N-linked glycosylation alteration in E0 and E2 glycoprotein of classical swine fever virus and novel classical swine fever virus vaccine

Abstract

E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). E2 is involved in several functions including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Seven putative glycosylation sites in E2 were modified by site directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2, but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days post-infection suggesting that glycosylation of E2 could be modified for development of CSF live-attenuated vaccines. Additionally, a new developed virus, contained deletions of putative glycosylation sites N1 in E2 and N1 in E0 (6b), called N1E0/2v, induce a solid protection against the challenge at 3 and 28 days post-inoculation.

1 . An isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a classical swine fever virus (CSFV) that is genetically modified, the E2 glycoprotein of said CSFV having been modified by mutating a region of the E2 gene of the highly pathogenic strain Brescia thus altering the glycosylation pattern of the E2 glycoprotein and resulting in attenuation of CSFV such that when said modified CSFV infects a porcine animal it is unable to produce CSF in said animal. 
     
     
         2 . The isolated polynucleotide molecule of  claim 1 , wherein said region encodes the amino acid of position 116 of the CSFV E2 glycoprotein, resulting in attenuation of CSFV. 
     
     
         3 . The isolated polynucleotide molecule of  claim 1 , wherein said region encodes the amino acids of positions 75, 116, 121, 229, 260, and 297 of the CSFV E2 glycoprotein, resulting in attenuation of CSFV. 
     
     
         4 . The isolated polynucleotide molecule of  claim 1 , wherein said region encodes alterations of the N1 sites in CSFV E0 and E2 glycoproteins, resulting in attenuation of CSFV. 
     
     
         5 . The isolated polynucleotide molecule of  claim 1 , wherein said DNA sequence is SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, or a sequence homologous to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. 
     
     
         6 . The isolated polynucleotide molecule of  claim 1 , wherein said DNA sequence is SEQ ID NO: 1 or a sequence homologous to SEQ ID NO:1, said DNA sequence contains a mutation altering the glycosylation pattern of amino acid 116, wherein said mutation disables the encoded CSFV in its ability to produce CSF disease in said animal. 
     
     
         7 . The isolated polynucleotide molecule of  claim 1 , wherein said DNA sequence is SEQ ID NO: 2 or a sequence homologous to SEQ ID NO: 2, said DNA sequence contains a mutation altering the glycosylation pattern of the amino acids 75, 116, 121, 229, 260, and 297, wherein said mutation disables the encoded CSFV in its ability to produce CSF disease in said animal. 
     
     
         8 . The isolated polynucleotide molecule of  claim 1 , wherein said DNA sequence is SEQ ID NO: 3 or a sequence homologous to SEQ ID NO: 3, said DNA sequence contains a mutation altering the glycosylation pattern of the amino acids of the N1 sites in CSFV E0 and E2 glycoproteins, wherein said mutation disables the encoded CSFV in its ability to produce CSF disease in said animal. 
     
     
         9 . A plasmid capable of directly transfecting a suitable host cell and expressing a genetically modified CSFV from the suitable host cell so transfected, which plasmid comprises a) the DNA sequence of  claim 1 , and b) a promoter capable of transcribing said infectious RNA molecule in said suitable host cell. 
     
     
         10 . A method for generating a genetically modified CSFV, which method comprises transfecting a suitable host cell with a plasmid according to  claim 9  encoding the genetically modified CSFV and obtaining the genetically modified CSFV generated by the transfected host cell. 
     
     
         11 . A host cell transfected with the polynucleotide molecule of  claim 1 . 
     
     
         12 . A recombinant classical swine fever virus comprising DNA encoding CSFV E2 glycoprotein which has been modified by mutating a region of the E2 gene of the highly pathogenic strain Brescia, wherein said region encodes amino acid of position 116 of the CSFV E2 glycoprotein, a modification resulting in attenuation of CSFV. 
     
     
         13 . A recombinant classical swine fever virus comprising DNA encoding CSFV E2 glycoprotein which has been modified by mutating a region of the E2 gene of the highly pathogenic strain Brescia, wherein said region encodes amino acids of positions 75, 116, 121, 229, 260, and 297 of the CSFV E2 glycoprotein, a modification resulting in attenuation of CSFV. 
     
     
         14 . A recombinant classical swine fever virus comprising DNA encoding CSFV E0 and E2 glycoprotein which has been modified by mutating the N1 sites of the E0 and E2 gene of the highly pathogenic strain Brescia, a modification resulting in attenuation of CSFV. 
     
     
         15 . A recombinant classical swine fever virus comprising DNA encoding a mutated CSFV E2 glycoprotein having a sequence identified by SEQ ID NO:1, a modification resulting in attenuation of CSFV. 
     
