INFECTIOUS BURSA DISEASE (IBD) VACCINE

Abstract

Vaccines for poultry against infectious bronchitis and turkey rhinotracheitis are provided. The vaccines are adjuvanted with an oil emulsion containing an immunostimulatory oligonucleotide. Methods for using the vaccines are also provided.

Description

[001] Turkey Rhinotracheitis (TRT) is an upper respiratory tract infection of turkeys and chickens caused by pneumovirus. It is a highly contagious acute disease affecting turkeys of all ages. Clinical symptoms of TRT infection include pronounced, often frothy, nasal discharge, rales, dry noises, sneezing, and head shaking. Eye discharge or swelling of the infraorbital sinuses may also be observed in infected turkeys. Antibodies against the TRT virus (TRTV) have been detected in some flocks of chickens (both broilers and broilers / breeders) suffering from Swollen Head Syndrome (SHS). It is postulated that TRTV plays a role in the etiology of SHS and related respiratory problems.

[002] Infectious bronchitis (IB) is a coronavirus that causes disease only in chickens, although some other birds may be subclinically infected. Some serotypes are geographically restricted, but several serotypes commonly co-circulate within a geographic region. In recent years, a new IBV genotype, the QX strain, has become increasingly common in Asia and Europe. Morbidity is commonly close to 100%. Chicks may cough, sneeze, and exhibit tracheal rales for 10 to 14 days. Conjunctivitis and dyspnea may be observed, and sometimes facial swelling, particularly with concomitant bacterial infection of the sinuses. Chicks may appear depressed and huddle under heat lamps. Feed intake and weight gain are reduced. Infection with nephropathogenic strains may cause initial respiratory signs, followed by depression, ruffled feathers, wet droppings, increased water intake, and death.In layer systems, egg production can drop by up to 70%, and the eggs are often deformed, with thin, soft, rough and / or pale shells, and may be smaller and have watery albumen. In most cases, egg production and egg quality are affected. Petition 870260072051, dated 07 / 20 / 2026, page 6 / 49 2 / 34 return to normal, but this can take up to 8 weeks. In most outbreaks, mortality is 5%, although mortality rates are higher when the disease is complicated by concurrent bacterial infection. Nephropathogenic strains can induce interstitial nephritis with high mortality (up to 60%) in young chicks. Infection of young chicks can cause permanent damage to the oviduct, resulting in layers or breeders that never reach normal production levels.

[003] It is known that known vaccine strains of IB virus are insufficient to protect against infectious bronchitis caused by IB-QX and IB-QX-like viruses. See document WO2010017440.

[004] Infectious bursal disease (IBD, Gumboro) is a highly contagious immunosuppressive disease of chickens that is found worldwide and causes a major economic impact on egg and meat production. IBD vaccines form an important part of a Gumboro control strategy. The choice of vaccine to be administered depends on the type of chicken being vaccinated and the prevailing challenge situation.

[005] Inactivated antigens have generally been used in poultry vaccines. However, the manufacture of inactivated viruses is relatively expensive and therefore effective vaccines containing lower levels of the antigen would be desirable.

[006] Another need in the vaccination of domestic birds is closely related to the cost of the vaccination itself. It is economically advantageous to create a multivalent vaccine designed to prevent multiple diseases. These multivalent vaccines reduce the cost of vaccine administration. However, due to a well-known phenomenon of antigen interference, simply mixing antigens in the same dosage forms is often not an effective approach to creating multivalent vaccines. Petition 870260072051, dated 07 / 20 / 2026, page 7 / 49 3 / 34

[007] Therefore, there is a need for vaccines for domestic birds with reduced amounts of the antigen and / or multivalent vaccines. SUMMARY OF THE INVENTION

[008] The present invention provides in one aspect an immunogenic composition comprising an antigen component and an immunologically effective amount of 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, oil emulsion and, optionally, a sterol.

[009] In certain modalities, at least one TRT antigen is the K strain of TRT.

[010] In certain modalities, at least one IB antigen is at least one of the IB D1466 and IB QX antigens.

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

[012] In certain embodiments, the optionally present sterol is mixed with the immunostimulatory oligonucleotide.

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

[014] In certain embodiments, the aforementioned IBD antigen is an inactivated Lukert strain antigen, which may be present in the quantity of 107'5a 108TCID50.

[015] 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; an antigen Petition 870260072051, dated 07 / 20 / 2026, page 8 / 49 4 / 34 of Newcastle disease; a Peruvian rhinotracheitis antigen.

[016] In another aspect, the invention provides a vaccine comprising an antigen component and an effective amount of the adjuvant component, wherein the adjuvant component comprises an immunostimulatory oligonucleotide and an oil emulsion, and wherein the antigen component comprises: a TRT antigen; a Newcastle antigen; a Low Egg Lactation Syndrome (EDS) antigen; an IBK antigen; and a Coryza antigen.

[017] In certain embodiments, the TRT antigen comprises an inactivated Peruvian Rhinotracheitis virus, the Newcastle antigen comprises an inactivated Newcastle virus, the EDS antigen comprises the inactivated EDS virus, the IBK antigen comprises the inactivated IBK virus, and the Coryza antigen comprises a mixture of Coryza M, Coryza 221 and Coryza S bacterins.

[018] In certain modalities, the TRT antigen is present in the amount of 1Q6'00TCID50 to 106'50TCID50 per dose.

[019] In certain embodiments, the said oil emulsion is an O / W emulsion. DETAILED DESCRIPTION Definitions: [Q2Q]The terms 'about' or 'approximately', when used in connection with a measurable numerical variable, refer to the stated value of the variable and to all values ​​of the variable that are within the experimental error of the stated value (e.g., within the 95% confidence interval for the mean) or within 10% of the stated value, whichever is greater, unless 'about' is used in reference to time intervals in weeks where “about 3 weeks” is 17 to 25 days and about 2 to 4 weeks is 10 to 40 days. [Q21]The term consisting essentially of and similar as applied to the adjuvant formulations of the present invention refers to compositions that do not Petition 870260072051, dated 07 / 20 / 2026, p. 9 / 49 5 / 34 contain additional adjuvant or immunomodulatory agents in amounts in which said agent exerts measurable adjuvant or immunomodulatory effects.

[022] The terms essentially saponin-free, saponin-free and the like refer to a composition that does not contain saponin in amounts at which saponin exerts measurable adjuvant or immunomodulatory effects. In certain embodiments, essentially saponin-free compositions contain saponin in an amount insufficient to cause a systemic immune response, such as fever. In certain embodiments, essentially saponin-free compositions do not contain saponin or contain saponin at or below the detection limit.

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

[024] The term parenteral administration refers to the introduction of a substance, such as a vaccine, into an individual's body through or by means of a route other than the digestive tract. Parenteral administration includes subcutaneous, intramuscular, transcutaneous, intradermal, intraperitoneal, intraocular, and intravenous administration.

