Molecular farming of VLP therapeutics

Abstract

Provided herein are methods of harvesting virus-like particles (VLPs) from plant tissue, and compositions and methods of use thereof of the harvested VLPs.

Description

Atty. Dkt. No.: 114198-3560MOLECULAR FARMING OF VLP THERAPEUTICSCROSS-REFERENCE TO RELATED PATENT APPLICATION

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Serial No. 63 / 713,840, filed October 30, 2024, the content of which is incorporated by reference in its entirety.STATEMENT OF GOVERNMENT SUPPORT[00021 This invention was made with government support under CA274640 awarded by the National Institutes of Health and under NNX16AO69A awarded by National Aeronautics and Space Administration. The government has certain rights in the invention.BACKGROUND

[0003] Virus-like particles (VLPs) can be used to fight leading courses of death worldwide, for example cardiovascular diseases, cancer and infectious disease. However, it is currently difficult to scale production to match human needs. This is mainly because current VLPs are purified using high-speed centrifugation and gradient centrifugation which are difficult to scale. The contemporary method for purification of VLPs uses multiple rounds of ultracentrifugation as well as isopycnic ultracentrifugation to meet these purity requirements. However, ultracentrifugation and especially isopycnic ultracentrifugation are difficult to scale. Additionally, the use of large volumes of chloroform (or alternatively methanol) poses a safety risk for the operator. Further, human therapeutics require high purity.

[0004] There exists a need to produce VLPs to overcome these limitations. Further, there exists a need to produce VLPs in resource limited environments, such as in outer space.

[0005] Virus-like particles (VLPs) can be used to fight leading courses of death worldwide, for example cardiovascular diseases, cancer and infectious disease. However, it is currently difficult to scale production to match human needs. This is mainly because current VLPs are purified using high-speed centrifugation and gradient centrifugation which are difficult to scale. The contemporary method for purification of VLPs uses multiple rounds of ultracentrifugation as well as isopycnic ultracentrifugation to meet these purity requirements. However, ultracentrifugation and especially isopycnic ultracentrifugation are difficult to-1-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 scale. Additionally, the use of large volumes of chloroform (or alternatively methanol) poses a safety risk for the operator. Further, human therapeutics require high purity.

[0006] There exists a need to produce VLPs to overcome these limitations. Further, there exists a need to produce VLPs in resource limited environments, such as in outer space.SUMMARY OF THE DISCLOSURE

[0007] Purification of recombinant proteins from plants usually involves the disruption of the plant tissue, resulting in the release of large amounts of soluble and insoluble impurities. In contrast, animal cells and microbes often secrete recombinant proteins into the cultivation medium, which greatly facilitates purification. Here Applicant sought to overcome the current limitations of purification of recombinant proteins from plants.

[0008] Applicant provides herein a downstream process and a scalable purification process. In developing this method, Applicant systematically screened the following processes: acidic extraction conditions making use of the virions pH stability, ultrafiltration making use of the nanoparticle character and the virions size, and finally ion exchange chromatography based on the virion’s surface charge. The three processes were then combined into a multi-step protocol - in one embodiment a 7-step protocol - providing efficiency compared to the contemporary laboratory procedures involving various centrifugation steps. Furthermore, Applicant streamlined the removal of endotoxins by combining detergent-based endotoxin removal with ion exchange chromatography. The new ultrafiltration-based process reduced the number of unit operations by more than half and the processing time from ~20 hours to ~7 hours compared to centrifugation-based purification of CPMV. Importantly, toxic organic solvents, ultracentrifugation and isopycnic ultracentrifugation which are difficult to scale were omitted. Applicant used numerous characterization methods to validate CPMV’s structural integrity and biological activity.

[0009] Applicant’s methods to produce VLPs address and overcome the limitations of the state of the art. In one aspect, Applicant provides a novel purification process which omits the need for ultracentrifugation as well as the use of toxic chloroform. This method is scalable using ultrafiltration membranes, i.e. the process can be readily scaled-up by increasing the membrane area used for ultrafiltration.-2-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0010] According to one embodiment, the methods comprise, or consist essentially of, or yet further consist of: (i) extracting the VLPs from the plant tissue using an extraction solution, wherein the VLPs are collected in the eluate; (ii) purifying the VLPs collected in the extraction solution of step (i) through a series of separations based on size; and (iii) purifying the VLPs from step (ii) through separation based on charge. In one embodiment, the VLPs are harvested at room temperature. In another embodiment the VLPs are harvested at high heat, for example at about 70°C, at about 80°C, at about 90°C, at about 100°C, or over 100°C.

[0011] According to another embodiment, the methods comprise, or consist essentially of, or yet further consist of: (i) extracting the VLPs from the plant tissue using an acidic extraction solution collecting the VLP-containing eluate; (ii) filtering the eluate from step (i) through a series of filtration membranes with serially decreasing pore size and collecting a the VLPs, wherein the VLPs are collected in the eluate or the membrane; and (iii) separating the VLPs from the filtration of step (ii) with ion exchange chromatography to harvest the VLPs. In some embodiments, the extraction solution has a pH of about 4.0. In one aspect, the ion exchange chromatography is anion exchange chromatography. In another aspect, the ion exchange chromatography is cation exchange chromatography.

[0012] According to yet another embodiment, the methods comprise, or consist essentially of, or yet further consist of: (i) extracting the VLPs from plant tissue using a basic extraction solution collecting the VLP-containing first eluate; (ii) filtering the first eluate from step (i) with a series of ultrafiltration membranes with serially decreasing pore size and collecting the VLPs, wherein the VLPs are collected in the eluate or the membrane; and (iii) separating the VLPs from the filtration of step (ii) with ion exchange chromatography, thereby harvesting the VLPs. In one aspect, the ion exchange chromatography is anion exchange chromatography. In another aspect, the ion exchange chromatography is cation exchange chromatography.

[0013] In a further aspect, this disclosure provides a method of harvesting virus-like particles (VLPs) from plant tissue comprising (i) extracting the VLPs from the plant tissue using an extraction solution, wherein the VLPs are collected in the eluate; (ii) purifying the VLPs-3-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 collected in the extraction solution of step (i) through a series of separations based on size; and (iii) purifying the VLPs from step (ii) through separation based on charge, thereby harvesting the VLPs. Alternatively, the method of harvesting virus-like particles (VLPs) from plant tissue comprises: (i) extracting the VLPs from the plant tissue using an acidic extraction solution collecting the VLP-containing eluate; (ii) filtering the eluate from step (i) through a series of filtration membranes with serially decreasing pore size and collecting a the VLPs, wherein the VLPs are collected in the eluate or the membrane; and (iii) separating the VLPs from the filtration of step (ii) with ion exchange chromatography to harvest the VLPs, thereby harvesting the VLPs. Yet further, the method of harvesting virus-like particles (VLPs) from plant tissue comprises: (i) extracting the VLPs from plant tissue using a basic extraction solution collecting the VLP-containing first eluate; (ii) filtering the first eluate from step (i) with a series of ultrafiltration membranes with serially decreasing pore size and collecting the VLPs, wherein the VLPs are collected in the eluate or the membrane; and (iii) separating the VLPs from the filtration of step (ii) with ion exchange chromatography, thereby harvesting the VLPs.[00141 In one aspect, the extraction solution is acidic, e.g., having a pH in the range of about 3.5 to about 4.5, or about 4.0. Alternatively, the extraction solution can be basic, e.g., having a pH from about 8.5 to about 9.5, or alternatively about 9.0.

[0015] In one aspect, these methods are performed in the absence of an ultracentrifugation step, optionally an isopycnic ultracentrifugation step or in the absence of a chloroform or a methanol extraction step. In a further embodiment, the harvested VLPs have a net negative charge at the pH of the extraction and / or chromatography buffers, or alternatively, the harvested VLPs have a net positive charge at the pH of the extraction and / or chromatography buffers.

[0016] In one embodiment of these methods, VLPs are harvested from plant tissue that comprises or consists essentially of leaf tissue, stem tissue, and / or root tissue, or from an intact plant. When the VLP are extracted from an intact plant, the extraction in step (i) can be by vacuum infiltrating an apoplast with the extraction solution and collecting the extraction solution with centrifugation. In one aspect, the extraction solution has: a pH of about 2.5 to-4-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 about 4.5, optionally about 4.0 for acidic extraction, and further optionally wherein the VLP comprises Cowpea Chlorotic Mottle Virus (CCMV); or a pH of about 8.5 to about 9.5, optionally about 9.0, for basic extraction, and further optionally wherein the VLP comprises Cowpea Mosaic Virus (CPMV).

[0017] In some aspects, the series of membranes comprises, or consists essentially of, or yet further consists of a first membrane and a second membrane, or yet further a third membrane. In one embodiment, the first membrane has a pore size of about 0.1 pm to about 0.4 pm, optionally about 0.2 pm, or alternatively, the second membrane has a molecular weight cutoff of about 2000kDa, or about lOOOkDa, and yet further, the third membrane has a molecular weight cut-off selected from of about lOOkDa to about 500kDa, about 300kDa to about 500kDa, or about 300kDa, or about 500kDa. In one embodiment, the first membrane and second membrane average pores sizes are larger than the diameter of the VLP. In another embodiment, the first and second membranes have average pore sizes larger than the diameter of the VLP and the third membrane have an average pore size smaller than the diameter of the VLP.[OOlSj In one embodiment, the ion exchange chromatography is anion exchange chromatography, e.g., cation exchange chromatography.

[0019] The methods can be practiced in any gravity environment. In one aspect, it is practiced in a microgravity environment.

[0020] Also provided by this disclosure is a method of harvesting virus-like particles (VLPs) from plant tissue comprising, or consisting essentially of, or yet further consisting of: (i) extracting the VLPs from the plant tissue using an acidic extraction solution and collecting the VLPs in a first eluate; (ii) filtering the VLPs from the first eluate of step (i) with a first membrane having a pore size of about 0.20 pm to about 0.25 pm, optionally about 0.22 pm and collecting a second eluate; (iii) filtering the VLPs from the second eluate of step (ii) with a second membrane, having a molecular weight cutoff of about 800 kDa to about 1200 kDa, optionally about 1000 kDa and collecting a third eluate; (iv) filtering the third eluate VLPs from step (iii) with a third membrane, wherein the third membrane has a molecular weight cutoff of about 300 to about 500 kDa, and collecting the VLPs retained from the third-5-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 membrane; (v) separating the VLPs collected from the third membrane in step (iv) with ion exchange chromatography, and wherein the method is performed in any gravity environment, and optionally wherein the VLP is icosahedral shaped.

[0021] Further provided is a method of harvesting virus-like particles (VLPs) comprising, or consisting essentially of, or yet further consisting of: (i) extracting the VLPs from an intact plant or plant part using an acid extraction solution, and collecting the VLPs from a first eluate by vacuum infiltrating the apoplast from the intact plant or plant tissue with the extraction solution and collecting the first eluate from the apoplast with centrifugation; (ii) filtering the VLPs from the first eluate of step (i) with a first membrane having a pore size of about 0.20 pm to about 0.2 pm 0.25 pm, optionally about 0.22 pm and collecting a second eluate; (iii) filtering the VLPs from step (ii) with a second membrane, wherein the second membrane has a molecular weight cutoff of about lOOOkDa and collecting a third eluate; (iv) separating the VLPs from the third eluate in step (iii) with ion exchange chromatography, wherein the method is performed in any gravity environment.

[0022] This disclosure further provides a method of harvesting virus-like particles (VLPs) comprising, or consisting essentially of, or yet further consisting of: (i) extracting the VLPs from an intact plant or plant part using an acid extraction solution, and collecting the VLPs from a first eluate by vacuum infiltrating the apoplast from the intact plant or plant tissue with the extraction solution and collecting the first eluate from the apoplast with centrifugation; (ii) filtering the VLPs from the first eluate of step (i) with a first membrane having a pore size of about 0.20 pm to about 0.25 pm, optionally about 0.22 pm and collecting a second eluate; (iii) filtering the VLPs from step (ii) with a second membrane, wherein the second membrane has a molecular weight cutoff of about 1,000 kDa , and collecting the VLPs retained on the second membrane; (iv) separating the VLPs from the second membrane in step (iii) with ion exchange chromatography, wherein the method is performed in any gravity environment, optionally wherein the VLP is rod-shaped.

[0023] In another aspect, a method of harvesting virus-like particles (VLPs) from plant tissue is provided, the method comprising, or consisting essentially of, or yet further consisting of: (i) incubating the plant tissue in a heated extraction solution; (ii) collecting the VLPs in the-6-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 extraction solution; (iii) purifying the VLPs collected in the extraction solution of step (ii) through a series of separations based on size; and (iv) purifying the VLPs from step (iii) through separation based on charge.

[0024] In one aspect of these methods, the heated extraction solution is at a temperature of about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 80°C, about 90°C, about 100°C, or over 100°C.[00251 Also provided herein is a method of producing and harvesting virus-like particles (VLPs) wherein the VLPs are recombinant plant virus particles secreted into the apoplast of intact plant tissue, comprising, or consisting essentially of, or yet further consisting of: (i) culturing a plant transformed or infected with a VLP expression construct comprising a secretion signal peptide operably fused to a viral coat protein sequence; (ii) vacuum infiltrating the apoplast of intact plant tissue with an extraction buffer; (iii) centrifuging the infiltrated intact plant tissue under conditions sufficient to recover apoplastic fluid containing the secreted VLPs; and (iv) purifying the VLPs by ultrafiltration and / or ion exchange chromatography; wherein the plant tissue remains viable after harvesting. In one aspect, the harvesting is repeated at least twice from the same plant without loss of VLP integrity or plant viability. In these methods, the extraction buffer is selected from an acidic, neutral or alkaline buffer suitable for maintaining VLP integrity, optionally having a pH between 4.0- 9.0 depending on the VLP species.

[0026] Further provided is a method of harvesting virus-like particles (VLPs) in a microgravity environment, comprising, or consisting essentially of, or yet further consisting of: (i) producing VLPs in an intact plant cultivated under microgravity conditions; (ii) recovering VLPs from the apoplast via infiltration-centrifugation using low-mass, manual or syringe-operated equipment; (iii) purifying the recovered VLPs using ultrafiltration / diafiltration devices having a membrane area of less than 100 cm2and sequentially decreasing molecular weight cut-offs; and (iv) polishing the VLPs using gravity-driven or low-energy ion exchange chromatography. In one aspect, the plant is subjected to controlled reactive oxygen species (ROS) stress for greater than 10 days to increase VLP yield in microgravity by at least 20% relative to an untreated control.-7-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0027] In one embodiment, a method for purifying a functionalized virus-like particle is comprising a covalently attached functionalizing agent, e.g., functional cargo, fluorophore, targeting ligand, or therapeutic cargo, the method comprising, or consisting essentially of, or yet further consisting of: (i) contacting the VLP with the functionalizing agent under conditions suitable for covalent linkage; (ii) purifying the conjugated VLP by ultrafiltration / diafiltration using a membrane that retains the conjugated VLP and permits passage of unbound functionalizing agent; and (iii) recovering the purified conjugated VLP, wherein the recovery yield is at least 90% and free functionalizing agent is reduced to below detectable levels by fluorescence or absorbance analysis. Non-limiting examples of the functionalizing agent is fluorescein isothiocyanate (FITC) or an equivalent dye detectable by excitati on / emi s si on spectroscopy .

[0028] In another aspect, a method of removing endotoxin and 1,3-P-glucan contaminants from a plant-derived virus-like particle preparation is provided, the method comprising, or consisting essentially of, or yet further consisting of: (i) adding a non-ionic detergent to the preparation, wherein the detergent is present at 0.05-0.5% (v / v); (ii) loading the detergent-containing preparation onto a charged resin suitable for ion exchange chromatography under conditions binding the VLP; and (iii) eluting the VLP with a salt gradient; wherein endotoxin and P-glucan levels in the final preparation are reduced to below regulatory thresholds for clinical administration. Non-limiting examples of the non-ionic detergent is Triton X-l 14, and the wherein detergent and contaminants are separated from the VLP during the chromatography step.

[0029] In each of the disclose methods the VLP is derived from a plant virus, optionally wherein the VLP is a plant virus coat protein, optionally wherein the plant virus is Cowpea Mosaic Virus, Cowpea Chlorotic Mottle Virus, or Potato Virus X.

[0030] In one embodiment of these methods, when the virus-like particles are secreted into the apoplast of intact plant tissue, the method further comprises, or consists essentially of, or yet further consists of: (i) directing the VLPs to the apoplast by inclusion of a secretion signal peptide operably linked to a viral coat protein coding region; (ii) vacuum infiltrating the apoplast with an extraction buffer; (iii) centrifuging the infiltrated intact plant tissue to-8-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 recover apoplastic fluid containing the secreted VLPs; and (iv) purifying the VLPs by ultrafiltration and / or ion exchange chromatography; wherein the plant tissue remains viable after harvesting. The harvesting can be repeated from the same plant on at least two occasions without loss of VLP integrity or plant viability. In addition, the extraction buffer can selected from acidic, neutral, or alkaline buffers compatible with VLP integrity, and has a pH optimized to the stability profile of the target VLP.[0031 [ When the methods are performed in a microgravity environment, the method can comprise, or consist essentially of, or yet further consist of: (i) cultivating the plant under microgravity; (ii) recovering VLPs from the apoplast via infiltration-centrifugation using low-mass manual or syringe-operated equipment; (iii) purifying the recovered VLPs by sequential ultrafiltration / diafiltration using membranes having a total surface area less than 100 cm2and serially decreasing MWCOs; and (iv) polishing the VLPs using gravity-driven or low-energy ion exchange chromatography. In one aspect when the plant is subjected to controlled reactive oxygen species (ROS) stress for greater than 10 days, VLP yield in microgravity is increased by at least 20% relative to an untreated control.[00321 The methods can be further modified to utilized a functionalized VLP, by purifying a functionalized virus-like particle that comprises a covalently attached fluorophore, targeting ligand, or therapeutic cargo, by: (i) contacting the VLP with the functionalizing agent under conditions suitable for covalent linkage; (ii) ultrafiltration / diafiltration of the reaction mixture using a membrane that retains the conjugated VLP and passes unbound functionalizing agent; and (iii) recovering the purified conjugated VLP, wherein free functionalizing agent is reduced to below detectable levels optionally by fluorescence or absorbance analysis. Nonlimiting examples of a functionalizing agent is fluorescein isothiocyanate (FITC) or an equivalent dye detectable by excitation / emission spectroscopy.

[0033] The methods can be further modified to remove endotoxin and 1,3-P-glucan contaminants by: (i) adding a non-ionic detergent in a concentration of 0.05-0.5% (v / v) to the VLP preparation; (ii) loading the detergent-containing preparation onto a charged resin suitable for ion exchange chromatography under conditions binding the VLP; and (iii) eluting the VLP with a salt gradient; wherein in one aspect, the endotoxin and 1,3-P-glucan levels in-9-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 the final preparation are reduced below regulatory thresholds for clinical administration. In one embodiment, the non-ionic detergent is Triton X-l 14, and detergent and contaminants are separated from the VLP during the chromatography step.|0034] Further provided are the harvested VLP prepared by the methods of this disclosure, or a plurality of the harvested VLPs. The VLPs can further comprise a functionalizing or a therapeutic agent, such as a therapeutic RNA. The VLPs can further comprise a detectable or purification label for in vitro and / or in vivo use.

[0035] Compositions comprising the harvested VLPs or plurality of harvested VLPs are provided that further comprise a carrier, such as a pharmaceutically acceptable carrier or a stabilizer, preservative or a lyophilizing agent. The VLPs in the composition can further comprise a detectable or purification label for in vitro and / or in vivo use.

[0036] The harvested VLPs and compositions can be delivered to a cell, tissue or a subject in need of the VLPs by contacting or administering the VLPs or compositions to the cell, tissue or subject as appropriate. The subject can be an animal or mammal, or optionally a human patient. The cell or tissue can be an animal, mammalian or human cell or tissue, which can be contacted in vitro, ex vivo or in vitro.[0037| Kits comprising the VLPs and / or methods and instructions to reproduce the methods are further provided herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIGS. 1A - 1G: Optimization of pH precipitation and ultrafiltration for purification of CPMV from plant extracts. (FIGS. 1A - 1C) Analysis of blender extracts from CPMV infected cowpea leaves using alkaline, neutral, and acidic buffers by gel electrophoresis and staining with Coomassie Brilliant Blue (FIG. 1A - converted color image, FIG. IB - black and white image) or western blotting (FIG. 1C). Rabbit anti-CPMV antibodies and HRP-labeled goat anti-rabbit antibodies were used for western blots. (FIG. ID) Analysis of process samples from ultrafiltration with a 1000 kDa MWCO by gel electrophoresis and staining with Coomassie Brilliant Blue. (FIG. IE) Analysis of process samples from ultrafiltration with a 500 kDa MWCO by gel electrophoresis and staining with Coomassie Brilliant Blue. Gels are representative images from replicated experiments.-10-4911-2869-8997.1Atty. Dkt. No.: 114198-3560(FIG. IF, FIG. 1G) TEM images of retentates and permeates from ultrafiltration with a 1000 kDa MWCO (FIG. IF) or 500 kDa MWCO (FIG. 1G). Black arrows denote the large and small CPMV coat protein. Arrows denote non-cleaved small coat protein. Ret - ultrafiltration retentate, Per - ultrafiltration permeate, + - positive control (purified CPMV obtained through the conventional centrifuge-based purification protocol).

[0039] FIGS. 2A -2H: Optimization of anion exchange (AEX) chromatography for purification of CPMV. (FIGS. 2A-2F) Chromatograms from purification of CPMV by anion exchange chromatography and analysis of corresponding process samples by gel electrophoresis. After electrophoretic separation, gels were stained with Coomassie Brilliant Blue. Chromatography was performed with and AKTApure system, using 1.0 mL (FIG. 2A, FIG. 2B) or 3.0 mL columns (FIG. 2C, FIG. 2D), HiTrap Q Sepharose Fast Flow resin, and a flowrate of 0.5 mL / min. Ultrafiltration-purified samples were used as feed, optionally supplemented with 0.2% (v / v) Triton XI 14 before chromatography (FIG. 2E, FIG. 2F). 100 mM potassium phosphate (pH 7.0) was used as equilibration and wash buffer. For target protein elution, the same buffer was supplemented with 1.0 M sodium chloride. Gels are representative images from replicated experiments. (FIG. 2G, FIG. 2H) Endotoxin removal during ion exchange chromatography with Triton XI 14. Endotoxins in process samples before (FIG. 2G) and after (FIG. 2H) incorporating Triton XI 14 were quantified using the LAL endotoxin assay. Black arrows in FIG. 2F denote the large and small CPMV coat protein. Elu - elution fraction, FT - flowthrough, + - positive control.[0()40| FIGS. 3A - 3D: Process flow diagram and characterization of the UF / DF process in comparison with the centrifugation process. (FIG. 3A) Centrifugation-based purification of CPMV. The contemporary method for purification of CPMV is a 3-day process that relies on toxic organic solvents, ultracentrifugation and isopycnic ultracentrifugation to remove insoluble impurities, which his difficult to scale. (FIG. 3B) Scalable CPMV purification with the UF / DF process. In the UF / DF process ultracentrifugation has been omitted through two consecutive ultrafiltration steps. A 1000 kDa MWCO membrane is used to remove impurities that are larger than CPMV and a 300 kDa membrane is used to remove impurities that are smaller than CPMV. Alongside with these impurities, bulk water is removed from the process, thus reducing sample volumes and -11-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 the equipment footprint. (FIG. 3C) Analysis of process samples from purification of CPMV with the UF / DF process. Process samples were separated by gel electrophoresis and stained with Coomassie Brilliant Blue. Gels are representative images from replicated experiments. (FIG. 3D) Quantification of total soluble protein, endotoxins and beta-glucans in samples from the same process.

[0041] FIGS. 4A - 4H: Characterization of CPMV preparations from the centrifugation process in comparison with the UF / DF process. (FIG. 4A) Analysis of CPMV preparations from the centrifugation process and the UF / DF process by denaturing gel electrophoresis and staining with Coomassie Blue. (FIG. 4B, FIG. 4C) Analysis of the same samples by native gel electrophoresis followed by RNA staining (FIG. 4B) and protein staining (FIG. 4C). (FIG. 4D) TEM images of the same samples after uranyl acetate staining. (FIG. 4E, FIG. 4F) Size exclusion chromatography elusion profiles of CPMV preparations from the centrifugation process (FIG. 4E) and the UF / DF process (FIG. 4F). Size exclusion chromatography was performed with a Superose 6 Increase column, using 100 mM potassium phosphate buffer and a flowrate of 0.5 mL / min. (FIG. 4G, FIG. 4H) Characterization of CPMV preparations from the centrifugation process (FIG. 4G) and the UF / DF process (FIG. 4H) by DLS. DLS was performed with a Zetasizer Nano, using 100 mM potassium phosphate as buffer. Sizes are reported as the mean of three technical replicates with 12 analytical replicates each. PDI - poly dispersity index.

[0042] FIGS. 5A - 5E: Biological activity of CPMV from the UF / DF process in comparison with controls from the centrifugation process. (FIG. 5A) Treatment schedule. Female BALB / c mice were inoculated intradermally with A20 lymphoma cells (2* 105cells / mouse). After tumors reached a volume of ~30 mm3, mice received weekly doses of 20 pg CPMV, 100 pg CPMV, or PBS by intratumoral administration for three weeks. Tumor volumes were measured every other day. To reduce the number of mice that had to be sacrificed, control groups were shared with another study, which will be reported elsewhere (Simms et al., Structure-function relationship of S protein cleavage on the efficacy of cowpea mosaic virus as an intratumoral immunotherapy, in review). The schematic was created with BioRender.com. (FIG. 5B, FIG. 5C) Kaplan-Meier survival plots of all treatment groups. Statistical analysis on survival curves was performed using the log-rank -12-4911-2869-8997.1Atty. Dkt. No.: 114198-3560(Mantel-Cox) test

[0080] , (FIG. 5D, FIG. 5E) Change of tumor volumes during the treatment period. Data are means ± SD (n?5). Statistical significance in tumor volumes was analyzed by two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).|'<)043] FIGS. 6A - 6F: Chemokine and cytokine response after treatment with CPMV from the centrifugation process or the UF / DF process. (FIG. 6A) Treatment schedule. Female BALB / c mice were inoculated intradermally with A20 lymphoma cells (2* 105cells / mouse). After tumors reached a volume of ~60 mm3, mice received one dose of PBS or CPMV (100 pg); 24 h after treatment tumors were resected and extracted for MSD assays. The schematic was created with BioRender.com. (FIG. 6B) Heatmap representing chemokine / cytokine expression after i.t. injection of PBS or CPMV. Cytokine expression was measured in tumor extracts using an MSD U-PLEX assay. A protein concentration of 50 pg / well was used for MSD assays. (FIGS. 6C-6F) MSD data of individual chemokines / cytokines. Error bars represent the standard deviation from n=3 biological replicates. Statistical differences were analyzed by analysis of variance (ANOVA) using the software OriginPro with a post hoc Bonferroni test and a significance level of ? = 0.05. Lowercase letters indicate significance groups. Conditions that share the same letter were not significant different (p > 0.05).

[0044] FIGS. 7A - 7B: Schematic representation of size-based protein separation with ultrafiltration. Ultrafiltration is a purification method that uses membranes with a defined molecular weight cut-off to separate proteins based on their size. The method can be readily scaled-up by increasing the membrane area used for filtration. (FIG. 7A) Representative sample composition before separation by ultrafiltration. (FIG. 7B) Representative sample composition after separation by ultrafiltration. The sample fraction that is retained by the ultrafiltration membrane is referred to as retentate, whereas the sample fraction that passes the ultrafiltration membrane is referred to as permeate. If concentrating the sample is not desired, buffer can be fed into the system to maintain a constant retentate volume.

