Method for reducing virus shedding, tissue colonization, and lymphoid depletion caused by or associated with PCV2 infection

Abstract

The present invention in particular relates to a method for reducing virus shedding, colonization of lymphoid tissue, and / or lymphoid depletion caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, the method comprising administering orally to the pig an immunogenic composition comprising non-replicating PCV2 antigen. In one example, an immunogenic composition is administered via the oral route to a pig, wherein the immunogenic composition comprises a recombinantly produced and purified PCV2 ORF2 protein. This allows to simply reduce virus shedding, colonization of lymphoid tissue, and / or lymphoid depletion caused by associated with a PCV2 infection in the pigs, while simultaneously reducing the propensity for adverse reactions, eliminating the risk of injection site reactions, avoiding pain, and putting less stress on the animals.

Description

[0001] Method for reducing virus shedding, tissue colonization, and lymphoid depletion caused by or associated with PCV2 infection

[0002] SEQUENCE LISTING

[0003] This application contains a sequence listing in accordance with 37 C. F. R. 1.821 - 1.825. The Sequence Listing is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is “23-0072-US-1_ Sequence_Listing.xml”. The XML file is 2.82KB; it was created on 05 November 2024; and it is being submitted electronically via EFS-Web, concurrent with the filing of the specification.

[0004] INCORPORATION BY REFERENCE

[0005] All references cited herein, are incorporated by reference herein, in their entirety.

[0006] BACKGROUND OF THE INVENTION

[0007] TECHNICAL FIELD

[0008] The present invention relates to a method for reducing virus shedding, colonization of lymphoid tissue, and / or lymphoid depletion caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, the method comprising administering orally to the pig an immunogenic composition comprising non-replicating PCV2 antigen. In one example, an immunogenic composition is administered via the oral route to a pig, wherein the immunogenic composition comprises a recombinantly produced and purified PCV2 ORF2 protein. This allows to simply reduce virus shedding, colonization of lymphoid tissue, and / or lymphoid depletion caused by associated with a PCV2 infection in the pigs, while simultaneously reducing the propensity for adverse reactions, eliminating the risk of injection site reactions, avoiding pain, and putting less stress on the animals.

[0009] BACKGROUND INFORMATION

[0010] Viral infection remains an important health problem in animals with adverse economic consequences. For example, there are a number of viral pathogens that cause disease in economically important livestock animals such as pigs. Viruses infecting pigs include, for example, porcine circovirus type 2.

[0011] Porcine circovirus type 2 (PCV2) is a small (17-22 nm in diameter), icosahedral, non-enveloped DNA virus, which contains a single-stranded circular genome. PCV2 shares approximately 80% sequence identity with porcine circovirus type 1 (PCV1 ). However, in contrast with PCV1, which is generally non-virulent, swine infected with PCV2 exhibit a syndrome commonly referred to as Post-weaning Multisystemic Wasting Syndrome (PMWS). PMWS is clinically characterized by wasting, paleness of the skin, unthriftiness, respiratory distress, diarrhea, icterus, and jaundice. In some affected swine, a combination of all symptoms will be apparent while other swine will only have one or two of these symptoms. During necropsy, microscopic and macroscopic lesions also appear on multiple tissues and organs, with lymphoid organs being the most common site for lesions. A strong correlation has been observed between the amount of PCV2 nucleic acid or antigen and the severity of microscopic lymphoid lesions. Mortality rates for swine infected with PCV2 can approach 80%. In addition to PMWS, PCV2 has been associated with several other infections including pseudorabies, porcine reproductive and respiratory syndrome (PRRS), Glasser’s disease, streptococcal meningitis, salmonellosis, postweaning colibacillosis, dietetic hepatosis, and suppurative bronchopneumonia.

[0012] Several vaccines are available to reduce the impact of PCV2 infections in pigs. U. S. Patent No. 6,703,023 (US 6,703,023 B1) provides a DNA based vaccine for the prophylaxis of pigs against PMWS, wherein this and the following publications referred to herein are incorporated by reference in their entirety. In W02003049703 production of a live chimeric vaccine is described, comprising the non-pathogenic PCV1 virus in which, however, the ORF2 protein is replaced by the ORF2 protein of the pathogenic PCV2. WO199918214 and WO199929717 have provided several PCV2 strains and procedures for the preparation of a killed PVC2 vaccine. Preparation of subunit vaccines have also been described in WO199918214 and WO199929717. An effective ORF2 based subunit vaccine has been reported in W02006072065. Further ORF2 based subunit vaccines are described also in WO200728823 or WO2015051099.

[0013] Vaccination of pigs with conventional vaccines against viruses, such as PCV2, which is usually done by injection, may result in injection site reactions, increases the propensity for adverse reactions and is frequently a risk factor for side effects. Additionally, such vaccination is linked with pain and stress for the animals.

[0014] Wang etal. (J Virol Methods. 150(1 -2): 1-6 (2008)) used Lactococcus lactis as vehicle to deliver the capsid protein of PCV2 in an attempt to develop oral vaccine, and significant higher levels of PCV2-specific IgG in the sera of mice were observed upon oral administration of strain cultures expressing the PCV2 antigen. Xu et al. (Vet Microbiol. 157(3-4):294-303 (2012)) report on the use of attenuated Salmonella enterica serovar Typhimurium (S. typhimurium) as a transgenic vehicle for the development of oral vaccines against PCV2, wherein the Cap-encoding gene of PCV2 was amplified by PCR and cloned into expression vector pYA3341, which recombinant plasmid pYA3341-Cap was then transformed into attenuated S.typhimurium X4550. This immunogenicity of this modified live vaccine was then tested in mouse and swine models. The Cap protein expression did not affect the ability of Salmonella to colonize host tissues, and compared with serum of the control groups, significantly reduced viral burdens were found in the serum of swine immunized with X4550 / pYA3341-Cap bacteria.

[0015] WO2023234407A1 describes the use of a powder produced from baculovirus infected silkworm pupae which was fed three times in a 5-day / 7day cycle to pigs. For producing said powder a specific silkworm pupa strain (Showa x Kinshu) was infected with recombinant baculovirus encoding PCV2 ORF protein. Infected silkworm pupae were then stored frozen at -80°C and to prepare the powder, pupae were taken out from -80°C and powdered using a mill mixer.

[0016] However, as the compositions applied to animals in such approaches still include at least some living cells and / or larger organelles, and as the case may be infectious baculovirus, with expression and / or replicating activity, they bear at least to some extent an unforeseeable risk of harm or contamination, and make difficult the large scale production of vaccines with constant quality, safety and batch to batch consistency.

[0017] Thus, methods are desired which can be reliably and simply practiced and which mount to a reduction of PCV2 related manifestations such as PCV2 virus shedding, colonization of lymphoid tissue, and lymphoid depletion, in the pigs, while simultaneously result in less propensity for adverse reactions, do not have the risk of injection site reactions, avoid pain, and put less stress on the animals.

[0018] DESCRIPTION OF THE INVENTION

[0019] The solution to the above technical problems is achieved by the description and the embodiments characterized in the claims. Thus, the invention in its different aspects is implemented according to the claims.

[0020] The invention is based on the surprising finding that orally administering an immunogenic composition which comprises a recombinant PCV2 ORF2 protein to pigs resulted in the reduction of PCV2 shedding, reduced PCV2 colonization of lymphoid tissue and a reduction of lymphoid depletion associated with PCV2 infection in the pigs, when the pigs became infected with PCV2.

[0021] In a first aspect, the invention thus relates to a method for reducing

[0022] - virus shedding,

[0023] - colonization of lymphoid tissue, and / or

[0024] - lymphoid depletion

[0025] caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, the method comprising administering orally to the pig an immunogenic composition comprising non-replicating PCV2 antigen, and wherein said method is also termed “the method of the present invention” hereinafter.

[0026] Said immunogenic composition comprising non-replicating PCV2 antigen, which is also termed “the immunogenic composition according to the present invention” hereinafter, is preferably homogeneous. The term “homogeneous”, as used herein, in particular means substantially free of insoluble components.

[0027] Said non-replicating PCV2 antigen, which is also termed “the non-replicating PCV2 antigen according to the present invention” hereinafter, preferably comprises or is a recombinant PCV2 protein.

[0028] Said recombinant PCV2 protein, which is also termed “the recombinant protein according to the present invention” hereinafter, is preferably capable of forming a virus-like particle.

[0029] In one aspect, the method of the present invention is a method for reducing virus shedding caused by a PCV2 infection in pig, the method comprising administering orally to the pig an immunogenic composition according to the present invention. As used herein, the terms “virus shedding caused by a PCV2 infection”, “PCV2 shedding", and “PCV2 virus shedding” are equivalent. In another aspect, the method of the present invention is a method for reducing colonization of lymphoid tissue caused by a PCV2 infection in pig, the method comprising administering orally to the pig an immunogenic composition according to the present invention.

[0030] In a further aspect, the method of the present invention is a method for reducing lymphoid depletion associated with a PCV2 infection in pig, the method comprising administering orally to the pig an immunogenic composition according to the present invention.

[0031] In a preferred aspect, the method of the present invention is a method for reducing

[0032] - virus shedding,

[0033] - colonization of lymphoid tissue, and

[0034] - lymphoid depletion

[0035] caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, the method comprising administering orally to the pig an immunogenic composition according to the present invention.

[0036] The immunogenic composition according to the present invention comprises a non-replicating PCV2 antigen according to the present invention, and wherein the non-replicating PCV2 antigen is preferably a recombinant PCV2 protein.

[0037] According to a preferred aspect, the immunogenic composition according to the present invention is isotropic.

[0038] In one preferred aspect, the immunogenic composition according to the present invention is free of replicating PCV2 antigen.

[0039] In one aspect, the immunogenic composition according to the present invention is substantially free of a vector capable of expressing the non-replicating antigen.

[0040] In one aspect, the immunogenic composition according to the present invention is substantially free of infectious baculovirus. The term “infectious baculovirus” in particular relates to baculovirus which is capable to reproduce (replicate) in a host cell. More particularly, an infection with a virus, such as baculovirus, includes attachment of the virus to a host cell, entry of the virus into the cell, disassembly of the virion, replication and transcription of the viral genome, expression of viral proteins and assembly and release of new infectious viral particles. In one aspect, the immunogenic composition according to the present invention is substantially free of 30S ribosomal subunits.

[0041] In one aspect, the immunogenic composition according to the present invention is substantially free of 40S ribosomal subunits.

[0042] In one aspect, the immunogenic composition according to the present invention is substantially free of functional ribosomes. The term “functional” ribosomes”, as mentioned herein, in particular relates to ribosomes which are capable to perform biological protein synthesis (i.e. mRNA translation).

[0043] In one aspect, the immunogenic composition according to the present invention is substantially free of cellular material having a size larger than 2 pm.

[0044] In one aspect, the immunogenic composition according to the present invention is substantially free of cellular material having a size larger than 0.8 pm.

[0045] The term “cellular material”, as used herein, in particular refers to materials derived from, secreted by, and otherwise currently or previously associated with a cell.

[0046] In one aspect, the immunogenic composition according to the present invention is substantially free of organelles having a size of at least 1 pm.

[0047] In one aspect, the immunogenic composition according to the present invention is substantially free of nuclei.

[0048] In one aspect, the immunogenic composition according to the present invention is substantially free of cells.

[0049] In one aspect, the immunogenic composition according to the present invention is substantially free of insect cells.

[0050] In one aspect, the immunogenic composition according to the present invention is substantially free of bacterial cells.

[0051] In one aspect, the immunogenic composition according to the present invention is substantially free of cell tissue.

[0052] In one aspect, the immunogenic composition according to the present invention is substantially free of insect cell tissue. It is in particular understood that the wording “substantially free of [...]”, as used herein, in one aspect also encompasses “free of [...]” or “completely free of [...]”, respectively.

[0053] According to another preferred aspect the method comprising administering orally to the pig an immunogenic composition comprising non-replicating PCV2 antigen, as mentioned herein, is a method consisting of administering orally to the pig an immunogenic composition comprising non-replicating PCV2 antigen.

[0054] According to another preferred aspect the method comprising administering orally to the pig an immunogenic composition comprising non-replicating PCV2 antigen, as mentioned herein, is a method consisting of administering orally to the pig an immunogenic composition comprising non-replicating PCV2 antigen.

[0055] As used herein, the term “virus-like particle” refers to a structure resembling a virus particle but which is non-pathogenic, non-replicative, and non-infectious as it lacks all or part of the viral genome.

[0056] Preferably, the recombinant PCV2 protein according to the present invention is a recombinant baculovirus expressed protein.

[0057] The term “recombinant”, as used herein, is in particular understood to be equivalent to “recombinantly expressed”. Thus, for instance “recombinant PCV2 protein” is equivalent to “recombinantly expressed PCV2 protein”.

[0058] The non-replicating PCV2 antigen according to the present invention is preferably a recombinant PCV2 protein, in particular recombinant baculovirus expressed PCV2 protein. The term "recombinant PCV2 protein" or “recombinantly expressed PCV2 protein”, respectively, as used herein, in particular refers to a PCV2 protein which is produced by recombinant DNA techniques, wherein generally DNA encoding the expressed protein is inserted into a suitable expression vector which is in turn used to transform or, in the case of a virus vector, to infect a host cell to produce the heterologous protein. Thus, the term "recombinant PCV2 protein" or “recombinantly expressed PCV2 protein”, respectively, as used herein, particularly refers to a protein molecule that is expressed from a recombinant DNA molecule. "Recombinant DNA molecule" as used herein refers to a DNA molecule that is comprised of segments of DNA joined together by means of molecular biological techniques. Suitable systems for production of recombinant proteins include but are not limited to insect cells (e.g., baculovirus), prokaryotic systems (e.g., Escherichia coll), fungi (e.g., Myceliophthora thermophile, Aspergillus oryzae, Ustilago maydis), yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris), mammalian cells (e.g., Chinese hamster ovary, HEK293), plants (e.g., safflower), algae, avian cells, amphibian cells, fish cells, and cell-free systems (e.g., rabbit reticulocyte lysate).

[0059] According to a particularly preferred aspect, the immunogenic composition according to the present invention is free of an adjuvant.

[0060] According to a particular preferred aspect, the immunogenic composition according to the present invention consists of

[0061] a non-replicating PCV2 antigen; and

[0062] one or more veterinary-acceptable carriers.

[0063] According to a further preferred aspect, the immunogenic composition according to the present invention consists of

[0064] a non-replicating PCV2 antigen; and

[0065] one or more veterinary-acceptable carriers; and

[0066] at least one immunogenic substance different from said PCV2 antigen. According to another aspect, the immunogenic composition according to the present invention consists of

[0067] a non-replicating PCV2 antigen; and

[0068] one or more veterinary-acceptable carriers; and

[0069] at least one adjuvant.

[0070] According to still another aspect, the immunogenic composition according to the present invention consists of

[0071] a non-replicating PCV2 antigen; and

[0072] one or more veterinary-acceptable carriers; and

[0073] at least one adjuvant; and at least one immunogenic substance different from said PCV2 antigen.

[0074] In one aspect, the non-replicating PCV2 antigen according to the present invention is an isolated PCV2 antigen. As used herein, the term “isolated” in particular means that the antigen is in an environment different from the environment in which it can, or has been, respectively, produced.

[0075] In one aspect, the non-replicating PCV2 antigen according to the present invention is a purified PCV2 antigen. The term “purified", as used herein, in particular refers to material, e.g. PCV2 antigen, that has been isolated under conditions that reduce er eliminate the presence of unrelated materials, i.e., contaminants, including biological materials from which the material is obtained. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a ceil. As used herein, the term “substantially free” s used operationally, in the context of analytical testing of the material. Preferably, purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.

