Figure 4.

Spermatozoa efficiently transmit HIV-1 to DCs, macrophages, and CD4⁺ T cells. (A) DCs were incubated for 60 min at 37°C with HIV-1 containing 25 ng of p24, in the absence (gray bar) or presence of different numbers of spermatozoa (Sp; open bars). Cells were then washed four times to remove free virus, and the infection of DCs was revealed after 7 d by measuring the amount of p24 in cell supernatants. Black bars represent spermatozoa incubated alone with HIV-1 containing 25 ng p24. Results are the mean ± SEM of four experiments performed in duplicate. Asterisk represents statistical significance (P < 0.05 vs. DCs). (B) DCs (10⁵/200 µl) were incubated with HIV-1 BAL containing 25 ng p24 for 60 min at 37°C. Cells were then washed thoroughly to remove free virus and then DCs were cultured with or without 1.5 or 6.0 × 10⁶ spermatozoa. Infection of DCs was revealed after 7 d. The results are expressed as the mean ± SEM of four experiments performed in duplicate. (C) Spermatozoa suspended at different concentrations were incubated with HIV-1 BAL containing 25 ng p24/200 µl of spermatozoa suspension for 60 min at 37°C. Cells were then washed thoroughly, different numbers of HIV-1–treated spermatozoa were incubated with (open bars) or without DCs (black bars) over 7 d, and the infection of DCs was then analyzed. Results are the mean ± SEM of four to five experiments performed in duplicate. Asterisks represent statistical significance (P < 0.05 vs. spermatozoa cultured without DCs). (D) Spermatozoa (1.5 × 10⁶/200 µl) were incubated with the primary HIV-1 isolates 93BR020.1 or GARR-G4 containing 25 ng p24 for 60 min at 37°C, and then cells were washed thoroughly. Spermatozoa and DCs were co-cultured during 7 d at a spermatozoa/DC ratio of 10:1, and the infection of DCs was then analyzed. Results are the mean ± SEM of three experiments performed in duplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (E) Spermatozoa (1.5 × 10⁶/200 µl) were incubated with HIV-1 BAL containing 25 ng p24 for 60 min at 37°C, washed thoroughly, and co-cultured with 10⁵ DCs during 3, 7, and 12 d at a spermatozoa/DC ratio of 10:1. Infection of DCs was then analyzed. Black bars represent the amount of p24 found in the supernatants of HIV-1–treated spermatozoa cultured without DCs. Results are the mean ± SEM of four experiments performed in duplicate. Asterisks represent statistical significance (P < 0.05 vs. controls). (F) Spermatozoa (1.5 × 10⁶ /200 µl) were treated with 5 U/ml heparinase II for 60 min at 25°C, incubated with HIV-1 BAL containing 25 ng p24 for 60 min at 37°C, washed thoroughly, and co-cultured during 7 d with DCs at a spermatozoa/DC ratio of 10:1. Infection of DCs was then revealed as described in A. Black bars represent the levels of p24 antigen in the supernatants of HIV-1–treated spermatozoa cultured alone. Results are the mean ± SEM of four experiments performed in duplicate. The asterisk represents statistical significance (P < 0.05 vs. controls). (G) Spermatozoa (1.5 × 10⁶/200 µl) were incubated with 25 ng HIV-1 BAL or HIV-1 IIIB for 60 min at 37°C. Cells were then washed thoroughly and incubated with macrophages, the T cell line MT2, PHA plus IL-2–activated PBMCs, or purified CD3⁺ T cells activated by PHA plus IL-2 (1.5 × 10⁵ cells/200 µl). Spermatozoa treated with HIV-1 BAL were used in macrophage cultures, whereas spermatozoa treated with HIV-1 IIIB were used for MT2 cells, PBMCs, and purified T cells. Cellular infection was analyzed after 7 d of culture. Results are the mean ± SEM of three to four experiments performed in duplicate. Asterisks represent statistical significance (P < 0.05 vs. spermatozoa cultured alone). (H) Spermatozoa (1.5 × 10⁶/200 µl) were incubated with HIV-1 IIIB containing 25 ng of p24 for 60 min at 37°C, and then cells were washed thoroughly. Spermatozoa and DCs were co-cultured during 60 min at 37°C at a spermatozoa/DC ratio of 10:1. Cells were then treated with 1,000 U/ml trypsin for 15 min at 37°C, washed, and cultured with or without 3.0 × 10⁵ T cells purified from peripheral blood and activated with PHA plus IL-2. The amount of p24 antigen in cell supernatants was analyzed after 7 d of culture. Results are the mean ± SEM of three experiments performed in duplicate. The asterisk represents statistical significance (P < 0.05 vs. Sp + DCs).