Use of a PCV2 immunogenic composition for lessening clinical symptoms in pigs

[0001] The present application contains a list of sequences in paper form and in a computer readable form.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION [0002] The present disclosure relates to the use of an immunogenic composition comprising PCV2 porcine circovirus type 2 antigen for the treatment of several clinical symptoms (diseases). Preferably, these clinical symptoms are associated with PCV2 infection. More specifically, the present disclosure relates to an immune composition effective in inducing an immune response that reduces or reduces the severity of clinical symptoms associated with PCV2 infection. The immunological composition contains recombinantly produced PCV2 antigen. The antigen is PCV2 ORF2 protein. More specifically, the present disclosure relates to an immune composition effective in treating clinical symptoms associated with PCV2 infection in pigs receiving the immune composition and wherein the composition comprises expressed PCV2 ORF2 protein. Another aspect of the present disclosure relates to the use of any composition provided herein as a medicine, preferably as a veterinary medicine, and even more preferably, as a vaccine. In addition, the present disclosure also relates to the use of any of the compositions described herein for the manufacture of a medicament for reducing or reducing the severity of clinical symptoms associated with PCV2 infection. Preferably, the drug is intended to prevent PCV2 infection, even more preferably in pigs. Another aspect of the present disclosure relates to a method of making a medicament comprising a PCV2 immunogenic composition for treating many clinical symptoms.

DESCRIPTION OF THE PRIOR ART [0003] The porcine circovirus type 2 (PCV2) is a small (17-22 nm diameter) icosahedron-shaped DNA virus that contains a single-stranded circular genome. PCV2 has approximately 80% sequence identity with porcine circovirus type 1 (PCV1). However, unlike the PCV1 circovirus, which is generally non-virulent, a disease commonly known as 'post-weaning multisystemic wasting syndrome' has been detected in pigs infected with PCV2 circovirus (PCT). postweaning multisystemic wasting syndrome, PMWS). Clinical manifestations of PMWS are characterized by cachexia, pallor of the skin, coughing, respiratory disorders, diarrhea, jaundice and jaundice with cholestasis. Some diseased pigs may have a combination of all symptoms, while others may only have one or two of these symptoms in those affected by pig infection. During dissection, microscopic and macroscopic changes also appear in many tissues and organs, with lymphoid organs being the most common site of lesions. A strong correlation between the amount of PCV2 nucleic acid or antigen and the severity of microscopic lymphoid lesions was observed. The mortality rate of PCV2 infected pigs can reach up to 80%. In addition to PMWS, PCV2 is associated with several other infections, including Aujeszky's disease virus, Porcine reproductive and respiratory syndrome virus (PRRS), GLASER disease, streptococcal meningitis, salmonellosis, colibacillosis after weaning, dietary hepatitis and purulent inflammation. bronchi. However, studies to date have not confirmed that any of these clinical symptoms were in fact a direct result of PCV2 infection. Furthermore, it is not yet known if any of these clinical symptoms can be effectively reduced or cured with an active agent directed against PCV2.

[0004] Current approaches to the treatment of infections include the DNA-based vaccine described in US Patent Document No. 6,703,023. However, such vaccines have been ineffective in conferring protective immunity against PCV2 infection or reducing, reducing or treating any clinical symptoms associated therewith. . In addition, the vaccines described in the prior art focused only on preventing PCV2 infection in pigs without considering any further medical applications.

[0005] Accordingly, there is a need in the art for an immunogenic composition for the treatment of many clinical symptoms. In addition, there is a need in the art for an immunogenic composition that conserves protection against PCV2 infection, which can also be used to treat existing clinical symptoms associated with PCV2 infection.

DISCLOSURE OF THE INVENTION [0006] The present invention allows to solve the problems inherent in the prior art and provides clear progress in the prior art. The present invention provides the medical use (s) to the immunogenic composition (s) containing the PCV2 antigen.

[0007] In the present invention are provided:

[1] An immunogenic composition capable of eliciting or enhancing an immune response against PCV2 and containing the recombinantly produced PCV2 ORF2 protein for use in the prevention of generalized lymphadenopathy, lymphoid atrophy and / or multinucleated / giant histiocytes in pigs where the PCV ORF2 protein is single administration of pig.

[2] The immunogenic composition according to [1] for use according to [1], wherein the recombinantly produced PCV2 ORF2 protein is expressed with baculovirus PCV2 ORF2 protein.

[3] The immunogenic composition according to [1] or [2] for use according to [1] or [2], wherein the recombinantly produced PCV2 ORF2 protein was obtained by a method in which

- sensitive cells are infected with a recombinant baculovirus vector containing DNA coding sequences for PCV2 ORF2 protein,

- the PCV2 ORF2 polypeptide is expressed by recombinant baculovirus, and

- expressed PCV2 ORF2 polypeptide is recovered from the supernatant by filtration and inactivated.

[4] The immunogenic composition according to any one of [1] to [3] for use according to any one of [1] to [3], wherein the recombinantly produced PCV2 ORF2 protein was recovered from the supernatant of in vitro cultured cells, wherein said cells were infected with a recombinant baculovirus vector containing PCV2 ORF2 DNA and expressing PCV2 ORF2 protein, and wherein said cell culture was treated with about 2 to about 8 mM BEI to inactivate the baculovirus vector and an equivalent concentration of neutralizing agent.

[5] The immunogenic composition according to any one of [1] to [4] for use according to any one of [1] to [4], further comprising at least a portion of a recombinant baculovirus vector expressing said PCV2 ORF2 protein.

[6] The immunogenic composition according to [5] for use according to [5], further comprising a part of the cell culture supernatant.

[7] The immunogenic composition of any one of [1] to [6] for use according to any of [1] to [6], wherein said immunogenic composition is a vaccine.

[8] An immunogenic composition according to any one of [1] to [7] for use according to any one of [1] to [7], containing from about 0.3 to about 200 μg of recombinantly produced PCV2 ORF2 protein per dose.

[9] The immunogenic composition according to any one of [1] to [8] for use according to any one of [1] to [8], wherein the pig is a piglet 2 weeks old or older, but not older than 15 weeks old.

[10] The immunogenic composition according to any one of [1] to [9] for use according to any one of [1] to [9], wherein the immunogenic composition further comprises an adjuvant.

[11] The immunogenic composition according to [10] for use according to [10], wherein the adjuvant may include aluminum hydroxide and aluminum phosphate, saponins, a water-in-oil emulsion, an oil-in-water emulsion, a water-in-oil-in-water emulsion or where the adjuvant is a selected compound from polymers of acrylic or methacrylic acid and copolymers of maleic anhydride and alkenyl derivative.

[12] The immunogenic composition according to [10] or [11] for use according to [10] or [11], containing 100 μg to 10 mg of adjuvant per dose.

[13] The immunogenic composition according to any one of [1] to [12] for use according to any one of [1] to [12], wherein the immunogenic composition is substantially free of PCV2 antigens or polypeptides other than the PCV2 ORF2 protein.

[14] The immunogenic composition according to [4] for use according to [4] and additionally comprising the adjuvant mentioned in [11], wherein the immunogenic composition is stable for a period of 24 months.

[15] The immunogenic composition according to any one of [1] to [14] for use according to any one of [1] to [14], wherein the medicament is intended for intramuscular administration.

[16] The use of an immunogenic composition capable of eliciting or enhancing an immune response against PCV2 and containing the recombinantly produced PCV2 ORF2 protein for the manufacture of a medicament for preventing generalized lymphadenopathy, lymphoid atrophy and / or multinucleated / giant histiocytes in pigs where the PCV2 ORF2 protein is intended for single administration to pigs.

[17] The use of [16], wherein the recombinantly produced PCV2 ORF2 protein is expressed with baculovirus PCV2 ORF2 protein.

[18] The use according to [16] or [17], wherein the recombinantly produced PCV2 ORF2 protein was obtained by a method in which

- sensitive cells are infected with a recombinant baculovirus vector containing DNA coding sequences for PCV2 ORF2 protein,

- the PCV2 ORF2 polypeptide is expressed by recombinant baculovirus, and

- expressed PCV2 ORF2 polypeptide is recovered from the supernatant by filtration and inactivated.

[19] Use according to any one of [16] to [18], wherein the recombinantly produced PCV2 ORF2 protein was recovered from the supernatant of in vitro cultured cells, wherein said cells were infected with a recombinant baculovirus vector containing PCV2 ORF2 DNA and expressing PCV2 ORF2 protein, and wherein said culture cell was treated with about 2 to about 8 mM BEI to inactivate the baculovirus vector and with an equivalent concentration of neutralizing agent.

[20] The use according to any one of [16] to [19], wherein the composition additionally comprises at least a portion of a recombinant baculovirus vector expressing said PCV2 ORF2 protein.

[21] The use of [20], wherein the composition further comprises a portion of the cell culture supernatant.

[22] The use according to any one of [16] to [21], wherein the immunogenic composition is a vaccine.

[23] The use according to any one of [16] to [22], wherein the composition comprises from about 0.3 to about 200 μg of recombinantly produced PCV2 ORF2 protein per dose.

[24] Use according to any one of [16] to [23], wherein the pig is a piglet 2 weeks old or older, but not older than 15 weeks old.

[25] The use according to any one of [16] to [24], wherein the immunogenic composition further comprises an adjuvant.

[26] The use of [25], wherein the adjuvant may include aluminum hydroxide and aluminum phosphate, saponins, a water-in-oil emulsion, an oil-in-water emulsion, a water-in-oil-in-water emulsion, or wherein the adjuvant is a compound selected from polymers of acrylic or methacrylic acid and copolymers of maleic anhydride and alkenyl derivative.

[27] The use according to [25] or [26], wherein the composition contains 100 μg to 10 mg of adjuvant per dose.

[28] The use according to any one of [16] to [27], wherein the immunogenic composition is substantially free of PCV2 antigens or polypeptides other than the PCV2 ORF2 protein.

[29] The use of [19], wherein the immunogenic composition further comprises the adjuvant mentioned in [26] and is stable for a period of 24 months.

[30] The use according to any one of [16] to [29], wherein the drug is intended for intramuscular administration.

[0008] In general, no adverse effects or injection site reactions have been reported for any of the PCV2 antigen immunogenic compositions used in the present invention. Thus, the immunogenic compositions used in the present invention appear safe when administered to young piglets, preferably piglets not older than 15 weeks old, more preferably not older than 6 weeks old, even more preferably not older than 3 weeks old, most preferably not older than 2 weeks old . Alternatively, preferably, the immunogenic compositions of the present invention are administered within at least 2 and preferably within at least 3 weeks after exposure to virulent PVC. According to another embodiment, the immunogenic compositions used in the present invention for any medical use described herein are administered to piglets 3 weeks of age or older, preferably 2 weeks of age or older, most preferably but not older than 15 weeks.

[0009] Surprisingly, it has been found that the therapeutic use of the immunogenic compositions described below is effective in reducing the severity of various clinical symptoms in pigs. In particular, it has been discovered that the therapeutic use of the immunogenic compositions of the present invention, in particular compositions comprising the PCV2 ORF2 antigen, is effective in reducing or reducing the generalized lymph node enlargement, lymphoid tissue atrophy, and / or multinucleated / giant histiocytes in PCV2 infected pigs. In addition, the therapeutic use of the antigenic composition provided herein and comprising the PCV2 antigen, preferably ORF2 antigen, reduces the total circovirus load and its immunosuppressive effect, thereby resulting in a higher level of overall disease resistance and a reduced incidence of diseases and symptoms associated with PCV-2.

[0010] Thus, the present document describes the use of an immunogenic composition comprising PCV2 antigen, preferably recombinant PCV2 antigen and more preferably, the PCV2 ORF2 protein provided herein, for the preparation of a medicament for the prevention, reduction and / or reduction of generalized lymph node enlargement, lymphoid tissue atrophy and / or multinucleated / giant histiocytes in pigs. Preferably, said medicament is effective to prevent, reduce and / or reduce generalized lymphadenopathy, lymphoid tissue atrophy and / or multinucleated / giant histiocytes associated with PCV2 infections in pigs. Even more preferably, said medicament is effective to prevent, reduce and / or reduce generalized lymphadenopathy, lymphoid atrophy and / or multinucleated / giant histiocytes associated with PCV2 infections in pigs when administered to piglets not older than 15 weeks, more preferably not older than 6 weeks, even more preferably not older than 3 weeks, and most preferably not older than 2 weeks. Alternatively, preferably, the immunogenic compositions of the present invention are administered for at least 2 and preferably for at least 3 weeks exposure to virulent PVC.

[0011] Another aspect of the present disclosure relates to a method of treating generalized lymphadenopathy, lymphoid atrophy and / or multinucleated / giant histiocytes in pigs, comprising administering the immunogenic composition provided herein, to a pig, said immunogenic composition comprising PCV2 antigen, preferably recombinant PCV2 antigen, and more preferably PCV2 ORF2 protein. In yet another aspect, the present disclosure provides a method of treating generalized lymphadenopathy, lymphoid atrophy and / or multinucleated / giant histiocytes associated with PCV2 infection in pigs, comprising administering the immunogenic composition provided herein, to a pig, said immunogenic composition comprising an antigen PCV2, preferably a recombinant PCV2 antigen and more preferably PCV2 ORF2 protein. Preferably, said results of treatment in terms of limiting, reducing, preventing and / or curing generalized lymphadenopathy, lymphoid tissue atrophy and / or multinucleated / giant histiocytes in pigs receiving said immunogenic composition. According to an additional aspect, said methods of treatment further comprise administering said immunogenic composition to piglets not older than 15 weeks old, more preferably not older than 6 weeks old, even more preferably not older than 3 weeks old, and most preferably not older than 2 weeks old. Alternatively, preferably, the immunogenic compositions of the present invention are administered for at least 2 and preferably for at least 3 weeks of exposure to virulent PVC.

[0012] In addition, it has been discovered that the therapeutic use of an immunogenic composition comprising PCV2 antigen, preferably a recombinant PCV2 antigen, and most preferably the PCV2 ORF2 protein provided herein, can reduce or reduce the generalized lymph node enlargement in combination with one or more of the following symptoms in an affected pig: (1) interstitial pneumonia with swelling of interlobular fissures, (2) pallor of the skin or jaundice, (3) spotty atrophic cirrhosis, (4) stomach ulcers, (5) nephritis and (6) reproductive disorders, for example, abortion, stillbirth, fetal mummification, etc.

[0013] Thus, this document discloses the use of an immunogenic composition comprising PCV2 antigen, preferably a recombinant PCV2 antigen, and more preferably, the PCV2 ORF2 protein provided herein, for the preparation of a medicament for preventing, reducing and / or reducing the generalized lymphadenopathy in combination with one or more more of the following symptoms in pigs: (I) interstitial pneumonia with swelling of interlobular fissures, (2) pallor of the skin or jaundice, (3) spotty atrophic cirrhosis, (4) stomach ulcers, (5) nephritis and (6) reproductive disorders, e.g. abortion, childbirth dead fetus, fetal mummification etc., in pigs. Preferably, said medicament is effective to prevent, reduce and / or reduce the generalized lymphadenopathy in combination with one or more of the following symptoms associated with PCV2 infection in pigs: (1) interstitial pneumonia with swelling of interlobular gaps, (2) pallor of the skin or jaundice, (3) spotty atrophic cirrhosis, (4) stomach ulcers, (5) nephritis and (6) reproductive disorders, e.g. abortion, delivery dead fetus, fetal mummification etc. According to an additional aspect, said medicament is effective to prevent, reduce and / or reduce the generalized lymph node enlargement in combination with one or more of the following symptoms in pigs: (1) interstitial pneumonia with swelling of interlobular gaps, (2) pallor of the skin or jaundice, (3) spotty atrophic cirrhosis, (4) stomach ulcers, (5) nephritis and (6) reproductive disorders, e.g. abortion, delivery dead fetus, fetal mummification etc., in pigs when administered to piglets not older than 15 weeks old, more preferably not older than 6 weeks old, even more preferably not older than 3 weeks old, and most preferably not older than 2 weeks old. Alternatively, preferably, the immunogenic compositions of the present invention are administered for at least 2 and preferably for at least 3 weeks of exposure to virulent PVC.

[0014] In addition, the present disclosure also relates to a method of treating generalized lymphadenopathy in combination with one or more of the following symptoms in pigs: (1) interstitial pneumonia with swelling of interlobular gaps, (2) pallor of the skin or jaundice, (3) spotty atrophic cirrhosis, (4) stomach ulcers, (5) nephritis and (6) reproductive disorders, e.g. abortion, delivery dead fetus, fetal mummification etc., said method comprising administering an immunogenic composition comprising PCV2 antigen, preferably recombinant PCV2 antigen and more preferably PCV2 ORF2 protein provided herein. Preferably, the present disclosure also relates to a method of treating generalized lymphadenopathy in combination with one or more of the following symptoms associated with PCV2 infection in pigs: (1) interstitial pneumonia with swelling of interlobular gaps, (2) pallor of the skin or jaundice, (3) spotty atrophic cirrhosis, (4) stomach ulcers, (5) nephritis and (6) reproductive disorders, e.g. abortion, delivery dead fetus, fetal mummification etc., said method comprising administering to the pig an immunogenic composition comprising the PCV2 antigen, preferably a recombinant PCV2 antigen and more preferably the PCV2 ORF2 protein provided herein. Preferably, said treatment results in a reduction or reduction of the generalized lymphadenopathy and one or more of the following symptoms associated with PCV2 infection in pigs: (1) interstitial pneumonia with swelling of interlobular fissures, (2) pallor of the skin or jaundice, (3) atrophic spot cirrhosis, (4) gastric ulcers, (5) nephritis and (6) reproductive disorders, for example abortion, stillbirth, fetal mummification, etc. According to an additional aspect, said methods of treatment further comprise administering an immunogenic composition comprising PCV2 antigen, preferably recombinant PCV2 antigen and more preferably PCV2 ORF2 protein, provided herein, piglets not older than 15 weeks old, more preferably not older than 6 weeks old, more preferably not older than 3 weeks, and most preferably not older than 2 weeks. Alternatively, preferably, the immunogenic compositions of the present invention are administered for at least 2 and preferably for at least 3 weeks exposure to virulent PVC.

[0015] It has also surprisingly been found that the therapeutic use of an immunogenic composition comprising PCV antigen, preferably a recombinant PCV2 antigen and more preferably the PCV2 ORF2 protein provided herein, can also limit or reduce lesions similar to postoperative inflammatory atrophy, which is known to be usually associated with with Lawsonia intracellularis infection (ileitis).

[0016] Thus, one aspect of the present disclosure relates to the use of an immunogenic composition comprising PCV2 antigen, preferably a recombinant PCV2 antigen and more preferably the PCV2 ORF2 protein provided herein, for the preparation of a medicament for preventing, limiting the severity and / or reducing lesions similar to postoperative inflammatory atrophy. which is known to be usually associated with Lawsonia intracellularis infection in pigs. According to an additional aspect, said medicament is effective to prevent, reduce the severity and / or reduce lesions similar to postoperative inflammatory atrophy, which is known to be associated with Lawsonia intracellularis infection when administered to piglets not older than 15 weeks, more preferably not older than 6 weeks, even more preferably not older than 3 weeks, and most preferably not older than 2 weeks. Alternatively, preferably, the immunogenic compositions of the present invention are administered for at least 2 and preferably for at least 3 weeks of exposure to virulent PVC.

[0017] In addition, the present disclosure also relates to methods of treating lesions similar to postoperative inflammatory atrophy, which is known to be usually associated with Lawsonia intracellularis infection, said method comprising administering to the pig an immunogenic composition comprising PCV2 antigen, preferably recombinant PCV2 antigen and more preferably the PCV2 ORF2 protein provided herein. Preferably, said treatment results in a reduction or reduction in lesions similar to postoperative inflammatory atrophy, which is known to be associated usually with Lawsonia intracellularis infection. According to an additional aspect, the methods of treatment described above further comprise administering an immunogenic composition comprising PCV2 antigen, preferably recombinant PCV2 antigen, and more preferably PCV2 ORF2 protein provided herein, piglets not older than 15 weeks old, more preferably not older than 6 weeks old, even more preferably not older than 3 weeks, and most preferably not older than 2 weeks. Alternatively, preferably, the immunogenic compositions of the present invention are administered within at least 2 and preferably within at least 3 weeks of exposure to virulent PVC.

Immunogenic composition [0018] The immunogenic composition used in the present invention is effective in inducing an immune response against PCV2 and preventing, reducing and / or reducing the severity of clinical symptoms associated with PCV2 infection. The composition generally contains at least one PCV2 antigen.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The term "immunogenic composition" as used in the present invention refers to any antigen-containing composition

PCV2, wherein the composition can be used to prevent or treat a disease or condition associated with PCV2 infection in a patient. A preferred immunogenic composition may induce, stimulate or enhance an immune response against PCV2. The term thus includes both subunit immunogenic compositions as described below, as well as compositions containing all killed, or attenuated and / or inactivated PCV2.

[0020] The term "subunit immunogenic composition" as used in the present invention refers to a composition comprising at least one immunogenic polypeptide or antigen, but not all antigens derived from or homologous to the PCV2 antigen. Such compositions are essentially free of intact PCV2. Thus, the "subunit immunogenic composition" is obtained from at least partially purified or fractionated (preferably substantially purified) immunogenic PCV2 polypeptides, or recombinant analogs thereof. The subunit immunogenic composition may contain the subunit antigen or antigens of interest substantially free of other PCV2 antigens or polypeptides, or in fractionated form. A preferred immunogenic subunit composition comprises the PCV2 ORF2 protein described below.

[0021] "Immune or immune response" to a composition or vaccine means developing in the host a cellular immune response and / or antigen mediated response to the composition or vaccine of interest. Typically, an "immune response" includes, but is not limited to, one or more of the following effects: production or activation of antibodies, B-lymphocytes, helper T-lymphocytes, suppressor T-lymphocytes, and / or cytotoxic T-lymphocytes and / or T-lymphocytes directed specifically against the antigen or antigens contained in the composition or vaccine of interest. Preferably, the host will show either a therapeutic or protective immune response such that immunity to a new infection will be enhanced and / or the clinical severity of the disease reduced. Such protection can be demonstrated by reducing the number or severity, or the absence of one or more symptoms associated with PCV2 infections as described above.

[0022] The terms "immunogenic" protein or polypeptide or "antigen" as used in the present invention refer to the amino acid sequence that elicits the immune response described above. The "immunogenic" protein or polypeptide used in the present invention includes the full-length sequence of all PCV2 proteins, their analogs, or immunogenic fragments thereof. The term "immunogenic fragment" refers to a protein fragment comprising one or more epitopes and thus elicits the immune response described above. Such fragments can be identified using a variety of epitope mapping techniques known in the art. See, for example, Epitope

Mapping Protocols in Methods in Molecular Biology, vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes can be determined, for example, while synthesizing a large number of peptides on solid supports, peptides corresponding to parts of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the carriers. Such techniques are known in the art and described in, for example, US Patent No. 4,708871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3998-4002; Geysen et al. (1986) Molec. Immunol. 23: 709-715. Similarly, conformational epitopes are easily identified by determining the spatial conformation of amino acids, for example by X-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, above.

[0023] Also included are synthetic antigens, for example, polyepitopes, flanking epitopes, and other antigens obtained recombinantly or synthetically. See, for example, Bergmann et al. (1993) Eur. J. Immunol. 23: 2777-2781; Bergmann et al. (1996), J. Immunol. 157: 3242-3249; Suhrbier, A. (1997), Immunol. and Cell Biol. 75: 402-408; Gardner et al. (1998) 12th World AIDS Conference, Geneva, Switzerland, June 28 - July 3, 1998.

[0024] In a preferred embodiment of the present invention, an immunogenic composition is provided that induces an immune response and confers protective immunity against clinical signs of PCV2 infection. The composition comprises a polypeptide, or a fragment thereof, PCV2 ORF2 expressed as an antigenic component of the composition. The PCV2 DNA and ORF-2 protein used in the present invention for making compositions and the processes provided in the present invention is a highly conserved domain within PCV2 isolates, and thus, any PCV2 ORF-2 would be effective as a source of DNA and / or ORF polypeptide -2 PCV2 used in this document. A preferred PCV2 ORF-2 protein is a protein having the sequence SEQ ID NO: 11. A preferred PCV ORF-2 polypeptide is provided as SEQ ID NO: 5. However, it should be obvious to those skilled in the art that said sequence can vary by as much as 6-10% in terms of sequence homology and still retain antigenic properties that make it useful in immunogenic compositions. The antigenic characterization of the immune composition can, for example, be determined in an experiment to evaluate the immunity described in Example 4. In addition, the antigenic characteristics of the modified antigen are still maintained when the modified antigen confers at least 70%, preferably 80%, more preferably 90% protective immunity to the PCV2 ORF-2 protein encoded by the polynucleotide sequence SEQ ID NO: 3 or SEQ ID NO : 4. As used herein, the term "immunogenic composition" means a PCV2 ORF2 protein that elicits an "immune response" in host cells and / or an immune response mediated by an antibody to the PCV2 ORF2 protein.

Preferably, the immunogenic composition is capable of eliciting or enhancing an immune response against PCV2 thereby giving protective immunity to PCV2 infection and causing a reduction in the incidence, severity or prevention of one or more, and preferably all, clinical symptoms associated therewith.

[0025] In some embodiments, the immunogenic portions of the PCV2 ORF-2 protein are used as the antigenic component of the composition. The term "immunogenic portion" as used herein refers to the truncated and / or substituted form or fragments of PCV2 ORF-2 protein and / or polynucleotide, respectively. Preferably, such truncated and / or substituted forms, or fragments, will contain at least 6 adjacent amino acids from the full-length ORF-2 polypeptide. More preferably, the truncated or substituted forms or fragments will contain at least 5, preferably 8, more preferably 10, more preferably at least 15, and even more preferably at least 19 adjacent amino acids from the full-length PCV ORF-2 polypeptide. Two preferred sequences in this range are provided as SEQ ID NO: 9 and SEQ ID NO: 10. It is further understood that such sequences may form part of larger fragments and truncated forms.

[0026] Another preferred PCV2 ORF-2 polypeptide provided herein is encoded by the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4. However, it should be obvious to those skilled in the art that the sequence may exhibit differences of up to 6- 20% in sequence homology and still retain the antigenic properties that make it useful in immunogenic compositions. In some embodiments, truncated or substituted forms or fragments of said PCV2 ORF-2 polypeptide are used as the antigenic component in the composition. Preferably, such truncated or substituted forms or fragments contain at least 18 contiguous nucleotides from the full-length ORF-2 nucleotide sequence, e.g., SEQ ID NO: 3 or SEQ ID NO: 4. More preferably, the truncated or substituted forms or fragments will contain at least 30, more preferably at least 45, and even more preferably at least 57 adjacent nucleotides from the full-length ORF-2 nucleotide sequence, e.g., SEQ ID NO: 3 or SEQ ID NO: 4 .

