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  • br Materials and methods br


    Materials and methods
    Results The monoclonal antibody (mAb), 12G5, reacts specifically with the human fusin protein and recognizes the protein on many T-cell lines such as the SUP-T1 LY3009120 mg (Endres et al., 1996). As shown in Fig. 1, AMD3100 at 25 μg/ml completely inhibited the binding of the 12G5 mAb to CXCR-4 on SUP-T1 cells. This is in contrast with two other potent HIV inhibitors, the sulfated polysaccharide dextran sulfate (DS) (Baba et al., 1990) and the oligonucleotide AR177, also called T30177 or Zintevir (Ojwang et al., 1995), which were both ineffective at 25 μg/ml in blocking the binding of 12G5 mAb to CXCR-4. The CXC chemokine, stromal cell-derived factor 1 (SDF-1) (Bleul et al., 1996, Oberlin et al., 1996), the natural ligand for CXCR-4 competed, as expected, with the binding of the mAb 12G5 to its receptor (Fig. 1). In the following experiments, the compound AMD3100 was brought onto the cells in serial dilutions and washed away after a 15 min incubation period with the cells at room temperature before the 12G5 mAb was added (Fig. 2). As can be seen in Fig. 2, the compound strongly interacted with the CXCR-4 receptor and even when washed away, inhibited the binding of the mAb 12G5 as efficiently as when the compound was present during the whole incubation period with the mAb (compare with AMD3100 at 25 μg/ml in Fig. 1, and data not shown). This indicates that AMD3100 does not interfere with the mAb itself but binds directly to CXCR-4. From Fig. 2 it is also clear that even at a concentration of 0.2 μg/ml AMD3100 still interfered with the binding of mAb 12G5 to CXCR-4. Adding AMD3100 together with the mAb, at room temperature or at 4°C, blocked the binding of the mAb as efficiently as adding the compound 15 min. before the mAb (data not shown). This also points to a very strong and direct interaction of AMD3100 with the CXCR-4 receptor and internalization of CXCR-4 can therefore be excluded. The bicyclam JM2763, which is about 20 fold less potent than AMD3100 in inhibiting HIV-1 and HIV-2 replication (De Clercq et al., 1994), also proved about 20-fold less potent than AMD3100 in inhibiting the binding of 12G5 mAb to the SUP-T1 cells (Fig. 3). This demonstrates a direct correlation between the anti-HIV activity of the bicyclams and their interaction with CXCR-4. If AMD3100 specifically interacts with CXCR-4, then the compound should be active only against viruses which use CXCR-4 as coreceptor to enter the target cells. This is indeed the case. As shown in Table 1, AMD3100 is very active against T-tropic viruses (IC50 between 0.003 and 0.01 μg/ml), but not active against M-tropic viruses (IC50: >25 μg/ml). These virus strains preferentially use CCR-5, but some can also use CCR-2 and/or CCR-3 (but not CXCR-4) to enter the cells. In addition, AMD3100 is also not active against SIV, which uses CCR-5 as coreceptor (Table 1). Because AMD3100 interacts specifically with CXCR-4, the receptor for SDF-1 and HIV is using CXCR-4 as its coreceptor to enter the cells, then SDF-1 should interfere with the infectivity of wild-type, but not AMD3100-resistant virus. Therefore, MT-4 cells were infected with 100 CCID50 of the HIV-1 NL4-3 wild type (WT), NL4-3 AMD3100-resistant strain (De Vreese et al., 1996a) and NL4-3 DS-resistant strain (Esté et al., 1997b). SDF-1 was added to the cells at different concentrations starting from 2 μg/ml. At 4 days after infection the cells were analyzed for CD4 expression because productive infection of T-tropic viruses is generally accompanied by the disappearance of CD4 from the cell surface (Dalgleish et al., 1984). The uninfected MT-4 cells were 99% CD4+ (Schols et al., 1989) whereas only 57%, 45% and 37% of the NL4-3#WT, AMD3100-resistant and the DS-resistant virus infected cells expressed CD4 (Fig. 4, upper panels). As can be seen in the lower panels of Fig. 4, SDF-1 was totally protective against the NL4-3 WT and also against the DS-resistant strain at 2 μg/ml (99% of the cells expressed CD4). However, SDF-1 (at 2 μg/ml) had no activity at all against the AMD3100-resistant strain (only 40% of the cells expressed CD4). The p24 viral Ag levels in the supernatant of the untreated cells infected with NL4-3 WT virus, NL4-3 DS-resistant virus and the NL4-3 AMD3100-resistant virus (Fig. 4) were determined and measured 416 840, 423 857 and 604 029 pg/ml, respectively. The p24 Ag level was below the detection limit of the ELISA (<5 pg/ml) in the supernatant of the cells treated with SDF-1 (at 2 μg/ml) and infected with the NL4-3 WT virus or the NL4-3 DS-resistant virus, but in the supernatant of the NL4-3 AMD3100-resistant virus with SDF-1 (at 2 μg/ml) a value of 672 416 pg/ml of p24 Ag was measured. SDF-1 had an identical IC50 (50% inhibitory concentration) of 100 ng/ml for the NL4-3WT and the DS-resistant strain, as determined with the MTT-method (Pauwels et al., 1988). Thus, the replication of AMD3100-resistant virus was not inhibited by SDF-1, whereas the replication of the WT virus and the DS-resistant virus were equally sensitive to this chemokine.