porcine vascular cell, a porcine kidney cell, a porcine pancreas cell, or a porcine lung cell.Atty. Dkt. No. 650053.01243

[0077] Embodiment 7. The method of embodiment 6, wherein the porcine heart cell comprises a cardiomyocyte.

[0078] Embodiment 8. The method of embodiment 6, wherein the porcine liver cell comprises a hepatocyte, a Kupffer cell, a hepatic stellate cell, or a cholangiocyte.

[0079] Embodiment 9. The method of embodiment 6, wherein the porcine vascular cell comprises a vascular endothelial cell.

[0080] Embodiment 10. The method of embodiment 6, wherein the porcine kidney cell comprises a kidney epithelial cell, a podocyte, a mesangial cell, or a kidney endothelial cell.

[0081] Embodiment 11. The method of embodiment 6, wherein the porcine pancreas cell comprises an acinar cell, an alpha cell, a beta cell, or a pancreatic polypeptide (PP) cell.

[0082] Embodiment 12. The method of embodiment 6, wherein the porcine lung cell comprises a lung epithelial cell, a lung fibroblast, an alveolar macrophage, an alveolar type 2 (ATII) cell, an airway basal cell, a bronchioalveolar stem cell (BASC), a mast cell, an alveolar epithelial cell, or a bronchiolar epithelial cell.

[0083] Embodiment 13. The method of any one of the preceding embodiments, wherein the one or more compound comprises two or more compounds selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, or pritelivir.

[0084] Embodiment 14. A method of reducing or preventing viral activation in a xenotransplantation donor tissue or organ, the method comprising contacting the xenotransplantation donor tissue or organ with one or more compound selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, or pritelivir.

[0085] Embodiment 15. The method of embodiment 14. wherein the one or more compound is selected from brincidofovir, cidofovir, maribavir, artesunate, artemisinin dimer 838, or emetine.

[0086] Embodiment 16. The method of embodiment 14 or 15, wherein the therapeutically effective amount results in the donor tissue or organ being contacted with the compound at a concentration of about 1 nM to about 100 nM.

[0087] Embodiment 17. The method of any one of embodiments 14-16, wherein the donor tissue or organ comprises porcine heart tissue, porcine liver tissue, porcine vascular tissue, porcine kidney tissue, porcine pancreas tissue, or porcine lung tissue.Atty. Dkt. No. 650053.01243

[0088] Embodiment 18. The method of any one of embodiments 14-17, wherein the donor tissue or organ comprises a porcine heart, a porcine liver, a porcine blood vessel, a porcine kidney, a porcine pancreas, or a porcine lung.

[0089] Embodiment 19. The method of any one of embodiments 14-18, wherein the tissue or organ is located in a living recipient and the tissue or organ is connected to the recipient’s circulatory system.

[0090] Embodiment 20. The method of embodiment 19, wherein the recipient is a primate.

[0091] Embodiment 21. The method of embodiment 20, wherein the recipient is a baboon.

[0092] Embodiment 22. The method of embodiment 20, wherein the recipient is a human subject.

[0093] Embodiment 23. The method of any one of embodiments 14-22, wherein the one or more compound comprises two or more compounds selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, or pritelivir.

[0094] Embodiment 24. A method of reducing the risk of rejection of a xenotransplant tissue or organ in a subject in need thereof or preventing rejection of a xenotransplant tissue or organ in a subject in need thereof, the method comprising administering a therapeutically effective amount of one or more compound selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir. emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, or pritelivir.

[0095] Embodiment 25. The method of embodiment 24, wherein the one or more compound is selected from brincidofovir, cidofovir, maribavir, artesunate, artemisinin dimer 838, or emetine.

[0096] Embodiment 26. The method of embodiment 24 or 25, wherein the therapeutically effective amount results in the donor tissue or organ being contacted with the compound at a concentration of about 1 nM to about 100 nM.

[0097] Embodiment 27. The method of any one of embodiments 24-26, wherein the donor tissue or organ comprises porcine heart tissue, porcine liver tissue, porcine vascular tissue, porcine kidney tissue, porcine pancreas tissue, or porcine lung tissue.

[0098] Embodiment 28. The method of any one of embodiments 24-27, wherein the donor tissue or organ comprises a porcine heart, a porcine liver, a porcine blood vessel, a porcine kidney, a porcine pancreas, or a porcine lung.

[0099] Embodiment 29. The method of embodiment 28, wherein the recipient is a primate.

[0100] Embodiment 30. The method of embodiment 29, wherein the recipient is a baboon.Atty. Dkt. No. 650053.01243

[0101] Embodiment 31. The method of embodiment 29, wherein the recipient is a human subject.

[0102] Embodiment 32. The method of any one of embodiments 24-31, wherein the one or more compound comprises two or more compounds selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, or pritelivir.

[0103] Embodiment 33. A method of treating porcine cytomegalovirus (PCMV) infection in a subj ect in need thereof, the method comprising administering a therapeutically effective amount of one or more compound selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir. emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, or pritelivir to the subject.

[0104] Embodiment 34. The method of embodiment 33, wherein the subject is a pig.

[0105] Embodiment 35. The method of embodiment 33, wherein the subject is a primate.

[0106] Embodiment 36. The method of embodiment 35, wherein the subject is a baboon.

[0107] Embodiment 37. The method of embodiment 35, wherein the subject is a human subject.

[0108] Embodiment 38. The method of any one of embodiments 33-37, wherein the subject comprises porcine tissue.

[0109] Embodiment 39. The method of any one of embodiments 33 or 35-38, wherein the subject comprises a porcine xenograft.

