Plasmids and methods for peptide display and affinity-selection on virus-like particles of rna bacteriophages

Abstract

The present invention relates to a system and method for controlling peptide display valency on virus-like particles (VLPs), especially including MS2 VLPs. In this method, large amounts of wild-type and low quantities of single-chain dimer coat proteins may be produced from a single RNA. Valency is controlled in immunogen (vaccine) production by providing a system that allows the production of large amounts of wild-type and low quantities of single-chain dimer coating proteins from a single RNA, allowing facile adjustment of display valency levels on VLPs, especially MS2 VLPS over a wide range, from few than one-on average- to as many as ninety per particle. This facilitates the production of immunogens and vaccines, including VLPs exhibiting low valency. Nucleic acid constructs useful in the expression of virus-like particles are disclosed, comprised of a coat polypeptide of MS2 modified by insertion of a heterologous peptide, wherein the heterologous peptide is displayed on the virus-like particle and encapsidates MS2 niRNA. Nucleic acid constructs are also disclosed which are useful in the expression of virus-like particles comprised of a coat polypeptide of PP7 modified by insertion of a heterologous peptide, wherein the heterologous peptide is displayed on the virus-like particle and encapsidates PP7 mRNA.

1 . A population of virus-like particles (VLPs), wherein each VLP is (a) a VLP of a RNA bacteriophage, wherein a portion of said VLP comprises (b) a single chain dimer of a coat polypeptide of said RNA bacteriophage modified by insertion of a heterologous peptide, said dimer comprising an upstream and a downstream subunit, wherein said heterologous peptide is displayed on said VLP and (c) encapsidates said bacteriophage mRNA, said VLP exhibiting low valency. 
     
     
         2 . The population according to  claim 1 , wherein said heterologous peptide is at least four amino acids in length. 
     
     
         3 . The population according to  claim 1 , wherein a plurality of said coat polypeptide of said bacteriophage is a wild-type coat polypeptide and the remaining coat polypeptide comprises a single chain dimer of said coat polypeptide comprising a heterologous peptide, said bacteriophage being selected from the group consisting of MS2 and PP7. 
     
     
         4 . The population according to  claim 1 , wherein the heterologous peptide is inserted at a site in an AB loop in a downstream subunit of said coat polypeptide or in a carboxy-terminus of said RNA bacteriophage coat polypeptide. 
     
     
         5 . The population according to  claim 4  wherein said heterologous peptide is inserted at a site corresponding to the carboxy-terminus of said RNA bacteriophage coat polypeptide. 
     
     
         6 . The population according to  claim 1 , wherein said RNA bacteriophage is selected from the group consisting of MS2 or PP7, Qβ, R17, SP, PP7, GA, M11, MX1, AP205, PRR1, f4, Cb5, Cb12r, Cb23r, 7s and f2. 
     
     
         7 . The population according to  claim 1 , wherein said coat polypeptide is a single chain dimer and a downstream copy of said coat polypeptide is modified in the AB loop by insertion of a heterologous peptide having a length of at least 4 amino acids. 
     
     
         8 - 10 . (canceled) 
     
     
         11 . The population according to  claim 4 , wherein the heterologous peptide is inserted at a carboxy terminus of said dimeric protein or at a site in an AB-loop of said coat proteins corresponding to
 (a) amino acids 11-17 of MS-2, R17 and fr coat polypeptides;   (b) amino acids 10-16 of GA coat polypeptide;   (c) amino acids 10-17 of Qβ and SP coat polypeptides;   (d) amino acids 8-11 of PP7 coat polypeptides and   (e) amino acids 9-17 of PRR1 coat polypeptides.   
     
     
         12 . An isolated transcription unit comprising:
 a bacterial or bacteriophage promoter;   a coding sequence of an RNA bacteriophage single chain coat polypeptide dimer comprising an upstream subunit and a downstream subunit, a site for insertion of a heterologous peptide located in the downstream subunit;   a stop codon replacing a codon which encodes a first amino acid of the downstream subunit of said coat protein; and,   a bacterial or bacteriophage terminator.   
     
     
         13 - 19 . (canceled) 
     
     
         20 . An isolated transcription unit comprising a bacteriophage promoter, a coding sequence of an RNA bacteriophage single chain coat polypeptide dimer having a site for insertion of a heterologous peptide in said coding sequence in a downstream half of said dimer wherein said coding sequence comprises a codon-juggled sequence in an upstream half of said dimer and optionally, a bacteriophage terminator. 
     
