Covalently fused viral coat proteins for the display of target molecules

Abstract

A fusion protein comprising a target protein, a first recombinant viral coat protein, a second recombinant viral coat protein and a first linkage peptide is provided. The target protein is at N-terminus of the first recombinant viral coat protein. The first recombinant viral coat protein is linked to N-terminus of the first linkage peptide. The second recombinant viral coat protein is linked to C-terminus of the first linkage peptide. The first and second recombinant viral coat proteins are derived from the coat protein (CP) of alfalfa mosaic virus (AIMV). The fusion protein may further comprise a second linkage peptide between the target protein and the first recombinant viral coat protein. The fusion protein may form a virus like particle (VLP). The target protein may be displayed on the surface of the VLP. Also provided are methods for producing the fusion protein and the VLP as well as the uses of the fusion protein and/or the VLP.

Claims

1. A fusion protein comprising a target protein, a first recombinant viral coat protein, a second recombinant viral coat protein and a first linkage peptide, wherein the target protein is at N-terminus of the first recombinant viral coat protein, wherein the first recombinant viral coat protein is linked to N-terminus of the first linkage peptide, wherein the second recombinant viral coat protein is linked to C-terminus of the first linkage peptide, and (a) wherein the first recombinant viral coat protein comprises an amino acid sequence identical to SEQ ID NO: 1 or at least 80% identical to SEQ ID NO: 1 with one or more deletions, insertions or substitutions within residues 17-38 of SEQ ID NO: 1; (b) wherein the second recombinant viral coat protein comprises an amino acid sequence identical to SEQ ID NO: 1 or at least 80% identical to SEQ ID NO: 1 with one or more deletions, insertions or substitutions within residues 17-38 of SEQ ID NO: 1; or (c) wherein the first recombinant viral coat protein comprises an amino acid sequence identical to SEQ ID NO: 1 or at least 80% identical to SEQ ID NO: 1 with one or more deletions, insertions or substitutions within residues 17-38 of SEQ ID NO: 1, and the second recombinant viral coat protein comprises an amino acid sequence identical to SEQ ID NO: 1 or at least 80% identical to SEQ ID NO: 1 with one or more deletions, insertions or substitutions within residues 17-38 of SEQ ID NO: 1.

2. The fusion protein of claim 1, further comprising a second linkage peptide between the target protein and the first recombinant viral coat protein.

3. The fusion protein of claim 1, wherein the first linkage peptide consists of SEQ NO: 2.

4. The fusion protein of claim 2, wherein the first linkage peptide consists of SEQ NO: 2.

5. The fusion protein of claim 2, wherein the second linkage peptide consists of SEQ NO: 2.

6. The fusion protein of claim 1, wherein the first recombinant viral coat protein comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1 with a substitution at residues 17-38 of SEQ ID NO: 1, and the second recombinant viral coat protein comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1 with a deletion at residues 17-38 of SEQ ID NO: 1.

7. The fusion protein of claim 1, wherein the target protein is an agent selected from the group consisting of an immunogenic agent, a therapeutic agent, a diagnostic agent and an enzyme.

8. The fusion protein of claim 1, wherein the target protein is an immunogenic agent.

9. The fusion protein of claim 1, wherein the target protein is a therapeutic agent.

10. The fusion protein of claim 1, wherein the target protein is a diagnostic agent.

11. The fusion protein of claim 1, wherein the target protein is an enzyme.

12. A method for producing the fusion protein of claim 1, comprising (a) introducing a nucleic acid molecule into cells, wherein the nucleic acid molecule encodes the fusion protein, and (b) expressing the fusion protein in the cells, whereby the fusion protein is produced.

13. A composition comprising the fusion protein of claim 1.

14. A virus like particle formed by the fusion protein of claim 1, wherein the target protein is displayed on the surface the virus like particle.

15. A composition comprising the virus like particles of claim 14.

16. A method of producing virus like particles, comprising (a) introducing a nucleic acid molecule into a cell, an organism or a portion of the organism, wherein the nucleic acid molecule encodes the fusion protein of claim 1, (b) expressing the fusion protein in the cell, the organism or the portion of the organism, and (c) forming virus like particles by the fusion protein, whereby the virus like particles are produced, wherein the target protein is displayed on the surface of the virus like particles.

17. A method of inducing an immunological response in a subject, comprising administering to the subject an effective amount of the fusion protein of claim 8 or virus like particles formed by the fusion protein of claim 8.

18. A method of treating a disease or condition in a subject, comprising administering to the subject an effective amount of the fusion protein of claim 9 or virus like particles formed by the fusion protein of claim 9.

19. A method of detecting a biomarker in a sample from a subject, comprising contacting the sample with an effective amount of the fusion protein of claim 10 or virus like particles formed by the fusion protein of claim 10, wherein the diagnostic agent binds the biomarker.

20. A method of catalyzing a reaction by a reagent, comprising contacting the reagent with an effective amount of the fusion protein of claim 11 or virus like particles formed by the fusion protein of claim 11, wherein the enzyme catalyzes the reaction.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the amino acid sequences for the coat protein (CP) of alfalfa mosaic virus (AIMV) (SEQ ID NO: 1), a linkage peptide (SEQ ID NO: 2), a first recombinant viral coat protein (SEQ ID NO: 3), a second recombinant viral protein (SEQ ID NO: 4) and a fusion protein (SEQ ID NO: 5).

(2) FIG. 2 shows a schematic of the Pfs230 protein, highlighting the region of the protein displayed on a VLP (Pfs230-CP.sup.2).

(3) FIG. 3 shows electrophoretic characterization of Pfs230-CP.sup.2. The protein is analyzed by gel code Blue stained SDS-PAGE, and recognized by anti-His tag mAb, anti-230 2G5 mAb, and anti-230 2610 mAb.

(4) FIG. 4 shows characterization of the Pfs230-CP.sup.2 fusion protein as a virus-like particle. (A) Analytical size exclusion chromatography analysis. (B) Negatively stained transmission electron microscopy image of purified Pfs230-CP.sup.2 VLPs. (C) Cartoon image of the Pfs230-CP.sup.2 VLPs showing the levels of antigen display.

