WO2021211303A1 - Antiviral, bacteriophage-derived polypeptides and their use against viruses - Google Patents

Abstract

Disclosed herein are methods of using Chp peptides for treating a viral infection such as a coronavirus, including, for example, COVID-19, in a subject. Also disclosed are methods of using Chp peptides for inhibiting the growth, reducing the population, neutralizing the infectivity of, and/or killing a virus, such as a coronavirus, including, for example, COVID-19. Also disclosed are pharmaceutical compositions comprising an effective amount of a Chp peptide; and a pharmaceutically acceptable carrier for treating a viral infection, such as an infection caused by a coronavirus, such as COVID-19.

Description

ANTIVIRAL, BACTERIOPHAGE-DERIVED POLYPEPTIDES AND THEIR

USE AGAINST VIRUSES

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of, and relies on the filing date of, U.S. provisional patent application number 63/009,974, filed 14 April 2020, and U.S. provisional patent application number 63/121,373, filed 4 December 2020, the entire disclosures of which are incorporated herein by reference.

SEQUENCE LISTING

[002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on 1 April 2021, is named 0341.0026-00-304_ST25.txt and is 56,770 bytes in size.

FIELD OF THE DISCLOSURE

[003] The present disclosure relates to phage-derived amurin peptides viruses and the use of these peptides in killing viruses and combatting viral infection, including the novel SARS-CoV-2 vims, also known as COVID-19.

BACKGROUND OF THE DISCLOSURE

[004] Recently, a new class of antimicrobial compounds called Direct Lytic Agents (DLAs) have emerged as therapeutic agents for the treatment of serious and life-threatening infectious diseases, including various bacterial infections. The DLAs represent a new concept in antibiosis based on the use of bacteriophage-encoded proteins and peptides with potent lytic activities against a range of target organisms.

[005] In addition to the lysin family of cell wall hydrolase enzymes (with potent bacteriolytic activities) there is a second distinct group of DLAs, called amurins. Amurins represent a newly identified group of phage-encoded antimicrobial peptides having host lysis activity (Chamakura KR et ah, 2017. Mutational analysis of the MS2 lysis protein L. Microbiology 163:961-969). The term amurin describes a limited set of nonmuralytic (not “wall-destroying,” i.e., not based on peptidoglycan hydrolysis of the cell wall) lysis activities from both ssDNA and ssRNA phages ( Microviridae and Leviviridae, respectively). For example, the protein E amurin of phage fC174 (Family Microviridae , genus Microvirus) is a 91 amino acid membrane protein that causes lysis by inhibiting the bacterial translocase MraY, an essential membrane-embedded enzyme that catalyzes the formation of the murein precursor, Lipid I (Zheng Y et ah, 2009. Purification and functional characterization of phiX174 lysis protein E. Biochemistry 48:4999-5006). Additionally, the A2 capsid protein of phage z)b (Family Leviviridae , genus Allolevivirus ) is a 420-amino acid structural protein (and amurin) that causes lysis by interfering with MurA activity and dysregulating the process of peptidoglycan biosynthesis (Gorzelnik KV et al., 2016. Proc Natl Acad Sci U S A 113:11519-11524). Other non-limiting examples include the LysM amurin of phage M, which is a specific inhibitor of MurJ, the lipid II flippase of E. coli, and the protein L amurin of phage MS2 (Family Levivirdae, genus Levivirus), which is a 75 amino acid integral membrane protein and causes lysis in a manner requiring the activity of host chaperone DnaJ (Chamakura KR et al., 2017. J Bacteriol 199). A putative domain structure for the L-like amurins has been assigned and includes an internal leucylserine dipeptide immediately preceded by a stretch of 10-17 hydrophobic residues Some amurins have been described in detail, for example in PCT Published Application No. WO 2001/009382, but at best they constitute a basis for development of therapeutics and have not been developed into antibacterial or antiviral therapeutics.

[006] Other amurins derived from Chlamydia phage (Chp peptides) are also described, for example, in WO 2019/191598 and U.S. Provisional Application Serial Nos: 62/870,908 (filed 05 My 2019); 62/892,783 (filed 28 August 2019); 62/911,900 (filed 07 October 2019), 62/948052 (filed 13 December 2019), and 62/964743 (filed 23 January 2020), all of which are incorporated by reference in their entireties.

[007] Other antimicrobial peptides are known to display strong antiviral activity by integration into the viral envelope (Lakshmaiah Narayana and Chen, 2015, Peptides 72, 88-94; Ahmed et al., 2019 Viruses, 11:704). LL-37 and other peptides, for example, act both directly on the viral envelope, by perforation, and on the cell membrane, by saturation of the attachment receptors of the virus, heparan sulfates, as well as indirectly by immune-modulation. LL-37 is for example potent against influenza vims A, possibly by disrupting the viral membrane, and LL-37 expression in keratinocytes and B cells reduced the viral load of varicella zoster vims (Takiguchi et al., 2014; Wang et al., 2014). Furthermore, antibacterial peptides can also directly neutralize lipopolysaccharides (LPS) and inhibit the production of inflammatory cytokines (such as TNF-a, IL-6, and IL-8), control immune responses, and reduce inflammatory injury through the different immune regulation.

[008] There is a need to identify compounds with antiviral activity that can be used to treat viruses, especially in light of the recent emergence of pandemics induced by viruses, including coronaviruses, such as SARS-CoV-2.

SUMMARY OF THE DISCLOSURE

[009] This application discloses a novel class of phage lytic agents that are derived, for example, from Microviridae genomic sequences and are distinct from other such agents, including known lysins/artilysins and amurins. The phage lytic agents disclosed herein are referred to as Chlamydia phage (Chp) peptides, also referred to as “amurin peptides” (a functional definition not implying sequence similarity with amurins). Disclosed herein are various Chp peptides that have been identified, constituting a family of specific bacteriolytic proteins, as well as non-naturally occurring modified variants of those Chp peptides (corresponding to SEQ ID NOs. 81-91 and 94- 102).

[0010] One aspect of the disclosure is directed to a method for inhibiting the growth, reducing the population, neutralizing the infectivity of, and/or killing of at least one virus, such as a coronavirus (e.g. SARS-CoV-2), the method comprising contacting the virus with a composition comprising an effective amount of a Chp peptide. In certain embodiments, the Chp petide comprises (i) a Chp peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-4, 6-27, 54-66, 81-91 and 94-102 or active fragments thereof, or (ii) a modified Chp peptide having at least 80%, such as at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, or at least 99% sequence identity with at least one of SEQ ID NOs. 1-4, 6-27, 54-66, 81-91 and 94-102. In certain embodiments, the Chp peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 81-86 or active fragments thereof.

[0011] In other aspects, there is provided a method for inhibiting the growth, reducing the population, neutralizing the infectivity of, and/or killing of at least one virus, such as a coronavirus (e.g. SARS-CoV-2), the method comprising contacting the virus with a composition comprising an effective amount of (i) a Chp peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 1, 2, 4, 6, 8-16, 18-21, 23-26, 59-61, 63-65, 67, 81-91 and 94- 102 or active fragments thereof, or (ii) a modified Chp peptide having at least 80%, such as at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, or at least 99% sequence identity with at least one of SEQ ID NOs. 1, 2, 4, 6, 8-16, 18-21, 23-26, 59-61, 63-65, 67, 81-91 and 94- 102, wherein the modified Chp peptide inhibits said growth, reduces said population, neutralizes the infectivity of, and/or kills said at least one virus. In certain embodiments, the Chp peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 81, 87, 88, 89, 91, 97, 100 and 101 or active fragments thereof.

[0012] Also disclosed herein is a method for treating a viral infection, comprising administering a pharmaceutical composition comprising a Chp peptide to a subject diagnosed with, at risk for, or exhibiting symptoms of a viral infection. In certain embodiments, the Chp peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-4, 6-26, 54-67, 81-91 and 94-102, active fragments thereof, or a modified Chp peptide thereof, as disclosed herein. In certain embodiments, the viral infection is caused by a coronavirus, such as SARS-CoV-2.

[0013] Further disclosed herein is a method for preventing or treating a viral infection comprising co-administering to a subject diagnosed with, at risk for, or exhibiting symptoms of a viral infection, a combination of a first amount of a pharmaceutical composition comprising a Chp peptide and a second amount of an antiviral, wherein the first and the second amounts together are effective for preventing or treating the viral infection. In certain embodiments, the Chp peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-4, 6-26, 54-67, 81-91 and 94-102, active fragments thereof, or a modified Chp peptide thereof, as disclosed herein. In certain embodiments, the viral infection is caused by a coronavirus, such as SARS-CoV- 2.

[0014] In another aspect, the present disclosure is directed to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of (i) an isolated Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-4, 6-26, 54-67, 81-91 and 94-102 or active fragments thereof, or (ii) a modified Chp peptide having at least 80%, such as at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, at least 99% sequence identity with at least one of SEQ ID NOs. 1-4, 6-26, 54-67, 81-91 and 94-102, wherein the modified Chp peptide inhibits the growth, reduces the population, neutralizes the infectivity, and/or kills at least one vims, such as a coronavims. In certain embodiments, the coronavirus is SARS-CoV-2.

[0015] In another embodiment disclosed herein, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of an isolated Chp peptide selected from the group consisting of peptides Chpl, Chp 2, Chp3, Chp4, Chp6, Chp7, Chp8, Chp9, ChplO, Chpll, Chp 12, CPAR39, Gkhl, Gkh2, Unpl, Ecpl, Tmal, Ecp2, Ospl, Unp2, Unp3, Gkh3, Unp5, Unp6, Spil, Spi2, Ecp3, Ecp4, Lvpl, Lvp2, ALCES1, AVQ206, AVQ244, CDL907, AGT915, HH3930, Fen7875, SBR77, Bdpl, Unp4, and Myol or active fragments thereof.

[0016] In some embodiments, the Chp peptide is Chp2, Chp4, Chp6, Ecpl or Ecp2.

[0017] In another embodiment disclosed herein, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of an isolated Chp peptide selected from the group consisting of peptides Chp2-Ml, Chp2-Cys, Chp2-NC, Chp4::Chp2, Chp2-CAV, Ecpl-CAV, Chp6-Ml, Chpl0-Ml, Mse-Ml, Chp4-Ml, Chp7-Ml, Osp-Ml, Unp2-Ml, Unp3-Ml, Spi2-Ml, Ecp3-Ml, and Agtl-Ml or active fragments thereof.

[0018] In some embodiments, the Chp peptide is Chp2-Ml, Chp4-Ml, Ecpl -Ml, Chp6-Ml, ChplO-Ml, Unp2-Ml, Agtl-Ml, or Ecp3-Ml.

[0019] In various embodiments of the disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of (i) an isolated Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65; SEQ ID NO: 66; and SEQ ID NO: 67 or active fragments thereof.

[0020] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of (i) an isolated Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6, SEQ ID NO: 16; SEQ ID NO: 18; and SEQ ID NO: 54 or active fragments thereof. [0021] In various embodiments of the disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of (i) an isolated Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 81; SEQ ID NO: 82; SEQ ID NO: 83; SEQ ID NO: 84; SEQ ID NO: 85; SEQ ID NO: 86; SEQ ID NO: 87; SEQ ID NO: 88, SEQ ID NO: 89; SEQ ID NO: 90; SEQ ID NO: 91; SEQ ID NO: 95; SEQ ID NO: 96; SEQ ID NO: 97; SEQ ID NO: 98; SEQ ID NO: 99; SEQ ID NO: 100; SEQ ID NO: 101; and SEQ ID NO: 102, or active fragments thereof, or (ii) a modified Chp peptide having at least 80%, such as at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, at least 99% sequence identity with at least one of SEQ ID NOs. 81-91 and 95-102.