[0045] FIGS. 8A - 8B: CPMV purity and recovery depending on the extraction conditions. (FIG. 8A) Densitometric evaluation of CPMV extracts prepared with acidic, neutral, and alkaline extraction buffers. Blender extracts from CPMV-infected cowpea leaves-13-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 were separated by gel electrophoresis and stained with Coomassie Brilliant Blue before imaging with a FluorChem R system (ProteinSimple). Extracts were prepared from two different batches of leaves; results from the first batch were shown in Figure 1 in the main manuscript. Densitometric analysis was performed with the built-in software of the device. (FIG. 8B) Comparison of CPMV purities and recoveries for different extraction conditions. Error bars represent the standard deviation from two different batches of CPMV-infected leaves.

[0046] FIG. 9: Regeneration of ion exchange chromatography resins after purification of CPMV from plant extracts. Chromatography was performed with and AKTApure system, using 1.0 mL columns, HiTrap Q Sepharose Fast Flow resin, and a flowrate of 0.5 mL / min. Chromatography resins were regenerated by washing with 5 column volumes of 0.5 M sodium hydroxide and 1.0 M sodium chloride (in 100 mM potassium phosphate buffer, pH 7.0). Individually, neither 0.5 M sodium hydroxide nor 1.0 M sodium chloride (in 100 mM potassium phosphate buffer, pH 7.0) were sufficient to regenerate ion exchange resins. Only sequential washing with 0.5 M sodium hydroxide and 1.0 M sodium chloride (in this order) completely regenerated the chromatography resin.

[0047] FIGS. 10A - 10B: Comparison of different ultrafiltration membranes for purification of CPMV. Clarified extracts from CPMV infected cowpea leaves were subjected to ultrafiltration using poly ether sulfone membranes with a 500 kDa (FIG. 10A) and 300 kDa (FIG. 10B) molecular weight cut off. For all ultrafiltration experiments a membrane area of 50 cm2was used with a transmembrane pressure of 0.5 bar and a tangential flowrate of 40 mL / min. Process samples were separated by gel electrophoresis and stained with Coomassie Brilliant Blue before imaging with a FluorChem R system (ProteinSimple). Densitometric analysis was performed with the built-in software of the device. The recovery of CPMV increased from -80% to -90% when switching from a 500 kDa molecular weight cut off to a 300 kDa molecular weight cut off. Retentate volumes were kept constant during sampling to facilitate densitometric analysis of recoveries.10048] FIG. 11: Concentration of p-glucans measured in multiple CPMV preparations purified with the centrifugation method. CPMV preparations were analyzed for the-14-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 presence of P-glucans by the Nanotechnology Characterization Laboratory (NCL). The concentration of 1,3-beta-D-glucans was measured using a Glucatell kit according to the manufacturer’s recommendations. Samples were measured in triplicates.

[0049] FIGS. 12A - 12B: Comparison of the electrophoretic mobility of different CPMV preparations. Analysis of CPMV preparations from the centrifugation process by native gel electrophoresis followed by RNA staining (FIG. 12A) and protein staining (FIG. 12B). The two electrophoretic forms of CPMV, denoted as slow (s) and fast (f), result from cleavage of 24 C-terminal amino acids from the small CPMV coat protein. Additional bands with slower mobility have previously been attributed to aggregation [1], The electrophoretic mobility differed between batches, because the centrifugation process yielded variable CPMV preparations (i.e. mixtures of cleaned and non-cleaved CPMV).

[0050] FIGS. 13A - 13C: Step-wise purification of CCMV using pH-precipitation, UF / DF and ion exchange chromatography. (FIG. 13A) Analysis of blender extracts from CCMV-infected cowpea leaves prepared with a pH of 3.0-6.0. Samples were separated by gel electrophoresis and stained with Coomassie Brilliant Blue. (FIG. 13B) Analysis of process samples from CCMV purification by step-wise pH-precipitation, UF / DF, and ion exchange chromatography. CCMV-infected leaves were extracted at pH 4.0. Ultrafiltration was performed with a transmembrane pressure of 0.5 bar, using 1000 kDa and 300 kDa membranes to remove impurities that are larger and smaller than CCMV. Chromatography was performed with an AKTApure system, using prepacked 1.0 mL HiTrap Q Sepharose Fast Flow columns and a flow rate of 0.5 mL / min. Process samples were separated by gel electrophoresis and stained with Coomassie Brilliant Blue. (FIG. 13C) Chromatogram from the purification of CCMV by anion exchange chromatography. Black arrows denote the CCMV coat protein. Elu = elution, FT = flowthrough, + = positive control.

[0051] FIGS. 14A - 14B: Process flow diagram of CCMV purification with the UF / DF process in comparison with the centrifugation process. (FIG. 14A) Purification of CCMV with the centrifugation process. The contemporary method for purification of CCMV is a 3-day process that relies on toxic organic solvents, ultracentrifugation and isopycnic ultracentrifugation to remove impurities, which is difficult to scale. (FIG. 14B) Scalable-15-4911-2869-8997.1Atty. Dkt. No.: 114198-3560CCMV purification with the UF / DF process. In the UF / DF process ultracentrifugation has been omitted through two consecutive ultrafiltration steps. Specifically, a 1000 kDa MWCO membrane is used to remove impurities that are larger than CPMV. A 300 kDa membrane is used to remove impurities that are smaller than CPMV, alongside bulk water.

[0052] FIGS. 15A - 15H: Characterization of CCMV preparations purified via UF / DF or centrifugation method. (FIG. 15A-15C) Analysis of CCMV preparations from the centrifugation process and the UF / DF process by denaturing (FIG. 15A) and native (FIG. 15B, FIG. 15C) gel electrophoresis. Coomassie Brilliant Blue was used to stain protein, and GelRed was used to stain RNA. (FIG. 15D) TEM images of negatively stained (2% w / v uranyl acetate) CCMV from the same process. (FIG. 15E, FIG. 15F) Analysis of CCMV preparation from the centrifugation process (FIG. 15E) and the UF / DF process (FIG. 15F) by size exclusion chromatography (SEC). SEC was performed with a Superose 6 Increase column, using 0.1 M sodium acetate buffer, containing 0.01 M EDTA (pH 5.5). (FIG. 15G, FIG. 15H) Characterization of CCMV preparations from the centrifugation process (FIG. 15G) and the UF / DF process (FIG. 15H) by dynamic light scattering (DLS). DLS was performed with a Zetasizer Nano, using 0.1 M sodium acetate buffer, containing 0.01 M EDTA (pH 5.5). Sizes are reported as the mean of three technical replicates with 12 analytical replicates each. Black arrows denote the CCMV coat protein. PDI - poly dispersity index.

[0053] FIGS. 16A - 16H: Comparison of UF / DF and centrifugation for single-step purification of CCMV conjugation reactions. (FIGS. 16A-16C) Denaturing gel electrophoresis comparing native CCMV and CCMV-FITC purified by UF / DF or centrifugation. Protein was stained with Coomassie Brilliant Blue and detected at 302 / 590 nm excitation / emission, RNA was stained with GelRed and detected at 302 / 590 nm, the fluorophore was detected at 475 / 537 nm. (FIGS. 16D-16E) Native agarose gel electrophoresis comparing the same samples. Protein, RNA and the fluorophore were detected as described above; the images showing RNA and the fluorophore were overlaid. Black arrows denote the CCMV coat protein. (FIG. 16F) Recovery of CCMV-FITC after purification by UF / DF or centrifugation. The recovery of CCMV in each setting was determined by UV-Vis analysis, using the specific extinction coefficient of ECCMV = 5.85-16-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 mg / mL / cm. (FIGS. 16G-16H) SEC elution profiles of CCMV-FITC purified by centrifugation (FIG. 16G) or UF / DF (FIG. 16H). SEC was performed with a Superose 6 Increase column, using 0.1 M sodium acetate buffer, containing 0.01 M EDTA (pH 5.5). It should be noted that FITC is strongly quenched under acidic conditions

[0046] ,

[0054] FIGS. 17A - 17B: Comparison of different ultrafiltration membranes for purification of CCMV. Clarified extracts from CCMV infected cowpea leaves were subjected to ultrafiltration using poly ether sulfone membranes with a 500 kDa (FIG. 17A) and 300 kDa (FIG. 17B) molecular weight cut off. Experiments were carried out with a membrane area of 50 cm2, a transmembrane pressure of 0.5 bar and a tangential flowrate of 40 mL / min. Process samples were separated by gel electrophoresis and stained with Coomassie Brilliant Blue before imaging with a FluorChem R system (ProteinSimple). Densitometric analysis was performed with the built-in software of the device. The recovery of CCMV increased from <50% to >90% when switching from a 500 kDa molecular weight cut off to a 300 kDa molecular weight cut off. Black arrows denote the CCMV coat protein. + = CCMV positive control, UF / DF = ultrafiltration / diafiltration.

[0055] FIGS. 18A - 18B: Adaption of a scalable plant virus purification strategy based on stability, size and charge for CCMV. (FIG. 18A) Scalable purification of CPMV by utilizing the stability, size and charge of the plant virus for its purification [1], (FIG. 18B) Adaption of the same purification principle for CCMV. Unlike CPMV, which has a broad pH stability, CCMV disassembles under neutral and alkaline conditions and at high ionic strength. To accommodates these limitations, an updated scalable purification process was developed for CCMV. AEX = anion exchange chromatography, UF / DF = ultrafiltrati on / di afiltrati on .

[0056] FIGS. 19A - 19C: SEC elution profile of CCMV-FITC conjugation reactions before purification. CCMV-FITC conjugation reactions were analyzed by SEC using a Superose 6 Increase column and 100 mM sodium acetate buffer, containing 10 mM EDTA (pH 5.5). It should be noted that FITC is strongly quenched under acidic conditions [2], (FIGS. 19B-19C) Regeneration of the chromatography resin. Free FITC interacted strongly with the chromatography resin and did not elute. The fluorescence of FITC was strongly-17-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 quenched under acidic conditions (FIG. 19B), but was restored when replacing the acidic sodium acetate buffer with neutral PBS buffer (FIG. 19C). Black arrows denote free FITC.

[0057] FIGS. 20A - 20D: Strategy for non-disruptive CPMV extraction from leaves and analysis of apoplast eluates. (FIG. 20A). Elution of CPMV from the apoplast with the infiltration-centrifugation method. Leaves are submerged in buffer and subjected to vacuum to infiltrate the apoplastic space with buffer. The infiltrated buffer is recovered by centrifugation. Leaf cross sections are not drawn to scale. (FIGS. 20B-20C) Analysis of apoplast eluates and blender extracts by SDS-PAGE and staining with Coomassie Brilliant Blue (FIG. 20B) or western blotting (FIG. 20C). Apoplast eluates were collected from CPMV-infected plants and non-infected control plants. Rabbit anti-CPMV antibodies and HRP-labeled goat anti-rabbit antibodies were used for western blotting. Black arrows denote the large and small CPMV coat protein. (FIG. 20D) TEM images of apoplast eluates. Grids were negative-stained with 2.0% (w / v) uranyl acetate.

[0058] FIGS. 21A - 21D: Setup for simulating microgravity on the ground using a customized random positioning machine. (FIG. 21A, FIG. 21B) Plants were grown in stone wool blocks to simulate growth conditions on space crafts. Stone wool blocks were encased in 25x25x40 mm (L, W, H) plastic molds to simulate root zone hypoxia. (FIG. 21C) A water reservoir was attached to the plastic mold for irrigation. (FIG. 21D) The random positioning machine was housed in a walk-in growth chamber to allow precise control of temperature, humidity and light intensity during experiments.

[0059] FIGS. 22A - 22F: Effect of simulated space conditions on the plant host. (FIGS. 22A-22B) Simulation of microgravity with a random positioning machine. Local gravity vectors (FIG. 22A) were measured with an accelerometer; the mean gravity (FIG. 22B) was calculated from the local gravity vectors. (FIG. 22C, FIG. 22D) Plant morphology under simulated space conditions. Representative images of N. benthamiana (FIG. 22C) and black- eyed peas (FIG. 22D) after 5 days under simulated space conditions. (FIG. 22E, FIG. 22F) Plant physiology under simulated space conditions. The chlorophyll content was measured in primary (FIG. 22E) and secondary leaves (FIG. 22F) of wildtype and CPMV-infected black- eyed peas after 14 days under simulated space conditions. ROS treatment started 2 days-18-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 before infecting primary leaves with CPMV and continued until 2 days post infection (dpi) for a short-term treatment group and until 14 dpi for a long-term treatment group. ROS+= short-term ROS stress (2 days), ROS++= long-term ROS stress (14 days).

[0060] FIGS. 23A - 23B: Effect of simulated space conditions on CPMV. (FIG. 23A) Effect of short-term and long-term ROS stress on CPMV accumulation in primary leaves. ROS treatment started 2 days before infecting primary leaves with CPMV and continued until 2 days post infection (dpi) for a short-term treatment group and until 14 dpi for a long-term treatment group. Blender extracts were analyzed by gel electrophoresis and staining with Coomassie Brilliant Blue. CPMV was quantified with a commercial DAS-ELISA kit. (FIG. 23B) CPMV accumulation in secondary leaves from the same experiment. Letters indicate significance groups. Conditions that share the same letter were not significant different (p>0.05). ROS' = no ROS stress, ROS+= short-term ROS stress, ROS++= long-term ROS stress.

[0061] FIGS. 24A - 24G: Process scaleup, optimization and characterization. (FIG.24A) Process flow diagram for simplified purification of CPMV from plants using infiltration-centrifugation and UF / DF. (FIG. 24B) Analysis of process samples from the same process. Samples were separated by gel electrophoresis and stained with Coomassie Brilliant Blue. UF / DF retentates are concentrated two-fold compared to the other process samples. (FIG. 24C, FIG. 24D) Analysis of apoplast eluates from CPMV infected black- eyed peas using alkaline, neutral and acidic buffers by denaturing gel electrophoresis (FIG. 24C) and staining with Coomassie Brilliant Blue. Gels were imaged with a FluorChem R system (ProteinSimple) and densitometric analysis (FIG. 24D) was performed with the onboard software of the device. (FIG. 24E) Native gel electrophoresis of CPMV preparations followed by RNA staining (left) and protein staining (right), Lane 1 = Control CPMV purified from blender extracts; Lane 2 = empty, Lane 3 = CPMV purified from apoplast eluates. (FIG. 24F) Size exclusion chromatography and (FIG. 24G) TEM image of CPMV purified from apoplast eluates (CPMV was negatively stained with uranyl acetate for TEM imaging).-19-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0062] FIG. 25: Tracing of infiltration solution in leaves. Leaves of 1-week old black-eyed peas were infiltrated with deionized water containing 0.2 mg / mL cadmium selenide zinc sulfide (CdSe / ZnS) quantum dots (QDs) with a particle size of 20-22 nm and an emission peak of 450 nm. Infiltrated leaf sections from were placed onto glass slides and sealed with a 12 mm circular cover glass and nail polish. Slides were imaged using an AIR Confocal microscope and N-STORM Super-Resolution System at 60 / magnification. The signals of Chlorophyll (red) and carbon dots (blue) did not overlap, indicating that the plant tissue was not damaged.

[0063] FIGS. 26A - 26C: Effect of the incubation temperature on CPMV infections.(FIG. 26A) Symptoms of CPMV infection in primary leaves of black-eyed peas. Plants were incubated under regular gravity at 25°C after infection with CPMV. (FIG. 26B, FIG. 26C) Representative image of CPMV-infected black-eyed peas grown under simulated microgravity at 20°C (FIG. 26B) or regular gravity at 20°C (FIG. 26C). All plants were imaged 1 week after infection with CPMV. Plants that were incubated at 20°C after CPMV infection did not develop symptoms of viral infection.FIGS. 27A - 27D: Evaluation of heat treatment as an alternative pretreatment method using potato virus X (PVX) as a model VLP. (FIG. 27A) SDS-PAGE analysis of process samples after heat treatment by blanching; intact Nicotiana benthamiana leaves expressing PVX were submerged in phosphate buffered saline (PBS) and incubated at 55-60C for 5-10 minutes. After heat treatment, leaves were extracted in a blender and insoluble particles were removed by centrifugation (16000 x g for 30 min at 4C). Electrophoresis was performed with 4-12% Bis-Tris gels in MOPS buffer at 200 V for 45 min. After gel electrophoresis, proteins were stained with Coomassie Brilliant Blue and detected at 302 / 590 nm. Arrows indicate the coat protein of PVX. (FIG. 27B) Western blot analysis of the same samples as in (FIG. 27A) using rabbit anti-PVX antibodies and HRP-conjugated goat anti-rabbit antibodies. PVX tolerated blanching temperatures up to 60C for 5-10 minutes, whereas -80% of plant host cell proteins were removed under these conditions. (FIG. 27C) Transmission electron microscopy (TEM) images of PVX that was purified from leaves after heat treatment at 60C for 5 minutes. Purified TMV was loaded onto 400-mesh formvar / carbon supported copper grids (Electron Microscopy Sciences), and grids were charged with a PELCO EasiGlow system -20-4911-2869-8997.1Atty. Dkt. No.: 114198-3560(Ted Pella) and negative-stained with 2.0% (w / v) uranyl acetate (Agar Scientific). Grids were imaged using a JEM-1400 Plus electron microscope (Jeol Ltd.) at 15 000* magnification. (FIG. 27D) TEM images of PVX that was purified from leaves that were not subjected to heat treatment. Grids were prepared and imaged as in (FIG. 27C).DETAILED DESCRIPTION

[0064] The novel viral harvesting methods described herein use physical properties that are common in virus like particles (VLPs), namely high stability (e.g. towards extreme pH values or high temperature), large size compared to plant host cell proteins, and either positive or negative charge. This means that the production process can be easily transferred to other plant viruses with little modifications.

[0065] While some VLPs have been purified by ion exchange chromatography or ultrafiltration before, the combination of (i) acidic extraction, (ii) sequential ultrafiltration with different membranes and (iii) ion exchange chromatography is new. Additionally, plantbased expression systems are used, whereas mammalian cells or bacteria are used in the literature - plants offer an enhanced safety profile because they don’t support the replication of human pathogens.

[0066] Definitions[0067| As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting or separating or both limiting and separating the subject matter described.

[0068] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure in their entireties to more fully describe the state of the art to which this invention pertains. In some aspect, technical publications are referenced by a number making reference to a publication, the full bibliographic citation for the publication is found immediately preceding the claims.-21-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0069] This technology uses standard methods from organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA unless stated otherwise. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2ndedition (1989); Current Protocols In Molecular Biology (F. M. Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)).10070] As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.[00711 As used herein, the term “comprising” is intended to mean that the compounds, compositions and methods include the recited elements but not exclude others. “Consisting essentially of’ when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Alternatively, “consisting essentially of’ active agents intends that only the recited active agents are the sole active agents to achieve one or more diagnostic or therapeutic uses. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.

[0072] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0073] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).-22-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0074] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1, 5, or 10%. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0075] As used herein, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. The term “about” when used before a numerical designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations which may vary by (+) or (-) 15%, 10%, 5%, 3%, 2%, or 1 %.

[0076] “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0077] As used herein, the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term “mammal” includes both human and non-human mammals.[0078J The term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals including cell and tissue types. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be at any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human.

[0079] A “composition” as used herein, refers to an active agent, such as a compound as disclosed herein and a carrier, inert or active. The carrier can be, without limitation, solid such as a bead or resin, or liquid, such as phosphate buffered saline.-23-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0080] Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetraoligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid / antibody components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.

[0081] A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0082] “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text -24-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

[0083] The compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.

[0084] An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the functionalizing or as disclosed herein for any particular subject depends upon a variety of factors including the activity of the specific compound employed, bioavailability of the compound, the route of administration, the age of the animal and its body weight, general health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. In general, one will desire to administer an amount of the active agent or compound that is effective to achieve a serum level commensurate with the concentrations-25-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 found to be effective in vivo. These considerations, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks.

[0085] The term “contacting” means direct or indirect binding or interaction between two or more. A particular example of direct interaction is binding. A particular example of indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.

[0086] As used herein, the term “plant virus” includes viruses that infect plants or plant systems, e.g., leaves, root and / or stems. Plant viruses can be stably stored (and are stable without cold chain requirements). Plant viruses do not infect or replicate in mammalian cells, thus adding another layer of safety compared to oncolytic viral therapies. Non-limiting examples include tobacco mosaic virus (TMV), tobacco mold green mottle virus (TMGMV), physalis mottle virus like particle (PhMV), cowpea chlorotic mottle virus (CCMV), and cowpea mosaic virus (CPMV). Methods of replicating and producing viruses for therapeutic application are known in the art and described in WO 2022 / 221692, published October 20, 2022, and incorporated herein by reference.

[0087] The “apoplasf ’ is the network of cell walls and intercellular spaces in plants that allows for the movement of water, ions and small molecules outside the plasma membrane. It consist of non-living parts of the plants.

[0088] As used herein, the term “plant virus nanoparticle,” “plant viral nanoparticle” or “VLP” refers to a non-replicating, viral shell, derived from one or more plant viruses (e.g., one or more plant viruses described herein). VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from these proteins. As used herein, “capsid protein” and “coat protein” are used interchangeably.VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. VLPs can also be engineered, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more viral proteins that comprise, or consists-26-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 essentially of, or yet further consists of, a modification. Methods for producing VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. Further, VLPs can be isolated by known techniques, e.g., density gradient centrifugation and identified by characteristic density banding. See, for example, Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Viral. 68:4503-4505; Vincente, J Invertebr Pathol., 2011; Schneider Ohrum and Ross, Curr. Top. Microbial. Immunol., 354: 53073, 2012). As used herein, VLP intends naturally occurring (wild-type or native) VLP and engineered VLP, unless explicitly stated otherwise.[0089| In some embodiments, the plant virus nanoparticle is a virus belonging to the order Picornavirales (i.e. plant picornavirus). A plant picomavirus is a virus belonging to the family Secoviridae, which together with mammalian picornaviruses belong to the order of the Picornavirales. Plant picornaviruses are relatively small, nonenveloped, positive- stranded RNA viruses with an icosahedral capsid. In some embodiments, the virus particles are selected from the Comovirinae virus subfamily. The Comovirinae subfamily comprises over 60 species across at least 3 genera (Comovirus, Fabavirus, Nepovirus). Some Picornavirales have a monopartite genome, while others have a bipartite genome.

[0090] Exemplary plant viruses from the Comovirinae subfamily include, but are not limited to Broad bean wilt virus 1, Tobacco ringspot virus, Cowpea severe mosaic virus, Turnip ringspot virus, Broad bean wilt virus 2, Bean pod mottle virus (BPMV), Potato virus B, Tomato ringspot virus, and Rice tungro spherical virus. Additional information about plant picornaviruses can be found in Zell et al (2017) Journal of General Virology, 98: 2421-2422.10091] In a further embodiment, the virus particles are from the genus Comovirus. A preferred example of a comovirus is the cowpea mosaic virus particles. Other suitable plant virus includes, but is not limited to bean pod mottle virus (BPMV), cowpea severe mosaic virus or rice tungro spherical virus.

[0092] In some embodiments, the plant virus nanoparticle or VLP is derived from Cowpea mosaic virus (CPMV). CPMV is a non-enveloped plant virus that belongs to the Comovirus-27-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 genus. CPMV strains include, but are not limited to, SB (Agrawal, H.O. (1964). Meded. Landb. Hoogesch. Wagen. 64: 1) and Vu (Agrawal, H.O. (1964). Meded. Landb. Hoogesch. Wagen. 64: 1). Cowpea mosaic virus (CPMV) is a VLP and a plant-infecting member of the order Picornavirales, with a relatively simple, non-enveloped capsid that has been extensively studied and a positive-sense, single-stranded RNA genome. For CPMV, the genome is bipartite, with RNA-1 (6 kb) and RNA-2 (3.5 kb) being separately encapsidated. CPMV has an icosahedral capsid structure, which is ~30 nm in diameter and is formed from 60 copies each of a Large (L) and Small (S) coat protein. These two coat proteins are processed from a single RNA-2-encoded precursor polyprotein (VP60) by the action of the 24 K viral proteinase which is encoded by RNA-1. Thus, capsid assembly, as well as viral infection, is dependent on the presence of both genomic segments in an infected plant cell. In some embodiments, the VLP particles have been treated, prepared and / or inactivated by methods known in the art. In some instances, CPMV produces a large capsid protein and a small capsid protein precursor (which generates a mature small capsid protein). In some cases, CPMV capsid is formed from a plurality of large capsid proteins and mature small capsid proteins. In some cases, the large capsid protein is a wild-type large capsid protein, optionally expressed by SB or Vu strain. In other instances, the large capsid protein is a modified large capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the large capsid protein comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03599 (residues 460-833):|0093] MEQNLFALSLDDTSSVRGSLLDTKFAQTRVLLSKAMAGGDVLLDEYLYDVV NGQDFRATVAFLRTHVITGKIKVTATTNISDNSGCCLMLAINSGVRGKYSTDVYTICS QDSMTWNPGCKKNFSFTFNPNPCGDSWSAEMISRSRVRMTVICVSGWTLSPTTDVIA KLDWSIVNEKCEPTIYHLADCQNWLPLNRWMGKLTFPQGVTSEVRRMPLSIGGGAG ATQAFLANMPNSWISMWRYFRGELHFEVTKMSSPYIKATVTFLIAFGNLSDAFGFYE SFPHRIVQFAEVEEKCTLVFSQQEFVTAWSTQVNPRTTLEADGCPYLYAIIHDSTTGTI SGDFNLGVKLVGIKDFCGIGSNPGIDGSRLLGAIAQ (SEQ ID NO: 1), or an equivalent thereof.-28-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0094] In some cases, the mature small capsid protein is a wild-type mature small capsid protein, optionally expressed by SB or Vu strain. In other instances, the mature small capsid protein is a modified mature small capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the mature small capsid protein comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03599 (residues 834- 1022):|0095] GP VCAEASD VYSPCMIASTPPAPF SD VTAVTFDLINGKITPVGDDNWNTHIYN PPIMNVLRTAAWKSGTIHVQLNVRGAGVKRADWDGQVFVYLRQSMNPESYDARTF VISQPGSAMLNFSFDIIGPNSGFEFAESPWANQTTWYLECVATNPRQIQQFEVNMRFD PNFRVAGNILMPPFPLSTETPPL (SEQ ID NO: 2), or an equivalent thereof.[0096| In some embodiments, the plant virus nanoparticle or VLP is derived from Cowpea chlorotic mottle virus (CCMV). CCMV is a spherical plant virus that belongs to the Bromovirus genus. Several strains have been identified and include, but not limited to, Carl (Ali, et al., 2007. J. Virological Methods 141 :84-86), Car2 (Ali, et al., 2007. J. Virological Methods 141 :84-86, 2007), type T (Kuhn, 1964. Phytopathology 54:1441-1442), soybean (S) (Kuhn, 1968. Phytopathology 58: 1441-1442), mild (M) (Kuhn, 1979. Phytopathology 69:621-624), Arkansas (A) (Fulton, et al., 1975. Phytopathology 65: 741-742), bean yellow stipple (BYS) (Fulton, et al., 1975. Phytopathology 65: 741-742), R (Sinclair, ed. 1982. Compendium of Soybean Diseases. 2nded. The American Phytopathological Society, St. Paul. 104 pp.), and PSM (Paguio, et al., 1988. Plant Diseases 72(9): 768-770).

