Poultry vaccines and methods of protecting poultry

Abstract

There is provided a method of protecting an offspring of a hen from infectious bronchitis infection, the method comprising administering to the hen a vaccine comprising an inactivated infectious bronchitis virus supplemented with a water-in-oil emulsion and an immunostimulatory oligonucleotide.

Description

Technical Field

[0001] The present invention is generally in the field of poultry vaccines. Background Art

[0002] Commercial poultry farming can be broadly divided into three specializations: laying hen, breeder and broiler production. Animals in all of these categories are susceptible to a number of viral, bacterial and parasitic infections.

[0003] Avian infectious bronchitis (IB) is probably one of the most widespread poultry diseases in the world due to its highly contagious nature. It is caused by a gammacoronavirus that affects the respiratory, urinary and reproductive systems of chickens, resulting in different disease states depending on the tissue tropism of the invading virus strain. The diagnosis of infectious bronchitis is one of the challenges in controlling the disease, as clinical signs of the respiratory system are common in other poultry diseases.

[0004] IB is shed by infected chickens in respiratory discharge and feces, and it can be transmitted by aerosols, ingestion of contaminated feed and water, and contact with contaminated equipment and clothing. Naturally infected chickens and chickens vaccinated with live IBV vaccines may shed the virus intermittently for up to 20 weeks after infection. The incubation period is generally 24-48 hours, with peak respiratory shedding lasting 3-5 days after infection.

[0005] Morbidity may reach 100%. In one-day-old chickens, IB infection can permanently damage the oviduct, affecting egg production and egg quality during the production period.

[0006] In chickens younger than 4 weeks of age, IB manifests itself in the form of severe respiratory signs (sneezing, coughing and rales). Rhinitis and conjunctivitis, depression and crowding around heat sources have been observed.

[0007] Therefore, there is a need in the art for a vaccine that protects chickens during the first few days of life. Summary of the invention

[0008] In a first aspect, a method for protecting offspring of hens from IBV infection is provided, the method comprising administering to the hens a vaccine comprising an antigenic component and an adjuvant component, wherein the antigenic component comprises an inactivated IB virus and the adjuvant component consists of oil, a CpG-containing immunostimulatory oligonucleotide and optionally one or more emulsifiers, wherein further, the vaccine is a W / O emulsion.

[0009] In certain embodiments of this first aspect, the vaccine comprises about 10 6.7 EID 50 to about 10 7.9 EID 50In certain embodiments, the antigenic component further comprises one or more of an inactivated TRT virus and an inactivated IBD virus, and wherein the vaccine protects offspring from TRT infection and / or IBDV infection, respectively. In some embodiments, the vaccine contains about 10 5.5 TCID 50 to about 10 6.8 TCID 50 of the inactivated TRT virus and / or about 10 6.9 TCID 50 to about 10 8.2 TCID 50 Inactivated IBD virus.

[0010] In further embodiments of the methods applicable to any of the above embodiments, the antigenic component further comprises inactivated NDV virus and / or inactivated reovirus, and wherein the vaccine protects offspring from NDV infection and / or reovirus infection, respectively. In certain embodiments, the vaccine comprises about 10 7.8 EID 50 to about 10 9.1 EID 50 of the inactivated NDV virus and / or about 10 6.4 TCID 50 to about 10 7.9 TCID 50 of inactivated reovirus.

[0011] In a most preferred embodiment of the method according to the first aspect, the vaccine comprises:

[0012] a) About 10 per dose 6.9 EID 50 Inactivated IB (strain H120);

[0013] b) About 10 per dose 7.2 TCID 50 Lukert's inactivated IBD virus and approximately 10 2.95 EID 50 Inactivated IBD virus strain 28-1;

[0014] c) Approximately 10 5.8 TCID 50 Inactivated TRT virus from Clone K;

[0015] d) About 10 per dose 8.1 EID 50 The inactivated ND virus strain LaSota;

[0016] e) A total of about 10 6.9TCID 50 Inactivated reovirus strains 1733 and 2408.

[0017] In a subset of embodiments of any method applicable to this first aspect of the invention, the CpG-containing immunostimulatory oligonucleotide is a P-class immunostimulatory oligonucleotide, preferably comprising a phosphorothioate bond and / or an I- or J-modification at the 5' end. Most preferably, the CpG-containing immunostimulatory oligonucleotide comprises SEQ ID NO: 8.

[0018] In certain embodiments, the vaccine administered according to the method of the first aspect comprises from about 5 to about 20 μg of said CpG-containing immunostimulatory oligonucleotide per dose.

[0019] In a subset of the embodiments of the first aspect, the vaccine according to any embodiment of the first aspect is administered to hens that have been primed according to a regimen comprising:

[0020] a) At 1, 3 and 8 weeks of age – administration of live antigens against Newcastle disease virus, infectious bronchitis and infectious bursal disease virus;

[0021] b) At 6 and 10 weeks – administration of live turkey rhinotracheitis antigen;

[0022] c) At week 12 – Administer live reovirus antigen.

[0023] In a second aspect, the present disclosure provides a vaccine for vaccinating hens, the vaccine comprising an antigen component and an adjuvant component, wherein the antigen component comprises inactivated IB virus, and the adjuvant component consists of oil, CpG-containing immunostimulatory oligonucleotides and optionally one or more emulsifiers, wherein further, the vaccine is a W / O emulsion, and the vaccine is used to protect the offspring of the hen from IBV infection.

[0024] In certain embodiments of this second aspect, the vaccine comprises about 10 6.7 EID 50 to about 10 7.9 EID 50 In certain embodiments, the antigenic component further comprises one or more of an inactivated TRT virus and an inactivated IBD virus, and wherein the vaccine protects offspring from TRT infection and / or IBDV infection, respectively. In some embodiments of this second aspect, the vaccine contains about 10 5.5 TCID 50 to about 10 6.8 TCID 50 of the inactivated TRT virus and / or about 10 6.9 TCID 50to about 10 8.2 TCID 50 Inactivated IBD virus.

[0025] In further embodiments of the vaccine applicable to any embodiment of the second aspect, as described above, the antigenic component further comprises inactivated NDV virus and / or inactivated reovirus, and wherein the vaccine protects offspring from NDV infection and / or reovirus infection, respectively. In certain embodiments, the vaccine contains about 10 7.8 EID 50 to about 10 9.1 EID 50 of the inactivated NDV virus and / or about 10 6.4 TCID 50 to about 10 7.9 TCID 50 of inactivated reovirus.

[0026] In a most preferred embodiment of the vaccine according to the second aspect, the vaccine comprises:

[0027] a) About 10 per dose 6.9 EID 50 Inactivated IB (strain H120);

[0028] b) About 10 per dose 7.2 TCID 50 Lukert's inactivated IBD virus and approximately 10 2.95 EID 50 Inactivated IBD virus strain 28-1;

[0029] c) Approximately 10 5.8 TCID 50 Inactivated TRT virus from Clone K;

[0030] d) About 10 per dose 8.1 EID 50 The inactivated ND virus strain Lasota;

[0031] e) A total of about 10 6.9 TCID 50 Inactivated reovirus strains 1733 and 2408.

