Ultrasensitive HBsAg testing predicts HBsAg seroreversion outcomes: considerations for new and existing therapies

Guidance on treatment endpoints and study design for clinical trials aiming to achieve cure in chronic hepatitis B and D: Report from the 2022 AASLD-EASL HBV-HDV Treatment Endpoints ConferenceJournal of HepatologyVol. 79Issue 5PreviewRepresentatives from academia, industry, regulatory agencies, and patient advocacy groups convened under the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) in June 2022 with the primary goal of achieving consensus on chronic HBV and HDV treatment endpoints to guide clinical trials aiming to "cure" HBV and HDV. Conference participants reached an agreement on some key points. The preferred primary endpoint for phase II/III trials evaluating finite treatments for chronic hepatitis B (CHB) is a "functional" cure, defined as sustained HBsAg loss and HBV DNA less than the lower limit of quantitation (LLOQ) 24 weeks off-treatment. Full-Text PDF We recently read the AASLD and EASL 2022 HBV/HDV treatment endpoints and study design conference consensus statement with great interest[1]Ghany M.G. Buti M. Lampertico P. Lee H.M. Guidance on treatment endpoints and study design for clinical trials aiming to achieve cure in chronic hepatitis B and D: Report from the 2022 AASLD-EASL HBV/HDV treatment endpoints conference.J Hepatol. 2023; Google Scholar. AASLD and EASL recommended "functional cure" as the primary endpoint for new HBV therapeutics under development, defined as HBsAg loss(S-loss) using a conventional HBsAg assay(limit of detection 0.05IU/mL) and HBV DNA below the lower limit of quantification(<10IU/mL) at 24 weeks off-therapy[1]Ghany M.G. Buti M. Lampertico P. Lee H.M. Guidance on treatment endpoints and study design for clinical trials aiming to achieve cure in chronic hepatitis B and D: Report from the 2022 AASLD-EASL HBV/HDV treatment endpoints conference.J Hepatol. 2023; Google Scholar. Currently, predicting whether patients with therapy driven S-loss will undergo HBsAg seroreversion or maintain S-loss after therapy is discontinued, is difficult. Studies assessing functional cure durability in patients with nucleos(t)ide analog (NA) driven S-loss and consolidation periods have reported low rates of HBsAg seroreversion, however S-loss in NA treated patients is rare[2]Chi H. Wong D. Peng J. et al.Durability of Response After Hepatitis B Surface Antigen Seroclearance During Nucleos(t)ide Analogue Treatment in a Multiethnic Cohort of Chronic Hepatitis B Patients: Results After Treatment Cessation.Clinical Infectious Diseases. 2017; 65: 680-683Crossref Scopus (28) Google Scholar,[3]Kim G.A. Lim Y.S. An J. et al.HBsAg seroclearance after nucleoside analogue therapy in patients with chronic hepatitis B: clinical outcomes and durability.Gut. 2014; 63: 1325-1332Crossref PubMed Scopus (280) Google Scholar. HBsAg seroreversion has also been observed in patients with interferon induced S-loss with the majority of seroreversion events occurring in the first year after therapy removal[4]Wu Y. Liu Y. Lu J. et al.Durability of Interferon-induced Hepatitis B Surface Antigen Seroclearance.Clin Gastroenterol Hepatol. 2020; 18 (6.e2): 514Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar,[5]Pan C.Q. Li M.H. Yi W. et al.Outcome of Chinese patients with hepatitis B at 96 weeks after functional cure with IFN versus combination regimens.Liver Int. 2021; 41: 1498-1508Crossref Scopus (23) Google Scholar. Importantly, patients receiving more than 12 weeks of interferon consolidation treatment after S-loss were observed to have lower rates of HBsAg seroreversion[6]Li M. Sun F. Bi X. et al.Consolidation treatment needed for sustained HBsAg-negative response induced by interferon-alpha in HBeAg positive chronic hepatitis B patients.Virol Sin. 2022; 37: 390-397Crossref Scopus (7) Google Scholar. HBsAg seroreversion was also observed in clinical trials of new therapies. In a recent phase 2b study of Bepirovirsen for Chronic Hepatitis B7, only 17/40 patients with HBsAg loss at the end-of-therapy sustained it to end of 24weeks follow-up in the on-NA arm, with 16/40 patients at end-of-therapy in the not-on-NA arm sustaining it to end of 24 weeks follow-up; consequently, over 50% of participants who achieved HBsAg<0.05IU/mL by the end-of-treatment rebounded to HBsAg>0.05IU/mL by week 24 of follow-up. Combined, these findings suggest that residual amounts of HBsAg(<0.05IU/mL) might be present in patients exhibiting off-therapy HBsAg seroreversion and that more sensitive HBsAg assays might help identify which patients are more likely to serorevert. To address this question, we tested samples from patients who achieved S-loss while on NA/PEG-IFN therapy in the INACTIVE(NCT02992704) and SWAP(NCT01928511) research clinical trials using the HBsAg Next Qualitative assay(Abbott Laboratories) which has an analytical sensitivity at the cutoff of 0.005 IU/mL[8]Lim S.G. Yang W.L. Ngu J.H. et al.Switching to or Add-on Peginterferon in Patients on Nucleos(t)ide Analogues for Chronic Hepatitis B: The SWAP RCT.Clin Gastroenterol Hepatol. 2022; 20: e228-e250Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar,[9]Lou S. Taylor R. Pearce S. Kuhns M. Leary T. An ultra-sensitive Abbott ARCHITECT(®) assay for the detection of hepatitis B virus surface antigen (HBsAg).J Clin Virol. 