Chronic hepatitis B virus (HBV) infection is a global health threat. Mutations in the surface antigen of HBV (HBsAg) may alter its antigenicity, infectivity, and transmissibility. A patient positive for HBV DNA and detectable but low-level HBsAg in parallel with anti-HBs suggested the presence of immune and/or diagnostic escape variants. To support this hypothesis, serum-derived HBs gene sequences were amplified and cloned for sequencing, which revealed infection with exclusively non-wildtype HBV subgenotype (sgt) D3. Three distinct mutations in the antigenic loop of HBsAg that caused additional N-glycosylation were found in the variant sequences, including a previously undescribed six-nucleotide insertion. Cellular and secreted HBsAg was analyzed for N-glycosylation in Western blot after expression in human hepatoma cells. Secreted HBsAg was also subjected to four widely used, state-of-the-art diagnostic assays, which all failed to detect the hyperglycosylated insertion variant. Additionally, the recognition of mutant HBsAg by vaccine- and natural infection-induced anti-HBs antibodies was severely impaired. Taken together, these data suggest that the novel six-nucleotide insertion as well as two other previously described mutations causing hyperglycosylation in combination with immune escape mutations have a critical impact on in vitro diagnostics and likely increase the risk of breakthrough infection by evasion of vaccine-induced immunity.

O n 19 March 2023, one of the most prominent researchers in medical virology of the last six decades passed away in his 89th year of life: William Sidney (Bill) Robinson, Professor Emeritus of Medicine at Stanford University (1). All his numerous contributions to virology were highly important, but his discovery and characterization of the hepatitis B virus (HBV) DNA remain the most outstanding which has shaped all subsequent work on this important liver disease. For more than a hundred years, infectious hepatitis was a known and growing problem, but the agents of viral hepatitis remained com­ pletely elusive until the 1960s despite intensive research. Epidemiological observations allowed the distinction of two kinds of agents: one that was transmitted by a fecaloral route (type A) and another that was transmitted via blood through percutaneous routes (type B). There was no animal model and no cell culture or an in vitro test for these putative viral agents. The situation was so desperate that researchers performed targeted infection of humans, even of children (2). In 1965, the first diagnostical marker for one of the blood-transmitted forms of hepatitis (hepatitis B) was discovered later with serendipity by Nobel Prize winner Baruch Blumberg (at the Institute for Cancer Research, Philadelphia), who was hunting for genetic markers of malignant diseases (3). His “Australia” antigen was shown by Alfred Prince to be associated with hepatitis B in 1968 (4). But physical characterization of this antigen generated puzzling data. This antigen consisted of small spherical particles, but, in contrast to all other known small viruses, it had a variable diameter of 16–25 nm and it did not contain nucleic acid. These findings led Blumberg to the hypothesis that the Australia antigen would be an unusual infectious agent without nucleic acid like prions (3). It is probably not an accident that he was honored with the Nobel Prize together with Daniel Carleton Gajdusek, who had elucidated the agent of Kuru (a rare form of spongiform encephalopathy). Two major discoveries led stepwise to the true agent of hepatitis B (2). In 1971, David Dane found under the electron microscope in Australia antigen-positive sera among the already known, more numerous small particles, virus-like enveloped 42 nm particles

The National Reference Center (NRC) for hepatitis B viruses (HBV) and hepatitis D viruses (HDV) has been located at the Institute of Medical Virology of the Justus Liebig University (JLU) in Giessen, Germany, since its establishment in 2011. This paper describes the NRC's areas of activity and related experience. The NRC offers comprehensive consulting services on all diagnostic and clinical aspects of acute and chronic HBV and HDV infections for the Public Health Service (oGD), diagnostic laboratories, clinics, research institutes, and physicians in private practice. Uncertain diagnostic findings can be analyzed and interpreted and epidemiological correlations clarified with the HBV/HDV special diagnostics established at the NRC using state-of-the-art molecular, biochemical, and genetic laboratory tools. The NRC has access to a strain collection of many well-characterized and cloned HBV/HDV isolates, allowing comparative analysis and evaluation of antiviral resistance mutations and immune escape variants. Together with its national and international partner institutions, the NRC initiates and supervises, among other things, interlaboratory studies for the diagnosis of HBV resistance and immune escape for the establishment and validation of international World Health Organization (WHO) standards and for the improvement of quantitative HDV genome determination. The NRC actively participates in current recommendations and guidelines on HBV and HDV and the recommendations of medical societies. It also highlights current HBV/HDV-relevant aspects with contributions in the form of national and international lectures as well as original articles and comments in national and international journals.

ZusammenfassungDie ersten Impfversuche gegen das Hepatitis-B-Virus (HBV) erfolgten 1970, noch bevor die Natur des dafür verwendeten „Australia-Antigens“ bekannt war. Bald darauf wurde dieses Antigen als Hüllprotein des HBV erkannt (HBV Surface Antigen, HBsAg), dann aus HBV-haltigem Plasma gereinigt und später gentechnisch in Hefezellen hergestellt. Die hohe Wirksamkeit des HBsAg-Impfstoffs wurde vielfach bewiesen, insbesondere bei Neugeborenen von HBV-infizierten Müttern, die sonst fast immer chronische HBV-Träger werden. Auch bei älteren Kindern und Erwachsenen schützt die Impfung und wird seit 1984 weltweit angewendet, was zu einer ungefähr 10-fachen Abnahme der HBV-Infektionen bei den Geimpften geführt hat.Es gibt dennoch verschiedene Herausforderungen bei der Hepatitis-B-Impfung. Bei Neugeborenen von hochvirämischen Müttern kann die Impfung versagen. Bei verringerter Immunkompetenz kann die Bildung schützender Antikörper ausbleiben, aber auch bei Risikofaktoren wie höherem Alter, Rauchen oder Übergewicht. Frühe Impfstudien belegten, dass Impfstoffe mit dem HBsAg-Subtyp adw2 auch gegen HBV mit anderen HBsAg-Subtypen schützen, neuere Beobachtungen zeigen aber, dass die Schutzwirkung gegen heterologe Subtypen schwächer ist. Gelegentlich werden auch Escape-Mutationen beobachtet.Die meisten jetzigen Impfstoffe beruhen auf dem Kenntnisstand vor 40 Jahren und könnten wesentlich verbessert werden. Eine Einbeziehung der bislang fehlenden PräS-Domänen der HBV-Hülle in die Impfstoffe würde die wichtigsten schützenden T‑ und B‑Zellepitope einbringen. Die Expression in Säugerzellkulturen verbessert die native Faltung der neutralisierenden HBsAg-Epitope und die Verwendung von regional vorherrschenden HBsAg-Subtypen würde die Schutzwirkung erhöhen. Optimale Adjuvanzien oder Epitopträger könnten die Immunogenität auch für eine HBV-Immuntherapie steigern.

