Production of a parvovirus vaccine in plants as viral coat protein fusions

Abstract

The present invention relates to foreign peptide sequences fused to recombinant plant viral structural proteins and a method of their production. Fusion proteins are economically synthesized in plants at high levels by biologically contained tobamoviruses. The fusion proteins of the invention have are useful as antigens for inducing the production of antibodies having desired binding properties, e.g., protective antibodies, or for use as vaccine antigens for the induction of protective immunity against the parvovirus. Feline parvovirus epitopes were fused to the N-terminus of the TMV coat protein, expressed in Nicotiana plants, extracted, purified, characterized and administered to animals, resulting in protective immunity.

What is claimed is:  
     
         1 . A polynucleotide encoding a fusion protein capable of being expressed in a plant or a plant cell, comprising: 
 (a) a coding region that encodes said fusion protein and includes: 
 i. a plant viral coat protein from a single-stranded plus-sense RNA virus, and fused thereto,  
 ii. a peptide comprising amino acids MGSDGAVQPDGGQPAV (SEQ ID NO:1) or a fragment thereof; and  
   (b) a promoter functional in plants that is 5′ to the coding region.    
     
     
         2 . A polynucleotide according to  claim 1 , wherein the peptide is fused to the N-terminus of the plant viral coat protein.  
     
     
         3 . A polynucleotide according to  claim 1 , wherein the peptide is fused to the C-terminus of the plant viral coat protein.  
     
     
         4 . A polynucleotide according to  claim 1 , wherein said fusion protein is an internal fusion protein with respect to the coat protein.  
     
     
         5 . A polynucleotide according to  claim 1 , further comprising 
 (c) a fusion joint having a leaky stop codon from a single-stranded plus-sense RNA virus.    
     
     
         6 . A polynucleotide according to  claim 1 , wherein the peptide MGSDGAVQPDGGQPAV or fragment comprises an antigen.  
     
     
         7 . A polynucleotide according to  claim 1 , wherein the coat protein is a tobacco mosaic virus coat protein.  
     
     
         8 . A polynucleotide according to  claim 1  wherein the coat protein is a tobamovirus coat protein.  
     
     
         9 . A recombinant plant viral genome comprising a polynucleotide according to  claim 1 .  
     
     
         10 . A recombinant plant virus particle, comprising a genome according to  claim 9 .  
     
     
         11 . A recombinant plant virus having a coat protein encoded by a polynucleotide according to  claim 1 .  
     
     
         12 . A plant cell comprising a polynucleotide according to  claim 1 .  
     
     
         13 . A plant cell comprising a recombinant plant viral genome according to  claim 9 .  
     
     
         14 . A plant cell comprising a recombinant plant virus particle according to  claim 10 .  
     
     
         15 . A plant cell comprising a recombinant plant virus according to  claim 11 .  
     
     
         16 . A plant comprising a polynucleotide according to  claim 1 .  
     
     
         17 . A plant comprising a recombinant plant viral genome according to  claim 9 .  
     
     
         18 . A plant comprising a recombinant plant virus particle according to  claim 10 .  
     
     
         19 . A plant comprising a recombinant plant virus according to  claim 11 .  
     
     
         20 . A polynucleotide encoding a fusion protein capable of being expressed in a plant or a plant cell, comprising: 
 (a) a coding region that encodes said fusion protein and includes: 
 i. a plant viral coat protein from a single-stranded plus-sense RNA virus, and fused thereto,  
 ii. a peptide comprising amino acids MGQPDGGQPAVRNERAT (SEQ ID NO:2) or a fragment thereof; and  
   (b) a promoter functional in plants that is 5′ to the coding region.    
     
     
         21 . A polynucleotide according to  claim 20 , wherein the peptide is fused to the N-terminus of the plant viral coat protein.  
     
     
         22 . A polynucleotide according to  claim 20 , wherein the peptide is fused to the C-terminus of the plant viral coat protein.  
     
     
         23 . A polynucleotide according to  claim 20 , wherein said fusion protein is an internal fusion protein with respect to the coat protein.  
     
     
         24 . A polynucleotide according to  claim 20 , further comprising 
 (c) a fusion joint having a leaky stop codon from a single-stranded plus-sense RNA virus.    
     
     
         25 . A polynucleotide according to  claim 20 , wherein the peptide MGQPDGGQPAVRNERAT or a fragment comprises an antigen.  
     
     
         26 . A polynucleotide according to  claim 20 , wherein the coat protein is a tobacco mosaic virus coat protein.  
     
     
         27 . A polynucleotide according to  claim 20  wherein the coat protein is a tobamovirus coat protein.  
     
     
         28 . A recombinant plant viral genome comprising a polynucleotide according to  claim 20 .  
     
     
         29 . A recombinant plant virus particle, comprising a genome according to  claim 28 .  
     
     
         30 . A recombinant plant virus, wherein the coat protein is encoded by a polynucleotide according to  claim 20 .  
     
     
         31 . A plant cell comprising a polynucleotide according to  claim 20 .  
     
     
         32 . A plant cell comprising a recombinant plant viral genome according to  claim 28 .  
     
