Biological and synthetic molecules inhibiting respiratory syncytial virus infection

Abstract

The present invention relates to a peptide with a length of 25 amino acids or less comprising the sequence X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13 (SEQ ID No: 1) as well as to A peptide with a length of 25 amino acids or less comprising the sequence X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14 (SEQ ID No: 2). The present invention further relates to a nanostructure comprising a nucleic acid scaffold and at least two peptide moieties, wherein the sequence of each of the at least two peptide moieties is independently selected from the sequence of the peptide of the invention as well as pharmaceutical compositions, nucleic acids, methods and uses related thereto.

Claims

1. A peptide comprising a sequence selected from: TABLE-US-00005 (SEQIDNo:6) VVVSTTYLPHYFDN; (SEQIDNo:7) IVVSTTYLPHYFDN; (SEQIDNo:8) [2-Abu]VVSTTYLPHYFDN; (SEQIDNo:9) LV[Chg]STTYLPHYFDN; (SEQIDNo:10) LVVSTT[Tyr3-I]LPHYFDN; (SEQIDNo:11) [Chg][Cpa]VSTT[Tyr3,5-I2]LPHYFDN; or (SEQIDNo:12) [Chg][Nle][Chg]STTYLPHYFDN; wherein the peptide is no more than 25 amino acids in length, or a retro-inverso peptide thereof.

2. The peptide of claim 1, wherein the peptide is no more than 20 amino acids in length, or a retro-inverso peptide thereof.

3. The peptide of claim 1, wherein the peptide: comprises L-amino acids, D-amino acids, or a mixture thereof, comprises at least one backbone-modified amino acid, is a cyclic molecule, comprises at least one non-peptide moiety selected from a coupling group, a polyethylene glycol (PEG) moiety, a detectable label, a protective group, a lipid moiety and a sugar moiety, or comprises one or more post-translational modifications, and/or is covalently or non-covalently bound to a scaffold; or a retro-inverso peptide thereof.

4. The peptide of claim 1, consisting of a sequence selected from: TABLE-US-00006 (SEQIDNo:6) VVVSTTYLPHYFDN (SEQIDNo:7) IVVSTTYLPHYFDN (SEQIDNo:8) [-Abu]VVSTTYLPHYFDN (SEQIDNo:9) LV[Chg]STTYLPHYFDN (SEQIDNo:10) LVVSTT[Tyr3-I]LPHYFDN, (SEQIDNo:11) [Chg][Cpa]VSTT[Tyr3,5-I2]LPHYFDN,and (SEQIDNo:12) [Chg][Nle][Chg]STTYLPHYFDN, or a retro-inverso peptide thereof.

5. A pharmaceutical composition comprising the peptide of claim 1, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable adjuvants and/or excipients.

Description

FIGURE LEGEND

(1) FIG. 1: Scheme of the used inhibition experiments. RSV-plaque reduction assay on human epithelial cells (HEp-2). HEp-2 cells were seeded in 96-well plate 24 h prior to infection (left). Next day, cells were infected with RSV (middle, white arrow). Simultaneously, peptides at concentrations of 20 M were added to the cells (middle, black arrow). The inhibitory activity was determined 48 post infection by counting viral plaques after immunocytochemical staining using RSV-specific monoclonal antibody (right, black-checked arrow).

(2) FIG. 2: Antiviral activity of truncated peptides against RSV in vitro. The diagram shows on its y-axis the sequences of various differently truncated peptides, each represented by its own bar. The x-axis displays the infectivity in percent in comparison to the corresponding DMSO control. The level of infectivity represented by each bar results from the mean value of triplicates normalized to corresponding DMSO controls(standard deviation) SD.

(3) FIG. 3: Antiviral activity of LVVSTTYLPHYFDN peptide against RSV in vitro. In FIG. 3A, antiviral activity against RSV (ATCC VR-26Long strain) was determined. In the plaque reduction assay, cells were treated with peptide (top lane) or DMSO as a control (bottom lane). Peptide concentrations added to the cells were from the left to the right: 80 M, 40 M and 20 M. The inhibitory activity was determined 48 h post infection by counting viral plaques after immunocytochemical staining using RSV-specific monoclonal antibody. In FIG. 3B, antiviral activity of the peptide LVVSTTYLPHYFD against RSV (ATCC VR-26Long strain) was quantified. The x-axis of the diagram shows the peptide concentration in M (from the left to the right: 10 M, 20 M, 40 M and 80 M). The y-axis shows the infectivity of each sample in comparison to the infectivity of the DMSO control in percent. The inhibitory activity was determined 48 h post infection by counting viral plaques after immunocytochemical staining using RSV-specific monoclonal antibody. The level of infectivity represented by each bar results from the mean value of triplicates normalized to corresponding DMSO controls(standard deviation) SD.

