A new role for an old marker, HBsAg

Abstract

Hepatitis B surface antigen levels during the natural history of chronic hepatitis B: A perspective on AsiaJournal of HepatologyVol. 52Issue 4PreviewData from clinical trials suggest a potential role for on-treatment monitoring of serum HBsAg titres during interferon-alpha (pegIFN) therapy in predicting virological responses. However, baseline HBsAg titres during the natural history of chronic hepatitis B (CHB) have not been well-characterized. We aimed to define the serum HBsAg titres during the different phases of CHB in a cohort of Asian patients infected with either genotype B or C HBV. Full-Text PDF Hepatitis B surface antigen (HBsAg) levels in the natural history of hepatitis B virus (HBV)-infection: A European perspectiveJournal of HepatologyVol. 52Issue 4PreviewThe quantifiable level of HBsAg has been suggested as a predictor of treatment response in chronic hepatitis B. However, there is limited information on HBsAg levels considering the dynamic natural course of HBV-infection. This study aimed to determine HBsAg levels in the different phases of HBV-infection in European HBsAg-positive patients. Full-Text PDF Detection in the serum of the “Australia antigen”, namely hepatitis B surface antigen (HBsAg), was the Nobel prize discovery that identified hepatitis B virus (HBV) about 40 years ago; to this day HBsAg remains the hallmark of overt HBV infection [1Blumberg B.S. Alter H.J. Visnich S. A “new” antigen in leukemia sera.JAMA. 1965; 191: 541-546Crossref PubMed Scopus (899) Google Scholar, 2Rotman Y. Brown A.T. Hoofnagle J.H. Evaluation of the patient with hepatitis B.Hepatology. 2009; 49: S22-S27Crossref PubMed Scopus (38) Google Scholar]. HBsAg circulates in a wide array of particulate forms: competent virions (42 nm, Dane particles), 20 nm diameter filaments of variable length, and 20–22 nm spherical defective particles, corresponding to empty viral envelopes [[3]Seeger C. Mason W.S. Hepatitis B virus biology.Microbiol Mol Biol Rev. 2000; : 51-68Crossref PubMed Scopus (1188) Google Scholar]. Serum HBsAg results from the different combinations of three proteins (small, medium and large), either glycosylated or not, that are specified by a single open reading frame providing 3 carboxy-terminal colinear HBsAg proteins of different length. The small (S) protein (226 amino acids) is expressed at the highest levels, predominates in both virions and subviral particles and is secreted without cleavage of amino acid residues during translocation because of its self-assembling capacity with host-derived lipids in the cell ER [[4]Simon K. Lingappa V.R. Ganem D. Secreted hepatitis B surface antigen polypeptides are derived from transmembrane precursor.J Cell Biol. 1988; 107: 2163-2168Crossref PubMed Scopus (78) Google Scholar]. The middle (M) protein (containing 55 extra residues of the pre-S2 domain) is regulated by the same promoter and is similarly secreted, whereas the transcription of the large protein (L) is regulated by a specific but weaker promoter (pre-S1) [[5]De Medina T. Kaktor O. Shaul Y. The S promoter of HBV is regulated by positive and negative elements.Mol Cell Biol. 1988; : 2449-2455PubMed Google Scholar]. Hepatitis B virus large surface protein (L-HBs) containing both the pre-S2 region and the 108–119 additional residues of the pre-S1 domain, is an essential component of both virions and filaments, and represents 10–20% of their envelope proteins. In contrast, the L-HBs represents only 2% of the 22 nm spherical particles [6Stibbe W. Gerlich W.H. Structural relationship between minor and major proteins of hepatitis B surface antigen.J Virol. 1983; 46: 626-628Crossref PubMed Google Scholar, 7Heermann K.H. Goldmann U. Schwartz W. Seyffarth T. Baumgarten H. Gerlich W.H. Large surface proteins of HBV containing the Pre-s sequence.J Virol. 