抗击HBV耐药： HIV治疗和耐药的启示 广州市第八人民医院 唐小平 教授

HIV和HBV的病毒学特点 HIV HBV 单链RNA胞膜病毒 部分双链DNA胞膜病毒 依靠逆转录酶复制子代RNA 蛋白激酶在病毒蛋白合成中扮演重要角色 主要感染淋巴细胞，也广泛存在其它组织中 主要感染肝细胞，也存在于其它组织中 病毒RNA逆转录成DNA整合到宿主CD4+T淋巴细胞基因组中长期存在 病毒DNA形成稳定cccDNA 甚至整合到宿主细胞DNA中长期存在

HIV-1 复制周期

HBV病毒的复制周期 HBsAg 被膜 部分双链 DNA A(n) 感染性HBV病毒颗粒 (-)-DNA mRNA cccDNA 逆转录酶 Key message Following infection by the hepatitis B virus, the entire virion enters the hepatocyte. The partially double-stranded DNA converted to a complete double-stranded DNA, transported to the nucleus and supercoiled - the covalently closed circular DNA or cccDNA. The cccDNA is extremely stable and is the template from which the virus replicates and generates viral proteins. This is done through a full length messenger RNA template and 3 smaller mRNA templates. The viral proteins are produced from the 3 smaller mRNA transcript on the host endoplasmic reticulum. The HBV DNA polymerase then binds to the full length mRNA and orchestrates the formation of an encapulated mRNA/polymerase complex and subsequent replication. Within the nucleocaspid complex, the mRNA is reverse transcribed (RT) into a minus strand DNA by the DNA polymerase and the mRNA is simultaneously degraded. The DNA polymerase then produces a partially double-stranded DNA. Some of the partially double-stranded DNA migrates to the endoplasmic reticulum where it forms complete infectious HBV virions. Additionally, some of the partially double stranded DNA may migrate to the nucleus where it forms further copies of cccDNA thus amplifying and replenishing the cccDNA pool in the nucleus of the infected hepatocytes We have direct evidence that lamivudine exerts its action at both the reverse transcription and the DNA polymerase stages of HBV replication. Lamivudine acts as a chain terminator after incorporation into the growing DNA chain. Lamivudine may also exert its effect by inhibiting the completion of the double-stranded DNA and hence impacting on the formation of cccDNA either at initial infection or subsequent amplification of the cccDNA pool. Lai et al., J Med Virol 2000

HIV和HBV在基因组结构.复制特性和感染有一定的相似性 聚合酶RT区同源性高,特别是与dNTP结合的7个保守区域(包括A-G区),同源性更高 二. 具高复制能力,(HIV100亿/天, HBV1000亿/天) 1.病毒多聚酶和单链RNA存在,多聚酶无校正功能 2.高突变率（~105 碱基对/复制周期），因此以每天1010-12病毒生成，每一个碱基对都有可能发生突变, 同样,抗病毒治疗易产生耐药 3.同一病人中病毒复杂的基因型：准种 三. 宿主免疫应答不良形成慢性感染 1.相对平衡状态：高复制率和高清除率 2.HBV和HIV持续保存在宿主细胞内：不能彻底清除或治愈

抗HIV药物有多种类型， 分别作用于不同的靶位点 NRTI：核苷类逆转录酶抑制剂 (1987~) PI：蛋白激酶抑制剂(1995~) NNRTI：非核苷类逆转录酶抑制剂(1996~) Entry inhibitor：病毒黏附抑制剂(2003~) CCR5受体阻断剂 整合酶抑制剂

抗HIV药物的作用靶位点及新批准的药物 Mature virus 蛋白酶抑制剂 病毒黏附 抑制剂 Darunavir Tipranavir Maraviroc 病毒黏附 抑制剂 逆转录酶 抑制剂 Etravirine This is a summary of some of the recently approved new or novel antiretroviral agents, starting with the entry inhibitor maraviroc, a novel CCR5 antagonist; the next-generation NNRTI etravirine, which is active against most NNRTI-resistant viruses; and the integrase inhibitor raltegravir. Some newer PIs include darunavir and tipranavir, which were developed specifically for activity against PI-resistant virus. 整合酶 抑制剂 Raltegravir 7 7

