Poultry vaccines and methods for protecting poultry

Abstract

A method of protecting progeny of hens against infectious bronchitis infection is provided, the method comprising administering to the hens a vaccine comprising an inactivated infectious bronchitis virus and adjuvanted 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 technology]

[0002] Commercial poultry farming can be roughly divided into three specialties: layer chickens, breeders, and broiler production. Animals in all of these groups are vulnerable to many viral, bacterial, and parasitic infections.

[0003] Avian infectious bronchitis (IB) is perhaps one of the most widespread poultry diseases worldwide due to its highly contagious nature. It is caused by a gammacoronavirus that affects the respiratory, urinary, and reproductive systems of chickens, causing different disorders depending on the tissue tropism characteristics of the invading virus strain. Diagnosis of infectious bronchitis presents a challenge for disease control, as respiratory clinical signs are common in other poultry diseases.

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

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

[0006] In chickens up to 4 weeks of age, IB manifests in the form of severe respiratory signs (sneezing, coughing, and rasping). Rhinitis and conjunctivitis, depression, and congestion around heat sources have been observed.

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

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

[0009] In certain embodiments of this first aspect, the vaccine contains about 10 6.7 EID 50 ~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 the vaccine protects progeny from TRT infection and / or IBDV infection, respectively. In some embodiments, the vaccine comprises about 10 5.5 TCID 50 ~about 10 6.8 TCID 50 of the inactivated TRT virus, and / or about 10 6.9 TCID 50 ~about 10 8.2 TCID 50 Contains inactivated IBD virus.

[0010] In additional embodiments applicable to the method of any of the embodiments recited above, the antigen component further comprises an inactivated NDV virus and / or an inactivated reovirus, and the vaccine protects offspring from NDV infection and / or reovirus infection, respectively. In certain embodiments, the vaccine is administered at a concentration of about 10 7.8 EID 50 ~about 10 9.1 EID 50of the inactivated NDV virus, and / or about 10 6.4 TCID 50 ~about 10 7.9 TCID 50 Contains inactivated reovirus.

[0011] In a most preferred embodiment of the method according to the first aspect, the vaccine comprises: a) Approximately 10 per dose 6.9 EID 50 Inactivated IB (strain H120), b) Approximately 10 per dose 7.2 TCID 50 strain of Lukert inactivated IBD virus, and approximately 10 per dose 2.95 EID 50 Inactivated IBD virus strain 28-1, c) Approximately 10 5.8 TCID 50 Inactivated TRT virus of clone K, d) approximately 10 per dose 8.1 EID 50 Inactivated ND virus strain LaSota, e) Approximately 10 per total dose 6.9 TCID 50 These include inactivated reovirus strains 1733 and 2408.

[0012] In a subset of embodiments applicable to any of the methods of this first aspect of the invention, the CpG-containing immunostimulatory oligonucleotide is preferably a P class immunostimulatory oligonucleotide comprising phosphorothioate linkages and / or I or J modifications at the 5' end. Most preferably, the CpG-containing immunostimulatory oligonucleotide comprises SEQ ID NO:8.

[0013] In certain embodiments, the vaccine administered according to the method of this first aspect comprises about 5 to about 20 μg of the CpG-containing immunostimulatory oligonucleotide per dose.

[0014] In a subset of embodiments of this first aspect, the vaccine according to any of the embodiments of this first aspect comprises: a) 1, 3, and 8 weeks of age - Administration of live antigens against Newcastle disease virus, infectious bronchitis, and infectious bursal disease virus b) Weeks 6 and 10 - administration of live turkey rhinotracheitis antigen; c) Week 12 - administered to hens that have been primovaccinated according to a regimen that includes administration of live reovirus antigen.

[0015] In a second aspect, the present disclosure provides a vaccine for vaccinating hens, for use in protecting progeny of the hens against IBV infection, the vaccine comprising an antigen component and an adjuvant component, wherein the antigen component comprises inactivated IB virus, the adjuvant component consists of oil, CpG-containing immunostimulatory oligonucleotides, and optionally one or more emulsifiers, and further wherein the vaccine is a W / O emulsion.

[0016] In certain embodiments of this second aspect, the vaccine contains about 10 6.7 EID 50 ~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 the vaccine protects progeny from TRT infection and / or IBDV infection, respectively. In some embodiments of this second aspect, the vaccine comprises about 10 5.5 TCID 50 ~about 10 6.8 TCID 50 of the inactivated TRT virus, and / or about 10 6.9 TCID 50 ~about 10 8.2 TCID 50 Contains inactivated IBD virus.

[0017] In additional embodiments applicable to the vaccine of any of the embodiments of this second aspect, as listed above, the antigen component further comprises an inactivated NDV virus and / or an inactivated reovirus, and the vaccine protects offspring from NDV infection and / or reovirus infection, respectively. In certain embodiments, the vaccine protects offspring from about 10 7.8 EID 50 ~about 10 9.1 EID 50 of the inactivated NDV virus, and / or about 10 6.4 TCID 50 ~about 10 7.9 TCID 50 Contains inactivated reovirus.

[0018] In a most preferred embodiment of the vaccine according to the second aspect, the vaccine comprises: a) Approximately 10 per dose 6.9 EID 50 Inactivated IB (strain H120), b) Approximately 10 per dose 7.2 TCID 50 strain of Lukert inactivated IBD virus, and approximately 10 per dose 2.95 EID 50 Inactivated IBD virus strain 28-1, c) Approximately 10 5.8 TCID 50 Inactivated TRT virus of clone K, d) approximately 10 per dose 8.1 EID 50 Inactivated ND virus strain Lasota, e) Approximately 10 per total dose 6.9 TCID 50 These include inactivated reovirus strains 1733 and 2408.

