Peptides for use in therapy or prophylaxis of Herpesviridae-infections

Abstract

The present invention relates to new peptides the amino acid sequences of which are derived from HD-5, for use in the treatment and/or prevention of Herpesviridae infections, in particular for treating and/or preventing a Betaherpesviridae infection, such as a HCMV-infection.

Claims

1. Peptide for use in treating, inhibiting and/or preventing viral infections in mammals, wherein the viral infection is caused by a member of the family Herpesviridae, wherein the peptide comprises a sequence having the following formula I:
A.sub.1-B.sub.1-X.sub.1-C.sub.1-X.sub.2-Z.sub.1-B.sub.2-A.sub.2-Z.sub.2  (Formel I) wherein A.sub.1 und A.sub.2 are identical or different, and each A.sub.1 und A.sub.2 is a nonpolar amino acid having an aliphatic or basic side chain, and each is preferably Alanin or Glycin, B.sub.1 and B.sub.2 are identical or different, and each B.sub.1 and B.sub.2 is a polar amino acid and having a hydroxylated side chain, and is preferably selected from Serin, Threonin, or Tyrosin, C.sub.1 is an polar amino acid having a aromatic side chain, and is preferably Tyrosin, X.sub.1 and X.sub.2 each is Cystein, and Z.sub.1 and Z.sub.2 each is Arginine.

2. The peptide for use of claim 1, wherein the peptide has an amino acid sequence that comprises 9 successive amino acids having the sequence ATCYCRTGR (SEQ ID No. 1) or the reverse sequence of SEQ ID No. 1, RGTRCYCTA (SEQ ID No. 2) of the attached sequence listing, or having a sequence that has at least 70% sequence identity with SEQ ID No. 1 or 2 of the attached sequence listing.

3. The peptide for use of claim 1, wherein the peptide consists of the SEQ ID No. 1.

4. The peptide for use as claimed in any of claims 1 to 3, wherein the peptide is a chemically synthesized or a biologically expressed peptide.

5. The peptide for use as claimed in any of claims 1 to 4, wherein the member of the Herpesviridae family is a member of the Betaherpesviridae subfamily, and preferably is selected from at least one of the following: human Cytomegalovirus (HCMV; HHV-5), Herpes Simplex Virus 1 (HSV-1; HHV-1), Herpes Simplex Virus 2 (HSV-2; HHV-2), Varicella Zoster Virus (VZV; HHV-3), Epstein-Barr Virus (EBV; HHV-4), Human Herpes Virus 6 (HHV-6), Human Herpes Virus 7 (HHV-7), or Kaposi's sarcoma-Associated Herpesvirus (KSHV; HHV-8).

6. The peptide for use as claimed in claim 1, wherein the Cytomegalovirus is human Cytomegalovirus (HCMV).

7. The peptide for use as claimed in claim 6, wherein the HCMV is a multi-resistant HCMV strain.

8. The peptide for use as claimed in any of claims 1 to 7, wherein the peptide is used in combination with another anti-infective agent.

9. The peptide for use as claimed in any of claims 1 to 8, wherein the peptide is used in a concentration of at least 1 μM, preferably 10 μM, and preferably of at most 150 μM.

10. Peptidomimetic for use in treating, inhibiting and/or preventing viral infections in mammals, wherein the viral infection is caused by a member of the family Herpesviridae, and wherein the peptidomimetic is derived from the peptide as claimed in any of claims 1 to 9.

11. Pharmaceutical composition for use in treating, preventing and/or inhibiting a Herpesviridae infection, the Pharmaceutical composition comprising a peptide as claimed in any of claims 1 to 9 and/or the peptidomimetic as claimed in claim 10, and a pharmaceutically acceptable carrier, vehicle or diluent.

12. A method of treating, preventing, or inhibiting a Herpesviridae infection comprising administering a therapeutically effective amount of the peptide of any of claims 1 to 9, the peptidomimetic as claimed in claim 10, or the pharmaceutical composition of claim 11 to a subject.

