Potent Inhibition of HIV-1 Infectivity in Macrophages and Lymphocytes by a Novel CCR5 Antagonist

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Science  11 Apr 1997:
Vol. 276, Issue 5310, pp. 276-279
DOI: 10.1126/science.276.5310.276


The chemokine receptors CXCR4 and CCR5 have recently been shown to act as coreceptors, in concert with CD4, for human immunodeficiency virus–type 1 (HIV-1) infection. RANTES and other chemokines that interact with CCR5 and block infection of peripheral blood mononuclear cell cultures inhibit infection of primary macrophages inefficiently at best. If used to treat HIV-1–infected individuals, these chemokines could fail to influence HIV replication in nonlymphocyte compartments while promoting unwanted inflammatory side effects. A derivative of RANTES that was created by chemical modification of the amino terminus, aminooxypentane (AOP)–RANTES, did not induce chemotaxis and was a subnanomolar antagonist of CCR5 function in monocytes. It potently inhibited infection of diverse cell types (including macrophages and lymphocytes) by nonsyncytium-inducing, macrophage-tropic HIV-1 strains. Thus, activation of cells by chemokines is not a prerequisite for the inhibition of viral uptake and replication. Chemokine receptor antagonists like AOP-RANTES that achieve full receptor occupancy at nanomolar concentrations are strong candidates for the therapy of HIV-1–infected individuals.

RANTES, MIP-1α, and MIP-1β are β-chemokines that inhibit HIV-1 infection in vitro (1-4) by interacting with the HIV-1 coreceptor CCR5 (3, 4). Such coreceptor ligands are potentially useful in the treatment of HIV-infected individuals. Unfortunately, although these reagents consistently inhibit HIV-1 replication in peripheral blood mononuclear cells (PBMCs) (1, 3, 5, 6), they do not block infection of primary macrophage cultures (3, 6, 7), which indicates that their use could fail to influence HIV replication in nonlymphocyte cell types. Moreover, the chemotactic and leukocyte-activating properties of these β-chemokines may result in undesirable inflammatory responses. Recently, Arenzana-Seisdedos et al. (5) described a chemokine antagonist that inhibited HIV-1 infection of phytohemagglutinin (PHA)-activated PBMCs but lacked chemotactic and leukocyte-activating properties. Here, we tested two other RANTES receptor antagonists for their ability to inhibit HIV-1 infection of different cell types, including primary human macrophage cultures expressing CCR5. Met- RANTES has an additional methionine at the NH2-terminus and is a potent antagonist of RANTES signaling in a variety of cell types (8). Because the side chain of methionine is nearly isosteric with the n-pentyl group, we decided to prepare another RANTES derivative (which we called AOP-RANTES) by first generating an aldehyde-like group at the NH2-terminus of RANTES and then reacting with aminooxypentane (9).

AOP-RANTES could not induce chemotaxis of primary human monocytes (10) and inhibited monocyte chemotaxis induced by MIP-1β (Fig. 1, A and B). Because MIP-1β only binds CCR5, this result implies that AOP-RANTES acts as a functional antagonist on primary, CCR5-expressing monocytes. In Fig. 1C, which shows competition of 125I-labeled MIP-1α binding to CCR5-expressing HEK293 cells by RANTES, Met-RANTES, and AOP-RANTES (11), RANTES and Met-RANTES both displayed clear two-component displacement curves with a high-affinity site of 0.022 nM and a low-affinity site of 18 nM (for RANTES). Thus, not even at 100 nM could RANTES or Met-RANTES displace all of the MIP-1α bound to the CCR5 receptor. In contrast, AOP-RANTES totally displaced MIP-1α binding at 1 nM, with a median inhibitory concentration (IC50) of 0.072 nM. In addition, its high-affinity competition curve was classical antagonist-like, with a Hill coefficient of −1.02, which indicates binding to a single site.

Figure 1

Chemotactic and binding properties of RANTES, Met-RANTES, and AOP-RANTES. (A) Monocyte chemotaxis induced by RANTES (•) and AOP-RANTES (▴). AOP-RANTES elicited no significant migration of monocytes onto the lower surface of the membrane. (B) AOP-RANTES was incubated at various concentrations with 100 nM RANTES (•) and 10 mM MIP-1β (▴). Median effective concentration values for these agonists are usually 2 to 5 nM. The inhibition data shown are typical of that seen with five donors. (C) Competition of 125I-labeled MIP-1α binding to CCR5. Serial dilutions of RANTES (•), Met-RANTES (▪), and AOP-RANTES (▴) were tested for their capacity to compete with MIP-1α binding to CCR5-expressing HEK293 cells.

We tested RANTES, Met-RANTES, and AOP-RANTES for inhibition of HIV-1 replication in PBMCs stimulated with PHA and interleukin-2 (IL-2) (12). The HIV-1 strains first tested were SF-162, E80, and LAI. SF-162 and E80 are primary nonsyncytium-inducing (NSI), macrophage-tropic strains that use CCR5 as a coreceptor (13), whereas LAI is a prototype T cell line–adapted, syncytium-inducing (SI) virus that uses CXCR4 as a coreceptor (14). Relative to RANTES or Met-RANTES, AOP-RANTES was up to 10 times as potent in blocking infection of PBMCs by both SF-162 and E80 (Fig. 2). Complete inhibition of both strains was achieved with an AOP-RANTES concentration of <100 ng/ml, whereas inhibition with RANTES and Met-RANTES required >200 ng/ml and >400 ng/ml, respectively. None of the three ligands inhibited LAI.

