Vaccine against infectious bronchitis

Abstract

A poultry vaccine against infectious bronchitis and turkey rhinotracheitis is provided. The vaccine is adjuvanted with an oil emulsion containing immunostimulatory oligonucleotides. Methods of using the vaccine are also provided. [Selection diagram] None

Description

[Background technology]

[0001]

[0001] Turkey rhinotracheitis (TRT) is an upper respiratory tract infection of turkeys and chickens caused by a pneumovirus and is a highly contagious acute disease affecting turkeys of all ages. Clinical signs of TRT infection include prominent and often foamy nasal discharge, crunching, clicking, sneezing, and head shaking. Ocular discharge or swollen infraorbital sinuses may also be observed in infected turkeys. Antibodies to the TRT virus (TRTV) have been detected in some flocks (both pullets and pullets / breeder) affected by swollen head syndrome (SHS). TRTV has been hypothesized to play a role in the pathogenesis of SHS and the associated respiratory distress.

[0002]

[0002] Infectious bronchitis (IB) is a coronavirus that causes disease only in chickens, but may also cause subclinical infection in some other avian species. While some serotypes are geographically restricted, many serotypes commonly cocirculate within one geographic region. In recent years, a novel IBV genotype, the QX strain, has become increasingly widespread in Asia and Europe. Morbidity is generally close to 100%. Coughing, sneezing, and bronchoconstriction in chicks may persist for 10-14 days. Conjunctivitis and dyspnea may be present, sometimes with facial swelling, especially concomitant bacterial infection of the sinuses. Chicks may appear lethargic and hunched under lamp heaters. Feed consumption and weight gain are reduced. Infection with nephropathogenic strains produces early respiratory signs followed later by lethargy, ruffled feathers, wet droppings, increased water intake, and death. In laying hens, egg production may decrease by as much as 70%, with malformed eggs that are often small with thin, soft, coarse and / or pale shells, and watery albumen. In most cases, egg production and quality return to normal, but this may take up to 8 weeks. Mortality is 5% in most outbreaks, but can increase if bacterial infections complicate the disease. In young chickens, nephropathogenic strains can induce interstitial nephritis with high mortality (up to 60%). Infection in young chickens can cause permanent damage to the oviduct and result in laying or breeding hens not achieving standard production levels.

[0003]

[0003] Known vaccine strains of IB viruses have proven insufficient to protect against infectious bronchitis caused by IB-QX and IB-QX-like viruses, see WO2010017440.

[0004]

[0004] Infectious bursal disease (IBD, also known as Gumboro disease) is a highly contagious, immunosuppressive disease of chickens worldwide, with serious economic impacts on egg and meat production. IBD vaccines are an important part of gumboro control strategies. The choice of vaccine to be administered depends on the breed of chicken being vaccinated and the prevailing challenge conditions.

[0005]

[0005] Generally, inactivated antigens have been used in poultry vaccines, but the production of inactivated viruses is relatively costly, so effective vaccines with lower antigen levels are desirable.

[0006]

[0006] Another need in poultry vaccination is closely related to the cost of vaccination itself. It is economically advantageous to create multivalent vaccines designed to prevent multiple diseases. Such multivalent vaccines reduce the cost of vaccine administration, but simply mixing antigens in the same formulation is often an ineffective approach to creating multivalent vaccines due to the well-known phenomenon of antigen interference.

[0007]

[0007] Thus, 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 invention provides an immunogenic composition comprising an antigen component comprising at least one TRT antigen and at least one IB antigen, and an immunologically effective amount of an adjuvant component comprising an immunostimulatory oligonucleotide, an oil emulsion, and optionally, a sterol.

[0009]

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

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

[0010]

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

[0011]

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

[0012]

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

[0013]

[0014] In one embodiment, the IBD antigen is 7.5 ~10 8 TCID 50 The antigen may be present in an amount of 0.1 to 100 mg / mL.

[0014]

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

[0015]

[0016] In another aspect, the invention provides a vaccine comprising an antigen component and an effective amount of an adjuvant component, the adjuvant component comprising an immunostimulatory oligonucleotide and an oil emulsion, the antigen component comprising a TRT antigen, a Newcastle antigen, an Egg Drop Syndrome (EDS) antigen, an IBK antigen, and a Coryza antigen.

[0016]

[0017] In some 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 one embodiment, the TRT antigen is administered at a dose of 10 6.00 TCID 50 ~10 6.50 TCID 50 is present in an amount of

[0018]

[0019] In one embodiment, the oil emulsion is a W / O emulsion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019]

[0020] Definition:

[0021] The terms "about" or "approximately," when used in connection with a measurable, numerical variable, refer to the indicated variable value and all values ​​of the variable that are within experimental error (e.g., within a 95% confidence interval of the mean) of the indicated value or within 10% of the indicated value, whichever is greater, except when "about" is used in reference to time intervals of several weeks, where "about 3 weeks" is 17-25 days and about 2 to about 4 weeks is 10-40 days.