1984; : 396-402PubMed Google Scholar]. The complexity of HBsAg production and secretion is known since the early studies that showed a larger excess of both filaments and spherical subviral particles was present in highly viremic HBeAg positive carriers as compared to low viremic anti-HBe positive carriers, in whom the decline of filaments paralleled that of virions whereas spherical particles remained in moderate excess [8Dienes H.P. Gerlich W.H. Worsdorfer M. Gerken G. Bianchi L. Hess G. et al.Hepatic expression patterns of the large and middle hepatitis B surface proteins in viremic and non viremic chronic hepatitis B.Gastroenterology. 1990; 98: 1017-1023Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 9Lau J.Y.N. Bain V.G. Davies S.E. Alexander G.J.M. Williams R. Export of intracellular HBsAg in chronic hepatitis B virus infection is related to viral replication.Hepatology. 1991; 14: 416-421Crossref PubMed Scopus (33) Google Scholar]. Thus, subviral HBsAg particles exceed virions by a variable factor of 102–105 and can accumulate up to concentrations of several hundred micrograms per milliliter of serum [[3]Seeger C. Mason W.S. Hepatitis B virus biology.Microbiol Mol Biol Rev. 2000; : 51-68Crossref PubMed Scopus (1188) Google Scholar]. Quantification of HBsAg was introduced more than 20 years ago, but only recently has it been significantly improved by new automated quantitative assays [[10]Nguyen T. Desmond P. Locarnini S. The role of quantitative hepatitis B serology in the natural history and management of chronic hepatitis B.Hepatol Int. 2009; 3: S5-S15Crossref PubMed Scopus (47) Google Scholar]. Several studies suggest a new potential role of quantitative serum HBsAg in the prediction of virological response to antiviral therapy, at least in Peg-interferon treated patients [11Manesis E.K. Hadziyannis E.S. Angelopoulou O.P. Hadziyannis S.J. Prediction of treatment-related HBsAg loss in HBeAg-negative chronic hepatitis B: a clue from serum HBsAg levels.Antivir Ther. 2007; 12: 73-82PubMed Google Scholar, 12Brunetto M.R. Moriconi F. Bonino F. Lau G.K. Farci P. Yurdayin C. et al.Hepatitis B virus surface antigen levels: a guide to sustained response to peginterferon alfa-2a in HBeAg-negative chronic hepatitis B.Hepatology. 2009; 49: 1141-1150Crossref PubMed Scopus (396) Google Scholar, 13Moucari R. Mackiewicz V. Lada O. Ripault M.P. Castelnau C. Mrtinot-Peignoux M. et al.Early serum HBsAg drop: a strong predictor of sustained virological response to pegylated interferon alfa-2a in HBeAg-negative patients.Hepatology. 2009; 49: 1151-1157Crossref PubMed Scopus (389) Google Scholar]. HBsAg appears useful to identify non-responders as early as 12–24 weeks after the beginning of treatment and to tailor treatment duration in responders [12Brunetto M.R. Moriconi F. Bonino F. Lau G.K. Farci P. Yurdayin C. et al.Hepatitis B virus surface antigen levels: a guide to sustained response to peginterferon alfa-2a in HBeAg-negative chronic hepatitis B.Hepatology. 2009; 49: 1141-1150Crossref PubMed Scopus (396) Google Scholar, 13Moucari R. Mackiewicz V. Lada O. Ripault M.P. Castelnau C. Mrtinot-Peignoux M. et al.Early serum HBsAg drop: a strong predictor of sustained virological response to pegylated interferon alfa-2a in HBeAg-negative patients.Hepatology. 2009; 49: 1151-1157Crossref PubMed Scopus (389) Google Scholar]. The correlations between HBsAg and HBV-DNA kinetics are complex and variable in the different treatment settings; the kinetics of the two parameters are dissociated in lamivudine treated patients and relapsers to Peg-interferon, but parallel in sustained responders to Peg-interferon [12Brunetto M.R. Moriconi F. Bonino F. Lau G.K. Farci P. Yurdayin C. et al.Hepatitis B virus surface antigen levels: a guide to sustained response to peginterferon alfa-2a in HBeAg-negative chronic hepatitis B.Hepatology. 