Vaccine against infectious bronchitis

Abstract

Poultry vaccines against infectious bronchitis and turkey rhinotracheitis are provided. [Solution] The immunogenic composition comprises an antigen component and an adjuvant component, wherein: a) the antigen component comprises at least one TRT antigen and at least one IB antigen, and the at least one IB antigen comprises an IB QX antigen; and b) the adjuvant component comprises an immunostimulatory oligonucleotide and an oil emulsion.

Description

Background Art

[0001]

[0001] Turkey rhinotracheitis (TRT) is an upper respiratory tract infection of turkeys and chickens caused by a paramyxovirus, and is a highly contagious acute disease that affects turkeys of all ages. Clinical symptoms of TRT infection include prominent often frothy nasal discharge, rales, snicking, sneezing, and head shaking. Also, in infected turkeys, eye discharge or swollen infraorbital sinuses may be observed. Antibodies against the TRT virus (TRTV) have been detected in some chicken flocks (both young chickens and young chicken / breeder chickens) suffering from swollen head syndrome (SHS). TRTV is hypothesized to play a role in the etiology of SHS and associated respiratory distress.

[0002]

[0002] Infectious bronchitis (IB) is a coronavirus that causes disease only in chickens, although some other birds may be subclinically infected. While some serotypes are geographically restricted, many serotypes generally co-circulate within a single geographic region. In recent years, the QX strain, a novel IBV genotype, has been gradually spreading in Asia and Europe. The morbidity rate is generally close to 100%. Coughing, sneezing, and bronchoconstriction in chicks may persist for 10 to 14 days. Conjunctivitis and dyspnea may be seen, sometimes accompanied by facial swelling, especially simultaneous bacterial infection of the sinuses. Chicks may appear listless and huddled under a heat lamp. Feed consumption and weight gain decrease. Infection with nephropathogenic strains presents initial respiratory signs and subsequently leads to listlessness, ruffled feathers, wet droppings, increased water intake, and death. In laying hens, egg production may decrease by as much as 70%, and eggs are often misshapen, with thin, soft, rough, and / or pale shells, smaller in size, and watery egg whites. In most cases, egg production and egg quality usually return to normal, although the period required for this may be up to 8 weeks. In most outbreaks, the mortality rate is 5%, but it increases if the disease worsens with concurrent bacterial infection. In young chicks, nephropathogenic strains may induce interstitial nephritis with a high mortality rate (up to 60%). Infection in young chicks can cause permanent damage to the oviduct, resulting in laying hens or breeding hens not reaching standard production levels.

[0003]

[0003] Known vaccine strains of IB virus have been proven to be insufficient to protect against infectious bronchitis caused by IB-QX virus and IB-QX-like viruses. See International Publication No. WO 2010 / 017440.

[0004]

[0004] Infectious bursal disease (IBD, also known as Gumboro disease) is a highly contagious immunosuppressive disease found in chickens worldwide and has a significant economic impact on chicken egg and meat production. IBD vaccines form an important part of the Gumboro control strategy. The choice of vaccine to be administered depends on the type of chicken receiving the vaccination and the prevailing challenging situation.

[0005]

[0005] Generally, inactivated antigens have been used in poultry vaccines. However, since the production of inactivated viruses is relatively costly, effective vaccines with low antigen content levels are desired.

[0006]

[0006] Another need in poultry vaccination is closely related to the cost of the vaccination itself. Producing a multivalent vaccine designed to prevent multiple diseases is economically advantageous. Such multivalent vaccines reduce the cost of vaccine administration, but often do not work as an approach to producing multivalent vaccines simply by mixing antigens in the same dosage form due to the well-known phenomenon of antigen interference.

[0007]

[0007] Therefore, there is a need for poultry vaccines with reduced antigen and / or multivalent vaccine content. SUMMARY OF THE INVENTION

[0008]

[0008] In one aspect, the present invention provides an immunogenic composition comprising an antigen component and an immunologically effective amount of an adjuvant component. The antigen component comprises at least one TRT antigen and at least one IB antigen, and the adjuvant component comprises an immunostimulatory oligonucleotide, an oil emulsion, and optionally, a sterol.

[0009]

[0009] In certain embodiments, at least one TRT antigen is TRT strain K.

[0010] In certain embodiments, at least one IB antigen is at least one of the IB D1466 antigen and the IB QX antigen.

[0010]

[0011] In certain embodiments, the immunogenic composition of the present invention is non-liposomal and / or essentially saponin-free.

[0011]

[0012] In certain embodiments, optionally, the sterol is mixed with an immunostimulatory oligonucleotide.

[0012]

[0013] The present invention also provides a vaccine comprising an antigenic component and an effective amount of an adjuvant component, wherein the adjuvant component comprises an immunostimulatory oligonucleotide and an oil emulsion, and the antigenic component comprises an IBD antigen.

[0013]

[0014] In certain embodiments, the IBD antigen is an inactivated Lukert strain antigen that may be present in an amount of 10 7.5 ~10 8 TCID 50 .

[0014]

[0015] In certain embodiments, the vaccine is a multivalent vaccine comprising at least one of an antigen derived from a non-Lukert strain of IBD; an infectious bronchitis antigen; a reovirus antigen; a Newcastle disease antigen; an antigen of turkey rhinotracheitis.

[0015]

[0016] In another aspect, the present invention provides a vaccine comprising an antigenic component and an effective amount of an adjuvant component. The adjuvant component comprises an immunostimulatory oligonucleotide and an oil ema comprising a Newcastle antigen; an egg production decline syndrome (EDS) antigen; an IBK antigen; and a coryza antigen, and the antigen component contains a TRT antigen.

[0016]

[0017] In certain embodiments, the TRT antigen comprises an inactivated turkey rhinotracheitis virus, the Newcastle antigen comprises an inactivated Newcastle virus, the EDS antigen comprises an inactivated EDS virus, the IBK antigen comprises an inactivated IBK virus, and the coryza antigen comprises a mixture of coryza M bacterin, coryza 221 bacterin, and coryza S bacterin.

[0017]

[0018] In certain embodiments, the TRT antigen is present in an amount of 10 6.00 TCID 50 ~10 6.50 TCID 50 per dose.

[0018]

[0019] In certain embodiments, the oil emulsion is a W / O emulsion.

Mode for Carrying Out the Invention

[0019]

[0020] Definition:

[0021] The term "about" or "approximately", when used in connection with a measurable numerical variable, refers to either the greater of (i) a variable within the experimental error of the indicated value (e.g., within the 95% confidence interval of the mean) or within 10% of the indicated value, or (ii) all variables within the indicated variable value. However, this does not apply to time intervals of several weeks where "about" is used and "about 3 weeks" is taken as 17 - 25 days and about 2 - about 4 weeks is taken as 10 - 40 days.

[0020]

[0022] The term "consisting essentially of" and similar terms applied to the adjuvant formulations of the present invention refer to compositions that do not contain additional adjuvant agents or immunomodulatory agents in an amount such that the agent exhibits a measurable adjuvanting or immunomodulatory effect.

[0021]

[0023] The terms "essentially free of saponin", "substantially free of saponin" and similar terms refer to compositions that do not contain saponin in an amount such that the saponin exhibits a measurable adjuvanting or immunomodulatory effect. In certain embodiments, in the case of a composition that is essentially free of saponin, the saponin content is an amount that is not sufficient to cause a systemic immune response such as fever. In certain embodiments, in the case of a composition that is essentially free of saponin, the saponin content is zero or below the detection limit.

