A triple vaccine against avian parafowl cholera, avian encephalomyelitis virus, and fowlpox virus.

Abstract

This invention relates to immunogenic compositions comprising: a) one or more antigens of *Avianobacter paragallinarum* and one or more antigens of avian encephalomyelitis virus and one or more antigens of fowlpox virus; and b) a pharmaceutically acceptable carrier. Furthermore, this invention relates to methods for immunizing subjects, comprising administering the immunogenic composition of this invention to such subjects. Additionally, this invention relates to methods for treating or preventing clinical signs caused by *Avianobacter paragallinarum*, avian encephalomyelitis virus, and fowlpox virus in subjects in need, comprising administering to the subject a therapeutically effective amount of the immunogenic composition of this invention.

Description

Background Technology

[0001] Infectious coryza (IC) is a contagious upper respiratory tract disease affecting poultry. It is caused by *Avibacterium paragallinarum*, formerly known as *Haemophilus paragallinarum*. Common clinical signs of IC include rhinitis, facial swelling or edema, anorexia, stunted growth, decreased egg production, and some mortality. This disease is of great importance in the poultry industry.

[0002] Avian encephalomyelitis virus (AEV) is a single-stranded RNA (ssRNA) virus belonging to the family Picornaviridae. Infection can cause neurological signs in poultry (such as ataxia, rapid tremors of the head and neck), weakness, weight loss, increased mortality, and decreased egg production.

[0003] Folverine virus (FPV) is a DNA virus belonging to the genus Avipoxvirus in the family Poxviridae, which infects poultry. Folverine disease has a significant economic impact worldwide, with losses resulting from decreased egg production in laying hens, slowed growth rates in broilers, blindness, and, in some cases, death.

[0004] However, what is needed are effective combination vaccines that provide protection against multiple pathogens. Such combination vaccines are highly desirable in order to minimize the number of vaccination treatments required to confer protection against multiple pathogens (animal welfare), reduce administration costs, and increase acceptance and coverage.

[0005] However, interference complicates the development of multi-component vaccines. Specifically, interference refers to the observed weakening of the response to certain antigens when multiple antigens are administered, relative to the immune response observed when such antigens are administered individually. Interference can generally occur when multiple antigens are administered. However, interference is particularly problematic when inactivated vaccines are combined with live vaccines, as the live vaccine component may be affected by the inactivated vaccine component (which may contain pharmaceutically acceptable carriers).

[0006] Therefore, there is a need for effective combination vaccines that provide protection against multiple pathogens. Detailed Implementation

[0007] Before describing aspects of the invention, it must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “an antigen” includes multiple antigens, reference to “virus” refers to one or more viruses and their equivalents known to those skilled in the art, and so on. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although preferred methods, apparatus, and materials are now described, any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing cell lines, vectors, and methods as reported in the publications that can be used in conjunction with the invention. Nothing herein should be construed as an admission that the invention is not entitled to precede such disclosure by prior invention.

[0008] Composition of substances

[0009] This invention solves the inherent problems in the prior art and provides a significant advancement in the current level of technology.

[0010] Generally, the present invention provides immunogenic compositions comprising: a) one or more antigens of *Avianobacter paragallinarum* and one or more antigens of avian encephalomyelitis virus and one or more antigens of fowlpox virus; and b) a pharmaceutically acceptable carrier.

[0011] Furthermore, the present invention also provides an immunogenic composition comprising: a) one or more antigens of a vaccine of *Avianobacter paragallinarum* and one or more antigens of a modified live avian encephalomyelitis virus and one or more antigens of a modified live fowlpox virus; and b) a pharmaceutically acceptable carrier. Therefore, the immunogenic composition relates to a combination of a vaccine of *Avianobacter paragallinarum* and modified live avian encephalomyelitis virus and modified live fowlpox virus.

[0012] In addition, the present invention provides an immunogenic composition comprising: a) a vaccine of *Avianella paragallinarum* and modified live avian encephalomyelitis virus and modified live fowlpox virus; and b) a pharmaceutically acceptable carrier.

[0013] In addition, the present invention provides an immunogenic composition comprising a combined vaccine of *Avianella paragallinarum* and modified live avian encephalomyelitis virus and modified live fowlpox virus.

[0014] Favorably, the experimental data presented in this paper clearly provide evidence that the trivalent vaccine confers protection against avian coryza, avian encephalomyelitis, and fowlpox. Surprisingly, no interference was detected regarding the efficacy of the different vaccine components. In fact, a slight positive synergistic effect was even observed between the fowlpox and AE vaccines in the trivalent vaccine combination, which is unexpected.

[0015] The term "Avian bacillus paragallinarum" is well known to those skilled in the art. Avian bacillus paragallinarum is a Gram-negative bacterium belonging to the family Pasteuraceae. Infectious coryza (IC) is caused by Avian bacillus paragallinarum. Infectious coryza (IC) is an infectious upper respiratory tract disease affecting poultry.

[0016] The term "avian encephalomyelitis virus" is well known to those skilled in the art. Avian encephalomyelitis virus is a single-stranded RNA (ssRNA) virus belonging to the family Picornaviridae. Infection in poultry can lead to neurological signs (such as ataxia, rapid tremors of the head and neck), weakness, weight loss, increased mortality, and decreased egg production.

[0017] The term "fowlpox virus" is well known to those skilled in the art. Fowlpox virus (FPV), a member of the genus FPV, infects poultry. Fowlpox virus disease has a significant economic impact worldwide, with losses resulting from decreased egg production in laying hens, slowed growth rates, blindness, and, in some cases, death in broilers.

[0018] As used herein, “antigen” refers to (but is not limited to) a component that elicits an immune response in a host to a target immunogenic composition or vaccine containing such antigen or an immunogenic component thereof. An antigen or immunogenic component may be a whole microorganism (inactivated or modified live form), or any fragment or fraction thereof, which, if administered to a host, can elicit an immune response in the host. An antigen may be or may contain a whole live organism in its original form or an attenuated organism as in a so-called modified live vaccine (MLV). An antigen may further contain suitable elements of said organism (subunit vaccines), whereby these elements are generated by: disrupting the growth culture of the whole organism or such organism and subsequent purification steps to produce the desired structure, or by inducing a synthetic process through appropriate manipulation of a suitable system, such as, but not limited to, bacteria, insects, mammals, or other species, and optionally by subsequent isolation and purification procedures, or by using a suitable pharmaceutical composition, through direct incorporation of genetic material into the animal requiring the vaccine (polynucleotide vaccination). An antigen may be contained in a whole organism inactivated by appropriate methods in a so-called inactivated vaccine (KV). If the organism is bacteria, the inactivated vaccine is called a bacterial vaccine.

[0019] The term "immunogenic composition" refers to a composition comprising at least one antigen that elicits an immune response in a host to which the immunogenic composition is applied. Such an immune response may be a cell- and / or antibody-mediated immune response to the immunogenic composition of the present invention. Preferably, the immunogenic composition induces an immune response, and more preferably, confers protective immunity against one or more clinical signs of infection. The host is also described as an "object." Preferably, any host or object described or mentioned herein is poultry or birds.

[0020] Typically, an "immune response" includes, but is not limited to, one or more of the following effects: the generation or activation of antibodies specifically targeting one or more antigens included in the immunogenic composition of the present invention, B cells, helper T cells, suppressor T cells and / or cytotoxic T cells and / or γ-δ T cells. Preferably, the host will exhibit a protective immune response or a therapeutic response.

[0021] A “protective immune response” or “protective immunity” will be demonstrated by the following: a reduction or absence of clinical signs typically exhibited by the infected host, faster recovery time and / or a decrease in the duration of infectivity in the infected host’s tissues or body fluids or excretions or a decrease in pathogen titer.

[0022] An immunogenic composition is described as a "vaccine" in cases where the host exhibits a protective immune response such that resistance to a new infection would be enhanced and / or the clinical severity of the disease would be reduced.

[0023] In one particular aspect of the immunogenic composition according to the invention, one or more antigens of *Paragonimus paragallinarum* are vaccines of *Paragonimus paragallinarum*.

[0024] As used in this article, "vaccine" refers to inactivated bacteria. Therefore, the vaccine for *Paragonimus paragallinarum* refers to killed and inactivated *Paragonimus paragallinarum*.

[0025] In another specific aspect of the immunogenic composition according to the invention, the vaccine of *Paragonimus paragallinarum* is a completely inactivated vaccine of *Paragonimus paragallinarum*.

[0026] Any conventional inactivation method can be used for the purposes of this invention. Therefore, inactivation can be performed by chemical and / or physical treatments known to those skilled in the art. Preferred inactivation methods include the addition of a cyclized diethyleneimine (BEI), including a solution containing 2-bromoethylamine hydrobromide (BEA), said 2-bromoethylamine hydrobromide having been cyclized to a diethyleneimine (BEI). Preferred further chemical inactivating agents include, but are not limited to, Triton X-100, sodium deoxycholate, hexadecyltrimethylammonium bromide, β-propiolactone, thimerosal, phenol, and formaldehyde (formalin). However, inactivation may also include a neutralization step. Preferred neutralizing agents include, but are not limited to, sodium thiosulfate, sodium bisulfite, etc.

[0027] In another specific aspect of the immunogenic composition according to the invention, the vaccine of *Paragonimus paragallinarum* is a whole-cell vaccine of *Paragonimus paragallinarum* inactivated by formalin or thimerosal.

[0028] Preferred formalin inactivation conditions include formalin concentrations of about 0.02% (v / v)–2.0% (v / v), more preferably about 0.1% (v / v)–1.0% (v / v), even more preferably about 0.15% (v / v)–0.8% (v / v), even more preferably about 0.16% (v / v)–0.6% (v / v), and most preferably about 0.2% (v / v)–0.4% (v / v). The incubation time depends on the resistance of *Paragonimus paragallinarum*. Generally, the inactivation process continues until the growth of *Paragonimus paragallinarum* is undetectable in a suitable culture system.

[0029] Preferably, the inactivated avian bacillus of the present invention is formalin-inactivated, and preferably at the concentration described above.

[0030] Preferred thimerosal inactivation conditions include thimerosal concentrations of about 1% (v / v)–20% (v / v), more preferably about 3% (v / v)–17% (v / v), even more preferably about 5% (v / v)–15% (v / v), and most preferably about 8% (v / v)–12% (v / v). The incubation time depends on the resistance of *Paragonimus paragallinarum*. Generally, the inactivation process continues until *Paragonimus paragallinarum* growth is undetectable in a suitable culture system.

[0031] Preferably, the inactivated avian bacillus of the present invention is inactivated by thimerosal, and preferably at the concentration described above.

[0032] The inactivated vaccine component of the present invention can be incorporated into liposomes using known techniques, such as those described in Nature, 1974, 252, 252-254 or Journal of Immunology, 1978, 120, 1109-13. In another embodiment of the invention, the inactivated vaccine component can be conjugated to suitable biological compounds such as polysaccharides, peptides, proteins, etc., or combinations thereof.

