Chronic hepatitis B virus infection (CHB) afflicts 1.5-2 million Americans, about 300 million people globally and causes early death in about 1 million people annually. In the “BeNEG-DO” grant we are studying an intervention aimed at safely stimulating immune-mediated clinical cures in patients with CHB. While suppressive standard-of-care nucleos(t)ide analog antiviral therapy (AVT) rarely cures CHB and is usually taken indefinitely, we are investigating if stopping AVT taken for at least 192 weeks (3.7 yrs), can safely stimulate a successful immune response to the hepatitis B virus (HBV) when replication reactivates after AVT is stopped. Clinical cure of CHB occurs when the diagnostic viral protein, the “surface antigen” (HBsAg), becomes undetectable in blood, which is the principal outcome we are evaluating with the stop- treatment intervention, while closely monitoring safety. BeNEG-DO was inspired by a seminal proof of concept study conducted at the University of Athens which stopped AVT in patients with the common “e-antigen negative” form of CHB (HBeAg-CHB). The Athens study was the first to show that successful HBV immunity and clinical cures can be generated in substantial numbers of people who stopped AVT after ≥3.7 years of viral suppression and was achieved without safety concerns. In the currently funded BeNEG-DO grant, we proposed testing the findings of this Greek study in a much larger San Francisco population that is naturally enriched for Asians who comprise the world's greatest HBV reservoir. In parallel, we proposed cellular and molecular studies using baseline liver biopsy and serial blood samples to dissect immunological mechanisms of HBsAg clearance from blood versus persistence. The translational scientific component was in part driven by hypotheses based on our mouse model and human data; and it also contained a host genetic prospecting arm that sought evidence for signature gene expression patterns that both predict outcome and could point to unsuspected mechanisms of immunity. A key objective of the studies was to distinguish patients who are most or least likely to benefit from the treatment withdrawal intervention. The 2-center BeNEG-DO study was fully enrolled; 76 cases stopped AVT, and 30 controls remained on therapy. All cases have been off therapy for at least 2.5 years through 10/25/20. Already, we have validated that withdrawal from AVT can lead to unprecedented clinical cure rates and identified groups at previously unknown risk after stopping AVT. Specifically, 11 patients have already lost HBsAg, 24 more have progressively falling levels; 11 with latest HBsAg levels <5 IU/mL (range 0.05-3.7). The trajectory of the remaining participants is still undefined. This renewal application requests funding to continue the objectives outlined in the original study and to take advantage of latest integrated genomic and proteomic analytical approaches to determine which HBeAg-CHB patients can safely stop AVT and benefit, ...