virus-like platform for rapid vaccine discovery

Abstract

The invention is directed to virus-like particles (VLPs) of an RNA bacteriophage that (a) comprises a coat polypeptide of said phage modified by insertion of a heterologous peptide that is displayed on said VLP and (b) encapsidates said bacteriophage mRNA as well as populations of these VLPs, and their uses. The invention is further directed to VLPs that encapsidate heterologous substances, as well as populations of these VLPs and their uses.

1 - 9 . (canceled) 
     
     
         10 . An isolated transcription unit comprising a bacterial or bacteriophage promoter, a coding sequence of an RNA bacteriophage single chain coat polypeptide dimer with a site for insertion of a heterologous peptide in the downstream subunit of the dimer and a bacterial or bacteriophage terminator. 
     
     
         11 - 15 . (canceled) 
     
     
         16 . An isolated transcription unit comprising a bacteriophage promoter, a coding sequence of an RNA bacteriophage single chain coat polypeptide with a site for insertion of a heterologous peptide in said coding sequence and optionally a bacteriophage terminator. 
     
     
         17 . A method for constructing a library of virus-like particles, wherein each virus-like particle comprises a heterologous peptide in the upstream or downstream single-chain RNA bacteriophage coat polypeptide dimer comprising
 (a) providing a plurality of transcription units of  claim 10     (b) treating said transcription units of (a) with a restriction enzyme;   (c) inserting coding sequences for heterologous peptides into said transcription units to obtain a population of transcription units   (d) expressing said transcription units of (c) and   (e) isolating said library.   
     
     
         18 . The method according to  claim 17  wherein said transcription units are expressed in a coupled transcription translation system in vitro. 
     
     
         19 . The method according to  claim 17  wherein said heterologous peptide is at least four amino acid units in length. 
     
     
         20 . The method according to  claim 17 , wherein said transcription units are expressed in a coupled transcription translation system in vitro, wherein at least one cycle of coupled transcription/translation is conducted in a compartmentalized water/oil emulsion. 
     
     
         21 . A method for identifying a peptide having a property of interest comprising
 (a) providing a population of virus-like particles (VLPs), wherein each particle (a) is a VLP of an RNA bacteriophage, (b) comprises a single chain dimer coat polypeptide of said phage modified by insertion of a heterologous peptide, wherein said heterologous peptide is displayed on said VLP; (c) encapsidates said phage mRNA, and wherein said coat polypeptide comprises an upstream and downstream subunit;   (b) assaying heterologous peptides expressed on the VLPs in said population for the property of interest to identify the peptide having a property of interest.   
     
     
         22 . The method according to  claim 21 , wherein the population in (a) is obtained by expressing a transcription unit comprising a bacteriophage promoter and 3′ terminator, a coding sequence for a modified RNA bacteriophage coat polypeptide, wherein said modification is a heterologous peptide sequence at least 4 amino acids in length in a coupled transcription/translation system from a nucleic acid template in a compartmentalized water/oil emulsion and recovering said population from said water/oil emulsion. 
     
     
         23 . The method according to  claim 21 , wherein said peptide having a property of interest is an immunogenic peptide. 
     
     
         24 . The method according to  claim 21 , wherein said peptide having a property of interest is an immunogenic fragment of a self-antigen. 
     
     
         25 . The method according to  claim 22 , wherein said immunogenic fragment is a fragment of an immunogenic HIV peptide. 
     
     
         26 . The method according to  claim 21 , wherein said peptide having a property of interest is a mimitope. 
     
     
         27 . A method for isolating an immunogenic protein comprising:
 (a) identifying said immunogenic peptide from a population of VLPs according to the method of  claim 21 ;   b) amplifying said identified immunogenic peptide and   (c) isolating said immunogenic peptide.   
     
     
         28 . The method according to  claim 22 , wherein said immunogenic peptide is isolated by affinity selection. 
     
     
         29 - 33 . (canceled) 
     
     
         34 . An immunogenic composition comprising one or more VLPs of a MS-2 RNA bacteriophage and comprises a single chain dimer of the coat polypeptide of said phage, said coat polypeptide comprising an upstream and downstream subunit, wherein said upstream or downstream subunit is modified by insertion of an immunogenic heterologous peptide in either the upstream or downstream subunit of said dimer. 
     
     
         35 . The immunogenic composition of  claim 34 , wherein said heterologous peptide is a self-antigen or immunogenic fragment thereof. 
     
     
         36 . The immunogenic composition of  claim 34 , wherein said self-antigen is a peptide derived from the group consisting of ErbB-2, amyloid-beta, immunoglobulin E (IgE), gastrin, ghrelin, vascular endothelial growth factor (VEGF), interleukin (IL)-17, IL-23, IL-13, CCR5, CXCR4, nerve growth factor (NGF), angiotensin II, TRANCE/RANKL, or MUC-1. 
     
     
         37 . The immunogenic composition of  claim 34 , wherein said heterologous peptide is an HIV immunogenic peptide. 
     
     
         38 . The immunogenic composition of  claim 34 , wherein said heterologous peptide is inserted in the downstream subunit of said coat polypeptide. 
     
     
         39 . The immunogenic composition of  claim 34 , wherein said heterologous peptide is inserted into the AB-loop of said downstream subunit of said coat-polypeptide. 
     
     
         40 . The immunogenic composition of  claim 34  wherein said heterologous peptide is inserted into the N-terminus of said dimer of said coat polypeptide. 
     
     
         41 . The method according to  claim 17  wherein said virus-like particle comprises a heterologous peptide in the downstream single-chain RNA bacteriophage coat polypeptide dimer. 
     
     
         42 . The method according to  claim 28  wherein said identified peptide is amplified by reverse transcription and polymerase chain reaction of RNA isolated from the affinity selected VLPs.