Pharmaceutical compositions for the treatment of viral infections

Abstract

The present disclosure is directed to a pharmaceutical composition for use in the prevention and / or treatment of viral infection and / or disorders associated with viral infection caused by an enveloped virus, comprising: a) a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, representing peptide PLNC8 β, and / or b) a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2, representing peptide PLNC8 α.

Description

[Technical field]

[0001] The present disclosure relates to the prevention or treatment of viral infections and is directed to pharmaceutical compositions for use in the prevention or treatment of viral infections and disorders associated with viral infections caused by enveloped viruses, the pharmaceutical compositions comprising a peptide having at least 90% sequence identity to the amino acid sequence of the peptide plantaricin NC8 (PLNC8) β and / or a peptide having at least 90% sequence identity to the amino acid sequence of the peptide PLNC8 α. [Background technology]

[0002] Humans are constantly exposed to viruses, and the outcome of a viral infection depends on many factors, such as the type of virus and its virulent properties, the route of infection, and the immunological and inflammatory state and response of the host. The prognosis of infection can be fatal if the host is immunocompromised. 1 Only a few antiviral drugs exist, which are too expensive for widespread use, and due to the complexity of viral pathogenesis, treatment of viral infections today often involves reducing symptoms by targeting the host's inflammatory response. 2,3 Newly emerging viral pathogens, including SARS-CoV-2, are proliferating rapidly, highlighting our limited therapeutic resources for viral infections. No drugs have been approved to specifically inhibit SARS-CoV-2 infection and thereby treat COVID-19, which has resulted in the complete paralysis of many countries in recent years due to lockdowns to limit the transmission of the virus and manage the recovery of infected patients. 4,5 Thus, specific drugs against SARS-CoV-2 are still lacking, although several candidates are in preclinical / clinical trials. The majority of approved antiviral drugs are used to treat the human immunodeficiency virus (HIV) by primarily targeting nucleoside and non-nucleoside reverse transcriptases, viral protease activity, and viral entry. 6,7The development of novel antiviral compounds against multiple classes of viruses, as opposed to single pathogen therapeutics, would be more advantageous in treating a broad range of viral infections. 8,9 In addition, virions can be transformed, for example, by amphipathic peptides. 8,10 The development of directly destroying antiviral substances would ultimately increase efficacy by rapidly eliminating viruses and their subsequent pathogenicity, thereby limiting their dissemination to other organs and transmission between individuals. This is particularly interesting considering that vaccine development is a costly and time-consuming process and must be regularly updated, as RNA viruses can mutate and render vaccines ineffective. In addition, secondary bacterial infections associated with viral infections stand out as a serious threat to human health, with Staphylococcus aureus identified as one of the most common pathogens. 11,12 Therefore, potent broad-spectrum antiviral drugs are urgently needed to combat newly emerging viral pathogens, including respiratory viruses such as influenza viruses and coronaviruses.

[0003] Bacteriocins are small antimicrobial peptides produced by bacteria, including lactobacilli, that kill microorganisms, usually by membrane disruption. 13,14 Their high potency, broad-spectrum antibacterial activity, and beneficial effects on human tissues make bacteriocins attractive candidates for therapeutic applications in traditional medicine against bacterial infections. Plantaricin NC8 (PLNC8)αβ is a two-peptide bacteriocin expressed by a Lactobacillus plantarum strain. We previously reported that PLNC8αβ effectively inhibits and kills several bacterial pathogens, including Staphylococcus spp., and significantly enhances the efficacy of antibiotics. 15PLNC8αβ consists of an amphipathic peptide with a positive net charge that exhibits high affinity for negatively charged pathogen membrane structures, including phosphatidylglycerol-rich bacterial membranes. This interaction leads to rapid disruption of bacterial membrane integrity, permeabilization, and loss of homeostasis, ultimately killing the bacteria. Anionic phospholipids in eukaryotic cell membranes are oriented toward the cytoplasmic leaflet, an active process controlled by a group of enzymes named flippases (ATPases). Furthermore, the high cholesterol content (about 40%) contributes to membrane stability and fluidity, preventing membrane permeabilization by PLNC8αβ and other similar peptides. Indeed, the inventors have previously shown that PLNC8αβ does not exhibit cytotoxicity to human cells, but rather induces cell proliferation and the expression of several growth factors, including TGF-β1, IGF-1, and EGF. 16 .

[0004] Viruses are broadly divided into two classes: enveloped and non-enveloped viruses. Both classes use host cells for their replication. Virus entry into host cells includes membrane fusion of the viral envelope with the plasma membrane or by receptor-mediated endocytosis. Enveloped viruses, such as influenza viruses, coronaviruses, filoviruses, and flaviviruses, cover their protein capsids with lipid envelopes through budding from the host cell membrane. For many virus families, such as coronaviridae and flaviviridae, translation, replication, assembly, and budding occur specifically in the endoplasmic reticulum (ER), which are then processed in the Golgi apparatus to produce mature viruses that are then released through exocytosis. Thus, the lipid envelopes of these viruses are derived from the ER. The lipid composition of the ER membrane differs from that of the plasma membrane, for example, by the even distribution of anionic lipids (e.g., phosphatidylserine) in both leaflets of the ER membrane, whereas the plasma membrane is asymmetric, with all charged lipids oriented towards the cytosol. However, the ER membrane also contains cholesterol (about 8%), which confers stability and resistance to membrane-active amphipathic peptides. 17,18 .

[0005] The current pandemic caused by SARS-CoV-2 has caused a life-threatening condition known as COVID-19, for which a vaccine has been developed through a concerted effort. It remains to be determined to what extent the virus will continue to mutate into more virulent strains and whether newly developed vaccines will be effective against these mutant strains. While elucidating viral pathogenesis is an important step towards the development of new therapeutic substances, effective broad-spectrum antiviral compounds are urgently needed given the rapid emergence of new viral pathogens. Severe illness in patients with respiratory viral infections is often associated with underlying medical conditions such as cancer, diabetes, and cardiovascular disease. Infected patients must endure severe suffering and longer hospitalizations, which in turn are given special medical attention requiring further medication and care, ultimately resulting in significantly increased healthcare costs. Considering that vaccine development involves high costs and limited use due to viruses, such as influenza viruses, continuously evolving and mutating to cause annual seasonal epidemics, these challenges call for innovative action worldwide. One such strategy is to develop new broad-spectrum antiviral agents based on cationic peptides that act on common elements shared by many viruses to combat serious infections. Summary of the Invention

[0006] The objective of the present disclosure is to provide a novel broad-spectrum antiviral pharmaceutical composition. It is shown herein that plantaricin NC8 (PLNC8)αβ, as well as its subunits, i.e., two peptides PLNC8α and PLNC8β, exert antiviral activity and have promising properties as antiviral agents against various viruses belonging to different virus families.

[0007] More specifically, the present disclosure is directed to a pharmaceutical composition for use in the prevention and / or treatment of viral infections and / or disorders associated with viral infections caused by enveloped viruses, comprising a) a peptide having at least 90%, e.g., 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO:1 (i.e., PLNC8 β), and / or b) a peptide having at least 90%, e.g., 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO:2 (i.e., PLNC8 α).

