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

• Published in: Vaccines (Basel) 2021-09, Vol.9 (9), p.991
• Main Authors: Takova, Katerina, Koynarski, Tsvetoslav, Minkov, George, Toneva, Valentina, Mardanova, Eugenia, Ravin, Nikolai, Lukov, Georgi L., Zahmanova, Gergana
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Antigens; Capsid protein; Dengue fever; detection of serum antibodies; Developing countries; diagnostic antigen; Dilution; Disease transmission; Enzyme immunoassay; Enzyme-linked immunosorbent assay; Genomes; Hepatitis; hepatitis E virus; Hogs; Immunoassay; Immunoglobulin G; Infections; LDCs; Meat; Microscopy; Optimization; Oral administration; ORF2 capsid protein; plant molecular farming; Plants; Proteins; RNA polymerase; Sensitivity analysis; Serology; Swine; transient expression; Viruses; West Nile virus; Zoonoses
• ISSN: 2076-393X; 2076-393X
• DOI: 10.3390/vaccines9090991

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.