Rapamycin reduces CCR5 mRNA levels in macaques: potential applications in HIV-1 prevention and treatment
Gilliam, Bruce L; Heredia, Alonso; DeVico, Anthony; Le, Nhut; Bamba, Douty; Bryant, Joseph L; Pauza, C David; Redfield, Robert R
Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA.
Received 6 July, 2007
Accepted 17 July, 2007
G1 cytostatic drugs reduce CCR5 co-receptor expression and enhance the antiviral activity of a CCR5 antagonist in vitro. The administration of rapamycin, a G1 cytostatic agent, to three cynomolgous macaques led to decreased CCR5 messenger RNA expression in peripheral blood mononuclear cells and cervicovaginal tissue. These results support further clinical evaluation of G1 cytostatic agents such as rapamycin targeting the downregulation of CCR5 expression as a strategy for both the prevention and treatment of HIV infection.
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.
1. Berger EA, Murphy PM, Farber JM. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol 1999; 17:657–700.
2. Dean M, Carrington M, Winkler C, Huttley GA, Smith MW, Allikmets R, et al
. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study. Science 1996; 273:1856–1862.
3. Samson M, Libert F, Doranz BJ, Rucker J, Liesnard C, Farber CM, et al
. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 1996; 382:722–725.
4. Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R, et al
. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 1996; 86:367–377.
5. Michael NL, Louie LG, Rohrbaugh AL, Schultz KA, Dayhoff DE, Wang CE, et al
. The role of CCR5 and CCR2 polymorphisms in HIV-1 transmission and disease progression. Nat Med 1997; 3:1160–1162.
6. Van't Wout AB, Blaak H, Ran LJ, Brouwer M, Kuiken C, Schuitemaker H. Evolution of syncytium-inducing and nonsyncytium-inducing biological virus clones in relation to replication kinetics during course of human immunodeficiency virus infection. J Virol 1998; 72:5099–5107.
7. Wolinsky SM, Wike CM, Korber BTM, Hutto C, Parks WP, Rosenblum LL, et al
. Selective transmission of human immunodeficiency virus type-1 variants from mothers to infants. Science 1992; 255:1134–1137.
8. Zhu T, Wang N, Carr A, Nam DS, Moor-Jankowski R, Cooper DA, et al
. Genetic characterization of human immunodeficiency virus type 1 in blood and genital secretions: evidence for viral compartmentalization and selection during sexual transmission. J Virol 1996; 70:3098–3107.
9. Lederman MM, Veazey RS, Offord R, Mosier DE, Dufour J, Mefford M, et al
. Prevention of vaginal SHIV transmission in rhesus macaques through inhibition of CCR5. Science 2004; 306:485–487.
10. Veazey RS, Klasse PJ, Ketas TJ, Reeves JD, Piatak M Jr, Kunstman K, et al
. Use of a small molecule CCR5 inhibitor in macaques to treat simian immunodeficiency virus infection or prevent simian-human immunodeficiency virus infection. J Exp Med 2003; 198:1551–1562.
11. Ray N, Doms RW. HIV-1 coreceptors and their inhibitors. Curr Top Microbiol Immunol 2006; 303:97–120.
12. Dorr P, Westby M, Dobbs S, Griffin P, Irvine B, Macartney M, et al
. Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum antihuman immunodeficiency virus type 1 activity. Antimicrob Agents Chemother 2005; 49:4721–4732.
13. Maeda K, Nakata H, Koh Y, Miyakawa T, Ogata H, Takaoka Y, et al
. Spirodiketopiperazine-based CCR5 inhibitor which preserves CC-chemokine/CCR5 interactions and exerts potent activity against R5 human immunodeficiency virus type 1 in vitro
. J Virol 2004; 78:8654–8662.
14. Strizki JM, Xu S, Wagner NE, Wojcik L, Liu J, Hou Y, et al
. SCH-C (SCH 351125), an orally bioavailable, small molecule antagonist of the chemokine receptor CCR5, is a potent inhibitor of HIV-1 infection in vitro
and in vivo
. Proc Natl Acad Sci U S A 2001; 98:12718–12723.
15. Takashima K, Miyake H, Furuta RA, Fujisawa JI, Iizawa Y, Kanzaki N, et al
. Inhibitory effects of small-molecule CCR5 antagonists on human immunodeficiency virus type 1 envelope-mediated membrane fusion and viral replication. Antimicrob Agents Chemother 2001; 45:3538–3543.
16. Crabb C. GlaxoSmithKline ends aplaviroc trials. AIDS 2006; 20:641.
17. Heredia A, Davis C, Amoroso A, Dominique JK, Le N, Klingebiel E, et al
. Induction of G1 cycle arrest in T lymphocytes results in increased extracellular levels of beta-chemokines: a strategy to inhibit R5 HIV-1. Proc Natl Acad Sci U S A 2003; 100:4179–4184.
18. Heredia A, Amoroso A, Davis C, Le N, Reardon E, Dominique JK, et al
. Rapamycin causes down-regulation of CCR5 and accumulation of anti-HIV beta-chemokines: an approach to suppress R5 strains of HIV-1. Proc Natl Acad Sci U S A 2003; 100:10411–10416.
19. Dambrin C, Klupp J, Birsan T, Luna J, Suzuki T, Lam T, et al
. Sirolimus (rapamycin) monotherapy prevents graft vascular disease in nonhuman primate recipients of orthotopic aortic allografts. Circulation 2003; 107:2369–2374.
20. Mattapallil JJ, Douek DC, Hill B, Nishimura Y, Martin M, Roederer M. Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection. Nature 2005; 434:1093–1097.
21. Platt EJ, Wehrly K, Kuhmann SE, Chesebro B, Kabat D. Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1. J Virol 1998; 72:2855–2864.
22. Heredia A, Gilliam B, DeVico A, Le N, Bamba D, Flinko R, et al
. CCR5 density levels on primary CD4 T cells impact the replication and enfuvirtide susceptibility of R5 HIV-1. AIDS 2007; 21:1317–1322.
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