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Hepatitis E virus (HEV) is a pathogen of global significance, but the value of HEV-related markers in the diagnosis of hepatitis E remains controversial. Previous studies on hepatitis E profiles have been mainly cross-sectional and conducted among inpatients in large hospitals, and hepatitis E cases have been primarily defined by limited partial markers. In this community-based study, 4,110 active hepatitis cases from a population of nearly 600,000 were followed over 48 months and serial serum samples were collected. Both HEV pathogen (HEV RNA and antigen) and anti-HEV antibody markers were used to determine HEV infection status and the relationship between hepatitis and HEV infection. In total, 98 hepatitis E patients were identified and all available isolates from 58 patients belonged to HEV genotype 4. The mean age of the patients was 58.14 years, with an overwhelming proportion of males (70.4%). Hepatitis E accounted for 22.86% of active hepatitis cases with alanine aminotransferase levels ≥15.0-fold the upper limit of normal, suggesting the need to include HEV in routine testing for these patients. Ninety-two hepatitis E patients were positive for at least 2 of HEV antigen, anti-HEV IgM, and HEV RNA markers at presentation, and 90.22% of them were positive for HEV antigen and anti-HEV IgM. HEV antigen, HEV RNA, and anti-HEV IgM positivity were observed in 89.80%, 82.65%, and 93.88% of hepatitis E patients at presentation, respectively. However, only 57.14% of anti-HEV IgM positivity occurred in hepatitis E patients. These findings will advance our understanding of hepatitis E and improve diagnosis.

In individuals with underlying chronic liver disease (CLD), hepatitis E virus (HEV) infection is a potential trigger of acute-on-chronic liver failure. In this systematic review, seven electronic databases were searched. Pooled incidence rates with 95% confidence intervals (95% CIs) were calculated by the Freeman-Tukey double arcsine transformation method. The association between death or liver failure and HEV superinfection in CLD patients was estimated by the odds ratios (OR) with a 95% CI. A total of 18 studies from 5 countries were eligible for systematic review. The prevalence of acute HEV infection in hospitalized CLD patients with clinical manifestations of hepatitis was 13.6%, which was significantly higher than that in CLD patients from the community (pooled prevalence 1.1%). The overall rates of liver failure and mortality in CLD patients with HEV superinfection were 35.8% (95% CI: 26.7%-45.6%) and 14.3% (95% CI: 10.6%-18.5%), respectively, with the rates in cirrhotic patients being approximately 2-fold and 4-fold higher than those in noncirrhotic patients, respectively. The risks of liver failure (OR = 5.5, 95% CI: 1.5-20.1) and mortality (OR = 5.0, 95% CI: 1.9-13.3) were significantly higher in CLD patients with HEV superinfection than in those without HEV superinfection. HEV testing in hospitalized CLD patients is necessary due to the high prevalence of HEV infection observed in hospitalized CLD patients. HEV superinfection could accelerate disease progression in patients with underlying CLD and increase mortality in these patients. HEV vaccination is appropriate for patients with pre-existing CLD., (© 2022 John Wiley & Sons Ltd.)

Background & Aims: Rat hepatitis E virus (Orthohepevirus species C; HEV-C1) is an emerging cause of viral hepatitis in humans. HEV-C1 is divergent from other HEV variants infecting humans that belong to Orthohepevirus species A (HEV-A). This study assessed HEV-C1 antigenic divergence from HEV-A and investigated the impact of this divergence on infection susceptibility, serological test sensitivity, and vaccine efficacy., Methods: Immunodominant E2s peptide sequences of HEV-A and HEV-C1 were aligned. Interactions of HEV-C1 E2s and anti-HEV-A monoclonal antibodies (mAbs) were modeled. Recombinant peptides incorporating E2s of HEV-A (HEV-A4 p239) and HEV-C1 (HEV-C1 p241) were expressed. HEV-A and HEV-C1 patient sera were tested using antibody enzymatic immunoassays (EIA), antigen EIAs, and HEV-A4 p239/HEV-C1 p241 immunoblots. Rats immunized with HEV-A1 p239 vaccine (Hecolin), HEV-A4 p239 or HEV-C1 p241 peptides were challenged with a HEV-C1 strain., Results: E2s sequence identity between HEV-A and HEV-C1 was only 48%. There was low conservation at E2s residues (23/53; 43.4%) involved in mAb binding. Anti-HEV-A mAbs bound HEV-C1 poorly in homology modeling and antigen EIAs. Divergence resulted in low sensitivity of commercial antigen (0%) and antibody EIAs (10-70%) for HEV-C1 diagnosis. Species-specific HEV-A4 p239/HEV-C1 p241 immunoblots accurately differentiated HEV-A and HEV-C1 serological profiles in immunized rats (18/18; 100%) and infected-patient sera (32/36; 88.9%). Immunization with Hecolin and HEV-A4 p239 was partially protective while HEV-C1 p241 was fully protective against HEV-C1 infection in rats., Conclusions: Antigenic divergence significantly decreases sensitivity of hepatitis E serodiagnostic assays for HEV-C1 infection. Species-specific immunoblots are useful for diagnosing HEV-C1 and for differentiating the serological profiles of HEV-A and HEV-C1. Prior HEV-A exposure is not protective against HEV-C1. HEV-C1 p241 is an immunogenic vaccine candidate against HEV-C1., Lay Summary: Rat hepatitis E virus (HEV-C1) is a new cause of hepatitis in humans. Using a combination of methods, we showed that HEV-C1 is highly divergent from the usual cause of human hepatitis (HEV-A). This divergence reduces the capacity of existing tests to diagnose HEV-C1 and also indicates that prior exposure to HEV-A (via infection or vaccination) is not protective against HEV-C1., Competing Interests: Conflict of interest Siddharth Sridhar has received speaker’s honoraria from Abbott Laboratories Limited. The other authors declare no conflicts of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Efficacy evaluation through human trials is crucial for advancing a vaccine candidate to clinics. Next-generation sequencing (NGS) can be used to quantify B cell repertoire response and trace antibody lineages during vaccination. Here, we demonstrate this application with a case study of Hecolin®, the licensed vaccine for hepatitis E virus (HEV). Four subjects are administered the vaccine following a standard three-dose schedule. Vaccine-induced antibodies exhibit a high degree of clonal diversity, recognize five conformational antigenic sites of the genotype 1 HEV p239 antigen, and cross-react with other genotypes. Unbiased repertoire sequencing is performed for seven time points over six months of vaccination, with maturation pathways characterize for a set of vaccine-induced antibodies. In addition to dynamic repertoire profiles, NGS analysis reveals differential patterns of HEV-specific antibody lineages and highlights the necessity of the long vaccine boost. Together, our study presents a quantitative strategy for vaccine evaluation in small-scale human studies.