[0110] Embodiment 40. The method of any one of embodiments 33-39, wherein treating comprises reducing one or more sign or symptom of PCMV infection in the subject.EXAMPLES

[0111] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.Example 1 - Inhibition of porcine CMV infection with antiviral compounds

[0112] PCMV is common in pigs and manifests as pneumonia, rhinitis, and a high mortality rate in young piglets. Studies have shown variable activity of the available HCMV drugs against PCMV. In infected porcine fallopian tube cells, monitoring of PCMV viral load in supernatants revealed good inhibition with ganciclovir and cidofovir. However, in another in vitro study using a PCMV-infected porcine fallopian tube model, PCMV was resistant to ganciclovir, acyclovir, and leflunomide, while cidofovir and foscamet showed some antiviral activity. In clinical use. foscamet and cidofovir carry a high risk of nephrotoxicity and other organ toxi cities.Atty. Dkt. No. 650053.01243 An in-vivo pig-to-baboon xenotransplant study also demonstrated reduced activity of ganciclovir against both PCMV and BCMV. The activity of recently approved anti-viral agents, alone or in drug combination, has not been tested for PCMV. These include maribavir, letermovir, anti-HSV - pritelivir, and the lipid-conjugate analog of cidofovir (brincidofovir), approved only for smallpox.

[0113] The inventor has extensive experience in CMV basic research, including detailed assessments of in vitro activity of novel candidate antiviral agents against HCMV. In addition to the approved antiviral agents ganciclovir, foscamet, cidofovir (and brincidofovir), maribavir, letermovir, acyclovir, and pritelivir, the inventor proposed testing several antiviral agents discovered in our laboratory’ including the antimalarial agent, artesunate and artemisinin derived dimers. Artesunate is effective against HCMV in vitro, including drug resistant strains. It was reported to have some success in treating HCMV disease and a child with HHV-6 myocarditis. Its 50% inhibitory' concentration is ~3.8 pM and ~ 10 pM against HHV-6 and HCMV, respectively, in vitro, and it reduces early and late viral protein expression. The inventor have developed the plaque and qPCR assays for PCMV and have shown that a few agents are inhibitory. These include: brincidofovir, cidofovir, maribavir, artesunate, artemisinin-derived dimer, emetine (similar activity to HCMV). Compounds with some activity: ganciclovir, pritelivir (less active than against HCMV).

[0114] Methods

[0115] PT-K75 cells (derived from the turbinate mucosa of two newborn piglets) were obtained from ATCC (CRL-2528) and grown in Dulbecco’s modified Eagle medium (DMEM) containing 10% FBS in a 5% CO2 incubator at 37°C degrees.

[0116] Porcine cytomegalovirus (PCMV) was obtained from ATCC (VR-1499). The stock solution was diluted 1: 10 to infect one well of a 6-well plate. After approximately 21-30 days, the supernatant and cells were collected and mixed with 3 x 106non-infected PT-K75 cells in a T175 flask. After 30 days of coinfection, cells were harvested and aliquoted in 90% FBS with 10% glycerol at 1 x 106infected cells per vial and frozen at -80 until use. Infected cells were thawed immediately prior to use and mixed with non-infected cells.

[0117] The following compounds were tested: Ganciclovir (GCV), Maribavir (MBV), Brincidofovir, Cidofovir, Foscamet, Pritelivir, Letermovir, Artesunate-AS, artemisinin dimer 8383, Emetine

[0118] Antiviral screening assay by qPCR.Atty. Dkt. No. 650053.01243

[0119] Non-infected and infected cells were mixed at a ratio of 2:1 (non-infected to infected) and plated at 1.5 x 106total cells in a 6-well plate. The media was removed 24 hours after plating the infected cell mixture, and drugs were added at varying concentrations in 4% FBS in DMEM. At 10 days post-infection, the released virus in the media supernatant and cells scraped from each well were combined and transferred to 1.5 ml Eppendorf tubes prior to isolation with 5x MagMAX Pathogen RNA / DNA kit (4462359, Applied Biosystems). Quantitative Real-Time PCR was performed using PowerUp SYBR Green Master Mix (A25741, Applied Biosystems) with the following primers (Halecker et al., Nat Set Rep, 2022, 12:21545, which is incorporated by reference herein in its entirety):

[0120] PCMV DNA Polymerase: F 5 -ACT TCG TCG CAG CTC ATC TGA -3’ (SEQ ID NO: 1)

[0121] R 5 -ATT GTT GAT AAA GTC ACT CGT CTG C-3’ (SEQ ID NO: 2)

[0122] GAPDH (Sus scrofa): F 5’-ACA TGG CCT CCA AGG AGT AAG A-3’ (SEQ ID NO: 3)

[0123] R 5’-GAT CGA GTT GGG GCT GTG ACT-3’ (SEQ ID NO: 4)

[0124] Gene fragment blocks (gBlocks), double-stranded DNA fragments, were created using a conserved region of the PCMV DNA polymerase (black box, FIG. 1A) to generate a PCR standard.

[0125] The qPCR efficiency was 98.41% (the ratio of the number of target gene molecules at the end of a PCR cycle divided by the number of target molecules at the start of the same PCR cycle), and the slope was -3.36, close to the ideal slope of -3.33 (FIG. IB).

[0126] The inventor used different infectivity ratios and time points to optimize the qPCR assay further.

[0127] Antiviral screening by Plaque assay:

[0128] Non-infected and infected cells were mixed at a ratio of 100:1 and plated in a 24-well plate. The following day, DMEM was removed, and compounds were added in 4% DMEM + 4% CMC. After 10-14 days, plates were stained with cry stal violet

[0129] Cytotoxicity assay:

[0130] An MTT cytotoxicity assay was performed. Briefly, non-infected cells were treated with the compounds or equivalent amounts of DMSO for 10 days, and MTT was added to each well. The plates were incubated for 2-3 hours at 37°C, and the resulting conversion of the yellow solution to dark blue was quantified by absorbance at 560 nm.Atty. Dkt. No. 650053.01243

[0131] The inventor tested the effect of ganciclovir (GCV) on PCMV replication (FIG. 3).Based on qPCR, there was some activity of GCV; the ECso was 10-20 pM. Treatment was performed 18 hours post-infection (hpi) for all PCR assays, and the compound was replenished on day 5. On day 10, cells were removed, and the cell and supernatant mixture was transferred to a 1.5ml centrifuge tube. Virus DNA was isolated using the eMag nucleic acid extraction platform, and qPCR was run with PowerUp SYBR Green Master Mix (Cat# A25741, applied biosystems).