[0097] In some instances, the plant virus nanoparticle or VLP from CCMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type CCMV capsid, optionally expressed by Carl, Car2, type T, soybean (S), mild (M), Arkansas (A), bean yellow stipple (BYS), R, or PSM strain. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the CCMV capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03601 :-29-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0098] MSTVGTGKLTRAQRRAAARKNKRNTRVVQPVIVEPIASGQGKAIKAWTGYS VSKWTASCAAAEAKVTSAITISLPNELSSERNKQLKVGRVLLWLGLLPSVSGTVKSC VTETQTTAAASFQVALAVADNSKDVVAAMYPEAFKGITLEQLTADLTIYLYSSAALT EGDVIVHLEVEHVRPTFDDSFTPVY (SEQ ID NO: 3), or an equivalent thereof.

[0099] In some cases, the virus or VLP from CCMV is prepared by the method as described in Ali et al., “Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation,” Journal of Virological Methods 141 : 84-86 (2007).

[0100] In some embodiments, the plant virus nanoparticle or VLP is derived from Physalis mottle virus (PhMV). Physalis mottle virus (PhMV) is a +ssRNA virus from the family Tymoviridae that forms a ~30 nm-sized icosahedral capsid from 180 identical capsid proteins CPs, and can be recombinantly expressed and purified as a homogenous and stable VLP. In some instances, the plant virus nanoparticle or VLP from PhMV comprises, or consists essentially of, or yet further consists of, a plurality of coat proteins.

[0101] In some instances, the coat protein is a wild-type PhMV coat protein. In other instances, the coat protein is a modified coat protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the PhMV coat protein comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P36351 : MDSSEVVKVKQASIPAPGSILSQPNTEQSPAIVLPFQFEATTFGTAETAAQVSLQTADP ITKLTAPYRHAQIVECKAILTPTDLAVSNPLTVYLAWVPANSPATPTQILRVYGGQSF VLGGAISAAKTIEVPLNLDSVNRMLKDSVTYTDTPKLLAYSRAPTNPSKIPTASIQISG RIRLSKPMLIAN (SEQ ID NO: 4), or an equivalent of each thereof.

[0102] In some cases, the gene encoding the PhMV coat protein comprises or consists essentially of, or yet further consists of, the sequence as set forth in NC_003634.1 :5958- 6524 CP [organism=Physalis mottle virus] [NCBI GeneID=940246] : ATGGACTCTTCGGAAGTTGTCAAAGTCAAGCAGGCCTCCATCCCCGCCCCTGGCT CCATTCTCTCCCAGCCCAACACAGAACAATCACCTGCCATAGTTCTCCCTTTTCA GTTTGAAGCCACTACTTTCGGCACCGCTGAAACCGCAGCCCAAGTCTCTCTCCAGACTGCCGACCCCATTACCAAACTGACCGCCCCCTACCGACATGCTCAGATCGTCG-30-4911-2869-8997.1Atty. Dkt. No.: 114198-3560AGTGCAAAGCTATCCTCACTCCAACTGATCTTGCTGTCTCCAATCCCCTCACAGT CTACCTAGCATGGGTCCCCGCCAACTCCCCTGCCACTCCGACTCAAATACTGCGA GTCTACGGCGGTCAGTCTTTTGTTCTTGGCGGCGCCATCTCAGCCGCCAAAACCA TTGAGGTCCCCCTCAATCTTGACTCTGTCAACCGCATGTTGAAAGACAGCGTGAC CTACACTGACACCCCCAAGCTCCTTGCCTACTCAAGAGCCCCCACCAACCCCTCG AAAATCCCAACCGCTAGTATTCAGATCAGCGGTCGCATTCGGCTCTCCAAGCCAA TGCTGATAGCCAACTAA (SEQ ID NO: 5), or an equivalent thereof.

[0103] In some embodiments, the plant virus nanoparticle or VLP is derived from Sesbania mosaic virus (SeMV). SeMV is a positive stranded RNA virus that belongs to the genus Sobemovirus. In some instances, the virus or VLP from SeMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type SeMV capsid protein. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the SeMV capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID Q9EB06:

[0104] MAKRLSKQQLAKAIANTLETPPQPKAGRRRNRRRQRSAVQQLQPTQAGISM APSAQGAMVRIRNPAVSSSRGGITVLTHSELSAEIGVTDSIVVSSELVMPYTVGTWLR GVAANWSKYSWLSVRYTYIPSCPSSTAGSIHMGFQYDMADTVPVSVNQLSNLRGYV SGQVWSGSAGLCFINGTRCSDTSTAISTTLDVSKLGKKWYPYKTSADYATAVGVDV NIATPLVPARLVIALLDGSSSTAVAAGRIYCTYTIQMIEPTASALNN (SEQ ID NO: 6), or an equivalent thereof.

[0105] In some embodiments, the plant virus nanoparticle or VLP is derived from Tobacco Mosaic Virus (TMV). It is a member of the Virgaviridae family. This virus is among the first if not the first characterized virus and has been utilized as a model virus for decades for life science applications.

[0106] TMV has a rod-like appearance. Its capsid is made from 2130 molecules of coat protein and one molecule of genomic single strand RNA, 6400 bases long. The coat protein self-assembles into the rod-like helical structure (16.3 proteins per helix turn) around the-31-4911-2869-8997.1Atty. Dkt. No.: 114198-3560RNA, which forms a hairpin loop structure. The protein monomer consists of 158 amino acids which are assembled into four main alpha-helices, which are joined by a prominent loop proximal to the axis of the virion. Virions are -300 nm in length and -18 nm in diameter. The RNA is located at a radius of -4 nm and is protected from the action of cellular enzymes by the coat protein. X-ray fiber diffraction structure of the intact virus was studied based on an electron density map at 3.6 A resolution. Inside the capsid helix, near the core, is the coiled RNA molecule, which is made up of 6,395 ±10 nucleotides.

[0107] In some instances, the capsid protein is a wild-type TMV capsid protein. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some embodiments, a TMV coat protein comprises, or alternatively consists essentially of, or yet further consists of the sequence as set forth in the UniProtKB ID: P69687:MSYSITTPSQFVFLSSAWADPIELINLCTNALGNQFQTQQARTVVQRQFSEVWKPSPQ VTVRFPDSDFKVYRYNAVLDPLVTALLGAFDTRNRIIEVENQANPTTAETLDATRRV DDATVAIRSAINNLIVELIRGTGSYNRSSFESSSGLVWTSGPAT (SEQ ID NO: 7), or an equivalent thereof.

[0108] In some embodiments, the plant virus is Potato Virus X (PVX). PVX belongs to the Potexvirus genus of the family Flexivirida. PVX virions are described as flexible, thread-like bodies measuring approximately 500-515 nanometers in length and 13-15 nanometers in diameter. Each viral particle comprises around 1300-1350 helically folded identical CP subunits enclosing a 6.4 knt viral RNA, with each turn of the primary helix consisting of 8.9 CP subunits. In some instances, the capsid protein is a wild-type PVX capsid protein. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the coat protein comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProt ID Pl 7782:MSAPASTTQATGSTTSTTTKTAGATPATASGLFTIPDGDFFSTARAIVASNAVATNED LSKIEAIWKDMKVPTDTMAQAAWDLVRHCADVGSSAQTEMIDTGPYSNGISRARLA-32-4911-2869-8997.1Atty. Dkt. No.: 114198-3560AAIKEVCTLRQFCMKYAPVVWNWMLTNNSPPANWQAQGFKPEHKFAAFDFFNGVT NPAAIMPKEGLIRPPSEAEMNAAQTAAFVKITKARAQSNDFASLDAAVTRGRITGTT TAEAVVTLPPP (SEQ ID NO: 8).

[0109] As used herein, the term “detectable label” refers to any moiety that, when attached to a biomolecule such as a protein, peptide, nucleic acid, carbohydrate, lipid, or other analyte, permits the presence, location, or quantity of the molecule to be determined by visual, spectroscopic, radiographic, enzymatic, magnetic, or other analytical means. Nonlimiting examples of detectable labels include, without limitation, fluorescent compounds such as fluorescein or Alexa Fluor dyes, chemiluminescent or bioluminescent proteins such as luciferase, chromogenic enzymes such as horseradish peroxidase or alkaline phosphatase, radioisotopes such as32P or125I, and haptens such as biotin that can be detected through specific binding partners. As used herein, the term “purification label” refers to any moiety that, when associated with the molecule of interest, facilitates isolation or enrichment of that molecule from a complex mixture by exploiting a known and highly selective binding interaction with a solid phase or ligand. Purification labels include, without limitation, affinity tags such as a polyhistidine sequence for immobilized metal affinity chromatography, glutathione S-transferase for binding to glutathione matrices, FLAG epitope for immunoaffinity capture, Strep-tag for binding to Strep-Tactin resin, and protein A or protein G domains for binding immunoglobulins. In preferred embodiments, such labels are genetically fused to VLPs and may be removed after detection or purification if desired, for example by proteolytic cleavage or chemical treatment, without substantially affecting the structure or function of the molecule to which they were attached.

[0110] As used herein, the term “purification label” refers to any moiety, domain, sequence, chemical group, or molecular construct that is operatively linked to a molecule of interest and facilitates the selective isolation, enrichment, or recovery of such molecule from a heterogeneous mixture. A purification label achieves this by mediating a specific and high-affinity interaction with a corresponding capture reagent, ligand, or solid phase under conditions that permit binding of the labeled molecule while permitting non-labeled components to be removed. Purification labels include, without limitation, affinity tags such as a polyhistidine sequence for immobilized metal affinity chromatography, glutathione-33-4911-2869-8997.1Atty. Dkt. No.: 114198-3560S-transferase for binding to glutathione-functionalized matrices, FLAG epitope recognized by anti-FLAG antibodies, Strep-tag for specific interaction with Strep-Tactin resin, and protein A or protein G domains for binding the Fc region of immunoglobulins. In certain embodiments, purification labels are incorporated into VLPs or are chemically conjugated to VLPs. In other embodiments, the purification label may be joined to the VLP via a cleavable linker to permit removal of the label after the purification process, such that the recovered molecule retains its desired structure and activity. The term “purification label” encompasses naturally occurring sequences or motifs that confer affinity for a binding partner, as well as synthetically engineered tags optimized for increased stability, binding specificity, or ease of removal.[01111 As used herein, the term “stabilizer” refers to any agent, compound, excipient, or additive that, when incorporated into a composition, enhances or maintains the physical, chemical, or biological integrity of the active ingredient over a desired period of storage or use. A stabilizer may function by preventing or reducing degradation, denaturation, aggregation, oxidation, hydrolysis, precipitation, loss of bioactivity, or other processes that compromise the quality, safety, or efficacy of the composition. Stabilizers include, without limitation, buffers that maintain pH within a desired range, antioxidants such as ascorbic acid, methionine, or tocopherols, chelating agents such as ethylenediaminetetraacetic acid (EDTA) that reduce metal-catalyzed degradation, cryoprotectants and lyoprotectants such as sugars (e.g., sucrose, trehalose), polyols (e.g., mannitol, glycerol), and amino acids (e.g., glycine) that protect biomolecules during freezing, drying, or lyophilization, surfactants such as polysorbates that reduce surface adsorption and aggregation, and specific protein- or polymer-based carriers that stabilize labile macromolecules. In certain embodiments, the stabilizer is selected to be pharmaceutically acceptable and compatible with the intended route of administration and can be present as a single agent or in combination with one or more other stabilizers to achieve a synergistic effect. The term “stabilizer” encompasses agents naturally present in biological fluids, as well as synthetic or modified molecules designed to impart enhanced stability characteristics to the composition.

[0112] As used herein, the term “lyophilization agent” refers to any compound, excipient, or additive that is included in a pharmaceutical or biotechnological formulation to facilitate or-34-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 improve the process of lyophilization (freeze-drying) and / or to enhance the stability, solubility, or bioactivity of the product upon reconstitution. A lyophilization agent may act by protecting the active ingredient, which may be a protein, peptide, nucleic acid, carbohydrate, lipid, live microorganism, or small molecule drug, from structural or functional degradation during freezing and drying, and by promoting recovery of the desired properties upon addition of a suitable diluent. Lyophilization agents include, without limitation, cryoprotectants and lyoprotectants such as disaccharides (e.g., sucrose, trehalose), monosaccharides (e.g., glucose), sugar alcohols (e.g., mannitol, sorbitol, glycerol), polymers such as polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP), and amino acids such as glycine, arginine, or histidine that can form stabilizing interactions with the active ingredient. In certain embodiments, the lyophilization agent serves to maintain cake structure, reduce collapse, and control residual moisture content of the lyophilized product, thereby improving shelf life and reconstitution characteristics. The term “lyophilization agent” also encompasses mixtures of protective agents selected to achieve optimal glass transition temperature, isotonicity, or physical stability, including naturally occurring excipients, chemically modified derivatives, or synthetic compounds specifically engineered for use in lyophilization processes.10113] As used herein, the term “functionalizing agent” or “cargo” refers to any chemical, biochemical, or biological moiety that is associated with, conjugated to, encapsulated within, or otherwise incorporated into a delivery vehicle, carrier, or substrate to confer a desired property, activity, or function. Functionalizing agents include, but are not limited to, detectable labels (such as fluorescein isothiocyanate (FITC)), therapeutic agents (including nucleic acids such as RNA, small interfering RNA (siRNA), inhibitory RNA), peptides, proteins, enzymes, antibodies, and small organic or inorganic molecules. Such agents may provide diagnostic, prognostic, prophylactic, or therapeutic effects, and may act within or outside target cells, tissues, or organisms.

[0114] As used in the methods herein, “eluate” refers to a product of the extraction and filtration steps of the methods described herein. In the context of the filtering solutions through membranes or filters, the eluate may also be considered filtrate as it passes through the membrane, filter or column with the liquid.-35-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0115] As used in the methods herein, “extracting” refers to the process of removing the virus like particles from the plant. For example, an “extraction solution,” is added to the plant or plant tissue, and through physical and / or chemical steps, the VLPs are removed from plant tissue and collected in eluate. Exemplary extraction solutions include water, phosphate buffer saline (PBS), MES (2-(A-morpholino)ethanesulfonic acid) buffer, and KPO4 buffer, sodium acetate buffer, phosphate buffer. Acidic extraction solutions include, but are not limited to, MES and sodium acetate.

[0116] As used herein, the term “harvesting” intends to separate from or isolate a VLP from its native environment.

[0117] As used in the methods herein, “purifying” refers to the process of removing impurities from the VLPs, so that only the VLPs remains. As used herein the terms “purification”, “purifying”, or “separating” refer to the process of isolating one or more VLPs or polypeptides from a complex mixture, such as a cell lysate, host cell proteins, or a mixture of polypeptides. The purification, separation, or isolation need not be complete, i.e., some components of the complex mixture may remain with the one or more polypeptides after the purification process. However, the product of purification should be enriched for the one or more VLP or polypeptides relative to the complex mixture before purification and a significant portion of the other components initially present within the complex mixture should be removed by the purification process.

[0118] As used herein, “ultrafiltration” refers to filtration with membranes with small pore sizes. Membranes for ultrafiltration may range between about 0.01 micron-0.1 micron. The pore sizes for ultrafiltration membranes are commonly characterized with a molecular weight cutoff (MWCO) (Daltons). By way of non-limiting example, ultrafiltration membranes may have a MWCO of 2000kDa, lOOOkDa, 500kDa, 200kDa, 3kDa.

[0119] As used herein, “ultracentrifugation” refers to centrifugation speeds of greater than 60,000xg. Ultracentrifuges generally have rotor speeds ranging from 60,000xg to l,000,000xg. Non-ultracentrifuges have lower rotor speeds than ultracentrifuges.

[0120] As used herein, “microgravity environment” refers to an environment in which people or objects appear to be weightless. A microgravity environment has a very weak gravity. For -36-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 example, there is a virtual absence of gravity in outer space. A microgravity environment can be an environment having gravity less than Ixg.10121] As used herein, the term “administration” refers to the introduction, application, or delivery of a composition, formulation, or active agent to a desired environment or subject so as to permit contact of the VLP or composition with its intended target. “Administration or contacting in vitro” refers to delivering the VLP or composition to an artificial, controlled, or laboratory-based system outside a living organism, including but not limited to cultured cells, tissue explants, organoids, isolated biological fluids, purified biomolecules, reaction mixtures, bioreactor cultures, or diagnostic assay systems. By way of example, in vitro administration may comprise adding the VLP to a cell culture medium to determine efficacy, dosage or the delivery of an agent such as a therapeutic agent. “Administration in vivo” refers to delivering the composition to a living organism, including humans, non-human animals, plants, or other multicellular biological systems, by any effective route that achieves a desired local or systemic exposure. In vivo administration encompasses, without limitation, parenteral routes such as intravenous, intramuscular, subcutaneous, or intraperitoneal injection; enteral routes such as oral or intragastric delivery; topical routes such as dermal or mucosal application; inhalation into the respiratory tract; or implantation of sustained-release devices. In certain embodiments, administration in vivo may be performed for therapeutic, prophylactic, diagnostic, or experimental purposes, while in vitro administration may be used for screening, mechanistic investigation, formulation testing, or quality control. The terms “in vitro administration” and “in vivo administration” are intended to encompass both single and repeated delivery events, and may include the co-administration of multiple compositions or agents, simultaneous or sequentially, to achieve a desired effect.

[0122] Modes for Carrying out the Disclosure

[0123] Methods of Harvesting VLPs

[0124] Described herein are methods of harvesting virus-like particles (VLPs) from plant tissue.

[0125] According to one embodiment, the method comprises, or consists essentially of, or yet further consists of: (i) extracting the VLPs from the plant tissue using an extraction solution,-37-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 wherein the VLPs are collected in the eluate; (ii) purifying the VLPs collected in the extraction solution of step (i) through a series of separations based on size; and (iii) purifying the VLPs from step (ii) through separation based on charge. In one embodiment, the VLPs are harvested at room temperature. In another embodiment the VLPs are harvested at high heat, for example at about 50°C, at about 55°C, at about 60°C, at about 65°C, at about 70°C, at about 80°C, at about 90°C, at about 100°C, or over 100°C. In one aspect, the extraction solution has a pH of the isoelectric point of the VLP. The isoelectric point of the VLP is the pH where the VLP does not carry a net electrical charge. The isoelectric point, of CPMV, for example is approximately a pH of 4.

[0126] According to one embodiment, the method comprises, or consists essentially of, or yet further consists of: (i) incubating the plant tissue in a heated extraction solution; (ii) collecting the VLPs in the extraction solution; (ii) purifying the VLPs collected in the extraction solution of step (ii) through a series of separations based on size; and (iii) purifying the VLPs from step (ii) through separation based on charge. In one aspect, the heated extraction solution is at about 50°C, at about 55°C, at about 60°C, at about 65°C, at about 70°C, at about 80°C, at about 90°C, at about 100°C, or over 100°C.

[0127] According to another embodiment, the method comprises, or consists essentially of, or yet further consists of: (i) extracting the VLPs from the plant tissue using an acidic extraction solution collecting the VLP-containing eluate; (ii) filtering the eluate from step (i) through a series of filtration membranes with serially decreasing pore size and collecting a the VLPs, wherein the VLPs are collected in the eluate or the membrane; and (iii) separating the VLPs from the filtration of step (ii) with ion exchange chromatography to harvest the VLPs. In one aspect, the extraction solution has a pH of the isoelectric point of the VLP. In some embodiments, the extraction solution has a pH of 4.0. In some embodiments the extraction solution has a pH of approximately 3.5 to 4.5, or alternatively approximately 4.0. In another embodiment, the VLP is CPMV and the extraction solution has a pH of approximately 3.5 to 4.5, or alternatively approximately about 4.0. In one aspect, the ion exchange chromatography is anion exchange chromatography. In another aspect, the ion exchange chromatography is cation exchange chromatography. In one aspect, step (ii) of the-38-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 method takes place at about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about room temperature.

[0128] According to yet another embodiment, the method comprises, or consists essentially of, or yet further consists of: (i) extracting the VLPs from plant tissue using a basic extraction solution collecting the VLP-containing first eluate; (ii) filtering the first eluate from step (i) with a series of filtration membranes with serially decreasing pore size and collecting the VLPs, wherein the VLPs are collected in the eluate or the membrane; and (iii) separating the VLPs from the filtration of step (ii) with ion exchange chromatography, thereby harvesting the VLPs. In one aspect, the ion exchange chromatography is anion exchange chromatography. In another aspect, the ion exchange chromatography is cation exchange chromatography. In some embodiments the extraction solution has a pH of approximately 8.5 to 9.5, or alternatively approximately 9.0. In another embodiment, the VLP is CPMV and the extraction solution has a pH of approximately 8.5 to 9.5, or alternatively approximately about 9.0. In one aspect, the extraction solution has a pH of the isoelectric point of the VLP. In one aspect, step (ii) of the method takes place at about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about room temperature.

[0129] In some embodiments, the VLP are extracted from an intact plant in step (i) by vacuum infiltrating an apoplast with the extraction solution and collecting the extraction solution with centrifugation.

[0130] In the methods described herein, in some embodiments the series of membranes comprises a first membrane and a second membrane. In some embodiments the series of membrane comprises a first membrane, a second membrane, and a third membrane. In some embodiments the series of membranes includes greater than three membranes. In one aspect, the first and second membrane pore size are larger than the diameter of the VLP and the third membrane pore size is smaller than the diameter of the VLP.

[0131] The size of VLPs harvested can be increased or decreased by the selection of membranes for filtration. According to one embodiment, the first membrane has a pore size of about 0.1 pm to about 0.4 pm, optionally about 0.2 pm, and the second membrane has a molecular weight cut-off of about 2000 kDa to about lOOOkDa, optionally selected from-39-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 about 2000kDa, about 1500kDa, about 1250kDa, or about lOOOkDa. According to one embodiment, the first membrane has a pore size of about 0.1 pm to about 0.4 pm, optionally about 0.2 pm, and the second membrane has a molecular weight cut-off of about lOOkDa to about 600kDa, about 300 kDa to about 500kDa, optionally selected from about 300kDa, about 400kDa, about 500kDa.

[0132] According to one embodiment, the first membrane has a pore size of about 0.1 pm to about 0.4 pm, optionally about 0.2 pm, the second membrane has a molecular weight cut-off of about 2,000 kDa to about lOOOkDa, optionally selected from about 2000kDa, about 1500kDa, about 1250kDa, or about lOOOkDa, and the third membrane has a molecular weight cut-off selected from of about lOOkDa to about 600kDa, about 300kDa to about 500kDa, or about 300kDa, about 400kDa, or about 500kDa.According to yet another embodiment, the method comprises: (i) extracting the VLPs from the plant tissue using an acidic extraction solution and collecting the VLPs in a first eluate; (ii) filtering the VLPs from the first eluate of step (i) with a first membrane having a pore size of about 0.20 pm to about 0.2 pm 0.22 pm, optionally about 0.22 pm and collecting a second eluate; (iii) filtering the VLPs from the second eluate of step (ii) with a second membrane, having a molecular weight cutoff of about 800 kDa to about 1200 kDa, optionally about 1000 kDa and collecting a third eluate; (iv) filtering the third eluate VLPs from step (iii) with a third membrane, wherein the third membrane has a molecular weight cutoff of about 300 to about 500 kDa, and collecting the VLPs retained from the third membrane; and (v) separating the VLPs collected from the third membrane in step (iv) with ion exchange chromatography. In one aspect, the extraction solution has a pH of the isoelectric point of the VLP. In one aspect, steps (iii) and (iv) of the method take place at about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about room temperature.

[0133] According to another embodiment, the methods comprise: (i) extracting the VLPs from an intact plant or plant part using an acidic extraction solution, and collecting the VLPs from a first eluate by vacuum infiltrating the apoplast from the intact plant or plant tissue with the extraction solution and collecting the first eluate from the apoplast with centrifugation; (ii) filtering the VLPs from the first eluate of step (i) with a first membrane having a pore size of about 0.20 pm to about 0.25 pm, optionally about 0.22 pm and-40-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 collecting a second eluate; (iii) filtering the VLPs from step (ii) with a second membrane, wherein the second membrane has a molecular weight cutoff of about 1,000 kDa and collecting a third eluate; and (iv) separating the VLPs from the third eluate in step (iii) with ion exchange chromatography. In one aspect, the extraction solution has a pH of the isoelectric point of the VLP. In one aspect, step (iii) of the method takes place at about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about room temperature.[0134| According to some embodiments the VLP is derived from a plant virus. The VLP may be a plant virus coat protein. The plant virus may be rod shaped or icosahedral shaped. In some aspects, the plant virus is Cowpea Mosaic Virus, Cowpea Chlorotic Mottle Virus, or Potato Virus X. In some respects, the virus is a different plant virus.

[0135] According to some embodiments, the methods are performed in the absence of an ultracentrifugation or isopycnic ultracentrifugation step. In some aspects, an ultracentrifugation step is a centrifugation step of greater than 65,000g. In other aspects, an ultracentrifugation step is a centrifugation step of 100,000g to 1,000,000g, or greater than 1,000,000g.

[0136] According to some embodiments, the harvested VLPs have a net negative charge. According to some other embodiments, the harvested VLPs have a net positive charge. CPMV, for example, has a net negative charge.

[0137] In some aspects, in the plant tissue comprises or consists essentially of leaf tissue, stem tissue, and / or root tissue. In some aspects the plant tissue is from an intact plant, and in other aspects the plant tissue is from a plant part. The transient expression strategy used herein is not limited to a single plant species. For a multipurpose use (e.g. production of food and pharmaceuticals) also edible plants like lettuce can be used as expression hosts. In some aspects, the plant is Nicotiana benthamiana. In other aspects the plant is lettuce, corn, soybean, or another plant host.

[0138] In some aspects, the VLP is produced in plant apoplast. Multiple plant viruses can be produced in the apoplast, for example PVX, CPMV, or CCPM.-41-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0139] In some embodiments, the VLP are extracted from an intact plant in step (i) by vacuum infiltrating an apoplast with the extraction solution and collecting the extraction solution with centrifugation.

[0140] In some embodiments, the VLP are extracted from a plant part in step (i) by vacuum infiltrating an apoplast with the extraction solution and collecting the extraction solution with centrifugation.[01411 The methods described herein may be practiced in any environment. In one aspect the method is practiced in a microgravity environment.

[0142] In one aspect of the methods, the membranes are selected from polyethersulfone, regenerated cellulose, nylon, or polytetrafluorethylene (PTFE).10143] In one aspect of the methods, the ion exchange chromatography is selected from an anion exchange chromatography or cation exchange chromatography and can achieve a purity of the VLPs of greater than or equal to 99%.

[0144] Further provided is a method of harvesting virus-like particles (VLPs) from plant tissue, comprising: (i) extracting the VLPs from the plant tissue using an extraction solution, wherein the VLPs are collected in an eluate; (ii) purifying the VLPs collected in the eluate by sequential separation steps based on particle size, each step comprising ultrafiltration and / or diafiltration through one or more membranes; and (iii) purifying the VLPs obtained from step (ii) by separation based on particle charge using ion exchange chromatography, wherein the method is performed without a chloroform or methanol extraction step and without ultracentrifugation, and further wherein the plant tissue is leaf, stem, and / or root tissue of an intact plant, and step (i) comprises vacuum infiltrating an apoplast of the intact plant with the extraction solution and recovering said eluate by centrifugation at less than 1,000 x g.

