Hepatitis E Virus (HEV) ORF2 Antigen Levels Differentiate Between Acute and Chronic HEV Infection

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1 The Journal of Journal Infectious of Diseases Infectious Diseases Advance Access published May 27, 2016 MAJOR ARTICLE Hepatitis E Virus (HEV) ORF2 Antigen Levels Differentiate Between Acute and Chronic HEV Infection Patrick Behrendt, 1,2,3 Birgit Bremer, 3 Daniel Todt, 1 Richard J. P. Brown, 1 Albert Heim, 4 Michael P. Manns, 2,3 Eike Steinmann, 1 and Heiner Wedemeyer 2,3 1 TWINCORE, Center for Experimental and Clinical Infection Research, Institute for Experimental Infection Research, 2 German Center for Infection Research, 3 Department for Gastroenterology, Hepatology, and Endocrinology, Medical School Hannover, and 4 Institute of Virology Hannover Medical School, Germany Background. Hepatitis E virus (HEV) genotype 3 infections are frequent in Europe and North America, with acute and chronic courses described in the literature. HEV RNA detection by real-time polymerase chain reaction (PCR) is the gold standard for diagnosis. Recently, an anti-hev antigen (Ag) specific enzyme-linked immunosorbent assay (ELISA) directed against the HEV capsid became commercially available. The effectiveness of anti-hev Ag specific ELISA at detecting HEV genotype 3 infections remains undefined. Methods. The performance of anti-hevag ELISA was compared with that of real-time PCR, using sera from a cohort of acutely infected individuals, in addition to a cohort of chronically infected patients undergoing ribavirin therapy. Furthermore, virion properties were evaluated by density fractionation. Results. Anti-HEV Ag specific ELISA was less sensitive than real-time PCR at detection of HEV infection. Anti-HEV Ag specific ELISA revealed significantly higher HEV Ag in chronically infected individuals as compared to acutely infected patients, with high sensitivity and specificity to distinguish acute from chronic HEV infection. Of note, HEV Ag remained detectable for >100 days after HEV RNA clearance in ribavirin-treated patients with chronic HEV. Density gradients revealed the presence of membrane-associated virions in the sera, with a different distribution as compared to HEV RNA. Conclusions. The anti-hev Ag specific ELISA is less sensitive than HEV RNA real-time PCR but represents a useful tool to discriminate chronic from acute infection. Keywords. hepatitis E virus; antigen level; chronic infection; acute infection; virion properties. Hepatitis E virus (HEV) infection represents a significant global healthcare problem. While previously considered an infection occurring in developing nations, improved surveillance has revealed that a significant number of individuals in industrialized countries experience HEV infection during their lifetime [1 4]. In the United States and Europe, approximately 30% of the population is positive for anti-hev immunoglobulin G (IgG), indicating a previous HEV infection, with increased frequencies observed in older people [2, 4 6]. The route of HEV transmission in developing nations is commonly through contaminated water, whereas in developed countries ingestion of infected pork meat is believed to be the main risk factor for genotype 3 infections [7]. Additionally, transmission via contaminated blood products accounts for a significant proportion of infections in at-risk populations of patients, including those with renal disease, transplant recipients, and people requiring a blood transfusion [8]. The clinical course of HEV infection is usually mild Received 19 October 2015; accepted 7 April Correspondence: P. Behrendt, Abteilung für Gastroenterologie, Hepatologie, und Endokrinologie, Medizinische Hochschule Hannover, Carl-Neuberg-Str.1, Hannover, Germany (behrendt.patrick@mh-hannover.de). The Journal of Infectious Diseases The Author Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, journals.permissions@oup.com. DOI: /infdis/jiw161 and self-limiting in immunocompetent patients. However, severe cases of acute HEV infection have been described for pregnant women and patients with preexisting liver diseases [7]. Furthermore, immunocompromised individuals experience a prolonged course of disease, and chronic infections develop in around 60% of the cases [9]. Chronic hepatitis is thereby accompanied by an increased risk of severe liver damage, such as rapid development of cirrhosis [9 11]. A clinical challenge in the