Heredia, Alonso; Gilliam, Bruce; DeVico, Anthony; Le, Nhut; Bamba, Douty; Flinko, Robin; Lewis, George; Gallo, Robert C; Redfield, Robert R
HIV-1 entry into cells requires a receptor (CD4) and a coreceptor (mainly CCR5 or CXCR4) on the cell surface [1–4]. CCR5 serves as the main coreceptor for transmitting strains of HIV-1, and is the coreceptor predominantly used during the early stages of infection . Individuals who are homozygous for a Δ32 mutation in the CCR5 gene lack coreceptor expression and are generally resistant to infection . CCR5 density levels vary widely among normal individuals, with values ranging from ∼2 × 103 to 1 × 104 molecules/CD4 T cell [7–10]. Density of the CCR5 coreceptor is likely to be relevant in vivo because it has been correlated with RNA viral load  and progression to AIDS  in untreated, HIV-1 infected individuals.
In vitro studies by Platt et al. have demonstrated that a coreceptor density threshold of ∼2 × 103 CCR5 molecules/cell is required for efficient replication of R5 HIV-1 on cell lines expressing CD4 levels similar to those on primary T cells . In addition to their role in viral replication, studies in cell lines have demonstrated that CCR5 density levels also influence the antiviral activity of the fusion inhibitor Enfuvirtide (T-20) against R5 HIV-1 [13–15]. In the present study we have evaluated the effect of CCR5 coreceptor density on the replication and T-20 susceptibility of R5 HIV-1 in primary CD4 T cells.
Peripheral blood mononuclear cells (PBMC) were separated by ficoll-hypaque density gradient centrifugation. CD8 T cells were depleted using immunomagnetic beads (Dynal Biotech, Oslo, Norway). Cells were then cultured at 106 cells/ml in culture medium [RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics] containing recombinant human interleukin 2 (100 U/ml) (Roche, Indianapolis, Indiana, USA) for 6 days prior to infectivity assays and FACS analysis. These culture conditions were used to restore altered expression of CCR5 levels due to Ficoll treatment [9,16], and to provide stimulation conditions required for cellular infection .
The HI-JC HeLa clones HI-JC 10 and HI-JC57, which express similar densities of CD4 and CXCR4 but different CCR5 densities , were cultured in DMEM medium supplemented with 10% FBS and antibiotics.
The following viruses were used in infection experiments: HIV-1 IIIb, HIV-1 ADA, HIV-1 BaL, HIV-1 JRCSF, and HIV-1 CC1/85. HIV-1 IIIb is a T-cell line adapted lab strain that uses CXCR4 for entry into cells, while the rest are CCR5 using isolates.
Infections were carried out by exposing cells to virus in the presence or absence of different concentrations of T-20 for 2 h. Infected cells were washed and cultured in medium supplemented with the same drug concentration as during the infection step. Donor cells were infected using a virus multiplicity of infection (m.o.i) of 0.001, whereas the HI-JC clones were infected using an m.o.i of 0.01. Virus growth was monitored in culture supernatants by measuring p24 antigen levels by ELISA (NCI, Frederick, Maryland, USA) on day 4 after infection.
T-20 and viruses were obtained from the NIH AIDS Research and Reference Reagent Program (Germantown, Maryland, USA), except for HIV-1 isolate CC1/85 (obtained from J. Moore, Cornell University).
Quantitative FACS analysis
Quantification of the CD4 and CCR5 receptors was done using Quantibrite-PE bead standards (BD Biosciences) as previously described . The following antibody clones were used: CD4 (clone RPA-T4), CD3 (clone UCHT1) and CCR5 (clone 45531). All antibodies were from BD Biosciences (Mountainview, California, USA) except for the CCR5 antibody, which was from R&D Systems (Minneapolis, Minnesota, USA).
Data were analyzed using SPSS 11.5 software. Associations between coreceptor density levels, virus replication levels and T-20 sensitivity (50% inhibitory concentration; IC50) were tested using the non-parametric Spearman's rank correlation test.