[0008] Preferred aspects of the present disclosure are set forth in the following detailed description and dependent claims. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows a schematic representation of the known lipid composition and structure in the human plasma membrane and endoplasmic reticulum (ER) membrane. [Figure 2-1] FIG. 2A: Cryo-electron microscopy (cryo-EM) images of liposomes containing 5% phosphatidylserine, either with or without cholesterol, either left untreated (control) or treated with peptide L-PLNC8 αβ. [Figure 2-2] Figure 2B: Effect of peptides L-PLNC8 α, L-PLNC8 β, and L-PLNC8 αβ on liposomes containing 70% POPC and 30% cholesterol. Figure 2C: Effect of peptides L-PLNC8 α, L-PLNC8 β, and L-PLNC8 αβ on liposomes containing 91% POPC, 1% POPS, and 8% cholesterol. Figure 2D: Effect of peptides L-PLNC8 α, L-PLNC8 β, and L-PLNC8 αβ on liposomes containing 81% POPC, 11% POPS, and 8% cholesterol. [Diagram 3]Figure 3A: Effects on flavivirus LGTV after exposure to peptides L-PLNC8 αβ, scrambled S-PLNC8 αβ, and LL-37, as well as microscopy images (Sytox Green fluorescence) of untreated LGTV (control). Figure 3B: Graph of quantified LGTV plaques (focus forming units, FFU) after exposure to peptides at different concentrations and of untreated LGTV (control). Representative images of viral plaques are presented above the bars. [Figure 4] Figure 4A: Effects on LGTV after exposure to peptides L-PLNC8 α and L-PLNC8 β, as well as images of untreated LGTV (control). Figure 4B: Graph of quantified LGTV plaques after exposure to peptides and of untreated LGTV (control). Representative images of viral plaques are presented above the bars. [Diagram 5] Figure 5: Effects on LGTV after exposure to AD-PLNC8 α, D-PLNC8 β, and D-PLNC8 αβ, as well as images of untreated LGTV (control). Figure 5B: Graph of quantified LGTV plaques after exposure to peptides and of untreated LGTV (control). Representative images of viral plaques are presented above the bars. [Figure 6] FIG. 1 shows a transmission electron microscope (TEM) image visualizing intracellular LGTV particles. [Figure 7] Figure 7A: Graph of quantified flavivirus KUNV plaques after exposure to L-PLNC8 αβ, L-PLNC8 α, and L-PLNC8 β. Figure 7B: Graph of quantified KUNV plaques after exposure to D-PLNC8 αβ, D-PLNC8 α, and D-PLNC8 β. [Figure 8] 1 is a graph showing the antiviral activity of L- and D-enantiomers of PLNC8 αβ against KUNV at different viral titers. [Figure 9] 9A and 9B are graphs showing the effect of L- and D-enantiomers of PLNC8 αβ on KUNV viral replication (FIG. 9A) and extracellular virion accumulation (FIG. 9B). [Figure 10] 10A and 10B are graphs showing the antiviral activity of the L-enantiomers of PLNC8 αβ and PLNC8 β (FIG. 10A) and the D-enantiomers of PLNC8 αβ and PLNC8 β (FIG. 10B) against SARS-CoV-2. [Figure 11] 11A and 11B are graphs showing the antiviral activity of the L-enantiomers of PLNC8 αβ and PLNC8 β (FIG. 11A) and the D-enantiomers of PLNC8 αβ and PLNC8 β (FIG. 11B) against influenza A virus. [Figure 12] 12A and 12B are graphs showing the effects of the L-enantiomers of PLNC8 αβ and PLNC8 β (FIG. 12A) and the D-enantiomers of PLNC8 αβ and PLNC8 β (FIG. 12B) against HIV-1. [Figure 13] 13A and 13B are graphs showing the effect of the L-enantiomer of PLNC8 αβ and the D-enantiomer of PLNC8 αβ, respectively, on the morphology (FIG. 13A) and viability (FIG. 13B) of uninfected or KUNV-infected cells. [Figure 14] 1 is a graph showing the effect of L- and D-enantiomers of PLNC8 αβ on human lung cancer cells infected with KUNV. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present disclosure solves or at least alleviates the above-mentioned problems associated with currently available prophylactic and treatment measures against viral infections by providing a pharmaceutical composition for use in the prevention and / or treatment of viral infections and / or disorders associated with viral infections caused by enveloped viruses, the pharmaceutical composition comprising: a) a peptide having at least 90%, e.g., 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:1 (i.e., PLNC8 β), and / or b) a peptide having at least 90%, e.g., 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:2 (i.e., PLNC8 α).

[0011] A notable advantage of the pharmaceutical composition disclosed herein is that the peptides contained therein have been shown to have potent activity against enveloped viruses through their permeabilizing effect on the viral envelope. More specifically, the experimental section herein shows that permeabilization is brought about by the peptide's activity through electrostatic interaction with the anionic lipids of the envelope, thus specifically targeting extracellular virions without affecting human cell viability. This hypothesis has been tested and verified by experimental microbiological studies investigating the effect of PLNC8 αβ on different virus species, in combination with previous knowledge of the composition of different biological membranes.

[0012] Moreover, it is shown herein that the concentrations of PLNC8 αβ required to eliminate all infectious viral particles are in the nanomolar (nM) to micromolar (μM) range, which is surprisingly efficient considering the high cholesterol content (8-35%) in the lipid envelope of the virus. Therefore, PLNC8 αβ can be used as an effective antiviral agent independent of viral antigenic mutations because it targets the viral envelope structure. This structure is stable and independent of the viral coding genome and originates from the infected host cell, e.g., plasma membrane, endoplasmic reticulum, Golgi complex, and nuclear envelope. Hence, the structural and functional differences between the viral envelope and the host cell plasma membrane make the viral membrane an ideal target for antiviral therapy by using membrane-active amphipathic peptides such as PLNC8 αβ.

[0013] Previous results show that PLNC8 αβ is not cytotoxic to human cells 15,16suggested that it is unlikely that PLNC8 αβ has any antiviral properties because enveloped viruses replicate and bud from human cells. More specifically, because eukaryotic membranes contain cholesterol and have specific phospholipid composition and orientation, it is unlikely that amphipathic peptides including PLNC8 αβ have any antiviral properties against enveloped viruses because these viruses utilize the translation machinery within the cell and bud from the plasma membrane of human cells. Nevertheless, we hypothesized that PLNC8 αβ would indeed have antiviral activity. This hypothesis is based on the clear differences in the cholesterol content, phospholipid composition, and anionic charge of the outer leaflet between the plasma membrane and the cell organelles, such as the ER membrane. Figure 1 shows that the plasma membrane has a higher cholesterol content (35%) than the ER membrane (8%) and furthermore that the plasma membrane is asymmetric, with the extracellular leaflet composed of zwitterionic phospholipids and all anionic phospholipids oriented towards the cytosol, whereas the ER membrane is symmetric with both leaflets containing anionic phospholipids. In the experimental section herein, it is also shown that the difference between the plasma membrane and the ER membrane is a crucial determinant that allows PLNC8αβ to efficiently permeabilize the viral envelope without affecting cell viability.

[0014] The virus families studied herein have distinct replication pathways. They replicate and assemble in different cell types and subcellular sites, which may explain their variable susceptibility to the antiviral peptides tested herein. Indeed, viruses that use the ER / Golgi complex pathway, such as SARS-CoV-2 and flaviviruses, were found to be significantly more sensitive to PLNC8 αβ compared to viruses that acquire their lipid envelope from the plasma membrane, such as influenza A virus and HIV-1. Nevertheless, the results indicate that the tested peptides exert antiviral effects on distinct enveloped viruses belonging to several different virus families. Moreover, as shown herein, the antiviral effect is achieved by permeabilization of the viral envelope and is not dependent on the viral coding genome. As a result, the peptides can exert antiviral effects on any type of enveloped virus.

[0015] As stated above, the present disclosure is directed to a pharmaceutical composition for use in the prevention and / or treatment of viral infections and / or disorders associated with viral infections caused by enveloped viruses, comprising a peptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, respectively. It should be understood that such a peptide having at least 90% sequence identity must also be functionally equivalent to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, respectively, in terms of its antiviral effect or activity. The antiviral effect or activity of a peptide can be determined by performing any one or more of the experiments described in the experimental section below.

[0016] Also described herein are peptides for use in the prevention and / or treatment of viral infections and / or disorders associated with viral infections caused by enveloped viruses, where the peptides are a) peptides having at least 90%, e.g., 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 1, or b) peptides having at least 90%, e.g., 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 2. As mentioned above, it should be understood that such peptides having at least 90% sequence identity must also be functionally equivalent to the amino acid sequence of SEQ ID NO: 1 or 2, respectively, in terms of their antiviral effect or activity. Again, the antiviral effect or activity of the peptides can be determined by performing any one or more of the experiments described in the experimental section below.

[0017] Furthermore, it should be understood that the peptides described above can be formulated in any pharma- ceutically acceptable manner, e.g., the peptides can be in the form of a pharma-ceutically acceptable salt or solvate.

[0018] The above-mentioned peptides which are naturally composed of L-amino acids may contain at least one, e.g., two, three, four, etc., D-amino acid residue up to and including the total number of amino acid residues present in the peptide.

[0019] Thus, in the pharmaceutical compositions described herein, at least one amino acid residue of peptide (a) and / or (b) (as defined above) may be a D-amino acid residue, e.g., 2, 3, 4, etc. D-amino acid residues, up to the total number of amino acid residues of peptide (a) and / or (b) (as defined above). Alternatively, in the pharmaceutical compositions, at least about 3%, e.g., about 3.5%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the amino acid residues of peptide (a) and / or (b) (as defined above) may be D-amino acid residues.

