Packaging of positive-strand RNA virus replicon particles

Abstract

The invention generally relates to recombinant polynucleotides, positive-strand RNA virus (psRNAV) recombinant expression vectors, and packaging systems. The packaging systems are based on the expression of helper functions by coinfecting re-combinant poxvirus vectors comprising recombinant polynucleotides. Methods for obtaining psRNAV replicon particles using these packaging systems are disclosed. Immunogenic compositions and pharmaceutical formulations are provided that comprise replicon particles of the invention. Methods for generating an immune response or producing a pharmaceutical effect are also provided.

1 - 53 . (canceled)  
     
     
         54 . 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.    
     
     
         55 . 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.