The challenges of adopting immunological biomarkers in the management of chronic HBV infection

See Article, pages 525–538 See Article, pages 525–538 The outcome of most viral diseases is determined by the balance between viral replication and host antiviral immunity. The severity of virus-induced pathology is often directly proportional to the viral load, which is inversely proportional to the level of host antiviral immunity. In HBV infection, this paradigm holds when we compare HBV-infected patients who resolve infection to those who develop chronicity.[1]Bertoletti A. Ferrari C. Adaptive immunity in HBV infection.J Hepatol. 2016; 64: S71-S83Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar A coordinated virus-specific humoral and cellular immune response linked with a rapid disappearance of HBV virological parameters (HBV-DNA, HBsAg) characterizes both the animal models[2]Thimme R. Wieland S. Steiger C. Ghrayeb J. Reimann K.A. Purcell R.H. et al.CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection.J Virol. 2003; 77: 68-76Crossref PubMed Scopus (731) Google Scholar and the patients who became HBsAg negative after acute hepatitis B.[1]Bertoletti A. Ferrari C. Adaptive immunity in HBV infection.J Hepatol. 2016; 64: S71-S83Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar A defect of the antiviral immune response and the persistence of virological parameters instead defines chronic HBV (CHB).[1]Bertoletti A. Ferrari C. Adaptive immunity in HBV infection.J Hepatol. 2016; 64: S71-S83Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar This dichotomy represents the rationale that supported the development of HBV therapies that do not directly target HBV replication but are instead designed to restore directly or indirectly the anti-HBV immune response.[3]Fanning G.C. Zoulim F. Hou J. Bertoletti A. Therapeutic strategies for hepatitis B virus infection: towards a cure.Nat Rev Drug Disc. 2019; 18: 827-844https://doi.org/10.1038/s41573-019-0037-0Crossref PubMed Scopus (194) Google Scholar Different immunotherapies aim to activate innate immunity (i.e. TLR agonists), to overcome the functional defects of “exhausted” HBV-specific T and B cells (i.e. checkpoint inhibitors), to induce new HBV-specific T and B cells (therapeutic vaccines) or to elicit a functional recovery of HBV-specific immunity trough inhibition of HBsAg production (antisense oligonucleotides, small-interfering RNA). Most of them have been used in recent years alone or in combination to achieve HBV functional cure. The evaluation of their efficacy and a robust understanding of their mechanisms in vivo has often been hindered by the lack of detailed characterization of their impact on the immune components that they are designed to restore. One other consideration that slowed down the clinical management of these new therapies, is that immunological defects are heterogeneous in the broad population of patients with CHB and cannot be predicted only by patient selection based on inflammatory liver parameters (alanine aminotransferase) and/or virological quantification. For example, the quantity of intrahepatic HBV-specific T cells is independent of the inflammatory status of the liver.[4]Maini M.K. Boni C. Lee C.K. Larrubia J.R. Reignat S. Ogg G.S. et al.The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection.J Exp Med. 2000; 191: 1269-1280Crossref PubMed Scopus (663) Google Scholar Additionally, in HBeAg-negative patients with CHB, even though the frequency of circulating HBV-core and polymerase-specific CD8 T cells is inversely related to HBV replication,[5]Schuch A. Alizei E.S. Heim K. Wieland D. Kiraithe M.M. Kemming J. et al.Phenotypic and functional differences of HBV core-specific versus HBV polymerase-specific CD8+ T cells in chronically HBV-infected patients with low viral load.Gut. 2019; (gutjnl-2018-316641)https://doi.org/10.1136/gutjnl-2018-316641Crossref PubMed Scopus (70) Google Scholar,[6]Aliabadi E. Urbanek-Quaing M. Maasoumy B. Bremer B. Grasshoff M. Li Y. et al.Impact of HBsAg and HBcrAg levels on phenotype and function of HBV-specific T cells in patients with chronic hepatitis B virus infection.Gut. 2021; (gutjnl-2021-324646)https://doi.org/10.1136/gutjnl-2021-324646Crossref PubMed Google Scholar similar quantities of total circulating HBV-specific T cells are observed in HBeAg+ or HBeAg- patients with CHB and are characterized by different quantities of HBV DNA.[7]Bert N.L. Gill U.S. Hong M. Kunasegaran K. Tan D.Z.M. Ahmad R. et al.Effects of hepatitis B surface antigen on virus-specific and global T cells in patients with chronic hepatitis B virus infection.Gastroenterology. 