[0020] Pharmaceutical compositions disclosed herein for use in the prevention and / or treatment of viral infections and / or disorders associated with viral infections caused by enveloped viruses may contain about 1 nM to about 1000 μM, e.g., about 10 nM to about 100 μM, or, e.g., about 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, 10 μM, 50 μM, 100 μM, 500 μM, or 1000 μM, of peptides (a) and / or (b).

[0021] Pharmaceutical compositions disclosed herein for use in the prevention and / or treatment of viral infections and / or disorders associated with viral infections caused by enveloped viruses may contain peptides (a) and (b) in a molar ratio of about 1:1 to about 20:1, e.g., about 1:1 to about 10:1, e.g., about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1.

[0022] In the context of the pharmaceutical compositions disclosed herein for use in the prevention and / or treatment of viral infections and / or disorders associated with viral infections, viral infections can be selected from the following virus families: Coronaviridae, Flaviviridae, Herpesviridae, Orthomyxoviridae, Retroviridae, Paramyxoviridae, Filoviridae, and / or other viruses. It may be caused by an enveloped virus selected from the group consisting of Filoviridae, Pneumoviridae, Arteriviridae, Asfarviridae, Bunyaviridae, Hepadnaviridae, Poxviridae, Togaviridae, and Rhabdoviridae.

[0023] Thus, by way of non-limiting example, the enveloped virus may belong to the Coronaviridae family. Alternatively, by way of non-limiting example, the enveloped virus may belong to the Flaviviridae family. Alternatively, by way of non-limiting example, the enveloped virus may belong to the Herpesviridae family. Alternatively, by way of non-limiting example, the enveloped virus may belong to the Orthomyxoviridae family. Alternatively, by way of non-limiting example, the enveloped virus may belong to the Retroviridae family. Alternatively, by way of non-limiting example, the enveloped virus may belong to the Paramyxoviridae family. Alternatively, by way of non-limiting example, the enveloped virus may belong to the Filoviridae family. Alternatively, by way of non-limiting example, the enveloped virus may belong to the Pneumoviridae family. Alternatively, for non-limiting examples, the enveloped virus may belong to the Arteriviridae family. Alternatively, for non-limiting examples, the enveloped virus may belong to the Asfarviridae family. Alternatively, for non-limiting examples, the enveloped virus may belong to the Bunyaviridae family. Alternatively, for non-limiting examples, the enveloped virus may belong to the Hepadnaviridae family. Alternatively, for non-limiting examples, the enveloped virus may belong to the Poxviridae family. Alternatively, for non-limiting examples, the enveloped virus may belong to the Togaviridae family. Alternatively, for non-limiting examples, the enveloped virus may belong to the Rhabdoviridae family.

[0024] Alternatively, the viral infection may be caused by an enveloped virus selected from the group consisting of, for example, the following viral families: Coronaviridae, Flaviviridae, Herpesviridae, Arteriviridae, Asfarviridae, Bunyaviridae, Hepadnaviridae, and Poxviridae, the envelope of which is obtained from the endoplasmic reticulum, Golgi apparatus, or nuclear envelope of the infected cell.

[0025] Thus, by way of non-limiting example, an enveloped virus whose envelope is derived from the endoplasmic reticulum, the Golgi apparatus, or the nuclear envelope of an infected cell may belong to the Coronaviridae family. Alternatively, by way of non-limiting example, such an enveloped virus may belong to the Flaviviridae family. Alternatively, by way of non-limiting example, such an enveloped virus may belong to the Herpesviridae family. Alternatively, by way of non-limiting example, such an enveloped virus may belong to the Arteriviridae family. Alternatively, by way of non-limiting example, such an enveloped virus may belong to the Asfarviridae family. Alternatively, by way of non-limiting example, such an enveloped virus may belong to the Bunyaviridae family. Alternatively, by way of non-limiting example, such an enveloped virus may belong to the Hepadnaviridae family. Alternatively, by way of non-limiting example, such an enveloped virus may belong to the family Poxviridae.

[0026] Non-limiting examples of such enveloped viruses include Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2), Epstein-Barr Virus (EBV), Human Cytomegalovirus (HCMV), Varicella-Zoster Virus (VZV), Dengue Virus (DENV), Zika Virus (Zika virus), and / or other viruses that cause respiratory distress. These are ZIKV, West Nile Virus (WNV), Langat Virus (LGTV), and Tick-borne Encephalitis Virus (TBEV).

[0027] Thus, by way of non-limiting example, such an enveloped virus may be Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV).

[0028] Alternatively, as a non-limiting example, such an enveloped virus can be Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2).

[0029] Alternatively, as a non-limiting example, such an enveloped virus can be Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

[0030] Alternatively, as a non-limiting example, such an enveloped virus can be Herpes Simplex Virus 1 (HSV-1).

[0031] Alternatively, as a non-limiting example, such an enveloped virus can be Herpes Simplex Virus 2 (HSV-2).

[0032] Alternatively, by way of non-limiting example, such an enveloped virus can be the Epstein-Barr Virus (EBV).

[0033] Alternatively, as a non-limiting example, such an enveloped virus can be Human Cytomegalovirus (HCMV).

[0034] Alternatively, as a non-limiting example, such an enveloped virus can be Varicella-Zoster Virus (VZV).

[0035] Alternatively, as a non-limiting example, such an enveloped virus may be the Dengue Virus (DENV).

[0036] Alternatively, as a non-limiting example, such an enveloped virus can be the Zika Virus (ZIKV).

[0037] Alternatively, as a non-limiting example, such an enveloped virus may be the West Nile Virus (WNV).

[0038] Alternatively, as a non-limiting example, such an enveloped virus can be the Langat Virus (LGTV).

[0039] Alternatively, as a non-limiting example, such an enveloped virus can be Tick-borne Encephalitis Virus (TBEV).

[0040] Another non-limiting example of such an enveloped virus is Kunjin virus (KUNV), a subtype of the West Nile Virus (WNV) discussed above.

[0041] Viral infections that may be prevented and / or treated by use of the pharmaceutical compositions described herein may be respiratory tract infections (eg, upper or lower respiratory tract infections) and / or mucus layer infections.

[0042] A non-limiting example of an enveloped virus that can cause respiratory tract and / or mucus layer infection is Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV).

[0043] Another non-limiting example of an enveloped virus that can cause respiratory tract and / or mucus layer infection is Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2).

[0044] Another non-limiting example of an enveloped virus that can cause respiratory tract and / or mucus layer infection is Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

[0045] Another non-limiting example of an enveloped virus that can cause respiratory tract and / or mucus layer infection is Herpes Simplex Virus 1 (HSV-1).

[0046] Another non-limiting example of an enveloped virus that can cause respiratory tract and / or mucus layer infection is Herpes Simplex Virus 2 (HSV-2).

[0047] Another non-limiting example of an enveloped virus that can cause airway and / or mucus layer infection is the Epstein-Barr Virus (EBV).

[0048] Another non-limiting example of an enveloped virus that can cause respiratory tract and / or mucus layer infection is Human Cytomegalovirus (HCMV).

[0049] Another non-limiting example of an enveloped virus that can cause respiratory tract and / or mucus layer infection is Varicella-Zoster Virus (VZV).

[0050] Throughout this text, the expression "disorder associated with a viral infection" is intended to mean a disorder induced or caused by a viral infection. In other words, the disorder may alternatively be said to be a "viral-induced disorder."

[0051] The term "disorder" may alternatively be described as a "disease," a "medical complication," a "physical complication," or in short, a "complication," which terms may be used interchangeably herein.

[0052] The peptides and pharmaceutical compositions described herein have been shown to exert a direct antiviral effect on enveloped viruses.Therefore, the peptides and pharmaceutical compositions will exert a preventive or therapeutic effect on any damage induced by enveloped viruses.Thanks to their direct antiviral effect, it is possible to prevent or treat damage associated with viral infection, regardless of whether the damage occurs early or late in the chain of events induced by viral infection in the host organism, and regardless of whether the damage is caused directly by the physical interaction between the virus and its host cell, or is then caused by another damage caused by the underlying viral infection.