CCR5 density levels on donor CD4 T cells are correlated with R5 HIV-1 replication
FACS analysis and infectivity assays using the HIV-1 ADA (R5) and HIV-1 IIIb (X4) strains were carried out in CD8-depleted PBMC that had been stimulated with interleukin-2 (IL-2) for 6 days. Quantitative FACS analysis yielded a mean density value for CD4 of 19 836 molecules/cell (range, 15 336–24 782 molecules/cell), and a mean density value for CCR5 of 3951molecules/cell (range, 1979–7286 molecules/cell). These values are within the range of those reported in previous studies [7–10]. Virus replication levels (determined on day 4 after infection) were plotted against the receptor and coreceptor density levels obtained on the day of infection (Fig. 1). Replication levels of the R5 strain HIV-1 ADA varied ∼12-fold among different donors' cells, whereas replication levels of the X4 strain HIV-1 IIIb varied less than twofold. We observed a positive correlation between CCR5 density levels and R5 HIV-1 (ADA strain) replication (Spearman correlation, r, 0.55; P = 0.011), but not between CCR5 density and X4 replication (Fig. 1a,c). In addition, CD4 density levels did not correlate with either R5 or X4 replication (Fig. 1b,d). Similar results to those obtained with HIV-1 ADA were obtained with the other R5 HIV-1 strains BaL, JRCSF and CC1/85 (data not shown). Thus, these results indicate that replication of R5 HIV-1 in primary CD4 T cells is positively correlated with CCR5 density levels.
Physiological relevant levels of CCR5 density influence the antiviral activity of T-20 against R5 HIV-1 in cell lines
Previous studies in CCR5 transfected cell lines have demonstrated that the antiviral activity of T-20 against R5 HIV-1 is greater in cells with ‘low’ CCR5 density levels than in cells with ‘high’ density levels , but quantification of the CCR5 density level was not determined. In order to determine the role of physiologically relevant CCR5 density levels on T-20 activity, we carried out infectivity assays using the HeLa HI-JC clones 10 and 57, which express CCR5 density levels of ∼2 × 103 and ∼9 × 103 CCR5 molecules/cell, respectively, in the presence of ∼105 CD4 molecules/cell . Thus, these clones represent the approximate lower and upper limits of CCR5 expression found on primary CD4 T cells [7–10]. Cells were infected with R5 and X4 HIV-1 in the presence of different concentrations of T-20, and virus replication was determined on day 4 after infection (Fig. 2a,b). When the cell lines were infected with R5 HIV-1 (ADA strain), the T-20 IC50 value was sevenfold lower in the clone with low CCR5 density levels. In contrast, upon X4 (HIV-1 IIIb) infection, T-20 IC50 values were similar in the clones with low and high CCR5 density. A similar pattern of infection on the HI-JC clones was observed using the R5 HIV-1 strains BaL and JRCSF (data not shown). These results in cell lines suggest that variations on CCR5 density within the range observed in donor T cells influence the activity of T-20 against R5 HIV-1.
CCR5 density levels on donor CD4 T cells impact the antiviral activity of T-20 against R5 HIV-1
We next performed infectivity studies using CD8-depleted PBMC from donors with CCR5 density levels similar to those in the HI-JC clones (Fig. 2c,d). In donor cells with low CCR5 density (∼2 × 103 molecules/cell) and high CCR5 density (∼6 × 103 molecules/cell), the T-20 IC50 values for R5 (HIV-1 ADA) infection were 0.4 and 13 nM, respectively (Fig. 2c). These T-20 IC50 values in donor cells are within the range observed in PBMC from different donors . Similar results were obtained with the R5 HIV-1 strains BaL, JRCSF and CC1/85 (data not shown). In contrast, infection with X4 (HIV-1 IIIb) yielded similar T-20 IC50 values (IC50 values of 7 and 11 nM in donors with low and high CCR5 densities, respectively) (Fig. 2d). We next expanded these observations by determining T-20 IC50 values upon R5 (HIV-1 ADA) infection of CD8-depleted PBMC from multiple donors, and found a positive correlation (Spearman correlation, r, 0.84; P = 0.00004) between T-20 IC50 values and CCR5 density (Fig. 2e). Similar positive correlations were found with the R5 strains BaL, JRCSF and CC1/85 (data not shown). These results demonstrate that CCR5 density level variations among donors' CD4 T cells influence the antiviral activity of T-20 against R5 HIV-1. Moreover, they demonstrate a positive correlation between CCR5 density levels and decreased susceptibility to T-20.
