WO2005023290A2

WO2005023290A2 - Uses of interferons for the treatment of severe acute respiratory syndrome and other viral infections

Info

Publication number: WO2005023290A2
Application number: PCT/US2004/016201
Authority: WO
Inventors: Sidney Pestka; Thomas B. Lavoie; William A. Clark
Original assignee: Pestka Biomedical Laboratories, Inc.
Priority date: 2003-05-23
Filing date: 2004-05-21
Publication date: 2005-03-17
Also published as: AU2004270102B2; CN1832755A; EP1626734A2; TW200509961A; WO2005023290A3; AU2004270102A1; IL171991A0; JP2006526022A; CA2526428A1

Abstract

This invention provides a method of treating a subject afflicted with severe acute respiratory syndrome (SARS), comprising administering to the subject an amount of an interferon polypeptide effective to reduce the concentration of SARS-associated coronavirus particles in the subject, thereby treating the subject. This invention also provides methods of treating a subject infected with certain viruses and methods of reducing a subject’s risk of viral infection, comprising administering to the subject an interferon polypeptide. This invention further provides a method of treating a subject afflicted with influenza or reducing a subject’s risk of influenza virus infection, comprising administering to the subject an interferon polypeptide.

Description

Uses of Interferons for the Treatment of Severe Acute Respiratory Syndrome and other Viral Infections This application claims priority to U.S. Provisional Application No. 60/473,134, filed May 23, 2003, the contents of which are hereby incorporated by reference into this application.

Various references are referred to throughout this application, and the contents of each of these references is incorporated by reference into this application.

Background of the Invention Interferons (L Ns) are a well known family of cytokines secreted by a large variety of eukaryotic cells upon exposure to various stimuli (Zoon KC: Human Interferons: Structure and Function, p. 1-12. In: Interferon 8. Academic Press, London, 1987; Walter et al., Cancer Biotherm Radiopharm 1998 June; 13(3):143-54; Pestka, S., Biopolymers 2000; 55(4):254-287; Pestka, S., Methods in Enzymology, 78, 1981; Pestka, S., Methods in Enzymology, 79, 1981; Pestka, S., Methods in Enzymology, 119, 1986). The interferons have been classified by their chemical and biological characteristics. The interferons are divided into two groups designated Type I and Type II interferons (Pestka, S.; Langer; J.A.; Zoon, K.C.; Samuel, CE. Ann Rev Biochem 1987, 56, 727-777; Pestka, S., Biopolymers 2000; 55(4):254-287). LFN-γ, known also as immune interferon, is the only Type II interferon whereas the Type I human interferons consist of five classes: IFN-α, IFN-β, IFN-ω, LFN- and IFN-τ. There is only one human IFN-β and one human LFN-ω, but a family of multiple IFN-α species exists. IFN-τ is only found in ungulates; there is no human LFN-τ. The IFNs exhibit anti-viral, immunoregulatory, and antiproliferative activity. The clinical potential of interferons has been recognized.

Interferon was discovered by Isaacs and Lindenmann (Proc. Royal Soc. London, Ser B 147, 258, 1957). Efforts to purify and characterize human leukocyte interferon have led to the preparation of homogeneous leukocyte interferons (now called IFN-αs) derived from normal or leukemic (chronic myelogenous leukemia or "CML") donor leukocytes (Pestka, S., Biopolymers 2000; 55(4):254-287; Rubinstein, M.; Levy, W.P.; Moschera, J.A.; Lai, C.-Y.; Hershberg, R.D.; Bartlett, R.T.; Pestka, S. Arch Biochem Biophys 1981, 210, 307-318.). Homogeneous fibroblast interferon (now called IFN-β) was also purified to homogeneity (Friesen, H.-J.; Stein, S.; Evinger, M.; Familletti, P.C; Moschera, J.; Meienhofer, J.; Shively, J.; Pestka, S. Arch Biochem Biophys 1981, 206, 432-450). These interferons are a family of proteins characterized by a potent ability to confer a virus-resistant state in their target cells. In addition, interferon can inhibit cell proliferation, modulate immune responses and alter expression of proteins. These properties have prompted the clinical use of leukocyte interferon as a therapeutic agent for the treatment of viral infections and malignancies. During the past several decades a large number of human and animal interferons have been produced, identified, purified and cloned. Several of the interferon preparations have been prepared for clinical trial in both crude form, as well as in purified form. Several individual recombinant interferon-α species have been cloned and expressed. The proteins have then been purified by various procedures and formulated for clinical use in a variety of formulations. Most of the interferons in clinical use that have been approved by various regulatory agencies throughout the world are mixtures or individual species of human α interferon (Hu-IFN-α). In some countries Hu-IFN-β and γ have also been approved for clinical trial and in some cases approved for therapeutic use. The major thesis underlying clinical use of these interferons was that they were natural molecules produced by normal individuals. Indeed, the specific thesis was that all the interferons prepared for clinical use, be they natural- or recombinant-generated products, represented interferons that were produced naturally by normal people. This is true for a large number of interferons as well as specific growth factors, lymphokines, cytokines, hormones, clotting factors and other proteins that have been produced. Summary of the Invention

The present invention provides nucleic acids comprising a polynucleotide encoding at least a portion of an interferon polypeptide including an amino acid sequence shown in at least one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, or a fragment thereof. Thus, one aspect of the invention provides a polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding an Interferon polypeptide including an amino acid sequence in at least one of these SEQ LD NOs:; (b) a nucleotide sequence encoding a biologically active fragment of a polypeptide shown in at least one of these SEQ LD NOs; and (c) a nucleotide sequence complementary to at least one of any of the nucleotide sequences in (a) or (b) above. Further embodiments of the invention include nucleic acids that comprise a polynucleotide having a nucleotide sequence at least 95% identical, and more preferably at least 96% identical, and even more preferably at least 97% identical, and even more preferably at least 98% identical, and still more preferably at least 99.25% identical, to any of the nucleotide sequences in (a), (b) or (c), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b) or (c), above, and preferably to a polynucleotide shown in at least one of SEQ LD NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 or 85. The fragments include biologically active fragments of the interferon polypeptides. SEQ ID NOs. 1-26 are feline interferon sequences. SEQ ID NOs. 27-36 are rhesus interferon sequences. SEQ LD NOs. 37-86 are human interferon sequences. "Stringent hybridization conditions" is intended to include stringent wash conditions, and one skilled in the art would be able to determine such highly conditions, such as low salt and high temperatures. An example of low salt is 0.1 X SSC. An example of high temperature is 65-68 °C One embodiment comprises overnight incubation at 42°C in a solution comprising: 50% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 ug/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65°C. When used herein, "high stringency" also refers to conditions that (i) employ low ionic strength and high temperature for washing after hybridization, for example, O.l.times.SSC and 0.1% (w/v) SDS at 50. degree. C; (ii) employ during hybridization conditions such that the hybridization temperature is, 25. degree. C. lower than the duplex melting temperature of the hybridizing polynucleotides, for example 1.5. times. SSPE, 10% (w/v) polyethylene glycol 6000, 7% (w/v) SDS, 0.25 mg/ml fragmented herring sperm DNA at 65.degree. C; or (iii) for example, 0.5M sodium phosphate, pH 7.2, 5 mM EDTA, 7% (w/v) SDS (28) and 0.5% (w/v) BLOTTO at 70.degree. C; or (iv) employ during hybridizational denaturing agent such as formamide, for example, 50% (v/v) formamide with 5.times.SSC, 50 mM sodium phosphate (pH 6.5) and 5.times.Denhardt's solution at 42.degree. C; or (v) employ, for example, 50% (v/v) formamide, 5.times.SSC, 50 mM sodium phosphate (pH 6.8), 0.1% (w/v) sodium pyrophosphate, 5.times.Denhardt's solution, sonicated salmon sperm DNA (50 .mu.g/ml) and 10%. dextran sulphate at 42.degree. C. With respect to polypeptide, peptide and protein fragments, these may be biologically active fragments and may be of any length less than the full lengths of the sequences described in the sequence identifiers provided herein. In one embodiment, the fragment is at least 10 amino acids in length, hi another embodiment, the fragment is at least 20 amino acids in length, h other embodiments, the fragments may be at least 30, 40, 50, 60, 70, 80, 90 or 100 amino acids in length. In further embodiments, the fragments may be at least 110, 120, 130, 140, 150, 160, or 170 amino acids in length. With respect to nucleic acid fragments, these include those which may be useful as diagnostic probes and primers as discussed herein. Such sequences may those which specifically identify the interferon nucleotide sequences described herein. And they may be of any length. For example, they include those of at least about 15 nucleotides, and more preferably at least about 20 nucleotides, still more preferably at least about 30 nucleotides, and even more preferably, at least about 40 nucleotides in length. Of course, larger fragments such as those from 50-300 nucleotides in length are also useful according to the present invention as are fragments corresponding to most, if not all, of at least one of the nucleotide sequences shown in at least one of SEQ LD NOs provided herein. By a fragment at least 20 nucleotides in length, for example, is intended fragments which include 20 or more contiguous bases from at least one of the nucleotide sequences as shown in at least one of SEQ ID NOs provided herein.. Fragments of 50-300 nucleotides in length, include, for example, those which are about 50, 60, 70, 80, 90 or 100 bases in length. They may also include fragments which are about 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 bases in length. They may also include fragments which are about 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 bases in length. A polynucleotide which hybridizes to a "portion" of a polynucleotide includes a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides, and more preferably at least about 20 nucleotides, still more preferably at least about 30 nucleotides, and even more preferably about 30-70 nucleotides, including any length in between (e.g. 50 bases), of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below. In another aspect, the present invention provides nucleic acids comprising a polynucleotide encoding at least a portion of a feline Interferon polypeptide including an amino acid sequence shown in at least one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or a fragment thereof. Thus, one aspect of the invention provides a polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a feline Interferon polypeptide including an amino acid sequence in at least one of these SEQ LD NOs; (b) a nucleotide sequence encoding a biologically active fragment of a polypeptide shown in at least one of these SEQ ID NOs:; and (c) a nucleotide sequence complementary to at least one of any of the nucleotide sequences in (a) or (b) above. Further embodiments of the invention include nucleic acids that comprise a polynucleotide having a nucleotide sequence at least 95% identical, and more preferably at least 96% identical, and even more preferably at least 97% identical, and even more preferably at least 98% identical, and still more preferably at least 99.25% identical, to any of the nucleotide sequences described herein, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide described herein, and preferably to a polynucleotide shown in at least one of SEQ JD NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25. The present invention further provides a nucleic acid comprising a polynucleotide encoding at least a portion of a Rhesus Interferon polypeptide including an amino acid sequence shown in at least one of SEQ LD NO: 28, 30, 32, 34, 36 or a fragment thereof. Thus, one aspect of the invention provides a polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a Rhesus Interferon polypeptide including an amino acid sequence in at least one of these SEQ ID NOs; (b) a nucleotide sequence encoding a biologically active fragment of a polypeptide shown in at least one of these SEQ ID NOs; and (c) a nucleotide sequence complementary to any of the nucleotide sequences in (a) or (b) above. Further embodiments include nucleic acids that comprise a polynucleotide having a nucleotide sequence at least 95% identical, and more preferably at least 96% identical, and even more preferably at least 97% identical, and even more preferably at least 98% identical, and still more preferably at least 99.25% identical, to any of the nucleotide sequences in (a), (b) or (c), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b) or (c), above, and preferably to a polynucleotide shown in at least one of SEQ ID NO: 27, 29, 31, 33 or 35. The present invention also provides nucleic acids comprising a polynucleotide encoding at least a portion of a human Interferon polypeptide including an amino acid sequence shown in at least one of SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86 or a fragment thereof. Thus, one aspect of the invention provides an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a human Interferon polypeptide including an amino acid sequence in at least one of these SEQ ID NOs; (b) a nucleotide sequence encoding a biologically active fragment of a polypeptide shown in at least one of these SEQ LD NOs; and (c) a nucleotide sequence complementary to any of the nucleotide sequences in (a) or (b) above. Further embodiments of the invention include isolated nucleic acids that comprise a polynucleotide having a nucleotide sequence at least 95% identical, and more preferably at least 96% identical, and even more preferably at least 97% identical, and even more preferably at least 98% identical, and still more preferably at least 99.25% identical, to any of the nucleotide sequences in (a), (b) or (c), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b) or (c), above, and preferably to a polynucleotide shown in at least one of SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 or 85. ha another aspect, any of the nucleic acids of the present invention which encode Interferon polypeptides may include, but are not limited to, those encoding the amino acid sequence of the complete polypeptide, by itself; and the coding sequence for the complete polypeptide and additional sequences, such as those encoding an added secretory leader sequence, such as a pre-, or pro- or prepro- protein sequence. Also provided are the nucleic acids described herein together with additional, non-coding sequences, including, for example, but not limited to introns and non- coding 5' and 3¹ sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example-ribosome binding and stability of mRNA; and an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif. 91311), among others, many of which are commercially available. For instance, hexa-histidine as described by Gentz et al. provides for convenient purification of the fusion protein (Gentz et al. (1989) Proc. Natl. Acad. Sci. USA 86: 821-824). The "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al. (1984) Cell 37: 767). Other examples of fusion tags include the following: (1) MBP tag is a portion of maltose binding protein (vectors available from Roche™ (Example vector designation: pIVEX MBP), New England Biological (Example vector designation: pMAL-p2X); (2) HA tag is a portion of the hemagglutinin of human influenza virus (vectors available from RocheJ (Example vector designation: pIVEX HA-tag), BD Biosciences (pCMV-HA); (3) FLAG is a particular 8 amino acid epitope (vector from Sigma Chemical Co.J Example vector designation: gWiz/GFP); (4) CBP tag is a portion of calmodulin binding protein (vector available from StratageneJ: Example vector designation: pDual); and (5) GFP is a portion of green fluorescent protein (vector available from StratageneJ: Example vector designation: pDual). Other common epitope tags for creating fusion proteins: c-myc, GST, AU1, AU5, DDDDK, E epitope, E2 tag, Glu- Glu, SI, KT-3, T7 epitope tag, V5 epitope tag, VSV-G, BFP, CFY, YFP. As discussed below, other such fusion proteins include an Interferon fused to Fc at the N- or C- terminus. The present invention also provides recombinant vectors, which include the nucleic acids of the present invention, and host cells containing the recombinant vectors, as well as methods of making such vectors and host cells and for using them for production of interferon polypeptides or peptides by recombinant techniques. The invention further provides an interferon polypeptide comprising an amino acid sequence selected from: (a) the amino acid sequence of an interferon polypeptide including an acid sequence shown in at least one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 or 86; and (b) the amino acid sequence of a biologically active fragment of a polypeptide shown in at least one of these SEQ ID NOs. The present invention also include polypeptides having an amino acid sequence at least 95% identical, and more preferably at least 96% identical, and even more preferably at least 97% identical, and even more preferably at least 98% identical, and still more preferably at least 99.25% identical to those polypeptides described herein. Also provided are polypeptides having an amino acid sequence with at least 95% similarity, more preferably at least 96% similarity, and even more preferably 97% similarity, and even more preferably at least 98% similarity, and still more preferably at least 99.25% similarity to those described herein. Also provided are polypeptides having an amino acid sequence with at least 95%, 96%, 97%, 98% or 99.25% homology to those described herein. Polynucleotides encoding such polypeptides are also provided. An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which comprises the amino acid sequence of an epitope-b earing portion of an interferon polypeptide having an amino acid sequence described herein.. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of an interferon polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention. In another embodiment, the invention provides an antibody that binds specifically to an interferon polypeptide having an amino acid sequence described herein. Such antibody may be a monoclonal antibody. It may also be an antigen binding fragment, such as an Fab or Fab' fragment. The antibody may be chimeric, humanized or a fully human antibody. The antibody may be a feline antibody or a rhesus antibody. The invention further provides methods for isolating antibodies that bind specifically to an interferon polypeptide having an amino acid sequence as described herein. Such antibodies are useful therapeutically as described below. In another aspect, the invention further provides a feline interferon polypeptide comprising an amino acid sequence selected from: (a) the amino acid sequence of an Interferon polypeptide including an acid sequence shown in at least one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26; and (b) the amino acid sequence of a biologically active fragment of a polypeptide shown in at least one of these SEQ ID NOs. The present invention also include polypeptides having an amino acid sequence at least 95% identical, and more preferably at least 96% identical, and even more preferably at least 97% identical, and even more preferably at least 98% identical, and still more preferably. at least 99.25% identical to those polypeptides described herein. Also provided are polypeptides having an amino acid sequence with at least 95% similarity, more preferably at least 96% similarity, and even more preferably 97% similarity, and even more preferably at least 98% similarity, and still more preferably at least 99.25% similarity to those described herein. Also provided are polypeptides having an amino acid sequence with at least 95%, 96%, 97%, 98% or 99.25% homology to those described herein. Polynucleotides encoding such polypeptides are also provided.

In another aspect, the invention further provides a Rhesus interferon polypeptide comprising an amino acid sequence selected from: (a) the amino acid sequence of an Interferon polypeptide including an acid sequence shown in at least one of SEQ ID NO: 28, 30, 32, 34 or 36; and (b) the amino acid sequence of a biologically active fragment of a polypeptide shown in at least one of these SEQ ID NOs. The present invention also include polypeptides having an amino acid sequence at least 95% identical, and more preferably at least 96% identical, and even more preferably at least 97% identical, and even more preferably at least 98% identical, and still more preferably at least 99.25% identical to those polypeptides described herein. Also provided are polypeptides having an amino acid sequence with at least 95% similarity, more preferably at least 96% similarity, and even more preferably 97% similarity, and even more preferably at least 98% similarity, and still more preferably at least 99.25% similarity to those described herein. Also provided are polypeptides having an amino acid sequence with at least 95%, 96%, 97%, 98% or 99.25% homology to those described herein. Polynucleotides encoding such polypeptides are also provided.

