An anti-CCR5 monoclonal antibody and small molecule CCR5 antagonists synergize by inhibiting different stages of human immunodeficiency virus type 1 entry

Virology 352 (2006) 477 – 484 www.elsevier.com/locate/yviro

An anti-CCR5 monoclonal antibody and small molecule CCR5 antagonists synergize by inhibiting different stages of human immunodeficiency virus type 1 entry Diana Safarian a,1 , Xavier Carnec a,1 , Fotini Tsamis a , Francis Kajumo a,b , Tatjana Dragic a,⁎ a

Albert Einstein College of Medicine, Department of Microbiology and Immunology, 1300 Morris Park Avenue, Bronx, NY 10461, USA b Progenics Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, USA Received 30 March 2006; returned to author for revision 28 April 2006; accepted 11 May 2006 Available online 14 June 2006

Abstract HIV-1 coreceptors are attractive targets for novel antivirals. Here, inhibition of entry by two classes of CCR5 antagonists was investigated. We confirmed previous findings that HIV-1 isolates vary greatly in their sensitivity to small molecule inhibitors of CCR5-mediated entry, SCH-C and TAK-779. In contrast, an anti-CCR5 monoclonal antibody (PA14) similarly inhibited entry of diverse viral isolates. Sensitivity to small molecules was V3 loop-dependent and inversely proportional to the level of gp120 binding to CCR5. Moreover, combinations of the MAb and small molecules were highly synergistic in blocking HIV-1 entry, suggesting different mechanisms of action. This was confirmed by time course of inhibition experiments wherein the PA14 MAb and small molecules were shown to inhibit temporally distinct stages of CCR5 usage. We propose that small molecules inhibit V3 binding to the second extracellular loop of CCR5, whereas PA14 preferentially inhibits subsequent events such as CCR5 recruitment into the fusion complex or conformational changes in the gp120-CCR5 complex that trigger fusion. Importantly, our findings suggest that combinations of CCR5 inhibitors with different mechanisms of action will be central to controlling HIV-1 infection and slowing the emergence of resistant strains. © 2006 Elsevier Inc. All rights reserved. Keywords: HIV-1; CCR5; Coreceptor; Entry; Inhibitors; Small molecules; Monoclonal antibodies; Synergy; V3 loop

Introduction The two physiologically relevant HIV-1 entry coreceptors are the 7 transmembrane spanning G-coupled chemokine receptors CCR5 and CXCR4. CCR5 is considered a prime target for novel antivirals because it does not appear to be essential for normal immune function (Carrington et al., 1997; Liu et al., 1996). Numerous small molecule CCR5 antagonists and anti-CCR5 monoclonal antibodies (MAbs) have been characterized, and some are in clinical development (reviewed in Seibert and Sakmar, 2004). It has been shown that from the outset, HIV-1 strains differ significantly in their sensitivity to small molecule antagonists such as SCH-C and TAK-779 ⁎ Corresponding author. Fax: +1 718 430 8711. E-mail address: [email protected] (T. Dragic). 1 The authors have contributed equally to this study. 0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2006.05.016

(Rusert et al., 2005; Strizki et al., 2001; Trkola et al., 2002). Moreover, resistant strains are generated rapidly in vitro by evolving to use inhibitor-occupied CCR5 (Kuhmann et al., 2004; Marozsan et al., 2005). Resistance to an anti-CCR5 MAb has also been described, but the mechanism remains unclear (Aarons et al., 2001). As with all HIV-1 antivirals, therefore, this new class of inhibitors may be limited by variable efficacy and the emergence of resistant strains. HIV-1 entry into target cells is mediated by a cascade of binding and conformational events (reviewed in Berger et al., 1999; Weiss, 2003). The virus attaches to the cell surface by binding the CD4 receptor, which induces a coreceptor binding site on gp120. There are at least two contact points between gp120 and the CCR5 coreceptor, which may be established in temporally distinct stages (reviewed in (Hartley et al., 2005)). The second extracellular loop (ECL2) of CCR5 probably interacts with the crown of the gp120 V3 loop whereas the

