Vaccine comprising an attenuated pestivirus

Abstract

Attenuated pestiviruses, in particular attenuated BVDV, wherein at least one mutation is in the coding sequence for glycoprotein E ms and at least another mutation in the coding sequence for N pro which preferably leads to combined inactivation of the RNase activity residing in glycoprotein E ms in addition to the inactivation of the (hypothesized) immunomodulating activity residing in N pro . Methods for attenuating pestiviruses such as BVDV, nucleic acids encoding the pestiviruses, in particular BVDV, compositions and vaccines comprising the attenuated pestiviruses, in particular BVDV, of the invention.

1 . An attenuated classical swine fever virus (CSFV) having at least one mutation in the coding sequence for glycoprotein E ms  and at least another mutation in the coding sequence for N pro . 
     
     
         2 . The classical swine fever virus according to  claim 1 , wherein the mutation in the coding sequence for glycoprotein E ms  leads to inactivation of RNase activity residing in E ms  and/or the mutation in the coding sequence for N pro  leads to inactivation of the N pro . 
     
     
         3 . The classical swine fever virus according to  claim 1 , wherein the mutations are selected from the group of deletions, insertion mutations, and substitution mutations. 
     
     
         4 . The classical swine fever virus according to  claim 2 , wherein the mutations are selected from the group of deletions, insertion mutations, and substitution mutations. 
     
     
         5 . The classical swine fever virus according to  claim 1 , wherein the mutation(s) are deletions. 
     
     
         6 . The classical swine fever virus according to  claim 2 , wherein the mutation(s) are deletions. 
     
     
         7 . The classical swine fever virus according to  claim 1 , wherein the mutation(s) in the coding sequence for glycoprotein E ms  are located in the encoding nucleotide sequence corresponding to amino acids at position 295 to 307 and/or position 338 to 357. 
     
     
         8 . The classical swine fever virus according to  claim 1 , wherein the mutation in the coding sequence for glycoprotein E ms  is a deletion or substitution of the histidine at position 346. 
     
     
         9 . The classical swine fever virus according to  claim 1 , wherein the mutation(s) in the coding sequence for glycoprotein E ms  are located in the nucleotide sequence coding for the conserved E ms  sequence SLHGIWPEKIC (SEQ ID NO: 15) and/or RHEWNKHGWCNW (SEQ ID NO: 16). 
     
     
         10 . The classical swine fever virus according to  claim 1 , wherein the mutation(s) in the coding sequence for glycoprotein E rns  are two mutations located in the nucleotide sequence coding for the conserved E ms  sequence SLHGIWPEKIC (SEQ ID NO: 15) and/or RHEWNKHGWCNW (SEQ ID NO: 16). 
     
     
         11 . The classical swine fever virus according to  claim 1 , wherein the mutation in the coding sequence for glycoprotein E ms  is a single mutation located in the conserved E ms  sequence SLHGIWPEKIC (SEQ ID NO: 15) or RHEWNKHGWCNW (SEQ ID NO: 16). 
     
     
         12 . The classical swine fever virus according to  claim 1 , wherein the mutation(s) in the coding sequence for N pro  lead to an encoded polyprotein as characterized by the following formula:
   [N pro ] x −[PS] y −[C-term]
   wherein:   [N pro ] is the N pro  portion of the polyprotein, wherein x is the number of amino acids of the N pro  present in the polyprotein;   [PS] is a processing signal selected from the group consisting of: ubiquitin, LC3, SUMO-1, NEDD8, GATE-16 or GABA(A)RAP), Intein, picornavirus 3C, caridovirus 2A, or p15 of rabbit hemorrhagic disease virus;   [C-term] is the complete virus polyprotein except for N pro , but including the capsid (C)-protein and any other protein present in the virus polyprotein including the carboxyterminal NSSB;   y is 0 or 1, where 0 means [PS] is absent and 1 means [PS] is present; and   x is 0 to 12 amino acids if y is 0, or 0 to 168 amino acids if y is 1.   
     
     
         13 . The classical swine fever virus according to  claim 12 , wherein the mutation(s) in the coding sequence for N pro  lead to an encoded polyprotein as characterized by the following formula:
   [N pro ] 1 −[PS] 0 −[C-term].
   
     
     
         14 . The classical swine fever virus according to  claim 12 , wherein the mutation(s) in the coding sequence for N pro  lead to an encoded polyprotein as characterized by the following formula:
   [N pro ] 3 −[PS] 0 −[C-term].
   
     
     
         15 . The classical swine fever virus according to  claim 12 , wherein the mutation(s) in the coding sequence for N pro  lead to an encoded polyprotein as characterized by the following formula: [N pro ] 3 −[PS] 0 [−C-term] and the mutation in the coding sequence for glycoprotein E ms  is a single mutation located in the conserved E ms  sequence SLHGIWPEKIC (SEQ ID NO: 15) or RHEWNKHGWCNW (SEQ ID NO: 16). 
     
     
         16 . The classical swine fever virus according to  claim 12 , wherein the mutation(s) in the coding sequence for N pro  lead to an encoded polyprotein as characterized by the following formula:
   [N pro ] 4 −[PS] 0 −[C-term].
   
