RU2809084C2 - Immunogenic composition against subtype h5 avian flu virus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an immunogenic composition against H5 avian influenza virus containing the hemagglutinin protein of H5 avian influenza virus. Also the following is disclosed: a method of differentiating animals naturally infected with an avian influenza virus from animals vaccinated with the above immunogenic composition, comprising analyzing a sample of the animal in an immunoassay and/or genomic analytical assay for the presence of an avian influenza marker that is not present in the specified immunogenic composition, or analyzing the sample animal in an immunoassay and/or genomic assay for the presence of an avian influenza marker that is specific to said immunogenic composition.

EFFECT: invention is effective for the prevention and/or treatment of infections caused by avian influenza virus.

13 cl, 4 dwg, 10 tbl, 11 ex

Description

FIELD OF THE INVENTION

[0001] The present invention relates to the field of veterinary medicine, in particular to an immunogenic composition against the avian influenza virus subtype H5.

PREREQUISITES FOR CREATION OF THE INVENTION

[0002] Avian influenza virus (AIV) is a member of the influenza A virus and occurs naturally in wild waterfowl worldwide and can infect poultry and other birds, including mammals. Influenza A viruses are divided into subtypes based on two proteins on the surface of the virus: hemagglutinin (HA) and neuraminidase (NA). There are 18 known subtypes of AN and 11 known subtypes of NA. Many different combinations of HA and NA proteins are possible. For example, "H5N1 virus" means that the virus is subtype 5 HA and subtype 1 NA.

[0003] There are nine known subtypes of H5 viruses (H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8 and H5N9, referred to herein as “H5Nx”). Most H5 viruses identified worldwide in wild birds and poultry are low pathogenicity avian influenza viruses, but some H5 viruses are highly pathogenic avian influenza (HPAI) viruses. H5Nx viruses evolve rapidly, resulting in significant divergent antigenic variation between different clades, revealing evolutionary relationships between different H5 lineages. Infection of poultry with HPAI viruses can cause severe disease with high mortality.

[0004] In countries where the H5Nx virus is endemic, vaccination has been used to control the disease. The most common AIV vaccines are inactivated whole virus vaccines (inactivated vaccines), which are prepared by inactivating the whole virus and emulsifying with appropriate adjuvants. However, an inactivated vaccine may not provide broad protection against different clades. Due to the rapid evolution of AIV, humans have to constantly develop new vaccines against different clades. Moreover, large quantities of live AIVs are required to produce an inactivated vaccine and therefore biosafety is a major concern.

[0005] The HA protein is a receptor-binding and membrane-fusion glycoprotein of the influenza A virus. The HA protein is known to be capable of eliciting protective antibodies, and therefore researchers are interested in developing subunit vaccines based on recombinant HA protein. WO2007/019094 discloses an HA molecule containing an amino acid substitution at the receptor binding site that makes the HA molecule more antigenic compared to an HA molecule lacking the amino acid substitution at the receptor binding site. WO2008/052173 discloses the hemagglutinin protein of avian influenza virus subtype H5 (hereinafter referred to as "AIV subtype H5 HA protein" or "H5 HA protein") containing certain amino acid mutations, and vaccines containing adjuvanted H5 HA protein can provide protection against clinical disease , caused by the influenza A virus. However, the above information is not related to broader protection against influenza A viruses of various clades.

[0006] WO2013/148164 discloses a method for producing an optimized H5N1 and H1N1 influenza HA polypeptide from human H5N1 and swine H1N1 influenza isolates. An influenza virus-like particle (VLP) containing an optimized HA influenza polypeptide is also produced. Experimental results show that VLPs can induce a humoral immune response that can recognize influenza viruses from two different clades in mice, but the effectiveness of protection is only 40-60%.

[0007] There is a need to develop a vaccine that can provide broader, more effective, longer-lasting and earlier protection against AIV subtype H5 in poultry.

BRIEF DESCRIPTION OF THE INVENTION

[0008] This invention provides an immunogenic composition containing a hemagglutinin protein of an avian influenza virus subtype H5, the hemagglutinin protein containing: (a) amino acid residues 120N or 120S, 155N and 223N or 223S; and (b) one or more amino acid residues selected from the group consisting of: 61D, 871, 99A, 102A, 110N, 136S, 140D, 149S, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A , 226V, 243D, 268Y, 279A and 2981; wherein the numbering relative to amino acid residues is as indicated in SEQ ID NO: 1.

[0010] The present invention also provides a method for producing an immunogenic composition, which includes: (i) culturing cells containing an expression vector expressing the hemagglutinin protein of the avian influenza virus subtype H5; and (ii) harvesting the entire cell culture; where the hemagglutinin protein contains: (a) amino acid residues 120N or 120S, 155N and 223N or 223S; and (b) one or more amino acid residues selected from the group consisting of: 61D, 87I, 99A, 102A, 110N, 136S, 140D, 149S, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A , 226V, 243D, 268Y, 279A and 2981; wherein the numbering relative to amino acid residues is as indicated in SEQ ID NO: 1.

[0011] Also provided is an immunogenic composition according to the invention for use in the prevention and/or treatment of infections caused by an avian influenza virus, preferably H5Nx. In one embodiment, H5Nx is H5N1, H5N2 and/or H5N6. In one embodiment, H5Nx is H5N1. In one embodiment, H5Nx is H5N2. In one embodiment, H5Nx is H5N6. In one embodiment, H5Nx is H5N1 and H5N2. In one embodiment, H5Nx is H5N2 and H5N6.

[0012] Also provided is a method for differentiating animals naturally infected with AIV from animals vaccinated with an immunogenic composition in accordance with the invention.

[0013] The immunogenic composition in accordance with the invention can provide broader, more effective, longer-lasting and earlier protection to poultry. In one embodiment, the immunogenic composition according to the invention provides broader protection to poultry. In one embodiment, the immunogenic composition according to the invention provides more effective protection to poultry. In one embodiment, the immunogenic composition according to the invention provides long-term protection to poultry. In one embodiment, the immunogenic composition according to the invention provides early protection to poultry.

DETAILED DESCRIPTION OF THE INVENTION

[0014] In one aspect, the invention provides an immunogenic composition comprising an H5 HA protein, wherein the H5 HA protein comprises: (a) amino acid residues 120N or 120S, 155N and 223N or 223S; and (b) one or more amino acid residues selected from the group consisting of: 61D, 87I, 99A, 102A, 110N, 136S, 140D, 149S, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 226V , 243D, 268Y, 279A and 2981; wherein the numbering relative to amino acid residues is as indicated in SEQ ID NO: 1.

[0015] As used herein, the term “immunogenic composition,” also referred to as “vaccine,” refers to a composition that contains at least one antigen that elicits an immune response to the composition. The host will exhibit a therapeutic or protective immune response such that resistance to new infections will increase and/or the severity of clinical signs will decrease.

[0016] The amino acid sequence of the HA protein H5 is constructed through a series of HA amino acid sequence alignments, subsequent generation of consensus amino acid sequences, and analysis of the most frequently occurring residues at each position. As used herein, the terms “hemagglutinin protein of the invention,” “AIV subtype H5 HA protein,” and “H5 HA protein” are used interchangeably.

[0017] In this context, SEQ ID NO: 1 is the amino acid sequence of the HA protein of strain A/duck/China/E319-2/03, but does not contain the signal peptidan N- end (the amino acid sequence of the HA protein of strain A/duck/China/E319-2/03 was also published in WO2008/052173). SEQ ID NO: 1 is used as a standard HA protein sequence for determining the amino acid position of the HA protein in accordance with the present invention.

[0018] The numbering of amino acid positions of the HA protein in accordance with the present invention, as used herein, refers to the amino acid position presented in SEQ ID NO:l. For example, the designation "120N or 120S" means N or S at a position that corresponds to position 120 of the amino acid sequence SEQ ID NO:1. "155N" means N at a position that corresponds to position 155 of amino acid sequence SEQ ID NO:l. "223N or 223S" means N or S at a position that corresponds to position 223 of the amino acid sequence SEQ ID NO:1. "61D" means D at a position that corresponds to position 61 of amino acid sequence SEQ ID NO:1. "871" means I at a position that corresponds to position 87 of amino acid sequence SEQ ID NO:1. "99A" means A at a position that corresponds to position 99 of the amino acid sequence SEQ ID NO:1. "102A" means A at a position that corresponds to position 102 of amino acid sequence SEQ ID NO:1. "HON" means N at a position that corresponds to the position of the amino acid sequence SEQ ID NO:1. "136S" means S at a position that corresponds to position 136 of amino acid sequence SEQ ID NO:1. "140D" means D at a position that corresponds to position 140 of amino acid sequence SEQ ID NO:1. "149S" means S at a position that corresponds to position 149 of amino acid sequence SEQ ID NO:1. "156T" means T at a position that corresponds to position 156 of amino acid sequence SEQ ID NO:1. "157P" means P at a position that corresponds to position 157 of amino acid sequence SEQ ID NO:1. "170N" means N at a position that corresponds to position 170 of amino acid sequence SEQ ID NO:1. "172T" means T at a position that corresponds to position 172 of amino acid sequence SEQ ID NO:1. "178R" means R at a position that corresponds to position 178 of amino acid sequence SEQ ID NO:1. "190V" means V at a position that corresponds to position 190 of amino acid sequence SEQ ID NO:1. "191L" means L at a position that corresponds to position 191 of amino acid sequence SEQ ID NO:1. "200A" means A at a position that corresponds to position 200 of amino acid sequence SEQ ID NO:1. "226V" means V at a position that corresponds to position 226 of amino acid sequence SEQ ID NO:1. "243D" means D at a position that corresponds to position 243 of amino acid sequence SEQ ID NO:1. "268Y" means Y at a position that corresponds to position 268 of amino acid sequence SEQ ID NO:1. "279A" means A at a position that corresponds to position 279 of amino acid sequence SEQ ID NO:1. "2981" means I at a position that corresponds to position 298 of amino acid sequence SEQ ID NO:1. Methods for determining amino acid positions are known in the art, including, but not limited to, amino acid alignments performed using the BLAST program.

[0019] In one embodiment of the immunogenic composition in accordance with this invention, the H5 HA protein contains any of the amino acid residues selected from the group consisting of: 61D, 871, 99A, 102A, HON, 136S, HOD, 149S, 156T , 157P, 170N, 172T, 178R, 190V, 191L, 200A, 226V, 243D, 268Y, 279A, and 2981. In one embodiment, the H5 HA protein of the present invention contains two amino acid residues selected from the group consisting of : 61D, 871, 99A, 102A, HON, 136S, 140D, 149S, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 226V, 243D, 268Y, 279A and 2981. In one embodiment, the HA protein H5 in accordance with this invention contains three, four, five, ......, or all amino acid residues selected from the group consisting of: 61D, 871, 99A, 102A, HON, 136S, 140D, 149S, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 226V, 243D, 268Y, 279A, and 2981. The entire HA protein from naturally occurring AIV is approximately 568 amino acid residues in length, and all amino acid residues before amino acid 514 are located on the surface of the virus. The above-mentioned amino acid sites in accordance with the present invention are identified as located in the region of amino acids 60-300 HA of the protein, which is believed to contain the majority of immunogenic epitopes. In one embodiment, the H5 HA protein of the present invention further comprises amino acid residues 120N or 120S, 155N, and 223N or 223S. In one embodiment, the H5 HA protein of the present invention contains amino acid residues 120N, 155N and 223N. In one embodiment, the H5 HA protein of the present invention contains amino acid residues 120N, 155N and 223S. In one embodiment, the H5 HA protein of the present invention contains amino acid residues 120S, 155N and 223N. In one embodiment, the H5 HA protein of the present invention contains amino acid residues 120S, 155N and 223S.

[0020] In one embodiment of the immunogenic composition of the present invention, the H5 HA protein contains amino acid residues 61D, 871, 99A, 102A, 110N, 120N, 136S, 140D, 149S, 155N, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 223N, 226V, 243D, 268Y, 279A and 2981. In one embodiment, the H5 HA protein of the present invention contains amino acid residues 61D, 871, 99A, 102A, HON, 120S, 136S, 140D, 149S, 155N, 156 T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 223S, 226V, 243D, 268Y, 279A, and 2981. In one embodiment, the H5 HA protein of the present invention contains amino acid residues 61D, 871, 99A, 102A, HON, 120 S, 136S, 140D, 149S, 155N, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 223N, 226V, 243D, 268Y, 279A and 2981. In one embodiment, the H5 HA protein of the present invention contains amino acids 61D residues, 871, 99A, 102A, 110N, 120N, 136S, 140D, 149S, 155N, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 223S, 226V, 243D, 268 Y, 279A and 2981.

[0021] In one embodiment of the immunogenic composition of the present invention, the H5 HA protein contains the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, the H5 HA protein of the present invention contains the amino acid sequence set forth in SEQ ID NO: 5. In one embodiment The H5 HA protein of the present invention consists of the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, the H5 HA protein of the present invention consists of the amino acid sequence set forth in SEQ ID NO: 5.

[0022] As used herein, the term “H5 HA protein of the present invention” can mean an isolated form of the H5 HA protein or a non-isolated form of the H5 HA protein, for example, the H5 HA protein contained in a cell culture. In one embodiment of the immunogenic composition of the present invention, the H5 HA protein is a non-isolated form of the H5 HA protein. In one embodiment of the immunogenic composition of the present invention, the H5 HA protein is an isolated form of the H5 HA protein.

[0023] In one embodiment of the immunogenic composition of the present invention, the H5 HA protein is produced by expressing a nucleic acid molecule encoding the H5 HA protein.