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CCR5 amino-terminal domain (Nt) binds the V3 stem and bridging sheet (Cormier and Dragic, 2002; Cormier et al., 2001; Hartley et al., 2005). Once the gp120-coreceptor complex attains its final conformation, including dissociation of the gp120/gp41 heterodimer (Abrahamyan et al., 2003; Binley et al., 2003), it triggers the gp41 membrane fusion machinery (reviewed in LaBranche et al., 2001). We have previously shown that small molecules SCH-C and TAK-779 bind to a pocket in the transmembrane domain of CCR5 and non-competitively block gp120 binding to the coreceptor (Dragic et al., 2000; Tsamis et al., 2003). We also determined that neither SCH-C nor TAK-779 inhibit the gp120 interaction with the Nt (Cormier et al., 2001). Both molecules, however, block binding of several monoclonal antibodies that recognize epitopes in the second extracellular loop of CCR5 (Tsamis et al., 2003). Based on these and other findings, we had proposed that the small molecules alter the conformation of ECL2 such that it is no longer recognized by the V3 crown. Reeves et al. showed that the V3 loop modulates sensitivity to TAK-779 (Reeves et al., 2002). Kuhmann et al. described the gp120 of a SCH-C-resistant isolate that sustained multiple substitutions in the V3 crown, leading to a reduced affinity for CCR5 and an increased affinity for the CCR5 inhibitor complex (Kuhmann et al., 2004). The data from these groups therefore support the model that small molecule compounds disrupt CCR5 interactions with the V3 loop by an allosteric mechanism.

Numerous anti-CCR5 monoclonal antibodies (MAbs) have been described (Lee et al., 1999; Olson et al., 1999; Wu et al., 1997). The mechanism of entry inhibition by anti-CCR5 MAbs, however, is not well understood. We had previously shown that MAbs that recognize epitopes in the CCR5 Nt efficiently block gp120 binding to the coreceptor but do not potently inhibit viral entry (Olson et al., 1999). In contrast, two MAbs with epitopes lying in ECL2 are poor inhibitors of gp120 binding but highly efficient inhibitors of viral entry (Olson et al., 1999). We had suggested that these MAbs inhibit important post-gp120 binding steps, such as conformational changes in CCR5 or its oligomerization (Kuhmann et al., 2000). Notably, resistance to one of these anti-CCR5 MAbs (2D7) does not appear to map to a particular region of gp120 and its mechanism remains unclear (Aarons et al., 2001). In this study, we explored inhibition of HIV-1 entry by two small molecules and an anti-CCR5 MAb. Envelope glycoproteins from a panel of primary isolates were used to generate reporter pseudoviruses and inhibition of entry was tested in HeLa-CD4-CCR5 cells. We thus showed that sensitivity to SCH-C and TAK-779 over four orders of magnitude in our experimental system. In contrast, sensitivity of the same isolates to inhibition by an anti-CCR5 MAb (PA14) varied by no more than six-fold. Sensitivity to the small molecules was dependent on gp120 affinity for CCR5, and this was regulated by the V3 loop crown. Combinations of PA14 and SCH-C or TAK-779 were synergistic in blocking HIV-1 entry. The PA14 MAb was

Fig. 1. Sensitivity of primary isolates to SCH-C and PA14. (A) Target cells (HeLa-CD4-CCR5) were infected with HIV-1 reporter viruses pseudotyped with envelope glycoproteins of thirteen different isolates in the presence of a range of inhibitor concentrations. Non-linear curve fitting was used to determine IC50 values for each combination of compound and isolate. Values are means of three independent experiments. Target cells (HeLa-CD4-CCR5) were infected with HIV-1 pseudotypes comprising envelope glycoproteins of JR-FL, 92NG083 or 93TH966 in the presence of different concentrations of (B) SCH-C, (C) TAK-779 or (D) PA14. Luciferase activity in cell lysates was determined 48 h post-infection and % HIV-1pp entry was calculated as (RLU in the presence of inhibitor)/ (RLU in the absence of inhibitor) × 100. The three isolates generated similar RLU values in the absence of inhibitor, ranging from 104 to 106 depending on the experiment.

