Immunogenic composition against avian influenza virus subtype H5

Abstract

To provide immunogenic compositions comprising hemagglutinin protein of avian influenza virus H5 subtype, and methods for discriminating animals vaccinated with the immunogenic composition from animals naturally infected with AIV.SOLUTION: Disclosed is an immunogenic composition comprising hemagglutinin protein of avian influenza virus H5 subtype, where the hemagglutinin protein comprises one or more amino acid residues selected from the group consisting of amino acid residues: (a) 120N or 120S, 155N, 223N or 223S; and (b) 61D, 87I, 99A, 102A, 110N, 136S, 140D, 149S, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 226V, 243D, 268Y, 279A and 298I with numbering with reference to amino acid residues as set forth in SEQ ID NO:1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of veterinary medicine, in particular to immunogenic compositions against avian influenza viruses of the H5 subtype. [Background technology]

[0002] Avian influenza viruses (AIVs) belong to the influenza A virus type, occur naturally among 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). Eighteen HA subtypes and eleven NA subtypes are known. Many different combinations of HA and NA proteins are possible. For example, an "H5N1 virus" means that the virus has HA subtype 5 and NA subtype 1. Nine subtypes of H5 viruses are known (H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, and H5N9, herein referred to as "H5Nx"). Most H5 viruses identified worldwide in wild birds and poultry are low-pathogenic avian influenza viruses, while some H5-containing viruses belong to highly pathogenic avian influenza (HPAI) viruses. H5Nx viruses evolve rapidly, resulting in considerable antigenic variation among different clades that reveal the evolutionary relationships among different H5 lineages. Infection of poultry with HPAI viruses can cause severe disease with high mortality. In countries with endemic H5Nx viruses, vaccination is used to control the disease. Most common AIV vaccines are inactivated whole-virus vaccines (inactivated vaccines), which are prepared by inactivating the whole virus and emulsifying it with an appropriate adjuvant. However, inactivated vaccines cannot provide broad protection against different clades. Because AIVs evolve rapidly, people must constantly develop new vaccines against different clades. Furthermore, the production of inactivated vaccines requires large quantities of live AIVs, posing a serious biosafety concern.

[0003] The HA protein is a receptor-binding and membrane-fusion glycoprotein of influenza A viruses. It is known that HA proteins can induce protective antibodies, making researchers interested in developing subunit vaccines based on recombinant HA proteins. International Publication No. 2007 / 019094 discloses an HA molecule containing amino acid substitutions in its receptor-binding site, making it more antigenic compared to HA molecules lacking such substitutions. International Publication No. 2008 / 052173 discloses a hemagglutinin protein of avian influenza virus H5 subtype containing several amino acid mutations (hereinafter referred to as "AIV H5 subtype HA protein" or "H5 HA protein"), demonstrating that a vaccine containing adjuvanted H5 HA protein can provide protection from clinical disease caused by influenza A viruses. However, the above disclosure does not relate to broader protection against influenza A viruses of different clades. WO 2013 / 148164 discloses a method for producing optimized H5N1 and H1N1 influenza HA polypeptides based on human H5N1 and swine H1N1 influenza isolates. Influenza virus-like particles (VLPs) containing optimized influenza HA polypeptides have also been prepared. Experimental results show that VLPs can induce antibody responses in mice that can recognize two different clades of influenza viruses, but the protective efficacy is only 40-60%. There is a need to develop a vaccine that can provide broader, more effective, long-lasting and early-onset protection in poultry against AIV H5 subtypes. Summary of the Invention

[0004] The present invention provides an immunogenic composition comprising a hemagglutinin protein of avian influenza virus subtype H5, wherein the hemagglutinin protein comprises one or more amino acid residues selected from the group consisting of: (a) amino acid residues 120N or 120S, 155N, and 223N or 223S; and (b) 61D, 87I, 99A, 102A, 110N, 136S, 140D, 149S, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 226V, 243D, 268Y, 279A, and 298I, numbered with reference to the amino acid residues set forth in SEQ ID NO:1. The present invention also provides a method for preparing an immunogenic composition, comprising the steps of: (i) culturing cells containing an expression vector expressing a hemagglutinin protein of avian influenza virus subtype H5; and (ii) harvesting the whole cell culture, wherein the hemagglutinin protein comprises one or more amino acid residues selected from the group consisting of: (a) amino acid residues 120N or 120S, 155N, and 223N or 223S; and (b) 61D, 87I, 99A, 102A, 110N, 136S, 140D, 149S, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 226V, 243D, 268Y, 279A, and 298I, numbered with reference to the amino acid residues set forth in SEQ ID NO:1.

[0005] Also provided is an immunogenic composition of the present invention for use in the prevention and / or treatment of an infectious disease 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. Methods for distinguishing between animals naturally infected with AIV and animals vaccinated with the immunogenic compositions of the invention are also provided. The immunogenic compositions of the invention can provide broader, more effective, long-lasting, and early-onset protection in poultry. In one embodiment, the immunogenic compositions of the invention provide broader protection in poultry. In one embodiment, the immunogenic compositions of the invention provide more effective protection in poultry. In one embodiment, the immunogenic compositions of the invention provide long-lasting protection in poultry. In one embodiment, the immunogenic compositions of the invention provide early-onset protection in poultry. Another aspect of the present invention may be as follows. [1] An immunogenic composition comprising a hemagglutinin protein of an avian influenza virus H5 subtype, wherein the hemagglutinin protein is: Numbering with reference to the amino acid residues set forth in SEQ ID NO: 1: (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 298I An immunogenic composition comprising: [2] The hemagglutinin protein is (a) amino acid residues 61D, 87I, 99A, 102A, 110N, 120N, 136S, 140D, 149S, 155N, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 223N, 226V, 243D, 268Y, 279A, and 298I; (b) the amino acid sequence set forth in SEQ ID NO: 6; or (c) the amino acid sequence shown in SEQ ID NO: 5 The immunogenic composition according to [1], comprising: [3] The immunogenic composition described in [1] or [2], wherein the hemagglutinin protein comprises the amino acid sequence shown in SEQ ID NO: 6. [4] The immunogenic composition described in [1] or [2], wherein the hemagglutinin protein comprises the amino acid sequence shown in SEQ ID NO: 5. [5] The immunogenic composition according to any one of [1] to [4], wherein the hemagglutinin protein is contained in a cell culture, and the cell culture is prepared by culturing cells containing an expression vector capable of expressing the hemagglutinin protein defined in any one of [1] to [4]. [6] The immunogenic composition according to any one of [1] to [5], wherein the expression vector comprises a nucleic acid molecule encoding a hemagglutinin protein defined in any one of [1] to [4]. [7] The immunogenic composition described in [6], wherein the nucleic acid molecule is represented by SEQ ID NO: 10. [8] The immunogenic composition described in [6], wherein the nucleic acid molecule is represented by SEQ ID NO: 11. [9] The immunogenic composition according to any one of [5] to [8] above, wherein the expression vector is a baculovirus and the cell is an insect cell.

[10] The immunogenic composition according to any one of [5] to [9] above, wherein the cell culture is subjected to an inactivation step, preferably an inactivation step using binary ethyleneimine.

[11] The immunogenic composition according to any one of [5] to

[10] above, which comprises a part or all of the cell culture.

[12] The immunogenic composition according to any one of [1] to

[11] above, further comprising an adjuvant.

[13] The immunogenic composition according to

[12] , wherein the adjuvant is a water-in-oil emulsion.

[14] administered in a single dose or multiple doses; and / or administered subcutaneously or intramuscularly; and / or Administered to animals at 1 day of age or later, 7 days of age or later, 10 days of age or later, 15 days of age or later, or 21 days of age or later, The immunogenic composition according to any one of [1] to

[13] above.

[15] The immunogenic composition according to any one of [1] to

[14] above, for use in the prevention and / or treatment of an infectious disease caused by one or more of avian influenza viruses, preferably H5Nx, more preferably H5N1, H5N2, and H5N6.

[16] The immunogenic composition according to

[15] , which is effective in preventing and / or treating infections caused by one or more of avian influenza viruses, preferably H5Nx, more preferably H5N1, H5N2 and H5N6, after a single or multiple administration of the immunogenic composition.

[17] The immunogenic composition according to

[16] , which is effective in preventing and / or treating infections caused by one or more of avian influenza viruses, preferably H5Nx, more preferably H5N1, H5N2 and H5N6, after a single administration of the immunogenic composition.

[18] A method for distinguishing between an animal naturally infected with an avian influenza virus and an animal vaccinated with the immunogenic composition according to any one of [1] to

[14] , comprising: (a) analyzing a sample from an animal for the presence of avian influenza markers that are not present in the immunogenic composition but are present in the naturally infected animal using an immunoassay or enzyme-linked immunosorbent assay immunoassay and / or genomic analysis test; (b) determining whether the sample is positive or negative for the avian influenza marker; (c) correlating the test results with the condition of the test animals, and animals that are positive for the avian influenza markers are naturally infected animals, and animals that are negative for the avian influenza markers are animals that have been vaccinated with the immunogenic composition of any one of [1] to

[14] . A method comprising:

[19] A method for distinguishing between an animal naturally infected with an avian influenza virus and an animal vaccinated with the immunogenic composition according to any one of [1] to

[14] , comprising: (a) analyzing a sample from an animal for the presence of avian influenza markers specific for the immunogenic composition but not present in the naturally infected animal with an immunoassay or enzyme-linked immunosorbent assay immunoassay and / or genomic analysis test; (b) determining whether the sample is positive or negative for the avian influenza marker; (c) correlating the test results with the condition of the test animals, and animals that are positive for the avian influenza marker are animals that have been vaccinated with the immunogenic composition described in any one of [1] to

[14] above. A method comprising: DETAILED DESCRIPTION OF THE INVENTION

[0006] In one aspect, the invention provides an immunogenic composition comprising an H5 HA protein, wherein the H5 HA protein comprises one or more amino acid residues selected from the group consisting of: (a) amino acid residues 120N or 120S, 155N, and 223N or 223S; and (b) 61D, 87I, 99A, 102A, 110N, 136S, 140D, 149S, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 226V, 243D, 268Y, 279A, and 298I, numbered with reference to the amino acid residues set forth in SEQ ID NO:1.

