Polypeptides Having Immunoactivating Activity And Methods Of Producing The Same

Abstract

Isolated polypeptides are provided that comprise a cholera toxin B subunit variant having one or more modifications to increase the expression of the polypeptide in a plant cell. Nucleic acids sequences, vectors, and plant cells for expressing the cholera toxin B subunit variant polypeptides are also provided. Further provided are methods for producing the cholera toxin B subunit variant polypeptides that include the steps of transforming a plant cell with a nucleic acid encoding the cholera toxin B subunit variant polypeptides; expressing the variant polypeptides; and purifying the polypeptides. Still further provided are methods of isolating the variant polypeptides that include the steps of obtaining a plant cell expressing the cholera toxin B subunit variant polypeptides; extracting the cholera toxin B subunit variant polypeptides from the plant cell; and purifying the cholera toxin B subunit variant polypeptides. Methods of eliciting an immune response are also provided.

What is claimed is: 
     
         1 . An isolated nucleic acid molecule, comprising a sequence that encodes a polypeptide comprising a cholera toxin B subunit variant having one or more modifications to increase the expression of the polypeptide in a plant cell. 
     
     
         2 . An expression vector, comprising the nucleic acid molecule of  claim 1  operably linked to an expression cassette. 
     
     
         3 . A plant cell transfected with the vector of  claim 2 , or a progeny of the plant cell, wherein the cell or the progeny thereof expresses the polypeptide. 
     
     
         4 . A method of producing a cholera toxin B subunit variant polypeptide, comprising:
 transforming a plant cell with a nucleic acid encoding a cholera toxin B subunit variant polypeptide having one or more modifications to increase the expression of the variant polypeptide in the plant cell;   expressing the cholera toxin B subunit variant polypeptide in the plant cell; and   purifying the cholera toxin B subunit variant polypeptide.   
     
     
         5 . The method of  claim 4 , wherein the plant cell comprises a  Nicotiana  plant cell. 
     
     
         6 . The method of  claim 5 , wherein the Nicotiana plant cell is a  Nicotiana benthamiana  plant cell. 
     
     
         7 . The method of  claim 4 , wherein the nucleic acid encoding the cholera toxin B subunit variant comprises the sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. 
     
     
         8 . The method of  claim 4 , wherein the cholera toxin B subunit variant polypeptide comprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 25. 
     
     
         9 . The method of  claim 4 , wherein the one or more modifications comprise a secretory signal peptide selected from the group consisting of a rice alphaamylase secretory signal peptide, a  Nicotiana plumbagenifolia  calreticulin secretory signal peptide, an apple pectinase secretory signal peptide, and a barley alpha-amylase secretory signal peptide. 
     
     
         10 . The method of  claim 4 , wherein the one or more modifications comprise a secretory signal peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 18, 20, 22, and 24. 
     
     
         11 . The method of  claim 9 , wherein the secretory signal polypeptide comprises the rice alpha-amylase secretory signal peptide. 
     
     
         12 . The method of  claim 4 , wherein polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 26-29. 
     
     
         13 . The method of  claim 4 , wherein the one or more modifications comprise an endoplasmic reticulum retention signal having the amino acid sequence of SEQ IDNO: 31. 
     
     
         14 . The method of  claim 4 , wherein the cholera toxin B subunit variant polypeptide includes two or more N-linked glycosylation sequons. 
     
     
         15 . The method of  claim 4 , wherein the cholera toxin B subunit variant polypeptide comprises the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14. 
     
     
         16 . A method of isolating a cholera toxin B subunit variant polypeptide from a plant tissue, comprising:
 obtaining a plant cell expressing a cholera toxin B subunit variant polypeptide having one or more modifications to increase the expression of the polypeptide in a plant cell;   extracting the cholera toxin B subunit variant polypeptide from the plant cell; and   purifying the cholera toxin B subunit variant polypeptide from the plant cell.   
     
     
         17 . The method of  claim 16 , wherein the step of extracting the cholera toxin B subunit variant polypeptide from the plant cell comprises homogenizing the plant tissue in an aqueous buffer having an acidic pH. 
     
     
         18 . The method of  claim 17 , wherein the pH of the aqueous buffer is about 5. 
     
     
         19 . The method of  claim 16 , wherein purifying the cholera toxin B subunit variant polypeptide from the plant cell comprises purifying the variant polypeptide using chromatography. 
     
     
         20 . The method of  claim 16 , wherein the plant tissue comprises a  Nicotiana  plant tissue. 
     
     
         21 . The method of  claim 20 , wherein the plant tissue is a  Nicotiana benthamiana  plant tissue. 
     
     
         22 . The method of  claim 16 , wherein the cholera toxin B subunit variant polypeptide comprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 25. 
     
     
         23 . The method of  claim 16 , wherein the one or more modifications comprise a secretory signal peptide selected from the group consisting of a rice alphaamylase secretory signal peptide, a  Nicotiana plumbaginifoha  calreticulin secretory signal peptide, an apple pectinase secretory signal peptide, and a barley alpha-amylase secretory signal peptide. 
     
     
         24 . The method of  claim 16 , wherein the one or more modifications comprise a secretory signal peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 18, 20, 22, and 24. 
     
     
         25 . The method of  claim 23 , wherein the secretory signal peptide is the rice alpha-amylase secretory signal peptide. 
     
     
         26 . The method of  claim 16 , wherein the cholera toxin B subunit variant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 26-29. 
     
     
         27 . The method of  claim 16 , wherein the one or more modifications comprise an endoplasmic reticulum retention signal having the amino acid sequence of SEQ IDNO: 31. 
     
     
         28 . The method of  claim 16 , wherein the cholera toxin B subunit variant polypeptide includes two or more N-linked glycosylation sequons. 
     
     
         29 . The method of  claim 16 , wherein the cholera toxin B subunit variant polypeptide comprises the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14. 
     
     
         30 . A method for eliciting an immune response in a subject, comprising administering to a subject in need thereof an effective amount of a cholera toxin B subunit variant polypeptide having one or more modifications to increase the expression of the polypeptide in a plant cell. 
     
     
         31 . The method of  claim 30 , wherein administering an effective amount of the cholera toxin B subunit variant polypeptide increases an amount of IgG, IgA, IgM, effector T cells, regulatory T cells, or combinations thereof in a subject. 
     
     
         32 . The method of  claim 30 , wherein administering an effective amount of the cholera toxin B subunit variant polypeptide comprises orally administering the cholera toxin B subunit variant polypeptide. 
     
     
         33 . The method of  claim 30 , wherein the cholera toxin B subunit variant polypeptide comprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 25. 
     
     
         34 . The method of  claim 30 , wherein the cholera toxin B subunit variant polypeptide includes two or more N-linked glycosylation sequons. 
     
     
         35 . The method of  claim 34 , wherein the cholera toxin B subunit variant polypeptide comprises the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.