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found to inhibit CCR5 usage at a stage following the one that is sensitive to inhibition by the small molecules, and preceding gp41-triggered membrane fusion. Results Small molecule SCH-C and anti-CCR5 MAb PA14 exhibit different patterns of HIV-1 entry inhibition We compared the abilities of small molecule antagonists SCH-C and TAK-779 as well as anti-CCR5 MAb PA14 to inhibit entry of HIV-1 reporter viruses pseudotyped with the envelope glycoproteins of thirteen primary R5 isolates (HIV1pp). The readout of our assay was luciferase activity (relative light units, RLU) in target cell lysates (HeLa-CD4-CCR5), which is directly proportional to HIV-1pp entry. Importantly, the percentage of inhibition by the anti-CCR5 compounds was independent of the starting levels of entry when these were between 104 and 106 RLU, meaning that we were working within the linear range of the assay. Note that the standard deviation of our entry assay is ±20%. Half-maximal inhibitory concentrations for SCH-C (IC50) ranged over four orders of magnitude, between 2.9 × 10−12 M and 2.1 × 10−8 M (Fig. 1A). The mean IC50 for our set of test isolates was 6.8 × 10−9 M. Similarly, TAK-779 IC50s ranged between 1.0 × 10−11 M and 1.1 × 10−7 M with a mean of 2.9 × 10−8 M. Partial overlap of SCH-C and TAK-779 binding sites on CCR5 probably explains this similarity. In contrast, the IC50 values for PA14 were constrained within a single order of magnitude and varied between 1.2 × 10−9 M and 6.9 × 10−9 M, with a mean of 4.0 × 10−9 M (Fig. 1A). The differences in IC50 variances between PA14 and SCH-C as well as PA14 and TAK-779 were highly significant (P < 0.0001 as determined by a F test). From the initial set of thirteen test isolates, three were chosen for further studies. Entry mediated by envelope glycoproteins of 92NG083 and 93TH966 was similarly sensitive to all antiCCR5 compounds whereas JR-FL-mediated entry was less sensitive to the small molecules (Figs. 1B–D). The IC50s for SCH-C were 4.5 × 10−9 M for JR-FL, 8.2 × 10−10 M for 92NG083 and 8.0 × 10−10 M for 93TH966 (Fig. 1B). Likewise, TAK-779 exhibited similar IC50s for 92NG083 and 93TH966, which were 2.4 × 10−9 M and 3.1 × 10−9 M respectively (Fig. 1C). JR-FL was also less sensitive to TAK-779 with an IC50 of 2.0 × 10−8 M. JR-FL was therefore approximately five times less sensitive to SCH-C and eight times less sensitive to TAK779 than 92NG083 and 93TH966. In contrast, PA14 IC50 values were 1.9 × 10−9 M for JR-FL, 1.3 × 10−9 M for 92NG083, and 1.0 × 10−9 M for 93TH966 (Fig. 1D). These values were not statistically different. The V3 loop crown determines sensitivity to SCH-C and TAK-779 Based on previous findings by us and others, we hypothesized that the gp120 V3 loop would be the major determinant of HIV-1 sensitivity to small molecule inhibitors. Using pair-wise

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alignment, we determined that JR-FL and 92NG083 V3 loops are 86% identical and 94% similar. Differences include a T → I at the first position of the amino-terminal arm of the stem, as well as H → P, R → Q, T → A and E → D in the crown (Fig. 2A). JR-FL and 92TH966 V3 loops are 67% identical and 80% similar and differ by ten residues, including a I → T, H → T, R → Q, A → V, T → R and E → D in the crown (Fig. 2A). We note that the majority of V3 loop differences map to the crowns. Envelope glycoprotein chimeras were generated to test the role of these differences in sensitivity to small molecule inhibitors. The V3 loop of JR-FL gp120 was substituted for the V3 loop of 92NG083 to generate the chimera JR-FL(92NG083). Likewise, the V3 loop of 92NG083 was substituted for that of JR-FL to generate 92NG083(JR-FL). We also generated 93TH966 gp120 bearing the V3 crown of the JR-FL isolate. Inhibition by SCH-C and TAK-779 of HIV-1pp entry mediated by full-length chimeric envelope glycoproteins was measured. SCH-C inhibited entry mediated by JR-FL(92NG083) with an

Fig. 2. Inhibition of entry mediated by chimeric envelope glycoproteins with small molecules. (A) Amino acid differences between the V3 loops of JR-FL, 92NG083 and 93TH966 are in bold. The V3 crown is outlined. Target cells (HeLa-CD4-CCR5) were infected with HIV-1 pseudotypes comprising chimeric envelope glycoproteins JR-FL(92NG083), 92NG083(JR-FL) or 93TH966(JRFL) in the presence of different concentrations of (B) SCH-C or (C) TAK-779. Luciferase activity in infected cells was determined 48 h post-infection and % HIV-1pp entry was calculated as (RLU in the presence of inhibitor)/(RLU in the absence of inhibitor) × 100. Like the wild-type envelope glycoproteins, the chimeras also generated similar RLU values in the absence of inhibitor, ranging from 104 to 106.

