Expression of the Multimeric and Highly Immunogenic Brucella spp. Lumazine Synthase Fused to Bovine Rotavirus VP8d as a Scaffold for Antigen Production in Tobacco Chloroplasts

E Federico Alfano,Andrés Wigdorovitz,Ezequiel M Lentz,María J Dus Santos,Fernando A Goldbaum,Demian Bellido,Fernando F Bravo-Almonacid

doi:10.3389/fpls.2015.01170

Abstract

Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein which can accommodate foreign polypeptides or protein domains fused to its N-termini, markedly increasing their immunogenicity. The inner core domain (VP8d) of VP8 spike protein from bovine rotavirus is responsible for viral adhesion to sialic acid residues and infection. It also displays neutralizing epitopes, making it a good candidate for vaccination. In this work, the BLS scaffold was assessed for the first time in plants for recombinant vaccine development by N-terminally fusing BLS to VP8d and expressing the resulting fusion (BLSVP8d) in tobacco chloroplasts. Transplastomic plants were obtained and characterized by Southern, northern and western blot. BLSVP8d was highly expressed, representing 40% of total soluble protein (4.85 mg/g fresh tissue). BLSVP8d remained soluble and stable during all stages of plant development and even in lyophilized leaves stored at room temperature. Soluble protein extracts from fresh and lyophilized leaves were able to induce specific neutralizing IgY antibodies in a laying hen model. This work presents BLS as an interesting platform for highly immunogenic injectable, or even oral, subunit vaccines. Lyophilization of transplastomic leaves expressing stable antigenic fusions to BLS would further reduce costs and simplify downstream processing, purification and storage, allowing for more practical vaccines.

Highlights

Rotavirus is the main cause of severe gastroenteritis in newborn mammals and constitutes severe economic losses across the world (Saif and Fernandez, 1996)
We report for the first time the production of transplastomic plants expressing a recombinant fusion of Brucella spp. (BLS), a highly immunogenic decameric protein from Brucella spp, to an antigen
We have recently succeeded in producing transplastomic tobacco which expressed bovine rotavirus (BRV) C486 VP8∗ protein, an interesting target for vaccine development since it is involved in rotavirus infectivity and neutralization

Summary

Introduction

Rotavirus is the main cause of severe gastroenteritis in newborn mammals and constitutes severe economic losses across the world (Saif and Fernandez, 1996). According to a ten year study spanning from 1994 to 2003, group A bovine rotavirus (BRV) is the main cause of neonatal diarrhea in calves in Argentina, being responsible for substantial economic constraints concerning. The outer layer is in turn formed by two different proteins, VP7 and VP4 (Estes et al, 2001). The latter possess hemagglutinating activity and participates in viral adsorption (Kalica et al, 1983; Crawford et al, 1994). VP8 displays neutralizing epitopes which makes it a good candidate for recombinant vaccines (Ruggeri and Greenberg, 1991)

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Conclusion