     
         16 . A recombinant classical swine fever virus comprising DNA encoding a mutated CSFV E2 glycoprotein having a sequence identified by SEQ ID NO:2, a modification resulting in attenuation of CSFV. 
     
     
         17 . A recombinant classical swine fever virus comprising DNA encoding a mutated CSFV E2 glycoprotein having a sequence identified by SEQ ID NO:3, a modification resulting in attenuation of CSFV. 
     
     
         18 . A rationally designed live attenuated CSF vaccine comprising a recombinant classical swine fever virus according to any one of  claims 15 ,  16 , or  17 . 
     
     
         19 . A method of immunizing an animal against CSF, comprising administering to said animal, a vaccine comprising a recombinant classical swine fever virus according to any one of  claims 15 ,  16 , or  17 . 
     
     
         20 . A method of protecting an animal against CSF, comprising administering to said animal an amount of the vaccine of any one of  claims 15 ,  16 , or  17  effective to protect said animal from clinical CSF. 
     
     
         21 . A method of distinguishing animals infected with CSFV from animals vaccinated with the rationally designed live attenuated CSF vaccine of any one of  claims 15 ,  16 , or  17 , comprising: analyzing serum from an animal under evaluation in a competitive ELISA to determine if said serum inhibits binding of mAb 303. 
     
     
         22 . A strategy for producing an attenuated recombinant classical swine fever virus comprising:
 (a) identifying a glycosylation site in the E2 glycoprotein of the highly pathogenic strain Brescia;   (b) mutating the DNA encoding said glycosylated amino acid, whereby mutating said DNA results in an alteration in the glycosylation pattern of an amino acid characteristic of the CSFV virulence determinant: and   (c) achieving attenuation of CSFV.   
     
     
         23 . A method of producing an attenuated recombinant classical swine fever virus comprising DNA encoding a modified CSFV E2 glycoprotein, comprising:
 (a) mutating a region of the E2 gene of the highly pathogenic strain Brescia, wherein said region encodes amino acid 116 of the CSFV E2 glycoprotein, and whereby mutations in said DNA result in a change in the glycosylation pattern characteristic of CSFV E2 glycoprotein; and   (b) achieving attenuation of CSFV as a result of such modification.   
     
     
         24 . A method of producing an attenuated recombinant classical swine fever virus comprising DNA encoding a modified CSFV E2 glycoprotein, comprising:
 (a) mutating a region of the E2 gene of the highly pathogenic strain Brescia, wherein said region encodes amino acids of positions 75, 116, 121, 229, 260, and 297 of the CSFV E2 glycoprotein, and whereby mutations in said DNA result in a change in the glycosylation pattern characteristic of CSFV E2 glycoprotein; and   (b) achieving attenuation of CSFV as a result of such modification.   
     
     
         25 . A method of producing an attenuated recombinant classical swine fever virus comprising DNA encoding a modified CSFV E2 glycoprotein, comprising:
 (a) mutating the N1 sites of the E0 and E2 gene of the highly pathogenic strain Brescia, whereby mutations in said DNA result in a change in the glycosylation pattern characteristic of CSFV E0 and E2 glycoprotein; and   (b) achieving attenuation of CSFV as a result of such modification.   
     
     
         26 . A vaccine for protecting a porcine animal against infection by a CSFV, which vaccine comprises (a) a genetically modified CSFV encoded by an infectious RNA molecule encoded by the polynucleotide molecule according to  claim 1 , (b) said infectious RNA molecule, (c) said polynucleotide molecule in the form of a plasmid, or (d) a viral vector comprising said polynucleotide molecule, wherein the genetically modified CSFV is able to elicit an effective immunoprotective response against infection by a CSFV, in an amount effective to produce immunoprotection against infection by a CSFV; and a carrier acceptable for veterinary use. 
     
     
         27 . A method for preparing a genetically modified CSFV that is capable of eliciting an immunoprotective response in a mammal vaccinated therewith, which method comprises obtaining an isolated DNA encoding an infectious RNA molecule which encodes a wild-type CSFV; genetically mutating the DNA so as to obtain a mutated DNA encoding an infectious RNA molecule which encodes a genetically modified CSFV which virus is replication-defective and remains able to elicit an effective immunoprotective response against infection by the wild-type CSF virus in a mammal; placing said mutated DNA in a plasmid in an operable linkage to a promoter; transfecting a host cell with said plasmid; and expressing the genetically modified CSFV. 
     
     
         28 . A CSF vaccine comprising a genetically modified CSFV that is replication-defective and does not produce CSF disease in swine, wherein said virus is encoded by the polynucleotide of  claim 1 . 
     
     
         29 . A genetically modified CSFV, wherein the virus is encoded by the isolated polynucleotide molecule of  claim 1 .