[025] The purity percentage or “X percent pure”, when applied in the preparation of immunostimulatory oligonucleotides, refers to a population of oligonucleotide molecules comprising 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 shorter fragments of the named oligonucleotide present as impurities during the manufacture of the named sequence. Thus, if the sequence is manufactured by 3'-5' sequencing, the 5' truncations would comprise the remainder. As a non-limiting example, a 100μg preparation of 80% pure SEQ ID NO:8 comprises 80μg of SEQ ID NO: 8 and the remaining 20μg are shorter fragments of SEQ ID NO: 8 present in the preparation. Petition 870260072051, dated 07 / 20 / 2026, page 10 / 49 6 / 34

[026] The terms “therapeutically effective amount”, “immunologically effective amount” and “effective amount” refer to an amount of an antigen or adjuvant or vaccine that would induce an immune response in an individual receiving the antigen or adjuvant or vaccine that is adequate to prevent or reduce signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with a pathogen, such as a virus or bacterium. Humoral immunity or cell-mediated immunity or humoral and cell-mediated immunity may be induced. The immunogenicity and efficacy of a vaccine in an animal may be assessed, for example, indirectly by measuring antibody titers, lymphocyte proliferation assays or directly by monitoring signs and symptoms after challenge with the wild-type strain.The protective immunity conferred by a vaccine can be assessed by measuring, for example, the reduction in clinical signs such as mortality, morbidity, temperature, overall physical condition, and general health and performance of the individual. The amount of a vaccine that is therapeutically effective can vary depending on the particular adjuvant used, the particular antigen used, or the individual's condition, and can be determined by someone skilled in the art.

[027] During their lives, commercial laying hens and breeder chickens are vaccinated with a wide variety of different vaccines. These vaccines are primarily live attenuated vaccines. When the hens are ready to start laying, they are vaccinated with inactivated combination vaccines to boost immunity against infectious agents against which they have already received a vaccine and to induce immunity against other agents that may cause drops in egg production or other damage during the laying period. It is important that these vaccines are able to induce high antibody titers and that the duration of immunity is long, because the vaccines must protect Petition 870260072051, dated 07 / 20 / 2026, page 11 / 49 7 / 34 throughout the entire laying period, which ranges from approximately 20 weeks to 70 weeks of age. In order to achieve long-lasting immunity, it is necessary to formulate the antigens in an adjuvant, for example, a water-in-oil (W / O) emulsion.

[028] Thus, in general, the 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 (or, in some embodiments, essentially consists of, or in other embodiments, consists of) an immunostimulatory oligonucleotide, oil emulsion and, optionally, a sterol. Antigen component

[029] Different IB QX antigens are suitable for the present invention. In certain embodiments, the IB QX antigen is a complete inactivated virus. In other embodiments, the virus is a modified live virus. In still other embodiments, subunit vaccines may be used. For example, proteins present on the surface of the virus may be suitable, including, without limitation, protein S, protein E, protein M, or any combination thereof. In embodiments involving the use of the complete inactivated virus, the antigen may be used in amounts of 10³ to 10¹⁰ infectious units per dose, for example, 10⁴, 10⁵, 10⁶, 10⁷ infectious units per dose. In certain embodiments, the amount of inactivated IB QX virus per dose is between about 10⁵ and about 10⁸ infectious units per dose.

[030] In other embodiments, the antigen component comprises an IBD antigen, which, in certain embodiments, is an inactivated IBD Lukert virus.

[031] In certain modalities, the amount of IBD Lukert virus Petition 870260072051, dated 07 / 20 / 2026, p. 12 / 49 8 / 34 inactivated is between 107 and 108 TCID50 per dose, for example, 107'1, 107'2, 107.3, 107.4, 107'5, 107'6, 107'7, 107'8, 107.9 TCID50 per dose.

[032] In certain embodiments, in addition to the inactivated IBD Lukert virus, the vaccine antigen component comprises other antigens. For example, different strains of infectious bronchitis virus may be used, for example, IB M41 and / or IB D1466 and / or IB D274. Alternatively or additionally, the vaccine of the present invention may also comprise TRT, Newcastle disease (e.g., LaSota strain), EDS (egg laying syndrome), reovirus and infectious bursal disease virus antigens, avian influenza.

[033] In other respects, the invention provides a multivalent vaccine comprising a TRT antigen; a Newcastle antigen; a low egg laying syndrome (EDS) antigen; an IBK (infectious bronchitis virus) antigen; and a coryza antigen.

[034] In certain embodiments of the invention, the TRT antigen is present in the amount of 106'00TCID50 to 106'50TCID50 per dose, for example, 106'00TCID50 or 106'10TCID50 or 106'20TCID50 or 106'30TCID50 or 106·40TCID50 or 106'50TCID50.

[035] The viruses used in the vaccines of this invention can be attenuated or inactivated. Methods for inactivating and attenuating viruses are well known in the art. For example, the virus can be inactivated by culture passage. Methods of inactivation include, without limitation, exposing the virus to an effective amount of an inactivating chemical selected from formalin, beta propiolactone (BPL), binary ethyleneimine (BEI), or phenol.

[036] Coryza is caused by different strains of Haemophilus paragallinarum. Thus, in certain embodiments, the Coryza antigen comprises one or more strains of Haemophilus paragallinarum, for example, strain M, strain Z, strain 221 and the like. In other embodiments, a mixture of strains representing serovars A, B and C is used. Thus, strain 221 (Coryza 221) can be used as a strain Petition 870260072051, dated 07 / 20 / 2026, page 13 / 49 9 / 34 For Serovar A, the Spross strain (Coryza S) can be used as a Serovar B strain, and the Modesto strain (Coryza M) can be used as a Serovar C strain.

[037] One skilled in the art may realize that the titer of a virus can vary depending on the virus titration methodology, sometimes by about 30%. In this description, where doses are measured as an exponent of 10, the exponent can vary by 0.2. Thus, for example, the titer of 106'40TCID50 can encompass values ​​between 106'20TCID50 and 106'60TCID50. The same idea applies to titer ranges. For example, the titer of 106'00TCID50-106'50TCID50 covers the range from 105·80TCID50 to 106.70TCID50.

[038] In other embodiments, other antigens besides those mentioned above may be used, for example, Salmonella enteritidis, Salmonella typhimurium, Mycoplasma gallisepticum, Salmonella gallinarum, Pasteurella multocida. Similar to virus inactivation, bacteria may also be inactivated, for example, by exposure to an effective amount of an inactivating chemical selected from formalin, beta propiolactone (BPL), binary ethyleneimine (BEI) or phenol. Adjuvant component

[039] In general, the adjuvant component used in the immunogenic composition of the invention comprises immunostimulatory oligonucleotide, oil and, optionally, surfactant(s). In certain embodiments, the adjuvant component is free or essentially free of saponins and / or ISCOMs.

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

[041] Suitable immunostimulatory oligonucleotides include ODN (DNA base) oligonucleotides, ORN (RNA base) oligonucleotides or structures of Petition 870260072051, dated 07 / 20 / 2026, p. 14 / 49 10 / 34 ODN-ORN chimeric compounds, which may have modified skeletons including, without limitation, phosphorothioate modifications, halogenations, alkylation (e.g., ethyl- or methyl- modifications) and phosphodiester modifications. In some embodiments, polyinosinic-cytidylic acid or a derivative thereof (poly I:C) may be used.