[0022]

[0024] The term "immunostimulatory molecule" refers to a molecule that generates an immune response.

[0025] The term "parenteral administration" refers to introducing a substance such as a vaccine into the body of a subject via a route that does not include the gastrointestinal tract. Parenteral administration includes subcutaneous administration, intramuscular administration, transdermal administration, intradermal administration, intraperitoneal administration, intraocular administration, and intravenous administration.

[0023]

[0026] The percentage purity or "purity X percent" applied to immunostimulatory oligonucleotide formulations " refers to a population of oligonucleotide molecules containing X% of the specified oligonucleotide (e.g., SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8, etc.), and the remainder (i.e., the value obtained by subtracting X% from 100%) contains short fragments of the specified oligonucleotide that are present as impurities during the production of the specified sequence. Therefore, when this sequence is produced by 3'-5' sequencing, the remainder will be included in the 5'-truncated form. As a non-limiting example, out of 100 μg of a preparation of SEQ ID NO: 8 with 80% purity, 80 μg contains SEQ ID NO: 8, and the remaining 20 μg are fragments of SEQ ID NO: 8 that are present in the formulation.

[0024]

[0027] The terms "therapeutically effective amount", "immunologically effective amount", and "effective amount" refer to an amount of an antigen, adjuvant, or vaccine that is sufficient to induce an immune response in a subject to whom the antigen, adjuvant, or vaccine is administered to prevent or alleviate the signs or symptoms of a disease, including adverse health effects or complications caused by infection with a pathogen such as a virus or bacterium. Humoral immunity or cell-mediated immunity, or both humoral and cell-mediated immunity, may be induced. The immunogenicity and efficacy of a vaccine in an animal can be evaluated indirectly, for example, through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring of signs and symptoms after administration of the antigen by the wild-type strain. The protective immunity conferred by a vaccine can be evaluated, for example, by measuring a decrease in clinical signs such as mortality, morbidity, body temperature values, general physical condition, and the overall health and performance of the subject. A therapeutically effective amount of a vaccine may be determined by one of ordinary skill in the art, which may vary depending on the specific adjuvant used, the specific antigen used, or the condition of the subject.

[0025]

[0028] Commercial laying hens and breeding hens are vaccinated during their lifetime using a variety of vaccines. These vaccines are mainly live attenuated bacterial vaccines. For chickens, until they are ready to start laying eggs, they are vaccinated with inactivated combined vaccines to reinforce immunity against infectious pathogens they have already been vaccinated against, and to induce immunity against other pathogens that may cause chicken egg production or other damage during the breeding period. Since these vaccines can induce high antibody titers and the vaccines need to protect during the entire breeding period from approximately 20 weeks to 70 weeks, it is important that the duration of immunity is long. To achieve long-term immunity, it is necessary to formulate the antigen in an adjuvant, such as a water-in-oil (W / O) emulsion.

[0026]

[0029] Accordingly, generally, the present invention provides an immunogenic composition comprising an antigen component and an adjuvant component, wherein the antigen component comprises at least one TRT antigen and at least one IB antigen, and the adjuvant component comprises an immunostimulatory oligonucleotide, an oil emulsion, and optionally, a sterol (or consists essentially of these in some embodiments, or consists of these in other embodiments).

[0027] Antigen component

[0030] Different IB QX antigens are suitable for the present invention. In certain embodiments, the IB QX antigen is a completely inactivated virus. In other embodiments, the virus is a modified live virus. In still other embodiments, a subunit vaccine may be used. For example, proteins considered suitable for being present on the surface of the virus include, but are not limited to, S protein, M protein, E protein, or any combination thereof. In embodiments involving the use of inactivated whole virus, the antigen is 10 3 ~10 10 infection units, such as 10 4 、10 5 、106 and 10 7 may be used in terms of the amount of infectious units. In certain embodiments, the amount of IB QX inactivated virus is about 10 5 to about 10 8 infectious units per dose.

[0028]

[0031] The antigenic component includes an IBD antigen in a further embodiment and is an inactivated Lukert IBD virus in certain embodiments.

[0029]

[0032] In certain embodiments, the amount of inactivated Lukert IBD virus is 10 7 to 10 8 TCID 50 (e.g., 10 7.1 , 10 7.2 , 10 7.3 , 10 7.4 , 10 7.5 , 10 7.6 , 10 7.7 , 10 7.8 , 10 7.9 TCID 50 ) per dose.

[0030]

[0033] In certain embodiments, the antigenic component of the vaccine includes not only inactivated Lukert IBD virus but also other antigens. For example, different infectious bronchitis virus strains such as IB M41 and / or IB D1466 and / or IB D274 can be used. Alternatively or additionally, the vaccine of the present invention may also include antigens of TRT, Newcastle disease (e.g., LaSota strain), EDS (egg drop syndrome), reovirus, and infectious bursal disease virus, avian influenza.

[0031]

[0034] In other aspects, the present invention provides a multivalent vaccine including a TRT antigen; a Newcastle antigen; an egg drop syndrome (EDS) antigen; an IBK antigen (infectious bronchitis virus); and a coryza antigen.

[0032]

[0035] In certain embodiments of the present invention, the TRT antigen is 10 6.00 TCID 50 ~10 6.50 TCID 50 (e.g., 10 6.00 TCID 50 , 10 6.10 TCID 50 , 10 6.20 TCID 50 , 10 6.30 TCID 50 , 10 6.40 TCID 50 or 10 6.50 TCID 50 ) and is present in an amount of.

[0033]

[0036] The virus used in the vaccine of the present invention can be attenuated or inactivated. Methods for inactivating and attenuating the virus are well known in the art. For example, the virus may be inactivated by serial passage in culture. Examples of inactivation methods include, but are not limited to, exposing the virus to an effective amount of an inactivating chemical selected from formalin, β-propiolactone (BPL), binary ethyleneimine (BEI), or phenol.

[0034]

[0037] Coryza is caused by different Haemophilus paragallinarum strains. Thus, in certain embodiments, the coryza antigen comprises one or more Haemophilus paragallinarum strains (e.g., strain M, strain Z, strain 221, and the like). In other embodiments, a mixture of strains representing serotypes A, B, and C is used. Thus, strain 221 (coryza 221) may be used as a Serovar A strain, Spross (coryza S) strain may be used as a Serovar B stain, and Stord Modesto (coryza M) may be used as a Serovar C strain.

[0035]

[0038] A person skilled in the art may sometimes realize that depending on the methodology of virus titration, the virus titer may vary, sometimes by up to about 30%. In the present disclosure, when the dose is measured as an exponent of 10, the exponent may vary by 0.2 each time. Therefore, for example, for a titer of 10 6.40 TCID 50 it may include values between 10 6.20 TCID 50 and 10 6.60 TCID 50 . The same concept applies to the range of titers. For example, for a titer of 10 6.00 TCID 50 to 10 6.50 TCID 50 it includes the range of 10 5.80 TCID 50 to 10 6.70 TCID 50 .

[0036]

[0039] In other embodiments, in addition to the above antigens, other antigens may be used, such as Salmonella enteritidis, Salmonella typhimurium, Mycoplasma gallisepticum, Salmonella gallinarum, Pasteurella multocida. Similar to the case of inactivating the virus, for example, the bacteria can be inactivated by exposure to an effective amount of an inactivating chemical selected from formalin, β-propiolactone (BPL), binary ethyleneimine (BEI), or phenol.

