Methods for producing polynucleotide libraries in vaccinia virus/eukaryotic cells

Abstract

This disclosure provides an improved method of constructing a library of polynucleotides of interest in a poxvirus, vaccinia virus vector system, where the polynucleotides of interest encode polypeptides of interest. The method comprises constructing the library in the presence of an inhibitor of poxvirus assembly, e.g., rifampicin, which allows construction of libraries with higher complexity and diversity than previous methods.

What is claimed is: 
     
         1 . A library comprising a plurality of polynucleotides of interest made by a process comprising the steps of:
 (a) cleaving an isolated poxvirus genome to produce a first viral fragment and a second viral fragment, wherein the first fragment is nonhomologous with the second fragment;   (b) providing a population of transfer plasmids each comprising a polynucleotide of interest flanked by a 5′ flanking region and a 3′ flanking region, wherein the 5′ flanking region comprises a region homologous to the 3′ end of the first viral fragment and the 3′ flanking region comprises a region homologous to the 5′ end of the second viral fragment; and wherein the transfer plasmids are capable of homologous recombination with the first and second viral fragments such that a viable poxvirus genome is formed;   (c) introducing the transfer plasmids and the first and second viral fragments into a mammalian host cell permissive for poxvirus infectivity;   (d) adding an inhibitor of poxvirus assembly; and   (e) allowing the transfer plasmid and the first and second viral fragments to undergo homologous recombination, thereby producing a library of viable modified poxvirus genomes each comprising a heterologous nucleic acid.   
     
     
         2 . The library of  claim 1 , further comprising a step (f) recovering the library. 
     
     
         3 . The library of  claim 1 , wherein step (c) comprises transfecting the mammalian host cell with the transfer plasmids and the first and second viral fragments, and wherein the host cell is maintained in cell culture medium following transfection. 
     
     
         4 . The library of  claim 3 , wherein the inhibitor of poxvirus assembly is rifampicin (rifampin) or a derivative thereof. 
     
     
         5 . The library of  claim 4 , wherein the rifampicin or derivative thereof is added to the cell culture medium comprising the transfected cells at about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, or about 60 hours following transfection. 
     
     
         6 . The library of  claim 4 , wherein the rifampicin or derivative thereof is added to the cell culture medium at a concentration of about 30 μg/ml, about 40 μg/ml, about 50 μg/ml, about 60 μg/ml, about 70 μ/ml, about 80 μg/ml, about 90 μg/ml, about 100 μg/ml, about 110 μg/ml, about 120 μg/ml, or about 130 μg/ml. 
     
     
         7 . The library of  claim 4 , wherein the rifampicin or derivative thereof is allowed to remain cell culture medium for about one day, about two days, about three days, about four days or about five days. 
     
     
         8 . The method of  claim 7 , wherein the cell culture medium is changed following treatment with rifampicin or a derivative thereof, and the transfected host cells further cultured without rifampicin for about one day, about two days, or about three days. 
     
     
         9 . The library of  claim 1 , wherein the library comprises an increased number of independent modified poxvirus genomes than a library constructed in the absence of the inhibitor of poxvirus assembly. 
     
     
         10 . The library of  claim 9 , wherein the number of independent modified poxvirus genomes is increased by at least about one-fold, about five-fold, about ten-fold, about fifteen-fold, about twenty-fold, about twenty-five-fold, or about thirty-fold as compared to a library constructed in the absence of the inhibitor of poxvirus assembly. 
     
     
         11 . The library of  claim 1 , wherein the library comprises an increased virus titer than a library constructed in the absence of the inhibitor of poxvirus assembly. 
     
     
         12 . The library of  claim 1 , wherein the isolated poxvirus genome comprises a first recognition site for a first restriction endonuclease and a second recognition site for a second restriction endonuclease; and wherein the first and second viral fragments are produced by digesting the viral genome with the first restriction endonuclease and the second restriction endonuclease, and isolating the first and second viral fragments. 
     
     
         13 . The library of  claim 1 , wherein the isolated poxvirus genome is an isolated vaccinia virus genome. 
     
     
         14 . The library of  claim 13 , wherein said isolated vaccinia virus genome is a WR genome, a Modified Vaccinia virus Ankara (MVA) genome, or a modified derivative thereof. 
     
     
         15 . The library of  claim 12 , wherein the first and second restriction enzyme recognition sites are situated in a vaccinia virus HindIII J fragment. 
     
     
         16 . The library of  claim 13 , wherein the first restriction enzyme is NotI. 
     
     
         17 . The library of  claim 13 , wherein the second restriction enzyme site is ApaI. 
     
     
         18 . The library of  claim 13 , wherein the isolated vaccinia virus genome is a v7.5/tk virus genome or a vEL/tk virus genome. 
     
     
         19 . The library of  claim 13 , wherein the host cell is capable of packaging the modified vaccinia virus genomes into infectious vaccinia virus particles. 
     
     
         20 . The library of  claim 19 , wherein the transfer plasmids and the first and second viral fragments are introduced into a mammalian host cell comprising a helper virus, wherein the host cell is non-permissive for the production of infectious virus particles of the helper virus, but supports packaging the modified vaccinia virus genomes into infectious vaccinia virus particles. 
     
     
         21 . The library of  claim 20 , wherein the helper virus is a fowlpox virus. 
     
     
         22 . The library of  claim 13 , wherein the 5′ and 3′ flanking regions of the transfer plasmid are capable of homologous recombination with a vaccinia virus thymidine kinase gene. 
     
     
         23 . The library of  claim 22 , wherein the 5′ and 3′ flanking regions of the transfer plasmid are capable of homologous recombination with a vaccinia virus HindIII J fragment. 
     
     
         24 . The library of  claim 23 , wherein the transfer plasmid comprises an insert nucleic acid ligated into a plasmid selected from the group consisting of: (a) pVHE, (b) pVLE, (c) pVKE. 
     
     
         25 . The library of  claim 13 , wherein the plurality of polynucleotides of interest each comprise a coding region of a polypeptide of interest capable of expression in a vaccinia virus-infected cell. 
     
     
         26 . The library of  claim 23 , wherein the transfer plasmid further comprises a transcriptional control region in operable association with the polynucleotide of interest, and wherein the transcriptional control region functions in the cytoplasm of a vaccinia virus-infected cell. 
     
     
         27 . The library of  claim 26 , wherein the transcriptional control region comprises a poxvirus promoter. 
     
     
         28 . The library of  claim 27 , wherein the promoter is a vaccinia p7.5 promoter, a vaccinia pEL promoter, or a vaccinia MH-5 promoter. 
     
     
         29 . The library of  claim 25 , wherein the plurality of polynucleotides of interest each encode an antibody subunit polypeptide comprising an antibody heavy chain variable region or antigen-binding fragment thereof, an antibody light chain variable region or antigen-binding fragment thereof, or a combination thereof. 
     
     
         30 . The library of  claim 29 , wherein the antibody subunit polypeptide further comprises a constant region or fragment thereof, a signal peptide capable of directing cell surface expression or secretion of the antibody subunit polypeptide, or a combination thereof.