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Development of an intra- and intergenotypic HCV cell culture method to phenotype and assess antiviral susceptibilities and resistance development of HCV NS3 protease genes from HCV genotypes 1-6

机译:表型和基因型之间的HCV细胞培养方法的开发,以评估表型和评估HCV基因型1-6的HCV NS3蛋白酶基因的抗病毒敏感性和耐药性

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摘要

The development of specific antiviral drugs directly targeting the hepatitis C virusud(HCV) is clinically important, as the current standard interferon/ribavirin combinationudtreatment is only partially effective, expensive and often associated with severeudside effects. Inhibitors of the NS3 protease (PI) therefore represent a promising alternativeudor additional therapy. To date, the development and in vitro evaluation of PIs isudrestricted to the genotype 1/2 based replicon and the genotype 2a full length viral celludculture system. However, proteases of the different HCV genotypes vary substantiallyudin their amino acid sequence and secondary structure and require separate evaluationudof their efficacy before they go into clinical trials.udTo address this issue, a panel of intra- and intergenotypic recombinants based on the recombinantudinfectious clone Jc1 (pFK JFH1/J6/C-846) was developed in this work. Theudviability of these recombinants was assessed in the Huh7.5 cell culture system, whereudreplicating viruses were detected by HCV-NS5A immunostaining. Intergenotypic recombinantsudcontaining genotype 1a, 1b, 3a, 4a and 6a derived proteases were replicationuddefective, whereas the recombinant with genotype 5a derived protease replicatedudefficiently after acquiring cell culture adaptive mutations. The replacement ofudnot only the NS3 protease gene region, but also its cofactor NS4A, allowed the generationudof replication competent intra- and intergenotypic recombinants for all 6 majorudgenotypes. Replacing the NS3 protease of the recombinants with that of patientderivedudproteases also generated replicating recombinants, greatly expanding the paneludof intergenotypic recombinants available for phenotyping and PI evaluation. However,udintra- and intergenotypic recombinants showed substantial differences in theirudreplication kinetics, which may be influenced by naturally occurring polymorphismudbetween genotypes and the differential requirement of adaptive/attenuating cell cultureudmutations. Genotype 1a recombinants replicated very poorly, which may be dueudto incompatibilities between the type 1a NS3/4A protease and the type 2a backbone.ud50% inhibitory concentrations (IC50) of different PIs were measured using Foci FormingudUnits/ml (FFU/ml) reductions and replication inhibition assays. The different recombinantsudshowed consistent, genotype-associated differences in their susceptibilityudto the PI BILN 2061, with genotypes 2a, 3a and 5a derived recombinants showingudapproximately 100-fold lower susceptibility than genotype 1b, 4a and 6a derived recombinants.udThese observations are consistent with major differences in response ratesudfound in recent treatment trials of genotype 1, 2 and 3 infected patients. Differencesudin susceptibility were also observed for VX-950, with genotype 1b, 2a and 6a derivedudrecombinants being twice as susceptible than genotype 3a, 4a and 5a derivedudrecombinants. Passaging the intra- and intergenotypic recombinants under increasingudconcentrations of PI allowed the identification of PI resistance mutations. Resistanceudmutations to BILN 2061 mapped to the previously identified positions 156 and 168udwithin the NS3 protease, with a great diversity of amino acid substitutions observedudwithin each genotype. Reintroduction of the identified resistance mutations into theudoriginal recombinant viruses conferred increased resistance towards BILN 2061 andudsome mutations also affected replication kinetics of the recombinants. The developedudsystem will be of major value for the phenotypic characterisation of naturally occurringudand treatment induced resistance mutations within all 6 major HCV genotypes towardsuddifferent PIs. This will allow treatment response predictions for newly developed PIsudbefore they enter clinical trials and the development of individually tailored antiviraludtreatment regimes.
机译:直接针对丙型肝炎病毒/ ud(HCV)的特定抗病毒药物的开发在临床上具有重要意义,因为当前标准的干扰素/利巴韦林联合治疗/局部治疗仅部分有效,昂贵且通常与严重的/副作用有关。因此,NS3蛋白酶(PI)的抑制剂代表了有希望的替代方案或其他疗法。迄今为止,PIs的开发和体外评估仅限于基于基因型1/2的复制子和基因型2a全长病毒细胞/ udculture系统。但是,不同HCV基因型的蛋白酶在其氨基酸序列和二级结构上存在很大差异,在进行临床试验之前,需要对其功效进行单独评估。 ud为了解决这个问题,基于该技术的一组基因内和基因型重组体这项工作产生了重组/感染性克隆Jc1(pFK JFH1 / J6 / C-846)。在Huh7.5细胞培养系统中评估了这些重组体的生存能力,并通过HCV-NS5A免疫染色检测了复制病毒。基因型重组体包含基因型1a,1b,3a,4a和6a的蛋白酶具有复制缺陷,而具有基因型5a蛋白酶的重组体在获得细胞培养适应性突变后可以有效复制。不仅替换了NS3蛋白酶基因区域,而且替换了它的辅助因子NS4A,使得所有6种主要的/预算型的复制能力强大的基因型和基因型之间的复制成为可能。用患者来源的 u​​dp蛋白酶替代重组体的NS3蛋白酶也产生了可复制的重组体,大大扩展了可用于表型和PI评估的基因型间的重组体。然而,异位基因型和基因型间的重组体在其复制动力学上显示出实质性差异,这可能受到基因型之间自然发生的多态性和适应性/减毒细胞培养物/突变的不同需求的影响。基因型1a重组体复制非常差,这可能是由于 1a NS3 / 4A型蛋白酶和2a型主链之间的不相容性造成的。使用Foci Forming udUnits / ml(FFU)测量了不同PI的ud50%抑制浓度(IC50) / ml)减少和复制抑制试验。不同的重组体对PI BILN 2061的敏感性表现出一致的,与基因型相关的差异,其中基因型2a,3a和5a衍生的重组体的敏感性比基因型1b,4a和6a衍生的重组体低约100倍。观察结果与近期对基因型1、2和3感染患者进行的治疗试验中发现的应答率的主要差异一致。在VX-950中也观察到 udin敏感性,基因型1b,2a和6a衍生 udrebinbins的敏感性是基因型3a,4a和5a衍生 udrecombinant的两倍。在PI浓度升高/过高的情况下传递基因型内和基因型之间的重组体可以鉴定PI抗性突变。对BILN 2061的抗性突变位于NS3蛋白酶中先前鉴定的位置156和168,在每个基因型中具有很大的氨基酸取代多样性。将已鉴定的抗性突变重新引入原始的重组病毒中,赋予了对BILN 2061的抗性,并且 udsome突变也影响了重组体的复制动力学。所开发的 udsystem将对所有6种主要HCV基因型中针对不同PIs的自然 udand治疗诱导的耐药突变的表型表征具有重要价值。这将允许在新开发的PI进入临床试验之前开发治疗反应预测,并开发出个性化的抗病毒/治疗方案。

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    Imhof Ingrid;

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  • 年度 2010
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