[0020]

[0022] The term "consisting essentially of" and the like as applied to the adjuvant formulations of the invention refers to a composition that does not contain an additional adjuvanting or immunomodulating agent in an amount such that the formulation exerts a measurable adjuvanting or immunomodulating effect.

[0021]

[0023] The terms "essentially saponin-free," "substantially saponin-free," and the like, refer to a composition that does not contain saponin in an amount such that saponin exerts a measurable adjuvanting or immunomodulatory effect. In some embodiments, an essentially saponin-free composition contains an amount of saponin that is insufficient to induce a systemic immune response, such as fever. In some embodiments, an essentially saponin-free composition contains zero or below the limit of detection of saponin.

[0022]

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

[0025] The term "parenteral administration" refers to the introduction of a substance, such as a vaccine, into a subject's body by or through a route that does not involve the digestive tract. Parenteral administration includes subcutaneous, intramuscular, transdermal, intradermal, intraperitoneal, intraocular, and intravenous administration.

[0023]

[0026] Percent purity or "X percent purity" as applied to an immunostimulatory oligonucleotide formulation refers to a population of oligonucleotide molecules that contains X% of the designated oligonucleotide (e.g., SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, etc.), and the remainder (i.e., 100% minus X%) comprises short fragments of the designated oligonucleotide that are present as impurities during manufacture of the designated sequence. Thus, if the sequence is manufactured by 3' to 5' sequencing, the 5' truncated version will include the remainder. As a non-limiting example, 100 μg of an 80% pure preparation of SEQ ID NO:8 will contain SEQ ID NO:8 (80 μg), and the remaining 20 μg will be short 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 antigen, adjuvant or vaccine sufficient to elicit an immune response in a subject receiving the antigen, adjuvant or vaccine sufficient to prevent or reduce signs or symptoms of disease, including adverse health effects or complications caused by infection with a pathogen such as a virus or bacteria. Humoral or cellular immunity, or both humoral and cellular immunity, may be elicited. The immunogenicity and efficacy of a vaccine in an animal can be assessed indirectly, for example, through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring of signs and symptoms after challenge with a wild-type strain. Protective immunity conferred via a vaccine can be assessed, for example, by measuring reduction in clinical signs such as mortality, morbidity, temperature values, general physical condition, and the general health and performance of the subject. A therapeutically effective amount of a vaccine may vary depending on the particular adjuvant used, the particular antigen used, or the condition of the subject, or may be determined by one of skill in the art.

[0025]

[0028] Commercial layer and breeding hens are vaccinated during their lifetime with a wide variety of vaccines. These vaccines are primarily live attenuated vaccines. By the time chickens are ready to start laying, they are vaccinated with inactivated combination vaccines to boost their immunity against infectious agents they have already been vaccinated against and to induce immunity against other agents that may cause egg production or other damage during the rearing period. Long duration of immunity is important because these vaccines can induce high antibody titers and because the vaccine must provide protection for the entire rearing period, which may be from approximately 20 to 70 weeks. 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] Thus, generally, the invention provides immunogenic compositions comprising an antigen component and an adjuvant component, where the antigen component comprises at least one TRT antigen and at least one IB antigen, and the adjuvant component comprises (or in some embodiments consists essentially of, or in other embodiments consists of) an immunostimulatory oligonucleotide, an oil emulsion, and optionally, a sterol.

[0027] Antigenic components

[0030] Different IB QX antigens are suitable for the present invention. In some embodiments, the IB QX antigen is a fully inactivated virus. In other embodiments, the virus is a modified live virus. In yet other embodiments, subunit vaccines may be used. For example, proteins present on the surface of the virus that may be suitable include, but are not limited to, the S protein, the M protein, the E protein, or any combination thereof. In embodiments involving the use of an inactivated whole virus, the antigen is administered at 10 3 ~10 10 Infectious units, e.g. 10 4 , 10 5 , 10 6 , 10 7 It may be used in an amount of infectious units. In one embodiment, the amount of IB QX inactivated virus is about 10 per dose. 5 ~about 10 8 It is an infectious unit.

[0028]

[0031] The antigen component in a further embodiment comprises an IBD antigen, which in one embodiment is an inactivated Lukert IBD virus.