2020; 159: 652-664https://doi.org/10.1053/j.gastro.2020.04.019Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar The HBsAg level is also a poor indication of the anti-HBV immunological profile. Quantity and functionality of HBs-specific B cells are not linked with HBsAg quantity[8]Salimzadeh L. Bert N.L. Dutertre C.-A. Gill U.S. Newell E.W. Frey C. et al.PD-1 blockade partially recovers dysfunctional virus-specific B cells in chronic hepatitis B infection.J Clin Invest. 2018; 128: 4573-4587https://doi.org/10.1172/jci121957Crossref PubMed Scopus (0) Google Scholar and the quantity of HBs-specific T cells appears to be inversely correlated with duration of infection and not with HBsAg quantity.[7]Bert N.L. Gill U.S. Hong M. Kunasegaran K. Tan D.Z.M. Ahmad R. et al.Effects of hepatitis B surface antigen on virus-specific and global T cells in patients with chronic hepatitis B virus infection.Gastroenterology. 2020; 159: 652-664https://doi.org/10.1053/j.gastro.2020.04.019Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar Finally, the functionality of HBV-specific T cells is not fixed but can be modified by the modality of antigen presentation.[9]Bénéchet A.P. Simone G.D. Lucia P.D. Cilenti F. Barbiera G. Bert N.L. et al.Dynamics and genomic landscape of CD8+ T cells undergoing hepatic priming.Nature. 2019; 4 (1182–29)https://doi.org/10.1038/s41586-019-1620-6Crossref Scopus (80) Google Scholar,[10]Isogawa M. Chung J. Murata Y. Kakimi K. Chisari F.V. CD40 activation rescues antiviral CD8+ T cells from PD-1-mediated exhaustion.PLoS Path. 2013; 9e1003490https://doi.org/10.1371/journal.ppat.1003490Crossref PubMed Scopus (101) Google Scholar The result of this complexity is that if we exclude presence or absence of anti-HBs and anti-HBe antibodies, we do not make use of HBV-specific immunological parameters to categorize the heterogeneous CHB patient population and, as such, new immunotherapies are evaluated in patients with CHB who are likely to present marked differences in their anti-HBV immune status. In this issue of the Journal of Hepatology, the “Immune monitoring working group”, a consortium of experts of HBV immunology coordinated by Adam Gehring, provide an important guide to overcome these shortcomings and promote the use of well-defined immunological biomarkers to evaluate the efficacy of HBV treatments. In their article entitled “Guidance for immunological biomarker discovery in cure regimens for chronic hepatitis B virus infection” they offer detailed recommendations for incorporating different immunological assays into clinical research linked to the treatment of CHB. A clear description of the different cell populations that can be studied both in the blood or in the liver is provided and the parameters and the different methods (ELISPOT, intracellular cytokine staining, HLA-multimers, immune profiling and single-cell RNA sequencing) that can be used to measure the functionality of HBV-specific T and B cells, but also of other immune cell populations (natural killer, mucosal-associated invariant T, myeloid cells) are discussed. The problems of sample collection, processing and methods standardization are also highlighted. The overall aim is to boost the development of immunological biomarkers that should be used to guide therapeutic selection in patients with CHB and to analyze the mechanisms of action of novel immunotherapies. The readers of this article will however note that the authors are still agnostic about the best immunological markers that should be used to define, for example, the patients who are more likely to achieve functional cure after immune therapy. Indeed, as they pointed out, we do not know which immunological biomarker correlates with CHB functional cure and since novel HBV therapies are designed to target different mechanisms,[3]Fanning G.C. Zoulim F. Hou J. Bertoletti A. Therapeutic strategies for hepatitis B virus infection: towards a cure.Nat Rev Drug Disc. 2019; 18: 827-844https://doi.org/10.1038/s41573-019-0037-0Crossref PubMed Scopus (194) Google Scholar immunological markers might be treatment specific. The manuscript does emphasize the measurement of circulating or intrahepatic HBV-specific adaptive immunity since the role of HBV-specific T and B cells in viral control is well demonstrated, at least in patients who clear HBV after acute infection. However, the long-term persistence of HBV in patients with CHB alters the repertoire and function of HBV-specific B[8]Salimzadeh L. Bert N.L. Dutertre C.-A. Gill U.S. Newell E.W. Frey C. et al.PD-1 blockade partially recovers dysfunctional virus-specific B cells in chronic hepatitis B infection.J Clin Invest. 