The CCR5 co-receptor is known to play an important role in HIV-1 transmission . In population studies, individuals with alterations in CCR5 amino acid composition (i.e. those with a 32-nucleotide deletion known as Δ32) lack functional CCR5 receptor expression and have been demonstrated to have significant decreased susceptibility to HIV infection [2–5]. In addition, viruses that use CCR5 as the primary co-receptor are the dominant HIV variants responsible for human to human mucosal transmission [6–8]. These observations have led to studies evaluating the potential use of CCR5 receptor antagonists, formulated in microbicides, as a strategy to prevent HIV transmission [9,10]. Although encouraging, these studies have demonstrated that the concentrations of CCR5 antagonists required to block viral transmission in vivo are several orders of magnitude greater than concentrations required in vitro. Pilot studies in humans have also demonstrated the ability of CCR5 antagonists to reduce viral loads in chronically infected individuals . Toxicity concerns have, however, ended the clinical development of some CCR5 antagonists and delayed the development of others [12–16].
We recently reported that G1 cytostatic drugs reduce CCR5 co-receptor expression in both T cells and macrophages, and inhibit the replication of CCR5-using strains of HIV-1 in in-vitro experiments . Moreover, the downregulation of CCR5 expression by G1 cytostatic agents enhanced the antiviral activity of a prototype CCR5 antagonist . We now report on the results obtained from a proof-of-concept study assessing the in-vivo effects of the G1 cytostatic agent, rapamycin (a bacterial macrolide approved for the treatment of renal transplantation rejection in human) on CCR5 expression in cynomolgous macaques.
Three healthy, female cynomologus macaques (Macaca fascicularis) were treated with rapamycin oral solution (0.5 mg/kg per day) for 12 weeks (Fig. 1a). The dose of rapamycin was chosen based on data from in-vitro studies  and in-vivo studies  to attain a potential therapeutic target level of 1–10 ng/ml. The drug was diluted in orange juice and placed in the cages for the animals to drink. Rapamycin blood levels (determined by high-pressure liquid chromatography), lymphocyte subset counts (assessed by fluorescence-activated cell sorter) and blood peripheral blood mononuclear cell (PBMC) CCR5 RNA levels (determined by quantitative TaqMan RNA polymerase chain reaction, as previously described ) were evaluated in blood samples collected at baseline and weeks 1, 4, 8 and 12 of rapamycin treatment. In two animals, CCR5 RNA levels were also evaluated in RNA prepared from punch biopsies of cervicovaginal tissue at baseline and weeks 8 and 12 of rapamycin treatment (Fig. 1b).
The regimen was well tolerated by the macaques with no changes in weight noted during the study. Total white blood count and lymphocyte subset counts did not change significantly throughout the study. The median trough rapamycin blood levels for each of the macaques were 2.5, 3.3, and 3.8 ng/ml, respectively (range 1.0–5.3 ng/ml) throughout the period of rapamycin administration and no changes in dose were required. CCR5 expression as determined by flow cytometry did not change significantly with the administration of rapamycin in any of the macaques. Recent work has, however, demonstrated that flow cytometry may be insensitive to describe changes in CCR5 expression . Therefore, we measured CCR5 expression by quantifying CCR5 messenger RNA. Rapamycin treatment reduced CCR5 RNA levels in blood PBMC in all three animals (fold reductions of 3, 8 and 33-fold at week 12 compared with baseline) (Fig. 1c). In two of the animals in which PBMC samples were taken 4 weeks after rapamycin discontinuation, CCR5 RNA levels rebounded albeit to levels below baseline. CCR5 RNA levels in vaginal punch biopsies were evaluated in two of the animals (Fig. 1c). In both animals, rapamycin treatment resulted in a reduction of CCR5 RNA levels in the vaginal biopsies compared with baseline (levels were reduced 0.6 and sevenfold at week 12). After standardization of the assay, the third animal did not have adequate cervicovaginal tissue for analysis.
Previous studies have suggested that a threshold density level of CCR5 expression is required for the efficient infection of cell lines . Relatively small changes (three to sevenfold) in CCR5 density on primary cells in vitro, which can be induced by rapamycin, can have a large impact on the replication levels of R5 HIV-1 strains . Rapamycin has also been demonstrated to potentiate the antiviral activity of a prototype CCR5 antagonist in vitro. The results of this proof-of-concept study extend these data by demonstrating a similar response in vivo, which suggests that G1 cytostatic agents could have the potential to allow lower doses of CCR5 antagonists, perhaps decreasing toxicity, to be used in the prevention or treatment of HIV. Rapamycin-induced decreases in CCR5 expression could have implications for altering the susceptibility to HIV infection of individuals at risk of HIV infection. In addition, rapamycin could also play a potential role in HIV therapeutics, especially in early infection. These results confirm the ability of rapamycin to reduce CCR5 co-receptor expression in PBMC and vaginal tissue biopsies in vivo. This supports further clinical evaluation of the use of rapamycin, and perhaps others G1 cytostatic agents, to target and downregulate CCR5 expression as a strategy for both the prevention and treatment of HIV infection, particularly in combination with new approaches targeting viral entry, including CCR5 antagonists and neutralizing antibodies.
The first two authors contributed equally to this work.
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