DEFENSINS AS INHIBITORS OF SARS-COV-2 INFECTION AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/113,793, filed November 13, 2020, which is incorporated by reference herein in its entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readable sequence listing submitted concurrently herewith and identified as follows: One 5,194 Byte ASCII (Text) file named “Sequence_Listing_ST25.txt,” created on November 10, 2021.

FIELD OF THE INVENTION

The field of the invention relates generally to therapeutics and medicine. More specifically, the field of the invention relates to therapies for prevention and treatments of SARS-CoV-2 infection.

BACKGROUND OF THE INVENTION

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID- 19), has spread worldwide with more than 179 million cases and 3.8 million deaths as of late- June 2021 (https://coronavirus.jhu.edu/map.html (accessed on 23 June. 2021). The ability of the innate immune response to limit the magnitude of virus replication during acute infection is known to determine the severity of disease outcomes of coronaviruses (Park et al., Cell Host Microbe 2020, 27, 870-878; Channappanavar et al., Cell Host Microbe 2016, 19, 181-193; Channappanavar et al., J. Clin. Invest. 2019, 129, 3625-3639). Identification of mucosal factors against SARS-CoV-2 is critical for developing antiviral treatments and therapeutic options for COVID- 19. Defensins, antimicrobial peptides that play a crucial role in innate immunity, are abundant in neutrophils and are expressed by epithelial cells at mucosal sites (Ganz, Nat. Rev. Immunol. 2003, 3, 710-720; Selsted et al., Nat. Immunol. 2005, 6, 551-557; Lehrer et al., Immunol. Rev. 2012, 245, 84-112). Neutrophils have been shown to infiltrate the lung in response to SARS-CoV-2 infection in animal models (Bao et al., Nature 2020, 583, 830-833; Sun et al., Cell Host Microbe 2020, 28, 124-133; Rockx et al., Science 2020, 368, 101), suggesting their function in the control of virus replication, although dysregulation of neutrophils may contribute to hyperinflammation in severe COVID-19 diseases (Barnes et al., J. Exp. Med. 2020, 217, e20200652).

Human defensins are cationic peptides with [3-plcatcd sheet structures that are stabilized by three intramolecular disulfide bonds between cysteine residues (Ganz, Nat. Rev. Immunol. 2003, 3, 710-720; Selsted et al., Nat. Immunol. 2005, 6, 551-557; Lehrer et al., Immunol. Rev. 2012, 245, 84-112). These defensins are classified into the oc-, f>- and 9-defensins based on the disulfide links between conserved cysteine residues and their structure (Ganz, Nat. Rev. Immunol. 2003, 3, 710-720; Selsted et al., Nat. Immunol. 2005, 6, 551-557; Lehrer et al., Immunol. Rev. 2012, 245, 84-112). The effects of defensins on viral infection are defensin-, virus- and cell-type-specific (Chang et al., J. Innate Immun. 2009, 1, 413-420; 13. Ding et al., J. Innate Immun. 2009, 1, 413-420; Klotman etal., PLoS Pathog. 2017, 13, el 006446). Human oc-defensins including human neutrophil peptides (HNPs) 1-4 and human oc-defensins 5 and 6 (HD5 and HD6), which are mainly produced by neutrophils and intestinal Paneth cells, respectively, exhibit antiviral activities against enveloped and non-enveloped viruses [12]; however, evidence indicates that HD5 and HD6 can also promote viral infectivity of HIV and some adenoviruses (Chang et al., J. Innate Immun. 2009, 1, 413-420; Wilson et al., PLoS Pathog. 2017, 13, el006446; Klotman et al., J. Immunol. 2008, 180, 6176-6185). Human [ -defensins (HBDs) do not appear to affect virions, but these peptides are known to suppress viral replication by altering cellular functions [14]. Theta-defensins (9-defensins), which are cyclic octadecapeptides expressed in rhesus macaques and baboons, and retrocyclins (RC), which are synthesized based on human 9-defensin pseudogenes, also exhibit antiviral activities (Lehrer et al., J. Biol. Chem. 2012, 287, 27014-27019; Cole et al., Proc. Natl. Acad. Sci. USA 2002, 99, 1813- 1818). In addition to their effects on virus replication, defensins serve as immune modulators to regulate innate and adaptive immunity (Fruitwala et al., Semin Cell Dev. Biol. 2019, 88, 163-172; Yang et al., Curr. Pharm. Des. 2007, 13, 3131-3139). For example, defensins exhibit inflammatory and anti-inflammatory activities, and recruit and activate T cells and myeloid cells such as monocytes and dendritic cells (Fruitwala et al. , Semin Cell Dev. Biol. 2019, 88, 163-172; Yang et al., Curr. Pharm. Des. 2007, 13, 3131— 3139). Administration of rhesus 9-defensin-l (RTD-1) prevents death in MA15-SARS- CoV-infected mice by reducing pulmonary pathology. The mechanism is thought to involve immune modulation, as RTD-1 does not exhibit anti-SARS-CoV activity in vivo or in vitro (Wohlford-Lenane et al., J. Virol. 2009, 83, 11385-11390). Here, we have determined the effects of various human defensins and a 9-defensin analog RC101 on SARS-CoV-2 infection in vitro and their underlying mechanisms.

What is needed are effective therapies for the prevention and treatment of SARS- CoV-2 infection.

The foregoing description of the background is provided to aid in understanding the invention, and is not admitted being or describe prior art to the invention.

SUMMARY OF THE INVENTION

It is to be understood that both the foregoing general description of the invention and the following detailed description are exemplary, and thus do not restrict the scope of the invention.

The present inventors have determined the effects of various human defensins and a 9-defensin analog RC101 on SARS-CoV-2 infection using pseudotyped virus particles harboring SARS-CoV-2 spike proteins. As described herein, HNPs 1-3, HD5, and RC-101 inhibited SARS-CoV-2 infection by blocking viral entry. These findings indicate that defensins may contribute to virus suppression during acute infection, suggesting they be useful additives to the anti-virus arsenal.

In one aspect, the invention provides a method of reducing SARS-CoV-2 entry into cells comprising contacting the cells with an effective amount of a composition comprising a polypeptide comprising a defensin, an active fragment, an analog or derivative thereof.

In another aspect, the invention provides a method of reducing SARS-CoV-2 entry into cells comprising contacting the cells with an effective amount of a composition comprising a nucleic acid encoding a polypeptide comprising a defensin, an active fragment, an analog or derivative thereof.

In another aspect, the invention provides a method of treating or preventing SARS- CoV-2 infection in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising a polypeptide comprising a defensin, an active fragment, an analog or derivative thereof. In another aspect, the invention provides a method of treating or preventing SARS- CoV-2 infection in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising a nucleic acid encoding a polypeptide comprising a defensin, an active fragment, an analog or derivative thereof.

In some embodiments, the defensin is an oc-defensin, a ^-defensin, or a 9-defensin.

In some embodiments, the defensin or analog is selected from HNP1, HNP2, HNP3, HNP4, HD5, HD6, and RC-101.

In some embodiments, the composition is administered to a subject by topical, intravenous, subcutaneous, intramuscular, intracutaneous, transcutaneous, intrathecal, intranasal, inhalation, intra-arterial, rectal, intragastric, parenteral, or oral administration.

In some embodiments, the nucleic acid is delivered as a viral vector.

In some embodiments, the method further comprises administering an effective amount of an additional pharmaceutically active agent. In some embodiments, the method further comprises administering an effective amount of an agent that inhibits the interaction of angiotensin-converting enzyme 2 (ACE2) and SARS-CoV-2 spike protein. In some embodiments, the agent is an antibody. In some embodiments, the agent is a vaccine composition directed against Sars-CoV-2. In some embodiments, the vaccine comprises a nucleic acid encoding the spike protein or a fragment thereof. In some embodiments, the vaccine comprises a polypeptide comprising the spike protein or a fragment thereof. In some embodiments, the additional pharmaceutically active agent comprises remdesivir.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1. Human oc-defensins inhibit infection by pseudotyped SARS-CoV-2 virus. Pseudotyped luciferase reporter virus expressing SARS-CoV-2 S protein was incubated with or without HNPsl-4, HD5 or HD6 at 37°C for 1 h. Viruses with or without defensin treatment were used to infect HEK293T cells expressing hACE2 (A-F) as described in the Methods. Infected cells were cultured for three days before measuring luciferase activity. The significance of the differences between defensin-treated virions and mocked-treated controls was calculated by Student’s two-tailed, unpaired /=test; *p < 0.05. Data are means ± SD and are representative of four independent experiments.

FIG. 2. Theta defensin analog RC101 but not HBDs inhibits pseudotyped SARS- CoV-2 infection. Pseudotyped luciferase reporter viruses expressing SARS-CoV-2 S proteins were incubated with or without the indicated concentrations of HBD2, HBD5, HBD6 or RC101 at 37°C for 1 h followed by infection of HEK293T cells expressing hACE2 (A-D) as described in the Methods. Infected cells were cultured for three days before measuring luciferase activity. The effect of defensins on the virus in the presence of 10% FBS was also determined (E and F). Differences between defensin-treated virions and non-treated controls were calculated by Student’s two-tailed, unpaired /-test; *p < 0.05. Data are means ± SD and are representative of three independent experiments.

FIG. 3. Defensins inhibit infection by replication-competent SARS-CoV-2 viruses. Replication-competent SARS-CoV-2 viruses expressing mNeonGreen (at MOI of five) were incubated with HNP1 (A), HD5 (B) and RC101 (C) at different concentrations and at 37°C for 1 h before addition to Vero E6 cells for 2 h. Infected cells were then cultured in FluoroBrite media with 10% FBS for two days. The fluorescence from productive viral infections was measured using Biotek Cytation 5. The differences between defensin- treated virions and non-treated controls were calculated by Student’s two-tailed, unpaired /-test; *p < 0.05. Data are means ± SD and are representative of three independent experiments. FIG. 4. HNP1, HD5 and RC101 inhibit infection by pseudotyped SARS-CoV-2 variants. Pseudotyped luciferase reporter virus expressing SARS-CoV-2 S protein from the P.l variant (A) or the B.1.1.7 variant (B) was incubated with or without HNP1, HD5 and RC101 at 37°C for 1 h. Viruses were added to HeLa-hACE2 cells and incubated at 37°C for 2 h. Infected cells were cultured for two days before measuring luciferase activity. The significance of the differences between defensin-treated virions and mocked-treated controls was calculated by Student’s two-tailed, unpaired /-test; *p < 0.05. Data are means ± SD and are representative of three independent experiments.

FIG. 5. Defensins are ineffective after viral entry. (A) To determine the effect of defensins on target cell resistance to infection, HEK293T-hACE2 cells were incubated with defensins in the presence of FBS for 1 h, and then exposed to pseudotyped SARS- CoV-2 luciferase reporter virus without defensins for 2 h. Cells were washed and then cultured in fresh media for three days before measuring luciferase activity. (B) To determine the post-entry effect of defensins, HEK293T-hACE2 cells were infected with pseudotyped SARS-CoV-2 viruses for 2 h followed by treatment of infected cells with the indicated concentration of defensins. The differences between defensin-treated samples and untreated controls were calculated by Student’s two-tailed /-test; *p < 0.05. Data are means ± SD of triplicate samples and are representative of three independent experiments.

FIG. 6. The effect of HNP1 on viral attachment, spike RBD-hACE2 interaction and viral fusion. (A) Pseudotyped SARS-CoV-2 viruses were treated with HNP1 at different concentrations for 1 h at 37°C. The virus-def ensin mixture was added to HeLa- hACE2 cells, and cells were incubated at 4°C for 2 h. After washing off unbound viruses, cells were lysed, and cell-associated HIVp24 was determined as described in the Methods. (B) Immobilized spike RBD proteins on the plate were incubated with biotinylated hACE proteins that were pre-treated with defensins at different concentrations for 1 h. After incubation at 37°C for 1 h, the plate was washed and the bound hACE2 proteins were detected by incubation with alkylate phosphatase (AP)-conjugated streptavidin followed by the AP colorimetric assay as described in the Methods. For panels A and B, the differences between defensin-treated samples and untreated controls were calculated by Student’s two-tailed /-test; *p < 0.05. Data are means ± SD of triplicate samples and are representative of three independent experiments. (C) A schematic of Forster resonance energy transfer (FRET)-based BlaM assay is shown on the left panel. Pseudotyped SARS- CoV-2 viruses containing BlaM-Vpr proteins were incubated with Huh7.5-ACE2- TMPRSS2 cells at 16°C for 30 min at 1550 xg. After washing off unbound viruses, cells were treated with HNP1 at different concentrations and incubated at 37°C for 2 h for viral fusion. The BlaM substrate, CCF4-AM, was added to cells, which were incubated at 11°C overnight. The cytoplasmic BlaM activity from viral fusion was determined by measuring the ratio of blue (460 nm) to green (520 nm) fluorescence with excitation at 410 nm. The differences between defensin-treated samples and untreated controls were calculated by Student’s two-tailed /-test; *p < 0.05. Data are means ± SD of two independent experiments.