[0132] All compounds were tested in the same way. Table 1 provides a comparison of the PCMV and HCMV EC50. Note the listed ranges for HCMV, as activity7was tested using different viral strains and cells. Table 2 provides the EC50 of each drug from a PCR assay and a plaque assay. The CC50 is also provided for each drug.Table 1 - comparison of the PCMV and HCMV EC50.Compound PCMV ECso (qPCR) HCMV ECsoGCV 21 pM ± 7 0.2 - 5 pMMBV N / A 1-10 pMFoscamet 150 pM 30-300 pMBrincidofovir 16.8 nM± 1.2 1-3 nMCidofovir 1.6 pM ± 0.15 0.22- 0.51 pMEmetine 36 nM± 1.8 40 nMArtesunate 6.7 ±0.3 10 pMArtemisinin dimer 838 99 nM ± 14.3 40 nMPritelivir (HSV1) 3.3 pM ± 0.37 10 - 30 nMExample 2- Inhibition of Porcine Cytomegalovirus Replication with Antiviral Compounds

[0133] The ongoing global organ shortage for transplantation has generated significant scientific interest, leading to the development of xenotransplantation as a potential solution. Recent breakthroughs in immunology and biotechnology have paved the way for genetically engineered xenotransplantation. However, areas of concern related to infectious complications and immune responses require further study. Infection with porcine cytomegalovirus (PCMV) has been reported in pigs and in recipients of pig organs and may play a significant role in the outcome of xenotransplantation. We evaluated the activities of FDA-approved and investigational antivirals against PCMV replication in vitro. Agents with specific activity against human cytomegalovirus (HCMV), herpes simplex 1 and 2 (HSV-1 and HSV-2), andAtty. Dkt. No. 650053.01243 human herpesvirus 6 (HHV-6), as well as investigational antiviral anticellular agents, were tested using plaque reduction assays and quantitative PCR of the PCMV polymerase. The most efficacious DNA polymerase inhibitors for PCMV inhibition were Brincidofovir, Cidofovir, and Cyclopropavir. Maribavir had no activity against PCMV. Artemisinin monomer, artesunate, dimer 838, emetine, and digitoxin showed anti -PCMV activity at concentrations similar to those required for their anti-HCMV activity. The combination of artesunate with active PCMV inhibitors was additive. Finally, two investigational anti-HCMV compounds (MLS8554 and MLS8091) showed activity against PCMV. In summary, this extensive investigation of antiviral agents against PCMV reveals a pattern of inhibition similar to that of HHV-6. Additional assays and studies are needed to identify new antivirals for PCMV, including the evaluation of drug combinations.

[0134] Introduction

[0135] Significant recent advances have been made in the field of xenotransplantation, with the emergence of genetically modified pigs as optimal xenotransplant donors1’3. However, infection with porcine CMV (PCMV) remains a significant challenge for xenotransplantation4. PCMV reduces the survival time of pig xenotransplants in non-human primates, in association with high viral loads. Cells producing PCMV proteins and possibly infectious vims were found in many organs of the baboon recipient4,5. Importantly, during the first pig heart xenotransplantation into a human patient at the University of Maryland, PCMV was transmitted to the patient6. In addition, baboon CMV (BCMV) can cause severe infection during transplantation of pig donor organs into baboon recipients4, an essential step in developing safe and effective xenotransplantation protocols for eventual human recipients.

[0136] PCMV is common in pigs and causes pneumonia, rhinitis, and high mortality in young piglets. Latent PCMV may be challenging to detect in donor pre-transplant testing.

[0137] The PCMV genome is closely related to the human herpesviruses (HHV)-6A, 6B, and -77. It is only distantly related to the human cytomegalovirus (HCMV). Limited studies have been performed to identify antiviral agents that inhibit PCMV. The activity of recently approved antiviral agents, alone or in combination with other drugs, has not been tested for PCMV. These agents include maribavir, letermovir, the anti-herpes simplex virus (HSV) pritelivir, and the lipid-conjugate analog of cidofovir (brincidofovir)8’11, which is FDA-approved only for smallpox. Based on sequence similarity, several antiviral agents that reportedly inhibit HHV6 may also have activity against PCMV12’15. These include cyclopropavir and its analogs16, Benzimidazole analogs, and maribavir17. Other agents include the antimalarial agent artesunate.Atty. Dkt. No. 650053.01243 which is effective against HCMV, including drug-resistant strains, as well as HHV618 19There is an unmet need for safe and effective antiviral agents for both PCMV and BCMV. We aimed to test inhibitors of PCMV replication in vitro.

[0138] Materials and Methods

[0139] Compounds

[0140] Compounds known to inhibit HCMV or HHV6 with specific antiviral mechanisms or anti-cellular antiviral activities are listed in Table 1. GCV was dissolved in distilled water; all other compounds were dissolved in dimethyl sulfoxide (DMSO). Stock solutions (10 mM) were stored at -80°C. The final DMSO concentration in all experiments was 0.1%.

[0141] Cell culture, virus infection, and antiviral assays

[0142] PT-K75 porcine kidney epithelial cells (ATCC, CRL-2528), passage 10-16, were maintained in Dulbecco's modified Eagle medium (DMEM; Sigma- Aldrich, Cat# 5796-500mL) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA) and 1% penicillinstreptomycin (Gibco, Carlsbad. CA) at 37°C in a humidified 5% CO2 incubator. PCMV (ATCC VR-1499) was propagated in PT-K.75 cells. For plaque assays. 1 x 106uninfected PT-K.75 cells were mixed with 1 x 104PCMV-infected PT-K.75 cells (100:1 ratio) and seeded into 24-well plates (Coming; Durham, NC) in a final volume of 500 pL per well. Negative control wells contained only 1 x 106uninfected cells. After overnight incubation (-16 h), the medium was replaced with 500 LIL of overlay medium comprising a 1:1 mixture of 0.5% carboxy methyl cellulose (Sigma, St Louis, MO, Cat# M0512-100G) in DMEM and DMEM supplemented with 4% FBS, with or without the test compounds. Plates were incubated undisturbed at 37°C in 5% CO2 for 14 days. On day 14, the overlay medium was removed, and wells were washed once with phosphate-buffered saline (PBS). Cells were fixed with 100 pL of 4% paraformaldehyde (16% w / v stock; Alfa Aesar, Cat# 43368, Thermo Fisher Scientific. Ward Hill. MA) for 1 h at room temperature, washed again with PBS, and stained with 0.1% crystal violet (Sigma, Cat# C 158-50G), approximately three drops per well, for 20 min. Excess stain was removed under running tap water, and plates were air-dried. Plaques, including both typical and atypical forms, were manually counted the next day using a stereomicroscope.

