WO2008157203A2

WO2008157203A2 - Methods of protecting animals from avian influenza infection

Info

Publication number: WO2008157203A2
Application number: PCT/US2008/066645
Authority: WO
Inventors: Javaraiah Srinivas; Chengjin Huang; Michael A. Gill; Hsien-Jue Chu
Original assignee: Wyeth
Priority date: 2007-06-15
Filing date: 2008-06-12
Publication date: 2008-12-24
Also published as: WO2008157203A3

Abstract

The present invention provides methods of vaccinating non-avian, non-human animals against avian influenza. The methods of the invention comprise administering to the animal at least one dose of a vaccine composition comprising an immunologically-effective amount of an inactivated influenza virus. The influenza virus may contain any combination of hemagglutinin and neuraminidase subtypes. An exemplary inactivated influenza virus for use in a vaccine composition of the invention is a reverse genetics H5N3 virus. The animals to which the vaccine composition is administered may be any non-avian, non-human animal such as a dog, a cat or a pig.

Description

METHODS OF PROTECTING ANIMALS FROM AVIAN INFLUENZA INFECTION

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

[0001] The present invention relates to the fields of immunology and virology. More specifically, the invention relates to avian influenza vaccines.

BACKGROUND ART

[0002] Influenza viruses that are capable of infecting avian species are commonly referred to as "avian influenza" viruses. Of the three types of influenza viruses (A₁ B and C), only influenza A viruses are known to infect birds. Influenza A viruses are further classified on the basis of the hemagglutinin (HA) and neuraminidase (NA) subtypes expressed by the viruses. (For a more thorough background on influenza viruses, see U.S. Patent Appl. Publ. No. 2006/0204976, and Horimoto and Kawaoka, CHn. Microbiol. Rev. 74:129-149 (2001 ), the disclosures of which are incorporated by reference herein in their entireties).

[0003] Sixteen different hemagglutinin subtypes (designated H1 through H16) and nine different neuraminidase subtypes (designated N1 through N9) have been thus far identified. An influenza virus may therefore be classified based on the hemagglutinin and neuraminidase subtype it expresses in terms such as HxNy, wherein x is a number from 1 through 16 and y is a number from 1 through 9. All known subtypes of influenza A viruses can infect birds and are therefore properly considered avian influenza viruses.

[0004] Avian influenza viruses are known to infect non-avian species. For example, the influenza A strain H5N1 has been found to infect humans, causing severe clinical disease and mortality. (To et a/.; J. Med. Virol. 63:242-246 (2001)). Avian influenza strains have also been reported to infect mice, ferrets, dogs, cats (including domestic cats and big cats such as leopards and tigers) and pigs. (See, e.g., Keawcharoen et al. Emerging Infectious Diseases 10:2λ 89-2191 (2004); Kuiken et al., Science 306:241 (2004)). Thus, there exists a need in the art for methods of protecting a wide variety of animals from avian influenza infection.

BRIEF SUMMARY OF THE INVENTION

[0005] The present invention satisfies the aforementioned need in the art by providing methods of vaccinating non-human, non-avian animals against avian influenza. The vaccination methods of the present invention are useful, e.g., for protecting animals from the clinical signs and symptoms associated with infection by either low pathogenic or high pathogenic avian influenza viruses.

[0006] In accordance with the present invention, methods are provided wherein a non-avian, non-human animal is administered a vaccine composition comprising an immunologically-effective amount of an inactivated influenza virus. The influenza virus included within the vaccine composition may have any combination of hemagglutinin and neuraminidase subtype. For example, the virus may have any one of M ₁ H2, H3, H4, H5, H6, H7, H8, H9, H10, H11 , H12, H13, H14, H15 or H16, in combination with any one of N1 , N2, N3, N4, N5, N6, N7, N8 or N9. In certain exemplary embodiments, the inactivated influenza virus has an H5 hemagglutinin and an N1 , N2, N3, N4, N5, N6, N7, N8 or N9 neuraminidase.

[0007] The present invention is directed to the use of inactivated influenza viruses in vaccine compositions and thus specifically excludes methods that comprise infecting an animal with a virulent (live) avian influenza virus. For example, experimental studies in which an animal is challenged with a live virus to determine if the animal is susceptible to avian influenza virus infection and/or disease are not within the scope of the present invention.

[0008] In certain embodiments of the present invention, the non-avian, non- human animal to which the vaccine composition is administered is a dog, a cat or a pig. As demonstrated by the non-limiting working Examples presented below, an inactivated H5N3 influenza virus constructed by a reverse genetics methodology induced significant immune responses when administered to dogs, cats and pigs. These Examples confirm the broad applicability of the vaccination methods of the present invention for protecting a variety of animal species from avian influenza.

DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention provides methods for vaccinating non-avian, non- human animals against avian influenza. The methods of the invention comprise administering to an animal at least one dose of a vaccine composition comprising an immunogenically-effective amount of an inactivated influenza virus. The inactivated influenza may contain any hemagglutinin subtype and any neuraminidase subtype. For example, the virus may have an H5 hemagglutinin and an N1 , N2, N3, N4, N5, N6, N7, N8 or N9 neuraminidase.

INFLUENZA VIRUSES

[0010] In certain embodiments, the inactivated influenza virus contained within the vaccine composition of the invention is not an equine influenza virus or a canine influenza virus. For example, certain embodiments of the present invention specifically exclude the use of inactivated equine influenza viruses such as the viruses known as equine/Kentucky/04, equine/Wisconsin/03 or equine/Ohio/03, (International Patent Appl. Publ. WO2007/047728), equine/2/Miami/1/63, equine/Kentucky/1998, equine/Kentucky/15/2002, equine/Kentucky/1/1994, equine/Massachusetts/213/2003, equine/NewYork/1999, and/or equine/Newmarket/A2/1993. (U.S. Patent Appl. Publ. No. 2007/0082012). Alternatively, or additionally, certain embodiments of the present invention specifically exclude the use of inactivated canine influenza viruses such as the viruses known as canine/FL/04, canine/FL/03, canine/TX/04, canine/Jax/05, canine/Miami/05 (International Patent Application Publication No. WO2006/116082), New York/05 (International Patent Appl. Publ. WO2007/047728), A/canine/lowa/13628/2005 (U.S. Patent Appl. Publ. No. 2007/0098742), and/or canine/lowa/9A1/B5/08/D12 (U.S. Patent Appl. Publ. No. 2007/0082012). Particularly, where the animal to which the vaccine composition is administered is a canine, the inactivated influenza virus is not an H3N8 influenza virus.

