Poultry vaccines and methods of protecting poultry

Abstract

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

Description

ZP000430 POULTRY VACCINES AND METHODS OF PROTECTING POULTRY FIELD OF THE INVENTION

[0001] This invention is generally in the field of poultry vaccines. BACKGROUND

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

[0003] Avian infectious bronchitis (IB) is probably one of the most widespread poultry diseases around the world, due to its highly contagious nature. It is caused by a gamma coronavirus that affects the respiratory, urinary and reproductive systems of the chickens, causing different disorders depending on the tissue tropism characteristics of the invading viral strain. The diagnosis of Infectious Bronchitis is one the challenges to control the disease, as the clinical signs in the respiratory system are common in other poultry diseases.

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

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

[0006] In chickens up to the age of 4 weeks, 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] Accordingly, there is a need in the art in vaccines that protect the chickens in the first several days of life.SUMMARY OF INVENTION

[0008] In the first aspect, provided is a method of protecting offspring of a hen against IBV infection, the method comprising administering to the hen a vaccine comprising an antigen component and the adjuvant component, wherein the antigen component comprises an inactivated IB virus and the adjuvant component consist 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 contains about 106.7EID50to about 107.9EID50of said inactivated IB virus per dose. In certain embodiments, the antigen component further comprises one or more of an inactivated TRT virus and an inactivated IBD virus, and wherein said vaccine protects the offspring from TRT infection and / or IBDV infection, respectively. In some embodiments, the vaccine contains about 105.5TCID50to about106.8TCID50of said inactivated TRT virus and / or about 106.9TCID50to about 108.2TCID50of the inactivated IBD virus.

[0010] In additional embodiments, applicable to the methods of any embodiments recited above, the antigen component further comprises an inactivated NDV virus and / or an inactivated reovirus, and wherein said vaccine protects the offspring from NDV infection and / or reovirus infection, respectively. In certain embodiments, the vaccine comprises about 107.8EID50 to about 109.1EID50 of said inactivated NDV virus and / or about 106.4TCID50 to about 107.9TCID50 of the inactivated reovirus.

[0011] In the most preferred embodiments of the method according the first aspect, the vaccine comprises: a) about 106.9EID50 per dose of the inactivated IB (strain H120); b) about 107.2TCID50 per dose the inactivated IBD virus of strain Lukert and about 102.95EID50 per dose of the inactivated IBD virus strain 28-1; c) about 105.8TCID50 of the inactivated TRT virus of Clone K; d) about 108.1EID50 per dose of the inactivated ND virus strain LaSota; e) about 106.9TCID50 per dose in total of the 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 a P-class immunostimulatory oligonucleotide, preferably comprising phosphorothioate linkages and / or I- or J- modification at the 5’ end. Most preferably, said CpG-containing immunostimulatory oligonucleotide comprises SEQ ID NO: 8.

[0013] In certain embodiments, the vaccine administered in accordance with the methods of this first aspect comprises about 5 to about 20 µg of said 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 is administered to a hen that has been primovaccinated according to the regimen comprising: a) at weeks 1, 3, and 8 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 antigens Turkey Rhinotracheitis; c) at week 12 – administration of a live reovirus antigen.

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

[0016] In certain embodiments of this second aspect, the vaccine contains about 106.7EID50 to about 107.9EID50 of said inactivated IB virus per dose. In certain embodiments, the antigen component further comprises one or more of an inactivated TRT virus and an inactivated IBD virus, and wherein said vaccine protects the offspring from TRT infection and / or IBDV infection, respectively. In some embodiments of this second aspect, the vaccine contains about 105.5TCID50 to about106.8TCID50 of said inactivated TRT virus and / or about 106.9TCID50 to about 108.2TCID50 of the inactivated IBD virus.

[0017] In additional embodiments, applicable to the vaccines of any embodiments of this second aspect, as recited above, the antigen component further comprises an inactivated NDV virusand / or an inactivated reovirus, and wherein said vaccine protects the offspring from NDV infection and / or reovirus infection, respectively. In certain embodiments, the vaccine contains about 107.8EID50to about 109.1EID50of said inactivated NDV virus and / or about 106.4TCID50to about 107.9TCID50of the inactivated reovirus.

[0018] In the most preferred embodiments of the vaccine according to the second aspect, the vaccine comprises: a) about 106.9EID50per dose of the inactivated IB (strain H120); b) about 107.2TCID50per dose the inactivated IBD virus of strain Lukert and about 102.95EID50per dose of the inactivated IBD virus strain 28-1; c) about 105.8TCID50of the inactivated TRT virus of Clone K; d) about 108.1EID50per dose of the inactivated ND virus strain Lasota; e) about 106.9TCID50per dose in total of the 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 a P-class immunostimulatory oligonucleotide, preferably comprising phosphorothioate linkages and / or I- or J- modification at the 5’ end. Most preferably, said 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 said CpG-containing immunostimulatory oligonucleotide per dose.