The first experimental vaccinations against hepatitis B virus (HBV) were performed in 1970, even before the nature of the administered "Australia antigen" was known. Soon, it was realized that this antigen was the envelope protein (HBV surface antigen, HBsAg), and it was purified from HBV-containing human plasma. Later, it was produced in genetically engineered yeast cells. The excellent efficacy of the HBsAg vaccine was confirmed in numerous studies, particularly in newborns from HBV-infected mothers who almost always become chronic HBV carriers without vaccination. But the vaccine is also highly effective in older children and adults and has been applied worldwide since 1984, leading to a circa tenfold decrease of HBV infections in the vaccinated. Still, there are several challenges with hepatitis B vaccination. In newborns from mothers with very high virus load, the vaccine may fail. Recipients who are immunocompromised, older, smokers, or obese may not produce protective antibodies. Early studies suggested that the vaccine with HBsAg subtype adw2 also protected against infections by other subtypes, but recent observations show that the protection is weaker against heterologous subtypes. Occasionally, escape mutations may develop. Most current HB vaccines are based on the knowledge of 40 years ago and could be significantly improved. Inclusion of the currently neglected preS domains in the HBV envelope would add the most important protective T- and B-cell epitopes to the vaccines. Expression of the HBsAg in mammalian cell cultures would enhance the folding of neutralizing HBsAg epitopes. Use of the regionally prevalent HBsAg subtypes would increase the protection. Optimal adjuvants and epitope carriers may enhance the immunogenicity to the level necessary for immune therapy of chronic hepatitis B.

Zusammenfassung Das Nationale Referenzzentrum (NRZ) für Hepatitis-B-Viren (HBV) und Hepatitis-D-Viren (HDV) befindet sich seit seiner Gründung und Berufung im Jahr 2011 am Institut für Medizinische Virologie der Justus-Liebig-Universität Gießen (JLU). In diesem Beitrag werden die Tätigkeitsbereiche des NRZ und die damit verbundenen Erfahrungen beschrieben. Das NRZ bietet eine umfassende Beratungstätigkeit zu allen diagnostischen und klinischen Aspekten der akuten und chronischen Infektion mit HBV und HDV für den Öffentlichen Gesundheitsdienst (ÖGD), diagnostische Laboratorien, Kliniken, Forschungsinstitute und niedergelassene Ärzte. Unklare diagnostische Befunde können mit der am NRZ etablierten HBV/HDV-Spezialdiagnostik unter Verwendung von aktuellen molekularbiologischen, biochemischen und genetischen Untersuchungsmethoden analysiert, interpretiert und epidemiologische Zusammenhänge aufgeklärt werden. Das NRZ kann dabei auf eine umfangreiche Stammsammlung von vielen gut charakterisierten und klonierten HBV/HDV-Isolaten zurückgreifen, die eine vergleichende Analyse und Bewertung von antiviralen Resistenzmutationen und Immunescape-Varianten zulässt. Das NRZ initiiert und begleitet mit seinen nationalen und internationalen Partnerinstitutionen unter anderem Ringversuche zur Diagnostik der HBV-Resistenz, einschließlich Immunescape, zur Entwicklung und Validierung von internationalen Standards der Weltgesundheitsorganisation (WHO) und zur Optimierung der quantitativen HDV-Genombestimmung. Das NRZ beteiligt sich aktiv an aktuellen Empfehlungen und Leitlinien zu HBV und HDV sowie an Empfehlungen von medizinischen Fachgesellschaften. Es weist mit Beiträgen in Form von nationalen und internationalen Vorträgen sowie mit Originalarbeiten und Kommentaren in nationalen und internationalen Journalen auf aktuelle HBV/HDV-relevante Aspekte hin.

See Article, pages 64–73 See Article, pages 64–73 In this issue of the Journal of Hepatology, a collaboration headed by the laboratory of Jane McKeating describes two new sequence elements in the hepatitis B virus (HBV) DNA genome that mediate transcriptional activation, leading to enhanced production of the viral RNA pregenome, under low oxygen conditions.[1]Wing P.A.C. Liu P.J. Harris J.M. Magri A. Michler T. Zhuang X. et al.Hypoxia inducible factors regulate hepatitis B virus replication by activating the basal core promoter.J Hepatol. 2021; 75: 64-73https://doi.org/10.1016/j.jhep.2020.12.034Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar Transcriptional regulation of the HBV genome has been intensively studied since the late 1970s, and these studies have found numerous transcription factors regulating HBV gene expression.[2]Slagle B.L. Bouchard M.J. Hepatitis B virus X and regulation of viral gene expression.Cold Spring Harb Perspect Med. 2016 Jan 8; 6: a021402https://doi.org/10.1101/cshperspect.a021402Crossref PubMed Scopus (53) Google Scholar,[3]Meier-Stephenson V. Bremner W.T.R. Dalton C.S. van Marle G. Coffin C.S. Patel T.R. Comprehensive analysis of hepatitis B virus promoter region mutations.Viruses. 2018 Nov 1; 10: 603https://doi.org/10.3390/v10110603Crossref Scopus (7) Google Scholar Thus, one might assume that not much can still be discovered in this small, 3,200 base pair genome. Now, Wing et al.[1]Wing P.A.C. Liu P.J. Harris J.M. Magri A. Michler T. Zhuang X. et al.Hypoxia inducible factors regulate hepatitis B virus replication by activating the basal core promoter.J Hepatol. 2021; 75: 64-73https://doi.org/10.1016/j.jhep.2020.12.034Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar show otherwise. They show that cellular hypoxia-inducible factors (HIF-1α, -1β, and -2α) bind as heterodimers to 2 short hypoxia response elements (HREs) upstream of the basal core promoter (BC-Pro) and thereby enhance production of an mRNA encoding both the HBV core protein and the viral DNA polymerase. At the same time, this mRNA functions as the HBV pregenome (pg) RNA, i.e. as a template for reverse transcription by its own translation product, leading eventually to the replication of the viral DNA genome (see the figure). The authors justly point out that most in vitro studies on HBV transcription and replication were done under normal oxygen levels of 18%, yet the liver is overall hypoxic at variable levels of 4–8%, depending on its zonal structure. Now, an impressive group of 24 researchers from 9 institutes in 4 European countries has studied the role of oxygen in the regulation of HBV replication using a plethora of in vitro and in vivo systems. A DNA sequence analysis identified 2 conserved HREs in virtually all genomes from human HBV genotypes (A-J, except genotype H), HBV genomes isolated from archaeological samples, and HBV of apes. Interestingly, no HREs were found in HBV-like viruses of monkeys and rodents, with the exception of the woodchuck HBV (Fig. 1A in Ref. [1]Wing P.A.C. Liu P.J. Harris J.M. Magri A. Michler T. Zhuang X. et al.Hypoxia inducible factors regulate hepatitis B virus replication by activating the basal core promoter.J Hepatol. 