[0019] In a subset of embodiments applicable to any of the vaccines of this second aspect of the invention, the CpG-containing immunostimulatory oligonucleotide is preferably a P class immunostimulatory oligonucleotide comprising a phosphorothioate linkage and / or an I or J modification at the 5' end. Most preferably, the CpG-containing immunostimulatory oligonucleotide comprises SEQ ID NO:8.

[0020] In certain embodiments of this second aspect, the vaccine comprises about 5 to about 20 μg of the CpG-containing immunostimulatory oligonucleotide per dose.

[0021] In a subset of embodiments, the vaccine according to any of the embodiments of this second aspect comprises: a) 1, 3, and 8 weeks of age - administration of live antigens against Newcastle disease virus, infectious bronchitis virus, and infectious bursal disease virus b) Weeks 6 and 10 - administration of live turkey rhinotracheitis antigen; c) Week 12 - administered to hens that have been initially vaccinated according to a regimen that includes administration of live reovirus antigen. DETAILED DESCRIPTION OF THE INVENTION

[0022] The terms "about" or "approximately," when used in connection with a measurable, numerical variable, refer to the stated value of the variable and all values ​​of the variable that are within experimental error of the stated value (e.g., within a 95% confidence interval about the mean) or within 10 percent of the stated value, whichever is greater.

[0023] Terms such as "consisting essentially of" as applied to the adjuvant formulations of the invention refer to vaccines and other compositions that do not contain added adjuvants or immunomodulatory agents in amounts such that the agent exerts a measurable adjuvant or immunomodulatory effect.

[0024] The term "conventional prime vaccine" refers to a vaccine administered to hens when they are between 1 and about 100 days old. Conventional prime vaccines are generally modified live vaccines containing attenuated viruses. Several conventional prime vaccines are commercially available, including, but not limited to, POULVAC® TRT, POULVAC® BURSA F, New Ls Mass I, Coryza Gel, POULVAC® MAGNIPLEX, VAXXITEK® HVD-IBD, BIORAL® H120, CEVAC® BRON 120L, CEVAC® IBD, CEVAC® TRANSMUNE IBD, MEVAC® ND HB1, and BUR® 706R.

[0025] The term "hen" refers to a female chicken that is the mother of offspring for which protection is achieved by administering to said hen a vaccine disclosed herein. Prior to administration of the inactivated vaccine disclosed herein, the hen has undergone an initial vaccination.

[0026] The phrase "laying eggs" refers to hens vaccinated with the vaccine of the present invention laying eggs from which the protected offspring hatch.

[0027] The term "initial vaccination" as applied to a hen receiving a vaccine recited herein refers to vaccination of the hen with one or more doses when the hen is between 1 and about 98 days of age, preferably between 1 and about 91 days of age, more preferably between 1 and about 84 days of age, with a conventional prime vaccine containing the same antigens as those of the vaccine recited herein used to administer to the hen.

[0028] In certain embodiments, hens are initially vaccinated with two or more doses, preferably three doses, of live infectious bronchitis virus.

[0029] In another embodiment, the hens have been initially vaccinated with two or more doses, preferably three doses, of live infectious bronchitis virus, and a) one or more administrations, preferably two administrations, of live TRT virus; and b) has been initially vaccinated with at least one of two or more doses, preferably three doses, of live IBD virus;

[0030] In another embodiment, the hens have been initially vaccinated with two or more doses, preferably three doses, of live infectious bronchitis virus, and a) one or more administrations, preferably two administrations, of live TRT virus; and b) two or more administrations, preferably three administrations, of a live IBD virus; c) two or more doses, preferably three doses, of live Newcastle disease virus; d) has been initially vaccinated with at least one dose of live avian reovirus.

[0031] In certain embodiments, the hens are a) two or more administrations, preferably three administrations, of live infectious bronchitis virus; b) one or more administrations, preferably two administrations, of live TRT virus; c) two or more administrations, preferably three administrations, of live IBD virus; d) two or more administrations, preferably three administrations, of live Newcastle disease virus; e) has been initially vaccinated with at least one dose of live avian reovirus;

[0032] In a particular subset of embodiments, the hens are a) three doses of live infectious bronchitis virus; b) two doses of live TRT virus; c) three doses of live IBD virus; d) three doses of live Newcastle disease virus; e) have been initially vaccinated with a single dose of live avian reovirus;

[0033] In certain embodiments, the primary vaccinated hens are vaccinated against IBDV, IBV, Newcastle disease virus, TRT, and reovirus according to the following schedule: [Table 1]

[0034] Thus, before being vaccinated with a vaccine according to the 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, 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.

[0035] The term "same antigen" as used in the conventional prime vaccine and the vaccine used to administer to hens refers to the property of the antigen in the inactivated vaccine used to administer to hens to boost the immune response to the antigen in the conventional prime vaccine.

[0036] The term "parenteral administration" refers to the introduction of a substance, such as a vaccine, into the body of a subject through or by a route that does not involve the digestive tract. Parenteral administration includes subcutaneous, intramuscular, transdermal, intradermal, intraperitoneal, intraocular, and intravenous administration.

[0037] A purity percentage or "X percent pure" as applied to an immunostimulatory oligonucleotide preparation refers to a population of oligonucleotide molecules that contains X% of the named oligonucleotide (e.g., SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, etc.), with the remainder (i.e., 100% minus X%) comprising shorter fragments of the named oligonucleotide present as impurities during production of the named sequence. Thus, if the sequence is produced by 3'-5' sequencing, the 5' truncation will comprise the remainder. As a non-limiting example, a 100 μg preparation of 80% pure SEQ ID NO:8 would contain 80 μg of SEQ ID NO:8, with the remaining 20 μg being shorter fragments of SEQ ID NO:8 present in the preparation.