[0007] As used herein, the term "immunogenic composition," also referred to as "vaccine," refers to a composition comprising at least one antigen that elicits an immune response against the composition, the host mounting a therapeutic or protective immune response such that resistance to new infections is enhanced and / or the severity of clinical signs is reduced. The amino acid sequence of the H5 HA protein is designed through a series of HA amino acid sequence alignments, followed by generation of a consensus amino acid sequence and analysis of the most common residue at each position. As used herein, the terms "hemagglutinin protein of the present invention," "AIV H5 subtype HA protein," and "H5 HA protein" are interchangeable.

[0008] As used herein, SEQ ID NO: 1 represents the amino acid sequence of the HA protein of the A / duck / China / E319-2 / 03 strain, but lacking the amino-terminal signal peptide (the amino acid sequence of the HA protein of the A / duck / China / E319-2 / 03 strain was also disclosed in WO 2008 / 052173). SEQ ID NO: 1 is used as the standard sequence of the HA protein to determine the amino acid positions of the HA protein of the present invention.

[0009] As used herein, the numbering of amino acid positions in the HA protein of the invention refers to the amino acid positions set forth in SEQ ID NO:1. For example, the designation "120N or 120S" means N or S at the position corresponding to position 120 in SEQ ID NO:1. "155N" means N at the position corresponding to position 155 in SEQ ID NO:1. "223N or 223S" means N or S at the position corresponding to position 223 in SEQ ID NO:1. "61D" means D at the position corresponding to position 61 in SEQ ID NO:1. "87I" means I at the position corresponding to position 87 in SEQ ID NO:1. "99A" means A at the position corresponding to position 99 in SEQ ID NO:1. "102A" means A at the position corresponding to position 102 in SEQ ID NO:1. "110N" means N at the position corresponding to position 110 in SEQ ID NO:1. "136S" means S at the position corresponding to position 136 in SEQ ID NO:1. "140D" means D at the position corresponding to position 140 in SEQ ID NO:1. "149S" means S at the position corresponding to position 149 of SEQ ID NO:1. "156T" means T at the position corresponding to position 156 of SEQ ID NO:1. "157P" means P at the position corresponding to position 157 of SEQ ID NO:1. "170N" means N at the position corresponding to position 170 of SEQ ID NO:1. "172T" means T at the position corresponding to position 172 of SEQ ID NO:1. "178R" means R at the position corresponding to position 178 of SEQ ID NO:1. "190V" means V at the position corresponding to position 190 of SEQ ID NO:1. "191L" means L at the position corresponding to position 191 of SEQ ID NO:1. "200A" means A at the position corresponding to position 200 of SEQ ID NO:1. "226V" means V at the position corresponding to position 226 of SEQ ID NO:1. "243D" means D at the position corresponding to position 243 of SEQ ID NO:1. "268Y" means Y at the position corresponding to position 268 of SEQ ID NO: 1. "279A" means A at the position corresponding to position 279 of SEQ ID NO: 1. "298I" means I at the position corresponding to position 298 of SEQ ID NO: 1. Methods of determining amino acid positions are known in the art, including, but not limited to, amino acid alignments performed by using BLAST programs.

[0010] In one embodiment of the immunogenic composition of the invention, the H5 HA protein comprises any one of the 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 298I. In one embodiment, the H5 HA protein of the present invention comprises two 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 298I. In one embodiment, the H5 HA protein of the present invention comprises three, four, five, or all of the 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 298I. The entire HA protein from a naturally occurring AIV is approximately 568 amino acid residues long, and all amino acid residues upstream of amino acid 514 are located on the viral surface. The above amino acid sites of the present invention have been identified as being located within the region of amino acids 60-300 of the HA protein, which is believed to contain most of the 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 comprises amino acid residues 120N, 155N and 223N. In one embodiment, the H5 HA protein of the present invention comprises amino acid residues 120N, 155N and 223S. In one embodiment, the H5 HA protein of the present invention comprises amino acid residues 120S, 155N and 223N. In one embodiment, the H5 HA protein of the present invention comprises amino acid residues 120S, 155N and 223S.

[0011] In one embodiment of the immunogenic composition of the invention, the H5 HA protein comprises amino acid residues 61D, 87I, 99A, 102A, 110N, 120N, 136S, 140D, 149S, 155N, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 223N, 226V, 243D, 268Y, 279A and 298I. In one embodiment, the H5 HA protein of the invention comprises amino acid residues 61D, 87I, 99A, 102A, 110N, 120S, 136S, 140D, 149S, 155N, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 223S, 226V, 243D, 268Y, 279A and 298I. In one embodiment, the H5 HA protein of the invention comprises amino acid residues 61D, 87I, 99A, 102A, 110N, 120S, 136S, 140D, 149S, 155N, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 223N, 226V, 243D, 268Y, 279A and 298I. In one embodiment, the H5 HA protein of the invention comprises amino acid residues 61D, 87I, 99A, 102A, 110N, 120N, 136S, 140D, 149S, 155N, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 223S, 226V, 243D, 268Y, 279A and 298I.

[0012] In one embodiment of the immunogenic composition of the invention, the H5 HA protein comprises the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, the H5 HA protein of the invention comprises the amino acid sequence set forth in SEQ ID NO: 5. In one embodiment, the H5 HA protein of the invention consists of the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, the H5 HA protein of the invention consists of the amino acid sequence set forth in SEQ ID NO: 5. As used herein, the term "H5 HA protein of the present invention" can refer to an isolated form of H5 HA protein, or a non-isolated form of H5 HA protein, such as an 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 H5 HA protein.In one embodiment of the immunogenic composition of the present invention, the H5 HA protein is an isolated form of H5 HA protein. In one embodiment of the immunogenic composition of the invention, the H5 HA protein is prepared by expressing a nucleic acid molecule encoding the H5 HA protein.

[0013] In one embodiment, the nucleic acid molecule of the present invention can be further codon-optimized for expression in cells. As used herein, the term "codon-optimized nucleic acid molecule" refers to a nucleic acid molecule in which codons are selected to be optimal for expression in a specific system (such as a specific 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. In one embodiment, a nucleic acid molecule of the invention comprises the nucleic acid sequence set forth in SEQ ID NO: 10. In one embodiment, a nucleic acid molecule of the invention consists of the nucleic acid sequence set forth in SEQ ID NO: 10. In one embodiment, a nucleic acid molecule of the invention comprises the nucleic acid sequence set forth in SEQ ID NO: 11. In one embodiment, a nucleic acid molecule of the invention consists of the nucleic acid sequence set forth in SEQ ID NO: 11. The nucleic acid molecule of the present invention can be contained in an expression vector. In one embodiment, the expression vector of the present invention is a virus, a plasmid, a cosmid, or a phage. The vector can be composed of either DNA or RNA, preferably DNA.

[0014] Vectors and methods of making and / or using vectors (or recombinants) for expression are described in, among other places, U.S. Pat. Nos. 4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683, 5,494,807, 4,722,848, 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 2003; 1996; Moss, "Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety," PNAS USA 93: 11341-11348, October 1996; Smith et al., U.S. Pat. No. 4,745,051 (recombinant baculovirus); Richardson, CD (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 Infected Cells with a Baculovirus Vector," Molecular and Cellular Biology March 1984, Vol. 4, No. 3, p. 406; European Patent No. 0370573; U.S. Patent Application No. 920197, filed October 16, 1986; European Patent Publication No. 265785; U.S. 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; U.S. Patent Nos. 5,591,439 and 5,552,143; International Publication No. 98 / 00166; U.S. Patent Application Nos. 08 / 675,556 and 08 / 675,566, both filed and accepted on July 3, 1996 (recombinant adenovirus); Grunhaus et al., 1992, "Adenovirus as cloning vectors," Seminars in Virology (Vol. 3) p. 237-52, 1993;Ballay et al. EMBO Journal, vol. 4, p. 3861-65, Graham, Tibtech 8, 85-87, April, 1990;Prevec et al., J. Gen Virol. 70, 42434; PCT International Publication No. 91 / 11525; Felgner 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 U.S. Pat. Nos. 5,591,639, 5,589,466, and 5,580,859 and WO 90 / 11092, WO 93 / 19183, WO 94 / 21797, WO 95 / 11307, WO 95 / 20660; may be obtained by or similar to methods disclosed in Tang et al., Nature and Furth et al., Analytical Biochemistry relating to DNA expression vectors. WO 98 / 33510; Ju et al., Diabetologia, 41: 736-739, 1998 (lentiviral expression system); U.S. Pat. No. 4,945,050 to Sanford et al.; WO 90 / 01543 to Fischbachet et al. (Intracel); Robinson et al., Seminars in Immunology vol. 9, pp.271-283 (1997) (DNA vector systems); U.S. Patent No. 4,394,448 to Szoka et al. (methods for inserting DNA into living cells); U.S. Patent No. 5,677,178 to McCormick et al. (use of cytopathic viruses); and U.S. Patent No. 5,928,913 (vectors for gene delivery); and other references cited herein.

[0015] In one embodiment, the expression vector of the invention is a viral vector. In one embodiment, an expression vector of the invention is a herpesvirus, such as Pseudorabies virus, equine herpesvirus, or herpes simplex virus. In one embodiment, an expression vector of the invention is an adenovirus, such as porcine adenovirus. In one embodiment, an expression vector of the invention is a poxvirus, such as vaccinia virus, avian poxvirus, canarypoxvirus, or swinepoxvirus.

[0016] In one embodiment, the viral vector is a recombinant baculovirus. In one embodiment, the recombinant baculovirus of the invention is obtained from BaculoGold (BD Biosciences Pharmingen, San Diego, CA). In one embodiment, the recombinant baculovirus of the invention is obtained from Sapphire™ baculovirus (Allele Biotechnology). In one embodiment, the recombinant baculovirus of the invention comprises a nucleic acid molecule encoding an H5 HA protein of the invention. In one embodiment, the recombinant baculovirus of the invention comprises a nucleic acid molecule set forth in SEQ ID NO: 10. In one embodiment, the recombinant baculovirus of the invention comprises a nucleic acid molecule set forth in SEQ ID NO: 11. In one embodiment of the immunogenic composition of the present invention, the H5 HA protein is contained in a cell culture. The cell culture of the present invention can be a whole cell culture obtained from a cell culture method, including cells and culture medium containing the HA protein, or a portion of the cell culture, for example, a portion of the cell culture containing the HA protein obtained by filtration or any other separation step.