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IC50 of 8.1 × 10−10 M, whereas entry mediated by 92NG083 (JR-FL) and 93TH966(JR-FL) was inhibited similarly, with IC50s of 8.5 × 10−9 M and 9.2 × 10−9 M, respectively (Fig. 2B). Likewise, TAK-779 inhibited entry mediated by JR-FL (92NG083) more efficiently than entry mediated by 92NG083 (JR-FL) and 93TH966(JR-FL): IC50 values of 2.1 × 10−9 M, 4.8 × 10−8 M and 5.0 × 10−8 M, respectively (Fig. 2C). Switching V3 loops therefore switches sensitivity phenotypes to small molecule inhibitors because JR-FL bearing the V3 loop of 92NG083 is inhibited similarly to wild-type 92NG083. Likewise, 92NG083(JR-FL) and 93TH966(JR-FL) acquire the JR-FL sensitivity phenotype. Our observations confirm that differences in sensitivity to small molecules between these three isolates can be entirely attributed to differences in the V3 loop and especially its crown. The V3 crown modulates gp120 affinity for CCR5 We next determined how the V3 loop modulates sensitivity to small molecule inhibitors SCH-C and TAK-779. For this, JRFL, 92NG083, JR-FL(92NG083) and 92NG083(JR-FL) soluble gp120 proteins were generated by transient transfection of 293T cells and quantified in supernatants by Western blotting and ELISA (Fig. 3A and data not shown). Binding of the envelope

glycoproteins to CD4-IgG2 as measured by ELISA was comparable (data not shown). Binding of gp120/CD4-IgG2 complexes to CCR5-positive L1.2 cells was measured by flow cytometry, as previously described by us (Dragic et al., 1998). Background MFI levels, measured with mock-transfected supernatants and CD4-IgG2, ranged between 2 and 4. Similar values were obtained when parental L1.2 cells were used in the binding assay (data not shown). At similar concentrations of soluble envelope proteins (∼40 μg/ml), JR-FL gp120 binding generated a mean fluorescence intensity (MFI) of 620 (Fig. 3B). In contrast, gp120 of 92NG083 exhibited very weak binding with an MFI of only 20. The CCR5-binding phenotype of the chimeric gp120 proteins was completely inversed. In other words, 92NG083 (JR-FL) bound CCR5 to the same extent as JR-FL gp120 (MFI of 560) (Fig. 3B). In contrast JR-FL gp120 bearing the V3 loop of 92NG083 exhibited a profound loss of binding activity and now behaved like 92NG083 gp120 (MFI of 25) (Fig. 3B). Note that the MFI values obtained with 92NG083 and JR-FL (92NG083) gp120 were low, thereby limiting the dynamic range of the assay and making it difficult to generate a reliable binding curve. Nonetheless, our results clearly demonstrate a difference in CCR5 binding of the four gp120 proteins and indicate that the higher sensitivity of 92NG083 to SCH-C and

Fig. 3. Binding of wild-type and chimeric gp120 proteins to CCR5. (A) 293T cells were transiently transfected with constructs expressing different gp120 proteins, including JR-FL, 92NG083, JR-FL(92NG083) and 92NG083(JR-FL). Gp120 in supernatants were quantified by ELISA and adjusted to the same concentration, which was confirmed by Western blotting with the B12 MAb. (B) CCR5-expressing L1.2 cells were incubated with CD4-IgG2 (20 μg/ml) mixed with mock-transfected 293T supernatants (dotted line) or supernatants comprising different gp120 proteins (40 μg/ml) (solid line). Binding to CCR5 was detected by flow cytometry after labeling with a PE-labeled goat anti-human IgG2 antibody (1:100).