[0038] The terms "therapeutically effective amount," "immunologically effective amount," and "effective amount" refer to an amount of an antigen, adjuvant, or vaccine that will induce an immune response in a subject receiving the antigen, adjuvant, or vaccine that is sufficient to prevent or reduce signs or symptoms of disease, including adverse health effects or complications, caused by infection with a pathogen, such as a virus or bacteria. Humoral or cell-mediated immunity, or both humoral and cell-mediated immunity, may be induced. The immunogenicity and efficacy of a vaccine in an animal can be assessed indirectly, for example, through measurement of antibody titers, lymphocyte proliferation assays, or directly, through monitoring signs and symptoms after challenge with a wild-type strain. Protective immunity conferred by a vaccine can be assessed, for example, by measuring reductions in clinical signs, such as mortality, morbidity, body temperature, overall physical condition, and overall health and performance of the subject. The amount of a vaccine that is therapeutically effective may vary depending on the particular adjuvant used, the particular antigen used, or the condition of the subject, and can be determined by one of skill in the art.

[0039] The terms "vaccine," "vaccine enumerated herein," "vaccine disclosed herein," "killed vaccine," and the like specifically refer to an immunogenic composition that elicits a protective immune response in offspring and comprises the enumerated inactivated antigens, formulated as a water-in-oil emulsion containing oil, immunostimulatory CpG oligonucleotides, and optionally an adjuvant comprising (or consisting essentially of, or consisting of) one or more emulsifiers. This term specifically excludes traditional prime vaccines used for the initial vaccination of hens.

[0040] antigen The vaccines described herein preferably contain inactivated viral antigens. In certain embodiments, the vaccine contains inactivated infectious bronchitis virus (IB or IBV). Several IB strains are known, including but not limited to the Massachusetts strain, the Brazilian IBV variant strain (BR-01), M41, D1466, and ARK99, 793B, QX, GA08, VAR2, Beaudette, Holte, Gray, N1 / 62, VicS, TP / 64, L165, ARK99, B, UFMG / G, D3896, Moroccan-G / 8 and others such as 3, B1648, B4, IZO28 / 86, CA / Machado / 88, JP8127, 58HeN-93II, Qu_mv, Spain / 97 / 314, 40GDGZ-97I, variant 2, V13, CA / 1737 / 04, NGA / B401 / 2006, GA08, N1 / 88, DE / 072 / 92, N4 / 02, TC07-2. In a preferred embodiment, the strain is a Massachusetts strain (e.g., H120 or M41 or Ma5 or other isolate) 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 of the 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.

[0041] In other embodiments, the strain is of the 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.

[0042] In still other embodiments, the strain is of the 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.

[0043] Inactivated IB virus should be at least 10 per dose 6.4 EID 50 , more preferably at least about 10 per dose 6.7 EID 50 , more preferably at least about 10 per dose 6.8 EID 50 , more preferably about 10 per dose 6.9 EID 50 ~approximately 10 per dose 7.9 EID 50 , and even more preferably about 10 per dose 6.9 EID 50 ~approximately 10 per dose 7.3 EID 50 Thus, in a different embodiment, an inactivated IB virus, such as Massachusetts strain H120, may be present in an amount of 10 per dose. 6.4 EID 50 , or about 10 per dose 6.5 EID 50 , or about 10 per dose 6.6 EID50, or approximately 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 EID50 , 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 10 per dose 7.9 EID 50 may be present in the vaccine in an amount of

[0044] The amount of inactivated IB and other inactivated antigens present in the vaccines described herein is the amount per dose before inactivation of the antigen.

[0045] In addition to an IB antigen according to any of the embodiments described above, the vaccine may 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.

[0046] Thus, in certain embodiments, the vaccines described herein contain an inactivated IB virus according to any of the embodiments described above, as well as one or more of an inactivated TRT virus and an inactivated IBD virus.

[0047] Several 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, BUT1#8544, PL21, and TRT50. In a more preferred embodiment, the TRT strain is Clone K. The inactivated TRT virus is administered in a concentration of at least 10 per dose. 5.5 TCID 50 , more preferably at least 10 per dose 5.6 TCID 50 , more preferably at least 10 per dose 5.7 TCID 50 , more preferably about 10 per dose5.8 TCID 50 ~approximately 10 per dose 6.8 TCID 50 , and even more preferably about 10 per dose 5.8 TCID 50 ~approximately 10 per dose 6.4 TCID 50 may be present in an amount of

[0048] Several 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, Winterfield2512, Moulthrop G603, variant 1084-E, S706, VNJO, variant E, LIBDV, GP82, GM97, CH / 80, 228E, D78, MB, V877, and GBV-8. In other embodiments, the IBDV strain may 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 is at least 10 per dose. 6.9 TCID 50 , more preferably at least 10 per dose 7.0 TCID 50 , more preferably at least 10 per dose 7.1 TCID 50 , more preferably about 10 per dose 7.2 TCID 50 ~approximately 10 per dose 8.2 TCID 50 , and even more preferably about 10 per dose 7.2 TCID 50 ~approximately 10 per dose 7.6 TCID 50 may be present in an amount of

[0049] In a more preferred embodiment, the vaccine comprises at least two strains of IBDV, more preferably Lukert and 28-1. When inactivated IBDV viruses of both the Lukert and 28-1 strains are present in the vaccine, the Lukert strain IBD virus is present in 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 ~about 10 7.7 TCID 50 , or about 10 7.2 TCID 50 ~about 10 7.5 TCID 50 The inactivated IBD virus of strain 28-1 may be present in an amount of at least 10 per dose. 2.5 EID 50 ~about 10 3.5 EID 50 , or about 10 per dose 2.6 ~about 10 3.4 EID 50 , or about 10 2.7 ~about 10 3.3 EID 50 , or about 10 2.8 ~about 10 3.2 EID 50 , or about 10 2.9 ~about 10 3.1 EID 50 , or about 10 2.9 ~about 10 3.0 EID 50 , or about 10 2.95 EID 50 may be present in an amount of

[0050] In other embodiments, the vaccines described herein contain one or more of the inactivated IB viruses described above, as well as inactivated Newcastle disease virus and inactivated reovirus.

