Rotavirus-like particle production in plants

Abstract

A method of producing a rotavirus-like particle (RLP) in a plant is provided. The method comprises expressing within a host or host cell for example a plant, portion of a plant or plant cell one or more nucleic acid comprising one or more regulatory region operatively linked to a first, second and third nucleotide sequence, the regulatory region active in the host or host cell. The first nucleotide sequence encoding a first rotavirus protein, the second nucleotide sequence encoding a second rotavirus protein and the third nucleotide sequence encoding a third rotavirus protein. The first, second and third encode rotavirus protein NSP4 and VP2 or VP6 and VP4 or VP7. The host or host cell is incubated under conditions that permit the expression of the nucleic acids, so that NSP4 and either VP2 of VP6 and VP4 or VP7 are expressed, thereby producing the RLP. Hosts comprising the RLP, compositions comprising the RLP and method for using the composition are also provided.

What is claimed is: 
     
         1 . A method of increasing incorporation of VP4, VP7, or both VP4 and VP7 into a rotavirus like particle (RLP) comprising:
 a) providing a host or host cell comprising one or more nucleic acid comprising   a first nucleotide sequence encoding a first rotavirus protein, a second nucleotide sequence encoding a second rotavirus protein, a third nucleotide sequence encoding a third rotavirus protein and a fourth nucleotide sequence encoding a fourth rotavirus protein; the first, second, third and fourth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and   the first, second, third and fourth nucleotide sequence encoding one of rotavirus protein VP7, VP4, NSP4 and VP2 or VP6;   b) incubating the host or host cell under conditions that permit the expression of the one or more nucleic acid, so that each of the VP7, VP4, NSP4 and VP2 or VP6 are expressed, thereby producing the RLP with enhanced levels of VP4, VP7, or both VP4 and VP7 when compared to the level of VP4 or VP7 produced by a second host or host cell that expresses the one or more nucleic acid that does not comprise NSP4, under the same conditions.   
     
     
         2 . The method of  claim 1 , wherein the one or more nucleic acid further comprises a fifth nucleotide sequence encoding a fifth rotavirus protein, the first, second, third, fourth and fifth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and the first, second, third, fourth and fifth nucleotide sequence encoding one of rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and wherein each of VP2, VP4, VP6, VP7 and NSP4 are expressed from the one or more nucleic acid. 
     
     
         3 . The method of  claim 1 , wherein the host or host cell comprises insect cells, mammalian cells, plant, portion of a plant or plant cells. 
     
     
         4 . The method of  claim 1 , wherein the host or host cell comprises plant, portion of plant or plant cells. 
     
     
         5 . The method of  claim 1 , wherein the one or more nucleic acid comprises one or more expression enhancer. 
     
     
         6 . The method of  claim 5 , wherein the expression enhancer is selected from the group consisting of CPMV HT, CPMV 160, CPMV 160+ and CPMV HT+. 
     
     
         7 . The method of  claim 1 , further comprising the steps of:
 c) harvesting the host or host cell, and   d) purifying the RLPs from the host or host cell, wherein the RLPs range in size from 70-110 nm.   
     
     
         8 . An RLP produced by the method of  claim 2 , wherein the RLP is a triple layered RLP comprising rotavirus protein, the rotavirus protein consisting of VP2, VP4, VP6 and VP7. 
     
     
         9 . A composition comprising an effective dose of the RLP of  claim 8  for inducing an immune response in a subject, and a pharmaceutically acceptable carrier. 
     
     
         10 . A method of inducing immunity to a rotavirus infection in a subject, comprising administering the composition of  claim 9  to the subject. 
     
     
         11 . The method of  claim 10 , wherein the composition is administered to a subject orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously. 
     
     
         12 . The method of  claim 1 , wherein the first rotavirus protein, the second rotavirus protein, the third rotavirus protein and fourth rotavirus protein are obtained from any one rotavirus strain having a genotype G1 to G27. 
     
     
         13 . The method of  claim 1 , wherein the first rotavirus protein, the second rotavirus protein, the third rotavirus protein and the fourth rotavirus protein are obtained from a rotavirus strain having a genotype G1 or G4. 
     
     
         14 . A plant, portion of a plant, a plant cell, a plant matter, or a plant extract comprising an RLP produced by the method of  claim 1 . 
     
     
         15 . The method of  claim 1 , wherein the host or host cell comprises  Nicotiana benthamiana,  portion of  Nicotiana benthamiana  or  Nicotiana benthamiana  cells. 
     
     
         16 . The method of  claim 2 , wherein the nucleotide sequence encoding VP2 comprises from 80% to 100% identity with a nucleotide sequence as defined by SEQ ID NO: 21, the nucleotide sequence encoding VP4 comprises from 80% to 100% identity with a nucleotide sequence as defined by SEQ ID NO:32, the nucleotide sequence encoding VP6 comprises from 80% to 100% identity with a nucleotide sequence as defined by SEQ ID NO:27, the nucleotide sequence encoding VP7 comprises from 80% to 100% identity with a nucleotide sequence as defined by SEQ ID NO:37, the nucleotide sequence encoding NSP4 comprises from 80% to 100% identity with a nucleotide sequence as defined by SEQ ID NO:42. 
     
