Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic.
J Clin Microbiol. 2013 May;51(5):1517-27. doi: 10.1128/JCM.00092-13. Epub 2013 Mar 13.
CCR5 antagonists are a powerful new class of antiretroviral drugs that require a companion assay to evaluate the presence of CXCR4-tropic (non-R5) viruses prior to use in human immunodeficiency virus (HIV)-infected individuals. In this study, we have developed, characterized, verified, and prevalidated a novel phenotypic test to determine HIV-1 coreceptor tropism (VERITROP) based on a sensitive cell-to-cell fusion assay. A proprietary vector was constructed containing a near-full-length HIV-1 genome with the yeast uracil biosynthesis (URA3) gene replacing the HIV-1 env coding sequence. Patient-derived HIV-1 PCR products were introduced by homologous recombination using an innovative yeast-based cloning strategy. The env-expressing vectors were then used in a cell-to-cell fusion assay to determine the presence of R5 and/or non-R5 HIV-1 variants within the viral population. Results were compared with (i) the original version of Trofile (Monogram Biosciences, San Francisco, CA), (ii) population sequencing, and (iii) 454 pyrosequencing, with the genotypic data analyzed using several bioinformatics tools, i.e., the 11/24/25 rule, Geno2Pheno (2% to 5.75%, 3.5%, or 10% false-positive rate [FPR]), and webPSSM. VERITROP consistently detected minority non-R5 variants from clinical specimens, with an analytical sensitivity of 0.3%, with viral loads of ≥1,000 copies/ml, and from B and non-B subtypes. In a pilot study, a 73.7% (56/76) concordance was observed with the original Trofile assay, with 19 of the 20 discordant results corresponding to non-R5 variants detected using VERITROP and not by the original Trofile assay. The degree of concordance of VERITROP and Trofile with population and deep sequencing results depended on the algorithm used to determine HIV-1 coreceptor tropism. Overall, VERITROP showed better concordance with deep sequencing/Geno2Pheno at a 0.3% detection threshold (67%), whereas Trofile matched better with population sequencing (79%). However, 454 sequencing using Geno2Pheno at a 10% FPR and 0.3% threshold and VERITROP more accurately predicted the success of a maraviroc-based regimen. In conclusion, VERITROP may promote the development of new HIV coreceptor antagonists and aid in the treatment and management of HIV-infected individuals prior to and/or during treatment with this class of drugs.
CCR5 拮抗剂是一类强大的新型抗逆转录病毒药物，在用于感染人类免疫缺陷病毒 (HIV) 的个体之前，需要一种伴随的检测方法来评估是否存在 CXCR4 嗜性（非 R5）病毒。在这项研究中，我们开发了一种新的基于敏感细胞间融合检测的 HIV-1 核心受体嗜性（VERITROP）的表型检测方法，并对其进行了特征描述、验证和预验证。该方法构建了一个专有的载体，其中包含一个接近全长的 HIV-1 基因组，用酵母尿嘧啶生物合成 (URA3) 基因取代了 HIV-1 env 编码序列。使用一种创新的基于酵母的克隆策略，通过同源重组引入源自患者的 HIV-1 PCR 产物。然后，将表达 env 的载体用于细胞间融合检测，以确定病毒群中是否存在 R5 和/或非 R5 HIV-1 变体。结果与 (i) Trofile（Monogram Biosciences，旧金山，CA）的原始版本，(ii) 群体测序，和 (iii) 454 焦磷酸测序进行比较，使用几种生物信息学工具对基因型数据进行分析，即 11/24/25 规则、Geno2Pheno（2%至 5.75%、3.5%或 10%假阳性率 [FPR]）和 webPSSM。VERITROP 一致地从临床标本中检测到少数非 R5 变体，分析灵敏度为 0.3%，病毒载量为≥1,000 拷贝/ml，且来自 B 和非 B 亚型。在一项试点研究中，与原始 Trofile 检测相比，观察到 73.7%（56/76）的一致性，其中 20 个不一致结果中有 19 个对应于使用 VERITROP 检测到的而非原始 Trofile 检测到的非 R5 变体。VERITROP 和 Trofile 与群体和深度测序结果的一致性程度取决于用于确定 HIV-1 核心受体嗜性的算法。总体而言，当检测阈值为 0.3%时，VERITROP 与深度测序/Geno2Pheno 的一致性更好（67%），而 Trofile 与群体测序的一致性更好（79%）。然而，使用 Geno2Pheno 进行 454 测序，假阳性率为 10%，检测阈值为 0.3%，以及 VERITROP 更准确地预测了 maraviroc 类药物治疗的成功。总之，VERITROP 可以促进新型 HIV 核心受体拮抗剂的开发，并有助于在使用该类药物之前和/或期间治疗和管理 HIV 感染个体。