Background: Nepal is an endemic area for hepatitis E virus (HEV) epidemics. The research on viral hepatitis in Nepal is limited., Methods: Serum samples from 170 patients presenting with symptoms of hepatitis were collected from April to May 2014 in Biratnagar, Nepal, and then transported to Xiamen, China, for further evaluation. All samples were tested for HEV RNA, HEV antigen, anti-HEV IgM, anti-HEV IgG and anti-HBc IgM, anti-HCV IgG, and anti-HAV IgM., Results: Sixteen patients were identified as acute hepatitis E with the presence of ≥ 2 HEV acute phase markers (antigen, RNA, and anti-HEV IgM). HEV infection was the major cause of potential active viral hepatitis (59.2%, 16 of 27), followed by HBV (25.9%, 7 of 27, anti-HBc IgM positive), HAV (18.5%, 5 of 27, anti-HAV IgM positive), and HCV (3.7%, 1 of 27, anti-HCV antibodies). All 16 confirmed HE cases were positive for HEV antigen, while 5 cases were HEV RNA positive, as well as 15 anti-HEV IgM positive. The low positive rate of RNA might be related to the collection and/or the transportation of these samples., Conclusions: This study showed that HEV is a major cause of acute hepatitis in developing countries and regions. Application of immunoassay diagnostic kits, especially the HEV antigen tests, showed great potential for HE detection in these countries and regions.

All hepatitis E virus (HEV) variants reported to infect humans belong to the species Orthohepevirus A (HEV-A). The zoonotic potential of the species Orthohepevirus C (HEV-C), which circulates in rats and is highly divergent from HEV-A, is unknown. We report a liver transplant recipient with hepatitis caused by HEV-C infection. We detected HEV-C RNA in multiple clinical samples and HEV-C antigen in the liver. The complete genome of the HEV-C isolate had 93.7% nt similarity to an HEV-C strain from Vietnam. The patient had preexisting HEV antibodies, which were not protective against HEV-C infection. Ribavirin was an effective treatment, resulting in resolution of hepatitis and clearance of HEV-C viremia. Testing for this zoonotic virus should be performed for immunocompromised and immunocompetent patients with unexplained hepatitis because routine hepatitis E diagnostic tests may miss HEV-C infection. HEV-C is also a potential threat to the blood product supply.

Hepatitis E virus (HEV) is emerging as a potential threat to the safety of blood transfusions. In many countries and regions endemic for HEV, such as China, blood donors are not routinely tested for HEV infection. In this study, 11747 eligible blood donors were screened for anti-HEV immunoglobulin M (IgM)/immunoglobulin G (IgG) and HEV RNA and antigen in China. Twenty-four donors who were positive for both HEV antigen and RNA were followed for ≥ 70 days, and none of these donors reported clinical hepatitis or illness. At least 1 follow-up sample was provided by 17 donors, including 10 with viremia and/or antigenemia for ≥ 70 days and 3 with antigen and RNA positivity for >90 days. Fourteen of the 17 donors did not present with an obvious serologic response during the follow-up period. These results differed from previous reports, in which viremia lasted for 68 days and elicited an antibody response. These donors showed atypical HEV infection progression that differed from that of hepatitis E patients. The presence of these donors presents a challenge for transfusion transmission screening.

Hepatitis E virus (HEV) is one of the major pathogens that cause acute viral hepatitis. The human (genotypes 1 and 2) and zoonotic (genotypes 3 and 4) groups of HEV present different epidemiology and clinical features. In this study, we developed a classification method for rapidly classifying HEV into human or zoonotic groups that combines a general antigen test with a zoonotic group-specific antigen test. Evaluation of serial samples from HEV-infected rhesus monkeys indicated that HEV antigen-positive samples can be classified using the antigen-based classification method. The antigen-based classification method was evaluated further on 55 genotyped samples from acute hepatitis E patients, including 9 human and 46 zoonotic groups. The novel method was completely consistent with the sequencing results: 9/9 for the human groups (100%, 95% confidence interval [CI] 66.4-100%) and 46/46 for the zoonotic groups (100%, 95% CI 92.3-100%). This method was also successfully used for the clustering of some samples that could not be clustered by sequencing. Compared with the sequencing-based method, this method is less time-consuming, less expensive, and less technically complex and is therefore ideal for large numbers of samples. In conclusion, this study provides a convenient and sensitive method for classifying different groups of HEV, and it has potentially important public health applications, especially in underdeveloped areas that cannot afford the high cost of nucleic acid testing.

The hepatitis E virus (HEV) is one of the main causes of enterically transmitted hepatitis worldwide. Although the mortality rates associated with HEV are generally low, they can be up to 28% in HEV-infected pregnant women, and the elderly are more susceptible. The reasons for this selective severity are unclear, partially because there is no suitable, easy-to-use model in which to study HEV infection. Non-human primates and standard swine have been identified as being sensitive to infection with HEV and have been used for HEV infection studies. However, studies in these animals have been limited by high housing costs and the difficulty of manipulating these animals. In the current study, we established a model of HEV infection using Bama miniature swine. The model is easy to use and is sensitive to infections with HEV genotypes 3 and 4, which are classified as zoonotic HEVs. In this model, infection of Bama miniature swine with HEV genotypes 3 and 4 caused the typical features. All Bama miniature swine that were infected with HEV genotypes 3 and 4 exhibited significant HEV viremia, shedding, anti-HEV antibody responses and partial liver inflammation. Bama miniature swine may serve as an alternative to standard swine models for the study of zoonotic HEV infection and HEV genotype specificity research.

Hepatitis E virus (HEV) is the aetiological agent of enterically transmitted hepatitis. The traditional methods for evaluating neutralizing antibody titres against HEV are real-time PCR and the immunofluorescence foci assay (IFA), which are poorly repeatable and operationally complicated, factors that limit their applicability to high-throughput assays. In this study, we developed a novel high-throughput neutralizing assay based on biotin-conjugated p239 (HEV recombinant capsid proteins, a.a. 368-606) and staining with allophycocyanin-conjugated streptavidin (streptavidin APC) to amplify the fluorescence signal. A linear regression analysis indicated that there was a high degree of correlation between IFA and the novel assay. Using this method, we quantitatively evaluated the neutralization of sera from HEV-infected and vaccinated macaques. The anti-HEV IgG level had good concordance with the neutralizing titres of macaque sera. However, the neutralization titres of the sera were also influenced by anti-HEV IgM responses. Further analysis also indicated that, although vaccination with HEV vaccine stimulated higher anti-HEV IgG and neutralization titres than infection with HEV in macaques, the proportions of neutralizing antibodies in the infected macaques' sera were higher than in the vaccinated macaques with the same anti-HEV IgG levels. Thus, the infection more efficiently stimulated neutralizing antibody responses.