(5) FIG. 5 shows characterization of the Pfs25 protein as a CP.sup.2 fusion protein. (A) Gel code Blue stained SDS-PAGE of Pfs25-CP.sup.2 protein CMB-01053, and (B) Negatively stained transmission electron microscopy image of CMB-01053 displaying the Pfs25 protein on VLPs.

(6) FIG. 6 shows gel code Blue stained SDS-PAGE of CP.sup.2 displayed antigens, (A) H1 influenza CP.sup.2 fusion protein CMB-02039, (B) H3 influenza CP.sup.2 fusion protein CMB-02079, (C) H5 influenza CP.sup.2 fusion protein CMB-02011, (D) H7 influenza CP.sup.2 fusion protein CMB-02059 or (E) H1 influenza protein domain CP.sup.2 fusion protein CMB-02047 after primary column purification. (F) Negatively stained transmission electron microscopy images of (left to right) CMB-02039, CMB-02079, CMB-02011, CMB-02059, CMB-02011, and CMB-02059 displaying the influenza antigens on VLPs.

(7) FIG. 7 shows characterization of the Western Equine encephalitis virus domain as a CP.sup.2 fusion protein. A) Gel code Blue stained SDS-PAGE of Western Equine encephalitis virus domain CP.sup.2 fusion protein CMB-02412, and recognition by anti-His antibody, and anti-Western Equine encephalitis (WEE) virus antibody. (B) Negatively stained transmission electron microscopy image of CMB-02412 displaying the Western Equine encephalitis virus domain as a VLP.

(8) FIG. 8 shows characterization of the Eastern Equine encephalitis virus domain as a CP.sup.2 fusion protein. (A) Gel code Blue stained SDS-PAGE of Eastern Equine encephalitis virus domain CP.sup.2 fusion protein CMB-02383, and recognition by anti-His antibody, and anti-Eastern Equine encephalitis (EEE) virus antibody. (B) Negatively stained transmission electron microscopy image of CMB-02383 displaying the Eastern Equine encephalitis virus domain as VLPs.

(9) FIG. 9 shows characterization of the Eastern Equine encephalitis virus protein as a CP.sup.2 fusion protein. (A) Gel code Blue stained SDS-PAGE of Eastern Equine encephalitis virus CP.sup.2 fusion protein CMB-02380, and recognition by anti-His antibody, and anti-Eastern Equine encephalitis virus antibody. (B) Negatively stained transmission electron microscopy image of CMB-02380 displaying the Eastern Equine encephalitis virus protein as VLPs.

(10) FIG. 10 shows a first generation VLP design. (A) Schematic showing the design of the first generation A85 molecule. (B) SDS-PAGE gel of the purified A85 VLP. (C) Cartoon image of the A85 VLP showing the levels of antigen display.

(11) FIG. 11 shows second generation VLP designs. (A) Schematic showing the designs of the second-generation molecules. (B) Table summarizing the results of the second-generation molecules.

(12) FIG. 12 shows third generation VLP designs. (A) Schematic showing the designs of the third-generation molecules. (B) Table summarizing the results of the third-generation molecules. (ND=not determined).

(13) FIG. 13 shows comparison of the A85 VLP to the CMB-01053 VLP. (A) SDS-PAGE gel of the purified A85 and CMB-01053 VLPs. (B) Negatively stained transmission electron microscopy image of purified A85 and CMB-01053 VLPs. (C) Cartoon image of the A85 and CMB-1053 VLPs showing the levels of antigen display.

(14) FIG. 14 shows immunological comparison of VLPs formed by Pfs25-CP fusion proteins B29, B30 and CMB-01053. (A) Animal study design comparing Pfs25-CP fusion proteins. (B) Anti-Pfs25 IgG responses at day 56. (C) Anti-Pfs25 IgG responses at day 168. (D) Standard membrane feeding assay (SMFA) of samples obtained on days 56 (D.56) and 168 (D.168).

(15) FIG. 15 shows gel code blue stained PAGE and negatively stained transmission electron microscopy image of CP-CP VLPs displaying a variety of antigens: Pfs230 subdomain (top left panel), Yellow fever subdomain (top right panel), influenza subdomain (bottom left panel) and Eastern equine encephalitis virus domain (bottom right panel).

(16) FIG. 16 shows degradation sites in the N-terminus of AIMV CP. (A) In silico identification of trypsin and chymotrypsin sensitive sites of amino acids 2 to 50 of wild-type AIMV CP (SEQ ID NO: 1). (B) Amino acids 2 to 50 of wild-type AIMV CP (SEQ ID NO: 1) with identified sites of in planta digestion indicated by arrows. (C) In silico identification of trypsin and chymotrypsin sensitive sites in the corresponding region from the modified AIMV CP (corresponding to residues 1-42 of SEQ ID NO: 3), showing removal of most of the protease sensitive sites.

(17) FIG. 17 shows expression of fusion protein of SEQ ID NO: 5 in extract of Sf21 insect cells on Western blots, where M—Molecular weight marker; 1—AIMV CP standard; 2—non-transfected insect SF21 cells; 3—transfected insect SF21 cells extract, expressing CP-CP fusion; 4—HAI standard, containing 6×His tag.

(18) FIG. 18 shows VLPs collected by high-speed centrifugation analyzed by negatively stained transmission electron microscopy (TEM), confirming that fusion protein of SEQ ID NO: 5 expressed in insect cells can form VLPs.

(19) FIG. 19 shows expression of fusion protein of SEQ ID NO: 5 in extract of yeast (Pichia pastoris) cells analyzed by Western blots, where M—Molecular weight marker; 1—AIMV CP standard; 2—extract from non-induced yeast cells, containing CP-CP fusion; 3—extract from induced yeast cells, expressing CP-CP fusion; 4—HAI protein standard containing 6×His tag.

(20) FIG. 20 shows expression of fusion protein in extract of mammalian cells analyzed Western blots, where M—Molecular weight marker; 1, 2—AIMV CP standard; 3—extract from non-transfected mammalian cells; 4—extract from transfected mammalian cells, expressing CP-CP fusion; 5, 6—HAI protein standard, containing 6×His tag. Arrow indicates band corresponding to CP-CP fusion.