2021; 75: 64-73https://doi.org/10.1016/j.jhep.2020.12.034Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). The short, 6 base-pair HREs are located between the HBV enhancers I and II, upstream of the BC-Pro. Using HBV DNA fragments containing the HREs and the BC-Pro upstream of a reporter gene, the authors showed that HIFs activate transcription starting at the BC-Pro in transfected human HepG2 hepatoma cells. This also occurs in the context of the entire episomal HBV circular covalently closed (ccc) DNA, which was either introduced into HepG2 cells by transfection or generated after de novo HBV infection of infection-susceptible HepG2-NTCP cells. Since the effect of HIFs could have been indirect, the groups checked in well-controlled experiments whether HIF-1β indeed binds to the HREs in HBV cccDNA in HepG2 cells by chromatin immunoprecipitation (ChIP). The positive result was confirmed in HBV-infected human hepatocytes transplanted into humanized “FRGN” mice. Binding depended on the availability of HIF-1α, which had to be stabilized either by low oxygen (1%) or by an inhibitor of oxygen-dependent HIF-1α degradation (FG-4592). Expression of the HBV pg and/or preCore (pC) mRNA in infected HepG2-NTCP cells increased up to 30-fold at 1% oxygen within 3 days. Return to 18% oxygen led to a gradual decrease in pg/pC RNA to previous levels (Fig. 5 – original manuscript). Although hypoxia seems to enhance only the activity of the BC-Pro, this is sufficient to enhance production of complete enveloped HBV particles because the HBV surface proteins are produced in excess irrespective of oxygen levels. A strong increase of HBV production and release in 1% oxygen was indeed observed with HBV-transfected and HBV-transduced HepG2 cells (Fig. S2 – original manuscript). Although the results with the various hepatic cell systems are convincing, infection of primary human hepatocytes or spheroids / organoids with HBV virions would have been more physiological. The stimulatory effect of low oxygen was limited to the transcription of the pg or pC mRNA, confirming the strong effect of the HREs on the BC-Pro and not on the promoters regulating the preS/S or HBx genes (Fig. 6 – original manuscript). The question remains whether both HREs are necessary for hypoxic activation of HBV replication. It appears plausible that the upstream HRE I would possibly act also on the enhancer I and the X-Pro, but the data[1]Wing P.A.C. Liu P.J. Harris J.M. Magri A. Michler T. Zhuang X. et al.Hypoxia inducible factors regulate hepatitis B virus replication by activating the basal core promoter.J Hepatol. 2021; 75: 64-73https://doi.org/10.1016/j.jhep.2020.12.034Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar are not completely clear about the effect of hypoxia on HBx expression, whereas HRE II might act alone on the BC-Pro and enhancer II. Given the large variety of approaches used by the authors, the absence of an analysis of HBV DNA containing 1 or 2 mutationally inactivated HREs is somewhat surprising. A mutational approach could also have helped to better distinguish differences in the transcription of pg and pC mRNA. This distinction is highly relevant because the HBV-e-antigen (HBeAg) encoding pC mRNA does not contribute to expression of HBV core and polymerase proteins and cannot serve as a pregenome (see Fig. 1). Small changes in the BC-Pro/enhancer II can shift the start of transcription from pg to pC mRNA and vice versa.[4]Buckwold V.E. Xu Z. Chen M. Yen T.S. Ou J.H. Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication.J Virol. 1996 Sep; 70: 5845-5851https://doi.org/10.1128/JVI.70.9.5845-5851.1996Crossref PubMed Scopus (0) Google Scholar The question remains whether hypoxia can alter the ratio between the mRNAs because the reverse-transcription PCR methods used by the authors could not distinguish between these highly overlapping mRNAs. The data showing strong enhancement of HBV replication suggest, however, that HIFs enhance pg transcription. The most relevant question is, of course, does this also happen in patients with chronic HBV infection? The group does not offer a clear answer, but they have analyzed liver biopsies from 18 chronically HBV-infected individuals for the levels of HBV pg/pC mRNA. Unfortunately, all patients studied were HBeAg negative, and most had relatively low levels of HBV DNA and HBsAg in serum (Fig. 2 – original manuscript). Thus, these patients were probably not representative of the majority of patients with clinically relevant HBV infection. Nevertheless, they found a significant correlation with mRNA levels of HIF-controlled cellular genes over a wide range of HBV mRNA levels, suggesting regulation of HBV transcription by hypoxia-sensitive elements. A more informative but very demanding approach would be to sequence RNAs in single hepatocytes from various hepatic zones,[5]Halpern K.B. Shenhav R. Matcovitch-Natan O. Toth B. Lemze D. Golan M. et al.Single-cell spatial reconstruction reveals global division of labour in the mammalian liver.Nature. 2017 Feb 16; 542 (Epub 2017 Feb 6. Erratum in: Nature. 2017 Mar 30;543(7647):742): 352-356https://doi.org/10.1038/nature21065Crossref PubMed Scopus (368) Google Scholar e.g. with different oxygen levels. It is not currently possible to use patients with HBV for experiments involving manipulation of the expression of HIFs in vivo, but there exist several transgenic mouse models producing complete HBV particles. Suppression of HIF production with HIF-1β silencing siRNA in the mouse strain, HBV1.3.32, from F. Chisari’s lab[6]Guidotti L.G. Matzke B. Schaller H. Chisari F.V. High-level hepatitis B virus replication in transgenic mice.J Virol. 1995; 69: 6158-6169Crossref PubMed Google Scholar markedly reduced the production of HBV mRNAs, HBeAg, and the number of HBV core protein-expressing hepatocytes. However, a clear effect was observed only in male mice, whereas the results were ambiguous in female mice. In humans, HBV can replicate to very high levels irrespective of sex, casting doubt on the validity of these finding for humans. On the other hand, males are much more often affected by HBV-related hepatocellular carcinomas (HCCs, see below).[7]Li C.L. Li C.Y. Lin Y.Y. Ho M.C. Chen D.S. Chen P.J. et al.Androgen receptor enhances hepatic telomerase reverse transcriptase gene transcription after hepatitis B virus integration or point mutation in promoter region.Hepatology. 2019 Feb; 69: 498-512https://doi.org/10.1002/hep.302018Crossref PubMed Google Scholar While the authors discuss the sex differences of hypoxia responses in various animal species, including zebrafish, they do not mention the effect of androgens on HBV expression. Male HBV transgenic mice express HBV 2x more actively than female mice[8]Tian Y. Kuo C.F. Chen W.L. Ou J.H. Enhancement of hepatitis B virus replication by androgen and its receptor in mice.J Virol. 2012 Feb; 86: 1904-1910https://doi.org/10.1128/JVI.06707-11Crossref PubMed Scopus (35) Google Scholar and more often develop HCC.[9]Wu M.H. Ma W.L. Hsu C.L. Chen Y.L. Ou J.H. Ryan C.K. et al.Androgen receptor promotes hepatitis B virus-induced hepatocarcinogenesis through modulation of hepatitis B virus RNA transcription.Sci Transl Med. 2010 May 19; 2: 32ra35https://doi.org/10.1126/scitranslmed.3001143Crossref PubMed Scopus (143) Google Scholar These differences could be attributed to 2 androgen-responsive elements (AREs, Fig. 1) in enhancer I, at nucleotides 923 and 959 of HBV DNA.