Pcv2 orf2 carrier platform

Abstract

The present invention relates to an immunogen-carrier, wherein the immunogen-carrier is preferably a virus-like particle (VLP) composed of a plurality of a modified PCV2 ORF2 protein. In particular, the present invention belongs to the field of compliance markers and marker vaccines which allow for the differentiation between infected and vaccinated individuals. In particular, it relates to a compliance marker for vaccines including a subunit antigen, and a DIVA (Differentiating Infected from Vaccinated Animals) system which makes it possible to differentiate between animals infected with a pathogen and animals treated with a subunit antigen derived from said pathogen.

What is claimed is: 
     
         1 . A polypeptide selected from the group consisting of the following (a), (b), and (c):
 a. a PCV2 ORF2 protein characterized in that at least one amino acid residue in the BC loop is replaced by an amino acid sequence of interest;   b. a PCV2 ORF2 protein characterized in that an amino acid sequence of interest is inserted into the BC loop;   c. a combination of (a) and (b).   
     
     
         2 . The polypeptide of  claim 1 , wherein the BC loop is the region of the amino acid positions 58 to 66, and wherein the numbering of the amino acid positions refers to the amino acid sequence of wild type PCV2 ORF2 protein. 
     
     
         3 . The polypeptide of  claim 1 , wherein the amino acid sequence of interest is an amino acid sequence comprising or consisting of at least two amino acid residues or preferably of at least eight amino acid residues. 
     
     
         4 . The polypeptide of  claim 1 , wherein the amino acid sequence of interest comprises or consists of a heterologous amino acid sequence. 
     
     
         5 . The polypeptide of  claim 1 , wherein the amino acid sequence of interest is selected from the group consisting of an epitope of interest, a biological response modulator, a growth factor, a recognition sequence, a fusion protein, and wherein the epitope of interest is preferably an epitope of interest from an antigen or a veterinary pathogen or toxin and wherein the epitope of interest is a peptide encoded by the orf5 gene of PRRS virus and wherein the peptide encoded by the orf5 gene of PRRS virus preferably comprises or consists of the amino acid sequence of SEQ ID NO:6 or preferably comprises or consists of at least 8 consecutive amino acid residues of the sequence set forth in SEQ ID NO: 6. 
     
     
         6 . The polypeptide of  claim 1 , wherein the amino acid sequence of interest comprises or consists of an epitope of interest, and wherein the epitope of interest is preferably an amino acid sequence comprising or consisting of 8 to 25 amino acid residues. 
     
     
         7 . The polypeptide of  claim 1 , wherein in (a) at least one amino acid residue in the region of the amino acid positions 58 to 64 is replaced by an amino acid sequence of interest, and wherein the numbering of the amino acid positions refers to the amino acid sequence of wild type PCV2 ORF2 protein. 
     
     
         8 . The polypeptide of  claim 1 , wherein in (a) at least two amino acid residues in the BC loop are replaced by an amino acid sequence of interest. 
     
     
         9 . The polypeptide of  claim 1 , wherein in (a) two, three, four, five, six, or seven amino acid residues in the BC loop are replaced by an amino acid sequence of interest. 
     
     
         10 . The polypeptide of  claim 1 , wherein in (a)
 the six amino acid residues of the amino acid positions 58 to 63 are replaced by an amino acid sequence of interest, wherein the numbering of the amino acid positions refers to the amino acid sequence of wild type PCV2 ORF2 protein; and/or   the amino acid sequence of interest comprises or consists of an amino acid sequence consisting of 11 amino acid residues, and/or   the amino acid sequence of interest comprises or consists of the sequence of SEQ ID NO:5 or SEQ ID NO:7.   
     
     
         11 . The polypeptide of  claim 1 , wherein said polypeptide is a recombinant protein, preferably a recombinant baculovirus expressed protein. 
     
     
         12 . The polypeptide of  claim 1 , wherein said PCV2 ORF2 protein is a PCV2 subtype b (PCV2b) ORF2 protein or a PCV2 subtype a (PCV2a) ORF2 protein and/or wherein said PCV2 ORF2 protein comprises or consists of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3. 
     
     
         13 . The polypeptide of  claim 1 , wherein said polypeptide comprises or consists of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1. 
     
     
         14 . The polypeptide  claim 2 , wherein said wild type PCV2 ORF2 protein is the protein set forth in SEQ ID NO:2 or SEQ ID NO:3. 
     
     
         15 . An immunogenic composition containing the polypeptide of  claim 1 . 
     
     
         16 . A polynucleotide comprising a sequence which encodes the polypeptide of  claim 1 . 
     
     
         17 . A plasmid, preferably an expression vector, which comprises a polynucleotide comprising a sequence which encodes the polypeptide of  claim 1 . 
     
     
         18 . A cell comprising a plasmid, preferably an expression vector, which comprises a polynucleotide comprising a sequence which encodes the polypeptide of  claim 1 . 
     
     
         19 . A virus like particle composed of a plurality of the polypeptide of  claim 1 . 
     
     
         20 . A baculovirus containing a polynucleotide comprising a sequence which encodes the polypeptide of any one of  claims 1  to  14 . 
     
