DNA sequences, molecules, vectors and vaccines for feline calicivirus disease and methods for producing and using same

Abstract

Nucleotide sequences obtained from the genome of the capsid gene of FCV strain 2280 wherein the splicing sites have been modified, so as to be deleted or inactivated are provided. The nucleotide sequences include DNA sequences that encode polypeptides, such as the capsid gene of FCV strain 2280, whereby the coding sequences are capable of being transcribed in the nucleus of a eukaryotic organism without the DNA coding sequence being altered by the organism's natural splicing machinery and the amino acid structure of the expressed protein is not altered. The recombinant molecules may be incorporated into FHV-1 vectors that are used to infect cell cultures for use, inter alia, in the development of vaccines and, in particular vaccines for preventing or treating FCV disease. Also provided are methods for obtaining nucleotide sequences coding for polypeptides that are naturally transcribed in the cytoplasm of cells and, in particular, DNA sequences coding for the FCV capsid protein, which sequences are modified so as to permit the transcription thereof in the nucleus of eukaryotic cells without the modified coding sequence being altered by the eukaryotic cells' splicing machinery.

What is claimed is:  
     
         1 . A method for providing an isolated nucleotide sequence that is capable of being naturally transcribed in the cytoplasm of eukaryotic cells without being altered by splicing machinery found within the cells, the method comprising modifying a consensus splicing site of the DNA sequence.  
     
     
         2 . The method of  claim 1 , wherein the consensus splicing site is deleted.  
     
     
         3 . The method of  claim 1 , wherein the nucleotide sequence codes for the FCV capsid protein.  
     
     
         4 . The method of  claim 1 , wherein the nucleotide sequence comprises the FCV C gene of FCV strain 2280.  
     
     
         5 . The method of  claim 1 , wherein the consensus splicing site is a member selected from the group consisting of D1, D2, D3, D4, D5, D6, D7, D8, D9, Al, A2, A3 and A4.  
     
     
         6 . An isolated nucleotide sequence comprising a feline calicivirus C gene having at least one modified or deleted consensus splicing site.  
     
     
         7 . The isolated nucleotide sequence of  claim 6 , wherein said nucleotide sequence is capable of being transcribed in the nucleus of eukaryotic cells without being altered by splicing machinery found in the eukaryotic cells.  
     
     
         8 . An isolated nucleotide sequence obtained from the feline calicivirus C gene of FCV strain 2280 wherein the D1, D2, D3, D4 and D9 splicing sites have been deleted therefrom.  
     
     
         9 . An isolated nucleotide sequence obtained from the feline calicivirus C gene of FCV strain 2280 wherein the D9 splicing site has been deleted therefrom.  
     
     
         10 . An isolated nucleotide sequence obtained from the feline calicivirus C gene of FCV strain 2280 wherein the D1, D2, D3 and D4 splicing sites have been deleted therefrom.  
     
     
         11 . An isolated nucleotide sequence obtained from the feline calicivirus C gene of FCV strain 2280 wherein the D9 splicing site has been deleted therefrom and the D4, D5 and A2 splicing sites have been inactivated.  
     
     
         12 . An isolated nucleotide sequence obtained from the feline calicivirus C gene of FCV strain 2280 wherein the D4, D5 and A2 splicing sites have been inactivated.  
     
     
         13 . An isolated nucleotide sequence obtained from the feline calicivirus C gene of FCV strain 2280 wherein the D9 splicing site has been deleted therefrom and the D2, D3, D4, D5 and A2 splicing sites have been inactivated.  
     
     
         14 . An isolated nucleotide sequence obtained from the feline calicivirus C gene of FCV strain 2280 wherein the D2, D3, D4, D5 and A2 splicing sites have been inactivated.  
     
     
         15 . An isolated nucleotide sequence obtained from the feline calicivirus C gene of FCV strain 2280 wherein the D1, D2, D3, D4 and D9 splicing sites have been deleted therefrom and the D5 and A2 splicing sites have been inactivated.  
     
     
         16 . An isolated recombinant DNA molecule wherein a nucleotide sequence of  claim 6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 ,  13 ,  14  or  15  is fused to a DNA sequence coding for a polypeptide.  
     
     
         17 . The isolated recombinant DNA molecule of  claim 16 , wherein the polypeptide is the FCV capsid protein of FCV strain 2280.  
     
     
         18 . An expression vector capable of expressing a nucleotide sequence in the nucleus of eukaryotic cells.  
     
     
         19 . The expression vector of  claim 18 , comprising a live FHV-1 expression vector.  
     
     
         20 . The expression vector of  claim 18 , comprising a recombinant DNA molecule of claims  16  or  17 .  
     
     
         21 . A cell culture, comprising transformed cells infected with the recombinant (FHV-1) vector of claims  18 ,  19  or  20 .  
     
     
         22 . The cell culture of  claim 21 , wherein the transformed cells comprise CRFK cells.  
     
     
         23 . A vaccine comprising the expression vector of  claim 18 .  
     
     
         24 . A vaccine comprising the recombinant DNA molecule of  claim 16 ,  17  or  18 .  
     
     
         25 . A vaccine for preventing or treating feline calicivirus (FCV) disease, comprising the expression vector of  claim 18 .  
     
     
         26 . A vaccine for preventing or treating feline calicivirus (FCV) disease, comprising the recombinant DNA molecule of claims  16 ,  17  or  18 .