Method for using tobacco mosaic virus to overproduce peptides and proteins

Abstract

The invention describes compositions and methods of use in which an infectious modified Tobacco Mosaic Virus (TMV) virion comprising a coat protein (CP) or a movement protein (MP) gene is replaced with a nuclear inclusion protease (NIa) expression cassette for the expression of a heterologous peptide in a tobacco mosaic virus (TMV) host plant.

1 . A method for producing a heterologous peptide in a plant comprising: 
 a) infecting a suitable plant with a CP modified infectious clone of TMV having a wild type MP gene and a modified CP gene comprising a nucleotide sequence encoding a heterogeneous peptide;    b) culturing the plant under conditions to foster plant growth;    c) recovering the heterologous peptide from the leaves of the plant.    
     
     
         2 . The method of  claim 1  wherein the plant is a transgenic plant expressing a wild type TMV movement protein.  
     
     
         3 . The method of  claim 1  wherein the plant is further infected with a MP modified infectious clone of TMV having a wild type CP gene and a modified MP gene.  
     
     
         4 . The method of  claim 3  wherein the modified MP gene is inactivated.  
     
     
         5 . The method of  claim 4  wherein the modified MP gene is SEQ. ID NO. 3.  
     
     
         6 . The method of  claim 3  wherein the infecting is in a ratio of CP modified clone to MP-modified clone of 1:1 to 10:1.  
     
     
         7 . The method of  claim 1  wherein the infecting is by viral transcripts produced in vitro.  
     
     
         8 . The method of  claim 1  wherein the suitable plant is selected from the group consisting of lettuce, spinach, tomato, potato,  Nicotiana tabacum, N. glutinosa, N. sylvester, Phaseolus vulgaris  and  Chenopodium amaranticolor.    
     
     
         9 . The method of  claim 1  wherein the heterologous peptide is an antigenic epitope comprising from 10 to 20 amino acids inserted immediately after CP amino acid 154.  
     
     
         10 . The method of  claim 6  wherein the antigenic epitope is SEQ ID NO: 14.  
     
     
         11 . The method of  claim 6  wherein the antigenic epitope is selected from the group consisting of SEQ ID NOS: 10, 11, 12 and 13.  
     
     
         12 . The method of  claim 6  wherein the antigenic epitope is SEQ ID NO: 16.  
     
     
         13 . A method for producing an immune response stimulating peptide in a plant comprising: 
 a) infecting a suitable plant with a CP modified infectious clone of TMV having a wild type MP gene and a modified CP gene comprising a nucleotide sequence encoding an immune response stimulating peptide;    b) culturing the plant under conditions to foster plant growth;    c) recovering the heterologous peptide from the leaves of the plant.    
     
     
         14 . The method of  claim 12  wherein the plant is a transgenic plant expressing a wild type MP gene.  
     
     
         15 . The method of  claim 13  or  14  wherein the nucleotide sequence encodes SEQ ID NO: 16.  
     
     
         16 . The method of  claim 13  wherein the plant is further infected with a MP modified infectious clone of TMV having a wild type CP gene and a modified MP gene.  
     
     
         17 . The method of  claim 16  wherein the infecting is in a ratio of CP modified clone to MP-modified clone of 1:1 to 10:1.  
     
     
         18 . The method of  claim 17  wherein the MP gene is inactivated in the MP-modified clone.  
     
     
         19 . The method of  claim 17  wherein the MP gene is SEQ ID NO: 3.  
     
     
         20 . The method of  claim 14  wherein the infecting is by viral transcripts produced in vitro.  
     
     
         21 . The method of  claim 14  wherein the suitable plant is selected from the group consisting of lettuce, spinach, tomato, potato,  Nicotiana tabacum. N. glutinosa, N. sylvester, Phaseolus vulgaris  and  Chenopodium amaranticolor.    
     
     
         22 . The method of  claim 14  wherein the immune stimulating peptide is a viral antigenic epitope comprising from 10 to 20 amino acids inserted immediately after CP amino acid 154.  
     
     
         23 . The method of  claim 22  wherein the immune stimulating peptide is SEQ ID NO: 16.  
     
     
         24 . A method for producing a heterologous peptide in a plant comprising: 
 a) infecting a suitable plant with a CP modified infectious clone of TMV comprising a NIa based cassette vector comprising: 
 (1) a nucleotide sequence encoding a nuclear inclusion protein from tobacco etch virus and flanking self-cleavage sequences therefor, and  
 (2) nucleotide sequences encoding heterologous peptides flanking the self-cleavage sites;  
   b) culturing the plant under conditions to foster plant growth; and    c) recovering the heterologous peptide from the leaves of the plant.    
     