The hepatitis E virus (HEV) ORF2 encodes a single structural capsid protein. The E2s domain (amino acids 459-606) of the capsid protein has been identified as the major immune target. All identified neutralizing epitopes are located on this domain; however, a comprehensive characterization of antigenic sites on the domain is lacking due to its high degree of conformation dependence. Here, we used the statistical software SPSS to analyze cELISA (competitive ELISA) data to classify monoclonal antibodies (mAbs), which recognized conformational epitopes on E2s domain. Using this novel analysis method, we identified various conformational mAbs that recognized the E2s domain. These mAbs were distributed into 6 independent groups, suggesting the presence of at least 6 epitopes. Twelve representative mAbs covering the six groups were selected as a tool box to further map functional antigenic sites on the E2s domain. By combining functional and location information of the 12 representative mAbs, this study provided a complete picture of potential neutralizing epitope regions and immune-dominant determinants on E2s domain. One epitope region is located on top of the E2s domain close to the monomer interface; the other is located on the monomer side of the E2s dimer around the groove zone. Besides, two non-neutralizing epitopes were also identified on E2s domain that did not stimulate neutralizing antibodies. Our results help further the understanding of protective mechanisms induced by the HEV vaccine. Furthermore, the tool box with 12 representative mAbs will be useful for studying the HEV infection process., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Hepatitis E virus (HEV) is a serious public health problem that causes acute hepatitis in humans and is primarily transmitted through fecal and oral routes. The major anti-HEV antibody responses are against conformational epitopes located in a.a. 459-606 of HEV pORF2. All reported neutralization epitopes are present on the dimer domain constructed by this peptide. While looking for a neutralizing monoclonal antibody (MAb)-recognized linear epitope, we found a novel neutralizing linear epitope (L2) located in a.a. 423-437 of pORF2. Moreover, epitope L2 is proved non-immunodominant in the HEV-infection process. Using the hepatitis B virus core protein (HBc) as a carrier to display this novel linear epitope, we show herein that this epitope could induce a neutralizing antibody response against HEV in mice and could protect rhesus monkeys from HEV infection. Collectively, our results showed a novel non-immunodominant linear neutralizing epitope of hepatitis E virus, which provided additional insight of HEV vaccine., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Hepatitis E virus (HEV) infection has become relevant to blood transfusion practice because isolated cases of blood transmission have been reported and because HEV has been found to cause chronic infection and severe liver disease in immunocompromised patients., Study Design and Methods: We tested for immunoglobulin (Ig)G and IgM antibodies to the HEV and for HEV RNA in 1939 unselected volunteer US blood donors. Subsequently, we tested the same variables in pre- and serial posttransfusion samples from 362 prospectively followed blood recipients to assess transfusion risk., Results: IgG anti-HEV seroprevalence in the total 1939 donations was 18.8%: 916 of these donations were made in 2006 at which time the seroprevalence was 21.8% and the remaining 1023 donations were in 2012 when the seroprevalence had decreased to 16.0% (p < 0.01). A significant (p < 0.001) stepwise increase in anti-HEV seroprevalence was seen with increasing age. Eight of 1939 donations (0.4%) tested anti-HEV IgM positive; no donation was HEV RNA positive. Two recipients had an apparent anti-HEV seroconversion, but temporal relationships and linked donor testing showed that these were not transfusion-transmitted HEV infections., Conclusion: No transfusion-transmitted HEV infections were observed in 362 prospectively followed blood recipients despite an anti-HEV seroprevalence among donations exceeding 16%., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)

Objectives: Hepatitis E virus (HEV) is an emerging pathogen. We evaluated the seroprevalence of HEV infection among swine farmers and the general population in Taiwan, a nonendemic country., Methods: We conducted a cross-sectional seroepidemiologic study in rural Taiwan in 2012 and 2013. The study included swine farmers, health examination attendees, pregnant women, and students. A commercial enzyme-linked immunosorbent assay was used to detect immunoglobulin G (IgG) and IgM against HEV. Pertinent information was collected using a questionnaire., Results: In total, 660 participants were enrolled in the study, including 156 swine farmers, 314 health examination attendees, 100 pregnant women, and 90 students. IgG anti-HEV was detected in 29.5% of swine farmers, 11.5% of health examination attendees, 2% of pregnant women, and 1.1% of students. Two subjects were positive for IgM anti-HEV. Logistic regression analysis revealed that swine farmers had an approximately 3.5-fold increased risk (odds ratio [OR], 3.46; 95% confidence interval [CI], 1.91-6.27; p<0.0001) for being seropositive for IgG anti-HEV as compared to the general population. Age was positively associated with seropositive rate (OR, 1.07 per year; 95% CI, 1.05-1.09; p<0.0001)., Conclusion: HEV infection is prevalent in Taiwan. The seroprevalence of HEV infection is high in swine farmers and in the elderly population.

There is emerging evidence for an under-recognized hepatitis E virus (HEV) as a human pathogen. Among different reasons for this neglect are the unsatisfactory performance and under-utilization of commercial HEV diagnostic kits; for instance, the number of anti-HEV IgM kits marketed in China is about one-fifth of that of hepatitis A kits. Over the last two decades, substantial progress has been achieved in furthering our knowledge on the HEV-specific immune responses, antigenic features of HEV virions, and development of serological assays and more recently prophylactic vaccines. This review will focus on presenting the evidence of the importance of HEV infection for certain cohorts such as pregnant women, the key antigenic determinants of the virus, and immunogenicity and clinical efficacy conferred by a newly developed prophylactic vaccine. Robust immunogenicity, greater than 195-fold and approximately 50-fold increase of anti-HEV IgG level in seronegative and seropositive vaccinees, respectively, as well as impressive clinical efficacy of this vaccine was demonstrated. The protection rate against the hepatitis E disease and the virus infection was shown to be 100% (95% CI 75-100) and 78% (95% CI 66-86), respectively.

Along with the improvement of diagnostic techniques, hepatitis E has attracted increasing awareness in recent years. Hepatitis E virus infection leads to high mortality in pregnant women and patients with underlying liver disease. Several hepatitis E vaccine candidates have been designed and have proved their efficacy in animal models; two candidates have successfully undergone clinical trials. Having proved safe and effective in a large phase III trials, an Escherichia coli expressed particulate protein, HEV 239, has been registered in China and is now available for use in China., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

There have been increased attentions on HEV and its associated diseases in recent years as a result of an increased number of reports on autochthonous patients from many developed countries. Vaccine development and better disease management are expected from protective immunity with increased knowledge on the pathogenesis and virology of HEV. This review summarizes the current understanding of the HEV virology, the key neutralization sites (epitopes) on the surface of the viral capsid, the host humoral immune responses for HEV infection, and the protective immunity conferred by natural infection and vaccination. Recombinant VLPs were prepared to mimic the protective and neutralizing epitopes on the virion surface, thus being capable of eliciting protective immunity when injected to nonhuman primates or human volunteers during preclinical tests and clinical trials. Four markers-viral RNA, anti-HEV IgM, anti-HEV IgG, and low avidity of anti-HEV IgG-are important in the diagnosis of HEV infection, particularly for patients presenting with acute hepatitis symptoms. This toolbox of genomic and immunological assays is valuable in furthering our understanding of the time course of HEV infection and the subsequent hepatitis during preclinical and clinical development of an efficacious vaccine. Two vaccine candidates had shown good tolerability, high immunogenicity, and high efficacy against symptomatic and/or asymptomatic HEV infection. One of them has been licensed in China recently. However, many issues need to be resolved before new technological progresses can benefit the people who need them most., (Copyright © 2012 John Wiley & Sons, Ltd.)