[0011] Preferably, the inactivated influenza virus is an avian influenza virus. Exemplary inactivated influenza viruses that can be included in the vaccine compositions of the present invention include inactivated avian influenza viruses having hemagglutinin and neuraminidase combinations such as, e.g., H1 N1 , H2N3, H3N8, H4N6, H6N2, H7N7, H8N4, H9N2, H10N7, H11 N6, H12N5, H13N6, H5N1 , H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8 and H5N9.

[0012] In certain embodiments, the inactivated influenza virus is an influenza virus having an H5 hemagglutinin. The H5 hemagglutinin may be derived from any naturally occurring influenza virus having an H5 hemagglutinin subtype. An exemplary influenza virus from which an H5 hemagglutinin may be derived is A/Chicken/Vietnam/C58/04.

[0013] In certain exemplary embodiments, the inactivated influenza virus has an N1 neuraminidase or an N3 neuraminidase. If the inactivated influenza virus has an N1 neuraminidase, the N1 neuraminidase may be derived from, e.g., an influenza virus selected from the group consisting of: A/PuertoRico/8/34, A/WSN/33, and A/Tea I/HK/W312/97. If the inactivated influenza virus has an N3 neuraminidase, the N3 neuraminidase may be derived from, e.g., A/Duck/Germany/1215/73.

[0014] Other viruses from which the hemagglutinin or neuraminidase gene segments may be derived are discussed elsewhere herein and are well known in the art. (See, e.g., Horimoto and Kawaoka, CHn. Microbiol. Rev. 14: 129-149 (2001 )).

[0015] As used in the context of the present invention, a hemagglutinin or neuraminidase is "derived from" a particular virus if the hemagglutinin or neuraminidase is encoded by a polynucleotide that is cloned, reverse transcribed, amplified or otherwise artificially synthesized from the virus in question, or if the hemagglutinin or neuraminidase is obtained from the virus in question through a reassortment process. As a non-limiting example, a cDNA copy of an H5 hemagglutinin gene can be reverse transcribed from isolated RNA of a particular influenza virus strain and cloned into an expression vector. The expression vector expressing the H5 cDNA can then be expressed in a cell line along with other influenza genes (from the same or other influenza virus strains) to produce a recombinant influenza virus containing the H5 of the initial influenza virus strain. (See, e.g., U.S. Patent No. 6,951,754). In this example, the H5 is said to be "derived from" the initial influenza virus.

[0016] In certain embodiments of the present invention, the inactivated influenza virus is a naturally obtained influenza virus that is subjected to inactivating conditions. Naturally obtained influenza viruses may be obtained from, e.g., infected animals including birds and other animals that have been infected with avian influenza virus in the wild.

[0017] Alternatively, the inactivated influenza virus may be a recombinant influenza virus. Recombinant influenza viruses may be obtained using conventional egg-based methods in which two influenza strains with the desired features for a new vaccine (e.g., HA and/or NA subtype) are injected into an egg where their genes reassort naturally. Recombinant viruses can also be obtained by introducing multiple (e.g., two) influenza strains into tissue cultures where natural reassortment is allowed to occur.

[0018] Alternatively, recombinant influenza viruses for use in the context of the present invention may be obtained using recombinant DNA techniques which may or may not involve the use of helper viruses. Exemplary plasmid-based or "reverse genetics" techniques that can be used to make influenza virus strains for use in the context of the present invention are described, e.g., in U.S. Patent Nos. 6,649,372, 6,887,699, 6,951 ,754, and in U.S. Patent Appl. Publ. Nos. 2005/0003349, 2005/0037487 and 2006/0057116. For example, the "dual promoter" reverse genetics technique of U.S. Patent No. 6,951 ,754, and variations thereof, can be used to make recombinant influenza viruses that can be used in the context of the vaccination methods of the present invention.

[0019] An exemplary plasmid-based method that can be used in the context of the present invention comprises introducing a set of plasmids into a host cell in vitro. The set of plasmids comprises a first plasmid that expresses H5 hemagglutinin and a second plasmid that expresses N1 , N2, N3, N4, N5, N6, N7, N8 or N9 neuraminidase. The set of plasmids may additionally comprise one or more plasmids that express avian influenza PB1 , PB2, PA, NP₁ M1 , M2, NS1 and NS2 proteins. In certain embodiments, the PB1 , PB2, PA, NP, M1 , M2, NS1 and NS2 proteins are each expressed from a separate plasmid. Alternatively, more than one (e.g., 2, 3, 4, 5, 6, 7, or 8) of these proteins may be expressed from a single plasmid. After the set of plasmids is introduced into a host cell in vitro, virus particles are recovered from the host cell. The recovered virus particles are then inactivated. Using a plasmid- based system such as the one described immediately above, recombinant influenza viruses having any combination of hemagglutinin and neuraminidase subtype can be easily constructed and isolated.

[0020] Amino acid and nucleic acid sequences of all known influenza gene segments can be easily obtained for use in constructing the recombinant influenza viruses of the present invention. For example, the Medical College of Wisconsin provides a searchable database (Influenza Primer Design Resource, "IPDR") which allows users to search for NCBI accession numbers corresponding to the sequences of the gene segments (PB1 , PB2, PA, HA, NP, NA, MP and NS) of all published influenza strains. Using this or similar databases along with well known molecular biology techniques, a person of ordinary skill in the art could easily construct recombinant influenza viruses with any combination of gene segments (e.g., any HA and NA segments) for use in the vaccine compositions of the present invention. Table 1 provides a non-limiting list of exemplary viral sources of HA and NA gene segments for use in constructing the recombinant influenza viruses of the present invention along with the NCBI accession numbers for the listed gene segments. TABLE 1

[0021] The influenza viruses that are included in the vaccine compositions of the present invention are preferably inactivated. Exemplary inactivating conditions include, e.g., chemical inactivation using chemical inactivating agents such as binary ethyleneimine, beta-propiolactone, formalin, gluteraldehyde, sodium dodecyl sulfate, or the like or a mixture thereof. The influenza viruses of the invention may also be inactivated by, e.g., heat or psoralen in the presence of ultraviolet light.

