Production of a monoclonal antibody therapeutic against west nile virus in plants

1. A method for producing an isolated humanized E16 antibody (hu-E16) and fragments thereof against West Nile Virus (WNV), comprising:

a) transfecting a N. benthamiana plant cell with a nucleic acid molecule having SEQ ID NO:4, which encodes a heavy chain sequence of hu-E16, and a nucleic acid molecule having SEQ ID NO:6, which encodes a light chain sequence of hu-E16, wherein said heavy and light chain sequences of hu-E16 comprise N. benthamiana -optimized codons;

b) expressing said heavy and light sequences of hu-E16 in said transfected N. benthamiana plant cell;

c) growing said N. benthamiana plant; and

d) purifying the hu-E16 from the leaves of said N. benthamiana plant, wherein said hu-E16 neutralizes WNV in vitro and in vivo.

2. The method of claim 1 , wherein said purifying comprises:

a) ammonium sulfate precipitation comprising an initial precipitation at 25% ammonium sulfate saturation of a crude sample of N. benthamiana plant leaves expressing said light and heavy chain, followed by thorough mixing at 4° C., followed by a second ammonium sulfate precipitation at 50% ammonium sulfate saturation to produce a 50% ammonium sulfate pellet;

b) resuspending said 50% ammonium sulfate pellet in a buffer, applying said suspension to a Protein A affinity column, and eluting said column with 50 mM sodium citrate, pH 2.5; and

c) neutralizing the eluate from step (b) with 1M Tris-base to a final pH of 7.0 and subjecting said neutralized eluate to DEAE anion exchange chromatography,

wherein said DEAE anion exchange chromatography step produces a purified plant-derived preparation of fully assembled Hu-E16 to a greater than 95% purity level.

3. The method of claim 2 , wherein said method produces greater than 5 g of hu-E16 from 16 kg of plant material.

4. The method of claim 2 , wherein said hu-E16 purified from said N. benthamiana plant leaves protects against infection from WNV.

5. The method of claim 2 , wherein said hu-E16 purified from said N. benthamiana plant leaves ameliorates the symptoms of infection from WNV after infection by said virus.

6. The method of claim 1 , wherein the nucleic acid sequence encoding the variable domain of the heavy chain wherein the variable domain has a sequence of SEQ ID NO: 1.

7. The method of claim 1 , wherein the amino acid sequence of the variable domain of the heavy chain optimized heavy chain has a sequence of SEQ ID NO:2.

8. The method of claim 1 , wherein the variable heavy chain amino acid sequence of the optimized heavy chain has a sequence of SEQ ID NO:3.

9. The method of claim 1 , optimized heavy chain sequence has an amino acid sequence of SEQ ID NO:5.

10. The method of claim 1 , wherein the amino acid sequence of the light chain optimized light chain has a sequence of SEQ ID NO:7.

11. A preparation of a plant-derived hu-E16 antibody produced according to the method of claim 2 , said antibody comprising SEQ ID NO:5 and SEQ ID NO:7.