[0051] Several NDV strains are known, including, but not limited to, Lasota, F, B1, V4, V4-HR, I-2, Mukteswar, Komarov, LZ, Miyadera, AF2240, HER / 33, Texas, Ulster, H, and clone 30. In certain embodiments, the NDV strain is LaSota or another lentogenic strain.

[0052] Inactivated NDV virus was administered at a concentration of at least 10 per dose. 7.8 EID 50 , more preferably at least about 10 per dose 7.9 EID 50 , more preferably at least about 10 per dose 8.0 EID 50 , more preferably about 10 per dose 8.1 EID 50 ~approximately 10 per dose 9.1 EID 50 , and even more preferably about 10 per dose 8.1 EID 50 ~approximately 10 per dose 8.5 EID 50 may be present in an amount of

[0053] Multiple 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 is present in a total of at least 10 per dose. 6.4 TCID 50 , more preferably at least about 10 per dose 6.7 TCID 50 , more preferably at least about 10 per dose 6.8 TCID 50 , more preferably about 10 per dose 6.9 TCID 50 ~approximately 10 per dose 7.9 TCID 50, and even more preferably about 10 per dose 6.9 TCID 50 ~approximately 10 per dose 7.3 TCID 50 may be present in an amount of

[0054] In a more preferred embodiment, the vaccine contains two strains of inactivated reovirus: 1733 and 2408. These inactivated reoviruses are used in a total of at least 10 per dose. 6.4 TCID 50 , more preferably at least about 10 per dose 6.7 TCID 50 , more preferably at least about 10 per dose 6.8 TCID 50 , more preferably about 10 per dose 6.9 TCID 50 ~approximately 10 per dose 7.9 TCID 50 , and even more preferably about 10 per dose 6.9 TCID 50 ~approximately 10 per dose 7.3 TCID 50 may be present in an amount of

[0055] In a particular 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 a dose of approximately 10 6.9 EID 50 The IBDV antigen was present in an amount of strain Lukert (approximately 10 per dose). 7.2 TCID 50 ) and strain 28-1 (present in amounts of approximately 10 per dose 2.95 EID 50 The inactivated TRT virus is of the clone K strain and is present in an amount of approximately 10 per dose. 5.8 TCID 50 The inactivated Newcastle disease virus is of the LaSota strain and is present in an amount of approximately 10 per dose. 8.1 EID 50The inactivated reovirus antigen is present in an amount of about 10 per dose. 6.9 TCID 50 The virus contains inactivated reovirus strains 1733 and 2408 present in a total amount of 1733 and 2408.

[0056] Adjuvants The adjuvants of the vaccines recited herein and administered to hens comprise immunostimulatory CpG oligonucleotides, oil, and optionally one or more emulsifiers, so that the vaccines are water-in-oil (W / O emulsions).

[0057] In certain embodiments, the adjuvant consists essentially of or consists of immunostimulatory CpG oligonucleotides, oil, and optionally surfactant(s). In certain embodiments, the adjuvant consists of immunostimulatory CpG oligonucleotides, oil, and optionally surfactant(s).

[0058] 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 some embodiments, polyinosinic-cytidylic acid or its derivatives (poly I:C) may be used.

[0059] 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 specific base sequence contexts. (Hansel TT, Barnes PJ (eds): New Drugs for Asthma, Allergy and COPD. Prog Respir Res. Basel, Karger, 2001, vol. 31, pp. 229-232, which is incorporated herein by reference.) These CpG motifs are not found in eukaryotic DNA, where CG dinucleotides are suppressed and, if present, are usually methylated, but are present in bacterial DNA, where they confer immunostimulatory properties.

[0060] In selected embodiments, the adjuvants of the present invention utilize so-called P-class immunostimulatory oligonucleotides, more preferably modified P-class immunostimulatory oligonucleotides, and even more preferably E-modified P-class oligonucleotides. P-class immunostimulatory oligonucleotides are generally immunostimulatory CpG oligonucleotides characterized by the presence of a palindrome 6-20 nucleotides in length. P-class oligonucleotides have the ability to spontaneously self-assemble into concatemers either in vitro and / or in vivo. While these oligonucleotides are strictly single-stranded, the presence of the palindrome allows for the formation of concatemers or, in some cases, stem-and-loop structures. The total length of P-class immunostimulatory oligonucleotides is 19-100 nucleotides, e.g., 19-30 nucleotides, 30-40 nucleotides, 40-50 nucleotides, 50-60 nucleotides, 60-70 nucleotides, 70-80 nucleotides, 80-90 nucleotides, or 90-100 nucleotides.

[0061] In one aspect of the invention, the immunostimulatory oligonucleotide contains a 5' TLR activation domain and at least two palindromic regions, one palindromic region being a 5' palindromic region at least 6 nucleotides in length and connected, either directly or via a spacer, to a 3' palindromic region at least 8 nucleotides in length.