[0037] Adjuvant component

[0040] Generally, the adjuvant component used in the immunogenic composition of the present invention includes immunostimulatory oligonucleotides, oils, and optionally, surfactant(s). In certain embodiments, the adjuvant component does not contain or is essentially free of saponin and / or ISCOM.

[0038]

[0041] In certain embodiments, the adjuvant component consists essentially of an immunostimulatory oligonucleotide, an oil, and optionally, a surfactant(s). In certain embodiments, the adjuvant component consists of an immunostimulatory oligonucleotide, an oil, and optionally, a surfactant(s).

[0039]

[0042] Suitable immunostimulatory oligonucleotides include ODN (DNA-based), ORN (RNA-based) oligonucleotides, or chimeric ODN-ORN structures. These structures can have modified backbones including, but not limited to, phosphorothioate modification, halogenation, alkylation (e.g., ethyl or methyl modification), and phosphodiester modification. In some embodiments, polyinosinic-cytidylic acid or a derivative thereof (poly I:C) may be used.

[0040]

[0043] CpG oligonucleotides are characterized by the presence of unmethylated CG dinucleotides in the context of a specific base sequence (CpG motif). (This document is incorporated herein by reference: Hansel TT, Barnes PJ (eds.): New Drugs for Asthma, Allergy and COPD. Prog Respir Res. Basel, Karger, 2001, vol 31, pp 229-232). These CpG motifs are not found in eukaryotic DNA. In eukaryotic DNA, CG dinucleotides are suppressed and are usually methylated when present, whereas CG dinucleotides are present in bacterial DNA which confers immunostimulatory properties.

[0041]

[0044] In a selected embodiment, the adjuvant of the present invention utilizes so-called P-class immunostimulatory oligonucleotides, more preferably modified P-class immunostimulatory oligonucleotides, and even more preferably E-modified P-class oligonucleotides. P-class immunostimulatory oligonucleotides are CpG oligonucleotides characterized by the presence of a palindrome generally 6 to 20 nucleotides in length. P-class oligonucleotides have the ability to spontaneously self-organize into concatemers either in vitro and / or in vivo. These oligonucleotides are strictly single-stranded, but the presence of the palindrome allows for the formation of concatemers (or in some cases, stem-and-loop structures). The full length of P-class immunostimulatory oligonucleotides is 19 to 100 nucleotides, such as 19 to 30 nucleotides, 30 to 40 nucleotides, 40 to 50 nucleotides, 50 to 60 nucleotides, 60 to 70 nucleotides, 70 to 80 nucleotides, 80 to 90 nucleotides, 90 to 100 nucleotides.

[0042]

[0045] In one aspect of the present invention, the immunostimulatory oligonucleotide comprises a 5'TLR activation domain and at least two palindrome regions. One palindrome region is a 5' palindrome region at least 6 nucleotides in length, which is linked directly or via a spacer to a 3' palindrome region at least 8 nucleotides in length.

[0043]

[0046] P-class immunostimulatory oligonucleotides can be modified according to techniques known in the art. For example, J modification refers to iodinated nucleotides. E modification refers to ethylated nucleotide(s). Thus, an E-modified P-class immunostimulatory oligonucleotide is a P-class immunostimulatory oligonucleotide in which at least one nucleotide (preferably the 5'-nucleotide) is ethylated. Further modifications include attachment of 6-nitro-benzimidazole, O-methylation, modification with propenyl-dU, inosine modification, attachment of 2-bromovinyl (preferably to uridine).

[0044]

[0047] P-class immunostimulatory oligonucleotides may also include modified internucleotide linkages including, but not limited to, phosphodiester and phosphorothioate linkages. The oligonucleotides of the present invention may be synthesized from commercial sources or may be obtained.

[0045]

[0048] P-class oligonucleotides and modified P-class oligonucleotides are further disclosed in Published PCT Application International Publication No. WO 2008 / 068638 (published June 12, 2008). Suitable non-limiting examples of modified P-class immunostimulatory oligonucleotides are as follows (in SEQ ID NOs: 1-10, "*" refers to a phosphorothioate linkage and "-" refers to a phosphodiester linkage). In SEQ ID NOs: 11-14, all linkages are phosphodiester linkages.

[0046] SEQ ID NO: 1 5’T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G 3’ SEQ ID NO: 2 5’T*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3’ SEQ ID NO: 3 5’T*C*G*A*C*G*T*C*G*A*T* C*G*G*C*G*C*G*C*G*C*C*G*T 3’ SEQ ID NO:4 5’JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3’ SEQ ID NO:5 5’JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3’ SEQ ID NO:6 5’JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3’ SEQ ID NO:7 5’EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3’ SEQ ID NO:8 5’JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T 3’ SEQ ID NO:9 5’JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T 3’ SEQ ID NO:10 5’T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C_G*C*G*C*C*G 3’ SEQ ID NO:11 5’-UUGUUGUUGUUGUUGUUGUU-3’ SEQ ID NO:12 5’-UUAUUAUUAUUAUUAUUAUU-3’ SEQ ID NO:13 5’-AAACGCUCAGCCAAAGCAG-3’ SEQ ID NO:14 5’-dTdCdGdTdCdGdTdTdTdTrGrUrUrGrUrGrUdTdTdTdT-3’

[0049] The immunostimulatory oligonucleotides of the present invention can be chemically synthesized. Furthermore, the immunostimulatory oligonucleotides can be used at about 60% purity (homogeneity) or higher (e.g., about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98% or 100% purity).

[0047]

[0050] The amount of the P-class immunostimulatory oligonucleotides used in the adjuvant composition depends on the nature of the P-class immunostimulatory oligonucleotides used and the species intended.

[0048]

[0051] Sterols share a common chemical core. This chemical core is a steroid ring structure [plural available] to which a hydroxyl (OH) group is usually attached to carbon-3. The length of the hydrocarbon chain of the fatty acid substituent is variable (usually 16 to 20 carbon atoms) and can be saturated or unsaturated. Sterols typically contain one or more double bonds within the ring structure and also contain various substituents attached to the ring. Sterols and their fatty acid esters are essentially water-insoluble. Therefore, considering these chemical similarities, it is highly likely that sterols sharing this chemical core will have similar properties when used in the vaccine compositions of the present invention. Sterols are well-known in the art and it is possible to purchase commercial products of sterols. For example, cholesterol is disclosed in Merck Index, 12th Ed., p. 369. Suitable sterols include, but are not limited to, β-sitosterol, stigmasterol, ergosterol, ergocalciferol, cholesterol and their derivatives, for example, DC-cholesterol (3β-[N-(dimethylaminoethane)carbamoyl]cholesterol).

[0049]

[0052] A plurality of oils and combinations thereof are suitable for use in the present invention. These oils include, but are not limited to, animal oils, vegetable oils, and non-metabolizable oils. Non-limiting examples of vegetable oils suitable in the present invention are corn oil, peanut oil, soybean oil, coconut oil, olive oil, and phytosqualane. A non-limiting example of an animal oil is squalane. Suitable non-limiting examples of non-metabolizable oils include light mineral oil, linear or branched saturated oils, branched oils, and the like.