VACCINE DOSES, FORMULATIONS AND ADMINISTRATION REGIMENS

[0022] According to the present invention, a "dose" of a vaccine composition, is a quantity of vaccine composition that is administered at a particular point in time. A "dose" may also be a quantity of vaccine composition that is gradually administered to an animal using an extended release formulation and/or apparatus. According to the present invention, a dose is typically within the range of about 0.25 mL to about 2.0 mL. A dose of vaccine composition that can be administered to an animal in the context of the present invention can be a volume of vaccine composition of about, e.g., 0.25 mL, 0.30 mL, 0.35 mL, 0.40 mL, 0.45 mL, 0.50 mL, 0.55 mL, 0.60 mL, 0.65 mL, 0.70 mL, 0.75 mL, 0.80 mL, 0.85 mL, 0.90 mL, 0.95 mL, 1.0 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2.0 mL, etc.

[0023] In certain embodiments of the present invention, two or more doses of the vaccine composition are administered to an animal at different time points. For example, the present invention includes vaccination methods in which a first dose of vaccine composition is administered to the animal at a first time point, and then a second dose of vaccine composition is administered to the animal at a second time point (e.g., booster vaccination). The second time point may be between 1 and 90 days after the first time point. For example, in methods that involve multiple administrations of the vaccine composition to the animal, the second time point may be 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27 or more days after the first time point.

[0024] According to the present invention, an "immunologically-effective amount" of an influenza virus (e.g., an inactivated influenza virus) is an amount of influenza virus (usually expressed in terms of hemagglutinating units or "HA units") which will induce complete or partial immunity in a treated animal against subsequent challenge with a virulent strain of avian influenza virus. Complete or partial immunity can be assessed by observing, either qualitatively or quantitatively, the clinical symptoms of influenza virus infection in a vaccinated animal as compared to an unvaccinated animal after being challenged with a virulent strains of avian influenza virus. Where the clinical symptoms of influenza virus infection in a vaccinated animal after challenge are reduced, lessened or eliminated as compared to the symptoms observed in an unvaccinated animal after a similar or identical challenge, the amount of influenza virus that was administered to the vaccinated animal is regarded as an "immunologically-effective amount." [0025] Exemplary amounts of inactivated influenza virus that may be regarded as "immunologically-effective amounts" include amounts between about 1 HA unit per dose to about 1000 HA units per dose. For example, depending on the size and species of animal to which the vaccine is administered, an "immunologically-effective amount" of inactivated influenza virus may be, e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, or 500 HA units per dose.

[0026] Methods of determining HA units are known in the art. As used herein, an HA unit is defined as the reciprocal of the highest dilution of an influenza virus- containing sample which causes visible hemagglutination when combined with erythrocytes.

[0027] In addition to an immunogenically-effective amount of an inactivated influenza virus, the vaccine compositions of the present invention may also comprise a pharmacologically acceptable carrier. Exemplary pharmacologically acceptable carriers include water, saline, or phosphate or other suitable buffers. The vaccine compositions may also comprise an oil component. For example, when an oil component is included, the vaccine composition can be formulated as a water-in-oil or oil-in-water emulsion. Also contemplated are double emulsions, often characterized as water-in-oil-in-water emulsions. The oil may help to stabilize the formulation and further function as an adjuvant or enhancer. Suitable oils include, without limitation, white oil, Drakeoil, squalane or squalene, as well as other animal, vegetable or mineral oils, whether naturally-derived or synthetic in origin.

[0028] In addition, the vaccine compositions of the present invention may contain other suitable adjuvants available in the art. These can include, e.g., Carbopol, dimethyl dioctadecyl ammonium bromide (DDA), aluminum hydroxide and aluminum phosphate as well as other metal salts. [0029] The vaccine compositions of the present invention may, in certain embodiments, contain a lipopolysaccharide, e.g., a bacterial lipopolysaccharide. Bacterial lipopolysaccharide derived adjuvants may be purified and processed from bacterial sources, or alternatively they may be synthetic. For example, purified monophosphoryl lipid A is described in Ribi et al., "Immunology and lmmunopharmacology of Bacterial Endotoxins," Plenum Publ. Corp., NY, pp. 407- 419 (1986), and 3-O-Deacylated monophosphoryl or diphosphoryl lipid A derived from Salmonella sp. is described in GB 2220211 and U.S. Patent No. 4,912,094. Other purified and synthetic lipopolysaccharides have been described, e.g., in Hilgers et al., Int. Arch. Allergy. Immunol., 79(4): 392-396 (1986); Hilgers et al., Immunology, 60(1 ):141-146 (1987); and EP 0 549 074 B1 ). An exemplary bacterial lipopolysaccharide adjuvant is 3-O-Deacylated monophosphoryl lipid A (3D-MPL). An exemplary form of 3D-MPL is in the form of an emulsion having a small particle size less than 0.2 μm in diameter, and its method of manufacture is disclosed in WO 94/21292. Aqueous formulations comprising monophosphoryl lipid A and a surfactant have been described in WO98/43670.