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TAK-779 is related to its lower binding to the coreceptor, which is regulated by the V3 loop crown. Small molecules and anti-CCR5 MAb PA14 inhibit distinct stages of CCR5 usage Synergy studies were performed by investigating inhibition of entry mediated by the envelope glycoproteins of the JR-FL isolate with combinations of SCH-C or TAK-779 and PA14

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(Figs. 4A, B). Data were analyzed by the median effect principle (Chou and Rideout, 1991). The concentrations of single agents or their mixtures required to produce a given effect were quantitatively compared in a term known as the Combination Index (CI). A CI value greater than 1 indicates antagonism, CI ∼1 indicates an additive effect, and CI <1 indicates a synergistic effect wherein the presence of one agent enhances the effect of another. We found that at 90% entry inhibition the CI index for SCH-C and PA14 was 0.17, whereas at 50% entry inhibition the CI index was 0.063 (Table 1). Similarly, the CI indices for TAK-779 and PA14 were 0.57 and 0.25 at IC90 and IC50 respectively (Table 1). Combinations of small molecules and PA14 therefore synergistically inhibit JRFL envelope-mediated entry. This suggests that the compounds act through different mechanisms. To determine whether the small molecule inhibitor SCH-C and PA14 inhibit different stages of HIV-1 entry, we performed time course of inhibition experiments with the two compounds as well as with T20, an inhibitor of gp41-mediated fusion. Briefly, HIV-1pp comprising the JR-FL envelope glycoprotein was spinoculated onto target cells at 4 °C for 1 h. Unbound particles were removed by washing and inhibitors at IC90 (3 × 10−9 M PA14, 3 × 10−8 M SCH-C and 3 × 10−8 M of T20) or medium were added at 37 °C in order to initiate synchronized viral entry and define a t = 0. Inhibitors were then added at different time points until 3 h post-t = 0. The time required for half maximal inhibition (t1/2) of JR-FL pseudotype entry by SCH-C was 9 min (Fig. 4C). For PA14, however, the t1/2 was 32 min, meaning that there is a 23-min lag between the stage inhibited by SCH-C and the stage inhibited by PA14. Finally, the t1/2 of inhibition of gp41-mediated membrane fusion by T20 occurred within 46 min, marking it as the end-point of HIV-1 entry. Similar results, with slightly slower kinetics, were obtained for the 92NG083 isolate (data not shown). Note that t1/2 values were calculated using one-phase exponential association curve fitting. Discussion

Fig. 4. Time course of inhibition of HIV-1 entry with SCH-C, PA14 and T20. (A, B) Target cells (HeLa-CD4-CCR5) were infected with HIV-1 pseudotypes comprising envelope glycoproteins of JR-FL in the presence of different concentrations of SCHC, TAK-779, PA14 or equimolar combinations of small molecules and PA14. Luciferase activity in cell lysates was determined 48 h post-infection and %HIV-1pp entry was calculated as (RLU in the presence of inhibitor)/(RLU in the absence of inhibitor) × 100. (C) JR-FL pseudoparticles were pre-bound to target cells (HeLaCD4-CCR5) at 4 °C by spinoculation. After washing, cells were warmed to 37 °C and inhibitors added at different time points. Luciferase activity in infected cells was determined 48 h post-infection and %JR-FLpp entry was calculated (RLU with inhibitor at n′)/(RLU without inhibitor) × 100. Values are means of three independent experiments ±SD.

In this study, we characterized the mechanism of inhibition of HIV-1 entry by small molecule CCR5 antagonists and an anti-CCR5 MAb. Inhibition of entry of a panel of HIV-1 isolates ranged over four orders of magnitude for SCH-C and TAK-779. In contrast, PA14 IC50 values varied by no more than six-fold for the same set of isolates. A comparison of PA14 and SCH-C IC50 values in a scatter plot showed a weak correlation between the two sets of values (P = 0.0315 using Pearson's r test). In other words, isolates that were more sensitive to SCH-C also tended to be more sensitive to PA14. This correlation, however, was completely lost (P = 0.6593) when the three isolates least sensitive to SCH-C (IC50 values ≥10−8 M) were removed from calculations of the linear regression curve. In contrast, a very strong correlation was found between sensitivity to SCH-C and sensitivity to TAK-779 (P < 0.0001). Together, these analyses gave the first indication that small molecule CCR5 antagonists and the anti-CCR5 MAb PA14 inhibit HIV-1 entry by different mechanisms.