     
         21 . The isolated transcription unit of  claim 20 , wherein said codon-juggled coding sequence comprises a plurality of silent nucleotide substitutions inserted into an upstream half of said dimer. 
     
     
         22 . The isolated transcription unit of claim  19  or  20 , wherein said coding sequence of said single chain coat polypeptide dimer comprises a stop codon replacing a codon which encodes a first amino acid of the downstream subunit of said coat protein. 
     
     
         23 . The isolated transcription unit of  claim 21  wherein said stop codon is an amber (UAG), opal (UGA) or ochre)(UAA) stop codon. 
     
     
         24 . A method for constructing a library of virus-like particles, wherein each virus-like particle comprises a heterologous peptide in a downstream single-chain RNA of at least one single chain dimer, but not all bacteriophage coat polypeptide dimers in said virus-like particle comprising
 providing a plurality of transcription units of  claim 12 ;   (b) inserting coding sequences for heterologous peptides into said transcription units to obtain a population of transcription units;   (c) expressing said transcription units of (c); and,   (d) isolating said library.   
     
     
         25 . The method according to  claim 24 , wherein said transcription units are expressed in a coupled transcription translation system. 
     
     
         26 . (canceled) 
     
     
         27 . The method according to  claim 24 , further comprising expressing said transcription units in a coupled transcription translation system, and conducting at least one cycle of coupled transcription/translation in a compartmentalized water/oil emulsion. 
     
     
         28 - 33 . (canceled) 
     
     
         34 . A method for isolating an immunogenic protein comprising:
 (a) identifying said immunogenic peptide from a population of VLPs according to the method of  claim 24 ;   b) amplifying said identified immunogenic peptide; and,   (c) isolating said immunogenic peptide.   
     
     
         35 . The method according to  claim 34 , wherein said immunogenic peptide is isolated by affinity selection. 
     
     
         36 - 72 . (canceled) 
     
     
         73 . A method for constructing a library of virus-like particles, the method comprising:
 (a) providing a plurality of a nucleic acid constructs of claim  47 ;   (b) treating the nucleic acid constructs with a restriction enzyme;   (c) inserting coding sequences for a heterologous peptide into the nucleic acid constructs to obtain a population of transcription units; and   (d) expressing the transcription units and, optionally, isolating the library, wherein each particle comprises a coat polypeptide of MS2 modified by insertion of a heterologous peptide, and wherein the heterologous peptide is displayed on the virus-like particle and encapsidates MS2 mRNA.   
     
     
         74 . A method for identifying a peptide having a property of interest, the method comprising:
 (a) providing a population of the virus-like particles of claim  19 , wherein   (1) each particle comprises a coat polypeptide of MS2 modified by insertion of a heterologous peptide, and wherein   (2) the heterologous peptide is displayed on the virus-like particle and encapsidates MS2 mRNA;   (b) assaying heterologous peptides expressed on the virus-like particles for the property of interest.   
     
     
         75 . A method for isolating an immunogenic protein, the method comprising:
 (a) identifying the immunogenic peptide from a population of virus-like particles according to the method of  claim 73 ;   (b) amplifying the identified immunogenic peptide; and optionally   (c) isolating the immunogenic peptide.   
     
     
         76 . An immunogenic composition comprising one or more virus-like particles of claim  65 , wherein:
 (1) each particle comprises a coat polypeptide of MS2 modified by insertion of a heterologous peptide, and wherein   (2) the heterologous peptide is displayed on the virus-like particle and encapsidates MS2 mRNA.   
     
     
         77 - 91 . (canceled) 
     
     
         92 . A nucleic acid construct comprising:
 (a) a bacterial or bacteriophage promoter which is operably associated with a coding sequence of bacteriophage MS2 or PP7 single chain coat polypeptide dimer, wherein the coat polypeptide dimer coding sequence is modified to (1) define a first restriction site which is located in the downstream portion of the coat polypeptide dimer coding sequence and which is either positioned 5′ to, or located within, the sequence which defines the coat polypeptide dimer AB loop, and (2) to contain a nucleotide sequence (NNS) x , where N is any nucleotide, S is a guanosine nucleotide (G) or cytidine nucleotide (C), and x is an integer from 1 to 500; (b) a second restriction site positioned 3′ to the coat polypeptide dimer coding sequence;   (c) PCR primers positioned 5′ to the first restriction site and 3′ to the second restriction site;   (d) an antibiotic resistance gene; and   (e) a replication origin for replication in a prokaryotic cell.   
     