[0022] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of (i) an isolated Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 81; SEQ ID NO: 87, SEQ ID NO: 88; SEQ ID NO: 89; SEQ ID NO: 91; SEQ ID NO: 97; SEQ ID NO: 100; and SEQ ID NO: 101 or active fragments thereof, or (ii) a modified Chp peptide having at least 80%, such as at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, at least 99% sequence identity with at least one of SEQ ID NOs. 81, 87, 88, 89, 91, 97, 100, and 101.

[0023] In certain embodiments, the Chp peptide as disclosed herein or active fragments thereof contains at least one non-natural modification relative to the amino acid sequence of any one of SEQ ID NOs. 1-4, 6-26, 54-67, 81-91 and 94-102, such as SEQ ID NO: 94 or SEQ ID NO: 102, and in certain embodiments, the non-natural modification is selected from the group consisting of substitution modification, such as a substitution of an amino acid; an N-terminal acetylation modification; and a C-terminal amidation modification. In certain embodiments, the modified Chp peptide comprises at least one amino acid substitution, insertion, or deletion relative to the amino acid sequence of any one of SEQ ID NOs. 1-4, 6-27, 54-66, 81-91 and 94-102, wherein the modified Chp peptide inhibits the growth, reduces the population, neutralizes the infectivity of, and/or kills at least one vims, such as a coronavims. In certain embodiments, the at least one vims is SARS-CoV-2. In certain embodiments, the at least one amino acid substitution is a conservative amino acid substitution. In certain embodiments, the modified Chp peptide comprising at least one amino acid substitution relative to the amino acid sequence of any one of SEQ ID NOs. 1-4, 6-27, 54-66, 81-91 and 94-102 is a cationic peptide having at least one alpha helix domain. [0024] The pharmaceutical composition in some embodiments may be a solution, a suspension, an emulsion, an inhalable powder, an aerosol, or a spray. In some embodiments the pharmaceutical composition may also comprise one or more antibiotics suitable for the treatment of Gram-negative bacteria or acid-fast bacteria. In some embodiments, the pharmaceutical composition may also comprise one or more antivirals, such as hydroxychloroquine, oseltamivir (Tamiflu®), zanamivir, peramivir, remdesivir, and baloxavir. Optionally, the peptide Chpl is excluded such that the pharmaceutical composition does not comprise Chpl.

[0025] In certain embodiments, disclosed herein is a vector comprising a nucleic acid that encodes (i) a Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-4, 6-27, 54-66, 81-91 and 94-102 or active fragments thereof, or (ii) a Chp peptide having at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, or at least 99% sequence identity with at least one of SEQ ID NOs. 1-4, 6-27, 54-66, 81-91 and 94-102.

[0026] Also disclosed herein are recombinant expression vectors comprising a nucleic acid encoding (i) a Chp peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-4, 6-27, 54-66, 81-91 and 94-102 or active fragments thereof, or (ii) a modified Chp peptide having at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, or at least 99% sequence identity with at least one of SEQ ID NOs. 1-4, 6-27, 54-66, 81-91 and 94-102. In certain embodiments, the nucleic acid is operatively linked to a heterologous promoter. In certain embodiments, the nucleic acid encodes a Chp peptide comprising an amino acid sequence selected from the group consisting of the group consisting of SEQ ID NOs: 1-4, 6- 27, 54-66, 81-86 or active fragments thereof.

[0027] Further embodiments disclosed herein include an isolated host cell comprising the foregoing vectors. In some embodiments, the nucleic acid sequence is a cDNA sequence.

[0028] In yet another aspect, the disclosure is directed to isolated, purified nucleic acid encoding a Chp peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-4, 6-27, 54-66, 81-91 and 94-102 or active fragments thereof. Optionally, the nucleic acid is cDNA. In certain embodiments, the nucleotide sequence contains at least one non-natural modification, such as a mutation (e.g., substitution, insertion, or deletion) or a nucleic acid sequence encoding an N-terminal modification or a C-terminal modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A are three-dimensional models predicted by I-Tasser for structures of Chlamydia phage peptide (Chp) family members Chpl, Chp 2, Chp4, Chp5, Chp6, Chp7, Ecpl, Ecp2, and Osp 1. The human innate immune effector peptide LL-37 is included for comparison. Alpha helical structures are evident, and the top terminal is generally the N-terminal.

[0030] Figure IB shows the consensus secondary structure predictions for Chp2 (SEQ ID NO: 2) using JPRED4. The alpha-helices are indicated by the thick striped bar.

[0031] Figure 1C shows the consensus secondary structure predictions for Chp4 (SEQ ID NO: 4) using JPRED4. The alpha-helices are indicated by the thick striped bar.

[0032] Figure 2A is the rooted (UPGMA clustering method) phylogenetic tree of certain Chp family members generated from a ClustalW alignment.

[0033] Figure 2B is the unrooted (neighbor-joining clustering method) phylogenetic tree of certain Chp family members generated from a ClustalW alignment.

[0034] Figure 3 two photomicrographs showing rapid membrane perforation of a bacterial cell wall treated with an amurin peptide AMI.

DETAILED DESCRIPTION Definitions

[0035] As used herein, the following terms and cognates thereof shall have the following meanings unless the context clearly indicates otherwise:

[0036] “Carrier” refers to a solvent, additive, excipient, dispersion medium, solubilizing agent, coating, preservative, isotonic and absorption delaying agent, surfactant, propellant, diluent, vehicle and the like with which an active compound is administered. Such carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.

[0037] “Pharmaceutically acceptable carrier” refers to any and all solvents, additives, excipients, dispersion media, solubilizing agents, coatings, preservatives, isotonic and absorption delaying agents, surfactants, propellants, diluents, vehicles and the like that are physiologically compatible. The carrier(s) must be “acceptable” in the sense of not being deleterious to the subject to be treated in amounts typically used in medicaments. Pharmaceutically acceptable carriers are compatible with the other ingredients of the composition without rendering the composition unsuitable for its intended purpose. Furthermore, pharmaceutically acceptable carriers are suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are “undue” when their risk outweighs the benefit provided by the composition. Non-limiting examples of pharmaceutically acceptable carriers or excipients include any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, and emulsions such as oil/water emulsions and microemulsions. Suitable pharmaceutical carriers are described, for example, in Remington's Pharmaceutical Sciences by E.W. Martin, 18th Edition. The pharmaceutically acceptable carrier may be a carrier that does not exist in nature.

[0038] “Antibiotic” refers to a compound having properties that have a negative effect on bacteria, such as lethality or reduction of growth. An antibiotic can have a negative effect on any and all combinations of Gram-positive bacteria, Gram-negative bacteria, acid-fast bacteria, and non-acid fast bacteria. By way of example, an antibiotic can affect cell wall peptidoglycan biosynthesis, cell membrane integrity, or DNA or protein synthesis in bacteria. Nonlimiting examples of antibiotics active against Gram-negative bacteria include cephalosporins, such as ceftriaxone-cefotaxime, ceftazidime, cefepime, cefoperazone, and ceftobiprole; fluoroquinolones such as ciprofloxacin and levofloxacin; aminoglycosides such as gentamicin, tobramycin, and amikacin; piperacillin, ticarcillin, imipenem, meropenem, doripenem, broad spectrum penicillins with or without beta- lactamase inhibitors, rifampicin, polymyxin B, and colistin. Non-limiting examples of antibiotics active against acid-fast bacteria include isoniazid, rifampin, ethambutol, and pyrazinamide.

[0039] “Antiviral” refers to a compound having properties that have a negative effect on a vims, such as lethality or a reduction in ability to replicate. Nonlimiting examples of antivirals include hydroxychloroquine, oseltamivir (Tamiflu®), zanamivir, peramivir, remdesivir, and baloxavir. [0040] “Drug resistant” generally refers to a vims that is resistant to the antiviral activity of a drug.

[0041] “Effective amount” refers to an amount which, when applied or administered in an appropriate frequency or dosing regimen, is sufficient to prevent, reduce, inhibit, or eliminate viral growth, viral replication, or viral burden or to prevent, reduce, or ameliorate the onset, severity, duration, or progression of the disorder being treated (for example, a viral infection, such as COVID-19), prevent the advancement of the disorder being treated, cause the regression of the disorder being treated, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy, such as antiviral, antibiotic or bacteriostatic therapy.

[0042] “Co-administer” refers to the administration of two agents, such as a Chp peptide and an antiviral, antibiotic or any other antibacterial agent, in a sequential manner, as well as administration of these agents in a substantially simultaneous manner, such as in a single mixture/composition or in doses given separately, but nonetheless administered substantially simultaneously to the subject, for example at different times in the same day or 24-hour period. Such co-administration of Chp peptides with one or more additional antiviral or antibacterial agents can be provided as a continuous treatment lasting up to days, weeks, or months. Additionally, depending on the use, the co-administration need not be continuous or coextensive. [0043] “Subject” refers to a mammal, a plant, a lower animal, a single cell organism, or a cell culture. For example, the term “subject” is intended to include organisms, e.g., prokaryotes and eukaryotes, which are susceptible to or afflicted with viral infections, such as COVID-19, or bacterial infections, for example Gram-positive, Gram-negative bacterial infections, or acid-fast bacterial infections. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, e.g., a human suffering from, at risk of suffering from, or susceptible to infection by a virus, such as a coronavirus (e.g. SARS-CoV-2).

[0044] “Polypeptide” is used herein interchangeably with the term “peptide” and refers to a polymer made from amino acid residues and generally having at least about 30 amino acid residues. The term includes not only polypeptides in isolated form, but also active fragments and derivatives thereof, including modified variants. The term “polypeptide” also encompasses fusion proteins or fusion polypeptides comprising a Chp peptide as described herein and maintaining, for example a lytic function. Depending on context, a polypeptide can be a naturally occurring polypeptide or a recombinant, engineered, or synthetically produced polypeptide. A particular Chp peptide can be, for example, derived or removed from a native protein by enzymatic or chemical cleavage, or can be prepared using conventional peptide synthesis techniques (e.g., solid phase synthesis) or molecular biology techniques (such as those disclosed in Sambrook, J. et ah, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)) or can be strategically truncated or segmented yielding active fragments, maintaining, e.g., lytic activity against the same or at least one common target bacterium. [0045] “Fusion polypeptide” refers to an expression product resulting from the fusion of two or more nucleic acid segments, resulting in a fused expression product typically having two or more domains or segments, which typically have different properties or functionality. In a more particular sense, the term “fusion polypeptide” may also refer to a polypeptide or peptide comprising two or more heterologous polypeptides or peptides covalently linked, either directly or via an amino acid or peptide linker. The polypeptides forming the fusion polypeptide are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C- terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The term “fusion polypeptide” can be used interchangeably with the term “fusion protein.” The open-ended expression “a polypeptide comprising” a certain structure includes larger molecules than the recited structure, such as fusion polypeptides.

[0046] “Heterologous” refers to nucleotide, peptide, or polypeptide sequences that are not naturally contiguous. For example, in the context of the present disclosure, the term “heterologous” can be used to describe a combination or fusion of two or more peptides and/or polypeptides wherein the fusion peptide or polypeptide is not normally found in nature, such as for example a Chp peptide or active fragment thereof and a cationic and/or a polycationic peptide, an amphipathic peptide, a sushi peptide (Ding et al. Cell Mol Life Sci., 65(7-8): 1202-19 (2008)), a defensin peptide (Ganz, T. Nature Reviews Immunology 3, 710-720 (2003)), a hydrophobic peptide, and/or an antimicrobial peptide which may have enhanced lytic activity. Included in this definition are two or more Chp peptides or active fragments thereof. These can be used to make a fusion polypeptide with lytic activity.

[0047] “Active fragment” refers to a portion of a polypeptide that retains one or more functions or biological activities of the isolated polypeptide from which the fragment was taken, for example antiviral activity.

