Scalable Production of HPV16 L1 Protein and VLPs from Tobacco Leaves

PLoS One. 2016 Aug 12;11(8):e0160995. doi: 10.1371/journal.pone.0160995. eCollection 2016.

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.

MeSH terms

  • Capsid Proteins / biosynthesis*
  • Capsid Proteins / genetics
  • DNA, Viral / genetics
  • Genetic Engineering / adverse effects
  • Genetic Engineering / methods*
  • Nicotiana / genetics*
  • Oncogene Proteins, Viral / biosynthesis*
  • Oncogene Proteins, Viral / genetics
  • Plant Leaves / genetics*
  • Safety
  • Vaccines, Virus-Like Particle / biosynthesis*
  • Vaccines, Virus-Like Particle / genetics
  • Vaccines, Virus-Like Particle / immunology

Substances

  • Capsid Proteins
  • DNA, Viral
  • Oncogene Proteins, Viral
  • Vaccines, Virus-Like Particle
  • L1 protein, Human papillomavirus type 16

Grants and funding

This study was supported by Helmsley Trust Foundation and James Graham Brown Cancer Center, School of Medicine, University of Louisville to MZ, JJ and ABJ.