[8]Tian Y. Kuo C.F. Chen W.L. Ou J.H. Enhancement of hepatitis B virus replication by androgen and its receptor in mice.J Virol. 2012 Feb; 86: 1904-1910https://doi.org/10.1128/JVI.06707-11Crossref PubMed Scopus (35) Google Scholar,[9]Wu M.H. Ma W.L. Hsu C.L. Chen Y.L. Ou J.H. Ryan C.K. et al.Androgen receptor promotes hepatitis B virus-induced hepatocarcinogenesis through modulation of hepatitis B virus RNA transcription.Sci Transl Med. 2010 May 19; 2: 32ra35https://doi.org/10.1126/scitranslmed.3001143Crossref PubMed Scopus (143) Google Scholar It appears plausible that the AREs may cooperate with the HRE at nucleotide 1240, near enhancer I, in activating the BC-Pro, at least in mice (Fig. 3 and Fig. S1 – original manuscript). The discovery of highly active HREs in HBV DNA raises the question of whether hypoxia may have an effect on the development of HBV-associated HCCs. Most of these carcinomas carry several integrated HBV DNA fragments without BCPs, but probably with HREs and AREs. As the authors mention “… the introduction of HREs could regulate downstream targets such as oncogenes that could be activated in the hypoxic tumor environment.” Aside from this serious consequence, the HREs may also activate the promoters of the HBV preS/S and HBx genes in that context. Interestingly, the authors do not find any effect of hypoxia on expression of preS/S proteins from integrated HBV DNA fragments in 3 established human hepatoma cell lines. However, integration of HBV DNA into the genome of HBV-infected hepatocytes is a frequent event, independent of eventually later developing malignancy.[10]Mason W.S. Gill U.S. Litwin S. Zhou Y. Peri S. Pop O. et al.HBV DNA integration and clonal hepatocyte expansion in chronic hepatitis B patients considered immune tolerant.Gastroenterology. 2016 Nov; 151 (e4): 986-998https://doi.org/10.1053/j.gastro.2016.07.012Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar Non-malignant but expanding human hepatocytes containing HBV DNA fragments may still express in vivo preS-containing HBV surface or HBx proteins. These HBV proteins have a certain degree of oncogenicity which may finally contribute to a fully developed HCC.[11]Mason W.S. Jilbert A.R. Litwin S. Hepatitis B virus DNA integration and clonal expansion of hepatocytes in the chronically infected liver.Viruses. 2021; 13: 210https://doi.org/10.3390/v13020210Crossref PubMed Scopus (5) Google Scholar It still remains unclear whether expression of these integrated HBV genes is completely independent of the oxygen level and whether it may eventually become detrimental in premalignant cells within the hypoxic tumor environment. It also remains unclear whether the sharp difference between 1% and 18% oxygen reflects the natural variations in the environment of the HBV-infected liver. HREs have been found in at least 20 very different DNA virus species,[12]Liu P.J. Balfe P. McKeating J.A. Schilling M. Oxygen sensing and viral replication: implications for tropism and pathogenesis.Viruses. 2020 Oct 25; 12: 1213https://doi.org/10.3390/v12111213Crossref Scopus (3) Google Scholar but there is no apparent common biological feature which would correlate with the presence of HREs in viral genomes. HBV is the only virus species infecting Hominidae among numerous other species in the large Orthohepadnavirus genus. In spite of their hepatotropism, i.e. adaptation to moderate hypoxia, most orthohepadnaviruses did not evolve HREs, with the exception of human, ape and woodchuck HBV. The phylogenetic tree of the orthohepadnaviruses suggests that diversification of HBV into human and ape genotypes started 30 million years ago[13]Glebe D. Goldmann N. Lauber C. Seitz S. HBV evolution and genetic variability: impact on prevention, treatment and development of antivirals.Antiviral Res. 2021 Feb; 186: 104973https://doi.org/10.1016/j.antiviral.2020.104973Crossref PubMed Scopus (7) Google Scholar and was linked with the acquisition and conservation of HREs, with 1 exception: genotype H. No feature of genotype H points to a special role compared to the other 9 HBV genotypes A-J. In fact, genotype H is most closely related to genotype F,[13]Glebe D. Goldmann N. Lauber C. Seitz S. HBV evolution and genetic variability: impact on prevention, treatment and development of antivirals.Antiviral Res. 2021 Feb; 186: 104973https://doi.org/10.1016/j.antiviral.2020.104973Crossref PubMed Scopus (7) Google Scholar which has 2 HREs. It appears that the acquisition of HREs is just one of many elements in optimizing the coexistence of HBV with humans and apes. The fact that hypoxia induces DNase I, which may be a restriction factor for HBV replication,[14]Hallez C. Li X. Suspène R. Thiers V. Bouzidi M.S. Dorobantu C.M. et al.Hypoxia-induced human deoxyribonuclease I is a cellular restriction factor of hepatitis B virus.Nat Microbiol. 2019 Jul; 4: 1196-1207https://doi.org/10.1038/s41564-019-0405-xCrossref PubMed Scopus (7) Google Scholar could explain the fact that so many orthohepadnaviruses lack HREs. Given the enormous significance of HIFs in cancer and many other diseases, this work[1]Wing P.A.C. Liu P.J. Harris J.M. Magri A. Michler T. Zhuang X. et al.Hypoxia inducible factors regulate hepatitis B virus replication by activating the basal core promoter.J Hepatol. 2021; 75: 64-73https://doi.org/10.1016/j.jhep.2020.12.034Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar provides valuable information on oxygen-regulated pathogenic processes including HBV infection, but its diagnostic and therapeutic relevance has still to be unraveled. The author does not receive any financial support for his scientific activity. The author declares to have no conflict of interest. Please refer to the accompanying ICMJE disclosure forms for further details. The following is the supplementary data to this article: Download .pdf (.13 MB) Help with pdf files Hypoxia inducible factors regulate hepatitis B virus replication by activating the basal core promoterJournal of HepatologyVol. 75Issue 1PreviewHypoxia inducible factors (HIFs) are a hallmark of inflammation and are key regulators of hepatic immunity and metabolism, yet their role in HBV replication is poorly defined. HBV replicates in hepatocytes within the liver, a naturally hypoxic organ, however most studies of viral replication are performed under conditions of atmospheric oxygen, where HIFs are inactive. We therefore investigated the role of HIFs in regulating HBV replication. Full-Text PDF Open Access

In The Lancet Infectious Diseases, Anna Funk and colleagues1 present their systematic review and meta-analysis on the efficacy and safety of peripartum antiviral prophylaxis against the mother-to-child-transmission (MTCT) of hepatitis B virus (HBV). Peripartum antiviral prophylaxis is costly, unpractical, and should preferably be provided to mothers with a high risk of MTCT; hence, a simple method is required to recognise mothers who are at high risk prenatally. The gold standard to estimate HBV infectivity, and thus the risk of MTCT, is quantitative determination of HBV DNA in the plasma of the mother using PCR or similar techniques.

