A Proline-Rich Domain in the Genotype 4 Hepatitis E Virus ORF3 C-Terminus Is Crucial for Downstream V105DLP108 Immunoactivity

Abstract

The hepatitis E virus (HEV) is responsible for serious viral hepatitis worldwide. Animals are considered a reservoir of HEV, particularly pigs. While HEV infection in pigs and dogs is always asymptomatic, the virus causes high death rates in patients with pre-existing chronic liver disease and pregnant women in developing countries. HEV open reading frame 2 (ORF2) has been used as a diagnostic target to detect specific antibodies against HEV in serum samples. Recent research has additionally supported the potential utility of the ORF3 protein as a target in serum anti-HEV detection. However, the epitope distribution of ORF3 protein remains ambiguous. In the current study, we showed that continuous amino acid motif, VDLP, at the C-terminus of genotype 4 HEV ORF3 is a core sequence of the ORF3 protein epitope. Moreover, cooperative interaction with upstream elements is essential for its immunoactivity. Three proline residues (P99, P102 and P103) in the upstream proline-rich domain exerted significant effects on the immunocompetence of VDLP. ELISA results revealed that SAPPLPPVVDLP and SAPPLPPVVDLPQLGL peptides containing the identified VDLP epitope display weaker reactions with anti-HEV serum than the commercial ELISA kit. Our collective findings provide valuable information on the epitope distribution characteristics of HEV ORF3 and improve our understanding of the influence of the proline-rich domain on the immunoactivity of downstream amino acids in the C-terminal region.

Identification and characterization of two linear epitope motifs in hepatitis E virus ORF2 protein

“…Purified recombinant ORF2 protein was used as an antigen to immunize six 5-week old Balb/c mice, and mAbs were prepared using standard methodology [ 12 ]. All of the Balb/c mice were kept at 21 to 25°C on a 12 h light/dark cycle and were given free access to food and water.…”

“…Polyclonal antibody against ORF2 was used as a positive control and normal mouse serum as a negative control. Subsequent steps were conducted as described previously [ 12 ].…”

“…The prepared mAbs were also used to detect HEV ORF2 protein expressed in baby hamster kidney (BHK) cells transfected with pcDNA3.1-ORF2 (nucleotides 1240–2022) plasmids, by immunofluorescence assay (IFA). The detailed procedures were performed as described previously [ 12 ].…”

“…Candidate peptides (10 μg/well diluted in 0.1 M NaHCO3, pH 8.6) were coated onto ELISA plates overnight at 4°C. Subsequent steps were conducted as described previously [ 12 ]. (OD 450 Sample—OD 450 Blank)/ (OD 450 Negative control—OD 450 Blank) > 2 was determined as positive result.…”

Hepatitis Vaccines

“…Knowledge of the structure of the HEV genome has been essential for vaccine development [ 71 ]. The Open Reading Frame (ORF) 2 encodes the capsid protein against which all neutralizing antibodies are targeted ( Figure 6 ).…”