慢性乙型肝炎追求临床治愈之综合优化治疗策略

Comprehensive Optimization of the Therapy in Chronic Hepatitis B for Pursuing Cinical Cure

摘要

我国为慢性乙型肝炎病毒(hepatitis B virus, HBV)感染高流行地区,慢性HBV感染对我国人民健康造成了极大危害和巨大经济负担。由于主要传播途径、遗传背景和病毒基因型与欧美存在差异,我国80%以上的肝细胞癌(hepatocellular carcinoma, HCC)与HBV感染相关,每年新发HCC人数和因HCC死亡的人数接近全球死亡人数的1/2。抗病毒治疗是阻断或延缓HBV相关肝脏疾病进展和降低HCC发生的重要措施,患者长期良好结局的唯一指标是乙型肝炎表面抗原(hepatitis B surface antigen, HBsAg)消失,HBsAg消失成为慢性HBV患者抗病毒治疗的理想治疗目标。因抗病毒治疗效果受多因素影响,在现有抗病毒药物的基础上,为了提高HBsAg消失发生率,需要根据患者的情况进行优化治疗,包括优势患者选择、聚乙二醇干扰素(pegylated interferon, PEG-IFN)与核苷(酸)类似物[nucleos(t)ide analogues, NAs]联合治疗、延长治疗、间歇治疗和获得HBsAg消失后的巩固治疗。本文就慢性HBV感染抗病毒治疗追求HBsAg消失的优化治疗策略作一述评,为追求临床治愈提供参考。

Abstract

China is a high prevalence area for chronic Hepatitis B virus （CHB） infection， which has caused significant harm to public health and imposed a substantial economic burden. Due to differences in main transmission routes， genetic backgrounds， and virus genotypes between China and regions like Europe and the United States， more than 80% of hepatocellular carcinoma （HCC） occurrences in China are associated with chronic Hepatitis B （CHB）. Additionally， the number of new HCC cases and the number of patients who die from HCC each year in China account for nearly half of the global figures. Antiviral therapy is an important measure to block or delay the progression of HBV-related liver diseases and reduce the occurrence of HCC， and the only indicator that can bring long-term good outcomes for patients is the disappearance of HBsAg. HBsAg loss has become the ideal therapeutic point for chronic HBV antiviral therapy. As the efficacies of antiviral therapy is affected by multiple factors， in order to improve the incidence of HBsAg disappearance with existing antiviral drugs， it is necessary to optimize treatment according to the situation of patients， including advantageous patient selection， combination therapy of PEG-IFN and NA， extended therapy， intermittent therapy， and consolidation therapy after HBsAg disappearance. This article discussed the optimal treatment strategy of antiviral therapy for chronic HBV infection in pursuit of HBsAg disappearance， and provided reference for clinical practice.

关键词

慢性乙型肝炎 / HBsAg消失 / 临床治愈 / 抗病毒治疗

Key words

Chronic hepatitis B / HBsAg loss / Clinical cure / Antiviral therapy

慢性乙型肝炎病毒（hepatitis B virus, HBV）感染是严重影响我国人民健康的公共卫生问题。在慢性HBV感染的肝脏疾病发生与发展过程中，病毒复制激活了肝脏细胞坏死和肝脏组织炎症，导致肝脏疾病进展，而病毒及其抗原则可抑制免疫细胞功能^[1-4]，使慢性肝炎迁延不愈。在HBV感染的肝脏疾病发生和进展过程中，炎症引起的宿主DNA损伤、转录因子的激活和免疫细胞功能的抑制均可引起肝细胞癌（hepatocellular carcinoma, HCC）的发生^[5]。

以欧洲高含油量冬油菜Sollux和中国品种高油605选系Gaoyou杂交F₁衍生的282个DH (doubled haploid)系为试验材料。QTL定位图谱包含481个分子标记^[1], 覆盖甘蓝型油菜基因组19条连锁群^[2], \frac{\partial \theta }{\partial t}=0总长1948.6 cM, 标记间平均距离4.05 cM。

抗病毒治疗是阻断慢性HBV感染所致疾病进展和降低HCC发生率最重要的措施^[6]。慢性乙型病毒性肝炎（chronic hepatitis B, CHB）的抗病毒治疗在我国推广已有20余年，然而因HCC导致死亡的人数却逐年增加。当今我国HCC的患病人数和因HCC死亡的人数约占全球总人数的1/2^[7]，每年新发HCC病例46万 ~ 37万^[8-9]。如何有效降低HCC发生风险、早期诊断HCC、提高患者生存率和延长生存期是当前临床重要的研究任务。