In another aspect, the invention further provides a human interferon polypeptide comprising an amino acid sequence selected from: (a) the amino acid sequence of an Interferon polypeptide including an acid sequence shown in at least one of SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 or 86; and (b) the amino acid sequence of a biologically active fragment of at least one of a polypeptide shown in these SEQ ID NOs. The present invention also include polypeptides having an amino acid sequence at least 95% identical, and more preferably at least 96% identical, and even more preferably at least 97% identical, and even more preferably at least 98% identical, and still more preferably at least 99.25% identical to those polypeptides described herein. Also provided are polypeptides having an amino acid sequence with at least 95% similarity, more preferably at least 96% similarity, and even more preferably 97% similarity, and even more preferably at least 98% similarity, and still more preferably at least 99.25% similarity to those described herein. Also provided are polypeptides having an amino acid sequence with at least 95%, 96%, 97%, 98% or 99.25% homology to those described herein. Polynucleotides encoding such polypeptides are also provided.

In another aspect, the invention further provides compositions comprising any of the interferon polynucleotides or interferon polypeptides, described herein, for administration to cells in vitro, to cells ex vivo, and to cells in vivo, or to a multicellular organism. In certain particularly preferred embodiments of this aspect of the invention, the compositions comprise an interferon polynucleotide for expression of an interferon polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with loss or lack of activity of an endogenous interferon. The invention also provides for pharmaceutical compositions comprising interferon polypeptides and methods which may be employed, for instance, to treat or prevent immune system-related disorders such as viral infection, parasitic infection, bacterial infection, cancer, autoimmune disease, multiple sclerosis, lymphoma and allergy. Methods of treating individuals in need of interferon polypeptides are also provided, hi certain preferred embodiments, the subject pharmaceutical composition is a veterinary composition for administration to a non-human animal, preferably a non- human primate. Exemplary conditions which can be treated with an interferon include but are not limited to viral infections. Without limitation, treatment with interferon may be used to treat conditions which would benefit from inhibiting the replication of interferon-sensitive viruses. Viral infections which may be treated in accordance with the invention include severe acute respiratory syndrome (SARS), smallpox virus, cowpox virus, monkeypox virus, West Nile virus, vaccinia, coronavirus, hepatitis A, hepatitis B, hepatitis C, other non-A/non-B hepatitis, herpes virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex, human herpes virus type 6 (HHV-6), papilloma virus, poxvirus, picornavirus, adenovirus, rhinovirus, human T lymphotropic virus-type 1 and 2 (HTLV-1/-2), human rotavirus, rabies, retroviruses including human immunodeficiency virus (HIV), encephalitis, arterivirus, filovirus, reovirus, papovavirus, hepadnavirus, astroviras, coxsackie virus, orthomyxoviridae (influenza viruses), paramyxoviridae, echovirus, enterovirus, and respiratory syncytial virus. Of particular interest is the treatment of SARS, which is believed to be caused by novel coronaviruses named SARS-associated coronaviruses (Peiris et al., Lancet, April 19, 2003; 361(9366): 1319-25; Ksiazek et al., New England Journal of Medicine, April 30, 2003 under PubMed LD#12690092; Drosten et al., New England Journal of Medicine, April 10, 2003, under PubMed ID#12690091; Marra et al., Science, epublication on May 1, 2003 with PubMed ID#12730501. PubMed articles can be accessed by E># at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi? CMD ^:::Search&DB=PubMedκ The method of the invention can also be used to modify various immune responses. The interferon polypeptides described herein may be used for treating SARS and viral infections caused by the viruses described herein. These interferon polypeptides may also used in a prophylactic manner, such as by preventing the infection or preventing the subject from exhibiting symptoms associated with the infection. Subjects who may benefit from such preventive treatment include those with an elevated risk of being infected, such as subjects who have become exposed to the virus or to individuals who have been infected by or exposed to the virus. In one embodiment, interferons can be used as anti-viral agents. Interferons have been used clinically for the treatment of acquired immune disorders, viral hepatitis including chronic hepatitis B, hepatitis C, hepatitis D, papilloma viruses, herpes, viral encephalitis, and in the prophylaxis of rhinitis and respiratory infections. h another embodiment, an interferon can be used as anti-parasitic agents. The interferons may be used, for example, for treating Cryptosporidium parvum infection. In still another embodiment, Interferons can be used as anti-bacterial agents. Interferons have been used clinically for anti-bacterial therapy. For example, Interferons can be used in the treatment of multidrug-resistant pulmonary tuberculosis. In yet another embodiment, Interferons can be used as part of an immunotherapy protocol. The interferons of the present invention may be used clinically for immunotherapy or more particularly, for example, to prevent graft vs. host rejection, or to curtail the progression of autoimmune diseases, such as arthritis, multiple sclerosis, or diabetes. In another embodiment, interferons can be used as part of a program for treating allergies. In still another embodiment, Interferons can be used as vaccine adjuvants. Interferons may be used as an adjuvant or coadjuvant to enhance or stimulate the immune response in cases of prophylactic or therapeutic vaccination. In addition to the treatment of animals in general, the specific invention particularly contemplates the use of interferons for the treatment of primates as part of veterinary protocols. In one embodiment, the interferon is a Rhesus interferon. The specific invention also particularly contemplates the use of interferons for the treatment of cats as part of veterinarian protocols. In one embodiment, the interferon is a feline interferon. In certain embodiments, interferons are used to treat cats for viral infections. For instance, cats with Feline Immunodeficiency Virus (FIV) require support therapies in order to maintain normal health. Interferons can be used as part of a treatment of cats infected with FIV. Likewise, Interferons can be used as part of a treatment of cats infected with Feline Leukemia Virus (FeLV). The feline leukemia virus (FeLV) is the causative agent of the most important fatal infectious disease complex of American domestic cats today. Interferons can be used for treating feline panleukopenia. Also called feline infectious enteritis, feline "distemper," and feline ataxia or incoordination, feline panleukopenia is a highly contagious viral disease of cats characterized by its sudden onset, fever, inappetence (loss of appetite), dehydration, depression, vomiting, decreased numbers of circulating white blood cells (leukopenia), and often a high mortality rate. Intrauterine (within the uterus) infection may result in abortions, stillbirths, early neonatal deaths, and cerebellar hypoplasia (underdevelopment of the cerebellum) manifested by incoordination (ataxia) in kittens beginning at two to three weeks of age. All members of the cat family (Felidae) are susceptible to infection with feline panleukopenia virus (FPV), as are raccoons, coatimundis, and ringtails, in the family Procyoniclae.

Interferons can be used for treating cats infected with feline infectious peritonitis. Interferons can be used for treating cats infected with rabies. In other embodiments directed to feline care, interferons can be used in treating inflammatory airway disease (IAD). In still another embodiment, Interferons can be used to treat dogs or other household pets, (de Mari K, Maynard L, Eun HM, Lebreux B. Vet Rec. (2003) 152:105-8). In still another embodiment, interferons can be used to treat farm animals. In yet another embodiment, Interferons can be used to treat humans. This invention provides a method of treating a subject afflicted with severe acute respiratory syndrome, comprising administering to the subject an amount of an interferon polypeptide effective to reduce the concentration of SARS-associated coronavirus particles in the subject, thereby treating the subject. This invention provides a method of treating a subject infected with a virus selected from the group consisting of coronavirus, smallpox virus, cowpox virus, monkeypox virus, West Nile virus, vaccinia virus, respiratory syncytial virus, rhinovirus, arterivirus, filovirus, picornavirus, reovirus, retrovirus, papovavirus, herpesvirus, poxvirus, hepadnavirus, astrovirus, coxsackie virus, paramyxoviridae, orthomyxoviridae, echovirus, enterovirus, cardiovirus, togavirus, rhabdo virus, bunyavirus, arenavirus, bornavirus, adenovirus, parvovirus, and flavivirus, comprising administering to the subject an amount of an interferon polypeptide that is effective to reduce the concentration of virus particles in the subject, thereby treating the subject. This invention contemplates any of the treatment methods described herein also as methods for preventing the subject from becoming afflicted or infected, or as methods of reducing the subject's risk of a affliction or infection, or as protecting the subject against disorders/conditions related to a particular virus, or as preventing the subject from exhibiting symptoms associated with a viral infection. For instance, the above methods for treating subject infected with a virus may also be used to prevent the subject from becoming infected with the virus, or to reduce the subject risk of viral infection. This invention provides a method of reducing a subject's risk of viral infection comprising administering to the subject an interferon polypeptide. In one embodiment, this method comprises preventing the subject from being infected with the virus. In one embodiment, this method comprises preventing the subject from exhibiting symptoms associated with a viral infection. In one embodiment, this method comprises protecting the subject against disorders/conditions related to a particular virus. This protection may be conferred by preventing or lessening the severity of a disorder/condition resulting from the infection. In another embodiment, the protection may also be conferred by reducing the spread of infection to others by lessening the severity of a disorder/condition resulting from the infection in the patient. In another embodiment, the prevention or reduction of risk is effected by causing the subject's cells to become less susceptible to infection. The methods and embodiments described herein are not necessarily mutually exclusive. The viral infections include but are not limited to those caused by coronavirus, smallpox virus, cowpox virus, monkeypox virus, West Nile virus, vaccinia virus, respiratory syncytial virus, rhinovirus, arterivirus, filovirus, picornavirus, reovirus, retrovirus, papovavirus, herpesvirus, poxvirus, hepadnavirus, astro virus, coxsackie virus, paramyxoviridae, orthomyxoviridae, echovirus, enterovirus, cardiovirus, togavirus, rhabdovirus, bunyavirus, arenavirus, bornavirus, adenovirus, parvovirus, and flavivirus. This invention provides a method of treating a subject afflicted with influenza (orthomyxo virus), comprising administering to the subject an amount of an interferon polypeptide that is effective to reduce the concentration of influenza virus particles in the subject, wherein the interferon polypeptide comprises an amino acid sequence that is at least 95% identical, and more preferably at least 96% identical, and even more preferably at least 97% identical, and even more preferably at least 98% identical, and still more preferably at least 99.25% identical to at least one of the SEQ LD NOs described herein, thereby treating the subject.

The interferon polypeptides referred to herein, such as in the context of treatment and/or prevention, include but are not limited to LFN-α polypeptides, LFN-β polypeptides, IFN-γ polypeptides, and IFN-ω polypeptides (Zoon KC: Human Interferons: Structure and Function, p. 1-12. hi: Interferon 8. Academic Press, London, 1987; Walter et al., Cancer Biotherm Radiopharm 1998 June; 13(3):143-54; Pestka, S., Biopolymers 2000; 55(4):254-287; Biopolymers 2000; 55(4):254-287; Pestka, S., Methods in Enzymology, 78, 1981; Pestka, S., Methods in Enzymology, 79, 1981; Pestka, S., Methods in Enzymology, 119, 1986; Pestka, S., Langer; J.A., Zoon, KC, Samuel, CE. Ann Rev Biochem 1987, 56, 727-777). They also include but are not limited to human JJFN, murine LFN, feline IFN and rhesus LFN. In one embodiment of the interferon polypeptide described herein, the interferon polypeptide comprises an amino acid sequence that is at least at least 95% identical, and more preferably at least 96% identical, and even more preferably at least

97% identical, and even more preferably at least 98% identical, and still more preferably at least 99.25% identical to one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,

20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 68, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 or 86. Also provided are polypeptides having an amino acid sequence with at least 95% similarity, more preferably at least 96% similarity, and even more preferably 97% similarity, and even more preferably at least

98%o similarity, and still more preferably at least 99.25% similarity to those above. Also provided are polypeptides having an amino acid sequence with at least 95%, 96%, 97%, 98% or 99.25%) homology to those described herein. In one embodiment of the interferon polypeptide described herein, the interferon polypeptide comprises an amino acid sequence selected from the group consisting of SEQ LD NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 68, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 86. In one embodiment of the interferon polypeptide described herein, the interferon polypeptide is encoded by a nucleic acid, which nucleic acid comprises a nucleotide sequence selected from the group of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 and 85. In one embodiment of the interferon polypeptide described herein, the interferon polypeptide is encoded by a nucleic acid which hybridizes under high stringency to a nucleic acid comprising a sequence selected from the group SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 and 85. In one embodiment of the interferon polypeptide described herein, the interferon polypeptide is encoded by a nucleic acid which hybridizes under high stringency to a nucleic acid complementary to a sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 and 85.

This invention provides a method of preventing a subject from becoming afflicted with severe acute respiratory syndrome, comprising administering to the subject an interferon polypeptide. This invention provides a method of reducing the risk of a subject from becoming afflicted with severe acute respiratory syndrome, comprising administering to the subject an interferon polypeptide.

This invention also provides a method of preventing a subject from becoming afflicted with a syndrome caused by a virus selected from the group consisting of coronavirus, smallpox virus, cowpox virus, monkeypox virus, West Nile virus, vaccinia virus, respiratory . syncytial virus, rhinovirus, arterivirus, filovirus, picornavirus, reovirus, retrovirus, papovavirus, herpesvirus, poxvirus, hepadnavirus, astrovirus, coxsackie virus, paramyxoviridae, orthomyxoviridae, echovirus, enterovirus, cardiovirus, togavirus, rhabdovirus, bunyavirus, arenavirus, bornavirus, adenovirus, parvovirus, and flavivirus, comprising administering to the subject an amount of an interferon polypeptide. This invention provides a method of preventing a subject from becoming afflicted with influenza, comprising administering to the subject an amount of an Interferon polypeptide described herein. This invention provides a method of reducing the risk of a subject from becoming infected with influenza, comprising administering to the subject an amount of an Interferon polypeptide described herein. In one embodiment of the methods described herein, the subject is a human being. The subjects include but are not limited to dogs, cats, monkeys, and farm animals. This invention provides the use of an interferon polypeptide described herein for the preparation of a medicament for any of the methods of treatment, prevention, prophylaxis, and reduction of risk described herein. This invention provides a pharmaceutical package comprising an interferon composition with instructions for administering the composition to a subject. The instructions may include written and/or pictorial instructions. The instructions may be for any of the methods or treatment, prevention, prophylaxis, and reduction of risk described herein. The subject invention also contemplates functional antagonists, e.g., wherein one or more amino acid residues are different from the wild-type interferon, which inhibit one or more biological activities of the wild-type Interferon. Such antagonists can be used to treat disorders resulting from aberrant overexpression or other activation of an endogenous interferon. The functional antagonists may be formulated in a pharmaceutical preparation. The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a biological activity of an hiterferon polypeptide, which involves contacting a receptor which is enhanced by an Interferon polypeptide with the candidate compound in the presence of an Interferon polypeptide, assaying, for example, anti- viral activity in the presence of the candidate compound and an Interferon polypeptide, and comparing the activity to a standard level of activity, the standard being assayed when contact is made between the receptor and Interferon in the absence of the candidate compound. In this assay, an increase in activity over the standard indicates that the candidate compound is an agonist of Interferon activity and a decrease in activity compared to the standard indicates that the compound is an antagonist of Interferon activity. An additional aspect of the invention is related to a method for treating an animal in need of an increased level of interferon activity in the body comprising administering to such an animal a composition comprising a therapeutically effective amount of an isolated Interferon polypeptide of the invention or an agonist thereof. A still further aspect of the invention is a method for treating an animal in need of a decreased level of interferon activity in the body comprising, administering to such an animal a composition comprising a therapeutically effective amount of an Interferon antagonist. Preferred antagonists for use in the present invention are Interferon-specific antibodies. Administration of the described dosages may be every other day, but is preferably once or twice a week. Doses are usually administered over at least a 24 week period by injection. Administration of the dose can be intravenous, subcutaneous, intramuscular, or any other acceptable systemic method. Based on the judgment of the attending clinician, the amount of drug administered and the treatment regimen used will, of course, be dependent on the age, sex and medical history of the patient being treated, the neutrophil count (e.g. the severity of the neutropenia), the severity of the specific disease condition and the tolerance of the patient to the treatment as evidenced by local toxicity and by systemic side-effects. Dosage amount and frequency may be determined during initial screenings of neutrophil count. Conventional pharmaceutical formulations can be also prepared using the subject interferon compositions of the present invention. The formulations comprise a therapeutically effective amount of an Interferon polypeptide together with pharmaceutically acceptable carriers. For example, adjuvants, diluents, preservatives and/or solubilizers, if needed, may be used in the practice of the invention. Pharmaceutical compositions of interferon including those of the present invention may include diluents of various buffers (e.g., Tris-HCl, acetate, phosphate) having a range of pH and ionic strength, carriers (e.g., human serum albumin), solubilizers (e.g., Polyoxyethylene Sorbitan or TWEEN™ polysorbate), and preservatives (e.g., thimerosal, benzyl alcohol). See, for example, U.S. Pat. No. 4,496,537. The therapeutic amount of the interferon composition administered to treat the conditions described above is based on the interferon activity of the composition. It is an amount that is effective to significantly affect a positive clinical response. In the case of treatment for a viral infection, a positive clinical response may be indicated by a reduction in the concentration of virus particles in the subject, or more generally as a reduction in the symptoms of the infection. In the case of prophylactic treatment, a positive clinical response may be indicated, for example, by an absence of virus particles in the subject, by a reduction in the concentration of virus particles in the subject, or by maintaining the concentration of virus particles in the subject below the^ threshold concentration above which the subject exhibits the symptoms of the viral infection. As used herein, a prophylactic treatment includes preventing a subject from becoming afflicted with a disorder caused by a virus.