2009; 49: 1141-1150Crossref PubMed Scopus (396) Google Scholar, 13Moucari R. Mackiewicz V. Lada O. Ripault M.P. Castelnau C. Mrtinot-Peignoux M. et al.Early serum HBsAg drop: a strong predictor of sustained virological response to pegylated interferon alfa-2a in HBeAg-negative patients.Hepatology. 2009; 49: 1151-1157Crossref PubMed Scopus (389) Google Scholar]. Preliminary reports on HBsAg and HBV-DNA serum levels in untreated acute and chronic hepatitis B cases confirm such a discrepancy, and little is known about their relative variations along the highly dynamic chronic HBV infection [[14]Kuhns M. Busch M. New strategies for blood donor screening for hepatitis B virus: nucleic acid testing versus immunoassay methods.Mol Diagn Ther. 2006; 10: 77-91Crossref PubMed Scopus (78) Google Scholar]. In this issue of the Journal, two manuscripts provide new insights into serum HBsAg levels during chronic HBV infection in Asian and European cohorts of HBV carriers. The novelty of the works of Nguyen et al. [[15]Nguyen T. Thompson A.J.V. Bowden S. Croagh C. Bell S. Desmond P.V. et al.Hepatitis B surface antigen levels during the natural history of chronic hepatitis B: A perspective on Asia.J Hepatol. 2010; 52: 508-513Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar] and Jaroszewicz et al. [[16]Jaroszewicz J. Serrano B.C. Wursthorn K. Deterding K. Schlue J. Raupach R. et al.Hepatitis B surface antigen (HBsAg) levels in the natural history of hepatitis B virus (HBV)-infection: a European perspective.J Hepatol. 2010; 52: 514-522Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar] stems from their study on the correlations between HBsAg serum levels and the clinical and virologic features of chronic HBV carriers analysed in different phases of infection according to the most recent criteria. Overall, 434 chronic carriers were studied: 62 immune-tolerant carriers (IT), 103 HBeAg positive patients in the immune-clearance phase (IC), 118 HBeAg negative carriers in the non-/low replicative phase (LC), and 151 patients with HBeAg negative hepatitis (ENH). Two major findings are common in the two studies: (1) median HBsAg levels differ significantly during the 4 phases of HBV infection and decline progressively from IT (4.5–4.96 log 10 IU/ml in Asian and European carriers) to LC (2.86–3.09 log 10 IU/ml in Asian and European carriers); (2) HBsAg/HBV-DNA ratios are significantly higher in LC (1.05 Asian–1.17 European) as compared to all the others patients (ratios range 0.55–0.64). These findings entail that HBsAg secretion is highly dynamic and varies along chronic HBV infection both quantitatively and qualitatively. Accordingly, one intriguing and complex issue remains the correlation between HBsAg and HBV-DNA serum levels. In spite of an overall correlation in the European cohort (R = 0.75, p < 0.001), the two parameters show weaker or absent correlations when the different phases of HBV infection are analysed separately or by HBV genotype. A negative correlation is reported in genotype A HBeAg positive (IC, R = −0.24) and negative (ENH, R = −0.07) patients, a positive relation in genotype B, C and D ENH, in genotype B and C IT and in genotype A and D LC, but all the correlation coefficients are poor (R ranging from 0.29 to 0.57). On the contrary, in genotype B and C HBeAg positive patients (IC) HBsAg and HBV-DNA serum levels correlate significantly (p = 0.0001) and with better coefficient (R = 0.77), as well as in 12 European patients with acute hepatitis B (R = 0.79). These data suggest direct interactions between HBV genotype, HBsAg serum level and viral load. Accordingly, genotype-specific patterns of expression of intracellular and extracellular viral DNA and antigens were shown by transfection of Huh7 [[17]Sugiyama M. Tanaka Y. Kato T. Orito E. Ito K. Acharya S.K. et al.Influence of hepatitis B virus genotypes on the intrahepatic and extracellular expression of viral DNA and antigens.Hepatology. 