Characterization of Two Novel Linear B-Cell Epitopes in the Capsid Protein of Avian Hepatitis E Virus (HEV) That Are Common to Avian, Swine, and Human HEVs

ABSTRACTAntisera raised against the avian hepatitis E virus (HEV) capsid protein are cross-reactive with human and swine HEV capsid proteins. In this study, two monoclonal antibodies (MAbs) against the avian HEV capsid protein, namely, 3E8 and 1B5, were shown to cross-react with the swine HEV capsid protein. The motifs involved in binding both MAbs were identified and characterized using phage display biopanning, peptide synthesis, and truncated or mutated protein expression, along with indirect enzyme-linked immunosorbent assay (ELISA) and Western blotting. The results showed that the I/VPHD motif is a necessary core sequence and that P and H are two key amino acids for recognition by MAb 3E8. The VKLYM/TS motif is the minimal amino acid sequence necessary for recognition by MAb 1B5. Cross-reactivity between the two epitopes and antibodies against avian, swine, and human HEVs in sera showed that both epitopes are common to avian, swine, and human HEVs. In addition, amino acid sequence alignment of the capsid proteins revealed that the key motifs of both novel epitopes are the same in HEVs from different animal species, predicting that they may be common to HEV isolates from boars, rabbits, rats, ferrets, mongooses, deer, and camels as well. Protein modeling analysis showed that both epitopes are at least partially exposed on the surface of the HEV capsid protein. Protective capacity analysis demonstrated that the two epitopes are nonprotective against avian HEV infection in chickens. Collectively, these studies characterize two novel linear B-cell epitopes common to avian, swine, and human HEVs, which furthers the understanding of HEV capsid protein antigenic structure.IMPORTANCEMore and more evidence indicates that the host range diversity of hepatitis E virus (HEV) is a global public health concern. A better understanding of the antigenic structure of the HEV capsid protein may improve disease diagnosis and prevention. In this study, binding site mapping and localization as well as the antigenic biology of two novel linear B-cell epitopes common to several different species of HEV were characterized. These findings partially reveal the antigenic structure of the HEV capsid protein and provide potential applications for the development of diagnostics and interventions for HEV infection.

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Identification of B-cell epitopes in the capsid protein of avian hepatitis E virus (avian HEV) that are common to human and swine HEVs or unique to avian HEV

Journal of General Virology ◽

10.1099/vir.0.81393-0 ◽

2006 ◽

Vol 87 (1) ◽

pp. 217-223 ◽

Cited By ~ 48

Author(s):

H. Guo ◽

E.-M. Zhou ◽

Z. F. Sun ◽

X.-J. Meng ◽

P. G. Halbur

Keyword(s):

B Cell ◽

Hepatitis E Virus ◽

Genomic Sequence ◽

Rabbit Antiserum ◽

Hepatitis E ◽

Sequence Difference ◽

B Cell Epitopes ◽

Orf2 Protein ◽

Avian Hepatitis E Virus ◽

Avian Hev

Avian hepatitis E virus (avian HEV) was recently discovered in chickens from the USA that had hepatitis–splenomegaly (HS) syndrome. The complete genomic sequence of avian HEV shares about 50 % nucleotide sequence identity with those of human and swine HEVs. The open reading frame 2 (ORF2) protein of avian HEV has been shown to cross-react with human and swine HEV ORF2 proteins, but the B-cell epitopes in the avian HEV ORF2 protein have not been identified. Nine synthetic peptides from the predicted four antigenic domains of the avian HEV ORF2 protein were synthesized and corresponding rabbit anti-peptide antisera were generated. Using recombinant ORF2 proteins, convalescent pig and chicken antisera, peptides and anti-peptide rabbit sera, at least one epitope at the C terminus of domain II (possibly between aa 477–492) that is unique to avian HEV, one epitope in domain I (aa 389–410) that is common to avian, human and swine HEVs, and one or more epitopes in domain IV (aa 583–600) that are shared between avian and human HEVs were identified. Despite the sequence difference in ORF2 proteins between avian and mammalian HEVs and similar ORF2 sequence between human and swine HEV ORF2 proteins, rabbit antiserum against peptide 6 (aa 389–399) recognized only human HEV ORF2 protein, suggesting complexity of the ORF2 antigenicity. The identification of these B-cell epitopes in avian HEV ORF2 protein may be useful for vaccine design and may lead to future development of immunoassays for differential diagnosis of avian, swine and human HEV infections.

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

Journal of Virology ◽

10.1128/jvi.00251-18 ◽

2018 ◽

Vol 92 (13) ◽

pp. e00251-18 ◽

Cited By ~ 4

Author(s):

Yiyang Chen ◽

Baoyuan Liu ◽

Yani Sun ◽

Huixia Li ◽

Taofeng Du ◽

...

Keyword(s):

B Cell ◽

Capsid Protein ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Disease Diagnosis ◽

Antigenic Structure ◽

Human Populations ◽

Genotype 3 ◽

Genotype 4 ◽

B Cell Epitopes

ABSTRACTHepatitis 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 that625DFCP628,458PSRPF462, and407EPTV410peptides 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.IMPORTANCEGenotype 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 studiedin vitroandin vivo. 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.