     
         93 - 111 . (canceled) 
     
     
         112 . A method for identifying a peptide having a property of interest, the method comprising:
 (a) providing a population of the virus-like particles of claim  99 , wherein (1) each particle comprises a coat polypeptide of PP7 modified by insertion of a heterologous peptide, and wherein (2) the heterologous peptide is displayed on the virus-like particle and encapsidates PP7 mRNA;   (b) assaying heterologous peptides expressed on the virus-like particles for the property of interest.   
     
     
         113 . A method for isolating an immunogenic protein, the method comprising:
 (a) identifying the immunogenic peptide from a population of virus-like particles according to the method of  claim 112 ;   (b) amplifying the identified immunogenic peptide; and optionally   (c) isolating the immunogenic peptide.   
     
     
         114 - 145 . (canceled) 
     
     
         146 . A method for constructing a library of virus-like particles, the method comprising:
 (a) providing a plurality of a nucleic acid constructs according to claim  116 ;   (b) treating the nucleic acid constructs with a restriction enzyme;   (c) inserting coding sequences for a heterologous peptide into the nucleic acid constructs to obtain a population of transcription units; and   (d) expressing the transcription units and, optionally, isolating the library, wherein   each particle comprises a coat polypeptide of MS2 or PP7 modified by insertion of a heterologous peptide, and wherein the heterologous peptide is displayed on the virus-like particle and encapsidates MS2 or PP7 mRNA, respectively.   
     
     
         147 . A method for identifying a peptide having a property of interest, the method comprising:
 (a) providing a population of the virus-like particles of claim  132 , wherein (1) each particle comprises a coat polypeptide of MS2 or PP7 modified by insertion of a heterologous peptide, and wherein (2) the heterologous peptide is displayed on the virus-like particle and encapsidates MS2 or PP7 mRNA, respectively; and   (b) assaying heterologous peptides expressed on the virus-like particles for the property of interest.   
     
     
         148 - 151 . (canceled) 
     
     
         152 . A method for affinity selection of vaccine candidates, the method comprising:
 (a) constructing a population of virus-like particles (VLPs) displaying a random sequence or antigen fragment library of peptides, wherein each particle comprises a single chain dimer of a coat polypeptide of MS2 or PP7 modified by insertion of a heterologous peptide from said library of peptides, and wherein (2) the heterologous peptide is displayed on the virus-like particle and encapsidates MS2 or PP7 mRNA, respectively;   (b) incubating the population of VLPs from step (a) to an immobilized monoclonal or polyclonal antibody to allow binding of said VLPs to said antibody;   (c) washing away unbound VLPs and eluting bound VLPs from said antibody;   (d) denaturing said eluted VLPs to obtain RNA contained therein and copying said RNA into DNA and amplifying said DNA by PCR;   (e) obtaining the amplified DNA and recloning said DNA into a plasmid expression vector;   (f) introducing said expression vector into an expression cell to produce a population of VLPs and isolating said VLPs from said expression cells.   
     
     
         153 . The method according to  claim 152  wherein said population of VLPs obtained from said expression cells of step (f) is further purified by conducting iterative rounds of affinity selection comprising:
 (g) conducting steps (b)-(f) on said population of VLPs obtained from said expression cells at least one to ten additional times, preferably 2-6 or 2-4 additional times. 
 
     
     
         154 . A population of virus-like particles (VLPs) displaying a random sequence or antigen fragment library of peptides, each particle comprising a single chain dimer of a coat polypeptide of MS2 or PP7 modified by insertion of a heterologous peptide from said library of peptides, wherein the heterologous peptide is displayed on the virus-like particle and encapsidates MS2 or PP7 mRNA, respectively and wherein said heterologous peptide is inserted into the AB loop or the N- or C-terminus of the single chain dimer. 
     
     
         155 . A population of virus-like particles (VLPs) displaying an antigen fragment library of peptides derived from target antigens, each particle comprising a single chain dimer of a coat polypeptide of MS2 or PP7 modified by insertion of a heterologous peptide from said library of fragment peptides, wherein the heterologous peptide is displayed on the virus-like particle and wherein said virus-like particle encapsidates MS2 or PP7 mRNA, respectively and said fragment peptide is inserted into the AB loop or the N- or C-terminus of the single chain dimer.