[0030] The vaccine compositions of the present invention may, in certain embodiments, also contain saponins. Saponins are described in Lacaille-Dubois, M and Wagner H., "A review of the biological and pharmacological activities of saponins," Phytomedicine, vol 2, pp 363-386, (1996). Saponins are steroid or triterpene glycosides widely distributed in the plant and marine animal kingdoms. Saponins are noted for forming colloidal solutions in water which foam on shaking, and for precipitating cholesterol. When saponins are near cell membranes they create pore-like structures in the membrane which cause the membrane to burst. Haemolysis of erythrocytes is an example of this phenomenon, which is a property of certain, but not all, saponins. Saponins are known as adjuvants in vaccines for systemic administration. The adjuvant and haemolytic activity of individual saponins has been studied in the art. For example, Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina), and fractions thereof, are described in U.S. Patent No. 5,057,540 and in Kensil, Crit. Rev. Ther. Drug. Carrier. Syst., 12 (1- 2): 1-55 (1996); and in EP 0 362 279 B1. Particulate structures, termed Immune Stimulating Complexes (ISCOMS), comprising fractions of Quil A are haemolytic and have been used in the manufacture of vaccines (EP 0 109 942 B1 ; WO 96/11711 ; WO 96/33739). The haemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A) have been described as potent systemic adjuvants, and the method of their production is disclosed in U.S. Patent No. 5,057,540 and EP 0 362 279 B 1. Other saponins which have been used in systemic vaccination studies include those derived from other plant species such as Gypsophila and Saponaria (Bomford et a/., Vaccine 10(9):572-577 , (1992)).

[0031] Additional excipients may also be included in the vaccine compositions of the present invention, including, e.g., surfactants or other wetting agents or formulation aids. Surfactants can include the sorbitan mono-oleate esters (TWEEN® series), as well as the ethylene oxide/propylene oxide block copolymers (PLURONIC® series), as well as others available in the art. Additional non-ionic surfactants include Triton X-45, t-octylphenoxy polyethoxyethanol (Triton X-100), Triton X-102, Triton X-114, Triton X-165, Triton X-205, Triton X-305, Triton N-57, Triton N-101 , Triton N-128, Breij 35, polyoxyethylene-9-lauryl ether (laureth 9) and polyoxyethylene-9-stearyl ether (steareth 9).

[0032] Other compounds recognized as stabilizers or preservatives may also be included in the vaccine compositions of the present invention. These compounds include, without limitation, carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin or glucose and the like, as well the preservative formalin, for example.

[0033] The vaccine compositions of the present invention may also be formulated as a dry powder, substantially free of exogenous water, which may then be reconstituted by an end user prior to administration.

[0034] In certain embodiments, the vaccine compositions of the present invention contain one or more additional antigenic components in addition to the inactivated influenza virus. For instance, the vaccine composition may include multiple (e.g., 2, 3, 4, 5 or more) inactivated influenza viruses, each having a different combination of hemagglutinin and neuraminidase components. Alternatively, or additionally, the vaccine composition may include one or more DNA vaccine components (e.g., a vector that expresses a particular antigen in vivo), subunit vaccine components, or peptide or polypeptide antigenic components.

ADMINISTRATION OF THE VACCINE COMPOSITIONS TO ANIMALS

[0035] According to the methods of the present invention, the vaccine composition may be administered to any non-human animal, preferably a non-avian, non-human animal. In certain embodiments, the animal is a non-avian, non-human, non-equine animal. In certain other embodiments, the animal is a non-avian, non- human, non-canine animal. In yet other embodiments, the animal is a non-avian, non-human, non-equine, non-canine animal. In certain specific exemplary embodiments, the animal is a canine, equine, feline, porcine, or bovine animal. For example, the animal may be, e.g., a dog, horse, cat, gerbil, hamster, mouse, stoat, weasel, pig, rat or ferret. After being administered the vaccine composition of the present invention and then subsequently being challenged with a low pathogenic or high pathogenic avian influenza virus, the animal will be protected from the clinical symptoms of the infection.

[0036] According to the methods of the invention, any method of administration can be used to administer the vaccine composition to the animal. An exemplary method of administration is intradermal delivery. Any suitable device may be used for intradermal delivery in the context of the present invention. Exemplary short needle devices include those described in U.S. Patent Nos. 4,886,499, 5,190,521 , 5,328,483, 5,527,288, 4,270,537, 5,015,235, 5,141 ,496, and 5,417,662. Intradermal vaccines may also be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in WO99/34850 and EP1092444. Also suitable are jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis. Jet injection devices are described, for example, in U.S. Patent Nos. 5,480,381 , 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189, 5,704,911 , 5,383,851, 5,893,397, 5,466,220, 5,339,163, 5,312,335, 5,503,627, 5,064,413, 5,520,639, 4,596,556, 4,790,824, 4,941 ,880, and 4,940,460, as well as WO 97/37705 and WO 97/13537. Also suitable are ballistic powder/particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis. Additionally, conventional syringes may be used in the classical mantoux method of intradermal administration.

ISOLATED SERUM SAMPLES

[0037] In another aspect of the present invention, isolated serum samples are provided that exhibit hemagglutination inhibition (HI) titers of at least 10 when tested against an H5N3 avian influenza virus in a standard HI assay. The isolated serum samples are obtained from a non-avian, non-human animal. In certain embodiments, the isolated serum samples are obtained from a dog, a cat or a pig that has been administered at least one dose of an inactivated influenza virus having an H5 hemagglutinin and an N1 , N2, N3, N4, N5, N6, N7, N8 or N9 neuraminidase.

[0038] The expression "standard HI assay," as used herein means an assay in which a serum sample obtained from an animal, or a serial dilution thereof, is incubated with a constant amount (e.g., 2 to 20 HA units) of an inactivated avian influenza H5N3 antigen, the virus/serum mixture is incubated with an equal volume of 0.5% chicken red blood cell suspension, and then the samples are observed for hemagglutination. HI titer is defined as the reciprocal of the highest serum dilution causing inhibition of hemagglutination. For example, where the highest serum dilution causing inhibition of hemagglutination is 1/10, the HI titer is 10.

[0039] In certain exemplary embodiments, the isolated serum samples of the invention will exhibit an HI titer of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 460, 465, 470, 475, 480, 485, 490, 495, 500, or more when tested against an H5N3 avian influenza virus in a standard HI assay. [0040] The isolated serum samples of the invention may be obtained from an animal any time following the time at which the animal is administered a dose of inactivated influenza virus. For example, the samples may be obtained from the animal 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or more days after the animal has been administered the inactivated influenza virus.

