Capsid protein of Hepatitis E virus (HEV) is capable of self-assembly into virus-like particles (VLPs) when expressed in Nicotiana benthamiana plants. Such VLPs could be used as carriers of antigens for vaccine development. In this study, we obtained VLPs based on truncated coat protein of HEV bearing the M2e peptide of Influenza A virus or receptor-binding domain of SARS-CoV-2 spike glycoprotein (RBD). We optimized the immunogenic epitopes’ presentation by inserting them into the protruding domain of HEV ORF2 at position Tyr485. The fusion proteins were expressed in Nicotiana benthamiana plants using self-replicating potato virus X (PVX)-based vector. The fusion protein HEV/M2, targeted to the cytosol, was expressed at the level of about 300–400 μg per gram of fresh leaf tissue and appeared to be soluble. The fusion protein was purified using metal affinity chromatography under native conditions with the final yield about 200 μg per gram of fresh leaf tissue. The fusion protein HEV/RBD, targeted to the endoplasmic reticulum, was expressed at about 80–100 μg per gram of fresh leaf tissue; the yield after purification was up to 20 μg per gram of fresh leaf tissue. The recombinant proteins HEV/M2 and HEV/RBD formed nanosized virus-like particles that could be recognized by antibodies against inserted epitopes. The ELISA assay showed that antibodies of COVID-19 patients can bind plant-produced HEV/RBD virus-like particles. This study shows that HEV capsid protein is a promising carrier for presentation of foreign antigen.
Introduction. Chronic viral hepatitis C (CHC) is a ubiquitous infectious disease, a significant limitation of which WHO attributes to the use of a new highly effective antiviral therapy. Previously, two B-cell epitopes were identified in NS4a antigen of the hepatitis C virus (HCV). It was shown that certain titers of antibodies (ABs) to the extended C-terminal epitope (1687–1718 a.a.) can predict a high probability of achieving a sustained virological response (SVR) to standard therapy with pegylated interferon-α and ribavirin.The aim of the work was to determine immunoreactivity of two B-cell epitopes (middle and C-terminal) of NS4a antigen, and to estimate a possible association of ABs to them with the achievement of SVR after standard interferon therapy and treatment with direct antiviral drugs (DAAs) daclatasvir and sofosbuvir (velpanat).Materials and methods. Blood serum samples of patients with CHC (n = 113), of which 55 participants received standard interferon therapy, 50 received velpanate treatment, the remaining 8 received no therapy were examined. The middle B-cell epitope (positions 24–34 a.a.) of NS4a was synthesized by the solid-phase method, while the C-terminal epitope (34–54 a.a.) was obtained using genetically engineered techniques. Enzyme immunoassay (ELISA) testing of the sera collected before treatment was performed for the two selected epitopes according to the conventional methods.Results. The antibodies to the C-terminal epitope were detected significantly more frequently than those to the middle one (p = 0.01) when analyzing the blood sera of patients (n = 113). The presence of ABs to the C-terminal epitope in the serum samples of participants who completed standard interferon therapy was associated with the achievement of SVR (p = 0.0245). In the blood sera of participants who completed therapy with velpanate, an association of the presence of ABs to the C-terminal epitope with the achievement of SVR was also established (p < 0.0001). The presence of ABs to the middle B epitope was not associated with the achievement of SVR, regardless of the therapy used.Discussion. The observed difference in the immunoreactivity of the two B-cell determinants may be associated with the localization of the nearest Th-epitopes, the sensitivity of NS4a antigen to proteolytic enzymes, and the peculiarities of epitope presentation by antigen-presenting cells. However, it should be noted that the immunoreactivity of the middle B-epitope is poorly studied. Although the association of ABs to the C-terminal epitope with the achievement of SVR has been shown by several scientific teams, the detailed molecular mechanism of their influence on the effectiveness of therapy is unclear.Conclusion. In CHC, ABs to the C-terminal epitope of NS4a are produced more frequently than those to the median epitope. The presence of ABs to the C-terminal epitope is a predictive marker of a high probability of achieving SVR, regardless of the type of therapy and antibody titer.
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