[0027] "Sequence identity", as known in the art, refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely, a reference sequence and a given sequence, compared to a reference sequence. Sequence identity is determined by comparing a given sequence with a reference sequence after optimal sequence alignment to obtain the highest degree of sequence similarity, which is determined by matching the sequences of these sequences. After such alignment, sequence identity is determined based on the alignment of individual positions, eg, the sequences are "identical" at a particular position if the nucleotides and amino acids are identical at that position. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to obtain% sequence identity. Sequence identity can be easily calculated using known methods, including but not limited to those described in Computational Molecular Biology, Lesk, ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, DW . ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, ed., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., edited by M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math, 48: 1073 (1988 ), the disclosure of which is incorporated herein by reference. Preferred methods for determining sequence identity are intended to provide the greatest alignment of test sequences. Methods for determining sequence identity are contained in publicly available computer programs that determine the identity of a given sequence. Examples of such programs, but not limited to, include the GCG program package (Devereux, J. et al., Nucleic Acids Research, 12 (1): 387 (1984)), BLASTP, BLASTN and FASTA (Altschul, et al., J. Molec. Biol., 215: 403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (Manual BLAST, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, SF et al., J. Molec. Biol., 215: 403-410 ( 1990), the disclosure of which is incorporated herein by reference). These programs optimally match sequences using the default gap weights to obtain the highest degree of sequence identity between data and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence of at least, for example, 85%, preferably 90%, even more preferably 95% "sequence identity" with the reference nucleotide sequence, it should be understood that the nucleotide sequence of a given polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 15, preferably up to 10, even more preferably up to 5 point mutations for every 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence of at least 85%, preferably 90%, more preferably 95% identity to the reference nucleotide sequence, up to 15%, preferably 10%, even more preferably 5% of the nucleotide in the reference sequence can be subjected to deletion or substitution with another nucleotide, or several nucleotides up to 15%, preferably 10%, even more preferably, 5% of all nucleotides in the reference sequence can be inserted into the reference sequence. These reference sequence mutations may occur at the 5 'or 3' terminal positions of the nucleotide reference sequence or anywhere between these end positions, scattered singly among the residues in the reference sequence or in one or more adjacent groups within the reference sequence. Similarly, a polypeptide having a given amino acid sequence with at least, e.g., 85%, preferably 90%, more preferably 95% sequence identity with a reference amino acid sequence is understood to mean that the given amino acid sequence of the polypeptide is identical to the reference sequence, except that a given polypeptide sequence may contain up to 15, preferably up to 10, even more preferably up to 5 amino acid modifications for every 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence of at least 85%, preferably 90%, even more preferably 95% sequence identity with a reference amino acid sequence, up to 15%, preferably up to 10%, even more preferably up to 5% of the amino acid residues in the reference sequence can be deleted or substituted with another amino acid, or several amino acids up to 15%, preferably up to 10%, even more preferably, up to 5% of the total number of amino acid residues in the reference sequence can be inserted into the reference sequence. These reference sequence modifications may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between these end positions, scattered singly among residues in the reference sequence or in one or more adjacent groups within the reference sequence. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match in determining sequence identity.

[0028] "Sequence homology", as used herein, refers to a method for determining the similarity of two sequences. To determine sequence homology, two or more sequences are optimally matched and, if necessary, introduced breaks. However, in contrast to "sequence identity", conservative amino acid substitutions count as matching when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide with 95% sequence homology with a reference sequence, 85%, preferably 90%, even more preferably 95% of the amino acid or nucleotide residues in the reference sequence must match or contain a conservative substitution with another amino acid or nucleotide, or several amino acids or nucleotides constituting up to 15%, preferably up to 10%, even more preferably up to 5% of all amino acid residues or nucleotides, excluding conservative substitutions, in the reference sequence may be inserted into the reference sequence. Preferably, the homologous sequence comprises at least a strand of 50, more preferably at least 100, even more preferably at least 250, and even more preferably at least 500 nucleotides.

[0029] "Conservative substitution" refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide for similar characteristics or properties, including size, hydrophobicity, etc., such that overall functionality does not change significantly.

[0030] "Isolated" means modified by the "human hand" in relation to its natural state, i.e. if it occurs in nature, it is changed and / or removed from its original environment. For example, a polynucleotide or polypeptide naturally occurring in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from co-naturally occurring materials is "isolated," as used herein.

[0031] Thus, the immunogenic composition used in the present invention also relates to a composition comprising PCV2 ORF2 protein, wherein said PCV2 ORF2 protein is any of those described above. Preferably, the PCV2 ORF2 protein is any of the proteins described above. Preferably, said PCV2 ORF-2 protein is

i) a polypeptide comprising the sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11;

ii) any polypeptide that is at least 80% homologous to the i) polypeptide, iii) any immunogenic portion of the i) and / or ii) polypeptides, iv) an immunogenic portion iii) containing at least 10 adjacent amino acids contained in the SEQ sequence ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11;

v) a polypeptide that is encoded by DNA comprising the sequence SEQ ID NO: 3 or SEQ ID NO: 4;

vi) any polypeptide that is encoded by a polynucleotide that is at least 80% homologous to the polynucleotide v), vii) any immunogenic portion of the polypeptides encoded by the polynucleotide v) and / or vi), viii) the immunogenic portion vii), where the polynucleotide encoding said immunogenic portion consists of at least 30 contiguous nucleotides contained in the sequence SEQ ID NO: 3 or SEQ ID NO: 4.

[0032] Preferably, each of these immunogenic portions has the immunogenic properties of the PCV2 ORF-2 protein, which is encoded by SEQ ID NO: 3 or SEQ ID NO: 4.

[0033] According to an additional aspect, the PCV2 ORF2 protein is provided in the immune composition at an antigen incorporation level effective to induce the desired immune response, namely, reducing the incidence, reducing the severity, or preventing one or more clinical signs resulting from PCV2 infection. Preferably, the level of PCV2 ORF-2 protein is at least 0.2 μg antigen / ml final immunogenic composition (g / ml), more preferably from about 0.2 to about 400 μg / ml, even more preferably from about 0.3 to about 200 μg / ml, even more preferably from about 0.35 to about 100 μg / ml, even more preferably from about 0.4 to about 50 μg / ml, even more preferably from about 0.45 to about 30 μg / ml, even more preferably from about 0.6 to about 15 μg / ml, even more preferably from about 0.75 to about 8 μg / ml, even more preferably from about 1.0 to about 6 μg / ml, even more preferably from about 1.3 to about 3.0 μg / ml, even more preferably from about 1.4 to about 2.5 μg / ml, even more preferably from about 1.5 to about 2.0 μg / ml, and most preferably about 1.6 μg / ml.

[0034] According to a further aspect, the level of ORF-2 antigen is at least 0.2 μg / PCV2 ORF-2 protein, as described above, per final antigen composition dose (g / dose), more preferably from about 0.2 to about 400 μg / dose, even more preferably from about 0.3 to about 200 μg / dose, even more preferably from about 0.35 to about 100 μg / dose, even more preferably from about 0.4 to about 50 μg / dose, even more preferably from about 0.45 to about 30 μg / dose, even more preferably from about 0.6 to about 15 μg / dose, even more preferably from about 0.75 to about 8 μg / dose, even more preferably from about 1.0 to about 6 μg / dose, even more preferably from about 1.3 to about 3.0 μg / dose, even more preferably from about 1, 4 to about 2.5 μg / dose, even more preferably from about 1.5 to about 2.0 μg / dose, and most preferably about 1.6 μg / dose.

[0035] The PCV2 ORF-2 polypeptide used in the immunogenic composition of the present invention can be obtained by any recombinant technique. Preferred methods for obtaining the PCV2 ORF-2 polypeptide are described herein and are also provided in US Patent Application No. 11/034 797. Briefly, sensitive cells are infected with a recombinant viral vector containing PCV2 ORF-2 DNA coding sequences, the PCV2 ORF-2 polypeptide is expressed by the recombinant virus, and the PCV2 ORF-2 polypeptide after expression is recovered from the supernatant by filtration and inactivated in any conventional manner , preferably with binary ethyleneimine and then neutralized to stop the inactivation process.

[0036] The immunogenic composition as used herein also refers to a composition comprising i) any PCV2 ORF-2 protein described above, preferably at the concentrations described above, and ii) at least a portion of a viral vector expressing said PCV2 ORF-2 protein , preferably a recombinant baculovirus. In addition, the immunogenic composition may comprise i) any PCV2 ORF-2 protein described above, preferably at the concentrations described above, ii) at least a portion of a viral vector expressing said PCV2 ORF-2 protein, preferably a recombinant baculovirus, and iii) a portion of the cell culture supernatant.

[0037] The immunogenic composition as used herein also refers to a composition comprising i) any PCV2 ORF-2 protein described above, preferably at the concentrations described above, ii) at least a portion of the viral vector expressing said PCV2 ORF-2 protein, preferably recombinant baculovirus and iii) a part of cell culture where about 90% of the components have a size smaller than 1 μιτι.

[0038] The immunogenic composition as used herein also refers to a composition comprising i) any PCV2 ORF-2 protein described above, preferably at the concentrations described above, ii) at least a portion of a viral vector expressing said PCV2 ORF-2 protein, iii) a part of the cell culture, iv) an inactivating agent for inactivating the recombinant vector, preferably

BEI, where about 90% of components i) to iii) are smaller than 1 μιτι. Preferably, the BEI is present in concentrations effective to inactivate the baculovirus. Effective concentrations are described above.

[0039] The immunogenic composition as used herein also refers to a composition comprising i) any PCV2 ORF-2 protein described above, preferably at the concentrations described above, ii) at least a portion of a viral vector expressing said PCV2 ORF-2 protein, iii) part of the cell culture, iv) an inactivating agent to inactivate the recombinant viral vector, preferably BEI iv) a neutralizing agent to stop inactivation, mediated by an inactivating agent, where about 90% of the components i) to iii) are smaller than 1 μιτ. Preferably, the inactivating agent is BEI, the composition contains sodium thiosulfate in an equivalent amount of BEI.

[0040] The polypeptide is incorporated into a composition that can be administered to an animal susceptible to PCV2 infection. In preferred embodiments, the composition may also contain additional ingredients known to those skilled in the art (see also Remington's Pharmaceutical Sciences. (1990), 18th edition, Mack Publ., Easton). In addition, the composition may contain one or more veterinarily acceptable carriers. As used herein, the term "veterinary acceptable carrier" includes all and all solvents, dispersion media, coatings, auxiliaries, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption retarding agents and the like. In a preferred embodiment, the immunogenic composition comprises the PCV2 ORF-2 protein provided in the present invention, preferably at the concentrations described above, mixed with an adjuvant, preferably Carbopol, and physiological saline.

[0041] It should be obvious to those skilled in the art that the composition used herein may contain physiologically acceptable sterile injectable solutions. Isotonic aqueous solutions, such as, for example, physiological saline solutions or corresponding plasma protein solutions, are easily available for the preparation of a ready-to-use solution for injection or parenteral infusion. In addition, the immunogenic compositions and vaccines of the present invention may contain diluents, isotonic agents, stabilizers or auxiliaries. Diluents may include water, saline, dextrose, ethanol, glycerol and the like. Isotonic agents may include, but are not limited to, sodium chloride, dextrose, mannitol, sorbitol and lactose. Stabilizers include, but are not limited to, albumin and alkali salts of ethylenediaminetetraacetic acid.

[0042] "Adjuvants" as used herein may include aluminum hydroxide, aluminum phosphate, saponins e.g. Quil A, QS-21 (Cambridge Biotech Inc, Cambridge MA) and GPI-0100 (Galenica Pharmaceuticals, Inc, Birmingham , AL), water-in-oil emulsion, oil-in-water emulsion. The emulsion may be based on light paraffin oil (European pharmacopoeia type); isoprenoid oil such as squalene or squalene oil resulting from oligomerization of alkenes, in particular isobutene or decene; esters of acids or alcohols containing a linear alkyl group, in particular vegetable oils, ethyl oleate, propylene glycol di (caprylate / caprate), glycerol tri (caprylate / caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form an emulsion. Emulsifiers are preferably nonionic surfactants, in particular sorbitan, manniide esters (e.g. anhydromannitol oleate), glycol, polyglycerol, propylene glycol and oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene block copolymers, in particular Pluronic products, especially L121. See. Hunter et al., The Theory and Practical Application of Adjuvants (ed. Stewart-Tull, DES). JohnWiley and Sons, NY, pp. 5194 (1995) and Todd et al. Vaccine 15: 564-570 (1997).

[0043] For example, it is possible to use the SPT emulsion described on page 147 "Vaccine Design, The Subunit and Adjuvant Approach" edited by M. Powell and M. Newman, Plenum Press 1995, and the MF59 emulsion is described on page 183 same book.

[0044] Another example of an adjuvant is a compound selected from polymers of acrylic or methacrylic acid and copolymers of maleic anhydride and alkenyl derivative. Preferred adjuvants are polymers of acrylic or methacrylic acid that are crosslinked, especially with the use of polyalkenyl ethers of sugars or polyalcohols. These compounds are known as carbomers (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art may also refer to US Patent Document No. 2,909,462, where such acrylic polymers crosslinked with polyhydroxylated compounds with at least 3 hydroxyl groups, preferably not more than 8, are described, where the hydrogen atoms of at least three hydroxyl groups have been replaced by unsaturated aliphatic radicals containing at least 2 carbon atoms. Preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. Products sold under the name Carbopol are particularly suitable; (BF Goodrich, Ohio, United States). They are cross-linked with allyl sucrose or allylpentaerythritol. These include Carbopol 974P, 934P and 971P. The most preferred is the use of Carbopol, in particular the use of Carbopol 971P, preferably in an amount from about 500 μg to about 5 mg per dose, more preferably in an amount from about 750 μg to about 2.5 mg per dose, and most preferably in an amount of about 1 mg per dose.

[0045] Other suitable adjuvants include, but are not limited to, RIBI adjuvant system (Ribi Inc), block copolymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid amine adjuvant for high temperature E. coli enterotoxins (recombinant or other), cholera toxin, IMS 1314 or muramyl dipeptide among many others.

[0046] Preferably, the adjuvant is added in an amount of about 100 Pg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 100 μg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 500 μg to about 5 mg per dose. Even more preferably, the adjuvant is added in an amount of about 750 μg to about 2.5 mg per dose. Most preferably, the adjuvant is added in an amount of about 1 mg per dose.

[0047] In addition, the composition may contain one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" includes all and all solvents, dispersion agents, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption retarding agents and the like. Most preferably, the composition provided in the present invention comprises PCV2 ORF-2 protein recovered from a cell culture supernatant in vitro, where the cells are infected with a recombinant viral vector containing PCV2 ORF-2 DNA and expressing PCV2 ORF-2 protein and wherein said cell culture has been exposed to about 2 up to about 8 mM BEI, preferably about 5 mM BEI to inactivate the viral vector, and an equivalent concentration of neutralizing agent, preferably a sodium thiosulfate solution to a final concentration of about 2 to about 8 mM, preferably about 5 mM.

[0048] The present invention also relates to an immunogenic composition which comprises i) any of the PCV2 ORF-2 proteins described above, preferably in the concentrations described above, ii) at least part of the viral vector expressing said PCV2 ORF-2 protein, iii) part of the cell culture , iv) an inactivating agent for inactivating the recombinant viral vector, preferably BEI, iv) a neutralizing agent for stopping inactivation mediated by the inactivating agent, preferably sodium thiosulfate in amounts equivalent to BEI; and vi) a suitable adjuvant, preferably Carbopol 971 in the amounts described above; where about 90% of the components i) to iii) have a size smaller than 1 μιτι. According to a further aspect, said immunogenic composition further comprises a pharmaceutically acceptable salt, preferably phosphate, in physiologically acceptable concentrations. Preferably, the pH of said immunogenic composition is adjusted to physiological pH, i.e. between about 6.5 and 7.5.

[0049] The immunogenic composition as used herein also relates to a composition containing per ml i) at least 1.6 μg protein

PCV2 ORF-2 described above, ii) at least part of baculovirus expressing said PCV2 ORF-2 protein, iii) part of cell culture, iv) about 2 to 8 mM BEI, v) sodium thiosulfate in equivalent amounts of BEI and vi) 1 mg Carbopol 971 and vii) phosphate salt at physiologically acceptable concentration, where about 90% of the components i) to iii) are smaller than 1 μm and the pH of said immunogenic composition is adjusted to a value in the range of about 6.5 to 7.5.

[0050] Immunogenic compositions may further comprise one or more other immunomodulatory agents, such as, for example, interleukins, interferons or other cytokines. Immunogenic compositions may also contain gentamicin and Merthiolate. While the amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by one of ordinary skill in the art, compositions comprising from about 50 to about 2000 μg of adjuvant and preferably about 250 μg / ml dose of vaccine composition are contemplated in the present invention. In this regard, an immunogenic composition as used herein refers to a composition containing from about 1 μg / ml to about 60 μg / ml antibiotics, and more preferably less than about 30 μg / ml antibiotics.

[0051] The immunogenic composition as used herein refers to a composition comprising i) any of the PCV2 ORF-2 proteins described above, preferably at the concentrations described above, ii) at least a portion of a viral vector expressing said PCV2 ORF-2 protein, iii) a portion cell culture, iv) an inactivating agent to inactivate the recombinant viral vector, preferably BEI, and v) a neutralizing agent to stop inactivation, mediated by an inactivating agent, preferably sodium thiosulfate in amounts equivalent to BEI; vi) a suitable adjuvant, preferably Carbopol 971 in the amounts described above; vii) a pharmaceutically acceptable concentration of a saline-based buffer, preferably a phosphate salt, and viii) an antimicrobial active agent; where about 90% of the components i) to iii) have a size smaller than 1 μιτι.

[0052] It has surprisingly been found that the immunogenic composition containing the PCV2 ORF2 protein is very stable for a period of 24 months. Immunogenic compositions have also been found to be very effective in reducing the clinical symptoms associated with PCV2 infection. It has also been discovered that immunogenic compositions containing recombinant PCV2 ORF2 protein obtained from baculovirus expression described above are surprisingly more effective than immunogenic compositions containing whole PCV2 virus inactivated or isolated PCV2 ORF2 antigen. In particular, it was surprisingly found that the recombinant baculovirus expressed PCV2 ORF2 protein is effective at very low concentrations, i.e. concentrations up to 0.25 μg / dose. This unexpected high immunogenic potential of the PCV2 ORF2 protein is increased by Carbopol. Examples 1 to 3 disclose in detail the preparation of immunogenic compositions containing PCV2 ORF2.

[0053] The immunogenic composition used in the present invention also relates to Ingelvac® CircoFLEX ™ products, (Boehringer Ingelheim Vetmedica, Inc., St Joseph, MO, United States), CircoVac® (Merial SAS, Lyon, France), CircoVent (Intervet Inc. , Millsboro, DE, United States) or Suvaxyn PCV-2 One Dose® (Fort Dodge Animal Health, Kansas City, KA, United States).

Administration of the immunogenic composition [0054] The composition of the invention may be used transdermally, intratracheally or vaginally. Preferably the composition may be used intramuscularly or intranasally, most preferably intramuscularly. In the animal's body, it may be beneficial to use the pharmaceutical composition as described above by intravenous or direct injection into the target tissue. For systemic use, intravenous, intravascular, intramuscular, intranasal, intraarterial, intraperitoneal, oral or intrathecal administration is preferred. More local administration may be subcutaneous, intradermal, intracardiac, lung lobes, spinal, pulmonary or directly on or near the tissue being treated (connective, bone, muscle, nervous, epithelial tissue). Depending on the desired duration and effectiveness of the treatment, the compositions of the invention may be administered once or several times, also intermittently, e.g. daily for several days, weeks or months, and at different dosages.

[0055] Preferably, at least one dose of the immunogenic compositions described above is administered intramuscularly to a patient in need thereof. According to a further aspect, the PCV-2 antigen or immunogenic composition containing such PCV-2 antigen as described above is prepared and administered in one (1) ml per dose. Thus, in a further aspect, the present invention also relates to 1 ml of an immunogenic composition comprising PCV-2 antigen as described herein for limiting or reducing generalized lymphadenopathy, lymphoid tissue atrophy and / or multinucleated / giant histiocytes in PCV2 infected pigs.

[0056] According to an additional aspect, the present disclosure also relates to 1 ml of an immunogenic composition comprising the PCV-2 antigen described herein in limiting or reducing the generalized lymph node enlargement in combination with one or more of the following symptoms in pigs: (1) interstitial pneumonia with swelling of interlobular gaps, (2) pallor of the skin or jaundice, (3) spotty atrophic cirrhosis, (4) stomach ulcers, (5) nephritis and (6) reproductive disorders, e.g. abortion, childbirth dead fetus, fetal mummification.

[0057] According to an additional aspect, at least one additional administration of at least one dose of the immunogenic composition described above is for a patient in need thereof, wherein the second or any subsequent administration is performed at least 14 days after the initial or any previous administration. Preferably, the immunogenic composition is administered with an immune system stimulator. Preferably, said immune stimulator is administered at least twice. Preferably, the interval between the first and second or each subsequent administration of the immune stimulator is at least 3 days, more preferably at least 5 days, even more preferably at least 7 days. Preferably, the immune system stimulator is administered at least 10 days, preferably 15 days, more preferably 20, even more preferably at least 22 days after the initial administration of the immunogenic composition provided herein. A preferred immune stimulator is, for example, KLH hemocyanin, preferably emulsified with Freund's incomplete adjuvant (KLH / ICFA). However, it should be understood that any other immune system stimulator known to those skilled in the art may also be used. The term "immune stimulator" as used herein, means any agent or composition that can elicit an immune response, preferably without initiating or enhancing a specific immune response, such as an immune response against a specific pathogen. In addition, instructions are given for administering the appropriate dose of immune stimulator.

[0058] Furthermore, it has also surprisingly been found that the immunogenic potential of the immunogenic compositions used in the present invention, preferably those containing recombinant PCV2 ORF2 protein expressed in baculovirus, even more preferably in combination with Carbopol, can be further confirmed by administration of Ingelvac PRRS MLV vaccine (see Example 5). The clinical and disease symptoms of PCV2 are significantly greater if PRRS infection is present. However, the immunogenic compositions and vaccination strategies envisaged herein reduce this effect more than expected. In other words, an unexpected synergistic effect has been observed in animals, preferably pigs, treated with the immunogenic PCV2 ORF2 composition, according to this document, and the Ingelvac PRRS MLV vaccine (Boehringer Ingelheim).

BRIEF DESCRIPTION OF THE FIGURES [0059] Figure 1 is a block diagram of a preferred construction of recombinant PCV2 ORF2 baculovirus; and Figures 2a and 2b each show a flowchart of a method for making one of the compositions for use in accordance with the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS [0060] The following examples describe preferred materials and procedures of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods, devices and materials have been described. It should be understood, however, that these examples are for illustrative purposes only and should not be considered as limiting the general scope of the invention in any way.

EXAMPLE 1 [0061] This example compares the relative yields of ORF2 using the methods described herein with the yields of methods known in the art. In each of four 1000 ml flasks with a vane spinner flask, approximately 1.0 x 10 was sown.⁶ cells / ml Sf + cells in 300 ml of serum-free insect medium, Excell 420 (JRH Biosciences, Inc., Lenexa, KS). Stem cell culture is identified as SF + (Spodoptera frugiperda) stem cell culture Master Cell Stock, passage 19, Lot # N112-095W. Cells used to generate SF + stem cells were obtained from Protein Sciences Corporation, Inc., Meriden, CT. The SF + cell line for this example was limited to passages between 19 and 59. Other passages will be used for the purposes of the present invention, but to increase the scale of the process for large-scale production, at least 19 passages will be required and passages beyond 59 may affect expression, although this has not been studied. More specifically, the initial SF + cultures stored in liquid nitrogen were grown in Excell 420 medium (JRH Biosciences, Inc., Lenexa, KS) as a suspension in sterile culture flasks with a paddle mixing system, with continuous mixing. The cultures were grown in 100 to 250-ml culture flasks with a paddle mixing system with 25 to 150 ml Excell 420 without serum. When these cells were expanded to a density of 1.0-8.0 x 10⁶ cells / ml, they were separated into new vessels with a density of 0.5-1.5 x 10⁶ cells / ml. Further expansion cultures were grown in culture flasks with a paddle mixing system with a capacity of up to 36 liters or in stainless steel bioreactors with a capacity of up to 300 liters for a period of 2-7 days, at a temperature of 25-29 ° C.

[0062] After inoculation, the flasks were incubated at 27 ° C for four hours. Then, each flask was inoculated with a recombinant baculovirus containing the PCV2 ORF-2 gene (SEQ ID NO: 4). A recombinant baculovirus containing the PCV2 ORF-2 gene was generated as follows: The PCV2 ORF2 gene from the North American PCV2 strain was PCR amplified to incorporate Kozak 5 'sequence (SEQ ID NO: 1) and 3' EcoR1 site (SEQ ID NO: 2), cloned into vector pGEM-T-Easy (Promega, Madison, WI). It was then excised and subcloned into the transfer vector pVL1392 (BD Biosciences Pharmingen, San Diego, CA). The subcloned portion is shown here as SEQ ID NO: 7. Plasmid pVL1392 containing the PCV2 ORF2 gene was designated N47-064Y and then co-transfected with BaculoGold baculovirus DNA^® (BD Biosciences Pharmingen) to Sf + insect cells (Protein Sciences, Meriden, CT) to produce a recombinant baculovirus containing the PCV2 ORF2 gene. The new construct is included herein as SEQ ID NO: 8. The recombinant baculovirus containing the PCV2 ORF2 gene was purified on plates, and the Master Seed Virus (MSV) was propagated on the SF + cell line, aliquoted and stored at -70 ° C. By PCR-RFLP using baculovirus specific primers, MSV was positively identified as PCV2 ORF2 baculovirus. Insect cells were infected with PCV2 ORF2 baculovirus to generate MSV or Working Seed Virus expressing PCV2 ORF2 antigen as detected by polyclonal serum or monoclonal antibodies in the indirect immunofluorescence assay. Furthermore, the identity of PCV2 ORF2 baculovirus was confirmed by sequencing of the N-terminal amino acids. MSV ORF2 PCV2 baculovirus was also tested for purity according to 9 CFR 113.27 (c), 113.28 and 113.55. Each recombinant baculovirus inoculated in culture flasks with a paddle mixing system had differentiated multiples of infection (MOI). Flask 1 was inoculated with 7.52 ml seed with 0.088 MOI; flask 2 was inoculated with 3.01 ml seed with 0.36 MOI; flask 3 was inoculated with 1.5 ml seed with 0.18 MOI; and flask 4 was inoculated with 0.75 ml seed with 0.09 MOI. A schematic diagram illustrating the basic steps used to construct the recombinant PCV2 ORF2 baculovirus is shown herein as Figure 1.

