Chimeric alphavirus replicon particles

Abstract

Chimeric alphaviruses and alphavirus replicon particles are provided including methods of making and using same. Specifically, alphavirus particles are provided having nucleic acid molecules derived from one or more alphaviruses and structural proteins (capsid and/or envelope) from at least two or more alphaviruses. Methods of making, using, and therapeutic preparations containing the chimeric alphavirus particle, are disclosed.

What is claimed is:  
     
         1 . An alphavirus replicon RNA encoding nonstructural proteins from at least one alphavirus and one or more heterologous sequence(s), wherein said replicon encodes a modified nsP4 polypeptide and wherein said replicon does not comprise sequences encoding alphavirus structural proteins.  
     
     
         2 . The alphavirus replicon RNA of  claim 1 , wherein one or more amino acids in a region corresponding to amino acid residues 363 to 404 of nsP4, numbered relative to a wild-type SIN, are modified.  
     
     
         3 . The alphavirus replicon RNA of  claim 2 , wherein one or more amino acid residues in a region corresponding to amino acid residues 387 to 396, numbered relative to SIN, are modified.  
     
     
         4 . The alphavirus replicon of  claim 2 , wherein the modification comprises replacing one or more wild-type amino residues with a different amino acid.  
     
     
         5 . The alphavirus replicon of  claim 3 , wherein the modification comprises replacing one or more wild-type amino residues with a different amino acid.  
     
     
         6 . An alphavirus particle comprising the replicon of  claim 1 .  
     
     
         7 . An alphavirus particle comprising the replicon of  claim 2 .  
     
     
         8 . An alphavirus particle comprising the replicon of  claim 3 .  
     
     
         9 . The replicon particle according to  claim 1 , wherein said particle is a replicon particle and further wherein said RNA comprises, in 5′ to 3′ order (i) a 5′ sequence required for nonstructural protein-mediated amplification, (ii) a nucleotide sequence encoding alphavirus nonstructural proteins, (iii) a means for expressing a heterologous nucleic acid, (iv) the heterologous nucleic acid sequence, (v) a 3′ sequence required for nonstructural protein-mediated amplification, and (vi) a polyadenylate tract, wherein said heterologous nucleic acid sequence replaces an alphavirus structural protein gene.  
     
     
         10 . The alphavirus replicon RNA of  claim 1 , wherein said replicon sequences are derived from Sindbis virus (SIN).  
     
     
         11 . The alphavirus replicon RNA of  claim 1 , wherein said replicon sequences are derived from Venezuelan equine encephalitis virus (VEE).  
     
     
         12 . The alphavirus replicon RNA of  claim 1 , wherein said replicon sequences are derived from Semliki Forest virus (SFV).  
     
     
         13 . The alphavirus replicon particle according to  claim 1 , wherein heterologous nucleic acid sequence encodes for a therapeutic agent or an immunogen.  
     
     
         14 . A method for producing alphavirus replicon particles, comprising introducing into a host cell: 
 a) an alphavirus replicon RNA encoding nonstructural proteins from at least one alphavirus and one or more heterologous sequence(s), wherein said replicon encodes a modified nsP4 polypeptide and wherein said replicon does not comprise sequences encoding alphavirus structural proteins; and    b) at least one separate defective helper RNA(s) encoding alphavirus structural protein(s) absent from the replicon RNA;    wherein alphavirus replicon particles are produced.    
     
     
         15 . A method of generating an immune response in a mammal, the method comprising administering an alphavirus replicon according to  claim 13  to said mammal, thereby generating an immune response.