[0062] P class immunostimulatory oligonucleotides can be modified according to techniques known in the art. For example, J modification refers to an iodo-modified nucleotide. E modification refers to an ethyl-modified nucleotide(s). Thus, an E-modified P class immunostimulatory oligonucleotide is a P class immunostimulatory oligonucleotide in which at least one nucleotide (preferably the 5' nucleotide) is ethylated. Additional modifications include 6-nitro-benzimidazole attachment, O-methylation, proynyl-dU modification, inosine modification, and 2-bromovinyl attachment (preferably to uridine).

[0063] P class immunostimulatory oligonucleotides may also contain modified internucleotide linkages, including, but not limited to, phosphodiester and phosphorothioate linkages. The oligonucleotides of the invention may be synthesized or obtained from commercial sources.

[0064] P class oligonucleotides and modified P class oligonucleotides are further disclosed in published PCT application No. 2008 / 068638, published June 12, 2008. Suitable, non-limiting examples of modified P class immunostimulatory oligonucleotides are provided below (in SEQ ID NOS: 1-10, "*" indicates a phosphorothioate linkage and "-" indicates a phosphodiester linkage): [Table A]

[0065] The immunostimulatory oligonucleotides of the present invention can be chemically synthesized. Furthermore, the immunostimulatory oligonucleotides can be used with a purity (homogenity) of about 60% or greater (e.g., about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or 100% purity).

[0066] The amount of P class immunostimulatory oligonucleotide for use in the adjuvant composition will depend on the nature of the P class immunostimulatory oligonucleotide used and the intended species.

[0067] The vaccine of the present invention is formulated as a W / O emulsion. Multiple oils and combinations thereof are suitable for use in the present invention. These oils include, but are not limited to, animal oils, vegetable oils, and non-metabolizable oils. Non-limiting examples of vegetable oils suitable in the present invention are corn oil, peanut oil, soybean oil, coconut oil, olive oil, and phytosqualane. A non-limiting example of an animal oil is squalane. Non-limiting examples of suitable non-metabolizable oils include light mineral oils, linear or branched saturated oils, ramified oils, etc.

[0068] In one set of embodiments, the oil used in the adjuvant formulation of the present invention is a light mineral oil. As used herein, the term "mineral oil" refers to a mixture of liquid hydrocarbons obtained from petrolatum via distillation techniques. The term is synonymous with "liquid paraffin," "liquid petrolatum," and "white mineral oil." The term is also intended to include "light mineral oil," i.e., an oil similarly obtained by distillation of petrolatum but having a slightly lower specific gravity than white mineral oil. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990, pp. 788 and 1323). Mineral oil can be obtained from various commercial sources, e.g., JT Baker (Phillipsburg, Pa.), USB Corporation (Cleveland, Ohio). A preferred mineral oil is light mineral oil commercially available under the name DRAKEOL®. In another embodiment, a suitable oil includes mineral oil MARCOL® 52. MARCOL™ 52 is a refined mixture of liquid saturated hydrocarbons. It is a clear, water-white product containing no toxic impurities. It is obtained from petroleum by vacuum distillation with subsequent refining steps including final purification by catalytic hydrogenation.

[0069] Suitable emulsifiers for use in the emulsions of the present invention include natural biologically compatible emulsifiers and non-natural synthetic surfactants. Biologically compatible emulsifiers include phospholipid compounds or mixtures of phospholipids. A preferred phospholipid is phosphatidylcholine (lecithin), such as soybean or egg lecithin. Lecithin can be obtained as a mixture of phosphatides and triglycerides by washing crude vegetable oil with water and separating and drying the resulting hydrated gum. Refined products can be obtained by fractionating the mixture for acetone-insoluble phospholipids and glycolipids remaining after removing triglycerides and vegetable oil by acetone washing. Alternatively, lecithin can be obtained from a variety of commercial sources. Other suitable phospholipids include phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid, cardiolipin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidylethanolamine. Phospholipids can be isolated from natural sources or conventionally synthesized.

[0070] In additional embodiments, the emulsifiers used herein are free of lecithin or use lecithin in amounts that are not immunologically effective.

[0071] Suitable non-natural synthetic emulsifiers for use in the adjuvant formulations of the invention include sorbitan-based nonionic surfactants, such as fatty acid-substituted sorbitan surfactants (commercially available under the names SPAN® or ARLACEL®), polyethoxylated fatty acid esters of sorbitol (TWEEN®), polyethylene glycol esters of fatty acids from sources such as castor oil (EMULFOR®), polyethoxylated fatty acids (e.g., stearic acid, available commercially under the name SIMULSOL® M-53), polyethoxylated isooctylphenol / formaldehyde polymers (TYLOXAPOL®), polyoxyethylene fatty alcohol ethers (BRIJ®), polyoxyethylene nonphenyl ether (TRITON® N), polyoxyethylene isooctylphenyl ether (TRITON® X). Preferred synthetic surfactants are those available under the SPAN® and TWEEN® names, such as TWEEN®-80 (polyoxyethylene (20) sorbitan monooleate) and ARLACEL® 83V (sorbitan sesquioleate).

[0072] Generally speaking, the emulsifier(s) may be present in the vaccine in an amount of from 0.01% to 40% by volume, preferably from 0.1% to 15%, more preferably from 2% to 10%.

[0073] In a subset of embodiments, the volume percentage of the oil and oil-soluble emulsifier combined is at least 50% by volume, e.g., 50% to 95% by volume, preferably in an amount greater than 50% to 85%, more preferably in an amount greater than 50% to 60%, and more preferably in an amount of 53 to 58% v / v of the vaccine. Thus, for example and without limitation, the oil may be present in an amount of 45% and the lipid-soluble emulsifier would be present in an amount greater than 5% v / v. Thus, the volume percentage of the oil and oil-soluble emulsifier combined will be at least 50%.

