Chance and necessity of simultaneous HBsAg and anti-HBs detection in the serum of chronic HBsAg carriers
Authors: Maurizia Rossana Brunetto
Journal: Journal of Hepatology (2013)

N-glycosylation mutations within hepatitis B virus surface major hydrophilic region contribute mostly to immune escapeJournal of HepatologyVol. 60Issue 3PreviewHBV immune escape represents a challenge to prevention, diagnosis, and treatment of hepatitis B. Here, we analyzed the molecular and clinical characteristics of HBV immune escape mutants in a Chinese cohort of chronically infected patients. Full-Text PDF Patients who recover from acute hepatitis B clear surface antigen (HBsAg) from serum and anti-HBs becomes detectable as hallmark of HBV immune control. However, anti-HBs appear weeks after HBsAg clearance and neutralizing antibodies are unlikely to contribute to early viral control but to prevent viral spread from cells that remain infected after hepatitis resolution [[1]Rehermann B. Immune response in hepatitis B virus infection.Semin Liver Dis. 2003; 23: 21-23Crossref PubMed Scopus (97) Google Scholar]. This notion is supported by the evidence that chimpanzees resolving hepatitis B are protected from viral rechallenge [[2]Moss B. Smith G.L. Gerin J.L. Purcell R.H. Live recombinant vaccinia virus protects chimpanzees against hepatitis B.Nature. 1984; 311: 67-69Crossref PubMed Scopus (171) Google Scholar]. Similarly, in non-infected individuals an adequate anti-HBs production (>10 mUI/ml) indicates a protective immunity after vaccination [[1]Rehermann B. Immune response in hepatitis B virus infection.Semin Liver Dis. 2003; 23: 21-23Crossref PubMed Scopus (97) Google Scholar]. Several evidences suggest that anti-HBs are present also in chronic HBsAg carriers, but not detected because hidden in circulating immune-complexes [[3]Dienstag J.L. Concurrent hepatitis B surface antigen and antibody and the clonal selection theory of antibody diversity.Gastroenterology. 1987; 93: 899-904Abstract Full Text PDF PubMed Google Scholar]. Thus the simultaneous detection of HBsAg/anti-HBs (HBsAg/anti-HBs-co-detection) represents an atypical serologic profile puzzling virologists and clinicians since the late 1970s [3Dienstag J.L. Concurrent hepatitis B surface antigen and antibody and the clonal selection theory of antibody diversity.Gastroenterology. 1987; 93: 899-904Abstract Full Text PDF PubMed Google Scholar, 4Gerlich W.H. The enigma of concurrent Hepatitis, B surface Antigen (HBsAg) and antibodies to HBsAg.Clin Infect Dis. 2007; 44: 1170-1172Crossref PubMed Scopus (30) Google Scholar, 5Pondé R.A. The underlying mechanisms for the "simultaneous HBsAg and anti-HBs serological profile".Eur J Clin Microbiol Infect Dis. 2011; 30 ([Epub 2011 Apr 12]): 1325-1340https://doi.org/10.1007/s10096-011-1240-zCrossref PubMed Scopus (33) Google Scholar]. Initially, it was explained by heterologous antibodies against subtypes different from the infecting HBV subtype, as HBsAg subtypes d/y infections could associate with y/d anti-HBs subtypes [[6]Tabor E. Gerety R.J. Smallwood L.A. Barker L.F. Coincident hepatitis B surface antigen and antibodies of different subtypes in human serum.J Immunol. 1997; 118: 369-370Google Scholar]. The hypothesis of primary infection with 2 subtypes followed by clearance of only one of them was contradicted by the animal model where humoral response against the a determinant of HBsAg prevented the re-infection with different HBV subtypes [[3]Dienstag J.L. Concurrent hepatitis B surface antigen and antibody and the clonal selection theory of antibody diversity.Gastroenterology. 1987; 93: 899-904Abstract Full Text PDF PubMed Google Scholar]. In addition, ay anti-HBs was found in HBsAg carriers from Japan, where this subtype is uncommon [[7]Hayashi J. Noguchi A. Nakashima K. Morofuji M. Kashiwagi S. Frequency of concurrence of hepatitis B surface antigen and antibody in a large number of carriers in Okinawa, Japan.Gastroenterol Jpn. 1990; 25: 593-597PubMed Google Scholar]. Therefore, the hypothesis of a mixed infection was dismissed in favour of clonal selection of antibody diversity [[3]Dienstag J.L. Concurrent hepatitis B surface antigen and antibody and the clonal selection theory of antibody diversity.Gastroenterology. 1987; 93: 899-904Abstract Full Text PDF PubMed Google Scholar]. The high similarity between 2 HBV serotypes (i.e., HBsAg/ad and HBsAg/ay) could induce the clonal expansion not only of high-titre, high-affinity anti-a and anti-d, but also lower-titre, low-affinity heterotypic anti-y [3Dienstag J.L. Concurrent hepatitis B surface antigen and antibody and the clonal selection theory of antibody diversity.Gastroenterology. 1987; 93: 899-904Abstract Full Text PDF PubMed Google Scholar, 4Gerlich W.H. The enigma of concurrent Hepatitis, B surface Antigen (HBsAg) and antibodies to HBsAg.Clin Infect Dis. 2007; 44: 1170-1172Crossref PubMed Scopus (30) Google Scholar]. Nevertheless, the question remains why only a minority of HBsAg carriers exhibit this condition. After the introduction of molecular biology techniques the characterization of viral quasispecies in clinical settings of HBV infections despite vaccination or HBIg treatment showed that HBsAg/anti-HBs-co-detection was associated with viral strains carrying HBsAg mutations that escaped the anti-HBs neutralization [8Carman W.F. Zanetti A.R. Karayiannis P. Waters J. Manzillop G. Tanzi E. et al.Vaccine-induced escape mutant of hepatitis B virus.Lancet. 1990; 336: 325-329Abstract PubMed Scopus (936) Google Scholar, 9Alavian S.M. Carman W.F. Jazayeri S.M. HBsAg variants: diagnostic-escape and diagnostic dilemma.J Clin Virol. 