[0063] After inoculation with baculovirus, the flasks were incubated at 27 ± 2 ° C for 7 days, and also mixed at 100 rpm during this time. The flasks were fitted with vent plugs to allow air flow. Every 24 hours during the next 7 days, samples were taken from each flask. After extraction, each sample was centrifuged and both the pellet and the supernatant were separated and microfiltered through a 0.45-1.0 μίΓ membrane [0064] The resulting samples then have an amount of ORF2 quantified by ELISA. An ELISA was carried out with pig anti-PCV2 Pab IgG Prot. G purified capture antibodies (diluted 1: 250 in PBS) diluted to 1: 6000 in 0.05 M carbonate buffer (pH 9.6). 100 μΐ of antibody was then placed in wells of the microtiter plate, sealed and incubated overnight at 37 ° C. The plates were then washed three times with a washing solution that contained 0.5 ml Tween 20 (Sigma, St. Louis, MO), 100 ml 10X D-PBS (Gibco Invitrogen, Carlsbad, CA) and 899.5 ml distilled water. Then 250 μΐ blocking solution (5 g skimmed Carnation milk (Nestle, Glendale, CA) in 10 ml D-PBS QS supplemented with 100 ml distilled water) was added to each well. The next step was washing the test plate followed by the addition of pre-diluted antigen. Pre-diluted antigen is prepared by adding 200 μl diluent solution (0.5 ml Tween 20 in 999.5 ml D-PBS) to each well in a dilution plate. The sample was then diluted in a ratio of 1: 240 and 1: 480, and 100 μl of each of these diluted samples was added to one of the upper wells on the dilution plate (i.e. 100 μl of 1: 240 diluted solution was added to one upper well and the other 100 μl 1: 480 diluted solution). Then serial dilutions were made for the remainder of the plate by removing 100 μΐ from each subsequent well and transferring to the next well on the plate. The contents of each well were mixed well before each transfer. Washing of the test plate included washing the plate three times with wash buffer.

The plates were then sealed and incubated for one hour at 37 ° C, followed by three washes with wash buffer. The detection antibodies used were monoclonal antibodies against PCV ORF2. They were diluted 1: 300 in diluent solution, and 100 μΐ of the diluted detection antibody was then added to the wells. The plates were then sealed and incubated for one hour at 37 ° C, followed by three washes with wash buffer. The conjugate diluent was then obtained by adding normal rabbit serum (Jackson ImmunoResearch, West Grove, PA) to the diluent solution to obtain a 1% concentration. Goat anti-mouse (H + 1) -HRP antibody conjugate (Jackson Immunoresearch) was diluted in conjugate diluent to 1: 10,000. 100 μL of diluted antibody conjugate was added to each well. The plates were then sealed and incubated for 45 minutes at 37 ° C, followed by three washes with wash buffer. 100 μl of substrate (TMB peroxidase substrate, Kirkgaard and Perry Laboratories (KPL), Gaithersberg, MD) mixed with an equal volume of peroxidase B substrate (KPL) was added to each well. The plate was incubated at room temperature for 15 minutes.

Then 100 Pl of 1N HCl solution was added to all wells to stop the reaction. The plate was then subjected to the ELISA test by starting the reader. The results of this test are given in Table 1 below:

Table 1

<td>Day</td><td>Flask</td><td>ORF2 in pellets ^ g)</td><td>ORF2 in the supernatant ^ g)</td>

<td> 3</td><td> 1</td><td> 47,53</td><td> 12</td>

<td> 3</td><td> 2</td><td> 57,46</td><td> 22</td>

<td> 3</td><td> 3</td><td> 53,44</td><td> 14</td>

<td> 3</td><td> 4</td><td> 58,64</td><td> 12</td>

<td> 4</td><td> 1</td><td> 43,01</td><td> 44</td>

<td> 4</td><td> 2</td><td> 65,61</td><td> 62</td>

<td> 4</td><td> 3</td><td> 70,56</td><td> 32</td>

<td> 4</td><td> 4</td><td> 64,97</td><td> 24</td>

<td> 5</td><td> 1</td><td> 31,74</td><td> 100</td>

<td> 5</td><td> 2</td><td> 34,93</td><td> 142</td>

<td> 5</td><td> 3</td><td> 47,84</td><td> 90</td>

<td> 5</td><td> 4</td><td> 55,14</td><td> 86</td>

<td> 6</td><td> 1</td><td> 14,7</td><td> 158</td>

<td>Day</td><td>Flask</td><td>ORF2 in pellets ^ g)</td><td>ORF2 in the supernatant ^ g)</td>

<td> 6</td><td> 2</td><td> 18,13</td><td> 182</td>

<td> 6</td><td> 3</td><td> 34,78</td><td> 140</td>

<td> 6</td><td> 4</td><td> 36,88</td><td> 146</td>

<td> 7</td><td> 1</td><td> 6,54</td><td> 176</td>

<td> 7</td><td> 2</td><td> 12,09</td><td> 190</td>

<td> 7</td><td> 3</td><td> 15,84</td><td> 158</td>

<td> 7</td><td> 4</td><td> 15,19</td><td> 152</td>

[0065] These results indicate that when the incubation time is extended, the expression of ORF2 into the supernatant of the centrifuged cells and medium is greater than the expression in the pellets of the centrifuged cells and medium. Accordingly, this method provides an increase in ORF2 yield and a significant improvement over existing methods by allowing the continuation of ORF2 expression for at least 5 days, and recovering it from the supernatant, than allowing the continuation of ORF2 expression for less than 5 days and recovering ORF2 from cells.

EXAMPLE 2 [0066] This example provides data regarding the effectiveness of the invention claimed herein. A 1000 ml paddle culture flask was inoculated with Sf + approximately 1.0x10⁶ cells / ml in 300 ml Excell 420 medium. The flask was incubated at 27 ° C and mixed at 100 rpm. After 24 hours of incubation, the flask was inoculated with 10 ml of PCV2 / Bac p + 6 ORF2 virus (recombinant baculovirus containing the PCV2 ORF2 gene passaged an additional 6 times in Sf9 insect cells) with 0.1 MOI.

[0067] The flask was incubated at 27 ° C for 6 days. After incubation, the flask was centrifuged and three supernatant samples were taken and inactivated. The supernatant was inactivated by bringing its temperature to 37 ± 2 ° C. To the first sample of the supernatant, a 0.4 M solution of 2-bromoethyleneamine hydrobromide was added, which was cyclized in 0.3 N NaOH to obtain 0.2 M binary ethyleneimine (BEI) to a final concentration of 5 mM. 10 mM BEI was added to the second sample of supernatant. BEI was not added to the third supernatant sample. The samples were then mixed continuously for 48 hours. A 1.0 M sodium thiosulfate solution was added to a final minimum concentration of 5 mM to neutralize residual BEI. Then, by the same ELISA procedure as described in Example 1, the amount of ORF2 in each sample was quantified. The results are shown in Table 2 below:

Table 2

<td>A sample</td><td>ORF2 in the supernatant ^ g)</td>

<td> 1</td><td> 78,71</td>

<td>A sample</td><td>ORF2 in the supernatant ^ g)</td>

<td> 2</td><td> 68,75</td>

<td> 3</td><td> 83,33</td>

[0068] This example shows that BEI neutralization does not remove or degrade a significant amount of recombinant PCV2 ORF2 protein. This is evidenced by the fact that there is no large loss of ORF2 in the BEI supernatant or at elevated temperatures. Those skilled in the art will recognize that recovered ORF2 is a stable protein product.

EXAMPLE 3 [0069] This example shows that the present invention is scalable, from small to large scale production of PCV2 ORF2 recombinant. 5.0 x 10 was sown in the 20,000 ml Applikon bioreactor⁵ cells / ml SF + in 7000 ml ExCell 420 medium. The medium and cells were incubated at 27 ° C and mixed at 100 rpm for a further 68 hours. At 68 hours, 41.3ml ORV2 PCV2 MSV + 3 baculovirus was added to 7000 mL ExCell 420 medium. The resulting mixture was then added to the bioreactor. Over the next seven days, the mixture was incubated at 27 ° C and mixed at 100 rpm. Samples from the bioreactor were extracted every 24 hours starting from day 4 after infection, and each sample was centrifuged. Sample supernatants were preserved and then the amount of ORF2 was determined by SDS-PAGE densitometry. The results are shown in Table 3 below:

Table 3

<td>The day after infection</td><td>ORF2 in the supernatant ^ g)</td>

<td> 4</td><td> 29,33</td>

<td> 5</td><td> 41,33</td>

<td> 6</td><td> 31,33</td>

<td> 7</td><td> 60,67</td>

EXAMPLE 4 [0070] The example shows the efficacy studies of seven potential PCV2 vaccines and further determines the performance parameters when exposed to the virulent PCV2 strain. One hundred eight (108) colostrum-free piglets after cesarean section (CDCD), aged 9-14 days, were randomly assigned to 9 equal-sized groups. Table 4 shows the overall study design for this example.

Table 4. General Study Design

<td>Group</td><td>number pigs</td><td>Treatment</td><td>Day treatment</td><td>KLH / ICFA on days 21 and 27</td><td>Provocation virulent PCV2 on day 24</td><td>Section in day 49</td>

<td> 1</td><td> 12</td><td>PCV2 vaccine No. 1- (vORF 16 μg)</td><td> 0</td><td> +</td><td> +</td><td> +</td>

<td> 2</td><td> 12</td><td>PCV2 vaccine No. 2- (vORF 8 μg)</td><td> 0</td><td> +</td><td> +</td><td> +</td>

<td> 3</td><td> 12</td><td>PCV2 vaccine No. 3- (vORF 4 μg)</td><td> 0</td><td> +</td><td> +</td><td> +</td>

<td> 4</td><td> 12</td><td>PCV2 vaccine No. 4- (vORF 16 μg)</td><td> 0</td><td> +</td><td> +</td><td> +</td>

<td> 5</td><td> 12</td><td>PCV2 vaccine No. 5- (vORF 8 μg)</td><td> 0</td><td> +</td><td> +</td><td> +</td>

<td> 6</td><td> 12</td><td>PCV2 vaccine No. 6- (vORF 4 μg)</td><td> 0</td><td> +</td><td> +</td><td> +</td>

<td> 7</td><td> 12</td><td>PCV2 vaccine No. 7- (whole virus cells killed)</td><td> 0</td><td> +</td><td> +</td><td> +</td>

<td> 8</td><td> 12</td><td>None - provoked control group</td><td>ON</td><td> +</td><td> +</td><td> +</td>

<td> 9</td><td> 12</td><td>None - strictly negative control group</td><td>ON</td><td> +</td><td> -</td><td> +</td>

<td colspan="7">vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus showing ORF2 expression; whole virus cells killed = PCV2 virus was cultured in an appropriate cell culture</td>

[0071] Seven groups (groups 1-7) received a dose of PCV2 ORF2 polypeptide, one group served as a challenge control group and did not receive PCV2 ORF2, and the other group served as a strictly negative control group, and also did not receive PCV2 ORF2. On day 0, groups 1 to 7 were treated with the assigned vaccines. This piglet in group 7 was treated with a booster on day 14. Piglets after vaccination were observed for adverse reactions and injection site reactions and on day 19 the piglets were transferred to a second research center. In the second research center, groups 1-8 were placed in one building, while Group 9 was located in a separate building.

All pigs received KLH hemocyanin / incomplete Freund's adjuvant (ICFA) on days 21 and 27, on day 24, groups 1-8 were infected with virulent PCV2.

[0072] Blood samples were collected for PCV2 serological tests before and after challenge. Before challenge, body weight data was collected to determine daily weight gain (ADWG), and clinical symptoms, and nasal swabs were taken to determine nasal secretion for the presence of PCV2. On day 49, all surviving pigs were dissected, lung lesions were assessed, and selected tissues were retained in formalin for immunohistochemistry (IHC) at a later date.

Materials and methods [0073] The study was a partially blind vaccination challenge study conducted on CDCD pigs from 9 to 14 days of age on day 0. The inclusion criterion was PCV2 IFA titre in sows &lt;1000. In addition, the serological status of sows corresponded to that of the PRRS negative herd. The serological status of PCV2 was examined in twenty-eight (28) sows. Fourteen sows (14) had PCV2 titres &lt;1000 and were transferred to the first research center. One hundred ten (110) piglets after caesarean section were included in this study on day -4. On day -3, 108 CDCDs of pigs in the first test center were weighed, pierced, blocked by weight and assigned to 1 of 9 groups as shown in Table 4 above. If any animal meeting the inclusion criteria and included in the study was later excluded from it for any reason, the data obtained from it was subject to consultation by the Investigator and the Monitor of the study to determine their use in the final analysis. The documentation covered data which piglets were excluded and what was the reason for exclusion. Initially, no sows were turned off. A total of 108 of 110 possible pigs were randomly allocated to one of 9 groups on day -3. The two smallest pigs (Nos. 17 and 19) were not assigned to any group and were treated as additional, if necessary. During the study, several animals were removed. Pig No. 82 (Group 9) on Day -1, Pig No. 56 (Group 6) on Day 3, Pig No. 53 (Group 9) on Day 4, Pig No. 28 (Group 8) on Day 8, Pig No. 69 (Group 8) on day 7 and pig No. 93 (group 4) on day 9, found dead before infection. These six pigs were not included in the final analysis of the study. Pig No. 17 (one of the additional pigs) was assigned to group 9. The remaining additional pig No. 19 was excluded from the study.

[0074] The compositions administered to each group were as follows: Group 1 was intended to administer 1 ml of viral ORF2 (vORF2) containing 16 μg ORF2 / ml. This was done by mixing 10.24 ml of viral ORF2 (256 μg / 25 μg / ml = 10.24 ml of vORF2) with 3.2 ml of 0.5% Carbopol and 2.56 ml of phosphate buffered saline at pH 7.4. This resulted in 16 ml of preparation for group 1. Group 2 was intended for 1 ml vORF2 containing 8 μg vORF2 / ml. This was done by mixing 5.12 ml vORF2 (128 μg / 25 μg / ml = 5.12 ml vORF2) with 3.2 ml 0.5% Carbopol and 7.68 ml phosphate buffered saline at pH 7.4. This resulted in 16 ml of preparation for group 2. Group 3 was intended for 1 ml vORF2 containing 4 μg vORF2 / ml. This was done by mixing 2.56 ml vORF2 (64 μg / 25 μg / ml = 2.56 ml vORF2) with 3.2 ml 0.5% Carbopol and 10.24 ml phosphate buffered saline at pH 7.4. This is how 16 ml of the preparation for group 3 was created. Group 4 was given the administration of 1 ml of recombinant ORF2 (rORF2) containing 16 μg rORF2 / ml. This was done by mixing 2.23 ml rORF2 (512 μg / 230 μg / ml = 2.23 ml rORF2) with 6.4 ml 0.5% Carbopol and 23.37 ml phosphate buffered saline at pH 7.4. This resulted in 32 ml of preparation for group 4. Group 5 was given the administration of 1 ml rORF2 containing 8 μg rORF2 / ml. This was done by mixing 1.11 ml rORF2 (256 μg / 230 μg / ml = 1.11 ml rORF2) with 6.4 ml 0.5% Carbopol and 24.49 ml phosphate buffered saline at pH 7.4. 32 ml of the preparation for group 5 were prepared in this way. Group 6 was given the administration of 1 ml rORF2 containing 8 μg rORF2 / ml. This was done by mixing 0.56 ml rORF2 (128 μg / 230 μg / ml = 0.56 ml rORF2) with 6.4 ml 0.5% Carbopol and 25.04 ml phosphate buffered saline at pH 7.4. This is how 32 ml of the preparation for group 6 was created. Group 7 was given the administration of 2 ml of whole-cell PCV2 vaccine (PCV2 KV) containing MAX

PCV2 KV. This was done by mixing 56 ml PCV2 KV with 14 ml 0.5% Carbopol. This resulted in 70 ml of preparation for group 7. Finally, for group 8, KLH was administered at a concentration of 0.5 μg / ml or 1.0 μg / ml in a 2 ml dose. This was done by mixing 40.71 ml KLH (7.0 μg protein / ml by 0.5 μg / ml = 570 ml (7.0 μg / ml) (x) = (0.5) (570 ml)) with 244.29 ml phosphate buffered saline at pH 7.4 and 285 ml Freund's adjuvant. Table 5 describes the time frame for the key activities of this example.

Table 5. Activities during the study

<td>Examination Day</td><td>Activity during the study</td>

<td>-4, 0 to 49</td><td>General remarks on general health and clinical symptoms</td>

<td> -3</td><td>Weighting; Random group assignment; Taking blood samples from everyone pigs</td>

<td> 0</td><td>Health examination; Vaccine administration (IVP) No. 1-7 respectively groups 1-7,</td>

<td> 0-7</td><td>Observation in pigs for injection site reactions</td>

<td> 14</td><td>Group 7 support with PCV2 vaccine No. 7; Taking a blood sample from all pigs</td>

<td> 14-21</td><td>Observation in group 7 pigs for site reactions application</td>

<td> 16-19</td><td>Antibiotic treatment of all pigs (no data)</td>

<td> 19</td><td>Transfer of pigs from the first place to the second place</td>

<td> 21</td><td>Treatment of KLH / ICFA groups 1-9</td>

<td> 24</td><td>Sampling of blood and nasal secretions from all pigs; Weighting all pigs; Challenging groups 1-8 with PVCV2 infectious material</td>

<td>Examination Day</td><td>Activity during the study</td>

<td> 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47</td><td>Sampling of nasal secretions from all pigs</td>

<td> 27</td><td>Treatment of KLH / ICFA groups 1-9</td>

<td> 31</td><td>Blood sampling from all pigs</td>

<td> 49</td><td>Sampling of blood and nasal secretions from all pigs; Weighing all pigs; Section of all pigs; Pathological changes indicating jaundice and gastric ulcer were noted; Lung changes were assessed; Fresh and formalin-fixed tissue samples were retained; The in-life study phase has been completed.</td>

[0075] After the in-life study phase was completed, formalin-fixed tissues were examined by immunohistochemistry (IHC) to detect PCV2 antigen, blood samples were tested for PCV2 serology, nasal secretion samples were tested for PCV2 excretion and mean daily weight gain (ADWG) from day 24 to day 49 was determined.

[0076] The animals were housed in the first test facility in individual cages in five rooms from birth until about the 11th day of life (approximately day 0 of the study). Each room had an identical layout and consisted of individual stainless steel cages arranged with a supply of heated and filtered air supplied separately for each cage. Each room had separate heating and ventilation to prevent mutual air pollution between rooms. In the second research center, the animals were kept in two different buildings. Group 9 (strictly negative control group) was placed separately in the converted pig house before slaughter and groups 1-8 were placed in the converted piglet building. Each group was placed in a separate pen (11-12 pigs in the pen) and each pen provided approximately 3.0 square meters per pig. Each pen was placed on a platform made of plastic slats. Below the slats there was a tank to store faeces and waste. Each building had its own heating and ventilation systems, with low probability of mutual air pollution between buildings.

[0077] In the first research center, piglets were fed specially formulated milk from birth to about 3 weeks. All piglets ate solid foods, specially mixed portions up to the 19th day (about 4 1/2 weeks of age). In the second research center, all piglets were fed a customary non-medicated commercial mix appropriate for their age and weight, ad libitum. Water was also available ad libitum in both research centers.

[0078] All pigs tested received vitamin E on day -2, iron injections on day -1, and Naxcel^® (1.0 ml, IM in alternating hams) on days 16, 17, 18 and 19. In addition, for various health reasons, pig No. 52 (Group 9) was treated with iron injections on Day 3, Pig No. 45 (Group 6) was treated iron injections on day 11, pig No. 69 (Group 8) was treated with Naxcel^® on day 6, pig No. 74 (Group 3) was treated with dexamethasone and penicillin on Day 14 and pig No. 51 (Group 1) was treated with dexamethasone and penicillin on Day 13 and Naxcel^® on day 14

[0079] While in both research centers, the pigs remained under veterinary care. Animal health tests were conducted on day 0 as recorded on the Animal Health Card form. Prior to vaccination, all animals were in good health and were nourished as determined by observation on day 0. Prior to challenge, all test animals were observed for health and nutrition. Animal bodies and tissues were removed by smelting. Final removal of test animals was recorded in an Animal Disposition Record.

[0080] On day 0, the pigs assigned to groups 1-6 received 1.0 ml PCV2 vaccine No. 1-6, respectively, intramuscularly, in the left neck region using a 3.0 ml sterile Luer-lock syringe and a sterile 20g x ¥ 2 needle ". Pigs assigned to group 7 received 2.0 ml of PCV2 vaccine No. 7 intramuscularly in the left neck area using a 3.0 ml sterile Luer-lock syringe and a sterile 20g x ¥ 2 needle." On day 14, pigs assigned to group 7 received 2.0 ml of PCV2 vaccine No. 7 intramuscularly in the left neck region using a 3.0 ml sterile Luer-lock syringe and a sterile 20g x ¥ 2 "needle.

[0081] On day 21, all pigs tested received 2.0 ml KLH / ICFA intramuscularly in the right ham region using a sterile Luer-lock syringe and a sterile 20g x 1 "needle. On day 27, all pigs tested received 2.0 ml KLH / ICFA in the left ham region using a 3.0 ml sterile Luer-lock syringe and a sterile 20g x 1 "needle.

[0082] On day 24, pigs assigned to groups 1-8 received 1.0 ml of PCV2 ISUVDL challenge material (5.11 log10 TCID50 / ml), intramuscularly in the left neck region using a 3.0 ml sterile Luer-lock syringe and a sterile 20g x 1 "needles. An additional 1.0 ml of the same material was administered intranasally to each pig (0.5 ml on the nostril) using a 3.0 ml sterile Luer-lock syringe and nasal cannula.

[0083] Test pigs were observed daily for overall health and the occurrence of adverse effects on day -4 and from day 0 to day 19. Observations were recorded in the clinical observation protocol. All pigs tested were observed from day 0 to day 7 and group 7 was further observed from day 14 to 21 for injection site reactions. The average daily weight gain was determined by weighing each pig on a calibrated weight on days -3, 24 and 49, or on the day on which dead after challenge was found. Animal body weights were recorded on the Body Mass Form. Day -3 body weight was used to block pig body weight before randomization. Body weight measurement data obtained on day 24 and day 49 was used to determine the average daily weight gain (ADWG) for each pig during these time points. In the case of pigs that died after challenge and before day 49, ADWG was established as ADWG from day 24 to day of death.

[0084] To determine the PCV2 serology, all venous blood was taken from the venous orbital sinus on days -3 and 14. Blood was collected from each piglet from the venous orbital sinus by inserting a sterile capillary tube into the medial ligament of one eye and extending approximately 3.0 ml whole blood to a 4.0 ml tube with serum separator (SST). On days 24, 31 and 49, venous blood from each pig was collected from the anterior vena cava using a sterile 18g x 1 Vac "Vacutainer needle (Becton Dickinson and Company, Franklin Lakes, New Jersey), needle holder and 13 ml SST Vacutainer. at each time point, blood was recorded in the Sampling Register, blood in each SST was allowed to clot, each SST was then centrifuged and the serum collected. Collected serum was transferred to sterile, snap-closed tubes and stored at -70 ± 10 ° C for testing at a later date. Serum samples were tested for the presence of PCV2 antibodies by BIVI-R&amp;D staff.

[0085] Pigs were observed once a day from day 20 to day 49 for clinical signs and clinical observations were recorded in the Clinical Observation Register.

[0086] To test the secretion of PCV2 to the nose, on days 24, 25, and then every other odd test day on day 49 inclusive, a sterile dacron swab was inserted into the right or left nostril of each pig (one swab for one pig), as far as possible under aseptic conditions, held for a few seconds and then removed. Each swab was placed in a sterile snap-cap tube containing 1.0 ml EMEM medium with 2% IFBS, 500 units / ml penicillin, 500 μg / ml streptomycin and 2.5 μg / ml fungizone. The swab was broken off in a test tube that was sealed and properly labeled with the animal number, test number, date of collection, test day and the text "nasal swab". Closed snap tubes were stored at -40 ± 10 ° C until transported on ice to BIVISt Joseph. Nasal swab collection was recorded on the nasal swab sampling form. Quantitative virus (VI) isolation was carried out at BIVI-R&amp;D by examining nasal swab samples for the presence of PCV2. The results are presented as log10 values. A log value of 1.3 or less was considered negative, and any value greater than log 1.3 was considered positive.

[0087] In dead pigs (Nos. 28, 52, 56, 69, 82 and 93) in the first test center a section was performed to the extent necessary to establish the diagnosis of the cause of death. Pathological changes were recorded and the tissues of these pigs were not preserved. In the second research center, pigs were killed that died before day 49 (nos. 45, 23, 58, 35), pigs found dead on day 49 before euthanasia (nos. 2, 43) and euthanized pigs on day 49. In the Section Report Form all pathological changes and percentage of lung lobes with changes were noted.

[0088] A sample of tonsil, lung, heart, liver, mesenteric lymph nodes, kidneys and inguinal lymph node was taken from each pig out of 103 pigs sectioned in the second test center; placed in one container with 10% buffered formalin; and a second tissue sample from the same organs mentioned above was placed in sterile Whirl-pak bags (M-Tech Diagnostics Ltd, Thelwall, UK), and each of the Whirl-pak bags was placed on ice. Each container has been correctly marked. Collected samples were recorded on the Section Report Form. Next, formalin-fixed tissue samples and diagnostic form were submitted for IHC test. The IHC test was conducted according to ISU standard laboratory procedures for sample collection, sample preparation and preparation, and staining techniques. Fresh tissue in sterile Whirl-Paks bags were exported on ice to the Test Monitor for storage (70 ° ± 10 ° C) and possible future use. Formalin-fixed tissues were examined by a pathologist to detect PCV2 by IHC and scored in the following scoring system: 0 = none; 1 = scanty positive staining, several places; 2 = moderate positive staining, many places; and 3 = abundant positive staining, scattered throughout the tissue. Because the pathologist could not positively differentiate inguinal lymph nodes (LN) from mesentery, the results for these tissues were simply labeled as lymph nodes and the result given was the highest score for each of the two tissues for each animal.

Results [0089] The results for this example are given below. It should be noted that one pig from group 9 died before day 0 and 5 other pigs died after vaccination (1 pig from group 4; 1 pig from group 6, 2 pigs from group 8 and 1 pig from group 9). A post mortem study showed that all six pigs died of underlying infections that were not associated with vaccination or PMWS. In addition, no adverse reactions or injection site reactions were reported in any group.

[0090] The results for mean daily weight gain (ADWG) are shown below in Table 6. In group 9, the strictly negative control group had the highest ADWG value (1.06 ± 0.17 pounds / day), followed by group 5 (0.94 ± 0.22 pounds / day) that received one dose of 8 μg rORF2. Group 3, which received one dose of 4 μg vORF2, had the lowest ADWG (0.49 ± 0.21 pounds / day), followed by Group 7 (0.50 ± 0.15 pounds / day), which received 2 dose of vaccine killed.