[0050]

[0053] In a series of embodiments, the oil used in the adjuvant formulation of the present invention is light mineral oil. As used herein, the term "mineral oil" refers to a mixture of liquid hydrocarbons obtained by distillation techniques from petroleum jelly. This term is synonymous with "liquid paraffin", "liquid petrolatum" and "white mineral oil". This term is also intended to include "light mineral oil" (i.e., an oil obtained similarly by distillation of petrolatum but having a slightly lower specific gravity than white mineral oil). See, for example, Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990, at pages 788 and 1323). Mineral oil is available from various commercial sources, such as J.T. Baker (Phillipsburg, N.J.), USB Corporation (Cleveland, Ohio). A preferred mineral oil is a light mineral oil commercially available under the name DRAKEOL®. In another embodiment, a suitable oil includes mineral oil MARCOL™ 52. MARCOL™ 52 is a refined mixture of liquid saturated hydrocarbons, a clear and colorless water-white product free of toxic impurities, and is obtained from petroleum via vacuum distillation with subsequent purification steps (including final purification by catalytic hydrogenation).

[0051]

[0054] Emulsifiers suitable for use in the present emulsion include biologically compatible natural emulsifiers and non-natural synthetic surfactants. Biologically compatible emulsifiers include phospholipid compounds or mixtures of phospholipids. Preferred phospholipids are phosphatidylcholine (lecithin), such as soybean lecithin or egg lecithin. Lecithin can be obtained as a mixture of phosphatides and triglycerides by washing crude vegetable oil with water, separating the resulting hydrated gum, and drying it. A purified product can be obtained by fractionating the mixture of acetone-insoluble phospholipids and glycolipids remaining after removing triglycerides and vegetable oil by acetone washing. Alternatively, lecithin can also be obtained from various commercial sources. Other suitable phospholipids include phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid, cardiolipin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidylethanolamine. Phospholipids may be isolated from natural sources or synthesized conventionally.

[0052]

[0055] In a further embodiment, the emulsifier used herein does not contain lecithin or an immunologically ineffective amount of lecithin is used.

[0053]

[0056] Non-natural synthetic emulsifiers suitable for use in the adjuvant formulation of the present invention include sorbitan-based nonionic surfactants, such as fatty acid-substituted sorbitan surfactants (commercially available under the names SPAN® or ARLACEL®), fatty acid esters of polyethoxylated sorbitol (TWEEN®), polyethylene glycol esters of fatty acids from sources such as castor oil (EMULFOR®); Polyethoxylated fatty acids (e.g., stearic acid available under the name SIMULSOL® M-53), polyethoxylated isooctylphenol / formaldehyde polymers (TYLOXAPOL®), polyoxyethylene fatty alcohol ethers (BRIJ®); polyoxyethylene nonylphenyl ethers (TRITON® N), polyoxyethylene isooctylphenyl ethers (TRITON® X). Preferred synthetic surfactants are surfactants available under the names SPAN® and TWEEN®, for example, TWEEN® 80 (polyoxyethylene (20) sorbitan monooleate) and ARLACEL® 83V (sorbitan sesquioleate).

[0054]

[0057] The emulsifier(s) can generally be present in the vaccine composition in an amount of 0.01% to 40% (volume basis), preferably 0.1% to 15%, more preferably 2% to 10%.

[0055]

[0058] In a subset of embodiments, the volume percentages of the oil and the oil-soluble emulsifier together are at least 50% v / v of the vaccine composition, e.g., 50 - 95% v / v (volume basis); preferably in an amount of 50% v / v or more and 85% v / v or less, more preferably in an amount of 50 - 60 v / v%, more preferably in an amount of 55 - 65% v / v. Thus, for example, without limitation, the oil can be present in an amount of 45% v / v and the fat-soluble emulsifier can be present in an amount greater than 5% v / v. Accordingly, the volume percentages of both the oil and the oil-soluble emulsifier will be at least 50%.

[0056]

[0059] In yet another subset applicable to all vaccines of the present invention, the volume percentage of oil is greater than 40% v / v of the vaccine composition, for example, 40 to 90% v / v (volume basis); 40% v / v to 85% v / v; 43% to 60% v / v, 44 to 50% v / v. In certain embodiments, the emulsion contains at least 60% v / v of the oil phase and 40% v / v of the aqueous phase.

[0057]

[0060] Sometimes, especially in scaled-up commercial applications, it may be necessary to use a low-concentration antigen solution because it is impossible or impractical to concentrate the antigen. Thus, in some embodiments, the vaccine composition of the present invention contains an adjuvant formulation as described above, and the content of the oil phase in these adjuvant formulations is diluted, and the vaccine composition is an oil-in-water emulsion.

[0058]

[0061] In fact, it is possible to prepare an oil-in-water emulsion with an oil phase of less than 50% v / v.

[0059]

[0062] Briefly, first, the adjuvant formulation of the present invention is prepared as described above. In the adjuvant formulation, the oil phase occupies more than 50% v / v of the adjuvant formulation. The amounts of components other than the oil and emulsifier(s) are each scaled up based on the final target concentration and the desired dilution rate. For example, when attempting to prepare a vaccine composition containing 80% v / v of the adjuvant formulation, the amounts of components other than the oil are scaled up by a factor of 1.25 (1 / 0.8). The amount of the emulsifier (TWEEN® 80 and / or SPAN® 80 if present) does not necessarily need to be scaled up, but it is preferable to maintain the same volume ratio of the oil and emulsifier(s) in the adjuvant formulation and the final vaccine composition. preferable.

[0060]

[0063] Next, the antigen solution is added to the adjuvant formulation.

[0064] The integrity of the water-in-oil emulsion can be maintained as long as the dispersed spherical water droplets do not exist in a form more concentrated than the maximum packing fraction of the random packing of monodisperse droplets, i.e., 0.64. See Tadros, Emulsion Formation, Stability and Rheology, 1st ed. 2013, Wiley-VCH GmbH & Co KGaA. Conversely, this implies that the oil phase should not drop below 36% v / v as long as the total volume fraction occupied by the aqueous droplets does not exceed 0.64 (i.e., 64% v / v).

[0061]

[0065] In some embodiments, a suitable single dose of the adjuvant comprises an immunostimulatory oligonucleotide of about 0.1 μg to about 20 μg (e.g., 1 - 20 μg, 5 - 15 μg, 8 - 12 μg, or 10 μg), and a sterol (such as cholesterol) of up to about 50 μg (e.g., 0.5 - 20 μg, or 1 - 10 μg).

[0062]

[0066] In certain embodiments, the method for preparing the adjuvant component is as follows. a) Dissolve sorbitan sesquioleate and cholesterol (if any) in light mineral oil. Sterile filter the resulting oil solution.

[0063] b) Dissolve the immunostimulatory oligonucleotide and polyoxyethylene (20) sorbitan monooleate in the aqueous phase, thereby forming an aqueous solution. c) Add this aqueous solution to the oil solution while continuously homogenizing.

[0064]

[0067] The immunogenic composition of the present invention can be prepared by adding the antigen component to the aqueous phase and subsequently combining the aqueous phase with the oil phase. In other embodiments, the antigen component may be added to the adjuvant component after the preparation of the adjuvant component.

[0065]

[0068] The immunogenic composition may further comprise a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial agents, antifungal agents, isotonic agents, adsorption delaying agents, and the like. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the subject. The carrier is typically sterile and pyrogen-free and is selected based on the mode of administration to be used. As is well known to those skilled in the art, preferred formulations of pharmaceutically acceptable carriers containing the composition are approved by applicable regulations published by the United States (U.S.) Department of Agriculture, the U.S. Food and Drug Administration (FDA), or equivalent government agencies in countries outside the U.S. Thus, pharmaceutically acceptable carriers for commercial production of the composition are carriers that have already been approved or are scheduled to be approved by appropriate government agencies in the U.S. or foreign countries.

