Methods and compositions to produce vaccines against smallpox in plants

Abstract

Compositions and methods for producing plant-derived immunogenic compositions that confer immunity against smallpox in a subject are provided. Plants engineered to produce antigenic proteins as well as expression cassettes comprising nucleic acids encoding proteins thereof are described. Methods of immunizing subjects with plant-derived compositions are also provided.

What is claimed is: 
     
         1 . A transgenic plant comprising an expression cassette that includes a first polynucleotide encoding a first immunogenic protein and a second polynucleotide encoding a second immunogenic protein, wherein each of the first immunogenic protein and the second immunogenic protein is independently selected from the group consisting of an L1 protein, a B5 protein and an A33 protein, wherein the protein selected as the first immunogenic protein differs from the protein selected as the second immunogenic protein. 
     
     
         2 . The transgenic plant of  claim 1 , wherein the first polynucleotide includes a sequence with at least 90% identity to a reference sequence selected from the group consisting of SEQ ID NOS: 6-11 and 14-19. 
     
     
         3 . The transgenic plant of  claim 1 , wherein the second polynucleotide includes a sequence with at least 90% identity to a reference sequence selected from the group consisting of SEQ ID NOS: 6-11 and 14-19, wherein the SEQ ID NO of the sequence selected as the second polynucleotide differs from the SEQ ID NO of the sequence selected as the first polynucleotide. 
     
     
         4 . The transgenic plant of  claim 1 , further comprising a third polynucleotide encoding a third immunogenic protein, wherein the third immunogenic protein is selected from the group consisting of an L1 protein, a B5 protein and an A33 protein, and wherein the protein selected as the third immunogenic protein differs from the protein selected as the first immunogenic protein and the second immunogenic protein. 
     
     
         5 . The transgenic plant of  claim 4 , wherein the third polynucleotide includes a sequence with at least 90% identity to a reference sequence selected from the group consisting of SEQ ID NOS: 6-11 and 14-19, wherein the SEQ ID NO of the sequence selected as the third polynucleotide differs from the SEQ ID NO of the sequence selected as the second polynucleotide and the third polynucleotide. 
     
     
         6 . The transgenic plant of  claim 1 , wherein the plant is a crop plant selected from the group consisting of tomato, tobacco, carrot, pepper, eggplant, lettuce, oilseed rape, collard, broccoli, cauliflower, cabbage crops, cucumber, melon, pumpkin, squash, peanut, soybeans, corn, rice, barley, buckwheat, sugar cane, cotton, beans, cassava, potatoes, sweet potatoes and okra. 
     
     
         7 . The transgenic plant of  claim 1 , wherein the plant is a medicinal plant selected from the group consisting of  Caragana sinica, Codonopsis pilosulae, Hedyotis diffusa, Houttuynia cordata, Lonicera japonica, Morinda offcinalis, Oenothera odorata, Kalanchoe pinnata, Echinacea angustifolia, Calendula officinalis  and  Arthemis nobilis.    
     
     
         8 . An immunogenic composition comprising a transgenic plant of any of  claims 1 - 7 , and at least one of an adjuvant and a pharmaceutically acceptable carrier. 
     
     
         9 . An immunogenic composition comprising a first immunogenic protein and a second immunogenic protein, wherein each of the first immunogenic protein and the second immunogenic protein is derived from a transgenic plant and is independently selected from the group consisting of an L1 protein, a B5 protein and an A33 protein, wherein the protein selected as the first immunogenic protein differs from the protein selected as the second immunogenic protein. 
     
     
         10 . The immunogenic composition of  claim 9 , wherein the first immunogenic protein is encoded by a first polynucleotide comprising a sequence with at least 90% identity to a reference sequence selected from the group consisting of SEQ ID NOS: 6-11 and 14-19. 
     
     
         11 . The immunogenic composition of  claim 9 , wherein the second immunogenic protein is encoded by a second polynucleotide comprising a sequence with at least 90% identity to a reference sequence selected from the group consisting of SEQ ID NOS: 6-11 and 14-19, wherein the SEQ ID NO of the sequence selected as the second polynucleotide differs from the SEQ ID NO of the sequence selected as the first polynucleotide. 
     
     
         12 . The immunogenic composition of  claim 9 , further comprising a third immunogenic protein, wherein the third immunogenic protein is derived from a transgenic plant and is selected from the group consisting of an L1 protein, a B5 protein and an A33 protein, and wherein the protein selected as the third immunogenic protein differs from the protein selected as the first immunogenic protein and the second immunogenic protein. 
     
     
         13 . The immunogenic composition of  claim 12 , wherein the third immunogenic protein is encoded by a third polynucleotide comprising a sequence with at least 90% identity to a reference sequence selected from the group consisting of SEQ ID NOS: 6-11 and 14-19, wherein the SEQ ID NO of the sequence selected as the third polynucleotide differs from the SEQ ID NO of the sequence selected as the second polynucleotide and the third polynucleotide. 
     
     
         14 . The immunogenic composition of  claim 9  further comprising an adjuvant. 
     
     
         15 . The immunogenic composition of  claim 14 , wherein the adjuvant is selected from the group consisting of: Alum, an oil-in-water nannoemulsion (MF59™), a glycolipid monophosphoryl lipid A (MPL®), virus-like particles (VLP), cholera toxin B subunit (CTB), montanides ISA51 and ISA720, saponines Quil-A, ISCOM and QS-21, syntax adjuvant formulation (SAF), muramyl dipeptides (MDP), immunostimulatory oligonucleotides, TLR ligands,  Escherichia coli  heat-labile exotoxin, and lipid-based adjuvants. 
     
     
         16 . The immunogenic composition of  claim 9  further comprising a pharmaceutically acceptable carrier.