[0145] In one aspect, the extraction solution is acidic, having a pH in the range of about 3.5 to about 4.5, or about 4.0. In another aspect, the extraction solution is basic, having a pH in the range of about 8.5 to about 9.5, or about 9.0.-42-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0146] In one embodiment, the harvested VLPs have a net negative charge at the pH of the extraction and / or chromatography buffers. In another aspect, the harvested VLPs have a net positive charge at the pH of the extraction and / or chromatography buffers.|0147] In a further aspect, the VLPs are derived from a plant virus selected from the group consisting of: Cowpea mosaic virus (CPMV), Cowpea chlorotic mottle virus (CCMV), Potato virus X (PVX), Tobacco mosaic virus (TMV), Physalis mottle virus (PhMV), or Sesbania mosaic virus (SeMV).

[0148] In one embodiment of these methods, the series of membranes comprises: (a) a first membrane having a pore size between 0.1 pm and 0.4 pm; (b) a second membrane having a molecular weight cut-off (MWCO) between about 800 kDa and 1,200 kDa; and (c) optionally, a third membrane having an MWCO between about 300 kDa and 500 kDa. In another aspect, the membranes in step (ii) are selected from polyethersulfone, regenerated cellulose, nylon or polytetrafluoroethylene (PTFE). In a further aspect, the ion exchange chromatography in step (iii) is selected from anion exchange chromatography or cation exchange chromatography, and achieved purity of the VLPs is > 99%.

[0149] The method can be performed in a microgravity environment, wherein the environment having gravity greater or less than l*g.

[0150] Yet further provided are methods of harvesting virus-like particles from plant tissue comprising: (i) incubating the plant tissue in a heated extraction solution; (ii) collecting the VLPs in the extraction solution; (iii) purifying the VLPs by sequential membrane separations based on size; and (iv) purifying the VLPs from step (iii) by separation based on charge using ion exchange chromatography; wherein the extraction temperature is between about 50°C and about 100°C and the VLPs retain structural integrity.

[0151] In one aspect, of the disclosed methods, the purification steps reduce endotoxin concentration to less than 0.1 EU / mL or wherein purification steps reduce 1,3- -glucan concentration to less than 2 ng / mL.

[0152] In another aspect, a method of purifying a bioconjugated virus-like particle is provided comprising: (i) providing a reaction mixture of a plant virus-like particle and one or more conjugation reagents; and (ii) filtering the mixture by ultrafiltration through at least one -43-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 membrane having an MWCO between about 200 kDa and 500 kDa to retain the bioconjugated VLP and remove unconjugated reagent; wherein purity is increased to >95% bioconjugate.|0153] VLPs and Compositions

[0154] According to some embodiments, the VLPs harvested or a plurality of the harvested VLPs prepared by the methods described herein. Thus, provided herein are isolated VLPs or a plurality of VLPs prepared by the methods described herein. The isolated VLPs or one or more of the plurality can be detectably labeled, or linked or conjugated to a purification label or tag. The VLP may comprise a functionalizing or a therapeutic agent. In some embodiments, the therapeutic agent is an RNA molecule.

[0155] Further provided is a virus-like particle preparation comprising cowpea mosaic virus wherein the small coat protein is uniformly cleaved at its C-terminus, said preparation made by extracting at pH in the range of about 3.5 to about 4.5, optionally about 4.0, followed by ultrafiltration and ion exchange chromatography.

[0156] In some embodiments, the VLP, plurality, with or without a functionalizing or a therapeutic agent is part of a composition.

[0157] Compositions

[0158] Compositions, including pharmaceutical compositions comprising, consisting essentially of, or consisting of a component or a combination as described herein, can be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping, or lyophilization processes. The component or combination can be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries which facilitate processing of the component or combination provided herein into preparations which can be used pharmaceutically.

[0159] The component or combination of the present disclosure can be administered by parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intraci sternal injection or infusion, subcutaneous injection, or implant), oral, by inhalation spray nasal, vaginal, rectal,-44-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration.

[0160] In one embodiment, this technology relates to a composition comprising a component or a combination as described herein and a carrier.[01611 In another embodiment, this technology relates to a pharmaceutical composition comprising a component or a combination as described herein and a pharmaceutically acceptable carrier.10162] In another embodiment, this technology relates to a pharmaceutical composition comprising a therapeutically effective amount of a component or a combination as described herein and a pharmaceutically acceptable carrier.[01631 The pharmaceutical compositions for the administration of a component or a combination as disclosed herein can be conveniently presented in dosage unit form and can be prepared by any one of the methods well known in the art of pharmacy. The pharmaceutical compositions can be, for example, prepared by uniformly and intimately bringing the compounds provided herein into association with a liquid carrier, a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition, each component provided herein is included in an amount sufficient to produce the desired effect. For example, pharmaceutical compositions of the present technology may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, infusion, transdermal, rectal, and vaginal, or a form suitable for administration by inhalation or insufflation. In one aspect, administration is intraperitoneal.10164] For topical administration, the component or the combination can be formulated as solutions, gels, ointments, creams, suspensions, etc., as is well-known in the art.

[0165] Systemic formulations include those designed for administration by injection (e.g., subcutaneous, intravenous, infusion, intramuscular, intrathecal, or intraperitoneal injection) as well as those designed for transdermal, transmucosal, oral, or pulmonary administration.-45-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0166] Useful injectable preparations include sterile suspensions, solutions, or emulsions of the compounds provided herein in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing, and / or dispersing agents. The formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives.

[0167] Alternatively, the injectable formulation can be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, and dextrose solution, before use. To this end, the component or the combination provided herein can be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.

[0168] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

[0169] For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets, or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets can be coated by methods well known in the art with, for example, sugars, films, or enteric coatings.

[0170] Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the combination of compounds provided herein in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents (e.g., com starch or alginic acid); binding agents (e.g.-46-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 starch, gelatin, or acacia); and lubricating agents (e.g., magnesium stearate, stearic acid, or talc). The tablets can be left uncoated or they can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. They may also be coated by the techniques well known to the skilled artisan. The pharmaceutical compositions of the present technology may also be in the form of oil-in-water emulsions.

[0171] Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin, or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophore™, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring, and sweetening agents as appropriate.

[0172] Methods of Application

[0173] According to one aspect, described herein are methods of application of the VLP or compositions described herein to a cell, tissue, or subject in need. The method comprises, consists of, or consists essentially of contacting or administering to the cell, tissue, or subject in need the VLP or composition.[0174| The cell or tissue can be contacted in vivo or in vitro.[0175| Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a-47-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human.[0176j Streamlined Molecular Farming of VLP Therapeutics in Space

[0177] Pharmaceuticals help to protect the health of astronauts during mission, yet drugs expire faster in space compared to the ground. Additionally, long term space missions cannot frequently be resupplied with pharmaceuticals, because of long travel distances. On-demand biomanufacturing capacity in space would allow to overcome these problems. Plants have conventionally been studied in space in the context of air or water treatment and supply of food, but recombinant protein expression in plants has now sufficiently matured as a technology to also consider them for the supply of astronauts with pharmaceuticals or other high value products.

[0178] Plant VLPs have immunotherapeutic potential. Specifically, treatment with cowpea mosaic virus (CPMV) demonstrated efficacy in tumor mouse models and more importantly, in canine cancer patients. This cancer immunotherapy is not limited to a specific tumor type and eliminates tumors as well as distant metastasis. It also acts like a vaccine and prevents recurrence of the disease. CPMV nanoparticles can be used as intra-tumoral agents or for systemic administration. Notably these nanoparticles also serve as a platform technology for immunotherapy and therapeutic vaccine development more broadly targeting infections, cardiovascular diseases, and cancer.

[0179] A major drawback that currently limits a broad utilization of plant-based expression systems are complex purification processes, resulting from the presence of large quantities of soluble host cell proteins and insoluble debris after extraction. This is especially problematic in space, because there is no room for complex process equipment.

[0180] The ability of plants is utilized to secrete pharmaceuticals to the apoplast. From this cell compartment pharmaceuticals can be simply eluted with a technique termed infiltrationcentrifugation, thus omitting the need to disrupt plant tissue. Such processes are desirable in a resource-environment and constricted environment such as space. VLP therapeutics have tremendous potential as platform technology for treatment of cancer, cardiovascular diseases and infectious diseases, which are highly relevant in space, but are also leading causes of-48-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 death on earth. For example, VLPs based on cowpea mosaic virus (CPMV) have demonstrated high potency against tumors in cancer mouse models and more importantly in canine cancer patients.

[0181] Results show that the model VLP CPMV can be eluted from the apoplast of intact leaves. The purity of CPMV eluted from the apoplast was >80% (as measured by densitometric analysis) - this represented an >100-fold increase in purity compared to conventional extraction of CPMV VLPs from plant tissue with a blender. Analysis of infiltration-centrifugation eluates by transmission electron microscopy (TEM) confirmed that CPMV VLPs produced in the apoplast were intact. Next, Applicant investigated whether this approach can be combined with size-based purification of VLPs using ultrafiltration and / or with stability -based purification of VLPs, using an acidic pH (4.0) treatment - these methods utilize the size difference between CPMV VLPs (5600 kDa) and major plant host cell proteins such as RuBisCO (550 kDa), as well as the difference in stability to remove the latter. Applicant confirmed that VLPs purified by an acidic pH treatment and ultrafiltration remained structurally intact in comparison to contemporary produced controls (i.e. chloroform / butanol extraction, PEG precipitation and multiple rounds of ultracentrifugation), using for example dynamic light scattering, fast protein liquid chromatography and transmission electron microscopy (TEM). Moreover, Applicant confirmed the biological activity of produced VLPs using a standardized cytokine secretion assay. Taken together, these data confirm that VLP therapeutics can be rapidly purified with simple methods, thus facilitating adaption in low-resource environments such as space.

[0182] Infiltration-Centrifugation

[0183] A prerequisite for using plants as a production platform for pharmaceuticals in space are simple purification processes

[0035] because there is no room for complex purification equipment in spacecrafts. One approach that can tremendously simplify purification processes in plant-based expression systems is the ability of plants to secrete pharmaceuticals to the apoplast. Pharmaceuticals can be readily eluted from this cell compartment with a technique termed infiltration-centrifugation. Plant VLPs can be eluted from the apoplast. For example, the coat protein of (PVX) was the dominant viral protein in the apoplast of infected-49-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 tobacco plants. Similar to CPMV, PVX has a lot of potential for the development of VLP- based therapeutics. Antibodies are currently the dominant class of biopharmaceutical products. Similar to VLPs, full-size antibodies can be eluted from the apoplast by infiltrationcentrifugation.

[0184] The principles are exemplified below. First, plant tissue is submerged in buffer or water and vacuum is applied to infiltrate the apoplast with liquid. Next, target proteins that naturally accumulate in the apoplast or have been directed to the apoplast with signal sequences are eluted alongside the infiltrated liquid by centrifugation. Centrifugal forces <1000 x g and centrifugation times <10 minutes are recommended for this step. Because the plant tissue is not disrupted in the process, major host cell proteins such as Ribulose- 1,5- bisphosphate carb oxy lase / oxygenase (RuBisCO) are retained in the plant tissue. Prerequisites for implementing infiltration-centrifugation in space are (i) the ability to apply a vacuum to plants and (ii) a small-scale centrifuge.

[0185] Applicant designed apoplast expression vectors and confirmed that CPMV VLPs can be readily eluted from the apoplast using the infiltration-centrifugation technique.

[0186] The purity of CPMV in the apoplast eluate was >80% (as measured by densitometric analysis), even without any optimization. Data shows that CPMV VLPs can be extracted with an > 100-fold increase in purity compared to conventional extraction of CPMV VLPs from plant tissue with a blender. Moreover, analysis of infiltration-centrifugation eluates by transmission electron microscopy (TEM) confirmed that CPMV VLPs produced in the apoplast were intact. Taken together, these data confirm that a plant-based production process for VLPs based on infiltration-centrifugation is feasible.

[0187] It is important to note that the plant tissue remains intact, hence the plants could continue to grow and be utilized for other purposes such as recycling of air and water. Plant tissue is not disrupted by the infiltration-centrifugation method: when targeted to the cytosol instead of the apoplast, the fluorescent marker protein DsRed remained in the plant tissue during infiltration-centrifugation

[0050] ,

[0188] Orthogonal purification Strategies for VLPs-50-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0189] In the context of orthogonal purification strategies for CPMV VLPs, Applicant confirmed that CPMV tolerates an acidic precipitation step (pH 4.0), whereas >80% of the plant host cell proteins were removed. CPMV VLPs are very stable and tolerate extreme pH values such as 4.0 or 9.0 for at least two days. Moreover, Applicant confirmed that remaining plant host cell proteins can be removed by ultrafiltration while retaining the CPMV VLPs. TEM imaging of purified samples confirmed that VLPs remained intact after pH treatment and ultrafiltration.

[0190] Applicant confirmed the efficacy of CPMV immunotherapy in dogs with canine mammary tumors. Without wishing to be bound by theory, Applicant hypothesizes similar results would result in human tumors because the tumor size in dogs and humans are similar. Similar to mouse models, the therapy was efficient against primary tumors as well as distant tumors, that were not directly treated with CPMV in canine patients. Notably, all dogs that received CPMV prior surgical removal of tumors survived to this day, whereas dogs that only received surgery died from reoccurrence of the disease.

[0191] VLP Generation in Any Gravity Environment

[0192] Without wishing to be bound by theory, a production process can be realized in plantbased expression systems by eluting pharmaceuticals from the apoplast. This strategy omits the need to disrupt plant tissue and therefore the need for complex purification equipment. A production process with VLPs as model pharmaceuticals, which can be used as a potential countermeasure for treatment of radiation-induced carcinogenesis. Genetic constructs for VLP production are be designed modularly to facilitate generation of construct libraries, loading of custom RNA into VLPs, and rapid adaption of the proposed production process to other pharmaceuticals.

[0193] A simple, plant-based manufacturing process for on-demand production of VLP therapeutics can be used to expand medical capabilities during space flight.

[0194] Method to Optimize VLP Production

[0195] Canine mammary tumors have been successfully treated with 0.2 mg injections of CPMV VLPs - the projected human cumulative dose is 1 mg per dose. State of the art cultivation systems can provide up to 1 m2of farming area on spacecrafts. Accordingly, a -51-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 productivity of 1 mg CPMV / m2farming area is desired. An exemplary system uses N. benthamiana for the production of CPMV VLPs, because this host allows to combine high biomass yields with high productivities. Moreover, N. benthamiana is already established for the production of CPMV VLPs, and Nicotiana seeds showed good survival in space flight.

[0196] Applicant provides herein VLP production methods optimized on the DNA level by cloning expression enhancing elements into previously established genetic constructs for VLP expression to provide ideal combinations of untranslated regions (UTRs) and terminators. This strategy has previously increased productivities up to 25-fold in Nicotiana species. Additionally, co-expression of silencing suppressors such as the pl9 protein, which have increased recombinant protein yields in N. benthamiana several-fold can be utilized. VLP production is further optimized by identifying ideal timepoints for inoculating and harvesting VLPs.

[0197] The software SnapGene can be used to plan cloning strategies and polymerase chain reaction (PCR), restriction cloning and Gibson assembly to introduce expression enhancing elements into existing genetic constructs. Agrobacterium tumefaciens is used for testing genetic constructs in plants. VLP expression is quantified by enzyme-linked immunosorbent assay (ELISA) using specific anti-CPMV antibodies and known VLP concentrations as standards. An equivalent system mass (ESM) analysis based on the process mass intensity (PMI) is used to compare the production process described here with other options for an application in space. The structural integrity of VLPs produced in the apoplast can be verified by transmission electron microscopy (TEM) imaging, dynamic light scattering (DLS), size exclusion chromatography (SEC), and gel electrophoresis (native agarose gels and denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)), using contemporary produced controls. SEC can confirm that produced VLPs are not aggregated, broken or disassembled.[0198| Assessing process resilience

[0199] To assess and improve the resilience of the proposed production process, plants are subjected to temperatures in the range of 16-40°C during VLP production; temperatures up to ~37°C have been observed in space growth systems due to technical problems. Models are-52-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 established for the VLP yield and identify stable operation windows; stable process conditions have a low effect of influence factors (the cultivation conditions) on the response (productivity). The generated models allow Applicant to evaluate the effect of potential technical difficulties during space missions.

[0200] The stability of genetic constructs and VLPs under conditions that mimic space flight; space flight is characterized by exposure to cosmic radiation, microgravity, and extreme temperatures can be assessed, all of which can induce DNA damage.

[0201] For example, ground analog centrifuge to replicate the launch profile of (multistage) rockets that can deliver payloads to space. Applicant uses large-scale Random Positioning Machine to produce microgravity conditions on the ground; other than parabolic flights, which provide only ~25 second intervals of micro gravity, microgravity can be maintained for hours with Random Positioning Machines. Applicant uses UV light to mimic the effect on space radiation; UV radiation is the most deleterious factor for survival of microorganisms in space.

[0202] The stability of genetic constructs with purified plasmid DNA and with lyophilized Agrobacteria containing the genetic constructs is tested. Additionally, the stability of purified VLPs with and without cryoprotectants like sucrose is tested.

[0203] The integrity of genetic constructs is tested with sequencing as well as through transformation experiments. VLP integrity is analyzed using the aforementioned methods using freshly produced VLPs as reference material.

[0204] A simple protocol for VLP purification

[0205] Pharmaceuticals intended for human application require a very high purity, typically >99%. Typical contaminations in apoplast eluates that need to be removed to reach this purity threshold are plant host cell proteins, pigments, phenolics, and salts. Potential adverse effects of these contaminates in the human body are antigenicity and toxicity. The most critical potential contaminations in biologic manufacturing processes are endotoxins (lipopolysaccharides), which can cause serious side effects in humans, including fever and shock.-53-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0206] Applicant optimizes the infiltration-centrifugation method for VLPs to remove impurities: Acidic elution buffers are used to minimize native protein solubility; data confirms that CPMV VLPs tolerate extreme pHs of 4.0 and 9.0, whereas -80% of the plant host cell proteins precipitated under these conditions.

[0207] Applicant utilizes the size difference between VLPs and impurities for further purification: host cell proteins in the apoplast are typically <50 kDa, phenolics range up to -5 kDa and pigments like chlorophyll range up to -1 kDa, whereas CPMV VLPs have a molecular weight of 5600 kDa. The size difference is utilized using a two-step filtration process: First, Applicant uses 0.2 pm filters to remove insoluble impurities and bacteria from apoplast eluates. Second, Applicant uses ultrafiltration membranes with a 300kDa or 500kDa to retain CPMV VLPs cut-off to wash out host cell proteins, phenolics, pigments, and salts.

[0208] Prior to the second step, Applicant may optionally use an ultrafiltration membrane to remove large particles, the membrane having a molecular weight cut-off of about lOOOkDa to about 2000kDa, or alternatively about lOOOkDa or about 2000kDa, or alternatively lOOOkDa to 2000kDa, or yet further lOOOkDa or 2000kDa.

[0209] Filtration-based purification strategies can be realized with syringe filters (data not shown), thus facilitating an adaption in space.

[0210] Ion exchange chromatography is used as an orthogonal purification method, using small-scale columns that can be operated with a syringe or with a centrifuge. Ion exchange chromatography is well suited for the purification of VLPs, because their surface is charged. For instance, CPMV has a negatively surface charge.

[0211] The purity, identity and conformational integrity of purified VLPs is analyzed with established methods, including a combination of size exclusion chromatography (SEC), dynamic light scattering (DLS), transmission election microscopy (TEM), and gel electrophoresis (native agarose gels and denaturing SDS-PAGE), using contemporary produced CPMV VLPs as control. For nanoscale characterization of CPMV VLPs, mass spectroscopy analysis and total RNA sequencing is used.

[0212] Purity criteria-54-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0213] The purity threshold for an application of VLPs in humans is >99%; intact CPMV VLPs have a diameter of 28 nm, a zeta potential ((^CPMV) of -7.5 mV, an extinction coefficient of 8.1 mL mg-1 cm-1 and an;4260 280 ratio of 1.57. Encapsulation of RNA into the capsid is indicated by co-migration of RNA and protein on native agarose gels. The purity derived from total RNA sequencing is 95% with sequences aligning to CPMV RNA-1 and RNA-2. Out of the peptides identified during mass spectrometry analysis (LC-MS / MS), 96% align with the CPMV coat proteins. Purified CPMV preparations is also analyzed using the limulus amebocyte lysate (LAL) endotoxin assay to confirm the absence of lipopolysaccharides (LPS). Acceptable formulations are free of LPS (or contain no more than 5 EU kg-1 body weight dosing - as per FDA regulations).[02141 Efficacy and Safety Validation of Final VLP Products in vitro[02151 The CPMV VLPs produced in the apoplast are compared to controls produced with contemporary methods, for example mechanical inoculation of plants by hand rubbing, extraction in a blender, chloroform-butanol extraction, and isopycnic ultracentrifugation. Because CPMV was shown to be a multi-Toll-like-receptor (TLR) agonist, RAW-Blue reporter cells are used to validate the biological activity of VLPs; this assay has been established as a tool to assay the potency and the immunomodulatory nature of CPMV and other VLPs. Further, bone marrow derived macrophages and dendritic cells (DCs) are used to assay VLP-cell interactions: Confocal microscopy and flow cytometry are used to measure VLP uptake, and ELISA and flow cytometry are used to determine whether DCs are activated upon CPMV treatment.

[0216] Efficacy and Safety Validation of Final VLP Products in vivo

[0217] The safety and efficacy of CPMV VLPs produced in the apoplast is validated in vivo, using a melanoma tumor model - CPMV in situ vaccination is efficient against various cancer types, including melanoma: B16F10 cells (200.000 cells / mouse in a total volume of 30 pL) are administered by intra-dermal injection; Applicant treats mice with 0.1 mg CPMV weekly (3 treatments). Treatment efficacy is measured by monitoring the primary tumor as well as outgrowth of metastases; The metastatic disease burden is assessed through the tumor-55-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 foci number and tyrosinase expression in the lungs (tyrosinase-related protein 1 is melanocyte-specific and its expression in the lungs is restricted to B16F10 cells).

[0218] Tumors are collected at 24 hours post first and last dose for tumor microenvironment analysis. Cytokines are analyzed by luminex assays and innate and adaptive immune cell populations is characterized by flow cytometry. All animal experiments are carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals in Research.[0219| Plug-and-Play Readiness[022(>| Applicant establishes CPMV VLPs as a tool for drug delivery by loading custom RNAs into CPMV VLPs.

[0221] Plug-and-Play readiness facilitates the generation of construct libraries which enables astronauts to react to emerging medical problems. The emerging of new infectious diseases and variants thereof, most recently overserved during the COVID19 pandemic, has demonstrated the need for plug-and-play technologies to allow a rapid response to changing demands for pharmaceuticals.

[0222] VLPs that contain mRNA of the marker gene mCherry as well as siRNAs targeting GFP or FOXA1 are prepared as non-limiting examples for siRNA therapeutics. It has already been established that custom RNA can be loaded into CPMV VLPs by flanking it with the 5' and 3' untranslated regions (UTRs) of CPMV DNA-2. However, CPMV VLPs have not yet been utilized for drug delivery. The constructs described herein contain the necessary UTRs as well as restriction enzyme recognition sites to facilitate the rapid exchange of RNA cargos.

[0223] The identity and integrity of VLPs as well as their RNA cargo is validated applying similar techniques as above. Additionally, agarose gels are used to confirm the presence of RNA cargo in CPMV VLPs and total RNA sequencing to confirm the identity of the RNA cargo. Flow cytometry is used to confirm the activity of siRNA cargos, using GFP-expressing HeLa cells and the breast cancer cell line MCF-7 as targets

[0063] ,

[0224] Experimental

[0225] Materials and Methods

[0226] Experiment No. 1 - CPMV Propagation-56-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0227] Plant cultivation and CPMV propagation

[0228] Black-eyed peas (Vigna unguiculate no. 5, Morgan County Seeds) were seeded in 3- 1 / 4" square pots (Greenhouse Megastore), using ProMix BX soil (Greenhouse Megastore), and were maintained in a walk-in growth chamber (Conviron) at 25 / 22°C (day / night cycle), 60% relative humidity and -100,000 lux (16 h photo period) as previously described

[0041] , Seven days after seeding, primary leaves were mechanically inoculated with 40 pL / leaf of 0.1 mg / mL CPMV in 100 mM potassium phosphate buffer (pH 7.0) as previously described

[0041] , Inoculated plants were incubated for 1 hour at room temperature in the dark, before rinsing leaves with tap water and transferring plants back into the growth chamber. Primary leaves were harvested 14 days after inoculation and stored at -80°C until processing.

[0229] CPMV was purified from infected plant leaves using two protocols: the conventional, termed centrifuge-based purification protocol. The novel process for CPMV purification using ultrafiltration, termed UF / DF, is described below.

[0230] CPMV purification with the centrifugation process1'0231] Purification of CPMV with the centrifugation process (contemporary method)10232] Purification of CPMV from infected plant leaves with the centrifugation process was carried out as described in the literature

[0043] , In brief, leaves were extracted with 3 volumes of 0.1 M potassium phosphate buffer (pH 7.0) in a blender. The extract was filtered through a double layer of Miracloth (MilliporeSigma) and clarified by centrifugation at 18,500*g for 20 min at 4°C using an Avanti J-E centrifuge and a JLA 10.500 rotor (Beckman Coulter). The clarified plant extract was then mixed with 0.7 volumes of 1 : 1 (v / v) chloroform :butanol (ThermoFisher Scientific) by stirring on ice for 30 min, before centrifugation at 6,600*g for 10 min at 4°C using the same centrifuge and rotor as above. The aqueous phase was collected and subjected to PEG precipitation, using 0.2 M sodium chloride and 10% (w / v) PEG8000 (ThermoFisher Scientific); precipitation was induced by mixing for 2 h on ice. The precipitate was collected by centrifugation at 30,000*g for 15 min at 4°C, using an Avanti J- E centrifuge and a JLA 16.250 rotor (Beckman Coulter). The supernatant was discarded and the pellet was resuspended in 0.01 M potassium phosphate buffer (pH 7.0) by pipetting. The suspension was then separated on a 10-40% (w / v) sucrose gradient in Ultra-Clear tubes-57-4911-2869-8997.1Atty. Dkt. No.: 114198-3560(Beckman Coulter) by centrifugation at 150,000xg for 2 h at 4°C, using an Optima L-90k centrifuge and a SW32 Ti rotor (Beckman Coulter). Bands were visualized with a flashlight and the middle two bands were collected with a syringe and transferred into screw-top ultracentrifugation tubes (Beckman Coulter). The extracted fractions were then pelleted by ultracentrifugation at 212,500xg for 2 hours at 4°C, using an Optima L-90K ultracentrifuge and a 50.2 Ti rotor (Beckman Coulter). The pellet was resuspended in 0.1 M potassium phosphate buffer (pH 7.0) on a rotary shaker at 4°C overnight, yielding pure CPMV preparations.

[0233] Removal of endotoxins from CPMV preparations purified using the centrifugation method

[0234] Endotoxins were removed from CPMV preparations purified with the centrifugation method as previously described

[0044] , In brief, CPMV preparations with a concentration of ~2 mg / mL in (0.1 M potassium phosphate buffer, pH 7.0) were mixed with 2% (v / v) Triton X- 114 (in deionized water) at a 1 : 1 (v / v) ratio, followed by mixing on a rotary shaker at 4°C for 20 minutes. Samples were then incubated at 37°C for 10 min to induce phase separation of Triton X-l 14

[0044] , and phases were separated by centrifugation at 20,000xg for 10 min at room temperature. The upper phase was collected and concentrated with Amicon Ultra-4 centrifugal filters (MilliporeSigma) by centrifugation at 7,500xg for 15 min at room temperature. Pierce detergent removal spin columns (ThermoFisher Scientific) were used according to the manufacturer’s instructions to remove residual Triton X-l 14 from CPMV formulations. PD-10 desalting columns (Cytiva) were used according to the manufacturer’s instructions to formulate CPMV preparation into the final storage buffer (0.1 M potassium phosphate, pH 7.0).

