Abstract: Production of modified influenza viral proteins in plants is described. More specifically, producing and increasing influenza virus-like particle (VLP) production in plants, wherein the VLPs comprise the modified influenza viral proteins, such as modified influenza hemagglutinin (HA) is described. The HA protein may comprise an amino acid sequence comprising at least one substitution when compared to a corresponding wildtype amino acid sequence. Further provided are nucleic acids encoding the modified HA protein. Furthermore, methods of producing an influenza virus like particle (VLP) and methods of increasing yield of production of an influenza virus like particle (VLP) in a plant, portion of a plant, or a plant cell, are also provided.

Abstract: The present invention relates to the production of modified influenza viral proteins in plants. More specifically, the present invention relates to producing and increasing influenza virus-like particle (VLP) production in plants, wherein the VLPs comprise the modified influenza viral proteins, such as modified influenza hemagglutinin (HA). The HA protein may comprising an amino acid sequence comprising at least one substitution when compared to a corresponding wildtype amino acid sequence. Further provided are nucleic acid encoding the modified HA protein. Furthermore methods of producing an influenza virus like particle (VLP) and methods of increasing yield of production of an influenza virus like particle (VLP) in a plant, portion of a plant, or a plant cell, are also provided.

Abstract: Methods of preparing plant-derived proteins or suprastructure proteins, are provided. The method may comprise obtaining a plant, or plant matter comprising apoplast-localized proteins, or suprastructure proteins, producing a protoplast/spheroplast fraction and apoplast fraction from the plant or plant matter, and recovering the apoplast fraction. The apoplast fraction comprises plant-derived proteins or suprastructure proteins. Alternatively, the proteins, or suprastructure proteins, may be obtained from plant or plant matter comprising plant-derived proteins or suprastructure proteins, by digesting the plant matter using a cell wall degrading enzyme composition to produced a digested fraction. The digested fraction is filtered to produced a filtered fraction, and the plant-derived proteins or suprastructure proteins, are recovered from the filtered fraction.

Abstract: Methods of preparing plant-derived virus like particles (VLPs) are provided. The method may comprise obtaining a plant, or plant matter comprising apoplast-localized VLPs, producing a protoplast/spheroplast fraction and apoplast fraction from the plant or plant matter, and recovering the apoplast fraction. The apoplast fraction comprises plant-derived VLPs. Alternatively, VLPs may be obtained from plant or plant matter comprising plant-derived VLPs by digesting the plant matter using a cell wall degrading enzyme composition to produced a digested fraction. The digested fraction is filtered to produced a filtered fraction, and the plant-derived VLPs are recovered from the filtered fraction.

Abstract: A method for synthesizing influenza virus-like particles (VLPs) within a plant or a portion of a plant is provided. The method involves expression of influenza HA in plants and the purification by size exclusion chromatography. The invention is also directed towards a VLP comprising influenza HA protein and plant lipids. The invention is also directed to a nucleic acid encoding influenza HA as well as vectors. The VLPs may be used to formulate influenza vaccines, or may be used to enrich existing vaccines.

Abstract: A method of increasing the yield, stability, or both of an acid sensitive protein in a plant is provided. The method comprises introducing a first nucleic acid and a second nucleic acid into the plant, or portion of the plant. The first nucleic acid comprises a first regulatory region active in the plant and operatively linked to a nucleotide sequence encoding the acid sensitive protein. The second nucleic acid comprises a second regulatory region active in the plant and operatively linked to a nucleotide sequence encoding a channel protein, for example but not limited to a proton channel protein. The plant or portion of the plant is incubated under conditions that permit the expression of the nucleic acids, thereby increasing the yield of the acid sensitive protein when compared to the yield of the acid sensitive protein produced in the plant or portion of the plant produced under the same conditions, and in the absence of the proton channel protein.