[0041] In certain embodiments, the isolated serum sample is obtained from an animal after the animal has been administered two or more doses of the inactivated influenza virus. For example, the animal may be administered a first and a second dose of the inactivated influenza virus with the second dose being administered 7 to 21 days after the first dose is administered. In such embodiments, where two doses of the inactivated influenza virus are administered to the animal, the serum sample may be obtained at any time after the second dose is administered, such time points generally being referred to as "days post vaccination number two" or simply "DPV2." For instance, under circumstances in which two doses of the inactivated influenza virus are administered to the animal, the sample may be obtained from the animal 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more days after the second dose is administered (i.e., at 1 DPV2, 2DPV2, 3DPV2, 4DPV2, 5DPV2, 6DPV2, 7DPV2, 8DPV2, 9DPV2, 10DPV2, 11 DPV2, 12DPV2, 13DPV2, 14DPV2, 15DPV2, 16DPV2, 17DPV2, 18DPV2, 19DPV2, 20DPV2, 21 DPV2, 22DPV2, 23DPV2, 24DPV2, 25DPV2, 26DPV2, 27DPV2, 28DPV2, 29DPV2, 30DPV2 or more).

[0042] The serum samples of the present invention are useful for a variety of useful and practical purposes such as diagnostic reagents to assess for avian influenza vaccination or infection. For example, the samples can be used as standards in assays which measure the extent to which an animal (e.g., a dog, cat or pig) has generated an immune response against avian influenza after being vaccinated against avian influenza virus. The serum samples may also be used to compare the immune response generated in an. animal using a vaccine composition of the present invention to the immune response generated in an animal using a different avian influenza vaccine composition. [0043] The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in molecular biology and chemistry which are obvious to those skilled in the art in view of the present disclosure are within the spirit and scope of the invention.

EXAMPLES

EXAMPLE 1

CONSTRUCTION OF A RECOMBINANT (REVERSE GENETICS) H5N3 AVIAN

INFLUENZA VIRUS

[0044] An H5N3 avian influenza virus was constructed using a reverse genetics methodology described in Hoffmann et a/., Vaccine 20:3165-3170 (2002). Briefly, plasmids encoding the PB1 , PB2, PA, NP, M and NS genes of avian influenza virus strain A/PR/8/34 were constructed by RT-PCR. In addition, plasmids encoding the NA gene of avian influenza virus strain A/DK/Germany/1215/73 (H2N3) and the HA gene of avian influenza virus strain A/Chicken/Vietnam/c58/04 (H5N1 ) were constructed also by RT-PCR. The H5 gene was then modified by deletion of a polybasic amino acid region at the cleavage site between HA1 and HA2. A total of eight separate plasmids, each containing a separate avian influenza gene segment, were therefore constructed using RT-PCR.

[0045] Co-cultured 293T and MDCK cells (0.2 to 1 x 10⁶ cells of each cell line) were transfected with a DNA-lipid complex containing 1 μg of each plasmid and 10 μL of transit LT1 (Panvera, Madison, Wl), in a final volume of 1 mL of OPTIMEM-I (Invitrogen, Carlsbad, CA). (Vero cells can be used instead of co-cultured 293T and MDCK cells if desired). Transfection was carried out for 6 hours, at which time the DNA-lipid complexes were removed and replaced with fresh medium. The cells were incubated for an additional 24 hours, and 0.5 μg/mL of TPCK-treated trypsin was added. After 72 hours, the supernatant was removed from the cells, and 100 μL of the supernatant was injected into the allantoic cavity of 10 day old embryonated chicken eggs. Recombinant (reverse genetics) H5N3 viruses were recovered and analyzed from harvested allantoic fluid. This virus is referred to as "rg-H5N3."

EXAMPLE 2

INACTIVATION OF RECOMBINANT RG-H5N3 AVIAN INFLUENZA VIRUS

[0046] In this Example, the rg-H5N3 avian influenza virus of Example 1 was inactivated by formalin treatment.

[0047] The rg-H5N3 virus of Example 1 was adapted and propagated in MDCK cells using seed viral fluid material harvested from the allantoic fluid of embryonated chicken eggs as inoculum. The viral seed stock used as inoculum represented an X+3 passage. Three-hundred-eighty milliliters of the viral stock harvested from the infected MDCK cell monolayers was utilized in this inactivation kinetic study.

[0048] Two 10 mL samples were reserved as a "pre-inactivation," positive control sample. 3.8 mL of 1 :5 diluted formalin in sterile PBS (0.76 mL formalin + 3.04 mL PBS) was added to 380 mL of MDCK grown rg-H5N3 influenza virus stock while stirring to give a final concentration of 0.2% formalin in the mixture. The virus- formalin mixture was stirred for an additional 10 minutes at 37⁰C, and then all of the mixture was transferred to a new container. Two 10 mL samples were obtained at the time of transfer as the "0 timepoint." Subsequently, two-10 mL samples were taken at 1 , 2, 4, 8, 12, 24, 48 and 72 hours after the "0 timepoint." Each time a sample was obtained, 73 μL of sodium bisulfite solution was added to neutralize remaining formalin. Samples were then held at 4⁰C until all samples had been obtained.

[0049] The titration for the demonstration of complete inactivation of rg-H5N3 influenza virus was done according to a standard protocol. In brief, embryonated chicken eggs were inoculated with virus samples, the eggs were incubated and mortality recorded. At 3-4 days post-inoculation, the eggs were chilled and allantoic fluid was harvested. The fluid was tested for hemagglutinating activity and passed to a second set of eggs, which were also incubated for three days with testing of allantoic fluid harvest for hemagglutinating activity. The first time-point at which no live virus was detected was considered as the minimum time for which complete inactivation could be expected.

[0050] Results of inactivation kinetic study of rg-H5N3/04 reassorted virus grown in MDCK cells are presented in Table 2.

