Detection of Serum Antibodies to Hepatitis E Virus Based on HEV Genotype 3 ORF2 Capsid Protein Expressed in Nicotiana benthamiana

Abstract

BackgroundHepatitis E virus (HEV) causes epidemics in developing countries and is primarily transmitted through the fecal-oral route. There have been recent reports on the zoonotic spread of the virus, and several animal species, primarily pigs, have been recognized as reservoirs of HEV. Because of its possible spread, there is an urgent need of a method for the cost-effective production of HEV proteins that can be used as diagnostic antigens for the serological detection of anti-HEV antibodies.MethodsThe HEV open reading frame (ORF)2 protein was purified from plant tissue by using immobilized metal-anion chromatography (IMAC). The recombinant protein was used to develop an in-house ELISA for testing anti-HEV antibodies in both human and swine sera. Thirty-six serum samples collected from patients with serologically proven HEV infection with commercial kits were tested for anti-HEV IgG antibodies by using the plant-expressed protein. Forty-five serum samples collected from apparently healthy pigs in Bulgarian farms were also tested.ResultsWe confirmed the transient expression and purification of a truncated version of the HEV genotype 3 capsid protein in Nicotiana benthamiana and its usefulness as a diagnostic antigen. ELISA showed the presence of anti-HEV IgG antibodies in 29 of the 36 human samples. The in-house ELISA showed anti-HEV IgG antibodies in 34 of the 45 pigs.ConclusionsWe describe a method for the production of HEV ORF2 protein in N. benthamiana and the usefulness of this protein for the serological detection of anti-HEV antibodies in both humans and swine.

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].…”

“…The first immunological study of pigs in Bulgaria from 2017 reported over 75% seropositivity. A noncommercial ELISA test based on the HEV ORF2 110-610 capsid protein was used to detect anti-HEV IgG antibodies in domestic pigs [33]. Another study reports overall HEV seroprevalence of 60.3%.…”

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

“…Presently, all efforts aimed at the development of a Hepatitis E vaccine are focused on the ORF2 capsid protein, as it contains epitopes that can induce neutralizing antibodies [11,12]. The ORF2 capsid protein is also an appropriate candidate for the serological diagnosis of HEV [13]. The full-length HEV ORF2 capsid protein consists of 660 aa, with a molecular weight of 72 kDa.…”

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

“…Additionally, transient expression of truncated ORF2 (110-610 aa) sequence from swine HEV-3 with the highly efficient vector pEAQ-HT was performed in Nicotiana benthamiana plants for the first time [127,128]. 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].…”