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 pestivirus 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 , 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 . 
     
     
         2 . (canceled) 
     
     
         3 . The virus according to  claim 1 , wherein the mutations are selected from the group of deletions, insertion mutations, and substitution mutations. 
     
     
         4 - 14 . (canceled) 
     
     
         15 . The 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 SLHGIWPEKICTG and/or LQRHEWNKHGWCNWFHIEPW. 
     
     
         16 . The virus according to  claim 1 , wherein the mutation(s) in the coding sequence for glycoprotein E ms  located in the nucleotide sequence coding for the conserved E ms  sequence SLHGIWPEKIC and/or RHEWNKHGWCNR. 
     
     
         17 . The virus according to  claim 1 , wherein the mutation(s) in the coding sequence for glycoprotein E ms  are two mutations located in the nucleotide sequence coding for the conserved E ms  sequence SLHGIWPEKIC and/or RHENKHGWCNR. 
     
     
         18 . The 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 or RHEWNKHGWCNR. 
     
     
         19 . The 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 NS5B; 
 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. 
 
     
     
         20 . (canceled) 
     
     
         21 . The virus according to  claim 19 , 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]. 
     
     
         22 . (canceled) 
     
     
         23 . The virus according to  claim 19 , 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]. 
     
     
         24 . (canceled) 
     
     
         25 . The virus according to  claim 19 , 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 or RHEWNKHGWCNR. 
     
     
         26 . (canceled) 
     
     
         27 . The virus according to  claim 19 , 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]. 
     
     
         28 . (canceled) 
     
     
         29 . The virus according to  claim 19 , 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]. 
     
     
         30 . (canceled) 
     
     
         31 . The virus according to  claim 19 , 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]O—[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. 
     
     
         32 . (canceled) 
     
     
         33 . The virus according to  claim 19 , wherein the mutation(s) in the coding sequence for N pro  lead to an encoded polyprotein is characterized by the following formula:
   [N pro ] x -[PS] 1 -[C-term],   
       wherein PS is ubiquitin or LC3. 
     
     
         34 - 38 . (canceled) 
     
     
         39 . The virus according to  claim 19 , wherein the mutation(s) in the coding sequence for N pro  lead to an encoded polyprotein as characterized by the following formula:
   [N pro ] 22 -[PS] 1 -[C-term],   
       wherein PS is ubiquitin or LC3. 
     
     
         40 . (canceled) 
     
     
         41 . The virus according to  claim 21 , 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. 
     
     
         42 - 43 . (canceled) 
     
     
         44 . A composition comprising the virus according to  claim 1  and a solution. 
     
     
         45 . (canceled) 
     
     
         46 . The composition according to  claim 44 , which induces an immunological response in an animal. 
     
     
         47 . (canceled) 
     
     
         48 . The composition according to  claim 44 , which is a vaccine. 
     
     
         49 . (canceled) 
     
     
         50 . The composition according to  claim 48 , further comprising a pharmaceutically acceptable carrier or excipient. 
     
     
         51 - 54 . (canceled) 
     
     
         55 . A method for attenuating a pestivirus, 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. 
     
     
         56 . (canceled) 
     
     
         57 . The method according to  claim 55 , the method comprising:
 (a) reversely transcribing a wild type pestivirus 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 pro ; and   (d) incorporating the cDNA into a plasmid or into a DNA virus capable of directing the transcription of the pestivirus cDNA into RNA in vitro or upon infection of suitable cells.   
     
     
         58 - 59 . (canceled)