2013; 57: 201-208Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar]. These findings raise the question whether genetic heterogeneity of HBV and particularly of the S gene could also be present in other HBsAg/anti-HBs carriers.Serum HBsAg results from one open reading frame providing 3 carboxy-terminal collinear proteins of different length (small, medium, and large, SHBs, MHBs, LHBs). In addition to the S gene MHBs contains pre-S2 sequence (55 aa) and LHBs preS1 (108/119 aa depending on subtype) and preS2 sequences. PreS1 and PreS2 and the aa 100–169 domain of SHBs (Major Hydrophilic Region, MHR) are exposed on viral particles and highly immunogenic. The studies of HBsAg variants focused mainly on SHBs, because it is expressed at the highest levels, predominates in both virions and subviral particles and contains within MHR the "a" determinant (aa 124–147), main target of neutralizing antibodies. Two major loops are present in the "a" determinant (the 1st at aa 124–137; the 2nd at aa 139–147), defined by multiple potential disulfide bridges, between aa 139/147 (or 149) and 121/124; conserved cysteines at 124, 137, 139, and 149 are essential for conformation and antigenicity [[9]Alavian S.M. Carman W.F. Jazayeri S.M. HBsAg variants: diagnostic-escape and diagnostic dilemma.J Clin Virol. 2013; 57: 201-208Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar]. MHR is the principal anti-HBs binding site after natural infection, immunisation and HBIg prophylaxis, although areas up and downstream may also be important in neutralisation. Initially characterized vaccine and HBIg escape mutants showed glycine to arginine substitutions at position 145 (G145R); later other mutations between 120 and 208 were described in patients with active or passive immune-prophylaxis or CHB [[10]Yamamoto K. Horikita M. Tsuda F. Itoh K. Yoshihiro A. Yotsumoto S. et al.Mayumi M Naturally occurring escape mutants of hepatitis B virus with various mutations in the carriers seropositive for antibody to hepatitis B surface antigen.J Virol. 1994; 68: 2671-2676PubMed Google Scholar].Overall, in both Western and Asian cohorts a significantly higher S gene variability, particularly of MHR with higher number of aa substitutions was found in HBsAg/anti-HBs positive patients than in controls [11Lada O. Benhamou Y. Poynard T. Thibault V. Coexistence of hepatitis B surface antigen (HBs Ag) and anti-HBs antibodies in chronic hepatitis B virus carriers: influence of 'a' determinant variants.J Virol. 2006; 80: 2968-2975Crossref PubMed Scopus (134) Google Scholar, 12Colson P. Borentain P. Motte A. Henry M. Moal V. Botta-Fridlund D. et al.Clinical and virological significance of the co-existence of HBsAg and anti-HBs antibodies in hepatitis B chronic carriers.Virology. 2007; 367: 30-40Crossref PubMed Scopus (63) Google Scholar, 13Chen Y. Qian F. Yuan Q. Li X. Wu W. Guo X. et al.Mutations in hepatitis B virus DNA from patients with coexisting HBsAg and anti-HBs.J Clin Virol. 2011; 52: 198-203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 14Liu W. Hu T. Wang X. Chen Y. Huang M. Yuan C. et al.Coexistence of hepatitis B surface antigen and anti-HBs in Chinese chronic hepatitis B virus patients relating to genotype C and mutations in the S and P gene reverse transcriptase region.Arch Virol. 2012; 157: 627-634Crossref PubMed Scopus (32) Google Scholar, 15Shi Y. Wei F. Hu D. Li Q. Smith D. Li N. et al.Mutations in the major hydrophilic region (MHR) of hepatitis B virus genotype C in North China.J Med Virol. 2012; 84: 1901-1906Crossref PubMed Scopus (22) Google Scholar, 16Yu D.-M. Li X.-H. Mom V. Lu Z.-H. Liao X.-W. Han Y. Pichoud C. et al.N-glycosylation mutation within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.J Hepatol. 2014; 60: 515-522Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. Two French studies showed in the same patients a significantly higher number of aa changes in the "s" gene (aa substitution/100 aa: 4.55 vs. 1.66), even more evident in MHR (5.71–4.68 vs. 1.43–2.07) and "a" determinant (9.52–11.00 vs. 2.43–2.48). Aa substitutions were significantly more frequent at positions 120, 126, 129, 130, 144, and 165; G145R/T was the most frequent [11Lada O. Benhamou Y. Poynard T. Thibault V. Coexistence of hepatitis B surface antigen (HBs Ag) and anti-HBs antibodies in chronic hepatitis B virus carriers: influence of 'a' determinant variants.J Virol. 2006; 80: 2968-2975Crossref PubMed Scopus (134) Google Scholar, 12Colson P. Borentain P. Motte A. Henry M. Moal V. Botta-Fridlund D. et al.Clinical and virological significance of the co-existence of HBsAg and anti-HBs antibodies in hepatitis B chronic carriers.Virology. 2007; 367: 30-40Crossref PubMed Scopus (63) Google Scholar]. These findings suggested that emergence of "a" determinant variants could favour viral escape from immune neutralization. Most of Asian studies confirmed the European findings except for a higher prevalence of aa changes in the 1st loop (aa 124–137) of "a" determinant than in the 2nd (aa 137–145) [13Chen Y. Qian F. Yuan Q. Li X. Wu W. Guo X. et al.Mutations in hepatitis B virus DNA from patients with coexisting HBsAg and anti-HBs.J Clin Virol. 2011; 52: 198-203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 14Liu W. Hu T. Wang X. Chen Y. Huang M. Yuan C. et al.Coexistence of hepatitis B surface antigen and anti-HBs in Chinese chronic hepatitis B virus patients relating to genotype C and mutations in the S and P gene reverse transcriptase region.Arch Virol. 