[042] CpG oligonucleotides are characterized by the presence of an unmethylated CG dinucleotide in specific base sequence contexts (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 here by reference). These CpG motifs are not seen in eukaryotic DNA, in which CG dinucleotides are suppressed and, when present, usually methylated, but are present in bacterial DNA to which they confer immunostimulatory properties.

[043] In selected embodiments, the adjuvants of the present invention use a class P immunostimulatory oligonucleotide, more preferably modified class P immunostimulatory oligonucleotides, even more preferably E-modified class P oligonucleotides. Class P immunostimulatory oligonucleotides are CpG oligonucleotides characterized by the presence of palindromes, generally 6-20 nucleotides in length. Class P oligonucleotides have the ability to spontaneously self-assemble into concatemers in vitro and / or in vivo. These oligonucleotides are, in a strict sense, single-stranded, but the presence of palindromes allows the formation of concatemers or possibly stem-and-loop structures.The total length of immunostimulatory oligonucleotides of class P is between 19 and 100 nucleotides, for example, 19-30 nucleotides, 30-40 nucleotides, 40-50 nucleotides, 50-60 nucleotides, 60-70 nucleotides, 70-80 nucleotides, 80-90 nucleotides, or 90-100 nucleotides.

[044] In one aspect of the invention, the immunostimulatory oligonucleotide Petition 870260072051, dated 07 / 20 / 2026, page 15 / 49 11 / 34 contains a 5' TLR activation domain and at least two palindromic regions, a palindromic region being a 5' palindromic region of at least 6 nucleotides in length and linked to a 3' palindromic region of at least 8 nucleotides in length, either directly or through a spacer.

[045] Immunostimulatory oligonucleotides of class P can be modified according to techniques known in the art. For example, the J modification refers to nucleotides modified with iodine. The E modification refers to nucleotide(s) modified with ethyl. Thus, E-modified immunostimulatory oligonucleotides of class P are immunostimulatory oligonucleotides of class P in which at least one nucleotide (preferably the 5' nucleotide) is ethylated. Additional modifications include 6-nitro-benzimidazole linkage, O-methylation, prionyl-dU modification, inosine modification, 2-bromovinyl linkage (preferably uridine).

[046] Class P immunostimulatory oligonucleotides may also contain a modified internucleotide linkage including, without limitation, phosphodiester linkages and phosphorothioate linkages. The oligonucleotides of the present invention may be synthesized or obtained from commercial sources.

[047] Class P oligonucleotides and modified class P oligonucleotides are further disclosed in published PCT application no. WO2008 / 068638, published on 12 June 2008. Suitable non-limiting examples of modified class P immunostimulatory oligonucleotides are provided below (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. SEQ ID NO: 15' T*CG*T*CG*A*CG*A*T*CG*G*C*G*CG*C*G*C*C*G 3' SEQ ID NO: 25' T*CG*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' Petition 870260072051, dated 07 / 20 / 2026, p. 16 / 49 12 / 34 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*CG*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*CG*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: 115'-UUGUUGUUGUUGUUGUUGUU-3' SEQ ID NO: 125'-UUAUUAUUAUUAUUAUUAUU-3' SEQ ID NO: 135'-AAACGCUCAGCCAAAGCAG-3' SEQ ID NO: 145’-dTdCdGdTdCdGdTdTdTdTrGrUrUrGrUrGrUdTdTdTdT-3'

[048] The immunostimulatory oligonucleotides of the present invention can be synthesized chemically. Furthermore, the immunostimulatory oligonucleotides can be used with about 60% purity (homogeneity) or higher (for example, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, 98%, or 100% purity).

[049] The amount of class P immunostimulatory oligonucleotide for use in adjuvant compositions depends on the nature of the class P immunostimulatory oligonucleotide used and the intended species.

[050] Sterols share a common chemical core, which is a steroid ring structure[s], having a hydroxyl group (OH), usually attached Petition 870260072051, dated 07 / 20 / 2026, page 17 / 49 13 / 34 to carbon-3. The hydrocarbon chain of the fatty acid substituent varies in length, generally from 16 to 20 carbon atoms, and may be saturated or unsaturated. Sterols commonly contain one or more double bonds in the ring structure and also a variety of substituents attached to the rings. Sterols and their fatty acid esters are essentially insoluble in water. In view of these chemical similarities, it is thus 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 can be commercially acquired. For example, cholesterol is disclosed in the Merck Index, 12th Ed., p. 369. Suitable sterols include, without limitation, β-sitosterol, stigmasterol, ergosterol, ergocalciferol, cholesterol and its derivatives, such as, for example, DC-Cholesterol (3β-[N-(Dimethylaminoethane)carbamoyl]cholesterol).

[051] Multiple oils and combinations thereof are suitable for use in the present invention. These oils include, without limitation, animal oils, vegetable oils, as well as non-metabolizable oils. Non-limiting examples of suitable vegetable oils in the present invention are corn oil, peanut oil, soybean oil, coconut oil, olive oil, and phytosqualane. A non-limiting example of animal oils is squalane. Suitable non-limiting examples of non-metabolizable oils include light mineral oil, saturated linear or branched chain oils, branched oils, and the like.

[052] In a number of embodiments, the oil used in the adjuvant formulations of the present invention is a light mineral oil. As used herein, the term mineral oil refers to a mixture of liquid hydrocarbons obtained from petrolatum through a distillation technique. The term is synonymous with liquefied paraffin, liquid petrolatum, and white mineral oil. The term is also intended to include light mineral oil, i.e., oil that is similarly obtained by distillation of Petition 870260072051, dated 07 / 20 / 2026, page 18 / 49 14 / 34 Vaseline, but which has a slightly lower specific gravity than white mineral oil. See, for example, Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990, pages 788 and 1323). Mineral oil can be obtained from various commercial sources, for example, JT Baker (Phillipsburg, Pennsylvania), USB Corporation (Cleveland, Ohio). The preferred mineral oil is the commercially available light mineral oil under the name DRAKEOL®. In another embodiment, the suitable oil comprises MARCOL™52 mineral oil. MARCOL™52 is a purified mixture of saturated liquid hydrocarbons. It is a crystalline, water-white product that contains no toxic impurities. It is obtained from petroleum by vacuum distillation with subsequent refining steps, including a final purification by catalytic hydrogenation.

[053] Suitable emulsifiers for use in the present emulsions include biologically compatible natural emulsifiers and non-natural synthetic surfactants. Biologically compatible emulsifiers include phospholipid compounds or a mixture of phospholipids. Preferred phospholipids are phosphatidylcholines (lecithin), such as soy or egg lecithin. Lecithin can be obtained as a mixture of phosphatides and triglycerides by washing crude vegetable oils with water and separating and drying the resulting hydrated gums. A refined product can be obtained by fractionating the mixture into acetone-insoluble phospholipids and glycolipids remaining after removal of the triglycerides and vegetable oil by washing with acetone. Alternatively, lecithin can be obtained from various commercial sources.Other suitable phospholipids include phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid, cardiolipin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidylethanolamine. Phospholipids can be isolated from natural sources or conventionally synthesized.

