Porcine adenovirus type 3 genome

Abstract

The complete nucleotide sequence of the genome of porcine adenovirus type 3 (PAV-3) is provided. Methods for construction of infectious PAV genomes by homologous recombination in procaryotic cells are provided. Recombinant PAV viruses are obtained by transfection of mammalian cells with recombinant PAV genomes. The PAV-3 genome can be used as a vector for the expression of heterologous nucleotide sequences, for example, for the preparation and administration of subunit vaccines to swine or other mammals. In addition, PAV-3 vectors can be used for gene therapy and expression of heterologous polypeptides. PAV-3 genome sequences can also be used for diagnostic purposes, to detect the presence of PAV-3 DNA in a subject or biological sample.

1. A replication-defective recombinant PAV-3 vector, wherein said PAV-3 vector is capable of duplex formation under conditions of high stringency with the genome of PAV-3 as depicted in SEQ ID NO:1, or its complement, said vector comprising ITR sequences, packaging sequences, and at least one heterologous nucleotide sequence, and wherein the PAV-3 vector lacks E1 function. 
     
     
       2. The replication-defective recombinant PAV-3 vector according to  claim 1 , wherein the vector is deleted in the E1 region. 
     
     
       3. The replication-defective recombinant PAV-3 vector according to  claim 2 , wherein the vector is additionally deleted in a region selected from the group consisting of E2, E3, E4, L1, L2, L3, L4, L5, L6 and the region between E4 and the right the genome. 
     
     
       4. The replication-defective recombinant PAV-3 vector according to  claim 3 , wherein the vector is additionally deleted in more than one region selected from the group consisting of E2, E3, E4, L1, L2, L3, L4, L5, L6 and the region between E4 and the end of the genome. 
     
     
       5. The replication-defective recombinant PAV-3 vector according to  claim 1 , wherein the heterologous nucleotide sequence encodes a polypeptide selected from the group consisting of coagulation factors, growth hormones, cytokines, lymphokines, tumor-suppressing polypeptides, cell receptors, ligands for cell receptors, protease inhibitors, antibodies, toxins, immunotoxins, dystrophins, cystic fibrosis transmembrane conductance regulator (CFTR) and immunogenic polypeptides. 
     
     
       6. A method for producing a recombinant PAV-3 that comprises introducing the PAV-3 vector of  claim 1  into a helper cell line comprising E1 function and recovering virus from the infected cells. 
     
     
       7. A host cell comprising the vector of  claim 1 . 
     
     
       8. A method for producing a recombinant polypeptide, the method comprising:
 (a) providing a population of host cells according to  claim 7 , and  
 (b) growing said population of cells under conditions whereby the polypeptide is expressed.  
 
     
     
       9. A composition comprising the replication-defective recombinant PAV-3 vector of  claim 1 . 
     
     
       10. The composition according to  claim 9  further comprising a pharmaceutically acceptable vehicle. 
     
     
       11. A composition capable of inducing an immune response in a mammalian subject, said composition comprising a replication-defective recombinant PAV-3 vector according to  claim 1 , wherein the vector comprises a heterologous nucleotide sequence that encodes an immunogenic polypeptide; and a pharmaceutically acceptable vehicle. 
     
     
       12. The composition according to  claim 11 , wherein said immunogenic polypeptide is a pathogen antigen. 
     
     
       13. A recombinant PAV-3 vector comprising a PAV-3 genome capable of duplex formation under conditions of high stringency to the PAV-3 genome as depicted in SEQ ID NO:1, or a complement thereof and at least one heterologous nucleotide sequence, wherein the heterologous nucleotide sequence is inserted in a region selected from the group consisting of E1 region, the E3 region,  and the E4 region and the region between E4 and the right end  of the genome. 
     
     
       14. The recombinant PAV-3 vector of  claim 13 , comprising two or more heterologous nucleotide sequences. 
     
     
       15. The recombinant PAV-3 vector of  claim 14 , wherein the two or more heterologous nucleotide sequences are inserted at different insertion sites. 
     