[0032] In a subset of embodiments of any vaccine applicable to this second aspect of the invention, the CpG-containing immunostimulatory oligonucleotide is a P-class immunostimulatory oligonucleotide, preferably comprising a phosphorothioate bond and / or an I- or J-modification at the 5' end. Most preferably, the CpG-containing immunostimulatory oligonucleotide comprises SEQ ID NO: 8.

[0033] In certain embodiments of this second aspect, the vaccine comprises about 5 to about 20 μg of said CpG-containing immunostimulatory oligonucleotide per dose.

[0034] In a subset of the embodiments, the vaccine according to any embodiment of the second aspect is administered to hens that have been primed according to a regimen comprising:

[0035] a) At 1, 3 and 8 weeks of age – administration of live antigens against Newcastle disease virus, infectious bronchitis and infectious bursal disease virus;

[0036] b) At 6 and 10 weeks – administration of live turkey rhinotracheitis antigen;

[0037] c) At week 12 – Administer live reovirus antigen. DETAILED DESCRIPTION

[0038] When used in conjunction with a measurable numerical variable, the term "about" or "approximately" refers to the indicated value of the variable and all values ​​of the variable that are within the experimental error range of the indicated value (e.g., within a 95% confidence interval for the mean) or within 10% of the indicated value, whichever is greater.

[0039] The term "consisting essentially of" and the like as applied to the adjuvant formulations of the present invention refers to vaccines and other compositions that do not contain additional adjuvants or immunomodulators in amounts that would allow the agent to exert a measurable adjuvant or immunomodulatory effect.

[0040] The term "conventional priming vaccine" refers to a vaccine that is administered to hens when they are between 1 and about 100 days old. Conventional priming vaccines are typically modified live vaccines and contain attenuated viruses. A variety of conventional priming vaccines are commercially available and include, but are not limited to TRT, BURSA F, New Ls Mass I, Coryza Gel, MAGNIPLEX, HVD-IBD, H120, BRON 120L, IBD, TRANSMUNE IBD、MEVAC TM ND HB1, BUR TM 706R.

[0041] The term "hen" refers to a female chicken that is the mother of the offspring whose protection is achieved by administering the vaccine disclosed herein to the hen. Prior to administering the inactivated vaccine disclosed herein, the hen has been primed.

[0042] The phrase "laying eggs" refers to the laying of eggs by hens vaccinated with the vaccine of the present invention, from which the offspring to be protected hatch.

[0043] The term "primary vaccination" as applied to hens that have received the vaccines described herein means that the hens are vaccinated when they are 1 to about 98 days old, preferably 1 to about 91 days old, more preferably 1 to about 84 days old, with one or more doses of a conventional priming vaccine containing the same antigens as those used in the vaccines described herein to be administered to the hens.

[0044] In certain embodiments, the hens have been primed with two or more administrations, preferably three administrations, of live infectious bronchitis virus.

[0045] In other embodiments, the hens have been primed with two or more administrations of live infectious bronchitis virus, preferably three administrations, and also with at least one of:

[0046] a) one or more administrations, preferably two administrations, of live TRT virus; and

[0047] b) Two or more administrations, preferably three administrations, of live IBD virus.

[0048] In other embodiments, the hens have been primed with two or more administrations of live infectious bronchitis virus, preferably three administrations, and also with at least one of:

[0049] a) one or more administrations, preferably two administrations, of live TRT virus; and

[0050] b) two or more administrations, preferably three administrations, of live IBD virus;

[0051] c) two or more administrations, preferably three administrations, of live Newcastle disease virus;

[0052] d) at least one administration of live avian reovirus.

[0053] In certain embodiments, the hens have been primed by:

[0054] a) two or more administrations, preferably three administrations, of live infectious bronchitis virus;

[0055] b) one or more administrations, preferably two administrations, of live TRT virus;

[0056] c) two or more administrations, preferably three administrations, of live IBD virus;

[0057] d) two or more administrations, preferably three administrations, of live Newcastle disease virus;

[0058] e) at least one administration of live avian reovirus.

[0059] In a specific subset of the embodiments, the hens have been initially vaccinated by:

[0060] a) three administrations of live infectious bronchitis virus;

[0061] b) two administrations of live TRT virus;

[0062] c) three administrations of live IBD virus;

[0063] d) three administrations of live Newcastle disease virus;

[0064] e) One administration of live avian reovirus.

[0065] In certain embodiments, naive hens have been vaccinated against IBDV, IBV, Newcastle Disease Virus, TRT, and Reovirus according to the following schedule:

[0066] Table 1

[0067]

[0068] Therefore, before being vaccinated with the vaccine of the present invention, the hens have received at least one dose (more preferably two doses, and even more preferably three doses) of a modified live Newcastle disease virus vaccine, at least one dose (more preferably two doses, and even more preferably three doses) of a modified live IBDV virus vaccine, at least one dose (more preferably two doses, and even more preferably three doses) of a modified live IB virus vaccine, at least one dose (more preferably, two or three doses) of a modified live TRT virus vaccine and at least one dose of a modified live reovirus vaccine.

[0069] The term "same antigen" as used in conventional priming vaccines and vaccines for administration to hens refers to the identity of the antigen in the inactivated vaccine used for administration to hens to enhance the immune response to the antigen in the conventional priming vaccine.

[0070] The term "parenteral administration" refers to the introduction of a substance (such as a vaccine) into a subject's body by or by a route that does not include the digestive tract. Parenteral administration includes subcutaneous, intramuscular, transdermal, intradermal, intraperitoneal, intraocular, and intravenous administration.

[0071] As applied to immunostimulatory oligonucleotide preparations, percent purity or "X percent purity" refers to a population of oligonucleotide molecules that contains X% of a specified oligonucleotide (e.g., SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8, etc.), and the remainder (i.e., 100% minus X%) contains shorter fragments of the specified oligonucleotide that were present as impurities during the production of the specified sequence. Thus, if the sequence is made by 3'-5' sequencing, the 5'-truncation will contain the remainder. As a non-limiting example, a 100 μg preparation of 80% pure SEQ ID NO: 8 contains 80 μg of SEQ ID NO: 8, and the remaining 20 μg are shorter fragments of SEQ ID NO: 8 present in the preparation.