[0048] “Amphipathic peptide” refers to a peptide having both hydrophilic and hydrophobic functional groups. In certain embodiments, secondary structure may place hydrophobic and hydrophilic amino acid residues at opposite sides ( e.g ., inner side vs outer side when the peptide is in a solvent, such as water) of an amphipathic peptide. These peptides may in certain embodiments adopt a helical secondary structure, such as an alpha-helical secondary structure. [0049] “Cationic peptide” refers to a peptide having a high percentage of positively charged amino acid residues. In certain embodiments, a cationic peptide has a pKa-value of 8.0 or greater. The term “cationic peptide” in the context of the present disclosure also encompasses polycationic peptides that are synthetically produced peptides composed of mostly positively charged amino acid residues, such as lysine (Lys) and/or arginine (Arg) residues. The amino acid residues that are not positively charged can be neutrally charged amino acid residues, negatively charged amino acid residues, and/or hydrophobic amino acid residues.

[0050] “Hydrophobic group” refers to a chemical group such as an amino acid side chain that has low or no affinity for water molecules but higher affinity for oil molecules. Hydrophobic substances tend to have low or no solubility in water or aqueous phases and are typically apolar but tend to have higher solubility in oil phases. Examples of hydrophobic amino acids include glycine (Gly), alanine (Ala), valine (Val), Leucine (Leu), isoleucine (lie), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp).

[0051] “Augmenting” refers to a degree of activity of an agent, such as antiviral or antimicrobial activity, that is higher than it would be otherwise. “Augmenting” encompasses additive as well as synergistic (superadditive) effects.

[0052] “Synergistic” or “superadditive” refers to a beneficial effect brought about by two substances in combination that exceeds the sum of the effects of the two agents working independently. In certain embodiments the synergistic or superadditive effect significantly, i.e., statistically significantly, exceeds the sum of the effects of the two agents working independently. One or both active ingredients may be employed at a sub-threshold level, i.e., a level at which if the active substance is employed individually produces no or a very limited effect. The effect can be measured by assays such as the checkerboard assay, described here.

[0053] “Treatment” refers to any process, action, application, therapy, or the like, wherein a subject, such as a human being, is subjected to medical aid with the object of curing a disorder, eradicating a pathogen, or improving the subject’s condition, directly or indirectly. Treatment also refers to reducing incidence, alleviating symptoms, eliminating recurrence, preventing recurrence, preventing incidence, reducing the risk of incidence, improving symptoms, improving prognosis, or combinations thereof. “Treatment” may further encompass reducing the population, growth rate, or virulence of a virus, such as a coronavirus (e.g. SARS-CoV-2), in the subject and thereby controlling or reducing a viral infection in a subject or viral contamination of an organ, tissue, or environment. Thus “treatment” that reduces incidence may, for example, be effective to inhibit growth of the vims in a particular milieu, whether it be a subject or an environment. On the other hand, “treatment” of an already established infection refers to inhibiting the growth, reducing the population, neutralizing the infectivity of, killing, including eradicating, the vims responsible for an infection.

[0054] “Preventing” refers to the prevention of the incidence, recurrence, spread, onset or establishment of a disorder such as a viral infection. It is not intended that the present disclosure be limited to complete prevention or to prevention of establishment of an infection. In some embodiments, the onset is delayed, or the severity of a subsequently contracted disease or the chance of contracting the disease is reduced, and such constitute examples of prevention.

[0055] “Contracted diseases” refers to diseases manifesting with clinical or subclinical symptoms, such as the detection of fever or cough, as well as diseases that may be detected by growth of a viral pathogen ( e.g ., in culture) or detection of viral RNA (e.g., by known methods such as RT-PCR) when symptoms associated with such pathology are not yet manifest.

[0056] The term “derivative” in the context of a peptide or polypeptide or active fragments thereof is intended to encompass, for example, a polypeptide modified to contain one or more chemical moieties other than an amino acid that do not substantially adversely impact or destroy the lytic activity. The chemical moiety can be linked covalently to the peptide, e.g., via an amino terminal amino acid residue, a carboxy terminal amino acid residue, or at an internal amino acid residue. Such modifications may be natural or non-natural. In certain embodiments, a non-natural modification may include the addition of a protective or capping group on a reactive moiety, addition of a detectable label, such as antibody and/or fluorescent label, addition or modification of glycosylation, or addition of a bulking group such as PEG (pegylation) and other changes known to those skilled in the art. In certain embodiments, the non-natural modification may be a capping modification, such as N-terminal acetylations and C-terminal amidations. Exemplary protective groups that may be added to Chp peptides include, but are not limited to, t-Boc and Fmoc. Commonly used fluorescent label proteins such as, but not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and mCherry, are compact proteins that can be bound covalently or noncovalently to a Chp peptide or fused to a Chp peptide without interfering with normal functions of cellular proteins. In certain embodiments, a polynucleotide encoding a fluorescent protein may be inserted upstream or downstream of the Chp polynucleotide sequence. This will produce a fusion protein (e.g., Chp Peptide: :GFP) that does not interfere with cellular function or function of a Chp peptide to which it is attached. Polyethylene glycol (PEG) conjugation to proteins has been used as a method for extending the circulating half-life of many pharmaceutical proteins. Thus, in the context of Chp peptide derivatives, the term “derivative” encompasses Chp peptides chemically modified by covalent attachment of one or more PEG molecules. It is anticipated that pegylated Chp peptides will exhibit prolonged circulation half-life compared to the unpegylated Chp peptides, while retaining biological and therapeutic activity.

[0057] “Modified variant” refers to a Chp peptide wherein a non-naturally occurring modification has been made to the amino acid sequence that either enhances the lytic and/or antiviral activity or does not substantially adversely impact or destroy the lytic and/or antiviral activity of the Chp peptide. Exemplary modifications that may be made to modified variants include modifying an amino acid of the Chp peptide, such as a positively charged amino acid, from an L-form to a D-form; adding an amino acid residue or residues to the C-terminus and/or the N- terminus, forming fusion polypeptides, and forming charge array variants, wherein amino acid charges have been reordered.

[0058] “Percent amino acid sequence identity” refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, such as a specific Chp peptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available software such as BLAST or software available commercially, for example from DNASTAR. Two or more polypeptide sequences can be anywhere from 0-100% identical, or any integer value there between. In the context of the present disclosure, two polypeptides are “substantially identical” when at least 80% of the amino acid residues (such as at least about 85%, at least about 90%, at least about 92.5%, at least about 95%, at least about 98%, or at least about 99%) are identical. The term “percent (%) amino acid sequence identity” as described herein applies to Chp peptides as well. Thus, the term “substantially identical” will encompass mutated, truncated, fused, or otherwise sequence-modified forms of isolated Chp polypeptides and peptides described herein, and active fragments thereof, as well as polypeptides with substantial sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, or at least 99% identity as measured for example by one or more methods referenced above) as compared to the reference (wild type or other intact) polypeptide.

[0059] As used herein, two amino acid sequences are “substantially homologous” when at least about 80% of the amino acid residues (such as at least about 85%, at least about 90%, at least about 92.5%, at least about 95%, at least about 98%, or at least about 99%) are identical, or represent conservative substitutions. The sequences of the polypeptides of the present disclosure are substantially homologous when one or more, such as up to 10%, up to 15%, or up to 20% of the amino acids of the polypeptide, such as the Chp peptides described herein, are substituted with a similar or conservative amino acid substitution, and wherein the resulting peptides have at least one activity (e.g., antiviral effect) of the reference polypeptide, such as the Chp peptides disclosed herein.

[0060] As used herein, a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0061] “Inhalable composition” refers to pharmaceutical compositions of the present disclosure that are formulated for direct delivery to the respiratory tract during or in conjunction with routine or assisted respiration (e.g., by intratracheobronchial, pulmonary, and/or nasal administration), including, but not limited to, atomized, nebulized, dry powder, and/or aerosolized formulations. [0062] “Suitable” in the context of an antiviral being suitable for use against certain virus refers to an antiviral that was found to be effective against that vims even if resistance subsequently developed.

Microviridae phages

[0063] Members of the phage family Microviridae may be of particular interest as potential sources of anti-infective agents for several reasons. As disclosed herein, it has been found that a large subset of these phages, including those of the genus Chlamydiamicrovirus (Family Microvirus, subfamily Gokushovirinae), have no conserved amurin sequence and instead encode small, uncharacterized cationic peptides that appear to form the basis of a heretofore uncharacterized lytic system. Additionally, bacteriophages of the family Microviridae infect medically-relevant organisms, including members of the families Enterobacteriaceae, Pseudomonadaceae, and Chlamydiaceae (Doore SM et al, 2016. Virology 491:45-55.). They also lack amurins and instead, as disclosed herein, encode unique uncharacterized antimicrobial-like peptides (called amurin peptides) that have not been previously identified or had a function ascribed to them. It was reasoned that if the putative antimicrobial-like peptides act in a manner similar to previously described antimicrobial peptides (AMPs), they would then be predicted to enable “lysis from without” in a manner not possible with the amurins and their cytoplasmic targets.

[0064] Several different amurin peptides have now been distinguished for which there is an emerging understanding of a potent antimicrobial method of action, distinct from lysins, based on the targeted disruption of certain membrane bilayers, including the outer membrane of a broad range of Gram-negative pathogens including P. aeruginosa, K. pneumoniae, A. baumannii, E. coli, E. cloacae, S. maltophilia, and others. The disruption occurs by rapid formation of “holes” or “tears” in the outer membrane structure (see Figure 3). This activity results in a rapid bacteriolytic effect against a broad group of Gram-negative pathogens, as well as additional features include potent antibiofilm activity, synergy with a broad array of antibiotics, a low propensity for resistance, and no cross resistance.

[0065] Based on a bioinformatics analysis of all annotated Microviridae genomic sequences in GenBank (with a focus on phages that lack amurins), several novel and syntenic open reading frames were identified. They encode small cationic peptides with predicted alpha-helical structures similar to AMPs (but with amino acid sequences dissimilar to AMPs) from the innate immune systems of a variety of vertebrates. These peptides, collectively referred to as “Chp peptides” or “amurin peptides,” are primarily found in the Chlamydiamicrovirus genus and, to a lesser extent, in other related members of the subfamily Gokushovirinae. See, e.g., Tables 1 and 2 below. The Chp peptides from a range of Microviridae phages may exhibit 30-100% identity to each other and may have no or little homology with other peptides in the protein sequence database. See, e.g., Table 3 below. [0066] As used herein, “Chp peptides” refers to both naturally-occurring Chp peptides, non- naturally occurring modified variants thereof, and modified Chp peptides having at least one modification (e.g., substitution) as compared to a wild-type Chp peptide. Several of the Chp peptides disclosed herein exhibit notable sequence similarities to each other but are distinct from other known peptides in the sequence databases. Despite the unique sequences of the Chp peptides, they are all predicted to adopt alpha-helical structures similar to some previously described antimicrobial peptides (AMPs) of vertebrate innate immune systems (E.F. Haney et al, 2017, In Hansen PR (ed), Antimicrobial Peptides: Methods and Protocols, Methods in Molecular Biology, vol. 1548) but with no sequence similarity to such AMPs.

[0067] The Chp peptides are about 40-50 amino acids in length and are highly cationic, with predicted pi values of greater than 10 and net charges of around +16. The Chp peptides are also predicted to adopt a-helical structures in membrane environments, which may form the basis of their antimicrobial effects, akin to some AMPs, through processes such as pore formation. Unlike most AMPs, however, the Chp peptides are predicted to adopt distinctly kinked helices (driven by strategically placed proline residues) with unusual amphipathic characters defined by a strongly polar face and a second hydrophobic face with regularly interspersed polar residues. While not wishing to be bound by theory, it is believed that these structural features may form the basis by which the Chp peptides target biological membranes in a unique manner distinct from other previously described AMPs, which have not evolved in the context of the phage lytic mechanism. Notably, the Chp peptides do not have a general membrane permeabilizing activity, as evidenced by the absence of hemolytic effects against human red blood cells.