FIG. 7. The structure of defensins required for anti-SARS-CoV-2 activity. To determine the effect of linear defensins (A) or proHD5 (B) on viral infection, pseudotyped SARS-CoV-2 virus was incubated with indicated concentrations of [Abu]HNPl or [Abu]HD5 for 1 h before infection of HEK293T-hACE2 cells. Infected cells were cultured for three days before measuring luciferase activity. There was no difference between defensin-treated samples and untreated controls as calculated by Student’s two-tailed I-test. Data are means ± SD of triplicate samples and are representative of two independent experiments.

FIG. 8. The effect of viral titers and cell types on defensin-mediated SARS-CoV- 2 inhibition. (A) To determine the effect of virus titers on the antiviral activity of HNP1, indicated dilutions of serum-free pseudotyped SARS-CoV-2 viruses were incubated with 1 pg/mE (left) or 25 pg/mL (right) HNP1 at 37°C for 1 h before infection of HEK293T- hACE2 cells. The percentage of control was calculated using the formula: (RLU of defensin-treated samples / RLUs of average of untreated samples at the same virus titer) x 100. (D) To determine the effect of defensins on SARS-CoV-2 infection of intestinal and lung epithelial cells expressing endogenous hACE2 receptors, pseudotyped SARS-CoV-2 viruses were incubated with 25 pg/mL HNP1, HD5 or RC101 at 37°C for 1 h, then added to intestinal CaCo-2 cells (left) or lung epithelial A549 cells (right) at 37°C for 1.5 h. After washing off unbound viruses, infected cells were cultured for three days before measuring luciferase activities. The differences between defensin-treated samples and untreated controls were calculated using Student’s two-tailed ost; *p < 0.05. Data are means ± SD of triplicate samples and are representative of three independent experiments.

FIG. 9. Pseudotyped SARS-CoV-2 virus infection is dependent on hACE2 expression. (A) Cell lysates from HEK293T cells transfected with or without a plasmid expressing SARS-CoV-2 spike proteins were analyzed in a 4-12% gel. The identity of spike proteins was confirmed by Western Blot using anti-SARS-CoV S antibody. The blot was then stripped and probed using anti-b-actin antibody. (B) Quantitative RT-PCR analysis of hACE2 expression in HEK293T and hACE2 transfected HEK293T cells. (C). HEK293T cells or hACE2-expressing HEK293T cells were infected with pseudotyped SARS-CoV-2 viruses (-20 ng p24 per well for a 48-well plate) for 1 h. Cells were washed and then cultured for 3 days before measurement of luciferase activity in infected cells.

FIG 10. Defensins with anti-SARS-CoV-2 activity are not cytotoxic. hACE2- expressing HEK293T cells were treated for 24 hours with the indicated concentration of HNP1 (A), HD5 (B), HD6 (C), and RC101 (D). Cell viability was determined by MTSbased CellTiter 96® AQueous One Solution Cell Proliferation Assay. Data are mean ± SD of 4-6 samples, and are representative of two independent experiments.

FIG. 11. Human oc-defensins (A) and P-defensins (B).

FIG. 12. Infection of hamster cells by pseudotyped viruses expressing SARS-CoV- 2 spike proteins. BHK cells and HEK293T cells overexpressing hACE2 were infected by pseudotyped SARS-CoV-2 luciferase reporter viruses.

DETAILED DESCRIPTION OF THE INVENTION

Innate immunity during acute infection plays a critical role in disease severity of Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), and is likely to contribute to COVID- 19 disease outcomes. Defensins are highly abundant innate immune factors in neutrophils, epithelial cells, including intestinal Paneth cells, and exhibit antimicrobial and immune modulatory activities. As provided herein, the present inventors investigated the effects of human oc and P defensins and RC-101, a 9 defensin analog, on SARS-CoV-2 infection. Using pseudotyped viruses expressing SARS- CoV-2 Surface (Spike) proteins, it was found that human neutrophil peptides (HNPs) 1-3 exhibited potent anti-SARS-CoV-2 activity, and human defensin (HD) 5 and RC101 exhibited moderate activity. HNP4 and HD6 had weak anti-SARS-CoV-2 activity, whereas human b defensins (HBD2, HBD5 and HBD6) had no effect. Pretreatment of cells with HNP1, HD5, or RC101 provided significant protection against viral infection. These defensins did not have an effect when provided post infection. Without being bound by theory, it is believed that the defensins block viral entry. High virus titers overcame the effect of low levels of HNP1, suggesting that defensins act on the virion. HNP1, HD5, and RC101 also blocked viral infection of intestinal and lung epithelial cells. The protective effects of defensins reported here indicate that they can be useful as therapeutics to prevent and treat SARS-CoV-2 infection.

Reference will now be made in detail to embodiments of the invention which, together with the drawings and the following examples, serve to explain the principles of the invention. These embodiments describe the invention in sufficient detail to enable those skilled in the art to practice the invention, and it is understood that other embodiments may be utilized, and that structural, biological, and chemical changes may be made without departing from the spirit and scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used). The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one" and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of’ and/or “consisting of.” As used herein, the term "about" means at most plus or minus 10% of the numerical value of the number with which it is being used.

It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

One skilled in the art may refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques. These texts include Current Protocols in Molecular Biology (Ausubel et. al., eds. John Wiley & Sons, N.Y. and supplements thereto), Current Protocols in Immunology (Coligan et al., eds., John Wiley St Sons, N.Y. and supplements thereto), Current Protocols in Pharmacology (Enna et al., eds. John Wiley & Sons, N.Y. and supplements thereto) and Remington: The Science and Practice of Pharmacy (Lippincott Williams & Wilicins, 2Vt edition (2005)), for example.

As used herein, the terms "effective amount" or "therapeutically effective amount" are interchangeable and refer to an amount that results in an improvement or remediation of at least one symptom of the disease or condition. Those of skill in the art understand that the effective amount may improve the patient's or subject's condition, but may not be a complete cure of the disease and/or condition. In some embodiments, the term “effective amount” corresponds to an amount administered that reduces the entry of SARS-CoV-2 into cells. The “effective amount” can correspond to an amount administered to subjects or to cells directly.

The term "preventing" as used herein refers to minimizing, reducing or suppressing the risk of developing a disease state or parameters relating to the disease state or progression of other abnormal or deleterious conditions, including entry of SARS-CoV-2 into cells.

The terms "treating" and "treatment" as used herein refer to administering to a subject a therapeutically effective amount of a composition so that the subject has an improvement in the disease or condition. The improvement is any observable or measurable improvement. Thus, one of skill in the art realizes that a treatment may improve the patient's condition but may not be a complete cure of the disease. Treating may also comprise treating subjects at risk of developing a disease and/or condition.

In one embodiment, the invention provides a method of reducing SARS-CoV-2 entry into cells comprising contacting the cells with an effective amount of a composition comprising a polypeptide comprising a defensin, an active fragment, an analog or derivative thereof.

In another embodiment, the invention provides a method of reducing SARS-CoV- 2 entry into cells comprising contacting the cells with an effective amount of a composition comprising a nucleic acid encoding a polypeptide comprising a defensin, an active fragment, an analog or derivative thereof.

In another embodiment, the invention provides a method of treating or preventing SARS-CoV-2 infection in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising a polypeptide comprising a defensin, an active fragment, an analog or derivative thereof.

In another embodiment, the invention provides a method of treating or preventing SARS-CoV-2 infection in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising a nucleic acid encoding a polypeptide comprising a defensin, an active fragment, an analog or derivative thereof.

The SARS-CoV-2 that is to be treated or prevent is not necessarily limiting. In some embodiments, the SARS-CoV-2 is a variant selected from Alpha (B.1.1.7 and Q lineages), Beta (B.1.351 and descendent lineages), Gamma (P.l and descendent lineages), Epsilon (B.1.427 and B.1.429), Eta (B.1.525), Iota (B.1.526), Kappa (B.1.617.1), 1.617.3, Mu (B.1.621, B.1.621.1), Zeta (P.2) and Delta (B.1.617.2 and AY lineages).

Defensins are small cysteine-rich cationic proteins that are highly evolutionarily conserved, as found in vertebrates, invertebrates, as well as plants. These proteins possess biological activity against a wide spectrum of organisms such as bacteria, fungi and many enveloped and non-env eloped viruses. Generally, defensins consist of 18-45 amino acids including 6 (in vertebrates) to 8 conserved cysteine residues. Various immune cells, such as neutrophil granulocytes and almost all epithelial cells, contain these peptides as host cells, with a key function of killing phagocytized or extracellular microorganisms. Many defensins function by binding to the microbial cell membrane, and, once embedded, form pore-like membrane defects that allow efflux of essential ions and nutrients to destroy microbe integrity.

Defensins are cationic, trisulfide-containing antimicrobial peptides that are produced by leukocytes and various epithelia. They are subdivided into the oc, P, and 0- defensin subfamilies, which are distinguished by peptide size and different disulfide motifs. In humans, four oc-defensins (HNP1 to HNP4) have been isolated from neutrophils and two enteric oc-defensins (HD-5 and HD-6) are expressed by Paneth cells in crypts of the small intestine. The expression of HD5 has also been detected in the female urogenital tract. Three human ^-defensins (hBDl to hBD3) have been isolated from epithelial and nonepithelial cell types of various organs, and the expression of several others has been deduced by cDNA analysis or from analysis of the human genome. Numerous lines of evidence suggest that defensins provide an antimicrobial effector function in skin, the respiratory epithelium, the urogenital tract, and various leukocytes (i.e., neutrophils, monocytes, and NK cells). Furthermore, defensins activate cells involved in both the innate and the adaptive immune responses, suggesting that they operate within and link two branches of immunity.

9-defensins are cyclic octadecapeptides formed by the posttranslational splicing of two nonapeptides derived from 76-amino-acid oc-defensin-related precursors. Humans do not express 0-dcfcnsin peptides since the expression of 9-defensins ceased near the time that orangutans emerged in evolution due to a mutation that introduced a premature stop codon in the peptide precursor. As used herein the terms "Theta-defensins" or " 9- defensins" include members of the 9-defensin family of defensin proteins as found in various primate species, such as Old World monkeys and apes (examples being the rhesus monkey, olive baboon, siamang gibbon and orangutan), wherein 9-defensins precursors are expressed transcriptionally and processed via post-translational modification into 9- defensin. This further includes pseudogenes found in Great Apes (examples being human, chimpanzees, bonobos, and gorillas), wherein genetic modification of 9-defensin pseudogenes may be altered according to techniques readily known in the art, to allow expression of 9-defensin proteins in mammalian cells. This also includes 0-dcfcnsin proteins that may be identified in the same or different species, according to techniques readily known in the art, such as computer modeling techniques describing sequence homology or conserved structure in a comparison window of nucleic acid and/or amino acid sequences, or selective hybridization techniques using nucleic acid probes to identify homologous 9-defensins. 9-defensins may be isolated from endogenous sources, produced in autologous or heterologous cell lines, produced via peptide synthesis, or according to any available method known to one of skill in the art. Examples of 0-dcfcnsins include RTD-1, RTD-2, RTD-3, RTD-4, RTD-5, RTD-6, BTD-1, BTD-2, BTD-3, BTD-4, BTD- 5, BTD-6, BTD-7, BTD-8, BTD-9, BTD-10 or HTDp.

The term "analog," as applied to 0-dcfcnsins herein, are polypeptides and peptides that contain a core structure derived from a defensin, such as 9-defensin, that possesses anti-microbial or anti-viral activity. Examples include cyclic peptides containing one, two, three, four, or more disulfide bonds across multiple cysteine residues or substantially similar substitutes, wherein the analog can range in length from 8-24 amino acids, and contains a net positive charge.