     
       16. The recombinant PAV-3 vector of  claim 13 , wherein the heterologous nucleotide sequence encodes a polypeptide selected from the group consisting of coagulation factors, growth hormones, cytokines, lymphokines, tumor-suppressing polypeptides, cell receptors, ligands for cell receptors, protease inhibitors, antibodies, toxins, immunotoxins, dystrophins, cystic fibrosis transmembrane conductance regulator (CFTR) and immunogenic polypeptides. 
     
     
       17. A host cell comprising the vector of  claim 13 . 
     
     
       18. A method for producing a recombinant polypeptide, the method comprising:
 (a) providing a population of host cells according to  claim 17 , and  
 (b) growing said population of cells under conditions whereby the polypeptide is expressed.  
 
     
     
       19. A composition comprising the vector of  claim 13 . 
     
     
       20. The recombinant PAV-3 vector according to  claim 13  wherein the heterologous nucleotide sequence is inserted in the E1 region. 
     
     
       21. The recombinant PAV-3 vector according to  claim 13  wherein the heterologous nucleotide sequence is inserted in the E3 region. 
     
     
       22. The recombinant PAV-3 vector according to  claim 13  wherein the heterologous nucleotide sequence encodes an immunogenic polypeptide. 
     
     
       23. The recombinant PAV-3 vector according to  claim 22 , wherein said immunogenic polypeptide is a pathogen antigen. 
     
     
       24. The recombinant PAV-3 vector of  claim 13 , wherein said vector is replication competent. 
     
     
       25. A method for obtaining a recombinant PAV-3 comprising a heterologous nucleotide sequence inserted into a PAV-3 insertion site located in a region selected from the group consisting of the E 1  region, and the E 4  region of the PAV-   3  genome , the method comprising the steps of:
 (a) providing a PAV-3 genome capable of duplex formation under conditions of high stringency to the PAV-3 genome as depicted in SEQ ID NO: 1;  
 (b) providing a heterologous nucleotide sequence;  
 (c) linking the heterologous nucleotide sequence to guide sequences, the guide sequences being capable of duplex formation under conditions of high stringency to said PAV-3 genome sequences flanking the PAV-3 insertion site, or the complement of said sequences, such that guide sequences are present at both ends of the heterologous sequence;  
 (d) introducing the construct from step (c) into a cell together with the PAV-3 genome;  
 (e) allowing homologous recombination to occur between the two sequences from step (d) to generate a recombinant PAV-3 genome;  
 (f) purifying the recombinant PAV-3 genome;  
 (g) inserting the recombinant PAV-3 genome into a mammalian cell;  
 (h) culturing the mammalian cell under conditions wherein the recombinant PAV-3 genome is replicated and packaged; and  
 (i) optionally collecting the recombinant PAV-3 from the cell or the culture medium.  
 
     
     
       26. The method according to  claim 25  wherein the insertion site is located in a region of the PAV-3 genome selected from the group consisting of the E1 region, the E3 region, the E4 region and the region between E4 and the right end of the genome. 
     
     
       27. The method according to  claim 26    claim 25  wherein the PAV-3 genome is deleted in a region selected from the group consisting of the E1 region, the E3 region, the E4 region and the region between E4 and the right end of the genome. 
     
     
       28. A composition comprising a recombinant PAV-3 obtained according to the method of  claim 25 . 
     
     
       29. The method according to  claim 25  wherein said insertion site is E1. 
     
     
       30. The method according to  claim 25  wherein said insertion site is E3. 
     
     
       31. A method for eliciting an immune response in a mammalian host comprising administering a composition comprising a recombinant PAV-3 vector that expresses an immunogenic polypeptide  comprises a heterologous nucleic acid that encodes an immunogenic polypeptide inserted at an insertion site selected from the group consisting of the E 1  region, and the E 4  region of the PAV-   3  genome , wherein said PAV-3 vector is capable of duplex formation under conditions of high stringency to the PAV-3 genome as depicted in SEQ ID NO:1, or a complement thereof; and a pharmaceutically acceptable vehicle. 
     
