A Conserved Domain of HCV E2 Glycoprotein Interacts with Human CD81 and Induces Interferon-Gamma Secretion from Peripheral Blood Mononuclear Cells

¹ Department of Gastroenterology and Hepatology, The Affiliated Hospital of Southwest Jiaotong University, The Third People's Hospital of Chengdu, Chongqing Medical University, 610031 Chengdu, Sichuan, China

² Department of Critical Care Medicine, The Affiliated Hospital of Southwest Jiaotong University, The Third People's Hospital of Chengdu, Chongqing Medical University, 610031 Chengdu, Sichuan, China

³ Department of Infectious diseases, The Third Affiliated Hospital of Sun Yat-sen university, 510630 Guangzhou, Guangdong, China

^*Correspondence: wn9898@163.com (Zhiyan Dai)
^†These authors contributed equally.

Front. Biosci. (Landmark Ed) 2023, 28(10), 239; https://doi.org/10.31083/j.fbl2810239

Submitted: 2 December 2022 | Revised: 15 April 2023 | Accepted: 24 May 2023 | Published: 18 October 2023

This is an open access article under the CC BY 4.0 license.

Abstract

Background: Hepatitis C virus (HCV) infection is a global health threat to the public, and vaccines against it are not yet available. The HCV envelope glycoprotein E2 is a key target for anti-HCV vaccines. The majority of previous studies have focused on the hypervariable region and the glycosylation sites of the_HCV structural protein. This study aims to investigate a conserved domain of HCV E2 glycoprotein and explore its potential to induce an immune response against HCV. Methods: HCV E2 conserved domain (encompassing amino acids 505–702) was prepared in Escherichia coli (E. coli). Peripheral blood mononuclear cells (PBMCs) were isolated from patients with HCV or healthy controls. Interferon-gamma (IFN- $\gamma{}$ ) enzyme-linked immunosorbent spot assay was conducted to examine the HCV E2-specific immune response as reflected by IFN- $\gamma{}$ -secreting cells/10 ${}^{6}$ PBMCs. Results: HCV E2 conserved domain was highly conserved among 25 HCV subtypes, and its recombinant soluble production in E. coli was recognized by anti-HCV E2 monoclonal antibodies. This study characterized in vitro direct interaction between bacterially expressed HCV E2 conserved domain and human CD81 (hCD81). Furthermore, the recombinant HCV E2_conserved domain markedly induced the production of IFN- $\gamma{}$ by PBMCs from patients with HCV. Its stimulated specific immune response was significantly different from non-specific peptide controls or PBMCs isolated from healthy controls. Conclusions: HCV E2 conserved domain directly binds hCD81 and activates the production of IFN- $\gamma{}$ in the PBMCs of patients with HCV. Therefore, the conserved domain of HCV E2 glycoprotein may be a new candidate for developing an HCV vaccine.

Keywords

hepatitis C virus

E2 glycoprotein

conserved domain

hCD81

protein–protein interaction

interferon-gamma

immune response

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Fig. 1.

Analysis of hepatitis C virus E2 and hCD81 protein expression and purification. Prokaryotic expression plasmids were transformed into Escherichia coli strain BL21, and hepatitis C virus E2 or hCD81 protein (the large extracellular loop) expression was induced by propylthio- $\beta{}$ -galactoside and purified by nickel-nitrilotriacetic acid affinity chromatography. (A) 12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis of cell extract from the supernatant (Lanes 1–4) and precipitate samples (Lanes 5–8). Lane M: protein marker; Lane1: pET-22b (+) empty vector control; Lane 2: phenobarbital without propylthio- $\beta{}$ -galactoside induction; Lane 3: phenobarbital with propylthio- $\beta{}$ -galactoside induction for 4 h; Lane 4: untreated cells; Lane 5: pET-22b (+) empty vector control; Lane 6: phenobarbital without propylthio- $\beta{}$ -galactoside induction; Lane 7: phenobarbital with propylthio- $\beta{}$ -galactoside induction for 4 h; Lane 8: untreated cells. (B) 12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis of fusion protein. B1, 12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis of hepatitis C virus E2 protein. Lane M: protein marker; Lane1: pET-22b (+) empty vector control; Lane 2: fusion protein E2 expression without induction with propylthio- $\beta{}$ -galactoside; Lane 3: induction of fusion protein E2 expression by propylthio- $\beta{}$ -galactoside; B2, 12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis of hCD81 protein. Lane M: protein marker; Lanes1–3: induction of fusion protein hCD81 in different concentrations by propylthio- $\beta{}$ -galactoside; Lanes 4–5: fusion protein hCD81 expression without propylthio- $\beta{}$ -galactoside induction. (C) 12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis of fusion protein purification. C1, Hepatitis C virus E2 protein purification. Lane M: protein marker; Lane 1: his-tagged purified E2 protein in the elusion fraction; Lane 2: non-his-tagged protein as negative control; C2, hCD81 protein purification. Lane M: protein marker; Lanes 1: his-tagged purified hCD81 protein in the elusion fraction; Lane 2: non-his-tagged protein as negative control. (D) Western blot analysis of fusion protein expression. D1, Western blot analysis of hepatitis C virus E2 protein expression. Lane1: hepatitis C virus E2 protein detected by an anti-histidine monoclonal antibody; D2, Western blot analysis of hCD81 protein expression. Lane1: hCD81 protein detected by an anti-histidine monoclonal antibody. (E) Western blot analysis of fusion protein expression. E1, Western blot analysis of hepatitis C virus E2 protein expression. Lane1: hepatitis C virus E2 protein detected by anti-hepatitis C virus E2 monoclonal antibody; E2, Western blot analysis of hCD81 protein expression. Lane1: hCD81 protein detected by anti-hCD81 monoclonal antibody.

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