[0143] Quantitative PCR (qPCR) Assay

[0144] PT-K75 cells were seeded in 24-well plates at a density of 0.1 x 106mixed with 0.5 x 106PCMV-infected cells per well (2: 1 ratio). Three wells containing only 1 x 106non-infected PT-K.75 cells served as negative controls. On Day 1 (approximately 16 h post-seeding), cells were treated with either 4% fetal bovine serum (FBS)-containmg growth medium or with testAtty. Dkt. No. 650053.01243 compounds diluted in 4% FBS-containing medium. On Day 5, the media in the non-treated wells were replaced with fresh 4% FBS growth medium, and the media in the treated wells were replaced with fresh drug-containing 4% FBS medium. On Day 10, cells were scraped using sterile pipette tips, and the entire contents of each well (cells and supernatant) were collected into 1.5 mL microcentrifuge tubes. Viral DNA was extracted using the eMag™ Nucleic Acid Extraction Platform (Applied Biosystems, Lithuania) according to the manufacturer's protocol. Quantitative PCR was performed using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Cat# A25741) with primers targeting the PCMV DNA polymerase: F 5’-ACT TCG TCG CAG CTC ATC TGA -3’ (SEQ ID NO: 1), and R 5’-ATT GTT GAT AAA GTC ACT CGT CTG C-3'(SEQ ID NO: 2). (1) Cell GAPDH (Sus scrofa) primers were F 5’-ACA TGG CCT CCA AGG AGT AAG A-3' (SEQ ID NO: 3), and R 5 -GAT CGA GTT GGG GCT GTG ACT-3’ (SEQ ID NO: 4). Amplifications were carried out in triplicate, and relative viral genome quantification was determined using the ACt method.

[0145] Cell viability

[0146] An MTT assay (Thermo Fisher Scientific, Fair Lawn, NJ, Cat# 158992500) was performed. PT-K75 cells were seeded into 96-well plates at a density of 1.5 x io4cells per well in DMEM containing 4% FBS and 1% penicillin-streptomycin. On the following day (Day 1), test compounds were prepared at various concentrations in 4% FBS-containing DMEM, and 100 pL of each solution was added to the appropriate wells. On Day 11, without removing the culture medium, 20 pL of MTT solution (5 mg / mL) was added to each well. Plates were incubated at 37°C for 4 h to allow the formation of formazan crystals. After incubation, 100 pL of pre-warmed solubilization solution was added per well, followed by a 10-minute incubation at room temperature or 37° C. Wells were mixed thoroughly using a pipette to ensure complete dissolution of formazan. Absorbance was measured at 570 nm using a microplate reader.

[0147] Dose-response curves

[0148] To assess the antiviral activity of individual compounds, dose-response curves were generated. Cells were seeded in appropriate culture conditions and infected at a defined multiplicity of infection. Serial dilutions of each compound were prepared to establish a concentration range suitable for calculating inhibitory effects. Viral replication was quantified, and the half-maximal effective concentration (EC50) was determined by fitting the data to the following equation:

[0149] Log (fa / fu) =m log (D / EC50) or f„ = 1 / [1 +(EC5o / D)m], where f, and f< (equal to 1 -fa) represent the fractions of virus affected and unaffected by treatment, respectively; D is the drugAtty. Dkt. No. 650053.01243 concentration; ECso is the concentration required to achieve 50% inhibition of viral replication; and m corresponds to the slope of the dose-response curve.

[0150] Analysis of drug combination

[0151] Each compound was applied at a concentration twice its ECso, followed by a series of 2-fold dilutions. Drug combination effects on PCMV replication were assessed using the Bliss independence model, assuming independent mechanisms of action; the expected combined effect (FU 1+2) is calculated as the product of the individual effects of each compound, according to the equation:1 1

[0152] Ful+2= F01■ FU2=D1■ p2^50(1 / 1EC5O(2 /

[0153] In this model, D represents the drug concentration, m is the slope (Hill coefficient), and EC50 denotes the concentration required to achieve 50% inhibition of viral replication. The indices 1 and 2 refer to inhibitors 1 and 2, respectively. The combined effect of the two inhibitors, expressed as the fractional unaffected population (Fui+2), is calculated as the product of their individual effects (Fu, and Fu2). To evaluate the nature of the drug interaction, the observed fold inhibition is compared to the expected fold inhibition predicted by the Bliss model. A ratio greater than 1 indicates a synergistic effect, a ratio less than 1 suggests antagonism, and a ratio equal to 1 reflects an additive interaction.

[0154] Sequence alignments

[0155] The HCMV and PCMV genomes were aligned. The overall sequence homology was low (YY%). Based on the antivirals used and their known HCMV targets, alignments were generated for HCMV UL97, and its kinase domain, the HCMV polymerase UL54, the terminase subunit UL56, ULI 05 helicase, UL70 primase, and ULI 02 primase-associated factor.

[0156] Results

[0157] Antiviral Activity of FDA- Approved Agents Against PCMV

[0158] To establish a comprehensive profile of antiviral susceptibility for PCMV, we screened a panel of FDA-approved antiviral agents using both plaque reduction and quantitative PCR assay.

[0159] The 50% effective concentration (EC50) values and 50% cell viability (CC50) are summarized in Table 2.

