In vitro microbicidal activity of the nonnucleoside reverse transcriptase inhibitor (NNRTI) UC781 against NNRTI-resistant human immunodeficiency virus type 1

Abstract

The nonnucleoside reverse transcriptase inhibitor (NNRTI) UC781 is under development as a microbicide to prevent sexual transmission of the human immunodeficiency virus type 1 (HIV-1). However, NNRTI-resistant HIV-1 is increasingly prevalent in the infected population, and one of the concerns for NNRTI-based microbicides is that they will be ineffective against drug-resistant virus and may in fact selectively transmit NNRTI-resistant virus. We evaluated the microbicidal activity of UC781 against UC781-resistant (UCR), efavirenz-resistant (EFVR), and nevirapine-resistant (NVPR) strains in a variety of microbicide-relevant tests, including inactivation of cell-free virus, inhibition of cell-to-cell HIV-1 transmission, and the ability of UC781 pretreatment to protect cells from subsequent infection in the absence of exogenous drug. UC781 was 10- to 100-fold less effective against NNRTI-resistant HIV-1 compared to wild-type (wt) virus in each of these tests, with UC781 microbicidal activity against the various virus strains being wt > or = NVPR > UCR > or = EFVR. Breakthrough experiments using UC781-pretreated cells and mixtures of wt and NNRTI-resistant HIV-1 showed that UC781-pretreatment selected for NNRTI-resistant HIV-1. However, the efficacy of UC781 was dose dependent, and 25 microM UC781 provided essentially equivalent microbicidal activity against NNRTI-resistant and wt virus. The amount of UC781 in topical microbicide formulations under current development is approximately 100-fold greater than this concentration, so transmission of NNRTI-resistant virus may not be an issue at these microbicide formulation levels of UC781. Nonetheless, the reduced microbicidal activity of UC781 against NNRTI-resistant HIV-1 suggests that additional antiviral agents should be included in NNRTI-based microbicide formulations.

FIG. 1.

Inactivation of cell-free HIV-1 particles following exposure to different concentrations of UC781. NNRTI-resistant HIV (UCR, EFVR, and NVPR) and wt NL4-3 were incubated with the indicated concentrations of UC781 for 1 h at 37°C. Excess drug was then removed by repeated dilution and concentration by ultrafiltration as described in Materials and Methods. Infectivity was then assessed by addition of equivalent aliquots of the different virus strains (normalized for p24 content) to CEM cells. HIV-1 p24 antigen content of cell culture supernatants was measured 5 days postinfection. ▿, wt HIV-1 NL4-3; •, NVPR HIV-1; ○, UCR HIV-1; ▴, EFVR HIV-1. □, antiviral activity of the final virus wash medium. Data shown are the means ± standard deviation of triplicate determinations.

FIG. 2.

Effect of UC781 treatment on transmission of cell-associated HIV-1. H9 cells persistently infected with wt or UCR HIV-1 were incubated with 25 μM UC781 for 18 h at 37°C. Exogenous UC781 was removed from the treated cells and from the virions produced by the cells during the 18-h incubation. (A) Infectivity of the virions produced by persistently infected H9 cells incubated for 18 h in the absence (gray bars) or the presence (black bars) of 25 μM UC781. Data are the means ± standard deviation of at least three individual determinations. (B) Persistently infected H9 cells were incubated in the absence or presence of 25 μM UC781 for 18 h, and the cells were then washed several times to remove exogenous drug. The cells were then cultured in drug-free medium, and the virus produced during each sequential 24-h period was isolated and evaluated for the ability to infect CEM cells. ▿, wt HIV-1, cells not exposed to drug; ○, UCR HIV-1, cells not exposed to drug; ▾, wt HIV-1, cells pretreated with 25 μM UC781; •, UCR HIV-1, cells pretreated with 25 μM UC781. Data are the means ± standard deviation of at least three individual determinations. (C) Persistently infected H9 cells were incubated in the absence or the presence of 25 μM UC781 for 18 h, and the cells were then washed several times to remove exogenous drug. The cells were then cultured in drug-free medium. At 24-h intervals, cells were isolated and then cocultured with uninfected MT4 cells at a ratio of 10³ persistently infected H9 cells to 2 × 10⁵ uninfected MT4 cells. The extent of cell-to-cell transmission of virus was evaluated by measuring HIV-1 p24 antigen in cell supernatants obtained 4 days postinfection. Gray bars, infectivity of persistently infected H9 cells after 24 h of culture in drug-free medium; white bars, infectivity of persistently infected H9 cells after 48 h of culture in drug-free medium; black bars, infectivity of persistently infected H9 cells following 72 h of culture in drug-free medium. Data are the means ± standard deviation of at least three individual determinations.

FIG. 3.

Effect of UC781 pretreatment of cells on subsequent HIV-1 infection in the absence of exogenous drug. CEM cells were incubated with the indicated concentrations of UC781 for 18 h at 37°C, and then exogenous drug was removed by repeated washing with RPMI 1640 medium containing 10% FBS. The pretreated cells were then exposed to equivalent inocula of wt or NNRTI-resistant HIV-1 (normalized for p24). HIV-1 replication was evaluated by measuring p24 antigen in the culture supernatants 7 days postinfection. ▿, wt HIV-1 NL4-3; •, NVPR HIV-1; ○, UCR HIV-1; ▴, EFVR HIV-1. Data are the means ± standard deviation of triplicate determinations.

FIG. 4.

Effect of UC781 pretreatment of cells on subsequent infection by mixtures of wt and NNRTI-resistant HIV-1 in the absence of exogenous drug. CEM cells were incubated with 25 μM UC781 for 18 h at 37°C and then washed free of residual exogenous drug. Cells were infected with different ratios of wt NL4-3 and (A) UCR HIV-1, (B) EFVR HIV-1, or (C) NVPR HIV-1. Aliquots of cell culture supernatants were assessed for HIV-1 p24 antigen levels every 2 days postinfection. The ratios of wt to NNRTI-resistant HIV-1 in the inocula were as follows: •, wt only; ▿, 10% NNRTI resistant; ▪, 25% NNRTI resistant; ⋄, 50% NNRTI resistant; ▴, 75% NNRTI resistant; ○, 90% NNRTI resistant; •, 100% NNRTI-resistant. ▿, untreated cells infected with 100% wt or 100% NNRTI-resistant HIV-1. Data are the means ± standard deviation of triplicate determinations.