NL2027924B1 - Antimicrobial peptide for prevention and treatment of virusinfections - Google Patents

Abstract

The invention relates to an antimicrobial peptide for use in a method of preventing or treating viral infections. The invention further relates to a composition comprising an antimicrobial peptide and a pharmaceutically acceptable diluent or excipient, for use in a method of preventing or treating and/or virus—infection related disorders. The invention also relates to a method of treating a patient suffering from virus—infection related disorders, the method comprising administering a therapeutically effective amount of an antimicrobial peptide thereof the human or animal is affected by viruses and or disorders.

Description

ANTIMICROBIAL PEPTIDE FOR PREVENTION AND TREATMENT OF VIRUS INFECTIONS DESCRIPTION:

TECHNICAL FIELD OF THE INVENTION The invention relates to the prevention and treatment of virus infections with an antimicrobial peptide. The invention further relates to an antimicrobial peptide for use in the treatment or for use in the prevention of virus infections and to a pharmaceutical composition comprising such antimicrobial peptide.

BACKGROUND OF THE INVENTION With the recent emergence of a pandemic of Corona virus (SARS-CoV-2 virus causing Covid-19), the need for improved vaccines against viral infections has become an international priority. Although strategies for vaccine development have improved significantly over time, we are still facing major challenges in delivering those vaccines by 2020. New technologies of the next generation, as represented by real-time DNA sequencing, provide a lot of data on viral pathogens and host cells. This allows us to 'mine' the genomic sequence for putative vaccine candidates or targets, allowing for a rational approach to vaccine design. The problem here is that it takes a lot of time and research to achieve this. It is expected that the first vaccine against Covid-19 will not come on the market until the end of 2020. Unfortunately, we cannot prevent that for a lot of people this too late become available.

SUMMARY OF THE INVENTION The current invention provides an improved therapy option in that, in one aspect, it provides an antimicrobial peptide for use in a method of preventing or treating virus infections, wherein the antimicrobial peptide for use in a method of preventing or treating the virus infection in a human or animal subject, wherein the antimicrobial peptide consists of any one of the amino acid sequences RRRRSVQWCA [SEQ ID NO: 1}, GRRRRSVQWCA [SEQ ID NO: 3], ACWQVSRRRR [SEQ ID NO: 2}, ACWQVSRRRRG [SEQ ID NO: 4}, CGRRRRSVQWCA [SEQ ID NO: 5}, or a functional mutant thereof. Such antimicrobial peptide, derived from the protein human lactoferrin (hLF), has been shown to be very effective in combating virus infections.

The antimicrobial peptide further shows no toxicity or other side-effects when topically applied orally or topical in human subjects suffering from infection of viruses.

In addition to innate immunity, in which antimicrobial proteins and peptides are of great value, plays acquired immunity (the evocation of specific antiviral antibodies) a role against the protection of viruses. Under immune compromised conditions, such as diseases, diabetes, the sensitivity to these viral infections increases greatly. Strengthening our own defense system (first line of defense) could be another approach in the fight against this virus. These cells (called Host defense peptides - HDPs) are multifunctional inducers and effectors of our immunity. immunomodulatory activity also directly respond to viral infectious processes. Whose antiviral activity, demonstrated against both envelope containing and naked viruses, is in the early phase of infection, preventing the virus from entering the host cell. Binding to specific receptors, viral parts or both. A number of peptides can bind to these binding sites, greatly inhibiting the attachment of a virus to its “target” cell, the first stage of the virus infection. On the other hand, the peptide can bind directly to the envelope protein, thus inhibiting the infectivity of the virus.

It has been demonstrated in experimental research that these peptides are active against various viruses, including HCV, Hepatitis B, HIV, and HPV. The advantage of these peptides is that they are endogenous to the body, so they do not cause rejection or adverse effects to the host. Safety profile has been clinically tested in HSCT patients. Experimental research has shown that strong inhibition of viruses can be achieved with relatively low doses. It is also commercially attractive, due to the favourable production price of a peptide when compared to producing a whole protein such as human lactoferrine itself (hLF). However, the invention as found and claimed does not rule out that the peptide is conjugated to other molecules such as non-hLF peptides or proteins, sugar molecules, LPS, aluminium hydroxide etc. in order to influence its chemical, physical, immunogenic or metabolic properties. Thus, the restriction that the current peptide consists of one of the peptides as referred to here above and in the appended claims, does not rule out that molecules other than the natural residual parts of the hLF protein are coupled to the small peptide itself. Such coupling could for example be the result of a process that takes place after production of the peptide, or could be the result of a recombinant process wherein an expression system is used to express the peptide as claimed, coupled to another molecule (anything but the natural counterpart of the peptide as found in hLF). Typically the peptide is part of a polypeptide having a length of at maximum 200 amino acids, preferably less than 150, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or even 12 amino acids.

