In this study, we isolated an R5 HIV-1 virus highly resistant to the novel CCR5 antagonist TAK-652 through long-term culture of infected PBMCs. The phenotypic analysis revealed that the virus was highly resistant to TAK-652 and had partial cross-resistance to TAK-779, probably due to their structural similarities (Fig. and Table ). In contrast to TAK-779, TAK-220, a structurally different CCR5 antagonist, was equally inhibitory to the replication of the TAK-652-resistant virus and the wild type. A similar finding has been reported for SCH-C and vicriviroc (SCH-D) (25
). Although both compounds are structurally related, the subtype G Russian clinical isolate RU570, which was weakly susceptible to inhibition by SCH-C (IC50
> 1 μM), retained high susceptibility to vicriviroc. Furthermore, four amino acid changes in the V3 region of gp120 were necessary and sufficient to confer resistance to SCH-C (10
), whereas vicriviroc-resistant viruses had no amino acid changes in the V3 region (1
). Thus, Env amino acid changes responsible for resistance to CCR5 antagonists differ from one compound to another. It would be of special interest to determine whether TAK-652 has sufficient antiviral activity against TAK-220-resistant R5 HIV-1.
We have also conducted a long-term culture experiment with R5 HIV-1-infected PBMCs under the selection pressure of TAK-220. However, no escape virus could be obtained with escalating concentrations of TAK-220, even after 132 passages (data not shown). At present, the reason for such difficulty in inducing TAK-220-resistant viruses is unclear. Nishikawa and colleagues recently analyzed the binding sites for TAK-220 on human CCR5 and found that TAK-220 shares some interacting amino acid residues with TAK-779 but also requires distinct amino acid residues for its inhibitory effect on HIV-1 (18
). It is possible that the conformation of CCR5 might be more extensively altered by binding of TAK-220 to CCR5 than by binding of TAK-652. Nevertheless, TAK-652 has unique properties with which TAK-220 and other CCR5 antagonists are not endowed. TAK-652 has good oral bioavailability and a long plasma half-life in humans (2
). Therefore, it is assumed that TAK-652 is able to retain a plasma concentration sufficiently higher than that required for virus inhibition by once-daily administration at a reasonable dose. TAK-652 is a potent inhibitor of ligand binding not only to CCR5 but also to CCR2b, which has been observed for neither TAK-220, maraviroc, vicriviroc, nor aplaviroc (8
Amino acid changes in the Env region accumulated with an increasing period of cultivation (Fig. ). Among the amino acid changes, several changes did not seem to be attributable to the selection pressure by TAK-652 but were the consequences of in vitro passage of infected cells, since these changes could be identified not only for the escape viruses but also for the corresponding control viruses. In addition, there were some amino acid changes that were found only for the control viruses. Selection of viruses with certain amino acid changes despite the absence of compounds has been commonly observed after in vitro passages of primary isolates and may be attributed to better replication fitness of these viruses. Apart from the amino acid changes unrelated to TAK-652 resistance, there were amino acid changes identified only for the escape viruses, and they accumulated with an increased period of cultivation (Table ). In particular, a considerable gap in drug susceptibility was found between KK652-43
(Table ). In addition to the amino acid changes observed for KK652-43
, the three changes T306K (V3), M424T (C4), and V766A (gp41) were identified for KK652-56
. Furthermore, three amino acid changes, K221N (C2), Q309E (V3), and I769S (gp41), occurred in the Env region of the highly resistant virus KK652-67
. It has been reported that, unlike the resistance to reverse transcriptase and protease inhibitors, one amino acid change of the Env region does not bring about the resistance to CCR5 antagonists (10
). Trkola and colleagues have proposed two possible mechanisms that confer resistance to CCR5 antagonists on HIV-1 (29
). The first one is the increase of gp120 binding affinity to CCR5. In this case, the virus can compete more strongly with a CCR5 antagonist and infect target cells. The second one is the creation of a substantially different binding site on gp120 for CCR5. In this case, the virus is still able to infect the target cells, even when the binding site of gp120 is already occupied with a CCR5 antagonist. Since the two mechanisms may not be mutually exclusive and can act sequentially, further studies, including the introduction of site-directed mutations, are required to elucidate the amino acids responsible for the resistance to TAK-652.
In accordance with previous experiments by others (29
), no HIV-1 coreceptor switch from CCR5 occurred in the escape viruses in this study (Table ). Instead, the control virus, KKC-67
, could use both CCR5 and CXCR4 for infection. It is known that only a few amino acid changes, especially in the V3 region of gp120, can convert R5 HIV-1 to X4 HIV-1 (7
). Indeed, seven amino acid changes, including two heterogeneous changes, were found in the V3 region of KKC-67
, suggesting that the virus might be a heterogeneous mixture of R5 HIV-1 and X4 HIV-1 rather than a dual-tropic (R5X4) virus. This assumption was confirmed by the drug-swapping experiment with TAK-652 and AMD3100 (see Results for details). Since the original strain, KKWT
, is a clinical isolate from a treatment-naïve patient, it is not surprising that a small population of X4 HIV-1 existed in the infected cells and expanded during their long-term culture.
In conclusion, this study provides important information on TAK-652-resistant viruses, such as no cross-resistance to TAK-220 and no coreceptor switch to X4 HIV-1. While our experiments using a clinical isolate and a single PBMC donor may reflect the in vivo scenario of drug resistance better than those using a laboratory strain and multiple donors, it is possible that different mutants will be selected in individual experiments. Furthermore, in a clinical setting, CCR5 antagonists must be used in combination with existing antiretrovirals (28
), which may alter the pattern for TAK-652 resistance. Thus, the emergence of drug resistance should be further investigated and confirmed in clinical trials.