Table 6. Summary of the average daily weight gain (ADWG) for each group

<td>Group</td><td>Treatment</td><td>N</td><td>ADWG - pounds / day (Day 24 to day 49) or determined for pigs dead before 29</td>

<td> 1</td><td>vORF2-16 μg (1 dose)</td><td> 12</td><td>0.87 ± 0.29 pounds / day</td>

<td> 2</td><td>vORF2-8 μg (1 dose)</td><td> 12</td><td>0.70 ± 0.32 pounds / day</td>

<td> 3</td><td>vORF2-4 μg (1 dose)</td><td> 12</td><td>0.49 ± 0.21 pounds / day</td>

<td> 4</td><td>rORF2-16 μg (1 dose)</td><td> 11</td><td>0.84 ± 0.30 pounds / day</td>

<td> 5</td><td>rORF2-8 μg (1 dose)</td><td> 12</td><td>0.94 ± 0.22 pounds / day</td>

<td> 6</td><td>rORF2-4 μg (1 dose)</td><td> 11</td><td>0.72 ± 0.25 pounds / day</td>

<td> 7</td><td>KV (2 doses)</td><td> 12</td><td>0.50 ± 0.15 pounds / day</td>

<td> 8</td><td>A provoked group control</td><td> 10</td><td>0.76 ± 0.19 pounds / day</td>

<td> 9</td><td>strictly negative group control</td><td> 11</td><td>1.06 ± 0.17 pounds / day</td>

<td colspan="4">vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus showing ORF2 expression; whole virus cells killed = PCV2; the virus was grown in an appropriate cell culture</td>

[0091] PCV2 serological results are shown below in Table 7. All nine groups were PCV2 seronegative on day -3. On day 14, the groups receiving the vORF2 vaccine had the highest titer that ranged from 187.5 to 529.2. Pigs receiving killed viral vaccine had the next highest titers followed by rORF2 vaccine groups. Groups 8 and 9 remained seronegative at that time. On day 24 and day 31, pigs receiving the vORF2 vaccine still showed a strong serological response, followed by the group that received two doses of the killed viral vaccine. Pigs receiving rORF2 vaccines had a slower serological response and groups 8 and 9 remained seronegative. On day 49, pigs receiving the vORF2 vaccine, 2 doses of killed viral vaccine, and the lowest dose of rORF2 showed the strongest serological response. Pigs receiving 16 μg and 8 μg rORF2 vaccines had slightly higher IFA titers than infected control groups. Group 9 on day 49 showed a strong serological response.

Table 7. Summary data - PCV2 IFA titers for all groups

<td colspan="7">Average IFA titers</td>

<td>Group</td><td>Treatment</td><td>Day -3</td><td>Day 14</td><td>Day 24</td><td>Day 31 **</td><td>Day 49 ***</td>

<td> 1</td><td>vORF2-16 μg (1 dose)</td><td> 50,0</td><td> 529,2</td><td> 4400,0</td><td> 7866,7</td><td> 11054,5</td>

<td> 2</td><td>vORF2-8 μg (1 dose)</td><td> 50,0</td><td> 500,0</td><td> 3466,7</td><td> 6800,0</td><td> 10181,8</td>

<td colspan="7">Average IFA titers</td>

<td>Group</td><td>Treatment</td><td>Day -3</td><td>Day 14</td><td>Day 24</td><td>Day 31 **</td><td>Day 49 ***</td>

<td> 3</td><td>vORF2-4 μg (1 dose)</td><td> 50,0</td><td> 187,5</td><td> 1133,3</td><td> 5733,3</td><td> 9333,3</td>

<td> 4</td><td>rORF2-16 μg (1 dose)</td><td> 50,0</td><td> 95,5</td><td> 1550,0</td><td> 3090,9</td><td> 8000,0</td>

<td> 5</td><td>rORF2-8 μg (1 dose)</td><td> 50,0</td><td> 75,0</td><td> 887,5</td><td> 2266,7</td><td> 7416,7</td>

<td> 6</td><td>rORF2-4 μg (1 dose)</td><td> 50,0</td><td> 50,0</td><td> 550,0</td><td> 3118,2</td><td> 10570,0</td>

<td> 7</td><td>KV (2 doses)</td><td> 50,0</td><td> 204,2</td><td> 3087,5</td><td> 4620,8</td><td> 8680,0</td>

<td> 8</td><td>A provoked group control</td><td> 50,0</td><td> 55,0</td><td> 50,0</td><td> 50,0</td><td> 5433,3</td>

<td> 9</td><td>Strictly negative group control</td><td> 50,0</td><td> 59,1</td><td> 59,1</td><td> 54,5</td><td> 6136,4</td>

<td colspan="7">vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus showing ORF2 expression; whole virus cells killed = PCV2 virus was cultured in an appropriate cell culture * For calculation purposes &lt;100 IFA, the titre was designated as "50"; &gt; 6400 titers IFA was designated as "12800". ** Day of infection *** Section Day</td>

[0092] The results of post-challenge clinical observations are presented in Table 8 below. This summary of results includes observations for Abnormal Behavior, Breathing Disorder, Cough, and Diarrhea. Table 9 contains the results from the group summary of the total incidence of clinical symptoms and Table 10 contains the results from the group summary of the mortality rates after challenge. The most common clinical symptom observed in this study was abnormal behavior, which was assessed as mild to severe lethargy. In pigs receiving the lowest 2 vORF2 doses, pigs receiving 16 μg rORF2 and pigs receiving two doses of KV vaccine the incidence was> 27.3%. No abnormal behavior was observed in pigs receiving 8 μg rORF2 and in a strictly negative control group. None of the pigs in this study showed a breathing disorder. Cough was often seen in all groups (from 0 to 25%), as was diarrhea (0-20%). None of the observed clinical symptoms was a pathognomic symptom of PMWS.

[0093] The overall incidence of clinical symptoms varied between groups. The highest incidence of general clinical symptoms (> 36.4%) was observed in the group receiving any of the vORF2 vaccines, in the group receiving 16 μg rORF2, in the group receiving 2 doses of the KV vaccine and the challenged control group. In the strictly negative control group, the total group of clinical symptoms of 0%, 8.3% and 9.1%, respectively, in the group receiving 8 μg rORF2 and the group receiving 4 μg rORF2.

[0094] The overall mortality rates between groups were also varied. The group receiving 2 doses of the KV vaccine had the highest mortality rate (16.7%); while in all groups that received 4 μg vORF2, 16 μg rORF2 or μg rORF2 and the strictly negative control group, the mortality rate was 0%.

Table 8. Summary data for abnormal behavior, abnormal breathing, coughing and diarrhea for all groups

<td>Group</td><td>Treatment</td><td>N</td><td>Incorrect Behavior¹</td><td>Incorrect Behavior ²</td><td>Cough³</td><td>Diarrhea⁴</td>

<td> 1</td><td>vORF2-16 μg (1 dose)</td><td> 12</td><td> 2/12 (16,7%)</td><td> 0/12 (0%)</td><td> 3/12 (25%)</td><td> 2/12 (16,7%)</td>

<td rowspan="2"> 2</td><td>vORF2-8 μg (1</td><td rowspan="2"> 12</td><td> 4/12</td><td> 0/12</td><td> 1/12</td><td> 1/12</td>

<td>dose)</td><td> (33,3%)</td><td> (0%)</td><td> (8,3%)</td><td> (8,3%)</td>

<td rowspan="2"> 3</td><td>vORF2-4 μg (1</td><td rowspan="2"> 12</td><td> 8/12</td><td> 0/12</td><td> 2/12</td><td> 1/12</td>

<td>dose)</td><td> (66,7%)</td><td> (0%)</td><td> (16,7%)</td><td> (8,3%)</td>

<td rowspan="2"> 4</td><td>rORF2-16 μg (1</td><td rowspan="2"> 11</td><td> 3/11</td><td> 0/11</td><td> 0/11</td><td> 2/11</td>

<td>dose)</td><td> (27,3%)</td><td> (0%)</td><td> (0%)</td><td> (18,2%)</td>

<td> 5</td><td>rORF2-8 μg (1 dose)</td><td> 12</td><td> 0/12 (0%)</td><td> 0/12 (0%)</td><td> 1/12 (8,3%)</td><td> 0/12 (0%)</td>

<td> 6</td><td>rORF2-4 μg (1 dose)</td><td> 11</td><td> 1/11 (9,1%)</td><td> 0/11 (0%)</td><td> 0/11 (0%)</td><td> 0/12 (0%)</td>

<td> 7</td><td>KV (2 doses)</td><td> 12</td><td> 7/12 (58,3%)</td><td> 0/12 (0%)</td><td> 0/12 (0%)</td><td> 1/12 (8,3%)</td>

<td rowspan="2"> 8</td><td>provocation</td><td rowspan="2"> 10</td><td> 1/10</td><td> 0/10</td><td> 2/10</td><td> 2/10</td>

<td>control group</td><td> (10%)</td><td> (0%)</td><td> (20%)</td><td> (20%)</td>

<td> 9</td><td>Close negative control group</td><td> 11</td><td> 0/11 (0%)</td><td> 0/11 (0%)</td><td> 0/11 (0%)</td><td> 0/11 (0%)</td>

<td>vORF2</td><td colspan="2">= isolated viral</td><td>ORF2; rORF2 =</td><td colspan="2">recombinant baculovirus</td><td>showing</td>

<td colspan="7">ORF2 expression; whole virus cells killed = PCV2 virus was cultured in an appropriate culture</td>

<td colspan="2">cell</td><td></td><td></td><td></td><td></td><td></td>

<td colspan="7">¹total number of pigs in each group that showed any abnormal behavior</td>

<td colspan="3">for at least one day</td><td></td><td></td><td></td><td></td>

<td colspan="7">²total number of pigs in each group that showed any abnormal breathing</td>

<td colspan="4">for at least one day</td><td></td><td></td><td></td>

<td colspan="7">³total number of pigs in each group that has at least one cough</td>

<td>Group</td><td>Treatment</td><td>N</td><td>Incorrect Behavior¹</td><td>Incorrect Behavior ²</td><td>Cough³</td><td>Diarrhea⁴</td>

<td colspan="7">day⁴total number of pigs in each group who had diarrhea for at least one day</td>

Table 9. Summary data on the overall incidence of clinical symptoms for all groups

<td>Group</td><td>Treatment</td><td>N</td><td>Frequencies clinical signs in pigs¹</td><td>Frequency indicator occurrence</td>

<td> 1</td><td>vORF2-16 μg (1 dose)</td><td> 12</td><td> 5</td><td> 41,7%</td>

<td> 2</td><td>vORF2-8 μg (1 dose)</td><td> 12</td><td> 5</td><td> 41,7%</td>

<td> 3</td><td>vORF2-4 μg (1 dose)</td><td> 12</td><td> 8</td><td> 66,7%</td>

<td> 4</td><td>rORF2-16 μg (1 dose)</td><td> 11</td><td> 4</td><td> 36,4%</td>

<td> 5</td><td>rORF2-8 μg (1 dose)</td><td> 12</td><td> 1</td><td> 8,3%</td>

<td> 6</td><td>&gt; rORF2-4 μg (1 dose)</td><td> 11</td><td> 1</td><td> 9,1%</td>

<td> 7</td><td>KV (2 doses)</td><td> 12</td><td> 7</td><td> 58,3%</td>

<td> 8</td><td>A provoked group control</td><td> 10</td><td> 4</td><td> 40%</td>

<td> 9</td><td>Strictly negative control group</td><td> 11</td><td> 0</td><td> 0%</td>

<td colspan="5">vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus showing ORF2 expression; whole virus cells killed = PCV2 virus was cultured in an appropriate cell culture¹total number of pigs in each group that showed any clinical signs for at least one day</td>

Table 10. Summary data of mortality rates after challenge for all groups

<td>Group</td><td>Treatment</td><td>N</td><td>Fallen after provoking</td><td>Indicator mortality</td>

<td> 1</td><td>vORF2-16 μg (1 dose)</td><td> 12</td><td> 1</td><td> 8,3%</td>

<td> 2</td><td>vORF2-8 μg (1 dose)</td><td> 12</td><td> 1</td><td> 8,3%</td>

<td> 3</td><td>vORF2-4 μg (1 dose)</td><td> 12</td><td> 0</td><td> 0%</td>

<td> 4</td><td>rORF2-16 μg (1 dose)</td><td> 11</td><td> 0</td><td> 0%</td>

<td>Group</td><td>Treatment</td><td>N</td><td>Fallen after provoking</td><td>Indicator mortality</td>

<td> 5</td><td>rORF2-8 μg (1 dose)</td><td> 12</td><td> 0</td><td> 0%</td>

<td> 6</td><td>rORF2-4 μg (1 dose)</td><td> 11</td><td> 1</td><td> 9,1%</td>

<td> 7</td><td>KV (2 doses)</td><td> 12</td><td> 2</td><td> 16,7%</td>

<td> 8</td><td>A provoked group control</td><td> 10</td><td> 1</td><td> 10%</td>

<td> 9</td><td>Strictly negative group control</td><td> 11</td><td> 0</td><td> 0%</td>

<td colspan="3">vORF2 = isolated viral ORF2; Ror ORF2 expression; whole virus cells = cell killed</td><td colspan="2">F2 = recombinant baculovirus showing PCV2 virus was cultured in an appropriate culture</td>

[0095] The results of the secretion of nasal PCV2 are shown in Table 11 below. After challenge on day 24, one pig in group 7 noted the secretion of PCV2 on day 27. None of the other groups had secretion until day 33. The highest amount of secretion to The nose was recorded from day 35 to day 45. The lowest nasal PCV2 secretion (&lt;9.1%) was observed in the groups receiving any of the three vORF2 vaccines and the groups receiving 4 or 8 μg rORF2. The highest secretion rate (80%) was observed in the challenge control group (Group 8), followed by the strictly negative control group (Group 9) for which the secretion rate was 63.6%.

Table 11. Summary data of the PCV2 nasal secretion index for all groups

<td>Group</td><td colspan="3">Treatment</td><td>N</td><td>No pigs having been secreted for at least one day</td><td>Frequency indicator occurrence</td>

<td> 1</td><td>vORF2-16 dose)</td><td>Pg</td><td> (1</td><td> 12</td><td> 1</td><td> 8,3%</td>

<td> 2</td><td>vORF2-8 dose)</td><td>Pg</td><td> (1</td><td> 12</td><td> 1</td><td> 8,3%</td>

<td> 3</td><td>vORF2-4 dose)</td><td>Pg</td><td> (1</td><td> 12</td><td> 1</td><td> 8,3%</td>

<td> 4</td><td>rORF2-16 dose)</td><td>Pg</td><td> (1</td><td> 11</td><td> 2</td><td> 18,2%</td>

<td> 5</td><td>rORF2-8 dose)</td><td>Pg</td><td> (1</td><td> 12</td><td> 1</td><td> 8,3%</td>

<td> 6</td><td>rORF2-4</td><td>Pg</td><td> (1</td><td> 11</td><td> 1</td><td> 9,1%</td>

<td>Group</td><td>Treatment</td><td>N</td><td>No pigs which have undergone secretion for at least one day</td><td>Frequency indicator occurrence</td>

<td></td><td>dose)</td><td></td><td></td><td></td>

<td> 7</td><td>KV (2 doses)</td><td> 12</td><td> 5</td><td> 41,7%</td>

<td> 8</td><td>A provoked group control</td><td> 10</td><td> 8</td><td> 80%</td>

<td> 9</td><td>Strictly negative control group</td><td> 11</td><td> 7</td><td> 63,6%</td>

<td colspan="3">vORF2 = isolated viral OR ORF2 expression; whole cell cells killed</td><td colspan="2">F2; rORF2 = recombinant baculovirus showing virus = PCV2 virus was cultured in the appropriate culture</td>

[0096] Cumulative data on the group occurrence of jaundice, the occurrence of gastric ulcer, the group results of average lung lesions, and the group occurrence of lung lesions are presented below in Table 12. Six pigs died in the first place of the study in the post-vaccination phase (group 4, N = 1 , group 6, N = 1; group 8, N = 2; group 9, N = 2). Four of six pigs had fibrinous lesions in one or more body cavities, one pig (Group 6) had changes related to Clostridial disease, and no major lesions were observed in one pig (Group 9). None of the pigs that died during the post-vaccination phase had any changes associated with PMWS.

[0097] Post-challenge pigs were killed and euthanized pigs on day 49. During section, no jaundice or gastric ulceration was observed in any group. Regarding the mean% lung changes, group 9 had the lowest mean% lung changes (0%) followed by group 1 0.40 ± 0.50% and group 5 0.68 ± 1.15 %. Groups 2, 3, 7 and 8 had the highest mean% lung changes (> 7.27%). In each of these four groups there was one Pig with% lung lesions> 71.5%, which overstated the results for these four groups. With the exception of group 9, for which the value of lung lesions was 0%, the other 8 groups had a value of <36% lung lesions. Almost all reported lung changes were described as red / purple and consolidated.

Table 12. Aggregate data for group jaundice, gastric ulcer, group results of mean% lung changes, and group occurrence of noticed lung changes

<td>Group</td><td>Treatment</td><td>Jaundice</td><td>ulcers gastric</td><td>Medium % changes in lungs</td><td>Noticed frequency occurrence lung changes</td>

<td>Group</td><td>Treatment</td><td>Jaundice</td><td>ulcers gastric</td><td>Medium % changes in lungs</td><td>Noticed frequency occurrence lung changes</td>

<td> 1</td><td>vORF2-16 μg (1 dose)</td><td> 0/12 (0%)</td><td> 0/12 (0%)</td><td> 0,40 ± 0,50%</td><td> 10/12 (83%)</td>

<td> 2</td><td>vORF2-8 μg (1 dose)</td><td> 0/12 (0%)</td><td> 0/12 (0%)</td><td> 7,41 ± 20,2%</td><td> 10/12 (83%)</td>

<td> 3</td><td>vORF2-4 μg (1 dose)</td><td> 0/12 (0%)</td><td> 0/12 (0%)</td><td> 9,20 ± 20,9%</td><td> 10/12 (83%)</td>

<td> 4</td><td>rORF2-16 μg (1 dose)</td><td> 0/11 (0%)</td><td> 0/1 (0%)</td><td> 1,5 ± 4,74%</td><td> 4/11 (36%)</td>

<td> 5</td><td>rORF2-8 μg (1 dose)</td><td> 0/12 (0%)</td><td> 0/12 (0%)</td><td> 0,68 ± 1,15%</td><td> 9/12 (75%)</td>

<td> 6</td><td>rORF2-4 μg (1 dose)</td><td> 0/11 (0%)</td><td> 0/11 (0%)</td><td> 2,95 ± 5,12%</td><td> 7/11 (64%)</td>

<td> 7</td><td>KV (2 doses)</td><td> 0/12 (0%)</td><td> 0/12 (0%)</td><td> 7,27 ± 22,9%</td><td> 9/12 (75%)</td>

<td> 8</td><td>A provoked group control</td><td> 0/10 (0%)</td><td> 0/10 (0%)</td><td> 9,88 ± 29,2%</td><td> 8/10 (80%)</td>

<td> 9</td><td>Strictly negative control group</td><td> 0/11 (0%)</td><td> 0/11 (0%)</td><td> 0/11 (0%)</td><td> 0/11 (0%)</td>

<td colspan="6">vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus showing ORF2 expression; KV or whole virus cells killed = PCV2 virus was cultured in an appropriate cell culture</td>

[0098] Summary data for all IHC positive groups is shown in Table 13. Group 1 (vORF2 - 16 μg) and group 5 (rORF2 - 8 μg) had the lowest IHC positive index (16.7%). Group 8 (challenge control group) and group 9 (strictly negative control group) had the highest IHC positive rate, 90% and 90.9%, respectively.

Table 13. Summary data for all positive occurrence groups

IHC

<td>Group</td><td>Treatment</td><td>N</td><td>No pigs affected by co at least one tissue with result positive for PCV2</td><td>Frequency indicator occurrence</td>

<td> 1</td><td>vORF2-16 μg (1 dose)</td><td> 12</td><td> 2</td><td> 16,7%</td>

<td>Group</td><td>Treatment</td><td>N</td><td>No pigs which had at least one tissue positive for PCV2</td><td>Incidence rate indicator</td>

<td> 2</td><td>vORF2-8 μg (1 dose)</td><td> 12</td><td> 3</td><td> 25,0%</td>

<td> 3</td><td>vORF2-4 μg (1 dose)</td><td> 12</td><td> 8</td><td> 66,7%</td>

<td> 4</td><td>rORF2-16 μg (1 dose)</td><td> 11</td><td> 4</td><td> 36,3%</td>

<td> 5</td><td>rORF2-8 μg (1 dose)</td><td> 12</td><td> 2</td><td> 16,7%</td>

<td> 6</td><td>rORF2-4 μg (1 dose)</td><td> 11</td><td> 4</td><td> 36,4%</td>

<td> 7</td><td>KV (2 doses)</td><td> 12</td><td> 5</td><td> 41,7%</td>

<td> 8</td><td>provocation control group</td><td> 10</td><td> 9</td><td> 90%</td>

<td> 9</td><td>Strictly negative control group</td><td> 11</td><td> 10</td><td> 90,9%</td>

<td colspan="5">vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus showing ORF2 expression; KV or whole virus cells killed = PCV2 virus was cultured in an appropriate cell culture</td>

[0099] After the challenge, group 5, which received one 8 μg dose of rORF2 antigen, performed better than the other 6 vaccinated groups. Group 5 had the highest ADWG (0.94 ± 0.22 pounds / day), the lowest incidence of abnormal behavior (0%), the second lowest incidence of cough (8.3%), the lowest incidence of general clinical symptoms (8, 3%), the lowest mortality rate (0%), the lowest rate of secretion of PCV2 to the nose (8.3%), the second highest rate of average% lung changes (0.68 ± 1.15%), and the lowest rate of occurrence positive tissues (16.7%). Groups receiving different levels of rORF2 antigen generally outperformed those receiving different levels of vORF2, and the worst group receiving 2 doses of killed whole-cell PCV2 vaccine. Tables 14 and 15 contain aggregate data for all groups after challenge.

Table 14. Summary data for all groups after challenge - Part 1

<td>Group</td><td>N</td><td>Treatment</td><td>ADWG (Lb / day)</td><td>Incorrect behavior</td><td>Cough</td><td>total occurrence symptoms clinical</td>

<td> 1</td><td> 12</td><td>vORF2-16 μg (1 dose)</td><td> 0,87 ± 0,29</td><td> 2/12 (16,7%)</td><td> 3/12 (25%)</td><td> 41,7%</td>

<td> 2</td><td> 12</td><td>vORF2-8 μg (1 dose)</td><td> 0,70 ± 0,32</td><td> 4/12 (33,3%)</td><td> 1/12 (8,3%)</td><td> 41,7%</td>

<td> 3</td><td> 12</td><td>vORF2-4 μg (1 dose)</td><td> 0,49 ± 0,21</td><td> 8/12 (66,7%)</td><td> 2/12 (16,7%)</td><td> 66,7%</td>

<td> 4</td><td> 11</td><td>rORF2-16 μg (1 dose)</td><td> 0,84 ± 0,30</td><td> 3/11 (27,3%)</td><td> 0/11 (0%)</td><td> 36,4%</td>

<td> 5</td><td> 12</td><td>rORF2-8 μg (1 dose)</td><td> 0,94 ± 0,22</td><td> 0/12 (0%)</td><td> 1/12 (8,3%)</td><td> 8,3%</td>

<td> 6</td><td> 11</td><td>rORF2-4 μg (1 dose)</td><td> 0,72 ± 0,25</td><td> 1/11 (9,1%)</td><td> 0/11 (0%)</td><td> 9,1%</td>

<td> 7</td><td> 12</td><td>KV (2 doses)</td><td> 0,50 ± 0,15</td><td> 7/12 (58,3%)</td><td> 0/12 (0%)</td><td> 58,3%</td>

<td> 8</td><td> 10</td><td>provocation group control</td><td> 0,76 ± 0,19</td><td> 1/10 (10%)</td><td> 2/10 (20%)</td><td> 40%</td>

<td> 9</td><td> 11</td><td>Close negative group control</td><td> 1,06 ± 0,17</td><td> 0/11 (0%)</td><td> 0/11 (0%)</td><td> 0%</td>

<td colspan="7">vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus showing ORF2 expression; KV or whole virus cells killed = PCV2 virus was cultured in an appropriate cell culture</td>

Table 15. Summary data for all post-infection groups - Part 2

<td>Group</td><td>N</td><td>Treatment</td><td>Indicator mortality</td><td>Secretion to nose</td><td>Average% changes in lungs</td><td>Indicator occurrence of co at least one tissue with the result positive for PCV2</td>

<td> 1</td><td> 12</td><td>vORF2-16 μg (1 dose)</td><td> 8,3%</td><td> 8,3%</td><td> 0,40 ± 0,50%</td><td> 16,7%</td>

<td> 2</td><td> 12</td><td>vORF2-8 μg (1 dose)</td><td> 8,3%</td><td> 8,3%</td><td> 7,41 ± 20,2%</td><td> 25,0%</td>

<td> 3</td><td> 12</td><td>vORF2-4 μg (1 dose)</td><td> 0%</td><td> 8,3%</td><td> 9,20 ± 20,9%</td><td> 66,7%</td>

<td> 4</td><td> 11</td><td>rORF2-16 μg (1 dose)</td><td> 0%</td><td> 18,2%</td><td> 1,50 ± 4,74%</td><td> 36,3%</td>

<td> 5</td><td> 12</td><td>rORF2-8 μg (1 dose)</td><td> 0%</td><td> 8,3%</td><td> 0,68 ± 1,15%</td><td> 16,7%</td>

<td> 6</td><td> 11</td><td>rORF2-4 μg (1 dose)</td><td> 9,1%</td><td> 9,1%</td><td> 2,95 ± 5,12%</td><td> 36,4%</td>

<td> 7</td><td> 12</td><td>KV (2 doses)</td><td> 16,7%</td><td> 41,7%</td><td> 7,27 ± 22,9%</td><td> 41,7%</td>

<td> 8</td><td> 10</td><td>provocation group control</td><td> 10%</td><td> 80%</td><td> 9,88 ± 29,2%</td><td> 90%</td>

<td> 9</td><td> 11</td><td>Close negative group control</td><td> 0%</td><td> 63,6%</td><td> 0/11 (0%)</td><td> 90,9%</td>

<td colspan="7">vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus showing ORF2 expression; KV or whole virus cells killed = PCV2 virus was cultured in an appropriate cell culture</td>

[0100] The results of this study indicate that all further work on vaccine 5 should focus on the rORF2 vaccine. Generally, nasal PCV2 secretion was detected after challenge and vaccination with PCV2 vaccine reduced this secretion. Immunohistochemistry of selected lymphatic tissues also proved to be a good parameter for assessing vaccine efficacy, while no significant differences in the groups

ADWG, in clinical symptoms and pathological changes. This study was complicated by the fact that at some point during the study external PCV2 was introduced, as evidenced by the secretion of PCV2 to the nose, PCV2 seroconversion and positive IHC results for tissues in group 9, a strictly negative control group.

Discussion [0101] This study evaluated seven PCV2 vaccines that contained three different dose levels of vORF2 antigen given once on day 0, three different dose levels of rORF2 antigen given once on day 0, and one dose of killed whole PCV2 cell vaccine administered on day 0 and on day 14. In general, group 5, which received 1 dose of the vaccine containing 8 μg of rORF2 antigen, obtained the best results. Group 5 had the highest ADWG, the lowest incidence of abnormal behavior, the lowest incidence of abnormal breathing, the second lowest incidence of cough, the lowest incidence of general clinical symptoms, the lowest mortality rate, the lowest rate of secretion of PCV2 to the nose, the second lowest results of average% lung changes and the lowest rate of positive IHC for tissues.

