Ultra-short response-guided Hepatitis C treatment with sofosbuvir and daclatasvir: the SEARCH study HCV sequence data

Background

WHO has called for research into predictive factors for selecting persons who could be successfully treated with shorter durations of antiviral therapy for Hepatitis C. We evaluated early virological response as a means of shortening treatment and explored host, viral and pharmacokinetic contributors to treatment outcome.

Methods

Duration of sofosbuvir and daclatasvir (SOF/DCV) was determined according to day 2 (D2) virologic response for HCV genotype (gt) 1- or 6-infected adults in Vietnam with mild liver disease. Participants received 4 or 8 weeks of treatment according to whether D2 HCV RNA was above or below 500 IU/ml (standard duration is 12 weeks). Primary endpoint was sustained virological response (SVR12). Those failing therapy were retreated with 12 weeks SOF/DCV. Host IFNL4 genotype and viral sequencing was performed at baseline, with repeat viral sequencing if virological rebound was observed. Levels of SOF, its inactive metabolite GS-331007 and DCV were measured on day 0 and 28.

Findings

Of 52 adults enrolled, 34 received 4 weeks SOF/DCV, 17 got 8 weeks and one withdrew. SVR12 was achieved in 21/34 (62%) treated for 4 weeks, and 17/17 (100%) treated for 8 weeks. Overall, 38/51 (75%) were cured with first-line treatment (mean duration of 37 days). Despite a high prevalence of putative NS5A-inhibitor resistance-associated substitutions (RAS), all first-line treatment failures were cured after retreatment (13/13). We found no evidence treatment failure was associated with host IFNL4 genotype, viral subtype, baseline RAS or DCV levels. SOF metabolite levels were higher in those failing 4-week therapy.

Interpretation

Shortened SOF/DCV therapy with retreatment if needed, reduces DAA use while maintaining high cure rates. D2 virologic response alone does not adequately predict SVR12 with 4 weeks of treatment.

Methods
At screening, HCV genotype and subtype were determined using NS5B, Core and 5’ UTR sequencing, according to the method described by Chau et. al. To evaluate the impact of HCV subtypes and resistance-associated substitutions on treatment outcome, whole genome sequencing (WGS) was additionally performed on all enrolled participants’ virus at baseline, and upon virological rebound and at start of retreatment in participants failing therapy. WGS of the HCV viral genome was attained using Illumina MiSeq platform. The de novo assemblies nucleotide sequences were translated into amino acid and were aligned to H77 HCV reference (GenBank ID: NC_038882.1) and the NS5A and NS5B protein regions were extracted.  We only looked for RAS that were present in at least 15% of the reads in the sample and had a read count of greater than 10.

We used the Public Health England (PHE) HCV Resistance Group’s definition for resistance-associated substitutions (RAS). For genotype 1, we looked for RASs defined specifically for genotype 1 as they are well studied. For genotype 6, we looked for all RASs defined across all genotypes, as little work has been done on RASs in genotype 6.  

For DCV, we looked for 24R, 28T, 30E/K/T, 31M/V, 32L, 58D, and 93C/H/N/R/S/W in genotype 1 infection and additionally looked for 28S, 30R and 31F in genotype 6 infection. For SOF, we looked for 159F, 237G, 282T, 315H/N, 321A/I in genotype 1 infection and additionally looked for 289I in genotype 6 infection.  

In addition to viral sequencing, we evaluated host genetic polymorphisms within the interferon lambda 4 (IFNL4) gene of all participants at baseline. Genotyping of IFNL4 rs368234815 was performed on host DNA using the TaqMan® SNP genotyping assay and primers described previously with Type‐it Fast SNP Probe PCR Master Mix (Qiagen).