[0072] The terms "therapeutically effective amount", "immunologically effective amount" and "effective amount" refer to the amount of an antigen or adjuvant or vaccine that will induce an immune response in a subject receiving the antigen or adjuvant or vaccine, sufficient to prevent or reduce the signs or symptoms of a disease caused by infection with a pathogen such as a virus or bacteria, including adverse health effects or complications thereof. Humoral immunity or cell-mediated immunity, or both humoral immunity and cell-mediated immunity can be induced. The immunogenicity and efficacy of the vaccine in animals can be evaluated indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs and symptoms after attack with a wild-type virus strain. The protective immunity conferred by the vaccine can be evaluated by measuring, for example, a reduction in clinical signs, such as mortality, morbidity, body temperature, overall physical condition, and overall health and performance of the subject. The therapeutically effective amount of the vaccine can vary depending on the specific adjuvant used, the specific antigen used, or the condition of the individual, and can be determined by those skilled in the art.

[0073] The terms "vaccine", "vaccine described herein", "vaccine disclosed herein", "inactivated vaccine" and the like specifically refer to an immunogenic composition that elicits a protective immune response in offspring and comprises the inactivated antigen and is formulated as a W / O emulsion comprising an adjuvant comprising (or consisting essentially of or consisting of) oil, an immunostimulatory CpG oligonucleotide and optionally one or more emulsifiers. The term specifically excludes conventional priming vaccines used for the initial vaccination of hens.

[0074] antigen

[0075] The vaccines described herein preferably contain inactivated viral antigens. In certain embodiments, the vaccine contains inactivated infectious bronchitis virus (IB or IBV). A variety of IB strains are known, including but not limited to Massachusetts strain, Brazilian IBV variant strain (BR-01), M41, D1466 and other strains, such as ARK99, 793B, QX, GA08, VAR2, Beaudette, Holte, Gray, N1 / 62, VicS, TP / 64, L165, ARK99, B, UFMG / G, D3896, Moroccan-G / 83, B1648, B4, IZO In a preferred embodiment, the strain is a Massachusetts strain (e.g., H120 or M41 or Ma5 or other isolated strains) or another strain of the GI-1 lineage. In a most preferred set of embodiments, the strain is H120. In other embodiments, the strain is GI-2, GI-3, GI-4, GI-5, GI-6, GI-7, GI-8, GI-9, GI-10, GI-11, GI-12, GI-13, GI-14, GI-15, GI-16, GI -17, GI-18, GI-19, GI-20, GI-21, GI-22, GI-23, GI-24, GI-25, GI-26, GI-27, GII-1, GIII-1, GIV-1, GV-1 or GVI-1 lineage.

[0076] In other embodiments, the strain is GI-1, GI-2, GI-3, GI-4, GI-5, GI-6, GI-7, GI-8, GI-9, GI-10, GI-11, GI-12, GI-13, GI -14, GI-15, GI-16, GI-17, GI-18, GI-19, GI-20, GI-21, GI-22, GI-23, GI-24, GI-25, GI-26, GI-27 lineage.

[0077] In other embodiments, the strain is GI-1, GI-2, GI-3, GI-4, GI-5, GI-6, GI-7, GI-8, GI-9, GI-10, GI-11, GI-12, GI-13 , GI-14, GI-15, GI-16, GI-17, GI-18, GI-20, GI-21, GI-22, GI-23, GI-24, GI-25, GI-26, GI-27 lineage.

[0078] Inactivated IB virus can be administered at a dose of at least 10 6.4 EID 50 More preferably, at least about 10 6.7 EID 50 More preferably, at least about 10 6.8 EID 50 More preferably, at least about 10 6.9 EID 50 Up to about 10 per dose 7.9 EID 50 , even more preferably at least about 10 6.9 EID 50 Up to about 10 per dose 7.3 EID 50 Thus, in various embodiments, the inactivated IB virus (such as Massachusetts strain H120) may be present in an amount of 10 6.4 EID 50 , or about 10 per dose 6.5 EID 50 , or about 10 per dose 6.6 EID50, or about 10 per dose 6.7 EID 50 , or about 10 per dose 6.8 EID 50 , or about 10 per dose 6.9 EID 50 , or about 10 per dose 7.0 EID 50 , or about 10 per dose 7.1 EID 50 , or about 10 per dose 7.2 EID 50 , or about 10 per dose 7.3 EID 50 , or about 10 per dose 7.4 EID 50 , or about 10 per dose 7.5 EID 50 , or about 10 per dose 7.6 EID 50 , or about 10 per dose 7.7 EID 50, or about 10 per dose 7.8 EID 50 or about 10 per dose 7.9 EID 50 The amount present in the vaccine.

[0079] The amount of inactivated IB and other inactivated antigens present in the vaccines described herein is the amount of antigen per dose prior to inactivation.

[0080] In addition to the IB antigen according to any of the above embodiments, the vaccine may also contain one or more inactivated viruses selected from the group consisting of infectious bursal disease virus (IBDV or Gumboro), Newcastle disease virus (NDV), turkey rhinotracheitis virus (TRT or TRTV) and avian reovirus.

[0081] Thus, in certain embodiments, the vaccines described herein contain an inactivated IB virus according to any of the above embodiments and one or more of an inactivated TRT virus and an inactivated IBD virus.

[0082] A variety of TRT strains are known. In certain embodiments, the TRT strain is selected from the group consisting of Clone K, 119 / 95-BR, TRTV-BR, 1062, BUT 1#8544, PL 21, TRT 50. In a more preferred embodiment, the TRT strain is Clone K. The inactivated TRT virus can be administered at a dose of at least 10 5.5 TCID 50 More preferably, at least 10 5.6 TCID 50 More preferably, at least 10 5.7 TCID 50 More preferably, at least about 10 5.8 TCID 50 Up to about 10 per dose 6.8 TCID 50 , even more preferably at least about 10 5.8 TCID 50 Up to about 10 per dose 6.4 TCID 50 The amount exists.

[0083] A variety of IBDV strains are known. In certain embodiments, the IBDV strain is selected from the group consisting of Lukert, STC, Del-E, Rs593, GLS, 28-1, S-21, Delaware variant E, Delaware variant AL2, Delaware variant 15-4, ArkProvent, Winterfield 2512, Moulthrop G603, variant 1084-E, S706, VNJO, variant E, LIBDV, GP82, GM97, CH / 80, 228E, D78, MB, V877, GBV-8. In other embodiments, the IBDV strain can be selected from one or more of Winterfield 2512, Moulthrop G603, GBV-8, variant 1084-E, S706, VNJO, variant E, LIBDV, GP82, GM97, CH / 80, 228E, D78, MB, V877, Lukert, 28-1, GBV-8. The inactivated IBDV virus can be administered at a dose of at least 10 6.9 TCID 50 More preferably, at least 10 7.0 TCID 50 More preferably, at least 10 7.1 TCID 50 More preferably, at least about 10 7.2 TCID 50 Up to about 10 per dose 8.2 TCID 50 , even more preferably at least about 10 7.2 TCID 50 Up to about 10 per dose 7.6 TCID 50 The amount exists.