2012; 157: 627-634Crossref PubMed Scopus (32) Google Scholar, 15Shi Y. Wei F. Hu D. Li Q. Smith D. Li N. et al.Mutations in the major hydrophilic region (MHR) of hepatitis B virus genotype C in North China.J Med Virol. 2012; 84: 1901-1906Crossref PubMed Scopus (22) Google Scholar]. When HBV genotype C infected individuals were analysed the classic G145R substitution was significantly less frequent, with prevalent mutations at aa 126, 129, 130, 131, 133, and 134, confirming a major influence of genotype on selection of viral quasispecies [13Chen Y. Qian F. Yuan Q. Li X. Wu W. Guo X. et al.Mutations in hepatitis B virus DNA from patients with coexisting HBsAg and anti-HBs.J Clin Virol. 2011; 52: 198-203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 14Liu W. Hu T. Wang X. Chen Y. Huang M. Yuan C. et al.Coexistence of hepatitis B surface antigen and anti-HBs in Chinese chronic hepatitis B virus patients relating to genotype C and mutations in the S and P gene reverse transcriptase region.Arch Virol. 2012; 157: 627-634Crossref PubMed Scopus (32) Google Scholar, 15Shi Y. Wei F. Hu D. Li Q. Smith D. Li N. et al.Mutations in the major hydrophilic region (MHR) of hepatitis B virus genotype C in North China.J Med Virol. 2012; 84: 1901-1906Crossref PubMed Scopus (22) Google Scholar]. However, a potential role of immune suppression and antiviral therapy in European patients cannot be ruled out. Interestingly, some studies showed a significant variability of S gene at its CTL epitope (aa 87–95) that might contribute to HBV neutralization failure [[13]Chen Y. Qian F. Yuan Q. Li X. Wu W. Guo X. et al.Mutations in hepatitis B virus DNA from patients with coexisting HBsAg and anti-HBs.J Clin Virol. 2011; 52: 198-203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar]. The major mechanism hypothesised for HBsAg/anti-HBs-co-detection remained the change in primary sequence of "a" determinant that alters its antigenic structure making the anti-HBs response less effective. Nevertheless, the real impact of many mutations on HBsAg antigenicity has yet to be determined. Yu D-M et al. addressed this issue investigating the biological features of 3 MHR mutants, identified among 216 Chinese HBsAg/Anti-HBs carriers [[16]Yu D.-M. Li X.-H. Mom V. Lu Z.-H. Liao X.-W. Han Y. Pichoud C. et al.N-glycosylation mutation within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.J Hepatol. 2014; 60: 515-522Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. Authors confirmed a significantly higher heterogeneity of "s" gene in HBsAg/anti-HBs patients as compared to 182 controls, particularly within MHR (mutation prevalence 43.1% vs. 15.5%). Interestingly, 22% (47 of 216) of HBsAg/Anti-HBs carriers harbored new N-glycosylation sites in the MHR as compared to only 1 of 182 (0.5%) controls. The 8 potential N-glycosylation sites were all located within or before the 1st loop of HBsAg (aa substitutions 113, 114, 116, 123, 129, 130, 131, with an additional "N-X-T/S" motif by insertion between 114 and 115). They confirmed clustered mutations in 129, 130, and 131 and selected mutations in 129 and 131, together with the 3 aa insertions functionally studied. They showed firstly by PNGaseF treatment and site direct mutagenesis, that these mutations produced new N-Glycosylation sites, then by deglycosylation experiments that the anti-HBs binding capacity of the 3 mutated proteins was several times lower. Finally, trans-complementation experiments with replication competent/secretion defective HBV plasmid and mutated HBV-env constructs showed an increase secretion of virions. These findings contribute to a better understanding of the underlying biological mechanisms of HBsAg/anti-HBs-co-detection and confirm a major role of the interplay between virus and immune system. On the one hand the HBV replication pathway leads to mutations in the S gene prompting the selection of variants with better fitness once the specific immune response has developed [[16]Yu D.-M. Li X.-H. Mom V. Lu Z.-H. Liao X.-W. Han Y. Pichoud C. et al.N-glycosylation mutation within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.J Hepatol. 2014; 60: 515-522Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. On the other hand when both HBsAg and anti-HBs are detected in highly viremic, HBeAg positive, immune tolerant carriers the alternative possibility that the heterogeneity of the antibodies production could overcome the variability of the virus still holds true, as suggested by Zhang et al. [[17]Zhang J.M. Xu Y. Wang X.Y. Yin Y.K. Wu X.H. Weng X.H. et al.Coexistence of hepatitis B surface antigen (HBsAg) and heterologous subtype-specific antibodies to HBsAg among patients with chronic hepatitis B virus infection.Clin Infect Dis. 2007; 44: 1161-1169Crossref PubMed Scopus (82) Google Scholar]. However, this is a classical chicken and egg question since a single nucleotide change is enough to induce aa substitutions at position 120, 126, and 160 shifting subtype d to y, i to t and w to r [[5]Pondé R.A. The underlying mechanisms for the "simultaneous HBsAg and anti-HBs serological profile".Eur J Clin Microbiol Infect Dis. 2011; 30 ([Epub 2011 Apr 12]): 1325-1340https://doi.org/10.1007/s10096-011-1240-zCrossref PubMed Scopus (33) Google Scholar].In addition, Yu D-M et al. demonstrate the infectivity of the 129 mutant in one responder to HBV vaccine suggesting that individuals harbouring these mutated HBV strains could represent a risky reservoir for the spread of HBV infection, even in vaccinated individuals [[16]Yu D.-M. Li X.-H. Mom V. Lu Z.-H. Liao X.