Although the clinical dose will cause some level of side effects in some patients, the maximal dose for mammals including humans is the highest dose that does not cause unmanageable clinically-important side effects. For purposes of the present invention, such clinically important side effects are those which would require cessation of therapy due to severe flu-like symptoms, central nervous system depression, severe gastrointestinal disorders, alopecia, severe pruritus or rash. Substantial white and/or red blood cell and or liver enzyme abnormalities or anemialike conditions are also dose limiting. Naturally, the dosages of interferon may vary somewhat depending upon the formulation selected. In general, however, the interferon composition is administered in amounts ranging from about 100,000 to about several million IU/m² per day, based on the mammal's condition. The range set forth above is illustrative and those skilled in the art will determine the optimal dosing of Interferon selected based on clinical experience and the treatment indication.

The pharmaceutical compositions may be in the form of a solution, suspension, tablet, capsule, lyophilized powder or the like, prepared according to methods well known in the art. It is also contemplated that administration of such compositions will be chiefly by the parenteral route although oral or inhalation routes may also be used depending upon the needs of the artisan.

The term "isolated" as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. The term isolated also refers to a nucleic acid or peptide, polypeptide or protein that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an "isolated nucleic acid" is meant to include nucleic acid fragment which are not naturally occurring as fragments and would not be found in the natural state.

A preferred method for determining the best overall match between a query sequence and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci, 6:237-245 (1990)). The term "sequence" includes nucleotide and amino acid sequences. In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is presented in terms of percent identity. Preferred parameters used in a FASTDB search of a DNA sequence to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining Penalty=30, Randomization Group Length=0, and Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, and Window Size=500 or query sequence length in nucleotide bases, whichever is shorter. Preferred parameters employed to calculate percent identity and similarity of an amino acid alignment are:

150, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty=0.05, and Window Size=500 or query sequence length in amino acid residues, whichever is shorter.

Protein and/or nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85(8):2444-2448; Altschul et al., 1990, J. Mol. Biol. 215(3):403- 410; Thompson et al., 1994, Nucleic Acids Res. 22(2):4673-4680; Higgins et al., 1996, Methods Enzymol. 266:383-402; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Altschul et al, 1993, Nature Genetics 3:266-272).

In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST") which is well known in the art (see, e.g., Karlin and Altschul, 1990, Proc. Natl. Acad Sci. USA 87:2267-2268; Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al, 1993, Nature Genetics 3:266-272; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402). In particular, five specific BLAST programs are used to perform the following task: (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database;

(4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and

(5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al, 1992, Science 256:1443-1445; Henikoff and Henikoff, 1993, Proteins 17:49-61). Less preferably, the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).

The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably select those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268). The parameters used with the above algorithms may be adapted depending on the sequence length and degree of homology studied. In some embodiments, the parameters may be the default parameters used by the algorithms in the absence of instructions from the user.

In some embodiments, the FASTDB algorithm described in Brutlag et al. Comp. App. Biosci. 6:237-245, 1990, is used. In such analyses the parameters may be selected as follows: Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the sequence which hybridizes to the probe, whichever is shorter.

Using the above methods and algorithms such as FASTA with parameters depending on the sequence length and degree of homology studied, for example the default parameters used by the algorithms in the absence of instructions from the user, one can obtain nucleic acids encoding proteins having at least 99.25%, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80% or at least 75% homology to a protein encoded by a nucleic acid. In some embodiments, the homology levels can be determined using the "default" opening penalty and the "default" gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in: Atlas of Protein Sequence and Structure, Vol. 5, Supp. 3 (1978)).

Alternatively, the level of polypeptide homology may be determined using the FASTDB algorithm described by Brutlag et al. Comp. App. Biosci. 6:237-245, 1990. In such analyses the parameters may be selected as follows: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Window Size=Sequence Length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the homologous sequence, whichever is shorter.

The invention also encompasses sequences having a lower degree of identity than that described herein but having sufficient similarity so as to perform one or more of the same functions. Similarity is determined by conserved amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and He, interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent is found in Bowie et al., Science 247:1306-1310 (1990).

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes), h a preferred embodiment, at least 30%, 40%, 50%, 60%>, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al, Nucleic Acids Res. 12(1):387 (1984)) (available at http ://www. cg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C C Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). In the nucleotide and amino acid sequences described herein within the sequence listing, with respect to a nucleotide sequence, an "n" or "x" can refer to any nucleotide, whereas with respect to a protein sequence, an "n" or "x" can refer to any amino acid.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Brief Description of the Drawings Figure 1 : shows the antiviral activity of the feline IFNα species.

Figure 2: shows the antiviral activity of the Rhesus IFNα species.

Figure 3: shows the sequence of the PCR primers used to amplify the human IFNα species. Figure 4: shows the primer pairs (described in detail in Figure 3) used to identify each of the human IFNo; species.

Figure 5: Inhibition of SARS Coronavirus cytopathic effect by interferons. Interferon inhibits the cytopathic effect of SARS CoV in a dose dependent manner. This assay was performed by infecting monkey kidney FRhk-4 cells with a SARS clinical isolate (GZ50). A score of 3 indicates full viral killing of the cells by virus (thus, no protection by interferon) and a score of 0 indicates no killing of cells by virus (full protection by interferon). See Example 8.

Figure 6: Inhibition of SARS CoV replication by interferons. The effectiveness of IFN o2a and novel interferons in inhibiting SARS CoV (Urbani) cytopathic effect on African green monkey cells (Vero 76). The IC50 for viral inhibition was determined either by direct visual evaluation of live cells or by neutral red staining of live cells. See Example 9.

Figure 7: Inhibition of Respiratory Syncytial Virus replication by interferons. The ability of IFN to inhibit the replication of two clinical isolates of B group Respiratory Syncytial Virus (RSV) and two clinical isolates of A group RSV was tested in vitro. The IC50 was determined by serial dilution of the IFNs and the concentration which inhibited viral replication by 50% was determined immunoassay for viral proteins. For those samples where the bar extends to the top of the graph, IC50s were calculated to be greater than 2500 pg/ml and not readily determinable in this assay. See Example 10.

Figure 8: Effectiveness of interferons in protecting human lung fibroblasts from Influenza virus infection. Effectiveness of interferons at inhibiting Influenza growth in human lung fibroblasts in vitro. The IC50 for each interferon was determined and the IC50 value attained for IFN o2a was set to one. The ratio of the IC50 novel LFN I IC50 LFN o_2a was determined. In this graph a higher number indicates that the interferon is more potent than LFN o2a. See Example 11.

Figure 9: Protection of liver cells from Yellow Fever Virus infection: The effectiveness of interferons to protect liver cells from YFV infection as a model of Hepatitis C treatment was determined using HepG2 liver cells. The IC50 was determined colorimetrically. Two independent determinations were made. See Example 12.

Figure 10: Comparison of IFN o2a and novel IFNs in a variety of cells types with different viral challenges. The effectiveness of LFN o2a and novel interferons was tested on 5 different cell types and with 3 different viruses. In all cases the activity of IFN Q2 was set as one and if the IFN is more potent then the value is greater than on if less potent it is less than one. See Examples 7 and 13.

Detailed Description of the Invention I. Exemplary Preparations hi another, aspect, the present invention provides pharmaceutical preparations comprising Interferons, Interferon agonists or Interferon antagonists. The Interferons, Interferon agonists and/or Interferon antagonists for use in the subject method may be conveniently formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof. The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists. As used herein, "biologically acceptable medium" includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the activity of the compositions of the present invention, its use in the pharmaceutical preparation of the invention is contemplated. Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "deposit formulations". Pharmaceutical formulations of the present invention can also include veterinary compositions, e.g., pharmaceutical preparations of the compositions of the present invention suitable for veterinary uses, e.g., for the treatment of livestock, non- human primate, or domestic animals, e.g., dogs and cats. Rechargeable or biodegradable devices may also provide methods of introduction. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for sustained release at a particular target site. The preparations of the present invention may be given orally, parenterally, topically, intrathecally/intracerebroventricularly (ICV), intracranially, directly into the central nervous system (intracavitary), or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, controlled release patch, etc., administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. In some embodiment, oral and topical administrations may be preferred. In one embodiment, the interferon is delivered directly to nasopharyngeal mucosa. In one embodiment, the interferon is delivered directly to the lung epithelium. This may make these cells resistant to viruses rather than using these tissues as a vehicle for systemic delivery. In this case, systemic interferon will be minimal such that side effects will be minimal or eliminated. The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra- arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, the urinary bladder or other compartment of the body, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually. Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms such as described below or by other conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. hi general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. The term "treatment" is intended to encompass also prophylaxis, therapy, cure, and prevention of spread of infection. The patient receiving this treatment is any animal in need or animal vector

(source of virus), including primates, in particular humans, and other mammals such as equines, cattle, swine, rodents and sheep; and poultry and pets in general. The compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable and/or sterile carriers and can also be administered in conjunction with other agents. Non-limiting examples of such agents include antimicrobial agents such as penicillins, cephalosporins, aminoglycosides, and glycopeptides. Conjunctive therapy thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutic effects of the first administered one is not entirely disappeared when the subsequent is administered. II. Pharmaceutical Compositions While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). The compositions of the present invention may be formulated for administration in any convenient way for use in human or veterinary medicine. In certain embodiments, the compound included in the pharmaceutical preparation may be active itself, or may be a prodrug, e.g., capable of being converted to an active compound in a physiological setting.

Thus, another aspect of the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more of the compounds described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginally or intrarectally, for example, as a pessary, cream or foam. However, in certain embodiments the subject compounds may be simply dissolved or suspended in sterile water. In certain embodiments, the pharmaceutical preparation is non-pyrogenic, i.e., does not elevate the body temperature of a patient.

The phrase "therapeutically effective amount" as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal and thereby blocking the biological consequences of that pathway in the treated cells, at a reasonable benefit/risk ratio applicable to any medical treatment.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agonists from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. III. Exemplary Formulations The interferons of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, polymers, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. The subject interferons can be provided in formulations also including penetration enhancers, carrier compounds and/or transfection agents. The compositions of the invention also encompass any pharmaceutically acceptable salts, esters or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts and other bioequivalents. Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,NI-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al, "Pharmaceutical Salts," J. of Pharma Sci., 1977, 66,1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a "pharmaceutical addition salt" includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids.

A. Supramolecular complexes In certain embodiments, the formulations are as part of a "supramolecular complex." To further illustrate, the interferon can be contacted with at least one polymer to form a composite and then the polymer of the composite treated under conditions sufficient to fonn a supramolecular complex containing the interferon and a multi-dimensional polymer network. The polymer molecule may be linear or branched. Accordingly, a group of two or more polymer molecules may be linear, branched, or a mixture of linear and branched polymers. The composite may be prepared by any suitable means known in the art. For example, the composite may be formed by simply contacting, mixing or dispersing the interferon with a polymer. A composite may also be prepared by polymerizing monomers, which may be the same or different, capable of forming a linear or branched polymer in the presence of the expression construct. The composite may be further modified with at least one ligand, e.g., to direct cellular uptake of the expression constmct or otherwise effect tissue or cellular distribution in vivo of the expression constmct. The composite may take any suitable form and, preferably, is in the form of particles. In certain preferred embodiments, the subject interferons are formulated with cationic polymers. Exemplary cationic polymers include poly(L)lysine (PLL) and polyethylenimine (PEI). In certain preferred embodiments, the subject expression constructs are formulated with β-cyclodextrin containing polymers (β CD-polymers). βCD-polymers are capable of forming polyplexes with nucleic acids and transfecting cultured cells. The βCD-polymers can be synthesized, for instance, by the condensation of a diamino-cyclodextrin monomer A with a diimidate comonomer B. Cyclodextrins are cyclic polysaccharides containing naturally occurring D(+)-glucopyranose units in an α-(l,4) linkage. The most common cyclodextrins are α-cyclodextrins, β- cyclodextrins and γ-cyclodextrins which contain, respectively, six, seven or eight glucopyranose units. Exemplary cyclodextrin delivery systems which can be readily adapted for delivery of the subject interferon are described in, for example, the Gonzalez et al PCT application WO00/01734 and Davis PCT application WO00/33885. In certain embodiments, the supramolecular complexes are aggregated into particles, for example, fonnulations of particles having an average diameter of between 20 and 5000 nanometer (nm). In another embodiment, the particles have an average diameter of between 20 and 200 nm. In another embodiment, the particles have an average diameter of between 2 and 10 microns. Use of a particle size of between 2 and 10 microns may be used for example, for delivery to the lung.

B. Other cationic, non-lipid formulations In certain embodiments, the interferons are provided in cationic, non-lipid vehicles and formulated to be used in aerosol delivery via the respiratory tract. Formulations using poly(ethylenimine) and macromolecules such as dsRNA and dsRNA-encoding plasmids can result in a high level of pulmonary transfection and increased stability during nebulization. PEI-nucleic acid formulations can also exhibit a high degree of specificity for the lungs. In addition to formulating interferon with PEI, the invention also contemplates the use of cyclodextrin-modified polymers, such as cyclodextrin-modified poly(ethylenimine). In certain embodiments, the subject polymers have a stmcture of the formula:

wherein R represents, independently for each occurrence, H, lower alkyl, a

cyclodextrin moiety, or

m, independently for each occurrence, represents an integer from 2-10,000, preferably from 10 to 5,000, or from 100 to 1,000. In certain embodiments, R represents a cyclodextrin moiety for at least about