[0021] In a subset of embodiments, the vaccine according to any of the embodiments of this second aspect is administered to a hen that has been primovaccinated according to the regimen comprising: a) at weeks 1, 3, and 8 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 antigens Turkey Rhinotracheitis; c) at week 12 – administration of a live reovirus antigen.DETAILED DESCRIPTION

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

[0023] The term ‘consisting essentially of’ and the like as applied to the adjuvant formulations of the instant invention refers to vaccines and other compositions which do not contain additional adjuvanting or immunomodulating agents in the amounts at which said agent exert measurable adjuvanting or immunomodulating effects.

[0024] The term “conventional prime vaccines” refers to vaccines administered to hens when said hens were 1 to about 100 days old. Conventional prime vaccines are generally modified live vaccines and contain attenuated viruses. Multiple conventional prime vaccines are available commercially and include, without limitations, 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, BUR™ 706 R.

[0025] The term “hen” refers to a female chicken twho is the mother of the offspring whose protection is achieved by administering the vaccines disclosed herein to said hen. Prior to the administration of the inactivated vaccines disclosed herein, the hen has been primovaccinated.

[0026] The phrase “laying an egg” refers to the hen’s vaccinated with the vaccine of the invention laying an egg from which the offspring that is to be protected hatches.

[0027] The term “primovaccinated” as applied to a hen that has received the vaccines recited herein refers to a vaccination of said hen, at the time when said hen was 1 to about 98 days old, preferably 1 to about 91 day old, more preferably 1 to about 84 days old with one or more doses with conventional prime vaccines containing the same antigens as the antigens of the vaccines recited herein which is used to be administered to the hen.

[0028] In certain embodiments, the hen has been primovaccinated with two or more administrations of live Infectious Bronchitis virus, preferably three administrations.

[0029] In other embodiments, the hen has been primovaccinated with two or more administrations of live Infectious Bronchitis virus, preferably three administrations, and also with at least one of: a) one or more administrations of live TRT virus, preferably two administrations; and b) two or more administrations of live IBD virus, preferably three administrations.

[0030] In other embodiments, the hen has been primovaccinated with two or more administrations of live Infectious Bronchitis virus, preferably three administrations and also with at least one of: a) one or more administrations of live TRT virus, preferably two administrations; and b) two or more administrations of live IBD virus, preferably three administrations; c) two or more administrations of live Newcastle disease virus, preferably three administrations; d) at least one administration of live avian reovirus.

[0031] In certain embodiments, the hen has been primovaccinated with: a) two or more administrations of live Infectious Bronchitis virus, preferably three administrations; b) one or more administrations of live TRT virus, preferably two administrations; c) two or more administrations of live IBD virus, preferably three administrations; d) two or more administrations of live Newcastle disease virus, preferably three administrations; e) at least one administration of live avian reovirus.

[0032] In a specific subset of embodiments, the hen has been primovaccinated with: a) three administrations of live Infectious Bronchitis virus; b) two administrations of live TRT virus; c) three administrations of live IBD virus; d) three administrations of live Newcastle disease virus; e) one administration of live avian reovirus.

[0033] In certain embodiments, primovaccinated hens have been vaccinated against IBDV, IBV, Newcastle Disease Virus, TRT, and reovirus according to the following schedule:Table 1 Week Antigen 1 Newcastle Disease virus - Live

[0034] Thus, the hens have received at least one dose (more preferred, two doses, and even more preferred, three doses) of modified live Newcastle Disease virus vaccine, at least one dose (more preferred, two doses, and even more preferred, three doses) of modified live IBDV virus vaccine, at least one dose (more preferred, two doses, and even more preferred, three doses) of modified live IB virus, at least one dose (more preferably, two or three doses) of modified live TRT virus vaccine, and at least one dose of modified live reovirus vaccine prior to being vaccinated with the vaccine according to the invention.

[0035] The term “the same antigens” used in the conventional prime vaccines and the vaccines used for the administration to the hens refers to the property of the antigen in the inactivated vaccine used for the administration of to the hen to boost the immune response against the antigen in the conventional prime vaccine.

[0036] The term “parenteral administration” refers to the introduction of a substance, such as a vaccine, into a subject's body through or by way of a route that does not include the digestivetract. Parenteral administration includes subcutaneous, intramuscular, transcutaneous, intradermal, intraperitoneal, intraocular, and intravenous administration.

