The detection of HBsAg mutants expressed in vitro using two different quantitative HBsAg assays

The detection of HBsAg mutants expressed in vitro using two different quantitative HBsAg assays

Journal of Clinical Virology 54 (2012) 279–281 Contents lists available at SciVerse ScienceDirect Journal of Clinical Virology journal homepage: www...

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Journal of Clinical Virology 54 (2012) 279–281

Contents lists available at SciVerse ScienceDirect

Journal of Clinical Virology journal homepage:

Short communication

The detection of HBsAg mutants expressed in vitro using two different quantitative HBsAg assays Jens Verheyen a,∗ , Maria Neumann-Fraune a , Thomas Berg b , Rolf Kaiser a , Martin Obermeier b a b

Institute of Virology, University of Cologne, Cologne, Germany Medizinisches Labor Dr. Berg, Berlin, Germany

a r t i c l e

i n f o

Article history: Received 2 March 2012 Received in revised form 4 April 2012 Accepted 17 April 2012 Keywords: HBsAg Quantitative assays Mutants

a b s t r a c t Background: The quantification of hepatitis-B surface antigen (HBsAg) is useful to identify inactive carriers in chronically HBV infected patients and to predict interferon treatment outcome. Objective: To compare two quantitative HBsAg assays for the detection of HBsAg mutants expressed in vitro. Study design: HBsAg mutants (n = 35) were expressed in HuH7 cells and the supernatants were tested in two different quantitative HBsAg assays (Architect, Abbott and Elecsys, Roche). Results: All HBsAg mutants were detected by both assays, but in general the results of the Architect system were higher than those of the Elecsys system. The detection of HBsAg mutants in comparison to wild type was similar using both assays. However, HBsAg mutation T123A was under quantified by the Architect, whereas HBsAg mutations P142L, P142S and G145K yielded lower results in the Elecsys system. Conclusions: Even though HBsAg assays are optimised for the detection of HBsAg mutants, discrepant results were obtained for some HBsAg mutants in two quantitative HBsAg assays. These findings have to be considered when testing samples from one patient with two different quantitative HBsAg assays. © 2012 Elsevier B.V. All rights reserved.

1. Background Currently one third of the human population is thought to have been exposed to hepatitis-B virus (HBV) leading to about 400 million chronically infected patients worldwide. The presence of HBsAg, which is the surface protein of virions and subviral particles of HBV, indicates the infectious stage of patients in most cases. HBsAg mutants conferring immune escape to the neutralising anti-HBs have been found to be responsible for the failure of preventing HBV infection by vaccination.1 Even though the frequencies of HBsAg mutations largely varied in different studies, a great variety of HBsAg mutations has been detected worldwide. These mutations can also hamper the detection of HBsAg in routine diagnostic screening assays, since the detection of HBsAg in commercial assays depends on the binding of anti-HBs to the viral HBsAg. The detection of HBsAg is especially used for HBV screening, but most recently also the amount of HBsAg in the serum has been recognised as biomarker for prognosis and treatment response in chronic hepatitis B.2 The quantitative HBsAg assays in combination with HBV PCR are used to identify inactive carriers within chronically HBV-infected patients and to predict sustained virologic

∗ Corresponding author. Tel.: +49 221 478 7741; fax: +49 221 478 3904. E-mail address: [email protected] (J. Verheyen). 1386-6532/$ – see front matter © 2012 Elsevier B.V. All rights reserved.

response after the end of antiviral treatment. Therefore individualised treatment strategies of chronically HBV-infected patients might be applicable in the future.2 HBsAg levels are reported by all assays using international units per millilitre (IU/ml), which is suggestive of standardised results obtained by all assays. Indeed two recent studies found a good concordance between two assays for HBsAg quantification in clinical samples.3,4 However, these quantitative assays relay on the binding of specific sets of antibodies to the HBsAg during the capture and detection phases. Even though these sets of antibodies were optimised for detecting HBsAg mutants, it has remained unclear whether decreased binding affinities of HBsAg mutants might specifically influence the results of quantitative HBsAg assays. 2. Objective To compare two quantitative HBsAg assays for the detection of HBsAg mutants expressed in vitro. 3. Methods HBsAg wild type (genotype D) and mutants (n = 35) were expressed in HuH7 cells using an expression vector. The expression vector (078 pCHsAg An b-glob) was kindly provided by Prof. Ulrike Protzer (Technische Universität München, Munich, Germany). A


J. Verheyen et al. / Journal of Clinical Virology 54 (2012) 279–281

Table 1 Quantitative HBsAg levels of in vitro expressed HBsAg mutants using two different assays. Architect Abbott HBsAg (IU/ml) Wild type T118A P120G P120S P120T R122K T123A T126I T126V Q129G G130D G130E T131I T131K S132F Y134N K141E K141W P142L P142S S143L D144A D144E D144G D144H G145K G145R C147S F161L F161S E164D L173F V194F I195M W196L W196S