Background and aims: Peripartum transmission of hepatitis B virus (HBV) from an infected mother to the child can be prevented in most but not all cases by immediate vaccination of the newborn. The aim of this study was to compare the efficacy of two licensed hepatitis B vaccines, Engerix-B versus Sci-B-Vac, in preventing peripartum HBV transmission. Methods: A prospective multicenter randomized controlled study in 4 delivery centers was performed from 2009 to 2014. HBsAg positive pregnant women and their newborns were recruited at the delivery rooms. All newborns received Hepatitis B Immune Globulin within 10 h after birth, as well as active HBV vaccination at 0, 1 and 6 months of age. Maternal assessment at delivery included transaminases, blood count, international normalized ratio and viral status. Infants were tested for HBsAg, anti-HBc and anti-HBs at 12 months of age. Results: In the intention to treat (ITT), 171 infant and mother pairs fulfilled the study enrollment criteria and completed follow up, 82 received Engerix-B and 89 Sci-B-Vac. Maternal parameters and viral status were similar in both groups. At 12 months of age, the Sci-B-Vac group had lower HBsAg carriage rates (1/89, 1.1%) than the Engerix-B group (5/82, 6.1%) with borderline significance (risk difference of −0.05, 95% CI −0.11–0.007, t-test = 0.05), and borderline significance lower vaccine failure rates with anti-HBs < 10 mIU/mL in the Sci-B-Vac (2/89, 2.2%) than in the Engerix-B (8/82, 9.8%, p = 0.05). Higher seroprotection rates were found in the Sci-B-Vac group with all anti-HBs titer stratifications of >10 mIU/mL (p = 0.05), >100 mIU/mL (p = 0.05) and >1000 mIU/mL (p = 0.01). Active/passive vaccination was effective in 10/13 cases with maternal HBV DNA levels > 7 log10 IU/mL up to 9.5 log10 IU/mL, but failed in 3 cases for unknown reasons. Conclusion: Sci-B-Vac was superior to Engerix-B in preventing peripartum HBV transmission in neonates from HBsAg+ mothers and induces significantly higher anti-HBs levels. NIH registration number: NCT 01133184.

Chronic hepatitis B (CHB) infection is still a huge public health problem, although tremendous progress in prevention and therapy of this infection has been achieved in the last decades. Vaccination has reduced prevalence of CHB by more than 90 % in vaccinated young adults [[1]Global Hepatitis Report 2017. World Health Organization, Geneva2017Google Scholar]. Therapy of the still very numerous existing cases of CHB in adults (estimated 257 million worldwide) with nucleos/tide analogs and/or interferon can suppress replication of HBV and progression of the liver disease. An open problem is, however, the achievement of a permanent cure of CHB allowing for safe termination of antiviral therapy. The HBV genome persists in the liver and continues to express its genes in spite of inhibited replication or active immune reactions. One of the key factors potentially leading to suppression of HBV gene expression is an immune defense against HBV antigens by activated T cells. The main target of the T cells is the HBV core antigen (HBcAg), but the most sensitive marker of HBV gene expression is the HBV surface antigen (HBsAg) in the serum. Loss of serum HBsAg during CHB is generally accepted as marker of a so-called "functional cure" indicating an efficient immune response against HBV genome expression [2Lok AS Zoulim F Dusheiko G Ghany MG. Hepatitis B cure: From discovery to regulatory approval.J Hepatol. 2017; 67 (j): 847-861Summary Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 3Cornberg M. Lok A.S. Terrault N.A. Zoulim F. EASL-AASLD HBV Treatment Endpoints Conference Faculty. Guidance for design and endpoints of clinical trials in chronic hepatitis B - Report from the 2019 EASL-AASLD HBV Treatment Endpoints Conference Faculty.J Hepatol. 2020; 72: 539-557https://doi.org/10.1016/j.jhep.2019.11.003Summary Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 4European Association for the Study of the LiverEASL 2017 Clinical Practice Guidelines on the management of hepatitis B 464 virus infection.J Hepatol. 2017; 67: 370-398https://doi.org/10.1016/j.jhep.2017.03.021Summary Full Text Full Text PDF PubMed Scopus (2515) Google Scholar]. The article from Shue Xiong and colleagues in this issue of EBioMedicine [[5]Xiong S. et al.Longitudinal characterization of phenotypic profile of T cells in chronic hepatitis B identifies immune markers associated with HBsAg loss.EBioMedicine. 2021; 69 (July)https://doi.org/10.1016/j.ebiom.2021.103464Summary Full Text Full Text PDF PubMed Scopus (3) Google Scholar] followed the kinetics (i.e., "longitudinal") of many markers for T cell activity in blood mononuclear cells of 172 CHB patients before, during and after the loss of HBsAg for periods up to 60 weeks. Most patients, but not all, had received antiviral therapy. HBsAg loss during CHB is a slow process which cannot be accelerated very much by therapy with nucleos/tide therapy. Consequently, a weakness of the study is that 141 of the 172 patients retained HBsAg during the follow-up and only 6 showed HBsAg loss. As supplement, the study included 25 patients in whom HBsAg loss had been observed in the years before. Furthermore, the authors followed the HBsAg concentration in 19 patients without HBsAg loss and correlated its "rapid decrease" with the T cell parameters. The authors summarize their data on T cells as follows: "Collectively, these data show that the activated phenotype of T cells is associated with the intensities of HBsAg reduction and HBV-specific T cell responses during the course of rapid HBsAg decrease and loss." This is important and supports some current concepts for future immune stimulatory therapies, e.g., with a therapeutic vaccine [[6]Cornelius C Schöneweis K Georgi F Weber M Niederberger V Zieglmayer P Niespodziana K Trauner M Hofer H Urban S Valenta R. Immunotherapy With the PreS-based Grass Pollen Allergy Vaccine BM32 Induces Antibody Responses Protecting Against Hepatitis B Infection.EBioMedicine. 