[0029]

[0032] In one embodiment, the amount of inactivated Lukert IBD virus is 10 per dose. 7 ~10 8 TCID 50 (e.g., 10 per dose 7.1 , 107.2 , 10 7.3 , 10 7.4 , 10 7.5 , 10 7.6 , 10 7.7 , 10 7.8 , 10 7.9 TCID 50 ).

[0030]

[0033] In some 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 invention can 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 another aspect, the present invention provides a multivalent vaccine comprising 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 one embodiment of the invention, the TRT antigen is administered in an amount of 10 6.00 TCID 50 ~10 6.50 TCID 50 (For example, 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 ) in the presence of

[0033]

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

[0034]

[0037] Each coryza is caused by a different strain of Haemophilus paragallinarum. Thus, in some 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, strain Spross (Coryza S) may be used as a Serovar B stain, and Stord Modesto (Coryza M) may be used as a Serovar C strain.

[0035]

[0038] Those skilled in the art may realize that depending on the methodology of virus titration, the titer of the virus may vary, sometimes by as much as about 30%. In the present disclosure, if the dose is measured as an index of 10, the index may vary by 0.2. Thus, for example, a titer of 10 6.40 TCID 50 There are 10 6.20 TCID 50 ~10 6.60 TCID 50 The same concept applies to a range of potencies. For example, a potency of 10 6.00 TCID 50 ~10 6.50 TCID 50 There are 10 5.80 TCID 50 ~10 6.70TCID 50 The range is encompassed.

[0036]

[0039] In other embodiments, other antigens may be used in addition to those mentioned above, for example, Salmonella enteritidis, Salmonella typhimurium, Mycoplasma gallisepticum, Salmonella gallinarum, Pasteurella multocida. As with inactivating viruses, bacteria can be inactivated by exposure to an effective amount of an inactivating chemical selected from, for example, formalin, beta-propiolactone (BPL), binary ethyleneimine (BEI), or phenol.

[0037] Adjuvant components

[0040] Generally, the adjuvant components used in the immunogenic compositions of the invention comprise an immunostimulatory oligonucleotide, an oil, and, optionally, a surfactant(s). In some embodiments, the adjuvant components are free or essentially free of saponins and / or ISCOMs.

[0038]

[0041] In some embodiments, the adjuvant components consist essentially of an immunostimulatory oligonucleotide, an oil, and optionally, a surfactant(s).In some embodiments, the adjuvant components consist 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 its derivatives (poly I:C) may be used.

[0040]

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

[0041]

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

[0042]

[0045] In one embodiment of the invention, the immunostimulatory oligonucleotide comprises a 5' TLR activation domain and at least two palindromic regions, one of which is a 5' palindromic region at least 6 nucleotides in length linked, either directly or via a spacer, to a 3' palindromic 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 an iodo-modified nucleotide. E modification refers to an ethyl-modified 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 propynyl-dU, inosine modification, attachment of 2-bromovinyl (preferably to uridine).

[0044]

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

[0045]

[0048] P class oligonucleotides and modified P class oligonucleotides are further disclosed in published PCT application 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, "*" indicates a phosphorothioate linkage and "-" indicates a phosphodiester linkage): In SEQ ID NOS: 11-14, all linkages are phosphodiester linkages.

[0046] Allocation number 1 5'T*CG*T*CG*A*CG*A*T*CG*G*C*G*CG*C*G*C*C*G 3' Allocation number 2 5'T*CG*A*C*G*T*C*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' Array number 3 5'T*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*G*C*C*G*T 3' Allocation number 4 5'JU*CG*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' Allocation number 5 5'JU*CG*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3' Array number 6 5'JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*G*C*T 3' Allocation number 7 5'EU*CG*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' Allocation number 8 5'JU*CG*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T 3' Array number 9 5'JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*G*C*T 3' Allocation number 10 5'T*CG*T*CG*A*CG*A*T*CG*G*C*G*C_G*C*G*C*C*G 3' Allocation number 11 5'-UUGUUGUUGUUGUUGUUGUU-3' Allocation number 12 5'-UUAUUAUUAUUAUUAUUAUU-3' Allocation number 13 5'-AAACGCUCAGCCAAAGCAG-3' Arrangement number 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 greater (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 P class immunostimulatory oligonucleotide used in the adjuvant composition will depend on the nature of the P class immunostimulatory oligonucleotide used and the intended species.