13. The peptide for use as claimed in any of claims 1 to 9, the peptidomimetic as claimed in claim 10, the pharmaceutical composition of claim 11 or the method of claim 12, wherein the peptide, the peptidomimetic or the pharmaceutical composition is administered to a subject that it selected from an immuno-compromised subject, pregnant women, newborns, or infants.

14. The peptidomimetic as claimed in claim 10, the pharmaceutical composition of claim 11, or the method of claim 12, wherein the peptidomimetic or the pharmaceutical composition is used for treating, preventing or inhibiting an infection caused by a member of the Betaherpesviridae, and is preferably selected from at least one of the following: Cytomegalovirus (HCMV; HHV-5), Herpes Simplex Virus 1 (HSV-1; HHV-1), Herpes Simplex Virus 2 (HSV-2; HHV-2), Varicella Zoster Virus (VZV; HHV-3), Epstein-Barr Virus (EBV; HHV-4), Human Herpes Virus 6 (HHV-6), Human Herpes Virus 7 (HHV-7), or Kaposi' sarcoma-Associated Herpesvirus (KSHV; HHV-8).

Description

[0069] In the figures:

[0070] FIG. 1 (A) shows a diagram displaying the results of experiments treating HCMV-infected HFF with different concentrations of HD 5-derived peptides; HFF were infected with TB40E-ΔUL16-EGFP and treated with peptides in different concentrations. After 40 h of incubation, cells were fixed and stained with DAPI, and the infection rate was analysed (GFP+/DAPI+, n=1); (B) shows a diagram displaying results of the MTT assay; HFF were treated with peptides in different concentrations; after 40 h of incubation, MTT stock solution was added and crystals were dissolved; absorption was measured (n=3);

[0071] FIG. 2 (A) shows a diagram displaying the results of experiments where different cell types were infected with TB40E-ΔUL16-EGFP and treated with HD5 (1-9) in different concentrations. After 40 h of incubation cells were fixed and an antibody staining for IE-antigens was performed; afterwards, nuclei were stained with DAPI. The infection rate was analysed (IE+/DAPI+, n=3, THP-1 n=1); (B-D) shows a diagram displaying the results of experiments, were different cell types were treated with the different peptides testing their toxicity: HFF (B), ARPE (C) and macrophages (D) were seeded in a culture dish with a microelectrode network on the bottom; after 24 h cells were treated with peptides in different concentrations; impedance was measured via xCELLigence every 30 min for 72 h (n=3, macrophages n=1);

[0072] FIG. 3 shows a diagram displaying results of experiments testing HD5 (1-9) on different clinical HCMV-isolates; HFF were infected with different clinical HCMV isolates and TB40/E-ΔUL16-eGFP as reference strain with an MOI of 0.2 (A) or 0.1 (B) and treated with HD5 (1-9) (SEQ ID No. 1) in different concentrations. After 40 h incubation, cells were fixed and HCMV-infected cells identified by IE1/2 antigen staining. Nuclear staining was done with DAPI. Infection rates were measured by imaging with a microplate imager and automated counting of DAPI+ and IE1/2+ cells. The graphs show the calculated infection rate (IE1/2+/DAPI+, normalized to medium only, mean±SD from duplicate infections each).

[0073] FIG. 4 (A) shows a diagram displaying the results of experiments where HFF were infected with TB40E-ΔUL16-EGFP and treated with HD5 (1-9) and its derivatives; (B) HFF were infected with TB40E-ΔUL16-EGFP and treated with HD5 (1-9) and its derivatives in different concentrations; after 40 h of incubation, cells were fixed and an antibody staining for IE-antigens ware performed; afterwards nuclei were stained with DAPI; the infection rate was analysed (IE+/DAPI+, n=3); HD5 (1-9): ATCYCRTGR (SEQ ID No. 1); HD5 (1-13): ATCYCRTGRCATR (SEQ ID No. 3); ns: not significant, **** p<0.0001, *** p<0.001; and