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The JR-FL gp120 amino acid sequence is 71% identical and 85% similar to both 92NG083 and 93TH966 sequences. Using gp120 chimeras, we demonstrated that the difference in sensitivity to small molecule CCR5 antagonists is directly regulated by the V3 loops, especially the crown regions, of our three test isolates. Moreover, we showed that the V3 loop directly modulates the level of gp120 binding to CCR5 and that low binding correlates with high sensitivity to SCH-C and TAK-779. In other words, a gp120 that interacts weakly with CCR5 is more easily displaced from the coreceptor in the presence of the inhibitors. A similar conclusion was reached by Reeves et al. using various gp120 V3 loop chimeras (Reeves et al., 2002). This group also showed that gp120 affinity for CCR5 directly affects the kinetics of viral entry (Reeves et al., 2002, 2004). Our findings further support the model wherein the crown of the V3 loop interacts with ECL2 of CCR5, thereby determining the efficiency of entry inhibition. In contrast to the small molecules, inhibition of entry by anti-CCR5 MAb PA14 was similar for all test isolates and not a function of gp120 affinity or V3 loop sequence. We had previously shown that PA14 was approximately 10-fold less potent at inhibiting gp120 binding than anti-CCR5 MAbs that target the Nt. Conversely, PA14 was 10-fold more potent than these MAbs at inhibiting viral entry (Olson et al., 1999). Here, we demonstrated that PA14 inhibits a step in CCR5 usage that comes after the step inhibited by the small molecule antagonists and before gp41-mediated membrane fusion. The PA14 epitope has an unusual configuration in that it comprises both the first residue of the Nt (D2) and the first residue of ECL2 (R169) at the junction of ECL2 and TM4 (Olson et al., 1999). When bound to CCR5, therefore, this MAb may lie close and parallel to the plasma membrane and preferentially inhibit gp120-independent events, for example, oligomerization of CCR5 within a fusion complex. Based on our findings, we propose the following model of HIV-1 entry and inhibition: the V3 crown–ECL2 interaction is the very first step in CCR5 usage; 9 min elapse between CD4 binding and V3 crown–ECL2 interactions. Small molecule antagonists such as SCH-C and TAK-779 disfigure CCR5 ECL2 making it unrecognizable by the gp120 V3 loop crown. Based on our previous work, we propose that the second step to occur is the interaction between the CCR5 Nt and the V3 stem/bridging sheet. This stage of coreceptor usage is weakly inhibited by MAbs that recognize the CCR5 Nt, probably due to poor epitope accessibility in the context of the gp120-CCR5 complex during viral entry (Olson et al., 1999). Once gp120 is firmly tethered to CCR5, further conformational changes, protein modifications or oligomerization can occur. We propose that these are blocked by PA14 and that together with the second stage of entry they occur over 23 min. Another 14 min is required for maturation of the CCR5-gp120 complex, including gp120-gp41 dissociation, resulting in gp41-mediated membrane fusion. Note that the t1/ 2s we measure are not absolute and depend on the concentrations of receptors and envelope glycoproteins, as well as the relative affinities of these proteins for each other. However, a similar distribution of Δt1/2s was measured for isolate 92NG083 (T.D.,