In a second aspect, the invention provides a composition comprising an antimicrobial peptide and a pharmaceutically acceptable diluent or excipient, for use in a method of preventing or treating a virus infection in a human or animal subject, wherein the antimicrobial peptide consists of any one of the amino acid sequences RRRRSVQWCA {SEQ ID NO: 1], GRRRRSVQWCA {SEQ ID NO: 3}, ACWQVSRRRR {SEQ ID NO: 2], ACWQVSRRRRG {SEQ ID NO: 4}, CGRRRRSVQWCA [SEQ ID NO: 5}, or a functional mutant thereof. Such composition is preferably odourless and colourless in order to improve patients’ compliance.

In a third aspect, the invention provides a method of treating a patient suffering from virus infections, the method comprising administering a therapeutically effective amount of an antimicrobial peptide consists of any one of the amino acid sequences RRRRSVQWCA [SEQ ID NO: 1}, GRRRRSVQWCA [SEQ ID NO: 3], ACWQVSRRRR [SEQ ID NO: 2}, ACWQVSRRRRG [SEQ ID NO: 4], CGRRRRSVQWCA [SEQ ID NO: 5}, or a functional mutant thereof.

AMINO ACID SEQUENCES & SEQ ID NOs Amino acid sequence relating to human lactoferrin peptide 2-11 (hLF2- 11): Arg Arg Arg Arg Ser Val Gln Trp Cys Ala {SEQ ID NO: 1] Amino acid sequence relating to artificial reverse human lactoferrin peptide 2-11 (Reverse hLF2-11): Ala Cys Trp Gln Val Ser Arg Arg Arg Arg [SEQ ID NO: 2] Amino acid sequence relating to human lactoferrin peptide 1-11 (hLFI- 11): Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala {SEQ ID NO: 3} Amino acid sequence relating to artificial reverse human lactoferrin peptide 1-11 (Reverse hLF1-11): Ala Cys Trp Gln Val Ser Arg Arg Arg Arg Gly [SEQ ID NO: 4]

Artificial Amino acid sequence relating to human lactoferrin peptide 1-11 provided with an N-terminal Cysteine (cysteine - hLF1-11): Cys Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala [SEQ ID NO: 5]

DEFINITIONS The embodiments of the invention described herein can operate in combination and cooperation, unless specified otherwise.

Furthermore, the various embodiments, although referred to as “preferred” or “e.g.” or “for example” or “in particular” are to be construed as exemplary manners in which the invention may be implemented rather than as limiting the scope of the invention.

The term “comprising”, used in the claims, should not be interpreted as being restricted to the elements, compounds or steps listed thereafter; it does not exclude other elements or compounds or steps. It needs to be interpreted as specifying the presence of the stated features, integers, steps, compounds or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps, compounds or components, or groups thereof. Thus, the scope of the expression “a composition comprising A and B” should not be limited to a composition consisting only of compounds A and B, rather with respect to the present invention, the only enumerated compounds of the composition are A and B, and further the claim should be interpreted as including equivalents of those compounds.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the effect of hFL-11 peptide on HCV viremia in HCV-Trimera mice.

Figures 2 and 3 are graphs showing the effect of hFL-11 peptide on SARS-CoV-2 infection in VERO cells.

DETAILED DESCRIPTION OF THE INVENTION In a first embodiment, the invention provides an antimicrobial peptide for use in a method of preventing or treating virus infections, wherein the antimicrobial peptide comprises the amino acid sequence GRRRRSVQWCA or ACWQVSRRRRG,

or a functional mutant thereof. The antimicrobial peptide preferably comprises or consists of the peptide sequence GRRRRSVQWCA. WO2009028943 describes the antimicrobial effect of antimicrobial peptides comprising the sequence GRRRRSVQWCA and ACWQVSRRRRG. WO2009028943 further shows mutations 5 in said sequence, which show at least the same effect as the original sequence.

More in particular, WO2009028943 describes that the following mutations / deletions in a sequence of the invention are equally well or even better than the non-mutated sequences: at least one of the amino acids S, V, Q or W is deleted; one R and at least one of the amino acids S, V, Q or W is deleted; S, V or Q are substituted with a conservative or non-conservative amino acid, provided that (i) the substitute is not a negatively charged amino acid and (ii) S is not substituted with an N; SV, VQ or SQ are substituted with conservative or non-conservative amino acids, provided that none of the substitutes is a negatively charged amino acid; SVQ are substituted with conservative or non-conservative amino acids, provided that none of the substitutes is a negatively charged amino acid; W is replaced by another aromatic amino acid; Q or S is substituted by an aromatic amino acid and W is substituted by a neutral amino acid; C is switched with an amino acid in the SVQW sequence; at least one but no more than three of the R's are substituted by another positively charged amino acid; or the second, third or fourth R is substituted by an A.