     
         25 . The method of  claim 24  wherein the plant is a transgenic plant expressing a wild type TMV movement protein.  
     
     
         26 . The method of  claim 24  wherein the plant is further infected with a MP modified infectious clone of TMV having a wild type CP gene and a modified MP gene.  
     
     
         27 . The method of  claim 25  wherein NIa based cassette vector replaces the MP gene in the infectious clone.  
     
     
         28 . The method of  claim 24  wherein the infecting is by viral transcripts produced in vitro.  
     
     
         29 . The method of  claim 24  wherein the suitable plant is selected from the group consisting of lettuce, spinach, potato, tomato,  Nicotiana tabacum, N. glutinosa, N. sylvester, Phaseolus vulgaris  and  Chenopodium amaranticolor.    
     
     
         30 . The method of  claim 24  wherein the heterologous peptide is a viral antigenic epitope comprising from 5 to 20 amino acids.  
     
     
         31 . A method for creating an immune response in a mammal comprising administering to the mammal an immune response stimulating amount of a compound of  claim 13 .  
     
     
         32 . The method of  claim 31  wherein the administering is intraparenterally.  
     
     
         33 . The method of  claim 31  wherein the administering is intravenously.  
     
     
         34 . The method of  claim 31  wherein the administering is intragastricly.  
     
     
         35 . The method of  claim 32 ,  33  or  34  wherein the immune response comprises generation of IgG antibodies to the antigenic epitope.  
     
     
         36 . A nucleotide sequence encoding a modified infectious clone of TMV comprising a heterologous DNA sequence encoding a peptide of from about 5 to 20 amino acids wherein the heterologous DNA is inserted immediately after the codon encoding amino acid 154 of the wild type coat protein of TMV.  
     
     
         37 . The nucleotide sequence of  claim 36  wherein the modified infectious clone further comprises a nucleotide sequence encoding a wild type movement protein.  
     
     
         38 . The nucleotide sequence of  claim 37  wherein the heterologous DNA sequence encodes a viral antigenic epitope.  
     
     
         39 . The nucleotide sequence of  claim 38  wherein the heterologous DNA sequence encodes a viral antigenic epitope of HIV gp 120.  
     
     
         40 . The nucleotide sequence of  claim 39  wherein the heterologous DNA sequence encodes a viral antigenic epitope of influenza hemagglutinin 12CA5.  
     
     
         41 . The nucleotide sequence of  claim 39  wherein the heterologous DNA sequence encodes an antigenic epitope of murine zona pellucida.  
     
     
         42 . A nucleotide sequence encoding a modified infectious clone of TMV comprising a nucleotide sequence encoding a wild type coat protein and a dysfunctional movement protein.  
     
     
         43 . The nucleotide sequence of  claim 42  wherein the dysfunctional movement protein is SEQ ID NO: 3.  
     
     
         44 . A modified TMV virion comprising a heterologous peptide sequence of from about 5 to 20 amino acids inserted immediately following amino acid 154 of the wild type TMV coat protein.  
     
     
         45 . The virion of  claim 44  wherein the heterologous peptide sequence is an antigenic epitope of zona pellucida 3.  
     
     
         46 . The virion of  claim 44  wherein the antigenic epitope is SEQ ID NO: 16.  
     
     
         47 . The virion of  claim 44  wherein the antigenic epitope is SEQ ID NO: 14.  
     
     
         48 . A transgenic plant comprising the recombinant virions of  claim 44 ,  45 ,  46  or  47 .  
     
     
         49 . A nucleotide sequence encoding a modified infectious clone of TMV comprising a heterologous DNA sequence encoding a wild type CP and a NIa based cassette filter comprising DNA encoding: 
 a) the nuclear inclusion (NIa) protease,    b) multiple restriction endonuclease sites; and    c) self-cleavage sites for the protease, wherein the self-cleavage sites flank the protease and each restriction site, except at the termini of the nucleotide sequence;    wherein the cassette is inserted in the place of the nucleotide sequence encoding the wild type movement protein thereof.    
     
     
         50 . A transgenic plant comprising the infectious clone of  claim 48 .  
     
     
         51 . The method of  claim 24 ,  25 , wherein the plant is infected with TEV prior to step b).  
     
     
         52 . A method of contraception in a mammal comprising feeding the mammal sufficient virions according to  claim 45  or  46  to induce a contraceptive immune response in the mammal to the heterologous peptide.