Interestingly, group 4, which received a higher dose of rORF2 antigen than group 5, did not achieve such good or better results than group 5. Group 4 had slightly less ADWG, a higher incidence of abnormal behavior, a higher incidence of general clinical symptoms , higher nasal PCV2 secretion rate, higher mean% lung lesion scores, and higher tissue IHC positive rates than in group 5. No statistical analysis was carried out for these data, which could indicate that the differences between these two groups were not statistically significant, but there were trends that group 4 did not perform as well as group 5.

[0103] After vaccination, six pigs died in the first test center. Four of the six pigs were from groups 8 or 9 that did not receive any vaccine. None of the six pigs showed any changes associated with PMWS, no adverse effects or other events were reported, all seven vaccines appear to be safe when given around 11 days of age. In the post-vaccination study, pigs receiving one of the three dose levels of the vORF2 vaccine or the whole cell vaccine had the highest IFAT levels, while group 5 just before infection had the lowest IFAT levels among the vaccinated groups.

[0104] Although this is not formally proven, it is believed that the main route of transmission of PCV2 virus in young pigs shortly after weaning is direct oral-nasal contact and an effective vaccine that reduces the secretion of PCV2 to the nose under production conditions would help control the spread of infection. Groups receiving one of three doses of vORF2 antigens and the group receiving 8 μg rORF2 had the lowest level of PCV2 secretion into the nose (8.3%). As expected, the infected control group had the highest nasal discharge rate (80%).

[0105] Large pathological changes in pigs secondary to PMWS infected after PCV2 infection are usually made up of generalized lymphadenopathy in combination with one or more of the following symptoms: (1) interstitial pneumonia with swelling of interlobular fissures, (2) pallor of the skin or jaundice , (3) macular atrophic cirrhosis, (4) stomach ulcers, (5) nephritis, and (6) reproductive disorders, for example, abortion, delivery of a dead fetus, fetal mummification, etc. During the section, none of the groups had jaundice, hepatitis, nephritis and gastric ulcer, and lymphadenopathy was not specifically studied. The results of mean% lung changes varied between groups. The group receiving 16 μg vORF2 antigen had the lowest mean% lung lesions (0.40 ± 0.50%), followed by the group receiving 8 μg rORF2 (0.68 ± 1.15%). As expected, the provoked control group had the highest scores of mean% lung changes (9.88 ± 29.2%). In all four groups, the mean% lung lesion scores were elevated due to one pig in each of these groups that had very high lung lesion scores. Most lung lesions are described as red / purple and consolidated. Usually, lung lesions associated with PMWS are described as brown and not susceptible to interlobular edema. The lung lesions noted in this study were not associated with PCV2 infection or the presence of a second pulmonary infectious agent. In this study, the% lung lesion results probably do not reflect the true measure of the amount of lung infection caused by PCV2.

[0106] Other researchers have shown a direct relationship between the presence of PCV2 antigen and IHC and histopathological findings. Histopathological examination of selected tissues was not conducted in this study. Group 1 (16 μg vORF2) and group 5 (8 μg rORF2) had the lowest incidence of pigs with positive PCV2 antigen (8.3%), while in group 9 (strictly negative control group - 90.9%) and group 8 (infected control group - 90.0%) had the highest pig occurrences with positive results for PCV2 antigen. Due to the subjective nature of this study, IHC results are probably one of the best parameters for assessing the effectiveness of this vaccine.

[0107] Thus, based on the CDCD pig model after PCV2 challenge, a minimum protective dose (MPD) of 1 ml / 1 recombinant product with extracted PCV2 ORF2 extracted antigen (rORF2) was established. Of the three groups receiving different doses of rORF2 antigen, group 5 (8 μg rORF2 antigen) clearly showed the highest level of protection. Group 5 either achieved the best results or was assigned to the most favorable results for all parameters tested. When group 5 was compared with the other six vaccinated groups after infection, group 5 had the highest ADWG (0.94 ± 022 pounds / day), the lowest incidence of abnormal behavior (0%), and the second lowest incidence of cough (8.3) %), the lowest incidence of general clinical symptoms (8.3%), the lowest rate of mortality (0%), the lowest rate of secretion of PCV2 to the nose (8.3%), the second lowest value of mean% lung changes (0.68 ± 1.15%) and the lowest rate of positive IHC for tissues (16.7%).

[0108] Based on the CDCD pig model after PCV2 challenge, an MPD of 1 ml / L dose of conventional product, partially purified PCV2 ORF2 antigen (vORF2) was determined. Of the three groups receiving different doses of vORF2 antigen, group 1 (16 μg vORF2) showed the highest level of protection. Group 1 outperformed Groups 2 and 3 for ADWG, mean% lung lesions, and IHC. Groups 1 and 2 (8 μg vORF2 antigen) behaved the same in relation to the overall incidence of clinical symptoms, group 3 (4 μg vORF2 antigen) had the lowest mortality rate, and all three groups showed equivalent results relative to nasal secretion. In general, vORF vaccines did not show as well as rORF vaccines.

[0109] Based on the CDCD pig model after PCV2 challenge, the efficacy of a maximum dose of 2 ml / 2 dose of conventional killed PCV2 vaccine was determined. Of the seven vaccines evaluated in this study, the vaccine containing whole killed PCV2 cells was the worst. Piglets receiving two killed vaccines with whole, killed PCV2 cells showed the lowest ADWG, the second highest rate of abnormal behavior (58.3%), the second highest rate of the total incidence of clinical symptoms (58.3%), the highest rate of mortality (16.7% ), the second highest nasal secretion in value (41.7%), highest average% lung changes (9.88% ± 29.2%), high observed incidence of lung lesions (75%) and moderate tissue incidence of IHC (41.7%). However, this vaccine was still effective in inducing an immune response.

[0110] In yet another aspect of the present invention, nasal excretion of PCV2 virus was evaluated as an efficacy parameter and previous PCV2 efficacy parameters obtained from previous studies were confirmed. The results of this study indicate that nasal excretion of PCV2 virus occurs after intranasal challenge and that PCV2 vaccines reduce nasal excretion of PCV2 virus after infection with PCV2 virus. In addition, the results of this study and literature reports indicate that IHC should still be evaluated in future studies of the PCV2 vaccine.

[0111] An additional conclusion from this study is that lymph node enlargement is one of the features of PMWS. Another characteristic feature of PMWS is lymphoid atrophy and multinucleated / giant histiocytes. In addition, no adverse reactions or injection site reactions were reported for any of the 7 PCV2 vaccines and all 7 PCV2 vaccines appear to be safe when administered to young pigs.

EXAMPLE 5 [0112] The example shows the efficacy studies of eight potential PCV2 vaccines and confirms the PCV2 challenge parameters from previous infection studies after exposure to the virulent PCV2 strain. One hundred fifty (150) colostrum-free piglets after caesarean section (CDCD), aged 6-16 days, were randomly allocated to 10 equal-sized groups. Table 16 shows the overall study design for this example.

Table 16. General Study Design

<td>Group</td><td>number pigs</td><td>Treatment</td><td>Day treatment</td><td>KLH / ICFA on day 22 and 28</td><td>Provocation virulent PCV2 on 25</td><td>PRRSV MLV in day 46</td><td>Section in day 50</td>

<td> 1</td><td> 15</td><td>Vaccine PCV2 1- 16 μg rORF2-IMS 1314</td><td>0 and 14</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td> 2</td><td> 15</td><td>Vaccine PCV2 2- 16 μg vORF2-Carbopol</td><td>0 and 14</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td> 3</td><td> 15</td><td>Vaccine PCV2 3- 16 μg rORF2-Carbopol</td><td>0 and 14</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td> 4</td><td> 15</td><td>Vaccine PCV2 2- 16 μg vORF2-Carbopol</td><td> 0</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td> 5</td><td> 15</td><td>Vaccine PCV2 3-4 μg rORF2-Carbopol</td><td>0 and 14</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td> 6</td><td> 15</td><td>Vaccine PCV2 3- 1 μg rORF2-Carbopol</td><td>0 and 14</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td> 7</td><td> 15</td><td>Vaccine PCV2 3- 0.25 μg rORF2-Carbopol</td><td>0 and 14</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td> 8</td><td> 15</td><td>Vaccine PCV2 4&gt; 8,0 log KV-Carbopol</td><td>0 and 14</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td> 9</td><td> 15</td><td>provocation</td><td>ON</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td>Group</td><td>number pigs</td><td>Treatment</td><td>Day treatment</td><td>KLH / ICFA on day 22 and 28</td><td>Provocation virulent PCV2 on 25</td><td>PRRSV MLV in day 46</td><td>Section in day 50</td>

<td></td><td></td><td>control group</td><td></td><td></td><td></td><td></td><td></td>

<td> 10</td><td> 15</td><td>No-closely negative control group</td><td>ON</td><td> +</td><td> -</td><td> +</td><td> +</td>

<td colspan="3">vORF2 = isolated viral O ORF2 expression; KV killed the whole cell virus grown in a suitable ho</td><td colspan="5">RF2; rORF2 = recombinant baculovirus showing virus cells = PCV2; any cells</td>

[0113] The following preparations were given to each group. Vaccine PCV2 No. 1, administered to group 1 at a dose of 1 x 2 ml, is a high dose (16 μg / 2 ml dose) of inactivated recombinant ORF2 antigen with IMS 1314 adjuvant (16 μg rORF2 - IMS 1314).

The PCV2 vaccine No. 2 administered to Group 2 at a dose of 1 x 2 ml is a high dose (16 μg / 2 ml dose) of partially purified VIDO R-1 PCV2 ORF2 antigen with Carbopol adjuvant (16 μg vORF2 - Carbopol). Vaccine PCV2 No. 3, administered to group 3 at a dose of 1 x 2 ml, is a high dose (16 μg / 2 ml dose) of inactivated recombinant ORF2 antigen with Carbopol adjuvant (16 μg rORF2 - Carbopol). Vaccine PCV2 No. 4 administered to group 4 at a dose of 1 x 1 ml is a high dose (16 μg / 1 ml dose) of partially purified VIDO R-1 PCV2 ORF2 antigen with Carbopol adjuvant (16 μg vORF2 - Carbopol). Vaccine 5, administered to group 5 at a dose of 1 x 2 ml, is a dose of 4 μg / 2 ml of inactivated recombinant ORF2 antigen with Carbopol adjuvant (4 μg rORF2 - Carbopol). The PCV2 vaccine No. 6 administered in Group 6 at a dose of 1 x 2 ml is the dose

1 μg / 2 ml of inactivated recombinant ORF2 antigen with Carbopol adjuvant (1 μg rORF2 - Carbopol). Vaccine PCV2 No. 7, administered to Group 7 at a dose of 1 x 2 ml is a low dose (0.25 μg / 2 ml dose) of inactivated recombinant ORF2 antigen with Carbopol adjuvant (0.25 μg rORF2 - Carbopol). Vaccine PCV2 No. 8, administered to group 8 at a dose of 1 x 2 ml is a high dose (pre-inactivation titre> 8.0 log / 2 ml,) inactivated conventional killed PCV2 Struve antigen generated in VIDO R-1 with Carbopol adjuvant (> 8.0 log KV - Carbopol). On day 0, groups 1-8 were treated with the assigned vaccines. Groups 1-3 and 5-8 received booster doses of the respective vaccines again on Day 14. The effectiveness of a single dose of 16 μg vORF2 - Carbopol was investigated in group 4 who did not receive booster vaccination on day 14. Piglets were observed for adverse reactions and injection site reactions after both vaccinations. On the day the piglets were moved to the second research center, where groups 1-9 were combined and kept in one building and group 10 was placed in a separate building. All pigs received hemocyanin emulsified with incomplete Freund's adjuvant (KLH / ICFA) on days 22 and 28. On day 25, groups 1-9 were infected with about 4 logs of virulent PCV2 virus. By day 46, very few deaths occurred in the infected control group. In an attempt to immunostimulate pigs and increase the virulence of PCV2 challenged material, all groups were treated on Ingelvac on day 46^® PRRSV MLV (Porcine Respiratory Reproductive Vaccine Vaccine, Live attenuated virus).

[0114] Before and after the challenge, blood samples were taken for PCV2 serological tests. After the challenge, body weight data was collected to determine daily weight gain (ADWG) and clinical symptoms. On day 50, all surviving pigs were dissected, pathological changes recorded, lung changes assessed, and selected tissues retained in formalin for immunohistochemistry (IHC) at a later date to detect PCV2 antigen.

Materials and methods [0115] The study was a partially blind feasibility study of vaccination challenge in CDCD pigs aged 6 to 16 days of age on day 0. The inclusion criterion was PCV2 IFA titer in sows &lt;1000. In addition, sow serological status corresponded to this for a PRRS negative herd. The serological status of PCV2 was examined in sixteen (16) sows and in sixteen (16) sows PCV2 titres were &lt;1000 and transferred to the first research center. One hundred fifty (150) piglets after caesarean section were included in this study on day -3. On day -3, 150 CDCDs of pigs in the first test center were weighed, pierced, blocked by weight and randomly allocated to 1 out of 10 groups as shown above in Table 16. Blood samples were taken from all pigs. If any animal meeting the inclusion criteria and included in the study was later excluded from it for any reason, the data obtained from it was consulted by the investigator and the Monitor Monitor to determine their use in the final analysis. The documentation covered data which piglets were excluded and what was the reason for exclusion. None of the sows meeting the inclusion criteria selected for testing and transported to the first testing center were not excluded. No piglets were excluded from the study and no test animals were removed from the study before the end of the study. Table 17 describes the time frame for the key activities of this example.

Table 17. Activities during the study

<td>Day survey</td><td>Dates real</td><td>Activity during the study</td>

<td> -3</td><td> 4-04-03</td><td>Weighting; health examination; Random group assignment; Taking blood samples</td>

<td> -3, 0-21</td><td>4-04-03 4-07-03 to</td><td>Observation of general health and in terms of activities side effects after vaccination</td>

<td>Day survey</td><td>Dates real</td><td>Activity during the study</td>

<td></td><td> 5-27-03</td><td></td>

<td> 0</td><td> 4-07-03</td><td>Administering appropriate vaccines (IVP) to groups 1-8,</td>

<td> 0-7</td><td>4-07-03 to 4-14-03</td><td>Observation in pigs for site reactions application</td>

<td> 14</td><td> 4-21-03</td><td>Supportive administration of the appropriate vaccine (IVP) to groups 1-3 and 5-8; Taking a blood sample from all pigs</td>

<td> 14-21</td><td>4-21-03 to 4-28-03</td><td>Observation in pigs for site reactions application</td>

<td> 19-21</td><td>4-26-03 to 4-28-03</td><td>Antibiotic treatment of all pigs</td>

<td> 21</td><td> 4-28-03</td><td>Transfer of pigs from Struve, Inc. laboratories to Veterinary Resources, Inc. (VRI)</td>

<td> 22-50</td><td>4- 28-03 down 5- 27-03</td><td>Monitoring pigs for clinical signs after challenge</td>

<td> 22</td><td> 4-29-03</td><td>Treatment of KLH / ICFA groups 1-10</td>

<td> 25</td><td> 5-02-03</td><td>Sampling of blood and nasal secretions from all pigs; Weighing all pigs; challenge of groups 1-9 with PCV2 provoking material</td>

<td> 28</td><td> 5-05-03</td><td>Treatment of KLH / ICFA groups 1-10</td>

<td> 32</td><td> 5-09-03</td><td>Blood sampling from all pigs</td>

<td> 46</td><td> 5-23-03</td><td>Administer to all Ingelvac® PRRS MLV groups</td>

<td> 50</td><td> 5-27-03</td><td>Blood sampling; Weighing all pigs and autopsy of all pigs; Registration pathological changes; Lung changes were assessed; Fresh and formalin-fixed tissue samples were retained; The in-life study phase has been completed.</td>

[0116] At the end of the in-life study phase, formalin-fixed tissues were tested by immunohistochemistry (IHC) to detect PCV2 antigen, blood samples were tested for PCV2 serology, nasal secretion samples were tested for PCV2 excretion and the mean daily weight gain (ADWG) from day 25 to day 50 was determined.

[0117] The animals were housed in the first test facility in individual cages in seven rooms from birth until about the 11th day of life (approximately day 0 of the study). Each room had an identical layout and consisted of individual stainless steel cages arranged, with a supply of heated and filtered air supplied separately for each cage. Each room had separate heating and ventilation to prevent mutual air pollution between rooms. In the second research center, the animals were kept in two different buildings. Group 10 (strictly negative control group) was placed separately in the converted building for young pigs and groups 1-9 were placed in the converted building for pigs before slaughter. Each group was placed in a separate pen (1,415 pigs in the pen) and each pen provided an area of ​​approximately 2.3 square meters per pig. Groups 2, 4 and 8 were placed in three adjacent pens on one side of the alley and groups 1, 3, 5, 6, 7 and 9 were placed in six adjacent pens on the other side of the alley. Group separation was due to anxiety in the Study Monitor that vaccines administered to groups 2, 4 and 8 were not completely inactivated. Each pen was placed on a platform made of plastic slats. The trench located under the farms served as a tank to store excrement and waste. Each building had its own heating and ventilation systems, with low probability of mutual air pollution between buildings.

[0118] In the first research center, piglets were fed specially formulated milk from birth to about 3 weeks of age. All piglets ate solid foods, specially mixed portions up to 21 days (about 4 1/2 weeks of age). In the second research center, all piglets were fed a customary non-medicated commercial mix appropriate for their age and weight, ad libitum. Water was also available ad libitum in both research centers.

[0119] All pigs tested received 1.0 ml Naxcel^® intramuscularly in alternating hams on 19, 20 and 21. In addition, for various health reasons, pig No. 11 (Group 1) was treated with Naxcel^® intramuscularly on day 10, pig No. 13 (Group 10) was treated with 1 ml penicillin and 1 ml Predef^® 2x on day 10, pig No. 4 (Group 9) was treated with 1 ml of Naxcel^® intramuscularly on day 11, pig No. 1 (Group 1), No. 4 and 11 were each treated with 1.0 ml Naxcel^® on day 14

[0120] While in both research centers, pigs remained under veterinary care. Animal health tests were conducted on day -3 as recorded on the Animal Health Card form. Prior to vaccination, all animals were in good health and nutrition, as determined by observation on day 0. Before infection, all test animals were observed for good health and nutrition. Animal bodies and tissues were removed by smelting. Final removal of test animals was recorded in an Animal Disposition Record.

[0121] On days 0 and 14, pigs assigned to groups 1-3 and 5-8 received 2.0 ml, respectively, PCV2 vaccines No. 1-4, intramuscularly, in the right and left neck region, respectively, using a 3.0 ml sterile Luer syringe -lock and a 20g x ¥ "sterile needle. Pigs assigned to group 4 received 1.0 ml PCV2 vaccine No. 2 intramuscularly in the right neck region using a 3.0 ml sterile Luer-lock syringe and a 20g x ¥ 2" sterile needle only on 0.

[0122] On day 22, all pigs tested received 2.0 ml KLH / ICFA intramuscularly in the left neck region using a 3.0 ml sterile Luer-lock syringe and a 20g x 1 "sterile needle. On day 28, all pigs tested received 2.0 ml KLH / ICFA in the right region of the ham using a 3.0 ml sterile Luer-lock syringe and a sterile 20g x 1 "needle.

[0123] On day 25, pigs assigned to groups 1-9 received 1.0 ml of PCV2 ISUVDL challenge material (3.98 log10 TCID50 / ml), intramuscularly the right neck area using a 3.0 ml sterile Luer-lock syringe and sterile needle 20g x 1 ". An additional 1.0 ml of the same material was administered intranasally to each pig (0.5 ml on the nostril) using a 3.0 ml sterile Luer-lock syringe and nasal cannula.

[0124] On day 46, all test pigs received intramuscularly 2.0 ml of Ingelvac^®PRRS MLV, to the right neck area using a 3.0 ml sterile Luer-lock syringe and a sterile 20g x 1 ”needle. PRRSV MLV is given to increase the virulence of PCV2 challenge material.

[0125] Test pigs were observed daily for overall health and the occurrence of side effects on day -3 and from day 0 to day 21. Each pig was evaluated for normal or abnormal behavior, abnormal breathing or coughing. Observations were recorded in a clinical observation report. All pigs tested were observed from day 0 to day 7 and group 7 was further observed from day 14 to 21 for injection site reactions. The average daily weight gain was determined by weighing each pig on a calibrated weight on days -3, 25 and 50, or on the day when dead after infection was found. Animal body weights were recorded on the Body Mass Form. Body weight of -3 was used to block pigs prior to randomization. Body weight measurement data obtained on day 25 and day 50 was used to determine the average daily weight gain (ADWG) for each pig during these time points. In the case of pigs that died after infection and before day 50, ADWG was established as ADWG from day 25 to day of death.

[0126] To determine the PCV2 serology, all venous blood was collected from the orbital venous sinus on days -3 and 14. Blood was collected from each piglet from the venous orbital sinus by inserting a sterile capillary tube into the medial ligament of one eye and extending approximately 3.0 ml whole blood to a 4.0 ml tube with serum separator (SST). On days 25, 32 and 50, venous blood from each pig was collected from the anterior vena cava using a 20g x 1 ¥ "Vacutainer® sterile needle (Becton Dickinson and Company, Franklin Lakes, New Jersey), Vacutainer needle holder^® and 13 ml SST. Blood collected at each time point was recorded in the Sampling Register. Blood in each SST was allowed to clot, each SST was then centrifuged and the serum collected. Collected serum was transferred to sterile, snap-closed tubes and stored at -70 ± 10 ° C for testing at a later date. Serum samples were tested for the presence of PCV2 antibodies by BIVI-R&amp;D staff.

[0127] Pigs were observed once a day from day 22 to day 50 for clinical signs and the normal or abnormal behavior, breathing disorder or cough was assessed. Clinical observations were recorded in the Clinical Observations Register.

[0128] Pigs No. 46 (Group 1) and 98 (Group 9) died in the first research center. The diagnosis of the cause of both deaths was bleeding and no autopsy was performed in these pigs. In the second research center, pigs that died after infection and before day 50 and pigs euthanized on day 50 were carried out. In the Section Report Form all pathological changes were recorded and the percentage changes in part of the lung lobes were recorded.

[0129] A tonsil, lung, heart, and mesenteric lymph node tissue sample was taken from each pig subjected to the second test center and placed in one container with 10% buffered formalin; and a second tissue sample from the same organs mentioned above was placed in sterile Whirl-pak bags^® (M-Tech Diagnostics Ltd, Thelwall, UK), and each of the Whirl-pak pouches^® placed on ice. Each container has been correctly marked. Samples collected were recorded on the Section Report Form. Next, formalin-fixed tissue samples and diagnostic form were submitted for IHC test. The IHC test was conducted according to ISU standard laboratory procedures for sample collection, sample preparation and preparation, and staining techniques. Fresh tissue in sterile Whirl-Paks bags were exported on ice to the test monitor for storage (-70 ° ± 10 ° C) and possible future use.

[0130] Formalin-fixed tissues were examined by a pathologist for detection of PCV2 by IHC and evaluation in the following scoring system: 0 = none; 1 = scanty positive staining, several places; 2 = Moderate positive staining, many places; and 3 = abundant positive staining, scattered throughout the tissue. For analytical purposes, a "0" result was considered "negative" and a result greater than "0" was considered "positive".

Results [0131] The results of this example are given below. It was noted that pigs Nos. 46 and 98 died on days 14 and 25, respectively. These deaths were classified as deaths due to bleeding. Pig No. 11 (Group 1) was panting and breathing faster on Day 15. Except for these cases, all pigs showed normal behavior, breathing and coughing during this observation period, and no systemic side effects were seen in any group. There was no injection site reaction after vaccination on day 0. After vaccination on day 14, seven (7) of fourteen (14) pigs in group 1 (50.0%) had edema with '2' on day 15. Four (4) of fourteen (14) pigs in group 1 (28.6%) still had swelling with a score of "2" on day 16. None of the other groups had any injection site reactions after each vaccination.

[0132] The results for mean daily weight gain (ADWG) are presented below in Table 18. Pigs No. 46 and 98 who died of bleeding were excluded from the analysis of the results. Group 4, which received one dose of 16 μg vORF2 - Carbopol had the highest ADWG value (1.16 ± 0.26 lb / day), followed by groups 1, 2, 3, 5, 6 and 10, which had ADWG values ranging from 1.07 ± 0.23 pounds / day to 1.11 ± 0.26 pounds / day. Group 9 had the lowest ADWG value (0.88 ± 0.29 pounds / day), followed by groups 8 and 7 in which the ADWG values ​​were

0.93 ± 0.33 pounds / day and 0.99 ± 0.44 pounds / day.

Table 18. Summary of the average daily weight gain (ADWG) for each group

<td>Group</td><td>Treatment</td><td>N</td><td>ADWG - pounds / day (Day 25 to 50 day) or determined for pigs dead before day 50</td>

<td> 1</td><td>rORF2 16 μg - IMS 1314 2 dose</td><td> 14</td><td>1.08 ± 0.30 pounds / day</td>

<td> 2</td><td>vORF2 16 μg - Carbopol 2 dose</td><td> 15</td><td>1.11 ± 0.16 pounds / day</td>

<td> 3</td><td>rORF2 16 μg - Carbopol 2 doses</td><td> 15</td><td>1.07 ± 0.21 pounds / day</td>

<td> 4</td><td>vORF2 16 μg - Carbopol 1 dose</td><td> 15</td><td>1.16 ± 0.26 pounds / day</td>

<td> 5</td><td>rORF2 4 μg - Carbopol 1 dose</td><td> 15</td><td>1.07 ± 0.26 pounds / day</td>

<td> 6</td><td>rORF2 1 μg - Carbopol 2 dose</td><td> 15</td><td>1.11 ± 0.26 pounds / day</td>

<td> 7</td><td>rORF2 0.25 μg - Carbopol 2 doses</td><td> 15</td><td>0.99 ± 0.44 pounds / day</td>

<td> 8</td><td>KV&gt; 8,0 log -Carbopol 2 doses</td><td> 15</td><td>0.93 ± 0.33 pounds / day</td>

<td> 9</td><td>A provoked group control</td><td> 14</td><td>0.88 ± 0.29 pounds / day</td>

<td> 10</td><td>strictly negative group control</td><td> 15</td><td>1.07 ± 0.23 pounds / day</td>

<td colspan="2">vORF2 = isolated viral OR ORF2 expression; KV or killed whole k</td><td colspan="2">F2; rORF2 = recombinant baculovirus showing virus cells = PCV2;</td>

<td>Group</td><td>Treatment</td><td>N</td><td>ADWG - pounds / day (Day 25 to 50 day) or determined for pigs dead before day 50</td>

<td>virus</td><td colspan="3">grown in an appropriate cell culture</td>

[0133] PCV2 serological results are shown below in Table 19. All ten (10) groups were seronegative to PCV2 on day -3. On day 14, PCV2 titers remained low (range 50-113) for all ten (10) groups. On day 25, group 8, which received the killed whole cell viral vaccine, had the highest titre of PCV2 (4617), followed by group 2 that received 16 μg vORF2 Carbopol, group 4, which received a single dose of 16 μg vORF2 - Carbopol and group 3, which received 16 μg rORF2 - Carbopol, whose titers were 2507, 1920 and 1503, respectively. On day 32 (one week after challenge), titers for groups 1-6 and group 8 ranged from

2360 up to 7619; during group 7 (0.25 μg rORF2 - Carbopol), 9 (challenge control group), and 10 (Strictly negative control group) titers were 382, ​​129 and 78, respectively. On day 50 (day of section), all ten (10 ) groups showed high titers of PCV2 (> 1257).

