Development and Validation of a Genotypic Assay to Quantify CXCR4- and CCR5-Tropic Human Immunodeficiency Virus Type-1 (HIV-1) Populations and a Comparison to Trofile®

Viruses. 2024 Mar 27;16(4):510. doi: 10.3390/v16040510.

Abstract

HIV-1 typically infects cells via the CD4 receptor and CCR5 or CXCR4 co-receptors. Maraviroc is a CCR5-specific viral entry inhibitor; knowledge of viral co-receptor specificity is important prior to usage. We developed and validated an economical V3-env Illumina-based assay to detect and quantify the frequency of viruses utilizing each co-receptor. Plasma from 54 HIV+ participants (subtype B) was tested. The viral template cDNA was generated from plasma RNA with unique molecular identifiers (UMIs). The sequences were aligned and collapsed by the UMIs with a custom bioinformatics pipeline. Co-receptor usage, determined by codon analysis and online phenotype predictors PSSM and Geno2pheno, were compared to existing Trofile® data. The cost of V3-UMI was tallied. The sequences interpreted by Geno2pheno using the most conservative cut-off, a 2% false-positive-rate (FPR), predicted CXCR4 usage with the greatest sensitivity (76%) and specificity (100%); PSSM and codon analysis had similar sensitivity and lower specificity. Discordant Trofile® and genotypic results were more common when participants had specimens from different dates analyzed by either assay. V3-UMI reagents cost USD$62/specimen. A batch of ≤20 specimens required 5 h of technical time across 1.5 days. V3-UMI predicts HIV tropism at a sensitivity and specificity similar to those of Trofile®, is relatively inexpensive, and could be performed by most central laboratories. The adoption of V3-UMI could expand HIV drug therapeutic options in lower-resource settings that currently do not have access to phenotypic HIV tropism testing.

Keywords: CCR5 co-receptor; CXCR4 co-receptor; R5 virus; X4 virus; maraviroc.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural
  • Comparative Study
  • Validation Study

MeSH terms

  • Genotype
  • Genotyping Techniques* / methods
  • HIV Infections / virology
  • HIV-1 / genetics
  • HIV-1 / physiology
  • Humans
  • Male
  • RNA, Viral / genetics
  • Receptors, CCR5* / genetics
  • Receptors, CCR5* / metabolism
  • Receptors, CXCR4* / genetics
  • Receptors, CXCR4* / metabolism
  • Sensitivity and Specificity
  • Viral Tropism

Substances

  • CCR5 protein, human
  • CXCR4 protein, human
  • Receptors, CCR5
  • Receptors, CXCR4
  • RNA, Viral