Hepatitis E virus (HEV) was discovered during the Soviet occupation of Afghanistan in the 1980s, after an outbreak of unexplained hepatitis at a military camp. A pooled faecal extract from affected soldiers was ingested by a member of the research team. He became sick, and the new virus (named HEV), was detected in his stool by electron microscopy. Subsequently, endemic HEV has been identified in many resource-poor countries. Globally, HEV is the most common cause of acute viral hepatitis. The virus was not initially thought to occur in developed countries, but recent reports have shown this notion to be mistaken. The aim of this Seminar is to describe recent discoveries regarding HEV, and how they have changed our understanding of its effect on human health worldwide., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The first prophylactic vaccine, Hecolin®, against hepatitis E virus (HEV) infection and the HEV associated disease was approved by China's State Food and Drug Administration (SFDA) in December 2011. Key milestones during the 14-year HEV vaccine development are summarized in this commentary.  After years of innovative research the recombinant virus-like particle (VLP) based antigen with virion-like epitopes was successfully produced in E. coli production platform on a commercial scale. Safety and efficacy of this vaccine was demonstrated in a large scale phase III clinical trial.

To investigate the expression and localization of various functional domains of ORF1 polyprotein and ORF3 protein of hepatitis E virus in host cells, the coding sequences of the various functional domains (RdRp, HEL, MET, PLP, X) of ORF1 were separately cloned into pcDNA3. 1-GFP vectors for constructing the recombinant plasmids which were verified by enzyme digestion and sequencing. The exact expression of the fusion proteins were detected by Western Blot, and the distribution and localization were observed by the laser scanning confocal microscope(LSCM). In huh7 cells, GFP-RdRp proteins were found mainly in the nuclei, GFP-HEL proteins were distributed vesicularly around the nucleus, GFP-MET proteins were distributed granularly both in the nuclei and the cytoplasm, GFP-PLP proteins had polar distribution around the nucleus, and unknown GFP-X proteins were distributed uniformly both in the nuclei and the cytoplasm. Different localization of these proteins verified the previous data obtained from in vitro studies, providing a support for further research on the biological functions of various proteins coded by HEV genome.

Background: Seroprevalence data suggest that a third of the world's population has been infected with the hepatitis E virus. Our aim was to assess efficacy and safety of a recombinant hepatitis E vaccine, HEV 239 (Hecolin; Xiamen Innovax Biotech, Xiamen, China) in a randomised, double-blind, placebo-controlled, phase 3 trial., Methods: Healthy adults aged 16-65 years in, Jiangsu Province, China were randomly assigned in a 1:1 ratio to receive three doses of HEV 239 (30 microg of purified recombinant hepatitis E antigen adsorbed to 0.8 mg aluminium hydroxide suspended in 0.5 mL buffered saline) or placebo (hepatitis B vaccine) given intramuscularly at 0, 1, and 6 months. Randomisation was done by computer-generated permuted blocks and stratified by age and sex. Participants were followed up for 19 months. The primary endpoint was prevention of hepatitis E during 12 months from the 31st day after the third dose. Analysis was based on participants who received all three doses per protocol. Study participants, care givers, and investigators were all masked to group and vaccine assignments. This trial is registered with ClinicalTrials.gov, number NCT01014845., Findings: 11,165 of the trial participants were tested for hepatitis E virus IgG, of which 5285 (47%) were seropositive for hepatitis E virus. Participants were randomly assigned to vaccine (n=56,302) or placebo (n=56,302). 48,693 (86%) participants in the vaccine group and 48,663 participants (86%) in the placebo group received three vaccine doses and were included in the primary efficacy analysis. During the 12 months after 30 days from receipt of the third dose 15 per-protocol participants in the placebo group developed hepatitis E compared with none in the vaccine group. Vaccine efficacy after three doses was 100.0% (95% CI 72.1-100.0). Adverse effects attributable to the vaccine were few and mild. No vaccination-related serious adverse event was noted., Interpretation: HEV 239 is well tolerated and effective in the prevention of hepatitis E in the general population in China, including both men and women age 16-65 years., Funding: Chinese National High-tech R&D Programme (863 programme), Chinese National Key Technologies R&D Programme, Chinese National Science Fund for Distinguished Young Scholars, Fujian Provincial Department of Sciences and Technology, Xiamen Science and Technology Bureau, and Fujian Provincial Science Fund for Distinguished Young Scholars., (Copyright 2010 Elsevier Ltd. All rights reserved.)

p239 is a virus-like particle constituted from hepatitis E virus (HEV) recombinant proteins. It can be used as a surrogate for HEV and as an investigative tool to study cellular interactions because of its ability to adsorb to and penetrate HepG2 cellular membranes. Our objective was to use p239 to define the role of HEV capsid proteins during the early stages of infection. Pull-down and MALDI-TOF MS experiments identified three host-cell proteins, Grp 78/Bip, alpha-tubulin and heat-shock protein 90 (HSP90), and the latter was investigated further. Antibodies to p239 alone or HSP90 alone could detect p239 or HSP90, suggesting the formation of a complex between p239 and HSP90. In the HepG2 cell, geldanamycin (GA), an HSP90-specific inhibitor, blocked intracellular transportation of p239, but had no effect on the binding and cellular entry of p239, suggesting that HSP90 was important for HEV capsid intracellular transportation. RT-PCR results showed that the efficiency of wild-type HEV infection was inhibited significantly by GA treatment, suggesting the importance of HSP90 in virus infectivity. It was concluded that HSP90 plays a crucial role in the intracellular transportation of viral capsids in the early stage of HEV infection.

A point prevalence study of hepatitis E virus (HEV) in Chinese blood donors was conducted, and the prevalences of antibodies against HEV immunoglobulin G (IgG) and IgM among Chinese blood donors were 32.60% and 0.94%, respectively. HEV viremia was 0.07%.

The candidate recombinant hepatitis E vaccine, HEV 239, protect monkeys against infection by hepatitis E virus (HEV). The safety and immunogenicity of the vaccine for humans was assessed in a randomized controlled phase II clinical trial. The study was conducted in an endemic area of southern China and consisted of a dose scheduling, involving 457 adults and a dose escalation component involving 155 high school students. The results showed that the vaccine is safe and immunogenic for humans and suggest that it could prevent new HEV infection.

