Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV).
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ABSTRACT:
SUBMITTER: Pezzoni G
PROVIDER: S-EPMC8310276 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Little data are available on cellular immune responses during infection with hepatitis E virus (HEV). We therefore mapped CD4 T-cell epitopes in open reading frame (ORF)2 and ORF3 proteins of HEV using lymphocyte proliferation assays and overlapping peptide libraries. Proliferation of peripheral blood mononuclear cells from 40 patients with acute hepatitis E and 21 healthy controls with recombinant HEV ORF2 protein or pools of overlapping HEV ORF2/ORF3 peptides was measured. HLA-DQB1 and HLA-DRB1 alleles were also determined. Mononuclear cells from patients with hepatitis E more often showed significant proliferation on stimulation with recombinant ORF2 protein than controls (32/40 vs 7/21), and had higher median (range) stimulation indices [2.6 (0.9-15.2) vs 1.3 (0.6-12.9)]. Peptide pools corresponding to amino acids 73-156, 289-372, 361-444 and 505-588 of HEV ORF2 protein were associated with significant proliferation. Individual peptides in these pools did not show a clear pattern of stimulation. HEV ORF3 peptide pools did not induce proliferative responses. Lymphocyte proliferation in response to the peptide pool corresponding to amino acids 289-372 of HEV ORF2 protein was associated with presence of HLA-DRB1 allele 010X. These data on mapping of T-cell epitopes in HEV proteins may prove useful for designing HEV vaccines and for studying the immunopathogenesis of hepatitis E.
| S-EPMC2441432 | biostudies-literature
Project description:Hepatitis E virus (HEV), an enterically transmitted pathogen, is one of the major causes of acute hepatitis in humans worldwide, being responsible for outbreaks and epidemics in regions with suboptimal sanitary conditions, in many of which it is endemic. In industrialized countries, hepatitis E is rarely reported, but recent studies have revealed quite high human seroprevalence rates and the possibility of porcine zoonotic transmission. There is currently no specific therapy or licensed vaccine against HEV infection, and little is known about its intracellular growth cycle, as until very recently no efficient cell culture system has been available. In the present study, vaccinia viruses have been used to express recombinant HEV ORF2 proteins, allowing the study of their glycosylation patterns and subcellular localization. Furthermore, the expressed proteins have been shown to be good antigens for diagnostic purposes and to elicit high and long-lasting specific anti-HEV titers of antibodies in mice that are passively transferred to the offspring by both transplacental and lactation routes.
| S-EPMC3421641 | biostudies-literature
Project description: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.
| S-EPMC6532094 | biostudies-literature
Project description:The diagnosis of recent hepatitis E virus (HEV) infection depends on serologic testing for anti-HEV IgM; however, false-positive results may occur. In the present study, we cloned the ORF2 fragment of genotype 4 HEV and demonstrated that a subregion covering amino acids 459 to 607 in ORF2 forms the immunodominant B-cell epitopes, as it does in genotype 1 viruses. Truncation of several residues from either the N or C terminus of the polypeptide abolished the reactivity of anti-HEV from naturally infected persons. By the combination of high reactivity of the immunodominant polypeptide and poor reactivity of the truncated polypeptide, we established an indirect enzyme-linked immunosorbent assay (ELISA) to detect anti-HEV IgM. In this assay, all 37 sera that were HEV RNA positive reacted with the immunodominant polypeptide but not with the truncated one, and none of 159 sera from healthy persons reacted with either of the polypeptides. In retesting of 117 sera that originally tested positive for anti-HEV IgM, using a Genelabs kit, only 34 were positive and 83 were negative. Western blot analyses and other experiments strongly indicated that these 83 discordant sera were negative for anti-HEV IgM. Furthermore, among the 117 sera, 5 reacted with both the immunodominant and truncated polypeptides, with comparable optical densities at 450 nm. However, their reactivity was demonstrated to result from nonspecific binding. Together, the data indicate that the poor reactivity of a truncated ORF2 polypeptide can be used to exclude nonspecific binding in the detection of anti-HEV IgM.