[0235] CPMV purification with the UF / DF process

[0236] Extraction and pH treatment

[0237] Frozen leaves were homogenized in 3 volumes (3 v / m) of extraction buffer (Table 1) for 3 x 30 s with 30-s breaks between mixing cycles in a blender

[0045] , Glycine was used as buffer to produce extracts with a pH of 9.0, potassium phosphate was used to produce extracts with a pH of 7.0, and MES was used to produce extracts with a pH of 4.0 or 5.0. If -58-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 needed, extracts were re-adjusted to the desired pH after extraction by addition of 0.5 M hydrochloric acid or 0.5 M sodium hydroxide. Extracts were then filtered through two layers of Miracloth (Millipore Sigma) and clarified by centrifugation at 16000 x g for 30 minutes at 4°C, using a JLA- 16.250 rotor and an Avanti J-E centrifuge (Beckman Coulter).

[0238] Table 1: Extraction buffers used for homogenization.[0239| Ultrafiltration[0240| A Minimate bench-top tangential flow filtration system (Cytiva) was used for all ultrafiltration experiments with a transmembrane pressure of 0.5 bar and a tangential flow rate of 40 mL / min. Polyethersulfone membranes (Cytiva) with 50 cm2filter area and an MWCO of 300 kDa, 500 kDa or 1,000 kDa were fed with 4 mL / cm2clarified plant extract; buffers and extracts were 0.22 pm filtered before ultrafiltration to remove larger particles and aggregates that could damage the membranes. In a typical experiment 200 mL clarified plant extract was concentrated 4-fold before continuously feeding buffer with the same pH as the clarified extract into the system to maintain a constant volume. Retentates and permeates (FIG. 7) were sampled after every 50 mL permeate collected. Ultrafiltration membranes were regenerated by rinsing with 4 mL / cm2extraction buffer and 4 mL / cm2sodium hydroxide (1.0 M), followed by incubation in sodium hydroxide overnight. The sodium hydroxide solution was removed by rinsing with 4 mL / cm2deionized water and 4 mL / cm220% (v / v) ethanol, and then membranes were stored in 20% (v / v) ethanol at 4°C. Membrane fouling was assessed through the normalized water capacity, measured before the first use and after regeneration as previously described

[0046] ,]0241[ Ion exchange chromatography-59-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0242] After purification by ultrafiltration, CPMV was further polished by ion exchange chromatography (AEX) using an AKTApure system (Cytiva) and 1.0 mL columns prepacked with HiTrap Q Sepharose Fast Flow resin (Cytiva). Optionally, a column volume of 3.0 mL was used to assess scalability. The volumetric flow rate was 0.5 mL / min (0.8 m / h), corresponding to a contact time of 2 minutes. The temperature was controlled during all chromatography experiments and kept at 4°C. In a typical experiment, chromatography resins were equilibrated with 5 column volumes (CV) of equilibration buffer (100 mM potassium phosphate, pH 7.0), followed by loading 25 CV of ultrafiltration-purified sample, and washing with 5 CV of equilibration buffer. Bound CPMV was eluted with a 0-35% gradient (over 1.4 minutes) of elution buffer (100 mM potassium phosphate, 1.0 M sodium chloride, pH 7.0), using an auto sampler for fractionation collection. Chromatography resins were regenerated by washing with 5 CV equilibration buffer, 5 CV 0.5 M sodium hydroxide, 5 CV elution buffer (100 mM potassium phosphate, 1.0 M sodium chloride, pH 7.0) and 5 CV 20% (v / v) ethanol; columns were then stored in 20% (v / v) ethanol at 4°C.

[0243] Endotoxin removal during chromatography

[0244] Optionally, the non-ionic detergent Triton-Xl 14 was added to ultrafiltration-purified samples to assess the removal of endotoxins during chromatography: Briefly, Triton-Xl 14 was added to samples to a final concentration of 0.25% (v / v), followed by mixing on a rotary shaker at 4°C for 15 minutes, and purification by AEX chromatography as described above.

[0245] Protein quantification

[0246] Total soluble protein in process samples was quantified with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) using bovine serum albumin standards in the range of 25 to 2,000 mg / L.

[0247] Endotoxin and glucan quantification

[0248] The concentration of endotoxins in process samples was measured using a Pierce Chromogenic Endotoxin Quantification kit (Thermo Fisher Scientific) according to the manufacturer’s recommendations; samples were measured in duplicates. The concentration of 1,3-beta-D-glucans in process samples was measured using a Glucatell kit (Cape Cod) according to the manufacturer’s recommendations; samples were measured in duplicates.-60-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0249] Characterization of process samples and purified CPMV

[0250] UV-Vis spectroscopy

[0251] UV-Vis spectra of purified CPMV were measured using a NanoDrop Spectrophotometer (Thermo Fisher Scientific) in 100 mM potassium phosphate. CPMV was quantified through the absorbance at 260 nm (A260), using Beer’s law and an extinction coefficient of e = 8.1 mL mg-1cm-1

[0041] , UV-Vis spectroscopy was also used to measure the RNA-to-protein ratio in purified samples; intact CPMV is characterized by an A260 / A280 ratio of ~1.7

[0041] ,

[0252] SDS-PAGE and western blot

[0253] Process samples and purified CPMV were separated on 4-12% Bis-Tris gels in MOPS buffer (Thermo Fisher Scientific) at 200 V for 45 min, and subsequently stained with Coomassie Brilliant Blue or used for western blotting as previously described

[0022] , CPMV was detected with rabbit anti-CPMV antibodies (custom-made, Pacific Immunology) diluted 1 :500 in 5% (m / v) milk powder in PBST, and HRP-conjugated goat anti-rabbit antibodies (Thermo Fisher Scientific) diluted 1 : 5000 in the same buffer

[0047] , Gels were imaged with a FluorChem R system (ProteinSimple) and densitometric analysis was performed with the built-in software of the device.

[0254] Native agarose gel electrophoresis

[0255] Purified CPMV was analyzed at a concentration of 1.0 mg / mL on 1.2% (w / v) agarose gels in 10 mM potassium phosphate at 80 V for 40 min as previously described

[0048] , GelRed (Thermo Fisher Scientific) was used to visualize nucleic acids and Coomassie Brilliant Blue (0.25% w / v) was used to visualize proteins.

[0256] Dynamic light scattering

[0257] The hydrodynamic diameter of purified CPMV was measured using a Zetasizer Nano ZSP / Zen5600 (Malvern Panalytical) and a CPMV concentration of 1.0 mg / mL in 100 mM potassium phosphate buffer pH 7.0

[0258] Size exclusion chromatography-61-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0259] Purified CPMV was analyzed by size exclusion chromatography at a concentration of 1.0 mg / mL on an Akta Explorer FPLC (Cytiva), using a Superose 6 Increase 10 / 300 GL column (Cytiva), 100 mM potassium phosphate buffer and a flow rate of 0.5 mg / mL as previously described

[0048] , Fractions were eluted using an isocratic profile and the absorbance was measured at 260 nm (nucleic acids) and 280 nm (protein) as previously described

[0049] ,

[0260] Transmission electron microscopy

[0261] Purified CPMV (0.1 mg / mL in deionized water) was loaded onto 400-mesh formvar / carbon supported copper grids (Electron Microscopy Sciences); grids were charged with a PELCOeasiGlow system (Ted Pella) and negative-stained with 2.0% (w / v) uranyl acetate (Agar Scientific). Grids were imaged using a JEM-1400 Plus electron microscope (Jeol Ltd.) at 50,000* and 80,000* magnification.[02621 Activity validation in vivo

[0263] Animal experiments were carried out with 7-8 weeks old female BALB / c mice (Charles River, strain #028) as directed by the University of California San Diego’s Institutional Animal Care and Use Committee (IACUC). A20 cells (murine B cell lymphoma) were cultivated as previously described

[0049] and 200,000 cells (in 30 pL sterile PBS) were inoculated intradermally (i.d.) into the right flank of mice. Treatment began 10- 11 days after tumor inoculation when tumors reached a volume of ~30 mm3. CPMV preparations (n=10 mice per group) were administered intratumorally (i.t.) once a week for three consecutive weeks at doses of 20 pg or 100 pg in 20 pL sterile PBS; sterile PBS (20 pL, i.t.) was administered to a control group (n=10 mice). Tumor volume and survival rates were monitored, and mice were euthanized once the tumor volume exceeded 1,000 mm3. Tumor- free mice were monitored up to 70 days post-tumor inoculation, corresponding to ~6-7 weeks of tumor remission. Tumor volumes were calculated using Equation 1, where V is the tumor volume, L is the tumor length (longer dimension) and W is the tumor width (shorter dimension) as previously described

[0050] ,4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0265] Equation 1

[0266] Statistical significance in tumor volumes was analyzed by two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Kaplan-Meier plots were used to compare survival between groups. Statistical analysis on survival curves was performed using the logrank (Mantel-Cox) test

[0051] , Statistical tests were performed with the software GraphPad Prism.

[0267] Analysis of chemokine / cytokine response

[0268] The chemokine / cytokine response after treatment with CPMV was analyzed using a customizable U-PLEX MSD assay. A20 cells were cultivated as described above and 200,000 cells (in 30 pL sterile PBS) were inoculated intradermally (i.d.) into the right flank of female BALB / c mice (Charles River, strain #028). Treatment began when tumors reached a volume of -60 mm3. CPMV preparations were administered intratumorally (i.t.) at a dose of 100 pg; 20 pL sterile PBS was administered to a control group (n=3 mice per group). Tumors were harvested 24 h after treatment and extracted by sonication as recommended for MSD assays. The following chemokines / cytokines were analyzed according to the manufacturer’s recommendations: GM-CSF, IFNy, IL-2, IL-6, IL-12p70, IFN-P, MCP-1, MIP-la, TNF-a, IFN-a. The plate was read using a MESO Sector S 600MM instrument and analyzed with the software MSD Workbench 4.0. A protein concentration of 50 pg / well was used for MSD assays.

[0269] Results and Discussion

[0270] Use of CPMV’s broad range pH stability for extraction

[0271] Applicant tested alkaline, neutral and acidic extraction conditions for HCP removal, using MES (pH 4.0 and 5.0), phosphate (pH 7.0) or glycine (pH 9.0) extraction buffers (Figure 1A-C). Applicant analyzed process samples by SDS-PAGE and assessed the CPMV purity and recovery using densitometry (FIG. 8). The extraction pH had a strong impact on the purity of CPMV preparations after extraction (FIG. 8). Specifically, an extraction pH of 4.0 achieved a target protein purity of -60% by precipitating the majority of plant HCPs, albeit at the cost of a -30% reduced recovery of CPMV. In contrast, neutral and alkaline extraction buffers only achieved a purity of -30% and -40%, respectively. These results -63-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 were consistent with the literature

[0046] , An extraction pH of 5.0 also improved the CPMV purity (-45%) compared to neutral extraction buffers (-30%), but this pH is close to the isoelectric point of CPMV (5.5

[0055] ), which can lead to precipitation

[0056] , Applicant previously linked a reduced target protein recovery under acidic conditions to coprecipitation with plant HCPs, which form aggregates at pH values below 6.0.

[0046] ,

[0272] The extraction pH also had a strong effect on the cleavage of the small CPMV coat protein (S protein

[0058] ), with acidic conditions (pH < 5.0) promoting cleavage, likely through the activity of acidic proteases like serine proteases

[0059] , This finding is important, because conventional preparations of CPMV are heterogenous and contain a mixture of cleaved and non-cleaved S protein

[0048] , This heterogeneity is derived from an in-planta cleavage process removing 24 C-terminal amino acids of the S protein

[0060] , It is not documented whether the C-terminal cleavage of the S protein alters its biological activity - however from a regulatory perspective, avoiding any heterogeneity is desired. Given high-yielding CPMV acid extraction, resulting in homogeneous CPMV with uniformly cleaved S protein, Applicant chose acid extraction in subsequent experiments. Noteworthy, MES buffer cannot be metabolized by bacteria and eukaryotic cells

[0061] , thus adding an additional level of safety when handling plant extracts.

[0273] CPMV can be efficiently purified based on its size using ultrafiltration

[0274] After optimizing the CPMV extraction conditions, Applicant made use of their Targe’ nanoscale size for separation from the smaller plant HCPs. CPMV particles measure 30 nm in diameter with a molecular weight of 5.6* 106g / mol

[0062] , which exceeds the molecular weight of large oligomeric plant host cell proteins such as RuBisCO (5.6* 105g / mol

[0046] ) by one order of magnitude. This significant size difference enables the use of size-based purification methods such as ultrafiltration. Applicant tested the suitability of ultrafiltration for purification of CPMV with polyether sulfone (PES) membranes, using a membrane area of 50 cm2, a transmembrane pressure of 0.5 bar and a tangential flowrate of 40 mL / min. First, Applicant tested membranes with molecular weight cut-off (MWCO) of 500 kDa vs. 1000 kDa and MES buffer (pH 4.0), because the acidic pH is expected to reduce membrane fouling by conferring a positive charge to PES membranes

[0063] , Also at pH of 4.0, many-64-4911-2869-8997.1Atty. Dkt. No.: 114198-3560HCPs are removed (see above) and those that remain in the extract would be positively charged based on their isoelectric point

[0046] , thus increasing electrostatic repulsion between proteins and PES membranes and in turn reducing membrane fouling

[0063] ,

[0275] SDS-PAGE of process samples indicated that CPMV passed membranes with a 1000 kDa MWCO, but was retained by membranes with a 500 kDa MWCO (FIGS. ID - 1G).

[0276] This observation was exciting, because it allowed the removal of impurities that are larger and smaller than CPMV by combining both ultrafiltration steps. Specifically, plant HCPs were removed during the 500 kDa ultrafiltration step (FIG. IE) and lipid micelles were removed during the 1000 kDa ultrafiltration step (FIG. IF). The purity of CPMV increased to -98% (densitometric analysis) after ultrafiltration, which was in good agreement with previous studies with a similar setup

[0064] , Notably, not all CPMV passed through the 1000 kDa membrane, indicating potential for further optimization. The molecular mass of CPMV (5.6x 106g / mol) suggests that it should be retained by a 1000 kDa membrane, especially when considering that CPMV forms a hollow sphere, thus behaving larger than its molecular mass. Possible explanations for virus permeation during ultrafiltration include the presence of abnormally large pores in membranes that are not included in the main pore size distribution

[0065] or the deformation of viruses as they pass through the membrane

[0066] , The rigidity of the latter has been found to be inversely related to their triangulation number (T=3 for CPMV

[0067] ).

[0277] High-resolution separation of CPMV from remaining impurities can be achieved based on its charge using ion exchange chromatography]0278| Making use of the possibility to exchange buffers during ultrafiltration (diafiltration), Applicant formulated CPMV into its storage buffer, 0.1 M potassium phosphate buffer pH 7.0

[0048] , CPMV has an isoelectric point of 5.5

[0055] , and therefore a negative net charge at neutral pH - thus enabling a polishing step using ion exchange chromatography and a strong anion exchange resin (HiTrap Q Sepharose Fast Flow resin). Applicant used 1.0 mL chromatography columns, a flow rate of 0.5 mL / min, 0.1 M potassium phosphate buffer (pH 7.0) as the low-salt buffer, and 0.1 M potassium phosphate with 1.0 M NaCl (pH 7.0) as high salt eluent. Ultrafiltration-purified samples were used as feed. Even though phosphate is-65-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 known to interact with the stationary phase during anion exchange chromatography, this setup is commonly used for protein purification, including CPMV [68, 69], Applicant’s rationale for using this buffer was that competition with phosphate ions would minimize the interaction between impurities and the chromatography resin, thus further increasing the purity of CPMV.

[0279] Analysis of process samples by SDS-PAGE revealed that CPMV was efficiently captured by a strong anion exchange resin, i.e. no CPMV was detected in the flowthrough (FIGS. 2A - 2B) A 0-35% gradient (1.4 min) of the high salt eluent produced two distinct elution peaks (FIG. 2A). The first elution peak (Pl, P2) was characterized by an A260 / 280 absorbance ratio of ~1.7 and corresponded to CPMV (FIG. 2B,

[0048] ). No CPMV was detected in elution fractions when further increasing the eluent concentration (FIGS. 2A - 2B), indicating that this gradient was sufficient for complete elution of CPMV. The second elution peak (P3) was characterized by an inverted A260 / 280 absorbance ratio, likely corresponding to impurities such as host DNA and protein. The purity of CPMV increased from -98% after ultrafiltration to 99.9% after chromatography, thus matching the purity threshold for clinical application

[0036] ,

[0280] Unlike CPMV, impurities interacted strongly with the chromatography resin and were not completely removed by the high salt eluent alone. Applicant found that washing columns first with 0.5-1.0 M sodium hydroxide and then with the high salt eluent efficiently removed remaining impurities (FIG. 9). Applicant therefore continued to use this method for regeneration of ion exchange chromatography resins in all subsequent experiments.

[0281] The resolution of anion exchange chromatography increased during scaleup

[0282] To investigate the effect of a process scale-up on CPMV purification with anion exchange chromatography, Applicant repeated above experiments with a column volume of 3.0 mL instead of 1.0 mL; process samples were again analyzed by SDS-PAGE. Applicant found that the resolution increased during scale-up, separating a shoulder from the CPMV elution peak (FIGS. 2C - 2D). Similarly, the separation of CPMV (Pl, P2) and impurities (P3, P4) further improved. These findings indicate that the resolution during chromatography-66-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 will benefit from a process scale up, which is desirable for translation of the purification process.

[0283] Removal of endotoxins through detergents can be combined with ion exchange chromatography

[0284] Pharmaceutical products not only are required to have high protein purity levels, but must also be free of other contaminants, such as endotoxins (or contain no more than 5 EU / kg body weight dosing per FDA regulations). Endotoxins can cause severe adverse reactions, including shock

[0070] , While plants are principally free of endotoxins, these are frequent contaminants often introduced in the process - therefore monitoring is an essential requirement. The most common source of endotoxin contaminations in biological processes are bacteria, because endotoxins are a major component of the outer membrane of gramnegative bacteria

[0071] , However, even when bacteria are omitted from the production process, it is hard to completely avoid endotoxin contaminations because they are prevalent in the environment. In the laboratory a common source of endotoxin contaminations is deionized water, because distillation and deionizing columns do not remove endotoxin

[0072] ,

[0285] Endotoxin removal protocols have been established such as the use of commercial kits or Triton X-l 14 extraction - while effective, these methods are low yielding and cumbersome

[0073] , Furthermore, Triton X-l 14 can interfere with immunogenic profiling of protein preparations

[0044] , Therefore, to streamline the endotoxin removal step, Applicant investigated whether endotoxins could be removed through Triton X-l 14 treatment during ion exchange chromatography. The non-ionic detergent Triton X-l 14 should not bind to charged resins used for ion exchange chromatography, allowing to efficiently remove detergent and endotoxin simultaneously.10286] Applicant repeated above process but added 0.2% (v / v) Triton X-l 14 to samples before chromatography, followed by incubation on a rotary shaker (15 min at 4°C), and loading onto a 3.0 mL ion exchange column (HiTrap Q Sepharose Fast Flow resin). Analysis of process samples by SDS-PAGE showed that the CPMV elution peak (Pl, P2) remained unchanged, but in proportion the height of second elution peak (P3, P4) corresponding to impurities decreased (FIGS. 2E - 2F). This observation indicated that the second elution-67-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 peak (P3, P4) contained endotoxins and related impurities, which would be removed by the detergent. Noteworthy, endotoxins are not stained by Coomassie and these impurities were therefore not visible on Coomassie-stained SDS gels (FIGS. 2A - 2F). Indeed, LAL assays confirmed the presence of endotoxins and successful removal of such by implementing the Triton X-l 14 addition (FIGS. 2G - 2H). Overall, these data confirmed that endotoxins can be efficiently removed during ion exchange chromatography without significantly increasing the process time. Noteworthy, Applicant’s chromatography setup was housed in a fridge with a constant temperature of 4°C, so there was no risk of phase separation of Triton X-l 14 mixtures.

[0287] CPMV can be rapidly purified by combining pH treatment, ultrafiltration and ion exchange chromatography[0288[ After individually optimizing extraction, ultrafiltration and polishing steps via ion exchange chromatography, Applicant then combined these steps into a single process for scalable purification of CPMV from infected leaf material. Applicant replaced the 500 kDa ultrafiltration membrane in the final UF / DF process with a 300 kDa membrane, increasing the recovery of CPMV in this step from -80% to -90% (FIG. 10). Because ultrafiltration is the core step of the novel, scalable purification process, Applicant termed this process ‘UF / DF process’ - opposed to ‘centrifugation process’, which is the core step of the contemporary method for CPMV purification.

[0289] Whereas the centrifugation process for purification of CPMV consisted of 15 steps with 2 overnight steps making this a 3-day process (FIG. 3A) corresponding to -20 working hours, the UF / DF process only required 7 steps (FIG. 3B) completed in less than 7 hours (in a single day) to achieve a purity >99% (FIG. 3C); this was the same purity level as in the centrifugation process.

[0290] The reduced process time with the UF / DF process compared to the centrifugation process can be attributed to the removal of bulk water during ultrafiltration, reducing the sample volume that has to be processed. Additionally, three process steps needed for endotoxin removal in the centrifugation process were eliminated in the UF / DF process by combining endotoxin removal through Triton X-l 14 with ion exchange chromatography.-68-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0291] The process yield was 0.189±0.016 g / kg (n=3) in the UF / DF process, which was -50% lower than the yield of 0.5 g / kg reported for centrifugation process

[0074] . Noteworthy, the yield reported for the centrifugation process (0.5 g / kg) does not include CPMV losses during endotoxin removal, which can be as high as 40% (Applicant’s unpublished data). This corresponds to a final yield of 0.3 g / kg for the centrifugation process. In the context of good manufacturing practices, the advantages of the UF / DF process - scalability, reduced process time, particle homogeneity and consistent removal of contaminants - outweighs the reduced yield. However, from a process economics perspective the low yield represents a limitation, requiring further optimization.

[0292] Major yield improvements for the UF / DF process can be expected by optimizing the 1000 kDa ultrafiltration step, which accounted for -50% of CPMV losses based on densitometric analysis (FIG. 3C). Similar to the 300 kDa ultrafiltration step, the 1000 kDa filtration step can be further optimized by testing other membrane pore sizes. Another option for increasing the process yields is to replace PES ultrafiltration membranes with more hydrophilic regenerated cellulose membranes, which have been shown to minimize target protein losses through adsorption to the membrane

[0046] , Alternatively, the process pH can be optimized to increase the step recovery. Specifically, a pH close to the isoelectric point of a protein (the net charge is zero) has been shown to drastically improve transmission during ultrafiltration

[0075] ,

[0293] Endotoxins and beta-glucans are efficiently removed in the UF / DF process

[0294] As the last step of process characterization, Applicant analyzed samples from the UF / DF process for the presence of endotoxins (also referred to as lipopolysaccharides). Applicant expanded Applicant’s analysis to beta-glucans (FIG. 3D), which have potential immunostimulatory properties

[0076] and can cause symptomatic infusion reactions

[0077] , As such, these impurities are receiving increasing attention in GMP process

[0077] ,

[0295] Applicant found that the majority of endotoxins and beta-glucans were removed during ultrafiltration using a 300 kDa membrane followed by ion exchange chromatography. Specifically, the endotoxin starting concentration in plant extracts was 3818.6±51.6 EU / mL; it decreased to 6.0±0.8 EU / mL after ultrafiltration and to <0.1 EU / mL after ion exchange-69-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 chromatography, respectively. These results matched values reported in the literature

[0078] , The starting concentration of beta-glucans in plant extracts was 194.0±0.5 ng / mL and decreased to 17.7±6.7 ng / mL after ultrafiltration and to 1.4±0.1 ng / mL after ion exchange chromatography (FIG. 3D). These results were in agreement with literature reporting the fractionation of polysaccharides with ultrafiltration (tangential flow filtration)

[0079] ,

[0296] The efficient removal of beta-glucans in the UF / DF process is a major improvement compared to the centrifugation process, where Applicant observed highly variable betaglucan levels ranging from 3.0-131.2 ng / mg per preparation (FIG. 11).

[0297] Characterization of CPMV preparations obtained from the UF / DF process vs. the centrifugation process

[0298] CPMV preparations purified either by the centrifugation process and the UF / DF processes (see FIG. 3) were then characterized in detail to confirm purity and integrity using a combination of methods: UV-Vis spectroscopy, denaturing SDS-PAGE, native agarose gel electrophoresis, transmission electron microscopy (TEM), size exclusion chromatography (SEC) and dynamic light scattering (DLS).

[0299] UV-Vis spectra of purified CPMV were measured with a NanoDrop 2000 spectrophotometer. An A260 / 280 ratio of ~1.7 indicates intact and pure CPMV; an A260 / 280 ratio of ?1.8 indicates the presence of nucleic acids in CPMV, whereas an A260 / 280 ratio of 0.8 indicates the absence of nucleic acids

[0074] , CPMV preparations that were purified by the UF / DF process - pH treatment, ultrafiltration and ion exchange chromatography - showed an A260 / 280 ratio of ~1.7 (Table 2), thus confirming that the UF / DF purification process is compatible with RNA cargos. UV-Vis spectra matched Applicant’s typical CPMV preparations [22, 48], and there was no significant difference (?=0.05, p-value >0.05, two- sided two-sample Welch’s t-test).|0300] Table 2: Characterization of CPMV formulation with UV-Vis spectroscopy.-70-4911-2869-8997.1Atty. Dkt. No.: 114198-3560[03011 Next, Applicant analyzed the electrophoretic mobility of the CPMV coat proteins and CPMV virions using denaturing and native gel electrophoresis, respectively (Figure 4A-C). Denaturing gel electrophoresis confirmed the presence of the large and small CPMV coat proteins at 42?kDa and 24?kDa, respectively

[0022] , Protein impurities were not detectable. Native gel electrophoresis confirmed that the RNA and protein component of CPMV comigrated, indicating that nucleoprotein assembles were intact virions (FIGS. 4B - 4C). Noteworthy, only a single electrophoretic form of CPMV was observed in preparations from the UF / DF process

[0048] , indicating that particles were uniform. In contrast, the centrifugation process yielded variable CPMV preparations, showing different electrophoretic forms of CPMV (FIG. 12). TEM imaging confirmed that CPMV from both processes was uniform and intact (FIG. 4D). Similarly, SEC elution profiles were consistent with pure CPMV preparations, eluting at 1 l-12?mL from a Superose 6 Increase SEC column with an A260 / 280 ratio of ~1.7. There were no signs of aggregation, degradation, or impurities (FIGS. 4E - 4F). Finally, nanoparticle sizing was carried out using DLS (FIGS. 4G - 4H), which confirmed monodisperse 30 nm-sized particles with low poly dispersity indices of 0.06 (UF / DF process) and 0.02 (centrifugation process), which matched Applicant’s previous studies [22, 48], Overall, these characterization data indicated that CPMV preparations from both processes were not significantly different.

[0302] CPMV retained its biological activity when purified using the UF / DF process[03031 Next, Applicant compared the biological activity of CPMV preparations from both processes, using an A20 lymphoma mouse model. Tumors were established by injecting 2* 105A20 lymphoma cells intradermally (i.d.) into the right flank of female BALB / c mice

[0049] , and treatment began when tumors reached a volume of -30 mm3. Animals received-71-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 weekly doses of 20 pg CPMV, 100 jug CPMV, or PBS by intratumoral administration (i.t.) for three weeks (FIG. 5A). Treatment efficacy was measured by monitoring survival and tumor volumes. Applicant note that the 20 pg dose is lower than Applicant’s established dose of 100 pg CPMV resulting in a reduced biological activity.