[0134] On days 25, 32 and 50, group 3, which received two doses of 16 μg rORF2, Carbopol had higher antibody titers than group 1, which received two doses of 16 μg rORF2 - IMS 1314. On days, 25, 32 and 50 , group 2, which received two doses of 16 μg vORF2, had higher titers than group 4, which only received one dose of the same vaccine. Groups 3, 5, 6, 7, which received the decreasing concentration of rORF2 - Carbopol 16, 4, 1 and 0.25 μg, respectively, had reduced antibody titers on days 25 and 32, respectively.

Table 19. Summary data for PCV2 IFA titers for all groups

<td colspan="7">Average IFA titers</td>

<td>Group</td><td>Treatment</td><td>Day -3</td><td>Day 14 **</td><td>Day 25 ***</td><td>Day 32</td><td>Day **** 50</td>

<td> 1</td><td>rORF2 16 μg - IMS 1314 2 doses</td><td> 50</td><td> 64</td><td> 646</td><td> 3326</td><td> 4314</td>

<td> 2</td><td>vORF2 16 μg - Carbopol 2 doses</td><td> 50</td><td> 110</td><td> 2507</td><td> 5627</td><td> 4005</td>

<td> 3</td><td>rORF2 16 μg - Carbopol 2 doses</td><td> 50</td><td> 80</td><td> 1503</td><td> 5120</td><td> 6720</td>

<td> 4</td><td>vORF2 16 μg - Carbopol 1 dose</td><td> 50</td><td> 113</td><td> 1920</td><td> 3720</td><td> 1257</td>

<td> 5</td><td>rORF2 4 μg - Carbopol 1 dose</td><td> 50</td><td> 61</td><td> 1867</td><td> 3933</td><td> 4533</td>

<td> 6</td><td>rORF2 1 μg - Carbopol 2</td><td> 50</td><td> 70</td><td> 490</td><td> 2360</td><td> 5740</td>

<td colspan="7">Average IFA titers</td>

<td>Group</td><td>Treatment</td><td>Day -3</td><td>Day 14 **</td><td>Day 25 ***</td><td>Day 32</td><td>Day **** 50</td>

<td></td><td>dose</td><td></td><td></td><td></td><td></td><td></td>

<td> 7</td><td>rORF2 0.25 μg Carbopol 2 doses</td><td> 50</td><td> 73</td><td> 63</td><td> 382</td><td> 5819</td>

<td> 8</td><td>KV&gt; 8,0 log -Carbopol 2 doses</td><td> 50</td><td> 97</td><td> 4617</td><td> 7619</td><td> 10817</td>

<td> 9</td><td>A provoked group control</td><td> 50</td><td> 53</td><td> 50</td><td> 129</td><td> 4288</td>

<td> 10</td><td>strictly negative group control</td><td> 50</td><td> 50</td><td> 50</td><td> 78</td><td> 11205</td>

<td colspan="7">vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus showing ORF2 expression; KV or whole virus cells killed = PCV2 virus was cultured in an appropriate cell culture * For calculation purposes &lt;100 IFA, the titre was designated as "50"; &gt; 6400 titers IFA was designated as "12800". ** Provocation Day *** Section Day</td>

[0135] The results of post-challenge clinical observations are presented below. Table 20 includes observations for Abnormal Behavior, Abnormal Breathing, Cough and Diarrhea. Table 21 contains results from a group summary of the incidence of general clinical symptoms, and Table 22 contains results from a group summary of post-infection mortality rates. The incidence of abnormal behavior, respiratory distress and coughing after infection was low in pigs receiving 16 μg rORF2-IMS 1314 (group 1), 16 μg rORF2-Carbopol (group 3), 1 μg rORF2-Carbopol (group 6), 0.25 μg rORF2- Carbopol (group 7) and in pigs in the control group (group 9). The incidence of abnormal behavior, respiration and cough after challenge was zero in pigs receiving 16 μg vORF2-Carbopol (group 2), a single dose ^ g vORF2-Carbopol (group 4), 4 μg rORF2-Carbopol (group 5),> 8 log KV-Carbopol (group 8) and pigs from the strictly negative control group (group 10).

[0136] The overall incidence of clinical symptoms varied between groups. Pigs receiving 16 μg vORF2-Carbopol (Group 2), a single dose of 16 μg vORF2-Carbopol (Group 4) and pigs in the strictly negative control group (Group 10) showed a 0% incidence; pigs receiving 16 μg rORF2-Carbopol (group 3) and 1 μg rORF2-Carbopol (group 6) showed an incidence of 6.7%; pigs receiving 16 μg rORF2-IMS 1314 (group 1) had an overall incidence rate of 7.1%; pigs receiving 4 μg rORF2-Carbopol (Group 5), 0.25 μg rORF2Carbopol (Group 7), and the> 8 log KV vaccine showed an incidence rate of 13.3%; and pigs in the infected control group (group 9) showed an incidence of 14.3%.

[0137] General mortality rates between groups were also varied.

Group 8 receiving 2 doses of the KV vaccine had the highest mortality rate of 20.0%; then the results belonged to group 9, the infected control group and group 7, which received 0.25 μg rORF2-Carbopol and showed mortality of 14.3% and 13.3%, respectively. Group 4, which received one 16 μg dose of vORF2-Carbopol, showed a mortality rate of 6.7%. All other groups 1, 2, 3, 5, 6 and 10 were present

0% mortality rate.

Table 20. Summary data for abnormal behavior observation, abnormal

Breathing and coughing for all groups

<td>Group</td><td>Treatment</td><td>N</td><td>Incorrect Behavior¹</td><td>Incorrect Behavior²</td><td>Cough³</td>

<td> 1</td><td>rORF2 16 μg - IMS 1314 2 doses</td><td> 14</td><td> 0/14 (0%)</td><td> 0/14 (0%)</td><td> 1/14 (7,1%)</td>

<td> 2</td><td>vORF2 16 μg Carbopol 2 doses</td><td> 15</td><td> 0/15 (0%)</td><td> 0/15 (0%)</td><td> 0/15 (0%)</td>

<td> 3</td><td>rORF2 16 μg Carbopol 2 doses</td><td> 15</td><td> 0/15 (0%)</td><td> 0/15 (0%)</td><td> 1/15 (6,7%)</td>

<td> 4</td><td>vORF2 16 μg Carbopol 1 dose</td><td> 15</td><td> 0/15 (0%)</td><td> 0/15 (0%)</td><td> 0/15 (0%)</td>

<td> 5</td><td>rORF2 4 μg Carbopol 1 dose</td><td> 15</td><td> 1/15 (6,7%)</td><td> 1/15 (6,7%)</td><td> 0/15 (0%)</td>

<td> 6</td><td>rORF2 1 μg Carbopol 2 doses</td><td> 15</td><td> 0/15 (0%)</td><td> 0/15 (0%)</td><td> 1/15 (6,7%)</td>

<td> 7</td><td>rORF2 0.25 μg Carbopol 2 doses</td><td> 15</td><td> 0/15 (0%)</td><td> 1/15 (6,7%)</td><td> 1/15 (6,7%)</td>

<td> 8</td><td>KV&gt; 8,0 log -Carbopol 2 doses</td><td> 15</td><td> 1/15 (6,7%)</td><td> 1/15 (6,7%)</td><td> 0/15 (0%)</td>

<td> 9</td><td>A provoked group control</td><td> 14</td><td> 1/14 (7,1%)</td><td> 1/14 (7,1%)</td><td> 2/14 (14,3%)</td>

<td> 10</td><td>Strictly negative control group</td><td> 15</td><td> 0/15 (0%)</td><td> 0/15 (0%)</td><td> 0/15 (0%)</td>

<td>Group</td><td>Treatment</td><td>N</td><td>Incorrect Behavior¹</td><td>Incorrect Behavior²</td><td>Cough³</td>

<td colspan="6">¹Total number of pigs in each group that showed any abnormal behavior for at least one day²The total number of pigs in each group that showed any abnormal breathing for at least one day³Total number of pigs in each group that showed a cough for at least one day</td>

Table 21. Summary data on the overall incidence of clinical symptoms for all groups

<td>Group</td><td>Treatment</td><td>N</td><td>Frequencies clinical signs in pigs¹</td><td>Frequency indicator occurrence</td>

<td> 1</td><td>rORF2 16 μg - IMS 1314 2 doses</td><td> 14</td><td> 1</td><td> 7,1%</td>

<td> 2</td><td>vORF2 16 μg Carbopol 2 doses</td><td> 15</td><td> 0</td><td> 0,0%</td>

<td> 3</td><td>rORF2 16 μg Carbopol 2 doses</td><td> 15</td><td> 1</td><td> 6,7%</td>

<td> 4</td><td>vORF2 16 μg Carbopol 1 dose</td><td> 15</td><td> 0</td><td> 0,0%</td>

<td> 5</td><td>rORF2 4 μg - Carbopol 1 dose</td><td> 15</td><td> 2</td><td> 13,3%</td>

<td> 6</td><td>rORF2 1 μg - Carbopol 2 doses</td><td> 15</td><td> 1</td><td> 6,7%</td>

<td> 7</td><td>rORF2 0.25 μg Carbopol 2 doses</td><td> 15</td><td> 2</td><td> 13,3%</td>

<td> 8</td><td>KV&gt; 8,0log -Carbopol 2 doses</td><td> 15</td><td> 2</td><td> 13,3%</td>

<td> 9</td><td>A provoked group control</td><td> 14</td><td> 2</td><td> 14,3%</td>

<td> 10</td><td>Strictly negative control group</td><td> 15</td><td> 0</td><td> 0%</td>

<td colspan="5">vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus showing ORF2 expression; KV or whole virus cells killed = PCV2 virus was cultured in an appropriate cell culture¹total number of pigs in each group that showed any clinical signs by which</td>

<td>Group</td><td>Treatment</td><td>N</td><td>Frequencies clinical signs in pigs¹</td><td>Frequency indicator occurrence</td>

<td colspan="5">at least one day</td>

Table 22. Summary data on post-infection mortality rates for all groups

<td>Group</td><td>Treatment</td><td>N</td><td>dead infection</td><td>after</td><td>Mortality Rate</td>

<td> 1</td><td>rORF2 16 μg - IMS 1314 2 dose</td><td> 14</td><td colspan="2"> 0</td><td> 0,0%</td>

<td> 2</td><td>vORF2 16 μg - Carbopol 2 dose</td><td> 15</td><td colspan="2"> 0</td><td> 0,0%</td>

<td> 3</td><td>rORF2 16 μg - Carbopol 2 doses</td><td> 15</td><td colspan="2"> 0</td><td> 0,0%</td>

<td> 4</td><td>vORF2 16 μg - Carbopol 1 dose</td><td> 15</td><td colspan="2"> 1</td><td> 6,7%</td>

<td> 5</td><td>rORF2 4 μο - Carbopol 1 dose</td><td> 15</td><td colspan="2"> 0</td><td> 0,0%</td>

<td> 6</td><td>rORF2 1 pg - Carbopol 2 dose</td><td> 15</td><td colspan="2"> 0</td><td> 0,0%</td>

<td> 7</td><td>rORF2 0.25 after - Carbopol 2 dose</td><td> 15</td><td colspan="2"> 2</td><td> 13,3%</td>

<td> 8</td><td>KV&gt; 8,0 log -Carbopol 2 doses</td><td> 15</td><td colspan="2"> 3</td><td> 20,0%</td>

<td> 9</td><td>Provoked control group</td><td> 14</td><td colspan="2"> 2</td><td> 14,3%</td>

<td> 10</td><td>Strictly negative group control</td><td> 15</td><td colspan="2"> 0</td><td> 0,0%</td>

<td>vORF2 =</td><td colspan="2">isolated ORF2 viral; rORF2</td><td colspan="2">= recombined</td><td>baculovirus showing</td>

<td colspan="6">ORF2 expression; KV or whole virus cells killed = PCV2 virus was cultured in the appropriate</td>

<td colspan="2">cell culture</td><td></td><td></td><td></td><td></td>

[0138] A summary of the group data on the average percentage of lung changes and the initial diagnosis is given below in Table 23. In group 9, the infected control group, the highest percentage of lung changes occurred with an average of 10.81 ± 23.27%, then in group 7 who received 0.25 μg rORF2-Carbopol had an average of 6.57 ± 24.74%, in group 5 who received 4 μg rORF2-Carbopol there was an average of 2.88 ± 8.88%, and in group 8 who received the vaccine KV had an average of 2.01 ± 4.98%. The other six (6) groups had lower mean lung changes ranging from 0.11 ± 0.38% to 0.90 ± 0.15%.

[0139] The initial diagnosis of pneumonia varied among groups. Group 3, which received two doses of 16 μg rORF2-Carbopol, had the lowest pre-diagnosed pneumonia value of 13.3%. Group 9, a provoked control group with 50% pre-diagnosed pneumonia, followed by Group 10, a strictly negative control group and Group 2 who received two doses of 16 μg vORF2-Carbopol, in which the pre-diagnosed pneumonia was 46, respectively. 7% and 40%.

[0140] Groups 1, 2, 3, 5, 9 and 10 were initially diagnosed as 0% infected with PCV2; while group 8, which received two doses of the KV vaccine, had the highest rate of group initial diagnosis of PCV2 infection, which was 20%. Group 7, which received two 0.25 μg doses of rORF2-Carbopol, and group 4, which received one 16 μg dose of vORF2Carbopol had a preliminary diagnosis of PCV2 infection of 13.3% and 6.7% for each group, respectively.

[0141] Gastric ulcers were diagnosed in only one pig of Group 7 (6.7%); while the other 9 groups remained free from stomach ulcers.

Table 23. Summary data on group results of mean% lung changes and initial diagnoses

<td>Group</td><td>Treatment</td><td>N</td><td>Number of pigs that moult for at least one day</td><td>Frequency occurrence</td>

<td> 1</td><td>rORF2 16 μg - IMS 1314 2 dose</td><td> 15</td><td> 0</td><td> 0%</td>

<td> 2</td><td>vORF2 16 μg - Carbopol 2 dose</td><td> 15</td><td> 1</td><td> 6,7%</td>

<td> 3</td><td>rORF2 16 μg - Carbopol 2 doses</td><td> 15</td><td> 3</td><td> 20%</td>

<td> 4</td><td>vORF2 16 μg - Carbopol 1 dose</td><td> 15</td><td> 2</td><td> 13,3%</td>

<td> 5</td><td>rORF2 4 pg - Carbopol 1 dose</td><td> 15</td><td> 3</td><td> 20,0%</td>

<td> 6</td><td>rORF2 1 pg - Carbopol 2 dose</td><td> 15</td><td> 6</td><td> 40,0%</td>

<td> 7</td><td>rORF2 0.25 pg - Carbopol 2 dose</td><td> 15</td><td> 7</td><td> 46,7%</td>

<td> 8</td><td>KV&gt; 8,0log -Carbopol 2 doses</td><td> 15</td><td> 12</td><td> 80%</td>

<td> 9</td><td>Provoked control group</td><td> 14</td><td> 14</td><td> 100,0%</td>

<td> 10</td><td>Strictly negative group control</td><td> 15</td><td> 14</td><td> 93,3%</td>

<td>Group</td><td>Treatment</td><td>N</td><td>Number of pigs that moult for at least one day</td><td>Frequency occurrence</td>

<td colspan="5">vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus showing ORF2 expression; KV or whole virus cells killed = PCV2 virus was cultured in an appropriate cell culture</td>

[0142] Aggregate data for all IHC positive groups is presented below in Table 24. Group 1 (16 μg rORF2-IMS 1314) had the lowest IHC positive group index with a value of 0% PCV2 positive pigs followed by group 2 ( 16 μg vORF2 - Carbopol) and group 4 (single dose 16 μg vORF2 - Carbopol), which showed a group index of positive IHC results of 6.7% and 13.3%, respectively. Group 9 infected with an infected control group showed the highest positive IHC in 100% PCV2 positive pigs, followed by group 10, strictly negative control group and group 8 (KV vaccine) with values ​​of

93.3% and 80% pigs positive for PCV2.

Table 24. Summary data for all positive occurrence groups

IHC

<td>Group</td><td>Treatment</td><td>N</td><td>Number of pigs that moult for at least one day</td><td>Frequency occurrence</td>

<td> 1</td><td>rORF2 16 μg - IMS 1314 2 doses</td><td> 15</td><td> 0</td><td> 0%</td>

<td> 2</td><td>vORF2 16 μg - Carbopol 2 doses</td><td> 15</td><td> 1</td><td> 6,7%</td>

<td> 3</td><td>rORF2 16 μg - Carbopol 2 doses</td><td> 15</td><td> 3</td><td> 20,0%</td>

<td> 4</td><td>vORF2 16 μg - Carbopol 1 dose</td><td> 15</td><td> 2</td><td> 13,3%</td>

<td> 5</td><td>rORF2 4 μg - Carbopol 1 dose</td><td> 15</td><td> 3</td><td> 20,0%</td>

<td> 6</td><td>rORF2 1 μg - Carbopol 2 dose</td><td> 15</td><td> 6</td><td> 40,0%</td>

<td> 7</td><td>rORF2 0.25 μg Carbopol 2 doses</td><td> 15</td><td> 7</td><td> 46,7%</td>

<td> 8</td><td>KV&gt; 8,0 log -Carbopol 2 doses</td><td> 15</td><td> 12</td><td> 80%</td>

<td>Group</td><td>Treatment</td><td>N</td><td>Number of pigs that moult for at least one day</td><td>Frequency occurrence</td>

<td> 9</td><td>A provoked group control</td><td> 14</td><td> 14</td><td> 100,0%</td>

<td> 10</td><td>Strictly negative group control</td><td> 15</td><td> 14</td><td> 93,3%</td>

<td>vORF2</td><td>= isolated viral O</td><td>RF2;</td><td colspan="2">rORF2 = recombinant baculovirus showing</td>

<td colspan="5">ORF2 expression; KV or whole virus cells killed = PCV2 virus was cultured in the appropriate</td>

<td colspan="2">cell culture</td><td></td><td></td><td></td>

Discussion [0143] This example evaluated seven PCV2 vaccines that contained a high dose (16 μg) of rORF2 antigen with IMS 1314 adjuvant administered twice, a high dose (16 μg) of vORF2 antigen with Carbopol adjuvant administered as one dose to one group of pigs and twice in the second group of pigs, a high dose (16 μg) of rORF2 antigen with Carbopol adjuvant administered twice, a dose of 4 μg of rORF2 antigen with Carbopol adjuvant administered twice, a dose of 1 μg rORF2 antigen with Carbopol adjuvant administered twice, a low dose (0.25 μg) of rORF2 antigen with Carbopol adjuvant administered twice and a high dose (> 8 log) of killed PCV2 vaccine with Carbopol adjuvant.

In general, Group 1, which received two doses of 16 μg rORF2 - IMS 1314, obtained slightly better results from Groups 2 to 7, which received vaccines containing different levels of both vORF2 or rORF2 antigens with Carbopol adjuvant and much better than Group 8, which received two dose of whole cell PCV2 vaccine killed. Group 1 achieved the third highest ADWG (1.80 ± 0.30 pounds / day), lowest incidence of abnormal behavior (0%), lowest incidence of respiratory distress (0%), low incidence of cough (7.1 %), the low incidence of general clinical symptoms (7.1%) was included in three other groups with the lowest mortality rate (0%), showed the second lowest rate for the average% lung changes (0.15 ± 0) , 34%). the second lowest rate for pneumonia (21.4%), and the lowest rate for IHC positive tissue (0%). However, group 1 was the only group in which injection site reactions were observed which affected 50% of those vaccinated after 1 day of the second vaccination. Other vaccines administered to groups 2 to 7 performed better than killed whole cell vaccine and almost as good as the vaccine administered in group 1.

[0144] Group 8, which received two doses of killed PCV2 vaccine with Carbopol adjuvant, obtained the worst set of results among the remaining vaccine group. Group 8 had the lowest ADWG (0.93 ± 0.33 pounds / day), the second highest rate of abnormal behavior (6.7%), the highest rate of respiratory distress (6.7%), this group was included in three other groups with the highest overall clinical signs (13.3%), achieved the highest mortality rates of all groups (20%), and achieved the highest IHC positive tissue rates (80%) of all vaccine groups. There was no fear that she killed the whole-cell PCV2 vaccine had not been fully inactivated prior to administration to group 8, which could explain the poor results of this group. Unfortunately, the final data that could confirm this was not available. Generally, in the context of this example, conventional killed PCV2 vaccine does not help control PCV2-related disease.

[0145] As previously mentioned, no adverse effects related to the tested vaccines were reported, except for vaccines with IMS 1314 adjuvant. Injection site reactions were reported in 50.0% pigs on day 1 after the second dose of IMS 1314 vaccine and 28.6% pigs on day 2 after the second vaccination. No reaction was reported in any pigs receiving Carbopol as a vaccine adjuvant. Any further studies that would include pigs vaccinated with IMS 1314 adjuvanted vaccines should be closely monitored for injection site reactions.

[0146] All pigs were PCV2 seronegative on day -3 and only group 2 had a titre above 100 on day 14. On day 25 (infection day), group 8 had the highest PCV2 antibody titer (4619) followed by group 2 (2507 ). With the exception of groups 7, 9 and 10, all groups showed a strong immune response on day 32. By day 50, all groups, including groups 7, 9 and 10, showed a strong immune response.

[0147] One of the hallmarks of late stage PCV2 infection and the subsequent development of PMWS is the retardation of the growth of weaned piglets, and in severe cases weight loss is noted. The average daily weight gain of the groups is a quantitative method demonstrating a slowing of growth or weight loss. In this example, there were no major differences in ADWG between groups. Group 8 had the lowest ADWG 0.88 ± 0.29 pounds / day, while Group 4 had the highest ADWG 1.16 ± 0.26 pounds / day. In the context of this study, there were not enough differences between groups to base the future efficacy of vaccines on ADWG.

[0148] In addition to weight loss, the clinical symptoms associated with PMWS are dyspnoea, lethargy, pallor of the skin, and sometimes jaundice. In this example, abnormal breathing behavior and disorders and coughing are rarely observed for each group. As demonstrated in the study, no out-clinical outcomes were obtained in this infection and challenge model, and this is not a strong parameter on which the vaccine's efficacy can be based.

[0149] Generally, mortality rates were not high in this example and the lack of high mortality rates in the infected control group limits this parameter as the one on which the efficacy of the vaccine can be based. Before day 46, one of fifteen pigs died in groups 4 and 7, two of fourteen pigs died in group 9, and three of fifteen pigs died in group 8. Due to the fact that in group 9, the infected control group there were no clinical signs indicative of PCV2 and only two pigs died in this group before day 46, all pigs on day 46 were given the vaccine against Porcine Reproductive and Respiratory Syndrome (PRRSV) MLV. Earlier studies have used Ingelvac^® PRRS MLV as an immunostimulant to initiate PCV2-related PMWS disease and higher mortality was obtained in these earlier studies. Two deaths occurred shortly after administration of the PRRS vaccine on day 46 - one in group 4 on day 46 and one in group 7 on day 47 - which were probably not associated with PRRS vaccine administration. By day 50, in group 8, which received two doses of killed vaccine, the highest mortality rate (20%) followed, then in group 9 (infected control group) and in group 7 (0.25 μg rORF2 - Carbopol), with mortality rate respectively 14.3% and 13.3%. Generally, administration of the PRRS vaccine to the infection model delayed the post-infection follow-up in this example and did not significantly increase mortality rates.

[0150] Large pathological changes in pigs secondary to PMWS infected after PCV2 infection are usually made up of generalized lymphadenopathy in combination with one or more of the following symptoms: (1) interstitial pneumonia with swelling of interlobular fissures, (2) pallor of the skin or jaundice , (3) macular atrophic cirrhosis (4) gastric ulcers, (5) nephritis, and (6) reproductive disorders, for example, abortion, delivery of a dead fetus, fetal mummification, etc. During section (day 50), no jaundice, hepatitis and nephritis were reported in any of the groups. Gastric ulcers were reported in one pig of Group 7, but lymph node enlargement was not specifically studied. Based on the presence of lesions consistent with PCV2 infection, three groups had at least one pig initially diagnosed with PCV2 (PMWS). In group 8, which received two doses of the killed vaccine, 20% were pre-diagnosed with PCV2, while in group 7 and group 4, pre-diagnosed with PCV2 in 13.3% and 6.7%, respectively. During section, the results of mean% lung changes varied between groups. Groups 1, 2, 3, 4, 6 and 10 had low% lung lesions that ranged from 0.11 ± 0.38 to 0.90 ± 0.15%. As expected, group 9 of the challenge control group had the highest mean% lung lesions (10.81 ± 23.27%). In four groups, the mean% lung lesion scores were increased due to the presence of one to three pigs in each of these groups with very high lung lesion scores. Lung changes were red / purple and consolidated. Usually, lung lesions associated with PMWS are described as brown and not susceptible to interlobular edema. The lung lesions noted in this study were not associated with PCV2 infection or the presence of a second pulmonary infectious agent. As part of this study, the% lung lesion results probably do not reflect a true measure of the amount of lung infection caused by PCV2. Similarly, the initial diagnosis of pneumonia can also be exaggerated. Each pig with lung lesions, in some cases as small as 0.10%, was mentioned in the initial diagnosis of pneumonia. In this example, there were not sufficient differences between groups in terms of lesions and% lung lesion results on which the vaccine efficacy could be based.

[0151] The results of the IHC study showed the largest differences between the groups. Group 1 (16 μg rORF2 - IMS 1314) had the lowest IHC positive for PCV2 antigen (0%); while groups 9 and 10 had the highest positive IHC results with an incidence of 100% and 93.3%, respectively. Groups 3, 5, 6 and 7, which received 16, 4, 1 or 0.25 μg of rORF2 antigen with Carbopol adjuvant, respectively, had positive IHC results of 20%, 20%, 40% and 46.7% respectively. Group 2, which received two doses of 16 μg vORF2 with Carbopol adjuvant, had a positive IHC of 6.7%, while group 4, which received only one dose of the same vaccine, had a positive IHC of 13.3%. Due to the subjective nature of this study, IHC results are probably one of the best parameters for assessing the effectiveness of this vaccine.

[0152] Based on the CDCD pig model after PCV2 challenge, the minimum protective dose (MPD) of PCV2 rORF2 antigen with Carbopol adjuvant was established. Groups 3, 5, 6 and 7 each received two doses of rORF2 antigen with Carbopol adjuvant, but the level of rORF2 antigen was different for each group. Groups 3, 5, 6 and 7 each received 16, 4, 1 or 0.25 μg of the rORF2 antigen, respectively. Overall, with a decrease in rORF2 antigen levels, PCV2 antibody titers decreased and mortality rates increased, mean% lung changes, and tissue incidence were positive for IHC. Of the four groups receiving different levels of rORF2 - Carbopol groups 3 and 5, who received two doses of 16 and 4 μg of the rORF2 antigen, respectively, each had a positive IHC of only 20%, and both groups had similar antibody titers. Generally, based on positive IHC results, the minimum protective dose of rORF2 antigen administered twice approximately is 4 μg.