1%, more preferably at least about 2%, or at least about 3%, and up to about 5% or even 10%, of the nitrogen atoms that would be primary amines (i.e., bearing two occuπences of R that represent H) but for the cyclodextrin moieties. In certain embodiments, the cyclodextrin moieties make up at least about 2%, 3% or 4% by weight, up to 5%, 7%, or even 10% of the cyclodextrin-modified polymer by weight. hi certain embodiments, at least about 2%, 3% or 4% by weight, up to 5%, 7%, or even 10% of the ethylenimine subunits in the polymer are modified with a cyclodextrin moiety. Copolymers of poly(ethylenimine) that bear nucleophilic amino substituents susceptible to derivatization with cyclodextrin moieties can also be used to prepare cyclodextrin-modified polymers within the scope of the present invention. Exemplary cyclodextrin moieties include cyclic structures consisting essentially of from 7 to 9 saccharide moieties, such as cyclodextrin and oxidized cyclodextrin. A cyclodextrin moiety optionally comprises a linker moiety that forms a covalent linkage between the cyclic stmcture and the polymer backbone, preferably having from 1 to 20 atoms in the chain, such as alkyl chains, including dicarboxylic acid derivatives (such as glutaric acid derivatives, succinic acid derivatives, and the like), and heteroalkyl chains, such as oligoethylene glycol chains. C. Liposome Formulations In certain embodiments, the invention provides composition including interferons that are encapsulated or otherwise associated with liposomes. Liposomes, as used herein, refer to lipid vesicles having an outer lipid shell, typically formed on one or more lipid bilayers, encapsulating an aqueous interior. In a prefeπed embodiment, the liposomes are cationic liposomes composed of between about 20-80 mole percent of a cationic vesicle-forming lipid, with the remainder neutral vesicle- forming lipids and/or other components. As used herein, "vesicle-forming lipid" refers to any amphipathic lipid having hydrophobic and polar head group moieties and which by itself can form spontaneously into bilayer vesicles in water, as exemplified by phospholipids. A preferred vesicle-forming lipid is a diacyl-chain lipid, such as a phospholipid, whose acyl chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation. A cationic vesicle-forming lipid is one whose polar head group with a net positive charge, at the operational pH, e.g., pH 4-9. Typical examples include phospholipids, such as phosphatidylethanolamine, whose polar head groups are derivatized with a positive moiety, e.g., lysine, as illustrated, for example, for the lipid DOPE derivatized with L-lysine (LYS-DOPE) (Guo, et al., 1993). Also included in this class are the glycolipids, such as cerebrosides and gangliosides having a cationic polar head-group. Another cationic vesicle-forming lipid which may be employed is cholesterol amine and related cationic sterols. Exemplary cationic lipids include l,2-diolelyloxy-3- (trimethylamino) propane (DOTAP); N-[l-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl- N-hydroxyethylammonium bromide (DMPJE); N-[l-(2,3,-dioleyloxy)propyl]-N,N- dimethyl-N-hydroxy ethylammonium bromide (DORTE); N-[l-(2,3-dioleyloxy) ρropyl]-N,N,N-trimethylammonium chloride (DOTMA); 3β [N-(N',N- dimethylaminoethane) carbamoly] cholesterol (DC-Choi); and dimethyldioctadecylammom^'um (DDAB). The remainder of the liposomes are formed of neutral vesicle-forming lipids, meaning vesicle foπning lipids which have no net charge or which may include a small percentage of lipids having a negative charge in the polar head group. Included in this class of lipids are the phospholipids, such as phosphatidylcholine (PC), phosphatidyl ethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM), and cholesterol, cholesterol derivatives, and other uncharged sterols. The above-described lipids can be obtained commercially, or prepared according to published methods. Other lipids that can be included in the invention are glycolipids, such as cerebrosides and gangliosides. In one embodiment of the invention, the interferon-liposome complex includes liposomes having a surface coating of hydrophilic polymer chains, effective to extend the blood circulation time of the plasmid/liposome complexes. Suitable hydrophilic polymers include cyclodextrin (CD), polyethylene glycol (PEG), polylactic acid, polyglycolic acid, polyvinyl-pyπolid-one, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses, such as hydroxymethylcellulose or hydroxyethyl-cellulose. A preferred hydrophilic polymer chain is polyethyleneglycol (PEG), preferably as a PEG chain having a molecular weight between 500-10,000 daltons, more preferably between 1,000-5,000 daltons. The hydrophilic polymer may have solubility in water and in a non-aqueous solvent, such as chloroform. The coating is preferably prepared by including in the vesicle-forming lipids a phospholipid or other diacyl-chain lipid, derivatized at its head group with the polymer chain. Exemplary methods of preparing such lipids, and forming polymer coated liposomes therewith, have been described in U.S. Pat. Nos. 5,013,556, and 5,395,619, which are incorporated herein by reference. It will be appreciated that the hydrophilic polymer can be stably coupled to the lipid, or coupled through an unstable linkage which allows the polymer-coated plasmid- liposome complexes to shed or "release" the hydrophilic polymer coating during circulation in the bloodstream or after localization at a target site. Attachment of hydrophilic polymers, in particular polyethyleneglycol (PEG), to vesicle-forming lipids through a bond effective to release the polymer chains in response to a stimulus have been described, for example in WO 98/16202, WO 98/16201, which are hereby incorporated by reference, and by Kirpotin, D. et a. (FEBS Letters, 388:115-118 (1996). The releasable linkage, in one embodiment, is a chemically releasable linkage which is cleaved by administration of a suitable releasing agent or is cleaved under selective physiological conditions, such as in the presence of enzymes or reducing agents. For example, ester and peptide linkages are cleaved by esterase or peptidase enzymes. Disulfide linkages are cleaved by administration of a reducing agent, such as glutathione or ascorbate, or by a reducing agent present in vivo, such as cysteine, which is present in plasma and intracellularly. Other releasable linkages include pH sensitive bonds- and bonds which are cleaved upon exposure to glucose, light or heat. By way of an example, the hydrophilic polymer chains can be attached to the liposome by a pH sensitive bond, and the plasmid-liposome complexes are targeted to a site having a pH effective to cleave the bond and release the hydrophilic chains, such as a tumor region. Exemplary pH sensitive bonds include acyloxyalkyl ether, acetal and ketal bonds. Another example is where the cleavable bond is a disulfide bond, broadly intended herein to refer to sulfur- containing bonds. Sulfur-containing bonds can be synthesized to achieve a selected degree of lability and include disulfide bonds, mixed sulfide-sulfone bonds and sulfide- sulfoxide bonds. Of the three bonds, the disulfide bond is least susceptible to thiolysis and the sulfide-sulfoxide bond is most susceptible. Such releasable bonds are useful to tailor the rate of release of the hydrophilic polymer segment from the liposome complexes. For example, a very labile disulfide bond can be used for targeting to blood cells or endothelial cells, since these cells are readily accessible and a shorter liposome blood circulation lifetime is sufficient. At the other extreme, a long-lasting or hearty disulfide bond can be used when the target is tumor tissue or other organs where a longer liposome blood circulation lifetime is generally needed for the complexes to reach the desired target. The releasable bond attaching the hydrophilic polymer chains to the liposome is cleaved in vivo typically as a result of change in environment, such as when the liposomes reach a specific site with a slightly lower pH, such as a region of tumor tissue, or a site with reducing conditions, such as a hypoxic tumor. Reducing conditions in vivo can also be effected by administration of a reducing agent, such as ascorbate, cysteine or glutathione. The cleavable bond may also be broken in response to external stimuli, such as light or heat. In another embodiment, the liposome complexes include an affinity moiety or targeting ligand effective to bind specifically to target cells at which the therapy is aimed. Such moieties can be attached to the surface of the liposome or to the distal ends of hydrophilic polymer chains. Exemplary moieties include antibodies, ligands for specific binding to target cell surface receptors and the like, as described, for example, in PCT application Nos. WO US94/03103, WO 98/16202 and WO 98/16201. The moiety can also be a hydrophobic segment to facilitate fusion of the complex with a target cell. Polycationic condensing agents used to condense the interferon can be multiply charged cationic polymers, and are preferably biopolymers such as such as spermidine, spermine, polylysine, protamine, total histone, specific histone fractions such as HI, H2, H3, H4, and other polycationic polypeptides, but may also include biocompatible polymers, such as polymyxin B. It will be appreciated that these polycationic condensing agents can be used in free base or salt forms, for example, protamine sulfate and polylysine hydrobromide. In a preferred embodiment, the polycationic condensing agent is a histone, which, as referred to herein, includes total histone or specific histone fractions.

. άT hi certain embodiments, the hydrophobic segment in the polymer-lipid conjugate is a hydrophobic polypeptide sequence. Preferably, the polypeptide sequence consists of about 5-80, more preferably 10-50, most preferably 20-30, non-polar and/or aliphatic/aromatic amino acid residues. These sequences are active in triggering fusion of certain enveloped viruses with host cells and include parainfluenza vimses, such as Sendai, Simian Virus-5 (SV5), measles vims, Newcastle Disease Vims (NDV) and Respiratory Syncytial Vims (RSV). Other examples include human retrovimses, such as Human Immunodeficiency Virus-1 (HIV-1), the causative agent of AIDS, which infects cells by fusion of the vims envelope with the plasma membrane of the host cell. Fusion occurs at physiological (i.e., neutral) pH and is followed by injection of the viral genetic material (nucleocapsid) into the cytoplasmic compartment of the host cell.

D. Ligand-directed formulations In certain embodiments, the polymeric complexes, such as the supramolecular complexes, and liposomes of the subject invention can be associated with one or more ligands effective to bind to specific cell surface proteins or matrix on the target cell, thereby facilitating sequestration of the complex to target cells, and in some instances, enhancing uptake of the interferon by the cell. Merely to illustrate, examples of ligands suitable for use in targeting the supramolecular complexes and liposomes of the present invention to specific cell types are listed in Table 1 below. Table 1

Ligand Receptor Cell type

The present invention also contemplates the derivatization of the subject polymeric and liposomal complexes with ligands that promote transcytosis of the complexes. To further illustrate, a polymeric complex, such as a supramolecular complex, can be covalently linked to an internalizing peptide which drives the translocation of the complex across a cell membrane in order to facilitate intracellular localization of the interferon. h this regard, the internalizing peptide, by itself, is capable of crossing a cellular membrane by, e.g., transcytosis, at a relatively high rate. The internalizing peptide is conjugated, e.g., as covalent pendant group, to the polymer. hi one embodiment, the internalizing peptide is derived from the drosophila antepennepedia protein, or homologs thereof. The 60 amino acid homeodomain of the homeo-protein antepennepedia has been demonstrated to translocate through biological membranes and can facilitate the translocation of heterologous polypeptides to which it is couples. See for example Derossi et al. (1994) J Biol Chem 269:10444-10450; and

Perez et al. (1992) J Cell Sci 102:717-722. Recently, it has been demonstrated that fragments as small as 16 amino acids long of this protein are sufficient to drive intemalization. See Derossi et al. (1996) J Biol Chem 271:18188-18193. The present invention contemplates an interferon complex that is decorated with at least a portion of the antepennepedia protein (or homolog thereof) sufficient to increase the transmembrane transport of the decorated complex, relative to the undecorated complex, by a statistically significant amount. Another example of an internalizing peptide is the HIV transactivator (TAT) protein. This protein appears to be divided into four domains (Kuppuswamy et al. (1989) Nucl. Acids Res. 17:3551-3561). Purified TAT protein is taken up by cells in tissue culture (Frankel and Pabo, (1989) Cell 55:1189-1193), and peptides, such as the fragment coπesponding to residues 37 -62 of TAT, are rapidly taken up by cell in vitro (Green and Loewenstein, (1989) Cell 55: 1179-1188). The highly basic region mediates intemalization and targeting of the internalizing moiety to the nucleus (Ruben et al., (1989) J. Virol. 63:1-8). Peptides or analogs that include a sequence present in the highly basic region, such as CFIT ALGISYGRKKRRQRRRPPQGS, are conjugated to the polymer to aid in intemalization and targeting those complexes to the intracellular milieu. Another exemplary transcellular polypeptide can be generated to include a sufficient portion of mastoparan (T. Higashijima et al., (1990) J. Biol. Chem. 265 : 14176) to increase the transmembrane transport of the interferon complexes. Other suitable internalizing peptides can be generated using all or a portion of, e.g., a histone, insulin, transferrin, basic albumin, prolactin and insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II) or other growth factors. For instance, it has been found that an insulin fragment, showing affinity for the insulin receptor on capillary cells, and being less effective than insulin in blood sugar reduction, is capable of transmembrane transport by receptor-mediated transcytosis and can therefore serve as an internalizing peptide for the subject transcellular polypeptides. Prefeπed growth factor-derived internalizing peptides include EGF (epidermal growth factor)-derived peptides, such as CMHIESLDSYTC and CMYIEALDKYAC; TGF- beta (transforming growth factor beta)-derived peptides; peptides derived from PDGF (platelet-derived growth factor) or PDGF-2; peptides derived from IGF-I (insulin-like growth factor) or IGF-II; and FGF (fibroblast growth factor)-derived peptides. Another class of translocating/intemalizing peptides exhibits pH-dependent membrane binding. For an internalizing peptide that assumes a helical conformation at an acidic pH, the internalizing peptide acquires the property of amphiphilicity, e.g., it has both hydrophobic and hydrophilic interfaces. More specifically, within a pH range of approximately 5.0-5.5, an internalizing peptide forms an alpha-helical, amphiphilic stmcture that facilitates insertion of the moiety into a target membrane. An alpha-helix- inducing acidic pH environment may be found, for example, in the low pH environment present within cellular endosomes. Such internalizing peptides can be used to facilitate transport of interferon-complexes, taken up by an endocytic mechanism, from endosomal compartments to the cytoplasm. Yet other prefeπed internalizing peptides include peptides of apo-lipoprotein A- 1 and B; peptide toxins, such as melittin, bombolittin, delta hemolysin and the pardaxins; antibiotic peptides, such as alamethicin; peptide hormones, such as calcitonin, corticotrophin releasing factor, beta endorphin, glucagon, parathyroid hormone, pancreatic polypeptide; and peptides coπesponding to signal sequences of numerous secreted proteins, h addition, exemplary internalizing peptides may be modified through attachment of substituents that enhance the alpha-helical character of the internalizing peptide at acidic pH. Yet another class of internalizing peptides suitable for use within the present invention includes hydrophobic domains that are "hidden" at physiological pH, but are exposed in the low pH environment of the target cell endosome. Upon pH-induced unfolding and exposure of the hydrophobic domain, the moiety binds to lipid bilayers and effects translocation of the covalently linked complexes into the cell cytoplasm. Such internalizing peptides may be modeled after sequences identified in, e.g., Pseudomonas exotoxin A, clathrin, or Diphtheria toxin. Pore-forming proteins or peptides may also serve as internalizing peptides herein. Pore- forming proteins or peptides may be obtained or derived from, for example, C9 complement protein, cytolytic T-cell molecules or NK-cell molecules. These moieties are capable of forming ring-like structures in membranes, thereby allowing transport of attached complexes through the membrane and into the cell interior. Mere membrane intercalation of an internalizing peptide may be sufficient for translocation of the complexes across cell membranes. However, translocation may be improved by attaching to the internalizing peptide a substrate for intracellular enzymes (i.e., an "accessory peptide"). It is prefeπed that an accessory peptide be attached to a portion(s) of the internalizing peptide that protrudes through the cell membrane to the cytoplasmic face. The accessory peptide may be advantageously attached to one terminus of a translocating/internalizing moiety or anchoring peptide. An accessory moiety of the present invention may contain one or more amino acid residues. In one embodiment, an accessory moiety may provide a substrate for cellular phosphorylation (for instance, the accessory peptide may contain a tyrosine residue). An exemplary accessory moiety in this regard would be a peptide substrate for N-myristoyl transfefase, such as GNAAAARR (Eubanks et al., in: Peptides. Chemistry and Biology, Garland Marshall (ed.), ESCOM, Leiden, 1988, pp. 566-69). In this construct, an internalizing, peptide would be attached to the C-terminus of the accessory peptide, since the N-terminal glycine is critical for the accessory moiety's activity. This hybrid peptide, attached to a polymer complex, is N-myristylated and further anchored to the target cell membrane, e.g., it serves to increase the local concentration of the complex at the cell membrane. Suitable accessory peptides include peptides that are kinase substrates, peptides that possess a single positive charge, and peptides that contain sequences which are glycosylated by membrane-bound glycotransferases. Accessory peptides that are glycosylated by membrane-bound glycotransferases may include the sequence x-NLT- x, where "x" may be another peptide, an amino acid, coupling agent or hydrophobic molecule, for example. When this hydrophobic tripeptide is incubated with microsomal vesicles, it crosses vesicular membranes, is glycosylated on the luminal side, and is entrapped within the vesicles due to its hydrophilicity (C Hirschberg et al, (1987) Ann. Rev. Biochem. 56:63-87). Accessory peptides that contain the sequence x-NLT-x thus will enhance target cell retention of coπesponding complexes. As described above, the internalizing and accessory peptides can each, independently, be added to an interferon complex or liposome by chemical cross- linking or through non-covalent interaction (e.g., use of streptavidin-biotin conjugates, His₆-Ni interactions, etc). In certain instances, unstructured polypeptide linkers can be included between the peptide moieties and the polymeric complex or liposome. It is also contemplates that such internalizing and accessory peptides can be associated directly with an interferon, such as through a covalent linkage to a hydroxyl group on the backbone of the protein. In certain embodiments, the linkage is susceptible to cleavage under physiological conditions, such as by exposure to esterases, or simple hydrolysis reactions. Such compositions can be used alone or formulated in polymeric complexes or liposomes.

E. Respirable Interferons Another aspect of the invention provides aerosols for the delivery of interferon to the respiratory tract. The respiratory tract includes the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli. The upper and lower airways are called the conductive airways. The terminal bronchioli then divide into respiratory bronchioli which then lead to the ultimate respiratory zone, the alveoli, or deep lung. Herein, administration by inhalation may be oral and/or nasal, intratracheal (trans-stomal or via tracheostomy tube), or via a breathing assistance device such as a respirator. Examples of pharmaceutical devices for aerosol delivery include metered dose inhalers (MDIs), dry powder inhalers (DPIs), and air-jet nebulizers. Exemplary delivery systems by inhalation which can be readily adapted for delivery of the subject interferons are described in, for example, U.S. patents 5,756,353; 5,858,784; and PCT applications WO98/31346; WO98/10796; WO00/27359; WO01/54664; WO02/060412. Other aerosol formulations that may be used for delivering the interferons are described in U.S. Patents 6,294,153; 6,344,194; 6,071,497, and PCT applications WO02/066078; WO02/053190; WO01/60420; WO00/66206. The human lungs can remove or rapidly degrade hydrolytically cleavable deposited aerosols over periods ranging from minutes to hours. In the upper airways, ciliated epithelia contribute to the "mucociliary excalator" by which particles are swept from the airways toward the mouth. Pavia, D., "LungMucociliary Clearance," in Aerosols and the Lung: Clinical and Experimental Aspects, Clarke, S. W. and Pavia, D., Eds., Butterworths, London, 1984. In the deep lungs, alveolar macrophages are capable of phagocytosing particles soon after their deposition. Warheit et al. Microscopy Res. Tech., 26: 412-422 (1993); and Brain, J. D., "Physiology and Pathophysiology of Pulmonary Macrophages," in The Reticuloendothelial System, S. M. Reichard and J. Filkins, Eds., Plenum, New. York, pp. 315-327, 1985. The deep lung, or alveoli, are the primary target of inhaled therapeutic aerosols for systemic delivery of interferons. In prefeπed embodiments, particularly where systemic dosing with the interferon is desired, the aerosolized interferons are formulated as microparticles. Microparticles having a diameter of between 0.5 and ten microns can penetrate the lungs, passing through most of the natural barriers. A diameter of less than ten microns is required to bypass the throat; a diameter of 0.5 microns or greater is required to avoid being exhaled. In certain prefeπed embodiments, the subject interferons are formulated in a supramolecular complex, as described above, which have a diameter of between 0.5 and ten microns, which can be aggregated into particles having a diameter of between 0.5 and ten microns. hi other embodiments, the subject interferons are provided in liposomes or supramolecular complexes (such as described above) appropriately formulated for pulmonary delivery.