临床治愈，即检测不到HBV DNA、乙型肝炎e抗原（hepatitis B e antigen, HBeAg）阴性和乙型肝炎表面抗原（hepatitis B surface antigen, HBsAg）消失[伴或不伴乙型肝炎表面抗体（hepatitis B surface antibody, HBsAb）的产生]，患者的免疫细胞有效抑制病毒复制和清除病毒感染肝细胞，最大限度降低HCC的发生。获得临床治愈的患者HCC累计发生率仅1%左右^[10-11]。临床治愈已成为CHB推荐的治疗目标^[12]，也是当今新药开发的研究热点。本文就现有治疗药物如何追求慢性HBV感染的临床治愈作一述评，以供临床参考。

(1) $y=sqr{a^2+b_3}$

(2)

1 聚乙二醇干扰素与核苷（酸）类似物及不同作用机制药物联合治疗

病毒因素是影响抗病毒治疗效果最重要的因素。HBV复制和抗原的表达均决定于肝细胞内共价闭合环状DNA（covalently closed circular DNA, cccDNA）的数量及其转录活性，细胞内cccDNA是否显著减少或清除也反映是否临床治愈。通过治疗达到HBsAg消失的患者，肝细胞内cccDNA水平显著低于初次治疗和未达到治愈的患者^[13]，临床治愈患者肝内HBV整合数量显著低于HBeAg阳性和HBeAg阴性CHB组，27%的患者达到cccDNA清除，肝内cccDNA阴性或HBsAb >100 IU/mL的临床治愈患者均未发生HBsAg逆转^[14]。在慢性HBV自然感染中，由于HBV复制并不断补充cccDNA，难以自发发生HBV DNA转阴和病毒抗原的消失。在抗病毒治疗中，由于核苷（酸）类似物 [nucleos（t）ide analogues, NAs] 能有效地抑制病毒复制，而聚乙二醇干扰素（pegylated interferon, PEG-IFN）可抑制病毒复制、降低抗原的产生和刺激免疫细胞功能，为了更好发挥药物的作用和提高临床治愈发生率，往往需要将PEG-IFN与NAs或其他具有免疫调节功能的药物联合使用。初治CHB患者在PEG-IFN和富马酸替诺福韦酯（tenofovir disoproxil fumarate, TDF）联合治疗48周后，患者的HBsAg消失率为10.4%，显著高于PEG-IFN单药治疗患者的HBsAg消失率^[15]。在我国初治CHB患者的PEG-IFN+TDF与PEG-IFN对比效果的研究中，PEG-IFN单药治疗48周的HBsAg消失率仅有5.7%，而联合治疗组的HBsAg消失率为11.5%，差异显著^[16]。经NAs治疗后HBV DNA < 12 IU/mL的患者仍可在肝脏组织中检测到病毒的残余复制^[17]，提示经过NAs治疗的患者在追求临床治愈时也最好采用PEG-IFN+NAs的联合治疗。OSST研究显示，通过1 ~ 3年NAs治疗到达HBV DNA阴性的患者，在停止NAs治疗而转换成PEG-IFN单药治疗48周期间，有近20%的患者出现HBV DNA的返阳^[18]，New Switch研究显示，经NAs治疗获得HBV DNA阴性和HBeAg消失的HBeAg阳性CHB患者转换为PEG-IFN单药治疗48周和96周期间，分别有2.6%和7.3%出现HBV DNA > 2 000 IU/mL的反弹^[19]。OSST研究和New Switch研究的总体患者中，经过NAs治疗的患者转换PEG-IFN单药治疗48周时，HBsAg消失率分别仅有8.5%和14%；而New Switch研究中患者采用PEG-IFN单药治疗96周后，HBsAg消失率为20.7%。而在1项联合治疗研究中^[20]，采用NAs治疗获得HBV DNA阴性和HBeAg消失的患者，加用PEG-IFN治疗48周后HBsAg消失率达20.5%；联合治疗至96周时，HBsAg消失率为31.4%。经NAs治疗后获得HBV DNA阴性和HBeAg血清学转换的患者再联合PEG-IFN+TDF治疗48周后可使28.3%的患者获得临床治愈；而进一步联合PEG-IFN+TDF+粒细胞-巨噬细胞集落刺激因子+乙肝疫苗联合治疗的患者，经48周治疗后其HBsAg消失率可达到41.1%^[21]。同时，对于经NAs治疗的优势患者（HBV DNA和HBeAg阴性、HBsAg < 1 500 IU/mL），加用PEG-IFN联合治疗24周~ 72周，可使32.6% ~ 37.4%的患者获得临床治愈^[22-23]。即使是NAs治疗的应答不佳或低病毒血症（low-level viremia, LLV）的患者，联合治疗也可获得更高的HBsAg消失率。研究显示，NAs治疗6个月以上HBV DNA应答不佳患者，且属于HBsAg < 1 500 IU/mL的优势患者，转换为PEG-IFN单药治疗的HBsAg消失率为22.2%^[24]，而采用NAs治疗的LLV患者，联合PEG-IFN+NAs治疗48周后，可使97.5%的患者获得完全病毒应答，且有30.9%患者获得HBsAg消失^[25]。联合治疗不但可以提高HBsAg消失率，在采用NAs治疗的HBeAg阴性患者中，再采用PEG-IFN治疗还可以降低停止NAs治疗后病毒的反弹率^[26]。上述研究结果提示，无论是CHB初治患者还是已采用NAs治疗的患者，NAs能有效抑制病毒复制减少cccDNA补充，同时PEG-IFN能降低病毒抗原和刺激免疫细胞功能，那么PEG-IFN+NAs的联合治疗可显著提高HBsAg消失率，因此联合治疗是临床追求CHB临床治愈的重要方法。慢性乙肝防治指南也推荐，为最大限度地帮助已采用NAs治疗的患者获得临床治愈，推荐加用干扰素治疗^[12]。但也有研究显示，PEG-IFN联合NAs治疗与PEG-IFN单药治疗相比较，联合治疗并不能提高HBsAg消失率。如WEI等^[27]比较了PEG-IFN联合NAs治疗与PEG-IFN单药治疗HBeAg阳性和HBeAg阴性初治患者的治疗效果，结果显示，治疗48周后，PEG-IFN联合NAs治疗与PEG-IFN单药治疗的HBsAg消失率无差异。以上研究结果提示，提高HBsAg消失发生率的联合治疗更适合于经NAs治疗或非活动性HBsAg携带患者。