[0084] In a more preferred embodiment, the vaccine comprises at least two IBDV strains, more preferably Lukert and 28-1. If inactivated IBDV viruses of both Lukert and 28-1 are present in the vaccine, the Lukert strain IBD virus may be present in an amount of at least 10 6.9 TCID 50 , more preferably at least about 10 7.1 TCID 50 , more preferably at least about 10 7.1 TCID 50 to about 10 7.7 TCID 50 , or about 10 7.2 TCID 50 to about 10 7.5 TCID 50The inactivated IBD virus of strain 28-1 may be present in an amount of at least 10 2.5 EID 50 to about 10 3.5 EID 50 , or about 10 per dose 2.6 to about 10 3.4 EID 50 , or about 10 2.7 to about 10 3.3 EID 50 , or about 10 2.8 to about 10 3.2 EID 50 , or about 10 2.9 to about 10 3.1 EID 50 , or about 10 2.9 to about 10 3.0 EID 50 , or about 10 2.95 EID 50 The amount exists.

[0085] In other embodiments, the vaccines described herein contain an inactivated IB virus as described above and one or more of an inactivated Newcastle Disease Virus and an inactivated Reovirus.

[0086] A variety of NDV strains are known, including but not limited to Lasota, F, B1, V4, V4-HR, 1-2, Mukteswar, Komarov, LZ, Miyadera, AF2240, HER / 33, Texas, Ulster, H, Clone 30. In certain embodiments, the NDV strain is LaSota or another lentogenic strain.

[0087] Inactivated NDV virus can be administered at a dose of at least 10 7.8 EID 50 More preferably, at least about 10 7.9 EID 50 More preferably, at least about 10 8.0 EID 50 More preferably, about 10 8.1 EID 50 Up to about 10 per dose 9.1 EID 50 , even more preferably about 10 8.1 EID 50 Up to about 10 per dose 8.5 EID 50 The amount exists.

[0088] A variety of reovirus strains are known, including but not limited to 1017-1, 2408, 601G, 601SI, 916, 918, 919, OS161, R2 / TW, T6, 1733, S1133, 2177, SS412. In certain embodiments, the reovirus strain is selected from the group consisting of S1133, 2177, 1733, 2408, SS412. The inactivated reovirus can be administered at a dose of at least 10 6.4 TCID 50 More preferably, at least about 10 6.7 TCID 50 More preferably, at least about 10 6.8 TCID 50 More preferably, about 10 6.9 TCID 50 Up to about 10 per dose 7.9 TCID 50 , even more preferably about 10 6.9 TCID 50 Up to about 10 per dose 7.3 TCID 50 The total amount exists.

[0089] In a more preferred embodiment, the vaccine contains two strains of inactivated reovirus: 1733 and 2408. These inactivated reoviruses can be administered in a dose of at least 10 6.4 TCID 50 More preferably, at least about 10 6.7 TCID 50 More preferably, at least about 10 6.8 TCID 50 More preferably, about 10 6.9 TCID 50 Up to about 10 per dose 7.9 TCID 50 , even more preferably about 10 6.9 TCID 50 Up to about 10 per dose 7.3 TCID 50 The total amount exists.

[0090] In a specific set of embodiments, the vaccine contains all five inactivated antigens: IB virus, IBDV, TRT virus, NDV and reovirus. The inactivated IB virus is the H120 strain and is administered at about 10 6.9 EID 50 The IBDV antigens include strain Lukert (approximately 10 7.2 TCID 50 of each dose) and strain 28-1 (approximately 102.95 EID 50 The inactivated IBD virus is a Clone K strain and is present in an amount of about 10 5.8 TCID 50 The inactivated Newcastle disease virus is a LaSota strain and is present in an amount of about 10 8.1 EID 50 The inactivated reovirus antigen comprises about 10 6.9 TCID 50 The total amount of inactivated reovirus of strains 1733 and 2408 was present.

[0091] Adjuvant

[0092] The adjuvant for the vaccine described herein and administered to hens comprises an immunostimulatory CpG oligonucleotide, an oil, and optionally one or more emulsifiers, such that the vaccine is a water-in-oil (W / O emulsion).

[0093] In certain embodiments, the adjuvant consists essentially of, or consists of, an immunostimulatory CpG oligonucleotide, an oil, and an optional surfactant. In certain embodiments, the adjuvant consists of an immunostimulatory CpG oligonucleotide, an oil, and an optional surfactant.

[0094] Suitable immunostimulatory oligonucleotides include ODN (DNA-based) or chimeric ODN-ORN structures, which may have modified backbones, including but not limited to phosphorothioate modifications, halogenation, alkylation (e.g., ethyl or methyl modifications), and phosphodiester modifications. In certain embodiments, polyinosinic acid-cytidylic acid or derivatives thereof (poly I:C) may be used.

[0095] CpG oligonucleotides (also referred to as immunostimulatory CpG oligonucleotides or immunostimulatory CpG-containing oligonucleotides) are characterized by the presence of unmethylated CG dinucleotides (CpG motifs) in a specific base sequence environment. (Hansel TT, Barnes PJ (editor): 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 when present, are usually methylated, but are present in bacterial DNA, where they confer immunostimulatory properties to bacterial DNA.

[0096] In selected embodiments, the adjuvant of the present invention utilizes 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 immunostimulatory CpG oligonucleotides that are characterized by the presence of palindromes, typically 6-20 nucleotides in length. P-class oligonucleotides have the ability to spontaneously self-assemble into concatemers in vitro and / or in vivo. These oligonucleotides are all single-stranded in the strict sense, but the presence of palindromes allows the formation of concatemers or possibly stem-loop structures. The overall length of P-class immunostimulatory oligonucleotides is between 19 and 100 nucleotides, such as 19-30 nucleotides, 30-40 nucleotides, 40-50 nucleotides, 50-60 nucleotides, 60-70 nucleotides, 70-80 nucleotides, 80-90 nucleotides, 90-100 nucleotides.

[0097] In one aspect of the invention, the immunostimulatory oligonucleotide contains a 5' TLR activation domain and at least two palindromes, one palindrome being a 5' palindrome of at least 6 nucleotides in length and linked directly or through a spacer to a 3' palindrome of at least 8 nucleotides in length.

[0098] P class immunostimulatory oligonucleotides can be modified according to techniques known in the art. For example, J-modification refers to iodine-modified nucleotides. E-modification refers to ethyl-modified nucleotides. Therefore, the E-modified P class immunostimulatory oligonucleotides are P class immunostimulatory oligonucleotides in which at least one nucleotide (preferably 5' nucleotides) is ethylated. Other modifications include the connection of 6-nitro-benzimidazoles, O-methylation, modification with propynyl-dU, inosine modification, 2-bromovinyl connection (preferably connected to uridine).

[0099] P-class immunostimulatory oligonucleotides may also contain modified internucleotide linkages, including but not limited to phosphodiether linkages and phosphorothioate linkages. The oligonucleotides of the invention may be synthesized or obtained from commercial sources.