[0074] In yet another subset applicable to all vaccines of the invention, the volume percentage of oil is greater than 40% by volume of the vaccine, for example, 40% to 90%, 40% to 85%, 43% to 60%, 44% to 50% v / v, or 45% to 55% v / v, hi certain embodiments, the emulsion contains at least 48% v / v oil phase and 52% v / v aqueous phase.

[0075] Sometimes, concentrating antigens is not possible or feasible, especially in scaled-up commercial applications, and low-concentration antigen solutions must be used. Thus, in some embodiments, vaccines of the invention comprise the adjuvant formulations described above, wherein the content of the oily phase in these adjuvant formulations is diluted, and the vaccine is a water-in-oil emulsion.

[0076] In practice, it is possible to create water-in-oil emulsions 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 form more concentrated than the maximum packing ratio for random packing of monodisperse droplets, i.e., 0.64. Tadros, Emulsion Formation, Stability and Rheology, 1 st ed. 2013, Wiley-VCH GmbH & Co. KGaA. As long as the total volume fraction occupied by the aqueous droplets does not exceed 0.64, i.e., 64% v / v. Conversely, this suggests that the oily phase should not fall below 36% v / v.

[0077] In some preferred embodiments, a single dose of adjuvant will contain about 0.1 to about 20 μg (e.g., 1 to 20 μg, or about 5 to about 15 μg, or about 8 to about 12 μg, or about 10 μg) of immunostimulatory oligonucleotide and up to about 50 μg (e.g., 0.5 to 20 μg, or 1 to 10 μg) of a sterol, such as cholesterol.

[0078] In certain embodiments, the adjuvant component is prepared as follows: a) Dissolve sorbitan sesquioleate and cholesterol, if present, in light mineral oil. Sterile filter the resulting oil solution; b) dissolving the immunostimulatory oligonucleotide and polyoxyethylene(20) sorbitan monooleate in the aqueous phase, thus forming an aqueous solution; c) The aqueous solution is added to the oil solution under continuous homogenization.

[0079] The vaccines of the present invention may be prepared by adding the antigen component to the aqueous phase and subsequently combining the aqueous phase with the oil phase. In other embodiments, the antigen component may be added to the adjuvant component after the adjuvant component has been prepared.

[0080] The vaccines described herein may further comprise a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonicity agents, adsorption delaying agents, and the like. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the subject. Typically, carriers are sterile, pyrogen-free, and will be selected based on the mode of administration to be used. Those skilled in the art will recognize that preferred formulations for pharmaceutically acceptable carriers comprising the composition are pharmaceutical carriers approved by applicable regulations promulgated by the United States (US) Department of Agriculture or the US Food and Drug Administration, or equivalent governmental agencies in countries other than the United States. Thus, pharmaceutically acceptable carriers for commercial production of the composition are carriers that have already been approved or will be approved by the appropriate governmental agency in the United States or a foreign country.

[0081] Other components of the composition can include pharmaceutically acceptable excipients, such as carriers, solvents, and diluents, isotonicity agents, buffers, stabilizers, preservatives, vasoconstrictors, antibacterial agents, antifungal agents, etc. Typical carriers, solvents, and diluents include water, saline, dextrose, ethanol, glycerol, etc. Representative isotonicity agents include sodium chloride, dextrose, mannitol, sorbitol, lactose, etc. Useful stabilizers include gelatin, albumin, etc.

[0082] In a particular set of embodiments, the vaccine contains all five inactivated antigens: IB virus, IBDV, TRT virus, NDV, and reovirus. The inactivated IB virus is H120, with a concentration of about 10 per dose. 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 approximately 10 per dose. 7.2 TCID 50 The amount of inactivated 28-1 strain virus was approximately 10 2.95 EID 50 The inactivated TRT virus was clone K and was approximately 10 per dose. 5.8 TCID 50 The inactivated Newcastle disease virus is a LaSota strain virus and is present in an amount of approximately 10 per dose. 8.1 EID 50 The inactivated reovirus antigen is present in an amount of about 10 per dose. 6.9 TCID 50The vaccine contains inactivated reovirus strains 1733 and 2408 present in a total amount of 10 ...

[0083] method Generally, the vaccines of the present invention can be administered to hens via several routes, which are known to those skilled in the art and include, but are not limited to, intramuscular and subcutaneous injection.

[0084] In certain embodiments, hens are vaccinated at about 14 to about 22 weeks of age (e.g., at 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).

[0085] 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) prior to expected egg lay. This regimen ensures that immunity to the antigen(s) present in the vaccines recited herein is developed by the time of egg lay and persists throughout the egg laying period of the vaccinated hens.

[0086] The vaccines listed herein are administered to hens that have been initially vaccinated or primed with a conventional prime vaccine. In certain embodiments, the hens are initially vaccinated as described above under "Definitions." In more preferred embodiments, the hens are a) 1, 3, and 8 weeks of age - administration of live antigens against Newcastle disease virus, infectious bronchitis virus, and infectious bursal disease virus b) 6 and 10 weeks - administration of live turkey rhinotracheitis antigen; c) Week 12 - Primary vaccinated hens that have been primary vaccinated according to a regimen that includes administration of live reovirus antigen.

[0087] Several conventional prime vaccines containing different antigens are known in the art. For example, POULVAC® IB Primer contains a lyophilized live infectious bronchitis virus of the Massachusetts type. NOBILIS® IB H120 is a lyophilized live vaccine indicated for use as a primary vaccination of poultry against infectious bronchitis. This vaccine contains strain H120 Massachusetts type. Other IB Primers can also be used with the vaccine of the present invention.

[0088] Similarly, there are conventional prime vaccines that contain other antigens and combinations of antigens that may be present in the vaccines listed herein.

