Virologic Failure in Therapy-Naïve Subjects on Aplaviroc plus Lopinavir-Ritonavir: Detection of Aplaviroc Resistance Requires Clonal Analysis of Envelope

Abstract

The CCR100136 (EPIC) study evaluated the antiviral activity of the novel CCR5 entry inhibitor aplaviroc in combination with lopinavir-ritonavir in drug-naïve human immunodeficiency virus type 1-infected subjects. Although the trial was stopped prematurely due to idiosyncratic hepatotoxicity, 11 subjects met the protocoldefined virologic failure criteria. Clonal analyses of the viral envelope tropism, aplaviroc susceptibility, and env sequencing were performed on plasma at day 1 and at the time of virologic failure. Molecular evolutionary analyses were also performed. Treatment-emergent resistance to aplaviroc or lopinavir-ritonavir was not observed at the population level. However, aplaviroc resistance was detected prior to therapy at both the clonal and population levels in one subject with virologic failure and in six subjects in a minority (<50%) of clones at day 1 or at the time of virologic failure. Reduced aplaviroc susceptibility manifested as a 50% inhibitory concentration curve shift and/or a plateau. Sequence changes in the clones with aplaviroc resistance were unique to each subject and scattered across the envelope coding region. Clones at day 1 and at the time of virologic failure were not phylogenetically distinct. Two subjects with virologic failure had a population tropism change from CCR5-to dual/mixed-tropic during treatment. Virologic failure during a regimen of aplaviroc and lopinavir-ritonavir may be associated with aplaviroc resistance, only at the clonal level, and/or, infrequently, tropism changes.Although highly active antiretroviral therapy results in a profound and sustained reduction in plasma human immunodeficiency virus type 1 (HIV-1) RNA in many individuals, the side effects of currently available antiretroviral therapy (ART) and the emergence of multidrug-resistant viral strains continue to represent major challenges for the management of HIV infection (7,29). The discovery that the chemokine receptors CCR5 and CXCR4 function as coreceptors that mediate HIV-1 entry into CD4 ϩ host cells (2) has led to the development of coreceptor inhibitors, which are currently being tested for use in ART.Unlike other drug classes, resistance to CCR5 entry inhibitors (CCR5 EIs) could involve the use of drug-bound CCR5 or a change in coreceptor usage. Much of the data on CCR5 EI resistance come from in vitro passage and short-term monotherapy studies. An analysis of virus envelopes (Env) during passage studies with CCR5 EIs demonstrated the maintenance of R5 tropism in a setting where the cells utilized expressed both CCR5 and CXCR4 (17,34,38). Resistance or reduced susceptibility to aplaviroc (APL) was slow to emerge in passage studies; the characterization of CCR5-tropic (R5-tropic) HIV-1 isolates from long-term passage (Ͼ48 weeks) showed marginal increases in the change (n-fold) in the 50% inhibitory concentration (FCIC 50 ) values, the standard measure by which resistance to other antiretroviral classes is detected (16,17).Another measure of drug susceptibility that has been suggested for CCR...

HIV-1 Resistance to CCR5 Antagonists Associated with Highly Efficient Use of CCR5 and Altered Tropism on Primary CD4 + T Cells

“…Thus, there is some variability in the relative importance of the CCR5 NЈ-terminal domain and ECL2 in supporting infection by different HIV-1 strains (41). This underlying plas- ticity can lead to rare, unexpected outcomes: we identified a patient whose virus exhibited baseline resistance to all CCR5 antagonists tested, which likely contributed to this individual's subsequent virologic failure on an antiviral regimen that included aplaviroc (21,48). This finding poses two central questions: by what mechanism(s) does resistance to CCR5 antagonists occur, and what are the implications of drug resistance for viral tropism?…”

“…Resistant viruses that utilize drug-bound CCR5 have been identified following in vitro passaging with multiple CCR5 antagonists (1,2,22,33,36,51,56). Recently, we identified a panel of viral Envs able to use aplaviroc (APL)-bound CCR5 that were isolated from a patient (21,48). The Envs from this patient were cross resistant to the CCR5 antagonists AD101, TAK779, SCH-C, and maraviroc.…”

HIV Type 1 from a Patient with Baseline Resistance to CCR5 Antagonists Uses Drug-Bound Receptor for Entry

“…These data are slightly different than the initial characterization of Envs from P5, in which the magnitude of APL resistance was greater in CCR5-expressing cells, and some clones were able to utilize CXCR4 for entry. 26 One explanation for these differences is that the pseudoviruses were produced separately using different cloning vectors and may have different characteristics. Additionally, although both sets of assays were performed on the same cell line, namely U87=CD4=CCR5, the CD4 and CCR5 expression levels may have been different in cells cultured in two separate laboratories.…”

“…In vitro, viruses passaged in the presence of increasing concentrations of CCR5 antagonists have evolved to utilize drug-bound CCR5 for entry, and coreceptor switching is rarely observed. 34 The initial characterization of viruses from this patient by Kitrinos and colleagues 26 indicated that some Envs used only CCR5 to mediate infection, whereas others could also use CXCR4 to a limited extent, with relative light units close to the threshold of the assay employed. In this study, we examined the tropism of the cloned envs by using pseudotyped viruses on cell lines expressing CD4 and either CCR5 or CXCR4.…”

“…This patient was found to have R5 virus strains that were incompletely suppressed by APL both at baseline and after virologic failure. 26 To investigate the mechanism of APL resistance in P5, we obtained HIV env clones directly from plasma from baseline and week 12 time points by isolating RNA, synthesizing cDNA, and cloning full-length env genes from 10 independent PCR reactions. Input cDNA concentrations were minimized by titration to reduce chances of recombination during PCR.…”