2018; 128: 4573-4587https://doi.org/10.1172/jci121957Crossref PubMed Scopus (0) Google Scholar,[11]Burton A.R. Pallett L.J. McCoy L.E. Suveizdyte K. Amin O.E. Swadling L. et al.Circulating and intrahepatic antiviral B cells are defective in hepatitis B.J Clin Invest. 2018; 128: 4588-4603https://doi.org/10.1172/jci121960Crossref PubMed Scopus (0) Google Scholar and T cells.5Schuch A. Alizei E.S. Heim K. Wieland D. Kiraithe M.M. Kemming J. et al.Phenotypic and functional differences of HBV core-specific versus HBV polymerase-specific CD8+ T cells in chronically HBV-infected patients with low viral load.Gut. 2019; (gutjnl-2018-316641)https://doi.org/10.1136/gutjnl-2018-316641Crossref PubMed Scopus (70) Google Scholar, 6Aliabadi E. Urbanek-Quaing M. Maasoumy B. Bremer B. Grasshoff M. Li Y. et al.Impact of HBsAg and HBcrAg levels on phenotype and function of HBV-specific T cells in patients with chronic hepatitis B virus infection.Gut. 2021; (gutjnl-2021-324646)https://doi.org/10.1136/gutjnl-2021-324646Crossref PubMed Google Scholar, 7Bert N.L. Gill U.S. Hong M. Kunasegaran K. Tan D.Z.M. Ahmad R. et al.Effects of hepatitis B surface antigen on virus-specific and global T cells in patients with chronic hepatitis B virus infection.Gastroenterology. 2020; 159: 652-664https://doi.org/10.1053/j.gastro.2020.04.019Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar As a result, the quantitative and functional parameters that might define which patients with CHB are more likely to respond to a particular therapy are still unknown and need to be longitudinally studied and correlated with therapeutic success or failure. A preference for analyzing immunological markers ex vivo instead of after in vitro manipulation is also advised. This recommendation has a strong rationale. In vitro expansion alters the phenotype and function of immune cells. Thus, the methods proposed to define the characteristics of immune cells (phenotypic and functional analysis of HLA-tetramer+, single-cell analysis) might lose significance after in vitro manipulation. Nevertheless, in vitro expansion of HBV-specific T cells has been utilized by many research groups for rational reasons. HBV-specific immune cells (B and T) are present at notoriously low frequencies in the circulation of patients with CHB. In vitro expansion of specific T cell populations after stimulation with different HBV antigens often enables the determination of a wider repertoire of T cells specific for different antigens using a limited number of initial cells.[12]Boni C. Laccabue D. Lampertico P. Giuberti T. Viganò M. Schivazappa S. et al.Restored function of HBV-specific T cells after long-term effective therapy with nucleos(t)ide analogues.Gastroenterology. 2012; 143: 963-973.e9https://doi.org/10.1053/j.gastro.2012.07.014Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar,[13]Rehermann B. Lau D. Hoofnagle J.H. Chisari F.V. Cytotoxic T lymphocyte responsiveness after resolution of chronic hepatitis B virus infection.J Clin Invest. 1996; 97: 1655-1665https://doi.org/10.1172/jci118592Crossref PubMed Google Scholar This might be difficult to achieve with direct ex vivo analysis, which has to be performed with much larger quantities of cells.[5]Schuch A. Alizei E.S. Heim K. Wieland D. Kiraithe M.M. Kemming J. et al.Phenotypic and functional differences of HBV core-specific versus HBV polymerase-specific CD8+ T cells in chronically HBV-infected patients with low viral load.Gut. 2019; (gutjnl-2018-316641)https://doi.org/10.1136/gutjnl-2018-316641Crossref PubMed Scopus (70) Google Scholar In addition, the ability of HBV-specific T cells to proliferate in vitro represents by itself a functional read-out and currently the only proposed predictive immunological biomarker linked with a favorable clinical outcome (HBV viral control after nucleoside analogue withdrawal).[14]Rivino L. Bert N.L. Gill U.S. Kunasegaran K. Cheng Y. Tan D.Z. et al.Hepatitis B virus-specific T cells associate with viral control upon nucleos(t)ide-analogue therapy discontinuation.J Clin Invest. 