Quantitative Viral Outgrowth Assay for HIV-1

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.

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Sanz M., Weideman A.M., Ward A.R., Clohosey M.L., Garcia-Recio S., Selitsky S.R., Mann B.T., Iannone M.A., Whitworth C.P., Chitrakar A., Garrido C., Kirchherr J., Coffey A.R., Tsai Y.H., Samir S., Xu Y., Copertino D., Bosque A., Jones B.R., Parker J.S., Hudgens M.G., Goonetilleke N, & Soriano-Sarabia N. (2023). Aminobisphosphonates reactivate the latent reservoir in people living with HIV-1. bioRxiv.

Publication Preprint 2023

rCD4 (CD3+/CD4+/CD69−/ CD25−/ HLA−DR−) T cells

Assays Cd25 Cd4 cells Cells Cultured cells Dilution Drugs Efavirenz Elisa Hiv 1 Hla dr Infectious Protein Raltegravir Rcd4 Replication Stemcell T cells Virus Virus particles

Corresponding Organization : George Washington University

Other organizations : University of North Carolina at Chapel Hill, Cornell University

Top 5 similar protocols

Variable analysis

independent variables

Exposure to N-BPs PAM
Exposure to N-BPs Zol
Exposure to N-BPs ALN

dependent variables

HIV-1 p24 protein production (replication-competent virus as measure of infectious particles production)

control variables

Resting of rCD4 (CD3+/CD4+/CD69−/ CD25−/ HLA−DR−) T cells in the presence of antiretroviral drugs (10nM Raltegravir and 20nM Efavirenz)
Plating of cells in limiting dilution from 1×10^6 to 0.1×10^6 cells
Addition of isolated PHA-activated CD4 cells from uninfected individuals as targets of new infections to outgrow the virus

positive control

Exposure to PHA and IL-2

negative control

Untreated cells

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