*Trauma Death = Death of the embryo due to inoculation injury *^*HA results of pooled sample

^$Death of the embryo is not due to virus infection as the post-inoculation allantoic sample was negative for HA HA results of individual allantoic sample

[0051] The allantoic fluids of embryonated chicken eggs not inoculated with the virus remained as negative controls to validate the egg source used as free of extraneous avian influenza for virus titration process. The pre-formalin treated virus sample, utilized as a positive control, was HA positive after two continuous passages in embryonated eggs. The virus-formalin mixture samples taken at 0-hour time point (immediately after formalin addition) was also found to be HA positive after first and second passages in embryonated eggs. The virus samples at 1-hour and all subsequent post-formalin-treatment timepoints were negative for the presence of live influenza H5N3 following corresponding first and second passages in embryonated eggs. The complete inactivation of the rg-H5N3 influenza of MDCK grown virus mixed and incubated at 37⁰C with formalin at a final concentration of 0.2% is thus considered as the minimum period required for complete inactivation.

EXAMPLE 3

SEROLOGICAL ASSESSMENT OF IMMUNE RESPONSE TO RG-H5N3 VACCINE

IN CANINES

[0052] In this Example, the ability of a vaccine composition comprising inactivated rg-H5N3 avian influenza virus to induce an immune response in dogs was analyzed.

[0053] A vaccine composition containing rg-H5N3 was prepared by combining the ingredients as shown in Table 3:

TABLE 3

[0054] The fourteen dogs used in this study were housed in isolation facilities. There were seven dogs per room. During the study the animals were under veterinary care and fed a standard commercial diet with water and food available ad libitum. The housing was in compliance with applicable animal welfare regulations. Animals requiring medical care were treated as deemed necessary by the veterinary staff after consultation with the Study Investigator. Only normal and healthy dogs seronegative to influenza H5N3 at the time of vaccination as determined by HI assay (HI titer <10) were included in the study. The seronegative dogs in the study were randomly divided into treatment groups as shown in Table 4. TABLE 4

[0055] The animals in Group 1 were vaccinated with two doses of recombinant influenza H5N3 (rg-H5N3) vaccine by the subcutaneous (SC) route in the dorsal medial aspect of the neck at three weeks interval between vaccinations. The animals in Group 2 were non-vaccinated seronegative controls and not administered vaccine or placebo. The dogs were bled approximately 5 to 10 mL whole blood/animal, for serum on 0, 7 and 14 days post vaccination one (DPV1); 0, 7, 14 and 21 days post vaccination two (DPV2) to monitor the serological responses. Sera were tested in a hemagglutinin inhibition (HI) assay for rg-H5N3 specific antibodies. Briefly, two-fold serial diluted serum samples were incubated for 30 minutes with constant levels (4 to 8 HA units) of the rg-H5N3 antigen. The virus/serum mixture was then incubated with an equal volume of 0.5% chicken red blood cell suspension in U-bottom plates. After incubation for at least one hour at room temperature, the wells were visually observed for hemagglutination. The serum HI antibody titer to influenza H5N3 antigen was the highest serum dilution causing inhibition of hemagglutination.

[0056] The results of HI antibody titers of the dogs in the study are presented in

Table 5.

TABLE 5

[0057] On 0 DPV1 , prior to vaccination all of the dogs in the study were seronegative (HI titer <10) to avian H5N3 influenza virus. All 4 non-vaccinated control dogs remained seronegative from 0 DPV1 through 21 DPV2. Four of 9 (44%) vaccinated dogs showed seroconversion (at least 4-fold increase in HI titer) on 7 DPV2, 3 of 9 (33%) on 14 DPV2, and 2 of 9 (22%) on 21 DPV2. Although the seroconversion against the inactivated recombinant rg-H5N3 avian influenza vaccine in dogs was not very high at the current antigen concentration, all the vaccinated dogs in the study did show some specific serological responses (HI antibody >10) at one or the other time point following vaccination. The HI antibody titers in the vaccinates varied from 10 to 80 following SC vaccination of 2 doses at 3 weeks interval. The geometric mean titers (GMT) of HI antibodies in the vaccinates were 14, 18 and 11 respectively on 7, 14 and 21 DPV2. The results of the current serological studies demonstrate that the inactivated recombinant rg-H5N3 avian influenza virus vaccine induced an antibody response in dogs following SC vaccination with 2 doses at 3 weeks interval between vaccinations.

EXAMPLE 4

SEROLOGICAL ASSESSMENT OF IMMUNE RESPONSE TO RG-H5N3 VACCINE

IN CATS

[0058] In this Example, the ability of a vaccine composition comprising inactivated rg-H5N3 avian influenza virus to induce an immune response in cats was analyzed.

[0059] Three vaccine compositions containing rg-H5N3 were prepared by combining the ingredients as shown in Table 6:

TABLE 6

[0060] Twelve cats were divided into three groups (4 cats per group) and were vaccinated with 1 mL doses of test vaccines A, B and C, respectively. The dosage regimen for each test vaccine was the subcutaneous administration of two doses, three weeks apart. Injection sites were in dorsal aspect of the nape of the neck. No controls were used in this study since none of the SPF kittens were expected to be exposed to any live H5N3 virus.

[0061] The cats were bled for serum samples by venipuncture following anesthetization with ketamine at the following time points: prior to each vaccination, 14 days post the first vaccination (DPV1), 7 days post the second vaccination (DPV2), 14 DPV2 and 21 DPV2.

[0062] Serum antibodies to avian influenza virus were measured using the hemagglutination inhibition assay (HI) per standard method. The HI antibody titers were calculated as the reciprocal of the higher serum dilutions causing inhibition of hemagglutination of chicken red blood cells by the H5N3 antigen.

[0063] The results of HI antibody titers of the cats in the study are presented in

Table 7.

TABLE 7

[0064] All cats were susceptible to the H5N3 virus as demonstrated by a lack of hemagglutination inhibition or HI antibody titers (<10). On 7 DPV2, HI antibody titers greater than 40 were reached in all cats tested. The antibody titers were maintained high at both 14 DPV2 and 21 DPV2. The geometric mean titers (GMTs) for the full- dose vaccine (Vaccine A) were 135, 95 and 95 for serum samples collected on 7 DPV2, 14 DPV2 and 21 DPV2, respectively. The GMTs for the fractional doses (Vaccine B and Vaccine C) were between 57 and 80 for serum samples collected on 7-21 DPV2.