[0048]

[0051] Sterols share a common chemical core. This chemical core is a steroid ring structure(s) with a hydroxyl (OH) group usually attached to carbon-3. The hydrocarbon chain of the fatty acid substituent is variable in length (usually 16-20 carbon atoms) and can be saturated or unsaturated. Sterols usually contain one or more double bonds in the ring structure and also contain a variety of substituents attached to the ring. Sterols and their fatty acid esters are essentially water insoluble. Thus, in view of their chemical similarity, sterols sharing this chemical core are likely to 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 sterols commercially. For example, cholesterol is disclosed in the Merck Index, 12th Ed., p. 369. Suitable sterols include, but are not limited to, β-sitosterol, stigmasterol, ergosterol, ergocalciferol, cholesterol and derivatives thereof, such as DC-cholesterol (3β-[N-(dimethylaminoethane)carbamoyl]cholesterol).

[0049]

[0052] A number of oils and their combinations 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. Non-limiting examples of animal oils are squalane. Non-limiting examples of suitable non-metabolizable oils include light mineral oils, linear or branched saturated oils, branched oils, and the like.

[0050]

[0053] In one series of embodiments, the oil used in the adjuvant formulation of the present invention is a light mineral oil. As used herein, the term "mineral oil" refers to a mixture of liquid hydrocarbons obtained by distillation techniques from petrolatum. The term is synonymous with "liquid paraffin", "liquid petrolatum" and "white mineral oil". The term is also intended to include "light mineral oil" (i.e., oils similarly obtained by distillation of petrolatum, but with 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 oils are available from a variety of commercial sources, such as JT Baker (Phillipsburg, Pa.), 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 comprises mineral oil MARCOL™ 52. MARCO™ 52 is a refined mixture of liquid saturated hydrocarbons, a clear, water-white product free of toxic impurities, obtained from petroleum via vacuum distillation with subsequent purification steps, including final purification by catalytic hydrogenation.

[0051]

[0054] Suitable emulsifiers for use in the emulsion include biologically compatible natural emulsifiers and non-natural synthetic surfactants. Biologically compatible emulsifiers include phospholipid compounds or mixtures of phospholipids. A preferred phospholipid is 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 and isolating and drying the resulting hydrated gum. A purified product can be obtained by fractionating the mixture of acetone-insoluble phospholipids and glycolipids remaining after removing the triglycerides and vegetable oil by acetone washing. Alternatively, lecithin can be obtained from a variety of 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 may be conventionally synthesized.

[0052]

[0055] In a further embodiment, the emulsifier used herein does not include lecithin or uses a non-immunologically effective amount of lecithin.

[0053]

[0056] Suitable non-natural synthetic emulsifiers for use in the adjuvant formulations of the invention include sorbitan-based non-ionic surfactants, such as fatty acid substituted sorbitan surfactants (commercially available under the names SPAN® or ARLACEL®), polyethoxylated fatty acid esters of 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 ether (TRITON® N), polyoxyethylene isooctylphenyl ether (TRITON® X). Preferred synthetic surfactants are those available under the names SPAN® and TWEEN®, such as TWEEN® 80 (polyoxyethylene (20) sorbitan monooleate) and ARLACEL® 83V (sorbitan sesquioleate).

[0054]

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

[0055]

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

[0056]

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

[0057]

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

[0058]

[0061] In practice it is possible to make water-in-oil emulsions where the oil phase is less than 50% v / v.

[0059]

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

[0060]

[0063] The antigen solution is then added to the adjuvant formulation.

[0064] It is possible to maintain the integrity of a water-in-oil emulsion as long as the dispersed spherical aqueous droplets are not present 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. When we say that the total volume fraction occupied by the aqueous droplets should not exceed 0.64 (i.e.: 64% v / v), this inversely implies that the oily phase should not drop below 36% v / v.

[0061]

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

[0062]

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

[0063] b) The immunostimulatory oligonucleotide and polyoxyethylene (20) sorbitan monooleate are dissolved in the aqueous phase, thereby forming an aqueous solution. c) This aqueous solution is added to the oil solution with continuous homogenization.

[0064]

[0067] The immunogenic compositions of the present invention can be prepared by adding the antigen component to the aqueous phase, which is then combined with the oil phase. In other embodiments, the antigen component may be added to the adjuvant component after the adjuvant component is prepared.

[0065]

[0068] The immunogenic composition may further comprise a pharma- ceutically acceptable carrier. As used herein, "pharma- ceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial agents, antifungal agents, isotonic agents, adsorption retardants, and the like. The carrier(s) must be "acceptable" in the sense of being compatible with the other components of the composition and not harmful to the subject. Carriers are typically sterile and pyrogen-free and are selected based on the mode of administration to be used. As known to those skilled in the art, preferred formulations of pharma- ceutical acceptable carriers that comprise the composition include pharmaceutical carriers approved by applicable regulations promulgated by the United States (US) Department of Agriculture, the US Food and Drug Administration (FDA), or equivalent government agencies of countries other than the United States. Thus, pharma- ceutical acceptable carriers for commercial production of the composition are carriers that have already been approved or will be approved by the appropriate government agencies in the United States or foreign countries.