9-defensins can be bio-synthesized using head-to-tail splicing of two 9-amino-acid sequences derived from 9-defensin precursors. 9-defensins were first identified in neutrophils and monocytes of the rhesus monkey, with a subsequent phylogenetic survey revealing the existence of 9-defensin genes in other Old World monkeys and two apes (the siamang and orangutan), but the existence of 9-defensins in New World monkeys or prosimians has not yet been reported. Humans, chimpanzees, bonobos, and gorillas express 9-defensin pseudogenes in which the precursor mRNA contains a mutation producing a stop codon in the signal sequence, thus preventing translation of the 9-defensin precursor. Rhesus 9-defensin- 1 (RTD-1) is produced from the heterodimeric splicing of two 9- defensin precursors, proRTDla and proRTDlb. Homodimeric excision/ligation reactions involving proRTDla and proRTDlb were revealed by the isolation of RTD-2 and RTD-3. RTD-1, -2, and -3 have potent microbicidal activities against bacteria and fungi and have been known to possess antiviral activities against human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus (HSV). In some embodiments, the defensin is a 9-defensin from a primate. In some embodiments, the 9-defensin is from a rhesus monkey. In some embodiments, the 9- defensin is RTD-1, RTD-2, RTD-3, RTD-4, RTD-5, or RTD-6. In some embodiments, the 9-defensin is from an olive baboon. In some embodiments, the 9-defensin is BTD-1, BTD- 2, BTD-3, BTD-4, BTD-5, BTD-6, BTD-7, BTD-8, BTD-9 or BTD-10. In some embodiments, the 9-defensin is from a human. In some embodiments, the 9-defensin is human 9-defensin pseudogene (HTDp). In some embodiments, the 9-defensin is expressed in a siamang or orangutan.

In some embodiments, the defensin is an oc, P, or 9-defensin, an active fragment or analog thereof. In some embodiments, the defensin is of human origin.

In some embodiments, the defensin, active fragment, analog or derivative thereof is selected from HNP1, HNP2, HNP3, HNP4, HD5, HD6, and RC-101.

In some embodiments, HNP1 has an amino acid sequence corresponding to SEQ ID NO:1. In some embodiments, HNP2 has an amino acid sequence corresponding to SEQ ID NO:2. In some embodiments, HNP3 has an amino acid sequence corresponding to SEQ ID NO:3. In some embodiments, HNP4 has an amino acid sequence corresponding to SEQ ID NO:4. In some embodiments, HD5 has an amino acid sequence corresponding to SEQ ID NO:5. In some embodiments, HD6 has an amino acid sequence corresponding to SEQ ID NO:6.

RC-101 is described, e.g., in Owen et al., AIDS Res Hum Retroviruses, 20(11): 1157-65 (2004), which is incorporated herein by reference. RC-101 has an amino acid sequence corresponding to SEQ ID NO: 13.

In some embodiments, the defensin is an 9-defensin isolated from a mammal. In some embodiments, the 9-defensin is isolated from a primate. In some embodiments, the 9-defensin is isolated from a human. In some embodiments, the 9-defensin is purified from a biological sample obtained from a mammal. In some embodiments, the 9-defensin is purified from a biological sample obtained from a primate. In some embodiments, the 9- defensin is purified from a biological sample obtained from a human.

In some embodiments, the defensin, fragment, analog or derivative peptide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids in length. In some embodiments, the defensin, fragment, analog or derivative is over 24 amino acids in length. In some embodiments, the defensin, fragment, analog or derivative is 14 amino acids in length. In some embodiments, the defensin, fragment, analog or derivative contains at least 2 cysteine residues forming one disulfide bond. In some embodiments, the defensin, fragment, analog or derivative contains 4 cysteine residues forming 2 disulfide bonds. In some embodiments, the defensin, fragment, analog or derivative contains 6 cysteine residues forming 3 disulfide bonds. In some embodiments, the defensin, fragment, analog or derivative includes a synthetic amino acid. In some embodiments, the defensin, fragment, analog or derivative has a net positive charge. In some embodiments, defensin, fragment, analog or derivative may be encoded by a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 at least 98%, at least 99%, or more percentage identity to a polynucleotide that encodes any of the defensins or analogs as set forth in any of SEQ ID NOS:1-13. One of ordinary skill in the art can establish percentage identity according to methods known in the art, including establishing a comparison window between a reference sequence and a second polynucleotide sequence, to establish the degree of percentage identity.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid, for example, an amino acid analog. As used herein, the terms encompass amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

In some embodiments, the amino acid sequence of defensin, an active fragment, analog or derivative thereof comprises any of the defensins or analogs as set forth in any of SEQ ID NOS:1-13. In some embodiments, the defensin, active fragment, analog or derivative thereof comprises a fusion protein. In some embodiments, the active fragment, analog or derivative of defensin has at least 75%, at least 80%, at least 85% identity to any of the sequences as set forth in any of SEQ ID NOS: 1-13. In some embodiments, the defensin, active fragment, analog or derivative thereof has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 at least 98%, at least 99%, or more percent identity to any of the sequences as set forth in any of SEQ ID NOS:1-13.

A fragment is a polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned defensin, analog or derivative polypeptide. In some embodiments, a fragment may constitute at least about 25 contiguous amino acids identified in any of the sequences as shown in any of SEQ ID NOS: 1-13. In some embodiments, the fragment is at least about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 2425, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more amino acids in length.

In some embodiments the fragments include, for example, truncation polypeptides having the amino acid sequence of defensin, analogs, or derivatives except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. In some embodiments, fragments are characterized by structural or functional attributes. Active fragments are those that mediate protein activity, including those with a similar activity or an improved activity, or with a decreased activity.

Active fragments or derivatives of defensin, include polypeptides having an amino acid sequence at least 75% identical to any of the sequences as shown in any of SEQ ID NOS: 1-13 or fragments thereof with at least 75% identity to the corresponding fragment, all of which retain the biological activity. Included in this group are derivatives of the defined sequence and fragment. In some embodiments, the derivatives are those that vary from the reference by conservative amino acid substitutions, i.e., those that substitute a residue with another of like characteristics. Typical substitutions are among Ala, Vai, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr. In some embodiments, the polypeptides are derivatives in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids are substituted, deleted, or added in any combination.

The proteins comprising defensin, an active fragment, analog or derivative can be prepared in any suitable manner. Such polypeptides include recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

In some embodiments, the polypeptide can comprise one or more epitope tags and/or purification tags. The epitope tag is not limiting and can include a Myc tag, a FLAG tag, a hemagglutinin (HA) tag and/or combinations thereof. The purification tag is not limiting and can include a histidine tag (6x), a glutathione S-Transferase tag or a combination thereof.

In some embodiments, the polypeptide comprises an enzymatic cleavage site to further aid in purification and processing of the fusion protein. In some embodiments, the cleavage site is an enterokinase cleavage site.

In some embodiments, the fusion protein comprises a histidine tag (6x), a FLAG tag, a hemagglutinin (HA) tag, and/or an enterokinase cleavage site.

In some embodiments, a nucleotide sequence encoding a defensin, an active fragment, an analog or derivative thereof may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In some embodiments, however, the nucleic acid would comprise complementary DNA (cDNA). The term "cDNA" is intended to refer to DNA prepared using messenger RNA (mRNA) as template. The advantage of using a cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, non- or partially processed RNA template, is that the cDNA primarily contains coding sequences of the corresponding protein. There may be times when the full or partial genomic sequence is preferred, such as where the non-coding regions are required for optimal expression.

The organismal source of the defensin, active fragment, analog or derivative is not necessarily limiting. In some embodiments, the defensin, active fragment, analog or derivative nucleic acid sequence is derived from a mammal, bird, reptile or fish. In some embodiments, the defensin, active fragment, analog or derivative is of human origin. In some embodiments, the defensin, active fragment, analog or derivative is of Old World monkey origin (e.g., rhesus monkey). In some embodiments, the defensin, active fragment, analog or derivative is of ape origin (e.g., siamang or orangutan). In some embodiments, the defensin, analog or derivative is of dog, cat, horse, mouse, rat, guinea pig, sheep, cow, or pig origin. In some embodiments, nucleic acid molecules may be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Defensin, active fragments, analogs or derivative nucleic acids include natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications provide the desired effect (e.g., production of defensin protein in non-human expression systems).

In some embodiments, a particular nucleotide sequence encoding defensin, active fragment, analog or derivative polypeptide may be identical over its entire length to the coding sequence that encodes any of the sequences as shown in SEQ ID NOS: 1-13. In some embodiments, a particular nucleotide sequence encoding defensin, active fragment, analog or derivative polypeptide may be an alternate form of sequence due to degeneracy in the genetic code or variation in codon usage encoding any of the sequences as shown in SEQ ID NOS:1-13.

In some embodiments, the nucleic acid sequence of defensin, active fragment, analog or derivative contains a nucleotide sequence that is highly identical, at least 90% identical, with a nucleotide sequence encoding defensin, active fragment, analog or derivative polypeptide. In some embodiments, the nucleic acid sequence of defensin, active fragment, analog or derivative comprises a nucleotide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical with a nucleic acid sequence that encodes any of the sequences as shown in SEQ ID NOS:1-13.

When a polynucleotide of the invention is used for the recombinant production of defensin, active fragment, analog or derivative polypeptide, the polynucleotide may include the coding sequence for the full-length polypeptide or a fragment thereof, by itself; the coding sequence for the full-length polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro or prepro-protein sequence, or other fusion peptide portions. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.

In some embodiments, the nucleotide sequence encoding defensin, active fragment, analog or derivative includes nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to (a) a nucleotide sequence encoding defensin or an analog having the amino acid sequence of any of the sequences as shown in SEQ ID NOS: 1-13; or (b) a nucleotide sequence complementary to the nucleotide sequences in (a).

Conventional means utilizing known computer programs such as the BestFit program (Wisconsin Sequence Analysis Package, Version 10 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) may be utilized to determine if a particular nucleic acid molecule is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence encoding defensin or an analog having the amino acid sequence of any of the sequences as shown in SEQ ID NOS:1-13.

In some embodiments, the nucleotide sequence encoding defensin, active fragment, analog or derivative encodes an amino acid sequence of any of the sequences as shown in SEQ ID NOS: 1-13, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues are substituted, deleted or added, in any combination.

In some embodiments, the nucleotide sequences are at least 90% identical over their entire length to a polynucleotide encoding a defensin or analog having the amino acid sequence set out in any of the sequences as shown in SEQ ID NOS: 1-13, and polynucleotides which are complementary to such polynucleotides. In some embodiments, the polynucleotides are at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical.

In some embodiments, the defensin, active fragment, analog or derivative nucleic acid sequence has been optimized for expression in alternative host organisms (e.g., nonhuman). Although as described above, the genetic code is degenerate, so frequently one amino acid may be coded for by two or more nucleotide codons. Thus, multiple nucleic acid sequences may encode one amino acid sequence. Although this creates identical proteins, the nucleic acids themselves are distinct, and can have other distinct properties. As described herein, one aspect of the choice of codon usage can be (but is not limited to) the ability to express a protein in a non-native cell (e.g., a human protein in bacteria or yeast), or the level of expression in such cells. In order to obtain enough protein for purification, testing, and use in in vitro assays, in animal models, and eventually in clinical development, efficient protein expression in non-human systems is needed.

In some embodiments, the nucleic acid sequence further includes a nucleotide sequence encoding one or more of an epitope tag or a purification tag.

The term "epitope tag" as used herein in reference to nucleic acid molecules refers to nucleotides encoding peptide sequences that are recognized and bound by the variable region of an antibody or fragment. In some embodiments, the epitope tag is not part of the native protein. In some embodiments, the epitope tag is removable. In some embodiments, the epitope tag is not intrinsic to the protein's native biological activity. Examples of epitope tags include, but are not limited to Myc, HA and FLAG.

The term "purification tag" as used herein in reference to nucleic acid molecules refers to nucleotides encoding peptide sequences that facilitate the purification of the protein, but are generally not necessary for the protein's biological activity. In some embodiments, purification tags may be removed following protein purification. Examples of purification tags include, but are not limited to glutathione S-transferase (GST) or 6x- histidine (H6).

In some embodiments, the epitope tag is selected from Myc, HA and FLAG and combinations thereof. In some embodiments, the purification tag is one or more of glutathione-S -transferase (GST) or 6x-histidine (H6).

In some embodiments, the nucleic acid also encodes a cleavage site for a protease. In some embodiments, the cleavage site is an enterokinase target sequence, located downstream from one or more epitope and/or purification tags.

The present invention also relates to vectors that comprise the nucleic acids of the present invention, including cloning vectors and expression vectors, host cells which are genetically engineered with vectors of the invention and methods for the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention. Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, Escherichia coli, Streptomyces and Bacillus subtilis; fungal cells, such as yeast and Aspergillus; insect cells such as Drosophila S2 and Spodoptera Sf9; mammalian cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK-293 and Bowes melanoma. A great variety of expression systems can be used, including DNA or RNA vectors.