[0065] In chickens, HI antibody titers greater than 10 are correlated with protection against mortality upon H5N1 challenge, and titers greater than 40 are correlated with protection against both mortality and virus shedding upon H5N1 challenge (Kumar et al., Avian Diseases 50: 481-483 (2007)). In cats, vaccines containing 80-320 HA units of inactivated H5N3 antigen were found to induce HI titers greater than 40. Thus, it is expected that cats vaccinated with rg-H5N3 are protected against clinical symptoms and mortality caused by avian influenza infection.

EXAMPLE 5

SEROLOGICAL ASSESSMENT OF IMMUNE RESPONSE TO RG-H5N3 VACCINE

IN PIGS

[0066] In this Example, the ability of a vaccine composition comprising inactivated rg-H5N3 avian influenza virus to induce an immune response in pigs was analyzed.

[0067] A vaccine composition containing rg-H5N3 was prepared by combining the ingredients as shown in Table 8: TABLE 8

[0068] The twenty-two pigs were housed at isolation facilities. During the study the animals were under veterinary care and fed a standard commercial diet with water and food available ad libitum. The housing was in compliance with applicable animal welfare regulations. Animals requiring medical care were treated as deemed necessary by the veterinary staff after consultation with the Study Investigator. Only normal and healthy pigs seronegative to influenza H5N3 at the time of vaccination as determined by HI assay (HI titer <10) were included in the study. The seronegative pigs in the study were randomly divided into treatment groups as shown in Table 9:

TABLE 9

[0069] The animals in Group 1 were vaccinated with two doses of recombinant influenza H5N3 vaccine by the SC route in the dorsal medial aspect of the neck at three weeks interval between vaccinations. The animals in Group 2 were non- vaccinated seronegative controls and not administered vaccine or placebo. The pigs were bled approximately 5 to 10 mL whole blood/animal, for serum on 0, 7 and 14 days post vaccination one (DPV1 ); 0, 7, 14 and 21 days post vaccination two (DPV2) to monitor the serological response. Sera were tested in a HI assay as described in Example 3 above for recombinant influenza H5N3 specific antibodies.

[0070] The results of HI antibody titers of the pigs in the study are presented in Table 10. TABLE 10

[0071] On 0 DPV1 , prior to vaccination all of the pigs in the study were seronegative (HI titer <10) to avian H5N3 influenza virus. All 7 non-vaccinated control pigs remained seronegative from 0 DPV1 through 21 DPV2. One hundred percent (15 of 15) of the vaccinates showed good seroconversion (at least 4-fold increase in HI titer) against the inactivated recombinant rg-H5N3 avian influenza virus by 7 DPV2, and 14 of 15 (93%) vaccinates remained seropositve on 14 and 21 DPV2. The HI antibody titers in the vaccinates varied from 20 to 640 with GMT of 127, 133 and 121 respectively on 7, 14 and 21 DPV2. These HI antibody titration results strongly indicate that the inactivated recombinant rg-H5N3 influenza virus vaccine is highly immunogenic in inducing strong humoral immune response in pigs. The results of the current serological studies demonstrate that the inactivated recombinant rg-H5N3 avian influenza virus vaccine induced following SC vaccination with 2 doses at 3 weeks interval between vaccinations a significantly high HI antibody response in pigs. EXAMPLE 6

USE OF AN INACTIVATED REVERSE GENETICS AVIAN INFLUENZA VACCINE

TO PROTECT ANIMALS FROM MORTALITY AND CLINICAL SYMPTOMS

CAUSED BY HIGHLY PATHOGENIC AVIAN INFLUENZA INFECTION

[0072] In this example, dogs, cats and pigs are vaccinated with the inactivated rg-H5N3 virus of Examples 1 and 2 and are subsequently challenged with a highly pathogenic avian influenza virus strain.

[0073] For the dog challenge experiments, the vaccine is prepared as in Example 3. For the cat challenge experiments, the vaccine is prepared as in Example 4 (vaccine A). For the pig challenge experiments, the vaccine is prepared as in Example 5. For all experiments, the vaccines are formulated to contain 320 HA units of rg-H5N3 per dose.

[0074] A total of 20 of each animal are used for the challenge experiments. Ten of each animal (10 dogs, 10 cats and 10 pigs) are administered a first dose of vaccine by the subcutaneous route on day 0 (0 DPV1 ), and on the same day, ten of each animal are administered a placebo as controls. Three weeks later (21 DPV1 = 0 DPV2), the vaccinated animals are administered a second dose of vaccine by the subcutaneous route and the control animals are administered a second dose of placebo.

[0075] At 14 days after the second vaccination (14 DPV2) (a time point at which all animals have been previously shown to exhibit a significant serological response to the rg-H5N3 vaccine, see Examples 3, 4 and 5, above), all of the animals are administered a dose of the highly pathogenic avian influenza A/chicken/Vietnam/c58/04 strain ("high-path H5N1"). The amount of high-path H5N1 administered to each animal is an amount that is known (by previous experiments or extrapolation) to cause mortality in 100% of unvaccinated animals at 14 days post challenge (14 DPC). [0076] The challenged animals are observed daily for clinical signs of avian influenza infection. The vaccine is deemed protective if at least 50% of challenged, vaccinated animals survive and do not show clinical signs of avian influenza infection at 14 DPC. Preferably, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the challenged, vaccinated animals survive and do not show clinical signs of avian influenza infection at 14 DPC.

[0077] Variations on the above-described challenge experiments are also contemplated wherein, e.g., the animals are administered varying doses of rg-H5N3 prior to challenge, the animals are administered more than two doses of vaccine or only one dose of vaccine prior to challenge, the animals are challenged by multiple strains of avian influenza virus, the animals are administered multivalent vaccines comprising more than one type of inactivated H5 influenza virus, the animals are administered the vaccine after challenge, etc.

[0078] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, this invention is not limited to the particular embodiments disclosed, but is intended to cover all changes and modifications that are within the spirit and scope of the invention as defined by the appended claims.

[0079] All publications and patents mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patents are herein incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method of vaccinating an animal against avian influenza, said method comprising administering to the animal at least one dose of a vaccine composition comprising an immunologically-effective amount of an inactivated influenza virus; wherein said inactivated influenza virus has an H5 hemagglutinin and an N1 , N2, N3, N4, N5, N6, N7, N8 or N9 neuraminidase; and wherein the animal is a non-avian, non-human animal.