[0066]

[0069] Other components of the composition can include pharmaceutically acceptable excipients such as carriers, solvents and diluents, isotonic agents, buffers, stabilizers, preservatives, vasoconstrictors, antibacterial agents, antifungal agents, and the like. Typical carriers, solvents and diluents include water, physiological saline Water, dextrose, ethanol, glycerol, oils, and the like. Representative isotonic agents include sodium chloride, dextrose, mannitol, sorbitol, lactose, and the like. Useful stabilizers include gelatin, albumin, and the like.

[0067] Vaccine Administration

[0070] The vaccines of the present invention can generally be administered via multiple routes. Such routes are known to those skilled in the art and include, but are not limited to, intramuscular injection and subcutaneous injection.

[0068]

[0071] In one embodiment, the vaccine is administered about 3 to 7 weeks (e.g., about 4 to 6 weeks) before the expected egg laying date. This regimen ensures that immunity against IB QX develops by the egg laying period of the vaccinated hens and persists throughout that egg laying period.

[0069]

[0072] In a further embodiment, the vaccine of the present invention is administered as a booster vaccine to hens that have been primed with antigen. A plurality of IB primers are known in the art. For example, the POULVAC® IB primer contains a freeze-dried live bacterium, the Massachusetts type infectious bronchitis virus. Nobilis® IB H120 is a freeze-dried live bacterium vaccine used as a primary vaccination against infectious bronchitis. This vaccine contains the H120 type Massachusetts. Other IB primers can also be used in combination with the vaccine of the present invention.

[0070]

[0073] In the following non-limiting examples, the present invention will be further described.

Example

[0071] Example 1. Preparation of Vaccine

[0074] Table 1 shows an exemplary water-in-oil emulsion. The antigen is diluted in the aqueous phase to which CpG has also been added. The aqueous phase contains the antigen, CpG, and thimerosal and is mixed with the oil phase. After thorough mixing, a stable W / O emulsion is formed.

[0072]

Table 1

[0073] Example 2. Efficacy Assay of IB or TRT

[0075] The inventors used an inactivated IB M41 antigen (10 before inactivation per dose 7.2 EID50 ) or an inactivated TRT antigen (10 before inactivation per dose 5.3 TCID 50 ) and a W / O emulsion containing the same were produced. The CpG content in these emulsions was different or zero (SEQ ID NO: 8, 65% purity) respectively. These emulsions were tested in a potency test in chickens. The chickens were vaccinated at 4 weeks of age. Blood samples were collected 5 weeks after vaccination and the antibody titer against the antigen was tested by ELISA.

[0074]

[0076] The antibody titer against the turkey rhinotracheitis (TRT) virus was measured using an enzyme-linked immunosorbent assay (ELISA) in which the wells of a 96-well micro ELISA plate were coated with the antigen. After coating, standard negative serum and standard positive serum were added to the wells and the serum to be tested. All sera were tested in duplicate. As a control, all sera were also tested in duplicate in wells without the antigen. The reactivity of the antibody against the antigen was visualized by removing unbound antibodies and adding an antibody against the serum antibody conjugated with peroxidase. After removing unbound conjugated antibodies, peroxidase substrate orthophenylenediamino + H 2 O 2 was added. The presence of peroxidase was demonstrated by the development of a color reaction.

[0075]

[0077] Table 2 is a summary of the results of the potency test of the TRT antigen. Generally, the antibody titers were sufficient values and there were no non-responsive examples in any group. There was a positive effect of CpG on the antibody titer. The best results were obtained with 10 μg of CpG per dose, but a plus effect was also obtained with 1 μg of CpG per dose.

[0076]

Table 2

[0077]

[0078] Moreover, regarding the antibody response to IBM41, the positive effect of CpG was also identified (see Table 3). The dose of 1 μg of CpG per dose was already sufficient to substantially stimulate the antibody response. Without CpG, the antibody responses of 7 out of 15 chickens were less than the threshold of 2.60, while in the test with 1 μg of CpG per dose, all chickens responded. Further increase was obtained with 10 μg of CpG per dose.

[0078]

Table 3

[0079] Example 3. Potency assay of IB, ND, and EDS

[0079] A group of 10 four-week-old specific pathogen-free (SPF) chickens was vaccinated by intramuscular injection with a dose of 0.5 ml per dose. Five weeks after vaccination, blood samples were collected for serological examination. To measure the antibody titers against IB, ND, and EDS, ELISA (Idexx FlockCheck IBV antibody kit; Idexx, Maine, USA), hemagglutination inhibition test, and ELISA (Idexx FlockCheck NCD antibody kit; Idexx, Maine, USA) were used respectively.

[0080]

[0080] The vaccine is IBM41: 10 6.9 EID 50 / dose, Newcastle disease (ND): 10 8,1 EID 50 / dose, Egg drop syndrome (EDS): 256 HAu / dose is contained in water-in-oil emulsions containing different amounts of CpG respectively.

[0081]

[0081] A group of 10 four-week-old specific pathogen-free (SPF) chickens, 0.5 ml per dose The dosage of was vaccinated by intramuscular injection. Five weeks after vaccination, blood samples were collected for serological examination. To measure the antibody titers against IB, ND, and EDS, ELISA (Idexx FlockCheck IBV antibody kit; Idexx, Maine, USA), hemagglutination inhibition test, and ELISA (Idexx FlockCheck NCD antibody kit; Idexx, Maine, USA) were used respectively.

[0082]

[0082] The results of IB, ND, and EDS are shown in Tables 4 to 6 respectively.

[0083]

Table 4

[0084]

Table 5

[0085]

Table 6

[0086]

[0083] Results and Conclusions

[0084] For IB M41 and ND, there is a significant difference between the CpG-free formulation and the formulation containing 5 μg of CpG per dose. For ND containing 2.5 μg of CpG per dose, the antibody titer was also significantly improved. For EDS, there was no significant difference between the CpG-free formulation and the CpG-containing formulation, but numerically, the case of containing 5 μg of CpG per dose was better than the CpG-free case. When added in an amount of 5 μg per dose, CpG can obtain a stimulating effect on the antibody responses of IB M41, ND, and EDS. The effect of 2.5 μg of CpG per dose has limitations.

[0087]

[0085] When added in an amount of 5 μg per dose, CpG can obtain a stimulating effect on the antibody responses of IB M41, ND, and EDS. The effect of 2.5 μg of CpG per dose has limitations.

[0088] Example 4. Efficacy Test

[0086] A mixed vaccine was prepared that contained inactivated IB QX, inactivated IB D1466, and inactivated TRT antigen in different amounts per dose in a CpG 10 μg-containing W / O emulsion. These emulsions were used to vaccinate 14-week-old specific pathogen-free (SPF) laying hens. Half of each different vaccine group had been vaccinated with a live IB vaccine at 10 weeks of age. Toxic QX-like IB virus or toxic IB D1466 virus was antigen-administered to 26-week-old chickens. The productivity of chicken eggs was measured from 2 weeks before to 4 weeks after antigen administration. In addition, the antibody titer was measured by a serum neutralization test 7 weeks after vaccination.