[0037] Purity percentage or “X percent pure” as applied to the immunostimulatory oligonucleotide preparation refers to a population of oligonucleotide molecules comprising X% of the named oligonucleotide (e.g., SEQ ID NO: 1, SEQ ID: NO: 5, SEQ ID NO: 8, etc), and the remainder (i.e., 100% minus X%) comprises shorter fragments of the named oligonucleotide present as impurities during the manufacturing of the named sequence. Thus, if the sequence is manufactured by 3’-5’ sequencing, 5’-truncations would comprise the remainder. As a non- limiting example, a preparation of 100 µg of 80% pure SEQ ID NO: 8 comprises 80 µg of SEQ ID NO: 8 and the remaining 20 µg are 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 or an adjuvant or vaccine that would induce an immune response in a subject receiving the antigen or the adjuvant or the vaccine which is adequate to prevent or reduce signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with a pathogen, such as a virus or a bacterium. Humoral immunity or cell-mediated immunity or both humoral and cell-mediated immunity may be induced. The immunogenicity and efficacy of a vaccine in an animal may be evaluated, e.g., indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strain. The protective immunity conferred by a vaccine can be evaluated by measuring, e.g., reduction in clinical signs such as mortality, morbidity, temperature number, 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 skilled in the art.

[0039] The terms “vaccine”, “vaccine recited herein”, “vaccine disclosed herein” “inactivated vaccine” and the like specifically refer to an immunogenic composition that elicits protective immune response in the offspring and that comprises recited inactivated antigens and is formulated as a W / O emulsion comprising an adjuvant that comprises (or consists essentially of or consists of) an oil, an immunostimulatory CpG oligonucleotide an optionally one or moreemulsifiers. The term specifically excludes conventional prime vaccines that are used to primovaccinate the hen. Antigens

[0040] The vaccine described herein preferably contains inactivated virus antigens. In certain embodiments, the vaccine contains an inactivated Infectious Bronchitis virus (IB or IBV). Multiple IB strains have been known, including, without limitations, Massachusetts Strain, Brazilian IBV Variant Strain (BR-01), M41, D1466 and others 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 28 / 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-type strain (e.g.., H120 or M41 or Ma5 or other isolates) or another strain of GI-1 lineage. In the most preferred set embodiments, the strain is H120. In other embodiments, the strain is of 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 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 yet other embodiments, the strain is of 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] The inactivated IB virus may be present in the amount of at least 106.4EID50per dose, more preferably at least about 106.7EID50per dose, more preferably at least about 106.8EID50per dose, more preferably about 106.9EID50per dose to about 107.9EID50per dose, even more preferably about 106.9EID50per dose to about 107.3EID50per dose. Thus, in different embodiments, inactivated IB virus, such as Massachusetts strain H120 may be present in the vaccine in the amount of 106.4EID50per dose, or about 106.5EID50per dose, or about 106.6EID50 per dose, or about 106.7EID50per dose, or about 106.8EID50per dose, or about 106.9EID50per dose, or about 107.0EID50per dose, or about 107.1EID50per dose, or about 107.2EID50per dose, or about 107.3EID50per dose, or about 107.4EID50per dose, or about 107.5EID50per dose, or about 107.6EID50per dose, or about 107.7EID50per dose, or about 107.8EID50per dose, or 107.9EID50per dose.

[0044] The amounts of inactivated IB and the other inactivated antigens present in the vaccines described herein are pre-inactivation per-dose amounts of the antigens.

[0045] In addition to the IB antigens according to any of the embodiments described above, the vaccine may contain one or more inactivated virus selected from the group consisting 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 vaccine described herein contains the inactivated IB virus according to any of the embodiments described above and one or more of the inactivated TRT virus and the inactivated IBD virus.

[0047] Multiple TRT strains have been 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 more preferred embodiments, the TRT strain is Clone K. The inactivated TRT virus may be present in the amount of at least 105.5TCID50per dose, more preferably at least 105.6TCID50per dose, more preferably at least 105.7TCID50 per dose, more preferably about 105.8TCID50 per dose to about 106.8TCID50 per dose, even more preferably about 105.8TCID50 per dose to about 106.4TCID50 per dose.

[0048] Multiple IBDV strains have been 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, 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 may be present in the amount of at least 106.9TCID50 per dose, more preferably at least 107.0TCID50 per dose, more preferably at least 107.1TCID50 per dose, more preferably about 107.2TCID50 per dose to about 108.2TCID50 per dose, even more preferably about 107.2TCID50 per dose to about 107.6TCID50 per dose.

[0049] In more preferred embodiments, the vaccine comprises at least two strains of IBDV, more preferably, Lukert and 28-1. If inactivated IBDV viruses of both Lukert strain and 28-1 strain are present in the vaccine, Lukert strain IBD virus may be present in the amount of at least 106.9TCID50, more preferably at least about 107.1TCID50, more preferably, at least about 107.1TCID50to about 107.7TCID50, or about 107.2TCID50to about 107.5TCID50. Inactivated IBD virus of strain 28-1 may be present in the amount of at least 102.5EID50to about 103.5EID50per dose, or about 102.6to about 103.4EID50, or about 102.7to about 103.3EID50, or about 102.8to about 103.2EID50, or about 102.9to about 103.1EID50, or about 102.9to about 103.0EID50, or about 102.95EID50per dose.

[0050] In other embodiments, the vaccine described herein contains the inactivated IB virus as described above and one or more of the inactivated Newcastle Disease virus and the inactivated reovirus.