2006; 44: 915-924Crossref PubMed Scopus (227) Google Scholar]. In vitro, genotype A showed a sharp dissociation between HBsAg and HBV-DNA production, with the highest HBsAg secretion combined with the lowest HBV-DNA production in the culture medium [[17]Sugiyama M. Tanaka Y. Kato T. Orito E. Ito K. Acharya S.K. et al.Influence of hepatitis B virus genotypes on the intrahepatic and extracellular expression of viral DNA and antigens.Hepatology. 2006; 44: 915-924Crossref PubMed Scopus (227) Google Scholar]. Larger studies are needed to address the correlations between HBV genotypes and the dynamics of HBsAg serum levels during the different phases of HBV infection. In spite of the possible limitation of genotype interference, it is interesting to note that HBsAg and HBV-DNA serum levels showed their highest correlation in the early phases of immune clearance, namely in acute hepatitis B and in HBeAg positive CHB (IC) [15Nguyen T. Thompson A.J.V. Bowden S. Croagh C. Bell S. Desmond P.V. et al.Hepatitis B surface antigen levels during the natural history of chronic hepatitis B: A perspective on Asia.J Hepatol. 2010; 52: 508-513Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 16Jaroszewicz J. Serrano B.C. Wursthorn K. Deterding K. Schlue J. Raupach R. et al.Hepatitis B surface antigen (HBsAg) levels in the natural history of hepatitis B virus (HBV)-infection: a European perspective.J Hepatol. 2010; 52: 514-522Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar]. These findings, together with the parallel kinetics of HBsAg and HBV-DNA in patients responding to Peg-IFN treatment [12Brunetto M.R. Moriconi F. Bonino F. Lau G.K. Farci P. Yurdayin C. et al.Hepatitis B virus surface antigen levels: a guide to sustained response to peginterferon alfa-2a in HBeAg-negative chronic hepatitis B.Hepatology. 2009; 49: 1141-1150Crossref PubMed Scopus (396) Google Scholar, 13Moucari R. Mackiewicz V. Lada O. Ripault M.P. Castelnau C. Mrtinot-Peignoux M. et al.Early serum HBsAg drop: a strong predictor of sustained virological response to pegylated interferon alfa-2a in HBeAg-negative patients.Hepatology. 2009; 49: 1151-1157Crossref PubMed Scopus (389) Google Scholar], suggest that virion and HBsAg antigen production correlate better when they are concordantly inhibited by a strong immune-response resulting in the efficient control of viral replication, namely HBeAg/anti-HBe or HBsAg/anti-HBs seroconversion. If the immune clearance does not succeed in the complete control of HBV infection, leading to HBsAg clearance and anti-HBs seroconversion, HBV infection evolves into the low replicative phase or HBeAg negative CHB. In both conditions, the discrepancy between HBV-DNA and HBsAg production may increase for several reasons but it is primarily due to the production of defective particles that outnumber the virions during the low replicative phase, as already shown in the older studies, where lower amounts of serum L-HBs (the hallmark of the surface protein of the virion and filaments) were found in non-viremic as compared to viremic HBV carriers [8Dienes H.P. Gerlich W.H. Worsdorfer M. Gerken G. Bianchi L. Hess G. et al.Hepatic expression patterns of the large and middle hepatitis B surface proteins in viremic and non viremic chronic hepatitis B.Gastroenterology. 1990; 98: 1017-1023Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 9Lau J.Y.N. Bain V.G. Davies S.E. Alexander G.J.M. Williams R. Export of intracellular HBsAg in chronic hepatitis B virus infection is related to viral replication.Hepatology. 