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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
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  • A61P37/04—Immunostimulants
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  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/10—RNA viruses
  • C07K16/108—Orthomyxoviridae (F), e.g. influenza virus
• • C—CHEMISTRY; METALLURGY
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  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16151—Methods of production or purification of viral material

Definitions

• the present invention relates to a novel antiviral peptide and a fusion
• the fusion phage displays the sequence CNDFRSKTC on its surface protein P3 which binds to AIV H9N2 with an IC 50 value of less 1O 1 S pfu/100 ⁇ l.
• Avian influenza virus belongs to the family of Orthomyxoviridae which contains two genera, influenza A & B and influenza C (Lamb and Krug, 1996). These viruses are the major cause of morbidity and mortality among
• Influenza A viruses are enveloped with lipid bilayer and contain eight single-stranded, 0 segmented, negative sense RNAs. There are two glycoproteins present on the surface of the virions namely, haemagglutinin (HA) and neurammidase (NA), and one ion channel protein (M2). The glycoproteins are the major antigenic determinants of influenza viruses.
• the HA protein initiates the first step in the viral infection, which involves the attachment of viruses to the host cell surface 5 sialic acid receptors (Lamb and Krug, 1996).
• the NA protein participates in the release of mature virions from the host cells (Palese ef al., 1974). Therefore, in order to study the virus - host interaction and also to identify molecules that inhibit this process a bacteriophage displaying a specific peptide sequence was selected by its affinity to avian influenza virus strain 0 H9N2 using a phage display library.
• the preferred primary strategy for the prevention of influenza virus infection is annual vaccination among susceptible population. But the antiviral drugs play an important role in a comprehensive approach to control the illness and transmission (Hayden, 2006).
• adamantane derivatives amantadine and rimantadine
• NAls neuraminidase inhibitors
• zanarnivir and oseltarnivir neuraminidase inhibitors
• adamantane derivatives act by binding and blocking the function of influenza A virus M2 ion channel protein and thereby prevents the viral replication inside the host cell (Wang et al., 1993)). Due to single point mutations in M2 proteins, adamantane resistant strains have emerged (Hay et al., 1986).
• a population of recombinant phages displaying random disulfide constrained heptapeptide sequences was screened against the virus. Then the peptide displayed on the surface of the fusion phage was synthesized chemically.
• the isolated peptide has the sequence of NDFRSKT with an IC50 value of less than 100 ⁇ M
• the specificity of the recombinant phage against the influenza virus A H9N2 was proved by an antibody - phage competition assay, in which the phages were able to compete with polyclonal antibodies for binding site on the viral surface proteins.
• synthetic peptides based upon one of the isolated sequences, either in linear or cyclic conformation, such as the synthetic peptide CNDFRSKTC, are able to inhibit the propagation of NDV, thereby preventing disease and spread of infection.
• Table I shows the heptapeptide sequences obtained from three rounds of biopanning against AIV H9N2.
• Table 2 shows haernagglutination inhibition activities of synthetic peptides and fusion phages.
• Figure I shows the results of the competition between recombinant phages bearing specific heptapeptide sequence and polyclonal antibodies for AIV H9N2.
• Figure 2 shows the inhibition of ATV propagation with synthetic peptides in ovo.
• Figure 3 shows the inhibition of AFV propagation with fusion phages in ovo.
• Figure 4 shows the inhibition of AIV propagation with fusion phages in vitro.
• Figure 5 shows the effect of fusion phage on viral replication in vitro.
• Table I shows the heptapeptide sequences obtained from four rounds of biopanning against AIV. After 4 rounds of selection and amplification 20, 35 and 35 individual clones from the 2 nd , 3 rd and 4 th rounds, respectively, were sequenced.
• Table 2 shows the inhibitory ability of the cyclic and linear peptides against the haernaggluti nation activity of the avian influenza virus H9N2. Experiments were performed in triplicates. + in the presence of, - in the absence of.
• Figure I shows the results of the competition between recombinant phages bearing specific heptapeptide sequence and polyclonal antibodies for AIV. The polyclonal antibodies (pAb) inhibited the association of the fusion phage with ATV, suggesting that the phages may share some common binding sites with the antibody. Experiments were done in triplicates and the error bars represent the standard deviation of the mean. Series I - Fusion phage; Series 2 - Fusion phage with polyclonal Antibodies.
