Clonal analysis of HIV-1 genotype and function associated with virologic failure in treatment-experienced persons receiving maraviroc: Results from the MOTIVATE phase 3 randomized, placebo-controlled trials

Detailed clonal phenotypic/genotypic analyses explored viral-escape mechanisms during maraviroc-based therapy in highly treatment-experienced participants from the MOTIVATE trials. To allow real-time assessment of samples while maintaining a blind trial, the first 267 enrolled participants were selected for evaluation. At failure, plasma samples from 20/50 participants (16/20 maraviroc-treated) with CXCR4-using virus and all 38 (13 maraviroc-treated) with CCR5-tropic virus were evaluated. Of those maraviroc-treated participants with CXCR4-using virus at failure, genotypic and phenotypic clonal tropism determinations showed >90% correspondence in 14/16 at Day 1 and 14/16 at failure. Phylogenetic analysis of clonal sequences detected pre-treatment progenitor CXCR4-using virus, or on-treatment virus highly divergent from the Day 1 R5 virus, excluding possible co-receptor switch through maraviroc-mediated evolution. Re-analysis of pre-treatment samples using the enhanced-sensitivity Trofile® assay detected CXCR4-using virus pre-treatment in 16/20 participants failing with CXCR4-using virus. Post-maraviroc reversion of CXCR4-use to CCR5-tropic occurred in 7/8 participants with follow-up, suggesting selective maraviroc inhibition of CCR5-tropic variants in a mixed-tropic viral population, not emergence of de novo mutations in CCR5-tropic virus, as the main virologic escape mechanism. Maraviroc-resistant CCR5-tropic virus was observed in plasma from 5 treated participants with virus displaying reduced maximal percent inhibition (MPI) but no evidence of IC50 change. Env clones with reduced MPI showed 1–5 amino acid changes specific to each V3-loop region of env relative to Day 1. However, transferring on-treatment resistance-associated changes using site-directed mutagenesis did not always establish resistance in Day 1 virus, and key ‘signature’ mutation patterns associated with reduced susceptibility to maraviroc were not identified. Evolutionary divergence of the CXCR4-using viruses is confirmed, emphasizing natural selection not influenced directly by maraviroc; maraviroc simply unmasks pre-existing lineages by inhibiting the R5 virus. For R5-viral failure, resistance development through drug selection pressure was uncommon and manifested through reduced MPI and with virus strain–specific mutational patterns.本研究通过详尽的克隆表型与基因型分析，探究了来自MOTIVE试验的经多线抗逆转录病毒治疗失败受试者在接受马拉韦罗（maraviroc）治疗期间的病毒逃逸机制。为在保持盲法试验设计的前提下实现样本的实时评估，研究纳入了首批入组的267名受试者进行分析。在治疗失败时，研究对50名携带使用CXCR4共受体病毒的受试者中的20名（其中16名接受马拉韦罗治疗），以及全部38名携带嗜CCR5病毒的受试者（其中13名接受马拉韦罗治疗）的血浆样本进行了检测。在那些治疗失败时携带使用CXCR4共受体病毒且接受马拉韦罗治疗的受试者中，克隆嗜性的基因型与表型检测结果显示，在第1天和治疗失败时，分别有14/16的样本检测一致性超过90%。对克隆序列的系统发育分析发现了治疗前的CXCR4嗜性祖病毒，或是与第1天的R5病毒（嗜CCR5病毒）序列差异极大的治疗期间病毒，由此排除了通过马拉韦罗介导的病毒演化发生共受体转换的可能性。采用高灵敏度Trofile®检测对治疗前样本进行重新分析后，在20名因携带CXCR4嗜性病毒而治疗失败的受试者中，有16名在治疗前即检测到该类病毒。在获得随访数据的受试者中，有7/8的患者在接受马拉韦罗治疗后出现了CXCR4嗜性向CCR5嗜性的逆转，这表明病毒逃逸的主要机制是马拉韦罗对混合嗜性病毒群体中的CCR5嗜性变异株产生选择性抑制，而非CCR5嗜性病毒出现新发突变。在5名接受治疗的受试者血浆中检测到了马拉韦罗耐药的CCR5嗜性病毒，这类病毒表现出最大抑制百分比（maximal percent inhibition, MPI）降低，但未观察到半抑制浓度（IC50）的变化。与第1天的病毒株相比，MPI降低的Env克隆在env基因的V3环区域存在1-5个特异性氨基酸突变。然而，通过定点诱变引入治疗期间获得的耐药相关突变，并不总能使第1天的病毒株产生耐药性，且未发现与马拉韦罗易感性降低相关的关键“特征性”突变模式。研究证实了CXCR4嗜性病毒的演化差异，表明其演化是自然选择的结果，并未直接受马拉韦罗的影响；马拉韦罗仅通过抑制R5病毒，从而暴露了预先存在的病毒谱系。对于因R5病毒导致的治疗失败，通过药物选择压力产生耐药性的情况并不常见，其表现为MPI降低以及病毒株特异性的突变模式。