[0033] In another specific aspect of the immunogenic composition according to the invention, the fowlpox virus is a modified live fowlpox virus.

[0034] In another specific aspect of the immunogenic composition according to the invention, the modified live fowlpox virus is attenuated.

[0035] The term "attenuated" refers to a pathogen with reduced virulence compared to a wild-type isolate. In this invention, an attenuated virus is a virus whose virulence has been reduced such that it does not cause clinical signs of viral infection but can induce an immune response in target animals. It can also mean that clinical signs are reduced in morbidity or severity in animals infected with an attenuated virus compared to a "control group" of animals infected with a non-attenuated virus and not receiving the attenuated virus. In this context, the term "reduced / decreased" means a reduction of at least 10%, preferably 25%, even more preferably 50%, still more preferably 60%, even more preferably 70%, even more preferably 80%, even more preferably 90%, even more preferably 95%, and most preferably 100%, compared to a control group infected with a non-attenuated virus as defined above. Therefore, attenuated viral strains are suitable for incorporation into immunogenic compositions comprising modified live viruses.

[0036] In another specific aspect of the immunogenic composition according to the invention, the modified live fowlpox virus is non-recombinant.

[0037] As used herein, the term “non-recombinant” refers to an RNA or DNA genome (or RNA sequence, cDNA sequence, DNA sequence, or protein) that has only modifications that are indeed naturally present in the corresponding RNA or DNA genome (or RNA sequence, cDNA sequence, DNA sequence, or protein).

[0038] In another specific aspect of the immunogenic composition according to the invention, the fowlpox virus is recombinant.

[0039] As used herein, the term "recombinant" refers to an RNA or DNA genome (or RNA sequence, cDNA sequence, DNA sequence, or protein) having any modifications that are not naturally present in the corresponding RNA or DNA genome (or RNA sequence, cDNA sequence, DNA sequence, or protein). For example, an RNA or DNA genome (or RNA sequence, cDNA sequence, DNA sequence, or protein) is considered "recombinant" if it contains, for example, an insertion, deletion, inversion, relocation, or point mutation artificially introduced through human intervention. Thus, the RNA or DNA genome (or RNA sequence, cDNA sequence, DNA sequence, or protein) is not bound to all or part of the sequence (the amino acid sequence of the RNA, cDNA, DNA sequence, or protein) to which it binds in nature. When used in relation to viruses or bacteria, the term "recombinant" means a virus or bacterium produced through artificial manipulation of its genome. The terms "recombinant virus" or "recombinant bacteria" encompass genetically modified viruses or bacteria.

[0040] Recombinant fowlpox virus vaccines have been described in the prior art and have been shown to provide protection. Furthermore, such recombinant vaccines against fowlpox are commercially available, as exemplarily described. NDV or H7 subtype (from Boehringer Ingelheim), or FP LT+AE (from Ceva) is based on a modified live fowlpox vaccine.

[0041] In another specific aspect of the immunogenic composition according to the invention, the avian encephalomyelitis virus is a modified live avian encephalomyelitis virus.

[0042] In another specific aspect of the immunogenic composition according to the invention, the modified live avian encephalomyelitis virus is attenuated.

[0043] In another specific aspect of the immunogenic composition according to the invention, the modified live avian encephalomyelitis virus is non-recombinant.

[0044] In another specific aspect of the immunogenic composition according to the invention, the avian encephalomyelitis virus is recombinant.

[0045] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition effectively treats and / or prevents clinical signs caused by Avian bacillus paragallinarum in subjects in need.

[0046] The terms “treatment and / or prevention,” “clinical signs,” and “need” have been defined elsewhere. The term “infection” or “infected” refers to an object infected by a pathogen, namely avian paraavirhea, avian encephalomyelitis virus, or fowlpox virus.

[0047] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition is effective in treating and / or preventing clinical signs of avian rhinitis.

[0048] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition effectively treats and / or prevents clinical signs caused by avian encephalomyelitis virus infection in subjects in need.

[0049] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition effectively treats and / or prevents clinical signs caused by fowlpox virus infection in subjects in need.

[0050] In another specific aspect of the immunogenic composition according to the invention, the object is poultry or avian. The terms "avian" and "poultry" have been defined elsewhere.

[0051] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition is a vaccine.

[0052] The term "vaccine" has been defined elsewhere in this document. However, in cases where the host exhibits a protective immune response such that resistance to a new infection would be enhanced and / or the clinical severity of the disease would be reduced, an immunogenic composition is described as a "vaccine".

[0053] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition is formulated for single-dose administration.

[0054] Each dose is administered only once. The volume of a single dose has been defined elsewhere in this document.

[0055] Furthermore, it has been shown that a single dose of the immunogenic composition of the present invention is effective following such a single-dose administration of such an immunogenic composition.

[0056] In another specific aspect of the immunogenic composition according to the invention, the pharmaceutically acceptable carrier is selected from solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delay agents, adjuvants, immunostimulants, and combinations thereof.

[0057] The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delay agents, adjuvants, immunostimulants, and combinations thereof.

[0058] "Diluents" can include water, saline, dextrose, ethanol, glycerol, etc. Isotonic agents can include, in particular, sodium chloride, dextrose, mannitol, sorbitol, and lactose. Stabilizers include, in particular, alkali metal salts of albumin and ethylenediaminetetraacetic acid.

[0059] Preferably, the immunogenic composition further comprises a sucrose gelatin stabilizer.

[0060] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition contains a preservative.

[0061] Preferably, formaldehyde (formalin) or thimerosal is used as a preservative.

[0062] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition contains formaldehyde as a preservative.

[0063] Preferably, a pharmaceutically acceptable carrier is chitosan. Chitosan is a naturally occurring deacetylated polysaccharide derived from chitin in crustaceans (e.g., shrimp, crab), insects, and other invertebrates. Recently, Rauw et al. (2009, Vet Immunol Immunop 134:249–258) demonstrated that chitosan enhances the cellular immune response to Newcastle disease live vaccines and promotes their protective effect. Furthermore, Wang et al. (2012, Arch Virol (2012) 157:1451–1461) have shown results revealing the potential of chitosan as an adjuvant in live attenuated influenza vaccines.

[0064] Preferably, the immunogenic composition may further comprise one or more other immunomodulators, such as interleukins, interferons, or other cytokines. The amount and concentration of adjuvants and additives that may be used in the context of this invention can be readily determined by those skilled in the art.

[0065] In another specific aspect of the immunogenic composition according to the invention, the pharmaceutically acceptable carrier is an adjuvant.

[0066] In some aspects, the immunogenic compositions of the present invention contain adjuvants. As used herein, "adjuvant" may include aluminum hydroxide and aluminum phosphate, saponins such as QuilA, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsions, oil-in-water emulsions, and water-in-oil-in-water emulsions. Emulsions may be particularly based on light liquid paraffin oils (European pharmacopoeia); isoprene-like oils, such as squalane or squalene; oils resulting from oligomerization reactions of olefins, particularly isobutylene or decene; esters of acids or alcohols containing linear alkyl groups, more particularly vegetable oils, ethyl oleate, propylene glycol di(caprylate / caprylate), glyceryl tri(caprylate / caprylate), or propylene glycol dioleate; esters of branched fatty acids or alcohols, particularly isostearates. Oils are used in combination with emulsifiers to form emulsions. Emulsifiers are preferably nonionic surfactants, particularly esters of sorbitol, dimannitol (e.g., mannitol oleate), ethylene glycol, polyglycerol, propylene glycol, and oleic acid, isostearic acid, castor oil, or hydroxystearic acid, optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, particularly Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (ed. Stewart-Tull, DES), John Wiley and Sons, NY, pp. 51–94 (1995), and Todd et al., Vaccine 15:564–570 (1997). Exemplary adjuvants are the SPT emulsion described on page 147 of “Vaccine Design, The Subunit and Adjuvant Approach”, edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of the same book.

[0067] A further example of an adjuvant is a compound selected from polymers of acrylic acid or methacrylic acid, as well as copolymers of maleic anhydride and alkenyl derivatives. Advantageous adjuvant compounds are polymers of acrylic acid or methacrylic acid, particularly those crosslinked with polyolefin ethers of sugars or polyols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Those skilled in the art may also refer to U.S. Patent No. 2,909,462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least three hydroxyl groups, preferably no more than eight hydroxyl groups, wherein the hydrogen atoms of the at least three hydroxyl groups are replaced by unsaturated aliphatic groups having at least two carbon atoms. Preferred groups are those containing two to four carbon atoms, such as vinyl, allyl, and other olefinically unsaturated groups. The unsaturated group itself may contain other substituents, such as methyl. Products sold under the name Carbomer (BF Goodrich, Ohio, USA) are particularly suitable. They are crosslinked with allyl sucrose or allyl pentaerythritol. Among these, carbomer 974P, 934P, and 971P can be mentioned. The use of carbomer 971P is most preferred. Among the copolymers of maleic anhydride and alkenyl derivatives is copolymer EMA (Monsanto), which is a copolymer of maleic anhydride and ethylene. The solubility of these polymers in water results in an acidic solution preferably neutralized to physiological pH, in order to provide an adjuvant solution into which the immunogenic composition, immunological composition, or vaccine composition itself will be incorporated.

[0068] Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), block copolymers (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophospholipid A, avridin lipid-amine adjuvant, heat-labile enterotoxins from Escherichia coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide, or naturally occurring or recombinant cytokines or their analogues, or stimulants of endogenous cytokine release, etc.

[0069] The adjuvant is expected to be added in an amount of about 100 μg to about 10 mg per dose, preferably about 100 μg to about 10 mg per dose, more preferably about 500 μg to about 5 mg per dose, even more preferably about 750 μg to about 2.5 mg per dose, and most preferably about 1 mg per dose. Alternatively, the adjuvant may be at a concentration of about 0.01% to 50% by volume of the final product, preferably about 2% to 30%, more preferably about 5% to 25%, even more preferably about 7% to 22%, and most preferably 10% to 20%.

[0070] In another specific aspect of the immunogenic composition according to the invention, the pharmaceutically acceptable carrier is an adjuvant selected from: aluminum hydroxide, aluminum phosphate, saponins, water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant systems, block copolymers, SAF-M, monophospholipid A, afridin lipid-amine, heat-sensitive enterotoxins from Escherichia coli (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, mineral oil, and combinations thereof.

[0071] In another specific aspect of the immunogenic composition according to the invention, the pharmaceutically acceptable carrier is an adjuvant selected from mineral oil, water-in-oil emulsion, oil-in-water emulsion, oil-in-water emulsion, and combinations thereof.

[0072] Advantageously, the experimental data presented in this paper show that water-in-oil emulsions are suitable as adjuvants.

[0073] In another specific aspect of the immunogenic composition according to the invention, the pharmaceutically acceptable carrier is a water-in-oil emulsion adjuvant or a water-in-oil emulsion adjuvant containing mineral oil. Such water-in-oil emulsion adjuvants or water-in-oil emulsion adjuvants containing mineral oil are well known to those skilled in the art and are commercially available. Exemplarily, white mineral oil can be obtained from Citation... TM and Avatech TM get.