22.19 24.00 14.66 25.75 13.03 6.56 2.88 21.02 5.08 8.94 22.15 21.61 28.71 28.09 30.43 29.69 10.50 11.07 3.10 14.00 13.92 8.98 21.32 7.85 13.20 7.80 2.06 17.43 23.02 21.40 17.18 34.61 20.50 27.47 24.33 24.83

Elecsys Roche % (mutant/wt)

HBsAg (IU/ml)

% (mutant/wt)

108.16 66.07 116.04 58.72 29.56 12.98 94.73 22.89 40.29 99.82 97.39 129.38 126.59 137.13 133.80 47.32 49.89 13.97 63.09 62.73 40.47 96.08 35.38 59.49 35.15 9.28 78.55 103.74 96.44 77.42 155.97 92.38 123.79 109.64 111.90

11.47 11.69 5.06 7.96 4.13 3.38 9.42 10.69 2.53 5.33 9.81 10.93 16.01 15.80 15.81 15.97 8.03 5.24 0.50 3.23 5.89 5.45 9.36 4.91 9.46 1.43 1.05 13.44 12.87 9.37 8.11 16.23 10.81 13.84 12.30 13.83

101.87 44.12 69.36 35.96 29.50 82.14 93.15 22.06 46.50 85.50 95.31 139.54 137.77 137.82 139.21 69.99 45.64 4.32 28.14 51.32 47.54 81.62 42.80 82.43 12.51 9.16 117.14 112.21 81.71 70.66 141.50 94.21 120.63 107.23 120.54

panel of HBsAg mutants was generated by site-directed mutagenesis by modifying the wild type HBsAg gene (genotype D) in the expression vector. The selected HBsAg mutants were previously described in terms of immune and detection escape (HBsAg: 118–147) or in terms of HBV drug resistance (HBsAg: 161–196). The wild type and mutated HBsAg were tested by transfection of human hepatoma (HuH7) cells with pCHsAg plasmids using FuGene6 (Roche Diagnostics GmbH, Mannheim, Germany). The supernatants were collected after 72 h of incubation, and tested in the HBsAg quantification assays from Abbott (HBsAg, Architect) (detection limit: 0.05 IU/ml) and Roche (HBsAg II quant, Elecsys, Cobas 6000/e501) (detection limit: 0.05 IU/ml) without any dilution step. An aliquot of the same sample was tested in both systems to avoid any bias from the in vitro expression of HBsAg mutants. Since the level of HBsAg remained stable in repeatedly tested supernatants of the wild type, supernatants of each HBsAg mutant were tested once in each system. For each HBsAg mutant the relative amount of detected HBsAg was calculated in comparison to wild type HBsAg (HBsAg(mutant)/HBsAg(wt) × 100). 4. Results The supernatants obtained from cell culture expressing mutated and wild type HBsAg mutants were tested positive in both assays measuring between 1.05 IU/ml and 34.61 IU/ml of HBsAg (Table 1). However, the quantification of the HBsAg in the same supernatant yielded different results for all HBsAg mutants as well as for wild type comparing both assays. In general the results between both assays differed by the factor 2, so that most results of the Abbott Architect system were twice as high as the results of the Roche

Elecsys system. The negative controls consisting of cell culture medium without expressed HBsAg were tested negative in both assays. To compare the results from both systems for each HBsAg mutant the percent of the detected mutant HBsAg in relation to the wild type was calculated and compared between both assays. These standardised results showed high concordance in the detection of HBsAg mutants in comparison to the wild type. The relative detection rates of HBsAg mutants compared to wild type varied less than 20% for 24 of 35 mutants. Especially mutations in the HBsAg corresponding to mutations in the polymerase, that confer drug resistance, did not affect the detection of these mutants in both quantitative HBsAg assays. However, in four cases clear differences between both assays could be observed (Table 1, in bold). Whereas the HBsAg in the supernatant of HBV mutant T123A was quantified with 2.88 IU/ml (12.98% compared to wild type) in the Architect, the result in the Elecsys system was 9.42 IU/ml (82.14% compared to wild type). Vice versa results were obtained for the mutants P142L, P142S and G145K in the Architect system (3.10 IU/ml (13.97%), 14.00 IU/ml (63.09%), 7.80 IU/ml (35.15%)) and in the Elecsys system (0.50 IU/ml (4.32%), 3.23 IU/ml (28.14%), 1.43 IU/ml (12.51%)), respectively. 5. Discussion The level of HBsAg in the serum provides the transcriptional activity of HBV cccDNA and is used as biomarker in chronically infected patients with and without antiviral treatment. In this study the results of both quantitative HBsAg assays differed by the factor two for most HBsAg mutants and wild type. These discrepancies have not been reported previously, in contrast two studies reported