(4) FIG. 4: Alanine scan of LVVSTTYLPHYFDN peptide. The diagram shows on its y-axis the sequences of peptides, each represented by its own bar. The unchanged LVVSTTYLPHYFDN peptide is on top. Below are the sequences wherein from top to bottom positions 1 to 14 of LVVSTTYLPHYFDN were exchanged with an alanine (marked by an A). The x-axis displays the infectivity in percent in comparison to the corresponding DMSO control. The level of infectivity represented by each bar results from the mean value of triplicates normalized to corresponding DMSO controls(standard deviation) SD.

(5) FIG. 5: In vitro antiviral activity of peptides with unnatural amino acid modifications. The peptide sequences used are listed in the key on the top right of the diagram. The pattern in the square left of each sequence corresponds to the pattern used in the bars. The x-axis of the diagram shows the applied peptide concentrations of the respective peptide in M. The y-axis shows the infectivity of each sample in comparison to the infectivity of the corresponding DMSO control in percent. The level of infectivity represented by each bar results from the mean value of triplicates normalized to corresponding DMSO controlsSD.

(6) FIG. 6: Binding of novel DNA-Peptide construct to RSV. FIG. 6A shows a scheme of the used ELISA, progressing from the left to the right. The single compounds are not in a representative scale. TMB=3,3,5,5-Tetramethylbenzidine, HRP=Horseradish peroxidase. FIG. 6B shows the ELISA results of peptide 6 and peptide 12 coupled to DNA. The x-axis of the diagram represents the different samples, the y-axis shows the absorbance. Only Anti-RSV antibody and Anti-RSV antibody+RSV represent internal ELISA controls and lack components described in FIG. 6A. The other three samples have all components described in FIG. 6A. Each bar results from the mean values of triplicatesSD.

(7) FIG. 7: Effects of intranasal administration of peptides on RSV infection in vivo. The x-axis of the diagram shows the different mice groups, while the y-axis shows the virus load in mice lungs in RSV-copies per 45 ng of total RNA. Each data point represents a single mouse: The half-filled triangle pointing upwards each represent mock-treated control mouse, the filled triangles pointing downwards each represent a mouse, which received peptide 6 and the squares each represent a mouse, which received peptide 12. Geometric mean values are given as bars. Significant differences are calculated by One-Way ANOVA followed by Tukey test (*, P<0.05; **, P<0.01; ***, P<0.001). LOD: Limit of detection. In the present experiment, LOD was 50 RSV copies.

(8) FIG. 8: In vitro antiviral efficacy of RSV inhibitory cyclic peptide. Human epithelial cells (HEp-2) were seeded in 96-well cell culture plate (10.sup.4 cells/well) 24 h prior to the assay. Peptide, whose sequence is shown in the figure (cyclic peptide of peptide with sequence CETALVVSTTYLPHYFDNC; SEQ ID No: 46), or the respective amount of DMSO vehicle control was mixed with RSV, incubated for 10 min at 37 C. and then added to the cells. The final concentrations on cells were 0.625-80 M. The inhibitory activity was determined 48 h post infection by counting viral plaques. Shown are mean values of triplicates normalized to corresponding DMSO controlsSD.

(9) FIG. 9: Effect of pre-treatment of cells with RSV inhibitory peptide. Human epithelial cells (HEp-2) were seeded in 96-well cell culture plate (10.sup.4 cells/well) 24 h prior to the assay. Peptide, whose sequence is Ac-LVVSTTYLPHYFDN-NH2 (SEQ ID No: 5), or the respective amount of DMSO vehicle control was applied to cells at the indicated concentrations and incubated at 37 C. for 3 h. After incubation, cells were either left in the same medium or washed and the medium replaced with fresh one. Then, RSV was applied to the cells. The inhibitory activity was determined 48 h post infection by counting viral plaques. Shown are mean values of triplicates normalized to corresponding DMSO controlsSD.