Therefore, in a preferred embodiment, a functional mutant of the invention is chosen from the group consisting of: a deletion mutant in which at least one of the amino acids S, V, Q or W is deleted; a deletion mutant in which one R and at least one of the amino acids S, V, Q or W is deleted; a substitution mutant in which S, V or Q are substituted with a conservative or non-conservative amino acid, provided that (1) the substitute is not a negatively charged amino acid and (ii) S is not substituted with an N; a double substitution mutant in which SV, VQ or SQ are substituted with conservative or non-conservative amino acids, provided that none of the substitutes is a negatively charged amino acid; a triple substitution mutant in which SVQ are substituted with conservative or non-conservative amino acids, provided that none of the substitutes is a negatively charged amino acid; a substitution mutant in which W is replaced by another aromatic amino acid; a double substitution mutant in which Q or S is substituted by an aromatic amino acid and W is substituted by a neutral amino acid; a substitution mutant in which C is switched with an amino acid in the SVQW sequence; a substitution mutant in which at least one but no more than three of the R's are substituted by another positively charged amino acid; a substitution mutant in which the second, third or fourth R is substituted by an A; and any combination thereof.

Such antimicrobial peptide may be formulated in a pharmaceutical composition.

In a second embodiment, therefore, the invention provides a composition comprising an antimicrobial peptide and a pharmaceutically acceptable diluent or excipient, for use in a method of preventing or treating virus infections, wherein the antimicrobial peptide comprises the amino acid sequence GRRRRSVQWCA or ACWQVSRRRRG, or a functional mutant thereof.

The antimicrobial peptide preferably comprises or consists of the peptide sequence GRRRRSVQWCA.

Such composition is preferably odourless and, preferably colourless, in order to improve patients’ compliance.

A colourless and odourless composition can be applied at any time.

An antimicrobial peptide of the invention thus comprises the amino acid sequence RRRRSVQWCA or ACWQVSRRRR, or a functional mutant thereof.

In a preferred embodiment, an antimicrobial peptide of the invention consists of the sequence RRRRSVQWCA, GRRRRSVQWCA, ACWQVSRRRRG or ACWQVSRRRR, or a functional mutant thereof.

In a preferred embodiment, the antimicrobial peptide consists of GRRRRSVQWCA.

In one preferred embodiment, the peptide has been made cyclic.

Various methods of cyclization are known to the skilled person, e.g., cys-cys cyclization, backbone-cyclization (= amide bond formation), or thio-ether cyclization. cys-cys cyclization stems from formation of a disulfide (S-S) bond between the thiol side chains of two cysteine residues in a peptide.

One disadvantage of cys-cys cyclized peptides is the limited stability of the SS bond, especially under reductive conditions.

Amide bond cyclization is also frequently used for cyclic peptides.

The bond is chemically more stable than a cys-cys bond.

Backbone- amide cyclized peptides are in general prepared via head-to-tail cyclization or side- chain-to-side-chain cyclization.

Thio-ether cyclization is performed by reacting the side chain thiol group of a cysteine with the alpha-carbon atom of another amino acid.

The reacting amino acids may already be comprised within the sequence of a peptide according to the invention or may be added for this specific purpose.

In order for performing cys-cys cyclization in a peptide consisting of the sequence GRRRRSVQWCA, an additional cysteine may for instance be added.

CRRRRRSVQWCA may then be cyclized by reacting the thiol groups of both cysteines present.

A peptide according to the invention may further be glycosylated. The skilled person is aware how to chemically glycosylate a peptide as, e.g., described by Crich [15]. An antimicrobial peptide according to the invention may, further to the RRRRSVQWCA or ACWQVSRRRR sequence, or a functional mutant thereof, comprise other amino acids, preferably positively charged amino acids, such as arginine (R) or lysine (K).

An antimicrobial peptide according to the invention may, further to the QWCKQWCK sequence, or a functional mutant thereof, comprise other amino acids, preferably positively charged amino acids, such as arginine (R).

An antimicrobial peptide according to the invention may, where various modifications or a functional mutant thereof, consists a changed C -Terminal, such as Amidation or N-Terminal, such as Acetylation.

In a preferred embodiment, a composition for use according to the invention is provided, wherein the antimicrobial peptide is present in a concentration of between | mg/kg/bodyweight and 16 mg/kg/bodyweight, preferably between 2 mg/kg/bodyweight and 8 mg/kg/bodyweight.

The composition is preferably provided as either a “high concentration therapy” or a “low concentration therapy”, or both. With high and low concentration therapy is meant that the antimicrobial peptide is present in the composition for use according to the invention in a concentration of about 8 mg/kg or of about 640 mg, respectively.

The composition is preferably provided as either a “very high concentration therapy”. With very high concentration therapy is meant that the antimicrobial peptide is present in the composition for use according to the invention in a concentration of about 16 mg/kg/bodyweight, respectively.