unpublished results), suggesting that the different stages of entry are common to all HIV-1 isolates. Combinations of SCH-C and PA14 are highly synergistic inhibitors because they target different stages of HIV-1 entry. Our data also have implications regarding the emergence of resistant HIV-1 isolates. The V3 loop is one of the most variable regions of gp120, and it should therefore be expected that strains resistant to small molecule inhibitors such as SCH-C and TAK-779 will appear relatively quickly. Whether these isolates will remain R5, as suggested by Kuhmann et al., or resort to CXCR4 usage in vivo remains to be seen. On the other hand, we would predict that HIV-1 will have difficulty developing resistance to MAbs such as PA14 since this compound does not (primarily) inhibit the CCR5-gp120 interaction but preferentially inhibits post-binding steps. Future drug discovery programs should therefore strive to develop screens for identifying small molecules with similar properties. We recognize that this may be difficult as small molecules tend to act through allosteric mechanisms rather than by sterically blocking interactions. Ultimately, compounds such as PA14 or small molecules with differing cooperativity factors will stand the best chance of retaining long-term effectiveness in the clinic. Materials and methods Cell lines, antibodies and inhibitors HeLa-CD4-CCR5 cells (Vodicka et al., 1997) were cultured in standard DMEM-based medium supplemented with G418 (800 μg/ ml). L1.2-CCR5 cells (Wu et al., 1996) were cultured in RPMIbased medium supplemented with β-mercaptoethanol (100 μM) and G418 (800 μg/ml). CD4-IgG2 and anti-CCR5 MAb PA14 were generated by Progenics Pharmaceuticals, Inc. (Tarrytown, NY) as described previously (Allaway et al., 1995; Olson et al., 1999). Anti-gp120 MAb B12 (Moore et al., 1994) was generously provided by Dr. William Olson (Progenics Pharmaceuticals). The small-molecule CCR5 antagonists SCH-C and TAK-779 were synthesized as described previously (Tsamis et al., 2003). T20 was synthesized by American Peptide according to the described sequence (Kilby et al., 1998; Matthews et al., 2004). Single cycle HIV-1 entry assay NLluc+env− pseudoparticles were made as described (Dragic et al., 1998), using HIV-1 envelope glycoproteins from the Table 1 CI values based on fixed ratio compound combinations

SCH-C + PA14 TAK-779 + PA14

IC50

IC90

0.06 0.25

0.17 0.57

The concentrations of single agents or combinations required to produce halfmaximal and 90% inhibition of HIV-1 entry were quantitatively compared in a term known as the combination index (CI). Synergism is defined as a greaterthan-expected-additive effect, and antagonism is defined as less-than-expectedadditive effect.

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following R5 isolates: JR-FL, JR-CSF (O'Brien et al., 1990), ADA (Westervelt et al., 1991), BaL (Hwang et al., 1992), 93MW960, 92BR025, 93MW965, 92RW020, 93TH966 (Gao et al., 1996), 94IN476, 98IN012 (Rodenburg et al., 2001), 94NG114, 92NG083 (Gao et al., 1998), as well as chimeric envelope glycoproteins JR-FL(92NG083) and 92NG083(JRFL) (see below). Hela-CD4-CCR5 target cells (4 × 104) were incubated with virus-containing supernatants (100 ng/ml p24), plus or minus different concentrations of inhibitors for 16 h before medium was changed. After 48 h, cells were lysed, and luciferase activity (relative light units, RLU) was measured using a standard kit (Promega) as described (Dragic et al., 1998). Synergy studies were performed with fixed-ratio compound combinations. Target cells were treated either with individual anti-CCR5 compounds or with equimolar mixtures of PA14 and SCH-C or TAK-779, followed by infection with JR-FL pseudotypes. The concentrations of single agents or combinations required to produce half-maximal and 90% inhibition of HIV-1 entry were quantitatively compared in a term known as the Combination Index (CI). The CI method is based on the median-effect principle described by Chou (for review, see Reynolds and Maurer, 2005). We developed our own software to calculate CI values (Olson et al., 1999). D = Dm [fa/fu]1/m, where D is compound concentration, Dm is the median-effect dose (IC50), fa is the fraction of the response affected by the dose, and fu = 1 − fa. CI for any given fa value is then calculated * as (D1combo/D1single) + (D2combo/D2single) + (D1combo D2combo/ * D1singleD2single). For time course of inhibition experiments, HIV-1 pseudoparticles were chilled on ice, added to cells and spinoculated for 1 h at 2000 rpm at 4 °C. Cells were washed twice with PBS at 4 °C and medium at 37 °C, containing PA14 (3 × 10−9 M), SCH-C (3 × 10−8 M) or T20 (3 × 10−8 M) was added at different time points, from 0 to 3 h. After addition of MAbs, cells were kept at 37 °C for the duration of the experiment. Medium was changed 16 h after the first time point of inhibitor addition (t = 0). Luciferase activity was measured in cell lysates 48 h post-infection. Generation of V3 loop chimeras Chimeric gp120 proteins were generated by substituting V3 loop residues using the QuickChange Kit (Stratagene). Nucleotide sequencing was performed to ascertain the presence of the appropriate substitutions. Envelope glycoprotein sequences in the SV7D expression vector served as templates and were used to generate pseudoparticles comprising chimeric glycoproteins. Wild-type and chimeric gp120 sequences were also subcloned into the PPI4 expression vector for generating soluble proteins (Binley et al., 2000). Supernatants containing wild-type and mutant gp120 proteins were generated by transient transfection of 293T cells as described previously (Binley et al., 2000). Soluble gp120 was quantified by ELISA and verified by Western blotting with the B12 anti-gp120 MAb (0.5 μg/ml) as described (Cormier et al., 2001). Soluble gp120 proteins were also characterized