In the present study we have investigated the impact of donor CCR5 density on the replication levels and T-20 susceptibility of R5 HIV-1. These studies required purification of PBMC from whole blood and culture conditions that enable virus infection. We thus purified PBMC by Ficoll separation, depleted CD8 T cells, and cultured the cells in medium containing IL-2 for 6 days prior to further analysis. Cells were cultured for 6 days prior to FACS and infectivity assays to restore altered levels of CCR5 expression due to Ficoll treatment  and to provide stimulation conditions required for HIV-1 infection . These short-term IL-2 culture conditions do not upregulate CCR5 expression from baseline levels .
In infectivity assays, we found that CCR5 density levels on CD4 T cells are positively correlated with replication of R5 HIV-1. These results in vitro lend support to studies in vivo showing that CCR5 density levels on blood CD4 T cells are correlated with viral load and progression to AIDS in untreated, HIV-1 infected individuals [10,11].
We have also evaluated the effect of donor CCR5 density on the antiviral activity of T-20 against R5 HIV-1. It is thought that T-20 can target the viral envelope only during a kinetic window that opens by CD4 binding and closes soon after coreceptor engagement [13,21–24]. Studies in cell lines have demonstrated that both the affinity of the HIV-1 Env for coreceptor as well as coreceptor level expression influence the kinetics of fusion and therefore susceptibility of R5 HIV-1 to T-20 [13–15]. However, the relevance of these findings in primary CD4 T cells is unclear as cell lines generally express higher and more variable levels of CCR5 than primary cells . In the present study we demonstrate using both cell lines and primary cells that normally occurring CCR5 density levels on donors impact the antiviral activity of T-20 against R5 HIV-1 strains. Furthermore, we report a positive correlation between CCR5 density levels on CD4 T cells and decreased sensitivity of R5 HIV-1 strains to T-20. Together, these results suggest that information regarding CCR5 density in patients may prove helpful in assessing the antiviral activity of T-20 against R5 strains of HIV-1, which are generally present throughout the course of the disease and are less sensitive to T-20 than X4 strains [5,24,26–29]. In addition, these results help explain previous reports of increased antiviral potency of T-20 in the presence of a CCR5 antagonist , and suggest that the CCR5 entry inhibitors currently in clinical development may have an additional antiviral benefit by potentiating T-20 activity against R5 strains of HIV-1.
We thank Dave Kabat (Oregon Health and Science University, Portland, Oregon) for providing the HI-JC HeLa clones, and Paul Coppola for outstanding technical assistance.
1. Alkhatib G, Combadiere C, Broder CC, Feng Y, Kennedy PE, Murphy PM, et al. CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 1996; 272:1955–1958.
2. Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, et al. Identification of a major co-receptor for primary isolates of HIV-1. Nature 1996; 381:661–666.
3. Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima KA, et al. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 1996; 381:667–673.
4. Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 1996; 272:872–877.
5. Michael NL, Chang G, Louie LG, Mascola JR, Dondero D, Birx DL, et al. The role of viral phenotype and CCR-5 gene defects in HIV-1 transmission and disease progression. Nat Med 1997; 3:338–340.
6. 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.
7. Hladik F, Lentz G, Delpit E, McElroy A, McElrath MJ. Coexpression of CCR5 and IL-2 in human genital but not blood T cells: implications for the ontogeny of the CCR5+ Th1 phenotype. J Immunol 1999; 163:2306–2313.
8. Hladik F, Liu H, Speelmon E, Livingston-Rosanoff D, Wilson S, Sakchalathorn P, et al. Combined effect of CCR5-Delta32 heterozygosity and the CCR5 promoter polymorphism -2459 A/G on CCR5 expression and resistance to human immunodeficiency virus type 1 transmission. J Virol 2005; 79:11677–11684.
9. Lee B, Sharron M, Montaner LJ, Weissman D, Doms RW. Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages. Proc Natl Acad Sci USA 1999; 96:5215–5220.
10. Reynes J, Portales P, Segondy M, Baillat V, Andre P, Reant B, et al. CD4+ T cell surface CCR5 density as a determining factor of virus load in persons infected with human immunodeficiency virus type 1. J Infect Dis 2000; 181:927–932.
11. Reynes J, Portales P, Segondy M, Baillat V, Andre P, Avinens O, et al. CD4 T cell surface CCR5 density as a host factor in HIV-1 disease progression. AIDS 2001; 15:1627–1634.
12. 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.
13. Reeves JD, Gallo SA, Ahmad N, Miamidian JL, Harvey PE, Sharron M, et al. Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics. Proc Natl Acad Sci USA 2002; 99:16249–16254.