In one preferred embodiment, therefore, a composition for use according to the invention is provided, wherein the antimicrobial peptide is present in a concentration between 1 mg/kg/bodyweight and 4 mg/kg/bodyweight, most preferably of about 2,5 mg/kg/bodyweight. These lower concentrations are in particular useful for maintenance therapy after high dose therapy or for prevention of (new) infections.

The antimicrobial peptide for use according to the invention or composition for use according to the invention is preferably administered at least once daily. Alternatively, the antimicrobial peptide for use according to the invention or composition for use according to the invention is administered at least once every two days, more preferably at least once every two days for at least 2 weeks. More preferably, the peptide or composition is administered at least daily for at least two weeks, more preferably at least 1 month, more preferably for at least 2 months. Preferably, the peptide or composition is administered at least twice a day. More preferably, the peptide or composition is administered at least twice a day for at least 1 month by very serious virus infections.

An antimicrobial peptide as used in the invention has been shown to be broadly active towards a variety of viruses Non-limiting examples of fungi that can be treated with an antimicrobial peptide of the invention are, e.g., In one embodiment, the invention provides a method of treating a patient (either human or non-human) suffering from virus infection, the method comprising administering a therapeutically effective amount of an antimicrobial peptide comprising the sequence RRRRSVQWCA or ACWQVSRRRR, or a functional mutant thereof to human body affected by Viruses.

The embodiments of the peptide for use according to the invention, as illustrated here below and claimed in the appended claims, correspond to embodiments for the second and third aspect of the invention, viz. the composition and the method of treatment.

EXAMPLES Example 1 The composition comprising an antimicrobial peptide of the invention and application thereof The composition used comprises the antimicrobial peptide hLF1-11 (GRRRRSVQWCA) [SEQ ID NO: 3} and is a clear and colorless liquid in a (glass) bottle with a dropper or lotion spray / cream to facilitate application to the skin or hairs of human subjects. The molecular mass of antimicrobial peptide hLF1-11 (GRRRRSVQWCA) [SEQ ID NO: 3} is 1415.6 Da. The composition does not contain preservatives or perfume. The antimicrobial peptide is completely biodegradable. The solution is an aqueous solution comprising 0,01% acetic acid based on the volume of the peptide solution (the composition). The concentration was 3 mg/l antimicrobial peptide based on the total weight of the composition. The composition is suitable for use in the treatment of viruses in a human subject, that are affected by a viral infection.

Furthermore, the composition of the invention comprising the antimicrobial peptide is suitable for use in the treatment or the reduction of symptoms of skin (HPV)..

Moreover, the topical application of the composition comprising the antimicrobial peptide onto the skin of a human subject is suitable for stimulating the natural barrier of the skin (defense system) by preventing new damage by infectious microorganisms such as fungi, bacteria, parasites, mites, algae and viruses. Thus, part of the invention is the composition of the invention comprising the hLF(1-11) peptide [SEQ ID NO: 3] for use in maintaining, controlling, and/or improving the natural barrier function of the skin of a human subject, wherein the natural barrier function of the skin is the prevention or reduction of colonization of said skin by a microbe and/or the prevention or reduction of invasion of the skin and body of said human subject by a microbe such as a bacterium and/or a fungus.

Changes in the microbiota and changes in the immune system can disturb the equilibrium provided by the natural barrier function of the skin of a human subject, and may lead to a range of skin diseases, once the skin does not function properly anymore as the natural barrier against colonization of the skin by a microbe and/or against invasion of the human body by a microbe by entering the body through the skin.

The use of the composition comprising the antimicrobial peptide improves the desired and natural well-balanced mix of fungi and bacteria (skin flora), typically existing on a healthy skin. The composition has an immunological effect in the sense that topical application of the composition onto the skin of a human subject maintains or improves the barrier function against microbial infection of the body, which barrier function is provided for by a healthy skin, and application of the composition of the invention provides for an improved or maintained natural protection against colonization, invasion and infection by the fungi and or bacteria that cause dermatophytosis. The first visible improvements are usually observed after two weeks of twice daily topical application of the composition of the invention onto the inflicted skin which is infected by said fungus, fungi, bacterium and/or bacteria and viruses.

Tested viruses: All MIC values ( Cyto Pathologic Effect = CPE) for the following viruses were found to be between 40 — 640 microgram/mL - Human cytomegalovirus - Human Herpes Simplex virus-1 (HSV) - Human immuno deficiency virus - Hepatitis C virus (in vivo 1 and 4 mg/kg in mice) - Polio virus - Rota virus - Respiratory Syncytial virus (RSV) - Human Metapneumo virus (hMPV) - Adenovirus - Enterovirus - Epstein Barr virus - Corona virus (SARS-CoV-2 causing COVID-19)

PROTOCOLS Basic info Cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging), the effects of drugs and toxic compounds on the cells, and mutagenesis and carcinogenesis. It is also used in drug screening and development, and large scale manufacturing of biological compounds (e.g., vaccines, therapeutic proteins). The major advantage of using cell culture for any of the these applications is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells.