[0017] In one embodiment, the cell culture comprises an H5 HA protein comprising one or more amino acid residues selected from the group consisting of: (a) amino acid residues 120N or 120S, 155N, and 223N or 223S; and (b) 61D, 87I, 99A, 102A, 110N, 136S, 140D, 149S, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 226V, 243D, 268Y, 279A, and 298I, numbered with reference to the amino acid residues set forth in SEQ ID NO:1. In one embodiment, the cell culture comprises an H5 HA protein comprising 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 comprising the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, the cell culture comprises an H5 HA protein comprising the amino acid sequence set forth in SEQ ID NO: 5.

[0018] The immunogenic composition of the present invention further comprises an adjuvant. In one embodiment, the immunogenic composition is formulated in an adjuvant. As used herein, "adjuvant" can include aluminum hydroxide and aluminum phosphate, saponins such as Quil A, QS-21 (Cambridge Biotech Inc., Cambridge, Massachusetts), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Alabama), water-in-oil emulsions, oil-in-water emulsions, and water-in-oil-in-water emulsions. The emulsion may be based on light liquid paraffin oil (European Pharmacopoeia type); isoprenoids such as squalane or squalene; oils obtained by the oligomerization of alkenes, especially isobutene or decene; esters of acids or alcohols containing linear alkyl groups, more specifically vegetable oils, ethyl oleate, propylene glycol di(caprylate / caprate), glyceryl tri(caprylate / caprate), or propylene glycol dioleate; or esters of branched fatty acids or alcohols, especially isostearate. The oil is used in combination with an emulsifier to form the emulsion. The emulsifier is preferably a nonionic surfactant, especially sorbitan, mannides (e.g., anhydromannitol oleate), glycol, polyglycerol, propylene glycol, and optionally ethoxylated esters of oleic acid, isostearic acid, ricinoleic acid, or hydroxystearic acid, and polyoxypropylene-polyoxyethylene copolymer blocks, especially Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, DES), John Wiley and Sons, NY, pp51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).Exemplary adjuvants are the SPT emulsion described on page 147 of "Vaccine Design, The Subunit and Adjuvant Approach" edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of the same book.

[0019] Further examples of adjuvants are compounds selected from polymers of acrylic or methacrylic acid and copolymers of maleic anhydride and alkenyl derivatives. Advantageous adjuvant compounds are polymers of acrylic or methacrylic acid crosslinked with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Those skilled in the art can also refer to U.S. Pat. No. 2,909,462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least three, and preferably no more than eight, hydroxyl groups, in which at least three hydroxyl hydrogen atoms are replaced by unsaturated aliphatic groups having at least two carbon atoms. Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl, allyl, and other ethylenically unsaturated groups. The unsaturated groups themselves may contain other substituents, such as methyl. Products sold under the name Carbopol (BF Goodrich, Ohio, USA) are particularly suitable. These are crosslinked with allyl sucrose or allyl pentaerythritol. Among them, Carbopol 974P, 934P, and 971P are mentioned. The use of Carbopol 971P is most preferred. Among the copolymers of maleic anhydride and alkenyl derivatives, there is the copolymer EMA (Monsanto), which is a copolymer of maleic anhydride and ethylene. Dissolving these polymers in water gives an acid solution, which is preferably neutralized to physiological pH to obtain an adjuvant solution into which the immunogenic, immunological, or vaccine composition itself is incorporated.

[0020] Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), block copolymers (CytRx, Atlanta, GA), SAF-M (Chiron, Emeryville, CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from Escherichia coli (E. coli) (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide, or naturally occurring or recombinant cytokines or analogs thereof, or stimulators of endogenous cytokine release, and the like.

[0021] In one embodiment, the immunogenic composition is formulated into a water-in-oil emulsion with a suitable adjuvant. The adjuvant can include an oil and a surfactant. In one embodiment, the adjuvant is MONTANIDE™ ISA 71R VG (manufactured by Seppic Inc., catalog number: 365187). The adjuvant can be added in an amount of about 100 μg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 100 μg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 500 μg to about 5 mg per dose. Even more preferably, the adjuvant is added in an amount of 750 μg to about 2.5 mg per dose. Most preferably, the adjuvant is added in an amount of about 1 mg per dose. In one embodiment, the immunogenic composition of the present invention comprises, per dose, an oil phase containing about 7 parts adjuvant and an aqueous phase containing about 3 parts H5 HA protein of the present invention.

[0022] It has surprisingly been found that the immunogenic compositions of the present invention can provide broader protection in poultry. As used herein, the term "broader protection in poultry" encompasses broader protection against different AIV H5 subtypes. Such subtypes include, or according to further embodiments, consist of, H5N1, H5N2, and / or H5N6. According to further embodiments, the term "broader protection in poultry" also encompasses protection against different clades of AIV H5 subtypes. Such AIV H5 clades include, or according to further embodiments, consist of, 2.3.4.4, 2.3.2.1, 2.3.2.1d, 2.3.4.4d, and / or 7.2.

[0023] Furthermore, the protection provided by the immunogenic compositions of the present invention has an early onset and a long-lasting duration. As used herein, the term "early onset" or "early protection" means that partial protection against infectious AIV H5 virus is obtained 7 days after vaccination. Complete protection against infectious AIV H5 virus is obtained 14 days after vaccination. The term "long-lasting duration" or "long-lasting protection in poultry" encompasses a protective duration of at least 42 days after vaccination. The immunogenic compositions of the present invention can provide protection to chickens of different ages, even newborn chickens, i.e., chickens on day 1 of age. The immunogenic compositions of the present invention should not be limited to a particular route of administration, and the protective efficacy of the immunogenic compositions of the present invention is not affected by different routes of administration.

[0024] In another aspect, the present invention also provides a method of preparing an immunogenic composition, the method comprising: (i) culturing cells containing an expression vector capable of expressing an H5 HA protein; and (ii) harvesting the H5 HA protein or the whole cell culture containing the H5 HA protein, wherein the H5 HA protein comprises one or more amino acid residues selected from the group consisting of: (a) amino acid residues 120N or 120S, 155N, and 223N or 223S; and (b) 61D, 87I, 99A, 102A, 110N, 136S, 140D, 149S, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 226V, 243D, 268Y, 279A, and 298I, numbered with reference to the amino acid residues set forth in SEQ ID NO:1.

[0025] In one embodiment of the methods of the invention, the H5 HA protein comprises 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 comprises the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, the H5 HA protein comprises the amino acid sequence set forth in SEQ ID NO: 5. In one embodiment of the method of the present invention, the expression vector is a recombinant baculovirus comprising a nucleic acid molecule of the present invention. In one embodiment, the nucleic acid molecule of the present invention is set forth in SEQ ID NO: 10. In one embodiment, the nucleic acid molecule of the present invention is set forth in SEQ ID NO: 11. In one embodiment, the recombinant baculovirus is obtained from a commercially available product sold under the trademark Sapphire™ baculovirus (Allele Biotechnology). In one embodiment, the cell is an insect cell. In one embodiment, the insect cell is an SF+ cell. In one embodiment, the SF+ cell is a commercially available product sold by Protein Sciences Corporation (Meriden, Connecticut).

[0026] In one embodiment of the method of the present invention, the method comprises preparing a recombinant baculovirus comprising a nucleic acid molecule of the present invention. In one embodiment, the nucleic acid molecule of the present invention is set forth in SEQ ID NO: 10. In one embodiment, the nucleic acid molecule of the present invention is set forth in SEQ ID NO: 11. In one embodiment, the recombinant baculovirus is obtained from a commercially available product sold under the trademark Sapphire™ baculovirus (Allele Biotechnology). In one embodiment of the method of the present invention, the method comprises infecting a cell with a recombinant baculovirus of the present invention. In one embodiment, the cell is an insect cell. In one embodiment, the insect cell is an SF+ cell. In one embodiment, the SF+ cell is a commercially available product sold by Protein Sciences Corporation (Meriden, Connecticut).

[0027] In one embodiment of the method of the present invention, the method comprises the steps of preparing a recombinant baculovirus comprising a nucleic acid molecule of the present invention and infecting insect cells with the recombinant baculovirus. In one embodiment, the recombinant baculovirus is obtained from a commercially available product sold under the trademark Sapphire™ Baculovirus (Allele Biotechnology). In one embodiment, the nucleic acid molecule of the present invention is set forth in SEQ ID NO: 10. In one embodiment, the nucleic acid molecule of the present invention is set forth in SEQ ID NO: 11. In one embodiment, the insect cells are SF+ cells. In one embodiment, the SF+ cells are a commercially available product sold by Protein Sciences Corporation (Meriden, Connecticut).

[0028] In one embodiment of the method of the present invention, the method comprises the steps of (i) preparing a recombinant baculovirus comprising a nucleic acid molecule of the present invention; (ii) infecting insect cells with the recombinant baculovirus; (iii) culturing the insect cells in a culture medium; and (iv) harvesting the H5 HA protein of the present invention or the whole cell culture comprising the H5 HA protein of the present invention. In one embodiment, the recombinant baculovirus is obtained from a commercially available product sold under the trademark Sapphire™ Baculovirus (Allele Biotechnology). In one embodiment, the nucleic acid molecule of the present invention is set forth in SEQ ID NO: 10. In one embodiment, the nucleic acid molecule of the present invention is set forth in SEQ ID NO: 11. In one embodiment, the insect cells are SF+ cells. In one embodiment, the SF+ cells are a commercially available product sold by Protein Sciences Corporation (Meriden, Connecticut).

[0029] In one embodiment of the method of the present invention, the culture medium for culturing the cells of the present invention is determined by those skilled in the art. In one embodiment, the culture medium is a serum-free insect cell culture medium. In one embodiment, the culture medium is Ex-CELL 420 (Ex-CELL® 420 serum-free medium for insect cells, Sigma-Aldrich, catalog 14420C). 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 10 days, preferably from about 2 to about 10 days, more preferably from about 4 to about 9 days, and even more preferably from about 5 to about 8 days. In one embodiment, the conditions suitable for culturing the insect cells include a temperature between about 22 and 32°C, preferably about 24 to 30°C, more preferably about 25 to 29°C, even more preferably about 26 to 28°C, and most preferably about 27°C. In one embodiment of the method of the present invention, the method further comprises the step of inactivating the cell culture of the present invention. Any conventional inactivation method can be used for the purposes of the present invention, including but not limited to chemical and / or physical treatment.