     
         33 . A plant cell comprising a recombinant plant virus particle according to  claim 29 .  
     
     
         34 . A plant cell comprising a recombinant plant virus according to  claim 30 .  
     
     
         35 . A plant comprising a polynucleotide according to  claim 20 .  
     
     
         36 . A plant comprising a recombinant plant viral genome according to  claim 28 .  
     
     
         37 . A plant comprising a recombinant plant virus particle according to  claim 29 .  
     
     
         38 . A plant comprising a recombinant plant virus according to  claim 30 .  
     
     
         39 . An immunochemical reagent comprising a fusion protein capable of being produced in a plant or a plant cell, wherein the fusion protein comprises 
 (i) a plant viral coat protein from a single-stranded plus-sense RNA virus; and    (ii) a peptide comprising amino acids MGQPDGGQPAVRNERAT or a fragment thereof fused to the N-terminus of the coat protein.    
     
     
         40 . A vaccine for the protection of mammals against parvovirus comprising the immunochemical reagent of  claim 39 .  
     
     
         41 . A vaccine according to  claim 40  together with a pharmaceutically or veterinarially acceptable carrier or excipient.  
     
     
         42 . An immunochemical reagent comprising a recombinant plant virus, wherein at least one capsid of the coat protein is a fusion protein capable of being produced in a plant or a plant cell, wherein the fusion protein comprises 
 (i) a plant viral coat protein from a single-stranded plus-sense RNA virus; and    (ii) a peptide comprising amino acids MGSDGAVQPDGGQPAV or a fragment thereof fused to the N-terminus of the coat protein.    
     
     
         43 . A vaccine for the protection of mammals against parvovirus comprising an immunochemical reagent according to  claim 42 .  
     
     
         44 . A vaccine according to  claim 43  together with a pharmaceutically or veterinarially acceptable carrier.  
     
     
         45 . A vaccine according to  claim 40 , wherein the fusion protein is in a recombinant plant virus.  
     
     
         46 . A vaccine according to  claim 45  wherein said virus is a live virus.  
     
     
         47 . A vaccine according to  claim 43 , wherein the recombinant plant virus is a live virus.  
     
     
         48 . A vaccine composition comprising a live recombinant plant virus according to  claim 46  and a pharmaceutically or veterinarially acceptable carrier or excipient.  
     
     
         49 . A vaccine composition comprising a live recombinant plant virus according to  claim 47  and a pharmaceutically or veterinarially acceptable carrier or excipient.  
     
     
         50 . A method of making the polynucleotide of  claim 1 , comprising ligating an oligonucleotide encoding a peptide having the sequence MGSDGAVQPDGGQPAV or a fragment thereof to a viral coat protein gene.  
     
     
         51 . A method of making a recombinant plant viral genome comprising 
 (a) inserting an oligonucleotide encoding a peptide having the sequence MGSDGAVQPDGGQPAV or a fragment thereof into the genome of a single-stranded plus-sense RNA virus so that said oligonucleotide is fused in frame with a plant viral coat protein gene and under the control of a promoter functional in plants; or    (b) ligating the polynucleotide of  claim 1  to the genome of a single-stranded plus-sense RNA virus, thereby making said recombinant plant viral genome.    
     
     
         52 . A method of making the recombinant plant virus that encodes a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGSDGAVQPDGGQPAV or a fragment thereof, comprising the steps of: 
 (a) ligating DNA encoding the fusion protein which protein is capable of being expressed in a plant or a plant cell, to a DNA copy of the genome of a single-stranded plussense RNA virus;    (b) transcribing said ligated DNA to RNA; and    (c) infecting a host plant or plant cell with said transcribed RNA,    so that said plant or plant cell makes said recombinant plant virus.    
     
     
         53 . A method of making the recombinant plant virus that encodes a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGSDGAVQPDGGQPAV or a fragment thereof, comprising the steps of; 
 (a) ligating DNA encoding the fusion protein which protein is capable of being expressed in a plant or a plant cell, to a DNA copy of the genome of a single-stranded plussense RNA virus, under the control of a promoter that is functional in plants; and    (b) transforming or transfecting a host plant or plant cell with said ligated DNA, so that said DNA is expressed in said plant or plant cell, whereby said plant or plant cell makes said recombinant plant virus.    
     
     
         54 . A method of making a plant cell that produces a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGSDGAVQPDGGQPAV or a fragment thereof, comprising transforming or transfecting a host plant cell with the plant viral genome of  claim 51 .  
     
     
         55 . A method of making a plant cell that produces a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGSDGAVQPDGGQPAV or a fragment thereof, comprising infecting a host plant cell with the plant virus of  claim 52 .  
     
     
         56 . A method of making a plant cell that produces a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGSDGAVQPDGGQPAV or a fragment thereof, comprising infecting a host plant cell with the plant virus of  claim 53 .  
     
     
         57 . A method of making a plant that produces a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGSDGAVQPDGGQPAV or a fragment thereof, comprising transforming or transfecting a host plant with the plant viral genome of  claim 51 .  
     
