Recombinant influenza virus-like particles (vlps) produced in transgenic plants expressing hemagglutinin

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 plants 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.

1 . A nucleic acid comprising a nucleotide sequence encoding a type A influenza hemagglutinin (HA) and a protein disulfide isomerase signal peptide, operatively linked to a regulatory region active in a plant. 
     
     
         2 - 3 . (canceled) 
     
     
         4 . The nucleic acid of  claim 1 , wherein the type A influenza is a subtype selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16. 
     
     
         5 . The nucleic acid of  claim 1 , wherein the HA is from a type A influenza, selected from the group consisting of H1, H2, H3, H5, H6, H7 and H9. 
     
     
         6 . A method of producing influenza virus like particles (VLPs) in a plant comprising:
 a) introducing the nucleic acid of  claim 1  into the plant, or portion of the plant, and   b) incubating the plant or portion of the plant under conditions that permit the expression of the nucleic acid, thereby producing the VLPs.   
     
     
         7 . The method of  claim 6 , wherein in the step of introducing (step a), the nucleic acid is transiently expressed in the plant. 
     
     
         8 . The method of  claim 6 , wherein, in the step of introducing (step a), the nucleic acid is stably expressed in the plant. 
     
     
         9 . The method of  claim 6  further comprising a step of
 c) harvesting the host and purifying the VLPs.   
     
     
         10 . The method of  claim 6 , wherein, in the step of introducing (step a), a second nucleic acid comprising a nucleotide sequence encoding one or more than one chaperone proteins is introduced to the plant. 
     
     
         11 . The method of  claim 10 , wherein the one or more than one chaperon proteins is selected from the group consisting of Hsp40 and Hsp70. 
     
     
         12 . A method of producing influenza virus like particles (VLPs) in a plant comprising:
 a) providing a plant, or a portion of a plant, comprising the nucleic acid of  claim 1 , and   b) incubating the plant or portion of the plant under conditions that permit the expression of the nucleic acid, thereby producing the VLPs   
     
     
         13 . A plant comprising the nucleic acid of  claim 1 . 
     
     
         14 . The plant of  claim 13 , further comprising a nucleic acid comprising a nucleotide sequence encoding one or more than one chaperone proteins operatively linked to a regulatory region active in a plant. 
     
     
         15 . The plant of  claim 14 , wherein the one or more than one chaperone proteins is selected from the group consisting of Hsp40 and Hsp70. 
     
     
         16 . A virus like particle (VLP) produced by the method of  claim 6 , comprising an influenza virus hemagglutinin (HA) protein and one or more than one lipid derived from a plant. 
     
     
         17 . The virus like particle (VLP) of  claim 16 , wherein the type A influenza is a subtype selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16. 
     
     
         18 . The VLP of  claim 17 , wherein the HA is from a type A influenza, selected from the group consisting of H1, H2, H3, H5, H6, H7 and H9. 
     
     
         19 . A composition comprising an effective dose of the VLP of  claim 16  for inducing immunity to an influenza virus in a subject and a pharmaceutically acceptable carrier. 
     
     
         20 . A method of inducing immunity to an influenza virus infection in a subject, comprising administering the virus like particle of  claim 16 . 
     
     
         21 . The method of  claim 20 , wherein the virus like particle is administered to a subject orally, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or Subcutaneously. 
     
     
         22 . A virus like particle (VLP) produced by the method of  claim 6 , comprising an influenza virus HA bearing plant-specific N-glycans, or modified N-glycans. 
     
     
         23 . The virus like particle (VLP) of  claim 22 , wherein the type A influenza is a subtype selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16. 
     
     
         24 . The VLP of  claim 23 , wherein the HA is from a type A influenza, selected from the group consisting of H1, H2, H3, H5, H6, H7 and H9. 
     
     
         25 . A composition comprising an effective dose of the VLP of  claim 22  for inducing immunity to an influenza virus in a subject and a pharmaceutically acceptable carrier. 
     
     
         26 . A method of inducing immunity to an influenza virus infection in a subject, comprising administering the composition of  claim 25 . 
     
     
         27 . The method of  claim 26 , wherein the composition is administered to a subject orally, intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or Subcutaneously.