Optimization of immunomodulatory properties of genetic vaccines

Abstract

This invention provides methods for obtaining molecules that can modulate an immune response, and immunomodulatory molecules obtained using the methods. The molecules find use, for example, in the tailoring of an immune response induced by a genetic vaccine for a desired purpose.

1 - 46 . (canceled)  
     
     
         47 . A method of providing an immunomodulatory polynucleotide that has an optimized modulatory effect on an immune response, or encodes a polypeptide that has an optimized modulatory effect on an immune response, the method comprising creating a library of recombinant or non-stochastically generated progeny polynucleotides from a parental polynucleotide set, thereby providing an immunomodulatory polynucleotide.  
     
     
         48 . The method of  claim 47 , wherein the library of recombinant or non-stochastically generated progeny polynucleotides is optimized by directed evolution of the parental polynucleotides, such that polypeptides encoded by the optimized progeny polynucleotides are enhanced in their modulatory effect on an immune response.  
     
     
         49 . The method of  claim 48 , wherein said progeny polynucleotide whose modulatory effect on an immune response is optimized by directed evolution is introduced into a genetic vaccine vector.  
     
     
         50 . The method of  claim 48 , wherein said method of directed evolution is selected from the group consisting of codon site-saturation mutagenesis, amino acid site-saturation mutagenesis, gene site saturation mutagenesis, introduction of mutations by recombinant or non-stochastic polynucleotide reassembly methods, synthetic ligation polynucleotide reassembly, gene reassembly, oligonucleotide-directed saturation mutagenesis, in vivo reassortment of polynucleotide sequences having partial homology, naturally occurring recombination processes which reduce sequence complexity, and any combination thereof.  
     
     
         51 . The method of  claim 50 , wherein the method of directed evolution introduces at least at least one point mutation, addition, deletion, or chimerization, from one or more parental polynucleotides.  
     
     
         52 . The method of  claim 47 , further comprising screening said library for progeny polynucleotides which encode polypeptides optimized for their immunomodulatory effect as compared to the parental polynucleotides.  
     
     
         53 . The method of  claim 47 , wherein the optimized recombinant or non-stochastically generated polynucleotide encodes a polypeptide that interacts with a cellular receptor.  
     
     
         54 . The method of  claim 53 , wherein the cellular receptor is a macrophage scavenger receptor.  
     
     
         55 . The method of  claim 53 , wherein the cellular receptor is selected from the group consisting of a cytokine receptor and a chemokine receptor.  
     
     
         56 . The method of  claim 55 , wherein the chemokine receptor is CCR6.  
     
     
         57 . The method of  claim 53 , wherein the polypeptide acts as an agonist or antagonist of the receptor.  
     
     
         58 . The method of  claim 47 , wherein the library is screened by contacting replicable genetic packages, which express the encoded polypeptides of the optimized progeny polynucleotides as fusions with proteins displayed on the surface, with a plurality of cells that display the receptor.  
     
     
         59 . The method of  claim 58 , further comprising identifying cells that exhibit a modulation of an immune response by the receptor.  
     
     
         60 . The method of  claim 58 , wherein the replicable genetic package is selected from the group consisting of a bacteriophage, a cell, a spore, and a virus.  
     
     
         61 . The method of  claim 60 , wherein the replicable genetic package is an M13 bacteriophage and the protein is encoded by geneIII or geneVIII.  
     
     
         62 . The method of  claim 47 , further comprising introducing the optimized recombinant or non-stochastically generated polynucleotide into a genetic vaccine vector and administering the vector to a subject.  
     
     
         63 . The method of  claim 62 , wherein the peptide or polypeptide is an agonist or antagonist of the receptor.  
     
     
         64 . The method of  claim 47 , wherein the optimized recombinant or non-stochastically generated polynucleotide is inserted into an antigen-encoding nucleotide sequence of a genetic vaccine vector.  
     
     
         65 . The method of  claim 64 , wherein the optimized recombinant or non-stochastically generated polypeptide is introduced into a nucleotide sequence that encodes an HBsAg polypeptide.  
     
     
         66 . The method of  claim 47 , wherein the optimized recombinant or non-stochastically generated polynucleotide comprises a nucleotide sequence rich in unmethylated CpG.  
     
     
         67 . The method of  claim 47 , wherein the optimized recombinant or non-stochastically generated polynucleotide encodes a polypeptide that inhibits an allergic reaction.  
     
     
         68 . The method of  claim 67 , wherein the polypeptide is selected from the group consisting of interferon-alpha, interferon-beta, IL-10, IL-12, an antagonist of IL-4, an antagonist of IL-5, and an antagonist of IL-13.  
     
     
         69 . The method of  claim 47 , wherein the optimized recombinant polynucleotide encodes an antagonist of IL-10.  
     
     
         70 . The method of  claim 69 , wherein the antagonist of IL-10 is soluble or defective IL-10 receptor or IL-20/MDA-7.  
     
     
         71 . The method of  claim 47 , wherein the optimized recombinant or non-stochastically generated polynucleotide encodes a co-stimulator.  
     