2016; 11 (Sep): 58-67https://doi.org/10.1016/j.ebiom.2016.07.023Summary Full Text Full Text PDF PubMed Scopus (38) Google Scholar]. The study also confirmed the old observation that therapy with pegylated interferon alpha induces activation of T cells and HBsAg loss in many patients months after the therapy. Surprising is the finding that the marker of T cell exhaustion PD1 was enhanced in patients with therapy. The authors mention that PD1 may also be a marker T cell survival which finally may result in the formation of T memory cells and subsequent immune responses. The most universal marker of T cell activation seemed to be expression of HLA-DR which was found elevated in patients with rapid HBsAg decrease. Consistently, patients who developed anti-HBs after HBsAg loss had also enhanced HLA-DR. The findings on the HBcAg-specific CD8+ T cell response are somewhat puzzling. It was virtually absent in patients with constant HBsAg levels as expected, but it seemingly remained weak even during rapid HBsAg decrease. In contrast, the loss of HBsAg was accompanied or followed by enhanced HBcAg-specific CD8+ T cell responses. Possibly, the HBcAg-specific T cells were retained in the HBV-infected liver during the early phases of HBsAg elimination, and appeared later in the blood. Liver samples, were, however, not available for the study. A mainstay for the long-term prognosis of CHB is the HBsAg level. The study followed mainly patients with low levels of HBsAg (<1000 IU/mL) which were already close to HBsAg loss. Many of the tested markers of the global T cell activity were significantly correlated with the loss of HBsAg (Table 2), but the best predictors of HBsAg loss were the quantity of HBsAg itself and the percentage of HLA-DR+ CD8+ T cells. Combination of quantitative HBsAg with this or further T cell markers may improve the prediction, but considering the high technical effort of the T cell profiling, other approaches may be considered. According to EASL, HBV infection may proceed through different phases from the highly replicative, quasi-immune tolerant to the occult, HBsAg negative phase [[4]European Association for the Study of the LiverEASL 2017 Clinical Practice Guidelines on the management of hepatitis B 464 virus infection.J Hepatol. 2017; 67: 370-398https://doi.org/10.1016/j.jhep.2017.03.021Summary Full Text Full Text PDF PubMed Scopus (2515) Google Scholar]. The distinction between HBsAg positive or negative at a limit of detection of 0.1 IU/mL appears somewhat arbitrary. There are far more sensitive HBsAg assays [[7]Deguchi M Kagita M Yoshioka N et al.Evaluation of the highly sensitive chemiluminescent enzyme immunoassay "Lumipulse HBsAg-HQ" for hepatitis B virus screening.J Clin Lab Anal. 2018; 32: e22334https://doi.org/10.1002/jcla.22334Crossref PubMed Scopus (19) Google Scholar]. The transition from the inflammatory chronic hepatitis B to the asymptomatic inactive carrier state with lower but still present HBsAg is much more relevant than reaching the so-called functional cure which is in fact a form of occult HBV infection. The results of the study are very interesting concerning the T cell-related mechanisms of immune control of CHB, but the focus on the period of the last months before and after undetectability of HBsAg impairs its practical usefulness. There are many other serological markers of HBV activity or immunity than a qualitative HBsAg result at the limit of analytical sensitivity. One unjustified simplification in the diagnosis of HBV is the neglect of the middle and large HBs proteins with their preS2 and preS1 domains. Using rather simple quantitative ELISAs for preS1 and 2 antigens in serum, the natural course of CHB [[8]Pfefferkorn M Böhm S Schott T Deichsel D Bremer CM Schröder K et al.Quantification of large and middle proteins of hepatitis B virus surface antigen (HBsAg) as a novel tool for the identification of inactive HBV carriers.Gut. 2018; 67 (Nov): 2045-2053https://doi.org/10.1136/gutjnl-2017-313811Crossref PubMed Scopus (48) Google Scholar] or the outcome of antiviral therapies including interferon [[9]Pfefferkorn M Schott T Böhm S Deichsel D Felkel C Gerlich WH Glebe D Wat C Pavlovic V Heyne R Berg T van Bömmel F. Composition of HBsAg is predictive of HBsAg loss during treatment in patients with HBeAg-positive chronic hepatitis B.J Hepatol. 2021; 74 (Feb): 283-292https://doi.org/10.1016/j.jhep.2020.08.039Summary Full Text Full Text PDF PubMed Scopus (16) Google Scholar] could be rather dependably predicted. Irrespective of these remarks, the study gives interesting insights to the role of T cells in controlling HBV infection. Future, similar in-depth studies of immune cells in CHB should also be devoted to the role of B cells. The author declares no competing interests. Longitudinal characterization of phenotypic profile of T cells in chronic hepatitis B identifies immune markers associated with HBsAg lossThe onset of HBsAg decrease and subsequent loss in cHBV patients on treatment is associated with significant alterations of both CD4+ and CD8+ T cell phenotypes. Characterization of the T cell phenotype in cHBV patients may present predicative value for sAg-L. Full-Text PDF Open Access

An array of chronic inflammatory diseases, including metabolic diseases such as obesity and diabetes, are thought to be promoted by disturbance of the intestinal microbiota. Such diseases disproportionately impact low-income communities, which are frequently afflicted by chronic stress and increased density housing. Hence, we hypothesized that overcrowded housing might promote stress, microbiota dysbiosis, inflammation, and, consequently, metabolic diseases. We tested this hypothesis in a tractable murine model of social overcrowding (SOC), in which mice were housed at twice normal density. SOC moderately impacted behavior in some widely used assays (Open Field, Elevated Plus Maze and Light/Dark tests) and resulted in a stark increase in corticosterone levels. Such indices of stress were associated with mild chronic gut inflammation, hyperglycemia, elevations in colonic cytokines, and alterations in gut microbiota composition. All of these consequences of SOC were eliminated by broad spectrum antibiotics, while some (inflammation and hyperglycemia) were transmitted by microbiota transplantation from SOC mice to germfree mice housed at normal density. Altogether, these results suggest a central role for intestinal microbiota in driving stress, inflammation, and chronic diseases that are promoted by overcrowded housing.