management of immunocompromised patients with HEV infection is to differentiate between the acute and chronic course of infection. Approximately 60% of infected immunocompromised individuals experience a chronic infection, which might require treatment with ribavirin. Diagnosis of HEV infection is commonly based on the detection of anti-hev antibodies and/or detection of viral RNA by real-time polymerase chain reaction (PCR). Recently, an anti- HEV antigen (Ag) specific enzyme-linked immunosorbent assay (ELISA) became commercially available. In this study, we used a well-characterized panel of patient samples derived from genotype 3 infected individuals with acute or chronic HEV infection and subjected them to both real-time PCR and anti-hev Ag specific ELISA,tocompare the 2 diagnostic techniques. Because we found significant differences between the tested cohorts, we performed density fractionation to further characterize HEV particles in sera from HEV Ag Level and Infection Course JID 1

2 patients with chronic and those with acute infection. Overall, we observed a low sensitivity of the anti-hev Ag specific ELISA to detect an acute genotype 3 infection. However, all of the samples of the chronically infected patients displayed a high-positive result in the anti-hevag specific ELISA, thereby discriminating chronic from acute HEV infection. Virions from individuals with chronic versus those with acute HEV infection displayed similar results in the density fractionation, pointing to circulating membrane-associated viral particles. METHODS Samples and Patient s Characteristics The patients characteristics are depicted in Table 1. Patients samples were retrospectively analyzed. Samples were obtained between 2008 and For the approximate duration of infection in chronically infected individuals, the levels of transaminases were screened retrospectively, and the onset of infection was assumed when elevated levels of transaminases were detected for the first time. Anti-HEV Ag Specific ELISA Anti-HEV Ag specific ELISA (Wantai Biopharmaceutical, Beijing, China) was performed according to the manufacturer s instructions. In brief, 100 µl of serum was added to each well of the ELISA plate ( precoated with an anti-hev ORF2 antibody) and incubated for 1 hour at 37 C. After washing the wells with provided buffer, a second horseradish peroxidase conjugated monoclonal anti-hev ORF2 antibody was added, followed by further incubation for 30 minutes at 37 C. Thereafter, wells were washed a second time, chromogen solution, provided with the kit, was added, and wells were incubated for 15 minutes at 37 C. The reaction was stopped by addition of Stop-Solution, and ODs were immediately measured with an ELISA plate reader (Synergy 2, BioTek; OD 450/630 ). For each plate, triplicate wells with negative controls were measured. These values were used to determine the cut-off values in accordance to manufacturer instructions. Briefly, the ratio of the OD 450/630 value for each individual specimen sample to the cutoff (S/CO) was used to indicate HEV Ag status, with positivity defined as a ratio of >1.1), negativity defined as a ratio of <0.9, and a borderline result defined as a ratio of Quantitative Real-Time PCR HEV RNA from serum was quantified by real-time PCR as recently described [12]. For detection of viral RNA, we used the QuantiTect Virus + ROC Viral Kit (Qiagen) and the Lightcycler 480 II (Roche). Primer and probes had the following sequences: GGTGGTTTCTGGGGTGAC (forward), AGGGGTTGGTTG- GATGAA (reverse), and 6-FAM-TGATTCTCAGCCCTTCGC- BBQ ( probe), generated by TIB-MOLBIOL. Stability Assay To address whether HEV Ag and HEV RNA are stable at room temperature and whether differences arise due to the methods of sample collection, we incubated serum and blood in ethylenediaminetetraacetic acid (EDTA) containing tubes, collected from 2 different individuals, for up to 10 days at room temperature and tested for RNA and HEV Ag. In parallel, we performed this assay with serum and plasma that was immediately centrifuged and stored at room temperature or left uncentrifuged at room temperature to mimic prolonged shipping conditions. The uncentrifuged samples were aliquoted beforehand, and centrifugation was performed for each sample at the indicated time point to measure RNA or Ag content. For all other experiments, sera were used that had been immediately centrifuged and stored at 20 C or 80 C. Iodixanol