Development and Characterization of a Novel Single-Cycle Recombinant-Virus Assay To Determine Human Immunodeficiency Virus Type 1 Coreceptor Tropism

Abstract

Most human immunodeficiency virus type 1 (HIV-1) strains require either the CXCR4 or CCR5 chemokine receptor to efficiently enter cells. Blocking viral binding to these coreceptors is an attractive therapeutic target. Currently, several coreceptor antagonists are being evaluated in clinical trials that require characterization of coreceptor tropism for enrollment. In this report, we describe the development of an automated and accurate procedure for determining HIV-1 coreceptor tropism (Trofile) and its validation for routine laboratory testing. HIV-1 pseudoviruses are generated using full-length env genes derived from patient virus populations. Coreceptor tropism is determined by measuring the abilities of these pseudovirus populations to efficiently infect CD4 ؉ /U87 cells expressing either the CXCR4 or CCR5 coreceptor. Viruses exclusively and efficiently infecting CXCR4 ؉ /CD4 ؉ /U87 cells are designated X4-tropic. Conversely, viruses exclusively and efficiently infecting CCR5 ؉ /CD4 ؉ /U87 cells are designated R5-tropic. Viruses capable of infecting both CXCR4 ؉ /CD4 ؉ /U87 and CCR5 ؉ /CD4 ؉ /U87 cells are designated dual/mixed-tropic. Assay accuracy and reproducibility were established by evaluating the tropisms of well-characterized viruses and the variability among replicate results from samples tested repeatedly. The viral subtype, hepatitis B virus or hepatitis C virus coinfection, and the plasma viral load did not affect assay performance. Minority subpopulations with alternate tropisms were reliably detected when present at 5 to 10%. The plasma viral load above which samples can be amplified efficiently in the Trofile assay is 1,000 copies per ml of plasma. Trofile has been automated for high-throughput use; it can be used to identify patients most likely to benefit from treatment regimens that include a coreceptor inhibitor and to monitor patients on treatment for the emergence of resistant virus populations that switch coreceptor tropism.

HIV-1 Tropism Test Evaluation: Assessment and Clinical Implications

“…Among phenotypic assays, Trofile test has been the most widely used (as discussed by Whitcomb et al [8]), and it has been recently replaced by an equivalent with enhanced sensitivity (Trofile ES or ESTA) (as discussed by Trinh et al [9]). This strategy utilizes patient-derived env gene amplified by PCR and inserted into an expression vector.…”

Chemokine Receptors as Therapeutic Targets in HIV Infection

“…As a result, a number of subjects were misjudged to have R5 virus at screening, whereas they actually harbored Dual/Mixed (D/M) virus populations when they entered clinical trials of CCR5 inhibitors; these patients appeared to have a blunted virologic response and high rates of early virologic failure [152][153][154]. Application of an enhanced-sensitivity Trofile assay or "deep sequencing" and re-analysis of the data showed that virologic failure in a significant proportion of these patients stemmed from the expansion of pre-existing minority CXCR4-using variants that went undetected during the first round of testing [155][156][157]. Accurate determination of coreceptor usage is therefore necessary to optimize treatment strategies before the initiation of and during therapy with CCR5 inhibitors.…”

HIV Antiretroviral Drug Resistance

“…The Trophile ES assay (Monogram Biosciences) tests for CCR5 coreceptor trophism but requires a viral load of preferably greater than 1,000 copies/ml [44]. The newer Trophile DNA assay (Monogram Biosciences) however, may be used even if the viral load is undetectable, such as the case of a patient receiving an optimal drug regimen but needing to be switched because of an adverse drug effect.…”