(21) FIG. 21 shows in vitro VLP formation of a CP.sup.2 fusion. A CP.sup.2 fusion protein resolved on analytical size exclusion chromatography, pre and post in vitro particle formation, where virus-like particles (VLPs) resolve at ˜9 mL.

(22) FIG. 22 shows negative stained transmission electron microscopy images of VLPs formed from CP-CP produced in (A) plant cells, (B) yeast cells, and (C) insect cells.

(23) FIG. 23 shows analysis of the HRP-CP.sup.2 fusion protein. The HRP-CP.sup.2 fusion protein was isolated by IMAC chromatography and characterized by (A) Coomassie stained SDS-PAGE, (B) anti-His Western blot, (C) chemiluminescence assay to detect HRP activity, and D) transmission electron microscopy images.

DETAILED DESCRIPTION OF THE INVENTION

(24) The present invention is based on the discovery that novel fusion proteins having recombinant coat proteins (CPs) derived from alfalfa mosaic virus (AIMV) proteins expressed in tandem, linked by a flexible linker to allow the AIMV molecules to align side by side, can be expressed in various expression systems and used to form virus like particles (VLPs), either in vivo or in vitro. This technology provides an effective and productive tool for making VLPs to present a wide variety of target proteins.

(25) The present invention provides a fusion protein. The fusion protein comprises two or more recombinant coat proteins (CPs) derived from alfalfa mosaic virus (AIMV), which are referred to as recombinant AIMV CPs. The recombinant AIMV CPs may be the same or different. Two adjacent recombinant AIMV CPs may be linked via a linkage peptide. The recombinant AIMV CPs are aligned in a manner conducive to VLP formation by the fusion protein. The fusion protein may further comprise a target protein at the N-terminus of the recombinant AIMV CPs. In some embodiments, the fusion protein comprises two recombinant AIMV CPs, also referred to as CP-CP or CP.sup.2.

(26) The term “derived from” as used herein refers to an origin or source. The recombinant coat proteins (CPs) are derived from the coat protein (CP) of alfalfa mosaic virus (AIMV) (FIG. 1). The amino acid sequence of the AIMV CP is shown in FIG. 1. The recombinant AIMV CP may comprise the AIMV CP, in part or in whole, and may be a fragment or variant of the AIMV CP.

(27) The term “mutation” as used herein refers to a deletion, insertion or substitution of one or more amino acids. An amino acid substitution may be a conservative amino acid substitution. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

(28) For example, one or more trypsin sites at residues 6, 7, 11, 17, 18, 27 and/or 37 of the AIMV CP (SEQ ID NO: 1) may be mutated by, for example, a deletion, insertion or substitution. A chymotrypsin site at residue 22 of the AIMV CP (SEQ ID NO: 1) may be mutated by, for example, a deletion, insertion or substitution. One or more in planta digestion sites between residues 24 and 25, between residues 25 and 26, and/or between residues 37 and 38 of the AIMV CP (SEQ ID NO: 1) may be mutated by, for example, a deletion, insertion or substitution. Residues 17-38 of the AIMV CP (SEQ ID NO: 1) may be mutated by, for example, a deletion, insertion or substitution by a linkage peptide consisting of SEQ ID NO: 2.

(29) The term “subject” as used herein refers to a mammal, for example, a mouse or human. Preferably, the subject is a human. The subject may be a patient suffering from a disease or condition. The subject may be in need of induction of an immunological response, treatment of a disease or condition, or detection of a biomarker.

(30) The term “an effective amount” as used herein refers to an amount of a fusion protein, virus like particles (VLPs) formed by the fusion protein, or a composition comprising the fusion protein or the VLPs required to achieve a stated goal (e.g., induction of an immunological response, treatment of a disease or condition, detection of a biomarker or catalyzing a reaction). The effective amount of the fusion protein, the VLPs, or the composition may vary depending upon the stated goals, the physical characteristics of the subject, the nature and severity of the disease or disorder, the existence of related or unrelated medical conditions, the nature of the fusion protein, the VLPs or the composition, the means of administering the fusion protein, the VLPs or the composition to the subject, and the administration route. A specific dose for a given subject may generally be set by the judgment of a physician. The pharmaceutical composition may be administered to the subject in one or multiple doses.

(31) A recombinant AIMV CP is a polypeptide comprising an amino acid sequence derived from that of the AIMV CP (SEQ ID NO: 1), for example, at least about 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1. The recombinant AIMV CP may consist of an amino acid sequence identical to SEQ ID NO: 1 except one or more mutations. For example, the recombinant AIMV CP may consist of the amino acid sequence of SEQ ID NO: 3 or 4 (FIG. 1).

(32) A linkage peptide may be of any length permitting one or more desirable properties of the fusion protein. For example, the linkage peptide may be of a length permitting formation of a virus like particle (VLP) by the fusion protein or displaying a target protein on the surface of the VLP. The linkage peptide may have at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids, or about 1-50, 5-30 or 10-20 amino acids. The linkage peptide may consist of GGGGSGGGGSGGGGS (SEQ ID NO: 2).

(33) The target protein may be an agent selected from the group consisting of an immunogenic agent, a therapeutic agent, a diagnostic agent, and an enzyme. The target protein may be an immunogenic agent. The immunogenic agent may be Malaria antigens (e.g., Pfs230 and Pfs25), influenza antigens (e.g., ectodomain and sub-domain of haemagglutinin), yellow fever subdomain, Western Equine encephalitis virus (WEE) antigen, and Eastern Equine encephalitis virus (EEE) antigen. The target protein may be a therapeutic agent. The target protein may be a diagnostic agent. The target protein may be an enzyme.

(34) In one embodiment, the fusion protein comprises a first recombinant viral coat protein, a second recombinant viral coat protein and a first linkage peptide. The first recombinant viral coat protein is linked to N-terminus of the first linkage peptide. The second recombinant viral coat protein is linked to C-terminus of the first linkage peptide. The first recombinant viral coat protein comprises an amino acid sequence at least about 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1. The second recombinant viral coat protein comprises an amino acid sequence at least about 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1.