Density-Gradient Fractionation Density-gradient centrifugation was performed as recently described [13]. In brief, samples were divided into 2 1 ml aliquots. One aliquot was treated with NP40 (5% Tergitol solution, Sigma-Aldrich) and incubated for 1 hour at room temperature. The second aliquot remained untreated. Following incubation, both aliquots were fractionated by overnight centrifugation through a iodixanol step gradient (0% 40%) at g in a TH-641 swing-out rotor at 4 C, using a Sorvall Ultra WX80 centrifuge. Thereafter, ml fractions were collected, and levels of viral RNA and HEV Ag of each fraction were determined. Buoyant densities were analyzed using a refractometer. Statistical Analysis Data were analyzed using GraphPad Prism v6.0b (GraphPad software, La Jolla, California). Comparisons of different groups was performed using a Bonferroni-corrected multiple comparisons t test. Based on a receiver operating characteristic (ROC) curve, a putative threshold was set. RESULTS Anti-HEV Ag Specific ELISA Shows Less Sensitivity Than HEV RNA Specific PCR in the Majority of Tested Sera The sensitivity of the anti-hev Ag specific ELISA was compared to that of real-time PCR, using serial dilutions of sera from 4 individuals with chronic HEV genotype 3 infection. Figure 1 demonstrates a reduced sensitivity of the anti-hev Ag specific ELISAwhencomparedtoreal-timePCR:onlya single sample tested positive in the anti-hev Ag specific ELISA across the entire dilution range (Figure 1A). For 3 of 4 dilution series (Figure 1B D), HEV RNA levels of < copies/ml led to negative test results of anti-hev Ag specific ELISA, while the real-time PCR continued to display a linear reduction in levels. HEV Ag Is Stable at Room Temperature In clinical practice, patient blood samples often are collected in different test tubes (EDTA-lined tubes and serum columns). Additionally, long storage at room temperature may cause false-negative test results. Therefore, we performed a stability 2 JID Behrendt et al

3 Table 1. Patient Characteristics Patient, Infection Course S/ CO a Age, y Reason for Immunosuppression AST Level, U/I ALT Level, U/I GGT Level, U/I Bilirubin Level, µmol/l Creatinine Level, µmol/l INR Platelet Count, 1000 Platelets/µL Approximate Duration of Infection, mo. Chronic Lack of CD4 + T cells ND ND ND ND ND ND ND Heart transplantation Multilocular cystic ND 64 ND fibrosis Lung transplantation Heart transplantation ND ND ND ND ND ND ND Kidney transplantation Kidney transplantation > Kidney transplantation ND Kidney transplantation > Liver transplantation > Heart transplantation Kidney transplantation Heart transplantation Kidney transplantation Kidney transplantation > Liver transplantation > Heart transplantation Kidney transplantation Microscopic >12 polyangiitis HIV infection ND Heart transplantation Acute NA ND ND ND ND ND ND ND < NA < NA < NA < NA NA < NA < Kidney transplantation 59 ND < NA ND ND ND ND ND ND ND < NA < Liver transplantation Unclear Liver transplantation < NA < NA < NA < NA < NA <1 39 ND 66 Kidney transplantation <1 Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, glutamyl transpeptidase; HIV, human immunodeficiency virus; INR, international normalized ratio; NA, not applicable; ND, no data. a Defined as the ratio of the OD 450/630 for each individual specimen sample to the cutoff (S/CO), with a value of >1.1 indicating positivity, a value of <0.9 indicating negativity, and a value of indicating a borderline test result. assay with samples from 2 patients with chronic HEV infection (patients 5 and 6; Table 1). We tested RNA and HEV Ag levels in plasma samples (Figure 2A and 2B) and in serum samples (Figure 2C and 2D) obtained over time that were immediately centrifuged and stored at room temperature (Figure 2B and 2D) or left uncentrifuged at room temperature (Figure 2A and 2C). Anti-HEV Ag ELISA and real-time PCR showed no significant reduction over a period of 10 days regardless of sample preparation or storage duration. Chronically Infected Individuals Can Be Discriminated From Acutely Infected Patients on the Basis of Anti-HEV Ag Specific ELISAOD 450/630 Values Next, we compared the performance of the anti-hev Ag specific ELISA in 20 individuals with chronic and 17 with HEV Ag Level and Infection Course JID 3

