Scalable Production of HPV16 L1 Protein and VLPs from Tobacco Leaves.

Maryam Zahin,Heribert Warzecha,Nobuyuki Matoba,Adam Husk,Donald M Miller,Alfred Bennett Jenson,Shin-Je Ghim,Sujita Khanal,Hugh Mason,Joongho Joh,Craig Meyers

doi:10.1371/journal.pone.0160995

Maryam Zahin, Heribert Warzecha + Show 9 more

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https://doi.org/10.1371/journal.pone.0160995

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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.

Highlights

Over 95% of cervical cancers are caused by human papillomaviruses (HPVs)
Three constructed vectors p26212-16L1d22, p26212-TPL1d22, and p26212-TPL1F for protein expression in plants were designated as V1, V2, and V3, respectively (Fig 1A)
The L1 protein was designed to be accumulated in chloroplasts, which lead to the assembly of predominantly 35–55 nm virus-like particle (VLP) with maximal protein level (>2.5% of total soluble protein (TSP))

Summary

Introduction

Over 95% of cervical cancers are caused by human papillomaviruses (HPVs). Cervical cancer is the second most common HPV-associated cancer in women, with approximately one-half million new cervical cancer cases and almost 250,000 deaths each year worldwide [1]. The greatest burden of HPV-induced cancers occurs in developing countries. Seventy-five percent of PLOS ONE | DOI:10.1371/journal.pone.0160995. Production of HPV16 L1 VLPs in Tobacco Plants. DPI, days post infiltration; TSP, total soluble protein; MoAb, monoclonal antibody; PoAb, polyclonal antibody. Cervical cancers are caused by two HPV types, 16 and 18. HPV16 is responsible for the majority of anal, penile, vaginal, vulvar, and oropharyngeal cancers [2]

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