[0030] In one embodiment, the inactivation step comprises the addition of 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 comprises the addition of a solution of 2-bromoethyleneamine hydrobromide, which is cyclized in NaOH to form BEI. In one embodiment, the inactivation step is carried out at a temperature between 25 and 40°C, preferably between 28 and 39°C, more preferably between 30 and 39°C, and more preferably between 35 and 39°C. In one embodiment, the inactivation step is carried out for 24 to 72 hours, preferably between 30 and 72 hours, and more preferably between 48 and 72 hours. Generally, the inactivation step is carried out until replication of the viral vector is no longer detectable.

[0031] In one embodiment of the method of the present invention, the method further comprises a neutralization step following the inactivation step. The neutralization step involves adding an equivalent amount of an agent that neutralizes the inactivation agent in the solution. In one embodiment, the inactivation agent is BEI. In one embodiment, the neutralizing agent is sodium thiosulfate. In one embodiment, when the inactivation agent is BEI, an equivalent amount of sodium thiosulfate is added. For example, if BEI is added to a final concentration of 5 mM, a 1.0 M sodium thiosulfate solution is added to give a final minimum concentration of 5 mM to neutralize any residual BEI. In one embodiment, when the inactivation agent is BEI, the neutralization step involves adding sodium thiosulfate solution to a final concentration of 1 to 20 mM, preferably 2 to 10 mM, and more preferably 5 mM or 10 mM. In one embodiment, the neutralizing agent is added after the inactivation step is complete, meaning that viral vector replication is no longer detectable. In one embodiment, the neutralizing agent is added after the inactivation step has been performed for 24 hours. In one embodiment, the neutralizing agent is added after the inactivation step has been performed for 30 hours, in one embodiment, the neutralizing agent is added after the inactivation step has been performed for 48 hours, and in one embodiment, the neutralizing agent is added after the inactivation step has been performed for 72 hours.

[0032] The amount of H5 HA protein contained in the immunogenic composition of the present invention can be quantified by any conventional method known in the art, for example, by using a hemagglutination assay (see OIE Terrestrial Manual 2015, Chapter 2.3.1 & 2.3.2, Avian Influenza (infection with avian influenza viruses)). In one embodiment, the amount of H5 HA protein contained in the immunogenic composition of the present invention is determined by a hemagglutination test using chicken red blood cells, and the amount of H5 HA protein can be expressed as hemagglutination assay units (HAU).

[0033] The term "HAU" refers to a unit of activity that agglutinates red blood cells (RBCs), and 1 HAU of hemagglutinin protein is the smallest unit that causes agglutination when mixed with RBCs. HAU can be evaluated by a hemagglutination titer assay. In this assay, a sample containing hemagglutinin protein is serially diluted two-fold in a 96-well plate, and a PBS solution is added to the wells. The HAU value is the maximum dilution factor of the sample that results in complete agglutination of RBCs. For example, if the maximum dilution factor of a 25 μl sample is 256, the amount of hemagglutinin protein contained in the sample is 256 HAU per 25 μl. To induce 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 comprises at least 32 HAU of the H5 HA protein of the present invention per dose. In a further embodiment, the immunogenic composition of the present invention comprises at least 64 HAU of the H5 HA protein of the present invention per dose. In a further embodiment, the immunogenic composition of the present invention comprises at least 128 HAU of the H5 HA protein of the present invention per dose. In a further embodiment, the immunogenic composition of the present invention comprises at least 256 HAU of the H5 HA protein of the present invention per dose.

[0034] Those skilled in the art can determine the upper limit of the amount of H5 HA protein by simple routine testing. Generally, the upper limit used by those skilled in the art will be in the range of about 1024 HAU per dose, but may be higher. In a further embodiment, the immunogenic composition of the invention comprises at least 32-1024 HAU of the H5 HA protein of the invention per dose. In a further embodiment, the immunogenic composition of the invention comprises at least 64-1024 HAU of the H5 HA protein of the invention per dose. In a further embodiment, the immunogenic composition of the invention comprises at least 128-1024 HAU of the H5 HA protein of the invention per dose. In a further embodiment, the immunogenic composition of the invention comprises at least 256-1024 HAU of the H5 HA protein of the invention per dose. Compared to conventional whole virus inactivated vaccines, which are required to be produced at Biosafety Level 2 or 3 (BSL-2, BSL-3), the above manufacturing method for producing H5 HA protein is classified as Biosafety Level 1 requirement. In another aspect, the present invention also provides a method for preventing and / or treating an infectious disease caused by an AIV, the method comprising the step of administering an effective amount of the immunogenic composition of the present invention to a subject in need thereof. The present invention also provides the immunogenic composition of the present invention for use in preventing and / or treating an infectious disease caused by an AIV.

[0035] As used herein, the term "prevention" refers to a reduction in the occurrence or severity of clinical signs of influenza infection, including up to complete prevention of such clinical signs. The preventative protective efficacy of an immunogenic composition can be assessed based on the survival rate of subjects vaccinated against different AIV clades. In one embodiment, the protective efficacy of the immunogenic composition is increased by at least 10%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, more preferably at least 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 subjects who did not receive the vaccine of the present invention but were exposed to infectious levels of AIV. In one embodiment, the protective efficacy of the immunogenic composition is 100% in subjects vaccinated against different AIV clades.

[0036] In one embodiment, the subject in need thereof may be a poultry, even more preferably a bird, chicken, duck, turkey, etc. In one embodiment, the subject is a chicken or duck. In one embodiment, the AIV is of the H5 subtype. In one embodiment, the AIV is H5Nx. In one embodiment, the AIV is HPAI H5Nx, which is spreading primarily in Asia, the Middle East, and Africa. In one embodiment, the H5Nx is H5N1, H5N2, and / or H5N6. In one embodiment, the H5Nx is H5N1. In one embodiment, the H5Nx is H5N2. In one embodiment, the H5Nx is H5N6. In one embodiment, the H5Nx is H5N1 and H5N2. In one embodiment, the H5Nx is H5N2 and H5N6. In one embodiment, the AIV is clade 2, preferably clade 2.3, more preferably clades 2.3.4 and 2.3.2. In one embodiment, the AIV is clade 2.3.4.4, 2.3.2.1, 2.3.2.1d, or 2.3.4.4d. In one embodiment, the AIV is clade 7.2. H5Nx of subtypes H5N1, H5N2 and / or H5N6 are known in the art.

[0037] As used herein, the term "clade" refers to a biological group (e.g., species) that includes all descendants of a common ancestor. A / duck / Guangdong / 1 / 96 is considered the ancestor of all H5-lineage viruses and was designated clade 0. This nomenclature is currently used to distinguish variants of the H5 HA gene, and approximately 20 distinct clades of viruses have been officially identified to date. Beyond clade 0, nine additional clades (1–9) have been described and defined based on all deposited sequences. However, most viral clades have become extinct over time. H5-lineage viruses continue to circulate and evolve in poultry and wild birds in parts of Asia, the Middle East, and Africa. The predominant clade responsible for endemic infections in several different countries is clade 2, and further sublineages resulting from antigenic drift have been described as secondary, tertiary, quaternary, and even quintuin clades. The nomenclature and emerging clades can be found in WHO / OIE / FAO. Continuing progress towards a unified nomenclature for the highly pathogenic H5N1 avian influenza viruses: 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. Influenza Other Respiration Viruses 2012;6:1-5; 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.

[0038] For example, H5Nx of clade 2.3.4.4 is described in Lee et al., Emerging Infectious Diseases, 2016; Volume 22(7), pp.1283-1284 and Lee et al., Journal Veterinary Science 2017; Vol.(S1), pp.269-280. The avian influenza isolate A / common magpie / Hong Kong / 5052 / 2007 (H5N1) described in Smith et al., Emerging Infectious Diseases, 2009; Volume 15(3), pp.402-407 belongs to clade 2.3.2.1. Additional H5Nx isolates of clade 2.3.2.1, including A / chicken / India / CL03485 / 2011 (H5N1) or A / chicken / India / CA0302 / 2011 (H5N1), are described, for example, in Bhat et al., 2015, Microbial Pathogenesis; volume 88, pp. 87-93. Additional H5Nx isolates of subclades 2.3.2.1 and 2.3.4.4, including 2.3.4.4d, such as A / duck / Vietnam / HU1-1507 / 2014 (H5N6) or A / duck / Vietnam / HU1-1151 / 2014 (H5N6), are described, for example, in Nguyen et al., 2019, Scientific Reports 9; Article 7723, pp. 1-13. For example, H5Nx isolates of clade 7.2, including (A / chicken / Gansu / 62012(H5N1)), have been described and characterized, for example, in Liu et al., Journal of Virology 2016, Volume 90(21), pp. 9797-9804.

[0039] In one embodiment, the immunogenic compositions of the invention may be administered via subcutaneous (SC) or intramuscular (IM) administration. The administration time of the immunogenic composition of the present invention can be determined by those skilled in the art according to practical requirements. For example, for white broilers, whose lifespan is approximately 40 to 50 days, the immunogenic composition of the present invention can be administered once; for yellow broilers, whose lifespan is approximately 70 days, the immunogenic composition of the present invention can be administered twice; for chickens, such as breeder chickens, whose lifespan is greater than one year or longer, the immunogenic composition of the present invention can be administered multiple times. In one embodiment, the immunogenic composition of the present invention is administered in a single dose. In one embodiment, the immunogenic composition of the present invention is administered in multiple doses, such as two, three, four, five, six, seven, eight, nine, or ten doses. In one embodiment, the interval between two doses is approximately 4 to 6 weeks, such as 4, 5, or 6 weeks.

[0040] Those 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 can be first administered at 1 day of age or later, 7 days of age or later, 10 days of age or later, 15 days of age or later, or 21 days of age or later. In one embodiment, the immunogenic composition of the present invention is administered at 1 day of age or later, 7 days of age or later, 10 days of age or later, 15 days of age or later, or 21 days of age or later. In one embodiment, the immunogenic composition of the present invention is administered in a single dose from 1 day of age, 7 days of age, 10 days of age, 15 days of age, or 21 days of age. In one embodiment, the immunogenic composition of the present invention is administered in a single dose from 1 day of age.

