Varied immune responses of HBV-specific B cells in patients undergoing pegylated interferon-alpha treatment for chronic hepatitis B.

BACKGROUND & AIMS: The changes of HBV-specific B-cells in chronic hepatitis B (CHB) patients underwent pegylated interferon-alfa (PEG-IFNα) treatment and achieved functional cure remain unclear. We aimed to evaluate the alterations in HBV-specific B-cells during treatment and therefore explored the mechanism of functional recovery of HBsAg-specific B-cells. METHODS: We included 39 nucleos(t)ide analogues-treated CHB patients who received sequential combination therapy with PEG-IFNα and 8 treatment-naive CHB patients. HBV-specific B-cells were characterized ex vivo using fluorescent labeled HBsAg and HBcAg. The frequency, phenotype, and subsets of HBV-specific B-cells and follicular helper T cells (Tfh-cells) were detected using flow cytometry. The functionality of HBV-specific B-cells was quantified through ELISpot assays. RESULTS: During treatment, the fraction of activated memory B-cells (MBCs) among HBsAg-specific B-cells and the expression of IgG, CXCR3, and CD38 increased. Antibody-secretion capacity of HBsAg-specific B-cell was restored after treatment only in patients with a functional cure and it showed a positive correlation with serum hepatitis B surface antibody levels. The phenotype and function of HBsAg-specific B-cells differed between patients with and without functional cure. Patients with functional cure exhibited IgG+ classical MBCs and plasmablasts in HBsAg-specific B-cells. HBcAg-specific B-cells displayed both attenuated antibody secretion with reduced IgG expression and an IgM+ atypical type of MBCs after treatment, irrespective of with and without functional cure. The number of CD40L+ Tfh-cells increased after PEG-IFNα treatment and positively correlated with HBsAg-specific B-cell activation. CONCLUSIONS: After PEG-IFNα treatment, HBsAg- and HBcAg-specific B-cells exhibit various changes in antibody secretion. Their functional differences are reflected in the alterations in phenotypes and subtypes. The presence of CD40L+ Tfh-cells is associated with the active recovery of HBsAg-specific B-cells. IMPACT AND IMPLICATIONS: HBV-related complications and hepatocellular carcinoma remain the leading causes of mortality from chronic liver disease worldwide, and a cure is rarely achieved with antiviral therapies. Elucidating the immunological mechanisms underlying the functional cure of CHB patients offers a promising therapeutic strategy for viral clearance, such as therapeutic vaccine. We analyzed the alterations in HBV-specific B-cells in patients treated with PEG-IFNα and identified novel pathways for immunotherapeutic boosting of B cell immunity.

Prevalence of Hepatitis E Virus and Its Associated Outcomes among Pregnant Women in China.

Hepatitis E virus (HEV) is a significant public health concern worldwide. Pregnant women are at high risk of severe HEV infection. Various adverse outcomes in pregnant women related to HEV infection have been well documented in low-income and middle-income countries with poor sanitation. However, previous studies have provided inconsistent conclusions regarding the effects of HEV infection on the health of pregnant women and their infants in developed countries and contemporary China. In China, previous studies on HEV in pregnant women mainly focused on anti-HEV IgM and/or anti-HEV IgG. In this study, 4244 pregnant women were retrospectively analyzed for HEV-related markers. The positive rates of HEV antigen, HEV RNA, anti-HEV IgM, and anti-HEV IgG were 0.28%, 0.54%, 0.35%, and 10.49%, respectively. Among the 467 pregnant women who tested positive for at least one HEV-related marker, 92.93% (434) were positive for anti-HEV IgG only and 0.21% (1) were positive for HEV antigen, anti-HEV IgM, and anti-HEV IgG. Although the prevalence of anti-HEV IgG significantly increased with age, the prevalence of anti-HEV IgM, HEV RNA, and HEV antigen did not differ among pregnant women of different ages. Thirty-three pregnant women were positive for at least one of anti-HEV IgM, HEV antigen, and HEV RNA, and these individuals were recently or currently infected with HEV. None of the 33 pregnant women exhibited obvious clinical symptoms. Of the 33 pregnant women, 39.39% (13) experienced adverse fetal outcomes, including preterm birth, fetal distress, and low birth weight, the incidence of which was significantly higher than in pregnant women who were not recently or currently infected with HEV. These findings suggest that maternal HEV infection may impact the health of fetuses; thus, these results may contribute to the development of appropriate public health interventions for this population.

Profile of clinical characteristics and serologic markers of sporadic hepatitis E in a community cohort study.

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.

An Optimized FI-RSV Vaccine Effectively Protects Cotton Rats and BALB/c Mice without Causing Enhanced Respiratory Disease.

