Expression of HPV-11 L1 protein in transgenic Arabidopsis thaliana and Nicotiana tabacum

Thomas O Kohl,Edward P Rybicki,Neil D Christensen,Inga I Hitzeroth

doi:10.1186/1472-6750-7-56

Abstract

BackgroundWe have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Xanthi as potential sources for an inexpensive subunit vaccine.ResultsTransformation of plants was only achieved with the HPV-11 L1 gene with the C-terminal nuclear localization signal (NLS-) encoding region removed, and not with the full-length gene. The HPV-11 L1 NLS- gene was stably integrated and inherited through several generations of transgenic plants. Plant-derived HPV-11 L1 protein was capable of assembling into virus-like particles (VLPs), although resulting particles displayed a pleomorphic phenotype. Neutralising monoclonal antibodies binding both surface-linear and conformation-specific epitopes bound the A. thaliana-derived particles and – to a lesser degree – the N. tabacum-derived particles, suggesting that plant-derived and insect cell-derived VLPs displayed similar antigenic properties. Yields of up to 12 μg/g of HPV-11 L1 NLS- protein were harvested from transgenic A. thaliana plants, and 2 μg/g from N. tabacum plants – a significant increase over previous efforts. Immunization of New Zealand white rabbits with ~50 μg of plant-derived HPV-11 L1 NLS- protein induced an antibody response that predominantly recognized insect cell-produced HPV-11 L1 NLS- and not NLS+ VLPs. Evaluation of the same sera concluded that none of them were able to neutralise pseudovirion in vitro.ConclusionWe expressed the wild-type HPV-11 L1 NLS- gene in two different plant species and increased yields of HPV-11 L1 protein by between 500 and 1000-fold compared to previous reports. Inoculation of rabbits with extracts from both plant types resulted in a weak immune response, and antisera neither reacted with native HPV-11 L1 VLPs, nor did they neutralise HPV-11 pseudovirion infectivity. This has important and potentially negative implications for the production of HPV-11 vaccines in plants.

Highlights

We have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv
PCR screening of putative HPV-11 L1 nuclear localisation signal (NLS)- transgenic A. thaliana first generation (T1) and N. tabacum regenerated generation (R0) plants, using primer pair 4 as listed in Table 1, showed that 8/20 A. thaliana T1 generation and 4/15 N. tabacum R0 lines were positive for the gene
We suggest that an immune response to plant-derived HPV-11 L1 NLS- is elicited mainly against an immunodominant epitope that appears to be surface exposed in virus-like particles (VLPs)

Summary

Introduction

We have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Given a HPV-11 prevalence rate of 5–12% in normal women [6,7,8], a serious recent concern is the impact of increasing human immunodeficiency virus (HIV) infection rates, and the associated immunosuppression of HIVpositive individuals, on HPV-6 or 11 coinfections. Silverberg et al [10] have found the prevalence of HPV-6 and 11 to be up to 5.6 times higher in HIV-seropositive women, thereby increasing the prevalence of genital warts by a factor of 3.2. The associated morbidity and negative effects on quality of life are a major problem among HIV-infected women (L Denny, pers comm). The complications of HPV-11 coinfection in HIV-seropositive individuals necessitate the urgent development of a safe, efficacious and inexpensive vaccine against HPV-11

Objectives

Methods

Results

Discussion

Conclusion