An assay, specifically optimized to evaluate the anti-HIV activity of antiretrovirals by flow cytometry analysis, is described. As widely used anti-HIV agents, zidovudine (AZT), abacavir (ABC), 2',3'-dideoxyinosine (DDI), lamivudine (3TC), nevirapine (NVP), and efavirenz (EFV), and as drugs of recent approval raltegravir (RAL), etravirine (ETR), and rilpivirine (RPV), were utilized as reference drugs. HIV-1 NL4-3 virus was prepared by transfection of HEK293T cells with purified plasmid DNA and quantified by p24 antigen-capture assay. For infection, CEM-GFP cells were exposed to vehicle or to several concentrations of the drugs for 2 hr at 37 °C before HIV-1 NL4-3 was added to each sample. The adsorption was prolonged for 3 hr at 37 °C. After 72 hr of incubation, HIV-induced GFP expression in infected CEM-GFP cells was assessed by flow cytometry analysis and expressed as % positive cells. For comparison, p24 production in supernatants was assessed by a commercial ELISA kit. On the basis of IC50 values, the anti-HIV activity, as assayed by this method, was EFV > 3TC > AZT > NVP > DDI > ABC and ETR > RPV > RAL. The comparison between the IC50 values calculated through flow cytometry and p24 production revealed overlapping results, showing that the optimized protocol of CEM-GFP infection with HIV NL4-3 is a suitable method to perform quantitative, rapid and low-expensive screening tests to evaluate the in vitro effect of new candidate anti-HIV drugs.

Frezza, C., Grelli, S., Federico, M., Marino Merlo, F., Mastino, A., Macchi, B. (2016). Testing anti-HIV activity of antiretroviral agents in vitro using flow cytometry analysis of CEM-GFP cells infected with transfection-derived HIV-1 NL4-3. JOURNAL OF MEDICAL VIROLOGY, 88(6), 979-986 [10.1002/jmv.24418].

Testing anti-HIV activity of antiretroviral agents in vitro using flow cytometry analysis of CEM-GFP cells infected with transfection-derived HIV-1 NL4-3

GRELLI, SANDRO;MACCHI, BEATRICE
2016-06-01

Abstract

An assay, specifically optimized to evaluate the anti-HIV activity of antiretrovirals by flow cytometry analysis, is described. As widely used anti-HIV agents, zidovudine (AZT), abacavir (ABC), 2',3'-dideoxyinosine (DDI), lamivudine (3TC), nevirapine (NVP), and efavirenz (EFV), and as drugs of recent approval raltegravir (RAL), etravirine (ETR), and rilpivirine (RPV), were utilized as reference drugs. HIV-1 NL4-3 virus was prepared by transfection of HEK293T cells with purified plasmid DNA and quantified by p24 antigen-capture assay. For infection, CEM-GFP cells were exposed to vehicle or to several concentrations of the drugs for 2 hr at 37 °C before HIV-1 NL4-3 was added to each sample. The adsorption was prolonged for 3 hr at 37 °C. After 72 hr of incubation, HIV-induced GFP expression in infected CEM-GFP cells was assessed by flow cytometry analysis and expressed as % positive cells. For comparison, p24 production in supernatants was assessed by a commercial ELISA kit. On the basis of IC50 values, the anti-HIV activity, as assayed by this method, was EFV > 3TC > AZT > NVP > DDI > ABC and ETR > RPV > RAL. The comparison between the IC50 values calculated through flow cytometry and p24 production revealed overlapping results, showing that the optimized protocol of CEM-GFP infection with HIV NL4-3 is a suitable method to perform quantitative, rapid and low-expensive screening tests to evaluate the in vitro effect of new candidate anti-HIV drugs.
giu-2016
Pubblicato
Rilevanza internazionale
Articolo
Esperti anonimi
Settore BIO/14
Settore MED/07 - Microbiologia e Microbiologia Clinica
English
Con Impact Factor ISI
anti-HIV assay; antiviral drugs; drug resistance; green fluorescent protein (GFP); human immunodeficiency virus type 1 (HIV-1)
Frezza, C., Grelli, S., Federico, M., Marino Merlo, F., Mastino, A., Macchi, B. (2016). Testing anti-HIV activity of antiretroviral agents in vitro using flow cytometry analysis of CEM-GFP cells infected with transfection-derived HIV-1 NL4-3. JOURNAL OF MEDICAL VIROLOGY, 88(6), 979-986 [10.1002/jmv.24418].
Frezza, C; Grelli, S; Federico, M; Marino Merlo, F; Mastino, A; Macchi, B
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/162460
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