BACKGROUND: Despite considerable efforts toward vaccine development in past decades, no effective vaccines against respiratory syncytial virus (RSV) are available. Recently, we showed that an optimized formalin concentration can preserve prefusion protein (pre-F) on RSV-infected cells and protect mice against RSV infection without causing enhanced respiratory disease (ERD). Here, we sought to further stabilize pre-F on RSV virions by optimizing the production of FI-RSV. METHODS: Freshly produced RSV virions were treated with formalin under different concentrations to obtained an opti-FI-RSV vaccine with high pre-F level. Immunogenicity and safety of opti-FI-RSV were evaluated in Balb/c mice and cotton rats. RESULTS: Using 0.0156-0.1778% formalin, we successfully preserved pre-F on virions. This opti-FI-RSV exhibited improved immunogenicity and efficacy without causing ERD. Surprisingly, opti-FI-RSV, with a pre-F-dominant immunogen, still caused ERD after immunization with a suboptimal dose or when the neutralizing antibody titers declined. ERD was avoided by coadministering opti-FI-RSV with CpG + MPLA adjuvant, which subsequently induced a Th1-biasing immune response and, more importantly, significantly improved antibody avidity. CONCLUSIONS: Our study provides a new method to obtain a novel FI-RSV vaccine with a high pre-F level and may provide a reference for developing other inactivated vaccines. Our findings also emphasize that appropriate adjuvants are critical for nonreplicating vaccines.

Epidemiological Features, Risk Factors, and Disease Burden of Respiratory Viruses among Hospitalized Children with Acute Respiratory Tract Infections in Xiamen, China.

Well-established surveillance and monitoring systems for respiratory viruses need to be improved, and epidemiological data on respiratory viruses in China are scarce. This study aimed to investigate the epidemiological characteristics of respiratory viruses among hospitalized children aged ≤2 years with acute respiratory tract infections (ARTIs) in Xiamen, China, from October 2014 to September 2017. The clinical records of 7,248 children hospitalized for ARTIs were retrospectively analyzed. Respiratory syncytial virus (RSV) (22.3%) was the most common virus among hospitalized children aged ≤2 years, followed by parainfluenza (5.0%), adenovirus (3.5%), and influenza (1.7%). RSV-infected children had a higher disease burden, including a higher intensive care unit (ICU) admission rate (12.7%) and higher hospital charges ($635.36). Particularly, infants aged <6 months had the highest risk of RSV infection (odds ratio = 2.4; 95% CI, 1.9-2.9) and a higher ICU admission rate (12.1% vs. 4.5%, 4.6%) and hospital cost ($923.3 vs. $785.5, $811.7) than the other age groups. Therefore, infants aged 0-6 months, particularly premature infants and children with congenital diseases, should receive more attention. There is an urgent need to develop effective immunization strategies to protect these infants during the first 6 months of life and in the RSV season.

Molecular Evolution of Attachment Glycoprotein (G) and Fusion Protein (F) Genes of Respiratory Syncytial Virus ON1 and BA9 Strains in Xiamen, China.

Monitoring viral transmission and analyzing the genetic diversity of a virus are imperative to better understand its evolutionary history and the mechanism driving its evolution and spread. Especially, effective monitoring of key antigenic mutations and immune escape variants caused by these mutations has great scientific importance. Thus, to further understand the molecular evolutionary dynamics of respiratory syncytial virus (RSV) circulating in China, we analyzed nasopharyngeal swab specimens derived from hospitalized children ≤5 years old with acute respiratory tract infections (ARIs) in Xiamen during 2016 to 2019. We found that infants under 6 months of age (52.0%) were the main population with RSV infection. The prevalent pattern "BBAA" of RSV was observed during the epidemic seasons. RSV ON1 and BA9 genotypes were the dominant circulating strains in Xiamen. Interestingly, we observed four Xiamen-specific amino acid substitution combinations in the G protein and several amino acid mutations primarily occurring at antigenic sites Ø and V in the F protein. Our analyses suggest that introduction of new viruses and local evolution are shaping the diversification of RSV strains in Xiamen. This study provides new insights on the evolution and spread of the ON1 and BA9 genotypes at local and global scales. IMPORTANCE Monitoring the amino acid diversity of the RSV G and F genes helps us to find the novel genotypes, key antigenic mutations affecting antigenicity, or neutralizing antibody-resistant variants produced by natural evolution. In this study, we analyzed the molecular evolution of G and F genes from RSV strains circulating in Xiamen, China. These data provide new insights on local and global transmission and could inform the development of control measures for RSV infections.

Epidemiology of Respiratory Pathogens Among Children Hospitalized for Pneumonia in Xiamen: A Retrospective Study.

