Expression of protective antigens in transgenic chloroplasts and the production of improved vaccines

Abstract

Vaccines for conferring immunity in mammals to infective pathogens are provided, as well as vectors and methods for plastid transformation of plants to produce protective antigens and vaccines for oral delivery. The invention further provides transformed plastids having the ability to survive selection in both the light and the dark, at different developmental stages by using genes coding for two different enzymes capable of detoxifying the same selectable marker, driven by regulatory signals that are functional in proplastids as well as in mature chloroplasts. The invention utilizes antibiotic-free selectable markers to provide edible vaccines for conferring immunity to a mammal against Bacillus anthracis , as well as Yersina pestis . The vaccines are operative by parenteral administration as well. The invention also extends to the transformed plants, plant parts, and seeds and progeny thereof. The invention is applicable to monocot and dicot plants.

1 . An orally-administrable vaccine composition for conferring immunity to  Bacillus anthracis  to a mammal, said vaccine comprising plant material comprising anthrax protective antigen. 
     
     
         2 . The vaccine composition of  claim 1 , wherein said plant material comprises a plant plastid genome having a polynucleotide sequence encoding for anthrax protective antigen. 
     
     
         3 . The vaccine composition of  claim 1 , wherein said plant material comprises a plurality of plant cells homoplasmic with respect to plant plastids transformed to express anthrax protective antigen. 
     
     
         4 . The vaccine composition of  claim 1 , wherein said vaccine composition is heat-stable. 
     
     
         5 . A process for vaccinating a mammal against  Bacillus anthracis  comprising feeding to said mammal an effective amount of the vaccine composition of  claim 1 . 
     
     
         6 . A plant plastid comprising a plastid genome transformed with a DNA coding sequence for anthrax protective antigen. 
     
     
         7 . A plant cell homoplasmic with respect to plastids transformed to express anthrax protective antigen. 
     
     
         8 . A plant comprising a plastid according to  claim 6 . 
     
     
         9 . A vaccine for conferring immunity to  Bacillus anthracis  to a mammal comprising anthrax protective antigen expressed within a plant plastid, wherein said vaccine is free of both anthrax edema factor and anthrax lethal factor. 
     
     
         10 . A plastid transformation vector for stably transforming a plastid, said plastid vector comprising, as operably linked components, a first flanking sequence, a DNA sequence coding for a anthrax protective antigen capable of expression in a plastid, and a second flanking sequence. 
     
     
         11 . A process for producing an anthrax protective antigen comprising: integrating a plastid transformation vector according to  claim 10  into the plastid genome of a plant cell; growing said plant cell to thereby express said anthrax protective antigen. 
     
     
         12 . The process of  claim 11  wherein said anthrax protective antigen is competent to produce an immunogenic response in a mammal. 
     
     
         13 . An orally-administrable vaccine composition for conferring immunity to  Yershina pestis  to a mammal, said vaccine comprising plant material comprising V and F1 antigens of  Y. pestis.    
     
     
         14 . The vaccine composition of  claim 13 , wherein said plant material comprises a plant plastid genome having a genetic sequence encoding for V and F1 antigens of  Y. pestis.    
     
     
         15 . The vaccine composition of  claim 13 , wherein said plant material comprises a plurality of plant cells homoplasmic with respect to plant plastids transformed to express V and F1 antigens of  Y. pestis.    
     
     
         16 . The vaccine composition of  claim 13 , wherein said vaccine composition is heat-stable. 
     
     
         17 . A process for vaccinating a mammal against  Yershina pestis  comprising feeding to said mammal an effective amount of the vaccine composition of  claim 13 . 
     
     
         18 . A plant plastid comprising a plastid genome transformed with a DNA coding sequence for V and F1 antigens of  Y. pestis.    
     
     
         19 . A plant cell homoplasmic with respect to plastids transformed to express V and F1 antigens of  Y. pestis.    
     
     
         20 . A plant comprising a plastid according to  claim 18 . 
     
     
         21 . The plant plastid of  claim 18 , wherein said genome expresses V and F1 antigens as a fusion protein. 
     
     
         22 . A plastid transformation vector for stably transforming a plastid, said plastid vector comprising, as operably linked components, a first flanking sequence, a DNA sequence coding for V and F1 antigens of  Y. pestis  capable of expression in a plastid, and a second flanking sequence. 
     
     
         23 . A process for producing a V and F1 antigens of  Y. pestis  comprising: integrating a plastid transformation vector according to  claim 22  into the plastid genome of a plant cell; growing said plant cell to thereby express said V and F1 antigens. 
     
     
         24 . The process of  claim 23  wherein said V and F1 antigens are competent to produce an immunogenic response in a mammal. 
     
     
         25 . The vector of  claim 22 , wherein said V and F1 antigens are expressed as a fusion protein.