[054] In additional embodiments, the emulsifiers used herein do not include Petition 870260072051, dated 07 / 20 / 2026, page 19 / 49 15 / 34 lecithin or they use lecithin in an amount that is not immunologically effective.

[055] Synthetic non-natural emulsifiers suitable for use in the adjuvant formulations of the present invention include nonionic sorbitan-based surfactants, for example fatty acid-substituted sorbitan surfactants (commercially available under the names SPAN® or ARLACEL®), polyethoxylated sorbitol fatty acid esters (TWEEN®), polyethylene glycol fatty acid esters from sources such as castor oil (EMULFOR®); polyethoxylated fatty acid (for example, stearic acid available under the name SIMULSOL® M53), iso-octylphenol / formaldehyde polyethoxylated polymer (TYLOXAPOL®), polyoxyethylene fatty alcohol ethers (BRIJ®); polyoxyethylene nonphenyl ethers (TRITON® N), polyoxyethylene isooctylphenyl ethers (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).

[056] In general, the emulsifier(s) may 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%.

[057] In a subset of embodiments, the volumetric percentage of the oil and the oil-soluble emulsifier together is at least 50%, for example, 50% to 95% by volume; preferably, in an amount greater than 50% to 85%; more preferably, in an amount of 50% to 60%, and most preferably in an amount of 55-65% v / v of the vaccine composition. Thus, for example and without limitation, the oil may be present in an amount of 45% and the oil-soluble emulsifier would be present in an amount greater than 5% v / v. Thus, the volume percentage of the oil and the oil-soluble emulsifier together would be at least 50%. Petition 870260072051, dated 07 / 20 / 2026, page 20 / 49 16 / 34

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

[059] Sometimes it is impossible or impractical to concentrate the antigen, particularly in large-scale commercial applications, and low concentrations of antigen solutions have to be used. Thus, in some embodiments, the vaccine compositions of the present invention comprise the adjuvant formulations as described above, wherein the oil phase content in these adjuvant formulations is diluted and wherein the vaccine composition is a water-in-oil emulsion.

[060] In practice, it is possible to create a water-in-oil emulsion in which the oil phase is less than 50% v / v.

[061] In summary, first, the adjuvant formulation of the present invention is prepared as described above. In said adjuvant formulation, the oil phase comprises more than 50% v / v of the adjuvant formulation. The amounts of ingredients other than the oil and the emulsifier(s) are scaled respectively, based on the final target concentration and the desired dilution. For example, if it is intended to prepare a vaccine composition where the adjuvant formulation comprises 80% v / v, the amounts of ingredients other than the oil are scaled by a factor of 1.25 (1 / 0.8). The amounts of emulsifiers, if any (e.g., TWEEN®80 and / or SPAN®80), do not necessarily need to be scaled, but preferably the volume ratio between the oil and the emulsifier(s) is maintained in the same adjuvant formulation and in the final vaccine composition.

[062] The antigen solution is then added to the adjuvant formulation. Petition 870260072051, dated 07 / 20 / 2026, p. 21 / 49 17 / 34

[063] The integrity of the water-in-oil emulsion can be maintained as long as the dispersed spherical water droplets are not present in a form more concentrated than the maximum packing fraction for 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. Provided that the total volume fraction occupied by aqueous droplets does not exceed 0.64, i.e.: 64% v / v. On the other hand, this implies that the oil phase should not fall below 36% v / v.

[064] In some suitable embodiments, a dose of the adjuvant would contain between about 0.1 and about 20μg (e.g., 1-20μg or 5-15μg or 8-12μg or 10μg) of immunostimulatory oligonucleotide, up to about 50μg (e.g., 0.520μg or 1-10μg) of sterol, such as cholesterol.

[065] In certain forms, the adjuvant component is prepared as follows: a) Sorbitan sesquioleate and cholesterol, if present, are dissolved in light mineral oil. The resulting oil solution is sterilized by filtration; b) The immunostimulatory oligonucleotide and polyoxyethylene (20) sorbitan monooleate are dissolved in the water phase, thus forming the aqueous solution; c) The aqueous solution is added to the oily solution under continuous homogenization.

[066] The immunogenic composition of the present invention can be prepared by adding the antigen component to the water phase, followed by combining the water phase with the oil phase. In other embodiments, the antigen component can be added to the adjuvant component after the adjuvant component has been prepared.

[067] The immunogenic composition may further comprise a pharmaceutically acceptable vehicle. As used herein, a pharmaceutically acceptable vehicle Petition 870260072051, dated 07 / 20 / 2026, p. 22 / 49 18 / 34 acceptable includes any and all solvents, dispersing media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption retardants and the like. The vehicle(s) must be acceptable in the sense of being compatible with the other components of the compositions and not harmful to the individual. Typically, the vehicles will be sterile and pyrogen-free, and selected based on the route of administration to be used. It is well known to those skilled in the art that the preferred formulations for the pharmaceutically acceptable vehicle comprising the compositions are the pharmaceutical vehicles approved under the applicable regulations promulgated by the United States Department of Agriculture (US) or the US Food and Drug Administration, or equivalent governmental agency in a country outside the US.Therefore, a pharmaceutically acceptable vehicle for the commercial production of the compositions is a vehicle that is already approved or will be approved by the appropriate government agency in the U.S. or a foreign country.

[068] Other components of the compositions may include pharmaceutically acceptable excipients, such as vehicles, solvents and diluents, isotonic agents, buffering agents, stabilizers, preservatives, vasoconstrictors, antibacterial agents, antifungal agents and the like. Typical vehicles, solvents and diluents include water, saline solution, dextrose, ethanol, glycerol, oil and the like. Representative isotonic agents include sodium chloride, dextrose, mannitol, sorbitol, lactose and the like. Useful stabilizers include gelatin, albumin and the like. Vaccine Administration

[069] In general, the vaccines of the present invention can be administered via multiple routes. Such routes are known to those skilled in the art and include, without limitation, intramuscular and subcutaneous injections. Petition 870260072051, dated 07 / 20 / 2026, p. 23 / 49 19 / 34

[070] In certain modalities, vaccines are administered approximately 3-7 weeks (e.g., approximately 4-6 weeks) before expected laying. This regimen ensures that immunity to IB QX is developed at the time of laying and persists throughout the laying period of vaccinated hens.

[071] In further embodiments, the vaccine of the present invention is administered as a booster vaccine to chickens that have been initiated. Multiple IB primers are known in the art. For example, POULVAC® IB Primer comprises a lyophilized live Massachusetts-type Infectious Bronchitis virus. Nobilis® IB H120 is a live lyophilized vaccine indicated for use as primary vaccination of chickens against Infectious Bronchitis. This vaccine contains the Massachusetts-type H120 strain. Other IB primers may also be used with the vaccine of the present invention.