4 Figure 1. Comparison of hepatitis E virus (HEV) antigen (Ag) and HEV-specific polymerase chain reaction (PCR). Depicted are the serial dilution of the sera of 4 different, chronically infected individuals (genotype 3), with the levels of HEV RNA (real-time PCR; squares) and HEVAg (dots). The dashed line represents the cutoff of the anti-hevag specific enzyme-linked immunosorbent assay. A value of >1 is interpreted as HEV positive (black dots), and a value of <1 is interpreted as HEV negative (open dots). acute HEV infection. Thirteen patients with acute hepatitis A virus infection served as negative controls. As depicted in Figure 3A, the anti-hev Ag specific ELISA showed a reduced sensitivity for detecting HEVAg in acutely infected individuals. In this group, 64.7% of RNA-positive patients were positive according to the anti-hev Ag specific ELISA. In contrast, results of the anti-hev Ag specific ELISA was positive in all of the cases of chronic HEV infections. None of the chronically infected patients showed a false-negative HEV Ag result, suggesting a high reliability of the assay for this cohort. While 6 samples positive for anti cytomegalovirus immunoglobulin M (IgM) and 6 positive for anti Epstein-Barr virus IgM showed no crossreactivity (data not shown), one hepatitis A virus infected patient displayed a false-positive anti-hev Ag specific ELISA result, even after retesting. This sample repeatedly tested negative for HEV RNA (data not shown). Overall, the specificity was 92% and the sensitivity was 65% for acutely infected patients, compared with 100% for chronically infected patients. A Bonferroni corrected multiple comparisons t test was performed, which revealed significant elevation of S/CO values for acutely and chronically infected individuals, compared with hepatitis A virus infected controls. However, we could also observe a significant elevation in sera from chronically infected patients, compared with that from acutely infected patients (Figure 3A). To identify a detection threshold for distinguishing between acutely and chronically infected patients, we plotted a ROC curve (Figure 3B). An S/CO OD threshold of could discriminate the acutely and chronically infected groups with a sensitivity of 95.0% and a specificity of 88.24%. The OD of was still within the linear range of the ELISA, as determined by serial dilution (data not shown). Anti-HEV Ag Levels During the Natural Course of Acute Infection and During Efficient Ribavirin Treatment of Chronically Infected Individuals Recent studies revealed that ribavirin is a potent treatment option for individuals chronically infected with HEV. Usually, the HEV RNA load is used to determine the response to therapy. We retrospectively analyzed samples from 3 acutely infected patients during the natural course of a self-limiting infection. In addition, 3 chronically infected patients who cleared viral RNA during therapy with ribavirin were checked for HEV Ag content at different time points. As shown in Figure 4, HEV RNA loss was accompanied by a parallel loss of HEV Ag in acutely infected patients (Figure 4A). Chronically infected individuals displayed different kinetics: although HEV RNAwas undetectable in most of the patients shortly after initiation of antiviral therapy, HEV Ag remained detectable. Indeed, anti-hev Ag specific ELISA remained positive days after onset of therapy and HEV RNA clearance (Figure 4B). 4 JID Behrendt et al

5 Figure 2. Stability of hepatitis E virus (HEV) RNA and antigen. Among individuals with chronic infection, sera or blood specimens in ethylenediaminetetraacetic acid (EDTA) containing tubes were obtained and aliquoted on day 0 and were either centrifuged (B and D) or left uncentrifuged (A and C) and harvested. Throughout 10 days, vials were assayed for HEV RNA and antigen content daily. The values for each day were normalized to the level of RNA or antigen at day 0 in percentage. All individuals were infected with genotype 3. Figure 3. Antigen test results in real-life setting. A, Hepatitis E virus (HEV) antigen (Ag) specific enzyme-linked immunosorbent assay (ELISA) was performed with sera for individuals with acute (n = 17) or chronic (n = 20) genotype 3 infection (as determined by HEV polymerase chain reaction). Thirteen individuals with acute hepatitis A virus infection served as a negative control and tested negative for HEV RNA. The y-axes represents the ratio of OD 450/630 values of the tested samples over the control (S/CO). The dotted line represents the cutoff of the anti-hevag specific ELISA. A value of >1 is interpreted as