1991; 14: 416-421Crossref PubMed Scopus (33) Google Scholar]. The higher HBsAg/HBV-DNA ratios found by Nguyen and Jaroszewicz in LC are consistent with the previous data, suggesting a relatively lower production of virions versus subviral HBs antigens in this subset of carriers. This might depend on a stronger inhibition of pre-genome transcription from intrahepatic cccDNA as compared to the envelope protein mRNAs, or to the defective secretion of virions as a consequence of the emergence of HBV mutants, which are selected by the long lasting immune pressure on the HBsAg gene that may deregulate HBsAg expression with an asymmetric production of S, M and L proteins [18Wang H.-C. Wu H.-C. Chen C.-F. Fausto N. Lei H.-Y. Su I.-J. Different types of ground glass hepatocytes in chronic hepatitis B virus infection contains specific Pre-S mutants that may induce endoplasmic reticulum stress.Am J Pathol. 2003; 163: 2441-2449Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 19Khan N. Guarnieri M. Ahn S.H. Li J. Zhou Y. Bang G. et al.Modulation of hepatitis B virus secretion by naturally occurring mutations in the S gene.J Virol. 2004; : 3262-3270Crossref PubMed Scopus (64) Google Scholar]. Indeed an inverse relationship was reported between serum HBsAg levels and the intrahepatic cytoplasmic storage of HBsAg (ground glass cells) [[14]Kuhns M. Busch M. New strategies for blood donor screening for hepatitis B virus: nucleic acid testing versus immunoassay methods.Mol Diagn Ther. 2006; 10: 77-91Crossref PubMed Scopus (78) Google Scholar]. In fact, overexpression of L-HBs can cause S protein to accumulate in the Golgi complex and to inhibit the secretion of HBsAg particles, as a proper stoichiometry between L and S proteins is required for the secretion of HBsAg and virions [18Wang H.-C. Wu H.-C. Chen C.-F. Fausto N. Lei H.-Y. Su I.-J. Different types of ground glass hepatocytes in chronic hepatitis B virus infection contains specific Pre-S mutants that may induce endoplasmic reticulum stress.Am J Pathol. 2003; 163: 2441-2449Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 19Khan N. Guarnieri M. Ahn S.H. Li J. Zhou Y. Bang G. et al.Modulation of hepatitis B virus secretion by naturally occurring mutations in the S gene.J Virol. 2004; : 3262-3270Crossref PubMed Scopus (64) Google Scholar]. Finally a role of integrated HBV-DNA in the alteration of HBsAg secretion was also advocated [[20]Huang Z.-M. Yen B.T.S. Dysregulated surface gene expression from disrupted hepatitis B virus genomes.J Virol. 1993; : 7032-7040PubMed Google Scholar]. In conclusion, HBsAg serum levels are the resultant of the complex equilibrium between the virus and the host’s immune system as well as the product of the transcription of specific mRNAs rather than viral replication. Thus, we may speculate that serum HBsAg is the indirect expression of transcriptionally active cccDNA rather than total intrahepatic cccDNA. Of course, we cannot exclude a role of integrated HBV-DNA in HBsAg synthesis; however, the evidence that HBsAg serum levels decline significantly with the duration of HBV infection, when the amount of integrated HBV-DNA is supposed to increase, weakens such a hypothesis. The substantial variations of total serum HBsAg in the different phases of HBV infection proposes quantitative HBsAg as a new diagnostic tool for the characterization of the HBV carrier in combination with HBV-DNA. The two HBV markers providing complementary information on the status of HBV infection (Fig. 1) may be very useful in clinical practice to define the specific condition of the single HBV carrier during the highly dynamic phases of chronic HBV infection, just as latitude and longitude allow to define the ship’s position in the ocean. This will be of paramount importance to avoid the misclassification of an asymptomatic HBeAg negative CHB patient as an inactive carrier because of a single point serum test with normal transaminases and negative HBV-DNA caused by the typical intermittent disease profile of HBeAg negative CHB.