In other embodiments, this invention provides an isolated nucleic acid molecule of the invention operably linked to a heterologous promoter. The invention further provides an isolated nucleic acid molecule operably linked to a heterologous promoter, wherein said isolated nucleic acid molecule is capable of expressing a fusion protein comprising defensin, active fragment, analog or derivative when used to transform an appropriate host cell.

Methods for the production of polypeptides of the invention including culturing a host cells transfected with one or more of the vectors of the present invention under conditions promoting expression of the polypeptide encoded by the vector, and isolating the polypeptide so expressed from the cell culture.

Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Pat. Nos. 5,871,986 and 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC 2.0 from INVITROGEN and BACPACK baculovirus expression system from CLONTECH.

Other examples of expression systems include COMPLETE CONTROL Inducible Mammalian Expression System from STRATAGENE, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN, which carries the T-REX (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast P. methanolica. One of skill in the art would know how to manipulate a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

Primary mammalian cell cultures may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the expression construct, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture techniques are well documented.

One embodiment involves the use of gene transfer to immortalize cells for the production of proteins. The nucleic acid for the protein of interest may be transferred as described above into appropriate host cells followed by culture of cells under the appropriate conditions.

Examples of useful mammalian host cell lines are Vero and HeLa cells and cell lines of Chinese hamster ovary, W138, BHK, COS-7, HEK-293, HepG2, NIH3T3, RIN and MDCK cells. In addition, a host cell clone may be chosen that modulates the expression of the inserted sequences, or modifies and process the gene product in the manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.

A number of selection systems may be used including, but not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside G418; and hygro, that confers resistance to hygromycin.

As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include their progeny, which are any and all subsequent generations. It is understood that all progenies may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors. A host cell may be "transfected" or "transformed," which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes (e.g., bacteria or yeast), depending upon whether the desired result is replication of the vector or expression of part or all of the vector-encoded nucleic acid sequences. Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Bacterial cells used as host cells for vector replication and/or expression include DH5oc, JM109, and KCB, as well as a number of commercially available bacterial hosts such as SURE Competent Cells and SOLOPACK Gold Cells (STRATAGENE, La Jolla). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.

Examples of eukaryotic host cells for replication and/or expression of a vector include HeLa, NIH3T3, Jurkat, HEK-293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.

Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above-described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides. In some embodiments, the invention provides a viral vector encoding a defensin, active fragment, an analog or derivative thereof. In some embodiments, the viral vector comprises a nucleic acid sequence encoding defensin, active fragment, analog or derivative as provided herein. In some embodiments, the defensin, active fragment, analog or derivative is fused to an epitope tag. The epitope tag is not limiting, and in some embodiments is selected from the group consisting of Myc, FLAG, hemagglutinin (HA) and/or combinations thereof. In some embodiments, the defensin, active fragment, analog or derivative encodes a protein that is at least 90% identical to any of the sequences as shown in SEQ ID NOS: 1-13.

The viral vector is not limiting. In some embodiments, the viral vector will typically comprise a highly attenuated, non-replicative virus. Viral vectors include, but are not limited to, DNA viral vectors such as those based on adenoviruses, herpes simplex virus, avian viruses, such as Newcastle disease virus, poxviruses such as vaccinia virus, and parvoviruses, including adeno-associated virus; and RNA viral vectors, including, but not limited to, the retroviral vectors. Vaccinia vectors and methods useful in immunization protocols are described in U.S. Pat. No. 4,722,848. Retroviral vectors include murine leukemia virus, and lentiviruses such as human immunodeficiency virus. Naldini et al. (1996) Science 272:263-267. Replication-defective retroviral vectors harboring a nucleotide sequence of interest as part of the retroviral genome can be used. Such vectors have been described in detail. (Miller et al. (1990) Mol. Cell. Biol. 10:4239; Kolberg, R. (1992) J. NIH Res. 4:43; Cornetta et al. (1991) Hum. Gene Therapy 2:215).

Adenovirus and adeno-associated virus vectors useful in the invention may be produced according to methods already taught in the art. (See, e.g., Karlsson et al. (1986) EMBO 5:2377; Carter (1992) Current Opinion in Biotechnology 3:533-539; Muzcyzka (1992) Current Top. Microbiol. Immunol. 158:97-129; Gene Targeting: A Practical Approach (1992) ed. A. L. Joyner, Oxford University Press, NY). Several different approaches are feasible.

Alpha virus vectors, such as Venezuelan Equine Encephalitis (VEE) virus, Semliki Forest virus (SFV) and Sindbis virus vectors, can be used for efficient gene delivery. Replication-deficient vectors are available. Such vectors can be administered through any of a variety of means known in the art, such as, for example, intranasally or intratumorally. See Lundstrom, Curr. Gene Ther. 2001 1:19-29.

Additional literature describing viral vectors which could be used in the methods of the present invention include the following: Horwitz, M. S., Adenoviridae and Their Replication, in Fields, B., et al. (eds.) Virology, Vol. 2, Raven Press New York, pp. 1679- 1721, 1990); Graham, F. et al., pp. 109-128 in Methods in Molecular Biology , Vol. 7: Gene Transfer and Expression Protocols, Murray, E. (ed.), Humana Press, Clifton, N.J. (1991); Miller, et al. (1995) FASEB Journal 9:190-199, Schreier (1994) Pharmaceutica Acta Helvetiae 68:145-159; Schneider and French (1993) Circulation 88:1937-1942; Curiel, et al. (1992) Human Gene Therapy 3:147-154; WO 95/00655; WO 95/16772; WO 95/23867; WO 94/26914; WO 95/02697 (Jan. 26, 1995); and WO 95/25071.

In some embodiments, the viral vector is a retrovirus/lentivirus, adenovirus, adeno- associated virus, alpha virus, vaccinia virus or a herpes simplex virus.

The active agents can be administered in a variety of ways and is not particularly limiting. In some embodiments, the defensin agent is administered directly (topically), intravenously, subcutaneously, transcutaneously, intrathecally, intramuscularly, intracutaneously, intragastrically, intranasally, by nasal and/or oral inhalation, rectally, intra-arterially, parenterally, or orally.

In some embodiments, an effective amount of the defensin agent that is administered includes a dose of about 0.0001 nM to about 2000 pM. In some embodiments, amount administered is from about 0.01 nM to about 2000 pM; about 0.01 pM to about 0.05 pM; about 0.05 pM to about 1.0 pM; about 1.0 pM to about 1.5 pM; about 1.5 pM to about 2.0 pM; about 2.0 pM to about 3.0 pM; about 3.0 pM to about 4.0 pM; about 4.0 pM to about 5.0 pM; about 5.0 pM to about 10 pM; about 10 pM to about 50 pM; about 50 pM to about 100 pM; about 100 pM to about 200 pM; about 200 pM to about 300 pM; about 300 pM to about 500 pM; about 500 pM to about 1000 pM; about 1000 pM to about 1500 pM; and about 1500 pM to about 2000 pM. Of course, all of these amounts are exemplary, and any amount in-between these points is also expected to be of use in the invention.

In some embodiments, the active agent can be administered parenterally or alimentarily. Parenteral administrations include, but are not limited to intravenously, intradermally, transdermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally. See, e.g., U.S. Pat. Nos. 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety). Alimentary administrations include, but are not limited to orally, buccally, rectally, or sublingually.

In some embodiments, the administration of the therapeutic compounds and/or the therapies of the present invention may include systemic, local and/or regional administrations, for example, topically (dermally, transdermally), via catheters, implantable pumps, dermal patches, transdermal patches, etc. Alternatively, other routes of administration are also contemplated such as, for example, arterial perfusion, intracavitary, intraperitoneal, intrapleural, intraventricular and/or intrathecal. The skilled artisan is aware of determining the appropriate administration route using standard methods and procedures. Other routes of administration are discussed elsewhere in the specification and are incorporated herein by reference.

As is well known in the art, a specific dose level of active compounds for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy.

In some embodiments, the compound(s) or composition(s) can be administered to the subject once, such as by a single injection or deposition at or near the site of interest. In some embodiments, the compound(s) or composition(s) can be administered to a subject over a period of days, weeks, months or even years. In some embodiments, the compound(s) or composition(s) is administered at least once a day to a subject. Where a dosage regimen comprises multiple administrations, it is understood that the effective amount of the compound(s) or composition(s) administered to the subject can comprise the total amount of the compound(s) or composition(s) administered over the entire dosage regimen.

Combination therapy

The active agents of the present invention can be administered alone or in combination with one or more additional active pharmaceutical agents. In some embodiments, the active agents of the invention are administered with one or more additional active pharmaceutical agents that are useful to treat conditions associated with infection, such as a coronavirus infection. In some embodiments, the additional active agent can include an agent useful to treat a lung disease or inflammatory condition.

In some embodiments, such additional pharmaceutical agents can include one or more corticosteroids, exogenous surfactants, statins, beta-blockers, n-acetylcysteine, antiinflammatory agents, immunosuppressants, therapeutic antibodies, antibiotics, or antiviral agents.

In one embodiment, the additional pharmaceutical agent is remdesivir or an analog thereof.

In some embodiments, the additional pharmaceutical agent is a type 1 interferon, such as IFN01.

In some embodiments, the additional pharmaceutical agent comprises a Casl3d protein and guide RNAs-containing spacer sequences specifically complementary to the virus RNA genome. See, e.g., Nguyen et al. Cell Research (2020) 30:189-190, which is incorporated herein by reference.

In some embodiments, the additional agent is convalescent plasma from a patient previously infected with SARS-CoV-2.

In one embodiment, the active agent comprises chloroquine and/or hydroxychloroquine. In this embodiment, zinc and/or azithromycin (e.g., ZITHROMAX®) can optionally be administered in combination with, or in addition to, the chloroquine and/or hydroxychloroquine.

In some embodiments, the additional agent comprises HCQ (+/- azithromycin (AZT)), CQ (+/- AZT), an IL-6 receptor antagonist (e.g., Sarilumab, Siltuximab, Tocilizumab), Acetaminophen, an NS AID, an ACE inhibitor/ ARB, Heparin, Ritonavir/lopinavir, Baricitinib, Bemcentinib, Bevacizumab, Colchicine, Dexamethasone, EIDD-2801, Favipiravir, Baricitinib, Bemcentinib, Bevacizumab, Fingolimod, Hydroxychloroquine and azithromycin, sulfate Ivermectin, Leronlimab, Lopinavir and ritonavir, Methylprednisolone, Umifenovir, Oseltamivir, Umifenovir, and Galidesivir.

In some embodiments, the additional agent comprises a kinase inhibitor. In some embodiments, the kinase inhibitor is selected from the group consisting of acalabrutinib (Calquence), baricitinib (Olumiant), ruxolitinib (Jakafi), tofacitinib (Xeljanz) and combinations thereof.

In one embodiment, the active agent comprises a cathepsin L inhibitor, such as thiocarbazate SID26681509 and teicoplanin.

In one embodiment, the active agent comprises a TMPRSS2 protease inhibitor such as camostat mesylate.

In one embodiment, the additional pharmaceutical agent is one or more of the compounds disclosed by Wu et al., “Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods,” Acta Pharmaceutica Sinica B http s ://doi . org/ 10.1016/j - a sb . 020.02.008 which is incorporated by reference herein.

In some embodiments, the additional active agent is a PLpro inhibitor, such as Ribavirin Valganciclovir, P-Thymidine, Aspartame, Oxprenolol, Doxycycline, Acetophenazine, lopromide, Riboflavin, Reproterol, 2,2 '-Cyclocytidine, Chloramphenicol, Chlorphenesin carbamate, Levodropropizine, Cefamandole,Floxuridine, Tigecycline, Pemetrexed, 1 (+)-Ascorbic acid, Glutathione, Hesperetin, Ademetionine, Masoprocol, Isotretinoin, Dantrolene, Sulfasalazine, Silybin, Nicardipine, Sildenafil, Platycodin, D, Chrysin, Neohesperidin, Baicalin, Sugetriol-3,9-diacetate, (-)-Epigallocatechin gallate, Phaitanthrin D, 2-(3,4-Dihydroxyphenyl)-2-[[2-(3,4-dihydroxyphenyl)-3,4-dihy dro-5,7- dihydroxy-2H-l-benzopyran-3-yl]oxy]-3,4-dihydro-2H-l-benzopy ran-3,4,5,7-tetrol, 2,2- Di (3-indolyl)-3-indolone, (S)-(lS,2R,4aS,5R,8aS)-l-Formamido-l,4a-dimethyl-6- methylene-5-((E)-2-(2-oxo-2,5-dihydrofuran-3-yl)ethenyl)deca hydronaphthalen-2-yl-2- amino-3-phenylpropanoate, Piceatannol, Rosmarinic acid, and Magnolol.