2 PEG-IFN基础上的延长治疗

临床治愈患者的获得与病毒感染肝细胞及肝内cccDNA的清除程度相关^[13-14]，由于PEG-IFN治疗中病毒感染肝细胞清除半衰期差异巨大，多数患者为了追求HbsAg消失，需延长PEG-IFN治疗时间。HbeAg阴性的CHB初始患者PEG-IFN单药48周疗程仅有约 3.1%的HbsAg消失率，而在经PEG-IFN治疗获得HBsAg消失的患者中，58.3%的HBsAg消失发生在治疗72周至120周^[28]。在PEG-IFN+NAs联合初始治疗的患者中，HBeAg阳性和HBeAg阴性患者平均HBsAg消失发生时间分别为（127.7 ± 59.7）周和（75 ± 42.9）周^[29]。

非活动性表面抗原携带者和经NAs治疗的乙肝患者是追求HBsAg消失的优势人群。在采用NAs治疗患者的OSST研究中，虽然在前期NAs治疗中达到HBV DNA阴性，但转为PEG-IFN治疗48周后，仅有8.5%患者实现HBsAg消失^[18]。采用NAs治疗后获得HBV DNA和HBeAg阴性的患者，继续转为PEG-IFN治疗48周后发现HBsAg消失率为14.4%，同时延长治疗至96周可使HBsAg消失率提升至20.7%^[19]。还有研究显示，延长PEG-IFN+NAs联合治疗时间，可以将经NAs治疗患者的HBsAg消失率由48周疗程的20.5%提高至96周疗程的31.3%^[30]。经NAs治疗的优势患者（即检测不到HBV DNA且HBeAg阴性，HBsAg < 1 500 IU/mL），将后续PEG-IFN治疗时间由48周延长至96周时，可使HbsAg消失率由23.1%提高至47.1%^[31]。而对于非活动HBsAg携带者，采用PEG-IFN治疗可将HBsAg消失率由48周时的20.8%提高到96周时的44.7%，且具有显著性的差异^[32]。

延长治疗可显著提高获得早期应答优势患者的HBsAg消失率。在New Switch研究中，采用NAs治疗后再转换为PEG-IFN治疗，治疗96周后患者HBsAg消失率为20.7%，尽管较治疗48周时患者14.4%的HBsAg消失率有所提高，但差异无统计学意义；而获得早期应答（即治疗24周时HBsAg < 1 500 IU/mL）的优势患者，HbsAg消失率由48周时的37.0%提高至96周时58.7%，差异显著^[19]。经PEG-IFN治疗48周后HBsAg达到较低水平的患者（HBsAg在10 ~ 100 IU/mL之间），延长治疗可使31.0%的患者在96周时达到HBsAg消失^[33]。其他临床研究也表明，延长疗程可以使48周治疗时有HBsAg大幅下降的患者提高临床治愈率，而有早期HBsAg应答的患者，延长疗程可使临床治愈率更高，而治疗12周和24周时HBsAg的下降程度和水平是预测HBsAg消失的关键因素^[34-35]。

上述研究结果提示，延长治疗可提高HBsAg消失率，特别是非活动性HBsAg携带者和采用NAs治疗的优势患者，包括PEG-IFN治疗时病毒和抗原表达低，PEG-IFN治疗有早期应答的患者，最好联合采用PEG-IFN治疗，一方面持续抑制病毒复制，另一方面不断降低病毒抗原。慢性乙肝防治指南也推荐治疗效果好的患者可延长治疗以追求临床治愈^[12,36]。

