Packaging of positive-strand RNA virus replicon particles

An alphavirus replicon packaging system comprising:

• (a) a recombinant MVA comprising a first portion comprising a sequence encoding a T7 DNA-dependent RNA polymerase operatively linked to a vaccinia virus synthetic early/late promoter;
• and a second portion comprising a sequence encoding at least one psRNAV structural protein, operatively linked to a second heterologous promoter,
• wherein the at least one psRNAV structural protein is an alphavirus structural protein selected from an alphavirus Venezuelan equine encephalitis virus (VEE) capsid and an alphavirus VEE glycoprotein, but not all of the psRNAV VEE structural proteins and wherein the second heterologous promoter binds to said T7 DNA-dependent RNA polymerase;
• (b) a recombinant MVA comprising a first portion comprising a sequence encoding at least one psRNAV structural protein, operatively linked to a first heterologous promoter;
• and a second portion comprising a second heterologous promoter operatively linked to a psRNAV replicon comprising an psRNAV subgenomic promoter operatively linked to a sequence encoding at least one foreign polypeptide
• wherein the at least one psRNAV structural protein is an alphavirus structural protein selected from an alphavirus Venezuelan equine encephalitis virus (VEE) capsid and an alphavirus VEE glycoprotein, but not all of the psRNAV VEE structural proteins and wherein the first and second heterologous promoters both bind to a T7 DNA-dependent RNA polymerase;
• and
• wherein the alphavirus structural protein of (a) and the alphavirus structural protein of (b) are not the same.

An alphavirus replicon packaging system comprising:

• (a) a recombinant MVA comprising a first portion comprising a sequence encoding a T7 DNA-dependent RNA polymerase operatively linked to a vaccinia virus synthetic early/late promoter;
• and a second portion comprising a sequence encoding a replicon-like psRNAV helper RNA sequence operatively linked to a second heterologous promoter;
• wherein the replicon-like psRNAV helper RNA sequence of the second portion is an alphavirus helper RNA sequence comprising a sequence encoding an alphavirus structural protein selected from an alphavirus VEE capsid and an alphavirus VEE glycoprotein and wherein the second heterologous promoter binds to a T7 DNA-dependent RNA polymerase;
• (b) a recombinant MVA comprising a first portion comprising a sequence encoding a replicon-like psRNAV helper RNA sequence operatively linked to a first heterologous promoter;
• and a second portion comprising a second heterologous promoter operatively linked to a psRNAV replicon comprising a psRNAV subgenomic promoter operatively linked to a sequence encoding at least one foreign polypeptide;
• wherein the replicon-like psRNAV helper RNA sequence of the first portion is an alphavirus helper RNA sequence comprising a sequence encoding an alphavirus structural protein selected from an alphavirus VEE glycoprotein and an alphavirus VEE capsid;
• and wherein the first and second heterologous promoters both bind to a T7 DNA dependent RNA polymerase;
• and
• wherein the alphavirus structural protein of (a) and the alphavirus structural protein of (b) are not the same.