Administration of interferon for prophylaxis against or treatment of pathogenic infection

<u style="single">Cross-reference of related applications</u> This application claims priority to US Provisional Patent Application No. 61 / 185,261, which is incorporated herein by reference in its entirety.

The present invention relates to the treatment or prevention of diseases or disorders caused by biological or chemical agents in a subject (eg, a mammal such as a human).

There is a range of emerging viruses that are regional, pandemic, artificially modified, or weaponized. To date, there is no widespread antiviral therapy that effectively prevents or treats infections caused by these viruses. Centers for Disease Control and Prevention (US) Centers for Disease Control and Prevention (CDC; Rotz et al., "CDC Emerging Infectious" According to Diseases, Vol. 8, No. 2, 2002), there are six Category A threats, including smallpox caused by the smallpox virus (smallpox), as well as phylloviruses such as the Ebola virus. , Bunyaviruses such as Hantavirus, and viral hemorrhagic fever caused by Arenaviruses such as Lassavirus. Category A factors are most likely to cause large casualties and have a detrimental effect on public health. Biological agents that can spread on a large scale and cause disease, but generally have a lower mortality rate, are classified as Category B threats. Several viral threats have been identified as Category B threats; this includes viral encephalitis, such as Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), and Western equine encephalitis virus (WEEV). (These are all alpha viruses) are included. There are also many emerging Category C threats, including diseases caused by Nipah and orthohantavirus.

In addition to the CDC list, the US Department of Health and Human Services (HHS) lists viruses under its Public Health Emergency Medical Countermeasures Enterprise (PHEMCE) program. Arenaviridae (eg, funinvirus and lassavirus), Filoviridae (eg, Ebola virus and Marburg virus), Poxviridae (natural poxvirus and monkeys) Poxvirus), and Orthomyxoviridae (eg, influenza A viruses such as H5N1 and H1N1 viruses) are listed. Obviously, it is not feasible to vaccinate the entire population against all strains of these viral factors. In fact, large-scale public vaccination against the threat of bioterrorism, such as anthrax, has failed.

Clinical and commercial use of interferon alfa (IFN-α) (eg, Roferon A®, Intron A®, Pegasys®, Peg Intron®, etc.), for example, malignant melanoma Has been successful in treating various cancers, including hairy cell leukemia, non-Hodgkin's lymphoma, AIDS-related Kaposi's sarcoma, and infectious diseases such as severe acute respiratory syndrome (SARS), chronic hepatitis B, and chronic hepatitis C. .. IFN-α is a type I interferon that binds to the IFN-α receptor.

IFN-α is one of the earliest cytokines released by antigen-presenting cells as part of the innate immune response. IFN-α is directly involved in the responsiveness of NK and T cells that drive the subsequent immune response. Since IFN-α responds early in the immune cascade, its main role has been suggested to induce a priming state during the initial response to infection, with low-dose IFN-α improving protection from viral attack. Has been shown to bring about.

IFN-α as a recombinant human therapeutic agent has a high cGMP production cost, is hindered by its short half-life in vivo, and is produced in a non-glycosylated form. IFN-α has an initial distribution half-life of 7 minutes and a β half-life of 2-5 hours. Due to this rapid decay, frequent injections, usually three times a week, are required to maintain therapeutic levels. Therefore, at a retail price of $ 2,500 per dose, recombinant human IFN-α is too costly to be used as a widespread antiviral drug in anti-bioterrorism or military operations.

To alleviate this rapid biodegradation, a PEGylated form of IFN-α has been developed, which has a half-life of several days instead of hours, thus reducing the number of injections to once a week. Will be done. However, the PEGylation process has been shown to reduce the activity of IFN-α, and the cost of producing PEG-IFN-α is even higher than IFN-α.

Currently, for pre-exposure prophylaxis that protects against infectious diseases, such as viral threats (eg, viral biological weapons used in terrorist incidents, or epidemics), or for post-exposure prophylaxis that responds accordingly. There is a need for widespread antiviral drugs that can be administered.

In a first aspect, the invention comprises a composition comprising a vector having a nucleic acid molecule encoding interferon (IFN) and a pharmaceutically acceptable excipient, wherein the composition is a dry lyophilized powder. , Gel, or liquid, and the composition is stable at room temperature for at least 1 week. In one embodiment, the interferon is substantially identical (eg, at least about) to IFN alpha (IFN-α; eg, consensus IFN-α; eg, shown in SEQ ID NO: 11) or the sequence set forth in SEQ ID NO: 11. 75%, 80%, 85%, 90%, 95%, 97%, or 99% or more identical)). In another embodiment, the vector is a viral vector (eg, an adenovirus vector (eg, an adenovirus 5 strain (Ad5) vector)). In another embodiment, the adenovirus vector (eg, Ad5 vector) contains a deletion of all or part of the E1 and E3 genes, thereby making it replication deficient. In yet another embodiment, the vector is a non-viral vector.

In another embodiment of the first aspect of the invention, when IFN is expressed in vivo after administration of the composition of the first aspect of the invention, the pathogen (eg, human) in the mammal (eg, human) to which the composition is administered. For example, a protective immune response against bacteria, viruses, fungi, or parasites occurs, or infections by pathogens are treated in mammals. In another embodiment, the expression of IFN in vivo after administration of the composition of the first aspect of the invention results in a protective response against autoimmune disease in the mammal (eg, human) to which the composition is administered.

In another embodiment of the first aspect of the invention, the nucleic acid molecule of the vector is the SV40 promoter, CMV promoter, adenovirus early and late promoters, metallothioneine gene (MT-1) promoter, Laus sarcoma virus (RSV) promoter, And operably linked to a promoter selected from the human ubiquitin C (UbC) promoter, or the vector is a signal sequence, polyadenylation sequence, and enhancer, upstream activation sequence, which promotes expression of a nucleic acid molecule encoding interferon. And one or more of the transcription termination factors. In yet another embodiment, excipients present in the composition in an amount ranging from 1% to 90% by weight (eg, in an amount in the range of 5% to 30% by weight) include fructose, maltose, and the like. Galactose, glucose, D-mannose, sorbose, lactose, sucrose, trehalose, cellobiose, raffinose, meregitos, maltdextrin, dextran, starch, mannitol, xylitol, xylose, maltitol, lactitol, xylitol Sorbitol, sorbitol, pyranosyl sorbitol, myo-inositol, glycine, CaCl₂, Hydroxyectine, ectoine, gelatin, di-mio-inositol phosphate (DIP), cyclic 2,3 diphosphoglycerate (cDPG), 1,1-di-glycerol phosphate (DGP), β-mannosyl glycerate ( It is selected from one or more of firoin), β-mannosyl glyceramide (filoin A), and proline betaine.

In a preferred embodiment, the excipient is an IFN-encoding delivery medium (eg, Ad5-IFN delivery medium) at room temperature for a long period of time (eg, longer than a week, and preferably longer than a year or more). It is capable of stabilizing a loss of viral titer or biological activity (eg, if the delivery medium is non-viral) to less than 20%. Non-limiting examples of such excipients include, for example, trehalose, sorbitol, sucrose, mannitol, glycine, CaCl.₂, Hydroxiectoin, ectoine, phylloin and gelatin.

In yet other embodiments, the composition may be formulated for aerosol delivery; it is stable at room temperature for at least 1 month (eg, 1 year or more); and mixed with a pharmaceutically acceptable liquid. Can form a liquid or gel.

In a second aspect, the invention relates to the lung or nasal mucosa of a subject (eg, mammals such as primates, dogs, cats, cows, horses, pigs, goats, rats, mice, or humans, or birds). , One or more times (eg, within a month or more or a year or more, eg 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or as needed) in an amount By administering the composition of the first aspect of the invention, the subject in need (eg, mammals such as primates, dogs, cats, cows, horses, pigs, goats, rats, mice, or humans, or birds) ) Features a method of preventing or treating infection, autoimmune disease, or cancer by a biological agent (eg, an infectious pathogen such as a bacterium, virus, fungus, or parasitic). In one embodiment, the vector targets lung or nasal epithelial cells after said administration. In yet another embodiment, when the vector is transfected into a target cell, in the cell of interest an IFN-α such as interferon (IFN; eg, consensus IFN-α (conIFN-α; eg, shown in SEQ ID NO: 11)). Is expressed, IFN acts locally and / or is secreted by cells into the subject's bloodstream. In other embodiments, the composition comprises an IFN-encoding adenovirus 5 strain (Ad5) vector and the composition contains at least about 1 × 10 of the Ad5 vector per dose.³~ About 1 × 10¹⁴Included in an amount in the range of individual virus particles.

In yet another embodiment of the second aspect of the invention, the subject is exposed to the pathogen prior to exposure (eg, at least about 15-30 minutes before exposure to the pathogen, preferably at least about 1 before exposure to the pathogen. , 2, 4, 6, 8, 10, 15, 20, or 24 hours before, and more preferably at least about 1-2 weeks before exposure to the pathogen) to receive the composition, or subject to pathogen After exposure (eg, immediately after exposure to the pathogen, at least about 15-30 minutes after exposure to the pathogen, or at least about 1, 2, 4, 6, 8, 10, 15, 20 after exposure to the pathogen. , 24, 48, or 72 hours later, or more. In other embodiments, the pathogen is a bacterium, virus, fungus, or parasite.

In other embodiments, the subject receives administration of the composition of the first aspect of the invention before or after the onset of an autoimmune disease or cancer, or symptoms thereof.

In yet another embodiment of the second aspect of the invention, the composition may be inhaled as a lyophilized powder (eg, as a non-reconstituted powder) or a pharmaceutically acceptable liquid (eg, water). Alternatively, it may be mixed with physiological saline and inhaled as an aerosol mist. In other embodiments, the aerosol mist comprises droplets with a diameter greater than 2 μm. In yet another embodiment, prior to administration of the composition of the first aspect of the invention, the subject is tested to see if the subject is exposed to a pathogen, is symptomatic of an autoimmune disease, or has cancer. It is determined whether or not it has. In another embodiment, after administration of the composition of the first aspect of the invention, the method comprises the step of measuring the serum IFN level of the subject and the serum IFN level of less than about 1000 IU / ml, preferably about. If less than 500 IU / ml, more preferably less than 100 IU / ml, for example in the range of about 0.0001 to about 250 IU / ml, further comprises the step of administering an additional dose of the composition. In other embodiments, serum IFN levels are from about 100 IU / ml to about 5.0 × 10 after administration of the compositions of the invention.⁵It is in the range of IU / ml, preferably in the range of about 200 to 10,000 IU / ml, more preferably in the range of about 250 to 5,000 IU / ml. In other embodiments, the subject is administered at least 2 doses (eg, 3, 4, 5, 6, 7, 8, 9, and 10 doses) of the composition. Preferably, the composition exposes the subject to at least about 24 hours, 36 hours, 48 hours, or 72 hours from infection by the pathogen, preferably at least about 1, 2, 3, 4, or 5 weeks, and more preferably at least. Defend for about 2, 6, 12, 18 or 24 months or more. In other embodiments, administration of the composition of the first aspect of the invention alleviates or attenuates symptoms associated with an autoimmune disease, or determines tumor size or cancerous cell count as determined using standard methods. 20, 40, 60, 80, or 100% reduction (eg, at least 20, 40, 60, 80, 90, or 95% of treated subjects enter complete remission in which all evidence of tumor or cancer disappears ). Desirably, the tumor or cancer does not recur, or recurs after at least 5, 10, 15, or 20 years.

In other embodiments, the composition is administered as a liquid or gel. The composition may be administered by the subject or by another person, such as a healthcare professional.

In another embodiment of the second aspect of the invention, after administration of the composition of the first aspect of the invention, the method determines the level of IFN-induced response as correlating with the activity of IFN in the subject. Includes more steps. For example, the method uses double-stranded RNA (dsRNA) -dependent protein kinase R (PKR), 2'-5'-oligoadenylate synthetase (2'-5'-OAS), IFN-induced Mx protein, and tryptophan degradation. Enzymes (see, eg, Pfefferkorn, Proc.Natl.Acad.Sci.USA 81: 908-912, 1984), adenosine deaminase (ADAR1), IFN activating gene 20 (ISG20), p56, ISG15, mGBP2 , GBP-1, APOBEC protein, viperine, or other factors (eg, Zhang et al., J. Virol., 81: 11246-11255, 2007, and US Patent No. 1 incorporated herein by reference in their entirety. It may include the step of determining or measuring the upregulation or activity of (see 7,442,527).

A third aspect of the present invention features an apparatus comprising the composition of any embodiment of the first aspect of the present invention. Preferably, the device is a) a container containing the composition; b) a nozzle for delivering the composition to the pulmonary or nasal mucosa of interest; c) a mechanical delivery pump for delivering the composition to the nozzle. A pump that fluidly connects the nozzle and the container when the pump is driven; d) A drive mechanism for operating the mechanical delivery pump (eg, driving the delivery pump at a predetermined pressure or flow rate). Can trigger) and include. The delivery pump may also include a liquid delivery pump for delivering a predetermined volume of the composition in liquid form or a powder delivery pump for delivering a predetermined amount of composition in powder form. In one embodiment, the nozzle may be configured to deliver an aerosol (eg, mist) or jet. The device used in the third aspect of the present invention is described herein.

A fourth aspect of the invention is a first container having the composition of any embodiment of the first aspect of the invention, a second container having a pharmaceutically acceptable liquid, and the present invention. A first container using the device of any embodiment of the third aspect, and optionally, for treating or inhibiting infection by a pathogen, an autoimmune disease or its symptoms, or cancer, eg, for a subject. Instructions for delivery of the contents of, or for combining the contents of the first and second containers to form a composite composition, and then using the device to deliver the composite composition. It features a kit that includes and. In one embodiment of all aspects of the invention, the vector is a recombinant viral vector containing a nucleic acid molecule encoding a cytokine (eg, interferon alpha (IFN-α) such as consensus IFN-α) (eg, Ad5, etc.). Adenoviral vector); a biological agent by administering the composition to a subject (eg, a mammal such as a primate, dog, cat, cow, horse, pig, goat, rat, mouse, or human, or a bird). Can protect against attacks or treat biological infections. The biological agent may be an infectious agent such as a bacterium, virus, fungus, or parasite.

In one embodiment of all aspects of the invention, the bacteria are Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae, Brucella. , Burkholderia mallei, Yersinia pestis, and Bacillus anthracis.

In one embodiment of all aspects of the invention, the virus is selected from members of the Flaviviridae family (eg, members of the Flaviviridae, Pestivirus and Hepacivirus). This includes hepacivirus C virus, yellow fever virus; Gadgets Gully virus, Kadam virus, Kasanur forest disease virus, Langat virus, Omusk hemorrhagic fever virus, Poissan virus, Royal Farm. Tick-borne viruses such as Farm virus, Karshi virus, tick-borne encephalitis virus, Neudoerfl virus, Sofjin virus, leaping disease virus and Negisivirus; Meaban virus, Saumarez Seabird tick-borne viruses such as Reef virus and Tyuleniy virus; Aroa virus, Deng virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray valley encephalitis Virus, St. Louis encephalitis virus, Usutu virus, Westnile virus, Yaounde virus, Kokobera virus, Bagaza virus, Ilheus virus, Israel turkey meningoencephalomyelitis virus, Untaya Virus, Tempus virus, Jika virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus, Vessel Mosquito-borne viruses such as Subron virus, yellow fever virus; and Entebbe bat virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukarasa bat virus, Carey Island virus, Dakar bat Viruses without known mediators such as Montana myelitis leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, and Cell fusing agent virus Is included.

In another embodiment of all aspects of the invention, the virus is selected from members of the Arenaviridae family, which includes Ippy virus, Lassavirus (eg, Josiah strain, LP strain). , Or GA391 strain, lymphocytic choriomyelitis virus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexal virus, guanaritovirus, funin virus, Latino ) Virus, Machupovirus, Oliveros virus, Parana virus, Pitindevirus, Pirital virus, Savior virus, Takaribe virus, Tamiami virus, Whitewater Arroyo ) Virus, Chaparet virus, and Lujo virus are included.

In yet another embodiment of all aspects of the invention, the virus is a member of the Bunyaviridae family (eg, the genus Hantavirus, the genus Nairovirus, the genus Orthobunyavirus, and Selected from the genus Phlebovirus), it includes Huntan virus, Shinnonbre virus, Dugbe virus, Bunyanbella virus, Rift Valley fever virus, Lacros virus, Punta Toro. Includes virus (PTV), California encephalitis virus, and Crimea-Congo hemorrhagic fever (CCHF) virus.

In yet another embodiment of all aspects of the invention, the virus is a member of the family Filoviridae (including Ebola viruses (eg, Zaire strains, Sudan strains, Cote d'Ivoire strains, Reston strains, and Uganda strains) and Marburg virus (including, for example, Angola, Ci67, Musoke, Popp, Ravn and Lake Victoria); members of the Togaviridae family (eg, Alpha) Members of the genus Alphavirus) (This includes Venezuelan encephalitis virus (VEE), Eastern horse encephalitis virus (EEE), Western horse encephalitis virus (WEE), Sindbis virus, Ruin virus, Semuliki forest virus, Los River virus , Bermaforest virus, Onyonnyon virus, and Chikungunya virus); Members of the Poxviridae family (eg, members of the Orthopoxvirus genus) (This includes natural poxvirus, monkey poxvirus) , And vaccinia virus); members of the herpesvirus family (including simple herpesviruses (HSV; types 1, 2, and 6), human herpesviruses (eg, types 7 and 8), cytomegalo Virus (CMV), Epstein-Bal virus (EBV), varicella herpes virus, and Kaposi sarcoma-related herpesvirus (KSHV)); members of the Orthomyxoviridae family (which includes H5N1 togaviridae virus or Influenza viruses such as H1N1 togaviridae (including types A, B, and C); members of the Coronaviridae family (including the Severe Acute Respiratory Syndrome (SARS) virus);Members of the Rhabdoviridae family (which includes mad dog disease virus and bullous stomatitis virus (VSV)); Paramyxoviridae members (which include human respiratory polynuclear virus (RSV)), Newcastle disease virus, Hendra virus, Nipavirus, measles virus, bovine epidemic virus, canine temper virus, Sendai virus, human paramyxovirus (eg, types 1, 2, 3, and 4), rhinovirus, and mumps virus. Includes); Members of the Picornaviridae family (including poliovirus, human astroviruses (types A, B, C, and D), hepatitis A virus, and coxsackie virus); Members of the Hepadnaviridae family (which includes the hepatitis B virus); members of the Papillamoviridae family (which includes the human papillomavirus); members of the Parvoviridae family (which includes the human papillomavirus). This includes adeno-related viruses); members of the Astroviridae family (which includes Astroviridae); members of the Polyomaviridae family (which includes JC virus, BK virus, and SV40 virus included); members of the Calciviridae family (which includes nowalk virus); members of the Reoviridae family (which includes rotavirus); and retrovirus family Members of (Retroviridae), including human immunodeficiency viruses (HIV; eg, types 1 and 2), and human T lymphocyte tropic viruses types I and II (HTLV-1 and HTLV-2, respectively). Is selected from).Members of the Paramyxoviridae family (including human respiratory polynuclear virus (RSV), Newcastle disease virus, Hendra virus, nipavirus, measles virus, bovine epidemic virus, inudistemper virus, Sendai virus, human paramyxoviridae virus) (For example, types 1, 2, 3, and 4), rhinovirus, and mumpsvirus); members of the Picornaviridae family (including poliovirus, human enterovirus (type A, type A,) B, C, and D), hepatitis A virus, and coxsackie virus); members of the Hepadnaviridae family (which includes hepatitis B virus); Papillamoviridae ) Members (which include human papillomavirus); Parvoviridae members (which include adeno-related viruses); Astroviridae members (which include astroviruses) Members of the Polyomaviridae family (which includes JC virus, BK virus, and SV40 virus); Members of the Calciviridae family (which includes Norwalk virus); Members of the Reoviridae family (which includes rotavirus); and members of the Retroviridae family (which include human immunodeficiency viruses (HIV; eg, types 1 and 2), and humans. It is selected from T lymphocyte tropic viruses type I and type II (including HTLV-1 and HTLV-2, respectively).Members of the Paramyxoviridae family (including human respiratory polynuclear virus (RSV), Newcastle disease virus, Hendra virus, nipavirus, measles virus, bovine epidemic virus, inudistemper virus, Sendai virus, human paramyxoviridae virus) (For example, types 1, 2, 3, and 4), rhinovirus, and mumpsvirus); members of the Picornaviridae family (including poliovirus, human enterovirus (type A, type A,) B, C, and D), hepatitis A virus, and coxsackie virus); members of the Hepadnaviridae family (which includes hepatitis B virus); Papillamoviridae ) Members (which include human papillomavirus); Parvoviridae members (which include adeno-related viruses); Astroviridae members (which include astroviruses) Members of the Polyomaviridae family (which includes JC virus, BK virus, and SV40 virus); Members of the Calciviridae family (which includes Norwalk virus); Members of the Reoviridae family (which includes rotavirus); and members of the Retroviridae family (which include human immunodeficiency viruses (HIV; eg, types 1 and 2), and humans. It is selected from T lymphocyte tropic viruses type I and type II (including HTLV-1 and HTLV-2, respectively).For example, types 1 and 2), and human T-lymphotropic viruses types I and II (including HTLV-1 and HTLV-2, respectively) are selected.For example, types 1 and 2), and human T-lymphotropic viruses types I and II (including HTLV-1 and HTLV-2, respectively) are selected.Members of the Picornaviridae family (including poliovirus, human astroviruses (types A, B, C, and D), hepatitis A virus, and coxsackie virus); hepadonavirus family ( Hepadnaviridae) members (which include hepatitis B virus); Papillamoviridae members (which include human papillomavirus); Parvoviridae members (which are accompanied by adenovirus) Viruses included); Members of the Astroviridae family (which includes Astroviridae); Members of the Polyomaviridae family (which include JC virus, BK virus, and SV40 virus) ); Members of the Calciviridae family (which includes the Norwalk virus); Members of the Reoviridae family (which includes the Rotavirus); and members of the Retroviridae family. Selected from human immunodeficiency viruses (HIV; eg, types 1 and 2) and human T lymphocyte-tropic viruses types I and II (HTLV-1 and HTLV-2, respectively). Virus.Members of the Picornaviridae family (including poliovirus, human astroviruses (types A, B, C, and D), hepatitis A virus, and coxsackie virus); hepadonavirus family ( Hepadnaviridae) members (which include hepatitis B virus); Papillamoviridae members (which include human papillomavirus); Parvoviridae members (which are accompanied by adenovirus) Viruses included); Members of the Astroviridae family (which includes Astroviridae); Members of the Polyomaviridae family (which include JC virus, BK virus, and SV40 virus) ); Members of the Calciviridae family (which includes the Norwalk virus); Members of the Reoviridae family (which includes the Rotavirus); and members of the Retroviridae family. Selected from human immunodeficiency viruses (HIV; eg, types 1 and 2) and human T lymphocyte tropic viruses types I and II (HTLV-1 and HTLV-2, respectively). Virus.Members of the Polyomaviridae family (which includes JC virus, BK virus, and SV40 virus); Members of the Calciviridae family (which includes Norwalk virus); Reoviridae (Reoviridae) members (which include Rotavirus); and Retroviridae members (which include human immunodeficiency viruses (HIV; eg, types 1 and 2)), and human T lymphocytes. It is selected from tropotropic viruses type I and type II (including HTLV-1 and HTLV-2, respectively).Members of the Polyomaviridae family (which includes JC virus, BK virus, and SV40 virus); Members of the Calciviridae family (which includes Norwalk virus); Reoviridae (Reoviridae) members (which include Rotavirus); and Retroviridae members (which include human immunodeficiency viruses (HIV; eg, types 1 and 2)), and human T lymphocytes. It is selected from tropotropic viruses type I and type II (including HTLV-1 and HTLV-2, respectively).

In yet other embodiments of all aspects of the invention, the fungi are Aspergillus, Blastomyces dermatitidis, Candida, Coccidioides immitis, Cryptococcus neofol. Cryptococcus neoformans, Histoplasma capsulatum var.capsulatum, Paracoccidioides brasiliensis, Sporothrix sporothrix sporothrix schenckii It may be Absidia corymbifera, Rhizomucor pusillus, or Rhizopus arrhizus.

In another embodiment of all aspects of the invention, the parasites are Toxoplasma gondii, Plasmodium falciparum, P. vivax, P. ovale. ), P. malariae, Trypanosoma spp., And Legionella spp.