背景

世界卫生组织（WHO）呼吁开展相关研究，以筛选出可通过更短疗程抗病毒疗法成功治愈的丙型肝炎患者。本研究评估了早期病毒学应答作为缩短治疗疗程的可行性，并探索了宿主、病毒及药代动力学因素对治疗结局的影响。

方法

针对越南境内感染丙型肝炎病毒（Hepatitis C virus, HCV）基因型1或6型且伴有轻度肝病的成年患者，索磷布韦/达卡他韦（sofosbuvir and daclatasvir, SOF/DCV）的治疗疗程依据第2天（D2）的病毒学应答结果确定。根据D2时HCV核糖核酸（HCV RNA）水平是否高于500国际单位/毫升（IU/ml），受试者分别接受4周或8周的治疗（标准疗程为12周）。本研究的主要终点为持续病毒学应答12周（sustained virological response, SVR12）。治疗失败的患者将接受12周的SOF/DCV补救治疗。研究在基线时对宿主IFNL4基因型进行检测并完成病毒测序，若观察到病毒学反弹则再次进行病毒测序。分别在第0天和第28天检测索磷布韦（SOF）、其无活性代谢产物GS-331007以及达卡他韦（DCV）的血药浓度。

研究结果

本研究共纳入52名成年受试者，其中34人接受4周SOF/DCV治疗，17人接受8周治疗，1人退出研究。接受4周治疗的受试者中，21/34（62%）达到SVR12；接受8周治疗的受试者全部（17/17，100%）达到SVR12。整体而言，51名完成一线治疗的受试者中38人（75%）获得治愈，平均治疗时长为37天。尽管NS5A抑制剂耐药相关替换（resistance-associated substitutions, RAS）的推定突变检出率较高，但所有一线治疗失败的受试者在接受补救治疗后均获得治愈（13/13）。本研究未发现治疗失败与宿主IFNL4基因型、病毒亚型、基线RAS或DCV血药浓度存在关联。接受4周治疗后失败的受试者，其SOF代谢产物的血药水平更高。

研究解读

按需辅以补救治疗的短疗程SOF/DCV方案，可在维持高治愈率的同时减少直接抗病毒药物（direct-acting antiviral, DAA）的使用量。仅依靠D2病毒学应答，无法充分预测4周疗程治疗后的SVR12结局。

补充方法

筛查阶段，本研究依据Chau等人报道的方法，通过NS5B、Core及5’非翻译区（5’ UTR）测序确定HCV基因型及亚型。为评估HCV亚型与RAS对治疗结局的影响，本研究对所有纳入受试者的基线病毒、治疗失败受试者的病毒学反弹样本以及补救治疗开始时的病毒样本额外开展全基因组测序（whole genome sequencing, WGS）。HCV全基因组测序采用Illumina MiSeq测序平台完成。将从头组装的核苷酸序列翻译为氨基酸序列，并与H77 HCV参考序列（GenBank收录号：NC_038882.1）进行比对，随后提取NS5A与NS5B蛋白区域序列。本研究仅统计样本中至少15%的测序读段（reads）携带、且读段计数大于10的RAS。

本研究采用英国公共卫生署（Public Health England, PHE）HCV耐药研究组定义的RAS判定标准。对于基因型1，本研究仅检索针对基因型1的特异性RAS，因其已有充分研究；对于基因型6，本研究检索所有基因型中已定义的RAS，因为目前针对基因型6 RAS的研究较少。

针对DCV，本研究在基因型1感染患者中检索24R、28T、30E/K/T、31M/V、32L、58D及93C/H/N/R/S/W位点突变，同时在基因型6感染患者中额外检索28S、30R及31F位点突变。针对SOF，本研究在基因型1感染患者中检索159F、237G、282T、315H/N、321A/I位点突变，同时在基因型6感染患者中额外检索289I位点突变。

除病毒测序外，本研究在基线时对所有受试者的宿主干扰素λ4（interferon lambda 4, IFNL4）基因区域的遗传多态性进行分析。采用TaqMan®单核苷酸多态性（single nucleotide polymorphism, SNP）基因分型检测试剂盒，结合既往报道的引物与Type-it Fast SNP Probe PCR Master Mix（Qiagen公司产品），对宿主DNA中IFNL4 rs368234815位点进行基因分型。