目前共23种独特的抗HIV药物被FDA批准上市 DLV NVP ddC ABC TDF d4T ZDV ddI EFV 3TC FTC ’87 ’88 ’89 ’90 ’91 ’92 ’93 ’94 ’95 ’96 ’97 ’98 ’99 ’00 ’01 ’02 ’03 ’04 ’05 ’06 ’07 NRTI SQR NFV LPV/r ATV Speaking Notes: Once again you see all the ARVs currently approved for use in the US market, this time mapped by their year of release. Note that from 1987 to 1995, the first 5 ARV drugs developed were all nucleoside analogues. Thus, when the idea of combining drugs together arose, the first combinations utilized drugs that were then available, i.e., a pair of nucleosides: either zidovudine (Retrovir) and zalcitabine (Hivid), didanosine (Videx), or lamivudine (Epivir), or the combination of lamivudine (Epivir) and stavudine (Zerit). NNRTI FPV DRV PI APV RTV TPV Entry inhibitor T-20 MVC IDV

目前有的抗HIV药物 Nucleoside Analogs ddI, didanosine, VidexEC ddC, zalcitabine, Hivid AZT, zidovudine, Retrovir d4T, stavudine, Zerit 3TC, lamivudine, Epivir abacavir, Ziagen FTC, emtricitabine, Emtriva Nucleotide Analogs: tenofovir, Viread Non Nucleoside RT inhibitors delavirdine, Rescriptor nevirapine, Viramune efavirenz, Sustiva etravirine, Intelence Protease Inhibitors 13. indinavir, Crixivan +/-r 14. saquinavir Invirase +r 15. ritonavir, Norvir 16. nelfinavir, Viracept 17. lopinavir +r, Kaletra 18. atazanavir, Reyataz +/-r 19. fos-amprenavir, Lexiva +/-r 20. tipranavir, Aptivus +r 21. darunavir, Prezista +r Fusion Entry Inhibitors 22. enfuvirtide, Fuzeon CCR5 Entry inhibitors 23. maraviroc, Selzentry Integrase inhibitors 24. raltegravir, Isentress

HIV耐药 原发耐药：ART治疗前存在耐药株 1. ART治疗前,其他治疗方案已选择出单药或多药耐药株 2. 耐药病毒株的传播 NRTI: 5.5-12.4%3 NNRTI: 1.9-8.1%3 PI: 2.7-6.6% 3 继发耐药：治疗过程中由于药物选择性压力存在而筛选出来的耐药。耐多药问题较常见。 Wheeler W, et al 14th conference on Retrovirueses and Opportunistic Infections Feb 25-28 2007 Los Angeles, CA Abstract 648 SPREAD Programme, AIDS. 2008; 22(5): 625-635 Martin S. et al Clinical Infectious Diseases 2008； 47: 266-85

Secondary versus Primary (Transmitted) Drug Resistance Failing Therapy Rx Secondary DR Rx HIV+ person not on treatment Primary DR HIV Drug resistance comes about in 2 ways. Secondary drug resistance comes about as a result of treatment therapy Primary resistance comes about through the transmission of the HIV resistant virus to another person. The monitoring and prevention of primary drug resistance is a public health issue in which prevention and early identification become essential interventions. WT Wild Type Virus Drug Resistant Virus 11

中国HIV/AIDS 的原发耐药 It’s as high as 15-27.8% among some high risk population (FBD and IDU)

1. Scott M. Hammer, et al JAMA Aug 6, 2008 Vol 300, No.5 555-570 抗HIV药物的治疗 起始HIV基因型耐药及药物敏感性分析(如果条件容许) 采用高效抗逆转录病毒HAART治疗 (联合治疗) 联合用药方案 2 NRTIs ＋ 1 NNRTI 2 NRTIs ＋ 1 PI 1. Scott M. Hammer, et al JAMA Aug 6, 2008 Vol 300, No.5 555-570

耐药的检测方法 表型耐药（Phenotyping） 在不同药物浓度下(RT/PI)检测病毒的复制能力。 基因耐药（Genotyping） 检测病毒RT和PI基因突变

临床上耐药的检测 Indications for use of resistance testing have greatly expanded Genotype preferred Treatment naive: acute or chronic infection Early virologic failure Patient no longer on therapy Phenotype, virtual phenotype, or combined phenotype/genotype preferred High-level resistance to NRTIs or PIs on genotype Multiple regimen failure with limited treatment options In summary, the indications for use of resistance testing in clinical practice have greatly expanded. Genotype testing is typically preferred in treatment-naive patients who have either acute or chronic infection, for cases of early virologic failure, or for patients who are no longer on therapy. By contrast, phenotype, virtual phenotype, and the combined phenotype/genotype tests are preferred when patients have high-level resistance to NRTIs or PIs on genotype or when they have had multiple regimen failures with limited treatment options and for whom interpretation of resistance tests has become too complex. The optimal selection of subsequent regimens requires integration of resistance data with the patient’s treatment history, as well as with results from clinical studies.

