Chronic hepatitis B affects around 257 million people worldwide. Current hepatitis B virus (HBV) therapeutic paradigms remain anchored to nucleos(t)ide analog (NA) for sustained viral suppression. The residual infectivity of HBV under NA therapy remains a critical unresolved issue in clinical hepatology while discrepant conclusions from cellular and humanized murine models [1,2]. This controversy has profound implications for patient management, public health strategies, and therapeutic goal setting in HBV treatment.
Ethical restrictions preclude direct human experimentation to resolve this biological paradox. However, two decompensated cirrhosis patients with persistent viremia (HBV DNA 914–3,120 IU/mL) undergoing auxiliary partial orthotopic liver transplantation (APOLT) provided unique chance to resolve this dilemma. The two enrolled patients presented with decompensated HBV-related cirrhosis complicated by recurrent variceal hemorrhage refractory to endoscopic intervention. Living donor liver transplantation was necessi-tated by critical organ shortage. However, preoperative evaluations indicated that the donor derived graft (hereafter 'graft') would be too small for the recipients using left lobe graft (with graft-to-recipient weight ratio [GRWR] <0.6), but the donor is not safe while using right lobe graft (remnant liver volume <35%). Thus, APOLT was considered to ensure donor safety and prevent small for size syndrome.
After APOLT, esophagogastric varices and refractory ascites regression confirmed by gastroscope and CT scan (Supplementary Fig. 1A, 1B). No instances of small-for-size syndrome, acute cellular rejection, or surgical morbidity (Clavien-Dindo ≥III) occurred in recipients. Recipients’ liver function successfully restored to normal (Supplementary Fig. 1C, 1D). All donors achieved complete hepatic regen-eration without complications. Sequential recipient native liver (hereafter 'native liver') resection was performed at 10–12 weeks post-APOLT following confirmed graft regenerative hypertrophy (GRWR >0.8) to prevent graft infection and native liver neoplasia. The situation of HBV infection has also been further studied.
Throughout the 97-day (Patient 1) and 71-day (Patient 2) graft-native liver coexistence under continuous NA therapy, Patient 1’s native liver exhibited concurrent 2+ HBsAg and 3+ HBcAg staining intensity (Fig. 1A, upper panel), associated with high intrahepatic cccDNA loads (7,075 copies/105 cells) and persistent HBeAg (24.13→16.91 S/CO) for 97 days with persistent serum viremia (914–851 IU/mL). Patient 2 underwent rapid HBeAg clearance (10.46→1.51 S/CO in 14 days) accompanied by HBV DNA decline (3,120→<20 IU/mL) but 3+ HBsAg and negative HBcAg staining intensity (Fig. 1A, lower panel). Crucially, both patients’ allografts remained virologically naive throughout the observation period, showing no HBsAg/HBcAg expression (IHC score 0, Fig. 1A, left panel) and undetectable cccDNA (<1 copy/105 cells).
Post-transplant serological trajectories revealed two distinct phases (Fig. 1B). During the graft-native liver coexistence phase, both patients exhibited progressive HBsAg decline (Patient 1: higher than 250→101.04 IU/mL; Patient 2: higher than 250→154.77 IU/mL) without HBsAb seroconversion (<10 mIU/mL) despite HBIG administration (Fig. 1B, green arrow, 4,000 IU intraoperatively + 2,000 IU/day ×7 days). Strikingly, complete native liver resection triggered immediate HBsAg clearance (Fig. 1B, red arrow) followed by sustained virological remission (HBsAg <0.05 IU/mL, HBV DNA <20 IU/mL) through follow-up.
Longitudinal nanopore sequencing uncovered dynamic shifts in HBV transcription sources. At APOLT, cccDNA-de-rived transcripts dominated (Patient 1: 85.8%; Patient 2: 49.2%) in native livers, producing intact pgRNA and Pre-C RNA (Fig. 1C, patient 1 pre). At completion hepatectomy, integrated HBV DNA contributions increased significantly, cccDNA-derived transcripts decreased (Patient 1: 56.5%; Patient 2: 11.7%) in native livers, However, residual cccDNA activity persisted with intact pgRNA detected (Fig. 1C, patient 1 post).
In Patient 2, serum HBV DNA became undetectable (<20 IU/mL) by week 2 post-APOLT, while HBsAg persisted at 78.32–154.77 IU/mL during coexistence period. Transcriptomic profiling revealed 2.1/2.4 kb HBsAg mRNA derived from integrated HBV DNA (Fig. 1C, patient 2 pre and patient 2 post), accounting for residual antigenemia in native liver during virological suppression. This integrated transcriptional activity resolved completely following native liver excision, confirming integrated genomes as a source of HBsAg independent of active replication.
While NAs prophylaxis effectively reduces perinatal transmission [1,2], this clinical scenario provides limited mechanistic insights as placental trophoblast layers restrict HBV virion passage, with vertical transmission primarily occurring in high-replicative mothers (HBeAg+, HBV DNA >106 IU/mL), Thus, mother-to-child transmission models are confounded by complex maternal-fetal interface dynamics rather than directly reflecting virion infectivity. APOLT overcomes these limitations through its unique anatomical configuration - direct vascular continuity between graft and native liver compartments [3] permits longitudinal virological surveillance in an in vivo human system.
The complete absence of graft infection despite prolonged coexistence with replicative native livers (cccDNA 544.8–7,075 copies/105 cells) and persistent low-level viremia provides definitive clinical validation of NA’ capacity to block HBV transmission. Notably, the delayed HBsAg seroconversion until native liver excision implicates integrated HBV DNA as the sustaining reservoir for antigen production, consistent with recent findings on viral persistence mechanisms.
This biological paradox—transcriptional activity without infectivity—fundamentally reshapes the paradigm of HBV management under NA therapy. While our data suggest HBIG sparing regimens merit investigation in select APOLT recipients with sustained virological suppression, this strategy requires validation in larger cohorts with extended follow-up. The substantial cost differential (~$15,000/decade [4,5]) never theless highlights the impor tance of developing biomarker-driven HBIG stewardship protocols.
While our findings provide the first human evidence of NA-mediated transmission blockade, the study's generalizability is constrained by the small cohort size inherent to APOLT's rarity. Patients with higher baseline viremia (>104 IU/mL) or NA resistance mutations might exhibit different transmission dynamics. Future multi-center registries tracking APOLT outcomes across HBV genotypes and treatment histories will help validate these observations.
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