title: Biochemical and biophysical characterization of maize-derived HBsAg for the development of an oral vaccine.
abstract: Although a vaccine against hepatitis B virus (HBV) has been available since 1982, it is estimated that 600,000 people die every year due to HBV. An affordable oral vaccine could help alleviate the disease burden and to this end the hepatitis B surface antigen (HBsAg) was expressed in maize. Orally delivered maize material induced the strongest immune response in mice when lipid was extracted by CO2 supercritical fluid extraction (SFE), compared to full fat and hexane-extracted material. The present study provides a biochemical and biophysical basis for these immunological differences by comparing the active ingredient in the differently treated maize material. Purified maize-derived HBsAg underwent biophysical characterization by gel filtration, transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-CD, and fluorescence. Gel filtration showed that HBsAg forms higher-order oligomers and TEM demonstrated virus-like particle (VLP) formation. The VLPs obtained from SFE were more regular in shape and size compared to hexane or full fat material. In addition, SFE-derived HBsAg showed the greatest extent of α-helical structure by far UV-CD spectrum. Fluorescence experiments also revealed differences in protein conformation. This work establishes SFE-treated maize material as a viable oral vaccine candidate and advances the development of the first oral subunit vaccine.
authors.affiliation: Roy J. Carver Department of Biochemistry Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
authors.forename: Shweta
authors.initials: S
authors.lastname: Shah
authors.affiliation: Applied Biotechnology Institute, Cal Poly Tech Park, San Luis Obispo, CA 93407, USA.
authors.forename: Celine A
authors.initials: CA
authors.lastname: Hayden
authors.affiliation: Applied Biotechnology Institute, Cal Poly Tech Park, San Luis Obispo, CA 93407, USA.
authors.forename: Maria E
authors.initials: ME
authors.lastname: Fischer
authors.affiliation: Roy J. Carver Department of Biochemistry Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA. Electronic address: gururao@iastate.edu.
authors.forename: A Gururaj
authors.initials: AG
authors.lastname: Rao
authors.affiliation: Applied Biotechnology Institute, Cal Poly Tech Park, San Luis Obispo, CA 93407, USA. Electronic address: jhoward@appliedbiotech.org.
authors.forename: John A
authors.initials: JA
authors.lastname: Howard
mesh_terms: D000284:Administration, Oral; D000595:Amino Acid Sequence; D000818:Animals; D025924:Chromatography, Supercritical Fluid; D006514:Hepatitis B Surface Antigens / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017325:Hepatitis B Vaccines / Q000008:administration & dosage / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D051379:Mice; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D030821:Plants, Genetically Modified; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D022462:Vaccines, Edible / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics; D058425:Vaccines, Virus-Like Particle / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics; D003313:Zea mays / Q000235:genetics*
chemical_list: D006514:Hepatitis B Surface Antigens; D017325:Hepatitis B Vaccines; D010446:Peptide Fragments; D022462:Vaccines, Edible; D058425:Vaccines, Virus-Like Particle
keywords: Hepatitis B surface antigen; Mucosal vaccine; Supercritical fluid extraction; TEM; Virus-like particle; Zea mays