[0074] FIG. 5 (A) shows a diagram displaying the results of experiments where HFF were infected with TB40E-ΔUL16-EGFP and treated with HD5 (1-9) in three different concentrations; in the first condition there was no preincubation of virus and peptide; in the second condition the peptide in the corresponding concentrations was preincubated with the peptide, in the third condition the peptide was preincubated with the virus in 100 μM and then diluted to 10 μM; preincubation was 1 h at 37° C.; after 40 h of incubation, cells were fixed and an antibody staining for IE-antigens ware performed; afterwards nuclei were stained with DAPI; the infection rate was analysed (IE+/DAPI+, n=3); (B) in the first condition, HFF were preincubated with peptide in different concentrations 3 h at 37° C., in the second condition the peptide was added at the time of infection and in the third condition the peptide was added 3 h after infection; HFF were infected with TB40E-ΔUL16-EGFP and after 40 h of incubation cells were fixed and an antibody staining for IE-antigens ware performed; afterwards nuclei were stained with DAPI; the infection rate was analysed (IE+/DAPI+, n=3).

EXAMPLES

[0075] Materials and Methods

[0076] Infection Assays:

[0077] For the infection assays, the cells were sown in 96-well plates. Human foreskin fibroblasts (HFF) and ARPE-19 cells (Adult Retinal Pigment Epithelial cell line-19) were seeded with 10 000 cells in 200 μl medium per well, macrophages with 20 000 cells in 200 μl medium per well and THP with 50 000 cells in 200 μl per well. The cells were incubated overnight at 37° C. and 5% CO.sub.2 and treated the following day. A medium change to P/S-free medium was performed to avoid interaction between the antibiotics used and the peptide sequences. Afterwards the corresponding peptides were added in different concentrations and immediately afterwards the virus, the final volume was 100 μl per well. The virus of the strain TB40/E-ΔUL16-eGFP was used. TB40-ΔUL16-eGFP is a derivative of the strain TB40, in which the majority of the open reading frame UL16 has been replaced by the open reading frame of eGFP, essentially as described in detail for a homologous mutant of strain AD169 (Tischer et al., “Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli”, Biotechniques, (2006), 40(2):191-7). TB40-delUL16-eGFP expresses eGFP under the control of the early UL16 promotor and has previously been used for FACS analyses (Sinzger et al., “Macrophage cultures are susceptible to lytic productive infection by endothelial-cell-propagated human cytomegalovirus strains and present viral IE1 protein to CD4+ T cells despite late downregulation of MHC class II molecules”, J. Gen. Virol., (2006), 87:1853-62). Depending on the experiment, different MOI (“multiplicity of infection”) were used, some of which were titrated beforehand. For HFF and ARPE-19, an absolute infection rate of 80% was targeted. For myeloid cells such a high infection rate could not be achieved, therefore an absolute infection rate of 40% was aimed at. The required volume of the virus stock was calculated using the titer according to the following formula:

Volume in ml=(1/(titer in IU/ml))×(cell number in well×MOI)

[0078] After a further 40 h incubation period, the cells were fixed with 150 μl 2% paraformaldehyde (PFA) in PBS per well (10 min at 37° C., 20 min at room temperature or overnight in the refrigerator) and permeabilized with 200 μl-20° C. cold 90% methanol in water for 20 min in the refrigerator. Then an antibody staining on IE-positive cells was performed, as this allows a very precise quantification of the infection rate. This was done with HCMV IE E13 as first antibody, of which 100 μl per well of a 1:1000 dilution in PBS were incubated for 90 min with the cells. As second antibody Goat anti-Mouse IgG (H+L) Alexa Fluor 594 was used, of which per well 100 μl of a 1:2000 dilution in PBS for 60 min were incubated with the cells. Finally, nuclear staining with DAPI was performed by leaving 100 μl per well of a 1:20 000 dilution of the DAPI stock solution (2 mg DAPI+1 ml PBS) in PBS for 8 min at room temperature on the cells. Between each step, the cells were washed three times with PBS. In the last step the PBS was left on the cells and the plate was stored at 4° C. in the refrigerator. Images were taken in the Cytation™ reader (BioTek Instruments), for evaluation. The infection rate was calculated by relating the number of IE positive signals to the number of DAPI stained nuclei. To determine the relative infection rate, the corresponding infection rate was related to the mean infection rate of the untreated or solvent-treated HCMV-infected cells. Technical duplicates or triplicates were used in the experiments.