[0089]

[0087] As seen in the data shown in Table 4, a clear antibody response could be detected by ELISA after the first antigen stimulation with live bacteria. The antibody titer against IB was also calculated by the SN test after vaccination with the inactivated vaccine. In the SN test, antibodies against IB QX and IB D1466 were specifically detected. No cross-reaction was observed at all, but antibodies against IB QX and IB D1466 could not be distinguished using ELISA.

[0090]

[0088] Also, as shown by the data in Table 7, after antigen administration, a significant decrease in the production of chicken eggs due to the harmful IB QX antigen-administered virus becomes extremely clear in non-vaccinated chickens. When vaccinated with only the live vaccine, only the inactivated vaccine, or both, protective immunity results against the decrease in egg production caused by the IB QX antigen-administered virus.

[0091]

Table 7

[0092]

[0089] Furthermore, unlike non-QX strains of IB, the inactivated IB QX antigen was previously thought to be almost impossible to induce antibody titers in chickens after vaccination. For example, currently marketed products containing antigens from other non-QX strains of infectious bronchitis (e.g., IB M41, IB D274, or IB D1466) are adjuvanted with CpG-free oil emulsions and further induce sufficient protection. In contrast, as shown in Table 8, CpG-free oil emulsions are insufficient to induce neutralizing antibodies, but the antibody response is strengthened by adding CpG. Previously, it was thought that it was almost impossible to induce antibody titers in chickens after vaccination with the inactivated IB QX antigen, which is different from non-QX strains of IB. For example, currently marketed products containing antigens from other non-QX strains of infectious bronchitis (e.g., IB M41, IB D274, or IB D1466) are adjuvanted with CpG-free oil emulsions and further induce sufficient protection. In contrast, as shown in Table 8, CpG-free oil emulsions are insufficient to induce neutralizing antibodies, but the antibody response is strengthened by adding CpG.

[0093]

Table 8

[0094]

[0090] At the same time, as described above, it has been proven that known vaccine strains of IB virus are insufficient to protect against infectious bronchitis caused by IB-QX virus and IB-QX-like viruses. See WO2010017440. Surprisingly, the vaccine of the present invention induced a distinct antibody response against the inactivated IB QX antigen.

[0095] Example 5. Vaccines against Infectious Bronchitis, Newcastle Disease, TRT, Infectious Bursal Disease, and Reovirus

[0091] In this experiment, newly hatched specific pathogen-free (SPF) Leghorn chickens were used in a mixed male and female group. The chickens were freely given standard feed and water.

[0096]

[0092] On day 0, these birds were inoculated by eye drop with a vaccine containing the Massachusetts 1 strain They were seeded and vaccinated against infectious bronchitis. On the 28th day after the experiment, POULVAC® REO and POULVAC® TRT vaccines were administered to the wings and eyes of these birds at the recommended dosages respectively. On the 49th day after the experiment, the experimental vaccine was administered to these birds. Groups T01, T02, T04, and T05 had 32 birds per group, and groups T03, T06, and T07 had 13 birds per group.

[0097]

Table 9

[0098]

[0093] All antigens used in T01, T02, T04, and T05 were inactivated in formaldehyde.

[0099]

[0094] Groups T01 - T05 received 0.5 ml intramuscular injection in the breast area. Groups T06 and T07 were treated according to the manufacturer's protocol.

[0100]

[0095] On the 70th and 77th days, blood was collected from these birds for serological analysis. Analyses were performed by serum neutralization tests (IB, IBD, Reo), HAI tests (Newcastle), and ELISA (TRT). Serological data for T01, T02, T04, and T05 were analyzed using a general linear mixed model with repeated measures. Appropriate logarithmic transformations were applied. Fixed effects brought about by treatment (time point, and treatment for each time point interaction); as well as random effects (animal terms) brought about by block, animals within the block, and treatment were included in the model.

[0101]

[0096] (Inverse-transformed) least squares means and 90% confidence intervals were reported along with the raw data ranges. When the main effect of treatment (i.e., treatment for each time interaction) was significant (P ≤ 0.10), comparisons between all treatment groups at each time point were performed and reported. Treatment groups T03, T06, and T07 were summarized with respect to geometric mean, standard error, and range.

[0102]

[0097] The results are shown in Table 10.

[0103]

Table 10

[0104]

[0098] These results demonstrate that adding CpG to the W / O emulsion nearly tripled the immune response to IBDV. Looking at the results from a different perspective, these results show a decrease in the IBD Lukert dose. 10 8 TCID 50 ~10 7.5 TCID 50 (Approximately a 1 / 3 decrease), and as a result of adding CpG to the formulation, the response to IBD was enhanced (when comparing T01 and T05). Similarly, the responses to Newcastle, reovirus, and TRT were also enhanced, and the reaction to IB was not statistically reduced.

[0105]

[0099] Furthermore, protective titers were generated by the responses to all viruses (the protective titer for IB was 20, for Newcastle was 16, for IBD was 32, and for reovirus was 16). The efficiency of the TRT vaccine is measured based on the seroconversion rate. When the seroconversion rate exceeded 70%, it indicated that the vaccine was effective.

[0106]

[0100] In the above experiment, both CpG-containing formulations (groups T01 and T02) were effective against TRT (seroconversion rate exceeded 90%). In contrast, CpG-deficient formulations (groups T04 and T05) were not effective against TRT (seroconversion rate was 62.5% or less).

[0107]

[0101] Group T06 was used as a positive control for IBDV. As shown in Table 10, the titer induced by vaccine T02 was equivalent to the titer induced by the positive control. Group T07 was used as a positive control for TRT. As shown in Table 10, experimental vaccines T01 and T02 induced higher TRT titers than T07.

[0108]

[0102] That is, by adding CpG to the W / O emulsion, it became possible to produce a pentavalent vaccine effective against bronchitis, Newcastle disease, TRT, infectious bursal disease (IBD or Gumboro), and reovirus. The formulation without CpG was not effective against TRT. In addition, the titers against Newcastle, reovirus, and IBD were lower for the CpG-free formulation than for the CpG-containing formulation.

[0109] Example 6. Vaccine against Infectious Bronchitis, Coryza, Egg Production Decline Syndrome, Newcastle Disease, and TRT

[0103] In this experiment, newly hatched specific pathogen-free (SPF) Leghorn chickens were used in a male-female mix. The chickens were given free access to standard feed and water.

[0110]

[0104] On day 0, these birds were inoculated with a Massachusetts 1 strain-containing vaccine by eye drop to inoculate a vaccine against infectious bronchitis. On day 14 after the experiment, the POULVAC® TRT vaccine was administered by intraocular injection at the recommended dose for each. On day 35 after the experiment, the experimental vaccine was administered by intramuscular injection.

[0111]

[0105] Table 11 shows the experimental vaccine compositions and control vaccine compositions used in these experiments. Each group included 56 birds.

[0112] [Table 11]

[0113]

[0106] All of the antigens used in T01, T02, T04, and T05 were inactivated in formaldehyde.

[0114]

[0107] For serological analysis, blood was collected from these birds on days 56 and 70. A 10% two-sided significance level was used for the analysis.

[0115]

[0108] Serological data for T01, T03, T04, T05, and T06 were analyzed using a general linear mixed model with repeated measures. An appropriate logarithmic transformation was applied. Fixed effects resulting from treatment (time point and treatment for each time point interaction); as well as random effects resulting from block, animals within the block, and treatment (animal terms) were included in the model.