(10) FIG. 10: In vitro antiviral efficacy of peptide-DNA trimers against RSV infection. DNA trimeric scaffolds which carry three peptide inhibitor arms were synthesized to target all three RSV-F subunits at once. To test the antiviral activity of constructed Peptide [Chg][Cpa]VSTT[Tyr3,5-I2]LPHYFDN (SEQ ID No: 11)-DNA trimers in comparison with unmodified DNA-timer (without bound peptides), human epithelial cells (HEp-2) were seeded in 96-well cell culture plate (10.sup.4 cells/well) 24 h prior to the assay. DNA constructs, or the respective amount of DMSO vehicle control was mixed with RSV, incubated for 10 min at 37 C. and then added to the cells. The final concentrations on cells were 0.009-1.25 M. The inhibitory activity was determined 48 h post infection by counting viral plaques. Shown are mean values of triplicates normalized to corresponding DMSO controlsSD.

(11) FIG. 11: In vitro prophylactic effect of RSV inhibitory peptide on HEp-2 cells. Human epithelial cells (HEp-2) were seeded in 96-well cell culture plate (10.sup.4 cells/well) 24 h prior to the assay. Cells were treated either with peptide, whose sequence is shown in the figure (SEQ ID No: 9), at concentration of 20 M or the respective amount of DMSO vehicle control at the indicated time points prior to infection with RSV. The inhibitory activity was determined 48 h post infection by counting viral plaques. Shown are mean values of triplicatesSD.

(12) FIG. 12: Detection of aggregation via dynamic light scattering. A) Measurement of Peptide [Chg][Cpa]VSTT[Tyr3,5-I2]LPHYFDN (SEQ ID No: 11). Shown are measurements at concentrations of 50 M (curve 1) and two times 100 M (curve 2 and 3) in 1PBS, 10 mM MgCl.sub.2, 0.05% Tween20 buffer displayed as size distribution by number. B) Measurement of Peptide [Chg][Cpa]VSTT[Tyr3,5-I2]LPHYFDN (SEQ ID No: 11) attached to 30 bp double-stranded DNA. Shown are measurements at concentrations of 0.12 M (curve 1) and 1 M (curve 2) in 1PBS, 10 mM MgCl.sub.2, 0.05% Tween20 buffer displayed as size distribution by number.

EXAMPLES

Example 1Rational Design and In Vitro Screening of Peptide Inhibitors

(13) To identify the minimal peptide sequence required for inhibition of RSV, a series of 20 peptides with overlapping sequences were designed to target the antigenic site () on the prefusion conformation of F protein.

(14) The designed peptides were synthesized and screened for their inhibitory activity against RSV in a cell culture-based plaque reduction assay as illustrated in FIG. 1. In detail, Anti-RSV activity was assessed in plaque reduction assay as follows: HEp-2 cells were seeded in a 96-well plate 24 h prior to infection. Next day, cells were infected with RSV (ATCC VR-26Long strain). Simultaneously, peptides at concentrations of 20 M were added to the cells. The inhibitory activity was determined 48 h post infection by counting viral plaques after immunocytochemical staining using RSV-specific monoclonal antibody.

(15) A 14 amino acid long peptide that has the following sequence: LVVSTTYLPHYFDN was identified to be able to inhibit infection with RSV on human epithelial cells (FIG. 2).

Example 2Determination of Anti-RSV Activity Based on Peptide Concentration

(16) Anti-RSV activity was assessed in plaque reduction assay. HEp-2 cells were seeded in 96-well plate 24 h prior to infection. Next day, cells were infected with RSVLong (FIGS. 3A and 3B). Simultaneously, peptide at concentrations of 10-80 M was added to the cells. The inhibitory activity was determined 48 h post infection by counting viral plaques after immunocytochemical staining using RSV-specific antibody.

(17) A 14 amino acid long peptide that has the following sequence: LVVSTTYLPHYFDN was identified to be able to inhibit infection with RSV on human epithelial cells by >50% at a concentration of 20 M (FIG. 3B).