HIV联合抗病毒治疗,抗病毒能力明显增加

HIV-1 dynamics after interruption of HAART Davey RT, et al PNAS 1999; 96:15109-14 Long-term suppression of HIV-1 by HAART does not confer on the host the ability to ultimately control viral replication once drug therapy is withdrawn. How to deplete HIV virus from sequestered sites throughout the body ? Off HAART 约60—70年

cccDNA完全清除需要多长时间 核苷类似物抗病毒治疗耗竭病毒复制模板cccDNA需要14.5年 100% HBV DNA 0% PIFN IFN APC CTL NK Lymphocyte Th B cell HBV DNA 抑制病毒复制 诱导阶段 清除感染肝细胞 Slide 18.慢性丙型肝炎患者对干扰素治疗的病毒学应答反应过程 慢性丙型肝炎患者对干扰素治疗的病毒学应答反应特点是HCV-RNA下降分为两期。在第一期——诱导期——干扰素通过抑制HCV的细胞内复制而导致循环血液中的早期病毒清除。此外，游离病毒的降解也发生在IFN反应者的诱导阶段的早期。这一阶段对IFN的治疗应答是剂量依赖型的。 第二阶段，也就是维持阶段，大概始于开始治疗后14到28天，持续于整个治疗期间。在这一阶段，免疫系统开始清除HCV感染的细胞。IFN持续治疗的目的就是要·清除HCV感染细胞，这一作用依赖于疗程的长短。 1. Ferenci P et al. Viral Hep Rev. 1999;5:229-245. 2. Neumann AU et al. Science. 1998;282:103-107. 维持阶段 检测限 0% 直接抗病毒 免疫应答 核苷类似物抗病毒治疗耗竭病毒复制模板cccDNA需要14.5年 Florin,et,al. Romanian Journal of Gastroenterology,2005 Vol.14 No.4,373~377

同时加强依从性教育和管理 在开始HAART之前应与病人有充分的交流，让他们了解治疗的必要性、治疗后可能出现的不适、坚持规律用药和服药后必须进行定期检测的重要性，以及在发生任何不适时应及时与医务人员联系。同时要得到其家属或朋友的支持，以提高病人的依从性。

依从性与治疗成功的关系 依从性的百分比 患者VL低于400拷贝的百分比 70% 90-95% >95% 70-80% 80-90% From Peterson et al, 6th Conf ROI 1999 abstr #92

目前只有两类抗HBV药物：干扰素和核苷类似物 2008 and beyond… 1992 1998 2002 2005 2006 IFN alfa ADV ETV PegIFN alfa-2a LdT TDF Clevudine ADV, adefovir; ETV, entecavir; IFN, interferon; LAM, lamivudine; LdT, telbivudine; PegIFN alfa-2a, peginterferon alfa 2a. TDF, tenofovir disoproxil fumarate. There are currently 7 US Food and Drug Application (FDA)–approved medications available for the treatment of chronic hepatitis B. Interferon alfa, approved in the early 1990s, is given either daily or every other day. In 1998, lamivudine was approved, marking the beginning of the new era of oral therapy. The timeline continues with the approval in 2002 of adefovir, followed by both entecavir and peginterferon alfa 2a in 2005. In 2006, telbivudine was approved for treatment of hepatitis B by the FDA. Tenofovir was approved in August 2008, and there are other medications currently being tested, such as clevudine which is in phase III trials. Other clinical trials are testing the efficacy of combining more than 1 medication for the treatment of hepatitis B patients. 至2008年，共7种抗HBV药物被批准 21

HBV耐药问题 HBV抗病毒药物耐药发生率,Lmd治疗4年70%左右,Ldt 2年22%,ADV治疗5年29% 交叉耐药问题, 耐药会影响后续治疗 多药耐药问题 原发耐药问题？ 耐药株的传播及免疫计划的威胁