(35) In another embodiment, the fusion protein comprises a target protein, a first recombinant viral coat protein, a second recombinant viral coat protein and a first linkage peptide. The target protein is at N-terminus of the first recombinant viral coat protein. The first recombinant viral coat protein is linked to N-terminus of the first linkage peptide. The second recombinant viral coat protein is linked to C-terminus of the first linkage peptide. The first recombinant viral coat protein comprises an amino acid sequence at least about 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1. The second recombinant viral coat protein comprises an amino acid sequence at least about 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1.

(36) The first and second recombinant viral coat proteins may be aligned in a manner conducive to VLP formation by the fusion protein. The first linkage peptide may be of any length permitting formation of a virus like particle (VLP) by the fusion protein. For example, the first linkage peptide may have at least about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids, or about 1-50, 5-30 or 10-20 amino acids. The first linkage peptide may consist of SEQ ID NO: 2.

(37) Where the fusion protein comprises the target protein, the fusion protein may further comprise a second linkage peptide, wherein the target protein is linked to N-terminus of the second linkage peptide, and the first recombinant viral coat protein is linked to C-terminus of the second linkage peptide. The second linkage peptide may be of any length permitting display of the target protein on the surface of a VLP formed by the fusion protein. For example, the second linkage peptide may have at least about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids, or about 1-50, 5-30 or 10-20 amino acids. The second linkage peptide may consist of SEQ ID NO: 2.

(38) The first recombinant viral coat protein may comprise an amino acid sequence at least about 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1 with a mutation of one or more trypsin sites at, for example, residues 6, 7, 11, 17, 18, 27 and/or 37. The first recombinant viral coat protein may comprise an amino acid sequence at least about 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1 with a mutation of a chymotrypsin site at, for example, residue 22. The first recombinant viral coat protein may comprise an amino acid sequence at least about 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1 with a mutation of one or more in planta digestion sites, for example, between residues 24 and 25, between residues 25 and 26, and/or between residues 37 and 38.

(39) The first recombinant viral coat protein may comprise an amino acid sequence at least about 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1 with a deletion, insertion or substitution at residues 17-38. For example, residues 17-38 may be substituted with a third linkage peptide. The third linkage peptide may consist of SEQ ID NO: 2. The first recombinant viral coat protein may consist of SEQ ID NO: 3. The first recombinant viral coat protein may further comprise a mutation of one or more trypsin sites at, for example, residues 6, 7 and/or 11.

(40) The second recombinant viral coat protein may comprise an amino acid sequence at least about 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1 with a mutation of one or more trypsin sites at, for example, residues 6, 7, 11, 17, 18, 27 and/or 37. The second recombinant viral coat protein may comprise an amino acid sequence at least about 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1 with a mutation of a chymotrypsin site at, for example, residue 22. The second recombinant viral coat protein may comprise an amino acid sequence at least about 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1 with a mutation of one or more in planta digestion sites, for example, between residues 24 and 25, between residues 25 and 26, and/or between residues 37 and 38.

(41) The second recombinant viral coat protein may comprise an amino acid sequence at least about 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1 with a deletion, insertion or substitution at residues 17-38. The second recombinant viral coat protein may consist of SEQ ID NO: 4. Residues 17-38 of SEQ ID NO: 1 may be substituted with a fourth linkage peptide. The fourth linkage peptide may be of any length, for example, having at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids. The fourth linkage peptide may consist of SEQ ID NO: 2. The second recombinant viral coat protein may further comprise a mutation of one or more trypsin sites at, for example, residues 6, 7 and/or 11.

(42) In one embodiment, the fusion protein comprises SEQ ID NO: 5. In another embodiment, the fusion protein consists of SEQ ID NO: 5.

(43) For each fusion protein, a method for producing the fusion protein is provided. The method comprises introducing a nucleic acid molecule into cells, wherein the nucleic acid molecule encodes the fusion protein, and expressing the fusion protein in the cells. Thereby, the fusion protein is produced. The nucleic acid molecule may be introduced into the cells transiently or stably. The method may further comprise purifying the fusion protein from the cells. The cells may be any cells in which the fusion protein can be expressed. The cells may be in a plant or a portion of a plant. The cells may be yeast cells. The cells may be insect cells. The cells may be mammalian cells.

(44) For each fusion protein, a composition comprising the fusion protein is provided. The composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be an adjuvant.

(45) A virus like particle (VLP) formed by the fusion protein of the present invention is provided. Where the fusion comprises a target protein, the target protein is displayed on the surface the VLP. The VLP may be formed in a cell, an organism or a portion of an organism. The cell may be selected from the group consisting of a plant cell, a yeast cell, an insect cell and a mammalian cell. The cell may be in a plant or a portion thereof. The plant may be a Nicotiana species. The Nicotiana species may be selected from the group consisting of Nicotiana benthamiana and Nicotiana tabacum. The cell may be a yeast cell. The cell may be an insect cell. The cell may be a mammalian cell.

(46) A composition comprising the virus like particles (VLPs) of the present invention is provided. At least about 50%, 60%, 70%, 80%, 90%, 95% or 99% of the VLPs may have a diameter within less than about 50%, 40%, 30%, 20%, 10% or 5% of an average diameter of the VLPs. The composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be an adjuvant.

(47) A method of producing virus like particles (VLPs) is provided. The method comprises introducing a nucleic acid molecule into a cell, an organism or a portion of the organism, wherein the nucleic acid molecule encodes the fusion protein of the present invention, expressing the fusion protein in the cell, the organism or the portion of the organism, and forming virus like particles by the fusion protein. Thereby, the VLPs are produced. The nucleic acid molecule may be introduced transiently or stably into the cell, the organism or the portion of the organism. Where the fusion protein comprises a target protein, the target protein is displayed on the surface of the VLPs. At least about 50%, 60%, 70%, 80%, 90%, 95% or 99% of the VLPs may have a diameter within less than about 50%, 40%, 30%, 20%, 10% or 5% of an average diameter of the VLPs. The VLPs may be formed by at least about 50%, 60%, 70%, 80%, 90% or 95% of the fusion protein.

