Comparison of Human Immunodeficiency Virus Type 1 Tropism Profiles in Clinical Samples by the Trofile and MT-2 Assays

The recent availability of CCR5 antagonists as anti-human immunodeficiency virus (anti-HIV) therapeutics has highlighted the need to accurately identify CXCR4-using variants in patient samples when use of this new drug class is considered. The Trofile assay (Monogram Biosciences) has become the method that is the most widely used to define tropism in the clinic prior to the use of a CCR5 antagonist. By comparison, the MT-2 assay has been used since early in the HIV epidemic to define tropism in clinical specimens. Given that there are few data from direct comparisons of these two assays, we evaluated the performance of the plasma-based Trofile assay and the peripheral blood mononuclear cell (PBMC)-based MT-2 assay for the detection of CXCR4 use in defining the tropism of HIV isolates derived from clinical samples. The various samples used for this comparison were derived from participants of the Amsterdam Cohort Studies on HIV infection and AIDS who underwent consecutive MT-2 assay testing of their PBMCs at approximately 3-month intervals. This unique sample set was specifically selected because consecutive MT-2 assays had demonstrated a shift from negative to positive in PBMCs, reflecting the first emergence of CXCR4-using virus in PBMCs above the level of detection of the assay in these individuals. Trofile testing was performed with clonal HIV type 1 (HIV-1) variants (n = 21), MT-2 cell culture-derived cells (n = 20) and supernatants (n = 42), and plasma samples (n = 76). Among the clonal HIV-1 variants and MT-2 cell culture-derived samples, the results of the Trofile and MT-2 assays demonstrated a high degree of concordance (95% to 98%). Among consecutive plasma samples, detection of CXCR4-using virus was at or before the time of first detection by the MT-2 assay in 5/10 patients by the original Trofile assay and in 9/10 patients by the enhanced-sensitivity Trofile assay. Differences in the time to the first detection of CXCR4 use between the MT-2 assay (PBMCs) and the original Trofile assay (plasma) were greatly reduced by the enhanced-sensitivity Trofile assay, suggesting that sensitivity for the detection of minor CXCR4-using variants may be a more important determinant of discordant findings than compartmentalization. The similarities in performance of the enhanced-sensitivity Trofile and MT-2 assays suggest that either may be an appropriate methodology to define tropism in patient specimens.The outcome of exposure to human immunodeficiency virus type 1 (HIV-1) varies greatly between individuals. One of the factors determining this variability in outcome is the cellular tropism or viral phenotype (7, 23), as the pathogenesis of HIV-1 is critically influenced by the cell types that the virus is capable of infecting. HIV-1 requires two cellular receptors for entry: CD4 and one of a family of chemokine receptors (coreceptor). In vivo, the major coreceptors used by HIV-1 are CCR5 (1, 13, 14) and CXCR4 (15). Individual viruses are classified on the basis of their ability to use CCR5 (R5 variants) or CXCR4 (X4 variants), or both (R5X4 or dual variants) (2). Viral populations demonstrating the use of both receptors are designated dual/mixed (D/M), as they may contain any mixture of these three types (43). Before the identification of HIV coreceptors, viruses were classified on the basis of their ability to infect T-cell lines and/or macrophages and/or on the basis of their ability to induce syncytia (multinucleated giant cells) in MT-2 cells (16, 36) which do not express CCR5 (11). Thus, viruses that do not infect MT-2 cells are non-syncytium inducing and R5, while viruses that do infect MT-2 cells are syncytium inducing and either X4 or R5X4 (4). Determinants that govern coreceptor use have been mapped to the envelope gene, especially to the variable regions, and in particular the third variable (V3) region, the V3 loop (12, 17).Of note, the ability to induce syncytia in MT-2 cells reflects the viral coreceptor use and not necessarily the enhanced cytopathogenicity of X4 virus per se, as R5 virus can at least be equally cytopathogenic to its target cells (18, 26, 33). The differential pathogenicity of R5 and X4 viruses in vivo mainly lies in the coreceptor expression patterns of the host cells. CXCR4 is expressed on many more CD4⁺ cells in the body (including hematopoietic progenitor cells, thymocytes, naïve T