[0053] Thus, non-limiting examples of disorders associated with viral infection are virus-induced inflammation, virus-induced cell death, virus-induced tissue destruction, and combinations thereof. Further, non-limiting examples of virus-induced tissue destruction are damage to mucosal surfaces, pulmonary fibrosis, organ failure, and combinations thereof. More specifically, non-limiting examples of disorders associated with infection with respiratory viruses (influenza A virus and SARS-CoV-2) are cell death (caused by budding of virus particles), tissue destruction, fever, and cold symptoms, such as sore throat, excessive runny nose, congestion of upper airways, and coughing, as well as pneumonia and pulmonary fibrosis. Non-limiting examples of disorders associated with HIV infection are destruction of lymphocytes, and susceptibility to infection caused by pathogenic microorganisms. Non-limiting examples of disorders associated with viral infection, such as Herpes Simplex virus, are viral sepsis.

[0054] The pharmaceutical composition for use as disclosed herein can be administered locally to or near the site of infection.Those skilled in the art (e.g., medical practitioners) can easily understand how close to the site of infection it is appropriate and / or necessary to administer the pharmaceutical composition to achieve the desired medical effect of the composition.The pharmaceutical composition for use as disclosed herein can be administered topically, for example, locally to or near the site of infection.

[0055] Pharmaceutical compositions for use as disclosed herein may be formulated as a powder, solution, cream, gel, ointment, or in immobilized form as a coating on a medical device.

[0056] If the pharmaceutical composition is formulated as a powder, it can be administered, for example, by means of an inhaler (ie, a portable device for administering drugs that can be inhaled).

[0057] If the pharmaceutical composition is formulated as a solution, optionally the solution can be an aerosol and / or take the form of a nasal spray, mouth spray, or can be administered by inhaler.

[0058] When the pharmaceutical composition is formulated in an immobilized form as a coating on a medical device, optionally the medical device can be a face mask, an air filter, a nasal cannula device, or an endotracheal tube.

[0059] The present disclosure is also useful for medical devices that are at least partially coated with a pharmaceutical composition comprising the peptides of (a) and / or (b) as defined above.

[0060] The pharmaceutical compositions for use as disclosed herein contain, in addition to the peptides described above, one or more pharma- ceutically acceptable excipients.Non-limiting examples of such excipients are solubilizers, surfactants, bulking agents, viscosity enhancing agents, preservatives, vehicles, salts, sugars, and buffers.

[0061] Pharmaceutical compositions for use as disclosed herein may be formulated for administration in a single dose or in multiple doses, e.g., two, three, four, or five times daily, for 1 to 20 days, e.g., 3 to 20 days, or for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days.

[0062] Pharmaceutical compositions for use as disclosed herein contain, in addition to the peptides described above and optionally one or more pharma- ceutically acceptable excipients as described above, one or more additional antiviral agents.

[0063] Non-limiting examples of antiviral agents are antiviral agents intended for use in the prevention or treatment of infections caused by Herpes simplex, such as acyclovir or a functional equivalent thereof, and / or antiviral agents intended for use in the prevention or treatment of infections caused by influenza A or B, such as oseltamivir (Tamiflu) or a functional equivalent thereof, and / or antiviral agents intended for use in the prevention or treatment of infections caused by SARS-CoV-2, such as remdesivir or a functional equivalent thereof.

[0064] The present disclosure is also useful for a kit of parts comprising: (i) a pharmaceutical formulation comprising an antiviral agent, optionally in admixture with a pharma- ceutically acceptable excipient; and (ii) A peptide of (a) and / or (b) as further defined above, or a pharmaceutical composition comprising a peptide of (a) and / or (b) as further defined above.

[0065] The present disclosure is further useful in the context of an antiviral agent, as defined elsewhere herein, for use in a method for the prevention and / or treatment of a viral infection and / or a disorder associated with a viral infection, said use comprising the administration of said antiviral agent in combination with a pharmaceutical composition comprising a peptide of (a) and / or (b) as further defined above.

[0066] The present disclosure is additionally useful for a method for the prevention and / or treatment of a viral infection and / or a disorder associated with a viral infection, said method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a peptide of (a) and / or (b) as further defined above.

[0067] The present disclosure is also useful for a method for the prevention and / or treatment of a viral infection and / or a disorder associated with a viral infection, said method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an antiviral agent and a peptide of (a) and / or (b) as further defined above. Optionally, said antiviral agent and said pharmaceutical composition may be present in the same pharmaceutical formulation. EXAMPLES

[0068] Experimental Section material and method cell culture Monkey (Cercopithecus aethiops) epithelial kidney cells (Vero E6, ATCC, CCL-81), canine (Canis familiaris) epithelial kidney cells (MDCK, ATCC, NBL-2), and human lung cancer cells (A549, ATCC, CCL-185) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 1 g / L glucose (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS, Gibco) and 100 U / mL penicillin-streptomycin (PEST, Gibco) at 37°C and 5% CO2. Jurkat T cells (E6-1, ATCC) were maintained in RPMI 1640 medium (Fisher scientific, Austria) containing 1.5 mM L-glutamine (Invitrogen, USA) supplemented with 10% FBS. The cells were incubated in a stable environment of 95% air, 5% CO2, and 37°C. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient medium Ficoll-Paque™ Plus (Amersham Biosciences, Sweden) according to the manufacturer's instructions. Briefly, freshly collected blood from healthy donors was diluted with an equal volume of PBS, 4 ml was carefully layered on top of 3 ml Ficoll-Paque Plus, and then the tube was centrifuged at 300×g for 30 min at room temperature. PBMCs were collected from the interface and washed twice with PBS to remove excess Ficoll-Paque Plus and platelets. The cells were suspended in RPMI medium supplemented with 10% FBS and maintained at 95% air, 5% CO2, and 37°C for the following experiments.

[0069] Virus strains and propagation Vero cells were infected with Langat virus (LGTV), West Nile virus (WNV), Kunjin virus (KUNV), human immunodeficiency virus (HIV)-1 (subtype B, MN strain), influenza A virus (H1N1 / CA09pdm), or severe acute respiratory syndrome coronavirus 2 (β-SARS-CoV-2) at a multiplicity of infection (MOI) of 0.1 to induce viral growth. Infected cells were then grown in complete DMEM at 37°C and 5% CO2 until cytopathic effect (CPE) was observed 4–6 days postinfection. After observation of CPE, cell culture medium was semi-purified by ultracentrifugation against a 20% sucrose cushion at 150,000 × g and 4°C for 2.5 h. The virus-containing pellet was resuspended in complete DMEM medium and the virus concentration was quantified by performing a plaque assay.

[0070] peptide The peptides used in the experiments herein were purchased from GL Biochem (Shanghai) Ltd, China. Their names, amino acid sequences, molecular weights, and net charges at pH 7, as well as sequence numbers, are presented in Table 1 below. In the peptides designated L-PLNC8 α and L-PLNC8 β, 13 In the peptides designated D-PLNC8α and D-PLNC8β, all amino acid residues are in the D-configuration. Both L-PLNC8β and D-PLNC8β have the amino acid sequence of SEQ ID NO:1, whereas both L-PLNC8α and D-PLNC8α have the amino acid sequence of SEQ ID NO:2. Scrambled forms of PLNC8α and PLNC8β (S-PLNC8α and S-PLNC8β, respectively) 15(denoted as LL-37) were generated by randomly shifting amino acids in the native sequences to demonstrate that the order of amino acids is important for the activity of PLNC8α and PLNC8β. The human cathelicidin-derived peptide LL-37 is a known bactericidal peptide.

[0071] [Table 1]

[0072] Liposome preparation Liposomes were prepared by thin film hydration followed by extrusion. 19 Lipids Stock solutions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and cholesterol (Chol) were mixed in molar ratios of 95:0:5, 65:30:5, 70:0:30, 91:1:8, and 81:11:8. The solvent was slowly evaporated with a stream of nitrogen, and then the vials were dried overnight in a vacuum desiccator. The dried films were hydrated by the addition of 1 mL 5(6)-carboxyfluorescein solution. CF solutions, which represent the self-quenching concentrations of CF, were prepared with 50 mM CF dissolved in 10 mM PB buffer and 90 mM NaCl, followed by pH adjustment. The CF solution was dispensed onto the lipid cake and incubated for 10 min under gentle shaking (50 rpm on an orbital shaker), followed by vortexing for 1 min. Liposomes were extruded 21 times through a 0.1 um membrane (Nuclepore track-etched hydrophilic membrane) in a mini extruder (Avanti Polar Lipids, Inc.). Lipids were purified by gel filtration through a PD MiniTrap G-25 column with elution in PB buffer (10 mM).