[072] The invention will be further described in the following non-limiting examples. EXAMPLES Example 1. Vaccine Preparation

[073] An exemplary water-in-oil emulsion is provided in Table 1. The antigens are diluted in the water phase to which CpG is also added. The water phase comprises antigens, CpG, and thimerosal. This is mixed with the oil phase. After thorough mixing, a stable W / O emulsion is formed. Table 1. Composition of an O / W emulsion Aqueous phase (40%): 80 mL Stock Components Target Quantity (mL) Tween 80 100% purity 0.004 mL / dose 1.6 Thimerosal 1% CpG (SEQ ID NO: 8, 65% purity) Saline Solution 100% 20000 purity μg / mL 0.005 10 N / D mL / dose μg / dose of add 2.0 0.0 + antigens N / D - 0.5 mL 76.4 Petition 870260072051, dated 07 / 20 / 2026, p. 24 / 49 20 / 34 Subtotal I 80.0 Oil Phase Ληη , (60%) 120 mL Components Stock Target Quantity (g) Arlacel™ 83V Marcol™ 52 100% purity 100% purity 0.239 g / dose 0.023 g / dose 95.6 9.2 Subtotal 104.8 Final Emulsion Components Stock Target, v / v Volume (mL) Aqueous phase (40%) Oil phase (60%) 100% purity 100% purity 40% 60% 80.0 120.0 Total volume 200.0 mL Example 2. Power Tests for IB or TRT

[074] The inventors produced O / W emulsions containing inactivated IB M41 antigen (107·2EID50 before dose inactivation) or inactivated TRT antigen (105·3TCID50 before dose inactivation). These emulsions contained varying amounts of CpG or no CpG (SEQ ID NO: 8, 6 5% purity). The emulsions were tested in a potency test in chickens. Chickens were vaccinated at 4 weeks of age. Five weeks after vaccination, blood samples were collected and tested for antibody titers against the antigens by ELISA.

[075] Antibody titers against Peruvian rhinotracheitis virus (TRT) were determined using an enzyme-linked immunosorbent assay (ELISA), in which the antigen was coated into wells of 96-well ELISA microplates. After coating, negative and positive standard sera were added to the wells and to the sera to be tested. All sera were tested in duplicate. As a control, all sera were also tested in duplicate in antigen-free wells. Unbound antibodies were removed and the Petition 870260072051, dated 07 / 20 / 2026, page 25 / 49 21 / 34 Antibody reactivity to the antigen was visualized by adding antibodies against the serum antibodies to which the peroxidase had been conjugated. After removing unbound conjugated antibodies, the peroxidase substrate ortho-phenylenediamine + H2O2 was added. The presence of peroxidase was demonstrated by the development of a color reaction.

[076] The results of the TRT antigen potency test are summarized in Table 2. In general, antibody titers were satisfactory and there were no non-responsiveness in any of the groups. There was a positive effect of CpG on antibody titers. The best results were obtained with 10 μg of CpG per dose, but 1 μg of CpG per dose also had a positive effect. Table 2. Antibody titers in blood samples from chickens vaccinated 5 weeks prior at 4 weeks of age with inactivated TRT antigen formulated in an O / W emulsion containing different amounts of CpG. TRT log2 antibody titers by ELISA CpG / dose 0 μg 1 μg 10 μg 10.53 12.30 16.03 11.38 14.99 16.93 7.78 15.27 12.77 17.25 13.16 15.64 Individual titers 11.47 15.71 15.20 expressed as log2 titers 10.68 12.37 15.31 14.03 12.40 17.10 12.06 13.04 16.16 15.21 15.74 13.92 12.28 15.84 Average 12.27 13.89 15.49

[077] Furthermore, a positive effect of CpG was determined on the response of Petition 870260072051, dated 07 / 20 / 2026, p. 26 / 49 22 / 34 antibodies against IB M41, see Table 3. A dose of 1 μg of CpG per dose was sufficient to substantially stimulate the antibody response. Without CpG, 7 of the 15 chickens had an antibody response below the threshold of 2.60, while all chickens responded when 1 μg of CpG per dose was tested. A further increase was obtained with 10 μg of CpG per dose. Table 3. Antibody titers in blood samples from chickens vaccinated 5 weeks prior, at 4 weeks of age, with inactivated IB M41 antigen formulated in an O / W emulsion containing different amounts of CpG. IB Antibody Titrations by ELISA Group O / A + 1 μg O / A + 10 treatment O / A CpG / dose μg CpG / dose 3.65 4.06 3.79 2.08 3.12 3.32 2.49 3.97 3.89 2.84 2.80 3.85 0.00 3.14 3.76 3.19 2.62 3.59 Individual Titrations 0.00 3.54 3.59 expressed as Titration of 2.42 3.36 3.12 log to 2.48 2.99 3.42 3.33 3.42 3.94 2.74 3.73 3.76 2.86 3.18 4.11 3.23 3.91 3.86 2.83 -4.13 Petition 870260072051, dated 07 / 20 / 2026, page 27 / 49 23 / 34 Average 2.28 3.36 3.69 * Titrations greater than 396 (Log 10 titration = 2.60) are considered positive by the kit. Example 3. Power Tests for IB, ND, EDS

[078] Groups of 10 SPF chickens were vaccinated with 0.5 mL per dose intramuscularly at 4 weeks of age. Blood samples for serological testing were collected 5 weeks after vaccination. For measuring antibody titers against IB, ND, and EDS, an ELISA (Idexx FlockCheck IBV antibody kit; Idexx, Maine, USA), a hemagglutination inhibition test, and an ELISA (Idexx FlockCheck NCD antibody kit; Idexx, Maine, USA) were used, respectively. The vaccines contained: IB M41: 106.9 EID50 / dose, Newcastle disease (ND): 108'1EID50 / dose, Low egg laying syndrome (EDS): 256 HAu / dose, in water-in-oil emulsions containing different amounts of CpG.

[079] Groups of 10 SPF chickens were vaccinated with 0.5 mL per dose intramuscularly at 4 weeks of age. Blood samples for serological testing were collected 5 weeks after vaccination. For measuring antibody titers against IB, ND, and EDS, an ELISA (Idexx FlockCheck NCD antibody kit; Idexx, Maine, USA), a hemagglutination inhibition test, and an ELISA (Idexx FlockCheck NCD antibody kit; Idexx, Maine, USA) were used, respectively.

[080] The results for IB, ND and EDS are shown in Tables 4-6, respectively: Table 4. Antibody titers by ELISA IB days after vaccination Vaccine batch N Geometric mean 2log No. of responders Petition 870260072051, dated 07 / 20 / 2026, p. 28 / 49 24 / 34 Not vaccinated 10 3.0 0 (0%) No CpG 10 7.7 a* 4 (40%) 5 μg of CpG / dose 10 9.9 B 8 (80%) 2.5 μg of CpG / dose 10 7.3 A 3 (30%) Table 5. NCD antibody titers by ELISA 35 days after vaccination. Vaccine Lot N Geometric Mean Number of Responders Not vaccinated 10 34 0 (0%) No CpG 10 12532 A 10 (100%) 5 μg of CpG / dose 10 18421 B 10 (100%) 2.5 μg of CpG / dose 10 15689B 10 (100%) Table 6. HI antibody titers by EDS 35 days after vaccination Vaccine Lot N Geometric Mean Number of Responders Not vaccinated 10 0 0 (0%) No CpG 10 7.7 A 10 (100%) 5 μg CpG / dose 10 8.4 A 10 (100%) 2.5 μg CpG / dose 10 7.8 A 10 (100%) *Different letters in superscript mean that the differences are statistically significant. Results and conclusions

[081] For IB M41 and ND there are significant differences between the formulations without CpG and the formulation with 5 μg of CpG per dose. With ND with 2.5 μg of CpG per dose, antibody titers were also significantly improved. For EDS there were no significant differences between the formulations without CpG and the formulations with CpG, but numerically 5 μg of CpG per dose was better than no CpG.