[0068] In addition, several modified variants were derived from the identified Chp peptides. Based on the prediction that the Chp peptides possess AMP-like activities, the family members and modified variants were synthesized (Chp2 and Chp3 being identical amino acid sequences) for analysis in different Aspartate Aminotransferase (AST) assays. Based on minimum inhibitory concentration (MIC) values of 0.25-4 pg/mL in the presence of human serum, several Chp peptides have demonstrated superior serum activity compared to a group of up to 17 known AMPs tested (including innate immune effectors and derivatives thereof). Several Chp peptides have additionally demonstrated superior activity in pulmonary surfactant (Survanta®) in concentrations that are inhibitory to other known antibiotics, such as daptomycin. Furthermore, activity against a range of Gram-negative pathogens has been demonstrated, including several on the World Health Organization (WHO) and Centers for Disease Control (CDC) priority lists, including P. aeruginosa, E. coli, E. cloacae, K. pneumoniae, A. baumannii, and S. typhimurium. Likewise, activity against the acid-fast pathogen M. smegmatis has been demonstrated for several Chp peptides, and Chp2-Ml has been demonstrated to have anti-biofilm activity against biofilm comprising Stenotrophomonas species, such as Stenotrophomonas maltophilia.

[0069] One major drawback with the use of previously described AMPs as a treatment for invasive infections concerns toxicity to erythrocytes and a generalized membranolytic activity (i.e., hemolysis) (Oddo A. et ah, 2017. Hemolytic Activity of Antimicrobial Peptides. Methods Mol Biol 1548:427-435). Generally, this may be tested in vitro using a standardized assay for detecting the lysis of human red blood cells. Many of the Chp peptides disclosed herein exhibit no hemolytic activity against human red blood cells, in contrast to several AMPs described in the literature (as well as Triton X-100) to have hemolytic activity. In certain embodiments, the Chp peptides disclosed herein may only exhibit minimum hemolytic activity or no hemolytic activity against human red blood cells, as compared to AMPs. Another drawback of AMPs described in the literature concerns a loss of activity in the presence of human blood matrices and physiological salt concentrations (Mohanram H. et ah, 2016. Salt-resistant short antimicrobial peptides. Biopolymers 106:345-356). Certain Chp peptides are active in the presence of either human serum or plasma and/or active in growth media, such as Mueller Hinton broth and Casamino Acid medium, containing physiological salt concentrations. Although not wishing to be bound by theory, it is believed that the differences observed in activities of the Chp peptides and AMP peptides (in the literature) may be attributed to the distinct sources of the two types of agents, where the Chp peptides are from phage and the AMPs are based largely on innate immune effectors of vertebrate immune systems. The high activity of Chp peptides, the activity of Chp peptides in blood matrices, and/or the absence of hemolytic activity make them suitable for use in treating invasive diseases, such as those caused by viruses, including, for example, coronavimses, such as SARS-CoV-2. For example, in certain embodiments, the Chp peptides may be active in nanomolar quantities.

Polypeptides [0070] As demonstrated and explained herein, the Chp peptides described in this section, including wild-type Chp peptides, modified Chp peptides, derivatives, modified variants, or active fragments thereof, can be used in the pharmaceutical compositions and methods described herein.

[0071] In some embodiments, the Chp peptide is selected from at least one of Chpl (SEQ ID NO: 1), Chp2 (SEQ ID NO: 2), CPAR39 (SEQ ID NO: 3), Chp3 (SEQ ID NO: 54); Chp4 (SEQ ID NO: 4), Chp6 (SEQ ID NO: 6), Chp7 (SEQ ID NO: 7), Chp8 (SEQ ID NO: 8), Chp 9 (SEQ ID NO: 9), Chp 10 (SEQ ID NO: 10), Chp 11 (SEQ ID NO: 11), Chpl2 (SEQ ID NO: 12), Gkhl (SEQ ID NO: 13), Gkh2 (SEQ ID NO: 14), Unpl (SEQ ID NO: 15), Ecpl (SEQ ID NO: 16), Tmal (SEQ ID NO: 17), Ecp2 (SEQ ID NO: 18), Ospl (SEQ ID NO: 19), Unp2 (SEQ ID NO: 20), Unp3 (SEQ ID NO: 21), Gkh3 (SEQ ID NO: 22), Unp5 (SEQ ID NO: 23), Unp6 (SEQ ID NO: 24), Spil (SEQ ID NO: 25), Spi2 (SEQ ID NO: 26), Ecp3 (SEQ ID NO: 55), Ecp4 (SEQ ID NO: 56); Lvpl (SEQ ID NO: 57), Lvp2 (SEQ ID NO: 58), ALCES1 (SEQ ID NO: 59), AVQ206 (SEQ ID NO: 60), AVQ244 (SEQ ID NO: 61), CDL907 (SEQ ID NO: 62), AGT915 (SEQ ID NO: 63), HH3930 (SEQ ID NO: 64), Fen7875 (SEQ ID NO: 65), SBR77 (SEQ ID NO: 66), and Bdpl (SEQ ID NO: 67) or active fragments thereof having lytic activity.

[0072] In some embodiments, the Chp peptide is selected from at least one of Chp2-Ml (SEQ ID NO: 81), Chp2-Cys (SEQ ID NO: 82), Chp2-NC (SEQ ID NO: 83), Chp4::Chp2 (SEQ ID NO: 84), Chp2-CAV (SEQ ID NO: 85), Ecpl-CAV (SEQ ID NO: 86), Ecpl-Ml (SEQ ID NO: 87), Chp6-Ml (SEQ ID NO: 88), ChplO-Ml (SEQ ID NO: 89), Mse-Ml (SEQ ID NO: 90), Chp4-Ml (SEQ ID NO: 91), Chp2-SCR1 (SEQ ID NO: 92), Chp2-SCR1-Ml (SEQ ID NO: 93), Unp4 (SEQ ID NO: 94), Chp7-Ml (SEQ ID NO: 95), Ospl-Ml (SEQ ID NO: 96), Unp2-Ml (SEQ ID NO: 97), Unp3-Ml (SEQ ID NO: 98), Spi2-Ml (SEQ ID NO: 99), Ecp3-Ml (SEQ ID NO: 100), Agtl- M1 (SEQ ID NO: 101), and Myol (SEQ ID NO: 102) or active fragments thereof having lytic activity.

[0073] The Chp peptide may be a modified Chp peptide or active fragment thereof. In certain embodiments, the Chp peptide or active fragment thereof contains at least one non-naturally occurring modification relative to at least one of SEQ ID NOs. 1-4, 6-26, 54-67, 81-91, and 94- 102, such as at least one amino acid substitution, insertion or deletion.

[0074] The modified Chp peptides of the present disclosure are typically designed to retain an a- helix domain, the presence or absence of which can be readily determined using various software programs, such as Jpred4 (compio.dundee.ac.uk/jpred) and Helical Wheel (hael.net/helical.htm). [0075] In some embodiments, the a-helix domain spans most of the molecule. See, e.g., Chpl and Chp4 in Figure 1. In some embodiments, the a-helix domain is interrupted (see, e.g., Chp2 in Figure 1), and in some embodiments, the a-helix domain is truncated (see, e.g., Chp6 and Ospl in Figure 1). The a-helix domain of the Chp peptides of the present disclosure varies in size between about 3 and 32 amino acids, more typically between about 10 and 25 amino acid residues.

[0076] The modified Chp peptides of the present disclosure typically retain one or more functional or biological activities of the reference Chp peptide. In some embodiments, the modification improves the antiviral activity of the Chp peptide. Typically, the modified Chp peptide has improved in vitro antiviral activity (e.g., in buffer and/or media) in comparison to the reference Chp peptide. In other embodiments, the modified Chp peptide has improved in vivo antiviral activity (e.g., in an animal infection model). In some embodiments, the modification improves the antiviral activity of the Chp peptide in the absence and/or presence of human serum and/or pulmonary surfactant.

[0077] In some embodiments, Chp peptides disclosed herein or variants or active fragments thereof are capable of inhibiting the growth of, or reducing the population of, neutralizing the infectivity of, or killing a virus, such as a coronavirus (e.g., SARS-CoV-2).

[0078] In certain embodiments, the modified Chp peptide comprises a polypeptide sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 92.5%, such as at least 95%, such as at least 98%, or such as at least 99% sequence identity with the amino acid sequence of at least one Chp peptide selected from the group consisting of SEQ ID NOs. 1-4, 6- 26, 54-67, 81-91 and 94-102 or an active fragment thereof, wherein the modified Chp peptide inhibits the growth, reduces the population, neutralizes the infectivity of, and/or kills a virus, such as a coronavirus (e.g., SARS-CoV-2), optionally in the presence of human serum and/or pulmonary surfactant.

[0079] In some embodiments, the Chp peptide is selected from (i) at least one of Chpl (SEQ ID NO: 1), Chp2 (SEQ ID NO: 2), CPAR39 (SEQ ID NO: 3), Chp3 (SEQ ID NO: 54); Chp4 (SEQ ID NO: 4), Chp6 (SEQ ID NO: 6), Chp7 (SEQ ID NO: 7), Chp8 (SEQ ID NO: 8), Chp 10 (SEQ ID NO: 10), Chp 11 (SEQ ID NO: 11), Ecpl (SEQ ID NO: 16), Ecp2 (SEQ ID NO: 18), Ecp3 (SEQ ID NO: 55), Ecp4 (SEQ ID NO: 56), Ospl (SEQ ID NO: 19), Unp2 (SEQ ID NO: 20), Gkh3 (SEQ ID NO: 22), Unp5 (SEQ ID NO: 23), Unp6 (SEQ ID NO: 24), Spil (SEQ ID NO: 25), Lvpl (SEQ ID NO: 57), ALCES1 (SEQ ID NO: 59), AVQ206 (SEQ ID NO: 60), CDL907 (SEQ ID NO: 62), AGT915 (SEQ ID NO: 63), SBR77 (SEQ ID NO: 66), and Bdpl (SEQ ID NO: 67), or active fragments thereof, or (ii) a modified Chp peptide having at least 80%, such as at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, or at least 99% sequence identity with at least one of SEQ ID NOs. 1-4, 6-8, 10, 11, 16, 18, 19, 21-25, 54-57, 59, 60, 62, 63, and 66, wherein the modified Chp peptide inhibits the growth, reduces the population, neutralizes the infectivity of, and/or kills a virus, such as a coronavims (e.g., SARS-CoV-2), optionally in the presence of human serum and/or pulmonary surfactant.

[0080] In some embodiments, the Chp peptide is selected from (i) at least one of Chp2-Ml (SEQ ID NO: 81), Chp2-Cys (SEQ ID NO: 82), Chp2-NC (SEQ ID NO: 83), Chp4::Chp2 (SEQ ID NO: 84), Chp2-CAV (SEQ ID NO: 85), Ecpl-CAV (SEQ ID NO: 86), Ecpl-Ml (SEQ ID NO: 87), Chp6-Ml (SEQ ID NO: 88), ChplO-Ml (SEQ ID NO: 89), Chp4-Ml (SEQ ID NO: 91), Chp7- M1 (SEQ ID NO: 95), Ospl-Ml (SEQ ID NO: 96), Unp2-Ml (SEQ ID NO: 97), Unp3-Ml (SEQ ID NO: 98), Ecp3-Ml (SEQ ID NO: 100), and Agtl-Ml (SEQ ID NO: 101) or active fragments thereof, or (ii) a modified Chp peptide having at least 80%, such as at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, or at least 99% sequence identity with at least one of SEQ ID NOs. 81-89, 91, 95-98, 100, and 101, wherein the modified Chp peptide inhibits the growth, reduces the population, neutralizes the infectivity of, and/or kills a virus, such as a coronavims (e.g., SARS-CoV-2), optionally in the presence of human serum and/or pulmonary surfactant. [0081] In some embodiments, the Chp peptide of the present disclosure is a derivative of one of the reference Chp peptides that has been chemically modified. A chemical modification includes but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties. Chemical modifications can occur anywhere in a Chp peptide, including the amino acid side chains, as well as the amino or carboxyl termini. For example, in certain embodiments, the Chp peptide comprises an N-terminal acetylation modification. In certain embodiments, the Chp peptide or active fragment thereof comprises a C-terminal amidation modification. Such modifications can be present at more than one site in a Chp peptide.