[0089] The antigens in these vaccines are attenuated, and the vaccines are administered according to a specific schedule to hens that are 1 day to about 100 days old (14-15 weeks) old. The hens can be 1 day to about 91 days old, 1 day to about 84 days old, 1 day to about 77 days old, or 1 day to about 70 days old.

[0090] In a most preferred embodiment, as described above, the vaccine administered in 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, with a concentration of about 10 per dose. 6.9 EID 50 The IBDV antigen is present in an amount of about 10 per dose. 7.2 TCID 50 strain Lukert, present in an amount of about 10 2.95 EID 50 The inactivated IBD virus is strain 28-1, present in an amount of approximately 10 per dose. The inactivated TRT virus is clone K, present in an amount of approximately 10 per dose. 5.8 TCID 50 The inactivated Newcastle disease virus is present in an amount of about 10 per dose. 8.1 EID 50 The inactivated reovirus antigens include inactivated reovirus strains 1733 and 2408, present in amounts of approximately 10 per dose. 6.9 TCID 50 The vaccine is present in a total amount of about 49.9% light mineral oil, about 5.85% ARLACEL® (sorbitan sesquioleate), about 3.25% TWEEN® 80, and about 10 μg of CpG solution (SEQ ID NO: 8) per dose. Such a vaccine has recently been approved in Brazil under the name POULVAC® MATERNAVAC ULTRA 5. Preferably, the vaccine is administered to hens about 2-4 weeks before egg laying, and the hens have been initially vaccinated as described in Table 1.

[0091] The following examples are presented as illustrative embodiments and should not be construed as limiting the scope of the present invention. Many variations, modifications, and other uses and applications of the present invention will be apparent to those skilled in the art. [Example]

[0092] Example 1 Effect of maternal vaccination on antibody levels in offspring The study used 300 Novogen White breeder chickens (100 per group) + 36 male chickens (10 per group + 6 spares). Birds showing any signs of disease or physical abnormalities were excluded from the study. Birds were individually identified by numbered rings on their wings.

[0093] The animals were kept at 80.0 m throughout the experimental period. 2 The chickens were kept in a shed with dimensions of 1.20 × 1.10 × 1.85 m, housed in boxes (3.20 × 1.10 × 1.85 m), and housed at a density of 6.25 hens / m 2 Each box housed 20 females and 2 males, for a total of 5 boxes per treatment group.

[0094] The boxes were mounted on a concrete floor with hexagonal plastic chicken coops and equipped with nipple drinkers (one for every 10 hens) and bell-type feeders (one for every 20 hens). The diet was formulated to meet the nutritional requirements of the birds. Water provided to the animals was adequate for consumption by the birds.

[0095] The floor was covered with shaving bedding. Temperature control was performed using fans and curtain controls to ensure the birds' thermal comfort according to their age. Daily temperature and humidity were recorded using a digital thermo-hygrometer.

[0096] The amount of light provided (hours / day) was appropriate for the category and age of the animals (approximately 10-16 hours / day).

[0097] As the start of laying approached, individual nests (one nest for every five hens) were placed 15 cm above the ground to reduce soiling inside.

[0098] The hens were initially vaccinated according to Table 1 and divided into three groups: The first group was vaccinated with placebo at week 14. The second group was vaccinated at 14 weeks of age with POULVAC® MATERNAVAC ULTRA 5 vaccine containing: a) 10 per dose 6.9 EID 50 Inactivated IB of strain H120 in the amount of b) 10 per dose 7.2 TCID 50 strain Lukert in an amount of 10 per dose 2.95 EID 50 Inactivated IBD virus of strain 28-1 in an amount of c) 10 per dose 5.8 TCID 50 Inactivated TRT virus of clone K in an amount of d) 10 per dose 8.1 EID 50 Inactivated ND virus of strain Lasota in the amount of e) 10 per dose 6.9 TCID 50 Inactivated reovirus antigen (inactivated reovirus strains 1733 and 2408) in a total amount.

[0099] The vaccine was formulated as a W / O emulsion containing 49.9% light mineral oil, 5.85% ARLACEL® (sorbitan sesquioleate), 3.25% TWEEN® 80, and 10 μg of CpG solution (SEQ ID NO: 8) per dose.

[0100] The vaccine was administered intramuscularly (0.5 mL).

[0101] A third group was vaccinated at 14 weeks of age with a commercial vaccine containing inactivated infectious bronchitis virus, inactivated infectious bursal disease virus, infectious turkey rhinotracheitis virus, inactivated Newcastle disease virus, and inactivated reovirus formulated as a water-in-oil (W / O) emulsion. The commercial vaccine was administered intramuscularly (0.5 mL).

[0102] The hens laid eggs approximately two weeks after the last vaccination, and the chickens hatched three weeks later. Blood was collected from one-day-old chickens (60 per group) to determine the level of antibodies against the antigens in the vaccine. Antibody levels were determined using commercially available ELISA kits (IDEXX IBD Ab kit, IDEXX REO Ac kit, BIOCHEK CK119 IBV, IDEXX NDV Ac, BIOCHEK CK120 ART).

[0103] result The results are summarized in Table 2. [Table 2]

[0104] Serological comparisons showed that chicks from breeders receiving POULVAC® MATERNAVAC ULTRA 5 exhibited mean log2 anti-IB antibody titers above the protective cutoff, whereas chicks from breeders receiving the commercial vaccine or placebo had anti-IB antibody titers below the protective cutoff.

[0105] Furthermore, chicks from breeders receiving POULVAC® MATERNAVC ULTRA 5 displayed significantly higher mean log2 antibody titers than 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). Numerical differences between POULVAC® MATERNAVC ULTRA 5 and competitor vaccines were detected for NDV (11.75 and 11.45, respectively) and REO (10.68 and 10.45), but no significant differences were observed for these fractions (95% CI).

