Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope

Abstract

The Hepatitis E virus (HEV) is a causative agent of acute hepatitis, mainly transmitted by the fecal-oral route or zoonotic. Open reading frame (ORF) 2 encodes the viral capsid protein, which is essential for virion assembly, host interaction, and inducing neutralizing antibodies. In this study, we investigated whether full-length and N- and C-terminally modified versions of the capsid protein transiently expressed in N. benthamiana plants could assemble into highly-immunogenic, virus-like particles (VLPs). We also assessed whether such VLPs can act as a carrier of foreign immunogenic epitopes, such as the highly-conserved M2e peptide from the Influenza virus. Plant codon-optimized HEV ORF2 capsid genes were constructed in which the nucleotides coding the N-terminal, the C-terminal, or both parts of the protein were deleted. The M2e peptide was inserted into the P2 loop after the residue Gly556 of HEV ORF2 protein by gene fusion, and three different chimeric constructs were designed. Plants expressed all versions of the HEV capsid protein up to 10% of total soluble protein (TSP), including the chimeras, but only the capsid protein consisting of aa residues 110 to 610 (HEV 110–610) and chimeric M2 HEV 110–610 spontaneously assembled in higher order structures. The chimeric VLPs assembled into particles with 22–36 nm in diameter and specifically reacted with the anti-M2e antibody.

Efficient Production of Chimeric Hepatitis B Virus-Like Particles Bearing an Epitope of Hepatitis E Virus Capsid by Transient Expression in Nicotiana benthamiana

“…The nucleotide sequences of the whole HEV genotype 3 nucleoprotein (HEV ORF2 1–660) [ 25 ] codon-optimized for N. benthamiana were used as templates for PCR amplification of HEV ORF2 551–607 to construct the pEAQ-HT:HBcHEV ORF2 551–607 ( Figure 2 ). Primers were designed to incorporate SalI and AseI restriction sites and long flexible linkers ((GGS)4) at 5’ and 3’ ends of the HEV ORF2 551–607 region.…”

“…Amplification of DNA fragment for the production of the HEV ORF2 501–607, was carried out by PCR. The target HEV ORF2 551–607 sequences were amplified using the synthetic master gene HEV ORF2 1–660 [ 25 ]. The PCR fragment was flanked by Sal I and Ase I restriction sites (New England Biolabs, Ipswich, MA, USA), which were used for sub-cloning of the PCR fragment.…”

“…The major anti-HEV antibody responses are against epitopes located at 459–606 aa of HEV ORF2 [ 18 ] and are mainly conformational [ 19 ]. Full-length and partially truncated versions of the capsid protein have been expressed in different systems (bacteria, yeast, insect cells, mammalian cells, and plants) to generate virus-like particles (VLPs) for vaccine design [ 20 , 21 , 22 , 23 , 24 , 25 , 26 ]. However, there can be issues of yield and product heterogeneity using this approach and alternatives are required.…”

Increasing Hepatitis E Virus Seroprevalence in Domestic Pigs and Wild Boar in Bulgaria

“…There is a need for the production of HEV immunogenic proteins, which can be used for development of serological tests for detection of the HEV infection. In our previous study, we described a method for the production of recombinant HEV open reading frame (ORF) 2 protein in Nicotiana benthamiana plants and the successful use of this protein for serological detection of anti-HEV antibodies in both humans and swine [33,56]. Other studies have also demonstrated the efficacy of rORF2 gt 3 produced in various expression systems in the detection of anti-HEV serum Ab [57,58].…”

Progress in the Production of Virus-Like Particles for Vaccination against Hepatitis E Virus

“…The truncated protein was purified in high amounts of up to 100 mg/kg FWT, and could be used as a diagnostic antigen [95]. Attempts were also made to produce HEV VLPs and chimeric M2-HEV VLPs using varying lengths of ORF2 [129]. The immunogenicity and VLP assembly properties of the transiently expressed ORF2 in plants remain to be determined.…”