[0160] Ganciclovir demonstrated moderate activity, with EC50 values of 22.74 ± 10.37 pM (PCR) and 9.45 ± 0.37 pM (plaque assay), while maintaining low cytotoxicity (CC50 > 500 pM),Atty. Dkt. No. 650053.01243 resulting in a favorable selectivity index (FIG. 3). The investigational compound cyclopropavir exhibited robust anti-PCMV activity with ECso values of 2.13 ± 0.31 pM (PCR) and 2.58 ± 0.23 pM (plaque), demonstrating approximately 10-fold greater potency than ganciclovir. Similarly, hPMPA showed activity with a PCR ECso of 8.37 ± 3.3 pM. Notably, foscamet failed to inhibit PCMV replication at concentrations up to 500 pM (Table 2).

[0161] Among DNA polymerase inhibitors, brincidofovir demonstrated the most potent activity with ECso values of 0.01 ± 0.00 pM in both PCR and plaque assays, representing approximately 100-fold greater potency than cidofovir (PCR ECso: 1.65 ± 0.16 pM; plaque ECso: 5.14 ± 0.21 pM), FIG. 4.

[0162] Differential Activity of Terminase and Kinase Inhibitors

[0163] Maribavir, a benzimidazole riboside that targets the HCMV UL97 kinase, showed no detectable anti-PCMV activity under standard culture conditions with 4% FBS (ECso >30 pM). When serum concentrations were reduced, maribavir demonstrated limited activity, with EC50 values of 16.02 ± 1.79 pM (1% FBS) and 20.84 ± 3.41 pM (0.5% FBS), suggesting potential differences in the PCMV kinase-binding site compared to HCMV UL97 (FIG. 4).

[0164] Similarly, letermovir, which targets the HCMV terminase complex, showed no activity against PCMV at concentrations up to 3 pM, indicating species-specific differences in the viral terminase complex. Acyclovir, as expected given its requirement for the HSV thymidine kinase activation, showed no activity at concentrations up to 30 pM. Pritelivir, a helicase-primase inhibitor approved for the treatment of HSV, demonstrated moderate activity with a PCR ECso of 3.33 ± 0.37 pM; however, plaque assay results were inconclusive at higher concentrations (10-50 pM).

[0165] Potent Inhibition by Anticellular Antiviral Agents

[0166] Compounds targeting cellular pathways essential for viral replication demonstrated remarkable potency against PCMV (FIGs. 5, 6). The artemisinin dimer 838 exhibited strong activity, with ECso values of 0.09 ± 0.00 pM (PCR) and 0.12 ± 0.00 pM (plaque assay), but a narrow therapeutic window (CCso: 0.04 ± 0.05 pM). The monomeric artemisinin, artesunate, exhibited ECso values of 7.15 ± 0.66 pM (PCR) and 5.67 ± 1.25 pM (plaque), with CCso: 117.6 ± 12.40 pM, resulting in a selectivity index of approximately 20.

[0167] Emetine, a protein synthesis inhibitor, demonstrated potent anti-PCMV activity with ECso values of 0.03 ± 0.00 pM in both assays, though cytotoxicity (CCso: 0.17 ± 0.01 pM) limited its therapeutic index to approximately 6-fold. Digitoxin, a cardiac glycoside that inhibits Na+ / K+-ATPase, showed the most potent activity among all tested compounds with ECso valuesAtty. Dkt. No. 650053.01243 of 0.010 ± 0.48 pM (PCR) and 0.006 ± 0.00 pM (plaque), maintaining a reasonable selectivity index with CCso of 0.20 ± 0.08 pM.

[0168] Activity of Novel Investigational Compounds

[0169] Two investigational compounds with previously reported anti-HCMV activity were evaluated against PCMV (FIG. 7). MLS8091 demonstrated moderate activity, with EC50values of 0.70 ± 0.18 μM (PCR) and varying plaque assay results (5.53 ± 0.15 μM or >50 μM, depending on conditions), while maintaining low cytotoxicity (CC50> 100 μM). MLS8554 exhibited consistent activity, with EC50values of 6.91 ± 2.68 μM (PCR) and 3.69 ± 0.13 μM (plaque assay), along with favorable cytotoxicity profiles (CC50> 100 μM).

[0170] Combination Studies Demonstrate Additive Effects

[0171] To explore potential synergistic interactions, we evaluated combinations of artesunate with other active PCMV inhibitors using the Bliss independence model (FIG. 8). Drug combinations were tested at concentrations ranging from 0.25 to 2 times their respective EC50values. The combination of artesunate with brincidofovir, cidofovir, or cyclopropavir resulted in additive effects, with observed fold-inhibition ratios approximating 1.0 across multiple concentration combinations. No evidence of antagonism was observed among any of the tested combinations, supporting the potential for combination therapy.

[0172] The fractional inhibition curves demonstrated that combining artesunate with DNA polymerase inhibitors maintained activity over a broader concentration range than either agent alone, potentially enabling dose-reduction strategies to minimize toxicity while maintaining antiviral efficacy.

[0173] Dose-Response Relationships and Therapeutic Indices

[0174] A comprehensive dose-response analysis revealed distinct patterns of PCMV inhibition across different compound classes. DNA polymerase inhibitors generally exhibited Hill coefficients (m) between 1.5 and 2.0, indicating cooperative binding or multiple binding sites. In contrast, anticellular agents exhibited steeper dose-response curves with Hill coefficients greater than 2.5, indicating more switch-like inhibition patterns consistent with threshold effects on cellular pathways.