3 PEG-IFN间隙治疗

由于CHB的治疗效果受病毒、宿主和药物等多种因素的影响，采用干扰素抗病毒治疗时不同个体病毒感染肝细胞和cccDNA清除半衰期差异巨大。虽然上述的研究均显示延长治疗可提高HBsAg消失率，但提高效果也有限，即使是具有HBsAg早期应答的患者。将具有HBsAg早期应答患者的疗程由48周延长至96周时，HBsAg消失率仅比48周提高约20%^[19,31,33]。即便优势患者都能通过延长治疗获得HBsAg消失，但采用NAs治疗或非活动HBsAg携带者在延长PEG-IFN治疗至96周时，HBsAg消失率仅比48周时分别提高10.9%和14.9%^[20,32]。有研究显示，即便在优势和早期应答患者的PEG-IFN治疗过程中，HBsAg的下降常会进入平台期，即后一个时间点的HBsAg水平较前一个时间点的HBsAg水平下降幅度 < 0.5 log IU/mL。而在平台期后，HBsAg也难以在随后的治疗中继续下降^[28]。为了探讨其原因，有研究对PEG-IFN治疗前、PEG-IFN治疗达到平台期及平台期后停止PEG-IFN治疗3 ~ 6个月的NK细胞和CD8⁺T细胞的功能进行了研究。

3.1. The interaction of carbon surface and NaHCO₃

The thermal properties of the hydrochar and NaHCO₃ mixtures were analyzed to reveal the activation mechanism involving thermal stability and gaseous product distribution. A more obvious adsorption band, assigned to the CO₂ at 2450-2250 cm⁻¹ during the temperature 700-800 °C, was observed according to TG-IR spectra (Fig. S1 in Appendix A) after the introduction of NaHCO₃, indicating an intense mutual reaction between the carbonate and organic components of the hydrochars. For NaHCO₃, the thermogravimetric analysis curves (Fig. S2 in Appendix A) shifted towards lower temperatures in the presence of hydrochar, which was likely due to the effect of the surface chemical properties of hydrochar on the volatilization activation energy [41]. DSC analysis (Fig. 1(a)) shows the heat changes of NaHCO₃ and their correlation with the ash of manure hydrochar and cellulose- (H_cellulose), lignin- (H_lignin), and protein- (H_protein) based hydrochar. Notably, the decomposition of NaHCO₃ was significantly boosted by the above monocomponent hydrochars, with the decline of the melting points from 845.65 to 771.41 °C. At the same time, the introduction of ash, which altered the thermal properties of molten salts by forming new eutectic systems, exhibited slight impacts [32]. These changes demonstrate the occurrence of oxidation/reduction chemical reactions during activation, primarily induced by the OFGs of the hydrochar, which accelerate the transformation of Na species to the corresponding atomic forms. The rich, accessible OFGs served as active sites for cation-anion interactions, which could alter the NaHCO₃ atomization mechanisms, leading to the advanced evaporation of oxides and Na.

The detailed morphology and pore distribution of all samples were characterized by SEM, BET, and TEM analyses (Figs. S5 and S6 and Table S2 in Appendix A and Fig. 1(c)). A larger surface area, ranging from 133.11 to 844.62 m²·g⁻¹, was obtained on activated hydrochars compared to precursors that possessed an almost nonporous nature (0.92-2.59 m²·g⁻¹). The original HC and HP textures were reshaped through Na-salt corrosion into rough, porous, and hierarchical structures, which generated rich functional groups that acted as interfacial active sites. The cross-stacking of the striped graphite layers on AHP-800 indicates the presence of a uniform aromatic cluster. With excellent mobility to enclose feedstocks and catalytic performance, the liquid-state carbonate caused more expanded meso- and micro-pores and the formation of worm-like structures with intricate micro-tunnels on AHC-800 and AHP-800, offering additional adsorption sites and dispersive forces for NH₃ storage.