[0076] Methods for purification are well-known in the art. For example, proteins can be purified by various methods including, without limitation, filtration, preparative disc-gel electrophoresis and soelectric focusing; affinity, HPLC, reversed-phase HPLC, size exclusion, ion exchange and partition chromatography; precipitation and salting-out chromatography; extraction; and countercurrent distribution. For some purposes, it is preferable to produce the protein in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a poiyhistidine sequence, or a sequence that specif icaliy binds to an antibody, such as FLAG and GST. The polypeptide can then be purified from a crude lysate of the host ceil by chromatography on an appropriate solid-phase matrix. Alternatively, antibodies produced against the protein or against peptides derived therefrom can be used as purification reagents. Celis can be purified by various techniques, including centrifugation, matrix separation (e.g., nylon wool separation), panning and other mmunoselection techniques, depletion (e.g., complement depletion of contaminating cells), and ceil sorting (e.g., fluorescence activated cell sorting (FACS)). Other purification methods are possible and contemplated herein. A purified material may contain iess than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components, media, proteins, or other nondesirable components or impurities (as context requires), with which it was originally associated.

[0077] In one aspect, the non-replicating PCV2 antigen according to the present invention is uniformly distributed within the immunogenic composition according to the present invention.

[0078] In one aspect, the non-replicating PCV2 antigen according to the present invention is freely dissolved in the immunogenic composition according to the present invention.

[0079] In one aspect, the non-replicating PCV2 antigen according to the present invention is quantitatively dissolved in the immunogenic composition according to the present invention. In one aspect, the non-replicating PCV2 antigen according to the present invention is not expressed or reproduced in the immunogenic composition according to the present invention. In one aspect, the non-replicating PCV2 antigen according to the present invention has been expressed in a cell culture system.

[0080] In one aspect, the non-replicating PCV2 antigen according to the present invention is capable of forming a virus-like particle.

[0081] In another preferred aspect, the immunogenic composition according to the present invention is a liquid.

[0082] In another aspect, the immunogenic composition according to the present invention is clear. As used herein the term “clear” in particular means a solution or a gel composition containing no or substantially no turbidity when visually judged. More particular, “clear” means a solution or gel composition substantially free of particles larger than 2 pm (optionally 1 pm).

[0083] In another aspect, the immunogenic composition according to the present invention is transparent. As used herein the term “transparent” in particular means capable of transmitting light. More particular, “transparent” means that the composition allows passage of 50% or more, preferably of 60% or more, and most preferably of 70% or more, of light in a wavelength range of 400 nm to 800 nm on average (i.e., the average transmittance is 50% or higher, preferably 60% or higher, and most preferably 70% or higher).

[0084] Preferably, the immunogenic composition according to the present invention additionally contains a gel composition, which is also termed “the gel composition according to the present invention” hereinafter. According to one preferred aspect the immunogenic composition according to the present invention is a gel.

[0085] The immune response, as mentioned herein, is preferably a protective immune response. Preferably, the herein mentioned immune response is an immune response against a porcine circovirus type 2 (PCV2). As used herein, the terms “porcine circovirus type 2”, “PCV2” and “PCV2 virus”, respectively, are equivalent.

[0086] As used herein, the term “PCV2 antigen” means a substance derived from a PCV2 virus or a component thereof or both of them, the substance being capable of inducing an immune response.

[0087] The term "non-replicating PGV2 antigen” in particular relates to molecules derived from a PCV2 virus, such as proteins, carbohydrates, lipids or nucleic acids, or are complex combinations thereof, more or less pure. When prepared from a virus, non-repiicating antigen can refer to an intact but killed (i.e. non-replicative) PCV2 virus, or can be a part thereof such as an extract, fraction, homogenate, or sonicate. Also, a non-repiicating PCV2 antigen can be a nucleic acid based, or recombinant product, such as an expression vector or an expressed protein, or the product of an in vitro expression system. All these are well-known in the art.

[0088] Most preferably, the non-repiicating PCV2 antigen according to the present invention is a recombinant PCV2 protein. The term “PCV2 protein” encompasses any protein that is derived from a PCV2 virus. Specific examples include the PCV2 capsid protein (PCV2 ORF2 protein), and the PCV2 repiicase (PCV2 Rep protein).

[0089] Preferably, the herein mentioned non-repiicating PCV2 antigen is a PCV2 protein capable of forming a virus-like particle.

[0090] In particular, the non-replicating PCV2 antigen according to the present invention is a viruslike particle. Said virus-like particle preferably comprises, or is respectively composed of a plurality of, a PCV2 protein capable of forming a virus-like particle.

[0091] Most preferably, the non-replicating PCV2 antigen according to the present invention is a viruslike particle comprising a recombinant PCV2 protein. Said virus-like particle, which is hereinafter also termed “the virus-like particle according to the present invention”, preferably comprises a recombinant protein (being also termed, as mentioned above, “the recombinant protein according to the present invention”). Preferably, the non-replicating PCV2 antigen according to the present invention is a recombinant PCV2 protein, or a virus-like particle comprising a recombinant PCV2 protein. The immunogenic composition according to the present invention has been preferably obtained by a procedure comprising the steps of

[0092] (1) permitting infection of susceptible cells in culture with a vector comprising a nucleic acid sequence encoding the recombinant PCV2 protein according to the present invention, wherein said recombinant protein is expressed by said vector,

[0093] (2) thereafter recovering said recombinant protein and / or virus-like particle comprising said recombinant protein from the cell culture, wherein preferably cell debris is separated from the recombinant protein and / or said virus-like particles via a separation step, preferably including a micro filtration through at least one filter, preferably two filters, wherein the at least one filter preferably has a pore size larger than the recombinant protein and / or viruslike particle comprising said recombinant protein, in particular has a pore size of about 1 to about 20 pm and / or about 0.1 pm to about 4 pm.

[0094] In particular the microfiltration in step (2) includes or consists of:

[0095] - a micro filtration through one or more filters having a pore size of about 2 pm to about 15 pm, and / or

[0096] - a micro filtration through one or more filters having a pore size of about 0.8 pm to about 1.0 pm.

[0097] Such a process is, for instance, set out in WO2015051099 (e.g. in Examples 1 to 3), or in WO 2019191005 (e.g. in Example 1, under “Production of PCV2 ORF2 protein - Upstream processing").

[0098] After step (2), as described above, the solution comprising the recombinant protein and / or said virus-like particles is preferably concentrated by removing an amount of solvent from the solution.

[0099] In particular, the solution comprising the recombinant protein and / or said virus-like particles is subsequently processed by continuous diafiltration.

[0100] In said process it is in particular preferred that the solution resulting from step (2) is further processed by a method comprising the steps of: (a) (i) inactivating the vector included in the solution resulting from step (2) by adding an inactivating agent to said solution;

[0101] (ii) neutralizing the inactivating agent by adding a neutralizing agent to the mixture resulting from step (i);

[0102] (b) concentrating the recombinant protein and / or said virus-like particles in the mixture resulting from step (a)(ii) by removing a portion of solvent from the mixture, and

[0103] (c) processing the solution resulting from step (b) by continuous diafiltration such that the concentration of the neutralized inactivating agent and / or the concentration of the neutralizing agent is decreased in the process solution.

[0104] Preferably, in step (b) the removing of a portion of the solvent from said mixture consists of or comprises filtering said mixture with at least one filter, wherein said at least one filter preferably comprises a filter membrane.

[0105] In step (b) said concentrating preferably comprises

[0106] - feeding the mixture into a filter system containing at least one filter, wherein the at least one filter comprises a filter membrane having a membrane pore size allowing the neutralized inactivating agent and / or the neutralizing agent to pass through while retaining the recombinant protein and / or said quaternary structures in the bulk flow,

[0107] - discharging the permeate comprising the neutralized inactivating agent and / or the neutralizing agent.

[0108] In step (c) said continuous diafiltration preferably comprises

[0109] - feeding the solution into a filter system containing at least one filter, wherein the at least one filter comprises a filter membrane having a membrane pore size allowing the neutralized inactivating agent and / or the neutralizing agent to pass through while retaining the recombinant protein and / or said quaternary structures in the bulk flow,

[0110] - discharging the permeate comprising the neutralized inactivating agent and / or the neutralizing agent,

[0111] - adding a liquid to the bulk flow at a rate equal to the permeate flow, wherein the liquid is different from said solvent. Said filter membrane preferably has an average pore size that is smaller than the recombinant protein and / or said virus-like particles, and / or wherein the filter membrane has a molecular weight cut off of between about 200 kDa and about 500 kDa.

[0112] “Inactivating agent”, for purposes of the present invention, refers to any agent that can be used in any conventional inactivation method. Inactivation can be performed by chemical and / or physical treatments which are known to the person skilled in the art. Preferred inactivating agents include cyclized binary ethylenimine (BEI) including a solution of 2-bromoethyleneamine hydrobromide (BEA), which has been cyclized to binary ethylenimine (BEI). Preferred further chemical inactivation agents comprise but are not limited to Triton X-100, Sodium deoxycholate, Cetyltrimethylammonium bromide, p-Propiolactone, Thimerosal, Phenol and Formaldehyde (Formalin). The term “inactivating agent”, as used herein, thus in particular relates to a chemical agent capable of modifying a vector by chemical reaction such that the vector is rendered unable to replicate.

[0113] Preferably, the inactivating agent is an aziridine compound, in particular binary ethylenimine (BEI) and / or the inactivating agent is added in a molar excess in relation to the vector, and in particular in a molar excess in relation to the nitrogenous bases of the vector DNA base pairs reacting with BEI.

[0114] “Neutralizing agent”, for purposes of the present invention, refers to any agent capable of neutralizing the inactivating agents as herein described such that the inactivating agent is no longer capable of inactivating a vector. If an aziridine compound is used for the inactivation, then preferably a nucleophile which opens the three-membered ring is used for the neutralization. The agent that neutralizes the inactivating agent is preferably sodium thiosulfate, sodium bisulfite and the like.

[0115] It is in particular preferred that the neutralizing agent is sodium thiosulfate and / or that the neutralizing agent is added in a molar excess in relation to the inactivating agent.

[0116] “Neutralized inactivating agent”, as described herein, in particular relates to the product or the products resulting from the chemical reaction of the inactivating agent with the neutralizing agent, e.g. to the products of the chemical reaction of BEI with thiosulfate or an other nucleophile.

[0117] Any conventional chemical inactivation method can be used for inactivating the vector. In preferred forms, for the chemical treatment, the temperature is brought to between about 32°C - 42°C, more preferably between abou*Q / | or' - 40°C, and most preferably between about 35°C - 39°C. Preferred inactivation methods include the addition of cyclized binary ethylenimine (BEI), preferably in a concentration of about 1 to about 20 mM, preferably of about 2 to about 10 mM, still more preferably of about 2 to about 8 mM, still more preferably of about 3 to about 7 mM, most preferably of about 5 mM. For example the inactivation includes the addition of a solution of 2-bromoethyleneamine hydrobromide (BEA), preferably of about 0.4 M, which has been cyclized to 0.2 M binary ethylenimine (BEI) in 0.3 N NaOH, to the fluids to give a final concentration of about 5mM BEI. Preferably, the fluids are then stirred continuously for 2 - 96 hours. After inactivation is completed a sodium thiosulfate solution, preferably at 1.0 M is added to neutralize any residual BEI, and the inactivated / neutralized harvest fluids can be stored frozen at - 40°C or below or between about 1°C - 7°C. Preferably, the sodium thiosulfate is added in equivalent amount as compared to the BEI added prior to for inactivation. For example, in the event BEI is added to a final concentration of 5 mM, a 1.0 M sodium thiosulfate solution is added to give a final minimum concentration of 5 mM to neutralize any residual BEI.

[0118] Thus, in step (iii) the neutralizing agent is preferably added in an equivalent amount as compared to the amount of inactivating agent added in step (ii). In the context of ranges, as described herein, it is in particular understood that “y - z” is equivalent to “y-z”, and both are equivalent to “y to z”, respectively, such that, for instance, “0.1 - 1.0” is equivalent to “0.1-1.0”. Such processing following step (2), as described above, is for instance set out in W02019191005 (e.g. Example 1, under “Production of PCV2 ORF2 protein - Downstream processing”).

[0119] Preferably, the recombinant PCV2 protein according to the present invention is a recombinant PCV2 capsid protein.

[0120] In particular, the non-replicating PCV2 antigen according to the present invention is a recombinant PCV2 ORF2 protein, wherein said PCV2 ORF2 protein preferably comprises or consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, still more preferably at least 99% or in particular 100% sequence identity with the sequence of SEQ ID NO:1.

[0121] According to another preferred aspect, the non-replicating PCV2 antigen according to the present invention is selected from the group consisting of recombinant PCV2 subtype a (PCV2a) ORF2 protein, recombinant PCV2 subtype b (PCV2b) ORF2 protein, recombinant PCV2 subtype c (PCV2c) ORF2 protein, recombinant PCV2 subtype d (PCV2d) ORF2 protein, recombinant PCV2 subtype e (PCV2e) ORF2 protein, recombinant PCV2 subtype f (PCV2f) ORF2 protein, recombinant PCV2 subtype g (PCV2g) ORF2 protein and recombinant PCV2 subtype h (PCV2h) ORF2 protein.

[0122] The terms “PCV2a”, “PCV2b”, “PCV2c”, “PCV2d”, “PCV2e”, “PCV2f”, “PCV2g”, “PCV2h”, as used herein, in particular relate to the established PCV2 genotype classification which is described in: Franzo G & Segales J. PLos One 13(12):e0208585 (2018) and Link EK et al. Virol J. 18(1)70 (2021).

[0123] In particular it is preferred that the recombinant PCV2 protein, as described herein, is a recombinant PCV2 ORF2 protein being 233 or 234 amino acid residues in length.

[0124] According to one specific aspect, the non-replicating PCV2 antigen according to the present invention is a recombinant PCV2 genotype a (PCV2a) ORF2 protein.

[0125] Preferably, the herein mentioned recombinant PCV2a ORF2 protein comprises or consists of an amino acid sequence having at least at least 95%, preferably at least 98%, more preferably at least 99% or in particular 100% sequence identity with the sequence of SEQ ID NO:1. In particular it is preferred that the recombinant PCV2a protein, as described herein, is a recombinant PCV2a ORF2 protein being 233 amino acid residues in length.

[0126] According to another specific aspect, the non-replicating PCV2 antigen according to the present invention is a recombinant PCV2 genotype d (PCV2d) ORF2 protein.

[0127] Preferably, the herein mentioned PCV2d ORF2 protein comprises or consists of an amino acid sequence having at least at least 95%, preferably at least 98%, more preferably at least 99% or in particular 100% sequence identity with the sequence of SEQ ID NO:2.

[0128] In particular it is preferred that the recombinant PCV2d protein, as described herein, is a recombinant PCV2d ORF2 protein being 233 amino acid residues in length.

[0129] Regarding the term “at least 90%”, as mentioned in the context of the present invention, it is understood that said term preferably relates to “at least 91 %”, more preferably to “at least 92%”, still more preferably to “at least 93%” or in particular to “at least 94%”.

[0130] Regarding the term “at least 95%” as mentioned in the context of the present invention, it is understood that said term preferably relates to “at least 96%”, more preferably to “at least 97%”, still more preferably to “at least 98%” or in particular to “at least 99%”. Regarding the term “at least 99%” as mentioned in the context of the present invention, it is understood that said term preferably relates to “at least 99.2%”, more preferably to “at least 99.4%”, still more preferably to “at least 99.6%” or in particular to “at least 99.8%”.

[0131] More particular, the term “at least 99% sequence identity” refers to 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity.

[0132] The term “having 100% sequence identity”, as used herein, is understood to be equivalent to the term “being identical”.