[0175] Correlation Between Plaque Reduction and Viral DNA Quantification

[0176] A strong correlation was observed between plaque reduction and qPCR-based viral DNA quantification for most compounds (r2= 0.87), validating both assay systems. Notable exceptions included pritelivir and MLS8091, where plaque assay results showed greater variability, potentially due to compound precipitation or cellular effects not captured by DNAAtty. Dkt. No. 650053.01243 quantification. The consistency between assays for nucleoside analogs supports their direct effect on viral DNA synthesis, while the modest discordance for some agents may reflect additional effects on viral spread or plaque formation independent of DNA replication.Table 2 - EC50 and CC50 of each drugTABLE 2 PCMV antiviral activity and cell viabilityPlaque EC50Compound Drug PCR EC50(μM) Plaque EC50(μM) CC50(μM) Ganciclovir(GCV) 22.74 ± 10.37 9.45 ± 0.37 >500μM FDA-approved Foscarnet N / A >500μM N / A Active Brincidofovir 0.01 ± 0.00 0.01 ± 0.00 >1.2 antivirals Cidofovir 1.65 ± 0.16 5.14 ± 0.21 >300Pntelivir 3.33 ± 0.37 >10 or >50 N / A Cyclopropavir 2.13 ± 0.31 2.58 ± 0.23 63.17 ± 17.7 hPMPA 8.37 ± 3.3MBV(4%FBS) N / A >30pM N / A MBV FDA-approved(1%FBS) N / A 16.02 ± 1.79 N / A InactiveMBVantivirals(0.5%FBS) N / A 20.84 ± 3.41 N / A Acyclovir N / A >30pM N / A LTV N / A >3pM N / A Active Artesunate 7.15 ± 0.66 5.67 ± 1.25 117.6 ± 12.40 Antiviral Dimer 838 0.09 ± 0.00 0.12 ± 0.00 0.01 ± 0.01 Anticellular Emetine 0.03 ± 0.00 0.03± 0.00 0.17 ± 0.01 agents Digitoxin 0.010± 0.48 0.006± 0.00 0.20 ± 0.08 Active 5.53 ± 0.15 or Investigational MLS8091 0.70 ± 0.18 >50 >100 Agents MLS8554 6.91 ± 2.68 3.69 ± 0.13 >100

[0177] Discussion

[0178] Xenotransplantation is gaining increasing importance as the global demand for organs continues to grow. In 2025, the FDA approved the transplantation of genetically modified animal organs into patients with end-stage kidney failure. Recently, two pig-to-human kidney transplants in brain-dead human recipients were performed. Although renal function restoration was achieved in the kidney xenografts from genetically modified minipigs, reactivation ofAtty. Dkt. No. 650053.01243 PCMV led to significant immunopathological changes in the recipients20. Pig-to-human lung xenotransplantation was recently reported in a brain-dead male human recipient following a brain hemorrhage21, and a pig liver was xenotransplanted to a brain-dead patient22.

[0179] Early studies already warned that PCMV infection could result in early rejection of kidney grafts in a pig-to-baboon xenotransplantation model5. Recently published reports on the dysfunction of the first human heart xenotransplant6-23also indicated that PCMV infection and reactivation may lead to endothelial cell damage in xenografts.

[0180] This report presents detailed in vitro testing of approved and investigational antiviral agents against PCMV. Several FDA-approved agents showed good activity against PCMV, specifically cidofovir and brincidofovir. Although GCV showed some activity, its EC50was lower than that of HCMV. Cyclopropavir exhibited a lower EC50than GCV, consistent with its enhanced activity against HCMV. Other FDA-approved agents, including acyclovir, LTV, PTV, and FOS, showed no activity against PCMV. Antiviral anticellular agents, such as artemisinin derivatives, emetine, and digitoxin, exhibited potent PCMV inhibition, and a combination therapy of artesunate resulted in additive effects. The results support both unique and shared targets of HCMV and PCMV, with a similar pattern of inhibition reported for HHV-6.

[0181] Studies have shown variable activity of the available HCMV drugs against PCMV24,25. In infected porcine fallopian tube cells, monitoring of PCMV viral load in supernatants revealed good inhibition with ganciclovir and cidofovir24. However, in another in vitro study using a PCMV -infected porcine fallopian tube model, PCMV was relatively resistant to ganciclovir, acyclovir, and leflunomide, while cidofovir and foscamet showed some antiviral activity25. In clinical use, foscarnet and cidofovir carry a high risk of nephrotoxicity and other organ toxicities26-27. An in vivo pig-to-baboon xenotransplant study also demonstrated reduced activity of ganciclovir against both PCMV and BCMV25. Higher doses of ganciclovir are highly toxic and cause severe bone marrow suppression28. The in vitro efficacy of GCV for HHV-6 is poor29, possibly due to low levels of phosphorylation by the HHV-6 U69 kinase and reduced susceptibility of the DNA polymerase30.

[0182] Brincidofovir, a prodrug of cidofovir, is active against a wide range of DNA viruses. It had good activity against HHV-6A in vitro, with an EC50of 3 nM (Williams-Aziz, 2005; Keith, 2004). It has also shown excellent activity in animal studies (Bidanset 2004, Quenelle 2004). Our data also reveal excellent activity against PCMV,

[0183] Cyclopropavir (CPV), a guanine nucleoside analog structurally similar to GCV, exhibits potent activity against many herpesviruses, particularly against HHV-6A and HHV-6B, withAtty. Dkt. No. 650053.01243 EC50values of 7.8 and 0.7 μM, respectively. CPV also demonstrated strong in vivo activity in a murine CMV model, resulting in reduced mortality in severe combined immunodeficient (SCID) mice and decreased viral replication in human fetal tissue implanted in SCID mice infected with HCM31. Its mechanism of action is similar to that of GCV, being initially phosphorylated by the CMV UL97 kinase, which results in the inhibition of viral DNA synthesis. It is more effective than GCV in vitro32, consistent with a study showing that purified pUL97 phosphorylated CPV 45-fold more extensively than GCV33. A surrogate assay for UL97 kinase activity revealed that similar to MBV, CPV inhibited the activity of UL9734. Thus, CPV is unique in that it inhibits DNA synthesis and the normal activity of the UL97 kinase.

[0184] In vitro studies testing a wide range of acyclic nucleoside analogs (S)-3-deaza-HPMPA, also referred to as 9-(S)-[3-hydroxy-2-(phosphonomethoxy) propyl] -3-deazaadenine or 3-deaza-HPMPA, have shown promising results with an EC50of 1.4 μM (Reymen 1995, Naesens 2006).

[0185] The antimalarial agent artesunate reportedly inhibits HCMV1235, including drugresistant strains of HCMV36,37. Artesunate was used in the treatment of a child with HHV-6 viral myocarditis38. Our studies showed similar EC50concentrations against PCMV as those reported for HCMV and HHV6. Artesunate has been in extended clinical use for the treatment of malaria, demonstrating its safety for human use (Gomes 2008).