[0041] In another aspect, the present invention also provides a method for distinguishing between animals naturally infected with AIV and animals vaccinated with a vaccine or immunogenic composition of the present invention. The present invention also provides the use of an immunogenic composition of the present invention in the preparation of a medicament for distinguishing between animals naturally infected with AIV and animals vaccinated with AIV.

[0042] In one embodiment, the method comprises the steps of: a) analyzing a sample from an animal with an immunological and / or genomic test, which is an enzyme immunoassay or enzyme-linked immunosorbent assay or agar gel precipitation assay or Western blot assay, for the presence of avian influenza markers that are not present in the immunogenic composition but are present in naturally infected animals; b) determining whether the sample is positive or negative for the avian influenza markers; and c) correlating the test results with the status of the tested animal, wherein animals that are positive for the avian influenza markers are naturally infected animals and animals that are negative for the avian influenza markers are animals that have been vaccinated with the immunogenic composition or vaccine of the present invention. In one embodiment, the method comprises the steps of: a) analyzing a sample from an animal with an immunological test, which may be an enzyme immunoassay or an enzyme-linked immunosorbent assay, and / or a genomic analysis test, for the presence of avian influenza markers that are specific for the immunogenic composition but not present in naturally infected animals; b) determining whether the sample is positive or negative for the avian influenza markers; and c) correlating the test results with the status of the tested animal, wherein animals that are positive for the avian influenza markers are animals that have been vaccinated with the immunogenic composition or vaccine of the present invention. In one embodiment, the animal may be a poultry, even more preferably a bird, chicken, duck, turkey, etc. In one embodiment, the animal is a chicken or duck.

[0043] In one embodiment, the AIV is of the H5 subtype. In one embodiment, the AIV is H5Nx. In one embodiment, the AIV is HPAI H5Nx, which is spreading primarily in Asia, the Middle East, and Africa. In one embodiment, the H5Nx is H5N1, H5N2, and / or H5N6. In one embodiment, the H5Nx is H5N1. In one embodiment, the H5Nx is H5N2. In one embodiment, the H5Nx is H5N6. In one embodiment, the H5Nx is H5N1 and H5N2. In one embodiment, the H5Nx is H5N2 and H5N6. In one embodiment, the AIV is clade 2, preferably clade 2.3, more preferably clades 2.3.4 and 2.3.2. In one embodiment, the AIV is clade 2.3.4.4 or 2.3.2.1, 2.3.2.1d or 2.3.4.4d. In one embodiment, the AIV is clade 7.2.

[0044] Animals vaccinated with a vaccine or immunogenic composition comprising the H5 HA protein of the present invention will only have an antibody response to this specific antigen and will have no responses to other viral components. A further advantage of the present invention is that it lends itself to the DIVA concept by using a specific ELISA to distinguish AIV-infected from vaccinated animals. In another aspect, the present invention also provides a kit comprising the H5 HA protein, nucleic acid molecule, vector, cell, or vaccine or immunogenic composition of the present invention. In one embodiment, the kit can be used in poultry, even more preferably birds, such as chickens, ducks, and turkeys. In one embodiment, when chickens are vaccinated, the H5 HA protein, vaccine, or immunogenic composition of the present invention can be used for vaccination at 1 day of age or later, 7 days of age or later, 10 days of age or later, 15 days of age or later, or 21 days of age or later. In one embodiment, the immunogenic composition of the present invention is administered in a single dose from 1 day of age, 7 days of age, 10 days of age, 15 days of age, or 21 days of age. In one embodiment, the immunogenic composition of the present invention is administered in a single dose from 1 day of age.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs at the time of filing. The meaning and scope of the terms should be clear, but in case of potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular. In this specification, the use of "or" means "and / or" unless otherwise stated. Furthermore, the use of the term "including" and other forms such as "comprises" and "comprised" is not limiting. All patents and publications mentioned herein are incorporated herein by reference.

[0046] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of virology, molecular biology, microbiology, recombinant DNA technology, protein chemistry and immunology which are within the skill of those in the art and are explained fully in the literature. For example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Vols. I, II and III, Second Edition (1989); DNA Cloning, Vols. I and II (DN Glover ed. 1985); Oligonucleotide Synthesis (MJ Gait ed. 1984); Nucleic Acid Hybridization (BD Hames & SJ Higgins eds. 1984);Animal Cell Culture (RK Freshney ed. 1986);Immobilized Cells and Enzymes (IRL press, 1986);Perbal, B., A Practical Guide to Molecular Cloning (1984);the series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);Protein purification methods - a practical approach (ELV Harris and S. Angal, eds., IRL Press at Oxford University Press); and Handbook of Experimental See Immunology, Vols. I-IV (DM Weir and CC Blackwell eds., 1986, Blackwell Scientific Publications).

[0047] It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. As used herein and in the appended claims, it should be noted that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antigen" includes a mixture of two or more antigens, reference to "a carrier" includes a mixture of two or more carriers, and so forth.

[0048] Terms The following clauses are also set forth herein and are part of the disclosure of the present invention: 1. An immunogenic composition comprising a hemagglutinin protein of avian influenza virus subtype H5, wherein the hemagglutinin protein is: Numbering with reference to the amino acid residues set forth in SEQ ID NO: 1: (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 298I An immunogenic composition comprising:

[0049] 2. Hemagglutinin protein (a) amino acid residues 61D, 87I, 99A, 102A, 110N, 120N, 136S, 140D, 149S, 155N, 156T, 157P, 170N, 172T, 178R, 190V, 191L, 200A, 223N, 226V, 243D, 268Y, 279A, and 298I; (b) the amino acid sequence set forth in SEQ ID NO: 6; or (c) the amino acid sequence shown in SEQ ID NO: 5 2. The immunogenic composition of clause 1, comprising:

[0050] 3. The immunogenic composition of clause 1 or 2, wherein the hemagglutinin protein comprises the amino acid sequence set forth in SEQ ID NO:6. 4. The immunogenic composition of clause 1 or 2, wherein the hemagglutinin protein comprises the amino acid sequence set forth in SEQ ID NO:5. 5. An immunogenic composition according to any one of clauses 1 to 4, wherein the hemagglutinin protein is contained in a cell culture, and the cell culture is prepared by culturing cells containing an expression vector capable of expressing the hemagglutinin protein defined in any one of clauses 1 to 4. 6. The immunogenic composition of any one of clauses 1 to 5, wherein the expression vector comprises a nucleic acid molecule encoding a hemagglutinin protein as defined in any one of clauses 1 to 4. 7. The immunogenic composition of clause 6, wherein the nucleic acid molecule is set forth in SEQ ID NO: 10. 8. The immunogenic composition of clause 6, wherein the nucleic acid molecule is set forth in SEQ ID NO: 11. 9. The immunogenic composition of any one of clauses 5 to 8, wherein the expression vector is a baculovirus and the cell is an insect cell.

[0051] 10. The immunogenic composition according to clauses 5 to 9, wherein the cell culture is subjected to an inactivation step, preferably an inactivation step with binary ethyleneimine. 11. The immunogenic composition of any one of clauses 5 to 10, comprising part or all of a cell culture. 12. The immunogenic composition of any one of clauses 1 to 11, further comprising an adjuvant. 13. The immunogenic composition of clause 12, wherein the adjuvant is a water-in-oil emulsion. 14. Administered in single or multiple doses; and / or administered subcutaneously or intramuscularly; and / or Administered to animals at 1 day of age or later, 7 days of age or later, 10 days of age or later, 15 days of age or later, or 21 days of age or later, 14. The immunogenic composition according to any one of clauses 1 to 13.

[0052] 15. The immunogenic composition of any one of clauses 1 to 14, administered to animals at or after 1 day of age, at or after 7 days of age, at or after 10 days of age, at or after 15 days of age or at or after 21 days of age. 16. The immunogenic composition according to any one of clauses 1 to 14, which is administered to animals from the age of 21 days. 17. The immunogenic composition according to any one of clauses 1 to 14, which is administered to animals from the age of 15 days. 18. The immunogenic composition of any one of clauses 1 to 14, which is administered to animals from 7 days of age. 19. The immunogenic composition of any one of clauses 1 to 14, which is administered to animals from 1 day of age.

[0053] 20. The immunogenic composition of any one of clauses 1 to 14, which is administered to animals from 1 day of age. 21. The immunogenic composition according to any one of clauses 1 to 20 for use in the prevention and / or treatment of an infection caused by one or more of the avian influenza viruses, preferably H5Nx, more preferably H5N1, H5N2 and H5N6. 22. The immunogenic composition according to any one of clauses 1 to 20 for use in the prevention and / or treatment of an infection caused by the H5N1 avian influenza virus. 23. The immunogenic composition according to any one of clauses 1 to 20 for use in the prevention and / or treatment of an infectious disease caused by the H5N2 avian influenza virus. 24. The immunogenic composition according to any one of clauses 1 to 20 for use in the prevention and / or treatment of an infection caused by the H5N6 avian influenza virus.

[0054] 25. The immunogenic composition of any one of clauses 1 to 20 for use in the prevention and / or treatment of an infection caused by an H5Nx avian influenza virus of clade 2, preferably clade 2.3, more preferably clade 2.3.4 or 2.3.2. 26. The immunogenic composition of any one of clauses 1 to 20 for use in the prevention and / or treatment of an infection caused by a clade 2 H5Nx avian influenza virus. 27. The immunogenic composition of any one of clauses 1 to 20 for use in the prevention and / or treatment of an infection caused by an H5Nx avian influenza virus of clade 2.3. 28. The immunogenic composition of any one of clauses 1 to 20 for use in the prevention and / or treatment of an infection caused by an H5Nx avian influenza virus of clade 2.3.4. 29. The immunogenic composition of any one of clauses 1 to 20 for use in the prevention and / or treatment of an infection caused by an H5Nx avian influenza virus of clade 2.3.4.4.

[0055] 30. The immunogenic composition of any one of clauses 1 to 20 for use in the prevention and / or treatment of an infection caused by an H5Nx avian influenza virus of clade 2.3.4.4d. 31. The immunogenic composition of any one of clauses 1 to 20 for use in the prevention and / or treatment of an infection caused by an H5Nx avian influenza virus of clade 2.3.2. 32. The immunogenic composition of any one of clauses 1 to 20 for use in the prevention and / or treatment of an infection caused by an H5Nx avian influenza virus of clade 2.3.2.1. 33. The immunogenic composition of any one of clauses 1 to 20 for use in the prevention and / or treatment of an infection caused by an H5Nx avian influenza virus of clade 2.3.2.1d. 34. The immunogenic composition of any one of clauses 1 to 20 for use in the prevention and / or treatment of an infection caused by an H5Nx avian influenza virus of clade 7.2.