3.2. Ash transformation during the activation process

Distinct inorganic compounds mingled in the excrement during the manure harvesting process in livestock production (Fig. S7 in Appendix A). XRF analysis suggested that the hydrothermal treatment facilitated the transfer of soluble components in manure, such as Na- and K-containing salts, into the aqueous phase. Si salts (44.38%) dominated the inorganic phase of CM, whereas more P (34.33%) and Ca (40.01%) salts were observed in the PM hydrochar. Without adding activator, the ash contents of livestock manure increased after pyrolysis (43.09%-62.05%) (Fig. 2(a)), indicating the slim impact of acidic washing on ash removal. This can be attributed to the dehydrogenation and deoxidation of organic matter, resulting in a more compact carbon mantle after thermal treatment, significantly deterring total ash dissolution [54]. A carbon surface dominated by C, O, and N was observed through SEM-EDS (Fig. 2(b) and Table S3 in Appendix A), whereas the ash signals decreased as the pyrolysis temperature increased, which was inconsistent with the variation in the ash content described above. XRD analysis was performed to provide deeper scanning for the crystal morphology of the unactivated hydrochar (Fig. 2(c)). In addition to the characteristic peak of SiO₂, the appearance of diffraction peaks of acid-soluble substances, such as calcium iron oxide and K₂O, suggests that a large amount of ash was still embedded in the internal layer of the carbon structure, strongly demonstrating the protective model of carbon towards ash in manure hydrochar. Metal nanoparticles exhibit much better-ordered carbon structures with straight stacked graphitic layers via autocatalytic reactions with local organics [55].

该研究结果显示，在PEG-IFN治疗过程中，具有杀伤功能的NK细胞亚群逐渐耗竭^[37]，具有病毒清除能和强杀伤作用的CD8⁺T细胞亚群（CD8⁺ CD38^dim）频数下降，而功能损伤的CD8⁺T细胞（CD8⁺ CD38^high）频数上升^[38]；同时在停止PEG-IFN治疗3 ~ 6个月后，具有杀伤效应的NK细胞亚群和病毒清除能力的CD8⁺T细胞亚群频数得到恢复。可见，当PEG-IFN治疗中HBsAg下降进入平台期时，可暂停PEG-IFN治疗3 ~ 6个月以恢复免疫细胞功能。笔者团队既往研究显示，进入HBsAg下降平台期的患者停止干扰素治疗3 ~ 6个月（间歇期）后再加用干扰素治疗，可使19.8%患者获得HBsAg消失；而在间歇期后再进行干扰素治疗的早期应答患者中，有44.4%获得HBsAg消失，且再次治疗时的HBsAg基线和再次治疗期12周后的早期HBsAg应答为独立相关因素^[39]。但干扰素治疗期间出现HBsAg下降平台期的患者，即使是HBsAg达到较低水平的患者，也不应一味地延长治疗。因为这种间歇治疗模式可提高HBsAg消失发生率，但其作用机制还需更多的队列研究证实。

4 HBsAg消失后的巩固治疗

临床治愈是一种宿主对HBV良好的免疫控制状态，并不是病毒的完全清除^[13]。经PEG-IFN治疗获得HBsAg消失的患者，有8.20% ~ 14.3%在停止治疗后出现HBsAg返阳，少数患者出现HBV DNA返阳^[40-42]。虽然HBsAg返阳和HBV DNA返阳与患者的免疫功能相关，但一些临床因素可以预测患者在停止治疗后HBsAg和HBV DNA是否返阳或是否能维持阴性应答。研究显示，干扰素治疗获得HBsAg消失后，继续巩固治疗12 ~ 24周与停药后持久HBsAg阴性应答显著相关^[40]。还有研究显示，在HBsAg消失后伴HBsAb的产生（最好> 100 mIU/L）也是预测持久性功能治愈的因素^[42-44]；同时HBsAb水平联合其他指标，如联合低乙型肝炎核心相关抗原（hepatitis B core-related antigen， HBcrAg）含量可提高持久性HBsAg阴性应答能力^[45]。HBeAg阳性患者停止干扰素治疗时，如果HBeAg仍为阳性，这也是预测停药后HBsAg返阳的独立因素^[40,42]。而对于HBsAg消失时未同时产生低水平HBsAb或HBsAb的患者，延长干扰素治疗可显著提高HBsAb产生率和提高其水平^[40,44,46]。而关于HBsAg消失后是否可采用乙肝疫苗注射以刺激HBsAb产生的问题，有研究显示，乙肝疫苗注射后可以增加HBsAg产生率^[47]。既往也有研究显示，接种乙肝疫苗并不会影响患者的HBsAg阳性率，但经疫苗接种产生了HBsAg的患者，特别是HBsAg ≥ 100 mIU/L的患者，该指标有助于维持随访期间HBsAg持续阴性^[43]。以上研究提示，有的患者可通过乙肝疫苗注射产生HBsAb，有的却不能。鉴于乙肝疫苗接种安全且价格经济，可适当建议HBsAg消失且在巩固治疗期间无HBsAb产生或水平较低的患者接种乙肝疫苗。

