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.

The human immunodeficiency virus type 1 (HIV-1) integrase mutations N155H and Q148R(H)(K) that reduce susceptibility to the integrase inhibitor raltegravir have been identified in patients failing treatment regimens containing raltegravir. Whether these resistance mutations occur individually or in combination within a single virus genome has not been defined, nor do we fully understand the impact of these primary mutations and other secondary mutations on raltegravir susceptibility and viral replication capacity. To address these important questions, we investigated the raltegravir susceptibility and replication capacity of viruses containing mutations at positions 155 and 148 separately or in combination with secondary mutations selected in subjects failing treatment regimens containing raltegravir. Clonal analysis demonstrated that N155H and Q148R(H)(K) occur independently, not in combination. Viruses containing a Q148R(H)(K) mutation generally displayed larger reductions in raltegravir susceptibility than viruses with an N155H mutation. Analysis of site-directed mutants indicated that E92Q in combination with N155H resulted in a higher level of resistance to raltegravir than N155H alone. Viruses containing a Q148R(H) mutation together with a G140S mutation were more resistant to raltegravir than viruses containing a Q148R(H) mutation alone; however, viruses containing G140S and Q148K were more susceptible to raltegravir than viruses containing a Q148K mutation alone. Both N155H and Q148R(H)(K) mutations reduced the replication capacity, while the addition of secondary mutations either improved or reduced the replication capacity depending on the primary mutation. This study demonstrates distinct genetic pathways to resistance in subjects failing raltegravir regimens and defines the effects of primary and secondary resistance mutations on raltegravir susceptibility and replication capacity.

In human immunodeficiency virus type 1 (HIV-1) subtype B, CXCR4 coreceptor use ranges from ϳ20% in early infection to ϳ50% in advanced disease. Coreceptor use by non-subtype B HIV is less well characterized. We studied coreceptor tropism of subtype A and D HIV-1 collected from 68 pregnant, antiretroviral drug-naive Ugandan women (HIVNET 012 trial). None of 33 subtype A or 10 A/D-recombinant viruses used the CXCR4 coreceptor. In contrast, nine (36%) of 25 subtype D viruses used both CXCR4 and CCR5 coreceptors. Clonal analyses of the nine subtype D samples with dual or mixed tropism revealed heterogeneous viral populations comprised of X4-, R5-, and dual-tropic HIV-1 variants. In five of the six samples with dual-tropic strains, V3 loop sequences of dual-tropic clones were identical to those of cocirculating R5-tropic clones, indicating the presence of CXCR4 tropism determinants outside of the V3 loop. These dual-tropic variants with R5-tropic-like V3 loops, which we designated "dual-R," use CCR5 much more efficiently than CXCR4, in contrast to dual-tropic clones with X4-tropic-like V3 loops ("dual-X"). These observations have implications for pathogenesis and treatment of subtype D-infected individuals, for the association between V3 sequence and coreceptor tropism phenotype, and for understanding potential mechanisms of evolution from exclusive CCR5 use to efficient CXCR4 use by subtype D HIV-1.Human immunodeficiency virus type 1 (HIV-1) infection requires interactions between the viral envelope (Env) surface glycoprotein (gp120), the cellular receptor (CD4), and a coreceptor (e.g., CCR5 and/or CXCR4) (36). CCR5, the most commonly used coreceptor, is present on primary T cells and macrophages. In contrast, CXCR4 is expressed on many cell types, including thymocytes, primary T cells, and macrophages (13). CXCR4-using viruses can induce formation of syncytia when cultured on the CXCR4-bearing MT2 cell line (syncytiuminducing, SI viruses) (3,8,21,33,48). SI or CXCR4-using viruses are typically found in individuals with advanced disease (2,9,16,18,19). However, it is not clear whether CXCR4 use precedes and causes more rapid disease progression or is merely the consequence of a change in target cell availability. The recent development of HIV-1 entry inhibitors that target CCR5 has heightened interest in coreceptor usage (44).Several surveys of coreceptor tropism were reported recently. Brumme et al. (6) studied almost 1,000 antiretroviral drug naive HIV-1-infected patients. CXCR4-using virus was detected in 18% of those individuals, more than 99% of which were also able to use CCR5 and were thus categorized as dualor mixed-tropic (DM). CXCR4 use was associated with decreased survival in univariate, but not multivariate, analyses. There was a statistically nonsignificant trend toward increased CXCR4 use in non-subtype B viruses (7 of 13 [54%] for nonsubtype B versus 143 of 675 [21%] for subtype B; C. J. Brumme and P. R. Harrigan, unpublished data). Moyle et al. (37) evaluated predictive factors for coreceptor use am...

Many studies have demonstrated that the third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is a major determinant of coreceptor tropism. Other regions in the surface gp120 subunit of Env can modulate coreceptor tropism in a manner that is not fully understood. In this study, we evaluated the effect of env determinants outside of V3 on coreceptor usage through the analysis of (i) patient-derived env clones that differ in coreceptor tropism, (ii) chimeric env sequences, and (iii) site-directed mutants. The introduction of distinct V3 sequences from CXCR4-using clones into an R5-tropic env backbone conferred the inefficient use of CXCR4 in some but not all cases. Conversely, in many cases, X4-and dual-tropic env backbones containing the V3 sequences of R5-tropic clones retained the ability to use CXCR4, suggesting that sequences outside of the V3 regions of these CXCR4-using clones were responsible for CXCR4 use. The determinants of CXCR4 use in a set of dual-tropic env sequences with V3 sequences identical to those of R5-tropic clones mapped to the gp41 transmembrane (TM) subunit. In one case, a single-amino-acid substitution in the fusion peptide of TM was able to confer CXCR4 use; however, TM substitutions associated with CXCR4 use varied among different env sequences. These results demonstrate that sequences in TM can modulate coreceptor specificity and that env sequences other than that of V3 may facilitate efficient CXCR4-mediated entry. We hypothesize that the latter plays an important role in the transition from CCR5 to CXCR4 coreceptor use.

Background Blood-based methods using cell-free DNA (cfDNA) are under development as an alternative to existing screening tests. However, early-stage detection of cancer using tumor-derived cfDNA has proven challenging because of the small proportion of cfDNA derived from tumor tissue in early-stage disease. A machine learning approach to discover signatures in cfDNA, potentially reflective of both tumor and non-tumor contributions, may represent a promising direction for the early detection of cancer. Methods Whole-genome sequencing was performed on cfDNA extracted from plasma samples ( N = 546 colorectal cancer and 271 non-cancer controls). Reads aligning to protein-coding gene bodies were extracted, and read counts were normalized. cfDNA tumor fraction was estimated using IchorCNA. Machine learning models were trained using k-fold cross-validation and confounder-based cross-validations to assess generalization performance. Results In a colorectal cancer cohort heavily weighted towards early-stage cancer (80% stage I/II), we achieved a mean AUC of 0.92 (95% CI 0.91–0.93) with a mean sensitivity of 85% (95% CI 83–86%) at 85% specificity. Sensitivity generally increased with tumor stage and increasing tumor fraction. Stratification by age, sequencing batch, and institution demonstrated the impact of these confounders and provided a more accurate assessment of generalization performance. Conclusions A machine learning approach using cfDNA achieved high sensitivity and specificity in a large, predominantly early-stage, colorectal cancer cohort. The possibility of systematic technical and institution-specific biases warrants similar confounder analyses in other studies. Prospective validation of this machine learning method and evaluation of a multi-analyte approach are underway. Electronic supplementary material The online version of this article (10.1186/s12885-019-6003-8) contains supplementary material, which is available to authorized users.