Baldick Carl J, Eggers Betsy J, Fang Jie, Levine Steven M, Pokornowski Kevin A, Rose Ronald E, Yu Cheng-Fang, Tenney Daniel J, Colonno Richard J
Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492, USA.
J Hepatol. 2008 Jun;48(6):895-902. doi: 10.1016/j.jhep.2007.12.024. Epub 2008 Feb 21.
BACKGROUND/AIMS: The efficacy of anti-viral therapy for chronic hepatitis B virus (HBV) is lost upon the emergence of resistant virus. Using >500 patient HBV isolates from several entecavir clinical trials, we show that phenotypic susceptibility correlates with genotypic resistance and patient virologic responses.
The full-length HBV or reverse transcriptase gene was amplified from patient sera, sequenced, and cloned into an HBV expression vector. Entecavir susceptibilities of individual virus clones and patient quasispecies populations were analyzed in conjunction with the sequenced resistance genotype and the patient's virologic response.
Entecavir susceptibility decreased approximately 8-fold for isolates with various constellations of lamivudine resistance substitutions. The spectrum of additional substitutions that emerged during therapy at residues rtT184, rtS202, or rtM250 displayed varying levels of entecavir susceptibility according to the specific resistance substitutions and the proportion of resistant variants in the quasispecies. Phenotypic analyses of samples associated with virologic breakthrough confirmed the role of these residue changes in entecavir resistance. Additional longitudinal phenotypic analyses showed that decreased susceptibility correlated with both genotypic resistance and increased circulating HBV DNA.
HBV phenotypic analysis provides additional insight as part of a resistance monitoring program that includes genotypic analysis and quantification of circulating virus.
背景/目的：耐药病毒出现后，慢性乙型肝炎病毒（HBV）抗病毒治疗的疗效会丧失。利用来自几项恩替卡韦临床试验的500多份患者HBV分离株，我们发现表型敏感性与基因型耐药性及患者病毒学反应相关。
从患者血清中扩增全长HBV或逆转录酶基因，进行测序，并克隆到HBV表达载体中。结合测序的耐药基因型和患者的病毒学反应，分析单个病毒克隆和患者准种群体对恩替卡韦的敏感性。
对于具有各种拉米夫定耐药替代组合的分离株，恩替卡韦敏感性降低了约8倍。在治疗期间rtT184、rtS202或rtM250位点出现的其他替代谱，根据特定的耐药替代和准种中耐药变异体的比例，显示出不同水平的恩替卡韦敏感性。与病毒学突破相关的样本的表型分析证实了这些位点变化在恩替卡韦耐药中的作用。进一步的纵向表型分析表明，敏感性降低与基因型耐药性和循环HBV DNA增加均相关。
HBV表型分析作为耐药监测计划的一部分，包括基因型分析和循环病毒定量，提供了额外的见解。
