Alphavirus replicon particle

Abstract

Provided is an alphavirus replicon particle (ARP), which comprises (i) alphavirus structural proteins comprising capsid and/or envelope, and (ii) an alphavirus replicon comprising a polynucleotide encoding alphavirus non-structural proteins nsp1, nsp2, nsp3 and nsp4 and at least one gene of interest wherein at least one of capsid, and E3 and E2 in the envelope comprise one or more amino acid alteration but E1 in the envelope comprises no amino acid alteration.

What is claimed is: 
     
         1 . An alphavirus replicon particle (ARP), which comprises
 (i) alphavirus structural proteins comprising capsid and/or envelope, and   (ii) an alphavirus replicon comprising a polynucleotide encoding alphavirus non-structural proteins nsp1, nsp2, nsp3 and nsp4 and at least one gene of interest wherein at least one of capsid, and E3 and E2 in the envelope comprise one or more amino acid alteration but E1 in the envelope comprises no amino acid alteration.   
     
     
         2 . The ARP of  claim 1 , wherein the alphavirus structural proteins comprise one or more alterations in the alphavirus capsid protein Nuclear Localization Signal (NLS). 
     
     
         3 . The ARP of  claim 2 , wherein one or more of lysine or arginine in the alphavirus capsid protein Nuclear Localization Signal is substituted with asparagine or alanine. 
     
     
         4 . The ARP of  claim 1 , wherein the alphavirus structural proteins comprise one or more alterations in the alphavirus E2 protein. 
     
     
         5 . The ARP of  claim 1 , wherein the alphavirus structural proteins comprise one or more alterations at the furin site in alphavirus E3 protein. 
     
     
         6 . The ARP of  claim 1 , wherein the alphavirus is a CHIKV or VEEV. 
     
     
         7 . The ARP of  claim 6 , wherein the CHIKV is CHIKV strain 37997 or strain OPY-1. 
     
     
         8 . The ARP of  claim 6 , wherein the VEEV is VEEV strain TC-83. 
     
     
         9 . The ARP of  claim 1 , wherein the gene of interest encodes an antigen. 
     
     
         10 . A method for preparing alphavirus replicon particles, comprising the steps of co-transfecting cells with
 i) a vector comprising a polynucleotide encoding alphavirus non-structural protein nsp1, nsp2, nsp3 and nsp4, and at least one gene of interest,   ii) a vector comprising a polynucleotide encoding an alphavirus capsid protein, and   iii) a vector comprising a polynucleotide encoding an alphavirus E3-52-6K-E1,   wherein at least one of the capsid, E3 and E2 comprises one or more amino acid alteration but E1 comprises no amino acid alteration,   culturing the transfected cells, and   purifying the ARPs from the cell culture.   
     
     
         11 . The method of  claim 10 , wherein the alphavirus structural proteins comprise one or more alterations in the alphavirus capsid protein Nuclear Localization Signal (NLS). 
     
     
         12 . The method of  claim 11 , wherein one or more of lysine or arginine in the alphavirus capsid protein Nuclear Localization Signal is substituted with asparagine or alanine. 
     
     
         13 . The method of  claim 10 , wherein the alphavirus structural proteins comprise one or more alternations in the alphavirus E2 protein. 
     
     
         14 . The method of  claim 13 , wherein the alphavirus structural proteins comprise one or more alterations at the furin site in alphavirus E3 protein. 
     
     
         15 . The method of  claim 10 , wherein the alphavirus is a CHIKV or VEEV. 
     
     
         16 . The method of  claim 15 , wherein the CHIKV is CHIKV strain 37997 or strain OPY-1. 
     
     
         17 . The method of  claim 15 , wherein the VEEV is VEEV strain TC-83. 
     
     
         18 . The method of  claim 10 , wherein the gene of interest encodes an antigen. 
     
     
         19 . An alphavirus replicon particle (ARP), which comprises
 (i) CHIKV structural proteins comprising capsid and/or envelope, and   (ii) a VEEV replicon comprising a polynucleotide encoding VEEV non-structural proteins nsp1, nsp2, nsp3 and nsp4 and at least one gene of interest.   
     
     
         20 . The ARP of  claim 19 , wherein the CHIKV is CHIKV strain 37997 or strain OPY-1, and VEEV is VEEV strain TC-83.