[0066]

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

[0067] Vaccine administration

[0070] Vaccines of the present invention generally can be administered via a number of routes, which are known to those of skill in the art and include, but are not limited to, intramuscular and subcutaneous injection.

[0068]

[0071] In one embodiment, the vaccine is administered about 3-7 weeks (e.g., about 4-6 weeks) prior to expected laying date. This regimen ensures that immunity to IB QX is developed by the time vaccinated hens lay and persists throughout the laying season.

[0069]

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

[0070]

[0073] The invention is further described in the following non-limiting examples. EXAMPLES

[0071] Example 1. Vaccine Preparation

[0074] Table 1 shows an exemplary water-in-oil emulsion. The antigen is diluted in the aqueous phase to which CpG is also 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. Potency Assay of IB or TRT

[0075] We used inactivated IB M41 antigen (10 before inactivation per dose) 7.2 EID 50 ) or inactivated TRT antigen (10 5.3 TCID 50 ) were prepared. The CpG content in these emulsions was either different or zero (SEQ ID NO: 8, 65% purity). These emulsions were tested in efficacy studies in chickens. Chickens were vaccinated at 4 weeks of age. Five weeks after vaccination, blood samples were taken to test antibody titers against the antigen by ELISA.

[0074]

[0076] Antibody titers against Turkey Rhinotracheitis (TRT) virus were measured using an enzyme-linked immunosorbent assay (ELISA) in which the antigen was coated onto the wells of a 96-well micro ELISA plate. After coating, standard negative and positive sera were added to the wells and sera to be tested. All sera were tested in duplicate. As a control, all sera were also tested in duplicate in wells that did not contain antigen. Antibody reactivity against the antigen was visualized by removing unbound antibody and adding antibody against serum antibody conjugated with peroxidase. After removing unbound conjugated antibody, the peroxidase substrate orthophenylenediamino+H2O2 was added. The presence of peroxidase was demonstrated by the appearance of a color reaction.

[0075]

[0077] Table 2 summarizes the efficacy study results of the TRT antigen. In general, antibody titers were adequate, and there were no non-responders in either group. There was a positive effect of CpG on antibody titers. The best results were obtained with 10 μg CpG per dose, but 1 μg CpG per dose also had a positive effect.

[0076] [Table 2]

[0077]

[0078] We also identified a positive effect of CpG on the antibody response to IB M41 (see Table 3). A dose of 1 μg CpG per dose was already sufficient to substantially stimulate the antibody response. Without CpG, 7 out of 15 chickens had an antibody response below the threshold of 2.60, whereas in the test with 1 μg CpG per dose, all chickens responded. A further increase was obtained with 10 μg CpG per dose.

[0078] [Table 3]

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

[0079] Groups of 10 4-week-old specific pathogen-free (SPF) chickens were vaccinated intramuscularly with a dose of 0.5 ml per dose. Five weeks after vaccination, blood samples were collected for serological testing. ELISA (Idexx FlockCheck IBV antibody kit; Idexx, Maine, USA), hemagglutination inhibition test, and ELISA (Idexx FlockCheck NCD antibody kit; Idexx, Maine, USA) were used to measure antibody titers against IB, ND, and EDS, respectively.

[0080]

[0080] The vaccine is IB M41: 10 6.9 EID 50 / dose, Newcastle Disease (ND): 10 8,1 EID 50 / dose, Egg Drop Syndrome (EDS): 256 HAu / dose in a water-in-oil emulsion containing different amounts of CpG.

[0081]

[0081] Groups of 10 4-week-old specific pathogen-free (SPF) chickens were vaccinated intramuscularly with a dose of 0.5 ml per dose. Five weeks after vaccination, blood samples were collected for serological testing. ELISA (Idexx FlockCheck IBV antibody kit; Idexx, Maine, USA), hemagglutination inhibition test, and ELISA (Idexx FlockCheck NCD antibody kit; Idexx, Maine, USA) were used to measure antibody titers against IB, ND, and EDS, respectively.

[0082]

[0082] Tables 4 to 6 show the IB, ND and EDS results, 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 formulations without CpG and those with 5 μg CpG per dose. The ND with 2.5 μg CpG per dose also improved antibody titers significantly. For EDS, there was no significant difference between the formulations without CpG and those with CpG, but the 5 μg CpG per dose was numerically better than the formulation without CpG.