[0056] 35. The immunogenic composition of any one of clauses 1 to 34, which is administered in a single dose. 36. The immunogenic composition of any one of clauses 1 to 34, administered as a single dose, said single dose being effective in the prevention and / or treatment of an infection caused by an avian influenza virus. 37. The immunogenic composition of any one of clauses 1-34, administered as a single dose, said single dose being effective in the prevention and / or treatment of infection caused by one or more of H5N1, H5N2 and H5N6. 38. The immunogenic composition of any one of clauses 1 to 34, administered as a single dose, said single dose being effective in the prevention and / or treatment of infection caused by any of the H5Nxs cited in clauses 22 to 34.

[0057] 39. A method for distinguishing between animals naturally infected with an avian influenza virus and animals vaccinated with an immunogenic composition according to any one of clauses 1 to 13, comprising: (a) analyzing the animal sample for the presence of avian influenza markers that are not present in the immunogenic composition but are present in the naturally infected animal using an immunoassay or enzyme-linked immunosorbent assay immunoassay and / or genomic analysis test; (b) determining whether the sample is positive or negative for the avian influenza marker; (c) correlating the test results with the status of the tested animals, and animals testing positive for avian influenza markers are naturally infected animals, and animals testing negative for avian influenza markers are animals vaccinated with the immunogenic composition of any one of clauses 1 to 13. A method comprising:

[0058] 40. A method for distinguishing between animals naturally infected with an avian influenza virus and animals vaccinated with an immunogenic composition according to any one of clauses 1 to 13, comprising: (a) analyzing the animal sample with an immunological test, which may be an enzyme immunoassay or an enzyme-linked immunosorbent assay, and / or a genomic analysis test, for the presence of avian influenza markers that are specific for the immunogenic composition but that are not present in naturally infected animals; (b) determining whether the sample is positive or negative for the avian influenza marker; (c) correlating the test results with the status of the tested animals, and animals that are positive for avian influenza markers are animals that have been vaccinated with the immunogenic composition according to any one of clauses 1 to 13. A method comprising:

[0059] Sequence overview The following sequences are hereby detailed and disclosed in the present invention: SEQ ID NO: 1: Amino acid sequence of the HA protein of the duck / China / E319-2 / 03 strain, but lacking the amino-terminal signal peptide. SEQ ID NO: 2: Amino acid sequence of H5Con1. SEQ ID NO: 3: Amino acid sequence of H5Con3. SEQ ID NO: 4: Amino acid sequence of H5Con5. SEQ ID NO: 5: Amino acid sequence of H5Con5Mut. SEQ ID NO: 6: Amino acid sequence of positions 60 to 300 of H5Con5Mut. SEQ ID NO: 7: Optimized nucleic acid sequence of H5Con1. SEQ ID NO: 8: Optimized nucleic acid sequence of H5Con3. SEQ ID NO: 9: Optimized nucleic acid sequence of H5Con5. SEQ ID NO: 10: Optimized nucleic acid sequence of H5ConMut. SEQ ID NO: 11: Optimized nucleic acid sequence encoding the amino acid sequence of positions 60 to 300 of H5Con5Mut. The following drawings form part of the present invention and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0060] [Figure 1] FIG. 1 shows that the H5Con1 sequence was generated by a sequential method from 444 sequences isolated between 1996 and 2012. [Figure 2] FIG. 1 shows that the H5Con3 sequence was generated by a single-round method from 297 sequences of specific clades isolated between 1996 and 2012. [Figure 3] FIG. 1 shows that the H5Con5 sequence was generated by the single-round method from 196 sequences isolated between 2005 and 2012. [Figure 4] FIG. 1 shows the construction of transfer plasmid pVL1393-H5Con5Mut. [Example]

[0061] The following examples are included to further describe the invention described herein and to demonstrate embodiments of the invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples below represent techniques discovered by the inventors to work well in the practice of the invention and can therefore be considered to constitute modes for its practice. However, those skilled in the art will, in light of this disclosure, appreciate that many changes can be made in the specific embodiments disclosed and still obtain like or similar results within the spirit and scope of the invention.

[0062] Example 1 Generation of H5 HA consensus amino acid sequence and its mutants Construction of H5N1 influenza HA consensus amino acid sequence 1 To generate H5N1 influenza HA consensus amino acid sequence 1 (H5Con1), we collected and analyzed 444 avian H5N1 influenza HA amino acid sequences isolated in China between 1996 and 2012. The 444 HA amino acid sequences consist of 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 the 444 amino acid sequences isolated between 1996 and 2012. First, 13 tier 1 consensus amino acid sequences were generated, each representing an individual clade using (1) 36 clade 0 sequences; (2) 31 clade 2.2 sequences; (3) 8 clade 2.3.2 sequences; (4) 49 clade 2.3.2.1 sequences; (5) 2 clade 2.3.3 sequences; (6) 210 clade 2.3.4 sequences; (7) 18 clade 2.4 sequences; (8) 6 clade 2.5 sequences; (9) 9 clade 3 sequences; (10) 5 clade 4 sequences; (11) 8 clade 5 sequences; (12) 3 clade 6 sequences; (13) 34 clade 7 sequences; and (14) 21 clade 9 sequences. The final consensus amino acid sequence was a tier 2 consensus amino acid sequence generated by aligning and analyzing all 13 tier 1 consensus amino acid sequences using MEGA 5.0 software. This final consensus amino acid sequence was designated as H5Con1 (SEQ ID NO:2).

[0063] Construction of H5N1 influenza HA consensus sequence 3 To generate H5N1 influenza HA consensus amino acid sequence 3 (H5Con3), we collected and analyzed 297 avian H5N1 influenza HA sequences isolated in China between 1996 and 2012. The 297 HA amino acid sequences consisted of only specific clades: clade 2.3.2.1, clade 2.3.4, and clade 7. Figure 2 illustrates the one-round method for generating H5Con3 from the 297 amino acid sequences of specific clades isolated between 1996 and 2012. The final consensus amino acid sequence was generated by aligning and analyzing (1) 49 sequences from clade 2.3.2.1, (2) 214 sequences from clade 2.3.4, and (3) 34 sequences from clade 7 using MEGA 5.0 software. This final consensus amino acid sequence was designated H5Con3 (SEQ ID NO: 3).

[0064] Construction of H5N1 influenza HA consensus amino acid sequence 5 To generate H5N1 influenza HA consensus amino acid sequence 5 (H5Con5), we collected and analyzed 196 avian H5N1 influenza HA amino acid sequences isolated in China between 2005 and 2012. The 196 HA amino acid sequences were 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 the one-round method for generating H5Con5 from the 196 amino acid sequences isolated between 2005 and 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). The strategies for generating H5Con1, H5Con3 and H5Con5 are summarized in Table 1.

[0065] TIFF0007770358000001.tif59150 Construction of 5 mutants of the H5N1 influenza HA consensus amino acid sequence To generate the H5N1 influenza HA consensus amino acid sequence 5 mutant (H5Con5Mut), two amino acid mutations were introduced into the amino acid sequence of H5Con5. At amino acid positions 120 and 223, both serines (S) were mutated to asparagines (N), i.e., S120N and S223N. The amino acid sequence of H5Con5Mut is shown by SEQ ID NO:5.

[0066] Analysis of H5Con1, H5Con3 and H5Con5 Table 2 shows the percent identity of the consensus amino acid sequences of H5Con1, H5Con3, and H5Con5 to sequences of circulating clades. Table 3 further shows the amino acid usage of each of the consensus amino acid sequences of H5Con1, H5Con3, and H5Con5 at some predicted antigenic sites in the H5 HA protein.

[0067] Table 2. Summary of % identity between the consensus amino acid sequences of H5Con1, H5Con3, and H5Con5 and sequences from prevalent clades TIFF0007770358000002.tif46148

[0068] Table 3. Summary of amino acid usage of each of the consensus amino acid sequences of H5Con1, H5Con3, and H5Con5 at predicted antigenic sites of the H5 HA protein. TIFF0007770358000003.tif48158

[0069] It can be seen from Tables 2 and 3 that H5Con1, H5Con3 and H5Con5 have different amino acid sequences compared to the sequences of each of the circulating clades and also have several amino acid changes in antigenic sites between the three different circulating clades.

[0070] Example 2 Optimization of nucleic acid sequences of H5Con1, H5Con3, H5Con5, and H5Con5Mut, construction of recombinant baculovirus expression system, and expression of H5Con1, H5Con3, H5Con5, and H5Con5Mut in insect cells Optimization of the nucleic acid sequences of H5Con1, H5Con3, H5Con5, and H5Con5Mut To optimize for better expression, each of the amino acid sequences of H5Con1, H5Con3, and H5Con5 identified in Example 1 was reverse-inferred to the corresponding nucleic acid sequence using Vector NTI software and optimized for expression in insect cells in a baculovirus expression system (Allele Biotechnology, catalog ABP-BVP-10002). The optimized nucleic acid sequences of H5Con1, H5Con3, and H5Con5 were synthesized using GenScript (GenScript, New Jersey, USA). The optimized nucleic acid sequences of H5Con1, H5Con3, and H5Con5 are shown in SEQ ID NOs: 7 to 9, respectively. The optimized nucleic acid sequence of H5Con5Mut was generated based on the optimized nucleic acid sequence of H5Con5 using a site-directed mutagenesis kit (QuickChange Site-directed Mutagenesis Kit, catalog no. 200518, Agilent). The optimized nucleic acid sequence of H5ConMut is shown in SEQ ID NO: 10.

[0071] Construction of recombinant baculovirus expression systems and expression of H5Con1, H5Con3, H5Con5, and H5Con5Mut in insect cells Each of the optimized nucleic acid sequences of H5Con1, H5Con3, H5Con5, and H5Con5Mut prepared above was inserted into the baculovirus expression system transfer vector pVL1393 included in the Sapphire™ Baculovirus DNA and Transfection Kit (Allele Biotechnology, Cat. ABP-BVD-10002) to generate transfer plasmids designated as pVL1393-H5Con1, pVL1393-H5Con3, pVL1393-H5Con5, and pVL1393-H5Con5Mut, respectively. Figure 4 exemplarily shows the construction of the transfer plasmid pVL1393-H5Con5Mut.