In another embodiment of all aspects of the invention, autoimmune diseases include systemic autoimmune diseases and organ-specific autoimmune diseases. Typical examples of autoimmune diseases are insulin-dependent diabetes (also known as type 1 diabetes), systemic erythematosus, rheumatoid arthritis, Hashimoto's disease, alopecia alopecia, tonic dermatomyositis, antiphospholipid syndrome, Autoimmune Azison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Bechet's disease, bullous vesicles, cardiomyopathy, ceriax prue-dermatomyositis, chronic fatigue immunodeficiency syndrome (CFIDS), chronic inflammatory demyelination Sexual polyneuropathy, Churg-Strauss syndrome, scarring dermatomyositis, CREST syndrome, cold agglutinosis, Crohn's disease, discoid lupus, ulcerative colitis, psoriatic arteritis, essential mixed cryoglobulinemia, fibers Myopathy-fibromyositis, Graves' disease, Gillan Valley, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, juvenile arteritis, squamous lichen, lupus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, severe myasthenia, asthma vulgaris, malignant anemia, nodular polyarteritis, polychondritis, polyglandular syndrome, rheumatic polymyositis, Polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Reynaud phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, sclerosis, Schegren's syndrome, Stiffman's syndrome, David's disease, hyperan arteritis , Temporal arteritis / giant cell arteritis, ulcerative colitis, vasculitis, vasitis, keratomyositis, and Wegener's granulomatosis.

In another embodiment of all aspects of the invention, the cancers include melanoma, clear cell sarcoma, head and neck cancer, bladder cancer, breast cancer, colon cancer, ovarian cancer, endometrial cancer, gastric cancer, pancreatic cancer, renal cancer, Cancers such as prostate cancer, salivary adenocarcinoma, lung cancer, liver cancer, skin cancer, and brain cancer can be mentioned.

In yet another embodiment of all aspects of the invention, the compositions and methods of the first, second, third, and fourth aspects of the invention are adjuvant therapeutic agents or regimens, such as antibodies or antibody fragments. (For example, recombinant, humanized, chimeric, or monoclonal antibody or fragment), microbial antigen, cytokine or growth factor, hormone, coagulation factor, drug resistant or antiviral resistant polypeptide, antitoxin, antioxidant, receptor or It further comprises the step of administering, or expressing, a polypeptide such as a ligand, immunomodulator, detectable label, cellular factor, or vaccine with a vector (eg, a viral vector). In other embodiments, the antibody or antibody fragment may be a single chain antibody (scFv), Fab, Fab'2, scFv, SMIP, diabodies, Nanobodies, aptamers, or domain antibodies. In yet other embodiments, the cytokine or growth factor is tumor necrosis factor alpha (TNF-α), TNF-β, IFN-β, IFN-γ, interleukin 1 (IL-1), IL-1β, interleukin. 2-14, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), RANTES, MIP-1α), transformed growth factor β (TGF-β), platelet-derived growth factor ( It may be PGDF), insulin-like growth factor (IGF), epithelial growth factor (EGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), erythropoetin (EPO), or thrombopoetin (TPO). Hormones include angiotensinogen, angiotensin, parathyroid hormone (PTH), basic fibroblast growth factor 2, luteinizing hormone, follicular stimulating hormone, adrenocorticotropic hormone (ACTH), vasopressin, oxytocin, somatostatin, gastrin, kore It may be cystokinine, leptin, atrial sodium diuretic peptide, epinephrine, norepinephrine, dopamine, calcitonin, or insulin. Coagulation factors are factor VII, factor VIII, factor It may be IX or fibrinogen. Enzymes are butyrylcholine esterase (BChE), adenosine deaminase, glucocerebrosidase, α1 antitrypsin, viral thymidin kinase, hypoxanthin phosphoribosyl transferase, manganese superoxide dismutase (Mn-SOD), catalase, copper-zinc superoxide dismutase ( It may be CuZn-SOD), extracellular superoxide dismutase (EC-SOD), glutathione reductase, phenylalanine hydroxylase, nitrogen monoxide dismutase, or paraoxinase. The receptor or ligand is the T cell receptor (TCR), LDL receptor, surface-bound immunoglobulin, soluble CD4, cystic fibrosis transmembrane conductance receptor (CFTR), or F._CIt may be a receptor. Immunomodulators include CTLA-4, VCP, PLIF, LSF-1, Nip, CD200, uromodulin, CD40L (CD154), FasL, CD27L, CD30L, 4-1BBL, CD28, CD25, B7.1, B7.2, Alternatively, it may be OX40L. The detectable label may be green fluorescent protein (GFP). Cellular factors are cytochrome b, ApoE, ApoC, ApoAI, MDR, tissue plasminogen activator (tPA), urokinase, hirudin, β-globin, α-globin, HbA, ras, src, or bcl. May be good. The polypeptide may be a cellular protein that acts as an antigen thereby causing an immune response against a biological or chemical agent in the subject. Vaccines are, for example, bacterial, viral, fungal, or parasitic vaccines known in the art to treat one or more of the bacteria, viruses, fungi, or parasitic factors described herein. There may be. For example, the vaccine is Pseudomonas. aeruginosa), Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae, Bruscella, Burkholderia mallei, Ersina pestis (Yers) Bacteria selected from Bacillus anthracis; viruses selected from Flaviviridae members (eg, members of Flaviviridae, Pestivirus, and Hepacivirus) (Hepacivirus C virus, yellow fever virus; Gadgets Gully virus, Kadam virus, Kasanur forest disease virus, Langat virus, Omusk hemorrhagic fever virus, Poissan virus, Royal Farm (Royal) Tick-borne viruses such as Farm virus, Karshi virus, tick-borne encephalitis virus, Neudorfl virus, Sofjin virus, leprosy virus and Negishi virus; Meaban virus, Saumarez Seabird tick-borne viruses such as Reef virus and Tyuleniy virus; Aroa virus, Deng virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray valley encephalitis Virus, St. Louis encephalitis virus, Usutu virus, Westnile virus, Yaounde virus, Kokobera virus, Bagaza virus, Ilheus virus, Israel turkey meningoencephalomyelitis virus, Untaya Virus, Tempus virus, Dika virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus, Vesselsbron virus, yellow fever virus and other mosquito-borne viruses; and Entebbe bat virus, Yokose virus , Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukarasa bat virus, Carey Island Island) virus, Dakar bat virus, Montana myitis white encephalitis virus, Phnom pen (Phnom) Viruses without known mediators such as Penh) bat virus, Rio Bravo virus, Tamana bat virus, and cell fusion factor virus; viruses selected from members of the Arenaviridae family (to this) Ippy virus, Lassa virus (eg, Josiah strain, LP strain, or GA391 strain), lymphocytic choriomyelitis virus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexal virus, Guanarit virus, Funin virus, Latino virus, Machupo virus, Oliveros virus, Parana virus, Pitindevirus, Pirital virus, Savior virus, Takaribe virus, Tamiami virus, Whitewater Arroyo (Whitewater) Includes Arroyo virus, Chaparet virus, and Lujo virus); Members of the Bunyaviridae family (eg, Hantavirus, Nairovirus, Orthobunyavirus) ), And a virus selected from the genus Phlebovirus (which includes Hantern virus, Shinnonbre virus, Dugbe virus, Bunyanbella virus, Rift Valley fever virus, Lacros virus, Puntatro. (Punta Toro) virus (PTV), California encephalitis virus, and Crimea-Congo hemorrhagic fever (CCHF) virus); viruses selected from members of the Filoviridae family (eg, Ebola virus (eg, Zeil strain) , Sudan, Cote d'Ivoire, Reston, and Uganda) and Marburg virus (eg, Angola, Ci67, Musoke, Popp, Ravn, and Lake Victoria). Members of the Togaviridae family (eg, members of the Alphavirus genus) (for example, Venezuelan lauma encephalitis virus (VEE), Eastern horse encephalitis virus (EEE), Western horse encephalitis virus (WEE)) , Sindbis virus, eczema virus, semuliki forest virus, loss river virus, verma forest virus, onyonnyon virus, and chikungunya virus); members of the Poxviridae family (eg, Orthopoxvirus) Members of) (This includes natural poxvirus, monkey poxvirus, and vaccinia virus); members of the Herpesviridae family (which include simple herpesviruses (HSV; types 1, 2, and 6)). (Types), human herpesviruses (eg, types 7 and 8), cytomegalovirus (CMV), Epstein-var virus (EBV), varicella herpes virus, and capo-sarcoma-related herpesvirus (KSHV)); Members of the Orthomyxoviridae family (including influenza viruses such as H5N1 triinfluenza virus or H1N1 porcine influenza (types A, B, and C)); members of the Coronaviridae family (Coronaviridae) This includes the Severe Acute Respiratory Syndrome (SARS) virus);Members of the Rhabdoviridae family (which includes mad dog disease virus and bullous stomatitis virus (VSV)); Paramyxoviridae members (which include human respiratory astrovirus (RSV)), Newcastle disease virus, Hendra virus, Nipavirus, measles virus, bovine epidemic virus, canine temper virus, Sendai virus, human parainfluenza virus (eg, types 1, 2, 3, and 4), rhinovirus, and mumps virus. Includes); Members of the Picornaviridae family (including poliovirus, human astroviruses (types A, B, C, and D), hepatitis A virus, and coxsackie virus); Members of the Hepadnaviridae family (which includes the hepatitis B virus); Papillamoviridae members (which include the human papillomavirus); Parvoviridae members (which include the human papillomavirus) This includes adeno-related viruses); members of the Astroviridae family (which includes Astroviridae); members of the Polyomaviridae family (which includes JC virus, BK virus, etc.)Members of the Picornavirus family (including polyomaviridae, human astroviruses (A, B, C, and D), hepatitis A virus, and coxsackie virus); Hepadnavirus family ( Members of Hepadnaviridae) (which includes hepatitis B virus); Members of the family Papillamoviridae (which includes human papillomavirus); Members of the family Parvoviridae (which accompanies adenovirus) Viruses included); Members of the Astroviridae family (which includes Astroviridae); Members of the Polyomaviridae family (This includes JC virus, BK virus,Members of the Picornavirus family (including polyomaviridae, human astroviruses (A, B, C, and D), hepatitis A virus, and coxsackie virus); Hepadnavirus family ( Members of Hepadnaviridae) (which includes hepatitis B virus); Members of the family Papillamoviridae (which includes human papillomavirus); Members of the family Parvoviridae (which accompanies adenovirus) Viruses included); Members of the Astroviridae family (which includes Astroviridae); Members of the Polyomaviridae family (This includes JC virus, BK virus, And SV40 virus); members of the Calciviridae family (which includes the nowalk virus); members of the Reoviridae family (which includes the rotavirus); and retroviruses Members of the family (Retroviridae), including human immunodeficiency viruses (HIV; eg, types 1 and 2), and human T lymphocyte tropic viruses types I and II (HTLV-1 and HTLV-2, respectively). Included); or Aspergillus, Blastomyces dermatitidis, Candida, Cocccidioides immitis, Cryptococcus neoformans, Cryptococcus neoformans, Cryptococcus neoformans (Histoplasma capsulatum var.capsulatum), Paracocccidioides brasiliensis, Sporothrix schenckii, Zygomycetes spp. Fungi selected from pusillus, and Rhizopus arrhizus; or Toxoplasma gondii, Plasmodium falciparum, P. vivax, P. ovare (P. vivax). ovale), P. malariae, Trypanosoma spp., And Legionella spp.) Can be targeted for parasites selected from.

In yet another embodiment of all aspects of the invention, the vector (eg, a viral vector) expresses one or more oligonucleotides capable of inhibiting viral replication or infection, such as RNA interference (RNAi) molecules. Can be modified as The RNAi molecule may be a small interfering RNA (siRNA) or a short hairpin RNA (shRNA) molecule.

In another embodiment of all aspects of the invention, the subject has been or is suspected of being exposed to a biological or chemical agent prior to receiving the pharmaceutical composition of the invention. In another embodiment of all aspects of the invention, the subject has been diagnosed with or presents with an autoimmune disease or cancer prior to receiving administration of the pharmaceutical composition of the invention. The subject may be administered a single dose or multiple doses of the pharmaceutical composition of the invention. In another embodiment of all aspects of the invention, the pharmaceutical composition of the invention protects a subject (eg, a mammal such as a human) as a prophylactic, eg, a vaccine-type prophylactic. Before exposure to biological or chemical agents (eg, just before exposure, eg, at least about 5, 10, or 30 minutes before exposure, or preferably at least about 1, 2, 3, 4, or at least about 1, 2, 3, 4, or 5 hours before, more preferably at least about 6, 24, 36, 48, or 72 hours before exposure, and more preferably at least about 1, 2, 3, or 4 weeks before exposure) , Or may be administered before the diagnosis of an autoimmune disease or cancer, or before the onset of its symptoms. The pharmaceutical composition of the present invention is intravenously, intramuscularly, orally, parenterally, intraperitoneally, intraarterially, transdermally, sublingually, nasally to a subject. It can be administered orally, orally, liposomes, fat, optimally, intraocularly, subcutaneously, intrathecally, locally or locally. In a preferred embodiment, the pharmaceutical composition is administered to the pulmonary or intranasal mucosa of the subject. If the delivery medium composition encoding IFN is a viral vector, the subject is at least about 1x10.³Viral particles (vp) / dose or 1x10¹~1×10¹⁴vp / dose, preferably 1x10³~1×10¹²vp / dose, and more preferably 1x10⁵~1×10¹⁰vp / dose can be administered. If the delivery medium composition encoding IFN is a non-viral vector, the subject is at least about 1x10.¹Molecule / dose, eg 1x10¹~1×10¹⁵Molecule / dose, preferably 1x10³~1×10¹⁰Molecule / dose, and more preferably 1x10⁴~1×10⁸A molecular / dose non-viral delivery vector can be administered.

In other embodiments of all aspects of the invention, expression of a heterologous protein (eg, an IFN such as consensus IFN-α) in a subject (as determined by measuring serum levels) is 1 week, 1 month, Occurs for more than 2 or 6 months. In yet another embodiment, the effect of expression of interferon (eg, IFN-α such as consensus IFN-α) is determined using surrogate markers on interferon expression (as discussed herein) for 1 week. Occurs for more than 1 month, 2 months, 6 months or 1-2 years.

In another embodiment of all aspects of the invention, the pharmaceutical composition of the invention is combined with one or more adjuncts that enhance or prolong the prophylactic or therapeutic effect of interferon (eg, consensus IFN-α) treatment. Can be administered to the subject. Auxiliary agents may be, for example, cytokines, antiviral agents, antibacterial agents, antifungal agents, antiparasitic agents, immunostimulators, or immunized vaccines. In another embodiment, the pharmaceutical composition of the invention comprises an IFN expression vector (eg, an Ad5 vector encoding IFN-α), a vaccine, and a pharmaceutically acceptable carrier. Fast-acting (eg, 1), when administered within at least 24 hours after exposure (eg, 1, 2, 4, 6, 8, 10, 12, 15, or 18 hours) or even within only 15-30 minutes after exposure. ,&gt; 80% (eg, 85%, 90%, 95%, or 99% or more (eg, 100%) expressed) efficacy (eg, measured by viability). In another embodiment. , Vaccines are viral vaccines (eg, Ebola vaccine (eg, Ebola Zeil vaccine Ad-CAGoptZGP; Richardson et al. (PloS) 4: e5308, 2009)). In another embodiment, the pharmaceutical compositions of the invention can be combined with an IFN expression vector (eg, an Ad5 vector encoding IFN-α), either individually or as a vaccine (eg, an Ebola vaccine (eg, Ebola zyle vaccine Ad-CAGoptZGP;). Includes pharmaceutically acceptable carriers that are administered in combination with viral vaccines) such as Richardson et al. (See PLoS 4: e5308, 2009)). For example, the pharmaceutical compositions of the invention are within 15-30 minutes of the vaccine, or within 1, 2, 4, 8, 10, 12, 24, 48, or 72 hours of the vaccine, or 1-2 weeks after the vaccine. Administered within.

In yet another embodiment of all aspects of the invention, the vector (eg, a viral vector such as an Ad5 vector) is administered with a pharmaceutically acceptable carrier or excipient.

<u style="single">Definition</u> The term "about" is used herein to mean a value that is ± 10% of the value described.

As used herein, "administering" means a method of giving a dosage of a pharmaceutical composition to a subject. The compositions utilized in the methods described herein are selected from, for example, parenteral, skin, transdermal, eyeball, inhalation, oral, sublingual, transtongue, nasal, rectal, topical, and oral. It can be administered by the route. Parenteral administration includes intraarterial, intravenous, intraperitoneal, subcutaneous, and intramuscular administration. The preferred method of administration may depend on a variety of factors (eg, the components of the composition administered and the severity of the condition being treated).

A "therapeutic amount" ameliorates, inhibits, or ameliorate the symptoms of a subject's condition or disorder in a clinically relevant manner (eg, one or more viruses or viruses). Whether to improve, inhibit, or ameliorate one or more symptoms of infection by a strain, or after infection, or to improve or treat an autoimmune disease or cancer, or one or more of its symptoms , Or the amount of composition administered to ameliorate). Any improvement in the subject is considered sufficient to achieve treatment. Preferably, a therapeutically sufficient amount alleviates or inhibits the development of a viral infection or one or more symptoms (eg, symptoms caused by infection with at least one and preferably two or more viruses or virus strains). Or an amount to prevent, or the severity of one or more infectious symptoms, or the length of time a subject suffers from it (eg, at least 10% compared to a control subject not treated with the compositions of the invention). , 20%, or 30%, more preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, 95%, 99%, or more). Sufficient amounts of the pharmaceutical composition used to carry out the methods described herein (eg, treatment of one or more viral infections) include the method of administration and the age, weight, and subject to be treated. Depends on general health. The doctor or researcher can determine the appropriate amount and medication regimen.

"Host", "subject" or "patient" means any organism such as mammals (eg, primates, dogs, cats, cows, horses, pigs, goats, rats, and mice) or birds; preferably. The organism is a human. The host may also be a domestic animal (eg, an agricultural animal) or a companion animal (eg, a pet).

"Inducing an immune response" means one or more viruses or strains of a virus (eg, two, three, four, or more) or more in a subject to which a pharmaceutical composition (eg, a vaccine) has been administered. It is meant to elicit a humoral response (eg, antibody production) or cellular response (eg, activation of T cells, macrophages, neutrophils, and natural killer cells) to a viral strain.

As used herein, "interferon" or "IFN" is IFN-α (eg, IFN-α-1a; as a whole, incorporated herein by reference in US Patent Application Publication No. 200707274950. ), IFN-α-1b (SEQ ID NOs: 1 and 2), IFN-α-2a (see WO 07/044083, incorporated herein by reference in its entirety) and IFN-α-2b (SEQ ID NOs: 3 and 4)), Consensus IFN-α (SEQ ID NO: 11), IFN-β (eg, described in US Pat. No. 7,238,344, incorporated by reference in its entirety; IFN -β-1a, as described in US Pat. No. 6,962,978; incorporated by reference in its entirety) and IFN-β-1b (US Pat. Nos. 4,588,585; 4,959,314; As described in Specifications 4,737,462; and 4,450,103; as a whole, incorporated by reference; see also SEQ ID NOs: 5 and 6), IFN-γ (see, eg, SEQ ID NOs: 7 and 8). And IFN-τ (as described in US Pat. No. 5,738,845 and US Patent Application Publication No. 20040247565 and 20070243163; as a whole, incorporated by reference; SEQ ID NO: 9 and Substantially identical to all or part of the sequence of interferon (eg, human interferon), such as (see also 10) (eg, at least 70%, 75%, 80%, 85%, 90%, 95%, 96). Refers to a peptide or protein having an amino acid sequence (%, 97%, 98%, 99%, or even 100% identical).

The term "interferon alpha" or "IFN-α" as used herein refers to a family of highly homologous species-specific proteins that inhibit viral replication and cell proliferation and regulate the immune response. .. Typical suitable interferon alphas include, but are not limited to, recombinant interferon alpha-2a, recombinant interferon alpha-2b, recombinant interferon alpha-2c, alpha 2 interferon, and consensus alpha interferon, eg, the book by reference. These include those described in US Pat. No. 4,897,471 and 4,695,623, which are incorporated herein by reference (particularly, Examples 7, 8 or 9 thereof).

A "pharmaceutical composition" is an immune response against at least one virus (eg, at least two, three, four, or more different viruses or virus strains) suitable for administration to a subject. A drug having therapeutic or biological activity (eg, incorporated into a viral vector) capable, or treating an autoimmune disorder or cancer, or reducing or ameliorating one or more symptoms of an autoimmune disorder or cancer. All or one of cytokines (eg, interferons such as IFN-α (eg, consensus IFN-α)) that are or are independent of the viral vector (eg, integrated into liposomes, microparticles, or nanoparticles). Any composition comprising at least one nucleic acid molecule encoding a portion). For the purposes of the present invention, pharmaceutical compositions suitable for delivery of agents having therapeutic or biological activity include, for example, tablets, gel caps, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels. Hydrogels, oral gels, pastes, eye drops, ointments, creams, ointments, swallows, delivery devices, suppositories, enemas, injections, implants, sprays, or aerosols. Any of these formulations can be prepared by a method well known and recognized in the art. For example, Remington: "The Science and Practice of Pharmacy" (21st edition), editors ARGennaro, Lippincott Williams &amp; Wilkins, 2005, and "Encyclopedia of Pharmaceutical Technology", editors J. Swarbrick, Informa Healthcare, 2006 (each each). (Incorporated by reference herein).

A "pharmaceutically acceptable diluent, excipient, carrier, or adjuvant" is a diluent that is physiologically acceptable to a subject while maintaining the therapeutic properties of the pharmaceutical composition administered together. Means an excipient, carrier, or adjuvant. An example of a pharmaceutically acceptable carrier is saline. Other physiologically acceptable diluents, excipients, carriers, or adjuvants and their formulations are known to those of skill in the art.

"Recombinant" in the context of a vector, eg, a viral vector, refers to IFN in a heterologous nucleic acid sequence (eg, a viral genome (eg, a replication-deficient Ad5 genome), eg, using recombinant nucleic acid technology to introduce changes into the vector. Means an in vitro engineered vector (eg, a viral genome integrated into one or more delivery media (eg, plasmid, cosmid, etc.)) to introduce (eg, conIFN-α). Examples of recombinant viral vectors of the invention include all or part of the adenovirus (eg, adenovirus 5 strain (Ad5)) genome and, for example, a cytokine gene sequence such as the interferon-α gene (eg, consensus IFN-α). A vector containing a nucleic acid sequence for all or part of (sequence).

"Room temperature" means a temperature of about 5 ° C to about 30 ° C, especially about 10 ° C to about 27 ° C (eg, about 23 to 27 ° C).

When used in connection with the comparison of a polynucleotide or polypeptide sequence with a reference sequence, the term "substantially identical" or "substantially identical" means that the polynucleotide or polypeptide sequence is a reference sequence. Means having the same sequence as, or having a certain proportion of nucleotide or amino acid residues that are the same at the corresponding positions in the reference sequence when the two sequences are optimally aligned. For example, amino acid sequences that are "substantially identical" to the reference sequence are measured using BLAST or BLAST 2.0 sequence comparison algorithms with default parameters, or by manual alignment and visual inspection (eg, NCBI website). Reference), when compared and aligned for maximum agreement over the entire length of the reference sequence, at least about 60% identity with the reference sequence, preferably 65%, 70%, 75%, 80%, 85%, Have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher percentages of identity (up to 100%).