[0186] MBV did not inhibit PCMV. and even under reduced serum conditions, the EC50was higher than that reported for HCMV17. This may indicate a difference in the binding site of the kinase for MBV.

[0187] Artesunate (AS) was reported to be successful in treating HCMV disease37and a child with HHV-6 myocarditis38. Its 50% inhibitory concentration is ~3.8 μM and ~ 10 μM against HHV-6 and HCMV, respectively, in vitro, and it reduces both early and late viral protein expression39. In a report of 117 patients who received an allogeneic hematopoietic stem cell transplant, AS was used in 25 patients. It was active against CMV, with a complete clearance in 20% of cases40. We and others have reported on the anti-HCMV activity of artemisinin monomers and dimers, with the latter showing improved inhibition12,13’36,37’40. The EC50of artesunate and dimer 838 against PCMV was similar to the level of HCMV inhibition, suggesting a shared cellular target.

[0188] Cardiac glycosides, specifically digitoxin, have been reported to inhibit HCMV and HSV-1 in vitro at nanomolar concentrations41’44. Their activity against HHV-6 has not beenAtty. Dkt. No. 650053.01243 reported. Digitoxin inhibited PCMV, suggesting the activation of shared cellular pathways by the Na, K-ATPase pump.

[0189] Emetine also exhibited a similar level of PCMV inhibition to previously reported effects on HCMV45.

[0190] Finally, two compounds that were recently reported to inhibit HCMV were tested and found to be active against PCMV. MLS8554 reportedly inhibits HCMV, MCMV, HSV-1, and HSV-215, and was also active against PCMV. MLS8091, which has shown HCMV inhibition at around the time of GCV, but was also active against GCV-resistant HCMV, was active against PCMV.

[0191] The effectiveness of antiviral therapy remains to be investigated. In the most recent heart xenotransplant recipient, GCV, val-ACV, and cidofovir were used, and after the second dose of cidofovir, the viral load was decreased6. However, this could have been confounded by the fact that the patient also received a human IgG preparation, which likely contained antibodies against HHV-6 that cross-react with GPCMV.

[0192] In summary, this detailed investigation identifies several inhibitors for future in vivo studies. We have used one viral strain and one cell line: therefore, future studies should include additional PCMV strains and cell lines, as well as assays to identify additional PCMV inhibitors and combination regimens.

[0193] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0194] Citations to a number of patent and non-patent references may be made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is anAtty. Dkt. No. 650053.01243 inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.References1. Yamada, K., et al. Marked prolongation of porcine renal xenograft survival in baboons through the use of alphal,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nat Med 11, 32-34 (2005). 2. Griesemer, A. D., et al. Results of gal-knockout porcine thymokidney xenografts.Am J Transplant 9, 2669-2678 (2009).3. Eisenson. D. L., Hisadome, Y. & Yamada. K. Progress in Xenotransplantation:Immunologic Barriers, Advances in Gene Editing, and Successful Tolerance Induction Strategies in Pig-To-Primate Transplantation. Front Immunol 13, 899657 (2022).4. Mueller, N. J., et al. Activation of cytomegalovirus in pig-to-primate organ xenotransplantation. J Virol 76, 4734-4740 (2002).5. Yamada, K., et al. Porcine cytomegalovirus infection is associated with early rejection of kidney grafts in a pig to baboon xenotransplantation model. Transplantation 98, 411-418 (2014).6. Griffith, B. P., et al. Genetically Modified Porcine-to-Human Cardiac Xenotransplantation. N Engl J Med 387, 35-44 (2022).7. Halecker, S., et al. How, where and when to screen for porcine cytomegalovirus (PCMV) in donor pigs for xenotransplantation. Sci Rep 12, 21545 (2022).8. Chemaly, R. F., Hill, J. A., Voigt, S. & Peggs, K. S. 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[0195] SequencesAtty. Dkt. No. 650053.01243 SEQ ID NO Sequence1 ACT TCG TCG CAG CTC ATC TGA2 ATT GTT GAT AAA GTC ACT CGT CTG C3 ACA TGG CCT CCA AGG AGT AAG A4 GAT CGA GTT GGG GCT GTG ACT

Claims

Atty. Dkt. No. 650053.01243CLAIMS1. A method of inhibiting infection of a cell by porcine cytomegalovirus (PCMV), the method comprising contacting the cell with one or more compound selected from ganciclovir, maribavir, foscarnet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir. MLS8091. digitoxin, or MLS8544.

2. The method of claim 1, wherein the one or more compound is brincidofovir, cidofovir, cyclopropavir, artesunate, artemisinin dimer 838, emetine, MLS8091, or MLS8544.

3. The method of claim 1, wherein contacting comprises contacting the cell with the one or more compound at a concentration of about 1 nM to about 100 nM.

4. The method of claim 1, wherein the cell comprises a porcine cell.

5. The method of claim 1, wherein the cell comprises a porcine heart cell, a porcine liver cell, a porcine vascular cell, a porcine kidney cell, a porcine pancreas cell, or a porcine lung cell.

6. The method of claim 4, wherein the porcine heart cell comprises a cardiomyocyte.

7. The method of claim 4, wherein the porcine liver cell comprises a hepatocyte, a Kupffer cell, a hepatic stellate cell, or a cholangiocyte.

8. The method of claim 4, wherein the porcine vascular cell comprises a vascular endothelial cell.

9. The method of claim 4, wherein the porcine kidney cell comprises a kidney epithelial cell, a podocyte, a mesangial cell, or a kidney endothelial cell.

10. The method of claim 4, wherein the porcine pancreas cell comprises an acinar cell, an alpha cell, a beta cell, or a pancreatic polypeptide (PP) cell.

11. The method of claim 4, wherein the porcine lung cell comprises a lung epithelial cell, a lung fibroblast, an alveolar macrophage, an alveolar type 2 (ATII) cell, an airway basal cell, a bronchioalveolar stem cell (BASC), a mast cell, an alveolar epithelial cell, or a bronchiolar epithelial cell.Atty. Dkt. No. 650053.01243 12. The method of claim 1, wherein the one or more compound comprises two or more compounds selected from ganciclovir maribavir, foscamet, brincidofovir. cidofovir. emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, MLS8091, or MLS8544.