2. The method of claim 1 , wherein the animal, after being administered said at least one dose of said vaccine composition, is protected from clinical symptoms of infection with a high pathogenic avian influenza strain.

3. The method of claim 1 , wherein said neuraminidase is an N3 neuraminidase.

4. The method of claim 1 , wherein said H5 hemagglutinin is derived from A/Chicken/Vietnam/C58/04.

5. The method of claim 3, wherein said N3 neuraminidase is derived from A/Duck/Germany/1215/73.

6. The method of claim 1 , wherein said neuraminidase is an N1 neuraminidase.

7. The method of claim 6, wherein said N1 neuraminidase is derived from an influenza virus selected from the group consisting of A/PuertoRico/8/34, A/WSN/33, and A/Teal/HK/W312/97.

8. The method of claim 1 , wherein the animal is a dog.

9. The method of claim 1 , wherein the animal is a cat.

10. The method of claim 1 , wherein the animal is a pig.

11. The method of claim 1 , wherein said immunologically-effective amount is between 10 and 1000 hemagglutinating units (HA units) of said inactivated influenza virus per dose.

12. The method of claim 11 , wherein said immunologically-effective amount is between 50 and 500 HA units of said inactivated influenza virus per dose.

13. The method of claim 12, wherein said immunologically-effective amount is about 80 HA units of said inactivated influenza virus per dose.

14. The method of claim 12, wherein said immunologically-effective amount is about 160 HA units of said inactivated influenza virus per dose.

15. The method of claim 12, wherein said immunologically-effective amount is about 320 HA units of said inactivated influenza virus per dose.

16. The method of claim 1 , wherein said inactivated influenza virus is a recombinant influenza virus obtained by a method comprising:

(a) introducing a set of plasmids into a host cell in vitro, wherein said set of plasmids comprises a first plasmid that expresses H5 hemagglutinin, a second plasmid that expresses N1 , N2, N3, N4, N5, N6, N7, N8 or N9 neuraminidase, and one or more additional plasmids that express avian influenza PB1 , PB2, PA, NP, M1 , M2, NS1 and NS2 proteins;

(b) recovering virus particles from said host cell; and

(c) inactivating said virus particles.

17. The method of claim 16, wherein said avian influenza PB1 , PB2, PA, NP, M1, M2, NS1 and NS2 proteins are expressed from separate plasmids.

18. A method of vaccinating an animal against avian influenza, said method comprising administering to the animal at least one dose of a vaccine composition comprising an immunologically-effective amount of an inactivated influenza virus; wherein said inactivated influenza virus has an H5 hemagglutinin and an N3 neuraminidase; and wherein said animal is a dog, cat or pig.

19. The method of claim 18, wherein the animal, after being administered said at least one dose of said vaccine composition, is protected from clinical symptoms of infection with a high pathogenic avian influenza strain.

20. The method of claim 18, wherein said H5 hemagglutinin is derived from A/Chicken/Vietnam/C58/04.

21. The method of claim 18, wherein said N3 neuraminidase is derived from A/Duck/Germany/1215/73.

22. The method of claim 18, wherein said immunologically-effective amount is between 10 and 1000 hemagglutinating units (HA units) of said inactivated influenza virus per dose.

23. The method of claim 22, wherein said immunologically-effective amount is between 50 and 500 HA units of said inactivated influenza virus per dose.

24. The method of claim 23, wherein said immunologically-effective amount is about 80 HA units of said inactivated influenza virus per dose.

25. The method of claim 23, wherein said immunologically-effective amount is about 160 HA units of said inactivated influenza virus per dose.

26. The method of claim 23, wherein said immunologically-effective amount is about 320 HA units of said inactivated influenza virus per dose.

27. The method of claim 18, wherein said inactivated influenza virus is a recombinant influenza virus obtained by a method comprising:

(a) introducing a set of plasmids into a host cell in vitro, wherein said set of plasmids comprises a first plasmid that expresses H5 hemagglutinin, a second plasmid that expresses N3 neuraminidase, and one or more additional plasmids that express avian influenza PB1 , PB2, PA, NP, M1 , M2, NS1 and NS2 proteins;

(b) recovering virus particles from said host cell; and

(c) inactivating said virus particles.

28. The method of claim 27, wherein said avian influenza PB1 , PB2, PA, NP, M1 , M2, NS1 and NS2 proteins are expressed from separate plasmids.

29. The method of claim 18, wherein the animal is a dog.

30. The method of claim 18, wherein the animal is a cat.

31. The method of claim 18, wherein the animal is a pig.

32. An isolated serum sample obtained from an animal, wherein said sample exhibits a hemagglutination inhibition (HI) titer of at least 10 when tested against an H5N3 avian influenza virus in a standard HI assay; wherein said animal is a dog, a cat or a pig that has been administered at least one dose of an inactivated influenza virus having an H5 hemagglutinin and an N1 , N2, N3, N4, N5, N6, N7, N8 or N9 neuraminidase.

33. The isolated serum sample of claim 32, wherein said sample exhibits an HI titer of at least 20 when tested against an H5N3 avian influenza virus in a standard HI assay.

34. The isolated serum sample of claim 33, wherein said sample exhibits an HI titer of at least 320 when tested against an H5N3 avian influenza virus in a standard HI assay.

35. The isolated serum sample of claim 32, wherein said sample is obtained from said animal at least seven days after said animal has been administered said inactivated influenza virus.

36. The isolated serum sample of claim 32, wherein said animal has been administered a first and a second dose of said inactivated influenza virus, wherein said second dose is administered 7 to 21 days after said first dose is administered.

37. The isolated serum sample of claim 36, wherein said sample is obtained from said animal at least seven days after said second dose is administered (7DPV2).

38. The isolated serum sample of claim 37, wherein said sample is obtained from said animal at least 14 days after said second dose is administered (14DPV2).

39. The isolated serum sample of claim 38, wherein said sample is obtained from said animal at least 21 days after said second dose is administered (21DPV2).