Viral Propagation Protecol Propagation in Cell Culture A number of ATCC viruses are propagated in cell culture. Typically, propagation hosts are grown in tissue culture vessels (such as T flasks) using media and reagents specified for the host cell line. Most cell lines are seeded the day prior to setting up an infection and should not be seeded more than two days in advance, nor passaged more than 9 times prior to infection. In addition to setting up cells for infection, negative control cells should also be set up to monitor cellular health.

1. Identify the recommended propagation host as indicated on the product sheet. Plan to use the propagation host.

2. Prepare the cell growth medium for growing the host cell line.

3. Prepare the virus growth medium as recommended on the product sheet.

4. One to two days prior to inoculation seed the host cells. Be sure to include a vessel that will not be inoculated with virus to serve as a negative control.

5. Allow cells to reach the appropriate confluency.

6. Prior to thawing the frozen virus stock check the virus titer listed on the certificate of analysis. Quickly thaw the virus in a 37°C water bath.

7. Dilute the virus stock in the appropriate volume of viral growth medium.

8. Remove the cell growth medium from the cell culture flasks.

9. Inoculate the diluted virus to provide an optimal MOI as indicated on the product sheet.

10. Incubate tissue cultures under the appropriate temperature and atmospheric conditions for the recommended incubation period. 11, After the recommended incubation time period, check the flask for cytopathic effects (CPE) when applicable. Tissue Culture Infectious Dose (TCID) Viral titer can be determined in vitro by calculating the infectious dose. For tissue culture-adapted strains, this calculation is ascertained through an endpoint dilution assay in cell culture. The most reproducible endpoint of the dilution assay is the dilution of the virus that will produce a pathological change in 50% of the cell cultures inoculated. This number is expressed as 50% the infectious dose, or TCIDso, which is analogous to the calculation for lethal dose 50. The accuracy of this method is related to the number of replicates at each dilation”.

1. Prepare a cell culture plate with the recommended cell line for viral propagation (preferably the same cells that the virus being tested was grown in).

2. Incubate the plate under the appropriate conditions for cell growth until cells reach an optimum density for infection.

3. Prepare viral dilutions in base medium.

4. Remove cell growth medium from plate, the monolayer may be washed with remove any inhibitory agents.

5. Inoculate at least 3 wells with each dilution. Be sure to inoculate wells with base medium to serve as negative controls. Use a fresh, sterile pipette for each dilution.

6. Allow the plate to incubate for 1 to 2 hours under conditions suitable for virus adsorption.

7. Add viral growth medium and incubate the plate under conditions suitable for the virus and observe all wells daily. Record results for each well daily.

8. The endpoint is determined when the CPE or immunofluorescence assay (IFA) read-out appear the same per dilution for 3 separate readings.

9, The titer is calculated using the method of Reed and Muench. A titer expressed as 10(3.0)TCIDso/0.2 mL in 3 days in XXXX cell line may be translated as: 0.2 mL of virus diluted at 1:1000 will infect 50% of the cells in 3 days when using XXXX cell line. XXXX cell line can be HeLa cells, MRC-5 or Vero cell lines.

Example 2 Effect of hLF1-11 peptide on HCV viremia in HCV-Trimera mice Evaluation of the effects of hLF1-11 peptide on established viremia in the HCV - Trimera mouse Model. This study consists of three experimental groups:

1 Control, saline treated animals (i.p.: Q.D.; n=16). 2 Treatment with 1 mg/kg/day of hLF1-11 peptide (i.p.; Q.D.; n=15). 3 Treatment with 4 mg/kg/day of hLF1-11 peptide (i.p.; Q.D.; n=16).

Treatment with saline solution of hLFI-11 peptide or with saline (control) was given from day 7 to day 20 post transplantation (total of 14 days). Bleeds were taken at day 21 (one day post treatment completion). Sera samples were further evaluated.

Results: Day 21 post transplantation Group Mean viral load + SE % of HCV - positive mice (HCV-RNA copies/ml serum)

1. Control, saline treated Animals (i.p.; Q.D.; n=16). 6.55x10" + 1.0x10" 88 (14/16)

2. Treatment with 1 mg/kg/day of hLFI-11 (i.p.; Q.D.; n=15). 7,83x10° + 2.0x10° 53 (8/15)

3. Treatment with 4 mg/kg/day of hLF1-11 (i.p.; Q.D.; n=15). 3.28x10° £ 5.0x107 19 (3/16) The results are also summarized in figure 1. The percentages of HC V-positive mice are given in parentheses and the viral loads are given as Mean Viral Load + SE. Differences in percentages of HCV-positive animals between control and treated groups of mice are compared using Fisher’s exact test analysis. Differences in viral loads between control and treated groups of mice (group pairs) are compared by the non-parametric Mann-Whitney U test.

