Identification and characterization of persistent intracellular human immunodeficiency virus type 1 integrase strand transfer inhibitor activity

Abstract

Pharmacokinetic and pharmacodynamic considerations significantly impact infectious disease treatment options. One aspect of pharmacodynamics is the postantibiotic effect, classically defined as delayed bacterial growth after antibiotic removal. The same principle can apply to antiviral drugs. For example, significant delays in human immunodeficiency virus type 1 (HIV-1) replication can be observed after nucleoside/nucleotide reverse transcriptase inhibitor (N/NtRTI) removal from culture medium, because these prodrugs must be anabolized into active, phosphorylated forms once internalized into cells. A relatively new class of anti-HIV-1 drugs is the integrase strand transfer inhibitors (INSTIs), and the INSTIs raltegravir (RAL) and elvitegravir (EVG) were tested here alongside positive N/NtRTI controls tenofovir disoproxil fumarate (TDF) and azidothymidine (AZT), as well as the nonnucleoside reverse transcriptase inhibitor negative control nevirapine (NVP), to assess potential postantiviral effects. Transformed and primary CD4-positive cells pretreated with INSTIs significantly resisted subsequent challenge by HIV-1, revealing the following hierarchy of persistent intracellular drug strength: TDF > EVG ∼ AZT > RAL > NVP. A modified time-of-addition assay was moreover developed to assess residual drug activity levels. Approximately 0.8% of RAL and 2% of initial EVG and TDF 1-h pulse drug levels persisted during the acute phase of HIV-1 infection. EVG furthermore displayed significant virucidal activity. Although there is no reason to suspect obligate intracellular modification, this study nevertheless defines significant intracellular persistence of prototype INSTIs. Ongoing second-generation formulations should therefore consider the potential for significant postantiviral effects among this drug class. Combined intracellular persistence and virucidal activities suggest potential pre-exposure prophylaxis applications for EVG.

FIG. 1.

Functional windows of antiviral drug action. (A) At the indicated times, 10-fold EC₉₅ adjusted levels of RAL, EVG, AZT, or NVP were added to HIV-Luc-infected cultures. Cells were lysed at 48 h postinfection, and luciferase activities are expressed as the percentage of the response in non-drug-treated cells. (B) Results of an experiment similar to that in panel A, except cells were treated with 10× the EC₉₅ of RAL, 100× the EC₉₅ of RAL, 10× the EC₉₅ of EVG, or 100× the EC₉₅ of EVG at the indicated times. Numbers indicate the time (in h) required for 50% inhibition compared to the no-drug control (dashed lines).

FIG. 2.

Functional intracellular persistence of HIV-1 inhibitors. Cells exposed to 100× the EC₉₅ levels of each compound for 24 h were washed to deplete extracellular drug, cultured for various time periods (0, 2, 4, 8, or 12 h), exposed to HIV-Luc, and then further cultured for 48 h prior to cell lysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to the no-drug control (based on Student's t test).

FIG. 3.

Virucidal activities of anti-HIV-1 compounds. (A) Normalized HIV-Luc infectivities following standard 2-h absorption, shortened 2-min absorption, or 2 min of MNP-mediated infection. (B) HIV-Luc was preincubated with the indicated level of drug for 1 h prior to 2 min of MNP-mediated infection. Extensively washed cultures were incubated in the absence of drug for 2 days prior to cell lysis and luciferase assays. The NN-luc control virus was omitted from the virucidal activity assays. **, P < 0.01; ***, P < 0.001 compared to the no-drug control (based on Student's t test). RLU, relative light units.

FIG. 4.

TOA pulse assay. RAL, EVG, TDF, or NVP (100× the EC₉₅ adjusted) was removed from infected cultures at the indicated time points after a 1-h pulse. Thereafter, rewashed cells were incubated prior to lysis at 48 h postinfection. Results shown are the percent infectivity in comparison to no-drug controls.