[0073] Carboxyfluorescein (CF) release assay To study the efficacy of peptides on liposome membrane disruption, liposome fluorescence upon peptide addition was measured using a microplate reader (Tecan Infinite M1000 Pro, λ ex = 485 nm, λ em Liposomes were diluted to a final concentration of 25 μM in 10 mM PB buffer and plated in a 96-well plate in a final volume of 200 μL at 10 -5 ~10 2 The liposomes were incubated with PLNC8α, PLNC8β, and PLNC8α / β at 10 μM concentrations. Due to the self-quenching concentration of carboxyfluorescein (CF) in the interior of the liposomes, any fluorescence in the liposome-peptide system could be attributed to liposome membrane disruption. Fluorescence was monitored before peptide addition (F0) and continuously for 30 min. Total CF release (F T ) was achieved by the addition of 1% Triton X-100. Instantaneous CF release (%) was calculated using the formula 100 × (F-F0) / (F T -F0), where F is the instantaneous fluorescence.

[0074] Flavivirus inhibition assay The antiviral activity of PLNC8 αβ was determined using plaque assays. A crystal violet-based plaque assay was performed to quantify KUNV, and an immunofocus plaque assay was performed to quantify LGTV. Briefly, a 90% confluent layer of Vero cells was infected with a series of virus dilutions in DMEM, either untreated or pre-exposed to PLNC8 αβ (forms and ratios as shown in the Examples below), for 1 h at 37°C, followed by overlaying the cells with DMEM supplemented with 1.2% Avicel (FMC), 2% HI-FBS (Gibco), 1× non-essential amino acids (Gibco), and 1% PEST (Gibco). After 3-5 days, the overlay was removed and the cells were fixed with methanol for 20 min before proceeding with the plaque assay. For immunofocus assay, fixed cells were blocked with 2% BSA (Fitzgerald) for 10 min and then labeled with mouse anti-E antibody (1:1000, anti-TBEV-E, United States Army Medical Research, Institute of Infectious Diseases, Fort Detrick, Frederick, MD, USA), followed by addition of anti-mouse secondary HRP polymer (1:100) for 1 h at 37 °C, and finally KPL TrueBlue peroxidase substrate (Seracare) for 15 min at room temperature (RT). For crystal violet-based plaque assay, fixed cells were stained with 2% crystal violet (Sigma), 20% methanol (Fisher), and 0.1% ammonium oxalate (Sigma) solution. The number of viral plaques was quantified, and representative images of wells were captured on an Olympus SZX9 at 10× magnification and processed using the software ImageJ.

[0075] SARS-CoV-2 virus inhibition assay Antiviral peptide activity against the SARS-CoV-2 virus was quantified using a SARS-CoV-2 virus neutralization assay. 20~22Briefly, peptides (L-PLNC8 αβ and D-PLNC8 αβ at 1:1 molar ratio) were diluted in DMEM-0.1% FCS and PEST and incubated with the selected virus dose of 100 plaque-forming units (PFU) / 0.1 ml for 30 min at 37°C, followed by transfer of the peptide-virus mixture to semi-confluent Vero E6 cells (85-95% confluency). Untreated virus samples were also included. The peptide-virus suspension was kept with the cells for 1 h, after which the cells were washed and agarose gel (2%) was added, followed by addition of 200 μl DMEM with 5% FCS on top of the gel. The cell cultures were kept at 37°C for 72-96 h before the assay was terminated. The cells were rinsed with sterile PBS and then fixed for 45 min. Cells were stained with 2% crystal violet (Merck, Sigma-Aldrich, Stlm, Sweden) for 45 min, after which the wells were emptied, washed twice with sterile PBS and dried. Plaques were counted in a plate microscope. The neutralizing titer end-point dilution at which no viral plaques were visible (i.e., 0 PFU) was defined as the protective dose of the antiviral peptide.

[0076] Influenza A virus inhibition assay Influenza A virus neutralization assay 23was used to quantify antiviral peptide activity against influenza A virus. Briefly, DMEM supplemented with 0.1% FCS and PEST with or without peptide was inoculated with the selected virus dose of 100 PFU / 0.1 ml and incubated at 37°C for 30 min, after which the peptide-virus mixture and untreated virus samples were transferred to semi-confluent MDCK cells. The peptide-virus suspension was kept with the cells for 1 h, after which the cells were washed, agarose gel (2%) was added, and 200 μl DMEM with 5% FCS was placed on top of the gel. The cell cultures were kept at 37°C for 72-96 h before the assay was terminated. The cells were rinsed with sterile PBS and then fixed for 45 min. The cells were stained with 2% crystal violet (Merck, Sigma-Aldrich, Sthlm, Sweden) for 45 min, after which the wells were emptied, washed twice with sterile PBS, and dried. Plaques were counted in a plate microscope. The neutralizing titer end point dilution at which no viral plaques were visible (i.e., 0 PFU) was defined as the protective dose of the antiviral peptide.

[0077] HIV-1 neutralization assay HIV-1 neutralization assays were performed as previously described. 24,25 , was performed. Briefly, the viral isolate was derived from HIV-1 subtype B MN strain, a subtype B strain (http: / / www.hiv.lanl.gov). Antiviral peptides were diluted in RPMI 1640 supplemented with 5% FCS and PEST in a 96-well tissue culture plate (Costar, 260860, ThermoFisher Scientific). Each peptide concentration was diluted to 50 tissue cell culture infectious doses (TCID 50 ) and incubated at 37°C for 1 hour, followed by 10 5 Human PBMCs / 0.1 ml were added and incubated with phytohemagglutinin (PHA) and rIL-2 (200-02, PeproTech) or (10 5c / 0.1 ml) Jurkat T cells were activated. Cells were incubated for 120 min at 37°C with 5% CO2 in air and washed twice with RPMI 1640 containing 5% FCS before cells were fed with fresh medium. After 3 days, 50% of the medium was replaced and the presence of HIV-1 p24 antigen in the culture medium was measured by capture ELISA for HIV-1 subtype B on days 5-7 of culture. p24 background was determined for each plate and subtracted from all wells. Percentage of neutralization was determined as [1-(mean p24 OD in the presence of test medium / mean p24 OD in the absence of test serum)] x 100.

[0078] cytotoxicity Cytotoxicity was determined by assessing lactate dehydrogenase (LDH) activity in cell culture supernatants using an LDH Cytotoxicity Assay Kit (Thermo Scientific™) according to the manufacturer's instructions. The assay principle is based on the fact that intracellular LDH is released by damaged cells and ultimately converts the chemicals used in the assay into a colored formazan product, which can be measured at 490 nm and whose level is directly proportional to the amount of LDH released.

[0079] Reverse transcription PCR (RT-PCR) RT-PCR was used to determine the expression levels of several human genes and KUNV mRNA levels in response to KUNV infection with or without L-PLNC8 αβ or D-PLNC8 αβ. Briefly, human lung cancer cells (A549) were infected with KUNV for 1 h, followed by treatment with 10 μM L- or D-PLNC8 αβ for 48 h. RNA was extracted using RNeasy® Plus Micro Kit (Qiagen, USA) according to the manufacturer's recommendations. Reverse transcription was performed using Maxima® First Strand cDNA Synthesis Kit (Fermentas, Sweden). Thermal cycling conditions for SYBR Green (Fermentas) consisted of a denaturation step at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. Gene expression was analyzed using a 7900 HT real-time PCR instrument (Applied Biosystems). The obtained Ct values ​​were normalized to GAPDH. Relative quantification of gene expression was determined by using the ΔΔCt method. For each sample, ΔCt was calculated by subtracting the Ct of GAPDH from the Ct of the gene of interest. ΔΔCt was calculated by subtracting the ΔCt of the control sample from the ΔCt of each treated sample. Fold change was calculated using the formula 2. -ΔΔCt It was derived by using

[0080] Microscopy The fluorescent dye Sytox® Green was used to examine membrane permeabilization caused by PLNC8 αβ. This fluorophore can only cross damaged membranes and fluoresce upon binding to nucleic acids. Briefly, flaviviruses (LGTV or KUNV, 10 5(1000 cells) were either left untreated or exposed to L-PLNC8 αβ, D-PLNC8 αβ, or scrambled (S)-PLNC8 αβ at a 1:1 molar ratio (see Table 1), or LL-37 for 2 hours. Samples were then fixed with 4% PFA, followed by the addition of Sytox Green for 5 minutes, and images were captured on an Olympus BX41 at 40× magnification. Images were processed and analyzed using the software ImageJ.