(48) Where the VLPs are formed in the cell, the organism or the portion of the organism, the method may further comprise purifying the VLPs from the cell, the organism or the portion of the organism.

(49) Where the VLPs are not formed inside the cell, the organism or the portion of the organism, the method may further comprise purifying the fusion protein from the cell, the organism or the portion of the organism before the VLPs are formed.

(50) A method of inducing an immunological response in a subject is provided. The method comprises administering to the subject an effective amount of a fusion protein comprising an immunogenic agent as the target protein according to the present invention or VLPs formed by the fusion protein. The subject may be a patient in need of the induction of the immunological response.

(51) A method of treating a disease or condition in a subject is provided. The method comprises administering to the subject an effective amount of a fusion protein comprising a therapeutic agent as the target protein according to the present invention or VLPs formed by the fusion protein. The subject may be a patient who suffers from the disease and condition.

(52) A method of detecting a biomarker in a sample from a subject is provided. The method comprises contacting the sample with an effective amount of a fusion protein comprising a diagnostic agent as the target protein according to the present invention or VLPs formed by the fusion protein. The diagnostic agent binds the biomarker. The biomarker may be an indicator of a disease or condition from which the subject is suspected of suffering.

(53) A method of catalyzing a reaction by a reagent is provided. The method comprises contacting the reagent with an effective amount of a fusion protein comprising an enzyme as the target protein according to the present invention or VLPs formed by the fusion protein. The enzyme catalyzes the reaction. The reaction may be catalyzed in vitro or in vivo.

(54) The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate.

Example 1. Antibodies to Plant-Produced P. falciparum Sexual Stage Proteins Exhibit Transmission Blocking Activity

(55) Transmission blocking vaccines (TBV) are considered a critical component in the overall strategy for control and eventually elimination of malaria worldwide. Sexual-stage proteins expressed by Plasmodium falciparum, Pfs230 and Pfs25, are the main transmission blocking antigens moving through clinical trial development. Antibodies generated upon vaccination with either of these results in interruption of sporogonic development in the mosquito, and transmission to the next host. Using a plant based transient expression system, we have produced Pfs25 and Pfs230 fused to various carrier proteins in Nicotiana benthamiana, purified and characterized the proteins, and evaluated the vaccine candidates in animal models for generation of transmission reducing activity (TRA)/transmission blocking activity (TBA). The Pfs25 and Pfs230 vaccine candidates are expressed at high levels, and induced TBA that persist up to 6 months post immunization. These data demonstrate the potential of the new malaria vaccine candidates, and supports the feasibility of expressing Plasmodium antigens in a plant-based system.

(56) The incidents of malaria have declined over the last half decade, with an estimated reduction of 50-75% in endemic areas (2015 WHO World Malaria Report). The combined use of mosquito nets, artemisinin based therapies, and residual spraying have reduced malaria related deaths down to an estimated 400,000 deaths in 2015. These tools and a malaria vaccine, RTS,S/ASO1, are considered key interventions to reach the newly set goal of a 90% reduction in malaria mortality rates by 2030. Additional therapeutic vaccines that focus on breaking the cycle of parasite transmission, such as transmission-blocking vaccines, are also considered key areas of importance.

(57) These alternative vaccines reduce or block transmission of the malaria parasite from the mosquito to the person and will play an important role in reducing transmission burden in highly endemic areas. The leading transmission blocking antigens are Pfs25 and Pfs230. Pfs25 is not expressed by the parasite when it is in the human host, and as such the immune response is completely driven by active immunization. Alternatively, Pfs230 is expressed by the parasite when it is in the human host during blood stage infection, and thus its immunogenicity is naturally boosted and maintained during the infection cycles. Pre-clinical animal studies have shown that immunization with Pfs230 candidates generate TBA and non-immunized convalescent malaria patients have naturally acquired anti-Pfs230 antibodies.

(58) Pfs230 Construct Designs

(59) Pfs230 primary sequence schematic (FIG. 2). Cysteine motif (CM) domains are essentially as described by Williamson et al (1995) Mol Biochem Parasitol, 75(1): 33-42. The amino terminus portion (444-730) was expressed with a carboxyl terminal 6×His tag and KDEL sequence (Pfs230-AM). One putative N-Linked glycosylation site was mutated (N.fwdarw.Q). The Pfs230-AM sequence was also expressed as a fusion to the amino terminus Alfalfa Mosaic Virus coat protein (CP). This construct was unstable, leading to Pfs230 antigen degradation. A novel design was generated where the Pfs230-AM molecule is fused to the amino terminus of a tandem CP-CP fusion where the CPs are spaced with a flexible linker. This construct, Pfs230-CP.sup.2, self-assembled into a virus like particle (VLP) and displayed the Pfs230 antigen on the VLPs surface with minimal Pfs230 degradation.

(60) Purification and Characterization of Pfs230-CP.sup.2 VLPs

(61) The Pfs230-CP.sup.2 DNA sequence was cloned into plant expression vectors, and infiltrated into plants. After four days of incubation, the plants were harvested, homogenized, and the Pfs230-CP.sup.2 VLPs purified by IMAC chromatography, followed by a particle formation step where the Pfs230-CP.sup.2 protein self-assemble into the VLPs (FIGS. 4A and B). The non-covalently linked peptides show two main protein bands on SDS-PAGE and are recognized in Western blot by anti-His and two anti-Pfs230 monoclonal antibodies (mAb) 2G5 and 2B10 (FIG. 3). Negative stained transmission electron microscopy shows the anticipated VLP shaped particles (FIG. 4B), that elute from SEC at the anticipate size of ˜2 M Dalton (FIG. 4A).

(62) Pfs230 Animal Study Design

(63) The Pfs230 vaccine candidate Pfs230-CP.sup.2 VLP was tested in a preliminary rabbit immunogenicity study. Five rabbits were immunized with 10 μg of Pfs230 at days 0 and 28 (Table 1).