"Treatment" means administering the pharmaceutical composition of the present invention for prophylactic and / or therapeutic purposes. Prophylactic treatment is, for example, infection by a particular biological condition, eg, a bacterium, virus, fungus, or parasite, although not yet ill (eg, the subject has already been exposed to an infectious agent but is absent). It can be administered to subjects who are symptomatic or whose level of exposure to infectious agents may be unknown), or who are predisposed to or at risk of developing an autoimmune disease or cancer. Therapeutic treatment is, for example, to improve or stabilize the condition of a subject who is already affected by contact with a biological agent (eg, a patient who is already infected with a pathogenic virus), or It can be administered to subjects who already have an autoimmune disease or cancer. Thus, in the claims and embodiments described herein, treatment is administration to a subject for therapeutic or prophylactic purposes. In some cases, treatment can ameliorate the symptoms of the disorder (eg, infection by a pathogen such as a virus, autoimmune disease, and cancer) or the symptoms of the disorder when compared to an equivalent untreated control, or one of the disorders. The progression, severity, or frequency of one or more symptoms is measured, for example, by any standard technique, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%. , 80%, 90%, 95%, or 100% reduction. For example, to measure the symptoms of an infection, for example, a blood test that examines the antibody or antigen itself against the pathogen; a culture of a sample of blood, body fluid, or other material taken from the area of infection; a cerebrospinal fluid test by lumbar puncture. Amplification of nucleic acid material from pathogens by polymerase chain reaction (PCR) technology; Detection of increased temporal lobe swelling by magnetic resonance imaging (MRI)) may be used. Symptoms of a pathogen infection can vary from mild to severe and can depend on which part of the body is affected, the type of pathogen, and the age and general health of the affected person, eg, fever. , Muscle pain, cough, squeeze, nasal discharge, sore throat, headache, cold, diarrhea, vomiting, rash, weakness, dizziness, subcutaneous bleeding, visceral bleeding, or bleeding from body openings such as mouth, eyes, or ears, shock , Nervous system dysfunction, dementia, seizures, renal (kidney) dysfunction, personality changes, cervical stiffness, dehydration, seizures, drowsiness, limb paralysis, confusion, back pain, loss of sensation, bladder and intestinal dysfunction, and coma Or drowsiness that can progress to death. In some cases, treatment can result in inhibition of pathogen infection, treatment of infection, and / or remission of infectious symptoms (eg, hemorrhagic fever). If improvement or lack of one or more infectious symptoms is detected, it indicates successful treatment. Treatment can also be confirmed by the absence of a pathogen (eg, a virus) in the subject being treated, or by the inability to detect its presence.

For the treatment or prevention of autoimmune diseases, for example, decreased autoimmune levels, decreased autoreactive T cell levels, increased target cells (eg β-pancreatic islet cells), and fatigue, depression, susceptibility to cold, weight gain. , Weakness, constipation, insomnia, irritation, weight loss, startled eyes, muscle tremor, rash, painful or swollen joints, sun sensitivity, coordination disorders, and improvement of paralysis. Can be done.

For treatment or alleviation of cancer, a decrease in tumor size or number of cancer cells, a delay or prevention of an increase in tumor size or cancer cell growth, an increase in disease-free survival between the disappearance of a tumor or other cancer and its recurrence, Prevention of the first or subsequent onset of the tumor or other cancer, or reduction of adverse symptoms associated with the tumor or other cancer can be scaled. In the desired embodiment, the percentage of tumor or cancerous cells that survive after treatment is measured using any standard assay (eg, Caspase assay, TUNEL and DNA fragmentation assay, Cell permeability assay, and Anexin V assay). When done, it is at least 20, 40, 60, 80, or 100% lower than the initial number of tumor cells or cancerous cells. Desirably, the reduction in tumor cell number or cancerous cell number induced by administration of the agents of the present invention is at least 2, 5, 10, 20, or 50 times greater than the reduction in nontumor or non-cancerous cell number. There are many. Desirably, the methods of the invention result in a 20, 40, 60, 80, or 100% reduction in tumor size or cancerous cell count when determined using standard methods. Desirably, at least 20, 40, 60, 80, 90, or 95% of the treated subjects will reach complete remission in which all evidence of the tumor or cancer disappears. Desirably, the tumor or cancer does not recur, or recurs after at least 5, 10, 15, or 20 years.

Subjects treated by the methods described herein (eg, subjects infected with or at risk of becoming infected with bacteria, viruses, fungi, or parasites) have been diagnosed by a healthcare professional as having such a condition. Can be a person. Diagnosis can be performed by any suitable means. Subjects with reduced onset of infection may or may not have received such a diagnosis. One of ordinary skill in the art may have subject to standard testing for the subject treated according to the invention, or there is one or more risk factors (eg, exposure to biological agents such as viruses) without testing. Therefore, you will understand that it may be identified as high risk.

A "viral vector" is one or more genes derived from a viral species, such as an adenovirus species (eg, Ad5), capable of transmitting one or more heterologous genes of viral or non-viral sources to a host or subject. Means a composition containing. The nucleic acid material of a viral vector may be encapsulated, for example, in a lipid membrane or by a structural protein (eg, a capsid protein) that may contain one or more viral polypeptides (eg, glycoproteins). A viral vector can be used to infect cells of interest (eg, nasal epithelium), which in turn facilitate the translation of one or more heterologous genes into a protein product (eg, IFN-α) of the viral vector.

Alternatively, administration of a viral vector to a subject infects one or more cells of the subject and then promotes expression of one or more heterologous genes in the viral vector, resulting in pathogens (eg, bacteria, viruses, fungi). , Or parasites), or the immune response that treats infections by pathogens can be stimulated (directly or indirectly).

The term "vaccine", as used herein, is defined as a material used to elicit and immunize an immune response after administration of a vaccine to a subject.

As used herein, the term "virus" is defined as an infectious agent that cannot grow or replicate outside the host cell and infects mammals (eg, humans) or birds.

Other features and advantages of the present invention will become apparent from the detailed description and claims.

<figref num="1">FIG. 1 is a table providing comparative amino acid sequences for human leukocyte interferon subtypes and consensus human leukocyte interferon.</figref><figref num="2">FIG. 2 is a schematic diagram showing the insertion of a nucleic acid molecule encoding consensus interferon alpha (conINF-α) into an adenovirus vector.</figref><figref num="3">FIG. 3 outlines the delivery of the Ad5-conIFN-α construct of the invention to the patient's nasal epithelial cells, the expression of the conIFN-α nucleic acid molecule in the cells, and the release of the IFN polypeptide into the patient's bloodstream. It is a figure.</figref><figref num="4">FIG. 4 is a schematic diagram showing the advantages of the Ad5-conIFN-α construct of the present invention.</figref><figref num="5">FIG. 5 is a table summarizing the results of experiments (in the animal model shown) to treat or prevent viral infections shown using the compositions of the invention.</figref><figref num="6">FIG. 6 is a graph showing the effect of intranasal (IN) Ad5-IFNα treatment on survival outcomes in hamsters attacked with the Punta Toro virus (PTV). Animals in each group were treated with the indicated amounts of Ad5-IFNα or empty vector viral particles once 24 hours prior to IN infusion of PTV. Ribavirin treatment was ip once daily for 6 days starting 4 hours before PTV infection.^*P &lt;0.05,^**P &lt;0.01 (compared to PBS vehicle placebo treated animals).^a&lt;0.001 (compared to EV treated animals).</figref><figref num="7">7A and 7B are graphs showing the effect of IN Ad5-IFNα treatment on survival outcome in mice attacked with WEE virus. The animals in each group are 10 as in the group outlined in Example 9 below.⁷It was treated with PFU Ad5-IFNα and attacked with WEE virus by IN infusion. IFNα B / D was given daily as a positive control group.</figref><figref num="8">8A and 8B are graphs showing the effect of IN Ad5-IFNα treatment on survival outcomes in mice attacked with SARS virus. Figure 8A shows the results of prophyly axis: animals in each group are as in the group outlined in Example 10 below.⁶Figure 8B, treated with PFU Ad5-IFNα and attacked with SARS virus by IN infusion, shows the results of treatment: animals in each group are as outlined in Example 10 below.⁶Or 10⁵It was treated with PFU Ad5-IFNα and attacked with SARS virus by IN infusion. Poly IC / LC was used as the positive control group and saline was used as the negative control.</figref><figref num="9">9A and 9B are graphs showing the effect of IN Ad5-IFNα treatment on survival outcomes in mice attacked with the YF virus. Figure 9A shows the results of the dose range for prophylaxis: Animals were treated with Ad5-IFNα as outlined in Example 11 below and attacked with YF virus by IN infusion. Complete protection was observed at the two highest doses, and a dose-response curve was obtained for the lower doses. Figure 9B shows the results of treatment: animals in each group were 5 × 10 as in the group outlined in Example 11 below.⁷It was treated with PFU Ad5-IFNα and attacked with SARS virus by IN infusion. Complete survival was observed in the -4 hour and + 1 dpi groups, with decreased survival in the other groups in correlation with treatment delays.</figref><figref num="10">10A and 10B are graphs showing the effect of IN Ad5-IFNα treatment on survival outcomes in ZEBOV-attacked mice. Figure 10A shows the results of mouse treatment: animals attacked with 100 LD50 EBOV and after 30 minutes were treated with Ad5-IFNα by either the IM or IN pathway. 10 for both routes of administration⁷Full defense was observed with PFU. Figure 10B shows the results of guinea pig treatment: animals attacked with 100 LD50 EBOV and after 30 minutes were IN treated with Ad5-IFNα. 2 × 10⁸Full defense was observed with PFU.</figref><figref num="11">FIG. 11 is a graph showing the effect of IN Ad5-IFNα treatment on survival outcome in mice attacked with pitindevirus. Animals were treated with Ad5-IFNα as outlined in Example 13 below and attacked with PCV by IN infusion. Complete protection was observed at the highest dose and a dose-response curve was obtained at lower doses.</figref><figref num="12">FIG. 12 is a graph showing the effect of IN Ad5-IFNα treatment in combination with the Ad-EBOV vaccine on the survival outcome of EBOV-attacked mice. Animals were treated with Ad5-IFNα as outlined in Example 14 below and attacked with PCV by IN infusion. Complete protection was observed at the highest dose and a dose-response curve was obtained at the lower dose.</figref>

Specific description of the invention

The present invention prevents diseases or disorders caused by infectious agents (eg, infectious agents such as viruses, bacteria, fungi, and parasites) in a subject (eg, mammals such as humans) (before or after exposure). And the composition and method for treatment. The infectious agent may be naturally occurring, may be formulated for use as a biological agent, or may be adapted for use as a biological agent. The invention also features the use of the compositions of the invention to treat or alleviate one or more symptoms of autoimmune disease and cancer in a subject (eg, a mammal such as a human).

The compositions of the present invention can be used, for example, as widespread prophylaxis or treatment to protect against or treat infections by several different infectious pathogens, particularly viral factors. Of particular note, the compositions of the present invention are preferably exposed for pre-exposure prophylaxis (eg, 1-30 minutes prior to exposure to an infectious agent (eg, 15-30 minutes)), preferably 1, 2 , 3, 4, 5, 6-12, 24-72 hours, or 1-6 weeks or more before exposure (eg, at least 2 weeks), and for post-exposure prophylaxis or treatment (eg, infection) Immediately after exposure to a sex factor, eg, 1-30 minutes after exposure (eg, 15-30 minutes), or 1, 2, 3, 4, 5, 6-12, 24, 48, or 72 hours after exposure. , Or within 1-2 weeks). Accordingly, the compositions of the present invention provide advantages in the prevention or treatment of a subject, for example, in preparation for or thereafter exposure to an infectious agent (eg, a virus in a bioterrorist attack, etc.). These benefits include both long-lasting and rapid defense as needed.

To avoid rapid decay of conventional IFN-α protein-based drugs in vivo, the compositions of the invention are delivery vectors capable of delivering IFN-encoding nucleic acid molecules (eg, adenovirus vectors (eg, adenovirus vectors). For example, a human such as IFN (eg, consensus IFN-α) by using a viral vector such as adenovirus 5 (Ad5) delivery platform) and thereby transfecting or transfecting the delivery vector. It drives continuous in situ production of IFN-α). Production of IFN continues in transduced or transfected cells (eg, over the life of the cell).

For example, a nucleic acid molecule encoding IFN-α is inserted into a replication-deficient Ad5 virus, and then the Ad5-IFN-α vector is delivered to the subject (eg, a mammal such as a human). In one embodiment, delivery of the viral vector is intranasal. Intranasal administration of the compositions of the invention suppresses recognition of the Ad5 vector by the host immune system and thus typically avoids any pre-existing immunity that the subject may exhibit against the delivery vector itself. In addition, intranasal administration eliminates the need for needles, which makes it easier, for example, when a bioterrorist attack requires mass administration to the public or when medical facilities are not readily available. It enables less invasive administration. The compositions of the present invention can also be delivered to the lung system (eg, upper and / or lower respiratory tract) by mouth-to-lung delivery.

The compositions of the present invention also provide the advantages of long-term storage and long shelf life. The compositions of the present invention can be stored at room temperature for a considerable period of time (eg, at least 1 week and up to 1 year or more). Alternatively, the compositions of the present invention can be prepared at temperatures in the range of 30 ° C to 55 ° C (eg, at 45 ° C) for a reasonable period of time (eg, at least 2-3 days, 1-3 weeks, 1-6. Can be stored for months and up to 1 year or more). In one embodiment, the compositions of the invention are in powder form when stored at temperatures in the range 30 ° C to 55 ° C. In yet another embodiment, the compositions of the invention are frozen in either powder or liquid form (eg, at a temperature of at least less than 4 ° C (eg, in the range 0 ° C to 20 ° C)). Can be saved. For example, the composition may be an unstabilized liquid formulation (eg, stabilizers such as trehalose, sorbitol, sucrose, mannitol, glycine, CaCl).₂, Hydroxiectoin, ectoine, phyllone and gelatin can be cryopreserved at all, or with only one or several species).

In one embodiment, the compositions of the invention are stored as stable lyophilized powders. The powder may be used directly (eg, in powder form without any kind of reconstitution) or reconstituted shortly before use (eg, a hydration medium such as saline or water, preferably sterile, Alternatively, it may be administered (using any other pharmaceutically acceptable hydration medium), eg, as an aqueous mist. Reconstitution of the compositions of the invention in powder form is possible where clean water is available, such as in medical facilities or military rear ladders. Alternatively, the powder composition of the present invention can be reconstituted into a gel form. Nasal gels are highly viscous concentrated solutions or suspensions. The advantages of nasal gel are that it reduces post-nasal drip due to high viscosity, reduces the effect on taste due to reduced swallowing, reduces the formulation that flows out of the nose, and stimulates the use of analgesic / slippery excipients. And better absorption due to mucosal targeting.

The compositions of the invention in powder form are provided in kits with vials of sterile hydration medium (eg, water or saline) that can be used for powder reconstruction (eg, formation of liquids or gels). be able to. When water is used as the hydration medium, the composition of the invention is the subject to which the composition is administered under the conditions of the composition in its final form (eg, pH, volumetric molar osmotic concentration, or ion concentration). It can be formulated to contain a reagent (eg, a buffer) that is adjusted to be suitable or tolerable, but it is not essential.

Administration of the compositions of the invention in powder form is likely to occur, for example, in emerging economies, expeditionary military operations, and in situations requiring urgent action. Effective for compositions of the invention that are not formulated to exhibit a long shelf life at room temperature or at higher temperatures (eg, compositions of the invention that exhibit a shelf life of less than one week when stored at room temperature). To extend the period, the composition is preferably stored at a temperature in the range of -20 ° C to about 20 ° C. These compositions are formulated with excipients that do not stabilize the Ad5-IFN delivery medium such that the period of time that can remain unchanged at room temperature unless refrigerated is, for example, 1 week to less than 1 month. May be good.

The compositions of the present invention (eg, Ad5-IFNα construct) have hitherto been representative viruses of the family of important togaviridae, eg, Filoviridae, in animal models of human diseases such as mouse, guinea pig, and hamster models. ) (Eboravirus, Zaire strain), Flaviviridae (yellow fever), Arenaviridae (Pitinde), Bunyaviridae (Punta Toro), Coronaviridae (Coronaviridae) SARS) has been successful in testing against attacks by the Togaviridae (VEEV and WEEV); see Figure 5. The compositions of the present invention have a good therapeutic profile and a good preventive window, and the data show complete protection for 21 days and partial protection at subsequent time points. The compositions of the present invention are fast-acting and impart both therapeutic and prophylactic effects to the recipient within minutes to hours; the effects of the compositions of the present invention are days and even numbers after administration. Effectiveness lasts for months.

<u style="single">The composition of the present invention</u> The compositions of the present invention include a delivery vector containing a nucleic acid molecule encoding a cytokine (eg, IFN such as conIFN-α). The compositions of the invention are of any route of administration (eg, routes of administration described herein, such as by nasal inhalation and / or oral inhalation for delivery to the upper and / or lower respiratory tract). Can be formulated for. The composition may be administered in single or multiple doses to the subject in need before or after exposure to an infectious pathogen, or before the diagnosis of an autoimmune disease or cancer, or after the onset of its symptoms. The compositions of the invention may also further comprise a secondary agent (as a nucleic acid molecule expressed by a cell of interest, or as a polypeptide or drug), or as discussed below, one or more additional agents. It may be administered in combination with a therapeutic regimen (eg, vaccine).

<u style="single">interferon</u> Used for pre-exposure prophylaxis or post-exposure treatment of pathogen infections (eg, viral, bacterial, fungal, or parasite infections), or for the treatment of autoimmune diseases or cancer (or one or more of its symptoms) The composition of the present invention comprises a delivery vector containing a nucleic acid molecule encoding IFN. Nucleic acid molecules include human IFN-α (eg, IFN-α-1a, IFN-α-1b, IFN-α-2a, IFN-α-2b, and consensus IFN-α (conIFN-α); Figure 1), Substantially identical (eg, at least 70%, 75%, 80%) sequence of human IFN-β (eg, IFN-β-1a and IFN-β-1b), human IFN-γ), or IFN-τ , 85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100% identical) interferon, or bioactivity similar to or similar to interferon (eg, human IFN) At least 50%, 60%, 70% of the activity of -α, human IFN-β, human IFN-γ, IFN-τ, or conIFN-α (SEQ ID NOS: 2, 4, 6, 8, 10, and 11 respectively). , 75%, 80%, 85%, 90%, 95%, or 100%). The nucleic acid molecule has the sequence shown in any one of SEQ ID NOs: 1, 3, 5, 7, or 9 corresponding to human IFN-α, human IFN-β, human IFN-γ, or IFN-τ, respectively. Or the nucleic acid molecule may be one of SEQ ID NOs: 1, 3, 5, 7, or 9 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, It may have sequences with 98%, 99%, or even 100% identity.

The biological activity of interferons of the invention includes, for example, virus-plaque reduction assays, inhibition of cell proliferation, regulation of functional cell activity, regulation of cell differentiation, and assays that measure IFN-mediated immunomodification, and IFN responses. When the promoter region of the sex gene is linked to a heterologous reporter gene, such as firefly luciferase or alkaline phosphatase, and transfected into an IFN-sensitive cell line, and thus a stably transfected cell line is exposed to IFN, the dose of IFN Can be confirmed using a reporter gene assay that directly correlates with and increases the expression of the reporter gene product (eg, Balducci et al., Appl. Microbiol. 11: 310-314, 1963; McNeil, J. Immunol. .Methods 46: 121-127, 1981; and Meager et al., J.Immunol.Methods See pp. 261: 21-36, 2002). Other assays for measuring IFN activity include double-stranded RNA (dsRNA) -dependent protein kinase R (PKR), 2'-5'-oligoadenylate synthetase (2'-5'-OAS), and IFN induction. Sex Mx protein, tryptophan degrading enzyme (see, eg, Pfefferkorn, Proc.Natl.Acad.Sci.USA 81: 908-912, 1984), adenosine deaminase (ADAR1), IFN activating gene 20 (ISG20). , P56, ISG15, mGBP2, GBP-1, APOBEC protein, viperine, or other factors (eg, Zhang et al., J. Virol., 81: 11246-11255, 2007). , And US Pat. No. 7,442,527, which is incorporated herein by reference in its entirety).

Interferon alpha (IFN-α), when used herein, for example, as illustrated by Pfeffer et al. (Cancer Res. 58: 2489-2499, 1998), has antiviral activity, cell proliferation and differentiation. Refers to cytokines with multiple biological activities, including regulation and immunomodulation. In one embodiment of the invention, IFN-α may be selected from, for example, IFN-α2a, IFN-α2b, IFN-α2c, and consensus IFN-α (conIFN-α) (see FIG. 4 and eg, reference). (See US Pat. No. 4,695,623, incorporated herein by.). In one embodiment, IFN-α is conIFN-α.

Unlike the compositions of the present invention, recombinant human IFN, in particular rhconIFN-α, which has been final approved and marketed as Infergen® for the treatment of chronic hepatitis C, is made by fermentation of prokaryotes. Therefore, it is not glycosylated. In addition, Infergen® is formulated for injection to patients.

<u style="single">Viral vector</u> In the invention described herein, interferon (eg, IFN-α such as conIFN-α) may be formulated for delivery using a viral vector containing a nucleic acid molecule encoding interferon. For example, adenovirus (eg, Ad2, Ad5, Ad9, Ad15, Ad17, Ad19, Ad20, Ad22, Ad26, Ad27, Ad28, Ad30, or Ad39; see Figure 2 for example), rhabdovirus (eg, bullous). Stomatitis virus), retroviruses (eg, Miller, Curr.Top.Microbiol.Immunol.158: 1-24, 1992; Salmons and Gunzburg, Human Gene Therapy 4: 129-141, 1993; and Miller et al., Methods in Enzymology 217: 581-599, 1994), Adeno-related vectors (Carter, Curr. Opinion) Biotech. 3: 533-539, 1992; and Muzcyzka, Curr.Top.Microbiol.Immunol.158: 97-129, outlined in 1992), Poxvirus, Herpesvirus Vector, and Sindbis Viral Vector Including (eg, Jolly, Cancer Gene Therapy 1: 51-64, 1994; Latchman, Molec.Biotechnol. 2: 179-195, 1994; Johanning et al., Nucl.Acids Res. 23: 1495-1501. , 1995; Berencsi et al., J.Infect.Dis.183: 1171-1179, 2001; Rosenwirth et al., Vaccine 19: 166-1670, 2001; Kittlesen et al., J.Immunol.164: 4204-4121. , 2000; Brown et al., Gene Ther. 7: 16801689, 2000; Kanesa-thasan et al., Vaccine 19: 483-491, 2000; and Sten Drug 60: 249-271, see Viral Vectors Generally Considered in 2000), any suitable viral vector system can be used. A composition comprising such a vector and an acceptable excipient is also a feature of the present invention.

Ad5 is a subtype 5 virus of the Adenoviridae family C. The virus is naturally occurring and usually causes mild upper respiratory tract infections in children. Ad5 can be used as a delivery platform for delivering genetic information and making human interferon in situ. Typically, Ad5 is replicate-deficient (due to specific gene deletions; eg, all or part of the E1 or E3 gene). Ad5 vectorized vaccines have been widely approved for clinical research in the past. Ad5 is widely used as a vector delivery system in clinical trials. As of June 2010, there are 29 ongoing clinical trials using Ad5 vectorized delivery of biopharmacy / drug. Adenovirus 5-based vectors exhibit an excellent safety profile. Compared to conventional vaccines such as live attenuated vaccines, which are vaccines in which the pathogenic virus is partially incapacitated by chemical or thermal treatment prior to injection, the Ad5 vector restores the Ad5 system and causes disease. It has the additional advantage that there is no risk of causing it. In addition, Ad5 is a live vaccine that has been shown to provide rapid immune defense. Ad5-based vectors that deliver cytokine genes to provide protection against biological weapons are described, for example, in US Pat. Nos. 6,565,853 and 6,936,257 (both incorporated herein by reference). Are listed.

Intravenous or intramuscular administration of drugs indicated for bioweapon defensive medicine using the Ad5 system disrupts the vector before the body's immune system recognizes this viral vector and the gene is delivered to the host cell. Therefore, it has not been successful so far. This has recently occurred in Merck's HIV-1 vaccine clinical trial, which was discontinued early because the trial was ineffective (see Robb, Lancet 372, 2008). Intranasal administration of the compositions of the invention (eg, the Ad5 vector encoding IFN) avoids this problem by avoiding immune targeting of the Ad5 vector by the body, as discussed herein.

Viral vectors can be constructed using prior art known to those of skill in the art. For example, a viral vector may contain at least one sequence encoding a heterologous gene (eg, consensus IFN-α), which is a regulatory that induces its expression in cells (eg, epithelial cells such as nasal or lung epithelial cells). It is under the control of the sequence. Suitable amounts for vector-mediated delivery of heterologous genes can be readily determined by one of ordinary skill in the art based on the information provided herein.

For delivery of IFN-α using adenovirus vectors, for example, Ahmed et al. (J. Interferon Cytokine Res. 21: 399408, 2001), Zhang et al. (Proc. Natl. Acad. Sci. USA 93: 4513-4518) P. 1996), Ahmed (Hum. Gene Ther. 10: 77-84, 1999), and Santodonato et al. (Cancer Gene Ther. 8: 63-72, 2001). For delivery of IFN-α using retroviral vectors, see, for example, Tuting et al. (Gene Ther. 4: 1053-1060, 1997) and Mecchia et al. (Gene Ther. 7: 167-179, 2000). Are listed.