-W. Han Y. Pichoud C. et al.N-glycosylation mutation within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.J Hepatol. 2014; 60: 515-522Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. However, such an event appears to occur only in specific clinical settings such as immune-compromised individuals who deserve a special attention and careful monitoring. Another case was a HBsAg/HBeAg positive immune tolerant patient, with initially undetectable anti-HBs, who turned positive at the time of transition to immune elimination phase, when the 131 mutant became detectable. According to Authors the mutant was horizontally transmitted at that time, but, alternatively, we may speculate that the variant was selected and emerged when the specific adaptive anti-HBV immune response was mounted [[16]Yu D.-M. Li X.-H. Mom V. Lu Z.-H. Liao X.-W. Han Y. Pichoud C. et al.N-glycosylation mutation within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.J Hepatol. 2014; 60: 515-522Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. This finding further supports the view that immune selection plays a pivotal role and suggests that the prevalence of HBsAg/anti-HBs-co-detection should increase in CHB patients. Actually, its reported prevalence is extremely variable, ranging from 2.5% to over 30% [5Pondé R.A. The underlying mechanisms for the "simultaneous HBsAg and anti-HBs serological profile".Eur J Clin Microbiol Infect Dis. 2011; 30 ([Epub 2011 Apr 12]): 1325-1340https://doi.org/10.1007/s10096-011-1240-zCrossref PubMed Scopus (33) Google Scholar, 11Lada O. Benhamou Y. Poynard T. Thibault V. Coexistence of hepatitis B surface antigen (HBs Ag) and anti-HBs antibodies in chronic hepatitis B virus carriers: influence of 'a' determinant variants.J Virol. 2006; 80: 2968-2975Crossref PubMed Scopus (134) Google Scholar, 12Colson P. Borentain P. Motte A. Henry M. Moal V. Botta-Fridlund D. et al.Clinical and virological significance of the co-existence of HBsAg and anti-HBs antibodies in hepatitis B chronic carriers.Virology. 2007; 367: 30-40Crossref PubMed Scopus (63) Google Scholar, 13Chen Y. Qian F. Yuan Q. Li X. Wu W. Guo X. et al.Mutations in hepatitis B virus DNA from patients with coexisting HBsAg and anti-HBs.J Clin Virol. 2011; 52: 198-203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 14Liu W. Hu T. Wang X. Chen Y. Huang M. Yuan C. et al.Coexistence of hepatitis B surface antigen and anti-HBs in Chinese chronic hepatitis B virus patients relating to genotype C and mutations in the S and P gene reverse transcriptase region.Arch Virol. 2012; 157: 627-634Crossref PubMed Scopus (32) Google Scholar, 15Shi Y. Wei F. Hu D. Li Q. Smith D. Li N. et al.Mutations in the major hydrophilic region (MHR) of hepatitis B virus genotype C in North China.J Med Virol. 2012; 84: 1901-1906Crossref PubMed Scopus (22) Google Scholar, 16Yu D.-M. Li X.-H. Mom V. Lu Z.-H. Liao X.-W. Han Y. Pichoud C. et al.N-glycosylation mutation within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.J Hepatol. 2014; 60: 515-522Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 17Zhang J.M. Xu Y. Wang X.Y. Yin Y.K. Wu X.H. Weng X.H. et al.Coexistence of hepatitis B surface antigen (HBsAg) and heterologous subtype-specific antibodies to HBsAg among patients with chronic hepatitis B virus infection.Clin Infect Dis. 2007; 44: 1161-1169Crossref PubMed Scopus (82) Google Scholar]; the changes of diagnostic criteria overtime could explain at least in part such variability, but patient heterogeneity seems a major cause. Accordingly, the HBsAg/anti-HBs prevalence is lower (2.5–5%) in HBsAg carriers identified by population screening, but increases up to 30% in CHB patients. Shields et al. showed that 63% of CHB patients were HBsAg/anti-HBs positive as compared to 14% of asymptomatic carriers [[18]Shiels M.T. Taswell H.F. Czaja A.J. Nelson C. Swenke P. Frequency and significance of concurrent hepatitis B surface antigen and antibody in acute and chronic hepatitis B.Gastroenterology. 1987; 93: 675-680Abstract Full Text PDF PubMed Google Scholar]. Furthermore, liver disease was shown to progress more frequently to HCC in Korean HBsAg/anti-HBs positive CHB patients who had also a higher prevalence of pre-S mutations (42.4% vs. 20.4%) [19Jang J.S. Kim H.S. Kim H.J. Shin W.G. Kim K.H. Lee J.H. et al.Association of concurrent hepatitis B surface antigen and antibody to hepatitis B surface antigen with hepatocellular carcinoma in chronic hepatitis B virus infection.J Med Virol. 2009; 81: 1531-1538Crossref PubMed Scopus (39) Google Scholar, 20Seo S.I. Choi H.S. Choi B.Y. Kim H.S. Kim H.Y. Jang M.K. Coexistence of hepatitis B surface antigen and antibody to hepatitis B surface may increase the risk of hepatocellular carcinoma in chronic hepatitis B virus infection: a retrospective cohort study.J Med Virol. 