E2 is a recombinant hepatitis E virus capsid protein including its main antigenic determinants but lacking of the particle assembling domain. P239 was the C-terminal extending protein of E2 and could self-assemble to form virus like particles, which might serve as mimicry of virions both structurally and antigenically. We previously used yeast two-hybrid system to screen proteins interacting with E2 based on a human hepatocyte cDNA library. One candidate was identified as the segment (aa388-437) of cytochrome P450 2A6 protein, which is predominantly expressed in liver and important for metabolization. Some studies have demonstrated that hepatitis virus infection may altered cell metabolic clearance of coumrarin which were rapidly matebolised by CYP2A6. In this research, we demonstrated that the protein interaction between HEV capsid proteins and CYP2A6 by pull-down and co-immunoprecipitation. It was also found that their interaction could decrease the CYP2A6 catalytic activity when p239 was incubated within the CYP2A6-transfected Huh7 cells. These results suggested that CYP2A6 might be related to the pathological process when HEV invaded host cells.

Western blot, capture-PCR, blocking ELISA and synthetic polypeptides were used to systematically study the recognition epitopes on HEV ORF2 of 23 anti-HEV monoclonal antibodies(McAbs) which were previously generated in our laboratory directed against HEV ORF2. Results showed that seven McAbs recognized linear epitopes that located at aa408-458 of HEV ORF2 and 16 conformation-dependent McAbs, most of which recognized the surface epitopes of native HEV, located at aa459-606 of HEV ORF2. The systematical study of the recognition epitopes of anti-HEV McAbs on HEV ORF2 provides important information for the investigation of virus receptor and HEV infection mechanism, as well as its vaccine and diagnostics development.

Objective: To isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography (IMAC)., Methods: Phage antibody library was allowed to mix with hex-His tagged expressed HEV specific antigen, NE2, in solution for adequate binding before affinity resin for hex-His was added. The non-specific phage antibodies were removed by extensive washing and the specific bound phage antibodies could then be eluted to infect TG1 or repeat the binding process for subsequent rounds of purification. The specificity of the selected human antibodies were tested by antigen competitive ELISA, human sera blocking ELISA, scFv expression, and sequence analysis., Results: His-NE2 specific recombinant phages were successfully enriched after panning procedure. Two individual phage clones, 126 and 138, showed 50% inhibition in NE2 antigen competition ELISA and obvious blocking effect by HEV positive serum in blocking ELISA. Soluble scFv of 126, 138 bound to NE2 specifically., Conclusion: Two specific human phage antibodies against hepatitis E virus (HEV) from phage display library were isolated by immobilized metal affinity chromatography. The immobilized metal affinity chromatography applied to phage antibody selection was a helpful supplement to the selection in solution.

HEV is classified into H (human) group and Z (zoonosis) group according to its compatible host. H group contains genotype 1 and genotype 2 HEV isolates which infect human only; Z group contains genotype 3 and genotype 4 HEV isolates which infect both human and animals. After analysis of amino acid sequences between ORF2 aa368 and aa606, four group-conserved sites that were all located in the neutralization region of ORF2 were identified. They are aa483, aa492, aa497 and aa599. Mutation analysis and capture PCR were then performed on these sites with a group of monoclonal antibodies. Results showed that the difference of the aa497 between H and Z groups was responsible for the maintenance of their group-specific immunodominant epitopes, probably through confirmation-dependent epitope changes. Thus, aa497 and its related change on the surface structure of HEV may play important roles in host selection by H and Z groups of HEV.

By using Western blot and immunofluorescence assays, the recombinant HEV capsid protein p239 was found specifically attached to the HepG2 cell surface and entered to the cytoplasm with the increase of incubation temperature. Pre-mixture of wild-type HEV with p239 blocked the infectivity of the virus on primary cultured human hepatocytes and HepG2 cells, indicating that p239 and HEV competed the same targeting site on these cells. These data provide evidence that p239 has a similar cell surface structure with wild-type HEV.

The candidate particulate hepatitis E vaccine, HEV 239, has been shown to be an efficacious vaccine in primates, and clinical study to date shows it to be safe and immunogenic for humans. The antigenicity of HEV 239 is virtually identical to its N-terminal 26 amino acids truncated protein, E2, which is not particulate but soluble. However, HEV 239 is over 200 times more immunogenic than E2. In present study, several events underlying this dramatic immunogenicity difference have been addressed. (1) HEV 239 can efficiently evoke a vigorous and predominant T cell response while E2 cannot induce detectable T cell response; (2) the dominant T cell epitopes in HEV 239 are identified, and both are also contained integrally in E2; (3) priming mice with Th epitope peptide can partially rescue the weak immunogenicity of E2 in alum adjuvant and (4) HEV 239 but not E2 can induce significant antibody response in athymic mice, which indicates that HEV 239 can directly activate B cell more efficiently. These results contribute to a better understanding of the mechanisms involved in the significant high immunogenicity of particulate antigen and may provide knowledge for the rational design and development of future vaccines.

A dominant H-2d restricted Th epitope P34 was found to be contained in recombinant particulate hepatitis E virus (HEV) vaccine HEV 239. In this paper, the cellular immune response induced in P34 immunized BALB/c mice were studied and the priming effect of P34 was characterized. Groups of BALB/c mice were subcutaneously (s. c.) immunized with P34, splenocytes were then stimulated with P34 and HEV 239 protein, cellular immune response was assayed by IFN-gamma-ELISPOT, flow cytometry and T cell proliferation experiments. Results showed that P34 immunized BALB/c splenocytes responsed to P34 and HEV 239 protein stimulation in IFN-gamma-ELISPOT, flow cytometry and T cell proliferation experiments. After depletion of the CD4+ T cells from the immunized splenocytes by magnetic separation, the response decreased to the background level while almost no influence was observed after CD8 + T cells depletion which showed that the cells responsible for IFN-gamma secretion were mainly CD4+ T cells. Then mice were primed with P34 and boosted with its vector protein, E2, the E2 specific antibody titer were assayed. Results showed that after P34 priming, some of the 10 microg, 20 microg E2 boosted mice could develop anti-E2 antibody 1 week later and all the mice had detectable antibody 3 weeks after boosting. In the control peptide P18 priming group, even after boosting with 20 microg E2, anti-E2 antibody couldn't be detected until the end of this experiment. The results showed that priming with P34 epitope could increase the immunogenicity of its vector protein, E2, in BALB/c mice.

The dimeric form of the recombinant peptide (E2), comprising amino acid 394-606 of the capsid protein of hepatitis E virus (HEV), is strongly recognized by HEV reactive human serum, and when used as a vaccine, it protects rhesus monkeys against experimental HEV infection. In this work, the relationship of E2 to HEV has been probed using three murine monoclonal antibodies, 8C11, 13D8 and 8H3, all of which react predominantly against the E2 dimer, and can effect immune capture of the virus as well. 8C11 and 8H3 were further found to neutralize HEV infectivity in animals. Cross-blocking patterns between these antibodies discerned two spatially separate antigenic domains, one identified by 8C11 and 13D8, and the other, by 8H3. Kinetic studies using BIAcore biosensor suggest that the epitope to which 8H3 is directed is partially masked, and thus has limited access by the native antibody. However, this is not the case with the smaller Fab. Access to the 8H3 epitope was enhanced by the binding of 8C11, and inhibited by the binding of 13D8 to a distal site on the peptide. Similar to the effects of binding 8H3 to E2, 8C11 was found to enhance immune capture by 8H3, while 13D8 was inhibitory. Moreover, 8C11 and 8H3 act synergistically to neutralize HEV infectivity. The parallel cross-reaction patterns that these antibodies exhibit against the peptide and the virus, respectively, implicate two interacting conformationally dependent neutralization sites on the HEV particle. These sites might cooperate in the adsorption and penetration of the HEV virus.