[082] CpG has a stimulating effect on antibody responses to IB M41, ND and EDS if added in an amount of 5 pg per dose. The effect of 2.5 μg CpG per dose is limited. Example 4. Effectiveness test Petition 870260072051, dated 07 / 20 / 2026, p. 29 / 49 25 / 34

[083] Combination vaccines were produced containing different amounts of inactivated QX IB antigens, inactivated D1466 IB, and inactivated TRT in an O / W emulsion containing 10 μg per CpG dose. These emulsions were used to vaccinate SPF layers at 14 weeks of age. Half of the different vaccine groups were vaccinated at 10 weeks of age with live IB vaccines. At 26 weeks of age, the hens were challenged with virulent QX-like IB virus or virulent D1466 IB virus. Egg production was measured between 2 weeks before and 4 weeks after challenge. In addition, antibody titers were measured 7 weeks after vaccination using a serum neutralization test.

[084] As can be seen from the data presented in Table 4 after vivo initiation, a clear antibody response can be detected by ELISA. After vaccination with the inactivated vaccines, antibody titers against IB were also determined by SN tests. The SN test specifically detects antibodies against IB QX and IB D1466. No cross-reactivity was observed, as no distinction can be made between antibodies against IB QX and IB D1466 using ELISA.

[085] The data in Table 7 also show that after the challenge there is a very clear drop in egg production by the virulent IB QX challenge virus in hens that had not been vaccinated. Vaccination with live vaccines only, with inactivated vaccines only, or both results in protective immunity against the low egg production caused by the IB QX challenge virus. Table 7. Mean antibody titers for IB, 7 weeks after initiation of live vaccines (D98) and 7 weeks after administration of the inactivated vaccine (D147) and protection against low egg laying after IB QX or IB D1466 challenges. Gr. Vaccines Average titration of arithmetic antibodies IB ELISA Average titrations of Low posture Petition 870260072051, dated 07 / 20 / 2026, page 30 / 49 26 / 34 D98 D147 SNT antibodies D147 *** of eggs by IB QX or IB D1466 Live * Inactivated s # Titration Responsive s Titration Responsive IB D1466 IB QX 10.75 1 + High dose 5385 A 40 / 40 1 18620A (20 / 20) 1 9.25 A (10, 10, A(11, 12, 9, 8) 10, -3.9% 10) QX 2 + Low dose 5235 AB 40 / 40 1 15532B (20 / 20) 1 9.25 A (8, 10, 9, 8) 9.75 AB(10, 9, 10, 2.5% 10) QX 3 + None m 4161 B 40 / 40 1 872 C (2 / 20) 2 4 B (4, 4, 4, 4) 7.75 B (10, 8, 6, 7) -3.2% QX 4 - High dose 22 A 0 / 40 1 10520A (20 / 20) 1 9.5 A(9, 12, 8, 8.25 A (6, 8, -0.8% 9) 9, 10) QX 5 - Low dose 27 A 0 / 40 1 8850 A (20 / 20) 1 8.75 A (9, 12, 9, 8) 9.5 A (10, 11.9, 8) 1.8% QX 6 - None 36 A 1 / 40 1 123 B (1 / 20) 2 4.25 B (5, 4, 4, 4.25 B (4, 4, -24.0% 4) 4, 5) QX 11.5 A 7 + High dose 14652 B 20 / 20 1 9.75 A (9, 9, (11, 11, 10, 11) 12, 5.7% 12) D1466 8 + Low dose 16457A 20 / 20 1 9 A (8, 9, 10, 9) 10 A (8, 10, 11, 13.8% 11) D1466 9 + None 1139 C 20 / 20 1 4.5 B(4, 4, 5, 5) 7 B(7, 7, 8, 0.8% m 6) D1466 10 - High dose 9552 A 20 / 20 1 8.5 A(8, 8, 10,9 A(8, 9, 10, 0% D1466, Petition 870260072051, dated 07 / 20 / 2026, p. 31 / 49 27 / 34 Dose 11 - low None 12 - m 7762 A 59 B 20 / 20 1 0 / 20 2 8) 8.75 A (7, 9, 10, 9) 4.25 B (4, 5, 4, 4) 9) 8.5 A (8, 8, 9, 9) 4.25 B (4, 4, 5, 4) 2.3% D1466 -8.3% D1466 # High dose: 10 8.0 EID 50IB QX and 10 6·8 EID 50 IB D1466, low dose: 10 74 EID 50 IB QX and 10 6'2EID5oIB D1466; 5'3TCID5oTRT per dose at 112 days of age. The cutoff for a positive response was > 5. The limit of

[086] Furthermore, it was previously believed that an inactivated IB QX antigen was hardly able to induce antibody titers in chickens after vaccination, unlike non-QX IB strains. For example, currently marketed products containing antigens from other non-QX infectious bronchitis strains (e.g., IB M41, or IB D274, or IB D1466) are adjuvanted with oil emulsion without CpG and yet provide sufficient protection. In contrast, as demonstrated in Table 8, oil emulsion without CpG is insufficient to induce neutralizing antibodies, while the addition of CpG results in a robust antibody response. Table 8. Antibody responses in 4-week-old SPF chickens against IB QX antigens inactivated in a water-in-oil emulsion containing CpG. IB QX strain 10 log EID 5 per dose Inactivating CpG per dose Serological test......on.... days after vaccination ELISA at 35 ELISA at 49 ELISA at 85 SN test at 35 L1148 8.0 Formalin 2.477 2.928 3.573 0.0 L1148 8.0 Formalin 5 μδ 3.460 3.924 4.047 5.0 L1148 8.0 BPL 2.179 2.444 2.861 3.0 Petition 870260072051, dated 07 / 20 / 2026, pp. 32 / 49 28 / 34 L1148 8.0 BPL 5 μg 3.203 3.809 3.939 4.4 L1148 7.2 Formalin 1.395 0.976 2.314 0.8 L1148 7.2 BPL 1.940 2.185 3.104 0.0 1449-2 7.2 Formalin 1.628 2.994 3.397 3.0 1449-2 7.2 BPL 2.531 2.769 3.439 3.7 1449-2 7.2 BPL 5 μg 2.777 3.191 3.621 4.3 None Placebo 1.793 0.000 1.191 0.0

[087] At the same time, as noted above, known IB virus vaccine strains have proven insufficient to protect against infectious bronchitis caused by IB-QX-like viruses and IB-QX. See document WO2010017440. Surprisingly, the vaccines of the present invention elicited a clear antibody response against inactivated IB QX antigens. EXAMPLE 5. Vaccine against Infectious Bronchitis, Newcastle Disease, TRT, Infectious Bursal Disease and Reovirus

[088] Newborn mixed-sex SPF Leghorn chickens were used in this experiment. The chickens were fed a standard diet with water ad libitum (at will).