[0153] The antigenicity of recombinant antigens (rORF2) and VIDO R-1 (vORF2) PCV2 was assessed. Group 2 received two doses of 16 μg vORF2 and group 3 received two doses of 16 μg rORF2. Both vaccines were adjuvanted with Carbopol. Both vaccines proved safe and both showed a 0% mortality rate. In Group 2, PCV2 antibody titers were 2507 on day 25, while in Group 3, PCV2 antibody titers were 1503. Group 3 had a lower mean% lung lesion score than group 2 (0.11 ± 0.38% versus 0.90 ± 0.15%), but group 2 had a lower IHC positive tissue incidence rate than group 3 (6, 7% compared to 20%). In general, both vaccines had similar antigenic properties, but vORF2 was associated with slightly better IHC results.

[0154] The utility of two different adjuvants was determined (Carbopol and IMS 1314). Both groups 1 and 3 received two doses of the vaccine containing 16 μg of rORF2 antigen, but group 1 received IMS 1314 adjuvanted antigen, while group 3 received Carbopol adjuvanted antigen. They were basically the same in both groups

ADWG, essentially the same incidence of clinical symptoms after infection, the same mortality rate, and essentially the same results of mean% lung changes; but Group 1 had an IHC positive tissue rate of 0%, while in Group 3 this was 20%. However, group 3 that received the Carbopol adjuvanted vaccine had higher titers of PCV2 IFAT on days 25, 32 and 50 than group 1 that received the IMS 1314 adjuvanted vaccine. In general, although the PCV2 vaccine with the IMS 1314 adjuvant gave better IHC results, it did not predominantly provide better protection against PCV2 infection and elicited injection site reactions. While the PCV2 vaccine with Carbopol adjuvant gave as good results as the vaccine with IMS 1314 adjuvant, it did not cause any side effects.

[0155] The possibility of obtaining PCV2 ORF2 as a product containing 1 dose in 1 ml was determined. Both groups 2 and 4 received 16 μο vORF2 vaccine with Carbopol adjuvant on day 0, but group 2 received a second dose on day 14. Group 4 had slightly higher ADWG and lower mean% lung lesion scores than in group 2, but group 2 had higher IFAT PCV2 titers on days 25, 32 and 50, and a slightly lower incidence of positive tissue for IHC. All results for these two groups were similar. Overall, one dose of vORF2 with Carbopol adjuvant was similar to two doses of the same vaccine.

[0156] The disclosure further includes: A method (I.) for reducing or reducing the severity of clinical symptoms associated with PCV2 infection, reducing the total porcine circovirus load of an animal, and / or limiting the immunosuppressive effect of porcine circovirus infection in pigs comprising administering porcine circovirus type 2 antigen and wherein said antigen specifically comprises a coded protein by a DNA sequence showing at least 80% sequence identity to porcine circovirus type 2 ORF2.

[0157] In any of the aforementioned embodiments of the method (1.), said porcine circovirus type 2 antigen is in particular a recombinant bovulovirus expressed antigen ORF2 and / or said porcine circovirus type 2 antigen is specifically prepared and administered in one (1) ) ml per dose.

[0158] In any of the aforementioned embodiments of the method (I.), said clinical symptoms are specifically selected from the group consisting of generalized lymphadenopathy, lymphoid atrophy and multinucleated / giant histiocytes, and wherein said generalized lymphadenopathy, tissue wasting lymphoid and / or multinucleated / giant histiocytes are in particular a combination with another symptom selected from the group consisting of interstitial pneumonia with swelling of interlobular fissures, pallor of the skin or jaundice, spotty atrophic cirrhosis, gastric ulcer, nephritis, lesions similar to postoperative inflammatory atrophy and reproductive disorders.

[0159] In any of the aforementioned embodiments of method (I.), said administration is in particular carried out on pigs younger than 15 weeks old and / or said administration is especially carried out on pigs not older than 3 weeks old, preferably not older than 3 weeks 2-week.

[0160] In any of the aforementioned embodiments of method (I.), said administration in particular is accomplished within about 3 weeks of exposure to the antigen of the virulent porcine circovirus type 2.

[0161] In any of the aforementioned embodiments of method (I.), said composition in particular further comprises at least one additional component selected from the group consisting of veterinarily acceptable carriers, pharmaceutically acceptable carriers and immunomodulatory agents.

[0162] In any of the aforementioned embodiments of the method (I.), said administration is in particular selected from the group consisting of intradermal, intratracheal, intravaginal, intramuscular, intranasal, intravenous, intravascular, intraarterial, intraperitoneal, oral, intrathecal, subcutaneous administration , intradermal, intracardial, lung lobes, spinal or pulmonary and / or wherein the administration of said porcine circovirus type 2 antigen in particular shows no adverse effects or reactions at the injection site.

[0163] The disclosure further includes:

[0164] A method (II.) For improving the level of overall resistance to pig diseases comprising the administration of porcine circovirus type 2 porcine circovirus antigen.

[0165] The disclosure further includes:

[0166] A method for producing a medicament for reducing or reducing the severity of clinical symptoms associated with PCV2 infection, limiting the total porcine circovirus load of an animal, or reducing the immunosuppressive effect of porcine circovirus infection, said method comprising the steps of obtaining a porcine circovirus antigen and combining said antigen with veterinarily acceptable carriers , pharmaceutically acceptable carriers or immunomodulatory agents, wherein said antigen particularly comprises a polypeptide encoded by porcine circovirus type 2 ORF2.

[0167] The disclosure further includes:

[0168] The use of (I.) of porcine circovirus type 2 antigen for the preparation of a medicament for reducing or reducing the severity of clinical symptoms associated with PCV2 infection, limiting the total porcine circovirus load of an animal and / or limiting the immunosuppressive effect of porcine circovirus infection in pigs where said drug it is given to a pig wherein said antigen specifically comprises a protein encoded by a DNA sequence having at least 80% sequence identity with porcine circovirus type 2 ORF2.

[0169] In any of the aforementioned embodiments of use (I.), said porcine circovirus type 2 antigen is in particular a recombinant bovulovirus expressed antigen ORF2 and / or said porcine circovirus type 2 antigen is specifically prepared and administered in one ( 1) ml per dose.

[0170] In any of the aforementioned embodiments of use (I.), said clinical symptoms are specifically selected from the group consisting of generalized lymphadenopathy, lymphoid atrophy and multinucleated / giant histiocytes, and wherein said generalized lymphadenopathy, lymphoid atrophy and / or multinucleated / giant histiocytes are in particular a combination with another symptom selected from the group consisting of interstitial pneumonia with swelling of interlobular fissures, pallor of the skin or jaundice, spotty atrophic cirrhosis, gastric ulcer, nephritis, similar lesions inflammatory postoperative atrophy and reproductive disorders.

[0171] In any of the above-mentioned embodiments of use (I.), said administration is in particular carried out on pigs younger than 15 weeks old and / or said administration is carried out especially on pigs not older than 3 weeks old, preferably not older than 3 weeks The 2-week and / or said administration in particular is carried out within approximately 3 weeks of exposure to the antigen of the virulent porcine circovirus type 2.

[0172] In any of the aforementioned embodiments of use (I.), said composition in particular further comprises at least one additional component selected from the group consisting of veterinarily acceptable carriers, pharmaceutically acceptable carriers and immunomodulatory agents.

[0173] In any of the aforementioned embodiments of use (I.), said administration is in particular intradermal, intratracheal, intravaginal, intramuscular, intranasal, intravenous, intravascular, intraarterial, intraperitoneal, oral, intrathecal, subcutaneous, intradermal, intracardial, intrathecal lung, spinal or pulmonary lobes and / or administration of said porcine circovirus type 2 antigen in particular shows no adverse effects or reactions at the injection site.

[0174] The disclosure further includes:

[0175] The use of (II) porcine circovirus type 2 antigen for the preparation of a medicament for improving the level of overall resistance to swine disease.

LIST OF SEQUENCES [0176] <110> Roof, Mike Eichmeyer, Mark Nitzel, Greg

Schaeffer, Merrill Hayes, Phillip <120> APPLICATION OF PVC IMMUNOGENIC COMPOSITION TO REDUCE CLINICAL SYMPTOMS IN PIGS <130> 34816-CIP1 <150> Unknown <151> 2005-12-30 <160> 11 <170> Patent In version 3.3 <210> 1 <211> 8 <212> DNA <213> Artificial <220>

<223> This is a modified Kozak sequence <400> 1 ccgccatg 8 <210> 2 <211> 6 <212> DNA <213> Artificial <220>

<223> This is the sequence of recombinant Eco R1 <400> 2 gaattc 6 <210> 3 <211> 713 <212> DNA <213> Porcine circovirus <400> 3

<td>cagctatgac</td><td>gtatccaagg</td><td>aggcgttacc</td><td>gcagaagaag</td><td>acaccgcccc</td><td>cgcagccatc</td><td> 60</td>

<td>ttggccagat</td><td>cctccgccgc</td><td>cgcccctggc</td><td>tcgtccaccc</td><td>ccgccaccgc</td><td>taccgttgga</td><td> 120</td>

<td>gaaggaaaaa</td><td>tggcatcttc</td><td>aacacccgcc</td><td>tctcccgcac</td><td>Ctteggatat</td><td>actgtggaga</td><td> 180</td>

<td>aggaaaaatg</td><td>gcatcttcaa</td><td>cacccgcctc</td><td>tccćgcacct</td><td>tcggatatac</td><td>tgtgacgact</td><td> 240</td>

<td>ttgttccccc</td><td>gggagggggg</td><td>accaacaaaa</td><td>tctctatacc</td><td>ctttgaatac</td><td>tacagaataa</td><td> 300</td>

<td>gaaaggttaa</td><td>ggttgaattc</td><td>tggccctgct</td><td>cccccatcac</td><td>ccagggtgat</td><td>aggggagtgg</td><td> 360</td>

<td>gctccactgc</td><td>tgttattcta</td><td>gatgataact</td><td>ttgtaacaaa</td><td>ggccacagcc</td><td>ctaacctatg</td><td> 420</td>

<td>acccatatgt</td><td>aaactactcc</td><td>tcccgccata</td><td>caatccccca</td><td>acccttctcc</td><td>taccactccc</td><td> 480</td>

<td>gttacttcac</td><td>acccaaacct</td><td>gttcttgact</td><td>ccactattga</td><td>ttacttccaa</td><td>ccaaataaca</td><td> 540</td>

<td>aaaggaatca</td><td>gctttggctg</td><td>aggctacaaa</td><td>cctctagaaa</td><td>tgtggaccac</td><td>gtaggcctcg</td><td> 600</td>

<td>gcactgcgtt</td><td>cgaaaacagt</td><td>aaatacgacc</td><td>aggactacaa</td><td>tatccgtgta</td><td>accatgtatg</td><td> ' 660</td>

<td>tacaattcag</td><td>agaatttaat</td><td>cttaaagacc</td><td>ccccacttaa</td><td>accctaaatg</td><td>aat</td><td> 713</td>

<210> 4 <211> 713 <212> DNA <213> Pig circovirus <400> 4

<td>ccgccatgac</td><td>gtatccaagg</td><td>aggcgttacc</td><td>gcagaagaag</td><td>acaccgcccc</td><td>cgcagccatc</td><td> 60</td>

<td>ttggccagat</td><td>cctccgccgc</td><td>cgcccctggc</td><td>tcgtccaccc</td><td>ccgccaccgc</td><td>taccgttgga</td><td> 120</td>

<td>gaaggaaaaa</td><td>tggcatcttc</td><td>aacacccgcc</td><td>tctcccgcac</td><td>ctteggatat</td><td>actgtcaagg</td><td> 180</td>

<td>ctaccacagt</td><td>cacaacgccc</td><td>tcctgggcgg</td><td>tggacatgat</td><td>gagatttaat</td><td>attgacgact</td><td> 24 0</td>

<td>ttgttccccc</td><td>gggagggggg</td><td>accaacaaaa</td><td>tctctatacc</td><td>ctttgaatac</td><td>tacagaataa</td><td> 300.</td>

<td>gaaaggttaa</td><td>ggttgaattc</td><td>tggccctgct</td><td>cccccatcac</td><td>ccagggtgat</td><td>aggggagtgg</td><td> 360</td>

<td>gctccactgc</td><td>tgttattcta</td><td>gatgataact</td><td>ttgtaacaaa</td><td>ggccacagcc</td><td>ctaacctatg</td><td> 4 20</td>

<td>acccatatgt</td><td>aaactactcc</td><td>tcccgccata</td><td>caatccccca</td><td>acccttctcc</td><td>taccactccc</td><td> 480</td>

<td>gttacttcac</td><td>acccaaacct</td><td>gttcttgact</td><td>ccactattga</td><td>ttacttccaa</td><td>ccaaataaca</td><td> 540</td>

<td>aaaggaatca</td><td>gctttggctg</td><td>aggctacaaa</td><td>cctctagaaa</td><td>tgtggaccac</td><td>gtaggcctcg</td><td> 600</td>

<td>gcactgcgtt</td><td>cgaaaacagt</td><td>aaatacgacc</td><td>aggactacaa</td><td>tatccgtgta</td><td>accatgtatg</td><td> 660</td>

<td>tacaattcag</td><td>agaatttaat</td><td>cttaaagacc</td><td>ccccacttga</td><td>accctaagaa</td><td>ttc</td><td> 713</td>

<210> 5 <210> 233 <211> 233 <212> PRT <213> Pig circovirus <400> 5

<img file="PL2371383T3_D0001.tif" />

25 30 · ' ·.

Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg · 40 45 ·· "· · ·.

Leu Ser Arg Thr Phe Gly Tyr Thr Val Lys Ala Thr Thr Val 'Thr Thr

55 60 . .

Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asp Asp Phe Val

70 75 80

Pro Pro Gly Gly Gly Thr Asn Lys Ile Ser Ile Pro Phe Glu Tyr Tyr

-90 95

Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr

100 105 110 .

Gin Gly Asp Arg Gly Val Gly Cheese Thr Ala Val Ile Leu Asp Asp Asn

115 120 125

Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr

130 135-140

Cheese Ser Arg His Thr Ile Pro Gin Pro Phe Ser Tyr His Cheese Arg Tyr

145 · 150 155 160

Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gin 'Pro and 165 170 175

Asn Asn Lys Arg Asn Gin Leu Trp Leu Arg Leu Gin Thr Ser Arg Asn

180 185 190 74

Val Asp.His.Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Lys Tyr Asp

195 200 205

Gln Asp Tyr Asn Ile Arg Val Thr Met Tyr Val Gln Phe Arg Glu Phe

210 215 220

Asn Leu Lys Asp Pro Pro Leu Lys Pro - ^: .

225 '230 <210> 6 <211> 233 <212> PRT <213> Pig circovirus <400> 6

Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg. Pro Arg

5 10 15 Cheese His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro

25 .30 ' ·

Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg

40 .45

Leu Ser Arg Thr Phe Gly Tyr Thr Val Lys Ala Thr Thr Val Thr Thr

55 60

Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn ile Asp Asp Phe val. 70 75 80

<img file="PL2371383T3_D0002.tif" />

<210> 7 <211> 756 <212> DNA <213> Artificial <220>

<223> This sequence comes from the open reading frame 2 of the porcine circovirus type 2, together with a part from the pGEM T-easy <400> 7

<td>gcggccgcgg</td><td>gaattcgatc</td><td>cgccatgacg</td><td>tatccaagga</td><td>ggcgttaccg</td><td>cagaagaaga</td><td> 60</td>

<td>caccgccccc</td><td>gcagccatct</td><td>tggccagatc</td><td>ctccgccgcc</td><td>gcęcctggct</td><td>cgtccacccc</td><td> 120</td>

<td>cgccaccgct</td><td>accgttggag</td><td>aaggaaaaat</td><td>ggcatcttca</td><td>acacccgcct</td><td>ctcccgcacc</td><td> 180</td>

<td>ttcggatata</td><td>ctgtcaaggc</td><td>taccacagtc</td><td>acaacgccct</td><td>cctgggcggt</td><td>ggacatgatg</td><td> 240</td>

<td>agatttaata</td><td>ttgacgactt</td><td>tgttcccccg</td><td>ggagggggga</td><td>ccaacaaaat</td><td>ctctataccc</td><td> 300</td>

<td>tttgaatact</td><td>acagaataag</td><td>aaaggttaag</td><td>gttgaattct</td><td>ggccctgctc</td><td>ccccatcacc</td><td> 360</td>

<td>cagggtgata</td><td>ggggagtggg</td><td>ctccactgct</td><td>gttattctag</td><td>atgataactt</td><td>tgtaacaaag</td><td> 420</td>

<td>gccacagccc</td><td>taacctatga</td><td>cccatatgta</td><td>aactactcct</td><td>cccgccatac</td><td>aatcęcccaa</td><td> 480</td>

<td>cccttctcct</td><td>accactccćg</td><td>ttacttcaca</td><td>cccaaacctg</td><td>ttcttgactc</td><td>cactattgat</td><td> 540</td>

<td>tacttccaac</td><td>caaataacaa</td><td>aaggaatcag</td><td>ctttggctga</td><td>ggctacaaac</td><td>ctctagaaat</td><td> 600</td>

<td>gtggaccacg</td><td>taggcctcgg</td><td>cactgcgttc</td><td>gaaaacagta</td><td>aatacgacca</td><td>ggactacaat</td><td> 660</td>

<td>atccgtgtaa</td><td>ccatgtatgt</td><td>acaattcaga</td><td>gaatttaatc</td><td>ttaaagaccc</td><td>cccacttgaa</td><td> 720</td>

<td>ccctaagaat</td><td>tctatcacta</td><td>gtgaattcgc</td><td>ggccgc</td><td></td><td> 756</td><td></td>

<210> 8 <211> 10387 <212> DNA <213> Artificial <220>

<223> This sequence comes from the open reading frame 2 of the porcine circovirus type 2, together with a part from the pGEM T-easy <400> 8

<td>aagctttact</td><td>cgtaaagcga</td><td>gttgaaggat</td><td>catatttagt</td><td>tgcgtttatg</td><td>agataag ^ m.p.</td><td> 60</td>

<td>gaaagćacgt</td><td>gtaaaatgtt</td><td>tcccgcgcgt</td><td>tggcacaact</td><td>atttacaatg</td><td>cggccaagtt</td><td> 120</td>

<td>ataaaagatt</td><td>ctaatctgat</td><td>atgttttaaa</td><td>acacctttgc</td><td>ggcccgagtt</td><td>gtttgcgtac</td><td> 180</td>

<td>gtgactagcg</td><td>aagaagatgt</td><td>gtggaccgca</td><td>gaacagatag</td><td>taaaacaaaa</td><td>ccctagtatt</td><td> 240</td>

<td>ggagcaataa</td><td>tcgatttaac</td><td>caacacgtct</td><td>aaatattatg</td><td>atggtgtgca</td><td>ttttttgcgg</td><td> 300</td>

<td>gcgggcctgt</td><td>tatacaaaaa</td><td>aattcaagta</td><td>cctggccaga</td><td>ctttgccgcc</td><td>tgaaagcata</td><td> 360</td>

<td>gttcaagaat</td><td>ttattgacac</td><td>ggtaaaagaa</td><td>tttacagaaa</td><td>agtgtCccgg</td><td>catgttggtg</td><td> 420</td>

<td>ggcgtgcact</td><td>gcaćacacgg</td><td>tattaatcgc</td><td>accggttaca</td><td>tggtgtgcag</td><td>atatttaatg</td><td> 480</td>

<td>cacaccctgg</td><td>gtattgcgcc</td><td>gcaggaagcc</td><td>atagatagat</td><td>tcgaaaaagc</td><td>cagaggtcac</td><td> 540</td>

<td>aaaattgaaa</td><td>gacaaaatta</td><td>cgttcaagat</td><td>ttattaattt</td><td>aattaatatt</td><td>atttgcattc</td><td> 600</td>

<td>tttaacaaat</td><td>actttatcct</td><td>attttcaaat</td><td>tgttgcgctt</td><td>ęttccagcga</td><td>accaaaacta</td><td> 660</td>

<td>tgcttcgctt</td><td>gctccgttta</td><td>gcttgtagcc</td><td>gatcagtggc</td><td>gttgttccaa</td><td>tcgacggtag</td><td> 720</td>

<td>gattaggccg</td><td>gatattctcc</td><td>accacaatgt</td><td>tggcaacgtt</td><td>gatgttacgt</td><td>ttatgctttt</td><td> 780</td>

<td>ggttttccac</td><td>gtacgtcttt</td><td>tggccggtaa</td><td>tagccgtaaa</td><td>cgtagtgccg</td><td>tcgcgcgtca</td><td> 840"</td>

<td>cgcacaacac</td><td>cggatgtttg</td><td>cgcttgtccg</td><td>cggggtattg</td><td>aaccgcgcga</td><td>tccgacaaat</td><td> 900</td>

<td>ccaccacttt</td><td>ggcaactaaa</td><td>tcggtgacct</td><td>gcgcgtcttt</td><td>tttctgcatt</td><td>atttcgtctt</td><td> 960</td>

<td>tcttttgcat</td><td>ggtttcctgg</td><td>aagccggtgt</td><td>acatgcggtt</td><td>tagatcagtc</td><td>atgacgcgcg</td><td> 1020</td>

<td>tgacctgcaa</td><td>atctttggcc</td><td>tcgatctgct</td><td>tgtccttgat</td><td>ggcaacgatg</td><td>cgttcaataa</td><td> 1080</td>

<td>actcttgttt</td><td>tttaacaagt</td><td>tcctcggttt</td><td>tttgcgccac</td><td>caccgcttgc</td><td>agcgcgtttg</td><td> 1140</td>

<td>tgtgctcggt</td><td>gaatgtcgca</td><td>atcagcttag</td><td>tcaccaactg</td><td>tttgctctcc</td><td>tcctcccgtt</td><td> 1200</td>

<td>gtttgatcgc</td><td>gggatcgtac</td><td>ttgccggtgc</td><td>agagcacttg</td><td>aggaattact</td><td>tcttctaaaa</td><td> 1260</td>

<td>gccattcttg</td><td>taattctatg</td><td>gcgtaaggca</td><td>atttggactt</td><td>cataatcagc</td><td>tgaatcacgć</td><td> 1320</td>

<td>cggatttagt</td><td>aatgagcact</td><td>gtatgcggct</td><td>gcaaatacag</td><td>cgggtcgccc</td><td>cttttcacga</td><td> 1380</td>

<td>cgctgttaga</td><td>ggtagggccc</td><td>ccattttgga</td><td>tggtctgctc</td><td>aaataacgat</td><td>ttg.tatttat</td><td> 1440</td>

<td>tgtctacatg</td><td>aacacgtata</td><td>gctttatcac</td><td>aaactgtata</td><td>ttttaaactg</td><td>ttagcgacgt</td><td> 1500</td>

<td>ccttggccac</td><td>gaaccggacc</td><td>tgttggtcgc</td><td>gctctagcac</td><td>gtaccgcagg</td><td>ttgaacgtat</td><td> 1560</td>

<td>cttctccaaa</td><td>tttaaattct</td><td>ccaattttaa</td><td>cgcgagccat</td><td>tttgatacac</td><td>gtgtgtcgat</td><td> 1620</td>

<td>tttgcaacaa</td><td>ctattgtttt</td><td>ttaacgcaaa</td><td>ctaaacttat</td><td>tgtggtaagc</td><td>aataattaaa</td><td> 1680</td>

<td>tatgggggaa</td><td>catgcgccgc</td><td>tacaacactc</td><td>gtcgttatga</td><td>acgcagacgg</td><td>cgccggtctc</td><td> 1740</td>

<td>ggcgcaagcg</td><td>gctaaaacgt</td><td>gttgcgcgtt</td><td>caacgcggca</td><td>aacatcgcaa</td><td>aagccaatag ·</td><td> 1800</td>

<td>tacagttttg</td><td>atttgcatat</td><td>taacggcgat</td><td>tttttaaatt</td><td>atcttattta</td><td>ataaatagtt</td><td> 1860</td>

<td>atgacgccta</td><td>caactccccg</td><td>cccgcgttga</td><td>ctcgctcjcac</td><td>ctcgagćagt</td><td>tcgttgacgc</td><td> 1920</td>

<td>cttcctccgt</td><td>gtggccgaac</td><td>acgtcgagcg</td><td>ggtggtcgat-</td><td>gaccagcggc</td><td>gtgćcgcacg</td><td> 1980</td>

<td>cgacgcacaa</td><td>gtatctgtac</td><td>accgaatgat</td><td>cgtcgggcga</td><td>aggcacgtcg</td><td>gcctcęaagt</td><td> 20 40</td>

<td>ggcaatattg</td><td>gcaaattcga</td><td>aaatątatac</td><td>agttgggttg</td><td>tttgcgcata</td><td>tctategtgg</td><td> 2100</td>

<td>cgttgggcat</td><td>gtacgtccga</td><td>acgttgattt</td><td>gcatgcaagc</td><td>cgaaattaaa</td><td>tcattgęgat</td><td> 2160</td>

<td>tagtgcgatt</td><td>aaaacgttgt</td><td>acatcctcgc</td><td>ttttaatcat</td><td>gccgtcgatt</td><td>aaatcgcgca</td><td> 2220</td>

<td>atcgagtcaa</td><td>gtgatcaaag</td><td>tgtggaataa</td><td>tgttttcttt</td><td>gtattcccga</td><td>gtcaagcgca</td><td> 2280</td>

<td>gcgcgtattt</td><td>taacaaacta</td><td>gccatcttgt</td><td>aagttagttt</td><td>catttaatgc</td><td>aactttatcc</td><td> 2340</td>

<td>aataatatat</td><td>tatgtatcgc</td><td>acgtcaagaa</td><td>ttaacaatgc</td><td>gcccgttgtc</td><td>gcatctcaac</td><td> 2400</td>

<td>acgactatga</td><td>tagagatcaa</td><td>ataaagcgcg</td><td>aattaaatag</td><td>cttgcgacgc</td><td>aacgtgcacg</td><td> 2460</td>

<td>atctgtgcac</td><td>gcgttccggc</td><td>acgagctttg</td><td>attgtaataa</td><td>gtttttacga</td><td>agcgatgaca</td><td> 2520</td>

<td>tgacccccgt</td><td>agtgacaacg</td><td>atcacgccca</td><td>aaagaactgc</td><td>cgactacaaa</td><td>attaccgagt</td><td> 2580</td>

<td>atgtcggtga</td><td>cgttaaaact</td><td>attaagccat</td><td>ccaatcgacc</td><td>gttagtcgaa</td><td>tcaggaccgc</td><td> 2640</td>

<td>tggtgcgaga</td><td>agęcgcgaag</td><td>tatggcgaat</td><td>gcatcgtata</td><td>acgtgtggag</td><td>tccgctcatt</td><td> 2700</td>

<td>agagcgtcat</td><td>gtttagacaa</td><td>gaaagctaca</td><td>tatttaattg</td><td>atcccgatga</td><td>ttttattgat</td><td> 2760</td>

