Viral Production in CD4+ T Cells

Healthy donor’s CD4⁺ T cells were activated by PHA-L (5 μg/ml) for 48 h and cultured in RPMI medium supplemented with 10% FBS and IL-2 at 20 U/ml (complete medium) for an additional 48 h before use in the VOA. To evaluate infectious viral production after platelet–CD4⁺ T-cell interaction, HIV-containing platelets from InRs were added to 10⁵ activated healthy donor’s CD4⁺ T cells cultured in complete medium with or without 20 μg/ml polybrene to facilitate fusion of viral envelope and host cell membrane (16 (link)). The platelet:T cell ratio employed was 2:1. After 7 days of coculture, medium was replaced and freshly prepared 10⁵ healthy donor’s activated CD4⁺ T cells were added to the T cells that interacted with HIV-containing platelets to improve viral propagation for an additional 7 days. At days 0, 3, 7, and 14 of the VOA assay, culture medium was collected and replaced by fresh medium. Collected media were analyzed by HIV p24 capsid protein ELISA (Innotest HIV Antigen mAb, FUJIREBIO) to measure the cumulative viral production.