[0074] In another specific aspect of the immunogenic composition according to the invention, the mineral oil comprises or consists of saturated aliphatic hydrocarbons and alicyclic hydrocarbons. Experimental data provided herein show that mineral oils comprising or consisting of saturated aliphatic hydrocarbons and alicyclic hydrocarbons are suitable. However, it must be understood that other mineral oils would also be suitable.

[0075] In another specific aspect of the immunogenic composition according to the invention, the antigens of *Avianobacterium paragallinarum*, avian encephalomyelitis virus, and fowlpox virus work synergistically.

[0076] Advantageously, the experimental data presented in this paper clearly provide evidence that when combined with an inactivated coryza vaccine (Avian bacillus paragallinarum) containing a pharmaceutically acceptable carrier, there is no detectable interference with the efficacy of live attenuated AE (avian encephalomyelitis) and FP (fowlpox) vaccines. Surprisingly, in the trivalent vaccine combination, there is even a slight positive synergistic effect between the efficacy of the fowlpox and AE vaccines.

[0077] The combination of the present invention is advantageous in that it minimizes the number of immunizations (vaccination treatments) required to confer protection against avian coryza, avian encephalomyelitis and fowlpox, reducing application costs and increasing acceptance and coverage.

[0078] As used herein, the term "synergistically" means that the immune response induced by the immunogenic composition (trivalent combination) of the present invention is increased compared to a reference monovalent or bivalent immunogenic composition comprising modified live avian encephalomyelitis virus and / or modified live fowlpox virus. Preferably, the immune response is increased by at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 75%, and most preferably at least 100% compared to the reference monovalent or bivalent immunogenic composition comprising modified live avian encephalomyelitis virus and / or modified live fowlpox virus. How to measure the immune response is within the general knowledge of those skilled in the art. In particular, it is clear to those skilled in the art that the comparison is made between the cell-mediated immune response of the target immunogenic composition and a reference cell-mediated immune response, or between the antibody-mediated immune response of the target immunogenic composition and an antibody-mediated immune response of the reference composition, but neither the cell-mediated immune response of the target immunogenic composition nor the antibody-mediated immune response of the reference composition, nor the other way around. Furthermore, cell-mediated immune responses can be measured, for example, by measuring the activation of cytotoxic T cells via the target immunogenic composition / antigen. Antibody-mediated immune responses can be measured, for example, by measuring the amount of antigen-specific antibodies generated in animals due to administration of an immunogenic composition containing such antigens. Further, immune responses can be measured using clinical parameters. Example 1 described herein illustrates how to determine the antibody titer (ELISA) for pox lesions or avian encephalomyelitis virus.

[0079] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition comprises 10 1 Up to 10 5 EID 50 Avian encephalomyelitis virus / dosage.

[0080] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition comprises 10 1 Up to 10 3 EID 50 Avian encephalomyelitis virus / dosage.

[0081] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition comprises 10 1 Up to 10 5 EID 50 avian pox virus / dosage.

[0082] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition comprises 10 1 Up to 10 3 EID 50 avian pox virus / dosage.

[0083] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition comprises 10 2 Up to 10 15 CFU dosage of avian paraguinea before inactivation.

[0084] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition comprises 10 5 Up to 10 10 CFU dosage of avian paraguinea before inactivation.

[0085] Modified live avian encephalomyelitis virus vaccines have been described extensively in the prior art. Calnek et al. (1961) described the Calnek vaccine strain and the van Roekel vaccine strain. Furthermore, the van Roekel strain is described in the accession number... VR-2058 (adapted to chicken embryos) is deposited at the American Tissue Culture Collection.

[0086] Modified live avian encephalomyelitis virus vaccines have been described in the prior art and have been shown to provide protection. Furthermore, such vaccines against avian encephalomyelitis virus are commercially available, as exemplarily described. AE1143 or AE+Pox (from MSD) AE+FP MLV (from Boehringer Ingelheim), or Tremor L or Poximmune AEL (from Ceva).

[0087] Furthermore, the source of any avian encephalomyelitis virus strain is within the general knowledge of those skilled in the art. Avian encephalomyelitis virus strains can be obtained from research institutions. Additionally, the Van Roekel strain has the following registry number: VR-713 (wild-type strain) is deposited at the U.S. Center for Tissue Culture Collection. Furthermore, avian encephalomyelitis virus strains can be isolated from the wild. Methods for isolating and characterizing avian encephalomyelitis virus strains are well known to those skilled in the art. Moreover, avian encephalomyelitis virus has been sequenced, and its genome sequence is available (exemplarily Marvil et al. 1999: J. Gen. Virol. 80: 653-62; or EMBL database accession number AJ225173). Therefore, the viral genome can be generated by synthesizing its sequence and, subsequently, by applying a reverse genetics system.

[0088] A modified live vaccine can be readily obtained through continuous passage of the aforementioned avian encephalomyelitis virus strain. This can be done effortlessly by those skilled in the art; however, US 5,208,023 provides further guidance in this regard.

[0089] In another specific aspect of the immunogenic composition according to the invention, the avian encephalomyelitis virus is selected from: the Calnek1733 strain and the egg-adapted Van Roekel strain.

[0090] In another specific aspect of the immunogenic composition according to the invention, the avian encephalomyelitis virus is the Calnek1733 strain.

[0091] Modified live fowlpox vaccines have been extensively described in the prior art. Mocket et al. (1990) exemplarily described modified live HP1 and HP193 fowlpox virus strains (Avian Pathology, 19:613-625), and Diallo et al. (1998) described the S vaccine strain (FPV30), M vaccine strain (FPV31), Steggeles vaccine strain (FPV32, 33, and 34), and Webster's vaccine strain (FPV30), as well as other strains and isolates. Ariyoshi et al. (2003) described the F132-c vaccine strain (J.Vet.Med.Sci.65(10):1127-1130). The fowlpox vaccine strain Beaudette is deposited at the U.S. Center for Tissue Cultures under accession number ATCC VR-229. KEM-7 and Gibbs vaccine strains have also been described in the prior art and used in commercially available fowlpox vaccines.

[0092] Modified live fowlpox vaccines have been described in the prior art and have been shown to provide protection. Furthermore, such vaccines against fowlpox are commercially available, as exemplarily described. AE+Pox (from MSD) AE+FP MLV (from Boehringer Ingelheim), or FP L or Poximmune AEL (from Ceva).

[0093] Furthermore, the source of any fowlpox virus strain is within the general knowledge of those skilled in the art. Fowlpox strains can be obtained from research institutions. Additionally, fowlpox strains can be isolated from the wild. Methods for isolating and characterizing fowlpox strains are well known to those skilled in the art. Furthermore, fowlpox viruses have been sequenced, and their genome sequences are available. Therefore, the viral genome can be generated by synthesizing its sequence and by applying a reverse genetics system. Modified live vaccines can be readily obtained through continuous passage of the fowlpox strain.

[0094] In another specific aspect of the immunogenic composition according to the invention, the fowlpox virus is selected from: HP1 and HP193 virus strains, S vaccine strain (FPV30), M vaccine strain (FPV31), Steggeles vaccine strain (FPV32, 33 and 34), Webster vaccine strain (FPV30), F132-c vaccine strain, Beaudette vaccine strain, KEM-7 vaccine strain and Gibbs vaccine strain.

[0095] In another specific aspect of the immunogenic composition according to the invention, the fowlpox virus is selected from: Weybridge vaccine strain, Beaudette vaccine strain, KEM-7 vaccine strain and Gibbs vaccine strain.

[0096] In another specific aspect of the immunogenic composition according to the invention, the fowlpox virus is the Beaudette vaccine strain.

[0097] For *Avianobacter paragallinarum*, three distinct serogroups A, B, and C, and nine serotypes (A1-A4, B-1, and C1-C4) have been described (Blackall et al. 1990; *Journal of Clinical Microbiology*, June 1990, pp. 1185-1187). Furthermore, different reference strains have been identified: strain 221 (serotypes A-A1), strain 2403 (serotypes A-A2), strain E-3C (serotypes A-A3), strain HP14 (serotypes A-A4), strain H-18 (serotypes C-C1), strain Modesto (serotypes C-C2), strain SA-3 (serotypes C-C3), strain HP60 (serotypes C-C4), and strain 2671 (serotypes B-B1). Further, *Avianobacter paragallinarum* strains are available at accession number... 29545、 29975 and 29976 is deposited at the U.S. Tissue Culture Collection.

[0098] Inactivated avian parafowl cholera vaccines (exemplary vaccines containing serogroups A+B+C or A+C) have been described in the prior art and have been shown to provide protection. Furthermore, such vaccines against avian coryza are commercially available, exemplarily... Coryza (from MSD) AC Plus Bact KV (from Boehringer Ingelheim), or CORYZA K (from Ceva).

[0099] Furthermore, the source of any strain of *Avianobacter paragallinarum* is within the general knowledge of those skilled in the art. *Avianobacter paragallinarum* strains can be obtained from research institutions. Additionally, *Avianobacter paragallinarum* strains can be isolated from the wild. Methods for isolating and characterizing *Avianobacter paragallinarum* strains are well known to those skilled in the art. Inactivated vaccines can be readily obtained by inactivation methods (e.g., using β-propiolactone, thimerosal, phenol, or formalin).

[0100] In another specific aspect of the immunogenic composition according to the invention, *Paragonimus westermani* is selected from serogroups A, B, and C.

[0101] In another specific aspect of the immunogenic composition according to the invention, *Paragonimus avianus* comprises strains of *Paragonimus avianus* serogroups A and C.

[0102] In another specific aspect of the immunogenic composition according to the invention, *Paragonimus paragallinarum* comprises strains of *Paragonimus paragallinarum* serogroups A, B, and C.

[0103] In another specific aspect of the immunogenic composition according to the invention, *Avianobacter paragallinarum* comprises strains selected from the following: A1 (O83), B1 (Spross / classic), 4143 (B1 variant), San Francisco 27 ​​(B1 variant), and C2 (Modesto).

[0104] In another specific aspect of the immunogenic composition according to the invention, *Paragonimus paragallinarum* comprises strains A1 (O83), B1 (Spross / classic), 4143 (B1 variant), San Francisco 27 ​​(B1 variant), and C2 (Modesto). Advantageously, it has been shown that the triple combination is effective when such *Paragonimus paragallinarum* strains are included.

[0105] Reagent test kit

[0106] When necessary, the composition may be contained in a packaging or dispenser device, which may contain one or more unit dosage forms containing the active ingredient. The packaging may, for example, comprise metal or plastic foil, such as blister packs. The packaging or dispenser device may be accompanied by instructions for use, preferably for the intended recipient, particularly poultry. Associated with such containers may be a notification in the form prescribed by a government agency regulating the manufacture, use, or sale of the drug or biological product, reflecting approval for human use by the manufacturing, using, or selling agency.