OBJECTIVES: To investigate the etiology of common respiratory pathogens in children < 2 years of age hospitalized with pneumonia in Xiamen from 2014 to 2017. METHODS: The medical records of 5581 children with pneumonia were retrospectively reviewed. Direct immunofluorescent test was used for respiratory virus testing. Bacteria were detected by conventional culture method. The results of pathogen detection at admission were analyzed as well as the clinical outcomes of children. RESULTS: The burden of hospitalized children with pneumonia was highest among infants < 6 months old (58.2%). Respiratory syncytial virus (RSV) was the most common respiratory virus (26.0%) followed by parainfluenza (4.8%) and adenovirus (3.2%). Haemophilus influenzae was the most common bacteria detected (16.6%) followed by Moraxella catarrhalis (13.4%), Staphylococcus aureus (13.0%), Streptococcus pneumoniae (12.3%), Escherichia coli (5.1%) and Klebsiella pneumoniae (4.8%). Notably, RSV and K. pneumoniae were detected more frequently in severe pneumonia (35.0% and 10.9%) versus mild pneumonia (25.6% and 4.6%), with higher rates of ICU admissions, longer hospital stays and higher hospital costs compared to those infected with other respiratory pathogens. CONCLUSIONS: Among children < 2 years of age hospitalized with pneumonia in Xiamen, RSV was the most common respiratory virus, while H. influenzae and S. pneumoniae remained the predominant bacterial pathogens detected. Considering the low implementation rate of vaccines against pneumococcal and Hib pneumonia in China, there is an urgent need to increase both vaccination rates to reduce pneumococcal and Hib disease burden.

Quantitative evaluation of protective antibody response induced by hepatitis E vaccine in humans.

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.

Lot-to-lot consistency study of an Escherichia coli-produced bivalent human papillomavirus vaccine in adult women: a randomized trial.

An Escherichia. coli-produced HPV-16/18 bivalent vaccine has been proved to be well-tolerated and highly efficacious against diseases associated with vaccine HPV types. As a part of the multi-center, randomized, double-blind phase III clinical trial, this lot-to-lot consistency study aimed to assess the safety and immunogenicity consistency of this novel HPV vaccine, which is also one of the objectives of the phase III trial. A total of 3689 healthy women aged 18-45 years were enrolled and randomly assigned 1:1:1 to three lots of the HPV vaccine groups. The primary outcomes were the IgG antibody level at 1 month after the last dose (month 7). In the immunogenicity per-protocol set (PPS), almost all of the participants seroconverted at month 7 and remained seropositive at month 42. For each paired comparison of the three lot groups, the two-sides of 90% CIs of GMC ratios for both IgG and neutralizing antibodies for HPV-16 and HPV-18 at month 7 were within the equivalence interval [0.5, 2]. Lot consistency was also demonstrated at month 42. The majority of recorded solicited reactions were mild or moderate. The incidences of solicited reactions of Lot 2 and Lot 3 were slightly higher than Lot 1. However, the incidences of solicited reactions of ≥ grade 3 and solicited reactions by symptoms were all similar among the three lot groups. None of the SAEs was considered related to vaccination by the investigator. In conclusion, this study demonstrates lot-to-lot consistency of the 3 consecutive lots of the E. coli-produced HPV-16/18 bivalent vaccine.

The Optimal Concentration of Formaldehyde is Key to Stabilizing the Pre-Fusion Conformation of Respiratory Syncytial Virus Fusion Protein.

BACKGROUND: To date, there is no licensed vaccine available to prevent respiratory syncytial virus (RSV) infection. The valuable pre-fusion conformation of the fusion protein (pre-F) is prone to lose high neutralizing antigenic sites. The goals of this study were to stabilize pre-F protein by fixatives and try to find the possibility of developing an inactivated RSV vaccine. METHODS: The screen of the optimal fixative condition was performed with flow cytometry. BALB/c mice were immunized intramuscularly with different immunogens. The serum neutralizing antibody titers of immunized mice were determined by neutralization assay. The protection and safety of these immunogens were assessed. RESULTS: Fixation in an optimal concentration of formaldehyde (0.0244%-0.0977%) or paraformaldehyde (0.0625%-1%) was able to stabilize pre-F. Additionally, BALB/c mice inoculated with optimally stabilized pre-F protein (opti-fixed) induced a higher anti-RSV neutralization (9.7 log2, mean value of dilution rate) than those inoculated with unstable (unfixed, 8.91 log2, p < 0.01) or excessively fixed (exce-fixed, 7.28 log2, p < 0.01) pre-F protein. Furthermore, the opti-fixed immunogen did not induce enhanced RSV disease. CONCLUSIONS: Only the proper concentration of fixatives could stabilize pre-F and the optimal formaldehyde condition provides a potential reference for development of an inactivated RSV vaccine.

Application of Hepatitis E Virus-Related Markers on Samples from a Developing Country.