[0051] Multiple NDV strains have been known, including, without limitations, Lasota, F, B1, V4, V4-HR, I-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.

[0052] The inactivated NDV virus may be present in the amount of at least 107.8EID50 per dose, more preferably at least about 107.9EID50per dose, more preferably at least about 108.0EID50per dose, more preferably about 108.1EID50 per dose to about 109.1EID50 per dose, even more preferably about 108.1EID50 per dose to about 108.5EID50 per dose.

[0053] Multiple reovirus strains have been known including without limitations 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 may be present in total the amount of at least 106.4TCID50 per dose, more preferably at least about 106.7TCID50 per dose, more preferably at least about 106.8TCID50 per dose, more preferably about 106.9TCID50 per dose to about 107.9TCID50 per dose, even more preferably about 106.9TCID50 per dose to about 107.3TCID50 per dose.

[0054] In more preferred embodiments, the vaccine contains two strains of inactivated reovirus: 1733 and 2408. These inactivated reoviruses may be present in total the amount of at least 106.4TCID50 per dose, more preferably at least about 106.7TCID50 per dose, more preferably at leastabout 106.8TCID50per dose, more preferably about 106.9TCID50per dose to about 107.9TCID50per dose, even more preferably about 106.9TCID50per dose to about 107.3TCID50per dose.

[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 H120 strain and is present in the amount of about 106.9EID50per dose. The IBDV antigen comprises inactivated IBD virus of strain Lukert (present in the amount of about 107.2TCID50per dose) and strain 28-1 (present in the amount of about 102.95EID50per dose). The inactivated TRT virus is of Clone K strain and is present in the amount of about 105.8TCID50per dose. The inactivated Newcastle Disease virus is of LaSota strain and is present in the amount of about 108.1EID50per dose. The inactivated reovirus antigen comprises inactivated Reovirus viruses of strains 1733 and 2408, present in the total amount of about 106.9TCID50per dose. Adjuvants

[0056] The adjuvants of the vaccines recited herein and administered to the hens comprise immunostimulatory CpG oligonucleotide, oil and optionally one or more emulsifiers, so that the vaccine is a water-in-oil (W / O emulsion).

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

[0058] Suitable immunostimulatory oligonucleotides include ODN (DNA-based), or chimeric ODN-ORN structures, which may have modified backbone including, without limitations, phosphorothioate modifications, halogenations, alkylation (e.g., ethyl- or methyl- modifications), and phosphodiester modifications. In some embodiments, poly inosinic -cytidylic acid or derivative thereof (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 an unmethylated CG dinucleotide in specific base-sequence contexts (CpG motif). (Hansel TT, Barnes PJ (eds): New Drugs for Asthma, Allergy and COPD. Prog Respir Res. Basel, Karger, 2001, vol 31, pp 229-232, which is incorporated herein by reference). These CpG motifs are not seen ineukaryotic DNA, in which CG dinucleotides are suppressed and, when present, usually methylated, but are present in bacterial DNA to which they confer immunostimulatory properties.

[0060] In selected embodiments, the adjuvants of the instant invention utilize a so-called P-class immunostimulatory oligonucleotide, more preferably, modified P- class immunostimulatory oligonucleotides, even more preferably, E-modified P-class oligonucleotides. P-class immunostimulatory oligonucleotides are immunostimulatory CpG oligonucleotides characterized by the presence of palindromes, generally 6-20 nucleotides long. The P-Class oligonucleotides have the ability to spontaneously self-assemble into concatamers either in vitro and / or in vivo. These oligonucleotides are, in a strict sense, single-stranded, but the presence of palindromes allows for formation of concatamers or possibly stem-and-loop structures. The overall length of P- class immunostimulatory oligonucleotides is between 19 and 100 nucleotides, e.g., 19-30 nucleotides, 30-40 nucleotides, 40-50 nucleotides, 50-60 nucleotides, 60-70 nucleotides, 70-80 nucleotides, 80-90 nucleotides, 90-100 nucleotides.

[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 of at least 6 nucleotides in length and connected to a 3' palindromic region of at least 8 nucleotides in length either directly or through a spacer.

[0062] The P-class immunostimulatory oligonucleotides may be modified according to techniques known in the art. For example, J-modification refers to iodo-modified nucleotides. E-modification refers to ethyl-modified nucleotide(s). Thus, E-modified P-class immunostimulatory oligonucleotides are P-class immunostimulatory oligonucleotides, wherein at least one nucleotide (preferably 5’ nucleotide) is ethylated. Additional modifications include attachment of 6-nitro-benzimidazol, O-Methylation, modification with proynyl-dU, inosine modification, 2-bromovinyl attachment (preferably to uridine).