[0024] In one embodiment, the nucleic acid molecule of the present invention may be further codon optimized for expression in cells. The term “codon-optimized nucleic acid molecule” as used herein means a nucleic acid molecule selected such that the codons are optimal for expression in a particular system (such as a particular species or group of species). Codon optimization does not change the amino acid sequence of the encoded protein. In one embodiment, the nucleic acid molecule of the present invention is codon-optimized for expression in insect cells.

[0025] In one embodiment, the nucleic acid molecule of the present invention comprises the nucleic acid sequence set forth in SEQ ID NO: 10. In one embodiment, the nucleic acid molecule of the present invention consists of the nucleic acid sequence set forth in SEQ ID NO: 10. In one embodiment In an embodiment, the nucleic acid molecule of the present invention comprises the nucleic acid sequence set forth in SEQ ID NO: 11. In one embodiment, the nucleic acid molecule of the present invention consists of the nucleic acid sequence set forth in SEQ ID NO: 11.

[0026] The nucleic acid molecule of the present invention may be contained in an expression vector. In one embodiment, the expression vector of the present invention is a virus, plasmid, cosmid or phage. The vector may consist of DNA or RNA, preferably DNA.

[0027] Vectors and methods for creating and/or using vectors (or recombinants) for expression may be methods or similar to methods disclosed in: US Patent Nos. 4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683, 5,494,807, 4,722,84 8, 5,942,235, 5,364,773, 5,762,938, 5,770,212, 5,942,235, 382,425, PCT publications WO 94/16716, WO 96/39491, WO 95/30018; Paoletti, "Applications of pox virus vectors to vaccination: An update," PNAS USA 93: 11349 - 11353, October 1996; Moss, "Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety," PNAS USA 93: 11341 - 11348, October 1996; Smith et al., US Patent No. 4,745,051 (recombinant baculovirus); Richardson, S.D. (editor), Methods in Molecular Biology 39, "Baculovirus Expression Protocols" (1995 Humana Press Inc.); Smith et al., “Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector,” Molecular and Cellular Biology, December 1983, vol. 3, no. 12, pp. 2156-2165; Pennock et al., "Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infective Cells with a Baculovirus vector," Molecular and Cellular Biology March 1984, vol. 4, No. 3, p. 406; EPA0 370 573; US Application No. 920,197, filed October 16, 1986; European Patent Publication No. 265785; US Patent No. 4,769,331 (recombinant herpesvirus); Roizman, "The function of herpes simplex virus genes: A primer for genetic engineering of novel vectors," PNAS USA 93:11307 - 11312, October 1996; Andreansky et al., "The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors," PNAS USA 93: 11313 - 11318, October 1996; Robertson et al., “Epstein-Barr virus vectors for gene delivery to B lymphocytes,” PNAS USA 93: 11334 - 11340, October 1996; Frolov et al., "Alphavirus-based expression vectors: Strategies and applications," PNAS USA 93: 11371 - 11377, October 1996; Kitson et al., J. Virol. 65, 3068 - 3075, 1991; US Patent Nos. 5,591,439, 5,552,143; WO 98/00166; issued applications US serial. Nos. 08/675,556 and 08/675,566, both filed July 3, 1996 (recombinant adenovirus); Grunhaus et al., 1992, "Adenovirus as cloning vectors," Seminars in Virology (vol. 3) pp. 237-52, 1993; Ballay et al. EMBO Journal, vol. 4, pp. 3861 - 65, Graham, Tibtech 8, 85 - 87, April, 1990; Prevec et al., J. Gen. Virol. 70, 42434; PCT WO 91/11525; Feigner et al. (1994), J. Biol. Chem. 269, 2550 - 2561, Science, 259: 1745 - 49, 1993; and McClements et al., “Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease,” PNAS USA 93: 11414 - 11420, October 1996. ; and US Patent Nos. 5,591,639, 5,589,466 and 5,580,859, as well as WO 90/11092, W093/19183, W094/21797, WO95/11307, WO95/20660; Tang et al., Nature, and Furth et al., Analytical Biochemistry, regarding DNA expression vectors, among others. See also WO 98/33510; Ju et al., Diabetologia, 41: 736 - 739, 1998 (lentiviral expression system); Sanford et al., US Patent No. 4,945,050; Fischbachet et al. (Intracel); WO 90/01543; Robinson et al., Seminars in Immunology vol. 9, pp. 271-283 (1997), (DNA vector systems); Szoka et al., US Patent No. 4,394,448 (method of incorporating DNA into living cells); McCormick et al., US Patent No. 5,677,178 (use of cytopathic viruses); and US Patent No. 5,928,913 (gene delivery vectors); as well as in other documents cited in this description.

[0028] In one embodiment, the expression vector of the present invention is a viral vector.

[0029] In one embodiment, the expression vector of the present invention is a herpes virus, such as Aujeszky's disease virus, equine herpes virus, or herpes simplex virus. In one embodiment, the expression vector of the present invention is an adenovirus, such as a porcine adenovirus. In one embodiment, the expression vector of the present invention is a poxvirus, such as vaccinia virus, avipoxvirus, canarypox virus (canarypox virus), or swinepox virus.

[0030] In one embodiment, the viral vector is a recombinant baculovirus. In one embodiment, said recombinant baculovirus of the present invention is obtained from BaculoGold (BD Biosciences Pharmingen, San Diego, Calif.). In one embodiment, the specified recombinant baculovirus of the present invention is obtained from Sapphire™ Baculovirus (Allele Biotechnology). In one embodiment, said recombinant baculovirus of the present invention contains a nucleic acid molecule that encodes the H5 HA protein of the present invention. In one embodiment, said recombinant baculovirus of the present invention comprises a nucleic acid molecule as set forth in SEQ ID NO: 10. In one embodiment, said recombinant baculovirus of the present invention comprises a nucleic acid molecule as set forth in SEQ ID NO: 11.

[0031] In one embodiment of the immunogenic composition of the present invention, the H5 HA protein is contained in a cell culture. The cell culture according to the present invention may be a whole cell culture obtained by a cell culture process including a cell and a culture medium, or a portion of a cell culture containing the HA protein, such as a portion of a cell culture containing the HA protein obtained by filtration or any other step divisions.

[0032] In one embodiment, the cell culture contains the H5 HA protein, wherein the H5 HA protein contains: (a) amino acid residues 120N or 120S, 155N and 223N or 223S; and (b) one or more amino acid residues selected from the group consisting of: 61D, 871, 99A, 102A, HON, 136S, 140D, 149S, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A , 226V, 243D, 268Y, 279A and 2981; wherein the numbering regarding amino acid residues is as presented in SEQ ID NO:l. In one embodiment, the cell culture contains the H5 HA protein, wherein the H5 HA protein contains amino acid residues 61D, 871, 99A, 102A, 110N, 120N, 136S, 140D, 149S, 155N, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 223N, 226V, 243D, 268Y, 279A, and 2981. In one embodiment, the cell culture comprises an H5 HA protein, wherein the H5 HA protein comprises the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, the cell culture comprises The H5 HA protein, wherein the H5 HA protein contains the amino acid sequence set forth in SEQ ID NO: 5.

[0033] The immunogenic composition of the present invention further contains an adjuvant. In one embodiment, the immunogenic composition is formulated in an adjuvant. "Adjuvants" as used herein may include aluminum hydroxide and aluminum phosphate, saponins, e.g. Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsion can be based, in particular, on light liquid paraffin oil (European Pharmacopoeia type); isoprenoid oil such as squalane or squalene; oil obtained from the oligomerization of alkenes, in particular isobutene or decene; esters of acids or alcohols containing a linear alkyl group, in particular vegetable oils, ethyl oleate, di-(caprylate/caprate) propylene glycol, tri-(caprylate/caprate) glycerol or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular esters of isostearic acid. The oil is used in combination with emulsifiers to form an emulsion. Emulsifiers are preferably non-ionic surfactants, in particular esters of sorbitan, mannide (eg anhydromannitoleate), glycol, polyglycerol, propylene glycol and oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated. polyoxypropylene-polyoxyethylene copolymer blocks, particularly Pluronic products, especially L121. See Hunter et al., Theory and Practical Application of Aduvants (Ed. Stewart-Tull, DES), John Wiley and Sons, NY, pp. 51 - 94 (1995) and Todd et al., Vaccine 15: 564 - 570 ( 1997). Examples of adjuvants are SPT emulsion, described on page 147 of the book "Vaccine Design, The Subunit and Adjuvant Approach", edited by M. Powell and M. Newman, Plenum Press, 1995, and MF59 emulsion, described on page 183 of the same book.

[0034] Another example of an adjuvant is a compound selected from polymers of acrylic or methacrylic acid and copolymers of maleic anhydride and an alkenyl derivative. Preferred adjuvants are polymers of acrylic or methacrylic acid that are cross-linked, especially with polyalkenyl esters of sugars or polyhydric alcohols. These compounds are known as carbomer (Phameuropa Vol. 8, No. 2, June 1996). Those skilled in the art may also refer to US Patent No. 2,909,462, which discloses such acrylic polymers cross-linked to a polyhydroxylated compound having at least 3 hydroxyl groups, preferably no more than 8, with at least hydrogen atoms three hydroxyls are replaced by unsaturated aliphatic radicals containing at least 2 carbon atoms. Preferred radicals are those containing from 2 to 4 carbon atoms, for example vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals themselves may contain other substituents, such as methyl. Products sold under the name Carbopol; (BF Goodrich, Ohio, USA) is particularly suitable. They are cross-linked with allyl sucrose or allyl pentaerythritol. Among them we can mention Carbopol 974P, 934P and 971P. The most preferable is to use Cabopol 971P. Among the copolymers of maleic anhydride and an alkenyl derivative are EMA copolymers (Monsanto), which are copolymers of maleic anhydride and ethylene. Dissolution of these polymers in water results in an acidic solution which will be neutralized, preferably to physiological pH, to produce an adjuvant solution into which the immunogenic, immunological or vaccine composition itself will be incorporated.

[0035] Additional suitable adjuvants include, but are not limited to, RIBI adjuvant system (Ribi Inc.), block copolymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid amine adjuvant , heat labile enterotoxin from E. coli (recombinant or other), cholera toxin, IMS 1314 or muramyl dipeptide, or naturally occurring or recombinant cytokines or analogs thereof, or stimulators of endogenous cytokine release, among many others.

[0036] In one embodiment, the immunogenic composition is formulated into a water-in-oil emulsion with a suitable adjuvant. Said adjuvant may contain oils and surfactants. In one embodiment, said adjuvant is MONTANIDE™ ISA 71R VG (manufactured by Seppic Inc, Cat no: 365187). The adjuvant may be added in an amount of from about 100 μg to about 10 mg per dose. Even more preferably, said adjuvant is added in an amount of from about 100 μg to about 10 mg per dose. Even more preferably, said adjuvant is added in an amount of from about 500 μg to about 5 mg per dose. Even more preferably, said adjuvant is added in an amount of from about 750 μg to about 2.5 mg per dose. Most preferably, said adjuvant is added in an amount of approximately 1 mg per dose. In one embodiment, the immunogenic composition of the present invention contains approximately 7 parts of an oil phase containing an adjuvant and approximately 3 parts of an aqueous phase containing the H5 HA protein of the present invention, per dose.

[0037] Surprisingly, it has been found that the immunogenic composition of the present invention can lead to broader protection of poultry. As used herein, the term “broader poultry protection” covers broad protection against the various H5 AIV subtypes. Such subtypes include or, in a further embodiment, consist of H5N1, H5N2 and/or H5N6. According to a further embodiment, the term “broader poultry protection” also covers protection against various clades of AIV H5 subtypes. Such clades AIV H5 comprise or, in a further embodiment, consist of 2.3.4.4, 2.3.2.1, 2.3.2.1d, 2.3.4.4d and/or 7.2.

[0038] In addition, the protection provided by the immunogenic composition of the present invention has an early onset and a long period. As used herein, the term “early onset period” or “early onset of protection” means that partial protection against infectious AIV H5 virus can be obtained 7 days after vaccination. Complete protection against the infectious AIV H5 virus is achieved 14 days after vaccination. The term "long-term" or "long-term protection of poultry" covers a period of protection of at least 42 days after vaccination.

[0039] The immunogenic composition of the present invention can provide protection to chickens at different ages, even those that have just hatched, that is, the first day of life. The immunogenic composition of the present invention should not be limited to a particular route of administration, and different routes of administration do not affect the protective effectiveness of the immunogenic composition of the present invention.

[0040] In another aspect, the invention also relates to a method for producing an immunogenic composition, which includes: (i) culturing cells containing an expression vector capable of expressing the H5 HA protein; and (ii) collecting the H5 HA protein or a whole cell culture containing the H5 HA protein, wherein the H5 HA protein comprises: (a) amino acid residues 120N or 120S, 155N and 223N or 223S; and (b) one or more amino acid residues selected from the group consisting of: 61D, 871, 99A, 102A, 110N, 136S, HOD, 149S, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A , 226V, 243D, 268Y, 279A and 2981; in this case, the numbering regarding amino acid residues is as presented in SEQ ID NO: 1.

[0041] In one embodiment of the method of the present invention, the H5 HA protein contains amino acid residues 61D, 871, 99A, 102A, 110N, 120N, 136S, 140D, 149S, 155N, 156T, 157P, 170N, 172T, 178R, 190V, 191L , 200A, 223N, 226V, 243D, 268Y, 279A and 2981. In one embodiment, the H5 HA protein contains the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, the H5 HA protein contains the amino acid sequence set forth in SEQ ID NO: 5.

