Rcd4 cd3 cd4 cd69 cd25 hla dr t cells

HIV-1 p24 protein production (replication-competent virus as measure of infectious particles production) was analyzed in quantitative viral outgrowth assays (QVOA) (19 (link), 29 (link)) after rCD4 T cells’ ex vivo exposure to N-BPs PAM and Zol. Briefly, magnetically isolated rCD4 (CD3+/CD4+/CD69-/CD25-/HLA-DR-) T cells (Stemcell Technologies) were rested in the presence of antiretroviral drugs (10nM Raltegravir and 20nM Efavirenz). Cells were washed, counted and plated in limiting dilution from 1x10⁶ to 0.1x10⁶ cells. Cells were treated with 5U/mL IL-2 as the negative control or exposed 24 hours to PHA and IL-2, or to N-BPs PAM, Zol and ALN. After washing, isolated PHA-activated CD4 cells from uninfected individuals were added twice during the 19-day culture as targets of new infections to outgrowth the virus. After 15 days of culture, supernatants were harvested and HIV-1p24 ELISA performed and confirmed at day 19 of culture. The frequency of infection was calculated and expressed as infectious units per million (IUPM) cultured cells, as explained in Statistical Methods.

HIV-1 p24 protein production (replication-competent virus as measure of infectious particles production) was analyzed in quantitative viral outgrowth assays (QVOA) (19 (link), 41 (link)) after rCD4 T cells’ ex vivo exposure to N-BPs PAM and Zol. Briefly, magnetically isolated rCD4 (CD3+/CD4+/CD69−/ CD25−/ HLA−DR−) T cells (Stemcell Technologies) were rested in the presence of antiretroviral drugs (10nM Raltegravir and 20nM Efavirenz). Cells were washed, counted and plated in limiting dilution from 1×10⁶ to 0.1×10⁶ cells. Cells were left untreated or exposed 24 hours to PHA and IL-2, or to N-BPs PAM, Zol and ALN. After washing, isolated PHA-activated CD4 cells from uninfected individuals were added twice during the 19-day culture as targets of new infections to outgrowth the virus. After 15 days of culture, supernatants were harvested and HIV-1p24 ELISA performed and confirmed at day 19 of culture. The frequency of infection was calculated and expressed as infectious units per million (IUPM) cultured cells, as explained in Statistical Methods.