In some embodiments, the additional active agent is a 3CLpro inhibitor, such as Lymecycline, Chlorhexidine, Alfuzosin, Cilastatin, Famotidine, Almitrine, Progabide, Nepafenac, Carvedilol, Amprenavir, Tigecycline, Demeclocycline, Montelukast, Carminic acid, Mimosine, Flavin mononucleotide, Lutein, Cefpiramide, Phenethicillin, Candoxatril, Nicardipine, Estradiol valerate, Pioglitazone, Conivaptan, Telmisartan, Doxycycline, Oxytetracycline, (IS ,2R,4aS ,5R,8aS)- 1 -Formamido- 1 ,4a-dimethyl-6- methylene-5-((E)-2-(2-oxo-2,5-dihydrofuran-3-yl)ethenyl)deca hydronaphthalen-2-yl 5- ((R)-l,2-dithiolan-3-yl) pentanoate, Betulonal, Chrysin-7-O-P-glucuronide, Andrographiside, (lS,2R,4aS,5R,8aS)-l-Formamido-l,4a-dimethyl-6-methylene-5-( (E)- 2-(2-oxo-2,5-dihydrofuran-3-yl)ethenyl)decahydronaphthalen-2 -yl 2-nitrobenzoate, 20- Hydroxy-3 ,4- seco-friedelolactone-27 -oic acid, (S )-( 1 S ,2R,4aS ,5R, 8aS )- 1 -Formamido-

I,4a-dimethyl-6-methylene-5-((E)-2-(2-oxo-2,5-dihydrofura n-3- yl)ethenyl)decahydronaphthalen-2-yl-2-amino-3-phenylpropanoa te, Isodecortinol, Cerevisterol, Hesperidin, Neohesperidin, Andrograpanin, 2-((lR,5R,6R,8aS)-6-Hydroxy- 5-(hydroxymethyl)-5,8a-dimethyl-2-methylenedecahydronaphthal en-l-yl)ethyl benzoate, Cosmosiin, Cleistocaltone A, 2,2-Di(3-indolyl)-3-indolone, Biorobin, Gnidicin, Phyllaemblinol, Theaflavin 3,3'-di-O-gallate, Rosmarinic acid, Kouitchenside I, Oleanolic acid, Stigmast-5-en-3-ol, and Deacetylcentapicrin, Berchemol,

In some embodiments, the additional active agent is an RNA-dependent RNA polymerase inhibitor, such as Valganciclovir, Chlorhexidine, Ceftibuten, Fenoterol, Fludarabine, Itraconazole, Cefuroxime, Atovaquone, Chenodeoxycholic acid, Cromolyn, Pancuronium bromide, Cortisone, Tibolone, Novobiocin, Silybin, Idarubicin, Bromocriptine, Diphenoxylate, Benzylpenicilloyl G, Dabigatran etexilate, Betulonal, Gnidicin, 20,3O0-Dihydroxy-3,4-seco-friedelolactone-27-lactone, 14-Deoxy-

I I,12-didehydroandrographolide, Gniditrin, Theaflavin 3,3'-di-O-gallate, (R)-

((lR,5aS,6R,9aS)-l,5a-Dimethyl-7-methylene-3-oxo-6-((E)-2 -(2-oxo-2,5-dihydrofuran-

3-yl)ethenyl)decahydro-lH-benzo [c] azepin- l-yl)methyl 2-amino-3-phenylpropanoate, 20-Hydroxy-3,4-seco-friedelolactone-27-oic acid, 2-(3,4-Dihydroxyphenyl)-2-[[2-(3,4- dihydroxyphenyl)-3 ,4-dihydro-5 ,7 -dihydroxy-2H- 1 -benzopyran-3 -yl] oxy] -3 ,4-dihydro- 2H-l-benzopyran-3,4,5,7-tetrol, Phyllaemblicin B, 14-Hydroxycyperotundone,

Andrographiside, 2-((lR,5R,6R,8aS)-6-Hydroxy-5-(hydroxymethyl)-5,8a- dimethyl-2-methylenedecahydronaphthalen- l-yl)ethyl benzoate, Sugetriol-3,9-diacetate, Baicalin, (lS,2R,4aS,5R,8aS)-l-Formamido-l,4a-dimethyl-6-methylene-5-( (E)-2-(2-oxo- 2,5-dihydrofuran-3-yl)ethenyl)decahydronaphthalen-2-yl 5-((R)-l,2-dithiolan-3-yl) pentanoate, l,7-Dihydroxy-3-methoxyxanthone, l,2,6-Trimethoxy-8-[(6-O-0-d- xylopyranosyl-0-d-glucopyranosyl)oxy]-9H-xanthen-9-one, l,8-Dihydroxy-6-methoxy-2- [(6-O-0-d-xylopyranosyl-0-d-glucopyranosyl)oxy]-9H-xanthen-9 -one, and 8-(0-d- Glucopyranosyloxy)-l,3,5-trihydroxy-9H-xanthen-9-one.

In some embodiments, the additional pharmaceutically active agent comprises one or more anti-SARS-CoV-2 antibodies. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibodies are polyclonal antibodies, such as those obtained from convalescent serum of recovered patients. In some embodiments, the antibody targets the spike protein. In some embodiments, the antibody targets the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments, dual affinity retargeting antibodies (DART)), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific and trispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity.

In some embodiments, the antibody therapy is selected from bamlanivimab, etesevimab, casirivimab, imdevimab, sotrovimab and combinations thereof. In some embodiments, the antibody therapy is a combination of bamlanivimab plus etesevimab or casirivimab plus imdevimab.

The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.

Single or multiple administrations of the compositions that are disclosed herein can be administered depending on the dosage and frequency as required and tolerated by the patient. The dosage can be administered once, but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. Compositions

The present invention also contemplates therapeutic methods employing compositions comprising the active substances disclosed herein. Preferably, these compositions include pharmaceutical compositions comprising a therapeutically effective amount of one or more of the active compounds or substances along with a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable" carrier means a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. Examples of pharmaceutically acceptable antioxidants include, but are not limited to, water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like; oil soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, a-tocopherol and the like; and the metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

In some embodiments, the total daily dose of the active compounds of the present invention administered to a subject in single or in divided doses can be in amounts, for example, from 0.01 to 25 mg/kg body weight or more usually from 0.1 to 15 mg/kg body weight. Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. In general, treatment regimens according to the present invention comprise administration to a human or other mammal in need of such treatment from about 1 mg to about 1000 mg of the active substance(s) of this invention per day in multiple doses or in a single dose of from 1 mg, 5 mg, 10 mg, 100 mg, 500 mg or 1000 mg.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water, isotonic solutions, or saline. Such compositions may also comprise adjuvants, such as wetting agents; emulsifying and suspending agents; sweetening, flavoring and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

The injectable formulation can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

In some embodiments, the active agents of the present invention can be administered as a nanoparticle formulation.

In order to prolong the effect of a drug, it is often desirable to slow the absorption of a drug from subcutaneous or intramuscular injection. The most common way to accomplish this is to inject a suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug becomes dependent on the rate of dissolution of the drug, which is, in turn, dependent on the physical state of the drug, for example, the crystal size and the crystalline form. Another approach to delaying absorption of a drug is to administer the drug as a solution or suspension in oil. Injectable depot forms can also be made by forming microcapsule matrices of drugs and biodegradable polymers, such as polylactide-polyglycoside. Depending on the ratio of drug to polymer and the composition of the polymer, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly orthoesters and poly anhydrides. The depot injectables can also be made by entrapping the drug in liposomes or microemulsions, which are compatible with body tissues.

Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter and polyethylene glycol, which are solid at ordinary temperature but liquid at the rectal temperature and will, therefore, melt in the rectum and release the drug.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, gelcaps and granules. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings and other release-controlling coatings.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferably, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Dosage forms for topical or transdermal administration of a compound of this invention further include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. Transdermal patches have the added advantage of providing controlled delivery of active compound to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel. The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

In one embodiment, the therapeutic compound is delivered transdermally. The term "transdermal delivery" as used herein means administration of the pharmaceutical composition topically to the skin wherein the active ingredient or its pharmaceutically acceptable salts, will be percutaneously delivered in a therapeutically effective amount.

In some embodiments, the composition to be applied transdermally further comprises an absorption enhancer. The term " absorption enhancer" as used herein means a compound which enhance the percutaneous absorption of drugs. These substances are sometimes also referred to as skin-penetration enhancers, accelerants, adjuvants and sorption promoters. Various absorption enhancers are known to be useful in transdermal drug delivery. U.S. Pat. Nos. 5,230,897, 4,863,970, 4,722,941, and 4,931,283 disclose some representative absorption enhancers used in transdermal compositions and for topical administration. In some embodiments, the absorption enhancer is N-lauroyl sarcosine, sodium octyl sulfate, methyl laurate, isopropyl myristate, oleic acid, glyceryl oleate or sodium lauryl sulfoacetate, or a combination thereof. In some embodiments, the composition contains on a weight/volume (w/v) basis the absorption enhancer in an amount of about 1-20%, 1-15%, 1-10% or 1-5%. In some embodiments, to enhance further the ability of the therapeutic agent(s) to penetrate the skin or mucosa, the composition can also contain a surfactant, an azone-like compound, an alcohol, a fatty acid or ester, or an aliphatic thiol. In some embodiments, the invention provides a transdermal patch comprising an effective amount of the therapeutic compound for treating or preventing COVID-19 disease. In some embodiments, the transdermal patch further comprises an absorption enhancer.

In some embodiments, the transdermal composition can further comprise one or more additional excipients. Suitable excipients include without limitation solubilizers (e.g., C2-C8 alcohols), moisturizers or humectants (e.g., glycerol [glycerin], propylene glycol, amino acids and derivatives thereof, polyamino acids and derivatives thereof, and pyrrolidone carboxylic acids and salts and derivatives thereof), surfactants (e.g., sodium laureth sulfate and sorbitan monolaurate), emulsifiers (e.g., cetyl alcohol and stearyl alcohol), thickeners (e.g., methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol and acrylic polymers), and formulation bases or carriers (e.g., polyethylene glycol as an ointment base). As a nonlimiting example, the base or carrier of the composition can contain ethanol, propylene glycol and polyethylene glycol (e.g., PEG 300), and optionally an aqueous liquid (e.g., isotonic phosphate-buffered saline).

The method of the present invention employs the compounds identified herein for both in vitro and in vivo applications. For in vivo applications, the invention compounds can be incorporated into a pharmaceutically acceptable formulation for administration. Those of skill in the art can readily determine suitable dosage levels when the invention compounds are so used.

Exemplary pharmaceutically acceptable carriers include carriers suitable for oral, intravenous, subcutaneous, intramuscular, intracutaneous, and the like administration. Administration in the form of creams, lotions, tablets, dispersible powders, granules, syrups, elixirs, sterile aqueous or non-aqueous solutions, suspensions or emulsions, and the like, is contemplated.

For the preparation of oral liquids, suitable carriers include emulsions, solutions, suspensions, syrups, and the like, optionally containing additives such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents, and the like. For the preparation of fluids for parenteral administration, suitable carriers include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of nonaqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized, for example, by filtration through a bacteria- retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile water, or some other sterile injectable medium immediately before use. The active compound is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.

The treatments may include various "unit doses." Unit dose is defined as containing a predetermined quantity of the therapeutic composition calculated to produce the desired responses in association with its administration, e.g., the appropriate route and treatment regimen. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. Also of importance is the subject to be treated, in particular, the state of the subject and the protection desired. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.

The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one COVID-19 disease drug or related compounds or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

The compounds and compositions of the invention such as COVID-19 therapeutics can comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraventricularly, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, nasal or oral inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

The compounds and compositions of the invention such as COVID-19 therapeutics can be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like. Further in accordance with the present invention, the compounds and composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition can be combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.

In a specific embodiment of the present invention, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of pharmaceutical lipid vehicle compositions that include compounds or compositions of the invention such as COVID- 19 therapeutics, one or more lipids, and an aqueous solvent. As used herein, the term "lipid" will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term "lipid" is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.

One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the COVID- 19 therapeutics may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic and/or prophylactic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

In some embodiments of the present invention, the compounds and compositions of the invention, such as COVID-19 therapeutics are formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.

In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. See, e.g., U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792,451, each specifically incorporated herein by reference in its entirety. The tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small intestines, the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells. A syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

For oral administration the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally administered formulation. For example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively, the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.

Additional formulations that are suitable for other modes of alimentary administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, poly alkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.