[0304] PBS-treated mice were euthanized 31-33 days after tumor inoculation, when tumors reached a volume of 1,000 mm3(endpoint). At the same time, CPMV-treated mice showed a significantly improved survival (p<0.01 low dose, p<0.0001 high dose), and significantly reduced tumor volume (p<0.0001) for both doses (FIGS. 5B - 5E). Tumor growth curves were not significantly different between the CPMV groups (p>0.05), but survival was significantly higher when treated CPMV formulations from the UF / DF process compared to the centrifugation process, both at the low dose (p<0.0056) and at the high dose (p=0.0297). Six weeks after the last treatment with CPMV from the centrifugation process, 10% of the mice that received a low dose, and 40% of the mice that received a high dose were tumor free (FIG. 5B and FIG. 5C). These results matched a previous B-cell lymphoma study, reporting that 50% of mice were tumor free

[0049] , Treatment with CPMV from the UF / DF process demonstrated strong efficacy at both doses, with 80% of the mice that received a low dose, and 90% of the mice that received a high dose remaining tumor-free after the same time (FIGS. 5B - 5C) These results were in good agreement with Applicant’s previous studies [1], confirming that CPMV retained its biological activity when subjected to pH treatment, ultrafiltration and ion exchange chromatography.[03051 CPMV from both processes induces a similar chemokine / cytokine response[0306J To further characterize potential differences in biological activity of CPMV from the centrifugation process and the UF / DF process, Applicant next analyzed the chemokine and cytokine response after treatment. Applicant used the same A20 lymphoma mouse model and established tumors as described above. When tumors reached a volume of ~60 mm3, mice received a single dose of PBS or 100 pg CPMV by intratumoral administration (FIG. 6A). Applicant notes that tumors were harvested 24 hours after injection of PBS or CPMV, because they started shrinking rapidly after treatment. Tumors were extracted by sonication-72-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 and Applicant analyzed the concentration of cytokines and chemokines in extracts with a customizable U-PLEX MSD assay.

[0307] Applicant found that treatment with CPMV from both processes induced similar chemokine / cytokine profiles, but CPMV from the UF / DF process induced a more potent immune response (FIG. 6B). Specifically, Applicant measured significantly higher concentrations of IFN-a, IFN-P, IL-6 and MCP-1 compared to treatment with PBS (FIG. 6C). Albeit not significant, Applicant also noticed a trend toward increased concentrations of GM-CSF and IL-12-p70. MCP-1 induces the recruitment of monocytes and dendritic cells

[0081] , whereas type I IFN (IFN-a and IFN-P) is a key component of antigen-specific immunity against viruses

[0073] , These results were consistent with the mode of action of CPMV, i.e. the activation of the innate immune system through the activation of pattern recognition receptors and stimulation of antiviral responses [81, 82], Differences in chemokine / cytokine concentrations can be explained with the early harvest time, considering that the activation of the adaptive immune system takes longer than 24 hours.

[0308] Overall these data confirm that CPMV from both process acts through the same mechanism of action. In Applicant’s analysis the only difference was the potency of both formulations.

[0309] Experimental Conclusions

[0310] Toward translation of drug candidates, besides demonstrating efficacy and mechanism of action of an identified lead candidate, the development of scalable and GMP-compatible processes for manufacturing is an important goal. Here, Applicant developed a scalable purification process for the immunotherapy candidate CPMV. By making use of the virions’ properties, Applicant systemically screened suitable conditions for extraction, purification, and polishing. By exploiting the pH stability of CPMV, -80% of plant host cell proteins were removed during acidic extraction (pH range 4.0-5.0). Optimizing the extraction step also resulted in homogeneous CPMV formulations with cleaved S protein. This finding is important, because CPMV preparations produced with contemporary methods are heterogenous and contain a mixture of particles with cleaved and non-cleaved S protein

[0048] , This elimination of heterogeneity provides enhanced quality assurance and is expected to-73-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 streamline the regulatory approval processes. It is not clear whether there are differences in biological activity as a function of the cleavage state of the S protein. Applicant’s efficacy study comparing CPMV purified by centrifugation-based vs. UF / DF methods indicated an enhanced performance of CPMV purified using the UF / DF methods presented here. While treatment with CPVM from both methods induced a similar chemokines / cytokine response, CPMV from the UF / DF method was more potent as indicate by a U-PLEX MSD assay.[03111 The UF / DF process - in particular, the combination of ultrafiltration using a 1000 kDa and 300 kDa MWCO membrane efficiently allowed removal of impurities larger and smaller than CPMV while simultaneously removing bulk water from the process. The removal of bulk water is desirable to reduce process volumes, the equipment footprint and accelerating subsequent purification steps

[0083] , Finally, anion exchange chromatography was found effective for polishing as it enabled high-resolution separation of CPMV from remaining impurities. The resolution increased during chromatography scaleup, i.e. the robustness improved with the process scale, which will facilitate translation of Applicant’s CPMV manufacturing process.[0312| Together, the presented UF / DF process significantly reduced processing time from a 3-day and ~20 working hours to a ~7-hour process that can be completed in single day.

[0313] Considering that the costs of new therapies may be a major determinate of whether these therapies will ultimately become available to patients

[0084] , the development of cost effective non-laborious processes is highly desirable

[0085] , In Applicant’s process Applicant have reduced direct labor, which accounts for -15% of cost of goods (depending on the product

[0086] ) by more than half. Additionally, Applicant have replaced ultracentrifugation, which is cost-ineffective due to high costs for instruments, maintenance and consumables

[0085] with cost-effective ultrafiltration. These improvements are conductive for the translation of the new production process.

[0314] The UF / DF process yielded CPMV particles with matched particle properties but improved purity (streamlined and more consistent removal of LPS and P-glucan) and improved homogeneity (cleaved S). The UF / DF process exploits virion’s properties, pH stability, size, and surface charge, and thus can be adapted and customized for other VLPs.-74-4911-2869-8997.1Atty. Dkt. No.: 114198-3560The process does not degrade or remove the RNA of the nucleoprotein assembly and thus would be applicable also for purification of VLPs with custom RNA cargos

[0087] ,

[0315] Experiment No.: 2

[0316] CCMV Propagation

[0317] Plant cultivation and CCMV propagation10318] Black-eyed peas (Vigna unguiculate no. 5, Morgan County Seeds) were seeded in ProMix BX soil (Greenhouse Megastore) using 3 ’A” square pots (Greenhouse Megastore). Plants were cultivated in a walk-in growth chamber (Conviron) at 25 / 22°C (day / night cycle), 60% relative humidity, and -100,000 lux (16 h photo period) as previously described

[0023] , Seven days post-seeding, primary leaves were inoculated mechanically using 40 pL / leaf of 0.1-0.2 mg / mL CCMV in 0.01 M sodium phosphate supplemented with 0.01 M magnesium chloride, (pH 6.0), as previously described [24, 25], After inoculation, the plants were incubated in a dark room at room temperature for 1 hour. They were then rinsed with tap water and returned to the growth chamber. After 14 days post-inoculation, the primary leaves were harvested and stored at -80°C until further processing. CCMV was purified from infected plant leaves using two protocols: the traditional purification protocol, designated as centrifuge-based, is described in detail within the Supporting Information and in ref.

[0026] , The novel process for CCMV purification using ultrafiltration, designated as UF / DF, is described below. The UF / DF process is comprised of three steps: extraction and pH precipitation, ultrafiltration, and ion exchange chromatography.10319] Extraction and pH precipitation

[0320] Frozen leaves were homogenized in 3 volumes per leaf mass (3 v / m) of extraction buffer (0.1 M MES) with a blender for 3, 30-second intervals, with 30-second breaks between blending intervals; Applicant used 0.1 M MES to prepare acidic extraction buffers with a pH of 3.0, 4.0, 5.0 and 6.0. After blending, the pH of the homogenate was re-adjusted to the extraction buffer pH using either 0.5 M hydrochloric acid or 0.5 M sodium hydroxide. For clarification, the homogenate was then filtered through two layers of Miracloth (Millipore Sigma), before centrifugation at 16,000*g for 20 minutes at 4°C to clarify the extract, using a JLA-16.250 rotor and an Avanti J-E centrifuge (Beckman Coulter).-75-4911-2869-8997.1Atty. Dkt. No.: 114198-3560[03211 Ultrafiltration

[0322] All ultrafiltration experiments were performed using a Minimate bench-top tangential flow filtration system (Cytiva), with a transmembrane pressure of 0.5 bar and a flow rate of 40 mL / min, using 50 cm2polyethersulfone membranes (Cytiva). Prior to ultrafiltration, all buffers and extracts were sterile filtered through 0.22 pm bottle top filters (Thermo Fisher) to remove larger impurities. Clarified and sterile filtered plant extracts were purified by sequential ultrafiltration through a 1000 kDa MWCO membrane and a 300 kDa MWCO membrane

[0020] ; during the 1000 kDa filtration step CCMV accumulates in the permeate, whereas during the 300 kDa ultrafiltration step CCMV accumulates in the retentate. The 300 kDa ultrafiltration step was used to replace the extraction buffer (0.1 M MES) with chromatography buffer (0.1 M sodium phosphate, pH 5.8). The ratio of extract to membrane area was kept constant at 4 mL / cm2in all experiments. In a typical experiment, 200 mL of the pH-adjusted and clarified extract was concentrated 4* before continuously adding buffer with the same pH as the extract to maintain a constant retentate volume. Retentates and permeates were sampled every 50 mL of permeate collected. Membranes used for ultrafiltration were regenerated by rinsing with 4 mL / cm2extraction buffer and 4 mL / cm2sodium hydroxide (1.0 M). The membranes were left to incubate overnight in sodium hydroxide solution, which was then removed by rinsing with 4 mL / cm2deionized water and 4 mL / cm220% (v / v) ethanol. The membranes were then stored in 20% (v / v) ethanol at 4°C. Membrane fouling was assessed through the normalized water capacity, measured before the first use and after regeneration as previously described

[0027] ,

[0323] Ion exchange chromatography

[0324] After purification by ultrafiltration, samples were further purified by ion exchange chromatography (AEX) using an AKTApure system (Cytiva) and 1.0 mL columns containing HiTrap Q Sepharose Fast Flow resin (Cytiva). The volumetric flow rate on the column was 0.5 mL / min (0.8 m / h), corresponding to a contact time of 2 minutes. The temperature for these experiments was kept at a constant 4°C. In a typical experiment the chromatography resin was equilibrated in 5 column volumes (CV) of equilibration buffer (0.1 M sodium phosphate, pH 5.8). After equilibration the columns were loaded with 25 CV of-76-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 ultrafiltration-purified sample, followed by washing with 5 CV of equilibration buffer. The CCMV bound to the column was then eluted with a 0-100% gradient (over 1.4 minutes) of elution buffer (0.1 M sodium phosphate, 0.35 M sodium chloride, pH 5.8); fractions were collected with an autosampler. The columns were regenerated by washing with 5 CV equilibration buffer, 5 CV of 0.5 M sodium hydroxide, 5 CV regeneration buffer (0.1 M potassium phosphate, 1.0 M sodium chloride, pH 7.0), and 5 CV of 20% (v / v) ethanol as previously described

[0020] , The columns were stored in 20% ethanol (v / v) and kept at 4°C. Applicant note that the sodium phosphate elution buffer is prone to precipitation when stored at 4°C and has to be prepared freshly.

[0325] Desalting

[0326] After chromatography, CCMV was formulated into CCMV storage buffer (Table 3), using PD-10 desalting columns (Cytiva) as previously described

[0020] ,

[0327] Table 3: Buffers used for storage and characterization of CCMV[0328| Endotoxin and glucan quantification

[0329] The concentration of endotoxins in purified samples was measured with an Endosafe Nexgen-PTS spectrophotometer (Charles River Laboratories), using (0.05-5 EU / mL) Endosafe LAL cartridges according to the manufacturer’s recommendations (Charles River Laboratories). LAL Reagent Water (Charles River Laboratories) was used to dilute samples. Applicant used an endotoxin limit of 35.7 EU / mg for assays as previously established

[0028] ,

[0330] Protein quantification-n-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0331] The total soluble protein concentration in samples was quantified via a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) using bovine serum albumin standards within a concentration of 25 to 2,000 mg / L.|0332] Characterization of purified CCMV|0333] UV-Vis spectroscopy

[0334] UV-Vis spectra of CCMV were measured with a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific) using CCMV characterization buffer (Table 3). The concentration of CCMV was quantified using Beer’s law and an extinction coefficient of e = 5.85 mL mg-1cm-1

[0023] , The RNA-to-protein ratio in purified samples was also measured via UV-Vis with intact CCMV characterized by an A260 / A280 ratio of ~1.7

[0023] ,

[0335] SDS-PAGE and western blot analysis

[0336] Purified CCMV was analyzed on 4-12% Bis-Tris gels in MOPS buffer (Thermo Fisher Scientific) at 200 V for 45 mins followed by staining with Coomassie Brilliant Blue or used for western blotting as described before

[0029] , Rabbit anti-CCMV antibodies (custom- made, Pacific Immunology), and HRP-conjugated goat anti-rabbit antibodies (Thermo Fisher Scientific) were used to detect CCMV, and were diluted 1 :500 in 5% (m / v) milk powder in PBST, and 1 :5000 in the same buffer, respectively

[0030] , Gels and western blots were imaged using a FluorChem R system (ProteinSimple) and a densitometric analysis was performed using the device’s on board software.

[0337] Native agarose gel and electrophoresis

[0338] Purified CCMV samples were analyzed on 1.2% (w / v) agarose gels at a concentration of 1.0 mg / mL in 0.01 M sodium phosphate at 80 V for 40 minutes. GelRed (Thermo Fisher Scientific) was used to stain nucleic acids; Coomassie Brilliant Blue (0.25% w / v) was used to stain proteins.

[0339] Dynamic light scattering

[0340] A Zetasizer Nano ZSP / Zen5600 (Malvern Panalytical) was used to measure the hydrodynamic diameter of the purified CCMV at a concentration of 1.0 mg / mL in CCMV characterization buffer (Table 3).-78-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0341] Size exclusion chromatography[0342 j Purified CCMV samples were analyzed by size exclusion chromatography using an AKTApure system (Cytiva) and a Superose 6 Increase 10 / 300 GL column (Cytiva), using CCMV characterization buffer (Table 3) and a flow rate of 0.5 mg / mL

[0028] , An isocratic profile was used to elute fractions, and the absorbance was measured at 260 nm (nucleic acid) and 280 nm (protein), as previously described

[0031] ,[0343| Transmission electron microscopy[0344| Samples of purified CCMV at a concentration of 0.1 mg / mL in deionized water, were placed onto a 400-mesh formvar / carbon supported copper grids (Electron Microscopy Sciences). The grids were charged with a PELCOeasiGlow system (Ted Pella), and were negative-stained using 2.0% (w / v) uranyl acetate (Agar Scientific). The grids were then imaged by a JEM-1400 Plus electron microscope (Jeol Ltd.) at 50,000* and 80,000* magnification.

[0345] Bioconjugation of CCMV

[0346] CCMV was conjugated to fluorescein isothiocyanate (FITC) dissolved in ethanol by addition of 2000 equivalents of FITC per CCMV and incubation in 0.3 mL buffer (0.1 M sodium acetate, 0.001 M EDTA, pH 5.5) at 4°C overnight. This corresponds to a 3.7-fold molar excess per solvent-exposed lysine residue (180 subunits per CCMV * 3 lysine residues per subunit

[0032] ). While the optimal pH range for conjugation reactions using FITC is between pH 8.0 and 9.0, the CCMV conjugation reaction was performed at pH 5.8 because CCMV is unstable at neutral or alkaline pH. CCMV-FITC was purified either by ultracentrifugation or by UF / DF. Purification by ultracentrifugation was performed by pelleting CCMV-FITC at 210,000 *g for 1 h at 4°C over a 30% sucrose cushion, using a 50.2 Ti rotor and an Optima L-90K centrifuge (Beckman Coulter). Purification by UF / DF was performed with a 300 kDa MWCO polyethersulfone membrane, using a Minimate bench-top tangential flow filtration system (Cytiva), a transmembrane pressure of 0.5 bar and a flow rate of 50 mL / min. Bioconjugation reactions were purified for 30 min by UF / DF. The CCMV-FITC samples were characterized using the methods as describe above.

[0347] Experiment No. 2 - Results and discussion-79-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0348] Stability-based purification of CCMV using acidic extraction

[0349] CCMV has been reported to be stable in a pH range of 3.0-6.0

[0033] , Taking advantage of CCMV’s stability under acidic conditions, Applicant tested extraction buffers with a pH of 3.0, 4.0, 5.0, and 6.0. Applicant chose this pH range based on the stability range of CCMV and Applicant’s prior work showing that plant host cell proteins (HCPs) can be removed during acidic extraction

[0020] , Applicant analyzed process samples by gel electrophoresis and found that extraction at pH 3.0 achieved the highest purity (79%), but at the cost of losing -20% of CCMV during extraction (FIG. 13A). The -20% loss of CCMV may be attributed to precipitation of CCMV when passing through its isoelectric point of 3.7 during pH adjustments

[0034] , Extraction at pH 4.0 also resulted in a high purity (66%), but restored the recovery to -100%. Extraction buffers with a pH of 5.0 and 6.0 achieved a lower purity of 49% and 30%, which was consistent with the literature

[0027] , Thus, Applicant proceeded with extraction of CCMV at pH 4.0.

[0350] Size-based purification of CCMV using ultrafiltration

[0351] Once the extraction conditions for CCMV were optimized, Applicant then took advantage of CCMV’s Targe’ nanoscale size, measuring 28 nm in diameter, to separate it from the smaller plant HCPs. Specifically, CCMV has a molecular mass of 2.0* 104g / mol

[0035] , but behaves larger than its molecular mass, because it forms a ‘hollow’ sphere that contains the viral RNA. This size difference was leveraged to enable size-based purification methods such as ultrafiltration. Applicant tested the efficacy of ultrafiltration for the purification of CCMV using polyethersulfone (PES) membranes, using a membrane area of 50 cm2, a flow rate of 40 mL / min, and a transmembrane pressure of 0.5 bar. Initially, Applicant tested membranes with a molecular weight cut-off (MWCO) of 1000 kDa, 500 kDa and 300 kDa using MES buffer with a pH of 4.0. Applicant hypothesized that the PES membrane and remaining HCPs would both be positively charged under these conditions, thus increasing the electrostatic repulsion between both, and reducing membrane fouling

[0036] , Analysis of process samples by SDS-PAGE showed that CCMV passed the 1000 kDa and the 500 kDa membranes, but it was retained by the 300 kDa membrane (FIG. 17). These results were consistent with the purification of CPMV by UF / DF, which resembles CCMV in-80-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 structure and size

[0020] , By leveraging this effect, Applicant were able to remove impurities both larger (using a 1000 kDa MWCO membrane) and smaller (using a 300 kDa MWCO membrane) than CCMV (FIG. 13B), alongside bulk water. The recovery of CCMV was -80% in the 1000 kDa UF / DF step and -90% in the 300 kDa UF / DF step, which was consistent with Applicant’s previous work

[0020] ,

[0352] Charge-based purification of CCMV using ion exchange chromatography

[0353] To facilitate subsequent polishing via chromatography, Applicant formulated CCMV into 0.1 M sodium phosphate buffer pH 5.8 during UF / DF. Because CCMV has an isoelectric point of 3.7

[0034] , virions will have a net negative charge in this buffer, thus enabling the use of anion exchange chromatography (AEX) as final polishing step. Applicant used 1.0 mL chromatography columns (HiTrap Q Sepharose Fast Flow resin), a flow rate of 0.5 mL / min, 0.1 M sodium phosphate buffer pH 5.8 as the low salt buffer, and 0.1 M sodium phosphate buffer containing 0.35 M sodium chloride pH 5.8 as the high salt eluent. Ultrafiltration- purified samples were used as feed for the AEX step.

[0354] Analysis by SDS-PAGE revealed that CCMV was efficiently captured by the resin, i.e. only traces of CCMV were detected in the flowthrough and wash fractions (FIG. 13B). A 0-100% gradient over a duration of 1.4 minutes of the high salt eluent produced two peaks: the first elution peak was characterized by an A260 / 280 ratio of -1.7, which corresponds to CCMV (FIG. 1C

[0037] ), the second elution peak exhibited an inverted A260 / 280 absorbance ratio, corresponding to plant host cell proteins. After ion exchange chromatography, the purity of CCMV increased from -95% after ultrafiltration to 99.9% after chromatography, thus meeting the purity threshold for clinical application

[0038] , Further improvements of the resolution during chromatography can be expected during scaleup, as previously demonstrated

[0020] ,

[0355] Because some plant HCPs interacted strongly with the chromatography resin, Applicant used a previously established protocol for regeneration of chromatography columns by washing with 0.5 M sodium hydroxide and 0.1 M potassium phosphate containing 1.0 M sodium chloride (pH 7.0

[0020] ). This method for regenerating ion exchange chromatography columns was used for all subsequent experiments.-81-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0356] Noteworthy, Applicant did not measure endotoxin concentrations above the projected safety threshold of 35.7 EU / mg

[0028] in CCMV preparations even without the use of detergents or endotoxin removal columns. The effect appeared to be linked to the acidic pH during purification, because Applicant measured endotoxin concentrations above the threshold in a similar process, featuring a neutral pH during purification

[0020] , A possible explanation for this effect is the inactivation of endotoxins through structural changes under acidic conditions as previously postulated in the literature

[0039] ,

[0357] Combining acidic extraction, ultrafiltration and ion exchange chromatography for scalable CCMV purification

[0358] After individually optimizing extraction, ultrafiltration, and ion exchange chromatography, Applicant combined these steps into a single process for scalable purification of CCMV from plant extracts. Consistently with Applicant’s previous work

[0020] , Applicant termed the novel CCMV purification process ‘UF / DF process’ whereas the traditional method for CCMV purification was termed ‘centrifugation process’ (FIG. 14).

[0359] Traditionally, the centrifugation process for CCMV purification consists of 13 steps, including one overnight step, making this a 2-3 day process

[0026] , However, Applicant found that Applicant can modify Applicant’s previously established CPMV protocol

[0020] and tailor it to CCMV to purify it at an expedited rate (FIG. 18). The novel process has 7 steps, can be completed in ~7 hours and achieved a purity >99%, which matched the purity level of the centrifugation process. The yield of the novel CCMV purification process was 0.22±0.01 g / kg, which is comparable to other state of the art processes for purification of CCMV

[0040] , The reduction in time required for the UF / DF process compared to the centrifugation method is primarily due to the removal of bulk water during ultrafiltration, thus reducing the sample volume that needs to be processed in subsequent process steps.

[0360] Characterization of CCMV preparations comparing the UF / DF and centrifugation process

[0361] CCMV preparations from the UF / DF process and the centrifugation process were characterized in detail to confirm purity and integrity using the following methods: native agarose gel electrophoresis, transmission electron microscopy (TEM), size exclusion-82-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 chromatography (SEC), and dynamic light scattering (DLS). The UV-Vis spectra of the purified CCMV were measured using a NanoDrop 2000 spectrophotometer. An A260 / 280 ratio of ~1.7 indicates intact and pure CCMV

[0037] , whereas an A260 / 280 ratio of -0.7 indicates the absence of nucleic acids

[0041] , CCMV preparations from the UF / DF process (pH treatment, ultrafiltration, and ion exchange chromatography) showed an A260 / 280 ratio of -1.7, thus indicating that the produced CCMV particles were intact and pure.[0362 J To further characterize the CCMV preparations from both processes, Applicant next analyzed the electrophoretic mobility of CCMV coat proteins and CCMV virions using denaturing and native gel electrophoresis, respectively (FIGS. 15A - 15C). Denaturing gel electrophoresis confirmed the presence of CCMV coat proteins at 20.3 kDa

[0042] ; impurities such as HCPs were not detected. Native agarose gel confirmed that both the protein and RNA components of CCMV co-migrated, indicating that the CCMV virions were intact and contained RNAFIGS. 15B - 13C). This observation is important, because CCMV has shown utility for gene delivery applications

[0022] , Because the CCMV RNA was not lost during purification by UF / DF and anion exchange chromatography, these methods could also be used for scalable purification CCMV preparations after in vitro RNA encapsulation, which typically involves disassembly and reassembly of the virions.

[0363] TEM imaging confirmed that the CCMV particles were intact and uniform (FIG. 15D). Additionally, SEC elution profiles aligned with that of pure CCMV, eluting at 11-12 mL from a Superose 6 Increase column, with an A260 / 280 ratio of -1.7. No signs of aggregation, impurities, or degradation were present (FIGS. 15E - 15F). Finally, DLS then confirmed the presence of monodisperse particles with a diameter of -28 nm, and poly dispersity indices of 0.142 (centrifugation process) and 0.301 (UF / DF process) (FIGS. 15G - 15H). Overall, this data indicated that there was no significant difference between the virus produced by the two different processes.[03641 Utility of UF / DF for clean-up of bioconjugation reactions

[0365] Various types of bioconjugation techniques have been established for plant viruses and viral nanoparticles in general, allowing functionalization post-harvest

[0043] ; modifications include the labeling with fluorophores to enable tracking and imaging, or addition of-83-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 functional cargos such as therapeutics or targeting ligands. In many cases multi-step conjugations are explored, requiring purification of the intermediates and final bioconjugate. The methods used are often based on centrifugation or size-exclusion chromatography

[0044] , thus matching the methods used to isolate the plant virus nanoparticles from the production host in the first place. To reduce time, costs, and enable scale-up, Applicant tested the suitability ofUF / DF in the clean-up of bioconjugates.

[0366] As a testbed Applicant used CCMV conjugated with fluorescein isothiocyanate (FITC), a fluorescent dye. Using a 20 mL scale, Applicant conjugated 20 mgs of CCMV with FITC using a 2000-fold excess of FITC and overnight incubation at 4°C. Applicant note that the optimal pH for lysine conjugation with isothiocyanates is alkaline, but Applicant used acidic conditions (pH 5.8) because CCMV loses integrity at neutral or alkaline pH

[0045] , A low conjugation efficiency was unproblematic, because the aim of this experiment was to optimize purification (i.e. removal of free dye) rather than conjugation.

[0367] The reaction was then cleaned-up using either UF / DF or centrifugation. Denaturing and non-denaturing gel electrophoresis confirmed covalent attachment of the FITC label to CCMV (Figure 4A-C), i.e. the fluorescent signal was co-localized with the CCMV coat protein or CCMV particles, respectively. Noteworthy, free FITC was efficiently removed during purification by UF / DF, whereas free FITC was still present after purification via centrifugation - highlighting that additional clean-up steps are required when using the contemporary purification method (FIGS. 16D - 16E).[0368J Applicant then analyzed the recovery of CCMV-FITC quantitatively comparing UF / DF vs centrifugation (FIG. 16F). Purification by UF / DF recovered 66.3±0.2% of CCMV-FITC conjugates free of impurities, whereas purification by centrifugation only recovered 47.7±0.2% of CCMV-FITC containing excess free FITC. These results were consistent with the presence of CCMV in the supernatants removed after centrifugation (FIG. 16). The recovery of CCMV-FITC after UF / DF increased to 94.9±0.3% when washing the UF / DF membrane with fresh buffer, indicating that some CCMV remained loosely bound to the ultrafiltration membrane (FIG. 16F ). Lastly, SEC confirmed that purified CCMV-FITC conjugates remained intact using either purification method (FIGS. 16G - 16H). Both-84-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 preparations showed a single elution peak and no aggregates or degradation products. Applicant note that FITC is strongly quenched under acidic conditions used for the chromatography

[0046] , resulting in a reduced signal during SEC. However, the fluorescence of FITC was readily restored when the pH was changed to neutral conditions.