? LMV 耐药影响后续治疗效果 LMV单药治疗 X A181T/V ADV T184S ETV LdT/克里夫定 M204V/I V214A/Q215S X LdT/克里夫定 ADV LdT T184S ETV T184G/S202I/M250V L80V/I A194T S. Locarnini el at. Journal of Hepatology 2007, S1, V46, S192; Abstract 505

所以应重视HBV耐药管理策略 挽救治疗 预测耐药 预防耐药 目前观念是“耐药管理的时间关口前移” 从挽救治疗,预测耐药”前移”到预防耐药

耐药管理时间关口前移 抗病毒药物治疗 临床耐药 生化学突破 病毒学突破 预防耐药 预测耐药 早期挽救治疗 晚期挽救治疗 耐药管理前移 ALT 病毒学突破 HBV DNA 杂交 PCR 时间 6 12 月数 预防耐药 预测耐药 早期挽救治疗 晚期挽救治疗 耐药管理前移 Santantonio et al, 2002

预防耐药胜于治疗耐药 预测耐药和挽救治疗需要准确的HBV DNA监测和及时修正治疗方案---临床实际---目前仍在采用晚期挽救治疗 耐药突变可以长期保存---cccDNA存在耐药突变报道 耐药突变可能导致与后续治疗药物的交叉耐药 医学界通用原则：预防胜于治疗

如何预防耐药 没有病毒复制 ＝ 没有耐药发生 方案一.初始单药治疗,使用强效和耐药率 最低的药物, 获得最大限度和持续的病毒抑制 方案二.初始联合治疗,联合两种疗效或作用机制不同药物,提高持续抗病毒的能力,最大限度的减低耐药(但目前暂不实施)

HBV联合抗病毒治目前暂不能实施 三. 其他现实问题: 一. HBV药物： 目前只针对HBV多聚酶一个靶位点,只有单一的抗病毒药物 目前仍无长期良好的临床数据 三. 其他现实问题: 1. 费用增加：cost-effectiveness 2. 交叉耐药 3. 联合治疗,药物毒性：长期安全性 缺乏妊娠安全性数据

如何避免耐药 高耐药基因学屏障 狭窄的潜在复制区 对依从性可容性大　 29

高耐药基因学屏障 基因屏障（GB）* 药物类别 无增强剂的PI 1 NNRTI NRTI 融合酶抑制剂 有增强剂的PI 3–8 0 MU ARV药物耐药 病毒复制逐渐增强 0 MU 1 MU 0 MU 1 MU 2 MU 3 MU 4 MU ARV药物耐药，病毒复制逐渐增强 30

举例: 初治患者中的病毒野生株：比较Kaletra 和 EFV 的pK 狭窄的选择压力区 举例: 初治患者中的病毒野生株：比较Kaletra 和 EFV 的pK 最后剂量 Day 1 Day 2 Kaletra 药物浓度 Efavirenz IC变异株 选择压力区 IC野生株 Days 第2天，长半衰期药物仍存在低浓度（单一治疗） NNRTI低基因屏障和长半衰期对耐药发展 的关联 Adapted from Taylor S, et al. 11th CROI, San Francisco 2004, #131 31

依从性可容性大 1634例患者(1996–2003) 根据处方计算和统计依从性>95% 或<95% 606例患者(37%)可检测出病毒 结论：依从性<95%时， 非增强PIs和NNRTIs与病毒血症关联 但与增强Pis无关 Hazard ratios Boosted PI NNRTI PI 0.5 1 1.5 2 2.5 3 Gross R, et al. 13th CROI, Denver 2006, #533 32

小结: HIV耐药处理的经验告诉我们什么？ HBV耐药病毒株传播是可能的，必须高度重视耐药问题，以免造成严重公共卫生问题 起始有条件做基因型或表型检测，选择敏感和高效抗病毒药物, 增加耐药屏障 长期（终身？）治疗 提高依从性：HBV治疗中出现病毒学突破30%与依从性有关 核苷类药物单药使用易引起耐药，开发多靶位点抗病毒药物，联合不同靶位点药物是抗击HBV耐药的重要策略 目前抗HBV药物只有两类，要着眼长期抑制HBV复制的治疗目标，根据正确耐药管理策略,保证长期抗病毒的疗效

谢 谢 !