(i). Polymers for forming Microparticles. h addition to the supramolecular complexes described above, a number of other polymers can be used to form the microparticles. As used herein, the term "microparticles" includes microspheres (uniform spheres), microcapsules (having a core and an outer layer of polymer), and particles of iπegular shape. Polymers are preferably biodegradable within the time period over which release of the interferon is desired or relatively soon thereafter, generally in the range of one year, more typically a few months, even more typically a few days to a few weeks. Biodegradation can refer to either a breakup of the microparticle, that is, dissociation of the polymers forming the microparticles and/or of the polymers themselves. This can occur as a result of change in pH from the carrier in which the particles are administered to the pH at the site of release, as in the case of the diketopiperazines, hydrolysis, as in the case of pόly(hydroxy acids), by diffusion of an ion such as calcium out of the microparticle, as in the case of microparticles formed by ionic bonding of a polymer such as alginate, and by enzymatic action, as in the case of many of the polysaccharides and proteins. In some cases linear release may be most useful, although in others a pulse release or "bulk release" may provide more effective results. Representative synthetic materials are: diketopiperazines, poly(hydroxy acids) such as poly(lactic acid), ρoly(glycolic acid) and copolymers thereof, polyanhydrides, polyesters such as polyorthoesters, polyamides, polycarbonates, polyalkylenes such as polyethylene, polypropylene, polyethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly vinyl compounds such as polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyπolidone, polyvinylacetate, and poly vinyl chloride, polystyrene, polysiloxanes, polymers of acrylic and methacrylic acids including poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyurethanes and co-polymers thereof, celluloses including alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellullose triacetate, and cellulose sulphate sodium salt, poly(butic acid), poly(valeric acid), and poly(lactide-co-caprolactone). Natural polymers include alginate and other polysaccharides including dextran and cellulose, collagen, albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. As used herein, chemical derivatives thereof refer to substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art. Bioadhesive polymers include bioerodible hydrogels described by H. S. Sawhney, C P. Pathak and J .A. Hubell in Macromolecules, 1993, 26, 581-587, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, and polyacrylates. To further illustrate, the matrices can be formed of the polymers by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art. Methods developed for making microspheres for drug delivery are described in the literature, for example, as described by Mathiowitz and Langer, J. Controlled Release 5, 13-22 (1987); Mathiowitz, et al., Reactive Polymers 6, 275-283 (1987); and Mathiowitz, et al, J. Appl. Polymer Sci. 35, 755-774 (1988). The selection of the method depends on the polymer selection, the size, external morphology, and crystallinity that is desired, as described, for example, by Mathiowitz, et al., Scanning Microscopy 4,329-340 (1990); Mathiowitz, et al., J. Appl. Polymer Sci. 45, 125-134 (1992); and Benita, et al., J. Pharm. Sci. 73, 1721-1724 (1984). In solvent evaporation, described for example, in Mathiowitz, et al, (1990), Benita, and U.S. Pat. No. 4,272,398 to Jaffe, the polymer is dissolved in a volatile organic solvent. The interferons, either in soluble form or dispersed as fine particles, is added to the polymer solution, and the mixture is suspended in an aqueous phase that contains a surface active agent such as poly(vinyl alcohol). The resulting emulsion is stiπed until most of the organic solvent evaporates, leaving solid microspheres. In general, the polymer can be dissolved in methylene chloride. Several different polymer concentrations can be used, for example, between 0.05 and 0.20 g/ml. After loading the solution with drag, the solution is suspended in 200 ml of vigorously stirring distilled water containing 1% (w/v) poly(vinyl alcohol) (Sigma Chemical Co., St. Louis, Mo.). After four hours of stirring, the organic solvent will have evaporated from the polymer, and the resulting microspheres will be washed with water and dried overnight in a lyophilizer. Microspheres with different sizes (1-1000 microns, though less than 10 microns for aerosol applications) and morphologies can be obtained by this method which is

However, labile polymers such as polyanhydrides may degrade due to exposure to water. For these polymers, hot melt encapsulation and solvent removal may be prefeπed. In hot melt encapsulation, the polymer is first melted and then mixed with the solid particles of interferon, preferably sieved to appropriate size. The mixture is suspended in a nou-miscible solvent such as silicon oil and, with continuous stirring, heated to 5°C above the melting point of the polymer. Once the emulsion is stabilized, it is cooled until the polymer particles solidify. The resulting microspheres are washed by decantation with petroleum ether to give a free-flowing powder. Microspheres with diameters between one and 1000 microns can be obtained with this method. The external surfaces of spheres prepared with this technique are usually smooth and dense. This procedure is useful with water labile polymers, but is limited to use with polymers with molecular weights between 1000 and 50000. In spray drying, the polymer is dissolved in an organic solvent such as methylene chloride (0.04 g/ml). A known amount of interferon is suspended (if insoluble) or co-dissolved (if soluble) in the polymer solution. The solution or the dispersion is then spray-dried, Microspheres ranging in diameter between one and ten microns can be obtained with a moiphology which depends on the selection of polymer. Hydrogel microspheres made of gel-type polymers such as alginate or polyphosphazines or other dicarboxylic polymers can be prepared by dissolving the polymer in an aqueous solution, suspending the material to be incorporated into the mixture, and extruding the polymer mixture through a microdroplet forming device, equipped with a nitrogen gas jet. The resulting microspheres fall into a slowly stirring, ionic hardening bath, as described, for example, by Salib, et al., Pharmazeutische Industrie 40-111 A, 1230 (1978). The advantage of this system is the ability to further modify the surface of the microspheres by coating them^ with polycationic polymers such as polylysine, after fabrication, for example, as described by Lim, et al., J. Pharm. Sci. 70, 351-354 (1981). For example, in the case of alginate, a hydrogel can be formed by ionically crosslinking the alginate with calcium ions, then crosslinking the outer surface of the microparticle with a polycation such as polylysine, after fabrication. The microsphere particle size will be controlled using various size extruders, polymer flow rates and gas flow rates. Chitosan microspheres can be prepared by dissolving the polymer in acidic solution and crosslinking with tripolyphosphate. For example, carboxymethylcellulose (CMC) microspheres are prepared by dissolving the polymer in an acid solution and precipitating the microspheres with lead ions. Alginate/polyethylenimine (PEI) can be prepared to reduce the amount of carboxyl groups on the alginate microcapsules.

(ii). Pharmaceutical Compositions. The microparticles can be suspended in any appropriate pharmaceutical carrier, such as saline, for administration to a patient, hi the most prefeπed embodiment, the microparticles will be stored in dry or lyophilized form until immediately before administration. They can then be suspended in sufficient solution, for example an aqueous solution for administration as an aerosol, or administered as a dry powder. (Hi). Targeted Administration. The microparticles can be delivered to specific cells, especially phagocytic cells and organs. Phagocytic cells within the Peyer's patches appear to selectively take up microparticles administered orally. Phagocytic cells of the reticuloendothelial system also take up microparticles when administered intravenously. Endocytosis of the microparticles by macrophages in the lungs can be used to target the microparticles to the spleen, bone marrow, liver and lymph nodes. The microparticles can also be targeted by attachment of ligands, such as those described above, which specifically or non-specifically bind to particular targets. ' Examples of such ligands also include antibodies and fragments including the variable regions, lectins, and hormones or other organic molecules having receptors on the surfaces of the target cells.

(iv). Storage of the Microparticles. hi the prefeπed embodiment, the microparticles are stored lyophilized. The dosage is determined by the amount of encapsulated interferon, the rate of release within the pulmonary system, and the pharmacokinetics of the compound.

(v). Delivery of Microparticles. The microparticles can be delivered using a variety of methods, ranging from administration directly into the nasal passages so that some of the particles reach the pulmonary system, to the use of a powder instillation device, to the use of a catheter or tube reaching into the pulmonary tract. Dry powder inhalers are commercially available, although those using hydrocarbon propellants are no longer used and those relying on the intake of a breath by a patient can result in a variable dose. Examples of suitable propellants include hydro fluoroalkane propellants, such as 1,1,1,2- tetrafluoroethane (CF3CH2F) (HFA-134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane (CF3CHFCF3) (HFA-227), perfluoroethane, monochloroifluoromethane, 1,1 difluoroethane, and combinations thereof.

F. Medical Device Coatings for release of Interferon Another aspect of the invention relates to coated medical devices. For instance, in certain embodiments, the subject invention provides a medical device having a coating adhered to at least one surface, wherein the coating includes the subject polymer matrix and an interferon. Such coatings can be applied to surgical implements such as screws, plates, washers, sutures, prosthesis anchors, tacks, staples, electrical leads, valves, membranes. The devices can be catheters, implantable vascular access ports, blood storage bags, blood tubing, central venous catheters, arterial catheters, vascular grafts, intra-aortic balloon pumps, heart valves, cardiovascular sutures, artificial hearts, a pacemaker, ventricular assist pumps, extracorporeal devices, blood filters, hemodialysis units, hemoperfusion units, plasmapheresis units, and filters adapted for deployment in a blood vessel. In some embodiments according to the present invention, monomers for forming a polymer are combined with an interferon and are mixed to make a homogeneous dispersion of the interferon in the monomer solution. The dispersion is then applied to a stent or other device according to a conventional coating process, after which the crosslinking process is initiated by a conventional initiator, such as UV light, h other embodiments according to the present invention, a polymer composition is combined with an interferon to form a dispersion. The dispersion is then applied to a surface of a medical device and the polymer is cross-linked to fonn a solid coating. In other embodiments according to the present invention, a polymer and an interferon are combined with a suitable solvent to form a dispersion, which is then applied to a stent in a conventional fashion. The solvent is then removed by a conventional process, such as heat evaporation, with the result that the polymer and interferon (together forming a sustained-release drag delivery system) remain on the stent as a coating. An analogous process may be used where the interferon is dissolved in the polymer composition. In some embodiments according to the invention, the system comprises a polymer that is relatively rigid, h other embodiments, the system comprises a polymer that is soft and malleable. In still other embodiments, the system includes a polymer that has an adhesive character. Hardness, elasticity, adhesive, and other characteristics of the polymer are widely variable, depending upon the particular final physical form of the system, as discussed in more detail below. Embodiments of the system according to the present invention take many different foπns. In some embodiments, the system consists of the interferon suspended or dispersed in the polymer. In certain other embodiments, the system consists of an interferon and a semi solid or gel polymer, which is adapted to be injected via a syringe into a body. In other embodiments according to the present invention, the system consists of an interferon and a soft flexible polymer, which is adapted to be inserted or implanted into a body by a suitable surgical method. In still further embodiments according to the present invention, the system consists of a hard, solid polymer, which is adapted to be inserted or implanted into a body by a suitable surgical method. In further embodiments, the system comprises a polymer having the interferon suspended or dispersed therein, wherein the interferon and polymer mixture forms a coating on a surgical implement, such as a screw, stent, pacemaker, etc. In particular embodiments according to the present invention, the device consists of a hard, solid polymer, which is shaped in the form of a surgical implement such as a surgical screw, plate, stent, etc., or some part thereof. In other embodiments according to the present invention, the system includes a polymer that is in the form of a suture having the interferon dispersed or suspended therein. In some embodiments according to the present invention, provided is a medical device comprising a substrate having a surface, such as an exterior surface, and a coating on the exterior surface. The coating comprises a polymer and an interferon dispersed in the polymer, wherein the polymer is permeable to the interferon or biodegrades to release the interferon. In certain embodiments according to the present invention, the device comprises an interferon suspended or dispersed in a suitable polymer, wherein the interferon and polymer are coated onto an entire substrate, e.g., a surgical implement. Such coating may be accomplished by spray coating or dip coating. In other embodiments according to the present invention, the device comprises an interferon and polymer suspension or dispersion, wherein the polymer is rigid, and forms a constituent part of a device to be inserted or implanted into a body. For instance, in particular embodiments according to the present invention, the device is a surgical screw, stent, pacemaker, etc. coated with the interferon suspended or dispersed in the polymer. In Other particular embodiments according to the present invention, the polymer in which the interferon is suspended forms a tip or a head, or part thereof, of a surgical screw. In other embodiments according to the present invention, the polymer in which interferon is suspended or dispersed is coated onto a surgical implement such as surgical tubing (such as colostomy, peritoneal lavage, catheter, and intravenous tubing). In still further embodiments according to the present invention, the device is an intravenous needle having the polymer and interferon coated thereon. As discussed above, the coating according to the present invention comprises a polymer that is bioerodible or non bioerodible. The choice of bioerodible versus non- bioerodible polymer is made based upon the intended end use of the system or device. In some embodiments according to the present invention, the polymer is advantageously bioerodible. For instance, where the system is a coating on a surgically implantable device, such as a screw, stent, pacemaker, etc., the polymer is advantageously bioerodible. Other embodiments according to the present invention in which the polymer is advantageously bioerodible include devices that are implantable, inhalable, or injectable suspensions or dispersions of interferon in a polymer, wherein the further elements (such as screws or anchors) are not utilized. In some embodiments according to the present invention wherein the polymer is poorly permeable and bioerodible, the rate of bioerosion of the polymer is advantageously sufficiently slower than the rate of interferon release so that the polymer remains in place for a substantial period of time after the interferon has been released, but is eventually bioeroded and resorbed into the suπounding tissue. For example, where the device is a bioerodible suture comprising the interferon suspended or dispersed in a bioerodible polymer, the rate of bioerosion of the polymer is advantageously slow enough that the interferon is released in a linear manner over a period of about three to about 14 days, but the sutures persist for a period of about three weeks to about six months. Similar devices according to the present invention include surgical staples comprising an interferon suspended or dispersed in a bioerodible polymer. In other embodiments according to the present invention, the rate of bioerosion of the polymer is advantageously on the same order as the rate of interferon release. For instance, where the system comprises an interferon suspended or dispersed in a polymer that is coated onto a surgical implement, such as an orthopedic screw, a stent, a pacemaker, or a non-bioerodible suture, the polymer advantageously bioerodes at such a rate that the surface area of the interferon that is directly exposed to the suπounding body tissue remains substantially constant over time. In other embodiments according to the present invention, the polymer vehicle is permeable to water in the suπounding tissue, e.g. in blood plasma. In such cases, water solution may permeate the polymer, thereby contacting the interferon. The rate of dissolution may be governed by a complex set of variables, such as the polymer's permeability, the solubility of the interferon, the pH, ionic strength, and protein composition, etc. of the physiologic fluid In some embodiments according to the present invention, the polymer is non- bioerodible. Non bioerodible polymers are especially useful where the system includes a polymer intended to be coated onto, or form a constituent part, of a surgical implement that is adapted to be permanently, or semi permanently, inserted or implanted into a body. Exemplary devices in which the polymer advantageously forms a permanent coating on a surgical implement include an orthopedic screw, a stent, a prosthetic joint, an artificial valve, a permanent suture, a pacemaker, etc. There is a multiplicity of different stents that may be utilized following percutaneous transluminal coronary angioplasty. Although any number of stents may be utilized in accordance with the present invention, for simplicity, a limited number of stents will be described in exemplary embodiments of the present invention. The skilled artisan will recognize that any number of stents may be utilized in connection with the present invention. In addition, as stated above, other medical devices may be utilized. A stent is commonly used as a tubular structure left inside the lumen of a duct to relieve an obstruction. Commonly, stents are inserted into the lumen in a non-expanded form and are then expanded autonomously, or with the aid of a second device in situ. A typical method of expansion occurs through the use of a catheter-mounted angioplasty balloon which is inflated within the stenosed vessel or body passageway in order to shear and dismpt the obstructions associated with the wall components of the vessel and to obtain an enlarged lumen. The stents of the present invention may be fabricated utilizing any number of methods. For example, the stent may be fabricated from a hollow or formed stainless steel tube that may be machined using lasers, electric discharge milling, chemical etching or other means. The stent is inserted into the body and placed at the desired site in an unexpanded form. In one exemplary embodiment, expansion may be effected in a blood vessel by a balloon catheter, where the final diameter of the stent is a function of the diameter of the balloon catheter used. It should be appreciated that a stent in accordance with the present invention may be embodied in a shape-memory material, including, for example, an appropriate alloy of nickel and titanium or stainless steel. Structures formed from stainless steel may be made self-expanding by configuring the stainless steel in a predetermined manner, for example, by twisting it into a braided configuration. In this embodiment after the stent has been formed it may be compressed so as to occupy a space sufficiently small as to permit its insertion in a blood vessel or other tissue by insertion means, wherein the insertion means include a suitable catheter, or flexible rod. On emerging from the catheter, the stent may be configured to expand into the desired configuration where the expansion is automatic or triggered by a change in pressure, temperature or electrical stimulation. Regardless of the design of the stent, it is preferable to have the interferon applied with enough specificity and a sufficient concentration to provide an effective dosage in the lesion area, h -this regard, the "reservoir size" in the coating is preferably sized to adequately apply the interferon at the desired location and in the desired amount. In an alternate exemplary embodiment, the entire inner and outer surface of the stent may be coated with the interferon in therapeutic dosage amounts. It is, however, important to note that the coating techniques may vary depending on the interferon. Also, the coating techniques may vary depending on the material comprising the stent or other intraluminal medical device. The intraluminal medical device comprises the sustained release drag delivery coating. The interferon coating may be applied to the stent via a conventional coating process, such as impregnating coating, spray coating and dip coating. In one embodiment, an intraluminal medical device comprises an elongate radially expandable tubular stent having an interior luminal surface and an opposite exterior surface extending along a longitudinal stent axis. The stent may include a permanent implantable stent, an implantable grafted stent, or a temporary stent, wherein the temporary stent is defined as a stent that is expandable inside a vessel and is thereafter retractable from the vessel. The stent configuration may comprise a coil stent, a memory coil stent, a Nitinol stent, a mesh stent, a scaffold stent, a sleeve stent, a permeable stent, a stent having a temperature sensor, a porous stent, and the like. The stent may be deployed according to conventional methodology, such as by an inflatable balloon catheter, by a self-deployment mechanism (after release from a catheter), or by other appropriate means. The elongate radially expandable tubular stent may be a grafted stent, wherein the grafted stent is a composite device having a stent inside or outside of a graft. The graft may be a vascular graft, such as an ePTFE graft, a biological graft, or a woven graft. The interferon may be incorporated onto or affixed to the stent in a number of ways. In the exemplary embodiment, the interferon is directly incorporated into a polymeric matrix and sprayed onto the outer surface of the stent. The interferon elutes from the polymeric matrix over time and enters the suπounding tissue. The interferon preferably remains on the stent for at least three days up to approximately six months, and more preferably between seven and thirty days. In certain embodiments, the polymer according to the present invention comprises any biologically tolerated polymer that is permeable to the interferon and while having permeability such that it is not the principal rate determining factor in the rate of release of the interferon from the polymer. In some embodiments according to the present invention, the polymer is non- bioerodible. Examples of non-bioerodible polymers useful in the present invention include poly(ethylene-co-vinyl acetate) (EVA), polyvinylalcohol and polyurethanes, such as polycarbonate-based polyurethanes. In other embodiments of the present invention, the polymer is bioerodible. Examples of bioerodible polymers useful in the present invention include polyanhydride, polylactic acid, polyglycolic acid, polyorthoester, polyallcylcyanoacrylate or derivatives and copolymers thereof. The skilled artisan will recognize that the choice of bioerodibility or non-bioerodibility of the polymer depends upon the final physical form of the system, as described in greater detail below. Other exemplary polymers include polysilicone and polymers derived from hyaluronic acid. The skilled artisan will understand that the polymer according to the present invention is prepared under conditions suitable to impart permeability such that it is not the principal rate determining factor in the release of the interferon from the polymer. Moreover, suitable polymers include naturally occurring (collagen, hyaluronic acid, etc.) or synthetic materials that are biologically compatible with bodily fluids and mammalian tissues, and essentially insoluble in bodily fluids with which the polymer will come in contact, h addition, the suitable polymers essentially prevent interaction between the interferon dispersed/suspended in the polymer and proteinaceous components in the bodily fluid. The use of rapidly dissolving polymers or polymers highly soluble in bodily fluid or which permit interaction between the interferon and proteinaceous components are to be avoided in certain instances since dissolution of the polymer or interaction with proteinaceous components would affect the constancy of drug release. Other suitable polymers include polypropylene, polyester, polyethylene vinyl acetate (PVA or EVA), polyethylene oxide (PEO), polypropylene oxide, polycarboxylic acids, polyalkylacrylates, cellulose ethers, silicone, poly(dl-lactide-co glycolide), various Eudragrits (for example, NE30D, RS