• Figure 2 shows the results of determination of the IC50 values of the synthetic peptides in ovo against AIV H9N2 propagation. Peptide concentration needed to inhibit 50% of the virus growth was determined using different concentrations of peptides. Experiments were done in triplicates and the error bars represent the standard deviation of the mean.
• Figure 3 shows the results Of IC 50 values of fusion phage FP-Pi against AIV H9N2 virus propagation in ovo. Experiments were done in triplicates and the error bars represent the standard deviation of the mean. Series I - Fusion phage FP-Pl; Series 2 - Wild phage as control. Figure 4 shows the results of antiviral activity of the fusion phage FP-Pi in vitro. Experiments were done in triplicates and the error bars represent the standard deviation of the mean. Series I - FP-P 1; Series 2 - Wild Phage M 13 as control.
• Figure 5 shows the results of the effect of the fusion phage FP-Pi on viral replication.
• MDCK cells were inoculated with untreated (o value) or FP-P I treated virus, and viral titers were determined on supernatants 72 hpi.
• Examples I to 5 are the conventional materials and methods which are prerequisites of the invention.
• phage display technology random peptides are usually displayed on the surface of bacteriophage molecules as fusion protein by inserting synthetic oligonucleotides in the gene encoding the coat proteins.
• a collection of these recombinant phages are known as phage display library, allows the screening of vast numbers of peptide sequences against a target by an in vitro selection procedure known as biopanning (Parmley et ah, 1988 and Smith et ah, 1993).
• biopanning Parenter et ah, 1988 and Smith et ah, 1993.
• the target used was whole AIV H9N2 virus particles and the library employed was the disulfide constrained library obtained from New England Biolabs, Inc.
• the displayed peptide molecules are flanked by a cysteine residue to the gpIII protein of phage M13.
• the cysteine residues help the peptides to attain a fixed cyclic shape, in the absence of reducing agents.
• This disulfide constrained library useful for targets whose interacting components (native ligands) have discontinuous binding sites (conformational epitope) (Hoess et al., 1994); in which amino acids are brought from different positions in a polypeptide to form an essential contact area.
• disulfide constrained cyclic peptide library is more useful in selecting high affinity ligands rather than a linear peptide library (O'Neil et aL,, 1992; Gho et ah, 1997).
• the avian influenza virus contains two surface glycoproteins namely haernagglutinin (HA) and neuraminidase (NA) which is responsible for virus entry and exit into and from the host cell.
• HA haernagglutinin
• NA neuraminidase
• Figure I summarizes the major steps involved in the selection of these ligands. First, the whole virus particles were directly attached to the surface of a high binding microliter plate well. The library was then added into the well to allow the recombinant phage particles to bind with the virus particles. The unbound phages were washed out and the bound phages were eluted at low pH.
• the eluted phages were then amplified in the bacterium, Escherichia coli and the amplified phages were used in second round of biopanning. This procedure was repeated for four times. After four successive rounds of affinity selection and amplification, a subset of the selected phages was grown up individually and tile identity of the peptides that bind to core particles were obtained by sequencing tile gpIII gene carrying the insertion.
• the stringency of selection was increased by (i) performing the biopanning at room temperature (28'C), (ii) shortening the time of binding to I h to select for ligands with rapid on rates (Ko,), (iii) washing the wells thoroughly (10 times) to remove low affinity binding phages, and (iv) repeating 4 rounds of biopanning to enrich high affinity binding clones.
• 47% of phages analysed from the fourth round panning carried the fusion peptide sequence NDFRSKT, 10.5% containing
• fusion peptide refers to amino acid sequence genetically encoded by a bacteriophage and physically linked to a coat protein of the phage.
• the claimed fusion peptides contain amino acid sequences NDFRSKT, but it is not limited to: (i) amino acid sequence which is shorter or longer than the claimed amino acid sequences; (ii) variations in the amino acid sequences, particularly amino acid substitutions within the same category as described below; (iii) amino acid sequences sharing at least 60% homology with those of the claimed fusion peptides and; (iv) either linear or constrained conformation.
• AIV possesses two surface glycoproteins, HA and NA, which protrude from the viral lipid bilayer membrane. These glycoproteins are essential for the entry and release of viruses into and outside of the host cells respectively. Since the phages bearing the sequences NDFRSKT bound to the virion in solution, these sequences may, to a certain extent, resemble a region on the host cell receptor that interacts with the intact virion.
• the surface glycoproteins HA and NA are two important proteins that elicit antibody production in the host.