[0100] P-class oligonucleotides and modified P-class oligonucleotides are further disclosed in published PCT application number WO2008 / 068638, published on June 12, 2008. Suitable non-limiting examples of modified P-class immunostimulatory oligonucleotides are provided below (in SEQ ID NOs 1-10, "*" refers to a phosphorothioate bond, and "-" refers to a phosphodiester bond).

[0101] SEQ ID NO:1 5'T*CG*T*CG*A*CG*A*T*CG*G*C*G*CG*C*G*C*C*G 3'

[0102] SEQ ID NO: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'

[0103] 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'

[0104] 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'

[0105] 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'

[0106] 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'

[0107] SEQ ID NO:7 5'EU*CG*A*C*G*T*C*G*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3'

[0108] SEQ ID NO: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'

[0109] 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'

[0110] SEQ ID NO:10 5'T*CG*T*CG*A*CG*A*T*CG*G*C*G*CG*C*G*C*C*G*T 3'

[0111] Immunostimulatory oligonucleotides of the present invention can be chemically synthesized. In addition, immunostimulatory oligonucleotides can be used with about 60% purity (homogeneity) or higher (e.g., about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98% or 100% purity).

[0112] The amount of P-class immunostimulatory oligonucleotide for use in the adjuvant composition depends on the nature of the P-class immunostimulatory oligonucleotide used and the intended species.

[0113] Vaccine of the present invention is formulated as W / O emulsion. Various oils and combinations thereof are suitable for the present invention. These oils include, but are not limited to, animal oils, vegetable oils and non-metabolism oils. Non-limiting examples of vegetable oils suitable for the present invention are corn oil, peanut oil, soybean oil, coconut oil, olive oil and plant squalane (phytosqualane). Non-limiting examples of animal oils are squalane. Suitable non-limiting examples of non-metabolism oils include light mineral oils, straight or branched saturated oils, branched oils, etc.

[0114] In one set of embodiments, the oil used in the adjuvant formulation of the present invention is light mineral oil. As used herein, the term "mineral oil" refers to a mixture of liquid hydrocarbons obtained from vaseline via distillation techniques. The term is synonymous with "liquefied paraffin," "liquid petrolatum," and "white mineral oil." The term is also intended to include "light mineral oil," which is similarly obtained by distilling vaseline, but has a slightly lower specific gravity than white mineral oil. See, e.g., Remington's Pharmaceutical Sciences, 18th edition (Easton, Pa.: Mack Publishing Company, 1990, pages 788 and 1323). Mineral oil can be obtained from a variety of commercial sources, such as JT Baker (Phillipsburg, Pa.), USB Corporation (Cleveland, Ohio). Preferred mineral oils are those that can be named Commercially available light mineral oil. In another embodiment, suitable oils include mineral oil MARCOL TM 52. MARCOL TM 52 is a purified mixture of liquid saturated hydrocarbons. It is a crystal clear water-white product free of toxic impurities. It is obtained from petroleum by vacuum distillation, with subsequent refining stages including final purification by catalytic hydrogenation.

[0115] Emulsifiers used in the emulsions of the present invention include natural biocompatible emulsifiers and non-natural synthetic surfactants. Biocompatible emulsifiers include phospholipid compounds or mixtures of phospholipids. Preferred phospholipids are phosphatidylcholine (lecithin), such as soybean or egg lecithin. Lecithin can be obtained as a mixture of phospholipids and triglycerides by washing crude vegetable oil with water, and separating and drying the resulting hydrated gum. Refined products can be obtained by fractionating acetone-insoluble phospholipids and glycolipids in the mixture after removing triglycerides and vegetable oils by acetone washing. 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 separated from natural sources or conventionally synthesized.

[0116] In other embodiments, the emulsifier used herein does not include lecithin, or lecithin is used in an amount that is not immunologically effective.

[0117] Non-natural synthetic emulsifiers suitable for use in the adjuvant formulations of the present invention include sorbitan-based nonionic surfactants, such as fatty acid-substituted sorbitan surfactants (which may be referred to as or Commercially available), fatty acid esters of polyethoxylated sorbitan Polyethylene glycol esters of fatty acids from sources such as castor oil Polyethoxylated fatty acids (e.g., M-53 obtained stearic acid), polyethoxylated isooctylphenol / formaldehyde polymer Polyoxyethylene fatty alcohol ether Polyoxyethylene nonylphenyl ether ( N), polyoxyethylene isooctylphenyl ether ( X). Preferred synthetic surfactants are those which may be named and Surfactants such as -80 (polyoxyethylene (20) sorbitan monooleate) and ARLACEL TM 83V (sorbitan sesquioleate).

[0118] In general, the emulsifier may be present in the vaccine in an amount of 0.01% to 40%, preferably 0.1% to 15%, more preferably 2% to 10% by volume.

[0119] In a subset of embodiments, the volume percentage of oil and oil-soluble emulsifier combined is at least 50% by volume, such as 50% to 95%; preferably, in an amount greater than 50% to 85%; more preferably, in an amount of 50% to 60%, and more preferably in an amount of 53-58% v / v of the vaccine. Thus, for example, but not limited to, the oil may be present in an amount of 45%, and the fat-soluble emulsifier will be present in an amount greater than 5% v / v. Thus, the volume percentage of oil and oil-soluble emulsifier combined will be at least 50%.

[0120] In another subset of all vaccines applicable to the present invention, the volume percentage of oil exceeds 40% of the vaccine by volume, such as 40% to 90%; 40% to 85%; 43% to 60%, 44-50% v / v or 45-55% v / v. In certain embodiments, the emulsion contains at least 48% v / v oil phase and 52% v / v aqueous phase.

[0121] Sometimes, it is impossible or impractical to concentrate the antigen, particularly in expanded commercial applications, and a low concentration antigen solution must be used. Therefore, in some embodiments, the vaccine of the present invention comprises an adjuvant formulation as described above, wherein the content of the oil phase in these adjuvant formulations is diluted, and wherein the vaccine is a water-in-oil emulsion.

[0122] In practice, it is possible to create a water-in-oil emulsion in which the oil phase is less than 50% v / v. The integrity of the water-in-oil emulsion can be maintained as long as the dispersed spherical water droplets are not present in a more concentrated form than the maximum packing fraction of random packing of monodisperse droplets, i.e. 0.64. See Tadros, Emulsion Formation, Stability and Rheology, 1st edition, 2013, Wiley-VCH GmbH & Co KGaA. As long as the total volume fraction occupied by the water droplets does not exceed 0.64, i.e.: 64% v / v. Conversely, this means that the oil phase should not be reduced to less than 36% v / v.

[0123] In some suitable embodiments, a dose of adjuvant will contain between about 0.1 and about 20 μg (e.g., 1-20 μg, or about 5 to about 15 μg, or about 8 to about 12 μg, or about 10 μg) of the immunostimulatory oligonucleotide, up to about 50 μg (e.g., 0.5-20 μg, or 1-10 μg) of a sterol, such as cholesterol.