     
         21 . A cell, preferably an insect cell, comprising a baculovirus which contains a polynucleotide comprising a sequence which encodes the polypeptide of any one of  claims 1  to  14 . 
     
     
         22 . Use of:
 the polypeptide of any one of  claims 1  to  14 ,   the immunogenic composition of  claim 15 ,   the polynucleotide of  claim 16 ,   the virus like particle of  claim 19 ,   the baculovirus of  claim 20 ,   the plasmid of  claim 17 , and/or   the cell of  claim 18  or  21  for the preparation of a medicament, preferably of a vaccine.   
     
     
         23 . The polypeptide of any one of  claims 1  to  14  or immunogenic composition of  claim 15  for use in a method for the treatment or prevention of an infection with PCV2, the reduction, prevention or treatment of clinical signs caused by an infection with PCV2, or the prevention or treatment of a disease caused by an infection with PCV2. 
     
     
         24 . The polypeptide of any one of  claims 1  to  14  or immunogenic composition of  claim 15  for use according to  claim 23 , wherein the infection with PCV2 is an infection with PCV2 subtype b (PCV2b) and/or with PCV2 of a subtype other than subtype 2b. 
     
     
         25 . The polypeptide of any one of  claims 1  to  20  or immunogenic composition of  claim 15  for use according to  claim 23  or  24 , wherein the infection with PCV2 is an infection with PCV2 of a subtype other than subtype 2b. 
     
     
         26 . The polypeptide of any one of  claims 1  to  14  or immunogenic composition of  claim 15  for use according to  claim 24 , wherein the infection with PCV2 is a concurrent infection with (i) PCV2b and (ii) PCV2 of a subtype other than subtype 2b. 
     
     
         27 . The polypeptide of any one of  claims 1  to  14  or immunogenic composition of  claim 15  for use according to any one of  claims 24  to  26 , wherein the infection with PCV2 of a subtype other than subtype 2b is an infection with PCV2 subtype a (PCV2a) and/or PCV2 subtype c (PCV2c). 
     
     
         28 . The polypeptide of any one of  claims 1  to  14  or immunogenic composition of  claim 15  for use according to any one of  claims 24  to  27 , wherein the infection with PCV2 of a subtype other than subtype 2b is an infection with PCV2a. 
     
     
         29 . The polypeptide of any one of  claims 1  to  14  or immunogenic composition of  claim 15  for use according to any one of  claims 23  to  28 , wherein the infection with PCV2 is a concurrent infection with (i) PCV2b and (ii) PCV2a. 
     
     
         30 . The polypeptide of any one of  claims 1  to  14  or immunogenic composition of  claim 15  for use according to any one of  claims 23  to  29 , wherein said infection with PCV2b is an infection with a PCV2 comprising a polypeptide that is at least 94% or preferably at least 95% identical to the sequence of SEQ ID NO:2 or comprising a polynucleotide which comprises a sequence encoding a polypeptide that is at least 94% or preferably at least 95% identical to the sequence of SEQ ID NO:2. 
     
     
         31 . The polypeptide of any one of  claims 1  to  14  or immunogenic composition of  claim 15  for use according to any one of  claims 23  to  30 , wherein said infection with PCV2a is an infection with a PCV2 comprising a polypeptide that is at least 94% or preferably at least 95% identical to the sequence of SEQ ID NO:3 or comprising a polynucleotide which comprises a sequence encoding a polypeptide that is at least 94% or preferably at least 95% identical to the sequence of SEQ ID NO:3. 
     
     
         32 . The polypeptide of any one of  claims 1  to  14  or immunogenic composition of  claim 15  for use according to any one of  claims 23  to  31 , wherein the treatment or prevention of an infection with PCV2 is based on or comprises or consists of the induction of an immune response against said PCV2, said clinical signs are selected from the group consisting of lymphoid depletion, lymphoid inflammation, positive IHC for PCV2 antigen of lymphoid tissue, viremia, nasal shedding, pyrexia, reduced average daily weight gain, lung inflammation, positive IHC for PCV2 antigen of lung tissue, or said disease is PMWS. 
     
     
         33 . The polypeptide of any one of  claims 1  to  14  or immunogenic composition of  claim 15  for use according to 24 to 32, wherein the treatment or prevention of an infection with PCV2 of a subtype other than 2b is based on or comprises or consists of the induction of an immune response against said PCV2 of a subtype other than 2b or the concurrent induction of an immune response against said PCV2 of a subtype other than 2b and PCV2b. 
     
     
         34 . A method for the treatment or prevention of an infection with PCV2, the reduction, prevention or treatment of clinical signs caused by an infection with PCV2, or the prevention or treatment of a disease caused by an infection with PCV2, comprising administering the polypeptide of any one of  claims 1  to  14  or the immunogenic composition of  claim 15  to an animal, wherein said infection with PCV2 is preferably the infection with PCV2 according to any one of  claims 24  to  32 . 
     