     
     
         17 . The classical swine fever virus according to  claim 12 , wherein the mutation(s) in the coding sequence for N pro  lead to an encoded polyprotein as characterized by the following formula:
   [N pro ] 6 −[PS] 0 −[C-term].
   
     
     
         18 . The classical swine fever virus according to  claim 12 , wherein the mutation(s) in the coding sequence for N pro  lead to an encoded polyprotein as characterized by the following formula:
   [N pro ] 4 −[PS] 0 −[C-term],
   
       wherein [C-term]* is [C-term] wherein in the C-protein the amino acid at position 2 is changed from D to N. 
     
     
         19 . The classical swine fever virus according to  claim 12 , wherein the mutation(s) in the coding sequence for N pro  leads to an encoded polyprotein is characterized by the following formula:
   [N pro ] x −[PS] 1 −[C-term],
   wherein PS is ubiquitin or LC3.   
     
     
         20 . The classical swine fever virus according to  claim 12 , wherein mutation(s) in the coding sequence for N pro  leads to an encoded polyprotein as characterized by a formula selected from the group consisting of:
   M−[PS] 0 −[C-term];
     MEL−[PS] 0 −[C-term];
     MELN−[PS] 0 −[C-term];
     MELNH−[PS] 0 −[C-term];
     MELHF−[PS] 0 −[C-term];
     MELNHFE−[PS] 0 −[C-term];
     MELNHFEL−[PS] 0 −[C-term];
     MELNHFELL−[PS] 0 −[C-term];
     MELNHFELLY−[PS] 0 −[C-term];
     MELNHFELLYK−[PS] 0 −[C-term]; and
     MELNHFELLYKT−[PS] 0 −[C-term].
   
     
     
         21 . The classical swine fever virus according to  claim 12 , wherein the mutation(s) in the coding sequence for N pro  leads to an encoded polyprotein as characterized by the following formula:
   [N pro ] x −[PS] 0 −ME−[PS] 0 −[C-term*],
   
       wherein [C-term]* is [C-term] wherein in the C-protein the amino acid at position 2 is changed from D to N. 
     
     
         22 . The classical swine fever virus according to  claim 12 , wherein the mutation(s) in the coding sequence for N pro  leads to an encoded polyprotein as characterized by the following formula:
   [N pro ] 22 −[PS] 1 −[C-term],
   wherein PS is ubiquitin or LC3.   
     
     
         23 . The classical swine fever virus according to one of  claims 12 , wherein the [PS] 0  is replaced by [PS] 1 , and wherein the PS is selected from the group of consisting of: ubiquitin, LC3, SUMO-1, NEDD8, GATE-16, GABA(A)RAP, intein, picornavirus 3C, caridovirus 2A, and p15 of rabbit hemorrhagic disease virus. 
     
     
         24 . A composition comprising the virus according to  claim 1  and a solution. 
     
     
         25 . The composition according to  claim 24 , which induces an immunological response in an animal. 
     
     
         26 . The composition according to  claim 24 , which is a vaccine. 
     
     
         27 . The composition according to  claim 26 , further comprising a pharmaceutically acceptable carrier or excipient. 
     
     
         28 . A nucleic acid molecule comprising the nucleic acid encoding a classical swine fever virus according to  claim 1 , or a fragment, functional variant, variant based on the degenerative nucleic acid code, fusion molecule, or a chemical derivative thereof. 
     
     
         29 . The nucleic acid molecule according to  claim 28 , wherein the nucleotide molecule is DNA. 
     
     
         30 . The nucleic acid molecule according to  claim 29 , wherein the nucleotide molecule is RNA. 
     
     
         31 . A method for attenuating a classical swine fever virus, wherein at least one mutation in the coding sequence for glycoprotein E ms  and at least another mutation in the coding sequence for N pro  is generated in a pestivirus. 
     
     
         32 . The method according to  claim 31 , the method comprising:
 (a) reversely transcribing a wild type classical swine fever to obtain a cDNA;   (b) cloning the cDNA;   (c) introducing mutations selected from deletions, insertion mutations, and/or substitution mutations into the cDNA, wherein the mutations are located in the coding sequence encoding glycoprotein E ms  and the protease N xo ; and   (d) incorporating the cDNA into a plasmid or into a DNA virus capable of directing the transcription of the classical swine fever virus cDNA into RNA in vitro or upon infection of suitable cells.   
     
     
         33 . A method of treatment of disease caused by classical swine fever virus, the method comprising administering to an animal in need thereof an effective amount of the attenuated classical swine fever virus according to  claim 1 . 
     
     
         34 . A method for attenuating a classical swine fever virus, wherein at least one mutation in the coding sequence for glycoprotein E ms  and at least another mutation in the coding sequence for N pro  is generated in the classical swine fever virus and wherein the attenuated virus does not cross the placenta in animals infected with the attenuated virus. 
     
     
         35 . The method according to  claim 34 , the method comprising:
 (a) reversely transcribing a wild type classical swine fever virus to obtain a cDNA;   (b) cloning the cDNA;   (c) introducing mutations selected from deletions, insertion mutations, and/or substitution mutations into the cDNA, wherein the mutations are located in the coding sequence encoding glycoprotein E ms  and the protease NP pro ; and   (d) incorporating the cDNA into a plasmid or into a DNA virus capable of directing the transcription of classical swine fever virus cDNA into RNA in vitro or upon infection of suitable cells.