[0042] In one embodiment of the method of the present invention, the expression vector is a recombinant baculovirus containing a nucleic acid molecule of the present invention. In one embodiment, the nucleic acid molecule of the present invention is presented in SEQ ID NO: 10. In one embodiment, the nucleic acid molecule of the present invention is presented in SEQ ID NO: 11 In one embodiment, the recombinant baculovirus is obtained from a commercial product sold under the trademark Sapphire™ Baculovirus (Allele Biotechnology). In one embodiment, the cells are insect cells. In one embodiment, the insect cells are SF+ cells. In one embodiment, said SF+ cells are a commercial product sold by Protein Sciences Corporation (Meriden, CT).

[0043] In one embodiment of the method of the present invention, the method includes the step of producing a recombinant baculovirus containing a nucleic acid molecule of the present invention. In one embodiment, the nucleic acid molecule of the present invention is presented in SEQ ID NO: 10. In one embodiment, the nucleic acid molecule of the present invention is presented in SEQ ID NO: 11. In one embodiment, the recombinant baculovirus is obtained from a commercial product sold under the trademark Sapphire ™ Baculovirus (Allele Biotechnology).

[0044] In one embodiment of the method of the present invention, the method includes the step of infecting (infecting) cells with a recombinant baculovirus of the present invention. In one embodiment, said cells are insect cells. In one embodiment, said insect cells are SF+ cells. In one embodiment, said SF+ cells are a commercial product sold by Protein Sciences Corporation (Meriden, CT).

[0045] In one embodiment of the method of the present invention, the method includes producing a recombinant baculovirus containing a nucleic acid molecule of the present invention, and infecting insect cells with the recombinant baculovirus. In one embodiment, said recombinant baculovirus is obtained from a commercial product sold under the trademark Sapphire™ Baculovirus (Allele Biotechnology). In one embodiment, the nucleic acid molecule of the present invention is presented in SEQ ID NO: 10. In one embodiment, the nucleic acid molecule of the present invention is presented in SEQ ID NO: 11. In one embodiment, said insect cells are SF+ cells. In one embodiment, said SF+ cells are a commercial product sold by Protein Sciences Corporation (Meriden, CT).

[0046] In one embodiment of the method of the present invention, the method includes: (i) producing a recombinant baculovirus containing a nucleic acid molecule of the present invention; (ii) infection of insect cells with a recombinant baculovirus; (iii) culturing these insect cells in a culture medium; and (iv) collecting the H5 HA protein of the present invention or a whole cell culture containing the H5 HA protein of the present invention. In one embodiment, the recombinant baculovirus is obtained from a commercial product sold under the trademark Sapphire™ Baculovirus (Allele Biotechnology). In one embodiment, the nucleic acid molecule of the present invention is presented in SEQ ID NO: 10. In one embodiment, the nucleic acid molecule of the present invention is presented in SEQ ID NO: 11. In one embodiment, said insect cells are SF+ cells. In one embodiment, said SF+ cells are a commercial product sold by Protein Sciences Corporation (Meriden, CT).

[0047] In one embodiment of the method of the present invention, the culture medium for culturing the cells of the present invention will be determined by those skilled in the art. In one embodiment, the culture medium is a serum-free insect cell medium. In one embodiment, the culture medium is Ex-CELL 420 (Ex-CELL® 420 Serum-Free Insect Cell Medium, Sigma-Aldrich, Cat. 14420C).

[0048] In one embodiment of the method of the present invention, insect cells are cultured under conditions suitable for expression of the H5 HA protein. In one embodiment, the insect cells are incubated for a period of up to ten days, preferably from about two days to about ten days, more preferably from about four days to about nine days, and even more preferably from about five days to about eight days. In one embodiment, conditions suitable for culturing insect cells include a temperature of about 22-32°C, preferably about 24-30°C, more preferably about 25-29°C, even more preferably about 26-28°C, and most preferably around 27°C.

[0049] In one embodiment of the method of the present invention, the method further includes the step of inactivating the cell culture of the present invention. For the purposes of the present invention, any suitable inactivation method can be used, including, but not limited to, chemical and/or physical treatments.

[0050] In one embodiment, the inactivation step includes adding cyclized binary ethyleneimine (BEI), preferably at a concentration of about 1 to about 20 mM, preferably about 2 to about 10 mM, more preferably about 5 mM or 10 mM. In one embodiment, the inactivation step includes adding a solution of 2-bromoethyleneamine hydrobromide, which undergoes cyclization to form BEI in NaOH.

[0051] In one embodiment, the inactivation step is carried out at a temperature in the range of 25-40°C, preferably in the range of 28-39°C, more preferably in the range of 30-39°C, more preferably in the range of 35-39°C. In one embodiment, the inactivation step is carried out for 24-72 hours, preferably for 30-72 hours, more preferably for 48-72 hours. Typically, the inactivation step is performed until no viral replication is detected. vector.

[0052] In one embodiment of the method of the present invention, the method further includes a neutralization step after the inactivation step. The neutralization step involves adding an equivalent amount of agent that neutralizes the inactivating agent in solution. In one embodiment, the inactivating agent is BEI. In one embodiment, the neutralizing agent is sodium thiosulfate. In one embodiment, when the inactivating agent is BEI, an equivalent amount of sodium thiosulfate will be added. For example, if BEI is added to a final concentration of 5 mM, 1.0 M sodium thiosulfate solution is added to obtain a final minimum concentration of 5 mM to neutralize any residual BEI. In one embodiment, the neutralization step includes adding sodium thiosulfate solution to a final concentration of 1 to 20 mM, preferably 2 to 10 mM, more preferably 5 mM or 10 mM when the inactivating agent is BEI. In one embodiment, the neutralizing agent is added after completion of the inactivation step, which means that no replication of the viral vector can be detected. In one embodiment, the neutralizing agent is added after the inactivation step has been carried out for 24 hours. In one embodiment, the neutralizing agent is added after the inactivation step has been carried out for 30 hours. In one embodiment, the neutralizing agent is added after the inactivation step has been carried out for 48 hours. In one embodiment, the neutralizing agent is added after the inactivation step has been carried out for 72 hours.

[0053] The amount of H5 HA protein contained in the immunogenic composition of the present invention can be quantified using any conventional methods known in the art, for example, hemagglutination assay (see OIE Terrestrial Manual 2015, chapter 2.3. 1 & 2.3.2 , Avian Influenza (infection with the avian influenza virus)). In one embodiment, the amount of H5 HA protein contained in the immunogenic composition of the present invention is determined using a chicken red blood cell hemagglutination test, and the amount of H5 HA protein may be expressed in hemagglutinin assay units (HAU).

[0054] The term "HAU" refers to a red blood cell agglutination activity unit (RBC), and 1 HAU of hemagglutinin protein refers to the minimum unit that causes agglutination when hemagglutinin is mixed with RBC. HAU can be assessed using a hemagglutination titer assay. In this assay, a sample containing hemagglutinin protein is serially diluted twofold in a 96-well plate and RBC solution is added to the wells. The HAU value represents the highest dilution factor of the sample that results in complete red blood cell (RBC) agglutination. For example, if the highest dilution factor of a 25 µL sample is 256, the amount of hemagglutinin protein contained in the sample is 256 HAU per 25 µL.

[0055] To elicit an effective immune response according to the present invention, the amount of H5 HA protein contained in the immunogenic composition of the present invention is at least 32 HAU per dose. Thus, the immunogenic composition of the present invention contains at least 32 HAU per dose of the H5 HA protein of the present invention. In a further aspect, the immunogenic composition of the present invention contains at least 64 HAU per dose of the H5 HA protein of the present invention. In a further aspect, the immunogenic composition of the present invention contains at least 128 HAU per dose of the H5 HA protein of the present invention. In a further aspect, the immunogenic composition of the present invention contains at least 256 HAU per dose of the H5 HA protein of the present invention.

[0056] One skilled in the art can determine the upper limit of the amount of HA protein H5 simply by routine testing. In general, the upper limit used by one skilled in the art will be in the range of approximately 1024 HAU per dose, but may also be higher. In a further aspect, the immunogenic composition of the present invention contains at least 32 to 1024 HAU per dose of the H5 HA protein of the present invention. In a further aspect, the immunogenic composition of the present invention contains at least 64 to 1024 HAU per dose of the H5 HA protein of the present invention. In a further aspect, the immunogenic composition of the present invention contains at least 128 to 1024 HAU per dose of the H5 HA protein of the present invention. In a further aspect, the immunogenic composition of the present invention contains at least 256 to 1024 HAU per dose of the H5 HA protein of the present invention.

[0057] Compared to conventional inactivated whole virus vaccines, which must be produced under Biosafety Level 2 or 3 (BSL-2, BSL-3), the above method for producing H5 HA protein is categorized as a Biosafety Level 1 requirement.

[0058] In another aspect, the invention also provides a method of preventing and/or treating infections caused by AIV, comprising administering an effective amount of an immunogenic composition of the present invention to a subject in need thereof. The invention also provides an immunogenic composition of the present invention for use in the prevention and/or treatment of infections caused by AIV.

[0059] As used herein, the term “prevention” refers to reducing the frequency or severity of clinical symptoms of influenza infection, up to and including completely preventing such clinical symptoms. The effectiveness of an immunogenic composition for prophylactic protection can be assessed based on the survival of the vaccinated subject against various AIV clades. In one embodiment, the protection efficiency of the immunogenic composition is increased by at least 10%, more preferably by at least 20%, even more preferably by at least 30%, even more preferably by at least 40%, more preferably by at least by 50%, even more preferably at least 60%, even more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95% and most preferably 100% compared to a subject who did not receive a vaccine of the present invention but was exposed to infectious levels of AIV. In one embodiment, the effectiveness of protection of the immunogenic composition is 100% in a vaccinated subject against various clades of AIV.

[0060] In one embodiment, the subject in need may be poultry, even more preferably poultry, chicken, duck, turkey, or the like. In one embodiment, the subject is a chicken or duck.

[0061] In one embodiment, AIV is subtype H5. In one embodiment, the AIV is H5Nx. In one embodiment, the AIV is HPAI H5Nx, which circulates primarily in Asia, the Middle East and Africa. In one embodiment, H5Nx is H5N1, H5N2 and/or H5N6. In one embodiment, H5Nx is H5N1. In one embodiment, H5Nx is H5N2. In one embodiment, H5Nx is H5N6. In one embodiment, H5Nx is H5N1 and H5N2. In one embodiment, H5Nx is H5N2 and H5N6. In one embodiment, AIV is clade 2, preferably clade 2.3, and most preferably clade 2.3.4 and 2.3.2. In one embodiment, AIV is clade 2.3.4.4, 2.3.2.1, 2.3.2.Id, or 2.3.4.4d. In one embodiment, AIV is clade 7.2.

[0062] H5Nx subtypes H5N1, H5N2 and/or H5N6 are well known in the art.

[0063] As used herein, the term “clade” refers to a group of biological taxa (such as species) that includes all descendants of a common ancestor. A/goose/Guangdong/1/96 (A/goose/Guangdong/1/96) was considered the ancestor of all H5 lineage viruses and was designated clade 0. This nomenclature is now used to distinguish groups of H5 HA gene variants, until now it was About 20 different clades of the virus have been officially identified. In addition to clade 0, an additional 9 clades (1-9) have been described and defined based on all deposited sequences. However, over time, most viral clades became extinct. The H5 lineage virus continued to circulate among domestic and wild birds in parts of Asia, the Middle East and Africa and continued to evolve. The most dominant clade, resulting in endemic infections in several different countries, is the clade 2 viruses, and further sublineages resulting from antigenic drift (variation) have been described as clades 2, 3, 4 and even 5. th order. The nomenclature and resulting clade can be seen in WHO/OIE/FAO. Progress towards a unified nomenclature for highly pathogenic H5N1 avian influenza viruses continues: divergence of clade 2.2 viruses. Influenza Other Respiration Viruses. Letter. 2009; 3: 59-62; WHO/OIE/FAO. Continued evolution of highly pathogenic avian influenza A (H5N1): updated nomenclature. (Continuing evolution of highly pathogenic avian influenza A (H5N1): updated nomenclature) Influenza Other Respiration Viruses 2012; 6:15; and WHO-OIE-FAO HNEWG. Revised and updated nomenclature for highly pathogenic avian influenza A (H5N1) viruses. Influenza Other Respiration Viruses 2014; 8: 384-388.

[0064] For example, H5Nx clade 2.3.4.4 is described in Lee et al., Emerging Infectious Diseases, 2016; vol 22(7) pp 1283-1284 and in Lee et al., Journal Veterinary Science 2017; volume (SI), pp. 269-280. Avian influenza A/magpie/Hong Kong/5052/2007 (H5N1) isolate described in Smith et al., Emerging Infectious Diseases, 2009; Volume 15(3), pp. 402-407, belongs to clade 2.3.2.1. Subsequent H5Nx isolates of clade 2.3.2.1, including A/chicken/India/CE03485/2011 (H5N1) or A/chicken/India/CAO2/2011 (H5N1), are described, for example, in Bhat et al., 2015, Microbial Pathogenesis; volume 88, pp. 87-93. Subsequent H5Nx lining 2.3.2.1, as well as 2.3.4.4, including 2.3.4.4d, such as, for example, A/duck/Vietnam/NSH-1507/2014 (H5N6) or A/duck/Vietnam/HU1-1151/2014 (H5N6) are described in Nguyen et al., 2019, Scientific Reports 9; Article 7723, pp. 1-13. H5Nx clade 7.2 isolates, including, for example, (A/chicken/Gansu/62012 (H5N1)) are described and characterized, for example, in Liu et al., Journal of Virology 2016, volume 90(21), pp. 9797-9804.

[0065] In one embodiment, the immunogenic composition of the present invention can be administered by subcutaneous (S.C.) or intramuscular (I.M.) administration.

