Figure 3.

Capture of HIV-1 by spermatozoa is mainly mediated through HS. (A) The expression of HS on spermatozoa was analyzed by flow cytometry using anti–HS-specific antibodies (clone 10E4). Isotype control (gray histogram) and 10E4 labeled (open histogram) are depicted. A representative experiment (n = 7) is shown. (B) Spermatozoa (1.5 × 10⁶/200 µl) were incubated with HIV-1 BAL containing 25 ng of p24 for 60 min at 37°C in the absence or presence of 10 and 100 U/ml heparin, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of six experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (C) Spermatozoa were treated with 5 U/ml heparinase II for 60 min at 25°C or 1,000 U/ml trypsin for 15 min at 37°C. Then the expression of HS was analyzed by flow cytometry. The results are expressed as the MFI ± SEM of five experiments performed in duplicate. Asterisks represent statistical significance (P < 0.05 vs. the expression of HS in controls). (D) Spermatozoa were treated with 1 and 5 U/ml heparinase II for 60 min at 25°C. Then their ability to capture HIV-1 was assayed as described for Fig. 2 A. Results are the mean ± SEM of five experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (E) Spermatozoa were treated with 5 U/ml heparinase III for 60 min at 25°C. Then the expression of the neoepitope 3G10 was analyzed by flow cytometry. Isotype (gray histogram) and 3G10-labeled untreated (control) or heparinase III–treated spermatozoa (open histograms) are shown. Isotype controls were similar for untreated and heparinase III–treated spermatozoa. A representative experiment (n = 4) is shown. (F) Spermatozoa (1.5 × 10⁶/200 µl) were incubated with different primary HIV-1 isolates containing 25 ng of p24 for 60 min at 37°C in the absence or presence of 100 U/ml heparin or 5 mg/ml mannan, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of three to five experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (G) HIV-1 pseudotypes were produced as described in Materials and methods. Spermatozoa (1.5 × 10⁶/200 µl) were incubated with HIV-1 env⁻ or HIV-1 env⁺ pseudotypes containing 40 ng of p24 for 60 min at 37°C in the absence or presence of 5 mg/ml mannan or 100 U/ml heparin, washed thoroughly, lysed, and assayed for p24 antigen by ELISA. Results are the mean ± SEM of three to four experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (H) The expression of syndecans 1–4 was analyzed by flow cytometry. Gray histograms correspond to isotype controls. In each case, a representative experiment (n = 3–6) is shown.