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Identification of an antigenic domain in the N-terminal region of avian hepatitis E virus (HEV) capsid protein that is not common to swine and human HEVs

Journal of General Virology ◽

10.1099/vir.0.069021-0 ◽

2014 ◽

Vol 95 (12) ◽

pp. 2710-2715 ◽

Cited By ~ 7

Author(s):

Lizhen Wang ◽

Yani Sun ◽

Taofeng Du ◽

Chengbao Wang ◽

Shuqi Xiao ◽

...

Keyword(s):

Amino Acid ◽

Capsid Protein ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Amino Acid Residues ◽

Antigenic Domains ◽

Avian Hepatitis E Virus ◽

Avian Hev ◽

Selection Of ◽

Antigenic Domain

The antigenic domains located in the C-terminal 268 amino acid residues of avian hepatitis E virus (HEV) capsid protein have been characterized. This region shares common epitopes with swine and human HEVs. However, epitopes in the N-terminal 338 amino acid residues have never been reported. In this study, an antigenic domain located between amino acids 23 and 85 was identified by indirect ELISA using the truncated recombinant capsid proteins as coating antigens and anti-avian HEV chicken sera as primary antibodies. In addition, this domain did not react with anti-swine and human HEV sera. These results indicated that the N-terminal 338 amino acid residues of avian HEV capsid protein do not share common epitopes with swine and human HEVs. This finding is important for our understanding of the antigenicity of the avian HEV capsid protein. Furthermore, it has important implications in the selection of viral antigens for serological diagnosis.

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

Journal of Virology ◽

10.1128/jvi.02205-18 ◽

2019 ◽

Vol 93 (11) ◽

Author(s):

Huixia Li ◽

Mengnan Fan ◽

Baoyuan Liu ◽

Pinpin Ji ◽

Yiyang Chen ◽

...

Keyword(s):

Viral Infection ◽

Capsid Protein ◽

Hepatitis E Virus ◽

Organic Anion ◽

Hepatitis E ◽

Host Cells ◽

Cell Culture System ◽

Organic Anion Transporting Polypeptide ◽

Avian Hepatitis E Virus ◽

Avian Hev

ABSTRACT Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease in chickens. Due to the absence of a highly effective cell culture system, there are few reports about the interaction between avian HEV and host cells. In this study, organic anion-transporting polypeptide 1A2 (OATP1A2) from chicken liver cells was identified to interact with ap237, a truncated avian HEV capsid protein spanning amino acids 313 to 549, by a glutathione S-transferase (GST) pulldown assay. GST pulldown and indirect enzyme-linked immunosorbent assays (ELISAs) further confirmed that the extracellular domain of OATP1A2 directly binds with ap237. The expression levels of OATP1A2 in host cells are positively correlated with the amounts of ap237 attachment and virus infection. The distribution of OATP1A2 in different tissues is consistent with avian HEV infection in vivo. Finally, when the functions of OATP1A2 in cells are inhibited by its substrates or an inhibitor or blocked by ap237 or anti-OATP1A2 sera, attachment to and infection of host cells by avian HEV are significantly reduced. Collectively, these results displayed for the first time that OATP1A2 interacts with the avian HEV capsid protein and can influence viral infection in host cells. The present study provides new insight to understand the process of avian HEV infection of host cells. IMPORTANCE The process of viral infection is centered around the interaction between the virus and host cells. Due to the lack of a highly effective cell culture system in vitro, there is little understanding about the interaction between avian HEV and its host cells. In this study, a total of seven host proteins were screened in chicken liver cells by a truncated avian HEV capsid protein (ap237) in which the host protein OATP1A2 interacted with ap237. Overexpression of OATP1A2 in the cells can promote ap237 adsorption as well as avian HEV adsorption and infection of the cells. When the function of OATP1A2 in cells was inhibited by substrates or inhibitors, attachment and infection by avian HEV significantly decreased. The distribution of OATP1A2 in different chicken tissues corresponded with that in tissues during avian HEV infection. This is the first finding that OATP1A2 is involved in viral infection of host cells.

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Mapping of linear B cell epitopes on open reading frames 2- and 3-encoded proteins of hepatitis E virus using synthetic peptides

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.1993.tb06176.x ◽

1993 ◽

Vol 109 (2-3) ◽

pp. 251-255 ◽

Cited By ~ 20

Author(s):

Pierre Coursaget ◽

Yves Buisson ◽

Nathalie Depril ◽

Pierre Cann ◽

Martine Chabaud ◽

...

Keyword(s):

B Cell ◽

Synthetic Peptides ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Open Reading Frames ◽

B Cell Epitopes ◽

Encoded Proteins ◽

Reading Frames ◽

Linear B

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Human linear B-cell epitopes encoded by the hepatitis E virus include determinants in the RNA-dependent RNA polymerase.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.89.9.3855 ◽

1992 ◽

Vol 89 (9) ◽

pp. 3855-3858 ◽

Cited By ~ 34

Author(s):

M. Kaur ◽

K. C. Hyams ◽

M. A. Purdy ◽

K. Krawczynski ◽

W. M. Ching ◽

...