[0082] Furthermore, one or more side groups, or terminal groups of a Chp peptide or active fragment thereof may be protected by protective groups known to the person ordinarily- skilled in the art.

[0083] In some embodiments, the Chp peptides or active fragments thereof are conjugated to a duration enhancing moiety. In some embodiments, the duration enhancing moiety is polyethylene glycol. Polyethylene glycol (“PEG”) has been used to obtain therapeutic polypeptides of enhanced duration (Zalipsky, S., Bioconjugate Chemistry, 6:150-165 (1995); Mehvar, R., J. Pharm. Pharmaceut. Sci., 3: 125- 136 (2000), which is herein incorporated by reference in its entirety). The PEG backbone, (CH2CH2-0-)n, wherein n is a number of repeating monomers, is flexible and amphiphilic. When attached to another chemical entity, such as a Chp peptide or active fragment thereof, PEG polymer chains can protect such polypeptides from immune response and other clearance mechanisms. As a result, pegylation can lead to improved efficacy and safety by optimizing pharmacokinetics, increasing bioavailability, and decreasing immunogenicity and dosing amount and/or frequency.

[0084] In certain embodiments, the Chp peptide is a modified variant wherein the positive amino acids (arginine, lysine, and histidine), which naturally appear in their L-isoform, have been replaced by the same amino acid in the D-isoform. It has been shown with a different antimicrobial protein derived from sapesin B that variants containing D-isoform amino acids may exhibit higher antimicrobial activity. See, e.g., Manabe et ak, Scientific Reports (2017); DOI:10.1038/srep43384. In certain embodiments, the Chp peptide is a modified variant wherein an amino acid residue or residues have been added to the C-terminus, the N-terminus, or both the C-terminus and the N- terminus. For example, in certain embodiments, a cysteine may be added to the C-terminus and/or the N-terminus. In certain embodiments, residues that are known to confer stability to alpha-helices and/or to promote activity in the presence of salt may be added to the C-terminus and/or the N- terminus. See, e.g., Park et ak, Helix stability confers salt resistance upon helical antimicrobial peptides, J. Biol. Chem. (2004); 279(14):13896-901. In yet further embodiments, the Chp peptide is a modified variant that is a charge array variant, wherein the amino acids have been reordered based on their charges to maintain amphipathic helical structures. In still further embodiments, the amino acid residues may be scrambled to create the modified variant which may, in certain embodiments, act as a control peptide.

[0085] In some embodiments, the Chp peptides disclosed herein or active fragments thereof exhibit antiviral activity in the presence and/or absence of human serum. For example, a MIC value (i.e., the minimum concentration of peptide sufficient to suppress at least 80% of the viral growth compared to control) may be determined for a Chp peptide or active fragment thereof and compared to, e.g., a compound inactive in human serum, e.g., T4 phage lysozyme or artilysin GN126. T4 phage lysozyme is commercially available, e.g. from Sigma- Aldrich, Inc. GN126 corresponds to Art-175, which is described in the literature and is obtained by fusing AMP SMAP- 29 to GN lysin KZ144. See Briers et al. 2014, Antimicrob, Agents Chemother. 58:3774-3784, which is herein incorporated by reference in its entirety.

[0086] In some embodiments, the Chp peptides disclosed herein or active fragments thereof exhibit antiviral activity in the presence and/or absence of pulmonary surfactant. As with for assessing the activity in human serum, a MIC value may be determined for a Chp peptide or active fragment thereof in pulmonary surfactant or a suitable substitute (e.g., Survanta®) and optionally compared to a compound exhibiting reduced activity in pulmonary surfactant and/or Survanta®, such as daptomycin.

[0087] In some embodiments, the Chp peptides disclosed herein or active fragments thereof show low toxicity against erythrocytes. Any methodology known in the art may be used to assess the potential for hemolytic activity of the present Chp peptides or active fragments thereof.

[0088] In some embodiments, the Chp peptides disclosed herein are active as antivirals. Antiviral activity may be evaluated by any means known in the art, including, for example, by viral neutralization assays.

Polynucleotides

Chp peptides and active fragments thereof

[0089] In one aspect, the present disclosure is directed to an isolated polynucleotide comprising a nucleic acid molecule encoding a Chp peptide or active fragments thereof having lytic activity. As used herein “lytic activity” encompasses the ability of a Chp peptide to kill a virus, reduce the population of a vims, neutralize viral infectivity, or inhibit viral growth e.g., by penetrating the viral envelope in the presence or absence of human serum.

[0090] In certain embodiments, the nucleic acid molecule encodes a Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID

NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21;

SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID

NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61; SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65; SEQ ID NO: 66, and SEQ ID NO: 67 or active fragments thereof.

[0091] In certain embodiments, the nucleic acid molecule encodes a Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 81; SEQ ID NO: 82; SEQ ID

NO: 83; SEQ ID NO: 84; SEQ ID NO: 85; SEQ ID NO: 86; SEQ ID NO: 87; SEQ ID NO: 88;

SEQ ID NO: 89; SEQ ID NO: 90; SEQ ID NO: 91; SEQ ID NO: 92; SEQ ID NO: 93; SEQ ID

NO: 94; SEQ ID NO: 95; SEQ ID NO: 96; SEQ ID NO: 97; SEQ ID NO: 98; SEQ ID NO: 99;

SEQ ID NO: 100; SEQ ID NO: 101; and SEQ ID NO: 102 or active fragments thereof.

[0092] In some embodiments, the isolated polynucleotides of the present disclosure comprise a nucleic acid molecule that encodes a modified Chp peptide, e.g., a Chp peptide containing one or more insertions, deletions and/or amino acid substitutions in comparison to a reference Chp peptide. Such reference Chp peptides include any one of SEQ ID NOs. 1-4, 6-26, 54-67, and 81- 102. In certain embodiments, the modified Chp peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to a reference Chp polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NOs. 1-4, 6-26, 54- 67, and 81-102.

[0093] In some embodiments, the nucleic acid molecules of the present disclosure encode an active fragment of the Chp peptides or modified Chp peptides disclosed herein. The term “active fragment” refers to a portion of a full-length Chp peptide, which retains one or more biological activities of the reference peptide. Thus, an active fragment of a Chp peptide or modified Chp peptide, as used herein, inhibits the growth, or reduces the population, neutralizes the infectivity of, and/or kills a virus, such as a coronavims (e.g., SARS-CoV-2), in the absence or presence of, or in both the absence and presence of, human serum and/or pulmonary surfactant. Typically, the active fragments retain an a-helix domain. In certain embodiments, the active fragment is a cationic peptide that retains an a-helix domain.

Vectors and Host Cells

[0094] In another aspect, the present disclosure is directed to a vector comprising an isolated polynucleotide comprising a nucleic acid molecule encoding any of the Chp peptides or active fragments thereof disclosed herein or a complementary sequence of the present isolated polynucleotides. In some embodiments, the vector is a plasmid or cosmid. In other embodiments, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral vector. In some embodiments, the vector can autonomously replicate in a host cell into which it is introduced. In some embodiments, the vector can be integrated into the genome of a host cell upon introduction into the host cell and thereby be replicated along with the host genome.

[0095] In some embodiments, particular vectors, referred to herein as “recombinant expression vectors” or “expression vectors”, can direct the expression of genes to which they are operatively linked. A polynucleotide sequence is “operatively linked” when it is placed into a functional relationship with another nucleotide sequence. For example, a promoter or regulatory DNA sequence is said to be “operatively linked” to a DNA sequence that codes for an RNA and/or a protein if the two sequences are operatively linked, or situated such that the promoter or regulatory DNA sequence affects the expression level of the coding or structural DNA sequence. Operatively linked DNA sequences are typically, but not necessarily, contiguous.

[0096] In some embodiments, the present disclosure is directed to a vector comprising a nucleic acid molecule that encodes a Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID

NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23;

SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID

NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 60; SEQ ID NO: 61;

SEQ ID NO: 62; SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65; SEQ ID NO: 66, and SEQ ID NO: 67, or active fragments thereof.

[0097] In some embodiments, the present disclosure is directed to a vector comprising a nucleic acid molecule that encodes a Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 81; SEQ ID NO: 82; SEQ ID NO: 83; SEQ ID NO: 84; SEQ ID NO: 85; SEQ ID NO: 86; SEQ ID NO: 87; SEQ ID NO: 88; SEQ ID NO: 89; SEQ ID NO: 90; SEQ ID NO: 91; SEQ ID NO: 92; SEQ ID NO: 93; SEQ ID NO: 94; SEQ ID NO: 95; SEQ ID NO: 96; SEQ ID NO: 97; SEQ ID NO: 98; SEQ ID NO: 99; SEQ ID NO: 100; SEQ ID NO: 101; and SEQ ID NO: 102 or active fragments thereof.

[0098] Generally, any system or vector suitable to maintain, propagate or express a polypeptide in a host may be used for expression of the Chp peptides disclosed herein or active fragments thereof. The appropriate DN A/poly nucleotide sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory (2001). Additionally, tags can also be added to the Chp peptides or active fragments thereof to provide convenient methods of isolation, e.g., c-myc, biotin, poly-His, etc. Kits for such expression systems are commercially available.

[0099] A wide variety of host/expression vector combinations may be employed in expressing the polynucleotide sequences encoding the Chp peptides disclosed herein or active fragments thereof. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. Examples of suitable vectors are provided, e.g., in Sambrook et al, eds., Molecular Cloning: A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory (2001). Such vectors include, among others, chromosomal, episomal and virus derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.

[00100] Furthermore, the vectors may provide for the constitutive or inducible expression of the Chp peptides or active fragments thereof of the present disclosure. Suitable vectors include but are not limited to derivatives of SV40 and known bacterial plasmids, e.g., E. coll plasmids colEl, pCRl, pBR322, pMB9 and their derivatives, plasmids such as RP4, pBAD24 and pBAD- TOPO; phage DNAS, e.g., the numerous derivatives of phage A, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 D plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like. Many of the vectors mentioned above are commercially available from vendors such as New England Biolabs Inc., Addgene, Takara Bio Inc., ThermoFisher Scientific Inc., etc. [00101] Additionally, vectors may comprise various regulatory elements (including promoter, ribosome binding site, terminator, enhancer, various cis-elements for controlling the expression level) wherein the vector is constructed in accordance with the host cell. Any of a wide variety of expression control sequences (sequences that control the expression of a polynucleotide sequence operatively linked to it) may be used in these vectors to express the polynucleotide sequences encoding the Chp peptides or active fragments thereof of the present disclosure. Useful control sequences include, but are not limited to: the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3 -phosphogly cerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast-mating factors, E. coli promoter for expression in bacteria, and other promoter sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. Typically, the polynucleotide sequences encoding the Chp peptides or active fragments thereof are operatively linked to a heterologous promoter or regulatory element.

[00102] In another aspect, the present disclosure is directed to a host cell comprising any of the vectors disclosed herein including the expression vectors comprising the polynucleotide sequences encoding the Chp peptides or active fragments thereof of the present disclosure. A wide variety of host cells are useful in expressing the present polypeptides. Non-limiting examples of host cells suitable for expression of the present polypeptides include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture. While the expression host may be any known expression host cell, in a typical embodiment the expression host is one of the strains of E. coli. These include, but are not limited to commercially available E. coli strains such as Top 10 (ThermoFisher Scientific, Inc.), DH5a (Thermo Fisher Scientific, Inc.), XLI-Blue (Agilent Technologies, Inc.), SCSllO (Agilent Technologies, Inc.), JM109 (Promega, Inc.), LMG194 (ATCC), and BL21 (Thermo Fisher Scientific, Inc.).