[0107] Therefore, the present invention provides a kit comprising the immunogenic composition as described herein.

[0108] In one particular aspect of the kit according to the invention, a vaccine of *Avianella paragallinarum*, a modified live avian encephalomyelitis virus, and a modified live fowlpox virus are contained in one container.

[0109] In one particular aspect of the kit according to the invention, a vaccine of *Avianella paragallinarum*, a modified live avian encephalomyelitis virus, and a modified live fowlpox virus are contained in one or two containers.

[0110] In one particular aspect of the kit according to the invention, the vaccine of *Avianella paragallinarum*, the modified live avian encephalomyelitis virus, and the modified live fowlpox virus are in separate containers.

[0111] In one particular aspect of the kit according to the invention, a vaccine of *Avianella paragallinarum* is contained in one container; and modified live avian encephalomyelitis virus and modified live fowlpox virus are contained in one container.

[0112] In one particular aspect of the kit according to the invention, the kit further includes instructions for treating and / or preventing poultry diseases.

[0113] In one particular aspect of the kit according to the invention, the kit further includes instructions for treating and / or preventing poultry diseases.

[0114] In one particular aspect of the kit according to the invention, the kit further includes instructions for the treatment and / or prevention of avian rhinitis and / or avian encephalomyelitis virus and / or fowlpox virus.

[0115] In another specific aspect of the kit according to the invention, the kit further comprises a dispenser capable of administering the vaccine to animals, poultry, or livestock.

[0116] Furthermore, according to a further aspect, the specification includes information on at least one dose of the combined vaccine that can be repeatedly administered.

[0117] Treatment

[0118] Furthermore, the present invention provides a method for immunizing a subject, comprising administering to such a subject an immunogenic composition as described herein.

[0119] The term "immunity" refers to active immunization, which involves applying an immunogenic composition to a subject to be immunized, thereby evoking an immune response against an antigen included in such an immunogenic composition.

[0120] Preferably, the immunization leads to a reduction in the incidence of a specific infection in the flock, or a reduction in the severity of clinical signs caused by or associated with a specific infection.

[0121] Furthermore, immunization of the desired subjects with the accompanying immunogenic composition results in the prevention of infection of the subjects from the specific infection. Even more preferably, immunization results in an effective and durable immune response against the specific infection. It should be understood that the time period will last for more than one month, preferably more than two months, more preferably more than three months, more preferably more than four months, more preferably more than five months, and more preferably more than six months. It should be understood that immunization may not be effective in all immunized subjects. However, this terminology requires that a significant proportion of subjects in the flock be effectively immunized.

[0122] Preferably, in this context, a group of subjects is envisioned who would typically (i.e., without immunity) develop clinical signs usually caused by or associated with a specific infection. It can be readily determined by those skilled in the art whether the subjects in said group have been effectively immunized. Preferably, immunization should be effective if, compared to subjects who were not immunized or immunized with immunogenic compositions available prior to the invention but subsequently infected (by *Avianella paragallinarum* and / or avian encephalomyelitis virus and / or fowlpox virus), clinical signs in at least 33%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, more preferably at least 95%, and most preferably 100% of the subjects in a given group are reduced in morbidity or severity by at least 10%, more preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably 100%.

[0123] Furthermore, the present invention provides a method for treating or preventing clinical signs caused by avian encephalomyelitis virus in subjects in need, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition as described herein.

[0124] Furthermore, the present invention provides a method for treating or preventing clinical signs caused by fowlpox virus in subjects in need, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition as described herein.

[0125] Furthermore, the present invention provides a method for treating or preventing clinical signs caused by fowlpox virus and avian encephalomyelitis virus in subjects in need, the method comprising administering to the subject a therapeutically effective amount of an immunogenic agent as described herein.

[0126] Furthermore, the present invention provides a method for treating or preventing clinical signs caused by Avianella paragallinarum in subjects in need, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition as described herein.

[0127] Furthermore, the present invention provides a method for treating or preventing clinical signs caused by fowlpox virus, avian encephalomyelitis virus and avian parafowl bacillus in subjects of need, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition as described herein.

[0128] The term "treatment or prevention" refers to a reduction in the incidence of a specific infection (caused by *Avianella paragallinarum* and / or avian encephalomyelitis virus and / or fowlpox virus) in a flock of birds, or a reduction in the severity of clinical signs caused by or associated with a specific infection. Therefore, the term "treatment or prevention" also refers to a reduction in the number of birds becoming infected (= a reduction in the incidence of a specific infection) in a flock of birds whose subjects have received an effective amount of an immunogenic composition as provided herein, compared to a flock whose subjects have not received such an immunogenic composition; or a reduction in the severity of clinical signs usually associated with or caused by a specific infection; or a reduction in viral shedding after infection; or prevention or reduction of egg production decline in infected laying hens.

[0129] "Treatment or prevention" generally refers to the administration of an effective amount of the immunogenic composition of the present invention to a subject or group of subjects who need or can benefit from such treatment / prevention. The term "treatment" means the administration of an effective amount of the immunogenic composition once at least some of the subjects or group of subjects have been infected, and wherein such subjects have exhibited some clinical signs caused by or related to such infection. The term "prevention" means administration to a subject prior to any infection of such subjects, or in cases where at least none of the subjects or group of subjects have exhibited any clinical signs caused by or related to infection. The terms "prophylaxis" and "preventing" are used interchangeably in this application.

[0130] As used herein, the term "effective amount" means, but is not limited to, the amount of antigen that elicits or is capable of eliciting an immune response in an object. Such effective amounts can reduce the incidence of a particular infection in a flock of birds, or alleviate the severity of the clinical signs of a particular infection (caused by Avianella paragallinarum and / or avian encephalomyelitis virus and / or fowlpox virus).

[0131] Preferably, compared with untreated or treated subjects with immunogenic compositions available prior to the present invention but subsequently infected, clinical signs are reduced in morbidity or severity by at least 10%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%.

[0132] As used herein, the term "clinical signs" refers to the signs of infection in an object caused by *Avianella paragallinarum* and / or avian encephalomyelitis virus and / or fowlpox virus. The clinical signs of infection depend on the pathogen selected.

[0133] Examples of such clinical signs caused by avian parafowl cholera infection include, but are not limited to, rhinitis, facial swelling or edema, anorexia, slowed growth rate, decreased egg production, lethargy, and increased mortality.

[0134] Examples of such clinical signs caused by avian encephalomyelitis virus infection include, but are not limited to, ataxia, rapid tremors of the head and neck, weakness, weight loss, increased mortality, and decreased egg production.

[0135] Examples of such clinical signs caused by fowlpox virus infection include, but are not limited to, pox; spreading eruptions, scabs, and lesions on the comb and dewlap; lesions in the mouth, pharynx, larynx, and trachea; decreased egg production; slowed growth rate; blindness; and increased mortality.

[0136] Clinical signs include, but are not limited to, those that can be directly observed in living animals. Examples of clinical signs that can be directly observed in living animals include pox; spreading eruptions, crusting, and lesions on the crown and dewlap; lesions in the mouth, pharynx, larynx, and trachea; ruffled feathers; weight loss; slowed growth rate; decreased appetite; lameness; lethargy; emaciation and weakness, etc.

[0137] Preferably, compared with untreated subjects or subjects treated with immunogenic compositions available prior to this invention but subsequently infected, the treated subjects exhibited reduced clinical signs of morbidity or severity, including shorter duration of bacteremia, shorter duration of viremia, lower bacterial load, lower viral load, reduced mortality, reduced tremor, reduced ataxia, reduced weakness, reduced weight loss, reduced decrease in egg production, reduced lesions, reduced anorexia, or combinations thereof.

[0138] As used herein, the terms “need” or “required” mean that the administration / treatment is associated with the enhancement or improvement of health or clinical signs, or any other positive medical effect on the health of the subject receiving the immunogenic composition according to the invention.

[0139] Furthermore, the present invention provides a method for reducing mortality in subjects in need compared with non-immunized control subjects of the same species, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition as described herein.

[0140] Furthermore, the present invention provides a method for reducing tremors in a desired subject compared to a non-immunized control group of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition as described herein.

[0141] Furthermore, the present invention provides a method for reducing the decline in egg production in desired subjects compared with non-immunized control subjects of the same species, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition as described herein.

[0142] Furthermore, the present invention provides a method for reducing acne or acne lesions in a subject in need compared with a non-immunized control group of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition as described herein.

[0143] As shown in the examples, the immunogenic compositions provided herein have been proven effective in treating and preventing fowlpox virus and avian encephalomyelitis virus infections, and also in treating and preventing avian parafowl cholera infection.

[0144] The terms “reducing”, “reduced”, “reduction”, or “lowering” are used interchangeably in this application. The term “reduction” means a reduction of clinical signs by at least 10%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%, compared to untreated (non-immunized) but subsequently infected subjects.

[0145] The terms "mortality rate, tremor, decreased egg production, reduction in pox or pox lesions" mean that, compared with a non-immunized control group of the same species, the clinical signs are reduced by at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%. How to measure the clinical signs / parameters is within the general knowledge of those skilled in the art.

[0146] In one aspect of the invention, the object is poultry.

[0147] The term "birds" is well known to those skilled in the art. The term "birds" encompasses all birds, including poultry.

[0148] In one aspect of the invention, the object is poultry.

[0149] The term "poultry" is well known to those skilled in the art. The term "poultry" includes chickens, turkeys, quails, pheasants, guinea fowl, geese, and ducks. Further, the term "chicken" includes broilers, laying hens, and breeding stock used for both, also referred to as breeder chickens.

[0150] In one aspect of the invention, the object is selected from chicken, turkey, quail or pheasant.

[0151] In one aspect of the invention, the object is a chicken.

[0152] In one aspect of the invention, the immunogenic composition is applied once.

[0153] It should be understood that a single dose is administered only once. As shown in the examples, the immunogenic compositions provided herein have proven effective after a single dose is administered to a subject in need.

[0154] The dose-volume ratio per poultry depends on the route of vaccination and the age of the poultry.

[0155] Vaccines for intramuscular or subcutaneous administration may contain a dose in a volume of 30 μl to 1000 μl. Preferably, a single dose has a total volume of about 30 μl to 1000 μl, more preferably about 100 μl to 800 μl, even more preferably about 100 μl, 200 μl to 700 μl, wherein single doses of 200 μl, 300 μl, 4000 μl, 500 μl, 600 μl, 700 μl or 800 μl are most preferred.

[0156] In one aspect of the invention, the immunogenic composition is administered in two or more doses.

[0157] However, the immunogenic composition may be administered in two or more doses, wherein the first dose is administered before the second (enhanced) dose is administered.

[0158] In a preferred aspect of the two-dose regimen, the first and second doses of the immunogenic composition are administered in the same amount. Preferably, each dose is the preferred amount specified above. In addition to the first and second dose regimen, an alternative embodiment includes further subsequent doses. For example, a third, fourth, or fifth dose may be administered in these aspects. Preferably, the subsequent third, fourth, and fifth dose regimens are administered in the same amount as the first dose, wherein the time frame between the doses coincides with the time arrangement between the first and second doses mentioned above.