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for CD4-IgG2 binding as described previously (Cormier et al., 2001). Binding of gp120/CD4-IgG2 complexes to cell-surface CCR5 L1.2-CCR5 cells (5 × 105) were incubated with gp120containing supernatant (4 × 10−7 M) and CD4-IgG2 (2 × 10−7 M) for 1 h at 37 °C, as described previously (Olson et al., 1999). Gp120/CD4-IgG2 bound to cells was revealed by flow cytometry analysis of the mean fluorescence intensity (MFI) after addition of a phycoerythrin-conjugated goat anti-human IgG2 (Pharmingen). Acknowledgments We wish to thank Dr. William Olson of Progenics Pharmaceuticals for generously providing us with critical reagents for this study, including PA14, TAK-779, SCH-C and CD4-IgG2. This work was supported by NIH grant AI43847 to T.D. This work was also supported in part by the NIAID Centers for AIDS Research grant AI051519 to Albert Einstein College of Medicine. References Aarons, E.J., Beddows, S., Willingham, T., Wu, L., Koup, R.A., 2001. Adaptation to blockade of human immunodeficiency virus type 1 entry imposed by the anti-CCR5 monoclonal antibody 2D7. Virology 287 (2), 382–390. Abrahamyan, L.G., Markosyan, R.M., Moore, J.P., Cohen, F.S., Melikyan, G.B., 2003. Human immunodeficiency virus type 1 Env with an intersubunit disulfide bond engages coreceptors but requires bond reduction after engagement to induce fusion. J. Virol. 77 (10), 5829–5836. Allaway, G.P., Davis-Bruno, K.L., Beaudry, G.A., Garcia, E.B., Wong, E.L., Ryder, A.M., Hasel, K.W., Gauduin, M.C., Koup, R.A., McDougal, J.S., al., a.e., 1995. Expression and characterization of CD4-IgG2, a novel heterotetramer that neutralizes primary HIV type 1 isolates. AIDS Res. Hum. Retroviruses 11, 533–539. Berger, E.A., Murphy, P.M., Farber, J.M., 1999. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease [In Process Citation]. Annu. Rev. Immunol. 17, 657–700. Binley, J.M., Sanders, R.W., Clas, B., Schuelke, N., Master, A., Guo, Y., Kajumo, F., Anselma, D.J., Maddon, P.J., Olson, W.C., Moore, J.P., 2000. A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. J. Virol. 74 (2), 627–643. Binley, J.M., Cayanan, C.S., Wiley, C., Schulke, N., Olson, W.C., Burton, D.R., 2003. Redox-triggered infection by disulfide-shackled human immunodeficiency virus type 1 pseudovirions. J. Virol. 77 (10), 5678–5684. Carrington, M., Kissner, T., Gerrard, B., Ivanov, S., O'Brien, S.J., Dean, M., 1997. Novel alleles of the chemokine-receptor gene CCR5. Am. J. Hum. Genet. 61 (6), 1261–1267. Chou, T.C., Rideout, D.C., 1991. Synergism and Antagonism in Chemotherapy. Academic Press, New York. Cormier, E.G., Dragic, T., 2002. The crown and stem of the V3 loop play distinct roles in human immunodeficiency virus type 1 envelope glycoprotein interactions with the CCR5 coreceptor. J. Virol. 76 (17), 8953–8957. Cormier, E.G., Tran, D.N., Yukhayeva, L., Olson, W.C., Dragic, T., 2001. Mapping the determinants of the CCR5 amino-terminal sulfopeptide interaction with soluble human immunodeficiency virus type 1 gp120CD4 complexes. J. Virol. 75 (12), 5541–5549.

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