Recombinant alphavirus-based vectors with reduced inhibition of cellular macro-molecular synthesis

Abstract

Isolated nucleic acid molecules are disclosed, comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.

1 . An alphavirus vector construct comprising:
 a 5′ promoter sequence capable of initiating in vivo the 5′ synthesis of alphavirus RNA from cDNA,   a sequence which initiates transcription of alphavirus RNA following the 5′ promoter,   a nucleic acid molecule which operably encodes all four alphaviral nonstructural proteins,   an alphavirus RNA polymerase recognition site,   a junction region promoter,   an internal ribosomal entry site (IRES), and   a 3′ polyadenylation site,   
       wherein the nucleic acid molecule encodes at least one altered alphaviral nonstructural protein (nsP) which, when operatively incorporated into a recombinant alphavirus particle, has a reduced level of vector-specific RNA synthesis as compared to wild-type. 
     
     
         2 . The alphavirus vector construct of  claim 1  wherein the recombinant alphavirus particle has the same or greater level of protein encoded by RNA transcribed from the junction region promoter as compared to wild-type recombinant alphavirus particle. 
     
     
         3 . The alphavirus vector construct of  claim 1  wherein the nucleic acid molecule encodes at least one nsP selected from the group consisting of Sindbis (SIN), Venezuelan equine encephalitis (VEE), Semliki forest virus (SFV), and Ross River virus (RRV). 
     
     
         4 . The alphavirus vector construct of  claim 1  wherein the altered nsP is an nsP2 protein which comprises serine at the amino acid position encoded by the codon comprising nucleotide 3855 of SEQ ID NO:101. 
     
     
         5 . The alphavirus vector construct of  claim 3  wherein the nsP2 protein which further comprises leucine at the amino acid position encoded by the codon comprising nucleotide 1971 of SEQ ID NO:101. 
     
     
         6 . The alphavirus vector construct of  claim 4  wherein the nucleic acid molecule further encodes:
 an nsP1 protein which comprises lysine at the amino acid position encoded by the codon comprising nucleotide 120 of SEQ ID NO:101; 
 an nsP3 protein which comprises valine at the amino acid position encoded by the codon comprising nucleotide 4339 of SEQ ID NO:101 and phenylalanine at the amino acid position encoded by the codon comprising nucleotide 4864 of SEQ ID NO:101; and 
 an nsP4 protein which comprises histidine at the amino acid position encoded by the codon comprising nucleotide 5854 of SEQ ID NO:101. 
 
     
     
         7 . The alphavirus vector construct of  claim 1  wherein the altered alphavirus nonstructural protein is an nsP4 protein which comprises histidine at the amino acid position encoded by the codon comprising nucleotide 5854 of SEQ ID NO:101. 
     
     
         8 . The alphavirus vector construct of  claim 6  wherein the nucleic acid molecule further encodes an nsP3 protein which comprises valine at the amino acid position encoded by the codon comprising nucleotide 4339 of SEQ ID NO:101 and phenylalanine at the amino acid position encoded by the codon comprising nucleotide 4864 of SEQ ID NO:101. 
     
     
         9 . The alphavirus vector construct of  claim 1  wherein the junction region promoter is modified to reduce or prevent viral transcription. 
     
     
         10 . A eukaryotic layered vector initiation system (ELVIS) comprising:
 a 5′ promoter sequence capable of initiating in vivo the 5′ synthesis of alphavirus RNA from cDNA,   a sequence which initiates transcription of alphavirus RNA following the 5′ promoter,   a nucleic acid molecule which operably encodes all four alphaviral nonstructural proteins,   an alphavirus RNA polymerase recognition site,   a junction region promoter,   an internal ribosomal entry site (IRES), and   a 3′ polyadenylation site,   
       wherein the nucleic acid molecule encodes at least one altered alphaviral nonstructural protein (nsP) which, when operatively incorporated into a recombinant alphavirus particle, has a reduced level of vector-specific RNA synthesis as compared to wild-type. 
     
     
         11 . The eukaryotic layered vector initiation system of  claim 1  wherein the recombinant alphavirus particle has the same or greater level of protein encoded by RNA transcribed from the junction region promoter as compared to wild-type recombinant alphavirus particle. 
     
     
         12 . The eukaryotic layered vector initiation system of  claim 1  wherein the nucleic acid molecule encodes at least one nsP selected from the group consisting of Sindbis (SIN), Venezuelan equine encephalitis (VEE), Semliki forest virus (SFV), and Ross River virus (RRV). 
     
     
         13 . The eukaryotic layered vector initiation system of  claim 1  wherein the altered nsP is an nsP2 protein which comprises serine at the amino acid position encoded by the codon comprising nucleotide 3855 of SEQ ID NO:101. 
     
     
         14 . The eukaryotic layered vector initiation system of  claim 3  wherein the nsP2 protein which further comprises leucine at the amino acid position encoded by the codon comprising nucleotide 1971 of SEQ ID NO:101. 
     
     
         15 . The eukaryotic layered vector initiation system of  claim 4  wherein the nucleic acid molecule further encodes:
 an nsP1 protein which comprises lysine at the amino acid position encoded by the codon comprising nucleotide 120 of SEQ ID NO:101; 
 an nsP3 protein which comprises valine at the amino acid position encoded by the codon comprising nucleotide 4339 of SEQ ID NO:101 and phenylalanine at the amino acid position encoded by the codon comprising nucleotide 4864 of SEQ ID NO:101; and 
 an nsP4 protein which comprises histidine at the amino acid position encoded by the codon comprising nucleotide 5854 of SEQ ID NO:101. 
 
     
     
         16 . The eukaryotic layered vector initiation system of  claim 1  wherein the altered alphavirus nonstructural protein is an nsP4 protein which comprises histidine at the amino acid position encoded by the codon comprising nucleotide 5854 of SEQ ID NO:101. 
     
     
         17 . The eukaryotic layered vector initiation system of  claim 6  wherein the nucleic acid molecule further encodes an nsP3 protein which comprises valine at the amino acid position encoded by the codon comprising nucleotide 4339 of SEQ ID NO:101 and phenylalanine at the amino acid position encoded by the codon comprising nucleotide 4864 of SEQ ID NO:101. 
     
     
         18 . The eukaryotic layered vector initiation system of  claim 7  wherein the junction region promoter is modified to reduce or prevent viral transcription. 
     
     
         19 . A method of stimulating an immune response in a mammal comprising administering to the mammal the eukaryotic layered vector initiation system of  claim 10 . 
     
     
         20 . A method of stimulating an immune response in a mammal comprising administering to the mammal the recombinant alphavirus particle of  claim 1 .