HEV positive, and a value of <1 is interpreted as HEV negative. The dashed line depicts the discrimination of acute and chronic samples at a putative threshold level, as calculated in panel B. Differences in means were tested with a Bonferroni corrected t test. ***P <.001. B, Receiver operating characteristic curve displaying the true-positive rate against the false-positive rate at various threshold settings to discriminate between acutely and chronically infected individuals. The open circle represents 95.0% sensitivity (95% confidence interval [CI], 75.13% 98.54%) and 88.24% specificity (95% CI, 63.56% 98.54%) at an OD 450/630 of (area under the curve, [95% CI, ]; likelihood ratio, 8.075; P <.0001). Abbreviation: S/CO, ratio of OD 450/630 values of the tested samples over the control. HEV Ag Level and Infection Course JID 5

6 Figure 4. Course of hepatitis E virus (HEV) infection. A, Sera from acutely infected individuals were tested for HEV antigen over time. B, HEV antigen levels in sera during treatment (indicated by a horizontal bar) from chronically infected individuals. Solid circles represent HEV RNA positive samples, and open circles represent HEV RNA negative samples. The dashed lines represent the cutoff of the antigen test. A value of >1 is interpreted as HEV positive, and a value of <1 is interpreted as negative. All individuals were infected with genotype 3. Abbreviations: RBV, ribavirin; S/CO, ratio of OD 450/630 values of the tested samples over the control. Virions From Sera of Chronically and Acutely Infected Individuals Are Membrane Associated HEV is commonly acknowledged as a naked virus. However, recent data indicated that it may circulate while wrapped in cellular membranes [14]. In theory, these membranes might influence the performance of the anti-hevag specific ELISA. The anti-hev monoclonal antibodies used in the anti-hev Ag specific ELISAaredirectedagainstthecapsid protein (ORF2). However, ORF2 capsid would be occluded from monoclonal antibody recognition in the case of membrane-enveloped viral particles. To address whether differences in membranous shielding of virus particles might cause the distinct differences in performance of the anti-hev Ag specific ELISA in patients with chronic versus those with acute infection, we performed density gradients of serum from infected individuals. Additionally, we added the detergent NP40 to disrupt potential viral particle associated membranes. As depicted in Figure 5, in both tested cohorts (acutely and chronically infected individuals), viral RNA levels peaked at a density of approximately g/ml. In all patients, treatment of the samples with NP40 shifted the peak of viral RNA to a density of approximately g/ml, suggesting a potent disruption of membrane structures. Again, HEV Ag levels were higher in chronically infected patients (Figure 5B). Notably, HEV Ag was also detectable in density fractions in which no viral RNA was present. 6 JID Behrendt et al

7 Figure 5. Iodixanol density-gradient fractionation. Plasma from 3 individuals with acute (A) and 3 with chronic (B) hepatitis E virus (HEV) genotype 3 infection were either treated with NP40 or left untreated for 1 hour. Thereafter, iodixanol density-gradient fractionation was performed. Ten fractions were removed and separately analyzed for RNA (the number of copies was normalized to the total HEV RNA level as a percentage) and HEV antigen (OD 450/630 ) content. Density was determined by a refractometer. DISCUSSION Besides causing acute infections, HEV, according to multiple studies, causes chronic infections, defined on the basis of HEV RNA positivity for >3 months [15]. Current treatment options are either reduction of immunosuppression, which leads to relief in about 30% of the cases [9], or a 3 6-month course of ribavirin, which is often associated with side effects, particularly anemia, which occurs in 29% of cases [16]. Testing for HEV in immunocompetent individuals is commonly performed by detection of anti-hev IgG/IgM, whereas in immunosuppressed hosts HEV RNA quantification is currently considered as the gold standard [11]. In this work, we tested the effectiveness of a recently released anti-hev Ag specific ELISA in the detection of genotype 3 infections. Our study demonstrates that anti-hevag specific ELISA has a sensitivity of 65% and a specificity of 92% in detecting an ongoing HEV infection in a real-life cohort. Recent reports for testing of acute genotype 4 infections further support these results (78% concordance of HEV RNA and HEV Ag among sera) [17]. Another study, from India, reported HEV Ag detection in 12 of 20 HEV RNA positive cases, demonstrating a specificity of 86.1% and a sensitivity of 60% [18]. In our study, comparison of chronically infected individuals with acutely infected patients revealed drastically increased ODs in chronically infected patients, while most of the positive samples from acutely infected patients displayed significantly lower HEV Ag Level and Infection Course JID 7