2018; 105: 18-25Crossref PubMed Scopus (33) Google Scholar,[10]Kuhns M.C. Holzmayer V. McNamara A.L. Sickinger E. Schultess J. Cloherty G.A. Improved detection of early acute, late acute, and occult Hepatitis B infections by an increased sensitivity HBsAg assay.J Clin Virol. 2019; 118: 41-45Crossref PubMed Scopus (16) Google Scholar. Stored serum samples from 36 individuals enrolled in the INACTIVE(n=16) and SWAP(n=20) studies were selected for HBsAg Next testing based on the identification of S-loss(HBsAg<0.05IU/mL) at the end of treatment(EOT). Samples that remained HBsAg <0.05 IU/mL at 24-week follow-up timepoints were also tested with HBsAg Next. Since both studies had well-defined EOT and 24-week post therapy removal timepoints, a combined analysis was conducted. Among the 36 total participants with on-therapy S-loss, 11(30.5%) had HBsAg seroreversion at 24-weeks of follow-up, and 10/11(90.9%) of these individuals were HBsAg Next reactive(S/CO≥1.0) at EOT(Figure 1). The remaining 25 participants maintained S-loss at 24-weeks of follow-up, and 19/25(76.0%) were HBsAg Next non-reactive(S/CO<1.0) at EOT. Contingency analysis(p=0.0003, Fisher's exact test) revealed a HBsAg Next reactive result Relative Risk (95% CI) for HBsAg seroreversion of 12.50(2.476-72.14). Of the 20 patients who were HBsAg Next non-reactive at EOT, seroreversion (>0.05 IU/mL) occurred in 1/20 patients at the 24-week follow-up timepoint(Figure 1). Longer follow-up was available for many of the patients with EOT HBsAg Next non-reactive results and identified a further 1/18 and 1/15 additional individuals with HBsAg seroreversion at 48-week and 2–3-year timepoints, respectively. Functional cure is a primary endpoint goal of finite duration therapy for chronic HBV patients[1]Ghany M.G. Buti M. Lampertico P. Lee H.M. Guidance on treatment endpoints and study design for clinical trials aiming to achieve cure in chronic hepatitis B and D: Report from the 2022 AASLD-EASL HBV/HDV treatment endpoints conference.J Hepatol. 2023; Google Scholar however, patients who achieve S-loss while on therapy may serorevert once therapy has concluded. Using a highly sensitive HBsAg assay we have shown that HBsAg was still detectable at EOT in 90.9% of patients who exhibited 24-week HBsAg seroreversion after treatment discontinuation, while most who were HBsAg Next non-reactive maintained S-loss at last follow-up of 2-3 years. These observations may explain why some patients, but not others, exhibit HBsAg seroreversion after treatment discontinuation in other therapeutic studies[4]Wu Y. Liu Y. Lu J. et al.Durability of Interferon-induced Hepatitis B Surface Antigen Seroclearance.Clin Gastroenterol Hepatol. 2020; 18 (6.e2): 514Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar,[5]Pan C.Q. Li M.H. Yi W. et al.Outcome of Chinese patients with hepatitis B at 96 weeks after functional cure with IFN versus combination regimens.Liver Int. 2021; 41: 1498-1508Crossref Scopus (23) Google Scholar,[7]Yuen M.F. Lim S.G. Plesniak R. et al.Efficacy and Safety of Bepirovirsen in Chronic Hepatitis B Infection.N Engl J Med. 2022; 387: 1957-1968Crossref PubMed Scopus (47) Google Scholar. As new therapeutics are developed which result in high rates of on-therapy S-loss it may be necessary to achieve HBsAg levels below 0.005 IU/mL to increase the chances of sustained S-loss after discontinuation. Furthermore, it may be necessary to administer therapy until HBsAg<0.005 IU/mL is reached to achieve functional cure, as currently defined. The original functional cure definition was based on HBsAg assays with sensitivities of 0.05 IU/ml, as these were the best available assays, but this level may not have biological significance nor correspond with durability but is associated with clinical benefits and has clinical significance. The recent Bepirovirsen phase 2b RCT[7]Yuen M.F. Lim S.G. Plesniak R. et al.Efficacy and Safety of Bepirovirsen in Chronic Hepatitis B Infection.N Engl J Med. 2022; 387: 1957-1968Crossref PubMed Scopus (47) Google Scholar demonstrated that HBsAg loss is not durable in over half the patients based on end-of-therapy HBsAg<0.05 IU/ml. Our study of NA and IFN treated patients indicates that the current threshold of HBsAg<0.05 IU/mL may be insufficient to identify durable HBsAg loss, whereas HBsAg <0.005IU/mL provides a high predictive value for durable HBsAg loss, and this may help explain the seroreversion observed with Bepirovirsen. However, since this is a small study, larger studies and longer follow-up periods are needed for confirmation, and validation in the Bepirovirsen phase 2B study would be beneficial. The current functional cure endpoint of HBsAg loss based on a threshold of 0.05 IU/mL or lower may need to be reconsidered as new agents achieve higher rates of HBsAg loss. MA, VH, MS, and GC are employees and shareholders of Abbott Laboratories. This work was supported by Abbott Diagnostics Division Research and Development funding. MA: Conceptualization, data analysis, manuscript writing; VH: Data collection; MS: Data analysis and statistics; SGL: Patient recruitment, study design, critical revision; GC: conceptualization, critical revision.