cells, and monocytes) than CCR5, which is mostly found on memory T cells and macrophages (3, 6). Thus, CXCR4 use potentially allows the virus access to a large and critical pool of target cells affecting T-cell ontogeny, a fact that may be relevant to the accelerated CD4⁺ T-cell decline associated with the emergence of CXCR4-using virus (5). Therefore, detection of the emergence of CXCR4-using virus has potential value for predicting pathogenesis, monitoring disease progression, and making treatment decisions. Moreover, the detection of CXCR4-using variants is required for the optimal use of the recently available CCR5 antagonists as HIV therapeutics, as the activity of CCR5 antagonists in patients is attenuated in the presence of CXCR4-using HIV (27).Traditionally, the MT-2 assay has served as a tool for the determination of tropism on the basis of the expression of CXCR4 (and not CCR5) on the cell surface. There are a wealth of data correlating the detection of CXCR4-using virus by the MT-2 assay and accelerated disease progression in treatment-naïve individuals (7, 8, 23, 29, 32). Two widely used versions of the MT-2 assay exist: one in which the patient''s cells are directly cocultured with the MT-2 cells (25) and another in which virus stocks are first generated from the patient''s cells by coculture with seronegative phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) (22). The latter approach may limit the sensitivity of the assay, as MT-2 cells are more sensitive to infection by CXCR4-using virus than PBMCs. Moreover, PBMC passaging of patient samples prior to MT-2 inoculation has the potential to alter the relative proportions of viral subpopulations present in the original sample (42). Although the MT-2 assay is not technically challenging, the need for viable patient cells (either fresh or cryopreserved) and biosafety level 3 laboratories has limited its widespread implementation.By comparison, the Trofile assay (Monogram Biosciences) was utilized in the MOTIVATE trials, which led to the approval of maraviroc, the first in the class of CCR5 antagonists (19). This assay, specifically adapted for high-throughput testing of patient samples, is available in 35 countries; and with more than 54,000 samples tested to date, the assay is the only clinically validated test available for the screening of patients considering CCR5 antagonist therapy (9, 19, 21, 28, 30, 44). The Trofile assay evaluates the coreceptor use of recombinant luciferase-reporter viruses pseudotyped with a population (or clones) of patient-derived HIV envelopes in a single-cycle infection assay. The original version of the assay was validated to detect low-level X4 or R5 variants with 100% sensitivity when those variants comprised 10% of a mixed HIV envelope population and with 85% sensitivity when those variants comprised 5% of a population (43). Clinical trial data indicated that an improved sensitivity for the detection of low-level CXCR4-using variants might improve the selection of patients for CCR5 antagonist therapy (19, 20). Therefore, an enhanced version of the Trofile assay with an approximately 30-fold increased sensitivity for the detection of low-level X4 variants was developed and validated to detect low-level X4 variants with 100% sensitivity when the variants comprised as little as 0.3% of a mixed envelope population (31). Retrospective analyses of two studies evaluating CCR5 antagonist activity among individuals with R5 tropism defined by the original Trofile assay demonstrated improved outcomes among those with R5 tropism defined by the enhanced-sensitivity Trofile assay (34, 35). The assay with enhanced sensitivity replaced the original Trofile assay in the clinic in June 2008.In this study we explored the performance of the Trofile assay for the detection of HIV-1 tropism profiles in clinical specimens derived from individuals in whom consecutive MT-2 assays at 3-month intervals had detected the emergence of CXCR4-using virus in PBMCs for the first time. The samples tested were plasma, clonal HIV-1 variants, and cells and cell-free supernatants derived from MT-2 cell cultures. Testing of clonal HIV-1 variants and samples derived from MT-2 cell cultures allowed a comparison of the tropism characterization of homogeneous viral populations. Testing of consecutive patient samples allowed interrogation of the performance of both the MT-2 assay (with PBMCs) and the Trofile assay (with plasma) near the limits of detection for CXCR4-using variants.