Figure 5. CD36-specific antibodies induce a potent, rapid, and long-lasting inhibition of HIV-1 release from macrophages. (A) Schematic representation of the experimental design. (B and C) Quantification of p24 Gag present in the overnight culture supernatant harvested as indicated in A (B), and in the corresponding cell lysates (C). (D) Total p24 Gag found in the supernatant + the cell lysates. (E) Cell viability was measured at the end of the experiment with the CellTiter-Glo kit. (F and G) Quantification of p24 Gag released from primary macrophages treated overnight with the indicated Abs. In F, all antibodies were used at 1 µg/ml. In G, the IgM specific for CD36 (SMΦ) was used, like its isotype control, at 20 µg/ml, while the FA6-156 and its isotype control were used at 10 µg/ml. (H and I) Titration of the effect of CD36-specific mAb (clone IVC7) on HIV-1–infected macrophages on the amounts of secreted (H) and cell-associated (I) p24 Gag. (J) Infectivity of the virions on the reporter cell line TZM-bl produced by the macrophages subjected to the anti-CD36 mAb treatment was evaluated using the same amount of p24 Gag (10 ng/ml; see Materials and methods). (K) Quantification of p24 Gag in the supernatant of HIV-1 NLAD8-infected macrophages treated at 7 d p.i. with an anti-CD36 mAb (FA6-152) or its isotype control for the indicated time. (L) Quantification of the p24 Gag produced by HIV-1 NLAD8–infected macrophages in a 24-h time window before (–) or after (+) antibody washout. 6-d-infected macrophages were washed and exposed to the indicated antibodies for 24 h. Then cells were washed out and incubated in compete medium for another 24 h. Supernatant collected before and after antibody washout were stored at −20°C for at least 1 d before p24 Gag quantification. Representative experiments are presented in B–L. All the experiment have been reproduced at least three times with three different donors, except for H and I, which have been performed once (similar results are shown in Fig. 6 I). Data are shown as mean ± SEM of triplicates. One-way ANOVA with Tukey’s multiple comparison test was used as a statistical test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).
Figure 5.

CD36-specific antibodies induce a potent, rapid, and long-lasting inhibition of HIV-1 release from macrophages. (A) Schematic representation of the experimental design. (B and C) Quantification of p24 Gag present in the overnight culture supernatant harvested as indicated in A (B), and in the corresponding cell lysates (C). (D) Total p24 Gag found in the supernatant + the cell lysates. (E) Cell viability was measured at the end of the experiment with the CellTiter-Glo kit. (F and G) Quantification of p24 Gag released from primary macrophages treated overnight with the indicated Abs. In F, all antibodies were used at 1 µg/ml. In G, the IgM specific for CD36 (SMΦ) was used, like its isotype control, at 20 µg/ml, while the FA6-156 and its isotype control were used at 10 µg/ml. (H and I) Titration of the effect of CD36-specific mAb (clone IVC7) on HIV-1–infected macrophages on the amounts of secreted (H) and cell-associated (I) p24 Gag. (J) Infectivity of the virions on the reporter cell line TZM-bl produced by the macrophages subjected to the anti-CD36 mAb treatment was evaluated using the same amount of p24 Gag (10 ng/ml; see Materials and methods). (K) Quantification of p24 Gag in the supernatant of HIV-1 NLAD8-infected macrophages treated at 7 d p.i. with an anti-CD36 mAb (FA6-152) or its isotype control for the indicated time. (L) Quantification of the p24 Gag produced by HIV-1 NLAD8–infected macrophages in a 24-h time window before (–) or after (+) antibody washout. 6-d-infected macrophages were washed and exposed to the indicated antibodies for 24 h. Then cells were washed out and incubated in compete medium for another 24 h. Supernatant collected before and after antibody washout were stored at −20°C for at least 1 d before p24 Gag quantification. Representative experiments are presented in B–L. All the experiment have been reproduced at least three times with three different donors, except for H and I, which have been performed once (similar results are shown in Fig. 6 I). Data are shown as mean ± SEM of triplicates. One-way ANOVA with Tukey’s multiple comparison test was used as a statistical test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

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