     
         72 . The method of  claim 71 , wherein the co-stimulator is B7-1 (CD80) or B7-2 (CD86).  
     
     
         73 . The method of  claim 72 , wherein the screening step involves selecting variants with altered activity through CD28 or CTLA-4.  
     
     
         74 . The method of  claim 71 , wherein the co-stimulator is CD1, CD40, CD154 (ligand for CD40) or CD150 (SLAM).  
     
     
         75 . The method of  claim 71 , wherein the co-stimulator is a cytokine.  
     
     
         76 . The method of  claim 75 , wherein the cytokine is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, GM-CSF, G-CSF, TNF-alpha, IFN-alpha, IFN-gamma, and IL-20 (MDA-7).  
     
     
         77 . The method of  claim 76 , wherein the library of recombinant or non-stochastically generated polynucleotides is screened by testing the ability of cytokines encoded by the recombinant or non-stochastically generated polynucleotides to activate cells which contain a receptor for the cytokine.  
     
     
         78 . The method of  claim 77 , wherein the cells contain a heterologous nucleic acid that encodes the receptor for the cytokine.  
     
     
         79 . The method of  claim 76 , wherein the cytokine is interleukin-12 and screening is performed by growing mammalian cells which contain the genetic vaccine vector in a culture medium and detecting whether T cell proliferation or T cell differentiation is induced by contact with the culture medium.  
     
     
         80 . The method of  claim 76 , wherein the cytokine is interferon-gamma.  
     
     
         81 . The method of  claim 80 , wherein the library is screened by contacting replicable genetic packages, which express the encoded polypeptides of the optimized progeny polynucleotides as fusions with proteins displayed on the surface, with a plurality of B cells that display the receptor.  
     
     
         82 . The method of  claim 81 , further comprising identifying phage library members that are capable of inhibiting proliferation of the B cells.  
     
     
         83 . The method of  claim 76 , wherein the immune response of interest is differentiation of T cells to T H 1 cells.  
     
     
         84 . The method of  claim 83 , wherein said immune response of interest is screened by contacting a population of T cells with the cytokines encoded by the members of the library of recombinant polynucleotides and identifying library members that encode a cytokine that induces the T cells to produce IL-2 and interferon-gamma.  
     
     
         85 . The method of  claim 75 , wherein the cytokine encoded by the optimized recombinant or non-stochastically generated polynucleotide exhibits reduced immunogenicity compared to a cytokine encoded by a non-optimized polynucleotide.  
     
     
         86 . The method of  claim 85 , wherein the reduced immunogenicity is detected by introducing a cytokine encoded by the recombinant or non-stochastically generated polynucleotide into a subject and determining whether an immune response is induced against the cytokine.  
     
     
         87 . The method of  claim 77 , wherein the cell is tested for ability to costimulate an immune response.  
     
     
         88 . The method of  claim 47 , wherein the optimized recombinant polynucleotide encodes a cytokine antagonist.  
     
     
         89 . The method of  claim 88 , wherein the cytokine antagonist is selected from the group consisting of a soluble cytokine receptor, a transmembrane cytokine receptor having a defective signal sequence, IL-10R and IL-4R.  
     
     
         90 . The method of  claim 47 , wherein the optimized recombinant or non-stochastically generated polynucleotide encodes a polypeptide capable of inducing a predominantly T H 1 immune response.  
     
     
         91 . The method of  claim 47 , wherein the optimized recombinant or non-stochastically generated polynucleotide encodes a polypeptide capable of inducing a predominantly T H 2 immune response.  
     
     
         92 . The method of  claim 47 , wherein said optimized modulatory effect on an immune response is a decrease in an unwanted modulatory effect on an immune response.  
     
     
         93 . The method of  claim 92 , wherein said method generates a molecule having a decreased ability to elicit an immune response from a host recipient of said molecule.  
     
     
         94 . The method of  claim 93 , wherein said recipient can be a human or an animal host.  
     
     
         95 . The method of  claim 94 , wherein said method generates a molecule having decreased antigenicity with respect to at least one host recipient of said molecule.  
     
     
         96 . The method of  claim 95 , wherein said recipient can be a human or an animal host.  
     
     
         97 . The method of  claim 47 , wherein said optimized modulatory effect on an immune response is both a decrease in a first unwanted modulatory effect on an immune response and an increase in a second desirable modulatory effect on an immune response.  
     
     
         98 . The method of  claim 97 , wherein the first and the second recipient hosts can be the same or different.  
     
     
         99 . The method of  claim 97 , wherein each of the first and the second recipient hosts can be human or animal.  
     
     
         100 . The method of  claim 97 , wherein said method generates a molecule having both a decreased ability to elicit a first immune response from a first host recipient of said molecule and an increased ability to elicit a second immune response from a second host recipient of said molecule.  
     
     
         101 . The method of  claim 100 , wherein the first and the second recipient hosts can be the same or different.  
     
     
         102 . The method of  claim 100 , wherein each of the first and the second recipient hosts can be a human or animal.  
     
     
         103 . The method of  claim 97 , wherein said method generates a molecule having both a first decreased antigenicity with respect to at least one host recipient of said molecule and a second decreased antigenicity with respect to at least one host recipient of said molecule.  
     
