Avipox recombinants expressing foot and mouth disease virus genes

Abstract

The present invention relates to modified poxyiral vectors and to methods of making and using the same. In particular, the invention relates to recombinant avipox that expresses gene products of foot and mouth disease virus (FMDV), and to compositions or vaccines that elicit immune responses directed to FMDV gene products and which can confer protective immunity against infection by FMDV.

1 - 30 . (canceled) 
     
     
         31 . A method of producing a recombinant avipox vector comprising at least one nucleic acid molecule encoding one or more foot-and-mouth disease virus (FMDV) antigen(s), comprising the steps of:
 (a) linearizing a donor plasmid with a restriction endonuclease, wherein the donor plasmid comprises restriction endonuclease cleavage sites or a multiple cloning site; and   (b) ligating at least one nucleic acid molecule comprising   (i) a nucleic acid sequence encoding one or more FMDV antigen(s),   (ii) a viral promoter sequence, and   (iii) insertion sequences flanking (i) and (ii) that have complementary restriction endonuclease cleavage sites to the donor plasmid at FMDV antigens,   thereby producing the recombinant avipox vector.   
     
     
         32 . The method of  claim 31 , further comprising the steps of:
 (c) introducing the vector into a cell permissive for replication of the vector; and   (d) isolating the vector from the cell.   
     
     
         33 . The method of  claim 31 , wherein the avipox is ALVAC. 
     
     
         34 . The method of  claim 31 , wherein the avipox is fowlpox. 
     
     
         35 . The method of  claim 31 , wherein the antigen comprises at least one of FMDV VP1, VP2, VP3, VP4, 2A, 2B, and 3C. 
     
     
         36 . The method of  claim 31 , wherein the nucleic acid sequence encoding one or more FMDV antigen(s) is a cDNA encoding FMDV P1 region and a cDNA encoding FMDV 3C protease. 
     
     
         37 . The method of  claim 31 , wherein the promoter sequence is selected from the group consisting of H6 vaccinia promoter, I3L vaccinia promoter, 42K poxyiral promoter, 7.5K vaccinia promoter and Pi vaccinia promoter. 
     
     
         38 . The method of  claim 37 , wherein the promoter is the H6 vaccinia promoter, which is mutated such that expression levels of the FMDV antigens are decreased compared with expression levels of the FMDV antigens under a wild type H6 vaccinia promoter. 
     
     
         39 . The method of  claim 31 , wherein the vector comprises a C6 insertion locus, and wherein flanking sequences of the C6 insertion locus promote homologous recombination of the FMDV antigens with the C6 insertion locus. 
     
     
         40 . The method of  claim 39 , wherein the flanking sequences comprise C6L and C6R open reading frames of avipox. 
     
     
         41 . The method of  claim 31 , wherein the vector comprises a F8 insertion locus, and wherein flanking sequences of the F8 insertion locus promote homologous recombination of the FMDV antigens with the F8 insertion locus. 
     
     
         42 . The method of  claim 41 , wherein the flanking sequences comprise F8L and F8R open reading frames of avipox. 
     
     
         43 . The method of  claim 31 , wherein the vector further comprises a reporter gene. 
     
     
         44 . The method of  claim 43 , wherein the reporter gene is selected from the group consisting of neomycin resistance gene, ampicillin resistance gene, lacZ (□-galactosidase), luciferase, and green fluorescent protein (GFP). 
     
     
         45 . The method of  claim 32 , wherein the cell permissive for growth of the vector is a chicken embryonic fibroblast.