Characterization of Three Novel Linear Neutralizing B-Cell Epitopes in the Capsid Protein of Swine Hepatitis E Virus

Abstract

Hepatitis E virus (HEV) causes liver disease in humans and is thought to be a zoonotic infection, with domestic animals, including swine and rabbits, being a reservoir. One of the proteins encoded by the virus is the capsid protein. This is likely the major immune-dominant protein and a target for vaccination. Four monoclonal antibodies (MAbs), three novel, 1E4, 2C7, and 2G9, and one previously characterized, 1B5, were evaluated for binding to the capsid protein from genotype 4 swine HEV. The results indicated that DFCP, PSRPF, and EPTV peptides on the capsid protein comprised minimal amino acid sequence motifs recognized by 1E4, 2C7, and 2G9, respectively. The data suggested that 2C7 and 2G9 epitopes were partially exposed on the surface of the capsid protein. Truncated genotype 4 swine HEV capsid protein (sp239, amino acids 368 to 606) can exist in multimeric forms. Preincubation of swine HEV with 2C7, 2G9, or 1B5 before addition to HepG2 cells partially blocked sp239 cell binding and inhibited swine HEV infection. The study indicated that 2C7, 2G9, and 1B5 partially blocked swine HEV infection of rabbits better than 1E4 or normal mouse IgG. The cross-reactivity of antibodies suggested that capsid epitopes recognized by 2C7 and 2G9 are common to HEV strains infecting most host species. Collectively, MAbs 2C7, 2G9, and 1B5 were shown to recognize three novel linear neutralizing B-cell epitopes of genotype 4 HEV capsid protein. These results enhance understanding of HEV capsid protein structure to guide vaccine and antiviral design. Genotype 3 and 4 HEVs are zoonotic viruses. Here, genotype 4 HEV was studied due to its prevalence in human populations and pig herds in China. To improve HEV disease diagnosis and prevention, a better understanding of the antigenic structure and neutralizing epitopes of HEV capsid protein are needed. In this study, the locations of three novel linear B-cell recognition epitopes within genotype 4 swine HEV capsid protein were characterized. Moreover, the neutralizing abilities of three MAbs specific for this protein, 2C7, 2G9, and 1B5, were studied and Collectively, these findings reveal structural details of genotype 4 HEV capsid protein and should facilitate development of applications for the design of vaccines and antiviral drugs for broader prevention, detection, and treatment of HEV infection of diverse human and animal hosts.

Synthetic Peptides Containing Three Neutralizing Epitopes of Genotype 4 Swine Hepatitis E Virus ORF2 induced Protection against Swine HEV Infection in Rabbit

“…Of these MAbs, 2C7 and 2G9 could partially inhibit genotype 4 swine HEV infection of HepG2 cells in vitro and of rabbits in vivo. In addition, MAb 1B5, which recognizes an epitope common to HEV isolates that infect various host species (including birds), also showed partial neutralizing ability both in vitro and in vivo [16,22]. These results infer that peptides recognized by these MAbs may themselves generate naturalizing anti-HEV antibodies in vivo after immunization.…”

“…During necropsy, liver tissues were collected for routine histological examination. Swine HEV truncated ORF2 protein sp239 (aa 368-606) and ORF3 protein were expressed separately in Escherichia coli BL21 (DE3) host cells and purified as described previously [22].…”

“…These results suggest that slight structural diversity exists among ORF2 proteins of the four major HEV genotypes. Notably, few published reports have described antibody-neutralizing epitopes residing within genotype 4 swine HEV ORF2 protein [22].…”

Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection

“…The procedure for inducing the expression of ap237 without any tags was based on modified methods described previously by Chen et al (50). Briefly, the expression of ap237 was induced by adding 1.0 mM IPTG for 6 to 8 h at 37°C.…”

“…KX227751), and genotype 4 swine HEV (CHN-SD-sHEV; GenBank accession no. KE176351), respectively, and were all expressed and purified as previously described by Chen et al (50). Another negative-control protein, the porcine reproductive and respiratory syndrome virus (PRRSV) N protein, was also expressed and purified according to the same procedures as the ones described above.…”