[0087]

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

[0088] Example 4. Efficacy Test

[0086] Combination vaccines were made containing different amounts of inactivated IB QX, inactivated IB D1466, and inactivated TRT antigens in a W / O emulsion containing 10 μg CpG per dose. These emulsions were used to vaccinate 14-week-old specific pathogen-free (SPF) laying hens. Half of the different vaccine groups had been vaccinated with a live IB vaccine at 10 weeks of age. Chickens were challenged at 26 weeks of age with either virulent QX-like IB virus or virulent IB D1466 virus. Egg production was measured 2 weeks before and 4 weeks after challenge. In addition, antibody titers were measured in a seroneutralization test 7 weeks after vaccination.

[0089]

[0087] As can be seen from the data presented in Table 4, after live priming a clear antibody response could be detected by ELISA. After vaccination with the inactivated vaccine the antibody titers against IB were also calculated by the SN test, which specifically detects antibodies against IB QX and IB D1466. Although no cross-reactivity was observed, the antibodies against IB QX and IB D1466 could not be distinguished using ELISA.

[0090]

[0088] Additionally, after challenge, egg production reduction by the harmful IB QX challenge virus is quite evident in unvaccinated chickens, as shown by the data in Table 7. Vaccination with live vaccine alone, inactivated vaccine alone, or both results in protective immunity against egg production reduction caused by the IB QX challenge virus.

[0091] [Table 7]

[0092]

[0089] Moreover, inactivated IB QX antigens, unlike non-QX strains of IB, were previously thought to be nearly incapable of eliciting antibody titers in chickens following 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 still elicit sufficient protection. In contrast, as shown in Table 8, CpG-free oil emulsions are insufficient to elicit neutralizing antibodies, but the antibody response is robust when CpG is added.

[0093] [Table 8]

[0094]

[0090] At the same time, as mentioned above, known vaccine strains of IB viruses have proven insufficient to protect against infectious bronchitis caused by IB-QX and IB-QX-like viruses, see WO2010017440. Surprisingly, the vaccine of the present invention induced a clear antibody response against the inactivated IB QX antigen.

[0095] Example 5. Infectious bronchitis, Newcastle disease, TRT, infectious bursal disease, and Vaccine against reovirus

[0091] Newly hatched specific pathogen free (SPF) Leghorn breeder chickens of mixed sex were used in this study. Chickens were fed a standard diet and water ad libitum.

[0096]

[0092] On day 0, the birds were vaccinated against infectious bronchitis by eye drop with the vaccine containing the Massachusetts 1 strain. On day 28 after the experiment, the birds were vaccinated with the recommended doses of POULVAC® REO and POULVAC® TRT vaccines in the wings and eyes, respectively. On day 49 after the experiment, the birds were vaccinated with the experimental vaccines. 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 to T05 received 0.5 ml intramuscular injections in the mammary region. Groups T06 and T07 were treated according to the manufacturer's protocol.

[0100]

[0095] Blood was taken from the birds on days 70 and 77 for serological analysis. Analysis was performed by serum neutralization tests (IB, IBD, Reo), HAI tests (Newcastle) and ELISA (TRT). A general linear mixed model with repeated measures was used to analyze the serological data for T01, T02, T04 and T05. Appropriate logarithmic transformations were applied. Fixed effects of treatment (time point and treatment by time point interaction); as well as random effects of block, animal within block and treatment (animal term) were included in the model.

[0101]

[0096] Least squares means (back transformed) and 90% confidence intervals were reported along with ranges for the raw data. Comparisons between all treatment groups at each time point were performed and reported when the main effect of treatment (i.e., treatment by time interaction) was significant (P < 0.10). Treatment groups T03, T06, and T07 were summarized in terms of geometric means, standard errors, and ranges.

[0102]

[0097] The results are shown in Table 10.

[0103] [Table 10]

[0104]

[0098] These results demonstrate that the addition of CpG to the W / O emulsion increased the immune response to IBDV by approximately three-fold. From a different perspective, these results indicate a reduction in the IBD Lukert dose. 8 TCID 50 ~10 7.5 TCID 50 (approximately 1 / 3 reduction), and the addition of CpG to the formulation resulted in an enhanced response to IBD (compared to T01 and T05), as well as enhanced responses to Newcastle, Reovirus, and TRT, with no statistically reduced response to IB.

[0105]

[0099] Furthermore, responses against all viruses produced protective titers (protective titer for IB: 20, Newcastle: 16, IBD: 32, Reovirus: 16). Efficacy of the TRT vaccine is measured by seroconversion rate. A seroconversion rate of more than 70% indicated the vaccine was effective.

[0106]

[0100] In the above experiments, both CpG-containing formulations (T01 and T02 groups) were effective against TRT (seroconversion rates were greater than 90%). In contrast, the CpG-depleted formulations (T04 and T05 groups) were not effective against TRT (seroconversion rates were less than 62.5%).