[0072] pVL1393-H5Con1, pVL1393-H5Con3, pVL1393-H5Con5, and pVL1393-H5Con5Mut were then cotransfected with linearized wild-type Sapphire™ baculovirus DNA (Sapphire™ Baculovirus DNA and Transfection Kit; Allele Biotechnology, Catalog ABP-BVD-10002) into sf9 insect cells (Invitrogen, Catalog No. B825-01, Lot No. 1030672) to obtain rescued recombinant baculoviruses. The recombinant baculoviruses containing the optimized nucleic acid sequences of H5Con1, H5Con3, H5Con5, and H5Con5Mut were designated rBacH5Con1, rBacH5Con3, rBacH5Con5, and rBacH5Con5Mut, respectively. Then, transfected sf9 cells containing each of the recombinant baculoviruses were obtained. The transfected sf9 cells prepared above were cultured in a 27°C incubator for 4 days, and the cell culture supernatant was harvested. To obtain pure recombinant viruses, the harvested supernatant was then subjected to plaque purification. Three rounds of plaque purification were performed, and viral plaques containing rBacH5Con1, rBacH5Con3, rBacH5Con5, and rBacH5Con5Mut were picked after the third round of plaque purification to obtain purified rBacH5Con1, rBacH5Con3, rBacH5Con5, and rBacH5Con5Mut.

[0073] Each of the purified rBacH5Con1, rBacH5Con3, rBacH5Con5, and rBacH5Con5Mut was further propagated in suspension culture insect cell line SF+ cells (Ex-CELL® 420 serum-free medium for insect cells, Sigma-Aldrich, catalog 14420C) in shake flasks. Briefly, SF+ cells (Protein Sciences Corporation, Meriden, CT) were cultured in shake flasks at 10 6 The purified rBacH5Con1, rBacH5Con3, rBacH5Con5, and rBacH5Con5Mut were inoculated into SF+ cells (Protein Sciences, Inc., Meriden, CT) at MOIs of 0.01 to 1.0. The inoculated SF+ cells were cultured in EX-CELL 420 medium at 27°C with a shaking speed of 80 to 120 rpm for 3 to 7 days. The cell culture suspensions containing the expressed H5Con1, H5Con3, H5Con5, and H5Con5Mut, respectively, were later harvested for the next step. The harvested cell culture suspensions were 0.5 to 1.5 x 10 6 The cells contained numbers between 0.01 and 0.1 cells / ml.

[0074] Example 3 Determining HAU of cell culture suspensions The HAU of cell culture suspensions containing H5Con1, H5Con3, H5Con5, and H5Con5Mut, respectively, was tested by hemagglutination assay (see OIE Terrestrial Manual 2015, Chapter 2.3.1 & 2.3.2, Avian Influenza (infection with avian influenza viruses)) using two-fold serial dilutions. Briefly, 10 ml of RBCs (Southern Regent Plant, Zhejiang Province) were centrifuged at 500 g for 20 minutes, and 1 volume of 4% centrifuged RBCs was added to 3 volumes of PBS to prepare 1% RBCs. 25 μl of PBS was dispensed into wells 1 to 11 of a 96-well plate. 25 μl of each cell culture suspension was added to wells 1 to 11 and then serially diluted 1:2 to 1:2048. 25 μl of 1% RBC was added to all wells and incubated at room temperature for approximately 40 minutes. Negative wells appeared as a dot in the center of the well, while positive results formed a uniform reddish color throughout the well. The dilution endpoint corresponded to the maximum dilution of the sample that resulted in complete agglutination of RBCs. The results showed that the HAU of the cell culture suspensions of H5Con1, H5Con3, H5Con5, and H5Con5Mut was greater than 71.1 HAU / 25 μl (corresponding to 256 HAU / 90 μl or 256 HAU / dose).

[0075] Example 4 Preparation of immunogenic compositions The harvested cell culture suspension with HAU >256 / dose was further subjected to binary ethyleneimine (BEI) treatment by adding 10 mM BEI solution at 37°C for 72 h to inactivate the infectivity of baculovirus, and then 10 mM sodium thiosulfate solution was added at 4°C to neutralize the BEI residue. After inactivation, the HAU of the cell culture suspensions was assessed again, and inactivated cell culture suspensions with an HAU greater than 256 / dose were used as the active antigen component for the subsequent emulsification procedure with adjuvant.

[0076] The immunogenic compositions of H5Con1, H5Con3, H5Con5, and H5Con5Mut were prepared 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 compositions was the commercially available adjuvant MONTANIDE™ ISA 71R VG (manufactured by Seppic Inc., catalog number 365187), and the aqueous phase contained each of the inactivated cell culture suspensions of H5Con1, H5Con3, H5Con5, and H5Con5Mut. For each dose (0.3 ml) of immunogenic composition, approximately 3 parts aqueous phase (90 μl) was added to approximately 7 parts oil phase (210 μl) and dispersed under low shear (approximately 11,000 rpm for 1 minute) at room temperature, followed by a high shear (16,000 rpm) for 2 minutes in an ice-water bath. A water-in-oil emulsion was prepared using a Miccra disperser (disperser, catalog number: Miccra D-9; disperser head, catalog number: DS-14 / P). For each of the H5Con1, H5Con3, H5Con5, and H5Con5Mut immunogenic compositions, the amount of H5Con1, H5Con3, H5Con5, and H5Con5Mut was at least 256 HAU / dose.

[0077] Example 5 Cross-reactivity testing and selection for H5Con1, H5Con3, H5Con5 and H5Con5Mut SPF chicken embryonated eggs were purchased from SPAFAS Jinan. After hatching, SPF chickens were randomly selected and reared in designated isolators. Cross-reactivity tests of H5Con1, H5Con3, H5Con5, and H5Con5Mut were performed using Re-4 to Re-8 and 03H5 (MutK+).

[0078] 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 commercially available products manufactured by Harbin Weike Biotechnology Development Co., Ltd. 03H5(MutK+) is a baculovirus-expressed HA protein from the A / Duck / China / E319-2 / 03 strain, which further contains the following amino acid changes: S120N, D150N, S223N, and K328 (see International Publication No. 2013024113). 03H5(MutK+) was formulated into an immunogenic composition using the same method as H5Con1, H5Con3, H5Con5, and H5Con5Mut.

[0079] Ten specific pathogen-free (SPF) chickens, aged 10 days, were each vaccinated with one dose (0.3 ml) of the above Re-4 to Re-8 and immunogenic compositions via subcutaneous injection. The vaccinated chickens were constantly housed in an isolator. Serum samples from each chicken were collected before vaccination, 2 weeks after vaccination, and 3 weeks after vaccination. Furthermore, by using the hemagglutination inhibition (HI) test (see OIE Terrestrial Manual 2015, Chapter 2.3.4, Avian Influenza (infection with avian influenza viruses)), serum samples prepared from a pool of 10 chickens within the same vaccination group were tested to determine the cross-HI reactivity of H5Con1, H5Con3, H5Con5, and H5Con5Mut. The HI test assessed the cross-reactivity of H5Con1, H5Con3, H5Con5, and H5Con5Mut with antigens from six 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).

[0080] The cross-HI reactivity scores for Re-4 to Re-8, 03H5(MutK+), H5Con1, H5Con3, H5Con5, and H5Con5Mut are shown in Table 4. In Table 4, "+" indicates the number of heterologous antigens from different clades with which the tested serum could cross-react. Sera scored with "++++" showed cross-reactivity with four different clades, sera scored with "+++" showed cross-reactivity with three different clades, sera scored with "++" showed cross-reactivity with two different clades, and immune sera scored with "+" showed cross-reactivity with only one clade. Scores ranging from "+" to "++++" indicate more extensive cross-reactivity. Antigens yielding a "++++" score were selected for further experiments.

[0081] TIFF0007770358000004.tif113150 Based on the cross-reactivity results, it can be determined that H5Con5Mut has broader cross-reactivity.

[0082] Example 6 Viral exposure and protective efficacy studies The protective efficacy of 03H5MutK+ (clade 2.3.2), H5Con3, and H5Con5Mut was further tested in this example. Re6+7+8, 03H5MutK+ (clade 2.3.2), H5Con3, and H5Con5Mut were evaluated in the protective efficacy test. Re6+7+8 is a trivalent vaccine (a mixture of Re6+Re7+Re8) against clades 2.3.2.1, 7.2, and 2.3.4.4, and was purchased from Harbin Weike Biotechnology Development Co., Ltd. Re6+7+8 was used as a positive control vaccine for protective efficacy against challenge viruses of each clade. Three different clades of highly pathogenic avian influenza (HPAI) H5Nx virus stocks were prepared and used for challenge studies: 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 one dose (0.3 ml) of immunogenic compositions containing Re6+7+8, as well as O3H5MutK+ (clade 2.3.2), H5Con3, and H5Con5Mut (each with at least 256 HAU / dose equivalent). Vaccinated chickens were housed in an ABSL3 (animal biosafety level 3) facility.

[0083] Three weeks (21 days) after vaccination, chickens were challenged by nasal drop with one of three H5Nx challenge viruses per test. The challenge dose for each chicken was 6 Log 10 The EID50 (EID50 = Embryonic Infective Dose 50%, which means the amount of infectious virus that causes infection in 50% of inoculated embryonated eggs) was used. Chickens were monitored for 2 weeks after challenge, and chicken mortality and morbidity were recorded daily. Cotton swabs of the trachea and cloaca of each chicken were collected 3, 5, and 7 days after challenge. Viral shedding was tested in the cotton swab samples via chicken embryo-based virus isolation.

[0084] Evaluation of protective efficacy against each challenge virus was based on the following criteria: 1) whether 100% of vaccinated chickens survived (0% mortality) during the 2-week monitoring period; Three vaccination / challenge animal studies were performed and Table 5 shows the protective efficacy of Re6+7+8, 03H5MutK+ (clade 2.3.2), H5Con3 and H5Con5Mut.