[0124] In certain embodiments, the adjuvant component is prepared as follows:

[0125] a) dissolving sorbitan sesquioleate and cholesterol (if any) in light mineral oil. Sterile filter the resulting oil solution;

[0126] b) dissolving the immunostimulatory oligonucleotide and polyoxyethylene (20) sorbitan monooleate in the aqueous phase to form an aqueous solution;

[0127] c) Add the aqueous solution to the oil solution under continuous homogenization.

[0128] The vaccine of the present invention can be prepared by adding the antigen component to the aqueous phase, followed by combining the aqueous phase with the oil phase. In other embodiments, the antigen component can be added to the adjuvant component after the adjuvant component is prepared.

[0129] Vaccines described herein may further include a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delay agents, etc. In the sense that it is compatible with the other components of the composition and harmless to the subject, the carrier must be "acceptable". Typically, the carrier will be sterile and pyrogen-free, and is selected based on the mode of administration to be used. It is well known to those skilled in the art that the preferred formulations of the pharmaceutically acceptable carrier constituting the composition are those pharmaceutical carriers approved in the applicable regulations promulgated by the U.S. Department of Agriculture or the U.S. Food and Drug Administration or the equivalent government agencies of non-U.S. countries. Therefore, the pharmaceutically acceptable carrier for commercial production of compositions is a carrier that has been approved by or will be approved by the appropriate government agencies of the United States or foreign countries.

[0130] Other components of the composition may include pharmaceutically acceptable excipients such as carriers, solvents and diluents, isotonic agents, buffers, stabilizers, preservatives, vasoconstrictors, antibacterial agents, antifungal agents, etc. Typical carriers, solvents and diluents include water, saline, glucose, ethanol, glycerol, etc. Representative isotonic agents include sodium chloride, glucose, mannitol, sorbitol, lactose, etc. Useful stabilizers include gelatin, albumin, etc.

[0131] In a specific set of embodiments, the vaccine contains all five inactivated antigens: IB virus, IBDV, TRT virus, NDV and reovirus. The inactivated IB virus is H120 and is administered at about 10 6.9 EID 50 The IBDV antigens include inactivated IBD viruses of strain Lukert and strain 28-1. The amount of inactivated Lukert strain virus is about 10 7.2 TCID 50 , and the amount of inactivated 28-1 strain virus is about 10 2.95 EID 50 The inactivated TRT virus was clone K and was dosed at about 10 5.8 TCID 50The inactivated Newcastle disease virus is a LaSota strain virus and is present in an amount of about 10 8.1 EID 50 The inactivated reovirus antigen comprises inactivated reovirus strains 1733 and 2408 and is present in an amount of about 10 6.9 TCID 50 The vaccine is formulated to contain 49.9% light mineral oil, 5.85% (Sorbitan sesquioleate), 3.25% 80 and 10 μg of a W / O emulsion of a CpG solution (SEQ ID NO: 8). This type of vaccine has recently been developed in Brazil under the name MATERNAVAC ULTRA 5 is approved. Preferably, the vaccine is administered to hens about 2 to 4 weeks prior to egg laying, wherein the hens have been primed as described in Table 1.

[0132] method

[0133] Generally, the vaccine of the present invention can be administered to hens via a variety of routes. Such routes are known to those of ordinary skill in the art and include, but are not limited to, intramuscular and subcutaneous injections.

[0134] In certain embodiments, hens are vaccinated at about 14 to about 22 weeks of age (e.g., about 15 weeks of age, or about 16 weeks of age, or about 17 weeks of age, or about 18 weeks of age, or about 19 weeks of age, or about 20 weeks of age, or about 21 weeks of age, or 22 weeks of age).

[0135] In certain embodiments, the vaccine is administered about 1 to about 26 weeks (e.g., about 1 to about 6 weeks, or about 1 to about 10 weeks, or about 2 to about 13 weeks, or about 2 to about 6 weeks, or about 2 to about 4 weeks, or about 2 to about 26 weeks, or about 6 to about 26 weeks, or about 10 to about 26 weeks, or about 13 to about 26 weeks) before expected egg laying. This regimen ensures that immunity to the antigens present in the vaccines described herein is generated at the time of egg laying and persists throughout the egg-laying period of the vaccinated hens.

[0136] The vaccines described herein are administered to hens that have been primed or primed with a conventional prime vaccine. In certain embodiments, the hens are primed as described in the above "definitions". In a more preferred embodiment, the hens are primed, and the hens have been primed according to the following protocol:

[0137] a) At 1, 3 and 8 weeks of age – administration of live antigens against Newcastle disease virus, infectious bronchitis and infectious bursal disease virus;

[0138] b) At weeks 6 and 10 – administration of live turkey rhinotracheitis antigen;

[0139] c) At week 12 – Administer live reovirus antigen.

[0140] A variety of conventional priming vaccines containing different antigens are known in the art. For example, IB Primer contains lyophilized live Massachusetts infectious bronchitis virus. IB H120 is a live, lyophilized vaccine indicated for use as a primary vaccination of poultry against infectious bronchitis. The vaccine contains the Massachusetts strain H120. Other IB primers may also be used with the vaccine of the present invention.

[0141] Similarly, there are conventional priming vaccines that contain other antigens and combinations of antigens that may be present in the vaccines described herein.

[0142] The antigens in these vaccines are attenuated, and the vaccines are administered to hens from 1 day to about 100 days (14-15 weeks) old according to a specific schedule. The hens can be from 1 day to about 91 days old, or from 1 day to about 84 days old, or from 1 day to about 77 days old, or from 1 day to about 70 days old.

[0143] In a most preferred embodiment, as described above, the vaccine administered to the methods disclosed herein (or for the uses described herein) comprises all five inactivated antigens: IB virus, IBDV, TRT virus, NDV, and Reovirus. The inactivated IB virus is H120 and is administered at about 10 6.9 EID 50 The IBDV antigen is present in an amount of about 10 7.2 TCID 50 The strain Lukert is present in an amount of about 10 2.95 EID 50 The inactivated IBD virus of strain 28-1 was present in an amount of about 10 μg / ml. The inactivated TRT virus was Clone K and was administered at about 10 μg / ml per dose. 5.8 TCID 50 The inactivated Newcastle disease virus is present in an amount of about 10 8.1 EID 50 The inactivated reovirus antigen comprises inactivated reovirus strains 1733 and 2408 and is administered at a dose of about 10 6.9 TCID 50 The vaccine is formulated as a W / O emulsion containing about 49.9% light mineral oil, about 5.85% (sorbitan sesquioleate), about 3.25% 80 and about 10 μg of CpG solution (SEQ ID NO: 8). This type of vaccine has recently been developed in Brazil under the name MATERNAVAC ULTRA 5 is approved. Preferably, the vaccine is administered to hens about 2 to 4 weeks prior to egg laying, wherein the hens have been primed as described in Table 1.