5 小结

抗病毒治疗虽然可延缓肝脏疾病进展，但只有临床治愈才能为患者带来长期良好临床结局和最大限度降低HCC发生，因此临床治愈已经成为CHB抗病毒治疗的目标。由于抗病毒治疗受多因素影响，在追求临床治愈的过程中，医者需要根据患者的具体情况进行治疗方案综合优化。若需要有效抑制病毒复制、降低病毒抗原和刺激免疫细胞功能时，PEG-IFN联合NAs治疗可显著提高HBsAg消失率。对于采用PEG-IFN治疗的患者，多数患者需要通过延长治疗才能获得临床治愈，特别是优势人群和早期应答患者，因为延长治疗可显著提高HBsAg消失率。由于PEG-IFN治疗期间可引起具有病毒杀伤功能的免疫细胞耗竭，常常导致PEG-IFN治疗期间HBsAg下降进入平台期，其后的继续治疗难以使HBsAg持续下降。为了恢复免疫细胞功能，可暂停干扰素治疗3 ~ 6个月后再进行PEG-IFN治疗，可使再次治疗的早期应答患者获得HBsAg消失。临床治愈是宿主对HBV感染极佳的免疫控制状态，但在停止PEG-IFN治疗后会有极少的患者出现HBsAg返阳，甚至HBV DNA返阳。为减少上述指标在停药后再次出现阳性，可在获得HBsAg消失后进行确认，并继续采用PEG-IFN进行巩固治疗12 ~ 24周；同时高水平的HBsAb可以预测停药后HBsAg持久阴性应答。HBsAg消失时无HBsAb产生的患者可延长PEG-IFN巩固治疗时间，或通过接种乙肝疫苗以期刺激产生HBsAb。总之，为了最大限度获得临床治愈，可通过优势患者的选择、及时预测疗效、采用PEG-IFN+NAs联合治疗、进行有效患者的延长治疗和间歇治疗、获得HBsAg消失后的巩固治疗和促进HBsAb的产生等治疗策略来得到持久性临床治愈。

参考文献

[1] TANG R, LEI Z, WANG X, et al. Hepatitis B envelope antigen increases Tregs by converting CD4⁺CD25^- T cells into CD4⁺CD25⁺Foxp3⁺ Tregs[J]. Exp Ther Med, 2020, 20(4): 3679-3686.

[2] YANG F, YU X, ZHOU C, et al. Hepatitis B e antigen induces the expansion of monocytic myeloid-derived suppressor cells to dampen T-cell function in chronic hepatitis B virus infection[J]. PLoS Pathog, 2019, 15(4): e1007690.

[3] HOOGEVEEN RC, ROBIDOUX MP, SCHWARZ T, et al. Phenotype and function of HBV-specific T cells is determined by the targeted epitope in addition to the stage of infection[J]. Gut, 2019, 68(5): 893-904.

[4] SCHUCH A, SALIMI ALIZEI E, HEIM K, et al. Phenotypic and functional differences of HBV core-specific versus HBV polymerase-specific CD8⁺T cells in chronically HBV-infected patients with low viral load[J]. Gut, 2019, 68(5): 905-915.

[5] LI YT, WU HL, LIU CJ. Molecular mechanisms and animal models of HBV-related hepatocellular carcinoma: with emphasis on metastatic tumor antigen 1[J]. Int J Mol Sci, 2021, 22(17): 9380.

[6] LIN CL, KAO JH. Development of hepatocellular carcinoma in treated and untreated patients with chronic hepatitis B virus infection[J]. Clin Mol Hepatol, 2023, 29(3): 605-622.

[7] RUMGAY H, ARNOLD M, FERLAY J, et al. Global burden of primary liver cancer in 2020 and predictions to 2040[J]. J Hepatol, 2022, 77(6): 1598-1606.

[8] 秦叔逵, 卢实春, 邱文生, 中国原发性肝癌临床登记调查（CLCS）2022年生存分析更新报告[C]. 第25届全国临床肿瘤学大会暨2022年CSCO学术会议, 2022, 26-27.

[9] HAN BF, ZHENG RS, ZENG HM, et al. Cancer incidence and mortality in China, 2022[J]. J Natl Cancer Cent, 2024, 4(1): 47-53.

[10] ANDERSON RT, CHOI HSJ, LENZ O, et al. Association between seroclearance of hepatitis B surface antigen and long-term clinical outcomes of patients with chronic hepatitis B virus infection: systematic review and meta-analysis[J]. Clin Gastroenterol Hepatol, 2021, 19(3): 463-472.

[11] YIP TC, WONG VW, TSE YK, et al. Similarly low risk of hepatocellular carcinoma after either spontaneous or nucleos(t)ide analogue-induced hepatitis B surface antigen loss[J]. Aliment Pharmacol Ther, 2021, 53(2): 321-331.

[12] 中华医学会肝病学分会, 中华医学会感染病学分会. 慢性乙型肝炎防治指南（2022年版）[J]. 中华肝脏病杂志, 2022, 30(12): 1309-1331.

[13] GAO N, GUAN G, XU G, et al. Integrated HBV DNA and cccDNA maintain transcriptional activity in intrahepatic HBsAg-positive patients with functional cure following PEG-IFN-based therapy[J]. Aliment Pharmacol Ther, 2023, 58(10): 1086-1098.