In one embodiment, the Ad5 vector comprises an intermediate-early promoter of CMV and a nucleic acid molecule encoding a human interferon alpha consensus sequence under transcriptional regulation of the salvirus 40 (SV40) polyadenylation sequence. In another embodiment, the human Ad5 vector comprises an E1 and E3 deletion that makes it a replication defect. The Ad5-IFN-α vector may be further stabilized with polysaccharide and electrolyte excipients during lyophilization and storage, as described herein. Since adenovirus is vulnerable to heat stress and it is difficult to manage the cold chain in the field, the temperature stability of the composition of the present invention is a great advantage. We have developed a systematic method for stabilizing virus-based vaccines, including adenovirus, based on a novel unique vector approach (eg, Kueltzo et al., J. Pharm. Sci. 92: 1805 ~. 1820, 2003; Fan et al., J.Pharm.Sci.94: 1893-1911, 2005; Ausar et al., Mol.Pharm.2: 491-499, 2005; and Rexroad et al., J.Pharm. See Sci.95: pp. 237-247, 2005). Multiple assays are then used to test for the ability of the virus to stabilize against physical and chemical degradation pathways that result in loss of activity (eg, physicochemical integrity, biological activity, etc.). Identify the form.

Increased expression levels of transfected nucleic acid molecules (eg, conIFN-α sequences) in host cells (eg, epithelial cells such as nasal or lung epithelial cells) are open frames that can function in selected expression hosts. It can be promoted by operably linking a nucleic acid molecule to an expression control sequence. Expression control sequences useful for eukaryotic host cells may be natural or exogenous to the nucleic acid molecule to be expressed and to the delivery vector. Examples of expression control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, enhancers, upstream activation sequences, signal peptide sequences, and transcription termination factors. Expression control sequences include, for example, SV40 (eg, early and late promoters of SV40), bovine papillomavirus, adenovirus (eg, early and late promoters of adenovirus), cytomegalovirus (CMV; eg, early human cytomegalovirus). Gene promoters), MT-1 (metallothioneine gene) promoters, rous sarcoma virus (RSV) promoters, and human ubiquitin C (UbC) promoters. To further enhance expression in mammalian cells, synthetic intron sequences can be inserted into the non-transcriptional region of the nucleotide sequence encoding the IFN-α polypeptide.

Other vector elements that can be used in the practice of the present invention include signal peptides. This sequence is typically located at 5'of the protein-encoding gene and is therefore added to the amino terminus of the protein during expression. The presence or absence of the signal peptide depends on the expression host cell used to produce the IFN-α polypeptide and the preference for producing the secretory product (ie, the expression of the IFN-α polypeptide is intracellular or extracellular). Different (depending on). In one embodiment, IFN-α (eg, conIFN-α) is secreted from the host cell during expression. The signal peptide may be homologous or heterologous to the IFN-α polypeptide or to the host cell.

A nucleic acid molecule is "operably linked" to another nucleic acid molecule when they are functionally aligned. This means that the appropriate molecule (eg, a transcriptional activator) binds to one or more regulatory sequences linked in a manner that regulates the expression of one or more regulatory sequences, genes, or nucleic acid molecules. .. For example, if pre-sequences or secretory leaders contribute to the secretion of mature proteins, they are operably linked to promoters. If a promoter affects the transcription of the coding sequence, the promoter is operably linked to the coding sequence. When the ribosome binding site is in a position that can be read as a coding sequence, the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means in contact with the linked nucleic acid molecule and secretory leader in the reading frame.

<u style="single">Non-viral vector</u> For the treatment or prevention of pathogen infections (eg, viral infections) by introducing therapeutic nucleic acid molecules (eg, nucleic acid molecules encoding IFN-α) into cells, or for the treatment or alleviation of autoimmune disease or cancer symptoms. Non-viral techniques can also be used. For example, interferons such as heterologous genes (eg, IFN-α (eg, consensus IFN-α)), lipofection (eg, Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413, 1987; Ono et al., Neuroscience Letters, p. 17: 259, 1990; Brigham et al., Am.J.Med.Sci. 298: 278, 1989; Staubinger et al., Methods in Enzymology 101: 512, 1983). Mucoid-polylysine conjugate (eg, Wu et al., Journal of Biological Chemistry 263: 14621, 1988; Wu et al., Journal of Biological Chemistry 264: 16985, see 1989), or then preferably by microinjection under surgical conditions (see, eg, Wolff et al., Science 247: 1465, 1990) (see, eg, 1990). , Epithelial cells such as nasal or lung epithelial cells). Gene transfer can also be achieved by the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes, microparticles, or nanoparticles are also beneficial for the delivery of nucleic acid molecules (eg, nucleic acid molecules encoding IFN-α) or proteins to cells or patients to stimulate an immune response against pathogens (eg, viruses). There is a possibility. For other virus-independent IFN-α delivery methods, see, for example, Coleman et al., Hum.Gene Ther. 9: 2223-2230, 1998, and Horton et al., Proc.Natl.Acad.Sci.USA 96: Pp. 1553 to 1558, 1999).

<u style="single">A method for preventing or treating a pathogen infection using the composition of the present invention.</u> The pharmaceutical compositions of the present invention can be used as gene therapy and / or gene vaccines for treating or inhibiting infection by pathogens such as bacteria, viruses, fungi, and parasites. In particular, the compositions of the present invention are viruses (eg, members of the Flaviviridae family (eg, members of the genus Flaviviridae, the genus Pestivirus, and members of the genus Hepacivirus). Hepacivirus C virus, yellow fever virus; Gadgets Gully virus, Kadam virus, Kasanur forest disease virus, Langat virus, Omsk hemorrhagic fever virus, Poissan virus, Royal Farm virus, Tick-borne viruses such as Karshi virus, tick-borne encephalitis virus, Neudorfl virus, Sofjin virus, leprosy virus and Negishi virus; Meaban virus, Saumarez Seabird tick-borne viruses such as Reef virus and Tyuleniy virus; Aroa virus, Deng virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray valley encephalitis Virus, St. Louis encephalitis virus, Usutu virus, Westnile virus, Yaounde virus, Kokobera virus, Bagaza virus, Ilheus virus, Israel turkey meningoencephalomyelitis virus, Untaya Virus, Tempus virus, Jika virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus, Vessel Mosquito-borne viruses such as Subron virus, yellow fever virus; and Entebbe bat virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukarasa bat virus, Carey Island virus, Dakar bat Virus, Montana myitis White encephalitis virus, Phnom Penh) bat virus, Rio Bravo virus, Tamana bat virus, and viruses without known mediator phalanges such as cell fusion factor virus); members of the Arenaviridae family (this includes) Ippy virus, Lassa virus (eg, Josiah strain, LP strain, or GA391 strain), lymphocytic choriomyelitis virus (LCMV), Mobala virus, Mopeia virus, flax Paris virus, Flexal virus, Guanarit virus, Funin virus, Latino virus, Machupo virus, Oliveros virus, Parana virus, Pitindevirus, Pirital virus, Savia Virus, Takaribe virus, Tamiami virus, Whitewater Arroyo (Whitewater) Includes Arroyo virus, Chaparet virus, and Lujo virus); Members of the Bunyaviridae family (eg, Hantavirus, Nairovirus, Orthobunyavirus) ), And members of the genus Phlebovirus) (including Huntan virus, Shinnonbre virus, Dugbe virus, Bunyanbella virus, Lift Valley fever virus, Lacros virus, Punta Toro) virus (PTV), California encephalitis virus, and Crimea-Congo hemorrhagic fever (CCHF) virus); members of the Filoviridae family (including Ebola virus (eg, Zaire strain, Sudan strain, Cote d'Ivoire) Strains, Reston Strains, and Uganda Strains) and Marburg Viruses (including, for example, Angola Strains, Ci67 Strains, Musoke Strains, Popp Strains, Ravn Strains, and Lake Victoria Strains); Togavirus Members of the family (Togaviridae) (eg, members of the genus Alphavirus) (for example, Venezuelan lauma encephalitis virus (VEE), Eastern horse encephalitis virus (EEE), Western horse encephalitis virus (WEE), Sindbis virus, Includes ruin virus, semuliki forest virus, loss river virus, verma forest virus, onyonnyon virus, and chikungunya virus); members of the Poxviridae family (eg, members of the Orthopoxvirus genus) ( This includes natural poxvirus, monkey poxvirus, and vaccinia virus); members of the Herpesviridae family (which include simple herpesviruses (HSV; types 1, 2, and 6), human herpes. Viruses (eg, types 7 and 8), cytomegalovirus (CMV), Epstein bar virus (EBV), varicella herpes virus, and Kaposi sarcoma-related herpesvirus (KSHV)); Members of Orthomyxoviridae) (including influenza viruses such as H5N1 triinfluenza virus or H1N1 porcine influenza (types A, B, and C)); members of the Coronaviridae family (which are severely acute) Respiratory Syndrome (SARS) virus included);Members of the Rhabdoviridae family (which includes mad dog disease virus and bullous stomatitis virus (VSV)); Paramyxoviridae members (which include human respiratory astrovirus (RSV)), Newcastle disease virus, Hendra virus, Nipavirus, measles virus, bovine epidemic virus, canine temper virus, Sendai virus, human paramyxovirus (eg, types 1, 2, 3, and 4), rhinovirus, and mumps virus. Includes); Members of the Picornaviridae family (including poliovirus, human astroviruses (types A, B, C, and D), hepatitis A virus, and coxsackie virus); Members of the Hepadnaviridae family (which includes hepatitis B virus); Papillamoviridae members (which include the human papillomavirus); Parvoviridae members (which include the human papillomavirus) This includes adeno-related viruses); members of the Astroviridae family (which includes Astroviridae); members of the Polyomaviridae family (which includes JC virus, BK virus, and SV40 virus included); members of the Calciviridae family (which includes nowalk virus); members of the Reoviridae family (which includes rotavirus); and retrovirus family Members of (Retroviridae), including human immunodeficiency viruses (HIV; eg, types 1 and 2), and human T lymphocyte tropic viruses types I and II (HTLV-1 and HTLV-2, respectively). Can be used to treat (pre-exposure or post-exposure) infection by).For example, for the treatment (pre-exposure or post-exposure) of infections with human T-lymphotropic viruses types I and II (including HTLV-1 and HTLV-2, respectively))). Can be used.

The pharmaceutical composition comprises a vector encoding an IFN (eg, IFN-α such as conIFN-α), which can be administered in vivo or ex vivo.

IFN-α is one of the earliest cytokines released from antigen-presenting cells as part of the innate immune response and is directly involved in the responsiveness of NK and T cells that drive subsequent immune responses. NK cells are one of the first cells to appear in the early antiviral immune response, whose sole function is killing. In addition, IFN-α appears to be the major cytokine that mediates the proliferation of CD8 + T cells. Since IFN-α responds early in the immune cascade, its main role has been suggested to induce a priming state during the initial response to infection, with low-dose IFN-α improving defense against viral attack. It has been shown to bring about (see, eg, Brassard et al., J. Leuk. Biol. 71: 565-581, 2002).

In addition, interferon induces the expression of MX protein, which is a 7-80 kDa protein with GTPase activity that affects viral replication by interfering with the transcription of influenza and other negative RNA viruses (ie). , MX protein inhibits viral RNA polymerase) (Acheson, "Fundamentals of Molecular Virology" J. Wiley and Sons, Hoboken NJ, 2007).

Interferon also induces the expression of ribonuclease L, which degrades viral (and host) mRNA and thus results in inhibition of viral replication by suppressing viral protein synthesis (Acheson, 2007). Thus, expression of IFN-α in a subject's transduced / transfected cells (eg, epithelial cells), in part, stimulates the subject's immune response and is subject to pathogen (eg, viral) infection. By activating these and other pathways that protect before and after exposure, it provides prevention and / or treatment of pathogen infections.

The pharmaceutical compositions of the present invention act through a two-step process, namely administration and expression. For example, after intranasal administration, the Ad5 virus invades epithelial cells of the upper and / or lower respiratory tract and carries IFN-α nucleic acid molecules to the nucleus. The IFN-α nucleic acid molecule is then transcribed, the resulting mRNA is translated, post-translated and modified by glycosylation, and expressed on the cell surface as mature IFN-α cytokines. The adenovirus itself is replication-deficient and therefore does not replicate. When expressed on the cell surface, IFN-α functions in the same manner as naturally in situ produced IFN-α.

Thus, by administering the vector to a subject (eg, by intranasal or transpulmonary administration) in the dosage and form discussed herein (eg, as aerosol powder, liquid mist, or gel), in vivo. Vectors can be used for transduction or transfection of cells of interest in (eg, epithelial cells such as nasal or lung epithelial cells) to provide prevention and / or treatment of pathogenic infections. Alternatively, cells may be removed from the subject and the IFN-encoding vector may be transduced or transfected with exovivo, or the cells may be returned to the subject to provide prevention and / or treatment of pathogen infection. In one embodiment, the cells of interest are removed and treated with Exobibo with the Ad5-IFN-α vector of the invention. The cells are then administered to the patient before or after exposure to treat or inhibit pathogen infection. Preferably at least about 1x10⁴~ About 10 × 10⁶Individual cells are treated and reintroduced into the subject.

In one embodiment, when a sufficient amount of the pharmaceutical composition is administered to the subject, at least about 0.0001 to 5.0 × 10⁵IU / ml, preferably about 0.0002 ~ 2.0 × 10⁵IU / ml, and most preferably about 0.0005 ~ 1.0 × 10⁵Expressed from IU / ml transfected / transfected cells, maximum blood levels of IFN-α are achieved (eg, NIBSC codes: 94/784 and 94/786; WHO International Standard for INTERFERON ALPHA (human). Leukocyte origin); dated February 14, 2008; Meager et al., J. Immunol.Methods 257: 17-33, 2001; and Mire-Sluis et al., J. Interferon Cytokine See Res. 16: 637-643, 1996). In another embodiment, the circulating IFN-α amount is from about 100 IU / ml to 1,000 IU / ml (eg, about 250 IU / ml). Preferably, the circulating concentration of IFN-α is maintained within this range for at least 1-15 days, or at least 1, 2, 3, or 4 weeks, or at least 2-6 months. The expression level of IFN-α can be determined, for example, by measuring the amount of IFN-α in the serum of the subject (eg, Forti et al., J. Clin. Microbiol. 21: 689-693, 1985. checking). In other embodiments, the antiviral effect of IFN-α is at least 1, 2, 3, or 4 weeks, more preferably at least 2, 4, or 6 months, and most preferably 1 year or more. It remains manifest in the subject over time. The antiviral effects of IFN-α are double-stranded RNA (dsRNA) -dependent protein kinase R (PKR), 2'-5'-oligoadenylate synthetase (2'-5'-OAS), IFN-induced Mx protein. , Tryptophan-degrading enzymes (see, eg, Pfefferkorn, Proc.Natl.Acad.Sci.USA 81: 908-912, 1984), adenosine deaminase (ADAR1), IFN activation gene 20 (ISG20), p56, It can be determined by measuring the upregulation or activity of ISG15, mGBP2, GBP-1, APOBEC protein, viperine, or other factors (eg, Zhang et al., J. Virol., 81: 11246-11255, See 2007). Assays for measuring the antiviral effect of IFN-α can be found, for example, in US Pat. No. 7,442,527, which is incorporated herein by reference in its entirety.

When a pharmaceutical composition containing an IFN-α delivery vector (eg, Ad5 delivery vector) is administered to, for example, nasal or lung epithelial cells, the nucleic acid molecule encoding IFN-α is integrated into the cell. These cells then produce IFN-α until death or apoptosis over the course of their lifespan, and thus months, days, compared to hours of exogenously administered rhIFN-α. Alternatively, human IFN-α can be expressed that lasts for several weeks or longer (eg, about 1 to 15 days, 1 to 4 weeks, or 2 to 6 months). Moreover, for example, the IFN produced from the Ad5-hIFN vector is complete, unlike rhIFN-α (ie, Infergen® (Alfacon; DIN 2239832)), which is currently commercially prepared by eukaryotic fermentation. Is glycosylated to. In addition, the therapeutic effect of IFN-α (eg, antiviral effect) is at least 1, 2, 3, or 4 weeks, more preferably at least 2, 4, or 6 months, and most preferably 1 year or more. And get.

The naturally occurring IFN-α is glycosylated. Most rhIFN preparations are made by prokaryotic fermentation and are not glycosylated. Due to the location of the glycosylation site, there is no risk of interfering with receptor binding with the addition of glycosylation. However, glycosylated IFN-α and non-glycosylated IFN-α can have different pharmacokinetics, and as with human granulocyte-macrophage colony-stimulating factor, glycosylation affects protein stability. Acceptable (GM-CSF; Adolf et al. (See Biochem. J. 276: 511-518, 1991). In addition, the immunogenicity of rhIFN-α can be affected by the lack of glycosylation. Gribben et al. Reported that antibodies to this protein were produced in 4 of 16 patients who received rhGM-CSF produced in yeast; these antibodies were exposed to recombinant factors. Reacted with an epitope that was but should have been protected by glycosylation (Gribben et al., Lancet) See 335: 434-437, 1990). The induction of antibodies against non-glycosylated rhIFN-α after long-term treatment in patients has been described and it is speculated that native IFN-α may be less immunogenic than recombinant proteins (Figlin). And Itri, Semin. Hematol. 25: 9-15, 1988, and Galton et al., Lancet 2: 572-573, 1989).

Although there is evidence that glycosylation using all forms of IFN (eg, α, β, ω, γ) does not affect a particular antiviral / biological activity of the protein (Bocci, Trends Biochem Sci 8). : 432-434, 1983, and Adolf et al., Biochem J. 276: pp. 511-518, 1991), it seems that glycosylation of IFN may be important for other reasons. A study dedicated to various translational methods for producing fully glycosylated hIFNA in exobibo (see, eg, Rossmann et al., Prot. Exp. Purif. 7: 335-342, 1996), And patents filed in protection of those methods (see, eg, U.S. Pat. Nos. 7,445,774; 7,338,654; 7,311,903; and 7,129,390). is there. Therefore, glycosylation is clearly a desirable component in IFN. The pharmaceutical compositions of the invention that deliver a vector that promotes insitu expression of fully glycosylated hIFN have the same level of therapeutic activity (eg, anti-glycosylation) as currently administered rhIFN polypeptides lacking glycosylation. It is likely to result in a protein that is more stable and has a lower immunogenic effect while maintaining (viral activity).

Expression of IFN-α (eg, conIFN-α) in cells of a subject transfected / transduced with the delivery vector of the invention provides the subject with fast-acting protection against pathogen infections (eg, viral infections). The IFN-α delivery vector of the present invention is fast-acting because the Ad5 vector integrates into epithelial cells (eg, nasal or lung epithelial cells) and reaches the nucleus from the cell surface within 30 minutes. The IFN-α delivery vector of the present invention, for example, when administered intranasally, has a large surface area (100-200 square cm), so that the Ad5 delivery vector has millions of upper and / or lower respiratory tract epithelial cells. It is especially effective because it can invade the nasal cavity. After integration, epithelial cells begin to produce IFN-α (eg conIFN-α) as if it were endogenous to the cell; IFN-α is expressed on the cell surface and secreted into the host circulation. To.

Expression of IFN-α typically occurs within 24 hours or less (eg, as early as 3 hours) after administration of the delivery vector. This result is particularly apparent when a rapid therapeutic response is preferred (eg, in a public outbreak of virus or in a situation where a pathogen is intentionally released (eg, against military personnel deploying on the front line)). It is beneficial. The IFN-α delivery vectors of the invention are rapid in response to situations where public sector medical personnel, as well as military planners and others, are threatened with various operations that may have uncertainties regarding the presence of infectious agents. Provide the ability to act on. For example, today's military planners would not deploy to areas with endemic pathogen risk without proper vaccination. This significantly reduces the ability of the military, law enforcement agencies, or regional emergency coordinators (LECs) to respond quickly to global threats. The pharmaceutical compositions of the present invention can be used to mitigate these risks and reduce the time to response to pathogen exposure or outbreaks.

The compositions of the present invention may be administered in single or multiple doses separately or simultaneously with other therapies for pathogen infections (eg, vaccines) or as monotherapy. The compositions of the present invention may also contain additional therapeutic agents, although not essential. These additional therapeutic agents are encoded as nucleic acid molecules in the same or different delivery vectors (eg, viral vectors) and can also be expressed as polypeptides with IFN, or as polypeptides or drugs with the compositions of the invention. , For example, can be administered as a single pharmaceutical composition or in separate pharmaceutical compositions.

The compositions of the invention are administered to a subject (eg, a human) before or after exposure to a pathogen infection (eg, a viral infection) to treat, prevent, or ameliorate one or more symptoms of the pathogen infection in the subject. It can inhibit its progression or reduce its severity. Examples of symptoms of pathogenic infections, especially viral infections, that can be treated using the compositions of the invention include fever, muscle pain, cough, squeeze, nasal discharge, sore throat, headache, cold, diarrhea, vomiting. , Rash, weakness, dizziness, subcutaneous bleeding, visceral bleeding, or bleeding from body openings such as mouth, eyes, or ears, shock, nervous system dysfunction, dementia , seizures, renal (kidney) dysfunction, personality changes, Neck stiffness, dehydration, seizures, drowsiness, limb paralysis, confusion, back pain, loss of sensation, bladder and intestinal dysfunction, and drowsiness that can progress to coma or death. These symptoms, and their resolution during treatment, can be measured, for example, by a physician during a physical examination or by other examinations and methods known in the art.

The dose of the composition of the invention (eg, a delivery vector encoding an IFN, a virus or other number) or the number of treatments using the composition of the invention will determine the severity, occurrence, or progression of the pathogen infection in the patient. It can be increased or decreased based on (eg, based on, for example, the severity of one or more symptoms of viral infection).

