Mucosal human defensins 5 and 6 antagonize the anti-HIV activity of candidate polyanion microbicides

Abstract

Defensins are highly abundant antimicrobial peptides in the female genital mucosa. We have previously shown that human defensins 5 and 6 (HD5 and HD6), produced by cervicovaginal epithelial cells, significantly enhance HIV infectivity in vitro. Candidate polyanion microbicides, including PRO 2000, cellulose sulfate and carrageenan, failed to protect women against HIV infection in large-scale clinical trials, but the molecular basis of ineffectiveness was not clear. We hypothesized that mucosal host factors such as HD5 an HD6 may alter the activity of polyanion microbicides against HIV. Our results demonstrated that HD5 and HD6 but not their linear analogs antagonized the anti-HIV activity of PRO 2000, cellulose sulfate and carrageenan in vitro. Polyanion microbicides also reduced the HIV-enhancing effect of these defensins. We conclude that mucosal host factors could negatively impact the efficacy of topical microbicides against HIV, and their impact on the activity of candidate microbicides needs to be considered during the preclinical evaluation.

Fig. 1

Effect of candidate polyanion microbicides on HIV infection of HeLa-CD4-CCR5 cells in the presence of HD5 and 6. HIV-1_JR-FL pseudotyped luciferase reporter virus was incubated with PRO 2000 (a), CS (b) and iota-carrageenan (c) in the presence (right panels) or absence (left panels) of HD5 or HD6 at 20 μg/ml for 1 h at 37°C. The mixture of virus with various treatments was added to HeLa-CD4-CCR5. The HIV infection assay was performed as described in the Methods section. Data are means ± SD of triplicate samples and represent 4 independent experiments. The difference between defensin-treated samples versus untreated samples in the presence of the same concentration of inhibitors was analyzed by the two-tailed, paired Student's t test (∗ p < 0.05). d The effect of PRO 2000 (1 μg/ml), CS (10 μg/ml) and iota-carrageenan (100 μg/ml) on HIV infection in the presence of linear analogs of HD5 and HD6 (20 μg/ml) with identical charges was determined. Samples without defensin treatment were included as a control (untreated). The untreated control for carrageenan contained DMSO (1%) that was used as a solvent. The difference between the untreated control and samples in [Abu]HD5 or [Abu]HD6 is not significant (⁺p > 0.05). Data are means ± SD of triplicate samples and represent 2 independent experiments. RLU = Relative light units.

Fig. 1

Effect of candidate polyanion microbicides on HIV infection of HeLa-CD4-CCR5 cells in the presence of HD5 and 6. HIV-1_JR-FL pseudotyped luciferase reporter virus was incubated with PRO 2000 (a), CS (b) and iota-carrageenan (c) in the presence (right panels) or absence (left panels) of HD5 or HD6 at 20 μg/ml for 1 h at 37°C. The mixture of virus with various treatments was added to HeLa-CD4-CCR5. The HIV infection assay was performed as described in the Methods section. Data are means ± SD of triplicate samples and represent 4 independent experiments. The difference between defensin-treated samples versus untreated samples in the presence of the same concentration of inhibitors was analyzed by the two-tailed, paired Student's t test (∗ p < 0.05). d The effect of PRO 2000 (1 μg/ml), CS (10 μg/ml) and iota-carrageenan (100 μg/ml) on HIV infection in the presence of linear analogs of HD5 and HD6 (20 μg/ml) with identical charges was determined. Samples without defensin treatment were included as a control (untreated). The untreated control for carrageenan contained DMSO (1%) that was used as a solvent. The difference between the untreated control and samples in [Abu]HD5 or [Abu]HD6 is not significant (⁺p > 0.05). Data are means ± SD of triplicate samples and represent 2 independent experiments. RLU = Relative light units.

Fig. 2

HD5 and HD6 antagonized the anti-HIV activity of polyanionic polymers in PBLs. The effect of defensins on polyanionic polymers was determined using PHA-activated PBLs. HIV infection was performed as described in the method section. Data are means ± SD of triplicate samples and represent three experiments using PBLs from different donors. ∗ p < 0.05, defensin-treated samples versus untreated samples in the presence of the same concentration of inhibitors. RLU = Relative light units.