[14] GAN W, GAO N, GU L, et al. Reduction in intrahepatic cccDNA and integration of HBV in chronic hepatitis B patients with a functional cure[J]. J Clin Transl Hepatol, 2023, 11(2): 314-322.

[15] AHN SH, MARCELLIN P, MA X, et al. Hepatitis B surface antigen loss with tenofovir disoproxil fumarate plus peginterferon Alfa-2a: week 120 analysis[J]. Dig Dis Sci, 2018, 63(12): 3487-3497.

[16] HU CG, SONG YD, TANG CR, et al. Effect of pegylated interferon plus tenofovir combination on higher hepatitis B surface antigen loss in treatment-naive patients with hepatitis B e antigen-positive chronic hepatitis B: a real-world experience[J]. Clin Ther, 2021, 43(3): 572-581.e3.

[17] YU T, ZHANG M, ZHANG H, et al. Evidence of residual ongoing viral replication in chronic hepatitis B patients successfully treated with Nucleos(t)ide analogues[J]. J Infect Dis, 2023, 227(5): 675-685.

[18] NING Q, HAN MF, SUN YT, et al. Switching from entecavir to PegIFN Alfa-2a in patients with HBeAg-positive chronic hepatitis B: a randomised open-label trial (OSST trial)[J]. J Hepatol, 2014, 61(4): 777-784.

[19] HU P, SHANG J, ZHANG W, et al. HBsAg loss with peg-interferon Alfa-2a in hepatitis B patients with partial response to nucleos(t)ide analog: new switch study[J]. J Clin Transl Hepatol, 2018, 6(1): 25-34.

[20] WU D, YAN WM, TAN DM, et al. Combination of NA, PEG-IFN alpha-2b and GM-CSF enhanced HBsAb production in NA experienced CHB patients(the anchor a study): an interim analysis[J]. J Hepatol, 2020, 73:S860.

[21] JIA H, YU G, YU J, et al. Immunomodulatory and antiviral therapy improved functional cure rate in CHB patients with high HBsAg level experienced NA[J]. J Clin Transl Hepatol, 2023, 11(5): 1003-1010.

[22] WU FP, YANG Y, LI M, et al. Add-on pegylated interferon augments hepatitis B surface antigen clearance vs continuous nucleos(t)ide analog monotherapy in Chinese patients with chronic hepatitis B and hepatitis B surface antigen≤1500 IU/mL: an observational study[J]. World J Gastroenterol, 2020, 26(13): 1525-1539.

[23] ZHANG W, XING M, SUN W, et al. Early clinical efficacy of pegylated interferon treatment in patients with different phases of chronic HBV infection: a real-world analysis[J]. J Viral Hepat, 2023, 30(5): 427-436.

[24] 李明慧, 胡蕾苹, 张璐, HBeAg阳性慢性乙型肝炎用核苷（酸）类似物治疗部分病毒学应答患者转换聚乙二醇干扰素α-2a治疗的研究. 中华肝脏病杂志, 2015, 23(11):826-831.

[25] CHEN XY, REN S, LU JF, et al. SAT382 Combination therapy in HBeAg-negative chronic hepatitis B patients with low-level viremia to nucleos (tide) analogues[J]. J Hepatol, 2022, 77: S844.

[26] LI FH, QU LH, LIU YH, et al. PegIFN alpha-2a reduces relapse in HBeAg-negative patients after nucleo(s)tide analogue cessation: a randomized-controlled trial[J]. J Hepatol, 2024

[27] WEI N, ZHENG B, CAI H, et al. Systematic review and meta-analysis: de novo combination of nucleos(t)ide analogs and pegylated interferon alpha versus pegylated interferon alpha monotherapy for the functional cure of chronic hepatitis B[J]. Front Pharmacol, 2024, 15: 1403805.

[28] LI MH, ZHANG L, LU Y, et al. Early serum HBsAg kinetics as predictor of HBsAg loss in patients with HBeAg-negative chronic hepatitis B after treatment with pegylated interferonα-2a[J]. Virol Sin, 2021, 36(2): 311-320.

[29] BAO XL, GUO J, XIONG F, et al. Clinical characteristics of chronic hepatitis B cured by peginterferon in combination with nucleotide analogs[J]. Int J Infect Dis, 2020, 96: 562-566.

[30] HAN MF, WU D, TAN DM, et al. Combination/sequential therapy with ETV, Peg-IFN alpha-2b and GMCSF enhanced HBsAg loss and appearance of HBsAb in NA suppressed CHB patients(the Anchor A study): an interim analysis[C]. Hepatology, AASLD ABSTRACTS, 2017,13A.

[31] GAO ZL, ZHU X, Lin BL, et al. The Optimizing Treatment of Peg Interferon Alfa in Hbeag Negative Chronic Hepatitis B Patients with Low Level HBsAg: A Multicenter Real World Study(Interferon Cure Study, I CURE Study)[C]. Hepatology, AASLD ABSTRACTS (Poster), 2018, 246A.

