Hepatitis B virus (HBV) persistently infected about 250 million people worldwide, and a curative treatment remains an unmet medical need. Among many approaches to treat chronic hepatitis B (CHB), therapeutic vaccines have been developed for two decades, but none have yielded promising results in clinical trials. Therefore, dissection of HBV clearance mechanisms during therapeutic vaccination in appropriate models, which could give rise to new curative therapies, is urgently needed. Growing evidence indicates that prolonged and intensive exposure of antigen-specific T cells to viral antigens is a major cause of T cell exhaustion, and decreases anti-HBV immunity efficacy of therapeutic vaccination. HBV X protein (HBx) is expressed at low levels, and the understanding of its immunogenicity and potential in therapeutic CHB vaccines is limited. HBV genome sequences from CHB patients were cloned into a pAAV plasmid backbone and transfected into immunocompetent mouse hepatocytes through hydrodynamic injection. Mice carrying > 500 IU/mL serum HBV surface antigen (HBs) for more than 4 weeks were considered HBV carriers mimicking human CHB and received 3 doses of weekly HBx vaccine by subcutaneous immunization. Serum HBV clearance was evaluated by monitoring serum HBs and HBV-DNA titers. Residual HBV in the liver was evaluated by western blotting for HBV core antigen. The splenic antigen-specific T cell response was quantified by a 15-mer overlapping peptide-stimulated interferon-γ enzyme-linked immunospot assay. Blood and hepatic immune cells were quantified by flow cytometric analysis. Our HBx-based vaccine induced systemic HBx-specific CD4+ and CD8+ T cell responses in HBV carrier mice and demonstrated significant HBs and HBV-DNA elimination. The protective effect persisted for at least 30 days without additional booster immunization. Different infiltrating myeloid cell subsets, each with distinctive roles during immune-mediated HBV clearance, were found in the liver of vaccinated mice. During vaccine therapy, inflammatory monocyte depletion resulted in sustained HBV clearance inhibition, whereas phagocytic monocyte-derived macrophage and Kupffer cell elimination resulted in only transient inhibition of vaccine-induced HBV clearance. We report the potential role of HBx as a major immunogen in an HBV therapeutic vaccine and the significance of a liver-infiltrating monocyte subset during hepatic viral clearance.

中文翻译：

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基于HBV X蛋白的治疗性疫苗可通过动员单核细胞向HBV携带者肝中浸润来加速病毒抗原清除

乙型肝炎病毒（HBV）持续感染着全球约2.5亿人，治愈性医疗仍未满足医疗需求。在许多治疗慢性乙型肝炎（CHB）的方法中，治疗性疫苗已开发了二十年，但在临床试验中均未产生令人满意的结果。因此，迫切需要在适当的模型中剖析治疗性疫苗接种过程中的HBV清除机制，这可能会引起新的治疗方法。越来越多的证据表明，抗原特异性T细胞长期和密集地暴露于病毒抗原是T细胞衰竭的主要原因，并降低了治疗性疫苗的抗HBV免疫效力。HBV X蛋白（HBx）的表达水平较低，对其免疫原性和治疗性CHB疫苗潜力的了解有限。将来自CHB患者的HBV基因组序列克隆到pAAV质粒主链中，并通过流体动力注射转染到具有免疫功能的小鼠肝细胞中。携带大于500 IU / mL血清HBV表面抗原（HBs）超过4周的小鼠被认为是模仿人CHB的HBV携带者，并通过皮下免疫每周接受3剂HBx疫苗。通过监测血清HBs和HBV-DNA滴度评估血清HBV清除率。通过蛋白质印迹法评估HBV核心抗原在肝脏中的残留HBV。通过15聚体重叠肽刺激的干扰素-γ酶联免疫斑点测定法定量脾抗原特异性T细胞应答。通过流式细胞术分析定量血液和肝免疫细胞。我们的基于HBx的疫苗在HBV携带者小鼠中诱导了系统性HBx特异性CD4 +和CD8 + T细胞应答，并显示出明显的HBs和HBV-DNA消除。在没有额外的加强免疫的情况下，保护作用持续了至少30天。在接种小鼠的肝脏中发现了不同的浸润性髓样细胞亚群，每个亚群在免疫介导的HBV清除过程中均具有独特的作用。在疫苗治疗期间，炎性单核细胞耗竭导致持续的HBV清除抑制，而吞噬单核细胞衍生的巨噬细胞和库普弗细胞的清除仅导致疫苗诱导的HBV清除的短暂抑制。我们报告了HBx作为HBV治疗性疫苗中的主要免疫原的潜在作用，以及在肝病毒清除过程中肝浸润性单核细胞亚群的重要性。