[0159] Immunogenic compositions are preferably administered topically or systemically. Suitable routes of administration are conventionally used, such as oral or parenteral, including intranasal, intravenous, intradermal, percutaneous, intramuscular, intraperitoneal, subcutaneous, inhalation, intraovarian, via spray, via drinking water, or via eye drops. However, depending on the nature and mode of action of the compound, immunogenic compositions may also be administered via other routes. These other routes include intradermal, intravenous, intravascular, intraarterial, intraperitoneal, intrathecal, intratracheal, intradermal, intracardiac, intralobar, intramedullary, intrapulmonary, rectal, and vaginal administration. However, most preferably, the immunogenic composition is administered subcutaneously, intramuscularly, orally, intraovarianly, via spray, via drinking water, or via eye drops.

[0160] In one aspect of the invention, the immunogenic composition is administered subcutaneously, intramuscularly, intradermally, orally, or via eye drops.

[0161] In one aspect of the invention, the immunogenic composition is administered subcutaneously or intramuscularly.

[0162] Typically, vaccines contain 10 1 Up to 10 8 EID 50 (50% oocyte infection dose) / dose concentration, preferably 10 1 Up to 10 5 EID 50 / concentration of dose, and more preferably 10 1 Up to 10 3 EID 50 / Dose concentration of avian encephalomyelitis virus.

[0163] In one aspect of the invention, the immunogenic composition comprises 10 1 Up to 10 5 EID 50Avian encephalomyelitis virus / dosage.

[0164] In one aspect of the invention, the immunogenic composition comprises 10 1 Up to 10 3 EID 50 Avian encephalomyelitis virus / dosage.

[0165] Typically, vaccines contain 10 1 Up to 10 8 EID 50 (50% oocyte infection dose) / dose concentration, preferably 10 1 Up to 10 5 EID 50 / concentration of dose, and more preferably 10 1 Up to 10 3 EID 50 / Dose concentration of fowlpox virus.

[0166] In one aspect of the invention, the immunogenic composition comprises 10 1 Up to 10 5 EID 50 avian pox virus / dosage.

[0167] In one aspect of the invention, the immunogenic composition comprises 10 1 Up to 10 3 EID 50 avian pox virus / dosage.

[0168] Typically, vaccines contain 10 1 Up to 10 20 The concentration of CFU (colony forming units) / dose is preferably 10. 2 Up to 10 15 EID 50 / concentration of dose, and more preferably 10 5 Up to 10 10 EID 50 / The concentration of *Bacillus paragenomicum* before inactivation.

[0169] In one aspect of the invention, the immunogenic composition comprises 10 2 Up to 10 15 CFU dosage of avian paraguinea before inactivation.

[0170] In one aspect of the invention, the immunogenic composition comprises 10 5 Up to 10 10 CFU dosage of avian paraguinea before inactivation.

[0171] In one aspect of the invention, the immunogenic composition comprises 10 2 Up to 103 EID 50 Avian encephalitis virus / dosage, 10 2 Up to 10 3 EID 50 fowlpox virus / dosage, and 10 7 Up to 10 9 CFU dosage of avian paraguinea before inactivation.

[0172] In one aspect of the invention, the immunogenic composition is applied to subjects after 2 weeks of age, 3 weeks of age, 4 weeks of age, 6 weeks of age, or 8 weeks of age.

[0173] In one aspect of the invention, the immunogenic composition is applied to subjects older than 2 weeks of age.

[0174] In one aspect of the invention, the immunogenic composition is applied to subjects aged 3 weeks and older.

[0175] In one aspect of the invention, the immunogenic composition is applied to subjects aged 6 weeks and older.

[0176] In one aspect of the invention, the immunogenic composition is applied to subjects 5 to 8 weeks before the onset of egg production.

[0177] In one aspect of the invention, the immunogenic composition is applied to subjects aged 3 weeks and older.

[0178] As shown in the examples, when applied to 3-week-old poultry, the immunogenic compositions provided herein have proven to be safe and effective.

[0179] In one aspect of the invention, the method results in improvements in efficacy parameters selected from the following, compared to a non-immunized control group of the same species: shorter duration of bacteremia, shorter duration of viremia, lower bacterial load, lower viral load, reduced mortality, reduced tremor, reduced ataxia, reduced weakness, reduced weight loss, reduced decrease in egg production, reduced lesions, reduced anorexia, or a combination thereof.

[0180] The terms “viral load” or “bacterial load” are well known to those skilled in the art. The term viral load (bacterial load) may be used interchangeably herein with the term viral titer (bacterial titer). Viral load (bacterial load) or viral titer (bacterial titer) is a measure of the severity of an active viral (bacterial) infection and can be determined by methods known to those skilled in the art. Determination may be based on, for example, detection of viral (bacterial) proteins by antibodies binding to viral (bacterial) proteins, and further or alternative detection by amplification methods such as RT-PCR of viral (bacterial) RNA. Exemplarily, viral load (bacterial load) or viral titer (bacterial titer) can be calculated by estimating the viral (bacterial) viability in the bodily fluids involved, such as the number of RNA copies per milliliter of plasma.

[0181] The terms “reducing,” “reduced,” “reduction,” or “lower” or “shorter” mean that, compared with a non-immunized control group of the same species, the efficacy parameters (duration of bacteremia, duration of viremia, bacterial load, viral load, mortality, tremor, ataxia, weakness, weight loss, decreased egg production, damage, anorexia) are reduced by at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%. How to measure the improvement in efficacy parameters is within the general knowledge of those skilled in the art.

[0182] In one aspect of the invention, treatment or prevention leads to a reduction or prevention of mortality compared to an untreated control group of the same species.

[0183] In one aspect of the invention, treatment or prevention leads to the prevention or reduction of tremor compared to an untreated control group of the same species.

[0184] In one aspect of the invention, treatment or prevention leads to a reduction or decrease in egg production compared to an untreated control group of the same species.

[0185] In one aspect of the invention, treatment or prevention leads to the prevention or reduction of acne or acne lesions compared to an untreated control group of the same species.

[0186] The present invention further provides an immunogenic composition comprising the following combination:

[0187] -Vaccine of avian parafowl cholera, and

[0188] - Modified live avian encephalomyelitis virus, and

[0189] - Modified live fowlpox virus; and

[0190] - Water-in-oil emulsion adjuvants or water-in-oil emulsion adjuvants containing mineral oil.

[0191] The present invention further provides an immunogenic composition comprising the following combination:

[0192] -with 10 5 Up to 10 10 CFU of pre-inactivated *Avianella paragallinarum* / dose of *Avianella paragallinarum* vaccine, and

[0193] -with 10 1 Up to 10 3 EID 50 Avian encephalomyelitis virus / dosage-modified live avian encephalomyelitis virus, and

[0194] -with 10 1 Up to 10 3 EID 50 avianpox virus / dosage-modified live avianpox virus; and

[0195] - Water-in-oil emulsion adjuvants or water-in-oil emulsion adjuvants containing mineral oil.

[0196] The present invention further provides an immunogenic composition comprising the following combination:

[0197] -Vaccine of avian parafowl cholera, and

[0198] - Modified live avian encephalomyelitis virus, and

[0199] - Modified live fowlpox virus; and

[0200] - A water-in-oil emulsion adjuvant containing mineral oil, wherein the mineral oil comprises or is composed of saturated aliphatic hydrocarbons and alicyclic hydrocarbons.

[0201] The present invention further provides an immunogenic composition comprising the following combination:

[0202] -with 10 5 Up to 10 10 EID 50 The inactivated strain of *Avianella paragallinarum* / dose of *Avianella paragallinarum* vaccine, and

[0203] -with 10 1 Up to 10 3 EID 50 Avian encephalomyelitis virus / dosage-modified live avian encephalomyelitis virus, and

[0204] -with 101 Up to 10 3 EID 50 avianpox virus / dosage-modified live avianpox virus; and

[0205] - A water-in-oil emulsion adjuvant containing mineral oil, wherein the mineral oil comprises or is composed of saturated aliphatic hydrocarbons and alicyclic hydrocarbons.

[0206] In one aspect of the invention, the immunogenic composition is a vaccine.

[0207] In one aspect of the invention, the immunogenic composition comprises 10 2 Up to 10 3 EID 50 Avian encephalomyelitis virus / dosage.

[0208] In one aspect of the invention, the immunogenic composition comprises 10 2 Up to 10 3 EID 50 avian pox virus / dosage.

[0209] In one aspect of the invention, the immunogenic composition comprises 10 7 Up to 10 9 CFU dosage of avian paraguinea before inactivation.

[0210] The present invention further provides immunogenic compositions as described herein for therapeutic use.

[0211] The present invention further provides immunogenic compositions as described herein, which can be used as immunogens or vaccines.

[0212] The present invention further provides immunogenic compositions as described herein, which are used as pharmaceuticals.

[0213] The present invention further provides the use of the immunogenic compositions described herein for the manufacture of pharmaceuticals.

[0214] The present invention further provides a method for preparing an immunogenic composition, comprising:

[0215] a.) Provide a vaccine for *Avianobacter pylori*; and

[0216] b.) Provide modified live avian encephalomyelitis virus; and

[0217] c.) Provide modified live fowlpox virus;

[0218] d.) Combining components a) through c) to form a trivalent composition; and

[0219] e.) Obtain the trivalent composition; and

[0220] f.) Add a pharmaceutically acceptable carrier.

[0221] The term “obtain” can include harvesting, separating, purifying and / or formulating (e.g., finishing and / or blending).

[0222] In one particular aspect of the method for preparing the immunogenic composition according to the invention, the pharmaceutically acceptable carrier is an adjuvant.

[0223] In another specific aspect of the method for preparing the immunogenic composition according to the invention, the pharmaceutically acceptable carrier is an adjuvant selected from: aluminum hydroxide, aluminum phosphate, saponins, water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant systems, block copolymers, SAF-M, monophospholipid A, afridin lipid-amine, heat-sensitive enterotoxins from Escherichia coli (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, mineral oil, and combinations thereof.

[0224] In another specific aspect of the method for preparing the immunogenic composition according to the invention, the pharmaceutically acceptable carrier is an adjuvant selected from mineral oil, water-in-oil emulsions, oil-in-water emulsions, oil-in-water emulsions, and combinations thereof.

[0225] In another specific aspect of the method for preparing the immunogenic composition according to the invention, the pharmaceutically acceptable carrier is a water-in-oil emulsion adjuvant or a water-in-oil emulsion adjuvant containing mineral oil.

[0226] In another specific aspect of the method for preparing the immunogenic composition according to the invention, the mineral oil comprises or consists of saturated aliphatic hydrocarbons and alicyclic hydrocarbons.

[0227] In another specific aspect of the method for preparing the immunogenic composition according to the invention, the immunogenic composition comprises 10 1 Up to 10 5 EID 50 Avian encephalomyelitis virus / dosage.