[0107]

[0101] Group T06 was used as a positive control for IBDV. As shown in Table 10, the titers induced by vaccine T02 were comparable to those 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] Thus, the addition of CpG to a W / O emulsion allowed the generation of a pentavalent vaccine with efficacy against bronchitis, Newcastle disease, TRT, infectious bursal disease (IBD or gumboro), and reovirus. The formulation without CpG was ineffective against TRT. In addition, the titers against Newcastle, reovirus, and IBD were lower for the formulation without CpG than for the formulation with CpG.

[0109] Example 6. Vaccines against infectious bronchitis, coryza, egg drop syndrome, Newcastle disease, and TRT

[0103] Newly hatched specific pathogen free (SPF) Leghorn breeder chickens of mixed sex were used in this study. Chickens were fed a standard diet and water ad libitum.

[0110]

[0104] On day 0, the birds were vaccinated against infectious bronchitis by eye drop with a vaccine containing the Massachusetts 1 strain. On day 14, the POULVAC® TRT vaccine was administered intraocularly at the respective recommended doses. On day 35, the experimental vaccines were administered by intramuscular injection.

[0111]

[0105] The experimental and control vaccine compositions used in these studies are shown in Table 11. Each group contained 56 birds.

[0112] [Table 11]

[0113]

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

[0114]

[0107] Blood was collected from the birds for serological analysis 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 analysed using a general linear mixed model with repeated measures. Appropriate logarithmic transformations were applied. Fixed effects of treatment (time point and treatment by time point interaction); as well as random effects of block, animal within block and treatment (animal terminology) were included in the model.

[0116]

[0109] Least squares means (back transformed) and 90% confidence intervals were reported along with ranges for the raw data. Where the main effect of treatment (i.e. treatment by time interaction) was significant (P < 0.10), comparisons between all treatment groups at each time point were performed and reported. T02 was summarised serologically in terms of geometric means, standard errors and ranges.

[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 emulsions increased the immune response to TRT measured by ELISA by 2-3 times. Looking at the results in terms of seroconversion rates, the groups treated with the formulations without CpG (therapeutic agents T03 and T05) did not demonstrate an efficient TRT seroconversion response rate (below 57%). In contrast, the groups treated with the formulations T04 and T06 (both containing CpG) did not demonstrate an efficient seroconversion rate (above 75%). Even with a reduction in the TRT antigen dose of approximately 1 / 3 (compared to 1 for T04 and T05), the addition of CpG compensates for the reduced antigen dose.

[0120]

[0112] Furthermore, responses against all viruses produced protective titers (protective titer against IB: 20, against Newcastle: 16, against Coryza: 5, against EDS: 18). The SN or HAI titers induced by formulations T04 and T06 (containing CpG) were usually approximately twice as high as those induced by formulations T03 and T05 (without CpG).

[0121]