[0133] As used herein, it is in particular understood that the term “sequence identity with the sequence of SEQ ID NO: X” is equivalent to the term “sequence identity with the sequence of SEQ ID NO: X over the length of SEQ ID NO: X” or to the term “sequence identity with the sequence of SEQ ID NO: X over the whole length of SEQ ID NO: X”, respectively. In this context, “X” is any integer selected from 1 and 2 so that “SEQ ID NO: X” represents any of the SEQ ID NOs mentioned herein.

[0134] The term "immunogenic composition" refers to a composition that comprises at least one antigen, which elicits an immune response in the host to which the immunogenic composition is administered. Such immune response can be a cellular and / or antibody-mediated immune response to the immunogenic composition according to the present invention. The host is also described as "pig". Preferably, any of the hosts or subjects described or mentioned herein is a porcine animal.

[0135] The term "pig", as used herein, is in particular understood to be equivalent to “swine”, and more particular relates to a domestic pig (Sus domesticus) and most particularly relates to a piglet or a sow.

[0136] Usually, an "immune response" includes but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or gamma-delta T cells, directed specifically to an antigen or antigens included in the immunogenic composition according to the present invention. Preferably, the host will display either a protective immune response or a therapeutic response. A "protective immune response" will be demonstrated by either a reduction or lack of one or more clinical signs normally displayed by an infected host, a quicker recovery time and / or a lowered duration of infectivity or lowered pathogen titer in the tissues or body fluids or excretions of the infected host. For instance, in order to asses a protective immune response against PCV2, PCV2 specific T cell immunity can be evaluated, following the protocol of Koinig HC etal. Vet Res. 46:20 (2015). As it is known, and described therein, multi-functional T cells, which simultaneously produce IFN-y, interleukin-2 (IL-2) and TNF-a, are strongly correlated with protection, and the appearance of IFN-y / TNF-a co-producing T cells after PCV2 vaccination correlated with prevention of viremia after PCV2 vaccination. Furthermore, astrong correlation is observed between the amount of PCV2 nucleic acid and the severity of PCV2 systemic disease (PCV2-SD) histopathological lesions (Segales J. Virus Res. 164(1 -2):10-19 (2012).

[0137] Standard procedures for determining virus shedding, colonization of lymphoid tissue, and lymphoid depletion, caused by or associated with a PCV2 infection are described, for instance, in Opriessnig et a / . Vaccine. 35(2):248-254 (2017).

[0138] In case where the host displays a protective immune response such that resistance to new infection will be enhanced and / or the clinical severity of the disease reduced, the immunogenic composition is described as a "vaccine".

[0139] An "antigen" as described herein refers to, but is not limited to, components which elicit an immune response in a host to an immunogenic composition or vaccine of interest comprising such antigen or an immunologically active component thereof. In particular, the term “antigen” as used herein refers to a protein or protein domain, which, if administered to a host, can elicit an immune response in the host.

[0140] The "inducing of an immune response", “induce an immune response”, or “elicit an immune response”, respectively, generally involves the administration of an effective amount of the immunogenic composition according to the present invention to a pig or herd of pigs in need of or that could benefit from the treatment / prophylaxis caused thereby.

[0141] The term "treatment and / or prophylaxis" refers to the lessening of the incidence of the particular pathogen infection in a herd or the reduction in the severity of one or more clinical signs caused by or associated with the particular pathogen infection. Thus, the term "treatment and / or prophylaxis" also refers to the reduction of the number of animals in a herd that become infected with the particular pathogen (= lessening of the incidence of the particular pathogen infection) or to the reduction of the severity of one or more clinical signs normally associated with or caused by an infection with the pathogen in a group of animals which animals have received an effective amount of the immunogenic composition as provided herein in comparison to a group of animals which animals have not received such immunogenic composition.

[0142] The term "treatment" refers to the administration of the effective amount of the immunogenic composition once the pig or at least some animals of the herd is / are already infected with such pathogen and wherein such animals already show some clinical signs caused by or associated with such pathogen infection. The term "prophylaxis" refers to the administration to a pig prior to any infection of such pig with a pathogen or at least where such animal or all of the animals in a group of animals do not show one or more clinical signs caused by or associated with the infection by such pathogen.

[0143] The term "an effective amount" as used herein means, but is not limited to an amount of PCV2 antigen, in particular of the proteins or virus-like particles of the present disclosure, that induces or is able to induce an immune response in a pig. Such effective amount is able to lessen the incidence of the particular pathogen infection in a herd or to reduce the severity of one or more clinical signs and other manifestations of the particular pathogen infection. Preferably, one or more clinical signs or other manifestations are lessened in incidence or severity by at least 10%, more preferably by at least 20%, still more preferably by at least 30%, even more preferably by at least 40%, still more preferably by at least 50%, even more preferably by at least 60%, still more preferably by at least 70%, even more preferably by at least 80%, still more preferably by at least 90%, and most preferably by at least 95% in comparison to pigs that are not treated but subsequently infected by the particular pathogen. Thus, the method of the present invention in particular comprises orally administering an immunogenic composition comprising an effective amount of a non-replicating PCV2 antigen to a pig.

[0144] The term "clinical signs" as used herein refers to signs of infection of a pig from the particular pathogen. The clinical signs of infection depend on the pathogen selected. Examples for such clinical signs, caused by a PCV2 infection, include but are not limited to wasting or weight loss, pallor of the skin, respiratory distress, diarrhea, and occasionally, icterus. Other manifestations of a PCV2 infection are virus schedding, colonization of lymphoid tissue and lymphoid depletion.

[0145] Reducing the incidence of or reducing the severity of one or more clinical signs and reduction of other hallmarks caused by or being associated with the PCV2 infection in a pig can be reached by the oral administration of one or more doses of the immunogenic composition according to the present invention to a pig. In one aspect, the immunogenic composition according to the present invention comprises at least 10 pg of the non-replicating PCV2 antigen according to the present invention per dose of said immunogenic composition.

[0146] In one aspect, the immunogenic composition comprises at least 15 pg of the non-replicating PCV2 antigen per dose of the immunogenic composition.

[0147] In one aspect, the immunogenic composition comprises at least 30 pg of the non-replicating PCV2 antigen per dose of the immunogenic composition.

[0148] In one aspect, the immunogenic composition comprises at least 50 pg of the non-replicating PCV2 antigen per dose of the immunogenic composition.

[0149] In one aspect, the immunogenic composition comprises at least 100 pg of the non-replicating PCV2 antigen per dose of the immunogenic composition.

[0150] In one aspect, the immunogenic composition comprises at least 150 pg of the non-replicating PCV2 antigen per dose of the immunogenic composition.

[0151] In one aspect, the immunogenic composition comprises at least 200 pg of the non-replicating PCV2 antigen per dose of the immunogenic composition.

[0152] In one aspect, the immunogenic composition comprises at least 250 pg of the non-replicating PCV2 antigen per dose of the immunogenic composition.

[0153] In another aspect, the immunogenic composition according to the present invention comprises 10 to 800 pg of the non-replicating PCV2 antigen per dose.

[0154] In another aspect, the immunogenic composition according to the present invention comprises 15 to 800 pg of the non-replicating PCV2 antigen per dose.

[0155] In another aspect, the immunogenic composition according to the present invention comprises 30 to 800 pg of the non-replicating PCV2 antigen per dose.

[0156] In another aspect, the immunogenic composition according to the present invention comprises 50 to 800 pg of the non-replicating PCV2 antigen per dose.

[0157] In another aspect, the immunogenic composition according to the present invention comprises 100 to 800 pg of the non-replicating PCV2 antigen per dose. In another aspect, the immunogenic composition according to the present invention comprises 150 to 800 pg of the non-replicating PCV2 antigen per dose.

[0158] In another aspect, the immunogenic composition according to the present invention comprises 200 to 800 pg of the non-replicating PCV2 antigen per dose.

[0159] In another aspect, the immunogenic composition according to the present invention comprises 250 to 800 pg of the non-replicating PCV2 antigen per dose.

[0160] In another aspect, the immunogenic composition according to the present invention comprises 300 to 800 pg of the non-replicating PCV2 antigen per dose.

[0161] In a further aspect, the immunogenic composition according to the present invention comprises up to 800 pg of the non-replicating PCV2 antigen per dose.

[0162] In a further aspect, the immunogenic composition according to the present invention comprises up to 700 pg of the non-replicating PCV2 antigen per dose.

[0163] In a further aspect, the immunogenic composition according to the present invention comprises up to 600 pg of the non-replicating PCV2 antigen per dose.

[0164] In a further aspect, the immunogenic composition according to the present invention comprises up to 500 pg of the non-replicating PCV2 antigen per dose.

[0165] In a further aspect, the immunogenic composition according to the present invention comprises up to 400 pg of the non-replicating PCV2 antigen per dose.

[0166] In a further aspect, the immunogenic composition according to the present invention comprises up to 300 pg of the non-replicating PCV2 antigen per dose.

[0167] It is understood that the wording “the immunogenic composition comprises [...] of the nonreplicating PCV2 antigen per dose of the immunogenic composition” or “the immunogenic composition comprises [...] of the non-replicating PCV2 antigen per dose”, respectively, is in particular equivalent to the wording “per dose of the immunogenic composition the antigenic composition comprises [...] of the non-replicating PCV2 antigen” or to the wording “the immunogenic composition is formulated to allow administration of [...] of the non-replicating PCV2 antigen per dose”, respectively. The term “per dose” or “per dose of the immunogenic composition”, respectively, as used herein, in particular means “per dose of the immunogenic composition which is to be administered to a pig”, in order to achieve a desired effect, namely to induce a protective immune response, in the pig.

[0168] Preferably, in particular when administered by oral drench, one dose of the immunogenic composition has a volume of about 1 mL.

[0169] The gel composition according to the present invention is in particular a gel composition suitable for oral administration.

[0170] The term "gei composition", as used herein, refers to a suitable liquid, semi-solid or solid material, which includes an amount of one or more gelling agents effective for gelling the composition.

[0171] According to another aspect a further preferred aspect, the gel composition according to the present invention comprises water. In particular, said gel composition is a hydrogel composition.

[0172] Gel compositions according to the present invention, which can be used in the preparation of the immunogenic composition according to the present invention, are readily available. In one example, the commercially available Underline® gel concentrate (Animal Science Products, Nacogdoches, TX (USA)) is used as the gel composition included in the immunogenic composition according to the present invention, but other gel compositions are suitable as well. According to a further preferred aspect, the immunogenic composition according to the present invention is liquid.

[0173] In one aspect, the immunogenic composition according to the present invention is viscous. In another aspect, the immunogenic composition according to the present invention has a viscosity of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 cPs (mPa s) or higher.

[0174] In one aspect, the immunogenic composition according to the present invention has a viscosity of at least 150 mPa s (at least 150 cP).

[0175] In another aspect, the immunogenic composition according to the present invention has a viscosity of at least 100 mPa s (at least 100 cP). In one aspect, the immunogenic composition according to the present invention has a viscosity of at least 50 mPa s or at least 50 cP. In another aspect, the immunogenic composition according to the present invention has a viscosity between 50 cP (50 mPa s) and 150 cP (150 mPa s). In yet another aspect, the immunogenic composition according to the present invention has a viscosity between 50 cP (50 mPa s) and 350 cP (350 mPa s).

[0176] In one aspect, the immunogenic composition according to the present invention has a viscosity of at least 25 mPa s or at least 25 cP. In another aspect, the immunogenic composition according to the present invention has a viscosity between 25 cP (25 mPa s) and 150 cP (150 mPa s). In yet another aspect, the immunogenic composition according to the present invention has a viscosity between 25 cP (25 mPa s) and 350 cP (350 mPa s).

[0177] The measurement unit of viscosity is Pa s (pascal second) or mPa s (millipascal second). The conventional measurement unit is cP (centipoise). A centipoise is one millipascal-second (1 cP = 10-3Pa-s = 1 mPa-s).

[0178] According to another aspect, the immunogenic composition according to the present invention is solid. The term "solid" in this context in particular relates to a gel having some resilience or dimensional stability, in contrast to "semi-solid" gels which are smearable and do not have such dimensional stability.

[0179] The gel composition according to the present invention preferably comprises one or more flavoring agents. It is understood that, as used herein, the wording “comprises or consists of one or more flavoring agents” is in particular encompassed by the wording “comprises a flavoring agent”.

[0180] The term "flavoring agent" as used herein refers to one or more compounds or mixtures that improve the palatability and / or taste in animals, preferably in pigs. In particular, the flavoring agent is, or the one or more flavoring agents are, respectively, capable of attracting pigs. Flavoring agents are well known to the person skilled in the art. Flavoring agents include but are not limited to nutritive and non-nutritive sweeteners, flavor additives, by-products and alternative ingredients. By way of example suitable flavorants include but are not limited to sucrose, glucose, sodium saccharin, sodium cyclamate, xylitol, perillartien, sucralose, D-tryptophan, aspartame, dihydrochalcones and the like, artificial fruit flavoring (e.g., strawberry flavoring), plasma protein (e.g., spray-dried plasma protein), cheese and cheese-like flavorings, dried milk, chocolate and chocolate by-products. Preferably, the one or more flavoring agents comprise or consists of a flavoring agent giving the immunogenic composition according to the present invention a particular taste and / or a flavoring agent giving the immunogenic composition according to the present invention a particular smell. In particular, the one or more flavoring agents comprise or consist of a flavoring agent capable to be tasted by a pig, such as a sweetener, and / or a flavoring agent capable to be smelled by a pig.

[0181] In particular, the one or more flavoring agents comprise or consists of

[0182] a sweetener, and

[0183] a flavor additive capable to be to be smelled by pigs.

[0184] The sweetener, as mentioned herein, is preferably selected from the group of sucrose, glucose, sodium saccharin, sodium cyclamate, xylitol, perillartine, sucralose, D-tryptophan, aspartame, dihydrochalcones.

[0185] The flavor additive, as mentioned herein, is preferably selected from the group consisting of artificial fruit flavoring (e.g., strawberry flavoring), and cheese and cheese-like flavorings. Most preferably, the gel composition according to the present invention comprises a sweetener and an artificial fruit flavoring

[0186] The at least one flavoring agent is preferably incorporated in the immunogenic composition according to the present invention at a concentration of 0.1% to 0.5% by weight.

[0187] In one aspect, the gel composition according to the present invention further comprises one or more colorants.

[0188] The term "colorant" also may be used in the compositions of the described invention to provide visual cues to the piglets and / or visual verification to animal caretakers that the composition is present, uniformly applied and appropriately adherent. Preferably, the gel composition according to the present invention further comprises one or more colorants capable of attracting pigs. Colorants are well known to the person skilled in the art and include pigments or dyes or a combination thereof. Suitable colorants include, but are not limited to FD& C colorants such as FD& C Blue No. 1, FD& C Blue No. 2, FD& C Green No. 3, Orange B, Citrus Red No. 2, FD& C Red No. 2, FD& C Red No. 3, FD& C Red No.40, FD& C Yellow No. 5 and FD& C Yellow No.6. FD& C Blue No. 1 is the dye named Brilliant blue FCF, which is also denoted by E number E133.

[0189] FD& C Blue No. 2 is the dye named indigotine which is also denoted by E number E132. FD& C Green No. 3 is the dye Fast Green FCF, which is also denoted by E number El 43. FD& C Red No. 2 is the dye Amaranth which is also denoted by E number E1 3,

[0190] FD& C Red No. 3 is the dye erythrosine, which is also denoted by E number E127, FD& C Red No.40 is the dye Allura Red AC, which is also denoted by E number E1 9.

[0191] FD& C Yellow No. 5 is the dye Tartrazine, which is also denoted by E number E102, FD& C Yellow No.6 is the dye Sunset yellow FCF, which is also denoted by E number 110. The gel composition according to the present invention preferably comprises at least one colorant selected from the group consisting of Brilliant blue FCF, indigotine, Fast Green FCF, Amaranth, erythrosine, Allura Red AC, Tartrazine, Sunset yellow FCF.

