Expression of immunogenic hepatitis B surface antigens in transgenic plants

Abstract

Plant expression vectors comprising at least two expression cassettes are provided which function to reduce transcriptional silencing of polynucleotide expression. Further, novel plant expression vectors for expression of immunogenic polypeptides, including HBsAg, are provided. The plant expression vectors can be used to produce immunogenic polypeptides, including HBsAg, in edible plant tissues. The edible plant tissues can be used to elicit an immune response in humans and animals when the plant tissues are consumed.

We claim:  
     
         1 . A plant cell transformed with a plant expression vector comprising two expression wherein the first expression cassette comprises a first polynucleotide sequence encoding an antigen polypeptide and wherein the second expression cassette comprises a second polynucleotide sequence encoding said antigen polypeptide sequence encoded by said first polynucleotide sequence, wherein said first polynucleotide sequence is non-identical to said second polynucleotide sequence.  
     
     
         2 . The plant cell of  claim 1  wherein virus like particles are assembled in the cell.  
     
     
         3 . The plant cell of  claim 1  wherein the plant cell is selected from the group consisting of tomato, potato, banana and carrot cells.  
     
     
         4 . The plant cell of  claim 1  wherein the plant expression vector is integrated into the nuclear genome of the plant cell.  
     
     
         5 . A plant seed comprising a plant expression vector comprising two expression cassettes, wherein the first expression cassette comprises a first polynucleotide sequence encoding an antigen polypeptide and wherein the second expression cassette comprises a second polynucleotide sequence encoding said antigen polypeptide sequence encoded by said first polynucleotide sequence, wherein said first polynucleotide sequence is non-identical to said second polynucleotide sequence.  
     
     
         6 . A transgenic plant cell comprising a polynucleotide comprising a nucleic acid sequence encoding a hepatitus B surface antigen (HBsAg) operably linked to: 
 (i) a plant functional promoter;    (ii) a translation enhancement sequence; and    (iii) a termination sequence wherein the polynucleotide lacks an untranslated region between the translation enhancement sequence and the HBsAg encoding sequence.    
     
     
         7 . The transgenic plant cell of  claim 6  wherein a virus like particle is assembled in the cell.  
     
     
         8 . The plant cell of  claim 6  wherein the plant cell is selected from the group consisting of tomato, potato, banana and carrot cells.  
     
     
         9 . The plant cell of  claim 6  wherein the plant expression vector is integrated into the nuclear genome of the plant cell.  
     
     
         10 . A plant seed comprising a plant expression vector comprising a polynucleotide comprising a nucleic acid sequence encoding a hepatitus B surface antigen (HBsAg) operably linked to: 
 (i) a plant functional promoter;    (ii) a translation enhancement sequence; and    (iii) a termination sequence wherein the polynucleotide lacks an untranslated region between the translation enhancement sequence and the HBsAg encoding sequence.    
     
     
         11 . An immunogenic composition comprising the plant cell of  claim 1  or  6 .  
     
     
         12 . The immunogenic composition of  claim 11  wherein the plant cell is present in a plant tissue selected from the group consisting of a fruit, leaf, tuber, plant organ, seed protoplast, and callus.  
     
     
         13 . The immunogenic composition of  claim 11  comprising juice or extract of the plant cell.  
     
     
         14 . The immunogenic composition of  claim 11  further comprising an adjuvant.  
     
     
         15 . The immunogenic composition of  claim 14  wherein the adjuvant is expressed as a fusion protein with an antigen of the immunogenic composition.  
     
     
         16 . A method of eliciting an immune response in mammal comprising the step of administering the composition of  claim 11  to a mammal, wherein an immune response is elicited.  
     
     
         17 . The method of  claim 16  wherein the composition is administered orally.  
     
     
         18 . The method of  claim 16  wherein the administration comprises consuming the transgenic plant cell.  
     
     
         19 . The method of  claim 16  wherein the polypeptide is administered by a technique selected from the group consisting of intramuscular, oral, intradermal, intraperitoneal, subcutaneous, and intranasal.  
     
     
         20 . The method of  claim 16  further comprising the step of administering an adjuvant.  
     
     
         21 . The method of  claim 20  wherein the adjuvant is selected from the group consisting of cholera toxin (CT),  E. coli  heat labile toxin (LT), anti-idiotypic antibody 2F10, colonization factor, shiga-like toxin and intimin.  
     
     
         22 . The method of  claim 16  wherein the immune response elicited is selected from the group of immune responses consisting of the humoral; mucosal; cellular; humoral and mucosal; humoral and cellular; mucosal and cellular; and humoral, mucosal and cellular immunogenic compositions.  
     
     
         23 . A method of isolating a recombinant HBsAg polypeptide expressed in a plant material comprising the step of subjecting the plant material to a detergent having a concentration of greater than 0.1% and less than 0.5%, whereby a recombinant HBsAg is isolated.  
     
     
         24 . A transgenic plant or plant cell that, when consumed as a foodstuff in four or less feedings, elicits an immune response comprising anti-HBsAg serum antibodies of greater than 50 mIU/mL in a mammal.  
     
     
         25 . The transgenic plant or plant cell of  claim 24 , wherein the immune response is a primary immune response.  
     
     
         26 . The transgenic plant cell of  claim 24 , wherein the plant cell comprises: 
 a) an Agrobacterium cell transformed with a plant expression vector comprising two expression cassettes, wherein the first expression cassette comprises a first polynucleotide sequence encoding an antigen polypeptide and wherein the second expression cassette comprises a second polynucleotide sequence encoding said antigen polypeptide sequence encoded by said first polynucleotide sequence, wherein said first polynucleotide sequence is non-identical to said second polynucleotide sequence from  claim 1 , or    b) an Agrobacterium cell transformed with an expression vector comprising a nucleic acid sequence encoding a hepatitus B surface antigen (HBsAg) operably linked to: 
 (i) a plant functional promoter;  
 (ii) a translation enhancement sequence; and  
 (iii) a termination sequence wherein the polynucleotide lacks an untranslated region between the translation enhancement sequence and the HBsAg encoding sequence further comprising a helper Ti plasmid.  
   
     
     
         27 . A transgenic plant or plant cell that, when consumed as a foodstuff in four or less feedings, elicits an anti-HBsAg boosting immune response that increases serum anti-HBsAg antibody levels at least four-fold or to levels greater than 500 mIU/mL in a mammal.  
     
     
         28 . The transgenic plant or plant cell of  claim 27 , wherein the plant cell comprises the plant cell of  claim 1  or  6 .