(18) Thus, the peptide that has the following sequence: LVVSTTYLPHYFDN was selected as a lead peptide candidate for developing inhibitors of RSV infection.

Example 3Structure-Activity Relationship Study

(19) In order to investigate the role of individual amino acids in the inhibitory function, an alanine-scanning mutagenesis of the lead peptide was performed (FIG. 4).

(20) In detail, anti-RSV activity was assessed in plaque reduction assay. HEp-2 cells were seeded in 96-well plate 24 h prior to infection. Next day, cells were infected with RSV (100 PFU/well). Simultaneously, peptides at concentrations of 20 M were added to the cells. Viral plaques were counted after immunocytochemical staining using RSV-specific antibody at 48 h after infection.

(21) As shown in FIG. 4, 7 key aa-residues involved in the inhibitory activity against RSV were identified. Significant reduction in RSV inhibition was observed when these residues were substituted with alanine, whereas substitution of the remaining 7 amino acids did not alter the activity of the native peptide activity.

Example 4Incorporation of Non-Natural Amino Acids

(22) The main problems in using peptides as antivirals are their low stability and rapid degradation by proteases which can limit their therapeutic value. To improve the stability of the lead peptide, different non-natural amino acids were introduced at multiple positions into its sequence. Unnatural amino acid-modified peptides are more resistant to proteolytic cleavage and offer an increase in the in vivo half-life and activity.

(23) Next, anti-RSV activity was assessed in plaque reduction assay. HEp-2 cells were seeded in 96-well plate 24 h prior to infection. Peptides at concentrations of 10-80 M were incubated with RSV for 10 min at 37 C. and then added to the cells. Viral plaques were counted after immunocytochemical staining using RSV-specific antibody at 48 h after infection. FIG. 5 shows the mean values of triplicates, normalized to corresponding DMSO controlsSD (FIG. 5).

(24) It was found that antiviral activity of the analog peptides with different unnatural amino acids substitution was preserved (FIG. 5). Several selected peptide candidates were further tested in vivo.

(25) Binding of newly developed peptides 6 (Ac-[Chg][Cpa]VSTT[Tyr3,5-I2]LPHYFDN-NH2) and 12 (Ac-[Chg][Nle][Chg]STTYLPHYFDN-NH2) to active RSV was verified by enzyme-linked immunosorbent assay (FIG. 6).

(26) FIG. 6A illustrates the scheme of the used ELISA. It has to be noted that the single compounds are not in a representative scale. ELISA results of peptide 6 and peptide 12 coupled to DNA are shown in FIG. 6B and revealed that RSV was strongly interacting with the DNA-peptide construct but not to the DNA (30 bp DNA). Unspecific binding of RSV and/or RSV-antibody to neutravidin was excluded by using the two other controls.

Example 5In Vivo Evaluation

(27) In addition, the ability of designed peptides to provide protection in an in vivo challenge experiment was tested. Female BALB/c mice were infected with RSV and treated simultaneously with 150 g of peptide. Five days post infection, viral loads in mice lungs were quantified. Treatment with both tested peptides (FIG. 7) led to significant reduction (100-1000 fold) of viral load in the lungs of treated mice when compared to mock-treated control.

(28) In detail, 9 weeks old female BALB/c mice (n=5 per group) were inoculated via intranasal route simultaneously with 10.sup.6 PFU/mouse of RSVLong strain (ATCC VR-26Long strain) and 150 g of peptide 6 or 12. Control mice received RSV with DMSO-PBS. Mice were sacrificed at day 5 postinfection, and their lungs were collected. Viral load in lungs was quantified by RT-qPCR.

(29) The results (FIG. 7) revealed that the designed peptides are effective and inhibit RSV replication in vivo.

Example 6In Vitro Antiviral Efficacy of RSV Inhibitory Cyclic Peptide

(30) Human epithelial cells (HEp-2) were seeded in 96-well cell culture plate (10.sup.4 cells/well) 24 h prior to the assay. Peptide, whose sequence is shown in FIG. 8 (cyclic peptide CETALVVSTTYLPHYFDNC; SEQ ID No: 46), or the respective amount of DMSO vehicle control was mixed with RSV, incubated for 10 min at 37 C. and then added to the cells. The final concentrations on cells were 0.625-80 M. The inhibitory activity was determined 48 h post infection by counting viral plaques. The results are shown in FIG. 8. Shown are mean values of triplicates normalized to corresponding DMSO controlsSD.