[00103] There are several advantages of using E. coli as a host system including: fast growth kinetics, where under the optimal environmental conditions, its doubling time is about 20 min (Sezonov et ah, J. Bacterial. 189 8746-8749 (2007)), easily achieved high density cultures, easy and fast transformation with exogenous DNA, etc. Details regarding protein expression in E. coli, including plasmid selection as well as strain selection are discussed in detail by Rosano, G. and Ceccarelli, E., Front Microbial., 5: 172 (2014).

[00104] Efficient expression of the present Chp peptides or active fragments thereof depends on a variety of factors such as optimal expression signals (both at the level of transcription and translation), correct protein folding, and cell growth characteristics. Regarding methods for constructing the vector and methods for transducing the constructed recombinant vector into the host cell, conventional methods known in the art can be utilized. While it is understood that not all vectors, expression control sequences, and hosts will function equally well to express the polynucleotide sequences encoding Chp peptides or active fragments thereof of the present disclosure, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this disclosure.

[00105] Chp peptides or active fragments thereof of the present disclosure can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography can also be employed for Chp peptide purification.

[00106] Alternatively, the vector system used for the production of Chp peptides or active fragments of the present disclosure may be a cell-free expression system. Various cell-free expression systems are commercially available, including, but are not limited to those available from Promega, LifeTechnologies, Clonetech, etc.

[00107] As indicated above, there is an array of choices when it comes to protein production and purification. Examples of suitable methods and strategies to be considered in protein production and purification are provided in WO 2017/049233, which is herein incorporated by reference in its entirety and further provided in Structural Genomics Consortium et al., Nat. Methods., 5(2): 135-146 (2008).

Pharmaceutical Compositions

[00108] The compositions of the present disclosure can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, tampon applications emulsions, aerosols, sprays, suspensions, lozenges, troches, candies, injectants, chewing gums, ointments, smears, time-release patches, liquid absorbed wipes, and combinations thereof.

[00109] Administration of the compositions of the present disclosure or pharmaceutically acceptable forms thereof may be topical, i.e., the pharmaceutical composition may be applied directly where its action is desired (for example directly to a wound), or systemic. In turn, systemic administration can be enteral or oral, i.e., the composition may be given via the digestive tract, parenteral, i.e., the composition may be given by other routes than the digestive tract such as by injection or inhalation. Thus, the Chp peptides of the present disclosure and compositions comprising them can be administered to a subject orally, parenterally, by inhalation, topically, rectally, nasally, buccally, via an implanted reservoir, or by any other known method. The Chp peptides of the present disclosure or active fragments thereof can also be administered by means of sustained release dosage forms.

[00110] For oral administration, the Chp peptides of the present disclosure or active fragments thereof can be formulated into solid or liquid preparations, for example tablets, capsules, powders, solutions, suspensions, and dispersions. The composition can be formulated with excipients such as, e.g., lactose, sucrose, com starch, gelatin, potato starch, alginic acid, and/or magnesium stearate.

[00111] For preparing solid compositions such as tablets and pills, a Chp peptide of the present disclosure or active fragments thereof may be mixed with a pharmaceutical excipient to form a solid pre-formulation composition. If desired, tablets may be sugar coated or enteric coated by standard techniques. The tablets or pills may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can include an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[00112] The topical compositions of the present disclosure may further comprise a pharmaceutically or physiologically acceptable carrier, such as a dermatologically or an otically acceptable carrier. Such carriers, in the case of dermatologically acceptable carriers, may be compatible with skin, nails, mucous membranes, tissues, and/or hair, and can include any conventionally-used dermatological carrier meeting these requirements. In the case of otically acceptable carriers, the carrier may be compatible with all parts of the ear. Such carriers can be readily selected by one of ordinary skill in the art. Carriers for topical administration of the compositions of the present disclosure include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene and/or polyoxypropylene compounds, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol, and water. In formulating skin ointments, the active components of the present disclosure may be formulated, for example, in an oleaginous hydrocarbon base, an anhydrous absorption base, a water-in-oil absorption base, an oil-in-water water-removable base, and/or a water-soluble base. In formulating otic compositions, the active components of the present disclosure may be formulated, for example, in an aqueous polymeric suspension including such carriers as dextrans, polyethylene glycols, polyvinylpyrrolidone, polysaccharide gels, gellan gums such as Gelrite®, cellulosic polymers such as hydroxypropyl methylcellulose, and carboxy- containing polymers such as polymers or copolymers of acrylic acid, as well as other polymeric demulcents. The topical compositions according to the present disclosure may be in any form suitable for topical application, including aqueous, aqueous-alcoholic or oily solutions; lotion or serum dispersions; aqueous, anhydrous or oily gels; emulsions obtained by dispersion of a fatty phase in an aqueous phase (O/W or oil-in-water) or, conversely, (W/O or water-in-oil); microemulsions or alternatively microcapsules, microparticles or lipid vesicle dispersions of ionic and/or nonionic type; creams; lotions; gels; foams (which may use a pressurized canister, a suitable applicator, an emulsifier, and an inert propellant); essences; milks; suspensions; and patches. Topical compositions of the present disclosure may also contain adjuvants such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preserving agents, antioxidants, solvents, fragrances, fillers, sunscreens, odor-absorbers, and dyestuffs. In a further aspect, the topical compositions disclosed herein may be administered in conjunction with devices such as transdermal patches, dressings, pads, wraps, matrices, and bandages capable of being adhered to or otherwise associated with the skin or other tissue of a subject, being capable of delivering a therapeutically effective amount of one or more Chp peptide or active fragment thereof as disclosed herein. [00113] In one embodiment, the topical compositions of the present disclosure additionally comprise one or more components used to treat topical bums. Such components may include, but are not limited to, a propylene glycol hydrogel; a combination of a glycol, a cellulose derivative, and a water soluble aluminum salt; an antiseptic; an antibiotic; and a corticosteroid. Humectants such as solid or liquid wax esters; absorption promoters such as hydrophilic clays or starches; viscosity building agents; and skin-protecting agents may also be added. Topical formulations may be in the form of rinses such as mouthwash. See, e.g., W02004/004650.

[00114] The compositions of the present disclosure may also be administered by injection of a therapeutic agent comprising the appropriate amount of a Chp peptide or active fragment thereof and a carrier. For example, the Chp peptide or active fragment thereof can be administered intramuscularly, intrathecally, subdermally, subcutaneously, or intravenously to treat infections by a vims, such as a coronavims (e.g., SARS-CoV-2). The carrier may be comprised of distilled water, a saline solution, albumin, a serum, or any combinations thereof. Additionally, pharmaceutical compositions of parenteral injections can comprise pharmaceutically acceptable aqueous or nonaqueous solutions of Chp peptides as disclosed herein or active fragments thereof in addition to one or more of the following: pH buffered solutions, adjuvants (e.g., preservatives, wetting agents, emulsifying agents, and dispersing agents), liposomal formulations, nanoparticles, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.

[00115] In cases where parenteral injection is the chosen mode of administration, an isotonic formulation may be used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers can include gelatin and albumin. A vasoconstriction agent can be added to the formulation. The pharmaceutical preparations according to this type of application may be provided sterile and pyrogen free.

[00116] The diluent may further comprise one or more other excipient such as ethanol, propylene glycol, an oil, or a pharmaceutically acceptable emulsifier or surfactant.

[00117] In another embodiment, the compositions of the present disclosure are inhalable compositions. The inhalable compositions of the present disclosure can further comprise a pharmaceutically acceptable carrier. In one embodiment, the Chp peptides of the present disclosure or active fragments thereof may be formulated as a dry, inhalable powder. In specific embodiments, an inhalation solution comprising Chp peptides or active fragments thereof may further be formulated with a propellant for aerosol delivery. In certain embodiments, solutions may be nebulized.

[00118] A surfactant can be added to an inhalable pharmaceutical composition of the present disclosure in order to lower the surface and interfacial tension between the medicaments and the propellant. Where the medicaments, propellant and excipient are to form a suspension, a surfactant may or may not be used. Where the medicaments, propellant and excipient are to form a solution, a surfactant may or may not be used, depending, for example, on the solubility of the particular medicament and excipient. The surfactant may be any suitable, non-toxic compound which is non-reactive with the medicament and which reduces the surface tension between the medicament, the excipient and the propellant and/or acts as a valve lubricant.

[00119] Examples of suitable surfactants include, but are not limited to: oleic acid; sorbitan trioleate; cetyl pyridinium chloride; soya lecithin; polyoxyethylene (20) sorbitan monolaurate; polyoxyethylene (10) stearyl ether; polyoxyethylene (2) oleyl ether; polyoxypropylene- polyoxy ethylene ethylene diamine block copolymers; polyoxyethylene (20) sorbitan monostearate; polyoxyethylene(20) sorbitan monooleate; polyoxypropylene-polyoxyethylene block copolymers; castor oil ethoxylate; and combinations thereof.

[00120] Examples of suitable propellants include, but are not limited to: dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, and carbon dioxide. [00121] Examples of suitable excipients for use in inhalable compositions include, but are not limited to: lactose, starch, propylene glycol diesters of medium chain fatty acids; triglyceride esters of medium chain fatty acids, short chains, or long chains, or any combination thereof; perfluorodimethylcyclobutane; perfluorocyclobutane; polyethylene glycol; menthol; lauroglycol; diethylene glycol monoethylether; polyglycolized glycerides of medium chain fatty acids; alcohols; eucalyptus oil; short chain fatty acids; and combinations thereof.

[00122] In some embodiments, the compositions of the present disclosure comprise nasal applications. Nasal applications include applications for direct use, such as nasal sprays, nasal drops, nasal ointments, nasal washes, nasal injections, nasal packings, bronchial sprays and inhalers, as well as applications for indirect use, such as throat lozenges and mouthwashes or gargles, or through the use of ointments applied to the nasal nares or the face, and any combination of these and similar methods of application. [00123] In another embodiment, the pharmaceutical compositions of the present disclosure comprise a complementary agent, including one or more antimicrobial agents and/or one or more conventional antibiotics and/or one or more conventional antiviral agents. In order to accelerate the treatment of the infection, or augment the antimicrobial effect, the therapeutic agent containing a Chp peptide of the present disclosure or active fragment thereof may further include at least one complementary agent that can also potentiate the antimicrobial activity of the peptide. The complementary agent may be one or more antibiotics used to treat Gram-negative bacteria or one or more antibiotics used to treat acid-fast bacteria. In one embodiment, the complementary agent is an antibiotic or antimicrobial agent used for the treatment of infections caused by P. aeruginosa. In one embodiment, the complementary agent is an antibiotic or antimicrobial agent used for the treatment of infections caused by M. tuberculosis, and in one embodiment, the complementary agent is an antibiotic or antimicrobial agent used for the treatment of infections caused by non tuberculosis mycobacteria. The complementary agent may be one or more antiviral agents typically used to treat a viral infection, such as, for example, hydroxychloroquine, oseltamivir (Tamiflu®), zanamivir, peramivir, remdesivir, and baloxavir.

[00124] The compositions of the present disclosure may be presented in unit dosage form and may be prepared by any methods well known in the art. The amount of active ingredients that can be combined with a carrier material to produce a single dosage form will vary depending, for example, upon the host being treated, the duration of exposure of the recipient to the infectious bacteria, the size and weight of the subject, and the particular mode of administration. The amount of active ingredients that can be combined with a carrier material to produce a single dosage form may, for example, be that amount of each compound which produces a therapeutic effect. In certain embodiments, out of one hundred percent, the total amount may range from about 1 percent to about ninety-nine percent of active ingredients, such as from about 5 percent to about 70 percent, or from about 10 percent to about 30 percent.

