title: Scalable Production of HPV16 L1 Protein and VLPs from Tobacco Leaves.
abstract: Cervical cancer is the most common malignancy among women particularly in developing countries, with human papillomavirus (HPV) 16 causing 50% of invasive cervical cancers. A plant-based HPV vaccine is an alternative to the currently available virus-like particle (VLP) vaccines, and would be much less expensive. We optimized methods to express HPV16 L1 protein and purify VLPs from tobacco (Nicotiana benthamiana) leaves transfected with the magnICON deconstructed viral vector expression system. L1 proteins were extracted from agro-infiltrated leaves using a series of pH and salt mediated buffers. Expression levels of L1 proteins and VLPs were verified by immunoblot and ELISA, which confirmed the presence of sequential and conformational epitopes, respectively. Among three constructs tested (16L1d22, TPL1d22, and TPL1F), TPL1F, containing a full-length L1 and chloroplast transit peptide, was best. Extraction of HPV16 L1 from leaf tissue was most efficient (> 2.5% of total soluble protein) with a low-salt phosphate buffer. VLPs were purified using both cesium chloride (CsCl) density gradient and size exclusion chromatography. Electron microscopy studies confirmed the presence of assembled forms of HPV16 L1 VLPs. Collectively; our results indicated that chloroplast-targeted transient expression in tobacco plants is promising for the production of a cheap, efficacious HPV16 L1 VLP vaccine. Studies are underway to develop plant VLPs for the production of a cervical cancer vaccine.
authors: James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, United States of America.
authors: Maryam
authors: M
authors: Zahin
authors: James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, United States of America.|Department of Medicine, University of Louisville, Louisville, Kentucky, United States of America.
authors: Joongho
authors: J
authors: Joh
authors: James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, United States of America.|Department of Biochemistry and Molecular Genetics, University of Louisville, Louisville, Kentucky, United States of America.
authors: Sujita
authors: S
authors: Khanal
authors: James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, United States of America.|Owensboro Cancer Research Program, Owensboro, Kentucky, United States of America.
authors: Adam
authors: A
authors: Husk
authors: Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, Arizona, United States of America.
authors: Hugh
authors: H
authors: Mason
authors: Plant Biotechnology and Metabolic Engineering, Technische Universita¨t Darmstadt, Schnittspahnstrasse 3-5, 64287, Darmstadt, Germany.
authors: Heribert
authors: H
authors: Warzecha
authors: James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, United States of America.
authors: Shin-Je
authors: SJ
authors: Ghim
authors: James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, United States of America.|Department of Medicine, University of Louisville, Louisville, Kentucky, United States of America.
authors: Donald M
authors: DM
authors: Miller
authors: James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, United States of America.|Owensboro Cancer Research Program, Owensboro, Kentucky, United States of America.|Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky, United States of America.
authors: Nobuyuki
authors: N
authors: Matoba
authors: James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, United States of America.
authors: Alfred Bennett
authors: AB
authors: Jenson
mesh_terms: D036022:Capsid Proteins / Q000096:biosynthesis* / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005818:Genetic Engineering / Q000009:adverse effects / Q000379:methods*; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis* / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics*; D012449:Safety; D014026:Nicotiana / Q000235:genetics*; D058425:Vaccines, Virus-Like Particle / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral; D009856:Oncogene Proteins, Viral; D058425:Vaccines, Virus-Like Particle; C063317:L1 protein, Human papillomavirus type 16