     
         58 . A method of making a plant that produces a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGSDGAVQPDGGQPAV or a fragment thereof, comprising infecting a host plant with the plant virus of  claim 52 .  
     
     
         59 . A method of making a plant that produces a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGSDGAVQPDGGQPAV or a fragment thereof, comprising infecting a host plant with the plant virus of  claim 53 .  
     
     
         60 . A method of making the polynucleotide of  claim 20 , comprising ligating an oligonucleotide encoding a peptide having the sequence MGQPDGGQPAVRNERAT or a fragment thereof to a viral coat protein gene.  
     
     
         61 . A method of making a recombinant plant viral genome comprising 
 (a) inserting an oligonucleotide encoding a peptide having the sequence MGQPDGGQPAVRNERAT or a fragment thereof into the genome of a single-stranded plus-sense RNA virus so that said oligonucleotide is fused in frame with a plant viral coat protein gene and under the control of a promoter functional in plants; or    (b) ligating the polynucleotide of  claim 1  to the genome of a single-stranded plus-sense RNA virus thereby making said recombinant plant viral genome.    
     
     
         62 . A method of making the recombinant plant virus that encodes a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGQPDGGQPAVRNERAT or a fragment thereof, comprising the steps of; 
 (a) ligating DNA encoding the fusion protein which protein is capable of being expressed in a plant or. a plant cell, to a DNA copy of the genome of a single-stranded plussense RNA virus;    (b) transcribing said ligated DNA to RNA; and    (c) infecting a host plant or plant cell with said transcribed RNA,    so that said plant or plant cell makes said recombinant plant virus.    
     
     
         63 . A method of making the recombinant plant virus that encodes a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGQPDGGQPAVRNERAT or a fragment thereof, comprising the steps of; 
 (a) ligating DNA encoding the fusion protein which protein is capable of being expressed in a plant or a plant cell, to a DNA copy of the genome of a single-stranded plussense RNA virus, under the control of a promoter that is functional in plants; and    (b) transforming or transfecting a host plant or plant cell with said ligated DNA, so that said DNA is expressed in said plant or plant cell,    whereby said plant or plant cell makes said recombinant plant virus.    
     
     
         64 . A method of making a plant cell that produces a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGQPDGGQPAVRNERAT or a fragment thereof, comprising transforming or transfecting a host plant cell with the plant viral genome of  claim 61 .  
     
     
         65 . A method of making a plant cell that produces a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGQPDGGQPAVRNERAT or a fragment thereof, comprising infecting a host plant cell with the plant virus of  claim 62 .  
     
     
         66 . A method of making a plant cell that produces a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGQPDGGQPAVRNERAT or a fragment thereof, comprising infecting a host plant cell with the plant virus of  claim 63 .  
     
     
         67 . A method of making a plant that produces a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGQPDGGQPAVRNERAT or a fragment thereof, comprising transforming or transfecting a host plant with the plant viral genome of  claim 61 .  
     
     
         68 . A method of making a plant that produces a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGQPDGGQPAVRNERAT or a fragment thereof, comprising infecting a host plant with the plant virus of  claim 62 .  
     
     
         69 . A method of making a plant that produces a fusion protein that includes a plant viral coat protein from a single-stranded plus-sense RNA virus and a peptide comprising amino acids MGQPDGGQPAVRNERAT or a fragment thereof, comprising infecting a host plant with the plant virus of  claim 63 .  
     
     
         70 . A method of isolating a virus, comprising: 
 (a) Homogenizing virus-containing plant tissue in Na 2 S 2 O 5 ;    (b) Straining the homogenate to obtain green juice;    (c) Adjusting the pH of the green juice to 5.0 with acid;    (d) Heating the green juice to about 47° C. for a period of about 5 minutes followed by cooling to about 5° C.;    (e) Centrifuging the green juice at about 6000×g for about 3 minutes to obtain a supernatant and pellet;    (f) Precipitating the supernatant in polyethylene glycol and NaCl to obtain a precipitate;    (g) Resuspending the precipitate in water at a concentration of about 1 mg per ml;    (h) Extracting the precipitate in chloroform and butanol and centrifuging the extract    (i) Recovering and lyophilizing the aqueous phase of the centrifuged material;    (j) Resuspending the lyophilized material at a concentration of about 5 to about 10 mg per ml water.    
     
     
         71 . A method of isolating a virus, comprising: 
 (a) Grinding virus-containing plant material in a buffer to obtain a homogenate;    (b) Straining the homogenate to obtain green juice, and adding thereto polyethyleneimine to a concentration of about 0.1% (v/v)    (c) Stirring at about 4° C. for about 30 minutes followed by centrifuging; at about 3000×g for about 5 minutes to obtain a supernatant;    (d) Precipitating the supernatant in polyethylene glycol and NaCl to obtain a precipitate;    (e) Resuspending the precipitate in water at a concentration of about 1 mg per ml;    (f) Extracting the resuspended material in chloroform and butanol and centrifuging the extracted material;    (g) Recovering and lyophilizing the aqueous phase; and    (h) Resuspending the lyophilized material at a concentration of about 5 to about 10 mg per ml water.