| S-EPMC2832410 | biostudies-literature
Project description:BackgroundHepatitis E virus (HEV) causes epidemics in developing countries and is primarily transmitted through the fecal-oral route. There have been recent reports on the zoonotic spread of the virus, and several animal species, primarily pigs, have been recognized as reservoirs of HEV. Because of its possible spread, there is an urgent need of a method for the cost-effective production of HEV proteins that can be used as diagnostic antigens for the serological detection of anti-HEV antibodies.MethodsThe HEV open reading frame (ORF)2 protein was purified from plant tissue by using immobilized metal-anion chromatography (IMAC). The recombinant protein was used to develop an in-house ELISA for testing anti-HEV antibodies in both human and swine sera. Thirty-six serum samples collected from patients with serologically proven HEV infection with commercial kits were tested for anti-HEV IgG antibodies by using the plant-expressed protein. Forty-five serum samples collected from apparently healthy pigs in Bulgarian farms were also tested.ResultsWe confirmed the transient expression and purification of a truncated version of the HEV genotype 3 capsid protein in Nicotiana benthamiana and its usefulness as a diagnostic antigen. ELISA showed the presence of anti-HEV IgG antibodies in 29 of the 36 human samples. The in-house ELISA showed anti-HEV IgG antibodies in 34 of the 45 pigs.ConclusionsWe describe a method for the production of HEV ORF2 protein in N. benthamiana and the usefulness of this protein for the serological detection of anti-HEV antibodies in both humans and swine.
| S-EPMC5409023 | biostudies-literature
Project description:BACKGROUND:Hepatitis E virus (HEV) is a common cause of acute viral hepatitis (AVH) in many developing countries. In Egypt, HEV seroprevalence is among the highest in the world; however, only a very limited number of Egyptian HEV sequences are currently available. OBJECTIVES:The objectives were to determine the HEV genotype(s) currently circulating in Egypt. STUDY DESIGN:AVH patients without serologic evidence of hepatitis A, B, and C viruses were evaluated for possible HEV infection using serologic assays for anti-HEV IgM and anti-HEV IgG and real-time PCR for HEV RNA. Stool suspensions from suspected cases were inoculated into rhesus macaques to confirm the presence of HEV. Sequence analysis was utilized to determine HEV genotype. RESULTS:Of 287 subjects with AVH enrolled, 58 had serologic evidence of acute HEV infection. Stool samples for two of these patients were repeatedly positive for HEV RNA by real-time PCR. Macaques experimentally inoculated with these human stools also developed viremia. Sequence analysis of open reading frame (ORF) 1 demonstrated that these isolates belonged to HEV genotype 1 and were 3.9-9.5% divergent from other genotype 1 isolates. ORF2 was 5.3-8.7% divergent from previously reported Egyptian isolates. CONCLUSIONS:This study strongly suggests that genotype 1 HEV related to other North African isolates is circulating in acute symptomatic patients in Egypt. Further evaluation of genotypic variability is underway in this highly endemic cohort and is considered an important component of our increased understanding of HEV pathogenesis.
| S-EPMC2753377 | biostudies-literature
Project description:What happens in cells infected with HEV is largely unknown. We used a recently established genotype 3 HEV cell culture system and profiled the host responses by RNA-seq.