[0144] The following examples are presented as illustrative embodiments, but should not be considered to limit the scope of the present invention. Many changes, variations, modifications and other uses and applications of the present invention will be obvious to those skilled in the art.

[0145] Examples

[0146] Example 1: Effect of maternal vaccination on antibody levels in offspring

[0147] Three hundred 300 Novogen White breeder hens (100 / group) + 36 roosters (10 / group + 6 extra roosters) were used in the study. Birds showing any signs of disease or physical abnormality were excluded from the study. The birds were individually identified using numbered rings on their wings.

[0148] During the entire experimental period, the animals were housed in a 2 The hens are kept in boxes (3.20 x 1.10 x 1.85 m) and are allowed to be 6.25 hens / m 2 In each box, 20 hens and 2 roosters were housed, for a total of 5 boxes per treatment group.

[0149] The boxes were mounted on a concrete floor with hexagonal plastic coops equipped with nipple drinkers (one for every 10 hens) and bell feeders (one for every 20 hens). The feed was formulated to meet the nutritional requirements of the birds. The water provided to the animals was sufficient for the birds' consumption.

[0150] The floor is covered with wood shavings litter. Temperature control is performed using fans and curtain management to ensure thermal comfort for the birds according to their age. A digital thermo-hygrometer is used to record daily temperature and humidity.

[0151] The amount of light provided (hours / day) is sufficient for the species and age of the animal (approximately 10-16 hours / day).

[0152] Towards the start of egg laying, individual nests (one opening for every 5 hens) were installed at a height of 15 cm from the ground to reduce dirtiness inside.

[0153] The hens were initially vaccinated according to Table 1 and divided into three groups. The first group was vaccinated with a placebo at 14 weeks of age. The second group was vaccinated with MATERNAVAC ULTRA 5 vaccine, which contains:

[0154] a) 10 per dose 6.9 EID 50 The amount of inactivated IB of strain H120;

[0155] b) 10 per dose 7.2 TCID 50 The amount of strain Lukert and each dose is 10 2.95 EID 50 An amount of inactivated IBD virus of strain 28-1;

[0156] c) 10 per dose 5.8 TCID 50 The amount of inactivated TRT virus of Clone K;

[0157] d) 10 per dose 8.1 EID 50 The amount of inactivated ND virus of strain Lasota;

[0158] e) 10 per dose 6.9 TCID 50 The total amount of inactivated reovirus antigen (inactivated reovirus strains 1733 and 2408).

[0159] The vaccine is formulated as a W / O emulsion containing 49.9% light mineral oil, 5.85% (Sorbitan sesquioleate), 3.25% 80 and 10 μg of CpG solution (SEQ ID NO: 8).

[0160] The vaccine was administered intramuscularly (0.5 mL).

[0161] The third group was vaccinated at 14 weeks of age with a commercial vaccine formulated as a water-in-oil (W / O) emulsion containing inactivated infectious bronchitis virus, inactivated infectious bursal disease virus, infectious turkey rhinotracheitis virus, inactivated Newcastle disease virus, and inactivated reovirus. The commercial vaccine was administered intramuscularly (0.5 mL).

[0162] Hens lay eggs about 2 weeks after the last vaccination, and chickens hatch after 3 weeks. Blood is collected from one-day-old chickens (60 per group) to determine the level of antibodies against the antigens in the vaccine. The level of antibodies is determined by commercial ELISA kits (IDEXX IBD Ab kit, IDEXX REO Ac kit, BIOCHEK CK119 IBV, IDEXX NDV Ac, BIOCHEK CK120 ART).

[0163] result

[0164] The results are summarized in Table 2

[0165]

[0166] Serological comparison showed that Chickens from breeders that received MATERNAVAC ULTRA 5 presented mean log2 anti-IB antibody titers above the protection cutoff, whereas in chickens from breeders that received the commercial vaccine or placebo, anti-IB antibody titers were below the protection cutoff.

[0167] In addition, from accepting Chickens from the MATERNAVC ULTRA 5 breeders presented significantly higher mean log2 antibody titers than the competitor and placebo for IBDV (13.00, 12.17, and 9.33, respectively), IBV (10.93, 9.5, and 8.41), and TRT (13.72, 12.41, and 11.95). There were numerical differences between MATERNAVC ULTRA 5 and the competitor vaccine, but no significant differences were observed for these scores (95% CI).

[0168] These data demonstrate that compared to the commercial vaccines tested in this example Advantages of MATERNAVAC ULTRA5 in protecting offspring against infectious bronchitis, IBDV and TRT.

[0169] All publications (both patent publications and non-patent publications) cited in this specification are indicative of the technical level of those skilled in the art to which the invention pertains. All of these publications are incorporated herein by reference in their entirety, to the same extent as if each individual publication was expressly and individually indicated to be incorporated by reference.

[0170] Although the present invention has been described herein with reference to specific 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 many modifications may be made to the illustrative embodiments and other arrangements may be designed without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

1. A method for protecting the offspring of a hen from IBV infection, the method comprising administering to the hen a vaccine comprising an antigen component and an adjuvant component, wherein the antigen component comprises an inactivated IB virus, and the adjuvant component consists of oil, a CpG-containing immunostimulatory oligonucleotide and optionally one or more emulsifiers, wherein further, the vaccine is a W / O emulsion.

2. The method of claim 1, wherein each dose of the vaccine contains about 10 6.7 EID 50 to about 10 7.9 EID 50 of the inactivated IB virus.

3. The method of claim 1 or 2, wherein the antigenic component further comprises one or more of an inactivated TRT virus and an inactivated IBD virus, and wherein the vaccine protects the offspring from TRT infection and / or IBDV infection, respectively.

4. The method of claim 3, wherein the antigenic component comprises the inactivated TRT virus and the inactivated IBD virus, and wherein the vaccine protects the offspring from both TRT infection and IBDV infection.

5. The method according to claim 3 or 4, wherein the vaccine contains about 10 5.5 TCID 50 to about 10 6.8 TCID 50 of the inactivated TRT virus and / or about 10 6.9 TCID 50 to about 10 8.2 TCID 50 of the inactivated IBD virus.

6. The method of claim 3 or 4, wherein the vaccine comprises about 10 6.9 TCID 50 to about 10 7.7 TCID 50 The amount of inactivated IBD virus strain Lukert and about 10 2.5 EID 50 to about 10 3.5 EID 50 amount of inactivated IBD virus strain 28-1.

7. The method according to any one of claims 1 to 6, wherein the antigenic component further comprises inactivated NDV virus and / or inactivated reovirus, and wherein the vaccine protects the offspring from NDV infection and / or reovirus infection, respectively.

