Table_1_Identification and Mapping of HBsAg Loss-Related B-Cell Linear Epitopes in Chronic HBV Patients by Peptide Array.xlsx

Identification of immunogenic targets against hepatitis B virus (HBV)-encoded proteins will provide crucial advances in developing potential antibody therapies. In this study, 63 treatment-naïve patients with chronic HBV infection and 46 patients who achieved hepatitis B surface antigen loss (sAg loss) following antiviral treatment were recruited. Moreover, six patients who transitioned from the hepatitis B e antigen-positive chronic infection phase (eAg+CInf) to the hepatitis phase (eAg+CHep) were enrolled from real-life clinical practice. Additionally, telbivudine-treated eAg+CHep patients and relapsers or responders from an off-treatment cohort were longitudinally studied. The frequencies and function of B cells were assessed by flow cytometry. We devised a peptide array composed of 15-mer overlapping peptides of HBV-encoded surface (S), core (C), and polymerase (P) proteins and performed a screening on B-cell linear epitopes with sera. Naïve B cells and plasmablasts were increased, whereas total memory, activated memory (AM), and atypical memory (AtM) B cells were reduced in sAg- patients compared with sAg+ patients. Importantly, longitudinal observations found that AtM B cells were associated with successful treatment withdrawal. Interestingly, we identified six S-specific dominant epitopes (S33, S34, S45, S76, S78, and S89) and one C-specific dominant epitope (C37) that reacted with the majority of sera from sAg- patients. Of note, more B-cell linear epitopes were detected in CHep patients with alanine aminotransferase (ALT) flares than in nonflare CInf patients, and five B-cell linear epitopes (S4, S5, S10, S11, and S68) were overwhelmingly recognized by ALT flare patients. The recognition rates of epitopes on C and P proteins were significantly increased in CHep patients relative to CInf patients. Strikingly, a statistically significant elevation in the number of positive epitopes was observed when ALT nonflare patients shifted into the flare phase. Moreover, S76 identified at baseline was confirmed to be associated with a complete response after 48 weeks of telbivudine therapy. Taken together, we identified several functional cure-related B-cell linear epitopes of chronic HBV infection, and these epitopes may serve as vaccine candidates to elicit neutralizing antibodies to treat HBV infection.

靶向乙型肝炎病毒（hepatitis B virus, HBV）编码蛋白的免疫原性靶点鉴定，将为潜在抗体疗法的开发带来关键性进展。本研究纳入了63例初治慢性HBV感染患者，以及46例经抗病毒治疗后实现乙型肝炎表面抗原（hepatitis B surface antigen, sAg）转阴的患者。此外，本研究从真实世界临床实践中纳入了6例从乙型肝炎e抗原阳性慢性感染期（hepatitis B e antigen-positive chronic infection phase, eAg+CInf）进展至肝炎期（hepatitis phase, eAg+CHep）的患者。另外，本研究还对接受替比夫定（telbivudine）治疗的eAg+CHep患者，以及停药队列中的复发者与应答者进行了纵向研究。本研究通过流式细胞术（flow cytometry）评估了B细胞的频率与功能，设计了一套包含HBV编码的表面（S）、核心（C）及聚合酶（P）蛋白15个氨基酸重叠肽段的肽芯片，并利用患者血清对B细胞线性表位进行筛选。与乙型肝炎表面抗原阳性（sAg+）患者相比，乙型肝炎表面抗原阴性（sAg-）患者的初始B细胞（naïve B cells）与浆母细胞（plasmablasts）比例升高，而总记忆性B细胞、活化记忆性（activated memory, AM）B细胞及非典型记忆性（atypical memory, AtM）B细胞比例降低。重要的是，纵向观察结果显示，非典型记忆性B细胞与治疗成功停药相关。有趣的是，本研究鉴定出6条S蛋白特异性显性表位（S33、S34、S45、S76、S78及S89）与1条C蛋白特异性显性表位（C37），这些表位可与大部分sAg-患者的血清发生反应。值得注意的是，相较于非发作性慢性感染期（CInf）患者，出现丙氨酸氨基转移酶（alanine aminotransferase, ALT）发作的肝炎期（CHep）患者可检测到更多的B细胞线性表位；且5条B细胞线性表位（S4、S5、S10、S11及S68）可被ALT发作患者大量识别。相较于慢性感染期（CInf）患者，肝炎期（CHep）患者的C蛋白与P蛋白表位识别率显著升高。值得关注的是，当ALT非发作患者进展至发作阶段时，其阳性表位数量出现具有统计学意义的升高。此外，基线水平鉴定出的S76表位被证实与替比夫定治疗48周后的完全应答相关。综上，本研究鉴定出若干与慢性HBV感染功能性治愈相关的B细胞线性表位，这些表位可作为候选疫苗以诱导中和抗体用于HBV感染的治疗。