In further embodiments, the compounds and compositions of the invention, such as COVID-19 therapeutics can be administered via a parenteral route. As used herein, the term "parenteral" includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, transdermally, intramuscularly, intraarterially, intraventricularly, intrathecally, subcutaneous, or intraperitoneally. See, e.g., U.S. Pat. Nos. 6,7537,514; 6,613,308; 5,466,468; 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).

In some embodiments, the therapeutic compound is administered intrathecally. In some embodiments, the compound is administered intrathecally via an implantable pump. In one embodiment, the implantable pump comprises a SynchroMed™ II pump that stores and delivers medication into the intrathecal space (Medtronic).

Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, dimethyl sulfoxide (DMSO), polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.

In other embodiments, the compounds and compositions of the invention may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder. Ointments include all oleaginous, adsorption, emulsion and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture. Transdermal administration of the present invention may also comprise the use of a "patch". For example, the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.

In certain embodiments, the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described, e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms. All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

EXAMPLES

EXAMPLE 1 - Human Defensins Inhibit SARS-CoV-2 Infection 1 by Blocking Viral Entry

To assess the effect of defensins on SARS-CoV-2 infection, pseudotyped luciferase virus particles expressing SARS-CoV-2 surface (spike, S) proteins were treated with or without defensins for 1 h before infection of HEK293T-hACE2 cells, as described in the Methods. Viral infection was determined by measuring the luciferase activity in cells at day three post-infection. Note that defensins were not added back after attachment. HNPs 1-3 blocked SARS-CoV-2 infection, achieving approximately 50% suppression at 1 pg/inL (290 nM) (Figure 1A-C). Because HNPs 1-3 differ by only a single (N-terminal) amino acid residue, it is not surprising that all three peptides had similar effects. HNP4, a low-abundance peptide in neutrophils and less known for its antiviral activity, was substantially less potent (Figure ID). Two intestinal defensins (HD5 and HD6) were also studied. HD5 exhibited significant anti-SARS-CoV-2 activity, achieving 60% suppression at 12.5 pg/mL (3.45 pM), whereas HD6 only blocked SARS-CoV-2 infection at the highest concentration tested (50 pg/mL, 13 pM) (Figure IE and IF). The antiviral activity of defensins was not associated with any observed cytotoxicity (not shown). Of note, the concentrations of HNPs 1-3 and HD5 employed in these experiments were within the physiological ranges of these defensins in neutrophils and in intestinal tissues and lumen (Ganz, Nat. Rev. Immunol. 2003, 3, 710-720; Ghosh et al., Nat. Immunol. 2002, 3, 583— 590). Unlike human a-defensins, [3-defensins HBD2, HBD5 and HBD6 did not effectively inhibit viral infection (Figure 2A-C). However, the 9-defensin analog RC101 exhibited a similar level of inhibition of SARS-CoV-2 infection (50% inhibition at 6.4 pg/mL, 3.4 pM) as HNPs 1-3 and HD5 (Figure 2D). The human a-defensins as well as RC101 largely maintained their antiviral activities in the presence of serum (Figure 2E and F).

We then determined whether defensins blocked replication-competent SARS-CoV- 2 viruses. Replication-competent SARS-CoV-2 viruses expressing mNeonGreen were incubated with HNP1, HD5 and RC101 at different concentrations for 1 h before infection of Vero E6 cells. In agreement with results from pseudotyped SARS-CoV-2 viruses (Figure 1), HNP1, HD5 and RC101 suppressed viral infection in a dose-dependent manner (Figure 3).

SARS-CoV-2 variants, including P.l and B.1.1.7 from Brazil and the United Kingdom, respectively, are highly transmissible and increase the risk of death (Burki et al., Lancet 2021, 397, 462; Faria et al., Science 2021, 372, 815-821; Volz et al., Cell 2021, 184, 64-75. el l; Washington et al., Cell 2021, 184, 2587-2594.e7). We determined the effect of defensins on pseudotyped viruses expressing spike proteins from the P.l and B.1.1.7 variants. HNP1, HD5 and RC101 suppressed the P.l variant in a dose-dependent manner (Figure 4A). HNP1, HD5 and RC101 exhibited moderate antiviral activity against the B.1.1.7 variant (Figure 4B). HNP1 and HD5 at 50 pg/mL, which inhibited the earlier Wuhan strain by 96% (Figure 1), suppressed Pl infection by 67% and 72%, respectively, suggesting that the emerged variant was more resistant to host antimicrobial peptides. Similarly, HNP1 and HD5 at 50 pg/mL inhibited B.l.1.7 infection by 58% and 32%, respectively. Although RC101 suppressed infection by both variants, it was also less potent against the B.l.1.7 variant (Figure 4).

Defensins Inhibit Viral Entry

We determined whether pre-treatment of cells with defensins provided protection against the virus. Cells were treated with defensins for 1 h, washed and then infected with pseudotyped SARS-CoV-2 virus. Pretreatment of cells with high concentrations of HNP1, HD5 and RC-101 partially blocked viral infection (Figure 5A). To determine whether defensins exhibited an effect after viral entry, cells were infected with pseudotyped SARS- CoV-2 viruses for 1.5 h and then treated with HNP1, HD5 or RC101 for three days. None of the tested defensins affected infection after viral entry (Figure 5B). These results suggested that defensins block SARS-CoV-2 infection at the step of viral entry.

The step of viral entry includes viral attachment and the binding of spike proteins through the RBD to hACE2, followed by fusion between the viral and cellular membranes. To assess the effect of HNP1 on viral attachment, pseudotyped SARS-CoV-2 viruses with or without HNP1 pretreatment were incubated with HeLa-hACE2 cells at 4°C for 2 h. After washing to remove unbound viruses, cells were lysed, and the level of cell associated HIVp24 was determined. HNP1 did not have a significant impact on viral attachment except that the high concentration (50 |lg/mL) of HNP1 suppressed viral attachment by 35% (Figure 6A). We did not find that HNP1 interfered with the interaction between spike RBD and hACE2 proteins (Figure 6B). In contrast, HNP1 at high concentrations (16.7 and 50 |lg/mL) slightly promoted the interaction between hACE2 and spike RBD proteins. We then assessed the effect of HNP1 on viral fusion. Pseudotyped viruses expressing SARS- CoV-2 spike D614G proteins (in the UK B.1.1.7 variant) were packaged with BlaM-Vpr. Virus binding to cells was augmented by low-speed centrifugation at 16°C. After washing off unbound viruses, cells were incubated in medium with or without defensins at 37°C to allow viral fusion to occur. In the absence of defensins, [3-lactamase, released into the cytoplasm from the viruses containing BlaM-Vpr proteins, cleaved the CCF4-AM substrate (Figure 6C, left panel), resulting in an increase in fluorescent signals at 460 nm. We found that HNP1 blocked viral fusion in a dose-dependent manner (Figure 6C, right panel).

Native Structure (Disulfide Bonding) of Defensins Is Required to Inhibit SARS- CoV-2 Infection

The structure of defensins is critical for their effects on viruses (Klotman et al., J. Immunol. 2008, 180, 6176-6185; Smith et al., PLoS Pathog. 2010, 6, el000959). To determine whether this was true of SARS-CoV-2 as well, we examined the anti-SARS- CoV-2 activity of [Abu]HNPl and [Abu]HD5, which are identically charged, but unstructured analogs of HNP1 and HD5 (Wu et al., J. Pept. Res. 2004, 64, 118-125). We treated pseudotyped SARS-CoV-2 virus with these linear defensins before adding the virus to cells and then determined infection at day three post-infection. In contrast to the antiviral activity of HNP1 or HD5 (Figure 1), the linear analogs [Abu]HNPl and [Abu]HD5 did not have an inhibitory effect on SARS-CoV-2 infection (Figure 7A). These results indicate that the antiviral effect of defensins requires native disulfide bonding and properly folded proteins. HD5 is synthesized as a propeptide (ProHD5) in Paneth cells in the small intestine and in epithelial cells of the genital tract, and is processed to the mature peptide by trypsin or neutrophil proteases (Ghosh et al., Nat. Immunol. 2002, 3, 583-590; Porter et al., Infect. Immun. 2005, 73, 4823-4833). The unprocessed pro-HD5 exhibited no anti-SARS-CoV-2 activity (Figure 7B), which is in agreement with prior studies reporting reduced or absent antimicrobial activity of proHD5 compared to processed HD5 (Ghosh et al., Nat. Immunol. 2002, 3, 583-590; Porter et al., Infect. Immun. 2005, 73, 4823-4833).

The effect of virus titers and cell types on defensin-mediated viral infection

We have previously shown that the direct effect of HNP1 on HIV virions was abolished by an increase in the number of virus particles (Chang et al. , J. Clin. Invest. 2005, 775, 765-773). To determine whether increasing the virus titer affected the antiviral activity of HNP1, different titers of serum-free pseudotyped SARS-CoV-2 were incubated with 1 or 25 g/mL HNP1 at 37°C for 1 h and then added to cells. Viral infection was determined at day three post- infection. At 25 pg/mL, HNP1 inhibited viral infection regardless of the virus titer. At 1 pg/mL, however, HNP1 displayed an inhibitory effect on low concentrations of the virus, but this effect was lost when the viral titer was high (Figure 8A). This result suggests that HNP1 interacts directly with the virus, and is depleted at high viral concentrations (Figure 8A). Finally, we examined whether the SARS-CoV-2 inhibitory effect of defensins was cell-type-dependent. Intestinal epithelial cells (Caco-2) express moderate levels of endogenous hACE2, and lung epithelial cells (A549) have near undetectable levels of hACE2 but a high abundance of SARS-CoV-2 alternative receptors, CD147 and tyro sine-protein kinase receptor UFO (AXL) (Xu etal., Viruses 2021, 13, 953). We found that HNP1, HD5 and RC101 exhibited anti-SARS-CoV-2 activity in both Caco- 2 and A549 cells (Figure 8B), indicating that the inhibitory effect of defensins on viral infection was not receptor- specific or cell-type-dependent.

Discussion The ability of the innate immune response to reduce viral replication is known to impact the severity of coronavirus-associated disease outcomes. Defensins are highly abundant antimicrobial peptides produced by neutrophils and epithelial cells, and play a critical role in innate immunity. Here, we showed that human oc-defensins including HNPsl-3 and HD5 as well as 0-defensin analog RC-101 suppressed SARS-CoV-2 infection at the step of viral entry. HNP1, HD5 and RC101 also blocked infection by the Brazil P.l and the UK B.l.1.7 variants. Linear unstructured defensins did not inhibit viral infection. Furthermore, the antiviral activity of defensins was also found for infections of epithelial cells which did not overexpress hACE2.

Our results showed that the anti- SARS- activity of defensins was specific for defensin subtypes. HBDs are generally more cationic and less hydrophobic than human a- defensins. Differential anti-SARS-CoV-2 activities among HBDs and lectin-like HNPsl- 3, HD5 and RC101 suggest that hydrophobic binding properties of these defensins may play a critical role in their viral inhibition. ProHD5 exhibits reduced lectin-like properties, which is consistent with the fact that the pro-region functionally inhibits defensin activity, and presumably contributes to the loss of antiviral activity (Ghosh et al., Nat. Immunol. 2002, 3, 583-590). We also found differential anti-SARS-CoV-2 activities between HD5 and HD6 (Figure 1), which is not surprising as these two intestinal defensins exhibit distinct structures and properties, particularly in the case of glycan-mediated viral attachment (Lehrer et al., Immunol. Rev. 2012, 245, 84-112; Rapista et al., Retrovirology 2011, 8, 45).