     
       32. The method of  claim 31  wherein said PAV-3 vector lacks E1 function. 
     
     
       33. The method of  claim 32  wherein said PAV-3 vector is additionally deleted in a region selected from the group consisting of E2, E3, E4, L1, L2, L3, L4, L5, L6 and the region between E4 and the right end of the genome. 
     
     
       34. The method according to  claim 31  wherein said immunogenic polypeptide is a pathogen antigen. 
     
     
       35. A method for obtaining a full-length genomic clone of a PAV-3 genome, the method comprising:
 (a) providing two or more cloned segments of the PAV-3 genome, wherein said segments are capable of duplex formation under conditions of high stringency to the PAV-3 genome as depicted in SEQ ID NO:1, or a complement thereof, and wherein the cloned segments of the PAV-3 genome, taken together, represent the entire PAV-3 genome;    (b) introducing the two or more cloned segments of the PAV-3 genome into a cell;    (c) allowing homologous recombination to occur within the cell between the two or more cloned segments of the PAV-3 genome to generate a full-length PAV-3 genome; and    (d) optionally purifying the full-length PAV-3 genome from the cell.    
     
     
       36. The method of  claim 35  wherein said cell is procaryotic cell. 
     
     
       37. The method of  claim 36  wherein bacterial cell is E. coli. 
     
     
       38. A method for obtaining a recombinant PAV-3 comprising a heterologous nucleotide sequence inserted into a PAV-3 insertion site located in a region selected from the group consisting of the E 1  region and the E 4  region of the PAV-   3  genome , comprising the steps of
 a) introducing a recombinant plasmid into a host cell in combination with a PAV-3 genome, wherein said plasmid comprises a heterologous nucleotide sequence flanked by nucleotide sequences that are capable of duplex formation under conditions of high stringency to PAV-3 nucleotide sequences flanking the PAV-3 insertion site, or the complement of said sequences, and wherein the PAV-3 genome is capable of duplex formation under conditions of high stringency to the PAV-3 genome as depicted in SEQ ID NO:1, or a complement thereof,  
 b) allowing homologous recombination to occur between the plasmid and the PAV-3 genome thereby generating a recombinant PAV-3 comprising said heterologous nucleotide sequences;  
 c) isolating said recombinant PAV-3;  
 d) introducing said isolated PAV-3 into a mammalian cell permissive for growth of said PAV-3;  
 e) culturing said mammalian cell under conditions suitable for PAV-3 replication and packaging; and  
 f) optionally, collecting said recombinant PAV-3 produced from step e).  
 
     
     
       39. The method of  claim 38  wherein said PAV-3 vector lacks E1 function. 
     
     
       40. The method of  claim 39  wherein said PAV-3 vector is additionally deleted in a region selected from the group consisting of E2, E3, E4, L1, L2, L3, L4, L5, L6 and the region between E4 and the right end of the genome. 
     
     
       41. The replication-defective recombinant PAV-3 vector according to  claim 1  wherein the heterologous nucleotide sequence encodes an immunogenic polypeptide. 
     
     
       42. The replication-defective PAV-3 vector according to  claim 41 , wherein said immunogenic polypeptide is a pathogen antigen. 
     
     
       43. A vaccine for protecting a mammalian host against infection comprising a recombinant PAV-3 vector comprising a PAV-3 genome capable of duplex formation under conditions of high stringency to the PAV-3 genome as depicted in SEQ ID NO:1, or a complement thereof, and at least one heterologous nucleotide sequence encoding an immunogenic polypeptide inserted into the PAV-   3  insertion site located in a region selected from the group consisting of the E 1  region, and the E 4  region of the PAV -   3  genome wherein the insertion renders the PAV -   3  replication defective , and a pharmaceutically acceptable excipient. 
     
     
       44. The vaccine of  claim 43  wherein said immunogenic polypeptide is a pathogen antigen.