A diagnostic HIV-1 tropism system based on sequence relatedness

Abstract

Key clinical studies for HIV coreceptor antagonists have used the phenotyping-based Trofile test. Meanwhile various simpler-to-do genotypic tests have become available that are compatible with standard laboratory equipment and Web-based interpretation tools. However, these systems typically analyze only the most prominent virus sequence in a specimen. We present a new diagnostic HIV tropism test not needing DNA sequencing. The system, XTrack, uses physical properties of DNA duplexes after hybridization of single-stranded HIV-1 env V3 loop probes to the clinical specimen. Resulting "heteroduplexes" possess unique properties driven by sequence relatedness to the reference and resulting in a discrete electrophoretic mobility. A detailed optimization process identified diagnostic probe candidates relating best to a large number of HIV-1 sequences with known tropism. From over 500 V3 sequences representing all main HIV-1 subtypes (Los Alamos database), we obtained a small set of probes to determine the tropism in clinical samples. We found a high concordance with the commercial TrofileES test (84.9%) and the Web-based tool Geno2Pheno (83.0%). Moreover, the new system reveals mixed virus populations, and it was successful on specimens with low virus loads or on provirus from leukocytes. A replicative phenotyping system was used for validation. Our data show that the XTrack test is favorably suitable for routine diagnostics. It detects and dissects mixed virus populations and viral minorities; samples with viral loads (VL) of <200 copies/ml are successfully analyzed. We further expect that the principles of the platform can be adapted also to other sequence-divergent pathogens, such as hepatitis B and C viruses.

FIG 1

(A) Typical electropherogram depicting the test principle. Four types of mass peaks are resolved: 80-bp molecular weight marker (MWM); single-stranded V3 probe (ss-probe); hybrid of patient-derived V3 sequence and probe (duplex). Residual double-stranded probe material serves as the ds-control. PM, perfect match; 1 and 2 indicate relative peak distances for tropism calculation. (B) Representative section of the list of V3 loop nucleotide sequences after grouping according to their relatedness to the probe candidate in the top line: HIV-1_JR-CSF.

FIG 2

Dendrogram of 655 HIV-1 sequences depicting the maximal relative relationship to one another (Los Alamos database and data from the Swiss HIV Cohort Study). Blue letters, R5-tropic viruses; red letters, X4-tropic isolates. Lobes of related sequences correspond with subtypes/groups (capital letters); the group O branch with 4 isolates is truncated. Yellow arrow indicates arbitrary reference sequence HIV-1_JR-CSF.

FIG 3

XTrack analysis of one prototypic R5-tropic and one X4-tropic virus at position “sample duplex.” MW, double-stranded molecular weight marker; ss-probe, labeled single-stranded probe; ds-probe, double-stranded probe.

FIG 4

Degree of agreement between the three systems, based on the data in Table 5. Bars indicate either the overall agreement between the three systems (“all”) or between XTrack and TrofileES, or XTrack and Geno2Pheno, or Geno2Pheno and TrofileES, as indicated. Percent agreement is given above each bar.