[0079] Infection Assays with Clinical Isolates

[0080] The antiviral activity of HD5 (1-9) (SEQ ID No. 1) in concentrations from 1.56 μM to 100 μM against infection with clinical HCMV isolates was investigated on HFF. In addition to the laboratory-adapted strain TB40/E-ΔUL16-eGFP, which served as reference strain, a breast milk-derived strain, an amniotic fluid-isolated strain and a multidrug-resistant viral isolate from leukocytes of a recipient after the third stem cell transplantation were used. The therapy-naïve strain from cell free milk whey (H1241-2016) was derived from a mother of a preterm infant 10 weeks postpartum during end of viral reactivation. The amnion fluid derived virus strain (H2497-2011) was isolated following termination of pregnancy based on severe fetal brain damage. The multidrug resistant CMV isolate (H815-2006) is already described (Gohring et al., “Dynamics of coexisting HCMV-UL97 and UL54 drug-resistance associated mutations in patients after haematopoietic cell transplantation”, J. Clin. Virol. 57(1):43-49, 2013). This viral isolate showed the canonical UL97 mutation L595S and an UL54 mutation V715M, leading to drug resistance against GCV, 1050=31.5 μM, and CDV, IC50=795 μM. IC50 value against PFA was 1.8 μM. All viral isolates were primarily HFF-adapted and propagated in vitro with at least 10 passages to get TCID values of cell free viral supernatants of 105 to 106/ml.

[0081] MTT:

[0082] In this test the cell viability of HFF was tested after treatment with the peptides HNP4, HNP4 (1-11), HNP4 (1-11mod), HD5, HD5 (1-9), HD5 (1-9mod), HD5 (1-13), HD5 (1-28), HD5 (7-32), HD5 (10-27), HD5 (10-32) and HD5 (26-32) in different concentrations. The assay is based on the reduction of the yellow water-soluble dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in blue-violet water-insoluble 2,3,5-Triphenyltetrazolium chloride with the aid of the pyridine-containing reduction equivalents NADH (reduced form of nicotinamide adenine dinucleotide) and NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) (Berridge et al. “Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction”, Arch. Biochem. Biophys. 303(2): 474-82 (1993)). For the screening tests 10 000 HFF in 200 μl medium per well were sown in 96-well plates. The cells were incubated overnight at 37° C. and 5% CO.sub.2 and treated the following day. A medium change to P/S-free medium was performed to avoid interaction between the antibiotics used and the peptide sequences. Afterwards, the peptides were added, such, that peptide concentrations of 7.5 μM and 75 μM were obtained analogous to the screening tests for antiviral activity. The final volume was 100 μl per well. The plate was incubated for 40 h, followed by a media change to 90 μl P/S and phenol red free medium. Then 10 μl per well of MTT stock solution (5 mg MTT+1 ml phenol red free DMEM) were added and carefully resuspended. After a further 3 h incubation period, the medium was removed and the crystals were dissolved in 100 μl 0.04 M hydrochloric acid each in isopropanol per well 10 min on the shaker. The absorption was then measured in the Cytation™ reader at 570 and 650 nm. For evaluation, the mean absorption value measured in empty wells was subtracted from all measured absorption values. To determine absolute absorption, the absorption values of the reference wavelength 650 nm were subtracted from the values at 570 nm. To determine the relative absorption, the corresponding value was related to the mean value of the absorption of the HFF treated with 0,01% acetic acid. Technical triplicates were used.

[0083] Impedance Measurement:

[0084] The “xCELLigence” system (ACEA Biosciences Inc.) was used for a more detailed toxicity study. The principle is based on a measurement of the electrical resistance caused by adhesive cells on the bottom of a 96-well plate equipped with microelectrodes. Cell proliferation causes an increase in resistance due to the partial isolation of the electrodes, while events leading to altered cell morphology or cell detachment lead to reduced resistance (Diemert et al. “Impedance measurement for real time detection of neuronal cell death”, J Neurosci Methods, 203(1):69-77, 2012)). The experiments were performed with HFF, ARPE-19 and macrophages, the appropriate cell numbers were determined in a preliminary experiment without treatment with peptides.