2013; https://doi.org/10.1002/jmv.23779Crossref Scopus (33) Google Scholar]. Yu et al. found a higher prevalence of N-glycosylation mutation in HCC patients as compared to controls (53% vs. 0%, p <0.001). Overall these findings suggest that HBsAg/anti-HBs-co-detection is linked to a long lasting immune clearance phase that favours the selection of highly variable viral quasispecies not only in the S gene, but also in regions of the genome with relevant oncogenic potential.In conclusion the possible clinical implications of HBsAg/anti-HBs-co-detection are different according to the immune competence of the chronic HBsAg carrier as indicated in Table 1. However, not all studies confirm the correlation between HBsAg/anti-HBs-co-detection and severity of liver disease, thus its clinical significance remains to be better characterized in larger prospective cohort studies.Table 1Biological and clinical implications of HBsAg/anti-HBs-co-detection. Open table in a new tab Conflict of interestM.R.B. is part of the speaker's bureau of Abbot, BMS, Gilead, Roche, MSD and Novartis. Patients who recover from acute hepatitis B clear surface antigen (HBsAg) from serum and anti-HBs becomes detectable as hallmark of HBV immune control. However, anti-HBs appear weeks after HBsAg clearance and neutralizing antibodies are unlikely to contribute to early viral control but to prevent viral spread from cells that remain infected after hepatitis resolution [[1]Rehermann B. Immune response in hepatitis B virus infection.Semin Liver Dis. 2003; 23: 21-23Crossref PubMed Scopus (97) Google Scholar]. This notion is supported by the evidence that chimpanzees resolving hepatitis B are protected from viral rechallenge [[2]Moss B. Smith G.L. Gerin J.L. Purcell R.H. Live recombinant vaccinia virus protects chimpanzees against hepatitis B.Nature. 1984; 311: 67-69Crossref PubMed Scopus (171) Google Scholar]. Similarly, in non-infected individuals an adequate anti-HBs production (>10 mUI/ml) indicates a protective immunity after vaccination [[1]Rehermann B. Immune response in hepatitis B virus infection.Semin Liver Dis. 2003; 23: 21-23Crossref PubMed Scopus (97) Google Scholar]. Several evidences suggest that anti-HBs are present also in chronic HBsAg carriers, but not detected because hidden in circulating immune-complexes [[3]Dienstag J.L. Concurrent hepatitis B surface antigen and antibody and the clonal selection theory of antibody diversity.Gastroenterology. 1987; 93: 899-904Abstract Full Text PDF PubMed Google Scholar]. Thus the simultaneous detection of HBsAg/anti-HBs (HBsAg/anti-HBs-co-detection) represents an atypical serologic profile puzzling virologists and clinicians since the late 1970s [3Dienstag J.L. Concurrent hepatitis B surface antigen and antibody and the clonal selection theory of antibody diversity.Gastroenterology. 1987; 93: 899-904Abstract Full Text PDF PubMed Google Scholar, 4Gerlich W.H. The enigma of concurrent Hepatitis, B surface Antigen (HBsAg) and antibodies to HBsAg.Clin Infect Dis. 2007; 44: 1170-1172Crossref PubMed Scopus (30) Google Scholar, 5Pondé R.A. The underlying mechanisms for the "simultaneous HBsAg and anti-HBs serological profile".Eur J Clin Microbiol Infect Dis. 2011; 30 ([Epub 2011 Apr 12]): 1325-1340https://doi.org/10.1007/s10096-011-1240-zCrossref PubMed Scopus (33) Google Scholar]. Initially, it was explained by heterologous antibodies against subtypes different from the infecting HBV subtype, as HBsAg subtypes d/y infections could associate with y/d anti-HBs subtypes [[6]Tabor E. Gerety R.J. Smallwood L.A. Barker L.F. Coincident hepatitis B surface antigen and antibodies of different subtypes in human serum.J Immunol. 1997; 118: 369-370Google Scholar]. The hypothesis of primary infection with 2 subtypes followed by clearance of only one of them was contradicted by the animal model where humoral response against the a determinant of HBsAg prevented the re-infection with different HBV subtypes [[3]Dienstag J.L. Concurrent hepatitis B surface antigen and antibody and the clonal selection theory of antibody diversity.Gastroenterology. 1987; 93: 899-904Abstract Full Text PDF PubMed Google Scholar]. In addition, ay anti-HBs was found in HBsAg carriers from Japan, where this subtype is uncommon [[7]Hayashi J. Noguchi A. Nakashima K. Morofuji M. Kashiwagi S. Frequency of concurrence of hepatitis B surface antigen and antibody in a large number of carriers in Okinawa, Japan.Gastroenterol Jpn. 1990; 25: 593-597PubMed Google Scholar]. Therefore, the hypothesis of a mixed infection was dismissed in favour of clonal selection of antibody diversity [[3]Dienstag J.L. Concurrent hepatitis B surface antigen and antibody and the clonal selection theory of antibody diversity.Gastroenterology. 1987; 93: 899-904Abstract Full Text PDF PubMed Google Scholar]. The high similarity between 2 HBV serotypes (i.e., HBsAg/ad and HBsAg/ay) could induce the clonal expansion not only of high-titre, high-affinity anti-a and anti-d, but also lower-titre, low-affinity heterotypic anti-y [3Dienstag J.L. Concurrent hepatitis B surface antigen and antibody and the clonal selection theory of antibody diversity.Gastroenterology. 1987; 93: 899-904Abstract Full Text PDF PubMed Google Scholar, 4Gerlich W.H. The enigma of concurrent Hepatitis, B surface Antigen (HBsAg) and antibodies to HBsAg.Clin Infect Dis. 2007; 44: 1170-1172Crossref PubMed Scopus (30) Google Scholar]. Nevertheless, the question remains why only a minority of HBsAg carriers exhibit this condition. After the introduction of molecular biology techniques the characterization of viral quasispecies in clinical settings of HBV infections despite vaccination or HBIg treatment showed that HBsAg/anti-HBs-co-detection was associated with viral strains carrying HBsAg mutations that escaped the anti-HBs neutralization [8Carman W.F. Zanetti A.R. Karayiannis P. Waters J. Manzillop G. Tanzi E. et al.Vaccine-induced escape mutant of hepatitis B virus.Lancet. 1990; 336: 325-329Abstract PubMed Scopus (936) Google Scholar, 9Alavian S.M. Carman W.F. Jazayeri S.M. HBsAg variants: diagnostic-escape and diagnostic dilemma.J Clin Virol. 