[0081] Transmission electron microscopy (TEM) was used to visualize cells infected with LGTV and exposed to L-PLNC8αβ or S-PLNC8αβ. Briefly, LGTV (5×10 4 (100 pieces) were either left untreated or exposed to L-PLNC8αβ or S-PLNC8αβ at a final concentration of 10 μM for 1 h. The suspension was then added to Vero cells and incubated for 1 h, followed by washing with PBS and adding fresh medium. After 24 h of incubation, cells were washed with PBS and fixed with 4% glutaraldehyde solution in 0.1 M phosphate buffer, pH 7.3. Specimens were washed in 0.1 M phosphate buffer, postfixed in 2% osmium tetroxide in 0.1 M phosphate buffer for 2 h, and embedded in LX-112 (Ladd, Burlington, Vermont, USA). Ultrathin sections (approximately 50–60 nm) were cut with a Leica ultracut UCT / Leica EM UC 6 (Leica, Wien, Austria). Sections were contrasted with uranyl acetate followed by lead citrate and examined on a Hitachi HT 7700 (Tokyo, Japan). Digital images were acquired by using a Veleta camera (Olympus Soft Imaging Solutions, GmbH, Munster, Germany).

[0082] Liposomes containing 5% 2-oleoyl-1-palmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (POPG) with or without cholesterol were exposed to 1 μM PLNC8 αβ for 2 min. Cryo-EM specimens were prepared by applying 3 μl liposome mixture onto a Quantifoil grid 2 / 2 coated with a 2 nm carbon layer. Images were collected on a Talos Arctica 200 kV microscope (Thermo Fischer Scientific) using a Falcon3 electron direct detector in linear mode at a magnification corresponding to a pixel size of 5 Å.

[0083] [Example 1] Our previous results showed that cholesterol-containing phospholipid bilayers are resistant to the membrane-disrupting activity of PLNC8αβ. 15 These results were confirmed herein by using cryo-electron microscopy (cryo-EM) to show the intense PLNC8 αβ-induced deformation of cholesterol-free liposomes compared to unaffected, resistant cholesterol-containing liposomes (Figure 2A). Figure 2A shows cryo-EM images of liposomes containing 5% phosphatidylserine, without cholesterol (POPC:POPS 95:5; left hand column) and with 30% cholesterol (POPC:POPS:Chol 65:5:30; right hand column), respectively. Liposomes were either left untreated (control; upper row) or exposed to L-PLNC8 αβ at a final concentration of 1 μM for 2 min (lower row). Liposomes without cholesterol were deformed by the peptide (black arrowheads) and no longer associated with the carbon delineating the edge of the grid, indicating that they had lost their charge. Thus, the 30% cholesterol content stabilized the phospholipid membrane for PLNC8 αβ. In summary, L-PLNC8 αβ was able to induce significant deformation of liposomes that did not contain cholesterol, whereas cholesterol-containing liposomes were not affected and maintained their round shape.

[0084] Figure 2B shows that liposomes with lipid composition POPC:POPS:Chol (70:0:30), which mimics the eukaryotic plasma membrane, are resilient to PLNC8 αβ. Figure 2C shows that by using lipid composition POPC:POPS:Chol (91:1:8), reducing the amount of cholesterol and introducing a minimal amount of negative charge, the liposome model becomes more sensitive to PLNC8 αβ. Figure 2D shows that liposomes with even higher negative charge by using lipid composition POPC:POPS:Chol (81:11:8), which mimics the eukaryotic ER membrane, are highly sensitive to PLNC8 αβ.

[0085] [Example 2] We first demonstrated the antiviral activity of PLNC8 αβ by targeting the flavivirus Langat (LGTV), which uses the rough ER for translation, assembly, and budding. 26 The test was performed with LGTV (1 × 10 5(1:1) were either left untreated (control) or exposed to L-PLNC8 αβ (1:1), scrambled S-PLNC8 αβ (1:1), or LL-37 at final concentrations of 5 μM, 10 μM, and 20 μM. Samples were fixed with 4% PFA after 2 h exposure, followed by addition of Sytox Green for 5 min. Images were captured at 40× magnification using an Olympus BX41. Scale bar is 200 μm. Figure 3A shows that full-length L-PLNC8 αβ caused rapid permeabilization of the viral envelope, whereas neither the PLNC8 αβ scrambled (S) variant (S-PLNC8 αβ; see Table 1) nor the human cathelicidin-derived peptide LL-37 (a known bactericidal peptide) caused permeabilization. Additionally, LGTV was incubated in cell culture medium containing the indicated concentrations of L-PLNC8 αβ, S-PLNC8 αβ, or LL-37 for 1 h at room temperature. The suspension was then added to Vero cells and incubated for 1 h. Overlay medium was poured onto the cells, and the plates were incubated for 3 days. Viral load was quantified by performing a plaque assay. Concentrations shown are in μM. Representative images of viral plaques are presented above the bars in FIG. 3B. In the representative images, the lack of viral plaque staining indicates efficient clearance of virions for L-PLNC8 αβ. Quantification of viral load after 1 h exposure of LGTV to peptides showed that it was dose-dependently reduced by L-PLNC8 αβ, resulting in >99% clearance of infectious virions (FIG. 3B). The results indicate that the antiviral activity of PLNC8 αβ is specific, since the effect was completely abolished when the sequence was scrambled. In conclusion, viral particles are rapidly permeabilized by L-PLNC8 αβ, forming large aggregates and reducing the viral load by >99.9%.

[0086] [Example 3] Next, LGTV (1×10 51000 ng / ml) were either left untreated (control) or exposed to PLNC8α or PLNC8β at a final concentration of 20 μM. FIG. 4A shows that LGTV was permeabilized by PLNC8β but not L-PLNC8α (2 h after treatment, Sytox Green staining). Scale bar is 200 μm. Additionally, LGTV was incubated in cell culture medium containing either PLNC8α or PLNC8β at 20 μM for 1 h at room temperature and subsequently infected into Vero cells for 1 h. Viral load was quantified by performing an immunofocus-based plaque assay. The results are shown in FIG. 4B. Viral particles are not permeabilized by PLNC8α but are rapidly permeabilized by PLNC8β, resulting in a reduction in viral load by >99%. Representative images of viral plaques are presented above the bars, and visualization of single viral plaques for L-PLNC8β shows a substantial reduction in viral load. However, optimal antiviral activity was achieved when both peptides were present, as shown in Figures 3A and 3B: L-PLNC8αβ significantly reduced the concentration required to efficiently clear infectious virions compared with PLNC8β alone, thus highlighting the importance of PLNC8α.

[0087] [Example 4] The findings described in Examples 2 and 3 prompted us to determine whether the antiviral activity of PLNC8 αβ is mediated through binding to a receptor on the surface of the virion, which we investigated by using the D-enantiomer of the peptide, which should be unable to bind to protein targets. Interestingly, the D-enantiomer of PLNC8 αβ exhibited potent antiviral properties, resulting in rapid permeabilization (Figure 5A) and efficient clearance (Figure 5B) of LGTV.

[0088] More specifically, the flavivirus LGTV (1 × 10 5LGTV particles were exposed to D-PLNC8α, D-PLNC8β at a final concentration of 20μM, or D-PLNC8αβ at final concentrations of 5μM, 10μM, and 20μM (1:1). Figure 5A shows that LGTV particles were permeabilized by D-PLNC8β and D-PLNC8αβ after 2h of exposure, whereas D-PLNC8α was not efficient. Scale bar is 200μm. Furthermore, LGTV was incubated for 1h at room temperature in cell culture medium containing the indicated concentrations of D-PLNC8αβ, either alone or together at a molar ratio of 1:1. The suspension was then used to infect Vero cells for 1h, followed by quantification of the viral load using an immunofocus assay. The indicated concentrations in Figure 5B are μM. Representative images of viral plaques are also presented above the bars in Figure 5B, and the reduced viral plaque staining indicates efficient clearance of virions for D-PLNC8 αβ. In conclusion, the D-enantiomer of PLNC8 αβ is efficient against the enveloped flavivirus LGTV. Viral particles were rapidly permeabilized by D-PLNC8 αβ, forming large aggregates and reducing the viral load by >99.9%. D-PLNC8 β was more efficient than D-PLNC8 α in permeabilizing LGTV. The results showed that PLNC8 αβ did not bind to a specific receptor on the flavivirus Langat, suggesting that the binding is triggered by electrostatic interactions between cationic peptides and anionic structures on the virion, such as phospholipids in the viral envelope.

