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(12) United States Patent (10) Patent No.: US 6,753,407 B2 Noga Et Al

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(12) United States Patent (10) Patent No.: US 6,753,407 B2 Noga Et Al USOO6753407B2 (12) United States Patent (10) Patent No.: US 6,753,407 B2 Noga et al. (45) Date of Patent: Jun. 22, 2004 (54) ANTIMICROBIAL PEPTIDES ISOLATED WO WO 94/21672 9/1994 FROM FISH OTHER PUBLICATIONS (75) Inventors: Edward J. Noga, Raleigh, NC (US); Umaporn Silphaduang, Fuquay-Varina Robinette, D., et al., Antimicrobial activity in the Skin of the NC (US) s s channel catfish Ictalurus punctatus. characterization of broad-spectrum histone-like antimicrobial proteins, CMLS (73) Assignee: North Carolina State University, Cellular and Molecular Life Sciences, vol. 54, pp. 467-475 Raleigh, NC (US) (1998). - Yu, K., et al., Relationship between the tertiary Structures of (*) Notice: Subject to any disclaimer, the term of this mastoparan B and its analogs and their lytic activities patent is extended or adjusted under 35 Studied by NMR spectroscopy, J. Peptide Res., vol. 55, pp. U.S.C. 154(b) by 0 days. 51-62 (2000). Boman, Hans, Peptide Antibiotics and Their Role in Innate (21) Appl. No.: 09/929,788 Immunity, Annual Review of Immunology, vol. 13, pp. 61-92 (22) Filed: Aug. 14, 2001 (1995). O O Silphaduang, et al., Peptide Antibiotics in Mast Cell of Fish, (65) Prior Publication Data Nature, vol. 414, No. 6861, pp. 268-269 (Nov. 15, 2001). US 2003/0083247 A1 May 1, 2003 Primary Examiner-Lynette R. F. Smith Related U.S. Application Data ASSistant Examiner Robert A. Zeman (60) Provisional application No. 60/225,354, filed on Aug. 15, (74) Attorney, Agent, or Firm- Myers Bigel Sibley & 2000. Sajovec (51) Int. Cl. ................................................ A61K 38/00 (57) ABSTRACT (52) U.S. Cl. .................... 530/326; 530/300; 424/184.1; 424/1845; 514/2; 514/13; 514/21 Antimicrobial peptides (endobiotic peptides), isolated from (58) Field of Search ................................. 530/300, 326; fish are described. Such endobiotic peptides may be isolated 424/1841, 185.1, 514/2, 13, 21 as 22 amino acid peptides having molecular weights of about 2500 Da from the gills of hybrid striped bass (Morone (56) References Cited SaxitilisXMorone chrysops). Antibodies that bind such pep tides and methods of using Such peptides are also described. FOREIGN PATENT DOCUMENTS WO WO 89/001.99 1/1989 2 Claims, No Drawings US 6,753,407 B2 1 2 ANTMICROBAL PEPTIDES SOLATED A further aspect of the invention is a method of treating FROM FISH StreSS in fish, comprising administering an endobiotic pep tide as described above to a fish in an amount effective to RELATED APPLICATIONS treat or combat StreSS therein. This application claims the benefit of U.S. Provisional A further aspect of the invention is a nucleic acid (e.g., a Application Serial No. 60/225,354, filed Aug. 15, 2000, the DNA) that encodes a peptide as described above. disclosure of which is incorporated by reference herein in its A further aspect of the invention is a method of treating entirety. StreSS in fish, comprising administering a nucleic acid to the fish (e.g., by injecting the nucleic acid into muscle of the GOVERNMENT SUPPORT fish) in an amount effective to treat or combat stress therein. This invention was made with Government support under A further aspect of the invention is a method of monitor University of North Carolina Sea Grant NA86-RG-0036 and ing fish health, comprising the steps of: (a) collecting a United States Department of Commerce Grant NA90AA biological Sample from a fish; and (b) detecting the level of DSG-062. The Government has certain rights to this inven at least one endogenous endobiotic peptide in the Sample, tion. 15 wherein lower levels of endobiotic peptides indicate decreased health in the fish. Examples of suitable endobiotic FIELD OF THE INVENTION peptides include, but are not limited to, the peptides The present invention relates to two families of novel described above. antimicrobial peptides exhibiting therapeutic antimicrobial A further aspect of the invention is a method of monitor properties, to antibodies that specifically bind to one of the ing freshness of a fish food product, the method comprising families of the novel peptides, and to methods of monitoring detecting the level of at least one endogenous endobiotic and improving health in aquaculture Species and of preser peptide, wherein lower levels of endobiotic peptides indicate Vation of Seafood. decreased freshness in the fish food product. Examples of BACKGROUND OF THE INVENTION 25 Suitable endobiotic peptides include, but are not limited to, the peptides described above. Infectious diseases are often the most Serious impediment A further aspect of the present invention is a method of to the Success of the commercial aquaculture industry in the Screening for compounds useful for treating StreSS in fish, the United States and worldwide. The epithelial Surfaces of fish, Such as the Skin, gills and alimentary tract, provide first method comprising the steps of: (a) administering a test contact with potential pathogens. It has been reported that compound to a fish; (b) collecting a biological Sample from Several kinds of endogenous antimicrobial peptides exist in the fish; and (c) detecting the level of at least one endog the skin, Stomach and blood of amphibians, mammals and enous endobiotic peptide in the sample, wherein higher insects. Examples include cecropins (insects), defensins levels of endobiotic peptide in the fish as compared to those (mammals, insects), and magainins (frogs) (See discussion found in the absence of administration of the test compound infra, Detailed Description of Preferred Embodiments). 