2018; 128: 668-681https://doi.org/10.1172/jci92812Crossref PubMed Scopus (0) Google Scholar As such, I am personally of the opinion that the in vitro method of immunological analysis could still offer, in select circumstances, some advantages and should not only be seen as a souvenir of the past. Leaving this specific argument to the limited numbers of human HBV immunology experts, one other problem that this review exposed, is the overall technical complexity of the assays proposed. Most of the methods discussed (HLA-tetramer staining, intracellular cytokine staining, single-cell RNA analysis) necessitate highly specialized personnel and equipment. Even a relatively simpler assay such as Fluorospot, still requires separation of peripheral blood mononuclear cells from blood, which is a time-consuming step that needs to be performed with highly standardized procedures. The article recommendations are therefore mainly directed to the limited number of research laboratories actively involved in the development of new treatments and will certainly help to set up a correct experimental system to facilitate the discovery of the biomarker(s) linked to HBV cure. Nevertheless, I am also wondering whether the inherent complexity of the assays currently used to evaluate HBV-specific antiviral immunity represents the actual problem that has prevented their wide use in clinical practice. The consequence, as I wrote above, is that selection of therapies for patients with CHB is not based on robust HBV-specific immune parameters, which has limited the proper measurement of the potential efficacy of immune therapies. Perhaps a different way forward is to also develop more widely applicable, point of care, immunological tests that might allow for the much easier and more generalized measurement of immunological parameters in patients with CHB. On this aspect, the recent COVID-19 pandemic has demonstrated that the research community can develop such rapid and easy-to-use assays. Direct SARS-CoV-2 antigen diagnostic tests[15]Brümmer L.E. Katzenschlager S. Gaeddert M. Erdmann C. Schmitz S. Bota M. et al.Accuracy of novel antigen rapid diagnostics for SARS-CoV-2: a living systematic review and meta-analysis.Plos Med. 2021; 18e1003735https://doi.org/10.1371/journal.pmed.1003735Crossref PubMed Scopus (74) Google Scholar enable point of care analysis of viral presence that individuals can self-administer, the presence of neutralizing antibodies against SARS-CoV-2 can be tested by surrogate neutralization tests that do not require any infectious system[16]Tan C.W. Chia W.N. Qin X. Liu P. Chen M.I.-C. Tiu C. et al.A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction.Nat Biotec. 2020; 395: 470-476https://doi.org/10.1038/s41587-020-0631-zCrossref Scopus (487) Google Scholar and SARS-CoV-2-specific T cell functionality can be rapidly measured directly in whole blood.[17]Tan A.T. Lim J.M.E. Bert N.L. Kunasegaran K. Chia A. Qui M.D.C. et al.Rapid measurement of SARS-CoV-2 spike T cells in whole blood from vaccinated and naturally infected individuals.J Clin Invest. 2021; 131https://doi.org/10.1172/jci152379Crossref Google Scholar Similar approaches translated to HBV might facilitate the wider and more frequent quantitative analysis of immunological parameters and would enable the stratification of patients based on their likelihood of benefitting from different immunotherapeutic approaches. Top-down and bottom-up approaches can co-exist and might provide the most rational approach to test new immunotherapies and define with precision their mechanism of action. This work was funded by Singapore Ministry of Health’s National Medical Research Council under its Singapore Translational Research Investigator Award (MOH-000019). Antonio Bertoletti (AB) declares the following relationship with commercial entities developing therapeutics for HBV treatment. He acted as a consultant and served on the advisory boards of Assembly Biosciences, Gilead Sciences, Roche, Janssen, Glaxo-SmithKline, Vir, Molecular Therapies and HUMABS BioMed. AB is also a co-founder of LION TCR pte. ltd. a biotech company developing T cell receptors for treatment of virus-related cancers and chronic viral diseases. Please refer to the accompanying ICMJE disclosure form for further details. The following are the supplementary data to this article: Download .pdf (.18 MB) Help with pdf files Multimedia component 1 Immunological biomarker discovery in cure regimens for chronic hepatitis B virus infectionJournal of HepatologyVol. 77Issue 2PreviewThere have been unprecedented advances in the identification of new treatment targets for chronic hepatitis B that are being developed with the goal of achieving functional cure in patients who would otherwise require lifelong nucleoside analogue treatment. Many of the new investigational therapies either directly target the immune system or are anticipated to impact immunity indirectly through modulation of the viral lifecycle and antigen production. While new viral biomarkers (HBV RNA, HBcAg, small, middle, large HBs isoforms) are proceeding through validation steps in clinical studies, immunological biomarkers are non-existent outside of clinical assays for antibodies to HBs, HBc and HBe. Full-Text PDF Open Access