[0063] The P-class immunostimulatory oligonucleotides may also contain a modified internucleotide linkage including, without limitations, phosphodiesther linkages and phosphorothioate linkages. The oligonucleotides of the instant 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. WO2008 / 068638, published on Jun. 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). SEQ ID NO: 1 5' T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G 3' SEQ ID NO: 2 5' T*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' SEQ ID NO: 3 5' T*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3' SEQ ID NO: 4 5' JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' SEQ ID NO: 5 5' JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C* G*T 3' SEQ ID NO: 6 5' JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C* G*T 3' SEQ ID NO: 7 5' EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' SEQ ID NO: 8 5' JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C* G*T 3' SEQ ID NO: 9 5' JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C* G*T 3' SEQ ID NO: 10 5' T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G*T 3'

[0065] The immunostimulatory oligonucleotides of the instant invention may be chemically synthesized. Further, the immunostimulatory oligonucleotides may be used at about 60% purity (homogeneity) or greater (e.g., about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or 100% purity).

[0066] The amount of P-class immunostimulatory oligonucleotide for use in the adjuvant compositions depends upon the nature of the P-class immunostimulatory oligonucleotide used and the intended species.

[0067] The vaccines of the invention are formulated as W / O emulsions. Multiple oils and combinations thereof are suitable for use of the instant invention. These oils include, without limitations, animal oils, vegetable oils, as well as non-metabolizable oils. Non-limiting examples of vegetable oils suitable in the instant invention are corn oil, peanut oil, soybean oil, coconut oil, olive oil, and phytosqualane. Non-limiting example of animal oils is squalane. Suitable non- limiting examples of non-metabolizable oils include light mineral oil, straight chained or branched saturated oils, ramified oils, and the like.

[0068] In a set of embodiments, the oil used in the adjuvant formulations of the instant invention is a light mineral oil. As used herein, the term "mineral oil" refers to a mixture of liquid hydrocarbons obtained from petrolatum via a distillation technique. The term is synonymous with "liquefied paraffin", "liquid petrolatum" and "white mineral oil." The term is also intended to include "light mineral oil," i.e., oil which is similarly obtained by distillation of petrolatum, but which 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, at pages 788 and 1323). Mineral oil can be obtained from various commercial sources, for example, J. T. Baker (Phillipsburg, Pa.), USB Corporation (Cleveland, Ohio). Preferred mineral oil is light mineral oil commercially available under the name DRAKEOL®. In another embodiment, the suitable oil comprises mineral oil MARCOL™ 52. MARCOL™ 52 is a purified mixture of liquid saturated hydrocarbons. It is a crystal clear, water-white product that contains no toxic impurities. It is obtained from petroleum by vacuum distillation with subsequent refining stages including an ultimate purification by catalytic hydrogenation.

[0069] Emulsifiers suitable for use in the present emulsions include natural biologically compatible emulsifiers and non-natural synthetic surfactants. Biologically compatible emulsifiers include phospholipid compounds or a mixture of phospholipids. Preferred phospholipids are phosphatidylcholines (lecithin), such as soy or egg lecithin. Lecithin can be obtained as a mixture of phosphatides and triglycerides by water-washing crude vegetable oils, and separating and drying the resulting hydrated gums. A refined product can be obtained by fractionating the mixture for acetone insoluble phospholipids and glycolipids remaining after removal of the triglycerides and vegetable oil 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. The phospholipids may be isolated from natural sources or conventionally synthesized.

[0070] In additional embodiments, the emulsifiers used herein do not include lecithin, or use lecithin in an amount which is not immunologically effective.

[0071] Non-natural, synthetic emulsifiers suitable for use in the adjuvant formulations of the present invention include sorbitan-based non-ionic surfactants, e.g. fatty-acid-substituted sorbitan surfactants (commercially available under the name SPAN® or ARLACEL®), fatty acid esters of polyethoxylated sorbitol (TWEEN®), polyethylene glycol esters of fatty acids from sources such as castor oil (EMULFOR®); polyethoxylated fatty acid (e.g., stearic acid available under the name SIMULSOL® M-53), polyethoxylated isooctylphenol / formaldehyde polymer (TYLOXAPOL®), polyoxyethylene fatty alcohol ethers (BRIJ®); polyoxyethylene nonphenyl ethers (TRITON® N), polyoxyethylene isooctylphenyl ethers (TRITON® X). Preferred synthetic surfactants are the surfactants available under the name SPAN® and TWEEN®, such as TWEEN®-80 (Polyoxyethylene (20) sorbitan monooleate) and ARLACELTM83V (Sorbitan Sesquioleate)).

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

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

[0074] In yet another subset, applicable to all vaccines of the invention, volume percentage of the oil is over 40%, e.g., 40% to 90% by volume; 40% to 85%; 43% to 60%, 44-50% v / v, or 45-55% v / v of the vaccine. In certain embodiments, the emulsions contain at least 48% v / v oil phase and 52% v / v aqueous phase.

[0075] Sometimes, it is impossible or impracticable to concentrate the antigen, particularly in scaled up commercial applications, and low-concentrations of antigen solutions have to be used. Thus in some embodiments, the vaccines of the instant invention comprise the adjuvant formulations as described above, wherein the content of the oily phase in these adjuvant formulations is diluted and wherein the vaccine is a water-in-oil emulsion.