[0085] On the first day 10 000 HFF or ARPE-19 or 20 000 macrophages per well were sown. First, 50 μl of the corresponding medium were added to the wells of the 96-well plate, the spaces between the wells were filled with 100 μl PBS each to prevent dehydration. Then a blank value was measured and the cells were added in corresponding numbers in 150 μl medium each, the final volume was 200 μl per well. The plate was placed in the “xCELLigence” and the impedance was measured at 37° C. and 5% CO.sub.2 over 24 h every 30 min. The plate was then removed and a media change and treatment with the peptides were performed. The peptide concentrations corresponded to a 1:2 dilution series from 100 μM to 1.56 μM. A positive control was performed with 10% tritone. Afterwards, the resistance at the bottom of the 96-well plate was measured every 30 min for a further 72 h. The resistance of the tritone was measured with a positive control of 10% tritone. From the measured impedance values, the RTCA software calculated the so-called cell index according to the following formula:

ZI(t)=(R(f.sub.n,t))−R(f.sub.n,t.sub.0))/Z.sub.n

[0086] ZI(t)=cell index at time t

[0087] R(f.sub.n, t)=measured impedance at frequency f.sub.n at time t

[0088] R(f.sub.n, t.sub.0)=measured impedance at frequency f.sub.n at time t.sub.0 (background measurement with medium without cells)

[0089] Z.sub.n=Corresponding frequency factor to frequency f.sub.n

[0090] To determine the normalized cell index, the cell index at time t was divided by the value of the measurement after 24 hours. The measurement after 24 h was the last before the treatment with the peptides, so the comparability of the values was ensured independent of the total cell count.

[0091] Experiments with Zebrafish

[0092] To test HD5 (1-9) (SEQ ID No. 1) for toxicity and to assess its effect on embryonic development in zebrafish, embryos obtained from natural crosses of wildtype TE fish were used. After mating, embryos were incubated at 28° C. until 6 h postfertilization (hpf). Triplicates of five embryos each were then placed in wells of a 96-well plate containing 200 μl of different peptide concentrations. HD5 (1-9) (SEQ ID No. 1) dissolved in PBS was diluted in normal embryo medium (250 mg/L Instant Ocean salt, 1 mg/L methylene blue in reverse osmosis water adjusted to pH 7 with NaHCO.sub.3) (Muller et al., “Differential diffusivity of Nodal and Lefty underlies a reaction-diffusion patterning system”; Science 336 (6082):721-724, 2012). The concentrations tested were 25 μM, 75 μM, 125 μM and 250 μM, and the concentration of PBS solvent was adjusted for each control group. At peptide concentrations of 75 μM and above we observed granulae in some wells, which could represent peptide accumulations. As a control, embryos were additionally incubated in 25 μg/ml cycloheximide (C4859, Sigma-Aldrich). After 13 hpf, 24 hpf and 48 hpf a microscopic phenotype analysis was performed. For each time point embryos were automatically imaged using an ACQUIFER Imaging Machine. For the phenotype analysis at 48 hpf, the larvae were manually dechorionated and anaesthetized with 2% tricaine methane-sulfonate (A5040-25G, Sigma-Aldrich). Images were acquired on an Axio Zoom.V16 microscope (ZEISS).

[0093] Results

[0094] In FIG. 1, the results of MTT-assays showing that defensin-derived peptides have antiviral activity against HCMV and don't show toxicity in an MTT assay are shown. 10 000 HFF were seeded into each well of a 96-well-plate containing 200 μl of medium. Cells were incubated overnight at 37° C. and 5% CO2. Next day medium was changed to medium without Penicillin/Streptomycin (P/S). Cells were infected with TB40E-ΔUL16-EGFP (MOI 0,5) and treated with peptides in concentrations of 7.5 μM and 75 μM in a volume of 100 μl. After 40 h of incubation cells were fixed with 2% PFA in PBS, permeabilised with 90% methanol in H.sub.2O and stained with DAPI. Infection rate was analysed (GFP+/DAPI+, normalized to solvent control, n=1).