[0192] Preferably, the gel composition according to the present invention comprises the colorants Brilliant blue FCF and Tartrazine. Thus, the at least one colorant, as mentioned herein, is preferably a mixture of Brilliant blue FCF and Tartrazine. Thus, in a preferred aspect, the gel composition according to the present invention has a green color.

[0193] According to another preferred aspect, the gel composition according to the present invention comprises water and / or an adhesion enhancing agent and / or a pH adjusting agent and / or a stabilizer.

[0194] In one aspect, the at least one adhesion enhancing agent is a hydrophilic polymer or copolymer that is linear or branched, crosslinked, is not biodegradable, or is selected from the group consisting of maltodextrins, hemicellulose extract, hemicellulose, xanthan, guar, pectins, gums, guar derivatives, chitosan, dextran, carrageenans, starch, polyethylene glycol, albumin, cellulose ethers, hyaluronic acid, carboxymethyihydroxyethyiceilulose, hydroxypropylmethyi cellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethyi cellulose (HEC), carboxy methyl cellulose (CMC), gelatins, vinyl acetates, polyvinyl pyrrolidone, polyvinyl pyrrolidonevinyl acetate copolymers, polyvinyl alcohols, polyphosphoesters, N-(2-hydroxypropyl) methacryl amide (HPMA) copolymers, polyacryiic acids, polyacrylamides, polyoxazoiines, divinyi ether-maleic anhydride, polyphosphazenes, including derivatives and substitutions and salts of any of the foregoing, and combinations thereof.

[0195] In one aspect, the at least one adhesion enhancing agent is one or more adhesion enhancing agents based on starch and / or hemicellulose.

[0196] In another aspect, the at least one adhesion enhancing agent(s) is or are maltodextrins and / or hemicellulose extract.

[0197] In one aspect, the gel composition according to the present invention comprises maltodextrin. In one aspect, the gel composition according to the present invention comprises hemicellulose extract.

[0198] Preferably, the herein mentioned hemicellulose extract comprises xanthan.

[0199] In some aspects, in the immunogenic composition according to the present invention the at least one adhesion enhancing agent is in a final concentration (w / v) of about 0.1%, 0.2%, 03%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, or 49%, or any ranges therebetween including, for example, from about 0.5% to 15% w / v, from about 0.5% to 10% w / v, from about 0.5% to 5% w / v, from about 0.5% to 2% w / v.

[0200] In one aspect, in the immunogenic composition according to the present invention the adhesion enhancing agent is in a final concentration (w / v) of about 0.5% to 15% w / v.

[0201] According to a further preferred aspect, the gel composition according to the present invention comprises one or more pharmaceutically acceptable carriers, in particular selected from the group consisting of stabilizers, pH adjusting compositions, alcohols, glycols, glycerols, and glycerines, lanolin and derivatives thereof, fatty acids and derivatives thereof, fatty alcohols and derivatives thereof, and fatty esters and derivatives thereof, or combinations thereof. Preferably, the one or more pharmaceutically acceptable carriers are selected from the group consisting of propylene glycol (PG), propylene glycol monolaurate (PGML), propylene glycol capryiate, polyethylene glycol monolaurate (PEGML), glycerol monolaurate (GML), methyl formamide (DMF), allantoin, urazole, N, N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), decylmethylsulfoxide, lecithin, polynxvlalycerides, the 1 -substituted azacycloheptan- 2-ones, particularly 1-n-dodecylcyclazacycloheptan-2-one, alcohols, and oils safe for porcine consumption such as vegetable oils.

[0202] In particular, the gel composition according to the present invention comprises a stabilizer, and wherein the stabilizer is preferably propylene glycol.

[0203] A "pH adjusting agent" is typically added to bring the pH of the composition to the desired value. Desirable pH values are between about 6 to about 8. The immunogenic compositions of the present invention therefore may be formulated to have a pH value that ranges between about 6 and about 8, or about 6.5 and about 7.5. Suitable pH adjusting agents include, but are not limited to, one or more adipic acids, glycines, citric acids, calcium hydroxides, magnesium aluminometasilicates, disodium phosphate, sodium phosphate, potassium phosphate, potassium chloride, sodium citrate, calcium lactate, sodium succinate, sodium glutamate, sodium bicarbonate, and potassium bicarbonate, and combinations thereof.

[0204] As used herein, "stabilizer" is an agent that helps stabilize the active agent in the immunogenic composition according to the present invention. The stabilizer includes but is not limited to reducing agents. Stabilizers that may be used include sodium thiosulfate, sodium metabisulfite, sodium bisulfite, sodium sulfite, sulphur dioxide, ammonium bisulfite, and ammonium thiosulfate. Sodium thiosulfate is preferred as it possess a high neutralization ability and is considered safe and not corrosive.

[0205] The term "stabilizer" also encompasses chelating agents. Chelating agents are optionally added to the gel composition according to the present invention to enhance the preservative or preservative system. Preferred chelating agents are mild agents, such as, for example, ethylenediaminetetraacetic acid (EDTA), EDTA derivatives, or any combination thereof. Suitable stabilizers or preservatives for use in the immunogenic compositions of the present invention include, without limitation, one or more alkanols, disodium EDTA (ethylenediamine tetraacetate), EDTA salts, EDTA fatty acid conjugates, isothiazolinone, parabens such as methylparaben and propylparaben, propylene glycols, sorbates, urea derivatives such as diazolindinyl urea, or any combinations thereof.

[0206] in some aspects of the invention, in the immunogenic composition according to the present invention the at least one pharmaceutically acceptable carrier is in a final concentration (v / v) of about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,

[0207]

[0208] any ranges therebetween including, for example, about 5% to about 10%, about 10% to about 15%, about 12% to about 13%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, and about 45% to about 50%.

[0209] In one aspect of the present invention the gel composition according to the present invention comprises water, an adhesion enhancing agent and a stabilizer.

[0210] In one aspect, the gel composition according to the present invention comprises water, an adhesion enhancing agent and a stabilizer and a pH adjusting agent.

[0211] In one aspect, the gel composition according to the present invention comprises water, maltodextrins, hemicellulose extract, and propylene glycol.

[0212] In another aspect, the gel composition according to the present invention comprises water, maltodextrins, cellulose, a gum and a stabilizer, preferably the stabilizer is propylene glycol. In one aspect, the gel composition according to the present invention comprises water, maltodextrins, hemicellulose extract, propylene glycol and artificial coloring.

[0213] According to a particularly preferred aspect, the gel composition according to the present invention comprises or consists of:

[0214] - water,

[0215] - maltodextrins,

[0216] - propylene glycol,

[0217] - hemicellulose extract,

[0218] - one or more colorants, and

[0219] - one or more flavoring agents.

[0220] According to another preferred aspect, the immunogenic composition according to the present invention comprises or contains one or more veterinary-acceptable carriers.

[0221] In one aspect of the present invention the one or more veterinary-acceptable carriers is a diluent. “Diluent” can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali salts of ethylenediaminetetraacetic acid, among others. In one aspect of the present invention the one or more veterinary-acceptable carriers is a physiologic buffer.

[0222] In one aspect of the present invention the one or more veterinary-acceptable carriers is phosphate buffered saline.

[0223] Preferably, the one or more veterinary-acceptable carriers are selected from the group consisting of solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and combinations thereof.

[0224] Preferably, the immunogenic composition according to the present invention further comprises sucrose gelatin stabilizer.

[0225] Preferably, the immunogenic composition can further include one or more other immunomodulatory agents such as, e.g. interleukins, interferons, or other cytokines.

[0226] According to another aspect, the immunogenic composition according to the present invention comprises or contains an adjuvant. It is understood, in the context of the present invention, that the person skilled in the art will preferably choose an adjuvant suitable for oral administration. Thus, the immunogenic composition according to the present invention preferably comprises or contains an adjuvant suitable for oral administration.

[0227] According to a further aspect, the immunogenic composition according to the present invention comprises or contains at least one immunogenic substance different from the non-replicating PCV2 antigen according to the present invention.

[0228] In one aspect, the non-replicating PCV2 antigen according to the present invention is a recombinant PCV2a ORF2 protein and said at least one immunogenic substance different from said PCV2 antigen is a recombinant PCV2d ORF2 protein, and wherein preferably said PCV2a ORF2 protein comprises or consists of an amino acid sequence having at least at least 95%, preferably at least 98%, more preferably at least 99% or in particular 100% sequence identity with the sequence of SEQ ID NO:1, and said PCV2d 0RF2 protein comprises or consists of an amino acid sequence having at least at least 95%, preferably at least 98%, more preferably at least 99% or in particular 100% sequence identity with the sequence of SEQ ID NO:2.

[0229] According to a particular preferred aspect, the immunogenic composition according to the present invention comprises said PCV2a ORF2 protein and said PCV2d ORF2 protein in a molar ratio of about 1:1.

[0230] In one aspect, the immunogenic composition according to the present invention is administered by oral drench to the pig

[0231] The invention further provides methods for reducing

[0232] - virus shedding,

[0233] - colonization of lymphoid tissue, and / or

[0234] - lymphoid depletion

[0235] caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, wherein the methods comprise the steps of:

[0236] adding an immunogenic composition comprising non-replicating PCV2 antigen into the liquid food or drinking water of the pig, and

[0237] allowing the pig to self-administer the liquid food or drinking water to which the immunogenic composition has been added.

[0238] Most preferably, in this context of the present invention, the immunogenic composition according to the present invention is added into the drinking water of the pig.

[0239] The term “self-administering” or “self-administer”, as used herein, in particular means that the pig drinks the liquid food or drinking water, and thereby orally consumes the immunogenic composition according to the present invention which has been added to the liquid food or drinking water.

[0240] Thus, the term “self-administer”, as used herein, is in particular equivalent to “orally self-administer”. All of the above-mentioned methods for reducing virus shedding, colonization of lymphoid tissue and / or lymphoid depletion caused by associated with a PCV2 infection in a pig will also be termed “the methods according to the present invention” hereinafter.

[0241] According to one aspect the methods according to the present invention consist of orally administering only one dose of said immunogenic composition to said pig or, respectively, said immunogenic composition is added only once into the liquid food or drinking water of the pig. According to another preferred aspect, in the methods according to the present invention the pig is not pre-vaccinated with a vaccine against PCV2.

[0242] In some embodiments of the present invention, the pig is a PCV2 vaccination naive pig. In one aspect, in the methods according to the present invention the reduction of

[0243] virus shedding,

[0244] - colonization of lymphoid tissue, and / or

[0245] - lymphoid depletion,

[0246] caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig is obtained by the administration of the immunogenic composition.

[0247] In another aspect, in the methods according to the present invention the reduction of virus shedding,

[0248] - colonization of lymphoid tissue, and / or

[0249] - lymphoid depletion,

[0250] caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig is obtained without a further administration of a vaccine against PCV2 to the pig.

[0251] In a further preferred aspect of the methods according to the present invention, the pig is a piglet or a sow.

[0252] The invention further provides the use of a liquid antigen concentrate for reducing

[0253] virus shedding, colonization of lymphoid tissue, and / or

[0254] - lymphoid depletion

[0255] caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, wherein 1 mL of said concentrate comprises about 100 to 800 pg of non-replicating PCV2 antigen, in particular of any of the non-replicating PCV2 antigens described herein in the context of the present invention, and wherein preferably said concentrate is diluted in drinking water or in liquid food of said pig. Said non-replicating PCV2 antigen is preferably a non-replicating PCV2 antigen according to the present invention.

[0256] Thus, said liquid antigen concentrate is preferably an immunogenic composition according to the present invention comprising a non-replicating PCV2 antigen in the specified concentration. In particular, said liquid antigen concentrate is hence an immunogenic composition according to the present invention, wherein 1 mL of said immunogenic composition comprises about 100 to 800 pg of a non-replicating PCV2 antigen according to the present invention.

[0257] Preferably, said concentrate, which is also termed the “liquid antigen concentrate according to the present invention” hereinafter, allows to simply establish a defined volume of drinking water or liquid food containing a desired concentration of the non-replicating PCV2 antigen depending on the self-administering pig and its environment. For example, pigs generally drink 8 to 12 % of their body weight per day, depending on the environmental temperature. Thus, the volume of drinking water consumed over a certain time per pig may be calculated (e.g. on the basis of pre-measured drinking water intake during a certain time period of the previous day around the time of planned vaccination), and an appropriate volume of drinking water containing sufficient antigen can be provided in order to ensure that pigs self-administer at least the desired dose of the antigen within a given time.

[0258] In one aspect, 1 mL of the liquid antigen concentrate according to the present invention comprises about 150 to 800 pg of the non-replicating PCV2 antigen.

[0259] In one aspect, 1 mL of the liquid antigen concentrate according to the present invention comprises about 200 to 800 pg of the non-replicating PCV2 antigen.

[0260] In one aspect, 1 mL of the liquid antigen concentrate according to the present invention comprises about 250 to 800 pg of the non-replicating PCV2 antigen. In one aspect, 1 mL of the liquid antigen concentrate according to the present invention comprises about 300 to 800 pg of the non-replicating PCV2 antigen.

[0261] In one aspect, 1 mL of the liquid antigen concentrate according to the present invention comprises about 400 to 800 pg of the non-replicating PCV2 antigen.

[0262] In one aspect, 1 mL of the liquid antigen concentrate according to the present invention comprises about 500 to 800 pg of the non-replicating PCV2 antigen.

[0263] In one aspect, 1 mL of the liquid antigen concentrate according to the present invention comprises about 600 to 800 pg of the non-replicating PCV2 antigen.

[0264] In one aspect, 1 mL of the liquid antigen concentrate according to the present invention comprises about 700 to 800 pg of the non-replicating PCV2 antigen.

[0265] The term “liquid”, as used herein, has the ordinary meaning in the art and in particular relates to a respective state of matter at room temperature (25°C) and atmospheric pressure (760 mmHg). A liquid, as mentioned herein, in particular relates to a substance being liquid. The invention further provides a method for enabling the self-immunization against PCV2 by a pig, comprising the step of diluting a liquid antigen concentrate comprising non-replicating PCV2 antigen according to the present invention, in the drinking water of the pig, wherein preferably said self-immunization results in the reduction of

[0266] virus shedding,

[0267] colonization of lymphoid tissue, and / or

[0268] lymphoid depletion

[0269] caused by or associated with a PCV2 infection in said pig,

[0270] and wherein said concentrate is in particular a liquid antigen concentrate according to the present invention.

[0271] The invention also provides the use of a container containing drinking water to which a liquid antigen concentrate comprising non-replicating PCV2 antigen according to the present invention, has been added, for reducing

[0272] virus shedding, colonization of lymphoid tissue, and / or

[0273] lymphoid depletion

[0274] caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, wherein the container is preferably placed in the housing environment of the pig, and wherein said container is also termed the “container of the present invention” hereinafter. In particular the container according to the present inention is a container containing drinking water to which a liquid antigen concentrate according to the present invention has been added.

[0275] Said container can be any container used for providing drinking water to a pig. Preferably, said container is selected from the group consisting of a bowl, a vessel and a barrel and / or said container is located in the housing environment of a pig to be vaccinated.

[0276] The invention also relates to a kit for use in a method for reducing

[0277] virus shedding,

[0278] colonization of lymphoid tissue, and / or

[0279] lymphoid depletion

[0280] caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, wherein the kit comprises

[0281] a liquid antigen concentrate comprising non-replicating PCV2 antigen, in particular nonreplicating PCV2 antigen according to the present invention; and

[0282] instructions to dilute said liquid antigen concentrate in drinking water or in liquid food to be consumed by the pig. Preferably, said liquid antigen concentrate is a liquid antigen concentrate according to the present invention.