In this article of EBioMedicine, Rudolf Valenta and colleagues report on the antibody response to the hepatitis B virus (HBV) component induced by their experimental vaccine BM32 [[1]Tulaeva I. Cornelius C. Zieglmayer P. et al.Quantification, epitope mapping and genotype cross-reactivity of hepatitis B preS-specific antibodies in subjects vaccinated with different dosage regimens of BM32.EBioMedicine. 2020; https://doi.org/10.1016/j.ebiom.2020.102953Summary Full Text Full Text PDF PubMed Scopus (3) Google Scholar]. Originally, the primary purpose of this vaccine was protection against allergenic peptides of grass pollen. The combination of allergy-related peptides with the preS domain of HBV may appear strange. However, in view of this and a previous paper the approach appears logical [[2]Cornelius C. Schöneweis K. Georgi F. et al.Immunotherapy with the preS-based grass pollen allergy vaccine BM32 induces antibody responses protecting against hepatitis B infection.EBioMedicine. 2016; 11: 58-67Summary Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. The first paper on the use of preS protein as carrier, in this case for a cat allergy antigen explained the rational as follows: "This approach is based on the selection of allergen-derived peptides that lack IgE reactivity and IgE-mediated allergenic activity and exhibit reduced T-cell reactivity. Coupled to a non–allergen-related carrier, they should then lead to a vaccine that induces allergen-specific IgG with T-cell help from carrier-derived epitopes."[[3]Niespodziana K. Focke-Tejkl M. Linhart B. et al.A hypoallergenic cat vaccine based on Fel d 1-derived peptides fused to hepatitis B PreS.J Allergy Clin Immunol. 2011; 127: 1562-1570Summary Full Text Full Text PDF PubMed Scopus (81) Google Scholar] Generation of such a vaccine can be well achieved by expression of fused allergen- and carrier-encoding DNA sequences. It would appear plausible to use an immunogenic carrier which has already safely and successfully been used like the current "recombinant" HBV vaccine. However, the group selected the preS domain of the large HBV surface protein (LHBs) as carrier. LHBs was the last surface protein which was identified in the HBV envelope. "PreS" does not mean a precursor function for a protein, it is just upstream of the S gene [[4]Gerlich W.H. Glebe D. Kramvis A. Magnius L.O Peculiarities in the designations of hepatitis B virus genes, their products, and their antigenic specificities: a potential source of misunderstandings.Virus Genes. 2020; 56: 109-119Crossref PubMed Scopus (8) Google Scholar]. Initially, preS was neglected as component of HB vaccines. In contrast, the small surface protein of HBV (SHBs) has been used since decades as extremely effective prophylactic vaccine [[5]WHO. Global Hepatitis Report2017. [accessed on August 16, 2020]. Available from: http://www.who.int/hepatitis/publications/global-hepatitisreport2017/en/Google Scholar]. SHBs vaccines have a certain weakness with regard to the presentation of T cell epitopes because the highly hydrophobic, complex-folded and strongly disulfide-crosslinked SHBs protein is not well processed in antigen-presenting cells. The detection of the small hydrophilic preS2 domain in the middle-sized HBV surface protein (MHBs) suggested to increase the immunogenicity of HBsAg vaccines by inclusion of the preS2 domain which could indeed overcome non-responsiveness to SHBs in certain mouse strains by increased induction of T helper cells [[6]Milich D.R. McLachlan A. Thornton G.B T-cell recognition of pre-S regions of HBsAg can bypass nonresponse to the S region.Adv Exp Med Biol. 1987; 225: 233-239Crossref PubMed Scopus (5) Google Scholar]. PreS2-containing vaccines have been used in humans for several years but they are no longer used [[7]Gerlich W.H Prophylactic vaccination against hepatitis B: achievements, challenges and perspectives.Med Microbiol Immunol. 2015; 204: 39-55Crossref PubMed Scopus (62) Google Scholar]. The entire preS domain (i.e. preS1 and preS2, as in BM32) of LHBs finally was shown to contain an attachment site of HBV to hepatic cells. This discovery immediately suggested to use the preS attachment peptide as prophylactic immunogen. Thereafter, at least two vaccines containing preS and SHBs were developed and were superior to the standard vaccines in large trials [[7]Gerlich W.H Prophylactic vaccination against hepatitis B: achievements, challenges and perspectives.Med Microbiol Immunol. 2015; 204: 39-55Crossref PubMed Scopus (62) Google Scholar]. However, these preS vaccines are currently not on the market. One disadvantage of these vaccines is the expression in mammalian cell lines whereas the SHBs vaccine can be inexpensively produced in yeast. The Valenta group recognized the preS protein as preferable carrier for anti-allergen vaccines because it can be easily expressed in E. coli and provides the needed T cell epitopes. In this article of EBioMedicine, the intensity and fine specificity of the antibody response to preS are described in detail. PreS contains several strong T cell epitopes and B cell epitopes within the HBV attachment and accessory domain. Thus, it is not surprising that BM32 induces high antibody titers against the attachment and accessory domain. In the preceding article, the group provided proof of principle for the HBV infectivity-neutralizing capacity of the BM32 antibody response in a small number of human recipients [[2]Cornelius C. Schöneweis K. Georgi F. et al.Immunotherapy with the preS-based grass pollen allergy vaccine BM32 induces antibody responses protecting against hepatitis B infection.EBioMedicine. 2016; 11: 58-67Summary Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. Now, they present quantitative data on the concentration of attachment and accessory domain-specific IgG1 and IgG4 antibodies from 76 recipients of BM32 and used for their immune assays peptides covering these sites derived from the 8 known HBV genotypes A-H. No significant differences in the amounts of antibodies against preS peptides from the various genotypes were noted although BM32 contains the preS only from subgenotype A2 and some variability exists between the genotypes in the region preS(1–51). The amounts of induced antibodies, >10 µg/ml up to 1.8 mg/ml, suggest that they may neutralize infectious inocula in exposed patients. But confirmation of this hypothesis requires much more work, e.g. in vitro neutralization assays using susceptible hepatic cell lines, in vivo assays in humanized mice and finally larger trials in humans. However, the authors put emphasis on BM32 as a candidate for immunotherapy of chronic HBV infection (CHB). An effective immunotherapeutic agent would be highly desirable in view of the difficulties to reach a permanent or so-called "functional cure" of CHB by current HBV therapies with reverse transcriptase inhibitors [[8]Revill P.A. Chisari F.V. Block J.M. et al.A global scientific strategy to cure hepatitis B [published correction appears in Lancet Gastroenterol Hepatol 2019; 4(7): e7].Lancet Gastroenterol Hepatol. 