[0106] These data demonstrate the superiority of POULVAC® MATERNAVAC ULTRA 5 in protecting offspring against infectious bronchitis, IBDV, and TRT compared to the commercially available vaccines tested in this example.

[0107] All publications, both patent and non-patent, cited in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and all such publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0108] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications can be made to the exemplary embodiments and that other arrangements can be devised without departing from the spirit and scope of the invention as defined by the following claims.

Claims

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

2. The vaccine is administered in an amount of about 10 per dose. ６．７ EID ５０ ~about 10 ７．９ EID ５０ 2. The method of claim 1, wherein the inactivated IB virus comprises:

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

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

5. The vaccine is about 10 ５．５ T.C.I.D. ５０ ~about 10 ６．８ T.C.I.D. ５０ of the inactivated TRT virus, and / or about 10 ６．９ T.C.I.D. ５０ ~about 10 ８．２ T.C.I.D. ５０ The method according to claim 3 or 4, wherein the inactivated IBD virus comprises:

6. The vaccine is administered in an amount of about 10 per dose. ６．９ T.C.I.D. ５０ ~about 10 ７．７ T.C.I.D. ５０ Inactivated IBD virus strain Lukert in an amount of about 10 ２．５ EID ５０ ~about 10 ３．５ EID ５０ The method of claim 3 or 4, comprising inactivated IBD virus strain 28-1 in an amount of

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

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

9. The vaccine is about 10 ７．８ EID ５０ ~about 10 ９．１ EID ５０ of the inactivated NDV virus, and / or about 10 ６．４ T.C.I.D. ５０ ~about 10 ７．９ T.C.I.D. ５０ The method of claim 7 or 8, comprising administering to a patient an inactivated reovirus comprising:

10. The vaccine a) about 10 per dose ６．９ EID ５０ Inactivated IB (strain H120), b) about 10 per dose ７．２ T.C.I.D. ５０ strain Lukert, and about 10 per dose ２．９５ EID ５０ the inactivated IBD virus strain 28-1 of c) about 10 ５．８ T.C.I.D. ５０ the inactivated TRT virus of clone K of d) about 10 per dose ８．１ EID ５０ Inactivated ND virus strain Lasota, e) about 10 per total dose ６．９ T.C.I.D. ５０ 10. The method of claim 1, comprising inactivated reovirus strains 1733 and 2408 of the present invention.

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

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

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

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

8.

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

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

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

19. The female chickens are a) 1, 3, and 8 weeks of age - Administration of live antigens against Newcastle disease virus, infectious bronchitis, and infectious bursal disease virus b) Weeks 6 and 10 - administration of live antigen turkey rhinotracheitis; c) Week 12 - The method of any one of claims 1 to 18, wherein the mouse has been primovaccinated according to a regimen comprising administration of a live reovirus antigen.

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

21. 1. A vaccine for vaccinating hens, for use in protecting offspring of said hens against IBV infection, said vaccine comprising an antigen component and an adjuvant component, said antigen component comprising an inactivated IB virus, said adjuvant component consisting of an oil, a CpG-containing immunostimulatory oligonucleotide, and optionally one or more emulsifiers, and further wherein said vaccine is a W / O emulsion.

22. The vaccine is administered in an amount of about 10 per dose. ６．７ EID ５０ ~about 10 ７．９ EID ５０ 22. The vaccine of claim 21, comprising the inactivated IB virus of

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

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

25. The vaccine is about 10 ５．５ T.C.I.D. ５０ ~about 10 ６．８ T.C.I.D. ５０ of the inactivated TRT virus, and / or about 10 ６．９ T.C.I.D. ５０ ~about 10 ８．２ T.C.I.D. ５０ 25. The vaccine of claim 23 or 24, comprising the inactivated IBD virus of

26. The vaccine is administered in an amount of about 10 per dose. ６．９ T.C.I.D. ５０ ~about 10 ７．７ T.C.I.D. ５０ Inactivated IBD virus strain Lukert in an amount of about 10 ２．５ EID ５０ ~about 10 ３．５ EID ５０ The method of claim 23 or 24, comprising inactivated IBD virus strain 28-1 in an amount of

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

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

29. The vaccine is about 10 ７．８ EID ５０ ~about 10 ９．１ EID ５０ of the inactivated NDV virus, and / or about 10 ６．４ T.C.I.D. ５０ ~about 10 ７．９ T.C.I.D. ５０ 29. The vaccine of claim 27 or 28, comprising the inactivated reovirus of

30. The vaccine a) about 10 per dose ６．９ EID ５０ Inactivated IB (strain H120), b) about 10 per dose ７．２ EID ５０ strain Lukert, and about 10 per dose ２．９５ EID ５０ the inactivated IBD virus strain 28-1 of c) about 10 per dose ５．８ EID ５０ the inactivated TRT virus of clone K of d) about 10 per dose ８．１ EID ５０ Inactivated ND virus (strain Lasota), e) about 10 per total dose ６．９ EID ５０ 30. The vaccine of any one of claims 21 to 29, comprising the inactivated reovirus (inactivated reovirus strains 1733 and 2408).

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

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

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

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

38. 38. The vaccine of any one of claims 21 to 37, wherein the hens are vaccinated about 14 days before laying eggs.

39. The female chickens are a) 1, 3, and 8 weeks of age - administration of live antigens against Newcastle disease virus, infectious bronchitis, and infectious bursal disease virus b) Weeks 6 and 10 - administration of live turkey rhinotracheitis antigen; c) Week 12 - The vaccine of any one of claims 21 to 38, which has been initially vaccinated according to a regimen comprising administration of a live reovirus antigen.

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

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