14. Reeves JD, Miamidian JL, Biscone MJ, Lee FH, Ahmad N, Pierson TC, et al. Impact of mutations in the coreceptor binding site on human immunodeficiency virus type 1 fusion, infection, and entry inhibitor sensitivity. J Virol 2004; 78:5476–5485.
15. Platt EJ, Durnin JP, Kabat D. Kinetic factors control efficiencies of cell entry, efficacies of entry inhibitors, and mechanisms of adaptation of human immunodeficiency virus. J Virol 2005; 79:4347–4356.
16. Lee B, Doranz BJ, Rana S, Yi Y, Mellado M, Frade JM, et al. Influence of the CCR2-V64I polymorphism on human immunodeficiency virus type 1 coreceptor activity and on chemokine receptor function of CCR2b, CCR3, CCR5, and CXCR4. J Virol 1998; 72:7450–7458.
17. Kinter AL, Poli G, Fox L, Hardy E, Fauci AS. HIV replication in IL-2-stimulated peripheral blood mononuclear cells is driven in an autocrine/paracrine manner by endogenous cytokines. J Immunol 1995; 154:2448–2459.
18. Salkowitz JR, Bruse SE, Meyerson H, Valdez H, Mosier DE, Harding CV, et al. CCR5 promoter polymorphism determines macrophage CCR5 density and magnitude of HIV-1 propagation in vitro. Clin Immunol 2003; 108:234–240.
19. Ketas TJ, Klasse PJ, Spenlehauer C, Nesin M, Frank I, Pope M, et al. Entry inhibitors SCH-C, RANTES, and T-20 block HIV type 1 replication in multiple cell types. AIDS Res Hum Retroviruses 2003; 19:177–186.
20. Bleul CC, Wu L, Hoxie JA, Springer TA, Mackay CR. The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc Natl Acad Sci USA 1997; 94:1925–1930.
21. Furuta RA, Wild CT, Weng Y, Weiss CD. Capture of an early fusion-active conformation of HIV-1 gp41. Nat Struct Biol 1998; 5:276–279.
22. Kliger Y, Shai Y. Inhibition of HIV-1 entry before gp41 folds into its fusion-active conformation. J Mol Biol 2000; 295:163–168.
23. Melikyan GB, Markosyan RM, Hemmati H, Delmedico MK, Lambert DM, Cohen FS. Evidence that the transition of HIV-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion. J Cell Biol 2000; 151:413–423.
24. Derdeyn CA, Decker JM, Sfakianos JN, Zhang Z, O'Brien WA, Ratner L, et al. Sensitivity of human immunodeficiency virus type 1 to fusion inhibitors targeted to the gp41 first heptad repeat involves distinct regions of gp41 and is consistently modulated by gp120 interactions with the coreceptor. J Virol 2001; 75:8605–8614.
25. Choudhry V, Zhang MY, Harris I, Sidorov IA, Vu B, Dimitrov AS, et al. Increased efficacy of HIV-1 neutralization by antibodies at low CCR5 surface concentration. Biochem Biophys Res Commun 2006; 348:1107–1115.
26. Doranz BJ, Baik SS, Doms RW. Use of a gp120 binding assay to dissect the requirements and kinetics of human immunodeficiency virus fusion events. J Virol 1999; 73:10346–10358.
27. Doranz BJ, Orsini MJ, Turner JD, Hoffman TL, Berson JF, Hoxie JA, et al. Identification of CXCR4 domains that support coreceptor and chemokine receptor functions. J Virol 1999; 73:2752–2761.
28. Hoffman TL, Canziani G, Jia L, Rucker J, Doms RW. A biosensor assay for studying ligand-membrane receptor interactions: binding of antibodies and HIV-1 Env to chemokine receptors. Proc Natl Acad Sci USA 2000; 97:11215–11220.
29. Yuan W, Craig S, Si Z, Farzan M, Sodroski J. CD4-induced T-20 binding to human immunodeficiency virus type 1 gp120 blocks interaction with the CXCR4 coreceptor. J Virol 2004; 78:5448–5457.
30. Tremblay CL, Giguel F, Kollmann C, Guan Y, Chou TC, Baroudy BM, et al. Anti-human immunodeficiency virus interactions of SCH-C (SCH 351125), a CCR5 antagonist, with other antiretroviral agents in vitro. Antimicrob Agents Chemother 2002; 46:1336–1339.
© 2007 Lippincott Williams & Wilkins, Inc.