The effect of hFL-11 peptide on HCV viremia in HCV-Trimera mice is shown in the graph of Figure 1. Conclusions: I. In this experiment and under specific conditions hLF1-11 peptide shows anti-HCV activity in the HCV- Trimera mouse model. This means that the peptide can be used for treating an existing infection. Starting the administration before the infection actually occurs thus is useful per se in preventing the infection (i.e. at least preventing unhindered infection as if no protection was available).

2. In this experiment and under specific conditions hLF1-11 peptide did not cause observable toxic effects in the mice.

3. Results should be confirmed in repeated experiments in order to decide how to proceed (dose range). Blood samples should be taken one day post- treatment completion and 3-5 days later (post effect).

4. Itis suggested to use, as negative control, a non-active peptide (scrambled peptide (MW= 1,5 KDa).

Example 3 Effect of hLF1-11 peptide on hMPV and RSV infections This study consists two times of three experimental groups: 1 Control, saline treated animals (i.p.: Q.D.; n=5). 2 Treatment with 1 mg/kg/day of hLF1-11 peptide (i.p.; Q.D.; n=5). 3 Treatment with 4 mg/kg/day of hLF1-11 peptide (i.p.; Q.D.; n=5). Treatment with saline solution of hLFI-11 peptide or with saline (control) was given from day -1 to day 11 post transplantation (total of 12 days). Bleeds were taken at day 12 (one day post treatinent completion). Sera samples were further evaluated. Standard protocols

Viruses: Virus stocks tor human metapneumovirus (hMPV) and respiratory syncytia virus (RSV) were grown on a Vero cell line. After harvesting, both viruses were titrated on Vero cells.

Animals: SPF (specific pathogen free) female Balb/C mice (6-10 weeks old). Animals are kept in filtertop cages and are given standard food and water ad libitum. Animals are left to acclimatize to their new environment for one week prior to start of the experiment.

Infection of mice: Mice are anesthesized by inhalation of isoflurane N20/02 and intranasally infected with either hMPV or RSV (5x10° TCIDS0 in a total volume of 50 pl). Animals are subsequently placed on a heated plate to recover from anesthesia. Each day, animals are observed for signs of illness or discomfort and the body weight of each individual animal is measured (starting three days prior to infection). At the desired timepoints post infection, animals are sacrificed and euthanised by CO2/02. Treatment with antivirals: Starting 1 day prior to virus infection, animals are injected i.p. daily with antiviral preparation (total volume 100 pl). Analysis: From the sacrificed animals, the trachea and lungs are harvested and homogenized. From these tissue preparations, virus titrations are performed to determine the number of infectious virus particles in the lungs. RNA isolated from the same material will be used to determine the number of viral RNA copies. Both virus titration and quantification of the viral RNA serve as a measure for the replication of the virus in the lungs. Results: Day 12 post transplantation Group 1 Mean viral load + SE (RSV-RNA copies/ml serum)

1. Control, saline treated Animals (i.p.; Q.D.; n=5). 5.55x10° + 1.0x10°

2. Treatment with 1 mg/kg/day of hLF1-11 (i.p.; Q.D.; n=5). 3,99x10° 2 2.0x10°

3. Treatment with 4 mg/kg/day of hLF1-11 (i.p.; Q.D.; n=5). 4.16x10* £ 5.0x 10° Group 2 Mean viral load + SE (hMPV-RNA copies/ml serum)

1. Control, saline treated Animals (i.p.; Q.D.; n=5). 5.71x10° + 1.0x10°

2. Treatment with 1 mg/kg/day of hLFI-11 (i.p.; Q.D.; n=5). 7.83x10* +2.0x10*

3. Treatment with 4 mg/kg/day of hLF1-11 (i.p.; Q.D.; n=5). 1.28x10% + 4.0x107 The percentages of RSV-positive mice and mHPV mice are given in parentheses and the viral loads are given as Mean Viral Load + SE.

Differences in percentages of mHPV or RSV-positive animals between control and treated groups of mice are compared using Fisher’s exact test analysis. Differences in viral loads between control and treated groups of mice (group pairs) are compared by the non-parametric Mann-Whitney U test. Conclusions: I. In this experiment and under specific conditions hLF1-11 peptide shows anti-RSV and anti-mHPV activity in the Balb/C mouse model.

2. In this experiment and under specific conditions hLF1-11 peptide did not cause observable toxic effects in the mice.

3. Results should be confirmed in repeated experiments in order to decide how to proceed (dose range). Blood samples should be taken one day post-treatment completion and 3-5 days later (post effect).