8. The method of claim 7, wherein the antigenic component comprises inactivated NDV virus and inactivated reovirus, and wherein the vaccine protects the offspring from NDV infection and reovirus infection.

9. The method of claim 7 or 8, wherein the vaccine comprises about 10 7.8 EID 50 to about 10 9.1 EID 50 of the inactivated NDV virus and / or about 10 6.4 TCID 50 to about 10 7.9 TCID 50 of the inactivated reovirus.

10. The method according to any one of claims 1 to 9, wherein the vaccine comprises: a) About 10 per dose 6.9 EID 50 The inactivated IB (strain H120); b) About 10 per dose 7.2 TCID 50 Lukert's inactivated IBD virus and approximately 10 2.95 EID 50 Inactivated IBD virus strain 28-1; c) Approximately 10 5.8 TCID 50 Inactivated TRT virus from Clone K; d) About 10 per dose 8.1 EID 50 The inactivated ND virus strain Lasota; e) A total of about 10 6.9 TCID 50 Inactivated reovirus strains 1733 and 2408.

11. The method according to any one of claims 1 to 10, wherein the CpG-containing immunostimulatory oligonucleotide is a P-class immunostimulatory oligonucleotide.

12. The method of claim 11, wherein the P-class immunostimulatory oligonucleotide comprises a phosphorothioate bond.

13. The method of claim 11 or claim 12, wherein the P-class immunostimulatory oligonucleotide is 5'I-modified or 5'J-modified.

14. The method according to any one of claims 1 to 13, wherein the immunostimulatory oligonucleotide comprises SEQ ID NO:

8.

15. The method of any one of claims 1 to 14, wherein the vaccine contains about 5 to about 20 μg of the CpG-containing immunostimulatory oligonucleotide per dose.

16. The method of any one of claims 1 to 15, wherein the oil is a light mineral oil.

17. The method of any one of claims 1 to 16, wherein the vaccine comprises about 49.9% oil v / v.

18. The method according to any one of claims 1 to 17, wherein the hens are vaccinated about 14 days prior to laying eggs.

19. The method according to any one of claims 1 to 18, wherein the hens have been initially vaccinated according to a regimen comprising: a) At 1, 3 and 8 weeks of age – administration of live antigens against Newcastle disease virus, infectious bronchitis and infectious bursal disease virus; b) At 6 and 10 weeks – administration of live turkey rhinotracheitis antigen; c) At week 12 – Administer live reovirus antigen.

20. The method of any one of claims 1 to 19, wherein the hen is about 14 to about 22 weeks old.

21. A vaccine for vaccinating hens, the vaccine comprising an antigen component and an adjuvant component, wherein the antigen component comprises inactivated IB virus, and the adjuvant component consists of oil, a CpG-containing immunostimulatory oligonucleotide and optionally one or more emulsifiers, wherein further, the vaccine is a W / O emulsion, and the vaccine is used to protect the offspring of the hen from IBV infection.

22. The vaccine of claim 21, wherein each dose of the vaccine contains about 10 6.7 EID 50 to about 10 7.9 EID 50 of the inactivated IB virus.

23. The vaccine of claim 21 or 22, wherein the antigenic component further comprises one or more of inactivated TRT virus and inactivated IBD virus, and wherein the vaccine protects the offspring from TRT infection and / or IBDV infection, respectively.

24. The vaccine of claim 23, wherein the antigenic component comprises the inactivated TRT virus and the inactivated IBD virus, and wherein the vaccine protects the offspring from both TRT infection and IBDV infection.

25. The vaccine according to claim 23 or 24, wherein the vaccine contains about 10 5.5 TCID 50 to about 10 6.8 TCID 50 of the inactivated TRT virus and / or about 10 6.9 TCID 50 to about 10 8.2 TCID 50 of the inactivated IBD virus.

26. The method of claim 23 or 24, wherein the vaccine comprises about 10 6.9 TCID 50 to about 10 7.7 TCID 50 The amount of inactivated IBD virus strain Lukert and about 10 2.5 EID 50 to about 10 3.5 EID 50 amount of inactivated IBD virus strain 28-1.

27. The vaccine according to any one of claims 21 to 26, wherein the antigenic component further comprises inactivated NDV virus and / or inactivated reovirus, and wherein the vaccine protects the offspring from NDV infection and / or reovirus infection, respectively.

28. The vaccine of claim 27, wherein the antigenic component comprises inactivated NDV virus and inactivated reovirus, and wherein the vaccine protects the offspring from NDV infection and reovirus infection.

29. The vaccine according to claim 27 or 28, wherein the vaccine contains about 10 7.8 EID 50 to about 10 9.1 EID 50 of the inactivated NDV virus and / or about 10 6.4 TCID 50 to about 10 7.9 TCID 50 of the inactivated reovirus.

30. The vaccine according to any one of claims 21 to 29, wherein the vaccine comprises: a) About 10 per dose 6.9 EID 50 The inactivated IB (strain H120); b) About 10 per dose 7.2 EID 50 Lukert's inactivated IBD virus and approximately 10 2.95 EID 50 Inactivated IBD virus strain 28-1; c) About 10 per dose 5.8 EID 50 Inactivated TRT virus from Clone K; d) About 10 per dose 8.1 EID 50 Inactivated ND virus (strain Lasota); e) A total of about 10 6.9 EID 50 of inactivated reovirus (inactivated reovirus strains 1733 and 2408).

31. A vaccine according to any one of claims 21 to 30, wherein the CpG-containing immunostimulatory oligonucleotide is a P-class immunostimulatory oligonucleotide.

32. The vaccine of claim 31 , wherein the P-class immunostimulatory oligonucleotide comprises a phosphorothioate bond.

33. The vaccine of claim 31 or 32, wherein the P-class immunostimulatory oligonucleotide is 5'I-modified or 5'J-modified.

34. The vaccine of any one of claims 21 to 33, wherein the immunostimulatory oligonucleotide comprises SEQ ID NO:

8.

35. The vaccine of any one of claims 21 to 34, wherein the vaccine contains about 5 to about 20 μg of the CpG-containing immunostimulatory oligonucleotide per dose.

36. The vaccine of any one of claims 21 to 35, wherein the oil is light mineral oil.

37. The vaccine of any one of claims 21 to 36, wherein the vaccine comprises about 49.9% oil v / v.

38. The vaccine of any one of claims 21 to 37, wherein the hen is vaccinated about 14 days prior to laying eggs.

39. A vaccine according to any one of claims 21 to 38, wherein the hens have been initially vaccinated according to a regimen comprising: a) At 1, 3 and 8 weeks of age – administration of live antigens against Newcastle disease virus, infectious bronchitis and infectious bursal disease virus; b) At weeks 6 and 10 – administration of live turkey rhinotracheitis antigen; c) At week 12 – Administer live reovirus antigen.

40. The vaccine of any one of claims 21 to 39, wherein the hen is about 14 to about 22 weeks old.

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