and RL PO), polyalkyl- alkyacrylate copolymers, polyester-polyurethane block copolymers, polyether- polyurethane block copolymers, polydioxanone, poly-(/?-hydroxybutyrate), polylactic acid (PLA), polycaprolactone, polyglycolic acid, and PEO-PLA copolymers. The coating of the present invention may be formed by mixing one or more suitable monomers and a suitable interferon, then polymerizing the monomer to form the polymer system. In this way, the interferon is dissolved or dispersed in the polymer. In other embodiments, the interferon is mixed into a liquid polymer or polymer dispersion and then the polymer is further processed to form the inventive coating. Suitable further processing may include crosslinking with suitable crosslinking interferons, further polymerization of the liquid polymer or polymer dispersion, copolymerization with a suitable monomer, block copolymerization with suitable polymer blocks, etc. The further processing traps the interferon in the polymer so that the interferon is suspended or dispersed in the polymer vehicle. Any number of non-erodible polymers may be utilized in conjunction with the interferon. Film-forming polymers that can be used for coatings in this application can be absorbable or non-absorbable and must be biocompatible to minimize irritation to the vessel wall. The polymer may be either biostable or bioabsorbable depending on the desired rate of release or the desired degree of polymer stability, but a bioabsorbable polymer may be prefeπed since, unlike biostable polymer, it will not be present long after implantation to cause any adverse, chronic local response. Furthermore, bioabsorbable polymers do not present the risk that over extended periods of time there could be an adhesion loss between the stent and coating caused by the stresses of the biological environment that could dislodge the coating and introduce further problems even after the stent is encapsulated in tissue. Suitable film-forming bioabsorbable polymers that could be used include polymers selected from the group consisting of aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylenes oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amido groups, poly(anhydrides), polyphosphazenes, biomolecules and blends thereof. For the purpose of this invention aliphatic polyesters include homopolymers and copolymers of lactide (which includes lactic acid d-,1- and meso lactide), e-caprolactone, glycolide (including glycolic acid), hydroxybutyrate, hydroxyvalerate, para-dioxanone, trimethylene carbonate (and its alkyl derivatives), l,4-dioxepan-2-one, l,5-dioxepan-2- one, 6,6-dimethyl-l,4-dioxan-2-one and polymer blends thereof. Poly(iminocarbonate) for the purpose of this invention include as described by Kemnitzer and Kohn, in the Handbook of Biodegradable Polymers, edited by Domb, Kost and Wisemen, Hardwood Academic Press, 1997, pages 251-272. Copoly(ether-esters) for the purpose of this invention include those copolyester-ethers described in Journal of Biomaterials Research, Vol. 22, pages 993-1009, 1988 by Cohn and Younes and Cohn, Polymer Preprints (ACS Division of Polymer Chemistry) Vol. 30(1), page 498, 1989 (e.g. PEO/PLA). Polyalkylene oxalates for the purpose of this invention include U.S. Pat. Nos. 4,208,511; 4,141,087; 4,130,639; 4,140,678; 4,105,034; and 4,205,399 (incorporated by reference herein). Polyphosphazenes, co-, ter- and higher order mixed monomer based polymers made from L-lactide, D,L-lactide, lactic acid, glycolide, glycolic acid, para-dioxanone, trimethylene carbonate and e-caprolactone such as are described by Allcock in The Encyclopedia of Polymer Science, Vol. 13, pages 31-41, Wiley Intersciences, John Wiley & Sons, 1988 and by Vandorpe, Schacht, Dejardin and Lemmouchi in the Handbook of Biodegradable Polymers, edited by Domb, Kost and Wisemen, Hardwood Academic Press, 1997, pages 161-182 (which are hereby incorporated by reference herein). Polyanhydrides from diacids of the form HOOC — C₆H₄-O-(CH₂)_m-O-C6H -COOH where m is an integer in the range of from 2 to 8 and copolymers thereof with aliphatic alpha-omega diacids of up to 12 carbons. Polyoxaesters polyoxaamides and polyoxaesters containing amines and/or amido groups are described in one or more of the following U.S. Pat. Nos. 5,464,929; 5,595,751; 5,597,579; 5,607,687; 5,618,552; 5,620,698; 5,645,850; 5,648,088; 5,698,213 and 5,700,583; (which are incorporated herein by reference). Polyorthoesters such as those described by Heller in Handbook of Biodegradable Polymers, edited by Domb, Kost and Wisemen, Hardwood Academic Press, 1997, pages 99-118 (hereby incorporated herein by reference). Film-forming polymeric biomolecules for the purpose of this invention include naturally occurring materials that may be enzymatically degraded in the human body or are hydrolytically unstable in the human body such as fibrin, fibrinogen, collagen, elastin, and absorbable biocompatable polysaccharides such as chitosan, starch, fatty acids (and esters thereof), glucoso- glycans and hyaluronic acid. Suitable film-forming biostable polymers with relatively low chronic tissue response, such as polyurethanes, silicones, poly(meth)acrylates, polyesters, polyalkyl oxides (polyethylene oxide), polyvinyl alcohols, polyethylene glycols and polyvinyl pyπolidone, as well as, hydrogels such as those formed from crosslinked polyvinyl pyrrolidinone and polyesters could also be used. Other polymers could also be used if they can be dissolved, cured or polymerized on the stent. These include polyolefins, polyisobutylene and ethylene-alphaolefin copolymers; acrylic polymers (including methacrylate) and copolymers, vinyl halide polymers and copolymers, such as polyvinyl chloride; polyvinyl ethers, such as polyvinyl methyl ether; polyvinylidene halides such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile, polyvinyl ketones; polyvinyl aromatics such as polystyrene; polyvinyl esters such as polyvinyl acetate; copolymers of vinyl monomers with each other and olefins, such as etheylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins and ethylene- vinyl acetate copolymers; polyamides, such as Nylon 66 and polycaprolactam; alkyd resins; polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxy resins, polyurethanes; rayon; rayon-triacetate, cellulose, cellulose acetate, cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers (i.e. carboxymethyl cellulose and hydroxyalkyl celluloses); and combinations thereof. Polyamides for the pmpose of this application would also include polyamides of the form -NH-(CH₂)_n-CO- and NH-(CH₂)_x-NH-CO-(CH₂)_y-CO, wherein n is preferably an integer in from 6 to 13; x is an integer in the range of form 6 to 12; and y is an integer in the range of from 4 to 16. The list provided above is illustrative but not limiting. The polymers used for coatings can be film-forming polymers that have molecular weight high enough as to not be waxy or tacky. The polymers also should adhere to the stent and should not be so readily deformable after deposition on the stent as to be able to be displaced by hemodynamic stresses. The polymers molecular weight will be high enough to provide sufficient toughness so that the polymers will not to be mbbed off during handling or deployment of the stent and must not crack during expansion of the stent. In certain embodiments, the polymer has a melting temperature above 40°C, preferably above about 45°C, more preferably above 50°C and most preferably above 55°C Coating may be formulated by mixing one or more of the therapeutic interferons with the coating polymers in a coating mixture. The interferon may be present as a liquid, a finely divided solid, or any other appropriate physical form. Optionally, the mixture may include one or more additives, e.g., nontoxic auxiliary substances such as diluents, carriers, excipients, stabilizers or the like. Other suitable additives may be formulated with the polymer and interferon. For example, hydrophilic polymers selected from the previously described lists of biocompatible film forming polymers may be added to a biocompatible hydrophobic coating to modify the release profile (or a hydrophobic polymer may be added to a hydrophilic coating to modify the release profile). One example would be adding a hydrophilic polymer selected from the group consisting of polyethylene oxide, polyvinyl pyπolidone, polyethylene glycol, carboxymethyl cellulose, hydroxymethyl cellulose and combination thereof to an aliphatic polyester coating to modify the release profile. Appropriate relative amounts can be determined by monitoring the in vitro and/or in vivo release profiles for the therapeutic interferons. The thickness of the coating can determine the rate at which the interferon elutes from the matrix. Essentially, the interferon elutes from the matrix by diffusion through the polymer matrix. Polymers are permeable, thereby allowing solids, liquids and gases to escape therefrom. The total thickness of the polymeric matrix is in the range from about one micron to about twenty microns or greater. It is important to note that primer layers and metal surface treatments may be utilized before the polymeric matrix is affixed to the medical device. For example, acid cleaning, alkaline (base) cleaning, salinization and parylene deposition may be used as part of the overall process described. To further illustrate, a poly(ethylene-co-vinylacetate), polybutyhnethacrylate and interferon solution may be incorporated into or onto the stent in a number of ways. For example, the solution may be sprayed onto the stent or the stent may be dipped into the solution. Other methods include spin coating and RF plasma polymerization. In one exemplary embodiment, the solution is sprayed onto the stent and then allowed to dry. In another exemplary embodiment, the solution may be electrically charged to one polarity and the stent electrically changed to the opposite polarity, h this manner, the solution and stent will be attracted to one another, h using this type of spraying process, waste may be reduced and more precise control over the thickness of the coat may be achieved. In another exemplary embodiment, the interferon may be incorporated into a film-forming polyfluoro copolymer comprising an amount of a first moiety selected from the group consisting of polymerized vinylidenefluoride and polymerized tetrafluoroethylene, and an amount of a second moiety other than the first moiety and which is copolymerized with the first moiety, thereby producing the polyfluoro copolymer, the second moiety being capable of providing toughness or elastomeric properties to the polyfluoro copolymer, wherein the relative amounts of the first moiety and the second moiety are effective to provide the coating and film produced therefrom with properties effective for use in treating implantable medical devices. In one embodiment according to the present invention, the exterior surface of the expandable tubular stent of the intraluminal medical device of the present invention comprises a coating according to the present invention. The exterior surface of a stent having a coating is the tissue-contacting surface and is biocompatible. The "sustained release interferon delivery system coated surface" is synonymous with "coated surface", which surface is coated, covered or impregnated with a sustained release interferon delivery system according to the present invention. Examples of delivery systems (controlled release, sustained release, slow release) have been described in applicants' U.S. Serial No. 10/193,654, published as US2003/0017169, and PCT/USOl/50355, published as WO 02/053174, the contents of which are incorporated by reference into this application. In an alternate embodiment, the interior luminal surface or entire surface (i.e. both interior and exterior surfaces) of the elongate radially expandable tubular stent of the intraluminal medical device of the present invention has the coated surface. The interior luminal surface having the inventive sustained release interferon delivery system coating is also the fluid contacting surface, and is biocompatible and blood compatible.

IV. Variant Interferon Polypeptides It is anticipated that certain mutant forms (or variants) of the Interferon polypeptides of the invention may act as agonist or antagonists. While not wishing to be bound to any particular theory, it is well known that mutant forms of protein signaling factors are capable of binding to the appropriate receptor and yet not capable of activating the receptor. Such mutant proteins act as antagonists by displacing the wild-type proteins and blocking the normal receptor activation. Additionally, it is well known that one or more amino acid substitutions can be made to many proteins in order to enhance their activity in comparison to wildtype forms of the protein. Such agonists may have, for example, increased half-life, binding affinity, stability, or activity in comparison to the wildtype protein. There are many well known methods for obtaining mutants (or variants) with a desired activity. Methods for generating large pools of mutant/variant proteins are well known in the art. In one embodiment, the invention contemplates using Interferon polypeptides generated by combinatorial mutagenesis. Such methods, as are known in the art, are convenient for generating both point and tmncation mutants, and can be especially useful for identifying potential variant sequences (e.g., homologs) that are functional in a given assay. The purpose of screening such combinatorial libraries is to generate, for example, Interferon variants or homologs that can act as either agonists or antagonists. Thus, combinatorially derived variants can be generated to have an increased potency relative to a naturally occurring form of the protein. Likewise, Interferon variants can be generated by the present combinatorial approach to act as antagonists, in that they are able to mimic, for example, binding to other extracellular matrix components (such as receptors), yet not induce any biological response, thereby inhibiting the action of Interferon polypeptides or Interferon agonists. Moreover, manipulation of certain domains of Interferon by the present method can provide domains more suitable for use in fusion or chimeric proteins, for example, domains demonstrated to have specific useful properties. To further illustrate the state of the art of combinatorial mutagenesis, it is noted that the review article of Gallop et al. (1994) J Med Chem 37:1233 describes the general state of the art of combinatorial libraries as of the earlier 1990's. In particular, Gallop et al state at page 1239 "[screening the analog libraries aids in determining the minimum size of the active sequence and in identifying those residues critical for binding and intolerant of substitution", hi addition, the Ladner et al. PCT publication WO90/02809, the Goeddel et al. U.S. Patent 5,223,408, and the Markland et al. PCT publication WO92/15679 illustrate specific techniques which one skilled in the art could utilize to generate libraries of variants which can be rapidly screened to identify variants/fragments which possess a particular activity. These techniques are exemplary of the art and demonstrate that large libraries of related variants/truncants can be generated and assayed to isolate particular variants without undue experimentation. Gustin et al. (1993) Virology 193:653, and Bass et al. (1990) Proteins: Structure, Function and Genetics 8:309-314 also describe other exemplary techniques from the art which can be adapted as a means for generating mutagenic variants of the Interferon polypeptides of the invention. Indeed, it is plain from the combinatorial mutagenesis art that large scale mutagenesis of Interferon proteins, without any preconceived ideas of which residues were critical to the biological function, can generate wide aπays of variants having equivalent biological activity. Alternatively, such methods can be used to generate a wide aπay of variants having enhanced activity or antagonistic activity. Indeed, it is the ability of combinatorial techniques to screen billions of different variants by high throughout analysis that removes any requirement of a priori understanding or knowledge of critical residues. V. Antibody antagonists It is anticipated that some antibodies can act as Interferon antagonists.