<u style="single">use</u> IFN is known to be effective against a wide range of pathogens, especially viruses. Therefore, the pharmaceutical composition of the present invention is referred to as a "widespread antiviral drug". Viruses for which the compositions of the present invention can be used include: members of the Flaviviridae family (eg, Flaviviridae, Pestivirus, and Hepacivirus). (Members of) (This includes hepacivirus, flaviviridae; Gadgets Gully virus, Kadam virus, Kasanur forest disease virus, flaviviridae, omsk hemorrhagic fever virus, poissan virus, royal Tick-borne viruses such as Royal Farm virus, Karshi virus, tick-borne encephalitis virus, Neudoerfl virus, Sofjin virus, leaping disease virus and Negisi virus; Meaban virus, Somarezu Reef (Saumarez Seabird tick-borne viruses such as Reef virus and Tyuleniy virus; Aroa virus, Deng virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray valley encephalitis Virus, St. Louis encephalitis virus, Usutu virus, Westnile virus, Yaounde virus, Kokobera virus, Bagaza virus, Ilheus virus, Israel turkey meningoencephalomyelitis virus, Untaya Virus, Tempus virus, Jika virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus, Vessel Mosquito-borne viruses such as Subron virus, yellow fever virus; and Entebbe bat virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukarasa bat virus, Carey Island virus, Dakar bat Virus, Montana myitis White encephalitis virus, Phnom Penh) bat virus, Rio Bravo virus, Tamana bat virus, and viruses without known mediator phalanges such as cell fusion factor virus); members of the Arenaviridae family (this includes) Ippy virus, Lassa virus (eg, Josiah strain, LP strain, or GA391 strain), lymphocytic choriomyelitis virus (LCMV), Mobala virus, Mopeia virus, flax Paris virus, Flexal virus, Guanarit virus, Funin virus, Latino virus, Machupo virus, Oliveros virus, Parana virus, Pitindevirus, Pirital virus, Savia Virus, Takaribe virus, Tamiami virus, Whitewater Arroyo (Whitewater) Includes Arroyo virus, Chaparet virus, and Lujo virus); Members of the Bunyaviridae family (eg, Hantavirus, Nairovirus, Orthobunyavirus) ), And members of the genus Phlebovirus) (including Huntan virus, Shinnonbre virus, Dugbe virus, Bunyanbella virus, Lift Valley fever virus, Lacros virus, Punta Toro) virus (PTV), California encephalitis virus, and Crimea-Congo hemorrhagic fever (CCHF) virus); members of the Filoviridae family (including Ebola virus (eg, Zaire strain, Sudan strain, Cote d'Ivoire) Strains, Reston Strains, and Uganda Strains) and Marburg Viruses (including, for example, Angora Strains, Ci67 Strains, Musoke Strains, Popp Strains, Ravn Strains, and Lake Victoria Strains); Togavirus Members of the family (Togaviridae) (eg, members of the genus Alphavirus) (including Venezuelan lauma encephalitis virus (VEE), Eastern horse encephalitis virus (EEE), Western horse encephalitis virus (WEE), Sindbis virus, Includes ruin virus, semuliki forest virus, loss river virus, verma forest virus, onyonnyon virus, and chikungunya virus); members of the Poxviridae family (eg, members of the Orthopoxvirus genus) ( This includes natural poxvirus, monkey poxvirus, and vaccinia virus); members of the Herpesviridae family (which include simple herpesviruses (HSV; types 1, 2, and 6), human herpes. Viruses (eg, types 7 and 8), cytomegalovirus (CMV), Epstein bar virus (EBV), varicella herpes virus, and Kaposi sarcoma-related herpesvirus (KSHV)); Members of Orthomyxoviridae) (including influenza viruses such as H5N1 triinfluenza virus or H1N1 porcine influenza (types A, B, and C)); members of the Coronaviridae family (which are severely acute) Respiratory Syndrome (SARS) virus included);Members of the Rhabdoviridae family (which includes mad dog disease virus and bullous stomatitis virus (VSV)); Paramyxoviridae members (which include human respiratory astrovirus (RSV)), Newcastle disease virus, Hendra virus, Nipavirus, measles virus, bovine epidemic virus, canine temper virus, Sendai virus, human paramyxovirus (eg, types 1, 2, 3, and 4), rhinovirus, and mumps virus. Includes); Members of the Picornaviridae family (including poliovirus, human astroviruses (types A, B, C, and D), hepatitis A virus, and coxsackie virus); Members of the Hepadnaviridae family (which includes hepatitis B virus); Papillamoviridae members (which include human papillomavirus); Parvoviridae members (which include human papillomavirus) This includes adeno-related viruses); members of the Astroviridae family (which includes Astroviridae); members of the Polyomaviridae family (which includes JC virus, BK virus, and SV40 virus included); members of the Calciviridae family (which includes nowalk virus); members of the Reoviridae family (which includes rotavirus); and retrovirus family Members of (Retroviridae), including human immunodeficiency viruses (HIV; eg, types 1 and 2), and human T lymphocyte tropic viruses types I and II (HTLV-1 and HTLV-2, respectively). ).Members of the Paramyxoviridae family (including human respiratory astrovirus (RSV), Newcastle disease virus, Hendra virus, nipavirus, measles virus, bovine epidemic virus, inudistemper virus, Sendai virus, human paramyxoviridae virus) (For example, types 1, 2, 3, and 4), rhinovirus, and mumpsvirus); members of the Picornaviridae family (including poliovirus, human enterovirus (type A, type A,) B, C, and D), hepatitis A virus, and coxsackie virus); members of the Hepadnaviridae family (which includes hepatitis B virus); Papillamoviridae ) Members (which include human papillomavirus); Parvoviridae members (which include adeno-related viruses); Astroviridae members (which include astroviruses) Members of the Polyomaviridae family (which includes JC virus, BK virus, and SV40 virus); Members of the Calciviridae family (which includes Norwalk virus); Members of the Reoviridae family (which includes rotavirus); and members of the Retroviridae family (which include human immunodeficiency viruses (HIV; eg, types 1 and 2), and humans. T lymphocyte-tropic viruses type I and type II (including HTLV-1 and HTLV-2, respectively).Members of the Paramyxoviridae family (including human respiratory astrovirus (RSV), Newcastle disease virus, Hendra virus, nipavirus, measles virus, bovine epidemic virus, inudistemper virus, Sendai virus, human paramyxoviridae virus) (For example, types 1, 2, 3, and 4), rhinovirus, and mumpsvirus); members of the Picornaviridae family (including poliovirus, human enterovirus (type A, type A,) B, C, and D), hepatitis A virus, and coxsackie virus); members of the Hepadnaviridae family (which includes hepatitis B virus); Papillamoviridae ) Members (which include human papillomavirus); Parvoviridae members (which include adeno-related viruses); Astroviridae members (which include astroviruses) Members of the Polyomaviridae family (which includes JC virus, BK virus, and SV40 virus); Members of the Calciviridae family (which includes Norwalk virus); Members of the Reoviridae family (which includes rotavirus); and members of the Retroviridae family (which include human immunodeficiency viruses (HIV; eg, types 1 and 2), and humans. T lymphocyte-tropic viruses type I and type II (including HTLV-1 and HTLV-2, respectively).For example, types 1 and 2), and human T-lymphotropic viruses types I and II (including HTLV-1 and HTLV-2, respectively).For example, types 1 and 2), and human T-lymphotropic viruses types I and II (including HTLV-1 and HTLV-2, respectively).For example, types 1 and 2), and human T-lymphotropic viruses types I and II (including HTLV-1 and HTLV-2, respectively).For example, types 1 and 2), and human T-lymphotropic viruses types I and II (including HTLV-1 and HTLV-2, respectively).Members of the Papillamoviridae family (which includes human polyomaviridae); Parvoviridae members (which include adeno-associated viruses); Astroviridae members (which include) Includes Astrovirus); Members of the Polyomaviridae family (including JC virus, BK virus, and SV40 virus); Members of the Calciviridae family (This includes nowalk viruses) Members of the Reoviridae family (which includes Rotavirus); and members of the Retroviridae family (which include human immunodeficiency viruses (HIV; eg, types 1 and 2)). Type), and human T lymphocytes type I and type II (including HTLV-1 and HTLV-2, respectively).Members of the Papillamoviridae family (which includes human papillomavirus); Parvoviridae members (which include adeno-associated viruses); Astroviridae members (which include) Includes Astrovirus); Members of the Polyomaviridae family (including JC virus, BK virus, and SV40 virus); Members of the Calciviridae family (This includes nowalk viruses) Members of the Reoviridae family (which includes Rotavirus); and members of the Retroviridae family (which include human immunodeficiency viruses (HIV; eg, types 1 and 2)). Type), and human T lymphocytes type I and type II (including HTLV-1 and HTLV-2, respectively).

Intended for the pharmaceutical composition of the present invention and in collaboration with the Division of Microbiology and Infectious Disease (DMID), a division of the National Institute of Allergy and Infectious Disease (NIAID). Specific indications that are currently being evaluated or have been evaluated include dengue and Punta. Toro) (BSL-2 surrogate for Rift Valley fever), monkeypox, influenza A (H5N1 and H1N1), SARS, yellow fever, pitinde (BSL-2 surrogate for Lassa fever), western horse encephalitis, Venezuelan encephalitis, and West Nile virus can be mentioned. From a broader perspective, the IFN-α delivery vector and pharmaceutical compositions containing it are effective against at least the following viridae: Alphaviridae, Filoviridae, Flaviviridae ), Orthomyxoviridae, Bunyaviridae, Arenaviridae, Herpesviridae, Hepadnaviridae, Coronaviridae, and Poxviridae. Poxviridae) (see Examples).

A significant proportion of the human population has been exposed to many adenovirus strains, including Ad5. Therefore, the immune system of any potential recipient of the pharmaceutical composition of the present invention is likely to have "meeted" Ad5 in the past and can be rapidly equipped with an immune response to it. Seem. This was the MRK Ad5 HIV-1 gag / pol / nef HIV vaccine tested in HIV-negative patients in a 2008 Phase II clinical trial. The results of this study, in which injections were used, were "ineffective", meaning no protection was found: the level of infection in the inoculated subjects was the same as in the non-inoculated subjects (Buchbinder et al., Lancet 372: 1881-1893, 2008). Ad5-positive serum status was significantly associated with acquisition (Robb, Lancet 372: 18571858, 2008), and vaccine design is at the "heart of research failure" (White, Lancet). 373: 805, 2009). Therefore, the Ad5 vectorized vaccine was considered useless because immunity is likely to already exist. In fact, all military personnel are actively vaccinated with Ad4 and Ad7 vaccines during the medical preparation of basic training after enlistment.

To avoid existing immunity to the delivery vector, the IFN-α delivery vector of the invention, and pharmaceutical compositions containing it, are administered, for example, by a transpulmonary or intranasal route that avoids problems with existing immunity to the delivery vector. can do. This is due to the lack of contact between the vector (eg, adenovirus vector (eg, Ad5)) and the immune system (eg, immune components in the blood) because the vector integrates directly into, for example, epithelial cells after administration. It is thought that. Functionally, these epithelial cells act as a barrier to cells and antibodies of the immune system. Therefore, the delivery vector is not exposed to the circulation; only IFN is released into the bloodstream, leaving no trace of the vector (see Figure 3).

<u style="single">A method for preventing or treating an autoimmune disease or cancer using the composition of the present invention.</u> The pharmaceutical compositions of the present invention can also be used as gene therapy and / or gene vaccines for treating or alleviating one or more symptoms of autoimmune diseases and cancer. The mechanism of action of the compositions of the invention described above applies equally to their use in this context.

Interferon exhibits both antiviral and antiproliferative activity. IFN-α is currently approved in the United States and other countries for the treatment of hairy cell leukemia, genital warts, Kaposi's sarcoma, and chronic non-A and non-B hepatitis. Two IFN-αs are licensed for therapeutic use: ROFERON -A Trademarked Interferon Alpha-2a and INTRON A Trademarked Interferon Alpha- 2b. The amino acid sequences of ROFERON -A and INTRON A differ at a single position, but are otherwise identical to the amino acid sequences of alpha interferon subtype 2 (subtype A).

In addition to the indications indicated on the label, IFN-α alone or in combination with chemotherapeutic agents can cause chronic myelogenous leukemia, multiple myeloma, superficial bladder cancer, skin cancer (basal cell carcinoma and malignancy). It has been used or evaluated in various other cell proliferation disorders including melanoma), renal cell carcinoma, ovarian cancer, low-grade lymphocytic lymphoma and cutaneous T-cell lymphoma, and glioma. IFN-α may be effective in combination with other chemotherapeutic agents for the treatment of solid tumors resulting from lung cancer, colorectal cancer and breast cancer (Rosenberg et al., "Principles and Applications of Biologic Therapy", "Cancer: Principles and Practices" Of Oncology, 3rd Edition, Devita et al., pp. 301-547 (1989), Balmer DICP, Ann Pharmacother 24, pp. 761-768 (1990)).

BETA SERON (Schering Corp's recombinant interferon beta-1b) was the first drug specifically indicated for the treatment of MS. In primary clinical trials, BETASERON was found to be effective in reducing the number and severity of exacerbations or recurrences in MS patients, as well as reducing evidence of intracerebral MS activity by magnetic resonance imaging (MRI). It was. Importantly, the results of this study were only for the relapsing-remitting patient group because the study did not include other forms of MS. In addition, the study has not demonstrated a beneficial effect of the drug on the ultimate disability of MS in the 2-3 years of the study, and the efficacy of the drug is significantly impaired by its side effects. U.S. Pat. Nos. 7,105,154; 5,372,808; 5,846,526; 6,204,022; 6,060,450; and 6,361,769 are also autoimmune diseases and It describes the use of IFN therapy to treat cancer; each of these documents is incorporated herein by reference). U.S. Pat. No. 7,442,380 describes the treatment of autoimmune diseases caused by viral infections with interferon.

Therefore, by administering the composition of the present invention (eg, Ad5-IFNα) to a subject (eg, human), an autoimmune disease (eg, multiple sclerosis, type I diabetes, lupus, Addison's disease, myasthenia gravis) in the subject is administered. Disease and amyotrophic lateral sclerosis) or one or more symptoms of cancer can be treated or alleviated. Examples of symptoms of autoimmune disease that can be treated or alleviated using the compositions of the invention include elevated autoimmune levels, elevated autoreactive T cell levels, target cells (eg β pancreatic islet cells). ) Decrease, fatigue, depression, sensitivity to cold, weight gain, muscle weakness, constipation, insomnia, irritation, weight loss, startled eyes, muscle tremor, rash, painful or swollen joints, sunlight Sensitivity, impaired coordination, and paralysis. These symptoms, and their resolution during treatment, can be measured, for example, by a physician in a physical examination or by other examination or method known in the art.

The dose of the composition of the invention (eg, delivery vector encoding IFN, viral or other number) or the number of treatments using the composition of the invention determines the severity, occurrence, or progression of the disease or condition in the patient. Can be increased or decreased based on.

<u style="single">Additional treatment regimen</u> If desired, the subject may also receive additional treatment regimens. For example, additional therapeutic agents may be mixed with the pharmaceutical compositions described herein in a single formulation at a concentration known to be effective for such therapeutic agents. Additional therapeutic agents may also be delivered separately. Different routes of administration may be used if the agents are present in separate pharmaceutical compositions. Particularly useful therapeutic agents include, for example, antiviral agents, immunostimulators, and other immunized vaccines. When treating cancer with the compositions of the present invention, particularly useful additional therapeutic agents include, for example, camptothecin, homocamptothecin, corhitin, thiocorhitin, combretastatin, drastatin, doxorubicin, methotrexate, podophylrotoxin, lysoxin. , Rezoxin D, Taxol, Paclitaxel, CC1065, and chemotherapeutic agents such as mitanthinoids.

In some cases, the pharmaceutical composition and additional therapeutic agents may be at least 1 hour, 2 hours, 4 hours, 6 hours, 10 hours, 12 hours, 18 hours, 24 hours, 3 days, 7 days, 14 days. Or, it is administered one month later. The dosage and frequency of administration of each component can be controlled independently. The additional therapeutic agents described herein may be mixed, for example, in conventional pharmaceutically acceptable carriers with additional active or inert ingredients. The pharmaceutical carrier may be a non-toxic substance with any compatibility suitable for administration of the compositions of the invention to a subject. Pharmaceutically acceptable carriers include, for example, water, saline, buffers and other compounds described, for example, in the Merck Index, Merck &amp; Co., Rahway, New Jersey. Sustained release formulations or devices can also be used for continuous administration. Additional treatment regimens may include other therapies, including lifestyle changes for the subject being treated.

<u style="single">Antiviral agent</u> As an additional therapeutic agent, antiviral agents may be used in combination with the vaccine or in separate doses. Exemplary antiviral agents are abacavir, aciclovir, acyclovir, adehovir, amantazine, amprenavir, albidol, atazanavir, atripla, bribdin, sidohovir, combivir, darunavir, delavirdine, didanosin, docosanol, edoxin, Efavirenz, emtricitabine, enfuvirtide, entecavir, invasion inhibitors, famcyclovir, fixed dose combination), homivirsen, phosamprenavir, foscalnet, phosphonet, fusion inhibitor, gancyclovir, gadacil, ivacitabine, immunovir, idoxuridine, imikimod, indinavir, inosin, integrase inhibitor, type III interferon , Type II interferon, type I interferon, interferon, lamivudine, ropinavir, robilide, MK-0518, malaviloc, moroxydin, nerfinavir, nevirapine, nexavir, nucleoside analogs, oseltamivir, pencyclovir, peramivir, pleconaryl, , Protease inhibitors, reverse transcriptase inhibitors, ribavirin, limantazine, ritonavir, saquinavir, stubdin, synergistic activators, tenofovir, tenofovir disoproxil, tipranavir, trifluidine, tridivir, tromantazine, tubada, balaccyclovir, balgancyclovir, bicriviroc Nevirapine, lamivudine, zarcitabin, saquinavir, and zidovudine. Exemplary antiviral agents are described, for example, in US Pat. Nos. 6,093,550 and 6,894,033, incorporated herein by reference.

<u style="single">Antibacterial agent</u> The compositions of the invention (eg, Ad5-IFNα) are combined with antibacterial agents such as antibiotics (eg, one or more penicillins, cephalosporins, aminoglycosides, macrolides, sulfa compounds, fluoroquinolones, or tetracyclines). Can be administered. Other examples of antibacterial agents include penicillin G, penicillin V, methicillin, naphthylin, oxacillin, cloxacillin, dicloxacillion, ampicillin, amoxicillin, bacampicillin, cyclacillin, carbenicillin indanyl, ticalcillin, mezulocillin, methicillin, mezulocillin. , Cefapirin, cefrazin, cephalexin, cefadoroxyl, cefamandra nafate, cefloxim, cefoniside, cefolinide, cefaclor, cefoxitin, cefotetan, cefmethazole, cephataxis, cefataxime, ceftyzoxime, ceftriaxion Rate, ethylsuccinate, estrate, lactobionate, gluceptate, azithromycin, clarislomycin oxytetracycline, demecrocycline, doxicycline, minocycline, amoxicillin sulfate, gentamycin sulfate, intrathecal, canamycin sulfate, netylmycin sulfate, sulfate Streptomycin, tobramycin sulfate, neomycin sulfate, sulfadiazine, sulfamethizole, sulfisoxazole, sulfisoxazoleacetyl, sulfamethoxazole, trisulfapyrimidine, phenazopyridine, erythromycin ethylsuccinate, trimetprim, cyprof Loxacin, cyprofloxacin hydrochloride, enoxacin, romefloxacin hydrochloride, norfloxacin, ophroxacin, vancomycin hydrochloride, teikoplanin, rifampin, metronidazole, metronidazole hydrochloride, polmyxin, bacitracin, methenamine, methenamine horseurate₄, Dixtran sulfate, dideoxycytidine, dideoxyinosine, didihydrodideoxythymidine, foscarnet sodium, fusidic acid, HPA-23, isoprinosine, peniciramine, peptide T, ribavirin, rifabutin, didanosin, zalcitabine and the like.

<u style="single">Immunostimulant</u> The immunogenicity of the pharmaceutical composition of the present invention can be significantly improved when the composition of the present invention (eg, Ad5-IFNα) is co-administered with an immunostimulatory agent or an adjuvant. Exemplary immunostimulants include aluminum phosphate, aluminum hydroxide, QS21, Quil A (and its derivatives and components), calcium phosphate, calcium hydroxide, zinc hydroxide, glycolipid analogs, and octodecyl amino acids. Esters), muramildipeptides, polyphosphazenes, lipoproteins, ISCOM matrices, DC-Chol, DDA, cytokines, and other adjuvants and derivatives thereof.

<u style="single">Immunization vaccine</u> In some cases, it may be desirable to combine the compositions of the invention with compositions that induce a protective response against other viruses. For example, the compositions of the invention (eg Ad5-IFNα) can be an immunized vaccine, such as a vaccine against influenza, malaria, tuberculosis, smallpox, measles, rubella, mumps, or any other vaccine known in the art. At the same time, it can be administered separately or continuously.

For example, the vaccine can be, for example, a bacterial vaccine, a viral vaccine, a fungal vaccine, or a parasitic vaccine, respectively, known in the art for treating bacterial, viral, fungal, or parasitic substances. The vaccines are Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae, Bruscella, Burkholderia mall, Burkholderia malle. Yersinia pestis and Bacillus Bacteria selected from anthracis; viruses selected from members of the Flaviviridae family (eg, members of the Flaviviridae, Pestivirus, and Hepacivirus). Hepacivirus C virus, yellow fever virus; Gadgets Gully virus, Kadam virus, Kasanur forest disease virus, Langat virus, Omusk hemorrhagic fever virus, Poissan virus, Royal Farm virus, Tick-borne viruses such as Karshi virus, tick-borne encephalitis virus, Neudorfl virus, Sofjin virus, leprosy virus and Negishi virus; Meaban virus, Saumarez Seabird tick-borne viruses such as Reef virus and Tyuleniy virus; Aroa virus, Deng virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray valley encephalitis Virus, St. Louis encephalitis virus, Usutu virus, Westnile virus, Yaounde virus, Kokobera virus, Bagaza virus, Ilheus virus, Israel turkey meningoencephalomyelitis virus, Untaya Virus, Tempus virus, Jika virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus, Vessel Mosquito-borne viruses such as Subron virus, yellow fever virus; and Entebbe bat virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukarasa bat virus, Carey Island virus, Dakar bat Virus, Montana myitis White encephalitis virus, Phnom Penh) Includes viruses without known mediator phalanges such as bat virus, Rio Bravo virus, Tamana bat virus, and cell fusion factor virus); selected from members of the Arenaviridae family Viruses (including Ippy virus, Lassa virus (eg, Josiah strain, LP strain, or GA391 strain), lymphocytic choriomyelitis virus (LCMV), Mobala virus, Mopeia (eg, Josiah strain, LP strain, or GA391 strain) Mopeia virus, Amapari virus, Flexal virus, Guanalit virus, Funin virus, Latino virus, Machupo virus, Oliveros virus, Parana virus, Pitindevirus, Pirital ( Pirital virus, Savior virus, Takaribe virus, Tamiami virus, Whitewater Arroyo (Whitewater) Includes Arroyo virus, Chaparet virus, and Lujo virus); Members of the Bunyaviridae family (eg, Hantavirus, Nairovirus, Orthobunyavirus) ), And a virus selected from the genus Phlebovirus (which includes Hantern virus, Shinnonbre virus, Dugbe virus, Bunyanbella virus, Rift Valley fever virus, Lacros virus, Puntatro. (Punta Toro) virus (PTV), California encephalitis virus, and Crimea-Congo hemorrhagic fever (CCHF) virus); members of the Filoviridae family (including Ebola virus (eg, Zaire strain, Sudan strain, Cote d'Ivoire) Strains, Reston Strains, and Uganda Strains) and Marburg Viruses (including, for example, Angora Strains, Ci67 Strains, Musoke Strains, Popp Strains, Ravn Strains, and Lake Victoria Strains); Togavirus Members of the family (Togaviridae) (eg, members of the genus Alphavirus) (including Venezuelan lauma encephalitis virus (VEE), Eastern horse encephalitis virus (EEE), Western horse encephalitis virus (WEE), Sindbis virus, Includes ruin virus, semuliki forest virus, loss river virus, verma forest virus, onyonnyon virus, and chikungunya virus); members of the Poxviridae family (eg, members of the Orthopoxvirus genus) ( This includes natural poxvirus, monkey poxvirus, and vaccinia virus); members of the Herpesviridae family (which include simple herpesviruses (HSV; types 1, 2, and 6), human herpes. Viruses (eg, types 7 and 8), cytomegalovirus (CMV), Epstein bar virus (EBV), varicella herpes virus, and Kaposi sarcoma-related herpesvirus (KSHV)); Members of Orthomyxoviridae) (including influenza viruses such as H5N1 triinfluenza virus or H1N1 porcine influenza (types A, B, and C)); members of the Coronaviridae family (which are severely acute) Respiratory Syndrome (SARS) virus included);Members of the Rhabdoviridae family (which includes mad dog disease virus and bullous stomatitis virus (VSV)); Paramyxoviridae members (which include human respiratory astrovirus (RSV)), Newcastle disease virus, Hendra virus, Nipavirus, measles virus, bovine epidemic virus, canine temper virus, Sendai virus, human paramyxovirus (eg, types 1, 2, 3, and 4), rhinovirus, and mumps virus. Includes); Members of the Picornaviridae family (including poliovirus, human astroviruses (types A, B, C, and D), hepatitis A virus, and coxsackie virus); Members of the Hepadnaviridae family (which includes the hepatitis B virus); members of the Papillamoviridae family (which includes the human papillomavirus); members of the Parvoviridae family (which includes the human papillomavirus). This includes adeno-related viruses); members of the Astroviridae family (which includes Astroviridae); members of the Polyomaviridae family (which includes JC virus, BK virus, and SV40 virus included); members of the Calciviridae family (which includes nowalk virus); members of the Reoviridae family (which includes rotavirus); and retrovirus family Members of (Retroviridae), including human immunodeficiency viruses (HIV; eg, types 1 and 2), and human T lymphocyte tropic viruses types I and II (HTLV-1 and HTLV-2, respectively). Virus selected from);Or Aspergillus, Blastomyces dermatitidis, Candida, Cocccidioides immitis, Cryptococcus neoformans, Cryptococcus neoformans, Histoplasma caps .capsulatum), Paracoccidioides brasiliensis, Sporothrix schenckii, Zygomycetes spp., Absidia corymbifera and Absidia corymbifera Fungus selected from Rhizopus arrhizus; or Toxoplasma gondii, Plasmodium falciparum, P. vivax, P. ovale, P. It can target parasites selected from P. malariae, Trypanosoma spp., And Legionella spp.capsulatum), Paracoccidioides brasiliensis, Sporothrix schenckii, Zygomycetes spp., Absidia corymbifera, Absidia corymbifera, Absidia corymbifera Fungi selected from Rhizopus arrhizus; or Toxoplasma gondii, Plasmodium falciparum, P. vivax, P. ovale, P. ovale. It can target parasites selected from P. malariae, Trypanosoma spp., And Legionella spp.capsulatum), Paracoccidioides brasiliensis, Sporothrix schenckii, Zygomycetes spp., Absidia corymbifera, Absidia corymbifera, Absidia corymbifera Fungi selected from Rhizopus arrhizus; or Toxoplasma gondii, Plasmodium falciparum, P. vivax, P. ovale, P. ovale. It can target parasites selected from P. malariae, Trypanosoma spp., And Legionella spp.) Can be targeted for parasites selected from.) Can be targeted for parasites selected from.