[089] On day 0, the birds were vaccinated against Infectious Bronchitis with a vaccine containing a Massachusetts strain via ocular administration. On day 28 of the experiment, the birds received the TRT POULVAC® REO and POULVAC® vaccines at manufacturer-recommended doses in the wings and eyes of the birds, respectively. On day 49 of the experiment, the birds received the experimental vaccines. Groups T01, T02, T04, and T05 had 32 birds per group. Groups T03, T06, and T07 had 13 birds per group. Table 9. Compositions of experimental vaccines Antigen Component Group Adjuvant T01 IB (106.9 EID (egg infectious dose)50), Reo 1733 and Reo2408 (106.9 TCID50 total), IBD Lukert (107.5 TCID50), IBD 28-1 (102.95 TCID50) Newcastle (108.1 EID50), W / O Emulsion (mineral oil ~49.9% v / v, emulsifiers (TWEEN®80 and ARLACEL® total ~9.1% v / v) + 10 μg of Petition 870260072051, dated 07 / 20 / 2026, pp. 33 / 49 29 / 34 TRT (106.40 TCID50) CpG (SEQ ID NO: 8, 65% purity) T02 IB (106.9 EID50), Reo 1733 and Reo2408 (106.9 TCID50 total), IBD Lukert (108.0 TCID50), IBD 28-1 (102.95 TCID50) avian pseudopest (108.1 EID50), TRT (106.40 TCID50) As in T01 T03 Negative control (without antigen) W / O emulsion as in T01 - without CpG T04 As in T01 W / O emulsion as in T01 - without CpG T05 As in T02 W / O emulsion as in T01 - without CpG T06 MATERNAVAC® DUO - IBDV + Reo Commercial Product T07 MSD-Nobilis (TRT reference) (IB, Newcastle, IBDV, TRT) Commercial Product MSD vaccine trade name: Nobilis®RT+IBmulti+G+ND

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

[091] Groups T01-T05 received a 0.5 ml intramuscular injection in the breast area. Groups T06 and T07 were treated according to the manufacturers' protocols.

[092] Blood was collected from birds on days 70 and 77 for serological analysis. Analysis was performed using serum neutralization test (IB, IBDV, Reo), HAI test (Newcastle) and ELISA (TRT). Serological data for T01, T02, T04 and T05 were analyzed using a general linear mixed model with repeated measures. The appropriate log transformation was applied. The model included fixed effects of treatment, time point and treatment-by-time point interaction and random effects of block and animal within block and treatment (animal term).

[093] Least squares means (back-transformed) and 90% confidence intervals were reported along with the range of raw data. If the main treatment effect or the treatment-by-time interaction was significant (P < 0.10), then comparisons between all treatment groups at each Petition 870260072051, dated 07 / 20 / 2026, pp. 34 / 49 30 / 34 time points were conducted and reported. Treatment groups T03, T06, and T07 were summarized with geometric means, standard errors, and ranges.

[094] The results are given in Table 10. Table 10. Antibody titration, back-transformed geometric means Bronchitis Newcastle IBD Reo TRT conversion, % TRT* ​​D70 D77 D7 0 D77 D70 D77 D70 D77 D7 0 D7 7 D70 D77 T0 1 99a 83a 89a 99a 4401 a 3701b 484a 374ab 90.6 93.7 3684 to 5598b T0 2 92a 76a 88a 109a 6499 to 9190a 641a 505a 96, 8 100 6155 to 7971a T0 3 10 9 0 0 0 0 1 1 - - 0 0 T0 4 160 to 144a 23b 29b 528c 1367c 222b 258bc 40, 6 46, 8 68c 123b T0 5 175 a 102a 21b 27b 970b 2201b c 168b 208c 62, 5 62, 5 342a 445c T0 6 13 16 0 0 3527 7845 713 712 - - 0 0 T0 7 371 9 206 9 243 460 3005 7437 0.5 0.5 - - 24 239 Statistical analysis was performed by comparing the titrations elicited by treatments T01, T02, T04, and T05. Different letters indicate a significant difference (p^0.1). TRT* indicates that the titration was obtained using ELISA. All other results were obtained using serum neutralization tests (IB, IBDV, Reo) or HAI (Newcastle) tests.

[095] These results demonstrate that the addition of CpG to the O / W emulsion resulted in an almost 3-fold increase in immune response to VDBI. Looking at the results from a different perspective, these results demonstrate that decreasing the dose of Lukert IBD from 108TCID50 to 107.5TCID50 (a reduction of approximately three times) and adding CpG (compare T01 and T05) to the formulation resulted in increased responses to IBD. Responses to Newcastle disease, reovirus, and TRT were also increased, and the response to IB was not statistically decreased.

[096] Furthermore, responses to all viruses resulted in titers of Petition 870260072051, dated 07 / 20 / 2026, pp. 35 / 49 31 / 34 protection (protection titer for IB is 20, protection titer for Newcastle is 16, protection titer for IBD is 32, protection titer for reovirus is 16). The effectiveness of the TRT vaccine is measured by seroconversion. Seroconversion above 70% indicated that the vaccine was effective.

[097] In the experiments above, the formulations containing CpG (groups T01 and T02) were both effective against TRT (over 90% seroconversion). In contrast, the formulations without CpG (groups T04 and T05) were not effective against TRT (seroconversion of 62.5% and below).

[098] Group T06 was used as a positive control for IBDV. As shown in Table 10, the titers induced by vaccine T02 were comparable with the titers 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.

[099] In summary, the addition of CpG to the O / W emulsion allowed the creation of an effective pentavalent vaccine against bronchitis, Newcastle disease, TRT, infectious bursal disease (IBD or Gumboro), and reovirus. Formulations without CpG were not effective against TRT. Furthermore, titers against Newcastle disease, reovirus, and IBD were lower in formulations without CpG than in formulations with CpG. Example 6. Vaccine against infectious bronchitis, coryza, low egg production syndrome, Newcastle disease and TRT.

[0100] Newborn mixed-sex SPF Leghorn chickens were used in this experiment. The chickens were fed a standard diet with water ad libitum.

[0101] On day 0, the birds were vaccinated against Infectious Bronchitis with a vaccine containing 1 Massachusetts strain via ocular administration. On day 14 of the experiment, the birds received TRT POULVAC® vaccines in doses recommended by the manufacturer intraocularly. On day 35 of the experiment, the Petition 870260072051, dated 07 / 20 / 2026, pp. 36 / 49 32 out of 34 experimental vaccines were administered via intramuscular injections.