2013; 57: 201-208Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar]. These findings raise the question whether genetic heterogeneity of HBV and particularly of the S gene could also be present in other HBsAg/anti-HBs carriers. Serum HBsAg results from one open reading frame providing 3 carboxy-terminal collinear proteins of different length (small, medium, and large, SHBs, MHBs, LHBs). In addition to the S gene MHBs contains pre-S2 sequence (55 aa) and LHBs preS1 (108/119 aa depending on subtype) and preS2 sequences. PreS1 and PreS2 and the aa 100–169 domain of SHBs (Major Hydrophilic Region, MHR) are exposed on viral particles and highly immunogenic. The studies of HBsAg variants focused mainly on SHBs, because it is expressed at the highest levels, predominates in both virions and subviral particles and contains within MHR the "a" determinant (aa 124–147), main target of neutralizing antibodies. Two major loops are present in the "a" determinant (the 1st at aa 124–137; the 2nd at aa 139–147), defined by multiple potential disulfide bridges, between aa 139/147 (or 149) and 121/124; conserved cysteines at 124, 137, 139, and 149 are essential for conformation and antigenicity [[9]Alavian S.M. Carman W.F. Jazayeri S.M. HBsAg variants: diagnostic-escape and diagnostic dilemma.J Clin Virol. 2013; 57: 201-208Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar]. MHR is the principal anti-HBs binding site after natural infection, immunisation and HBIg prophylaxis, although areas up and downstream may also be important in neutralisation. Initially characterized vaccine and HBIg escape mutants showed glycine to arginine substitutions at position 145 (G145R); later other mutations between 120 and 208 were described in patients with active or passive immune-prophylaxis or CHB [[10]Yamamoto K. Horikita M. Tsuda F. Itoh K. Yoshihiro A. Yotsumoto S. et al.Mayumi M Naturally occurring escape mutants of hepatitis B virus with various mutations in the carriers seropositive for antibody to hepatitis B surface antigen.J Virol. 1994; 68: 2671-2676PubMed Google Scholar]. Overall, in both Western and Asian cohorts a significantly higher S gene variability, particularly of MHR with higher number of aa substitutions was found in HBsAg/anti-HBs positive patients than in controls [11Lada O. Benhamou Y. Poynard T. Thibault V. Coexistence of hepatitis B surface antigen (HBs Ag) and anti-HBs antibodies in chronic hepatitis B virus carriers: influence of 'a' determinant variants.J Virol. 2006; 80: 2968-2975Crossref PubMed Scopus (134) Google Scholar, 12Colson P. Borentain P. Motte A. Henry M. Moal V. Botta-Fridlund D. et al.Clinical and virological significance of the co-existence of HBsAg and anti-HBs antibodies in hepatitis B chronic carriers.Virology. 2007; 367: 30-40Crossref PubMed Scopus (63) Google Scholar, 13Chen Y. Qian F. Yuan Q. Li X. Wu W. Guo X. et al.Mutations in hepatitis B virus DNA from patients with coexisting HBsAg and anti-HBs.J Clin Virol. 2011; 52: 198-203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 14Liu W. Hu T. Wang X. Chen Y. Huang M. Yuan C. et al.Coexistence of hepatitis B surface antigen and anti-HBs in Chinese chronic hepatitis B virus patients relating to genotype C and mutations in the S and P gene reverse transcriptase region.Arch Virol. 2012; 157: 627-634Crossref PubMed Scopus (32) Google Scholar, 15Shi Y. Wei F. Hu D. Li Q. Smith D. Li N. et al.Mutations in the major hydrophilic region (MHR) of hepatitis B virus genotype C in North China.J Med Virol. 2012; 84: 1901-1906Crossref PubMed Scopus (22) Google Scholar, 16Yu D.-M. Li X.-H. Mom V. Lu Z.-H. Liao X.-W. Han Y. Pichoud C. et al.N-glycosylation mutation within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.J Hepatol. 2014; 60: 515-522Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. Two French studies showed in the same patients a significantly higher number of aa changes in the "s" gene (aa substitution/100 aa: 4.55 vs. 1.66), even more evident in MHR (5.71–4.68 vs. 1.43–2.07) and "a" determinant (9.52–11.00 vs. 2.43–2.48). Aa substitutions were significantly more frequent at positions 120, 126, 129, 130, 144, and 165; G145R/T was the most frequent [11Lada O. Benhamou Y. Poynard T. Thibault V. Coexistence of hepatitis B surface antigen (HBs Ag) and anti-HBs antibodies in chronic hepatitis B virus carriers: influence of 'a' determinant variants.J Virol. 2006; 80: 2968-2975Crossref PubMed Scopus (134) Google Scholar, 12Colson P. Borentain P. Motte A. Henry M. Moal V. Botta-Fridlund D. et al.Clinical and virological significance of the co-existence of HBsAg and anti-HBs antibodies in hepatitis B chronic carriers.Virology. 2007; 367: 30-40Crossref PubMed Scopus (63) Google Scholar]. These findings suggested that emergence of "a" determinant variants could favour viral escape from immune neutralization. Most of Asian studies confirmed the European findings except for a higher prevalence of aa changes in the 1st loop (aa 124–137) of "a" determinant than in the 2nd (aa 137–145) [13Chen Y. Qian F. Yuan Q. Li X. Wu W. Guo X. et al.Mutations in hepatitis B virus DNA from patients with coexisting HBsAg and anti-HBs.J Clin Virol. 2011; 52: 198-203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 14Liu W. Hu T. Wang X. Chen Y. Huang M. Yuan C. et al.Coexistence of hepatitis B surface antigen and anti-HBs in Chinese chronic hepatitis B virus patients relating to genotype C and mutations in the S and P gene reverse transcriptase region.Arch Virol. 2012; 157: 627-634Crossref PubMed Scopus (32) Google Scholar, 15Shi Y. Wei F. Hu D. Li Q. Smith D. Li N. et al.Mutations in the major hydrophilic region (MHR) of hepatitis B virus genotype C in North China.J Med Virol. 