2019; 4: 545-558Summary Full Text Full Text PDF PubMed Scopus (131) Google Scholar]. However, these therapies suppress production of infectious HBV particles. Thus, it is open whether the very good production of potentially neutralizing anti-HBV antibodies induced by BM32 would add much benefit to the current therapies, even if immunotherapy with BM32 could overcome the immune tolerance to HBV surface antigens in CHB patients. However, the HBV T cell epitopes of BM32 may help to build an antiviral cellular immune response which could support reaching the desired "functional cure". PreS by itself is weakly immunogenic, being a small, non-conformational, monomeric protein. PreS has recently been coupled to highly immunogenic ferritin particles and led as therapeutic vaccine to functional cure in a CHB-mouse model [[9]Wang W. Zhou X. Bian Y. et al.Dual-targeting nanoparticle vaccine elicits a therapeutic antibody response against chronic hepatitis B.Nat Nanotechnol. 2020; 15: 406-416Crossref PubMed Scopus (39) Google Scholar]. However, for an anti-allergy vaccine a more moderate immunogenicity may be preferable and even for an CHB immunotherapy an overly immunogenic vaccine may precipitate severe immune pathogenesis. Comparing BM32 with other potential pre-clinical candidates for CHB immunotherapy, it has the huge advantage that it has practically passed phase I and II trials in healthy human populations. But it remains to be seen whether it can become a true candidate for clinical trials in CHB patients. More realistic may be the complementation of the current prophylactic HBV vaccines by BM32 because there are numerous SHBs vaccine recipients with advanced age >40 years or weakened immune competence who do not produce sufficient anti-HBs antibodies. Another aspect is the genotype bias of the standard vaccines most of which contain SHBs of genotype A2 although 99% of the HBV infections worldwide have other genotypes. Vaccine producers consider HBV genotypes not relevant for the worldwide vaccination campaign, but several examples of reduced HBV genotype cross-protection exist [[7]Gerlich W.H Prophylactic vaccination against hepatitis B: achievements, challenges and perspectives.Med Microbiol Immunol. 2015; 204: 39-55Crossref PubMed Scopus (62) Google Scholar]. Recently, even a gradual selection of mutated HBV strains heterologous to the major vaccine strains was reported from China [[10]Ye H. Teng J. Lin Z. et al.Analysis of HBsAg mutations in the 25 years after the implementation of the hepatitis B vaccination plan in China.Virus Genes. 2020; https://doi.org/10.1007/s11262-020-01773-1Crossref Scopus (2) Google Scholar]. In that respect the broad genotype reactivity of the BM32 induced anti-preS response is highly relevant. Dr. Gerlich wrote the commentary. Dr. Gerlich has nothing to disclose. Quantification, epitope mapping and genotype cross-reactivity of hepatitis B preS-specific antibodies in subjects vaccinated with different dosage regimens of BM32BM32 induces high levels of IgG1 and IgG4 antibodies against the receptor binding sites of all eight HBV genotypes and hence might be suitable for therapeutic HBV vaccination. Full-Text PDF Open Access

The nomenclature of the hepatitis B virus (HBV) genes and their products has developed stepwise, occasionally in an erratic way, creating many misunderstandings, especially among those who do not know the structure of HBV and its genome in detail. One of the most frequent misunderstandings, even presented in leading journals, is the designation of HBV "e"-antigen as envelope or early antigen. Another problem area are the so-called "pre" regions in the HBV genome present upstream of both the core and the surface genes of HBV, inadvertently suggesting that they may be a part of corresponding precursor proteins. Misnomers and misclassifications are frequent in defining the subgenotypes and serological subtypes of HBV. Even the well-established terminology for HBV surface (HBs) or HBV core (HBc) antigen deviates from the conventional virological nomenclature for viral envelopes or capsid proteins/antigens, respectively. Another matter of undesirable variability between publications is the numbering of the nucleotides and the graphical representation of genomic maps. This editorial briefly explains how the nomenclature evolved, what it really means, and suggests how it could be adapted to today's knowledge.

Background & Aims: During treatment of chronic HBV infections, loss or seroconversion of the HBV surface antigen (HBsAg) is considered a functional cure. HBsAg consists of the large (LHBs), middle (MHBs), and small surface protein (SHBs) and their relative proportions correlate strongly with disease stage. Our aim was to assess the association between HBsAg composition and functional cure during treatment. Methods: A total of 83 patients were retrospectively analyzed. HBsAg loss was achieved by 17/64 patients during nucleos(t)ide analogue (NA) treatment and 3/19 patients following treatment with pegylated interferon-alfa2a (PEG-IFN) for 48 weeks. Sixty-three patients without HBsAg loss were matched as controls. LHBs, MHBs and SHBs were quantified in sera collected before and during treatment. Results: Before treatment, median MHBs levels were significantly lower in patients with subsequent HBsAg loss than in those without (p = 0.005). During treatment, MHBs and LHBs proportions showed a fast decline in patients with HBsAg loss, but not in patients with HBV e antigen seroconversion only or patients without serologic response. MHBs became undetectable by month 6 of NA treatment in all patients with HBsAg loss, which occurred on average 12.8 +/- 8.7 (0-52) months before loss of total HBsAg. Receiver-operating characteristic analyses revealed that the proportion of MHBs was the best early predictor of HBsAg loss before NA treatment (AUC = 0.726, p = 0.019). In patients achieving HBsAg loss with PEG-IFN, the proportions of MHBs and LHBs showed similar kinetics. Conclusion: Quantification of HBsAg proteins shows promise as a novel tool to predict early treatment response. These assessments may help optimize individual antiviral treatment, increasing the rates of functional cure in chronically HBV-infected patients. Lay summary: The hepatitis B surface antigen (HBsAg) is a key serum marker for viral replication. Loss of HBsAg is considered stable remission, which can be achieved with antiviral treatments. We have investigated whether the ratios of the different components of HBsAg, namely the large (LHBs) and medium (MHBs) HBsAg during different treatments are associated with the occurrence of HBsAg loss. We found that LHBs and MHBs decrease earlier than total HBsAg before HBsAg loss and we propose LHBs and MHBs as promising novel biomarker candidates for predicting cure of HBV infection. (C) 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Der HBsAg-Verlust oder die Serokonversion (SC) während der Behandlung mit Nukleos(t)id-analoga (NA) oder pegyliertem Interferon-alfa (PEG-IFN) wird als funktionelle Heilung angesehen. HBsAg besteht aus dem großen (L), mittleren (M) und kleinen (S)HBs. Bei Patienten im inaktiven Trägerstatus zeigt sich ein stark verminderter Anteil von LHBs und MHBs im Vergleich zu anderen Krankheitsstadien. Wir haben untersucht, ob bei Patienten, die unter NA- bzw. IFN-basierter Therapie eine funktionelle Heilung erreichen, eine frühzeitige Änderung der HBsAg-Zusammensetzung erkennbar ist.