[0066] The time of administration of the immunogenic composition of the present invention can be determined by one skilled in the art in accordance with practical requirements. For example, with regard to white broilers, whose life expectancy is about 40-50 days, the immunogenic composition of the present invention can be administered as a single dose; As for yellow broilers, whose life expectancy is about 70 days, the immunogenic composition of the present invention can be administered in two doses; and for chickens, such as breeding chickens, whose lifespan is more than one year or longer, the immunogenic composition of the present invention can be administered in multiple doses. In one embodiment, the immunogenic composition of the present invention is administered as a single dose. In one embodiment, the immunogenic composition of the present invention is administered in multiple doses, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses. In one embodiment, the interval between two doses is about 4-6 weeks, such as 4 weeks, 5 weeks, or 6 weeks.

[0067] One skilled in the art can also determine when to administer the first dose according to practical requirements. For example, when vaccinating chickens. In one embodiment, the immunogenic composition of the present invention may be first administered on or after day 1 of life, on or after day 7 of life, on or after day 10 of life, on or after day 15 of life, or on or after day 21 of life. In one embodiment, the immunogenic composition of the present invention is administered on or after day 1 of life, on or after day 7 of life, on or after day 10 of life, on or after day 15 of life, or on or after day 21 of life. In one embodiment, the immunogenic composition of the present invention is administered from day 1 of life, from day 7 of life, from day 10 of life, from day 15 of life, or from day 21 of life in a single dose. In one embodiment, the immunogenic composition of the present invention is administered once from day 1 of life.

[0068] In another aspect, the invention also relates to a method for differentiating animals naturally infected with AIV from animals vaccinated with a vaccine or immunogenic composition of the present invention. The invention also provides the use of the immunogenic composition of the present invention for the preparation of an agent for differentiating between animals naturally infected with AIV and vaccinated animals.

[0069] In one embodiment, the method includes: a) analyzing an animal sample in an immunoassay and/or genomic assay for the presence of an avian influenza marker that is not present in the immunogenic composition but is present in naturally infected animals, where the immunoassay represents is an enzyme-linked immunosorbent assay or enzyme-linked immunosorbent assay, or agar gel precipitation assay, or Western blot assay; b) determining whether a sample is positive or negative for an avian influenza marker; and c) correlating the test results with respect to the status of the animal being tested, with an animal that is positive for the avian influenza marker being a naturally infected animal and an animal that is negative for the avian influenza marker. marker of avian influenza is an animal vaccinated with the immunogenic composition or vaccine of the present invention.

[0070] In one embodiment, the method includes: a) testing an animal sample in an immune and/or genomic assay for the presence of an avian influenza marker that is specific to the immunogenic composition but is not present in a naturally infected animal, wherein the immunoassay is an enzyme-linked immunosorbent assay assay or enzyme-linked immunosorbent assay, b) determining whether the sample is positive or negative for an avian influenza marker, and c) correlating the test results with the status of the animal being tested, where an animal that is positive for an avian influenza marker is an animal that has been vaccinated with an immunogenic composition or vaccine of the present invention.

[0071] In one embodiment, the animal may be poultry, even more preferably poultry, chicken, duck, turkey, or the like. In one embodiment, the animal is a chicken or a duck.

[0072] In one embodiment, AIV is subtype H5. In one embodiment, said AIV is H5Nx. In one embodiment, said AIV is HPAI H5Nx, which circulates primarily in Asia, the Middle East and Africa. In one embodiment, H5Nx is H5N1, H5N2 and/or H5N6. In one embodiment, H5Nx is H5N1. In one embodiment, H5Nx is H5N2. In one embodiment, H5Nx is H5N6. In one embodiment, H5Nx is H5N1 and H5N2. In one embodiment, H5Nx is H5N2 and H5N6.

[0073] In one embodiment, AIV is clade 2, preferably clade 2.3, and more preferably clade 2.3.4 and 2.3.2. In one embodiment, AIV is clade 2.3.4.4 or 2.3.2.1, 2.3.2.1d or 2.3.4.4d. In one embodiment, AIV is clade 7.2.

[0074] Animals vaccinated with a vaccine or immunogenic composition containing the H5 HA protein of the present invention will have an antibody response only against that specific antigen, while having a negative response to other viral components. Another advantage of the present invention is that it is useful for the concept of DIVA with specific ELISA for differentiating AIV-infected animals from vaccinated animals.

[0075] In another aspect, the invention also provides kits containing the H5 HA protein of the present invention, a nucleic acid molecule, vector, cell or vaccine, or immunogenic composition of the present invention. In one embodiment, the kits can be used for a subject such as poultry, even more preferably poultry, chicken, duck, turkey, and the like. In one embodiment, when a chicken is vaccinated, the H5 HA protein, vaccine or immunogenic composition of the present invention can be used for vaccination on or after day 1 of life, on or after day 7 of life, or on or after day 10 of life. , on the 15th day of life or later, or on the 21st day of life or later. In one embodiment, the immunogenic composition of the present invention is administered from the 1st day of life, from the 7th day of life, from the 10th day of life, from the 15th day of life, or from the 21st day of life in a single dose. In one embodiment, the immunogenic composition of the present invention is administered once on day 1 of life.

[0076] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention relates at the time of filing. The meaning and scope of the terms must be clear; however, in the event of any hidden ambiguity, the definitions given herein take precedence over any dictionary or other definitions provided. In addition, unless the context otherwise requires, terms in the singular shall include the plural and terms in the plural shall include the singular. In this specification, the use of “or” means “and/or” unless otherwise indicated. In addition, the use of the term “including” as well as other forms such as “includes” and “included” is not limiting. All patents and publications mentioned herein are incorporated herein by reference.

[0077] In practice, in accordance with the present invention, unless otherwise indicated, conventional methods of virology, molecular biology, microbiology, recombinant DNA technology, protein chemistry and immunology will be used, which are within the competence of specialists in the field. Such methods are fully explained in the literature. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, volumes I, II and III, second edition (1989); DNA Cloning, volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins ed. 1984); Animal Cell Culture (R. K. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL press, 1986); Perbal, V., A Practical Guide to Molecular Cloning (1984); series Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Protein purification methods a practical approach (E.L.V. Harris and S. Angal, eds., IRL Press at Oxford University Press); and Handbook of Experimental Immunology, volumes I-IV (D. M. Weir and S. S. Blackwell eds., 1986, Blackwell Scientific Publications).

[0078] It should also be understood that the terminology used herein is intended only to describe specific embodiments of the present invention and is not intended to be limiting. It should be noted that, as used in this specification and the accompanying claims, the singular forms include plural terms and concepts unless the content clearly indicates otherwise. Thus, for example, a reference to “antigen” includes a mixture of two or more antigens, a reference to “carrier” includes mixtures of two or more carriers, and the like.

POINTS

[0079] The following items are also described in this specification and disclosure portion of the present invention:

[0080] 1. An immunogenic composition containing the hemagglutinin protein of the avian influenza virus subtype H5, where the hemagglutinin protein contains:

(a) amino acid residues 120N or 120S, 155N, and 223N or 223S; And

(b) one or more amino acid residues selected from the group consisting of: 61D, 871, 99A, 102A, 110N, 136S, 140D, 149S, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 226V, 243D, 268Y, 279A and 2981; wherein the numbering regarding amino acid residues is as presented in SEQ ID NO:l

[0081] 2. The immunogenic composition according to claim 1, where the hemagglutinin protein contains:

(a) amino acid residues 61D, 871, 99A, 102A, 110N, 120N, 136S, 140D, 149S, 155N, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 223N, 226V , 243D, 268Y, 279A and 2981;

(b) the amino acid sequence shown in SEQ ID NO: 6; or

(c) the amino acid sequence shown in SEQ ID NO: 5.

[0082] 3. The immunogenic composition of claim 1 or 2, wherein the hemagglutinin protein contains the amino acid sequence set forth in SEQ ID NO: 6.

[0083] 4. The immunogenic composition of claim 1 or 2, wherein the hemagglutinin protein contains the amino acid sequence set forth in SEQ ID NO: 5.

[0084] 5. The immunogenic composition according to any one of claims 14, wherein the hemagglutinin protein is contained in a cell culture, and the cell culture is obtained by culturing cells containing an expression vector capable of expressing the hemagglutinin protein as described in any of claims 1-4.

[0085] 6. The immunogenic composition according to any one of claims 1 to 5, wherein the expression vector contains a nucleic acid molecule encoding a hemagglutinin protein as described in any of claims 1 to 4.

[0086] 7. An immunogenic composition at position 6, wherein the nucleic acid molecule is represented in SEQ ID NO: 10.

[0087] 8. An immunogenic composition at position 6, wherein the nucleic acid molecule is shown in SEQ ID NO: 11.

[0088] 9. The immunogenic composition according to any one of claims 5-8, wherein the expression vector is a baculovirus and the cells are insect cells.

[0089] 10. The immunogenic composition of claims 5-9, wherein the cell culture is subjected to an inactivation step, preferably a binary ethyleneimine inactivation step.

[0090] 11. The immunogenic composition according to any one of claims 5-10, where the immunogenic composition contains part or all of the cell culture.

[0091] 12. The immunogenic composition according to any one of claims 1-11, where the immunogenic composition further comprises an adjuvant.

[0092] 13. The immunogenic composition of claim 12, wherein the adjuvant is a water-in-oil emulsion.

[0093] 14. The immunogenic composition according to any one of paragraphs 1 to 13, where the immunogenic composition is administered in a single or multiple administration; and/or

the immunogenic composition is administered subcutaneously or intramuscularly; and/or the immunogenic composition should be administered to the animal on the 1st day of life or older, on the 7th day of life or older, on the 10th day of life or older, on the 15th day of life or older, or on the 21st day life or older.

[0094] 15. The immunogenic composition according to any one of claims 1-14, wherein the immunogenic composition should be administered to the animal on the 1st day of life or older, on the 7th day of life or older, on the 10th day of life or older, on 15 1st day of life or older, or 21st day of life or older.

[0095] 16. The immunogenic composition according to any one of paragraphs 1 to 14, where the immunogenic composition should be administered to the animal from the 21st day of life.

[0096] 17. The immunogenic composition according to any one of paragraphs 1-14, where the immunogenic composition should be administered to the animal from the 15th day of life.

[0097] 18. The immunogenic composition according to any one of paragraphs 1-14, where the immunogenic composition should be administered to the animal from the 7th day of life.

[0098] 19. The immunogenic composition according to any one of paragraphs 1-14, where the immunogenic composition should be administered to the animal from the 1st day of life.

[0099] 20. The immunogenic composition according to any one of paragraphs 1-14, where the immunogenic composition should be administered to the animal from the 1st day of life.

[00100] 21. An immunogenic composition according to any one of claims 1-20 for use in the prevention and/or treatment of infections caused by an avian influenza virus, preferably H5Nx, more preferably one or more of H5N1, H5N2 and H5N6.

[00101] 22. An immunogenic composition according to any one of claims 1-20 for use in the prevention and/or treatment of infections caused by the H5N1 avian influenza virus.

[00102] 23. An immunogenic composition according to any one of claims 1-20 for use in the prevention and/or treatment of infections caused by the H5N2 avian influenza virus.

[00103] 24. An immunogenic composition according to any one of claims 1-20 for use in the prevention and/or treatment of infections caused by the H5N6 avian influenza virus.

[00104] 25. An immunogenic composition according to any one of claims 1-20 for use in the prevention and/or treatment of infections caused by H5Nx avian influenza virus clade 2, preferably clade 2.3 and more preferably clade 2.3.4 or 2.3.2.

[00105] 26. An immunogenic composition according to any one of claims 1-20 for use in the prevention and/or treatment of infections caused by H5Nx clade 2 avian influenza virus.

[00106] 27. An immunogenic composition according to any one of claims 1-20 for use in the prevention and/or treatment of infections caused by H5Nx avian influenza virus clade 2.3.

[00107] 28. An immunogenic composition according to any of claims 1-20 for use in the prevention and/or treatment of infections caused by H5Nx avian influenza virus clade 2.3.4.

[00108] 29. An immunogenic composition according to any one of claims 1-20 for use in the prevention and/or treatment of infections caused by H5Nx avian influenza virus clade 2.3.4.4.

[00109] 30. An immunogenic composition according to any one of claims 1-20 for use in the prevention and/or treatment of infections caused by H5Nx avian influenza virus clade 2.3.4.4d.

[00110] 31. An immunogenic composition according to any of paragraphs 1-20 for use in the prevention and/or treatment of infections caused by H5Nx avian influenza virus clade 2.3.2.

[00111] 32. An immunogenic composition according to any one of claims 1-20 for use in the prevention and/or treatment of infections caused by H5Nx avian influenza virus clade 2.3.2.1.

[00112] 33. An immunogenic composition according to any one of claims 1-20 for use in the prevention and/or treatment of infections caused by H5Nx avian influenza virus clade 2.3.2.1d.

[00113] 34. An immunogenic composition according to any one of claims 1-20 for use in the prevention and/or treatment of infections caused by H5Nx avian influenza virus clade 7.2.

[00114] 35. The immunogenic composition according to any one of claims 1-34, wherein the immunogenic composition is administered in a single administration.

[00115] 36. The immunogenic composition according to any one of claims 1-34, wherein the immunogenic composition is administered as a single dose and wherein said single dose is effective in the prevention and/or treatment of infections caused by the avian influenza virus.

[00116] 37. The immunogenic composition of any one of claims 1 to 34, wherein the immunogenic composition is administered as a single dose and wherein said single dose is effective in preventing and/or treating infections caused by one or more of H5N1, H5N2 and H5N6.

[00117] 38. The immunogenic composition of any one of claims 1 to 34, wherein the immunogenic composition is administered as a single dose and wherein said single dose is effective in preventing and/or treating infections caused by any of the H5Nx as described in claims 22 to 34 .