Examples of vaccines known in the art that can be administered in combination with the compositions of the invention (eg, the Ad5-IFNα construct described herein) are AVA (BioThrax) for charcoal and VAR for varicella. (Varivax) and MMRV (ProQuad); DTaP (Daptacel, Infanrix, Tripedia), Td (Decavaca, Generic), DT (-Generic-), Tdap (Boostrix, Adacel), DTaP-IPV (Kinrix), DTaP- HepB-IPV (Pediarix), DTaP-IPV / Hib (Pentacel), and DTaP / Hib (TriHIBit); HepA (Havrix, Vaqta) and HepA-HepB (Twinrix) for hepatitis A; HepB (Engerix-) for hepatitis B B, Recombivax HB), Hib-HepB (Comvax), DTaP-HepB-IPV (Pediarix), and HepA-HepB (Twinrix); / Hib (TriHIBit), and DTaP-IPV / Hib (Pentacel); HPV4 (Gardasil) and HPV2 (Cervarix) against human papillomavirus (HPV); TIV (Afluria, Agriflu, FluLaval, Fluarix, Fluvirin, Fluzone) and LAIV against influenza (FluMist); JE (Ixiaro and JE-Vax) for Japanese encephalitis (JE); MMR (MMR II) and MMRV (ProQuad) for measles; MCV4 (Menactra), MPSV4 (Menomune), and MODC (Menveo) for meningococcal vaccine ); MMR for Mumps (MMR) II) and MMRV (ProQuad); DTaP (Daptacel, Infanrix, Tripedia), Tdap (Adacel, Boostrix), DTaP-IPV (Kinrix), DTaP-HepB-IPV (Pediarix), DTaP-IPV / Hib (Pentacel) for pertussis , And DTaP / Hib (TriHIBit); PCV7 (Prevnar), PCV13 (Prevnar13), and PPSV23 (Pneumovax 23) for bacterial pneumonia; Polio (Ipol), DTaP-IPV (Kinrix), DTaP-HepB-IPV ( Pediarix), and DTaP-IPV / Hib (Pentacel); Mad dog disease (Imovax Rabies and RabAvert); RV1 (Rotarix) and RV5 (RotaTeq) for Rotavirus; MMR (MMR) for wind rash II) and MMRV (ProQuad); ZOS (Zostavax) for herpes zoster; Vaccinia for vaccination and monkey vaccination (ACAM2000, Dryvax); DTaP (Daptacel, Infanrix, Tripedia) for tetanus, Td (Decavac, generic), DT (- Generic-), TT (-generic-), Tdap (Boostrix, Adacel), DTaP-IPV (Kinrix), DTaP-HepB-IPV (Pediarix), DTaP-IPV / Hib (Pentacel), and DTaP / Hib (TriHIBit) BCG for tuberculosis (TB) (TICE BCG, Mycobax); Oral intestinal typhos (Vivotif) and Typhim Vi for intestinal typhoid; and YF (YF-Vax) for yellow fever.

<u style="single">Ebola vaccine</u> Ad-CAGoptZGP is a vaccine that uses the adenovirus 5 skeleton and encodes the surface protein of Ebola virus (Richardson et al. (PLoS). 4: e5308, 2009)). Early versions of this vaccine have been shown to protect mice, guinea pigs and non-human primates from the inherently lethal attack of the Zaire Ebola virus. Ad-CAGoptZGP incorporates three improvements: codon optimization of gene inserts, inclusion of consensus Kozak sequences, and rearrangement of the CAG promoter. Transfection or transduction of Ad-CAGoptZGP into cells results in high expression of Ebola glycoprotein from these cells, a functional dose that is approximately 100-fold lower than that of other adenovirus-based Ebola vaccine constructs. Is possible, and the time to immunization is faster. Finally, Ad-CAGoptZGP has the ability to induce full protection (partial protection against guinea pigs) against mice given 30 minutes after attack, while the previous vaccine did not work after exposure. The advantage of this vaccine is its sustained immunity.

In one embodiment, the pharmaceutical compositions of the invention (eg, the Ad5-IFNα construct described herein) can be administered separately or sequentially at the same time as the Ad-CAGopt ZGP Ebola vaccine. Preferably, one or both of the agents are formulated for intranasal or transpulmonary administration. The experimental data of the present inventors show a high synergistic effect when, for example, Ad5-IFNα and Ad-CAGoptZGP are combined (whether they are administered in a single composition or in separate compositions). Regardless; see, eg, Example 14 herein). Specifically, complete cure was seen 30 minutes after exposure to ZEBOV in both mouse and guinea pig models, with no weight loss. As seen in Table 1, we benefit from both rapid expression of Ad5-IFNα (3 hours) and long-lasting protection of Ad-CAGopt ZGP in order to maximize the protective effect of both components. Expect to get. The combination of immunostimulatory agent and Ebola vaccine contributes to highly effective intensive therapy, and broad-spectrum antiviral drugs make this combination an excellent treatment option.

<tables num="1"><img id="000002" he="105" wi="159" file="JP5908397B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

The combination of Ad5-IFNα and Ad-CAGoptZGP also provides rapid onset of therapeutic and prophylactic effects as well as sustained protection against reinfection. The combination of Ad5-IFNα and Ad-CAGoptZGP (either individually or in combination) promotes direct stimulation of the innate immune system within 1-10 hours (eg, within 3 hours), eg, the recipient. It acts to nullify the viral hemorrhagic fever virus present in the body. Fast onset to defense is one of the many benefits of combination therapy. The combination of Ad5-IFNα and Ad-CAGoptZGP also works rapidly and fully at a single dose, however multiple doses of one or both of the drugs as needed (eg, 2, 3, 4, or 5 doses). May be administered.

<u style="single">Expeditionary and storage stability</u> To minimize logistical constraints, the combination of Ad5-IFNα and Ad-CAGoptZGP can be formulated to be storage stable and durable with respect to expeditability. Depending on the formulations described herein, if necessary, above 35 ° C for longer than, for example, 30 to 90 days (eg, at least 60 days), and even at temperatures as high as 90 ° C, for 30 minutes to 5 hours. It is possible to deploy one or more drugs over a short period of time (eg, at least 1 hour).

<u style="single">Filovirus efficacy data</u> Ad5-IFNα and Ad-CAGoptZGP have been tested separately and in combination in animal models of well-characterized filovirus infection (Zaire Ebola; ZEBOV), respectively. In the mouse test, 10⁴~10⁶Administration of Ad-CAGopt ZGP in the range of plaque forming units (PFU) is fully protective, and 10⁷All mice treated or pretreated with PFU Ad5-IFNα survived, indicating that weight loss was negligible.

Similar results were obtained from a guinea pig model of lethal ZEBOV infection, here 2 x 10⁸Intranasal delivery of PFU mAd5-IFNα resulted in 100% survival and slight weight loss in treated animals compared to 100% mortality in untreated animals. Ten¹⁰PFU's Ad-CAGopt ZGP had a survival rate of 33%, while the combination of Ad5-IFNα and Ad-CAGopt ZGP had a survival rate of 100% and no weight loss. Given the susceptibility of guinea pigs to ZEBOV, these results are particularly striking. The study also evaluated the efficacy of once-daily injections of recombinant IFNα protein and noted some survival benefits (Figure 10B).

<u style="single">Preparation and administration of the pharmaceutical composition of the present invention</u> The compositions used in the methods described herein are, for example, from parenteral, dermal, transdermal, eyeball, inhalation, oral, sublingual, peritongue, nasal, rectal, topical, and oral administration. It can be formulated for administration by the route of choice. Administration may be, for example, by intranasal release. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, and intramuscular administration. Parenteral, intranasal or intraocular administration is sterile, including, for example, aqueous suspensions, isotonic saline, pharmacologically compatible dispersants and / or solubilizers such as propylene glycol or polyethylene glycol. It may be provided using an injectable solution, a lyophilized powder formulation, and a gel formulation. The preferred method of administration may depend on a variety of factors (eg, the components of the composition administered and the severity of the condition being treated). Suitable formulations for oral or nasal administration are from effective amounts of compositions, capsules, sachets, tablets, or gels dissolved in a liquid solution, such as a diluent (eg, water, saline, or PEG-400). Each comprises a predetermined amount of the IFN delivery medium of the composition of the invention. The pharmaceutical composition may also be, for example, an aerosol formulation for inhalation into the respiratory tract. The aerosol formulation may be mixed with a pressurized pharmaceutically acceptable propellant (eg, dichlorodifluoromethane, propane, or nitrogen). In particular, administration by inhalation involves, for example, sorbitan trioleate or an aerosol containing oleic acid, for example with trichlorofluoromethane, dichlorofluoromethane, dichlorotetrafluoroethane, or any other biologically compatible propellant. It can be achieved by using it.

The immunogenicity of the compositions of the present invention can be significantly improved when co-administered with an immunostimulatory agent or adjuvant. Suitable adjuvants known to those skilled in the art include, for example, aluminum phosphate, aluminum hydroxide, QS21, Quil A (and derivatives and components thereof), calcium phosphate, calcium hydroxide, zinc hydroxide, glycolipid analogs, amino acids. Included are octodecyl esters, muramyl dipeptide, polyphosphazene, lipoproteins, ISCOM matrix, DC-Chol, DDA, cytokines, and other adjuvants and derivatives thereof.

In some cases, it may be desirable to combine the compositions of the invention with compositions that elicit a protective response against other viruses. For example, the compositions of the invention are administered separately or sequentially at the same time as other immunized vaccines, such as vaccines against influenza, malaria, tuberculosis, or any other vaccine known in the art. be able to.

The pharmaceutical compositions according to the invention described herein are to be released immediately after administration (eg, targeted delivery) or after administration using a controlled release or sustained release formulation. It may be formulated to be released after an arbitrary predetermined time. The compositions, alone or in combination, (i) have a small difference between the narrow therapeutic index (eg, the plasma concentration leading to an adverse side effect or toxic response and the plasma concentration leading to a therapeutic effect; in general, the therapeutic index TI is lethal. Median quantity (LD)₅₀) Median effective dose (ED₅₀); (Ii) a narrow absorption window at the site of release (eg, gastrointestinal tract); or (iii) a short biological half-life (and thus frequent during the day to maintain therapeutic levels). If administration is required), administration of the pharmaceutical composition in the release controlled or sustained release formulation is useful.

Many measures can be pursued to achieve release control or sustained release in which the release rate exceeds the metabolic rate of the pharmaceutical composition. For example, release control can be achieved by appropriately selecting the formulation parameters and ingredients, eg, the appropriate release control composition and coating. Suitable formulations are known to those of skill in the art. Examples include single-unit or multiple-unit tablets or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes.

The compositions of the invention may be administered to provide pre-exposure prophylaxis or after the subject has been exposed to a pathogen such as a virus. The composition may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35, 40, 45, 50, 55, or 60 minutes before exposure. Administered 2, 4, 6, 10, 15, or 24 hours ago, 2, 3, 5, or 7 days ago, 2, 4, 6 or 8 weeks ago, or even 3, 4, or 6 months ago Often, or 15-30 minutes after exposure to a pathogen (eg a viral pathogen) or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 20, 24, 48, 72 hours It may be administered to the subject later or more later.

When treating an autoimmune disease or cancer, the compositions of the invention may be administered to a subject prior to the onset or definitive diagnosis of symptoms, or after the diagnosis or manifestation of symptoms. For example, the composition may be used, for example, immediately after diagnosis or clinical recognition of symptoms, or 2, 4, 6, 10, 15, or 24 hours after diagnosis or detection of symptoms, 2, 3, 5, or 7 days. It may be administered after 2, 4, 6 or 8 weeks, or even after 3, 4, or 6 months.

The composition may be sterilized by conventional sterilization techniques or may be sterilized and filtered. The resulting aqueous solution may be packaged for use as is or lyophilized, and the lyophilized formulation may be administered in powder form or in combination with a sterile aqueous carrier prior to administration. The pH of the formulation is typically 3-11, more preferably 5-9 or 6-8, and most preferably 7-8, eg 7-7.5. The resulting solid form composition may be packaged in multiple single dose units, each with a fixed amount of an IFN delivery vector (eg, Ad5 conIFN-α delivery vector) and, if necessary, one or more. Immunomodulators are included in a sealed package of tablets or capsules, or a suitable dry powder inhaler (DPI) capable of administering one or more doses.

<u style="single">Nasal or pulmonary delivery</u> Nasal or transpulmonary administration has several advantages over, for example, oral, intravascular or intramuscular administration. In particular, intranasal or transpulmonary routes of administration are less harsh for adenoviral vector systems. For example, less proteolytic enzymes are present in the nasal epithelium and the environment has a more neutral pH (ie, less acidic). Also, virus delivery vectors in the nasal or pulmonary mucosa, as compared to the intestine, where changes in the contents of the intestinal lumen are large and therefore the ability of the vector to transduce / transfect cells in the environment concerned will be greater. It seems that the uptake of the particles is more consistent. In addition, the nasal mucosa is a highly permeable mucosal site with sufficient water.

Thus, in one embodiment, the IFN-α delivery vector of the invention and the pharmaceutical composition comprising it will be delivered by the intranasal or transpulmonary route, eg, in lyophilized powder form, in aerosol liquid form, or in gel form. Will be done. These routes of administration avoid recognition of, for example, the Ad5 vector by the host immune system, thus avoiding any pre-existing immunity that the host may have. In addition, intranasal and transpulmonary delivery can be readily administered when mass distribution is required.

Transpulmonary and / or intranasal administration of the compositions of the present invention comprises providing, for example, mist (aqueous or granular) to the lungs (upper respiratory tract and / or lower respiratory tract) or nasal epithelium, respectively. This dosage form has a number of advantages over traditional needle injection. First, it does not involve the use of needles, which means that it is "painless" and therefore has good patient compliance. Secondly, since self-administration is possible by transpulmonary administration and intranasal administration, the doctor's time is saved, the use of equipment is not required, and the patient's anxiety is eliminated. Third, the use of sugar or salt-based placebo powders or solutions facilitates painless dosing training. Fourth, there is no risk of medical problems caused by, for example, needle-mediated bacterial / viral contamination or other problems due to unclean injection sites. Fifth, the dispersion of aerosols or powders allows the vaccine to be applied more closely and more uniformly. Sixth, based on the characteristics of the dosing device, the particle size of the vaccine can be controlled so that effective attachment occurs, for example, in the upper and / or lower respiratory tract. In addition, needled administration was typically trained to ensure that the infused drug was properly delivered to the correct compartment of the body (ie, intravenous when compared to intramuscular). You need a medical professional. For the preparation of aerosolized adenovirus vectors, see, for example, US Pat. No. 6,0P, incorporated herein by reference http://patft.uspto.gov/netacgi/nph- Parser? Sectl = PTOl &amp; Sect2 = HITOFF &amp; d = PALL &amp; p = l &amp; u =% 2Fnetahtml% 2FPTQ% 2 Fsrchnum.htm &amp; r = l &amp; f = G &amp; l = 50 &amp; sl = 7,097,827.PN. &amp; OS = PN / 7,097,827 &amp; RS = PN /- hO http://patft.uspto.gov/netacgi/nph- Parser? Sectl = PTOl &amp; Sect2 = HITOFF &amp; d = PALL &amp; p = l &amp; u =% 2Fnetahtml% 2FPTO% 2 Fsrchnum.htm &amp; r = l &amp; f = G &amp; l = 50 &amp; sl = 7.097,827.PN. &amp; OS = PN / 7.097.827 &amp; RS = PN /- It is described in h2 No. 7,097,827.

Suitable formulations for use in jet or ultrasonic nebulizers typically include vectors (eg, Ad5-conIFN-α vectors), eg, about 0.01-25 mg of vector per mL of solution, preferably about 0.1-. Contain in aqueous medium at a concentration of 10 mg / mL. The formulation may also contain buffers and monosaccharides (eg, to stabilize proteins and regulate osmolality), and / or human serum albumin in a concentration range of 0.1-10 mg / ml. Examples of buffers that can be used are sodium acetate, citrate and glycine. Preferably, the buffer may have a composition and molarity suitable for adjusting the solution to a pH in the range of 3-9. Generally, a molar concentration of buffer of 1 mM to 50 mM is suitable for this purpose. Examples of excipients, typically in amounts ranging from 1% to 90% by weight of the formulation (eg, 1% to 50% by weight, more preferably 5% to 30% by weight), include, for example, fructose. , Monosaccharides such as maltose, galactose, glucose, D-mannose, sorbitol; disaccharides such as lactose, sucrose, trehalose, cellobiose; polysaccharides such as raffinose, meregitos, maltodextrin, dextran, starch; mannitol, xylitol, xylose, Arditol such as martitol, lactitol, xylitol sorbitol (glucitol), sucrose, pyranosyl sorbitol, myoinositol; and glycine, CaCl₂, Hydroxyectine, ectoine, gelatin, di-mio-inositol phosphate (DIP), cyclic 2,3 diphosphoglycerate (cDPG), 1,1-di-glycerol phosphate (DGP), β-mannosyl glycerate ( Phylloin), β-mannosyl glyceramide (phylloin A), proline betaine and / or its derivatives and combinations thereof.

The nebulizer formulation may also contain a surfactant to reduce or prevent surface-induced aggregation of the composition components caused by atomization of the solution in forming the aerosol. Various conventional surfactants can be used, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitan fatty acid esters. The amount generally ranges from 0.001% to 4% by weight of the formulation. A particularly preferred surfactant for the purposes of the present invention is polyoxyethylene sorbitan monooleate.

Specific formulations and methods for producing suitable dispersions of the liquid particles of the present invention are described, for example, in WO 94/200069, US Pat. No. 5,915,378, US Pat. No. 5,960,792, US Pat. 5,957,124, US Pat. No. 5,934,272, US Pat. No. 5,915,378, US Pat. No. 5,855,564, US Pat. No. 5,826,570, and US Pat. No. 5,522,385 (each of which). Incorporated herein by reference).

The compositions of the invention (eg, an adenovirus vector containing a nucleic acid molecule encoding interferon (eg, Ad5-conIFN-α)) are selectively administered intranasally. The Ad5 virus is extremely efficient at delivering genes to the epithelial cells of the nasal mucosa. Mucosal administration is efficient because it stimulates both systemic and mucosal immunity at the entrance (eg, Gutierro et al., Vaccine 20: 2181-2190, 2002; and Patel et al., J. Infect. Dis. 196: See S413-420, 2007). In addition, the use of live Ad5 virus for the delivery of IFN provides an additional immunostimulatory pathway and thus acts as an adjuvant to ensure maximum efficacy is achieved. Therefore, delivery of the compositions of the invention to a site where an infectious agent (eg, a virus) invades would reduce the required dose. Special instruments have been developed to effectively deliver aerosol droplets (diameter&gt; 2 μm) to this compartment (eg, Mucosal Atomization Device (MAD300), Wolfe Tory). See Medical). The size of the droplets (or powdered particles) is important because aerosols smaller than 1 μm can penetrate further into the respiratory tract and cause harmful effects.

The compositions of the present invention can also be delivered in powder form, for example using a metered dose inhaler. The powder may be lyophilized and may also contain stabilizers such as human serum albumin (HSA). Typically more than 0.5% (w / w) of HSA is added. In addition, if necessary, to enhance one or more features (eg, to facilitate the application of powder from the device, to extend the shelf life of the vaccine composition, or to extend the shelf life of the vaccine composition, or to the composition of the vaccine during sucrose drying. One or more of the following may be added to the formulation as excipients (to improve the stability of the product): monosaccharides such as fructose, alditol, galactose, glucose, D-mannose, sorbitol; lactose, sucrose, Disaccharides such as trehalose, cellobiose; polysaccharides such as raffinose, meregitos, maltodextrin, dextran, starch, etc .; Such as alditol; and glycine, CaCl₂, Hydroxyectine, ectoine, gelatin, di-mio-inositol phosphate (DIP), cyclic 2,3 diphosphoglycerate (cDPG), 1,1-di-glycerol phosphate (DGP), β-mannosyl glycerate ( Philoin), β-mannosyl glyceramide (Philoin A), proline betaine and / or derivatives and combinations thereof. The amount added to the pharmaceutical product is about 0.01 to 200% (w / w), preferably about 1 to 50% (w / w), and more preferably about 5 to 30% (w / w) of the existing vector. It can be a range. The next such product is lyophilized and ground to the desired particle size. The powder particles must have aerodynamic properties in the nasal cavity and lungs, which is less than 50 μm, preferably 1.5-10 μm, more preferably 1.8-7.0 μm, and most preferably about 2.0-4 μm in diameter center. Approximately 1 g / cm with value²Corresponds to particles with a density of. The average particle diameter can be measured using conventional instruments such as Cascade Impactor (Andersen, Ga.).

The dry powder formulation of the present invention conveniently initially contains a vector (eg, an adenovirus vector containing a nucleic acid molecule encoding interferon (eg, Ad5-conIFN-α) or another nucleic acid construct of the present invention) in aqueous solution. It can be formulated by suspending. The relative amount of the vector and any additive excipient material may depend on the desired final ratio of the vector to the excipient. Conveniently, the ratio of the vector to the excipient is in the range of about 2: 1 to 1: 100 (vector: excipient), preferably 1: 1 to 1:10, and is the total solid in the aqueous suspension. Concentrations are generally less than 5% by weight and more generally less than 3% by weight.

The powder may be suspended in the propellant by using a surfactant. The propellant is a chlorofluorocarbon, hydrochlorofluorocarbon, hydrofluorocarbon, or hydrocarbon, such as trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or a combination thereof. It may be any conventional material used for this purpose, such as. Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid can also be useful as a surfactant. The mixture is then loaded into the delivery device.

In the case of the composition of the present invention containing a viral vector, it is usually desirable to buffer the aqueous solution in order to enhance the activity of the viral vector after drying. Buffers or pH regulators typically include salts prepared from, for example, organic acids or bases. Typical buffers include citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid organic acid salts, tris, tromethamine hydrochloride, or phosphate buffers.

Additional polymeric excipients / additives that can be included in the formulations of the compositions of the invention include Ficoll (polymeric sugars) such as, for example, polyvinylpyrrolidone, derivatized celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose. ), Hydroxyl starch, dextrin (eg, cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin and sulfobutyl ether-β-cyclodextrin), polyethylene glycol, and pectin.

The powder compositions of the present invention used in such devices include International Publication No. 96/32149, International Publication No. 97/41833, and International Publication No. 98/29096, and US Pat. No. 7,482,024. ; http://patft.uspto.gov/netacgi/nph- Parser? Sectl = PTOl &amp; Sect2 = HITOFF &amp; d-PALL &amp; p = l &amp; u =% 2Fnetahtml% 2FPTO% 2 Fsrchnum.htm &amp; r = l &amp; f = G &amp; l = 50 &amp; sl = 7.481.212.PN. &amp; OS = PN / 7.481.212 &amp; RS = PN /- hO http://patft.uspto.gov/netacgi/nph- Parser? Sectl = PTOl &amp; Sect2 = HITOFF &amp; d = PALL &amp; p = l &amp; u =% 2Fnetahtml% 2FPTO% 2 Fsrchnum.htm &amp; F = l &amp; f = G &amp; l = 50 &amp; sl = 7,481.212.PN. &amp; OS = PN / 7,481,212 &amp; RS = PN /- h2 No. 7,481,212; No. 7,371,373; No. 6,303,582; No. 6,001,336; No. 5,997,848; No. 5,993,783; No. 5,985,248; Generated by the method disclosed in the specification 5,976,574; the specification 5,922,354; the specification 5,785,049; and the US Pat. No. 5,654,007 (each of which is incorporated herein by reference). And / or can be delivered. The powder form is also described, for example, in a pre-filled dosing device, eg, US Pat. No. 5,437,267; 6,068,199; 6,715,485; 5,994,314; 7,235,391. It can also be administered using the devices described in the same No. 6,398,774, each of which is incorporated herein by reference. The powder may have a moisture content of generally less than about 20% by weight, usually less than about 10% by weight, and preferably less than about 6% by weight. Such low water content solids tend to exhibit higher stability during packaging and storage.