     
         17 . The method of  claim 2 , wherein the VP2 comprises an amino acid sequence having from 80% to 100% identity with the amino acid sequence as defined by SEQ ID NO:24, the VP4 comprises an amino acid sequence having from 80% to 100% identity with the amino acid sequence as defined by SEQ ID NO:34, the VP6 comprises an amino acid sequence having from 80% to 100% identity with the amino acid sequence as defined by SEQ ID NO:29, the VP7 comprises an amino acid sequence having from 80% to 100% identity with the amino acid sequence as defined by SEQ ID NO: 39, the NSP4 comprises an amino acid sequence having from 80% to 100% identity with the amino acid sequence as defined by SEQ ID NO:44. 
     
     
         18 . A method of increasing incorporation of VP4, VP7, or both VP4 and VP7 in a rotavirus like particle (RLP) comprising:
 a) introducing into a host or host cell one or more nucleic acid comprising   a first nucleotide sequence encoding a first rotavirus protein, a second nucleotide sequence encoding a second rotavirus protein, a third nucleotide sequence encoding a third rotavirus protein, and a fourth nucleotide sequence encoding a fourth rotavirus protein the first, second, third and fourth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and   the first, second, third and fourth nucleotide sequence encoding one of rotavirus protein VP7, VP4, NSP4 and VP2 or VP6;   b) incubating the host or host cell under conditions that permit the expression of the one or more nucleic acid, so that each of VP7, VP4, NSP4 and VP2 or VP6 are expressed, thereby producing the RLP with enhanced levels of VP4, VP7, or both VP4 and VP7 when compared to the level of VP4 or VP7 produced by a second host or host cell that expresses the one or more nucleic acid that does not comprise NSP4, under the same conditions.   
     
     
         19 . The method of  claim 18 , wherein the one or more nucleic acid further comprises a fifth nucleotide sequence encoding a fifth rotavirus protein, the first, second, third, fourth and fifth nucleotide sequence being operatively linked to one or more regulatory region active in the host or host cell; and
 the first, second, third, fourth and fifth nucleotide sequence encoding one of rotavirus protein VP2, VP4, VP6, VP7 or NSP4 and wherein each of VP2, VP4, VP6, VP7 and NSP4 are expressed from the one or more nucleic acid.   
     
     
         20 . The method of  claim 18 , wherein the host or host cell comprises insect cells, mammalian cells, plant, portion of a plant or plant cells. 
     
     
         21 . The method of  claim 18 , wherein the host or host cell comprises plant, portion of plant or plant cells. 
     
     
         22 . The method of  claim 18 , wherein the one or more nucleic acid comprises one or more expression enhancer. 
     
     
         23 . The method of  claim 22 , wherein the expression enhancer is selected from the group consisting of CPMV HT, CPMV 160, CPMV 160+ and CPMV HT+. 
     
     
         24 . The method of  claim 18 , further comprising the steps of:
 c) harvesting host or host cell, and   d) purifying the RLPs from the host or host cell, wherein the RLPs range in size from 70-110 nm.   
     
     
         25 . An RLP produced by the method of  claim 19 , wherein the RLP is a triple layered RLP comprising rotavirus protein, the rotavirus protein consisting of VP2, VP4, VP6 and VP7. 
     
     
         26 . A composition comprising an effective dose of the RLP of  claim 25  for inducing an immune response in a subject, and a pharmaceutically acceptable carrier. 
     
     
         27 . A method of inducing immunity to a rotavirus infection in a subject, comprising administering the composition of  claim 26  to the subject. 
     
     
         28 . The method of  claim 27 , wherein the composition is administered to a subject orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously. 
     
     
         29 . The method of  claim 18 , wherein the first rotavirus protein, the second rotavirus protein, the third rotavirus protein and fourth rotavirus protein are obtained from any one rotavirus strain having a genotype G1 to G27. 
     
     
         30 . The method of  claim 18 , wherein the first rotavirus protein, the second rotavirus protein, the third rotavirus protein and the fourth rotavirus protein are obtained from a rotavirus strain having a genotype G1 or G4. 
     
     
         31 . A plant, portion of a plant, a plant cell, a plant matter, or a plant extract comprising an RLP produced by the method of  claim 18 . 
     
     
         32 . The method of  claim 18 , wherein the host or host cell comprises  Nicotiana benthamiana,  portion of  Nicotiana benthamiana  or  Nicotiana benthamiana  cells. 
     
     
         33 . The method of  claim 19 , wherein the nucleotide sequence encoding VP2 comprises from 80% to 100% identity with a nucleotide sequence as defined by SEQ ID NO: 21, the nucleotide sequence encoding VP4 comprises from 80% to 100% identity with a nucleotide sequence as defined by SEQ ID NO:32, the nucleotide sequence encoding VP6 comprises from 80% to 100% identity with a nucleotide sequence as defined by SEQ ID NO:27, the nucleotide sequence encoding VP7 comprises from 80% to 100% identity with a nucleotide sequence as defined by SEQ ID NO:37, the nucleotide sequence encoding NSP4 comprises from 80% to 100% identity with a nucleotide sequence as defined by SEQ ID NO:42. 
     
     
         34 . The method of  claim 19 , wherein the VP2 comprises an amino acid sequence having from 80% to 100% identity with the amino acid sequence as defined by SEQ ID NO:24, the VP4 comprises an amino acid sequence having from 80% to 100% identity with the amino acid sequence as defined by SEQ ID NO:34, the VP6 comprises an amino acid sequence having from 80% to 100% identity with the amino acid sequence as defined by SEQ ID NO:29, the VP7 comprises an amino acid sequence having from 80% to 100% identity with the amino acid sequence as defined by SEQ ID NO: 39, the NSP4 comprises an amino acid sequence having from 80% to 100% identity with the amino acid sequence as defined by SEQ ID NO:44.