[00118] 39. A method of differentiating animals naturally infected with an avian influenza virus from animals vaccinated with an immunogenic composition according to any of paragraphs 1-13, including: (a) analyzing a sample of the animal in an immunoassay and/or genomic analytical assay for the presence of an avian marker influenza that is not present in the immunogenic composition but is present in a naturally infected animal, where the immunoassay is an enzyme-linked immunosorbent assay or an enzyme-linked immunosorbent assay,

(b) determining whether the sample is positive or negative for an avian influenza marker, and

(c) correlation of the test results with the status of the test animal, where an animal that is positive for the avian influenza marker is a naturally infected animal, and an animal that is negative for the avian influenza marker is an animal vaccinated with the immunogenic composition of any of paragraphs 1 to 13.

[00119] 40. A method for differentiating animals naturally infected with an avian influenza virus from animals vaccinated with an immunogenic composition according to any of paragraphs 1-13, including:

(a) testing an animal sample in an immunoassay and/or a genomic assay for the presence of an avian influenza marker that is specific to the immunogenic composition but is not present in a naturally infected animal, where the immunoassay is an enzyme-linked immunosorbent assay or an enzyme-linked immunosorbent assay,

(b) determining whether the sample is positive or negative for an avian influenza marker, and

(c) correlation of the test results with the status of the test animal, where an animal positive for the avian influenza marker is an animal vaccinated with an immunogenic composition according to any of paragraphs 1 to 13.

SEQUENCE OVERVIEW

[00120] The following sequences are described and disclosed in detail in the present invention:

[00121] SEQ ID NO: 1: Amino acid sequence of the HA protein of duck/China/E319-2/03 strain, which does not contain a signal peptide at the N-terminus.

[00122] SEQ ID NO: 2: Amino acid sequence of H5Con1.

[00123] SEQ ID NO: 3: Amino acid sequence of H5Con3.

[00124] SEQ ID NO: 4: Amino acid sequence of H5Con5.

[00125] SEQ ID NO: 5: Amino acid sequence of H5Con5Mut.

[00126] SEQ ID NO: 6: Amino acid sequence from position 60 to 300 of H5Con5Mut.

[00127] SEQ ID NO: 7: Optimized H5Con1 nucleic acid sequences.

[00128] SEQ ID NO: 8: Optimized H5Con3 nucleic acid sequences.

[00129] SEQ ID NO: 9: Optimized H5Con5 nucleic acid sequences.

[00130] SEQ ID NO: 10: Optimized H5ConMut Nucleic Acid Sequences.

[00131] SEQ ID NO: 11: Optimized nucleic acid sequence encoding the amino acid sequence from positions 60 to 300 of H5Con5Mut.

BRIEF DESCRIPTION OF DRAWINGS

[00132] The following drawings form part of the present invention and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein.

[00133] Figure 1: The HSConl sequence was generated by a sequential method from 444 sequences isolated from 1996 to 2012.

[00134] Figure 2: The H5Con3 sequence was generated using a one-cycle method from 297 clade-specific sequences isolated from 1996 to 2012.

[00135] Figure 3: The H5Con5 sequence was generated using a one-round method from 196 sequences isolated from 2005 to 2012.

[00136] Figure 4: Construction of transfer plasmid pVL1393-H5Con5Mut.

EXAMPLES

[00137] The following examples are included to further illustrate the invention described herein and to demonstrate embodiments of the present invention. It will be understood by those skilled in the art that the methods disclosed in the examples that follow represent methods that the inventors have found to function well in the practice of the present invention, and thus may be considered constituting methods for its application. However, those skilled in the art should, in light of the present disclosure, appreciate that many changes could be made to the specific disclosed embodiments and still achieve a similar or similar result that is within the spirit and scope of the present invention.

[00138] Example 1: Generation of HA H5 consensus amino acid sequences and their mutants

[00139] Construction HA of the H5N1 influenza consensus amino acid sequence 1

[00140] To create the HA consensus amino acid sequence 1 of influenza H5N1 (H5Con1), 444 HA amino acid sequences of the H5N1 influenza virus isolated in China from 1996 to 2012 were collected and analyzed. 444 HA amino acid sequences include clade 0, clade 2.2, clade 2.3.2, clade 2.3.2.1, clade 2.3.4, clade 2.4, clade 2.5, clade 3, clade 4, clade 5, clade 6, clade 7 and clade 9. Figure 1 shows the sequential method for generating H5Con1 from 444 amino acid sequences isolated from 1996 to 2012. First, 13 level 1 consensus amino acid sequences were generated, and each level represents a distinct clade using (1) 36 clade 0 sequences; (2) 31 sequences of clade 2.2; (3) 8 sequences of clade 2.3.2; (4) 49 sequences of clade 2.3.2.1, (5) 2 sequences of clade 2.3.3; (6) 210 sequences of clade 2.3.4; (7) 18 sequences of clade 2.4; (8) 6 sequences of clade 2.5; (9) 9 sequences of clade 3; (10) 5 sequences of clade 4; (11) 8 sequences of clade 5; (12) 3 sequences of clade 6; (13) 34 sequences of clade 7; and (14) 21 clade 9 sequences. The final consensus amino acid sequence is a level 2 consensus amino acid sequence that was obtained by aligning and analyzing all 13 level 1 consensus amino acid sequences using MEGA 5.0 software. This final consensus amino acid sequence was designated H5Conl (SEQ ID NO: 2).

[00141] Construction of the HA consensus amino acid sequence 3 of influenza H5N1

[00142] To create the H5N1 influenza consensus amino acid sequence HA 3 (H5Con3), 297 H5N1 influenza amino acid sequence HAs isolated in China from 1996 to 2012 were collected and analyzed. The indicated 297 HA amino acid sequences consist only of certain clades, i.e. clade 2.3.2.1, clade 2.3.4, and clade 7. Figure 2 shows the one-cycle method for generating H5Con3 from 297 clade-specific amino acid sequences isolated from 1996 to 2012. The final consensus amino acid sequence was obtained by alignment and analysis of (1) 49 clade 2.3.2.1 sequences, (2) 214 clade 2.3.4 sequences; and (3) 34 clade 7 sequences with MEGA 5.0 software. This final consensus amino acid sequence was designated H5Con3 (SEQ ID NO: 3).

[00143] Construction of HA consensus amino acid sequence 5 of influenza H5N1

[00144] To create the H5N1 influenza virus consensus amino acid sequence HA 5 (H5Con5), 196 H5N1 influenza virus HA amino acid sequences isolated in China from 2005 to 2012 were collected and analyzed. The indicated 196 HA amino acid sequences are taken from clades 0, 2.2, 2.3.1, 2.3.2, 2.3.2.1, 2.3.4, 2.4, 7 and 9. Figure 3 shows a one-cycle method for generating H5Con5 from 196 amino acid sequences isolated since 2005 to 2012. A total of 196 amino acid sequences were aligned simultaneously using MEGA 5.0 software. The final consensus amino acid sequence was designated H5Con5 (SEQ ID NO: 4).

[00145] The strategies for creating H5Con1, H5Con3 and H5Con5 are summarized in Table 1.

[00147] Construction of a mutant HA of the consensus amino acid sequence 5 of the H5N1 influenza virus

[00148] To create the H5N1 influenza virus consensus amino acid sequence 5 mutant HA (H5Con5Mut), two amino acid mutations were introduced into the H5Con5 amino acid sequence. At amino acid positions 120 and 223, both serines (S) were changed to asparagines (N), i.e. S120N and S223N. The amino acid sequence of H5Con5Mut is shown in SEQ ID NO: 5.

[00149] Analysis of H5Conl, H5Con3 and H5Con5

[00150] Table 2 shows the percentage identity between the consensus amino acid sequences of H5Con1, H5Con3 and H5Con5 and sequences from the predominant clades. Table 3 also shows the amino acid usage of each of the consensus amino acid sequences of H5Con1, H5Con3 and H5Con5 in some predicted antigenic sites of the H5 HA protein.

[00153] From Tables 2 and 3, it can be seen that H5Con1, H5Con3 and H5Con5 differ in amino acid sequences compared to sequences from each of the predominant clades, and also have some amino acid changes at antigenic sites among the three different predominant clades.

[00154] Example 2: Optimization of H5Con1, H5Con3, H5Con5 and H5Con5Mut nucleic acid sequences, creation of recombinant baculovirus expression systems and expression of H5Con1, H5Con3, H5Con5 and H5Con5Mut in insect cells

[00155] Optimization of nucleic acid sequences H5Con1, H5Con3. H5Con5 and H5Con5Mut

[00156] To optimize for best expression, each of the amino acid sequences of HSConl, H5Con3 and H5Con5, as identified in Example 1, were reverse engineered into the corresponding nucleic acid sequence using Vector NTI software and optimized for expression in insect cells of the baculovirus expression system ( Allele Biotechnology, CAT ABP-BVP-10002). The optimized nucleic acid sequences of H5Con1, H5Con3 and H5Con5 were synthesized by GenScript (GenScript, NJ, USA). These optimized nucleic acid sequences of H5Con1, H5Con3 and H5Con5 are shown in SEQ ID NO: 7-9, respectively. The optimized H5Con5Mut nucleic acid sequence was generated using a site-directed mutagenesis kit (QuickChange Site-Directed Mutagenesis kit, catalog no. 200518, Agilent) based on the optimized H5Con5 nucleic acid sequence. The optimized nucleic acid sequence of H5ConMut is provided in SEQ ID NO: 10.

[00157] Construction of recombinant baculovirus expression systems and expression of H5Con1, H5Con3, H5Con5 and H5Con5Mut in insect cells.

[00158] Each of the optimized nucleic acid sequences H5Con1, H5Con3, H5Con5 and H5Con5Mut, as described above, was inserted into the pVL1393 baculovirus expression system transfer vector, which was included in the Sapphire™ Baculovirus DNA and Transfection Kit (Allele Biotechnology, CAT ABP-BVD -10002) to obtain transfer plasmids designated pVL1393-H5Conl, PVL1393-H5Con3, PVL1393-H5Con5, and pVL1393-H5Con5Mut, respectively. Figure 4 shows the construction of the transfer plasmid pVL1393-H5Con5Mut as an example.

[00159] Each of pVL1393-H5Conl, PVL1393-H5Con3, PVL1393-H5Con5, and pVL1393-H5Con5Mut was then cotransfected with linearized wild-type Sapphire™ baculovirus DNA (Sapphire™ Baculovirus DNA and Transfection Kit; Allele Biotechnology, CAT ABP-BVD-1 0002) into cells sf9 insects (Invitrogen, Cat #B825-01, Lot #1030672) so as to obtain isolated recombinant baculoviruses. These recombinant baculoviruses containing optimized nucleic acid sequences H5Con1, H5Con3, H5Con5 and H5Con5Mut were designated rBacH5Conl, rBacH5Con3, rBacH5Con5 and rBacH5Con5Mut, respectively. And transfected sf9 cells containing each of the recombinant baculoviruses were obtained.

[00160] The transfected sf9 cells obtained above were cultured at 27°C in an incubator for four days and the cell culture supernatant was collected. To obtain pure recombinant virus, the collected supernatant was then subjected to plaque cloning. Three rounds of plaque cloning were carried out, and viral plaques containing rBacH5Con1, rBacH5Con3, rBacH5Con5 and rBacH5Con5Mut, respectively, were collected after the third round of plaque cloning, so as to obtain purified rBacH5Conl, rBacHSConl, rBacH5Con3, rBacH5Con3, rBacH5Con3 and rBacH5Con5.

[00161] Each of the above purified rBacHSCon1, rBacH3Con3, rBacH5Con5 and rBacH5Con5Mut was further propagated in a suspension culture of SF+insect cell line (Ex-CELL® 420 serum-free insect cell medium, Sigma-Aldrich, Cat. 14420C) in shake flasks . Briefly, SF+ cells (Protein Sciences Corporation, Meriden, CT) were seeded in a shake flask at a density of 106 cells/ml, and each of the above purified rBacH5Conl, rBacH5Con3, rBacH5Con5, and rBacH5Con5Mut was inoculated into SF+ cells (Protein Sciences, Inc., Meriden , CT) with a multiplicity of infection from MOI=0.01 to MOI=1. Inoculated SF+ cells were cultured in EX-CELL 420 medium at 27°C with a shaking speed of 80-120 rpm. for 3-7 days, and for the next step, cell culture suspensions containing expressed H5Con1, H5Con3, H5Con5 and H5Con5Mut, respectively, were collected. The collected cell culture suspensions contained cell counts in the range of 0.5-1.5×10 ⁶ cells/ml.

[00162] Example 3: HATJ Determination of Cell Culture Suspensions

[00163] The HAU of cell culture suspensions containing H5Con1, H5Con3, H5Con5 and H5Con5Mut, respectively, was examined using a hemagglutination assay (see OIE Terrestrial Manual 2015, chapter 2.3.1 & 2.3.2, Avian Influenza) with the two-fold serial dilution method. Briefly, 10 mL of red blood cells (from Southern Regent Plant, Zhejiang Province) were centrifuged at 500 rpm. for 20 min, and 1 volume of 4% centrifuged erythrocytes was added to 3 volumes of PBS to obtain 1% erythrocytes. 25 μl of PBS was added to wells 1 to 11 of a 96-well plate. 25 μl of each of the cell culture suspensions was added to well 1-well 11 and then serially diluted from 1:2 to 1:2048 times. 25 μl of 1% red blood cells were added to all wells and incubated at room temperature for approximately 40 minutes.