Mechanical devices designed for pulmonary and / or nasal delivery of the compositions of the present invention include, but are not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are included. It is well known to those skilled in the art. Specific examples of commercially available devices suitable for carrying out the present invention are Mallinckrodt, Inc., St. Louis, Mo., Ultravent nebulizers manufactured by the United States; Mucosal Atomization Device (eg, MAD300), Wolfe Tory Medical; Acorn II Nebulizer manufactured by Marquest Medical Products, Englewood, Colo., USA; Ventolin metered dose inhaler manufactured by Glaxo Inc., Research Triangle Park, NC, USA; OptiNose, Oslo, Norway OptiNose equipment manufactured; Spinhaler powder inhaler Nektar manufactured by Fisons Corp., Bedford, Mass., USA Therapeutics, Inc., San Carlos, Calif., US "standing cloud" device; Alkermes, Cambridge, Mass., AIR inhalers manufactured by the US; and Aradigm AERx pulmonary drug delivery system manufactured by Corporation, Hayward, Calif., USA. Also, for example, the delivery device described in US Pat. Nos. 5,522,378; 5,775,320; 5,934,272; and 5,960,792; US Pat. No. 6,715,485; 7,347,201; and OptiNose device in 7,481,218; and US Patent Application Publication No. 2004/0112378; No. 2005/0072430; No. 2004/0112379; No. 2004 / 0149289, 2005/0028812; 2008/0163874; 2008/0161771; 2008/0223363; 2005/0235992; See also 2006/0096589; 2006/0169278; 2007/0039614; and 2007/0186927; and U.S. Pat. No. 7,669,597. That thing.

The compositions of the present invention can also be formulated as an intranasal carrier in the form of a nasal gel, cream, paste or ointment that provides more lasting contact with the surface of the nasal mucosa. These formulations may have viscosities of, for example, about 10 to about 250,000 centipoise (cps), or about 2500 to 100,000 cps, or about 5,000 to 50,000 cps or higher. Such carrier viscous formulations may be based solely on, by way of example, alkyl cellulose and / or other high viscosity biocompatible carriers known in the art (see, eg, Remington cited above). Preferred alkyl celluloses are, for example, methyl celluloses at concentrations ranging from about 5 to about 1000 mg or more per 100 ml of carrier. A more preferred concentration of methylcellulose is, by way of example, from about 25 to about mg per 100 ml of carrier. The carrier containing the IFN delivery medium of the present invention can also be immersed in a cloth material such as gauze that can be attached to the surface of the nasal mucosa and allow the delivery medium to permeate there.

Examples of gel formulations that can be used to prepare the compositions of the present invention are also described, for example, in the US patent http://patft.uspto.gov/netacgi/nph- Parser? Sectl = PTOl &amp; Sect2 = HITOFF &amp; d = PALL &amp; p = l &amp; u =% 2Fnetahtml% 2FPTO% 2 Fsrchnum.htm &amp; r = l &amp; f = G &amp; l = 50 &amp; sl = 7,538,122.PN. &amp; OS = PN / 7,538.122 &amp; RS = PN /- hO http://patft.uspto.gov/netacgi/nph- Parser? Sectl = PTOl &amp; Sect2 = HITOFF &amp; d = PALL &amp; p = l &amp; u-% 2Fnetahtml% 2FPTO% 2 Fsrchnum.htm &amp; r = l &amp; f = G &amp; l = S0 &amp; sl = 7,538,122.PN. &amp; OS ^ PN / 7,538.122 &amp; RS = PN /- h2 No. 7,538,122; http://patft.uspto.gov/netacgi/nph- Parser? Sect1 = PTO1 &amp; Sect2 = HITQFF &amp; d = PALL &amp; p = &amp; u =% 2Fnetahtml% 2FPTO% 2 Fsrchnmn.htm &amp; i = l &amp; f = G &amp; l = 50 &amp; sl = 7,387J88.PN. &amp; OS = PN / 7,387,788 &amp; RS = PN /- h2 http://patft.uspto.gov/netacgi/nph- Parser? Sectl = PTOl &amp; Sect2 = HITOFF &amp; d = PALL &amp; p = l &amp; u =% 2Fnetahtml% 2FPTO% 2 Fsrchnum.htm &amp; r = l &amp; f = G &amp; l = 50 &amp; sl = 7,387J88.PN. &amp; OS = PN / 7,387J88 &amp; RS = PN /- h2, No. 7,387,788; http://patft.uspto.gov/netacgi/nph- Parser? Sectl-PTOl &amp; Sect2 = HITOFF &amp; d = PALL &amp; p = l &amp; u =% 2Fnetalitml% 2FPTO% 2 Fsrchnum.htm &amp; r = l &amp; f = G &amp; l = 50 &amp; sl = 7,166,575.PN. &amp; OS = PN / 7,166,575 &amp; RS = PN /- hO http://patftuspto.gov/netacgi/nph- Parser? Sectl = PTOl &amp; Sect2 = HITOFF &amp; d = PALL &amp; p = l &amp; u =% 2Fnetahtml% 2FPTO% 2 Fsrchmim.htm &amp; r = l &amp; f = G &amp; l = 50 &amp; sl = 7.166.S75.PN. &amp; OS = PN / 7.166.575 &amp; RS = PN /- h2, No. 7,166,575; http://patft.uspto.gov/netacgi/nph- Parser? Sectl = PT01 &amp; Sect2 = HITOFF &amp; d = PALL &amp; p = l &amp; u =% 2Fnetahtml% 2FPTO% 2 Fsrchnum.htm &amp; r = l fef = G &amp; l = 50 &amp; sl = 6,413,539.PN. &amp; OS ^ PN / 6,413,539 &amp; RS = PN /- hO http://patft.uspto.gov/netacgi/nph- Parser? Sectl = PTOl &amp; Sect2 = HITOFF &amp; d-PALL &amp; p = l &amp; u =% 2Fnetalitml% 2FPTO% 2 Fsrchnum.htm &amp; r = l &amp; f = G &amp; l = 50 &amp; sl = 6.413,539.PN. &amp; OS = PN / 6,413.539 &amp; RS = PN /- h2 Also described in 6,413,539; and 6,004,583; each of which is incorporated herein by reference. The gel preparation of the present invention may further contain a permeation enhancer (penetration accelerator). Permeation enhancers include, but are not limited to, sulfoxides such as dimethyl sulfoxide and decylmethyl sulfoxide; sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride, poroxamer (231, 182, 184), tween. Surfactants such as (20, 40, 60, 80) and lecithin; 1-substituted azacycloheptan-2-one, especially 1-n-dodecylcyclazacycloheptan-2-one; lauryl alcohol, myristyl alcohol, oleyl Fatty acids such as alcohols; fatty acids such as lauric acid, oleic acid and valerate; fatty acid esters such as isopropyl myristate, isopropyl palmitate, methylpropionate, and ethyl oleate; polyols and esters thereof, such as propylene glycol, Ethylene glycol, glycerol, butanediol, polyethylene glycol, and polyethylene glycol monolaurates, amides and other nitrogen compounds such as urea, dimethyl acetamide (DMA), dimethyl formamide (DMF), 2-pyrrolidone, 1-methyl-2. -Pyrrolidone, ethanolamine, diethanolamine and triethanolamine, terpen; alkanone, and organic acids, especially salicyl acid and salicylate, citric acid and succinic acid. Permeation accelerator is about 0. 1 ~ about 30% w / w can be present. Preferred permeation enhancers are fatty alcohols and fatty acids. The gel composition may also include buffers such as carbonate buffers, citric acid buffers, phosphate buffers, acetate buffers, hydrochloric acid, lactic acid, tartaric acid, inorganic and organic bases. The buffer may be present in a concentration of about 1 to about 10 weight percent, more preferably about 2 to about 5 depending on the type of buffer used, as will be appreciated by those skilled in the art. It is a weight percent concentration. However, the concentration of one or more buffers can vary and the buffer may be replaced with up to 100% water in the composition.

<u style="single">Dosing</u> The pharmaceutical compositions of the present invention can be administered in therapeutically effective amounts that provide immunogenicity and / or protective action against infection by pathogens such as viruses. For example, if the composition contains a viral vector (eg, an Ad5-based vector) that encodes an IFN (eg, IFN-α such as conIFN-α), at least about 1x10.³Viral particles (vp) / dose or 1x10¹~1×10¹⁴vp / dose, preferably 1x10³~1×10¹²vp / dose, and more preferably 1x10⁵~ 1 × 10 11vp / dose (eg 1.5 ~ 3.0 × 10)⁸The vp / ml) viral vector provides a therapeutically effective amount of IFN after expression in the host cell. A single viral particle encodes a viral and non-viral protein (eg, including viral and non-structural proteins and non-endogenous IFNs) and contains a protective membrane containing protein subunits (eg, a lipid-based envelope or protein). Includes one or more nucleic acid molecules (either DNA or RNA) surrounded by a base capsid). The number of virus particles can be measured, for example, based on the dissolution of vector particles followed by absorbance measurements at 260 nm (see, eg, Steel, Curr. Opin. Biotech. 10: 295-297, 1999). ).

If the composition is a non-viral vector containing a nucleic acid molecule encoding an IFN (eg, IFN-α such as conIFN-α), the subject is at least about 1x10.¹Molecule / dose, eg 1x10¹~1×10¹⁵Molecule / dose, preferably 1x10³~1×10¹⁰Molecule / dose, and more preferably 1x10⁴~1×10⁸A molecular / dose non-viral delivery vector must be administered. A single nucleic acid molecule in a non-viral vector is administered, for example, in one or more nucleic acid molecules in the form of a plasmid, cosmid, yeast or bacteriophage (eg, DNA or RNA), and in naked form. , Or a bacteriophage that is surrounded by or complexed with a protective substance (eg, a lipid or lipid-based envelope, peptide, and polymer).

The dosage administered is the subject to be treated (eg, age, weight, immune system capacity, and overall health of the subject to be treated), the mode of administration (eg, as solid or liquid), the method of administration (eg, as solid or liquid). , By injection, inhalation, dry powder propellant), and depending on the target cell (eg, epithelial cells such as vascular epithelial cells, nasal epithelial cells, or lung epithelial cells). The composition is preferably administered in an amount that provides sufficient levels of IFN expression to elicit an immune response without the treatment causing excessive adverse physiological effects in the host.

In addition, single or multiple doses of the compositions of the invention may be given to the subject (before or after exposure) (eg, one dose or two or more doses). For example, subjects who are particularly susceptible to viral infections, for example, may require multiple treatments to establish and / or maintain protection against the virus. The level of induced immunity provided by the pharmaceutical compositions described herein can be monitored, for example, by measuring neutralization secretion and serum antibody levels. Dosings can then be adjusted or repeated as needed to maintain the desired level of protection, eg, against viral infections.

Alternatively, therapeutic efficacy can be determined by monitoring the level of IFN-α expressed in the subject (eg, human) after administration of the composition of the invention (eg, Ad5-IFN-α vector). For example, blood or lymph of interest can be tested for IFN-α levels, eg, using standard assays known in the art (eg, human interferon from Pestka Biomedical Laboratories (PBL), Piscataway, New Jersey). See Alpha Multispecies Assay Kit (Product No. 41105) and Human Interferon Alpha Serum Sample Kit (Product No. 41110)). The efficacy of treatment is also double-stranded RNA (dsRNA) -dependent protein kinase R (PKR), 2'-5'-oligoadenylate synthetase (2'-5'-OAS), IFN-induced Mx protein, tryptophan. Degrading enzymes (eg Pfefferkorn, Proc.Natl.Acad.Sci.USA 81: 908-912, see 1984), adenosine deaminase (ADAR1), IFN activating gene 20 (ISG20), p56, ISG15, mGBP2, GBP-1, APOBEC protein, viperin, or other factors, etc. It can also be determined by monitoring the level of expression or activation of IFN-α upregulators in (eg, Zhang et al., J. Virol., 81: 11246-11255, 2007, and as a whole by reference. See US Pat. No. 7,442,527, incorporated herein by reference).

A single intranasal dose of the compositions of the invention provides protection from infectious agents (eg, viral factors) prior to exposure. This is a dramatic improvement over the weekly or even multiple daily doses required for current IFN-α treatment. In addition, a single dose administered directly after exposure to viral or other infectious factors (eg, within 24 hours) may serve as a treatment according to the invention. Due to the effectiveness of a single dose of the compositions of the present invention, the need for follow-up and re-treatment or re-vaccination of the treated subject, which is typically a challenge in a pandemic or bioterrorism attack followed by widespread panic. Is gone.

A single intranasal dose of the composition of the invention can also be used to achieve treatment in a subject to be treated for an autoimmune disease or cancer. If desired, multiple doses (eg, 2, 3, 4, 5 doses or higher) may also be administered to such subjects.

<u style="single">Storage stability</u> Pharmaceutical formulations of the compositions of the invention (eg, formulations containing the Ad5-conIFN-α delivery vector) demonstrate long shelf life, which provides advantages over other adenovirus, antiviral, or vaccine products. .. In particular, the Ad5-based IFN-α delivery vectors of the invention that can be manufactured and lyophilized (freeze dried) can be stored at room temperature for at least about 1, 2, 3, or 4 weeks, preferably at least about 1, 2. , 3, 4, 5, 6, 12, or 18 months, more preferably at least 20 months, even more preferably at least about 22 months, and most preferably at least about 24 months. This is crucial for the military and in developing countries where the public health sector cannot guarantee refrigeration of drugs. The shelf life of the compositions of the present invention can be extended by storing at 4 ° C.

The shelf life of the adenovirus vector-containing compositions of the present invention can be determined, for example, by measuring the adenovirus vector titer (see, eg, Croyle et al., Gene Therapy 8: 1281-1290, 2001) or IFN. Can be assessed by assessing the biological activity (eg, the ability to transfect cells and express biologically active IFN) of a delivery medium containing (eg, viral or non-viral delivery medium). .. In one embodiment, the compositions of the invention are less than 20%, more preferably less than 10%, and most preferably less than 5% of their original titer (or bioactivity) after storage at room temperature for at least 12 months. Indicates loss of. In other embodiments, the compositions of the invention are less than 40%, more preferably less than 30%, and most preferably 20% of their original titer (or bioactivity) after storage at room temperature for at least 24 months. Indicates a loss of less than.

The pharmaceutical formulations of the compositions of the present invention are also stored at temperatures in the range of about 30 ° C to about 55 ° C (eg, about 45 ° C) for at least about 1-15 days or 2-4 weeks or even more. Indicates a shelf life of at least about 2-6 months. In one embodiment, the composition to be preserved is in dry, non-reconstituted powder form. Preferably, the compositions of the invention stored at temperatures in the range of about 30 ° C to about 55 ° C have the original titer (or organism) when stored for a period of time in the range of 1 week to 2 months. Shows less than 40% (more preferably less than 30%, 20%, or less than 10%, and most preferably less than 5%) of activity).

In another embodiment, when the pharmaceutical formulation of the composition of the present invention is stored frozen (eg, at a temperature in the range below 4 ° C (eg, 0 ° C to about -1900 ° C)). At least about 1, 2, 3, or 4 weeks, preferably at least about 1, 2, 3, 4, 5, 6, 12, or 18 months, more preferably at least 20 months, even more preferably at least about 22 months, And most preferably at least about 24 months. In this embodiment, the composition can be stored as an unstabilized frozen solution. Preferably, the compositions of the invention, which are stored at temperatures below 4 ° C (eg, 0 ° C to about -20 ° C), have the original titer when stored for a period ranging from 2 months to 2 years. Indicates a loss of less than 40% (more preferably less than 30%, 20%, or less than 10%, and most preferably less than 5%) of valence (or biological activity).

Advantages of long-term stability and shelf life of the compositions of the present invention include: a) Ease of storage of the composition due to the absence of a cold chain, thereby stabilizing electricity. Increased likelihood that the composition can be disseminated and stored in unavailable areas (eg, Third World economic countries and disaster or conflict zones), and less "materials" that must be transported or used in areas without refrigeration. Therefore, the tempo of military action is improved; b) forward deployment is possible if the drug can be placed in the backpack of a soldier or the rear of a WHO disaster transport vehicle; c) because the loss due to defrosting is reduced. Less drug waste; and d) The composition does not need to include refrigeration means for storage, making the use of Strategic National Stockpile (SNS) storage space warehouses more cost effective.

Other advantages of the Ad5-based IFN-α delivery vector of the present invention are shown in FIG.

<u style="single">kit</u> The invention also includes a kit comprising an IFN-α delivery vector of the invention in lyophilized powder form and a vial of a hydration medium (eg, sterile water or saline) that can be used to reconstitute the powder. provide. In another embodiment, the kit comprises a container of the IFN-α delivery vector of the invention in lyophilized powder form and a separate delivery device capable of releasing the contents of the container during administration in combination with the container. .. The kit also contains a container of the IFN-α delivery vector of the invention in lyophilized powder form and, if desired, a hydration medium (eg, sterile water or saline) that can be used to reconstitute the powder. The vial may include a delivery device (eg, by transpulmonary or intranasal administration) that can be used to release the IFN-α delivery vector as a powder or reconstituted liquid in aerosol form. The kits of the invention are optionally instructions for the practice of any of the methods described herein, including methods of treatment or prevention, and use for the use of any composition specified herein. Includes instructions and / or instructions for operating any equipment, system, equipment, or component described herein, as well as packaging materials.

The following examples illustrate the present invention. These are not intended to limit the invention in any way.

<u style="single">Example 1: Effectiveness of pre-exposure and post-exposure protection against Western equine encephalitis virus and Venezuelan equine encephalitis virus</u> Western equine encephalitis virus, an arthropod (mosquito) vector alpha virus classified as a Category B pathogen by the US Centers for Disease Control and Prevention (CDC) using the Ad5-IFN-α delivery vector It has been shown to provide both pre- and post-exposure protection against (WEEV; Wu et al., Virology 369: 206-213, 2007). In this study, mice were injected intramuscularly 10⁷Inoculated with PFU Ad5-mIFNA and attacked by various WEEV strains at a series of time points. Ad5-mIFNA showed complete protection when administered 24 hours, 48 hours, and 1 week before exposure, and 38% protection when treated 13 weeks before exposure. A single inoculation 6 hours after the attack delayed the progression of the WEEV infection, providing approximately 60% protection.

Similar results were obtained in a test using the Venezuelan equima encephalitis virus (VEEV). VEEV is a more infectious virus, with intramuscular administration of Ad5-IFN-α resulting in complete protection against 10LD50 (not tested at other time points) when administered 24 hours before exposure, and 100LD50. A survival rate of 75% was obtained. In this case, Ad5-IFN showed no protection when administered after exposure (O'Brien et al., J. Gen. Virol. 90: 874-882, 2009).

<u style="single">Example 2: Use of the composition of the present invention</u> Pre-exposure (post-event) prevention: The compositions of the invention are used, for example, as single-dose widespread antiviral preventative medical measures against the risk of exposure to the threat of virus-based biological weapons or the threat of endemic viral threats. be able to.

<u style="single">Military or law enforcement operations</u> The compositions of the present invention can be used as a precaution for military, law enforcement, or regional emergency coordinator (LEC) personnel exposed to virus-based biological weapons threats during operation. The determination of administration of the composition of the invention (eg, an Ad5 delivery vector containing a nucleic acid molecule encoding conIFN-α and formulated as a lyophilized powder for delivery to the nasal mucosa) to a soldier is made, for example, a. ) Presence of identifiable biological weapons (as aerosols or surface contaminants on the device) as measured by biosensors, b) Information that such virus-based weapons have been or can be deployed by enemies, Or c) it may be based on soldier contact with an affected sentinel or d) a victim who is expected to exhibit symptoms of a viral disease.

<u style="single">Exposure during study</u> Similar scenarios are presented by researchers or manufacturers who are routinely exposed to pathogenic viruses or other biological threats due to the nature of the work itself, and for further precautions against device or protocol errors. To. The compositions of the present invention can also be used as a prophylaxis (pre-exposure or post-exposure) for such individuals.

<u style="single">Example 3: Medical chain</u> The compositions of the present invention come into contact with persons engaged in the medical chain, such as doctors, nurses, cleaners, and other patients who have or may have a viral or bacterial infection. It can be administered prophylactically to a person. Due to the widespread nature of the compositions of the present invention, they can be administered to a subject before obtaining information about the biopathogen and even when there is no time to reliably identify the viral pathogen. The compositions of the invention are also beneficial when the virus mutates during a pandemic, making established vaccines ineffective or less protective.

<u style="single">Example 4: Public health</u><u style="single">Ring treatment immediately after exposure</u> If the patient is found to have come into contact with a viral threat within the preceding 24 hours, it comprises a nucleic acid molecule encoding the composition of the invention (eg, IFN-α (eg, conIFN-α)) and is delivered nasally or pulmonary. For example, an Ad5 delivery vector formulated as an aerosol powder or liquid mist) can be administered as a post-exposure treatment. If desired, the compositions of the invention can be administered, for example, as a "ring" treatment to all susceptible individuals within a predetermined range around the outbreak of the infection. Ring treatments control outbreaks by treating and monitoring people around each infected person.

<u style="single">Treatment of suspected exposure</u> The compositions of the invention are administered to people who are considered to have been exposed ("worried well") because the side effects of IFN are minimal, even if no exposure to biological threats is confirmed. can do. For example, a cranberry grower in Massachusetts gets sick after being bitten by a mosquito. For example, because of the risk of a local epidemic of eastern equine encephalitis (EEE), the person is administered the composition of the invention, for example by nasal or pulmonary delivery (eg, as an aerosol powder or liquid mist). Signs of improvement can be observed prior to farming near the Cranberry Wetland.

<u style="single">Post-exposure prophylaxis</u> At the population level, if the spread of the viral threat is known or is believed to have occurred, the compositions of the invention are administered, for example by nasal or pulmonary delivery (eg, as aerosol powder or liquid mist). , Can prevent the spread of virus threats. In this case, the intervention is performed without knowing the infection status of the recipient, and therefore the preventive and therapeutic functions are likely to be applied.

<u style="single">Example 5: Veterinary indication of Ad5 vectorized IFN</u> The broad-spectrum antiviral potential of interferon polypeptides is well recognized in veterinary medicine. In fact, oral administration of IFN is associated with pneumonia in Thoroughbred racehorses (Akai et al., J. Equine Sci. 19: 91, 2008) and bovine respiratory disease (BRDC; Cummins et al., J. Inf. &amp; Cyto.Res. 19: 907, 1999) and in the general treatment of equine respiratory disease (Moore et al., Can. Vet. J. 45: 594, 2004). Intranasal or transpulmonary delivery of Ad5-IFN can overcome the current constraints of repeated doses and high costs. An intranasal delivery system for horses that can be used to administer the compositions of the invention is described, for example, in US Pat. No. 6,398,774, which is incorporated herein by reference. The use of Ad5-IFN production systems has been shown to be safe and effective in laboratory animals (see, eg, Wu et al., Virology 369: 206, 2007).

Other veterinary indications include treatment or prevention of pandemics caused by pathogens such as Rift Valley fever, treatment or prevention of endemic pathogens, and treatment or prevention of intentionally released pathogens. Treatment or prevention in this regard prevents or reduces the potential for catastrophic loss of animals in the food chain.

<u style="single">Example 6: Ad5-VEE / WEE / EEE Horse Vaccine</u> So far, vaccination is the only effective means of treating the highly infectious mosquito-borne encephalitis alpha virus. All horses in North America are at risk and vaccination is recommended. Currently commercially available trivalent vaccines manufactured by conventional techniques require multiple injections and booster immunizations per year to provide protection. The "live vaccine" approach using adenovirus provides a safe means of providing rapid and sustained protection using a single intranasal administration.