2019-10-31 | GSE88731 | GEO
Project description:Genotype 4 hepatitis E virus (HEV) is a zoonotic pathogen transmitted to humans through food and water. Previously, three genotype 4 swine HEV ORF2 peptides (407EPTV410, 410VKLYTS415, and 458PSRPF462) were identified as epitopes of virus-neutralizing monoclonal antibodies that partially blocked rabbit infection with swine HEV. Here, individual and tandem fused peptides were synthesized, conjugated to keyhole limpet hemocyanin (KLH), then evaluated for immunoprotection of rabbits against swine HEV infection. Forty New Zealand White rabbits were randomly assigned to eight groups; groups 1 thru 5 received three immunizations with EPTV-KLH, VKLYTS-KLH, PSRPF-KLH, EPTVKLYTS-KLH, or EPTVKLYTSPSRPF-KLH, respectively; group 6 received truncated swine HEV ORF2 protein (sp239), and group 7 received phosphate-buffered saline. After an intravenous swine HEV challenge, all group 7 rabbits exhibited viremia and fecal virus shedding by 2-4 weeks post challenge (wpc), seroconversion by 4-9 wpc, elevated alanine aminotransferase (ALT) at 2 wpc, and severe liver lymphocytic venous periphlebitis. Only 1-2 rabbits/group in groups 1-4 exhibited delayed viremia, fecal shedding, seroconversion, increased ALT levels, and slight liver lymphocytic venous periphlebitis; groups 5-6 showed no pathogenic effects. Collectively, these results demonstrate that immunization with a polypeptide containing three genotype 4 HEV ORF2 neutralizing epitopes completely protected rabbits against swine HEV infection.
| S-EPMC7348971 | biostudies-literature
Project description:Viral protein expression in Escherichia coli (E. coli) is a powerful tool for structural/functional studies as well as for vaccine and diagnostics development. However, numerous factors such as codon bias, mRNA secondary structure and nucleotides distribution, have been indentified to hamper this heterologous expression.In this study, we combined computational and biochemical methods to analyze the influence of these factors on the expression of different segments of hepatitis E virus (HEV) ORF 2 protein and hepatitis B virus surface antigen (HBsAg). Three out of five HEV antigens were expressed while all three HBsAg fragments were not. The computational analysis revealed a significant difference in nucleotide distribution between expressed and non-expressed genes; and all these non-expressing constructs shared similar stable 5'-end mRNA secondary structures that affected the accessibility of both Shine-Dalgarno (SD) sequence and start codon AUG. By modifying the 5'-end of HEV and HBV non-expressed genes, there was a significant increase in the total free energy of the mRNA secondary structures that permitted the exposure of the SD sequence and the start codon, which in turn, led to the successful expression of these genes in E. coli.This study demonstrates that the mRNA secondary structure near the start codon is the key limiting factor for an efficient expression of HEV ORF2 proteins in E. coli. It describes also a simple and effective strategy for the production of viral proteins of different lengths for immunogenicity/antigenicity comparative studies during vaccine and diagnostics development.
| S-EPMC5686824 | biostudies-literature
Project description:Hepatitis E virus (HEV) is responsible for hepatitis E, which represents a global public health problem. HEV genotypes 3 and 4 are reported to be zoonotic, and animals are monitored for HEV infection in the interests of public hygiene and food safety. The development of novel diagnostic methods and vaccines for HEV in humans is thus important topics of research. Opening reading frame (ORF) 2 of HEV includes both linear and conformational epitopes and is regarded as the primary candidate for vaccines and diagnostic tests. We investigated the precise location of the HEV epitopes in the ORF2 protein. We prepared four monoclonal antibodies (mAbs) against genotype 4 ORF2 protein and identified two linear epitopes, G438IVIPHD444 and Y457DNQH461, corresponding to two of these mAbs using phage display biopanning technology. Both these epitopes were speculated to be universal to genotypes 1, 2, 3, 4, and avian HEVs. We also used two 12-mer fragments of ORF2 protein including these two epitopes to develop a peptide-based enzyme-linked immunosorbent assay (ELISA) to detect HEV in serum. This assay demonstrated good specificity but low sensitivity compared with the commercial method, indicating that these two epitopes could serve as potential candidate targets for diagnosis. Overall, these results further our understanding of the epitope distribution of HEV ORF2, and provide important information for the development of peptide-based immunodiagnostic tests to detect HEV in serum.
| S-EPMC5619941 | biostudies-literature