(64) TABLE-US-00001 TABLE 1 Rabbit immunogenicity study design. Rabbit Pfs230 dose (μg) at # Vaccine Route Adjuvant day 0 and 28 5 Pfs230-CP.sup.2 IM Alhydrogel 10 VLP
Transmission Blocking Activity of Immunized Serum

(65) Serum was taken at 56 days post immunization and tested in an ex vivo standard membrane feeding assay (SMFA) to examine transmission blocking activity of immunized rabbit sera. Purified immunoglobulin (IgG) was combined (final concentration 3.75 mg/mL) with the gametocyte containing blood meal fed to mosquitos. The anti-Pfs25 mAb 4B7 was used as a positive control. The Pfs230-CP.sup.2 vaccine candidate resulted in high levels of transmission blocking activity in the serum of immunized rabbits (Table 2).

(66) TABLE-US-00002 TABLE 2 SMFA results from rabbits immunized with Pfs230-CP.sup.2 vaccine candidate. Average % inhibition Sample name oocyts estimate 95% CI Lo 95% CI Hi p-value mAb-4B7 control 3.0 93.8 85.6 97.3 0.001 Pfs230-CP.sup.2 0.0 100.0 98.8 100.0 0.001 VLP 0.1 99.7 99.0 100.0 0.001 0.1 99.7 99.0 100.0 0.001 0.2 99.6 98.7 100.0 0.001 0.0 100.0 99.7 100.0 0.001

Example 2. Other Examples of Application of the CP-CP Format

(67) Malaria Pfs25 Antigen

(68) The malaria Pfs25 antigen was expressed as a CP.sup.2 fusion (CMB-01053) resulting in a single polypeptide, which self-assembled into VLP particles (FIG. 5). By contrast, when a single CP fusion was expressed, the protein had high levels of cleavage, resulting in lower than anticipated decoration of Pfs25.

(69) Influenza Antigens—H1 Subtype

(70) The ectodomain of haemagglutinin was expressed as a CP.sup.2 fusion resulting in a stable recombinant protein (FIG. 6A). Construct code: CMB-02039. The influenza CP.sup.2 constructs showed better expression and recovery compared to the single CP fusions.

(71) Influenza Antigens—H3 Subtype

(72) The ectodomain of haemagglutinin was expressed as a CP2 fusion resulting in a stable recombinant protein (FIG. 6B). Construct code: CMB-02079. The influenza CP.sup.2 constructs showed better expression and recovery compared to the single CP fusions.

(73) Influenza Antigens—H5 Subtype

(74) The ectodomain of haemagglutinin was expressed as a CP.sup.2 fusion resulting in a stable recombinant protein (FIG. 6C). Construct code: CMB-02011. The influenza CP.sup.2 constructs showed better expression and recovery compared to the single CP fusions.

(75) Influenza Antigens—H7 Subtype

(76) The ectodomain of haemagglutinin was expressed as a CP.sup.2 fusion resulting in a stable recombinant protein (FIG. 6D). Construct code: CMB-02059. The influenza CP.sup.2 constructs showed better expression and recovery compared to the single CP fusions.

(77) Influenza Antigens—H1 Subtype

(78) Sub-domain of haemagglutinin (HA3) was expressed as a CP.sup.2 fusion resulting in a stable recombinant protein (FIG. 6E). Construct code: CMB-02047. The influenza CP.sup.2 constructs showed better expression and recovery compared to the single CP fusions.

(79) Western Equine Encephalitis Virus (EEV) Antigen

(80) A sub-domain of the Western strain of EEV E2 glycoprotein was expressed as a CP.sup.2 fusion resulting in a stable recombinant protein (FIG. 7). Construct code: CMB-02412. These constructs were either poorly or not expressible as single CP fusions.

(81) Eastern Equine Encephalitis Virus (EEV) Antigen

(82) A sub-domain of the Eastern strain of EEV E2 glycoprotein was expressed as a CP.sup.2 fusion resulting in a stable recombinant protein (FIG. 8). Construct code: CMB-02383. These constructs were either poorly or not expressible as single CP fusions.

(83) Eastern Equine Encephalitis Virus (EEV) Antigen

(84) The ectodomain of the Eastern strain of EEV E2 glycoprotein was expressed as a CP.sup.2 fusion resulting in a stable recombinant protein (FIG. 9). Construct code: CMB-02380. These constructs were either poorly or not expressible as single CP fusions.

Example 3. First Generation VLP

(85) To increase the immunogenicity of the malaria transmission blocking Pfs25 vaccine antigen, the antigen was displayed on a VLP, by fusion to the amino terminus of the alfalfa mosaic virus (AIMV) coat protein (CP) (FIG. 10A). This first generation Pfs25-VLP was called A85, and was shown to form stable VLPs, be highly immunogenic, and effective at preventing the transmission of malaria in animal models. This molecule was tested through a Phase 1 clinical trial, where it showed no adverse reactivity. However, the VLPs effectiveness was hampered by in planta cleavage events within the CP molecule, resulting in considerable loss of the Pfs25 antigen from the VLP (FIG. 10B-C).

Example 4. Second Generation VLPs

(86) To address this antigen loss, work was performed to identify the sites of cleavage within the CP molecule. Amino terminal sequencing was performed, and the sites of CP cleavage were identified to be at positions 24 and 36 of the CP molecule. A third possible AIMV CP cleavage site was identified in the published literature at position 26.

(87) Three different versions of the CP molecule were generated to prevent CP degradation, and to increase the antigen display on the VLP (FIG. 11). B29 The three identified cleavage sites were point mutated to alanine. B30 The 22-amino acid region containing the sites of degradation was removed, generating a shorter d22CP molecule. B64 The 22-amino acid region containing the sites of degradation was replaced with a flexible linker (FL1), comprised of the amino acids GGGGSGGGGSGGGGSGG.

(88) The constructs B30 and B64 had much higher protein recovery, and were purified as a single protein without degradation. However, these molecules did not form VLPs. The purified B29 protein had some protein degradation, but could successfully form VLPs. Compared to the original A85 molecule, the B29 molecule had more Pfs25 antigens displayed per VLP, resulting in improved immunogenicity and malaria transmission blocking ability.

(89) From this work, it was determined that 1. It was possible to improve the antigen display on the VLP through protein design of the CP molecule. 2. The identified 22-amino acid region contained all the in planta CP molecule cleavage sites. 3. If the protein was just an antigen-CP fusion protein, such as B30 or B64, it may not form VLPs. However, if there was additional antigen free CP present, through for example, internal cleavage of the antigen-CP fusion protein, then VLP formation was possible.