[0113] Without being limited thereto, the present invention includes the following aspects: [Aspect 1] An immunogenic composition comprising an antigen component and an adjuvant component, a) the antigen component comprises at least one TRT antigen and at least one IB antigen, the at least one IB antigen comprising an IB QX antigen; b) the immunogenic composition, wherein the adjuvant component comprises an immunostimulatory oligonucleotide and an oil emulsion. [Aspect 2] 2. The immunogenic composition according to embodiment 1, wherein the oil emulsion is a W / O emulsion. [Aspect 3] 3. The immunogenic composition of embodiment 1 or 2, further comprising a sterol. [Aspect 4] The immunogenic composition of embodiment 3, wherein the sterol is cholesterol. [Aspect 5] 5. The immunogenic composition according to embodiment 3 or 4, which is essentially free of saponin. [Aspect 6] The immunogenic composition of any one of aspects 3 to 5, wherein the sterol is mixed with the immunostimulatory oligonucleotide. [Aspect 7] 7. The immunogenic composition according to any one of aspects 1 to 6, which does not comprise liposomes. [Aspect 8] The immunogenic composition according to any one of the preceding aspects, wherein the at least one IB antigen further comprises the IB D1466 antigen. [Aspect 9] 9. The immunogenic composition according to any one of the preceding aspects, wherein the antigen component further comprises at least one of the following antigens: IB D1466 antigen, TRT antigen, Newcastle Disease antigen, EDS antigen and IB M41 antigen. [Aspect 10] 9. The immunogenic composition according to any one of the preceding aspects, wherein the antigen component further comprises at least one of an IB M41 antigen, an IB D274 antigen, a Newcastle disease LaSota strain antigen, an EDS antigen, and a TRT antigen. [Aspect 11] 11. A method for preventing TRT in a poultry animal, comprising administering to said poultry animal an immunogenic composition according to any one of aspects 1 to 10. [Aspect 12] A method for preventing IB in a poultry animal, the method comprising administering to said poultry animal an immunogenic composition according to any one of aspects 1 to 10. [Aspect 13] 13. The method according to embodiment 11 or 12, wherein the poultry animal is a chicken. [Aspect 14] A method according to any one of aspects 11 to 13, comprising administering a primer vaccine prior to administration of the immunogenic composition according to aspects 1 to 10. [Aspect 15] 15. The method of embodiment 14, wherein the primer vaccine comprises live infectious bronchitis virus strain H120 Massachusetts. [Aspect 16] A method for preventing IB in a poultry animal, comprising administering to said poultry animal a single dose of the immunogenic composition according to aspects 1-10. [Aspect 17] A vaccine comprising an antigen component and an adjuvant component, the adjuvant component comprising an immunostimulatory oligonucleotide and an oil emulsion, and the antigen component comprising an IBD antigen. [Aspect 18] 18. The vaccine of embodiment 17, wherein the IBD antigen is an inactivated Lukert strain antigen. [Aspect 19] The IBD antigen is 7.5 ~10 8 TCID 50 19. The vaccine according to embodiment 17 or 18, wherein the vaccine is present in an amount of [Aspect 20] The antigen component is a) antigens derived from non-Lukert strains of IBD; b) infectious bronchitis antigen; c) reovirus antigen; d) Newcastle disease antigen; e) Turkey rhinotracheitis antigen 20. The vaccine of any one of aspects 18 to 19, further comprising at least one of the following: [Aspect 21] 21. The vaccine of embodiment 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 a non-Lukert strain of inactivated IBD virus, the Newcastle disease antigen comprises an inactivated Newcastle virus, and the turkey rhinotracheitis antigen comprises an inactivated turkey rhinotracheitis virus. [Aspect 22] A vaccine comprising an antigen component and an adjuvant component, the adjuvant component comprising an immunostimulatory oligonucleotide and an oil emulsion, the antigen component comprising a) TRT antigen; b) with Newcastle antigen; c) Egg Drop Syndrome (EDS) antigens; d) IBK antigen; e) Coryza antigen and The vaccine comprising: [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) A vaccine according to aspect 22, wherein said Coryza antigens comprise a mixture of Coryza M antigen, Coryza 221 antigen and Coryza S antigen. [Aspect 24] The TRT is 10 6.00 TCID 50 ~10 6.50 TCID 50 24. The vaccine according to embodiment 22 or 23, wherein the vaccine is present in an amount of [Aspect 25] 25. The vaccine according to any one of aspects 17 to 24, wherein the oil emulsion is a W / O emulsion. [Aspect 26] 26. The vaccine of any one of aspects 17 to 25, wherein the oil emulsion comprises mineral oil. [Aspect 27] 27. The vaccine of any one of aspects 17 to 26, wherein the immunostimulatory oligonucleotide comprises SEQ ID NO:8. [Aspect 28] 28. The vaccine of any one of aspects 17 to 27, wherein the immunostimulatory oligonucleotide is present in an amount of 2.5 to 20 μg per dose. [Aspect 29] 29. The vaccine of any one of aspects 17-28, wherein the adjuvant consists essentially of the immunostimulatory oligonucleotide, the oil, and one or more emulsifying agents. All publications (both patent and non-patent publications) cited in this specification are herein fully incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. All publications (both patent and non-patent publications) cited in this specification are herein fully incorporated by reference 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 particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that many modifications can be made to the illustrative 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 for intramuscular administration 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 an oil emulsion comprising an oil and optionally one or more emulsifiers, wherein the CpG-containing immunostimulatory oligonucleotide is present in an amount of 1-20 μg per dose; The immunogenic composition.

2. The immunogenic composition of claim 1 , wherein the oil emulsion is a W / O emulsion.

3. The immunogenic composition of claim 1 or 2, wherein the immunostimulatory oligonucleotide comprises SEQ ID NO:

8.

4. The immunogenic composition of claim 3, wherein the immunostimulatory oligonucleotide is present in an amount of 2.5 to 20 μg per dose.

5. The immunogenic composition of claim 3 or 4, wherein the immunostimulatory oligonucleotide is present in an amount of 5 to 15 μg per dose.

6. The immunogenic composition of any one of claims 3 to 5, wherein the immunostimulatory oligonucleotide is present in an amount of 8 to 12 μg per dose.

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 intramuscularly 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 intramuscularly administering to said poultry animal an immunogenic composition according to claims 1 to 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 a single dose of the immunogenic composition of claims 1 to 10 intramuscularly to said poultry animal.

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