[0283] Most preferably, the herein-mentioned pig is a piglet.

[0284] EXAMPLES

[0285] The following examples are only intended to illustrate the present disclosure. They shall not limit the scope of the claims in any way. EXAMPLE 1

[0286] Results of Oral PCV2 Vaccination Study

[0287] Objective:

[0288] To investigate if Porcine Circovirus type 2 (PCV2) virus like particle (VLP) is immunogenic when given orally, thus being an oral vaccine to prevent against PCV2 disease in swine. Methods:

[0289] To investigate the immunogenicity of Porcine Circovirus type 2, virus like particle, oral vaccine for swine, an experiment was conducted with two treatment groups. SPF pigs, being around six weeks of age at study day 0, were randomly assigned to treatment group 1 ) Non-Vaccinated Control and 2) Oral VLP Vaccine group. These groups were comprised of 7 and 10 pigs, respectively.

[0290] Pigs in each treatment group were split into two pens. On study day 0, pigs in the Oral VLP Vaccine group received, via oral drench, a 1 mL, 300 pg / mL dose of Porcine Circovirus, type 2d, killed baculovirus vector expressed virus like particle, i.e. recombinant baculovirus expressed PCV2d ORF2 protein (SEQ ID NO:2 (corresponds to SEQ ID NO:1 of WO2015051099)). The PCV2d ORF2 protein had been produced as described in WO2015051099 (Examples 1 to 3), and was then further subjected to a downstream processing as set out in WO 2019191005 (Example 1, without mixing with a carbomer solution (adjuvant)).

[0291] This vaccine did not contain adjuvant. At study day 0, prior to vaccination, at study day 14 and study day 23 each pig had individual blood drawn for serum collection and antibody measurement. At 23 days post vaccination, all pigs had blood drawn for peripheral blood mononuclear cell (PBMC) extraction to evaluate PCV2 specific T cell immunity, following the protocol of Koinig HC et al. Vet Res. 46:20 (2015). The quantification of IgM in serum was performed with the INGEZIM Circovirus IgG / IgM ELISA kit. At study day 22 oral fluids were also collected to evaluate anti-PCV2 IgA, IgG and IgM antibodies. Quantification of IgG in serum, along with IgA, IgM and IgG in oral fluids was performed following the methods described in Prickett et a / . Transbound Emerg Dis. 58(2):121-7 (2011). The antigen used for the Prickett et al. (2011 ) assay was the same PCV2d antigen used to immunize pigs. All blood samples were also submitted to test for PCV2 viremia by PCR. Results:

[0292] PCV2 viremia was not detected in any pig. The oral vaccine was well tolerated, and no adverse events were observed. Antibody results revealed that the Oral VLP Vaccine led to an increase in anti-PCV2 IgM antibody levels, an effect not observed in the Non-Vaccinated Control group (Figure 1). While both groups had similar IgM antibody levels on study day 0 prior to vaccination, antibody levels in the Oral VLP Vaccine group were significantly higher than the Non-Vaccinated Control group at 14 (p= 0.01) and 23 days post vaccination (p=0.03) (Figure 1). In addition to IgM, a significant increase of anti-PCV2 IgG serum antibody level was also observed with oral vaccination. At 23 days post vaccination, the Oral VLP Vaccine group had an average S / P value of 0.665 while the Non-Vaccinated Control group had around a tenfold less antibody concentration with an average S / P value of 0.066 (p=0.034) (Figure 2). To further evaluate immune response, specific anti-PCV2 antibodies were measured in oral fluids following vaccination. This analysis revealed much higher quantities of anti-PCV2 antibodies of the classes of IgA, IgG and IgM of pigs in the Oral VLP Vaccine group (Figure 3). The unexpected observation of a substantive increase of IgA is indicative of a strong mucosal immune response induced by the orally administered PCV2 subunit vaccine. Also, the observation of IgG was surprising, as it could not be expected that an orally applied PCV2 subunit vaccine could lead to a significant serological response.

[0293] Analysis of cellular immunity revealed that the pigs in the Oral VLP vaccine group developed PCV2 antigen specific T cells while pigs in the Non-Vaccinated Control group did not. At 23 days post vaccination, the Oral VLP vaccine group had PCV2 antigen specific interferon gamma and tumor necrosis factor producing CD4+ T cells, while the Non-Vaccinated Control group did not (Figure 4).

[0294] Conclusion:

[0295] These results demonstrate that the oral administration of Porcine Circovirus, type 2, virus like particle was immunogenic in pigs, inducing both cellular and humoral immune response, thus being a method and vaccine to prevent disease caused by PCV2 in pigs.

[0296] EXAMPLE 2

[0297] Results of PCV2 VLP Gel Administration Study

[0298] Objective: To investigate if Porcine Circovirus type 2 (PCV2) virus like particle (VLP) is immunogenic when administered to pigs by a gel, thus being an oral and mucosal vaccine to prevent against PCV2 associated disease in swine.

[0299] Methods:

[0300] To investigate the immunogenicity of Porcine Circovirus type 2, virus like particle, administered by gel for swine, an experiment was conducted with two treatment groups.

[0301] SPF pigs being, on study day 0, about 6 weeks of age were randomly assigned to treatment group 1) Non-Vaccinated Control and 2) Gel VLP Vaccine group. These groups were comprised of 7 and 10 pigs, respectively.

[0302] On study day 0, pigs in the Gel VLP Vaccine group were provided an immunogenic composition according the present invention such that for each pig, 100 mL of finally prepared Gel VLP vaccine containing a total of 300 pg of recombinant baculovirus expressed PCV2d ORF2 protein (SEQ ID NO:2 (corresponds to SEQ ID NO:1 of WO2015051099)), said PCV2 antigen herein being also termed “Porcine Circovirus, type 2d, killed baculovirus vector expressed virus like particle” or “PCV2 VLP”, respectively, was made available. The PCV2d ORF2 protein had been produced as described in WO2015051099 (Examples 1 to 3), and was then further subjected to a downstream processing as set out in WO 2019191005 (Example 1, without mixing with a carbomer solution (adjuvant)).

[0303] The finally prepared Gel VLP Vaccine was manufactured by mixing commercially available Underline® gel concentrate (Animal Science Products, Nacogdoches, TX (USA)) - which has the ingredients: water, maltodextrins, propylene glycol, hemicellulose extract, artificial color, natural and artificial flavor - according to the manufacturer's specifications with PCV2 VLP in the following ratios: 1 part Underline® gel concentrate was mixed with 2 parts of cold PCV2 VLP liquid.

[0304] The Gel VLP Vaccine was placed into the housing of the pigs by adding a total of 500 mL thereof (comprising 1,500 pg PCV2 VLP) per pen containing five pigs. This composition was added to two bowls (gruel feeders) per pen to allow for the pigs to consume the gel vaccine mixture on their own. This vaccine did not contain adjuvant. At 23 days post vaccination, all pigs had blood drawn for peripheral blood mononuclear cell (PBMC) extraction to evaluate PCV2 specific T cell immunity, following the protocol of Koinig HC etal. Vet Res. 46:20 (2015). All blood samples were also submitted to test for PCV2 viremia by PCR.

[0305] Results: PCV2 viremia was not detected in any pig. The pigs immediately were attracted to the gel VLP mixture and consumed it entirely. This allowed for the immunization of pigs against PCV2 without needing to individually handle each pig, drastically reducing the labor and time needed to immunize the animals. This vaccine composition was well tolerated, and no adverse events were observed.

[0306] Analysis of cellular immunity revealed that the pigs in the Gel VLP Vaccine group developed PCV2 antigen specific T cells while pigs in the Non-Vaccinated Control group did not. At 23 days post vaccination, the Oral VLP vaccine group had PCV2 antigen specific interferon gamma and tumor necrosis factor producing CD4+T cells, while the Non-Vaccinated Control group did not (Figure 5).

[0307] Conclusion:

[0308] Porcine Circovirus, type 2, virus like particle when combined with a semi-solid, liquid, gel formulation for consumption was immunogenic to pigs, thus being a method and vaccine to prevent disease caused by PCV2 in pigs.

[0309] EXAMPLE 3

[0310] Results of Oral PCV2 Vaccine Efficacy Evaluation

[0311] Objective:

[0312] The objective of this study was to evaluate the efficacy of orally administered Porcine Circovirus type 2 (PCV2) virus like particle (VLP) containing PCV type 2a and PCV type 2d VLPs against a PCV2d challenge in swine.

[0313] Methods:

[0314] To investigate the efficacy of Porcine Circovirus type 2, virus like particle, oral vaccine for swine, an experiment was conducted with four treatment groups. These treatment groups consisted of a placebo group as well as groups receiving different concentrations of PCV2 VLP. Caesarian Derived, Colostrum Deprived Colostrum deprived (CDCD) pigs, being around three weeks of age at study day 0, were randomly assigned to treatment groups: T01 Placebo, non-vaccinated and challenged; T02 Oral 1x, vaccinated and challenged; T03 Oral 5x vaccinated and challenged; T04 Oral 10x vaccinated and challenged. Each group was comprised of 25 pigs. Pigs in each treatment group were mixed among pens. On study day 0, pigs in the Oral vaccinated groups received, via oral drench, a 1 mL dose of different concentrations of antigen. Treatment T02 Oral 1x, received 15pg PCV type 2a and 15pg PCV type 2d VLP; Treatment T03 Oral 5x received 75 pg PCV type 2a and 75 pg PCV type 2d VLP and Treatment T04 Oral 10x received 150 pg PCV type 2a and 150 pg PCV type 2d VLP. These antigens were (i) Porcine Circovirus, type 2d, killed baculovirus vector expressed virus like particle, i.e. recombinant baculovirus expressed PCV2d ORF2 protein (SEQ ID NO:2 (corresponds to SEQ ID NO:1 of WO2015051099)), which had been produced as described above in Example 1, and (II) Porcine Circovirus, type 2a, killed baculovirus vector expressed virus like particle, i.e. recombinant baculovirus expressed PCV2a ORF2 protein (SEQ ID NO:1 (corresponds to SEQ ID NO:1 of WO 2019191005)), which had been produced as described in Example 1 of WO 2019191005 (under “Production of PCV2 ORF2 protein - Upstream processing”), and was then further subjected to a downstream processing as set out in WO 2019191005 (Example 1, without mixing with a carbomer solution (adjuvant)). The placebo group received saline solution with the same route and volume as the oral vaccinated groups on study day 0.

[0315] Animals were challenged on study day 28. Challenge consisted of 1.0 mL intramuscular (IM) administration in the left neck and 1.0 mL of the same material intranasally (IN) into one nostril using a sterile syringe. This challenge targeted a dose of 5.0 logi0TCID5o Porcine Circovirus type 2d / 2 mL per pig. Blood was collected prior to vaccination (study day -2), prior to challenge (study day 27) as well as weekly post challenge on study days 35, 42 and 49. The study ended on day 49, twenty-one days post challenge. PCV2 viremia was quantified in serum of the collected blood samples by PCR.

[0316] Results:

[0317] Prior to challenge, PCV2 viremia was not detected in any pig (Figure 6). The T01 Placebo group developed PCV2 viremia after challenge which peaked at study day 49 with an average of 7.48 logic PCV2 genomic copies per mL. The oral administration of PCV2 VLP had a clear protective effect in reducing the consequences of PCV2 infection as evidenced by a significant reduction of viremia. This reduction of viremia occurred in all three orally vaccinated treatment groups which were administered different concentrations of PCV2 VLP antigen (Figure 6). At study day 49, the T02 Oral 1x group led to a 1.676 log PCV2 genomic copies / mL reduction of viremia compared to the T01 Placebo group (Figure 6). The effect of PCV2 VLP antigen concentration was also observed, as groups which were administered higher antigen concentrations had lower levels of viremia. This further highlights the immunogenic and protective effect of orally administered PCV2 VLP antigen. On both study day 42 and 49, there was a significant difference among PCV2 levels in serum of T02 Oral 1x and T04 Oral 10x groups. These differences were of 2.390 logic and 1.951 logw PCV2 genomic copies per mL, respectively. The T03 Oral 5x group had average viremia levels that were intermediary to those of the T02 Oral 1x and T04 Oral 10x treatment groups at all three time points post challenge (Figure 6). The T04 Oral 10x group had the lowest levels of viremia. A lower level of PCV2 viremia was found between the T04 Oral 10x and the T01 Placebo group at all time points following challenge (Figure 6). The difference in PCV2 viremia levels among both treatments was of 1.435 log, 3.432 log and 3.627 logw PCV2 genomic copies / mL at study days 35, 42 and 49, respectively.

[0318] Conclusion:

[0319] These results demonstrate that the oral administration of Porcine Circovirus, type 2, virus like particle mitigated infection and thus was protective against infection, thus being a method and vaccine to prevent disease caused by PCV2 in pigs.

[0320] EXAMPLE 4

[0321] Additional Results of PCV2 Vaccine Efficacy Evaluation

[0322] Objective:

[0323] The objective of this study was to evaluate the efficacy of orally administered Porcine Circovirus type 2 (PCV2) virus like particle (VLP) containing PCV type 2a and PCV type 2d VLPs against a PCV2d challenge in swine. The objective was to evaluate if vaccination could reduce viral shedding, viral colonization and lymphoid depletion.

[0324] Methods:

[0325] Fecal samples as well as samples of tracheobronchial lymph node (TBLN), mesenteric lymph node (MLN), and external iliac lymph node (ILN) collected at necropsy of the experiment of Example 3 above were used for this investigation. Fecal samples were collected prior to vaccination (study day -2), prior to challenge (study day 27) as well as weekly post challenge on study days 35, 42 and 49. These samples were frozen after being collected and were thawed and tested by PCR to evaluate levels of PCV2 virus. PCV2 qPCR was conducted according to standard operating procedure: MagMAX-96 Pathogen RNA / DNA kit (Applied Biosystem™, Waltham, MA, USA) with a KingFisher™ Flex 96 Deep-Well Magnetic Particle Processor (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract the DNA from fecal swab samples according to the manufacturer's instructions. DNA extracts were used to detect the conserved region of the PCV2 Replicase gene (ORF1) using TaqMan™ Fast Virus 1-step Master Mix (Life Technologies, Carlsbad, CA, USA) with forward primer 5'-GACTGTWGAGACTAAAGGTGGAACTGTA-3' and reverse primer 5'-GCTTCTACACCTGGGACAGCA-3' with the probe 5'- / 56-FAM / - CCCGTTGGAATGGT / 3MGBEc / -3'. The qPCR was performed (7500 Fast Real-Time PCR System, Applied Biosystems®, Foster City, CA, USA) with the following cycling conditions: one cycle at 50°C for 5 min, one cycle at 95°C for 20 sec, 40 cycles at 95°C for 3 s and 60°C for 30 s. Samples with Ct values <37 were considered positive. PCV2 quantitative RT-rtPCR was conducted under standard operating procedures using a standard curve of known genomic copies / mL of a synthetic target to calculate quantities based on Ct value. All samples were controlled appropriately for PCV2 using standard positive, negative, and internal validated controls. The TBLN, MLN and ILN samples were placed in formalin and paraffin embedded following standard processing protocols after being collected at necropsy on study day 49 when all pigs were necropsied. These tissues were processed using standard procedures for histology with hematoxylin and eosin (H& E) staining and immunohistochemistry (IHC) for the PCV2 virus. Fixed tissues were stored in 10% neutral buffered formalin and were routinely processed and embedded in paraffin in an automated tissue processor according to standard operating procedure: Sections were cut at 6 pm and stained with hematoxylin and eosin (HE) for routine histological examination and assessment of lymphoid depletion. PCV2 antigen detection was performed by PCV2 IHC using the Leica Bond RX system (Leica Biosystems, Wetzlar, Germany) according to standard operating procedure: All paraffin-embedded tissues were deparaffinized in xylene, rehydrated in gradients of ethanol followed by peroxidase blocking step for 5 min (BOND Polymer Refine Detection System, Leica Biosystems, Wetzlar, Germany). Primary antibody against PCV2 at 1:1000 dilution (Rabbit Polyclonal ORF-2) was incubated for 15 min at room temperature (and followed by polymer incubation for 8 min, Diaminobenzidine (DAB) chromogen for 10 min and counterstained with hematoxylin (BOND Polymer Refine Detection System, Leica Biosystems, Wetzlar, Germany). All samples were tested and controlled appropriately for PCV2 using validated standard positive controls. Each H& E and IHC slide was be scored by a pathologist blinded to treatment group following the scoring system outlined in Table 1. All scores were compiled and averaged by tissue to evaluate the severity of lymphoid depletion and PCV2 lymphoid colonization. All shedding data was logic transformed and averaged to evaluate differences in viral shedding between treatment groups. These treatment groups and samples came from the same experiment of Example 3 above and were: T01 Placebo, non-vaccinated and challenged; T02 Oral 1x, vaccinated and challenged; T03 Oral 5x vaccinated and challenged; T04 Oral 10x vaccinated and challenged. Each group was comprised of 25 pigs.