[0095] Further, 10 000 HFF were seeded into each well of a 96-well-plate containing 200 μl of medium. Cells were incubated overnight at 37° C. and 5% CO.sub.2. Next day medium was changed to medium without P/S and cells were treated with peptides in concentrations of 7.5 μM and 75 μM in a volume of 100 μl. After 40 h of incubation medium was changed and 10 μl of MTT stock solution was added. 3 h later medium and stock solution were removed and crystals were dissolved in 100 μl of 0.04 M hydrochloric acid in isopropyl. Absorption was measured (normalized to solvent control, n=3), and the results are shown in FIG. 1B: (ns: not significant, **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05, upper symbol: 75 μM, lower symbol: 7.5 μM).

[0096] As can be seen (see, FIG. 1A), HD5 (1-9), a peptide according to the invention has an excellent antiviral effect in a concentration of 75 μM as compared to other tested peptides. Also, in un-infected corresponding cells (see FIG. 1B); there was no negative effect of this peptide as compared to other peptides, even in a concentration of 75 μM.

[0097] In FIG. 2, the results of experiments showing that HD5 (1-9) (=SEQ ID No. 1) inhibits HCMV infection of different cell types from a concentration of 50 μM and shows no cytotoxicity up to 150 μM are shown.

[0098] Different cell types were seeded into a 96-well-plate, each well containing 200 μl of medium (HFF and ARPE-19: 10 000 cells per well, THP-1: 50 000 cells per well) (THP-1 is a monocyte cell type); all cell types are obtainable, e.g. at the ATCC (American Type Culture Collection). Cells were incubated overnight at 37° C. and 5% CO2. Next day medium was changed to medium without P/S. Cells were infected with TB40E-ΔUL16-EGFP (HFF and ARPE-19: MOI 0,5; THP-1: MOI 10) and treated with peptide in concentrations from 1.56 μM to 100 μM in a volume of 100 μl. After 40 h of incubation cells were fixed with 2% PFA in PBS and permeabilised with 90% methanol in H.sub.2O. Afterwards an antibody staining for IE-antigens was performed and nuclei were stained with DAPI. Infection rate was analysed and the results are shown in FIG. 2A (IE+/DAPI+, normalized to medium control, HFF and ARPE-19: n=3, THP-1: n=1). As can be taken from FIG. 2A, HD5 (1-9) displayed a HCMV-infection inhibitory effect on different infected cell types from a concentration of 50 μm.

[0099] FIG. 2B to D show the results for HFF (B), ARPE (C) and macrophages (D), which were seeded in a culture dish with a microelectrode network on the bottom (HFF and ARPE-19: 10 000 cells per well, macrophages: 20 000 cells per well, volume of 200 μl). Cells were incubated 24 h at 37° C. and 5% CO2. Next day medium was changed to medium without P/S and cells were treated with peptide in concentrations from 1.56 μM to 100 μM in a volume of 100 μl. Cells with peptide were incubated 72 h at 37° C. and 5% CO2. Impedance was measured via xCELLigence every 30 min, cell index was calculated from measured impedance and normalized to cell index after 24 h (HFF and ARPE-19: n=3, macrophages: n=1). As can be taken from FIG. 2, a peptide of the invention, i.e. HD5 (1-9) does not show any cytotoxicity up to 150 μM on the tested different cell types.

[0100] Next, a peptide of the invention, i.e. HD5 (1-9) was tested on clinical HCMV isolates. 10 000 HFF were seeded into each well of a 96-well-plate containing 200 μl of medium. Cells were incubated overnight at 37° C. and 5% CO.sub.2. Next day medium was changed to medium without P/S. Cells were infected with different clinical HCMV isolates (MOI 0,2) and treated with peptide in concentrations from 1.56 μM to 100 μM in a volume of 100 μl. After 40 h of incubation cells were fixed with 2% PFA in PBS and permeabilised with 90% methanol in H.sub.2O. Afterwards an antibody staining for IE-antigens was performed and nuclei were stained with DAPI. Infection rate was analysed (IE+/DAPI+, normalized to medium control, n=1).