     
         35 . Use of the polypeptide of any one of  claims 1  to  14  or the immunogenic composition of  claim 15  for the preparation of a medicament for the treatment or prevention of an infection with PCV2, the reduction, prevention or treatment of clinical signs caused by an infection with PCV2 or for the treatment or prevention of a disease caused by an infection with PCV2, wherein said infection with PCV2 is preferably the infection with PCV2 according to any one of  claims 24  to  32 . 
     
     
         36 . The method of  claim 34  or the use of  claim 35 , wherein the treatment or prevention of an infection with PCV2 is based on or comprises or consists of the induction of an immune response against said PCV2, or wherein the prevention, reduction or treatment of an infection with PCV2 of a subtype other than subtype 2a is based on or comprises or consists of the induction of an immune response against said PCV2 of a subtype other than subtype 2a or the concurrent induction of an immune response against said PCV2 of a subtype other than subtype a and PCV2a, said clinical signs are selected from the group consisting of lymphoid depletion, lymphoid inflammation, positive IHC for PCV2 antigen of lymphoid tissue, viremia, nasal shedding, pyrexia, reduced average daily weight gain, lung inflammation positive IHC for PCV2 antigen of lung tissue, or said disease is PMWS. 
     
     
         37 . The polypeptide of any one of  claims 1  to  14  or immunogenic composition of  claim 15  for use according to any one of  claims 23  to  33 , or the method of  claim 34  or  36 , or the use according to  claim 35  or  36 , wherein said polypeptide or said immunogenic composition is administered only once. 
     
     
         38 . A method of producing the polypeptide of any one of  claims 1  to  14 , comprising transfecting a cell with the plasmid of  claim 27 . 
     
     
         39 . A method of producing the polypeptide of any one of  claims 1  to  14 , comprising infecting a cell, preferably an insect cell, with the baculovirus of  claim 20 . 
     
     
         40 . A method of determining whether an individual has received an immunogenic composition containing the polypeptide of any one  claims 1  to  14 ,
 wherein said method comprises determining in a biological sample obtained from said individual the presence or absence of one or more markers showing that the individual has received said amino acid sequence of interest, 
 and wherein the presence of said one or more markers in said biological sample indicates that said individual has received said immunogenic composition. 
 
     
     
         41 . The method of  claim 40 , wherein said one or more markers showing that the individual has received the amino acid sequence of interest are antibodies specific for said amino acid sequence of interest. 
     
     
         42 . The method of  claim 40  or  41 , comprising the steps of:
 contacting the biological sample with a capture reagent immobilized to a solid support, wherein the immobilized capture reagent is capable of binding said one or more markers, and 
 determining the presence or absence of said one or more markers bound to the capture reagent, wherein the presence of said one or more markers bound to the capture reagent is indicative for the presence of said one or more markers in said biological sample. 
 
     
     
         43 . The method of any one of  claims 40  to  42 , wherein said method comprises determining in said biological sample the presence or absence of said one or more markers, wherein said markers are antibodies specific for said amino acid sequence of interest, and wherein said method comprises the steps of:
 a. contacting the biological sample with a capture reagent immobilized to a solid support, wherein the capture reagent is selected from the group consisting of:
 i. a protein comprising the amino acid sequence of interest, 
 ii. a peptide comprising or consisting of the amino acid sequence of interest; 
 
 b. separating the biological sample from the immobilized capture reagent; 
 c. contacting the immobilized capture reagent-antibody complex with a detectable agent that binds to the antibody of the reagent-antibody complex; and 
 d. measuring the level of antibody bound to the capture reagent using a detection means for the detectable agent. 
 
     
     
         44 . The method of  claim 43 , wherein the measuring step (D) further comprises a comparison with a standard curve to determine the level of antibody bound to the capture reagent 
     
     
         45 . The method of  claim 43  or  44 , wherein said detectable agent that binds to the antibody of the reagent-antibody complex is a detectable antibody, preferably a labelled secondary antibody 
     
     
         46 . The method of any one of  claims 40  to  45  further comprising the step of determining in said biological sample the presence of one or more analytes selected from the group consisting of
 antibodies specific for a polypeptide comprising or consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3. 
 
     
     
         47 . The method of any one of  claims 40  to  46 , wherein the individual is a pig or a rabbit. 
     
     
         48 . The method of any one of  claims 40  to  47 , wherein the biological sample has been isolated from a pig. 
     
     
         49 . The method of any one of  claims 40  to  48 , wherein the biological sample is selected from the group consisting of whole blood, blood plasma, serum, urine, and oral fluids. 
     
     
         50 . The method of any one of  claims 40  to  49 , wherein the immobilized capture reagent is coated on a microtiter plate. 
     
     
         51 . A kit for determining whether an individual has received an immunogenic composition containing the polypeptide of  claim 1 , wherein said kit contains one or more capture reagents immobilized to a solid support, wherein the one or more immobilized capture reagents are capable of binding antibodies specific for said amino acid sequence of interest. 
     
     
         52 . The kit of  claim 51 , wherein the capture reagent is selected from the group consisting of:
 i. a protein comprising the amino acid sequence of interest; and   ii. a peptide comprising or consisting of the amino acid sequence of interest.