The hepatitis E virus (HEV) capsid consists of a single structural protein, a portion of which is engaged in isosahedral contact to form a basal shell, and another portion in dimeric contact to form the homodimers protruding from the shell. Previous studies revealed that homodimers of the truncated HEV capsid proteins, E2 (amino acids 394-606) and p239 (amino acids 368-606), model dominant antigenic determinants of HEV. Immunization with these proteins protected rhesus monkeys against the virus, and three monoclonal antibodies against the homodimers could neutralize HEV infectivity and/or immune-capture of the virus. Furthermore, homodimers of p239 further interact to form particles of 23 nm diameter, rendering it an efficacious candidate vaccine. In light of this we postulate that the interactions involved in the formation of the homodimers and particles might be similar to those involved in assembly of the virus capsid. Presently, mutational analysis was carried out to identify these sites of interactions. The site of dimeric interactions was located to a cluster of six hydrophobic amino acids residues, Ala597, Val598, Ala599, Leu601, and Ala602; furthermore, the site involved in particle formation was located at amino acids 368-394. The possibility that these sites are also involved in assembly of the virus capsid is supported by the fact that they are located at two major and highly conserved hydrophobic regions of the HEV structural protein.

Background: Understanding the genotype and clinical features of the hepatitis E virus (HEV) are important for understanding its characteristics, for evaluating region-specific diagnostic assays, and producing vaccines., Objectives: To investigate the epidemiology and the genotypes of HEV among outpatients and inpatients in the Department of Infectious Diseases of Tongji Hospital in Wuhan, China., Methods: Clinical data were elicited from the hospital records of patients who were clinically diagnosed with acute hepatitis between January 2000 and August 2004 (4920 patients). Of these cases, 120 patients with anti-HEV-IgM, IgG-positive were selected to analysis. Conserved genomic sequences of open reading frame 2 (345 bp) in the HEV gene were detected using polymerase chain reaction, 25 of which were cloned and sequenced. Clustal X and Mega software were used for phylogenetic analysis of genotypes strains., Results: The HEV infection rate is gradually increasing in Wuhan. The number of male patients was 3.3-fold greater than the number of female patients found in clinical investigations. People aged 30-59 years are more susceptible to infection, and people are more susceptible in March-June. Twenty-five isolates shared the same genotype, genotype IV, with 82.61-98.55% nucleotide identity. This genotype had 76.52-81.74%, 70.43-73.04%, 76.52-81.16%, and 84.35-88.70% homology with the nucleotide sequence of HEV genotypes I-IV, respectively. Phylogenetic analysis suggested that these 25 isolates represented at least three different subtypes, but there were no significant differences found in the epidemiological features or liver function of patients with the three subtypes., Conclusions: HEV sequences isolated from patients in Wuhan belong to different subtypes of HEV genotype IV.

Aim: To weaken the immunogenicity of the neutralizing monoclonal antibody (mAb) 13D8 against hepatitis E virus and express its scFv., Methods: The V(L) and V(H) genes were cloned by RT-PCR from hybridoma cells producing mouse mAb. And then V(H)-linker-V(L) fragment (scFv) was constructed and cloned into vector pTO-T7. The scFv protein was expressed in E.coli. The activity of expressed scFv was detected by ELISA and Western blot., Results: SDS-PAGE analysis showed that the scFv was highly expressed mostly in the form of inclusion body in E.coli, and the yield was up to 26.8% of the total bacteria protein. The results of indirect ELISA and Western blot showed that the expressed scFv could bind specifically to a recombinant protein in OFR2 region of HEV (NE2). The result of competitive ELISA demonstrated that the epitope recognized by the scFv was the same as that by mAb 13D8., Conclusion: The scFv constructed from V(H) and V(L) genes of mAb 13D8 with immunological activity was successfully expressed.

Aim: To select the peptide mimicking the neutralization epitope of hepatitis E virus which bound to non-type-specific and conformational monoclonal antibodies (mAbs) 8C11 and 8H3 fromed 7-peptide phage display library, and expressed the peptide recombinant with HBcAg in E.coli, and to observe whether the recombinant HBcAg could still form virus like particle (VLP) and to test the activation of the recombinant polyprotein and chemo-synthesized peptide that was selected by mAb 8H3., Methods: 8C11 and 8H3 were used to screen for binding peptides through a 7-peptide phage display library. After 4 rounds of panning, monoclonal phages were selected and sequenced. The obtained dominant peptide coding sequences was then synthesized and inserted into amino acid 78 to 83 of hepatitis B core antigen (HBcAg), and then expressed in E.coli. Activity of the recombinant proteins was detected by Western blotting, VLPs of the recombinant polyproteins were tested by transmission electron microscopy and binding activity of the chemo-synthesized peptide was confirmed by BIAcore biosensor., Results: Twenty-one positive monoclonal phages (10 for 8C11, and 11 for 8H3) were selected and the inserted fragments were sequenced. The DNA sequence coding for the obtained dominant peptides 8C11 (N'-His-Pro-Thr-Leu-Leu-Arg-Ile-C', named 8C11A) and 8H3 (N'-Ser-Ile-Leu-Pro- Tyr-Pro-Tyr-C', named 8H3A) were then synthesized and cloned to the HBcAg vector, then expressed in E.coli. The recombinant proteins aggregated into homodimer or polymer on SDS-PAGE, and could bind to mAb 8C11 and 8H3 in Western blotting. At the same time, the recombinant polyprotein could form virus like particles (VLPs), which could be visualized on electron micrograph. The dominant peptide 8H3A selected by mAb 8H3 was further chemo-synthesized, and its binding to mAb 8H3 could be detected by BIAcore biosensor., Conclusion: These results implicate that conformational neutralizing epitope can be partially modeled by a short peptide, which provides a feasible route for subunit vaccine development.

