No abstract

Several classes of microbicides are being evaluated for the prevention of sexual HIV transmission.In vivo, the infectious dose and viral source involved in transmission remain uncertain and it is likely that women will use microbicides both before and after high-risk HIV exposure. Therefore, we evaluated HIV entry inhibitors (EIs) and reverse transcriptase inhibitors (RTIs) for their ability to block cell-free and cell-associated HIV-1 infection in co-cultures of monocyte-derived dendritic cells (MO-DC) and CD4 + T-cells using settings of pre-and post-exposure prophylaxis. In the pre-exposure assay, where compound was present before, during and 24 h after infection, all tested EIs (BMS806, TAK779 and T20) and RTIs (PMPA, TMC120 and UC781) blocked infection with 10 -4 multiplicity of infection (MOI) of cell-free virus at a dose between 100 and 10,000 nM, dependent on the compound used. At 10 -3 MOI, however, only T20 and the RTIs completely blocked infection. Furthermore, in experiments with cell-associated virus, EIs were ineffective, whereas RTIs actively blocked infection with similar potency as in the experiments with cell-free virus. In the post-exposure assay, where compound was added 2 h after infection and remained present for 24 h, EIs were inactive whereas RTIs blocked cellfree and cell-associated viral infections equally efficiently. Moreover, post-exposure prophylaxis initiated 24 h after infection with cell-free or cellassociated HIV-1 was still effective with 1,000 nM of TMC120. Both EIs and RTIs were non-cytotoxic at any tested concentration for MO-DC and CD4 + T-cells in co-culture. Our study shows that RTIs are potent inhibitors of cell-free and cell-associated virus used either in pre-or post-exposure settings. It highlights that parameters such as viral input, viral source, the time of compound addition and the target cells should be considered in microbicides evaluation.

Conceptually, blocking human immunodeficiency virus type 1 (HIV-1) integration is the last possibility for preventing irreversible cellular infection. Using cocultures of monocyte-derived dendritic cells and CD4؉ T cells, which represent primary targets in sexual transmission, we demonstrated that blocking integration with integrase strand transfer inhibitors (InSTIs), particularly L-870812, could consistently block cell-free and cell-associated HIV-1 infection. In a pretreatment setting in which the compound was present before and during infection and was afterwards gradually diluted during the culture period, the naphthyridine carboxamide L-870812 blocked infection with the cell-free and cell-associated HIV-1 Ba-L strain at concentrations of, respectively, 1,000 and 10,000 nM. The potency of L-870812 was similar to that of the nucleotide reverse transcriptase inhibitor R-9-(2-phosphonylmethoxypropyl) adenine (PMPA) but one or two orders of magnitude lower than those of the nonnucleoside reverse transcriptase inhibitors UC781 and TMC120. In contrast, the diketo acid RDS derivative InSTIs showed clear-cut but weaker antiviral activity than L-870812. Moreover, L-870812 completely blocked subtype C and CRFO2_AG primary isolates, which are prevalent in the African heterosexual epidemic. Furthermore, the addition of micromolar concentrations of L-870812 even 24 h after infection could still block both cell-free and cell-associated Ba-L, opening the prospect of postexposure prophylaxis. Finally, an evaluation of the combined activity of L-870812 with either T20, zidovudine, PMPA, UC781, or TMC120 against replication-deficient HIV-1 Ba-L (env) pseudovirus suggested synergistic activity for all combinations. Importantly, compounds selected for the study by using the coculture model were devoid of acute or delayed cytotoxic effects at HIV-blocking concentrations. Therefore, these findings provide evidence supporting consideration of HIV-1 integration as a target for microbicide development.

Microbicides based on nonnucleoside reverse transcriptase inhibitors (NNRTIs) are currently being developed to protect women from HIV acquisition through sexual contact. However, the large-scale introduction of these products raises two major concerns. First, when these microbicides are used by undiagnosed HIV-positive women, they could potentially select for viral resistance, which may compromise subsequent therapeutic options. Second, NNRTI-based microbicides that are inactive against NNRTI-resistant strains might promote the selective transmission of these viruses. In order to address these concerns, drug resistance was selected in vitro by the serial passage of three viral isolates from subtypes B and C and CRF02_AG (a circulating recombinant form) in activated peripheral blood mononuclear cells (PBMCs) under conditions of increasing concentrations of three NNRTIs (i.e., TMC120, UC781, and MIV-160) that are currently being developed as candidate microbicides. TMC120 and MIV-160 displayed a high genetic barrier to resistance development, whereas resistance to UC781 emerged rapidly, similarly to efavirenz and nevirapine. Phenotypically, the selected viruses appeared to be highly cross-resistant to current first-line therapeutic NNRTIs (i.e., delavirdine, nevirapine, and efavirenz), although they retained some susceptibility to the more recently developed NNRTIs lersivirine and etravirine. The ability of UC781, TMC120, and MIV-160 to inhibit the in vitro-selected NNRTI-resistant viruses was also limited, although residual activity could be observed for the candidate microbicide NNRTI MIV-170. Interestingly, only four p2/p7/p1/p6/PR/RT/INT recombinant NNRTI-resistant viruses (i.e., TMC120-resistant VI829, EFV-resistant VI829, MIV-160-resistant VI829, and EFV-resistant MP568) showed impairments in replicative fitness. Overall, these in vitro analyses demonstrate that due to potential cross-resistance, the large-scale introduction of single-NNRTI-based microbicides should be considered with caution.