[0228] In another specific aspect of the method for preparing the immunogenic composition according to the invention, the immunogenic composition comprises 10 1 Up to 10 3 EID 50 Avian encephalomyelitis virus / dosage.

[0229] In another specific aspect of the method for preparing the immunogenic composition according to the invention, the immunogenic composition comprises 10 1 Up to 10 5 EID50 avian pox virus / dosage.

[0230] In another specific aspect of the method for preparing the immunogenic composition according to the invention, the immunogenic composition comprises 10 1 Up to 10 3 EID 50 avian pox virus / dosage.

[0231] In another specific aspect of the method for preparing the immunogenic composition according to the invention, the immunogenic composition comprises 10 2 Up to 10 15 CFU dosage of avian paraguinea before inactivation.

[0232] In another specific aspect of the method for preparing the immunogenic composition according to the invention, the immunogenic composition comprises 10 5 Up to 10 10 CFU dosage of avian paraguinea before inactivation.

[0233] Technical solution

[0234] This article also describes the following technical solutions:

[0235] 1. An immunogenic composition comprising: a) one or more antigens of *Avianobacter paragallinarum* and one or more antigens of avian encephalomyelitis virus and one or more antigens of fowlpox virus; and b) a pharmaceutically acceptable carrier.

[0236] 2. An immunogenic composition comprising: a) one or more antigens of a vaccine of *Avianella paragallinarum* and one or more antigens of a modified live avian encephalomyelitis virus and one or more antigens of a modified live fowlpox virus; and b) a pharmaceutically acceptable carrier.

[0237] 3. An immunogenic composition comprising: a) a vaccine of *Avianella paragallinarum* and modified live avian encephalomyelitis virus and modified live fowlpox virus; and b) a pharmaceutically acceptable carrier.

[0238] 4. An immunogenic composition comprising a combined vaccine of *Avianella paragallinarum* and modified live avian encephalomyelitis virus and modified live fowlpox virus.

[0239] 5. The immunogenic composition of technical solution 1, wherein one or more antigens of *Paragonimus paragallinarum* are vaccines of *Paragonimus paragallinarum*.

[0240] 6. The immunogenic composition of any one of technical solutions 2 to 5, wherein the bacterium paragallinarum vaccine is a completely inactivated bacterium paragallinarum vaccine.

[0241] 7. The immunogenic composition of any one of technical solutions 2 to 6, wherein the *Avianobacter paragallinarum* vaccine is a formaldehyde-inactivated or thimerosal-inactivated whole-cell vaccine of *Avianobacter paragallinarum*.

[0242] 8. The immunogenic composition of technical solution 1, wherein the fowlpox virus is a modified live fowlpox virus.

[0243] 9. An immunogenic composition of any one of technical claims 2 to 4 and 6 to 8, wherein the modified live fowlpox virus is attenuated.

[0244] 10. An immunogenic composition of any one of claims 2 to 4 and 6 to 9, wherein the modified live fowlpox virus is non-recombinant.

[0245] 11. An immunogenic composition of any one of claims 2 to 4 and 6 to 9, wherein the modified live fowlpox virus is recombinant.

[0246] 12. The immunogenic composition of technical solution 1, wherein the avian encephalomyelitis virus is a modified live avian encephalomyelitis virus.

[0247] 13. The immunogenic compositions of technical solutions 2 to 4 and 6 to 12, wherein the modified live avian encephalomyelitis virus is attenuated.

[0248] 14. An immunogenic composition of any one of technical claims 2 to 4 and 6 to 13, wherein the modified live avian encephalomyelitis virus is non-recombinant.

[0249] 15. An immunogenic composition of any one of technical claims 2 to 4 and 6 to 13, wherein the modified live avian encephalomyelitis virus is recombinant.

[0250] 16. An immunogenic composition of any one of technical solutions 1 to 15, wherein the immunogenic composition effectively treats and / or prevents clinical signs caused by avian paragallinar infection in subjects in need.

[0251] 17. An immunogenic composition of any one of technical solutions 1 to 16, wherein the immunogenic composition effectively treats and / or prevents clinical signs of avian coryza.

[0252] 18. An immunogenic composition of any one of technical claims 1 to 17, wherein the immunogenic composition effectively treats and / or prevents clinical signs caused by avian encephalomyelitis virus infection in subjects in need.

[0253] 19. An immunogenic composition of any one of claims 1 to 18, wherein the immunogenic composition effectively treats and / or prevents clinical signs caused by fowlpox virus infection in subjects in need.

[0254] 20. An immunogenic composition of any one of claims 16 to 19, wherein the target is poultry or domestic fowl.

[0255] 21. An immunogenic composition of any one of technical solutions 1 to 20, wherein the immunogenic composition is a vaccine.

[0256] 22. An immunogenic composition of any one of claims 1 to 21, wherein the immunogenic composition is formulated for single-dose administration.

[0257] 23. An immunogenic composition of any one of technical solutions 1 to 3 and 5 to 19, wherein the pharmaceutically acceptable carrier is selected from solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delay agents, adjuvants, immunostimulants, and combinations thereof.

[0258] 24. An immunogenic composition of any one of claims 1 to 23, wherein the immunogenic composition comprises a preservative.

[0259] 25. An immunogenic composition of any one of technical solutions 1 to 24, wherein the immunogenic composition comprises formaldehyde as a preservative.

[0260] 26. An immunogenic composition of any one of claims 1 to 3 and 5 to 25, wherein the pharmaceutically acceptable carrier is an adjuvant.

[0261] 27. An immunogenic composition of any one of claims 1 to 3 and 5 to 26, wherein the pharmaceutically acceptable carrier is an adjuvant selected from: aluminum hydroxide, aluminum phosphate, saponins, water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant systems, block copolymers, SAF-M, monophospholipid A, afridin lipid-amine, heat-sensitive enterotoxins from Escherichia coli (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, mineral oil, and combinations thereof.

[0262] 28. An immunogenic composition of any one of technical solutions 1 to 3 and 5 to 27, wherein the pharmaceutically acceptable carrier is an adjuvant selected from mineral oil, water-in-oil emulsion, oil-in-water emulsion, oil-in-water emulsion, and combinations thereof.

[0263] 29. An immunogenic composition of any one of technical claims 1 to 3 and 5 to 28, wherein the pharmaceutically acceptable carrier is a water-in-oil emulsion adjuvant or a water-in-oil emulsion adjuvant containing mineral oil.

[0264] 30. An immunogenic composition of any one of technical solutions 27 to 29, wherein the mineral oil comprises or is composed of saturated aliphatic hydrocarbons and alicyclic hydrocarbons.

[0265] 31. An immunogenic composition of any one of technical solutions 1 to 30, wherein the antigens of *Avianobacterium paragallinarum*, avian encephalomyelitis virus, and fowlpox virus act synergistically.

[0266] 32. The immunogenic composition of any one of technical solutions 1 to 31, wherein the immunogenic composition comprises 10 1 Up to 10 5 EID 50 Avian encephalomyelitis virus / dosage.

[0267] 33. The immunogenic composition of any one of technical solutions 1 to 32, wherein the immunogenic composition comprises 10 1 Up to 10 3 EID 50 Avian encephalomyelitis virus / dosage.

[0268] 34. An immunogenic composition according to any one of technical solutions 1 to 33, wherein the immunogenic composition comprises 10 1 Up to 10 5 EID 50 avian pox virus / dosage.

[0269] 35. An immunogenic composition according to any one of technical solutions 1 to 34, wherein the immunogenic composition comprises 10 1 Up to 10 3 EID 50 avian pox virus / dosage.

[0270] 36. An immunogenic composition according to any one of technical solutions 1 to 35, wherein the immunogenic composition comprises 10 2 Up to 10 15 CFU dosage of avian paraguinea before inactivation.

[0271] 37. An immunogenic composition according to any one of technical solutions 1 to 36, wherein the immunogenic composition comprises 10 5 Up to 10 10 CFU dosage of avian paraguinea before inactivation.

[0272] 38. An immunogenic composition of any one of technical solutions 1 to 37, wherein the avian encephalomyelitis virus is selected from: Calnek 1733 strain and egg-adapted Van Roekel strain.

[0273] 39. An immunogenic composition of any one of technical solutions 1 to 38, wherein the avian encephalomyelitis virus is the Calnek 1733 strain.

[0274] 40. An immunogenic composition of any one of technical claims 1 to 39, wherein the fowlpox virus is selected from: HP1 and HP193 virus strains, S vaccine strain (FPV30), M vaccine strain (FPV31), Steggeles vaccine strain (FPV32, 33 and 34), Webster vaccine strain (FPV30), F132-c vaccine strain, Beaudette vaccine strain, KEM-7 vaccine strain and Gibbs vaccine strain.

[0275] 41. An immunogenic composition of any one of technical solutions 1 to 40, wherein the fowlpox virus is selected from: Weybridge vaccine strain, Beaudette vaccine strain, KEM-7 vaccine strain and Gibbs vaccine strain.

[0276] 42. An immunogenic composition of any one of technical claims 1 to 41, wherein the fowlpox virus is the Beaudette vaccine strain.

[0277] 43. An immunogenic composition of any one of technical solutions 1 to 42, wherein the *Avianella paragallinarum* is selected from serogroup A, serogroup B and serogroup C.

[0278] 44. An immunogenic composition of any one of technical solutions 1 to 43, wherein the *Paragonimus paragallinarum* comprises strains of *Paragonimus paragallinarum* serogroups A and C.

[0279] 45. An immunogenic composition of any one of technical solutions 1 to 44, wherein the *Paragonimus paragallinarum* comprises strains of *Paragonimus paragallinarum* serogroups A, B and C.

[0280] 46. ​​An immunogenic composition of any one of technical claims 1 to 45, wherein the *Avianella paragallinarum* comprises strains selected from the following: A1 (O83), B1 (Spross / classic), 4143 (B1 variant), San Francisco 27 ​​(B1 variant), and C2 (Modesto).

[0281] 47. An immunogenic composition of any one of technical solutions 1 to 46, wherein the *Avianobacterium paragallinarum* comprises strains A1 (O83), B1 (Spross / classic), 4143 (B1 variant), San Francisco 27 ​​(B1 variant), and C2 (Modesto).

[0282] 48. A kit comprising an immunogenic composition of any one of claims 1 to 47.

[0283] 49. The kit according to technical solution 48, wherein the avian paraavirhea vaccine, the modified live avian encephalomyelitis virus, and the modified live fowlpox virus are contained in one container.

[0284] 50. The kit according to technical solution 48, wherein the avian paraavirhea vaccine, the modified live avian encephalomyelitis virus, and the modified live fowlpox virus are in one or two containers.

[0285] 51. The kit according to technical solution 48, wherein the avian paraavirhea vaccine, the modified live avian encephalomyelitis virus, and the modified live fowlpox virus are in separate containers.

[0286] 52. The kit according to technical solution 48, wherein the avian paragallinarum vaccine is in one container; and the modified live avian encephalomyelitis virus and the modified live fowlpox virus are in one container.