2012; 84: 1901-1906Crossref PubMed Scopus (22) Google Scholar]. When HBV genotype C infected individuals were analysed the classic G145R substitution was significantly less frequent, with prevalent mutations at aa 126, 129, 130, 131, 133, and 134, confirming a major influence of genotype on selection of viral quasispecies [13Chen Y. Qian F. Yuan Q. Li X. Wu W. Guo X. et al.Mutations in hepatitis B virus DNA from patients with coexisting HBsAg and anti-HBs.J Clin Virol. 2011; 52: 198-203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 14Liu W. Hu T. Wang X. Chen Y. Huang M. Yuan C. et al.Coexistence of hepatitis B surface antigen and anti-HBs in Chinese chronic hepatitis B virus patients relating to genotype C and mutations in the S and P gene reverse transcriptase region.Arch Virol. 2012; 157: 627-634Crossref PubMed Scopus (32) Google Scholar, 15Shi Y. Wei F. Hu D. Li Q. Smith D. Li N. et al.Mutations in the major hydrophilic region (MHR) of hepatitis B virus genotype C in North China.J Med Virol. 2012; 84: 1901-1906Crossref PubMed Scopus (22) Google Scholar]. However, a potential role of immune suppression and antiviral therapy in European patients cannot be ruled out. Interestingly, some studies showed a significant variability of S gene at its CTL epitope (aa 87–95) that might contribute to HBV neutralization failure [[13]Chen Y. Qian F. Yuan Q. Li X. Wu W. Guo X. et al.Mutations in hepatitis B virus DNA from patients with coexisting HBsAg and anti-HBs.J Clin Virol. 2011; 52: 198-203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar]. The major mechanism hypothesised for HBsAg/anti-HBs-co-detection remained the change in primary sequence of "a" determinant that alters its antigenic structure making the anti-HBs response less effective. Nevertheless, the real impact of many mutations on HBsAg antigenicity has yet to be determined. Yu D-M et al. addressed this issue investigating the biological features of 3 MHR mutants, identified among 216 Chinese HBsAg/Anti-HBs carriers [[16]Yu D.-M. Li X.-H. Mom V. Lu Z.-H. Liao X.-W. Han Y. Pichoud C. et al.N-glycosylation mutation within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.J Hepatol. 2014; 60: 515-522Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. Authors confirmed a significantly higher heterogeneity of "s" gene in HBsAg/anti-HBs patients as compared to 182 controls, particularly within MHR (mutation prevalence 43.1% vs. 15.5%). Interestingly, 22% (47 of 216) of HBsAg/Anti-HBs carriers harbored new N-glycosylation sites in the MHR as compared to only 1 of 182 (0.5%) controls. The 8 potential N-glycosylation sites were all located within or before the 1st loop of HBsAg (aa substitutions 113, 114, 116, 123, 129, 130, 131, with an additional "N-X-T/S" motif by insertion between 114 and 115). They confirmed clustered mutations in 129, 130, and 131 and selected mutations in 129 and 131, together with the 3 aa insertions functionally studied. They showed firstly by PNGaseF treatment and site direct mutagenesis, that these mutations produced new N-Glycosylation sites, then by deglycosylation experiments that the anti-HBs binding capacity of the 3 mutated proteins was several times lower. Finally, trans-complementation experiments with replication competent/secretion defective HBV plasmid and mutated HBV-env constructs showed an increase secretion of virions. These findings contribute to a better understanding of the underlying biological mechanisms of HBsAg/anti-HBs-co-detection and confirm a major role of the interplay between virus and immune system. On the one hand the HBV replication pathway leads to mutations in the S gene prompting the selection of variants with better fitness once the specific immune response has developed [[16]Yu D.-M. Li X.-H. Mom V. Lu Z.-H. Liao X.-W. Han Y. Pichoud C. et al.N-glycosylation mutation within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.J Hepatol. 2014; 60: 515-522Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. On the other hand when both HBsAg and anti-HBs are detected in highly viremic, HBeAg positive, immune tolerant carriers the alternative possibility that the heterogeneity of the antibodies production could overcome the variability of the virus still holds true, as suggested by Zhang et al. [[17]Zhang J.M. Xu Y. Wang X.Y. Yin Y.K. Wu X.H. Weng X.H. et al.Coexistence of hepatitis B surface antigen (HBsAg) and heterologous subtype-specific antibodies to HBsAg among patients with chronic hepatitis B virus infection.Clin Infect Dis. 2007; 44: 1161-1169Crossref PubMed Scopus (82) Google Scholar]. However, this is a classical chicken and egg question since a single nucleotide change is enough to induce aa substitutions at position 120, 126, and 160 shifting subtype d to y, i to t and w to r [[5]Pondé R.A. The underlying mechanisms for the "simultaneous HBsAg and anti-HBs serological profile".Eur J Clin Microbiol Infect Dis. 2011; 30 ([Epub 2011 Apr 12]): 1325-1340https://doi.org/10.1007/s10096-011-1240-zCrossref PubMed Scopus (33) Google Scholar]. In addition, Yu D-M et al. demonstrate the infectivity of the 129 mutant in one responder to HBV vaccine suggesting that individuals harbouring these mutated HBV strains could represent a risky reservoir for the spread of HBV infection, even in vaccinated individuals [[16]Yu D.-M. Li X.-H. Mom V. Lu Z.-H. Liao X.-W. Han Y. Pichoud C. et al.N-glycosylation mutation within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.J Hepatol. 