[32] CAO Z, LIU Y, MA L, et al. A potent hepatitis B surface antigen response in subjects with inactive hepatitis B surface antigen carrier treated with pegylated-interferon alpha[J]. Hepatology, 2017, 66(4): 1058-1066.

[33] 闫一杰, 王晓晓, 曹振环, 48周HBsAg低水平的HBeAg阴性慢性乙型肝炎患者是获得HBsAg清除的优势人群[J]. 中华肝脏病杂志, 2018, 26(11): 813-818.

[34] 梁晗, 王晨, 朱鹏飞, 核苷（酸）类似物经治慢性乙型肝炎患者加用聚乙二醇干扰素α治疗的临床治愈效果研究[J]. 中华肝脏病杂志, 2023, 31(12): 1297-1305.

[35] 贾瑞, 王文鑫，周志平, 核苷（酸）类似物经治慢性乙型肝炎患者序贯联合聚乙二醇干扰素α治疗48～96周的疗效及影响因素分析[J]. 中华肝脏病杂志, 2023, 31(12): 1290-1296.

[36] 中华医学会感染病学分会, 中华医学会肝病学分会. 慢性乙型肝炎防治指南(2019年版)[J]. 中华肝脏病杂志, 2019, 27(12): 938-961.

[37] BI X, XIE S, WU S, et al. Changes of natural killer cells' phenotype in patients with chronic hepatitis B in intermittent interferon therapy[J]. Front Immunol, 2023, 14: 1116689.

[38] LIN Y, SHEN G, XIE S, et al. Dynamic changes of the proportion of HLA-DR and CD38 coexpression subsets on T lymphocytes during IFN-based chronic hepatitis B treatment[J]. Front Immunol, 2022, 13: 1116160.

[39] LI M, XIE S, BI X, et al. An optimized mode of interferon intermittent therapy help improve HBsAg disappearance in chronic hepatitis B patients[J]. Front Microbiol, 2022, 13: 960589.

[40] LI MH, SUN FF, BI XY, et al. Consolidation treatment needed for sustained HBsAg-negative response induced by interferon-alpha in HBeAg positive chronic hepatitis B patients[J]. Virol Sin, 2022, 37(3): 390-397.

[41] WU Y, LIU Y, LU J, et al. Durability of interferon-induced hepatitis B surface antigen seroclearance[J]. Clin Gastroenterol Hepatol, 2020, 18(2): 514-516.e2.

[42] ZHANG W, CHEN J, SUN W, et al. The impact of hepatitis B surface antigen seroconversion on the durability of functional cure induced by pegylated interferon alpha treatment[J]. Virol J, 2024, 21(1): 243.

[43] JIANG S, CAI M, ZHANG Z, et al. The potential effect of HBV vaccination on off-treatment HBsAg reversion after interferon-induced HBsAg clearance[J]. Hum Vaccin Immunother, 2023, 19(1): 2161254.

[44] GAO N, YU H, ZHANG J, et al. Role of hepatitis B surface antibody in seroreversion of hepatitis B surface antigen in patients achieving hepatitis B surface antigen loss with pegylated interferon-based therapy[J]. J Viral Hepat, 2022, 29(10): 899-907.

[45] HUANG D, WU D, WANG P, et al. End-of-treatment HBcrAg and HBsAb levels identify durable functional cure after Peg-IFN-based therapy in patients with CHB[J]. J Hepatol, 2022, 77(1): 42-54.

[46] LI MH, YI W, ZHANG L, et al. Predictors of sustained functional cure in hepatitis B envelope antigen-negative patients achieving hepatitis B surface antigen seroclearance with interferon-alpha-based therapy[J]. J Viral Hepat, 2019, 26(): 32-41.

[47] LIU Y, REN S, MA L, . Clinical study of hepatitis B vaccine in achieving hepatitis B surface antibody seroconversion in patients with functional cure[J]. Braz J Infect Dis, 2023, 27(6): 103703.

[48] 刘定富, 蔡怀武. 甘蓝型油菜特长荚突变体的发现和鉴定. 湖北农学院学报, 1994, 4(2): 1-4.

[49] Liu D F, Cai H W. Detection and identification of specially-long pod mutant in Brassica napus L. J Hubei Agric Coll, 1994, 4(2): 1-4 (in Chinese with English abstract)

基金资助

国家重点研发计划项目(2023YFC2308105)

北京市卫生健康委高层次公共卫生技术人才建设项目学科带头人(-03-26)

北京市医院管理局临床医学发展专项经费资助项目(XMLX 202127)

北京市医院管理中心“登峰”人才培养计划(DFL20241803)

首都健康研发专项(2022-1-2172)