Detailed mechanisms of defensin-mediated inhibition of SARS-CoV-2 fusion remain to be determined. Analyses of specific steps during viral entry revealed that HNP1 had a weak inhibitory effect on viral attachment and no effect on the binding of the spike RBD domain to hACE2, but significantly inhibited SARS-CoV-2 spike protein-mediated viral fusion. Defensins have also been shown to block viral fusion of HIV (Ding et al., J. Innate Immun. 2009, 1, 413-420; Xu et al., Front. Immunol. 2020, 11, 764), but specific mechanisms of action were not reported. Defensins may alter protein domains of spike proteins important for viral fusion (Huang et al., Acta Pharmacol. Sin. 2020, 41, 1141— 1149). Additionally, these peptides may suppress viral entry by aggregating virions or by affecting the lipid bilayer structure of the viral membrane (Dugan et al., J. Biol. Chem. 2008, 283, 31125-31132). Several host factors including TMPRSS2 and TMPRSS4, neutrophilin- 1 and heparan sulfate interact with spike proteins and promote SARS-CoV-2 infection (Hoffmann et al., Cell 2020, 181, 271-280 e8; Cantuti-Castelvetri et al., Science 2020, 370, 856; Zang et al., Sci. Immunol. 2020, 5, eabc3582; Daly et al., Science 2020, 370, 861; Clausen et al., Cell 2020, 183, 1043-1057.el5; Zamorano Cuervo et al., Elife 2020, 9 , e61390). Thus, defensins may inhibit viral entry by acting on these host factors. For example, serine protease TMPRSS2 has been shown to prime SARS-CoV-2 proteins for hACE2-dependent viral entry (Hoffmann etal., Cell 2020, 181, 271-280 e8). Since RC101 is known to inhibit Japanese encephalitis virus protease, investigations into the effects of defensins on serine protease are warranted (Rothan et al., BMC Infect. Dis. 2012, 12, 314). Additionally, alternative receptors including CD147 and AXL for SARS-CoV-2 viral entry have been reported, particularly in cells with a low abundance of hACE2 (Xu et al. , Viruses 2021, 13, 953; Wang et al., Signal Transduct. Target. Ther. 2020, 5, 283; Wang et al., Cell Res. 2021, 31, 126-140). We have shown that pseudotyped viruses expressing SARS-CoV- 2 spike proteins enter A549 cells through CD 147 in a spike RBD-independent manner (Xu et al., Viruses 2021, 13, 953). Similarly, AXL mediates SARS-CoV-2 infection of lung epithelial cells through interactions with the N-terminal domain of SARS-CoV-2 spike proteins (Wang et al., Cell Res. 2021, 31, 126-140). We found that HNP1 did not impact the binding of RBD to hACE2 but suppressed pseudotyped SARS-CoV-2 infection of A549 cells, suggesting that defensins may interfere with the binding of spike proteins to alternative receptors such as CD 147.

Defensins are known for exhibiting both pro-inflammatory and anti-inflammatory activities, and they can suppress viral infection by modulating immune cell activities (Klotman et al., PLoS Pathog. 2017, 73, el 006446; Fruitwala et al., Semin Cell Dev. Biol. 2019, 88, 163-172). Therefore, although HBDs did not block viral entry, these peptides may inhibit viruses by promoting host restriction factors in specific cell types. Studies on the role of defensins in the regulation of viral infection through immune modulation using innate immune cells will offer insights into immune-modulatory functions of defensins relevant to SARS-CoV-2 infection and disease outcomes.

Defensins represent one category of antimicrobial peptides (Guam-Guerra et al., Clin. Immunol. 2010, 135, 1-11). Antimicrobial peptides are evolutionarily conserved molecules for defense, which are produced by bacteria, insects, plants and vertebrates (Guam-Guerra et al., Clin. Immunol. 2010, 135, 1-11; Lehrer et al., Curr. Opin. Immunol. 1999, 11, 23-27). More than 3000 antimicrobial peptides have been reported (Huan et al., Front. Microbiol. 2020, 11, 582779). Considering their versatile properties including antimicrobial activities and immune functions, the development of natural or synthetic antimicrobial peptides against SARS-CoV-2 and other viruses may help combat emerging pathogens in the future.

Our findings have clinical relevance. Individuals differ in their defensin repertoire, which may influence disease outcomes in response to SARS-CoV-2 infection (Machado et al., Front. Immunol. 2015, 6, 115; Hollox etal., Genome Res. 2008, 18, 1686-1697; Lehrer et al., Curr. Opin. Hematol. 2007, 14, 16-21; Taudien et al., BMC Genom 2004, 5, 92). Older adults and people with hypertension have dysfunctional neutrophils, and they have higher mortality in response to SARS-CoV-2 infection (Butcher et al., Immunology 2000, 100, 411-416; Wenisch etal., J. Leukoc. Biol .2000, 67, 40-45; Obama et al., Hypertension 2014, 63, 1171-1172; Liu et al., Am. J. Hypertens. 2015, 28, 1339-1346; Li et al., Clin. Res. Cardiol. 2020, 109, 531-538; Yang et al., Int. J. Infect. Dis. 2020, 127, 104371; Wu et al., JAMA 2020, 323, 1239-1242; Xie et al., Jama Netw Open 2020, 3, e205619). It is possible that in these high-risk groups, defensin levels are insufficient to block virus replication during acute infection and are insufficient to control inflammation mediated by phagocytes in the aftermath of infection, which may provide a partial explanation for severe disease outcomes in these groups. Furthermore, HNPs form stable complexes with lipoprotein (a) and low-density lipoproteins, which are elevated in people with hypertension (Bdeir et al., Blood 1999, 94, 2007-2019). Thus, studies on the effects of lipoproteins on HNP-mediated SARS-CoV-2 inhibition will likely offer insights into COVID-19 disease pathogenesis.

In summary, our findings provide insights into the function of human oc-defensins in innate immunity against SARS-CoV-2 infection. Our findings show that oc-defensins and their analog RC101 are inhibitors of SARS-CoV-2 infection. Understanding the mechanisms by which defensins and their analogs block SARS-CoV-2 infection offers new therapeutic avenues.

Materials and Methods Reagents, Plasmids, and Cell Lines

Defensins and their analogs were chemically synthesized, folded and verified as described previously (Wu et al., J. Pept. Res. 2004, 64, 118-125). To construct linear unstructured analogs of HNP1 and HD5, [Abu]HNPl and [Abu]HD5, the six cysteine residues were replaced by isosteric a- aminobutyric acid (Abu). Recombinant HD5 propeptides (aa 20-94) were synthesized using a baculovirus/insect cell culture system (Porter et al., Infect. Immun. 1997, 65, 2389-2395). Recombinant SARS-CoV-2 spike receptor-binding domain (RBD) and hACE2 proteins, expressed in HEK293T cells, were purchased from RayBiotech (Peachtree Comers, GA, USA). The CCF4-AM P-lactamase substrate (GeneBLAzer in vivo detection kit) was purchased from Invitrogen (Carlsbad, CA, USA).

The construct for full-length SARS-CoV-2-Wuhan-Hu-l surface (spike) (GenBank accession number QHD43416) was codon-optimized for humans and synthesized with Kozak-START GCCACC ATG and STOP codons and with 5' Nhel/3'Apal sites for subcloning into the pcDNA3.1(+) vector (Thermo Fisher Scientific, Waltham, MA, USA) (Wu et al., Nature 2020, 579, 265-269). Plasmids encoding spike proteins of B.1.1.7 and Pl variants were kindly provided by Dennis Burton (The Scripps Research Institute, La Jolla, CA, USA). The packaging HIV-1 pR9AEnv (from Chris Aiken at Vanderbilt University), pMM310 expressing P-lactamase-Vpr chimera (BlaM-Vpr, provided by Michael Miller at Merck Research Laboratories and now available from NIH AIDS Research and Reference Program), pMDG-VSV-G plasmid expressing VSV-G (from J. Young at Roche Applied Science, Mannheim, Germany), psPAX2 lentiviral packaging vector and pcRev vector have been described previously (Sood et al., J. Biol. Chem. 2017, 292, 6014-6026). pCAGGS- SARS-CoV-2 S D614G (cat. #156421) and pWPLIRES-Puro- Ak-ACE2-TMPRSS2 (cat. #154987) expression vectors were obtained from Addgene (Watertown, MA, USA).

HEK293T/17, Vero E6, Caco-2 and A549 cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). HEK293T-hACE2 cells and HeLa-hACE2 cells were kindly provided by Hyeryun Choe (The Scripps Research Institute, Jupiter, Florida, USA) [26] and by Dennis Burton, respectively (Rogers et al., Science 2020, 369, 956-963; Moore et al., J. Virol. 2004, 78, 10628-10635). Huh7.5 was obtained from Dr. Charles Rice (Rockefeller University) through Apath LLC (Brooklyn, NY, USA). To generate Huh7.5-ACE2-TMPRSS2 cells for viral fusion assay, Huh7.5 cells grown at 60% confluency in six- well plates were transduced with single-cycle infectious VSV-G pseudotyped lentiviral viruses (0.5 ng p24/well) encoding for ACE2-TMPRSS2 by centrifugation at 16°C for 30 min at 1550 xg. After 24 h transduction, cells were transferred in a 10-cm tissue culture dish in the presence of 2 pg/mL puromycin.

Cell Culture

HEK 293T, HEK293T-hACE2, HeLa-hACE2, Caco-2 and A549 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat- inactivated fetal bovine serum. For HEK293T/17 cells, the growth medium was supplemented with 0.5 mg/mL of G418 sulfate (Mediatech, Inc). Huh7.5-hACE2- TMPRSS2 cells were cultured in complete media with 2 g/mL puromycin. Production of Pseudotyped Viruses

Replication-defective HIV-1 luciferase-expressing reporter viruses pseudotyped with SARS-CoV-2 S proteins were produced by co-transfection of a plasmid encoding the envelope-deficient HIV-1 NL4-3 virus with the luciferase reporter gene (pNL4-3.Luc.R- E- or pNL4-3.Luc.R+E- from N. Landau, New York University) and a pcDNA3.1 plasmid expressing the SARS-CoV-2 glycoprotein into HEK 293T cells, which were seeded at 6- 7xl0 ⁶ in a 10-cm dish and cultured overnight, using Lipofectamine 3000 (Thermo Fisher Scientific) as described previously (Xu et al., Viruses 2021, 13, 953). The supernatant was collected 48 h after transfection and filtered. Virus stocks were analyzed for HIV-1 p24 antigen by the AlphaLISA HIV p24 kit (PerkinElmer). Because serum is known to reduce the effect of defensins on the virion, viruses were produced under serum-free conditions, or for viruses generated in 10% FBS, these were diluted in serum-free medium (to a final FBS concentration of 3%) (Daher et al., J. Virol. 1986, 60, 1068-1074; Chang et al., J. Clin. Invest. 2005, 775, 765-773). To produce serum-free SARS-CoV-2 pseudotyped viruses, the culture media were replaced by DMEM without serum at 24 h after transfection, and the cells cultured for an additional 24 h prior to collecting viruses. Virus stocks contained approximately 200 ng/mL of HIV p24 proteins.

For infection assays, cells plated at 2xl0 ⁴ or 5xl0 ⁴ cells/well in a 96-well or 48- well plate, respectively, were cultured overnight. Pseudotyped SARS-CoV-2 luciferase reporter viruses were incubated with defensins at 37°C for 1 h. The defensin-virus mixture was then added to cells. After 1-2 h viral attachment, infected cells were cultured in media with 10% FBS for 48-72 h before measuring luciferase activity using Luciferase Substrate Buffer (Promega Inc). Luciferase activity (relative light units; RLU.) reflecting viral infection was measured on a 2300 EnSpire Multilabel Plate Reader (PerkinElmer, Waltham, MA). Results obtained using different titers of viruses are reported as the average percentage of the control calculated using the formula: (RLU of treated cells / RLUs of untreated cells) x 100.

To generate BlaM-Vpr harboring pseudotyped SARS-CoV-2 viruses for the viral fusion assay, HEK293T/17 cells grown at 75% confluency in a 10-cm dish were transfected with pCAGGS-SARS-CoV-2 S D614G (4 pg), pR9AEnv (4 pg), BlaM-Vpr (2 pg) and pcRev (0.5 pg) using the JetPRIME transfection reagent (Polyplus-transfection, Illkirch- Graffenstaden, France). Transfected cells were incubated for 14h at 37°C and then cultured in a fresh growth medium for an additional 36 h. The viral supernatants were filtered through 0.45 pm polyethersulfone filters (VWR, Radnor, PA, USA) and concentrated 5x using Lenti-X™ Concentrator (Clontech, Mountain View, CA, USA). To produce the transducing VSV-G pseudotyped viruses for generating Huh7.5-hACE2-TMPRSS2 cells, HEK293T/17 cells were transfected with pMDG-VSV-G (1.5 pg), psPAX2 (3 pg) and ACE2-TMPRSS2 expression vectors (4 pg). Viruses were prepared as described above. Replication-Competent Virus Infection

Replication-competent SARS-CoV-2 viruses expressing mNeonGreen, kindly provided by Pei- Yong Shi at the University of Texas Medical Branch, Galveston, TX, USA, were propagated in Vero E6 cells as described previously (Xie et al., Cell Host Microbe 2020, 27, 841-848 e3). Experiments were performed in a biosafety level 3 laboratory with personal protection equipment including powered air-purifying respirators (Breathe Easy, 3M), Tyvek suits, aprons, sleeves, booties and double gloves. Virus titers were determined by plaque assays in Vero E6 cells as described previously (Xu et al., Pathogens 2021, 10, 272). For the infection assay, Vero E6 cells at 1.5 xl0 ⁴ cells/well in a black 96-well glass plate (Greiner, Monroe, NC, USA) were incubated overnight. Cells were exposed to viruses (30 ml) with or without defensin treatment at a multiplicity of infection (MOI) of five for 1 h followed by the addition of 100 pL FluoroBrite medium containing 2% FBS. Fluorescence from productive viral infections was monitored at 48 h after infection using a Biotek Cytation 5 multi-mode plate reader.