Example 5. Third Generation VLPs

(90) Eight different 3rd-generation VLP constructs were developed to further improve antigen display on the VLP (FIG. 12). CMB-01045 The B64 molecule was modified to replace the FL1 with a longer flexible linker (FL2) comprised of the amino acids GGGGSAAALGAAGGGGSAAGTSAAGGGGSAAALGAA CMB-01047 The first 27-amino acids of the CP molecule were removed and replaced with the FL2 linker. CMB-01049 The B64 molecule was modified to replace the FL1 with an alpha helical linker (AH) comprised of the amino acids EAAAKEAAAKEAAAKEAAAKEAAAKEAAAK CMB-01075 The B64 molecule was modified by the addition of a Kex2P self-cleavage site after the FL1. The Kex2P site contained the amino acids IGKRGIGKRGIGKRG CMB-01053 The B64 molecule was modified by the addition of a second FL1 and d22CP to the C-terminus of the molecule as described in Example 2 (FIG. 5). CMB-01063 The CMB-01045 molecule was modified by the addition of a second FL1 and d22CP to the C-terminus of the molecule. CMB-01067 The B64 molecule was modified by the addition of a second d22CP and FL1 to the N-terminus of the molecule. CMB-01071 The B64 molecule was modified by the addition of a second d22CP and FL2 to the N-terminus of the molecule.

(91) The constructs CMB-01047, CMB-01075, CMB-01067, and CMB-01071 produce very low levels of protein, thereby preventing analysis of fusion to total protein ratios, or particle formation. The constructs CMB-01045 and CMB-01049 produced protein, but limited amounts of VLPs. The two CP.sup.2 VLP molecules, CMB-01053 and CMB-01063 both produced protein, which formed into VLPs, with the CMB-01053 molecule having the highest expression. The CMB-01053 protein was purified as a single protein without degradation (FIG. 13A). Compared to the original A85 VLP, this newly developed molecule allowed for much higher levels of protein production and formed VLPs with greatly improved antigen display (FIG. 13B-C).

(92) Three of the Pfs25 molecules were compared in a mouse animal study. The CMB-01053 VLP was compared to both the B30 molecule and the B29 VLP over a range of low Pfs25 antigen doses (FIG. 14A). The proteins were administered intramuscularly with alhydrogel adjuvant. At day 56 of the study (FIG. 14B), the anti-Pfs25 IgG titers of the B30 group was significantly lower than the IgG titers from either B29 or CMB-01053 VLP (p<0.05) at the 0.003 μg dose level. At day 168 of the study (FIG. 14C), the B30 group at a 0.003 μg dose had significantly lower anti-Pfs25 IgG titers than the B29 group (p<0.05), while at the 0.03 μg Pfs25 dose level the CMB-01053 group had significantly higher anti-Pfs25 IgG titers than the B29 VLP group (p<0.01). This is consistent with the CMB-01053 VLP not just displaying more Pfs25 antigens per molecule, but also displaying the antigens more effectively. Statistics were performed using Kruskal-Wallis analysis followed by Dunn's multiple comparison test, with *=p<0.05 and **=p<0.01.

(93) To determine if the molecules have transmission blocking activity, serum was collected at days 56 and 168 post immunization and analyzed in an ex vivo standard membrane feeding assay (SMFA). All three Pfs25 molecules, including the CMB-01053 VLP, demonstrated malaria transmission blocking activity (FIG. 14D), with only the B30 group at the 0.003 μg dose show a statistically significant difference in SMFA levels, p<0.0001 at day 56, and p<0.05 at day 168. Statistics were performed using a one-way ANOVA followed by Tukey's multiple comparison test.

(94) Additional antigens were tested on the CP.sup.2 design developed for CMB-01053. Antigens successfully displayed on the CP.sup.2VLP include the malaria antigen Pfs230, a yellow fever antigen, several influenza antigens, and an Eastern equine encephalitis virus antigen (FIG. 15).

Example 6. Degradation Sites in the N-Terminus of AIMV

(95) The in silico digestion analysis for trypsin and chymotrypsin sensitive sites was performed using the program “PeptideCutter” at ExPASy, the SIB Bioinformatics Resources Portal. In planta cleavage sites of AIMV were identified from purified AIMV particles using N-terminal sequencing.

(96) Potential sites of degradation in the N-terminus of the AIMV protein were identified using the program PeptideCutter (FIG. 16A), revealing many potential trypsin and chymotrypsin sensitive sites.

(97) AIMV particles were purified and N-terminally sequenced, identifying sites where the AIMV protein is cleaved in planta (FIG. 16B). Information from the N-terminal sequencing and in silico digestion was used to identify the amino acids within the AIMV molecule to replace with the flexible linker. This modified AIMV sequence has a greatly reduced number of protease sensitive sites (FIG. 16C).

Example 7. Expression of Fusion Proteins in Insect Cells, Yeast Cells, and Mammalian Cells

(98) A fusion protein consisting of SEQ ID NO: 5 was expressed in insect cells (FIGS. 17 and 18), yeast cells (FIG. 19), and mammalian cells (FIG. 20) using the methods described below. All expressed fusion proteins formed or are expected to form virus-like particles.

(99) Baculoviral Expression System.

(100) The gene encoding a CP-CP fusion protein was optimized for expression in insect cells and synthetized by GeneArt (ThermoFisher Scientific). The gene was subcloned into BamHI/HindIII-digested baculovirus transfer vector pFastBac1 (ThermoFisher Scientific) and the subcloned sequence was verified. The virus was subsequently propagated using the Bac-to-Bac Baculovirus Expression System according to the manufacturer's protocol. Briefly, the resulting vector carrying the CP.sup.2 gene was transformed into E. coli DH10Bac cells containing the baculovirus genome (bacmid DNA). Transposition occurred between pFastBac1 and the bacmid to generate a recombinant bacmid with CP.sup.2 gene. Positive clones were selected and the recombinant bacmid was isolated and transfected into Spodoptera frugiperda (Sf21) cells for propagation of recombinant baculovirus.