[00164] Negative wells appeared as dots in the center of the wells, and positive results formed a uniform reddish color throughout the entire well. The end point of dilution corresponds to the greatest dilution of the sample resulting in complete agglutination of red blood cells. The results showed that the HAU of HSConl, H5Con3, H5Con5 and H5Con5Mut cell culture suspensions was greater than 71.1 HAU/25 μl (corresponding to 256 HAU/90 μl or 256 HAU/dose).

[00165] Example 4: Preparation of immunogenic compositions

[00166] The collected cell culture suspensions with HAU greater than 256/dose were further treated with binary ethyleneimine (BEI) by adding 10 mM BEI solution at 37°C for 72 hours to inactivate baculovirus infectivity. A 10 mM sodium thiosulfate solution was then added at 4°C to neutralize the BEI residue.

[00167] After inactivation, the HAU of the cell culture suspensions was again assessed, and the inactivated cell culture suspensions with HAU greater than 256 per dose were used as the active antigenic ingredient for use in the next emulsification procedure with adjuvants.

[00168] The immunogenic compositions HSConl, H5Con3, H5Con5 and H5Con5Mut were formulated as water-in-oil emulsions. Briefly, water-in-oil emulsions are two-phase systems consisting of a continuous oil phase and a dispersed aqueous phase, with the aqueous phase dispersed as small droplets in the oil phase. The oil phase used to prepare the immunogenic composition was the commercially available adjuvant MONTANIDETM ISA 71R VG (manufactured by Seppic Inc, catalog no. 365187), while the aqueous phase contained each of the inactivated cell culture suspensions HSConl, H5Con3, H5Con5 and H5Con5Mut. For each dose (0.3 mL) of the immunogenic composition, approximately 3 parts of the aqueous phase (90 μL) was added to approximately 7 parts of the oil phase (210 μL) and dispersed at low shear (approximately 11,000 rpm for 1 minute) at room temperature and then at a high shear rate of 16,000 rpm. for 2 minutes in an ice-water bath. Miccra dispersant (dispersant, catalog number Miccra D-9, dispersant head, catalog number DS-14/P) was used to prepare water-in-oil emulsions. For each immunogenic composition H5Con1, H5Con3, H5Con5 and H5Con5Mut, the amount of HSCon1, H5Con3, H5Con5 and H5Con5Mut was at least 256 HAU/dose.

[00169] Example 5: Cross-reactivity tests and selection for H5Con1, H5Con3, H5Con5 and H5Con5Mut

[00170] SPF embryonated chicken eggs were purchased from SPAFAS Jinan. After hatching, SPF chicks were randomly selected and kept in special isolators. In the cross-reactivity tests of H5Con1, H5Con3, H5Con5 and H5Con5Mut, Re-4 to Re-8 and 03H5 (MutK+) were used to evaluate the reactivity tests of H5Con1, H5Con3, H5Con5 and H5Con5Mut.

[00171] Re-4 to Re-8 are inactivated whole AIV (H5 subtype) vaccines against clades 7.2, 2.3.4, 2.3.2.1, 7.2 and 2.3.4.4, respectively, and are commercial products from Harbin Weike Biotechnology. Development Co., Ltd. 03H5 (MutK+) is an HA protein expressed by baculovirus from strain A/duck/China/EZ 19-2/03, in addition to the following amino acid substitutions: S120N, D150N, S223N and an additional K328 (see WO2013024113Al). 03H5 (MutK+) was formulated into an immunogenic composition using the same methods as H5Con1, H5Con3, H5Con5 and H5Con5Mut.

[00172] Ten special pathogen free (SPF) chickens aged 10 days were vaccinated with one dose (0.3 ml) of the above compositions Re-4 to Re-8 and the immunogenic composition, respectively, by subcutaneous injection. Vaccinated chickens were constantly kept in isolation wards. Serum samples from individual chickens were collected before vaccination, two weeks after vaccination and three weeks after vaccination. In addition, serum samples prepared from a pool of 10 chickens from one vaccinated group were tested to determine the HI cross-reactivity of H5Con1, H5Con3, H5Con5 and H5Con5Mut using a hemagglutination inhibition (HI) assay (see OIE Terrestrial Manual 2015, Chapter 2.3. 4, Avian Influenza (avian influenza virus infection) The HI assay assessed the cross-reactivity of HSConl, H5Con3, H5Con5 and H5Con5Mut with 6 different clades (rBac03H5 represents clade 2.3.2, Re4 represents clade 7.2, Re5 represents clade 2.3.4, Re6 represents clade 2.3.2.1, Re7 represents clade 7.2, and Re8 represents clade 2.3.4.4.) antigens.

[00173] The HI cross-reactivity rates of Re-4 - Re-8, 03H5 (MutK+), HSCon1, H5Con3, H5Con5 and H5Con5Mut are shown in Table 4. In Table 4, “+” indicates the number of heterologous antigens derived from different clades, per which the test sera may cross-react. Sera scored “++++” showed cross-reactivity with 4 different clades, sera scored “+++” showed cross-reactivity with 3 different clades, sera scored “++” showed cross-reactivity with 2 different clades, and immune sera scored “+” showed cross-reactivity with only 1 clade. A score of “+” to “++++” indicates greater cross-reactivity. Antigens scoring “++++” were selected for the following experiments.

[00175] Based on the cross-reactivity results, it can be determined that H5Con5Mut has a wider cross-reactivity.

[00176] Example 6: Virus detection and protection effectiveness studies

[00177] The protection effectiveness of 03H5MutK+(clade 2.3.2), H5Con3 and H5Con5Mut was further tested in this example. Defense efficacy studies evaluated Re6+7+8, 03H5MutK+(clade 2.3.2), H5Con3, and H5Con5Mut. Re6+7+8 is a trivalent vaccine against clade 2.3.2.1, clade 7.2 and clade 2.3.4.4 (Re6+Re7+Re8 mixture) purchased from Harbin Weike Biotechnology Development Co., Ltd. Re6+7+8 was used as a positive control vaccine to ensure protection against each clade of control virus. Stock mixtures of three different clades of highly pathogenic avian influenza (HPAI) H5Nx viruses were prepared and used for challenge tests: 1) strain 383 (clade 2.3.2.1), 2) strain 14079 (clade 2.3.4.4), and 3) strain 13147 (clade 7.2). SPF chickens were vaccinated subcutaneously at 21 days of age with a single dose (0.3 ml) of Re6+7+8 and an immunogenic composition containing 03H5MutK+ (clade 2.3.2), H5Con3 and H5Con5Mut, each with comparable HAU of at least 256/ dose, respectively. Vaccinated chickens were kept in ABSL3 (animal biosafety level 3) facilities.

[00178] Three weeks (21 days) after vaccination, chickens were challenged via nasal drops with one of three H5Nx control viruses per test. The challenge dose for each chick was 6Logi ₁₀ EID50 (EID50=50% embryo challenge dose, meaning the amount of infectious virus that causes infection in 50% of the inoculated embryonated eggs). Chicks were observed for two weeks after infection, and chick mortality and morbidity were recorded daily. Cotton swab swabs of the trachea and cloaca of each chicken were collected at 3, 5, and 7 days postinfection. Virus recovery was studied using cotton swab samples by isolating the virus from chicken embryos.

[00179] The evaluation of the effectiveness of protection against each infecting virus was based on the following criteria: 1) whether 100% of vaccinated chicks would survive the two-week monitoring period (0% mortality).

[00180] Three animal vaccination/challenge trials were conducted and Table 5 shows the protective efficacy of Re6+7+8, 03H5MutK+(clade 2.3.2), H5Con3 and H5Con5Mut.

[00182] Chickens vaccinated with an immunogenic composition containing H5Con5Mut have been shown to exhibit 100% protection when challenged with HPAI viruses of clade 2.3.4.4, clade 2.3.2.1 and clade 7.2. That is, compared to conventional vaccines such as the Re6+Re7+Re8 mixture, the immunogenic composition containing H5Con5Mut per se provided superior and broader protection against three different AIV clades simultaneously.

[00183] Example 7: Studies on the effectiveness of protection against various HPAI subtypes H5

[00184] The effectiveness of protecting H5Con5Mut against various H5 HPAI subtypes was further tested in this example.

[00185] Groups 1a - 5a were defined as immunization groups that received H5Con5Mut, and groups lb - 5b were defined as control groups that received HPAI challenge only. Each group contained 12 10-day-old SPF chicks. On test day (D0), chickens in groups 1a - 5a were vaccinated subcutaneously with one dose (0.3 ml) of an immunogenic composition containing H5Con5Mut. At 21 days post immunization (dpi), chickens in all groups were challenged intranasally with different HPAI subtypes H5, and the challenge dose was 6Log ₁₀ EID _50/200 μl/chicken. During the 14 days after infection, morbidity and mortality of chickens in each group were calculated daily.

[00186] Clinical symptoms of HAPI include low mood, uneven plumage, significantly reduced appetite, cough, nasal and eye discharge, facial swelling, cyanosis, diarrhea, neurological symptoms. The occurrence of at least these clinical symptoms was used to calculate incidence. Mortality was calculated by counting dead chicks. % protection was calculated by dividing the number of chicks protected by the total number of chicks tested.

[00187] The H5 HPAI subtypes used for infection were as follows:

Control strains were obtained from South China Agricultural University, Wushan Road, Tianhe District, Guangzhou, Guangdong Province, China, and are representative isolates of H5N1 (Clades 2.3.3.1(d) and 2.3.4.4), H5N2 (Clade 7.2), and H5N6 (Clade 2.3.4.4(d)) described above.

[00188] The observed protection effectiveness of H5Con5Mut against various H5 AIV subtypes is summarized in Table 6.

[00190] Table 6 showed that in each control group, challenge at 21 days post-immunization was effective as 100% mortality was observed within 14 weeks post-challenge, and the H5Con5Mut group showed 100% protection when chickens were challenged with H5N1, H5N2 and H5N6. The data demonstrated that H5Con5Mut provided superior and broader protection against various H5 HPAI subtypes.

[00191] Example 8: H5Con5Mut Onset of Protection Studies

[00192] The onset of protection of H5Con5Mut from HPAI challenge was further examined in this example.

[00193] Groups 1a and 2a were defined as immunization groups that received H5Con5Mut, and groups lb and 2b were defined as control groups that were challenged with HPAI (strain 14079) only. Each group consisted of 12 10-day-old SPF chicks and the tests were repeated twice. On test day (DO), chickens in groups 1a and 2a were vaccinated subcutaneously with one dose (0.3 ml) of an immunogenic composition containing H5Con5Mut. 7 days after immunization (dpi), chickens in groups 1a and lb were intranasally infected with strain 14079. 14 days after immunization (dpi), chickens in groups 2a and 2b were intranasally infected with strain 14079. The challenge dose was 6Log ₁₀ EID ₅₀ /200 μl/ chicken During the 14 days after infection, morbidity and mortality of chickens in each group were calculated daily. The observed onset of H5Con5Mut protection is summarized in Table 7.

[00195] Table 7 shows that H5Con5Mut induced protection as early as day 7 postimmunization, with 100% protection by day 14 postimmunization. The data demonstrated that H5Con5Mut provides rapid and early protection against HPAI challenge.

[00196] Example 9: H5Con5Mut Duration of Protection Studies

[00197] The duration of protection of H5Con5Mut from HPAI challenge was further examined in this example.

[00198] Groups 1-3 were defined as immunization groups that received H5Con5Mut, and group 4 was defined as a control group that received HPAI (strain 14079) challenge only. Each group consisted of 12 10-day-old SPF chicks and the experiments were performed twice. On test day (D0), chickens in groups 1-3 were vaccinated subcutaneously with one dose (0.3 ml) of an immunogenic composition containing H5Con5Mut. At 28 days post immunization (dpi), 35 dpi, and 42 dpi, chickens in groups 1–3 were intranasally administered strain 14079, respectively. In the control group, chickens were intranasally injected with strain 14079 28 days after immunization. The challenge dose was 6Log ₁₀ EID ₅₀ /200 µl/chicken. During the 14 days after infection, morbidity and mortality of chickens in each group were calculated daily. The observed duration of H5Con5Mut protection is summarized in Table 8.

[00200] Table 8 shows that H5Con5Mut provides 100% protection for at least 42 days after immunization. The data showed that H5Con5Mut provided long-lasting and effective protection against HPAI.

[00201] Example 10: Protection Study on Chicks of Different Ages

[00202] The protection of H5Con5Mut in chickens of different ages against HPAI challenge was further examined in this example.

[00203] Groups 1-2 were defined as the immunization groups that received H5Con5Mut, and Group 3 was defined as the control group that was challenged with HPAI (strain 14079) only. Each group consisted of 12 SPF chicks, and the experiments were performed twice. In group 1, chickens at the age of 1 day were vaccinated subcutaneously with one dose (0.3 ml) of an immunogenic composition containing H5Con5Mut. In group 2, chickens at the age of 10 days were vaccinated subcutaneously with one dose (0.3 ml) of an immunogenic composition containing H5Con5Mut. 21 days after immunization, chickens in groups 1-3 were intranasally administered strain 14079, respectively. The challenge dose was 6Log ₁₀ EID ₅₀ /200 µl/chicken. During the 14 days after infection, morbidity and mortality of chickens in each group were calculated daily. The observed protection of H5Con5Mut in chickens of different ages is summarized in Table 9.

[00205] Table 9 shows that H5Con5Mut provides 100% protection against HPAI infection in day-old chicks. The data showed that H5Con5Mut provided superior chick protection even in newly hatched chicks.

[00206] Example 11: Study of Protection by Different Routes of Administration

[00207] The protection of chickens by H5Con5Mut under different routes of administration was further examined in this example.