[0101] HD5(1-9) Inhibits Multiresistant, Primary HCMV Isolates

[0102] HCMV TB40/E is a lab-adapted strain that might differ from primary HCMV isolates in terms of cellular tropism, infectivity or cytopathic properties. We hence tested whether HD5 (1-9) (SEQ ID No. 1) is also active against primary HCMV isolates from different compartments of patients including amniotic fluid, breast milk and leukocytes (FIG. 3). Of note, the leukocyte isolate from a stem cell transplant recipient is genotypically (mutations UL97 L595S, UL54 V715M) and phenotypically resistant against GCV (IC50>30 μM), PFA (1050=795 μM), and CDV (1050=1.8 μM) (Gohring et al., 2013), while both isolates from amniotic fluid and breast milk were therapy-naive. We infected HFF cells at MOIs of 0.2 (FIG. 3A) and 0.1 (FIG. 3B) in the presence of increasing amounts of HD5 (1-9) (SEQ ID No. 1). Similar to our previous results, HD5 (1-9) (SEQ ID No. 1) inhibited HCMV infection efficiently at a concentration of 100 μM in HFF. Importantly, not only infection with the lab-adapted TB40/E strain was blocked, but also infection with the primary isolates was sensitive towards inhibition by HD5(1-9) (SEQ ID No. 1). Hence, HD5 (1-9) (SEQ ID No. 1) is a peptide inhibitor active against primary as well as multiresistant HCMV strains.

[0103] As can be taken from FIG. 3, HD5 (1-9) also inhibited infection with all clinical isolates tested starting at a concentration of 50 μM.

[0104] Next, amino acid substitutions of a peptide according to the invention (HD5 (−9) were tested in view of alterations in their antiviral effects. In FIG. 4, the results of experiments testing the antiviral activity of HD5 (1-9) and its derivatives comprising different amino acid substitutions on TB40E-ΔUL16-EGFP infected HFF cells are shown.

[0105] 10 000 HFF were seeded into each well of a 96-well-plate containing 200 μl of medium. Cells were incubated overnight at 37° C. and 5% CO.sub.2. Next day medium was changed to medium without P/S and cells were infected with TB40E-ΔUL16-EGFP (MOI 0,2). Cells were treated with HD5 (1-9) and its derivatives in concentrations from 1.56 μM to 100 μM (A) and HD5 (1-13) and its derivative (B) in concentrations of 7.5 μM and 75 μM in a volume of 100 μl. After 40 h of incubation cells were fixed with 2% PFA in PBS and permeabilised with 90% methanol in H.sub.2O. Afterwards an antibody staining for IE-antigens was performed and nuclei were stained with DAPI. Infection rate was analysed (IE+/DAPI+, normalized to medium control, n=3). HD5 (1-9): ATCYCRTGR, (SEQ ID NO. 1) HD5 (1-13): ATCYCRTGRCATR (SEQ ID No. 3) ns: not significant, **** p<0.0001, *** p<0.001.

[0106] As can be taken from FIG. 4A, the Arginine and Cysteine residues in a peptide according to the invention are crucial for the antiviral effect of HD5 (1-9), since alternations in these amino acids lead to a diminished, if not abolished, antiviral activity.

[0107] Next, experiments for identifying the specific mechanism of the antiviral effect of the peptides of the invention were assessed. In FIG. 5, a diagram displaying results showing that HD5 (1-9) has an antiviral effect against HCMV infection by directly interacting with the cells is shown.