Abstract: A method of producing a virus like particle (VLP) in a plant is provided. The method comprises introducing a first nucleic acid and a second nucleic acid into the plant, or portion of the plant. The first nucleic acid comprises a first regulatory region active in the plant and operatively linked to a nucleotide sequence encoding a structural virus protein. The second nucleic acid comprises a second regulatory region active in the plant and operatively linked to a nucleotide sequence encoding a channel protein, for example but not limited to a proton channel protein. The plant or portion of the plant is incubated under conditions that permit the expression of the nucleic acids, thereby producing the VLP.

Abstract: A method of producing a virus like particle (VLP) in a plant comprising modified hemagglutinin is provided. The method comprises introducing a nucleic acid comprising a regulatory region active in the plant and operatively linked to a nucleotide sequence encoding a modified influenza hemagglutinin (HA) protein into the plant, or portion of the plant, the modified HA protein comprises a modified proteolytic loop. Followed by incubating the plant or portion of the plant under conditions that permit the expression of the nucleic acids, thereby producing the VLP. The modified proteolytic loop may comprise one or more protease cleavage sites exhibiting reduced or abolished cleavage by a protease. The nucleotide sequence encoding the HA may be selected from the group consisting of B HA, C, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16. Also described is a virus like particle (VLP) produced by the method, and plants expressing the VLP.

Abstract: A method of producing a virus like particle (VLP) in a plant, and compositions comprising VLPs, are provided. The method involves introducing a nucleic acid comprising a regulatory region active in the plant and operatively linked to a chimeric nucleotide sequence encoding, in series, an ectodomain from a virus trimeric surface protein or fragment thereof, fused to an influenza transmembrane domain and cytoplasmic tail, into the plant, or portion of the plant, the ectodomain is from a non-influenza virus trimeric surface protein and heterologous with respect to the influenza transmembrane domain, and the cytoplasmic tail. The plant or portion of the plant are incubated under conditions that permit the expression of the nucleic acid, thereby producing the VLP. A VLP produced by this method are also provided.

Abstract: A method of producing a virus like particle (VLP) in a plant, and compositions comprising VLPs, are provided. The method involves introducing a nucleic acid comprising a regulatory region active in the plant and operatively linked to a chimeric nucleotide sequence encoding, in series, an ectodomain from a virus trimeric surface protein or fragment thereof, fused to an influenza transmembrane domain and cytoplasmic tail, into the plant, or portion of the plant, the ectodomain is from a non-influenza virus trimeric surface protein and heterologous with respect to the influenza transmembrane domain, and the cytoplasmic tail. The plant or portion of the plant are incubated under conditions that permit the expression of the nucleic acid, thereby producing the VLP. A VLP produced by this method are also provided.

Abstract: A method of producing a picornavirus-like particle (PVLP) in a plant is provided. The method comprises introducing a first nucleic acid and a second nucleic acid into the plant, portion of the plant, or a plant cell. The first nucleic acid comprising a first regulatory region active in the plant operatively linked to a nucleotide sequence encoding a polyprotein. The second nucleic acid comprises a second regulatory region active in the plant and operatively linked to a nucleotide sequence encoding one or more protease. The plant, portion of the plant, or plant cell is incubated under conditions that permit the expression of the nucleic acids, thereby producing the PVLP. A PVLP comprising the polyprotein is also provided.

Abstract: A method of producing a virus like particle (VLP) in a plant comprising modified hemagglutinin is provided. The method comprises introducing a nucleic acid comprising a regulatory region active in the plant and operatively linked to a nucleotide sequence encoding a modified influenza hemagglutinin (HA) protein into the plant, or portion of the plant, the modified HA protein comprises a modified proteolytic loop. Followed by incubating the plant or portion of the plant under conditions that permit the expression of the nucleic acids, thereby producing the VLP. The modified proteolytic loop may comprise one or more protease cleavage sites exhibiting reduced or abolished cleavage by a protease. The nucleotide sequence encoding the HA may be selected from the group consisting of B HA, C, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16. Also described is a virus like particle (VLP) produced by the method, and plants expressing the VLP.