• the phages were able to bind to one of the mimotopes and therefore inhibit the binding of antibodies raised against the virion.
• Figure I shows that the recombinant phages displaying the peptide NDFRSKT, selected from biopanning experiment, were able to compete with the antibodies for the binding site on AIV.
• the number of phages bound to the virus coated wells reduced dramatically as a result of competition between these two molecules for the same binding site on the virus.
• the recombinant phage molecules may serve as a potential diagnostic agent for the AIV infection.
• AIV can easily be propagated in the allantoic fluid of embryonated chicken eggs. Therefore, the virus and the propagation system provide a simple model to assess directly the antiviral activities of a particular compound or peptide in vivo.
• the amount of virus present in the allantoic fluids, before and after the antiviral compounds treatment, is determined by hemagglutination titer. Injection of the claimed peptide either in cyclic or linear form and the fusion phage displaying the peptide reduced the ATV titer in the allantoic fluid ( Figures 2 and 3).
• the cyclic peptide showed higher IC 50 value at a peptide concentration lower than 100 ⁇ M to the linear peptide.
• cyclization can avoid the digestion of the peptides by exopeptidases.
• synthetic peptides claimed herein can be used as therapeutic agents to treat, control and also to eradicate the disease.
• MDCK cell lines are effective medium for the propagation of avian influenza viruses in vitro.
• the in vitro antiviral property of the fusion phages against the virus multiplication were checked in these cell lines.
• the inhibition of viral replication was indirectly checked by the cell viability, before and after the treatment with the fusion phage molecules.
• the cell viability increases nearly 2 fold after the infection with phage treated viruses to the untreated viruses ( Figure 4).
• the reduction in viral titer was further confirmed by haemagglutination test of the supernatants ( Figure 5). This experiment proved the antiviral property of the fusion phage molecules in vitro.
• AIV subtype H9N2 was injected into a 9 days old specific pathogen free embryonated chicken eggs. After 3 days of incubation at 37 0 C, the allantoic fluid was harvested and clarified by centrifugation (30 min, 35,000 x g, 4 0 C. The virus was purified from the clarified supernatants by 3O%-6o% sucrose gradient ultracentrifugation (3.5 h, 285,000 x g, 4 0 C and was used for the studies below.
• Biopanning of purified ATV The protein concentration of virus was measured using the Bradford assay (1976).
• AIV (15 ⁇ g/ml) was coated onto a microtiter plate well [Na 2 CO 3 /NaHCO 3 buffer (0.1 M, pH 9.6)]. Streptavidin and BSA were used as positive and negative controls respectively. The experiment was carried out at room temperature (about 25 0 C). Phages from a disulfide constrained y-mer phage display library (New England Biolabs, USA) were diluted to I X 10" pfu in TBS [50 mM Tris-HCI (pH 7.5), 150 mM NaCl; 110 ⁇ l] and added into each coated well. The mixtures were incubated for 30 min.
• Phage titration method was adapted from Sambrook et al. (1989).
• Sequencing of the selected phages Single plaques from all 4 rounds of panning were picked from (Luria Broth) LB plates (used in output titration) and grown in LB media. Single stranded DNA of the phages was extracted as described by Sambrook et al, (1989). Sequencing of inserts was done using the ABI automated sequencer by First Base Laboratories Sdn Bhd, Kuala lumpur.
• Antibody competition assay Wells were coated with AIV (15 ⁇ g/ml) and polyclonal antibodies (1:500 dilution; 100 ⁇ l) raised against AIV were added with a series of different phage concentrations (io9 - 1O 1 S pfu; 100 ⁇ l). The mixtures were incubated for I hour and washed 6 X with Tris-buffered saline - Tween (TBST). Bound phages were eluted and titered as described above.
• MDCK cells were inoculated with medium alone or phage treated (10 8 - 1O 1 S pfu / 100 ⁇ l) or non-treated virus (multiplicity of infection [MoI] 0.05) for I hr at 37 0 C. Following adsorption, monolayers were washed and incubated in EMEM containing 5% fetal bovine serum (FBS). Cytopathic effect was monitored by light microscopy and quantitated by XTT Cell Viability Assay Kit (Biotium, USA).
• MDCK cells were inoculated with phage-treated or untreated virus at an MOI of 0.05, supernatants were collected at 72 hpi, and viral titers were determined by HA titer.
• the IC 50 value was estimated by interpolation of the dose-response curve.

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