Background and aims: After completion of NAP-based combination therapy with pegIFN and follow-up in the REP 301-LTF study (3.5 years) and REP 401 study (48 weeks), combined HBV outcomes were functional cure (HBsAg <0.05 IU/mL, HBV DNA target not detected, normal ALT [FC]) in 18/52 (35%), virologic control (HBV DNA ≤2000 IU/mL, normal ALT [VC]) in 19/52 (36%) and rebound (R) in 15/52 (29%) of participants. The goal of this study was to analyze the relationship between HBV outcome and experimental virologic markers of HBV infection. Methods: Frozen serum samples (n = 1153) from all 52 participants in the REP 301/REP 301-LTF and REP 401 studies were analyzed by the following: 1. Abbott ARCHITECT HBsAg NEXT (analytical sensitivity 0.005 IU/ mL). 2. Abbott research use only (RUO) assays for HBsAg isoforms (large, medium, small). 3. Abbott RUO assay for HBsAg/anti-HBs immune complexes (HBsAg IC). 4. Abbott RUO assay for pregenomic HBV RNA (pgRNA). 5. Fujirebio HBcrAg (LLOQ 3log10 U/mL). Results: All participants experiencing HBsAg loss (<0.05 IU/mL) during therapy (28/52) rapidly became negative with HBsAg Next. HBsAg <0.005 IU/mL was confirmed at the end of follow-up in 18/18 functional cure and 1/1 virologic control participants with previous HBsAg <0.05 IU/mL. A more rapid reduction of S-HBsAg relative to the reduction of other HBsAg isoforms occurred in 39/40 participants with HBsAg decline >2 log10 from baseline, consistent with targeting SVPs. At the end of follow-up, HBsAg isoforms were detectable in 0/18 (FC), 17/19 (VC) and 15/15 (R) participants. HBsAg IC (relative luminescence units or RLU) were in the positive range in 30/52 participants at baseline and at the end of follow-up in 0/18 (FC), 5/19 (VC) and 10/15 (R) participants. At baseline, HBV RNA and HBcrAg were present in 42 and 34 participants. HBV RNA loss on therapy occurred in 5/ 14 (FC), 5/16 (VC) and 1/12 (R) participants. HBcrAg < LLOQ on therapy occurred in 5/9 (FC), 7/15 (VC) and 3/10 (R) participants. At the end of follow-up, both HBV RNA and HBcrAg were > LLOQ in 1/18 (FC), 15/19 (VC) and 13/15 (R) participants. Conclusion: Functional cure of HBV infection following NAP-based combination therapy is profound, with HBsAg <0.005 IU/mL and both HBV RNA and HBcrAg < LLOQ. NEXT negativity and the absence of HBsAg IC RLUs in the positive range at the end of follow-up in FC participants suggests efficient reduction in integrated HBV DNA.