Abstract: A method for synthesizing chimeric influenza virus-like particles (VLPs) within a plant or a portion of a plant is provided. The method involves expression of chimeric influenza HA in a plant or a portion of a plant. The invention is also directed towards a VLP comprising chimeric influenza HA protein and plants lipids. The invention is also directed to a nucleic acid encoding chimeric influenza HA as well as vectors. The VLPs may be used to formulate influenza vaccines, or may be used to enrich existing vaccines.

Abstract: A method of producing a picornavirus-like particle (PVLP) in a plant is provided. The method comprises introducing a first nucleic acid and a second nucleic acid into the plant, portion of the plant, or a plant cell. The first nucleic acid comprising a first regulatory region active in the plant operatively linked to a nucleotide sequence encoding a polyprotein. The second nucleic acid comprises a second regulatory region active in the plant and operatively linked to a nucleotide sequence encoding one or more protease. The plant, portion of the plant, or plant cell is incubated under conditions that permit the expression of the nucleic acids, thereby producing the PVLP. A PVLP comprising the polyprotein is also provided.

Abstract: A method for synthesizing influenza virus-like particles (VLPs) within a plant or a portion of a plant is provided. The method involves expression of influenza HA in plants and the purification by size exclusion chromatography. The invention is also directed towards a VLP comprising influenza HA protein and plant lipids. The invention is also directed to a nucleic acid encoding influenza HA as well as vectors. The VLPs may be used to formulate influenza vaccines, or may be used to enrich existing vaccines.

Abstract: Methods of recovering plant-derived proteins or suprastructure proteins, are provided. The method may comprise obtaining a plant, or plant matter comprising apoplast-localized proteins, or suprastructure proteins, loosening the cell wall to produce a plant or plant matter having a loosened cell wall, thus allowing the release of apoplastic content through the cell wall to produce an apoplastic content fraction from the plant or plant matter, and recovering the apoplastic content fraction. The apoplastic content fraction comprises plant-derived proteins or suprastructure proteins.

Abstract: A method for synthesizing influenza virus-like particles (VLPs) within a plant or a portion of a plant is provided. The method involves expression of a novel influenza HA protein in plants and its purification The invention is also directed towards a VLP comprising influenza HA protein and plants lipids. The invention is also directed to a nucleic acid encoding improved influenza HA as well as vectors. The VLPs may be used to formulate influenza vaccines, or may be used to enrich existing vaccines.

Abstract: A method for synthesizing influenza virus-like particles (VLPs) within a plant or a portion of a plant is provided. The method involves expression of influenza HA of type A/California/04/09 in plants and the purification by size exclusion chromatography. The invention is also directed towards a VLP comprising influenza HA protein of type A/California/04/09 and plants lipids. The invention is also directed to a nucleic acid encoding influenza HA of type A/California/04/09 as well as vectors. The VLPs may be used to formulate influenza vaccines, or may be used to enrich existing vaccines.

Abstract: A method for synthesizing influenza virus-like particles (VLPs) within a plant or a portion of a plant is provided. The method involves expression of influenza HA in plants and the purification by size exclusion chromatography. The invention is also directed towards a VLP comprising influenza HA protein and plant lipids. The invention is also directed to a nucleic acid encoding influenza HA as well as vectors. The VLPs may be used to formulate influenza vaccines, or may be used to enrich existing vaccines.

Abstract: A method for synthesizing influenza virus-like particles (VLPs) within a plant or a portion of a plant is provided. The method involves expression of influenza HA in plants and the purification by size exclusion chromatography. The invention is also directed towards a VLP comprising influenza HA protein and plant lipids. The invention is also directed to a nucleic acid encoding influenza HA as well as vectors. The VLPs may be used to formulate influenza vaccines, or may be used to enrich existing vaccines.