[0287] 53. A kit according to any one of technical claims 48 to 52, wherein the kit further comprises instructions for treating and / or preventing poultry diseases.

[0288] 54. A kit according to any one of technical claims 48 to 53, wherein the kit further comprises instructions for treating and / or preventing poultry diseases.

[0289] 55. A kit according to any one of technical claims 48 to 54, wherein the kit further comprises instructions for treating and / or preventing avian coryza and / or avian encephalomyelitis virus and / or fowlpox virus.

[0290] 56. A method for immunizing a subject, comprising administering to such a subject an immunogenic composition of any one of technical solutions 1 to 47.

[0291] 57. A method for treating or preventing clinical signs caused by avian encephalomyelitis virus in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 47.

[0292] 58. A method for treating or preventing clinical signs caused by fowlpox virus in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 47.

[0293] 59. A method for treating or preventing clinical signs caused by fowlpox virus and avian encephalomyelitis virus in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 47.

[0294] 60. A method for treating or preventing clinical signs caused by *Avianobacterium paragallinarum* in a subject, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 47.

[0295] 61. A method for treating or preventing clinical signs caused by fowlpox virus, avian encephalomyelitis virus and avian parafowl bacillus in a subject of need, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 47.

[0296] 62. A method for reducing mortality in subjects in need compared with a non-immunized control group of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 47.

[0297] 63. A method for reducing tremors in a desired subject compared to a non-immunized control group of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 47.

[0298] 64. A method for reducing the decline in egg production in a desired subject compared with a non-immunized control group of the same species, said method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 47.

[0299] 65. A method for reducing acne or acne lesions in a desired subject compared with a non-immunized control group of the same species, said method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 47.

[0300] 66. An immunogenic composition of any one of claims 1 to 47, wherein the method of immunizing a subject comprises administering a therapeutically effective amount of the immunogenic composition to the subject.

[0301] 67. An immunogenic composition according to any one of claims 1 to 47, in a method for treating or preventing clinical signs caused by fowlpox virus and / or avian encephalomyelitis virus and / or avian parafowl bacillus in a subject of need, the method comprising administering a therapeutically effective amount of the immunogenic composition to the subject.

[0302] 68. An immunogenic composition according to any one of claims 1 to 47, used in a method for reducing mortality and / or tremor and / or decreased egg production and / or pox or pox lesions in subjects of need, compared with a non-immunized control group of the same species, wherein the method comprises administering a therapeutically effective amount of the immunogenic composition to the subject.

[0303] 69. The method or use of any one of technical solutions 56 to 68, wherein the object is a bird.

[0304] 70. The method or use of any one of technical solutions 56 to 69, wherein the object is poultry.

[0305] 71. The method or use of any one of technical solutions 56 to 70, wherein the object is selected from chicken, turkey, quail or pheasant.

[0306] 72. The method or use of any one of technical solutions 56 to 71, wherein the object is a chicken.

[0307] 73. The method or use of any one of technical solutions 56 to 72, wherein the immunogenic composition is applied once.

[0308] 74. The method or use of any one of technical claims 56 to 72, wherein the immunogenic composition is administered in two or more doses.

[0309] 75. The method or use of any one of technical solutions 56 to 74, wherein the immunogenic composition is administered subcutaneously, intramuscularly, intradermally, orally, or via eye drops.

[0310] 76. The method or use of any one of technical solutions 56 to 75, wherein the immunogenic composition is administered subcutaneously or intramuscularly.

[0311] 77. The method or use of any one of technical solutions 56 to 76, wherein the immunogenic composition comprises 10 1 Up to 10 5 EID 50 Avian encephalomyelitis virus / dosage.

[0312] 78. The method or use of any one of technical solutions 56 to 77, wherein the immunogenic composition comprises 10 1 Up to 10 3 EID 50 Avian encephalomyelitis virus / dosage.

[0313] 79. The method or use of any one of technical solutions 56 to 78, wherein the immunogenic composition comprises 10 1 Up to 10 5 EID 50 avian pox virus / dosage.

[0314] 80. The method or use of any one of technical solutions 56 to 79, wherein the immunogenic composition comprises 10 1 Up to 10 3 EID 50 avian pox virus / dosage.

[0315] 81. The method or use of any one of technical solutions 56 to 80, wherein the immunogenic composition comprises 10 2 Up to 10 15 CFU dosage of avian paraguinea before inactivation.

[0316] 82. The method or use of any one of technical solutions 56 to 81, wherein the immunogenic composition comprises 10 5 Up to 10 10 CFU dosage of avian paraguinea before inactivation.

[0317] 83. The method of any one of technical solutions 56 to 82, wherein the immunogenic composition is applied to subjects after 2 weeks of age, 3 weeks of age, 4 weeks of age, 6 weeks of age, or 8 weeks of age.

[0318] 84. The method of any one of technical solutions 56 to 83, wherein the immunogenic composition is applied to subjects aged 3 weeks or older.

[0319] 85. The method of any one of technical claims 56 to 84, wherein the method results in an improvement in efficacy parameters selected from the following, compared with a non-immunized control group of the same species: shorter duration of bacteremia, shorter duration of viremia, lower bacterial load, lower viral load, reduced mortality, reduced tremor, reduced ataxia, reduced weakness, reduced weight loss, reduced decrease in egg production, reduced lesions, reduced anorexia, or a combination thereof.

[0320] 86. An immunogenic composition comprising the following combination:

[0321] -Vaccine of avian parafowl cholera, and

[0322] - Modified live avian encephalomyelitis virus, and

[0323] - Modified live fowlpox virus; and

[0324] - Water-in-oil emulsion adjuvants or water-in-oil emulsion adjuvants containing mineral oil.

[0325] 87. An immunogenic composition comprising the following combination:

[0326] -with 10 5 Up to 10 10 CFU of pre-inactivated *Avianella paragallinarum* / dose of *Avianella paragallinarum* vaccine, and

[0327] -with 10 1 Up to 10 3 EID 50 Avian encephalomyelitis virus / dosage-modified live avian encephalomyelitis virus, and

[0328] -with 10 1 Up to 10 3 EID 50 avianpox virus / dosage-modified live avianpox virus; and

[0329] - Water-in-oil emulsion adjuvants or water-in-oil emulsion adjuvants containing mineral oil.

[0330] 88. An immunogenic composition comprising the following combination:

[0331] -Vaccine of avian parafowl cholera, and

[0332] - Modified live avian encephalomyelitis virus, and

[0333] - Modified live fowlpox virus; and

[0334] - A water-in-oil emulsion adjuvant containing mineral oil, wherein the mineral oil comprises or is composed of saturated aliphatic hydrocarbons and alicyclic hydrocarbons.

[0335] 89. An immunogenic composition comprising the following combination:

[0336] -with 10 5 Up to 10 10 EID 50 The inactivated strain of *Avianella paragallinarum* / dose of *Avianella paragallinarum* vaccine, and

[0337] -with 10 1 Up to 10 3 EID 50 Avian encephalomyelitis virus / dosage-modified live avian encephalomyelitis virus, and

[0338] -with 10 1 Up to 10 3 EID 50 avianpox virus / dosage-modified live avianpox virus; and

[0339] - A water-in-oil emulsion adjuvant containing mineral oil, wherein the mineral oil comprises or is composed of saturated aliphatic hydrocarbons and alicyclic hydrocarbons.

[0340] 90. An immunogenic composition of any one of technical solutions 86 to 89, wherein the immunogenic composition is a vaccine.

[0341] 91. The immunogenic composition of any one of technical solutions 86 to 90, wherein the immunogenic composition comprises 10 2 Up to 10 3 EID 50 Avian encephalomyelitis virus / dosage.

[0342] 92. The immunogenic composition of any one of technical solutions 86 to 91, wherein the immunogenic composition comprises 10 2 Up to 10 3 EID 50 avian pox virus / dosage.

[0343] 93. The immunogenic composition of any one of technical solutions 86 to 92, wherein the immunogenic composition comprises 10 7 Up to 10 9 CFU dosage of avian paraguinea before inactivation.

[0344] Production methods

[0345] 94. A method for preparing an immunogenic composition, comprising:

[0346] a.) Provide a vaccine for *Avianobacter pylori*; and

[0347] b.) Provide modified live avian encephalomyelitis virus; and

[0348] c.) Provide modified live fowlpox virus;

[0349] d.) Combining components a) through c) to form a trivalent composition; and

[0350] e.) Obtain the trivalent composition; and

[0351] f.) Add a pharmaceutically acceptable carrier.

[0352] 95. A method for preparing the immunogenic composition of technical solution 94, wherein the pharmaceutically acceptable carrier is an adjuvant.

[0353] 96. A method for preparing the immunogenic composition of technical solution 94 or 95, wherein the pharmaceutically acceptable carrier is an adjuvant selected from the following: aluminum hydroxide, aluminum phosphate, saponins, water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant systems, block copolymers, SAF-M, monophospholipid A, afridin lipid-amine, heat-sensitive enterotoxins from Escherichia coli (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, mineral oil, and combinations thereof.

[0354] 97. A method for preparing an immunogenic composition according to any one of technical solutions 94 to 96, wherein the pharmaceutically acceptable carrier is an adjuvant selected from mineral oil, water-in-oil emulsion, oil-in-water emulsion, oil-in-water emulsion, and combinations thereof.

[0355] 98. A method for preparing an immunogenic composition according to any one of technical solutions 94 to 97, wherein the immunogenic composition comprises 10 1 Up to 10 5 EID 50 Avian encephalomyelitis virus / dosage.

[0356] 99. A method for preparing an immunogenic composition according to any one of technical solutions 94 to 98, wherein the immunogenic composition comprises 10 1 Up to 10 3 EID 50 Avian encephalomyelitis virus / dosage.

[0357] 100. A method for preparing an immunogenic composition according to any one of technical solutions 94 to 99, wherein the immunogenic composition comprises 10 1 Up to 10 5 EID 50 avian pox virus / dosage.

[0358] 101. A method for preparing an immunogenic composition according to any one of technical solutions 94 to 100, wherein the immunogenic composition comprises 10 1 Up to 10 3 EID 50 avian pox virus / dosage.

[0359] 102. A method for preparing an immunogenic composition according to any one of technical solutions 94 to 101, wherein the immunogenic composition comprises 10 2 Up to 10 15 CFU dosage of avian paraguinea before inactivation.

[0360] 103. A method for preparing an immunogenic composition according to any one of technical solutions 94 to 102, wherein the immunogenic composition comprises 10 5 Up to 10 10 CFU dosage of avian paraguinea before inactivation. Attached Figure Description

[0361] Figure 1 Percentage of chickens per day with pox lesions (on the wings and / or thighs) after attack. PC = positive control; NC = negative control.

[0362] Figure 2 Mean AE ELISA titer / treatment group. PC = positive control; NC = negative control.

[0363] Example

[0364] The following embodiments illustrate specific implementations of the present invention. These embodiments are merely illustrative and should not be construed as limiting the scope or basic principles of the invention.