13. A method of inhibiting reactivation of porcine cytomegalovirus (PCMV) in a porcine cell infected with PCMV, the method comprising contacting the cell with one or more compound selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, MLS8091, digitoxin, or MLS8544.

14. The method of claim 12, wherein the one or more compound is brincidofovir, cidofovir, artesunate, artemisinin dimer 838, emetine, cyclopropavir, MLS8091, digitoxin, or MLS8544.

15. The method of claim 12, wherein contacting comprises contacting the cell with the compound at a concentration of about 1 nM to about 100 nM.

16. The method of claim 12, wherein the cell comprises a porcine cell.

17. The method of claim 12, wherein the cell comprises a porcine heart cell, a porcine liver cell, a porcine vascular cell, a porcine kidney cell, a porcine pancreas cell, or a porcine lung cell.

18. The method of claim 16, wherein the porcine heart cell comprises a cardiomyocyte.

19. The method of claim 16, wherein the porcine liver cell comprises a hepatocyte, a Kupffer cell, a hepatic stellate cell, or a cholangiocyte.

20. The method of claim 16, wherein the porcine vascular cell comprises a vascular endothelial cell.

21. The method of claim 16, wherein the porcine kidney cell comprises a kidney- epithelial cell, a podocyte, a mesangial cell, or a kidney endothelial cell.

22. The method of claim 16, wherein the porcine pancreas cell comprises an acinar cell, an alpha cell, a beta cell, or a pancreatic polypeptide (PP) cell.Atty. Dkt. No. 650053.01243 23. The method of claim 16, wherein the porcine lung cell comprises a lung epithelial cell, a lung fibroblast, an alveolar macrophage, an alveolar type 2 (ATII) cell, an airway basal cell, a bronchioalveolar stem cell (BASC), a mast cell, an alveolar epithelial cell, or a bronchiolar epithelial cell.

24. The method of claim 12, wherein the one or more compound comprises two or more compounds selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, MLS8091, digitoxin, or MLS8544.

25. A method of reducing or preventing viral activation in a xenotransplantation donor tissue or organ, the method comprising contacting the xenotransplantation donor tissue or organ with one or more compound selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, MLS8091, digitoxin, or MLS8544.

26. The method of claim 24, wherein the one or more compound is brincidofovir, cidofovir. artesunate, artemisinin dimer 838. emetine, cyclopropavir, MLS8091, digitoxin, or MLS8544.

27. The method of claim 24, wherein the donor tissue or organ is contacted with the one or more compound at a concentration of about 1 nM to about 100 nM.

28. The method of claim 24, wherein the donor tissue or organ comprises porcine heart tissue, porcine liver tissue, porcine vascular tissue, porcine kidney tissue, porcine pancreas tissue, or porcine lung tissue.

29. The method of claim 24, wherein the donor tissue or organ comprises a porcine heart, a porcine liver, a porcine blood vessel, a porcine kidney, a porcine pancreas, or a porcine lung.

30. The method of claim 24, wherein the tissue or organ is located in a living recipient and the tissue or organ is connected to the living recipient’s circulatory system.

31. The method of claim 29, wherein the recipient is a primate.

32. The method of claim 30, wherein the recipient is a baboon.Atty. Dkt. No. 650053.01243 33. The method of claim 30, wherein the recipient is a human subject.

34. The method of claim 24, wherein the one or more compound comprises two or more compounds selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir. cyclopropavir, MLS8091, digitoxin, or MLS8544.

35. A method of reducing the risk of rejection of a xenotransplant tissue or organ in a subject in need thereof or preventing rejection of a xenotransplant tissue or organ in a subject in need thereof, the method comprising administering an amount of one or more compound selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate, artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, MLS8091, digitoxin, or MLS8544.

36. The method of claim 34, wherein the one or more compound is brincidofovir, cidofovir, artesunate, artemisinin dimer 838, emetine, cyclopropavir, MLS8091, digitoxin, or MLS8544.

37. The method of claim 34, wherein the donor tissue or organ is contacted with the one or more compound at a concentration of about 1 nM to about 100 nM.

38. The method of claim 34, wherein the donor tissue or organ comprises porcine heart tissue, porcine liver tissue, porcine vascular tissue, porcine kidney tissue, porcine pancreas tissue, or porcine lung tissue.

39. The method of claim 34, wherein the donor tissue or organ comprises a porcine heart, a porcine liver, a porcine blood vessel, a porcine kidney, a porcine pancreas, or a porcine lung.

40. The method of claim 38, wherein the recipient is a primate.

41. The method of claim 39, wherein the recipient is a baboon.

42. The method of claim 39, wherein the recipient is a human subject.

43. The method of claim 34, wherein the one or more compound comprises two or more compounds selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir,Atty. Dkt. No. 650053.01243 emetine, artesunate, artemisinin, artemisinin dimer 838. emetine, pritelivir. cyclopropavir, MLS8091, digitoxin, or MLS8544.

44. A method of treating porcine cytomegalovirus (PCMV) infection in a subj ect in need thereof, the method comprising administering an amount of one or more compound selected from ganciclovir, maribavir, foscamet, brincidofovir, cidofovir, emetine, artesunate. artemisinin, artemisinin dimer 838, emetine, pritelivir, cyclopropavir, MLS8091, digitoxin, or MLS8544 to the subject.

45. The method of claim 44, wherein the one or more compound is brincidofovir. cidofovir, artesunate, artemisinin dimer 838, emetine, cyclopropavir, MLS8091, or MLS8544.

46. The method of claim 44, wherein the subject is a pig.

47. The method of claim 44, wherein the subject is a primate.

48. The method of claim 46, wherein the subject is a baboon.

49. The method of claim 46, wherein the subject is a human subject.

50. The method of claim 44, wherein the subject comprises porcine tissue.

51. The method of claim 44, wherein the subj ect comprises a porcine xenograft.

52. The method of claim 44, wherein treating comprises reducing one or more sign or symptom of PCMV infection in the subject.

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