[0076] In practice, it is possible to create a water-in-oil emulsion wherein the oily phase is less than 50% v / v. Water-in-oil emulsion’s integrity can be maintained as long as the dispersed spherical water droplets are not present in a more concentrated form than the maximum packing fraction for random packing of monodisperse droplets, i.e.: 0.64. See Tadros, Emulsion Formation, Stability and Rheology, 1sted.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 implies that the oily phase should not drop below 36% v / v.

[0077] In some embodiments suitable, one dose of the adjuvant would 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 immunostimulatory oligonucleotide, up to about 50 μg (e.g., 0.5-20 μg, or 1-10 μg) of the sterol such as cholesterol.

[0078] In certain embodiments, the adjuvant component is prepared as follows: a) Sorbitan Sesquioleate, and cholesterol, if any, are dissolved in light mineral oil. The resulting oil solution is sterile filtered; b) The immunostimulatory oligonucleotide and Polyoxyethylene (20) sorbitan monooleate are dissolved in aqueous phase, thus forming the aqueous solution; c) The aqueous solution is added to the oil solution under continuous homogenization.

[0079] The vaccines of the instant invention may 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 may be added to the adjuvant component after the adjuvant component is prepared.

[0080] The vaccines described herein may further comprise a pharmaceutically acceptable carrier. As used herein, "a pharmaceutically-acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. The carrier(s) must be "acceptable" in the sense of being compatible with the other components of the compositions and not deleterious to the subject. Typically, the carriers will be sterile and pyrogen-free, and selected based on the mode of administration to be used. It is well known by those skilled in the art that the preferred formulations for the pharmaceutically acceptablecarrier which comprise the compositions are those pharmaceutical carriers approved in the applicable regulations promulgated by the United States (US) Department of Agriculture or US Food and Drug Administration, or equivalent government agency in a non-US country. Therefore, the pharmaceutically accepted carrier for commercial production of the compositions is a carrier that is already approved or will be approved by the appropriate government agency in the US or foreign country.

[0081] Other components of the compositions can include pharmaceutically acceptable excipients, such as carriers, solvents, and diluents, isotonic agents, buffering agents, stabilizers, preservatives, vaso-constrictive agents, antibacterial agents, antifungal agents, and the like. Typical carriers, solvents, and diluents include water, saline, dextrose, ethanol, glycerol, and the like. Representative isotonic agents include sodium chloride, dextrose, mannitol, sorbitol, lactose, and the like. Useful stabilizers include gelatin, albumin, and the like.

[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 and is present in the amount of about 106.9EID50 per dose. The IBDV antigen comprises inactivated IBD virus of strain Lukert and strain 28-1. The amount of the inactivated Lukert strain virus is about 107.2TCID50 per dose, and the amount of the inactivated 28-1 strain virus is about 102.95EID50. The inactivated TRT virus is Clone K and is present in the amount of about 105.8TCID50 per dose. The inactivated Newcastle Disease virus is LaSota strain virus and is present in the amount of about 108.1EID50 per dose. The inactivated reovirus antigen comprises inactivated Reovirus strains 1733 and 2408, present in the total amount of about 106.9TCID50 per dose. This vaccine is formulated as a W / O emulsion containing 49.9% of light mineral oil, 5.85% of ARLACEL® (Sorbitan sesquioleate), 3.25% of TWEEN®80 and 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, this vaccine is administered to the hens about 2 to 4 weeks prior to laying eggs, wherein the hen has been primovaccinated as described in Table 1.Methods

[0083] Generally, the vaccines of the instant invention may be administered to hens via multiple routes. Such routes are known to persons of ordinary skill in the art and include, without limitations intramuscular and subcutaneous injections.

[0084] In certain embodiments, the hens of about 14 to about 22 weeks of age are vaccinated (e.g., about 15 weeks, or about 16 weeks, or about 17 weeks or about 18 weeks, or about 19 weeks or about 20 weeks or about 21 weeks or 22 weeks of age).

[0085] In certain embodiments, the vaccines are administered at 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 the expected lay. This regimen ensures that the immunity to the antigen(s) present in the vaccines recited herein is developed by the time of lay and persists throughout the laying period of the vaccinated hens.

[0086] The vaccines recited herein are administered to the hens that have been primovaccinated or primed with conventional prime vaccines. In certain embodiments, the hens are primovaccinated as described in the “definitions” above. In more preferred embodiments, the hens are primovaccinated hen has been primovaccinated according to the regimen comprising: a) at weeks 1, 3, and 8 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 – administration of a live reovirus antigen.

[0087] Multiple conventional prime vaccines containing different antigens are known in the art. For example, POULVAC® IB Primer comprises a freeze-dried, Massachusetts type, live Infectious Bronchitis virus. NOBILIS® IB H120 is a live freeze-dried vaccine indicated for use as a primary vaccination of fowls against Infectious Bronchitis. This vaccine contains strain H120 type Massachusetts. Other IB primers may also be used with the vaccine of the instant invention.