(101) For large-scale expression of CP.sup.2 protein, Sf21 cells were infected at 5×10.sup.6 cells/mL with virus at an optimal multiplicity of infection (MOI) of 2.5 in Sf-900™ II SFM cell medium and incubated at 27° C. with mixing at 160 rpm. Forty-eight hours post-infection, the insect cells were collected by centrifugation at 500×g for 10 minutes, after which the cells were solubilized in 100 mM pyrophosphate buffer, pH6.5, sonicated and clarified by centrifugation at 20,000×g for 20 minutes to remove cell debris. Supernatant was centrifuged at 60,000 RPMs in Ti70 rotor for two hours. Pellet was dissolved in 100 mM pyrophosphate buffer, pH6.5 and submitted for TEM.

(102) Yeast Expression System.

(103) The gene encoding a CP.sup.2 fusion protein was optimized for expression in yeast cells and synthetized by GeneArt (ThermoFisher Scientific). The gene was subcloned into BamHI/EcoRI-digested yeast vector pPIC3.5 (ThermoFisher Scientific) and the subcloned sequence was verified. The recombinant plasmid was linearized with SalI and then transformed into Pichia pastoris GS115 by electroporation. The transformants were plated on MD (minimal dextrose) plates without histidine and incubated at 30° C. for 2-3 days. Colonies were analyzed by PCR, using gene specific primers. Positive colonies were propagated and fusion protein expression was induced with methanol.

(104) Mammalian Expression System.

(105) The gene encoding a CP.sup.2 fusion protein was optimized for expression in mammalian cells and synthetized by GeneArt (ThermoFisher Scientific). The gene was subcloned into HindIII/XhoI-digested yeast vector pcDNA3.1 (ThermoFisher Scientific) and sequence verified. Plasmid DNA was used for transfection of Vero cells using LIPOFECTAMINE™ 2000. After two days of incubation cells were precipitated by centrifugation at 1000 g for 5 minutes.

(106) Western Blot Analysis and TEM.

(107) To characterize expression of a fusion protein in mammalian, insect or yeast cells, cells were disrupted by boiling for 10 minutes in 1× Laemmli sample buffer, when protein samples were separated on a 10% SDS-PAGE gel, transferred onto a polyvinylidene difluoride membrane, and probed with a mouse anti tetra-His mAb (Qiagen) or rabbit anti-AIMV CP polyclonal antibodies (Fraunhofer). HRP-conjugated rabbit anti-mouse or goat anti-rabbit Abs were used as secondary antibodies, respectively.

(108) Particle formation was evaluated by negative stained transmission electron microscopy. The images of the negatively stained particles were captured using a Zeiss LIBRA 120 transmission electron microscope.

Example 8. In Vitro Particle Formation

(109) An antigen-CP.sup.2 fusion protein was purified using IMAC chromatography, and the eluted protein was concentrated using centrifugal spin concentrators to ˜10 mg/mL. Analytical size exclusion chromatography was performed using an SRT-1000 column (Sepax) at 1 mL/min. In vitro particle formation was performed by dialysis of concentrated protein into 80 mM sodium pyrophosphate buffer pH 6.0.

(110) To show that it is possible to form AIMV based VLPs in vitro, an antigen-CP.sup.2 fusion protein was expressed in plants and purified by IMAC chromatography. The eluted protein was concentrated, and in vitro particle formation performed by dialysis into 80 mM sodium pyrophosphate buffer pH 6.0. Protein samples pre and post particle formation were analyzed for the presence of VLPs by analytical size exclusion chromatography, where VLPs resolve at ˜9 mL (FIG. 21). No VLP associated peak was observed in the pre particle formation conditions, but a clear VLP associated peak was observed in the post particle formation conditions, confirming the in vitro formation of VLPs.

Example 9. Particle Formation from CP-CP Produced in Multiple Protein Production Systems

(111) The CP.sup.2 fusion protein without an attached antigen was expressed in plants (Nicotiana benthamiana), yeast (Pichia pastoris), and insect cells (Spodoptera frugiperda), and purified using IMAC chromatography. The eluted protein was concentrated using centrifugal spin concentrators, and formed into particles by dialysis into 80 mM sodium pyrophosphate buffer pH 6.0. Particle formation was confirmed by negative stained transmission electron microscopy analysis.

(112) The CP.sup.2 fusion protein was successfully expressed and purified from plant, yeast, and insect protein expression systems. To confirm that the protein produced in the different expression systems could successfully form into particles, the IMAC elution's were concentrated, and formed into particles by buffer exchange into 80 mM sodium pyrophosphate buffer pH 6.0. Negatively stained transmission electron microscopy analysis confirmed the presence of particles (FIG. 22A-C).

Example 10. Enzyme Display on a CP.SUP.2 .Molecule

(113) In addition to displaying antigens, the CP.sup.2 molecule can also display other types of proteins, such as enzymes. Horseradish peroxidase C1 (HRP) (EC number 1.11.1.7) uses hydrogen peroxide to oxidize both organic and inorganic compounds. The HRP protein was fused to the N-terminus of the CP.sup.2 molecule, generating HRP-CP.sup.2 fusion protein CMB-03057, and expressed in plants. The protein was isolated using IMAC chromatography against a C-terminal His-tag.

(114) The presence of the HRP-CP.sup.2 fusion protein in the 300 mM imidazole IMAC fraction was detected using Coomassie stained SDS-PAGE (FIG. 23A), and confirmed by Western blotting against the C-terminal His-tag (FIG. 23B). The samples were analyzed for HRP activity using a chemiluminescence assay, where protein fractions and buffers were incubated with HRP substrate, and light emission detected using a GeneGnome HR camera (Syngene) (FIG. 23C). Only the 300 mM imidazole IMAC fraction, containing the HRP-CP.sup.2 fusion protein, displayed HRP activity, confirming both that the HRP-CP.sup.2 fusion protein was produced and that the enzyme was correctly folded as a functional enzyme. The HRP-CP.sup.2 sample was analyzed by negative stained transmission electron microscopy, which confirmed the presence of particles (FIG. 23D).

(115) All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and/or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.