35 indicate the compound is useful in treating StreSS in the fish. These peptides exhibit antimicrobial activity against a broad Examples of Suitable endobiotic peptides include, but are spectrum of organisms. This ability to exhibit broad not limited to, the peptides described above. Spectrum activity provides the advantages of nonspecificity DETAILED DESCRIPTION OF THE and rapid response. These advantages enable the host to PREFERRED EMBODIMENT delay or prevent microbial colonization. Enhancement of 40 nonspecific defenses exhibiting a broad range of activity The present invention will now be described more fully against numerous pathogens may be a cost-effective method hereinafter with reference to the accompanying figures, in of controlling disease epidemics that inhibit Successful which preferred embodiments of the invention are shown. aquacultural endeavors. 45 This invention may, however, be embodied in different Therefore, it is an objective of this invention to provide a forms and should not be construed as limited to the embodi better understanding of the mechanisms responsible for ments Set forth herein. Rather these embodiments are pro disease resistance in aquatic animals and to use this knowl Vided So that this disclosure will be thorough and complete, edge to reduce epidemic disease. Additionally, this invention and will fully convey the scope of the invention to those skilled in the art. may aid in the development of peptide antibiotics for the 50 treatment of human diseases. Other features and advantages Unless otherwise defined, all technical and Scientific of the present invention will be more apparent in the detailed terms used herein have the same meaning as commonly description set forth below. understood by one of ordinary skill in the art to which the invention belongs. All publications, patent applications, SUMMARY OF THE INVENTION 55 patents, and other references mentioned herein are incorpo A first aspect of the present invention is an antimicrobial rated by reference in their entirety. compound or endobiotic peptide isolated from fish. The Amino acid Sequences disclosed herein are presented in compound may be selected from the group consisting of the amino to carboxy direction, from left to right. The amino peptides having an amino acid Sequence Selected from the and carboxy groups are not presented in the Sequence. group consisting of: SEQID NO: 1; SEQ ID NO: 2; SEQ ID 60 Amino acids are represented herein in by Single letter code. NO:3, or SEQ ID NO: 4. “Amino acid Sequence' as used herein, refers to an A further aspect of the present invention is a pharmaceu oligopeptide, peptide, polypeptide, or protein Sequence, and tical formulation comprising a compound as described fragment thereof, and to naturally occurring or Synthetic above in a pharmaceutically acceptable carrier. molecules. Where "amino acid Sequence” is recited herein to A further aspect of the present invention is an antibody 65 refer to an amino acid Sequence of a naturally occurring (e.g., a monoclonal antibody) that specifically binds to a protein molecule, amino acid Sequence, and like terms, are compound as described above. not meant to limit amino acid Sequence to the complete, US 6,753,407 B2 3 4 native amino acid Sequence associated with the recited predisposing factor for infectious disease in fish (Meyer, F protein molecule. (1970) Seasonal Fluctuations in the Incidence of Disease on As used herein, the term “antibody” refers to all types of Fish Farms. In: Snieszko, S (ed) A Symposium on Diseases immunoglobulins, including IgG, IgM, IgA, Ig), and IgE. of fishes and shellfishes. Special Publication no 5, American Of these IgM and IgG are particularly preferred. The anti Fisheries Society, Washington, D.C.; Walters, G and Plumb, bodies may be monoclonal or polyclonal and may be of any J (1980).J. Fish Biol. 17: 177–185; Barton (1997) Stress in Species of origin including (for example) mouse, rat, rabbit, Finfish: Past, Present, and Future-A Historical Perspective. horse, or human, or may be chimeric antibodies. See, e.g., In: Iwana et al. (eds) Fish Stress and Health in Aquaculture. M. Walker et al., Molec. Immunol. 26, 403–11 (1989). Soc. Exper. Biol. Seminar Series 62: 1-34, Cambridge Antibodies that bind to the peptides of Endobiotic Family 1 University Press, New York). A number of stressors com and/or Endobiotic Family 2 can be prepared using intact monly associated with aquaculture can adversely impact or peptides or fragments containing Small peptides of interest impair the fish immune System, including crowding (Klinger as the immunizing antigen.
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