[0369] Noteworthy, PES membranes used here for purification of CCMV-FITC conjugates are susceptible to dimethyl sulfoxide (DMSO), which is often used to dissolve dyes like FITC. To prevent damage to UF / DF membranes, dyes for labelling can also be dissolved in ethanol as done here. Alternatively, solvent-resistant membrane materials such as polytetrafluoroethylene (PTFE), nylon or hydrophilic regenerated cellulose (RC) can be used instead of PESU

[0047] .

[0370] In summary, purification of CCMV bioconjugation reactions by UF / DF removed free dye more efficiently than centrifugation and improved the recovery of CCMV-FITC conjugates up to 95%. Noteworthy, purification of CCMV-FITC by UF / DF was also faster than purification by centrifugation, reducing the processing time from 60 minutes to 30 minutes. These data confirm that UF / DF can be used as a scalable alternative for purification of functionalized plant viruses - the ability to scale conjugation reactions and their purification will further facilitate the translation of these materials.

[0371] Experimental Conclusions

[0372] Plant virus nanotechnology has matured into a platform technology that receives increasing attention in different areas of science, engineering, and technology

[0048] , With plant virus-derived nanomaterials entering the translational pathway and commercialization, the ability to produce these materials in a robust and scalable manner becomes increasingly important. Applicant established and optimized a protocol for the scalable purification of cowpea chlorotic mottle virus (CCMV), building on a previously established ultrafiltration / diafiltration (UF / DF) process

[0020] , By systematically utilizing the properties of CCMV virions, Applicant optimized extraction, ultrafiltration, and ion exchange chromatography for purification of CCMV. Specifically, an acidic buffer (pH 4.0) allowed to remove -80% of plant host cell proteins during extraction while recovering CCMV at near 100% yield. Two consecutive UF / DF steps, using a 1000 kDa and 300 kDa molecular weight-85-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 cut-off, removed impurities both larger and smaller than CCMV. Alongside impurities, bulk water was removed from the process during UF / DF, which reduced sample volumes, therefore reducing equipment footprints and accelerating successive purification steps

[0049] , Anion exchange chromatography was used to separate remaining impurities from the CCMV. When combining these steps, the process time for purification of CCMV was reduced from ~2-3 days with the centrifugation process to -7 hours with the UF / DF process. The UF / DF process yielded CCMV preparations that matched the properties of CCMV preparations from the centrifugation process. Purified CCMV virions were intact and contained RNA, thus enabling scalable purification of CCMV for gene delivery applications

[0022] ,

[0373] In Experiment No. 1, Applicant demonstrated utility of the UF / DF purification process for cowpea mosaic virus (CPMV). Minor tweaks enabled to transfer the method to CCMV, indicating that this method could find broad utility for the scalable purification of different plant-virus nanomaterials and their bioconjugates. The only major difference when purifying CPMV and CCMV with the UF / DF process was the process pH, which was neutral for CPMV and acidic for CCMV. Interestingly this modification led to efficient endotoxin removal even without the use of detergents like Triton X-100. A likely explanation are structural changes under acidic conditions, facilitating the removal of endotoxins during UF / DF and chromatography.

[0374] Importantly, Applicant found that the UF / DF method not only shortened processing time and improved the recovery of CCMV-FITC up to -95%, but also removed free FITC more efficiently. These data highlight that UF / DF can be viable alternative for purification of bioconjugation reactions, enabling the scale-up of these reactions. The ability to scalable the purification of CCMV and other plant virus-bioconjugates will further accelerate the translation of these nanomaterials.

[0375] Experiment No. 3 - Propagation and Purification From Apoplasts

[0376] Materials and methods

[0377] Plant cultivation

[0378] Nicotiana benthamiana (in-house seeds) and black-eyed peas (Vigna unguiculata no. 5, Morgan County Seeds) were seeded in 25x25x40 mm (L, W, H) stone wool blocks (Hort -86-4911-2869-8997.1Atty. Dkt. No.: 114198-3560Americas) and grown in an Al 000 chamber (Conviron) at 25 / 22°C (day / night cycle), 60% relative humidity and -100,000 lux (16 h photo period) as previously described

[0043] , Stonewool blocks were placed in RooTrimmer 1020 Trays (Greenhouse Megastore) constantly filled with 1 inch of tap water. Black-eyed peas received fertilizer (JR Peters, cat. 77860) once at a concentration of 0.5 g / L after seeding. N. benthamiana plants received the same fertilizer and concentration after seeding and then every 2 weeks of cultivation. N. benthamiana plants were grown for 4 weeks before they were used for experiments whereas black-eyed peas were grown for 1 week.10379] Simulated space conditions

[0380] Microgravity

[0381] Microgravity conditions were simulated with a custom-built random positioning machine (RPM). The device consists of two aluminum frames, which can be rotated independently using two RMD-L-7025 electrical engines. A raspberry pi (RPM) with a custom control software was used to rotate biological samples around three axes, resulting in randomization of the influence of gravity. An accelerometer (Adafruit LSM6DS3TRC) was used to monitor the acceleration of the samples, which were mounted in the center of the device to minimize residual acceleration. The RPM was operated at an average angular velocity of 7° / s for all experiments.

[0382] For plant growth experiments the RPM was housed in a walk-in growth chamber (MTS144, Conviron) to allow control of temperature, humidity and photo periods. In default experiments a temperature of 25 / 22°C (day / night cycle), 60% relative humidity and -100,000 lux (16 h photo period) were used as described above

[0043] , The position of the light source was outside of the RPM, thus preventing plants from orienting themselves based on the angle of light incidence. For all experiments static ground controls (1 xg) were grown under the same conditions.

[0383] Plants were infected with CPMV by mechanical inoculation of primary leaves after 2 days of exposure to simulated microgravity as previously described

[0043] , After infection with CPMV, plants were cultivated under simulated gravity conditions for 2 weeks before analyzing the effects on morphology, chlorophyll content and CPMV accumulation.-87-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0384] Oxidative stress[0385J The effect of oxidative stress on plant growth and CPMV yields was tested by spraying plants with hydrogen peroxide

[0044] , Plants were treated with 100 pM hydrogen peroxide in deionized water 3 times a day by spraying (~1.5 mL hydrogen peroxide per plant)

[0045] , starting 2 days before infecting plants with CPMV. Short-term oxidative stress was simulated by treating plants for 2 days after infection with CPMV; long-term oxidative stress was simulated by treatment with hydrogen peroxide for 14 days after infection with CPMV. Optionally, ROS stress was combined with temperature stress by cultivating plants at a temperature of 30°C instead of 25 °C for the entire duration of the experiment.

[0386] Chlorophyll measurements

[0387] Chlorophyll measurements were used to assess the physiological status of plants under simulated space conditions. The leaf chlorophyll content was estimated nondestructively using a SP AD-502 chlorophyll meter (Minolta) after 2 weeks of exposure to simulated space conditions (i.e., microgravity and optional oxidative stress and change in temperature). The average function of the device was used to calculate the chlorophyll content from 6 distinct spots on the top side of leaves, avoiding major veins. Before each use, the chlorophyll meter was calibrated according to the manufacturer’s instructions.]0388| Extraction with a blender

[0389] Extracts of whole leaves were prepared with a blender, using 3 volumes (3 v / m) of extraction buffer (0.1 M potassium phosphate, pH 7.0) as previously described

[0046] , Before analysis extracts were filtered through two layers of Miracloth (MilliporeSigma), and clarified by centrifugation at 16000*g for 30 min at 4°C, using an Avanti J-E centrifuge and a JLA 16.250 rotor (Beckman Coulter).

[0390] Infdtration-centrifugation[039.1] The infiltration-centrifugation technique was used to extract apoplastic fluid from plant leaves

[0023] , Briefly, freshly harvested leaves were infiltrated with buffer (Table 4) by applying vacuum (70 cm Hg) for 2 minutes, followed by a rapid release of the vacuum

[0047] , Leaves were then carefully dried with paper towels and the infiltrated buffer was recovered-88-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 by centrifugation at lOOOxg for 10 minutes at room temperature

[0023] , The eluate was either analyzed directly or further purified by ultrafiltration for scale-up experiments.

[0392] Table 4: Buffers used for infiltration-centrifugation.

[0393] Ultrafiltration / diafiltration (UF / DF)10394] All ultrafiltration experiments were carried out with a Minimate bench-top tangential flow (TFF) filtration system (Cytiva), using 50 cm2300 kDa polyethersulfone membranes (Cytiva), a transmembrane pressure of 0.5 bar and a flow rate of 40 mL / min. Sterile filtered extraction buffer (0.1 M potassium phosphate, pH 7.0) was used for all ultrafiltration experiments.10395] Ultrafiltration membranes were regenerated by rinsing with 4 mL / cm2extraction buffer and 4 mL / cm2sodium hydroxide (1.0 M), followed by incubation overnight as previously described

[0048] , Residual sodium hydroxide was removed by washing with 4 mL / cm2deionized water and 4 mL / cm220% (v / v) ethanol. The latter was also used for storage of membranes.

[0396] Analysis of process samples

[0397] Bicinchoninic acid (BCA) Assay

[0398] The concentration of total soluble protein in process samples was quantified with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions, using 8 bovine serum albumin standards in the range of 25 to 2,000 mg / L. Samples and standards were measured in triplicates.

[0399] Enzyme-Linked Immunosorbent Assay (ELISA)-89-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0400] The concentration of CPMV in process samples was quantified with a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) kit (Agdia) according to the manufacturer’s instructions, using six CPMV standards with a concentration of 0.0 to 0.6 pg / mL. Deviating from the manufacturer’s instructions, plates were blocked with 200 pL of 3% (w / v) BSA before adding standards or samples (100 pL) to the wells. All incubation steps were carried out at 25°C on a rotary shaker at 100 rpm for 1 h. Between incubation steps, plates were washed three times with 200 pL PBS-T (PBS, pH 7.4 with 0.05% (v / v) Tween 20). Samples and standards were measured in triplicates.10401] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

[0402] Process samples were separated on 4-12% Bis-Tris gels in MOPS buffer (Thermo Fisher Scientific) at 200 V for 45 min. Gels were subsequently stained with Coomassie Brilliant Blue or used for western blotting as previously described

[0042] , CPMV was detected using a rabbit anti-CPMV primary antibody and an HRP-conjugated goat anti-rabbit secondary antibody

[0048] , The primary antibody was diluted 1 :500 in 5% (m / v) milk powder in PBST and the secondary antibody was diluted 1 :5000 in the same buffer

[0049] , Gels were scanned with a FluorChem R system (ProteinSimple) and densitometric analysis was performed with the on-board software of the device.

[0403] Transmission electron microscopy (TEM)

[0404] Process samples were analyzed by TEM using 400-mesh formvar / carbon supported copper grids (Electron Microscopy Sciences) that were charged with a PELCOeasiGlow system (Ted Pella). Grids were negatively stained with 2.0% (w / v) uranyl acetate (Agar Scientific) and imaged at 20,000* or 80,000* magnification and 80 KeV using a JEOL 1400Plus transmission electron microscope (Jeol Ltd.) as previously described

[0048] ,

[0405] Experimental Results and Discussion

[0406] CPMV nanoparticles can be eluted from the apoplast without disrupting plant tissue1 407] While accumulation of CPMV has not been reported in the apoplast, some plant viruses, such as potato virus X have been found in the extracellular space [50, 51] - thus Applicant hypothesized that - if Applicant can confirm accumulation of CPMV in the-90-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 apoplast - CPMV could also be purified from the apoplast without disrupting the plant tissue. To test Applicant’s hypothesis, Applicant infected black-eyed peas no. 5 with wildtype CPMV through mechanical inoculation and extracted the apoplastic fluid from infected leaves using the infiltration-centrifugation method

[0023] , The method has two steps: first, leaves were submerged in buffer (0.1 M potassium phosphate, pH 7.0) and subjected to vacuum. The vacuum is then rapidly removed to drive infiltration of the buffer into the apoplastic space (FIG. 20A). Second, the infiltrated buffer was recovered from the leaves by centrifugation (FIG. 20A). Applicant termed these process samples ‘eluates’, as opposed to ‘extracts’ when disrupting plant tissue with a blender.

[0408] Applicant analyzed eluates from the infiltration-centrifugation method in comparison with blender extracts by SDS-PAGE and staining with Coomassie Blue (FIG. 20B) and western blotting (FIG. 20C). Indeed, the small (24 kDa) and large (42 kDa) CPMV coat proteins were the dominant proteins in apoplast eluates from CPMV infected leaves. Western blotting with anti-CPMV antibodies confirmed the identity of the small land large coat proteins. When comparing blender extracts and apoplast eluates, Applicant found that the purity of CPMV increased ~200-fold, i.e. from -0.1% to -20% (based on densitometric analysis), because major plant host cell proteins (HCPs) were absent in apoplast eluates (FIG. 20B). This observation highlights that the plant tissue was not damaged during the infiltration-centrifugation method - because this would release intracellular proteins such as RuBisCO and other HCPs, as is seen in the process samples from the extracts. To verify that the process was not damaging only accessing the apoplast, Applicant infiltrated leaves with buffer containing blue carbon dots as a tracer. Confocal imaging of the leaves showed the chlorophyll inside the plant cells (red) and carbon dots (blue) excluded from plant cells, i.e. within the apoplast. did not overlap, indicating that the carbon dots did not mix with the content of the plant cells (FIG. 25).[04091 SDS-PAGE revealed a greater number of HCPs in eluates from control vs CPMV- infected plants (FIG. 20B), indicating that infection with CPMV changed the expression of plant HCPs in the apoplast. This observation is consistent with literature investigating the effect of potato virus X infections on the apoplast proteome of N. benthamiana

[0051] , The suppression of HCP expression further simplifies the purification of virus particles from the -91-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 apoplast and is thus synergistic with Applicant’s aim to simplify plant-based purification processes.

[0410] Importantly, imaging by transmission electron microscopy (TEM) confirmed that CPMV particles eluted from the apoplast were intact (FIG. 20D). These data demonstrate that it is possible to eluate intact CPMV from the apoplast without breaking the plant tissue. To Applicant’s knowledge, this is the first time that intact CPMV particles have been reported in and isolated from the apoplast.

[0411] Simulated space conditions affect plant morphology and plant physiology

[0412] After establishing that intact CPMV can be eluted from the apoplast, Applicant next investigated how the space environment affects this process. Major stressors that affect plants in space include altered gravity and space radiation

[0052] , both of which generate reactive oxygen species (ROS) in plants

[0053] , To simulate microgravity on the ground, Applicant customized a random positioning machine (RPM) for plants (FIG. 21). The mean effective gravity in the RPM was 0.067xg (FIGS. 21 A - 21B), and thus comparable with similar setups for earth-bound spaceflight experiments

[0054] , The light source was mounted outside of the RPM, because light acts as a substitute for gravity in growth regulation

[0055] , Optionally, plants were sprayed with hydrogen peroxide (100 pM, 3 times per day

[0045] ) - this mimics ROS stress that plants experience during spaceflight due to radiation

[0044] , ROS treatment started 2 days before infecting primary leaves with CPMV and continued until 2 days post infection (dpi) for a short-term treatment group, and until 14 dpi for a long-term treatment group.

[0413] First, Applicant grew non-infected control plants to assess plant growth and health. When comparing the morphology of black-eyed pea plants that were subjected to simulated space conditions with l *g controls, Applicant found that the simulated space environment induced a compact, spherical morphology (FIGS. 22C - 22D). The effect was reproducible also when growing N benthamiana (FIGS. 22C - 22D). This effect was interesting because it can potentially be exploited to grow plants into a more compact shape, thus allowing to accommodate more plants per growth area, and ultimately saving mission resources.-92-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0414] Next, Applicant assessed the effects of simulated microgravity and ROS stress on the plant physiology using the chlorophyll content in leaves as an indicator of photosynthetic function. Applicant found that chlorophyll levels in plants grown under microgravity were slightly reduced compared to 1 *g controls, but the effect was not significant in primary (p=0.45) or secondary (p=0.31) leaves (FIGS. 22E - 22F) and thus consistent with the literature. This finding indicated that microgravity alone had a limited impact on plant health and that additional stressors may be required to elicit significant physiological changes

[0056] , Infection with CPMV significantly reduced the chlorophyll content in primary leaves (t-test, a=0.05, pO.OOOl, n=3), and secondary leaves (t-test, a=0.05, p=0.0023, n=3) compared to non-infected control plants. The reduction was strongest when combining CPMV infections with ROS stress; secondary leaves were most impacted (FIGS. 22E - 22F). Because only primary leaves were directly infected with CPMV, Applicant concluded that ROS stress promoted spreading of CPMV infections to secondary leaves.

[0415] Overall, these data indicated that plant growth under microgravity and ROS stress affected the severity of CPVM infections in plants, especially in secondary leaves. Applicant next investigated this effect in more detail by quantifying CPMV in primary and secondary leaves.

[0416] CPMV accumulation increased under simulated space conditions

[0417] Because ROS stress appeared to affect the severity of CPMV-infections, Applicant next investigated the accumulation levels of CPMV under ROS stress. Applicant’s hypothesis was that an increased susceptibility of plants towards viruses under space conditions could be exploited to increase the production yields of CPMV. Applicant simulated short-term and long-term ROS stress as described above, and also tested ambient (25°C) and elevated temperatures (30°C), because temperature fluctuations have been observed in space growth systems due to technical problems

[0057] , Applicant conducted these experiments under regular gravity (1 xg) to assess the potential utility under terrestrial conditions.

[0418] Applicant used a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) to quantify CPMV in primary and secondary leaves

[0058] , CPMV was extracted with a blender to assess the total CPMV accumulation. CPMV accumulation levels-93-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 in primary leaves in control plants were in the range of 20 g / L (5 g / kg leaves) and thus on the upper end of values reported under terrestrial conditions

[0058] , Constant exposure to elevated temperatures (30°C) reduced CPMV accumulation levels in primary and secondary leaves compared to ambient (25°C) controls (FIG. 22). Short-term ROS stress also resulted in decreased CPMV accumulation in both primary and secondary leaves at 25°C and at 30°C (FIG. 23). In contrast, long-term ROS stress resulted in increased CPMV accumulation in both primary and secondary leaves (FIG. 23). The effect was significant in secondary leaves, but not in primary leaves (FIG. 23), indicating that continuous ROS stress promoted spreading of CPMV in plants.

[0419] The observed temperature-dependence of virus-host interactions is consistent with the literature

[0059] , and has previously been linked to the activity of heat shock proteins

[0060] , Noteworthy, the effect of elevated temperatures on virus accumulation appears to be time dependent

[0059] , Specifically, short-term exposure to elevated temperatures often increased virus accumulation, whereas long-term exposure to elevated temperatures reduced virus accumulation in plants

[0059] , The optimal temperature for maximizing CPMV yields is likely around 25°C, because Applicant observed reduced yields at 30°C comparted to 25°C (FIG. 23) and symptoms of viral infection were not detected when plants were incubated at 20°C (FIG. 26)

[0420] Reduced CPMV accumulation levels after short-term exposure to ROS stress can likely be explained with the activation of antioxidant and pathogenesis-related genes in plants at low concentrations of hydrogen peroxide [61-63], For example, ROS stress induces peroxidase-mediated cross-linking of cell wall proteins, thus reinforcing the latter against pathogen infections. Second, hydrogen peroxide is thought to induce defense genes during pathogen infection

[0064] , In the literature, low concentrations of exogenously applied hydrogen peroxide have been documented to prime plants against various abiotic [65, 66] and biotic stressors [67-69], Applicant have observed a similar effect when studying antibacterial polymers for protection of plant health, where Applicant also observed short-term ROS stress, and increased resistance to pathogen infections (Palomino et al., Polynorbornene Spray Coating to Enhance Plant Health, in review). Overall these data indicate that short-term-94-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 exposure to ROS species can promote the resistance of plants towards CPMV and potentially other plant pathogens.

[0421] A potential explanation for the increased accumulation of CPMV after long-term exposure to ROS stress is that ROS may promote viral replication by mediating the oxidation of viral factors

[0061] , Additionally, the reduced activity of ribonucleotide reductase, which catalyzes the rate limiting step of DNA synthesis

[0070] , under oxidative conditions may favor the replication of RNA viruses

[0061] , CPMV is an RNA virus

[0071] , which is consistent with this theory.

[0422] On board of spacecrafts, plants likely experience long-term oxidative stress

[0044] , and Applicant’s data suggest that this will increase CPMV yields during space flight. From a process resilience perspective, plant molecular farming of CPMV is therefore well suited for space applications. This finding also has practical implications for plant molecular farming on the ground because exogenous treatment with reactive oxygen species (ROS) can easily be automated, thus allowing to increase CPMV yields under terrestrial conditions.

[0423] Elution of CPMV from the apoplast is scalable

[0424] To assess the potential for scalability, Applicant next tested the simplified production process with more than a dozen plants at a time (grown under 1 xg). To remove remaining impurities in apoplast eluates, Applicant combined the infiltration-centrifugation method with ultrafiltration / diafiltration (UF / DF) to ensure high purity (typically >99%

[0072] ), which is a prerequisite for the clinical application of plant virus-based therapeutics. The resulting process had 5 steps which can be completed in less than 2 hours (FIG. 24A).

[0425] Applicant found that UF / DF with a 300 kDa molecular weight cut-off (MWCO) membrane retained the majority of CPMV whereas impurities were washed out in the permeate (FIG. 24B). These results were consistent with the size of common impurities in the apoplast. For instance, HCPs in the apoplast are typically <50 kDa

[0073] , phenolics range up to ~5 kDa

[0074] and pigments like chlorophyll range up to ~1 kDa

[0075] - all are orders of magnitude smaller than CPMV.

[0426] The purity of CPMV preparations from infiltration-centrifugation and UF / DF was >99%, thus matching the purity threshold for clinical application. The process yield was 0.49 -95-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 mg from 100 g leaves, which is low compared to purification of CPMV by blender extraction and UF / DF

[0048] , Thus, there is room for optimization and a need to increase the process yield. For example, buffer recovery from leaves was -50% (Table 2), this indicates that this step could be optimized to maximize recovery, i.e. through optimization of the technique or buffers used for infiltration-centrifugation.

[0427] Making use of the broad pH stability of CPMV, Applicant tested acidic (pH 4.0), neutral (pH 7.0) and alkaline (pH 9.0) buffers for elution of CPMV from the apoplast (FIGS. 24B - 24C). Applicant found that alkaline extraction buffers (pH 9.0) achieved a higher CPMV purity (85%), compared to neutral (20%) or acidic elution buffers (24%). This observation was surprising at first, because acidic buffers removed plant HCPs efficiently from extracts of whole plants

[0048] , However, unlike the cytosol of plant cells which has a neutral or slightly alkaline pH

[0076] , the apoplast is an acidic environment with a pH as low as 4.5 [77, 78], HCPs in this plant cell compartment should thus be accustomed to acidic conditions. This explains why HCPs are not removed when using acidic extraction buffers.Acidic extraction conditions were accompanied by a reduced recovery of CPMV (FIGS. 24B - 24C), which was consistent with Applicant’s previous work

[0048] , This effect can likely be attributed to a reduced solubility of CPMV close to its isoelectric point of 5.5

[0079] , Vice versa, the higher recovery of CPMV with alkaline buffers might be attributed to an increased (net negative) surface charge, increasing electrostatic repulsion and thus reducing protein aggregation and minimizing non-specific interactions.[0428| Table 4: Infiltration-centrifugation process metrics during scale-up.-96-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0429] Characterization of CPMV after elution from the apoplast and purification by UF / DF

[0430] CPMV preparations from the infiltration-centrifugation and UF / DF purification process (FIG. 23A) were characterized to confirm the purity and integrity of the particles, using native agarose gel electrophoresis, size exclusion chromatography (SEC), and transmission electron microscopy (TEM). Native gel electrophoresis confirmed the colocalization of the viral RNA and CPMV coat protein, indicating that CPMV particles were intact (FIG. 23E). Typical CPMV preparations obtained from blender extracts contain a slow and fast electrophoretic form of CPMV reflecting a mixture of particles with cleaved and uncleaved S protein

[0080] , CPMV purified from the apoplast predominantly consisted of the fast electrophoretic form, which was consistent with the cleavage of the S protein under acidic conditions

[0048] - the apoplast is an acidic environment as discussed above. SEC revealed the typical elution profiles of intact CPMV preparations eluting at ~12 mL from a Superose 6 Increase column with an A260 / 280 ratio of ~1.7

[0048] , There were no signs of aggregation, degradation, or impurities, indicating that the latter were efficiently removed during UF / DF with a 300 kDa MWCO membrane (FIG. 23F). TEM imaging confirmed that CPMV obtained from the infiltration-centrifugation and UF / DF purification processes was uniform and intact (FIG. 23G). Overall, this data confirmed that Applicant’s simplified purification process can produce intact and pure CPMV.

[0431] Experimental Conclusions

[0432] Applicant found that intact particles of cowpea mosaic virus (CPMV) can be eluted from the apoplast without disrupting the plant tissue. To the best of Applicant’s knowledge, this is the first time this strategy has been used to purify CPMV (or other plant viruses) from plants. Remaining impurities in apoplast eluates were readily removed with ultrafiltration / diafiltration (UF / DF) by utilizing the size difference between CPMV and impurities. Specifically, CPMV has a molecular mass of 5.6x 103 kDa

[0084] , thus exceeding the molecular mass of impurities like host cell proteins (<50 kDa

[0073] ), phenolics (-5 kDa

[0074] ) or pigments (-1 kDa

[0075] ) by more than one order of magnitude.

[0433] Other than CPMV, viral particles of potato virus X (PVX) have also been detected in the apoplast

[0051] , indicating that elution from the apoplast and subsequent purification by-97-4911-2869-8997.1Atty. Dkt. No.: 114198-3560UF / DF is not limited to CPMV. Further, strategies to secrete pharmaceutically relevant proteins such as antibodies to the apoplast have been developed

[0024] , indicating a broader utility of the simplified purification process.

[0434] Because the plant tissue was not destroyed when extracting CPMV with the infiltration-centrifugation method, repeated harvest cycles are possible, thus offering a high level of utility. Moreover, the process was successfully transferred to more than a dozen plants at a time and thus deemed scalable.

[0435] Whereas space flight-induced stress has so far mainly been perceived as a threat for plants, Applicant’s data show that it can also be an opportunity to increase productivities in the context of plant molecular farming. Specifically, Applicant found that CPMV accumulation in plants decreased after short-term exposure to ROS stress but increased after long-term exposure to ROS stress. The duality of the role of ROS in resistance and susceptibility of plants to viral infections is consistent with the literature

[0061] , Applicant’s findings also have practical implications for plant molecular farming on the ground, because timed exposure to ROS stress could be used to protect plants from pathogens or increase yields in the context of plant molecular farming. Conversely to terrestrial stressors which typically decrease recombinant protein expression in plants because they can induce stress- related proteins that compete with the recombinant protein machinery [87-89], space-flight induced stress improved the accumulation of CPMV in plants. The improved process resilience is highly desirable for applications in space and on the ground. For instance, resilience in pharma production is one of the main arguments in the EU horizon 2020 program and the Horizon Europe 2025-2027 program, which is also aligned with the 2022 National Biotechnology & Biomanufacturing Initiative (NBBI) in the US.