[0116]

[0109] (Inverse-transformed) Least-squares means and 90% confidence intervals were reported along with the range of the raw data. When the main effect of treatment (i.e., treatment for each time interaction) was significant (P ≤ 0.10), comparisons between all treatment groups at each time point were performed and reported. T02 was summarized serologically in terms of geometric mean, standard error, and range.

[0117]

[0110] Table 12 summarizes the results of the experimental groups (T03 - T06).

[0118] [Table 12]

[0119]

[0111] These results demonstrate that the addition of CpG to the W / O emulsion increased the immune response against TRT measured by ELISA by 2- to 3-fold. Looking at the results from the perspective of the seroconversion rate, in the groups treated with the formulations without CpG (therapeutic agents T03 and T05), an efficient TRT seroconversion response rate was not demonstrated (57% or less). In contrast, in the groups treated with formulations T04 and T06 (both containing CpG), an efficient seroconversion rate (75% or more) was not demonstrated. Even if the dose of the TRT antigen was reduced by about 1 / 3-fold, the addition of CpG could compensate for the reduction in the antigen dose (compared with T04 and T05).

[0120]

[0112] Furthermore, protective titers were generated by the response against all viruses (the protective titer against IB was 20, the protective titer against Newcastle was 16, the protective titer against Coryza was 5, and the protective titer against EDS was 18). The SN titers or HAI titers induced by formulations T04 and T06 (containing CpG) were generally about 2-fold those induced by formulations T03 and T05 (without CpG).

[0121]

[0113] Non-limitingly, the present invention includes the following aspects [Aspect 1] An immunogenic composition comprising an antigen component and an adjuvant component, a) the antigen component includes at least one TRT antigen and at least one IB antigen, and the at least one IB antigen includes an IB QX antigen; b) the adjuvant component includes an immunostimulatory oligonucleotide and an oil emulsion, the immunogenic composition. [Aspect 2] The immunogenic composition according to Aspect 1, wherein the oil emulsion is a W / O emulsion. [Aspect 3] The immunogenic composition according to Aspect 1 or 2, further comprising a sterol. [Aspect 4] The immunogenic composition according to aspect 3, wherein the sterol is cholesterol. [Aspect 5] The immunogenic composition according to aspect 3 or 4, which is essentially free of saponin. [Aspect 6] The immunogenic composition according to any one of aspects 3 to 5, wherein the sterol is mixed with the immunostimulatory oligonucleotide. [Aspect 7] The immunogenic composition according to any one of aspects 1 to 6, which does not contain liposomes. [Aspect 8] The immunogenic composition according to any one of aspects 1 to 7, wherein the at least one IB antigen further comprises the IB D1466 antigen. [Aspect 9] The immunogenic composition according to any one of aspects 1 to 8, wherein the antigen component further comprises at least one of the IB D1466 antigen, the TRT antigen, the Newcastle disease antigen, the EDS antigen, and the IB M41 antigen. [Aspect 10] The immunogenic composition according to any one of aspects 1 to 8, wherein the antigen component further comprises at least one of the IB M41 antigen, the IB D274 antigen, the Newcastle disease LaSota strain antigen, the EDS antigen, and the TRT antigen. [Aspect 11] A method for preventing TRT in poultry animals, the method comprising administering the immunogenic composition according to aspects 1 to 10 to the poultry animals. [Aspect 12] A method for preventing IB in poultry animals, the method comprising administering the immunogenic composition according to aspects 1 to 10 to the poultry animals. [Aspect 13] The method according to aspect 11 or 12, wherein the poultry animal is a chicken. [Aspect 14] The method according to any one of aspects 11 to 13, which comprises administering a primer vaccine before administering the immunogenic composition according to aspects 1 to 10. [Aspect 15] The method according to aspect 14, wherein the primer vaccine comprises the live infectious bronchitis virus strain H120 Massachusetts type. [Aspect 16] A method for preventing IB in poultry animals, the method comprising administering a single dose of the immunogenic composition according to aspects 1 to 10 to the poultry animals. [Aspect 17] A vaccine comprising an antigen component and an adjuvant component, wherein the adjuvant component comprises an immunostimulatory oligonucleotide and an oil emulsion, and the antigen component comprises an IBD antigen. [Aspect 18] The vaccine according to aspect 17, wherein the IBD antigen is an inactivated Lukert strain antigen. [Aspect 19] The IBD antigen is 10 7.5 ~10 8 TCID 50 The vaccine according to aspect 17 or 18, which is present in an amount of. [Aspect 20] The antigen component a) an antigen derived from a non-Lukert strain of IBD; b) an infectious bronchitis antigen; c) a reovirus antigen; d) a Newcastle disease antigen; e) a turkey rhinotracheitis antigen The vaccine according to any one of aspects 18 to 19, further comprising at least one of. [Aspect 21] The infectious bronchitis antigen comprises an inactivated infectious bronchitis virus, the reovirus antigen comprises an inactivated reovirus, the antigen derived from a non-Lukert strain of IBD comprises an inactivated IBD virus of the non-Lukert strain, the Newcastle disease antigen comprises an inactivated Newcastle virus, and the turkey rhinotracheitis antigen comprises an inactivated turkey rhinotracheitis virus. The vaccine according to aspect 20. [Aspect 22] A vaccine comprising an antigen component and an adjuvant component, wherein the adjuvant component comprises an immunostimulatory oligonucleotide and an oil emulsion, and the antigen component comprises a) a TRT antigen; b) a Newcastle antigen; c) an egg drop syndrome (EDS) antigen; d) an IBK antigen; e) a colibacillosis antigen and the vaccine. [Aspect 23] a) The TRT antigen comprises an inactivated turkey rhinotracheitis virus; b) The Newcastle antigen comprises an inactivated Newcastle virus; c) The EDS antigen comprises an inactivated EDS virus; d) The IBK antigen comprises an inactivated EDS virus; e) The colibacillosis antigen comprises a mixture of a colibacillosis M antigen, a colibacillosis 221 antigen, and a colibacillosis S antigen, the vaccine according to aspect 22. [Aspect 24] The TRT is present in an amount of 10 6.00 TCID 50 ~10 6.50 TCID 50 per dose, the vaccine according to aspect 22 or 23. [Aspect 25] The oil emulsion is a W / O emulsion, the vaccine according to any one of aspects 17 to 24. [Aspect 26] The oil emulsion contains a mineral oil, the vaccine according to any one of aspects 17 to 25. [Aspect 27] The immunostimulatory oligonucleotide contains SEQ ID NO: 8, the vaccine according to any one of aspects 17 to 26. [Aspect 28] The immunostimulatory oligonucleotide is present in an amount of 2.5 to 20 μg per dose, the vaccine according to any one of aspects 17 to 27. [Aspect 29] The vaccine according to any one of aspects 17 to 28, wherein the adjuvant consists essentially of the immunostimulatory oligonucleotide, the oil, and one or more emulsifiers. All publications (both patent publications and non-patent publications) cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. All publications (both patent publications and non-patent publications) cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0122]

[0114] Although the invention has been described with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the invention. Accordingly, it should be understood that many modifications can be made to the exemplary embodiments and other arrangements can be devised without departing from the spirit and scope of the invention as defined by the following claims.