[0102] Table 11 illustrates the compositions of experimental and control vaccines used in these experiments. Each group contained 56 birds. Table 11. Experimental vaccine compositions Antigen Group Adjuvant T01 None None (Saline) T02 POULVAC® MIX 6 (IBK (106.90 EID50), Coryza M, Coryza 221, Coryza S (total 106.88 EID50)), EDS* (80.00 HA Units), Newcastle (108.45 EID50) Proprietary T03 IBK (106.90 EID50), TRT (106.00 TCID50), Coryza M, Coryza 221, Coryza S (total 106.88 EID50), EDS (88.00 HA Units), Newcastle (108.43 EID50) W / O Emulsion (mineral oil ~51.6% v / v, emulsifiers (TWEEN®80 and ARLACEL® total ~8.4%) v / v) T04 IBK (106.90 EID50), TRT (106.00 TCID50), Coryza M, Coryza 221, Coryza S (total 106.88 EID50), EDS (88.00 HA Units), Newcastle (108.43 EID50) As in T03 + 10 μg of CpG (SEQ ID NO: 8, 65% purity) T05 IBK (106.90 EID50), TRT (106.50 TCID50), Coryza M, Coryza 221, Coryza S (total 106.88 EID50), EDS (88.00 HA Units), Newcastle (108.43 EID50) As in T03 T06 IBK (106.90 EID50), TRT (106.50 TCID50), Coryza M, Coryza 221, Coryza S total 1O6'88EID5o), EDS (88.00 HA Units), Newcastle (108.43 EID50) As in T04 * EDS refers to low egg laying syndrome, IBK refers to infectious bronchitis.

[0103] All antigens used in T01, T02, T04, and T05 were inactivated in formaldehyde.

[0104] Blood was collected from the birds on days 56 and 70 for serological analysis. A ten percent two-sided significance level was used in the analysis.

[0105] Serological data for T01, T03, T04, T05, and T06 were analyzed using a general linear mixed model with repeated measures. The appropriate log transformation was applied. The model included fixed effects of treatment, time point, and treatment-by-time point and effects interaction. Petition 870260072051, dated 07 / 20 / 2026, pp. 37 / 49 33 / 34 random block and animal within block and treatment (animal deadline).

[0106] Least squares means (half transformed back) and 90% confidence intervals were reported along with the range of the raw data. If the main treatment effect or the treatment-by-time interaction was significant (P < 0.10), then comparisons between all treatment groups at each time point were performed and reported. Serology for T02 was summarized with geometric means, standard errors, and ranges.

[0107] The results for experimental groups (T03-T06) are summarized in Table 12. Table 12. Antibody Titrations GR P Bronchitis, SN EDS, HAI Coryza, SN Newcastle, HAI TRT, seroconversion % TRT, ELISA D56 D70 D56 D70 D5 6 D70 D56 D70 D56 D70 D56 D70 T0 78± 49± 40± 111± 7±1 9± 15± 40± 39 57 1527 b 1738c 3 14b 9 b 7 b 21 b 1 b 2 4 b T0 288± 141± 71± 148± 11± 18± 66± 80± 86 95 3199 a 4708 a 4 44a 25 a 14 a 30 ab 1 a 2 a 7 b 9 a T0 70± 44± 40± 103± 5± 8± 21± 37± 34 45 1184c 1279d 5 12 b 8 b 7 b 18 b 1 bc 1 b 2c 4 b T0 183± 94± 91± 216± 6± 23± 92± 100± 75 86 2964 a 3641 b 6 33 a 18 a 19 a 44 a 1 b 3 a 10 a 11 a Different letters indicate a significant difference (p<0.1).

[0108] These results demonstrate that the addition of CpG to the O / W emulsion resulted in a 2- to 3-fold increase in the immune response to TRT as measured by ELISA. Analyzing the results from a seroconversion perspective, the groups treated with formulations without CpG (treatments T03 and T05) did not demonstrate an efficient seroconversion response to TRT (57% or less). In contrast, the groups treated with formulations T04 and T06, both containing CpG, demonstrated efficient seroconversion (75% and above). Even if the TRT antigen dose decreased approximately threefold, the addition of CpG more than compensated for the lower antigen dose (compare T04 and T05). Petition 870260072051, dated 07 / 20 / 2026, pp. 38 / 49 34 / 34

[0109] Furthermore, responses to all viruses resulted in protective titers (the protective titer for IB is 20, the protective titer for Newcastle is 16, the protective titer for Coryza is 5, the protective titer for EDS is 18). The SN titers or HAI titers induced by formulations T04 and T06 (containing CpG) were generally about twice as high as the titers induced by formulations T03 and T05 (without CpG).

[0110] All publications cited in the descriptive report, whether patent publications or non-patent publications, are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

[0111] Although the invention has been described herein with reference to particular embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the present invention. Therefore, it should be understood that numerous modifications can be made to the illustrative embodiments and that other configurations can be conceived without departing from the spirit and scope of the present invention, as defined by the following claims.

Claims

1. Vaccine CHARACTERIZED in that it comprises an antigen component and an adjuvant component, wherein the adjuvant component essentially consists of an immunostimulatory oligonucleotide containing CpG and an oil emulsion, and wherein the antigen component comprises an infectious bursal disease (IBD) virus antigen, wherein said IBD virus antigen is an inactivated IBD virus, wherein said CpG is a class P CpG, wherein said oil emulsion is a water-in-oil emulsion comprising light mineral oil, and wherein said IBD antigen is an inactivated IBD virus and is present in the amount of 107'0 to 108'0 of TCID50, and wherein the antigen further comprises at least one of: 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.

2. Vaccine, according to claim 1, CHARACTERIZED in that said infectious bursal disease (IBD) virus antigen is an inactivated Lukert strain virus.

3. Vaccine, according to claim 1 or 2, CHARACTERIZED in that the IBD antigen is present in the amount of 107·1, 107·2, 107·3, 107.4, or 107.5 of TCID50.

4. Vaccine, according to claim 3, CHARACTERIZED in that the infectious bronchitis antigen comprises an inactivated infectious bronchitis virus, the reovirus antigen comprises an inactivated reovirus, the non-Lukert strain IBD-derived antigen comprises an inactivated IBD virus of a non-Lukert strain, the Newcastle disease antigen comprises an inactivated Newcastle virus and the Peruvian rhinotracheitis antigen comprises an inactivated Peruvian rhinotracheitis virus.

5. Vaccine, according to any one of claims 1 to 4, CHARACTERIZED in that said immunostimulatory oligonucleotide containing CpG comprises phosphorothioate linkages.

6. Vaccine, according to any one of claims 1 to 5, CHARACTERIZED in that said immunostimulatory oligonucleotide comprises SEQ ID NO:

8.

7. Vaccine, according to any one of claims 1 to 6, CHARACTERIZED in that the immunostimulatory oligonucleotide is present in the amount of 2.5 to 20 μg per dose.

8. Vaccine, according to any one of claims 1 to 7, CHARACTERIZED in that the adjuvant consists of an immunostimulatory oligonucleotide, oil and, optionally, one or more emulsifiers.