[0085] TIFF0007770358000005.tif78150 Chickens vaccinated with an immunogenic composition containing H5Con5Mut demonstrated 100% protection when challenged with HPAI viruses of clades 2.3.4.4, 2.3.2.1, and 7.2. That is, compared to conventional vaccines such as the Re6+Re7+Re8 mixture, the immunogenic composition containing H5Con5Mut itself simultaneously provided superior and broader protection against AIVs of three different clades.

[0086] Example 7 Protective efficacy testing against different HPAI H5 subtypes The protective efficacy of H5Con5Mut against different HPAI H5 subtypes was further tested in this example. Groups 1a to 5a were designated as immunized groups administered with H5Con5Mut, and groups 1b to 5b were designated as control groups that received only HPAI challenge. Each group contained 12 10-day-old SPF chickens. On the test day (D0), chickens in groups 1a to 5a were vaccinated via the subcutaneous route with one dose (0.3 ml) of an immunogenic composition containing H5Con5Mut. After 21 days post-immunization (dpi), chickens in all groups were challenged intranasally with different HPAI H5 subtypes, with a challenge dose of 6 Log 10 EID 50 / 200 μl / chicken. Morbidity and mortality rates were calculated daily for each group of chickens for 14 days after challenge.

[0087] Clinical signs of HAPI include insufficient spirits, ruffled feathers, significantly reduced appetite, coughing, nasal and ocular discharge, facial swelling, cyanosis, diarrhea, and neurological symptoms. The appearance of at least three clinical signs was used to calculate morbidity. Mortality was calculated by counting dead chickens. % protection was calculated by dividing the number of chickens protected by the total number of chickens tested. The HPAI H5 subtypes used for challenge were as follows:

[0088] TIFF0007770358000006.tif46150 The exposure strains were obtained from South China Agriculture University, Wushan Road, Tianhe District, Guangzhou, Guangdong Province, China, and are representative isolates of the H5N1 (clades 2.3.3.1(d) and 2.3.4.4), H5N2 (clade 7.2), and H5N6 clades (2.3.4.4(d)) mentioned above.

[0089] The observed protective efficacy of H5Con5Mut against different AIV H5 subtypes is summarized in Table 6. TIFF0007770358000007.tif137150

[0090] Table 6 shows that exposure at 21 dpi was effective, as 100% mortality was observed in each control group during the 14 weeks post-challenge, and H5Con5Mut provided 100% protection when chickens were challenged with H5N1, H5N2, and H5N6. This data demonstrated that H5Con5Mut provides better and broader protective efficacy against different HPAI H5 subtypes.

[0091] Example 8 H5Con5Mut defense launch test The protective initiation of H5Con5Mut against HPAI challenge was further tested in this example. Groups 1a and 2a were designated as immunized groups administered with H5Con5Mut, while groups 1b and 2b were designated as control groups that were only exposed to HPAI (14079 strain). Each group contained 12 10-day-old SPF chickens, and the experiment was repeated twice. On the test day (D0), chickens in groups 1a and 2a were vaccinated via the subcutaneous route with one dose (0.3 ml) of an immunogenic composition containing H5Con5Mut. Seven days post-immunization (dpi), chickens in groups 1a and 1b were challenged intranasally with the 14079 strain. Fourteen days post-immunization (dpi), chickens in groups 2a and 2b were challenged intranasally with the 14079 strain. The exposure dose was 6 Log 10 EID 50 / 200 μl / chicken. Morbidity and mortality rates were calculated daily for each group of chickens for 14 days after challenge. The observed onset of protection for H5Con5Mut is summarized in Table 7.

[0092] Table 7 demonstrates that H5Con5Mut induces protection as early as 7 dpi, with 100% protection at 14 dpi. This data demonstrates that H5Con5Mut provides rapid, early-onset protection against HPAI challenge.

[0093] Example 9 H5Con5Mut defense period test The duration of protection of H5Con5Mut against HPAI challenge was further examined in this example.

[0094] Groups 1 to 3 were designated as immunized groups administered with H5Con5Mut, and group 4 was designated as a control group that received only HPAI challenge (14079 strain). Each group contained 12 10-day-old SPF chickens, and the experiment was conducted twice. On the test day (D0), chickens in groups 1 to 3 were vaccinated subcutaneously with one dose (0.3 ml) of an immunogenic composition containing H5Con5Mut. After 28 dpi, 35 dpi, and 42 dpi, chickens in groups 1 to 3 were intranasally challenged with the 14079 strain, respectively. In the control group, chickens were intranasally challenged with the 14079 strain after 28 dpi. The exposure dose was 6 Log10 EID 50 / 200 μl / chicken. Morbidity and mortality rates were calculated daily for each group of chickens for 14 days after challenge. The observed duration of protection for H5Con5Mut is summarized in Table 8.

[0095] Table 8 showed that H5Con5Mut provided 100% protection lasting at least 42 dpi. This data demonstrated that H5Con5Mut provides long-lasting, effective protection against HPAI challenge.

[0096] Example 10 Protection tests in chickens of different ages The protection of H5Con5Mut in chickens of different ages against HPAI challenge was further tested in this example.

[0097] Groups 1 and 2 were designated as immunized groups administered with H5Con5Mut, and Group 3 was designated as a control group that received only HPAI challenge (14079 strain). Each group contained 12 SPF chickens, and the experiment was conducted twice. In Group 1, 1-day-old chickens were vaccinated subcutaneously with one dose (0.3 ml) of an immunogenic composition containing H5Con5Mut. In Group 2, 10-day-old chickens were vaccinated subcutaneously with one dose (0.3 ml) of an immunogenic composition containing H5Con5Mut. 21 dpi, chickens in Groups 1 to 3 were each challenged intranasally with the 14079 strain. The challenge dose was 6 Log10 EID 50 / 200 μl / chicken. Morbidity and mortality rates were calculated for each group of chickens daily for 14 days after challenge. The observed protection of H5Con5Mut in chickens of different ages is summarized in Table 9.

[0098] Table 9 showed that H5Con5Mut provided 100% protection against HPAI challenge to 1-day-old chickens. This data demonstrated that H5Con5Mut provides excellent protection in chickens even from neonatal age.

[0099] Example 11 Protection studies using different routes of administration The protection of H5Con5Mut in chickens via different routes of administration was further tested in this example. Groups 1 and 2 were designated as immunized groups administered with H5Con5Mut, and group 3 was designated as a control group that received only HPAI challenge (14079 strain). Each group contained 12 10-day-old SPF chickens, and the experiment was conducted twice. On the test day (D0), chickens in groups 1 and 2 were vaccinated via the subcutaneous route with one dose (0.3 ml) of an immunogenic composition containing H5Con5Mut via subcutaneous (SC) or intramuscular (IM) administration. 21 dpi, chickens in groups 1 to 3 were each challenged intranasally with the 14079 strain. The challenge dose was 6 Log10 EID 50 / 200 μl / chicken. Morbidity and mortality rates were calculated for each group of chickens daily for 14 days after challenge. The observed protection of H5Con5Mut in chickens via different administration routes is summarized in Table 10.

[0100] TIFF0007770358000011.tif62150

[0101] Table 10 shows that H5Con5Mut provided excellent protection in chickens via both SC and IM routes, and the protective efficacy of H5Con5Mut is not limited to a specific administration route. The flexible administration method allows H5Con5Mut to be suitable for chickens at different developmental stages, such as neonatal chickens with little muscle mass.

[0102] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described with reference to illustrative embodiments, those skilled in the art will recognize that changes can be made to the compositions and methods and the steps or the order of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the following claims.

Claims

1. A method for distinguishing animals naturally infected with avian influenza virus among a group of animals vaccinated with an immunogenic composition, comprising: (a) analyzing a sample from an animal for the presence of avian influenza markers that are not present in the immunogenic composition but are present in the naturally infected animal using an immunoassay or enzyme-linked immunosorbent assay immunoassay and / or genomic analysis test; (b) determining whether the sample is positive or negative for the avian influenza marker; (c) correlating the test results with the status of the test animals, and animals that test positive for the avian influenza markers are naturally infected animals. Including, The immunogenic composition comprises a hemagglutinin protein of an avian influenza virus subtype H5, wherein the hemagglutinin protein is (i) the amino acid sequence set forth in SEQ ID NO: 6; or (ii) the amino acid sequence shown in SEQ ID NO: 5 A method comprising:

2. A method for distinguishing animals that have not been naturally infected with avian influenza virus from a group of animals vaccinated with an immunogenic composition, comprising: (a) analyzing a sample from an animal for the presence of avian influenza markers specific for said immunogenic composition but not present in said naturally infected animal by an immunological test, which may be an enzyme immunoassay or an enzyme-linked immunosorbent assay, and / or a genomic analysis test; (b) determining whether the sample is positive or negative for the avian influenza marker; (c) correlating the test results with the status of the test animals, and animals that test positive for the avian influenza markers are animals that are not naturally infected with avian influenza virus. Including, The immunogenic composition comprises a hemagglutinin protein of an avian influenza virus subtype H5, wherein the hemagglutinin protein is (i) the amino acid sequence set forth in SEQ ID NO: 6; or (ii) the amino acid sequence shown in SEQ ID NO: 5 A method comprising:

3. The method of claim 1 or 2, wherein the hemagglutinin protein is contained in a cell culture, the cell culture is prepared by culturing cells containing an expression vector, and the expression vector includes a nucleic acid molecule encoding a hemagglutinin protein having the amino acid sequence shown in SEQ ID NO: 6 or 5.

4. The method of claim 3, wherein the nucleic acid molecule is set forth in SEQ ID NO: 10 or 11.

5. The method according to any one of claims 3 to 4, wherein the expression vector is a baculovirus and the cell is an insect cell.

6. The method according to any one of claims 3 to 5, wherein the cell culture is subjected to an inactivation step.

7. The method of any one of claims 3 to 6, wherein the immunogenic composition comprises part or all of the cell culture.

8. The method of any one of claims 1 to 7, wherein the immunogenic composition further comprises an adjuvant, preferably wherein the adjuvant is a water-in-oil emulsion.

9. 9. The method of any one of claims 1 to 8, wherein the animal vaccinated with the immunogenic composition is selected from an animal that is 1 day or older, 7 days or older, 10 days or older, 15 days or older or 21 days old, preferably wherein the animal is a chicken.

10. A method according to any one of claims 1 to 9, wherein the avian influenza virus is an avian influenza virus H5Nx, preferably wherein the H5Nx is selected from one or more of H5N1, H5N2 and H5N6.

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