Dosage and Administration

[00125] Dosages administered may depend on a number of factors such as the activity of infection being treated; the age, health and general physical condition of the subject to be treated; the activity of a particular Chp peptide or active fragment thereof; the nature and activity of the antibiotic if any with which a Chp peptide or active fragment thereof according to the present disclosure is being paired; and the combined effect of such pairing. In certain embodiments, effective amounts of the Chp peptide or active fragment thereof to be administered may fall within the range of about 1-50 mg/kg (or 1 to 50 mcg/ml). In certain embodiments, effective amounts of the Chp peptide or active fragment thereof to be administered may fall within the range of about 1-50 mg/mL, such as within the range of about 1-10 mg/mL, about 1 mg/mL, or about 10 mg/mL. In certain embodiments, the Chp peptide or active fragment thereof may be administered 1-4 times daily for a period ranging from 1 to 14 days. The antibiotic if one is also used may be administered at standard dosing regimens or in lower amounts in view of any synergism. All such dosages and regimens, however, (whether of the Chp peptide or active fragment thereof or any antibiotic administered in conjunction therewith) are subject to optimization.

[00126] In some embodiments, time exposure to the Chp peptides disclosed herein or active fragments thereof may influence the desired concentration of active peptide units per ml. Carriers that are classified as “long” or “slow” release carriers (such as, for example, certain nasal sprays or lozenges) may possess or provide a lower concentration of peptide units per ml but over a longer period of time, whereas a “short” or “fast” release carrier (such as, for example, a gargle) may possess or provide a high concentration peptide units (meg) per ml but over a shorter period of time. There are circumstances where it may be desirable to have a higher unit/ml dosage or a lower unit/ml dosage.

[00127] For the Chp peptides or active fragments thereof of the present disclosure, the therapeutically effective dose may be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model can also be used to achieve a desirable concentration range and route of administration. Obtained information can then be used to determine the effective doses, as well as routes of administration, in humans. Dosage and administration can be further adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Additional factors that may be taken into account include the severity of the disease state; age, weight and gender of the patient; diet; desired duration of treatment; method of administration; time and frequency of administration; drug combinations; reaction sensitivities; tolerance/response to therapy; and the judgment of a treating physician.

[00128] A treatment regimen can entail administration daily (e.g., once, twice, thrice, etc. daily), every other day (e.g., once, twice, thrice, etc. every other day), semi- weekly, weekly, once every two weeks, once a month, etc. In one embodiment, treatment can be given as a continuous infusion. Unit doses can be administered on multiple occasions. Intervals can also be irregular as indicated by monitoring clinical symptoms. Alternatively, the unit dose can be administered as a sustained release formulation, in which case less frequent administration may be used. Dosage and frequency may vary depending on the patient. It will be understood by one of skill in the art that such guidelines will be adjusted for localized administration, e.g., intranasal, inhalation, rectal, etc., or for systemic administration, e.g., oral, rectal (e.g., via enema), intramuscular (i.m.), intraperitoneal (i.p.), intravenous (i.v.), subcutaneous (s.c.), transurethral, and the like.

Methods

[00129] The Chp peptides and active fragments thereof of the present disclosure can be used in vivo, for example, to treat viral infections, including viral infections caused by a coronavirus, such as COVID-19, as well as in vitro, for example to reduce the level of viral contamination on, for example, a surface, e.g., of a medical device.

[00130] In one aspect, the present disclosure is directed to a method of treating a viral infection caused by a virus, such as a coronavirus (e.g., SARS-CoV-2), comprising administering to a subject diagnosed with, at risk for, or exhibiting symptoms of a viral infection, a pharmaceutical composition as described herein described.

[00131] In certain embodiments, the subject has an underlying medical condition, such as hypertension, diabetes, lung disease, auto-immune disease, or non-viral infection. In certain embodiments, non-viral infection is meant to include respiratory tract infections (RTIs), such as respiratory tract infections in patients having cystic fibrosis (CF), lower respiratory tract infections, such as acute exacerbation of chronic bronchitis (ACEB), acute sinusitis, community-acquired pneumonia (CAP), hospital-acquired pneumonia (HAP) and nosocomial respiratory tract infections; sexually transmitted diseases, such as gonococcal cervicitis and gonococcal urethritis; urinary tract infections; acute otitis media; sepsis including neonatal septisemia and catheter- related sepsis; osteomyelitis; tuberculosis, and non-tuberculosis mycobacteria infections. In some embodiments, the Chp peptides and active fragments thereof of the present disclosure are used to treat a subject at risk for acquiring an infection due to a virus, such as a coronavirus (e.g., COVID- 19). Subjects at risk for acquiring a viral infection include, for example, the elderly or immunocompromised patients. [00132] In another aspect, the present disclosure is directed to a method of preventing or treating a viral infection comprising co-administering to a subject diagnosed with, at risk for, or exhibiting symptoms of a viral infection, a combination of a first effective amount of the composition containing an effective amount of a Chp peptide or active fragment thereof as described herein, and a second effective amount of an antiviral suitable for the treatment of a virus. In certain aspects, the present disclosure is directed to a method of preventing or treating a viral infection comprising co-administering to a subject diagnosed with, at risk for, or exhibiting symptoms of a viral infection, a combination of a first effective amount of the composition containing an effective amount of a Chp peptide or active fragment thereof as described herein, and a second effective amount of an antiviral suitable for the treatment of a vims.

[00133] The Chp peptides and active fragments thereof of the present disclosure can be co administered with standard care antivirals or with antivirals of last resort, individually or in various combinations as within the skill of the art. In certain embodiments, the antiviral is chosen from hydroxychloroquine, oseltamivir (Tamiflu®), zanamivir, peramivir, remdesivir, and baloxavir. [00134] In yet another aspect, the present disclosure is directed to a method of inhibiting the growth, or reducing the population, neutralizing the infectivity of, or killing of a vims, such as a coronavims (e.g., SARS-CoV-2), the method comprising contacting the vims with a composition containing an effective amount of a Chp peptide or active fragment thereof as described herein, wherein the Chp peptide or active fragment thereof inhibits the growth, or reduces the population, neutralizes the infectivity of, or kills at least one vims.

[00135] In some embodiments, inhibiting the growth, or reducing the population, neutralizing the infectivity of, or killing a vims, such as a coronavims (e.g., SARS-CoV-2) comprises contacting the vims with the Chp peptides or active fragments as described herein, wherein the vims is present on a surface of e.g., medical devices, floors, stairs, walls and countertops in hospitals and other health related or public use buildings and surfaces of equipment in operating rooms, emergency rooms, hospital rooms, clinics, and bathrooms and the like. [00136] Examples of medical devices that can be protected using the Chp peptides or active fragments thereof described herein include but are not limited to tubing and other surface medical devices, such as urinary catheters, mucous extraction catheters, suction catheters, umbilical cannulae, contact lenses, intrauterine devices, intravaginal and intraintestinal devices, endotracheal tubes, bronchoscopes, dental prostheses and orthodontic devices, surgical instruments, dental instruments, tubings, dental water lines, fabrics, paper, indicator strips (e.g., paper indicator strips or plastic indicator strips), adhesives (e.g., hydrogel adhesives, hot-melt adhesives, or solvent-based adhesives), bandages, tissue dressings or healing devices and occlusive patches, and any other surface devices used in the medical field. The devices may include electrodes, external prostheses, fixation tapes, compression bandages, and monitors of various types.

Examples

[00137] Bioinformatic studies. All proteins were identified in annotated GenBank database entries for all Microviridae and Leviviridae genomes. The accession number for each Chp group peptide is indicated in Tables 1 and 2 below. Blastp analyses were performed using the UniProt server, available at uniprot.org/blast/. Protein secondary structure predictions were performed using JPRED4, available at www.compbio.dundee.ac.uk/jpred/index, and I-Tasser, available at www.zhanglab.ccmb.med.umich.edu/I-TASSER/. Phylogenetic analyses were performed using ClustalW Multiple Sequence Alignment tools, available at www.genome.jp/tools-bin/clustalw. Predicted molecular weights and isoelectric points were determined using the ExPASy Resource Portal, available at web.expasy.org/compute_pi/. Example 1: Identification of Chp peptides

[00138] Having knowledge of certain poorly described bacteriophage

( Chlamydiamicroviridae ) that specifically infect and kill the Gram-negative bacteria Chlamydia, published genomes of these organisms were studied, initially looking to identify novel lysins, although no lysin-like sequences nor any sequences similar to previously described amurins were observed. Chlamydia do not utilize peptidoglycans (a known target of lysins) in their structures as abundantly as other bacteria, but rather Chlamydia generally only use peptidoglycans during division. Therefore, the question arose as to what the target of Chlamydia phage was. It was postulated that the mechanism by which Chlamydia phage invade their target may be different from the ones previously known, and their target may be different and focused on lipopolysaccharide (LPS), a main constituent of the outer membrane of Gram-negative bacteria and an obstacle to penetration by lysins of the outer membrane.

[00139] The published genomes of Chlamydiamicrovirus were studied with a view to identifying syntenic loci, i.e., similar genes in the same position in a genome of a group of genetically related phages, which suggested similar function. Small highly cationic peptides were identified that had a very similar molecular charge profile to previously identified antimicrobial peptides (AMPs). While the Chlamydia phage sequences had no protein sequence similarity to AMPs, lysins, or to known amurin proteins (such as Protein A2, protein E and others), the overall positive charge was a prominent feature. Using bioinformatic techniques as described above (JPRED and iTASSAR), structural predictions were conducted that revealed the presence of alpha helices, a hallmark feature of many AMPs. The alpha helices, the overall charge, the conservation among Chlamydia, and the related Gram-negative bacteria phage genomes all suggested that these proteins may represent a family of previously uncharacterized phage lytic polypeptides and that they may define a previously undescribed phage lytic mechanism. The fact that they were predicted to be small in size and soluble (based on their charge profile) also meant that, once synthesized, they would likely be readily amenable to testing by simply adding them to susceptible bacteria cultures.

[00140] Based on the foregoing, 12 conserved sequences within syntenic loci were extracted from the Microviridae genomes in the GenBank database and specifically from the Chlamydiamicroviruses genomes (as well as some other viruses described below). The 12 conserved sequences were annotated only as hypothetical, uncharacterized or non-stmctural proteins and encoded small (putatively) cationic proteins predicted to adopt alpha-helical structures. These 12 sequences are set forth in Table 1. One of the peptides in Table 1, Chp5 was synthesized to have a molecular charge different from Chp4 by replacing arginines and lysines, which are positively charged, with negatively charges amino acid residues. Chp5 was predicted to be inactive. While these peptides exhibit no sequence similarity to other lytic or antimicrobial proteins, they are predicted to adopt alpha-helical structures (for examples, see Figure 1) similar to subsets of the large family of antibacterial agents AMPs. It was postulated that Chp peptides perform the host lysis function for the phages from which they are derived.

[00141] Based on the foregoing considerations, further study of genomes of other phages

(related to the Chlamydiamicroviruses , in the same family, Microviridae) that infect Gram negative bacteria, as well as other uncharacterized sources that presented with the same synteny and charge profile, yielded 29 additional peptides listed in Table 2. Together, all 41 peptides (excluding Chp5) form a related family of novel phage lytic agents. They are all from Microviridae sources, with the exception of Myol (SEQ ID NO: 102), which is from Microbacterium.

[00142] Furthermore, certain of these peptides were modified to synthesize novel variants.

Notably, for Chp2, the L-form of each of the positively charged amino acids (arginine and lysine) was substituted for the D-form of that amino acid, to see if the D-form may enhance antibacterial activity.

