Background HBsAg can be derived from intrahepatic cccDNA and integrated HBV DNA (iDNA). We examined the iDNA from liver tissues of 24 HBeAg(+) and 32 HBeAg(−) treatment-naive CHB participants.

Methods Liver tissues were obtained from the North American Hepatitis B Research Network (HBRN). For cccDNA analysis, DNA was heat-denatured and digested by plasmid-safe ATP-dependent DNase to remove rcDNA and iDNA prior to qPCR. For iDNA detection, total DNA was subjected to HBV hybridization-targeted next generation sequencing (HBV-NGS) assay. The HBV-host junction sequences were identified by ChimericSeq. Comparison of HBV cccDNA and iDNA with serum and intrahepatic virological parameters were assessed.

Results Intrahepatic cccDNA, serum HBV DNA, HBV RNA, HBcrAg and qHBsAg were higher among the HBeAg(+) participants. Among the HBeAg(+) samples, 87% had positive intrahepatic HBcAg staining compared to 13% of HBeAg(−) samples (p<0.0001). HBsAg staining, in contrast, was present in over 85% of both HBeAg(+) and (−) livers. 23 (95.8%) HBeAg(+) participants had ≤50% iDNA of total HBV DNA whereas 25 (78.1%) HBeAg(−) participants had >50% iDNA in their livers. The iDNA junction-breakpoint distributions for the HBeAg(+) group were random with 15.9% localized to the DR2-DR1 region. In contrast, 52.4% of the iDNA were clustered at DR2-DR1 region among the HBeAg(−) participants. Microhomology-mediated end joining (MMEJ) patterns of dslDNA HBV integration was more frequent in HBeAg (+) livers.

Conclusion Serum RNA and HBcrAg reflect the intrahepatic cccDNA concentrations. HBeAg(−) CHB participants had high levels of intrahepatic iDNA and HBsAg despite lower cccDNA levels suggesting that iDNA is the primary source of HBsAg in HBeAg(−) CHB.

Conflict of interest statement for all authors: DL: Research support-Gilead; consultant-GSK, Gilead, Abbott Diagnostics, Aligos YS, YZ: JBS Science equity holders HG: stock shares of Arbutus, consultant: Aligos, Genentech, LyGenesis RS: Research support: Roche, Abbott RC: Research grants to institution: Abbvie, Janssen, BMS, Boehringer, GSK GC: Abbott employee and shareholder EK, WK, DK, MG, AH, YL, HL: No COI to report

National Institutes of Health : R56AI179574 to DL, YS, and HG; R01AI110762, R01AI150255, R21AI179929 to HG; U01DK082919-010 to DL; U01CA275648 to YS; R43AI167169 to SL, R01AI155140 to RTC.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The patient tissue specimens were collected through the HBRN Adult Cohort Study (NCT01263587), which includes 21 clinical sites across the US and Canada. The study protocols were approved by each local institutional review board. A list of the 21 clinical sites is provided below: Los Angeles, California, United States, 90048 Cedars Sinai Medical Center Los Angeles, California, United States, 90095 University of California Los Angeles San Francisco, California, United States, 94115 California Pacic Medical Center San Francisco, California, United States, 94143 University of California San Francisco Honolulu, Hawaii, United States, 96813 The Queen's Medial Center Bethesda, Maryland, United States, 20892 NIH Clinical Center Boston, Massachusetts, United States, 02114 Massachusetts General Hospital Boston, Massachusetts, United States, 02215 Beth Israel Deaconess Medical Center Ann Arbor, Michigan, United States, 48109 University of Michigan Health System Minneapolis, Minnesota, United States, 55455 University of Minnesota Rochester, Minnesota, United States, 55905 Mayo Clinic Saint Louis, Missouri, United States, 63104 Saint Louis University Saint Louis, Missouri, United States, 63108 Washington University Chapel Hill, North Carolina, United States, 27599 University of North Carolina Durham, North Carolina, United States, 27710 Duke University Medical Center Dallas, Texas, United States, 75246 Baylor University Medical Center Dallas, Texas, United States, 75390 University of Texas Southwestern Richmond, Virginia, United States, 23298 Virginia Commonwealth University Medical Center Seattle, Washington, United States, 98101 Virginia Mason Medical Center Seattle, Washington, United States, 98104 Harborview Medical Center Ontario Locations Toronto, Ontario, Canada Toronto Western Hospital Liver Centre

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

Grant support: National Institutes of Health : R56AI179574 to DL, YS, and HG; R01AI110762, R01AI150255, R21AI179929 to HG; U01DK082919-010 to DL; U01CA275648 to YS; R43AI167169 to SL, R01AI155140 to RTC.

Disclosures: Conflict of interest statement for all authors:

DL: Research support-Gilead; consultant-GSK, Gilead, Abbott Diagnostics, Aligos

YS, YZ: JBS Science equity holders

HG: stock shares of Arbutus, consultant: Aligos, Genentech, LyGenesis

RS: Research support: Roche, Abbott

RC: Research grants to institution: Abbvie, Janssen, BMS, Boehringer, GSK

GC: Abbott employee and shareholder

EK, WK, DK, MG, AH, YL, HL: No COI to report

Data transparency statement: Study materials will be made available to other researchers. Data will be open-access and available publicly after patent filing and publication.

Study materials will be made available to other researchers. Data will be open-access and available publicly after patent filing and publication.