[0088] Similarly, conventional prime vaccines exist that contain other antigens and antigen combinations that may be present in the vaccines recited herein.

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

[0090] In the most preferred embodiment, as noted above, the vaccine administered to the methods disclosed herein (or the vaccine for the use described herein) comprises all five inactivated antigens: IB virus, IBDV, TRT virus, NDV and reovirus. The inactivated IB virus is H120 and is present in the amount of about 106.9EID50 per dose. The IBDV antigen comprises inactivated IBD virus of strain Lukert, present in the amount of about 107.2TCID50 per dose, and strain 28-1, present in the amount of about 102.95EID50. The inactivated TRT virus is Clone K and is present in the amount of about 105.8TCID50 per dose. The inactivated Newcastle Disease virus is Lasota strain virus present in the amount of about 108.1EID50 per dose. The inactivated reovirus antigen comprises inactivated Reovirus strains 1733 and 2408 and are present in the total amount of about 106.9TCID50 per dose. This vaccine is formulated as a W / O emulsion containing about 49.9% of light mineral oil, about 5.85% of ARLACEL® (Sorbitan sesquioleate), about 3.25% of 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, this vaccine is administered to the hens about 2 to 4 weeks prior to laying eggs, wherein the hen has been primovaccinated as described in Table 1.

[0091] The following examples are presented as illustrative embodiments, but should not be taken as limiting the scope of the invention. Many changes, variations, modifications, and other uses and applications of this invention will be apparent to those skilled in the art. EXAMPLES Example 1. Effect of maternal vaccination on antibody levels in the offsprings

[0092] Three hundred 300 Novogen White breeders (100 / group) + 36 roosters (10 / group + 6 extras) were used in the study. Birds that showed any sign of disease or physical abnormalitywould be excluded from the study. The birds were individually identified with a numbered ring on the wing.

[0093] During the entire experimental period, the animals were kept in a shed measuring 80.0 m2and housed in boxes (3.20 x 1.10 x 1.85 m), allowing a density of 6.25 hens / m2. In each box, 20 females and 2 males were housed, in total there were 5 boxes per treatment group.

[0094] The boxes were mounted on a concrete floor with a hexagonal plastic chicken coop, equipped with a nipple drinker (1 for every 10 hens) and a bell-type feeder (1 for every 20 hens). The feed was formulated to meet the nutritional requirements of the birds. The water provided to the animals was adequate for consumption by the birds.

[0095] The floor was covered with shavings bedding. Temperature control was carried out using fans and curtain management to ensure the thermal comfort of the birds according to their age. Daily temperature and humidity were recorded using a digital thermos-hygrometer.

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

[0097] Close to the beginning of laying, individual nests were installed (1 mouth for every 5 hens) at a height of 15 cm from the ground to reduce dirtiness on the inside.

[0098] The hens were primovaccinated according to Table 1 and split into three groups. The first group was vaccinated with a placebo at 14 weeks. The second group was vaccinated at 14 weeks of age with POULVAC® MATERNAVAC ULTRA 5 vaccine containing: a) the inactivated IB of strain H120 in the amount of 106.9EID50 per dose; b) the inactivated IBD virus of strain Lukert in the amount of 107.2TCID50 per dose and strain 28-1 in the amount of 102.95EID50 per dose; c) the inactivated TRT virus of Clone K in the amount of 105.8TCID50 per dose; d) the inactivated ND virus of strain Lasota in the amount of 108.1EID50 per dose; e) the inactivated reovirus antigen (inactivated reoviruses strains 1733 and 2408) in the total amount of 106.9TCID50 per dose.

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

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

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

[0102] The hens laid eggs approximately 2 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 levels of the antibodies to the antigens in the vaccines. The levels of the antibodies were determined by commercial ELISAs kits (IDEXX IBD Ab kit, IDEXX REO Ac kit, BIOCHEK CK119 IBV, IDEXX NDV Ac, BIOCHEK CK120 ART). Results

[0103] The results are summarized in Table 2 Cutoff / Group T01 (Placebo) T02 (POULVAC® COMMERICAL MATERNAVC ULTRA VACCINE 7 1 5 5

[0104] The serological comparison showed that chicks originated from breeders that received POULVAC® MATERNAVAC ULTRA 5 presented the mean log2 anti IB antibody titers above the protection cutoff, whereas in chicks originated from the breeders that received the commercial vaccine or placebo anti-IB antibody titers were below the protection cutoff.

[0105] Moreover, chicks originated from breeders that received POULVAC® MATERNAVC ULTRA 5 presented the mean log2 antibody titers significantly higher 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). Numerical differences between POULVAC® MATERNAVC ULTRA 5 and the competitor vaccine 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 the offspring protection against Infectious Bronchitis, IBDV, and TRT compared to the commercial vaccine tested in this example.