8 values. This led to a sensitivity of 100% (20 of 20) among anti- HEV Ag specific ELISA results for chronically HEV infected individuals. In ROC analysis, we observed that, in our small cohort, the likelihood of a chronic infection was increased 8-fold when the OD of the serum samples for HEV Ag was > However, this value reflects the test results that we obtained in our center and might differ at other sites depending on the handling protocol and technical devices. Additionally, very recently the anti-hev Ag specific ELISA has been provided with a diluent, which likely has an impact on the results and this threshold. During the course of infection, clearance of HEV is defined on the basis of the undetectability of HEV RNA. Interestingly, we found prolonged detectability of HEV Ag when compared to HEV RNA levels in individuals successfully treated with ribavirin during chronic hepatitis. Contrastingly, the decrease of HEV Ag and HEV RNA levels in acutely infected patients was concomitant. Data from India also revealed, although to a lesser extent, that acute genotype 1 infections displayed prolonged HEV Ag detectability over time despite the absence of RNA: while HEV RNA was detected in only 54% of cases by days 4 7, HEV Ag remained detectable in 88% of patients [19]. In contrast, HEV Ag became undetectable 4 weeks earlier than HEV RNA during acute infection with genotype 4 HEV [20]. Whether this ongoing detection of HEV Ag in our ribavirintreated cohort reflects ongoing viral replication or is caused by a long half-life of HEV Ag is unclear. In approximately 20% of cases, initial treatment with ribavirin fails or a relapse of infection after the end of treatment can be observed [16]. Therefore, HEV Ag could potentially serve as a marker to control for successful treatment, although further patient trials are required to determine this. Previous reports indicate that HEV particles might circulate in the blood while wrapped by cellular membranes [14]. This may influence the performance of the anti-hev Ag specific ELISA and explain the differences observed between chronically and acutely infected individuals. By performing density gradients with or without the addition of a detergent, we showed that most HEV virions circulate as membrane-associated particles. However, we were unable to detect any significant differences between chronically and acutely infected individuals. We observed that viral RNA titers peaked at a density of approximately g/ml for enveloped particles and g/ml for nonenveloped particles. Intriguingly, we found, that the HEVAg content was detectable throughout all fractions of the gradients, suggesting the presence of distinct fragments of the viral capsid proteins with different densities. Therefore, we conclude, that the presence of ORF2 HEVAg does not necessarily correlate with infectious virions. The underlying mechanism for this observation still needs to be evaluated in future studies. In conclusion, we show for the first time differences in the level of HEV Ag in sera from individuals with acute versus those with chronic HEV genotype 3 infection, which could be diagnostically useful for the decision to initiate antiviral therapy. Additionally, we revealed that HEV virions likely circulate as membrane-associated particles. However, during infection, capsid structures without HEV RNA content can be found at different densities of sera from infected patients. Notes Financial support. This work was supported by the German Center for Infection Research clinical leave stipend to P. B.), the Helmholtz Center for Infection Research (to E. S.), and the German Ministry for Education and Research, through the German Indonesian Anti Infectives Cooperation (grant 16GW0105 to E. S.). Potential conflicts of interest. All authors: No reported conflicts. 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