Production of Hepatitis E Virus capsid protein by high cell density culture in recombinant E. coli has been studied in 10L and 30L fermentors. The effects of different factors on growth and producing recombinant protein of E. coli have been studied by batch culture, such as different media, the ratio of phosphate and Magnesium sulfate. Comparison of fermentation performance for recombinant E. coli in different fed-methods culture has been investigated by fed-batch culture. The effects of inducing at different stages of growth and time of inducing on growth and producing recombinant protein, also obtained by fed-batch culture. At last, the solubility of inclusion body in different urea concentrations also has been obtained by fed-batch culture. The results show that the concentration of phosphate and Magnesium sulfate in the optimal media is 80mmol/L and 20mmol/L in batch culture respectively, that induction with 1.0mmol/L IPTG at mid log phase (about 45 OD at 600nm) is suitable for growth and recombinant protein expression, the cells were approaching stationary growth phase and the maximum cell OD at 600nm of 80 was achieved in 5h of fed-batch culture, and the expression level is 29.74%. The results also indicate that the solubility of inclusion body in 4mol/L urea solution induced at 37 degrees C reaches 14mg/mL, over 80% inclusion body was resolved. The culture process achieved in 10L fermentor could be successfully scaled up to 30L fenmentor with good reproducibility.

An E. coli expressed recombinant antigen NE2 was reported to aggregate into homo-oligomer, and can induce protective antibodies on rhesus monkey, but its immunogenicty was much weak after being purified. In this study, three N-terminal extension mutant of NE2 were expressed in E. coli, one of which named HEV 239 was found to aggregate into particle. HEV 239 antigen had good reactivity with sera of hepatitis E patients. The reactivity of HEV 239 against neutralization monoclonal antibody 8C11 was similar as NE2 antigen, while the reactivity of it against another neutralization monoclonal antibody 8H3 is much better than NE2 antigen, which indicated better antigenicity of HEV 239 than NE2. The diameter of purified HEV 239 particulate antigen was between 15 nm to 30 nm. The ED50 of immunization of HEV 239 particle adsorbed by aluminum adjuvant to BALB/c mice was between 0.08 microg to 0.25 microg. In contrast, the seraconversion rate of mice immunized by NE2 antigen adsorbed by aluminium adjuvant was only 25% on 60 microg vaccination. These results suggested that HEV 239 antigen particle has better immunogenicity as well as antigenicity than those of NE2 antigen, so it is a better vaccine candidate against HEV.

Objective: To evaluate the serological markers and biological marker in the diagnosis of hepatitis E infection in a rhesus monkey model., Methods: 86 rhesus monkeys had been infected with different doses of HEV. Hence, they were taken sequential blood samples at intervals up to 86 weeks for 4 hepatitis E virus (HEV) specific antibody assays (E2-IgM, E2-IgG, GL-IgG, and YES-IgG), and nucleic acid assay., Results: All the animals produced E2-IgG and all but one also produced E2-IgM and excreted the virus in stool, whereas positive rate of GL-IgG and YES IgG were low and correlated with virus level. Hepatitis occurred over a period of 4 weeks (between 3 an 7 weeks) after infection. Virological marker occurred mainly during incubation period and declined rapidly after onset of hepatitis. Seroconversion of E2-IgM occurred before onset of hepatitis in 70% monkeys and declined rapidly up to 50% of peak value after 4 weeks. E2-IgM seroconversion was closely paralleled by E2-IgG; however, E2-IgG persisted in all animals for the entire duration of experiment of up to 86 weeks. Production of GL-IgG and YES-IgG was delayed by one week after the E2 antibodies, these antibodies showed a transient occurrence and seroprevalence declined to 50% of the peak value over a period of 12 weeks., Conclusion: E2-IgM might be used as a suitable acute hepatitis E marker, and E2-IgG as a suitable epidemiological marker. The seroconversion or titer elevation of GL-IgG and YES-IgG antibodies probably used to confirm the infection. The viral markers are optional for early diagnosis.

Hepatitis E is a main cause of acute viral hepatitis in developing countries where it occurs as sporadic cases and in epidemics form. The causative agent, hepatitis E virus, is transmitted primarily by the fecal-oral route. The approximately 7.5 kb positive-sense single-strand RNA genome includes three open reading frames (ORFs), one of which (ORF2) is postulated to encode the major viral capsid protein (pORF2) of 660 amino acid residues. We earlier showed that a bacterially expressed peptide, designated as NE2, located from amino acid residues 394 to 606 of ORF2, was found to aggregate into homodimer to at least hexamer. To understand the interface domains within this peptide vital for dimerization and formation of major neutralizing epitopes, NE2 protein underwent terminal-truncated and site-directed mutation. The hydrophobic region, ORF2 aa597-aa602 (AVAVLA), played a key role in oligomerization. Any amino acid residue of this region replaced with glutamic acid residue, the peptide can not refold as homodimer and/or oligomer. The immunoreactivities of these mutant peptides, blotted with anti-HEV neutralizing monoclonal antibody (8C11) and convalescent human sera, show associated to the formation of homodimer. The intermolecular contact region on homodimer was investigated by chemical cross-linking of two site-directed cysteines. When the alanine on aa597 site mutated with cysteine, two different homodimers were found in SDS-PAGE analysis. One (42kD) can be disassociated with 8mol/L urea, which is postulated to form by virtue of hydrophobic interaction, and the other (60kD) falls apart with the reductant DTT present. The exact conformation, generating the cross-linking reaction of cysteines, was further investigated by induced-oxidation on monomer and hydrophobic homodimer of A597C protein with GSH/GSSG. And the results revealed, it is the conformation of hydrophobic homodimer that induces the disulfide bond come into being, instead of the one of monomer. So the aa597 site was verified to be located on interface domain of hydrophobically interacting homodimeric complex. To evaluate the biological significance of hydrophobicity of interface domain, we searched natural variations as to the region on all available databases with NCBI blast program. All variations on these amino acid residues kept higher hydrophobicity, which suggests that the hydrophobic domain is critical for the assemblage and propagation of HEV. NE2 N-terminal deletions up to aa458 had no effect on dimerization and took no exact part in formation of major neutralizing epitopes, but the fragment may act as helper for the formation of major neutralizing epitopes on NE2. Interestingly, the C-terminus aa605-aa660 of ORF2 can also act as helper instead of the N-terminus of NE2. This study suggests an interface domain of NE2 might be vital for HEV capsomer assembly and formation of major neutralizing epitopes. These results may offer clues to the rational design of recombinant anti-HEV vaccine.