Antibodies can have extraordinary affinity and specificity for particular epitopes. The binding of an antibody to its epitope on a protein may antagonize the function of that protein by competitively or non-competitively inhibiting the interaction of that protein with other proteins necessary for proper function. Antibodies with Interferon antagonist activity can be identified in much the same way as other Interferon antagonists. For example, candidate antibodies can be administered to cells expressing a reporter gene, and antibodies that cause decreased reporter gene expression are antagonists. In one variation, antibodies of the invention can be single chain antibodies (scFv), comprising variable antigen binding domains linked by a polypeptide linker. Single chain antibodies are expressed as a single polypeptide chain and can be expressed in bacteria and as part of a phage display library. In this way, phage that express the appropriate scFv will have Interferon antagonist activity. The nucleic acid encoding the single chain antibody can then be recovered from the phage and used to produce large quantities of the scFv. Construction and screening of scFv libraries is extensively described in various publications (U.S. Patents 5,258,498; 5,482,858; 5,091,513; 4,946,778; 5,969,108; 5,871,907; 5,223,409; 5,225,539). G. Human IFN-α alleles and protein products

Table 2 below illustrates the common alleles of the human interferon α family of genes/proteins and was constructed based on Pestka, S. (1983) Arch Biochem Biophys 221:1-37; Diaz, M.O., Pomykala, H.M., Bohlander, S.K., Maltepe, E., Malik, K., Brownstein, B., and Olopade, O.I. (1994) Genomics 22:540-52; and Pestka, S. (1986) Meth. Enzymol 119:3-14, and reviewed in Krause, CD., Lunn, C.A., Izotova, L.S., Mirochnitchenlco, O., Kotenko, S.V., Lundell, D.J., Narala, S.K., and Pestka, S. (2000) J Biol Chem. 275:22995-3004. Table 2

Note: ψ indicates pseudogene Allelic variants of human interferon-or genes and interferon-α mutants have been reported in the following applications: WO 2002/095067; WO 02/079249; WO 02/101048; WO 02/095067; WO 02/083733; WO 02/086156; WO 2002/083733; WO 03/000896; WO 02/101048; WO 02/079249; WO 03/000896; WO 2004/022593; WO 2004/022747; WO 03/023032; WO 2004/022593. See also the following publications: (1) Kim et al, Cancer Lett. 2003 Jan 28; 189(2): 183-8; (2) Hussain et al., J Interferon Cytokine Res. 2000 Sep;20(9):763-8; (3) Hussain et al., J Interferon Cytokine Res. 1998 Jul;18(7):469-77; (4) Nyman et al., Biochem J. 1998 Jan 15;329 ( Pt 2):295-302; (5) Golovleva et al, J Interferon Cytokine Res. 1997 Oct;17(10):637-45; (6) Hussain et al, J Interferon Cytokine Res. 1997 Sep;17(9):559-66; (7) Golovleva, I., Saha, N, Beckman, L. Hum Hered. 1997 Jul-Aug;47(4): 185-8; (8) Golovleva et al, 1997 Apr;18(4):645-7; (9) Kita et al, J Interferon Cytokine Res. 1997 Mar;17(3):135-40; (10) Golovleva et al, Am J Hum Genet. 1996 Sep;59(3):570-8; (11) Hussain et al, J Interferon Cytokine Res. 1996 Jul;16(7):523-9 ; (12) Linge et al, Biochim Biophys Acta. 1995 Dec 27;1264(3):363-8 ; (13) Gewert et al, J Interferon Cytokine Res. 1995 May;15(5):403-6; (14) Lee et al, J Interferon Cytokine Res. 1995 Apr;15(4):341-9; (15) Kaluz et al, Acta Virol. 1994 Apr;38(2):101-4; (16) Emanuel et al, J Interferon Res. 1993 Jun;13(3):227-31; (17) Kaluz et al, Acta Virol. 1993 Feb;37(l):97-100; (18) Shekhter et al, Dold Akad Nauk SSSR. 1990;314:998-1001; (19) Li et al, Sci China B. 1992 Feb;35(2):200-6.

Exemplification The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1: Isolation of Feline IFNa Clones Feline IFNα clones were isolated by PCR amplification of genomic DNA from a cat lung cell line (AKD) using standard methods. Nine distinct sequences were isolated and designated Fe-IFN-αA (SEQ LD NO: 9), Fe-IFN-αB (SEQ ID NO: 11), Fe-IFN-αC (SEQ ID NO: 13), Fe-IFN-αD (SEQ ID NO: 15), Fe-IFN-αE (SEQ ID NO: 17), Fe-LFN-αF (SEQ ID NO: 19), Fe-LFN-αG (SEQ ID NO: 21), Fe-IFN-αH (SEQ LD NO: 23), and Fe-LFN-αl (SEQ ID NO: 25). Amino acid sequences coπesponding to each of these are also provided: Fe-IFN-αA (SEQ ID NO: 10), Fe-IFN-αB (SEQ ID NO: 12), Fe-LFN-αC (SEQ ID NO: 14), Fe-IFN-αD (SEQ ID NO: 16), Fe-LFN-αE (SEQ TD NO: 18), Fe-IFN-αF (SEQ LD NO: 20), Fe-IFN-αG (SEQ LD NO: 22), Fe- IFN-αH (SEQ ID NO: 24), and Fe-IFN-αl (SEQ ID NO: 26). PCR was performed using standard procedure. Two rounds of amplification from genomic DNA were performed. Flanking primers used to amplify the feline sequences are: 5' primer: 5'-CTCTTCCTTCTTGGTGGCCCTG-3' 3' primer: 5'-GTGATGAGTCAGTGAGAATCATTTC-3'

Example 2: Antiviral Activity of the Feline IFNa Species The antiviral activity of the subject interferon species was measured using a cytopathic effect assay (CPE). Briefly, serial dilutions of interferon were incubated with test cells for 1 to 4 hours at 37 C. Vims was then added to the cells and incubated for 16 hours at 37 C. The surviving cells were visualized by uptake of crystal violet stain, and the dilution of interferon at which approximately 50% of the cells survive viral infection was deteπnined. Figure 1 summarizes the results of these experiments which demonstrate that feline LFN-αA, LFN-αB, LFN-αC, LFN-αD, IFN-αE, LFN-αF, IFN-αG, and LFN-αl each possess antiviral activity as measured by CPE. The activity of feline IFN-αH was not determined in this assay. In this particular experiment, the test cells were AKD feline lung cells and the vims was vesicular stomatitis vims (VSV).

Example 3: Isolation of Rhesus IFNa Clones Rhesus monkey IFNα clones were isolated by PCR amplification of genomic DNA from a Rhesus monkey kidney cell line (LLCMK-2) using standard methods. Two separate primer pairs were used to amplify sequences. Using the first primer pair, one sequence was isolated and designated Rh-IFN-α4b (SEQ ID 29). The amino acid sequence coπesponding to the Rh-IFN-α4b nucleic acid sequence is designated in SEQ ID NO: 30. PCR was performed using standard procedure. Two rounds of amplification from genomic DNA were performed. Flanking primers used to amplify this Rhesus sequence are: 5' primer: 5'-CTTCAGAGAACCTGGAGCC-3' 3' primer: 5 '-AATCATTTCCATGTTGAACCAG-3 '

Three additional Rhesus LFNα clones were isolated by PCR amplification of genomic DNA from a Rhesus monkey kidney cell line (LLCMK-2) using standard methods and a second primer pair: Rh-IFN-αDl (SEQ ID NO: 31), Rh-IFN-αD2 (SEQ ID NO: 33), and Rh-LFN-αD3 (SEQ ID NO: 35). Amino acid sequences coπesponding to each of these are also provided: Rh-IFN-αDl (SEQ ID NO: 32), Rh-LFN-αD2 (SEQ ID NO: 34), and Rh-IFN-αD3 (SEQ ID NO: 36). PCR was performed using standard procedures. Two rounds of amplification from genomic DNA were performed. Flanking primers used to amplify these Rhesus sequences are: 5' primer: 5 '-AGAAGCATCTGCCTGCAATATC-3 ' 3' primer: 5'-GCTATGACCATGATTACGAATTC-3'

Example 4: Antiviral Activity of the Rhesus IFNa Species The antiviral activity of the subject interferon species was measured using a cytopathic effect assay (CPE). Briefly, serial dilutions of interferon were incubated with test cells for 1 to 4 hours at 37°C. Vims was then added to the cells and incubated for 16 hours at 37°C The surviving cells were visualized by uptake of crystal violet stain, and the dilution of interferon at which approximately 50% of the cells survive viral infection was determined. Figure 2 summarizes the results of experiments which demonstrate that Rhesus

IFN-α4b possesses antiviral activity as measured by an anti-viral activity assay (CPE).

The activities of Rhesus IFN-αDl, IFN-αD2, and IFN-αD3 were not determined in this assay. This assay was performed using as test cells either Madin-Darby bovine kidney endothelial cells (MDBK) or African green monkey kidney cells (Vero) infected with VSV.

Example 5: Isolation of Human IFNa Clones Eighteen human interferon-α species were isolated in accordance with the procedures described in U.S. Patents 5,789,551, 5,869,293, and 6,001,589. Briefly, human genomic DNA was analyzed by PCR using standard methods. The primers used in this analysis are described in Figures 3 and 4. The eighteen human interferon-α species identified using this approach are: hu- LFN-αOOl (SEQ LD No: 37), hu-LFN-α002 (SEQ ID No: 39), hu-LFN-α003 (SEQ ID No: 41), hu-IFN-α004 (SEQ ID No: 43), hu-LFN-α005 (SEQ ID No: 45), hu-LFN-α006 (SEQ TD No: 47), hu-IFN-α007 (SEQ ID No: 49), hu-IFN-α008 (SEQ ID No: 51), hu- IFN-α009 (SEQ LD No: 53), hu-LFN-αOlO (SEQ ID No: 55), hu-LFN-αOll (SEQ LD No: 57), hu-IFN-α012 (SEQ ID No: 59), hu-IFN-α013 (SEQ ID No: 61), hu-LFN-α014 (SEQ ED No: 63), hu-LFN-α015 (SEQ ED No: 65), hu-IFN-α016 (SEQ ID No: 67), hu- LFN-α017 (SEQ LD No: 69), hu-IFN-α018 (SEQ ID No: 71). Amino acid sequences coπesponding to each of these are also provided: IFN-αOOl (SEQ ID No: 38), hu-EFN- α002 (SEQ ID No: 40), hu-IFN-α003 (SEQ ID No: 42), hu-LFN-α004 (SEQ ID No: 44), hu-IFN-α005 (SEQ ID No: 46), hu-IFN-α006 (SEQ ID No: 48), hu-LFN-α007 (SEQ ED No: 50), hu-IFN-α008 (SEQ ID No: 52), hu-IFN-α009 (SEQ ID No: 54), hu- LFN-αOlO (SEQ ED No: 56), hu-IFN-αOll (SEQ ED No: 58), hu-EFN-α012 (SEQ ED No: 60), hu-LFN-α013 (SEQ ID No: 62), hu-E?N-α014 (SEQ ID No: 64), hu-LFN-α015 (SEQ ID No: 66), hu-IFN-α016 (SEQ ID No: 68), hu-tFN-α017 (SEQ ID No: 70), hu- LFN-α018 (SEQ ID No: 72). Additionally, hu-EFN- αOOl and hu-EFN- α012 were back translated using optimal E. coli codons and designated hu-LFN-α001-BT (SEQ ID No: 73) and hu-EFN- α012-BT (SEQ LD No: 75). Amino acid sequences coπesponding to each of these are also provided: hu-LFN-αOOl-BT (SEQ ID No: 74) and hu-IFN-α012-BT (SEQ ID No: 76).

Example 6: Isolation of Human IFNa Variants During the constmction of expression vectors containing the human FNa species described in detail above, the following clones containing mutations were generated. These LFNα variants can be tested for activity. EFNα variants can contain silent substitutions, and thus have identical activity to the wild type IFNα species. Alternatively, a variant may contain a substitution that alters the activity of the polypeptide. The substitution may increase, enhance or augment the activity, and thus be an EFNa agonist. Additionally, the substitution may decrease or interfere with the activity, and thus be an IFNα antagonist. Nucleic acid sequences for the variant species are provided: hu-IFN-α019 (SEQ ID No: 77), hu-IFN-α020 (SEQ TD No: 79), hu-EFN-α021 (SEQ D No: 81), hu-LFN- α022 (SEQ TD No: 83), and hu-IFN-α023 (SEQ ED No: 85). Amino acid sequences coπesponding to each of these are also provided: hu-IFN-α019 (SEQ LD No: 78), hu- EFN-α020 (SEQ LD No: 80), hu-IFN-α021 (SEQ ID No: 82), hu-EFN-α022 (SEQ LD No: 84), and hu-IFN-α023 (SEQ ID No: 86).

Example 7: Antiviral Activity of the Human IFNa Species ' The antiviral activity of the human EFNα species was also determined using the

CPE assay, as outlined in detail above. The assay was performed using the following test cell and vims combinations: MDBK test cells with VSV; human epithelial squamous (HEP-2) cells with VSV; mouse connective tissue fibroblasts (L929) with EMC; human lung squamous (H226) cells with VSV; and human lung fibroblasts with influenza vims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. Various publications are cited throughout this application. The contents of these publications are hereby incorporated by reference into this application. See Figure 10 and Example 13 for more elaboration.

Example 8: Inhibition of SARS Coronavirus cytopathic effect by interferons.

Materials and Methods: To evaluate the anti-SARS-CoV activity of interferons, FRhk-4 cells (5xl0³ per well) in MEM medium supplemented with 10% fetal bovine serum (FBS, Invitrogen) were seeded into 96-well cell culture plates and cultured overnight. Cells were then incubated for one hour with varied concentrations of different forms of interferons dissolved in 100 Dl of MEM medium. They were then infected with SARS-CoV (GZ50 strain) and cultured. At 24 hours after infection with SARS-CoV, the degree of protection offered cells against SARS-CoV viral cytopathic effects was determined by observing and recording cell morphology under phase contrast microscope. Each experiment was done in triplicates and repeated at least three times. The umnfected cells were flattened whereas the SARS-CoV infected cells became refractile and rounded up. The degree of SARS-CoV induced cytopathic effect were graded from 0 (no CPE); + 1 (weak CPE); + 2 (medium CPE); ± 3 (strong CPE, no protective agent added).

Cells were seeded in 96-well dishes sixteen hours before infection. The conditioned medium from infected cells were diluted at 10-fold serial in MEM with 1% FBS and used for infecting cells. Ten wells were used for each dilution and two wells were used as control without vims infection. CPE was recorded under phase-contrast microscopy at 24 hours post-infection and the infectious viral titer was calculated according to standard protocol (50% tissue culture infectious dose).

Results: See Figure 5. The effectiveness of EFN α2a and novel interferons was tested in vitro in an assay measuring inhibition of SAR CoV, isolate GZ50, cytopathic effect on FRhk-4 monkey kidney cells. As can be seen in Figure X, a concentration of 20 ng/ml of EFN alpha 2b does not fully inhibit the ability of the SARS coronavims to kill these cells in culture. EFN α012 is more active at protecting these cells and at 20 ng/ml is fully protective with an IC50 between 0.8 and 0.16 ng/ml. EFN α023 is even more potent giving- complete protection at 4 ng/ml and an IC50 between 0.8 and 0.16 ng/ml.

Example 9: Inhibition of SARS CoV replication by interferons. Materials and Methods: Cells and vims: African green monkey kidney cells (Vero 76) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were routinely grown in minimal essential medium (MEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Hyclone Laboratories; Logan UT, USA). For antiviral assays, the serum was reduced to 2% and gentamicin was added to medium at a final concentration of 50 μg/ml.

Severe acute respiratory syndrome coronavims (SARS CoV), strain Urbani (200300592), was obtained from the CDC and routinely passaged in Vero 76 cells.

Cytopathic Effect (CPE) Inhibition Assay. The protocol of Barnard et al. 2001 (Barnard, D.L., Stowell, V.D., Seley, K.L., Hegde, V.R., Das, S.R., Rajappan, V.P., Schneller, S.W., Smee, D.F., & Sidwell, R.W. 2001, Antiviral Chemistry & Chemotherapy 12:221-230) was used. Compounds were tested at varying concentrations (four single log 10 or seven 1/2 log 10 dilutions) one to two times in this assay and the cell viability was then verified spectrophotometrically by neutral red (NR) uptake assay on the same plate (see below). Virus (multiplicity of infection [MOI] = 0.001) and compound were added in equal volumes to 80-90% confluent cell monolayers in 96-well tissue culture plates. In wells where interferons were evaluated, then cells were pretreated with the interferon for 24 h prior to viras exposure. The MOI used was such that 100% of the cells in the viras controls showed cytopathic effects (CPE) within 3-5 days. The plates were incubated at 37°C until the cells in the viras control wells showed complete viral CPE as observed by light microscopy. Each concentration of drag was assayed for inhibition of viral CPE in triplicate and for cytotoxicity in duplicate. Six wells per microplate were set aside as uninfected, untreated cell controls and six wells received viras in medium only per microplate and represented controls for viras replication. Alferon was included as a positive control drug for each set of compounds tested. For all CPE-based assays, the 50% effective concentrations of interferon (IC50) were calculated by linear regression analysis of the means of the CPE ratings expressed as percentages of untreated, uninfected controls for each concentration.

Neutral Red (NR) Uptake Assay of CPE Inhibition and Compound Cytotoxicity. This assay was done on the same CPE inhibition test plates described above to verify the inhibitory activity and the cytotoxicity observed by visual observation. The usual coπelation between visual and NR assays in our hands has been greater than 95% (Barnard, D.L., Stowell, V.D., Seley, K.L., Hegde, V.R., Das, S.R., Rajappan, V.P., Schneller, S.W., Smee, D.F., & Sidwell, R.W. 2001, Antiviral Chemistry & Chemotherapy 12:221-230). The NR assay was performed using a modified method of Cavenaugh et al. (Cavenaugh, P.R. Jr., Moskwa,, P.S., Donish, W.H., Pera, P.J., Richardson, D. & Andrese, A.P. (1990) Investigational New Drags 8:347-354) as described by Barnard et al. (Barnard, D.L., Sidwell, R.W., Xiao, W., Player, M.R., Adah, S. & Toπence, P.F. (1999) Antiviral Research 41:119-134).

Briefly, medium was removed from each well of a plate, 0.034% NR added to each well of the plate and the plate incubated for 2 hr at 37°C in the dark. The NR solution was removed from the wells, rinsed and the remaining dye extracted using ethanol buffered with Sδrenson's citrate buffer. Absorbances at 540 nm 450 nm were read with a microplate reader (Bio-Tek EL 1309; Bio-Tek Instruments, hie, Winooski, VT). Absorbance values were expressed as percentages of untreated controls, and IC50 values were calculated as described above.

Results: See Figure 6. The effectiveness of EFN o2a and novel interferons in inhibiting SARS CoV were tested in vitro in another assay of SARS CoV inhibition. In these experiments a different viral strain, Urbani, and cell line, African green monkey cell (Vero 76), were used. When measured by neutral red uptake, only EFN α012 was more effective than EFN c_2a at protecting cells. The visual measurement demonstrates that all three of the novel EFNs shown (EFN α003, α023 and α012) were more effective than EFN α_2a with EFN α012 again demonstrating the highest antiviral activity.