Keyword(s):

Rna Polymerase ◽

B Cell ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Rna Dependent Rna Polymerase ◽

B Cell Epitopes ◽

Linear B

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The putative capsid protein of the newly identified avian hepatitis E virus shares antigenic epitopes with that of swine and human hepatitis E viruses and chicken big liver and spleen disease virus

Journal of General Virology ◽

10.1099/0022-1317-83-9-2201 ◽

2002 ◽

Vol 83 (9) ◽

pp. 2201-2209 ◽

Cited By ~ 73

Author(s):

G. Haqshenas ◽

F. F. Huang ◽

M. Fenaux ◽

D. K. Guenette ◽

F. W. Pierson ◽

...

Keyword(s):

Capsid Protein ◽

Disease Virus ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Cross Reactivity ◽

Putative Open Reading Frame ◽

Orf2 Protein ◽

Avian Hepatitis E Virus ◽

Avian Hev ◽

Antigenic Epitopes

We recently identified a novel virus, designated avian hepatitis E virus (avian HEV), from chickens with hepatitis–splenomegaly (HS) syndrome in the USA. We showed that avian HEV is genetically related to swine and human HEVs. Here we report the antigenic cross-reactivity of the putative open reading frame 2 (ORF2) capsid protein of avian HEV with those of swine and human HEVs and the Australian chicken big liver and spleen disease virus (BLSV). The region encoding the C-terminal 268 amino acid residues of avian HEV ORF2 was cloned into expression vector pRSET-C. The truncated ORF2 protein was expressed in E. coli as a fusion protein and purified by affinity chromatography. Western blot analysis revealed that the avian HEV ORF2 protein reacted with antisera against the Sar-55 strain of human HEV and with convalescent antisera against swine HEV and the US2 strain of human HEV, as well as with antiserum against BLSV. Convalescent sera from specific-pathogen-free chickens experimentally infected with avian HEV also reacted with the recombinant capsid proteins of swine HEV and Sar-55 human HEV. Antisera against the US2 human HEV also reacted with recombinant ORF2 proteins of both swine HEV and Sar-55 human HEV. The antigenic cross-reactivity of the avian HEV putative capsid protein with those of swine and human HEVs was further confirmed, for the most part, by ELISA assays. The data indicate that avian HEV shares certain antigenic epitopes in its putative capsid protein with swine and human HEVs, as well as with BLSV. The results have implications for HEV diagnosis and taxonomy.

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Protection of chickens against avian hepatitis E virus (avian HEV) infection by immunization with recombinant avian HEV capsid protein

Vaccine ◽

10.1016/j.vaccine.2006.09.038 ◽

2007 ◽

Vol 25 (15) ◽

pp. 2892-2899 ◽

Cited By ~ 21

Author(s):

H. Guo ◽

E.M. Zhou ◽

Z.F. Sun ◽

X.J. Meng

Keyword(s):

Capsid Protein ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Avian Hepatitis E Virus ◽

Avian Hev

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Linear B-Cell Epitopes in Human Norovirus GII.4 Capsid Protein Elicit Blockade Antibodies

Vaccines ◽

10.3390/vaccines9010052 ◽

2021 ◽

Vol 9 (1) ◽

pp. 52

Author(s):

Hassan Moeini ◽

Suliman Qadir Afridi ◽

Sainitin Donakonda ◽

Percy A. Knolle ◽

Ulrike Protzer ◽

...

Keyword(s):

B Cell ◽

Capsid Protein ◽

Immune Escape ◽

Solvent Accessibility ◽

Effective Vaccine ◽

Blood Group Antigens ◽

Human Norovirus ◽

B Cell Epitopes ◽

Blocking Rate ◽

Linear B

Human norovirus (HuNoV) is the leading cause of nonbacterial gastroenteritis worldwide with the GII.4 genotype accounting for over 80% of infections. The major capsid protein of GII.4 variants is evolving rapidly, resulting in new epidemic variants with altered antigenic potentials that must be considered for the development of an effective vaccine. In this study, we identify and characterize linear blockade B-cell epitopes in HuNoV GII.4. Five unique linear B-cell epitopes, namely P2A, P2B, P2C, P2D, and P2E, were predicted on the surface-exposed regions of the capsid protein. Evolving of the surface-exposed epitopes over time was found to correlate with the emergence of new GII.4 outbreak variants. Molecular dynamic simulation (MD) analysis and molecular docking revealed that amino acid substitutions in the putative epitopes P2B, P2C, and P2D could be associated with immune escape and the appearance of new GII.4 variants by affecting solvent accessibility and flexibility of the antigenic sites and histo-blood group antigens (HBAG) binding. Testing the synthetic peptides in wild-type mice, epitopes P2B (336–355), P2C (367–384), and P2D (390–400) were recognized as GII.4-specific linear blockade epitopes with the blocking rate of 68, 55 and 28%, respectively. Blocking rate was found to increase to 80% using the pooled serum of epitopes P2B and P2C. These data provide a strategy for expanding the broad blockade potential of vaccines for prevention of NoV infection.

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