[0108] 10 000 HFF were seeded into each well of a 96-well-plate containing 200 μl of medium. Cells were incubated overnight at 37° C. and 5% CO.sub.2. Next day medium was changed to medium without P/S. Cells were infected with TB40E-ΔUL16-EGFP (MOI 0,2) and treated with peptide in concentrations from 1.56 μM to 100 μM in two different conditions: in the first condition there was no preincubation of virus and peptide, in the second condition the peptide in the corresponding concentrations was preincubated with the virus. In an additional condition the peptide was preincubated with the virus in 100 μM and then diluted to 10 μM in a volume of 100 μl. Preincubation was 1 h at 37° C. After 40 h of incubation cells were fixed with 2% PFA in PBS and permeabilised with 90% methanol in H.sub.2O. Afterwards an antibody staining for IE-antigens was performed and nuclei were stained with DAPI. Infection rate was analysed (IE+/DAPI+, normalized to medium control, n=3).

[0109] Also, in another experiment, 10 000 HFF were seeded into each well of a 96-well-plate containing 200 μl of medium. Cells were incubated overnight at 37° C. and 5% CO.sub.2. Next day medium was changed to medium without P/S. Cells were infected with TB40E-ΔUL16-EGFP (MOI 0,2) and treated with peptide in concentrations from 1.56 μM to 100 μM in two different conditions: In the first condition HFF were preincubated with peptide in a volume of 60 μl 3 h at 37° C., peptide was concentrated in such a way, that after infection in a total volume of 100 μl concentrations from 1.56 μM to 100 μM were reached. In the second condition the peptide was added at the time of infection and in the third condition the peptide was added 3 h after infection. After 40 h of incubation cells were fixed with 2% PFA in PBS and permeabilised with 90% methanol in H.sub.2O. Afterwards an antibody staining for IE-antigens was performed and nuclei were stained with DAPI. Infection rate was analysed (IE+/DAPI+, normalized to medium control, n=3).

[0110] With the results displayed in FIG. 5, a direct interaction of the peptide of the invention with the cells was shown, rather than an inhibitory effect on the virus as it is the case with the presently available antiviral substances used for treating HCMV-infections.

[0111] In additional experiments, UL328 (encoding for pp150) was GFP (green fluorescence protein)-tagged, UL100 (encoding for gM) was mCherry-tagged. This allowed discrimination between enveloped particles, which were EGFP and mCherry-positive and appeared yellow, and non-enveloped particles, which were only EGFP-positive and appeared green.

[0112] HFF were preincubated with peptide in the corresponding concentration 1 h at 37° C. and then infected TB40-BAC.sub.KL7-UL32EGFP-UL 100mCherry. After 6 h of incubation, cells were fixed and stained with DAPI.

[0113] The results achieved with these experiments (data not shown) showed that HD5 (1-9) blocks the attachment of viral particles.

[0114] Effect of HD5(1-9) on Embryonic Development

[0115] As a first test of HD5 (1-9) (SEQ ID No. 1) toxicity in vivo, as well as to elucidate potential effects of the peptide on embryonic development, experiments with zebrafish embryos were performed (He et al., 2014). Embryos were treated with HD5 (1-9) (SEQ ID No. 1) at concentrations of 25 μM, 75 μM, 125 μM and 250 μM starting at 6-7 h post fertilization (hpf), which is the time point recommended by pharmaceutical company-utilized assays to assess toxic effects of compounds on early embryonic development (Ball et al., 2014). Phenotypes were analyzed at 13.24 and 48 hpf (data not shown). At the highest peptide concentration of 250 μM, half of the embryos had died by 13 hpf, indicating that excess of HD5 (1-9) (SEQ ID No. 1) can affect embryonic development. Impaired development at 250 μM peptide exposure was also evident over the whole observation period, leading to the death of 11 embryos and 4 embryos having severe developmental delays by 48 hpf. Reduction of the peptide concentration to 125 μM reduced the negative effects on embryonic development, with approximately half of the embryos having no alterations (data not shown). Furthermore, all embryos treated with 25 μM or 75 μM of HD5 (1-9) (SEQ ID No. 1) developed normally, except for one embryo that had died by 13 hpf due to unknown reasons. Altogether, HD5 (1-9) (SEQ ID No. 1) concentrations of 25 μM-75 μM, which are higher than the IC50 of ˜40 μM, do not affect zebrafish embryonic development or show toxicity in vivo.