[0089] [Example 5] Furthermore, the antiviral effect of L-PLNC8 αβ was visualized under transmission electron microscopy (TEM) by the lack of clearly visible intracellular virus-induced single-membrane vesicles in LGTV-infected cells and samples pretreated with scrambled PLNC8 αβ (Figure 6). More specifically, the antiviral effect of L-PLNC8 αβ was visualized by the lack of clearly visible intracellular virus-induced single-membrane vesicles in LGTV-infected cells and samples pretreated with scrambled PLNC8 αβ (Figure 6). 5(10 μM) were pretreated with 10 μM L-PLNC8αβ or S-PLNC8αβ for 1 h prior to infection of Vero cells, followed by washing and addition of fresh medium. After 24 h of incubation, cells were fixed with 4% glutaraldehyde solution, processed, and visualized using TEM. Black arrowheads point to the formation of large aggregates with L-PLNC8αβ, and white arrowheads point to the presence of virus-induced single-membrane vesicles within the lumen of the rough ER.

[0090] Interestingly, exposure of LGTV to L-PLNC8 αβ resulted in the formation of large, electron-dense aggregates, which may be the result of rapid and efficient permeabilization of virions, since these large aggregates were visible under a fluorescent microscope in response to L-PLNC8 αβ but not to the scrambled peptide (Figure 3).

[0091] [Example 6] The antiviral activity of PLNC8 αβ against enveloped viruses was examined using Kunjin virus (KUNV), a flavivirus closely related to LGTV. Like LGTV, it sheds its lipid envelope from the ER. KUNV (1 × 10 5(Figure 7A) or D-enantiomers (Figure 7B) of PLNC8α, PLNC8β at a final concentration of 20 μM or PLNC8αβ at a final concentration of 0.1 μM, 1 μM, 5 μM, 10 μM, and 20 μM (1:1 molar ratio) for 1 h at room temperature. The suspension was then added to Vero cells and incubated for 1 h, followed by quantification of the viral load by performing a plaque assay. Concentrations shown are in μM. Representative images of viral plaques are presented above the bars. These images show enhanced staining of cells, indicative of cell survival, as a result of efficient clearance of virions in a dose-dependent manner for L-PLNC8αβ and D-PLNC8αβ, respectively. Viral particles were rapidly permeabilized and formed large aggregates by both the L- and D-enantiomers of PLNC8 αβ (data not shown), resulting in a reduction in viral load by >99.9%. Similarly, PLNC8 β alone was found to be more efficient than PLNC8 α in clearing KUNV (Figure 7).

[0092] [Example 7] Furthermore, PLNC8 αβ efficiently eliminated KUNV even at low peptide concentrations and regardless of the initial viral load. KUNV at multiplicities of infection (MOI) of 0.1, 0.01, and 0.001 were exposed to increasing concentrations of L-PLNC8 αβ or D-PLNC8 αβ (1:1) for 1 h, followed by infection of Vero cells and quantification of viral load. Final peptide concentrations of 0.1 μM and 1 μM reduced viral load by 80% and 95%, respectively, while concentrations of ≧10 μM completely eliminated all virions (FIG. 8). Thus, it has been shown that both the L-enantiomer and the D-enantiomer of PLNC8 αβ can reduce viral load even at low concentrations (0.01 μM).

[0093] [Example 8] The following experiment was designed to examine the antiviral activity of PLNC8 αβ on cells infected with KUNV to determine whether the peptide could affect viral replication, intracellular viral load, and extracellular virion accumulation. Human lung cancer cells (A549) were infected with KUNV for 1 h, followed by removing the cell culture medium and adding fresh medium (DMEM supplemented with 2.5% FBS) containing L- or D-PLNC8 αβ at a final concentration of 10 μM. After 48 h, the cell culture medium was collected and the cells were harvested. Viral replication was quantified by RT-PCR. A single dose of L-PLNC8 αβ or D-PLNC8 αβ reduced KUNV mRNA levels by 78% and 87%, respectively (FIG. 9A). Interestingly, both enantiomers were equally efficient in counteracting virus-induced cytotoxicity, since the number of viable cells was increased compared to infected and untreated cells (control; white bars). PLNC8 αβ reduced the extracellular virion load by 70-80% (Figure 9B). Extracellular virions were quantified in cell culture medium. The substantial reduction in viral mRNA levels and intracellular virions could be the result of efficient clearance of extracellular virions, i.e., prevention of viral dissemination between cells. However, whether the peptide can cross the plasma membrane, interfere with viral replication and / or target intracellular viruses is currently under investigation. Thus, it has been shown that PLNC8 αβ targets mature extracellular viruses and causes a substantial reduction in extracellular virions.

[0094] Our observations of the antiviral properties of PLNC8 αβ prompted us to determine its activity against other enveloped viruses, such as SARS-CoV-2.

[0095] [Example 9] SARS-CoV-2 virus (1 × 10 3(100 ml) were either left untreated (data not shown) or exposed to the L-enantiomer (FIG. 10A) or D-enantiomer (FIG. 10B) of PLNC8 β or PLNC8 αβ (1:1) at the indicated concentrations for 1 h at 37° C. The suspensions were then used to infect Vero cells and the viral load was quantified by performing a plaque assay. Both enantiomers of PLNC8 αβ caused a substantial reduction in the viral load in a dose-dependent manner. We show that both enantiomers of PLNC8 αβ were remarkably efficient against SARS-CoV-2 and caused a substantial reduction in the viral load in a dose-dependent manner (FIG. 10). Regarding the SARS-CoV-2 virus concentrations used, a 50% reduction in PFU with the L-form of PLNC8 αβ was achieved at 0.01 μM, whereas L-PLNC8 β alone required 0.9-1 μM (Figure 10A). The D-form of PLNC8 αβ was more efficient against SARS-CoV-2, achieving a 50% reduction at a final concentration of 0.005 μM, whereas D-PLNC8 β alone required 1.4 μM (Figure 10B).

[0096] The lipid envelope of flaviviruses is derived from the ER, whereas coronaviruses utilize a vesicle-tubule transport system between the ER and the Golgi apparatus, i.e., the ER-Golgi intermediate compartment (ERGIC). 27~29 The membrane properties of the ER and Golgi apparatus are similar in that both expose anionic lipids in their outer leaflets and contain low levels of cholesterol compared to the plasma membrane. 17,18 The hypothesis of this study is based on these membrane properties and knowledge explaining the apparent membrane activity of PLNC8 αβ, in addition to our previous results showing that PLNC8 αβ does not exhibit cytotoxic effects. The hypothesis was tested by examining the antiviral activity of PLNC8 αβ against enveloped viruses that acquire their lipid barrier from the plasma membrane, e.g., influenza A virus and HIV-1.

[0097] [Example 10] Influenza A virus (1 × 10 3 10 μM) were pretreated with the L-enantiomer (FIG. 11A) or D-enantiomer (FIG. 11B) of PLNC8 β or PLNC8 αβ (1:1) for 1 h at 37°C. The suspension was used to infect Vero cells, and the viral load was subsequently quantified. L-PLNC8 αβ caused a 50% reduction in influenza A PFU at 20 μM (FIG. 11A), while 10 μM of D-PLNC8 αβ was sufficient to cause the same reduction in PFU (FIG. 11B). Higher concentrations of PLNC8 β alone were required to inhibit influenza A, but the D type was more effective than the L type. Influenza A was inhibited by 50% at 19 μM of the D type and >20 μM of the L type of PLNC8 β, respectively.

[0098] In conclusion, it has been shown that clearance of influenza A requires 100-1000-fold higher peptide concentrations to achieve an antiviral effect comparable to that of peptides against flaviviruses and SARS-CoV-2.