[0365] Example 1:

[0366] Developing vaccines against avian infectious coryza and modified live vaccines against fowlpox and avian encephalomyelitis at home. Interference with the efficacy of poultry

[0367] Research Objectives

[0368] To determine the interference of inactivated infectious avian coryza vaccine (Avian bacillus parageli) with the efficacy of live attenuated vaccines against fowlpox (FP) and avian encephalomyelitis (AE) in poultry.

[0369] Experimental Design

[0370] For SPF laying hens (21 days old at D0), they were randomly assigned to four treatment groups (TG):

[0371] • TG01 (20 animals): Vaccinated with a live AE-FP vaccine (trivalent vaccine) incorporated into an inactivated rhinitis vaccine, followed by FP challenge. This group was designed to detect interference of the rhinitis vaccine on the efficacy of the AE-FP vaccine.

[0372] • TG02 (20 animals): Vaccinated with the AE-FP live vaccine (bivalent vaccine), followed by FP challenge. This group was intended to demonstrate the efficacy of the live AE-FP vaccine.

[0373] • TG03 (10 animals): Unvaccinated but exposed to FP virus (FVP). This group was used to determine challenge with FPV and thus validate animal experiments (challenge control).

[0374] • TG04 (5 animals): Unvaccinated, but inoculated with sterile water. This group was used to study the effectiveness of controlling antibody deficiency against AE and FPV, as well as the lack of vaccinia development.

[0375] The experimental unit was the treatment group (TG).

[0376] Vaccinated animals received a single dose of the vaccine tested in different combinations. The routes of vaccination were the registered vaccine routes (Table 1). Challenge was completed three weeks post-vaccination; TG01, TG02, and TG03 were administered via wing netting and feather pouch method using pathogenic fowlpox strains, and TG04 (control group) was inoculated with sterile water. Chickens were observed daily thereafter until the end of the study, and the appearance of clinical signs of fowlpox was examined between days 29 and 35. At the end of the study, chickens were euthanized by inhalation of an O2-CO2 gas mixture. Blood samples were obtained from all chickens at days 0, 21, and 42. The presence or absence of antibodies against AE in the blood was examined.

[0377] Table 1. Experimental Design of the Study

[0378]

[0379] *For ELISA AE

[0380] **For ELISA AE

[0381] ***or even earlier, when chickens were euthanized for animal welfare reasons

[0382] The main parameters in this study are:

[0383] 1. Antibody response against AE virus determined by using a standard commercially available ELISA test;

[0384] 2. Use a hemagglutination inhibition test to determine the antibody titer against rhinitis A;

[0385] 3. The presence or absence of pox development at the inoculation site (pterygoid and lateral thigh).

[0386] Materials and methods

[0387] Table 2. Bivalent vaccines against avian encephalomyelitis and fowlpox

[0388]

[0389] Table 3. Trivalent vaccines against avian infectious coryza, avian encephalomyelitis, and fowlpox

[0390]

[0391] Table 4. Attack Materials

[0392] Generic name of active ingredient FPV Manufacturer GD (Gezondheidsdienst voor Dieren, Deventer, Netherlands) efficacy <![CDATA[10 7.1 EID 50 / vial (1mL)<!-- 24 --> ]]> Storage conditions <-60℃

[0393] Table 5. Culture Media

[0394] Generic name of active ingredient sterile water Manufacturer GD Storage conditions 2-8℃

[0395] Table 6. Details of Vaccination and Attack Procedures

[0396]

[0397] sc = subcutaneous

[0398] Table 7. Vaccine administration

[0399]

[0400] sc = subcutaneous

[0401] Table 8. Application of offensive materials

[0402]

[0403] observe

[0404] Following the attack, chickens were observed twice throughout the day for clinical signs. All signs and mortality rates were recorded and stored in the study file.

[0405] sampling

[0406] Blood samples for serological testing were obtained by puncturing the pterygoid vein on days 0, 21, and 42. Blood was collected in serum gel tubes and allowed to coagulate at room temperature for at least 2 hours. Serum was then collected by centrifugation (4700g; 10–15 min; room temperature) and aliquoted and stored at <-16°C for further processing.

[0407] Blood coagulation inhibition test

[0408] The presence of antibodies against coryza serotype A was evaluated in the serum of chickens obtained from D42 using the Kume strain A1 antigen and a hemagglutination inhibition assay. Serial dilutions of serum were prepared in PBS using a round-bottom microtiter plate. Equal volumes of hemagglutination units (50 μl, 4 units) and 0.75% formalinized chicken erythrocytes (50 μl) were added to each cup. After incubation at room temperature for 45 minutes, the result was read as the maximum serum dilution for complete hemagglutination inhibition. Formalinized chicken erythrocytes were prepared by incubating chicken erythrocytes (washed twice in PBS) with PBS containing 3% formaldehyde at 4°C for 18 hours. Subsequently, the erythrocytes were washed once in PBS and resuspended in PBS to prepare a 50% suspension, which was then diluted to 0.75% immediately before the assay was completed (Jacobs et al., 1992).

[0409] result

[0410] A. Rhinitis A (Avian bacillus paragallinarum)

[0411] None of the chickens in groups TG02, TG03, and TG04, which were not vaccinated with inactivated rhinitis vaccine, showed detectable levels of antibodies against rhinitis serotype A. This demonstrates the high specificity of the test.

[0412] In group TG01 (live AE+FP added to the inactivated rhinitis vaccine), antibody levels against rhinitis serotype A were detected at the expected levels (data not shown). These results indicate that there is no indication of negative interference with the rhinitis vaccine components, as expected. For any or both of the live vaccine components, any negative interference is predominantly expected.

[0413] B. Fungal pox.

[0414] The results of the vaccination were shown Figure 1 (Percentage of chickens with fowlpox lesions after attack).

[0415] The study was valid because the negative control group (TG04) showed no pox lesions, while all chickens in the positive control group (TG03) developed pox lesions. The same conclusion was reached for the avian encephalitis portion of the study, as the unvaccinated groups (TG03 and TG04) remained ELISA negative, while the two AE-vaccinated groups (TG01 and TG02) became AE antibody-positive.

[0416] Figure 1 The data shows the protection levels against pox in two groups vaccinated with live attenuated AE-FP vaccine. All unvaccinated chickens attacked showed pox lesions within a week or longer; most vaccinated chickens (approximately 75%) did not show any pox lesions after attack; approximately 25% of the chickens showed minor lesions (classified as small (size 2-3 mm)) for about 1 to 3 days. The protection level was 96% in TG01 (inactivated coryza + AE-FP vaccine) and 92% in TG02 (live AE-FP vaccine), so the protection level in TG01 was slightly higher. At 21 days, no lesions were presented in either vaccination group, while 89% of the TG03 chickens still showed pox lesions.

[0417] These results, surprisingly, showed no indication of a negative interference between the inactivated rhinitis vaccine and the efficacy of the live attenuated FP vaccine. However, it was even surprising that a slight positive synergistic effect on the efficacy of the vaccinia vaccine was observed in the trivalent vaccine combination.

[0418] C. Avian encephalomyelitis (AE) virus

[0419] Table 9 and Figure 2 The mean AEELISA titers for all groups are shown on the day of vaccination and at 21 and 42 days post-vaccination.

[0420] Table 9. Mean AE ELISA titer / treatment group

[0421]

[0422] The development of the average titer of the AE component is shown in Figure 2 In the group vaccinated with the live attenuated AE-FP vaccine (containing an inactivated rhinitis vaccine) at 21 and 42 days post-vaccination, the percentage of chickens positive for AE antibodies via ELISA (TG01) was 81% and 95%, respectively. The percentage of positive chickens vaccinated with the live attenuated AE-Pox vaccine (TG02) was 57% and 90% on the same days. Therefore, the percentage of antibody-positive chickens was slightly higher in TG01. These results indicate that there is no negative interference with the efficacy of the live attenuated AE-FP vaccine by the inactivated rhinitis vaccine. However, it is surprising that even a slight positive synergistic effect exists regarding AE vaccine efficacy (an increased percentage of AE antibody-positive chickens) in the trivalent vaccine combination.

[0423] in conclusion

[0424] In conclusion, it can be observed that when inactivated coryza vaccine (Avian bacillus paragallinarum) is added, there is no detectable interference with the efficacy of live attenuated AE (avian encephalomyelitis) and FP (fowlpox) vaccines. The data presented in this paper show that animals vaccinated with the trivalent vaccine were protected against fowlpox challenge and developed high antibody titers against AE. Surprisingly, even a slight positive synergistic effect exists between the efficacy of the fowlpox and AE vaccines in the trivalent vaccine combination. When combining vaccines to ultimately obtain generally more complex vaccine compositions, negative effects on vaccine efficacy are often expected. In particular, this is anticipated when combining a modified live vaccine with a bacterial vaccine containing an adjuvant, as adjuvants can interfere with the activity of modified live vaccine components.

Claims

1. An immunogenic composition comprising: a) a fully inactivated vaccine of *Avibacterium paragallinarum* and attenuated live avian encephalomyelitis virus and attenuated live fowlpox virus; and b) a pharmaceutically acceptable vector, wherein the avian encephalomyelitis virus is the Calnek1733 strain, the fowlpox virus is the Beaudette strain, and the fully inactivated vaccine of *Avibacterium paragallinarum* is a fully inactivated vaccine of *Avibacterium paragallinarum* containing serogroups A, B, and C.

2. The immunogenic composition of claim 1, wherein the immunogenic composition is a vaccine.

3. The immunogenic composition of any one of claims 1 to 2, wherein the pharmaceutically acceptable carrier is an adjuvant.

4. The immunogenic composition of any one of claims 1 to 2, wherein the pharmaceutically acceptable carrier is an adjuvant selected from mineral oil, water-in-oil emulsion, oil-in-water emulsion, oil-in-water emulsion, and combinations thereof.

5. A kit comprising the immunogenic composition of any one of claims 1 to 4.

6. The kit according to claim 5, wherein the avian paraavirhea vaccine, the attenuated live avian encephalomyelitis virus, and the attenuated live fowlpox virus are in one or two containers.

7. Use of the immunogenic composition of any one of claims 1 to 4 in the preparation of a kit for the prevention of clinical signs caused by infection with Avianella paragallinarum, avian encephalomyelitis virus and fowlpox virus in chickens in need.

8. Use of the immunogenic composition of any one of claims 1 to 4 in the preparation of a kit for reducing mortality, tremors or decreased egg production in chickens in need compared with unimmunized control chickens.

9. Use according to any one of claims 7 to 8, wherein the immunogenic composition is administered once.

10. Use according to any one of claims 7 to 8, wherein the immunogenic composition is in the form of subcutaneous, intramuscular, or intradermal administration.

11. Use according to any one of claims 7 to 8, wherein the vaccine of *Avianella paragallinarum*, the attenuated live avian encephalomyelitis virus and the attenuated live fowlpox virus in the kit are in one or two containers.

Images