Method for the purification of alphavirus replicon particles

Abstract

Methods of production and purification for viruses and virus-derived vectors, including those related to alphaviruses, are disclosed. In one aspect, methods of purification that subject alphavirus replicon particle preparations to one or more steps of chromatographic purification, such as using an ion exchange resin, are provided. Also disclosed are methods of characterizing alphavirus replicon particles and utilizing these materials for vaccines and gene-based therapeutics.

1 . A method for purifying alphavirus replicon particles comprising: 
 a) contacting a preparation containing alphavirus replicon particles with a tentacle ion exchange resin, under conditions and for a time sufficient to bind to said resin;    b) removing the portion of the preparation which is not bound to said ion exchange resin from said ion exchange resin;    c) eluting the bound alphavirus replicon particles from said ion exchange resin; and    d) recovering said replicon particles.    
     
     
         2 . The method according to  claim 1  wherein said tentacle ion exchange resin is a cationic exchange resin.  
     
     
         3 . The method according to  claim 1  wherein said tentacle ion exchange resin is an anionic exchange resin.  
     
     
         4 . A method for purifying alphavirus replicon particles comprising at least two chromatographic purification steps wherein said purification steps are selected from the group consisting of ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography.  
     
     
         5 . The method according to  claim 4  wherein said ion exchange chromatography is performed using a tentacle ion exchange resin.  
     
     
         6 . The method according to  claim 4  wherein a first purification step is ion exchange chromatography and a second purification step is size exclusion chromatography.  
     
     
         7 . An alphavirus replicon particle preparation made according to any one of claims  4 - 6 .  
     
     
         8 . An alphavirus replicon particle preparation according to  claim 7  wherein said preparation comprises an immunogenic composition capable of expressing an antigen derived from a pathogenic agent.  
     
     
         9 . The alphavirus replicon particle preparation according to  claim 8  wherein said pathogenic agent is selected from the group consisting of viruses, bacteria, fungi, parasites, and cancerous cells.  
     
     
         10 . An alphavirus replicon particle preparation according to  claim 7  wherein said alphavirus replicon particle preparation comprises a therapeutic.  
     
     
         11 . An alphavirus replicon particle preparation according to  claim 10  wherein said alphavirus replicon particle preparation expresses a lymphokine, cytokine, or chemokine.  
     
     
         12 . An alphavirus replicon particle preparation according to  claim 7  wherein said lymphokine, cytokine or chemokine is selected from the group consisting of IL-2, IL-10-, IL-12, gamma interferon, GM-CSF, macrophage inflammatory protein (MIP)3α, MIP3β, and secondary lymphoid tissue chemokine (SLC).  
     
     
         13 . A method for stimulating an immune response within a warm-blooded animal, comprising administering to a warm-blooded animal the alphavirus replicon particle preparation of  claim 7 .  
     
     
         14 . The method according to  claim 13  wherein said alphavirus replicon particle preparation expresses a lymphokine, cytokine, or chemokine.  
     
     
         15 . The method according to  claim 14  wherein said lymphokine, cytokine or chemokine is selected from the group consisting of IL-2, IL-10-, IL-12, gamma interferon, GM-CSF, macrophage inflammatory protein (MIP)3α, MIP3β, and secondary lymphoid tissue chemokine (SLC).  
     
     
         16 . A method of producing alphavirus replicon particles comprising: 
 a) infecting alphavirus packaging cells with a seed stock of alphavirus replicon particles;    b) incubating the infected packaging cells in a bioreactor, under conditions and for a time sufficient to permit the production of alphavirus replicon particles; and    c) harvesting culture supernatants containing said replicon particles.    
     
     
         17 . A method according to  claim 16  wherein said bioreactor is an external component bioreactor.  
     
     
         18 . A method according to  claim 16  wherein said bioreactor is a suspension culture bioreactor.  
     
     
         19 . A method according to  claim 16  wherein said bioreactor is a hollow fiber bioreactor.  
     
     
         20 . A method for producing alphavirus replicon particles comprising: 
 a) transfecting alphavirus packaging cells with a DNA-based alphavirus replicon or eukaryotic layered vector initiation system;    b) incubating the transfected packaging cells in a bioreactor, under conditions and for a time sufficient to permit the production of alphavirus replicon particles; and    c) harvesting culture supernatants containing said replicon particles.    
     
     
         21 . A method for producing alphavirus replicon particles comprising: 
 a) transfecting alphavirus packaging cells with an alphavirus RNA vector replicon transcribed in vitro;    b) incubating the transfected packaging cells in a bioreactor, under conditions and for a time sufficient to permit the production of alphavirus replicon particles; and    c) harvesting culture supernatants containing said replicon particles.    
     
     
         22 . A method for detecting multiple recombination events in a population of alphavirus replicon particles comprising: 
 a) providing a nucleic acid substrate suitable for detecting multiple recombination events, said substrate derived from said population of alphavirus replicon particles;    b) reacting said nucleic acid substrate with at least one first reaction mixture comprising an oligonucleotide complementary to an alphavirus nonstructural protein gene and an oligonucleotide complementary to an alphavirus structural protein gene, wherein said structural protein is either a capsid protein or a non-capsid structural protein, under conditions suitable and for a time sufficient to permit amplification of said nucleic acid substrate to form a first reaction product;    c) reacting said first reaction product with a second reaction mixture comprising an oligonucleotide complementary to an alphavirus capsid protein gene and an oligonucleotide complementary to a non-capsid alphavirus structural protein gene, under conditions suitable and for a time sufficient to permit amplification of said nucleic acid template to form a second reaction product; and    d) determining the presence or absence of said second reaction product.    
     
     
         23 . A method for detecting multiple recombination events in a population of alphavirus replicon particles comprising: 
 a) providing a nucleic acid substrate suitable for detecting multiple recombination events, said substrate derived from said population of alphavirus replicon particles.    b) reacting said nucleic acid substrate with a first reaction mixture comprising an oligonucleotide complementary to an alphavirus nonstructural protein gene and an oligonucleotide complementary to an alphavirus capsid protein gene, under conditions suitable and for a time sufficient to permit amplification of said nucleic acid substrate to form a first reaction product;    c) reacting said first reaction product with a second reaction mixture comprising an oligonucleotide complementary to an alphavirus capsid protein gene and an oligonucleotide complementary to a non-capsid alphavirus structural protein gene, under conditions suitable and for a time sufficient to permit amplification of said nucleic acid template to form a second reaction product; and    d) determining the presence or absence of said second reaction product.    
     
     
         24 . A method for detecting multiple recombination events in a population of alphavirus replicon particles comprising: 
 a) providing a nucleic acid substrate suitable for detecting multiple recombination events, said substrate derived from said population of alphavirus replicon particles;    b) reacting said nucleic acid substrate with a first reaction mixture comprising an oligonucleotide complementary to an alphavirus nonstructural protein gene and an oligonucleotide complementary to a non-capsid alphavirus structural protein gene, under conditions suitable and for a time sufficient to permit amplification of said nucleic acid substrate to form a first reaction product,    c) reacting said first reaction product with a second reaction mixture comprising an oligonucleotide complementary to an alphavirus capsid protein gene and an oligonucleotide complementary to a non-capsid alphavirus structural protein gene, under conditions suitable and for a time sufficient to permit amplification of said nucleic acid template to form a second reaction product; and    d) determining the presence or absence of said second reaction product.