4. Itis suggested to use, as negative control, a non-active peptide (scrambled peptide (MW= 1,5 kDa).

Example 4 Synergistic effect of hLF1-11 peptide with ivermectin on Covid-19 infections Experimental outline: Pre-incubation of cells with hLF1-11 peptide and scrambled peptide (crtl): SARS CoV-2 inoculum was mixed with different hLF1-11 peptide concentrations (final concentrations 25 and 100 pg/ml), ivermectin (200 ug/ml) and combination of hLF1-11 and ivermectin or 0.01% acetic acid, and infected onto Vero cells at MOI 0.1 for 1 hr @ 37C for 3hrs. Then SARS pseudoviruses were added to the pre-incubated treated cells to test the effect of hLF and combination with ivermectin on SARS pseudovirus infection. Initial infection was done in DMEM without any supplements. Infection was done on Vero FM cells seeded in standard 24-well cell culture plates. After 1 hr incubation, viral inoculum was discarded, cells washed with PBS and replaced with infection medium containing the same concentration of peptide or acetic acid. Infection medium consists of DMEM containing 2% FBS, 20 mM HEPES and 1% antibiotics. Virus (dilution & quantification) protocol: virus titer patient 1 passage 3: 9.8x 106; 1:100 dilution, 50 ul virus and 4950 ul medium. MOI 0,1; 10,000 cells per well. Virus will be quantified by titration and/or qRT-PCR. As shown in Figures 2 and 3, the number of GFP expressing cells decreased sharply with increasing concentration of LF. Because cell entry of SARS CoV-2 leads to GFP expression, this result suggests that the infection of Vero cells by SARS CoV-2 can be dramatically inhibited in the presence of hLF1-11 and or ivermectin combined. Figures 2 and 3 show the antiviral activity of hLF1-11, Ivermectin, and a combination thereof, as a potent inhibitor of the SARS-CoV-2 virus. This was evaluated in an experiment wherein Vero cells were infected with SARS-CoV-2 clinical isolate (MOI =

0.1) for 3 h prior to addition of vehicle control (scrambled peptide), hLF1-11 and/or Ivermectin at the indicated concentrations.

Samples were taken at 2 days post infection for quantitation of viral load using real-time PCR of cell associated virus (A) or supernatant (B). IC50 values were determined in subsequent experiments at 48 h post infection using the indicated concentrations of Ivermectin (treated at 2 h post infection as per A/B). Results represent mean + SD (n = 3). Example 5 Antiviral activity of hLF1-11 peptide

Standard protocols Cell cultures: Vero cells, MA 104 cells, HeLA, MRC-5 or and HEp-2 were grown and maintained in minimum essential medium (MEM) supplemented with 10% fe-

tal bovine serum and 1% of different antibiotics PSA at 37°C in a humidified 5% CO, atmosphere.

Virus strains: AdenoV-5, HSV-typel, Cytomegalo and Rotavirus were propagated and titrated in Vero,

MA 104, HeLa, MRC-5 or and HEp-2 respectively.

Assays: Cytotoxicity: cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay.

Vero, HeLa, MRC-5, HEp-2, and or MA 104 cells were grown in 96-well microtiter plates for 24 h at 37°C in a humidified 5% CO; atmosphere.

Before incubation, media was replaced with fresh MEM containing serial dilutions of the peptides (100, 200 and 400 pg/mL) respectively.

After 72 (Rotavirus), 96 (HSV-type 1), and 120 h (Adenovirus and CMV) of incubation, cytotoxicity was assessed and expressed as CCso (concentration that reduced the absorbance of treated cells by 50% when compared to control - untreated cells). All assays were performed in triplicate.

Percentages of protection were calculated as [(A-B)/(C-B) x 100}, where A, B, and C indicate the absorbance of the peptides, virus and control cells, respectively.

The calculated ECs value was defined as the concentration that reduced the absorbance of infected cells to 50% when compared to infected cells and control cells. To determine whether the compounds inhibited virus replication by affecting their adsorption or penetration on the host cells another strategy was adopted by pretreatment assay; cells were grown as described above. Plates were incubated for 3 -6h with hLF1-11 peptide prior to virus infection. Percentages of inhibition of the different tested viruses by hLF1-11 peptide, obtained by MTT assay Adenovirus Cytomegalo vrius HSV-typel Rotavirus Conc. % Conc. % Conc % Conc. % hiF1-11 100 100 100 100 Direct 24.60 £5.70 18.66 + 4.67 24.06 + 13.19 18.66 + 4.67 Pre-incubated 36.67 16.61 31.69 + 14.09 23.77+5.70 31.69 + 14.09 hLF1-11 200 200 200 200 Direct 31.73 £ 5,46 32.14 £9.36 34.28 + 7.40 32.14 19.36 Pre-incubated 52.25 +9.27 51.41 £20.92 59.14 + 12.46 51.41 + 20.92 hiF1-11 400 400 400 400 Direct 61.54 £ 13.60 69.40 + 13.13 58.50 8.26 56.65 t 12.58 Pre-incubated 83.72 £15.59 87.46 + 27.42 70.91+ 12.46 85.17 £17.18 Conc: concentrations are expressed in pg/mL; %: results are expressed as mean t SDM of three separate experiments. Direct; and pre-treatment assay {cells were pre-incubated 3 - 6h prior to virus} infection. Conclusions:

1. In this experiment and under specific conditions hLF1-11 peptide shows antiviral activity against Adenovirus, Cytomegalovirus, HSV-type 1 and Rotavirus.