[0099] [Example 11] HIV-1 (1 × 10 3 1000 cells) were treated with the L-enantiomer (Figure 12A) or D-enantiomer (Figure 12B) of PLNC8 β or PLNC8 αβ (1:1) for 1 h at 37 °C. The suspension was then used to infect isolated primary PBMCs, followed by quantification of viral load. HIV-1 was shown to be less sensitive to PLNC8 αβ, since a final concentration of 50 μM caused approximately 20% inhibition for the D-enantiomer. However, the 50% reduction concentration could not be calculated with respect to the HIV-1 viral titer (TCID50 / 0.1 ml) used (Figures 12A-B). In conclusion, elimination of HIV-1 requires 1000 μM to achieve an effect comparable to that of flaviviruses and SARS-CoV-2. 3 ~10 4 It has been shown that a 2-fold increase in peptide concentration is required.

[0100] [Example 12] Furthermore, the host cell inflammatory response was analyzed after infection of human cells with KUNV in the presence or absence of PLNC8 αβ. The results in Table 2 below show that gene expression of 12 genes (tlr3, tlr9, nod1, nod2, rankl, c-fos, c-jun, il-1β, il-6, tnf-α, cxcl8, and ccl-5) was slightly altered by peptides L-αβ and D-αβ, respectively. Human lung cancer cells (A549) were infected with KUNV for 1 hour and subsequently treated with 10 μM L- or D-PLNC8 αβ for 48 hours. Gene expression of inflammatory markers was analyzed by RT-PCR. KUNV induced gene expression of all analyzed inflammatory markers except nod1 and rankl, and the presence of peptides altered these effects by promoting cell viability. Furthermore, microscopy analysis demonstrated that both enantiomers of PLNC8 αβ counteracted KUNV-induced cytotoxicity (FIG. 13A). Quantitative cytotoxicity data are presented in FIG. 13B.

[0101] [Table 2]

[0102] [Example 13] In the following experimental setup, three doses of PLNC8 αβ at three different concentrations (0.1 μM, 1 μM, and 10 μM) were administered to KUNV-infected cells once every 24 h. After 72 h, the viral load was quantified and showed that final concentrations of L-PLNC8 αβ at 0.1 μM, 1 μM, and 10 μM caused 40%, 60%, and >90% inhibition of extracellular virions, respectively (Figure 14). The D-form of PLNC8 αβ was significantly more potent than the L-form at lower concentrations (0.1 μM and 1 μM), causing an 80-96% reduction in the number of infectious virions.

[0103] [Example 14] In vivo experiments will be performed as follows: The development of formulations with PLNC8 αβ will be mainly focused on topical application, a non-invasive and effective approach. The route of administration will be intranasal to cover the largest possible area of ​​epithelial surfaces. Transgenic hACE2 mice (Mus musculus), hamsters (Mesocricetus auratus), and / or ferrets (Mustela putorius) will be used as animal models with intranasal infection with SARS-CoV-2, which has been shown to develop symptoms similar to humans with COVID-19. Animal experiments will be performed under strict regulations of the Ethics Committee for Animal Experimentation and with all appropriate ethical approvals. The antiviral activity of the novel peptides will be tested after intranasal administration for prophylactic (administration before SARS-CoV-2 infection) and therapeutic (administration after SARS-CoV-2 infection) purposes. Changes in health status, including body temperature, weight, movement and posture, possible skin changes, appetite, bowel function, and respiration, will be described daily. Blood samples will be collected during the experiment to determine the effects on the immune system, inflammatory response, hematological and chemical changes, and viral titers using standardized laboratory techniques (flow cytometry, multiplex analysis, ELISA, RT-PCR, immunohistochemistry, plaque assay). In addition, nasal secretions and lung lavage fluids will also be collected. These results will provide a proof of concept for the therapeutic effect of preventing and / or treating viral infections with the antiviral peptide PLNC8 αβ.

[0104] References [Table 3-1]

[0105] [Table 3-2]

Claims

1. 1. A pharmaceutical composition for use in the prevention and / or treatment of viral infections and / or disorders associated with viral infections caused by an enveloped virus, comprising: a) a peptide having at least 90%, e.g., 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO:1, and / or b) a peptide having at least 90%, e.g., 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO:

2.

23. A pharmaceutical composition comprising:

2. The pharmaceutical composition for use according to claim 1, wherein at least one amino acid residue of the peptides (a) and / or (b) is a D-amino acid residue.

3. 3. A pharmaceutical composition for use according to claim 1 or 2, comprising from about 1 nM to about 1000 μM, such as from about 10 nM to about 100 μM, of the peptides (a) and / or (b).

4. 4. The pharmaceutical composition for use according to any one of claims 1 to 3, comprising peptides (a) and (b) in a molar ratio of about 1:1 to about 20:1, such as about 1:1 to about 10:1, such as about 1:

1.

5. 5. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the viral infection is caused by an enveloped virus selected from the group consisting of the following viral families: Coronaviridae, Flaviviridae, Herpesviridae, Orthomyxoviridae, Retroviridae, Paramyxoviridae, Filoviridae, Pneumoviridae, Arteriviridae, Asfarviridae, Bunyaviridae, Hepadnaviridae, Poxviridae, Togaviridae, and Rhabdoviridae.

6. The viral infection is caused by an enveloped virus, for example an enveloped virus selected from the group consisting of the following viral families: Coronaviridae, Flaviviridae, Herpesviridae, Arteriviridae, Asfarviridae, Bunyaviridae, Hepadnaviridae, and Poxviridae, the envelope of which is obtained from the endoplasmic reticulum, Golgi apparatus, or nuclear envelope of the infected cell, and the viral infection is caused by an enveloped virus, for example an enveloped virus selected from the group consisting of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2), Middle East Respiratory Syndrome Coronavirus (SERS-CoV-3), Severe Acute Respiratory Syndrome Coronavirus (SERS-CoV-4), Severe Acute Respiratory Syndrome Coronavirus (SERS-CoV-5), Severe Acute Respiratory Syndrome Coronavirus (SERS-CoV-6), Severe Acute Respiratory Syndrome Coronavirus (SERS-CoV-7), Severe Acute Respiratory Syndrome Coronavirus (SERS-CoV-8), Severe Acute Respiratory Syndrome Coronavirus (SERS-CoV-9), Severe Acute Respiratory Syndrome Coronavirus (SERS-CoV-10), Severe Acute Respiratory Syndrome Coronavirus (SERS-CoV-11), Severe Acute Respiratory Syndrome Coronavirus (SERS-CoV-12), Severe Acute Respiratory Syndrome Coronavirus (SERS-CoV-13), Severe Acute Respiratory Syndrome Coronavirus (SERS-CoV-14), Severe Acute Respiratory Syndrome Coronavirus (SERS-CoV-15), Severe Acute Respiratory Syndrome Coronavirus (SERS-CoV-16), Severe Acute Respiratory Syndrome Coronavirus (SERS-CoV-17), Severe Ac The disease may be caused by any of the following: Human Immunodeficiency Virus (HERS-CoV), Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2), Epstein-Barr Virus (EBV), Human Cytomegalovirus (HCMV), Varicella-Zoster Virus (VZV), Dengue Virus (DENV), Zika Virus (ZIKV), West Nile Virus (WNV), Langat Virus (LGTV), and Tick-borne Encephalitis Virus (TBEV), of which the West Nile Virus is a genotype that is not specifically defined as a genotype. Virus (WNV) is a subtype of Kunjin virus (KunjinThe pharmaceutical composition for use according to any one of claims 1 to 5, wherein the composition is selected from the group consisting of :

7. The pharmaceutical composition for use according to any one of claims 1 to 6, wherein said viral infection is a respiratory tract infection and / or a mucus layer infection.

8. A pharmaceutical composition for use according to any one of claims 1 to 7, which is administered locally, for example topically, to or near the site of infection.

9. 9. The pharmaceutical composition for use according to any one of claims 1 to 8, formulated as a powder, solution, cream, gel, ointment or in immobilized form as a coating on a medical device, said solution may be an aerosol and / or take the form of a nasal or oral spray, said medical device may be a face mask, air filter, nasal cannula device, or an endotracheal tube.

10. 10. The pharmaceutical composition for use according to any one of claims 1 to 9, formulated for administration in a single dose or multiple doses, for example 2, 3, 4 or 5 times daily for 1 to 20 days.

11. 11. The pharmaceutical composition for use according to any one of claims 1 to 10, wherein the disorder associated with viral infection is selected from the group consisting of viral induced inflammation, viral induced cell death, viral induced tissue destruction, and combinations thereof, and the viral induced tissue destruction may be selected from the group consisting of damage to mucosal surfaces, pulmonary fibrosis, organ failure, and combinations thereof.

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