Claims

1. An immunogenic composition comprising an antigen component and an adjuvant component, a) said antigenic components comprise at least one turkey rhinotracheitis (TRT) antigen and at least one infectious bronchitis (IB) antigen, said at least one IB antigen comprising an IB QX inactivated virus; b) the adjuvant component consists essentially of a CpG-containing immunostimulatory oligonucleotide and a water-in-oil emulsion comprising mineral oil and optionally one or more emulsifiers, wherein the CpG-containing immunostimulatory oligonucleotide is present in an amount of 5-15 μg per dose, wherein at least one nucleotide in the CpG-containing immunostimulatory oligonucleotide is iodine-modified, and wherein the immunostimulatory oligonucleotide comprises a 5' TLR activation domain and at least two palindrome regions, wherein one of the palindrome regions is a 5' palindrome region at least 6 nucleotides in length linked, directly or via a spacer, to a 3' palindrome region at least 8 nucleotides in length; The immunogenic composition.

2. 2. The immunogenic composition of claim 1, wherein the water-in-oil emulsion comprises fatty acid esters of fatty acid substituted sorbitan surfactants and polyethoxylated sorbitol surfactants.

3. 3. The immunogenic composition of claim 1 or 2, wherein the water-in-oil emulsion comprises polyoxyethylene (20) sorbitan monooleate and sorbitan sesquioleate.

4. The immunogenic composition of any one of claims 1 to 3, wherein the immunostimulatory oligonucleotide comprises a phosphorothioate bond.

5. The immunogenic composition of any one of claims 1 to 4, wherein the immunostimulatory oligonucleotide is present in an amount of 8 to 12 μg per dose.

6. The immunogenic composition of any one of claims 1 to 5, wherein the immunostimulatory oligonucleotide comprises SEQ ID NO:

8.

7. The immunogenic composition of any one of claims 1 to 6, which does not contain liposomes.

8. The immunogenic composition of any one of claims 1 to 7, wherein the at least one IB antigen further comprises the IB D1466 antigen.

9. The immunogenic composition according to any one of claims 1 to 8, wherein the antigen component further comprises at least one of an IB D1466 antigen, a TRT antigen, a Newcastle disease antigen, an EDS antigen and an IBM M41 antigen.

10. The immunogenic composition according to any one of claims 1 to 8, wherein the antigen component further comprises at least one of IBM M41 antigen, IB D274 antigen, Newcastle disease LaSota strain antigen, EDS antigen, and TRT antigen.

11. A method for preventing TRT in a poultry animal, comprising administering to said poultry animal an immunogenic composition according to claims 1 to 10.

12. A method for preventing IB in a poultry animal, comprising administering to said poultry animal an immunogenic composition according to claims 1-10.

13. The method of claim 11 or 12, wherein the poultry animal is a chicken.

14. The method according to any one of claims 11 to 13, comprising administering a primer vaccine prior to administering the immunogenic composition according to claims 1 to 10.

15. 15. The method of claim 14, wherein the primer vaccine comprises live infectious bronchitis virus strain H120 Massachusetts.

16. A method for preventing IB in a poultry animal, comprising administering to said poultry animal a single dose of the immunogenic composition of claims 1-10.

17. 1. A vaccine comprising an antigen component and an adjuvant component, said adjuvant component consisting essentially of a CpG-containing immunostimulatory oligonucleotide and a water-in-oil emulsion, said water-in-oil emulsion comprising mineral oil and optionally one or more emulsifiers, and said antigen component comprising an infectious bursal disease (IBD) antigen, wherein said IBD antigen is 10 ７．０ ~10 ７．５ T.C.I.D. ５０ and wherein The vaccine, wherein at least one nucleotide in the CpG-containing immunostimulatory oligonucleotide is iodine-modified, the CpG-containing immunostimulatory oligonucleotide comprises a 5' TLR activation domain and at least two palindrome regions, one of the palindrome regions being a 5' palindrome region at least 6 nucleotides in length linked, either directly or via a spacer, to a 3' palindrome region at least 8 nucleotides in length, and present in an amount of 2.5-20 μg per dose.

18. 18. The vaccine of claim 17, wherein the IBD antigen is an inactivated Lukert strain antigen.

19. The IBD antigen is 10 ７．２ T.C.I.D. ５０ 19. The vaccine of claim 17 or 18, wherein the vaccine is present in an amount of

20. The antigen component is a) antigens derived from non-Lukert strains of IBD; b) infectious bronchitis antigen; c) Reovirus antigens; d) Newcastle disease antigen; e) Turkey rhinotracheitis antigen 20. The vaccine of claim 18 or 19, further comprising at least one of:

21. 21. The vaccine of claim 20, wherein the infectious bronchitis antigen comprises an inactivated infectious bronchitis virus, the reovirus antigen comprises an inactivated reovirus, the antigen derived from a non-Lukert strain of IBD comprises an inactivated non-Lukert strain of IBD virus, the Newcastle disease antigen comprises an inactivated Newcastle virus, and the turkey rhinotracheitis antigen comprises an inactivated turkey rhinotracheitis virus.

22. 1. A vaccine comprising an antigen component and an adjuvant component, said adjuvant component consisting essentially of a CpG-containing immunostimulatory oligonucleotide and a water-in-oil emulsion, said water-in-oil emulsion comprising mineral oil and optionally one or more emulsifiers, said antigen component a) a TRT antigen comprising an inactivated turkey rhinotracheitis virus; b) a Newcastle antigen comprising an inactivated Newcastle virus; c) an Egg Drop Syndrome (EDS) antigen, comprising an inactivated EDS virus; d) an inactivated infectious bronchitis virus (IBK) antigen; e) Coryza antigens, including a mixture of Coryza M antigen, Coryza 221 antigen, and Coryza S antigen; wherein at least one nucleotide in the CpG-containing immunostimulatory oligonucleotide is iodine modified, the CpG-containing immunostimulatory oligonucleotide comprises a 5' TLR activation domain and at least two palindromic regions, one of the palindromic regions being a 5' palindromic region at least 6 nucleotides in length linked, directly or via a spacer, to a 3' palindromic region at least 8 nucleotides in length, and present in an amount of 2.5-20 μg per dose. The vaccine.

23. 23. The vaccine of claim 22, wherein the CpG-containing immunostimulatory oligonucleotide is present in an amount of 10 μg per dose.

24. The TRT is 10 per dose ６．００ T.C.I.D. ５０ ~10 ６．５０ T.C.I.D. ５０ 24. The vaccine of claim 22 or 23, wherein the vaccine is present in an amount of

25. 25. The vaccine of any one of claims 17 to 24, wherein the water-in-oil emulsion comprises fatty acid esters of fatty acid substituted sorbitan surfactants and polyethoxylated sorbitol surfactants.

26. 26. The vaccine of any one of claims 17 to 25, wherein the water-in-oil emulsion comprises polyoxyethylene (20) sorbitan monooleate and sorbitan sesquioleate.

27. The vaccine of any one of claims 17 to 22, 24 to 26, wherein the CpG-containing immunostimulatory oligonucleotide is present in an amount of 5 to 15 μg per dose.

28. The vaccine of any one of claims 17 to 22, 24 to 26, wherein the CpG-containing immunostimulatory oligonucleotide is present in an amount of 8 to 12 μg per dose.

29. The vaccine of any one of claims 17 to 28, wherein the adjuvant consists of the CpG-containing immunostimulatory oligonucleotide, the oil, and one or more emulsifying agents.

30. The vaccine of any one of claims 17-29, wherein the CpG-containing immunostimulatory oligonucleotide contains one or more phosphorothioate linkages.

31. The vaccine of any one of claims 17 to 29, wherein the CpG-containing immunostimulatory oligonucleotide comprises SEQ ID NO:8.

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