Viral Attachment Assay

HeLa-hACE2 cells seeded at 5xl0 ⁴ per well in 48-well plates were cultured overnight. Pseudotyped viruses were pretreated with or without defensins at 37°C for 1 h. Cells were incubated with viruses at 4°C for 2 h, washed with cold PBS three times, and lysed with 100 ml of 1% Triton X-100. Cell- associated HIV p24 was determined by AlphaLISA HIV p24 kit (PerkinElmer).

SARS-CoV-2 RBD and hACE Binding Assay

Greiner MicroIon 200 plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with recombinant SARS-CoV-2 RBD proteins at 50 ng/well in 0.1 M bicarbonate buffer pH 9.6 at 4°C for 24 h. The plates were blocked with 2% bovine serum albumin in PBS (w/ v) for 30 min at 37°C, washed with PBS, and incubated with biotinylated hACE2 proteins (0.1 mg/mL) that were pre-treated with defensins at 37°C for 1 h. After washing, the bound hACE2 proteins were detected by incubating with alkaline phosphatase- conjugated streptavidin at 37°C for 1 h. Plates were washed and the binding of hACE2 proteins to SARS-CoV-2 RBD was detected by adding 1 mg/mL of r-nitrophenol in DEA buffer at pH 9.8, and measuring the resulting signal at 405 nm.

Virus-Cell Fusion Assay

The virus-cell fusion assay was performed as described previously (de la Vega et al., Retrovirology 2011, 8, 99). Briefly, pseudotyped SARS-CoV-2 viruses containing a 0- lactamase-Vpr chimera (BlaM-Vpr) were diluted in the medium without FBS (0.5 ng p24/well), bound to target cells by centrifugation at 16°C for 30 min at 1550 xg. Unbound viruses were removed and a growth medium containing or lacking defensins was added. Virus-cell fusion was initiated by incubating the samples at 37°C for 2 h. Cells were then loaded with the CCF4-AM fluorescent substrate and incubated overnight at 11°C. The cytoplasmic BlaM activity (ratio of blue to green fluorescence) was measured using the SpectraMaxi3 fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA). Cytotoxicity Assay

HEK 293T-hACE2 cells were plated in 96-well plates at 5000 cells per well and then treated with various concentrations of defensins for 24 h. Cell viability was analyzed using MTS-based CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA).

Statistical Analysis

Statistical comparisons were performed using a two-tailed Independent-Samples t- test; p < 0.05 was considered significant. Prism 8 (GraphPad Software, LLC, San Diego, CA, USA) was used for these analyses.

EXAMPLE 2 - Determining the effect of defensins on SARS-CoV-2 suppression and immune modulation in Syrian hamster models.

Animal models including mice, hamsters, ferrets, and non-human primates have been used for SARS and SARS-CoV-2 infection (Rapista et al., Retro virology, 2011 8(1):45; Roberts et al., Virus Res, 2008, 133(l):20-32; Takayama et al., Trends Pharmacol Sci, 2020 41(8):513-517). Hamsters are an excellent model animal for SARS-CoV-2 infection studies as they can be infected by SARS-CoV-2 isolates from humans, can transmit viruses to other hamsters, and they exhibit a consist viral load in the respiratory tract, virus-dose-dependent lung pathology and weight loss, and age-dependent recovery (Chan et al., Clin Infect Dis. 2020; Imai et al., Proc Natl Acad Sci U S A, 2020, 117(28):16587-16595; Sia et al., Nature, 2020, 583(7818):834-838). Unlike mice, hamsters have neutrophil defensins with conserved amino acid residues (Mak et al., Comp Biochem Physiol B Biochem Mol Biol, 1996, 115(1): 135-141).

The effect of HNP1, HD5, and RC101 on viral load, immune and pathological response, and disease outcomes in SARS-CoV2-infected animals can be determined as described below. HBDs can also be included in such in vivo studies.

Determining the effect of HaNPs on SARS-CoV-2 infection.

Similar to humans, hamsters have 4 neutrophil peptides. We can determine the anti- SARS-CoV-2 activity of HaNPl, 2, 3, 4 in vitro. We can synthesize and fold the peptides and linearized analogs in which the six cysteine residues are replaced by isosteric a- aminobutyric acid (Abu) (Wu et al., J Pept Res, 2004, 64(3): 118- 125). HaNPs are incubated with pseudotyped SARS-CoV-2 viruses followed by infection of HeLa-hACE2 cells and BHK cells (a hamster cell line not overexpressing hACE2), which are susceptible to pseudotyped SARS-CoV-2 infection (Fig. 8). The effect of HaNPs on SARS-CoV-2 viruses can be examined using replication competent viruses with or without GFP which will be monitored by GFP fluorescence or plaque assay, respectively (Xie et al., Cell Host Microbe, 2020, 27(5):841-848 e843).

Determining the kinetics of defensin and immune profiles and disease outcomes in Syrian hamsters after intranasal challenge with high or low dose SARS-CoV-2.

We can first define defensin levels, immune response, and disease outcomes in adult hamsters in response to high and low dose SARS-CoV-2 (Table 1) as described in Imai et al using female animals (Imai et al., Proc Natl Acad Sci U S A, 2020, 117(28): 16587-16595). Here, both male and female animals are used. Golden Syrian hamsters (Mesocricetus auratus) can be purchased from Envigo (formerly Covance) and allowed to acclimatize for a week before infection at the BSL3 animal facility. Adult animals (5 months old) are infected intranasally with the SARS-CoV-2 USA WA1/2020 isolate at 10 ³ PFU (low infectious dose) or 10 ⁶ PFU (high infectious dose) (Table 1). Six animals per group per time points are used, based on published data and based on statistical power calculations from our previous studies in rabbits, which, as a similarly out-bred species are expected to produce similar variability (Chan et al., Clin Infect Dis. 2020; Imai et al., Proc Natl Acad Sci U S A, 2020, 117(28): 16587- 16595; Sia et al., Nature, 2020, 583(7818):834-838). Animals are handled as per the Animal Welfare Act of AALAC and per the Rutgers IACUC norms.

Group A: female; B: male

Table 1. Animal numbers to be used to assess defensin levels and immune response for each dose of SARS-CoV-2. Hamsters are infected intranasally with SARS-CoV-2 at 10 ³ PFU or 10 ⁶ PFU. The immune profiles in blood and tissues are assessed at indicated time points after treatment (Day 0 before infection).

Methods

Animal health examination: Physiological measurements, including breathing rate, activity on spin-wheels, body weight and temperature, food and water intake, fecal/urine output, general well-being of SARS-CoV-2 infected and control animals are measured and recorded daily from the day of infection until the experimental end point.

Sample collection: Blood, nasal swabs, nasal turbinate, trachea, lungs, lymph nodes, liver, spleen, kidney, intestine, genital tissues, brain, and feces are collected at time points as indicated (Table 2). Tissue samples are divided into two parts: fixed tissues for immunohistochemistry (IHC), and frozen tissues for plaque assay and RNAseq.

Table 2. Schematic representation of exemplary experimental design for hamster

Viral load: Virus titers in nasal swabs and blood are analyzed by RT-PCR. Virus titers in nasal turbinate and tissues including lungs and gut tissues (jejunum, ileum, and colon) are determined by RT-PCR and by plaque assays in Vero cells.

Histopathology and IHC analysis: Tissue sections are stained with H&E (pathology), Trichrome (fibrosis), and immunohistochemistry to determine compositions and locations of immune cells including lymphocytes (CD3, CD4, CD8), monocytes/macrophages (CD 14, CD68), and neutrophils (myeloperoxidase, MPO).

Defensins, immune mediators, Abs in blood and tissues: Abs can be generated against HaNPs (Lampire Biological Laboratories, Inc) to determine the levels of HaNPs in serum and tissues. The levels of cytokines involved in the cytokine storm during CO VID- 19 in humans can also be determined. IL-6, TNFa, MCP-1, IFNy, IP-10, MIP-la, IL-10, IL- 12, IL- 17, IL-2, IL-4, G-CSF in blood and lung tissue homogenates are determined by specific ELISA kits (ELISAGenie; G-BioSciences; Abeam). Mucosal IgA and IgG as well as IgG in blood are determined by ELISA. Anti-SARS-CoV-2 spike and nucleoprotein in sera are determined by ELISA. The neutralization ability of sera against SARS-CoV-2 virus are determined. The level of IFNa/b are determined. We can also assess interferon- inducible genes (ISGs) including Mxl, OAS1, ISG14, and IFIT1 by RT-PCR as markers for a type I IFN response. We can assess IFNI, a type III IFN, and IFNe, a unique mucosal type I IFN, which protects against viral infection (Fung et al., Science, 2013, 339(6123): 1088-1092; Park et al., Nat Methods, 2008, 5(10):877-879). IFNe is induced by TNFa and may contribute to SARS-CoV-2 pathogenesis (Couret et al., Cell Biosci, 2017, 7:57). Due to the lack of Abs against these proteins in hamsters, we can use single-molecule fluorescence in situ hybridization (smFISH) developed by Dr. Sanjay Tyagi (Raj et al. 2008).

Global transcriptome analysis. We can assess global transcriptomes of lung and gut tissues of hamsters by RNA-seq analysis on an Illumina HiSeq 3000 (performed at the Genomics Core Facility at Rutgers, NJMS). Samples pre- and post-infection at selected timepoints in each group based on viral loads, immune profiles, and disease outcomes can be analyzed. Immune networks can be analyzed as we have described to identify alterations in genes/pathways in response to SARS-CoV-2 infection (Tasker et al., JCI Insight, 2016, l(20):e88255). bioinformatics analyses to identify immune attributes/pathways associated with disease severity can be conducted, after which selected genes can be validated by quantitative RT-PCR analysis.

Determining the effect of human defensins on SARS-CoV-2 infection and immune modulation.

Rhesus theta defensin (RTD-1), which does not affect viral replication, prevents death in a mouse model of SARS coronavirus pulmonary disease by modulating the immune response (Wohlford-Lenane etal., J Virol, 2009, 83(21): 11385-11390). Similarly, HNP1 has been shown to protect mice against Klebsiella pneumoniae and Staphylococcus aureus by promoting infiltration of leucocytes (Welling et al., J Clin Invest, 1998, 102(8): 1583-1590). These studies support the feasibility of studying defensins in small animal models. The effect of HNP1, HD5, and RC101 on protecting animals against SARS-CoV-2 infection can be tested by known methods and as described herein. We can also test HBD2 and HBD3 to address their abilities of modulating SARS-CoV-2 infection by immune modulation. Adult hamsters can be used for the initial study, and aged animals can be used to address the role of defensins in high-risk groups. Defensins can be administrated 15 min prior to intranasal infection with replication competent SARS-CoV- 2 (10 ³ PFUs) as described by Wohlford-Lenane et al (Wohlford-Lenane et al., J Virol, 2009, 83(21): 11385-11390). A second dose of defensins at day 1 post-infection can be administered as suggested (Wohlford-Lenane et al., J Virol, 2009, 83(21): 11385-11390). Physiological characteristics, body weights and other symptoms can be recorded daily, and blood, nasal swabs, nasal turbinate, and fecal samples can be collected daily. Tissue samples for IHC/smFISH or RNAseq can be collected at different time points (Table 2) as previously described. Viral loads in nasal samples and blood can be determined by RT- PCR. Infectious virus particles in lung tissue homogenates can be assessed by plaque assays. The levels of immune mediators in blood (serum) and lung tissues can be determined by ELISA or RT-PCR (tissues). Tissue pathology and immune cell profiles in tissues by H&E and IHC can be assessed as previously described. Transcriptome analysis can be performed to gain insights into the immune pathways important for defensin- mediated regulation of SARS-CoV-2 infection in vivo as previously described.

To determine whether defensins can control viral infection and reduce disease symptoms in infected animals by modulating immune responses, hamsters are infected intranasally with SARS-CoV-2 (10 ³ PFUs). HNP1, HD5, and/or RC101 are then administered i.p. 24 h or 3 days post-infection as described (Welling et al., J Clin Invest, 1998, 102(8): 1583- 1590). Viral loads, immune response, and disease outcomes are assessed as previously described.

As described herein, these defensins should block virus replication in vivo, modulate immune responses, and/or reduce severity of disease. While not wishing to be bound by theory, it is possible that early treatment (day 1 post-infection) will have a better outcome than later treatment (e.g., day 3 post-infection).

While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.