[0326] Table 1. Scoring metric for the evaluation of lymphoid depletion and lymphoid colonization.

[0327]

[0328] Results:

[0329] Both lymphoid colonization and depletion are measures of the severity of PCV2 infection in swine (Segales J. Virus Res. 164(1 -2):10-19 (2012)). This study clearly demonstrates the protective effect that the oral administration of PCV2 VLP had to mitigate infection and disease. Figure 7 shows that all vaccinated groups receiving PCV2 VLP in all concentrations led to reduction of PCV2 colonization of ILN, TBLN and MLN. This is remarkable as it clearly shows that the pigs developed a protective immune response to lessen the colonization of the virus in the pig. An additive effect was observed with the increase of vaccinal antigen. In these tissues, the greater the oral PCV2 VLP concentration administered, the less colonization was detected. The results encountered for lymphoid depletion followed a similar pattern to that observed for PCV2 colonization. All vaccinated pigs had a decrease in the severity of lymphoid depletion in ILN, TBLN and MLN (Figure 8). Again, a dose dependent response was observed, as the levels of lymphoid depletion decreasorl ineach group with increasing oral PCV2 VLP concentration administered. These results of PCV2 colonization and associated pathology in the swine target species clearly show that the oral administration of PCV2 VLP not only induces an immune response but that this immune response is protective.

[0330] To evaluate if the oral administration of PCV2 VLP would have an impact over virus shedding, PCR was performed of fecal samples collected at different timepoints of the trial. Prior to challenge, fecal PCV2 shedding was not detected in any pig (Figure 9). The T01 Placebo group began shedding PCV2 after challenge which peaked at study day 42 with an average of 7.94 logw PCV2 genomic copies per gram. The oral administration of PCV2 VLP had a clear protective effect in reducing the consequences of PCV2 infection as evidenced by a significant reduction of virus shedding. This reduction of shedding occurred in all three orally vaccinated treatment groups which were administered different concentrations of PCV2 VLP antigen (Figure 9). At study day 42, a 0.68, 1.6 and 2.14 log PCV2 genomic copies / g reduction of shedding compared to the T01 Placebo group was observed in the T02 Oral 1x, T03 Oral 5x and T04 Oral 10x groups, respectively (Figure 9). Again, the effect of PCV2 VLP antigen concentration was observed, as groups which were administered higher antigen concentrations had lower levels of fecal viral shedding. This further highlights the immunogenic and protective effect of orally administered PCV2 VLP antigen. In addition to study day 42, at study day 35 and 49 all of the vaccinated groups had a lower level of virus shedding as compared to the placebo group (Figure 9).

[0331] Conclusion:

[0332] These results demonstrate that the oral administration of Porcine Circovirus, type 2, virus like particle mitigated infection as shown by a reduction of viral colonization, viral associated pathology (lymphoid depletion) and shedding and thus was protective against infection and disease, thus being a method and vaccine to prevent disease caused by PCV2 in pigs.

[0333] LIST OF FIGURES:

[0334] Figure 1 (“Fig. 1 ”): Average S / P value of PCV2 specific IgM in Negative Control and Oral VLP Vaccine groups at 0, 14 and 23 days post vaccination, dpv = days post vaccination different letters indicate statistical significance within each time point (p<0.05). Within the pairs of bars shown in the chart, the left bar is designated “Non-vaccinated Control” and the right bar is designated “Oral VLP vaccine”.

[0335] Figure 2 (“Fig. 2”). Average S / P value of PCV2 specific IgG in Negative Control and Oral VLP Vaccine groups at 0 days post vaccination and 14 days post vaccination, dpv = days post vaccination, different letters indicate statistical significance within each time point (p<0.05). Within the pairs of bars shown in the chart, the left bar is designated “Non-vaccinated Control” and the right bar is designated “Oral VLP vaccine".

[0336] Figure 3 (“Fig. 3”): Average S / P value of PCV2 specific IgA, IgG and IgM in Negative Control and Oral VLP Vaccine groups at 22 days post vaccination quantified in oral fluids. Within the pairs of bars shown in the chart, the left bar is designated “Non-vaccinated Control” and the right bar is designated “Oral VLP vaccine".

[0337] Figure 4 (“Fig. 4”): Percentage of interferon gamma (IFNg) and tumor necrosis factor alpha (TNFa) positive PCV2 antigen specific CD4+ T cells measured in blood of pigs at 23 days post vaccination.

[0338] Figure 5 (“Fig. 5”): Percentage of interferon gamma (IFNg) and tumor necrosis factor alpha (TNFa) positive PCV2 antigen specific CD4+T cells measured in blood of pigs at 23 days post vaccination.

[0339] Figure 6 (“Fig. 6”): Average PCV2 genomic copies of PCV2 in serum from different treatment groups and different timepoints.

[0340] Figure 7 (“Fig. 7”): Evaluation of PCV2 lymphoid colonization among different lymphoid tissues and treatment groups. ILN = iliac lymph node; TBLN = tracheobronchial lymph node; MLN = mesenteric lymph node.

[0341] Figure 8 (“Fig. 8”): Evaluation of lymphoid depletion among different lymphoid tissues and treatment groups. ILN = iliac lymph node; TBLN = tracheobronchial lymph node; MLN = mesenteric lymph node.

[0342] Figure 9 (“Fig. 9”): Average PCV2 genomic copies of PCV2 in feces from different treatment groups and different timepoints.

[0343] Sequences included in the sequence listing: SEQ ID N0:1

[0344] SEQ ID N0:1 corresponds to the sequence of a PCV2a ORF2 protein (being 233 amino acid residues in length). SEQ ID NO:1 has the amino acid sequence (in the one letter code for amino acid residues, from N- to C-terminus, including an N-terminal methionine residue (M) at amino acid position 1):

[0345] MTYPRRRYRRRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLSRTFGYTVKAT TVTTPSWAVDMMRFNIDDFVPPGGGTNKISIPFEYYRIRKVKVEFWPCSPITQGDRGVGS TAVILDDNFVTKATALTYDPYVNYSSRHTIPQPFSYHSRYFTPKPVLDSTIDYFQPNNKR NQLWLRLQTSRNVDHVGLGTAFENSKYDQDYNIRVTMYVQFREFNLKDPPLEP

[0346] SEQ ID NO:2

[0347] SEQ ID NO:2 corresponds to the sequence of a PCV2d ORF2 protein (being 234 amino acid residues in length). SEQ ID NO:2 has the amino acid sequence (in the one letter code for amino acid residues, from N- to C-terminus, including an N-terminal methionine residue (M) at amino acid position 1):

[0348] MTYPRRRFRRRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLSRTIGYTVKKT TVTTPSWNVDMMRFNINDFLPPGGGSNPLTVPFEYYRIRKVKVEFWPCSPITQGDRGVGS TAVILDDNFVTKANALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDRTIDYFQPNNKR NQLWLRLQTTGNVDHVGLGTAFENSIYDQDYNIRITMYVQFREFNLKDPPLNPK

[0349] The following clauses are also disclosed herein. Thus, the present disclosure further includes aspects as featured by the following clauses:

[0350] 1. A method for reducing

[0351] virus shedding,

[0352] - colonization of lymphoid tissue, and / or

[0353] - lymphoid depletion,

[0354] caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, the method comprising administering orally to the pig an immunogenic composition comprising non-replicating PCV2 antigen. 2. The method according to clause 1, wherein said method is a method for reducing virus shedding caused by a PCV2 infection in a pig, the method comprising administering orally to the pig an immunogenic composition comprising nonreplicating PCV2 antigen.

[0355] 3. The method according to clause 1 or 2, wherein said method is a method for reducing colonization of lymphoid tissue caused by a PCV2 infection in a pig, the method comprising administering orally to the pig an immunogenic composition comprising non-replicating PCV2 antigen.

[0356] 4. The method according to any one of clauses 1 to 3, wherein said method is a method for reducing lymphoid depletion associated with a PCV2 infection in a pig, the method comprising administering orally to the pig an immunogenic composition comprising non-replicating PCV2 antigen.

[0357] 5. The method according to clause 1, wherein said method is a method for reducing virus shedding,

[0358] - colonization of lymphoid tissue, and

[0359] - lymphoid depletion

[0360] caused by or associated with a PCV2 infection in a pig, the method comprising administering orally to the pig an immunogenic composition comprising nonreplicating PCV2 antigen.

[0361] 6. The method according to any one of clauses 1 to 5, wherein said immunogenic composition is isotropic.

[0362] 7. The method according to any one of clauses 1 to 6, wherein the immunogenic composition is free of replicating PCV2 antigen.

[0363] 8. The method according to any one of clauses 1 to 7, wherein the immunogenic composition is substantially free of a vector capable of expressing the nonreplicating antigen.

[0364] 9. The method according to any one of clauses 1 to 8, wherein the immunogenic composition is substantially free of infectious baculovirus. 10. The method according to any one of clauses 1 to 9, wherein the immunogenic composition is substantially free of 30S ribosomal subunits.

[0365] 11. The method according to any one of clauses 1 to 10, wherein the immunogenic composition is substantially free of 40S ribosomal subunits.

[0366] 12. The method according to any one of clauses 1 to 11, wherein the immunogenic composition is substantially free of functional ribosomes.

[0367] 13. The method according to any one of clauses 1 to 12, wherein the immunogenic composition is substantially free of cellular material having a size larger than 2 pm.

[0368] 14. The method according to any one of clauses 1 to 13, wherein the immunogenic composition is substantially free of cellular material having a size larger than 0.8 pm.

[0369] 15. The method according to any one of clauses 1 to 14, wherein the immunogenic composition is substantially free of organelles having a size of at least 1 pm. 16. The method according to any one of clauses 1 to 15, wherein the immunogenic composition is substantially free of nuclei.

[0370] The method according to any one of clauses 1 to 16, wherein the immunogenic composition is substantially free of cells.

[0371] The method according to any one of clauses 1 to 17, wherein the immunogenic composition is substantially free of insect cells.

[0372] 19. The method according to any one of clauses 1 to 18, wherein the immunogenic composition is substantially free of bacterial cells.

[0373] 20. The method according to any one of clauses 1 to 19, wherein the immunogenic composition is substantially free of cell tissue.

[0374] 21. The method according to any one of clauses 1 to 20, wherein the immunogenic composition is substantially free of insect cell tissue.

[0375] 22. The method according to any one of clauses 1 to 21, wherein the immunogenic composition consists of a non-replicating PCV2 antigen; and

[0376] one or more veterinary-acceptable carriers.

[0377] The method according to any one of clauses 1 to 21, wherein the immunogenic composition consists of

[0378] a non-replicating PCV2 antigen; and

[0379] one or more veterinary-acceptable carriers; and

[0380] at least one adjuvant.

[0381] The method according to any one of clauses 1 to 21, wherein the immunogenic composition consists of

[0382] a non-replicating PCV2 antigen; and

[0383] one or more veterinary-acceptable carriers; and

[0384] at least one immunogenic substance different from said PCV2 antigen.

[0385] The method according to any one of clauses 1 to 21, wherein the immunogenic composition consists of

[0386] a non-replicating PCV2 antigen; and

[0387] one or more veterinary-acceptable carriers; and

[0388] at least one adjuvant; and

[0389] at least one immunogenic substance different from said PCV2 antigen.

[0390] The method according to any one of clauses 1 to 25, wherein the non-replicating PCV2 antigen is an isolated PCV2 antigen.

[0391] The method according to any one of clauses 1 to 26, wherein the non-replicating PCV2 antigen is a purified PCV2 antigen.

[0392] The method according to any one of clauses 1 to 27, wherein said non-replicating PCV2 antigen is uniformly distributed within the immunogenic composition. 29. The method according to any one of clauses 1 to 28, wherein said non-replicating PCV2 antigen is freely dissolved in said immunogenic composition.

[0393] 30. The method according to any one of clauses 1 to 29, wherein said non-replicating PCV2 antigen is quantitatively dissolved in said immunogenic composition.

[0394] 31. The method according to any one of clauses 1 to 30, wherein said non-replicating PCV2 antigen is not expressed or reproduced in the immunogenic composition.

[0395] 32. The method according to any one of clauses 1 to 31, wherein said non-replicating PCV2 antigen has been expressed in a cell culture system.

[0396] 33. The method according to any one of clauses 1 to 32, wherein said non-replicating PCV2 antigen is capable of forming a virus-like particle.

[0397] 34. The method according to any one of clauses 1 to 33, wherein the non-replicating PCV2 antigen comprises or is a recombinant PCV2 protein.

[0398] 35. The method according to any one of clauses 1 to 34, wherein the non-replicating PCV2 antigen is a virus-like particle.

[0399] 36. The method according to any one of clauses 1 to 35, wherein the non-replicating PCV2 antigen is a virus-like particle comprising a recombinant PCV2 protein. 37. The method according to any one of clauses 1 to 36, wherein the non-replicating PCV2 antigen is a recombinant PCV2 protein, or a virus-like particle comprising a recombinant PCV2 protein, and wherein the immunogenic composition has been obtained by a procedure comprising the steps of

[0400] (1) permitting infection of susceptible cells in culture with a vector comprising a nucleic acid sequence encoding said recombinant protein, wherein said recombinant protein is expressed by said vector,

[0401] (2) thereafter recovering said recombinant protein and / or virus-like particle comprising said recombinant protein from the cell culture, wherein preferably cell debris is separated from the recombinant protein and / or said virus-like particles via a separation step, preferably including a micro filtration through at least one filter, preferably two filters, wherein the at least one filter preferably has a pore size larger than the recombinant protein and / or virus-like particle comprising said recombinant protein, in particular has a pore size of about 1 to about 20 pm and / or about 0.1 pm to about 4 pm.

[0402] 38. The method according to clause 37, wherein the microfiltration in step (2) includes or consists of:

[0403] - a micro filtration through one or more filters having a pore size of about 2 pm to about 15 pm, and / or

[0404] - a micro filtration through one or more filters having a pore size of about 0.8 pm to about 1.0 pm.

[0405] 39. The method according to clause 37 or 38, wherein, after step (2), the solution comprising the recombinant protein and / or said virus-like particles is concentrated by removing an amount of solvent from the solution.

[0406] 40. The method according to any one of clauses 37 to 39, wherein the solution comprising the recombinant protein and / or said virus-like particles is subsequently processed by continuous diafiltration.