Objective: To understand the infectivity and pathogenicity of the plasma of hepatitis E virus (HEV) viremia to primate animals., Methods: RNA fragment of HEV genotype IV was detected on one healthy donor who was positive for anti-HEV IgM and negative for anti-HEV IgG. Then 10 ml plasma from above donor was transfused to rhesus monkey to observe its infectivity and pathogenicity., Results: Acute hepatitis E was developed in rhesus monkey who accept HEV RNA positive plasma. It was confirmed by virological, immunological, biochemical and histopathological data., Conclusion: Acute hepatitis E can be induced by plasma transfusion of HEV viremia, which indicate the possibility of transfusion transmitted hepatitis E

We have evaluated four hepatitis E virus (HEV) specific antibody assays, using sequential samples taken from 86 rhesus monkeys at intervals for up to 86 weeks after they had been infected with different doses of HEV. The animals are a common experimental model of hepatitis E. The large collection of sequential samples used avoids uncertainties encountered in previous studies regarding the precise infection status of study subjects and minimizes bias due to the individuality of response to infection. One assay (YES IgG) was produced with synthetic peptides; the others (E2 IgM, E2 IgG, and GL IgG) were produced with recombinant antigens. The results were compared with the viral RNA contents of the serum and stool samples and the occurrence of these virological and immunological markers in the course of the infection was temporally related to the development of hepatitis. Diagnostic utility of the markers was assessed according to their response rates and prevalence at different times in the course of infection. All the animals produced E2 IgG and developed viremia and all but one also produced E2 IgM and excreted the virus in stool, whereas response rates for the other antibodies were lower and decreased with virus dose. Hepatitis occurred over a period of 4 weeks between 3 and 7 weeks after infection. Virological activity occurred mainly during the incubation period and the prevalence of viral markers declined rapidly after the onset of hepatitis. Production of the E2 antibodies immediately preceded the onset of hepatitis, and this was followed about one week later by production of the other antibodies. Seroprevalence E2 IgM reached a peak value 3 weeks after the onset of hepatitis, whereas seroprevalence of GL IgG and YES IgG peaked after the disease had subsided. E2 IgG persisted in all animals for the entire duration of the experiment of up to 86 weeks and possibly beyond and, thus, can serve as a useful epidemiological marker of HEV infection., (Copyright 2003 Wiley-Liss, Inc.)

Hepatitis E is an acute hepatitis casused by hepatitis E virus (HEV) in developing countries, where it occurs as cases sporadic and in epidemics form. The causative agent, hepatitis E virus, is transmitted primarily by the fecal-oral route. HEV is icosahedron non-enveloped virus, and its genome is a single-stranded, positive-sense, 3'-polyadenylated RNA about 7.5 kb in length. It contains three open reading frames (ORFs). Of which ORF1 codes for a polyprotein of 1693 amino acids and contain domains homologous to a viral methyltransferase, a papainlike cysteine protease, an RNA helicasre, and an RNA-dependent RNA polymerase, besides the most hypervariable region of the HEV genome. And ORF3 codes for a 123-amino-acide-long polypeptide with unknown function. While the major viral capsid protein (pORF2, ORF2 codes) of 660 amino acid was showed to contain the protective epitope. The bacterially expressed polypeptide disignated as NE2 has been proved to be a protective antige. And the anti-NE2 monoclonal antibodies (mAb) was screend, two of these mAbs 8C11 and 8H3 were showed to be against separate conformational neutralization epitope of hepatitis E virus (HEV). And these two mAb were used to screen for binding peptides from a 7-peptides phage display library. After four rounds of panning, tweenty-one positive monoclonal phages (11 for 8C11, and 10 for 8H3) were selected and the inserted fragments were sequenced. The DNA sequence coding for the obtained dominant peptide 8C11 (N'-His-Pro-Thr-Leu-Leu-Arg-Ile-C', named 8C11A) and 8H3 (N'-Ser-Ile-Leu-Pro-Tyr-Pro-Tyr-C', named 8H3A) were then synthesized and cloned to insert between amino acid 78 to 83 of hepatitis B core antigen (HBcAg), then expressed in E. coli. The recombinant proteins aggregate into homodimer or polymer on SDS-PAGE, and could bind with mAb 8C11 and 8H3 in Western blotting. Respectively, the recombinant protein C8C11A showed to be dimer mainly, which can bind with mAb 8C11. The monomer and dimer of C8H3A are in the same amount on SDS-PAGE, but only the dimer could bind with mAb 8H3 on Western blotting. The renatured recombinant proteins were all showed to aggregate into virus like particles which were similar as HBcAg on transmission electron micrograph. The dominant peptide 8H3A (N'-Ser-Ile-Leu-Pro-Tyr-ProTyr-C') that selected out by mAb 8H3 was further chemo-synthesized, and its binding activity was confirmed by BIAcore biosensor. The result showed that this 7-peptide can bind with mAb 8H3 in a big Ka and Kd form, which means the binding is not stable. These results implicated that conformational dependent neutralization epitope could be partially modeled by short peptide, which provided a feasible route for subunit vaccine development.

Evidence that hepatitis E is zoonotic is accumulating. Serum samples were collected from pigs, cattle, and goats from various regions of China to determine whether they had been infected with hepatitis E virus (HEV). An in-house enzyme immunoassay (EIA) and reverse transcriptase-polymerase chain reaction (RT-PCR) with primers from open reading frame (ORF) 2 were used to detect anti-HEV antibodies and HEV RNA. The mean positivity rates of anti-HEV antibody for pigs and cattle were 78.8% and 6.3% but none of the goat sera were positive. Pigs may be more susceptible to infection with HEV than cattle or goats. Five of 263 pig sera were positive for HEV RNA and four of these five were also positive for anti-HEV. The PCR products (nt 6007-6354) were cloned and sequenced and compared to other HEV sequences in the nucleotide databases. The five sequences shared 83-93% identity to each other at the nucleotide level and 74-79%, 73-74%, 73-78%, and 83-99% identity to HEV genotypes 1, 2, 3, and 4, respectively. They were closely related to human isolates of HEV genotype 4. Phylogenetic analyses also place these swine sequences in HEV genotype 4, resembling most closely viruses isolated from Chinese patients with acute hepatitis. These data support the hypothesis that sporadic hepatitis E in China is zoonotic., (Copyright 2002 Wiley-Liss, Inc.)

Abstract: It was evaluated its antigenicity, immunogenicity and efficacy of a candidate recombinant hepatitis E virus (HEV) vaccine, referred hitherto as HEV 239 vaccine. The vaccine peptide has a 26 amino acids extension from the N terminal of another peptide, E2, of the HEV capsid protein, which has been shown to protect monkeys against HEV infection previously. The vaccine peptide is similar as E2 in that: first, the vaccine peptide migrates predominantly as dimer in SDS-PAGE and it is dissociated into monomers by heating; second, its dimeric form of which predominantly recognized by HEV reactive human serum; and third, it shows the same pattern of reaction as E2 with a panel of eight monoclonal antibodies that had been raised against E2. In contrast to E2, the vaccine peptide aggregates to form particles of 13nm mean radius, and consequently, it is more than 240 times more immunogenic than E2. Using alum as adjuvant, immunizing dose determined in mice was 80–250ng for the vaccine and >60μg for E2. Rhesus monkeys twice vaccinated with a 10μg or a 20μg formulation of this vaccine showed essentially the same antibody response, whereas the response to a 5μg formulation was delayed but reached similar antibody levels. All the three vaccine formulations afford complete protection against infection with 104 genome equivalent dose of the homologous genotype 1 virus. At higher virus dose of 107, the same vaccine formulation partially protected against the infection and completely protected against hepatitis. The efficacy of the vaccine was essentially the same for the homologous genotype 1 virus and heterologous genotype 4 virus. [Copyright &y& Elsevier]