<u style="single">Example 7: Co-administration of Ad5-IFN with one or more secondary antivirals</u> The Ad5-IFN delivery vehicle (eg, encoding conIFN-α or another IFN described herein) is pharmaceutically acceptable for intranasal administration in combination with antihistamines and neuraminidase inhibitors. It can be formulated with an excipient. The composition can be administered to the subject prior to viral exposure or within 48 hours of exposure. Antihistamines help reduce nasal congestion or obstruction caused by any nasal obstruction, eg, viral infection or rhinitis, thereby dispersing Ad5-IFN and neuraminidase inhibitors and epithelium of the upper and / or lower respiratory tract. Maximize its absorption by. Examples of such antihistamines can be H1 antagonists such as fexofenadine or loratadine. Neuraminidase inhibitors such as zanamivir (Relenza®, GlaxoSmithKline) are potent selective inhibitors of viral neuraminidase glycoproteins that are important for viral replication of, for example, influenza A and B viruses and other viruses. The net effect of the combination of these three drugs is improved virus protection, where IFN elicits a broad immune response, neuraminidase inhibitors block the release of virus from infected cells, and antihistamines nasal. Ensure or improve delivery of the drug to the epithelium.

Alternatively, the Ad5-IFN delivery vector can be administered intranasally as a separate composition, with antihistamines and neuraminidase inhibitors (eg, oseltamivir phosphate (Tamiflu®, Roche Pharma)). It can be administered orally in separate or single compositions (see, eg, US Pat. No. 6,605,302, incorporated herein by reference).

<u style="single">Example 8: Prevention or treatment of the Punta Toro virus (Bunyaviridiae)</u> Rift Valley fever virus (RVFV) is an arthropod-borne viral fever that causes direct infection in humans and livestock. The mode of transmission is by being bitten by infectious Aedes or Culex mosquitoes. Mechanical infections with aerosols or infected blood have been reported in persons who work with, handle, or process livestock or contaminated carcasses. People of all ages, both male and female, are susceptible and infected with RVFV can develop retinitis, encephalitis, or hepatitis with fatal bleeding (Heyman, American Public Health Association, Washington DC, 2008). .. A recent outbreak in Kenya killed 118 people with a case fatality rate of 29% (CDC, Morb.Motal.Wkly.Rep.56: 73-76, 2007). There is no approved vaccine or effective treatment for RVFV. Reflecting the concerns of public health officials, RVFV is the National Institute for Allergic and Infectious. It is classified as a Category A pathogen by Diseases and has a "Dual Agent" status by the Department of Health and Human Services and the US Department of Agriculture. ..

There is a strong need for effective measures that are highly stable, easy to administer, and induce long-lasting defensive immunity. Working directly with the RVFV is extremely restrictive and requires an advanced BSL-3 + facility, so we recently used the Syrian hamster in the intranasal (IN) respiratory pathway Punta Toro. A virus (PTV) infection model has been established. PTV is a BSL-2 surrogate of RVFV that causes disease in hamsters that model RVFV infection and disease progression in humans (Gowen et al., Antiviral Res. 77: 215-224, 2008).

The purpose of this experiment was to evaluate Ad5-IFNα as a prophylactic agent that abolished exposure to PTV. The route of Ad5-IFNα exposure was by intranasal (IN) -a proposed route of administration in humans-which stimulates respiratory mucosal surface delivery. Ten⁸、10⁷, And 10⁶A dose of PFU Ad5-IFNα (n = 15) was administered 24 hours prior to PTV infection attack. The doses selected were scaled to the hamster model based on previous studies demonstrating high levels of protection and based on typical dose extrapolation using body surface area. As shown in Figure 6, 100% survival compared to ribavarin treatment, empty vector treatment, and placebo control when Ad5-IFNα was administered at the dose shown at least 24 hours prior to attack by PTV. It became.

In addition, we present the airways and subcutaneously in mice treated with Ad5-IFNα as a) prophylaxis before attack (up to 21 days before attack) and b) up to +48 hours after exposure. Demonstrated significant protection against both PTV attack infections.

<u style="single">Example 9: Prevention or treatment of western horse encephalitis (Togaviridae)</u> Western horse encephalitis belongs to the genus Alphavirus of the Togavirus family, which corresponds to a group of emerging mosquito-borne, severely neuropathogenic pathogens in domestic and humans. WEEV is a local disease in western North America that is maintained in nature throughout a cycle involving wild birds as host and Culex tarsalis mosquitoes as vectors (Wu et al., Virology 369: 206-213, 2007). ), The overall case lethality rate is 3% -8% depending on age.

As a weapon, WEEV can be easily transmitted via the aerosol pathway, with case fatality rates as high as 40% in laboratory accidents (Hanson et al., Science 158: 1283-1286, 1967). A closely related virus-Venezuelan Encephalitis Virus-was weaponized by the United States and the former Soviet Union to spray aerosols as a battlefield incapacitating agent. It was predicted that biological weapons attacks in areas inhabited by vector horses and mosquitoes could cause epidemics (Eitzen et al., Medical Management of Biological Casualties, 3rd Edition, US Army Medical Research Institute for Infectious Diseases (US. Department of Defense by Army Medical Research Institute of Infectious Disease, Fort Detrick, Frederick MD Published for Defense), 1998). Concerns about these viruses, which continue to exist as existing biological weapons, and the lack of safe and effective vaccines or antiviral drugs raise public health concerns, and these viruses are listed as Category B bioterrorism threats by the CDC. (CDC, Centers for Disease Control and Prevention; Public Health Assessment of Potential Biological Terrorsm Agents, Volume 8, 2010).

This study used 140 female Balb / c mice (10 / group) and was divided into two trials; each used a total of 70 mice. The first study examined efficacy against the WEEV California strain and the second study examined efficacy against the WEEV CBA 87 strain. The following treatment groups were used for both trials: Group 1-5: (Days-21, -14, -7, -1 or +4 hours, respectively) 10⁷Single IN treatment with PFU Ad5-IFNα Group 6-IFNα B / D (recombinant mouse) 2 × 10 once daily from 0 to 8 days starting 4 hours before attack⁷IU / kg Group 7-Control: Attack untreated

Lethal dose of 2.5 x 10 in all mice in Test 1³Intranasal attack on day 0 with pfu WEEV California strain and 500 pfu WEEV CBA87 strain in Study 2, followed for clinical signs of disease for 14 days, euthanasia upon moribund / morbidity I let you. Administration of Ad5-IFNα (mice) provided complete protection in all animals in the prophylaxis window, and survival rates were 100% (California) and 70% (CBA87) in the +4 hour treatment group (Figure 7A). And Figure 7B).

<u style="single">Example 10: Prevention or treatment of severe acute respiratory syndrome (Coronaviridae)</u> SARS has recently emerged in the human population as a fatal respiratory disease. Severely affected patients develop acute respiratory distress syndrome, which corresponds to diffuse alveolar injury at autopsy. The newly discovered coronavirus SCV has been identified as the leading cause of SARS. Patients with SARS have been experimentally treated with a combination of ribavirin, oseltamivir, antibiotics and corticosteroids and have been partially successful. Treatment with recombinant human interferon (Alfacon®) has been shown to be clinically promising.

50 μl Ad5-IFNα (mice, 10) once in groups of 10 mice at 14, 7, 5, or 3 days prior to virus exposure (PVE)⁶PFU) was administered IN. In addition, 50 μl of Ad5-IFNα (mice) (10) once in groups of 10 mice at 6, 12, and 24 hours after virus exposure.⁶PFU or 10⁵PFU) was administered IN. In both experiments, poly ICLC was given at 1 mg / kg via the IN pathway 24 hours before virus exposure and 8 hours after virus exposure, served as a positive control to control viral infection, and 15 mice were fed. At each time point, treatment with buffered physiological saline was used as a placebo control. Animal mortality was recorded for up to 21 days after exposure to the virus.

As shown in FIGS. 8A and 8B, treatment with Ad5-IFNα (mice) provided complete protection for all animals in the treatment group.

<u style="single">Example 11: Prevention or treatment of yellow fever virus (Flaviviridae)</u> Yellow fever (YF) is an acute infectious viral disease characterized by jaundice and bleeding symptoms with a case fatality rate of 20-50%. YF is transmitted by mosquitoes in urban areas, typically Aede spp, and in forest areas by Haemogogus spp or Sabethes spp, and is carried by humans or primates. .. YF has an endemic zone between 15 degrees north and 10 degrees south, including 33 African countries, 9 South African countries and Caribbean islands, with a total population of over 500 million (Heymann, "Control". of Communicable Disease Manual, American Public Health Association, Washington, DC, 2008). Effective vaccines are available, but immunization coverage is variable, ranging from 30-95% in Africa. There is no approved treatment.

For hamsters (15-20 animals / group), 0.1 ml diluted virus (10 CCID)₅₀/ Animal) was injected intraperitoneally (IP). Ad5-IFNα is 1 × 10⁸、5×10⁷、5×10⁶Or 5x10⁵、1.25×10⁶The dose of PFU / animal was administered by intranasal infusion once every 4 hours. Mortality was observed daily for 21 days and body weight was recorded at 0, 3, and 6 dpi. Liver tissue was removed from 5 animals in each group by necropsy for virus titer measurement at 4 dpi. In the second study, animals were 5 x 10 at -4 hours, or days after infection (dpi) +1 day, +2 or +3 days.⁷PFU IN Ad5-IFNα was administered and the same controls as in the previous experiment were used.

Ad5-IFNα 1 × 10⁸pfu and 5x10⁷Full protection of hamsters was observed at the top two doses of pfu (Fig. 9A). There was a dose response in which lower doses resulted in higher mortality, however, these groups had significantly improved survival and delayed mortality curves compared to the subjects. Overall, all Ad5-IFNα doses provided significant protection compared to empty adenovirus vector controls, with similar or higher potency compared to positive control potency. 5 × 10⁷Using the Ad5-IFNα dose of PFU, complete survival was observed on day +1 and 90% survival at + 2 dpi (Fig. 9B).

<u style="single">Example 12: Treatment of Ebola virus (Filoviridae)</u> Ebola was first recognized in 1976 with two simultaneous outbreaks in Sudan and Zaire, where more than 600 people were affected, with 55% and 90% case fatality rates, respectively. Human-to-human contact is ensured through direct contact with blood, secretions, organs, or semen from an infected person. Nosocomial infections were frequent, and virtually everyone infected from contaminated needles died. Despite extensive research, no natural animal holdings for Ebola are yet known. There is no approved vaccine or effective treatment for filovirus infection (Heymann, "Control of Communicable Disease Manual", American Public Health Association, Washington, DC, 2008).

Ebola virus is considered a Category A bioterrorist factor by the CDC (CDC, 2010, above) and is a top priority public health biological threat (PHEMCE), Public Health Emergency Medical Countermeasures Enterprise, Health. &amp; Human Services, Washington DC, 2007). Such factors can be easily transmitted or transmitted from person to person, resulting in high mortality, potentially significant impacts on public health, and require special attention to public health preparation. And poses a risk to national security.

Here, Ad5-IFNα was tested in a mouse and guinea pig model of the Ebola virus Zaire strain (ZEBOV). 1000 x LD by intraperitoneal (IP) injection of a group of 10 mice₅₀Attacked with the mouse-adaptive Ebola virus. After 30 minutes, a single dose of 1 × 10 per mouse by either the IM route (50 μl for each hind limb) or the IN route (50 μl)⁷IFU (infection unit) mAd5-IFNα was administered. Control mice were IM-injected with phosphate buffered saline (PBS) (50 μl for each hind limb). A complete survival effect was observed by administration of mouse mAd5-IFNα by either route, and there was no significant weight loss in the treatment group compared with the control (Fig. 10A).

After successful mouse testing, Ad5-IFNα was tested in a guinea pig (GP) model of the Ebola virus Zaire strain (ZEBOV). The GP model more closely mimics the pathophysiology of disease in humans, and animals are more sensitive to attack, making it a more difficult model to achieve positive results. 8 Hartley guinea pigs 100 × LD by IP injection₅₀Attacked with ZEBOV adapted to guinea pigs. After 30 minutes, 2 x 10 per guinea pig in 2 animals⁸PFU Ad5-IFNα was administered IN. In addition, recombinant IFN protein was administered to 3 GPs daily for 6 days to evaluate the therapeutic potential of the protein alone, while 3 animals were untreated and served as a negative control group. All animals treated with Ad5-IFNα survived compared to 66% of the interferon protein group, while all control animals died (Fig. 10B).

<u style="single">Example 13: Prevention of pitindevirus (Arenaviridae)</u> Arenaviruses cause acute viral illness, progressing to severe multiple organ dysfunction in 20% of patients, with inpatient mortality rates of up to 15%. The disease becomes severe during pregnancy, with a fetal loss rate of up to 80% and a associated high frequency of maternal mortality. Arenaviruses are serologically divided into the Old World (eg Lassa fever) and the New World (eg Machubo or Funin). Lassa fever has the greatest impact on public health due to hemorrhagic fever, with more than 100,000 cases of regional epidemics in West Africa and 5,000 deaths each year (Fischer-Hoch et al., J. Virol). .74: 6777-6783, 2000). The mode of transmission is through aerosols, by direct contact with contaminated rodent excrement, or from person to person by pharyngeal secretions, semen or urine.

The arenavirus is considered a Category A bioterrorist factor by the CDC (CDC, 2010, above) and is the preferred public health biological weapons threat (PHEMCE, 2007, above). Such factors can be easily transmitted or transmitted from person to person, resulting in high mortality, potentially significant impacts on public health, and require special attention to public health preparation. And poses a risk to national security. Pitindevirus (PCV) is a New World arenavirus that is extremely pathogenic in hamsters but non-pathogenic in humans (Buchmeier et al., Infect. Immun. 9: 821-823, 1974). PCV infection in hamsters is a well-characterized animal model that develops fulminant disease through vascular leak syndrome, which increases systemic viral titers, and finally leads to terminal shock death. The distribution of viral antigens within infected hosts (Connolly et al., AJTrop.Med.Hyg.4; pp. 10-24, 1993) mimics the disease symptoms reported in human arenavirus cases (Walker et al., Am.J.Path. 107: 349-356, 1982), can be safely used under BSL-2 conditions (Gowen and Holbrook, Antiviral Res. 78: 79-90, 2007).

Ad5-IFNα was tested in a hamster model infected with pitindevirus. 1 day before attack, 10 animals per hamster by IN route to a group of 10 animals⁸、10⁷Or 10⁶A single dose of any of PFU's Ad5-IFNα was administered (200 μl). LD by intraperitoneal (IP) injection of animals₉₅Attacked with a hamster-adaptive PCV. Control mice were treated with phosphate buffered saline (PBS) IN (100 μl per nostril). A complete survival effect was observed with the highest dose of Ad5-IFNα, and a dose-dependent decrease in survival was observed at lower levels (Fig. 11).

<u style="single">Example 14: Treatment with a combination "quick-acting vaccine" for Ebola (Filoviridae)</u><u style="single">Ad5-IFNα given with the vaccine</u> Since Jenner in 1796, vaccines have been the basis of effective infectious disease prevention. Vaccines are cost-effective, easy to administer, generally safe and durable. However, in the face of the biological weapons threat, widespread national vaccine campaigns have received considerable opposition. Prejudice against vaccination is the result of the public balancing the risk of a low-probability bioweapon threat with the definite adverse effects of the vaccine on a small number of patients. In fact, even the first and only infectious disease vaccination campaign ever eradicated was canceled about 30 years ago, despite the president's support for vaccination of police and health care workers. .. The second public health issue is time dilation. Vaccines provide gradual (often 7-21 days) protection against vaccination and booster immunization. This time delay has fatal consequences or infection with the most pathogenic viral biological weapons. Therefore, current public health vaccination strategies and stockpiles are aimed at disease mitigation and prevention of secondary infections and disease epidemics. Those infected at Ground Zero only receive symptomatic care. We propose to use Ad5-IFNα and vaccines to radically change this disease management paradigm to include treatment and prevention. In addition, existing vaccine stockpiles can be repurposed here and used as part of a "quick-acting vaccine".

It is clear that Ad5-IFNα can act as both prophylactic and therapeutic. In this example, we combine Ad5-IFNα (which acts as a type of adjuvant) with a standard vaccine to form a "quick-acting vaccine." The advantage of this method is great. Ad5-IFNα acts as an immune system stimulant, which has the following advantages; a) Ad5-IFNα can be administered with a vaccine to protect the host from viral damage as long as the vaccine is functioning. And b) Ad5-IFNα stimulates the immune system to make the response to the vaccine faster or stronger, thus allowing faster establishment of protective antibody levels.

In the case of Ebola, the inventors described Ad5-IFNα as an Ad5 vectorized Ebola glycoprotein vaccine (Ad-CAGoptZGP; Richardson et al., 2009, supra; Croyle et al., PLoS 3: 1-9, 2008). ), And demonstrated the method and effect of a fast-acting vaccine. Ad5-IFNα (2 × 10) against 6 guinea pigs 30 minutes after 1000LD50 attack by ZEBOV⁸Vaccine containing PFU (10)⁹Or 10¹⁰Infection unit) was administered by IN administration. This combination treatment resulted in 100% survival of the animals (Fig. 12). Ad5-IFNα alone survived 50% of the attacked animals, and vaccine alone survived only 30% in the 1/10 model of attack. Therefore, the synergistic action of the two elements survives animals that would not have been individually saved from the attack by Ebola.

Given this data, Ad5-IFNα has significant potential to act as a vaccine adjuvant for a wide range of vaccines, thereby accelerating time to defense in both prophylactic and therapeutic models.

<u style="single">Example 15: Vaccine stability</u> The present inventors have developed a highly durable storage-stable preparation of the combination therapy Ad5-IFNα + Ad-CAGoptZGP. Preliminary test data from the present inventors show that the Ad5 vector is stable and there is no noticeable loss of activity at 37 ° C for 84 days and at 100 ° C for at least 1 hour ( ASM 2010).

<u style="single">Example 16: Safety data</u> There is a large amount of clinical data showing that the Ad5 vector system and recombinant human IFN are individually safe (including when administered using multiple multiple doses). In addition, in Germany, the single use of Ad-CAGoptZGP has been successful in treating a researcher's suspected Ebola infection. The patient developed fever and headache, commonly associated with antiviral vaccines, but achieved complete recovery.

The doses of Ad5-IFNα + Ad-CAGoptZGP as assessed in the mouse and guinea pig ZEBOV models discussed above demonstrate safety even at the highest expected doses. In our experience to date, at lower doses of Ad5-IFNα (as low as 1/1000) used in animal models of other diseases (eg Punta Toro, WEE, and SARS). Even if there is, it shows excellent efficacy. This result, taking into account the synergistic relationship of the two factors (Ad5-IFNα + Ad-CAGoptZGP), would be substantially effective against pathogen infections, such as Ebola infection, even at lower doses. Is shown.

To date, more than 60 clinical trials using Ad5 as a gene delivery vector have provided a solid toxicity research framework for the Ad5-IFNα-containing compositions of the present invention (eg, Ad5-IFNα and Ad-CAGoptZGP). Provided (including combination with). For example, in humans, the combination of Ad5-IFNα and Ad-CAGopt ZGP is 1.0 × 10 for a 70 kg person.⁶~1.0×10¹²(For example, 1.6x10⁹Dosings in the range of PFU) are expected to provide therapeutic and prophylactic effects against the attack or exposure of pathogens (eg, viral factors). In the animal model test, the present inventors have successfully tested the combination of Ad5-IFNα and Ad-CAGoptZGP with a virus particle (vp) to PFU ratio of 10: 1. For example, for a 50: 1 virus particle (vp) to PFU ratio, a higher dosing range limit is expected, with a dose of 8x10.¹⁰Can be vp.

<u style="single">Safety of replication-deficient Ad5 vector</u> The safety of the replication-deficient Ad5 vector has been confirmed in a dose-escalation study involving 12 patients with Ad5 delivered intranasally (2 x 10).⁷~2×10¹⁰PFU / Patients; see Knowles et al., NEJ Med. 333: 823-831, 1995). At the highest dose, the adverse effects were considered moderate (ear pain and mucosal hypersensitivity) and resolved within 3 weeks. Recently, dose-limiting toxicity of 2 × 10 in a pilot phase I safety study¹²In vp, it was pointed out that repeated doses of the Ad5 vector were well tolerated (see Keedy et al., J. Clin. Oncol. 26: 4166-4171, 2008). The NIH Recombinant DNA Advisory Committee (NIH Report, Hum.Gene Ther. 13: 3-13, 2002) states that the safety limit for replication-deficient Ad5 vectors is non-toxic. 7 × 10¹³Reported as vp. Using these previous studies, we predict that the effective dose of combination therapies such as Ad5-IFNα and Ad-CAGoptZGP will be at least 1-2 orders of magnitude lower than the safe dose low threshold for Ad5 administration.

<u style="single">Interferon safety</u> Interferon is used clinically and safely for the treatment of hepatitis C and SARS, where high dose side effects can be flu-like symptoms such as elevated body temperature, headache, myalgia, convulsions, and dizziness. In some cases, thinning hair and depression have also been observed. For high-risk melanoma, maximum capacity is used daily for 1 month (4.5 x 10)⁵U / kg) (see Jonasch et al., Cancer J. 6: 1390145, 2000), followed by half doses used three times a week for 48 weeks. The IFN level in the bloodstream obtained 12 hours after injection can be estimated to be approximately 230 U / mL (see Cantel et al., J. Gen. Virol. 22: 453-455, 1974). Serum IFN levels measured in our mouse model were 250 U / mL (see Wu et al., Virology 369: 206-213, 2007). Again, this comparison shows that our maximum expected dose results in serum IFN levels consistent with those found in patients receiving antiviral therapy.

<u style="single">Other embodiments</u> Although the present invention has been described in connection with a particular embodiment thereof, further modifications are possible, and the present application generally follows the principles of the present invention and is related to the present invention. It is intended to embrace any modification, use, or adaptation of the invention, including such deviations from the present disclosure, which are within the scope of known or common practice in the art, which may be applicable to such features. That will be understood.

All publications and patent applications described herein are by reference to the same extent as if each individual publication or patent application was specifically and individually indicated as being incorporated by reference in its entirety. Incorporated in the specification.

<u style="single">appendix</u><u style="single">Interferon Alpha 1b-IFNA1</u>Nucleotide: NCBI Reference Sequence: NM_024013.1 Human (Homo sapiens) (SEQ ID NO: 1)<chemistry num="1"><img id="000003" he="95" wi="160" file="JP5908397B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

Amino acid: NCBI reference sequence: NP_076918.1 Human (Homo sapiens) (SEQ ID NO: 2)<chemistry num="2"><img id="000004" he="27" wi="154" file="JP5908397B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

<u style="single">Interferon Alpha 2b-IFNA2</u>Nucleotide: NCBI Reference Sequence: NM_000605.3 Human (Homo sapiens) (SEQ ID NO: 3)<chemistry num="3"><img id="000005" he="137" wi="154" file="JP5908397B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

Amino Acids: NCBI Accession No. AAP20099 Human (Homo sapiens) (SEQ ID NO: 4)<chemistry num="4"><img id="000006" he="37" wi="154" file="JP5908397B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

<u style="single">Interferon beta 1a-IFNB1</u>Nucleotide: NCBI Reference Sequence: NM_002176.2 Human (Homo sapiens) (SEQ ID NO: 5)<chemistry num="5"><img id="000007" he="89" wi="152" file="JP5908397B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

Amino acid: NCBI reference sequence: NP_002167.1 Human (Homo sapiens) (SEQ ID NO: 6)<chemistry num="6"><img id="000008" he="29" wi="152" file="JP5908397B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

<u style="single">Interferon gamma-IFNG</u>Nucleotide: NCBI Reference Sequence: NM_000619.2 Human (Homo sapiens) (SEQ ID NO: 7)<chemistry num="7"><img id="000009" he="149" wi="152" file="JP5908397B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

Amino acid: NCBI reference sequence: NP_000610.2 Human (Homo sapiens) (SEQ ID NO: 8)<chemistry num="8"><img id="000010" he="22" wi="152" file="JP5908397B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

<u style="single">Interferon Tau-IFNT</u>Nucleotide: NCBI Reference Sequence: NM_001015511.2 Bos taurus (SEQ ID NO: 9)<chemistry num="9"><img id="000011" he="112" wi="154" file="JP5908397B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

Amino Acid: GenBank: AAK53058.1 Bos taurus (SEQ ID NO: 10)<chemistry num="10"><img id="000012" he="23" wi="154" file="JP5908397B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>

<u style="single">Consensus interferon (conIFN-α)</u>Amino acid: (SEQ ID NO: 11)<chemistry num="11"><img id="000013" he="22" wi="154" file="JP5908397B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>