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.

Long-term HEV carriers without antibody seroconversion among eligible immunocompetent blood donors.

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.

An optimized high-throughput fluorescence plate reader-based RSV neutralization assay.

A licensed vaccine for respiratory syncytial virus (RSV) has yet to be developed, and a reliable and repeatable neutralizing assay is indispensable for vaccine development. Here, we demonstrated an optimized high-throughput RSV neutralization assay that utilizes a fluorescence plate reader (reader) as a substitute for flow cytometry to detect fluorescent signals in RSV-A2 mKate-infected cells. Furthermore, this study tested the influence of virus input and infectivity on the neutralizing assay and highlighted critical factors (together with a suggested protocol) for obtaining stable data using this assay.

Structural basis of respiratory syncytial virus subtype-dependent neutralization by an antibody targeting the fusion glycoprotein.

A licensed vaccine for respiratory syncytial virus (RSV) is unavailable, and passive prophylaxis with the antibody palivizumab is restricted to high-risk infants. Recently isolated antibodies 5C4 and D25 are substantially more potent than palivizumab, and a derivative of D25 is in clinical trials. Here we show that unlike D25, 5C4 preferentially neutralizes subtype A viruses. The crystal structure of 5C4 bound to the RSV fusion (F) protein reveals that the overall binding mode of 5C4 is similar to that of D25, but their angles of approach are substantially different. Mutagenesis and virological studies demonstrate that RSV F residue 201 is largely responsible for the subtype specificity of 5C4. These results improve our understanding of subtype-specific immunity and the neutralization breadth requirements of next-generation antibodies, and thereby contribute to the design of broadly protective RSV vaccines.

Classification of human and zoonotic group hepatitis E virus (HEV) using antigen detection.

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.

Discovery of a Prefusion Respiratory Syncytial Virus F-Specific Monoclonal Antibody That Provides Greater In Vivo Protection than the Murine Precursor of Palivizumab.

Palivizumab, a humanized murine monoclonal antibody that recognizes antigenic site II on both the prefusion (pre-F) and postfusion (post-F) conformations of the respiratory syncytial virus (RSV) F glycoprotein, is the only prophylactic agent approved for use for the treatment of RSV infection. However, its relatively low neutralizing potency and high cost have limited its use to a restricted population of infants at high risk of severe disease. Previously, we isolated a high-potency neutralizing antibody, 5C4, that specifically recognizes antigenic site Ø at the apex of the pre-F protein trimer. We compared in vitro and in vivo the potency and protective efficacy of 5C4 and the murine precursor of palivizumab, antibody 1129. Both antibodies were synthesized on identical murine backbones as either an IgG1 or IgG2a subclass and evaluated for binding to multiple F protein conformations, in vitro inhibition of RSV infection and propagation, and protective efficacy in mice. Although 1129 and 5C4 had similar pre-F protein binding affinities, the 5C4 neutralizing activity was nearly 50-fold greater than that of 1129 in vitro In BALB/c mice, 5C4 reduced the peak titers of RSV 1,000-fold more than 1129 did in both the upper and lower respiratory tracts. These data indicate that antibodies specific for antigenic site Ø are more efficacious at preventing RSV infection than antibodies specific for antigenic site II. Our data also suggest that site Ø-specific antibodies may be useful for the prevention or treatment of RSV infection and support the use of the pre-F protein as a vaccine antigen.IMPORTANCE There is no vaccine yet available to prevent RSV infection. The use of the licensed antibody palivizumab, which recognizes site II on both the pre-F and post-F proteins, is restricted to prophylaxis in neonates at high risk of severe RSV disease. Recommendations for using passive immunization in the general population or for therapy in immunocompromised persons with persistent infection is limited because of cost, determined from the high doses needed to compensate for its relatively low neutralizing potency. Prior efforts to improve the in vitro potency of site II-specific antibodies did not translate to significant in vivo dose sparing. We isolated a pre-F protein-specific, high-potency neutralizing antibody (5C4) that recognizes antigenic site Ø and compared its efficacy to that of the murine precursor of palivizumab (antibody 1129) matched for isotype and pre-F protein binding affinities. Our findings demonstrate that epitope specificity is an important determinant of antibody neutralizing potency, and defining the mechanisms of neutralization has the potential to identify improved products for the prevention and treatment of RSV infection.

The Bama miniature swine is susceptible to experimental HEV infection.

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.

A high-throughput neutralizing assay for antibodies and sera against hepatitis E virus.

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.

A Comprehensive Study of Neutralizing Antigenic Sites on the Hepatitis E Virus (HEV) Capsid by Constructing, Clustering, and Characterizing a Tool Box.

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.

A novel linear neutralizing epitope of hepatitis E virus.

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.