Analysis of HBsAg Immunocomplexes and cccDNA Activity During and Persisting After NAP-Based Therapy

Abstract

Therapy with nucleic acid polymers (NAPs), tenofovir disoproxil fumarate (TDF), and pegylated interferon (pegIFN) achieve high rates of HBsAg loss/seroconversion and functional cure in chronic hepatitis B virus (HBV) infection. The role of hepatitis B surface antigen (HBsAg) seroconversion and inactivation of covalently closed circular DNA (cccDNA) in establishing functional cure were examined. Archived serum from the REP 401 study was analyzed using the Abbott ARCHITECT HBsAg NEXT assay (Chicago, IL), Abbott research use-only assays for HBsAg immune complexes (HBsAg ICs), circulating HBV RNA, and the Fujirebio assay for hepatitis B core-related antigen (HBcrAg; Malvern, PA). HBsAg became < 0.005 IU/mL in 23 participants during NAP exposure, which persisted in all participants with functional cure. HBsAg IC declined during lead-in TDF monotherapy and correlated with minor declines in HBsAg. Following the addition of NAPs and pegIFN, minor HBsAg IC increases (n = 13) or flares (n = 2) during therapy were not correlated with HBsAg decline, hepatitis B surface antibody (anti-HBs) titers, or alanine aminotransferase. HBsAg IC universally declined during follow-up in participants with virologic control or functional cure. Universal declines in HBV RNA and HBcrAg during TDF monotherapy continued with NAP + pegIFN regardless of therapeutic outcome. At the end of therapy, HBV RNA was undetectable in only 5 of 14 participants with functional cure but became undetectable after removal of therapy in all participants with functional cure. Undetectable HBV RNA at the end of therapy in 5 participants was followed by relapse to virologic control or viral rebound. Conclusion: Anti-HBs-independent mechanisms contribute to HBsAg clearance during NAP therapy. Inactivation of cccDNA does not predict functional cure following NAP-based therapy; however, functional cure is accompanied by persistent inactivation of cccDNA. Persistent HBsAg loss with functional cure may also reflect reduction/clearance of integrated HBV DNA. Clinicaltrials.org number NCT02565719.

© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.

Figures

FIG. 1

Baseline virologic parameters are not correlated with HBsAg response or therapeutic outcome in the REP 401 study. Differences between baseline HBsAg (A), HBsAg IC (B), ALT (C), HBV DNA (D), HBV pgRNA (E), and HBcrAg (F) in participants experiencing viral rebound (n = 11), virologic control (n = 15), or functional cure (FC, n = 14) after removal of therapy are presented. No significant difference in any baseline parameter between therapeutic outcomes was observed as determined by analysis of variance. Baseline levels of HBsAg IC were not correlated with baseline HBsAg (G), baseline ALT (H), or HBsAg reduction from baseline (I) during therapy. Abbreviations: FC, functional cure; VC, virologic control; VR, viral rebound.

FIG. 2

Analysis of HBsAg decline in the REP 401 study through high‐sensitivity HBsAg NEXT assay. (A) Design of the REP 401 trial. (B) qHBsAg response (top row) for all control (left) and experimental (middle left) participants during the first 48 weeks of treatment, for all participants achieving HBsAg ( 19 ) Abbreviations: CTL, control; EXP, experimental.

FIG. 3

Antiviral responses during TDF monotherapy in the REP 401 study. HBsAg IC decline during TDF monotherapy (A) was statistically significant and universal except for participants 01‐042 and 02‐003. Increases in declines in HBV DNA, HBV RNA, and HBcrAg were significantly greater than for HBsAg (B).

FIG. 4

Analysis of HBsAg IC dynamics in the REP 401 study. Anti‐HBs (top row) and ALT dynamics (bottom row) are provided for comparison with HBsAg IC dynamics (middle row). Individual responses for all control (left) and experimental (middle left) participants during the first 48 weeks of treatment, for all participants achieving HBsAg ( 19 ) Anti‐HBs and HBsAg IC responses in all participants experiencing viral rebound during follow‐up are presented in Supporting Fig. S2.

FIG. 5

Analysis of HBsAg (top two rows) and HBsAg IC (bottom row) in participants (n = 15) with increase in HBsAg IC (≥ 1.5× baseline) during NAP therapy. Individual responses for all control and experimental participants during the first 48 weeks of therapy (left), for participants during NAP combination therapy (middle) and during follow‐up (left) are provided. Individual responses for the 2 participants experiencing “flares” in HBsAg IC during therapy (01‐007 and 02‐050) are highlighted in bold for all virologic markers examined. Treatment paradigms and proportions of control, experimental, REP 2139‐Mg, and REP 2165‐Mg are indicated at the top (see also Fig. 2A). qHBsAg responses were previously published.( 19 )

FIG. 6

Analysis of HBV RNA and HBcrAg dynamics in the REP 401 study. HBV‐DNA dynamics (top row) are provided for comparison with HBV RNA (middle row) and HBcrAg (bottom row). Individual responses for all control and experimental participants during the first 48 weeks of therapy (left), for all participants during NAP combination therapy (middle) and maintaining HBV DNA ( 19 )