     
         104 . The method of  claim 92 , wherein said first and said second modulatory effect on an immune response are evolved for respectively a first and a second module on the same multimodule vaccine vector.  
     
     
         105 . The method of  claim 104 , wherein said module is selected from the group of modules consisting of an antigen coding sequence, a polyadenylation sequence, a sequence coding for a co-stimulatory molecule, a sequence coding for an inducible repressor or transactivator, a eukaryotic origin of replication, a prokaryotic origin of replication, a sequence coding for a prokaryotic marker, an enhancer, a promoter, an operator, an intron, or derivative fragments or analogs thereof, and any combination thereof.  
     
     
         106 . The method of  claim 47 , wherein the optimized modulatory effect on an immune response is comprised of an increase in the stability of the immunomodulatory (IM) polynucleotide or polypeptide encoded thereby.  
     
     
         107 . The method of  claim 106 , wherein said method generates a molecule having an increased stability ex vivo.  
     
     
         108 . The method of  claim 106 , wherein said method generates a molecule having increased stability in vivo, with respect to any means of biological elimination or degradation, upon administration to a host recipient.  
     
     
         109 . The method of  claim 47 , wherein the immunomodulatory (IM) polynucleotide or polypeptide encoded thereby has an optimized modulatory effect on an immune response in an animal or human host recipient.  
     
     
         110 . The method of  claim 109 , wherein said method generates an optimized genetic vaccine for any human and/or non-human recipients.  
     
     
         111 . A method of providing an optimized recombinant or non-stochastically generated polynucleotide that has a modulatory effect on an immune response said method comprising recombinant or non-stochastically reassembling at least two parental template polynucleotides, each of which encodes a molecule that is involved in modulating an immune response, thereby providing a library of recombinant or non-stochastically generated polynucleotides.  
     
     
         112 . The method of  claim 111 , wherein the first and second parental templates differ from each other in two or more nucleotides.  
     
     
         113 . The method of  claim 111 , further comprising screening the library to identify at least one optimized recombinant or non-stochastically generated polynucleotide that exhibits through the encoded molecule an enhanced ability to modulate an immune response in comparison to a parental polynucleotide from which the library was created.  
     
     
         114 . The method of  claim 111 , wherein an optimized recombinant or non-stochastically generated polynucleotide is subjected to at least one further round of recombinant or non-stochastic reassembly with at least one additional polynucleotide to produce additional working libraries of recombinant polynucleotides.  
     
     
         115 . The method of  claim 114 , wherein said additional working libraries are screened to identify at least one further optimized recombinant or non-stochastically generated polynucleotide which encodes a polypeptide that has been optimized for its immunomodulatory effect when compared to the parental polynucleotide from which the library was created.  
     
     
         116 . A method of providing an optimized polynucleotide that encodes an accessory molecule that improves the transport or presentation of antigens by a cell, said method comprising creating a library of recombinant or non-stochastically generated polynucleotides by subjecting to optimization by recombinant or non-stochastic directed evolution a parental polynucleotide set in which is encoded all or part of the accessory molecule.  
     
     
         117 . The method of  claim 116 , further comprising screening the library to identify an optimized recombinant or non-stochastically generated progeny polynucleotide that encodes a recombinant molecule that confers upon a cell an increased or decreased ability to transport or present an antigen on a surface of the cell as compared to an accessory molecule encoded by template polynucleotides not subjected to the recombinant or non-stochastic reassembly.  
     
     
         118 . The method of  claim 116 , wherein said method of directed evolution is selected from the group consisting of codon site-saturation mutagenesis, amino acid site-saturation mutagenesis, gene site saturation mutagenesis, introduction of mutations by recombinant or non-stochastic polynucleotide reassembly methods, synthetic ligation polynucleotide reassembly, gene reassembly, oligonucleotide-directed saturation mutagenesis, in vivo reassortment of polynucleotide sequences having partial homology, naturally occurring recombination processes which reduce sequence complexity, and any combination thereof.  
     
     
         119 . The method of  claim 116 , wherein said method generates an optimized molecule for any human and/or non-human recipients.  
     
     
         120 . The method of  claim 116 , further comprising forming a library of vectors by introducing the library of recombinant or non-stochastically generated polynucleotides into a genetic vaccine vector that encodes an antigen.  
     
     
         121 . The method of  claim 120 , wherein the library of vectors is introduced into mammalian cells.  
     
     
         122 . The method of  claim 121 , wherein said cells that exhibit increased or decreased immunogenicity to the antigen are identified.  
     
     
         123 . The method of  claim 116 , wherein the accessory molecule comprises a proteasome or a TAP polypeptide.  
     
     
         124 . The method of  claim 116 , wherein the accessory molecule comprises a cytotoxic T-cell inducing sequence.  
     
     
         125 . The method of  claim 124 , wherein the cytotoxic T-cell inducing sequence is obtained from a hepatitis B surface antigen.  
     
     
         126 . The method of  claim 116 , wherein the accessory molecule comprises an immunogenic agonist sequence.