[0407] 41. The method according to any one of clauses 37 to 40, wherein the solution resulting from step (2) is further processed by a method comprising the steps of: (a) (i) inactivating the vector included in the solution resulting from step (2) by adding an inactivating agent to said solution;

[0408] (ii) neutralizing the inactivating agent by adding a neutralizing agent to the mixture resulting from step (i);

[0409] (b) concentrating the recombinant protein and / or said virus-like particles in the mixture resulting from step (a)(ii) by removing a portion of solvent from the mixture, and

[0410] (c) processing the solution resulting from step (b) by continuous diafiltration such that the concentration of the neutralized inactivating agent and / or the concentration of the neutralizing agent is decreased in the process solution.

[0411] 42. The method according to clause 41, wherein in step (b) the removing of a portion of the solvent from said mixture consists of or comprises filtering said mixture with at least one filter, wherein said at least one filter preferably comprises a filter membrane. 43. The method according to clause 41 or 42, wherein in step (b) said concentrating comprises

[0412] - feeding the mixture into a filter system containing at least one filter, wherein the at least one filter comprises a filter membrane having a membrane pore size allowing the neutralized inactivating agent and / or the neutralizing agent to pass through while retaining the recombinant protein and / or said quaternary structures in the bulk flow,

[0413] - discharging the permeate comprising the neutralized inactivating agent and / or the neutralizing agent.

[0414] 44. The method according to any one of clauses 41 to 44, wherein in step (c) said continuous diafiltration comprises

[0415] - feeding the solution into a filter system containing at least one filter, wherein the at least one filter comprises a filter membrane having a membrane pore size allowing the neutralized inactivating agent and / or the neutralizing agent to pass through while retaining the recombinant protein and / or said quaternary structures in the bulk flow,

[0416] - discharging the permeate comprising the neutralized inactivating agent and / or the neutralizing agent,

[0417] - adding a liquid to the bulk flow at a rate equal to the permeate flow, wherein the liquid is different from said solvent.

[0418] 45. The method according to any one of items 42 to 44, wherein the filter membrane has an average pore size that is smaller than the recombinant protein and / or said virus-like particles, and / or wherein the filter membrane has a molecular weight cut off of between about 200 kDa and about 500 kDa.

[0419] 46. The method according to any one of clauses 34 to 45, wherein the recombinant PCV2 protein is capable of forming a virus-like particle.

[0420] 47. The method according to any one of clauses 34 to 46, wherein the recombinant PCV2 protein is a recombinant PCV2 capsid protein.

[0421] 48. The method according to any one of clauses 34 to 47, wherein the recombinant PCV2 protein is a recombinant Pr'' / O9RF2 protein. The method according to clause 48, wherein said recombinant PCV2 ORF2 protein comprises or consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, still more preferably at least 99% or in particular 100% sequence identity with the sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0422] The method according to any one of clauses 34 to 49, wherein said recombinant PCV2 protein is a recombinant PCV2 ORF2 protein being 233 or 234 amino acid residues in length.

[0423] The method according to any one of clauses 34 to 50, wherein the recombinant PCV2 protein is selected from the group consisting of recombinant PCV2 subtype a (PCV2a) ORF2 protein, recombinant PCV2 subtype b (PCV2b) ORF2 protein, recombinant PCV2 subtype c (PCV2c) ORF2 protein, recombinant PCV2 subtype d (PCV2d) ORF2 protein, recombinant PCV2 subtype e (PCV2e) ORF2 protein, recombinant PCV2 subtype f (PCV2f) ORF2 protein, recombinant PCV2 subtype g (PCV2g) ORF2 protein and recombinant PCV2 subtype h (PCV2h) ORF2 protein.

[0424] The method according to any one of clauses 34 to 51, wherein the recombinant PCV2 protein is a recombinant PCV2 subtype a (PCV2a) ORF2 protein.

[0425] The method according to clause 51 or 52, wherein said recombinant PCV2a ORF2 protein comprises or consists of an amino acid sequence having at least 95%, preferably at least 98%, more preferably at least 99% or in particular 100% sequence identity with the sequence of SEQ ID NO:1.

[0426] The method according to any one of clauses 51 to 53, wherein said recombinant PCV2a ORF2 protein is 233 amino acid residues in length.

[0427] The method according to any one of clauses 34 to 51, wherein the recombinant PCV2 protein is a recombinant PCV2 subtype d (PCV2d) ORF2 protein.

[0428] The method according to clause 51 or 55, wherein said recombinant PCV2d ORF2 protein comprises or consists of an amino acid sequence having at least 95%, preferably at least 98%, more preferably at least 99% or in particular 100% sequence identity with the sequence of SEQ ID NO:2. 57. The method according to any one of clauses 51, and 55 to 56, wherein said recombinant PCV2d protein is 234 amino acid residues in length.

[0429] 58. The method according to any one of clauses 34 to 57, wherein said recombinant PCV2 protein is a recombinant baculovirus expressed protein.

[0430] 59. The method according to any one of clauses 1 to 58, wherein the immunogenic composition comprises at least 10 pg of the non-replicating PCV2 antigen per dose.

[0431] 60. The method according to any one of clauses 1 to 59, wherein the immunogenic composition comprises at least 15 pg of the non-replicating PCV2 antigen per dose. 61. The method according to any one of clauses 1 to 60, wherein the immunogenic composition comprises at least 30 pg of the non-replicating PCV2 antigen per dose.

[0432] 62. The method according to any one of clauses 1 to 61, wherein the immunogenic composition comprises at least 50 pg of the non-replicating PCV2 antigen per dose.

[0433] 63. The method according to any one of clauses 1 to 62, wherein the immunogenic composition comprises at least 100 pg of the non-replicating PCV2 antigen per dose.

[0434] 64. The method according to any one of clauses 1 to 63, wherein the immunogenic composition comprises at least 150 pg of the non-replicating PCV2 antigen per dose.

[0435] 65. The method according to any one of clauses 1 to 64, wherein the immunogenic composition comprises at least 200 pg of the non-replicating PCV2 antigen per dose.

[0436] 66. The method according to any one of clauses 1 to 65, wherein the immunogenic composition comprises at least 250 pg of the non-replicating PCV2 antigen per dose.

[0437] 67. The method according to any one of clauses 1 to 66, wherein one dose of the immunogenic composition has a volume of about 1 mL. The method according to any one of clauses 1 to 67, wherein said immunogenic composition comprises or contains one or more veterinary-acceptable carriers. The method according to any one of clauses 1 to 68, wherein said one or more veterinary acceptable carriers are selected from the group consisting of solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents.

[0438] The method according to any one of clauses 1 to 21, 23, and 25 to 69, wherein the immunogenic composition comprises or contains at least one adjuvant.

[0439] The method composition according to any one of clauses 1 to 21, and 24 to 70, wherein the immunogenic composition comprises or contains at least one immunogenic substance different from said non-replicating PCV2 antigen.

[0440] The method according to any one of clauses 24 to 71, wherein said non-replicating PCV2 antigen is a recombinant PCV2a ORF2 protein, and wherein the at least one immunogenic substance different from said PCV2 antigen is a recombinant PCV2d ORF2 protein.

[0441] The method according to clause 72, wherein said PCV2a ORF2 protein comprises or consists of an amino acid sequence having at least at least 95%, preferably at least 98%, more preferably at least 99% or in particular 100% sequence identity with the sequence of SEQ ID NO:1,

[0442] and wherein said PCV2d ORF2 protein comprises or consists of an amino acid sequence having at least at least 95%, preferably at least 98%, more preferably at least 99% or in particular 100% sequence identity with the sequence of SEQ ID NO:2.

[0443] The method according to any one of clauses 1 to 73, wherein the immunogenic composition is a liquid.

[0444] The method according to any one of clauses 1 to 74, wherein the immunogenic composition additionally contains a gel composition.

[0445] The method according to any one of clauses 1 to 73, and 75, wherein the immunogenic composition is a gel. 77. The method according to any one of clauses 1 to 76, wherein said immunogenic composition is clear.

[0446] 78. The method according to any one of clauses 1 to 77, wherein said immunogenic composition is transparent.

[0447] 79. The method according to any one of clauses 75 to 78, wherein the gel composition is a gel composition suitable for oral administration.

[0448] 80. The method according to any one of clauses 75 to 79, wherein said gel composition comprises water.

[0449] 81. The method according to any one of clauses 75 to 80, wherein said gel composition is a hydrogel composition.

[0450] 82. The method according to any one of clauses 75 to 81, wherein said gel composition comprises one or more flavoring agents.

[0451] 83. The method according to any one of clauses 75 to 82, wherein the one or more flavoring agents are selected from the group consisting of sucrose, glucose, sodium saccharin, sodium cyclamate, xylitol, perillartien, sucralose, D-tryptophan, aspartame, dihydrochalcones, artificial fruit flavoring (e.g., strawberry flavoring), plasma protein (e.g., spray-dried plasma protein), cheese and cheese-like flavorings, dried milk, chocolate and chocolate by-products.

[0452] 84. The method according to any one of clauses 75 to 83, wherein the one or more flavoring agents are capable of attracting pigs.

[0453] 85. The method according to any one of clauses 75 to 84, wherein said gel composition comprises one or more colorants, in particular one or more colorants capable of attracting pigs.

[0454] 86. The method according to any one of clauses 75 to 85, wherein the one or more colorants are, selected from the group consisting of FD& C Blue No. 1, FD& C Blue No. 2, FD& C Green No. 3, Orange B, Citrus FD& C Red No. 2, FD& C Red No. 2, FD& C Red No. 3, FD& C Red No.40, FD& C Yellow No. 5 and FD& C Yellow No.6.

[0455] 87. The method according to any one of clauses 75 to 86, wherein said gel composition comprises maltodextrin. 88. The method according to any one of clauses 75 to 87, wherein said gel composition comprises hemicellulose extract.

[0456] 89. The method according to any one of clauses 75 to 88, wherein said gel composition comprises propylene glycol.

[0457] 90. The method according to any one of clauses 75 to 89, wherein said gel composition comprises or consists of:

[0458] - water,

[0459] - maltodextrins,

[0460] - propylene glycol,

[0461] - hemicellulose extract,

[0462] - one or more colorants, and

[0463] - one or more flavoring agents.

[0464] 91. The method according to any one of clauses 1 to 90, wherein the immunogenic composition is administered by oral drench to the pig.

[0465] 92. A method for reducing

[0466] virus shedding,

[0467] - colonization of lymphoid tissue, and / or

[0468] - lymphoid depletion

[0469] caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, wherein the method comprises the steps of:

[0470] adding an immunogenic composition comprising non-replicating PCV2 antigen into the liquid food or drinking water of the pig, and

[0471] allowing the pig to self-administer the liquid food or drinking water to which the immunogenic composition has been added. The method according to clause 92, wherein said immunogenic composition is an immunogenic composition as specified in any one of clauses 6 to 25, 37 to 45, and 59 to 90.

[0472] The method according to clause 92 or 93, wherein said non-replicating PCV2 antigen is a non-replicating PCV2 antigen as specified in any one of clauses 26 to 37.

[0473] The method according to any one of clauses 92 to 94, wherein said non-replicating PCV2 antigen is a recombinant PCV2 protein as specified in any one of clauses 46 to 58.

[0474] An immunogenic composition comprising non-replicating PCV2 antigen for use in a method for reducing

[0475] virus shedding,

[0476] - colonization of lymphoid tissue, and / or

[0477] - lymphoid depletion

[0478] caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, wherein the method comprises orally administering said immunogenic composition to said pig.

[0479] The immunogenic composition for use according to clause 96, wherein said immunogenic composition is an immunogenic composition as specified in any one of clauses6 to 25, 37 to 45, and 59 to 91.

[0480] The immunogenic composition for use according to clause 96 or 97, wherein said non-replicating PCV2 antigen is a non-replicating PCV2 antigen as specified in any one of clauses 26 to 37.

[0481] The immunogenic composition for use according to any one of clauses 96 to 98, wherein said non-replicating PCV2 antigen is a recombinant PCV2 protein as specified in any one of clauses 46 to 58.

[0482] Use of an immunogenic composition comprising non-replicating PCV2 antigen in the preparation of a medicament "se in a method for reducing virus shedding,

[0483] - colonization of lymphoid tissue, and / or

[0484] - lymphoid depletion

[0485] caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, wherein the method comprises orally administering said immunogenic composition to said pig.

[0486] 101. Use according to clause 100, wherein said immunogenic composition is an immunogenic composition as specified in any one of clauses 6 to 25, 37 to 45, and 59 to 91.

[0487] 102. Use according to clause 100 or 101, wherein said non-replicating PCV2 antigen is a non-replicating PCV2 antigen as specified in any one of clauses 26 to 37. 103. Use according to any one of clauses 100 to 102 wherein said non-replicating PCV2 antigen is a recombinant protein as specified in any one of clauses 46 to 58. 104. The method according to any one of clauses 1 to 91, the immunogenic composition for use according to any one of clauses 96 to 99, or the use according to any one of clauses 100 to 103, wherein the method consists of orally administering only one dose of said immunogenic composition to said pig.

[0488] 105. The method according to any one of clauses 92 to 95, wherein said immunogenic composition is added only once into the liquid food or drinking water of the pig.

[0489] 106. The method according to any one of clauses 1 to 95, 104 and 105, the immunogenic composition for use according to any one of clauses 96 to 99, and 104, or the use according to any one of clauses 100 to 104, wherein the pig is not pre-vaccinated with a vaccine against PCV2.

[0490] 107. The method according to any one of clauses 1 to 95, and 104 to 106, the immunogenic composition for use according to any one of clauses 96 to 99, 104 and 106, or the use according to any one of clauses 100 to 104, and 106, wherein the pig is a PCV2 vaccination naive pig. The method according to any one of clauses 1 to 95, and 104 to 107, the immunogenic composition for use according to any one of clauses 96 to 99, 104, 106, and 107, or the use according to any one of clauses 100 to 104, 106, and 107, wherein said reducing of virus shedding,

[0491] - colonization of lymphoid tissue, and / or

[0492] - lymphoid depletion

[0493] caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig is obtained by said administration of said immunogenic composition.

[0494] The method according to any one of clauses 1 to 95, and 104 to 108, the immunogenic composition for use according to any one of clauses 96 to 99, 104, and 106 to 108, or the use according to any one of clauses 100 to 104, and 106 to 108, wherein said reducing of

[0495] virus shedding,

[0496] - colonization of lymphoid tissue, and / or

[0497] - lymphoid depletion

[0498] caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig is obtained without a further administration of a vaccine against PCV2 to the pig.

[0499] The method according to any one of clauses 1 to 95, and 104 to 110, the immunogenic composition for use according to any one of clauses 96 to 99, 104, and 106 to 110, or the use according to any one of clauses 100 to 104, and 106 to 110, wherein the immunogenic composition comprises 10 to 800 pg of said nonreplicating PCV2 antigen per dose.

[0500] The method according to clause 110, the immunogenic composition for use according to clause 110, or the use according to clause 110, wherein the immunogenic composition comprises 15 to 800 pg of said non-replicating PCV2 antigen per dose. 112. The method according to clause 110, the immunogenic composition for use according to clause 110, or the use according to clause 110, wherein the immunogenic composition comprises 30 to 800 pg of said non-replicating PCV2 antigen per dose.

[0501] 113. The method according to clause 110, the immunogenic composition for use according to clause 110, or the use according to clause 110, wherein the immunogenic composition comprises 50 to 800 pg of said non-replicating PCV2 antigen per dose.

[0502] 114. The method according to clause 110, the immunogenic composition for use according to clause 110, or the use according to clause 110, wherein the immunogenic composition comprises 100 to 800 pg of said non-replicating PCV2 antigen per dose.

[0503] 115. The method according to clause 110, the immunogenic composition for use according to clause 110, or the use according to clause 110, wherein the immunogenic composition comprises 150 to 800 pg of said non-replicating PCV2 antigen per dose.