[00208] Groups 1-2 were defined as the immunization groups that received H5Con5Mut, and group 3 was defined as the control group that received HPAI (strain 14079) challenge only. Each group consisted of 12 10-day-old SPF chicks and the experiments were performed twice. On test day (D0), chickens in groups 1-2 were vaccinated subcutaneously with one dose (0.3 ml) of an immunogenic composition containing H5Con5Mut, by subcutaneous (SC) or intramuscular (IM) administration. 21 days after vaccination, chickens in groups 1-3 were intranasally injected with strain 14079, respectively. The challenge dose was 6Log ₁₀ EID ₅₀ /200 µl/chicken. During the 14 days after infection, morbidity and mortality of chickens in each group were calculated daily. The observed protection of H5Con5Mut in chickens by different routes of administration is summarized in Table 10.

[00210] Table 10 shows that H5Con5Mut provides excellent protection to chicks both when administered subcutaneously S.C. and when administered intramuscularly, and the effectiveness of protection of H5Con5Mut is not limited to a particular route of administration. The flexible route of administration allowed H5Con5Mut to be suitable for chicks with different growth status, such as newly hatched chicks with little muscle mass.

[00211] All compositions and methods disclosed and claimed herein can be made and performed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described by way of illustrative embodiments, those skilled in the art will appreciate that variations may be applied to the compositions and methods, as well as in the steps or sequence of steps of the method described herein. without departing from the concept, spirit and scope of the present invention. More specifically, it will be appreciated that certain agents that are both chemically and physiologically related may be substituted for the agents described herein and the same or similar results may be achieved. All such like substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the following claims.

--->

LIST OF SEQUENCES

<110> BÖHRINGER INGELHEIM VETMEDIKA GMBH

<130> P19-0002-WO-2

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 551

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of the HA protein strain

duck/China/E319-2/03, but without the N-terminal signal peptide.

<400> 1

Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val

1 5 10 15

Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile

20 25 30

Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys

35 40 45

Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn

50 55 60

Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val

65 70 75 80

Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn

85 90 95

Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu

100 105 110

Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser

115 120 125

Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Ser Ser Ser Phe Phe

130 135 140

Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asp Ala Tyr Pro Thr Ile

145 150 155 160

Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp

165 170 175

Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Arg Leu Tyr Gln

180 185 190

Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg

195 200 205

Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly

210 215 220

Arg Met Asp Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn

225 230 235 240

Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile

245 250 255

Val Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Val Glu Tyr Gly

260 265 270

Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser

275 280 285

Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys

290 295 300

Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser

305 310 315 320

Pro Gln Arg Glu Arg Arg Arg Lys Arg Gly Leu Phe Gly Ala Ile Ala

325 330 335

Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly

340 345 350

Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu

355 360 365

Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile

370 375 380

Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn

385 390 395 400

Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly

405 410 415

Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu

420 425 430

Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr

435 440 445

Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn

450 455 460

Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser

465 470 475 480

Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg

485 490 495

Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr

500 505 510

Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu

515 520 525

Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser

530 535 540

Leu Gln Cys Arg Ile Cys Ile

545 550

<210> 2

<211> 552

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of H5Con1 (without signal

peptide)