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a high concordance for both assays in testing clinical samples.3,4 Nevertheless in one of these studies the Elecsys HBsAgII assay tended to slightly detect higher levels of HBsAg than the Architect assay, especially in HBV genotype A-infected patients.4 Since in this study HBsAg mutants were expressed in vitro and measured in the cell culture supernatants matrix effects could not be excluded but were also not suggestive due to the negative controls. Whether or not the genetic background of genotype D in this study was responsible for the discrepant results warrants further analyses. However, it should be noted that all detected levels of HBsAg in the supernatants were in the lower detection range of both assays, which might partly explain the observed differences in this study. In contrast, the relative detection rates of HBsAg mutants (HBsAg(mutant)/HBsAg(wild type)) using both assays provided highly comparable results for the majority of tested HBsAg mutants. Therefore, the detection of most HBsAg mutants was comparable to wild type in both assays. Clearly distinguished detection rates were observed for the relative detection rates of four HBsAg mutants. Whereas mutant T123A seemed to be underquantified in the Architect system, mutants P142S, P142L and G145K yielded lower results using the Elecsys system. The underquantification of HBsAg mutant T123A by the Architect did not surprise since this mutation was recently found to be responsible for false negative results in clinical samples tested with the Architect HBsAg (Qual) assay.5 The HBsAg mutant T123A expressed in vitro was in fact tested positive in this study using the Architect assay but was concordantly less well detected than with the Elecsys system. Even though this mutation was initially selected in HBV-HIV-1 coinfected patients infected with HBV genotype E, the conformation of the HBsAg seemed to be significantly changed in the background of genotype E and genotype D. Accordingly HBsAg levels of mutants P142S, P142L and G145K seemed to be underrated by the Roche system, even though the detection of these mutants also detected in clinical HBV isolates by the qualitative Roche HBsAg assay has been proven previously.6,7 Even though the prevalence of most tested HBsAg mutants was low (<1%) in clinical HBV isolates, the overall number of HBV isolates carrying at least one HBsAg mutation ranged from 5% to 40% in three European studies.8–10 Distinguished detection rates of HBsAg mutants by two quantitative HBsAg assays might lead to divergent results, which would not hold true when testing these samples with the same assay. Discrepant results of quantitative HBsAg assays for detecting a minority of HBsAg mutants is neither surprising nor a major draw back for routine diagnostics. However, it has to be considered that


discrepant results using two different quantitative HBsAg assays for the same sample might partly be explained by the variability of HBV in the HBsAg. Funding This study was funded by a grant of BMBF (HOPE-project, AZ: 01ES0822). Competing interests None. Ethical approval Not required. References 1. Carman WF. The clinical significance of surface antigen variants of hepatitis B virus. J Viral Hepat 1997;4(Suppl. 1):11–20. 2. Chan HL, Thompson A, Martinot-Peignoux M, Piratvisuth T, Cornberg M, Brunetto MR, et al. Hepatitis B surface antigen quantification: why and how to use it in 2011—a core group report. J Hepatol 2011;55:1121–31. 3. Sonneveld MJ, Rijckborst V, Boucher CA, Zwang L, Beersma MF, Hansen BE, et al. A comparison of two assays for quantification of hepatitis B surface antigen in patients with chronic hepatitis B. J Clin Virol 2011;51:175–8. 4. Wursthorn K, Jaroszewicz J, Zacher BJ, Darnedde M, Raupach R, Mederacke I, et al. Correlation between the Elecsys HBsAg II assay and the Architect assay for the quantification of hepatitis B surface antigen (HBsAg) in the serum. J Clin Virol 2011;50:292–6. 5. Geretti AM, Patel M, Sarfo FS, Chadwick D, Verheyen J, Fraune M, et al. Detection of highly prevalent hepatitis B virus coinfection among HIV-seropositive persons in Ghana. J Clin Microbiol 2010;48:3223–30. 6. Jia JD, Hong M, Wei L, Zhang XX, Mao YL, Wang LL, et al. Multicentre evaluation of the Elecsys hepatitis B surface antigen II assay for detection of HBsAg in comparison with other commercially available assays. Med Microbiol Immunol 2009;198:263–9. 7. Muhlbacher A, Weber B, Burgisser P, Eiras A, Cabrera J, Louisirirotchanakul S, et al. Multicenter study of a new fully automated HBsAg screening assay with enhanced sensitivity for the detection of HBV mutants. Med Microbiol Immunol 2008;197:55–64. 8. Avellon A, Echevarria JM. Frequency of hepatitis B virus ‘a’ determinant variants in unselected Spanish chronic carriers. J Med Virol 2006;78:24–36. 9. Servant-Delmas A, Mercier M, El Ghouzzi MH, Girault A, Bouchardeau F, Pillonel J, et al. National survey of hepatitis B virus (HBV) polymorphism in asymptomatic HBV blood donors from 1999 to 2007 in France. Transfusion (Paris) 2010;50:2607–18. 10. Roque-Afonso AM, Ferey MP, Ly TD, Graube A, Costa-Faria L, Samuel D, et al. Viral and clinical factors associated with surface gene variants among hepatitis B virus carriers. Antivir Ther 2007;12:1255–63.