Development and Optimization of an Enzyme Immunoassay to Detect Serum Antibodies against the Hepatitis E Virus in Pigs, Using Plant-Derived ORF2 Recombinant Protein

Abstract

Hepatitis E is an emerging global disease, mainly transmitted via the fecal–oral route in developing countries, and in a zoonotic manner in the developed world. Pigs and wild boar constitute the primary Hepatitis E virus (HEV) zoonotic reservoir. Consumption of undercooked animal meat or direct contact with infected animals is the most common source of HEV infection in European countries. The purpose of this study is to develop an enzyme immunoassay (EIA) for the detection of anti-hepatitis E virus IgG in pig serum, using plant-produced recombinant HEV-3 ORF2 as an antigenic coating protein, and also to evaluate the sensitivity and specificity of this assay. A recombinant HEV-3 ORF2 110-610_6his capsid protein, transiently expressed by pEff vector in Nicotiana benthamiana plants was used to develop an in-house HEV EIA. The plant-derived HEV-3 ORF2 110-610_6his protein proved to be antigenically similar to the HEV ORF2 capsid protein and it can self-assemble into heterogeneous particulate structures. The optimal conditions for the in-house EIA (iEIA) were determined as follows: HEV-3 ORF2 110-610_6his antigen concentration (4 µg/mL), serum dilution (1:50), 3% BSA as a blocking agent, and secondary antibody dilution (1:20 000). The iEIA developed for this study showed a sensitivity of 97.1% (95% Cl: 89.9–99.65) and a specificity of 98.6% (95% Cl: 92.5–99.96) with a Youden index of 0.9571. A comparison between our iEIA and a commercial assay (PrioCHECK™ Porcine HEV Ab ELISA Kit, ThermoFisher Scientific, MA, USA) showed 97.8% agreement with a kappa index of 0.9399. The plant-based HEV-3 ORF2 iEIA assay was able to detect anti-HEV IgG in pig serum with a very good agreement compared to the commercially available kit.

Plant‐based expression and characterization of SARS‐CoV‐2 virus‐like particles presenting a native spike protein

“…The plantderived S protein in the S and EMS samples was successfully used as coating antigen to detect anti-SARS-CoV2 IgG in serum samples from COVID-19 recovered patients (Figure 6), which shows that plants produced antigenically relevant protein. In this regard it is worth noting that plant-produced diagnostic antigens from other viruses have shown high sensitivity and specificity (Siew et al, 2021;Takova al., 2021), demonstrating the utility of plant expression systems. The data also suggest that induction of an immune response in the human body might also be possible with the plant-made VLPs reported here, as previously demonstrated with a modified S protein by Ward et al (2021).…”

Plant-Derived Recombinant Vaccines against Zoonotic Viruses

“…VLP formation was also examined by atomic force and electron microscopy. The collected data confirmed self-assembled VLP particles with very heterogeneous sizes [95,312]. The immunogenicity of the plant-produced HEV 110-610 protein was examined in mice, and it was concluded that immunizations with the recombinant protein induced high titers of specific IgG antibodies in comparison to the negative control group [311].…”

“…The transient expression approach was used in conjunction with the highly productive vectors pEAQ-HT and pEff, based on the Cowpea mosaic virus (CPMV) and the potato virus X (PVX), respectively. When a truncated ORF2 (110-610 aa) sequence, cloned into the pEAQ-HT vector, was expressed, it resulted in a protein production rate of 100 µg/g of FW [310], whereas the same sequence cloned into the pEff vector yielded up to 200 µg/g of FW [311,312]. Both products confirmed that the truncated HEV capsid proteins expressed in plants are suitable for diagnostic purposes [310,312].…”

“…When a truncated ORF2 (110-610 aa) sequence, cloned into the pEAQ-HT vector, was expressed, it resulted in a protein production rate of 100 µg/g of FW [310], whereas the same sequence cloned into the pEff vector yielded up to 200 µg/g of FW [311,312]. Both products confirmed that the truncated HEV capsid proteins expressed in plants are suitable for diagnostic purposes [310,312]. VLP formation was also examined by atomic force and electron microscopy.…”

High-Yield Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope and Receptor Binding Domain of SARS-CoV-2 in Plants Using Viral Vectors

“…Examination of the HEV/RBD samples by electron and atomic force microscopy revealed VLPs with size of 30–40 nm, while dynamic light scattering detected ~40 nm particles and aggregates 200–300 nm in size ( Table 1 ). In comparison, the HEV 110-610 capsid protein without any fusion partners formed smaller particles with an estimated size of 18–26 nm ( Table 1 and [ 71 ]).…”