[0504] 116. The method according to clause 110, the immunogenic composition for use according to clause 110, or the use according to clause 110, wherein the immunogenic composition comprises 200 to 800 pg of said non-replicating PCV2 antigen per dose.

[0505] 117. The method according to clause 110, the immunogenic composition for use according to clause 110, or the use according to clause 110, wherein the immunogenic composition comprises 250 to 800 pg of said non-replicating PCV2 antigen per dose.

[0506] 118. The method according to clause 110, the immunogenic composition for use according to clause 110, or the use according to clause 110, wherein the immunogenic composition comprises 300 to 800 pg of said non-replicating PCV2 antigen per dose.

[0507] 119. The method according to any one of clauses 1 to 95, and 104 to 118, the immunogenic composition for use according to any one of clauses 96 to 99, 104, and 106 to 118, or the use according to any one of clauses 100 to 104, and 106 to 118, wherein the immunogenic composition comprises up to 800 pg of said nonreplicating PCV2 antigen per dose.

[0508] 120. The method according to clause 119, the immunogenic composition for use according to clause 119, or the use according to clause 119, wherein the immunogenic composition comprises up to 700 pg of said non-replicating PCV2 antigen per dose.

[0509] 121. The method according to clause 119, the immunogenic composition for use according to clause 119, or the use according to clause 119, wherein the immunogenic composition comprises up to 600 pg of said non-replicating PCV2 antigen per dose.

[0510] 122. The method according to clause 119, the immunogenic composition according to clause 119, or the use according to clause 119, wherein the immunogenic composition comprises up to 500 pg of said non-replicating PCV2 antigen per dose.

[0511] 123. The method according to clause 119, the immunogenic composition for use according to clause 119, or the use according to clause 119, wherein the immunogenic composition comprises up to 400 pg of said non-replicating PCV2 antigen per dose.

[0512] 124. The method according to clause 119, the immunogenic composition for use according to clause 119, or the use according to clause 119, wherein the immunogenic composition comprises up to 300 pg of said non-replicating PCV2 antigen per dose.

[0513] 125. Use of a liquid antigen concentrate for reducing

[0514] virus shedding,

[0515] - colonization of lymphoid tissue, and / or

[0516] - lymphoid depletion caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, wherein 1 mL of said concentrate comprises about 100 to 800 pg of non-replicating PCV2 antigen, and wherein preferably said concentrate is diluted in drinking water or in liquid food of said pig.

[0517] 126. The use according to clause 125, wherein 1 mL of said concentrate comprises about 150 to 800 pg of non-replicating PCV2 antigen.

[0518] 127. The use according to clause 125, wherein 1 mL of said concentrate comprises about 200 to 800 pg of non-replicating PCV2 antigen.

[0519] 128. The use according to clause 125, wherein 1 mL of said concentrate comprises about 250 to 800 pg of non-replicating PCV2 antigen.

[0520] 129. The use according to clause 125, wherein 1 mL of said concentrate comprises about 300 to 800 pg of non-replicating PCV2 antigen.

[0521] 130. The use according to clause 125, wherein 1 mL of said concentrate comprises about 400 to 800 pg of non-replicating PCV2 antigen.

[0522] 131. The use according to clause 125, wherein 1 mL of said concentrate comprises about 500 to 800 pg of non-replicating PCV2 antigen.

[0523] 132. The use according to clause 125, wherein 1 mL of said concentrate comprises about 600 to 800 pg of non-replicating PCV2 antigen.

[0524] 133. The use according to clause 125, wherein 1 mL of said concentrate comprises about 700 to 800 pg of non-replicating PCV2 antigen.

[0525] 134. The use according to any one of clauses 125 to 133, wherein said non-replicating PCV2 antigen is a non-replicating PCV2 antigen as specified in any one of clauses 26 to 37, and / or wherein said non-replicating PCV2 antigen is baculovirus expressed.

[0526] 135. The use according to any one of clauses 125 to 134, wherein said non-replicating PCV2 antigen is a recombinant PCV2 protein as specified in any one of clauses 46 to 58. 136. Method for enabling the self-immunization against PCV2 by a pig, comprising the step of diluting a liquid antigen concentrate comprising non-replicating PCV2 antigen in the drinking water of the pig,

[0527] wherein said self-immunization results in the reduction of

[0528] virus shedding,

[0529] - colonization of lymphoid tissue, and / or

[0530] - lymphoid depletion

[0531] caused by or associated with a PCV2 infection in said pig.

[0532] 137. Use of a container containing drinking water to which a liquid antigen concentrate comprising non-replicating PCV2 antigen has been added for reducing

[0533] virus shedding,

[0534] - colonization of lymphoid tissue, and / or

[0535] - lymphoid depletion

[0536] caused by or associated with a PCV2 infection in a pig, wherein the container is preferably placed in the housing environment of the pig.

[0537] 138. The use according to clause 137, wherein said container is selected from the group consisting of bowl, vessel and barrel.

[0538] 139. The use according to clause 127 or 138, wherein said container is located in the housing environment of a pig to be vaccinated.

[0539] 140. Kit for use in a method for reducing

[0540] virus shedding,

[0541] - colonization of lymphoid tissue, and / or

[0542] - lymphoid depletion

[0543] caused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, wherein the kit comprises - a liquid antigen concentrate comprising non-replicating PCV2 antigen; and - instructions to dilute said liquid antigen concentrate in drinking water or in liquid food to be consumed by the pig.

[0544] The method according to clause 136, the use according to any one of clauses 137 to 139, or the kit according to clause 140, wherein said non-replicating PCV2 antigen is a non-replicating PCV2 antigen as specified in any one of clauses 26 to 37, and / or wherein said non-replicating PCV2 antigen is baculovirus expressed. The method according to clause 136 or 141, the use according to any one of clauses 137 to 139, and 141, or the kit according to clause 140 or 141, wherein said non-replicating PCV2 antigen is a recombinant PCV2 protein as specified in any one of clauses 46 to 58.

[0545] The method according to any one of clauses 136, 141, and 142, the use according to any one of clauses 137 to 139, 141, and 142, or the kit according to any one of clauses 140 to 142, wherein said liquid antigen concentrate is the liquid antigen concentrate according to any one of clauses 125 to 135.

[0546] The method according to any one of clauses 136, and 141 to 143, the use according to any one of clauses 125 to 135, 137 to 139, and 141 to 143, or the kit according to any one of clauses 140 to 143, wherein said liquid antigen concentrate is an immunogenic composition as specified in any one of clauses 6 to 25, 37 to 45, 68 to 71, and 74 to 78.

[0547] The method according to any one of clauses 1 to 95, 104 to 124, 136, and 141 to 144, the immunogenic composition for use according to any one of clauses 96 to 99, 104, and 106 to 124, the use according to any one of clauses 100 to 104, 106 to 135, 137 to 139, and 140 to 144, or the kit according to any one of clauses 140 to 144, wherein said pig is a piglet.

[0548] The method according to any one of clauses 1 to 95, 104 to 124, 136, and 141 to 144, the immunogenic composition for use according to any one of clauses 96 to 99, 104, and 106 to 124, the use according to any one of clauses 100 to 104, 106 to 135, 137 to 139, and 140 to 144, or the kit according to any one of clauses 140 to 144, wherein said pig is a sow.

Claims

CLAIMS:

1. A method for reducing- virus shedding,- colonization of lymphoid tissue, and / or- lymphoid depletioncaused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, the method comprising administering orally to the pig an immunogenic composition comprising non-replicating PCV2 antigen.

2. A method for reducing- virus shedding,- colonization of lymphoid tissue, and / or- lymphoid depletioncaused by or associated with a PCV2 infection in a pig, wherein the method comprises the steps of:adding an immunogenic composition comprising non-replicating PCV2 antigen into the drinking water of the pig or liquid food of the pig, andallowing the pig to self-administer the drinking water, or liquid food, to which the immunogenic composition has been added.

3. An immunogenic composition comprising non-replicating PCV2 antigen for use in a method for reducing- virus shedding,- colonization of lymphoid tissue, and / or- lymphoid depletioncaused by or associated with a PCV2 infection in a pig,wherein the method comprises orally administering said immunogenic composition to said pig.

4. The method according to claim 1 or 2, or the immunogenic composition for use according to claim 3, wherein the immunogenic composition is free of replicating PCV2 antigen, and / or.wherein the immunogenic composition is substantially free of cells, and / or wherein the immunogenic composition is substantially free of cellular material having a size larger than 2 pm, and / orwherein the immunogenic composition is homogenous, and / orwherein the immunogenic composition is isotropic.

5. The method according to any one of claims 1, 2, and 4, or the immunogenic composition for use according to claim 3 or 4, wherein the immunogenic composition has been obtained by a procedure comprising the steps of(1) permitting infection of susceptible cells in culture with a vector comprising a nucleic acid sequence encoding said recombinant protein, wherein said recombinant protein is expressed by said vector,(2) thereafter recovering said recombinant protein and / or virus-like particle comprising said recombinant protein from the cell culture, wherein preferably cell debris is separated from the recombinant protein and / or said virus-like particles via a separation step, preferably including a micro filtration through at least one filter, preferably two filters, wherein the at least one filter preferably has a pore size larger than the recombinant protein and / or virus-like particle comprising said recombinant protein, in particular has a pore size of about 1 to about 20 pm and / or about 0.1 pm to about 4 pm.

6. The method according to any one of claims 1, 2, 4, and 5, or the immunogenic composition for use according to any one of claim 3 to 5, wherein the immunogenic composition consists of- a non-replicating PCV2 antigen; and- one or more veterinary-acceptable carriers,or wherein the immunogenic composition consists of- a non-replicating PCV2 antigen; and- one or more veterinary-acceptable carriers;- and optionally at least one adjuvant and / or optionally at least one immunogenic substance different from said PCV2 antigen.

7. The method according to any one of claims 1, 2, and 4 to 6, or the immunogenic composition for use according to any one of claims 3 to 6, wherein the immunogenic composition additionally contains a gel composition, in particular a hydrogel composition,and wherein optionally the gel composition comprises one or more flavoring agents,and wherein the one or more flavoring agents are preferably capable of attracting pigs,and / or wherein the one or more flavoring agents are preferably selected from the group consisting of sucrose, glucose, sodium saccharin, sodium cyclamate, xylitol, perillartien, sucralose, D-tryptophan, aspartame, dihydrochalcones, artificial fruit flavoring (e.g., strawberry flavoring), plasma protein (e.g., spray-dried plasma protein), cheese and cheese-like flavorings, dried milk, chocolate and chocolate by-products.

8. The method according to any one of claims 1, 2, and 4 to 6, or the immunogenic composition for use according to any one of claims 3 to 7, wherein the immunogenic composition comprisesat least 10 pg of the non-replicating PCV2 antigen per dose, orat least 15 pg of the non-replicating PCV2 antigen per dose, orat least 30 pg of the non-replicating PCV2 antigen per dose, orat least 50 pg of the non-replicating PCV2 antigen per dose, orat least 100 ng of the non-replicating PCV2 antigen per dose, orat least 150 pg of the non-replicating PCV2 antigen per dose, orat least 200 pg of the non-replicating PCV2 antigen per dose, orat least 250 pg of the non-replicating PCV2 antigen per dose.

9. The method according to any one of claims 1, 2, and 4 to 8, or the immunogenic composition for use according to any one of claims 3 to 8, wherein said nonreplicating PCV2 antigen comprises or is a recombinant PCV2 protein, and wherein the recombinant PCV2 protein is preferably capable of forming a virus-like particle.

10. The method according to any one of claims 1, 2, and 4 to 9, or the immunogenic composition for use according to any one of claims 3 to 9 wherein said nonreplicating PCV2 antigen is- a virus-like particle, in particular a virus-like particle comprising a recombinant PCV2 protein.

11. The method according to claim 9 or 10, or the immunogenic composition for use according to claim 9 or 10, wherein the recombinant PCV2 protein is- a recombinant PCV2 ORF2 protein,wherein said PCV2 ORF2 protein preferably comprises or consists of an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, still more preferably at least 99% or in particular 100% sequence identity with the sequence of SEQ ID NO:1 or SEQ ID NO:2;and / or wherein said recombinant PCV2 protein is a recombinant baculovirus expressed protein.

12. The method according to any one of claims 6 to 11, or the immunogenic composition for use according to any one of claims 6 to 11, wherein said one or more veterinary-acceptable carriers are selected from the group consisting of solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents.

13. The method according to any one of claims 1, and 4 to 12, or the immunogenic composition for use according to any one of claims 3 to 12, wherein the immunogenic composition is administered by oral drench to the pig.

14. The method according to any one of claims 1, 2, and 4 to 13, or the immunogenic composition for use according to any one of claims 3 to 13, wherein the immunogenic composition comprises 10 to 800 pg of the non-replicating PCV2 antigen per dose.

15. Use of a liquid antigen concentrate for reducing- virus shedding,- colonization of lymphoid tissue, and / or- lymphoid depletioncaused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, wherein 1 mL of said concentrate comprises about 100 to 800 pg of non-replicating PCV2 antigen, preferably about 300 to 800 pg of non-replicating PCV2 antigen, and wherein preferably said concentrate is diluted in the drinking water, or liquid food, of said pig.

16. A method for enabling the self-immunization against PCV2 by a pig, comprising the step of diluting a liquid antigen concentrate comprising non-replicating PCV2 antigen in the drinking water of the pig,wherein said self-immunization results in the reduction of- virus shedding,- colonization of lymphoid tissue, and / or- lymphoid depletioncaused by or associated with a PCV2 infection in said pig.

17. Use of a container containing drinking water to which a liquid antigen concentrate comprising non-replicating PCV2 antigen has been added for reducing- virus shedding,- colonization of lymphoid tissue, and / or- lymphoid depletioncaused by or associated with a porcine circovirus type 2 (PCV2) infection in a pig, wherein preferably the container is placed in the housing environment of the pig.

18. The use according to claim 17, wherein said container is selected from the group consisting of- a bowl containing drinking water to which a liquid antigen concentrate comprising non-replicating PCV2 antigen has been added,- a vessel containing drinking water to which a liquid antigen concentrate comprising non-replicating PCV2 antigen has been added, and- a barrel containing drinking water to which a liquid antigen concentrate comprising non-replicating PCV2 antigen has been added;and / or wherein said container is located in the housing environment of a pig to be immunized.

19. Kit for use in a method for reducing- virus shedding,- colonization of lymphoid tissue, and / or- lymphoid depletioncaused by or associated with a PCV2 infection in a pig, wherein the kit comprises - a liquid antigen concentrate comprising non-replicating PCV2 antigen; and - instructions to dilute said liquid antigen concentrate in drinking water or liquid food to be consumed by the pig.

20. The method according to claim 16, the use according to claim 17 or 18, or the kit according to claim 19, wherein said concentrate comprises about 100 to 800 pg of non-replicating PCV2 antigen, preferably about 300 to 800 pg of non-replicating PCV2 antigen.

21. The use according to claim 15, 17, 18, and 20, the method according to claim 16 or 20, or the kit according to claim 19 or 20, wherein said concentrate is an immunogenic composition as specified in any one of claims 4 to 8, and 12.

22. The use according to claim 15, 17, 18, 20, and 21, the method according to any one of claims 16, 20, and 21, or the kit according to any one of claims 19 to 21, wherein said non-replicating PCV2 antigen comprises a recombinant PCV2 protein as specified in claim 11.

23. The method according to any one of claims 1, 2, 4 to 14, 16, and 20 to 22, the immunogenic composition for use according to any one of claims 3 to 14, the use according to any one of claims 15, 17, 18, and 20 to 22, or the kit according to any one of claims 19 to 22, wherein said pig is a piglet or a sow.

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