[0436] In sum, this disclosure provides a scalable, non-laborious, orthogonal purification processes for virus-like particles (VLPs) from plants that omit ultracentrifugation, toxic organic solvents, and complex multi-day workflows. The disclosure systematically leverages intrinsic physical / chemical properties of plant viruses — such as broad pH stability, nanoscale size, and charged surfaces — to enable simplified purification in any environment, including resource-limited and microgravity environments (e.g. spaceflight). In several embodiments,-98-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 the methods combines: controlled extraction conditions (acidic or basic, tuned to the VLP pH stability range) to precipitate most host cell proteins while preserving intact virions; sequential ultrafiltration / diafiltration steps with different molecular weight cut-offs to remove impurities both larger and smaller than the target VLP, while reducing bulk water volume; ion exchange chromatography to perform a final polishing step, removing remaining contaminants including endotoxins and P-glucans to achieve >99% purity.

[0437] The methods yields high-purity, intact plant-derived VLPs in a single day, suitable for therapeutic applications (including RNA-loaded VLP drug delivery), and is transferable to multiple plant viruses (e.g. CPMV, CCMV, PVX). It is designed to be adaptable to microgravity and other constrained settings via equipment-light procedures such as infiltration-centrifugation.[0438| Current state-of-the-art methods for purifying plant-derived VLPs, such as chloroform / butanol extraction, PEG precipitation, and multiple rounds of high-speed or isopycnic ultracentrifugation, have significant drawbacks:

[0439] Scalability limitations: ultracentrifugation and gradient separations are impractical or impossible to scale for large-volume biomanufacturing.[0440| Process complexity and duration: multi-day, multi-step workflows with high labor demand (e.g. 15+ unit operations over 2-3 days).

[0441] Safety hazards: use of toxic organic solvents (chloroform, methanol), which pose risks to operators and require stringent handling / disposal.

[0442] Suitability in constrained environments: existing methods are unsuited for low- resource conditions, including outer space or field deployment, due to bulky equipment, high energy demand, and need for cold chains.

[0443] Regulatory compliance: difficulty in reproducibly achieving the >99% purity and consistent removal of contaminants (e.g. endotoxins, P-glucans) required for human therapeutics.

[0444] The disclosure invention solves the technical bottleneck of producing therapeutic- grade plant VLPs rapidly, safely, and scalably without reliance on equipment or solvents that-99-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 make current processes slow, cost-ineffective, unsafe, or impractical in microgravity or low- resource settings. It enables plug-and-play manufacturing of VLP -based nanomedicines for on-Earth and space missions.|0445] Clauses

[0446] Clause 1. A method of harvesting virus-like particles (VLPs) from plant tissue comprising: (i) extracting the VLPs from the plant tissue using an extraction solution, wherein the VLPs are collected in the eluate; (ii) purifying the VLPs collected in the extraction solution of step (i) through a series of separations based on size; and (iii) purifying the VLPs from step (ii) through separation based on charge, thereby harvesting the VLPs.

[0447] Clause 2. A method of harvesting virus-like particles (VLPs) from plant tissue comprising: (i) extracting the VLPs from the plant tissue using an acidic extraction solution collecting the VLP-containing eluate; (ii) filtering the eluate from step (i) through a series of filtration membranes with serially decreasing pore size and collecting a the VLPs, wherein the VLPs are collected in the eluate or the membrane; and (iii) separating the VLPs from the filtration of step (ii) with ion exchange chromatography to harvest the VLPs, thereby harvesting the VLPs.

[0448] Clause 3. A method of harvesting virus-like particles (VLPs) from plant tissue comprising: (i) extracting the VLPs from plant tissue using a basic extraction solution collecting the VLP-containing first eluate; (ii) filtering the first eluate from step (i) with a series of ultrafiltration membranes with serially decreasing pore size and collecting the VLPs, wherein the VLPs are collected in the eluate or the membrane; and (iii) separating the VLPs from the filtration of step (ii) with ion exchange chromatography, thereby harvesting the VLPs.

[0449] Clause 4. The method of any of clauses 1-3, wherein the method is performed in the absence of an ultracentrifugation step, optionally an isopycnic ultracentrifugation step.

[0450] Clause 5. The method of any one of clauses 1-4, wherein the method is performed in the absence of a chloroform or a methanol extraction step.-100-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0451] Clause 6. The method of any of clauses 2-5, wherein the harvested VLPs have a net negative charge.

[0452] Clause 7. The method of any of clauses 3-5, wherein the harvested VLPs have a net positive charge.

[0453] Clause 8. The method of any one of clauses 1-7, wherein the plant tissue comprises or consists essentially of leaf tissue, stem tissue, and / or root tissue.

[0454] Clause 9. The method of any one of clauses 1-7, wherein the plant tissue is from an intact plant.

[0455] Clause 10. The method of any of clauses 1-9, wherein the VLP are extracted from an intact plant in step (i) by vacuum infiltrating an apoplast with the extraction solution and collecting the extraction solution with centrifugation.

[0456] Clause 11. The method of any of clauses 1-10, wherein the extraction solution has: a pH of about 2.5 to about 4.5, optionally about 4.0 for acidic extraction, and further optionally wherein the VLP comprises Cowpea Chlorotic Mottle Virus (CCMV); or a pH of about 8.5 to about 9.5, optionally about 9.0, for basic extraction, and further optionally wherein the VLP comprises Cowpea Mosaic Virus (CPMV).

[0457] Clause 12. The method of any of clauses 2-11, wherein the series of membranes comprises a first membrane and a second membrane.

[0458] Clause 13. The method of clause 12, further comprising a third membrane.

[0459] Clause 14. The method of clause 12 or 13, wherein the first membrane has a pore size of about 0.1 pm to about 0.4 pm, optionally about 0.2 pm.

[0460] Clause 15. The method of any of clauses 12-14, wherein the second membrane has a molecular weight cut-off of about 2000kDa, or about lOOOkDa.

[0461] Clause 16. The method of any of clauses 13-15, wherein the third membrane has a molecular weight cut-off selected from of about lOOkDa to about 500kDa, about 300kDa to about 500kDa, or about 300kDa, or about 500kDa.-101-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0462] Clause 17. The method of clause 12, wherein the first membrane and second membrane average pores sizes are larger than the diameter of the VLP.

[0463] Clause 18. The method of clause 13, wherein the first and second membranes have average pore sizes larger than the diameter of the VLP and the third membrane have an average pore size smaller than the diameter of the VLP.

[0464] Clause 19. The method of any of clauses 1-18, wherein the VLP is derived from a plant virus, optionally wherein the VLP is a plant virus coat protein, optionally wherein the plant virus is Cowpea Mosaic Virus, Cowpea Chlorotic Mottle Virus, or Potato Virus X.

[0465] Clause 20. The method of any of clauses 2-19, wherein the ion exchange chromatography is anion exchange chromatography.

[0466] Clause 21. The method of any of clauses 2-19, wherein the ion exchange chromatography is cation exchange chromatography.

[0467] Clause 22. A method of generating plant virus-like particles, according to the method depicted in Figure IB.

[0468] Clause 23. The method of any of clauses 1-22, wherein the method is practiced in any gravity environment.

[0469] Clause 24. The method of any of clauses 1-22, wherein the method is practiced in a microgravity environment.

[0470] Clause 25. An isolated VLP or a population of VLPs harvested by the method of any one of clauses 1-24.

[0471] Clause 26. The VLPs of clause 25 and a functionalizing agent or a therapeutic agent.

[0472] Clause 27. The VLP of clause 26, wherein the therapeutic agent is an RNA.

[0473] Clause 28. A composition comprising the VLPs of any of clauses 25 to 27 and a carrier.

[0474] Clause 29. The composition of clause 28, wherein the carrier is a pharmaceutically acceptable carrier.-102-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0475] Clause 30. A delivery method comprising administering to one or more of a cell, a tissue or a subject in need thereof the VLPs of any of clauses 25-27 or the composition of claim 28 or 29.

[0476] Clause 31. The method of clause 30, wherein the subject is a mammal, optionally a human.

[0477] Clause 32. A method of harvesting virus-like particles (VLPs) from plant tissue comprising: (i) extracting the VLPs from the plant tissue using an acidic extraction solution and collecting the VLPs in a first eluate; (ii) filtering the VLPs from the first eluate of step (i) with a first membrane having a pore size of about 0.20 pm to about 0.25 pm, optionally about 0.22 pm and collecting a second eluate; (iii) filtering the VLPs from the second eluate of step (ii) with a second membrane, having a molecular weight cutoff of about 800 kDa to about 1200 kDa, optionally about 1000 kDa and collecting a third eluate; (iv) filtering the third eluate VLPs from step (iii) with a third membrane, wherein the third membrane has a molecular weight cutoff of about 300 to about 500 kDa, and collecting the VLPs retained from the third membrane; (v) separating the VLPs collected from the third membrane in step (iv) with ion exchange chromatography, and wherein the method is performed in any gravity environment, optionally wherein the VLP is icosahedral shaped.

[0478] Clause 33. A method of harvesting virus-like particles (VLPs) comprising: (i) extracting the VLPs from an intact plant or plant part using an acid extraction solution, and collecting the VLPs from a first eluate by vacuum infiltrating the apoplast from the intact plant or plant tissue with the extraction solution and collecting the first eluate from the apoplast with centrifugation; (ii) filtering the VLPs from the first eluate of step (i) with a first membrane having a pore size of about 0.20 pm to about 0.2 pm 0.22 pm, optionally about 0.22 pm and collecting a second eluate; (iii) filtering the VLPs from step (ii) with a second membrane, wherein the second membrane has a molecular weight cutoff of about lOOOkDa and collecting a third eluate; (iv) separating the VLPs from the third eluate in step (iii) with ion exchange chromatography, wherein the method is performed in any gravity environment.

[0479] Clause 34. A method of harvesting virus-like particles (VLPs) comprising: (i) extracting the VLPs from an intact plant or plant part using an acid extraction solution, and-103-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 collecting the VLPs from a first eluate by vacuum infiltrating the apoplast from the intact plant or plant tissue with the extraction solution and collecting the first eluate from the apoplast with centrifugation; (ii) filtering the VLPs from the first eluate of step (i) with a first membrane having a pore size of about 0.20 pm to about 0.25 pm, optionally about 0.22 pm and collecting a second eluate; (iii) filtering the VLPs from step (ii) with a second membrane, wherein the second membrane has a molecular weight cutoff of about 1,000 kDa , and collecting the VLPs retained on the second membrane; (iv) separating the VLPs from the second membrane in step (iii) with ion exchange chromatography, wherein the method is performed in any gravity environment, optionally wherein the VLP is rod-shaped.

[0480] Clause 35. An isolated harvested VLP or a population of harvested VLPs prepared by the method of any one of claims clause 32-34.[04811 Clause 36. A method of harvesting virus-like particles (VLPs) from plant tissue comprising: (i) incubating the plant tissue in a heated extraction solution; (ii) collecting the VLPs in the extraction solution; (ii) purifying the VLPs collected in the extraction solution of step (ii) through a series of separations based on size; and (iii) purifying the VLPs from step (ii) through separation based on charge.[0482| Clause 37. The method of clause 36, wherein the heated extraction solution is at a temperature of about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 80°C, about 90°C, about 100°C, or over 100°C.

[0483] Clause 38. A method of producing and harvesting virus-like particles (VLPs) wherein the VLPs are recombinant plant virus particles secreted into the apoplast of intact plant tissue, comprising: (i) culturing a plant transformed or infected with a VLP expression construct comprising a secretion signal peptide operably fused to a viral coat protein sequence; (ii) vacuum infiltrating the apoplast of intact plant tissue with an extraction buffer; (iii) centrifuging the infiltrated intact plant tissue under conditions sufficient to recover apoplastic fluid containing the secreted VLPs; and (iv) purifying the VLPs by ultrafiltration and / or ion exchange chromatography; wherein the plant tissue remains viable after harvesting.

[0484] Clause 39. The method of clause 38, wherein harvesting is repeated at least twice from the same plant without loss of VLP integrity or plant viability.-104-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0485] Clause 40. The method of clause 38 or 39, wherein the extraction buffer is selected from an acidic, neutral or alkaline buffer suitable for maintaining VLP integrity, optionally having a pH between 4.0-9.0 depending on the VLP species.

[0486] Clause 41. A method of harvesting virus-like particles (VLPs) in a microgravity environment, comprising: (i) producing VLPs in an intact plant cultivated under microgravity conditions; (ii) recovering VLPs from the apoplast via infiltration-centrifugation using low- mass, manual or syringe-operated equipment; (iii) purifying the recovered VLPs using ultrafiltration / diafiltration devices having a membrane area of less than 100 cm2and sequentially decreasing molecular weight cut-offs; and (iv) polishing the VLPs using gravity- driven or low-energy ion exchange chromatography.

[0487] Clause 42. The method of clause 41, wherein the plant is subjected to controlled reactive oxygen species (ROS) stress for greater than 10 days to increase VLP yield in microgravity by at least 20% relative to an untreated control.

[0488] Clause 43. A method for purifying a functionalized virus-like particle comprising a covalently attached fluorophore, targeting ligand, or therapeutic cargo, the method comprising: (i) contacting the VLP with the functionalizing agent under conditions suitable for covalent linkage; (ii) purifying the conjugated VLP by ultrafiltration / diafiltration using a membrane that retains the conjugated VLP and permits passage of unbound functionalizing agent; and (iii) recovering the purified conjugated VLP, wherein the recovery yield is at least 90% and free functionalizing agent is reduced to below detectable levels by fluorescence or absorbance analysis.[0489[ Clause 44. The method of clause 43, wherein the functionalizing agent comprises fluorescein isothiocyanate (FITC), a therapeutic agent, a detectable label, a purification label or an equivalent FITC dye detectable by excitation / emission spectroscopy.

[0490] Claus 45. A method of removing endotoxin and l,3-[3-glucan contaminants from a plant-derived virus-like particle preparation comprising: (i) adding a non-ionic detergent to the preparation, wherein the detergent is present at 0.05-0.5% (v / v); (ii) loading the detergentcontaining preparation onto a charged resin suitable for ion exchange chromatography under conditions binding the VLP; and (iii) eluting the VLP with a salt gradient; wherein endotoxin-105-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 and P-glucan levels in the final preparation are reduced to below regulatory thresholds for clinical administration.

[0491] Clause 46. The method of clause 45, wherein the non-ionic detergent is Triton X-l 14, and wherein detergent and contaminants are separated from the VLP during the chromatography step.

[0492] Clause 47. The method of any one of clauses 38-46, wherein the VLP is derived from a plant virus, optionally wherein the VLP is a plant virus coat protein, optionally wherein the plant virus is Cowpea Mosaic Virus, Cowpea Chlorotic Mottle Virus, or Potato Virus X.

[0493] Clause 48. A kit for producing functionalizing a virus-like particles in plants, comprising: (i) a plant transformation vector including an expression cassette for CPMV coat proteins and untranslated regions suitable for cargo encapsidation; and (ii) instructions for assembling a selected cargo into the vector and producing cargo loaded VLPs in plants using infiltrati on-centrifugati on harve sting .

[0494] Clause 49. The kit of clause 48, wherein the cargo comprises RNA.

[0495] Equivalents

[0496] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0497] The present technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed.-106-4911-2869-8997.1Atty. Dkt. No.: 114198-3560

[0498] Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology.

[0499] It should be understood that although the present invention has been specifically disclosed by certain aspects, embodiments, and optional features, modification, improvement and variation of such aspects, embodiments, and optional features can be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure.

[0500] The present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0501] In addition, where features or aspects of the present technology are described in terms of Markush groups, those skilled in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.[0502 [ All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entireties, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0503] Other aspects are set forth within the following claims.

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Claims

1. Atty. Dkt. No.: 114198-3560WHAT IS CLAIMED IS:

1. A method of harvesting virus-like particles (VLPs) from plant tissue comprising:(i) extracting the VLPs from the plant tissue using an extraction solution, wherein the VLPs are collected in the eluate;(ii) purifying the VLPs collected in the extraction solution of step (i) through a series of separations based on size; and(iii) purifying the VLPs from step (ii) through separation based on charge, thereby harvesting the VLPs.

2. A method of harvesting virus-like particles (VLPs) from plant tissue comprising:(i) extracting the VLPs from the plant tissue using an acidic extraction solution collecting the VLP-containing eluate;(ii) filtering the eluate from step (i) through a series of filtration membranes with serially decreasing pore size and collecting a the VLPs, wherein the VLPs are collected in the eluate or the membrane; and(iii) separating the VLPs from the filtration of step (ii) with ion exchange chromatography to harvest the VLPs, thereby harvesting the VLPs.

3. A method of harvesting virus-like particles (VLPs) from plant tissue comprising:(i) extracting the VLPs from plant tissue using a basic extraction solution collecting the VLP- containing first eluate;(ii) filtering the first eluate from step (i) with a series of ultrafiltration membranes with serially decreasing pore size and collecting the VLPs, wherein the VLPs are collected in the eluate or the membrane; and(iii) separating the VLPs from the filtration of step (ii) with ion exchange chromatography, thereby harvesting the VLPs.

4. The method of any of claims 1-3, wherein the method is performed in the absence of an ultracentrifugation step, optionally an isopycnic ultracentrifugation step.-119-4911-2869-8997.1Atty. Dkt. No.: 114198-35605. The method of any one of claims 1-4, wherein the method is performed in the absence of a chloroform or a methanol extraction step.

6. The method of any of claims 2-5, wherein the harvested VLPs have a net negative charge.

7. The method of any of claims 3-5, wherein the harvested VLPs have a net positive charge.

8. The method of any one of claims 1-7, wherein the plant tissue comprises or consists essentially of leaf tissue, stem tissue, and / or root tissue.

9. The method of any one of claims 1-7, wherein the plant tissue is from an intact plant.

10. The method of any of claims 1-9, wherein the VLP are extracted from an intact plant in step (i) by vacuum infiltrating an apoplast with the extraction solution and collecting the extraction solution with centrifugation.

11. The method of any of claims 1-10, wherein the extraction solution has: a pH of about 2.5 to about 4.5, optionally about 4.0 for acidic extraction, and further optionally wherein the VLP comprises Cowpea Chlorotic Mottle Virus (CCMV); or a pH of about 8.5 to about 9.5, optionally about 9.0, for basic extraction, and further optionally wherein the VLP comprises Cowpea Mosaic Virus (CPMV).

12. The method of any of claims 2-11, wherein the series of membranes comprises a first membrane and a second membrane.

13. The method of claim 12, further comprising a third membrane.

14. The method of claim 12 or 13, wherein the first membrane has a pore size of about 0.1 pm to about 0.4 pm, optionally about 0.2 pm.

15. The method of any of claims 12-14, wherein the second membrane has a molecular weight cut-off of about 2000kDa, or about lOOOkDa.

16. The method of any of claims 13-15, wherein the third membrane has a molecular weight cut-off selected from of about lOOkDa to about 500kDa, about 300kDa to about 500kDa, or about 300kDa, or about 500kDa.-120-4911-2869-8997.1Atty. Dkt. No.: 114198-356017. The method of claim 12, wherein the first membrane and second membrane average pores sizes are larger than the diameter of the VLP.

18. The method of claim 13, wherein the first and second membranes have average pore sizes larger than the diameter of the VLP and the third membrane have an average pore size smaller than the diameter of the VLP.

19. The method of any of claims 1-18, wherein the VLP is derived from a plant virus, optionally wherein the VLP is a plant virus coat protein, optionally wherein the plant virus is Cowpea Mosaic Virus, Cowpea Chlorotic Mottle Virus, o Potato Virus X.

20. The method of any of claims 2-19, wherein the ion exchange chromatography is anion exchange chromatography.

21. The method of any of claims 2-19, wherein the ion exchange chromatography is cation exchange chromatography.

22. A method of generating plant virus-like particles, according to the method depicted in Figure IB.

23. The method of any of claims 1-22, wherein the method is practiced in any gravity environment.

24. The method of any of claims 1-22, wherein the method is practiced in a microgravity environment.

25. An isolated VLP or a population of VLPs harvested by the method of any one of claims 1-24.

26. The VLPs of claim 25 and a functionalizing or a therapeutic agent.

27. The VLP of claim 26, wherein the therapeutic agent is an RNA.

28. A composition comprising the VLPs of any of claims 25 to 27 and a carrier.

29. The composition of claim 28, wherein the carrier is a pharmaceutically acceptable carrier.

30. A delivery method comprising administering to one or more of a cell, a tissue or a subject in need thereof the VLPs of any of claims 25-27 or the composition of claim 28 or 29.-121-4911-2869-8997.1Atty. Dkt. No.: 114198-356031. The method of claim 30, wherein the subject is a mammal, optionally a human.

32. A method of harvesting virus-like particles (VLPs) from plant tissue comprising:(i) extracting the VLPs from the plant tissue using an acidic extraction solution and collecting the VLPs in a first eluate;(ii) filtering the VLPs from the first eluate of step (i) with a first membrane having a pore size of about 0.20 pm to about 0.25 pm, optionally about 0.22 pm and collecting a second eluate;(iii) filtering the VLPs from the second eluate of step (ii) with a second membrane, having a molecular weight cutoff of about 800 kDa to about 1200 kDa, optionally about 1000 kDa and collecting a third eluate;(iv) filtering the third eluate VLPs from step (iii) with a third membrane, wherein the third membrane has a molecular weight cutoff of about 300 to about 500 kDa, and collecting the VLPs retained from the third membrane;(v) separating the VLPs collected from the third membrane in step (iv) with ion exchange chromatography, and wherein the method is performed in any gravity environment, optionally wherein the VLP is icosahedral shaped.

33. A method of harvesting virus-like particles (VLPs) comprising:(i) extracting the VLPs from an intact plant or plant part using an acid extraction solution, and collecting the VLPs from a first eluate by vacuum infiltrating the apoplast from the intact plant or plant tissue with the extraction solution and collecting the first eluate from the apoplast with centrifugation;(ii) filtering the VLPs from the first eluate of step (i) with a first membrane having a pore size of about 0.20 pm to about 0.2 pm 0.22 pm, optionally about 0.22 pm and collecting a second eluate;(iii) filtering the VLPs from step (ii) with a second membrane, wherein the second membrane has a molecular weight cutoff of about lOOOkDa and collecting a third eluate;(iv) separating the VLPs from the third eluate in step (iii) with ion exchange chromatography,-122-4911-2869-8997.1Atty. Dkt. No.: 114198-3560 wherein the method is performed in any gravity environment.

34. A method of harvesting virus-like particles (VLPs) comprising:(i) extracting the VLPs from an intact plant or plant part using an acid extraction solution, and collecting the VLPs from a first eluate by vacuum infiltrating the apoplast from the intact plant or plant tissue with the extraction solution and collecting the first eluate from the apoplast with centrifugation;(ii) filtering the VLPs from the first eluate of step (i) with a first membrane having a pore size of about 0.20 pm to about 0.25 pm, optionally about 0.22 pm and collecting a second eluate;(iii) filtering the VLPs from step (ii) with a second membrane, wherein the second membrane has a molecular weight cutoff of about 1,000 kDa , and collecting the VLPs retained on the second membrane;(iv) separating the VLPs from the second membrane in step (iii) with ion exchange chromatography, wherein the method is performed in any gravity environment, optionally wherein the VLP is rod-shaped.

35. An isolated harvested VLP or a population of harvested VLPs prepared by the method of any one of claims claim 32-34.

36. A method of harvesting virus-like particles (VLPs) from plant tissue comprising:(i) incubating the plant tissue in a heated extraction solution;(ii) collecting the VLPs in the extraction solution;(ii) purifying the VLPs collected in the extraction solution of step (ii) through a series of separations based on size; and(iii) purifying the VLPs from step (ii) through separation based on charge.

37. The method of claim 36, wherein the heated extraction solution is at a temperature of about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 80°C, about 90°C, about 100°C, or over 100°C.-123-4911-2869-8997.1Atty. Dkt. No.: 114198-356038. A method of producing and harvesting virus-like particles (VLPs) wherein the VLPs are recombinant plant virus particles secreted into the apoplast of intact plant tissue, comprising:(i) culturing a plant transformed or infected with a VLP expression construct comprising a secretion signal peptide operably fused to a viral coat protein sequence;(ii) vacuum infiltrating the apoplast of intact plant tissue with an extraction buffer;(iii) centrifuging the infiltrated intact plant tissue under conditions sufficient to recover apoplastic fluid containing the secreted VLPs; and(iv) purifying the VLPs by ultrafiltration and / or ion exchange chromatography; wherein the plant tissue remains viable after harvesting.

39. The method of claim 38, wherein harvesting is repeated at least twice from the same plant without loss of VLP integrity or plant viability.

40. The method of claim 38 or 39, wherein the extraction buffer is selected from an acidic, neutral or alkaline buffer suitable for maintaining VLP integrity, optionally having a pH between 4.0-9.0 depending on the VLP species.

41. A method of harvesting virus-like particles (VLPs) in a microgravity environment, comprising:(i) producing VLPs in an intact plant cultivated under microgravity conditions;(ii) recovering VLPs from the apoplast via infiltration-centrifugation using low-mass, manual or syringe-operated equipment;(iii) purifying the recovered VLPs using ultrafiltration / diafiltration devices having a membrane area of less than 100 cm2and sequentially decreasing molecular weight cut-offs; and(iv) polishing the VLPs using gravity-driven or low-energy ion exchange chromatography.

42. The method of claim 41, wherein the plant is subjected to controlled reactive oxygen species (ROS) stress for greater than 10 days to increase VLP yield in microgravity by at least 20% relative to an untreated control.

43. A method for purifying a functionalized virus-like particle comprising a covalently attached fluorophore, targeting ligand, or therapeutic cargo, the method comprising:-124-4911-2869-8997.1Atty. Dkt. No.: 114198-3560(i) contacting the VLP with the functionalizing agent under conditions suitable for covalent linkage;(ii) purifying the conjugated VLP by ultrafiltration / diafiltration using a membrane that retains the conjugated VLP and permits passage of unbound functionalizing agent; and(iii) recovering the purified conjugated VLP, wherein the recovery yield is at least 90% and free functionalizing agent is reduced to below detectable levels by fluorescence or absorbance analysis.

44. The method of claim 43, wherein the functionalizing agent comprises fluorescein isothiocyanate (FITC), a therapeutic agent, a detectable label, a purification label or an equivalent FITC dye detectable by excitation / emission spectroscopy.

45. A method of removing endotoxin and 1,3-P-glucan contaminants from a plant-derived virus-like particle preparation comprising:(i) adding a non-ionic detergent to the preparation, wherein the detergent is present at 0.05- 0.5% (v / v);(ii) loading the detergent-containing preparation onto a charged resin suitable for ion exchange chromatography under conditions binding the VLP; and(iii) eluting the VLP with a salt gradient; wherein endotoxin and P-glucan levels in the final preparation are reduced to below regulatory thresholds for clinical administration.

46. The method of claim 45, wherein the non-ionic detergent is Triton X-l 14, and wherein detergent and contaminants are separated from the VLP during the chromatography step.

47. The method of any one of claims 38-46, wherein the VLP is derived from a plant virus, optionally wherein the VLP is a plant virus coat protein, optionally wherein the plant virus is Cowpea Mosaic Virus, Cowpea Chlorotic Mottle Virus, o Potato Virus X.

48. A kit for producing functionalizing a virus-like particles in plants, comprising:(i) a plant transformation vector including an expression cassette for CPMV coat proteins and untranslated regions suitable for cargo encapsidation; and-125-4911-2869-8997.1Atty. Dkt. No.: 114198-3560(ii) instructions for assembling a selected cargo into the vector and producing cargo loaded VLPs in plants using infiltration-centrifugation harvesting.

49. The kit of claim 48, wherein the cargo comprises RNA.-126-4911-2869-8997.1

Images