<td>aaattgaccc</td><td>taactccata</td><td>cacggtattc</td><td>tacaatggcg</td><td>gggttttggt</td><td>caaaatttcć</td><td> 2820</td>

<td>ggactgcgat</td><td>tgtacatgct</td><td>gttaacggct</td><td>ccgcccacta</td><td>ttaatgaaat</td><td>taaaaattćc</td><td> 2880</td>

<td>aattttaaaa</td><td>aacgcagcaa</td><td>gagaaacatt</td><td>tgtatgaaag</td><td>aatgcgtaga</td><td>aggaaagaaa</td><td> 2940</td>

<td>aatgtcgtcg</td><td>acatgctgaa</td><td>caacaagatt</td><td>aatatgcctc</td><td>cgtgtataaa</td><td>aaaaatattg</td><td> 3000</td>

<td>aacgatttga</td><td>aagaaaacaa</td><td>tgtaccgcgc</td><td>ggcggtatgt</td><td>acaggaagag</td><td>gtttatacta</td><td> 3060</td>

<td>aactgttaca</td><td>ttgcaaacgt</td><td>ggtttcgtgt</td><td>gccaagtgtg</td><td>aaaaccgatg</td><td>tttaatcaag</td><td> 3120</td>

<td>gctctgacgc</td><td>atttctacaa</td><td>ccacgactcc</td><td>aagtgtgtgg</td><td>gtgaagtcat</td><td>geatctttta</td><td> 3180</td>

<td>atcaaatccc</td><td>aagatgtgta</td><td>taaaccacca</td><td>aactgccaaa</td><td>aaatgaaaac</td><td>tgtcgacaag</td><td> .3240</td>

<td>ctctgtccgt</td><td>ttgctggcaa</td><td>ctgcaagggt</td><td>ctcaatccta</td><td>tttgtaatta</td><td>ttgaataata</td><td> 3300</td>

<td>aaacaattat</td><td>aaatgctaaa</td><td>tttgtttttt</td><td>attaacgata</td><td>ćaaaccaaac</td><td>gcaacaagaa</td><td> 3360</td>

<td>catttgtagt</td><td>attatctata</td><td>attgaaaacg</td><td>cgtagttata</td><td>atcgctgagg</td><td>taątatttaa</td><td> . 3420</td>

<td>aatcattttc</td><td>aaatgattca</td><td>cagttaattt</td><td>gcgacaatat</td><td>aattttattt</td><td>tcacataaac</td><td> 3480</td>

<td>tagacgęctt</td><td>gtcgtcttct</td><td>tcttcgtatt</td><td>ccttctcttt</td><td>ttcatttttc</td><td>tcctcataaa</td><td> 3540</td>

<td>aattaacata</td><td>gttattatcg</td><td>tatccatata</td><td>tgtatctatc</td><td>gtatagagta</td><td>aattttttgt</td><td> 3600</td>

<td>tgtcataaat</td><td>atatatgtct</td><td>tttttaatgg</td><td>ggtgtatagt</td><td>accgctgcgc</td><td>atagtttttc</td><td> 3660</td>

<td>tgtaatttac</td><td>aacagtgcta</td><td>ttttctggta</td><td>gttcttcgga</td><td>gtgtgttgct</td><td>ttaattatta</td><td> 3720</td>

<td>aatttatata</td><td>atcaatgaat</td><td>ttgggatcgt</td><td>cggttttgta</td><td>caatatgttg</td><td>ccggcatagt</td><td> 3780</td>

<td>acgcagcttc</td><td>ttctagttca</td><td>attacaccat</td><td>tttttagęag</td><td>caccggatta</td><td>acataacttt</td><td> 384 0</td>

<td>ccaaaatgtt</td><td>gtacgaaccg</td><td>ttaaacaaaa</td><td>acagttcacc</td><td>tcccttttct</td><td>atactattgt</td><td> 3900</td>

<td>ctgcgagcag</td><td>ttgtttgttg</td><td>ttaaaaataa</td><td>cagccattgt</td><td>aatgagacgc</td><td>acaaactaat</td><td> 3960</td>

<td>atcacaaact</td><td>ggaaatgtct</td><td>atcaatatat</td><td>agttgctgat</td><td>atcatggaga</td><td>taattaaaat</td><td> 4020</td>

<td>gataaccatc</td><td>tcgcaaataa</td><td>ataagtattt</td><td>tactgttttc</td><td>gtaacagttt</td><td>tgtaataaaa</td><td> 4080</td>

<td>aaacctataa</td><td>atattccgga</td><td>ttattcatac</td><td>cgtcccacca</td><td>tcgggcgcgg</td><td>atcagatctg</td><td> 4140</td>

<td>cagcggccgc</td><td>gggaattcga</td><td>tccgccatga</td><td>cgtatccaag</td><td>gaggcgttac</td><td>cgcagaagaa</td><td> 4200</td>

<td>gacaccgccc</td><td>ccgcagccat</td><td>cttggccaga</td><td>tcctccgccg</td><td>ccgcccctgg</td><td>ctcgtccacc</td><td> 4260</td>

<td>cccgccaccg</td><td>ctaccgttgg</td><td>agaaggaaaa</td><td>atggcatctt</td><td>caacacccgc</td><td>ctctcccgca</td><td> 4320</td>

<td>ccttcggata</td><td>tactgtcaag</td><td>gctaccacag</td><td>tcacaacgcc</td><td>ctcctgggcg</td><td>gtggacatga</td><td> 4380</td>

<td>tgagattfaa</td><td>tattgacgac</td><td>tttgttcccc</td><td>cgggaggggg</td><td>gaccaacaaa</td><td>atctctatac</td><td> 4440</td>

<td>cctttgaata</td><td>Ctacagaata</td><td>agaaaggtta</td><td>aggttgaatt</td><td>ćtggccćtgc</td><td>tcccccatca</td><td> 4500</td>

<td>cccagggtga</td><td>taggggagtg</td><td>ggctccactg</td><td>ctgttattet</td><td>agatgataac</td><td>tttgtaacaa</td><td> 4560</td>

<td>aggccacagc</td><td>cctaacctat</td><td>gacccatatg</td><td>taaactactc</td><td>ctcccgccat</td><td>acaatccccc</td><td> 4620</td>

<td>aacccttctc</td><td>ctaccactcc</td><td>cgttacttca</td><td>cacccaaacc</td><td>tgttcttgac</td><td>tccactattg</td><td> 4680</td>

<td>attacttcca</td><td>accaaataac</td><td>aaaaggaatc</td><td>agctttggct</td><td>gaggctącaa</td><td>acctctagaa</td><td> 4740</td>

<td>atgtggacca</td><td>cgtaggectc</td><td>ggcactgcgt</td><td>tcgaaaacag</td><td>taaatacgac</td><td>caggactaca</td><td> 4800</td>

<td>atatccgtgt</td><td>aaccatgtat</td><td>gtacaattca</td><td>gagąatttaa</td><td>tcttaaagac</td><td>cccccacttg</td><td> 4860</td>

<td>aaccctaaga</td><td>attctatcac</td><td>tagtgaattc</td><td>gcggccgccg</td><td>gccgctccag</td><td>aattctagaa</td><td> 4920</td>

<td>ggtacccggg</td><td>atcctttcct</td><td>gggacccggc</td><td>aagaaccaaa</td><td>aactcactct</td><td>cttcaaggaa</td><td> 4980</td>

<td>atccgtaatg</td><td>ttaaacccga</td><td>cacgatgaag</td><td>cttgtcgttg</td><td>gątggaaagg</td><td>aaaagagttc</td><td> 5040</td>

<td>tacagggaaa</td><td>cttggacccg</td><td>cttcatggaa</td><td>gacagcttcc</td><td>ccattgttaa</td><td>cgaccaagaa</td><td> 5100</td>

<td>gtgatggatg</td><td>ttttccttgt</td><td>tgtcaacatg</td><td>cgtcccacta</td><td>gacccaaccg</td><td>ttgttacaaa</td><td> 5160</td>

<td>ttcctggccc</td><td>aacacgctct</td><td>gcgttgcgac</td><td>cccgactatg</td><td>tacctcatga</td><td>cgtgattagg</td><td> 5220</td>

<td>atcgtcgagc</td><td>cttcatgggt</td><td>gggcagcaac</td><td>aacgagtacc</td><td>gcatcagcct</td><td>ggctaagaag</td><td> 5280</td>

<td>ggcggcggct</td><td>gcccaataat</td><td>gaaccttcac</td><td>tctgagtaca</td><td>ccaactcgtt</td><td>cgaacagttc</td><td> 5340</td>

<td>atcgatcgtg</td><td>tcatctggga</td><td>gaacttctac</td><td>aagcccatcg</td><td>tttacatcgg</td><td>taccgactct</td><td> 5400</td>

<td>gctgaagagg</td><td>aggaaattct</td><td>ccttgaagtt</td><td>tccctggtgt</td><td>tcaaagtaaa</td><td>ggagtttgca</td><td> 5460</td>

<td>cćagacgcac</td><td>ctctgttcac</td><td>tggtccggcg</td><td>tattaaaaca</td><td>cgatacattg</td><td>ttattagtac</td><td> 5520</td>

<td>atttattaag</td><td>cgctagattc</td><td>tgtgcgttgt</td><td>tgatttacag</td><td>acaattgttg</td><td>tacgtatttt</td><td> 5580</td>

<td>aataattcat</td><td>taaatttatś</td><td>atctttaggg</td><td>tggtatgtta</td><td>gagcgaaaat</td><td>caaatgattt</td><td> 5640</td>

<td>tcagcgtctt</td><td>tatatctgaa</td><td>tttaaatatt</td><td>aaatcctcaa</td><td>tagatttgta</td><td>aaataggttt</td><td> 5700</td>

<td>cgattagttt</td><td>caaacaaggg</td><td>ttgtttttcc</td><td>gaaccgatgg</td><td>ctggactatc</td><td>taatggattt</td><td> 5760</td>

<td>tcgctcaacg</td><td>ccacaaaact</td><td>tgccaaatct</td><td>tgtagcagca</td><td>atctagcttt</td><td>gtcgatattc</td><td> 5820</td>

<td>gtttgtgttt</td><td>tgttttgtaa</td><td>taaaggttcg</td><td>acgtcgttca</td><td>aaatattatg</td><td>cgcttttgta</td><td> 5880</td>

<td>tttctttcat</td><td>cactgtcgtt</td><td>agtgtacaat</td><td>tgactcgacg</td><td>taaacacgtt</td><td>aaataaagct</td><td> 5940</td>

<td>tggacatatt</td><td>taacatcggg</td><td>cgtgttagct</td><td>ttattaggcc</td><td>gattatcgtc</td><td>gtcgtcęcaa</td><td> 6000</td>

<td>ccctcgtcgt</td><td>tagaagttgc</td><td>ttccgaagac</td><td>gattttgcca</td><td>tagccacacg</td><td>acgcctatta</td><td> 6060</td>

<td>attgtgtcgg</td><td>ctaacacgtc</td><td>cgcgatcaaa</td><td>tttgtagttg</td><td>agctttttgg</td><td>aattatttct</td><td> 6120</td>

<td>gattgcgggc</td><td>gtttttgggc</td><td>gggtttcaat</td><td>ctaactgtgc</td><td>ccgattttaa</td><td>ttcagacaac</td><td> 6180</td>

<td>acgttagaaa</td><td>gcgatggtgc</td><td>aggcggtggt</td><td>aacatttcag</td><td>acggcaaatc</td><td>tactaatggc</td><td> 6240</td>

<td>ggcggtggtg</td><td>gagctgatga</td><td>taaatctacc</td><td>atcggtggag</td><td>gcgcaggcgg</td><td>ggctggcggc</td><td> 6300</td>

<td>ggaggcggag</td><td>gcggaggtgg</td><td>tggcggtgat</td><td>gcagacggcg'</td><td>gtttaggctc</td><td>aaatgtctct</td><td> 6360</td>

<td>ttaggcaaca</td><td>cagtcggcac</td><td>ctcaactatt</td><td>gtactggttt</td><td>cgggćgccgt</td><td>ttttggtttg</td><td> 6420</td>

<td>accggtctga</td><td>gacgagtgcg</td><td>atttttttcg</td><td>tttctaatag</td><td>cttccaacaa</td><td>ttgttgtctg</td><td> 6480</td>

<td>tcgtctaaag</td><td>gtgcagcggg</td><td>ttgaggttcc</td><td>gtcggcattg</td><td>gtggagcggg</td><td>cggcaattca</td><td> 6540</td>

<td>gacatcgatg</td><td>gtggtggtgg</td><td>tggtggaggc</td><td>gctggaatgt</td><td>taggcacggg</td><td>agaaggtggt</td><td> 6600</td>

<td>ggcggcggtg</td><td>ccgccggtat</td><td>aatttgttct</td><td>ggtttagttt</td><td>gttcgcgcac</td><td>gattgtgggc</td><td> 6660</td>

<td>accggcgcag</td><td>gcgccgctgg</td><td>ctgcacaaćg</td><td>gaaggtcgtc</td><td>tgcttcgagg</td><td>cagcgcttgg</td><td> 6720</td>

<td>ggtggtggca</td><td>attcaatatt</td><td>ataattggaa</td><td>taeaaatcgt</td><td>aaaaatctgc</td><td>tataagęatt</td><td> 6780</td>

<td>gtaatttcgc</td><td>tatcgtttac</td><td>cgtgccgata</td><td>tttaacaacc</td><td>gctcaatgta</td><td>agcaattgta</td><td> 6840</td>

<td>ttgtaaagag</td><td>attgtctcaa</td><td>gctcgccgca</td><td>cgccgataac</td><td>aagccttttc</td><td>atttttacta</td><td> 6900</td>

<td>cagcattgta</td><td>gtggcgagac</td><td>aćttcgctgt</td><td>cgtcgacgta</td><td>catgtatgct</td><td>ttgttgtcaa</td><td> 6960</td>

<td>aaacgtcgtt</td><td>ggcaagcttt</td><td>aaaatattta</td><td>aaagaacatc</td><td>tctgttcagc</td><td>accactgtgt</td><td> 7020</td>

<td>tgtcgtaaat</td><td>gttgtttttg</td><td>ataatttgcg</td><td>cttccgcagt</td><td>atcgacacgt</td><td>tcaaaaaatt</td><td> 7080</td>

<td>gatgcgcatc</td><td>aattttgttg</td><td>ttcctattat</td><td>tgaataaata</td><td>agattgtaca</td><td>gattcatatc</td><td> 7140</td>

<td>tacgattcgt</td><td>catggccacc</td><td>acaaatgcta</td><td>Cgctgcaaac</td><td>gctggtacaa</td><td>ttttacgaaa</td><td> 7200</td>

<td>actgcaaaaa</td><td>cgtcaaaact</td><td>cggtataaaa</td><td>taatcaacgg</td><td>gcgctttggc</td><td>aaaatatcta</td><td> 7260</td>

<td>ttttatcgca</td><td>caagcccact</td><td>agcaaattgt</td><td>atttgcagaa</td><td>aacaatttcg</td><td>gcgcacaatt</td><td> 7320</td>

<td>ttaacgctga</td><td>cgaaataaaa</td><td>gttcaccagt</td><td>taatgagcga</td><td>ccacccaaat</td><td>tttataaaaa</td><td> 7380</td>

<td>tctattttaa</td><td>tcacggttcc</td><td>atcaacaacc</td><td>aagtgatcgt</td><td>gatggactac</td><td>attgactgtc</td><td> 7440</td>

<td>ccgatttatt</td><td>tgaaacacta</td><td>caaattaaag</td><td>gcgagctttc</td><td>gtaccaactt</td><td>gttagcaata</td><td> 7500</td>

<td>ttattagaca</td><td>gctgtgtgąa</td><td>gcgctcaacg</td><td>atttgcącaa</td><td>gcacaatttc</td><td>atacacaacg</td><td> 7560</td>

<td>acataaaact</td><td>cgaaaatgtc</td><td>ttatatttcg</td><td>aagcacttga</td><td>tcgcgtgtat</td><td>gtttgcgatt</td><td> 7620</td>

<td>acggattgtg</td><td>caaacacgaa</td><td>aactcactta</td><td>gcgtgcacga</td><td>cggcacgttg</td><td>gagtatttta</td><td> 7680</td>

<td>gtccggaaaa</td><td>aattćgacac</td><td>acaactatgc</td><td>acgtttcgtt</td><td>tgactggtac</td><td>gcggcgtgtt</td><td> 7740</td>

<td>aacatacaag</td><td>ttgctaacgt</td><td>aatcatggtc</td><td>atagctgttt</td><td>cctgtgtgaa</td><td>attgttatcc</td><td> 7800</td>

<td>gctcacaatt</td><td>ccacacaaca</td><td>tacgagccgg</td><td>aagcataaag</td><td>tgtaaagcęt</td><td>ggggtgccta</td><td> 7860</td>

<td>atgagtgagc</td><td>taactcacat</td><td>taattgcgtt</td><td>gcgctcactg</td><td>cccgctttcc</td><td>agtcgggaaa</td><td> 7920</td>

<td>cctgtcgtgc</td><td>cagctgcatt</td><td>aatgaatcgg</td><td>ccaacgcgcg</td><td>gggagaggcg</td><td>gtttgcgtat</td><td> 7980</td>

<td>tgggcgctct</td><td>tccgcttcct</td><td>cgctcactga</td><td>ctcgctgcgc</td><td>tcggtcgttc</td><td>ggctgcggcg</td><td> 8040</td>

<td>agcggtatca</td><td>gctcactcaa</td><td>aggcggtaat</td><td>acggttatcc</td><td>acagaatcag</td><td>gggataacgc</td><td> 8100</td>

<td>aggaaagaac</td><td>atgtgagcaa</td><td>aaggccagca</td><td>aaaggccagg</td><td>aaccgtaaaa</td><td>aggccgcgtt</td><td> 8160</td>

<td>gctggcgttt</td><td>ttccataggc</td><td>tccgcccccc</td><td>tgacgagcat</td><td>cacaaaaatc</td><td>gacgctcaag</td><td> 8220</td>

<td>tcagaggtgg</td><td>cgaaacccga</td><td>caggactata</td><td>aagataccag</td><td>gcgtttcccc</td><td>ctggaagctc</td><td> 8280</td>

<td>ćctcgtgcgc</td><td>tctcctgttc</td><td>cgaccctgcc</td><td>gcttaccgga</td><td>tacctgtccg</td><td>cctttctccc</td><td> 834 0</td>

<td>ttcgggaagc</td><td>gtggcgcttt</td><td>ctcatagctc</td><td>acgctgtagg</td><td>tatctcagtt</td><td>cggtgtaggt</td><td> 84 00</td>

<td>cgttcgctcc</td><td>aagctgggct</td><td>gtgtgcacga</td><td>accccccgtt</td><td>cagcccgacc</td><td>gctgcgcctt</td><td> 8460</td>

<td>atccggtaac</td><td>tatcgtcttg</td><td>agtccaaccc</td><td>ggtaagacac</td><td>gacttatcgc</td><td>cactggcagc</td><td> 8520</td>

<td>agccactggt</td><td>aacaggatta</td><td>gcagagcgag</td><td>gt.atgtaggc</td><td>ggtgctacag</td><td>agttcttgaa</td><td> 8580</td>

<td>gtggtggcct</td><td>aactacggct</td><td>acactagaag</td><td>gacagtattt</td><td>ggtatctgcg</td><td>ctctgctgaa</td><td> 8640</td>

<td>gccagttacc</td><td>ttcggaaaaa</td><td>gagttggtag</td><td>ctcttgatcc</td><td>ggcaaacaaa</td><td>ccaccgctgg</td><td> 8700</td>

<td>tagcggtggt</td><td>ttttttgttt</td><td>gcaagcagca</td><td>gattacgcgc</td><td>agaaaaaaag</td><td>gatctcaaga</td><td> 8760</td>

<td>agatcctttg</td><td>atcttttcta</td><td>cggggtctga</td><td>cgctcagtgg</td><td>aacgaaaact</td><td>cacgttaagg</td><td> 8820</td>

<td>gattttggtc</td><td>atgagattat</td><td>caaaaaggat</td><td>cttcacctag</td><td>atccttttaa</td><td>attaaaaatg</td><td> 8880</td>

<td>aagttttaaa</td><td>tcaatctaaa</td><td>gtatatatga</td><td>gtaaacttgg</td><td>tctgacagtt</td><td>accaatgctt</td><td> 8940</td>

<td>aatcagtgag</td><td>gcacctatct</td><td>cagcgatctg</td><td>tctatttcgt</td><td>tcatccatag</td><td>ttgcctgact</td><td> 9000</td>

<td>ccccgtcgtg</td><td>tagataacta</td><td>cgatacggga</td><td>gggcttacca</td><td>tctggcccca</td><td>gtgctgcaat</td><td> 9060</td>

<td>gataccgcga</td><td>gacccacgct</td><td>caccggctcc</td><td>agatttatca</td><td>gcaataaacc</td><td>agccagccgg</td><td> 9120</td>

<td>aagggccgag</td><td>cgcagaagtg</td><td>gtcctgcaac</td><td>tttatccgcc</td><td>tccatccagt</td><td>ctattaattg</td><td> 9180</td>

<td>ttgccgggaa</td><td>gctagagtaa</td><td>gtagttcgcc</td><td>agttaatagt</td><td>ttgcgcaacg</td><td>ttgttgccat</td><td> 9240</td>

<td>tgctacaggc</td><td>atcgtggtgt</td><td>cacgctcgtc</td><td>gtttggtatg</td><td>gcttcattca</td><td>gctccggttc</td><td> 9300</td>

<td>ccaacgatca</td><td>aggcgagtta</td><td>catgatcccc</td><td>catgttgtgc</td><td>aaaaaagcgg</td><td>ttagctcctt</td><td> 9360</td>

<td>cggtcctccg</td><td>atcgttgtca</td><td>gaagtaagtt</td><td>ggccgcagtg</td><td>ttatcactca</td><td>tggttatggc</td><td> 9420</td>

<td>agcactgcat</td><td>aattctctta</td><td>ctgtcatgcc</td><td>atccgtaaga</td><td>tgcttttctg</td><td>tgąętggtga</td><td> 94 80</td>

<td>gtactcaacc</td><td>aagtcattct</td><td>gagaatagtg</td><td>tatgcggcga</td><td>ccgagttgct</td><td>cttgcccggć</td><td> 9540</td>

<td>gtcąatacgg</td><td>gataataccg</td><td>cgccacatag</td><td>cagaacttta</td><td>aaągtgctca</td><td>tcattggąaa</td><td> 9600</td>

<td>acgttcttcg</td><td>gggcgaaaac</td><td>tctcaaggat</td><td>cttaccgctg</td><td>ttgagatcca</td><td>gttćgatgta</td><td> 9660</td>

<td>acccactcgt</td><td>gcacccaaćt</td><td>gatcttcagc</td><td>atcttttact</td><td>ttćaęęagcg</td><td>tttctgggtg</td><td> 9720</td>

<td>agcaaaaaca</td><td>ggaaggcaaa</td><td>atgccgcaaa</td><td>aaagggaata</td><td>agggcgacac</td><td>ggaaatgttg</td><td> 9780</td>

<td>aatactcata</td><td>ctcttccttt</td><td>ttcaatatta</td><td>ttgaagcatt</td><td>tatcagggtt</td><td>attgtctcat</td><td> 9840</td>

<td>gagcggatac</td><td>atatttgaat</td><td>gtatttagaa</td><td>aaataaacaa</td><td>ataggggttc</td><td>ćgćgcacatt</td><td> 9900</td>

<td>tccccgaaaa</td><td>gtgccacctg</td><td>acgtctaaga</td><td>aaccattatt</td><td>atcatgacat</td><td>taacctataa</td><td> 9960</td>

<td>aaataggcgt</td><td>atcacgaggc</td><td>cctttcgtct</td><td>cgcgcgtttc</td><td>ggtgatgacg</td><td>gtgaaaacct</td><td> 10020</td>

<td>ctgacacatg</td><td>cagctcccgg</td><td>agacggtcac</td><td>agcttgtctg</td><td>taagcggatg</td><td>ccgggagcag</td><td> 10080</td>

<td>acaagcccgt</td><td>cagggcgcgt</td><td>cagcgggtgt</td><td>tggcgggtgt</td><td>cggggctggc</td><td>ttaactatgc</td><td> 10140.</td>

<td>ggcatcagag</td><td>cagattgtac</td><td>tgagagtgca</td><td>ccatatgcgg</td><td>tgtgaaatac</td><td>cgcacagatg</td><td> 10200</td>

<td>cgtaaggaga</td><td>aaataccgca</td><td>tcaggcgcća</td><td>ttcgccattc</td><td>aggctgcgca</td><td>actgttggga</td><td> 10260</td>

<td>agggcgatcg</td><td>gtgcgggcct</td><td>cttcgętatt</td><td>acgccagctg</td><td>gcgaaagggg</td><td>gatgtgćtgc</td><td> 10320</td>

<td>aaggcgatta</td><td>agttgggtaa</td><td>cgccagggtt</td><td>ttcccagtca</td><td>cgacgttgta</td><td>aaacgacggc</td><td> 10380</td>

cagtgcc 10387 _: <210> 9 <211> 20 <212> PRT <213> Cirkowirus świni <400> 9

Ser Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His His Pro Pro Ser 1 5 10 '15

His Leu Gly Gin <210> 10 <211> 19 <212> PRT <213> Cirkowirus świni <400> 10

Pro Arg His His Tyr Arg Pro Arg Arg Lys Asn Gly Ile Phe Asn Thr

5 10 .15

Thr Leu Ser · <210> 11 <211> 233 <212> PRT <213> Sztuczna <220>

<223> Jest to sekwencja aminokwasowa otwartej ramki odczytu 2 cirkowirusa świni typu 2 <400> 11

Met Thr Tyr Pro Arg Arg Arg Tyr Arg Arg Arg Arg His Arg Pro Arg1 · · 5 . 10 15

Ser His Leu Gly Gin Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro 20 25 ' 30

Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg

40 45

Leu Ser Arg Thr Phe Gly Tyr Thr Val Lys Ala Thr Thr Val Arg Thr

55 60

Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile Asp Asp Phe Val

70 75 80

Pro Pro Gly Gly Gly Thr Asn Lys Ile Ser Ile Pro Phe Glu Tyr Tyr

90 95

Arg Ile Lys Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr • · ' 100 _ ' 105 · 110 .

Gin Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn 115 120 ' . 125 .

• z

Phe Val Thr Lys Ala Thr Ala Leu Thr Tyr Asp Pro Tyr Vai Asn Tyr • 130 135 140

Ser Ser Arg His Thr Ile Pro Gin Pro Phe Ser Tyr His Ser Arg Tyr

145 150 155 160

Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp Tyr Phe Gin Pro

165 170 175

Asn Asn Lys Arg Asn Gin Leu Trp Leu Arg L‘eu Gin Thr Ser Arg Asn

180 185 190

Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp

<img file="PL2371383T3_D0003.tif" />

Boehringer Ingelheim Vetmedica, Inc., Stany Zjednoczone

Pe nomocent:

Z-13730 / B1