2014; 60: 515-522Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. However, such an event appears to occur only in specific clinical settings such as immune-compromised individuals who deserve a special attention and careful monitoring. Another case was a HBsAg/HBeAg positive immune tolerant patient, with initially undetectable anti-HBs, who turned positive at the time of transition to immune elimination phase, when the 131 mutant became detectable. According to Authors the mutant was horizontally transmitted at that time, but, alternatively, we may speculate that the variant was selected and emerged when the specific adaptive anti-HBV immune response was mounted [[16]Yu D.-M. Li X.-H. Mom V. Lu Z.-H. Liao X.-W. Han Y. Pichoud C. et al.N-glycosylation mutation within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.J Hepatol. 2014; 60: 515-522Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. This finding further supports the view that immune selection plays a pivotal role and suggests that the prevalence of HBsAg/anti-HBs-co-detection should increase in CHB patients. Actually, its reported prevalence is extremely variable, ranging from 2.5% to over 30% [5Pondé R.A. The underlying mechanisms for the "simultaneous HBsAg and anti-HBs serological profile".Eur J Clin Microbiol Infect Dis. 2011; 30 ([Epub 2011 Apr 12]): 1325-1340https://doi.org/10.1007/s10096-011-1240-zCrossref PubMed Scopus (33) Google Scholar, 11Lada O. Benhamou Y. Poynard T. Thibault V. Coexistence of hepatitis B surface antigen (HBs Ag) and anti-HBs antibodies in chronic hepatitis B virus carriers: influence of 'a' determinant variants.J Virol. 2006; 80: 2968-2975Crossref PubMed Scopus (134) Google Scholar, 12Colson P. Borentain P. Motte A. Henry M. Moal V. Botta-Fridlund D. et al.Clinical and virological significance of the co-existence of HBsAg and anti-HBs antibodies in hepatitis B chronic carriers.Virology. 2007; 367: 30-40Crossref PubMed Scopus (63) Google Scholar, 13Chen Y. Qian F. Yuan Q. Li X. Wu W. Guo X. et al.Mutations in hepatitis B virus DNA from patients with coexisting HBsAg and anti-HBs.J Clin Virol. 2011; 52: 198-203Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 14Liu W. Hu T. Wang X. Chen Y. Huang M. Yuan C. et al.Coexistence of hepatitis B surface antigen and anti-HBs in Chinese chronic hepatitis B virus patients relating to genotype C and mutations in the S and P gene reverse transcriptase region.Arch Virol. 2012; 157: 627-634Crossref PubMed Scopus (32) Google Scholar, 15Shi Y. Wei F. Hu D. Li Q. Smith D. Li N. et al.Mutations in the major hydrophilic region (MHR) of hepatitis B virus genotype C in North China.J Med Virol. 2012; 84: 1901-1906Crossref PubMed Scopus (22) Google Scholar, 16Yu D.-M. Li X.-H. Mom V. Lu Z.-H. Liao X.-W. Han Y. Pichoud C. et al.N-glycosylation mutation within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.J Hepatol. 2014; 60: 515-522Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 17Zhang J.M. Xu Y. Wang X.Y. Yin Y.K. Wu X.H. Weng X.H. et al.Coexistence of hepatitis B surface antigen (HBsAg) and heterologous subtype-specific antibodies to HBsAg among patients with chronic hepatitis B virus infection.Clin Infect Dis. 2007; 44: 1161-1169Crossref PubMed Scopus (82) Google Scholar]; the changes of diagnostic criteria overtime could explain at least in part such variability, but patient heterogeneity seems a major cause. Accordingly, the HBsAg/anti-HBs prevalence is lower (2.5–5%) in HBsAg carriers identified by population screening, but increases up to 30% in CHB patients. Shields et al. showed that 63% of CHB patients were HBsAg/anti-HBs positive as compared to 14% of asymptomatic carriers [[18]Shiels M.T. Taswell H.F. Czaja A.J. Nelson C. Swenke P. Frequency and significance of concurrent hepatitis B surface antigen and antibody in acute and chronic hepatitis B.Gastroenterology. 1987; 93: 675-680Abstract Full Text PDF PubMed Google Scholar]. Furthermore, liver disease was shown to progress more frequently to HCC in Korean HBsAg/anti-HBs positive CHB patients who had also a higher prevalence of pre-S mutations (42.4% vs. 20.4%) [19Jang J.S. Kim H.S. Kim H.J. Shin W.G. Kim K.H. Lee J.H. et al.Association of concurrent hepatitis B surface antigen and antibody to hepatitis B surface antigen with hepatocellular carcinoma in chronic hepatitis B virus infection.J Med Virol. 2009; 81: 1531-1538Crossref PubMed Scopus (39) Google Scholar, 20Seo S.I. Choi H.S. Choi B.Y. Kim H.S. Kim H.Y. Jang M.K. Coexistence of hepatitis B surface antigen and antibody to hepatitis B surface may increase the risk of hepatocellular carcinoma in chronic hepatitis B virus infection: a retrospective cohort study.J Med Virol. 2013; https://doi.org/10.1002/jmv.23779Crossref Scopus (33) Google Scholar]. Yu et al. found a higher prevalence of N-glycosylation mutation in HCC patients as compared to controls (53% vs. 0%, p <0.001). Overall these findings suggest that HBsAg/anti-HBs-co-detection is linked to a long lasting immune clearance phase that favours the selection of highly variable viral quasispecies not only in the S gene, but also in regions of the genome with relevant oncogenic potential. In conclusion the possible clinical implications of HBsAg/anti-HBs-co-detection are different according to the immune competence of the chronic HBsAg carrier as indicated in Table 1. However, not all studies confirm the correlation between HBsAg/anti-HBs-co-detection and severity of liver disease, thus its clinical significance remains to be better characterized in larger prospective cohort studies. Conflict of interestM.R.B. is part of the speaker's bureau of Abbot, BMS, Gilead, Roche, MSD and Novartis. M.R.B. is part of the speaker's bureau of Abbot, BMS, Gilead, Roche, MSD and Novartis.