Optimizing a Human Papillomavirus Type 16 L1-Based Chimaeric Gene for Expression in Plants.

Inga I Hitzeroth,Edward P Rybicki,Marcus Graf,Aleyo Chabeda,Mark P Whitehead

doi:10.3389/fbioe.2018.00101

Inga I Hitzeroth, Edward P Rybicki + Show 3 more

Open Access

https://doi.org/10.3389/fbioe.2018.00101

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Abstract

Human papillomaviruses (HPVs) are the causative agents of cervical cancer, the fourth most prevalent cancer in women worldwide. The major capsid protein L1 self-assembles into virus-like particles (VLPs), even in the absence of the minor L2 protein: such VLPs have successfully been used as prophylactic vaccines. There remains a need, however, to develop cheaper vaccines that protect against a wider range of HPV types. The use of all or parts of the L2 minor capsid protein can potentially address this issue, as it has sequence regions conserved across several HPV types, which can elicit a wider spectrum of cross-neutralizing antibodies. Production of HPV VLPs in plants is a viable option to reduce costs; the use of a L1/L2 chimera which has previously elicited a cross-protective immune response is an option to broaden cross-protection. The objective of this study was to investigate the effect of codon optimization and of increasing the G+C content of synthetic L1/L2 genes on protein expression in plants. Additionally, we replaced varying portions of the 5′ region of the L1 gene with the wild type (wt) viral sequence to determine the effect of several negative regulatory elements on expression. We showed that GC-rich genes resulted in a 10-fold increase of mRNA levels and 3-fold higher accumulation of proteins. However, the highest increase of expression was achieved with a high GC-content human codon-optimized gene, which resulted in a 100-fold increase in mRNA levels and 8- to 9-fold increase in protein levels. Changing the 5′ end of the L1 gene back to its wt sequence decreased mRNA and protein expression. Our results suggest that the negative elements in the 5′ end of L1 are inadvertently destroyed by changing the codon usage, which enhances protein expression.

Highlights

Cervical cancer is the fourth most common cancer among women worldwide (Ferlay et al, 2015), and the causal association between Human papillomavirus (HPV) infection and cervical cancer has been well described
The wt ChiF has 38% GC content and the native HPV codon usage was preserved; in the SAF2 back-translated gene the GC content was kept similar to the wt type gene, but tobacco (N. benthamiana) preferred codons were used
The HPV-16 L1 gene was human codon optimized for a gene with 62% GC content

Summary

Introduction

Cervical cancer is the fourth most common cancer among women worldwide (Ferlay et al, 2015), and the causal association between Human papillomavirus (HPV) infection and cervical cancer has been well described (zur Hausen et al, 1981; zur Hausen, 1996). Three multivalent HPV prophylactic vaccines based on L1 virus-like proteins (VLPs) have been licensed, and are highly effective in the prevention of vaccine-type infections and associated disease (Schiller et al, 2008; Joura et al, 2015; Toh et al, 2015; Signorelli et al, 2017). Despite the success of these vaccines, the cervical cancer burden remains high. These vaccines are expensive and show type-restrictive prophylactic efficacy. The L2 N-terminal region contains broadly cross-neutralizing epitopes, L2 is subdominant to L1 as an antigen, and the use of L1+L2 VLPs in vaccination does not confer more cross-protection in animals compared to use of L1 VLPs only (Roden et al, 2000). Increasing the density of these sequences at the surface of the particles in a different context than the immunodominent L1 pentamer would almost certainly significantly increase their immunogenicity

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