<400> 2

Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val

1 5 10 15

Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile

20 25 30

Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys

35 40 45

Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn

50 55 60

Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val

65 70 75 80

Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn

85 90 95

Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu

100 105 110

Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser

115 120 125

Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser Ser Phe Phe

130 135 140

Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Ala Tyr Pro Thr Ile

145 150 155 160

Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp

165 170 175

Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln

180 185 190

Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg

195 200 205

Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly

210 215 220

Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn

225 230 235 240

Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile

245 250 255

Val Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Leu Glu Tyr Gly

260 265 270

Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser

275 280 285

Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys

290 295 300

Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser

305 310 315 320

Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile

325 330 335

Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr

340 345 350

Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys

355 360 365

Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser

370 375 380

Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe

385 390 395 400

Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp

405 410 415

Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met

420 425 430

Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu

435 440 445

Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly

450 455 460

Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu

465 470 475 480

Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala

485 490 495

Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly

500 505 510

Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala

515 520 525

Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly

530 535 540

Ser Leu Gln Cys Arg Ile Cys Ile

545 550

<210> 3

<211> 568

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of H5Con3

<400> 3

Met Glu Lys Ile Val Leu Leu Leu Ala Ile Val Ser Leu Val Lys Ser

1 5 10 15

Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val

20 25 30

Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile

35 40 45

Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys

50 55 60

Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn

65 70 75 80

Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val

85 90 95

Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn

100 105 110

Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu

115 120 125

Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser

130 135 140

Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Asn Pro Ser Phe Phe

145 150 155 160

Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro Thr Ile

165 170 175

Lys Lys Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp

180 185 190

Gly Ile His His Pro Asn Asp Glu Ala Glu Gln Thr Lys Leu Tyr Gln

195 200 205

Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg

210 215 220

Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly

225 230 235 240

Arg Met Asp Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn

245 250 255

Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile

260 265 270

Val Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Val Glu Tyr Gly

275 280 285

Asn Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser

290 295 300

Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys

305 310 315 320

Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser

325 330 335

Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile

340 345 350

Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr

355 360 365

Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys

370 375 380

Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser

385 390 395 400

Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe

405 410 415

Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp

420 425 430

Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met

435 440 445

Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu

450 455 460

Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly

465 470 475 480

Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu

485 490 495

Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala

500 505 510

Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly

515 520 525

Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala

530 535 540

Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly

545 550 555 560

Ser Leu Gln Cys Arg Ile Cys Ile

565

<210> 4

<211> 567

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of H5Con5

<400> 4

Met Glu Lys Ile Val Leu Leu Leu Ala Ile Val Ser Leu Val Lys Ser

1 5 10 15

Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val

20 25 30

Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile

35 40 45

Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys

50 55 60

Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn

65 70 75 80

Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val

85 90 95

Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn

100 105 110

Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu

115 120 125

Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser

130 135 140

Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Asn Pro Ser Phe Phe

145 150 155 160

Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro Thr Ile

165 170 175

Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp

180 185 190

Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Arg Leu Tyr Gln

195 200 205

Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg

210 215 220

Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly

225 230 235 240

Arg Met Asp Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn

245 250 255

Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile

260 265 270

Val Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Val Glu Tyr Gly

275 280 285

Asn Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser

290 295 300

Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys

305 310 315 320

Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser

325 330 335

Pro Gln Arg Glu Arg Arg Arg Lys Arg Gly Leu Phe Gly Ala Ile Ala

340 345 350

Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly

355 360 365

Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu

370 375 380

Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile

385 390 395 400

Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn

405 410 415

Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly

420 425 430

Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu

435 440 445

Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr

450 455 460

Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn

465 470 475 480

Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser

485 490 495

Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg

500 505 510

Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr

515 520 525

Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu

530 535 540

Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser

545 550 555 560

Leu Gln Cys Arg Ile Cys Ile

565

<210> 5

<211> 568

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence of Con5Mut

<400> 5

Met Glu Lys Ile Val Leu Leu Leu Ala Ile Val Ser Leu Val Lys Ser

1 5 10 15

Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val

20 25 30

Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile

35 40 45

Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys

50 55 60

Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn

65 70 75 80

Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val

85 90 95

Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn

100 105 110

Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu

115 120 125

Lys Ile Gln Ile Ile Pro Lys Asn Ser Trp Ser Asp His Glu Ala Ser

130 135 140

Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Thr Pro Ser Phe Phe

145 150 155 160

Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro Thr Ile

165 170 175

Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp

180 185 190

Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln

195 200 205

Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg

210 215 220

Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Asn Gly

225 230 235 240

Arg Met Asp Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn

245 250 255

Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile

260 265 270

Val Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Val Glu Tyr Gly

275 280 285

Asn Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser

290 295 300

Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys

305 310 315 320

Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser

325 330 335

Pro Gln Arg Glu Arg Arg Arg Arg Lys Arg Gly Leu Phe Gly Ala Ile

340 345 350

Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr

355 360 365

Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys

370 375 380

Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser

385 390 395 400

Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe

405 410 415

Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp

420 425 430

Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met

435 440 445

Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu

450 455 460

Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly

465 470 475 480

Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu

485 490 495

Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala

500 505 510

Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly

515 520 525

Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala

530 535 540

Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly

545 550 555 560

Ser Leu Gln Cys Arg Ile Cys Ile

565

<210> 6

<211> 241

<212>PRT

<213> Artificial sequence

<220>

<223> Amino acid sequence from position 60 to 300

H5Con5Mut

<400> 6

Leu Asp Gly Val Lys Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly

1 5 10 15

Trp Leu Leu Gly Asn Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu

20 25 30

Trp Ser Tyr Ile Val Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr

35 40 45

Pro Gly Asn Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg

50 55 60

Ile Asn His Phe Glu Lys Ile Gln Ile Ile Pro Lys Asn Ser Trp Ser

65 70 75 80

Asp His Glu Ala Ser Ser Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly

85 90 95

Thr Pro Ser Phe Phe Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn

100 105 110

Thr Tyr Pro Thr Ile Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp

115 120 125

Leu Leu Val Leu Trp Gly Ile His His Pro Asn Asp Ala Ala Glu Gln

130 135 140

Thr Lys Leu Tyr Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser

145 150 155 160

Thr Leu Asn Gln Arg Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val

165 170 175

Asn Gly Gln Asn Gly Arg Met Asp Phe Phe Trp Thr Ile Leu Lys Pro

180 185 190

Asn Asp Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu

195 200 205

Tyr Ala Tyr Lys Ile Val Lys Lys Gly Asp Ser Ala Ile Met Lys Ser

210 215 220

Glu Val Glu Tyr Gly Asn Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly

225 230 235 240

Ala

<210> 7

<211> 1707

<212> DNA

<213> Artificial sequence

<220>

<223> Optimized H5Con1 nucleic acid sequences

<400> 7

atggaaaaaa tcgtgctgct cttggctatt gtcagtttgg ttaagtcgga ccagatctgc

60

attggatacc acgccaacaa ctctaccgaa caagtcgaca ctatcatgga gaagaacgtt

120

acagtgacgc acgctcagga tatcctggag aagacccata acggaaaact gtgtgacctc

180

gatggtgtga agcctttgat cctgcgtgac tgctctgtgg ctggttggct gctgggaaac

240

cctatgtgtg atgagttcat caacgtcccc gaatggtctt acattgttga gaaggctaac

300

ccagccaacg acctctgcta cccgggtaac ttcaacgatt acgaggaatt gaagcacttg

360

ctgagtagga tcaaccattt cgaaaagatc cagatcatcc caaagtccag ctggtccgac

420

cacgaggctt cttcaggagt gagttcggcc tgtccgtacc agggcaagtc cagcttcttc

480

cgtaacgtgg tctggctgat taagaaaaac aacgcctacc caacaatcaa gaggtcatac

540

aacaacacga accaccaaga cctcttggtg ctgtggggta tccaccatcc aaacgatgct

600

gccgagcaga ctcgtctcta ccaaaacccg accacttaca tctccgtcgg caccagcact

660

ttgaaccaga gactggttcc gaagatcgca acacgctcca aagtgaacgg tcaaagcggc

720

aggatggact tcttctggac gatcctgaag cctaacgatg ctattaactt cgaatccaac

780

ggaaacttca tcgcacccga gtacgcgtac aagattgtca agaaaggtga ctctgcaatc

840

atgaaatcag agctggaata cggtaactgc aacacaaagt gtcaaacgcc catgggcgcg

900

atcaactctt caatgccttt ccacaacatc catcccctca caattggcga gtgccctaag

960

tacgttaaaa gtaacagact cgtgttggct accggattgc gtaactcgcc ccagagggaa

1020

cgccgtagga agaaacgcgg actgttcggt gctatcgccg gtttcattga gggtggctgg

1080

caaggcatgg tggacggctg gtacggatac caccatagta acgaacaggg atcgggttac

1140

gcagcggaca aggagtctac ccaaaaagct atcgatggtg tgactaacaa ggtcaactca

1200

atcattgaca aaatgaacac tcagttcgag gccgtcggca gagaattcaa caacctcgag

1260

agacgcatcg aaaacttgaa caagaaaatg gaagacggat tcctggatgt ctggacctac

1320

aacgctgagc tgctcgttct gatggagaac gaacgcactc tcgacttcca cgattccaac

1380

gtgaagaacc tctacgacaa agtcagactg caactccgcg ataacgctaa ggaattgggc

1440

aacggatgct tcgagttcta ccataagtgc gacaacgagt gtatggaatc cgtccgcaac

1500

ggcacatacg attacccaca gtacagcgag gaagctcgtc tcaagaggga ggaaatttct

1560

ggcgttaaat tggagtcaat cggaacatac caaatcctgt ccatttacag cacggttgca

1620

agttcgttgg cactggcgat catggtggcg ggcctcagct tgtggatgtg ctctaacgga

1680

tcactgcagt gccgcatctg tatttaa

1707

<210> 8

<211> 1707

<212> DNA

<213> Artificial sequence

<220>

<223> Optimized H5Con3 nucleic acid sequences

<400> 8

atggaaaaga tcgtgctgct cttggcaatt gtctccttgg ttaagagcga ccagatctgc

60

attggatacc acgcgaacaa cagcacagaa caagtcgaca cgatcatgga gaagaacgtt

120

acagtgacgc acgcacagga tatcctggag aaaacccata acggaaagct gtgtgacctc

180

gatggtgtga agcctttgat cctgcgcgac tgctctgtcg ctggttggct gctcggcaac

240

ccaatgtgtg atgagttcat caacgtcccg gaatggtcat acattgttga gaaggcaaac

300

cctgcgaacg acctctgcta ccccggtaac ttcaacgatt acgaggaatt gaagcacttg

360

ctgtctcgta tcaaccattt cgaaaagatc cagatcatcc caaagtccag ctggagtgac

420

cacgaggcat cgctgggagt gtcttcagcg tgtccttacc aaggtaaccc cagtttcttc

480

aggaacgtgg tctggctcat taagaaaaac aacacatacc cgacgatcaa gaaatcctac

540

aacaacacta accaccaaga cctcttggtg ctgtggggta tccaccatcc taacgatgag

600

gctgaacaga caaagctcta ccaaaacccc accacttaca tctctgtcgg cacctcaact

660

ttgaaccaga gactggttcc caaaatcgct acacgcagta aggtgaacgg tcaatcgggc

720

cgtatggact tcttctggac gatcctgaag ccaaacgatg ccattaactt cgaatccaac

780

ggaaacttca tcgctccgga gtacgcctac aaaattgtca agaaaggtga ctccgctatc

840

atgaagagcg aggttgaata cggtaactgc aacaccaaat gtcaaactcc aatcggcgcc

900

attaacagtt cgatgccatt ccacaacatc catccgctga caattggcga gtgccctaag

960

tacgttaaat ctaacaagct cgtgttggct accggattgc gtaactcacc ccagagggaa

1020

cgccgtagga agaaacgcgg actgttcggt gcaatcgcgg gtttcattga gggtggctgg

1080

caaggcatgg tggacggctg gtacggatac caccattcta acgaacaggg atcaggttac

1140

gctgccgaca aagagtccac ccaaaaggca atcgatggtg tgactaacaa agtcaacagc

1200

atcattgaca agatgaacac ccagttcgag gcggtcggca gagaattcaa caacctcgag

1260

agacgcatcg aaaacttgaa caagaaaatg gaagacggat tcctggatgt ctggacctac

1320

aacgctgagc tgctcgttct gatggagaac gaacgcactc tcgacttcca cgattcaaac

1380

gtgaaaaacc tctacgacaa ggtcagactg caactccgcg ataacgccaa ggaattgggc

1440

aacggatgct tcgagttcta ccataagtgc gacaacgagt gtatggaaag tgtgcgtaac

1500

ggcacttacg attaccccca gtactcggag gaagcccgtc tcaaaaggga ggaaatttcc

1560

ggcgtcaagt tggagagcat cggaacatac caaatcctgt ctatttactc aacggttgct

1620

tccagcttgg ctctggccat catggtggcc ggcctctcct tgtggatgtg cagtaacgga

1680

tcgctgcagt gcaggatctg tatttaa

1707

<210> 9

<211> 1707

<212> DNA

<213> Artificial sequence

<220>

<223> Optimized H5Con5 nucleic acid sequences

<400> 9

atggaaaaga tcgtgctgct cttggctatt gtctctctgg ttaaatcaga ccagatctgc

60

attggatacc acgccaacaa ctccacagaa caagtcgaca cgatcatgga gaagaacgtt

120

acagtgacgc acgctcagga tatcctcgag aaaacccata acggaaagct gtgtgacctc

180

gatggtgtga agcctttgat cctgagggac tgcagtgtcg ccggttggct gctcggcaac

240

ccaatgtgtg atgagttcat caacgtcccg gaatggtcct acattgttga gaaggctaac

300

cctgccaacg acctctgcta ccccggtaac ttcaacgatt acgaggaatt gaagcacttg

360

ctgtctagaa tcaaccattt cgaaaagatc cagatcatcc caaagtccag ctggtcagac

420

cacgaggctt cttcaggagt gagttcggcc tgtccttacc aaggtactcc cagcttcttc

480

aggaacgtgg tctggctgat taagaaaaac aacacatacc cgacgatcaa gagatcttac

540

aacaacacta accaccaaga cctcttggtg ctgtggggta tccaccatcc taacgatgct

600

gccgagcaga caaagctcta ccaaaacccc accacttaca tcagtgtcgg cacctcgact

660

ttgaaccagc gcctggttcc caaaatcgca acacgtagta aggtgaacgg tcaatcgggc

720

aggatggact tcttctggac gatcctcaag ccaaacgatg ctattaactt cgaaagcaac

780

ggaaacttca tcgcaccgga gtacgcgtac aaaattgtca agaaaggtga ctccgcaatc

840

atgaagagcg aggttgaata cggtaactgc aacaccaaat gtcaaactcc aatcggcgcg

900

attaactcca gcatgccatt ccacaacatc catccgctga caattggcga gtgccctaag

960

tacgttaaat ctaacaagct cgtgttggcc accggattga ggaactcacc cccagagaa

1020

cgccgtagga gaaagcgcgg actgttcggt gctatcgccg gtttcattga gggtggctgg

1080

caaggcatgg tggacggctg gtacggatac caccattcta acgaacaggg atcaggttac

1140

gcagcggaca aagagtccac ccaaaaggct atcgatggtg tgactaacaa agtcaacagc

1200

atcattgaca agatgaacac ccagttcgag gccgtcggcc gcgaattcaa caacctcgag

1260

cgccgtatcg aaaacttgaa caagaaaatg gaagacggat tcctggatgt ctggacctac

1320

aacgctgagc tgctcgttct gatggagaac gaacgtactc tcgacttcca cgattcaaac

1380

gtgaaaaacc tctacgacaa ggtccgcctg caactccgtg ataacgctaa ggaattgggc

1440

aacggatgct tcgagttcta ccataagtgc gacaacgagt gtatggaaag tgtgcgcaac

1500

ggcacttacg attaccccca gtactcggag gaagctaggt tgaaaagaga ggaaatttcc

1560

ggcgtcaagt tggagagcat cggaacatac caaatcctga gtatttactc gacggttgca

1620

tcttcattgg cactggcgat catggtggcg ggcctctcct tgtggatgtg ctccaacgga

1680

agcctgcagt gccgtatctg tatttaa

1707

<210> 10

<211> 1704

<212> DNA

<213> Artificial sequence

<220>

<223> Optimized nucleic acid sequence

H5ConMut

<400> 10

atggaaaaga tcgtgctgct cttggctatt gtctctctgg ttaaatcaga ccagatctgc

60

attggatacc acgccaacaa ctccacagaa caagtcgaca cgatcatgga gaagaacgtt

120

acagtgacgc acgctcagga tatcctcgag aaaacccata acggaaagct gtgtgacctc

180

gatggtgtga agcctttgat cctgagggac tgcagtgtcg ccggttggct gctcggcaac

240

ccaatgtgtg atgagttcat caacgtcccg gaatggtcct acattgttga gaaggctaac

300

cctgccaacg acctctgcta ccccggtaac ttcaacgatt acgaggaatt gaagcacttg

360

ctgtctagaa tcaaccattt cgaaaagatc cagatcatcc caaagaatag ctggtcagac

420

cacgaggctt cttcaggagt gagttcggcc tgtccttacc aaggtactcc cagcttcttc

480

aggaacgtgg tctggctgat taagaaaaac aacacatacc cgacgatcaa gagatcttac

540

aacaacacta accaccaaga cctcttggtg ctgtggggta tccaccatcc taacgatgct

600

gccgagcaga caaagctcta ccaaaacccc accacttaca tcagtgtcgg cacctcgact

660

ttgaaccagc gcctggttcc caaaatcgca acacgtagta aggtgaacgg tcaaaatggc

720

aggatggact tcttctggac gatcctcaag ccaaacgatg ctattaactt cgaaagcaac

780

ggaaacttca tcgcaccgga gtacgcgtac aaaattgtca agaaaggtga ctccgcaatc

840

atgaagagcg aggttgaata cggtaactgc aacaccaaat gtcaaactcc aatcggcgcg

900

attaactcca gcatgccatt ccacaacatc catccgctga caattggcga gtgccctaag

960

tacgttaaat ctaacaagct cgtgttggcc accggattga ggaactcacc cccagagaa

1020

cgccgtagga gaaagcgcgg actgttcggt gctatcgccg gtttcattga gggtggctgg

1080

caaggcatgg tggacggctg gtacggatac caccattcta acgaacaggg atcaggttac

1140

gcagcggaca aagagtccac ccaaaaggct atcgatggtg tgactaacaa agtcaacagc

1200

atcattgaca agatgaacac ccagttcgag gccgtcggcc gcgaattcaa caacctcgag

1260

cgccgtatcg aaaacttgaa caagaaaatg gaagacggat tcctggatgt ctggacctac

1320

aacgctgagc tgctcgttct gatggagaac gaacgtactc tcgacttcca cgattcaaac

1380

gtgaaaaacc tctacgacaa ggtccgcctg caactccgtg ataacgctaa ggaattgggc

1440

aacggatgct tcgagttcta ccataagtgc gacaacgagt gtatggaaag tgtgcgcaac

1500

ggcacttacg attaccccca gtactcggag gaagctaggt tgaaaagaga ggaaatttcc

1560

ggcgtcaagt tggagagcat cggaacatac caaatcctga gtatttactc gacggttgca

1620

tcttcattgg cactggcgat catggtggcg ggcctctcct tgtggatgtg ctccaacgga

1680

agcctgcagt gccgtatctg tatt

1704

<210> 11

<211> 723

<212> DNA

<213> Artificial sequence

<220>

<223> Optimized nucleic acid sequence,

coding

amino acid sequence from position 60 to 300 H5Con5Mut

<400> 11

ctcgatggtg tgaagccttt gatcctgagg gactgcagtg tcgccggttg gctgctcggc

60

aacccaatgt gtgatgagtt catcaacgtc ccggaatggt cctacattgt tgagaaggct

120

aaccctgcca acgacctctg ctaccccggt aacttcaacg attacgagga attgaagcac

180

ttgctgtcta gaatcaacca tttcgaaaag atccagatca tcccaaagaa tagctggtca

240

gaccacgagg cttcttcagg agtgagttcg gcctgtcctt accaaggtac tcccagcttc

300

ttcaggaacg tggtctggct gattaagaaa aacaacacat acccgacgat caagagatct

360

tacaacaaca ctaaccagga agacctcttg gtgctgtggg gtatccacca tcctaacgat

420

gctgccgagc agacaaagct ctaccaaaac cccaccactt acatcagtgt cggcacctcg

480

actttgaacc agcgcctggt tcccaaaatc gcaacacgta gtaaggtgaa cggtcaaaat

540

ggcaggatgg acttcttctg gacgatcctc aagccaaacg atgctattaa cttcgaaagc

600

aacggaaact tcatcgcacc ggagtacgcg tacaaaattg tcaagaaagg tgactccgca

660

atcatgaaga gcgaggttga atacggtaac tgcaacacca aatgtcaaac tccaatcggc

720

gcg

723

<---

Claims (20)

1. An immunogenic composition against the H5 avian influenza virus subtype, containing the hemagglutinin protein of the H5 avian influenza virus, in which the hemagglutinin protein contains the amino acid sequence specified in SEQ ID NO: 6; or the amino acid sequence set forth in SEQ ID NO: 5. 2. The immunogenic composition according to claim 1, in which the hemagglutinin protein is contained in a cell culture, which is obtained by cultivating cells containing an expression vector encoding the specified hemagglutinin protein. 3. The immunogenic composition according to claim 2, in which the nucleic acid molecule is presented in SEQ ID NO: 10 or SEQ ID NO: 11. 4. Immunogenic composition according to any one of paragraphs. 2, 3, in which the expression vector is a baculovirus and the cells are insect cells. 5. Immunogenic composition according to claims. 2-4, in which the cell culture is subjected to an inactivation step, preferably an inactivation step using binary ethyleneimine. 6. Immunogenic composition according to any one of paragraphs. 2-5, where the immunogenic composition contains part or all of the cell culture. 7. Immunogenic composition according to any one of paragraphs. 1-6, where the immunogenic composition further contains an adjuvant. 8. Immunogenic composition according to claim 7, in which the adjuvant is a water-in-oil emulsion. 9. Immunogenic composition according to any one of paragraphs. 1-8, where the immunogenic composition is administered in a single or multiple administration; and/or the immunogenic composition is administered subcutaneously or intramuscularly; and/or the immunogenic composition should be administered to the animal on or after day 1 of life, on or after day 7 of life, on or after day 10 of life, on or after day 15 of life, or on day 21 of life or older. 10. Immunogenic composition according to any one of paragraphs. 1-9 for use in the prevention and/or treatment of infections caused by an avian influenza virus, preferably an H5Nx virus, more preferably one or more of H5N1, H5N2 and H5N6. 11. An immunogenic composition according to claim 10, wherein the immunogenic composition is effective in the prevention and/or treatment of infections caused by an avian influenza virus, preferably an H5Nx virus, more preferably one or more of H5N1, H5N2 and H5N6, after administration of a single dose or multiple doses immunogenic composition. 12. A method for differentiating animals naturally infected with the avian influenza virus from animals vaccinated with an immunogenic composition according to any one of paragraphs. 1-9, including: (a) testing an animal sample in an immunoassay and/or genomic assay for the presence of an avian influenza marker that is not present in the immunogenic composition but is present in a naturally infected animal, where the immunoassay is an enzyme-linked immunosorbent assay or an enzyme-linked immunosorbent assay, (b) determining whether the sample is positive or negative for an avian influenza marker, and (c) correlation of the test results with the status of the test animal, where an animal that is positive for an avian influenza marker is a naturally infected animal, and an animal that is negative for an avian influenza marker is an animal vaccinated with an immunogenic composition according to any one of paragraphs. 1-9. 13. A method for differentiating animals naturally infected with the avian influenza virus from animals vaccinated with an immunogenic composition according to any one of paragraphs. 1-9, including: (a) testing an animal sample in an immunoassay and/or a genomic assay for the presence of an avian influenza marker that is specific to the immunogenic composition but is not present in a naturally infected animal, where the immunoassay is an enzyme-linked immunosorbent assay or an enzyme-linked immunosorbent assay, (b) determining whether the sample is positive or negative for an avian influenza marker, and (c) correlation of the test results with the status of the test animal, where the animal positive for the avian influenza marker is an animal vaccinated with the immunogenic composition according to any one of paragraphs. 1-9.