(31) The results indicate that cyclization of the peptides of the invention does not negatively affect in vitro antiviral activity.

Example 7Effect of Pre-Treatment of Cells with RSV Inhibitory Peptide

(32) Human epithelial cells (HEp-2) were seeded in 96-well cell culture plate (10.sup.4 cells/well) 24 h prior to the assay. Peptide, whose sequence is Ac-LVVSTTYLPHYFDN-NH2 (SEQ ID No: 5), or the respective amount of DMSO vehicle control was applied to cells at the indicated concentrations and incubated at 37 C. for 3 h. After incubation, cells were either left in the same medium or washed and the medium replaced with fresh one. Then, RSV was applied to the cells. The inhibitory activity was determined 48 h post infection by counting viral plaques. The results are shown in FIG. 9. Shown are mean values of triplicates normalized to corresponding DMSO controlsSD.

(33) The results demonstrate that the peptides of the invention are useful for the prophylactic treatment of an RSV infection.

Example 8In Vitro Antiviral Efficacy of Peptide-DNA Trimers Against RSV Infection

(34) DNA trimeric scaffolds which carry three peptide inhibitor arms were synthesized to target all three RSV-F subunits at once. To test the antiviral activity of constructed Peptide [Chg][Cpa]VSTT[Tyr3,5-I2]LPHYFDN (SEQ ID No: 11)-DNA trimers in comparison with unmodified DNA-timer (without bound peptides), human epithelial cells (HEp-2) were seeded in 96-well cell culture plate (10.sup.4 cells/well) 24 h prior to the assay. DNA constructs, or the respective amount of DMSO vehicle control was mixed with RSV, incubated for 10 min at 37 C. and then added to the cells. The final concentrations on cells were 0.009-1.25 M. The inhibitory activity was determined 48 h post infection by counting viral plaques. The results are shown in FIG. 10. Shown are mean values of triplicates normalized to corresponding DMSO controlsSD.

(35) The results demonstrate that the nanostructures of the invention exhibit antiviral activity for RSV and are useful for the treatment, prophylactic treatment or amelioration of an RSV infection.

Example 9In Vitro Prophylactic Effect of RSV Inhibitory Peptide on HEp-2 Cells

(36) Human epithelial cells (HEp-2) were seeded in 96-well cell culture plate (10.sup.4 cells/well) 24 h prior to the assay. Cells were treated either with peptide, whose sequence is shown in FIG. 12 (SEQ ID No: 9), at concentration of 20 M or the respective amount of DMSO vehicle control at the indicated time points prior to infection with RSV. The inhibitory activity was determined 48 h post infection by counting viral plaques. The results are shown in FIG. 11. Shown are mean values of triplicatesSD.

(37) The results demonstrate that the peptides of the invention are useful for the prophylactic treatment of an RSV infection. In particular, the data demonstrate that the peptides are stable for long periods of time in biological conditions. The peptides are therefore useful for in vivo applications in the human and mammal body, for therapeutic and prophylactic purposes.

Example 10Detection of Aggregation Via Dynamic Light Scattering

(38) A) Measurement of Peptide [Chg][Cpa]VSTT[Tyr3,5-I2]LPHYFDN (SEQ ID No: 11). The results are shown in FIG. 12A.

(39) Shown are measurements at concentrations of 50 M (curve 1) and two times 100 M (curve 2 and 3) in 1PBS, 10 mM MgCl.sub.2, 0.05% Tween20 buffer displayed as size distribution by number.

(40) B) Measurement of Peptide [Chg][Cpa]VSTT[Tyr3,5-I2]LPHYFDN (SEQ ID No: 11) attached to 30 bp double-stranded DNA. The results are shown in FIG. 12A. Shown are measurements at concentrations of 0.12 M (curve 1) and 1 M (curve 2) in 1PBS, 10 mM MgCl.sub.2, 0.05% Tween20 buffer displayed as size distribution by number.

(41) The results indicate that attachment of the peptides to the DNA scaffold in the nanostructures of the invention may increase bioavailability of the peptides in vivo, through elimination of aggregation.

REFERENCES

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