[0107] All publications cited in the specification, both patent publications and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein fully incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being 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 may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

CLAIMS 1. A method of protecting offspring of a hen against IBV infection, the method comprising administering to the hen a vaccine comprising an antigen component and the adjuvant component, wherein the antigen component comprises an inactivated IB virus and the adjuvant component consist 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 said vaccine contains about 106.7EID50to about 107.9EID50of said inactivated IB virus per dose.

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 wherein said vaccine protects the offspring from TRT infection and / or IBDV infection, respectively.

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

5. The method of claim 3 or 4, wherein said vaccine contains about 105.5TCID50 to about 106.8TCID50 of said inactivated TRT virus and / or about 106.9TCID50 to about 108.2TCID50 of the inactivated IBD virus.

6. The method of claim 3 or 4, wherein the vaccine comprises an inactivated IBD virus strain Lukert in the amount of about 106.9TCID50 to about 107.7TCID50 and an inactivated IBD virus strain 28-1 in the amount of about 102.5EID50 to about 103.5EID50 per dose.

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

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

9. The method of claim 7 or 8, wherein said vaccine contains about 107.8EID50to about 109.1EID50of said inactivated NDV virus and / or about 106.4TCID50to about 107.9TCID50of the inactivated reovirus.

10. The method according to any one of claims 1-9 wherein said vaccine comprises: a) about 106.9EID50per dose of the inactivated IB (strain H120); b) about 107.2TCID50per dose the inactivated IBD virus of strain Lukert and about 102.95EID50per dose of the inactivated IBD virus strain 28-1; c) about 105.8TCID50of the inactivated TRT virus of Clone K; d) about 108.1EID50per dose of the inactivated ND virus strain Lasota; e) about 106.9TCID50per dose in total of the inactivated reovirus strains 1733 and 2408.

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

12. The method according to claim 11, wherein said P-class immunostimulatory oligonucleotide comprises phosphorothioate linkages.

13. The method according to claim 11 or claim 12, wherein said P-class immunostimulatory oligonucleotide is 5’ I-modified or 5’ J-modified.

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

8.

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

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

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

18. The method according to any one of claims 1-17, wherein said hen is vaccinated about 14 days before laying an egg.

19. The method according to any one of claims 1-18 wherein said hen has been primovaccinated according to the regimen comprising:a) at weeks 1, 3, and 8 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 antigens Turkey Rhinotracheitis; c) at week 12 – administration of a live reovirus antigen.

20. The method according to any one of claims 1-19 wherein said hen is about 14 to about 22 weeks of age.

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

22. The vaccine of claim 21, wherein said vaccine contains about 106.7EID50 to about 107.9EID50 of said inactivated IB virus per dose.

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

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

25. The vaccine of claim 23 or 24, wherein said vaccine contains about 105.5TCID50 to about106.8TCID50 of said inactivated TRT virus and / or about 106.9TCID50 to about 108.2TCID50 of the inactivated IBD virus.

26. The method of claim 23 or 24, wherein the vaccine comprises an inactivated IBD virus strain Lukert in the amount of about 106.9TCID50 to about 107.7TCID50 and an inactivated IBD virus strain 28-1 in the amount of about 102.5EID50 to about 103.5EID50 per dose.

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

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

29. The vaccine of claim 27 or 28, wherein said vaccine contains about 107.8EID50to about 109.1EID50of said inactivated NDV virus and / or about 106.4TCID50to about 107.9TCID50of the inactivated reovirus.

30. The vaccine according to any one of claims 21-29 wherein said vaccine comprises: a) about 106.9EID50per dose of the inactivated IB (strain H120); b) about 107.2EID50per dose the inactivated IBD virus of strain Lukert and about 102.95EID50per dose of the inactivated IBD virus strain 28-1; c) about 105.8EID50 per dose of the inactivated TRT virus of Clone K; d) about 108.1EID50 per dose of the inactivated ND virus (strain Lasota); e) about 106.9EID50per dose of total of the inactivated reovirus (inactivated reoviruses strains 1733 and 2408).

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

32. The vaccine according to claim 31, wherein said P-class immunostimulatory oligonucleotide comprises phosphorothioate linkages.

33. The vaccine according to claim 31 or 32, wherein said P-class immunostimulatory oligonucleotide is 5’ I-modified or 5’ J-modified.

34. The vaccine according to any one of claims 21-33 wherein said immunostimulatory oligonucleotide comprises SEQ ID NO:

8.

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

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

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

38. The vaccine according to any one of claims 21-37, wherein said hen is vaccinated about 14 days before laying an egg.

39. The vaccine according to any one of claims 21-38, wherein said hen has been primovaccinated according to the regimen comprising: a) at weeks 1, 3, and 8 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 – administration of a live reovirus antigen.

40. The vaccine according to any one of claims 21-39 wherein said hen is about 14 to about 22 weeks of age.

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