[00143] This modification resulted in Chp2-Ml (SEQ ID. NO: 81). Similar D-form variants were created from the native Chp peptides or modified variants of Chp peptides to arrive at Ecpl- M1 (SEQ ID NO: 87), Chp6-Ml (SEQ ID NO: 88), ChplO-Ml (SEQ ID NO: 89), Mse-Ml (SEQ ID NO: 90), Chp4-Ml (SEQ ID NO: 91), Chp2-SCR-Ml (SEQ ID NO: 93), Chp7-Ml (SEQ ID NO: 95), Osp-Ml (SEQ ID NO: 96), Unp2-Ml (SEQ ID NO: 97), Unp3-Ml (SEQ ID NO: 98), Spi2-Ml (SEQ ID NO: 99), Ecp3-Ml (SEQ ID NO: 100), and Agtl-Ml (SEQ ID NO: 101). [00144] Likewise for Chp2, a cysteine residue was added to the C-terminus to arrive at Chp2-Cys (SEQ ID NO: 82), and additional residues previously shown to confer alpha-helix stability and promote activity in the presence of salt were added to both the C-terminus and the N- terminus to arrive at Chp2-NC (SEQ ID NO: 83). Park et ah, Helix stability confers salt resistance upon helical antimicrobial peptides , J. Biol. Chem. (2004); 279(14): 13896-901.

[00145] Chp4::Chp2 (SEQ ID NO: 84) is a fusion peptide comprising alpha helices from Chp4 (SEQ ID NO: 4) and Chp2 (SEQ ID NO: 2). Chp2-CAV (SEQ ID NO: 85) and Ecpl-CAV (SEQ ID NO: 86) are charge array variants, wherein various amino acid charges were reordered to maintain amphipathic helices. Chp2-SCR1 (SEQ ID NO: 92) is a modified variant of Chp2 (SEQ ID NO: 2), wherein the amino acid residues have been scrambled to create a control peptide. [00146] Thus, a complete list of all Chp family members (including certain features of each peptide) is provided in Table 1, Table 2, and Table C. Included in this group are peptides Chpl- 4 and 6-12 and CPAR39, which are derived from 11 different Chlamydiamicroviruses and are described in Table 1; peptides Chp2 and Chp3 are two identical peptides from two different phages. As stated above, Chp5 is a modified derivative of Chp4 generated by the replacement of all positively charged amino acids, including arginines and lysines, with negatively charged amino acids, including glutamine and glutamic acid. The additional members of the Chp family were identified by homology with the Chlamydiamicrovirus proteins and are described in Table 2 (“Additional Chp family members”). The additional Chp family members are not from Chlamydiamicrovirus sources but from putative Microviridae and Microbacterium phage sources. Table C provides several modified variants of Chp peptides, including D-form variants and charge array variants as discussed above. In Table C, amino acids that are italicized and in bold indicate amino acid residues that have been changed from the L-form to the D-form.

[00147] Table 1 - Chlamydia phage (Chp)-derived lytic agents

[00148] Table 2 - Additional Chp family members

[00149] Table C - Modified Chp peptide family members

[00150] Additional information regarding the protein sequence homologies of several Chp family members is provided in Table 3. Chpl, Bdpl, Lvpl, and Lvp2 are the only Chp family members for which a predicted activity is indicated in the GenBank annotation. Chpl (GenBank sequence NP_044319.1) is annotated as a DNA binding protein, although no data are provided to support this, and the annotation is inconsistent with a putative role in host lysis. Overall, the Chp proteins are 39-100% identical to each other and are not homologous to other peptides in the protein sequence database. Rooted and unrooted phylogenetic trees showing certain members of the Chp family are indicated in Figures 2A and 2B, respectively.

[00151] Table 3 - Annotations and similarities of Chp family proteins

Example 2: Synthesis of the Chp peptides [00152] All Chp peptides were synthesized by GenScript, NJ, USA with capping [N- terminal acetylation (Ac) and C-terminal amidation (Nth)] on a fee-for- service basis. GenScript assessed the purity of each peptide by high performance liquid chromatography (HPLC) and mass spectrometry (MS). GenScript also performed a solubility test for all peptides and determined the net peptide content (NPC%) using a Vario MICRO Organic Elemental Analyzer. With the exception of Chp5, Lvpl, and Lvp2, all peptides were soluble in water and were suspended at a concentration of either 1 mg/mL, 5 mg/mL or 10 mg/mL. Chp5 and Lvpl were suspended in DMSO at a concentration of 10 mg/mL; Lvp2 was suspended in DMSO at a concentration of 2 mg/mL. The solubility of Ecpl-CAV was not determined. Control peptides RI18, RP-1, WLBU2, BAC3, GN-2 amp, GN-3 amp, GN-4 amp, GN-6 amp, and Bac8c were also synthesized at GenScript as above. All additional peptides were commercial products purchased from either GenScript or Anaspec.

Example 3 - Cytopathic Effect (CPE) Assay

[00153] The antiviral activity of Chp peptides was investigated using a 3-day CPE assay. Human coronavims OC43 (HCoV-OC43), an ssRNA virus of the same genus (i.e., Betacoronavims) as SARS-CoV-19, was obtained from ATCC (VR-1558), and African Green Monkey kidney cells (Vero cell line ATC CRL-1586) were used. Chp2-Ml (also known as AM2) was evaluated first.

[00154] The CPE assay protocol was developed from Baer et al., Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems, J. VlS . EXP. 2014; 93:e52065. Namely, Vero cells were plated at 1.2e5 cells per well of a 24-well polystyrene dish and incubated overnight at 37 °C (5% CO2) in MEM + 10% Fetal Bovine Serum to 90% confluency. Prior to infection, serial 2-fold dilutions (from 64 pg/mL to 0.0625 pg/mL) of each of the Chp peptides were co-incubated (in duplicate) with a single concentration of vims (PFUs = 6e4) for 1 hour at room temperature.

[00155] After 1 hour, each vims/peptide combination (200 pL) was added (in duplicate) to the Vero cells, which were pre-washed twice with PBS. As controls, peptides alone (at each concentration), virus alone, and buffer alone (with no vims or peptides) were used. Additionally, Chp5 was used as a control amurin, as it is known to have no antimicrobial activity. [00156] Cells were infected (MOI = 0.05) for 1 hour at 33 °C before removal of the extracellular virus/peptide, over-layed with 0.3% top agarose/MEM + 2% Fetal Bovine Serum and incubated for 72 hours at 33 °C with 5% CO2.

[00157] Monolayers were then visualized by microscopy and islands of CPE were enumerated. Each area of CPE corresponds to one plaque forming unit (PFU). The median Tissue Culture Infectious Dose (TCID50) was determined, signifying the concentration at which 50% of the cells were infected, as compared to an untreated control.

[00158] 19 CPE lesions were observed per dish for vims preincubated with Chp2-Ml at 64 mg/mL, as compared to >5000 CPE lesions per dish for a control with no peptide (vims only). The TCID50 was 1 mg/mL. No inhibition was observed for Chp5 at any concentration, including 0 mg/mL, 2 mg/mL, 8 mg/mL, and 64 mg/mL. Chp2-Ml alone and Chp5 alone had no visible impact on viability and did not include CPEs.

[00159] After the CPE induced with each treatment group (in duplicate) was visually quantified, the percent inhibition as compared to an untreated (no peptide) control was calculated. Pre-incubation of HCoV-OC43 for 1 hour with 1 mg/mL Chp2-Ml resulted in a greater than 50% reduction in CPE. The TCID50 for the negative control Chp5, however, was greater than 64 mg/mL. The results are shown below in Table 4.

[00160] Table 4 - Percent Inhibition of HCoV-OC43 with Chp2-Ml and Chp5

[00161] Further testing with Chp2 (also known as AMI) and ChplO-Ml (also known as AM3) revealed that, at a Multiplicity of Infection (MOI) of 0.005, the TCID50 for Chp2, Chp2- Ml, and ChplO-Ml was 0.5 mg/mL, while the TCID50 for control peptides WLBU2 and LL-37 was 16 mg/mL and 8 mg/mL, respectively.

[00162] Based on these results for Chp2, Chp2M-l, and ChplO-Ml, the inhibition study was expanded to include 10 additional Chp peptides: Spi2-M2, Alcesl, Chp8, Ecpl-Ml, Ecp3- Ml, Hhl, Gsk3, Ospl, Unp2-Ml, and Unp3. As discussed above, a percent inhibition in the CPE assay format (MOI = 0.005) was compared to an untreated (no peptide) control and the TCID50 values were determined. The results are shown below in Table 5.

[00163] Table 5 - Chp Peptide Activity

[00164] As shown in Table 5, Chp2, Chp2-Ml, and ChplO-Ml exhibited the highest level of activity. Several known lysins were also evaluated, including pp296, GN316, GN370, and GN428. The TCID50 values for each of the 4 lysins was calculated to be >64 mg/mL, evidencing that the tested lysins did not exhibit any anti-viral activity. Accordingly, certain Chp peptides, including Chp2, Chp2-Ml, and ChplO, are distinguishable from other direct lytic agents based on their in vitro anti- viral activity.

Prophetic Example:

[00165] Amurin peptides and modified variants thereof (such as the Chp peptides disclosed herein as SEQ ID NOs: 1-4, 6-26, 54-67, 81-91, and 95-102) are evaluated for the ability to inactivate SARS-CoV-2 in a viral neutralization assay. Other assays may be used to quantify CPE or cytotoxicity, including, for example, MTT and luciferase assays. If neutralization is observed at appropriately low amurin peptide concentrations, additional characterizations may be considered to further evaluate in vitro activity and in vivo efficacy to support further development.

Claims

What is claimed is:

1. A pharmaceutical composition comprising: an effective amount of (i) an isolated Chp peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-4, 6-26, 54-67, 81-91 and 94-102 or active fragment thereof, or (ii) a modified Chp peptide having 80% sequence identity with the amino acid sequence of at least one of SEQ ID NOs. 1-4, 6-26, 54-67, 81-91 and 94-102, wherein the modified Chp peptide inhibits the growth, reduces the population, or kills at least one virus; and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, wherein the Chp peptide has an amino acid sequence is SEQ ID NO: 2 or active fragments thereof and contains at least one non-natural modification.

3. The pharmaceutical composition of claim 2, wherein the non-natural modification is selected from the group consisting of substitution modifications, N-terminal acetylation modifications, and C-terminal amidation modifications.

4. The pharmaceutical composition according to claim 1, wherein the amino acid sequence is selected from the group consisting of SEQ ID NO: 81, SEQ ID NO: 89, and active fragments thereof.

5. The pharmaceutical composition according to any of the preceding claims, which is a solution, a suspension, an emulsion, an inhalable powder, an aerosol, or a spray.

6. The pharmaceutical composition according to any of the preceding claims, further comprising one or more antivirals suitable for the treatment of a viral infection.

7. A method of inhibiting the growth, reducing the population, neutralizing the infectivity of, or killing of at least one virus, such as a coronavirus, the method comprising contacting the virus with a composition comprising an effective amount of (i) a Chp peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-4, 6-26, 54- 67, 81-91 and 94-102 or active fragments thereof, or (ii) a modified Chp peptide having 80% sequence identity with the amino acid sequence of at least one of SEQ ID NOs. 1-4, 6-26, 54-67, 81-91 and 94-102, said Chp peptide or modified Chp peptide having lytic activity for a period of time sufficient to inhibit said growth, reduce said population, neutralize said infectivity of, or kill said at least one virus.

8. The method of claim 7, wherein the amino acid sequence is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 81, SEQ ID NO: 89, and active fragments thereof.

9. A method of preventing or treating a viral infection caused by at least one virus, such as a coronavirus, comprising administering to a subject diagnosed with, at risk for, or exhibiting symptoms of a viral infection, a pharmaceutical composition according to any one of claims 1-6.

10. The method of any one of claims 7-9, wherein the at least one virus is a coronavirus.

11. The method of claim 10, wherein the coronavirus is SARS-CoV-2.

12. The method of claim 10, wherein the coronavirus is HCoV-OC43.

13. The method of any one of claims 7-12, further comprising administering to the subject an antiviral suitable for the treatment of a viral infection.