Enterovirus D68 virus-like particles expressed in Pichia pastoris potently induce neutralizing antibody responses and confer protection against lethal viral infection in mice

Chao Zhang,Xueyang Zhang,Wei Zhang,Wenlong Dai,Jing Xie,Liping Ye,Hongli Wang,Huan Chen,Qingwei Liu,Sitang Gong,Lanlan Geng,Zhong Huang

doi:10.1038/s41426-017-0005-x

Chao Zhang, Xueyang Zhang + Show 10 more

Open Access

https://doi.org/10.1038/s41426-017-0005-x

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Abstract

Enterovirus D68 (EV-D68) has been increasingly associated with severe respiratory illness and neurological complications in children worldwide. However, no vaccine is currently available to prevent EV-D68 infection. In the present study, we investigated the possibility of developing a virus-like particle (VLP)-based EV-D68 vaccine. We found that co-expression of the P1 precursor and 3CD protease of EV-D68 in Pichia pastoris yeast resulted in the generation of EV-D68 VLPs, which were composed of processed VP0, VP1, and VP3 capsid proteins and were visualized as ~30 nm spherical particles. Mice immunized with these VLPs produced serum antibodies capable of specifically neutralizing EV-D68 infections in vitro. The in vivo protective efficacy of the EV-D68 VLP candidate vaccine was assessed in two challenge experiments. The first challenge experiment showed that neonatal mice born to the VLP-immunized dams were fully protected from lethal EV-D68 infection, whereas in the second experiment, passive transfer of anti-VLP sera was found to confer complete protection in the recipient mice. Collectively, these results demonstrate the proof-of-concept for VLP-based broadly effective EV-D68 vaccines.

Highlights

Enterovirus D68 (EV-D68) is a virus belonging to the Enterovirus genus of the Picornaviridae family[1]
In order to express EV-D68 virus-like particle (VLP) in P. pastoris, three constructs were made: pEV-D68-001 and pEV-D68-002 carried only one single P1 and one single 3CD expression cassette, respectively, while pEV-D68-003 contained both cassettes to co-express P1 and 3CD (Fig. 1a)
Our results demonstrated for the first time that yeastproduced EV-D68 VLP could induce effective protective

Summary

Introduction

Enterovirus D68 (EV-D68) is a virus belonging to the Enterovirus genus of the Picornaviridae family[1]. The genome of EV-D68 is a positivesense single-stranded RNA of ~7.4 kb and contains a single open reading frame (ORF) that encodes a large polyprotein[1,2]. This polyprotein can be processed to produce three precursor proteins, P1, P2, and P3. A crystal structure of EV-D68 virus shows that the EV-D68 viral capsid is made of 60 copies of each of VP1, VP2, VP3, and VP4 subunit proteins and it possesses structural features typical for enteroviruses, such as threefold propeller-like protrusions, star-shaped five-fold plateaus, narrow depressions (canyons) surrounding each plateau, and hydrophobic pockets inside VP1 and directly beneath the canyon floor[3]. Recent studies have identified neuron-specific intercellular adhesion molecule-5 (ICAM-5/telencephalin) and sialic acid as two cellular receptors for EV-D684–6

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