Figure 2

Inhibition of HIV-1 infection of PBMCs with RANTES (•), Met-RANTES (▪), and AOP-RANTES (▴). RANTES, Met-RANTES, and AOP-RANTES inhibited the NSI viruses SF-162 (left) and E80 (center), which use CCR5, but not LAI (right), which uses CXCR4. LAI infectivity, but not SF-162 infectivity, was reduced 50% using stromal cell–derived factor 1 at 3.2 μg/ml.

We then compared the effect of RANTES, Met-RANTES, and AOP- RANTES in inhibiting a wider range of HIV-1 strains. Three additional primary NSI macrophage-tropic strains (M23, M53, and SL-2) were blocked by AOP-RANTES, with equivalent concentrations of RANTES and Met-RANTES showing consistent but weaker inhibition. None of these proteins blocked infection by HIV-1 strains that can use CXCR4, including the T cell line–adapted strains LAI and RF (13,14) as well as 2076 and 2044, which are primary viruses that infect macrophages as well as T cell lines (Table 1). Both 2076 and 2044 can use CXCR4, although 2076 can use CCR5 as well (13).

Table 1

RANTES and RANTES antagonists inhibit infection of PBMC cultures by NSI, but not SI, HIV-1 strains.

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Previous studies have reported that RANTES (or a mixture of RANTES, MIP-1α, and MIP-1β) does not block infection of primary macrophage cultures by macrophage-tropic HIV-1 strains (3, 6, 7). RANTES, AOP-RANTES, and Met- RANTES were tested for their effect on HIV-1 replication in macrophages. Macrophage cultures were prepared as described (13, 15, 16). RANTES and Met-RANTES (200 and 800 ng/ml) had little effect on macrophage replication by the primary NSI macrophage-tropic strains, SF-162, E80, or M23 (Fig.3A), whereas a fourth strain, SL-2, was blocked by RANTES but not by Met-RANTES. This result indicates that particular strains are likely to be more sensitive to chemokine inhibition than others. In contrast, AOP-RANTES efficiently inhibited macrophage replication of all four strains tested. Similar results were obtained with further macrophage preparations from separate donors (17).

Figure 3

Effect of AOP-RANTES on HIV-1 infection of primary macrophage cultures and CCR5-expressing CCC-CD4 and HeLa-CD4 cells. (A) AOP-RANTES, but not RANTES or Met-RANTES, inhibited SF-162, M23, and E80 infection of primary human macrophage cultures. SL-2 infection was blocked by both RANTES and AOP-RANTES. (B) AOP-RANTES, but not RANTES or Met-RANTES, inhibited SF-162 infection of CCR5+ CCC-CD4 and HeLa-CD4 cells. CCR5 is the only coreceptor present on CCC-CD4 and HeLa-CD4 for SF-162, which cannot infect the CCR5 parental counterparts. Arrows denote columns that do not register on vertical scale. The means of triplicate samples from one experiment are shown; similar results were obtained in additional experiments (17).

It is possible that alternative coreceptors on macrophages could allow HIV to bypass CCR5 and escape inhibition by RANTES and Met-RANTES on macrophages. We therefore tested the effect of RANTES, Met-RANTES, and AOP-RANTES on SF-162 infection of cat kidney CCC cells stably expressing human CD4 (18) and transiently expressing transfected CCR5 (13, 19), as well as on HeLa cells that stably expressed both CD4 and CCR5 (Fig. 3B) (20). CCC and HeLa cells that express CD4 (CCC-CD4 and HeLa-CD4 cells) do not usually express coreceptors for primary NSI viruses (13); therefore, when CCR5 is expressed on these cell types, it is the only coreceptor available for such strains. Replication of SF-162 in CCR5-expressing CCC-CD4 cells was efficiently blocked only with AOP-RANTES. Similar results were obtained with HeLa-CD4 cells stably expressing CCR5 (20); SF-162 infection was efficiently inhibited by AOP-RANTES, whereas RANTES and Met-RANTES reduced SF-162 infection by <50%. The inability of RANTES and Met-RANTES to efficiently block infection of CCR5+ HeLa-CD4 or CCC-CD4 cells therefore cannot be attributed to the presence of alternative SF-162 coreceptors that do not bind (and cannot be blocked by) RANTES or Met-RANTES.

We currently do not understand why RANTES inhibits HIV-1 NSI infection of PBMCs yet does not reliably block infection of primary macrophage cultures (3, 6, 7). Only one of four strains tested here (SL-2) was consistently inhibited by RANTES, although we have observed weak inhibition of other strains if lower amounts of input infectivity are used. We recently observed a similar phenomenon with a CXCR4-specific monoclonal antibody, 12G5 (21). This monoclonal antibody inhibited HIV infection and fusion of CXCR4+ RD/CD4 (a rhabdomyosarcoma cell line), yet failed to block infection of most CD4+ T cell lines and infection of at least one cell line expressing CXCR4 as the sole coreceptor (22). Together, these results indicate that other factors influence the presentation of coreceptors at the cell surface, and these affect the efficiency with which ligands can inhibit virus infection.

Amino-terminal derivatives of RANTES such as AOP-RANTES and Met- RANTES, which bind with high affinity to the CCR5 receptor yet fail to induce chemotaxis signaling, act as antagonists. Moreover, although Met-RANTES has preserved the two-component interaction mode with the CCR5 receptor (showing even a larger low-affinity component than RANTES itself), the AOP modification has succeeded in creating a classical antagonist with a monocomponent high-affinity binding mode. The total occupancy of CCR5 receptors achieved by the AOP-RANTES antagonist, even at nanomolar concentrations, is probably the reason why AOP-RANTES is a more potent inhibitor of HIV strains that use CCR5 than is RANTES. AOP-RANTES or similarly modified chemokines (or even nonpeptide antagonists) that achieve full receptor occupancy at low concentrations are therefore suitable candidates for treatment of HIV-infected individuals.


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