Example 10: Inhibition of Respiratory Syncytial Virus replication by interferons. Materials and Methods: Human embryonic diploid fibroblast cells were grown to confluence in flat-bottomed 96-well plates in Eagles MEM incorporating 5% foetal bovine serum (FBS). Cell monolayers were then infected with different clinical isolates of RSV at low MOI (about 0.01) and vims left to adsorb at room temperature for 1 hour. Virus was removed, monolayers washed with PBS and replaced with MEM plus 2% FBS that contained serial dilutions of various interferons, with drag-free controls. After 3 days incubation at 36°C, cells were fixed in 0.05% glutaraldehyde/PBS and blocked with 5%BSA/PBS for 1 hour. After washing, monolayers received diluted mouse monoclonal antibody (cross reactive with A & B sub-types) and incubated for lhour at 36°C). After washing, monolayers were incubated with Protein-A-HRP conjugate at the recommended concentration for 1 hour at 36°C After washing, substrate was added and the reaction stopped by addition of diluted sulphuric acid. Absorbance values were measured at 450nm. Data were plotted and 50% end-points determined. Results: See Figure 7. The effectiveness of EFN α2a and novel interferons was tested in an in vitro model of Respiratory Syncytial Viras (RSV) infections. For most of the isolates, EFN α012 was more potent than EFN o_2a with the exception of VS1511 whereas EFN α023 was definitively more potent on all strains tested. IFN α003 and αOOl were not highly potent in this assay system.

Example 11: Effectiveness of interferons in protecting human lung fibroblasts from

Influenza virus infection.

Materials and Methods: In order to determine the appropriate viras titer to be used on the assay, a viras sensitivity assay was performed. Duplicate rows of 100 μl of cells are plated onto a 96 well microtiter plate at a concentration of 2.0E+5 human lung fibroblasts (HLF) cells/ml. The microtiter plate is placed at 37°C with 5.0% CO₂ overnight. On the second day, influenza is titered out on a separate microtiter plate. 1 :2 serial dilutions are carried out across the plate for a total of 24 dilutions. Fifty μl of these vims dilutions is then added to the microtiter plate of cells and are placed into the incubator overnight. On day three, the plates are stained with crystal violet and allowed to dry. In order to obtain the working dilution of viras to be used in this assay, an endpoint 3 wells to the right of the last well where 100% killing of the cells occurs is used. In this case, a dilution of influenza of 1 :20 from our working stocks of viras was determined to the best dilution.

To find the interferon titer, the interferon is first diluted to approximately 8000 U/ml in 90% DMEM, 10% FBS. Ten μl of this solution is then added to 190 μl of media on a 96 well microtiter plate. Two-fold dilutions are done across the plate for a total of twelve dilutions of interferon. A human EFN alpha A laboratory standard of known concentration is also diluted 1 :20 in the first well and serially diluted across the plate. HLF cells are counted and diluted to a concentration of 2.0E+5 cells/ml. One hundred μl of these cells is then added to each well. The microtiter plate is then placed on a platform rocker and allowed to rock for 10 minutes. The plate is then placed in the incubator (37°C, 5.0% CO₂) overnight. On day 2, influenza is diluted to the predetermined concentration, here 1:20, and 50 μl is added to each well. Again, the plate is placed in the incubator overnight. The plates are then stained with crystal violet and allowed to dry. In order to determine the interferon titer, an endpoint where 50% killing has occuπed is determined. This endpoint is then compared to the endpoint of the human EFN alpha A lab standard.

Results: See Figure 8. The effectiveness of novel interferons was tested in vitro in an influenza model using WSN/A strain of virus (H1N1) and normal human lung fibroblasts (HLF). The concentration of EFN required for 50% inhibition of the viral cytopathic effect was determined and is expressed as relative potency. En this assay EFN α003 was equivalent to EFNo2a while EFN αOOl was less potent. EFN α012 was more than 20 times as potent as EFN o2a.

Example 12: Effectiveness of interferons in protecting liver cells from Yellow Fever Virus infection.

Materials and Methods: Flat bottomed 96 well microtiter plates coated with 0.1%) collagen type 1 diluted 1 in 40 in PBS were seeded with HepG2 cells. The cells were grown to sub-confluent monolayers prior to the addition of the putative antiviral agents. Compounds were added in 6 replicates to wells B2 to G2 and 3 fold dilutions were prepared across the plates (B2 toBlO, C2 to CIO, etc). No compound was added to Column 11.

Compounds were incubated in the presence of cells for 27 hours. Yellow fever vims strain 17D (Stock 6) was added to cells in rows B, C and D, 50 μl per well to provide 500 pfu/well. When sufficient killing in the virally infected cells had occuπed the viable cells were measured by adding 25 μl per well of XTT and PMS to each well, and plates were incubated for 1 hour at 37°C. Plates were cooled to room temperature for 10 minutes before a plate sealer was applied and plates were analyzed for spectrophotometric absorbance at 450nm.

Results: See Figure 9. The effectiveness of novel interferons to protect HepG2 liver cells from killing by Yellow Fever Vims was compared to EFN o2a. IFN α012 was slightly more effective than EFN α2a while EFN α003, and α023 were significantly more effective than EFN o2a. Since YFV is closely related to Hepatitis C viras, this may serve as a model of Hepatitis C treatment.

Example 13: Comparison of IFN (x2a and novel IFNs in a variety of cells types with different viral challenges.

Materials and Methods: Antiviral Assay Protocols:

HEp-2 cells/ VSV

En order to determine the appropriate viras titer to be used on the assay, a viras sensitivity assay was done first. Duplicate rows of 100 μl of cells are plated onto a 96 well microtiter plate at a concentration of 3.0E+5 HEp-2 cells/ml. The microtiter plate is placed at 37°C with 5.0% CO₂ overnight. On the second day, VSV is titered out on a separate microtiter plate. 1 :2 serial dilutions are carried out across the plate for a total of 24 dilutions. Fifty μl of these vims dilutions is then added to the microtiter plate of cells and are placed into the incubator overnight. On day three, the plates are stained with crystal violet and allowed to dry. In order to obtain the working dilution of viras to be used in this assay, an endpoint 3 wells to the right of the last well where 100% killing of the cells occurs is used. In this case, a dilution of VSV of 1:150 from our working stocks of viras was determined to be the best dilution.

To determine the interferon IC50, the interferon is first diluted to approximately 8000 U/ml in 90% DMEM, 10% FBS. Ten μl of this solution is then added to 190 μl of media on a 96 well microtiter plate. Two-fold dilutions are done across the plate for a total of twelve dilutions of interferon. A human EFN alpha A laboratory standard, of known concentration, is also diluted 1 :20 in the first well and serially diluted across the plate. HEP-2 cells are counted and diluted to a concentration of 3.0E+5 cells/ml. One hundred μl of these cells is then added to each well. The microtiter plate is then placed on a platform rocker and allowed to rock for 10 minutes. The plate is then placed in the incubator (37°C, 5.0% CO₂) overnight. On day 2, VSV is diluted to the predetermined concentration, here 1:150, and 50 μl is added to each well. Again, the plate is placed in the incubator overnight. The plates are then stained with crystal violet and allowed to dry. h order to determine the interferon titer, an endpoint where 50% killing has occuπed is determined. This endpoint is then compared to the endpoint of the human EFN alpha A lab standard.

H226 cells/ VSV

En order to determine the appropriate vims titer to be used on the assay, a viras sensitivity assay was done first Duplicate rows of 100 μl of cells are plated onto a 96 well microtiter plate at a concentration of 2.5E+5 H226 cells/ml. The microtiter plate is placed at 37°C with 5.0% CO₂ overnight. On the second day, VSV is titered out on a separate microtiter plate. 1 :2 serial dilutions are carried out across the plate for a total of 24 dilutions. Fifty μl of these viras dilutions is then added to the microtiter plate of cells and are placed into the incubator overnight. On day three, the plates are stained with crystal violet and allowed to dry. En order to obtain the working dilution of vims to be used in this assay, an endpoint 3 wells to the right of the last well where 100% killing of the cells occurs is used, hi this case, a dilution of VSV of 1:1000 from our working stocks of vims was determined to be the best dilution.

To determine the interferon IC50, the interferon is first diluted to approximately 8000 U/ml in 90% RPMI, 10% FBS. Ten μl of this solution is then added to 190 μl of media on a 96 well microtiter plate. Two-fold dilutions are done across the plate for a total of twelve dilutions of interferon. A human EFN alpha A laboratory standard, of known concentration, is also diluted 1 :20 in the first well and serially diluted across the plate. H226 cells are counted and diluted to a concentration of 2.5E+5 cells/ml. One hundred μl of these cells is then added to each well. The microtiter plate is then placed on a platform rocker and allowed to rock for 10 minutes. The plate is then placed in the incubator (37°C, 5.0% CO₂) overnight. On day 2, VSV is diluted to the predetermined concentration, here 1:1000, and 50 μl is added to each well. Again, the plate is placed in the incubator overnight. The plates are then stained with crystal violet and allowed to dry. In order to determine the interferon titer, an endpoint where 50% killing has occuπed is determined. This endpoint is then compared to the endpoint of the human EFN alpha A lab standard.

HLF cells/ Influenza

In order to determine the appropriate vims titer to be used on the assay, a vims sensitivity assay was done first. Duplicate rows of 100 μl of cells are plated onto a 96 well microtiter plate at a concentration of 2.0E+5 HLF cells/ml. The microtiter plate is placed at 37°C with 5.0% CO₂ overnight. On the second day, influenza is titered out on a separate microtiter plate. 1 :2 serial dilutions are carried out across the plate for a total of 24 dilutions. Fifty μl of these viras dilutions is then added to the microtiter plate of cells and are placed into the incubator overnight. On day three, the plates are stained with crystal violet and allowed to dry. En order to obtain the working dilution of viras to be used in this assay, an endpoint 3 wells to the right of the last well where 100% killing of the cells occurs is used. In this case, a dilution of influenza of 1:20 from our working stocks of viras was determined to the best dilution.

To determine the interferon IC50, the interferon is first diluted to approximately 8000 U/ml in 90% DMEM, 10% FBS. Ten μl of this solution is then added to 190 μl of media on a 96 well microtiter plate. Two-fold dilutions are done across the plate for a total of twelve dilutions of interferon. A human EFN alpha A laboratory standard, of known concentration, is also diluted 1:20 in the first well and serially diluted across the plate. HLF cells are counted and diluted to a concentration of 2.0E+5 cells/ml. One hundred μl of these cells is then added to each well. The microtiter plate is then placed on a platform rocker and allowed to rock for 10 minutes. The plate is then placed in the incubator (37°C, 5.0% CO₂) overnight. On day 2, influenza is diluted to the predetermined concentration, here 1:20, and 50 μl is added to each well. Again, the plate is placed in the incubator overnight. The plates are then stained with crystal violet and allowed to dry. In order to determine the interferon titer, an endpoint where 50% killing has occuπed is determined. This endpoint is then compared to the endpoint of the human EFN alpha A lab standard.

L cells/EMCV

In order to determine the appropriate viras titer to be used on the assay, a viras sensitivity assay was done first. Duplicate rows of 100 μl of cells are plated onto a 96 well microtiter plate at a concentration of 3.0E+5 L cells/ml. The microtiter plate is placed at 37°C with 5.0% CO₂ overnight. On the second day, EMCV is titered out on a separate microtiter plate. 1:2 serial dilutions are carried out across the plate for a total of 24 dilutions. Fifty μl of these vims dilutions is then added to the microtiter plate of cells and are placed into the incubator overnight. On day three, the plates are stained with crystal violet and allowed to dry. In order to obtain the working dilution of vims to be used in this assay, an endpoint 3 wells to the right of the last well where 100% killing of the cells occurs is used, this case, a dilution of EMCV of 1:2000 from our working stocks of viras was determined to the best dilution.

To determine the interferon IC50, the interferon is first diluted to approximately 8000 U/ml in 90% MEM, 10% FBS. Ten μl of this solution is then added to 190 μl of media on a 96 well microtiter plate. Two-fold dilutions are done across the plate for a total of twelve dilutions of interferon. A human EFN alpha A laboratory standard, of known concentration, is also diluted 1 :20 in the first well and serially diluted across the plate. HEP-2 cells are counted and diluted to a concentration of 3.0E+5 cells/ml. One hundred μl of these cells is then added to each well. The microtiter plate is then placed on a platform rocker and allowed to rock for 10 minutes. The plate is then placed in the incubator (37°C, 5.0% CO₂) overnight. On day 2, EMCV is diluted to the predetermined concentration, here 1:2000, and 50 μl is added to each well. Again, the plate is placed in the incubator overnight. The plates are then stained with crystal violet and allowed to dry. En order to determine the interferon titer, an endpoint where 50% killing has occuπed is deteπnined. This endpoint is then compared to the endpoint of the human IFN alpha A lab standard.

Results: See Figure 10. The effectiveness of novel interferons to protect a number of cells lines from viral challenge was determined. The cell lines were a bovine line, MDBK, a murine line, L cells, and 3 human lines, HEP-2, H226 and HLF. LFN α012 and α023 were better than EFN α2a on all human lines tested (EFN α023 was not tested in the HLF/Flu assay). Additionally EFN α012 demonstrated significant activity (~70 fold more potent than EFN o2a) on murine cells where EFN α2a has very little effect. This opens the possibility of performing murine models of infection more readily with this EFN than others. The differences in relative efficacy of these EFNs in different vims/cell pairs suggests that selected EFNs or combinations of selected EFNs may be prefeπed for treatment of specific viral infections. For example, EFN α003 is significantly more potent than EFN c_2a at protecting HEP-2 cells from VSV infection whereas EFN α003 has potency similar to that of EFN o2a in the HLF/Flu viras assay. It should also be noted that while EFN α021 and EFN G018 did not demonstrate highly potent antiviral activity in the assays presented herein, it is clear that another virus/cell combination may be highly responsive to these EFNs. Alternatively, it is contemplated that these EFNs may be useful as EFN antagonists is certain conditions where modulation of endogenous EFN activity is waπanted.

Claims

CLAIMS:

1. A method of treating a virus-infected subject or reducing the subject's risk of viral infection, comprising admimstering to the subject an interferon polypeptide comprising an amino acid sequence at least 95% identical to one of SEQ ED NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 68, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 or 86, and is not a naturally occurring interferon allele, wherein the viras is selected from the group consisting of severe acute respiratory syndrome associated coronavirus, influenza, coronavirus, smallpox viras, cowpox viras, West Nile viras, vaccinia viras, respiratory syncytial viras, rhino viras, arteriviras, filoviras, picornaviras, reoviras, retroviras, papovaviras, herpesviras, poxviras, hepadnaviras, astroviras, coxsackie viras, paramyxovirus, orthomyxovirus, echovims, enterovirus, cardiovirus, togavirus, rhabdo viras, bunyaviras, arenaviras, bomavims, adenovims, parvovirus, and flavivirus, thereby treating the virus-infected subject or reducing the subject's risk of viral infection.

2. The method of claim 1, wherein the interferon polypeptide comprises an amino acid sequence selected from the group consisting of SEQ TD NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 68, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 86.

3. The method of claim 1, wherein the interferon polypeptide is encoded by a nucleic acid, which nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ED NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 and 85.

4. The method of claim 1, wherein the interferon polypeptide is encoded by a nucleic acid which hybridizes under high stringency to a nucleic acid comprising a sequence selected from the group consisting of SEQ TD NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 and 85.

5. The method of claim 1, wherein the interferon polypeptide is encoded by a nucleic acid which hybridizes under high stringency to a nucleic acid complementary to a sequence selected from the group consisting of SEQ ED NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 and 85.

6. The method of claim 1 or 2, wherein the subject is a human being, non-human primate, feline, canine, or farm animal.

7. The method of claim 1 or 2, wherein the interferon is administered nasally, orally, parenterally, topically, rectally, by injection, inhalation, eye lotion, ointment, suppository, controlled release patch, infusion, or inhalation.

8. The method of claiml or 2, wherein the interferon is administered to the subject's nasopharyngeal mucosa or lung epithelium.

9. The method of claim 1 or 2, wherein the amount of the interferon polypeptide administered is an amount effective to reduce the concentration of the viras particles in the subject, thereby treating the subject.

10. The method of claim 1 or 2, wherein the amount of the interferon polypeptide administered is an amount effective to prevent or reduce an increase in the concentration of virus particles in the subject, thereby prophylactically treating the subject.

11. A pharmaceutical package comprising an interferon composition with instructions for administering the composition to a subject, wherein (i) the instructions are for treating a virus-infected subject or reducing the subject's risk of viral infection, wherein the viras is selected from the group consisting of severe acute respiratory syndrome associated coronavirus; influenza, coronavims, smallpox viras, cowpox viras, West Nile viras, vaccinia viras, respiratory syncytial viras, rhinoviras, arteriviras, filoviras, picomavims, reovirus, retrovims, papovaviras, herpesviras, poxviras, hepadnaviras, astroviras, coxsackie viras, paramyxovirus, orthomyxoviras, echovirus, enterovirus, cardiovirus, togavirus, rhabdoviras, bunyaviras, arenaviras, bomavims, adenovirus, parvoviras, and flavivims, and (ii) the interferon polypeptide comprises an amino acid sequence that is at least 95% identical to one of SEQ ED NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 68, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 or 86.

12. The pharmaceutical package of claim 11, wherein the interferon composition comprises an interferon selected from the group consisting of SEQ TD NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 68, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 or 86.