2. Results should be confirmed in repeated experiments in order to decide how to proceed (dose range, cytotoxicicy).

3. Itis suggested to use, as negative control, a non-active peptide (scrambled peptide (MW= 1,5 KDa).

SEQLTXT

SEQUENCE LISTING <110> CBMR Scientific Nanoscience B.V. <120> ANTIMICROBIAL PEPTIDE FOR TREATMENT AND CONTROLLING VIRUS INFECTIONS <130> P.CBMR/NL-3195 <140> NL2025294 <141> 2020-04-07 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Homo sapiens <400> 1 Arg Arg Arg Arg Ser Val Gln Trp Cys Ala 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Reverse hLF2-11 <400> 2 Ala Cys Trp Gln Val Ser Arg Arg Arg Arg 1 5 10 <210> 3 <211> 11 <212> PRT <213> Homo sapiens <400> 3 Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> Pagina 1

SEQLTXT

<223> Reverse hLF1-11

<400> 4

Ala Cys Trp Gln Val Ser Arg Arg Arg Arg Gly

1 5 10

<210> 5

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> cysteine - hLF1-11

<400> 5

Cys Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala

1 5 10

<210> 6

<211> 4

<212> PRT

<213> Artificial Sequence

<400> 6

Ser Val Gln Trp

1

<210> 7

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 7

Cys Arg Arg Arg Arg Arg Ser Val Gln Trp Cys Ala

1 5 10 Pagina 2

Claims (8)

CONCLUSIONS An antimicrobial peptide for use in a method of preventing or treating a viral infection in a human or animal, wherein the antimicrobial peptide consists of one of the amino acid sequences RRRRSVQWCA {SEQ ID NO: 1}, GRRRRSVQWCA [SEQ ID NO: 3] , ACWQVSRRRR [SEQ ID NO: 2}, ACWOVSRRRRG {SEQ ID NO: 4], CGRRRRSVQWCA [SEQ ID NO: 5], or a functional mutant thereof. Antimicrobial peptide for use according to claim 1, characterized in that the virus infection is selected from infection with human cytomegalovirus, human herpes simplex virus-1, human immunodeficiency virus, hepatitis C virus, polio virus, rotavirus, respiratory syncytial virus. virus, Human Metapneumovirus, Adenovirus, Enterovirus, Epstein Barr virus, Coronavirus (SARS-CoV-2). Antimicrobial peptide for use according to claim 1 or 2, characterized in that the virus infection is selected from infection with human cytomegalovirus, human herpes simplex virus-1, hepatitis C virus, rotavirus, respiratory syncytial virus, human metapneumovirus, Adenovirus, Corona virus (SARS-CoV-2). Antimicrobial peptide for use according to any one of the preceding claims, characterized in that the virus is Coronavirus (SARS-CoV-2). Antimicrobial peptide for use according to claim 4, characterized in that the peptide is used in combination with ivermectin. Antimicrobial peptide for use according to any one of the preceding claims, characterized in that the peptide is GRRRRSVQWCA or ACWQVSRRRRG. A composition comprising an antimicrobial peptide and a pharmaceutically acceptable diluent or excipient for use in a method of preventing or treating a viral infection in a human or animal, wherein the antimicrobial peptide consists of one of the amino acid sequences RRRRSVQWCA [SEQ ID NO : 1], GRRRRSVQWCA [SEQ ID NO: 3}, ACWQVSRRRR [SEQ ID NO: 2}, ACWQVSRRRRG [SEQ ID NO: 4], CGRRRRSVQWCA [SEQ ID NO: 5}, or a functional mutant thereof. A method of treating a patient suffering from viral infections, the method comprising administering a therapeutically effective amount of an antimicrobial peptide to patients afflicted with viral infections, wherein the antimicrobial peptide consists of one of the amino acid sequences RRRRSVQWCA [SEQ ID NO: 1], GRRRRSVQWCA [SEQ ID NO: 3}, ACWQVSRRRR [SEQ ID NO: 2}, ACWQVSRRRRG [SEQ ID NO: 4}, CGRRRRSVQWCA [SEQ ID NO: 5], or a functional mutant thereof.